FI92602B - Process for the preparation of -2 ', 3'-dideoxyinosine by enzymatic deamination - Google Patents

Description

92602

Process for the preparation of β-2 ', 3'-dideoxyinosine by enzymatic deamination

The present invention relates to an improved process for the preparation of β-2 *, 3 * -dideoxyinosine.

Typically, 2 ', 3'-dideoxycytidine (ddC) is synthesized from 2'-deoxycytidine (Samukov, VV; Ofitserov, VI Bioorg. Khim. 1983, 9, 132. Prisbe, EJ; Martin, JC Synth. Commun. 1985, 5 15, 401) from. This is a common method for synthesizing 2,31-dideoxynucleosides. However, the starting materials required for this synthesis are very expensive and are not available in bulk. In addition, the reagents required for this deoxygenation are quite expensive.

U.S. Patent 028,817, filed March 20, 1987, discloses a process for producing 2 ', 3'-dideoxynucleosides of the following formula: wherein B is a purine or pyrimidine base and R is H or hydrogen. 25 protecting group, and the process comprises the steps of (a) converting γ-carboxy-t-butyrolactone to 5-O-hydroxy-protecting group-methyl-t-butyrolactone, (b) converting the intermediate from step (a) to 5-O-hydroxy-protecting group-methyl-2 ' , 3'-dideoxypipofuranose, (c) converting intermediate 30 of step (b) to 1'-O-activating group-5-O-hydroxy protecting group-methyl-2 ', 3 * -dideoxypentofuranose, (d) converting intermediate 1 of step (c) -deleting group-5-O-hydroxy protecting group to 2 ', 3'-dideoxypentofuranose, (e) the intermediate of step (d) reacts with an activated purine or pyrimidine base, and (f) recovering the dideoxynucleoside from step (e). The resulting 2,9602 product contains a mixture of B and α anomers which can be separated using chromatography and crystallization methods well known in the art to which this invention pertains.

There is a need in the art to improve the process so that the generally active or more active B-anomer of 2 *, 3'-dideoxynucleosides can be selectively obtained without expensive and time-consuming chromatographic or crystallization separations of B- and α-anomers.

In summary, the present invention comprises a method

selectively producing B-2 ', 3'-dideoxyinosine of formula A

15 ao-v ° J

wherein I is a purine base, hypoxanthine, and wherein 2 ', 3'-dideoxyadenosine is produced, which is then subjected to enzymatic deamination and replacement of the adenosine base with inosine.

The invention is characterized in that it comprises the following steps: a) converting 4-carboxy-4-butyrolactone having

formula B

HOOC

k ° r 30 \ _ / (B)

To a 5-O-hydroxy protecting group-methyl-tert-butyrolactone of formula C

35 92602 3 RO-i o ^ Cr 5 wherein R is a hydroxy protecting group, reacting a compound of formula B with a carboxyl group reducing agent and then reacting the resulting hydroxymethyl group with a hydroxy protecting group reagent;

b) converting the intermediate of step (a) of formula C, 5-O-hydroxy protecting group methyl-2,3-dideoxy-pentofuranose of formula D

R0-I / 0 \ 20 wherein R is a hydroxy protecting group by reacting a compound of formula C with a carbonyl group reducing agent;

c) converting the intermediate of step (b) of formula D to a 1-O-activating group-5-O-hydroxy protecting group-methyl-2,3-dideoxypentofuranose of formula E

25

RO -1 (N

\ Y ~ 0R

Wherein R is a hydroxy protecting group and A is an O-activating group which is an alkylcarbonyl group by reacting a compound of formula D with an acylating agent corresponding to group A above; D) modifying the intermediate from step (c), a compound of formula E, by reacting it with a compound of formula MX 4 92602

with its compound 1-leaving group-5-O-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of formula F

Wherein R is a hydroxy protecting group and X is a leaving group which is Cl or Br, by reacting a compound of formula E with a compound of formula MX wherein M is (CH3) 3Si; e) reacting an intermediate of formula (F) of step (d) with an activated adenine derivative wherein the base, adenine, is activated by reacting the C-6 amino group and the N-9 nitrogen atom of the base of said base with an activating compound which is silylating or an acylating or benzoylating agent in the presence of a Brönsted acid or a Lewis acid and a solvent which is a polar or non-polar solvent; and f) subjecting the intermediate of step (e) to a chemical reaction capable of replacing the 5-O-hydroxy protecting group to give an anomeric mixture of B- and α-2 ', 3'-dideoxyadenosine; «G) contacting a mixture of β- and α-2 ', 3'-dideoxyadenosine from step (f) with an enzyme, adenosine deaminase, to selectively replace the amino group at the 6-position of the purine (adenosine) ring system with B-2 2', 3 in '-dideoxyadenosine with a hydroxyl group, whereby adenosine is converted to inosine; and h) recovering dideoxyinosine from step (g) above.

Scheme I below shows the process of this invention for the preparation of B-2 ', 3'-di-92602 5 deoxyinosine starting from 5-O-hydroxy protecting group methyl-γ-butyrolactone.

Scheme I

5 ,, O ~ ^ 0y «» »* ,, ογγ ° Η i? β, 0 - ^ / ° \ ^ ΟΑί B'SiMt, *

ίο _ \ / -, ΙΟπ / VI

αϊ, ο silyladenine 7? 1.1. Nf 4a> · ^ ~ £> 4b 20? * Apa____ -Töl ~ ^ j · * A 6 3b

ok OH

* 25 NH, i "" OO '·

In Scheme I and Actual Working Examples 30, steps (d) and (e) are conveniently and more preferably performed in one vessel by sequential addition of a halogenating agent and an activated adenine derivative to 1-O-acetyl-5-O-benzyl-2,3-pentofuranose of formula 3.

The starting material γ-carboxy-tert-butyrolactone can be preferably obtained from glutamic acid using standard methods used in the chemical literature (Taniguchi, M .; Koga, K .; Yamada, S. Tetrahedron 1974, 30, 3547).

The combination of chemical reactions produces a suitably capped 2,3-dideoxypentafuranose, 3. This compound and its derivatives are known from the literature.

Thus, the conversion of the starting γ-carboxy-β-butyrolactone to 5-O-hydroxy-protecting group-methyl-butyrolactone, step (a), requires reduction of the β-carboxy group to the hydroxymethyl group as a result of reaction with the hydroxy-protecting group-10 reagent. For example, reduction of the N-carboxy group and protection of the resulting N-hydroxymethyl group with a benzyl group (PhCH2-) or a benzoyl group [PhC (O) -], step (a), was performed by sequential reaction of the starting material of formula I with BH3SMe2, PhCH2Br3.4. or with PhC (O) Cl 15. The use of a benzoyl group is more advantageous than the use of a benzyl group because the removal of the next benzoyl protecting group has been found to be easier and thus better product yields are obtained.

The primary alcohol functional group may be protected as an ether, such as trialkyl or dialkylaryl or diarylalkyl or triarylsilyl, unsubstituted or substituted benzyl, unsubstituted or substituted alkyl or allyl ether, or as an ester, such as benzoyl, pivaloyl, mesitoyl -25 as an unsubstituted or substituted acetyl or carbonate thiester [See "Protective Groups in Organic Synthesis," TW Greene, John Wiley, New York (1981) for a more detailed presentation of protecting groups and their chemistry]. In an even more preferred embodiment, a 5-benzoyl group was used as the hydroxy protecting group for the primary alcohol because, as described above, the subsequent removal of the benzoyl protecting group has been found to be easier and results in a higher product yield. The 5-O-benzoyl protecting group can be easily replaced by hydrolysis under basic conditions, for example by treatment with methanol saturated with ammonia.

Conversion of the 5-O-hydroxy protecting group-butyrolactone to the 5-O-hydroxy protecting group-2,3-dideoxypentafuranose of formula 2 in step (b) was accomplished by reacting an intermediate of formula 1 with NaH and HCO 2 Et: and then HCl (Spry, DV; Bhala, AR Heterocycles, 1985, 23, 1901).

It should be apparent to those skilled in the art that any of the reducing agents may be used in performing either or both of steps (a) and (b) independently (i.e., sequentially) or simultaneously. Other useful reducing agents in addition to BH 3 * SMe 2 for use in the second or both steps (a) and (b) as described above are [(CH 3) 2 CHCH (CH 3) 2] BH (diamylamiborane), NaBH 4 and LiCl or AlCl 3 or BH3, LiAlH4, LiAlH (OMe) 3, LiAlH (Ot-Bu) 3 and the like. It is particularly preferred to use disiamylborane in step (b) to reduce the carbonyl group of the lactone ring.

Step c, which converts an intermediate of formula 2 to an intermediate of formula 3 and has an O-activating group at the C (1) position in the 2,3-dideoxypentafuranose ring systems, is performed with any reagent effective to convert this 1- a hydroxy group which can be easily replaced by Cl or Brr by reaction with HCl or HBr. Such groups which can be easily substituted include O-activation groups which are alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups in which the alkyl moiety may be unsubstituted or unsubstituted. The β-alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group, and the substituent on the alkyl or aryl moiety may have 1 to 3 groups selected from halogen or C 1-3 alkoxy groups. Even more preferably, this activating group is the corresponding (with the above) acetoxy or benzyloxy group and most preferably acetoxy. Step (c) was performed well using acetic anhydride / pyridine reagent.

Step (d), replacing the 1-O-activating group-5 functional group with a leaving group which is Cl or Br, can be performed by treatment with 3 HCl, HBr or halotrialkylsilane of formula R 3 SiX, wherein R is alkyl and X is halogen, more preferably (CH3) 3SiBr in CH2Cl2 at low temperature to give Furano-10 silyl halides present in solution as a mixture of anomers (a and B).

In step (e) of the preparation process of this invention, the intermediate of formula F from step (d) may react with an appropriately activated adenosine base, such adenosine base being activated by reaction with well known activators to give a silylated, acetylated or benzoylated base, a suitable polar or in the presence of a non-polar solvent and optionally in the presence of a Lewis acid such as, for example, boron halide, aluminum halide, titanium halide, stannous chloride, zinc halide, trimethylsilyl bromide, iodide, triflate or any other well known species , which is used in glycosylation reactions, or in the presence of a Bronsted acid such as hydrochloride, 2'5-bromide or iodide. Particularly useful in this step (e) is the use of silylated adenosine in the presence of non-polar solvents such as benzene, toluene, chloroform, dichloromethane, dichloroethane or carbon tetrachloride. Examples of useful polar solvents include, for example, tetrahydrofuran and dioxane, as well as nitriles such as acetonitrile, dimethylformamide and dimethyl sulfoxide. An example of a useful process for performing step (e) is disclosed in Brundidge et al., U.S. Patent 4,625,020, which discloses the incorporation of silylated pyrimidines by trapping active hydrogens of hydroxy and amino groups with silyl groups such as trimethylsilyl using 2-deoxy- 2-fluoriarabinofuranosyylihalogenidia.

Alternatively, in another embodiment of this invention, when the group "A" from step (c) is a gun-5 style, the intermediate of step (c) may be reacted with an activated adenine derivative in the presence of a Lewis acid as in step (e) to give 5-O-hydroxy -protecting the group-2 ', 3'-dideoxyadenosine intermediate without a separate step (d).

As mentioned above, in yet another more preferred embodiment of the present invention, the compound of formula 3 1-O-acetyl-5-O-benzoyl-2,3-pentofuranose from step (c) was first combined with bromotrimethylsilane and then with bis-silyladenine to give one. In step -15 without separation of the 1-bromo-5-O-benzoyl-2,3-pentofuranose intermediate from step (d) 5'-O-benzoyl-2 ', 3'-dideoxyadenosine as a product from the combined steps (d) and (e) ).

In the process of the present invention, in step (f), 5'-O-benzoyl-2 ', 3'-dideoxyadenosine was chemically reacted to remove the 5-O-protecting group. Suitable methods for removing this protecting group are well known in the art of the present invention [see for example: "Protective Groups in Organic Synthesis", T.W.

: 25 Greene, John Wiley, New York, (1981), cited above]. When the protecting group is a benzyl group, it can be easily removed catalytically by hydrogenation (H2 / PdCl2). Even more preferably, 5'-O-benzoyl-2 ', 3'-dideoxyadenosine was treated with methanol saturated with ammonia to give 2', 3'-dideoxyadenosine as a mixture of α- and β-anomers.

In the process of this invention, in step (g), a mixture of β- and α-2 ', 3'-dideoxyadenosines from step (f) was combined with the enzyme, adenosine deaminase (ADA) isolated from calf spleen, in a neutral aqueous medium (Secrist , JA et al., J. Med. Chem. 1987, 30, 746). This enzyme selectively catalyzes the deamination of B-2 ', 3'-dideoxyadenosine to quantitatively yield B-2', 3'-dideoxyinosine. Although calf spleen adenosine deaminase (ADA) was used in the present 5 example, all adenosine aminohydrolase preparations ("deaminase", EC 3.5.4.4) are believed to be suitable. Thus, it is within the scope of this invention to use any adenosine aminohydrolase preparations (or "deaminase preparations") that are capable of selectively deaminating the B-2 ', 3'-dideoxyadenosine anomer, thus, in addition to using the free enzyme in a suitable medium such as the inert aqueous mixture disclosed herein. in the medium, an enzyme, ADA, bound to a suitable carrier, such as, for example, the oxirane-acrylic polymer carrier Eupergit c (Rohm Pharma Gmb), can be used, and ADA can be bound to the polymer using conventional methods.

The 2 ', 3'-dideoxyinosine product was preferably recovered by collecting the reaction mixture from step (g), removing insoluble reagents and additives by filtration through Celite and purifying the resulting product by column chromatography, for example on a silica gel column, eluting with 1-5% chloroform in methanol.

The use of an enzyme, adenosine deaminase, according to the method of the present invention, has the advantage that the resulting anomers (a and B) do not need to be separated using expensive and time consuming chromatographic and crystallization methods hitherto well known in the art.

The B-2 ', 3'-dideoxyinosine anomer is shown because it is the active anomer or is at least more active as an antiviral agent than the α-anomer.

The following examples, in which the compounds are numbered, referring to Reaction Scheme I, illustrate a few representative embodiments of the present invention 92602 11 and are not intended to limit the scope of the invention. All parts and percentages are by weight and temperatures are in degrees Celsius unless otherwise indicated.

Example 1 (D) -5-O-Benzyl-2,3-dimethylpentofuranose (2) 200 ml of a 0.5 M solution of 0.5 M disiamylborane in THF at 0 ° C was added dropwise under nitrogen to a solution of 155.1 g (0.069 mol) of (D) -5'-benzoyloxy-5-hydroxymethyl-10-butyrolactone in 50 ml of dry tetrahydrofuran. After 20 minutes at 0 ° C, the reaction mixture was heated to 22 ° C. The reaction mixture was stirred at 22 ° C for 16 hours, then 12 mL of water was added slowly and heated to reflux for 30 minutes.

The reaction mixture was cooled to 0 ° C and then 24 mL of 30% hydrogen peroxide was added slowly, maintaining the pH between 7-8 with 1N NaOH. After the addition, the reaction mixture was evaporated under reduced pressure on a rotary evaporator at 30 ° C to give an oily residue. This was partitioned between 500 ml of dichloro-20 methane and 150 ml of water. The aqueous layer was extracted with 2 x 100 mL of dichloromethane and then the combined organic phases were washed with 50 mL of water, dried over anhydrous sodium sulfate and evaporated to an oil. Yield 15.3 g (100%) of 5-O-benzoyl-2,3-dideoxypentofuranose. 1 H-NMR was consistent with structure and the oil was used directly in the next step.

Example 2 1-O-Acetyl-5-O-benzoyl-2,3-dideoxypentofuranose (3) A mixture of 5-O-benzoyl-2,3-dideoxypentofuranose (15.3 g, 0.069 mol) ) In 32 ml of pyridine and 16 ml of acetic anhydride, stirred at 22 ° C for 4 hours and then diluted with 500 ml of dichloromethane and 100 g of ice were added. The reaction mixture was then washed with 3 x 100 mL of 1N hydrochloric acid, 3 x 100 mL of saturated aqueous sodium bicarbonate and 100 mL of saturated brine. The organic layer was dried over sodium sulfate and evaporated to give a pale yellow oil. This could be used as such or chromatographed on silica gel with 35-60% EtOAc / hexane-5a. Yield 15.9 g (87%) of 1-O-acetyl-5-O-benzoyl-2,3-dideoxypentofuranose. Its NMR was consistent with structure.

Example 3 5'-O-Benzoyl-21,31-dideoxyadenosine (4) 1-O-acetyl-5-O-benzoyl-2,3-pentofuranose 10 (0.522 g, 0.00198 mol) was dissolved in 5 ml of 1 , 2-dichloroethane. To this was added 276 μΐ (1.2 eq.) Of trimethylsilyl bromide. This was stirred at 22 ° C for 15 minutes before 11.9 ml of a solution of 0.2M bis-silyladenine in 1,2-dichloroethane was added. The solution was stirred for 88 15 hours at 22 ° C. The reaction was then quenched by cooling to 0 ° C, and then the mixture was poured into 40 ml of cold saturated aqueous sodium bicarbonate solution. The reaction mixture was partitioned between 200 mL of dichloromethane and 2 x 40 mL of cold saturated aqueous sodium bicarbonate solution and 40 mL of saturated brine. The organic layer was dried and evaporated to give a colorless oil which was chromatographed on silica gel eluting with 8% methanol in methylene chloride. The fractions were collected to give 420 mg of 5'-0-25 benzoyl-2 ', 3'-dideoxyadenosine as a mixture of anomers (63%).

Example 4 2 ', 3 * -Dideoxyinosine (6) This mixture of anomers (1.66 g) was treated with 30 ml of methanol saturated with ammonia. This was sealed and stirred at 18 ° C for 48 hours.

TLC showed the reaction to be complete. The solvent was evaporated and replaced with 170 ml of fresh methanolic ammonia solution. After 48 hours, TLC again showed the reaction to be complete. Thus, the solvent was evaporated and ethanol (5 ml) was added. There was thus obtained colorless crystals, which were collected and washed with 5 ml of 95% ethanol to give 1.20 g (95%) of 2 ', 3'-dideoxyadenosine 5a together with its α-anomer 5b. 1: 1 by 1 H-NMR. The mixture of α + 8-2 ', 3'-dideoxyadenosine-5 was dissolved in 50 ml of deionized water, and to this solution was added 10 mg of adenosine deaminase (type II, Sigma; 9 units / mg → 9 pmol / min). The solution was stirred at 20 ° C and the reaction was monitored by HPLC. After 2 hours 30 minutes, another 10 mg of adenosine deamin-10 nase was added. HPLC showed that the reaction was almost complete after 3 hours. The reaction mixture was then concentrated (1-2 mL) at 35 ° C to give an oil. The oil was collected and slowly diluted using 2 x 1.5 mL portions of methanol to give white crystals. After 15 minutes, the material was filtered and the colorless crystals were washed with 2 x 1.5 mL portions of methanol to give 2 ', 3'-dideoxyinosine (120 mg, 48%). The mother liquor was treated as above to give an additional 29 mg (12%) of good quality product which was characterized using 1 H-NMR. The NMR spectrum was consistent with structure.

Claims (11)

92602 14 A process for the selective production of β-2 ', 3'-dideoxyinosine of the formula A 5 NN (A) HOCH, O O, characterized in that it comprises the following steps: a) modification of the β-carboxy- n-butyrolactone of formula B HOOC 20 / (B) Process according to Claim 1, characterized in that the reducing agent used in steps (a) and (b) is BH3 (B2H6), BH3SMe2, [(CH3CHCH (CH3)] 2BH, NaBH4, NaBH4 and either LiCl or AlCl3 or BF3, LiAlH4 , LiAlH- (0Me) 3 25 or LiAlH (OBu-t) 3 Process according to Claim 1, characterized in that the reducing agent used in step (b) is [(CH 3) 2 CHCH (CH 3)] 2BH Process according to Claim 1, characterized in that the hydroxy-protecting group used in step (a) is a benzoyl group. Process according to Claim 1, characterized in that the intermediate of step (c) reacts with (CH 3) 3 SiBr, which then acts as a compound of the formula MX. 92602 17 To a 5-O-hydroxy-protecting group-methyl-N-butyrolactone of the formula C25 -R-xyr 30 wherein R is a hydroxy-protecting group by reacting a compound of formula B with a carboxyl group reducing agent and then reacting the obtained hydroxymethyl group with a hydroxy-protecting group reagent; b) converting an intermediate of step (a) of formula 35 into a C, 5-O-hydroxy protecting group methyl-2,3-dideoxy-pentofuranose of formula D 92602 15 R0-I / O-O-OH wherein R is a hydroxy protecting group, by reacting a compound of formula C with a carbonyl group reducing agent; c) converting the intermediate of step (b) of formula D, 1-O-activating group-5-O-hydroxy protecting group-methyl-2,3-dideoxypentofuranose of formula E RO -1 Q Ύ y ^ 0A Wherein N is a hydroxy-protecting group and A is an O-activating group which is an alkylcarbonyl group by reacting a compound of formula D with an acylating agent corresponding to group A above; d) converting the intermediate from step (c), a compound of formula E, by reacting it with a compound of formula MX to give a 1-leaving group-5-O-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose having the formula Wherein R is a hydroxy-protecting group and X is a leaving group which is Cl or Br, by reacting a compound of formula E with a compound of formula MX wherein M is 35 (CH 3) 3 Si; 92602 16 e) reacting an intermediate of formula F of step (d) with an activated adenine derivative wherein the base, adenine, is activated by reacting the C-6 amino group and the N-9 nitrogen atom of the base of said base with an activating compound which is silylating or an acylating or benzoylating agent in the presence of a Brönsted acid or a Lewis acid and a solvent which is a polar or non-polar solvent; and f) subjecting the intermediate of step (e) to a chemical reaction capable of replacing the 5-O-hydroxy protecting group to give an anomeric mixture of β- and α-2 ', 3'-dideoxyadenosine; g) contacting a mixture of β- and α-2 ', 3'-dideoxyadenosine from step (f) with an enzyme, adenosine deaminase, to selectively replace the amino group at the 6-position of the adenosine ring system with B-2', 3'-di- in deoxyadenosine with a hydroxyl group, whereby adenosine is converted to inosine; and h) recovering dideoxyinosine from step (g) above. Process according to Claim 1, characterized in that in step (e) the adenosine base compound, activated by reaction with a halotrialkylsilane acting as a silylating agent in which the halo is bromine 5 or iodine, reacts with a 1-leaving group-5-O-protecting group-2 With a ', 3'-dideoxypentafuranose intermediate of formula F from step (d) to give a 5'-O-hydroxy protecting group-2', 3 ', - dideoxyadenosine intermediate. Process according to Claim 6, characterized in that reaction step (e) is carried out in a non-polar solvent which is CHCl 3, CH 2 Cl 2, C 1 CH 2 -CH 2 Cl or CCl 4. Process according to Claim 1, characterized in that the reactions in steps (d) and (e) are carried out in a single vessel by successively adding a halogenating agent and an activated adenosine derivative to the intermediate of step (c). Process according to Claim 8, characterized in that the halogenating agent is bromotrimethylsilane and the activated adenosine derivative is bis-silyladenine. Process according to Claim 1, characterized in that in step (f) the intermediate according to step (e), in which the protecting group is benzoyl, is reacted with methanol saturated with ammonia, the 5-O-hydroxy protecting group being replaced. Process according to Claim 1, characterized in that the enzyme, adenosine deaminase, is used in step (g) as a solution in a neutral aqueous medium or as a bound enzyme preparation. 92602 18