Hydrocodone bitartrate is morphinan-6-one, 4,5-epoxy-3-methoxy-17-methyl-, (5α)-, [R-(R*,R*)]-2,3-dihydroxybutanedioate (1:1), hydrate (2:5); also known as 4,5α-Epoxy-3-methoxy-17-methylmorphinan-6-one tartrate (1:1) hydrate (2:5); a fine white crystal or crystalline powder, which is derived from the opium alkaloid, thebaine; and may be represented by the following structural formula:

Hydrocodone Bitartrate
C₁₈H₂₁N0₃•C₄H₆0₆•2.5 H₂0
Molecular weight = 494.5

Purdue’s hydrocodone bitartrate tablets granted priority review designation

Purdue Pharma has been granted priority review designation by the US Food and Drug Administration (FDA) for its hydrocodone bitartrate extended-release tablets for treatment of chronic pain.

The once-daily, single-entity pain medication was formulated to incorporate abuse-deterrent properties designed to make the product more difficult to manipulate for misuse or abuse by various routes of administration.http://www.pharmaceutical-technology.com/news/newspurdues-hydrocodone-bitartrate-tablets-granted-priority-review-designation-4313765?WT.mc_id=DN_News

SORAFENIB

SYNTHESIS http://newdrugapprovals.org/2014/06/26/bayer-healthcare-has-obtained-approval-from-the-japanese-ministry-of-health-labour-and-welfare-mhlw-for-its-nexavar-sorafenib-for-treatment-of-patients-with-unresectable-differentiated-thyroid-ca/

Bayer receives Canadian approval for Nexavar to treat differentiated thyroid cancer

Health Canada has approved Bayer’s Nexavar (sorafenib tablets) for treatment of patients with locally advanced or metastatic, progressive, differentiated (papillary/follicular/Hurthle cell) thyroid carcinoma, refractory to radioactive iodine.

Nexavar’s approval in Canada is supported by a positive outcome from the Phase III DECISION (‘stuDy of sorafEnib in loCally advanced or metastatIc patientS with radioactive Iodine refractory thyrOid caNcer’) trial.

http://www.pharmaceutical-technology.com/news/newsbayer-receives-canadian-approval-nexavar-treat-differentiated-thyroid-cancer-4313077?WT.mc_id=DN_News

FOOTBALL BRAZIL 2014

Glenmark Generics receives final ANDA approval for Telmisartan Tablets

Mumbai, India, July 8, 2014

Glenmark Generics Inc., USA a subsidiary of Glenmark Generics Limited has been granted final abbreviated new drug approval (ANDA) from the United States Food and Drug Administration (US FDA) for Telmisartan Tablets. Glenmark will commence distribution of the product immediately.
Telmisartan Tablets are Glenmark’s generic version of Boehringer Ingelheim’s Micardis®. Telmisartan is indicated for the treatment of hypertension.

The approval is for the 20mg, 40mg and 80mg tablets. For the 12 month period ending March 2014, Telmisartan garnered annual sales of USD 250 Million according to IMS Health.

http://www.business-standard.com/content/b2b-pharma/glenmark-receives-usfda-approval-for-telmisartan-tablets-114070900982_1.html

Glenmark receives USFDA approval for telmisartan tablets

Telmisartan, which is the generic version of Boehringer Ingelheim’s Micardis, garnered annual sales of $ 250 million for the 12 month period ending March 2014

By Frederic L. “Rick” Sax, M.D., global head for the Center for Integrated Drug Development, Quintiles.

The biopharmaceutical manufacturing industry has used quality by design (QbD) principles for decades. The essence of QbD is designing with the end in mind (in this case, the efficient manufacture of a high-quality drug product). This approach emphasizes that the operative word in QbD is not quality, but design.

read all at

http://www.pharmaceuticalonline.com/doc/how-to-apply-qbd-principles-in-clinical-trials-0001

\

http://www.google.nl/patents/US8173841

XEN-D0501

Xention (Originator)

XEN-D0501, a novel TRPV1 antagonist, is being developed to treat overactive bladder.

in phase 2 Chronic obstructive pulmonary disease (COPD)

Ario Kicks Off Efficacy Trial of Chronic Idiopathic Cough Drug
Ario Pharma Ltd, the biopharmaceutical company developing innovative new treatments for respiratory disease, announced that it has commenced a Phase 2a study of its oral TRPV1 antagonist, XEN-D0501, for the treatment and prevention of cough in patients with chronic idiopathic cough (CIC).http://www.dddmag.com/news/2014/07/ario-kicks-efficacy-trial-chronic-idiopathic-cough-drug?et_cid=4039308&et_rid=523035093&type=cta

Letrozole boosts fertility in women with PCOS, says study
Penn State College of Medicine’s nationwide study showed that Letrozole resulted in higher birth rates in women with polycystic ovary syndrome (PCOS) than the current preferred infertility treatment drug, Clomiphine citrate. http://www.pharmaceutical-technology.com/news/newsletrozole-boosts-fertility-women-pcos-says-study-4314880?WT.mc_id=DN_News

Letrozole (INN, trade name Femara) is an oral non-steroidal aromatase inhibitor for the treatment of hormonally-responsive breast cancer after surgery.

Uses

FDA-approved use

Femara 2.5 mg oral tablet

Letrozole is approved by the United States Food and Drug Administration (FDA) for the treatment of local or metastatic breast cancer that is hormone receptor positive or has an unknown receptor status in postmenopausal women.^[2]

4-[alpha (4-cyanophenyl)-l-(l,2,4-triazoly)-methyl]- benzonitrile

Systematic (IUPAC) name
4,4′-((1H-1,2,4-triazol-1-yl)methylene)dibenzonitrile
Clinical data
Trade names	Femara
AHFS/Drugs.com	monograph
MedlinePlus	a698004
Licence data	US FDA:link
Pregnancy cat.	D (US)
Legal status	Schedule VII (CA) POM (UK) ℞-only (US)
Routes	Oral
Pharmacokinetic data
Bioavailability	99.9%
Protein binding	60%, mainly to albumin
Metabolism	pharmacologically-inactive carbinol metabolite (4,4΄-methanol-bisbenzonitrile)[1]
Half-life	2 days[1]
Excretion	Kidneys[1]
Identifiers
CAS number	112809-51-5
ATC code	L02BG04
PubChem	CID 3902
DrugBank	DB01006
ChemSpider	3765
UNII	7LKK855W8I
KEGG	D00964
ChEBI	CHEBI:6413
ChEMBL	CHEMBL1444
Chemical data
Formula	C17H11N5
Mol. mass	285.303 g/mol

Off-label uses

Letrozole has been used for ovarian stimulation by fertility doctors since 2001 because it has fewer side-effects than clomiphene (Clomid) and less chance of multiple gestation. A Canadian study presented at the American Society of Reproductive Medicine 2005 Conference suggests that letrozole may increase the risk of birth defects. A more detailed ovulation induction follow-up study found that letrozole, compared with a control group of clomiphene, had significantly lower congenital malformations and chromosomal abnormalities at an overall rate of 2.4% (1.2% major malformations) compared with clomiphene 4.8% (3.0% major malformations).^[3] Despite this, India banned the usage of letrozole in 2011, citing potential risks to infants.^[4] In 2012, an Indian parliamentary committee said that the drug controller office colluded with letrozole’s makers to approve the drug for infertility in India and also stated that letrozole’s use for infertility was illegal worldwide;^[5] however, such off-label uses are legal in many countries such as the US and UK.^[6][7]

The anti-estrogen action of letrozole has been shown to be useful in pretreatment for termination of pregnancy, in combination with misoprostol. It can be used in place of mifepristone, which is expensive and unavailable in many countries.^[8]

Letrozole is sometimes used as a treatment for gynecomastia, although it is probably most effective at this if caught in an early stage (such as in users of anabolic steroids).^[9][10]

Some studies have shown that letrozole can be used to promote spermatogenesis in male patients suffering from nonobstructive azoospermia.^[11]

Letrozole has also been shown to delay the fusing of the growth plates in mice.^[12] When used in combination with growth hormone, letrozole has been shown effective in one adolescent boy with a short stature.^[13]

Letrozole has also been used to treat endometriosis.^[14]

Mechanism of action

Estrogens are produced by the conversion of androgens through the activity of the aromatase enzyme. Estrogens then bind to an estrogen receptor, which causes cells to divide.

Letrozole prevents the aromatase from producing estrogens by competitive, reversible binding to the heme of its cytochrome P450 unit. The action is specific, and letrozole does not reduce production of mineralo- or corticosteroids.

Contraindications

Letrozole is contraindicated in women having a pre-menopausal hormonal status, during pregnancy and lactation.^[15]

Adverse effects

The most common side effects are sweating, hot flashes, arthralgia (joint pain), and fatigue.^[15]

Generally, side effects include signs and symptoms of hypoestrogenism. There is concern that long term use may lead to osteoporosis,^[2] which is in certain patient populations such as post-menopausal women or osteoporotics, bisphosphonates may also be prescribed.

Interactions

Letrozole inhibits the liver enzyme CYP2A6, and to a lesser extent CYP2C19, in vitro, but no relevant interactions with drugs like cimetidine and warfarin have been observed.^[15]

Comparison with tamoxifen

Tamoxifen is also used to treat hormonally-responsive breast cancer, but it does so by interfering with the estrogen receptor. However, letrozole is effective only in post-menopausal women, in whom estrogen is produced predominantly in peripheral tissues (i.e. in adipose tissue, like that of the breast) and a number of sites in the brain.^[16] In pre-menopausal women, the main source of estrogen is from the ovaries not the peripheral tissues, and letrozole is ineffective.

In the BIG 1–98 Study, of post-menopausal women with hormonally-responsive breast cancer, letrozole reduced the recurrence of cancer, but did not change survival rate, compared to tamoxifen.^[17][18]

Synthesis

Letrozole can by synthesized from 4-cyanobenzyl bromide, triazole, and 4-fluorobenzonitrile:^[19]

………………………………………..

http://www.google.com/patents/EP2212301B1?cl=en

• Letrozole, chemically known as 4-[alpha (4-cyanophenyl)-1-(1,2,4-triazoly)-methyl]-benzonitrile, and represented by formula (I),

  is a therapeutically and commercially important non-steroidal aromatase inhibitor, which is widely used for adjuvant treatment of hormonally responsible breast cancer in postmenopausal women. Estrogens are produced by the conversion of androgen through the activity of aromatase enzyme, the suppression of estrogen biosynthesis in peripheral tissues and in the cancer tissue itself can therefore be achieved by specifically inhibiting the aromatase enzyme.

• [0003]
  1. Bowman et al. were the first to disclose Letrozole in US 4,978,672 , and US 5,352,795 and reported two methods for synthesis of Letrozole, the chemistry for Method-1 is summarized in Scheme-1.
• [0004]
  The Method-1 for synthesis of Letrozole as disclosed by Bowman et al. in US 4,978,672 , and US 5,352,795 and as summarized in Scheme-1, comprises reaction of alpha-bromo-4 tolunitrile or 4-bromomethyl benzonitrile (II) with 1H-1, 2,4-triazole (III), in a mixture of chloroform and acetonitrile as solvent at reflux temperature for 15 hours to give 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV), which on reaction with 4-flurobenzonitrile (VI) in the presence of potassium t-butoxide and in N, N-dimethylformamaide, gives crude Letrozole (I), which is recrystallized from 95% ethanol or a mixture of ether and ethyl acetate to give pure Letrozole (I):
• [0005]
  As would be evident from Examples 9, 25, and 26 of US 4,978,672 , and US 5,352,795 , in the step reaction of alpha-bromo-4 tolunitrile or 4-bromomethyl benzonitrile (II) with 1H-1, 2,4-triazole (III), as per Method-1, Scheme-I, in addition to the desired 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) an appreciable amount of isomeric 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V) is also formed in the reaction, which necessitates separation of the two isomers by column chromatography, subsequent to which the separated pure 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) is reacted with 4-flurobenzonitrile (VI) to give Letrozole. Example 25 of US 4,978,672 , and US 5,352,795 further report that Letrozole obtained after recrystalization from 95% ethanol has a melting point of 181° – 183°C, while Example 26 reports that Letrozole obtained after recrystalization from a mixture of ether and ethyl acetate has a melting point of 184°- 185° C.
• [0006]
  The major disadvantage and limitation of the Method-1 disclosed in US 4,978,672 , and US 5,352,795 is that it leads to formation of appreciable amounts of the unwanted isomer i.e. 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V), calling for tedious chromatographic techniques for its separation from the desired isomer i.e. 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV), which is expected to result in considerable loss and low yield of the desired isomer. Such a method, obviously, cannot be expected to be economically or commercially viable. Further, nowhere in the Specifications and Experimental Descriptions of US 4,978,672 , and US 5,352,795 there is any mention about the yield and purity of Letrozole obtained by the method described therein.
• [0007]
  The second method, Method-2, reported by Bowman et al. in US 4,978,672 , and US 5,352,795 is summarized in Scheme-II, which comprises of reaction of N-tert.butyl-4-bromo benzamide (1) with n-butyllithium and ethyl formate to give Bis- (4-N-tert.butyl carbamoylphenyl) methanol (2), which on reaction with thionyl chloride gives 4-(alpha-chloro-4′cyanobenzyl)benzonitrile (3). Reaction of 4-(alpha-chloro-4′cyanobenzyl) benzonitrile (3) with 1H-1,2,4-triazole (III) gives Letrozole (I).
• [0008]
  The major disadvantage and limitation of the Method-2 disclosed in US 4,978,672 , and US 5,352,795 , as evident from Examples 3, 5 and 28, described therein, is that first of all it utilizes corrosive and hazardous n-butyllithium and thionyl chloride; which require special storage, handling and disposal as well as calls for cryogenic temperatures of -60° C and higher temperatures of about 160° C, which collectively renders the method unsafe and industrially and commercially not of particular viability. Further, as in the case of Method-1, nowhere in the Specifications and Experimental Descriptions of US 4,978,672 , and US 5,352,795 there is any mention about the yield and purity of Letrozole obtained by the Method-2 described therein. Furthermore, the reaction of 4-(alpha-chloro-4′cyanobenzyl) benzonitrile (3) with 1,2,4-triazole (III) would most likely result in formation of the corresponding isomer along with the desired Letrozole, which would involve tedious purification techniques for its separation.
• [0009]
  Improvements over the methods disclosed by Bowman et al. in US 4,978,672 , and US 5,352,795 are the subject matter of the following reports, viz.
• [0010]
  2. Wadhwa et al. in US 2005/0209294 A1 , recite a method for synthesis of the intermediate 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV), comprising reaction of alpha-bromo-4 tolunitrile or 4-bromomethyl benzonitrile (II) with a salt of 1H-1,2,4-triazole, preferably an alkali metal salt of 1H-1,2,4-triazole (4), in a suitable solvent at a temperature of between 10° to 15° C, followed by crystallization of the isolated product. The chemistry is summarized in Scheme-III.
• [0011]
  Wadhwa et al. in US 2005/0209294 A1 , while stating that the method disclosed by Bowman et al. in US 4,978,672 , and US 5,352,795 is not selective in that it produces the undesired isomeric 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V) in about 50%, which as mentioned hereinbefore requires tedious chromatographic separation techniques for its removal, emphasize that by virtue of utilization of an alkali metal salt of 1H-1, 2,4-triazole (4), the desired 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) is obtained in >96% selectivity, thereby circumventing the utilization of tedious chromatographic techniques for its purification. Wadhwa et al., further state that the said intermediate i.e. 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV), obtained by their method can be converted to Letrozole of US Pharmacopoeial Quality, through conventional procedure.
• [0012]
  While the method disclosed by Wadhwa et al. in US 2005/0209294 A1 , reportedly affords the intermediate 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) in >96% selectivity and further, reportedly does away with chromatographic techniques in its isolation, however, the entire Specification and the Experimental Description given in Example-1 therein, is silent about the actual yield and purity of not only the intermediate 4-[1-(1,2,4-triazolyl) methyl]-benzonitrite (IV) but also that of Letrozole obtained by the method. The industrial or commercial viability of the method, therefore, cannot be commented, in view of insufficient disclosure.
• [0013]
  3. Kompella et al. in WO 2005/047269 A1 , disclose a method for separation of the Letrozole precursor, 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV) from its isomer, 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V), comprising treating a solution of the mixture of the two isomeric compounds (IV) and (V) in dichloromethane or chloroform with isopropylalcohol hydrochloride, followed by addition of isopropyl ether, wherein the hydrochloride salt of the undesired 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V) precipitates out, which is removed by filtration. Basification of the filtrate, followed by evaporation of solvent and isolation of the residue from hexane or petroleum ether affords the desired 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV). The method is summarized in Scheme-IV.
• [0014]
  The required isomer is obtained in 47-61 % yield and a purity of about 99%.
• [0015]
  4. In another variant of the Method-1 of Bowman et al., an improved regiospecific method disclosed by Patel et al. in US 2006/0128775 A1 for synthesis of Letrozole is summarized in Scheme-V.
• [0016]
  The method disclosed by Patel et al. in US 2006/0128775 A1 utilizes 4-amino-1, 2,4-triazole (5), instead of 1H-1, 2,4-triazole (III) or an alkali metal salt of 1H-1, 2,4-triazole (4), as utilized by Bowman et al. in US 4,978,672 , and US 5,352,795 and Wadhwa et al. in US 2005/0209294 A1 respectively, for reaction with alpha-bromo-4 tolunitrile or 4-bromomethyl benzonitrile (II) to give 4-[(4-amino-1,2,4-triazolium-1-yl)methyl]benzonitrile bromide (6), which on diazotisation leads to the required intermediate, 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV), further reaction of which with 4-flurobenzonitrile (VI) gives crude Letrozole, which is recrystallized from polar or non-polar solvents to give pure Letrozole (I).
• [0017]
  The method of Patel et al. in US 2006/0128775 A1 , in the first place provides an elegant regiospecific synthesis of Letrozole in that it like the method of Wadhwa et al. in US 2005/0209294 A1 , minimizes the formation of the undesired isomeric 4-[1-(1,3,4-triazolyl) methyl]-benzonitrile (V) and also does away with tedious chromatographic separation techniques.
• [0018]
  The method of Patel et al. in US 2006/0128775 A1 , albeit, as evident from Example-1, described therein, reportedly gives Letrozole of 99.90% HPLC purity, however, gives Letrozole of the said purity only in an overall yield of 34%, which renders it of not being an particularly economic process. Secondly, the method comprises of an additional step of deamination of the intermediate compound (6), which in turn calls for a diazotization step, through utilization of sodium nitrite, which is hazardous and explosive, more suitable to small scale preparations rather than industrial manufacture. The method, hence, might not be particularly amenable for industrial scale-up and manufacture.
• [0019]
  5. MacDonald et al. in US 2007/0066831 A1 , report another variant of the methods disclosed by Bowman et al. in US 4,978,672 , and US 5,352,795 and Wadhwa et al. in US 2005/0209294 A1 in that the said method, as summarized in Scheme-VI comprises:

  1. a) Reaction of alpha-bromo-4 tolunitrile or 4-bromomethyl benzonitrile (II) with an alkali metal salt of 1H-1,2,4-triazole (4), in presence of a solvent selected from the group consisting of diemthylacetamide, N-methyl-2-pyrrolididone, or a mixture thereof, at a temperature of about -20° to 0°C to give 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV);
  2. b) Extracting the impurities form intermediate compound (IV), in a two phase system, comprising an aqueous phase and a water-immiscible phase; and
  3. c) Reacting compound (IV) with 4-flurobenzonitrile (VI), in presence of a solvent selected from the group consisting of dimethylformamide, diemthylacetamide, N-methyl-2-pyrrolididone, and tetrahydrofuran or a mixture thereof and a base selected from sodium bis(trimethylsilyl)amide, hexyl lithium, butyl lithium, lithium didsopropylamide, alkoxide or mixtures thereof.
• [0020]
  US 2007/0066831 A1 further, states that the steps (a) and (b) could be combined together resulting in a one-pot synthesis of Letrozole.
• [0021]
  In the first place, it might be mentioned herein that the chemistry disclosed by Macdonald et al. in US 2007/0066831 A1 is a nominal variation of the method disclosed by Wadhwa et al. in US 2005/0209294 A1 , in that uses specific solvents such as diemthylacetamide, and N-methyl-2-pyrrolididone for formation of compound (IV) and again utilizes the same solvents for obtaining Letrozole from compound (IV), in addition to use of specific lithium containing bases, most of which are hazardous and expensive, requiring special precautions during storage, handling and disposal.
• [0022]
  6. In yet another variation, Radhakrishnan et al. in WO 2007/039912 provide a method for synthesis of Letrozole, as summarized in Scheme-VII, which is a one-pot synthesis comprising reaction of compounds (II) and (4) to give compound (IV), which without isolation and on further reaction with compound (VI) gives Letrozole.
• [0023]
  The major disadvantage with the method is that is still does not obliterate the use of chromatographic separation/purification of Letrozole.
• [0024]
  7. Haider et al. in WO 2007/054964 A2 provide an improvement, as summarized in Scheme- VIII, over Method-1 disclosed by Bowman et al. in US 4,978,672 and US 5,352.795 . in that the improvement comprises of selective removal of the isomeric 4-[1-(1,3,4-triazolyl)methyl]-benzonitrile (V), formed in the reaction of compound (II) and (III) in isopropanol as solvent, through a method of extraction, which provides the desired 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV), of >99% purity, and relatively free of the isomeric impurity (V).
• [0025]
  The method of extraction, as taught by Haider et al. in WO 2007/054964 A2 comprises repeated extraction of the reaction medium containing mixture of the desired 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV) and the undesired 4-[1-(1,3,4-triazolyl)methyl]-benzonitrile (V) with water and a water-immiscible solvent to afford the pure 4-[1-(1,2,4-triazolyl)methyl]-benzonitrile (IV) in the organic phase, which is then further converted to Letrozole (I) of >99% purity by conventional methods. Haider et al. also teach a process for conversion of the mixture of the desired 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) and the undesired 4-[1-(1,3,4-triazolyl)methyl]-benzonitrile (V) to Letrozole, from which the isomeric form of Letrozole i.e. Isoletrozole (9) so formed is removed by repeated crystallization to afford Letrozole (I) of >99% purity. -
• [0026]
  It might be noted that the method of Haider et al., primarily is one for purification of the intermediate 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV) as well as Letrozole (I) for removal of the corresponding isomeric impurities and as such does not provide any inputs for controlling or minimization of the formation of the isomeric 4-(1-(1,3,4-triazolyl)methyl]-benzonitrile (V) in the reaction. Secondly, the method of extraction as well as purification taught by Haider et al. is tedious, comprising multiple extractions, with multiple solvents and this coupled with the fact that it does not provide any improvement in controlling or minimization of the formation of the isomeric 4-[1-(1,3,4-triazolyl)methyl]-benzonitrile (V) in the reaction, leads to significant losses, thereby resulting in rather low yields of Letrozole (I). The method, therefore, is not of commercial significance.
• [0027]
  8. Pizzocaro et al. in WO 2007/090464 A1 , a process for preparation of Letrozole (I), as summarized in Scheme-IX, characterized in that it teaches either simultaneous addition of a solution of 4-[1-(1,2,4-triazolyl) methyll-benzonitrile (IV) and a solution of 4-fluorobenzonitrile (VI) in an aprotic dipolar solvent to a solution of an alkali metal alkoxide in the same aprotic dipolar solvent or addition of an unique solution in an aprotic dipolar solvent comprising of compounds (IV) and (VI) to aprotic dipolar solvent, and reacting at a temperature of between -20 to + 40° C.
• [0028]
  The method of Pizzocaro et al., in addition to involving adherence to several critical parameters like temperature, flow rate, etc. moreover, does not provide any details of the yields and purity of Letrozole, obtained by the methods described therein.
• [0029]
  9. Srinivas et al. WO 2007/107733 A1 recite a further variation of Method-1 disclosed by et al. in US 4,978,672 and US 5,352,795 , for synthesis of Letrozole, substantially free from its isomeric impurity, which is summarized in Scheme-X. The method comprises reacting 4-bromomethylbenzonitrile (II), with 1H 1,2,4-triazole (III) in an organic solvent in presence of cesium carbonate and precipitation of 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile (IV), thus formed from the reaction medium using a suitable organic solvent. The intermediate (IV) is further converted to Letrozole by reaction with 4-fluorobenzonitrile (VI) in presence of an organic solvent and silicon amine, which are lithium, sodium, or potassium disilazanes or monosilazane.
• [0030]
  The method utilizes sensitive and expensive silicon compounds like lithium hexamethyldisilazane, which requires highly controlled reaction conditions.
• [0031]
  10. Hasson et al. in US 2007/0112203 A1 , provide a method, as summarized in Scheme-XI, for purification of a mixture containing Letrozole (I) and its isomeric impurity i.e. Isoletrozole (IX), which is an extension of Method-2 disclosed by Bowman et al. in US 4,978,672 and US 5,352,795 . The method takes advantage of the rapid oxidation of Isoletrozole (9) to 4,4′-dicyclobenzophenone (10), in comparison to Letrozole (I), the oxidized compound (10), being easily separable from Letrozole, can be removed by crystallization, affording pure Letrozole. The Letrozole product, in turn is prepared by Method-2 disclosed by Bowman et al. in US 4,978,672 and US 5,352,795 .
• [0032]
  From the Enabling Disclosures of Hasson et al. in US 2007/0112203 A1 , it could be seen that the method of oxidative purification of Letrozole, does not provide the said Letrozole, free of the Isoletrozole impurity (IX), directly and in fact, about 1 to 4% of Isoletrozole (IX) remains in the product, which is further removed by successive crystallizations to provide Letrozole (I) of 99.9% purity.

  It is also noted that Letrozole to some extent also undergoes oxidation, albeit slowly, resulting in formation of additional impurities. Removal of such impurities, coupled with the task of removal of Isoletrozole (IX) and 4,4-dicyclobenzophenone (10) results in significant yield loss, rendering the method not particularly attractive, economically.

• [0033]
  11. Palle et al. in US 2007/0100149 A1 , recite an alternate method for synthesis of Letrozole, as summarized in Scheme-XII.
• [0034]
  The method of Palle et al. comprises reacting 4,4-(hydroxymethylene)bis benzonitrile (12), in turn obtained from 4,4-dibromobenzophenone (11), with p-toluenesulfonyl chloride to give the corresponding p-tolenesulfonate (13), which on reaction with 1H 1,2,4-triazole (III), gives crude Letrozole, which is further purified by successive chromatography and crystallization.
• [0035]
  The yield of the p-tolenesulfonate (13), in the key step is only 21%, indicative of formation of large amount of impurities in the said step. Further, the overall yield of Letrozole obtained by the method is only about 14%, which would render the method not viable commercially.
• [0036]
  12. Friedman et al. in US 2007/0112202 A1 , provide an extension of Method-2 disclosed by Bowman et al. in US 4,978,672 and US 5,352,795 , which is summarized in Scheme-XIII.
• [0037]
  US 2007/0112202 A1 reports synthesis of Letrozole by the abovementioned method in 54-56% yield and having a HPLC purity 99.4%, which may not suit Pharmacopoeial standards, which suggests that the product obtained requires further purification, which, incidentally, is acknowledged by Friedman et al., who state that single purification using various solvents does not give Letrozole of acceptable purity, and hence multiple purifications are required to achieve the same. Needless to mention, this would result in significant loss of the precious product. Further, the novelty and inventiveness of the method is in question, since Bowman et al. in US 4,978,672 and US 5,352,795 have disclosed the same chemistry earlier.
• [0038]
  13. Agarwal et al. in WO 2007/074474 A1 recite a synthesis of Letrozole, utilizing novel intermediates, the chemistry of which is summarized in Scheme-XIV.
• [0039]
  The method is lengthy and the reported overall yield of Letrozole appears to be only 9-11%.

………………………………………….

http://www.google.com/patents/EP2212301B1?cl=en

The process for preparation of 4-[1-(1,2,4-triazolyl) methyl]-benzonitrile hydrochloride of formula (VII) and Letrozole of formula (I), both having a purity of ≥99% as per the present invention is schematically represented in Scheme-XV.

Reference Example – 3 Preparation of 4-[1-(4-cyanophenyl)-1-(1,2,4-trinzol-1yl)methyl]benzonitrile (Letrozole, I)

• [0101]
  To a mixture of potassium tertiarybutoxide (635.92 gm; 5.66 mol) and N,N-dimethylformamide (3.75 Lt), under an atmosphere of nitrogen and cooled to a temperature of -20° to -25°C, was added 4-(1H-1,2,4-triazol-1-ylmethyl)benzonitrile hydrochloride (VII, as obtained in Reference Examples 1 or 2; 250 gm; 1.13 mol) within 5 minutes and was stirred for 60 minutes at -20°C to -25°C. To the mixture was added 4-fluoro benzonitrile (VI, 150.9 gm; 1.24 mol) within 5 minutes and the mass agitated for an hour at-20°C to -25°C. After completion of the reaction, pH of the mixture was adjusted to between 6.0 to 6.5 by addition of 50% aqueous hydrochloric acid, maintaining the temperature between -20°C to 0°C. After the addition of the hydrochloric acid solution, the reaction mass was stirred for additional 30 minutes and filtered. To the filtrate was added ethyl acetate and water and the ethyl acetate layer was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under vacuum to give a residual solid amounting to 179 gm (55%) of Letrozole (I), having a purity of 83%.
• [0102]
  The solid was chromatogaphed over silica gel (60-120 mesh) using n-Hexane and ethyl acetate as eluent to give Letrozole (100.5 gm; 56%), having a purity of 99%.
• [0103]
  The material (100 gm) was further dissolved in ethyl acetate (1.6 Lt) at 70° to 75°C, and the solution was filtered hot. The filtrate was evaporated under vacuum till the volume was between 200 to 220 ml. The solution was cooled to 0° to 5°C for 4 hours, and the solid filtered, washed with cold ethyl acetate and dried to give Letrozole (I, 95 gm; 95%), having a purity of 99.6%.

Example – 3 Preparation of 4-[1-(4-cyanophenyl)-1-(1,2,4-triazol-1-yl)methyl]benzonitrile (Letrozole, I)

• [0104]
  To a mixture of potassium tertiarybutoxide (635.92 gm; 5.66 mol) and N,N-dimethylformamide (3.75 Lt). under an atmosphere of nitrogen and cooled to a temperature of -20° to -25°C, was added 4-(1H-1,2,4-triazol-1-ylmethyl)benzonitrile hydrochloride (VII, as obtained in Examples 1 or 2; 250 gm; 1.13 mol) within 5 minutes and was stirred for 60 minutes at -20°C to -25°C. To the mixture was added 4-fluoro benzonitrile (VI, 150.9 gm; 1.24 mol) within 5 minutes and the mass agitated for an hour at -20°C to -25°C. After completion of the reaction, pH of the mixture, was adjusted to between 6.0 to 6.5 by addition of 50% aqueous hydrochloric acid, maintaining the temperature between -20°C to 0°C. After the addition of the hydrochloric acid solution, the reaction mass was stirred for additional 30 minutes and filtered. To the filtrate was added ethyl acetate and water and the ethyl acetate layer was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under vacuum to give a residual solid amounting to 244 gm (75%) of Letrozole (I), having a purity of 99%.
• [0105]
  The material (244 gm) was further dissolved in ethyl acetate (500 ml) at 70° to 75°C, and the solution was filtered hot. The filtrate was cooled to 0° to 5°C for 4 hours, and the solid filtered, washed with cold ethyl acetate and dried to give Letrozole (I, 221 gm; 98.6%), having a purity of 99.7%.

…………………………………………..

http://www.google.com/patents/EP2212301A1?cl=en

Reference Example – 3 Preparation of4-ll-(4-cyanophenyl)-l-(l,2,4-triazol-l-yl)methyl]benzonitrile (Letrozole, I)

To a mixture of potassium tertiarybutoxide (635.92 gm; 5.66 mol) and N₅N- dimethylformamide (3.75 Lt), under an atmosphere of nitrogen and cooled to a temperature of -20° to -25 °C, was added 4-(lH-l,2,4-triazol-l-ylmethyl)benzonitrile hydrochloride (VII, as obtained in Reference Examples 1 or 2; 250 gm; 1.13 mol) within 5 minutes and was stirred for 60 minutes at -20°C to -25°C. To the mixture was added 4-fluoro benzonitrile (VI, 150.9 gm; 1.24 mol) within 5 minutes and the mass agitated for an hour at – 20°C to -25°C. After completion of the reaction, pH of the mixture was adjusted to between 6.0 to 6.5 by addition of 50% aqueous hydrochloric acid, maintaining the temperature between -20⁰C to 0°C. After the addition of the hydrochloric acid solution, the reaction mass was stirred for additional 30 minutes and filtered. To the filtrate was added ethyl acetate and water and the ethyl acetate layer was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under vacuum to give a residual solid amounting to 179 gm (55%) of Letrozole (I), having a purity of 83%.

The solid was chromatogaphed over silica gel (60-120 mesh) using n-Hexane and ethyl acetate as eluent to give Letrozole (100.5 gm; 56%), having a purity of 99%.

The material (100 gm) was further dissolved in ethyl acetate (1.6 Lt) at 70° to 75°C, and the solution was filtered hot. The filtrate was evaporated under vacuum till the volume was between 200 to 220 ml. The solution was cooled to 0° to 5°C for 4 hours, and the solid filtered, washed with cold ethyl acetate and dried to give Letrozole (I, 95 gm; 95%), having a purity of 99.6%. Example – 3 Preparation of4-[l-(4-cyanophenyl)-l-(l,2,4-triazol-l-yl)methyl]benzonitrile (Letrozole, I)

To a mixture of potassium tertiarybutoxide (635.92 gm; 5.66 mol) and N,N- dimethylformamide (3.75 Lt). under an atmosphere of nitrogen and cooled to a temperature of -20° to -25°C, was added 4-(l H-l ,2,4-triazol-l -ylmethyl)benzonitrile hydrochloride (VII. as obtained in Examples 1 or 2; 250 gm; 1.13 mol) within 5 minutes and was stirred for 60 minutes at -20°C to -25°C. To the mixture was added 4-fluoro benzonitrile (VI, 150.9 gm; 1.24 mol) within 5 minutes and the mass agitated for an hour at -20°C to -25°C. After completion of the reaction, pH of the mixture_Jwas adjusted to between 6.0 to 6.5 by addition of 50% aqueous hydrochloric acid, maintaining the temperature between -20°C to 0°C. After the addition of the hydrochloric acid solution, the reaction mass was stirred for additional 30 minutes and filtered. To the filtrate was added ethyl acetate and water and the ethyl acetate layer was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under vacuum to give a residual solid amounting to 244 gm (75%) of Letrozole (I), having a purity of 99%.

The material (244 gm) was further dissolved in ethyl acetate (500 ml) at 70° to 75°C, and the solution was filtered hot. The filtrate was cooled to 0° to 5°C for 4 hours, and the solid filtered, washed with cold ethyl acetate and dried to give Letrozole (I, 221 gm; 98.6%), having a purity of 99.7%.

…………………………

http://www.google.com/patents/EP1945618A2?cl=en

Aromatase is an enzyme, which effects aromatisation of ring A in the metabolic formation of various steroid hormones. Various cancers, for example, breast cancer is dependent upon circulating steroid hormones, which have an aromatic ring A. Such cancers can be treated by removing the source of ring A aromatised steroid hormones, for example by the combination of oophorectomy and adrenalectomy. An alternative way of obtaining the same effect is by administering a chemical compound, which inhibits the aromatisation of the steroid ring A.

Letrozole is a non-steroidal antineoplastic, claimed to inhibit the aromatase (oestrogen synthase) activity. It is useful in the treatment of advanced breast cancer in postmenopausal women.

The growth of some cancers of the breast are stimulated or maintained by estrogens. Treatment of breast cancer thought to be hormonally responsive (i.e., estrogen and/or progesterone receptor positive or receptor unknown) has included a variety of efforts to decrease estrogen levels (ovariectomy, adrenalectomy, hypophysectomy) or inhibit estrogen effects (antiestrogens and progestational agents). These interventions lead to decreased tumor mass or delayed progression of tumor growth in some women.

In postmenopausal women, estrogens are mainly derived from the action of the aromatase enzyme, which converts adrenal androgens (primarily androstenedioήe and testosterone) to estrone and estradiol. The suppression of estrogen biosynthesis in peripheral tissues and in the cancer tissue itself can therefore be achieved by specifically inhibiting the aromatase enzyme.

Letrozole is a nonsteroidal competitive inhibitor of the aromatase enzyme system; it inhibits the conversion of androgens to estrogens. In adult tumor bearing females, Letrozole is as effective as ovariectomy in reducing uterine weight, elevating serum LH, and causing the regression of estrogen-dependent tumors. In contrast to ovariectomy, treatment with Letrozole does not lead to an increase in serum FSH. Letrozole selectively inhibits gonadal steroidogenesis but has no significant effect on adrenal mineralocorticoid or glucocorticoid synthesis. l Letrozole inhibits the aromatase enzyme by competitively binding to the heme of the cytochrome P450 subunit of the enzyme, resulting in a reduction of estrogen biosynthesis in all tissues. Treatment of women with Letrozole significantly lowers serum estrone, estradiol and estrone sulfate and has not been shown to significantly affect adrenal corticosteroid synthesis, aldosterone synthesis, or synthesis of thyroid hormones. Description of prior art

Synthesis of Letrozole is reported in US Patent No. 4,978,672 and EP 236,940. In the above patents the synthesis of Letrozole starts with 4-bromomethylbenzonitrile (1), which undergoes condensation with 1,2,4-triazole (2) to form 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3) as an intermediate. The compound of structural formula (3) is purified by column chromatography to remove 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4) and followed by reaction with 4-fluorobenzonitrile (5) to

SCHEME – 1

afford Letrozole (6).

In the above process, the undesired intermediate 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4) is formed during the course of the preparation 4-[(l,2,4- triazol-l-yl)methyl]benzonitrile (3) in 10%w/w to 30%w/w. The undesired impurity 4- [(l,3,4-triazol-l-yl)methyl]benzonitrile (4) present with 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3), further on reaction with 4-fluorobenzonitrile (5) leads to the formation of another impurity 4-[l-(4-cyanophenyl)-l-(l,3,4-triazol-l- yl)methyl]benzonitrile (7) in approximately same ratio.

To control the formation of impurity of structural formula (7), it is required to make intermediate of structural formula (3) in its pure form. The separation of desired compound from isomeric impurities is of great importance. In basic patents US 4,978,672 and EP 236,940; chromatographic technique is used to isolate intermediate (3) from its mixture with regioisomer (4). Chromatography has its own limitations on commercial scale; it is an expensive and time consuming operation at plant scale, which also consumes lots of solvent and is hazardous for environment.

To overcome the above problems, purification of intermediate (3) is reported in PCT application WO 2005/047269 via the hydrochloride salt formation of the mixture of product (3) along with regioisomer (4). Selective crystallisation of regioisomer as hydrochloride using approximately 8.5 volume diisopropyl ether, filtering the resultant and then isolation of the intermediate (3) as pure product from the filtrate in approximately 60% overall yield. Finally washing the product with hexane or petroleum ether. In above PCT application, highly flammable solvents like diisopropyl ether, hexane and petroleum ether are used in process, which require high level of precautions and are never safe to handle on plant scale.

Another process reported in PCT application WO 2004/076409, discloses the different route to prepare the pure intermediate (3). The said patent discloses a reaction of 4-bromomethylbenzonitrile (1) with 4-amino-l,2,4-triazole (8) to give quaternary ammonium salt (9), which undergoes diazotisation reaction to give 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3) in approximately 59% molar yield. The process is complicated and involves lengthy steps and tedious operations. Objects of the invention

It is therefore, an important object of the present invention to provide a process for the preparation of Letrozole which avoids the use of highly flammable solvents and is safe and smooth.. Summary of the invention

To overcome the problems in the use of highly flammable solvents, complicated and lengthy steps and tedious operations; we have opted a simple and novel process for the purification of Letrozole intermediate (3), which is free from its regioisomer (4) and other related impurities.

Purification of intermediate (3) to remove its regioisomer (4) using crystallization method to achieve desired level of purity is unsuccessful. We have planned to go for extraction of intermediate (3) selectively from the mixture with regioisomer (4) in aqueous layer using a suitable solvent.

LETROZOLE

In order to obtain the pure Letrozole (6), we have planned to get intermediate (3) in its pure form and free from its regioisomer (4). For the said purpose, we have used solvent extraction method using suitable solvent system and selectively extract the desired intermediate 4-[(l,2,4-triazol-l-yl)methyl]benzonitrile (3), from a mixture with regioisomer 4-[(l,3,4-triazol-l-yl)methyl]benzonitrile (4). The control of the regioisomer at intermediate level leads its control at the final stage. Therefore, in an embodiment, the present invention relates to Letrozole (6) with its regioisomer 4-[l-(4-cyanophenyl)-l-(l,3,4-triazol-l-yl)methyl]benzonitrile (7), preferably, less than 0.3%w/w, more preferably, less than 0.1%w/w and most preferably, below the quantitation limit.

In another feature, the present invention provides an improved process for the preparation of Letrozole with its regioisomer 4-[l-(4-cyanophenyl)-l-(l,3,4-triazol-l- yl)methyl]benzonitrile (7), preferably, less than 0.3%w/w, more preferably, less tjian 0.1%w/w and most preferably, below the quantitation limit.

In another feature, the present invention provides 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3) with its regioisomer 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4), preferably, less than 0.3%w/w, more preferably, less than 0.1%w/w and most preferably, below the quantitation limit.

SCHEME – 4

(5)

In yet another feature, the present invention provides an improved process for the preparation of 4-[(l,2,4-triazol-l-yl)methyl]benzonitrile (3) with its regioisomer A- [(l,3,4-triazol-l-yl)methyl]benzonitrile (4), preferably, less than 0.3%w/w, more preferably, less than 0.1%w/w and most preferably, below the quantitation limit.

In order to obtain Letrozole (6) in purer form and free from its regioisomer (7) and other related impurities; intermediate 4-[(l,2,4-triazol-l-yl)methyl]benzonitrile (3) is to be prepared in its pure form, free from its regioisomer 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4) and other related impurities. Thus, the main aspect of the present invention relates to the preparation of Letrozole (6) with its regioisomer (7) preferably less than 0.3%, more preferably less than 0.1% and most preferably below quantitation limit. For this purpose intermediate 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3) is required to be of the same purity level. Another aspect of the present invention relates to the preparation of 4-[(l,2,4-triazol-l- yl)methyl]benzonitrile (3) with its regioisomer 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4) preferably less than 0.3%, more preferably less than 0.1% and most preferably below quantitation limit.

It has been also found that during the preparation of Letrozole intermediate A- [(l,2,4-triazol-l-yl)methyl]benzonitrile (3) another impurity is formed, which is characterized as the quaternary salt (10). To control the formation of this quaternary salt, mole ratio of 1,2,4-triazole is optimized preferably from 1.5 mole to 4.4 mole equivalents and more preferably to 3.0 mole equivalents with respect to A- bromomethylbenzonitrile (1). Thus, another aspect of the present invention relates to the preparation of Letrozole (6) with quaternary salt (10) preferably less than 0.1% and more preferably below quantitation limit. Another important aspect of the present invention relates to the preparation of 4-

[(l,2,4-triazol-l-yl)methyl]benzonitrile (3) in the pure form, free from its regioisomer 4-[(l,3,4-triazol-l-yl)methyl]benzonitrile (4). The purification of 4-[(l,2,4-triazol-l- yl)methyl]-benzonilrilc (3) takes place by its selective extraction from a mixture with its regioisomer 4-[(l,3,4-triazol-l-yl)methyl]benzonitrile (4) by using suitable solvents and/or mixture of solvents.

According to another aspect of the present invention Letrozole intermediate A- [(l,2,4-triazol-l-yl)methyl]benzonitrile (3) is prepared with its regioisomer 4-[(l,3,4- triazol-l-yl)methyl]benzonitrile (4) less than 30% and followed by the preparation of Letrozole enriched with its regioisomer (7), which is removed by using crystallisation method using suitable solvent system.

Example – 3

4-[(l,2,4-triazol-l-yl)methyl]benzonitrile (S) with a mixture of 4-[(l,3,4-triazol-l- yl)methyl]benzonitrile (4) To a 250 mL three neck R. B. Flask fitted with a reflux condenser and a thermometer pocket, isopropanol (37.5 mL), p-cyanobenzylbromide (25 g), 1,2,4- triazole (26.4 g) and potassium carbonate (52.8 g) were charged to the reaction mixture at RT with stirring. The reaction mixture was heated to 60-65 ⁰C for 1.0 hr. The progress of reaction was monitored over TLC for the absence of p- cyanobenzylbromide. After completion, the reaction mixture was cooled down to RT and water (100 mL) was added to the reaction mixture and reaction mass was transferred to a one lit R. B. Flask containing water (275 mL). Cone. HCl (50 mL) was added very slowly to the reaction mass to adjust pH 7 – 8. The reaction mixture was extracted from dichloromethane (250 mL). Dichloromethane layer was distilled out at 50 ⁰C giving 21.0 gm viscous oily residue. The residue is crystallized from a mixture of IPA: Cyclohexane (1:10) to give 18 g of 4-[(l,2,4-triazol-l-yl)methyl]benzonitrile (3) with a mixture of its regioisomer (4). HPLC Purity ~ 85%; Regioisomer ~ 13%. Example – 4 4-[l-(4-cyanophenyl)-l-(l,2,4-triazol-l-yl)methyl]benzonitrile (6) & 4-[l-(4- cyanophenyl)-l-(l,3,4-triazol-l-yl)methyl]benzonitrile (7)

In a 250 mL three neck flask, equipped with thermometer pocket, mechanical stirrer and a guard tube, THF (50 mL) was charged at room temperature. Potassium tert-butoxide (12.3 gm) is added in small portions in 30 minutes. The solution was cooled to -15 ⁰C and a solution of product from example-3 (10 g) and p- fluorobenzonitrile (8.5 g) in THF (50 mL) is added very slowly to the reaction mixture in 4-5 hrs. Stir the reaction mixture at same temperature for 3 hrs. Progress of the reaction is monitored on TLC. Dichloromethane (200 mL) is added to the reaction mixture followed by the addition of acetic acid (7 g). Reaction mixture is added to another flask containing water (220 mL). pH of the reaction mixture is adjusted to 7-8 by addition of 5% sodium bicarbonate solution (180 mL). Dichloromethane layer is washed with water (200 mL), separated, filtered through hyfiow bed and distilled at a temperature below 50 ⁰C. The residue obtained was crystallized from Isopropanol (20 mL) to get the solid product (4.9 g). HPLC Purity: 89.6%. Regioisomer: 7.41%. Example – 5

Removal of regioisomer (7) from Letrozole (6)

To a 250 mL R. B. Flask crude Letrozole (4.5 g) was charged in methanol (115 mL) and heated to 60 ⁰C to dissolve completely and get clear solution. Methanol (approx. 100 mL) was distilled out and the solution was cooled to 25 – 30 ⁰C, and was stirred for 2 hrs at this temperature. Solid product was filtered and washed with methanol (10 mL x 2) to get solid product, which was dried in vacuum to get 3.5 gm of product.

HPLC Purity: 96.33%, Regioisomer: 3.4%

Using the same purification method, desired purity of Letrozole had been achieved containing acceptable level of regioisomer (7).

Following the above purification from methanol repeatedly, the Letrozole may be prepared with the desired limit of its regioisomer (7).

References

External links

• Femara website

St. John’s Wort (Hypericum perforatum) – This herb is often used to treat mild to moderate depression. It is especially helpful to patients who do not respond well to SSRI medication (selective serotonin reuptake inhibitors). This herb can limit the effectiveness of some prescription medications, though, so double check with your doctor before taking it. A 2009 systematic review of 29 international studies suggested that St. John’s Wort may be better than a placebo (an inactive substance that appears identical to the study substance) and as effective as standard prescription antidepressants for major depression of mild to moderate severity.

Hypericum perforatum
Scientific classification
Kingdom:	Plantae
(unranked):	Angiosperms
(unranked):	Eudicots
(unranked):	Rosids
Order:	Malpighiales
Family:	Hypericaceae
Genus:	Hypericum
Species:	H. perforatum
Binomial name
Hypericum perforatum L.

Hypericum perforatum, also known as St John’s wort, is a flowering plant species of the genus Hypericum and a medicinal herb that is sold over-the-counter as a treatment for depression.^[1][2] Other names for it include Tipton’s weed, rosin rose, goatweed, chase-devil, or Klamath weed.^[1] With qualifiers, St John’s wort is used to refer to any species of the genus Hypericum. Therefore, H. perforatum is sometimes called common St John’s wort or perforate St John’s wort to differentiate it. Hypericum is classified in the family Hypericaceae, having previously been classified as Guttiferae or Clusiaceae.^[3][4] Approximately 370 species of the genus Hypericum exist worldwide with a native geographical distribution including temperate and subtropical regions of Europe, Turkey, Ukraine, Russia, Middle East, India, andChina.

Botanical description

Translucent dots on the leaves

Hypericum perforatum is a yellow-flowering, stoloniferous or sarmentose, perennial herb indigenous to Europe. It has been introduced to many temperate areas of the world and grows wild in many meadows. The herb’s common name comes from its traditional flowering and harvesting on St John‘s day, 24 June. The genus name Hypericum is derived from the Greek words hyper (above) and eikon (picture), in reference to the plant’s traditional use in warding off evil by hanging plants over a religious icon in the house during St John’s day. Thespecies name perforatum refers to the presence of small oil glands in the leaves that look like windows, which can be seen when they are held against the light.^[1]

St John’s wort is a perennial plant with extensive, creeping rhizomes. Its stems are erect, branched in the upper section, and can grow to 1 m high. It has opposing, stalkless, narrow, oblong leaves that are 12 mm long or slightly larger. The leaves are yellow-green in color, with transparent dots throughout the tissue and occasionally with a few black dots on the lower surface.^[1] Leaves exhibit obvious translucent dots when held up to the light, giving them a ‘perforated’ appearance, hence the plant’s Latin name.

Its flowers measure up to 2.5 cm across, have five petals, and are colored bright yellow with conspicuous black dots. The flowers appear in broad cymes at the ends of the upper branches, between late spring and early to mid summer. The sepals are pointed, with glandular dots in the tissue. There are many stamens, which are united at the base into three bundles. The pollen grains are ellipsoidal.^[1]

When flower buds (not the flowers themselves) or seed pods are crushed, a reddish/purple liquid is produced.

• Full plant

• Seedlings

• Fruit

• Blossom

Ecology

St John’s wort reproduces both vegetatively and sexually. It thrives in areas with either a winter- or summer-dominant rainfall pattern; however, distribution is restricted by temperatures too low for seed germination or seedling survival. Altitudes greater than 1500 m, rainfall less than 500 mm, and a daily mean January (in Southern hemisphere) temperature greater than 24 degrees C are considered limiting thresholds. Depending on environmental and climatic conditions, and rosette age, St John’s wort will alter growth form and habit to promote survival. Summer rains are particularly effective in allowing the plant to grow vegetatively, following defoliation by insects or grazing.

The seeds can persist for decades in the soil seed bank, germinating following disturbance.^[5]

Invasive species

Although Hypericum perforatum is grown commercially in some regions of south east Europe, it is listed as a noxious weed in more than twenty countries and has introduced populations in South and North America, India, New Zealand, Australia, and South Africa.^[5] In pastures, St John’s wort acts as both a toxic and invasive weed.^[6] It replaces nativeplant communities and forage vegetation to the dominating extent of making productive land nonviable^{[citation needed]} or becoming an invasive species in natural habitats andecosystems. Ingestion by livestock can cause photosensitization, central nervous system depression, spontaneous abortion, and can lead to death. Effective herbicides for control of Hypericum include 2,4-D, picloram, and glyphosate. In western North America three beetles Chrysolina quadrigemina, Chrysolina hyperici and Agrilus hyperici have been introduced as biocontrol agents.

Medical uses

Major depressive disorder

St John’s wort is widely known as a herbal treatment for depression. In some countries, such as Germany, it is commonly prescribed for mild to moderate depression, especially in children and adolescents.^[7] Specifically, Germany has a governmental organization called Commission E which regularly performs rigorous studies on herbal medicine. It is proposed that the mechanism of action of St. John’s wort is due to the inhibition of reuptake of certain neurotransmitters.^[1] The best studied chemical components of the plant are hypericin and pseudohypericin.

An analysis of twenty-nine clinical trials with more than five thousand patients was conducted by Cochrane Collaboration. The review concluded that extracts of St John’s wort were superior to placebo in patients with major depression. St John’s wort had similar efficacy to standard antidepressants. The rate of side-effects was half that of newer SSRIantidepressants and one-fifth that of older tricyclic antidepressants.^[8] A report^[8] from the Cochrane Review states:

The available evidence suggests that the Hypericum extracts tested in the included trials a) are superior to placebo in patients with major depression; b) are similarly effective as standard antidepressants; and c) have fewer side-effects than standard antidepressants.

However the report also noted that some of the studies they reviewed may have been flawed or biased, as “results from German-language countries are considerably more favourable for Hypericum than trials from other countries”. The authors did not know the reason for this discrepancy.

Other medical uses

St John’s wort is being studied for effectiveness in the treatment of certain somatoform disorders. Results from the initial studies are mixed and still inconclusive; some research has found no effectiveness, other research has found a slight lightening of symptoms. Further study is needed and is being performed.

A major constituent chemical, hyperforin, may be useful for treatment of alcoholism, although dosage, safety and efficacy have not been studied.^[9][10] Hyperforin has also displayed antibacterial properties against Gram-positive bacteria, although dosage, safety and efficacy has not been studied.^[11] Herbal medicine has also employed lipophilic extracts from St John’s wort as a topical remedy for wounds, abrasions, burns, and muscle pain.^[10] The positive effects that have been observed are generally attributed to hyperforin due to its possible antibacterial and anti-inflammatory effects.^[10] For this reason hyperforin may be useful in the treatment of infected wounds and inflammatory skin diseases.^[10] In response to hyperforin’s incorporation into a new bath oil, a study to assess potential skin irritation was conducted which found good skin tolerance of St John’s wort.^[10]

A randomized controlled trial of St John’s wort found no significant difference between it and placebo in the management of ADHD symptoms over eight weeks. However, the St John’s wort extract used in the study, originally confirmed to contain 0.3% hypericin, was allowed to degrade to levels of 0.13% hypericin and 0.14% hyperforin. Given that the level of hyperforin was not ascertained at the beginning of the study, and levels of both hyperforin and hypericin were well below that used in other studies, little can be determined based on this study alone.^[12] Hypericin and pseudohypericin have shown both antiviral and antibacterial activities. It is believed that these molecules bind non-specifically to viral and cellular membranes and can result in photo-oxidation of the pathogens to kill them.^[1]

A research team from the Universidad Complutense de Madrid (UCM) published a study entitled “Hypericum perforatum. Possible option against Parkinson’s disease”, which suggests that St John’s wort has antioxidant active ingredients that could help reduce the neuronal degeneration caused by the disease.^{[13][14][15][16]}

Recent evidence suggests that daily treatment with St John’s wort may improve the most common physical and behavioural symptoms associated with premenstrual syndrome.^[17]

St John’s wort was found to be less effective than placebo, in a randomized, double-blind, placebo-controlled trial, for the treatment of irritable bowel syndrome.^[18]

St John’s wort alleviated age-related long-term memory impairment in rats.^[19]

Adverse effects and drug interactions

St John’s wort is generally well tolerated, with an adverse effect profile similar to placebo.^[20] The most common adverse effects reported are gastrointestinal symptoms, dizziness, confusion, tiredness and sedation.^[21][22] It also decreases the levels of estrogens, such as estradiol, by speeding up its metabolism, and should not be taken by women oncontraceptive pills as it upregulates the CYP3A4 cytochrome of the P450 system in the liver.^[23]

St John’s wort may rarely cause photosensitivity. This can lead to visual sensitivity to light and to sunburns in situations that would not normally cause them.^[20] Related to this, recent studies concluded that the extract reacts with light, both visible and ultraviolet, to produce free radicals, molecules that can damage the cells of the body. These can react with vital proteins in the eye that, if damaged, precipitate out, causing cataracts.^[24] Another study found that in low concentrations, St. John’s wort inhibits free radical production in both cell-free and human vascular tissue, revealing antioxidant properties of the compound. The same study found pro-oxidant activity at the highest concentration tested.^[25]

St John’s wort is associated with aggravating psychosis in people who have schizophrenia.^[26]

Consumption of St. John’s wort is discouraged for those with bipolar disorder. There is concern that people with major depression taking St. John’s wort may be at a higher risk for mania.^[27]

While St. John’s wort shows some promise in treating children, it is advised that it is only done with medical supervision. ^[27]

Pharmacokinetic interactions

St John’s wort has been shown to cause multiple drug interactions through induction of the cytochrome P450 enzymes CYP3A4 and CYP2C9, and CYP1A2 (females only). This drug-metabolizing enzyme induction results in the increased metabolism of certain drugs, leading to decreased plasma concentration and potential clinical effect.^[28] The principal constituents thought to be responsible are hyperforin and amentoflavone.

St John’s wort has also been shown to cause drug interactions through the induction of the P-glycoprotein (P-gp) efflux transporter. Increased P-gp expression results in decreased absorption and increased clearance of certain drugs, leading to lower plasma concentration and potential clinical efficacy.^[29]

For a complete list, see CYP3A4 ligands and CYP2C9 ligands. For further updating on interactions and appropriate management, see Herbological.com – St John’s Wort Interactions table (outdated since 2005).

Pharmacodynamic interactions

In combination with other drugs that may elevate 5-HT (serotonin) levels in the central nervous system (CNS), St John’s wort may contribute to serotonin syndrome, a potentially life-threatening adverse drug reaction.^[30]

Detection in body fluids

Hypericin, pseudohypericin, and hyperforin may be quantitated in plasma as confirmation of usage and to estimate the dosage. These three active substituents have plasma elimination half-lives within a range of 15–60 hours in humans. None of the three has been detected in urine specimens.^[31]

Chemical constituents

The plant contains the following:^[32][33]

• Flavonoids (e.g. epigallocatechin, rutin, hyperoside, isoquercetin, quercitrin, quercetin, amentoflavone, biapigenin, astilbin, myricetin, miquelianin, kaempferol, luteolin)
• Phenolic acids (e.g. chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, vanillic acid)
• Naphthodianthrones (e.g. hypericin, pseudohypericin, protohypericin, protopseudohypericin)
• Phloroglucinols (e.g. hyperforin, adhyperforin)
• Tannins (unspecified, proanthocyanidins reported)
• Volatile oils (e.g. 2-methyloctane, nonane, 2-methyldecane, undecane, α-pinene, β-pinene, α-terpineol, geraniol, myrcene, limonene, caryophyllene, humulene)
• Saturated fatty acids (e.g. isovaleric acid (3-methylbutanoic acid), myristic acid, palmitic acid, stearic acid)
• Alkanols (e.g. 1-tetracosanol, 1-hexacosanol)
• Vitamins & their analogues (e.g. carotenoids, choline, nicotinamide, nicotinic acid)
• Miscellaneous others (e.g. pectin, β-sitosterol, hexadecane, triacontane, kielcorin, norathyriol)

The naphthodianthrones hypericin and pseudohypericin along with the phloroglucinol derivative hyperforin are thought to be among the numerous active constituents.^{[1][34][35][36]}It also contains essential oils composed mainly of sesquiterpenes.^[1]

Mechanism of action

St. John’s wort (SJW), similarly to other herbal products, contains a whole host of different chemical constituents that may be pertinent to its therapeutic effects.^[32] Hyperforin andadhyperforin, two phloroglucinol constituents of SJW, is a TRPC6 receptor agonist and, consequently, it induces noncompetitive reuptake inhibitor of monoamines (specifically,dopamine, norepinephrine, and serotonin), GABA, and glutamate when it activates this receptor.^[2][37][38] It inhibits reuptake of these neurotransmitters by increasing intracellularsodium ion concentrations.^[2] Moreover, SJW is known to downregulate the β₁ adrenoceptor and upregulate postsynaptic 5-HT_1A and 5-HT_2A receptors, both of which are a type of serotonin receptor.^[2] Other compounds may also play a role in SJW’s antidepressant effects such compounds include: oligomeric procyanidines, flavonoids (quercetin),hypericin, and pseudohypericin.^{[2][39][40][41]}

In humans, the active ingredient hyperforin is a monoamine reuptake inhibitor which also acts as an inhibitor of PTGS1, Arachidonate 5-lipoxygenase, SLCO1B1 and an inducer ofcMOAT. Hyperforin is also a powerful anti-inflammatory compound with anti-angiogenic, antibiotic, and neurotrophic properties.^{[37][38][42][43]} Hyperforin also has an antagonistic effect on NMDA receptors, a type of glutamate receptor.^[42] According to one study, hyperforin content correlates with therapeutic effect in mild to moderate depression.^[44]Moreover, a hyperforin-free extract of St John’s wort (Remotiv) may still have significant antidepressive effects.^[45][46] The limited existing literature on adhyperforin suggests that, like hyperforin, it is a reuptake inhibitor of monoamines, GABA, and glutamate.^[47]

Livestock

Poisoning

In large doses, St John’s wort is poisonous to grazing livestock (cattle, sheep, goats, horses).^[6] Behavioural signs of poisoning are general restlessness and skin irritation. Restlessness is often indicated by pawing of the ground, headshaking, head rubbing, and occasional hindlimb weakness with knuckling over, panting, confusion, and depression. Mania and hyperactivity may also result, including running in circles until exhausted. Observations of thick wort infestations by Australian graziers include the appearance of circular patches giving hillsides a ‘crop circle’ appearance, it is presumed, from this phenomenon. Animals typically seek shade and have reduced appetite. Hypersensitivity to water has been noted, and convulsions may occur following a knock to the head. Although general aversion to water is noted, some may seek water for relief.

Severe skin irritation is physically apparent, with reddening of non-pigmented and unprotected areas. This subsequently leads to itch and rubbing, followed by further inflammation, exudation, and scab formation. Lesions and inflammation that occur are said to resemble the conditions seen in foot and mouth disease. Sheep have been observed to have face swelling, dermatitis, and wool falling off due to rubbing. Lactating animals may cease or have reduced milk production; pregnant animals may abort. Lesions onudders are often apparent. Horses may show signs of anorexia, depression (with a comatose state), dilated pupils, and injected conjunctiva.

Diagnosis[edit]

Increased respiration and heart rate is typically observed while one of the early signs of St John’s wort poisoning is an abnormal increase in body temperature. Affected animals will lose weight, or fail to gain weight; young animals are more affected than old animals. In severe cases death may occur, as a direct result of starvation, or because of secondary disease or septicaemia of lesions. Some affected animals may accidentally drown. Poor performance of suckling lambs (pigmented and non-pigmented) has been noted, suggesting a reduction in the milk production, or the transmission of a toxin in the milk.

Photosensitisation[edit]

Most clinical signs in animals are caused by photosensitisation.^[96] Plants may induce either primary or secondary photosensitisation:

• primary photosensitisation directly from chemicals contained in ingested plants
• secondary photosensitisation from plant-associated damage to the liver.

Araya and Ford (1981) explored changes in liver function and concluded there was no evidence of Hypericum-related effect on the excretory capacity of the liver, or any interference was minimal and temporary. However, evidence of liver damage in blood plasma has been found at high and long rates of dosage.

Photosensitisation causes skin inflammation by a mechanism involving a pigment or photodynamic compound, which when activated by a certain wavelength of light leads tooxidation reactions in vivo. This leads to lesions of tissue, particularly noticeable on and around parts of skin exposed to light. Lightly covered or poorly pigmented areas are most conspicuous. Removal of affected animals from sunlight results in reduced symptoms of poisoning.

See also[edit]

• Dietary supplement
• EU Food supplements directive
• List of plants poisonous to equines

References[edit]

Further reading[edit]

• British Herbal Medicine Association Scientific Committee (1983). British Herbal Pharmacopoeia. West Yorkshire: British Herbal Medicine Association. ISBN 0-903032-07-4.
• Müller, Walter (2005). St. John’s Wort and its Active Principles in Depression and Anxiety. Basel: Birkhäuser. doi:10.1007/b137619. ISBN 978-3-7643-6160-0.

External links

Wikispecies has information related to: Hypericum perforatum

Wikimedia Commons has media related to Hypericum perforatum.

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FDA accepts Kythera’s ATX-101 new drug application
Kythera Biopharmaceuticals’ new drug application (NDA) for its ATX-101, a submental contouring injectable drug, has been accepted for filing by the US Food and Drug Administration (FDA).

According to Kythera Biopharmaceuticals, the ATX-101 NDA will be subject to a standard review and will have a prescription drug user fee act (PDUFA) action date of 13 May 2015. The company submitted the NDA in May 2014.

http://www.pharmaceutical-technology.com/news/newsfda-accepts-kytheras-atx-101-new-drug-application-4316052?WT.mc_id=DN_News

cas 83-44-3, C₂₄ H₄₀ O₄

cas of Na salt….302-95-4

NSC-681065 , NSC 8797

OTHERS

5β-​Cholan-​24-​oic acid, 3α,​12α-​dihydroxy- (8CI); 17β-​[1-​Methyl-​3-​carboxypropyl]​-​etiocholane-​3α,​12α-​diol;

3α,​12α-​Dihydroxy-​5β-​cholan-​24-​oic acid;

3α,​12α-​Dihydroxy-​5β-​cholanic acid;

3α,​12α-​Dihydroxy-​5β-​cholanoic acid; 3α,​12α-​Dihydroxycholanic acid;

5β-​Cholanic acid-​3α,​12α-​diol;

5β-​Deoxycholic acid; 7-​Deoxycholic acid; ATX 101;

Cholerebic; Cholic acid, deoxy-; Cholorebic; Degalol; Deoxycholatic acid; Deoxycholic acid; Desoxycholic acid; Droxolan; NSC 8797; Pyrochol; Septochol

Deleted CAS Registry Numbers: 728917-​93-​9

University of California, Oakland (Originator)
LA BioMed (Originator)

LICENSE….

Kythera Biopharmaceuticals, Inc.

Rapid removal of body fat is an age-old ideal, and many substances have been claimed to accomplish such results, although few have shown results. ”Mesotherapy”, or the use of injectables for the removal of fat. is not widely accepted among medical practitioners due to safety and efficacy concerns, although homeopathic and cosmetic claims have been made since the 1950′s. Mesotherapy was originally conceived in Europe as a method of utilizing cutaneous injections containing a mixture of compounds for the treatment of local medical and cosmetic conditions. Although mesotherapy was traditionally employed for pain relief, its cosmetic applications, particularly fat and cellulite removal, have recently received attention in the United States. One such reported treatment for localized fat reduction, which was popularized in Brazil and uses injections of phosphatidylcholine, has been erroneously considered synonymous with mesotherapy. Despite its attraction as a purported “fat-dissolving” injection, there is little safety and efficacy data of these cosmetic treatments. See, Rotunda, A.M. and M.

olodney, Dermatologic Surgery 32:, 465-480 (2006) (“Mesotherapy and

Phosphatidy lcholine Injections: Historical Clarification and Review^**).

Recently published literature reports that the bile acid, DCA, and salts thereof, have fat removing properties when injected into fatty deposits in vivo. See, WO

2005/1 17900 and WO 2005/1 12942, as well as US2005/0261258; US2005/0267080; US2006/127468; and US20060154906, each of which is incorporated herein by reference in its entirety). Deoxycholate injected into fat tissue degrades fat cells via a cytolytic mechanism. Because deoxycholate injected into fat is rapidly inactivated by exposure to protein and then rapidly returns to the intestinal contents, its effects are spatially contained. As a result of this attenuation effect that confers clinical safety, fat removal typically require 4 – 6 sessions. This localized fat removal without the need for surgery is beneficial not only for therapeutic treatment relating to pathological localized fat deposits (e.g., dyslipidemias incident to medical intervention in the treatment of HIV), but also for cosmetic fat removal without the attendant risk inherent in surgery (e.g., liposuction). See, Rotunda et ai, Dermatol. Surgery 30: 1001-1008 (2004) (“Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution”) and Rotunda et al, J. Am. Acad. Dermatol. (2005 : 973-978) (“”Lipomas treated with subcutaneous deoxycholate injections”), both incorporated herein by reference in their entirety. US Patent Nos. 7,622,130 and

7,754,230 describe using DCA for fat removal.

In addition, many important steroids have a 12- -hydroxy-substituent on the C- ring of the steroid. Such compounds include, by way of example, bile acids such as DCA, cholic acid, lithocholic acid, and the like. Heretofore, such compounds were typically- recovered from bovine and ovine sources which provided a ready source of bile acids on a cost effective basis. However, with the recent discovery that pathogens such as prions can contaminate such sources, alternative methods for the synthesis of bile acids from plant sources or synthetic starting materials have become increasingly important. For example, DCA from animals in New Zealand are a source of bile acids for human use under US regulatory regimes, as long as the animals continue to remain isolated and otherwise free of observable pathogens. Such stringent conditions impose a limitation on the amount of suitable mammalian sourced bile acids and does not preclude the possibility that the bile acid will be free of such pathogens. US Patent Publication No.

8,242,294 relates to DCA containing less than 1 ppt ¹⁴C.

ATX-101, sodium deoxycholate for injection, is awaiting for approval in the U.S. for the reduction of localized submental fat. Phase II trials for the treatment of superficial lipomas have been completed at Kythera Biopharmaceuticals and Intendis. Treatment with ATX-101 is expected to significantly reduce the size of or eliminate lipomas and provide an effective non-surgical, minimally invasive treatment option for patients.

Licensed to Kythera from Los Angeles Biomedical Institute at Harbor-UCLA Medical Center in 2007, ATX-101 is also being evaluated by the company for aesthetic applications. Specifically, phase II trials are under way for the reduction of submental fat. In 2010, ATX-101 was licensed to Intendis by Kythera Biopharmaceuticals outside of the U.S. and Canada for the treatment of dermatological disorders. In 2010, the product was licensed by Kythera Biopharmaceuticals to Bayer outside Canada and the U.S., and in 2014, Kythera acquired those same rights from Bayer.

………………………………..

WO 2011075701

http://www.google.com/patents/WO2011075701A2?cl=en

Scheme 2

Conversion of Compound 24 to Compound 33:

The hydrogenation of compound 24 on 10.0 g scale using dry 10 % Pd/C (15 wt %) in ethyl acetate (20 parts) was added and applied about 50 psi hydrogen pressure and temperature raised to 70 °C. After reaching temperature 70 °C, observed increase of hydrogen pressure to about 60 psi, at these conditions maintained for 60 h. After 60 hours 0.6% of compound 24 and 2.75% of allylic alcohol were still observed, so further stirred for additional 12 h (observed 0.16% of allylic alcohol and 0.05% of compound 24). After work-up, the reaction provided 9.5g of residue.

Anther hydrogenation reaction on 25 g of compound 24 with above conditions for 76 h provided 24.5 g of residue.

Method A

10% Pd/C (900 mg) was added to a solution of compound 24 (2.0 g, 4.5 mmol) in EtOAc (150 mL) and the resulting slurry was hydrogenated in a Parr apparatus (50 psi) at 50 °C for 16 h. At this point the reaction was determined to be complete by TLC. The mixture was filtered through a small plug of Celite® and the solvent was removed under vacuum, providing compound 33 (1.6 g, 80% yield) as a white solid.

TLC: p-anisaldehyde charring, R_f for 33 = 0.36 and R_f for 25 = 0.32.

TLC mobile phase: 20% – EtOAc in hexanes. 1H NMR (500 MHz, CDC1₃): δ = 4.67-4.71 (m, 1H), 3.66 (s, 3H), 2.45-2.50 (t, J = 15 Hz, 2H), 2.22-2.40 (m, 1H), 2.01 (s, 3H), 1.69-1.96 (m, 9H), 1.55 (s, 4H), 1.25-1.50 (m, 8H), 1.07-1.19 (m, 2H), 1.01 (s, 6H), 0.84-0.85 (d, J= 7.0 Hz, 3H).

¹³C NMR (125 MHz, CDC1₃): δ = 214.4, 174.5, 170.4, 73.6, 58.5, 57.4, 51.3, 46.4, 43.9, 41.2, 38.0, 35.6, 35.5, 35.2, 34.8, 32.0, 31.2, 30.4, 27.4, 26.8, 26.2, 25.9, 24.2, 22.6,

21.2, 18.5,1 1.6,

Mass (m/z) = 447.0 [M⁺ + 1], 464.0 [M⁺ + 18].

IR (KBr) = 3445, 2953, 2868, 1731, 1698, 1257, 1029 cm^-1.

m.p. =142.2-144.4 °C (from EtOAc/hexanes mixture).

[α]_D = +92 (c = 1 % in CHCl₃).

ELSD Purity: 96.6%: Retention time = 9.93 (Inertsil ODS 3 V, 250 ^χ 4.6 mm, 5um, ACN:

0.1 % TFA in water (90: 10)

Method B

A slurry of 10% Pd/C (9 g in 180 mL of ethyl acetate) was added to a solution of compound 24 (36 g, 81 mmol) in EtOAc (720 mL) and the resulting slurry was treated with hydrogen gas (50 psi) at 45-50 °C for 16 h. (A total of 1080 mL of solvent may be used). At this point the reaction was determined to be complete by HPLC (NMT 1% of compound 24). The mixture was filtered through Celite® (10 g) and washed with ethyl acetate (900 mL). The filtrate was concentrated to 50% of its volume via vacuum distillation below 50 °C. To the concentrated solution was added pyridinium

chlorochromate (20.8 g) at 25-35 °C and the mixture was stirred for 2 h at 25-35 °C, when the reaction completed by HPLC (allylic alcohol content is NMT 1%).

The following process can be conducted if compound 24 content is more than 5%. Filter the reaction mass through Celite® (10 g) and wash with ethyl acetate (360 mL). Wash the filtrate with water (3 x 460 mL) and then with saturated brine (360 mL). Dry the organic phase over sodium sulphate (180 g), filter and wash with ethyl acetate (180 mL). Concentrate the volume by 50% via vacuum distillation below 50 °C. Transfer the solution to a clean and dry autoclave. Add slurry of 10% palladium on carbon (9 g in 180 mL of ethyl acetate). Pressurize to 50 psi with hydrogen and stir the reaction mixture at 45-50 °C for 16 h. Upon complete consumption of compound 24 by HPLC (the content of compound 24 being NMT 1%), the reaction mixture was filtered through Celite® (10 g) and the cake was washed with ethyl acetate (900 mL). The solvent was concentrated to dryness via vacuum distillation below 50 °C. Methanol (150 mL) was added and concentrated to dryness via vacuum distillation below 50 °C. Methanol (72 mL) was added to the residue and the mixture was stirred for 15-20 min at 10-15 °C, filtered and the cake was washed with methanol (36 mL). The white solid was dried in a hot air drier at 45-50 °C for 8 h to LOD being NMT 1 % to provide compound 33 (30 g, 83.1 % yield).

Conversion of Compound 33 to Compound 34:

Method A

A THF solution of lithium tri-tert-butoxyaluminum hydride (1 M, 22.4 mL, 22.4 mmol) was added drop wise to a solution of compound 33 (2.5 g, 5.6 mmol) in THF (25 mL) at ambient temperature. After stirring for an additional 4-5 h, the reaction was determined to be complete by TLC. The reaction was quenched by adding aqueous HCl (1 M, 10 mL) and the mixture was diluted with EtOAc (30 mL). The phases were separated and the organic phase was washed sequentially with water (15 mL) and saturated brine solution (10 mL). The organic phase was then dried over anhydrous Na₂S0₄ (3 g) and filtered. The filtrate was concentrated under vacuum and the resulting solid was purified by column chromatography [29 mm (W) ^x 500 mm (L), 60-120 mesh silica, 50 g], eluting with EtOAc/hexane (2:8) [5 mL fractions, monitored by TLC with p- anisaldehyde charring]. The fractions containing the product were combined and concentrated under vacuum to provide compound 34 (2.3 g, 91%) as a white solid.

TLC: p-anisaldehyde charring, R_f for 34 = 0.45 and R_f for 33 = 0.55.

TLC mobile phase: 30% – EtOAc in hexanes.

1H NMR (500 MHz, CDC1₃): δ = 4.68-4.73 (m, 1H), 3.98 (s, 1H), 3.66 (s, 3H), 2.34-2.40 (m, 1H), 2.21-2.26 (m, 1H), 2.01 (s, 3H), 1.75-1.89 (m, 6H), 1.39-1.68 (m, 16H), 1.00-1.38 (m, 3H), 0.96-0.97 (d, J= 5.5 Hz, 3H), 0.93 (s, 3H), 0.68 (s, 3H).

¹³C NMR (125 MHz, CDCI₃): δ = 174.5, 170.5, 74.1, 72.9, 51.3, 48.1, 47.2, 46.4, 41.7, 35.8, 34.9, 34.7, 34.0, 33.5, 32.0, 30.9, 30.8, 28.6, 27.3, 26.8, 26.3, 25.9, 23.4, 22.9, 21.3, 17.2, 12.6 Mass (m/z) = 449.0 [M⁺ + 1], 466.0 [M + 18].

IR ( Br) = 3621, 2938, 2866, 1742, 1730, 1262, 1 162, 1041, cm^-1.

m.p = 104.2-107.7 °C (from EtOAc).

[α]_D = +56 (c = 1% in CHCl₃).

ELSD Purity: 97.0%: Retention time = 12.75 (Inertsil ODS 3V, 250 ^χ 4.6 mm, 5um, ACN: Water (60:40)

Method B

A THF solution of lithium tri-rert-butoxyaluminum hydride (1 M, 107.6 mL, 107.6 mmol) was added over 1 h to a solution of compound 33 (30.0 g, 67 mmol) in dry THF (300 mL) at 0-5 °C. After stirring for an additional 4 h at 5-10 °C, the reaction was determined to be complete by HPLC (NMT 1% of compound 33). The reaction was cooled to 0-5 °C and quenched by adding 4N HCl (473 mL). The phases were separated. The aqueous layer was extracted with DCM (2 x 225 mL) and the combined organic phase was washed sequentially with water (300 mL) and saturated brine solution (300 mL). The organic phase was then was concentrated to dryness by vacuum distillation below 50 °C. Methanol (150 mL) was added to the residue and concentrated to dryness by vacuum distillation below 50 °C. Water (450 mL) was then added to the residue and the mixture was stirred for 15-20 min., filtered and the cake was washed with water (240 mL). The white solid was dried in a hot air drier at 35-40 °C for 6 h to provide compound 34 (30 g, 99.6%).

Conversion of Compound 34 to crude DCA:

Method A

A solution of LiOH (187 mg, 4.4 mmol) in H₂0 (2.0 mL) was added to a solution of compound 34 (500 mg, 1.1 1 mmol) in THF (8 mL) and MeOH (8 mL). The resulting mixture was stirred for 3-4 h at 50 °C. Upon complete disappearance of the starting material by TLC, the reaction mixture was concentrated under vacuum. A mixture of water (10 mL) and 3 N HCl (1 mL) were combined and cooled to 0 °C and then added to the crude product. After stirring for 1 h at 0 °C, the precipitated solids were filtered and then washed with water (10 mL) and hexane (20 mL). Drying under vacuum at room temperature provided deoxycholic acid (DCA, 400 mg, 91% yield) as a white solid. TLC: -anisaldehyde charring, R_f for DC A = 0.32 and R_f for 2.1a = 0.82.

TLC mobile phase: 10% – Methanol in DCM.

1H NMR (500 MHz, DMSO): δ = 11.92 (s, 1H), 4.44 (s, 1H), 4.19 (s, 1H), 3.77 (s, 1H), 3.35-3.36 (m, 1H), 2.19-2.21 (m, 1H), 2.08-2.10 (m, 1H), 1.73-1.80 (m, 4H), 1.43- 1.63 (m, 6H), 1.15-1.35 (m, 12H), 0.98-1.05 (m, 2H), 0.89-0.90 (d, J = 6.0 Hz, 3H),

0.83 (s, 3H), 0.58 (s, 3H).

¹³C NMR (125 MHz, DMSO): δ =174.8, 71.0, 69.9, 47.4, 46.1, 46.0, 41.6, 36.3, 35.6, 35.1, 34.9, 33.8, 32.9, 30.8, 30.7, 30.2, 28.6, 27.1, 27.0, 26.1, 23.5, 23.0, 16.9, 12.4.

Mass (m/z) = 393 [M⁺, + 1].

IR = 3363, 2933, 2863, 1694, 1453, 1372, 1042, cm^-1.

m.p. = 171.4-173.6 °C (from ethanol); 174-176 °C (Alfa Aesar) and 171-174 °C (Aldrich)

[<x]_D = +47 (c = 1% in EtOH ), +54° (c = 2% in ethanol) [Alfa Aesar]

ELSD Purity: 99.7%: Retention time = 5.25 (Inertsil ODS 3 V, 250 ^χ 4.6 mm, 5um, ACN:

0.1% TFA in water (90:10).

Method B

A 20% solution of NaOH (40 g, 270 mmol) in H₂0 (54 mL) was added to a solution of compound 34 (30 g, 67 mmol) in THF (120 mL) and MeOH (120 mL) at 0-5 °C. The resulting mixture was stirred for 4 h at 25-35 °C. Upon completion of reaction by HPLC (NMT 0.5% of compound 34 and intermediates), the solvent was removed via vacuum distillation below 50 °C. The residue was dissolve in water (300 mL) and washed with DCM (2 x 150 mL). The pH of aqueous layer was adjusted to 1-2 with 2N HCl (~ 173 mL). The solids were filtered, washed thoroughly with water (3 L) and dried by a hot air drier at 70-75 °C until the moisture content is less than 2% to provide deoxycholic acid (DCA, 26 g, 99% yield) as a white solid.

EXAMPLE 9

Deoxycholic acid (DCA) Purification

1. Solvent Selection

Two solvent systems were explored for further purification of DCA: • 10% Hexanes in EtOAc

• DCM

The following experiments have been conducted and the experimental results tabulated below.

 

* The DCA to be purified was dissolved in a mixture of methanol and DCM and then the methanol was removed by azeotropic distillation. The amount of methanol required to dissolve the crude DCA depends on how pure it is to begin with.

Typical crude material was—75% pure and could be dissolved at reflux using 10% methanol-DCA (by volume) using—20 mL per gram. With purer DCA, the percentage of methanol had to be increased to 15%.

Effective purification was achieved by crystallization of the product from DCM following dissolution in a mixture of methanol and DCM and azeotropic removal of the methanol via atmospheric distillation.

2. Solvent Quantity

Experiments have been conducted using different solvent volumes and the experimental results are tabulated below.

Excellent recoveries and product quality were obtained at all solvent levels.

3. Isolation Temperature

The following experiments have been conducted by varying the isolation temperature and the results are tabulated below:

 

Higher quality product was obtained when isolation is done at 25-30 °C as compared to 10-15 °C. Purification of DCA in 100 g Scale

The final purification procedure for this step is given below:

 

Crude DCA (110 g) was dissolved in 10% methanol in DCM (2.5 L) at reflux temperature. To this clear solution 2.5 L of dichloromethane was added at reflux temperature and then about 3.0 L of solvent was distilled at atmospheric pressure (GC analysis of reaction mass supernatant revealed the presence of about 3% of methanol). The reaction slurry was cooled to 20-25 °C and then stirred for 3-4 h. The mixture was filtered and the solids were washed with DCM (300 mL). The product was dried in a hot air oven at 50-55 °C for 6-8 h.

The above dried DCA was added to water (1.0 L) and then 10% sodium hydroxide solution (122 mL) was added resulting in a clear solution. This solution was filtered through 5μ filter paper. The filtrate was diluted with water (2.0 L), and the pH was adjusted to 1— 2 with 2N HCl solution (204 mL). The precipitated solids were stirred for 1 h, filtered and the solids were washed with additional water (7.0 L). After drying in a hot air oven at 70-75 °C for 16-20 h, purified DCA (~ 66 g with more than 99% purity by HPLC RI detection) was obtained as a white solid.

TLC: 7-Anisaldehyde charring, R_f for DCA = 0.32 and R_f for compound 34 = 0.82. Eluent = 10% methanol in DCM. 1H NMR (500 MHz, DMSO): δ = 11.92(s, 1H),4.44(s, 1H), 4.19(s, 1H), 3.77 (s, 1H), 3.36-3.35 (m, 1H), 2.21-2.19 (m, 1H), 2.10-2.08 (m, 1H), 1.80-1.73 (m, 4H), 1.63- 1.43(m, 6H), 1.35-1.15(m, 12H), 1.05-0.98(m, 2H), 0.90-0.89 (d, J = 6.0 Hz, 3H), 0.83 (s, 3H), 0.58 (s, 3H).

1 C NMR (125 MHz, DMSO): δ =174.8, 71.0, 69.9, 47.4, 46.1, 46.0, 41.6, 36.3, 35.6, 35.1, 34.9, 33.8, 32.9, 30.8, 30.7, 30.2, 28.6, 27.1, 27.0, 26.1, 23.5, 23.0, 16.9, 12.4.

Mass (m/z) = 393 [M⁺, + 1].

IR = 3363, 2933, 2863, 1694, 1453, 1372, 1042, cm^-1.

m.p. = 171.4-173.6 °C (from ethanol); 174-176 °C (Alfa Aesar) and 171-174 °C (Aldrich).

Recrystallization of Deoxycholic acid (DC A)

DCA obtained from Method B (26 g) above, was charged into a clean and dry flask. Methanol (65 mL) and DCM (585 mL) were added. The mixture was heated to reflux to obtain a clear solution. DCM (650 mL) was charged to the solution and the solvent was distilled atmospherically until 780 mL of solvent was collected. The mixture was assayed by GC to determine the solvent composition. If the methanol content is more than 2%, add DCM (200 mL) and distill atmospherically until 200 mL of distillate have been collected. (Check for the methanol content by GC). The reaction mixture was cooled over 1-2 h to 20-25 °C and stirred at this temperature for 3-4 h. The product was filtered and washed with DCM (81 mL), dried in a hot air drier at 50-55 °C for 8 h. The purity was determined by HPLC. If single max impurity is more than 0.1%, the above process is repeated.

The dried material from the above was charged in to a clean flask. Water (190 mL) was added and followed by 10% aqueous NaOH (3.18 g in 31.8 mL of water). The solution was filtered through 5μ filter paper and the filtrate was diluted with additional water (380 mL). The pH was adjusted to 1-2 with 2 N HCl (53 mL). The resulting solids was filtered, washed thoroughly with water (1.9 L), and dried in a hot air drier at 70-75 °C until the water content is below 1% to give DCA as a white solid (17 g, % of recovery: 65). EXAMPLE 10

Alternate method of Synthesis and purification of DCA from compound 33

Step la— Hydrogenation of methyl 3a-acetoxy-12-oxo—5fi-chol-9(ll)-en-24-oate (24)

 

Dry Pd/C (75.0 g, 25 wt %) was added to 24 (300.0 g, 0.7 mol) in EtOAc (7.5 L, 25 vol). The reaction mixture was heated to 45°— 50°C and pressurized to 50 psi of H₂. HPLC analysis after 21 hours indicated < 1.0% area under the curve (AUC) of 24 remained; 4.6% AUC of the allylic alcohol impurity 86 and 1 1.1% AUC of the 87 formed. The reaction mixture was cooled to 30° – 35°C, filtered over Hyflo® (300 g) and washed with EtOAc (7.5 L) to remove the catalyst. The resulting filtrate was

concentrated to about 6 L and taken forward without further manipulation (67.8% AUC by HPLC, 5.5% AUC of the allylic alcohol impurity 86 and 13.0% AUC of 87).

Step lb/c – Oxidation of allylic alcohol 86 and 87 and rehydrogenation of 24 to methyl 3a-acetoxy-12-oxo-5fi-cholan-24-oate (33)

Step lb – PCC oxidation of allylic alcohol 86 and 87

A slurry of PCC (149.1 g, 1.03 equiv.) in EtOAc (1.5 L) was added to the 33 solution from above at 20°— 25°C. The reaction was allowed to proceed for 3.5 hours where HPLC analysis showed that < 1% AUC of the allylic alcohol 86 and < 1% AUC of 87 remained. The reaction mixture was filtered over Hyflo® (300 g) and washed with EtOAc (3.0 L). The EtOAc filtrate was washed with deionized (DI) water (2 x 3.6 L) and brine (3.6 L), filtered over Hyflo® (300 g) and washed with EtOAc (3.0 L). The resulting filtrate was concentrated to -7.5 L and taken forward without further manipulation (77.7% AUC by HPLC containing 5.3% AUC of 24).

Step lc— Rehydrogenation of 24 to 33

Powder activated carbon DARCO (60 g, 20 wt %) was added to the crude 33 solution from above containing 24. The resulting slurry was heated to 45°— 50°C for 4 hours, cooled to 30°— 35°C and filtered over Celite®. The filter cake was washed with EtOAc (7.5 L), concentrated to -7.5 L and added to dry Pd/C (60.0 g, 20 wt %). The reaction mixture was heated to 45° – 50°C and pressurized to 50 psi of H₂ for 6 hours. HPLC analysis indicated < 1.0% AUC of 24 remained; 1.1% AUC of 86 impurity and < 1.0% AUC of 87 formed. The reaction was deemed complete and cooled to 30° – 35°C, filtered over Celite® and washed with EtOAc (7.5 L). The EtOAc filtrate was concentrated to—5 volumes and azeotroped with MeOH (2 x 4.5 L) back down to—5 volumes. The resulting slurry was diluted with DI water (2.4 L) and maintained at 20-25 °C. The slurry was filtered, washed with DI water (2 x 600 mL) and dried under vacuum at 40° – 50°C to yield 266 g (88%) of 33 (66.2% AUC by HPLC).

Step 2— Synthesis of 34

A solution of 33 (245 g, 0.5 mol) in THF (2.5 L) was cooled to 0° – 5°C and 1 M solution of Li(t-BuO)₃A1H (822.9 niL, 1.5 equiv.) was added while maintaining the temperature below 5°C. The reaction mixture was stirred at 5° – 10°C for 22 hours. Reaction may be complete in 2-4 hours. HPLC analysis indicated that the reaction was complete with < 1% of 33 remaining. The reaction was quenched with 4 M HCl (3.7 L) while maintaining the temperature below 20°C. The reaction mixture was extracted with CH₂CI₂ (2 x 2.5 L) and the combined organic phases were washed with DI water (2 x 2.5 L). The CH₂C1₂ phase was concentrated to afford 300 g (122%) of 34 (73.5% AUC by HPLC). 1H NMPv analysis indicated that 9.7 wt % of THF and 0.8 wt % of CH₂C1₂ remained.

Step 3 – Synthesis of DCA

A NaOH solution (87.6 g, 4 equiv.) in DI water (438.6 mL) was added to a solution of 34 (245 g, 0.5 mol) in MeOH (980 mL) and THF (475 mL) at 0° – 5°C. The reaction mixture was allowed to warm to 20° – 25°C. HPLC analysis showed that the reaction was complete after 1 hour with < 0.5% 34 and < 0.5% of the hydrolysis intermediates remaining. The reaction was diluted with DI water (2.5 L) and

concentrated to—10 volumes. The aqueous solution was washed with CH₂C1₂ (2 x 1.3 L) and adjusted to pH 1.7— 2.0 using 2 M HCl (1.6 L). A white slurry formed and was stirred at 20° – 25 °C for 1 hour. The slurry was filtered, washed with DI water (7 x 1 L) and dried under vacuum to yield 195 g (91%) of DCA (82.2% AUC by HPLC).

Step 4 – Purification of DCA

A solution of DCA obtained above (190 g, 0.48 mol) in MeOH (475 mL) and CH₂C1₂ (4275 mL) was heated to 35° – 40°C. The MeOH/CH₂Cl₂ was distilled out of the mixture while CH₂CI₂ (4740 mL) was added matching the rate of distillation. Analysis of the solvent composition by Ή NMR indicated 4.5 mol % of MeOH remained relative to CH₂C1₂. The slurry was allowed to cool to 20°— 25°C and held for 16 hours. The solids were isolated by filtration, washed with CH₂Cl₂ (600 mL) and dried under vacuum to yield 104 g (55%) of DCA (> 99% AUC by HPLC-RID and 98.7% AUC by HPLC- CAD).

The recrystallization was repeated by heating a mixture of DCA (103 g, 0.3 mol) in MeOH (359 mL) and CH₂C1₂ (1751 mL) to 35° – 40°C. The MeOH/CH₂Cl₂ was distilled out of the mixture while CH₂CI₂ (3760 mL) was added matching the rate of distillation. Analysis of the solvent composition by 1H NMR indicated 4.7 mol % of MeOH remained relative to CH₂C1₂. The slurry was allowed to cool to 20°— 25°C. After 1 hour, the solids were isolated by filtration, washed with CH₂CI₂ (309 mL) and dried under vacuum to afford 82 g (79%) of DCA (> 99% AUC by HPLC-RID and 99.3% AUC by HPLC-C AD).

To assess the effect of additional purification and reprocessing, the product was recrystallized a third time prior to the normal final water isolation step. The above sample of DCA (80 g, 0.2 mol) in MeOH (240 mL) and CH₂C1₂ (1400 mL) was heated to 35° – 40°C. The MeOH/CH₂Cl₂ was distilled out of the mixture while CH₂C1₂ (2000 mL) was added matching the rate of distillation. Analysis of the solvent composition by ^!H NMR indicated 6.7 mol % of MeOH remained relative to CH₂C1₂. The slurry was allowed to cool to 20° – 25°C. After 1 hour, the solids were isolated by filtration, washed with CH₂CI₂ (240 mL) and dried under vacuum to afford 72 g (89%) of DCA (99.7% AUC by HPLC-CAD).

The sample was slurried in DI water (840 mL) and diluted with a solution of

NaOH (14.0 g) in DI water (140 mL). The resulting solution was filtered over Celite® and washed with DI water (1.4 L). The filtrate was adjusted to pH 1.6 with 2 M HCl (—300 mL) resulting in a white precipitate which was held for 1 hour at 20°— 25°C. The product was isolated by filtration, washed with DI water (9.0°L) and dried under vacuum to afford 63 g (87%) of DCA (99.7% AUC by HPLC-CAD).

 

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WO 2013044119

http://www.google.com/patents/WO2013044119A1?cl=en

Scheme 10

Example 4: Converting Compound 129 To DCA

[0125| In Scheme 1 below, there is provided a scheme for the synthesis and purification of DCA from compound 1.

Scheme 10

A. Conversion of Compound 129 to Compound 130:

Method Al

[0126] 10% Pd/C (900 mg) was added to a solution of compound 129 (2.0 g, 4.5 mmol) in EtOAc (150 mL) and the resulting slurry was hydrogenated in a Parr apparatus (50 psi) at 50 °C for 16 h. At this point the reaction was determined to be complete by TLC. The mixture was filtered through a small plug of Celite® and the solvent was removed under vacuum, providing compound 130 (1.6 g, 80% yield) as a white solid.

TLC: -anisaldehyde charring, R_t for 130 = 0.36. TLC mobile phase: 20% – EtOAc in hexanes.

Ή NMR (500 MHz, CDCL): δ = 4.67-4.71 (m, 1 H), 3.66 (s, 3H), 2.45-2.50 (t, J = 15 Hz, 2H ), 2.22-2,40 (m, 1H), 2.01 (s, 3H). 1 ,69- 1 .96 (m, 9H), 1 ,55 (s, 4H), 1 ,25- 1.50 (m, 8H)₅ 1.07-1 . 19 (m. 2H), 1 .01 (s, 6H), 0.84-0.85 (d, J = 7.0 Hz, 3H).

¹³C NMR (125 MHz, CDC1₃): δ = 214.4, 174.5, 170.4, 73.6, 58,5, 57.4, 51.3, 46,4, 43.9, 41.2, 38.0, 35.6, 35.5, 35.2, 34.8, 32.0, 31 .2, 30.4, 27.4. 26.8, 26.2, 25.9, 24.2, 22.6, 21 .2, 18.5, 1 1.6,.

Mass (m/z) = 447.0 | \! + 1 ], 464.0 [M^f + 18]. IR ( Br) = 3445, 2953, 2868, 1731 , 1698, 1257, 1029 cm^“1 , m.p. = 142,2- 144.4 °C (from EtOAc/hexanes mixture). [a]_D = +92 (c = l % in CHCl₃).

ELSD Purity: 96.6%: Retention time = 9.93 (Inertsil ODS 3V, 250 * 4.6 mm, 5 urn, ACN: 0.1 % TFA in water (90: 10)

Method A2

[0127J A slurry of 10% Pd/C (9 g in 180 mL of ethyl acetate) was added to a solution of compound 129 (36 g, 81 mmol) in EtOAc (720 mL) and the resulting slurry was treated with hydrogen gas (50 psi) at 45-50 °C for 16 h. (A total of 1080 mL of solvent may be used). At this point the reaction was determined to be complete by HPLC (NMT 1 % of compound 129). The mixture was filtered through Cclite® (10 g) and washed with ethyl acetate (900 mL). The filtrate was concentrated to 50% of its volume via vacuum distillation below 50 °C. To the concentrated solution was added pyridinium

chlorochromate (20.8 g) at 25-35 °C and the mixture was stirred for 2 h at 25-35 °C, when the reaction completed by LIPLC (allylic alcohol content is NMT 1 %).

[0128] The following process can be conducted if compound 129 content is more than 5%. Filter the reaction mass through Celite® (10 g) and wash with ethyl acetate (360 mL). Wash the filtrate with water (3 x 460 mL) and then with saturated brine (360 mL). Dry the organic phase over sodium sulphate (180 g), filter and wash with ethyl acetate ( 180 mL). Concentrate the volume by 50% via vacuum distillation below 50 °C. Transfer the solution to a clean and dry autoclave. Add slurry of 10% palladium on carbon (9 g in 1 80 mL of ethyl acetate). Pressurize to 50 psi with hydrogen and stir the reaction mixture at 45-50 °C for 16 h.

[0129] Upon complete consumption of compound 129 by HPLC ( the content of compound 129 being NMT 1 %), the reaction mixture was filtered through Celite® ( 10 g) and the cake was washed with ethyl acetate (900 mL). The solvent was concentrated to dryness via vacuum distillation below 50 °C. Methanol (150 mL) was added and concentrated to dryness via vacuum distillation below 50 °C. Methanol (72 mL) was added to the residue and the mixture was stirred for 15-20 min at 10- 15 °C, filtered and the cake was washed with methanol (36 mL). The white solid was dried in a hot air drier at 45-50 °C for 8 h to LOD being NMT 1% to provide compound 230 (30 g, 83.1 % yield).

B. Conversion of Compound 130 to Compound 1 1.a

Method Bl

[0130J A THF solution of lithium tri-te -butoxyaluminum hydride (1 M. 22.4 mL, 22.4 mmol) was added drop wise to a solution of compound 130 (2.5 g, 5.6 mmol) in THF (25 mL) at ambient temperature. After stirring for an additional 4-5 h, the reaction was determined to be complete by TLC. The reaction was quenched by adding aqueous HQ (1 M, 10 mL) and the mixture was diluted with EtOAc (30 mL). The phases were separated and the organic phase was washed sequentially with water (15 mL) and saturated brine solution (10 mL). The organic phase was then dried over anhydrous Na₂SO-i (3 g) and filtered. The filtrate was concentrated under vacuum and the resulting solid was purified by column chromatography [29 mm (W) x 500 mm (L), 60-120 mesh silica, 50 g], eluting with EtOAc/hexane (2:8) [5 mL fractions, monitored by TLC with p- anisaldehyde charring]. The fractions containing the product were combined and concentrated under vacuum to provide compound 131. a (2.3 g, 91 %) as a white solid.

TLC: /7-anisaldehyde charring, R_f for 131. a = 0.45 and R_t for 130 = 0.55. TLC mobile phase: 30% – EtOAc in hexanes.

Ή NMR (500 MHz, CDC1₃): δ = 4.68-4.73 (m, 1 H), 3.98 (s, 1 H), 3.66 (s, 3H), 2.34-2.40 (m, 1H), 2.21-2.26 (m, 1H), 2.01 (s, 3H), 1.75-1.89 (m, 6H), 1.39-1.68 (m, 16H), 1.00-1.38 (m, 3H), 0.96-0.97 (d, J = 5.5 Hz, 3H), 0.93 (s, 3H), 0.68 (s, 3H).

¹³C NMR (125 MHz, CDCI3): δ = 174.5, 170.5, 74.1 , 72.9, 51.3, 48.1 , 47.2, 46.4, 41.7, 35.8, 34.9, 34.7, 34.0, 33.5, 32.0, 30.9, 30.8, 28.6, 27.3, 26.8, 26.3, 25.9, 23.4. 22.9, 21.3. 17.2, 12.6

Mass (m/z) = 449.0 [M⁺ + 1 ], 466.0 [M⁺ + 18].

IR (KBr) = 3621 , 2938, 2866, 1742, 1730, 1262, 1 162, 1041 , cm⁴. m.p = 104.2-107.7 °C (from EtOAc).

[<x]_D = +56 (c = 1% in CHCI3). ELSD Purity: 97.0%: Retention time = 12.75 (Inertsil ODS 3V, 250 ^χ 4.6 mm, 5 urn, ACN: Water (60:40)

Method B2

[0131 ] A THF solution of lithium tri-/er?-butoxyaluminum hydride (1 M, 107.6 mL, 107.6 mmol) was added over 1 h to a solution of compound 130 (30.0 g, 67 mmol) in dry THF (300 mL) at 0-5 °C. After stirring for an additional 4 h at 5-10 °C, the reaction was determined to be complete by HPLC (NMT 1% of compound 130). The reaction was cooled to 0-5 °C and quenched by adding 4N HC1 (473 mL). The phases were separated. The aqueous layer was extracted with DCM (2 x 225 mL) and the combined organic phase was washed sequentially with water (300 mL) and saturated brine solution (300 mL). The organic phase was then was concentrated to dryness by vacuum distillation below 50 °C. Methanol (150 mL) was added to the residue and concentrated to dryness by vacuum distillation below 50 °C. Water (450 mL) was then added to the residue and the mixture was stirred for 15-20 min., filtered and the cake was washed with water (240 mL). The white solid was dried in a hot air drier at 35-40 °C for 6 h to provide compound 131.a (30 g, 99.6%).

C. Conversion of Compound 131.a to crude DCA:

[01321 To a solution of 131. a in MeOH (4 vol) and THF (4 vol) was added a solution of NaOH (4.0 equiv) in DI water (5 M) maintaining the temperature below 20 °C. HPLC analysis after 20 hours at 20-25 °C indicated <0.5% AUC of 131.a and the two

intermediates remained. The reaction was deemed complete, diluted with DI water (10 vol) and concentrated to -10 volumes. The sample was azeotroped with 2-MeTHF (2 x 10 vol) and assayed by Ή NMR to indicate MeOH was no longer present. The rich aqueous phase was washed with 2-MeTHF (2 x 10 vol) and assayed by HPLC to indicate 0.3% AUC of the alcohol impurity remained. The aqueous phase was diluted with 2- MeTHF (10 vol ) and adjusted to pH = 1 .7-2.0 using 2 M HC1 (~4 vol ). The phases were separated and the 2-MeTHF phase was washed with DI water (2 x 10 vol). The 2- MeTHF phase was filtered over Celite and the filter cake was washed with 2-MeTHF (2 vol). The 2-MeTHF filtrate was distillated to -10 volumes and azeotroped with ^-heptane containing Statsafe™ 5000 (3 x 10 vol) down to -10 vol. The mixture was assayed by Ή N MR to indicate <5 mol% of 2-MeTHF remained relative to o-heptane. The slurry was held for a minimum of 2 hours at 20-25 °C and filtered. The filter cake was washed with //-heptane (2 x 10 vol) and conditioned under vacuum on the Niitsche filter with N₂ for a minimum of 1 hour to afford DCA-crude as white solids. Purity = 94.6% (by HPLC). HPLC analysis for DS-DCA (NMT 5% AUC).

D. Recrystallization of DCA

|0133] DCA-crude was diluted with 2 mol% MeOH in CH₂C1₂ (25 vol) and heated to 35—37 °C for 1 hour. The slurry was allowed to cool to 28-30 °C and filtered. The filter cake was washed with CITC (5 vol) and dried under vacuum at 40 °C to afford DCA. HPLC analysis for DS-DCA (NMT 0.15% AUC).

[0134] DCA was dissolved in 10% DI water/ EtOH (12 vol), polish filtered over Celite and washed with 10% DI water/ EtOH (3 vol). The resulting 15 volume filtrate was added to DI water (30 vol) and a thin white slurry was afforded. The slurry was held for 24 hours, filtered, washed with DI water (20 vol) and dried under vacuum at 40 °C to afford pure DCA. OVI analysis for CH₂C1₂. EtOH. ^-heptane, MeOH and MeTHF was conducted to ensure each solvent was below ICH guideline. KF analysis conducted (NMT 2.0%). Purity = 99.75% (by HPLC). Yield from DCA-crude = 59%.

……………………………

WO 2012174229

 http://www.google.com/patents/WO2012174229A2?cl=en

In Scheme 1 below, there is provided a scheme for the synthesis and purification of deoxycholic acid from compound 1.

Scheme 1

Conversion of Compound 1 to Compound 2:

[0043] The hydrogenation of compound 1 on 10.0 g scale using dry 10 % Pd/C (15 wt %) in ethyl acetate (20 parts) was added and applied about 50 psi hydrogen pressure and temperature raised to 70 °C. After reaching temperature 70 °C, observed increase of hydrogen pressure to about 60 psi, at these conditions maintained for 60 h. After 60 hours 0.6% of compound 2 and 2.75%> of allylic alcohol were still observed, so further stirred for additional 12 h (observed 0.16% of allylic alcohol and 0.05% of compound 2). After work-up, the reaction provided 9.5 g of residue.

[0044] Anther hydrogenation reaction on 25 g of compound 1 with above conditions for 76 h provided 24.5 g of residue.

Method A

[0045] 10% Pd/C (900 mg) was added to a solution of compound 1 (2.0 g, 4.5 mmol) in EtOAc (150 mL) and the resulting slurry was hydrogenated in a Parr apparatus (50 psi) at 50 °C for 16 h. At this point the reaction was determined to be complete by TLC. The mixture was filtered through a small plug of Celite® and the solvent was removed under vacuum, providing compound 2 (1.6 g, 80%> yield) as a white solid.

TLC: /?-anisaldehyde charring, R_f for 2 TLC mobile phase: 20% – EtOAc in hexanes.

1H NMR (500 MHz, CDC1₃): δ = 4.67-4.71 (m, 1H), 3.66 (s, 3H), 2.45-2.50 (t, J = 15 Hz, 2H), 2.22-2.40 (m, 1H), 2.01 (s, 3H), 1.69-1.96 (m, 9H), 1.55 (s, 4H), 1.25-1.50 (m, 8H), 1.07-1.19 (m, 2H), 1.01 (s, 6H), 0.84-0.85 (d, J= 7.0 Hz, 3H).

¹³C NMR (125 MHz, CDC1₃): δ = 214.4, 174.5, 170.4, 73.6, 58.5, 57.4, 51.3, 46.4, 43.9, 41.2, 38.0, 35.6, 35.5, 35.2, 34.8, 32.0, 31.2, 30.4, 27.4, 26.8, 26.2, 25.9, 24.2, 22.6, 21.2, 18.5,11.6,.

Mass (m/z) = 447.0 [M⁺ + 1], 464.0 [M⁺ + 18].

IR (KBr) = 3445, 2953, 2868, 1731, 1698, 1257, 1029 cm^“1.

m.p. =142.2-144.4 °C (from EtO Ac/hex anes mixture).

[a]_D = +92 (c = 1% in CHC1₃).

ELSD Purity: 96.6%: Retention time = 9.93 (Inertsil ODS 3V, 250 4.6 mm, 5 urn, ACN: 0.1% TFA in water (90: 10)

Method B

[0046] A slurry of 10%> Pd/C (9 g in 180 mL of ethyl acetate) was added to a solution of compound 1 (36 g, 81 mmol) in EtO Ac (720 mL) and the resulting slurry was treated with hydrogen gas (50 psi) at 45-50 °C for 16 h. (A total of 1080 mL of solvent may be used). At this point the reaction was determined to be complete by HPLC (NMT 1% of compound 1). The mixture was filtered through C elite® (10 g) and washed with ethyl acetate (900 mL). The filtrate was concentrated to 50% of its volume via vacuum distillation below 50 °C. To the concentrated solution was added pyridinium

chlorochromate (20.8 g) at 25-35 °C and the mixture was stirred for 2 h at 25-35 °C, when the reaction completed by HPLC (allylic alcohol content is NMT 1%).

[0047] The following process can be conducted if compound 1 content is more than 5%>. Filter the reaction mass through Celite® (10 g) and wash with ethyl acetate (360 mL). Wash the filtrate with water (3 x 460 mL) and then with saturated brine (360 mL). Dry the organic phase over sodium sulphate (180 g), filter and wash with ethyl acetate (180 mL). Concentrate the volume by 50% via vacuum distillation below 50 °C. Transfer the solution to a clean and dry autoclave. Add slurry of 10% palladium on carbon (9 g in 180 mL of ethyl acetate). Pressurize to 50 psi with hydrogen and stir the reaction mixture at 45-50 °C for 16 h.

[0048] Upon complete consumption of compound 1 by HPLC (the content of compound 1 being NMT 1%), the reaction mixture was filtered through Celite® (10 g) and the cake was washed with ethyl acetate (900 mL). The solvent was concentrated to dryness via vacuum distillation below 50 °C. Methanol (150 mL) was added and concentrated to dryness via vacuum distillation below 50 °C. Methanol (72 mL) was added to the residue and the mixture was stirred for 15-20 min at 10-15 °C, filtered and the cake was washed with methanol (36 mL). The white solid was dried in a hot air drier at 45-50 °C for 8 h to LOD being NMT 1% to provide compound 2 (30 g, 83.1 % yield).

Conversion of Compound 2 to Compound 3:

Method A

[0049] A THF solution of lithium tri-tert-butoxyaluminum hydride (1 M, 22.4 mL, 22.4 mmol) was added drop wise to a solution of compound 2 (2.5 g, 5.6 mmol) in THF (25 mL) at ambient temperature. After stirring for an additional 4-5 h, the reaction was determined to be complete by TLC. The reaction was quenched by adding aqueous HCl (1 M, 10 mL) and the mixture was diluted with EtO Ac (30 mL). The phases were separated and the organic phase was washed sequentially with water (15 mL) and saturated brine solution (10 mL). The organic phase was then dried over anhydrous Na₂S0₄ (3 g) and filtered. The filtrate was concentrated under vacuum and the resulting solid was purified by column chromatography [29 mm (W) x 500 mm (L), 60-120 mesh silica, 50 g], eluting with EtO Ac/hex ane (2:8) [5 mL fractions, monitored by TLC with p- anisaldehyde charring]. The fractions containing the product were combined and concentrated under vacuum to provide compound 3 (2.3 g, 91%) as a white solid.

TLC: /?-anisaldehyde charring, R_f for 3 = 0.45 and R_f for 2 = 0.55.

TLC mobile phase: 30% – EtO Ac in hexanes.

1H NMR (500 MHz, CDC1₃): δ = 4.68-4.73 (m, 1H), 3.98 (s, 1H), 3.66 (s, 3H), 2.34-2.40 (m, 1H), 2.21-2.26 (m, 1H), 2.01 (s, 3H), 1.75-1.89 (m, 6H), 1.39-1.68 (m, 16H), 1.00-1.38 (m, 3H), 0.96-0.97 (d, J= 5.5 Hz, 3H), 0.93 (s, 3H), 0.68 (s, 3H). ^ljC NMR (125 MHz, CDC1₃): δ = 174.5, 170.5, 74.1, 72.9, 51.3, 48.1, 47.2, 46.4, 41.7, 35.8, 34.9, 34.7, 34.0, 33.5, 32.0, 30.9, 30.8, 28.6, 27.3, 26.8, 26.3, 25.9, 23.4, 22.9, 21.3, 17.2, 12.6

Mass (m/z) = 449.0 [M⁺ + 1], 466.0 [M⁺ + 18].

IR (KBr) = 3621, 2938, 2866, 1742, 1730, 1262, 1162, 1041, cm^“1.

m.p = 104.2-107.7 °C (from EtOAc).

[a]_D = +56 (c = 1% in CHC1₃).

ELSD Purity: 97.0%: Retention time = 12.75 (Inertsil ODS 3V, 250 4.6 mm, 5 urn, ACN: Water (60:40)

Method B

[0050] A THF solution of lithium tri-tert-butoxyaluminum hydride (1 M, 107.6 mL, 107.6 mmol) was added over 1 h to a solution of compound 2 (30.0 g, 67 mmol) in dry THF (300 mL) at 0-5 °C. After stirring for an additional 4 h at 5-10 °C, the reaction was determined to be complete by HPLC (NMT 1% of compound 2). The reaction was cooled to 0-5 °C and quenched by adding 4N HC1 (473 mL). The phases were separated. The aqueous layer was extracted with DCM (2 x 225 mL) and the combined organic phase was washed sequentially with water (300 mL) and saturated brine solution (300 mL). The organic phase was then was concentrated to dryness by vacuum distillation below 50 °C. Methanol (150 mL) was added to the residue and concentrated to dryness by vacuum distillation below 50 °C. Water (450 mL) was then added to the residue and the mixture was stirred for 15-20 min., filtered and the cake was washed with water (240 mL). The white solid was dried in a hot air drier at 35-40 °C for 6 h to provide compound 3 (30 g, 99.6%).

Conversion of Compound 3 to crude DCA:

[0051] To a solution of 3 in MeOH (4 vol) and THF (4 vol) was added a solution of NaOH (4.0 equiv) in DI water (5 M) maintaining the temperature below 20 °C. HPLC analysis after 20 hours at 20-25 °C indicated <0.5% AUC of 3 and the two intermediates remained. The reaction was deemed complete, diluted with DI water (10 vol) and concentrated to ~10 volumes. The sample was azeotroped with 2-MeTHF (2 x 10 vol) and assayed by 1H NMR to indicate MeOH was no longer present. The rich aqueous phase was washed with 2-MeTHF (2 x 10 vol) and assayed by HPLC to indicate 0.3% AUC of the alcohol impurity remained. The aqueous phase was diluted with 2-MeTHF (10 vol) and adjusted to pH = 1.7-2.0 using 2 M HC1 (~4 vol). The phases were separated and the 2-MeTHF phase was washed with DI water (2 x 10 vol). The 2- MeTHF phase was filtered over Celite and the filter cake was washed with 2-MeTHF (2 vol). The 2-MeTHF filtrate was distillated to ~10 volumes and azeotroped with n-heptane containing Statsafe™ 5000 (3 ^x 10 vol) down to ~10 vol. The mixture was assayed by 1H NMR to indicate <5 mol% of 2-MeTHF remained relative to n-heptane. The slurry was held for a minimum of 2 hours at 20-25 °C and filtered. The filter cake was washed with n-heptane (2 x 10 vol) and conditioned under vacuum on the Nutsche filter with N₂ for a minimum of 1 hour to afford DCA-crude as white solids. Purity = 94.6% (by HPLC). HPLC analysis for DS-DCA (NMT 5% AUC).

Recrystallization of Deoxycholic acid (DCA)

[0052] DCA-crude was diluted with 2 mol% MeOH in CH₂C1₂ (25 vol) and heated to 35-37 °C for 1 hour. The slurry was allowed to cool to 28-30 °C and filtered. The filter cake was washed with CH₂C1₂ (5 vol) and dried under vacuum at 40 °C to afford DCA. HPLC analysis for DS-DCA (NMT 0.15% AUC).

[0053] DCA was dissolved in 10% DI water/ EtOH ( 12 vol), polish filtered over Celite and washed with 10% DI water/ EtOH (3 vol). The resulting 15 volume filtrate was added to DI water (30 vol) and a thin white slurry was afforded. The slurry was held for 24 hours, filtered, washed with DI water (20 vol) and dried under vacuum at 40 °C to afford pure DCA. OVI analysis for CH₂C1₂, EtOH, n-heptane, MeOH and MeTHF was conducted to ensure each solvent was below ICH guideline. KF analysis conducted (NMT 2.0%). Purity = 99.75% (by HPLC). Yield from DCA-crude = 59%.

……………………………..

old cut paste

http://clinicaltrials.gov/ct2/show/NCT01426373

The drug is sodium deoxycholate for injection, code-named ATX-101 was developed for the treatment of lipomas – benign tumors of subcutaneous adipose tissue, as well as other unwanted fatty growths, such as a double chin. This substance, which is a salt of one of the bile acids, emulsifies fats, destroying their excess deposits

 

ATX-101 (a first-in-class injectable drug being studied for the reduction of localized fat. ATX-101 is a proprietary formulation of deoxycholate  a well-studied endogenous compound that is present in the body), a facial injectable drug for the reduction of unwanted fat under the chin, or submental fat. V. Leroy Young, MD, FACS, presented the initial results at the American Society for Aesthetic Plastic Surgery (ASAPS) 45^th Annual Aesthetic Meeting in Vancouver, British Columbia, on May 4, 2012.

In August 2010 Bayer Consumer Care AG signed a licensing and development collaboration agreement with KYTHERA, thereby obtaining commercialization rights to ATX-101 outside the US and Canada. KYTHERA and Bayer are collaborating on the development of ATX-101 in Europe.

KYTHERA Biopharmaceuticals Inc. 02 MAR 3013,  announced positive interim results from a Phase IIIb multi-center open-label study (ATX-101-11-26) to evaluate the safety and efficacy of ATX-101 an investigational injectable drug for the reduction of unwanted submental fat (SMF) commonly known as double chin. The results presented at the Late Breaking Research Symposium at the 71st American Academy of Dermatology (AAD) Annual Meeting in Miami Beach Fla. found that ATX-101 is well-tolerated and may be effective in reducing SMF by both clinician and patient reported outcome measures. The ATX-101 global clinical development program has enrolled more than 2500 total patients of which more than 1500 have been treated with ATX-101.

“In my practice patients often request a non-surgical way to treat their submental fat or undesirable double chin” said investigator Susan Weinkle MD FAAD a board certified dermatologist and affiliate clinical professor at the University of South Florida. “For these patients double chin is often resistant to diet and exercise. The results of this study suggest that microinjections of ATX-101 can reduce submental fat without worsening skin laxity.”

ATX-101 is a proprietary synthetically-derived formulation of deoxycholic acid (DCA) a naturally-occurring molecule found in the body that aids in fat metabolism. In this open-label Phase IIIb study interim results three months after the last ATX-101 treatment found:

• Reduction of submental fat
  • 87 percent of patients achieved at least a one-grade improvement from baseline on the Clinician-Reported Submental Fat Rating Scale (CR-SMFRS)
  • Similarly 83 percent of patients achieved at least a one-grade improvement on the Patient-Reported Submental Fat Rating Scale (PR-SMFRS)
• 96 percent of patients had unchanged or improved skin laxity based on the clinician rated Submental Skin Laxity Grading Scale (SMSLG)
• 95 percent of patients were satisfied with treatment based on the Global Post Treatment Satisfaction Scale
• Adverse events were of mild to moderate intensity transient and primarily associated with the treatment area

Topline results from this study were announced in November 2012. As previously announced 71.3 percent of subjects had at least a one-grade improvement on the CR-SMFRS / PR-SMFRS composite and 14.0 percent had at least a two-grade improvement on the same composite measure.

These results are based on a multicenter 12-month open-label Phase IIIb study conducted at 21 sites across the United States evaluating 165 adults who received injections of ATX-101 for up to six treatments at four-week intervals. Patients received ATX-101 (2 mg/cm2) by subcutaneous microinjections directly into their SMF and were evaluated three months after their last treatment. The study population includes females (77.6 percent) and males (22.4 percent) with a mean age of 47 who report at least moderate SMF and dissatisfaction with the appearance of their chin. All Fitzpatrick Skin Types an industry standard scale to categorize skin tone are represented.

“We are pleased with these ATX-101 study results” said Patricia S. Walker M.D. Ph.D. chief medical officer KYTHERA Biopharmaceuticals Inc. “These results along with efficacy analyses in double-blind placebo-controlled studies support ATX-101 entering the market as potentially the first medical aesthetic drug approved for the reduction of submental fat.”

About ATX-101

ATX-101 is a potential first-in-class injectable drug candidate under clinical investigation for the reduction of unwanted submental fat. ATX-101 is a proprietary formulation of synthetic deoxycholic acid a well-characterized endogenous compound that is present in the body to promote the natural breakdown of dietary fat. ATX-101 is designed to be a locally-injected drug that causes proximal preferential destruction of adipocytes or fat cells with minimal effect on surrounding tissue. Based on clinical trials conducted to date ATX-101 has exhibited significant meaningful and durable results in the reduction of submental fat which commonly presents as an undesirable “double chin.” These results correspond with subject satisfaction measures demonstrating meaningful improvement in perceived chin appearance.

In August 2010 Bayer signed a licensing and collaboration development agreement with KYTHERA thereby obtaining development and commercialization rights to ATX-101 outside of the U.S. and Canada. Bayer recently completed two pivotal Phase III trials of ATX-101 in Europe for the reduction of submental fat. Topline results from these trials were reported in the second quarter of 2012. KYTHERA completed enrollment in its pivotal Phase III clinical program for ATX-101 in more than 1000 subjects randomized to ATX-101 or placebo in 70 centers across the United States and Canada in August 2012. The Company expects to release topline results in mid-2013.

About KYTHERA Biopharmaceuticals Inc.

KYTHERA Biopharmaceuticals Inc. is a clinical-stage biopharmaceutical company focused on the discovery development and commercialization of novel prescription products for the aesthetic medicine market. KYTHERA initiated its pivotal Phase III clinical program for ATX-101 in March 2012 and completed enrollment of more than 1000 patients randomized to ATX-101 or placebo in 70 centers across the U.S. and Canada in August 2012. KYTHERA also maintains an active research interest in hair and fat biology. Find more information at www.kytherabiopharma.com.

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GLENMARK SCIENTIST ,  INDIA
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10/07/2014

European Medicines Agency recommends 39 medicines for human use for marketing authorisation in first half of 2014

Thirty-nine medicines for human use were recommended for marketing authorisationby the European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) in the first half of 2014, compared with 44 in first half of 2013 and 33 in first half of 2012.

This figure includes a number of new innovative medicines with the potential to meet unmet medical needs, treat diseases for which no treatments were previously available or bring significant added benefit to patients over existing therapies. Among these medicines are the anticancer medicines Mekinist (trametinib) and Gazyvaro (obinutuzumab), the anti-inflammatory* Entyvio (vedolizumab), the anti-infective Daklinza (daclatasvir), as well as Translarna (ataluren) and Sylvant (siltuximab), which are both intended for the treatment of rare conditions.

In parallel, the number of medicines recommended for approval via the European Union centralised procedure based on generic or informed consent applications has decreased compared with the first half of 2013 (6 versus 13).

More than two in three applicants received scientific advice from the CHMP during the development phase of their medicine, and for innovative medicines four in five applicants received such advice. This is a significant increase compared with the first half of 2013 (when one in two applicants received scientific advice), and mirrors the growing number of requests for scientific advice received by the Agency.

Confirming the trend observed in the past few years, the number of new medicines intended for the treatment of rare diseases is steadily increasing, providing treatments for patients who often have only few or no options. In the first half of 2014, eight medicines were recommended for the treatment of rare diseases. This number includes three medicines for which the CHMP recommended conditional approval but whose applications were withdrawn by the sponsor prior to a final decision by the European Commission **.

Conditional approval is one of the Agency’s mechanisms to provide early patient access to medicines that fulfill unmet medical needs or address life-threatening diseases. The CHMP also used this mechanism for the recommendation of the first treatment for Duchenne muscular dystrophy (Translarna), a life-threatening condition.

The CHMP granted two positive opinions after an accelerated assessment for the medicines Sylvant and Daklinza; this mechanism aims to speed up the assessment of medicines that are expected to be of major public health interest particularly from the point of view of therapeutic innovation.

The CHMP also gave an opinion on the use of a new combination product in the treatment of hepatitis C virus (HCV) infection in a compassionate use programme (ledipasvir and sofosbuvir). These programmes are intended to give patients with a life-threatening, long-lasting or seriously disabling disease access to treatments that are still under development. The treatment paradigm of hepatitis C is currently shifting rapidly, with the development of several new classes of direct-acting antivirals. By recommending the conduct of three compassionate use programmes and the marketing authorisation of three new medicines for HCV infection over the past eight months, the Agency is actively supporting this shift which is expected to bring significant added benefit to patients.

Notes

* On Friday 11 July 2014 at 11:00 the statement, ‘the anti-infectives Entyvio (vedolizumab) and Daklinza (daclatasvir)’ was corrected to ‘the anti-inflammatory Entyvio (vedolizumab), the anti-infective Daklinza (daclatasvir)’.

** The CHMP had recommended a conditional approval for Vynfinit (vintafolide) and its companion diagnostics Folcepri (etarfolatide) and Neocepri (folic acid). After authorisation, the company was to provide confirmatory data from an ongoing study with Vynfinit. However, before the authorisation process could be completed by the European Commission, preliminary data from this study became available which showed that the study could not confirm the benefit of Vynfinit in ovarian cancer patients. Therefore, the company terminated the study and decided to withdraw the applications.

Crystallisations are frequent process steps in the manufacture of active pharmaceutical ingredients (APIs). They are the primary means of intermediate or product formation and separation to achieve the desired purity and form. These unit operations are complex processes which are difficult to control due to the interlinked chemical and physical effects. For example, chemical aspects such as salt and polymorph concerns are in the forefront of process research, but physical effects manifesting themselves on scale-up, due to equipment influences, can be equally important for the successful outcome of a campaign. Several operational parameters, such as temperature or impeller speed, need to be understood and controlled to achieve constant desupersaturation, consistent narrow particle size distribution around the desired mean, minimal attrition, and homogeneous growth conditions. This paper focuses on the equipment influence on crystallisations, relating it to first principles with respect to heat and momentum transfer, analysing it with computational fluid dynamics (CFD), and demonstrating its process impact using examples from recent development work. Dynamic process modelling and CFD are state-of-the-art engineering tools to identify process requirements and match them with equipment capabilities. The work reported here demonstrates how a semiquantitative application of these tools can lead to a controllable, robust process in an existing plant despite the time and resource limitations usually encountered in the industry.

http://pubs.acs.org/doi/full/10.1021/op040013n

Application of Process Modelling Tools in the Scale-Up of Pharmaceutical Crystallisation Processes

Beloranib

CAS   251111-30-5 (beloranib),529511-79-3 (beloranib hemioxalate)

(E)-(3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl 3-(4-(2-(dimethylamino)ethoxy)phenyl)acrylate

6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol

Mechanism of Action:methionine aminopeptidase 2 (MetAP2) inhibitor

Indication:Obesity US Patent : US6063812 Patent Exp Date: May 13, 2019

Originator: Chong Kun Dang (CKD) Pharma (종근당) Chong Kun Dang Pharm Corp

Developer: Zafgen Inc. (자프젠)Zafgen Corporation

Zafgen’s Prader-Willi syndrome therapy receives orphan drug designation in Europe The European Commission (EC) has granted orphan drug designation to US-based Zafgen for its beloranib for treating Prader-Willi syndrome. Beloranib is a potent inhibitor of Methionine aminopeptidase-2 that reduces hunger while stimulating the use of stored fat as an energy source (MetAP2). MetAP2 is an enzyme that modulates the activity of key cellular processes that control metabolism. http://www.pharmaceutical-technology.com/news/newszafgens-prader-willi-syndrome-therapy-receives-orphan-drug-designation-in-europe-4316842?WT.mc_id=DN_News

INTRODUCTION   Beloranib is an experimental drug candidate for the treatment of obesity. It was discovered by CKD Pharmaceuticals and is currently being developed by Zafgen. Beloranib, an analog of the natural chemical compound fumagillin, is an inhibitor of the enzyme METAP2. It was originally designed as angiogenesis inhibitor for the treatment of cancer. However, once the potential anti-obesity effects of METAP2 inhibition became apparent, the clinical development began to focus on these effects and beloranib has shown positive results in preliminary clinical trials for this indication. At such low doses, says Thomas E. Hughes, president and chief executive officer of Zafgen, toxicity concerns tend to evaporate, in part because so little opportunity exists to inhibit off-target proteins.

Zafgen, a small pharmaceutical company in Cambridge, Mass., sees high selectivity and low toxicity with its covalent molecule for treating obesity, beloranib hemioxalate, also known as ZGN-433. “You’re passing a wave of the molecule through the body,” he says. “It hits the different tissues, silences the target enzyme where it finds it, and then it goes away.” Zafgen’s drug candidate inhibits an enzyme called methionine aminopeptidase 2 (MetAP2), which had been of interest in oncology circles until it turned out to be a poor target for treating cancer in mice. However, animals treated with a MetAP2 inhibitor lost weight. Zafgen pursued the enzyme as a target for obesity. Its drug candidate contains a spiroepoxide that bonds with a histidine in the protein’s active site.

ZGN-433 has undergone a Phase I clinical trial, in which obese volunteers lost up to 2 lb per week. It will enter Phase II trials within a year, Hughes says, funded by $33 million the company raised from investors. With dosing of up to 2 mg twice per week, ZGN-433 reaches a maximum concentration in the body of just a few nanomolar for several hours before the body quickly eliminates it, Hughes says. During that time, the drug is much more likely to interact with MetAP2 than with anything else. “You’re flying under the radar of a lot of concerns,” he says. “Drug-drug interactions are not an issue. There’s just not enough inhibitor to go around.

The same is true for off-target inhibition: The chance of off-target toxicity is largely gone.” Proponents of covalent inhibitors are quick to point out that dozens of such drugs are already on the market. They include aspirin, the world’s most widely used medicine; penicillin and related antibiotics; and recently developed blockbusters such as Plavix, Prevacid, and Nexium. The drugs treat a broad range of conditions, and many have minimal side effects, even when taken for years. By one count, of the marketed drugs that inhibit enzymes, more than one-third work by covalent modification (Biochemistry, DOI: 10.1021/bi050247e).

6-O-(4-dimethylaminoethoxy) cinnamoyl fumagillol hemioxalate

Beloranib, ZGN-433, CKD-732
IUPAC name [(3R,6R,7S,8S)-7-methoxy-8-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-2-oxaspiro[2.5]octan-6-yl] (E)-3-[4-[2-(dimethylamino)ethoxy]phenyl]prop-2-enoate
Other names CKD-732; ZGN-433
Identifiers
CAS number	251111-30-5 , 529511-79-3 (hemioxalate)
PubChem	6918502
ChemSpider	26286923
UNII	FI471K8BU6
Jmol-3D images	Image 1
Properties
Molecular formula	C29H41NO6
Molar mass	499.64 g mol−1
Except where noted otherwise, data are given for materials in their standard state (at 25 °C (77 °F), 100 kPa)

Beloranib (previously known as CKD-732; ZGN-433), a methionine aminopeptidase 2 (MetAP2) inhibitor originally designed as an anticancer agent, is being developed by Zafgen as a first-in-class obesity therapy. Beloranib, a twice-daily injection, is discovered by korean company Chong Kun Dang (CKD) Pharmaceuticals and was licensed to Cambridge, MA-based startup Zafgen, Inc. Zafgen holds exclusive worldwide rights (exclusive of Korea) for development and commercialization of beloranib. Beloranib, an analog of  the antimicrobial agent fumagillin, is an inhibitor of the enzyme METAP2 involved in fatty acid production. It was originally designed as angiogenesis inhibitor for the treatment of cancer. However, once the potential anti-obesity effects of METAP2 inhibition became apparent, the clinical development began to focus on these effects.

Zafgen has chosen to develop beloranib not for the folks that need to shed a few pounds, but for severely obese people, and smaller groups of patients with rare and dangerous conditions. In January 2013, beloranib was granted orphan drug designation by the U.S. Food and Drug Administration to treat a rare genetic condition known as Prader-Willi Syndrome (PWS) that causes obesity through compulsive eating. Zafgen plans to seek the same designation for beloranib in craniopharyngioma (a rare benign brain tumor) related obesity as well. By going after these orphan indications, Zafgen can get onto the market quicker and cheaper than if it went straight for the larger obesity market. Zafgen recently completed two Phase 2a clinical trials evaluating beloranib’s ability to reduce body weight and to improve hyperphagia, one in PWS patients and one in severely obese patients. In its Phase 2a clinical trials, Zafgen observed reductions in body weight, body mass and body fat content in both patient populations and reductions in hyperphagia-related behaviors in PWS patients.

On June 19, 2014, Zafgen Inc. raised $96 million in its initial public offering (IPO) on the Nasdaq under the symbol “ZFGN” amid strong demand from investors. With its IPO cash, Zafgen plans to initiate its Phase 3 clinical program, consisting of two Phase 3 clinical trials, of beloranib in PWS patients, with the first Phase 3 trial to start in the second half of 2014, after finalizing the program design based on ongoing conversations with the FDA and certain European regulatory authorities. Zafgen is also planning a phase 2a trial in craniopharyngioma, and a Phase 2b trila in patients with severe obesity, all this year. The composition of matter patent (US6063812) on beloranib will each expire in May 2019.  Zafgen owns two issued U.S. patents relating to beloranib polymorph compositions of matter that will expire in 2031 and two issued U.S. patents to methods of treating obesity that will expire in 2029.   Beloranib is an experimental drug candidate for the treatment of obesity. It was discovered by CKD Pharmaceuticals and is currently being developed by Zafgen.^[1] Beloranib, an analog of the natural chemical compound fumagillin, is an inhibitor of the enzyme METAP2.^[2] It was originally designed as angiogenesis inhibitor for the treatment of cancer.^[3] However, once the potential anti-obesity effects of METAP2 inhibition became apparent, the clinical development began to focus on these effects and beloranib has shown positive results in preliminary clinical trials for this indication.^[4][5]

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http://www.google.com/patents/WO2005082349A1?cl=en

compound O-(4- dimethylaminoethoxycinnamoyl)fumagillol can be used in the form of a salt, e.g., acetate, lactate, benzoate, salicylate, mandelate, oxalate, methanesulfonate, or p- toluenesulfonate. Korean Patent No. 0357542 and its corresponding patents (U.S. Patent No. 6,063,812, Japanese Patent No. 3370985, and European Patent No. 1077964), filed by the present applicant, disclose fumagiUol derivatives, including the compounds used in the present invention. The composition of the present invention can be prepared in combination with pharmaceutically acceptable carriers commonly used in pharmaceutical formulations.

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http://www.google.com/patents/WO2012064838A1?cl=en

MetAP2 encodes a protein that functions at least in part by enzymatically removing the amino terminal methionine residue from certain newly translated proteins, such as, glyceraldehyde-3- phosphate dehydrogenase (Warder et al. (2008) J Proteome Res 7:4807). Increased expression of the MetAP2 gene has been historically associated with various forms of cancer. Molecules inhibiting the enzymatic activity of MetAP2 have been identified and have been explored for their utility in the treatment of various tumor types (Wang et al. (2003) Cancer Res 63:7861) and infectious diseases, such as, microsporidiosis, leishmaniasis, and malaria (Zhang et al. (2002) J. Biomed Sci. 9:34). Notably, inhibition of MetAP2 activity in obese and obese-diabetic animals leads to a reduction in body weight in part by increasing the oxidation of fat and in part by reducing the consumption of food (Rupnick et al. (2002) Proc Natl Acad Sci USA 99: 10730). [0003] 6-O-(4-Dimethylaminoethoxy)cinnamoyl fumagillol is a METAP2 inhibitor and is useful in the treatment of, e.g., obesity. 6-O-(4-Dimethylaminoethoxy)cinnamoyl fumagillol is characterized by formula I:

Example 1 [0060] Crystalline, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol was prepared as follows: [0061] Approximately 423 mg of amorphous gum/oil-like 6-O-(4- dimethylaminoethoxy)cinnamoyl fumagillol free base compound was dissolved in ca. 6 mL of diisopropylether (IPE). The solution was allowed to stir for ca. 24 hours at ambient temperature (18-22°C) during which time solid precipitated. The resulting solid was isolated by filtration and dried under vacuum at ambient for ca. 4 hours (yield 35.8 %).

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http://www.google.com/patents/WO1999059986A1?cl=en

Example 14 : 0-(4-dimethylaminocinnamoyl)fumagillol 1) To a solution of 4-dimethylaminocinnamic acid (950 mg) in toluene (20 ml), dipyridyl disulfide (1.64 g) and triphenyl phosphine (1.97 g) were added, and the mixture was stirred for 12 hours. 2) The resultant solution of 1) was added to fumagillol (500 mg) at room temperature. Sodium hydride (142 mg) was added thereto, and the reaction mixture was stirred for 30 minutes. After adding saturated ammonium chloride solution (20 ml), the reaction mixture was extracted with ethyl acetate (100 ml). The organic layer was washed with brine and dried over anhydrous magnesium sulfate. After filtering, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (eluent: ethyl acetate/ n-hexane = 1/2) to obtain yellow solid (470 mg). ‘H-NMR (CDCI₃) δ : 7.60 (d, IH, J=15.8Hz), 7.41 (d, 2H, J=8.9Hz), 6.67 (d, 2H, J=8.9Hz), 6.27 (d, IH, J=15.8Hz), 5.71 (m, IH), 5.22 (bit, IH), 3.70 (dd, IH, J=2.8, 11.0Hz), 3.45 (s, 3H), 3.02 (s, 6H), 3.01 (d, IH, J=4.3Hz), 2.63 (t, IH, J=6.3Hz), 2.56 (d, IH, J=4.3Hz), 2.41 – 1.81 (m, 6H), 1.75 (s, 3H), 1.67 (s, 3H), 1.22 (s, 3H), 1.15 – 1.06 (m, IH)

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Organic Letters, 16(3), 792-795; 2014

An efficient, two-step construction of highly complex alkaloid-like compounds from the natural product fumagillol is described. This approach, which mimics a biosynthetic cyclase/oxidase sequence, allows for rapid and efficient structure elaboration of the basic fumagillol scaffold with a variety of readily available coupling partners. Mechanistic experiments leading to the discovery of an oxygen-directed oxidative Mannich reaction are also described.

http://pubs.acs.org/doi/full/10.1021/ol4035269

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