PL202430B1 - The manner of distribution of enantiomers (1R, 2S)(1S,2R) of methyl ester of 1-amino-2-vinylocyclopropylocarboxyl acid - Google Patents

Abstract

Method for the separation of enantiomers from a mixture of (1R, 2S) / (1S, 2R) isomers of a methyl ester 1-amino-2-vinylcyclopropylcarboxylic acid and its protected derivatives, characterized in that it comprises the step of treating said mixture with an esterase to be prepared the corresponding enantiomer (1R, 2S).

Description

Description of the invention

The subject of the invention is a process for the separation of enantiomers from a mixture of (1R, 2S) / (1S, 2R) isomers of 1-amino-2-vinylcyclopropylcarboxylic acid methyl ester and its protected derivatives.

The term "1-aminocyclopropylcarboxylic acid (Acca) as used herein refers to a compound of the formula:

The term "ester, alone or in combination with another substituent," as used herein, means esters of a compound of Formula I in which the carboxyl functional group is replaced with an alkoxycarbonyl functional group:

wherein the R group of the ester is selected from alkyl.

The invention relates to the separation of mixtures of compounds of the general formula:

wherein R1 is not H, and C1 and C2 each represent an asymmetric carbon atom at the 1 and 2 positions of the cyclopropyl. Without excluding the existence of other possible asymmetric centers in other parts of the compounds of formula I, the presence of these two asymmetric centers means that the compounds of formula I can exist as racemic mixtures of diastereoisomers. As illustrated in the examples below, racemic mixtures can be prepared and then resolved into individual optical isomers, or these optical isomers can be prepared by chiral synthesis.

Thus, a compound of formula I may exist as a racemic mixture of diastereoisomers at carbon 1 when R1 on carbon 2 has a syn orientation to the carbonyl at position 1, representing the radical:

or a compound of formula I may exist as a racemic mixture of diastereoisomers when R1 in the 2nd position has the anti-orientation to the carbonyl at the 1-position, which is represented by the radical:

PL 202 430 B1

The racemic mixtures can then be separated into individual optical isomers.

The process of the invention relates to the separation of enantiomers from a mixture of (1R, 2S) / (1S, 2R) isomers of 1-amino-2-vinylcyclopropylcarboxylic acid methyl ester and its protected derivatives, which includes the step of treating said mixture with an esterase to generate the corresponding enantiomer. (1R, 2S).

_®

Preferably, in the above method as said esterase is used Alcalase ^®.

The invention allows to obtain a compound with a preferred R configuration of the carbon 1 atom from the mixture.

The introduction of the substituent (R1) on C2 affects activity when introduced such that C1l has the R configuration. For example, compounds in the (1R, 2S) and (1R, 2R) configuration show activities at concentrations of 25 and 82 nM, respectively. Compared to the analogous compound with unsubstituted cyclopropyl (475 nM), a significant increase in activity is observed. In addition, when carbon 1 has the R configuration, inhibition of the HCV NS3 protease is further enhanced by the substituent configuration (e.g., alkyl or alkylene) at carbon 2 of the cyclopropyl, e.g. a compound that has a R1 in the syn to carboxy position is more active (25 nM) than the "anti" enantiomer (82 nM). In special cases, the effect of the R and S configuration on C1 can be observed by comparing the compound (1R, 2S) and its corresponding isomer (1S, 2S), which have activities at concentrations of 6 nM and> 10 μΜ, respectively, which is a difference of more than 1500 times! !

Thus, the most preferred compound is represented by an optical isomer having R1 and a carbonyl in syn orientation in the following absolute configuration:

when R1 is, for example, ethyl and the asymmetric carbon atoms in positions 1 and 2 have the R, R configuration. A preferred embodiment is a compound in which carbon atom 1 has the R configuration,

and R1 is vinyl.

Protecting groups which can be used in the art and therefore for the compounds of the invention are mentioned, for example, in Greene, "Protective Groups in Organic Chemistry, John Wiley & Sons,

PL 202 430 B1

New York (1981) and Peptides: Analysis, Synthesis, Biology, Vol. 3, Academic Press, New York (1981), and are incorporated herein by reference.

The following examples illustrate the resolution of all the above mixtures of enantiomers at carbon 1 with the use of enzymes.

Examples

Temperatures are given in degrees Celsius. Solution percentages are weight to volume ratios, and solution ratios are volume to volume ratios, unless otherwise stated. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz spectrometer; chemical shifts (δ) are reported in parts per million. Flash chromatography was performed on silica gel (SiO2) according to Still's procedure (W.C. Still et al., J. Org. Chem., (1978), 43, 2923).

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Enzymatic resolution of methyl Boc-1-amino-2-ethylcyclopropylcarboxylate isomers (1R, 2R) / (1S, 2R)

* HPLC analysis using a Chiralcel® OD-H column ** Other esters are also acceptable (e.g. Et)

a) The mixture of the (1S, 2R) / (IR, 2R) enantiomers of 1-Boc-amino-2-ethylcarboxylic acid methyl ester according to Example 10 (0.31 g, 1.27 mmol) was dissolved in acetone (3 ml) and then diluted with water (7 ml) while stirring rapidly. The pH of the solution was adjusted to 7.5. with 0.05 M aqueous NaOH prior to the addition of the enzyme Alcalase ^® [2.4 L extract from Novo Nordisk Industrials] (300 mg). The pH was stabilized with NaOH during the incubation and a pH stat was used to monitor the addition of the NaOH solution. After 40 hours, the mixture was diluted with EtOAc and H 2 O (with 5 mL of saturated NaHCO 3 solution) and the phases were separated. The aqueous phase was acidified with 10% aqueous HCl and extracted with EtOAc, dried (MgSO4), filtered and concentrated to give acid 13a (48.5 mg). The absolute stereochemistry was determined using correlation.

b) treating the sample acid 13a with diazomethane in Et2O to give the methyl ester and then ana lysis _® HPLC using a chiral column [Chiralcel OD-H ^®, a mixture of 2.5% isopropanol / hexane, isocratic] showed the presence of the (S, R) 51: 1 aspect ratio.

P r z k ł a d 2

Enzymatic resolution of the (1R, 2S) / (1S, 2S) isomers of ethyl 1-amino-2-vinylcyclopropylcarboxylate

_® * HPLC analysis using a Chiralcel ^® OD-H column

a) The racemic derivative 17a (0.29 g, 1.14 mmol) was dissolved in acetone (5 ml) and diluted with H 2 O (10 ml). Before the addition of Alcalase ^® (300 mg) adjusted to pH 7.2 with Pople 202 430 B1 under a 0.2 N aqueous solution of NaOH. In order to keep the pH constant during the incubation, the NaOH solution was added via a pH-stat dosing over 9 days until the theoretical amount of base had been added. Performing the acid / base extraction as described in Example 1, the unhydrolyzed ester (0.15 g, 100%) and the hydrolyzed material (0.139 g, 95%) were isolated. Analysis of the unhydrolyzed ester by HPLC using a chiral column showed a 43: 1 ratio of the desired compound 17c with the (R, S) stereochemistry based on the chemical correlation.

Conditions for HPLC analysis: Chiralcel OD-H ^® column (4.6 mm x 25 cm), isocratic conditions using a mobile phase of 2.5% isopropanol / hexane.

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Enzymatic separation of (1R, 2S) -1-amino-2-vinylcyclopropanecarboxylic acid methyl ester hydrochloride

Preparation of N-Boc- (1R, 2S) -1-amino-2-vinylcyclopropane carboxylic acid methyl ester 20a:

The racemic ester 19f (0.200 g, 0.83 mmol) was dissolved in acetone (3 ml) and water (7 ml) was added. Was added 0.05 M NaOH (1 drop) to adjust the pH of the solution to about 8 et seq ę then added Alcalase ^® 2.4 L (Novo Nordisk Biochem, 0.3 g in one mL of water). The mixture was stirred vigorously at room temperature, maintaining the pH of the solution at 8, using an automatic dosing device. At the beginning of the 4th and 5th day of stirring, at a pH value of 8, additional enzyme solution (2 x 0.3 g) was added. After a total of 5 days, a total of 8.3 ml of 0.05 M NaOH solution had been used. The reaction mixture was diluted with EtOAc and water, and the organic phase was separated. After washing with brine, the organic extract was dried (MgSO4) and concentrated in vacuo. Compound 20a (0.059 g, 30% yield) was obtained as a clear oil.

Analysis ¹ H NMR identical to that of compound 19f; HPLC (Chiralcel ODH, 4.6 x 250 mm, isocratic 1% EtOH in hexane, flow rate 0.8 ml / min): (1R, 2S) -2 Rf 19.3 minutes (97%); (1S, 2R) -2 Rf 17.0 minutes (3%).

Preparation of (1R, 2S) -1-amino-2-vinylcyclopropane carboxylic acid methyl ester hydrochloride 20b:

Compound 20a (39.96 g, 165.7 mmol) was dissolved in dioxane (25 mL) and the solution was added dropwise to a stirred 4N solution of HCl in dioxane (Aldrich, 250 mL). After 45 minutes, TLC analysis showed complete deprotection. The volatiles were removed under reduced pressure and the residue was coevaporated twice with MeOH (2 x 100 mL). Ether (300 mL) and MeOH (10 mL) were added to the brown, oily residue, and the mixture was stirred overnight at room temperature, resulting in precipitation of a semi-solid. MeOH (15 ml) was added and stirring was continued for 6 hours at which time the yellowish solid was collected by filtration. The product was washed with 5% MeOH in ether (50 ml) and ether (2 x 50 ml) and dried in vacuo to afford compound 20b as a yellowish solid (22.60 g, 76% yield). The filtrates (including the washes) were concentrated in vacuo to provide additional 20b as a brown oil (7.82 g, 26% yield). Both fractions were pure enough for use in the synthesis of HCV protease inhibitors.

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[α] D ²⁵ + 38.2 ° (c 1.0, MeOH); ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.15 (broad s, 3H), 5.65 (ddd, J = 17.10.9 Hz, 1H), 5.36 (dd, J = 17, 1.5Hz, 1H), 5.19 (dd, J = 10, 1.5Hz, 1H), 3.74 (s, 3H), 2.50 (q, obscured by DMSO signal, J = 9Hz, 1H) , 1.86 (dd, J = 10.6 Hz, 1H), 1.64 (dd, J = 8.6 Hz, 1H).

Claims (2)

A method of separating enantiomers from a mixture of (1R, 2S) / (1S, 2R) isomers of 1-amino-2-vinylcyclopropylcarboxylic acid methyl ester and its protected derivatives, characterized by the step of treating said mixture with an esterase to produce the corresponding enantiomer (1R, 2S). _® 2. The method according to p. 1, characterized in that the said esterase is used Alcalase ^®.