WAY-315193

7-Fluoro-1-[(1S,2R)-1-(3-fluorophenyl)-2-hydroxy-3-(methylamino)propyl]-3,3-dimethylindolin-2-one Hydrochloride

Drugs that possess norepinephrine reuptake inhibition, either selectively or in combination with serotonin reuptake inhibition, have been used for multiple indications including major depressive disorder, attention deficit hyperactivity disorder, stress urinary incontinence, vasomotor symptoms, and pain disorders such as diabetic neuropathy and fibromyalgia.1 In the search for new candidates with improvements in both potency and selectivity, one of the lead compounds in the 1-(3-amino- 2-hydroxy-1-phenylpropyl)indolin-2-one series, WAY-315193 (1), was identified.2

Paper

Organic Process Research & Development 2009, 13, 880–887

Large-Scale Synthesis of a Selective Inhibitor of the Norepinephrine Transporter:
Mechanistic Aspects of Conversion of Indolinone Diol to Indolinone Aminoalcohol
and Process Implications
Asaf Alimardanov,* Alexander Gontcharov, Antonia Nikitenko, Anita W. Chan, Zhixian Ding, Mousumi Ghosh,
Mahmut Levent, Panolil Raveendranath,† Jianxin Ren, Maotang Zhou, Paige E. Mahaney,‡ Casey C. McComas,‡
Joseph Ashcroft, and John R. Potoski
Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965, U.S.A., and Wyeth Research, 500 Arcola Road,
CollegeVille, PennsylVania 19426, U.S.A.

TREATMENT OF GYNECOLOGICAL DISORDERS
WAY-315193 (Wyeth Pharmaceuticals)

Development of a scalable synthesis of WAY-315193 is described.
Use of LiHMDS as a base and Ti(O-i-Pr)4 as a Lewis acid was optimal for efficient and reproducible addition of indolinone anion to epoxyalcohol. Conversion of indolinone diol to indolinone aminoalcohol was achieved via monotosylationmethylamination.
The possibility of selective formation of the amidine side product, as well as its utilization for alternative selective preparation of the target aminoalcohol, was demonstrated.

The synthetic route used initially for preparation of 1 is shown in Scheme 1. The key step of the synthesis was the
Sharpless epoxidation of fluorocinnamic alcohol 3 which selectively introduced both relative and absolute configurations at the C-2 and C-3 positions. At the early stages of the project, allylic alcohol 3 was prepared in two steps from commercially available fluorocinnamic acid 2 by treatment with MeI in the presence of Cs2CO3 in acetone, followed by DIBAL reduction at -78 °C. The epoxide 4 was opened with the sodium salt of dimethylfluoroindolinone in DMF to afford the diol. The diol 6 was further elaborated into the final aminoalcohol hydrochloride 1 in 30-34% yield via tosylation with p-toluenesulfonyl chloride (TsCl) in pyridine, isolation of the intermediate monotosylate, treatment with MeNH2, and conversion to HCl salt. Dimethylfluoroindolinone was prepared by reduction and bis-methylation of 7-fluoroisatin by a process developed earlier as described in a prior publication.3

white solid (58% yield). Mp 209-212 °C.
[R]D25°)+10.7°.

1H NMR (D2O, 400 MHz) δ: 7.40-7.25 (m,3H), 7.16-6.97 (m, 4H), 5.47-5.25 (2H, broad m), 3.27-3.20
(2H, broad m), 2.76 (s, 3H), 1.37 (s, 3H), 1.24 (broad s, 3H).
ES+ MS, m/z 361 (MH+). Anal. Calc’d for C20H23ClF2N2O2:C, 60.53; H, 5.84; N, 7.06. Found: C, 60.43; H, 5.69; N, 6.84.
Sn content: <1 ppm. Enantiomeric purity: 99.1% ee. Chiral SFCanalysis conditions: column: Chiralcel OF 250 mm × 4.6 mm;mobile phase: 30% ethanol, 0.4% diethylamine in CO2; detection wavelength: 254 nm; 2 mL/min, 40 °C.

* Corresponding author. E-mail: alimara@wyeth.com.
† Deceased.
‡ Wyeth Research, Collegeville, PA.
(1) (a) For a review on norepinephrine reuptake inhibitors, see: Babu,R. P. K.; Maiti, S. N. Heterocycles 2006, 69, 539. (b) Krell, H. V.;Leuchter, A. F.; Cook, I. A.; Abrams, M. Psychosomatics 2005, 46,379. (c) Hajos, M.; Fleishaker, J. C.; Filipiak-Reisner, J. K.; Brown,M. T.; Wong, E. H. W. CNS Drug ReV. 2004, 10, 23. (d) McCormack,
P. L.; Keating, G. M. Drugs 2004, 64, 2567.
(2) Kim, C. Y.; Mahaney, P. E.; Trybulski, E. J.; Zhang, P.; Terefenko,E. A.; McComas, C. C.; Marella, M. A.; Coghlan, R. D.; Heffernan,G. D.; Cohn, S. T.; Vu, A. T.; Sabatucci, J. P.; Ye, F. Phenylaminopropanol
Derivatives and Methods of Their Use. U.S. Patent 7,517,899,2009.

(3) Wu, Y.; Wilk, B. K.; Ding, Z.; Shi, X.; Wu, C. C.; RaveendranathP.; Durutlic, H. Process for the Synthesis of Progesterone ReceptorModulators. U.S. Patent Publ. Appl. US 2007/027327, 2007.
(4) (a) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune,H.; Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765. (b) For a recent example of large-scale asymmetric epoxidation, see: Henegar,
K. E.; Cebula, M. Org. Process Res. DeV. 2007, 11, 354.

(5) (a) For indolinone deprotonation for epoxide opening, see: Proudfoot,J. R.; Regan, J. R.; Thomson, D. S.; Kuzmich, D.; Lee, T. W.;Hammach, A.; Ralph, M. S.; Zindell, R.; Bekkali, Y. Preparation ofPropanol and Propylamine Derivatives and Their Use as Glucocorticoid Ligands. WO 2004/063163, 2004. (b) Gillman, K.; Bocchino, D. M.
Preparation of Monosaccharides Prodrugs of Fluorooxindoles Useful in Treatment of Disorders Which are Responsive to the Opening of Potassium Channels. U.S. Patent Publ. Appl. US 2004/0152646, 2004.
(c) For amide deprotonation for epoxide opening, see: Albanese, D.; Landini, D.; Penso, M. Tetrahedron 1997, 53, 4787. (d) Chan, W. N.; Evans, J. M.; Hadley, M. S.; Herdon, H. J.; Jerman, J. C.; Morgan,H. K. A.; Stean, T. O.; Thompson, M.; Upton, N.; Vong, A. K. J. Med.Chem. 1996, 39, 4537.
(6) Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991, 56, 4218.
(7) (a) Smith, J. G. Synthesis 1984, 629. (b) Parker, R. E.; Isaacs, N. S.Chem. ReV. 1959, 59, 737.

//////////WAY-315193

Cetilistat, セチリスタット

• Molecular FormulaC₂₅H₃₉NO₃
• Average mass401.582 Da

Cetilistat is a drug designed to treat obesity. It acts in the same way as the older drug orlistat (Xenical) by inhibitingpancreatic lipase, an enzyme that breaks down triglycerides in the intestine. Without this enzyme, triglycerides from the diet are prevented from being hydrolyzed into absorbable free fatty acids and are excreted undigested.^[1]

In human trials, cetilistat was shown to produce similar weight loss to orlistat, but also produced similar side effects such as oily, loose stools, fecal incontinence, frequent bowel movements, and flatulence.^[2][3] It is likely that the same precautions would apply in that absorption of fat-soluble vitamins and other fat-soluble nutrients may be inhibited, requiring vitamin supplements to be used to avoid deficiencies.

Central obesity have an important impact on the development of risk factors for coronary heart disease, including dislipidemia, glucose intolerance, insulin resistance and hypertension. These factors contribute to building cardiovascular (CV) disease as a major cause of death. The approach to obesity therapy should be designed to reduce CV risk and mortality. Diet and lifestyle changes remain the cornerstones of therapy for obesity, but the resultant weight loss is often small and long-term success is uncommon and disappointing. Drug therapy is considered for individuals with a body mass index greater than 30 kg/m² or ranging from 25 to 30 kg/m² if they have comorbid conditions. Antiobesity agents can be helpful to some patients in achieving and maintaining meaningful weight loss, but yet our pharmaceutical tools are of limited effectiveness considering the magnitude of the problem. At the present, only two drugs, orlistat and sibutramine, are approved for long-term treatment of obesity and promote no more than 5 to 10% of weight loss.

Rimonabant, a cannabinoid-1 receptor antagonist, was withdrawn from the market because of concerns about its safety, including risk of suicidal and seizures, although very effective in promoting clinically meaningful weight loss, reduction in waist circumference, and improvements in several metabolic risk factors, rimonabant, a cannabinoid-1 receptor antagonist was withdrawn from the market because it concerns about its safety, including risk of suicidal and seizures. Fortunately, recent fundamental insights into the neuroendocrine mechanisms regulating body weight provide an expanding list of molecular targets for novel, rationally designed antiobesity drugs. In this review, the therapeutic potential of some antiobesity molecules in the development will be analyzed based on an understanding of energy homeostasis.

Cetilistat has completed Phase 1 and 2 trials in the West and is currently in Phase 3 trials in Japan where it is partnered with Takeda.^[4] Norgina BV has now acquired the full global rights to cetilistat from Alizyme after the latter went into administration.^[5]

A published phase 2 trial found cetilistat significantly reduced weight with and was better tolerated than orlistat.^[6

CLIP

Cetilistat (Oblean®)
Cetilistat is a selective pancreatic lipase inhibitor which was approved in Japan in September 2013
for the treatment of obesity. The drug was discovered by Alizyme PLC and later co-developed with
Takeda. Cetilistat demonstrated a lower incidence of adverse gastrointestinal events during a 12 week clinical trial, and the degree of weight loss associated with cetilistat is comparable to that of other approved antiobesity therapies.30 The most likely process-scale preparation of cetilistat is described below in Scheme. 4.31
Commercially available hexadecanol (21) was treated with phosgene in THF/toluene to give the
corresponding chloroformate (22), which was immediately subjected to commercial 2-amino-5-
methylbenzoic acid (23) in pyridine. Subsequent slow addition of methyl chloroformate at room
temperature resulted in the formation of cetilistat (IV), which was produced in 31% overall yield from
hexadecanol.31

REF FOR ABOVE ONLY

30  Kopelman, P.; Groot, G. d. H.; Rissanen, A.; Rossner, S.; Toubro, S.; Palmer, R.; Hallam, R.;
Bryson, A.; Hickling, R. I. Obesity 2010, 18, 108.
31. Hodson, H. F.; Downham, R.; Mitchell, T. J.; Carr, B. J.; Dunk, C. R.; Palmer, R. M. J. US
Patent 20030027821A1, 2003.

SYNTHESIS

Route 1

Route 2

http://www.google.co.in/patents/US7396952

Carbamic ester derivatives of the general formula (1) and especially (2-carboxy-4-methylphenyl)carbamic esters of the general formula (1′)

are suitable intermediates for active pharmaceutical ingredients.

Thus, for example, hexadecyl (2-carboxy-4-methylphenyl)carbamate as compound of the formula (1′) with R═C₁₆H₃₃ is disclosed as an intermediate in the preparation of 2-hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one of the formula (3)

from the originally published version of WO-A 00/40569.

2-Hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one of the formula (3) is described therein as potential active ingredient for the treatment of obesity and type II diabetes.

In this originally published version of WO-A 00/40569, two synthetic routes 1 and 2 are described for preparing 2-hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one (3), each of which starts from the 5-methyl-substituted anthranilic acid (4).

In the two-stage synthetic route 1, the 5-methyl-substituted anthranilic acid (4) is reacted with hexadecyl chloroformate (5) and subsequently with methyl chloroformate to give 2-hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one (3), although the overall yield obtained is only 31%.

The one-stage synthetic route 2 with an excess of pyridine affords 2-hexadecyl-oxy-6-methyl-4H-3,1-benzoxazin-4-one (3) in an even lower yield of 15%.

The starting compound which is required for both the synthetic routes 1 and 2, the 5-methyl-substituted anthranilic acid (4), is not easily obtainable, however.

It is prepared by the method described in J. Org. Chem. 1952, 17, 141. This starts from p-toluidine, which is reacted with chloral hydrate and hydroxylamine hydrochloride. The resulting oxime is cyclized with acid catalysis, and subsequently the ring is cleaved again by oxidation under basic conditions.

The disadvantages of this synthesis are the low yields and the fact that only very low concentrations can be used. For this reason, this synthetic route is unattractive for an industrial reaction.

Further alternative routes known in principle for obtaining anthranilic acids are as follows:

J. Org. Chem. 1978, 43, 220 and Chem. Ber. 1909, 42, 430 disclose initial nitration of 3-cyanotoluene, then reduction of the nitro group and subsequent hydrolysis of the nitrile to the carboxylic acid.

A disadvantage of this synthesis is that the nitration of 3-cyanotoluene does not proceed selectively and therefore a further purification step is necessary. This requires additional effort and reduces the yield.

The synthesis which is described in J. Chem. Soc. Perkin I, 1973, 2940 and which starts from 3-toluic acid with subsequent nitration and reduction of the nitro group also has the same disadvantage.

The synthesis which is disclosed in Monatsh. Chem. 1920, 41, 155 and starts from 2,4-dimethyl-1-nitrobenzene is likewise unsuitable because oxidation of the methyl group next to the nitro group does not proceed selectively and therefore an elaborate separation of isomers is necessary.

EP-A 0 034 292 discloses a process for preparing optionally substituted anthranilic acids which includes a transition metal-catalysed carbonylation reaction with carbon monoxide to give an anthranilic acid derivative. This carbonylation reaction takes place in an aqueous reaction medium containing a trialkylamine and a catalyst formed from palladium and a tertiary phosphine. The anthranilic acid derivatives can be obtained by eliminating the protective group. The precursors employed for the carbonylation are obtained starting from optionally substituted anilines as shown in principle in the reaction scheme below:

EP-A 0 034 292 describes this reaction sequence of acetylation (a), halogenation (b), carbonylation (c) and subsequent elimination of the acetyl group (d) as affording the optionally substituted anthranilic acids in good yields (>80%). However, the introduction of the acetyl group is a disadvantage. This is necessary because the free anilines give only poor yields in transition metal-catalysed carbonylation reactions because of pronounced complexation [J. Org. Chem. 1981, 46, 4614-4617].

WO-A 97/28118 discloses a comparable process.

Because of the diverse difficulties, described above, associated with the known processes for preparing optionally substituted anthranilic acids and the yields, which are only unsatisfactory and thus limiting for the overall process, of the subsequent synthetic routes 1 and 2, the object of the present invention was to provide an improved process for preparing carbamic ester derivatives of the general formula (1).

US7396952B2.

EXAMPLES Example 1 Synthesis of hexadecyl 4-methylphenylcarbamate

91 g (375 mmol) of 1-hexadecanol were added to a solution of 50 g (375 mmol) of p-tolyl isocyanate in 50 ml of toluene, and the resulting solution was heated under reflux for 8 h. After cooling to room temperature and stirring at this temperature for 12 h, the precipitated solid was filtered off. The colourless solid was washed twice with 10 ml of toluene each time and then dried in vacuo. 80 g (213 mmol, 57%) of the desired carbamate were obtained in the form of a colourless solid with a melting point of 75° C. The melting point agreed with literature data (75-76° C., Microchem J. 1962, 6, 179).

¹H-NMR (CDCl₃, 400 MHz): δ=0.88 ppm (t, J=7.3 Hz, 3H), 1.25-1.40 (m, 26 H), 1.66 (sext, J=6.9 Hz, 2H), 2.30 (s, 3H), 4.14 (t, J=6.9 Hz, 2H), 6.53 (br, 1 H), 7.10 (d, J=7.8 Hz, 2H), 7.25 (d, J=8.3 Hz, 2H). Elemental Analysis Showed: Calculated: C 76.8%, H 11.0%, N 3.7% Found: C 76.9%, H 11.2%, N 3.7%.

Example 2 Synthesis of hexadecyl (2-bromo-4-methylphenyl)carbamate

19 g (119 mmol) of bromine were added dropwise to a solution of 45 g (119 mmol) of the carbamate in 225 ml (235 g) of glacial acetic acid at room temperature over the course of 1 h, and then the resulting solution was stirred at room temperature for 1 h. After addition of a further 25 ml (26 g, 437 mmol) of glacial acetic acid, the reaction mixture was stirred at 40° C. for 5 h and then cooled to room temperature. The precipitated solid was filtered off and washed with 20 ml of glacial acetic acid. Drying in vacuo resulted in 40 g (88 mmol, 74%) of the desired bromo compound in the form of a colourless solid with a melting point of 57° C.

¹H-NMR (CDCl₃, 400 MHz): δ=0.93 ppm (t, J=6.6 Hz, 3H), 1.25-1.43 (m, 26 H), 1.73 (sext, J=6.8 Hz, 2H), 2.33 (s, 3H), 4.21 (t, J=6.7 Hz, 2H), 7.04 (br, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.37 (s, 1H), 8.02 (d, J=8.3 Hz, 1H). ¹³C-NMR (CDCl₃, 100 MHz): δ=14.2 ppm, 20.4, 22.7. 25.9, 29.0, 29.3, 29.4, 29.6 (2C), 29.7 (2C), 29.8 (4C), 32.0, 65.7, 112.5, 120.3, 129.0, 132.5, 133.5, 134.1, 153.5. Elemental Analysis Showed: Calculated: C 63.4%, H 8.9%, N 3.1% Found: C 63.6%, H 8.9%, N 3.1%.

Example 3 Synthesis of 2-hexadecyloxycarbonylamino-5-methylbenzoic acid

217.5 g (478.5 mmol) of hexadecyl (2-bromo-4-methylphenyl)carbamate, 0.5 g (0.7 mmol) of bis(triphenylphosphine)palladium dichloride and 2.5 g (9.3 mmol) of triphenylphosphine were introduced into an autoclave. The autoclave was closed, flushed with nitrogen and an oxygen-free solution of 78.1 g (565.3 mmol) of potassium carbonate in 400 ml of water is added. The autoclave is evacuated and then 2 bar of carbon monoxide are injected and heated to 115° C. The pressure is subsequently adjusted to 8 bar. After CO uptake ceases, the mixture is cooled to RT and 200 ml of toluene are added. The pH is adjusted to 2 with 2M aqueous HCl solution, and the organic phase is separated off. The aqueous phase is extracted anew with 100 ml of toluene, the organic phase is separated off, and the two toluene extracts are combined. Removal of the solvent in vacuo results in 154.9 g (369.2 mmol, 77%) of 2-hexadecyloxycarbonylamino-5-methylbenzoic acid in the form of a pale yellow-coloured solid.

¹H-NMR (CDCl₃, 400 MHz): δ=0.88 ppm (t, J=6.7 Hz, 3H), 1.24-1.40 (m, 26 H), 1.70 (sext, J=6.8 Hz, 2H), 2.33 (s, 3H), 4.17 (t, J=6.8 Hz, 2H), 7.38 (d, J=8.7 Hz, 1H), 7.90 (s, 1H), 8.35 (d, J=8.6 Hz, 1H). Signal of the NH proton not identifiable.¹³C-NMR (CDCl₃, 100 MHz): δ=14.1 ppm, 20.5, 22.7. 25.9, 29.0, 29.3, 29.4, 29.6 (2 C), 29.7 (6 C), 32.0, 65.5, 113.6, 119.0, 131.1, 131.8, 136.3, 140.1, 153.9, 172.5.

Example 4 Synthesis of 2-hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one

4.0 g (10.0 mmol) of 2-hexadecyloxycarbonylamino-5-methylbenzoic acid are introduced into 20 ml of pyridine at 0° C. under a nitrogen atmosphere, and 4.93 g (45.4 mmol) of ethyl chloroformate are added dropwise to the resulting solution at 0° C. over the course of 20 min. After the reaction mixture has been stirred at 0° C. for 1 h and at room temperature for 2 h it is added to 30 ml of ice-water. The solid is filtered off and dried in vacuo. 3.3 g (8.2 mmol, 82%) of 2-hexadecyloxy-6-methyl-4H-3,1-benzoxazin-4-one are obtained in the form of a pale yellow coloured solid with a melting point of 67° C. (literature: 72-73° C., WO 00/40569).

¹H-NMR (CDCl₃, 400 MHz): δ=0.86 ppm (t, J=6.6 Hz, 3H), 1.24-1.42 (m, 26 H), 1.75-1.82 (m, 2H), 2.40 (s, 3H), 4.41 (t, J=6.8 Hz, 2H), 7.30 (d, J=8.3 Hz, 1H), 7.51 (dd, J=8.2, 1.9 Hz, 1H), 7.90 (d, J=0.9 Hz, 1H).

The ¹H-NMR data agree with the literature data from WO-A 00/40569.

Patent

https://www.google.com/patents/CN104341370A?cl=en

cetirizine orlistat (2-methyl-6-firing sixteen -4H-3, 1- benzo ah winded -4- Korea, cetilistat) is a long-acting Alizyme developed and potent specific gastrointestinal lipase inhibitor, with the active serine site of the gastric and intestinal lumen gastric lipase and lipase membrane forms a covalent bond to inactivate the enzyme, and to reduce calorie intake, weight control therapeutic effect. The biggest advantage of the drug is not acting on the nervous system, does not affect other activity in the gastrointestinal tract, it is more secure than existing similar drugs orlistat. Its structural formula is as follows:

West Division for the benefit of his synthesis and intermediates have been described in U.S. Patent US2007232825 and US2003027821, domestic literature orlistat no cetirizine synthesis of relevant reports.

U.S. Patent US2007232825 2-amino-5-methyl-benzoic acid starting material, direct and vilify chloroformate cetyl alcohol vinegar into the ring, get cetirizine orlistat. The reaction byproducts and more difficult W purification needs over baby gel column, resulting in a low yield, suitable for mass industrialization. Directions are as follows:

Patent US2003027821 W toluene different acid vinegar as raw material to produce amino acid vinegar intermediate chloroformate, cetyl alcohol and vinegar reaction, after the desert generation essays glycosylation chloroformate caprolactone ring closure to give cetirizine orlistat. This method requires a great deal of glacial acetic acid, the presence of H waste discharge more harsh reaction conditions, equipment requirements, is not conducive to industrial production and other defects.

The present invention is a W under the technical program realization:

The following combination of embodiments of the present invention will be further described below.

(Sixteen essays firing oxygen-amino) -5- Preparation of 2-methyl benzoate desert vinegar; [0041] Example 1

4. 9g H phosgene will be added to 50 blood dichloromethane firing, the temperature was lowered to OC, a solution of 2-amino-5 Desert benzoic acid methyl ester (5g) and H hexylamine (13.8 blood) dichloro A firing (20 blood) solution, the addition was complete OC to maintain 15min, warmed to room temperature the reaction mix of football.

[0042] The 5. 26g cetyl alcohol was added to the reaction solution at room temperature the reaction of. After completion of the reaction, filtered and the filtrate was concentrated in vacuo spin dry, dry methanol residue fight starched coating, filtration, the filter cake is dried to constant weight. To give a white solid powder 9. Ig, namely 2- (sixteen essays firing oxygen-ylamino) -5-benzoic acid methyl ester desert; Yield; 85%.

2- (grilled oxygen sixteen essays) -5-methyl-benzoic acid methyl ester prepared; [0043] Example 2

Under nitrogen blanket IOg 2- (sixteen grilled oxygen essays) -5- desert benzoic acid methyl ester was dissolved in 1,4-dioxane (50mL) and water Qiao blood), and Ilg anhydrous carbonate Bell, 1.44g methacrylic acid test, 0. 731g Pd (dppf) 2Cl2, the mixture at 105C for 3 hours. Completion of the reaction, cool down, filtered and the filtrate spin dry, the residue of anhydrous methanol wash coating, the filter cake dried to give a gray solid 6. 5g, is 2- (xvi grilled oxygen essays) -5-methyl benzoic acid methyl ester in 75% yield.

2- (grilled oxygen sixteen essays) -5-methyl-benzoic acid; [0044] Example 3

The 7g 2- (sixteen grilled oxygen essays) -5-methyl-benzoic acid methyl ester was added to 35mL tetraammine clever furans and 7mL water mixture, adding ammonia oxidation in 20. Ig, 6 (TC reaction of the reaction is completed, the reaction mixture was concentrated, the residue was added 70mL of ice water, 6M hydrochloric suppression of 7, the filter cake was dried to constant weight to give a gray solid 6. 2g, namely 2- (sixteen firing oxygen-ylamino essays ) -5-methyl-benzoic acid, yield 92%.

Preparation of 2-methyl-6-firing sixteen -4H-3, 1- benzo Lai ah winded -4- (cetirizine Division him); 4 [0045] Example

The 66g 2- (XVI essays firing oxo-ylamino) -5-methylbenzoic acid in 330mL of information coincidence floating in an ice bath, was slowly added dropwise 45mL chloroformate caprolactone, after the addition was complete, naturally rise to room temperature The reaction of. After completion of the reaction, the reaction solution was poured into 700mL ice water, filtered, and the filter cake was dried to constant weight to give a gray solid 56g, that is, sixteen firing-6-methyl-2- -4H-3, 1- benzo Lai ah winded -4- (cetirizine orlistat), a yield of 85%. Mass spectrum shown in Figure 2, ESI-MS〇b / z): 402 [M + Tin +; X- ray diffraction as shown in (3 consistent with the data reported in FIG patent US2012101090), analyzed as shown in Table 1, Figure 1 FIG. 2 W and W Table 1 confirm that the product was obtained as cetirizine orlistat.

[0046] Table 1

CJPH  2015, Vol. 46 Issue (09): 946-947    DOI: 10.16522/j.cnki.cjph.2015.09.003

CLIP

http://amogsobgy.com/downloads/AkumentisChechwt/CurrOpinInvestigDrugs.pdf

CLICK ON IMAGE

Cetilistat, a new lipase inhibitor for the treatment of obesity – AMOGS

PATENT

SEE

http://www.google.co.in/patents/WO1999016758A1?cl=en

CLIP

Taken from Ayurajan

https://ayurajan.blogspot.in/2016_01_01_archive.html

Cetilistat | Inhibitor of Gastrointestinal Lipases | Inhibitor of Pancreatic Lipases | Anti-Obesity Drug

Cetilistat [2-(Hexadecyloxy)-6-methyl-4H-3,1-benzoxazin-4-one] is a novel highly lipophilic benzoxazinone that inhibits gastrointestinal (GI) and pancreatic lipases, and is chemically distinct from Orlistat [1].

Cetilistat: 2D and 3D Structure

Pancreatic lipase is the enzyme that breaks down triglycerides in the intestine. Inhibition of this enzyme ensures that triglycerides from the diet are prevented from being hydrolyzed into absorbable free fatty acids and are excreted undigested.

In Phase I clinical trials in healthy volunteers, Cetilistat increased faecal fat excretion and was well tolerated. Cetilistat produced a clinically and statistically significant weight loss in obese patients in this short-term 12-week study. This was accompanied by significant improvements in other obesity-related parameters. Cetilistat treatment was well tolerated. The risk-benefit demonstrated in this study in terms of weight loss vs intolerable GI adverse effects shows that Cetilistat merits further evaluation for the pharmacotherapy of obesity and related disorders.

The NDA submission is based on the results of three Phase 3 clinical trials in obese patients with type 2 diabetes and dyslipidemia: a 52-week placebo-controlled, double-blind study to evaluate the efficacy and safety, and two open-label studies to evaluate safety, 24-week and 52-week respectively. The results of the 52-week placebo-controlled, double-blind study demonstrate that Cetilistat 120mg three times daily is superior to placebo in the primary endpoint, with a mean reduction in body weight from baseline of -2.776% with Cetilistat versus -1.103% with placebo (p=0.0020). Greater reduction in HbA1c and low-density lipoprotein cholesterol were also observed in patients treated with Cetilistat, compared to placebo. In all these three studies, Cetilistat showed a good safety profile and was well tolerated.

Cetilistat was approved in Japan in September 2013 for the treatment of obesity. Cetilistat (Tradename: Oblean) is approved for a dosage of 120 mg three times a day for the treatment of obesity with complications.

The drug was discovered by UK based Alizyme PLC and in 2003 Takeda acquired the rights for development and commercialisation for Japan. Norgine acquired all rights to the product from Alizyme in October 2009 [3].

Cetilistat Synthesis

US20030027821A1: It appears to be the industrial process. The yields are in the range of 30-35%.

Identification:

1H NMR (Estimated) for Cetilistat

Experimental: 1H-NMR δH (400 MHz, CDCl₃) 0.87 (3H, t, J 6.8, CH₂CH₃), 1.24-1.45 (26H, m, 13×CH₂), 1.75-1.83 (2H, m, OCH₂CH₂), 2.41 (3H, s, ArCH₃), 4.41 (2H, t, J 6.7, OCH₂), 7.3 (1H, d, J 8.3, ArH), 7.51 (1H, dd, J 8.5, 2.0, ArH), 7.90 (1H, d, J 1.1, ArH); m/z (ES+) 402 (MH+); M Pt. 72-73° C.

Sideeffects: The most frequently experienced adverse events were those involving the gastrointestinal (GI) tract. The proportion of patients and the total number of GI adverse events reported in each of the active treatment groups were higher compared to the placebo group. However, GI adverse events were predominantly mild to moderate in intensity, with no evidence of a dose relationship.

The most frequently reported GI-related adverse events included increased defecation, soft stools, abdominal pain, flatulence and fatty/oily stool, which were all reported more frequently in the treatment arms compared to the placebo arm.

Faecal incontinence, flatus with discharge, oily evacuation and oily spotting occurred in only 1.8-2.8% of subjects in the active treatment arms and was not dose-related. Adverse events generally occurred on only one occasion and resolved rapidly.

Serum vitamin D, vitamin E and β-carotene levels were decreased significantly in the Cetilistat treatment arms. Generally, these reductions in vitamin levels did not take the levels outside the normal range and none required the use of vitamin supplements.

References FOR ABOVE ONLY

1. Kopelman, P.; et. al. Cetilistat (ATL-962), a novel lipase inhibitor: a 12-week randomized, placebo-controlled study of weight reduction in obese patients. Int J Obes (Lond) 2007, 31(3), 494-499.
2. Hodson, H.; et. al. 2-Oxy-benzoxazinone derivatives for the treatment of obesity.US20030027821A1
3. Cetilistat Approval (here).

References

1.  Yamada Y, Kato T, Ogino H, Ashina S, Kato K (2008). “Cetilistat (ATL-962), a novel pancreatic lipase inhibitor, ameliorates body weight gain and improves lipid profiles in rats”. Hormone and Metabolic Research. 40 (8): 539–43. doi:10.1055/s-2008-1076699. PMID 18500680.
2.  Kopelman, P; Bryson, A; Hickling, R; Rissanen, A; Rossner, S; Toubro, S; Valensi, P (2007). “Cetilistat (ATL-962), a novel lipase inhibitor: A 12-week randomized, placebo-controlled study of weight reduction in obese patients”. International journal of obesity (2005). 31 (3): 494–9. doi:10.1038/sj.ijo.0803446. PMID 16953261.
3.  Padwal, R (2008). “Cetilistat, a new lipase inhibitor for the treatment of obesity”. Current opinion in investigational drugs (London, England : 2000). 9 (4): 414–21. PMID 18393108.
4.  http://www.alizyme.com/alizyme/products/cetilistat/ Archived January 7, 2009, at the Wayback Machine.
5.  Norgine acquires cetilistat
6.  “Weight loss, HbA1c reduction, and tolerability of cetilistat in a randomized, placebo-controlled phase 2 trial in obese diabetics: comparison with orlistat (Xenical).”. Obesity. 18: 108–15. Jan 2010. doi:10.1038/oby.2009.155. PMID 19461584.
7. Japan PMDA.

セチリスタット
Cetilistat

C₂₅H₃₉NO₃ : 401.58
[282526-98-1]

Cetilistat

Systematic (IUPAC) name
2-(Hexadecyloxy)-6-methyl-4H-3,1-benzoxazin-4-one
Identifiers
CAS Number	282526-98-1
ATC code	none
PubChem	CID 9952916
ChemSpider	8128526
UNII	LC5G1JUA39
KEGG	D09208
ChEMBL	CHEMBL2103825
Chemical data
Formula	C25H39NO3
Molar mass	401.582 g/mol
SMILES CCCCCCCCCCCCCCCCOc2oc(=O)c1cc(C)ccc1n2

///////////////Cetilistat, ATL-962, ATL962, ATL 962, 2013-09-20, JAPAN, APPROVED,  Japan PMDA, 282526-98-1, セチリスタット

 SEE

Annual Reports in Medicinal Chemistry

BMS-442608

8-Azaspiro(4.5)decane-7,9-dione, 6-hydroxy-8-(4-(4-(2-pyrimidinyl)-1-piperazinyl)butyl)-, (6R)-

(6R)-6-Hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione

(R)-6-Hydroxybuspirone, UNII-93881477KV, CAS 477930-30-6,

Molecular Formula, C21-H31-N5-O3, Molecular Weight, 401.5079

BMS-442608 is a 5-HT1A partial agonist. BMS-442608 is the R-enantiomer. (R)-Enantiomer showed higher affinity and selectivity for the 5HT1A receptor compared to the (S)-enantiomer. (S)-Enantiomer has advantage of being cleared more slowly from blood compared to the (R)-enantiomer.

PAPER

Enantioselective α-Hydroxylation of 2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered Cytochrome P450 BM-3

http://pubs.acs.org/doi/abs/10.1021/ja061261x

Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the α-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee.

PATENT

US 20020193380

http://www.google.st/patents/US20020193380

PATENT

WO 2003009851

https://google.com/patents/WO2003009851A1?cl=en

PAPER

Tetrahedron: Asymmetry (2005), 16(16), 2711-2716.

Tetrahedron: Asymmetry

Volume 16, Issue 16, 22 August 2005, Pages 2711–2716

http://www.sciencedirect.com/science/article/pii/S0957416605005549

http://dx.doi.org/10.1016/j.tetasy.2005.07.020

Tetrahedron: Asymmetry (2005), 16(16), 2778-2783

http://dx.doi.org/10.1016/j.tetasy.2005.07.015

Abstract

PAPER

Enzyme and Microbial Technology (2006), 39(7), 1441-1450.

http://dx.doi.org/10.1016/j.enzmictec.2006.03.033

Abstract

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. RecombinantE. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.

PATENT

https://www.google.com/patents/US6686361

The present invention relates to methods of treating anxiety and depression using R-6-hydroxy-buspirone and pharmaceutical compositions containing R-6-hydroxy-buspirone.

Buspirone, chemically: 8-[4-[4-(2-pyrimidinyl)1-piperazinyl]butyl-8-azaspiro(4,5)-decane-7,9-dione, is approved for the treatment of anxiety disorders and depression by the United States Food and Drug Administration. It is available under the trade name BUSPAR® from Bristol-Myers Squibb Company.

Studies have shown that buspirone is extensively metabolized in the body. (See, for example, Mayol, et al., Clin. Pharmacol. Ther., 37, p. 210, 1985). One of the metabolites is 6-hydroxy-8-[4-[4-(2-pyrimidinyl)1-piperazinyl]butyl-8-azaspiro(4,5)-decane-7,9-dione having Formula I. This metabolite is also known as BMS 28674, BMS 442608, or

as 6-hydroxy-buspirone. This compound is believed to be the active metabolite of buspirone and its use in treating anxiety disorders and depression is disclosed in U.S. Pat. No. 6,150,365. The specific stereochemistry of 6-hydroxy-buspirone has not been described previously. Neither racemic 6-hydroxy-buspirone nor its enantiomers are commercially available at the present time.

Preclinical studies demonstrate that 6-hydroxy-buspirone, like buspirone, demonstrates a strong affinity for the human 5-HT1A receptor. In functional testing, 6-hydroxy-buspirone produced a dose-dependent anxiolytic response in the rat pup ultrasonic vocalization test, a sensitive method for assessment of anxiolytic and anxiogenic effects (Winslow and Insel, 1991, Psychopharmacology, 105:513-520).

Clinical studies in volunteers orally dosed with buspirone demonstrate that 6-hydroxy-buspirone blood plasma levels were not only 30 to 40 times higher but were sustained compared to buspirone blood plasma levels. The time course of 6-hydroxy-buspirone blood plasma levels, unlike buspirone blood plasma levels, correlate more closely with the sustained anxiolytic effect seen following once or twice a day oral dosing with buspirone.

Although buspirone is an effective treatment for anxiety disorders and depression symptomatology in a significant number of patients treated, about a third of patients get little to no relief from their anxiety and responders often require a week or more of buspirone treatment before experiencing relief from their anxiety symptomatology. Further, certain adverse effects are reported across the patient population. The most commonly observed adverse effects associated with the use of buspirone include dizziness, nausea, headache, nervousness, lightheadedness, and excitement. Also, since buspirone can bind to central dopamine receptors, concern has been raised about its potential to cause unwanted changes in dopamine-mediated neurological functions and a syndrome of restlessness, appearing shortly after initiation of oral buspirone treatment, has been reported in small numbers of patients. While buspirone lacks the prominent sedative effects seen in more typical anxiolytics such as the benzodiazepines, patients are nonetheless advised against operating potentially dangerous machinery until they experience how they are affected by buspirone.

It can be seen that it is desirable to find a medicament with buspirone’s advantages but which demonstrates more robust anxiolytic potency with a lack of the above described adverse effects.

Formation of 6-hydroxy-buspirone occurs in the liver by action of enzymes of the P450 system, specifically CYP3A4. Many substances such as grapefruit juice and certain other drugs; e.g. erythromycin, ketoconazole, cimetidine, etc., are inhibitors of the CYP3A4 isozyme and may interfere with the formation of this active metabolite from buspirone. For this reason it would be desirable to find a compound with the advantages of buspirone but without the drug—drug interactions when coadministered with agents affecting the activity level of the CYP3A4 isozyme.

R-6-hydroxy-buspirone may be prepared utilizing methods of synthesis and enantiomeric separation known to one skilled in the art. One method of preparation (Scheme 1) utilizes buspirone as a starting material to produce racemic 6-hydroxy-buspirone that is separated into the two enantiomers by chiral chromatographic techniques.

An improved one-step synthesis of racemic 6-hydroxy-buspirone is set forth in Scheme 2. Again, enantiomeric separation provides R-6-hydroxy-buspirone.

EXAMPLE 1 Preparation of 6-Hydroxy-buspirone

A. Di-4-nitrobenzyl Peroxydicarbonate (V)

Di-4-nitrobenzyl peroxydicarbonate was prepared using a modification of the literature procedure¹. Thus, to an ice-cold solution of 4-nitrobenzyl chloroformate (10.11 g, 4.7 mmol) in acetone (20 mL) was added dropwide over 30 min an ice-cold mixture of 30% H₂O₂ (2.7 mL, 24 mmol) and 2.35 N NaOH (20 mL, 47 mmol). The mixture was vigorously stirred for 15 min and then it was filtered and the filter-cake was washed with water and then with hexane. The resulting damp solid was taken up in dichloromethane, the solution was dried (Na₂SO₄) and then it was diluted with an equal volume of hexane. Concentration of this solution at 20° C. on a rotary evaptor gave a crystalline precipitate which was filtered, washed with hexane and dried in vacuo to give compound III (6.82 g, 74%) as pale yellow microcrystals, mp 104° C. (dec).

¹F. Strain, et al., J. Am. Chem. Soc., 1950, 72, 1254

Di-4-nitrobenzyl peroxydicarbonate was found to be a relatively stable material which decomposed as its melting point with slow gas evolution. In comparison, dibenzyl peroxydicarbonate² decomposed with a sudden vigorous expulsion of material from the melting point capillary.

²Cf. M. P. Gove, J. C. Vedaras, J. Org. Chem., 1986, 51, 3700

B. 6-(4-Nitrobenzyl peroxydicarbonatyl)-8-[4-[4-(2-pyrimidinyl)-piperazinyl]-butyl]-8-azaspiro[4.5]-7,9-dione (III)

To a solution of 8-[4-[4-(2-pyrimidinyl)-piperazinyl]-butyl]-8-azaspiro[4.5]-7,9-dione (buspirone: 10 g, 26 mmole) in dry THF (250 mL) was added LiN (Me₃Si)₂ (28.5 mL of a 1 M THF solution) at 78° C. and stirred for 3 h and then a solution of di-4-nitrobenzyl peroxydicarbonate (11.2 g) in dry THF (150 mL) was added dropwide over 1 h. Stirring was continued at −78° C. for 1 h.

The cooling bath was removed and the reaction solution was poured into a mixture of H₂O and EtOAc. The organic phase was separated and washed with H₂O and then brine. The organic base was dried and then evaporated to a viscous oil. Flash chromatography of this oil, eluting the silica column with MeCN-EtOAc (1:2) gave crude product which was washed with acetone, to remove unreacted buspirone, leaving 6.23 g of a white solid (46%) product (III).

C. 6-Hydroxy-8-[4-[4-(2-pyrimidinyl)-piperazinyl]-butyl]-8-azaspiro[4.5]-7,9-dione (I; 6-Hydroxy-buspirone)

A mixture of III (4.0 g; 6.9 mmole) and 10% Pd/C (about 1 g) in MeOH (100 mL) was hydrogenated in a Parr shaker at 40-45 psi for 1 h. The hydrogenation mixture was filtered through a Celite pad which was then washed with EtOAc. The filtrate was evaporated to a gum which was purified by flash chromatography through a silica gel column eluting with EtOAc to give 0.41 g of an off-white solid (I).

Anal. Calcd. for C₂₁H₃₁N₅O₃: C, 62.82; H, 7.78; N, 17.44. Found: C, 62.84; H, 7.81; N, 17.33.

EXAMPLE 2 Enantiomeric Separation

Preparative Chiral HPLC Purification Procedure for 6-hydroxy-buspirone

1.1 g 6-Hydroxy-buspirone is dissolved in 55 mL HPLC grade methanol (20 mg/mL). Repetitive 0.5 mL injections of the solution are made on a Chirobiotic-Vancomycin Chiral HPLC column, 22.1 mm×250 mm, 10 um particle size (Advanced Separation Technologies, Inc., Whippany, N.J.) equilibrated with a mobile phase of MeOH/acetic acid/triethylamine, 100/0.2/0.1, v/v/v, at a flow rate of 20 mL/minute. The UV trace is monitored at 236 nm. Each enantiomer (RTs˜10.9 and ˜13.4 minutes, respectively) is collected in ˜1000 mL of mobile phase and condensed separately under reduced pressure at 40° C. ˜2 mL of clear solution resulting from the evaporation of methanol is diluted with 5 mL of H₂O. The pH of these solutions is adjusted from 5 to ˜8 with NH₄OH, upon which a white precipitate is observed. The precipitates are centrifuged, and the aqueous layers extracted three times with equal volumes of methylene chloride. The methylene chloride layers are evaporated and any remaining solid is re-chromatographed. The centrifuged precipitates are washed three times with H₂O to remove any residual salts and air dried at room temperature.

The basic form of R-6-hydroxy-buspirone can be converted to the hydrochloride salt by treatment of an ethanol solution of R-6-hydroxy-buspirone with ethanolic HCl.

EXAMPLE 3 One-Step Synthesis of 6-Hydroxy-buspirone (I)

Buspirone (19.3 g, 50 mmole) was dissolved in dry THF (400 mL) and the resulting solution was cooled to −78° C. A solution of KN(SiMe₃)₂ in toluene (100 mL, 1 M) was added slowly. After the reaction mixture was stirred at −78° C. for 1 h, a solution of 2-(phenylsulfonyl)-3-phenyloxaziridine (Davis reagent, prepared according to literature method: F. A. Davis, et al., Org. Synth., 1988, 66, 203) (17.0 g, 65 mmole) in dry THF (150 mL, precooled to −78° C.) was added quickly via a cannular. After stirred for 30 mins at −78° C., the reaction was quenched with 1 N HCl solution (500 mL). It was extracted with EtOAc (3×500 mL). The aqueous layer was separated, neutralized with saturated sodium bicarbonate solution, and extracted with EtOAc (3×500 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a white solid residue which was subjected to column chromatography using CH₂Cl₂/MeOH/NH₄OH (200:10:1) as the eluent to give pure 6-hydroxy-buspirone (I, 7.2 g) and a mixture of buspirone and 6-hydroxy-buspirone (I). The mixture was purified by above column chromatography to afford another 3.3 g of pure 6-hydroxy-buspirone (I).

¹H NMR (CDCl₃) δ8.30 (d, J=4.7 Hz, 2H), 6.48 (t, J=4.7 Hz, 1H), 4.20 (s, 1H), 3.83-3.72 (m, 5H), 3.55 (s, 1H), 2.80 (d, J=17.5 Hz, 1H), 2.55-2.40 (m, 7H), 2.09-2.03 (m, 1H), 1.76-1.54 (m, 10 H), 1.41-1.36 (m, 1H), 1.23-1.20 (m, 1H).

EXAMPLE 4 5-HT1A Receptor Binding Assay

Membranes are prepared for binding using the human 5-HT1 A receptor expressed in HEK293 cells. Cells are collected and ruptured using a dounce homogenizer. The cells are spun at 18000×g for 10 minutes and the pellet is resuspended in assay buffer, frozen in liquid nitrogen and kept at −80° C. until the day of the assay.

A total of 30 ug protein is used per well. The assay is carried out in 96-deep-well plates. The assay buffer is 50 mM HEPES containing 2.5 mM MgCl₂ and 2 mM EGTA. The membrane preparation is incubated at 25° C. for 60 minutes with 0.1 nM to 1000 nM test compound and 1 nM 3H-8-OH-DPAT. 10 mM serotonin serves as blocking agent to determine non-specific binding. The reaction is terminated by the addition of 1 ml of ice cold 50 mM HEPES buffer and rapid filtration through a Brandel Cell Harvester using Whatman GF/B filters. The filter pads are counted in an LKB Trilux liquid scintillation counter. IC₅₀ values are determined using non-linear regression by Excel-fit.

EXAMPLE 5 Rat Pup Isolation-Induced Ultrasonic Vocalization Test

Harlan Sprague-Dawley rat pups (male and female) were housed in polycarbonate cages with the dam until 9-11 days old. Thirty minutes before testing, pups were removed from the dam, placed into a new cage with a small amount of home bedding and brought into the lab and placed under a light to maintain body temperature at 37° C. Pups were then weighed, sexed, marked and returned to the litter group until behavioral assessment. Testing took place in a Plexiglas recording chamber that contained a metal plate maintained at (18-20° C.) with a 5×5 cm grid drawn on the plate. A microphone was suspended 10 cm above the plate to record ultrasonic vocalizations. Ultrasonic calls were recorded using the Noldus UltraVox system providing online analysis of the frequency and duration of calls. The number of grid cells entered by the pup was also collected by visual scoring. Pups that failed to emit at least 60 calls during a 5 minute pretest session were excluded from pharmacological assessment. Immediately following the collection of the baseline measures, pups were injected with vehicle or drug subcutaneously at the nape of the neck and returned to its littermates. Thirty minutes later, pups were retested on each of the dependent measures (vocalization and grid cell crossings) to assess drug effects. Unless otherwise specified, each pup was used only once. Baseline differences and percent change from baseline for the frequency of ultrasonic vocalizations and grid cell crossings were analyzed using a one-way ANOVA. Bonferroni/Dunn post hoc comparisons were performed to assess the acute drug effects with vehicle control. Log-probit analysis was used to estimate the dose (milligrams per kilogram) of each agonist predicted to inhibit isolation-induced ultrasonic vocalizations by 50% (ID₅₀). All comparison were made with an experimental type I error rate (α) set at 0.05.

Doses for each drug were administered in an irregular order across several litters. R-6-hydroxy-buspirone and racemic 6-hydroxy-buspirone were dissolved in physiological saline (0.9% NaCl; vehicle). All injections were administered subcutaneously in a volume of 10 ml/kg. Doses of the drug refer to weight of the salt.

Clip

http://onlinelibrary.wiley.com/doi/10.1002/bdd.566/abstract?systemMessage=Due+to+essential+maintenance+the+subscribe%2Frenew+pages+will+be+unavailable+on+Wednesday+26+October+between+02%3A00+-+08%3A00+BST%2F+09%3A00+%E2%80%93+15%3A00++SGT%2F+21%3A00-+03%3A00+EDT.+Apologies+for+the+inconvenience.

Pharmacokinetics of 6-hydroxybuspirone and its enantiomers administered individually or following buspirone administration in humans

The objective of this study was to assess the pharmacokinetics of 6-hydroxybuspirone (6OHB) when given orally via three forms: racemate (BMS-528215), S-enantiomer (BMS-442606) and R-enantiomer (BMS-442608), versus following the administration of buspirone. A double-blind, randomized, four-period, four-treatment, crossover study balanced for residual effects in healthy subjects was conducted (n=20). Subjects received single 10 mg doses of each compound in a randomized fashion with pharmacokinetics determined over a 24 h period. There was a 4-day washout between each dosing period. All three forms of 6OHB (racemate, S-enantiomer and R-enantiomer) were well tolerated. There was nterconversion between enantiomers. The dominant enantiomer was the S-enantiomer no matter which form of 6OHB was administered. All three forms of 6OHB produced approximately 2- to 3-fold greater exposure to total 6OHB than did buspirone. All three forms produced equal exposure to 1-(2-pyrimidinyl)-piperazine (1-PP) which was approximately 30% less than the 1-PP exposure derived from buspirone administration. All three forms of 6OHB produced approximately 3-fold higher 6OHB:1-PP ratios and approximately 2.5-fold higher total 6OHB exposures than did buspirone administration. All compounds were well tolerated. There seemed to be no advantage of one of the enantiomers of 6OHB over the racemate. Therefore, the racemate was chosen for further clinical development. Copyright © 2007 John Wiley & Sons, Ltd.

REFERENCES

1: Dockens RC, Tran AQ, Zeng J, Croop R. Pharmacokinetics of 6-hydroxybuspirone and its enantiomers administered individually or following buspirone administration in humans. Biopharm Drug Dispos. 2007 Oct;28(7):393-402. PubMed PMID: 17668416.

///////////////BMS-442608, BMS 442608, (R)-6-Hydroxybuspirone, UNII-93881477KV, CAS 477930-30-6

c1cnc(nc1)N2CCN(CC2)CCCCN3C(=O)CC4(CCCC4)[C@H](C3=O)O

MW: 401.5079

S FORM

https://www.google.com/patents/US6821976

Onions contain a powerful cancer fighting compound Onions contain a powerful cancer fighting compound We review the study” Anti-cancer effects found in natural compound derived from onions ” This study was done in regard to Ovarian Cancer, but should have potential in a variety of cancers. * Tsuboki, J. et al. Onionin A inhibits ovarian […]

via Onions contain a powerful cancer fighting compound — ClinicalNews.Org

BMS-741672

N-((1R,2S,5R)-5-(Isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide BMS-741672

N-((lR,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6- (trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide

N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide;

CAS 1004757-96-3

PHASE 2, , Treatment of Type 2 Diabetes, Agents for Neuropathic Pain

Chemokine CCR2 (MCP-1 Receptor) Antagonists

• Originator Bristol-Myers Squibb
• Class Analgesics; Antihyperglycaemics
• Mechanism of Action CCR2 receptor antagonists

• Discontinued Diabetic neuropathies; Type 2 diabetes mellitus

Most Recent Events

• 10 Apr 2007 Preclinical trials in Inflammation in USA (unspecified route)

BMS-741672, 1 , is a highly selective CCR2 antagonist (IC₅₀ = 1.4 nM) featuring a complex array of four stereocenters. The key synthetic challenge was efficient assembly of the densely functionalized 1,2,4-triaminocyclohexane (TACH) core in a minimum number of linear steps.

N-((1R,2S,5R)-5-(Isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide BMS-741672

Mp 161.3 °C.

¹H NMR (400 MHz, CDCl₃) δ 9.50–9.20 (1H), 9.04 (s, 1H), 8.68 (s, 1H), 8.41 (d, J = 7.1 Hz, 1H), 7.87 (s, 1H), 5.04 (dt, J = 1.3, 7.3 Hz, 1H), 4.9 (m, 1H), 4.07 (dt, J = 3.7, 12.9 Hz, 1H), 3.53 (dt, J = 1.4, 9.9 Hz, 1H), 3.44–3.30 (m, 2H), 2.39 (dq, J = 13.6, 8.4 Hz, 1H), 2.26 (m, 1H), 2.21 (s, 3H), 2.17 (q, J = 2.9 Hz, 1H), 2.03–1.91 (m, 5H), 1.71–1.54 (m, 5H), 1.04 (s, br., 6H).

¹³C NMR (100 MHz, d₆-DMSO) δ 171.46, 169.49, 159.62, 156.92, 151.22, 129.28, 128.27 (q, ⁴J_CF = 3 Hz), 125.78 (q, ²J_CF = 32 Hz), 124.11 (q, ¹J_CF = 272 Hz), 121.57 (q, ³J_CF = 4 Hz), 114.33, 54.83, 53.54, 52.36, 47.34, 46.94, 43.13, 30.76, 30.24, 26.94, 26.38, 23.28, 20.87, 17.65 (br.), 16.73 (br.).

¹³C NMR (100 MHz, CDCl₃) δ 172.17. 170.73, 159.89, 156.91, 151.16, 128.68, 128.06 (q,⁴J_CF = 3.0 Hz), 127.25 (q, ²J_CF = 32 Hz), 123.98 (q, ¹J_CF = 272 Hz), 121.78 (q, ³J_CF = 4 Hz), 115.11, 54.89, 53.21, 52.40, 47.40, 46.98, 43.72, 30.84, 30.70, 29.96, 27.80, 23.55, 19.96, 17.70 (2C).

LCMS (ESI, pos.): 508 (16.8), 507 (66.2), 254 (5.0). HR-ESI(pos)-MS: calcd for C₂₅H₃₄F₃N₆O₂ 507.2690 [M + H]⁺, found 507.2694.

IR (KBr): ν = 3428 (m, br.), 2966, 1686 (s), 1635 (m), 1584 (s), 1540 (m), 1334 (m), 1307 (s), 1164 (m), 1121 (m), 870, 845.

[α]²⁰_D−187.9 (c 1.0, CHCl₃).

Anal. Calcd for C₂₅H₃₃F₃N₆O₂: C, 59.28; H, 6.57; F, 11.25; N, 16.59. Found: C, 59.21; H, 6.43; F, 11.07; N, 16.53.

PATENT

WO 2008014381

http://www.google.ch/patents/WO2008014381A2?cl=en&hl=de

EXAMPLE 1

N-((lR,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6- (trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide

[00212] Example 1, Step 1: (IR, 2S, 5R)-tert-Butyl 2-benzyloxycarbonylamino- 7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (89.6 g, 0.24 mol, see: P. H. Carter, et al. PCT application WO 2005/021500) was dissolved in ethyl acetate (1.5 L) and the resulting solution was washed with sat. NaHCCh (2 x 0.45 L) and sat. NaCl (I x 0.45 L). The solution was dried (Na₂SO₄) and then filtered directly into a 3 -necked 3 L round-bottom flask. The solution was purged with direct nitrogen injection before being charged with 10% Pd/C (13.65 g) under nitrogen atmosphere. The flask was evacuated and back-filled with hydrogen; this was repeated twice more. Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated, back-filled with nitrogen, and charged with fresh catalyst (6 g of 10% Pd/C). Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated and back-filled with nitrogen. The mixture was filtered through Celite; the filter pad was then washed with ethyl acetate. The filtrate (-1.6 L EtOAc volume) was diluted with acetonitrile (0.3 L) and charged sequentially with Z-N-Cbz- methionine (68 g, 0.24 mol), TBTU (77 g, 0.24 mol), and Ν,Ν-diisopropylethylamine (42 mL, 0.24 mol). The reaction was stirred at room temperature for 4 h, during which time it changed from a suspension to a clear solution. The reaction was quenched with the addition of sat. NH₄Cl (0.75 L) and water (0.15 L); the mixture was diluted further with EtOAc (0.75 L). The phases were mixed and separated and the organic phase was washed with sat. Na₂Cθ3 (2 x 0.9 L) and sat. NaCl (1 x 0.75 L). The solution was dried (Na₂SO₄), filtered, and concentrated in vacuo to give (IR,2S,5R)- tert-butyl 2-((5)-2-(benzyloxycarbonylamino)-4-

(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate as an oil, which was taken into the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺ = 406.3; [M+Naf = 528.3. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.36 (m, 5H), 5.11 (s, 2H), 4.32 (m, IH), 4.2 (m, IH), 4.0 (m, IH), 2.5 – 2.7 (m, 3H), 2.25 (m, IH), 2.11 (s, 3H), 2.05 (m, 4H), 1.9 (m, IH), 1.7 (m, 2H), 1.54 (s, 9H). Also present are EtOAc [1.26 (t), 2.03 (s), 4.12 (q)] and N,N,N,N-tetramethylurea [2.83

(^S)].

[00213] Example 1, Step 2: A sample of (1^,25,5^)- tert-butyl 2-((5)-2- (benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza- bicyclo[3.2. l]octane-6-carboxylate (0.24 mol assumed; see previous procedure) was dissolved in iodomethane (1,250 g) and stirred for 48 h at room temperature. The reaction was concentrated in vacuo. The residue was dissolved in dichloromethane and concentrated in vacuo. This was repeated twice more. The resultant sludge was dissolved in dichloromethane (0.4 L) and poured into a rapidly stirring solution of MTBE (4.0 L). The resultant yellow solids were collected via suction filtration and dried under high vacuum to afford the sulfonium salt (179 g). This material was taken into the next step without further purification. LC/MS for primary peak: [M- Me₂S+H]⁺ = 458.4; [M]⁺ = 520.4. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.35 (m, 5H), 5.09 (s, 2H), 4.33 (m, IH), 4.28 (m, IH), 3.98 (m, IH), 3.3 – 3.45 (m, 2H), 2.97 (s, 3H), 2.94 (s, 3H), 2.78 (m, IH), 2.0 – 2.3 (m, 4H), 1.7 (m, 2H), 1.52 (s, 9H). Also present are MTBE [1.18 (s), 3.2 (s)] and traces of N,N,N,N-tetramethylurea [2.81 (s)]. [00214] Example 1, Step 3: All of the sulfonium salt from the previous step (0.24 mol assumed) was dissolved in DMSO (2.0 L). The resultant solution was stirred under nitrogen at room temperature and charged with cesium carbonate (216 g) portionwise. The suspension was stirred at room temperature for 3 h and then filtered to remove the solids. The solution was divided into -0.22 L portions and worked up as follows: the reaction mixture (-0.22 L) was diluted with ethyl acetate (1.5 L) and washed successively with water (3 x 0.5 L) and brine (1 x 0.3 L). The organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The desired (\R,2S,5R)- tert-bvXyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-7-oxo-6- azabicyclo[3.2.1]octane-6-carboxylate (90.8 g, 83%) was obtained as a microcrystalline foam, free from tetramethyl urea impurity. LC/MS for primary peak: [M-Boc+H]⁺ = 358.4; [M+Na]⁺ = 480.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.12 (s, 2H), 4.35 (m, 2H), 4.2 (m, IH), 3.6 (m, IH), 3.3 (m, IH), 2.64 (m, IH), 2.28 – 2.42 (m, 2H), 2.15 (m, IH), 1.7 – 2.0 (m, 5H), 1.55 (s, 9H). If desired, this material can be isolated as a solid by dissolving in MTBE (1 volume), adding to heptane (3.3 volumes), and collecting the resultant precipitate.

[00215] Example 1, Step 4: A stirring solution of (\R,2S,5R)- tert-butyl 2-((S>3- (benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6- carboxylate (108 g, 0.236 mol) in THF (1 L) was charged with lithium hydroxide monohydrate (21.74 g, 0.519 mol). Water (0.3 L) was added slowly, such that the temperature did not exceed 20 ⁰C. The reaction was stirred at room temperature overnight and the volatiles were removed in vacuo. The pH was adjusted to -4 through the addition of IN HCl (450 mL) and NaH₂PO₄. The resultant white precipitates were collected by filtration and washed with water (2 x 1 L). The solid was dissolved in dichloromethane (1.5 L) and water (~ 1 L). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was dissolved in EtOAc (0.7 L) and the resultant solution was heated at reflux for 1 h. Solids separated after cooling to RT, and were collected via filtration. These solids were purified by recrystallization in isopropanol to afford the desired (\R,2S,5R)-2-((S)-3- (benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxylic acid as a white solid (104.5 g, 93% yield). LC/MS for primary peak: [M-tBu+H]⁺ = 420.2; [M-Boc+H]⁺ = 376.2; [M+H]⁺ = 476.2. ¹H-NMR (400 MHz, d₄-Me0H): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.35 (m, 2H), 3.71 (m, IH), 3.45 – 3.6 (m, 2H), 2.99 (m, IH), 2.41 (m, IH), 2.15 (m, IH), 2.0 (m, 2H), 1.6 – 1.9 (m, 4H), 1.46 (s, 9H).

[00216] Example 1, Step 5: A 3 L round bottom flask was charged with (lR,25′,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxylic acid (75.5 g, 0.158 mol), EDOHCl (33.5 g, 0.175 mol), 1 -hydroxybenzotriazole (23.6 g, 0.175 mol), and dichloromethane (1 L). The reaction was stirred at room temperature for 2 h, during which time it changed from a white suspension to a clear solution. Ammonia (gas) was bubbled into the solution until the pH was strongly basic (paper) and the reaction was stirred for 10 min; this ammonia addition was repeated and the reaction was stirred for an additional 10 min. Water was added. The organic phase was washed with sat. NaHCθ3, NaH₂PO₄, and brine before being concentrated in vacuo. The residue was slurried with acetonitrile (0.5 L) and then concentrated in to give (lR,2S,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxamide as a white solid (75.9 g, -100%), which was used in the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺ = 375.3; [M+H]⁺ = 475.4; [M-tBu+H]⁺ = 419.3. ¹H-NMR (400 MHz, Cl₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.25 (m, 2H), 3.70 (m, IH), 3.6 (m, IH), 3.45 (m, IH), 2.91 (m, IH), 2.38 (m, IH), 2.12 (m, IH), 1.9 – 2.05 (m, 2H), 1.65 – 1.9 (m, 4H), 1.46 (s, 9H).

[00217] Example 1, Step 6: The reaction was run in three equal portions and combined for aqueous workup. A 5 L, 3-necked round bottom flask was charged with (lR,2S,5R)-2-((5)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-l-yl)-5-(tert- butoxycarbonylamino)cyclohexanecarboxamide (25.3 g, 53 mmol), acetonitrile (1.9 L), and 2.6 L of water/ice. The mixture was stirred and cooled to 0 ⁰C. Iodobenzene diacetate (25.77 g, 80 mmol) was added and the reaction was stirred for 2 h; another 0.5 eq of iodobenzene diacetate was added. The reaction was stirred for 9 h (reaction temp < 10 ⁰C). The mixture was charged with 8 eq N,N-diisopropylethylamine and 2 eq acetic anhydride. Over the next thirty minutes, 4 eq N,N-diisopropylethylamine and 2 eq acetic anhydride were added every ten minutes, until the reaction had proceeded to completion (HPLC). The acetonitrile was removed in vacuo; some solid separated from the residue, and this was collected by filtration. The remaining residue was extracted with dichloromethane (3 L, then 1 L). The organic phase was washed sequentially with water, sat. NaHCθ3, and brine. The collected solids were added to the organic phase, along with activated carbon (15 g). The mixture was stirred for 30 minutes at 40 ⁰C before being filtered and concentrated in vacuo. The residue was dissolved in EtOAc (1 L), and the resultant solution was stirred at 75 ⁰C for 1 h before being allowed to cool to room temperature. A solid separated and was collected by filtration. This solid was purified further by recrystallization: it was first dissolved in 0.5 L CH₂CI₂, then concentrated in vacuo, then re-crystallized from 1 L EtOAc; this was repeated three times. The solids obtained from the mother liquors of the above were recrystallized three times using the same method. The combined solids were recrystallized twice more from acetonitrile (0.7 L) to provide 66 g (84%) of tert-bυXyl (lR,3R,45)-3-acetamido-4-((5)-3-(benzyloxycarbonylamino)-2- oxopyrrolidin-l-yl)cyclohexylcarbamate (purity >99.5% by HPLC). LC/MS for primary peak: [M+H]⁺ = 489.4; [M-tBu+H]⁺ = 433.3. ¹H-NMR (400 MHz, d₄– MeOH): δ 7.3 – 7.4 (m, 5H), 5.11 (s, 2H), 4.35 (m, IH), 4.15 (m, IH), 4.04 (m, IH), 3.8 (m, IH), 3.6 (m, 2H), 2.44 (m, IH), 2.12 (m, IH), 1.87 – 2.05 (m, 4H), 1.87 (s, 3H), 1.55 – 1.7 (m, 2H), 1.46 (s, 9H). The stereochemical fidelity of the Hofmann rearrangement was confirmed through X-ray crystal structure analysis of this compound, as shown in Figure 1. [00218] Example 1, Step 7: A stirring solution of tert-butyl (\R,3R,4S)-3- acetamido-4-((5′)-3 -(benzyloxycarbonylamino)-2-oxopyrrolidin- 1 – yl)cyclohexylcarbamate (66 g, 0.135 mol) in dichloromethane (216 mL) was charged with trifluoroacetic acid (216 mL). The reaction was stirred for 2 h at room temperature and concentrated in vacuo. The residue was dissolved in methanol and the resultant solution was concentrated in vacuo; this was repeated once. Benzyl («S)-l-((l«S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate was obtained as an oil and used directly in Step 8 below. LC/MS found [M + H]⁺ = 389.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.41 (br. s, IH), 4.15 (m, IH), 4.00 (t, J= 9.3 Hz, IH), 3.81 (t, J= 9.1 Hz, IH), 3.65 (q, J= 8.4 Hz, IH), 3.3 – 3.4 (m, IH), 2.45 (m, IH), 1.95 – 2.24 (m, 5H), 2.00 (s, 3H), 1.6 – 1.8 (m, 2H). [00219] Example 1, Step 8: A stirring solution of benzyl (S)- 1-(( \S,2R,4R)-2- acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (-0.135 mol) in methanol (675 mL) was charged sequentially with acetone (37.8 g, 4 eq), sodium acetate (33.2 g, 3 eq), and sodium cyanoborohydride (16.9 g, 2 eq). The mixture was stirred at room temperature for 6 h and filtered. The filtrate was dissolved in dichloromethane (1 L); this solution was washed with IN NaOH (1 L). The solids collected in the filtration were dissolved in IN NaOH (IL) at 0 ⁰C and then extracted with dichloromethane (1 L). The organic extracts were combined and extracted with aqueous HCl (200 mL IN HCl + 800 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then IN NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-I- ((lS,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3- ylcarbamate as an oil. LC/MS found [M + H]⁺ = 431.45. ¹H-NMR (400 MHz, d₄– MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.31 (m, IH), 4.24 (t, J= 9.4 Hz, IH), 4.11 (m, IH), 3.61 (t, J= 9.1 Hz, IH), 3.52 (q, J= 8.6 Hz, IH), 3.04 (br. s, IH), 2.96 (sep, J= 6.3 Hz, IH), 2.40 (m, IH), 2.15 (m, IH), 1.92 (s, 3H), 1.7 – 1.9 (m, 5H), 1.65 (m, IH), 1.12 (app. dd, J= 6.3, 1.1 Hz, 6H).

[00220] Example 1, Step 9 (See Alternative Step 9, below): A stirring solution of benzyl (S)-I -((lS’,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2- oxopyrrolidin-3-ylcarbamate (-115 mmol) in dichloromethane (600 mL) was cooled to 0 ⁰C and charged sequentially with formaldehyde (18.6 g, 37 wt% solution), triethylamine (23 mL), and sodium triacetoxyborohydride (28.7 g). The mixture was stirred at room temperature for 30 minutes and diluted with dichloromethane (up to 1.2 L). This solution was washed thrice with 500 mL sat. NaHCθ3 + NaOH (sat. NaHCO₃, pH to 11 w/ IN NaOH). The organic layer was extracted with aq. HCl (200 mL IN HCl + 600 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then IN NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (1.2 L). The organic extracts were combined, dried (Na2SO4), filtered, and concentrated in vacuo to give benzyl {S)-\-{{\S,2R,AR)-2- acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil, which was used directly in Step 10 below. LC/MS found [M + H]⁺ = 445.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3 – 7.4 (m, 5H), 5.12 (s, 2H), 4.33 (br s, IH), 4.25 (t, J= 9.2 Hz, IH), 4.11 (br s, IH), 3.5 – 3.6 (m, 2H), 2.77 (v br s, 2 H), 2.41 (m, IH), 2.26 (s, 3H), 2.0 – 2.1 (m, 2H), 1.92 (s, 3H), 1.7 – 1.9 (m, 5H), 1.10 (app. dd, J = 17, 6.4 Hz, 6H). [00221] Example 1, Step 10: To a solution of benzyl (S)- 1-(( 15″,2R,4R)-2- acetamido-4-(isopropyl(methyl)amino)-cyclohexyl)-2-oxopyrrolidin-3 -ylcarbamate (-0.115 mol) in methanol (600 mL) was added 10% Pd/C (6 g of 50% wet catalyst). The flask was evacuated and back-filled with hydrogen. The mixture was stirred under 1 atm H₂ for 2 h and the catalyst was removed by filtration through Celite. The filtrate was concentrated in vacuo to provide N-((li?,25,5i?)-2-((S)-3-amino-2- oxopyrrolidin-l-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide as an oil, which was taken on to the next step without further purification. LC/MS found [M + H]⁺ = 311.47. ¹H-NMR (400 MHz, (I₄-MeOH): δ 4.39 (br s, IH), 4.00 (m, IH), 3.3 –

3.5 (m, 4H), 2.73 (m, IH), 2.38 (m, IH), 2.25 (s, 3H), 2.0 – 2.2 (m, 3H), 1.94 (s, 3H),

1.6 – 1.75 (m, 4H), 1.07 (app. dd, J= 21, 6.4 Hz, 6H). [00222] Example 1, Step 11: To a solution of N-((lR,25′,5R)-2-((S)-3-amino-2- oxopyrrolidin-l-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide (~35 g, 0.115 mol) in isopropanol (600 mL) was added 4-chloro-6-(trifluoromethyl)quinazoline (32 g, 0.138 mol, 1.2 eq, see: P.H. Carter et al, PCT application WO 2005/021500). The mixture was stirred at room temperature overnight before being charged with triethylamine (46 g, 0.46 mol, 4 eq). The mixture was stirred at 60 ⁰C for 10 h. The solvent was removed under reduced pressure to give an oil. Azeotropic distillation with isopropanol was performed twice. The residue was dissolved in dichloromethane (600 mL) and extracted with water (250 mL, containing 4 eq acetic acid). Dichloromethane (600 mL) was added to the combined aqueous washes, and the mixture was cooled to 0 ⁰C. Aqueous NaOH (50% by weight) was added with stirring until the pH reached 11. The water layer was extracted with dichloromethane twice (2 x 600 mL). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo to give the amorphous free base of the title compound (99% purity by HPLC). LC/MS found [M+H]⁺ = 507.3. ¹H-NMR (400 MHz, U₄– MeOH): δ 8.82 (s, IH), 8.59 (s, IH), 8.05 (dd, J= 8.8, 1.8 Hz, IH), 7.9 (d, J= 8.7 Hz, IH), 5.28 (t, J= 8.6 Hz, IH), 4.58 (br s, IH), 4.06 (m, IH), 3.52 – 3.68 (m, 2H), 3.43 (m, IH), 2.76 (br s, IH), 2.55 (m, IH), 2.28 (s, 3H), 2.1 – 2.3 (m, 3H), 2.0 (s, 3H), 2.0 (m, IH), 1.65 – 1.8 (m, 3H), 1.09 (app. dd, J= 24, 6.4 Hz, 6 H).

Example 1, Alternative Step 9

[00223] Example 1, Alternative step 9a¹: To a hydrogenator were charged ethyl (7R,SS)-S-((S)- l-phenyl-ethylamino)-l,4-dioxa-spiro[4.5]decane-7-carboxylate A- toluenesulfonate salt I A (1417 g, 2.8 moles, c.f : WO2004098516, prepared analogous to US Pat.6,835,841), ethanol (200 proof, 11.4 L), and 10% Pd/C catalyst (50% wet, 284 g). The mixture was inerted with nitrogen, then pressurized with hydrogen gas (45 psig) and agitated vigorously at approx. 40 ⁰C until starting material was consumed (HPLC). The suspension was cooled, purged with nitrogen gas and the catalyst was removed by filtration while inerted. The spent catalyst was washed with ethanol (4.3 L). The filtrate and washings were combined and concentrated under vacuum to a volume of 2-3 L while maintaining the batch between 40°-60 ⁰C. Isopropyl acetate (5 L) was charged and the mixture was concentrated to a volume of ~2 L until most ethanol was removed (<0.5%) and residual moisture content was <l,000 ppm. Batch volume was adjusted to -7.5 L by the addition of isopropyl acetate. The mixture was heated to 80 ⁰C until clear, then cooled 65°-70 ⁰C. Seed crystals of 1 (5 g) were added and the batch was cooled to 50⁰C over 2 hours, then further cooled to 20 ⁰C over 4 hours and held for ~10 hours. The resulting slurry was filtered and the cake was washed with isopropyl acetate (2 L). The product was dried under vaccum at -35 ⁰C until volatiles were recduced below -1% (LOD). Ethyl (7R,85′)-8-amino-l,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 was obtained as a white, crystalline solid (936 g, 83% yield; HPLC purity: 99.8%). ¹H-NMR: (300MHz, CDCl₃) 8.14-7.89 (brs, 3H), 7.75 (d, J 9.0Hz, 2H), 7.15 (d, J 8.0Hz, 2H), 4.22-4.04 (m, 2H), 4.01-3.77 (m, 4H), 3.55-3.43 (m, IH,), 3.20-3.13 (m, IH), 2.40-2.27 (m, 4H), 2.21-1.94 (m, 2H), 1.81-1.51 (m, 3H), 1.23 (t, J 7.0Hz, 3H); HPLC: Waters Xterra MS C18 4.6 mm x 150 mm Ld., 3.5μm particle size, 0.05% NH40H (5% ACN, 95% H₂O, solvent A), to 0.05% NH₄OH (95% ACN, 5% H₂O, solvent B), 5% B to 20% B in 10 minutes, changed to 95% B in 25 minutes, and then changed to 5% B in 1 minute; 11.1 minutes (aminoester 1).

Example 1, Alternative Step 9a”: Aminoester 1 (63g, 0.16M, leq.; the product of reductive deprotection of a known compound – (See e.g. R. J. Cherney, WO 2004/098516 and G. V. Delucca & S. S. Ko, WO 2004/110993) was placed in a round bottom flask and MeCN (50OmL) was added. EDAC (33.1g, 0.17M, l. leq), HOBt-H₂O (21.2g, 0.16M, l.Oeq) and N-Cbz-Z-methionine (46.7g, 0.17M, 1.05eq) were then added followed by TEA (48.OmL, 0.35M, 2.2eq). An exotherm to 38 ⁰C was observed. The reaction mass was left to stir at RT. After 30mins, HPLC indicated complete conversion. The reaction mass was diluted with EtOAc (2.5L) and washed with KHCO₃ (4x500mL, 20wt% aq. solution) and brine (50OmL). The organic phase was separated, dried over MgSO₄ and concentrated. The residue was dissolved in TBME and reconcentrated to give ethyl (7R,85)-8- {(2S)-2-benzyloxycarbonylamino- 4-methylsulfanyl-butyr-yl-amino}-l,4-dioxa-spiro[4.5]decane-7-carboxylate 2 as a sticky semi-solid (76.2g, 98% yield, 93AP purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.36-7.30 (m, 5H), 7.03 (d, J9.0Hz, IH), 5.66 (d, J 8.0Hz, IH), 5.10 (s, 2H), 4.35- 4.25 (m, 2H), 4.19-4.04 (m, 2H,), 3.98-3.86 (m, 4H), 2.87-2.80 (m, IH), 2.55-2.45 (m, 2H), 2.18 (dd, J 14.0Hz, 7.0Hz, IH), 2.08 (s, 3H), 2.05-1.67 (m, 6H), 1.26 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. lO.Olmin (Compound 2, 93.1 AP). HRMS: m/z 495.2166 [CaIc: C₂₄H₃₅N₂O₇S 495.2165].

2 3 [00224] Example 1, Alternative Step 9b: Methionine amide 2 (75.Og, 0.15M) was dissolved in MeI (225mL, 3mL/g) – some off gassing was noted but no exotherm. The reaction mass was left to stir in the dark for 16.5h. After this time a thick light yellow precipitate had formed. The flask was then evacuated to 200mmHg and some of the MeI removed. The remaining material was slurried in TBME (50OmL), after a 30min stir-out the slurry was filtered, the cake washed with TBME (50OmL). NMR analysis of this material indicated a small amount of MeI remaining. The cake was re-slurried in TBME (50OmL), filtered, washed with TBME (50OmL) and dried under vacuum to give [(35)-3-benzyloxycarbonylamino-3-{(7R,85′)-7- ethoxycarbonyl-l,4-di-oxa-spiro[4.5]dec-8-ylcarbamoyl}-propyl]-dimethylsulfonium iodide 3 as a free flowing off-white solid (93.5g, 97%, 99 area% purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.75 (d, J 9.0Hz, IH), 7.38-7.27 (m, 5H), 6.40 (d, J 7.0Hz, IH), 5.10 (s, 2H), 4.76-4.65 (m, IH), 4.48-4.39 (m, IH), 4.14-3.85 (m, 6H), 3.84-7.73 (m, IH), 3.68-3.55 (m, IH), 3.21 (s, 3H), 3.12 (s, 3H), 2.90-2.83 (s, IH), 2.52-1.55 (m, 8H), 1.24 (t, J7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 2.45min (I-), 8.14min (Compound 3, 43.6AP, I^“ 54.6AP). HRMS: m/z 509.2341 [CaIc: C₂₅H₃₇N₂O₇S 509.2321].

[00225] Example 1, Alternative Step 9c: Cs₂CO₃ (61.5g, 0.19M, 1.5eq) was placed in an round bottom flask and anhydrous DMSO (2.4L) was added. Sulfonium salt 3 (80.Og, 0.13M, 1.Oeq) was then added portionwise. Once the addition was complete the reaction mass was left to stir in the dark for 2Oh. The reaction mass was then split in half and each half worked up separately: the reaction mass was diluted with EtOAc (2.0L) and washed with brine (2L), the organic phase was washed with brine (50OmL). The combined aq. layers were then washed EtOAc (50OmL). The combined organic phases were then washed with brine (3x750mL). The second half of the reaction mass was treated in an identical manner and the combined organics dried over MgSO₄ and concentrated to give ethyl (7R,8S)-8-{(3S>3- Benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl}-l,4-dioxa-spiro[4.5]decane-7- carboxylate 4 as a light colored oil (56.5g, 0.13M, -100 area-% purity) pure by NMR analysis. ¹H-NMR: (300MHz, CDCl₃) δ 7.38-7.30 (m, 5H), 5.37 (br d, J4.0Hz, IH), 5.11 (s, 2H), 4.27-4.18 (m, IH), 4.17-3.82 (m, 8H), 3.32 (td, J 10.0Hz, 60.0Hz, IH), 3.23 (q, J5.0Hz, IH), 2.63-2.57 (m, IH), 2.42-2.25 (m, 2H), 1.94-1.68 (m, 5H), 1.25 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro Cl 8 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 8.99min (Compound 5, produced on column, 4.2AP), 9.48 (Compound 4, 74.3AP). HRMS: m/z 447.2127 [CaIc: C₂₃H₃₁N₂O₇ 447.2131].

4 5

[00226] Example 1, Alternative Step 9d: Pyrrolidinone 4 (50.Og, 0.1 IM) was dissolved in acetone (50OmL) and IN HCl (50OmL) was added. The reaction mass was then heated to 65°C. After 20mins HPLC indicated complete reaction. The reaction mass was allowed to cool to RT and the acetone was removed on a rotary evaporator. During this distillation the product precipitated from solution as a white solid. This was isolated by filtration and the cake washed with water. The cake was then dried azeotropically with toluene (3x3OOmL) to give ethyl (\R,2S)-2-((3S)-3- Benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-oxo-cyclohexanecarboxylate 5 as a white solid (39.8g, 88%, 97 area-% purity). ¹H-NMR: (300MHz, CDCl₃) δ 7.37- 7.32 (m, 5H), 6.65 (br d, J4.0Hz, IH), 5.12 (s, 2H), 4.54-4.47 (m, IH), 4.34-4.26 (m, IH), 4.18 (dq, J 11.0Hz, 7.0Hz, IH), 4.09 (dq, J 11.0Hz, , 7.0Hz, IH), 3.36-3.20 (m, 3H), 2.70-2.35 (m, 6H), 2.05-1.96 (m, IH), 1.81 (quin., J l l.OHz, IH), 1.24 (t, J 7.0Hz, 3H). HPLC: YMC-Pack Pro C18 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 8.95min (Compound 5). HRMS: m/z 403.1864 [CaIc: C₂iH₂₇N₂O₆403.1869].

[00227] Example 1, Alternative Step 9e: Cyclohexanone 5 (22.5g, 0.06M, leq), DMSO (3OmL) and Ti(O-ZPr)₄ (33.7mL, 0.1 IM, 2.04eq) were placed in a round bottom flask. N-isopropyl-N-methylamine (11.6mL, 0.1 IM, 2.0eq) was then added in one portion. The mixture was left to stir for 30mins at room temperature before being cooled to <3°C in ice/water. MeOH (3OmL) was then added followed by the portionwise addition OfNaBH₄ (4.33g, 0.1 IM, 2.04eq) – temperature kept <8°C. 30mins after the addition was completed the reaction mass was diluted with methylene chloride (30OmL) and then NaOH (IN, 4OmL). The resulting slurry was filtered through Celite, and the cake washed with methylene chloride (10OmL). The resulting liquor was concentrated under reduced pressure and the residue dissolved in EtOAc (50OmL). This solution was extracted with IN HCl (2x400mL), the combined aqueous layers were then basified with Na₂CO3. Extraction with EtOAc (4x250mL) provided a clear and colorless organic phase which was dried over Na₂SO₄ and concentrated to give a white powder (24.6g, 96%, 7: 1 d.r.). This material was then slurried overnight in hexane (67OmL). The solid was isolated by filtration and dried under reduced pressure to give ethyl (lR,25′,5R)-2-((3S)-3-benzyloxycarbonylamino- 2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 as a while solid (20.9g, 81%, 24: 1 d.r.). ¹H-NMR: (300MHz, CDCl₃) δ 7.37-7.28 (m, 5H), 5.55 (d, J4.5, IH), 5.10 (s, 2H), 4.42 (q, J4.5, IH), 4.23-4.12 (m, IH), 4.08 (dq, J 10.5, 7.0, IH), 4.02 (dq, J 10.5, 7.0, IH), 3.84 (t, J9.0, IH), 3.46-3.36 (m, IH), 3.04 (septet, J6.5, IH), 2.86-2.80 (m, IH), 2.63-2.48 (m, 2H), 2.17 (s, 3H, Me), 2.10-1.63 (m, 7H), 1.22 (t, J 7.0, 3H), 1.00 (d, J 6.5, 3H), 0.97 (d, J 6.5, 3H). HPLC: YMC- Pack Pro C18 5μm 4.6 x 150 mm, 0.01M NH₄OAc (MeOH:water 20:80) to 0.01M NH₄OAc (MeOH:water:MeCN 20:5:75) 10 to 100% 15min gradient. 8.23 (Compound 6), 8.88 (5-e/«-Compound 6). HRMS: 460.2798 [CaIc: C₂₅H₃₈N₃O₅ 460.2811].

[00228] Example 1, Alternative Step 9f: The aminoester 6 (9.76 g, 2.12 mmol) was dissolved in 2N HCl (80 mL), then heated to -55 ⁰C under inert atmosphere. The reaction was stirred for 20 h, then cooled to room temperature. The reaction solution was washed twice with toluene (25 mL portions), neutralized to pH 6 – 7 by the addition of KOH pellets, then extracted eight times with methylene chloride (100 mL portions). The combined extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure to 50 mL total volume. The concentrated solution was then slowly added to methyl tert-butyl ether (300 mL) over 15 min in an addition funnel with vigorous stirring. The resulting white slurry was stirred at ambient temperature for Ih, then cooled to 0 ⁰C and stirred for Ih. The product was filtered, and washed twice with methyl tert-butyl ether (25 mL portions). Water from the wet cake was removed by azeotropic distillation with acetonitrile (300 mL). The product was dried under reduced pressure to provide (li?,25^r,5R)-2-((35′)-3-Benzyloxycarbonylamino-2- oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylic acid 7, (7.69 g, 84% yield) as a white foam. ¹H-NMR: (400 MHz, 50⁰C, CDCl₃) δ 7.44-7.32 (m, 5H), 6.10 (broad s, IH), 5.19 (app s, 2H), 4.42 (dd, J= 15.6, 7.8 Hz, IH), 4.29-4.23 (m, IH), 3.68-3.60 (m, 2H), 3.33-3.27 (m, 2H), 3.20 (broad s, IH), 2.99 (broad s, IH), 2.51 (s, 3H), 2.49-2.45 (m, 3H), 2.33-2.31 (m, IH), 2.00 (ddd, J= 9.0, 8.6, 3.9 IH), 1.95-1.78 (m, 2H), 1.36-1.21 (m, 6H). LCMS: m/z 432.20 [CaIc: C₂₃H₃₄N₃O₅ 432.25].

NHCbz

[00229] Example 1, Alternative Step 9g: Amino acid 7 (6.3g, 14.7mmol, l.Oeq) was dissolved in THF (8OmL) under N₂ and NaH (584mg, 14.7mmol, l.Oeq, 60wt% dispersion in mineral oil) was added portionwise. When the addition was complete, and the evolution of gas had ceased, the reaction mass was concentrated under reduced pressure and the resulting solid azeotroped with toluene (50 mL) to give a white solid (KF 0.59wt%). This solid was slurried in toluene (100 mL) under N₂and heated to 90⁰C. DPPA (3.32 mL, 15.3 mmol, 1.05 eq) was added dropwise over ~2min. After ~5min all the solids had dissolved, after lOmins precipitation of a white solid was observed. After 30mins HPLC analysis indicated complete reaction. The reaction mass was allowed to cool to RT before being filtered, the cake was washed with toluene. The liquors where then slowly added into ACOH/AC₂O (80/20, 168mL) solution at 90⁰C. After 45mins HPLC still indicated some isocyanate. At 1.15h , the reaction mass was cooled to RT and diluted with toluene (10OmL) and water (10OmL). The organic layer was removed and the toluene washed with IN HCl

(10OmL). The combined aq. phases were then basified with K₂Cθ3(s) and brought to pH 12 with NaOH (10N), keeping the temperature below 20⁰C. The aq layer was then extracted with methylene chloride (4xl50mL), the combined organic layers dried over K₂CO₃ and concentrated to give benzyl (S)-l-((lS,2R,4R)-2-acetamido-4- (isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate 8 as a white foam (4.5g, 70%, 94AP purity). The ¹H-NMR was identical to material obtained from the route described above (Example 1, Step 9). HPLC: YMC-Pack Pro Cl 8 5μm 4.6 x 150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% lOmin gradient. 7.20min (Compound 8), 7.85min (urea dimer). HRMS: 445.2809 [CaIc: C₂₄H₃₇N₄O₄ 445.2815].

Alternative Preparation of Example 1

2 3

[00230] Example 1, Alternative Preparation, Step 1: Ethyl (7R,85)-8-amino- l,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 (450. Ig), was combined with l-ethyl-3-(3-dimethyl-amino-propyl)carbo-diimide hydrochloride (236.3g), 1-hydroxy benzotriazole hydrate (171.9g), N-carbobenzyloxy-Z -methionine (333.4g) and acetonitrile (3.1 L). To the stirred mixture was added triethylamine (249.5g) below 30 ⁰C. Upon reaction completion (HPLC), the mixture was diluted with ethyl acetate (8.2 L) and washed with aqueous 25% potassium bicarbonate solution (2×4.5 L) followed by water (4.5 L). The organic phase was separated and concentrated under reduced pressure to obtain a solution of ethyl (7R,85)-8-((5)-2- benzyloxycarbonylamino-4-methylsulfanyl-butyrylamino)-l,4-dioxa- spiro[4.5]decane-7-carboxylate 2 (1.4 L). Methyl iodide (2.39 kg) was added, the vessel was shielded from light and the mixture was held under slow agitation for approx. 24 h. To the thick yellow precipitate was added methyl tert-butyl ether (2.7 L) and the mixture was held for approx. 1 h. The product was isolated by filtration and the cake was washed with methyl tert-butyl ether (2×1.4 L), then dried under vacuum, yielding [(5)-3-benzyloxy-carbonylamino-3-((7R,8«S’)-7-ethoxycarbonyl-l,4- dioxa-spiro[4.5]dec-8-ylcarbamoyl)-propyl]-dimethylsulfonium iodide 3 (671.4 g, -94% yield) as an off-white solid (HPLC purity 99.9%).

[00231] Example 1, Alternative Preparation, Step 2: Sulfonium salt 3 (619.4 g), and cesium carbonate (416.8 g) and anhydrous dimethyl sulfoxide (6.2 L) were combined in a reactor equipped with a scrubber to neutralize volatile sulfides.

Vigorous agitation was maintained until complete conversion was obtained (HPLC). Ethyl acetate (12.4 L) was added, followed by 20 % brine (3 L). The organic phase was separated, washed twice with brine (2×3 L) and evaporated to obtain a solution of ethyl (7R,8«S)-8-((«S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-l,4-dioxa- spiro[4.5]decane-7-carboxylate 4 in ethyl acetate (~0.8 L). Acetone (2.55 L) was added, followed by aqueous 0.5 M hydrochloric acid solution (2.3 L). With good mixing, the solution was heated to 50 to 60 ⁰C until conversion of 4 to ethyl (IR,2S)- 2-((5)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-oxo- cyclohexanecarboxylate 5 was complete (HPLC). The mixture was concentrated under reduced pressure while below 40 ⁰C, cooled to -30 ⁰C, and water (4.1 L) was added. The resulting slurry was cooled to 5 to 10 ⁰C and agitated for ~1 hour. The product was filtered and the cake was washed with water (2×2.5 L). Upon deliquoring, the cake was dried to a constant weight below 40 ⁰C in a vacuum oven. Cyclohexanone 5 (272g, 70% yield) was obtained (HPLC purity 98.7%).

[00232] Example 1, Alternative Preparation, Step 3: Cyclohexanone 5 (206 g) was dissolved in dichloromethane (1.1 L) and charged to a hydrogenator. Titanium tetraisopropoxide (218.2 g) and N-isopropyl N-methylamine (63.64 g) were added and the mixture was stirred at ambient temperature (23 to 25 ⁰C) for at least 5 h. Platinum catalyst (5% Pt/S/C, 15 g, approx. 7.5 % relative to 5) was added and hydrogenation was performed at -30 psig for at least 6 h, yielding a mixture of ethyl (lR,25′,5R)-2-((5)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl- methyl-amino)-cyclohexanecarboxylate 6 and its 5-epz-isomer (-7%). The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to approx. -600 mL. Wet ethyl acetate (-3% water, 2.0 L) was added with vigorous agitation over a period of at least 1.5 h. Stirring was continued for at least an additional 6 h. The slurry was filtered. Filter cake was washed with ethyl acetate (1.0 L) and discarded. The combined filtrate and washings were concentrated to -400 mL. Toluene (2.0 L) was added and the solution was washed with 2M aqueous hydrochloric acid (2 x 400 mL). The aqueous layer was warmed to 50° to 60 ⁰C for approx. 20 h or hydrolysis of 6 was deemed complete (HPLC). Aqueous sodium hydroxide solution was added to adjust to pH -10, and mixture was extracted with toluene (3×600 mL). The organic phase was discarded and pH was readjusted to ~6 by addition of aqueous hydrochloric acid. The aqueous phase was concentrated to -600 mL under reduced pressure and extracted with methylene chloride (at least 3×2.0 L). The combined methylene chloride layers were evaporated under reduced pressure and continuously replaced with THF to obtain a solution of (\R,2S,5R)-2- ((5^*)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)- cyclohexane carboxylic acid 7 (-148 g) in THF (-4 L). Seed crystals of 8 were added, followed by 25 % solution of sodium methoxide in methanol (81.24 g) below 25 ⁰C. The slurry was held for at least additional 16h with agitation. The product was isolated by filtration and the cake was washed with THF (4×200 mL) and dried to a constant weight in vacuo below 30 ⁰C. Dry (lR,25′,5R)-2-((5)-3-benzyloxycarbonyl- amino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl-amino)-cyclohexane-carboxylate sodium salt 8 was obtained (139g, -60% yield from 5).

[00233] Example 1, Alternative Preparation, Step 4: Aminoester sodium salt 8 (10Og), diphenyl phosphate (3.86g), tert-BuOH (1275 mL) and toluene (225 mL) were combined and heated to reflux under reduced pressure. Approx. 500 mL of distillate were collected and discarded while being continuously replaced with a solution of toluene in tert-BuOH. Vacuum was removed and distillate was switched to percolate through a column filled with molecular sieves and allowed to return to the vessel. After drying was complete, DPPA (52.4mL; dissolved in 60 mL toluene) was added slowly to the slurry at 80 ⁰C. Upon complete conversion (HPLC), tert- BuOH was removed by vacuum distillation and continuously replaced with toluene. The mixture was cooled to room temperature and washed twice with 10% aqueous K2HPO4 (lx800mL, 1×400 mL) and water (40OmL). The organic phase was heated and concentrated in vacuo to approx. 27OmL. Vacuum was removed and heptane (1.1 L) was added slowly at approx. 80 ⁰C, followed by seeds of 9 (~lg). The slurry was slowly cooled to room temperature and benzyl {(S)-l-[(lS,2R,4R)-2- tert- butoxycarbonylamino-4-(isopropyl-methyl-amino)-cyclo-hexyl]-2-oxo-pyrrolidin-3- yl} -carbamate 9 was isolated by filtration as a white solid (86.76g, 78% yield).

[00234] Example 1, Alternative Preparation, Step 5: The tert-Butyl carbamate 9 (5Og) was dissolved in Toluene (50OmL) and /-PrOH (15OmL). The resulting solution was then heated to 6O⁰C. Methanesulfonic acid (19.6mL) was added below 65°C. Upon reaction completion (HPLC), the mixture was cooled to RT and triethylamine (69.4mL) added slowly below 25°C. Acetic anhydride was then added below 25°C. After Ih acetic acid (25OmL) was added below 25°C. The toluene phase was discarded and 2-methyl-THF (50OmL) was added to the aqueous phase. The mixture was stirred vigorously and basified with NaOH (25% aqueous solution) to pH 12. The aqueous phase was discarded and the organic layer was washed with brine (25OmL). The organic layer was concentrated under reduced pressure and continuously replaced with /-PrOH. The solution was cooled and filtered to provide benzyl {(5′)-l-[(15^r,2R,4R)-2-acetylamino-4-(isopropyl-methyl-amino)-cyclohexyl]-2- oxo-pyrrolidin-3-yl} -carbamate 10 in /-PrOH solution which was used directly in the hydrogenation.

[00235] Example 1, Alternative Preparation, Step 6: To a solution containing acetamide 10 (~61g) in /-PrOH (-625 mL) was added 10% Pd/C wet catalyst (2.5 g) and the suspension was hydrogenated at 30 psig and approx. 25 ⁰C for at least 2 h. Upon completion (HPLC), the catalyst was removed by filtration and the filtrate was concentrated to approx. 550 mL. Water (8.8 mL) was added, followed by 5.6 N hydrochloric acid in /-PrOH solution (69.5 mL). The resulting slurry was held at room temperature overnight. The product was isolated by filtration and the cake was rinsed with /-PrOH (2×100 mL) and dried in vacuo to constant weight at -50 ⁰C to give N-[(li?,25^r,5R)-2-((5′)-3-amino-2-oxo-pyrrolidin-l-yl)-5-(isopropyl-methyl- amino)-cyclohexyl]-acetamide 11 (55.6 g, 97% yield) as its hydrochloric acid salt (73.6% free base assay, HPLC).

NH,

CL,

Example 1

[00236] Example 1, Alternative Preparation, Step 7: To 6-trifluoromethyl- quinazolin-4-ol 12 (20.1 g) in MeCN (400 mL) was added 5.5 M solution of sodium methoxide in methanol (17.0 mL). The resulting suspension was distilled under reduced pressure and continuously replaced by MeCN to remove methanol. To the slurry was added DMF (1.4 g), followed by oxalyl chloride (13.0 mL) below 50 ⁰C. Upon reaction completion (HPLC), excess reagent was removed under reduced pressure to give -400 mL of slurry. The mixture was cooled to room temperature and washed with 10 % aqueous K₂HPO₄ (lxl.O L, 1×0.5 L) to afford 4-chloro-6- trifluoromethyl-quinazoline 13 (-21.2 g) in approx. 450 mL of wet MeCN solution, which was used directly in the subsequent coupling reaction (HPLC purity 99.8 %). [00237] Example 1, Alternative Preparation, Step 8: To a mixture of acetamide 11 (28.5 g, HCl salt, 73.6% free base assay), acetonitrile (100 mL), N,N,-di-isopropyl- N-ethylamine (61 mL) at room temperature was added a solution of 13 (-21.2 g) in MeCN (-450 mL). The homogeneous mixture was held overnight. Upon reaction completion (HPLC), the mixture was concentrated in vacuo to approx. 125 mL. A 9.5% aqueous solution of acetic acid (240 mL) was added and the aqueous phase was extracted with methylene chloride. The aqueous phase was separated and methyl tert- butyl ether (450 mL) was added, followed by 2N aqueous lithium hydroxide solution to adjust to pH >11.5. The organic layer was separated, washed with water and filtered. Approx. half of the ether phase was diluted with methyl tert-bvAyl ether (-250 mL) and concentrated in vacuo. Heptane (45 mL) was added slowly below 60 ⁰C, followed by seed crystals of Example 1 (0.4 g). Additional heptane (125 mL) was added and the mixture was slowly cooled to room temperature and the resulting slurry was held overnight. The product was isolated by filtration, the cake was washed with heptane and dried in vacuo to constant weight to give N-((lR,25′,5R)-5- (isopropylamino)-2-((5′)-2-oxo-3-(6-(trifluoromethyl)-quin-azolin-4- ylamino)pyrrolidin-l-yl)cyclohexyl)acetamide 14 (15.0 g, 85% yield).

Crystallization Procedures for Example 1

[00238] Example 1, Production of bis-BSA salt and purification: The entirety of the amorphous free base from Example 1, Step 11 was dissolved in methanol (600 mL). The resultant solution was heated at 60 ⁰C and charged with benzenesulfonic acid (2.5 eq). The mixture was cooled to room temperature and the resultant white solid was collected by filtration to yield the bis-benzene sulfonic acid salt of the title compound (95 g, 86%). This material was >99% pure by HPLC. This material was further purified by re-crystallization from 80/20 EtOH/H₂θ, which provided the salt free from any residual methanol. HPLC purity = 99.8%. ¹H ΝMR (500 MHz, D₂O) δ ppm 8.75 (1 H, s), 8.66 (1 H, s), 8.25 (1 H, d, J=8.80 Hz), 7.90 (1 H, d, J=8.80 Hz), 7.75 (4 H, d, J=8.25 Hz), 7.43 – 7.57 (6 H, m), 5.42 (1 H, t), 4.33 – 4.44 (1 H, m), 4.09 – 4.19 (1 H, m), 3.83 – 3.91 (1 H, m), 3.74 – 3.83 (2 H, m), 3.61 (1 H, t, J=I 1.55 Hz), 2.75 (3 H, d, J=6.60 Hz), 2.61 – 2.70 (1 H, m), 2.31 – 2.44 (1 H, m), 2.20 – 2.27 (1 H, m), 2.17 (2 H, d, J=12.10 Hz), 1.94 – 2.04 (1 H, m, J=12.65 Hz), 1.90 – 1.95 (3 H, m), 1.72 – 1.91 (2 H, m), 1.37 (3 H, d, J=6.05 Hz), 1.29 (3 H, d, J=6.60 Hz). Differential scanning calorimetry utilized a heating rate of 10 °C/min and revealed a melting / decomposition endotherm with an onset temperature of 297.6 ⁰C and a peak temperature at 299.1 ⁰C. [00239] Example 1, Crystallization of the Free Base: A sample of the amorphous free base of N-((lR,25^r,5R)-5-(isopropyl(methyl)amino)-2-((5′)-2-oxo-3- (6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin- 1 -yl)cyclohexyl)acetamide ( 1 g) was dissolved in dichloromethane (5 mL). The solution was charged with heptane (30 mL) and then warmed to distill the dichloromethane. The solution was cooled to 40 ⁰C; a white solid precipitated. The suspension was heated to 90 ⁰C and stirred for 2 h. The suspension was cooled to room temperature and filtered to provide the pure free base of the title compound. No residual solvent was apparent by ¹H-NMR.

PATENT

US 7671062

http://google.com/patents/US7671062

or a pharmaceutically acceptable salt, solvate or prodrug, thereof, having an unexpected combination of desirable pharmacological characteristics. Crystalline forms of the present invention are also provided. Pharmaceutical compositions containing the same and methods of using the same as agents for the treatment of inflammatory diseases, allergic, autoimmune, metabolic, cancer and/or cardiovascular diseases is also an objective of this invention. The present disclosure also provides a process for preparing compounds of Formula (I), including N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide:

wherein R¹, R⁸, R⁹, R¹⁰, and

are as described herein. Compounds that are useful intermediates of the process are also provided herein.

1^st embodiment, the disclosure provides a process for preparing a compound of formula IV, or a salt thereof:

Example 1 N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide

Example 1, Step 1: (1R,2S,5R)-tert-Butyl 2-benzyloxycarbonylamino-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (89.6 g, 0.24 mol, see: P. H. Carter, et al. PCT application WO 2005/021500) was dissolved in ethyl acetate (1.5 L) and the resulting solution was washed with sat. NaHCO₃ (2×0.45 L) and sat. NaCl (1×0.45 L). The solution was dried (Na₂SO₄) and then filtered directly into a 3-necked 3 L round-bottom flask. The solution was purged with direct nitrogen injection before being charged with 10% Pd/C (13.65 g) under nitrogen atmosphere. The flask was evacuated and back-filled with hydrogen; this was repeated twice more. Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated, back-filled with nitrogen, and charged with fresh catalyst (6 g of 10% Pd/C). Hydrogen was bubbled through the solution for 30 min and then the reaction was stirred under 1 atm H₂ for 18 h. The flask was evacuated and back-filled with nitrogen. The mixture was filtered through Celite; the filter pad was then washed with ethyl acetate. The filtrate (˜1.6 L EtOAc volume) was diluted with acetonitrile (0.3 L) and charged sequentially with L-N-Cbz-methionine (68 g, 0.24 mol), TBTU (77 g, 0.24 mol), and N,N-diisopropylethylamine (42 mL, 0.24 mol). The reaction was stirred at room temperature for 4 h, during which time it changed from a suspension to a clear solution. The reaction was quenched with the addition of sat. NH₄Cl (0.75 L) and water (0.15 L); the mixture was diluted further with EtOAc (0.75 L). The phases were mixed and separated and the organic phase was washed with sat. Na₂CO₃ (2×0.9 L) and sat. NaCl (1×0.75 L). The solution was dried (Na₂SO₄), filtered, and concentrated in vacuo to give (1R,2S,5R)-tert-butyl 2-((S)-2-(benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate as an oil, which was taken into the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺=406.3; [M+Na]⁺=528.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.36 (m, 5H), 5.11 (s, 2H), 4.32 (m, 1H), 4.2 (m, 1H), 4.0 (m, 1H), 2.5-2.7 (m, 3H), 2.25 (m, 1H), 2.11 (s, 3H), 2.05 (m, 4H), 1.9 (m, 1H), 1.7 (m, 2H), 1.54 (s, 9H). Also present are EtOAc [1.26 (t), 2.03 (s), 4.12 (q)] and N,N,N,N-tetramethylurea [2.83 (s)].

Example 1, Step 2: A sample of (1R,2S,5R)-tert-butyl 2-((S)-2-(benzyloxycarbonylamino)-4-(methylthio)butanamido)-7-oxo-6-aza-bicyclo[3.2.1]octane-6-carboxylate (0.24 mol assumed; see previous procedure) was dissolved in iodomethane (1,250 g) and stirred for 48 h at room temperature. The reaction was concentrated in vacuo. The residue was dissolved in dichloromethane and concentrated in vacuo. This was repeated twice more. The resultant sludge was dissolved in dichloromethane (0.4 L) and poured into a rapidly stirring solution of MTBE (4.0 L). The resultant yellow solids were collected via suction filtration and dried under high vacuum to afford the sulfonium salt (179 g). This material was taken into the next step without further purification. LC/MS for primary peak: [M-Me₂S+H]⁺=458.4; [M]⁺=520.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.09 (s, 2H), 4.33 (m, 1H), 4.28 (m, 1H), 3.98 (m, 1H), 3.3-3.45 (m, 2H), 2.97 (s, 3H), 2.94 (s, 3H), 2.78 (m, 1H), 2.0-2.3 (m, 4H), 1.7 (m, 2H), 1.52 (s, 9H). Also present are MTBE [1.18 (s), 3.2 (s)] and traces of N,N,N,N-tetramethylurea [2.81 (s)].

Example 1, Step 3: All of the sulfonium salt from the previous step (0.24 mol assumed) was dissolved in DMSO (2.0 L). The resultant solution was stirred under nitrogen at room temperature and charged with cesium carbonate (216 g) portionwise. The suspension was stirred at room temperature for 3 h and then filtered to remove the solids. The solution was divided into ˜0.22 L portions and worked up as follows: the reaction mixture (˜0.22 L) was diluted with ethyl acetate (1.5 L) and washed successively with water (3×0.5 L) and brine (1×0.3 L). The organic phase was dried (Na₂SO₄), filtered, and concentrated in vacuo. The desired (1R,2S,5R)-tert-butyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6-carboxylate (90.8 g, 83%) was obtained as a microcrystalline foam, free from tetramethyl urea impurity. LC/MS for primary peak: [M-Boc+H]⁺=358.4; [M+Na]⁺=480.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.12 (s, 2H), 4.35 (m, 2H), 4.2 (m, 1H), 3.6 (m, 1H), 3.3 (m, 1H), 2.64 (m, 1H), 2.28-2.42 (m, 2H), 2.15 (m, 1H), 1.7-2.0 (m, 5H), 1.55 (s, 9H). If desired, this material can be isolated as a solid by dissolving in MTBE (1 volume), adding to heptane (3.3 volumes), and collecting the resultant precipitate.

Example 1, Step 4: A stirring solution of (1R,2S,5R)-tert-butyl 2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-7-oxo-6-azabicyclo[3.2.1]octane-6-carboxylate (108 g, 0.236 mol) in THF (1 L) was charged with lithium hydroxide monohydrate (21.74 g, 0.519 mol). Water (0.3 L) was added slowly, such that the temperature did not exceed 20° C. The reaction was stirred at room temperature overnight and the volatiles were removed in vacuo. The pH was adjusted to ˜4 through the addition of 1N HCl (450 mL) and NaH₂PO₄. The resultant white precipitates were collected by filtration and washed with water (2×1 L). The solid was dissolved in dichloromethane (1.5 L) and water (˜1 L). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was dissolved in EtOAc (0.7 L) and the resultant solution was heated at reflux for 1 h. Solids separated after cooling to RT, and were collected via filtration. These solids were purified by recrystallization in isopropanol to afford the desired (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid as a white solid (104.5 g, 93% yield). LC/MS for primary peak: [M-tBu+H]⁺=420.2; [M-Boc+H]⁺=376.2; [M+H]⁺=476.2. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.35 (m, 2H), 3.71 (m, 1H), 3.45-3.6 (m, 2H), 2.99 (m, 1H), 2.41 (m, 1H), 2.15 (m, 1H), 2.0 (m, 2H), 1.6-1.9 (m, 4H), 1.46 (s, 9H).

Example 1, Step 5: A 3 L round bottom flask was charged with (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxylic acid (75.5 g, 0.158 mol), EDC.HCl (33.5 g, 0.175 mol), 1-hydroxybenzotriazole (23.6 g, 0.175 mol), and dichloromethane (1 L). The reaction was stirred at room temperature for 2 h, during which time it changed from a white suspension to a clear solution. Ammonia (gas) was bubbled into the solution until the pH was strongly basic (paper) and the reaction was stirred for 10 min; this ammonia addition was repeated and the reaction was stirred for an additional 10 min. Water was added. The organic phase was washed with sat. NaHCO₃, NaH₂PO₄, and brine before being concentrated in vacuo. The residue was slurried with acetonitrile (0.5 L) and then concentrated in to give (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxamide as a white solid (75.9 g, ˜100%), which was used in the next step without further purification. LC/MS for primary peak: [M-Boc+H]⁺=375.3; [M+H]⁺=475.4; [M-tBu+H]⁺=419.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.35 (m, 5H), 5.11 (s, 2H), 4.25 (m, 2H), 3.70 (m, 1H), 3.6 (m, 1H), 3.45 (m, 1H), 2.91 (m, 1H), 2.38 (m, 1H), 2.12 (m, 1H), 1.9-2.05 (m, 2H), 1.65-1.9 (m, 4H), 1.46 (s, 9H).

Example 1, Step 6: The reaction was run in three equal portions and combined for aqueous workup. A 5 L, 3-necked round bottom flask was charged with (1R,2S,5R)-2-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)-5-(tert-butoxycarbonylamino)cyclohexanecarboxamide (25.3 g, 53 mmol), acetonitrile (1.9 L), and 2.6 L of water/ice. The mixture was stirred and cooled to 0° C. Iodobenzene diacetate (25.77 g, 80 mmol) was added and the reaction was stirred for 2 h; another 0.5 eq of iodobenzene diacetate was added. The reaction was stirred for 9 h (reaction temp<10° C.). The mixture was charged with 8 eq N,N-diisopropylethylamine and 2 eq acetic anhydride. Over the next thirty minutes, 4 eq N,N-diisopropylethylamine and 2 eq acetic anhydride were added every ten minutes, until the reaction had proceeded to completion (HPLC). The acetonitrile was removed in vacuo; some solid separated from the residue, and this was collected by filtration. The remaining residue was extracted with dichloromethane (3 L, then 1 L). The organic phase was washed sequentially with water, sat. NaHCO₃, and brine. The collected solids were added to the organic phase, along with activated carbon (15 g). The mixture was stirred for 30 minutes at 40° C. before being filtered and concentrated in vacuo. The residue was dissolved in EtOAc (1 L), and the resultant solution was stirred at 75° C. for 1 h before being allowed to cool to room temperature. A solid separated and was collected by filtration. This solid was purified further by recrystallization: it was first dissolved in 0.5 L CH₂Cl₂, then concentrated in vacuo, then re-crystallized from 1 L EtOAc; this was repeated three times. The solids obtained from the mother liquors of the above were recrystallized three times using the same method. The combined solids were recrystallized twice more from acetonitrile (0.7 L) to provide 66 g (84%) of tert-butyl (1R,3R,4S)-3-acetamido-4-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)cyclohexylcarbamate (purity>99.5% by HPLC). LC/MS for primary peak: [M+H]⁺=489.4; [M-tBu+H]⁺=433.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.11 (s, 2H), 4.35 (m, 1H), 4.15 (m, 1H), 4.04 (m, 1H), 3.8 (m, 1H), 3.6 (m, 2H), 2.44 (m, 1H), 2.12 (m, 1H), 1.87-2.05 (m, 4H), 1.87 (s, 3H), 1.55-1.7 (m, 2H), 1.46 (s, 9H). The stereochemical fidelity of the Hofmann rearrangement was confirmed through X-ray crystal structure analysis of this compound, as shown in FIG. 1.

Example 1, Step 7: A stirring solution of tert-butyl (1R,3R,4S)-3-acetamido-4-((S)-3-(benzyloxycarbonylamino)-2-oxopyrrolidin-1-yl)cyclohexylcarbamate (66 g, 0.135 mol) in dichloromethane (216 mL) was charged with trifluoroacetic acid (216 mL). The reaction was stirred for 2 h at room temperature and concentrated in vacuo. The residue was dissolved in methanol and the resultant solution was concentrated in vacuo; this was repeated once. Benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate was obtained as an oil and used directly in Step 8 below. LC/MS found [M+H]⁺=389.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.41 (br. s, 1H), 4.15 (m, 1H), 4.00 (t, J=9.3 Hz, 1H), 3.81 (t, J=9.1 Hz, 1H), 3.65 (q, J=8.4 Hz, 1H), 3.3-3.4 (m, 1H), 2.45 (m, 1H), 1.95-2.24 (m, 5H), 2.00 (s, 3H), 1.6-1.8 (m, 2H).

Example 1, Step 8: A stirring solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-aminocyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (˜0.135 mol) in methanol (675 mL) was charged sequentially with acetone (37.8 g, 4 eq), sodium acetate (33.2 g, 3 eq), and sodium cyanoborohydride (16.9 g, 2 eq). The mixture was stirred at room temperature for 6 h and filtered. The filtrate was dissolved in dichloromethane (1 L); this solution was washed with 1N NaOH (1 L). The solids collected in the filtration were dissolved in 1N NaOH (1 L) at 0° C. and then extracted with dichloromethane (1 L). The organic extracts were combined and extracted with aqueous HCl (200 mL 1N HCl+800 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then 1N NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil. LC/MS found [M+H]⁺=431.45. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.31 (m, 1H), 4.24 (t, J=9.4 Hz, 1H), 4.11 (m, 1H), 3.61 (t, J=9.1 Hz, 1H), 3.52 (q, J=8.6 Hz, 1H), 3.04 (br. s, 1H), 2.96 (sep, J=6.3 Hz, 1H), 2.40 (m, 1H), 2.15 (m, 1H), 1.92 (s, 3H), 1.7-1.9 (m, 5H), 1.65 (m, 1H), 1.12 (app. dd, J=6.3, 1.1 Hz, 6H).

Example 1, Step 9 (See Alternative Step 9, below): A stirring solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropylamino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (˜115 mmol) in dichloromethane (600 mL) was cooled to 0° C. and charged sequentially with formaldehyde (18.6 g, 37 wt % solution), triethylamine (23 mL), and sodium triacetoxyborohydride (28.7 g). The mixture was stirred at room temperature for 30 minutes and diluted with dichloromethane (up to 1.2 L). This solution was washed thrice with 500 mL sat. NaHCO₃+NaOH (sat. NaHCO₃, pH to 11 w/1N NaOH). The organic layer was extracted with aq. HCl (200 mL 1N HCl+600 mL water). The aqueous phase was basified with sat. NaHCO₃ (500 mL) and then 1N NaOH (100 mL) until pH 11. The aqueous phase was extracted with dichloromethane (1.2 L). The organic extracts were combined, dried (Na₂SO₄), filtered, and concentrated in vacuo to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate as an oil, which was used directly in Step 10 below. LC/MS found [M+H]⁺=445.4. ¹H-NMR (400 MHz, d₄-MeOH): δ 7.3-7.4 (m, 5H), 5.12 (s, 2H), 4.33 (br s, 1H), 4.25 (t, J=9.2 Hz, 1H), 4.11 (br s, 1H), 3.5-3.6 (m, 2H), 2.77 (v br s, 2H), 2.41 (m, 1H), 2.26 (s, 3H), 2.0-2.1 (m, 2H), 1.92 (s, 3H), 1.7-1.9 (m, 5H), 1.10 (app. dd, J=17, 6.4 Hz, 6H).

Example 1, Step 10: To a solution of benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)-cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate (0.115 mol) in methanol (600 mL) was added 10% Pd/C (6 g of 50% wet catalyst). The flask was evacuated and back-filled with hydrogen. The mixture was stirred under 1 atm H₂ for 2 h and the catalyst was removed by filtration through Celite. The filtrate was concentrated in vacuo to provide N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide as an oil, which was taken on to the next step without further purification. LC/MS found [M+H]⁺=311.47. ¹H-NMR (400 MHz, d₄-MeOH): δ 4.39 (br s, 1H), 4.00 (m, 1H), 3.3-3.5 (m, 4H), 2.73 (m, 1H), 2.38 (m, 1H), 2.25 (s, 3H), 2.0-2.2 (m, 3H), 1.94 (s, 3H), 1.6-1.75 (m, 4H), 1.07 (app. dd, J=21, 6.4 Hz, 6H).

Example 1, Step 11: To a solution of N-((1R,2S,5R)-2-((S)-3-amino-2-oxopyrrolidin-1-yl)-5-(isopropyl(methyl)amino)cyclohexyl)acetamide (˜35 g, 0.115 mol) in isopropanol (600 mL) was added 4-chloro-6-(trifluoromethyl)quinazoline (32 g, 0.138 mol, 1.2 eq, see: P. H. Carter et al., PCT application WO 2005/021500). The mixture was stirred at room temperature overnight before being charged with triethylamine (46 g, 0.46 mol, 4 eq). The mixture was stirred at 60° C. for 10 h. The solvent was removed under reduced pressure to give an oil. Azeotropic distillation with isopropanol was performed twice. The residue was dissolved in dichloromethane (600 mL) and extracted with water (250 mL, containing 4 eq acetic acid). Dichloromethane (600 mL) was added to the combined aqueous washes, and the mixture was cooled to 0° C. Aqueous NaOH (50% by weight) was added with stirring until the pH reached 11. The water layer was extracted with dichloromethane twice (2×600 mL). The combined organic extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo to give the amorphous free base of the title compound (99% purity by HPLC). LC/MS found [M+H]⁺=507.3. ¹H-NMR (400 MHz, d₄-MeOH): δ 8.82 (s, 1H), 8.59 (s, 1H), 8.05 (dd, J=8.8, 1.8 Hz, 1H), 7.9 (d, J=8.7 Hz, 1H), 5.28 (t, J=8.6 Hz, 1H), 4.58 (br s, 1H), 4.06 (m, 1H), 3.52-3.68 (m, 2H), 3.43 (m, 1H), 2.76 (br s, 1H), 2.55 (m, 1H), 2.28 (s, 3H), 2.1-2.3 (m, 3H), 2.0 (s, 3H), 2.0 (m, 1H), 1.65-1.8 (m, 3H), 1.09 (app. dd, J=24, 6.4 Hz, 6 H).

Example 1 Alternative Step 9

Example 1, Alternative step 9aⁱ: To a hydrogenator were charged ethyl (7R,8S)-8-((S)-1-phenyl-ethylamino)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1A (1417 g, 2.8 moles, c.f.: WO2004098516, prepared analogous to U.S. Pat. No. 6,835,841), ethanol (200 proof, 11.4 L), and 10% Pd/C catalyst (50% wet, 284 g). The mixture was inerted with nitrogen, then pressurized with hydrogen gas (45 psig) and agitated vigorously at approx. 40° C. until starting material was consumed (HPLC). The suspension was cooled, purged with nitrogen gas and the catalyst was removed by filtration while inerted. The spent catalyst was washed with ethanol (4.3 L). The filtrate and washings were combined and concentrated under vacuum to a volume of 2-3 L while maintaining the batch between 40°-60° C. Isopropyl acetate (5 L) was charged and the mixture was concentrated to a volume of ˜2 L until most ethanol was removed (<0.5%) and residual moisture content was <1,000 ppm. Batch volume was adjusted to ˜7.5 L by the addition of isopropyl acetate. The mixture was heated to 80° C. until clear, then cooled 65°-70° C. Seed crystals of 1 (5 g) were added and the batch was cooled to 50° C. over 2 hours, then further cooled to 20° C. over 4 hours and held for ˜10 hours. The resulting slurry was filtered and the cake was washed with isopropyl acetate (2 L). The product was dried under vaccum at ˜35° C. until volatiles were reduced below ˜1% (LOD). Ethyl (7R,8S)-8-amino-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 was obtained as a white, crystalline solid (936 g, 83% yield; HPLC purity: 99.8%). ¹H-NMR: (300 MHz, CDCl₃) 8.14-7.89 (brs, 3H), 7.75 (d, J 9.0 Hz, 2H), 7.15 (d, J 8.0 Hz, 2H), 4.22-4.04 (m, 2H), 4.01-3.77 (m, 4H), 3.55-3.43 (m, 1H,), 3.20-3.13 (m, 1H), 2.40-2.27 (m, 4H), 2.21-1.94 (m, 2H), 1.81-1.51 (m, 3H), 1.23 (t, J 7.0 Hz, 3H); HPLC: Waters Xterra MS C18 4.6 mm×150 mm i.d., 3.5 μm particle size, 0.05% NH4OH (5% ACN, 95% H₂O, solvent A), to 0.05% NH₄OH (95% ACN, 5% H₂O, solvent B), 5% B to 20% B in 10 minutes, changed to 95% B in 25 minutes, and then changed to 5% B in 1 minute; 11.1 minutes (aminoester 1).

Example 1, Alternative Step 9aⁱⁱ: Aminoester 1 (63 g, 0.16M, 1 eq.; the product of reductive deprotection of a known compound—(See e.g. R. J. Cherney, WO 2004/098516 and G. V. Delucca & S. S. Ko, WO 2004/110993) was placed in a round bottom flask and MeCN (500 mL) was added. EDAC (33.1 g, 0.17M, 1.1 eq), HOBt.H₂O (21.2 g, 0.16M, 1.0 eq) and N-Cbz-L-methionine (46.7 g, 0.17M, 1.05 eq) were then added followed by TEA (48.0 mL, 0.35M, 2.2 eq). An exotherm to 38° C. was observed. The reaction mass was left to stir at RT. After 30 mins, HPLC indicated complete conversion. The reaction mass was diluted with EtOAc (2.5 L) and washed with KHCO₃ (4×500 mL, 20 wt % aq. solution) and brine (500 mL). The organic phase was separated, dried over MgSO₄ and concentrated. The residue was dissolved in TBME and reconcentrated to give ethyl (7R,8S)-8-{(2S)-2-benzyloxycarbonylamino-4-methylsulfanyl-butyr-yl-amino}-1,4-dioxa-spiro[4.5]decane-7-carboxylate 2 as a sticky semi-solid (76.2 g, 98% yield, 93 AP purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.36-7.30 (m, 5H), 7.03 (d, J 9.0 Hz, 1H), 5.66 (d, J 8.0 Hz, 1H), 5.10 (s, 2H), 4.35-4.25 (m, 2H), 4.19-4.04 (m, 2H,), 3.98-3.86 (m, 4H), 2.87-2.80 (m, 1H), 2.55-2.45 (m, 2H), 2.18 (dd, J 14.0 Hz, 7.0 Hz, 1H), 2.08 (s, 3H), 2.05-1.67 (m, 6H), 1.26 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 10.01 min (Compound 2, 93.1 AP). HRMS: m/z 495.2166 [Calc: C₂₄H₃₅N₂O₇S 495.2165].

Example 1, Alternative Step 9b: Methionine amide 2 (75.0 g, 0.15M) was dissolved in MeI (225 mL, 3 mL/g)—some off gassing was noted but no exotherm. The reaction mass was left to stir in the dark for 16.5 h. After this time a thick light yellow precipitate had formed. The flask was then evacuated to 200 mmHg and some of the MeI removed. The remaining material was slurried in TBMF (500 mL), after a 30 min stir-out the slurry was filtered, the cake washed with TBMF (500 mL). NMR analysis of this material indicated a small amount of MeI remaining. The cake was re-slurried in TBMF (500 mL), filtered, washed with TBMF (500 mL) and dried under vacuum to give [(3S)-3-benzyloxycarbonylamino-3-{(7R,8S)-7-ethoxycarbonyl-1,4-di-oxa-spiro[4.5]dec-8-ylcarbamoyl}-propyl]-dimethylsulfonium iodide 3 as a free flowing off-white solid (93.5 g, 97%, 99 area % purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.75 (d, J 9.0 Hz, 1H), 7.38-7.27 (m, 5H), 6.40 (d, J 7.0 Hz, 1H), 5.10 (s, 2H), 4.76-4.65 (m, 1H), 4.48-4.39 (m, 1H), 4.14-3.85 (m, 6H), 3.84-7.73 (m, 1H), 3.68-3.55 (m, 1H), 3.21 (s, 3H), 3.12 (s, 3H), 2.90-2.83 (s, 1H), 2.52-1.55 (m, 8H), 1.24 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 2.45 min (I−), 8.14 min (Compound 3, 43.6 AP, I⁻54.6 AP). HRMS: m/z 509.2341 [Calc: C₂₅H₃₇N₂O₇S 509.2321].

Example 1, Alternative Step 9c: Cs₂CO₃ (61.5 g, 0.19M, 1.5 eq) was placed in an round bottom flask and anhydrous DMSO (2.4 L) was added. Sulfonium salt 3 (80.0 g, 0.13M, 1.0 eq) was then added portionwise. Once the addition was complete the reaction mass was left to stir in the dark for 20 h. The reaction mass was then split in half and each half worked up separately: the reaction mass was diluted with EtOAc (2.0 L) and washed with brine (2 L), the organic phase was washed with brine (500 mL). The combined aq. layers were then washed EtOAc (500 mL). The combined organic phases were then washed with brine (3×750 mL). The second half of the reaction mass was treated in an identical manner and the combined organics dried over MgSO₄ and concentrated to give ethyl (7R,8S)-8-{(3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl}-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4 as a light colored oil (56.5 g, 0.13M, ˜100 area-% purity) pure by NMR analysis. ¹H-NMR: (300 MHz, CDCl₃) δ 7.38-7.30 (m, 5H), 5.37 (br d, J 4.0 Hz, 1H), 5.11 (s, 2H), 4.27-4.18 (m, 1H), 4.17-3.82 (m, 8H), 3.32 (td, J 10.0Hz, 60.0 Hz, 1H), 3.23 (q, J 5.0 Hz, 1H), 2.63-2.57 (m, 1H), 2.42-2.25 (m, 2H), 1.94-1.68 (m, 5H), 1.25 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 8.99 min (Compound 5, produced on column, 4.2 AP), 9.48 (Compound 4, 74.3 AP). HRMS: m/z 447.2127 [Calc: C₂₃H₃₁N₂O₇ 447.2131].

Example 1, Alternative Step 9d: Pyrrolidinone 4 (50.0 g, 0.11M) was dissolved in acetone (500 mL) and 1N HCl (500 mL) was added. The reaction mass was then heated to 65° C. After 20 mins HPLC indicated complete reaction. The reaction mass was allowed to cool to RT and the acetone was removed on a rotary evaporator. During this distillation the product precipitated from solution as a white solid. This was isolated by filtration and the cake washed with water. The cake was then dried azeotropically with toluene (3×300 mL) to give ethyl (1R,2S)-2-((3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-oxo-cyclohexanecarboxylate 5 as a white solid (39.8 g, 88%, 97 area-% purity). ¹H-NMR: (300 MHz, CDCl₃) δ 7.37-7.32 (m, 5H), 6.65 (br d, J 4.0 Hz, 1H), 5.12 (s, 2H), 4.54-4.47 (m, 1H), 4.34-4.26 (m, 1H), 4.18 (dq, J 11.0 Hz, 7.0 Hz, 1H), 4.09 (dq, J 11.0 Hz, 7.0 Hz, 1H), 3.36-3.20 (m, 3H), 2.70-2.35 (m, 6H), 2.05-1.96 (m, 1H), 1.81 (quin., J 11.0 Hz, 1H), 1.24 (t, J 7.0 Hz, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 8.95 min (Compound 5). HRMS: m/z 403.1864 [Calc: C₂₁H₂₇N₂O₆403.1869].

Example 1, Alternative Step 9e: Cyclohexanone 5 (22.5 g, 0.06M, 1 eq), DMSO (30 mL) and Ti(O-iPr)₄ (33.7 mL, 0.11M, 2.04 eq) were placed in a round bottom flask. N-isopropyl-N-methylamine (11.6 mL, 0.11M, 2.0 eq) was then added in one portion. The mixture was left to stir for 30 mins at room temperature before being cooled to <3° C. in ice/water. MeOH (30 mL) was then added followed by the portionwise addition of NaBH₄ (4.33 g, 0.11M, 2.04 eq)—temperature kept <8° C. 30 mins after the addition was completed the reaction mass was diluted with methylene chloride (300 mL) and then NaOH (1N, 40 mL). The resulting slurry was filtered through Celite, and the cake washed with methylene chloride (100 mL). The resulting liquor was concentrated under reduced pressure and the residue dissolved in EtOAc (500 mL). This solution was extracted with 1N HCl (2×400 mL), the combined aqueous layers were then basified with Na₂CO₃. Extraction with EtOAc (4×250 mL) provided a clear and colorless organic phase which was dried over Na₂SO₄ and concentrated to give a white powder (24.6 g, 96%, 7:1 d.r.). This material was then slurried overnight in hexane (670 mL). The solid was isolated by filtration and dried under reduced pressure to give ethyl (1R,2S,5R)-2-((3S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 as a while solid (20.9 g, 81%, 24:1 d.r.). ¹H-NMR: (300 MHz, CDCl₃) δ 7.37-7.28 (m, 5H), 5.55 (d, J 4.5, 1H), 5.10 (s, 2H), 4.42 (q, J 4.5, 1H), 4.23-4.12 (m, 1H), 4.08 (dq, J 10.5, 7.0, 1H), 4.02 (dq, J 10.5, 7.0, 1H), 3.84 (t, J 9.0, 1H), 3.46-3.36 (m, 1H), 3.04 (septet, J 6.5, 1H), 2.86-2.80 (m, 1H), 2.63-2.48 (m, 2H), 2.17 (s, 3H, Me), 2.10-1.63 (m, 7H), 1.22 (t, J 7.0, 3H), 1.00 (d, J 6.5, 3H), 0.97 (d, J 6.5, 3H). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.01M NH₄OAc (MeOH:water 20:80) to 0.01M NH₄OAc (MeOH:water:MeCN 20:5:75) 10 to 100% 15 min gradient. 8.23 (Compound 6), 8.88 (5-epi-Compound 6). HRMS: 460.2798 [Calc: C₂₅H₃₈N₃O₅ 460.2811].

Example 1, Alternative Step 9f: The aminoester 6 (9.76 g, 2.12 mmol) was dissolved in 2N HCl (80 mL), then heated to ˜55° C. under inert atmosphere. The reaction was stirred for 20 h, then cooled to room temperature. The reaction solution was washed twice with toluene (25 mL portions), neutralized to pH 6-7 by the addition of KOH pellets, then extracted eight times with methylene chloride (100 mL portions). The combined extracts were dried (Na₂SO₄), filtered, and concentrated under reduced pressure to 50 mL total volume. The concentrated solution was then slowly added to methyl tert-butyl ether (300 mL) over 15 min in an addition funnel with vigorous stirring. The resulting white slurry was stirred at ambient temperature for Ih, then cooled to 0° C. and stirred for 1 h. The product was filtered, and washed twice with methyl tert-butyl ether (25 mL portions). Water from the wet cake was removed by azeotropic distillation with acetonitrile (300 mL). The product was dried under reduced pressure to provide (1R,2S,5R)-2-((3S)-3-Benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylic acid 7, (7.69 g, 84% yield) as a white foam. ¹H-NMR: (400 MHz, 50° C., CDCl₃) δ 7.44-7.32 (m, 5H), 6.10 (broad s, 1H), 5.19 (app s, 2H), 4.42 (dd, J=15.6, 7.8 Hz, 1H), 4.29-4.23 (m, 1H), 3.68-3.60 (m, 2H), 3.33-3.27 (m, 2H), 3.20 (broad s, 1H), 2.99 (broad s, 1H), 2.51 (s, 3H), 2.49-2.45 (m, 3H), 2.33-2.31 (m, 1H), 2.00 (ddd, J=9.0, 8.6, 3.9 1H), 1.95-1.78 (m, 2H), 1.36-1.21 (m, 6H). LCMS: m/z 432.20 [Calc: C₂₃H₃₄N₃O₅ 432.25].

Example 1, Alternative Step 9g: Amino acid 7 (6.3 g, 14.7 mmol, 1.0 eq) was dissolved in THF (80 mL) under N₂ and NaH (584 mg, 14.7 mmol, 1.0 eq, 60 wt % dispersion in mineral oil) was added portionwise. When the addition was complete, and the evolution of gas had ceased, the reaction mass was concentrated under reduced pressure and the resulting solid azeotroped with toluene (50 mL) to give a white solid (KF 0.59 wt %). This solid was slurried in toluene (100 mL) under N₂and heated to 90° C. DPPA (3.32 mL, 15.3 mmol, 1.05 eq) was added dropwise over ˜2 min. After ˜5 min all the solids had dissolved, after 10 mins precipitation of a white solid was observed. After 30 mins HPLC analysis indicated complete reaction. The reaction mass was allowed to cool to RT before being filtered, the cake was washed with toluene. The liquors where then slowly added into AcOH/Ac₂O (80/20, 168 mL) solution at 90° C. After 45 mins HPLC still indicated some isocyanate. At 1.15 h, the reaction mass was cooled to RT and diluted with toluene (100 mL) and water (100 mL). The organic layer was removed and the toluene washed with 1N HCl (100 mL). The combined aq. phases were then basified with K₂CO₃(s) and brought to pH 12 with NaOH (10N), keeping the temperature below 20° C. The aq layer was then extracted with methylene chloride (4×150 mL), the combined organic layers dried over K₂CO₃ and concentrated to give benzyl (S)-1-((1S,2R,4R)-2-acetamido-4-(isopropyl(methyl)amino)cyclohexyl)-2-oxopyrrolidin-3-ylcarbamate 8 as a white foam (4.5 g, 70%, 94AP purity). The ¹H-NMR was identical to material obtained from the route described above (Example 1, Step 9). HPLC: YMC-Pack Pro C18 5 μm 4.6×150 mm, 0.05% TFA (20% MeOH, 80% H₂O), to 0.05% TFA (20% MeOH, 80% MeCN), 0-100% 10 min gradient. 7.20 min (Compound 8), 7.85 min (urea dimer). HRMS: 445.2809 [Calc: C₂₄H₃₇N₄O₄445.2815].

Alternative Preparation of Example 1

Example 1, Alternative Preparation, Step 1: Ethyl (7R,8S)-8-amino-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4-toluenesulfonate salt 1 (450.1 g), was combined with 1-ethyl-3-(3-dimethyl-amino-propyl)carbo-diimide hydrochloride (236.3 g), 1-hydroxy benzotriazole hydrate (171.9 g), N-carbobenzyloxy-L-methionine (333.4 g) and acetonitrile (3.1 L). To the stirred mixture was added triethylamine (249.5 g) below 30° C. Upon reaction completion (HPLC), the mixture was diluted with ethyl acetate (8.2 L) and washed with aqueous 25% potassium bicarbonate solution (2×4.5 L) followed by water (4.5 L). The organic phase was separated and concentrated under reduced pressure to obtain a solution of ethyl (7R,8S)-8-((S)-2-benzyloxycarbonylamino-4-methylsulfanyl-butyrylamino)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 2 (1.4 L). Methyl iodide (2.39 kg) was added, the vessel was shielded from light and the mixture was held under slow agitation for approx. 24 h. To the thick yellow precipitate was added methyl tert-butyl ether (2.7 L) and the mixture was held for approx. 1 h. The product was isolated by filtration and the cake was washed with methyl tert-butyl ether (2×1.4 L), then dried under vacuum, yielding [(S)-3-benzyloxy-carbonylamino-3-((7R,8S)-7-ethoxycarbonyl-1,4-dioxa-spiro[4.5]dec-8-ylcarbamoyl)-propyl]-dimethylsulfonium iodide 3 (671.4 g, ˜94% yield) as an off-white solid (HPLC purity 99.9%).

Example 1, Alternative Preparation, Step 2: Sulfonium salt 3 (619.4 g), and cesium carbonate (416.8 g) and anhydrous dimethyl sulfoxide (6.2 L) were combined in a reactor equipped with a scrubber to neutralize volatile sulfides. Vigorous agitation was maintained until complete conversion was obtained (HPLC). Ethyl acetate (12.4 L) was added, followed by 20% brine (3 L). The organic phase was separated, washed twice with brine (2×3 L) and evaporated to obtain a solution of ethyl (7R,8S)-8-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-1,4-dioxa-spiro[4.5]decane-7-carboxylate 4 in ethyl acetate (˜0.8 L). Acetone (2.55 L) was added, followed by aqueous 0.5 M hydrochloric acid solution (2.3 L). With good mixing, the solution was heated to 50 to 60° C. until conversion of 4 to ethyl (1R,2S)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-oxo-cyclohexanecarboxylate 5 was complete (HPLC). The mixture was concentrated under reduced pressure while below 40° C., cooled to ˜30° C., and water (4.1 L) was added. The resulting slurry was cooled to 5 to 10° C. and agitated for ˜1 hour. The product was filtered and the cake was washed with water (2×2.5 L). Upon deliquoring, the cake was dried to a constant weight below 40° C. in a vacuum oven. Cyclohexanone 5 (272 g, 70% yield) was obtained (HPLC purity 98.7%).

Example 1, Alternative Preparation, Step 3: Cyclohexanone 5 (206 g) was dissolved in dichloromethane (1.1 L) and charged to a hydrogenator. Titanium tetraisopropoxide (218.2 g) and N-isopropyl N-methylamine (63.64 g) were added and the mixture was stirred at ambient temperature (23 to 25° C.) for at least 5 h. Platinum catalyst (5% Pt/S/C, 15 g, approx. 7.5% relative to 5) was added and hydrogenation was performed at ˜30 psig for at least 6 h, yielding a mixture of ethyl (1R,2S,5R)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexanecarboxylate 6 and its 5-epi-isomer (˜7%). The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure to approx. ˜600 mL. Wet ethyl acetate (˜3% water, 2.0 L) was added with vigorous agitation over a period of at least 1.5 h. Stirring was continued for at least an additional 6 h. The slurry was filtered. Filter cake was washed with ethyl acetate (1.0 L) and discarded. The combined filtrate and washings were concentrated to ˜400 mL. Toluene (2.0 L) was added and the solution was washed with 2M aqueous hydrochloric acid (2×400 mL). The aqueous layer was warmed to 50° to 60° C. for approx. 20 h or hydrolysis of 6 was deemed complete (HPLC). Aqueous sodium hydroxide solution was added to adjust to pH ˜10, and mixture was extracted with toluene (3×600 mL). The organic phase was discarded and pH was readjusted to ˜6 by addition of aqueous hydrochloric acid. The aqueous phase was concentrated to ˜600 mL under reduced pressure and extracted with methylene chloride (at least 3×2.0 L). The combined methylene chloride layers were evaporated under reduced pressure and continuously replaced with THF to obtain a solution of (1R,2S,5R)-2-((S)-3-benzyloxycarbonylamino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexane carboxylic acid 7 (˜148 g) in THF (˜4 L). Seed crystals of 8 were added, followed by 25% solution of sodium methoxide in methanol (81.24 g) below 25° C. The slurry was held for at least additional 16 h with agitation. The product was isolated by filtration and the cake was washed with THF (4×200 mL) and dried to a constant weight in vacuo below 30° C. Dry (1R,2S,5R)-2-((S)-3-benzyloxycarbonyl-amino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexane-carboxylate sodium salt 8 was obtained (139 g, ˜60% yield from 5).

Example 1, Alternative Preparation, Step 4: Aminoester sodium salt 8 (100 g), diphenyl phosphate (3.86 g), tert-BuOH (1275 mL) and toluene (225 mL) were combined and heated to reflux under reduced pressure. Approx. 500 mL of distillate were collected and discarded while being continuously replaced with a solution of toluene in tert-BuOH. Vacuum was removed and distillate was switched to percolate through a column filled with molecular sieves and allowed to return to the vessel. After drying was complete, DPPA (52.4 mL; dissolved in 60 mL toluene) was added slowly to the slurry at 80° C. Upon complete conversion (HPLC), tert-BuOH was removed by vacuum distillation and continuously replaced with toluene. The mixture was cooled to room temperature and washed twice with 10% aqueous K₂HPO₄ (1×800 mL, 1×400 mL) and water (400 mL). The organic phase was heated and concentrated in vacuo to approx. 270 mL. Vacuum was removed and heptane (1.1 L) was added slowly at approx. 80° C., followed by seeds of 9 (˜1 g). The slurry was slowly cooled to room temperature and benzyl {(S)-1-[(1S,2R,4R)-2-tert-butoxycarbonylamino-4-(isopropyl-methyl-amino)-cyclo-hexyl]-2-oxo-pyrrolidin-3-yl}-carbamate 9 was isolated by filtration as a white solid (86.76 g, 78% yield).

Example 1, Alternative Preparation, Step 5: The tert-Butyl carbamate 9 (50 g) was dissolved in Toluene (500 mL) and i-PrOH (150 mL). The resulting solution was then heated to 60° C. Methanesulfonic acid (19.6 mL) was added below 65° C. Upon reaction completion (HPLC), the mixture was cooled to RT and triethylamine (69.4 mL) added slowly below 25° C. Acetic anhydride was then added below 25° C. After 1 h acetic acid (250 mL) was added below 25° C. The toluene phase was discarded and 2-methyl-THF (500 mL) was added to the aqueous phase. The mixture was stirred vigorously and basified with NaOH (25% aqueous solution) to pH 12. The aqueous phase was discarded and the organic layer was washed with brine (250 mL). The organic layer was concentrated under reduced pressure and continuously replaced with i-PrOH. The solution was cooled and filtered to provide benzyl {(S)-1-[(1S,2R,4R)-2-acetylamino-4-(isopropyl-methyl-amino)-cyclohexyl]-2-oxo-pyrrolidin-3-yl}-carbamate 10 in i-PrOH solution which was used directly in the hydrogenation.

Example 1, Alternative Preparation, Step 6: To a solution containing acetamide 10 (˜61 g) in i-PrOH (˜625 mL) was added 10% Pd/C wet catalyst (2.5 g) and the suspension was hydrogenated at 30 psig and approx. 25° C. for at least 2 h. Upon completion (HPLC), the catalyst was removed by filtration and the filtrate was concentrated to approx. 550 mL. Water (8.8 mL) was added, followed by 5.6 N hydrochloric acid in i-PrOH solution (69.5 mL). The resulting slurry was held at room temperature overnight. The product was isolated by filtration and the cake was rinsed with i-PrOH (2×100 mL) and dried in vacuo to constant weight at ˜50° C. to give N-[(1R,2S,5R)-2-((S)-3-amino-2-oxo-pyrrolidin-1-yl)-5-(isopropyl-methyl-amino)-cyclohexyl]-acetamide 11 (55.6 g, 97% yield) as its hydrochloric acid salt (73.6% free base assay, HPLC).

Example 1, Alternative Preparation, Step 7: To 6-trifluoromethyl-quinazolin-4-ol 12 (20.1 g) in MeCN (400 mL) was added 5.5 M solution of sodium methoxide in methanol (17.0 mL). The resulting suspension was distilled under reduced pressure and continuously replaced by MeCN to remove methanol. To the slurry was added DMF (1.4 g), followed by oxalyl chloride (13.0 mL) below 50° C. Upon reaction completion (HPLC), excess reagent was removed under reduced pressure to give ˜400 mL of slurry. The mixture was cooled to room temperature and washed with 10% aqueous K₂HPO₄ (1×1.0 L, 1×0.5 L) to afford 4-chloro-6-trifluoromethyl-quinazoline 13 (˜21.2 g) in approx. 450 mL of wet MeCN solution, which was used directly in the subsequent coupling reaction (HPLC purity 99.8%).

Example 1, Alternative Preparation, Step 8: To a mixture of acetamide 11 (28.5 g, HCl salt, 73.6% free base assay), acetonitrile (100 mL), N,N,-di-isopropyl-N-ethylamine (61 mL) at room temperature was added a solution of 13 (˜21.2 g) in MeCN (˜450 mL). The homogeneous mixture was held overnight. Upon reaction completion (HPLC), the mixture was concentrated in vacuo to approx. 125 mL. A 9.5% aqueous solution of acetic acid (240 mL) was added and the aqueous phase was extracted with methylene chloride. The aqueous phase was separated and methyl tert-butyl ether (450 mL) was added, followed by 2N aqueous lithium hydroxide solution to adjust to pH>11.5. The organic layer was separated, washed with water and filtered. Approx. half of the ether phase was diluted with methyl tert-butyl ether (˜250 mL) and concentrated in vacuo. Heptane (45 mL) was added slowly below 60° C., followed by seed crystals of Example 1 (0.4 g). Additional heptane (125 mL) was added and the mixture was slowly cooled to room temperature and the resulting slurry was held overnight. The product was isolated by filtration, the cake was washed with heptane and dried in vacuo to constant weight to give N-((1R,2S,5R)-5-(isopropylamino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)-quin-azolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide 14 (15.0 g, 85% yield).

Crystallization Procedures for Example 1Example 1, Production of bis-BSA salt and purification: The entirety of the amorphous free base from Example 1, Step 11 was dissolved in methanol (600 mL). The resultant solution was heated at 60° C. and charged with benzenesulfonic acid (2.5 eq). The mixture was cooled to room temperature and the resultant white solid was collected by filtration to yield the bis-benzene sulfonic acid salt of the title compound (95 g, 86%). This material was >99% pure by HPLC. This material was further purified by re-crystallization from 80/20 EtOH/H₂O, which provided the salt free from any residual methanol. HPLC purity=99.8%. ¹H NMR (500 MHz, D₂O) δ ppm 8.75 (1H, s), 8.66 (1H, s), 8.25 (1H, d, J=8.80 Hz), 7.90 (1H, d, J=8.80 Hz), 7.75 (4H, d, J=8.25 Hz), 7.43-7.57 (6H, m), 5.42 (1H, t), 4.33-4.44 (1H, m), 4.09-4.19 (1H, m), 3.83-3.91 (1H, m), 3.74-3.83 (2H, m), 3.61 (1H, t, J=11.55 Hz), 2.75 (3H, d, J=6.60 Hz), 2.61-2.70 (1H, m), 2.31-2.44 (1H, m), 2.20-2.27 (1H, m), 2.17 (2H, d, J=12.10 Hz), 1.94-2.04 (1H, m, J=12.65 Hz), 1.90-1.95 (3H, m), 1.72-1.91 (2H, m), 1.37 (3H, d, J=6.05 Hz), 1.29 (3H, d, J=6.60 Hz). Differential scanning calorimetry utilized a heating rate of 10° C./min and revealed a melting/decomposition endotherm with an onset temperature of 297.6° C. and a peak temperature at 299.1° C.

Example 1, Crystallization of the Free Base: A sample of the amorphous free base of N-((1R,2S,5R)-5-(isopropyl(methyl)amino)-2-((S)-2-oxo-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-1-yl)cyclohexyl)acetamide (1 g) was dissolved in dichloromethane (5 mL). The solution was charged with heptane (30 mL) and then warmed to distill the dichloromethane. The solution was cooled to 40° C.; a white solid precipitated. The suspension was heated to 90° C. and stirred for 2 h. The suspension was cooled to room temperature and filtered to provide the pure free base of the title compound. No residual solvent was apparent by ¹H-NMR.

PAPER

A concise bulk synthesis of stereochemically complex CCR2 antagonist BMS-741672 is reported. A distinct structural feature is the chiral all-cis 1,2,4-triaminocyclohexane (TACH) core, which was assembled through consecutive stereocontrolled heterogeneous hydrogenations: efficient Pt-catalyzed reduction of a β-enaminoester, directed by (S)-α-methylbenzylamine as a low-cost chiral template, and reductive amination of a 3,4-cis-disubstituted cyclohexanone over sulfided Pt/C introduced a tert-amine, setting the third stereocenter in the all-cis cyclohexane core. The heterogeneous catalysts were recycled. Ester hydrolysis produced a γ-amino acid, isolated as its Na salt. A challenging Curtius reaction to introduce the remaining C–N bond at C-2 was strongly influenced by the presence of the basic tert-amine, providing a stereoelectronically highly activated isocyanate. Detailed mechanistic and process knowledge was required to enable clean trapping with an alcohol (t-BuOH) while avoiding formation of side products, particularly an unusual carbamoyl phosphate. Deprotection, N-acetylation, and uncatalyzed S_NAr coupling with known 4-chloroquinazoline provided the final product. The resulting 12-step synthesis was used to prepare 50 kg of the target compound in an average yield of 82% per step.

Stereoselective Bulk Synthesis of CCR2 Antagonist BMS-741672: Assembly of an All-cis (S,R,R)-1,2,4-Triaminocyclohexane (TACH) Core via Sequential Heterogeneous Asymmetric Hydrogenations

Patents

Bristol-Myers Squibb, Paul Biondi Senior Vice President, Head of Business Development

About Bristol-Myers Squibb

Bristol-Myers Squibb is a global biopharmaceutical company whose mission is to discover, develop and deliver innovative medicines that help patients prevail over serious diseases. For more information, please visit www.bms.com or follow us on Twitter at http://twitter.com/bmsnews.

//////////   ////////////////BMS-741672, BMS 741672, BRISTOL MEYER SQUIB, PHASE 2,  type 2 Diabetes, Neuropathic Pain, Bristol-Myers Squibb
CC(C)N(C)[C@H]1C[C@@H](NC(C)=O)[C@H](CC1)N4CC[C@H](Nc3ncnc2ccc(cc23)C(F)(F)F)C4=O

4-(6-chloro-1-oxo-3-thioxo-9,9a-dihydro-1H-imidazo[1,5-a]indol-2(3H)-yl)-2-(trifluoromethyl)-benzonitrile

WILL BE UPDATED………

Prostate cancer, one of the most malignant tumors worldwide, is the second leading cause of cancer deaths among men in America . Although androgen deprivation therapy (ADT) has been proved to be effective initially, the tumor will eventually progress and develop into the lethal castration resistant prostate cancer (CRPC) . The androgen receptor (AR) is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily and plays a critical role in the progression of normal prostate cells. However, overexpression of AR was found in most CRPC, which is essential for CRPC to adapt to the low levels of androgens. As AR contributes significantly to the resistance to castration, it has been recognized as an attractive target for the treatment of CRPC

Reagents and conditions: (i) HNO3 , H2SO4 , -5 oC, 3 h; (ii) SOCl2 , MeOH, reflux, 12 h; (iii) H2 , Pd/C, MeOH, rt, 12 h; (iv) (CH3CO)2O, TEA, 50 oC, 6 h; (v) H2 , Pd/C, MeOH, rt, 12 h; (vi) acetone, HCl (6 mol/L), -10 oC, 0.5 h, NaNO2 , H2O, -10 oC, 1 h, CuCl/CuBr/KI, 0 oC, 3 h; (vii) HCl, 50 oC, 3 h; (viii) SOCl2 , MeOH, reflux, 12 h; (ix) 2, DMF, TEA, 60 oC, 1 h.

4-(6-chloro-1-oxo-3-thioxo-9,9a-dihydro-1H-imidazo[1,5-a]indol-2(3H)-yl)-2-(trifluoromethyl)-benzonitrile (48c). It was obtained as a yellow solid

m.p. 220-222 oC;

1H-NMR (300 MHz，DMSO-d6): δ 8.40 (d, J = 8.1 Hz, 1H, Ar-H), 8.19 (s, 1H, Ar-H), 8.02-7.92 (m, 2H, Ar-H), 7.49-7.46 (m, 1H, Ar-H), 7.34-7.32 (m, 1H, Ar-H), 5.56 (t, J = 9.6 Hz, 1H, -CH-), 3.58 (d, J = 9.6 Hz, 2H, -CH2-) ppm;

13C-NMR (75 MHz, DMSO-d6): δ 184.1, 172.1, 142.3, 138.5, 136.8, 134.7, 131.9, 131.5, 128.0, 126.1 (q, J = 267.9 Hz, CF3), 117.2, 115.4, 66.9, 39.9 ppm;

IR (KBr): 3094, 2232, 1763, 1607, 1499, 1270, 1136, 1052, 998, 786 cm-1;

HRMS (ESI): m/z, calculated for C18H9ClF3N3OS 408.0180 (M + H)+ , found 408.0173.

Paper

A series of indoline thiohydantoin derivatives were synthesized and evaluated in vitro.The most potent compound 48c shows comparable ability with enzalutamide in proliferation inhibition of LNCaP cells.Compound 48c has less cytotoxic to AR-negative cells compared with Enzalutamide.

The bicalutamide-resistant mechanism was clarified and overcome by compound 48c.

European Journal of Medicinal Chemistry

Available online 22 October 2016

In Press, Accepted Manuscript — Note to users

http://dx.doi.org/10.1016/j.ejmech.2016.10.049

////////Prostate cancer, Androgen receptor, Antagonist, Indoline thiohydantoin derivatives, indoline thiohydantoin derivatives, enzalutamide, antiproliferative agents, prostate cancer

c1c(cc(c(c1)C#N)C(F)(F)F)N2C(C3Cc4ccc(cc4N3C2=S)Cl)=O

PAPER

HETEROCYCLES

Published online: 11th October, 2016

■ A Concise and Highly Efficient Synthesis of Praziquantel as an Anthelmintic Drug

Abstract

BMS-599793

(DS003, BMS-599793)

2-(1-(2-(4-methoxy-7-(pyrazin-2-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanoyl)piperidin-4- ylidene)-2-phenylethanenitrile

1 (DS003, BMS-599793) is a small molecule entry inhibitor that interferes with HIV infection by binding to the gp120 protein.1 The International Partnership for Microbicides (IPM) licensed 1 from Bristol-Myers Squibb (BMS) with the goal to develop it as a topical microbicide for use in resource-poor countries. Microbicides are vaginal dosage forms of potent inhibitors of HIV that women can use to prevent sexual transmission of HIV from male partners.

1 (a) Maddon, P. J.; Dalgleish, A. G.; McDougal, J. S.; Clapham, P. R.; Weiss, R. A.; Axel. R. Cell 1986, 47, 333-348. (b) McDougal, J. S.; Kennedy, M. S.; Sligh, J. M.; Cort, S. P.; Mawle, A.; Nicholson, J. K. Science 1986, 231, 382-385. (c) Moore, J. P.; Jameson, B. A.; Weiss, R. A.; Sattentau, Q. J. in Viral fusion mechanisms. ed. J. Bentz, CRC Press, Boca Raton, Fla. 1993, p. 233-289.

2-(1-(2-(4-methoxy-7-(pyrazin-2-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanoyl)piperidin-4- ylidene)-2-phenylethanenitrile (1, laboratory scale process). A flask was charged with acid 11 (9.29 g, 31.2 mmol), DIPEA (12.9 mL, 78 mmol), 4 (7.18 g, 36.3 mmol) and DMF (95 mL) subsequently. HATU (13.66 g, 35.9 mmol) was added to the reaction mixture over 10 minutes, which was accompanied by increase of internal temperature from 19 0C to 27 0C. After the reaction mixture was stirred at 25 0C for 3.5  h the HPLC analysis showed complete disappearance of acid 11. Ethanol (950 mL) was added and the resulting suspension was heated at reflux for 1 h. The mixture was then cooled to 25 0C and 1 was isolated by filtration and washed with ethanol (50 mL). The material was dried under vaccum at 50 0C to afford 10.58 g (71% yield) of 1 as a colorless solid.

1H NMR (300 MHz, CDCl3) 2.58-2.65 (m, 2H), 2.91-2.99 (m, 2H), 3.48-3.51(m, 1H), 3.68-3.78 (m, 2H), 3.95-3.99 (m, 1H), 4.11 (s, 3H), 7.27-7.46 (m, 5H), 8.16 (d, J = 5.1 Hz, 1H), 8.21-8.25 (m, 1H), 8.60 (s, 2H), 9.82 (d, J = 3.9 Hz, 1H), 11.75(br s, 1H).

LCMS: m/e 479.3 (M+H)+. Analysis by ICP-MS showed 16 ppm Pd, 79 ppm Fe, 102 ppm Zn. This material was found to be a mixture of two polymorphs: Form 1 and Form 2.

kilo-lab scale process including polymorph conversion

1 (84% yield) and 99.6% purity by HPLC. This material was a pure Form 1 polymorph.

1H NMR (400 MHz, CDCl3) 2.61 (t, J = 5.8 Hz, 1H), 2.65 (t, J = 5.9 Hz, 1H), 2.94 (t, J = 5.8 Hz, 1H), 2.99 (t, J = 5.9 Hz, 1H), 3.51 (t, J = 5.8 Hz, 1H), 3.72 (t, J = 5.8 Hz, 1H), 3.78 (t, J = 6.0 Hz, 1H), 3.98 (t, J = 6.0 Hz, 1H), 4.12, 4.12 (two s, 3H), 7.29-7.47 (m, 5H), 8.16, 8.18 (two s, 1H), 8.23, 8.25 (two d, J = 3.1 Hz,  1H), 8.60-8.63 (m, 2H), 9.83, 9.84 (two d, J = 1.4 Hz, 1H), 11.76, 11.78 (two br s, 1H);

13H NMR (100 MHz, CDCl3) 30.3, 31.0, 33.7, 34.3, 41.7, 42.0, 46.0, 46.3, 56.8, 111.4, 115.0 (2C), 117.7 (2C), 120.9, 124.2, 128.9 (2C), 129.0, 129.1 (2C), 131.9, 132.6 (2C), 133.9 (2C), 136.6, 142.2, 143.4, 143.7 (2C), 151.1, 151.3, 154.8, 166.4, 166.5, 185.6

Anal. Calcd for C27H22N6O3: C, 67.77; H, 4.63; N, 17.56. Found: C, 67.84; H, 4.64; N, 17.56.

1H NMRPREDICT

13C NMR PREDICT

Synthetic Process Development of BMS-599793 Including Azaindole Negishi Coupling on Kilogram Scale

Abstract

A new approach to the synthesis of 1 (DS003, BMS-599793), a small-molecule HIV entry inhibitor, is described. The initial medical chemistry route has been modified by rearranging the sequence of synthetic steps followed by replacement of the Suzuki coupling step by the Negishi conditions. Acylation of the resulting azaindole 7 under the Friedel–Crafts conditions is studied using monoesters of chlorooxalic acid in the presence of aluminum chloride. Polymorphism of 1is also investigated to develop conditions suitable for preparation of the desired Form 1 of the target compound. The new route is further optimized and scaled up to establish a new process that is applied to the synthesis of kilogram quantites of the target active pharmaceutical ingredient.

////////BMS-599793, BMS 599793

SOLIFENACIN, YM-905

MF C₂₃H₂₆O₂, a molecular weigt  362.4647

CAS 242478-37-1

242478-37-1 (Solifenacin )
242478-38-2 (Solifenacin Succinate)

Solifenacin succinate, YM-67905

CAS 242478-38-2

Usage Muscarinic M3 receptor antagoinst. Used in treatment of urinary incontinence.
Usage sedative
Usage Solifenacin succinate is a urinary antispasmodic of the antimuscarinic class.

(3R)-l-azabicyclo[2.2.2]oct-3-yl-(lS)-l-phenyl-3,4-dihydroisoquinoline-2-(lH)- carboxylate ((S)-phenyl-l_?2,3,4-tetrahydroisoquinoline-2-carboxylic acid 3(R)-quinuclidinyl ester) is known as solifenacin, also known as YM-905 (in its free base form) and YM-67905 (in its succinate form). Solifenacin has the molecular formula C₂₃H₂₆O₂, a molecular weight of 362.4647, and the following chemical structure:

C23H26N2O2 Exact Mass: 362.1994

MoI. Wt.: 362.4647 m/e: 362.1994 (100.0%), 363.2028 (25.6%), 364.2061 (3.1%) C, 76.21; H, 7.23; N, 7.73; O, 8.83

Solifenacin succinate is a urinary antispasmodic, acting as a selective antagonist to the M(3)-receptor. It is used as treatment of symptoms of overactive bladder, such as urinary urgency and increased urinary frequency, as may occur in patients with overactive bladder syndrome (OAB), as reviewed in Chilman-Blair, Kim et at., Drugs of Today, 40(4):343 – 353 (2004). Its crystalline powder is white to pale yellowish-white and is freely soluble at room temperature in water, glacial acetic acid, DMSO, and methanol. The commercial tablet is marketed under the trade name VESICAJRE®. As VESICARE®, it was approved by the FDA for once daily treatment of OAB and is prescribed as 5 mg and 10 mg tablets.

The drug was developed by Yamanouchi Pharmaceutical Co. Ltd. and disclosed in US. Patent No. 6,017,927 and its continuation, US. Patent No. 6,174,896.

Solifenacin succinate was first approved by the European Medicines Agency (EMA) on June 8, 2004, then approved by U.S. Food and Drug Administration (FDA) on Nov 19, 2004, and approved by Pharmaceuticals and Medical Devices Agency of Japan (PMDA) on April 20, 2006. It was developed and marketed as Vesicare^® by Astellas.

Solifenacin is a competitive muscarinic receptor antagonist. Muscarinic receptors play an important role in several major cholinergically mediated functions, including contractions of urinary bladder smooth muscle and stimulation of salivary secretion. By preventing the binding of acetylcholine to these receptors, solifenacin reduces smooth muscle tone in the bladder, allowing the bladder to retain larger volumes of urine and reducing the number of micturition, urgency and incontinence episodes. It is indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency.

Vesicare^® is available as tablet for oral use, containing 5 or 10 mg of Solifenacin succinate. The recommended dose is 5 mg once daily. If the 5 mg dose is well tolerated, the dose may be increased to 10 mg once daily.

(1S)-3,4-dihydro-1-phenyl-2-(1H)-isoquinolinecarboxylic acid (3R)-1- azabicyclo[2.2.2]oct-3-yl ester. succinate (Solifenacin succinate) (1)

Solifenacin succinate (1) as white crystalline powder. (104.50 g, 87% w/w yield based on in-put).

Chromatographic purity: 99.94 % (by HPLC). Chiral purity: 99.94% (by chiral HPLC). (1S, 3’S)- Diastereomer content: 0.06% (by chiral HPLC).

Mp 145-146 °C.

[α]D 25 (c=1, in Water): + 40.6°.

IR (KBr) (cm-1): 3282, 3024, 3007, 2964, 2937, 2881, 2607, 1722, 1685, 1579, 1491, 1227, 761, 751.

HRMS: m/z = 363.2071 [M + H] + . 1H NMR (DMSO-d6): δ 1.50 – 1.81 (m, 4H), 2.07 (m, 1H), 2.36 (s, 4H), 2.56 – 3.30 (m, 8H), 3.41 & 3.85 (2m, 2H), 4.79 (m, 1H), 6.27 (brs, 1H), 7.20 – 7.32 (m, 9H), 11.79 (brs, 2H).

13C NMR (DMSO-d6): δ 18.3 (CH2), 22.3 (CH2), 24.6 (CH), 27.7 (CH2), 30.2 (2xCH2), 38.9 (CH2), 45.1 (CH2), 46.0 (CH2), 54.1 (CH2), 57.3, 70.2, 126.2 (CH), 127.1 (CH), 127.2 (2xCH), 128.1 (CH), 128.4 (2xCH), 128.7 (CH), 134.7, 135.4, 154.3, 174.5.

Solifenacin (INN, trade name Vesicare) is a medicine of the antimuscarinic class and was developed for treating contraction of overactive bladder^[1] with associated problems such as increased urination frequency and urge incontinence.^[2] It is manufactured and marketed by Astellas, GlaxoSmithKline^[3] and Teva Pharmaceutical Industries.

Solifenacin is contraindicated for people with urinary retention, gastric retention, uncontrolled or poorly controlled closed-angle glaucoma, severe liver disease (Child-Pugh class C),^[4] and hemodialysis.^[2]

Long QT syndrome is not a contraindication although solifenacin, like tolterodine and darifenacin, binds to hERG channels of the heart and may prolong the QT interval. This mechanism appears to be seldom clinically relevant.^[5]

Side effects

The most common side effects of solifenacin are dry mouth, blurred vision, and constipation. As all anticholinergics, solifenacin may rarely cause hyperthermia due to decreased perspiration.^[4]

Interactions

Solifenacin is metabolized in the liver by the cytochrome P450 enzyme CYP3A4. When administered concomitantly with drugs that inhibit CYP3A4, such as ketoconazole, the metabolism of solifenacin is impaired, leading to an increase in its concentration in the body and a reduction in its excretion.^[4]

As stated above, solifenacin may also prolong the QT interval. Therefore, administering it concomitantly with drugs which also have this effect, such as moxifloxacin or pimozide, can theoretically increase the risk of arrhythmia.^[3]

Pharmacology

Mechanism of action

Solifenacin is a competitive cholinergic receptor antagonist, selective for the M₃ receptor subtype. The binding of acetylcholine to these receptors, particularly M₃, plays a critical role in the contraction of smooth muscle. By preventing the binding of acetylcholine to these receptors, solifenacin reduces smooth muscle tone in the bladder, allowing the bladder to retain larger volumes of urine and reducing the number of micturition, urgency and incontinence episodes. Because of a long elimination half life, a once-a-day dose can offer 24-hour control of the urinary bladder smooth muscle tone.^[2]

Pharmacokinetics

Peak plasma concentrations are reached 3 to 8 hours after absorption from the gut. In the bloodstream, 98% of the substance are bound to plasma proteins, mainly acidic ones. Metabolism is mediated by the liver enzyme CYP3A4 and possibly others. There is one known active metabolite, 4R-hydroxysolifenacin, and three inactive ones, the N–glucuronide, the N-oxide and the 4R-hydroxy-N-oxide. The elimination half-life is 45 to 68 hours. 69% of the substance, both in its original form and as metabolites, are excreted renally and 23% via the feces.^[2]

Chemistry

Like other anticholinergics, solifenacin is an ester of a carboxylic acid containing (at least) an aromatic ring with an alcohol containing a nitrogen atom. While in the prototype anticholinergic atropine the alcohol is tropine, solifenacin has another bicycle, quinuclidinyl alcohol.

The substance is a basic yellow oil, while the form used in tablets, solifenacin succinate, consists of white to slightly yellowish crystals.^[6]

Scheme 1 wherein the quinuclidinol reactant is available commercially. The overall synthesis as reported by Mealy, N., et al. in Drugs of the Future, 24 (8): 871-874 (1999) is depicted in Scheme 2:

Scheme 2

U.S. Patent No. 6,017,927 discloses another process for the preparation of solifenacin, wherein 3-quinuclidinyl chloroformate monohydrochloride is admixed with ( IR)-I -phenyl- 1,2,3,4-tetrahydroisoquinoline to obtain solifenacin, as seen below in Scheme 3:

Scheme 3

History

The compound was studied using animal models by the Yamanouchi Pharmaceutical Co., Ltd. of Tokyo, Japan. It was known as YM905 when under study in the early 2000s.^[7]

Society and culture

Economics

A 2006 cost-effectiveness study found that 5 mg solifenacin had the lowest cost and highest effectiveness among anticholinergic drugs used to treat overactive bladder in the United States, with an average medical cost per successfully treated patient of $6863 per year.^[8]

Chemically, solifenacin succinate is butanedioic acid, compounded with (1S)-(3R)-1-azabicyclo[2.2.2]oct-3-yl 3,4-dihydro-1-phenyl-2(1H)iso-quinolinecarboxylate (1:1) having an empirical formula of C₂₃H₂₆N₂O₂•C₄H₆O₄, and a molecular weight of 480.55. The structural formula of solifenacin succinate is:

Solifenacin succinate is a white to pale-yellowish-white crystal or crystalline powder. It is freely soluble at room temperature in water, glacial acetic acid, dimethyl sulfoxide, and methanol. Each VESIcare tablet contains 5 or 10 mg of solifenacin succinate and is formulated for oral administration. In addition to the active ingredient solifenacin succinate, each VESIcare tablet also contains the following inert ingredients: lactose monohydrate, corn starch, hypromellose 2910, magnesium stearate, talc, polyethylene glycol 8000 and titanium dioxide with yellow ferric oxide (5 mg VESIcare tablet) or red ferric oxide (10 mg VESIcare tablet).

Paper

http://shodhganga.inflibnet.ac.in/bitstream/10603/71060/10/10_chapter%202.pdf

PAPER

An Improved Process for the Preparation of Highly Pure Solifenacin Succinate via Resolution through Diastereomeric Crystallisation

An improved process for the preparation of solifenacin succinate (1) involving resolution through diastereomeric crystallization is described. (1S)-IQL derivative (5) is esterified to form (1S)-ethoxycarbonyl IQL derivative (6) which is condensed with (RS)-3-quinuclidinol (7) to form a solifenacin diastereomeric mixture (8); this is subjected to resolution through diastereomeric crystallization to produce solifenacin succinate (1), which is used for the treatment of an overactive bladder.

CLIP

http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-9-265

The piperidine scaffold also features in a recently discovered pharmaceutical, namely solifenacin (2.57, Vesicare), a competitive antagonist of the muscarinic acetylcholine receptor used in the treatment of an overactive bladder. This species was co-developed by Astellas and GSK scientists and consists of a chiral hydroisoquinoline linked to a (R)-quinuclidinol unit through a carbamate linkage (Figure 6). Upon protonation the tertiary amine of the quinuclidine is expected to resemble the ammonium substructure of muscarine (2.58) [76].

This molecule can be prepared by direct coupling of the (R)-quinuclidinol and tetrahydroisoquinoline carbamate partner (Scheme 28). The (R)-quinuclidinol (2.59) itself can be accessed from quinuclidone (2.60), and is most conveniently prepared by alkylation of ethyl isonicotinate (2.61) with ethyl bromoacetate (2.62) followed by full reduction of the pyridine ring therefore yielding the corresponding piperidine 2.63. A base-mediated Dieckmann cyclisation and Krapcho decarboxylation [77] then furnishes 2.60. Traditionally, the reduction of 2.60 to prepare 2.59 can be carried out under fairly mild hydrogenation conditions that ultimately produce racemic quinuclidinol. However, an improved approach makes use of a Noyori-type asymmetric reduction employing a BINAP ligated RuCl₂ and a chiral diamine to yield the desired (R)-quinuclidine in high yield and enantioselectivity [78].

The enantioselective synthesis of the tetrahydroisoquinoline fragment is achieved via an asymmetric addition of phenylethylzinc to the imine N-oxide 2.66 yielding the corresponding 3,4-dihydroisoquinoline-N-hydroxide 2.68. Further reductive cleavage of the hydroxylamine moiety followed by activation with 4-nitrophenyl chloroformate [79] yields the intermediate 2.69. In the last step of the sequence the addition of (R)-quinuclidinol generates solifenacin (2.57).

1. 76 Broadley, K. J.; Kelly, D. R. Molecules 2001, 6, 142–193.
   Return to citation in text:
2. 77  Daeniker, H. U.; Grob, C. A. Org. Synth. 1964, 44. doi:10.1002/0471264180.os044.30
   Return to citation in text:
3. 78  Arai, N.; Akashi, M.; Sugizaki, S.; Ooka, H.; Inoue, T.; Ohkuma, T. Org. Lett. 2010, 12, 3380–3383. doi:10.1021/ol101200z
   Return to citation in text:

PATENT

https://www.google.com/patents/EP2489666A2?cl=enhttps://www.google.com/patents/EP2489666A2?cl=en

Solifenacin {1(S)-Phenyl-1,2,3,4-tetrahydroisoquinolin-2-carboxylic acid 3(R)-quinuclidinyl ester or [(3R)-1-azabicyclo[2.2.2]oct-3-yl-(1S)-1-phenyl-3,4-dihydroisoquinoline-2-(1H)-carboxylate]}, also known as YM-905 (in its free base form) has the following structure.

Molecular formula of Solifenacin is C₂₃H₂₆N₂O₂ and its molecular weight is 362.5. Solifenacin and its salts are used as therapeutic agents for Pollakiuria and incontinence of urine due to hyperactive bladder, not as agents for curing hyperactive bladder itself but as therapeutic agents for suppressing the symptoms thereof.

The drug Solifenacin was first disclosed in US. Patent NoS. 6,017,927and 6,174,896 (CIP of US 6017927 ) (Yamanouchi Pharmaceuticals). Disclosed therein are compounds with the following general formula

A specific method for producing Solifenacin or its HCl salt is also disclosed, as depicted by the following scheme (Scheme-1).

In the reference cited above, the method of preparation of the Solifenacin base using sodium hydride and subsequent conversion of the base to the HCl salt is described, but no data is given for the purity of either the Solifenacin base or the salt. Solifenacin hydrochloride is disclosed particularly in Example-8 of the same patent and crystallization is carried out in a mixture of acetonitrile and diethyl ether. The melting point reported is 212-214 °C. This patent also discloses 3-quinuclidinyl-1-phenyl-1,2,3,4-tetrahydro-2-isoquinoline carboxylate mono oxalate (Example 1) which is the oxalate salt of racemic Solifenacin (M.P. 122-124 °C). The crystallization of the racemate oxalate salt is carried out in a mixture of isopropanol and isopropyl ether.

Polymorphism, the occurrence of different solid state forms, is a property of many molecules and molecular complexes. A single molecular entity may give rise to a variety of solid state forms having distinct crystal structures and physical properties such as melting point, powder X-ray diffraction pattern, infrared (IR) absorption fingerprint and different physicochemical properties. One solid state form may give rise to several polymorphic forms, which are different from one another in all the above properties.

Subsequently, a process for preparation of Solifenacin base and its salts, wherein succinate salt was obtained in high degree of optical purity for medicinal use, was described in EP 1714965 by Astellas Pharma. This document stated that the free base of Solifenacin has the following impurities,

The concentration of the impurities present in the base were as follows:

• Compound A	4.51%
  Compound B	2.33%
  Compound C	0.14%
  Compound D	0.32%
  Compound E	1.07%

WO 2008062282 discloses a process for the preparation of Solifenacin, which is shown below. (Scheme 6).

Example 1Preparation of (+)-(1S,3’R)-quinuclidin-3′-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (Solifenacin)

To the cooled solution of freshly prepared sodium methoxide (1.8 g), (R)-3-quinuclidinol HCl (6.4 g) was added under N₂ atmosphere. It was stirred at 5-30 °C for 30 min to 1 hrs. Distilled out the solvent at reduced pressure. To the semi-solid mass dry toluene was added. Reaction mixture was heated to reflux temp. and stirred for 1-3 h. During this process, traces of water and methanol were removed azeotropically by using Dean-Stark apparatus and was cooled to 60-70 °C. (S)- Ethyl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-caboxylate (10 g) dissolved in dry toluene and dry DMF were added. It was again heated to reflux temperature and stirred for 5-25 h while distilling off solvent to remove ethanol at intervals with addition of fresh quantity of dry solvent. It was cooled to room temperature.

Workup:

To the reaction mixture water and toluene were added. It was stirred for 10-15 min. and transferred into a separating funnel. Organic layer was collected. The product was extracted with 20 % aqueous HCl solution. It was basified with 40 % aqueous K₂CO₃ solution at 15-20 °C. The product was extracted with ethyl acetate. Both the extracts were combined and washed with brine solution. Organic layer was collected and dried over anhydrous sodium sulfate and solvent was distilled out at reduced pressure.

(+)-(1S,3’R)-quinuclidin-3′-yl-1-phenyl-1,2,3,4-tetrahydro-isoquinoline-2-carboxylate (Solifenacin) (6.6 g , 51 % yield) was obtained.

CLIP

https://www.researchgate.net/figure/235670427_fig2_Figure-6-Scheme5-Synthesis-of-the-chiral-drug-solifenacin

PATENT

WO2014067219A1.

PATENT

https://www.google.com/patents/EP2406257A1?cl=enhttps://www.google.com/patents/EP2406257A1?cl=en

Solifenacin succinate is the international common denomination for butanedioic acid compounded with (l S)-(3R)-l-azabicyclo[2.2.2]oct-3-yl-3,4-dihydro-l-phenyl-2(lH)-isoquinolinecarboxylate (1 : 1), having an empirical formula Of C₂SH_2ON₂O₂ .C₄H₆O₄ and the structure is represented in formula VI given below;

Solifeπacin and its pharmaceutically acceptable salts are first reported in US Patent No. 6,017,927 (927′), which disclosed two’ synthetic routes “Route-A and Route-B” for the preparation of (I RS, 3’RS)- Solifenacin and (I S, 3’RS)-Solifenacin as shown in Scheme- 1 :

Scheme 1 : Reported synthetic schemes in US’ 927

Both the routes have several drawbacks such as; a) Use of hazardous and pyrophoric reagent, NaH, in the process which is very difficult to handle and thus makes the process unsafe to handle at industrial level. The use of strong agent NaH also leads to racemization of the products and thus suffers to provide enantiomerically pure Solifencin; b) Use of ethylchloroformate to prepare ethyl carboxylate derivative in route A which is lachrymatory in nature; c) Ethylcarboxylate derivative produces ethanol as a by-product during trans-esterification reaction in the subsequent reaction that interferes in nucleophilic attack against Solifenacin in the presence of a base and hence it is necessary to remove ethanol from the reaction mixture in the form of azeotrope with toluene or the like simultaneously while carrying out the reaction, so as to control the reaction; d) Use of column chromatography for the purification of Solifenacin base, which makes the process industrially not feasible; f) The reaction requires longer time for the completion and hence turn around time of the batch in production makes it less attractive. International Patent Application No WO2005/075474 disclosed another synthetic route for the preparation of Solifenacin and Solifenacin succinate as shown in Scheme-2.

Scheme 2

The above route does not overcome the problems associated with the process disclosed in 927′ as the process described in this scheme also uses ethylchloroformate in the first step and produces ethanol as a by-product in the second step.

Yet another International Patent application no W02005/105795A1 discloses an improved process for preparing Solifenacin as represented in Scheme-3, wherein leaving group (Lv) can be lH-imidazole-1-yl, 2,5-dioxopyrrolidin-l-yloxy, 3-methyl-l H-imidazol-3-ium- l-yl or chloro and further condensation is carried out in the presence of sodium hydride as a base and a mixture of toluene and dimethylformamide or toluene alone as a reaction medium. The process described herein represents few draw backs such as, use of hazardous sodium hydride, use of chromatographic purifications, and use of moisture sensitive leaving groups (Lv) and hence handling of the reaction is difficult. Further the leaving groups used are expensive and thus making the process uneconomic.

Scheme 3 Hence, there is need of efficient process for producing Solifenacin and its succinate salt which is safe to handle, industrially feasible, and economically viable.

Example 1

PREPARATION OF SOLIFENACIN SUCCINATE OF FORMULA (VI);

To a stirred solution of (3R)-quinuclidin-3-ol (25 gm) in dimethylformamide (175 ml) was added bis-(4- dinitrophenyl) carbonate (83.83 gm) with stirring at 25-3O⁰C under nitrogen atmosphere. The reaction mass was stirred at 25-30⁰C for 2_r3. hours. Upon completion of this reaction by HPLC, ( IS)-I -phenyl- 1,2,3,4-tetrahydroisoquinoline (41.0 gm) was added to resultant brown colored reaction solution and further stirred at 25-3O⁰C for 3-4 hrs. After completion of the reaction (monitored by HPLC), the reaction solution was diluted with water (250 ml) and the pH of the solution was adjusted to 1-2 using concentrated hydrochloric acid. The resulting reaction solution was extracted with diisopropylether (300ml X 2) to separate the nitro-phenol.

The aqueous layer was then extracted with dichloromethane (300 ml) and dichloromethane layer was separated and diluted with 200 ml water. The pH of the biphasic mixture was adjusted to 9-10 with ammonium hydroxide and organic layer was separated, washed with water (200 ml X 2), and concentrated under vacuum to yield 57.0 gm (79%) of compound I as a syrup having HPLC purity of 98.8% and Chiral purity of 99.9%: Compound (I) was further dissolved in acetone (400 ml) and contacted with succinic acid (18.58 gm) at 25-30⁰C. and stirred for 30 min. Precipitated solid was filtered, washed with acetone (57 ml), and dried under vacuum to yield 53.0 gm solifenacin succinate of formula (VI) as a white crystalline solid; HPLC purity 99.93%; Chiral purity : 99.98%;

The ether layer comprising nitro-phenol was subjected to vacuum distillation to recover diisopropylether and nitro-phenol.

Example 2

PREPARATION OF SOLIFENACIN SUCCINATE OF FORMULA (VI);

To a stirred solution of (3R)-quinuclidin-3-ol (5 gm) in dry pyridine (30 ml) was added bis-(4- dinitrophenyl) carbonate (17.5 gm) with stirring at 25-3O⁰C under nitrogen atmosphere. The reaction mass was stirred at 25-30⁰C for 2-3 hours. Upon completion of the reaction by HPLC, ( IS)-I -phenyl – 1,2,3,4-tetrahydroisoquinoline (7.5 gm) was added to resultant brown colored reaction solution and further stirred at 25-3O⁰C for 3-4 hrs. After completion of the reaction (monitored by HPLC), the reaction solution was diluted with water (100 ml) and the pH of the solution was adjusted to 1-2 using concentrated hydrochloric acid. The resulting reaction solution was extracted with diisopropylether (60 ml X 2) to separate the nitro-phenol.

The aqueous layer was then extracted with dichloromethane (60 ml), and dichloromethane layer was separated and diluted with 40 ml of water. The pH of the biphasic mixture was adjusted to 9-10 with ammonium hydroxide and organic layer was separated, washed with water (40 ml X 2), and concentrated under vacuum to yield 10.0 gm (70.8%) of solifenacin of formula (I) as a syrup having HPLC purity of 97.9% and Chiral purity of 99.96%: Compound (I) was further dissolved in acetone (70 ml) and contacted with succinic acid (3.25 gm) at 25-30⁰C. and stirred for 30 min. Precipitated solid was filtered, washed with acetone (10 ml), and dried under vacuum to yield 8.5.0 gm solifenacin succinate of formula (VI) as a white crystalline solid; HPLC purity 99.78%; Chiral purity : 99.96%;

Example 3

PREPARATION OF SOLIFENACIN SUCCINATE OF FORMULA (VI):

(3/?)-quinuclidin-3-ol (1.0 gm) of was dissolved in tetrahydrofuran (15 ml) and dry pyridine (1.0 ml) with stirring. Bis-(4-dinitrophenyl) carbonate (3.82 gm) was added to the above solution at 25-30⁰C. After completion of the reaction, (lS)-l-phenyl-l,2,3,4-tetrahydroisoquinoline (1.5 gm) was added to the resulting brown reaction solution and then stirred till completion of the reaction. Upon completion of the reaction, the reaction solution was diluted with water (20 ml) and the pH of the solution was adjusted to 1 -2 using concentrated hydrochloric acid. The resulting solution was extracted with diisopropylether (12.0 ml X 2) to separate the nitro-phenol.

The aqueous layer was separated and further extracted with dichloromethane (12 ml X 2). The dichloromethane layer was diluted with water (8 ml) and pH of the resulting mixture was adjusted to 9-10 using ammonium hydroxide solution. The aqueous layer was separated from organic layer, washed with water (8 ml x 2) and concentrated to yield 1.5 gm (53.5%) solifenacin of Formula (I) having HPLC purity 96.47% ; chiral purity 99.10%; Compound (I) was dissolved in acetone (10.5 ml) and treated with 0.48 gm succinic acid at 25-30⁰C, and stirred for 30 minutes. The precipitated solid was filtered, washed with 1.0 ml acetone, and solid dried under vacuum yield 1.4 gm of compound VI having HPLC purity 99.86%; chiral purity: 99.93%.

Example 4

PREPARATION OF (3^-l-AZABICYCLO[2.2.21OCT-3-YL4-NITROPHENYL CARBONATE

OF FORMULA (TV);

To a stirred solution of (3i?)-quinuclidin-3-ol (1.0 gm) in dichloromethane (10 ml) was added Bis-(4- dinitrophenyl) carbonate (2.87 gm) at 25-30⁰C and the resulting brown solution was stirred at ambient temperature till the completion of reaction by HPLC. Dichloromethane was distilled off to get the residue that was diluted with water (10 ml) and was added concentrated hydrochloric acid till pH of the mixture is I to 2. The acidic solution was extracted with di-isopropylether (10 ml X 2) to separate out the nitro- phenol. The aqueous layer was then extracted with dichloromethane (20 ml) to separate the compound of formula (IV). The dicloromethane layer- comprising the compound of formula (IV) was further mixed with water (10ml) and pH was adjusted to 9-10 with ammonium hydroxide. The organic layer was then separated, washed with water, dried over sodium sulphate, and concentrated under vacuum to yield (3R)-I- azabicyclo[2.2.2]oct-3-yl4-nitrophenyl carbonate of formula (IV) as a syrup with around 46% yield (1.07 gm); HPLC purity: 87.27% by HPLC.

Example 5

PREPARATION OF SOLIFENACIN SUCCINATE OF FORMULA (VI)

To a stirred solution of (3R)-l-azabicyclo[2.2.2]oct-3-yl4-nitrophenyl carbonate (1.0 gm) of formula (IV) obtained as per Example 4 in pyridine (5 ml), (lS)-l-phenyl-l,2,3,4-tetrahydroisoquinoline (0.78 gm) was added and the resulting brown solution was stirred for 6 hrs. After completion of the reaction the solvent was distilled off and the residue obtained was diluted with 10 ml water, the pH of the resulting solution was adjusted to 1-2 using the concentrated hydrochloric acid and extracted with di-isopropylether (10 ml X 2) to separate out the nitro-phenol.

The aqueous layer was separated and further extracted with dichloromethane (20 ml) and obtained dichloromethane layer was mixed with water (10 ml) and pH of the resulting mixture was adjusted to 9- 10 using ammonium hydroxide. Layers were separated, the organic layer was washed with water, dried over sodium sulphate, and concentrated in vacuum to yield the 1.07 gm (89.43%) of compound solifenacin of formula (I) having HPLC purity 97.08% purity

Example 6

PREPARATION OF SOLIFENACIN SUCCINATE OF FORMULA (VD.

To a stirred solution of (3R)-quinuclidin-3-ol (Formula II, 100 gm) in dimethylformamide (400 ml), bis- (4-dinitrophenyl) carbonate (Formula III, 285.04 gm) was added with stirring at 25-30°C under nitrogen atmosphere. The reaction mass was stirred at 25-30°C for 2-3 hours. After completion of the reaction which was monitored by TLC, ( IS)-I -phenyl- 1, 2,3, 4-tetrahydroisoquinoline (Formula V, 171.44 gm) was added to the resultant brown colored reaction solution. The reaction mixture was further stirred at 25- 3O⁰C for 3-4 hrs. After completion of the reaction (by HPLC), the reaction solution was diluted with water (1000 ml) and the pH of the solution was adjusted to 1-2 using concentrated hydrochloric acid. The resulting reaction solution was extracted with diisopropylether (1000ml X 2) to separate the nitro-phenol. The aqueous layer was then mixed with dichloromethane (1000ml), the content was stirred, and dichloromethane layer was separated. Aqueous layer was re-extracted with dichloromethane (l OOOml).The combined dichloromethane was distilled off completely to obtain the residue. The residue was dissolved in water (1000 ml) and toluene (1000 ml) was added and the pH of the biphasic mixture was adjusted to 9-10 with ammonium hydroxide. The mixture was stirred and toluene layer was separated and aqueous layer was re-extracted with toluene (1000 ml). The combined toluene layer were washed with water (1000 ml) followed by solution of 0.5% sodium hydroxide (1000 ml X 2) and further washed with water (1000 ml). The toluene layer was distilled off completely to obtain the residue which was further dissolved in acetone (800 ml) and toluene 1080 ml). The solution was treated with Succininc acid (88.0 gm) and the mixture obtained was heated at 55-60⁰C for 30 min. The mixture was further cooled to 10-15⁰C, maintained for 60 min and filtered. The product was dried to afford Solifenacin Succinate (Formula VI) as white crystalline solid. Yield of the compound VI270 gm. HPLC purity : 99.85%: Chiral Purity: 99.99%

Example 7

Purification process for Solifenacin Succinate:

The wet material obtained from the example 6 was purified to improve chiral and chemical purity.. The wet material (270 gm) was dissolved in a mixture of water (700 ml) and toluene (700 ml) and stirred for 15 min. The pH of resulting mixture was adjusted to 9-10 using aqueous ammonia, stirred for 15-20 min and separated organic and aqueous layer. Aqueous layer was re-extracted with toluene (700 ml) and combined with the separated organic layer. The combined organic layer was washed with water (700 ml x 2) and distilled off completely to obtain the thick residue. The residue was dissolved in acetone (1600 ml), decolorized with activated charcoal, and treated with succininc acid (75.0 gm). The contents were heated at 55-60⁰C for 30 mih, cooled to 10-15°C, and maintained for 60 min. The crystalline solid obtained was filtered, and dried under vacuum (650-700 mm/Hg to afford Solifenaicn Succinate (Formula VI) as white crystalline solid. Yield: 250 gm (66.6%); HPLC purity: 99.95% and Chiral purity: 100.0%

PAPER

PATENT

https://www.google.com/patents/WO2007076116A2?cl=en

Scheme 4:

3-Quinuclidinol χ= halogen,

R=alkyl

Scheme 5

EXAMPLES Example 1 : Preparation of solifenacin succinate

A solution of (S)-l-phenyl-l,2,3,4-tetrahydroisoquinoline (C15H₁₅N) (16g), toluene (80ml), and diisopropylethylamine (DIPEA, 13.5g) was cooled to 0°C. Chloroethylchloro formate (C₃H₄CbO₂) (CECF, 13.0gr) was added dropwise, keeping the temperature between 0°-20°C. After stirring at room temperature for 1.5 hours, the mixture was filtered.

The filtrate was added to solution of (R)-quinuclidin-3-ol (C₇Hi₃NO) (11.6g) in toluene (80ml), DMF (16ml), and NaH (60%, 5.5g) at 80°C during 1 hour, and stirred at 95°-100°C for 17 hours. The mixture was cooled to room temperature, and THF (small amount) was added. A saturated NaCl solution (300ml) was added, and the phases were separated. The organic phase was acidified with 10% HCl solution, and the phases were separated. The aqueous phase was basified with K₂CO₃solution and extracted with ethyl acetate (EtOAc). The organic phase was filtered and evaporated to obtain solifenacin (SLF) (21.25g). The residue was dissolved in ethanol (EtOH) (100ml) and succinic acid (7.Og) was added. Seeding with SLF-succinate was performed, and the mixture was stirred at RT for 16 hours. The product was isolated by vacuum filtration, washed with EtOH (3x20ml), and dried in vacuum oven at 50° over night to obtain SLF-succinate (10.46g).

Example 2: Preparation of solifenacin succinate

Chloroethylchloroformate (CECF, 13.Og) is added dropwise to solution of (R)- quinuclidin-3-ol (11.6g) and diisopropylethylamine (DIPEA, 13.5g) in THF (150ml), keeping the temperature between 0°-20°C. The mixture is stirred at room temperature for several hours. Then (S)-l-phenyl-l,2,3,4-tetrahydroisoquinoline (16g) is added and the solution is stirred at room temperature for another 16 hours. The solution is diluted with EtOAc (350ml) and washed with a saturated NaCl solution (300ml). The organic phase is acidified with 10% HCl solution, and the phases are separated. The aqueous phase is basified with K.₂CO₃ solution and extracted with EtOAc. The organic phase is filtered and evaporated to obtain SLF. The residue is dissolved in EtOH (100ml), and succinic acid (7.Og) is added. Seeding with SLF-succinate is performed, and the mixture is stirred at RT for 16 hours. The product is isolated by vacuum filtration, washed with EtOH (3x20ml), and dried in vacuum oven at 50° over night to obtain SLF-succinate. Example 3: Preparation of solifenacin succinate

Chloroethylchloroformate (CECF, 13.Og) is added dropwise to solution of (R)- quinuclidin-3-ol (11.6g) and diisopropylethylamine (DIPEA, 13.5g) in Toluene (150ml), keeping the temperature between 0°-20°C. The mixture is stirred at room temperature for several hours and filtrated. Then (S)-l-phenyl-l,2,3,4-tetrahydroisoquinoline (16g) is added followed by addition of sodium hydride (60%, 5.5g) and the mixture is stirred at reflux for another 16 hours. The solution is diluted with EtOAc (350ml) and washed with a saturated NaCl solution (300ml). The organic phase is acidified with 10% HCl solution, and the phases are separated. The aqueous phase is basified with K2CO3 solution and extracted with EtOAc. The organic phase is filtered and evaporated to obtain SLF. The residue is dissolved in EtOH (100ml), and succinic acid (7.Og) is added. Seeding with SLF-succinate is performed, and the mixture is stirred at RT for 16 hours. The product is isolated by vacuum filtration, washed with EtOH (3x20ml), and dried in vacuum oven at 50° over night to obtain SLF-succinate.

CLIP

PATENT

1. WO9620194A1 / US6017927A.

2. J. Med. Chem. 2005, 48, 6597-6606.

3. WO2007076116A2.

PATENT

WO2010103529A1.

PATENT

WO2013147458A1.

 

References

Solifenacin

Systematic (IUPAC) name
1-azabicyclo[2.2.2]oct-3-yl (1R)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate
Clinical data
Trade names	Vesicare
AHFS/Drugs.com	Monograph
MedlinePlus	a605019
License data	US FDA: Solifenacin
Pregnancy category	AU: B3 US: C (Risk not ruled out)
Routes of administration	Oral
Legal status
Legal status	UK: POM (Prescription only) US: ℞-only
Pharmacokinetic data
Bioavailability	90%
Protein binding	98%
Metabolism	CYP3A4
Metabolites	Glucuronide, N-oxide, others
Biological half-life	45 to 68 hours
Excretion	Renal (69.2%) and fecal (22.5%)
Identifiers
CAS Number	242478-37-1
ATC code	G04BD08 (WHO)
PubChem	CID 154059
IUPHAR/BPS	7483
DrugBank	DB01591
ChemSpider	135771
UNII	A8910SQJ1U
KEGG	DG00481
ChEMBL	CHEMBL1734
Synonyms	YM905
Chemical data
Formula	C23H26N2O2
Molar mass	362.465 g/mol
SMILES[hide] C1CN2CCC1[C@H](C2)OC(=O)N3CCC4=CC=CC=C4[C@@H]3C5=CC=CC=C5

////////солифенацин , سوليفيناسين , 索利那新 , Solifenacin, YM 67905, YM 905

ACS INDUSTRY SYMPOSIUM

RECENT ADVANCES IN DRUG DEVELOPMENT IN PARTNERSHIP WITH DR. REDDY’S LABORATORIES HYDERABAD, INDIA

11-12 NOVEMBER 2016

click        http://acssymposium.org.in/

for registrations

PF-610355

• Molecular Formula C₃₄H₃₉N₃O₆S
• Average mass 617.755 Da

PF 610655, PF-00610355; PF-610,355

862541-45-5  cas

• Originator Pfizer
• Class Antiasthmatics; Benzeneacetamides; Sulfonamides
• Mechanism of Action Beta 2 adrenergic receptor agonists

• Orphan Drug StatusNo
• On Fast trackNo

Highest Development Phases

• Discontinued Asthma; Chronic obstructive pulmonary disease

Most Recent Events

• 11 Aug 2011 Discontinued – Phase-I for Asthma in Belgium (Inhalation)
• 11 Aug 2011 Discontinued – Phase-II for Asthma in Sweden (Inhalation)
• 11 Aug 2011 Discontinued – Phase-II for Asthma in Germany (Inhalation)

PF-610355 (also known as PF-00610355 or PF-610,355) is an inhalable^[1] ultra-long-acting β₂ adrenoreceptor agonist^[2] (ultra-LABA) that was investigated as a treatment of asthma and COPD by Pfizer.^[3] It utilizes a sulfonamide agonist headgroup, that confers high levels of intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in the solid state. Optimization of pharmacokinetic properties minimized systemic exposure following inhalation and reduced systemically-mediated adverse events.^[4] Its in vivo duration on action confirmed its potential for once-daily use in humans.^[5]

The investigation and development of PF-610355 were discontinued in 2011,^[6] likely for strategic and regulatory reasons.^[7]

PF-610355). Mp 197−199 °C.

1 H NMR (600 MHz, d6-DMSO) δ 0.89 (s, 3H), 0.91 (s, 3H), 2.54 (s, 2H), 2.59−2.68 (m, 2H), 2.89 (s, 3H), 3.44 (s, 2H), 4.31 (d, 2H), 4.42 (dd, 1H), 6.80−8.83 (m, 3H), 6.97−7.02 (m, 2H), 7.08−7.12 (m, 3H), 7.16 (t, 1H), 7.19 (d, 1H), 7.30 (t, 1H), 7.37−7.41 (m, 4H), and 8.50 (t, 1H).

13C NMR (151 MHz, d6-DMSO) δ 26.39, 26.64, 39.85, 42.22, 42.48, 46.36, 50.15, 52.71, 72.00, 115.14, 115.70, 123.76, 124.02, 124.29, 124.38, 124.76, 125.23, 126.49, 127.57, 127.63, 128.40, 128.71, 130.82, 131.13, 135.38, 135.73, 138.43, 139.94, 140.20, 149.76, 157.11, and 170.28.

HRMS (ESI): calcd (M + H)+ 618.2632, found 618.2641. Found: C, 65.70%; H, 6.33%; N, 6.71%; S, 5.24%. C34H39N3O6S·0.17H2O requires C, 65.78%; H, 6.39%; N, 6.77%; S, 5.16%

PAPER

Optimization of the Manufacturing Route to PF-610355 (2): Synthesis of the API

Abstract

PF-610355 is a novel inhaled β-2 adrenoreceptor agonist. Process development of the final intermediate and the API are discussed with emphasis on the control of physical properties and subsequent isolations. This includes development of a constant volume distillation and evaluation of Nutsche filtration, agitated filter drying, and centrifugation to prevent particle attrition. The optimized process employed to manufacture 100 kg of the API is described.

PAPER

Optimization of the Manufacturing Route to PF-610355 (1): Synthesis of Intermediate 5

Abstract

Tertiary carbinamine 5 is an isolated intermediate in the synthesis of a novel, inhaled β-2 adrenoreceptor agonist PF-610355. Process development for the key amide-formation and Ritter reactions, together with reaction understanding studies are discussed in context of the synthesis of 5. The optimized process employed to manufacture 140 kg of 5 is described, and was shown to have superior metrics to the preliminary commercial route.

References

PF-610355

Systematic (IUPAC) name
N-[(4′-Hydroxy-3-biphenylyl)methyl]-2-[3-(2-{[(2R)-2-hydroxy-2-{4-hydroxy-3-[(methylsulfonyl)amino]phenyl}ethyl]amino}-2-methylpropyl)phenyl]acetamide
Clinical data
Routes of administration	Inhalation
Legal status
Legal status	Development terminated
Identifiers
CAS Number	862541-45-5
ATC code	None
PubChem	CID 11505444
ChemSpider	9680243
UNII	ZH5SMU97AJ
ChEMBL	CHEMBL1240967
Chemical data
Formula	C34H39N3O6S
Molar mass	617.76 g·mol−1

///////////PF 610355