CA1335187C - Dideoxyinosine by enzymatic deamination of dideoxyadenosine - Google Patents
Dideoxyinosine by enzymatic deamination of dideoxyadenosineInfo
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Abstract
There is disclosed a novel process to selectively produce .beta.-2',3'-dideoxyinosine in high yields.
.beta.-2',3'-Dideoxyinosine so produced is useful as an antiviral and antibiotic agent.
.beta.-2',3'-Dideoxyinosine so produced is useful as an antiviral and antibiotic agent.
Description
DIDEOXYINOSINE BY ENZYMATIC DEAMINATION OF DIDEOXYADENOSINE
BACKGROUND OF THE INVENTION
Field of the Invention This invention relates to an improved process to produce B-2',3'-dideoxyinosine.
Background - ~elated References Typically, 2',3'-dideoxycytidine (ddC) is synthesized from 2'-deoxycytidine1'2. This is a general method for the synthesis of 2,3'-dideoxynucleosides. The starting materials for this synthesis are, however, extremely expensive and not available in bulk. The reagents required for this deoxygenation, furthermore, are also quite costly.
There is a process for producing 2',3'-dideoxynucleosides represented by the formula RO B
~5 ~
wherein B is a purine or pyrimidine base and R is ~ or a ~~
" .
~ 13~5187 hydroxy-protecting group by the steps of (a) converting a r-carboxy- ~ butyrolactone to a 5-0-hydroxy-protecting group-methyl-r-butyrolactone, (b) converting the intermediate from step (a) to the 5-0-hydroxy-protecting group-methyl-2',3'-dideoxypentofuranose, (c) converting the intermediate from step (b) to the 1-0-activating group -~-0-hydroxy-protecting group-methyl-2',3'-dideoxypento-furanose, (d) converting the intermediate from step (c) to the 1-leaving group-S-0-hydroxy-protecting group-2,'3'-dideoxypentofuranose, (e) reacting the intermediate from step (d) with an activated purine or pyrimidine base, dna (f) recovering the dideoxynucleoside from step (e). The resulting product comprises a mixture of B-and a-anomers which may be separated using chromatographic and crystallization techniques well-known in the field to which this invention relates.
There remains in the field a need for process improvements whereby the generally active, or more active, ~-anomer of 2',3'-dideoxynucleosides can be selectively obtained without the costly and time-consuming chromatographic or crystallization separation of B- and a-anomers.
SUMMARY OF THE INVENT~ON
In summary, this invention comprises a process for selectively producing B-2',3'-dideoxyinosine represented by the formula HO
~
Formula A
wherein I is the purine base, inosine, and wherein there is produced 2~,3~-dideoxyadenosine which is then subjected to enzymatic deamination and replacement of the adenosine base with inosine.
DETAILED DESCRIPTION OF THE I~ NTION
The present invention is a process for selectively producing B-2',3'-dideoxyinosine represented by the formula -~ Formula A
wherein I is the purine base, inosine, which comprises the steps of:
(a) converting a r'-carboxy-~butyrolactone of the formula HOOC
~0 Formula B
to a 5-O-hydroxy-protecting group-methyl-r-butyrolactone of the formula RO ~ o Formula C
wherein R is a hydroxy-protecting group;
(b) converting the intermediate from step (a~, Formula C, to the 5-O-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula RO
Formula D
wherein R is a hydroxy-protecting group;
(c) converting the intermediate from step (b), Formula ~, to the 1-O-activating-group-5-O-hydroxy-protecting 1 33~1 87 group-methyl-2,3-dideoxypentofuranose of the formula RO----~
~ OA
Formula E
wherein R is a hydroxy-protecting group and A is an O-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and Cl-C3 alkoxy groups;
(d) converting the intermediate from step (c), Formula E, by reaction with a compound of the formula MX to the 1-leaving-group-5-O-hydroxy-protecting group-methyl-2,3-di-deoxypentofuranose of the formula RO
~ X
Formula F
wherein R is a hydroxy-protecting group and X is a leaving group selected from Cl and Br and M is selected from H and (CH3)3Si;
(e) reacting the intermediate from step (d), Formula F, with an activated adenine derivative wherein the base, adenine, has been activated by means of reacting the pendant N-G
amino group and N-9 Nitrogen atom on the nucleus of adenine with an activating compound selected from silylating and acetylating and benzoylating agents, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of a solvent selected from polar and non-polar solvents;
(f) subjecting the intermediate from step (e) to chemical reaction effective to displace the 5-0-hydroxy-protecting group to obtain an anomeric mixture of ~- and ~-2',3'-dideoxyadenosine;
(g) contacting the mixture of ~- and ~-2',3'-dideoxyadenosine from step (f) with the enzyme, adenosine deaminase, especially preferably in a neutral aqueous medium, to effect displacement of the amino group pendant to the 6-position of the purine (adenine) ring system selectively in the ~-2',3'-dideoxyadenosine with a hydroxyl group whereby adenosine is converted to inosine; and (h) recovering the ~-2',3'-dideoxyinosine from step (e) above.
~, ~3 - 13~187 The process for producing B-2',3'-dideoxyinosine according to this invention starting from the 5-0-hydroxy-protecting group-methyl-r-butyrolactone,is outlined in Scheme I below.
Scheme I
R, e~ H ~~ OH
8 2 0~0 OAc 8 r Si M~
Ac~O silyl. ~denine B20~J
4 a j~ 4 b 2 5 HO~y 5a ~D~
H0y~ H~
5b OH
A = ~) I = N~NNl\>
- -- 1335l87 As is illustrated in Scheme I and in the actual working examples which follow, steps (d) and (e) are conveniently and more preferably carried-out in one pot by the sequential addition of a halogenating agent and an activated adenine derivative to 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 3.
The starting material, y-carboxy-~ butyrolactone may be conveniently obtained from glutamic acid by utilizing conventional techniques reported in the chemical literature3.
A combination of chemical reactions produce a suitably blocked 2,3-dideoxypentafuranose, 3. This compound and its derivatives are known in the literature.
Thus, the conversion of the starting material, r-carboxy-r-butyrolactone, to a 5-0-hydroxy-protecting group-methyl- butyrolactone, step (a), involves the reduction of the -carboxy group to a hydroxymethyl group followed by reaction with a hydroxy-protecting group reagent. For example, the reduction of the r-carboxy group and protection of the resulting r-hydroxymethyl group by means of the benzyl group (PhCH2-) or the benzoyl group (PhC(O)-), step (a), was acheived by the successive reaction of the starting compound of Formula 1 with BH3 SMe2 and - 13~5187 PhCH2Br3'4, or PhC(O)Cl. The use of the benzoyl group is preferred over the use of the benzyl group because the subsequent removal of the benzoyl protecting group has been found to proceed more readily and to provide higher yield of product.
The primary alcohol functional group may be protected as an ether, such as a trialkyl or dialkyl aryl or diaryl alkyl or triaryl silyl, unsubstituted and substituted benzyl, unsubstituted and substituted alkyl, or allyl ether, or as an ester, such as a benzoyl, mesitoyl, pivaloyl, unsubstituted or substituted acetyl, or a carbonate ester.
See "Protective Groups in Organic Synthesis," T. W. Greene, John Wiley, New York 1981 for more detailed description of protecting groups and the chemistry relating to the same.
In a more preferred embodiment, there was used as the hydroxy-protecting group for the primary alcohol group at the 5-position the benzoyl group because, as mentioned above, the subsequent removal of the benzoyl protecting group has been found to proceed more readily and to provide higher yield of product. The 5-0-benzoyl protecting group may be conveniently displaced by hydrolysis under basic conditions, for example, by treatment with methanol saturated with ammonia.
The conversion of the 5-0-hydroxy-protecting group- r-butyrGlactone, to the 5-0-hydroxy-protecting g group-2,3-dideoxypentafuranose having Formula 2 in step (b) was achieved by reacting the intermediate having Formula 1 with NaH and HCO2Et followed by HCl4.
It will be understood by those skilled in the art to which this invention relates that any one of a number of reducing agents may be used to carry out either or both of steps (a) and (b) independently (i.e., in succession) or at the same time. Other useful reducing agents besides BH3~SMe2 used in either or both of steps (a) and (b) as described above include [(CH3)2CHCH(CH3)2]BH
(disiamylborane), NaBH4 plus LiCl or AlCl3 or BF3, LiAlH4, LiAlH(OMe)3, LiAlH(O-t-Bu)3, and the like. The use of disiamylborane for the step (b) reduction of the lactone ring carbonyl group is especially preferred.
The step (c) conversion of the intermediate having Formula 2 to the intermediate having Formula 3 bearing an O-activating group at the C(l) position of the 2,3-dideoxypentafuranose ring system may be achieved by any reagent effective to convert this l-hydroxy group to a group which may be displaced readily by Cl or Br upon reaction with HCl or B r. Such a group which may be so displaced readily includes O-activating groups selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from l to 3 groups selected from halo and C1-C3 alkoxy groups. More preferably, this activating group is selected from corresponding (to the above) acetoxy and benzoyloxy groups and, most preferably, acetoxy. Step (c) was conveniently carried-out using acetic anhydride/pyridine reagent.
The step (d) displacement of the l-O-activating group functional group with a leaving group selected from Cl and Br may be achieved by treatment of 3 with one of HCl, HBr, and a halotrialkylsilane having the formula R3SiX where R is alkyl and X is halo, more preferably (CH3)3SiBr, in CH2Cl2 at low temperature to give the furanosyl halides, which are present in solution as a mixture of anomers (~ and ~).
In step (e) of the process according to this invention, the intermediate having Formula F from step (d) above may be reacted with a suitably activated adenosine base, wherein such adenosine base has been activated by reaction with well-known activating reagents to obtain a silylated, acetylated or benzoylated base, in the pr ~
. .
suitable polar or non-polar solvent and, optionally, in the presence of a Lewis acid such as, for example, boron halides, aluminum halides, titanium halides, tin (IV) S chloride, zinc halides, trimethylsilyl bromide, iodide, triflate and any other species well-known to be used in glycosylation reactions or in the presence of a Bronsted acid such as hydrogen chloride. bromide or iodide.
Especially useful in this step (e) is the use of silylated adenosine in the presence of non-polar solvents such as, for example, benzene, toluene, chloroform, dichloromethane, dichloroethane and carbon tetrachloride. Examples of useful polar solvents include others such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, dimethylformamide, and dimethylsulfoxide. An example of a useful procedure to carry out step (e) is disclosed in Brundidge et al., U.S.
Patent Patent 4,625,020 which discloses the coupling of silylated pyrimidines, wherein active hydrogens of hydroxy and amino groups are blocked by silyl groups such as the trimethylsilyl group, with a 2-deoxy-2-fluoroarabino-furanosyl halide.
Alternatively, in another embodiment according to this invention, when from step (c) the group "A" is acetyl, the intermediate from step (c) may be reacted with an activated adenine derivative in the presence of a Lewis acid as in 1335~87 step (e) so as to obtain the 5-0-hydroxy-protecting group-2',3'-dideoxyadenosine intermediate without proceeding through a separate step (d).
As mentioned above, in still another, more preferred embodiment according to this invention, the 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 3 from step (c) was contacted first with bromotrimethylsilane followed by a bis-silyl adenine to obtain in a single step, without isolation of the 1-bromo-5-0-benzoyl-2,3-pento-furanose intermediate as from step (d), to obtain 5'-0-benzoyl-2',3'-dideoxyadenosine as the product from the combined steps (d) and (e).
In step (f) of the process according to the present invention, the 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to chemical reaction to remove the 5-O-protecting group. Suitable procedures to remove this protecting group are well-known in the field to which this invention relates and examples are disclosed in "Protective Groups in Organic Synthesis" by T. W. Greene, John Wiley, New York, 1981 mentioned above. When the protecting group is the benzyl group, it can be easily removed by catalytic hydrogenation (H2/PdCl2). More preferably, 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to methanol saturated with ammonia to obtain 2',3'-dideoxyadenosine as a mixture of a- and B-anomers.
In step (g) of the process according to this invention, the mixture of ~- and ~-2',3'-dideoxyadenosine from step (f) was contacted with the enzyme, adenosine deaminase (ADA) 5, isolated from calf spleen, in a neutral aqueous medium. This enzyme selectively catalyzes the deamination of~-2'-3'-dideoxyadenosine to yield quantitatively ~-2',-3'-dideoxyinosine. Although adenosine deaminase (ADA) from calf spleen was used in the actual examples, it is believed that any preparation of adenosine aminohydrolase ("deaminase," EC 3.5.4.4) would be suitable.
Thus, the use of any preparation of adenosine aminohydrolase (or "deA~;n~e") effective to selectively deaminate the ~-2',3'-dideoxyadenosine anomer is within the scope of the process claimed as the present invention. Accordingly, in addition to the use of the free enzyme in an appropriate medium, such as in a neutral aqueous medium described herein, there may be used the enzyme, ADA, immobilized on an appropriate compatible substrate such as, for example, the oxirane-acrylic polymer substrate Eupergit C TM (Rohm Pharma Gmb). The ADA may be bound to the polymer using conventional techniques.
The 2',3'-dideoxyinosine product, was conveniently recovered by collecting the reaction mixture from step (e) or from the additional step wherein the 5-O-protecting group was removed, removing insoluble reactants and additives by filtration through Celite*, and purifying the resulting product by column chromatography, for example on a silica gel column using a mixture of about 1-5% methanol in chloroform as eluent.
* Trade-mark -14-.~ ~.
, 133~187 The use of the enzyme, adenosine deaminase, according to the process of the present invention affords the advantage whereby the resulting anomers (~ and a) need not be separated using costly and time-consuming chromatographic and crystallization techniques that heretofore have been well-known in the field to which this invention relates.
The ~-2',3'-dideoxyinosine anomer is desired because it is the active anomer or is, at least, substantially more active as an antiviral agent than is the a-anomer.
The following examples, wherein the compounds are numbered with reference to Scheme I, illustrate but a few representative embodiments of the process according to this invention and are not to be construed as limiting in scope.
All parts and percentages are by weight and temperatures are in degrees Celsius unless otherwise specified.
EXPERIMENTAL
Example 1:
(D)-5-O-Benzyl-2,3-dideoxypentofuranose. 2. To 200 mL of a 0.5 M solution of disiamylborane in THF solution at 0C was added dropwise under nitrogen a solution of 15.1 g (0.069 mole) of (D)-5'-benzoyloxy-5-hydroxymethylbutyrolactone, 1, in 50 mL of dry tetrahydrofuran. After 20 min at 0C the 1~35187 reaction was warmed to 22C. The reaction was stirred at 22C for 16 hrs, then worked up by slowly adding 12 mL of water and refluxing for 30 min. The reaction mixture was cooled to 0C followed by the slow addition of 24 mL of 30%
hydrogen peroxide maintaining the pH between 7-8 with lN
sodium hydroxide. After the addition the reaction was evaporated under reduced pressure on a rotovapor at 30C to an oily residue. This was partitioned between 500 mL
dichloromethane and 150 mL water. The aqueous layer was extracted wtih 2X 100 mL dichloromethane and then the combined organic layers were washed with 50 mL water, dried over anhydrous sodium sulfate and evaporated to an oil.
Yield = 15.3 g (100%) of 5-0-benzoyl-2,3-dideoxypento-furanose. The lH-NMR was consistent for structure and the oil was used directly in the next step.
Example 2:
1-0-Acetyl-5-0-benzoyl-2,3-dideoxypentofuranose. 3. A
solution of 5-0-benzoyl-2,3-dideoxypentofuranose (15.3 g, 0.069 mole) in 32 mL of pyridine and 16 mL of acetic anhydride was stirred at 22C for 4 hrs then diluted with 500 mL dichloromethane and 100 g of ice was added. The reaction mixture was then washed with 3 X 100 mL lN
hydrochloric acid 3 X 100 mL saturated aqueous sodium bicarbonate and 100 mL of brine. The organic layer was 133~187 dried over sodium sulfate and evaporated to yield a pale yellow oil. This could be used as is or chromatographed on silica gel 35% ~ 60% EtOAc/hexane. Yield: 15.9 g (87%) of 1-0-acetyl-5-0-benzoyl-2,3-dideoxy-pentofuranose. Its NMR was consistent for structure.
Example 3:
5'-O-benzoyl-2',3-dideoxyadenosine. 4.
1-0-acetyl-5-0-benzoyl-2,3-pentofuranose (0.522 g, 0.00198 mole) was dissolved in S mL of 1,2-dichloroethane. To this was added 276 ~L (1.2 eq) of trimethylsilylbromide. This was stirred at 22C for 15 min prior to the addition of 11.9 mL of a 0.2 M solution of bis-silyladenine in 1,2-dichloroethane. The solution was stirred for 88 hrs at 22C. The reaction was then worked up by cooling to 0C, followed by pouring into 40 mL of cold saturated aqueous sodium bicarbonate. The reaction mixture was partitioned between 200 mL dichloromethane and 2 X 40 mL cold saturated aqueous sodium bicarbonate and 40 mL brine. The organic layer was dried and evaporated to yield a colorless oil that was chromatographed on silica gel and eluted with 8%
methanol in methylene chloride. Fractions were collected and similar ones pooled to yield 420 mg 5'-0-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 170 mL of methanol saturated with ammonia.
This was tightly stoppered and stirred at 18C for 48 hrs.
TLC indicated that reaction was incomplete. The solvent was evaporated and replaced with 170 mL of fresh ammonia solution in methanol. TLC after an additional 48 hr implied the reaction was complete. Hence, the solvent was evaporated and ethanol was added (5 mL). This yielded colorless crystals which were collected and washed with (5 mL) of 95% ethanol to yield 1.20 g (95%) of 2',3'-dideoxyadenosine 5a together with its a anomer 5b in a 1:1 ratio by lH-NMR. The a+~ 2',3'-dideoxyadenosine mixture was dissolved in S0 mL of deionized water and to this solution was added 10 mg of adenosine deaminase (type II, from Sigma; 9 units/mg ~ .9 ~mole/min). The solution was stirred at 20C and reaction monitored by HPLC. After 2 hrs 30 min another 10 mg of adenosine de~m;n~se was added.
HPLC showed the reaction was nearly complete after 3 hrs.
The reaction was then concentrated (1-2 mL) at 35C to give an oil. The oil was scratched and slowly diluted with 2 X
1.5 mL portions of methanol which produced white crystals.
After 15 min the material was filtered and the colorless crystals washed with 2 X 1.5 mL portions of methanol to yield 2',3'-dideoxyinosine (120 mg, 48%). The mother liquor was treated as above to yield an additional 29 mg (12%) of good quality product, which was characterized by lH-NMR.
The nuclear magnetic resonance spectrum was consistent with the structure.
SUPPT-l~M~NTARY DISCLOSIJRE
The currently preferred embodiment of the invention comprises an efficient process for selectively producing ~ -(D)-2',3'-dideoxyinosine represented by the formula H O~<oy Formula A
wherein I is the purine base, inosine. The process involves producing an C~ anomeric mixture of (D)-2',3'-dideoxyadenosine, which is then subjected to selective enzymatic deAm;nAtion converting the adenosine base to inosine for only the ~3 -(D)-isomer which is readily isolable and easily purified by simple recrystallization.
19 _ ~T~ILED DESCRIPTION OF THE PRESENTLY
The present invention is an improved process for selectively producing ~-(D)-2'-3'-dideoxyinosine represented by the formula H ~ o I
Farmula R
wherein I is the purine base, inosine, which comprises the steps of:
1. preparing an anomeric mixture of a-and ~-(D)-2', 3'-dideoxyadenosine;
2. selectively deaminating the ~-~D)-2',3'-dideoxy-adenosine anomer by contact of the anomeric mixture of step 1. with the enzyme, adenosine deaminase; and 3. recovering the ~-(D)-2',3'-dideoxyinosine produced in step 2.
A typical process for producing the anomeric mixture used in step 1. comprises the steps of:
(a) converting (D)- ~-carboxy-~-butyrolactone of the formula HOOC
~ a Formula 1 - 2~ -.,~,.,~
~ CT-1907A
. 133~187 to a 5-0-hydroxy-protecting group-methyl-~-butyrolactone of the formula RO
~ 0 Formula 2 wherein R is a hydroxy-protecting group by reacting the Formula 1 compound with a carboxy group reducing agent followed by protecting the resulting hydroxymethyl group;
(b) converting the intermediate from step (a), Formula 2, to the 5-0-hydroxy-protecting group-methyl-2,3-dideoxy-(D)- pentofuranose of the formula RO~
OH
Formula 3 wherein R is a hydroxy-protecting group by reacting the Formula 2 compound with a carbonyl group reducing agent;
(c) converting the intermediate from step (b), Formula 3, to the 1-0-activating-group-5-0-hydroxy-protecting group-2,3-dideoxy-(D)- pentofuranose of the formula RO
_OR
Formula 4 ~ ' ~,, wherein R is a hydroxy-protecting group and A is an 0-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups, wherein the alkyl moiety may be an unsubstituted or substituted Cl-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and Cl-C3 alkoxy groups by reacting the Formula 4 compound with an acylating or sulfonating or carbonylating agents corresponding to group A above;
(d) converting the intermediate from step (c), Formula 4, by reaction with a compound having one of the formulas HX
and trimethylsilyl halide to the l-leaving-group-5-0-protecting group-2,3-dideoxy-(D)- pentofuranose of the formula RO
-~X
For~ula 5 wherein R is a hydroxy-protecting group and X is a leaving group selected from F, Cl, Br and I;
(e) reacting the intermediate from step (d), Formula 5, with an activated derivative of adenine, wherein the base, adenine, has been activated by means of reacting the pendant amino and hydroxy groups on the adenine nucleus with an activating compound selected from silylating and acetylating and benzoylating agents, and optionally, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of an inert organic solvent; and (-f) recover~ng the anomeric mixture of a-and ~-(D)-2'3'-dideoxyadenosine after cleavage of the 5-0-hydroxy-protecting group from the intermediate product of step (e) above.
The starting material, (D)-~-carboxy-~butyrolactone - -may be conveniently obtained from L-glutamic acid by utilizing conventional techniques reported in the chemical literature3.
A combination of chemical reactions produce a suitably blocked 2,3-dideoxypentofuranose, 3. This compound and its derivatives are known in the literature.
Thus, the conversion of the starting material, (D)--carboxy-~-butyrolactone, to a 5-0-hydroxy-protecting group-methyl-~-butyrolactone, step (a), involves the reduction of the ~-carboxy group to a hydroxymethyl group followed by reaction with a hydroxy-protecting group reagent. For example, the reduction of the ~-carboxy group and protection of the resulting ~-hydroxymethyl group by means of the benzyl group (PhCH2-), step (a), was achieved by the successive reaction of the starting compound of Formula 1 with BH3.SMe2 and PhCH2Br.
~ I - 23 -.~ .
,,,,,,. CT-1907A ' ~
The primary alcohol functional group may be protected as an ether, such as trialkyl or dialkyl aryl or diaryl alkyl or triaryl silyl, unsubstituted and substituted benzyl, unsubstituted and substituted alkyl, or allyl ether, or as an ester, such as a benzoyl, mesitoyl, pivaloyl, unsubstituted or substituted acetyl, or a carbonate ester.
See "Protective Groups in Organic Synthesis, "T.W. Greene, John Wiley, New York 1981 for more detailed description of protecting groups and the chemistry relating to the same.
In a more preferred embodiment, there was used as the hydroxy-protecting group for the primary alcohol group at the 5-position the benzyl group, and most preferably the benzoyl group, because of their stability and well-known preparation.
The conversion of the 5-0-hydroxy-protecting group-~- -butyrolactone, to the 5-0-hydroxy-protecting group-2,3-dideoxypentofuranose having Formula 3 in step (b) was achieved by reacting the intermediate having Formula 2 with NaH and HCO2Et followed by HCl.
It will be understood by those skilled in the art to which this invention relates that any one of a number of reducing agents may be used to carry out either or both of steps (a) and (b) independently (i.e., in succession) or at the same time. Other useful reducing agents besides BH3-SMe2 used in steps (a) and (b) as described above include NaBHg, NaBH4 plus LiC1 or AlCl3 or BF3, LiAlH4, LiAlH(OMe)3, LiAlH(O-t-Bu)3, (Sia)2BH (disiamyl borane) and 1~
, ~ 24 -~- CT-1907A
- 133il87 , other dialkylboranes, and the like. Disiamyl borane is preferred as the reducing agent because of its convenient handling and reactivity.
The step (c) conversion of the intermediate having Formula 3 to the intermediate having Formula 4 bearing an "activated" hydroxy group at the C(l) position of the 2,3-dideoxypentofuranose ring system may be achieved by any reagent effective to convert this hydroxy group to a group which may be displaced readily by Cl or Br upon reaction with HCl or HBr or, preferably, TMSBr or TMSCl (+) catalytic amount of TMSI. ("TMS" represents the trimethylsilyl group). Such a group which can be so displaced readily include 0-activating groups selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted Cl-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and C1-C3 alkoxy groups. More preferably, this activating group is selected from corresponding (to the above) acetoxy and benzoyloxy groups and, most preferably, acetoxy. Step (c) was conveniently carried-out using acetic anhydride/pyridine reagent.
The step (d) displacement of the l-0-activating group functional group with a leaving group selected from C1 and ~ CT-1907A
Br was achieved by treatment of 4 with HCl (or HBr) in CH2C12 at low temperature to give the furanosyl halides 5a and 5b, which are present in solution as a mixture of anomers ( a and ~
In step (e) of the process~according to this invention, the intermediate having Formula 5 from step (d) above was reacted with adenine activated by reaction with well-known activating reagents to obtain a silylated, acetylated or benzoylated or other acylated adenine, in the presence of a suitable polar or non-polar solvent and, optionally, in the presence of a Lewis acid such as, for example, boron halides, aluminum halides, titanium halides, tin (IV) chloride, zinc halides, trimethylsilyl bromide, iodide, triflate and any other species well-known to be used in glycosylation reactions or in the presence of a Bronsted acid such as hydrogen chloride, bromide or iodide.
Especially useful in this step (e) is the use of silylated adenine in the presence of non-polar solvents such as, for example, benzene, toluene, chloroform, dichloromethane, dichloroethane and carbon tetrachloride. Examples of useful polar solvents include others such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, dimethylformamide, and dimethylsulfoxide. An example of a useful procedure to carry out step (e) is disclosed in Brundidge et al., U.S.
Patent 4,625,020 which discloses the coupling of silylated pyrimidines, wherein active hydrogens of hydroxy and amino groups are blocked by silyl groups such as the trimethylsilyl group, with a 2-deoxy-2-fluoroarabino-furanosyl halide. As this referenced procedure was applied to step (e) an adenine base having all active amino hydrogens blocked by the trimethylsilyl group was reacted with an intermediate having Formula 5.
Thus treatment of intermediate having Formula 5 with a silylated adenine as defined above in CH2C12 and CHC13 gave protected (D)-2',3-dideoxyadenosine as a mixture of anomers.
Alternatively, in a variation, when in step (c) the group "A" is acetyl, the intermediate from step (c) may be reacted with an activated adenine derivative in the presence of a Lewis acid as in step (e) so as to obtain the 5-0-hydroxy-protecting group -2',3'-dideoxyadenosine product without proceeding through step (d).
As mentioned above, in still another, more preferred embodiment according to this invention, the 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 4 from step (c) was contacted first with bromotrimethylsilane followed by a bis-silyl adenine to obtain in a single step, without isolation of the l-bromo-5-0-benzoyl-2,3-pentofuranose intermediate as from step (d), to obtain 5'-0-benzoyl-2',3'-dideoxyadenosine as the product from the combined steps (d) and (e).
In step (f) of the process according to the present invention, the 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to chemical reaction to remove the 5-0-protecting group. Suitable procedures to remove this protecting group .~
i335187 are well-known in the field to which this invention relates and examples are disclosed in "Protective Groups in Organic Synthesis" by T.W. Greene, John Wiley, New York, 1981 mentioned above. More preferably, 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to methanol saturated with ammonia to obtain 2',3'-dideoxyadenosine as a mixture of a-and ~-anomers.
In step 2. of the claimed instant process according to this invention, the mixture of a-and ~-(D)-2',3'-dideoxy-adenosine from step 1. was contacted with the enzyme, adenosine deaminase (ADA), isolated from calf spleen, in a neutral aqueous medium. This enzyme selectively catalyzes the deamination of the ~-anomer of (D)-2',3'-dideoxy-adenosine to yield quantitatively ~-(D)-2',3'-dideoxy-inosine. Although adenosine deaminase (ADA) from calf spleen was used in the actual examples, it is believed that any preparation of adenosine aminohydrolase ("deaminase," EC
3.5.4.4) would be suitable. Thus, the use of any preparation of adenosine aminohydrolase (or "deaminase") effective to selectively deaminate the ~-2',3'-dideoxy-adenosine anomer is within the scope of the process claimed as the present invention. Accordingly, in addition to the use of the free enzyme in an appropriate medium, such as in a neutral aqueous medium described herein, there may be used the enzyme, ADA, immobilized on an appropriate compatible - . CT-1907A
substrate such as, for example, the oxirane-acrylic polymer substrate Eupergit C TM (Rohm Pharma Gmb). The ADA may be bound to the polymer using conventional techniques.
The (D)-2',3'-dideoxyinosine product, was~conveniently recovered by collecting the reaction mixture from step 2., removing insoluble reactants and additives by filtration through Celite, and purifying the resulting product by simple recrystallization, methanol being a preferred recrystallization solvent. In step 2., a catalytic to approximately equimolar to an excess amount of adenosine deaminase is added to the anomeric mixture of (D)-2',3'-dideoxyadenosine from step l. in a suitable solvent. While a variety of solvents may be used, polar solvents, e.g.
water or alcohol, are preferred. The reaction is essentially complete after a period of time which can range from less than an hour to several hours, depending on amount of enzyme, reaction condition, and the like. The reaction is preferably conducted for about 1 to 4 hours at about 20-25C.
In order to suppress contaminating amounts of deaminated a-anomer from appearing in the product, the progress of the reaction should be followed to determine the maximum contact time, according to procedure well-~nown to those skilled in the art, e.g. thin-layer chromatography or high pressure liquid chromatographic techniques.
_ zg _ ,. ~
- 13~187 - As an indication of the unobviousness of this selective enzymatic deamination step, it was determined that in the (L)-anomeric mixture, it was the a-anomer and not the ~-anomer that is favored by rate of enzymatic deamination.
Since adenosine deaminase tends to be a rather non-specific enzyme and quite reactive, evidenced by its action on modified substrates, it certainly would not be obvious beforehand that there would be an advantageous rate difference in deamination of the ~- and ~-anomers of (D)-2',3'-dideoxyadenosine.
The use of the enzyme, adenosine deaminase, according to the process of the present invention affords the advantage whereby the resulting anomers (~ and a) need not be separated using costly and time-consuming chromatographic and crystallization techniques that heretofore have been well-known in the field to which this invention relates.
The ~-(D)-2',3'-dideoxyinosine anomer is desired because it is the active anomer or is, at least, substantially more active as an antiviral agent than is the a-anomer.
The process for producing ~-(D)-2',3'-dideoxyinosine according to this invention starting from the 5-0-hydroxy-protecting group-methyl-~-butyrolactone, is outlined in Scheme I below.
. .
1~35187 Scheme I
--~o R,BII 1~20~,0H
B20~,0~c ~ d~nin~ yy ~ ~b 5 ~
~D~ I
H0~ H~
~b 6 _, 0~
>
~,..
CT-1907A ~
- The following examples, wherein the compounds are numbered with reference to Scheme I, illustrate but a few representative embodiments of the process according to this invention and are not to be construed as limiting in scope.
All parts and percentages are by weight and temperatures are in degrees Celsius unless otherwise specified.
.
EXPERIMENTAL
Example 1:
(D)-5-0-Benzyl-2,3-dideoxypentofuranose.(2) To 200 mL of a 0.5 M solution of disiamylborane in THF solution at 0C was added dropwise under nitrogen a solution of 15.1 g (0.069 mole) of (D)-5'-benzoyloxy-5-hydroxymethylbutyrolactone, 1, in 50 mL of dry tetrahydrofuran. After 20 min at 0 C the reaction was warmed to 22 C. The reaction was stirred at 22C for 16 hrs, then worked up by slowly adding 12 mL of water and refluxing for 30 min. The reaction mixture was cooled to 0C followed by the slow addition of 24 mL of 30%
hydrogen peroxide maintaining the pH between 7-8 with lN
sodium hydroxide. After the addition the reaction was evaporated under reduced pressure on a rotovapor at 30C to an oily residue. This was partitioned between 500 mL
dichloromethane and 150 mL water. The aqueous layer was extracted wtih 2X 100 mL dichloromethane and then the combined organic layers were washed with 50 mL water, dried ~ J
...,~, ` 1335187 over anhydrous sodium sulfate and evaporated to an oil.
Yield = 15.3 g (100%) of 5-0-benzoyl-2,3-dideoxypento-furanose. The H-NMR was consistent for structure and the oil was used directly in the next step.
Example 2:
l-O-Acetyl-5-0-benzoyl-2,3-dideoxypentofuranose. (3~ A
solution of 5-0-benzoyl-2,3-dideoxypentofuranose (15.3 g, 0.069 mole) in 32 mL of pyridine and 16 mL of acetic anhydride was stirred at 22C for 4 hrs then diluted with 500 mL dichloromethane and 100 g of ice was added. The reaction mixture was then washed with 3 X 100 mL lN
hydrochloric acid 3 X 100 mL saturated aqueous sodium bicarbonate and 100 mL of brine. The organic layer was dried over sodium sulfate and evaporated to yield a pale yellow oil. This could be used as is or chromatographed on silica gel 35% ---> 60% EtOAc/hexane. Yield: 15.9 g (87%) of 1-0-acetyl-5-0-benzoyl-2,3-dideoxy-pentofuranose. Its NMR was consistent for structure.
Example 3:
5'-0-benzoyl-2',3-dideoxyadenosine. (4) 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose (0.522 g, 0.00198 mole) was dissolved in 5 mL of 1,2-dichloroethane. To this was added 276 ~L (1.2 eq) of trimethylsilylbromide. This was stirFed at 22 C for 15 min prior to the addition of 11.9 - * ~
~- -;CT-1907A
mL of a 0.2 M solution of bis-silyladenine in~
1,2-dichloroethane. The solution was stirred for 88 hrs at 22 C. The reaction was then worked up by cooling to 0C, followed by pouring into 40 mL of cold saturated aqueous sodium bicarbonate. The reaction mixture was partitioned between 200 mL dichloromethane and 2 X 40 mL cold saturated aqueous sodium bicarbonate and 40 mL brine. The organic layer was dried and evaporated to yield a colorless oil that was chromatographed on silica gel and eluted with 8%
methanol in methylene chloride. Fractions were collected and similar ones pooled to yield 420 mg 5'-0-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 170 mL of methanol saturated with ammonia.
This was tightly stoppered and stirred at 18C for 48 hrs.
TLC indicated that reaction was incomplete. The solvent was evaporated and replaced with 170 mL of fresh ammonia solution in methanol. TLC after an additional 48 hr implied the reaction was complete. Hence, the solvent was evaporated and ethanol was added (5 mL). This yielded colorless crystals which were collected and washed with (5 mL) of 95% ethanol to yield 1.20 g (95%) of 2',3'-dideoxyadenosine 5a together with its a anomer 5b in a 1:1 ratio by lH-NMR. The a+~ 2',3'-dideoxyadenosine mixture ~ ~ - 34 -~ :". .~ i, was dissolved in 50 mL of deionized water and to this solution was added 10 mg of adenosine deaminase (type II, from Sigma; 9 units/mg ~ .9 ~mole/min). The solution was stirred at 20C and reaction monitored by HPLC, After 2 hrs 30 min another 10 mg of adenosine deaminase was added. HPLC
showed the reaction was nearly complete after 3 hrs. The reaction was then concentrated (1-2 mL) at 35C to give an oil. The oil was scratched and slowly diluted with 2 X 1.5 mL portions of methanol which produced white crystals.
After 15 min the material was filtered and the colorless crystals washed with 2 X 1.5 mL portions of methanol to yield 2',3'-dideoxyinosine (120 mg, 48%). The mother liquor was treated as above to yield an additional 29 mg (12%) of good quality product, which was characterized by lH-NMR.
The nuclear magnetic resonance spectrum was consistent with the structure.
s.
. ,,~3~.
REFERENCES: ~
1. Samukov, V.V.; Ofitserov, V.I. Bioorg. Khim. 1983, 9, 132.
2. Prisbe, E.J.;- Martin, J.C. synth. Commun. 1985, 15, 401.
3. Taniguchi, M.; Koga, K.; Yamada, S. Tetrahedron 1974, 30, 3547.
4. Spry, D. V.; Bhala, A. R. Heterocycles, 1985, 23, 1901.
5. Secrist, J. A. et al. J. Med. Chem. 1987, 30, 746.
BACKGROUND OF THE INVENTION
Field of the Invention This invention relates to an improved process to produce B-2',3'-dideoxyinosine.
Background - ~elated References Typically, 2',3'-dideoxycytidine (ddC) is synthesized from 2'-deoxycytidine1'2. This is a general method for the synthesis of 2,3'-dideoxynucleosides. The starting materials for this synthesis are, however, extremely expensive and not available in bulk. The reagents required for this deoxygenation, furthermore, are also quite costly.
There is a process for producing 2',3'-dideoxynucleosides represented by the formula RO B
~5 ~
wherein B is a purine or pyrimidine base and R is ~ or a ~~
" .
~ 13~5187 hydroxy-protecting group by the steps of (a) converting a r-carboxy- ~ butyrolactone to a 5-0-hydroxy-protecting group-methyl-r-butyrolactone, (b) converting the intermediate from step (a) to the 5-0-hydroxy-protecting group-methyl-2',3'-dideoxypentofuranose, (c) converting the intermediate from step (b) to the 1-0-activating group -~-0-hydroxy-protecting group-methyl-2',3'-dideoxypento-furanose, (d) converting the intermediate from step (c) to the 1-leaving group-S-0-hydroxy-protecting group-2,'3'-dideoxypentofuranose, (e) reacting the intermediate from step (d) with an activated purine or pyrimidine base, dna (f) recovering the dideoxynucleoside from step (e). The resulting product comprises a mixture of B-and a-anomers which may be separated using chromatographic and crystallization techniques well-known in the field to which this invention relates.
There remains in the field a need for process improvements whereby the generally active, or more active, ~-anomer of 2',3'-dideoxynucleosides can be selectively obtained without the costly and time-consuming chromatographic or crystallization separation of B- and a-anomers.
SUMMARY OF THE INVENT~ON
In summary, this invention comprises a process for selectively producing B-2',3'-dideoxyinosine represented by the formula HO
~
Formula A
wherein I is the purine base, inosine, and wherein there is produced 2~,3~-dideoxyadenosine which is then subjected to enzymatic deamination and replacement of the adenosine base with inosine.
DETAILED DESCRIPTION OF THE I~ NTION
The present invention is a process for selectively producing B-2',3'-dideoxyinosine represented by the formula -~ Formula A
wherein I is the purine base, inosine, which comprises the steps of:
(a) converting a r'-carboxy-~butyrolactone of the formula HOOC
~0 Formula B
to a 5-O-hydroxy-protecting group-methyl-r-butyrolactone of the formula RO ~ o Formula C
wherein R is a hydroxy-protecting group;
(b) converting the intermediate from step (a~, Formula C, to the 5-O-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula RO
Formula D
wherein R is a hydroxy-protecting group;
(c) converting the intermediate from step (b), Formula ~, to the 1-O-activating-group-5-O-hydroxy-protecting 1 33~1 87 group-methyl-2,3-dideoxypentofuranose of the formula RO----~
~ OA
Formula E
wherein R is a hydroxy-protecting group and A is an O-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and Cl-C3 alkoxy groups;
(d) converting the intermediate from step (c), Formula E, by reaction with a compound of the formula MX to the 1-leaving-group-5-O-hydroxy-protecting group-methyl-2,3-di-deoxypentofuranose of the formula RO
~ X
Formula F
wherein R is a hydroxy-protecting group and X is a leaving group selected from Cl and Br and M is selected from H and (CH3)3Si;
(e) reacting the intermediate from step (d), Formula F, with an activated adenine derivative wherein the base, adenine, has been activated by means of reacting the pendant N-G
amino group and N-9 Nitrogen atom on the nucleus of adenine with an activating compound selected from silylating and acetylating and benzoylating agents, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of a solvent selected from polar and non-polar solvents;
(f) subjecting the intermediate from step (e) to chemical reaction effective to displace the 5-0-hydroxy-protecting group to obtain an anomeric mixture of ~- and ~-2',3'-dideoxyadenosine;
(g) contacting the mixture of ~- and ~-2',3'-dideoxyadenosine from step (f) with the enzyme, adenosine deaminase, especially preferably in a neutral aqueous medium, to effect displacement of the amino group pendant to the 6-position of the purine (adenine) ring system selectively in the ~-2',3'-dideoxyadenosine with a hydroxyl group whereby adenosine is converted to inosine; and (h) recovering the ~-2',3'-dideoxyinosine from step (e) above.
~, ~3 - 13~187 The process for producing B-2',3'-dideoxyinosine according to this invention starting from the 5-0-hydroxy-protecting group-methyl-r-butyrolactone,is outlined in Scheme I below.
Scheme I
R, e~ H ~~ OH
8 2 0~0 OAc 8 r Si M~
Ac~O silyl. ~denine B20~J
4 a j~ 4 b 2 5 HO~y 5a ~D~
H0y~ H~
5b OH
A = ~) I = N~NNl\>
- -- 1335l87 As is illustrated in Scheme I and in the actual working examples which follow, steps (d) and (e) are conveniently and more preferably carried-out in one pot by the sequential addition of a halogenating agent and an activated adenine derivative to 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 3.
The starting material, y-carboxy-~ butyrolactone may be conveniently obtained from glutamic acid by utilizing conventional techniques reported in the chemical literature3.
A combination of chemical reactions produce a suitably blocked 2,3-dideoxypentafuranose, 3. This compound and its derivatives are known in the literature.
Thus, the conversion of the starting material, r-carboxy-r-butyrolactone, to a 5-0-hydroxy-protecting group-methyl- butyrolactone, step (a), involves the reduction of the -carboxy group to a hydroxymethyl group followed by reaction with a hydroxy-protecting group reagent. For example, the reduction of the r-carboxy group and protection of the resulting r-hydroxymethyl group by means of the benzyl group (PhCH2-) or the benzoyl group (PhC(O)-), step (a), was acheived by the successive reaction of the starting compound of Formula 1 with BH3 SMe2 and - 13~5187 PhCH2Br3'4, or PhC(O)Cl. The use of the benzoyl group is preferred over the use of the benzyl group because the subsequent removal of the benzoyl protecting group has been found to proceed more readily and to provide higher yield of product.
The primary alcohol functional group may be protected as an ether, such as a trialkyl or dialkyl aryl or diaryl alkyl or triaryl silyl, unsubstituted and substituted benzyl, unsubstituted and substituted alkyl, or allyl ether, or as an ester, such as a benzoyl, mesitoyl, pivaloyl, unsubstituted or substituted acetyl, or a carbonate ester.
See "Protective Groups in Organic Synthesis," T. W. Greene, John Wiley, New York 1981 for more detailed description of protecting groups and the chemistry relating to the same.
In a more preferred embodiment, there was used as the hydroxy-protecting group for the primary alcohol group at the 5-position the benzoyl group because, as mentioned above, the subsequent removal of the benzoyl protecting group has been found to proceed more readily and to provide higher yield of product. The 5-0-benzoyl protecting group may be conveniently displaced by hydrolysis under basic conditions, for example, by treatment with methanol saturated with ammonia.
The conversion of the 5-0-hydroxy-protecting group- r-butyrGlactone, to the 5-0-hydroxy-protecting g group-2,3-dideoxypentafuranose having Formula 2 in step (b) was achieved by reacting the intermediate having Formula 1 with NaH and HCO2Et followed by HCl4.
It will be understood by those skilled in the art to which this invention relates that any one of a number of reducing agents may be used to carry out either or both of steps (a) and (b) independently (i.e., in succession) or at the same time. Other useful reducing agents besides BH3~SMe2 used in either or both of steps (a) and (b) as described above include [(CH3)2CHCH(CH3)2]BH
(disiamylborane), NaBH4 plus LiCl or AlCl3 or BF3, LiAlH4, LiAlH(OMe)3, LiAlH(O-t-Bu)3, and the like. The use of disiamylborane for the step (b) reduction of the lactone ring carbonyl group is especially preferred.
The step (c) conversion of the intermediate having Formula 2 to the intermediate having Formula 3 bearing an O-activating group at the C(l) position of the 2,3-dideoxypentafuranose ring system may be achieved by any reagent effective to convert this l-hydroxy group to a group which may be displaced readily by Cl or Br upon reaction with HCl or B r. Such a group which may be so displaced readily includes O-activating groups selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from l to 3 groups selected from halo and C1-C3 alkoxy groups. More preferably, this activating group is selected from corresponding (to the above) acetoxy and benzoyloxy groups and, most preferably, acetoxy. Step (c) was conveniently carried-out using acetic anhydride/pyridine reagent.
The step (d) displacement of the l-O-activating group functional group with a leaving group selected from Cl and Br may be achieved by treatment of 3 with one of HCl, HBr, and a halotrialkylsilane having the formula R3SiX where R is alkyl and X is halo, more preferably (CH3)3SiBr, in CH2Cl2 at low temperature to give the furanosyl halides, which are present in solution as a mixture of anomers (~ and ~).
In step (e) of the process according to this invention, the intermediate having Formula F from step (d) above may be reacted with a suitably activated adenosine base, wherein such adenosine base has been activated by reaction with well-known activating reagents to obtain a silylated, acetylated or benzoylated base, in the pr ~
. .
suitable polar or non-polar solvent and, optionally, in the presence of a Lewis acid such as, for example, boron halides, aluminum halides, titanium halides, tin (IV) S chloride, zinc halides, trimethylsilyl bromide, iodide, triflate and any other species well-known to be used in glycosylation reactions or in the presence of a Bronsted acid such as hydrogen chloride. bromide or iodide.
Especially useful in this step (e) is the use of silylated adenosine in the presence of non-polar solvents such as, for example, benzene, toluene, chloroform, dichloromethane, dichloroethane and carbon tetrachloride. Examples of useful polar solvents include others such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, dimethylformamide, and dimethylsulfoxide. An example of a useful procedure to carry out step (e) is disclosed in Brundidge et al., U.S.
Patent Patent 4,625,020 which discloses the coupling of silylated pyrimidines, wherein active hydrogens of hydroxy and amino groups are blocked by silyl groups such as the trimethylsilyl group, with a 2-deoxy-2-fluoroarabino-furanosyl halide.
Alternatively, in another embodiment according to this invention, when from step (c) the group "A" is acetyl, the intermediate from step (c) may be reacted with an activated adenine derivative in the presence of a Lewis acid as in 1335~87 step (e) so as to obtain the 5-0-hydroxy-protecting group-2',3'-dideoxyadenosine intermediate without proceeding through a separate step (d).
As mentioned above, in still another, more preferred embodiment according to this invention, the 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 3 from step (c) was contacted first with bromotrimethylsilane followed by a bis-silyl adenine to obtain in a single step, without isolation of the 1-bromo-5-0-benzoyl-2,3-pento-furanose intermediate as from step (d), to obtain 5'-0-benzoyl-2',3'-dideoxyadenosine as the product from the combined steps (d) and (e).
In step (f) of the process according to the present invention, the 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to chemical reaction to remove the 5-O-protecting group. Suitable procedures to remove this protecting group are well-known in the field to which this invention relates and examples are disclosed in "Protective Groups in Organic Synthesis" by T. W. Greene, John Wiley, New York, 1981 mentioned above. When the protecting group is the benzyl group, it can be easily removed by catalytic hydrogenation (H2/PdCl2). More preferably, 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to methanol saturated with ammonia to obtain 2',3'-dideoxyadenosine as a mixture of a- and B-anomers.
In step (g) of the process according to this invention, the mixture of ~- and ~-2',3'-dideoxyadenosine from step (f) was contacted with the enzyme, adenosine deaminase (ADA) 5, isolated from calf spleen, in a neutral aqueous medium. This enzyme selectively catalyzes the deamination of~-2'-3'-dideoxyadenosine to yield quantitatively ~-2',-3'-dideoxyinosine. Although adenosine deaminase (ADA) from calf spleen was used in the actual examples, it is believed that any preparation of adenosine aminohydrolase ("deaminase," EC 3.5.4.4) would be suitable.
Thus, the use of any preparation of adenosine aminohydrolase (or "deA~;n~e") effective to selectively deaminate the ~-2',3'-dideoxyadenosine anomer is within the scope of the process claimed as the present invention. Accordingly, in addition to the use of the free enzyme in an appropriate medium, such as in a neutral aqueous medium described herein, there may be used the enzyme, ADA, immobilized on an appropriate compatible substrate such as, for example, the oxirane-acrylic polymer substrate Eupergit C TM (Rohm Pharma Gmb). The ADA may be bound to the polymer using conventional techniques.
The 2',3'-dideoxyinosine product, was conveniently recovered by collecting the reaction mixture from step (e) or from the additional step wherein the 5-O-protecting group was removed, removing insoluble reactants and additives by filtration through Celite*, and purifying the resulting product by column chromatography, for example on a silica gel column using a mixture of about 1-5% methanol in chloroform as eluent.
* Trade-mark -14-.~ ~.
, 133~187 The use of the enzyme, adenosine deaminase, according to the process of the present invention affords the advantage whereby the resulting anomers (~ and a) need not be separated using costly and time-consuming chromatographic and crystallization techniques that heretofore have been well-known in the field to which this invention relates.
The ~-2',3'-dideoxyinosine anomer is desired because it is the active anomer or is, at least, substantially more active as an antiviral agent than is the a-anomer.
The following examples, wherein the compounds are numbered with reference to Scheme I, illustrate but a few representative embodiments of the process according to this invention and are not to be construed as limiting in scope.
All parts and percentages are by weight and temperatures are in degrees Celsius unless otherwise specified.
EXPERIMENTAL
Example 1:
(D)-5-O-Benzyl-2,3-dideoxypentofuranose. 2. To 200 mL of a 0.5 M solution of disiamylborane in THF solution at 0C was added dropwise under nitrogen a solution of 15.1 g (0.069 mole) of (D)-5'-benzoyloxy-5-hydroxymethylbutyrolactone, 1, in 50 mL of dry tetrahydrofuran. After 20 min at 0C the 1~35187 reaction was warmed to 22C. The reaction was stirred at 22C for 16 hrs, then worked up by slowly adding 12 mL of water and refluxing for 30 min. The reaction mixture was cooled to 0C followed by the slow addition of 24 mL of 30%
hydrogen peroxide maintaining the pH between 7-8 with lN
sodium hydroxide. After the addition the reaction was evaporated under reduced pressure on a rotovapor at 30C to an oily residue. This was partitioned between 500 mL
dichloromethane and 150 mL water. The aqueous layer was extracted wtih 2X 100 mL dichloromethane and then the combined organic layers were washed with 50 mL water, dried over anhydrous sodium sulfate and evaporated to an oil.
Yield = 15.3 g (100%) of 5-0-benzoyl-2,3-dideoxypento-furanose. The lH-NMR was consistent for structure and the oil was used directly in the next step.
Example 2:
1-0-Acetyl-5-0-benzoyl-2,3-dideoxypentofuranose. 3. A
solution of 5-0-benzoyl-2,3-dideoxypentofuranose (15.3 g, 0.069 mole) in 32 mL of pyridine and 16 mL of acetic anhydride was stirred at 22C for 4 hrs then diluted with 500 mL dichloromethane and 100 g of ice was added. The reaction mixture was then washed with 3 X 100 mL lN
hydrochloric acid 3 X 100 mL saturated aqueous sodium bicarbonate and 100 mL of brine. The organic layer was 133~187 dried over sodium sulfate and evaporated to yield a pale yellow oil. This could be used as is or chromatographed on silica gel 35% ~ 60% EtOAc/hexane. Yield: 15.9 g (87%) of 1-0-acetyl-5-0-benzoyl-2,3-dideoxy-pentofuranose. Its NMR was consistent for structure.
Example 3:
5'-O-benzoyl-2',3-dideoxyadenosine. 4.
1-0-acetyl-5-0-benzoyl-2,3-pentofuranose (0.522 g, 0.00198 mole) was dissolved in S mL of 1,2-dichloroethane. To this was added 276 ~L (1.2 eq) of trimethylsilylbromide. This was stirred at 22C for 15 min prior to the addition of 11.9 mL of a 0.2 M solution of bis-silyladenine in 1,2-dichloroethane. The solution was stirred for 88 hrs at 22C. The reaction was then worked up by cooling to 0C, followed by pouring into 40 mL of cold saturated aqueous sodium bicarbonate. The reaction mixture was partitioned between 200 mL dichloromethane and 2 X 40 mL cold saturated aqueous sodium bicarbonate and 40 mL brine. The organic layer was dried and evaporated to yield a colorless oil that was chromatographed on silica gel and eluted with 8%
methanol in methylene chloride. Fractions were collected and similar ones pooled to yield 420 mg 5'-0-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 170 mL of methanol saturated with ammonia.
This was tightly stoppered and stirred at 18C for 48 hrs.
TLC indicated that reaction was incomplete. The solvent was evaporated and replaced with 170 mL of fresh ammonia solution in methanol. TLC after an additional 48 hr implied the reaction was complete. Hence, the solvent was evaporated and ethanol was added (5 mL). This yielded colorless crystals which were collected and washed with (5 mL) of 95% ethanol to yield 1.20 g (95%) of 2',3'-dideoxyadenosine 5a together with its a anomer 5b in a 1:1 ratio by lH-NMR. The a+~ 2',3'-dideoxyadenosine mixture was dissolved in S0 mL of deionized water and to this solution was added 10 mg of adenosine deaminase (type II, from Sigma; 9 units/mg ~ .9 ~mole/min). The solution was stirred at 20C and reaction monitored by HPLC. After 2 hrs 30 min another 10 mg of adenosine de~m;n~se was added.
HPLC showed the reaction was nearly complete after 3 hrs.
The reaction was then concentrated (1-2 mL) at 35C to give an oil. The oil was scratched and slowly diluted with 2 X
1.5 mL portions of methanol which produced white crystals.
After 15 min the material was filtered and the colorless crystals washed with 2 X 1.5 mL portions of methanol to yield 2',3'-dideoxyinosine (120 mg, 48%). The mother liquor was treated as above to yield an additional 29 mg (12%) of good quality product, which was characterized by lH-NMR.
The nuclear magnetic resonance spectrum was consistent with the structure.
SUPPT-l~M~NTARY DISCLOSIJRE
The currently preferred embodiment of the invention comprises an efficient process for selectively producing ~ -(D)-2',3'-dideoxyinosine represented by the formula H O~<oy Formula A
wherein I is the purine base, inosine. The process involves producing an C~ anomeric mixture of (D)-2',3'-dideoxyadenosine, which is then subjected to selective enzymatic deAm;nAtion converting the adenosine base to inosine for only the ~3 -(D)-isomer which is readily isolable and easily purified by simple recrystallization.
19 _ ~T~ILED DESCRIPTION OF THE PRESENTLY
The present invention is an improved process for selectively producing ~-(D)-2'-3'-dideoxyinosine represented by the formula H ~ o I
Farmula R
wherein I is the purine base, inosine, which comprises the steps of:
1. preparing an anomeric mixture of a-and ~-(D)-2', 3'-dideoxyadenosine;
2. selectively deaminating the ~-~D)-2',3'-dideoxy-adenosine anomer by contact of the anomeric mixture of step 1. with the enzyme, adenosine deaminase; and 3. recovering the ~-(D)-2',3'-dideoxyinosine produced in step 2.
A typical process for producing the anomeric mixture used in step 1. comprises the steps of:
(a) converting (D)- ~-carboxy-~-butyrolactone of the formula HOOC
~ a Formula 1 - 2~ -.,~,.,~
~ CT-1907A
. 133~187 to a 5-0-hydroxy-protecting group-methyl-~-butyrolactone of the formula RO
~ 0 Formula 2 wherein R is a hydroxy-protecting group by reacting the Formula 1 compound with a carboxy group reducing agent followed by protecting the resulting hydroxymethyl group;
(b) converting the intermediate from step (a), Formula 2, to the 5-0-hydroxy-protecting group-methyl-2,3-dideoxy-(D)- pentofuranose of the formula RO~
OH
Formula 3 wherein R is a hydroxy-protecting group by reacting the Formula 2 compound with a carbonyl group reducing agent;
(c) converting the intermediate from step (b), Formula 3, to the 1-0-activating-group-5-0-hydroxy-protecting group-2,3-dideoxy-(D)- pentofuranose of the formula RO
_OR
Formula 4 ~ ' ~,, wherein R is a hydroxy-protecting group and A is an 0-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups, wherein the alkyl moiety may be an unsubstituted or substituted Cl-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and Cl-C3 alkoxy groups by reacting the Formula 4 compound with an acylating or sulfonating or carbonylating agents corresponding to group A above;
(d) converting the intermediate from step (c), Formula 4, by reaction with a compound having one of the formulas HX
and trimethylsilyl halide to the l-leaving-group-5-0-protecting group-2,3-dideoxy-(D)- pentofuranose of the formula RO
-~X
For~ula 5 wherein R is a hydroxy-protecting group and X is a leaving group selected from F, Cl, Br and I;
(e) reacting the intermediate from step (d), Formula 5, with an activated derivative of adenine, wherein the base, adenine, has been activated by means of reacting the pendant amino and hydroxy groups on the adenine nucleus with an activating compound selected from silylating and acetylating and benzoylating agents, and optionally, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of an inert organic solvent; and (-f) recover~ng the anomeric mixture of a-and ~-(D)-2'3'-dideoxyadenosine after cleavage of the 5-0-hydroxy-protecting group from the intermediate product of step (e) above.
The starting material, (D)-~-carboxy-~butyrolactone - -may be conveniently obtained from L-glutamic acid by utilizing conventional techniques reported in the chemical literature3.
A combination of chemical reactions produce a suitably blocked 2,3-dideoxypentofuranose, 3. This compound and its derivatives are known in the literature.
Thus, the conversion of the starting material, (D)--carboxy-~-butyrolactone, to a 5-0-hydroxy-protecting group-methyl-~-butyrolactone, step (a), involves the reduction of the ~-carboxy group to a hydroxymethyl group followed by reaction with a hydroxy-protecting group reagent. For example, the reduction of the ~-carboxy group and protection of the resulting ~-hydroxymethyl group by means of the benzyl group (PhCH2-), step (a), was achieved by the successive reaction of the starting compound of Formula 1 with BH3.SMe2 and PhCH2Br.
~ I - 23 -.~ .
,,,,,,. CT-1907A ' ~
The primary alcohol functional group may be protected as an ether, such as trialkyl or dialkyl aryl or diaryl alkyl or triaryl silyl, unsubstituted and substituted benzyl, unsubstituted and substituted alkyl, or allyl ether, or as an ester, such as a benzoyl, mesitoyl, pivaloyl, unsubstituted or substituted acetyl, or a carbonate ester.
See "Protective Groups in Organic Synthesis, "T.W. Greene, John Wiley, New York 1981 for more detailed description of protecting groups and the chemistry relating to the same.
In a more preferred embodiment, there was used as the hydroxy-protecting group for the primary alcohol group at the 5-position the benzyl group, and most preferably the benzoyl group, because of their stability and well-known preparation.
The conversion of the 5-0-hydroxy-protecting group-~- -butyrolactone, to the 5-0-hydroxy-protecting group-2,3-dideoxypentofuranose having Formula 3 in step (b) was achieved by reacting the intermediate having Formula 2 with NaH and HCO2Et followed by HCl.
It will be understood by those skilled in the art to which this invention relates that any one of a number of reducing agents may be used to carry out either or both of steps (a) and (b) independently (i.e., in succession) or at the same time. Other useful reducing agents besides BH3-SMe2 used in steps (a) and (b) as described above include NaBHg, NaBH4 plus LiC1 or AlCl3 or BF3, LiAlH4, LiAlH(OMe)3, LiAlH(O-t-Bu)3, (Sia)2BH (disiamyl borane) and 1~
, ~ 24 -~- CT-1907A
- 133il87 , other dialkylboranes, and the like. Disiamyl borane is preferred as the reducing agent because of its convenient handling and reactivity.
The step (c) conversion of the intermediate having Formula 3 to the intermediate having Formula 4 bearing an "activated" hydroxy group at the C(l) position of the 2,3-dideoxypentofuranose ring system may be achieved by any reagent effective to convert this hydroxy group to a group which may be displaced readily by Cl or Br upon reaction with HCl or HBr or, preferably, TMSBr or TMSCl (+) catalytic amount of TMSI. ("TMS" represents the trimethylsilyl group). Such a group which can be so displaced readily include 0-activating groups selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups wherein the alkyl moiety may be an unsubstituted or substituted Cl-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and C1-C3 alkoxy groups. More preferably, this activating group is selected from corresponding (to the above) acetoxy and benzoyloxy groups and, most preferably, acetoxy. Step (c) was conveniently carried-out using acetic anhydride/pyridine reagent.
The step (d) displacement of the l-0-activating group functional group with a leaving group selected from C1 and ~ CT-1907A
Br was achieved by treatment of 4 with HCl (or HBr) in CH2C12 at low temperature to give the furanosyl halides 5a and 5b, which are present in solution as a mixture of anomers ( a and ~
In step (e) of the process~according to this invention, the intermediate having Formula 5 from step (d) above was reacted with adenine activated by reaction with well-known activating reagents to obtain a silylated, acetylated or benzoylated or other acylated adenine, in the presence of a suitable polar or non-polar solvent and, optionally, in the presence of a Lewis acid such as, for example, boron halides, aluminum halides, titanium halides, tin (IV) chloride, zinc halides, trimethylsilyl bromide, iodide, triflate and any other species well-known to be used in glycosylation reactions or in the presence of a Bronsted acid such as hydrogen chloride, bromide or iodide.
Especially useful in this step (e) is the use of silylated adenine in the presence of non-polar solvents such as, for example, benzene, toluene, chloroform, dichloromethane, dichloroethane and carbon tetrachloride. Examples of useful polar solvents include others such as tetrahydrofuran and dioxane, nitriles such as acetonitrile, dimethylformamide, and dimethylsulfoxide. An example of a useful procedure to carry out step (e) is disclosed in Brundidge et al., U.S.
Patent 4,625,020 which discloses the coupling of silylated pyrimidines, wherein active hydrogens of hydroxy and amino groups are blocked by silyl groups such as the trimethylsilyl group, with a 2-deoxy-2-fluoroarabino-furanosyl halide. As this referenced procedure was applied to step (e) an adenine base having all active amino hydrogens blocked by the trimethylsilyl group was reacted with an intermediate having Formula 5.
Thus treatment of intermediate having Formula 5 with a silylated adenine as defined above in CH2C12 and CHC13 gave protected (D)-2',3-dideoxyadenosine as a mixture of anomers.
Alternatively, in a variation, when in step (c) the group "A" is acetyl, the intermediate from step (c) may be reacted with an activated adenine derivative in the presence of a Lewis acid as in step (e) so as to obtain the 5-0-hydroxy-protecting group -2',3'-dideoxyadenosine product without proceeding through step (d).
As mentioned above, in still another, more preferred embodiment according to this invention, the 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose of Formula 4 from step (c) was contacted first with bromotrimethylsilane followed by a bis-silyl adenine to obtain in a single step, without isolation of the l-bromo-5-0-benzoyl-2,3-pentofuranose intermediate as from step (d), to obtain 5'-0-benzoyl-2',3'-dideoxyadenosine as the product from the combined steps (d) and (e).
In step (f) of the process according to the present invention, the 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to chemical reaction to remove the 5-0-protecting group. Suitable procedures to remove this protecting group .~
i335187 are well-known in the field to which this invention relates and examples are disclosed in "Protective Groups in Organic Synthesis" by T.W. Greene, John Wiley, New York, 1981 mentioned above. More preferably, 5'-0-benzoyl-2',3'-dideoxyadenosine was subjected to methanol saturated with ammonia to obtain 2',3'-dideoxyadenosine as a mixture of a-and ~-anomers.
In step 2. of the claimed instant process according to this invention, the mixture of a-and ~-(D)-2',3'-dideoxy-adenosine from step 1. was contacted with the enzyme, adenosine deaminase (ADA), isolated from calf spleen, in a neutral aqueous medium. This enzyme selectively catalyzes the deamination of the ~-anomer of (D)-2',3'-dideoxy-adenosine to yield quantitatively ~-(D)-2',3'-dideoxy-inosine. Although adenosine deaminase (ADA) from calf spleen was used in the actual examples, it is believed that any preparation of adenosine aminohydrolase ("deaminase," EC
3.5.4.4) would be suitable. Thus, the use of any preparation of adenosine aminohydrolase (or "deaminase") effective to selectively deaminate the ~-2',3'-dideoxy-adenosine anomer is within the scope of the process claimed as the present invention. Accordingly, in addition to the use of the free enzyme in an appropriate medium, such as in a neutral aqueous medium described herein, there may be used the enzyme, ADA, immobilized on an appropriate compatible - . CT-1907A
substrate such as, for example, the oxirane-acrylic polymer substrate Eupergit C TM (Rohm Pharma Gmb). The ADA may be bound to the polymer using conventional techniques.
The (D)-2',3'-dideoxyinosine product, was~conveniently recovered by collecting the reaction mixture from step 2., removing insoluble reactants and additives by filtration through Celite, and purifying the resulting product by simple recrystallization, methanol being a preferred recrystallization solvent. In step 2., a catalytic to approximately equimolar to an excess amount of adenosine deaminase is added to the anomeric mixture of (D)-2',3'-dideoxyadenosine from step l. in a suitable solvent. While a variety of solvents may be used, polar solvents, e.g.
water or alcohol, are preferred. The reaction is essentially complete after a period of time which can range from less than an hour to several hours, depending on amount of enzyme, reaction condition, and the like. The reaction is preferably conducted for about 1 to 4 hours at about 20-25C.
In order to suppress contaminating amounts of deaminated a-anomer from appearing in the product, the progress of the reaction should be followed to determine the maximum contact time, according to procedure well-~nown to those skilled in the art, e.g. thin-layer chromatography or high pressure liquid chromatographic techniques.
_ zg _ ,. ~
- 13~187 - As an indication of the unobviousness of this selective enzymatic deamination step, it was determined that in the (L)-anomeric mixture, it was the a-anomer and not the ~-anomer that is favored by rate of enzymatic deamination.
Since adenosine deaminase tends to be a rather non-specific enzyme and quite reactive, evidenced by its action on modified substrates, it certainly would not be obvious beforehand that there would be an advantageous rate difference in deamination of the ~- and ~-anomers of (D)-2',3'-dideoxyadenosine.
The use of the enzyme, adenosine deaminase, according to the process of the present invention affords the advantage whereby the resulting anomers (~ and a) need not be separated using costly and time-consuming chromatographic and crystallization techniques that heretofore have been well-known in the field to which this invention relates.
The ~-(D)-2',3'-dideoxyinosine anomer is desired because it is the active anomer or is, at least, substantially more active as an antiviral agent than is the a-anomer.
The process for producing ~-(D)-2',3'-dideoxyinosine according to this invention starting from the 5-0-hydroxy-protecting group-methyl-~-butyrolactone, is outlined in Scheme I below.
. .
1~35187 Scheme I
--~o R,BII 1~20~,0H
B20~,0~c ~ d~nin~ yy ~ ~b 5 ~
~D~ I
H0~ H~
~b 6 _, 0~
>
~,..
CT-1907A ~
- The following examples, wherein the compounds are numbered with reference to Scheme I, illustrate but a few representative embodiments of the process according to this invention and are not to be construed as limiting in scope.
All parts and percentages are by weight and temperatures are in degrees Celsius unless otherwise specified.
.
EXPERIMENTAL
Example 1:
(D)-5-0-Benzyl-2,3-dideoxypentofuranose.(2) To 200 mL of a 0.5 M solution of disiamylborane in THF solution at 0C was added dropwise under nitrogen a solution of 15.1 g (0.069 mole) of (D)-5'-benzoyloxy-5-hydroxymethylbutyrolactone, 1, in 50 mL of dry tetrahydrofuran. After 20 min at 0 C the reaction was warmed to 22 C. The reaction was stirred at 22C for 16 hrs, then worked up by slowly adding 12 mL of water and refluxing for 30 min. The reaction mixture was cooled to 0C followed by the slow addition of 24 mL of 30%
hydrogen peroxide maintaining the pH between 7-8 with lN
sodium hydroxide. After the addition the reaction was evaporated under reduced pressure on a rotovapor at 30C to an oily residue. This was partitioned between 500 mL
dichloromethane and 150 mL water. The aqueous layer was extracted wtih 2X 100 mL dichloromethane and then the combined organic layers were washed with 50 mL water, dried ~ J
...,~, ` 1335187 over anhydrous sodium sulfate and evaporated to an oil.
Yield = 15.3 g (100%) of 5-0-benzoyl-2,3-dideoxypento-furanose. The H-NMR was consistent for structure and the oil was used directly in the next step.
Example 2:
l-O-Acetyl-5-0-benzoyl-2,3-dideoxypentofuranose. (3~ A
solution of 5-0-benzoyl-2,3-dideoxypentofuranose (15.3 g, 0.069 mole) in 32 mL of pyridine and 16 mL of acetic anhydride was stirred at 22C for 4 hrs then diluted with 500 mL dichloromethane and 100 g of ice was added. The reaction mixture was then washed with 3 X 100 mL lN
hydrochloric acid 3 X 100 mL saturated aqueous sodium bicarbonate and 100 mL of brine. The organic layer was dried over sodium sulfate and evaporated to yield a pale yellow oil. This could be used as is or chromatographed on silica gel 35% ---> 60% EtOAc/hexane. Yield: 15.9 g (87%) of 1-0-acetyl-5-0-benzoyl-2,3-dideoxy-pentofuranose. Its NMR was consistent for structure.
Example 3:
5'-0-benzoyl-2',3-dideoxyadenosine. (4) 1-0-acetyl-5-0-benzoyl-2,3-pentofuranose (0.522 g, 0.00198 mole) was dissolved in 5 mL of 1,2-dichloroethane. To this was added 276 ~L (1.2 eq) of trimethylsilylbromide. This was stirFed at 22 C for 15 min prior to the addition of 11.9 - * ~
~- -;CT-1907A
mL of a 0.2 M solution of bis-silyladenine in~
1,2-dichloroethane. The solution was stirred for 88 hrs at 22 C. The reaction was then worked up by cooling to 0C, followed by pouring into 40 mL of cold saturated aqueous sodium bicarbonate. The reaction mixture was partitioned between 200 mL dichloromethane and 2 X 40 mL cold saturated aqueous sodium bicarbonate and 40 mL brine. The organic layer was dried and evaporated to yield a colorless oil that was chromatographed on silica gel and eluted with 8%
methanol in methylene chloride. Fractions were collected and similar ones pooled to yield 420 mg 5'-0-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 170 mL of methanol saturated with ammonia.
This was tightly stoppered and stirred at 18C for 48 hrs.
TLC indicated that reaction was incomplete. The solvent was evaporated and replaced with 170 mL of fresh ammonia solution in methanol. TLC after an additional 48 hr implied the reaction was complete. Hence, the solvent was evaporated and ethanol was added (5 mL). This yielded colorless crystals which were collected and washed with (5 mL) of 95% ethanol to yield 1.20 g (95%) of 2',3'-dideoxyadenosine 5a together with its a anomer 5b in a 1:1 ratio by lH-NMR. The a+~ 2',3'-dideoxyadenosine mixture ~ ~ - 34 -~ :". .~ i, was dissolved in 50 mL of deionized water and to this solution was added 10 mg of adenosine deaminase (type II, from Sigma; 9 units/mg ~ .9 ~mole/min). The solution was stirred at 20C and reaction monitored by HPLC, After 2 hrs 30 min another 10 mg of adenosine deaminase was added. HPLC
showed the reaction was nearly complete after 3 hrs. The reaction was then concentrated (1-2 mL) at 35C to give an oil. The oil was scratched and slowly diluted with 2 X 1.5 mL portions of methanol which produced white crystals.
After 15 min the material was filtered and the colorless crystals washed with 2 X 1.5 mL portions of methanol to yield 2',3'-dideoxyinosine (120 mg, 48%). The mother liquor was treated as above to yield an additional 29 mg (12%) of good quality product, which was characterized by lH-NMR.
The nuclear magnetic resonance spectrum was consistent with the structure.
s.
. ,,~3~.
REFERENCES: ~
1. Samukov, V.V.; Ofitserov, V.I. Bioorg. Khim. 1983, 9, 132.
2. Prisbe, E.J.;- Martin, J.C. synth. Commun. 1985, 15, 401.
3. Taniguchi, M.; Koga, K.; Yamada, S. Tetrahedron 1974, 30, 3547.
4. Spry, D. V.; Bhala, A. R. Heterocycles, 1985, 23, 1901.
5. Secrist, J. A. et al. J. Med. Chem. 1987, 30, 746.
6. Gillard, J. W.; Israel, M. Tet. Letters 1981, 513.
~ 36 -~.,
~ 36 -~.,
Claims (17)
1. A process for selectively producing .beta.-2',3'-dideoxyinosine represented by the formula Formula A
wherein I is the purine base, hypoxanthine, which comprises the steps of:
(a) converting a .gamma.-carboxy-.gamma.-butyrolactone of the formula Formula B
to a 5-0-hydroxy-protecting group-methyl-.gamma.-butyrolactone of the formula Formula C
wherein R is a hydroxy-protecting group, by reacting the compound of Formula B with carboxyl group reducing agent followed by reacting the resulting hydroxymethyl group with a hydroxy-protecting group reagent;
(b) converting the intermediate from step (a), Formula B, to the 5-0-hydroxy-protecting group-methyl-2,3-dideoxy-pentofuranose of the formula Formula D
wherein R is a hydroxy-protecting group, by reacting the compound of Formula C with a carbonyl group reducing agent;
(c) converting the intermediate from step (b), Formula D, to a 1-0-activating-group-5-0-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula Formula E
wherein R is a hydroxy-protecting group and A is an O-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups, wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and C1-C3 alkoxy groups, by reacting the compound of Formula D with an acylating or sulfonating or carbonylating agent corresponding to the group A above;
(d) converting the intermediate from step (c), Formula E, by reaction with a compound of the formula MX to the 1-leaving-group-5-0-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula Formula F
wherein R is a hydroxy-protecting group and X is a leaving group selected from Cl and Br by reacting the compound of Formula E with a compound of the formula MX wherein M is selected from H and (CH3)3Si;
(e) reacting the intermediate from step (d), Formula F
with an activated adenine derivative wherein the base, adenine, has been activated by means of reacting the pendant N-6 amino group and N-9 nitrogen atom on the nucleus of the given base with an activating compound selected from silylating and acetylating and benzyoylating agents, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of a solvent selected from polar and non-polar solvents; and (f) subjecting the intermediate from step (e) to chemical reaction effective to displace the 5-0-hydroxy-protecting group to obtain an anomeric mixture of .beta.-and .alpha.-2',3'-dideoxyadenosine;
(g) contacting the mixture of .beta.- and .alpha.-2',3'-dideoxyadenosine from step (f) with the enzyme, adenosine deaminase, to effect displacement of the amino group pendant to the 6-position of the purine (adenosine) ring system selectively in the .beta.-2',3'-dideoxyadenosine with a hydroxyl group whereby adenosine is converted to inosine; and (h) recovering the dideoxyinosine from step (e) above.
wherein I is the purine base, hypoxanthine, which comprises the steps of:
(a) converting a .gamma.-carboxy-.gamma.-butyrolactone of the formula Formula B
to a 5-0-hydroxy-protecting group-methyl-.gamma.-butyrolactone of the formula Formula C
wherein R is a hydroxy-protecting group, by reacting the compound of Formula B with carboxyl group reducing agent followed by reacting the resulting hydroxymethyl group with a hydroxy-protecting group reagent;
(b) converting the intermediate from step (a), Formula B, to the 5-0-hydroxy-protecting group-methyl-2,3-dideoxy-pentofuranose of the formula Formula D
wherein R is a hydroxy-protecting group, by reacting the compound of Formula C with a carbonyl group reducing agent;
(c) converting the intermediate from step (b), Formula D, to a 1-0-activating-group-5-0-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula Formula E
wherein R is a hydroxy-protecting group and A is an O-activating group selected from alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, arylthiocarbonyl, alkylsulfonyl, arylsulfonyl and carbonate groups, wherein the alkyl moiety may be an unsubstituted or substituted C1-C3 alkyl group and the aryl moiety may be an unsubstituted or substituted phenyl group and wherein the substituent on the alkyl and aryl moieties may be selected from 1 to 3 groups selected from halo and C1-C3 alkoxy groups, by reacting the compound of Formula D with an acylating or sulfonating or carbonylating agent corresponding to the group A above;
(d) converting the intermediate from step (c), Formula E, by reaction with a compound of the formula MX to the 1-leaving-group-5-0-hydroxy-protecting group-methyl-2,3-dideoxypentofuranose of the formula Formula F
wherein R is a hydroxy-protecting group and X is a leaving group selected from Cl and Br by reacting the compound of Formula E with a compound of the formula MX wherein M is selected from H and (CH3)3Si;
(e) reacting the intermediate from step (d), Formula F
with an activated adenine derivative wherein the base, adenine, has been activated by means of reacting the pendant N-6 amino group and N-9 nitrogen atom on the nucleus of the given base with an activating compound selected from silylating and acetylating and benzyoylating agents, in the presence of one of a Bronsted acid and a Lewis acid and in the presence of a solvent selected from polar and non-polar solvents; and (f) subjecting the intermediate from step (e) to chemical reaction effective to displace the 5-0-hydroxy-protecting group to obtain an anomeric mixture of .beta.-and .alpha.-2',3'-dideoxyadenosine;
(g) contacting the mixture of .beta.- and .alpha.-2',3'-dideoxyadenosine from step (f) with the enzyme, adenosine deaminase, to effect displacement of the amino group pendant to the 6-position of the purine (adenosine) ring system selectively in the .beta.-2',3'-dideoxyadenosine with a hydroxyl group whereby adenosine is converted to inosine; and (h) recovering the dideoxyinosine from step (e) above.
2. A process according to claim 1 wherein the reducing agent used in steps (a) and (b) is selected from BH3 (B2H6), BH3 SMe2, [(CH3)2CHCH(CH3)2]BH, NaBH4, NaBH4 plus one of LiCl and AlCl3 and BF3, LiAlH4, LiAlH(OMe)3, and LiAlH(O-?-BU)3.
3. A process according to claim 1 wherein the reducing agent used in step (b) is [(CH3)2CHCH(CH3)2]BH.
4. A process according to claim 1 wherein the hydroxy-protecting group used in step (a) is selected from unsubstituted and substituted benzyl, trialkylsilyl, alkylarylsilyl, unsubstituted and substituted alkyl, vinyl, benzoyl, mesitoyl, pivaloyl, unsubstituted and substituted acetoxy, and carbonate groups.
5. A process according to claim 5 wherein the hydroxy-protecting group is benzoyl.
6. A process according to claim 1 wherein the O-activating group used in step (c) is selected from alkylcarbonyl and arylcarbonyl.
7. A process according to claim 6 wherein the O-activating group used in step (c) is selected from acetyl and benzoyl.
8. A process according to claim 1 wherein the intermediate from step (c) is reacted with (CH3)3SiBr as the compound of formula MX.
9. A process according to claim 1 wherein in step (e) the adenine base component, activated by reaction with a halotrialkylsilane as the silylating agent wherein the halo is selected from bromo and iodo, is reacted with the 1-leaving group-5-0-protecting group-2',3'-dideoxypentafuranose intermediate having Formula F from step (d) to obtain the 5'-0-hydroxy-protecting group -2',3'-dideoxyadenosine intermediate.
10. A process according to claim 9 wherein the reaction in step (e) is carried out in a non-polar solvent selected from CHCl3, CH2Cl2, ClCH2-CH2Cl, and CCl4.
11. A process according to claim 1 wherein the reactions of steps (d) and (e) are carried out in one pot by the sequential addition of a halogenating agent and an activated adenine derivative to the intermediate from step (c).
12. A process according to claim 11 wherein the halogenating agent is bromotrimethylsilane and wherein the activated adenine derivative is a bis-silyladenine.
13. A process according to claim 1 wherein step (f) the intermediate from step (e), wherein the protecting group is benzoyl, is reacted with methanol saturated with ammonia to displace the 5-0-hydroxy-protecting group.
14. A process according to claim 1 wherein step (g) the enzyme, adenosine deaminase, is used in a form selected from a solution of the enzyme in a neutral aqueous medium and an immobilized enzyme preparation.
CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE
CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE
15. An improved method for selectively producing .beta.-(D)-2',3'-dideoxyinosine which comprises treatment of an .alpha.
-and .beta.-anomeric mixture of (D)-2',3'-dideoxyadenosine with adenosine deaminase while monitoring conversion of the preferntially deaminated .beta.-anomer so that reduction time is controlled minimizing .alpha.-anomer deamination, thereby producing .beta.-(D)-2',3'-dideoxyinosine which is recovered from the enzymatic reaction mixture.
-and .beta.-anomeric mixture of (D)-2',3'-dideoxyadenosine with adenosine deaminase while monitoring conversion of the preferntially deaminated .beta.-anomer so that reduction time is controlled minimizing .alpha.-anomer deamination, thereby producing .beta.-(D)-2',3'-dideoxyinosine which is recovered from the enzymatic reaction mixture.
16. The method of claim 1 wherein the adenosine deaminase is dissolved in a neutral aqueous medium for contact with (D)-2',3'-dideoxyadenosine anomeric mixture.
17. The method of claim 1 wherein an immobilized adenosine deaminase preparation is utilized in the deamination process.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US7484487A | 1987-07-17 | 1987-07-17 | |
| US074,844 | 1987-07-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1335187C true CA1335187C (en) | 1995-04-11 |
Family
ID=22122017
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000572184A Expired - Lifetime CA1335187C (en) | 1987-07-17 | 1988-07-15 | Dideoxyinosine by enzymatic deamination of dideoxyadenosine |
Country Status (8)
| Country | Link |
|---|---|
| KR (1) | KR930005870B1 (en) |
| AT (1) | AT395977B (en) |
| CA (1) | CA1335187C (en) |
| ES (1) | ES2007532A6 (en) |
| FI (1) | FI92602C (en) |
| GR (1) | GR1000483B (en) |
| LU (1) | LU87280A1 (en) |
| PT (1) | PT88017B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GR861255B (en) * | 1985-05-15 | 1986-09-16 | Wellcome Found | Therapeutic nucleosides |
| EP0285884A3 (en) * | 1987-03-20 | 1990-07-04 | Bristol-Myers Squibb Company | process to produce 2', 3'-dideoxynucleosides |
-
1988
- 1988-07-04 AT AT0173088A patent/AT395977B/en not_active IP Right Cessation
- 1988-07-08 GR GR880100455A patent/GR1000483B/en not_active IP Right Cessation
- 1988-07-13 FI FI883339A patent/FI92602C/en active IP Right Grant
- 1988-07-15 ES ES8802250A patent/ES2007532A6/en not_active Expired
- 1988-07-15 LU LU87280A patent/LU87280A1/en unknown
- 1988-07-15 PT PT88017A patent/PT88017B/en not_active IP Right Cessation
- 1988-07-15 KR KR1019880008865A patent/KR930005870B1/en not_active IP Right Cessation
- 1988-07-15 CA CA000572184A patent/CA1335187C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| FI92602B (en) | 1994-08-31 |
| GR1000483B (en) | 1992-07-30 |
| AT395977B (en) | 1993-04-26 |
| FI883339A0 (en) | 1988-07-13 |
| LU87280A1 (en) | 1989-03-08 |
| FI883339A (en) | 1989-01-18 |
| ES2007532A6 (en) | 1989-06-16 |
| KR930005870B1 (en) | 1993-06-25 |
| FI92602C (en) | 1994-12-12 |
| PT88017A (en) | 1989-06-30 |
| GR880100455A (en) | 1989-04-12 |
| KR900001858A (en) | 1990-02-27 |
| ATA173088A (en) | 1992-09-15 |
| PT88017B (en) | 1995-03-01 |
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