8-Azaspiro(4.5)decane-7,9-dione, 6-hydroxy-8-(4-(4-(2-pyrimidinyl)-1-piperazinyl)butyl)-, (6R)-
(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.
Enantioselective α-Hydroxylation of 2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered Cytochrome P450 BM-3
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.
Tetrahedron: Asymmetry (2005), 16(16), 2711-2716.
Preparation of (R)- and (S)-6-hydroxybuspirone by enzymatic resolution or hydroxylation
- Department of Process Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903, USA
6-Hydroxybuspirone is an active metabolite of the antianxiety drug buspirone. The (R)- and (S)-enantiomers of 6-hydroxybuspirone were prepared using an enzymatic resolution process. l-Amino acid acylase from Aspergillus melleus (Amano Acylase 30000) was used to hydrolyze racemic 6-acetoxybuspirone to (S)-6-hydroxybuspirone in 95% ee after 45% conversion. The remaining (R)-6-acetoxybuspirone with 88% ee was converted to (R)-6-hydroxybuspirone by acid hydrolysis. The ee of both enantiomers could be improved to 99% by crystallization as a metastable polymorph. (S)-6-Hydroxybuspirone was also obtained in 88% ee and 14.5% yield by hydroxylation of buspirone using Streptomyces antibioticus ATCC 14890.
Tetrahedron: Asymmetry (2005), 16(16), 2778-2783
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 (R)- or (S)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones 2 and 3 is described.
Enzyme and Microbial Technology (2006), 39(7), 1441-1450.
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.
|Inventors||Joseph P. Yevich, Robert F. Mayol, Jianqing Li,Frank Yocca|
|Original Assignee||Bristol-Myers Squibb Company|
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.
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.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US6150365||Jun 6, 2000||Nov 21, 2000||Bristol-Myers Squibb Company||Anxiety method|
|1||Mayol, et al., “Pharmacokinetics and Disposition of 14C-Buspirone HCI After Intravenous and Oral Dosing in Man,” Clin. Pharmacol. Ther., 37, p. 210, 1985.|
|2||*||Robichaud et al. in Annual Reports in Medicinal Chemistry, vol. 35,pp. 11-20 (2000).*|
|3||Winslow, et al., “Serotonergic modulation of the rat pup ultrasonic isolation call: studies with 5HT1 and 5HT2 subtype-selective agonists and antagonists,” Psychopharmacology, 105, pp. 513-520, 1991.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US20090023744 *||Jun 17, 2008||Jan 22, 2009||The General Hospital Corporation||Combination therapy for depression|
|WO2015197079A1||Jun 25, 2015||Dec 30, 2015||Contera Pharma Aps||Use of buspirone metabolites|
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