GSK2982772 is a potent and selective receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate for the Treatment of Inflammatory Diseases. GSK2982772 is, currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. GSK2982772 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. RIP1 has emerged as an important upstream kinase that has been shown to regulate inflammation through both scaffolding and kinase specific functions.
GSK-2982772, an oral receptor-interacting protein-1 (RIP1) kinase inhibitor, is in phase II clinical development at GlaxoSmithKline for the treatment of active plaque-type psoriasis, moderate to severe rheumatoid arthritis, and active ulcerative colitis. A phase I trial was also completed for the treatment of inflammatory bowel disease using capsule and solution formulations.
Originator GlaxoSmithKline
Class Antipsoriatics
Mechanism of Action Receptor-interacting protein serine-threonine kinase inhibitors
Highest Development Phases
Phase II Plaque psoriasis; Rheumatoid arthritis; Ulcerative colitis
Phase I Inflammatory bowel diseases
Most Recent Events
15 Dec 2016 Biomarkers information updated
01 Nov 2016 Phase-II clinical trials in Ulcerative colitis (Adjunctive treatment) in USA (PO) (NCT02903966)
01 Oct 2016 Phase-II clinical trials in Rheumatoid arthritis in Poland (PO) (NCT02858492)
PHASE 2 Psoriasis, plaque GSK
Inflammatory Bowel Disease, Agents for
Rheumatoid Arthritis, Treatment of
Antipsoriatics
Data Science and Informatics Leader | Innovation Advocate
GSK
University of North Carolina at Chapel Hill
He is a data scientist and innovator with experience in both early and late stages of drug development. his current role involves the late stage of drug product development. I’m leading a project to bring GSK’s large molecule process and analytical data onto our big data platform and develop new data analysis and modeling capabilities. Also, working within GSK’s Advanced Manufacturing Technology (AMT) initiative provides plenty of other opportunities to impact how we make medicines.
Previously as a computational chemist (i.e. a data scientist in drug discovery), he worked with scientists from many domains, including chemists, biologists, and other informaticians. he enjoys digging into all the computational aspects of life science research, and solving data challenges by exploiting adjacencies and connections – between diverse fields of knowledge, and the equally diverse scientists trained in them.
He has supported multiple drug discovery projects at GSK starting from target identification (“how should we modulate disease X?”) through to candidate selection and early clinical development (“let’s see if what we discovered can become a medicine”). Deriving insight by custom data integration is one of my specialties; recently he designed and implemented a platform for integrating data sets from multiple experiments that will be used by GSK screening scientists to find and combine hits.
A trained computer scientist and cheminformatician, he is an active member of the algorithms, data science and internal innovation communities at GSK, leading many of these efforts.
His Ph.D. work introduced new computational geometry techniques for structural bioinformatics and protein function prediction. I have touched on several other subject areas:
* data mining/machine learning (predictive modeling and graph mining), * computer graphics and augmented reality (one of the pioneers of projection mapping) * robotics (keen current interest and future aspiration)
Receptor-interacting protein- 1 (RIP1) kinase, originally referred to as RIP, is a TKL family serine/threonine protein kinase involved in innate immune signaling. RIPl kinase is a RHIM domain containing protein, with an N-terminal kinase domain and a C-terminal death domain ((2005) Trends Biochem. Sci. 30, 151-159). The death domain of RIPl mediates interaction with other death domain containing proteins including Fas and TNFR-1 ((1995) Cell 81 513-523), TRAIL-Rl and TRAIL-R2 ((1997) Immunity 7, 821-830) and TRADD ((1996) Immunity 4, 387-396), while the RHIM domain is crucial for binding other RHFM domain containing proteins such as TRIF ((2004) Nat Immunol. 5, 503-507), DAI ((2009) EMBO Rep. 10, 916-922) and RIP3 ((1999) J. Biol. Chem. 274, 16871-16875); (1999) Curr. Biol. 9, 539-542) and exerts many of its effects through these interactions. RIPl is a central regulator of cell signaling, and is involved in mediating both pro-survival and programmed cell death pathways which will be discussed below.
The role for RIPl in cell signaling has been assessed under various conditions
36560-36566), TRAIL ((2012) J .Virol. Epub, ahead of print), FAS ((2004) J. Biol. Chem. 279, 7925-7933)], but is best understood in the context of mediating signals downstream of the death receptor TNFRl ((2003) Cell 114, 181-190). Engagement of the TNFR by TNF leads to its oligomerization, and the recruitment of multiple proteins, including linear K63-linked polyubiquitinated RIPl ((2006) Mol. Cell 22, 245-257), TRAF2/5 ((2010) J. Mol. Biol. 396, 528-539), TRADD ((2008) Nat. Immunol. 9, 1037-1046) and cIAPs ((2008) Proc. Natl. Acad. Sci. USA. 105, 1 1778-11783), to the cytoplasmic tail of the receptor. This complex which is dependent on RIPl as a scaffolding protein (i.e. kinase
independent), termed complex I, provides a platform for pro-survival signaling through the activation of the NFKB and MAP kinases pathways ((2010) Sci. Signal. 115, re4).
Alternatively, binding of TNF to its receptor under conditions promoting the
deubiquitination of RIPl (by proteins such as A20 and CYLD or inhibition of the cIAPs) results in receptor internalization and the formation of complex II or DISC (death-inducing signaling complex) ((2011) Cell Death Dis. 2, e230). Formation of the DISC, which contains RIPl, TRADD, FADD and caspase 8, results in the activation of caspase 8 and the onset of programmed apoptotic cell death also in a RIPl kinase independent fashion ((2012) FEBS J 278, 877-887). Apoptosis is largely a quiescent form of cell death, and is involved in routine processes such as development and cellular homeostasis.
Under conditions where the DISC forms and RJP3 is expressed, but apoptosis is inhibited (such as FADD/caspase 8 deletion, caspase inhibition or viral infection), a third RIPl kinase-dependent possibility exists. RIP3 can now enter this complex, become phosphorylated by RIPl and initiate a caspase-independent programmed necrotic cell death through the activation of MLKL and PGAM5 ((2012) Cell 148, 213-227); ((2012) Cell 148, 228-243); ((2012) Proc. Natl. Acad. Sci. USA. 109, 5322-5327). As opposed to apoptosis, programmed necrosis (not to be confused with passive necrosis which is not programmed) results in the release of danger associated molecular patterns (DAMPs) from the cell.
These DAMPs are capable of providing a “danger signal” to surrounding cells and tissues, eliciting proinflammatory responses including inflammasome activation, cytokine production and cellular recruitment ((2008 Nat. Rev. Immunol 8, 279-289).
Dysregulation of RIPl kinase-mediated programmed cell death has been linked to various inflammatory diseases, as demonstrated by use of the RIP3 knockout mouse (where RIPl -mediated programmed necrosis is completely blocked) and by Necrostatin-1 (a tool inhibitor of RIPl kinase activity with poor oral bioavailability). The RIP3 knockout mouse has been shown to be protective in inflammatory bowel disease (including Ulcerative colitis and Crohn’s disease) ((2011) Nature 477, 330-334), Psoriasis ((2011) Immunity 35, 572-582), retinal-detachment-induced photoreceptor necrosis ((2010) PNAS 107, 21695-21700), retinitis pigmentosa ((2012) Proc. Natl. Acad. Sci., 109:36, 14598-14603), cerulein-induced acute pancreatits ((2009) Cell 137, 1100-1111) and Sepsis/systemic inflammatory response syndrome (SIRS) ((2011) Immunity 35, 908-918). Necrostatin-1 has been shown to be effective in alleviating ischemic brain injury ((2005) Nat. Chem. Biol. 1, 112-119), retinal ischemia/reperfusion injury ((2010) J. Neurosci. Res. 88, 1569-1576), Huntington’s disease ((2011) Cell Death Dis. 2 el 15), renal ischemia reperfusion injury ((2012) Kidney Int. 81, 751-761), cisplatin induced kidney injury ((2012) Ren. Fail. 34, 373-377) and traumatic brain injury ((2012) Neurochem. Res. 37, 1849-1858). Other diseases or disorders regulated at least in part by RIPl -dependent apoptosis, necrosis or cytokine production include hematological and solid organ malignancies ((2013) Genes
Dev. 27: 1640-1649), bacterial infections and viral infections ((2014) Cell Host & Microbe 15, 23-35) (including, but not limited to, tuberculosis and influenza ((2013) Cell 153, 1-14)) and Lysosomal storage diseases (particularly, Gaucher Disease, Nature Medicine Advance Online Publication, 19 January 2014, doi: 10.1038/nm.3449).
A potent, selective, small molecule inhibitor of RIP1 kinase activity would block RIP 1 -dependent cellular necrosis and thereby provide a therapeutic benefit in diseases or events associated with DAMPs, cell death, and/or inflammation.
A mixture of (S)-3-amino-5-methyl-2,3-dihydrobenzo[b][l,4]oxazepin-4(5H)-one, hydrochloride (4.00 g, 16.97 mmol), 5-benzyl-4H-l,2,4-triazole-3-carboxylic acid, hydrochloride (4.97 g, 18.66 mmol) and DIEA (10.37 mL, 59.4 mmol) in isopropanol (150 mL) was stirred vigorously for 10 minutes and then 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (T3P) (50% by wt. in EtOAc) (15.15 mL, 25.5 mmol) was added. The mixture was stirred at rt for 10 minutes and then quenched with water and concentrated to remove isopropanol. The resulting crude material is dissolved in EtOAc and washed with 1M HC1, satd. NaHC03 and brine. Organics were concentrated and purified by column chromatography (220 g silica column; 20-90% EtOAc/hexanes, 15 min.; 90%, 15 min.) to give the title compound as a light orange foam (5.37 g, 83%). 1H NMR (MeOH-d4) δ: 7.40 – 7.45 (m, 1H), 7.21 – 7.35 (m, 8H), 5.01 (dd, J = 11.6, 7.6 Hz, 1H), 4.60 (dd, J = 9.9, 7.6 Hz, 1H), 4.41 (dd, J = 11.4, 9.9 Hz, 1H), 4.17 (s, 2H), 3.41 (s, 3H); MS (m/z) 378.3 (M+H+).
Alternative Preparation:
To a solution of (S)-3-amino-5-methyl-2,3-dihydrobenzo[b][l,4]oxazepin-4(5H)-one hydrochloride (100 g, 437 mmol), 5-benzyl-4H-l,2,4-triazole-3-carboxylic acid hydrochloride (110 g, 459 mmol) in DCM (2.5 L) was added DIPEA (0.267 L, 1531 mmol) at 15 °C. The reaction mixture was stirred for 10 min. and 2,4,6-tripropyl-l, 3, 5,2,4,6-trioxatriphosphinane 2,4,6-trioxide >50 wt. % in ethyl acetate (0.390 L, 656 mmol) was slowly added at 15 °C. After stirring for 60 mins at RT the LCMS showed the reaction was complete, upon which time it was quenched with water, partitioned between DCM and washed with 0.5N HCl aq (2 L), saturated aqueous NaHC03 (2 L), brine (2 L) and water (2 L). The organic phase was separated and activated charcoal (100 g) and sodium sulfate
(200 g) were added. The dark solution was shaken for 1 h before filtering. The filtrate was then concentrated under reduced pressure to afford the product as a tan foam (120 g). The product was dried under a high vacuum at 50 °C for 16 h. 1H MR showed 4-5% wt of ethyl acetate present. The sample was dissolved in EtOH (650 ml) and stirred for 30 mins, after which the solvent was removed using a rotavapor (water-bath T=45 °C). The product was dried under high vacuum for 16 h at RT (118 g, 72% yield). The product was further dried under high vacuum at 50 °C for 5 h. 1H NMR showed <1% of EtOH and no ethyl acetate. 1H NMR (400 MHz, DMSO-i¾) δ ppm 4.12 (s, 2 H), 4.31 – 4.51 (m, 1 H), 4.60 (t, J=10.36 Hz, 1 H), 4.83 (dt, 7=11.31, 7.86 Hz, 1 H), 7.12 – 7.42 (m, 8 H), 7.42 – 7.65 (m, 1 H), 8.45 (br. s., 1 H), 14.41 (br. s., 1 H). MS (m/z) 378 (M + H+).
Crystallization:
(S)-5-Benzyl-N-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][l,4]oxazepin-3-yl)-4H-l,2,4-triazole-3-carboxamide (100 mg) was dissolved in 0.9 mL of toluene and 0.1 mL of methylcyclohexane at 60 °C, then stirred briskly at room temperature (20 °C) for 4 days. After 4 days, an off-white solid was recovered (76 mg, 76% recovery). The powder X-ray diffraction (PXRD) pattern of this material is shown in Figure 7 and the corresponding diffraction data is provided in Table 1.
The PXRD analysis was conducted using a PANanalytical X’Pert Pro
diffractometer equipped with a copper anode X-ray tube, programmable slits, and
X’Celerator detector fitted with a nickel filter. Generator tension and current were set to 45kV and 40mA respectively to generate the copper Ka radiation powder diffraction pattern over the range of 2 – 40°2Θ. The test specimen was lightly triturated using an agate mortar and pestle and the resulting fine powder was mounted onto a silicon background plate.
Table 1.
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Paper
Discovery of a first-in-class receptor interacting protein 1 (RIP1) kinase specific clinical candidate (GSK2982772) for the treatment of inflammatory diseases
J Med Chem 2017, 60(4): 1247
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
Synthesis of (<it>S</it>)-3-amino-benzo[<it>b</it>][1,4]oxazepin-4-one via Mitsunobu and S<INF>N</INF>Ar reaction for a first-in-class RIP1 kinase inhibitor GSK2982772 in clinical trials
Tetrahedron Lett 2017, 58(23): 2306 Harris, P.A. Identification of a first-in-class RIP1 kinase inhibitor in phase 2a clinical trials for immunoinflammatory diseases
ACS MEDI-EFMC Med Chem Front (June 25-28, Philadelphia) 2017, Abst
Harris, P. Identification of a first-in-class RIP1 kinase inhibitor in phase 2a clinical trials for immuno-inflammatory diseases
253rd Am Chem Soc (ACS) Natl Meet (April 2-6, San Francisco) 2017, Abst MEDI 313
Mechanism of Action Sodium-bile acid cotransporter-inhibitors
Highest Development Phases
Phase II Primary biliary cirrhosis; Pruritus; Type 2 diabetes mellitus
Phase I Cholestasis
Most Recent Events
01 Jan 2017 Phase-II clinical trials in Pruritus in USA (PO) (NCT02966834)
14 Nov 2016 GlaxoSmithKline completes a phase I trial for Cholestasis in Healthy volunteers in Japan (PO, Tablet) (NCT02801981)
11 Nov 2016 Efficacy, safety and pharmacodynamic data from a phase II trial in Primary biliary cirrhosis and Pruritus presented at The Liver Meeting® 2016: 67th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD-2016)
GSK2330672 , an ileal bile acid transport (iBAT) inhibitor indicated for diabetes type II and cholestatic pruritus, is currently under Phase IIb evaluation in the clinic. The API is a highly complex molecule containing two stereogenic centers, one of which is quaternary
GSK-2330672 is highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor for treatment of type 2 diabetes.
More than 200 million people worldwide have diabetes. The World Health Organization estimates that 1 .1 million people died from diabetes in 2005 and projects that worldwide deaths from diabetes will double between 2005 and 2030. New chemical compounds that effectively treat diabetes could save millions of human lives.
Diabetes refers to metabolic disorders resulting in the body’s inability to effectively regulate glucose levels. Approximately 90% of all diabetes cases are a result of type 2 diabetes whereas the remaining 10% are a result of type 1 diabetes, gestational diabetes, and latent autoimmune diabetes of adulthood (LADA). All forms of diabetes result in elevated blood glucose levels and, if left untreated chronically, can increase the risk of macrovascular (heart disease, stroke, other forms of cardiovascular disease) and microvascular [kidney failure (nephropathy), blindness from diabetic retinopathy, nerve damage (diabetic neuropathy)] complications.
Type 1 diabetes, also known as juvenile or insulin-dependent diabetes mellitus (IDDM), can occur at any age, but it is most often diagnosed in children, adolescents, or young adults. Type 1 diabetes is caused by the autoimmune destruction of insulin-producing beta cells, resulting in an inability to produce sufficient insulin. Insulin controls blood glucose levels by promoting transport of blood glucose into cells for energy use. Insufficient insulin production will lead to decreased glucose uptake into cells and result in accumulation of glucose in the bloodstream. The lack of available glucose in cells will eventually lead to the onset of symptoms of type 1 diabetes: polyuria (frequent urination), polydipsia (thirst), constant hunger, weight loss, vision changes, and fatigue. Within 5-10 years of being diagnosed with type 1 diabetes, patient’s insulin-producing beta cells of the pancreas are completely destroyed, and the body can no longer produce insulin. As a result, patients with type 1 diabetes will require daily administration of insulin for the remainder of their lives.
Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, occurs when the pancreas produces insufficient insulin and/or tissues become resistant to normal or high levels of insulin (insulin resistance), resulting in excessively high blood glucose levels. Multiple factors can lead to insulin resistance including chronically elevated blood glucose levels, genetics, obesity, lack of physical activity, and increasing age. Unlike type 1 diabetes, symptoms of type 2 diabetes are more salient, and as a result, the disease may not be diagnosed until several years after onset with a peak prevalence in adults near an age of 45 years. Unfortunately, the incidence of type 2 diabetes in children is increasing.
The primary goal of treatment of type 2 diabetes is to achieve and maintain glycemic control to reduce the risk of microvascular (diabetic neuropathy, retinopathy, or nephropathy) and macrovascular (heart disease, stroke, other forms of cardiovascular disease) complications. Current guidelines for the treatment of type 2 diabetes from the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) [Diabetes Care, 2008, 31 (12), 1 ] outline lifestyle modification including weight loss and increased physical activity as a primary therapeutic approach for management of type 2 diabetes. However, this approach alone fails in the majority of patients within the first year, leading physicians to prescribe medications over time. The ADA and EASD recommend metformin, an agent that reduces hepatic glucose production, as a Tier 1 a medication; however, a significant number of patients taking metformin can experience gastrointestinal side effects and, in rare cases, potentially fatal lactic acidosis. Recommendations for Tier 1 b class of medications include sulfonylureas, which stimulate pancreatic insulin secretion via modulation of potassium channel activity, and exogenous insulin. While both medications rapidly and effectively reduce blood glucose levels, insulin requires 1 -4 injections per day and both agents can cause undesired weight gain and potentially fatal hypoglycemia. Tier 2a recommendations include newer agents such as thiazolidinediones (TZDs pioglitazone and rosiglitazone), which enhance insulin sensitivity of muscle, liver and fat, as well as GLP-1 analogs, which enhance postprandial glucose-mediated insulin secretion from pancreatic beta cells. While TZDs show robust, durable control of blood glucose levels, adverse effects include weight gain, edema, bone fractures in women, exacerbation of congestive heart failure, and potential increased risk of ischemic cardiovascular events. GLP-1 analogs also effectively control blood glucose levels, however, this class of medications requires injection and many patients complain of nausea. The most recent addition to the Tier 2 medication list is DPP-4 inhibitors, which, like GLP-1 analogs, enhance glucose- medicated insulin secretion from beta cells. Unfortunately, DPP-4 inhibitors only modestly control blood glucose levels, and the long-term safety of DPP-4 inhibitors remains to be firmly established. Other less prescribed medications for type 2 diabetes include a-glucosidase inhibitors, glinides, and amylin analogs. Clearly, new medications with improved efficacy, durability, and side effect profiles are needed for patients with type 2 diabetes.
GLP-1 and GIP are peptides, known as incretins, that are secreted by L and K cells, respectively, from the gastrointestinal tract into the blood stream following ingestion of nutrients. This important physiological response serves as the primary signaling mechanism between nutrient (glucose/fat) concentration in the
gastrointestinal tract and other peripheral organs. Upon secretion, both circulating peptides initiate signals in beta cells of the pancreas to enhance glucose-stimulated insulin secretion, which, in turn, controls glucose concentrations in the blood stream (For reviews see: Diabetic Medicine 2007, 24(3), 223; Molecular and Cellular Endocrinology 2009, 297(1-2), 127; Experimental and Clinical Endocrinology & Diabetes 2001 , 109(Suppl. 2), S288).
The association between the incretin hormones GLP-1 and GIP and type 2 diabetes has been extensively explored. The majority of studies indicate that type 2 diabetes is associated with an acquired defect in GLP-1 secretion as well as GIP action (see Diabetes 2007, 56(8), 1951 and Current Diabetes Reports 2006, 6(3), 194). The use of exogenous GLP-1 for treatment of patients with type 2 diabetes is severely limited due to its rapid degradation by the protease DPP-4. Multiple modified peptides have been designed as GLP-1 mimetics that are DPP-4 resistant and show longer half-lives than endogenous GLP-1 . Agents with this profile that have been shown to be highly effective for treatment of type 2 diabetes include exenatide and liraglutide, however, these agents require injection. Oral agents that inhibit DPP-4, such as sitagliptin vildagliptin, and saxagliptin, elevate intact GLP-1 and modestly control circulating glucose levels (see Pharmacology & Therapeutics 2010, 125(2), 328; Diabetes Care 2007, 30(6), 1335; Expert Opinion on Emerging Drugs 2008, 13(4), 593). New oral medications that increase GLP-1 secretion would be desirable for treatment of type 2 diabetes.
Bile acids have been shown to enhance peptide secretion from the
gastrointestinal tract. Bile acids are released from the gallbladder into the small intestine after each meal to facilitate digestion of nutrients, in particular fat, lipids, and lipid-soluble vitamins. Bile acids also function as hormones that regulate cholesterol homeostasis, energy, and glucose homeostasis via nuclear receptors (FXR, PXR, CAR, VDR) and the G-protein coupled receptor TGR5 (for reviews see: Nature Drug Discovery 2008, 7, 672; Diabetes, Obesity and Metabolism 2008, 10, 1004). TGR5 is a member of the Rhodopsin-like subfamily of GPCRs (Class A) that is expressed in intestine, gall bladder, adipose tissue, liver, and select regions of the central nervous system. TGR5 is activated by multiple bile acids with lithocholic and deoxycholic acids as the most potent activators {Journal of Medicinal Chemistry 2008, 51(6), 1831 ). Both deoxycholic and lithocholic acids increase GLP-1 secretion from an enteroendocrine STC-1 cell line, in part through TGR5
{Biochemical and Biophysical Research Communications 2005, 329, 386). A synthetic TGR5 agonist INT-777 has been shown to increase intestinal GLP-1 secretion in vivo in mice {Cell Metabolism 2009, 10, 167). Bile salts have been shown to promote secretion of GLP-1 from colonic L cells in a vascularly perfused rat colon model {Journal of Endocrinology 1995, 145(3), 521 ) as well as GLP-1 , peptide YY (PYY), and neurotensin in a vascularly perfused rat ileum model {Endocrinology 1998, 139(9), 3780). In humans, infusion of deoxycholate into the sigmoid colon produces a rapid and marked dose responsive increase in plasma PYY and enteroglucagon concentrations (Gi/M993, 34(9), 1219). Agents that increase ileal and colonic bile acid or bile salt concentrations will increase gut peptide secretion including, but not limited to, GLP-1 and PYY.
Bile acids are synthesized from cholesterol in the liver then undergo conjugation of the carboxylic acid with the amine functionality of taurine and glycine. Conjugated bile acids are secreted into the gall bladder where accumulation occurs until a meal is consumed. Upon eating, the gall bladder contracts and empties its contents into the duodenum, where the conjugated bile acids facilitate absorption of cholesterol, fat, and fat-soluble vitamins in the proximal small intestine (For reviews see: Frontiers in Bioscience 2009, 74, 2584; Clinical Pharmacokinetics 2002,
41(10), 751 ; Journal of Pediatric Gastroenterology and Nutrition 2001 , 32, 407). Conjugated bile acids continue to flow through the small intestine until the distal ileum where 90% are reabsorbed into enterocytes via the apical sodium-dependent bile acid transporter (ASBT, also known as iBAT). The remaining 10% are deconjugated to bile acids by intestinal bacteria in the terminal ileum and colon of which 5% are then passively reabsorbed in the colon and the remaining 5% being excreted in feces. Bile acids that are reabsorbed by ASBT in the ileum are then transported into the portal vein for recirculation to the liver. This highly regulated process, called enterohepatic recirculation, is important for the body’s overall maintenance of the total bile acid pool as the amount of bile acid that is synthesized in the liver is equivalent to the amount of bile acids that are excreted in feces.
Pharmacological disruption of bile acid reabsorption with an inhibitor of ASBT leads to increased concentrations of bile acids in the colon and feces, a physiological consequence being increased conversion of hepatic cholesterol to bile acids to compensate for fecal loss of bile acids. Many pharmaceutical companies have pursued this mechanism as a strategy for lowering serum cholesterol in patients with dyslipidemia/hypercholesterolemia (For a review see: Current Medicinal Chemistry 2006, 73, 997). Importantly, ASBT-inhibitor mediated increase in colonic bile acid/salt concentration also will increase intestinal GLP-1 , PYY, GLP-2, and other gut peptide hormone secretion. Thus, inhibitors of ASBT could be useful for treatment of type 2 diabetes, type 1 diabetes, dyslipidemia, obesity, short bowel syndrome, Chronic Idiopathic Constipation, Irritable bowel syndrome (IBS), Crohn’s disease, and arthritis.
Certain 1 ,4-thiazepines are disclosed, for example in WO 94/18183 and WO 96/05188. These compounds are said to be useful as ileal bile acid reuptake inhibitors (ASBT).
Patent publication WO 201 1/137,135 dislcoses, among other compounds, the following compound. This patent publication also discloses methods of synthesis of the compound.
Patent publication WO 201 1/137,135 dislcoses general methods for preparing the compound. In addition, a detailed synthesis of the compound is disclosed in Example 26. J. Med. Chem, Vol 56, pp5094-51 14 (2013) also discloses a method for synthesising the compound.
The present invention discloses an improved synthesis of the compound.
The synthetic scheme of the present invention is depicted in Scheme 1 .
Treatment of 2-methoxyphenyl acetate with sulfur monochloride followed by ester hydrolysis and reduction with zinc gave rise to thiophenol (A). Epoxide ring opening of (+)-2-butyl-ethyloxirane with thiophenol (A) and subsequent treatment of tertiary alcohol (B) with chloroacetonitrile under acidic conditions gave chloroacetamide (C), which was then converted to intermediate (E) by cleavage of the chloroacetamide with thiourea followed by classical resolution with dibenzoyl-L-tartaric acid.
Benzoylation of intermediate (E) with triflic acid and benzoyl chloride afforded intermediate (H). Cyclization of intermediate (H) followed by oxidation of the sulfide to a sulphone, subseguent imine reduction and classical resolution with (+)-camphorsulfonic acid provided intermediate (G), which was then converted to intermediate (H). Intermediate (H) was converted to the target compound using the methods disclosed in Patent publication WO 201 1/137,135.
Scheme 1
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Dibenzoyl-L-tataric acid
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The present invention also discloses an alternative method for construction of the quaternary chiral center as depicted in Scheme 2. Reaction of intermediate (A) with (R)-2-ammonio-2-ethylhexyl sulfate (K) followed by formation of di-p-toluoyl-L-tartrate salt furnished intermediate (L).
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The present invention also discloses an alternative synthesis of intermediate (H) as illustrated in Scheme 3. Acid catalyzed cyclization of intermediate (F) followed by triflation gave imine (M), which underwent asymmetric reduction with catalyst lr(COD)2BArF and ligand (N) to give intermediate (O). Oxidation of the sulfide in intermediate (O) gave sulphone intermediate (H).
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The present invention differs from the synthesis disclosed in WO 201 1/137,135 and J. Med. Chem, Vol56, pp5094-51 14 (2013) in that intermediate (H) in the present invention is prepared via a new, shorter and more cost-efficient synthesis while the synthesis of the target compound from intermediate (H) remains unchanged.
Intermediate A: 3-Hydroxy-4-methoxythiophenol
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A reaction vessel was charged with 2-methoxyphenyl acetate (60 g, 0.36 mol), zinc chloride (49.2 g, 0.36 mol) and DME (600 mL). The mixture was stirred and S2CI2 (53.6 g, 0.40 mol) was added. The mixture was stirred at ambient temperature for 2 h. Concentrated HCI (135.4 mL, 1 .63 mol) was diluted with water (60 mL) and added slowly to the rxn mixture, maintaining the temperature below 60 °C. The mixture was stirred at 60 °C for 2 h and then cooled to ambient
temperature. Zinc dust (56.7 g, 0.87 mol) was added in portions, maintaining the temperature below 60 °C. The mixture was stirred at 20-60 °C for 1 h and then concentrated under vacuum to -300 mL. MTBE (1 .2 L) and water (180 mL) were added and the mixture was stirred for 10 min. The layers were separated and the organic layer was washed twice with water (2x 240 mL). The layers were separated and the organic layer was concentrated under vacuum to give an oil. The oil was distilled at 1 10-1 15 °C/2 mbar to give the title compound (42 g, 75%) as colorless oil, which solidified on standing to afford the title compound as a white solid. M.P. 41 -42 °C. 1 H NMR (500 MHz, CDCI3)$ ppm 3.39 (s, 1 H), 3.88 (s, 3H), 5.65 (br. S, 1 H), 6.75 (d, J – 8.3 Hz, 1 H), 6.84 (ddd, J – 8.3, 2.2, 0.6 Hz, 1 H), 6.94 (d, J – 2.2 Hz).
Intermediate E: (R)-5-((2-amino-2-ethylhexyl)thio)-2-methoxyphenol, dibenzoyl-L-tartrate salt
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A reaction vessel was charged with 3-hydroxy-4-methoxythiophenol (5.0 g, 25.2 mmol), (+)-2-butyl-2-ethyloxirane (3.56 g, 27.7 mmol) and EtOH (30 mL). The mixture was treated with a solution of NaOH (2.22 g, 55.5 mmol) in water (20 mL), heated to 40 °C and stirred at 40 °C for 5 h. The mixture was cooled to ambient temperature, treated with toluene (25 mL) and stirred for 10 min. The layers were separated and the organic layer was discarded. The aqueous layer was neutralized with 2N HCI (27.8 mL, 55.6 mmol) and extracted with toluene (100 mL). The organic layer was washed with water (25 mL), concentrated in vacuo to give an oil. The oil was treated with chloroacetonitrile (35.9 mL) and HOAc (4.3 mL) and cooled to 0 °C. H2SO4 (6.7 mL, 126 mmol, pre-diluted with 2.3 mL of water) was added at a rate maintaining the temperature below 10 °C. After stirred at 0 °C for 0.5 h, the reaction mixture was treated with 20% aqueous Na2CO3 solution to adjust the pH to
7-8 and then extracted with MTBE (70 ml_). The extract was washed with water (35 ml_) and concentrated in vacuo to give an oil. The oil was then dissolved in EOH (50 ml_) and treated with HOAc (10 ml_) and thiourea (2.30 g, 30.2 mmol). The mixture was heated at reflux overnight and then cooled to ambient temperature. The solids were filtered and washed with EtOH (10 ml_). The filtrate and the wash were combined and concentrated in vacuo, treated with MTBE (140 ml_) and washed successively with 10% aqueous Na2C03 and water. The mixture was concentrated in vacuo to give an oil. The oil was dissolved in MeCN (72 ml_), heated to -50 °C and then dibenzoyl-L-tartaric acid (9.0 g, 25.2 mmol) in acetonitrile (22 ml_) was added slowly. Seed crystals were added at -50 °C. The resultant slurry was stirred at 45-50 °C for 5 h, then cooled down to ambient temperature and stirred at ambient temperature overnight. The solids were filtered and washed with MeCN (2x 22 ml_). The wet cake was treated with MeCN (150 ml_) and heated to 50 °C. The slurry was stirred at 50 °C for 5 h, cooled over 1 h to ambient temperature and stirred at ambient temperature overnight. The solids were collected by filtration, washed with MeCN (2 x 20 ml_), dried under vacuum to give the title compound (5.5 g, 34% overall yield, 99.5% purity, 93.9% ee) as a white solid. 1 H NMR (500 MHz, DMSO-d6) δ ppm 0.78 (m, 6H), 1 .13 (m, 4H), 1 .51 (m, 2H), 1 .58 (q, J – 7.7 Hz, 2H), 3.08 (s, 2H), 3.75 (s, 3H), 5.66 (s, 2H), 6.88 (m, 2H), 6.93 (m, 1 H), 7.49 (m, 4H), 7.63 (m, 2H), 7.94 (m, 4H). EI-LCMS m/z 284 (M++1 of free base).
A suspension of (R)-5-((2-amino-2-ethylhexyl)thio)-2-methoxyphenol, dibenzoyl-L-tartrate salt (29 g, 45.2 mmol) in DCM (435 mL) was treated with water (1 16 mL) and 10% aqueous Na2C03 solution (1 16 mL). The mixture was stirred at ambient temperature until all solids were dissolved (30 min). The layers were separated. The organic layer was washed with water (2 x 60 mL), concentrated under vacuum to give (R)-5-((2-amino-2-ethylhexyl)thio)-2-methoxyphenol (free base) as an off-white solid (13.0 g, quantitative). A vessel was charged with TfOH (4.68 ml, 52.9 mmol) and DCM (30 mL) and the mixture was cooled to 0 °C. 5 g (17.6 mmol) of (R)-5-((2-amino-2-ethylhexyl)thio)-2-methoxyphenol (free base) was dissolved in DCM (10 mL) and added at a rate maintaining the temperature below 10 °C. Benzoyl chloride (4.5 mL, 38.8 mmol) was added at a rate maintaining the temperature below 10 °C. The mixture was then heated to reflux and stirred at reflux for 48 h. The mixture was cooled to 30 °C. Water (20 mL) was added and the mixture was concentrated to remove DCM. EtOH (10 mL) was added. The mixture was heated to 40 ° C, treated with 50% aqueous NaOH solution (10 mL) and stirred at 55 °C. After 1 h, the mixture was cooled to ambient temperature and the pH was adjusted to 6-7 with cone. HCI. The mixture was concentrated in vacuo to remove EtOH. EtOAc (100 mL) was added. The mixture was stirred for 5 min and the layers were separated. The organic layer was washed successively with 10% aqueous Na2CO3 (25 mL) and water (25 mL) and then concentrated in vacuo. The resultant oil was treated with DCM (15 mL). The resultant thick slurry was further diluted with DCM (15 mL) followed by addition of Hexanes (60 mL). The slurry was stirred for 5 min, filtered, washed with DCM/hexanes (1 :2, 2 x 10 mL) and dried under vacuum to give the title compound (7.67 g, 80%) as a yellow solid. 1 NMR (500 MHz, DMSO-d6) δ ppm 0.70 (t, 7.1 Hz, 3 H), 0.81 (t, 7.1 Hz, 3H), 1 .04-1 .27 (m, 8H), 2.74 (s, 2H), 3.73 (s, 3H), 6.91 (s, 1 H), 7.01 (s, 1 H), 7.52 (dd, J – 7.8, 7.2 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1 H), 7.67 (d, J = 7.8 Hz, 2H). EI-LCMS m/z 388 (M++1 ).
Intermediate G: (3R,5R)-3-butyl-3-ethyl-8-hydroxy-7-methoxy-5-phenyl-2,3,4,5-tetrahydrobenzo[f][1 ,4]thiazepine 1 ,1 -dioxide, (+)-camphorsulfonate salt
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A vessel was charged with (R)-(2-((2-amino-2-ethylhexyl)thio)-4-hydroxy-5-methoxyphenyl)(phenyl)methanone (1 .4 g, 3.61 mmol), toluene (8.4 ml_) and citric acid (0.035 g, 0.181 mmol, 5 mol%). The mixture was heated to reflux overnight with a Dean-Stark trap to remove water. The mixture was concentrated under reduced pressure to remove solvents. Methanol (14.0 ml_) and oxone (2.22 g, 3.61 mmol, 1 .0 equiv) were added. The mixture was stirred at gentle reflux for 2 h. The mixture was cooled to ambient temperature, and filtered to remove solids. The filter cake was washed with small amount of Methanol. The filtrate was cooled to 5 °C, and treated with sodium borohydride (0.410 g, 10.84 mmol, 3.0 equiv.) in small portions. The mixture was stirred at 5 °C for 2 h and then concentrated to remove the majority of solvents. The mixture was quenched with Water (28.0 ml_) and extracted with EtOAc (28.0 ml_). The organic layer was washed with brine, and then concentrated to remove solvents. The residue was dissolved in MeCN (14.0 ml_) and concentrated again to remove solvents. The residue was dissolved in MeCN (7.00 ml_) and MTBE (7.00 ml_) at 40 °C, and treated with (+)-camphorsulfonic acid (0.839 g, 3.61 mmol, 1 .0 equiv.) at 40 °C for 30 min. The mixture was cooled to ambient temperature, stirred for 2 h, and filtered to collect solids. The filter cake was washed with MTBE/MeCN (2:1 , 3 ml_), and dried at 50 °C to give the title compound (0.75 g, 32% overall yield, 98.6 purity, 97% de, 99.7% ee) as white solids. 1 NMR (400 MHz, CDCI3) δ ppm 0.63 (s, 3H), 0.88 (t, J – 6.9 Hz, 3H), 0.97 (m, 6H), 1 .29-1 .39 (m, 5H), 1 .80-1 .97 (m, 6H), 2.08-2.10 (m, 1 H), 2.27 (d, J – 17.3 Hz, 1 H), 2.38-2.44 (m, 3H), 2.54 (b, 1 H), 2.91 (b, 1 H), 3.48 (d, J – 15.4 Hz, 1 H), 3.79 (s, 3H), 4.05 (d, J – 17.2 Hz, 1 H), 6.45 (s, 1 H), 6.56 (s, 1 H), 7.51 -7.56 (m, 4H), 7.68 (s, 1 H), 7.79 (b, 2H), 1 1 .46 (b, 1 H). EI-LCMS m/z 404 (M++1 of free base).
Method 1 : A mixture of (3R,5R)-3-butyl-3-ethyl-8-hydroxy-7-methoxy-5-phenyl-2,3,4,5-tetrahydrobenzo[f][1 ,4]thiazepine 1 ,1 -dioxide, (+)-camphorsulfonate salt (0.5 g, 0.786 mmol), EtOAc (5.0 mL), and 10% of Na2C03 aqueuous solution (5 mL) was stirred for 15 min. The layers were separated and the aqueous layer was discarded. The organic layer was washed with dilute brine twice, concentrated to remove solvents. EtOAc (5.0 mL) was added and the mixture was concentrated to give a pale yellow solid free base. 1 ,4-Dioxane (5.0 mL) and pyridine (0.13 mL, 1 .57 mmol) were added. The mixture was cooled to 5-10 °C and triflic anhydride (0.199 mL, 1 .180 mmol) was added while maintaining the temperature below 15 °C. The mixture was stirred at ambient temperature until completion deemed by HPLC (1 h). Toluene (5 mL) and water (5 mL) were added. Layers were separated. The organic layer was washed with water, concentrated to remove solvents. Toluene (1 .0 mL) was added to dissolve the residue followed by Isooctane (4.0 mL). The mixture was stirred at rt overnight. The solids was filtered, washed with Isooctane (4.0 mL) to give the title compound (0.34 g, 81 %) as slightly yellow solids. 1 NMR (400 MHz, CDCI3) δ ppm 0.86 (t, J – 7.2 Hz, 3H), 0.94 (t, J – 7.6 Hz, 3H), 1 .12-1 .15 (m, 1 H), 1 .22-1 .36 (m, 3H), 1 .48-1 .60 (m, 2H), 1 .86-1 .93 (m, 2H), 2.22 (dt, J = 4.1 Hz, 12 Hz, 1 H), 3.10 (d, J – 14.8 Hz, 1 H), 3.49 (d, J – 14.8 Hz, 1 H), 3.64 (s, 3H), 6.1 1 (s, 1 H), 6.36 (s, 1 H), 7.38-7.48 (m, 5), 7.98 (s, 1 H).
Method 2: A mixture of (R)-3-butyl-3-ethyl-7-methoxy-5-phenyl-2,3-dihydrobenzo[f][1 ,4]thiazepin-8-yl trifluoromethanesulfonate (0.5 g, 0.997 mmol), ligand (N) (0.078 g, 0.1 10 mmol) and lr(COD)2BArF (0.127 g, 0.100 mmol) in DCM (10.0 mL) was purged with nitrogen three times, then hydrogen three times. The mixture was shaken in Parr shaker under 10 Bar of H2 for 24 h. The experiment described above was repeated with 1 .0 g (1 .994 mmol) input of (R)-3-butyl-3-ethyl-7-methoxy-5-phenyl-2,3-dihydrobenzo[f][1 ,4]thiazepin-8-yl
trifluoromethanesulfonate. The two batches of the reaction mixture were combined,
concentrated to remove solvents, and purified by silica gel chromatography
(hexanes:EtOAc =9:1 ) to give the sulfide (O) as slightly yellow oil (0.6 g, 40% yield, 99.7% purity). The oil (0.6 g, 1 .191 mmol) was dissolved in TFA (1 .836 mL, 23.83 mmol) and stirred at 40 °C. H202 (0.268 mL, 2.62 mmol) was added slowly over 30 min. The mixture was stirred at 40 °C for 2 h and then cooled to room temperature. Water (10 mL) and toluene (6.0 mL) were added. Layers were separated and the organic layer was washed successively with aqueous sodium carbonate solution and wate, and concentrated to dryness. Toluene (6.0 mL) was added and the mixture was concentrated to dryness. The residue was dissolved in toluene (2.4 mL) and isooctane (7.20 mL) was added. The mixture was heated to reflux and then cooled to room temperature. The mixture was stirred at room temperature for 30 min. The solid was filtered and washed with isooctane to give the title compound (0.48 g, 75%).
Intermediate L: (R)-5-((2-amino-2-ethylhexyl)thio)-2-methoxyphenol, di-p-toluoyl-L-tartrate salt
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To a mixture of (R)-2-amino-2-ethylhexyl hydrogen sulfate (1 1 .1 g, 49.3 mmol) in water (23.1 mL) was added NaOH (5.91 g, 148 mmol). The mixture was stirred at reflux for 2 h. The mixture was cooled to room temperature and extracted with MTBE (30.8 mL). The extract was washed with brine (22 mL), concentrated under vacuum and treated with methanol (30.8 mL). The mixture was stirred under nitrogen and treated with 3-hydroxy-4-methoxythiophenol (7.70 g, 49.3 mmol). The mixture was stirred under nitrogen at room temperature for 1 h. The mixture was concentrated under vacuum, treated with acetonitrile (154 mL) and then heated to 45 °C. To the stirred mixture was added (2R,3R)-2,3-bis((4-methylbenzoyl)oxy)succinic acid (19.03 g, 49.3 mmol). The resultant slurry was
stirred at 45 °C. After 2 h, the slurry was cooled to room temperature and stirred for 5 h. The solids were filtered, washed twice with acetonitrile (30 mL) and dried to give the title compound (28.0 g, 85%) as white solids. 1 NMR (400 MHz, DMSO-d6) δ (ppm): 0.70-0.75 (m, 6H), 1 .17 (b, 4H), 1 .46-1 .55 (m, 4H), 2.30 (s, 6H), 3.71 (s, 3H), 5.58 (s, 2H), 6.84 (s, 2H), 6.89 (s, 1 H), 7.24 (d, J – 1 1 .6 Hz, 4H), 7.76 (d, J – 1 1 .6 Hz, 4H).
A flask was charged with (R)-(2-((2-amino-2-ethylhexyl)thio)-4-hydroxy-5-methoxyphenyl)(phenyl)methanone (3.5 g, 9.03 mmol), citric acid (0.434 g, 2.258 mmol), 1 ,4-Dioxane (17.50 mL) and Toluene (17.50 mL). The mixture was heated to reflux with a Dean-Stark trap to distill water azetropically. The mixture was refluxed for 20 h and then cooled to room temperature. EtOAc (35.0 mL) and water (35.0 mL) were added and layers were separated. The organic layer was washed with aqueous sodium carbonate solution and concentrated to remove solvents to give crude imine as brown oil. The oil was dissolved in EtOAc (35.0 mL) and cooled to 0-5 °C. To the mixture was added triethylamine (1 .888 mL, 13.55 mmol) followed by slow addition of Tf2O (1 .831 mL, 10.84 mmol) at 0-5 °C. The mixture was stirred at room temperature for 1 h. Water was added and layers were separated. The organic layer was washed with brine, dried over Na2SO4 and concentrated under vacuum. The crude triflate was purified by silica gel chromatography
Journal of Medicinal Chemistry (2013), 56(12), 5094-5114.
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The apical sodium-dependent bile acid transporter (ASBT) transports bile salts from the lumen of the gastrointestinal (GI) tract to the liver via the portal vein. Multiple pharmaceutical companies have exploited the physiological link between ASBT and hepatic cholesterol metabolism, which led to the clinical investigation of ASBT inhibitors as lipid-lowering agents. While modest lipid effects were demonstrated, the potential utility of ASBT inhibitors for treatment of type 2 diabetes has been relatively unexplored. We initiated a lead optimization effort that focused on the identification of a potent, nonabsorbable ASBT inhibitor starting from the first-generation inhibitor 264W94 (1). Extensive SAR studies culminated in the discovery of GSK2330672 (56) as a highly potent, nonabsorbable ASBT inhibitor which lowers glucose in an animal model of type 2 diabetes and shows excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.
Example 26: 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl- 2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioic acid
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Method 1 , Step 1 : To a solution of (3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-5- phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepine-8-carbaldehyde 1 ,1 -dioxide (683 mg, 1 .644 mmol) in 1 ,2-dichloroethane (20 mL) was added diethyl 3- aminopentanedioate (501 mg, 2.465 mmol) and acetic acid (0.188 mL, 3.29 mmol). The reaction mixture was stirred at room temperature for 1 hr then treated with NaHB(OAc)3 (697 mg, 3.29 mmol). The reaction mixture was then stirred at room temperature overnight and quenched with aqueous potassium carbonate solution. The mixture was extracted with DCM. The combined organic layers were washed with H2O, saturated brine, dried (Na2SO4), filtered, and concentrated under reduced pressure to give diethyl 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5- phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioate (880 mg, 88%) as a light yellow oil: MS-LCMS m/z 603 (M+H)+.
Method 1 , Step 2: To a solution of diethyl 3-({[(3R,5R)-3-butyl-3-ethyl-7- (methyloxy)-l ,1 -dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8- yl]methyl}amino)pentanedioate (880 mg, 1 .460 mmol) in a 1 :1 :1 mixture of
THF/MeOH/H2O (30 mL) was added lithium hydroxide (175 mg, 7.30 mmol). The reaction mixture was stirred at room temperature overnight then concentrated under reduced pressure. H2O and MeCN was added to dissolve the residue. The solution was acidified with acetic acid to pH 4-5, partially concentrated to remove MeCN under reduced pressure, and left to stand for 30 min. The white precipitate was collected by filtration and dried under reduced pressure at 50°C overnight to give the title compound (803 mg, 100%) as a white solid: 1 H NMR (MeOH-d4) δ ppm 8.05 (s, 1 H), 7.27 – 7.49 (m, 5H), 6.29 (s, 1 H), 6.06 (s, 1 H), 4.25 (s, 2H), 3.60 – 3.68 (m, 1 H), 3.58 (s, 3H), 3.47 (d, J = 14.8 Hz, 1 H), 3.09 (d, J = 14.8 Hz, 1 H), 2.52 – 2.73 (m, 4H), 2.12 – 2.27 (m, 1 H), 1 .69 – 1 .84 (m, 1 H), 1 .48 – 1 .63 (m, 1 H), 1 .05 – 1 .48 (m, 5H), 0.87 (t, J = 7.4 Hz, 3H), 0.78 (t, J = 7.0 Hz, 3H); ES-LCMS m/z 547 (M+H) Method 2: A solution of dimethyl 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-
1 ,1 -dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8- yl]methyl}amino)pentanedioate (~ 600 g) in THF (2.5 L) and MeOH (1 .25 L) was cooled in an ice-bath and a solution of NaOH (206 g, 5.15 mol) in water (2.5 L) was added dropwise over 20 min (10-22°C reaction temperature). After stirring 20 min, the solution was concentrated (to remove THF/MeOH) and acidified to pH~4 with concentrated HCI. The precipitated product was aged with stirring, collected by filtration and air dried overnight. A second 600g batch of dimethyl 3-({[(3R,5R)-3- butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4- benzothiazepin-8-yl]methyl}amino)pentanedioate was saponified in a similar fashion. The combined crude products (~2 mol theoretical) were suspended in CH3CN (8 L) and water (4 L) and the stirred mixture was heated to 65°C. A solution formed which was cooled to 10°C over 2 h while seeding a few times with an authentic sample of the desired crystalline product. The resulting slurry was stirred at 10°C for 2 h, and the solid was collected by filtration. The filter cake was washed with water and air-dried overnight. Further drying to constant weight in a vacuum oven at 55°C afforded crystalline 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 – dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8- yl]methyl}amino)pentanedioic acid as a white solid (790 g).
Method 3: (3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-5-phenyl-2,3,4,5-tetrahydro- 1 ,4-benzothiazepine-8-carbaldehyde 1 ,1 -dioxide (1802 grams, 4.336 moles) and dimethyl 3-aminopentanedioate (1334 grams, 5.671 moles) were slurried in iPrOAc (13.83 kgs). A nitrogen atmosphere was applied to the reactor. To the slurry at 20°C was added glacial acetic acid (847 ml_, 14.810 moles), and the mixture was stirred until complete dissolution was observed. Solid sodium triacetoxyborohydride (1424 grams, 6.719 moles) was next added to the reaction over a period of 7 minutes. The reaction was held at 20°C for a total of 3 hours at which time LC analysis of a sample indicated complete consumption of the (3R,5R)-3-butyl-3-ethyl- 7-(methyloxy)-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepine-8-carbaldehyde 1 ,1 – dioxide. Next, water (20.36 kgs) and brine (4.8 kgs) were added to the reactor. The contents of the reactor were stirred for 10 minutes and then settled for 10 minutes. The bottom, aqueous layer was then removed and sent to waste. A previously prepared, 10% (wt/wt) aqueous solution of sodium bicarbonate (22.5 L) was added to the reactor. The contents were stirred for 10 minutes and then settled for 10 minutes. The bottom, aqueous layer was then removed and sent to waste. To the reactor was added a second wash of 10% (wt/wt) aqueous, sodium bicarbonate
(22.5 L). The contents of the reactor were stirred for 10 minutes and settled for 10 minutes. The bottom, aqueous layer was then removed and sent to waste. The contents of the reactor were then reduced to an oil under vacuum distillation. To the oil was added THF (7.15 kgs) and MeOH (3.68 kgs). The contents of the reactor were heated to 55°C and agitated vigorously until complete dissolution was observed. The contents of the reactor were then cooled to 25°C whereupon a previously prepared aqueous solution of NaOH [6.75 kgs of water and 2.09 kgs of NaOH (50% wt wt solution)] was added with cooling being applied to the jacket. The contents of the reactor were kept below 42°C during the addition of the NaOH solution. The temperature was readjusted to 25°C after the NaOH addition, and the reaction was stirred for 75 minutes before HPLC analysis indicated the reaction was complete. Heptane (7.66 kgs) was added to the reactor, and the contents were stirred for 10 minutes and then allowed to settle for 10 minutes. The aqueous layer was collected in a clean nalgene carboy. The heptane layer was removed from the reactor and sent to waste. The aqueous solution was then returned to the reactor, and the reactor was prepared for vacuum distillation. Approximately 8.5 liters of distillate was collected during the vacuum distillation. The vacuum was released from the reactor, and the temperature of the contents was readjusted to 25°C. A 1 N HCI solution (30.76 kgs) was added to the reactor over a period of 40 minutes. The resulting slurry was stirred at 25°C for 10 hours then cooled to 5°C over a period of 2 hours. The slurry was held at 5°C for 4 hours before the product was collected in a filter crock by vacuum filtration. The filter cake was then washed with cold (5°C) water (6 kgs). The product cake was air dried in the filter crock under vacuum for approximately 72 hours. The product was then transferred to three drying trays and dried in a vacuum oven at 50°C for 79 hours. The temperature of the vacuum oven was then raised to 65°C for 85 additional hours. The product was off-loaded as a single batch to give 2568 grams (93.4% yield) of intermediate grade 3-({[(3R,5R)-3- butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4- benzothiazepin-8-yl]methyl}amino)pentanedioic acid as an off-white solid.
Intermediate grade 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5- phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioic acid was dissolved (4690 g) in a mixture of glacial acetic acid (8850 g) and purified water (4200 g) at 70°C. The resulting solution was transferred through a 5 micron polishing filter while maintaining the temperature above 30°C. The reactor and filter were rinsed through with a mixture of glacial acetic acid (980 g) and purified water (470 g). The solution temperature was adjusted to 50°C. Filtered purified water (4230 g) was added to the solution. The cloudy solution was then seeded with crystalline 3-({[(3 5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl-2,3 ,4,5- tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioic acid (10 g). While maintaining the temperature at 50°C, filtered purified water was charged to the slurry at a controlled rate (1 1030 g over 130 minutes). Additional filtered purified water was then added to the slurry at a faster controlled rate (20740 g over 100 minutes). A final charge of filtered purified water (3780 g) was made to the slurry. The slurry was then cooled to 10°C at a linear rate over 135 minutes. The solids were filtered over sharkskin filter paper to remove the mother liquor. The cake was then rinsed with filtered ethyl acetate (17280 g) then the wash liquors were removed by filtration. The resulting wetcake was isolated into trays and dried under vacuum at 50°C for 23 hours. The temperature was then increased to 60°C and drying was continued for an additional 24 hours to afford crystalline 3-({[(3R,5R)-3-butyl-3-ethyl- 7-(methyloxy)-1 ,1 -dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8- yl]methyl}amino)pentanedioic acid (3740 g, 79.7% yield) as a white solid.
To a slurry of this crystalline 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 – dioxido-5-phenyl-2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8- yl]methyl}amino)pentanedioic acid (3660 g) and filtered purified water (3.6 L) was added filtered glacial acetic acid (7530 g). The temperature was increased to 60°C and full dissolution was observed. The temperature was reduced to 55°C, filtered, and treated with purified water (3.2 L). The solution was then seeded with crystalline 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl-2,3,4,5- tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioic acid (18 g) to afford a slurry. Filtered purified water was charged to the slurry at a controlled rate (9 L over 140 minutes). Additional filtered purified water was then added to the slurry at a faster controlled rate (18 L over 190 minutes). The slurry was then cooled to
10°C at a linear rate over 225 minutes. The solids were filtered over sharkskin filter paper to remove the mother liquor. The cake was then rinsed with filtered purified water (18 L), and the wash liquors were removed by filtration. The resulting wetcake was isolated into trays and dried under vacuum at 60°C for 18.5 hours to afford a crystalline 3-({[(3R,5R)-3-butyl-3-ethyl-7-(methyloxy)-1 ,1 -dioxido-5-phenyl- 2,3,4,5-tetrahydro-1 ,4-benzothiazepin-8-yl]methyl}amino)pentanedioic acid (3330 g, 90.8% yield) as a white solid which was analyzed for crystallinity as summarized below.
Paper
Cowan, D. J.; Collins, J. L.; Mitchell, M. B.; Ray, J. A.; Sutton, P. W.; Sarjeant, A. A.; Boros, E. E.Enzymatic- and Iridium-Catalyzed Asymmetric Synthesis of a Benzothiazepinylphosphonate Bile Acid Transporter InhibitorJ. Org. Chem.2013, 78 ( 24) 12726– 12734, DOI: 10.1021/jo402311e
A synthesis of the benzothiazepine phosphonic acid 3, employing both enzymatic and transition metal catalysis, is described. The quaternary chiral center of 3 was obtained by resolution of ethyl (2-ethyl)norleucinate (4) with porcine liver esterase (PLE) immobilized on Sepabeads. The resulting (R)-amino acid (5) was converted in two steps to aminosulfate 7, which was used for construction of the benzothiazepine ring. Benzophenone 15, prepared in four steps from trimethylhydroquinone 11, enabled sequential incorporation of phosphorus (Arbuzov chemistry) and sulfur (Pd(0)-catalyzed thiol coupling) leading to mercaptan intermediate 18. S-Alkylation of 18 with aminosulfate 7 followed by cyclodehydration afforded dihydrobenzothiazepine 20. Iridium-catalyzed asymmetric hydrogenation of 20 with the complex of [Ir(COD)2BArF] (26) and Taniaphos ligand P afforded the (3R,5R)-tetrahydrobenzothiazepine 30 following flash chromatography. Oxidation of 30 to sulfone 31 and phosphonate hydrolysis completed the synthesis of 3 in 12 steps and 13% overall yield.
Paper
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Scheme 1. Current Route to Chiral Intermediate 4 in the Synthesis of GSK2330672
Development of an Enzymatic Process for the Production of (R)-2-Butyl-2-ethyloxirane
†Synthetic Biochemistry, Advanced Manufacturing Technologies, ‡API Chemistry, ∥Protein and Cellular Sciences, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, United Kingdom
§API Chemistry, ⊥Synthetic Biochemistry, Advanced Manufacturing Technologies, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States
# Biotechnology and Environmental Shared Service, Global Manufacturing and Supply, GlaxoSmithKline, Dominion Way, Worthing BN14 8PB, United Kingdom
∇ Molecular Design, Computational and Modeling Sciences, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, Pennsylvania 19426, United States
An epoxide resolution process was rapidly developed that allowed access to multigram scale quantities of (R)-2-butyl-2-ethyloxirane 2 at greater than 300 g/L reaction concentration using an easy-to-handle and store lyophilized powder of epoxide hydrolase from Agromyces mediolanus. The enzyme was successfully fermented on a 35 L scale and stability increased by downstream processing. Halohydrin dehalogenases also gave highly enantioselective resolution but were shown to favor hydrolysis of the (R)-2 epoxide, whereas epoxide hydrolase from Aspergillus nigerinstead provided (R)-7 via an unoptimized, enantioconvergent process.
REFERENCES
1: Nunez DJ, Yao X, Lin J, Walker A, Zuo P, Webster L, Krug-Gourley S, Zamek-Gliszczynski MJ, Gillmor DS, Johnson SL. Glucose and lipid effects of the ileal apical sodium-dependent bile acid transporter inhibitor GSK2330672: double-blind randomized trials with type 2 diabetes subjects taking metformin. Diabetes Obes Metab. 2016 Jul;18(7):654-62. doi: 10.1111/dom.12656. Epub 2016 Apr 21. PubMed PMID: 26939572.
2: Wu Y, Aquino CJ, Cowan DJ, Anderson DL, Ambroso JL, Bishop MJ, Boros EE, Chen L, Cunningham A, Dobbins RL, Feldman PL, Harston LT, Kaldor IW, Klein R, Liang X, McIntyre MS, Merrill CL, Patterson KM, Prescott JS, Ray JS, Roller SG, Yao X, Young A, Yuen J, Collins JL. Discovery of a highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor (GSK2330672) for treatment of type 2 diabetes. J Med Chem. 2013 Jun 27;56(12):5094-114. doi: 10.1021/jm400459m. Epub 2013 Jun 6. PubMed PMID: 23678871.
09 Mar 2017 Phase-III clinical trials in Interstitial cystitis in United Kingdom, Poland, Latvia and Canada before March 2017 (PO) (EudraCT2016-000906-12) (NCT02858453)
04 Jan 2017 Aquinox Pharmaceuticals completes a phase I trial in Healthy volunteers in United Kingdom (NCT03185195)
07 Sep 2016 Phase-III clinical trials in Interstitial cystitis in Czech Republic, Hungary, Denmark (PO) (EudraCT2016-000906-12)
Rosiptor, also known as AQX-1125 is a potent and selective SHIP1 activator currently in clinical development.
AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient but not in SHIP1-deficient cells, reduced cytokine production in splenocytes, inhibited the activation of mast cells and inhibited human leukocyte chemotaxis.
AQX-1125 suppresses leukocyte accumulation and inflammatory mediator release in rodent models of pulmonary inflammation and allergy. As shown in the mouse model of LPS-induced lung inflammation, the efficacy of the compound is dependent on the presence of SHIP1. Pharmacological SHIP1 activation may have clinical potential for the treatment of pulmonary inflammatory diseases.
Dysregulated activation of the PI3K pathway contributes to inflammatory/immune disorders and cancer. Efforts have been made to develop modulators of PI3K as well as downstream kinases (Workman et al., Nat. Biotechnol. 24, 794-796, 2006; Simon, Cell 125, 647-649, 2006; Hennessy et al., Nat. Rev. Drug. Discov. 4, 988-1004, 2005; Knight et al., Cell 125, 733-747, 2006; Ong et al., Blood (2007), Vol. 110, No. 6, pp 1942-1949). A number of promising new PI3K isoform specific inhibitors with minimal toxicities have recently been developed and used mouse models of inflammatory disease (Camps et al., Nat. Med. 11, 936-943, 2005; Barber et al., Nat. Med. 11, 933-935, 2005) and glioma (Fan et al., Cancer Cell 9, 341-349, 2006). However, because of the dynamic interplay between phosphatases and kinases in regulating biological processes, inositol phosphatase activators represent a complementary, alternative approach to reduce PIP3 levels. Of the phosphoinositol phosphatases that degrade PIP3, SHIP1 is a particularly ideal target for development of therapeutics for treating immune and hemopoietic disorders because of its hematopietic-restricted expression (Hazen et al., Blood 113, 2924-2933, 2009; Rohrschneider et al., Genes Dev. 14, 505-520, 2000).
Small molecule SHIP1 modulators have been disclosed, including sesquiterpene compounds such as pelorol. Pelorol is a natural product isolated from the tropical marine sponge Dactylospongia elegans (Kwak et al., J. Nat. Prod. 63, 1153-1156, 2000; Goclik et al., J. Nat. Prod. 63, 1150-1152, 2000). Other reported SHIP1 modulators include the compounds set forth in PCT Published Patent Applications Nos. WO 2003/033517, WO 2004/035601, WO 2004/092100, WO 2007/147251, WO 2007/147252, WO 2011/069118, WO 2014/143561 and WO 2014/158654 and in U.S. Pat. Nos. 7,601,874 and 7,999,010.
One such molecule is AQX-1125, which is the acetate salt of (1S,3S,4R)-4-((3aS,4R,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1H-inden-5-yl)-3-(hydroxymethyl)-4-methylcyclohexanol (AQX-1125). AQX-1125 is a compound with anti-inflammatory activity and is described in U.S. Pat. Nos. 7,601,874 and 7,999,010, the relevant disclosures of which are incorporated in full by reference in their entirety, particularly with respect to the preparation of AQX-1125, pharmaceutical compositions comprising AQX-1125 and methods of using AQX-1125.
AQX-1125 has the molecular formula, C20H36NO2+.C2H3O2−, a molecular weight of 381.5 g/mole and has the following structural formula:
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AQX-1125 is useful in treating disorders and conditions that benefit from SHIP1 modulation, such as cancers, inflammatory disorders and conditions and immune disorders and conditions. AQX-1125 is also useful in the preparation of a medicament for the treatment of such disorders and conditions.
Synthetic methods for preparing AQX-1125 are disclosed in U.S. Pat. Nos. 7,601,874 and 7,999,010. There exists, therefore, a need for improved methods of preparing AQX-1125.
Dysregulated activation of the PI3K pathway contributes to
inflammatory/immune disorders and cancer. Efforts have been made to develop modulators of PI3K as well as downstream kinases (Workman et al., Nat. Biotechnol 24, 794-796, 2006; Simon, Cell 125, 647-649, 2006; Hennessy et al., Nat Rev Drug Discov 4, 988-1004, 2005; Knight et al., Cell 125, 733-747, 2006; Ong et al., Blood (2007), Vol. 110, No. 6, pp 1942-1949). A number of promising new PI3K isoform specific inhibitors with minimal toxicities have recently been developed and used in mouse models of inflammatory disease (Camps et al., Nat Med 1 1 , 936-943, 2005; Barber et ai, Nat Med 1 1 , 933-935, 2005) and glioma (Fan et al., Cancer Cell 9, 341-349, 2006). However, because of the dynamic interplay between phosphatases and kinases in regulating biological processes, inositol phosphatase activators represent a complementary, alternative approach to reduce PIP3 levels. Of the phosphoinositol phosphatases that degrade PIP3i SHIP1 is a particularly ideal target for development of therapeutics for treating immune and hemopoietic disorders because of its
hematopietic-restricted expression (Hazen et al., Blood 1 13, 2924-2933, 2009;
Rohrschneider et ai, Genes Dev. 14, 505-520, 2000).
Small molecule SHIP1 modulators have been disclosed, including
sesquiterpene compounds such as pelorol. Pelorol is a natural product isolated from the tropical marine sponge Dactylospongia elegans (Kwak et al., J Nat Prod 63, 1 153-1 156, 2000; Goclik et al., J Nat Prod 63, 1150-1152, 2000). Other reported SHIP1 modulators include the compounds set forth in PCT Published Patent Applications Nos. WO 2003/033517, WO 2004/035601 , WO 2004/092100, WO 2007/147251 , WO 2007/147252, WO 2011/069118, WO 2014/143561 and WO 2014/158654 and in U.S. Patent Nos. 7,601 ,874 and 7,999,010.
While significant strides have been made in this field, there remains a need for effective small molecule SHIP1 modulators.
One such molecule is the acetate salt of (1 S,3S,4 )-4-((3aS,4 ,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1 /-/-inden-5-yl)-3-(hydroxymethyl)-4-methylcyclohexanol (referred to herein as Compound 1). Compound 1 is a compound with anti-inflammatory activity and is described in U.S. Patent Nos. 7,601 ,874 and 7,999,010, the relevant disclosures of which are incorporated in full by reference in their entirety, particularly with respect to the preparation of Compound 1 ,
pharmaceutical compositions comprising Compound 1 and methods of using
Compound 1.
Compound 1 has the molecular formula, C2oH36N02+ · C2H302“, a molecular weight of 381.5 g/mole
The application is directed to crystalline forms of the acetate salt of (1S,3S,4R)-4-(3aS,4R,5S,7aS)-4-(aminomethyl)-7a- methyl-1-methyleneoctahydro-1H-inden-5-yl)-3-(hydroxymethyl) -4-methylcyclohexanol and processes for their preparation. The compound acts as a SHIP1 modulator and is thus useful in the treatment of cancer or inflammatory and immune disorders and conditions.
Process for the synthesis of substituted indene derivative (particularly AQX-1125 ) as a SH2-containing inositol 5-phosphatase 1 (SHIP1) modulator for treating cancer, inflammatory disorders and immune disorders. Aquinox Pharmaceuticals is developing AQX-1125 (phase III clinical trial in July 2017), a SHIP1 agonist, for the treatment of inflammatory diseases. For a prior filing see WO2016210146 , claiming novel crystalline forms of rosiptor acetate. In July 2017, Seenisamy and Chetia were associated with Syngene
Synthetic Method 1
In one aspect of the invention, AQX-1125 was prepared by the method described below in Reaction Scheme 1 where Pg1 is an oxygen-protecting group, Pg2 is a carbonyl protecting group, Lg1 is a leaving group and X is bromo or chloro:
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Reaction Scheme 1A:
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Synthetic Example 77
Step 11: Preparation of AQX-1125 from Compound 16
A. To a stirred solution of (1S,3S,4R)-4-((3aS,4R,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1H-inden-5-yl)-3-(hydroxymethyl)-4-methylcyclohexan-1-ol (Compound 16, 58.0 g, 0.180 mol, 1.0 eq, from Synthetic Example 76) in methanol (174 mL, 3 V) was added acetic acid (23.5 mL, 0.4 V) dropwise at 10° C. under a nitrogen atmosphere over 20 min. The reaction mixture was stirred at room temperature for 1 h. The solution was filtered to remove undissolved particles and washed with methanol (58 mL, 1 V). The filtrate was collected and evaporated at 35° C. to half the volume (˜125 mL). MTBE (348 mL, 6 V) was slowly added to the above concentrated mixture and the reaction stirred at 10° C. for 2 h. During the MTBE addition, slow precipitation of the product was observed. The solids were filtered and washed with MTBE (116 mL, 2V) to afford (1S,3S,4R)-4-((3aS,4R,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1H-inden-5-yl)-3-(hydroxymethyl)-4-methylcyclohexan-1-ol, acetic acid salt, (AQX-1125) as a white solid (50 g, yield 72.6%). 1H NMR (400 MHz, pyridine-d5): δ 5.85 (br s, 5H), 4.70 (s, 2H), 4.08 (dd, J=10.4, 2 Hz, 1H), 3.95-3.85 (m, 1H), 3.60-3.50 (m, 1H), 3.18 (d, J=14 Hz, 1H), 2.92-2.86 (m, 1H), 2.80 (d, J=13.6 Hz, 1H), 2.50-2.40 (m, 1H), 2.25-1.97 (m, 3H), 2.15 (s, 3H), 1.90-1.65 (m, 4H), 1.56-1.40 (m, 4H), 1.39-1.20 (m, 2H), 1.25 (s, 3H), 0.78 (s, 3H). LCMS: (Method A) 322.4 (M+1), Retention time: 1.95 min, HPLC (Method H): 95.5 area %, Retention time: 16.66 min.
Synthetic Example 66
Preparation of Compound 16 and AQX-1125
A. To a solution of 7a-methyl-5-((1S,2R,5S)-2-methyl-7-oxo-6-oxabicyclo[3.2.1]octan-2-yl)-1-methyleneoctahydro-1H-indene-4-carbaldehyde oxime (Compound 68, 100 mg, 0.30 mmol, from Synthetic Example 65) in 1,4-dioxane (5 mL) in a 25 mL RB flask fitted with reflux condenser was added a solution of lithium aluminum hydride (1 M in THF, 1.51 ml, 1.50 mmol) at RT under nitrogen and the reaction mass was stirred using a magnetic stirrer at 100° C. for 24 hours. Another lot of a solution of lithium aluminum hydride (1 M in THF, 1.51 ml, 1.50 mmol) was added and the reaction was further refluxed for 24 hours. Completion of the reaction was monitored by LCMS analysis.
B. The reaction mass was quenched by the drop-wise addition of saturated aq. sodium sulfate solution, filtered through a CELITE™ bed on glass frit funnel and concentrated by rotary evaporation to get a crude mass which was further purified by preparative HPLC to afford (1S,3S,4R)-4-((4R,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1H-inden-5-yl)-3-(hydroxymethyl)-4-methylcyclohexan-1-ol (Compound 16, 35 mg, 36% yield) as an off-white solid. 1H-NMR (400 MHz, CD3OD): δ 4.69 (s, 2H), 3.73 (br d, J=10.0 Hz, 1H), 3.52-3.45 (m, 1H), 3.22-3.15 (m, 1H), 3.05-2.98 (m, 1H), 2.62-2.55 (m, 1H), 2.38-2.25 (m, 1H), 2.20-2.15 (m, 1H), 1.95-1.81 (m, 6H), 1.62-1.25 (m, 10H), 1.10 (s, 3H), 0.86 (s, 3H). LCMS (Method A) m/z: 322.5 (M+1), Retention time: 2.06 min, Purity: 98.9 area % (ELSD). HPLC (Method A): Retention time: 2.70 min, Purity: 99.3 area %.
C. AQX-1125 was prepared from Compound 16 in the same manner as described above in Synthetic Example 16.
REFERENCES
1: Nickel JC, Egerdie B, Davis E, Evans R, Mackenzie L, Shrewsbury SB. A Phase II Study of the Efficacy and Safety of the Novel Oral SHIP1 Activator AQX-1125 in Subjects with Moderate to Severe Interstitial Cystitis/Bladder Pain Syndrome. J Urol. 2016 Sep;196(3):747-54. doi: 10.1016/j.juro.2016.03.003. PubMed PMID: 26968644.
2: Chuang YC, Chermansky C, Kashyap M, Tyagi P. Investigational drugs for bladder pain syndrome (BPS) / interstitial cystitis (IC). Expert Opin Investig Drugs. 2016;25(5):521-9. doi: 10.1517/13543784.2016.1162290. PubMed PMID: 26940379.
3: Leaker BR, Barnes PJ, O’Connor BJ, Ali FY, Tam P, Neville J, Mackenzie LF, MacRury T. The effects of the novel SHIP1 activator AQX-1125 on allergen-induced responses in mild-to-moderate asthma. Clin Exp Allergy. 2014 Sep;44(9):1146-53. doi: 10.1111/cea.12370. PubMed PMID: 25040039.
4: Stenton GR, Mackenzie LF, Tam P, Cross JL, Harwig C, Raymond J, Toews J, Wu J, Ogden N, MacRury T, Szabo C. Characterization of AQX-1125, a small-molecule SHIP1 activator: Part 1. Effects on inflammatory cell activation and chemotaxis in vitro and pharmacokinetic characterization in vivo. Br J Pharmacol. 2013 Mar;168(6):1506-18. doi: 10.1111/bph.12039. PubMed PMID: 23121445; PubMed Central PMCID: PMC3596654.
5: Stenton GR, Mackenzie LF, Tam P, Cross JL, Harwig C, Raymond J, Toews J, Chernoff D, MacRury T, Szabo C. Characterization of AQX-1125, a small-molecule SHIP1 activator: Part 2. Efficacy studies in allergic and pulmonary inflammation models in vivo. Br J Pharmacol. 2013 Mar;168(6):1519-29. doi: 10.1111/bph.12038. PubMed PMID: 23121409; PubMed Central PMCID: PMC3596655.
6: Croydon L. BioPartnering North America–Spotlight on Canada. IDrugs. 2010 Mar;13(3):159-61. PubMed PMID: 20191430.
The SHIP1 Pathway – Highlighting the Role of AQX-1125
AQX-1125 is our lead product candidate and has generated positive clinical data from three completed clinical trials, including two proof-of-concept trials, one in COPD and one in allergic asthma, demonstrating a favorable safety profile and anti-inflammatory activity. Overall, more than 100 subjects have received AQX-1125. Importantly, our clinical trial results were consistent with the drug-like properties and anti-inflammatory activities demonstrated in our preclinical studies. AQX-1125 is a once daily oral capsule with many desirable drug-like properties. We are currently investigating AQX-1125 in two Phase 2 clinical trials, one in COPD and one in BPS/IC.
Based on our three completed clinical trials, we have demonstrated that AQX-1125:
has desirable pharmacokinetic, absorption and excretion properties that make it suitable for once daily oral administration;
is generally well tolerated, exhibiting mild to moderate adverse events primarily related to gastrointestinal upset that resolve without treatment or long-term effects and are reduced by taking the drug candidate with food; and
has anti-inflammatory properties consistent with those exhibited in preclinical studies and exhibited activity in two trials using two distinct inflammatory challenges.
AQX-1125 is an activator of SHIP1, which controls the PI3K cellular signaling pathway. If the PI3K pathway is overactive, immune cells can produce an abundance of pro-inflammatory signaling molecules and migrate to and concentrate in tissues, resulting in excessive or chronic inflammation. SHIP1 is predominantly expressed in cells derived from bone marrow tissues, which are mainly immune cells. Therefore drugs that activate SHIP1 can reduce the function and migration of immune cells and have an anti-inflammatory effect. By controlling the PI3K pathway, AQX-1125 reduces immune cell function and migration by targeting a mechanism that has evolved in nature to maintain homeostasis of the immune system.
AQX-1125 has demonstrated compelling preclinical activity in a broad range of relevant inflammatory studies including preclinical models of COPD, asthma, pulmonary fibrosis, BPS/IC and inflammatory bowel disease (IBD). In these studies we have seen a meaningful reduction in the relevant immune cells that are the cells that cause inflammation, such as neutrophils, eosinophils and macrophages, and a reduction in the symptoms of inflammation, such as pain and swelling. The activity, efficacy and potency seen with AQX-1125 in most preclinical studies compare favorably to published results with corticosteroids. In addition, AQX-1125 demonstrated compelling activity in the smoke airway inflammation and Bleomycin Fibrosis models, which are known to be steroid refractory, or in other words, do not respond to corticosteroids. We believe this broad anti-inflammatory profile is not typical amongst drugs in development and supports the therapeutic potential for AQX-1125.
In addition to demonstrating strong in vitro and in vivo activity, AQX-1125 was also selected as a lead candidate based on its many desirable drug-like properties. The drug candidate is highly water soluble and does not require complex formulation for oral administration. AQX-1125 has low plasma protein binding, is not metabolized and is excreted unmetabolized in both urine and feces. After oral or intravenous dosing, AQX-1125 reaches high concentrations in respiratory, urinary and gastrointestinal tracts, all of which have mucosal surfaces of therapeutic interest. In humans, AQX-1125 has shown pharmacokinetic properties suitable for once-a-day dosing. In addition, the absorption of the drug candidate is equivalent whether taken with or without food.
MOLECULAR FORMULA C27H31FN4O8
MOLECULAR WEIGHT 558.6
SPONSOR Tetraphase Pharmaceuticals, Inc.
CODE DESIGNATION TP-434
CAS REGISTRY NUMBER 1207283-85-9
WHO NUMBER 9702
Eravacycline (TP-434) is a synthetic fluorocyclineantibiotic in development by Tetraphase Pharmaceuticals. It is closely related to the glycylglycine antibiotic tigecycline and the tetracycline class of antibiotics. It has a broad spectrum of activity including many multi-drug resistant strains of bacteria. Phase III studies in complicated intra-abdominal infections (cIAI) [1] and complicated urinary tract infections (cUTI)[2] were recently completed with mixed results. Eravacylcine has been designated as a Qualified Infectious Disease Product (QIDP), as well as for fast track approval by the FDA.[3]
Eravacylme is a tetracycline antibiotic that has demonstrated broad spectrum activity against a wide variety of multi-drug resistant Gram-negative, Gram-positive and anaerobic bacteria in humans. In Phase I and Phase II clinical trials, eravacycline also demonstrated a favorable safety and tolerability profile. In view of its attractive
pharmacological profile, synthetic routes to eravacycline and, in particular, synthetic routes that result in suitable quantities of eravacycline for drag development and manufacturing, are becoming increasingly important.
As described in International Publication No . WO 2010/017470, eravacycline is conveniently synthesized from 7-fluorosancycline, another tetracycline. 7-Fluorosancycline can be synthesized, in turn, from commercially available 7-ammosancycline or a protected derivative thereof. However, very few procedures for the conversion of (^-ammo-substituted tetracyclines, such as 7-aminosancycline, to C7-fiuoro-substituted tetracyclines, such as 7-fluorosancycline, have been reported, and those that have are not suitable to be deployed at production-scale.
Therefore, there is a need for improved processes, particularly improved production -scale processes, for converting C7-amino-substituted tetracyclines to C7-fluoro-substituted tetracyclines.
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Example 3. Preparation of Eravacycline From 9-Aminosancycline Using a Photolytic Fluorination
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[00158] Sancycline (0.414 g, 1.0 mmol) was dissolved in trifiuoroacetic acid (TFA). The solution was cooled to 0 °C. To the solution was added N-bromosuccinimide (NBS, 0.356 g, 2.1 mmol). The reaction was complete after stirring at 0 °C for 1 h. The reaction mixture was allowed to warm to rt. Solid NO3 (0.1 Ig, 0.11 mrnoi) was added and the reaction mixture was stirred at rt for 1 h. The reaction solution was added to 75 mL cold diethyl ether. The precipitate was collected by filtration and dried to give 0.46 g of compound 6. Compound 6 can then be reduced to compounds 7, 8, or 9 using standard procedures.
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13
[00159] 9-Aminosancycline (7, 1 g, 0233 mmol) was dissolved in 20 mL sulfuric acid and the reaction was cooled using an ice bath. Potassium nitrate (235 mg, 0.233 mmol) was added in several portions. After stirring for 15 min, the reaction mixture was added to 400 mL MTBE followed by cooling using an ice bath. The solid was collected by filtration. The filter cake was dissolved in 10 mL water and the pH of the aqueous solution was adjusted to 5.3 using 25% aqueous NaOH. The resulting suspension was filtered, and the filter cake was dried to give 1 g compound 10: MS (ESI) m/z 475.1 (M+l).
[00160] Compound 10 (1.1 g) was dissolved in 20 mL of water and 10 mL of acetonitrile. To the solution was added acyl chloride 3 (in two portions: 600 mg and 650 mg). The pH of the reaction mixture was adjusted to 3.5 using 25% aqueous NaOH. Another portion of acyl chloride (800 mg) was added. The reaction was monitored by HPLC analysis. Product 11 was isolated from the reaction mixture by preparative HPLC. Lyophilization gave 1.1 g of compound 11: MS (ESI) m/z 586.3 (M+l).
[00161] Compound 11 (1.1 g) was dissolved in methanol. To the solution was added concentrated HC1 (0.5 mL) and 10% Pd-C (600 mg). The reaction mixture was stirred under a hydrogen atmosphere (balloon). After the reaction was completed, the catalyst was removed by filtration. The filtrate was concentrated to give 1 g of compound 12: ‘H NMR (400 MHz, DMSO), 8.37 (s, 1H), 4.38-4.33 (m, 3H), 3.70 (br s, 2H), 3.30-2.60 (m, 1211), 2.36-2.12 (m, 2H), 2.05-1.80 (m, 4H), 1.50-1.35 (m, 1H); MS (ESI) m/z 556.3 (M+l).
[00162] Compound 12 (150 mg) was dissolved in 1 mL of 48% HBF4. To the solution was added 21 mg of NaN02. After compound 12 was completely converted to compound 13 (LC/MS m/z 539.2), the reaction mixture was irradiated with 254 nm light for 6 h while being cooled with running water. The reaction mixture was purified by preparative HPLC using acetonitrile and 0.05 N aqueous HCl as mobile phases to yield the compound 4 (eravacyclme, 33 mg) as a bis-HCl salt (containing 78% of 4 and 10% of the 7-H byproduct, by HPLC): MS (ESI) m/z 559.3 (M+l).
PAPER
Exploring the Boundaries of “Practical”: De Novo Syntheses of Complex Natural Product-Based Drug Candidates
Department of Chemistry and Biochemistry, University of California−Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States
This review examines the state of the art in synthesis as it relates to the building of complex architectures on scales sufficient to drive human drug trials. We focus on the relatively few instances in which a natural-product-based development candidate has been manufactured de novo, rather than semisynthetically. This summary provides a view of the strengths and weaknesses of current technologies, provides perspective on what one might consider a practical contribution, and hints at directions the field might take in the future.
PAPER
Journal of Medicinal Chemistry (2012), 55(2), 597-605
Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: A Potent, Broad Spectrum Antibacterial Agent
†Discovery Chemistry, ‡Microbiology, and §Process Chemistry R&D, Tetraphase Pharmaceuticals, 480 Arsenal Street, Watertown, Massachusetts 02472, United States
This and the accompanying report (DOI: 10.1021/jm201467r) describe the design, synthesis, and evaluation of a new generation of tetracycline antibacterial agents, 7-fluoro-9-substituted-6-demethyl-6-deoxytetracyclines (“fluorocyclines”), accessible through a recently developed total synthesis approach. These fluorocyclines possess potent antibacterial activities against multidrug resistant (MDR) Gram-positive and Gram-negative pathogens. One of the fluorocyclines, 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline (17j, also known as TP–434, 50th Interscience Conference on Antimicrobial Agents and Chemotherapy Conference, Boston, MA, September 12–15, 2010, poster F1–2157), is currently undergoing phase 2 clinical trials in patients with complicated intra-abdominal infections (cIAI).
A convergent route to eravacycline (1) has been developed by employing Michael–Dieckmann cyclization between enone 3 and a fully built and protected left-hand piece (LHP, 2). After construction of the core eravacycline structure, a deprotection reaction was developed, allowing for the isoxazole ring opening and global deprotection to be achieved in one pot. The LHP is synthesized from readily available 4-fluoro-3-methylphenol in six steps featuring a palladium-catalyzed phenyl carboxylation in the last step.
Eravacycline di-HCl salt (1) as a yellow solid: purity 97.9%; mp 197–199 °C dec. The spectral data matched those from original sample as reported in our previous publication.(3)
3 Ronn, M.; Zhu, Z.; Hogan, P. C.; Zhang, W.-Y.; Niu, J.; Katz, C. E.; Dunwoody, N.; Gilicky, O.; Deng, Y.;Hunt, D. K.; He, M.; Chen, C.-L.; Sun, C.; Clark, R. B.; Xiao, X.-Y.Org. Process Res. Dev.2013, 17, 838–845, DOI: 10.1021/op4000219
Process research and development of the first fully synthetic broad spectrum 7-fluorotetracycline in clinical development is described. The process utilizes two key intermediates in a convergent approach. The key transformation is a Michael–Dieckmann reaction between a suitable substituted aromatic moiety and a key cyclohexenone derivative. Subsequent deprotection and acylation provide the desired active pharmaceutical ingredient in good overall yield.
Free base of 7. HPLC (248 nm) showed 97.1% purity with 0.80% of the corresponding impurity from 19, 1.2% of epimer of 7, and 0.80% the corresponding impurity from 20 (102g, 88.7% yield) product as a yellow solid.
MS (ES) m/z calcd for +H: 559.2 (100.0), 560.2 (29.9), 561.2 (5.9); found: 559.3, 560.2, 561.3.
Give 7·2HCl (531 g containing 6.9% by weight water, ∼ 784 mmol). HPLC (248 nm) indicated a 96.6% purity with 0.64% of the corresponding impurity from 19, 1.3% of epimer of 7, and 1.3% the corresponding impurity from 20.
aDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, United States E-mail:pbaran@scripps.edu
bChemical Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, United States E-mail:martin.eastgate@bms.com
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Tetracycline (Myers/Tetraphase, 2005–2013). Myers’ convergent approach to the tetracyclines is a great example of how a scalable synthesis of a key intermediate en route to a natural product can fuel the discovery of entirely new drug candidates. These broad-spectrum polyketide antibiotics have been widely used in human and veterinary medicine, but, due to the development of tetracycline-resistant strains, there is an unmet need for novel tetracycline drugs. Pioneering work in this eld has been achieved by the Myers’ group, who published a landmark synthetic approach to the tetracycline class of antibiotics in 2005.16 Using this route, over 3,000 fully synthetic tetracyclines have been prepared to date. Central to their strategy was the synthesis of a highly versatile intermediate, AB enone 7, 17 which enabled the convergent construction of novel tetracycline antibiotics (Scheme 3, A).18 Naturally, the route to 7 had to be practical and amenable to large-scale synthesis and consequently, the synthetic approaches to this building block have become more and more practical and efficient with every new generation. In 2007, Myers published their rst practical and enantioselective approach to 7 (Scheme 3, B).17 The route started from 8, which can be accessed in multi-hundred gram amounts from commercially available 3-hydroxy-5-isoxazolecarboxylate (not shown) by O-benzylation followed by DIBAL reduction. In a three-step sequence, 8 was transformed into carbinol 9. In the key step of the sequence, 9 underwent an intramolecular Diels–Alder reaction to give a mixture of 4 diastereomeric cycloadducts, which, aer Swern oxidation, could be readily separated by ash column chromatography to afford 10. Finally, boron trichloride mediated opening of the oxabicyclic ring system and demethylation, followed by TBS protection of the tertiary hydroxyl-group, afforded 40 g of the AB enone 7 in 21% overall yield, over nine steps from commercial material. Slight modications of this route have allowed for the preparation of >20 kg batches of the AB enone. The availability of large-scale batches of 7 has both enabled the discovery and the development of eravacycline (11), the rst fully synthetic tetracycline analog in clinical development, from Tetraphase Pharmaceuticals. In their process route,19 the key Michael– Dieckmann cyclization between 7 and 12 was carried out on kg-scale and afforded 13 in 93.5% yield. This compound was transformed into eravacycline (11) in 3 more steps, including TBS-cleavage, hydrogenolysis and amide bond formation. Using this process, several kg of 11 have been prepared to date to support clinical studies. Finally, a third- and fourth-generation route to 7 has recently been published by Myers that is not only shorter than previous routes, but also amenable of structural modications of the AB-ring enone.20
16 M. G. Charest, C. D. Lerner, J. D. Brubaker, D. R. Siegel and A. G. Myers, Science, 2005, 308, 395–398. 17 J. D. Brubaker and A. G. Myers, Org. Lett., 2007, 9, 3523–3525. 18 (a) C. Sun, Q. Wang, J. D. Brubaker, P. M. Wright, C. D. Lerner, K. Noson, M. Charest, D. R. Siegel, Y.-M. Wang and A. G. Myers, J. Am. Chem. Soc., 2008, 130, 17913–17927; (b) X.-Y. Xiao, D. K. Hunt, J. Zhou, R. B. Clark, N. Dunwoody, C. Fyfe, T. H. Grossman, W. J. O’Brien, L. Plamondon, M. R¨onn, C. Sun, W.-Y. Zhang and J. A. Sutcliffe, J. Med. Chem., 2012, 55, 597–605; (c) R. B. Clark, M. He, C. Fyfe, D. Loand, W. J. O’Brien, L. Plamondon, J. A. Sutcliffe and X.-Y. Xiao, J. Med. Chem., 2011, 54, 1511–1528; (d) R. B. Clark, D. K. Hunt, M. He, C. Achorn, C.-L. Chen, Y. Deng, C. Fyfe, T. H. Grossman, P. C. Hogan, W. J. O’Brien, L. Plamondon, M. R¨onn, J. A. Sutcliffe, Z. Zhu and X.-Y. Xiao, J. Med. Chem., 2012, 55, 606–622; (e) C. Sun, D. K. Hunt, R. B. Clark, D. Loand, W. J. O’Brien, L. Plamondon and X.-Y. Xiao, J. Med. Chem., 2011, 54, 3704–3731. 19 M. Ronn, Z. Zhu, P. C. Hogan, W.-Y. Zhang, J. Niu, C. E. Katz, N. Dunwoody, O. Gilicky, Y. Deng, D. K. Hunt, M. He, C.-L. Chen, C. Sun, R. B. Clark and X.-Y. Xiao, Org. Process Res. Dev., 2013, 17, 838–845. 20 D. A. Kummer, D. Li, A. Dion and A. G. Myers, Chem. Sci., 2011, 2, 1710–1718. 21 (a) G. R. Pettit, Z. A. Chicacz, F. Gao, C. L. Herald, M. R. Boyd, J. M. Schmidt and J. N. A. Hooper, J. Org. Chem., 1993, 58, 1302–1304; (b) M. Kobayashi, S. Aoki, H. Sakai, K. Kawazoe, N. Kihara, T. Sasaki and I. Kitagawa, Tetrahedron Lett., 1993, 34, 2795–2798. 22 (a) J. Guo, K. J. Duffy, K. L. Stevens, P. I. Dalko, R. M. Roth, M. M. Hayward and Y. Kishi, Angew. Chem., Int. Ed., 1998, 37, 187–190; (b) M. M. Hayward, R. M. Roth, K. J. Duffy, P. I. Dalko, K. L. Stevens, J. Guo and Y. Kishi, Angew. Chem., Int. Ed., 1998, 37, 190–196; (c) I. Paterson, D. Y. K. Chen, M. J. Coster, J. L. Acena, J. Bach, ˜ K. R. Gibson, L. E. Keown, R. M. Oballa, T. Trieselmann, D. J. Wallace, A. P. Hodgson and R. D. Norcross, Angew. Chem., Int. Ed., 2001, 40, 4055–4060; (d) M. T. Crimmins, J. D. Katz, D. G. Washburn, S. P. Allwein and L. F. McAtee, J. Am. Chem. Soc., 2002, 124, 5661–5663; (e) M. Ball, M. J. Gaunt, D. F. Hook, A. S. Jessiman, S. Kawahara, P. Orsini, A. Scolaro, A. C. Talbot, H. R. Tanner, S. Yamanoi and S. V. Ley, Angew. Chem., Int. Ed., 2005, 44, 5433–5438. 23 A. B. Smith, T. Tomioka, C. A. Risatti, J. B. Sperry and C. Sfouggatakis, Org. Lett., 2008, 10, 4359–4362. 24 (a) U. Eder, G. Sauer and R. Wiechert, Angew. Chem., Int. Ed. Engl., 1971, 10, 496–497; (b) Z. G. Hajos and D. R. Parrish, J. Org. Chem., 1974, 39, 1615–1621; (c) B. List, Tetrahedron, 2002, 58, 5573–5590; (d) A. B. Northrup and D. W. C. MacMillan, Science, 2004, 305, 1752–1755; (e) A. B. Northrup, I. K. Mangion, F. Hettche and D. W. C. MacMillan, Angew. Chem., Int. Ed., 2004, 43, 2152– 2154. 25 A. B. Smith, C. Sfouggatakis, D. B. Gotchev, S. Shirakami, D. Bauer, W. Zhu and V. A. Doughty, Org. Lett., 2004, 6, 3637–3640. 26 A. B. Smith, C. A. Risatti, O. Atasoylu, C. S. Bennett, J. Liu, H. Cheng, K. TenDyke and Q. Xu, J. Am. Chem. Soc., 2011, 133, 14042–14053. 27 A. R. Carroll, E. Hyde, J. Smith, R. J. Quinn, G. Guymer and P. I. Forster, J. Org. Chem., 2005, 70, 1096–1099. 2W. J. O’Brien, L. Plamondon, M. R¨onn, C. Sun, W.-Y. Zhang and J. A. Sutcliffe, J. Med. Chem., 2012, 55, 597–605; (c) R. B. Clark, M. He, C. Fyfe, D. Loand, W. J. O’Brien, L. Plamondon, J. A. Sutcliffe and X.-Y. Xiao, J. Med. Chem., 2011, 54, 1511–1528; (d) R. B. Clark, D. K. Hunt, M. He, C. Achorn, C.-L. Chen, Y. Deng, C. Fyfe, T. H. Grossman, P. C. Hogan, W. J. O’Brien, L. Plamondon, M. R¨onn, J. A. Sutcliffe, Z. Zhu and X.-Y. Xiao, J. Med. Chem., 2012, 55, 606–622; (e) C. Sun, D. K. Hunt, R. B. Clark, D. Loand, W. J. O’Brien, L. Plamondon and X.-Y. Xiao, J. Med. Chem., 2011, 54, 3704–3731. 19 M. Ronn, Z. Zhu, P. C. Hogan, W.-Y. Zhang, J. Niu, C. E. Katz, N. Dunwoody, O. Gilicky, Y. Deng, D. K. Hunt, M. He, C.-L. Chen, C. Sun, R. B. Clark and X.-Y. Xiao, Org. Process Res. Dev., 2013, 17, 838–845. 20 D. A. Kummer, D. Li,
PAPER
Applications of biocatalytic arene ipso,ortho cis-dihydroxylation in synthesis
aDepartment of Chemistry, University of Bath, Bath, UK E-mail:S.E.Lewis@bath.ac.uk
10.1039/C3CC49694E
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In 2005, Myers and co-workers reported the first use of 4 in complex natural product total synthesis.13 From their previously reported building block 43, tricyclic diketone 59 was accessible in a further 7 steps (10% overall yield from benzoate, Scheme 7). Diketone 59 serves as a common precursor to the tetracycline AB-ring system and may be coupled with D-ring precursors such as 60 by a Michael–Dieckmann cascade cyclisation that forms the C-ring. Thus, after deprotection, the natural product ()-6-deoxytetracycline 61 is accessible in 14 steps and 7.0% overall yield from benzoate. Several points about the synthesis are noteworthy. The yield represents an improvement of orders of magnitude over the yields for all previously reported total syntheses of tetracyclines. Thus, for the first time, novel tetracycline analogues became accessible in useful quantities; union of 62 with 59 to access 63 is a representative example. Secondly, previous total syntheses of tetracyclines had been bedevilled by the difficulty of installing the C12a tertiary alcohol at a late stage.14c The Myers approach is conceptually distinct in that the C12a hydroxyl group is installed in the very first step, i.e. it is the hydroxyl group deriving from the microbial ipso hydroxylation. Finally, apart from the C12a stereocentre, all other stereocentres in the final tetracyclines are set under substrate control. Thus, all the stereochemical information in the final products may be considered ultimately to be of enzymatic origin. In the years following the Myers group’s initial disclosure, the methodology has been extended and improved to allow for the preparation of a greater diversity of novel tetracycline analogues.14 This has culminated in the development of eravacycline 65 (accessed from 59 and 64) by Tetraphase Pharmaceuticals.15 Eravacycline is indicated for treatment of multidrug-resistant infections and is currently in phase III trials.
13 (a) M. G. Charest, C. D. Lerner, J. D. Brubaker, D. R. Siegel and A. G. Myers, Science, 2005, 308, 395; (b) M. G. Charest, D. R. Siegel and A. G. Myers, J. Am. Chem. Soc., 2005, 127, 8292.
14 (a) J. D. Brubaker and A. G. Myers, Org. Lett., 2007, 9, 3523; (b) C. Sun, Q. Wang, J. D. Brubaker, P. M. Wright, C. D. Lerner, K. Noson, M. Charest, D. R. Siegel, Y.-M. Wang and A. G. Myers, J. Am. Chem. Soc., 2008, 130, 17913; (c) D. A. Kummer, D. Li, A. Dion and A. G. Myers, Chem. Sci., 2011, 2, 1710; (d) P. M. Wright and A. G. Myers, Tetrahedron, 2011, 67, 9853.
15 (a) R. B. Clark, M. He, C. Fyfe, D. Lofland, W. J. O’Brien, L. Plamondon, J. A. Sutcliffe and X.-Y. Xiao, J. Med. Chem., 2011, 54, 1511; (b) C. Sun, D. K. Hunt, R. B. Clark, D. Lofland, W. J. O’Brien, L. Plamondon and X.-Y. Xiao, J. Med. Chem., 2011, 54, 3704; (c) X.-Y. Xiao, D. K. Hunt, J. Zhou, R. B. Clark, N. Dunwoody, C. Fyfe, T. H. Grossman, W. J. O’Brien, L. Plamondon, M. Ro¨nn, C. Sun, W.-Y. Zhang and J. A. Sutcliffe, J. Med. Chem., 2012, 55, 597; (d) R. B. Clark, D. K. Hunt, M. He, C. Achorn, C.-L. Chen, Y. Deng, C. Fyfe, T. H. Grossman, P. C. Hogan, W. J. O’Brien, L. Plamondon, M. Ro¨nn, J. A. Sutcliffe, Z. Zhu and X.-Y. Xiao, J. Med. Chem., 2012, 55, 606; (e) M. Ronn, Z. Zhu, P. C. Hogan, W.-Y. Zhang, J. Niu, C. E. Katz, N. Dunwoody, O. Gilicky, Y. Deng, D. K. Hunt, M. He, C.-L. Chen, C. Sun, R. B. Clark and X.-Y. Xiao, Org. Process Res. Dev., 2013, 17, 838; ( f ) R. B. Clark, M. He, Y. Deng, C. Sun, C.-L. Chen, D. K. Hunt, W. J. O’Brien, C. Fyfe, T. H. Grossman, J. A. Sutcliffe, C. Achorn, P. C. Hogan, C. E. Katz, J. Niu, W.-Y. Zhang, Z. Zhu, M. Ro¨nn and X.-Y. Xiao, J. Med. Chem., 2013, 56, 8112.
WO 2017125557, Crystalline forms of eravacycline dihydrochloride or its solvates or hydrates. Also claims a process for the preparation of an eravacycline intended for oral or parenteral use, for the treatment of bacterial infections, preferably intra-abdominal and urinary tract infections caused by multidrug resistant gram negative pathogens. Follows on from WO2017097891 .
Tetraphase Pharmaceuticals is developing eravacycline, a fully synthetic fluorocycline antibiotic and the lead from a series of tetracycline analogs which includes TP-221 and TP-170, for treating bacterial infections.
Eravacycline is a tetracycline antibiotic chemically designated (4S,4aS,5aR,l2aS)-4-(Dimethylamino)-7-fluoro-3 ,10,12,12a-tetrahydroxy- 1,11 -dioxo-9-[2-(-pyrrolidin- 1 -yl)acetamido]-l,4,4a,5,5a,6,l l ,12a-octahydrotetracene-2-carboxamide and can be represented by the following chemical structure according to formula (I).
Eravacycline possesses antibacterial activity against Gram negative pathogens and Gram positive pathogens, in particular against multidrug resistant (MDR) Gram negative pathogens and is currently undergoing phase III clinical trials in patients suffering from complicated intraabdominal infections (cIAI) and urinary tract infections (cUTI).
WO 2010/017470 Al discloses eravacycline as compound 34. Eravacycline is described to be prepared according to a process, which is described in more detail only for related compounds.
The last step of this process involves column chromatography with diluted hydrochloric acid/ acetonitrile, followed by freeze drying.
WO 2012/021829 Al discloses pharmaceutically acceptable acid and base addition salts of eravacycline in general and a general process for preparing the same involving reacting eravacycline free base with the corresponding acids and bases, respectively. On page 15, lines 3 to 6, a lyophilized powder containing an eravacycline salt and mannitol is disclosed.
Xiao et. al. “Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: A Potent, Broad Spectrum Antibacterial Agent” J. Med. Chem. 2012, 55, 597-605 synthesized eravacycline following the procedure for compounds 17e and 17i on page 603. After preparative reverse phase HPLC, compounds 17e and 17i were both obtained as bis-hydrochloride salts in form of yellow solids.
Ronn et al. “Process R&D of Eravacycline: The First Fully Synthetic Fluorocycline in Clinical Development” Org. Process Res. Dev. 2013, 17, 838-845 describe a process yielding eravacycline bis-hydrochloride as the final product. The last step involves precipitation of eravacycline bis-hydrochloride salt by adding ethyl acetate as an antisolvent to a solution of eravacycline bis-hydrochloride in an ethanol/ methanol mixture. The authors describe in some detail the difficulties during preparation of the bis-hydrochloride salt of eravacycline. According to Ronn et al. “partial addition of ethyl acetate led to a mixture containing suspended salt and a gummy form of the salt at the bottom of the reactor. At this stage, additional ethanol was added, and the mixture was aged with vigorous stirring until the gummy material also became a suspended solid.” In addition, after drying under vacuum the solid contained “higher than acceptable levels of ethanol”. “The ethanol was then displaced by water by placing a tray containing the solids obtained in a vacuum oven at reduced pressure (…) in the presence of an open vessel of water.” At the end eravacycline bis-hydrochloride salt containing about 4 to 6% residual moisture was obtained. The authors conclude that there is a need for additional improvements to the procedure along with an isolation step suitable for large scale manufacturing.
It is noteworthy that eravacycline or its salts are nowhere described as being a crystalline solid and that the preparation methods used for the preparation of eravacycline are processes like lyophilization, preparative column chromatography and precipitation, which typically yield amorphous material.
The cumbersome process of Ronn et al. points towards problems in obtaining eravacycline bis-hydrochloride in a suitable solid state, problems with scaleability of the available production process as well as problems with the isolation and drying steps of eravacycline.
In addition, amorphous solids can show low chemical stability, low physical stability, hygroscopicity, poor isolation and powder properties, etc.. Such properties are drawbacks for the use as an active pharmaceutical ingredients.
Thus, there is a need in pharmaceutical development for solid forms of an active pharmaceutical ingredient which demonstrate a favorable profile of relevant properties for formulation as a pharmaceutical composition, such as high chemical and physical stability, improved properties upon moisture contact, low(er) hygroscopicity and improved powder properties.
The invention relates to crystalline eravacycline bis-hydrochloride and to a process for its preparation. Furthermore, the invention relates to the use of crystalline eravacycline bis-hydrochloride for the preparation of pharmaceutical compositions. The invention further relates to pharmaceutical compositions comprising an effective amount of crystalline eravacycline bis-hydrochloride. The pharmaceutical compositions of the present invention can be used as medicaments, in particular for treatment and/ or prevention of bacterial infections e.g. caused by Gram negative pathogens or Gram positive pathogens, in particular caused by multidrug resistant Gram negative pathogens. The pharmaceutical compositions of the present invention can thus be used as medicaments for e.g. the treatment of complicated intra-abdominal and urinary tract infection
^ Jump up to:abSolomkin, Joseph; Evans, David; Slepavicius, Algirdas; Lee, Patrick; Marsh, Andrew; Tsai, Larry; Sutcliffe, Joyce A.; Horn, Patrick (2016-11-16). “Assessing the Efficacy and Safety of Eravacycline vs Ertapenem in Complicated Intra-abdominal Infections in the Investigating Gram-Negative Infections Treated With Eravacycline (IGNITE 1) Trial: A Randomized Clinical Trial”. JAMA surgery. ISSN2168-6262. PMID27851857. doi:10.1001/jamasurg.2016.4237.
Tetraphase Pharmaceuticals Inc. (NASDAQ:TTPH) today announced that it will present two posters at IDWeek 2013 that examine the potential of its lead antibiotic candidate eravacycline to treat serious multi-drug resistant (MDR) infections. The first will highlight positive results of a Phase 1 study assessing the bronchopulmonary disposition safety and tolerability of eravacycline in healthy men and women; this study represents the first clinical assessment of eravacycline for potential use in treating pneumonia. The second poster will provide the results of a study that examined the activity of eravacycline in vitro against multiple Gram-negative and Gram-positive pathogens to set quality-control limits for monitoring eravacycline activity in future testing programs. http://www.pharmiweb.com/pressreleases/pressrel.asp?ROW_ID=79335#.Uk6MOoanonU
More: http://www.pharmiweb.com/pressreleases/pressrel.asp?ROW_ID=79335#.Uk6MOoanonU#ixzz2gkEMTtQm
Eravacycline (TP-434 or 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline) is a novel fluorocycline that was evaluated for antimicrobial activity against panels of recent aerobic and anaerobic Gram-negative and Gram-positive bacteria. Eravacycline showed potent broad spectrum activity against 90% of the isolates (MIC90) in each panel at concentrations ranging from ≤0.008 to 2 μg/mL for all species panels except Pseudomonas aeruginosa and Burkholderia cenocepacia (MIC90 values of 32 μg/mL for both organisms). The antibacterial activity of eravacycline was minimally affected by expression of tetracycline-specific efflux and ribosomal protection mechanisms in clinical isolates. Further, eravacycline was active against multidrug-resistant bacteria, including those expressing extended spectrum β-lactamases and mechanisms conferring resistance to other classes of antibiotics, including carbapenem resistance. Eravacycline has the potential to be a promising new IV/oral antibiotic for the empiric treatment of complicated hospital/healthcare infections and moderate-to-severe community-acquired infections.
Tetraphase’s lead product candidate, eravacycline, has also received an award from Biomedical Advanced Research and Development Authority (BARDA) that provides for funding to develop eravacycline as a potential counter-measure to certain biothreat pathogens. It is worth up to USD 67 million.
We are developing our lead product candidate, eravacycline, as a broad-spectrum intravenous and oral antibiotic for use as a first-line empiric monotherapy for the treatment of multi-drug resistant (MDR) infections, including MDR Gram-negative bacteria. We developed eravacycline using our proprietary chemistry technology. We completed a successful Phase 2 clinical trial of eravacycline with intravenous administration for the treatment of patients with complicated intra-abdominal infections (cIAI) and have initiated the Phase 3 clinical program.
Eravacycline is a novel, fully synthetic tetracycline antibiotic. We selected eravacycline for development from tetracycline derivatives that we generated using our proprietary chemistry technology on the basis of the following characteristics of the compound that we observed in in vitro studies of the compound:
potent antibacterial activity against a broad spectrum of susceptible and multi-drug resistant bacteria, including Gram-negative, Gram-positive, atypical and anaerobic bacteria;
potential to treat the majority of patients as a first-line empiric monotherapy with convenient dosing; and
potential for intravenous-to-oral step-down therapy.
In in vitro studies, eravacycline has been highly active against emerging multi-drug resistant pathogens like Acinetobacter baumannii as well as clinically important species ofEnterobacteriaceae, including those isolates that produce ESBLs or are resistant to the carbapenem class of antibiotics, and anaerobes.
Based on in vitro studies we have completed, eravacycline shares a similar potency profile with carbapenems except that it more broadly covers Gram-positive pathogens like MRSA and enterococci, is active against carbapenem-resistant Gram-negative bacteria and unlike carbapenems like Primaxin and Merrem is not active against Pseudomanas aeruginosa. Eravacycline has demonstrated strong activity in vitro against Gram-positive pathogens, including both nosocomial and community-acquired methicillin susceptible or resistantStaphylococcus aureus strains, vancomycin susceptible or resistant Enterococcus faecium andEnterococcus faecalis, and penicillin susceptible or resistant strains of Streptococcus pneumoniae. In in vitro studies for cIAI, eravacycline consistently exhibited strong activity against enterococci and streptococci. One of the most frequently isolated anaerobic pathogens in cIAI, either as the sole pathogen or often in conjunction with another Gram-negative bacterium, is Bacteroides fragilis. In these studies eravacycline demonstrated activity against Bacteroides fragilis and a wide range of Gram-positive and Gram-negative anaerobes.
Key Differentiating Attributes of Eravacycline
The following key attributes of eravacycline, observed in clinical trials and preclinical studies of eravacycline, differentiate eravacycline from other antibiotics targeting multi-drug resistant infections, including multi-drug resistant Gram-negative infections. These attributes will make eravacycline a safe and effective treatment for cIAI, cUTI and other serious and life-threatening infections for which we may develop eravacycline, such as ABSSSI and acute bacterial pneumonias.
Broad-spectrum activity against a wide variety of multi-drug resistant Gram-negative, Gram-positive and anaerobic bacteria. In our recently completed Phase 2 clinical trial of the intravenous formulation of eravacycline, eravacycline demonstrated a high cure rate against a wide variety of multi-drug resistant Gram-negative, Gram-positive and anaerobic bacteria. In addition, in in vitro studies eravacycline demonstrated potent antibacterial activity against Gram-negative bacteria, including E. coli; ESBL-producing Klebsiella pneumoniae; Acinetobacter baumannii; Gram-positive bacteria, including MSRA and vancomycin-resistant enterococcus, or VRE; and anaerobic pathogens. As a result of this broad-spectrum coverage, eravacycline has the potential to be used as a first-line empiric monotherapy for the treatment of cIAI, cUTI, ABSSSI, acute bacterial pneumonias and other serious and life-threatening infections.
Favorable safety and tolerability profile. Eravacycline has been evaluated in more than 250 subjects in the Phase 1 and Phase 2 clinical trials that we have conducted. In these trials, eravacycline demonstrated a favorable safety and tolerability profile. In our recent Phase 2 clinical trial of eravacycline, no patients suffered any serious adverse events, and safety and tolerability were comparable to ertapenem, the control therapy in the trial. In addition, in the Phase 2 clinical trial, the rate at which gastrointestinal adverse events such as nausea and vomiting that occurred in the eravacycline arms was comparable to the rate of such events in the ertapenem arm of the trial.
Convenient dosing regimen. In our recently completed Phase 2 clinical trial we dosed eravacycline once or twice a day as a monotherapy. Eravacycline will be able to be administered as a first-line empiric monotherapy with once- or twice-daily dosing, avoiding the need for complicated dosing regimens typical of multi-drug cocktails and the increased risk of negative drug-drug interactions inherent to multi-drug cocktails.
Potential for convenient intravenous-to-oral step-down. In addition to the intravenous formulation of eravacycline, we are also developing an oral formulation of eravacycline. If successful, this oral formulation would enable patients who begin intravenous treatment with eravacycline in the hospital setting to transition to oral dosing of eravacycline either in hospital or upon patient discharge for convenient home-based care. The availability of both intravenous and oral administration and the oral step-down will reduce the length of a patient’s hospital stay and the overall cost of care.
Additionally, in February 2012, Tetraphase announced a contract award from the Biomedical Advanced Research and Development Authority (BARDA) worth up to $67 million for the development of eravacycline, from which Tetraphase may receive up to approximately $40 million in funding. The contract includes pre-clinical efficacy and toxicology studies; clinical studies; manufacturing activities; and associated regulatory activities to position the broad-spectrum antibiotic eravacycline as a potential empiric countermeasure for the treatment of inhalational disease caused by Bacillus anthracis, Francisella tularensis and Yersinia pestis.\
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Process research and development of the first fully synthetic broad spectrum 7-fluorotetracycline in clinical development is described. The process utilizes two key intermediates in a convergent approach. The key transformation is a Michael–Dieckmann reaction between a suitable substituted aromatic moiety and a key cyclohexenone derivative. Subsequent deprotection and acylation provide the desired active pharmaceutical ingredient in good overall yield.
Process R&D of Eravacycline: The First Fully Synthetic Fluorocycline in Clinical Development
FDA Grants QIDP Designation to Eravacycline, Tetraphase’s Lead Antibiotic Product Candidate
– Eravacycline designated as a QIDP for complicated intra-abdominal infection (cIAI) and complicated urinary tract infection (cUTI) indications –
July 15, 2013 08:30 AM Eastern Daylight Time
WATERTOWN, Mass.–(BUSINESS WIRE)–Tetraphase Pharmaceuticals, Inc. (NASDAQ: TTPH) today announced that the U.S. Food and Drug Administration (FDA) has designated the company’s lead antibiotic product candidate, eravacycline, as a Qualified Infectious Disease Product (QIDP). The QIDP designation, granted for complicated intra-abdominal infection (cIAI) and complicated urinary tract infection (cUTI) indications, will make eravacycline eligible to benefit from certain incentives for the development of new antibiotics provided under the Generating Antibiotic Incentives Now Act (GAIN Act). These incentives include priority review and eligibility for fast-track status. Further, if ultimately approved by the FDA, eravacycline is eligible for an additional five-year extension of Hatch-Waxman exclusivity.
The U.S. Food and Drug Administration today approved Idhifa (enasidenib) for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) who have a specific genetic mutation. The drug is approved for use with a companion diagnostic, the RealTime IDH2 Assay, which is used to detect specific mutations in the IDH2 gene in patients with AML.
The U.S. Food and Drug Administration today approved Idhifa (enasidenib) for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) who have a specific genetic mutation. The drug is approved for use with a companion diagnostic, the RealTime IDH2 Assay, which is used to detect specific mutations in the IDH2 gene in patients with AML.
“Idhifa is a targeted therapy that fills an unmet need for patients with relapsed or refractory AML who have an IDH2 mutation,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “The use of Idhifa was associated with a complete remission in some patients and a reduction in the need for both red cell and platelet transfusions.”
AML is a rapidly progressing cancer that forms in the bone marrow and results in an increased number of abnormal white blood cells in the bloodstream and bone marrow. The National Cancer Institute at the National Institutes of Health estimates that approximately 21,380 people will be diagnosed with AML this year; approximately 10,590 patients with AML will die of the disease in 2017.
Idhifa is an isocitrate dehydrogenase-2 inhibitor that works by blocking several enzymes that promote cell growth. If the IDH2 mutation is detected in blood or bone marrow samples using the RealTime IDH2 Assay, the patient may be eligible for treatment with Idhifa.
The efficacy of Idhifa was studied in a single-arm trial of 199 patients with relapsed or refractory AML who had IDH2 mutations as detected by the RealTime IDH2 Assay. The trial measured the percentage of patients with no evidence of disease and full recovery of blood counts after treatment (complete remission or CR), as well as patients with no evidence of disease and partial recovery of blood counts after treatment (complete remission with partial hematologic recovery or CRh). With a minimum of six months of treatment, 19 percent of patients experienced CR for a median 8.2 months, and 4 percent of patients experienced CRh for a median 9.6 months. Of the 157 patients who required transfusions of blood or platelets due to AML at the start of the study, 34 percent no longer required transfusions after treatment with Idhifa.
Common side effects of Idhifa include nausea, vomiting, diarrhea, increased levels of bilirubin (substance found in bile) and decreased appetite. Women who are pregnant or breastfeeding should not take Idhifa because it may cause harm to a developing fetus or a newborn baby.
The prescribing information for Idhifa includes a boxed warning that an adverse reaction known as differentiation syndrome can occur and can be fatal if not treated. Sign and symptoms of differentiation syndrome may include fever, difficulty breathing (dyspnea), acute respiratory distress, inflammation in the lungs (radiographic pulmonary infiltrates), fluid around the lungs or heart (pleural or pericardial effusions), rapid weight gain, swelling (peripheral edema) or liver (hepatic), kidney (renal) or multi-organ dysfunction. At first suspicion of symptoms, doctors should treat patients with corticosteroids and monitor patients closely until symptoms go away.
Idhifa was granted Priority Review designation, under which the FDA’s goal is to take action on an application within six months where the agency determines that the drug, if approved, would significantly improve the safety or effectiveness of treating, diagnosing or preventing a serious condition. Idhifa also received Orphan Drugdesignation, which provides incentives to assist and encourage the development of drugs for rare diseases.
The FDA granted the approval of Idhifa to Celgene Corporation. The FDA granted the approval of the RealTime IDH2 Assay to Abbott Laboratories
Enasidenib (AG-221) is an experimental drug in development for treatment of cancer. It is a small molecule inhibitor of IDH2 (isocitrate dehydrogenase 2). It was developed by Agios Pharmaceuticals and is licensed to Celgene for further development.
Isocitrate dehydrogenase is a critical enzyme in the citric acid cycle. Mutated forms of IDH produce high levels of 2-hydroxyglutarate and can contribute to the growth of tumors. IDH1 catalyzes this reaction in the cytoplasm, while IDH2 catalyzes this reaction in mitochondria. Enasidenib disrupts this cycle.[1][2]
Development
The drug was discovered in 2009, and an investigational new drug application was filed in 2013. In an SEC filing, Agios announced that they and Celgene were in the process of filing a new drug application with the FDA.[3] The fast track designation allows this drug to be developed in what in markedly less than the average 14 years it takes for a drug to be developed and approved.[4]
Enasidenib (AG-221) is an experimental drug in development for treatment of cancer. It is a small molecule inhibitor of IDH2 (isocitrate dehydrogenase 2). It was developed by Agios Pharmaceuticals and is licensed to Celgene for further development.
An orally available inhibitor of isocitrate dehydrogenase type 2 (IDH2), with potential antineoplastic activity. Upon administration, AG-221 specifically inhibits IDH2 in the mitochondria, which inhibits the formation of 2-hydroxyglutarate (2HG). This may lead to both an induction of cellular differentiation and an inhibition of cellular proliferation in IDH2-expressing tumor cells. IDH2, an enzyme in the citric acid cycle, is mutated in a variety of cancers; It initiates and drives cancer growth by blocking differentiation and the production of the oncometabolite 2HG.
Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate (i.e., a-ketoglutarate). These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)-dependent isocitrate dehydrogenases, which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer.
IDH2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial) is also known as IDH; IDP; IDHM; IDPM; ICD-M; or mNADP-IDH. The protein encoded by this gene is the
NADP(+)-dependent isocitrate dehydrogenase found in the mitochondria. It plays a role in intermediary metabolism and energy production. This protein may tightly associate or interact with the pyruvate dehydrogenase complex. Human IDH2 gene encodes a protein of 452 amino acids. The nucleotide and amino acid sequences for IDH2 can be found as GenBank entries NM_002168.2 and NP_002159.2 respectively. The nucleotide and amino acid sequence for human IDH2 are also described in, e.g., Huh et al., Submitted (NOV-1992) to the
EMBL/GenBank/DDBJ databases; and The MGC Project Team, Genome Res.
14:2121-2127(2004).
Non-mutant, e.g., wild type, IDH2 catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate (a- KG) thereby reducing NAD+ (NADP+) to NADH (NADPH), e.g., in the forward reaction:
It has been discovered that mutations of IDH2 present in certain cancer cells result in a new ability of the enzyme to catalyze the NAPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). 2HG is not formed by wild- type IDH2. The production of 2HG is believed to contribute to the formation and progression of cancer (Dang, L et al, Nature 2009, 462:739-44).
The inhibition of mutant IDH2 and its neoactivity is therefore a potential therapeutic treatment for cancer. Accordingly, there is an ongoing need for inhibitors of IDH2 mutants having alpha hydroxyl neoactivity.
Mechanism of action
Isocitrate dehydrogenase is a critical enzyme in the citric acid cycle. Mutated forms of IDH produce high levels of 2-hydroxyglutarate and can contribute to the growth of tumors. IDH1 catalyzes this reaction in the cytoplasm, while IDH2 catalyzes this reaction in mitochondria. Enasidenib disrupts this cycle.[1][2]
Development
The drug was discovered in 2009, and an investigational new drug application was filed in 2013. In an SEC filing, Agios announced that they and Celgene were in the process of filing a new drug application with the FDA.[3] The fast track designation allows this drug to be developed in what in markedly less than the average 14 years it takes for a drug to be developed and approved.[4]
Example 1, Step 1: preparation of 6-trifluoromethyl-pyridine-2-carboxylic acid
Diethyl ether (4.32 L) and hexanes (5.40 L) are added to the reaction vessel under N2 atmosphere, and cooled to -75 °C to -65 °C. Dropwise addition of n-Butyl lithium (3.78 L in 1.6 M hexane) under N2 atmosphere at below -65 °C is followed by dropwise addition of dimethyl amino ethanol (327.45 g, 3.67 mol) and after 10 min. dropwise addition of 2-trifluoromethyl pyridine (360 g, 2.45 mol). The reaction is stirred under N2 while maintaining the temperature below -65 °C for about 2.0-2.5 hrs. The reaction mixture is poured over crushed dry ice under N2, then brought to a temperature of 0 to 5 °C while stirring (approx. 1.0 to 1.5 h) followed by the addition of water (1.8 L). The reaction mixture is stirred for 5-10 mins and allowed to warm to 5-10 °C. 6N HC1 (900 mL) is added dropwise until the mixture reached pH 1.0 to 2.0, then the mixture is stirred for 10-20 min. at 5-10 °C. The reaction mixture is diluted with ethyl acetate at 25-35 °C, then washed with brine solution. The reaction is concentrated and rinsed with n-heptane and then dried to yield 6-trifluoromethyl-pyridine-2-carboxylic acid.
Example 1, Step 2: preparation of 6-trifluoromethyl-pyridine-2-carboxylic acid methyl ester Methanol is added to the reaction vessel under nitrogen atmosphere. 6-trifluoromethyl- pyridine-2-carboxylic acid (150 g, 0.785 mol) is added and dissolved at ambient temperature. Acetyl chloride (67.78 g, 0.863 mol) is added dropwise at a temperature below 45 °C. The reaction mixture is maintained at 65-70 °C for about 2-2.5 h, and then concentrated at 35-45 °C under vacuum and cooled to 25-35 °C. The mixture is diluted with ethyl acetate and rinsed with saturated NaHC03 solution then rinsed with brine solution. The mixture is concentrated at temp 35-45 °C under vacuum and cooled to 25-35 °C, then rinsed with n-heptane and concentrated at temp 35-45 °C under vacuum, then degassed to obtain brown solid, which is rinsed with n-heptane and stirred for 10-15 minute at 25-35 °C. The suspension is cooled to -40 to -30 °C while stirring, and filtered and dried to provide 6-trifluoromethyl-pyridine-2-carboxylic acid methyl ester.
Example 1, Step 3: preparation of 6-(6-Trifluoromethyl-pyridin-2-yl)-lH-l,3,5-triazine-2,4-dione
1 L absolute ethanol is charged to the reaction vessel under N2 atmosphere and Sodium Metal (11.2 g, 0.488 mol) is added in portions under N2 atmosphere at below 50 °C. The reaction is stirred for 5-10 minutes, then heated to 50-55 °C. Dried Biuret (12.5 g, 0.122 mol) is added to the reaction vessel under N2 atmosphere at 50-55 °C temperature, and stirred 10-15 minutes. While maintaining 50-55 °C 6-trifluoromethyl-pyridine-2-carboxylic acid methyl ester (50.0 g, 0.244 mol) is added. The reaction mixture is heated to reflux (75-80 °C) and maintained for 1.5-2 hours. Then cooled to 35-40 °C, and concentrated at 45-50 °C under vacuum. Water is added and the mixture is concentrated under vacuum then cooled to 35-40 °C more water is added and the mixture cooled to 0 -5 °C. pH is adjusted to 7-8 by slow addition of 6N HC1, and solid precipitated out and is centrifuged and rinsed with water and centrifuged again. The off white to light brown solid of 6-(6-Trifluoromethyl-pyridin-2-yl)-lH-l,3,5-triazine-2,4-dione is dried under vacuum for 8 to 10 hrs at 50 °C to 60 °C under 600mm/Hg pressure to provide 6-(6-Trifluoromethyl-pyridin-2-yl)-lH-l,3,5-triazine-2,4-dione.
Example 1, Step 4: preparation of 2, 4-Dichloro-6-(6-trifluoromethyl-pyridin-2-yl)-l, 3, 5-triazine
POCI3 (175.0 mL) is charged into the reaction vessel at 20- 35 °C, and 6-(6-Trifluoromethyl-pyridin-2-yl)-lH-l,3,5-triazine-2,4-dione (35.0 g, 0.1355 mol) is added in portions at below 50 °C. The reaction mixture is de-gassed 5-20 minutes by purging with N2 gas. Phosphorous pentachloride (112.86 g, 0.542 mol) is added while stirring at below 50 °C and the resulting slurry is heated to reflux (105-110 °C) and maintained for 3-4 h. The reaction mixture is cooled to 50-55 °C, and concentrated at below 55 °C then cooled to 20-30 °C. The reaction mixture is rinsed with ethyl acetate and the ethyl acetate layer is slowly added to cold water (temperature ~5 °C) while stirring and maintaining the temperature below 10 °C. The mixture is stirred 3-5 minutes at a temperature of between 10 to 20 °C and the ethyl acetate layer is collected. The reaction mixture is rinsed with sodium bicarbonate solution and dried over anhydrous sodium sulphate. The material is dried 2-3 h under vacuum at below 45 °C to provide 2, 4-Dichloro-6-(6-trifluoromethyl-pyridin-2-yl)-l, 3, 5-triazine. Example 1, Step 5: preparation of 4-chloro-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoro-methyl)- pyridin-4-yl)-l,3,5-triazin-2-amine
A mixture of THF (135 mL) and 2, 4-Dichloro-6-(6-trifluoromethyl-pyridin-2-yl)-l, 3, 5-triazine (27.0 g, 0.0915 mol) are added to the reaction vessel at 20 – 35 °C, then 4-amino-2-(trifluoromethyl)pyridine (16.31 g, 0.1006 mol) and sodium bicarbonate (11.52 g, 0.1372 mol) are added. The resulting slurry is heated to reflux (75-80 °C) for 20-24 h. The reaction is cooled to 30-40 °C and THF evaporated at below 45 °C under reduced pressure. The reaction mixture is cooled to 20-35 °C and rinsed with ethyl acetate and water, and the ethyl acetate layer collected and rinsed with 0.5 N HC1 and brine solution. The organic layer is concentrated under vacuum at below 45 °C then rinsed with dichloromethane and hexanes, filtered and washed with hexanes and dried for 5-6h at 45-50 °C under vacuum to provide 4-chloro-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoro-methyl)- pyridin-4-yl)-l,3,5-triazin-2-amine.
Example 1, Step 6: preparation of 2-methyl-l-(4-(6-(trifluoromethyl)pyridin-2-yl)-6-(2-(trifluoromethyl)- pyridin-4-ylamino)-l,3,5-triazin-2-ylamino)propan-2-ol
THF (290 mL), 4-chloro-6-(6-(trifluoromethyl)pyridin-2-yl)-N-(2-(trifluoro-methyl)-pyridin-4-yl)-l,3,5-triazin-2-amine (29.0 g, 0.06893 mol), sodium bicarbonate (8.68 g, 0.1033 mol), and 1, 1-dimethylaminoethanol (7.37 g, 0.08271 mol) are added to the reaction vessel at 20-35 °C. The resulting slurry is heated to reflux (75-80 °C) for 16-20 h. The reaction is cooled to 30-40 °C and THF evaporated at below 45 °C under reduced pressure. The reaction mixture is cooled to 20-35 °C and rinsed with ethyl acetate and water, and the ethyl acetate layer collected. The organic layer is concentrated under vacuum at below 45 °C then rinsed with dichlorom ethane and hexanes, filtered and washed with hexanes and dried for 8-1 Oh at 45-50 °C under vacuum to provide 2-methyl-l-(4-(6-(trifluoromethyl)pyridin-2-yl)-6-(2-(trifluoromethyl)- pyridin-4-ylamino)-l,3,5-triazin-2-ylamino)propan-2-ol.
The U.S. Food and Drug Administration today approved Idhifa (enasidenib) for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) who have a specific genetic mutation. The drug is approved for use with a companion diagnostic, the RealTime IDH2 Assay, which is used to detect specific mutations in the IDH2 gene in patients with AML.
The U.S. Food and Drug Administration today approved Idhifa (enasidenib) for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) who have a specific genetic mutation. The drug is approved for use with a companion diagnostic, the RealTime IDH2 Assay, which is used to detect specific mutations in the IDH2 gene in patients with AML.
“Idhifa is a targeted therapy that fills an unmet need for patients with relapsed or refractory AML who have an IDH2 mutation,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “The use of Idhifa was associated with a complete remission in some patients and a reduction in the need for both red cell and platelet transfusions.”
AML is a rapidly progressing cancer that forms in the bone marrow and results in an increased number of abnormal white blood cells in the bloodstream and bone marrow. The National Cancer Institute at the National Institutes of Health estimates that approximately 21,380 people will be diagnosed with AML this year; approximately 10,590 patients with AML will die of the disease in 2017.
Idhifa is an isocitrate dehydrogenase-2 inhibitor that works by blocking several enzymes that promote cell growth. If the IDH2 mutation is detected in blood or bone marrow samples using the RealTime IDH2 Assay, the patient may be eligible for treatment with Idhifa.
The efficacy of Idhifa was studied in a single-arm trial of 199 patients with relapsed or refractory AML who had IDH2 mutations as detected by the RealTime IDH2 Assay. The trial measured the percentage of patients with no evidence of disease and full recovery of blood counts after treatment (complete remission or CR), as well as patients with no evidence of disease and partial recovery of blood counts after treatment (complete remission with partial hematologic recovery or CRh). With a minimum of six months of treatment, 19 percent of patients experienced CR for a median 8.2 months, and 4 percent of patients experienced CRh for a median 9.6 months. Of the 157 patients who required transfusions of blood or platelets due to AML at the start of the study, 34 percent no longer required transfusions after treatment with Idhifa.
Common side effects of Idhifa include nausea, vomiting, diarrhea, increased levels of bilirubin (substance found in bile) and decreased appetite. Women who are pregnant or breastfeeding should not take Idhifa because it may cause harm to a developing fetus or a newborn baby.
The prescribing information for Idhifa includes a boxed warning that an adverse reaction known as differentiation syndrome can occur and can be fatal if not treated. Sign and symptoms of differentiation syndrome may include fever, difficulty breathing (dyspnea), acute respiratory distress, inflammation in the lungs (radiographic pulmonary infiltrates), fluid around the lungs or heart (pleural or pericardial effusions), rapid weight gain, swelling (peripheral edema) or liver (hepatic), kidney (renal) or multi-organ dysfunction. At first suspicion of symptoms, doctors should treat patients with corticosteroids and monitor patients closely until symptoms go away.
Idhifa was granted Priority Review designation, under which the FDA’s goal is to take action on an application within six months where the agency determines that the drug, if approved, would significantly improve the safety or effectiveness of treating, diagnosing or preventing a serious condition. Idhifa also received Orphan Drugdesignation, which provides incentives to assist and encourage the development of drugs for rare diseases.
The FDA granted the approval of Idhifa to Celgene Corporation. The FDA granted the approval of the RealTime IDH2 Assay to Abbott Laboratories
3,4,6-Tridésoxy-3-(diméthylamino)-β-D-xylo-hexopyranoside de (3aS,4R,7S,9R,10R,11R,13R,15R,15aR)-1-{4-[4-(3-aminophényl)-1H-1,2,3-triazol-1-yl]butyl}-4-éthyl-7-fluoro-11-méthoxy-3a,7,9,11,13,15-hex améthyl-2,6,8,14-tétraoxotétradécahydro-2H-oxacyclotétradécino[4,3-d][1,3]oxazol-10-yle
Solithromycin exhibits excellent in vitro activity against a broad spectrum of Gram-positiverespiratory tractpathogens,[3][4] including macrolide-resistant strains.[5] Solithromycin has activity against most common respiratory Gram-(+) and fastidious Gram-(-) pathogens,[6][7] and is being evaluated for its utility in treating gonorrhea.
May 2011: Solithromycin is in a Phase 2 clinical trial for serious community-acquired bacterial pneumonia (CABP) and in a Phase 1 clinical trial with an intravenous formulation.[8]
September 2011 : Solithromycin demonstrated comparable efficacy to levofloxacin with reduced adverse events in Phase 2 trial in people with community-acquired pneumonia[9]
January 2015: In a Phase 3 clinical trial for community-acquired bacterial pneumonia (CABP), Solithromycin administered orally demonstrated statistical non-inferiority to the fluoroquinolone, Moxifloxacin.[10]
July 2015: Patient enrollment for the second Phase 3 clinical trial (Solitaire IV) for community-acquired bacterial pneumonia (CABP) was completed with results expected in Q4 2015.[11]
Oct 2015: IV to oral solithromycin demonstrated statistical non-inferiority to IV to oral moxifloxacin in adults with CABP.[12]
July 2016: Cempra Announces FDA Acceptance of IV and oral formulations of Solithera (solithromycin) New Drug Applications for in the Treatment of Community-Acquired Bacterial Pneumonia.[13]
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Structure
X-ray crystallography studies have shown solithromycin, the first fluoroketolide in clinical development, has a third region of interactions with the bacterial ribosome,[14] as compared with two binding sites for other ketolides.
Macrolide antibiotics, such like erythromycin, azithromycin, and clarithromycin, have proven to be safe and effective for use in treating human infectious diseases such as community-acquired bacterial pneumonia (CABP), urethritis, and other infections.
Because of the importance of macrolide antibiotics, there has been growing recent interest in this area as exemplified by the new fourth-generation macrolide solithromycin , which is developed by Cempra Pharmaceuticals as the first fluoroketolide antibiotic that has recently completed phase III clinical trials and demonstrates potent activity against the pathogens associated with CABP, including macrolide- and penicillin-resistant isolates of S. pneumoniaeis
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To date, all macrolide antibiotics are produced by chemical modification (semisynthesis) of erythromycin, a natural product produced on the ton scale by fermentation. Depicted are erythromycin and the approved semisynthetic macrolide antibiotics clarithromycin, azithromycin and telithromycin along with the dates of their FDA approval and the number of steps for their synthesis from erythromycin. The previous ketolide clinical candidate cethromycin and the current clinical candidate solithromycin are also depicted. It is evident that increasingly lengthy sequences are being employed in macrolide discovery efforts.
Nature, 533, 338–345 (19 May 2016), doi:10.1038/nature17967
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a, Graphical representation of the convergent synthesis of solithromycin, which was previously only accessible by semisynthesis. This route has been adapted for the synthesis of >30 novel ketolide antibiotic candidates, as well as the FDA-approved ketolide telithromycin. Downward, ‘Y’-shaped arrows signify convergent coupling reactions. b, Synthesis of solithromycin, reagents and conditions (subscripts L and R indicate left and right portions, respectively): (aL) lithium (1S,2R)-1-phenyl-2-(pyrrolidin-1-yl)-1-propanolate, 85%; (bL) CuSO4, sodium L-ascorbate, 95%; (aR) tBuLi, MgBr2, 81%; (bR) KH, MeI, 99%; (cR) H5IO6, 99%; (dR) ZnCl2, 91%; (eR) AgOTf, 70%; (fR) HF (aq.), 95%; (gR) Dess–Martin periodinane, 92%; (h) Cp2TiCl2, cyclopentylmagnesium bromide, 80%; (i) Dess–Martin periodinane, 97%; (j) Bu4NF, 95%; (k) 132 °C, 0.5 mM, PhCl, 66%; (l) KOtBu, FN(SO2Ph)2, 85%; (m) Im2CO, DBU; (n) imidazole hydrochloride, 60 °C, 87% over 2 steps.
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The residue was purified by column chromatography (3% methanol–dichloromethane + 0.3% 30% aqueous ammonium hydroxide) to provide solithromycin (170 mg, 87%) as a white powder.
Potential causes for the formation of synthetic impurities that are present in solithromycin (1) during laboratory development are studied in the article. These impurities were monitored by HPLC, and their structures are identified on the basis of MS and NMR spectroscopy. In addition to the synthesis and characterization of these seven impurities, strategies for minimizing them to the level accepted by the International Conference on Harmonization (ICH) are also described.
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PAPER
Identification, Characterization, Synthesis, and Strategy for Minimization of Potential Impurities Observed in the Synthesis of Solithromycin
Macrolide antibacterial agents are characterized by a large lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, are attached. The first generation macrolide, erythromycin, was soon followed by second generation macrolides clarithromycin and azithromycin. Due to widespread of bacterial resistance semi-synthetic derivatives, ketolides, were developed. These, third generation macrolides, to which, for example, belongs telithromycin, are used to treat respiratory tract infections. Currently, a fourth generation macrolide, solithromycin (also known as CEM-101 ) belonging to the fluoroketolide class is in the pre-registration stage. Solithromycin is more potent than third generation macrolides, is active against macrolide-resistant strains, is well-tolerated and exerts good PK and tissue distribution.
One route of synthesis of solithromycin is disclosed in WO2004/080391 A2. Said route is based on the synthetic strategy disclosed in Tetrahedron Letters, 2005, 46, 1483-1487. It is formally a 10 step linear synthesis starting from clarithromycin. Its characteristics are a late cleavage of cladinose, a hexose deoxy sugar which is in ketolides replaced with a keto group, and a 3-step building up of the side chain. Most intermediates are amorphous or cannot be purified by crystallization, hence chromatographic separations are required.
WO 2009/055557 A1 describes a process, in which the linker part of the side chain (azidobutyl) is synthesized separately thus making the synthesis more convergent. In addition, the benzoyl protection group instead of the acetyl is used to protect the 3-hydroxy group of the tetrahydropyran moiety. The linker part of the side chain is introduced using 4-azidobutanamine which is prepared through a selective Staudinger monoreduction of 1 ,4-diazidobutane.
WO 2014/145210 A1 discloses several routes of synthesis all based on the use of already fully constructed side chain building blocks, or its protected forms, which are reacted with an imidazoyl carbamate still containing the protected cladinose moiety. After the introduction of the side chain, the cladinose is cleaved and the aniline group protected for the oxidation of the hydroxy group and the fluorination. After fluorination and deprotection or unmasking of the aniline group, solithromycin is obtained.
Synthesis of crude solithromycin
A third aspect of the invention is a process for providing crude solithromycin (the compound of formula 5) through a convergent synthesis that combines both aforementioned building blocks, the macrolide building block (compound of formula 3) and the side chain building block 3-(1 -(4-aminobutyl)-1 H-1 ,2,3-triazol-4-yl)aniline prepared as discussed above.
As shown in Scheme 4, first, the compound of formula 3 and 3-(1-(4-aminobutyl)-1 H-1 ,2,3-triazol-4-yl)aniline are reacted in the presence of a strong base, for example, DBU, in a suitable solvent, for example, MeCN, to give the compound of formula 4. In the last step, the acetyl protecting group of the hydroxy group located on position β to the dimethylamino substituent is cleaved in methanol.
As discussed above, the fluorination is performed in the presence of acetyl group in the pyran part of the molecule and prior to the incorporation of the side chain, which allows that both exchanging the protection group in the pyran part of the molecule and masking or protecting the aniline moiety from oxidation caused under fluorinating conditions can be avoided.
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Scheme 4: Representation of the specific embodiment of the present invention.
In another aspect of the present invention a process is provided for purification of crude solithromycin.
Purification of crude solithromycin
Due to its properties, solithromycin is very difficult to purify. The amorphous material obtained after various syntheses is truly a challenge for further processing.
Chromatographic separation is very difficult and of poor resolution. Due to the basic polar functional groups, the compound and its related impurities all tend to “trail” on typical normal stationary phases that are considered suitable for industrial use, such as silica and alumina. Purification by crystallization is just as difficult unless the material is already sufficiently pure. Impurities inhibit its crystallization to such an extent that solutions in alcohols can be stable even in concentrations of several-fold above the saturation levels of the pure material. Such solutions refuse to crystallize even after weeks of stirring or cooling. Seeding with crystalline solithromycin has no effect and the added seeds simply dissolve. In addition, only limited purification is achieved in most solvents. Lower primary alcohols, particularly ethanol, are most efficient for purification by crystallization when this is possible, but give low recovery and the crystallization is most sensitive toward impurities, thus still demanding prior chromatographic separation.
Clearly an alternative method of purification would be advantageous. For this reason we developed a process for purification employing an acidic salt formation, for example solithromycin oxalate salt, freebasing back to solithromycin, and crystallization from ethanol (Scheme 5).
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Scheme 5: Representation of a particular embodiment of the present invention.
The formation of crystalline salts from crude solithromycin may be inhibited by impurities and is dependent on the solvent used. Only a limited number of acids gave useful precipitates from solutions of crude solithromycin. Of these, the precipitation of the oxalate salt from isopropyl acetate (‘PrOAc) or 2-methyltetrahydrofuran (MeTHF) was found to be most efficient in regard to yields, reaction times and purification ability. Precipitation of the citrate salt from MeTHF also significantly increased purity. However, impurities strongly inhibited crystal growth rates, the reaction thus required longer times compared to the oxalate salt formation. Crystallization of salts from solutions of impure solithromycin was also found possible with D-(-)-tartaric, dibenzoyl-d-tartaric, 2,4-dihydroxybenzoic, 3,5-dihydroxybenzoic, and (R)-(+)-2-pyrrolidinone-5-carboxylic acids using ethyl acetate, isopropyl acetate or MeTHF as solvents, or their mixtures with methyl f-butyl ether, but the efficiency and purification abilities were inferior to both the oxalate and the citrate salt.
Some acids, such as (R)-(-)-mandelic, L-(+)-tartaric, p-toluenesulfonic, benzoic, malonic, 4-hydroxybenzoic, (-)-malic, and (+)-camphor-10-sulfonic acid formed insoluble amorphous precipitates without improving purity. Many other acids were found unable of forming any precipitate from impure solithromycin under the conditions tested.
A solution of (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,5R,6R,7R,9R,13S,14R,Z)-14-ethyl-13-hydroxy-7-methoxy-3,5,7,9,11,13-hexamethyl-2,4,10-trioxooxacyclotetradec-11-en-6-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl acetate, 1 (313 g) in dichloromethane (2.22 L) was cooled to -25 °C. DBU (115 mL) followed by CDI (125 g) were added and temperature of the reaction was raised to 0°C. The completion of the reaction was followed by HPLC. Upon the completion, the pH of the reaction mixture was adjusted to 6 using 10% aqueous acetic acid. Layers were separated and organic layer was washed twice with water, dried over sodium sulphate and concentrated to afford compound 2 as white foam (HPLC purity: 90 area%).
Example 2: Synthesis of compound 3: (2R,3S,7R,9R, 10R, 11 R, 13S,Z)-10-(((2S,3R,4S,6R)-3- acetoxy-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-13- fluoro-9-methoxy-3,5,7,9, 11,13-hexamethyl-6, 12, 14-trioxooxacyclotetradec-4-en- 3-yl 1H-imidazole-1-carboxylate
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A solution of (2R,3S,7R,9R,10R,11 R,13R,Z)-10-(((2S,3R,4S,6R)-3-acetoxy-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-9-methoxy-3,5,7,9,11,13-hexamethyl-6,12,14-trioxooxacyclotetradec-4-en-3-yl 1H-imidazole-1-carboxylate, 2 in THF (9 L) was cooled to 0 °C. DBU (115 mL) followed by NFSI (211 g) were added and reaction mixture was stirred at 0°C
until completion (followed by HPLC). The reaction mixture was quenched with cold, diluted NaHC03 (3 L). DCM (2.5 L) was added and layers were separated. Aqueous layer was washed with additional DCM (1.5 L). Combined organic layers were washed with brine (2 L), dried over sodium sulphate, filtrated and concentrated. Crude material was suspended in /‘PrOAc (2.5 L). Undissolved material was filtered-off and filtrate was concentrated in vacuo to afford compound 3 as pale yellow foam (475 g, HPLC purity: 85 area%).
A solution of (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,5R,6R,7R,9R,13S,14R,Z)-14-ethyl-13-hydroxy-7-methoxy-3,5,7,9,1 1 , 13-hexamethyl-2,4, 10-trioxooxacyclotetradec-1 1-en-6-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl acetate, 1 (2.45 g) in THF (17 mL) was cooled to 0 °C. DBU (0.9 mL) followed by CDI (0.97 g) were added. The completion of the reaction was followed by HPLC. Upon the completion, reaction was diluted by addition of THF (34 mL). Temperature of the reaction was lowered to -10 °C. DBU (0.72 mL) was added followed by solution of NFSI (1.51 g) in THF (14 mL). Upon completion of the reaction, mixture was diluted with the addition of water/ZPrOAc (1 :4) mixture and layers were separated. Organic phase was washed with water (3 x 25 mL), dried over Na2S04, filtered and concentrated to afford compound 3 as a white foam (3.1 g, HPLC purity: 70 area%).
Example 4: Synthesis of 3-(1 -(4-aminobutyl)-1 H-1 ,2,3-triazol-4-yl)aniline
K2
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1 moi% CuS04(aq)
2-(4-Chlorobutyl)isoindoline-1 ,3-dione. A mixture of phthalimide potassium salt (1 134 g, 6.00 mol), potassium carbonate (209 g, 1.50 mol), 1 ,4-dichlorobutane (1555 g, 12.00 mol), and potassium iodide (51 g, 0.30 mol, 5 mol%) in 2-butanone (4.80 L) was stirred 3 days at reflux conditions. The reaction mixture cooled to 40 °C was filtered and the insoluble materials washed with 2-butanone (1.00 L). The filtrate was evaporated at 80 °C under reduced pressure. 2-Propanol (1.00 L) was added to the residue and the solvent removed under reduced pressure. The residue was then crystallized from 2-propanol (4.30 L) at 25 °C. The product was isolated by filtration and washed with 2-propanol (1.00 L). After drying at 40 °C and approximately 50 mbar, there was obtained a white powder (1 1 1 1 g): 95% assay by quantitative 1H NMR; MS (ESI) m/z = 238 [MH]+.
2-(4-(4-(3-Aminophenyl)-1 H-1 ,2,3-triazol-l -yl)butyl)isoindoline-1 ,3-dione. To a solution of 2-(4-chlorobutyl)isoindoline-1 ,3-dione (950 g, 4.00 mol) in DMSO (2.80 L) was added sodium azide (305 g) and the mixture stirred 4 h at 70 °C. The reaction temperature was reduced to 25 °C and there was added in this order water (0.80 L), ascorbic acid (43 g, 0.24 mol, 6 mol%), 0.5M CuS04(aq) (160 ml_, 2 mol%) and m-aminophenylacetylene (493 g, 4.00 mol). The resulting mixture was stirred 18 h at 40 °C, forming a thick yellow slurry, which was then cooled to 0 °C and slowly diluted with water (2.40 L). The product was isolated by filtration, washing the filter cake with water (3 χ 2.00 L) and a 1 : 1 (vol.) mixture of methanol and water (2.00 L). After drying at 50 °C and approximately 50 mbar the product was obtained as a yellow powder (1405 g): 85% assay by quantitative 1H NMR; MS (ESI) m/z = 362 [MH]+.
3-(1-(4-Aminobutyl)-1H-1,2,3-triazol-4-yl)aniline. To a stirred suspension of 2-(4-(4-(3-Aminophenyl)-1 H-1 ,2,3-triazol-1 -yl)butyl)isoindoline-1 ,3-dione (659 g, 1 .55 mol) in 1 -butanol (3.26 L) was added hydrazine hydrate (50-60%, 174 ml_). After stirring for 18 h at 60 °C there was added toluene (0.72 L) and 1 M NaOH(aq) (5.00 L). After stirring for 20 min at 60 °C, the aqueous phase was removed and the organic phase washed at this same temperature with 1 M NaOH(aq) (1 .00 L), saturated NaCI(aq) (2 χ 2.00 L), and concentrated under reduced pressure to 2/3 of the initial quantity, dried over anhydrous sodium sulfate (300 g) in the presence of Fluorisil (30 g), filtered and evaporated under reduced pressure at 70 °C. The residual 1 -butanol is removed azotropically by adding toluene and evaporation under reduced pressure (2 χ 0.50 L). The residue was dissolved in tetrahydrofuran (0.50 L). To this solution kept stirring at 25 °C was slowly added methyl t-butyl ether (0.50 L) at which point the mixture was seeded. Additional methyl t-butyl ether (0.50 L) was slowly added and the product isolated by filtration, washed with methyl t-butyl ether (0.50 L), and dried at 35 °C and approximately 50 mbar to give an amber colored powder (321 g): mp = 70-73 °C (DSC);
A solution of (2R,3S,7R,9R,10R, 1 1 R, 13S,Z)-10-(((2S,3R,4S,6R)-3-acetoxy-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-13-fluoro-9-methoxy-3,5,7,9, 1 1 , 13-hexamethyl-6, 12,14-trioxooxacyclotetradec-4-en-3-yl 1 H-imidazole-1-carboxylate (425 g) in acetonitrile (3.1 L) was cooled to 0 °C. 4-(1-(4-aminobutyl)-1 H-1 ,2,3-triazol-4-yl)aniline, 3 (213 g) followed by DBU (83 ml.) were added. The mixture was stirred at 0° C until completion of the reaction (followed by HPLC). Mixture of DCM and water was added (4 L, 1 :1 ) and the pH was adjusted to 6 with 10% aqueous acetic acid. Layers were separated and organic layer was washed with water (2 x 2 L), dried over sodium sulphate and concentrated. The crude material was suspended in EtOAc (2.5 L). Undissolved material was filtered off and filtrate was concentrated in vacuo to afford compound 4 as yellow foam (470 g, HPLC purity: 75 area%).
Example 11 : Purification of crude solithromycin (compound 5) via oxalate (2)
To isopropyl acetate (5.77 L) crude solithromycin (192 g, 72 area%) was added, afterwards the mixture was stirred at reflux and filtered to remove any insoluble material. The filtrate was then stirred at 55 °C and oxalic acid (14.91 g, 164 mmol) was added in one batch. The suspension was cooled to 20 °C in the course of 1 h, stirred for additional 1 h and the product was isolated by filtration, washed with isopropyl acetate (0.5 L), and dried at 40 °C under reduced pressure to give the oxalate salt (106 g): 87.81 area% by HPLC (UV at 228 nm). The evaporation of the filtrate gave a resinous material containing solithromycin that can be recovered by reprocessing (88 g, 61.13 area%).
The above oxalate salt (106 g) was dissolved in water (2.40 L) and washed with MeTHF (2 χ 1.00 L) and ethyl acetate (0.50 L). Aqueous ammonia (25%; 37 mL) was then added to the filtrate while stirring at 25 °C. The precipitated product was extracted with ethyl acetate two times (3.00 L and 0.50 L). The combined extracts were washed with water (0.50 L), dried over Na2S04 and evaporated under reduced pressure. To the residue ethanol (2 χ 0.4 L) was added and again evaporated to affect the solvent exchange. The residue was then dissolved in ethanol (300 mL). After stirring for 24 h at 25 °C, the crystallized product was isolated by filtration and drying at 40 °C under reduced pressure to give solithromycin as an off-white crystalline solid (42 g): 98.61 area% by HPLC (UV at 228 nm).
The filtrate was evaporated under reduced pressure to give a yellowish foam (29 g: 68.65 area% by HPLC (UV at 228 nm) that was used for reprocessing.
Crude solithromycin 5 (106 g, HPLC purity: 71 %) was suspended in /‘PrOAc (3 L) and heated to reflux, then filtered to remove undissolved material. Oxalic acid (8 g) was added to a filtrate at 60°C. Mixture was slowly cooled to RT and left stirring for additional 1 h. Precipitate was filtered off and dried in vacuo. Oxalate salt was suspended in water (1 .3 L) (if needed mixture was first filtered through Celite) then 25% aqueous ammonia (21 mL) was added and mixture was stirred additional 15 minutes. Precipitate was filtered off, rinsing with water. Wet solithromycin was dissolved it EtOAc (1 .8 L)/water (800 mL) solution. Layers were separated and organic phase was washed with water (2 x 250 mL), dried over Na2S04 and filtered. EtOAc was removed in vacuo to afford yellow foam.
Yellow foam was dissolved in EtOH (230 mL) and left stirring at RT until white precipitate fell out. White precipitate was dried in vacuo at room temperature to afford clean material (HPLC purity 97 area%).
A solution of (2S,3R,4S,6R)-2-(((3aS,4R,7S,9R, 10R, 1 1 R, 13R, 15R)-1 -(4-(4-(3-aminophenyl)-1 H-1 ,2,3-triazol-1 -yl)butyl)-4-ethyl-7-fluoro-1 1 -methoxy-3a,7,9, 1 1 , 13,15-hexamethyl-2,6,8, 14-tetraoxotetradecahydro-1 H-[1 ]oxacyclotetradecino[4,3-d]oxazol-10-yl)oxy)-4-(dimethylamino)-6- methyltetrahydro-2H-pyran-3-yl acetate, 4 (470 g) in methanol (4.7 L) was stirred at room temperature and completion of the reaction was followed by HPLC. Upon completion, the reaction mixture was concentrated to afford crude solithromycin 5 as orange foam (402 g, HPLC purity: 73 area%).
Jump up^Putnam, Shannon D.; Castanheira, Mariana; Moet, Gary J.; Farrell, David J.; Jones, Ronald N. (2010). “CEM-101, a novel fluoroketolide: antimicrobial activity against a diverse collection of Gram-positive and Gram-negative bacteria”. Diagnostic Microbiology and Infectious Disease. 66 (4): 393–401. PMID20022192. doi:10.1016/j.diagmicrobio.2009.10.013.
Jump up^Putnam, Shannon D.; Sader, Helio S.; Farrell, David J.; Biedenbach, Douglas J.; Castanheira, Mariana (2011). “Antimicrobial characterisation of solithromycin (CEM-101), a novel fluoroketolide: activity against staphylococci and enterococci”. International Journal of Antimicrobial Agents. 37 (1): 39–45. PMID21075602. doi:10.1016/j.ijantimicag.2010.08.021.
The U.S. Food and Drug Administration today approved Mavyret (glecaprevir and pibrentasvir) to treat adults with chronic hepatitis C virus (HCV) genotypes 1-6 without cirrhosis (liver disease) or with mild cirrhosis, including patients with moderate to severe kidney disease and those who are on dialysis. Mavyret is also approved for adult patients with HCV genotype 1 infection who have been previously treated with a regimen either containing an NS5A inhibitor or an NS3/4A protease inhibitor but not both.
The U.S. Food and Drug Administration today approved Mavyret (glecaprevir and pibrentasvir) to treat adults with chronic hepatitis C virus (HCV) genotypes 1-6 without cirrhosis (liver disease) or with mild cirrhosis, including patients with moderate to severe kidney disease and those who are on dialysis. Mavyret is also approved for adult patients with HCV genotype 1 infection who have been previously treated with a regimen either containing an NS5A inhibitor or an NS3/4A protease inhibitor but not both.
Mavyret is the first treatment of eight weeks duration approved for all HCV genotypes 1-6 in adult patients without cirrhosis who have not been previously treated. Standard treatment length was previously 12 weeks or more.
“This approval provides a shorter treatment duration for many patients, and also a treatment option for certain patients with genotype 1 infection, the most common HCV genotype in the United States, who were not successfully treated with other direct-acting antiviral treatments in the past,” said Edward Cox, M.D., director of the Office of Antimicrobial Products in the FDA’s Center for Drug Evaluation and Research.
Hepatitis C is a viral disease that causes inflammation of the liver that can lead to diminished liver function or liver failure. According to the Centers for Disease Control and Prevention, an estimated 2.7 to 3.9 million people in the United States have chronic HCV. Some patients who suffer from chronic HCV infection over many years may have jaundice (yellowish eyes or skin) and complications, such as bleeding, fluid accumulation in the abdomen, infections, liver cancer and death.
There are at least six distinct HCV genotypes, or strains, which are genetically distinct groups of the virus. Knowing the strain of the virus can help inform treatment recommendations. Approximately 75 percent of Americans with HCV have genotype 1; 20-25 percent have genotypes 2 or 3; and a small number of patients are infected with genotypes 4, 5 or 6.
The safety and efficacy of Mavyret were evaluated during clinical trials enrolling approximately 2,300 adults with genotype 1, 2, 3, 4, 5 or 6 HCV infection without cirrhosis or with mild cirrhosis. Results of the trials demonstrated that 92-100 percent of patients who received Mavyret for eight, 12 or 16 weeks duration had no virus detected in the blood 12 weeks after finishing treatment, suggesting that patients’ infection had been cured.
Treatment duration with Mavyret differs depending on treatment history, viral genotype, and cirrhosis status.
The most common adverse reactions in patients taking Mavyret were headache, fatigue and nausea.
Mavyret is not recommended in patients with moderate cirrhosis and contraindicated in patients with severe cirrhosis. It is also contraindicated in patients taking the drugs atazanavir and rifampin.
Hepatitis B virus (HBV) reactivation has been reported in HCV/HBV coinfected adult patients who were undergoing or had completed treatment with HCV direct-acting antivirals, and who were not receiving HBV antiviral therapy. HBV reactivation in patients treated with direct-acting antiviral medicines can result in serious liver problems or death in some patients. Health care professionals should screen all patients for evidence of current or prior HBV infection before starting treatment with Mavyret.
Glecaprevir (INN,[1] codenamed ABT-493) is a hepatitis C virus (HCV) nonstructural (NS) protein 3/4A protease inhibitor that was identified jointly by AbbVie and Enanta Pharmaceuticals. It is being developed as a treatment of chronic hepatitis C infection in co-formulation with an HCV NS5A inhibitor pibrentasvir. Together they demonstrated potent antiviral activity against major HCV genotypes and high barriers to resistance in vitro.[2]
On December 19, 2016, AbbVie submitted New Drug Application to U.S. Food and Drug Administration for glecaprevir/pibrentasvir (trade name Maviret) regimen for the treatment of all major genotypes (1–6) of chronic hepatitis C.[3]
The topic of starting materials has been a vexed topic for some period. Indeed concerns relating to lack of clarity and issues pertaining to practical implementation led the EMA in Sept 2014 to publish a reflection paper—Reflection on the requirements for selection and justification of starting materials for the manufacture of chemical active substances.(10) The paper sought to outline key issues as well as authority expectations; specific areas of interest identified included the following:
1.
Variance in interpretation between applicant and reviewer.
2.
The registration of short syntheses that employ complex custom-made starting materials.
3.
Lack of details preventing authorities being able to assess the suitability of a proposed registered starting material and its associated control strategy.
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While the consensus was that overall this provided a useful perspective of at least the EMA’s interpretation of ICH Q11(2) and requirements for starting…
BV-102; GLYX13, GLYX-13, in phase 3 clinical trials
Originator Northwestern University
Developer Allergan; Naurex
Class Amides; Antidepressants; Neuropsychotherapeutics; Oligopeptides; Small molecules
Mechanism of Action NR2B N-Methyl-D-Aspartate receptor agonists
Highest Development Phases
Phase III Major depressive disorder
Discontinued Bipolar depression; Neuropathic pain
Most Recent Events
01 Jan 2017 Allergan initiates enrolment in a phase III trial for Major depressive disorder (Adjunctive treatment) in USA (IV, Injection) (NCT03002077)
21 Dec 2016 Allergan plans a phase III trial for Major depressive disorder (Adjunctive treatment) in USA (IV, Injection) (NCT03002077)
01 Nov 2016 Phase-III clinical trials in Major depressive disorder (Adjunctive treatment, Prevention of relapse) in USA (IV) (NCT02951988)Image may be NSFW. Clik here to view.Image may be NSFW. Clik here to view.
It is disclosed that GLYX-13 (Rapastinel) acts as NMDA receptor partial agonist, useful for treating neurodegenerative disorders such as stroke-related brain cell death, convulsive disorders, and learning and memory. See WO2015065891 , claiming peptidyl compound. Naurex , a subsidiary of Allergan is developing rapastinel (GLYX-13) (in phase3 clinical trials), a rapid-acting monoclonal antibody-derived tetrapeptide and NMDA receptor glycine site functional partial agonist as well as an amidated form of NT-13, for treating depression.
On March 3, 2014, the U.S. FDA granted Fast Track designation to the development of rapastinel as an adjunctive therapy in treatment-resistant major depressive disorder.[3] As of 2015, the drug had completed phase II clinical development for this indication.[4] On January 29, 2016, Allergan (who acquired Naurex in July 2015) announced that rapastinel had received Breakthrough Therapydesignation from the U.S. FDA for adjunctive treatment of major depressive disorder.
Rapastinel belongs to a group of compounds, referred to as glyxins (hence the original developmental code name of rapastinel, GLYX-13),[5] that were derived via structural modification of B6B21, a monoclonal antibody that similarly binds to and modulates the NMDA receptor.[2][6][7] The glyxins were invented by Joseph Moskal, the co-founder of Naurex.[5] Glyxins and B6B21 do not bind to the glycine site of the NMDA receptor but rather to a different regulatory site on the NMDA receptor complex that serves to allosterically modulate the glycine site.[8] As such, rapastinel is technically an allosteric modulator of the glycine site of the NMDA receptor, and hence is more accurately described as a functional glycine site weak partial agonist.[8]
Apimostinel (NRX-1074), an analogue of rapastinel with the same mechanism of action but dramatically improved potency, is being developed by the same company as a follow-on compound to rapastinel.
Division of Organic Chemistry, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, India
b
Academy of Scientific and Innovative Research (AcSIR), CSIR, Pune 411008, India
Highlights
•
Structural mimics of rapastinel comprising silaproline is reported.
•
Sip introduction is expected to improve its pharmacokinetic profiles.
•
Standard peptide coupling strategy in the solution-phase is utilized for synthesis.
Abstract
Rapastinel (GLYX-13) is a C-amidated tetrapeptide drug under clinical development for adjunctive treatment of major depressive disorder (MDD). Rapastinel features two consecutive proline residues centered at the peptide sequence (Thr-Pro-Pro-Thr-NH2), which are detrimental to its biological activity. In this communication, we report the synthesis of very close structural analogues of rapastinel comprising silaproline (Sip) as proline surrogate. By virtue of its enhanced lipophilicity and metabolic stability, Sip introduction in the native rapastinel sequence is expected to improve its pharmacokinetic profiles.
Graphical abstract
This paper reports the synthesis of silaproline (Sip)-incorporated close structural mimics of potent antidepressant peptide drug rapastinel (GLYX-13).
Depression is the most common neuropsychiatric diseases, seriously affecting people’s health.In China With accelerated pace of life, increasing the incidence of depression was significantly higher social pressure.
[0003] Drug therapy is the primary means of treatment of depression.The main treatment drugs, including tricyclic antidepressants such as imipramine, amitriptyline and the like; selective serotonin reuptake inhibitors such as fluoxetine, sertraline and the like; serotonin / norepinephrine dual uptake inhibitors such as venlafaxine, duloxetine.However, commonly used drugs slow onset, usually takes several weeks to months, and there is not efficient and toxicity obvious shortcomings.
[0004] GLYX-13 is a new antidepressant, Phase II clinical study is currently underway.It does this by regulating the brain NMDA (N_ methyl -D- aspartate) receptors play a role, and none of them have serious side effects such as ketamine and R-rated, such as hallucinations and schizophrenia and so on.GLYX-13 can play a strong, fast and sustained antidepressant effects, the onset time of less than 24 hours, and the sustainable average of 7 days.As a peptide drug, GLYX-13 was well tolerated and safe to use.
[0005] GLYX-13 is a tetrapeptide having the sequence structure Thr-Pro-Pro-Thr, which is a free N-terminal amino group, C terminal amide structure.GLYX-13 synthesis methods include traditional methods of two solid-phase peptide synthesis and liquid phase peptide synthesis, because of its short sequence, the amount of solid phase synthesis of amino acids, high cost, and difficult to achieve a lot of preparation.A small amount of liquid phase amino acids, high yield can be prepared in large quantities.
The present invention can be further described by the following examples.
Preparation of r-NH2; [0013] Example 1 Four peptide H-Thr-Pr〇-P; r〇-Th
[0015] 500ml three flask was added Boc-Thr (tBu) -0H20g (0.073mol), anhydrous tetrahydrofuran (THF) 150ml, stirring to dissolve the solid.Ice-salt bath cooled to -10 ° C~_15 ° C, was added N- methylmorpholine 8ml, then l〇ml isobutyl chloroformate, keeping the temperature not higher than -10 ° C, after the addition was complete retention low temperature reaction 10min, then adding ammonia 20ml, ice bath reaction 30min, then at room temperature the reaction 8h.The reaction was stopped, water 300ml, 200ml ethyl acetate was added to extract the precipitate, washed with water 3 times.Dried over anhydrous sodium sulfate 6h.Filtered, and then the solvent was distilled off under reduced pressure to give a white solid 16. 6g, 83% yield.
[0016] The above product was dissolved in 50ml of trifluoroacetic acid or 2N hydrochloric acid / ethyl acetate solution was reacted at room temperature lh, the solvent was distilled off to give a white solid, i.e. amidated carboxyl threonine trifluoroacetic acid / hydrochloric acid salt H- Thr-NH 2. HC1.
[0017] 1.2 Pro – Preparation of threonine dipeptide fragment H-Pr〇-Thr-NH2 of
[0018] 500ml flask was added Boc-Pr〇 three-0H20g (0. 093mol), in anhydrous tetrahydrofuran (TH F) 200ml, stirring to dissolve solids, cooled to ice-salt bath -l〇 ° C~-15 ° C, added N- methylmorpholine 11ml, then dropwise isobutyl 13ml, keeping the temperature not higher than -10 ° C, keep it cool after the addition was complete the reaction 10min.H-Thr-NH2. HC114. 5g dissolved in 50ml of tetrahydrofuran, was added N- methyl morpholine 11ml.The above solution was added to the reaction mixture, the low temperature reaction 30min, then at room temperature the reaction 8h.The reaction was stopped, water 300ml, 200ml ethyl acetate was added to extract the precipitate, washed with water 3 times.Dried over anhydrous sodium sulfate 6h.Filtered and then evaporated under reduced pressure to give a white solid 25.7g, 82% yield.
[0019] The above product was dissolved in 100ml of 2N trifluoroacetic acid or hydrochloric acid / ethyl acetate solution was reacted at room temperature lh, the solvent was distilled off to give a white solid, i.e., proline – threonine dipeptide hydrochloride salt of H-Pr〇 -Thr-NH 2. HC1.
[0020] The above product was dissolved in 100ml of pure water, sodium carbonate solution was added to adjust the PH value, the precipitated white solid was filtered and dried in vacuo to give the desired product proline – threonine dipeptide fragment H-Pr square-Thr- NH223g.
Protected threonine [0021] 1.3 – Preparation of dipeptide fragment Boc-Thr (tBu) -Pr〇-0H of
[0022] Boc-Thr (tBu) -0H20g (0 · 073mol) was dissolved in dry tetrahydrofuran (THF) 150ml, stirring to dissolve the solid.Ice-salt bath cooled to -10 G~-15 ° C, was added N- methylmorpholine 8ml, then dropwise isobutyl 10ml, maintained at a temperature no higher than -10 ° C, kept cold reaction After dropping 10min.Proline methyl ester hydrochloride
PAPER
Journal of Medicinal Chemistry (1989), 32(10), 2407-11.
Threonylprolylprolylthreoninamide (HRP-7). The synthesis of HRP-7 was begun with 3 g of p-methylbenzhydrylamine-resin containing 1.41 mmol of attachment sites. The protected tetrapeptidyl-resin (1.63 g) was subjected to HF cleavage. Radioactivity was found in the 1% acetic acid extract (77%) and in the 5% extract (24%). These solutions were combined and lyophilized. Crude peptide (309 mg, 97%) was gel filtered on Sephadex G-15 (1.1 X 100 cm). Peptide eluting between 34 and 46 mL was pooled and lyophilized to yield 294 mg (95%, overall yield 92%) of homogeneous HRP-7.
Process for synthesizing dipyrrolidine peptide compounds (eg GLYX-13) is claimed.
An N-methyl-D-aspartate (NMDA) receptor is a postsynaptic, ionotropic receptor that is responsive to, inter alia, the excitatory amino acids glutamate and glycine and the synthetic compound NMDA. The NMDA receptor (NMDAR) appears to controls the flow of both divalent and monovalent ions into the postsynaptic neural cell through a receptor associated channel and has drawn particular interest since it appears to be involved in a broad spectrum of CNS disorders. The NMDAR has been implicated, for example, in neurodegenerative disorders including stroke-related brain cell death, convulsive disorders, and learning and memory.
NMDAR also plays a central role in modulating normal synaptic transmission, synaptic plasticity, and excitotoxicity in the central nervous system. The NMDAR is further involved in Long-Term Potentiation (LTP), which is the persistent strengthening of neuronal connections that underlie learning and memory The NMDAR has been associated with other disorders ranging from hypoglycemia and cardiac arrest to epilepsy. In addition, there are preliminary reports indicating involvement of NMDA receptors in the chronic neurodegeneration of Huntington’s, Parkinson’s, and Alzheimer’s diseases. Activation of the NMDA receptor has been shown to be responsible for post-stroke convulsions, and, in certain models of epilepsy, activation of the NMDA receptor has been shown to be necessary for the generation of seizures. In addition, certain properties of NMDA receptors suggest that they may be involved in the information-processing in the brain that underlies consciousness itself. Further, NMDA receptors have also been implicated in certain types of spatial learning.
[0003] In view of the association of NMDAR with various disorders and diseases, NMDA-modulating small molecule agonist and antagonist compounds have been developed for therapeutic use. NMDA receptor compounds may exert dual (agonist/antagonist) effect on the NMDA receptor through the allosteric sites. These compounds are typically termed “partial agonists”. In the presence of the principal site ligand, a partial agonist will displace some of the ligand and thus decrease Ca flow through the receptor. In the absence of the principal site ligand or in the presence of a lowered level of the principal site ligand, the partial agonist acts to increase Ca++ flow through the receptor channel.
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Example 2: Synthesis of GLYX-13
[00119] GLYX-13 was prepared as follows, using intermediates KSM-1 and KSM-2 produced in Example 1. The synthetic route for the same is provided in Figure 2.
Stage A – Preparation of (S)-N-((2S, 3R)-l-amino-3-hydroxy-l-oxobutan-2-yl)-l-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide (Compound XI)
[00120] In this stage, KSM -1 was reacted with 10%Pd/C in presence of methanol to produce a compound represented by Formula XI. The reaction was optimized and performed up to 4.0 kg scale in the production plant and observed consistent quality (>80% by HPLC%PA) and yields (80% to 85%).
[00121] The reaction scheme involved in this method is as follows:
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[00122] Raw materials used for this method are illustrated in Table 7 as follows:
Table 7.
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[00123] In stage A, 10% Palladium on Carbon (w/w, 50% wet) was charged into the pressure reactor at ambient temperature under nitrogen atmosphere. KSM-1 was dissolved in methanol in another container and sucked into above reactor under vacuum. Hydrogen pressure was maintained at 45-60 psi at ambient temperature for over a period of 5-6 hrs. Progress of the reaction mixture was monitored by HPLC for KSM-1 content; limit is not more than 5%.
Hyflow bed was prepared with methanol (Lot-II). The reaction mass was filtered through nutsche filter under nitrogen atmosphere and bed was washed with Methanol Lot-Ill. Filtrate was transferred into the reactor and distilled completely under reduced pressure at below 50 °C (Bath temperature) to get the syrup and syrup material was unloaded into clean and dry container and samples were sent to QC for analysis.
[00124] From the above reaction(s), 1.31 kg of compound represented by Formula XI was obtained with a yield of 89.31% and with a purity of 93.63%).
Stage B – Preparation of Benzyl (2S, 3R)-l-((S)-2-((S)-2-((2S, 3R)-I-amino-3-hydroxy-I- oxobutan-2-ylcarbamoyl) pyrrolidine-! -carbonyl) pyrrolidin-1 -yl)-3-hydroxy-l -oxobutan-2- ylcarbamate (Compound XII)
[00125] In this stage the compound represented by Formula XI obtained above was reacted with KSM-2 to produce a compound represented by Formula XII. This reaction was optimized and scaled up to 3.0 kg scale in the production plant and obtained 25% to 28% yields with UPLC purity (>95%).
[00126] The reaction scheme is as follows:
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[00127] Raw materials used for this method are illustrated in Table 8 as follows:
Table 8.
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[00128] Stage B: ethanol was charged into the reactor at 20 to 35 °C. Compound represented by Formula XI was charged into the reactor under stirring at 20 to 35 °C and reaction mass was cooled to -5 to 0°C. EDC.HC1 was charged into the reaction mass at -5 to 0 °C and reaction mass, was maintained at -5 to 0 °C for 10-15 minutes. N-Methyl morpholine was added drop wise to the above reaction mass at -5 to 0 °C and reaction mass was maintained at -5 to 0 °C for 10-15 minutes.
[00129] KSM-2 was charged into the reactor under stirring at -5 to 0 °C and reaction mass was maintained at -5 to 0 °C for 3.00 to 4.00 hours. The temperature of the reaction mass was raised to 20 to 35 °C and was maintained at 20 to 35 °C for 12 – 15 hours under stirring. (Note:
Monitor the reaction mass by HPLC for Stage A content after 12.0 hours and thereafter every 2.0 hours. The content of stage A should not be more than 2.0%). Ethanol was distilled out completely under vacuum at below 50 °C (Hot water temperature) and reaction mass was cooled to 20 to 35 °C. Water Lot-1 was charged into the residue obtained followed by 10% DCM-Isopropyl alcohol (Mixture of Dichloromethane Lot-1 & Isopropyl alcohol Lot-1 prepared in a cleaned HDPE container) into the reaction mass at 20 – 35 °C.
[00130] Both the layers were separated and the aqueous layer was charged into the reactor. 10%) DCM-Isopropyl alcohol (Mixture of Dichloromethane Lot-2 & Isopropyl alcohol Lot-2 prepared in a cleaned HDPE container) was charged into the reaction mass at 20 to 35 °C. Both the layers were separated and the aqueous layer was charged back into the reactor. 10%> IDCM-isopropyl alcohol (Mixture of Dichloromethane Lot-3 & Isopropyl alcohol Lot-3 prepared in a cleaned HDPE container) was charged into the reaction mass at 20 to 35 °C. Both the layers were separated and the aqueous layer was charged back into the reactor. 10%> DCM-Isopropyl alcohol (Mixture of Dichloromethane Lot-4 & Isopropyl alcohol Lot-4 prepared in a cleaned HDPE container) was charged into the reaction mass at 20 to 35 °C and separated both the layers. The above organic layers were combined and potassium hydrogen sulfate solution (Prepare a solution in a HDPE container by dissolving Potassium hydrogen sulfate Lot-1 in water Lot-2) was charged into the reaction mass at 20 to 35 °C. Separated both the layers and charged back organic layer into the reactor. Potassium hydrogen sulfate solution (Prepared a solution in a HDPE container by dissolving Potassium hydrogen sulfate Lot-2 in water Lot-3) was charged into the reaction mass at 20 to 35 °C. Separated both the layers and the organic layer was dried over Sodium sulfate and distilled out the solvent completely under vacuum at below 45 °C (Hot water temperature).
[00131] The above crude was absorbed with silica gel (100-200mesh) Lot-1 in
dichloromethane. Prepared the column with silica gel (100-200 mesh) Lot-2, and washed the silica gel bed with from Dichloromethane Lot-5 and charged the adsorbed compound into the column. Eluted the column with 0-10% Methanol Lot-1 in Dichloromethane Lot-5 and analyzed fractions by HPLC. Solvent was distilled out completely under vacuum at below 45 °C (Hot water temperature). Methyl tert-butyl ether Lot-1 was charged and stirred for 30 min. The solid was filtered through the Nutsche filter and washed with Methyl tert-butyl ether Lot-2 and
samples were sent to QC for complete analysis. (Note: If product quality was found to be less than 95%, column purification should be repeated).
[00132] From the above reaction(s), 0.575 kg of compound represented by Formula XII was obtained with a yield of 17% and with a purity of 96.28%).
Stage C – Preparation of Benzyl (S)-N-((2S, 3R)-l-amino-3-hydroxy-l-oxobutan-2-yl)-l-((S)-l- ((2R, 3R)-2-amino-3-hydroxybutanoyl) pyrrolidine-2 carbonyl) pyrrolidine-2-carboxamide (GLYX-13)
[00133] In this reaction step the compound of Formula XII obtained above was reacted with 10%oPd in presence of methanol to produce GLYX-13. This reaction was optimized and performed up to 2.8 kg scale in the production plant and got 40% to 45% of yields with UPLC purity >98%.
[00134] The reaction scheme involved in this method is as follows:
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i
[00135] Raw materials used for this method are illustrated in Table 9 as follows:
Table 9.
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[00136] In an exemplary embodiment of stage C, 10% Palladium Carbon (50% wet) was charged into the pressure reactor at ambient temperature under nitrogen atmosphere. Compound of Formula XII was dissolved in methanol in a separate container and sucked into the reactor under vacuum. Hydrogen pressure was maintained 45-60 psi at ambient temperature over a period of 6-8 hrs. Progress of the reaction was monitored by HPLC for stage-B (compound represented by Formula XII) content (limit is not more than 2%). If HPLC does not comply continue the stirring until it complies. Prepared the hyflow bed with methanol (Lot-II) and the reaction mass was filtered through hyflow bed under nitrogen atmosphere, and the filtrate was collected into a clean HDPE container. The bed was washed with Methanol Lot-Ill and the filtrate was transferred into the Rota Flask and distilled out the solvent completely under reduced pressure at below 50°C (Bath temperature) to get the crude product. The material was unloaded into clean HDPE container under Nitrogen atmosphere.
[00137] Neutral Alumina Lot-1 was charged into the above HDPE container till uniform mixture was formed. The neutral Alumina bed was prepared with neutral alumina Lot-2 and dichloromethane Lot-1 in a glass column. The neutral Alumina Lot-3 was charged and
Dichloromethane Lot-2 into the above prepared neutral Alumina bed. The adsorbed compound was charged into the column from op.no.11. The column was eluted with Dichloromethane Lot-2 and collect 10 L fractions. The column was eluted with Dichloromethane Lot-3 and collected 10 L fractions. The column was eluted with Dichloromethane Lot-4 and Methanol Lot-4 (1%) and collected 10 L fractions. The column was eluted with Dichloromethane Lot-5 and Methanol Lot-5 (2%) and collected 10 L fractions. The column was eluted with Dichloromethane Lot-6 and Methanol Lot-6 (3%) and collected 10 L fractions. The column was eluted with
Dichloromethane Lot-7 and Methanol Lot-7 (5%). and collected 10 L fractions. The column was eluted with Dichloromethane Lot-8 and Methanol Lot-8 (8%). and collected 10 L fractions. The column was eluted with Dichloromethane Lot-9 and Methanol Lot-9 (10%) and collected 10 L fractions. Fractions were analyzed by HPLC (above 97% purity and single max impurity >0.5% fractions are pooled together)
[00138] Ensured the reactor is clean and dry. The pure fractions were transferred into the reactor.
[00139] The solvent was distilled off completely under vacuum at below 45 °C (Hot water temperature). The material was cooled to 20 to 35°C. Charged Dichloromethane Lot- 10 and Methanol Lot- 10 into the material and stirred till dissolution. Activated carbon was charged into the above mixture at 20 to 35°C and temperature was raised to 45 to 50 °C.
[00140] Prepared the Hyflow bed with Hyflow Lot-2 and Methanol Lot-11 Filtered the reaction mass through the Hy-flow bed under nitrogen atmosphere and collect the filtrate into a clean FIDPE container. Prepared solvent mixture with Dichloromethane Lot-11 and Methanol Lot- 12 in a clean FIDPE container and washed Nutsche filter with same solvent. Charged filtrate in to Rota evaporator and distilled out solvent under vacuum at below 50°C. Dry the compound in Rota evaporator for 5 to 6 hours at 50°C, send sample to QC for Methanol content (residual solvent) which should not be more than 3000 ppm. The material was cooled to 20 to 35 °C and the solid material was unloaded into clean and dry glass bottle. Samples were sent to QC for complete analysis.
[00141] From the above reaction(s), 0.92 kg of Glyx-13 was obtained with a yield of 43.5% and with a purity of 99.73%.
^ Jump up to:abcHashimoto K, Malchow B, Falkai P, Schmitt A (August 2013). “Glutamate modulators as potential therapeutic drugs in schizophrenia and affective disorders”. Eur Arch Psychiatry Clin Neurosci. 263 (5): 367–77. PMID23455590. doi:10.1007/s00406-013-0399-y.
^ Jump up to:abcdMoskal JR, Burgdorf JS, Stanton PK, Kroes RA, Disterhoft JF, Burch RM, Amin Khan M (2016). “The Development of Rapastinel (Formerly GLYX-13); a rapid acting and long lasting antidepressant”. Curr Neuropharmacol. PMID26997507.
Jump up^Haring R, Stanton PK, Scheideler MA, Moskal JR (1991). “Glycine-like modulation of N-methyl-D-aspartate receptors by a monoclonal antibody that enhances long-term potentiation”. J. Neurochem. 57 (1): 323–32. PMID1828831. doi:10.1111/j.1471-4159.1991.tb02131.x.
Jump up^Moskal JR, Kuo AG, Weiss C, Wood PL, O’Connor Hanson A, Kelso S, Harris RB, Disterhoft JF (2005). “GLYX-13: a monoclonal antibody-derived peptide that acts as an N-methyl-D-aspartate receptor modulator”. Neuropharmacology. 49 (7): 1077–87. PMID16051282. doi:10.1016/j.neuropharm.2005.06.006.
^ Jump up to:abBurch RM, Amin Khan M, Houck D, Yu W, Burgdorf J, Moskal JR (2016). “NMDA Receptor Glycine Site Modulators as Therapeutics for Depression: Rapastinel has Antidepressant Activity without Causing Psychotomimetic Side Effects”. Curr Neuropharmacol. PMID26830963.
Jump up^Moskal, Joseph R.; Kuo, Amy G.; Weiss, Craig; Wood, Paul L.; O’Connor Hanson, Amy; Kelso, Stephen; Harris, Robert B.; Disterhoft, John F. (2005). “GLYX-13: A monoclonal antibody-derived peptide that acts as an N-methyl-d-aspartate receptor modulator”. Neuropharmacology. 49 (7): 1077–87. PMID16051282. doi:10.1016/j.neuropharm.2005.06.006.
Jump up^Stanton, Patric K.; Potter, Pamela E.; Aguilar, Jennifer; Decandia, Maria; Moskal, Joseph R. (2009). “Neuroprotection by a novel NMDAR functional glycine site partial agonist, GLYX-13”. NeuroReport. 20 (13): 1193–7. PMID19623090. doi:10.1097/WNR.0b013e32832f5130.
Jump up^Wood, Paul L.; Mahmood, Siddique A.; Moskal, Joseph R. (2008). “Antinociceptive action of GLYX-13: An N-methyl-D-aspartate receptor glycine site partial agonist”. NeuroReport. 19(10): 1059–61. PMID18580579. doi:10.1097/WNR.0b013e32830435c9.
LY2606368 is a small-molecule Chk-1 inhibitors invented by Array and being developed by Eli Lilly and Company. Lilly is responsible for all clinical development and commercialization activities. Chk-1 is a protein kinase that regulates the tumor cell’s response to DNA damage often caused by treatment with chemotherapy. In response to DNA damage, Chk-1 blocks cell cycle progression in order to allow for repair of damaged DNA, thereby limiting the efficacy of chemotherapeutic agents. Inhibiting Chk-1 in combination with chemotherapy can enhance tumor cell death by preventing these cells from recovering from DNA damage.
Originator Array BioPharma; Eli Lilly
Developer Eli Lilly; National Cancer Institute (USA)
Class Antineoplastics; Nitriles; Pyrazines; Pyrazoles; Small molecules
Phase II Breast cancer; Ovarian cancer; Small cell lung cancer; Solid tumours
Phase I Acute myeloid leukaemia; Colorectal cancer; Head and neck cancer; Myelodysplastic syndromes; Non-small cell lung cancer
Most Recent Events
10 Apr 2017 Eli Lilly completes a phase I trial for Solid tumours (Late-stage disease, Second-line therapy or greater) in Japan (NCT02514603)
10 Mar 2017 Phase-I clinical trials in Solid tumours (Combination therapy, Metastatic disease, Inoperable/Unresectable) in USA (IV) (NCT03057145)
22 Feb 2017 Khanh Do and AstraZeneca plan a phase H trial for Solid tumour (Combination therapy, Metastatic disease, Inoperable/Unresectable) in USA (NCT03057145)
Prexasertib (LY2606368) is a small molecule checkpoint kinase inhibitor, mainly active against CHEK1, with minor activity against CHEK2. This causes induction of DNA double-strand breaks resulting in apoptosis. It is in development by Eli Lilly. [1]
A phase II clinical trial for the treatment of small cell lung cancer is expected to be complete in December 2017.[2]
an aminopyrazole compound, or a pharmaceutically acceptable salt thereof or a solvate of the salt, that inhibits Chkl and is useful for treating cancers characterized by defects in deoxyribonucleic acid (DNA) replication, chromosome segregation, or cell division.
Chkl is a protein kinase that lies downstream from Atm and/or Atr in the DNA damage checkpoint signal transduction pathway. In mammalian cells, Chkl is phosphorylated in response to agents that cause DNA damage including ionizing radiation (IR), ultraviolet (UV) light, and hydroxyurea. This phosphorylation which activates Chkl in mammalian cells is dependent on Atr. Chkl plays a role in the Atr dependent DNA damage checkpoint leading to arrest in S phase and at G2M. Chkl phosphorylates and inactivates Cdc25A, the dual-specificity phosphatase that normally dephosphorylates cyclin E/Cdk2, halting progression through S-phase. Chkl also phosphorylates and inactivates Cdc25C, the dual specificity phosphatase that dephosphorylates cyclin B/Cdc2 (also known as Cdkl) arresting cell cycle progression at the boundary of G2 and mitosis (Fernery et al, Science, 277: 1495-1, 1997). In both cases, regulation of Cdk activity induces a cell cycle arrest to prevent cells from entering mitosis in the presence of DNA damage or unreplicated DNA. Various inhibitors of Chkl have been reported. See for example, WO 05/066163,
WO 04/063198, WO 03/093297 and WO 02/070494. In addition, a series of aminopyrazole Chkl inhibitors is disclosed in WO 05/009435.
However, there is still a need for Chkl inhibitors that are potent inhibitors of the cell cycle checkpoints that can act effectively as potentiators of DNA damaging agents. The present invention provides a novel aminopyrazole compound, or a pharmaceutically acceptable salt thereof or solvate of the salt, that is a potent inhibitor of Chkl . The compound, or a pharmaceutically acceptable salt thereof or a solvate of the salt, potently abrogates a Chkl mediated cell cycle arrest induced by treatment with DNA damaging agents in tissue culture and in vivo. Furthermore, the compound, or a pharmaceutically acceptable salt thereof or a solvate of the salt, of the present invention also provides inhibition of Chk2, which may be beneficial for the treatment of cancer. Additionally, the lack of inhibition of certain other protein kinases, such as CDKl, may provide a -2- therapeutic benefit by minimizing undesired effects. Furthermore, the compound, or a pharmaceutically acceptable salt thereof or a solvate of the salt, of the present invention inhibits cell proliferation of cancer cells by a mechanism dependent on Chkl inhibition.
A solution of tert-butyl 3-(2-(3-(5-bromopyrazin-2-ylamino)-lH-pyrazol-5-yl)-3- methoxyphenoxy)propylcarbamate (0.378 g, 0.730 mmol) and zinc cyanide (0.10 g, 0.870 mmol) in DMF (10 mL) is degassed with a stream of nitrogen for one hour and then -25- heated to 80 0C. To the reaction is added Pd(Ph3P)4 (0.080 g, 0.070 mmol), and the mixture is heated overnight. The reaction is cooled to room temperature and concentrated under reduced pressure. The residue is purified by silica gel chromatography (CH2Cl2/Me0H) to give 0.251 g (73%) of the title compound.
A 5 L flange-neck round-bottom flask equipped with an air stirrer rod and paddle, thermometer, pressure-equalizing dropping funnel, and nitrogen bubbler is charged with 5-(5-(2-hydroxy-6-methoxy-phenyl)-lH-pyrazol-3-ylamino)-pyrazine-2-carbonitrile (47.0 g, 152 mmol) and anhydrous THF (1.2 L). The stirred suspension, under nitrogen, is cooled to 0 0C. A separate 2 L 3 -necked round-bottom flask equipped with a large -28- magnetic stirring bar, thermometer, and nitrogen bubbler is charged with triphenylphosphine (44.0 g; 168 mmol) and anhydrous THF (600 mL). The stirred solution, under nitrogen, is cooled to 0 0C and diisopropylazodicarboxylate (34.2 g; 169 mmol) is added and a milky solution is formed. After 3-4 min, a solution of7-butyl-N-(3- hydroxypropyl)-carbamate (30.3 g, 173 mmol) in anhydrous THF (100 mL) is added and the mixture is stirred for 3-4 min. This mixture is then added over 5 min to the stirred suspension of starting material at 0 0C. The reaction mixture quickly becomes a dark solution and is allowed to slowly warm up to room temperature. After 6.5 h, more reagents are prepared as above using PPh3 (8 g), DIAD (6.2 g) and carbamate (5.4 g) in anhydrous THF (150 mL). The mixture is added to the reaction mixture, cooled to -5 0C and left to warm up to room temperature overnight. The solvent is removed in vacuo. The resulting viscous solution is loaded onto a pad of silica and product is eluted with ethyl acetate. The concentrated fractions are separately triturated with methanol and resulting solids are collected by filtration. The combined solids are triturated again with methanol (400 mL) and then isolated by filtration and dried in vacuo at 50 0C overnight to give 31.3 g of desired product. LC-ES/MS m/z 466.2 [M+ 1]+.
A 5 L flange-neck, round-bottom flask equipped with an air stirrer rod and paddle, thermometer, and air condenser with bubbler attached, is charged with tert-bvXyl 3-(2-(3- (5-cyanopyrazin-2-ylamino)-lH-pyrazol-5-yl)-3-methoxyphenoxy)propylcarbamate (30.9 g, 66.3 mmol) and ethyl acetate (3 L). The mechanically stirred yellow suspension is cooled to just below 10 0C. Then hydrogen chloride from a lecture bottle is bubbled in -29- vigorously through a gas inlet tube for 15 min with the ice-bath still in place. After 5 h the mixture is noticeably thickened in appearance. The solid is collected by filtration, washed with ethyl acetate, and then dried in vacuo at 60 0C overnight to give 30.0 g. 1H NMR (400 MHz, DMSO-d6) δ 2.05 (m, 2H), 2.96 (m, 2H), 3.81 (s, 3H), 4.12 (t, J = 5.8 Hz, 2H), 6.08 (br s, 3H), 6.777 (d, J = 8.2 Hz, IH), 6.782 (d, J = 8.2 Hz, IH), 6.88 (br s, IH), 7.34 (t, J = 8.2 Hz, IH), 8.09 (br s, IH), 8.55 (br s, IH), 8.71 (s, IH), 10.83 (s, IH), 12.43 (br s, IH). LC-ES/MS m/z 366.2 [M+lf.
Example 3 5 -(5 -(2-(3 -Aminopropoxy)-6-methoxyphenyl)- 1 H-pyrazol-3 -ylamino)pyrazine-2- carbonitrile
5-(5-(2-(3-Aminopropoxy)-6-methoxyphenyl)-lH-pyrazol-3-ylamino)pyrazine-2- carbonitrile dihydrogen chloride salt (3.0 g, 6.84 mmol) is suspended in 200 mL of CH2Cl2. 1 N NaOH is added (200 mL, 200 mmol). The mixture is magnetically stirred under nitrogen at room temperature for 5 h. The solid is collected by filtration and washed thoroughly with water. The filter cake is dried in vacuo at 50 0C overnight to give 2.26 g (90%) of the free base as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.81 (m, 2H), 2.73 (t, J = 6.2 Hz, 2H), 3.82 (s, 3H), 4.09 (t, J = 6.2 Hz, 2H), 6.76 (t, J = 8.2 Hz, 2H), 6.93 (br s, IH), 7.31 (t, J = 8.2 Hz, IH), 8.52 (br s, IH), 8.67 (s, IH). LC- MS /ES m/z 366.2 [M+ 1]+.
5-(5-(2-(3-aminopropoxy)-6-methoxyphenyl)-lH-pyrazol-3-ylamino)pyrazine-2- carbonitrile (1.0 g, 2.74 mmol) is suspended in MeOH (100 mL). A I M solution of methanesulfonic acid in MeOH (2.74 mL, 2.74 mmol) is added to the mixture dropwise with stirring. The solid nearly completely dissolves and is sonicated and stirred for 15 min, filtered, and concentrated to 50 mL. The solution is cooled overnight at -15 0C and the solid that forms is collected by filtration. The solid is dried in a vacuum oven overnight to give 0.938 g (74%) of a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.97 (m, 2H), 2.28 (s, 3H), 2.95 (m, 2H), 3.79 (s, 3H), 4.09 (t, J = 5.9 Hz, 2H), 6.753 (d, J = 8.4 Hz, IH), 6.766 (d, J = 8.4 Hz, IH), 6.85 (br s, IH), 7.33 (t, J = 8.4 Hz, IH), 7.67 (br s, 3H), 8.49 (br s, IH), 8.64 (s, IH), 10.70 (s, IH), 12.31 (s, IH). LC-ES/MS m/z 366.2 [M+l]+.
5-(5-(2-Hydroxy-6-methoxyphenyl)-lH-pyrazol-3-ylamino)pyrazine-2- carbonitrile (618 g, 1.62 mol) is slurried in tetrahydrofuran (6.18 L, 10 volumes) and chilled to -5 to 0 0C with an acetone/ice bath. Triethylamine (330 g, 3.25 mol) is added through an addition funnel over 30 – 40 min at -5 to 5 0C. The resulting slurry is stirred at -5 to 5 0C for 60 – 90 min. The insoluble triethylamine hydrochloride is filtered and the solution of the phenol ((5-(2-hydroxy-6-methoxyphenyl)-lH-pyrazol-3- ylamino)pyrazine-2-carbonitrile) collected in an appropriate reaction vessel. The cake is rinsed with THF (1.24 L). The THF solution of the phenol is held at 15 to 20 0C until needed.
Triphenylphosphine (1074 g, 4.05 mol) is dissolved at room temperature in THF (4.33 L). The clear colorless solution is cooled with an acetone/ice bath to -5 to 5 0C. Diisopropylazodicarboxylate (795 g, 3.89 mol) is added dropwise through an addition funnel over 40 – 60 min, keeping the temperature below 10 0C. The resulting thick white slurry is cooled back to -5 to 0 0C. tert-Butyl 3-hydroxypropylcarbamate (717g, 4.05 moles) is dissolved in a minimum of THF (800 mL). The tert-butyl 3- hydroxypropylcarbamate/THF solution is added, through an addition funnel, over 20 – 30 -35- min at -5 to 5 0C to the reagent slurry. The prepared reagent is stirred in the ice bath at -5 to 0 0C until ready for use.
The prepared reagent slurry (20%) is added to the substrate solution at 15 to 20 0C. The remaining reagent is returned to the ice bath. The substrate solution is stirred at ambient for 30 min, then sampled for HPLC. A second approximately 20% portion of the reagent is added to the substrate, stirred at ambient and sampled as before. Addition of the reagent is continued with monitoring for reaction completion by HPLC. The completed reaction is concentrated and triturated with warm methanol (4.33 L, 50 – 60 0C) followed by cooling in an ice bath. The resulting yellow precipitate is filtered, rinsed with cold MeOH (2 L), and dried to constant weight to provide 544 g (72%) of the title compound, mp 214 – 216 0C; ES/MS m/z 466.2 [M+l]+.
tert-Butyl 3-(2-(3-(5-cyanopyrazin-2-ylamino)-lH-pyrazol-5-yl)-3- methoxyphenoxy)propylcarbamate (1430 g, 3.07 mol) is slurried with acetone (21.5 L) in a 30 L reactor. Methanesulfonic acid (1484 g, 15.36 mol) is added through an addition funnel in a moderate stream. The slurry is warmed to reflux at about 52 0C for 1 to 3 h and monitored for reaction completion by HPLC analysis. The completed reaction is cooled from reflux to 15 to 20 0C over 4.5 h. The yellow slurry of 2-pyrazinecarbonitrile, 5-[[5-[-[2-(3-aminopropyl)-6-methoxyphenyl]-lH-pyrazol-3-yl]amino] dimesylate salt is filtered, rinsed with acetone (7 L) and dried in a vacuum oven. The dimesylate salt, (1608 g, 2.88 mol) is slurried in water (16 L). Sodium hydroxide (aqueous 50%, 228 g, 2.85 mol) is slowly poured into the slurry. The slurry is -36- heated to 60 0C and stirred for one hour. It is then cooled to 16 0C over 4 h and filtered. The wet filter cake is rinsed with acetone (4 L) and dried to constant weight in a vacuum oven at 40 0C to provide 833 g (94%) of 2-pyrazinecarbonitrile, 5-[[5-[-[2-(3- aminopropyl)-6-methoxyphenyl]-lH-pyrazol-3-yl]amino] monomesylate monohydrate. mp 222.6 0C; ES/MS m/z 366.2 [M+l]+.
Crude 2-pyrazinecarbonitrile, 5 -[ [5 – [- [2-(3 -aminopropyl)-6-methoxyphenyl]- IH- pyrazol-3-yl] amino] monomesylate monohydrate is purified using the following procedure. The technical grade 2-pyrazinecarbonitrile, 5-[[5-[-[2-(3-aminopropyl)-6- methoxyphenyl]-lH-pyrazol-3-yl] amino] mono mesylate mono hydrate (1221 g, 2.55 mol) is slurried in a solvent mixture of 1: 1 acetone/water (14.7 L). The solid is dissolved by warming the mixture to 50 – 55 0C. The solution is polish filtrated while at 50 – 55 0C through a 0.22μ cartridge filter. The solution is slowly cooled to the seeding temperature of about 42 – 45 0C and seeded. Slow cooling is continued over the next 30 – 60 min to confirm nucleation. The thin slurry is cooled from 38 to 15 0C over 3 h. A vacuum distillation is set up and the acetone removed at 110 – 90 mm and 20 – 30 0C. The mixture is cooled from 30 to 15 0C over 14 h, held at 15 0C for 2 h, and then filtered. The recrystallized material is rinsed with 19: 1 water/acetone (2 L) and then water (6 L) and dried to constant weight in a vacuum oven at 40 0C to provide 1024 g (83.9%) of the title compound, mp 222.6 0C; ES/MS m/z 366.2 [M+l]+. X-ray powder diffraction (XRPD) patterns may be obtained on a Bruker D8
Advance powder diffractometer, equipped with a CuKa source (λ=l.54056 angstrom) operating at 40 kV and 40 mA with a position-sensitive detector. Each sample is scanned between 4° and 35° in °2Θ ± 0.02 using a step size of 0.026° in 2Θ ± 0.02 and a step time of 0.3 seconds, with a 0.6 mm divergence slit and a 10.39 mm detector slit. Primary and secondary Soller slits are each at 2°; antiscattering slit is 6.17 mm; the air scatter sink is in place. -37-
Characteristic peak positions and relative intensities:
Differential scanning calorimetry (DSC) analyses may be carried out on a Mettler- Toledo DSC unit (Model DSC822e). Samples are heated in closed aluminum pans with pinhole from 25 to 350 0C at 10 °C/min with a nitrogen purge of 50 mL/min. Thermogravimetric analysis (TGA) may be carried out on a Mettler Toledo TGA unit (Model TGA/SDTA 85Ie). Samples are heated in sealed aluminum pans with a pinhole from 25 to 350 0C at 10 0C /min with a nitrogen purge of 50 mL/min.
The thermal profile from DSC shows a weak, broad endotherm form 80 – 1400C followed by a sharp melting endotherm at 222 0C, onset (225 0C, peak). A mass loss of 4% is seen by the TGA from 25 – 140 0C.
PATENT
US 20110144126
WO 2017015124
WO 2017100071
WO 2017105982
WO 2016051409
Clip
Kilogram-scale prexasertib monolactate monohydrate synthesis under continuous-flow CGMP conditions
Science 16 Jun 2017:
Vol. 356, Issue 6343, pp. 1144-1150
DOI: 10.1126/science.aan0745
science2017, 356, 1144
Kilogram-Scale Prexasertib Monolactate Monohydrate Synthesis under Continuous-Flow CGMP Conditions
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A multidisciplinary team from Eli Lilly reports the development and implementation of eight continuous unit operations for the synthesis of ca. 3 kg API per day under CGMP conditions (K. P. Cole et al., Science2017, 356, 1144). The recent drive toward more potent APIs that have a low annual demand (<100 kg) has made continuous synthesis a viable alternative to traditional batch processes with advantages which include reducing equipment footprint and worker exposure. In this report the authors describe the enablement of three continuous synthetic steps followed by a salt formation, using surge tanks between steps to allow each step to be taken offline if online PAT detects a loss in reaction performance. A combination of MSMPRs (mixed-suspension, mixed-product removal) vessels, plug-flow reactors, and dissolve-off filters were used to perform the chemistry, with an automated 20 L rotary evaporator used to concentrate process streams and perform solvents swaps. This paper gives an excellent account of the potential solutions to continuous API synthesis and is well worth a read for anyone contemplating such methodology.
Continuous crystallisation, shown here, and subsequent filtration have been the most difficult-to-develop part of the prexasertib production process
Eli Lilly has taken an important step away from traditional batch process drug manufacturing by using an industry-first continuous process to make a compound for phase I and II clinical trials. Workers at Lilly’s Kinsale site in Ireland, did three steps involved in producing cancer drug candidate prexasertib continuously, under current good manufacturing practice (CGMP) standards that ensure safety for human consumption.
Continuous processing relies on chemical and physical changes happening as substances flow through pipes. Isolated steps of this type are already well-established in the pharmaceutical industry. However, Lilly principal research scientist Kevin Cole stresses that a series including reaction and purification steps like this has not been demonstrated before. And the company wants to go much further.
‘We envision entire synthetic routes consisting of many reaction and separation unit operations being executed simultaneously in flow, with heavy reliance on design space understanding, process analytical technologies and process modelling to ensure quality,’ Cole says. ‘We think this will drastically change the environment for pharmaceutical manufacturing.’
The complex synthesis of prexasertib even requires the use of toxic hydrazine – used as a rocket fuel. As a result, and because of prexasertib’s toxicity, the drug was a good candidate to test out a comprehensive flow chemistry setup
In batch processes different chemical reaction and purification steps are typically done in large, costly vessels. However, this can be uneconomical when small amounts of drug molecules are needed for early stage clinical trials and, because drugs are getting more potent, increasingly in mainstream production.
By contrast, small volume continuous flow processing runs in more compact equipment in fume hoods. Flow systems can adapt to different processes, with cheap parts that can either be dedicated to specific drugs or readily replaced. The US Food and Drug Administration (FDA) has also been promoting continuous manufacturing because it integrates well with advanced process analytical technology. This helps pharmaceutical companies make high quality drugs with less FDA oversight.
Lilly chose prexasertib as its test case for such a process because it’s challenging to make. It is a chain of three aromatic rings, and one challenge comes because its central ring is formed using hydrazine. Hydrazine is used as a component in rocket fuel, and is also highly toxic. A second challenge comes from prexasertib itself, which, as a potent kinase inhibitor, is toxic to healthy cells, as well as cancerous ones, even at low doses. Lilly therefore wants to minimise its workers’ exposure.
Feeding the plant
Cole and his colleagues at Lilly’s labs in Indianapolis, US, have developed flow processes for three of the seven steps involved in prexasertib production. They start with the hydrazine step, which they could safely speed up by super-heating in the continuous process. After aqueous workup purification the solution of the two-ring intermediate solution runs into a ‘surge tank’. From there the solution flows intermittently into a rotary evaporator that removes solvents to concentrate it.
The second continuous flow step adds the third of prexasertib’s rings. In this case, the Lilly team purified the intermediate by crystallising it and filtering it out, washing away impurities. They could then redissolve the pure intermediate in formic acid, which also removes a protecting group, giving the desired prexasertib molecule. Automating this was probably the hardest part, Cole says. ‘Development of a predictive filtration model, equipment design and identification of formic acid as the solvent were keys to success,’ he explains. The final flow step then starts converting prexasertib to its final lactate salt form.
This coil of tubes forms a low-cost deprotection gas/liquid reactor Eli Lilly uses during continuous processing of prexasertib
After developing the processes and systems in Indianapolis, Lilly shipped them to be equipped in an existing facility at its Kinsale manufacturing site at the cost of €1 million (£870,000). Once the prexasertib system was installed, the company was able to make 3kg of raw material per day for clinical trials. Cole describes the level of manual intervention needed as ‘moderate’.
Klavs Jensen from the Massachusetts Institute of Technology calls the paper describing the work ‘terrific’. ‘This work marks an important milestone in the continuous manufacturing of pharmaceuticals by demonstrating the feasibility of producing a modern kinase inhibitor under CGMP conditions,’ he says.
Likewise, Brahim Benyahia from Loughborough University, UK, calls this achievement ‘very interesting’. ‘The paper is another example that demonstrates the benefits and feasibility of the integrated continuous approach in pharma,’ he says.
Cole adds that Lilly has several other similar projects in advanced stages of development intended for the €35 million small-volume continuous plant it recently built in Kinsale. ‘We are committed to continuous manufacturing as well as full utilisation of our new facility,’ he says.
Correction: This article was updated on 16 June 2017 to clarify the chronology of the completion of the Kinsale, Ireland plant
1: Lowery CD, VanWye AB, Dowless M, Blosser W, Falcon BL, Stewart J, Stephens J, Beckmann RP, Bence Lin A, Stancato LF. The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma. Clin Cancer Res. 2017 Mar 7. pii: clincanres.2876.2016. doi: 10.1158/1078-0432.CCR-16-2876. [Epub ahead of print] PubMed PMID: 28270495.
2: Zeng L, Beggs RR, Cooper TS, Weaver AN, Yang ES. Combining Chk1/2 inhibition with cetuximab and radiation enhances in vitro and in vivo cytotoxicity in head and neck squamous cell carcinoma. Mol Cancer Ther. 2017 Jan 30. pii: molcanther.0352.2016. doi: 10.1158/1535-7163.MCT-16-0352. [Epub ahead of print] PubMed PMID: 28138028.
3: Ghelli Luserna Di Rorà A, Iacobucci I, Imbrogno E, Papayannidis C, Derenzini E, Ferrari A, Guadagnuolo V, Robustelli V, Parisi S, Sartor C, Abbenante MC, Paolini S, Martinelli G. Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia. Oncotarget. 2016 Aug 16;7(33):53377-53391. doi: 10.18632/oncotarget.10535. PubMed PMID: 27438145; PubMed Central PMCID: PMC5288194.
REFERENCES
1: Zeng L, Beggs RR, Cooper TS, Weaver AN, Yang ES. Combining Chk1/2 inhibition with cetuximab and radiation enhances in vitro and in vivo cytotoxicity in head and neck squamous cell carcinoma. Mol Cancer Ther. 2017 Jan 30. pii: molcanther.0352.2016. doi: 10.1158/1535-7163.MCT-16-0352. [Epub ahead of print] PubMed PMID: 28138028.
2: Ghelli Luserna Di Rorà A, Iacobucci I, Imbrogno E, Papayannidis C, Derenzini E, Ferrari A, Guadagnuolo V, Robustelli V, Parisi S, Sartor C, Abbenante MC, Paolini S, Martinelli G. Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia. Oncotarget. 2016 Aug 16;7(33):53377-53391. doi: 10.18632/oncotarget.10535. PubMed PMID: 27438145; PubMed Central PMCID: PMC5288194.
3: King C, Diaz HB, McNeely S, Barnard D, Dempsey J, Blosser W, Beckmann R, Barda D, Marshall MS. LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms. Mol Cancer Ther. 2015 Sep;14(9):2004-13. doi: 10.1158/1535-7163.MCT-14-1037. PubMed PMID: 26141948.
4: Hong D, Infante J, Janku F, Jones S, Nguyen LM, Burris H, Naing A, Bauer TM, Piha-Paul S, Johnson FM, Kurzrock R, Golden L, Hynes S, Lin J, Lin AB, Bendell J. Phase I Study of LY2606368, a Checkpoint Kinase 1 Inhibitor, in Patients With Advanced Cancer. J Clin Oncol. 2016 May 20;34(15):1764-71. doi: 10.1200/JCO.2015.64.5788. PubMed PMID: 27044938.
Lifetime achievement award ……..WORLD HEALTH CONGRESS 2017 in Hyderabad, 22 aug 2017 at JNTUH KUKATPALLY. HYDERABAD, TELANGANA, INDIA, Award given by Dr. M Sunitha Reddy Head of the Department, Centre for Pharmaceutical Sciences, Institute of Science &Technology, JNTU-H, Kukatpally, Hyderabad, India
Class Amides; Antiparkinsonians; Benzothiazoles; Carboxylic acids; Morpholines; Piperidines; Small molecules
Mechanism of Action Adenosine A2A receptor antagonists
Highest Development Phases
Phase III Parkinson’s disease
Phase I Liver disorders
Most Recent Events
30 Jun 2017 Biotie Therapies plans a phase I trial in Healthy volunteers in Canada (NCT03200080)
30 Jun 2017 Phase-I clinical trials in Liver disorders (In volunteers) in USA (PO) (NCT03212313)
27 Apr 2017 Acorda Therapeutics initiates enrolment in a phase III trial for Parkinson’s disease in Germany (EudraCT2016-003961-25)(NCT03051607)
Biotie Therapies Holding , under license from Roche , is developing tozadenant (phase 3, as of August 2017) for the treatment of Parkinson’s disease.
SYN-115, a potent and selective adenosine A2A receptor antagonist, is in phase III clinical trials at Biotie Therapeutics for the treatment of Parkinson’s disease, as an adjunjunctive therapy with levodopa. Phase 0 trials were are underway at the National Institute on Drug Abuse (NIDA) for the treatment of cocaine dependency, but no recent development has been reported.
The A2A receptor modulates the production of dopamine, glutamine and serotonin in several brain regions. In preclinical studies, antagonism of the A2A receptor resulted in increases in dopamine levels, which gave rise to the reversal of motor deficits.
Originally developed at Roche, SYN-115 was acquired by Synosia in 2007, in addition to four other drug candidates with potential for the treatment of central nervous system (CNS) disorders. Under the terms of the agreement, Synosia was responsible for clinical development and in some cases commercialization, while Roche retained the right to opt-in to two preselected programs.
In 2010, the compound was licensed to UCB by Synosia Therapeutics for development and commercialization worldwide.
In February 2011, Synosia (previously Synosis Therapeutics) was acquired by Biotie Therapeutics, and in 2014, Biotie regained global rights from UCB.
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Representative examples of A2AAdoR antagonists.
Tozadenant, also known as 4-hydroxy-N-(4-methoxy-7-(4-morpholinyl)benzo[d]thiazol-2-yl)-4-methylpiperidine-l-carboxamide or SYN115, is an adenosine A2A receptor antagonist. The A2A receptor modulates the production of
dopamine, glutamine and serotonin in several brain regions. In preclinical studies, antagonism of the A2A receptor resulted in increases in dopamine levels, which gave rise to the reversal of motor deficits.
Tozadenant is currently phase III clinical trials for the treatment of Parkinson’s disease as an adjunctive therapy with levodopa. It has also been explored for the treatment of cocaine dependency.
Adenosine modulates a wide range of physiological functions by interacting with specific cell surface receptors. The potential of adenosine receptors as drug targets was first reviewed in 1982. Adenosine is related both structurally and metabolically to the bioactive nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP); to the biochemical methylating agent S-adenosyl-L-methione (SAM); and structurally to the coenzymes NAD, FAD and coenzyme A; and to RNA. Together adenosine and these related compounds are important in the regulation of many aspects of cellular metabolism and in the modulation of different central nervous system activities.
[0003]
The adenosine receptors have been classified as A1, A2A, A2B and A3receptors, belonging to the family of G protein-coupled receptors. Activation of aderosine receptors by adenosine initiates signal transduction mechanisms. These mechanisms are dependent on the receptor associated G protein. Each of the adenosine receptor subtypes has been classically characterized by the adenylate cyclase effector system, which utilises cAMP as a second messenger. The A1and A3 receptors, coupled with Gi proteins inhibit adenylate cyclase, leading to a decrease in cellular cAMP levels, while A2A and A2Breceptors couple to Gs proteins and activate adenylate cyclase, leading to an increase in cellular cAMP levels. It is known that the A1receptor system activates phospholipase C and modulates both potassium and calcium ion channels. The A3 subtype, in addition to its association with adenylate cyclase, also stimulates phospholipase C and activates calcium ion channels.
[0004]
The A1 receptor (326-328 amino acids) was cloned from various species (canine, human, rat, dog, chick, bovine, guinea-pig) with 90-95% sequence identify among the mammalian species. The A2Areceptor (409-412 amino acids) was cloned from canine, rat, human, guinea pig and mouse. The A2B receptor (332 amino acids) was cloned from human and mouse and shows 45% homology with the human A1 and A2A receptors. The A3 receptor (317-320 amino acids) was cloned from human, rat, dog, rabbit and sheep.
[0005]
The A1 and A2A receptor subtypes are proposed to play complementary roles in adenosine’s regulation of the energy supply. Adenosine, which is a metabolic product of ATP, diffuses from the cell and acts locally to activate adenosine receptors to decrease the oxygen demand (A1) or increase the oxygen supply (A2A) and so reinstate the balance of energy supply: demand within the tissue. The actions of both subtypes is to increase the amount of available oxygen to tissue and to protect cells against damage caused by a short term imbalance of oxygen. One of the important functions of endogenous adenosine is preventing damage during traumas such as hypoxia, ischemia, hypotension and seizure activity.
[0006]
Furthermore, it is known that the binding of the adenosine receptor agonist to mast cells expressing the rat A3 receptor resulted in increased inositol triphosphate and intracellular calcium concentrations, which potentiated antigen induced secretion of inflammatory mediators. Therefore, the A3 receptor plays a role in mediating asthmatic attacks and other allergic responses.
[0007]
Adenosine is a neurotransmitter able to modulate many aspects of physiological brain function. Endogenous adenosine, a central link between energy metabolism and neuronal activity, varies according to behavioral state and (patho)physiological conditions. Under conditions of increased demand and decreased availability of energy (such as hypoxia, hypoglycemia, and/or excessive neuronal activity), adenosine provides a powerful protective feedback mechanism. Interacting with adenosine receptors represents a promising target for therapeutic intervention in a number of neurological and psychiatric diseases such as epilepsy, sleep, movement disorders (Parkinson or Huntington’s disease), Alzheimer’s disease, depression, schizophrenia, or addiction. An increase in neurotransmitter release follows traumas such as hypoxia, ischemia and seizures. These neurotransmitters are ultimately responsible for neural degeneration and neural death, which causes brain damage or death of the individual. The adenosine A1agonists mimic the central inhibitory effects of adenosine and may therefore be useful as neuroprotective agents. Adenosine has been proposed as an endogenous anticonvulsant agent, inhibiting glutamate release from excitatory neurons and inhibiting neuronal firing. Adenosine agonists therefore may be used as antiepileptic agents. Furthermore, adenosine antagonists have proven to be effective as cognition enhancers. Selective A2A antagonists have therapeutic potential in the treatment of various forms of dementia, for example in Alzheimer’s disease, and of neurodegenerative disorders, e.g. stroke. Adenosine A2A receptor antagonists modulate the activity of striatal GABAergic neurons and regulate smooth and well-coordinated movements, thus offering a potential therapy for Parkinsonian symptoms. Adenosine is also implicated in a number of physiological processes involved in sedation, hypnosis, schizophrenia, anxiety, pain, respiration, depression, and drug addiction (amphetamine, cocaine, opioids, ethanol, nicotine, and cannabinoids). Drugs acting at adenosine receptors therefore have therapeutic potential as sedatives, muscle relaxants, antipsychotics, anxiolytics, analgesics, respiratory stimulants, antidepressants, and to treat drug abuse. They may also be used in the treatment of ADHD (attention deficit hyper-activity disorder).
[0008]
An important role for adenosine in the cardiovascular system is as a cardioprotective agent. Levels of endogenous adenosine increase in response to ischemia and hypoxia, and protect cardiac tissue during and after trauma (preconditioning). By acting at the A1 receptor, adenosine A1 agonists may protect against the injury caused by myocardial ischemia and reperfusion. The modulating influence of A2Areceptors on adrenergic function may have implications for a variety of disorders such as coronary artery disease and heart failure. A2Aantagonists may be of therapeutic benefit in situations in which an enhanced anti-adrenergic response is desirable, such as during acute myocardial ischemia. Selective antagonists at A2A Areceptors may also enhance the effectiveness of adenosine in terminating supraventricula arrhytmias.
[0009]
Adenosine modulates many aspects of renal function, including renin release, glomerular filtration rate and renal blood flow. Compounds which antagonize the renal affects of adenosine have potential as renal protective agents. Furthermore, adenosine A3 and/or A2Bantagonists may be useful in the treatment of asthma and other allergic responses or and in the treatment of diabetes mellitus and obesity.
[0010]
Numerous documents describe the current knowledge on adenosine receptors, for example the following publications:
EXAMPLE 14-Hydroxy-4-methyl-piperidine-1-carboxylic acid(4-methoxy-7-morpholin-4-yl-benzothiazol-2-yl)-amide (I)
[0065]
To a solution of (4-methoxy-7-morpholin-4-yl-benzothiazol-2-yl)-carbamic acid phenyl ester (3.2 g, 8.3 mmol) and N-ethyl-diisopropyl-amine (4.4 ml, 25 mmol) in trichloromethane (50 ml) is added a solution of 4-hydroxy-4-methyl-piperidine in trichloromethane (3 ml) and tetrahydrofurane (3 ml) and the resulting mixture heated to reflux for 1 h. The reaction mixture is then cooled to ambient temperature and extracted with saturated aqueous sodium carbonate (15 ml) and water (2×5 ml). Final drying with magnesium sulphate and evaporation of the solvent and recrystallization from ethanol afforded the title compound as white crystals (78% yield), mp 236° C. MS: m/e=407(M+H+).
Novel deuterated forms of tozadenant are claimed. Also claimed are compositions comprising them and method of modulating the activity of adenosine A2A receptor (ADORA2A), useful for treating Parkinson’s diseases. Represents new area of patenting to be seen from CoNCERT Pharmaceuticals on tozadenant. ISR draws attention towards WO2016204939 , claiming controlled-release tozadenant formulations.
This invention relates to deuterated forms of morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide compounds, and pharmaceutically acceptable salts thereof. This invention also provides compositions comprising a compound of this invention and the use of such compositions in methods of treating diseases and conditions that are beneficially treated by administering an adenosine A2A receptor antagonist.
Many current medicines suffer from poor absorption, distribution, metabolism and/or excretion (ADME) properties that prevent their wider use or limit their use in certain indications. Poor ADME properties are also a major reason for the failure of drug candidates in clinical trials. While formulation technologies and prodrug strategies can be employed in some cases to improve certain ADME properties, these approaches often fail to address the underlying ADME problems that exist for many drugs and drug candidates. One such problem is rapid metabolism that causes a number of drugs, which otherwise would be highly effective in treating a disease, to be cleared too rapidly from the body. A possible solution to rapid drug clearance is frequent or high dosing to attain a sufficiently high plasma level of drug. This, however, introduces a number of potential treatment problems such as poor patient compliance with the dosing regimen, side effects that become more acute with higher doses, and increased cost of treatment. A rapidly metabolized drug may also expose patients to undesirable toxic or reactive metabolites.
[3] Another ADME limitation that affects many medicines is the formation of toxic or biologically reactive metabolites. As a result, some patients receiving the drug may experience toxicities, or the safe dosing of such drugs may be limited such that patients receive a suboptimal amount of the active agent. In certain cases, modifying dosing intervals or formulation approaches can help to reduce clinical adverse effects, but often the formation of such undesirable metabolites is intrinsic to the metabolism of the compound.
[4] In some select cases, a metabolic inhibitor will be co- administered with a drug that is cleared too rapidly. Such is the case with the protease inhibitor class of drugs that are used to treat HIV infection. The FDA recommends that these drugs be co-dosed with ritonavir, an inhibitor of cytochrome P450 enzyme 3A4 (CYP3A4), the enzyme typically responsible for their metabolism (see Kempf, D.J. et al., Antimicrobial agents and chemotherapy, 1997, 41(3): 654-60). Ritonavir, however, causes adverse effects and adds to the pill burden for HIV patients who must already take a combination of different drugs. Similarly, the
CYP2D6 inhibitor quinidine has been added to dextromethorphan for the purpose of reducing rapid CYP2D6 metabolism of dextromethorphan in a treatment of pseudobulbar affect.
Quinidine, however, has unwanted side effects that greatly limit its use in potential combination therapy (see Wang, L et al., Clinical Pharmacology and Therapeutics, 1994, 56(6 Pt 1): 659-67; and FDA label for quinidine at http://www.accessdata.fda.gov).
[5] In general, combining drugs with cytochrome P450 inhibitors is not a satisfactory strategy for decreasing drug clearance. The inhibition of a CYP enzyme’s activity can affect the metabolism and clearance of other drugs metabolized by that same enzyme. CYP inhibition can cause other drugs to accumulate in the body to toxic levels.
[6] A potentially attractive strategy for improving a drug’s metabolic properties is deuterium modification. In this approach, one attempts to slow the CYP-mediated metabolism of a drug or to reduce the formation of undesirable metabolites by replacing one or more hydrogen atoms with deuterium atoms. Deuterium is a safe, stable, non-radioactive isotope of hydrogen. Compared to hydrogen, deuterium forms stronger bonds with carbon. In select cases, the increased bond strength imparted by deuterium can positively impact the ADME properties of a drug, creating the potential for improved drug efficacy, safety, and/or tolerability. At the same time, because the size and shape of deuterium are essentially identical to those of hydrogen, replacement of hydrogen by deuterium would not be expected to affect the biochemical potency and selectivity of the drug as compared to the original chemical entity that contains only hydrogen.
[7] Over the past 35 years, the effects of deuterium substitution on the rate of metabolism have been reported for a very small percentage of approved drugs (see, e.g., Blake, MI et al, J Pharm Sci, 1975, 64:367-91; Foster, AB, Adv Drug Res 1985, 14: 1-40 (“Foster”); Kushner, DJ et al, Can J Physiol Pharmacol 1999, 79-88; Fisher, MB et al, Curr Opin Drug Discov Devel, 2006, 9: 101-09 (“Fisher”)). The results have been variable and unpredictable. For some compounds deuteration caused decreased metabolic clearance in vivo. For others, there was no change in metabolism. Still others demonstrated increased metabolic clearance. The variability in deuterium effects has also led experts to question or dismiss deuterium modification as a viable drug design strategy for inhibiting adverse metabolism (see Foster at p. 35 and Fisher at p. 101).
[8] The effects of deuterium modification on a drug’s metabolic properties are not predictable even when deuterium atoms are incorporated at known sites of metabolism. Only by actually preparing and testing a deuterated drug can one determine if and how the rate of metabolism will differ from that of its non-deuterated counterpart. See, for example, Fukuto et al. (J. Med. Chem. 1991, 34, 2871-76). Many drugs have multiple sites where metabolism is possible. The site(s) where deuterium substitution is required and the extent of deuteration necessary to see an effect on metabolism, if any, will be different for each drug.
4-hydroxy-4-methyl-piperidine-1-carboxylic acid (4-methoxy-7-morpholin-4-yl-benzothiazol-2-yl)-amide FOR THE TREATMENT OF POST-TRAUMATIC STRESS DISORDER
The U.S. Food and Drug Administration today granted accelerated approval to benznidazole for use in children ages 2 to 12 years old with Chagas disease. It is the first treatment approved in the United States for the treatment of Chagas disease.
The U.S. Food and Drug Administration today granted accelerated approval to benznidazole for use in children ages 2 to 12 years old with Chagas disease. It is the first treatment approved in the United States for the treatment of Chagas disease.
Chagas disease, or American trypanosomiasis, is a parasitic infection caused by Trypanosoma cruzi and can be transmitted through different routes, including contact with the feces of a certain insect, blood transfusions, or from a mother to her child during pregnancy. After years of infection, the disease can cause serious heart illness, and it also can affect swallowing and digestion. While Chagas disease primarily affects people living in rural parts of Latin America, recent estimates are that there may be approximately 300,000 persons in the United States with Chagas disease.
“The FDA is committed to making available safe and effective therapeutic options to treat tropical diseases,” said Edward Cox, M.D., director of the Office of Antimicrobial Products in the FDA’s Center for Drug Evaluation and Research.
The safety and efficacy of benznidazole were established in two placebo-controlled clinical trials in pediatric patients 6 to 12 years old. In the first trial, approximately 60 percent of children treated with benznidazole had an antibody test change from positive to negative compared with approximately 14 percent of children who received a placebo. Results in the second trial were similar: Approximately 55 percent of children treated with benznidazole had an antibody test change from positive to negative compared with 5 percent who received a placebo. An additional study of the safety and pharmacokinetics (how the body absorbs, distributes and clears the drug) of benznidazole in pediatric patients 2 to 12 years of age provided information for dosing recommendations down to 2 years of age.
The most common adverse reactions in patients taking benznidazole were stomach pain, rash, decreased weight, headache, nausea, vomiting, abnormal white blood cell count, urticaria (hives), pruritus (itching) and decreased appetite. Benznidazole is associated with serious risks including serious skin reactions, nervous system effects and bone marrow depression. Based on findings from animal studies, benznidazole could cause fetal harm when administered to a pregnant woman.
Benznidazole was approved using the Accelerated Approval pathway. The Accelerated Approval pathway allows the FDA to approve drugs for serious conditions where there is unmet medical need and adequate and well-controlled trials establish that the drug has an effect on a surrogate endpoint that is reasonably likely to predict a clinical benefit to patients. Further study is required to verify and describe the anticipated clinical benefit of benznidazole.
The FDA granted benznidazole priority review and orphan product designation. These designations were granted because Chagas disease is a rare disease, and until now, there were no approved drugs for Chagas disease in the United States.
With this approval, benznidazole’s manufacturer, Chemo Research, S. L., is awarded a Tropical Disease Priority Review Voucher in accordance with a provision included in the Food and Drug Administration Amendments Act of 2007 that aims to encourage development of new drugs and biological products for the prevention and treatment of certain tropical diseases.
Benznidazole is an antiparasitic medication used in the treatment of Chagas disease.[2] While it is highly effective in early disease this decreases in those who have long term infection.[3] It is the first line treatment given its moderate side effects compared to nifurtimox.[1] It is taken by mouth.[2]
Benznidazole came into medical use in 1971.[2] It is on the World Health Organization’s List of Essential Medicines, the most effective and safe medicines needed in a health system.[7] It is not commercially available in the United States, but can be obtained from the Centers of Disease Control.[2] As of 2012 Laboratório Farmacêutico do Estado de Pernambuco, a government run pharmaceutical company in Brazil was the only producer.[8]
Medical uses
Benznidazole has a significant activity during the acute phase of Chagas disease, with a therapeutical success rate up to 80%. Its curative capabilities during the chronic phase are, however, limited. Some studies have found parasitologic cure (a complete elimination of T. cruzi from the body) in pediatric and young patients during the early stage of the chronic phase, but overall failure rate in chronically infected individuals is typically above 80%.[6]
However, some studies indicate treatment with benznidazole during the chronic phase, even if incapable of producing parasitologic cure, because it reduces electrocardiographic changes and a delays worsening of the clinical condition of the patient.[6]
Benznidazole has proven to be effective in the treatment of reactivated T. cruzi infections caused by immunosuppression, such as in people with AIDS or in those under immunosuppressive therapy related to organ transplants.[6]
Benznidazole can be used in children and infants, with the same 5–7 mg/kg per day weight-based dosing regimen that is used to treat adult infections.[9] Children are found to be at a lower risk of adverse events compared to adults, possibly due to increased hepatic clearance of the drug. The most prevalent adverse effects in children were found to be gastrointestinal, dermatologic, and neurologic in nature. However, the incidence of severe dermatologic and neurologic adverse events is lower in the pediatric population compared to adults.[10]
Studies in animals have shown that benznidazole can cross the placenta.[11] Due to its potential for teratogenicity, use of benznidazole in pregnancy is not recommended.[9]
Side effects tend to be common and occur more frequently with increased age.[12] The most common adverse reactions associated with benznidazole are allergic dermatitis and peripheral neuropathy.[1] It is reported that up to 30% of people will experience dermatitis when starting treatment.[11][13] Benznidazole may cause photosensitization of the skin, resulting in rashes.[1] Rashes usually appear within the first 2 weeks of treatment and resolve over time.[13] In rare instances, skin hypersensitivity can result in exfoliative skin eruptions, edema, and fever.[13]Peripheral neuropathy may occur later on in the treatment course and is dose dependent.[1] It is not permanent, but takes time to resolve.[13]
Other adverse reactions include anorexia, weight loss, nausea, vomiting, insomnia, and dysguesia, and bone marrow suppression.[1] Gastrointestinal symptoms usually occur during the initial stages of treatment and resolves over time.[13] Bone marrow suppression has been linked to the cumulative dose exposure.[13]
Contraindications
Benznidazole should not be used in people with severe liver and/or kidney disease.[12] Pregnant women should not use benznidazole because it can cross the placenta and cause teratogenicity.[11]
Pharmacology
Mechanism of action
Benznidazole is a nitroimidazole antiparasitic with good activity against acute infection with Trypanosoma cruzi, commonly referred to as Chagas disease.[11] Like other nitroimidazoles, benznidazole’s main mechanism of action is to generate radical species which can damage the parasite’sDNA or cellular machinery.[14] The mechanism by which nitroimidazoles do this seems to depend on whether or not oxygen is present.[15] This is particularly relevant in the case of Trypanosoma species, which are considered facultative anaerobes.[16]
Under anaerobic conditions, the nitro group of nitroimidazoles is believed to be reduced by the pyruvate:ferredoxin oxidoreductase complex to create a reactive nitro radical species.[14] The nitro radical can then either engage in other redox reactions directly or spontaneously give rise to a nitrite ion and imidazole radical instead.[15] The initial reduction takes place because nitroimidazoles are better electron acceptors for ferredoxin than the natural substrates.[14] In mammals, the principal mediators of electron transport are NAD+/NADH and NADP+/NADPH, which have a more positive reduction potential and so will not reduce nitroimidazoles to the radical form.[14] This limits the spectrum of activity of nitroimidazoles so that host cells and DNA are not also damaged. This mechanism has been well-established for 5-nitroimidazoles such as metronidazole, but it is unclear if the same mechanism can be expanded to 2-nitroimidazoles (including benznidazole).[15]
In the presence of oxygen, by contrast, any radical nitro compounds produced will be rapidly oxidized by molecular oxygen, yielding the original nitroimidazole compound and a superoxide anion in a process known as “futile cycling“.[14] In these cases, the generation of superoxide is believed to give rise to other reactive oxygen species.[15] The degree of toxicity or mutagenicity produced by these oxygen radicals depends on cells’ ability to detoxify superoxide radicals and other reactive oxygen species.[15] In mammals, these radicals can be converted safely to hydrogen peroxide, meaning benznidazole has very limited direct toxicity to human cells.[15] In Trypanosoma species, however, there is a reduced capacity to detoxify these radicals, which results in damage to the parasite’s cellular machinery.[15]
Benznidazole and other nitroimidazoles have been shown to decrease the rate of clearance of 5-fluorouracil (including 5-fluorouracil produced from its prodrugscapecitabine, doxifluridine, and tegafur).[19]While co-administration of any of these drugs with benznidazole is not contraindicated, monitoring for 5-fluorouracil toxicity is recommended in the event they are used together.[20]
^ Jump up to:abcCastro, José A.; de Mecca, Maria Montalto; Bartel, Laura C. (2006-08-01). “Toxic side effects of drugs used to treat Chagas’ disease (American trypanosomiasis)”. Human & Experimental Toxicology. 25 (8): 471–479. ISSN0960-3271. PMID16937919. doi:10.1191/0960327106het653oa.
^ Jump up to:abcdeEdwards, David I (1993). “Nitroimidazole drugs – action and resistance mechanisms. I. Mechanism of action”. Journal of Antimicrobial Chemotherapy. 31: 9–20. doi:10.1093/jac/31.1.9.
Jump up^Cheng, Thomas C. (1986). General Parasitology. Orlando, Florida: Academic Press. p. 140. ISBN0-12-170755-5.
^ Jump up to:abRaaflaub, J; Ziegler, WH (1979). “Single-dose pharmacokinetics of the trypanosomicide benznidazole in man”. Arzneimittelforschung. 29 (10): 1611–1614.
The U.S. Food and Drug Administration today approved Vabomere for adults with complicated urinary tract infections (cUTI), including a type of kidney infection, pyelonephritis, caused by specific bacteria. Vabomere is a drug containing meropenem, an antibacterial, and vaborbactam, which inhibits certain types of resistance mechanisms used by bacteria.
The U.S. Food and Drug Administration today approved Vabomere for adults with complicated urinary tract infections (cUTI), including a type of kidney infection, pyelonephritis, caused by specific bacteria. Vabomere is a drug containing meropenem, an antibacterial, and vaborbactam, which inhibits certain types of resistance mechanisms used by bacteria.
“The FDA is committed to making new safe and effective antibacterial drugs available,” said Edward Cox, M.D., director of the Office of Antimicrobial Products in the FDA’s Center for Drug Evaluation and Research. “This approval provides an additional treatment option for patients with cUTI, a type of serious bacterial infection.”
The safety and efficacy of Vabomere were evaluated in a clinical trial with 545 adults with cUTI, including those with pyelonephritis. At the end of intravenous treatment with Vabomere, approximately 98 percent of patients treated with Vabomere compared with approximately 94 percent of patients treated with piperacillin/tazobactam, another antibacterial drug, had cure/improvement in symptoms and a negative urine culture test. Approximately seven days after completing treatment, approximately 77 percent of patients treated with Vabomere compared with approximately 73 percent of patients treated with piperacillin/tazobactam had resolved symptoms and a negative urine culture.
The most common adverse reactions in patients taking Vabomere were headache, infusion site reactions and diarrhea. Vabomere is associated with serious risks including allergic reactions and seizures. Vabomere should not be used in patients with a history of anaphylaxis, a type of severe allergic reaction to products in the class of drugs called beta-lactams.
To reduce the development of drug-resistant bacteria and maintain the effectiveness of antibacterial drugs, Vabomere should be used only to treat or prevent infections that are proven or strongly suspected to be caused by susceptible bacteria.
Vabomere was designated as a qualified infectious disease product (QIDP). This designation is given to antibacterial products that treat serious or life-threatening infections under the Generating Antibiotic Incentives Now (GAIN) title of the FDA Safety and Innovation Act. As part of its QIDP designation, Vabomere received a priority review.
The FDA granted approval of Vabomere to Rempex Pharmaceuticals.
The U.S. Food and Drug Administration issued a historic action today making the first gene therapy available in the United States, ushering in a new approach to the treatment of cancer and other serious and life-threatening diseases
The U.S. Food and Drug Administration issued a historic action today making the first gene therapy available in the United States, ushering in a new approach to the treatment of cancer and other serious and life-threatening diseases.
The FDA approved Kymriah (tisagenlecleucel) for certain pediatric and young adult patients with a form of acute lymphoblastic leukemia (ALL).
“We’re entering a new frontier in medical innovation with the ability to reprogram a patient’s own cells to attack a deadly cancer,” said FDA Commissioner Scott Gottlieb, M.D. “New technologies such as gene and cell therapies hold out the potential to transform medicine and create an inflection point in our ability to treat and even cure many intractable illnesses. At the FDA, we’re committed to helping expedite the development and review of groundbreaking treatments that have the potential to be life-saving.”
Kymriah, a cell-based gene therapy, is approved in the United States for the treatment of patients up to 25 years of age with B-cell precursor ALL that is refractory or in second or later relapse.
Kymriah is a genetically-modified autologous T-cell immunotherapy. Each dose of Kymriah is a customized treatment created using an individual patient’s own T-cells, a type of white blood cell known as a lymphocyte. The patient’s T-cells are collected and sent to a manufacturing center where they are genetically modified to include a new gene that contains a specific protein (a chimeric antigen receptor or CAR) that directs the T-cells to target and kill leukemia cells that have a specific antigen (CD19) on the surface. Once the cells are modified, they are infused back into the patient to kill the cancer cells.
ALL is a cancer of the bone marrow and blood, in which the body makes abnormal lymphocytes. The disease progresses quickly and is the most common childhood cancer in the U.S. The National Cancer Institute estimates that approximately 3,100 patients aged 20 and younger are diagnosed with ALL each year. ALL can be of either T- or B-cell origin, with B-cell the most common. Kymriah is approved for use in pediatric and young adult patients with B-cell ALL and is intended for patients whose cancer has not responded to or has returned after initial treatment, which occurs in an estimated 15-20 percent of patients.
“Kymriah is a first-of-its-kind treatment approach that fills an important unmet need for children and young adults with this serious disease,” said Peter Marks, M.D., Ph.D., director of the FDA’s Center for Biologics Evaluation and Research (CBER). “Not only does Kymriah provide these patients with a new treatment option where very limited options existed, but a treatment option that has shown promising remission and survival rates in clinical trials.”
The safety and efficacy of Kymriah were demonstrated in one multicenter clinical trial of 63 pediatric and young adult patients with relapsed or refractory B-cell precursor ALL. The overall remission rate within three months of treatment was 83 percent.
Treatment with Kymriah has the potential to cause severe side effects. It carries a boxed warning for cytokine release syndrome (CRS), which is a systemic response to the activation and proliferation of CAR T-cells causing high fever and flu-like symptoms, and for neurological events. Both CRS and neurological events can be life-threatening. Other severe side effects of Kymriah include serious infections, low blood pressure (hypotension), acute kidney injury, fever, and decreased oxygen (hypoxia). Most symptoms appear within one to 22 days following infusion of Kymriah. Since the CD19 antigen is also present on normal B-cells, and Kymriah will also destroy those normal B cells that produce antibodies, there may be an increased risk of infections for a prolonged period of time.
The FDA today also expanded the approval of Actemra (tocilizumab) to treat CAR T-cell-induced severe or life-threatening CRS in patients 2 years of age or older. In clinical trials in patients treated with CAR-T cells, 69 percent of patients had complete resolution of CRS within two weeks following one or two doses of Actemra.
Because of the risk of CRS and neurological events, Kymriah is being approved with a risk evaluation and mitigation strategy (REMS), which includes elements to assure safe use (ETASU). The FDA is requiring that hospitals and their associated clinics that dispense Kymriah be specially certified. As part of that certification, staff involved in the prescribing, dispensing, or administering of Kymriah are required to be trained to recognize and manage CRS and neurological events. Additionally, the certified health care settings are required to have protocols in place to ensure that Kymriah is only given to patients after verifying that tocilizumab is available for immediate administration. The REMS program specifies that patients be informed of the signs and symptoms of CRS and neurological toxicities following infusion – and of the importance of promptly returning to the treatment site if they develop fever or other adverse reactions after receiving treatment with Kymriah.
To further evaluate the long-term safety, Novartis is also required to conduct a post-marketing observational study involving patients treated with Kymriah.
The FDA granted Kymriah Priority Review and Breakthrough Therapy designations. The Kymriah application was reviewed using a coordinated, cross-agency approach. The clinical review was coordinated by the FDA’s Oncology Center of Excellence, while CBER conducted all other aspects of review and made the final product approval determination.
The FDA granted approval of Kymriah to Novartis Pharmaceuticals Corp. The FDA granted the expanded approval of Actemra to Genentech Inc.
The U.S. Food and Drug Administration today approved Mylotarg (gemtuzumab ozogamicin) for the treatment of adults with newly diagnosed acute myeloid leukemia whose tumors express the CD33 antigen (CD33-positive AML). The FDA also approved Mylotarg for the treatment of patients aged 2 years and older with CD33-positive AML who have experienced a relapse or who have not responded to initial treatment (refractory).
The U.S. Food and Drug Administration today approved Mylotarg (gemtuzumab ozogamicin) for the treatment of adults with newly diagnosed acute myeloid leukemia whose tumors express the CD33 antigen (CD33-positive AML). The FDA also approved Mylotarg for the treatment of patients aged 2 years and older with CD33-positive AML who have experienced a relapse or who have not responded to initial treatment (refractory).
Mylotarg originally received accelerated approval in May 2000 as a stand-alone treatment for older patients with CD33-positive AML who had experienced a relapse. Mylotarg was voluntarily withdrawn from the market after subsequent confirmatory trials failed to verify clinical benefit and demonstrated safety concerns, including a high number of early deaths. Today’s approval includes a lower recommended dose, a different schedule in combination with chemotherapy or on its own, and a new patient population.
“We are approving Mylotarg after a careful review of the new dosing regimen, which has shown that the benefits of this treatment outweigh the risk,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “Mylotarg’s history underscores the importance of examining alternative dosing, scheduling, and administration of therapies for patients with cancer, especially in those who may be most vulnerable to the side effects of treatment.”
AML is a rapidly progressing cancer that forms in the bone marrow and results in an increased number of white blood cells in the bloodstream. The National Cancer Institute of the National Institutes of Health estimates that approximately 21,380 people will be diagnosed with AML this year and that 10,590 patients with AML will die of the disease.
Mylotarg is a targeted therapy that consists of an antibody connected to an anti-tumor agent that is toxic to cells. It is thought to work by taking the anti-tumor agent to the AML cells that express the CD33 antigen, blocking the growth of cancerous cells and causing cell death.
The safety and efficacy of Mylotarg in combination with chemotherapy for adults were studied in a trial of 271 patients with newly diagnosed CD33-positive AML who were randomized to receive Mylotarg in combination with daunorubicin and cytarabine or to receive daunorubicin and cytarabine without Mylotarg. The trial measured “event-free survival,” or how long patients went without certain complications, including failure to respond to treatment, disease relapse or death, from the date they started the trial. Patients who received Mylotarg in combination with chemotherapy went longer without complications than those who received chemotherapy alone (median, event-free survival 17.3 months vs. 9.5 months).
The safety and efficacy of Mylotarg as a stand-alone treatment were studied in two, separate trials. The first trial included 237 patients with newly diagnosed AML who could not tolerate or chose not to receive intensive chemotherapy. Patients were randomized to receive treatment with Mylotarg or best supportive care. The trial measured “overall survival,” or how long patients survived from the date they started the trial. Patients who received Mylotarg survived longer than those who received only best supportive care (median overall survival 4.9 months vs. 3.6 months). The second trial was a single-arm study that included 57 patients with CD33-positive AML who had experienced one relapse of disease. Patients received a single course of Mylotarg. The trial measured how many patients achieved a complete remission. Following treatment with Mylotarg, 26 percent of patients achieved a complete remission that lasted a median 11.6 months.
Common side effects of Mylotarg include fever (pyrexia), nausea, infection, vomiting, bleeding, low levels of platelets in the blood (thrombocytopenia), swelling and sores in the mouth (stomatitis), constipation, rash, headache, elevated liver function tests, and low levels of certain white blood cells (neutropenia). Severe side effects of Mylotarg include low blood counts, infections, liver damage, blockage of the veins in the liver (hepatic veno-occlusive disease), infusion-related reactions, and severe bleeding (hemorrhage). Women who are pregnant or breastfeeding should not take Mylotarg, because it may cause harm to a developing fetus or a newborn baby. Patients with hypersensitivity to Mylotarg or any component of its formulation should not use Mylotarg.
The prescribing information for Mylotarg includes a boxed warning that severe or fatal liver damage (hepatotoxicity), including blockage of veins in the liver (veno-occlusive disease or sinusoidal obstruction syndrome), occurred in some patients who took Mylotarg.
Mylotarg received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases.
The FDA granted the approval of Mylotarg to Pfizer Inc.
Gemtuzumab ozogamicin (marketed by Wyeth as Mylotarg) is a drug-linked monoclonal antibody (an antibody-drug conjugate) that was used to treat acute myelogenous leukemia from 2000 to 2010. It was withdrawn from market in June 2010 when a clinical trial showed the drug increased patient death and added no benefit over conventional cancer therapies.
Mechanism and side effects
Gemtuzumab is a monoclonal antibody to CD33 linked to a cytotoxic agent from the class of calicheamicins. CD33 is expressed in most leukemic blast cells but also in normal hematopoietic cells, the intensity diminishing with maturation of stem cells.
Gemtuzumab ozogamicin was created in a collaboration between Celltech and Wyeth that began in 1991.[1][2] The same collaboration later produced inotuzumab ozogamicin.[3] Celltech was acquired by UCB in 2004[4] and Wyeth was acquired by Pfizer in 2009.[5]
Within the first year after approval, the FDA required a black box warning be added to Gemtuzumab packaging. The drug was noted to increase the risk of veno-occlusive disease in the absence of bone marrow transplantation.[9] Later the onset of VOD was shown to occur at increased frequency in Gemtuzumab patients even following bone marrow transplantation.[10] The drug was discussed in a 2008 JAMA article, which criticized the inadequacy of postmarketing surveillance of biologic agents.[11]
A randomized phase 3 comparative controlled trial (SWOG S0106) was initiated in 2004 by Wyeth in accordance with the FDA accelerated-approval process. The study was stopped[when?] prior to completion due to worrisome outcomes. Among the patients evaluated for early toxicity, fatal toxicity rate was significantly higher in the gemtuzumab combination therapy group vs the standard therapy group. Mortality was 5.7% with gemtuzumab and 1.4% without the agent (16/283 = 5.7% vs 4/281 = 1.4%; P = .01).[7]
In June 2010, Pfizer withdrew Mylotarg from the market at the request of the US FDA.[12][13] However, some other regulatory authorities did not agree with the FDA decision, with Japan’s Pharmaceuticals and Medical Devices Agency stating in 2011 that the “risk-benefit balance of gemtuzumab ozogamicin has not changed from its state at the time of approval”.[14]
In early 2017 Pfizer reapplied for US and EU approval, based on a meta-analysis of prior trials and results of the ALFA-0701 clinical trial, an open-label Phase III trial in 280 older people with AML. [8]
Jump up^Niculescu-Duvaz, I (December 2000). “Technology evaluation: gemtuzumab ozogamicin, Celltech Group.”. Current opinion in molecular therapeutics. 2 (6): 691–6. PMID11249747.
Jump up^Damle, NK; Frost, P (August 2003). “Antibody-targeted chemotherapy with immunoconjugates of calicheamicin.”. Current opinion in pharmacology. 3 (4): 386–90. PMID12901947. doi:10.1016/S1471-4892(03)00083-3.
Jump up^Giles FJ, Kantarjian HM, Kornblau SM, Thomas DA, Garcia-Manero G, Waddelow TA, David CL, Phan AT, Colburn DE, Rashid A, Estey EH (2001). “Mylotarg (gemtuzumab ozogamicin) therapy is associated with hepatic venoocclusive disease in patients who have not received stem cell transplantation.”. Cancer. 92 (2): 406–13. PMID11466696. doi:10.1002/1097-0142(20010715)92:2<406::AID-CNCR1336>3.0.CO;2-U.
Jump up^Wadleigh M, Richardson PG, Zahrieh D, Lee SJ, Cutler C, Ho V, Alyea EP, Antin JH, Stone RM, Soiffer RJ, DeAngelo DJ (2003). “Prior gemtuzumab ozogamicin exposure significantly increases the risk of veno-occlusive disease in patients who undergo myeloablative allogeneic stem cell transplantation.”. Blood. 102 (5): 1578–82. PMID12738663. doi:10.1182/blood-2003-01-0255.
Developer The Medicines Company; US Department of Health and Human Services
Class Antibacterials; Pyrrolidines; Small molecules; Thienamycins
Mechanism of Action Beta lactamase inhibitors; Cell wall inhibitors
Highest Development Phases
Registered Urinary tract infections
Phase III Bacteraemia; Gram-negative infections; Pneumonia; Pyelonephritis
Most Recent Events
29 Aug 2017 Registered for Urinary tract infections (Treatment-experienced, Treatment-resistant) in USA (IV) – First global approval
29 Aug 2017 Updated efficacy and safety data from a phase III trial in Gram-negative infections released by The Medicines Company
09 Aug 2017 Planned Prescription Drug User Fee Act (PDUFA) date for Urinary tract infections (Treatment-experienced, Treatment-resistant) in USA (IV) is 2017-08-29
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Rapidly rising resistance to multiple antimicrobial agents in Gram-negative bacteria, commonly related to healthcare-associated infections, is an emerging public health concern in U.S. hospitals. While the cephalosporin class of β-lactams was the mainstay of treatment in the 1980s, the dissemination of extended-spectrum β-lactamases (ESBLs) over the past 2 decades has dramatically weakened the utility of this class and brought about a corresponding reliance on the carbapenems.(1) Although carbapenems are widely recognized as a safe and effective class of antimicrobials, carbapenem-resistant Enterobacteriaceae (CRE) due to the Klebsiellapneumoniaecarbapenemase (KPC) and other β-lactamases now threatens the usefulness of all β-lactam antibiotics.(2) The Centers for Disease Control (CDC) considers CRE to be an urgent antimicrobial resistance threat that now has been detected in nearly every U.S. state, with an alarming increase in incidence over the past 5 years.(3) The failure to develop antimicrobial agents to manage CRE threatens to have a catastrophic impact on the healthcare system.(4)
A proven strategy to overcome resistance to β-lactam antibiotics has been to restore their activity by combining them with an inhibitor of the β-lactamase enzymes responsible for their degradation. Examples of clinically important β-lactamase inhibitors (Figure 1) include clavulanic acid (combined with amoxicillin), sulbactam (with ampicillin), and tazobactam (with piperacillin). The KPC β-lactamase is poorly inhibited by these β-lactamase inhibitors, and thus, they have no usefulness in the treatment of infections due to CRE. More recently, the diazabicyclooctane inhibitors avibactam (NXL-104)(5) and relebactam (MK-7655)(6) have entered clinical development, in combination with ceftazidime and imipenem, respectively. Both compounds display a broad spectrum of β-lactamase inhibition that includes the KPC enzyme.
Image may be NSFW. Clik here to view.Next generation β-lactamase inhibitors recently approved or in clinical trials. A. Avibactam. B. Relebactam. C. Vaborbactam.
Vaborbactam (INN)[1] is a non-β-lactamβ-lactamase inhibitor discovered by Rempex Pharmaceuticals, a subsidiary of The Medicines Company. While not effective as an antibiotic by itself, it restores potency to existing antibiotics by inhibiting the beta-lactamase enzymes that would otherwise degrade them. When combined with an appropriate antibiotic it can be used for the treatment of gram-negative bacterial infections.[2]
According to a Medicines Company press release, as of June 2016 a combination of vaborbactam with the carbapenem antibiotic meropenem had met all pre-specified primary endpoints in a phase III clinical trial in patients with complicated urinary tract infections.[3] The company planned to submit an NDA to the FDAin early 2017.
Biochemistry
Carbapenemases are a family of β-lactamase enzymes distinguished by their broad spectrum of activity and their ability to degrade carbapenem antibiotics, which are frequently used in the treatment of multidrug-resistant gram-negative infections.[4] Carbapenemases can be broadly divided into two different categories based on the mechanism they use to hydrolyze the lactam ring in their substrates. Metallo-β-lactamases contain bound zinc ions in their active sites and are therefore inhibited by chelating agents like EDTA, while serine carbapenemases feature an active siteserine that participates in the hydrolysis of the substrate.[4] Serine carbapenemase-catalyzed hydrolysis employs a three-step mechanism featuring acylation and deacylation steps analogous to the mechanism of protease-catalyzed peptide hydrolysis, proceeding through a tetrahedral transition state.[4][5]
Boronic acids are unusual in their ability to reversibly form covalent bonds with alcohols such as the active site serine in a serine carbapenemase. This property enables them to function as transition state analogs of serine carbapenemase-catalyzed lactam hydrolysis and thereby inhibit these enzymes. Based on data from Hecker et al., vaborbactam is a potent inhibitor of a variety of β-lactamases, exhibiting a 69-nanomolar {\displaystyle K_{i}}Image may be NSFW. Clik here to view. against the KPC-2 carbapenemase and even lower inhibition constants against CTX-M-15 and SHV-12.[2]
Given their mechanism of action, the possibility of off-target effects brought about through inhibition of endogenous serine hydrolases is an obvious possible concern in the development of boronic acid β-lactamase inhibitors, and in fact boronic acids like bortezomib have previously been investigated or developed as inhibitors of various human proteases.[2] Vaborbactam, however, is a highly specific β-lactamase inhibitor, with an IC50 >> 1 mM against all human serine hydrolases against which it has been tested.[2] Consistent with its high in vitro specificity, vaborbactam exhibited a good safety profile in human phase I clinical trials, with similar adverse events observed in both placebo and treatment groups.[6] Hecker et al. argue this specificity results from the higher affinity of human proteases to linear molecules; thus it is expected that a boronheterocycle will have zero effect on them.
Antibiotics have been effective tools in the treatment of infectious diseases during the last half-century. From the development of antibiotic therapy to the late 1980s there was almost complete control over bacterial infections in developed countries. However, in response to the pressure of antibiotic usage, multiple resistance mechanisms have become widespread and are threatening the clinical utility of antibacterial therapy. The increase in antibiotic resistant strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs
[0003] Various bacteria have evolved β-lactam deactivating enzymes, namely, β-lactamases, that counter the efficacy of the various β-lactams. β-lactamases can be grouped into 4 classes based on their amino acid sequences, namely, Ambler classes A, B, C, and D. Enzymes in classes A, C, and D include active-site serine β-lactamases, and class B enzymes, which are encountered less frequently, are Zn-dependent. These enzymes catalyze the chemical degradation of β-lactam antibiotics, rendering them inactive. Some β-lactamases can be transferred within and between various bacterial strains and species. The rapid spread of bacterial resistance and the evolution of multi- resistant strains severely limits β-lactam treatment options available.
[0004] The increase of class D β-lactamase-expressing bacterium strains such as Acinetobacter baumannii has become an emerging multidrug-resistant threat. A. baumannii strains express A, C, and D class β-lactamases. The class D β-lactamases such as the OXA families are particularly effective at destroying carbapenem type β-lactam antibiotics, e.g., imipenem, the active carbapenems component of Merck’s Primaxin® (Montefour, K.; et al. Crit. Care Nurse 2008, 28, 15; Perez, F. et al. Expert Rev. Anti Infect. Ther. 2008, 6, 269; Bou, G.; Martinez-Beltran, J. Antimicrob. Agents Chemother. 2000, 40, 428. 2006, 50, 2280; Bou, G. et al, J. Antimicrob. Agents Chemother. 2000, 44, 1556). This has imposed a pressing threat to the effective use of drugs in that category to treat and prevent bacterial infections. Indeed the number of catalogued serine-based β- lactamases has exploded from less than ten in the 1970s to over 300 variants. These issues fostered the development of five “generations” of cephalosporins. When initially released into clinical practice, extended- spectrum cephalosporins resisted hydrolysis by the prevalent class A β-lactamases, TEM-1 and SHV-1. However, the development of resistant strains by the evolution of single amino acid substitutions in TEM-1 and SHV-1 resulted in the emergence of the extended- spectrum β-lactamase (ESBL) phenotype.
[0005] New β-lactamases have recently evolved that hydrolyze the carbapenem class of antimicrobials, including imipenem, biapenem, doripenem, meropenem, and ertapenem, as well as other β-lactam antibiotics. These carbapenemases belong to molecular classes A, B, and D. Class A carbapenemases of the KPC-type predominantly in Klebsiella pneumoniae but now also reported in other Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. The KPC carbapenemase was first described in 1996 in North Carolina, but since then has disseminated widely in the US. It has been particularly problematic in the New York City area, where several reports of spread within major hospitals and patient morbidity have been reported. These enzymes have also been recently reported in France, Greece, Sweden, United Kingdom, and an outbreak in Germany has recently been reported. Treatment of resistant strains with carbapenems can be associated with poor outcomes.
[0006] Another mechanism of β-lactamase mediated resistance to carbapenems involves combination of permeability or efflux mechanisms combined with hyper production of beta-lactamases. One example is the loss of a porin combined in hyperproduction of ampC beta-lactamase results in resistance to imipenem in Pseudomonas aeruginosa. Efflux pump over expression combined with hyperproduction of the ampC β-lactamase can also result in resistance to a carbapenem such as meropenem.
[0007] Because there are three major molecular classes of serine-based β- lactamases, and each of these classes contains significant numbers of β-lactamase variants, inhibition of one or a small number of β-lactamases is unlikely to be of therapeutic value. Legacy β-lactamase inhibitors are largely ineffective against at least Class A carbapenemases, against the chromosomal and plasmid-mediated Class C cephalosporinases and against many of the Class D oxacillinases. Therefore, there is a need for improved β-lactamase inhibitors.
The following compounds are prepared starting from enantiomerically pure (R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129, 4175-4177) in accordance with the procedure described in the above Example 1.
The following compounds are prepared starting from enantiomerically pure (R)-tert-butyl 3-hydroxypent-4-enoate (J. Am. Chem. Soc. 2007, 129, 4175-4177) in accordance with the procedure described in the above Example 1.
[0192] Chlorotrimethylsilane (4.6 mL, 36.3 mmol, 1.25 eq) was added to a solution of (R)-t-butyl 3-hydroxy-pent-4-enoate (1, 5 g, 29 mmol) and triethylamine (5.3 mL, 37.3 mmol, 1.3 eq) in dichloromethane (25 mL) keeping the temperature below 30 °C. After completion of the addition, the white heterogeneous mixture was stirred at rt for 20 minutes (TLC, GC, note 2) then quenched with MeOH (352 μί, 0.3 eq). After stirring at rt for 5 minutes, the white heterogeneous reaction mixture was diluted with heptane (25 mL). The salts were filtered off and rinsed with heptane (2 x 10 mL). The combined turbid filtrates were washed with a saturated solution of NaHC03 (2 x 25 mL) and concentrated to dryness. The residual oil was azeotroped with heptane (25 mL) to give a colorless oil that was used immediately.
[0193] A solution of bis-diphenylphosphino-ethane (46.3 mg, 0.2 mol%) and [Ir(COD)Cl]2 (39 mg, 0.1 mol%) in CH2C12 (5 mL) was added to a refluxing solution of crude TMS-protected pentenoate 7. Pinacol borane (9.3 mL,l .l eq) was added to the
refluxing solution. After stirring at reflux for 3 h, the reaction mixture was cooled to room temperature, concentrated to dryness and taken up in heptane (50 mL). The insolubles were filtered over Celite and rinse with heptane (10 mL).
Ethanolamine-boronic acid salt (10)
[0194] A mixture of fully protected boronate 8 (5.0 g, 13.4 mmol), 0.5 N HC1 (5 mL) and acetone (0.5 mL) was stirred vigorously at room temperature, providing intermediate 9. After complete consumption of the starting material, a solution of NaI04 (3.44 g, 1.2 eq) in water (15 mL) was added slowly keeping the temperature <30 °C. Upon the completion of the addition (30 min), the reaction mixture was allowed to cool to room temperature. After consumption of all pinacol, MTBE (5 mL) was added. After stirring at room temperature for 10 min, the white solids were filtered off and rinsed with MTBE (2 x 5 mL). The filtrate was partitioned and the aqueous layer was extracted with MTBE (10 mL). The combined organic extracts were washed sequentially with a 0.1 M NaHS03 solution (2 x 5 mL), a saturated NaHC03 solution (5 mL) and brine (5 mL). The organic layer was concentrated to dryness. The residue was taken up in MTBE (15 mL) and the residual salts filtered off. The filtrate was concentrated to dryness and the residue was taken up in MTBE (10 mL) and acetonitrile (1.7 mL). Ethanolamine (0.99 mL, 1.1 eq) was added. After stirring at room temperature for 1 hour, the heterogeneous mixture was stirred at 0 °C. After stirring at 0 °C for 2 hours, the solids were collected by filtration, rinsed with MTBE (2 x 5 mL), air dried then dried under high vacuum to give Compound 10 as a white granular powder.
Example 2 – Preparation of Beta-Lactamase Inhibitor (15)
[0195] The compound of Formula 15 was synthesized as shown in Scheme 4 below:
Scheme 4
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Synthesis of pinanediol boronate (12)
[0196] Ethanolammonium boronate 11 (15 g, 61.7 mmol) and pinanediol (10.5 g, 61.7 mmol, 1 eq) were suspended in MTBE (75 mL). Water (75 mL) was added and the yellow biphasic heterogeneous mixture was stirred at room temperature. After stirring for 2 hours at room temperature, some pinanediol was still present and stirring was continued overnight. The layers were separated and the organic layer was washed with brine, concentrated under reduced pressure and azeotroped with MTBE (2 x 30 mL). The residual oil was taken up in dichloromethane (40 mL). In another flask, TBSC1 (1 1.6 g, 77.1 mmol, 1.25 eq) was added to a solution of imidazole (9.66 g, 141.9 mmol, 2.3 eq) in dichloromethane (25 mL). The white slurry was stirred at room temperature. After 5 minutes, the solution of pinanediol boronate was added to the white slurry and the flask was rinsed with dichloromethane (2 x 5 mL). The heterogeneous reaction mixture was heated at reflux temeprature. After stirring at reflux for 8 hours, the reaction mixture was cooled to 30 °C and TMSC1 (330 \JL) was added. After stirring 30 minutes at 30 °C, MeOH (15 mL) was added. After stirring at room temperature overnight, the reaction mixture was washed sequentially with 0.5 N HC1 (115 niL), 0.5 N HC1 (60 n L) and saturated NaHC03 (90 niL). The organic layer was concentrated under reduced pressure and azeotroped with heptane (150 n L) to give 12 as a yellow oil (27.09 g, 94.1%) which was used without purification.
Synthesis of chloroboronate (13)
[0197] A solution of n-butyllithium (2.5 M in hexane, 29.6 niL, 74.1 mmol, 1.3 eq) was added to THF (100 mL) at -80 °C. The resulting solution was cooled to -100 °C. A solution of dichloromethane (14.6 mL, 228 mmol, 4 eq) in THF (25 mL) was added via syringe pump on the sides of the flask keeping the temperature < -95 °C. During the second half of the addition a precipitate starts to appear which became thicker with the addition of the remaining dichloromethane solution. After stirring between -100 and -95 °C for 30 min, a solution of 12 (26.59 g, 57 mmol) in THF (25 mL) was added by syringe pump on the sides of the flask while maintaining the batch temperature < -95 °C to give a clear yellow solution. After stirring between -100 and -95 °C for 30 min, a solution of zinc chloride (1 M in ether, 120 mL, 120 mmol, 2.1 eq) was added keeping the temperature < -70 °C. The reaction mixture was then warmed to room temperature (at about -18 °C the reaction mixture became turbid/heterogeneous). After stirring at room temperature for 2 hours, the reaction mixture was cooled to 15 °C and quenched with 1 N HC1 (100 mL). The layers were separated and the organic layer was washed sequentially with 1 N HC1 (100 mL) and water (2 x 100 mL), concentrated to oil and azeotroped with heptane (3 x 150 mL) to provide 13 as a yellow oil (30.03 g, 102%) which was used without purification.
Synthesis of (14)
[0198] LiHMDS (1 M in THF, 63 mL, 62.7 mmol, 1.1 eq) was added to a solution of 13 (29.5 g, 57 mmol) in THF (60 mL) while maintaining the batch temperature at < -65 °C. After stirring at -78 °C for 2 hours, additional LiHMDS (5.7 mL, 0.1 eq) was added to consume the remaining starting material. After stirring at -78 °C for 30 minutes, the tan reaction mixture was warmed to room temperature. After stirring at room temperature for one hour, the solution of silylated amine was added via cannula to a solution of HOBT ester of 2-thienylacetic acid in acetonitrile at 0 °C (the solution of HOBT ester was prepared by adding EDCI (16.39 g, 85.5 mmol, 1.5 eq) to a suspension of recrystallized 2-thienylacetic acid (9.73 g, 68.4 mmol, 1.2 eq) and HOBT.H20 (11.35 g, 74.1 mmol, 1.3 eq) in acetonitrile (10 mL) at 0 °C. The clear solution was stirred at 0 °C for 30 minutes prior to the addition of the silylated amine). After stirring at 0 °C for one hour, the heterogeneous reaction mixture was placed in the fridge overnight. Saturated aqueous sodium bicarbonate (80 mL) and heptane (80 mL) were added, and after stirring 30 minutes at room temperature, the layers were separated. The organic layer was washed with saturated aqueous sodium bicarbonate (2 x 80 mL) and filtered through Celite. The filtrate was concentrated under reduced pressure and the tan oil was azeotroped with heptane (3 x 1 10 mL). The residue was taken up in heptane (60 mL) and seeds were added. After stirring at room temperature for one hour, the reaction mixture became heterogeneous. After stirring 4 hours at 0 °C, the solids were collected by filtration and washed with ice cold heptane (3 x 20 mL), air dried then dried under high vacuum to give 14 as an off white powder (10.95 g, 31%).
Synthesis of (15)
[0199] A mixture of 14 (10 g, 16.1 mmol), boric acid (1.3 g, 20.9 mmol, 1.3 eq), dioxane (20 mL), and 1 M sulfuric acid (10 mL) was heated at 75 °C. After stirring 7 hours at 75 °C, the cooled reaction mixture was diluted with water (10 mL) and MTBE (30 mL) and the residual mixture was cooled to 0 °C. The pH was adjusted to 5.0 with a solution of 2 N NaOH (14 mL). The layers were separated and the aqueous layer was extracted with MTBE (2 x 30 mL) then concentrated to dryness. The residue was taken up in water (10 mL) and the solution was filtered through a 0.45 μηι GMF syringe filter. The flask and filter were rinsed with water (7.5 mL). The pH of the filtrate was lowered to 4.0 with 2 M H2SO4 and seeds (5 mg) were added. After stirring at room temperature for 10 minutes, the pH was lowered to 1.9 over 1 hour with 2 M H2S04 (total volume 3.5 mL). After stirring at room temperature for 2 hours, the solids were collected by filtration. The filtrate was recirculated twice to rinse the flask and the cake was washed with water (2 x 12 mL), air dried then dried under high vacuum to give 15 as a white powder (3.63 g, 76%).
PAPER
Journal of Medicinal Chemistry (2015), 58(9), 3682-3692
Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases
The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the β-lactam class of antimicrobials. A program was initiated to discover a new series of serine β-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine β-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiellapneumoniaecarbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.
The U.S. Food and Drug Administration today approved Vabomere for adults with complicated urinary tract infections (cUTI), including a type of kidney infection, pyelonephritis, caused by specific bacteria. Vabomere is a drug containing meropenem, an antibacterial, and vaborbactam, which inhibits certain types of resistance mechanisms used by bacteria.
The U.S. Food and Drug Administration today approved Vabomere for adults with complicated urinary tract infections (cUTI), including a type of kidney infection, pyelonephritis, caused by specific bacteria. Vabomere is a drug containing meropenem, an antibacterial, and vaborbactam, which inhibits certain types of resistance mechanisms used by bacteria.
“The FDA is committed to making new safe and effective antibacterial drugs available,” said Edward Cox, M.D., director of the Office of Antimicrobial Products in the FDA’s Center for Drug Evaluation and Research. “This approval provides an additional treatment option for patients with cUTI, a type of serious bacterial infection.”
The safety and efficacy of Vabomere were evaluated in a clinical trial with 545 adults with cUTI, including those with pyelonephritis. At the end of intravenous treatment with Vabomere, approximately 98 percent of patients treated with Vabomere compared with approximately 94 percent of patients treated with piperacillin/tazobactam, another antibacterial drug, had cure/improvement in symptoms and a negative urine culture test. Approximately seven days after completing treatment, approximately 77 percent of patients treated with Vabomere compared with approximately 73 percent of patients treated with piperacillin/tazobactam had resolved symptoms and a negative urine culture.
The most common adverse reactions in patients taking Vabomere were headache, infusion site reactions and diarrhea. Vabomere is associated with serious risks including allergic reactions and seizures. Vabomere should not be used in patients with a history of anaphylaxis, a type of severe allergic reaction to products in the class of drugs called beta-lactams.
To reduce the development of drug-resistant bacteria and maintain the effectiveness of antibacterial drugs, Vabomere should be used only to treat or prevent infections that are proven or strongly suspected to be caused by susceptible bacteria.
Vabomere was designated as a qualified infectious disease product (QIDP). This designation is given to antibacterial products that treat serious or life-threatening infections under the Generating Antibiotic Incentives Now (GAIN) title of the FDA Safety and Innovation Act. As part of its QIDP designation, Vabomere received a priority review.
The FDA granted approval of Vabomere to Rempex Pharmaceuticals.
Image may be NSFW. Clik here to view.Moxalactam synthesis
It has an azoloazine base structure, which represents a new structural class of non-nucleoside antiviral drugs.[1]
It was originally developed as a potential treatment for pandemic influenza strains such as H5N1, and most of the testing that has been done has focused on its anti-influenza activity.[2][3][4]
Yunona Holdings, was investigating riamilovir sodium dihydrate (triazavirin), a novel nucleoside inhibitor of human influenza virus A and B replication, for the potential oral treatment of influenza virus infection.
In November 2009, the company was seeking to outlicense the drug for development in the EU, presumed to be for use as a prescription medicine .
Negative-sense, single-stranded RNA viruses (ssRNA), such as ssRNA viruses belonging to the Order Mononegavirales such as viruses in the Rhabdoviridae family, in particular the Rabies virus, the Filoviridae family, in particular the Ebolavirus, and the Paramyxoviridae family, in particular the Measles virus, other ssRNA viruses belonging to unassigned families such as notably the
Arenaviridae family, the Bunyaviridae family and the Orthomyxoviridae family and other unassigned ssRNA viruses such as notably the Deltavirus, cause many diseases in wildlife, domestic animals and humans. These ssRNA viruses belonging to different families are genetically and antigenically diverse, exhibiting broad tissue tropisms and a wide pathogenic potential.
For example, the Filoviridae viruses belonging to the Order
Mononegavirales, in particular the Ebolaviruses and Marburgviruses, are among the most lethal and most destructive viruses in the world. Filoviridae viruses are of particular concern as possible biological weapons since they have the potential for aerosol dissemination and weaponization.
The Ebolavirus includes five species: the Zaire, Sudan, Reston, Ta‘i Forest and Bundibugyo Ebolaviruses. In particular the Zaire, Sudan and Bundibugyo Ebolavirus cause severe, often fatal, viral hemorraghic fevers in humans and nonhuman primates.
For more than 30 years, the Ebolavirus has been associated with periodic episodes of hemorrhagic fever in Central Africa that produce severe disease in
infected patients. Mortality rates in outbreaks have ranged from 50% for the Sudan species of the Ebolavirus to up to 90% for the Zaire species of the Ebolavirus ((Sanchez et al., Filoviridae: Marburg and Ebola Viruses, in Fields Virology, pages 1409-1448 (Lippincott Williams & Wilkins, Philadelphia)). In November 2007, during an outbreak in the Bundibugyo district of Uganda, near the border with the Democratic Republic of the Congo the fifth species of the Ebolavirus was discovered, the Bundibugyo species. Said outbreak resulted in a fatality rate of about 25% (Towner et al., PLoS Pathog., 4(11 ) :e1000212 (2008)). The Zaire species of the Ebolavirus has also decimated populations of wild apes in this same region of Africa (Walsh et al., Nature, 422:611-614 (2003)).
When infected with the Ebolavirus, the onset of illness is abrupt and is characterized by high fever, headaches, joint and muscle aches, sore throat, fatigue, diarrhea, vomiting, and stomach pain. A rash, red eyes, hiccups and internal and external bleeding may be seen in some patients. Within one week of becoming infected with the virus, most patients experience chest pains and multiple organ failure, go into shock, and die. Some patients also experience blindness and extensive bleeding before dying.
Another example of a negative sense single-stranded RNA envelope virus is the Morbilllivirus such as the Measles virus which is associated with Measles and the Lyssavirus such as the Rabies virus.
The Lyssavirus, belonging to the family Rhabdoviridae, includes eleven recognized species, in particular the Rabies virus which is known to cause Rabies. Rabies is an ancient disease with the earliest reports possibly dated to the Old World before 2300 B.C and remains a world health threat due to remaining lack of effective control measures in animal reservoir populations and a widespread lack of human access to vaccination. The Rabies virus is distributed worldwide among mammalian reservoirs including carnivores and bats. Each year there are many reported cases of transmission of the Rabies virus from animals to humans (e.g. by an animal bite). More than 50,000 people annually die of Rabies, particularly in Asia and Africa.
Thus, there remains a need for antiviral compounds which are effective for use in the treatment of the ssRNA virus infections different from the Influenza A and Influenza B virus infections
SYNTHESIS CONTRUCTED WITH 3 ARTICLES AS BELOW
RU 2340614 C2 20081210,
e-EROS Encyclopedia of Reagents for Organic Synthesis, 1-7; 2009,
European Journal of Medicinal Chemistry, 113, 11-27; 2016
Reaction of 3-nitro- and 3-ethoxycarbonyl-1,2,4-triazolo[5,1-c]-1,2,4-triazin-4-ones with 1-adamantanol (or 1-adamantyl nitrate) in concentrated sulfuric acid or with 1-bromoadamantane in sulfolane affords N-adamantyl derivatives. The adamantylation of 3-nitro-1,4-dihydro-7H-1,2,4-triazolo[5,1-c]-1,2,4-triazin-4-one yields a mixture of N8– and N1-isomers that undergo interconversion in concentrated sulfuric acid along intermolecular mechanism.
Nucleoside analogs containing hydroxybutyl, hydroxyethoxymethyl, allyloxymethyl, and propargyloxymethyl fragments were synthesized based on 1,2,4-triazolo[3,2-c][1,2,4]triazin-7-ones isosteric to purine bases. Some of the compounds obtained inhibit in vitro reproduction of influenza and respiratory syncytial virus infection.
The nucleophilic susbstitution of nitro group in [1,2,4]triazolo[5,1-c][1,2,4]triazinones upon treatment with cysteine and glutathione was studied as a model for the interaction with thiol groups of virus proteins, which mimics the metabolic transformations of antiviral drug Triazavirin® and its derivatives.
Step 1 : Diazotization of compound (B): A solution (solution [1], herein after) was prepared of 5.8 g (0.05 ) of 5-amino-3-mercapto-1 ,2,4-triazole in 6.7 ml of nitric acid (15 M) and 12 ml of water. Said solution [1] was refrigerated to -7°C . Then a 40% sodium nitrite solution was added to the solution [1] in portions of 0.5 mL to obtain a total amount of sodium nitrite equal to 3.8 g in the mixture.
Step 2: Condensation of diazonium compound with an a-nitroester:
To the resulting diazonium salt of step 1 , 8.54 ml of diethyl nitromaionate was added. After holding for five minutes, a cooled solution of sodium hydroxide was slowly added dropwise to the reaction mixture until the pH was between pH 9 and pH 10 (solution [2], herein after). The resulting solution [2] was stirred at 0°C for 1 hour and at room temperature for 2 hours.
Step 3: alkylation: To the solution [2] of step 2, 6.23 ml (0.1 moi) of methyl iodide was added. The mixture was stirred for 1 hour at room temperature and filtered. The resulting precipitate was successively crystallized from water and dried in air. The reaction scheme is depicted below in Scheme 1.
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SCHEME 1
The yield was 9.87 g (69%).
Physical and chemical characteristics of the sodium salt of 7-methylthio-3-nitro
[1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one sodium salt: yellow crystalline powder, soluble in water, acetone, dimethylsulfoxide, dimethylformamide. insoluble in chloroform; Tmelt = 300°C, H NMR spectrum, δ, ppm, solvent DMSO-d6: 2.62 (3H, s, SCH3); IR spectrum, n, cm“1: 3535 (OH), 1649 (C=0), 1505 (N02), 1367 (N02); found.. %: C – 20.86, H 2.51 , N 29.28;
C5H;N6Na05S; Calculated, %: C – 20.98, H 2.47, N 29.36.
Example 2: Synthesis of the sodium salt of 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one sodium salt
In this example the synthesis comprises 3 steps: in the first step 5-amino-3-mercapto-1.2,4-triazole (i.e. compound (B)) was prepared by condensation of aminoguanidine with a thio-derivative (thio ester) of formic acid, HC(=0)S-R, wherein -R was: methyl. In the second step 5-amino-3-mercapto-1 , 2,4-triazole was converted to the corresponding diazonium salt. In the third step this diazonium salt was reacted with an a-nitroester, 2-nitroacetoacetic ester, to form the 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one. The different steps are explained in more detail below.
Step 1 : Synthesis of compound (B): In a reaction flask equipped with a stirrer, reflux condenser, under inert gas (nitrogen, argon), 20 g (0.1 mol) of aminoguanidine and 7.6 g (0.1 mol) methylthio-formate was added to 400 ml of absolute pyridine. The reaction mixture was boiled for 4 hours at 115°C.
Subsequently the reaction mixture was transferred into distilled water and washed several times with water. The washed mixture was dried over a Nutsche filter under vacuum. Recrystallization was carried out from ethanol. The reaction scheme is depicted below in Scheme 2.
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SCHEME 2
The yield was 19.3 g (70%)
Step 2: Diazotation of compound (B): A solution (solution [3], herein after) was prepared of 26 g (0.1 mol) of 5-amino-3-mercapto-1 ,2,4-triazole (as obtained in step 1) in 32 ml of nitric acid (0.1 mol) and 200 ml of water. The solution was mixed and cooled to -5°C. In a separate recipient, a 0.1 M solution of sodium nitrite was prepared by dissolving 16 g of sodium nitrite in 100 ml of water. The sodium nitrite solution was put in the freezer until there was ice formation and subsequently the ice was crushed. Thereafter, the solution [3] and the sodium nitrite crushed ice were transferred into a 1 L reactor and stirred for 1 hour while the reactor temperature was kept at 0°C. The low temperature and the fact that the two reaction components are in different phases (i.e. liquid and solid) ensured a slow gradual progress of diazotization reaction at the phase interface. The end of the diazotization process was controlled by a iodine starch test (proof of the absence of sodium nitrite in a free state).
The rea
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SCHEME 3
Step 3: Condensation of the diazonium compound with an α-nitroester: A solution (solution [4], herein after) was prepared by mixing 17.5 g of methyl 2- nitro-acetoacetate in 300 mL of isopropanoi. The solution [4] was mixed with the diazonium salt of step 2. The mixture was cooled to 0°C. At 0°C, a 10% sodium hydroxide solution was added to the reaction mixture (to neutralize residual nitrite and acetate) until there was a marked alkaline reaction (pH between 8 and 9). The temperature was controlled and was kept below +5°C. The resulting mixture was stirred for 1 hour. The precipitate was filtered off and dried in air. The yield was 78%.
The reaction scheme is depicted in Scheme 4
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SCHEME 4
Example 3: Synthesis of the sodium salt of 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one
The synthesis of the sodium salt of 7-methylthio-3-nitro-1 ,2,4-triazolo [5,1-c]-1 ,2,4-triazin-7-one may be carried out as in Example 2, only in step 2 the aqueous alcohol solution is replaced by an alcohol with alkali (such as sodium hydroxide). The yield of the antiviral compound (A) (sodium salt of 7-methylthio-3-nitro [1 2, 4] triazolo [5,1-c] [1 , 2. 4] triazin -4 (1 H)-one) may increase to 83%. The reaction scheme is depicted below in Scheme 5:
Process for the preparation of 7-thio-substituted-3-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazin-4(1H)-one i.e. riamilovir sodium dihydrate is claimed. Also claimed are use of triazolo compounds for the treatment of ssRNA virus infections such as Zika virus and flavivirus, ssRNA viruses different from the Influenza A and Influenza B viruses and compositions comprising them. Along with concurrently published WO2017144709 claiming similar derivatives. Represents new area of interest from Doring International Gmbh and the inventors on this moiety.
Example 1 : One pot synthesis of the sodium salt of 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1 -cj [1, 2, 4] triazin -4 (1H)-one
Step 1: Diazotization of compound (B): A solution (solution [1], herein after) was prepared of 5.8 g (0.05 M) of 5-amino-3-mercapto-1 ,2,4-triazole in 6.7 ml of nitric acid (15 M) and 12 ml of water. Said solution [1] was refrigerated to -7°C . Then a 40% sodium nitrite solution was added to the solution [1] in portions of 0.5 mL to obtain a total amount of sodium nitrite equal to 3.8 g in the mixture.
Step 2: Condensation of diazonium compound with an a-nitroester: To the resulting diazonium salt of step 1 , 8.54 ml of diethyl nitromalonate was added. After holding for five minutes, a cooled solution of sodium hydroxide was slowly added dropwise to the reaction mixture until the pH was between pH 9 and pH 10 (solution [2], herein after). The resulting solution [2] was stirred at 0°C for 1 hour and at room temperature for 2 hours.
Step 3: alkylation: To the solution [2] of step 2, 6.23 ml (0.1 mol) of methyl iodide was added. The mixture was stirred for 1 hour at room temperature and
filtered. The resulting precipitate was successively crystallized from water and dried in air. The reaction scheme is depicted below in Scheme 1.
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SCHEME 1
The yield was 9.87 g (69%).
Physical and chemical characteristics of the sodium salt of 7-methylthio-3-nitro
[1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one sodium salt: yellow crystalline powder, soluble in water, acetone, dimethylsulfoxide, dimethylformamide, insoluble in chloroform; Tmei, = 300°C, 1H NMR spectrum, δ, ppm, solvent DMSO-d6: 2.62 (3H, s, SCH3); IR spectrum, n, cm“1: 3535 (OH), 1649 (CO), 1505 (N02), 1367 (N02); found, %: C – 20.86, H 2.51 , N 29.28; C5H7N6Na05S; Calculated, %: C – 20.98, H 2.47, N 29.36.
Example 2: Synthesis of the sodium salt of 7-methylthio-3-nitro [1, 2,
In this example the synthesis comprises 3 steps: in the first step 5-amino-3-mercapto-1 ,2,4-triazole (i.e. compound (B)) was prepared by condensation of aminoguanidine with a thio-derivative (thio ester) of formic acid, HC(=0)S-R, wherein -R was: methyl. In the second step 5-amino-3-mercapto-1 ,2,4-triazole was converted to the corresponding diazonium salt. In the third step this diazonium salt was reacted with an a-nitroester, 2-nitroacetoacetic ester, to form the 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one. The different steps are explained in more detail below.
Step 1 : Synthesis of compound (B): In a reaction flask equipped with a stirrer, reflux condenser, under inert gas (nitrogen, argon), 20 g (0.1 mo!) of
aminoguanidine and 7.6 g (0.1 mol) methylthio-formate was added to 400 ml of absolute pyridine. The reaction mixture was boiled for 4 hours at 115°C. Subsequently the reaction mixture was transferred into distilled water and washed several times with water. The washed mixture was dried over a Nutsche filter under vacuum. Recrystallization was carried out from ethanol. The reaction scheme is depicted below in Scheme 2.
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SCHEME 2
The yield was 19.3 g (70%)
Step 2: Diazotation of compound (B): A solution (solution [3], herein after) was prepared of 26 g (0.1 mol) of 5-amino-3-mercapto-1 ,2,4-triazole (as obtained in step 1 ) in 32 ml of nitric acid (0.1 mol) and 200 ml of water. The solution was mixed and cooled to -5°C. In a separate recipient, a 0.1 M solution of sodium nitrite was prepared by dissolving 16 g of sodium nitrite in 100 ml of water. The sodium nitrite solution was put in the freezer until there was ice formation and subsequently the ice was crushed. Thereafter, the solution [3] and the sodium nitrite crushed ice were transferred into a 1 L reactor and stirred for 1 hour while the reactor temperature was kept at 0°C. The low temperature and the fact that the two reaction components are in different phases (i.e. liquid and solid) ensured a slow gradual progress of diazotization reaction at the phase interface. The end of the diazotization process was controlled by a iodine starch test (proof of the absence of sodium nitrite in a free state).
The rea
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SCHEME 3
Step 3: Condensation of the diazonium compound with an a-nitroester: A solution (solution [4], herein after) was prepared by mixing 17.5 g of methyl 2-nitro-acetoacetate in 300 mL of isopropanol. The solution [4] was mixed with the diazonium salt of step 2. The mixture was cooled to 0°C. At 0°C, a 10% sodium hydroxide solution was added to the reaction mixture (to neutralize residual nitrite and acetate) until there was a marked alkaline reaction (pH between 8 and 9). The temperature was controlled and was kept below +5°C. The resulting mixture was stirred for 1 hour. The precipitate was filtered off and dried in air. The yield was 78%.
The reaction scheme is depicted in Scheme 4
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SCHEME 4
Example 3: Synthesis of the sodium salt of 7-methylthio-3-nitro [1, 2, 4] triazolo [5,1-c] [1, 2, 4] triazin -4 (1H)-one
The synthesis of the sodium salt of 7-methy I th io-3-nitro- 1 ,2, 4-triazolo [5,1-c]-1 ,2,4-triazin-7-one may be carried out as in Example 2, only in step 2 the aqueous alcohol solution is replaced by an alcohol with alkali (such as sodium hydroxide). The yield of the antiviral compound (A) (sodium salt of 7-methylthio-3-nitro [1 2, 4] triazolo [5,1-c] [1 , 2, 4] triazin -4 (1 H)-one) may increase to 83%.
The reaction scheme is depicted below in Scheme 5:
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SCHEME 5
References
Jump up^Rusinov VL, Sapozhnikova IM, Ulomskii EN, Medvedeva NR, Egorov VV, Kiselev OI, Deeva EG, Vasin AV, Chupakhin ON. Nucleophilic substitution of nitro group in nitrotriazolotriazines as a model of potential interaction with cysteine-containing proteins. Chemistry of Heterocyclic Compounds 2015;51(3):275-280. doi 10.1007/s10593-015-1695-4
Jump up^Loginova SIa, Borisevich SV, Maksimov VA, Bondarev VP, Kotovskaia SK, Rusinov VL, Charushin VN. Investigation of triazavirin antiviral activity against influenza A virus (H5N1) in cell culture. (Russian) Antibiotiki i Khimioterapiia. 2007;52(11-12):18-20. PMID19275052
Jump up^Karpenko I, Deev S, Kiselev O, Charushin V, Rusinov V, Ulomsky E, Deeva E, Yanvarev D, Ivanov A, Smirnova O, Kochetkov S, Chupakhin O, Kukhanova M. Antiviral properties, metabolism, and pharmacokinetics of a novel azolo-1,2,4-triazine-derived inhibitor of influenza A and B virus replication. Antimicrobial Agents and Chemotherapy. 2010 May;54(5):2017-22. doi: 10.1128/AAC.01186-09 PMID20194696
Jump up^Kiselev OI, Deeva EG, Mel’nikova TI, Kozeletskaia KN, Kiselev AS, Rusinov VL, Charushin VN, Chupakhin ON. A new antiviral drug Triazavirin: results of phase II clinical trial. (Russian). Voprosy Virusologii. 2012 Nov-Dec;57(6):9-12. PMID23477247
Jump up^Loginova SIa, Borisevich SV, Rusinov VL, Ulomskiĭ UN, Charushin VN, Chupakhin ON. Investigation of Triazavirin antiviral activity against tick-borne encephalitis pathogen in cell culture. (Russian). Antibiotiki i Khimioterapiia. 2014;59(1-2):3-5. PMID25051708