WO1998058073A1

WO1998058073A1 - Enzymatic kinetic resolution of an intermediate useful for preparing substituted tricyclics

Info

Publication number: WO1998058073A1
Application number: PCT/US1998/011501
Authority: WO
Inventors: William Brian Morgan; Jinchu Liu
Original assignee: Schering Corporation
Priority date: 1997-06-17
Filing date: 1998-06-15
Publication date: 1998-12-23
Also published as: AU8058398A; MY133532A; JP3421355B2; IN187248B; JP2000512862A; EP1002123A1; CA2293708C; CA2293708A1; AR015892A1; ZA985217B

Abstract

The invention relates to a process for preparing a substituted (6,11-dihydro-5H-benzo [5,6]cyclohepta[1,2-b]pyridin-11-yl)piperidine compound of formula ((+)-I) wherein: R, R?1, R2, R3 and R4¿ are independently selected from the group consisting of hydrogen, halo, C¿1?-C6 alkyl, amino, -OCH3, -OCF3 and CF3, and the dotted line represents an optional double bond; comprising: enzymatically catalyzing the acylation of a compound of the formula ((±)-II) wherein the variables are as defined above, and hydrolysing the product to obtain ((+)-I).

Description

ENZYMATIC KINETIC RESOLUTION OF AN INTERMEDIATE USEFUL FOR PREPARING SUBSTITUTED TRICYCLICS

BACKGROUND OF THE INVENTION

This invention provides an enzymatic process for preparing optically enriched intermediates useful in the preparation of substituted tricyclic compounds known as antihistamines and as inhibitors of famesyl protein transf erase (FPT). In particular, the process of this invention is useful in preparing intermediates useful in the preparation of FPT inhibitors disclosed, for example, in International Publication Number W095/10516, published April 20, 1995.

The use of enzymes for the synthesis of non-racemic chiral compounds is now well established. Since they are easy to use and readily available, hydrolases (proteases, esterases and lipases) have been used for the preparation of chirally pure molecules, under both aqueous and non-aqueous conditions. Enzyme catalyzed acylation reactions in non-aqueous solvents have been widely used for the kinetic resolution of racemic alcohols and amines. There are numerous examples in the literature of the selective acylation of a single enantiomer of a racemic primary amine: Scheme A:

^NH2 Hydrolase NHCOR³ NH₂ L *~ ϊ ₄- ^"

R¹ R² R³COOR⁴ R¹^ R² R¹"^ R²

(±) However, the enzymatic acylation of secondary and cyclic amines has been described less frequently: Scheme B:

COR'

H . N. Hydrolase H

JM. . N .

R¹ ^R R³COOR⁴ R^r " ² R¹ " R²

(±) (+)-Amide (-)-Amine

Most of the examples gleaned from the literature involve the acylation of chirally pure proline esters or amides catalyzed by alcalase (Chen et al, Biorg. Med. Chem. Lett, 4 (1994), p. 443), clostridiopeptidase B (Fortier et al, Biotechnol. Lett, 8 (1986), p. 777), α-chymotrypsin (Paradkar et al, J. Amer. Chem. Soc, 116 (1994), p. 5009), and aminoacyl-t-RNA synthetase (Nakajima et al, Int. J. Pept Protein Res., 28 (1986), p. 1986). Examples illustrating the enzymatic acylation of chiral secondary amines are shown in the following reaction schemes:

Scheme C:

ion

/

30% Conversion

(Gutman et al, Tet Lett, 33 (1992), p. 3943)

Scheme D:

PPL Catalyzed Acylation of 2-Hydroxymethylpiperidine

Enzyme Temp °C/Time h eep Converison

PPL RT/ 4 0.23 0.70 0.25 7

40/ 4 0.13 0.59 0.18 ^"4

0-5/ 30 0.39 0.51 0.43 4

(Asensio et al, Tet Lett., 32 (1991), p. 4197). Scheme E:

Enzymatic Acylation of 3-Hydroxymethylpiperidine

Solvent Enzyme Vinyl Acetate Time 2 3 mg/mmol equiv. h (Yield)(ee) (Yield)(ee)

Vinyl 300 50 9 1 29% (0.19) 69% (n/d) Acetate Acetonitrile 300 5 94 37% (<0.02) 42% (n/d) CH2CI2 1 00 2.5 7.5 68% (0)

(Herradon et al, S. Synlett (1995), p. 599).

Scheme F:

RT

Product R % yield ee

2 a methyl 24 0.27 2 b ethyl 31 0.31 2 c Al lyl 49 0.84

Conditions: Substrate, 1 mmoi; Carbonate, 1 mL;

Enzyme, 20 mg; RT, 45 h.

(Orsat, et al, J. Amer. Chem. Soc, 778 (1996), p. 712).

Scheme G:

96% ee (Orsat, et al, J. Amer. Chem. Soc, 118 (1996), p. 712).

Generally, the resolutions suffer from low reactivity and/or selectivity.

The reactions shown in schemes D and E probably occur by enzymatic acylation of the primary hydroxyl, followed by non-enzymatic intramolecular acyl transfer:

(In the above reaction Schemes A-H, the designation of the R substituents is for convenience in discussing those reactions, but does not correspond to the designation of the R substituents in the process claimed below.)

SUMMARY OF THE INVENTION

This invention provides a highly selective process for preparing a substituted (6,11 -dihydro-5H-benzo[5,6]cyclohepta[1 ,2-b]-pyridin-11 - yl)piperidine compound of the formula (+)-I

wherein:

R, R¹ , R², R³ and R⁴ are independently selected from the group consisting of hydrogen, halo, C-i-Cβ alkyl, amino, -OCH3, -OCF3 and CF3, and the dotted line represents an optional double bond; comprising:

(1)(a) enzymatically catalyzing the acylation of a compound of formula (±)-II, wherein the variables are as defined above, to obtain a compound of formula (+)-III

wherein the enzyme is a hydrolase and wherein the acylating agent is of the formula R⁵COOR⁶, wherein R⁵ is C1-C-15 alkyl, halo methyl, aryl, benzyl or benzyloxy, R⁶ is alkyl, C1-C6 alkenyl, -COR⁷, trifluoroethyl, -CH₂CH(OCOR⁷)CH2θCOR⁷, halo methyl or benzyl, and R⁷ is Cι-Cι₅ alkyl; and

(b) hydrolysing the compound of formula (+)-III;

(c) optionally converting an undesired isomer from step (a) wherein a double bond is present to the racemate by heating, and resubjecting the racemate to enzymatic acylation and hydrolysis; or

(2) enzymatically catalyzing the acylation of a compound of formula (±)-IIa, wherein R, R , R², R³ and R⁴ are as defined above and the bond is a single bond, with a hydrolase, and wherein the acylating agent is as defined above.

Preferred compounds of formula (+)-I made by this process are those wherein R³ is not hydrogen. Also preferred are compounds wherein R is halo. Still another group of preferred compounds is that wherein R¹ is hydrogen and R, R², and R³ are selected form the group consisting of halo. Halo is preferably Cl or Br.

DETAILED DESCRIPTION As used herein, the term "halo" means fluoro, chloro, bromo and iodo, with chloro and bromo being preferred.

As used herein , the term "aryl" means phenyl, naphthyl, substituted phenyl or substituted naphthyl, wherein "substituted" means 1-3 substituents indpendently selected form the group consisting of C-I-C6 alkyl, C-i-Cβ alkoxy, halo, NO2 and halo methyl.

Those skilled in the art recognize that suitable acylating enzymes may have opposing selectivity, and therefore may involve either direct or subtractive resolution. That is, some enzymes may acylate the desired isomer, requiring separation of the isomers, followed by hydrolysis to obtain the desired product (i.e., direct resolution, as claimed in step (1)), while others may acylate the undesired isomer, requiring only separation of the isomers (no hydrolysis) to obtain the desired isomer (i.e., subtractive resolution, as claimed in step (2)).

Commercially available enzymes suitable for use in the claimed process include Altus ChiroCLEC™ PC (Pseudomonas cepacia); Amano Lipase AY-30 (Candida rugosa); Meito Lipase MY (Candida rugosa), Meito Lipase AL (Achromobacter sp.), Meito Lipase QLC (Alcaligenes sp.) and Meito Lipase QLG (Alcaligenes sp.); Toyobo LIP- 300 and LIP-301 (Pseudomonas sp.); Novo SP435 and Novozym 435 (Candida antarctica lipase B); Boehringer Mannheim Lipase

(Pseudomonas sp.); and Boehringer Chirazyme™ L3 (Candida rugosa), Chirazyme™ L4 (Pseudomonas sp.) and Chirazyme™ L6 (Pseudomonas sp.).

Preferred enzymes are Toyobo LIP-300/301 , Altus Chiro CLEC™ PC, Boehringer Mannheim Lipase, Novo SP435 and Novozym 435.

Acylating agents of formula R⁵COOR⁶ are commercially available or can be prepared by known methods. Preferred acylating agents are trifluoroethyl acetate (TFEOAc), trifluoroethyl butyrate (TFEOBu), trifluoroethyl isobutyrate (TFEOiBu), trifluoroethyl benzoate (TFEBenz), triacetin and tributyrin.

The enzymatic acylation may be carried out in a solvent such as an alkyl acetate such as methyl acetate (MeOAc) or isopropyl acetate, t-butyl methyl ether (TBME), tetrahydrofuran (THF), acetone, acetonitrile, t-amyl alcohol, t-butyl alcohol, pyridine or dioxane. Alternatively, the acylating agent may serve as the solvent. A preferred acylating agent which may also act as the solvent is trifluoroethyl isobutyrate.

The reaction is carried out in a temperature range of 0 to 50°C, preferably at 25 to 30°C (e.g., ambient temperature). The reaction time ranges from 18 to 48 hours, with 24 hours being preferred. The enzyme is added at a ratio of about 1 : 2 times the amount of the starting material , preferably about 2 times the amount. The acylating agent is present at about 2 to 10 times the starting material, preferably about 3 times the amount of starting material when the enzyme is present at 2 times the amount of the starting material. The hydrolysis is carried out using standard procedures well known in the art. For example, the acylated compound is refluxed with an acid such as H2SO4. The desired isomer is then recovered by precipitating out by adding a base such as NaOH.

The reaction is preferably carried out under anhydrous conditions. The solvent, or acylating agent when used as the solvent, can be anhydrous, or the solution of the starting material in the solvent, or acylating agent when used as the solvent, can be dried by azeotropic distillation before the enzyme is added. The enzyme should be dried under vacuum before adding to the solution, preferably to <700 ppm water.

The undesired (-) isomer can be recovered from enzymatic isobutyrylation of racemic II. Heating (-)-ll in diphenyl ether or diethylene glycol dibutyl ether (5-15:1 , v:v) at 200-260°C, preferably 210°C, for 0.5- 26 hours results in complete racemization to racemic II which can be recovered in 77-95% yield with 93-99% purity. The recovered racemic II undergoes the enzymatic isobutyrylation under the same conditions as above.

Preferred embodiments of the claimed process are shown in the following reaction schemes: Scheme 1 :

Scheme 2:

Sche

Especially preferred embodiments of the claimed process are represented by the following reaction schemes: Scheme 1A:

Scheme 2A:

Previous methods used to resolve isomers to obtain compounds of formula I involved the resolution of a compound of formula (±)-IIa by chiral chromatography or chemical resolution using stiochiometric amounts of a resolving agent. The process claimed herein uses a biocatalyst to effect the resolution, the biocatalyst being reusable up to 15 times. Furthermore, compounds lib are chiral atropisomers at room temperature due to restricted rotation about the double bond. However, the isomers can be racemized at high temperatures. By carrying out the enzymatic resolution of lib, the undesired (-) isomer can be isolated, racemized at 200-260°C, preferably at 210°C, and then subjected to a further enzymatic acylation to increase throughput of (±)-IIIa. The products of this process are intermediates useful in the preparation of tricyclic compounds useful as famesyl protein trasnsferase inhibitors such as those disclosed in International Publication Number WO95/10516, published April 20, 1995.

The following tables show the results of varying the various parameters of the reactions. In most of the tables, the compound of formula I prepared by the process has the following substituents^". R and R³ are each bromo, R¹ and R⁴ are each hydrogen and R² is chloro; those skilled in the art will recognize that compounds with different R- group substitution are expected to react in a similar manner. In the tables and elsewhere in this application, the terms have the following meanings: ee_s is the enantiomeric excess of the unreacted starting material; eep is the enantiomeric excess of the product; c is the conversion (ee_s/(ee_s + ee_p)); E is the Enantiomeric Ratio: (ln[(1-ee_s) (1-c)/ln[(1+ee_s) (1-c)] or ln[1-c(1+ee_p)]/ln[1-c(1+ eep)]); Ac is acetyl, OAc is acetate, Me is methyl, Et is ethyl, Pr is propyl and TFE is trifluoroethyl. ENZYMATIC TRANSESTERIFICATION A. Screen Results

General Procedure: TFEOAc (0.06 mL, 20 equivs.) was added to a mixture of (±)-IIc (10 mg) and the enzyme (2-100 mg) in TBME (1.0 mL), except for Runs 1 and 4 which were run in the presence of CaCU3 (30- 40 mg) with MeOAc (1.0 mL) as both solvent and acylating agent. The reactions were shaken at 250 rpm at ambient temperature and monitored by thin layer chromatography. Reactions of interest were analyzed by chiral HPLC, the results of which are shown in Table 1.

Table 1. (±)-Hc Acetylation Screen: Results from 248 Enzyme Preparations

Run Enzyme Wt Time ee_s ee_p c E mg h

1 Altus ChiroCLEC PC 4.1 45 0.22 0.60 0.26 5

2 Amano Lipase AY 16 40 0.18 0.19 0.48 2

3 Meito Lipase MY 18 40 0.18 0.17 0.51 2

4 Toyobo LIP-300 7.0 45 0.22 0.66 0.25 6

5 Toyobo LIP-300 18 16 0.93 0.58 0.61 1 2

6 Boehringer Chirazyme L3 16 40 0.14 0.18 0.43 2

7 Boehringer Chirazyme L4 12 40 0.1 1 0.20 0.35 2

8 Boehringer Chirazyme L6 15 40 0.03 0.04 0.42 1

9 Meito Lipase AL 29.4 63.5 0.19 0.11 0.64 2

10 Meito Lipase QLC 53.6 63.5 0.55 0.24 0.69 3

1 1 Meito Lipase QLG 94.2 63.5 0.69 0.34 0.67 4

12 Boehringer Pseudomonas sp. 2 4 0.676 0.687 0.50 1 1

Conditions: Runs 2,3, 5-11 : (±)-IIc, 10 mg; Trifluoroethyl Acetate, 20 equiv; TBME, 1.0 mL; RT, 250 φm. Runs 1 ,4: (±)-IIc, 10 mg; CaCθ3, 30-40 mg; MeOAc, 1.0 mL (as solvent and acylating agent); RT; 250 rpm. Run12: (+)-IIc, 12 mg; Trifluoroethyl acetate, 5 equiv.; TBME, 1.0 mL; RT, 250 rpm B. The Effect of Solvent

General Procedure: TFEOAc (0.2 mL, 40 equivs.) was added to a mixture of (±)-IIc (19-25 mg) and Toyobo LIP-300 (19-25 mg) in the appropriate solvent (2.0 mL). The reactions were shaken at 250 rpm at +4°C and monitored by TLC and chiral HPLC. The results are in Table 2.

Table 2. The Effect of Solvent on the Acetylation of (±)-IIc Using Toyobo LIP-300

Run Solvent Time h ee_s ee_p

1 MeOAc 9 1 0.21 0.46 0.31 3

2 nPrOAc 9 1 0.04 0.03 0.60 1

3 TBME 9 1 0.65 0.64 0.50 9

4 Toluene 9 1 0.01 n/d n/d n/d

5 THF 9 1 0.39 0.51 0.43 4

6 Acetone 9 1 0.09 0.32 0.21 2

7 MeCN 2 6 0.24 0.77 0.24 1 0

9 1 0.32 0.66 0.32 7

8 CH₂CI₂ 9 1 0.01 n/d n/d n/d

9 tAmylOH 9 1 0.03 0.37 0.07 2

1 0 tBuOH 9 1 0.04 0.35 0.1 0 2

1 1 Pyridine 9 1 0.02 0.06 0.23 1

1 2 pDioxane 9 1 0.1 3 0.33 0.28 2

Conditions: (±)-IIc, 19-25 mg; Toyobo LIP-300, 19-25 mg; Trifluoroethyl Acetate, 40 equivs; Solvent, 2.0 mL; 250 rpm; +4°C.

C. The Effect of Acylating Agent

General Procedure: The acylating agent (20 equivs.) was added to a mixture of (+)-IIc (20 mg) and enzyme (19-26 mg) in TBME (2.0 mL), except for Run 8 which used MeOAc (2.0 mL) as solvent and aeylating agent, Run 11 which used 10 equivalents of acylating agent and Run 18 which used 88 equivalents of acylating agent. The reactions were shaken at 250 rpm at ambient temperature, except Run 11 which was shaken at +4°C, and the reactions monitored by TLC and chiral HPLC.

With the exception of Runs 4, 5, 10, 1 1 , 17, 18 and 19, all reactions were subjected to workup in which the product and the starting material were separated by preparative TLC and the enantiomeric excesses determined separately. The results are collected in Table 3.

Table 3. The Effect of Acylating Agent on the Acylation of (±)-IIc Using Toyobo LIP-300

Run Acylating Agent Time h ees ee_p

1 Trifluoroethyl Acetate 23.75 0.92 0.82 0.53 3 2

Isolated 0.91 0.84 0.52 3 5

2 Trifluoroethyl Acetate 1 5 0.92 0.79 0.54 2 8

1 6.5 0.95 0.80 0.54 3 3

Isolated

3 Trifluoroethyl Acetate 1 5 0.91 0.82 0.53 3 1

1 6.5 0.93 0.85 0.52 4 3

Isolated

4 Trifluoroethyl Chloroacetate 27.00 0.05 0.47 0.09 3

5 Trifluoroethyl Dichloroacetate 27.00 0.01 0.08 0.08 1

6 Trifluoroethyl Butyrate 27.00 0.87 0.90 0.49 5 3

Isolated 0.95 0.77 0.55 2 8

7 Trifluoroethyl Butyrate 1 5 0.68 0.88 0.44 3 3

1 7 0.77 0.87 0.47 34

Isolated

8 Trifluoroethyl Hexanoate 27.00 0.88 0.80 0.52 2 6

Isolated 0.94 0.66 0.59 1 7

9 Trifluoroethyl Laurate 27.00 0.39 0.83 0.32 1 6

Isolated 0.70 0.78 0.47 1 7

1 0 Methyl Acetate (neat) 23.75 0.07 0.42 0.1 4 3

1 1 lsopropenyl Acetate 7 0.1 2 0.28 0.31 2

1 2 Triacetin 23.75 0.37 0.89 0.30 2 4

Isolated 0.53 0.93 0.36 48

1 3 Triacetin 1 5 0.27 0.84 0.24 1 4

20.75 0.38 0.96 0.28 8 1

Isolated

1 4 Tributyrin 27.00 0.29 0.79 0.27 1 1

Isolated 0.55 0.94 0.37 60

1 5 Tributyrin 1 5 0.30 0.81 0.27 1 3

20.75 0.45 0.84 0.35 1 8

Isolated

1 6 Trifluoroethyl Benzyl Carbonate 1 5 0.86 0.71 0.55 1 6

20.75 0.90 0.61 0.59 1 2 isolated

1 7 Dibenzyl carbonate 1 5 0.06 n/d n/d n/d

1 8 Ethyl Butyrate 1 5 0.02 0.59 0_^04 4

1 9 Trifluoroethyl benzoate 26 0.235 0.990 0.1 92 250

Conditions: (±)-IIc, 20 mg; Toyobo LIP-300. 19-26 mg; TBME, 2 mL; Acylating agent, 20 equivs.; 250 φm; RT. Exceptions: Run 10, MeOAc as solvent and acylating agent; Run 11, Acylating agent 10 equivs. and +4°C; Run 18, 88 equivs. of acylating agent; Run 19, (±)-IIc, 10 mg; Acylating agent, 5 equivs. ENZYMATIC ISOBUTYRYLATION OF (+VIIc A. Enzyme Survey

General Procedure: A mixture of (±)-IIc (25 mg), TFEOiBu (0.04 mL, 5 equivs.), 4A molecular sieves (25-40 mg) and enzyme (6-27 mg) in TBME (1.0 mL) was shaken at ambient temperature and 250 rpm for 23.5 h. The reactions were monitored by chiral HPLC and the results are collected in Table 4. Table 4. Isobutyrylation of (±)-IIc with Various Enzyme Preparations in TBME

Run Source Enzyme ee_s ee_p

1 Sawa Immob. Lipase LIP-301 0.61 0 0.982 0.383 203 lot# 33580

2 Sawa Lipoprotein Lipase 0.245 0.982 0.200 1 43 LPL-701 lot#0514A

3 Toyobo Lipase (LIP-300) 0.71 2 0.985 0.41 9 278

4 Toyobo Lipoprotein Lipase 0.059 0.920 0.060 25 (LPL-31 1 ) Type A

5 Toyobo Lipoprotein lipase 0.267 0.979 0.21 4 1 23 ( L P L -701 )

6 Toyobo Lipoprotein lipase 0.079 0.932 0.078 3 1 (Type A)

7 Boehringer chirazyme L4 0.092 0.91 0 0.092 2 3

8 Boehringer chirazyme L6 0.260 0.977 0.21 0 1 1 0

9 Altus ChiroClec PC 0.053 0.908 0.055 22

1 0 Toyobo LIP-300 lot# 36510 0.724 0.987 0.423 335

1 1 Toyobo LPL-31 1 lot# 53250 0.037 0.922 0.038 2 6 Conditions: (±)-IIc (25 mg, 50mM), Trifluoroethyl isobutyrate (5 eq), Enzyme (6-27 mg), 4A Sieves (25-40 mg), TBME (1.0 mL), 250 rpm, RT, 23.5 h.

B. Effect of Solvent

General Procedure: For Runs 1-9, (±)-IIc (49-57 mg), 4A molecular sieves (47-59 mg) and Toyobo LIP-300 (50-55 mg) were suspended in the appropriate solvent (2.0 mL) and trifluoroethyl isobutyrate (0.08 mL,

5 equivs.) added, except for Runs 1-3 where the solvent was used as the acylating agent. The reactions were shaken at 250 rpm at ambient temperature for 22.5 h. For Runs 10-25, mixtures of (±)-IIc (70 mg), Toyobo LIP-300 (70 mg) and trifluoroethyl isobutyrate (5 equivs.), except Runs 17-23 which used solvent as acylating agent, in the appropriate solvent (2.0 mL) were shaken at 300 rpm and 30°C for 24 h.

The results of the chiral HPLC analysis are collected in Table 5.

Table 5. Effect of Solvent on the isoButyrylation of (±)-IIc Usinq Toyobo LIP-300

Run Solvent Acylating ee_s ee_p c E Agent

1 Trifluoroethyl None 0.445 0.947 0.320 57 isobutyrate

2 Ethyl isobutyrate None 0.106 0.881 0.107 18

3 Methyl isobutyrate None 0.032 n/d n/d n/d

4 TBME TFEOiBu 0.535 0.984 0.352 217

5 Toluene TFEOiBu 0.145 0.917 0.137 27

6 THF TFEOiBu 0.147 0.926 0.137 30

7 Acetone TFEOiBu 0.097 0.990 0.089 219

8 MeCN TFEOiBu 0.134 0.924 0.126 29

9 pDioxane TFEOiBu 0.086 >0.99 0.080 217

10 TBME TFEOiBu 0.618 0.973 0.388 137

11 10%Et₃N/TBME TFEOiBu 0.851- 0.936- 0.476- 83-

0.917 0.938 0.494 102

12 10% Toluene/TBME TFEOiBu 0.541 0.968 0.359 106

13 20% TolueneTBME TFEOiBu 0.447 0.967 0.316 93

14 30% TolueneTBME TFEOiBu 0.388 0.965 0.287 82

15 40% Toluene/TBME TFEOiBu 0.316 0.964 0.247 75

16 50% TolueneTBME TFEOiBu 0.241 0.966 0.200 72

17 Methyl isobutyrate neat None 0.022 n/d n/d n/d

18 Ethyl isobutyrate neat None 0.134 0.988 0.119 192

19 10% EtOiBu/TBME None 0.029 0.956 0.030 46

20 20% EtOiBu/TBME None 0.059 0.991 0.056 226

21 30% EtOiBu/TBME None 0.07 0.991 0.066 242

22 40% EtOiBuTBME None 0.076 0.991 0.071 251

23 50% EtOiBu/TBME None 0.092 0.991 0.085 251

24 TBME/Dried Enzyme TFEOiBu 0.856- 0.944- 0.476- 96-

0.943 0.946 0.499 130

25 20% Et₃N/TBME TFEOiBu 0.875- 0.872- 0.501- 42-

0.960 0.877 0.523 60

Conditions: Runs 1-9: (±)-IIc (50 mg, 50mM), Toyobo LIP-300 (50-55 mg), TFEOiBu (5 eq., except Runs 1-3 which used solvent as acylating agent), Solvent (2.0 mL), 4A Sieves (47-59 mg), 250 rpm, RT, 22.25 h.

Runs 10-25: (±)-IIc (70 mg, 75 mM), Toyobo LIP-300 (70 mg); Solvent (2.0 mL), TFEOiBu (5 equiv., except Runs 17-23 which used solvent as acylating agent), 30°C, 300 rpm, 24 h. ENZYMATIC RESOLUTION OF (±VIId

A. Acylation of (±)-IId Using ChiroCLEC PC

Table 6. Acetylation of (±)-IId using ChiroCLEC PC in Various Solvents

Run Solvent Time h ee_s ee_p Conversion

1 EtOAc 20.25 0.02 0 n/d n/d

2 PrOAc 20.25 0.00 0 n/d n/d

3 TBME 20.25 0.08 0.67 0.1 1 6

4 Acetone 20.25 0.07 0.48 0. 1 2 3

5 MeCN 3.75 0.56 0.83 0.40 1 8

5 MeCN 20.25 0.68 0.78 0.47 1 7

6 tAmyl Alcohol 20.25 0.02 0.45 0.05 3

7 Pyridine 20.25 0.01 0.20 0.03 2

8 3-Me-3- pentanol 20.25 0.09 1 .00 0.08 n/d

Conditions: (±)-IId (5-9 mg), TFEOAc (12-20 equiv.), CLEC PC (4.5-9.1 mg), Solvent (1.0 mL), RT, 250 φm.

B. Acylation of (±)-IId Using Toyobo LIP-300

Table 7. Acetylation of (±)-IId using Toyobo LIP-300 in Various Solvents

Run Reaction Time h ees eep Conversion E

MeOAc 29.75 0.27 >0.95 0.21 >10 0

2 PrOAc 29.75 0.02 n/d n/d n/d

3 TBME 29.75 0.39 0.69 0.36 8

4 Toluene 29.75 0.01 0.27 0.04 2

5 THF 29.75 0.00 n/d n/d n/d

6 Acetone 29.75 0.21 0.51 0.29_ 4

7 MeCN 2.25 0.1 5 0.82 0.1 5 1 2

4.75 0.31 0.91 0.26 3 0

21 .75 0.82 0.85 0.49 3 1

29.75 0.89 0.80 0.53 2 6 8 Dichloromethane 29.75 0.02 n/d n/d n/d

9 tAmyl Alcohol 29.75 0.04 0.38 0.1 1 2

1 0 Pyridine 29.75 0.02 n/d n/d n/d

1 1 Dioxane 29.75 0.1 2 n/d n/d n/d

1 2 MeOAc, neat 27.25 0.47 0.92 0.34 4 1

1 3 Trifluoroethyl Acetate, neat 27.25 0.1 7 0.49 0.25 3

1 4 isoPropenyl Acetate, neat 3 Complete reaction

Conditions: (±)-IId, 6.5-12.8 mg; TFEOAC, 0.08 mL, 30-50 equiv.; Enzyme, 2.8- 8.4 mg; Solvent, 2.0 mL; RT, 250 rpm., except Runs 12-14 which used solvent (2.0 mL) as acylating agent

C. Isobutyrylation of (±)-IId Using Toyobo LIP-300

Table 8 Isobutyrylation of (±)-IId Using Toyobo LIP-300 in Various Solvents

Run Solvent floe eep Conversion E

1 TBME 0.818 0.971 0.457 1 74

2 THF 0.474 0.889 0.348 27

3 Toluene 0.202 0.962 0. 1 73 6 3

4 MeCN 0.236 0.932 0.202 36

Conditions: (±)-IId, 50 mg; Toyobo LIP-300, 50 mg; TFEOiBu, 0.08 mL, 5 equivs. Solvent, 2.0 mL; 250 rpm; RT.

D. Acylation of (±)-IId Using NOVO SP435

Table 9. Acetylation of (±)-IId using Novo SP435 in Various Solvents/Temperatures

Run Reaction Time h ee_s ee_r Conversion

1 MeOAc 1 5.50 0.45 0.1 8 0.72 2

2 MeOAc 1 .00 0.68 0.70 0.49 1 1

3 PrOAc 1 5.5 0.1 7 0.61 0.22 5

4 TBME 40 0.09 >0.95 0.09 4 3

5 Toluene 40 0.05 0.85 0.06 1 3

6 THF 1 5.5 0.82 0.31 0.72 4

7 Acetone 1 5.5 0.33 0.78 0.30 1 1

8 Acetone 1 0.72 0.64 0.53 1 0

9 MeCN 1 5.5 0.24 0.02 0.91 1 1 0 MeCN 1 >0.95 0.43 0.69 8

1 1 MeCN, 0°C 1 .25 0.55 0.73 0.43 1 1

1 2 MeCN/NaHC0₃; -2 to -5°C 1 .5 0.39 0.77 0.34 1 1

1 3 Dichloromethane 40 0.07 >0.95 0.07 42

1 4 tAmylOH 15.5 0.02 >0.95 0.02 40

1 5 Pyridine 1 5.5 0.52 0.35 0.60 3

Conditions: Runs 1 ,3-7,9, 13-15: (±)-IId, 7-11 mg; TFEOAc, 50 mL, 14-25 equiv.; SP435.8-13 mg; Solvent, 2.0 mL, RT, 250 φm.

Runs 2, 8, 10-12: (±)-IId, 12-15 mg; TFEOAc, 100 mL, 25-31 equiv.(except Run 2 which used solvent as the acylating agent); SP 435, 4-7 mg; Solvent, 2.0 mL; 250 rpm.

Table 10. Acylation of (±)-IId with Various Acylating Agents catalyzed by Novo SP435 Run Solvent/ Acylating Time ee_s eep c E

Acylating Agent Agent h equiv.

1 MeOAc Neat 66.25 >0.95 0.09 0.91 n/d

2 MeOAc Neat 2.0 0.26 0.83 0.24 1 3

3 MeOAc Neat 1 .0 0.68 0.70 0.49 1 1

4 EtOAc Neat 66.25 >0.95 0.1 1 0.90 n/d

5 PrOAc Neat 66.25 0.74 0.47 0.61 6

6 iPropenylOAc (neat) Neat 66.25 0 0 1 .00 n/d

7 iPropenylOAc 1 2 66.25 0 0 1 .00 n/d

8 Acetic Anhydride 1 0 66.25 0 0 1 .00 n/d

9 TFEOAc 1 4 66.25 0.60 0.29 0.67 3

1 0 TFEOAc/Acetone -5°C 1 1 20.75 0.1 6 0.76 0.1 8 8

1 1 EtOAcCI Neat 66.25 0 0 0 n/d

1 2 TFEOAcCI 1 7 66.25 0 0 0 n/d

1 3 Propionic Anhydride 1 4 66.25 0 0 1 .00 n/d

1 4 Butyric Anhydride 1 7 66.25 0 0 1 .00 n/d

1 5 isoButyric Anhydride 1 7 66.25 0 0 1 .00 n/d

1 6 EtOBu Neat 66.25 0 0 0 n/d

1 7 TFEOBu 1 7 66.25 0.07 0.85 0.07 1 4

1 8 TFEOBu/MeCN 1 2 20.75 0.08 n/d n/d n/d

1 9 TFEOiBu 1 6 66.25 0 0 0 n/d

20 TFEOiBu/MeCN 1 1 20.75 0.04 n/d n/d n/d

2 1 MeOAσOMe 5 3 2.0 0.29 0.40 0.42 3

22 TFEHexanoate 25 5.0 0.29 0.83 0.26 1 4

2 3 TFELaurate 1 5 5.0 0.54 0.88 0.38 26

24 TFE2-MeButyrate 25 5.0 0 0 0 n/d

Conditions: Runs 1 , 4-9, 11-17, 19: (±)-IId ,4.9 mg; TBME or neat acylating agent, 1.0 mL; SP 435, 6.2-10.8 mg; 250 rpm; RT.

Runs 2, 21-24: (±)-Hd , 9-12 mg; MeCN or neat acylating agent, 2.0 mL; SP 435, 5-7 mg; CaCθ3, 42-33 mg, 250 φm, RT.

Runs 10, 18, 20: (±)-IId , 11 -16 mg;Solvent, 2.0 mL; SP 435, 6-8 mg; NaHCθ3, 34-37 mg (except Run 10), 250 rpm, RT.

Run 3: (±)-IId , 15 mg; Acylating agent/solvent, 2.0 mL; SP435, 4 mg; 250 rpm; RT E. Other Substrates

Substrate R1 R2 R4 R3 ee_s or eep or ⁵ [^«]²„⁵

1 NH₂ Cl H Br 0.770 0.953 0.447 9 8

2 H Me H OMe 0.520 0.989 0.345 31 1

3 H Cl NH₂ Br - 8.09 ° + 1 1 4.1 ° n/d n/d

(c 1 .484, (c 0.142,

MeOH) MeOH)

Conditions:

Run 1 : 1 , 5 mg; LIP-301 , 10 mg; Trifluoroethyl isobutyrate, 10 equivs.; TBME, 1.0 mL; 200 rpm; RT

Run 2: 2, 5.4 mg; LIP-301 , 16.6 mg; Trifluoroethyl isobutyrate, 20 equivs.; TBME, 1.0 mL; 200 rpm, RT.

Run 3: 3, 0.2 g; LIP-301 , 0.4 g; Trifluoroethyl isobutyrate, 10 equivs.; TBME, 4 mL;

200 rpm, RT.

Following is a detailed example of a preferred embodiment of the process of this invention.

EXAMPLE 1 A mixture of (±)-IIc (20 g, 42.7 mmol, 98% pure by assay) in

TBME (600 mL) was stirred at ambient temperature for 1 h, then filtered to remove a small amount of insoluble material. The solution was dried by azeotropic distillation; after 200 mL was distilled, a further 200 mL of TBME was added to the reaction mixture. After a total of 400 mL had been distilled, the moisture content (Karl-Fischer) of the solution was 214 ppm. Toyobo LIP-300 (40 g; 1282 ppm water) was added to the reaction mixture and stirred for 0.5 h; moisture content at this stage was 250 ppm. Trifluoroethyl isobutyrate (19.1 mL, 3 equivs.) was added and the mixture was stirred at ambient temperature. The reaction was terminated after 24 h. The enzyme was removed by filtration and washed with TBME (100 mL).

The combined filtrates were extracted with 0.5M H2SO4 (100 mL, 50 mL, 50 mL). The combined acidic extracts were added slowly (60 min) to a mixture of 50% NaOH (15 mL) and water (150 mL). The solid which precipitated was filtered and dried to give (-)-Ic (10.6 g, 96% pure by assay, 52.1% yield; 76.7% ee).

The reaction mixture was extracted with 6M H2SO4 (2 X 30 mL). The combined extracts were heated to reflux for 8 h, cooled to room temperature, then added slowly (90 min.) to a mixture of 50% NaOH (70 mL) and ice (170 g), maintaining the temperature at <40°C. The precipitated solid was filtered and dried to give (+)-Ic (8.8 g, 97% pure by assay, 43.4% yield; 98.4% ee).

Example 2

(-) II (27.30 g, 94% pure, 81.4% ee) was dissolved in diphenyl ether (137 mL) and heated at reflux under N2 for 40 min., by which time the ee was <1%.

The mixture was cooled to room temperature and diluted with TBME (500 mL). Analysis of this solution showed a solution yield of

95.8%. The solution was extracted with 0.5 M H2SO4 (2 X 218 mL) and the combined acidic extracts were added slowly over a period of 1 hour to a vigorously stirred mixture of 50% NaOH (45 mL) and water (405 mL). After stirring for 0.5 hours, the precipitated solid was collected by filtration and washed with water (820 mL) (26.11 g, 94.6% yield, 1.0% ee).

Example 3

Enzymatic Resolution: 1 st Cycle

Preparation of the R-isobutyramide (+)-lllb: (±)-llc (93.0 g, 0.2 mol) was dissolved in TBME (2.0 L) and stirred at room temperature for 1 h. The reaction mixture was filtered, the insoluble material washed with more TBME (~1.0 L), and the volume of filtrate adjusted to 2.9 L. The solution of (±)-llc was then dried by azeotropic distillation, removing 1.0 L of the solvent. The solution was cooled to room temperature and Toyobo LIP-301 (200 g) was added. After stirring at room temperature for 1 h, trifluoroethyl isobutyrate (90 mL, 0.56 mol) was added in one portion.

The reaction was stirred at room temperature under N₂ for 24 h. The enzyme was then removed by filtration and washed with TBME (0.9 L). The combined filtrates were extracted sequentially with three portions of 0.5 M H2SO4 (450 mL, 225 mL and 225 mL). These combined acidic extracts contained the unreacted (-)-llc. The organic layer was then extracted with two portions of 6M H2SO4 (135 mL and 135 mL). These combined acidic extracts contained the product isobutyramide (+)-lllb.

Isolation of (+)-lc: The combined 6M H2SO4 extracts were heated at reflux for 14 .5 h, then cooled to room temperature. The reaction mixture was then added slowly to a cold, vigorously stirred mixture of NH4OH (900 mL) and CH3CN (270 mL). The solid which precipitated was filtered, washed with water and dried (40.5 g, 43.5%; 0.960 ee). Isolation of (-)-llb: The combined 0.5 M H2SO4 extracts were added slowly to a cold, vigorously stirred mixture of NH4OH (450 mL) and CH3CN (270 mL). The solid which precipitated was filtered , washed with water and dried (40.5 g, 43.5%; 0.966 ee). Racemization of (-)-llb. Diphenyl ether (190 mL) was degassed under vacuum for 5-10 min and then purged with N2 for 5-10 min. (-)-llb (38 g, 81 mmol) was added and the mixture stirred under N2 and heated to 245°C. The reaction mixture was maintained at 245°C for 2 h, whereupon racemization was complete. After cooling to room temperature, the reaction mixture was diluted with TBME (570 mL) and filtered. The filtrate was extracted with two portions of 0.5 M H2SO4 (190 mL and 95 mL). The extracts were combined, charcoal (19 g) added, and the mixture heated to reflux for 1 h. After cooling, the mixture was filtered through Celite and the bed washed with 0.5 M H2SO4 (95 mL). The combined filtrates were added slowly to a cold, vigorously stirred mixture of NH OH (190 mL) and CH3CN (114 mL). The solid which precipitated was filtered, washed with water and dried (31.9 g, 84.0%). A similar procedure can be carried out using diethylene glycol dibutyl ether in place of diphenyl ether, and heating at 210°C for about 12 hours. Enzymatic Resolution: 2nd Cycle Racemized (±)-llc (30 g, 64 mmol) was dissolved in TBME (600 mL), filtered and the volume adjusted to 900 mL. The solution was then dried by azeotropic distillation, removing 300 mL solvent. The mixture was cooled and Toyobo LIP-301 (60 g; recovered from 1 st cycle above) was added. The mixture was stirred for 1 h, then trifluoroethyl isobutyrate (30 mL, 190 mmol) was added. After stirring at room temperature under N2 for 24 h, the reaction mixture was then filtered and the enzyme cake washed with TBME (300 mL). The combined filtrate was extracted with three portions of 0.5 M H2SO4 (150 mL, 75 mL and 75 mL) to remove the unreacted (-)-llc. The organic layer was then extracted with two portions of 6M H2SO4 (45 mL and 45 mL) which were combined and refluxed for 16 h. The cooled reaction mixture was then added slowly to a vigorously stirred, cold mixture of NH4OH (300 mL) and CH3CN (90 mL). The precipitated (+)-lc was filtered, washed with water and dried: (13 g, 43%; 0.986 ee).

Claims

We claim:

1. A process for preparing a substituted (6,11 -dihydro-5H-benzo- [5,6]cyclohepta[1 ,2-b]pyridin-11-yl)piperidine compound of the formula (+)-I

wherein:

R, R¹ , R², R³ and R⁴ are independently selected from the group consisting of hydrogen, halo, C-i-C╬▓ alkyl, amino, -OCH3, -OCF3 and CF3, and the dotted line represents an optional double bond; comprising:

(1)(a) enzymatically catalyzing the acylation of a compound of formula (┬▒)-II, wherein the variables are as defined above, to obtain a compound of formula (+)-III

wherein the enzyme is a hydrolase and wherein the acylating agent is of the formula R⁵COOR⁶, wherein R⁵ is C1-C15 alkyl, halo methyl, aryl, benzyl or benzyloxy, R⁶ is C1-C6 alkyl, Ci-C╬▓ alkenyl, -COR⁷, trifluoroethyl, -CH2CH(OCOR⁷)CH₂OCOR⁷, halo methyl or benzyl, and R⁷ is C₁-Ci5 alkyl; and

(b) hydrolysing the compound of formula (-t-)-IH;

(2) enzymatically catalyzing the acylation of a compound of formula (┬▒)-IIa, wherein R, R¹ , R², R³ and R⁴ are as defined above and the bond is a single bond, with a hydrolase, and wherein the acylating agent is as defined above.

2. A process of claim 1 wherein the enzyme is Toyobo LIP-300, Toyobo LIP-301 , Altus Chiro CLECΓäó PC or Novozym 435.

3. A process of claim 1 wherein the acylating agent is selected from the group consisting of trifluoroethyl acetate, trifluoroethyl butyrate, trifluoroethyl isobutyrate, trofluoroethyl benzoate, triacetin and tributyrin.

4. A process of claim 1 for preparing a compound of formula I wherein R, R² and R³ are halo and R¹ and R⁴ are hydrogen.

5. A process of claim 1 for preparing a compound of the formula

comprising enzymatically catalyzing the acylation a compound of the formula

using Toyobo LIP-300, Toyobo LIP-301 or Altus ChiroCLECΓäó PC as the enzyme and trifluoroethyl acetate, trifluoroethyl butyrate, trifluoroethyl isobutyrate, trifluoroethyl benzoate, triacetin or tributyrin as the acylating agent, followed by hydrolysis and optionally followed by reconversion of the undesired isomer to the racemate, and resubjecting the racemate to enzymatic acylation and hydrolysis.

6. A process of claim 1 wherein in step (c), the undesired isomer from step (a) is converted to the racemate by heating at 200-260┬░C in diphenyl ether or diethylene glycol dibutyl ether.

7. A process of claim 5 wherein the undesired isomer is converted to the racemate by heating at 200-260┬░C in diphenyl ether or diethylene glycol dibutyl ether.

8. A process of claim 1 for preparing a compound of the formula

comprising enzymatically catalyzing the acylation a compound of the formula

using Toyobo LIP-300, Toyobo LIP-301 or Altus ChiroCLECΓäó PC as the enzyme and trifluoroethyl acetate or trifluoroethyl isobutyrate as the acylating agent, followed by hydrolysis.

9. A process of claim 1 for preparing a compound of the formula

comprising enzymatically catalyzing the acylation a compound of the formula

using Novozyme SP435 as the enzyme and trifluoroethyl acetafe, trifluoroethyl butyrate, trifluoroethyl hexanoate, trifluoroethyl laurate or methyl acetate as the acylating agent.