CA1332942C - Synthesis of 2' ,3'-dideoxycytidine - Google Patents

Abstract

2',3'-Dideoxycytidine is prepared by subjecting an N-acyl-3'-halo-2'-acylcytidine to a reductive elimination reaction using a zinc-copper couple followed by reduction and removal of protecting groups. The N-acyl-3'-halo-2'-acylcytidine can be obtained by reacting N-acyl cytidine with a suitable halogenating agent, for example 2-acetoxy-isobutyryl bromide. 2'3'-Dideoxy-cytidine is used in processes of oligo-nucleotide sequencing and is of interest for treatment of AIDS and AIDS-related diseases.

Description

BACKGROUND OF THE INVENTION
The present invention relates to the synthesis of 2',3'-dideoxycytidine (hereafter sometimes referred to as ddC).

2',3'-Dideoxycytidine is the compound of the following formula I

The compound has been used for some years in processes of~oligo-nucleotide sequencing. More recently, ddC has come to be considered for use in the treatment of AIDS and AIDS-related diseases.
DISCUSSION OF THE PRIOR ART
Various processes are known for the synthesis of ddC.
Horwitz et al, in J. Org. Chem. 32, 817 (1967), disclose a synthesis starting from 2'-deoxycytidine. A disadvantage of this process is that the key starting material, 2'-deoxycytidine, is limited in supply and, consequently, very expensive. Another process, disclosed in Bio. Org. Khim. 9, 52 (1983), makes use of 2'-deoxycytidine protected at the amino group by means of a benzoyl group. This process therefore suffers from the disadvant-age of the first mentioned process, i.e. the limited supply of 2'-deoxycytidine. It also suffers from the disadvantage that the subsequent synthesis steps make use of carbon disulfide, which is highly flammable, methyl iodide, which is carcinogenic, and tributyl tin hydride, which is expensive and hard to handle.
In 1974, Japanese researchers published a study dealing with halogenations of uridine and cytidine derivatives [R.
Marumoto, M. Honjo; Chem. Pharm. Bull. 22, 128 (1974)]. While the reactions of uridine and cytidine with acetyl bromide in acetonitrile gave the expected 2'-brominated products, the corres-ponding reaction of N-acetylcytidine progressed in a different way. As one of the products, a 3'-brominated species was isolated in moderate yield. Catalytic reduction gave 3'-deoxycytidine together with a small amount (7%) of 2',3'-dideoxycytidine (presumably formed by a Pd-catalysed reductive elimination and subsequent hydrogenation). An intermediate obtained in the synthesis is a tri-acylated 3'-bromo compound of the following formula:

This compound was later used as substrate in an electrochemical reduction giving the corresponding diacetyl 2',3'-unsaturated cytidine derivative in good yield. [T. Adachi, T. Iwasaki, I. Inoue, and M. Miyoshi; J. Org. Chem. 44, 1404 (1979)]. The electrochemical conversion of the tri-acylated compound into the 2',3'-unsaturated cytidine derivative occurs smoothly in the laboratory but does not lend itself well to scale up for produc-tion of the unsaturated intermediate, and subsequent reduction toddC, on an industrial scale. The design of a suitable electro-chemical cell for large scale production would be prohibitively expensive and concentration limits may cause problems with electrochemical processes.
T.C. Jain et al, J. Org. Chem. 39, 30 (1974), disclose a process by which a halogenated adenosine derivative was converted, by reaction with chromous acetate in the presence of a base, to a mixture of deoxy- and 2',3'-unsaturated dideoxy derivatives of adenosine. So far as is known to the present applicant, this procedure has not been published using cytidine derivatives, presumably because of lack of a suitable precursor. M.J. Robins, et al, in Tetrahedron Letters 25, 367 (1984), disclose the reac-tion of a 3'-bromo derivative of adenosine with a zinc-copper couple in dimethylformamide, followed by reaction with ammonia and methanol to obtain the 2',3'-unsaturated dideoxy compound. This was reduced by hydrogenation in the presence of a palladium-on-carbon catalyst in ethanol, to yield dideoxyadenosine. Again, as far as applicant is aware this process has not been applied to cytidine.
SUMMARY OF THE INVENTION
The process of the invention avoids or alleviates the various disadvantages which arise with the above-mentioned processes. Thus, the present invention provides a process for preparing 2',3'-dideoxycytidine which comprises subjecting a compound of the formula II

II

wherein X is a halogen atom, preferably bromine or iodine: R and R2 are acyl groups and R3 is a protecting group, to a reductive elimination reaction with a zinc-copper couple to remove the halogen atom X and the acyloxy moiety oR2 in the 3'-position to yield the corresponding 2',3'-unsaturated dideoxy compound, removing the groups R1 and R3 and reducing the 2'-double bond, the removal of the groups R1 and R3 and the reduction being done in any order.
In another aspect the present invention provides a process for the preparation of 2',3'-dideoxycytidine comprising the steps of:
1. reductive elimination of bromoacetate derivative of 4-amino, 5'-hydroxy protected cytidine by treatment with zinc-copper couple to yield the corresponding 2',3'-didehydroderivative, 2. hydrogenating the 2',3'-didehydroderivative to yield 4-amino-5'-hydroxy protected 2',3'-dideoxycytidine, 3. removing the 4-amino and 5'-hydroxy protecting groups to yield 2',3'-dideoxycytidine.
In yet another aspect the present invention provides a process for the preparation of 2',3'-dideoxycytidine comprising the steps of:

4a a. protecting the 4-amino group of cytidine with a suitable protecting agent, b. reacting the 4-amino protected cytidine with unsubstituted 2-acetoxy-2-methyl-propanoyl bromide, c. reductively eliminating bromide and an acetoxy group from the bromoacetate derivative by treatment with a zinc-copper couple, d. hydrogenating the 2',3'-didehydroderivative, e. removing 4-amino and 5'-hydroxy protecting groups to yield 2',3'-dideoxycytidine.
Some intermediate compounds are novel and the invention extends to these novel compounds. Accordingly, the present invention provides a compound of the formula:

wherein R and R are both acyl.

The invention also includes a compound of the formula:

4b wherein R is acyl.
The invention also includes a compound of the formula:

wherein R1 is acyl.
DISCUSSION OF THE PREFERRED EMBODIMENTS
The reductive elimination reaction using the zinc-copper couple will proceed under a variety of conditions. It has been found to proceed when the ratio of Zn-Cu to substrate has values between about 2:1 and approx. 50:1, but a preferred range of ratios is approx. 3:1 to 4:1.. The reaction can be carried out in a variety of solvents, of which particular mention is made of dimethylformamide, dimethoxyethane, tetrahydrofuran and mixtures of ethyl acetate and water. Of these, tetrahydrofuran is preferred. To avoid acidic degradation, it is desirable that the reaction should be carried out in the presence of a mildly basic buffering agent. Preferred agents include sodium acetate, sodium citrate and basic phosphate buffers. The reaction is preferably carried out with mild cooling: a temperature in the range O to 10C is particularly preferred.
The product of the reductive elimination with the zinc-copper couple is the 2',3'-unsaturated compound. It is preferred not to isolate this compound from the reaction mixture but to proceed to the- next stage of the process. In a preferred embodiment the protecting groups are removed by treatment with a suitable base, for example sodium methoxide in methanol, methan-olic ammonia or potassium hydroxide in methanol followed by reduction of the 2', 3' double bond.
Suitable methods of reduction will be known to those skilled in the art and include catalytic hydrogenation. Suitable catalysts for the hydrogenation reaction include platinum group metals and palladised barium sulfate,Raney nickel is less pre-ferred. The reduction is preferably carried out by hydrogenation in the presence of a palladium-on-carbon catalyst.
After the reductive elimination but before the removal of the protective groups and reduction of the double bond there may be a treatment of the reaction mixture to remove any organic soluble zinc and copper salts. This is desirable because it has been found that the presence of such salts in the subsequent stages reduces the yield. One method of removing these salts which has been found to be satisfactory is to subject the reaction mixture to liquid-liquid extraction with an excess of aqueous sodium bicarbonate, sodium carbonate or potassium carbonate.
Alternatively or additionally the organic reaction mixture can be subjected to a washing proceduFe with a chelating agent, for ~ J~a~ ~ar~

example an aqueous solution of EDTA.
Any water-soluble form of EDTA can be used, for example the di- and tetra-sodium and potassium salts. The tetra-sodium salt is preferred.
The crude ddC obtained from the hydrogenation can be purified by chromatography or by batch treatment on a strong anionic exchange resin for instance Dowex~lX2-OH, Dowex~SBR-P(OH) or Amberlite lRA400. This is followed by crystallization. A
batch resin treatment is preferred.
In a preferred embodiment, the starting material of formula II is a 3'-bromo compound, i.e. X is bromine. The protecting group on the amino group, i.e. Rl, is an acyl group for example an acetyl, benzoyl, benzyloxycarbonyl or tert.-butoxycar-bonyl group. The protecting group on the 5'-hydroxyl group, i.e.
R3, is a group of formula A compound in which X is bromine, Rl and R2 are both acetyl and R3 is a group of the above formula is readily and cleanly obtained from N-acetyl-cytidine by reaction with 2-acetoxy-isobutyryl bromide. Other suitable halogenating agents are 2-acetoxy-isobutylryl iodide and other halides with structures similar to 2-acetoxy-isobutylryl bromide, for instance acetyl salicyloyl bromide or acetyl salicyloyl iodide. These compounds still yield ~ t~ c ~r~

a product in which X is a halogen and R2 is acetyl. Acetyl bromide is not preferred as a brominating agent because its use leads to production of a substantial quantity of the undesired 4-N,2',3',5'-tetraacetyl compound.
Suitable solvents for the halogenation reaction are inert organic solvents including acetonitrile, nitromethane, glacial acetic acid, chloroform, ethyl acetate, benzene, butyro-lactone and the like, at a temperature of about 25C to 150C, suitably the reflux temperature of the solvent. The reaction can also be carried out without solvent. The product obtained is substantially or completely free of any per-acylated species. In this respect, it differs from the product obtained by Marumoto, et al, as discussed above, when they used acetyl bromide. The absence of per-acylated species is of importance for the final purification of ddC.
3'-Halogenated cytidine compounds of formula II in which R3 is a group of formula are novel and constitute a further aspect of the invention.
Depending upon the conditions, this cyclic group may rearrange to the linear structure This compound is of course also novel and constitutes an aspect of the invention.
It will be seen that the preferred embodiment of the present process commences with cytidine which is protected at the amino group. Cytidine is a much more abundant material than 2'-deoxycytidine. Hence, Applicant's process has substantial advant-ages over the previously known processes for preparing ddC. As will be seen from the following Examples yields, based on cyti-dine, of about 25 to 40% of ddC can be obtained. In contrast, in the prior art ddC has been obtained from 2'-deoxycytidine (which is much less readily available than cytidine) and according to the literature yields, based on deoxycytidine, of about 25% have been obtained.
The process of the invention is further illustrated in the following Examples.
Example 1 (3'-Bromo Intermediate) To a suspension of 142.5 g of N-acetylcytidine (O.S moles) in 3.75 L of anhydrous acetonitrile is added 300 ml (420 g, 2 moles) of 2-acetoxyisobutyryl bromide over 10 minutes.
After approximately 15 minutes, solution occurs. The reaction mixture is stirred at room temperature over night. The mixture is poured into 10 L saturated sodium bicarbonate solution and the layers separated. The aqueous layer is extracted with three 1.5 L
portions of ethyl acetate. The organic phases are combined, washed with bicarbonate and dried over sodium sulfate. The drying agent is filtered off and the filtrate concentrated to dryness to give 247 g of a white, amorphous powder (95% crude yield). The material can be used in the next step without further purifica-tion. A sample, recrystallized from ethyl acetate shows the following nmr spectrum:
Solvent CDCL3; 10.5 (s, lH, 4-NH); 8.14 and 7.54 (2d, 2H, Cs-H and C6-H); 5.95 (d, lH, Cl'-H); 5.5 (S, lH, C2'-H), 4.4-4.65 (m, 3H, C4'-H, Cs"-H); 4.35 (d, lH, C3'-H), 2.3 (s, 3H, N-COCH3); 2.17 (s, 3H, 2'-OCOCH3); 2.07 (s, 3H, 2-acetoxy); 1.57 (d, 6H, geminal CH3).
Example 2 (Zu-Cu Reaction in DME) The 3'-brominated intermediate (42.7 g 0.08 mole) is dissolved in 750 ml of dimethoxyethane (DME). The solution is placed in an ice-water bath and 330 g (4.6 mole) of freshly pre-pared zinc-copper couple is added at once. The mixture is stirred at 0C for 1.5 hours and the progress of the reaction monitored by tlc. The 2',3' unsaturated product shows a characteristic purple color upon charring of the tlc plate. The reaction mixture was filtered through Celite~and the filter cake washed with 100 ml of DME. The filtrate is concentrated to 500 ml, 1 L of water is added and the product is extracted with ethyl acetate (4x700 ml).
The combined extracts are washed with saturated sodium bicarbonate solution (700 ml) and brine solution (600 ml) and dried over sodium sulfate. The drying agent is filtered off and the filtrate concentrated on a rotary evaporator to give 19.9 g of a yellowish glass (63.8%). The crude product is not stable at room tempera-~ 7ra~e ~

ture and should be processed promptly.
Example 3 (Zn-Cu Reaction in Ethyl Acetate/H2O) The crude 3'-brominated intermediate (259 gm, 0.5 mol) is dissolved in 750 ml ethyl acetate. After addition of 25 gm of sodium acetate and 50 ml of water, the mixture is cooled in an ice-bath. Under vigorous stirring, a pre-cooled, aqueous suspen-sion of freshly prepared zinc-copper couple is added and the mix-ture is stirred for approximately one hour. The reaction mixture is filtered through Celite and the filter cake is washe~ with 600 ml of a 2:1 mixture of ethyl acetate and water. To the com-bined filtrate is added 3 L of saturated aqueous sodium bicarbon-ate solution. A white precipitate forms which is removed by fil-tration. The filtrate, which consists of two layers, is extracted with ethyl acetate (4x300 ml). The combined organic layer is washed with 500 ml saturated aqueous sodium bicarbonate solution and with 500 ml of brine. After drying over sodium sulfate and removal of the drying agent, the filtrate is concentrated to give 91.2 g of a yellowish glass (approx. 50~ yield).
Example 4 (Zinc Copper Couple Reaction in Acetonitrile) To an acetonitrile solution of 5'-blocked cytidine bromoacetate (prepared from 1.75 mole of N-acetylcytidine) is added sodium acetate (100.5 g). The solution is treated at room temperature with zinc-copper couple prepared from zinc dust (251.6 g) and copper sulfate.5H20 (25.3 g). The resulting reac-tion mixture is stirred at room temperature for 12 hours. The reaction mixture is filtered through a Celite pad to remové un-soluble zinc salts and the filter cake washed in portions (1.5 L

total) with acetonitrile to remove traces of occluded product.
The filtrate and washes are combined and drowned into a stirred aqueous solution of sodium carbonate prepared from sodium carbon-ate (371.0 g) and water (5.0 kg). The drowned reaction is stirred overnight. The organic layer is separated and the aqueous layer extracted twice with lL portions of acetonitrile. The original organic layer and the extracts are combined and concentrated to give a green-brown aqueous oil. The oil is dissolved in methylene chloride (2 L) to give a clear organic layer and an aqueous layer.
EDTA tetra sodium salt (50.0 g) is added to the mixture and the mixture stirred until all of the salt dissolved. The stirring is stopped and the organic layer is separated. The organic layer is concentrated to dryness to yield 566 g of a light brown foam. The residue is dissolved in refluxing ethyl acetate (1.5 L) and then cooled to 4C. The crystallized solid is collected by suction filtration and the filter cake washed with cold (0C) ethyl ace-tate (160 ml) to remove residual colour. The filter cake is dried under vacuum to yield 210 g (35.6~) of 1-[5-0-(2-acetoxy,2-methyl propanoyl)-2,3-dideoxy-beta-D-glyceropent-2-enofuranosyl)cytosine as a white crystalline solid with mp 159-162C; nmr (CDC13) 1.53 (d, 6H, geminal CH3), 2.02 (s, 3H, COCH3) 4.22 (dd, lH, J=3.5, 12Hz, C5~H), 4.38 (dd, lH, J=4.5, 12Hz, C5~H], 5.03 (m, lH, C4'-H) 5.95 (m, 2H, aromatic OH and C'-H), 6.18 (m, lH, C3'-H), 7.04 (m, lH, C2'-H) 7.51 (d, lH, J=7.5 Hz, aromatic C-H) 6.5-8.4 (bs, 2H NH2).
Example 5 (Zn-Cu Reaction in tetrahydrofuran)*
To a THF-solution of the 3'-bromo-2'-O-acetate inter-*preferred embodiment mediate (60 kg, containing 8.4 kg intermediate) is added 930 g of sodium acetate. The mixture is cooled to 10C and 7 kg of freshly prepared zinc-copper couple is added under vigorous stirring.
Stirring is continued at 10C until tlc indicates complete conver-sion (usually 1-2 hours). The zinc-copper couple is filtered off and the filtrate passed directly into an excess of saturated aqueous bicarbonate. The white precipitate (bulk of the zinc salts and/or oxides) is filtered off. The two-phase system is extracted twice with ethyl acetate (47 kg and 30 kg). The com-bined organic phase is washed with aqueous EDTA solution (5 kg EDTA in 100 kg water) to remove residual metal ions. The organic phase is concentrated and taken up in anhydrous methanol (30 kg), sodium methoxide (0.3 kg) added and the mixture is stirred under nitrogen protection until tlc indicates complete deprotection.
One obtains a methanolic solution containing approximatey 2 kg (60%) of 2',3'-unsaturated cytidine, ready for hydrogenation.

xample 6 (Conversion of blocked 2'3'-unsaturated intermediate to ddC) A sample of crude, blocked, unsaturated intermediate (66 g, generated from 91 g of crude 3'-brominated compound), obtained by the reductive elimination with the zinc-copper couple in accordance with Example 2, 3, 4 or 5, is dissolved in 450 ml of anhydrous methanol. The solution is treated with 8.5 ml of a 25 of sodium methoxide in methanol and stirred at room temperature until deblocking is complete. The methanolic solution is hydro-genated at 40 psi using 5% Palladium on carbon (approx. 5 g dry weight). The catalyst is removed by filtration through Celite and ~ 7iao~

the filtrate neutralized with IRC-50 (H+) resin. The filtrate is concentrated to near dryness, the residue taken up in water (lO0 ml) and the solution is chromatographed on a column contain-ing Dowex lx2, hydroxide-form. Washing with distilled water elutes the product. The fractions containing ddC are combined and concentrated to dryness. Treatment of the residue with hot methanol followed by cooling, yields 14 g of 2'3'-dideoxycytidine (38~, based on N-acetylcytidine) with physical properties ident-ical to the ones reported in the literature.

Claims (17)

1. A process for preparing 2',3'-dideoxycytidine which comprises subjecting a compound of formula II

wherein X is a halogen atom, R1 and R2 are acyl groups and R3 is a protecting group, to a reductive elimination reaction with a zinc-copper couple to remove the halogen atom X and the acyloxy moiety OR2 to yield the corresponding 2',3'-unsaturated dideoxy compound, removing the groups R1 and R3 and reducing the 2'-double bond, the removal of the groups R1 and R3 and the reduction of the double bond being done in any order.

2. A process according to claim 1 wherein X is a bromine atom, R1 and R2 are acetyl groups and R3 is a group of formula or and the starting material is obtained by reacting N-acetylcytidine with 2-acetoxy isobutyryl bromide.

3. A process according to claim 2 wherein the reaction of N-acetylcytidine with 2-acetoxy isobutyryl bromide is carried out in acetonitrile.

4. A process according to claim 1, 2 or 3 wherein in the reductive elimination the ratio of the zinc-copper couple to substrate is between 2:1 and approximately 50:1.

5. A process according to claim 1, 2 or 3 wherein in the reductive elimination the ratio of the zinc-copper couple to substrate is between 3:1 and apprximately 4:1.

6. A process according to claim 1, 2 or 3 wherein the reductive elimination is carried out in a solvent selected from the group consisting of dimethylformamide, dimethoxyethane, acetonitrile, tetrahydrofuran and mixtures of ethyl acetate and water.

7. A process according to claim 1, 2 or 3 wherein the reductive elimination is carried out in tetrahydrofuran as solvent and in the presence of sodium acetate.

8. A process according to claim 1, 2 or 3 wherein after the reductive elimination the reaction mixture is treated with an excess of sodium bicarbonate to remove organic soluble zinc and copper salts prior to deprotection and reduction.

9. A process according to claim 1, 2 or 3 wherein after the reductive elimination the reaction mixture is washed with a chelating agent to remove organic soluble zinc and copper salts, prior to deprotection and reduction.

10. A process according to claim 1, 2 or 3 wherein deprotec-tion is carried out by reaction with sodium methoxide in methanol, followed by reduction by hydrogenation in the presence of a palladium-on-carbon catalyst.

11. A compound of formula wherein X is a halogen atom and R1 and R2 are both acyl groups.

12. A compound according to claim 11 wherein X is a bromine atom and R1 and R2 are both acetyl groups.

13. A process for the preparation of 2',3'-dideoxycytidine comprising the steps of:
1. reductive elimination of bromoacetate derivative of 4-amino, 5'-hydroxy protected cytidine by treatment with zinc-copper couple to yield the corresponding 2',3'-didehydroderivative, 2. hydrogenating the 2',3'-didehydroderivative to yield 4-amino-5'-hydroxy protected 2',3'-dideoxycytidine, 3. removing the 4-amino and 5'-hydroxy protecting groups to yield 2',3'-dideoxycytidine.

14. A process for the preparation of 2',3'-dideoxycytidine comprising the steps of:
a. protecting the 4-amino group of cytidine with a suitable protecting agent, b. reacting the 4-amino protected cytidine with unsubstituted 2-acetoxy-2-methyl-propanoyl bromide, c. reductively eliminating bromide and an acetoxy group from the bromoacetate derivative by treatment with a zinc-copper couple, d. hydrogenating the 2',3'-didehydroderivative, e. removing 4-amino and 5'-hydroxy protecting groups to yield 2',3'-dideoxycytidine.

15. A compound of the formula:

wherein R1 and R2 are both acyl.

16. A compound of the formula:

\

wherein R1 is acyl.

17. A compound of the formula:

wherein R1 is acyl.