U.S. patent application number 09/987845 was filed with the patent office on 2002-05-16 for method and compositions for the synthesis of dioxolane nucleosides with beta-configuration.
This patent application is currently assigned to BIOCHEM PHARMA INC.. Invention is credited to Bednarski, Krzysztof, Cimpoia, Alex, Mansour, Tarek.
Application Number | 20020058670 09/987845 |
Document ID | / |
Family ID | 10785435 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058670 |
Kind Code |
A1 |
Mansour, Tarek ; et
al. |
May 16, 2002 |
Method and compositions for the synthesis of dioxolane nucleosides
with beta-configuration
Abstract
The present invention relates to methods and compositions for
preparing biologically important nucleoside analogues containing
1,3-dioxolane sugar rings. In particular, this invention relates to
the stereoselective synthesis of the beta (cis) isomer by
glycosylating the base with an intermediate of formula (II) below a
temperature of about -10.degree. C. 1 wherein R.sub.1 and L are as
defined herein.
Inventors: |
Mansour, Tarek; (Montreal,
CA) ; Cimpoia, Alex; (Montreal, CA) ;
Bednarski, Krzysztof; (Laval, CA) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 600
WASHINGTON
DC
20036
US
|
Assignee: |
BIOCHEM PHARMA INC.
|
Family ID: |
10785435 |
Appl. No.: |
09/987845 |
Filed: |
November 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09987845 |
Nov 16, 2001 |
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09532739 |
Mar 22, 2000 |
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09532739 |
Mar 22, 2000 |
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09251431 |
Feb 17, 1999 |
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6069250 |
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09251431 |
Feb 17, 1999 |
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08849722 |
Jun 26, 1997 |
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5922867 |
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08849722 |
Jun 26, 1997 |
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PCT/CA96/00845 |
Dec 13, 1996 |
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Current U.S.
Class: |
514/263.2 ;
514/256; 514/269; 544/276; 544/277; 544/309 |
Current CPC
Class: |
Y02P 20/55 20151101;
C07D 405/04 20130101 |
Class at
Publication: |
514/263.2 ;
514/256; 514/269; 544/276; 544/277; 544/309 |
International
Class: |
A61K 031/522; A61K
031/513; C07D 473/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 1995 |
GB |
9525606.1 |
Claims
We claim:
1. A process for producing a .beta.-nucleoside analogue compound of
formula (III): 20and salts thereof, wherein R.sub.1 is a hydroxyl
protecting group; and R.sub.2 is a purine or pyrimidine base or an
analogue or derivative thereof, the process comprising
glycosylating said purine or pyrimidine base at a temperature below
about -10.degree. C., with an intermediate of formula (II):
21wherein L is halogen.
2. The process according to claim 1, wherein L is iodo.
3. The process according to claim 2, wherein R.sub.1 is benzyl.
4. The process according to claim 1, wherein R.sub.2 is selected
from the group consisting of 22wherein R.sub.3 is selected from the
group consisting of hydrogen, C.sub.1-6 alkyl and C.sub.1-6 acyl
groups; R.sub.4 and R.sub.5 are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, bromine, chlorine,
fluorine, and iodine; R.sub.6 is selected from the group of
hydrogen, halogen, cyano, carboxy, C.sub.1-6 alkyl, C.sub.1-6
alkoxycarbonyl, C.sub.1-6 acyl, C.sub.1-6 acyloxy, carbamoyl, and
thiocarbamoyl; and X and Y are independently selected from the
group of hydrogen, bromine, chlorine, fluorine, iodine, amino, and
hydroxyl groups.
5. The process according to claim 1, wherein R.sub.2 is 23wherein
R.sub.3 is selected from the group consisting of hydrogen,
C.sub.1-6 alkyl and C.sub.1-6 acyl groups; and R.sub.4 is selected
from the group consisting of hydrogen, C.sub.1-6 alkyl, bromine,
chlorine, fluorine, and iodine.
6. The process according to claim 5, wherein R3 is H or acetyl and
R4 is H or F.
7. The process according to claim 1, wherein the glycosylation
reaction occurs at a temperature below about -15.degree. C.
8. The process according to claim 1, wherein the glycosylation
reaction occurs at a temperature below about -20.degree. C.
9. The process according to claim 1, wherein the glycosylation
reaction occurs at a temperature below about -50.degree. C.
10. The process according to claim 1, wherein the glycosylation
reaction occurs at about -78.degree. C.
11. The process according to any one of claims 7 to 10 wherein L is
iodo.
12. The process according to claim 11 wherein R.sub.1 is
benzyl.
13. The process according to claim 1, wherein the compound of
formula (III) is subsequently deprotected to give a compound of
formula (I) 24wherein R.sub.2 is a purine or pyrimidine base or an
analogue or derivative thereof.
14. The process according to claim 1, wherein the intermediate of
formula (II) is prepared by reacting an intermediate of formula
(II') 25wherein L' is a leaving group; with a Lewis acid of formula
(IV) 26wherein R.sub.3, R.sub.4 and R.sub.5 are independently
selected from the group consisting of hydrogen; C.sub.1-20 alkyl
(e.g. methyl, ethyl, ethyl, t-butyl), optionally substituted by
halogens (F, Cl, Br, I), C.sub.6-20 alkoxy (e.g., methoxy) or
C.sub.6-20 aryloxy (e.g., phenoxy); C.sub.7-20 aralkyl (e.g.,
benzyl), optionally substituted by halogen, C.sub.1-20 alkyl or
C.sub.1-20 alkoxy (e.g., p-methoxybenzyl); C.sub.6-20 aryl (e.g.,
phenyl), optionally substituted by halogens, C.sub.1-20 alkyl or
C.sub.1-20 alkoxy; trialkylsilyl; fluoro; bromo; chloro and iodo;
and R.sub.6 is selected from the group consisting of halogen (F,
Cl, Br, I); C.sub.1-20 sulphonate esters, optionally substituted by
halogens (e.g., trifluoromethane sulphonate); C.sub.1-20 alkyl
esters, optionally substituted by halogen (e.g., trifluoroacetate);
polyvalent halides (e.g., triiodide); trisubstituted silyl groups
of the general formula (R.sub.3) (R.sub.4) (R.sub.5)Si (wherein
R.sub.3, R.sub.4, R.sub.5 are as defined above); saturated or
unsaturated selenenyl C.sub.6-20 aryl; substituted or unsubstituted
C.sub.6-20 arylsulphenyl; substituted or unsubstituted C.sub.1-20
alkoxyalkyl; and trialkylsiloxy.
15. The process according to claim 14, wherein the Lewis acid is
selected from TMSI and SiH.sub.2I.sub.2.
16. The process according to claim 15, wherein the Lewis acid is
TMSI.
17. The process according to claim 14, wherein L is iodo.
18. The process according to claim 17, wherein R.sub.1 is
benzyl.
19. The process according to any one of claims 14 to 18, wherein
the compound of formula (III) is subsequently deprotected to give a
compound of formula (I) 27wherein R.sub.2 is a purine or pyrimidine
base or an analogue or derivative thereof.
20. The process according to claim 19, wherein the glycosylation
reaction occurs at a temperature below about -15.degree. C.
21. The process according to claim 19, wherein the glycosylation
reaction occurs at a temperature below about -20.degree. C.
22. The process according to claim 19, wherein the glycosylation
reaction occurs at a temperature below about -50.degree. C.
23. The process according to claim 19, wherein the glycosylation
reaction occurs at about -78.degree. C.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for preparing nucleoside analogues containing dioxolane sugar
rings. In particular, the invention relates to the stereoselective
synthesis 1,3-dioxolane nucleosides having .beta. or cis
configuration.
BACKGROUND OF THE INVENTION
[0002] Nucleosides and their analogues represent an important class
of chemotherapeutic agents with antiviral, anticancer,
immunomodulatory and antibiotic activities. Nucleoside analogues
such as 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine
(ddI), 2',3'-dideoxycytidine (ddC),
3'-deoxy-2',3'-didehydrothymidine (d.sub.4T) and
(-)-2'-deoxy-3'-thiacytidine (3TC.TM.) are clinically approved for
the treatment of infections caused by the human immunodeficiency
viruses. 2'-Deoxy-2'-methylidenecytidine (DMDC, Yamagami et al.
Cancer Research 1991, 51, 2319) and 2'-deoxy-2',2'-difluorocytidine
(gemcytidine, Hertel et al. J. Org. Chem. 1988, 53, 2406) are
nucleoside analogues with antitumor activity. A number of C-8
substituted guanosines such as 7-thia-8-oxoguanosine (Smee et al.
J. Biol. Response Mod. 1990, 9, 24) 8-bromoguanosine and
8-mercaptoguanosine (Wicker et al. Cell Immunol. 1987, 106, 318)
stimulate the immune system and induce the production of
interferon. All of the above biologically active nucleosides are
single enantiomers.
[0003] Recently, several members of the 3'-heterosubstituted class
of 2',3'-dideoxynucleoside analogues such as 3TC.TM. (Coates et al.
Antimicrob. Agents Chemother. 1992, 36, 202), (-)-FTC (Chang et al.
J. Bio. Chem. 1992, 267, 13938-13942) (-)-dioxolane C (Kim et al.
Tetrahedron Lett. 1992, 33, 6899) have been reported to possess
potent activity against HIV and HBV replication and possess the
.beta.-L absolute configuration. (-)-Dioxolane C has been reported
to possess antitumor activity (Grove et al. Cancer Res. 1995, 55,
3008-3011). The dideoxynucleoside analogues (-)-dOTC and (-)-dOTFC
(Mansour et al. J. Med. Chem. 1995, 38, 1-4) were selective in
activity against HIV-1.
[0004] For a stereoselective synthesis of nucleoside analogues, it
is essential that the nucleobase be introduced predominately with
the desired relative stereochemistry without causing anomerization
in the carbohydrate portion. One approach to achieve this is to
modify the carbohydrate portion of a preassembled nucleoside by a
variety of deoxygenation reactions (Chu et al. J. Org. Chem. 1989,
54, 2217-2225; Marcuccio et al. Nucleosides Nucleotides 1992, 11,
1695-1701; Starrett et al. Nucleosides Nucleotides 1990, 9,
885-897, Bhat et al. Nucleosides Nucleotides 1990, 9, 1061-1065).
This approach however is limited to the synthesis of those
analogues whose absolute configuration resembles that of the
starting nucleoside and would not be practical if lengthy
procedures are required to prepare the starting nucleoside prior to
deoxygenation as would be the case for .beta.-L dideoxynucleosides.
An alternative approach to achieve stereoselectivity has been
reported which requires assembling the nucleoside analogue by a
reaction of a base or its synthetic precursor with the carbohydrate
portion under Lewis acid coupling procedures or SN-2 like
conditions.
[0005] It is well known in the art that glycosylation of bases to
dideoxysugars proceed in low stereoselectivity in the absence of a
2'-substituent on the carbohydrate rings capable of neighboring
group participation. Okabe et al. (J. Org. Chem. 1988, 53,
4780-4786) reported the highest ratio of .beta.:.alpha. isomers of
ddC of 60:40 with ethylaluminium dichloride as the Lewis acid.
However, with a phenylselenenyl substituent at the C-2 position of
the carbohydrate (Chu et al. J. Org. Chem. 1980, 55, 1418-1420;
Beach et al. J. Org. Chem. 1992, 57, 3887-3894) or a phenylsulfenyl
moiety (Wilson et al. Tetrahedron Lett. 1990, 31, 1815-1818) the
.beta.:.alpha. ratio increases to 99:1. To overcome problems of
introducing such substituents with the desired
.alpha.-stereochemistry, Kawakami et al. (Nucleosides Nucleotides
1992, 11, 1673-1682) reported that disubstitution at C-2 of the
sugar ring as in 2,2-diphenylthio-2,3-dideoxyribose affords
nucleosides in the ratio of .beta.:.alpha.=80:20 when reacted with
silylated bases in the presence of trimethylsilyltriflate (TMSOTf)
as a catalyst. Although this strategy enabled the synthesis of the
.beta.-anomer, removal of the phenylthio group proved to be
problematic.
[0006] Due to the limited generality in introducing the C-2
substituent stereoselectively, synthetic methodologies based on
electrophilic addition of phenyl sulfenyl halides or
N-iodosuccinimides and nucleobases to furanoid glycal intermediates
have been reported (Kim et al. Tetrahedron Lett. 1992, 33,
5733-5376; Kawakami et al. Heterocycles 1993, 36, 665-669; ; Wang
et al. Tetrahedron Lett. 1993, 34, 4881-4884; El-laghdach et al.
Tetrahedron Lett. 1993, 34, 2821-2822). In this approach, the
2'-substituent is introduced in situ however, multistep procedures
are needed for removal of such substituents.
[0007] SN-2 like coupling procedures of 1-chloro and 1-bromo
2,3-dideoxysugars have been investigated (Farina et al. Tetrahedron
Lett. 1988, 29, 1239-1242; Kawakami et al. Heterocycles 1990, 31,
2041-2053). However, the highest ratio of .beta. to .alpha.
nucleosides reported is 70:30 respectively.
[0008] In situ complexation of metal salts such as SnCl.sub.4 or
Ti(O-Pr).sub.2Cl.sub.2 to the .alpha.-face of the sugar precursor
when the sugar portion is an oxathiolanyl or dioxolanyl derivative
produces .beta.-pyrimidine nucleosides (Choi et al. J. Am. Chem.
Soc. 1991, 113, 9377-9379). Despite the high ratio of .beta.- to
.alpha.-anomers obtained in this approach, a serious limitation
with enantiomerically pure sugar precursor is reported leading to
racemic nucleosides (Beach et al. J. Org. Chem. 1992, 57,
2217-2219; Humber et al. Tetrahedron Lett. 1992, 32, 4625-4628;
Hoong et al. J. Org. Chem. 1992, 57, 5563-5565). In order to
produce one enantiomeric form of racemic nucleosides, enzymatic and
chemical resolution methods are needed. If successful, such methods
would suffer from a practical disadvantage of wasting half of the
prepared material.
[0009] As demonstrated in the above examples, the art lacks an
efficient method to generate .beta.-nucleosides. In particular,
with sugar precursors carrying a protected hydroxymethyl group at
C-4', low selectivity is encountered during synthesis of
.beta.-isomers or racemization problems occur. Specifically, the
art lacks a method of producing stereoselectively dioxolanes from
sugar intermediates carrying a C-2 protected hydroxymethyl moiety
without racemization. Therefore, a general stereoselective
synthesis of biologically active .beta.-nucleoside analogues is an
important goal.
[0010] International patent application publication no. WO92/20669
discloses a method of producing dioxolanes stereoselectively by
coupling sugar intermediates carrying C-2 ester moieties with
silylated nucleobases and subsequently reducing the C-2 ester group
to the desired hydroxymethyl group. However, over reduction
problems in the pyrimidine base have been disclosed (Tse et al.
Tetrahedron Lett. 1995, 36, 7807-7810).
[0011] Nucleoside analogues containing 1,3-dioxolanyl sugars as
mimetics of 2',3'-dideoxyfuranosyl rings have been prepared by
glycosylating silylated purine and pyrimidine bases with
1,3-dioxolanes containing a C-2 hydroxymethyl and C-4 acetoxy
substituents. The crucial coupling reaction is mediated by
trimethylsilytriflate (TMSOT.sub.f) or iodotrimethylsilane (TMSI)
and produces a mixture of .beta. and .alpha.-anomers in 1:1 ratio
(Kim et al. J. Med. Chem. 1992, 35, 1987-1995 and J. Med. Chem.
1993, 36, 30-37; Belleau et al. Tetrahedron Lett. 1992, 33,
6948-6952; and Evans et al. Tetrahedron Asymmetry 1992, 4,
2319-2322). By using metal salts as catalysts the .beta.-nucleoside
is favoured (Choi et al. J. Am. Chem. Soc. 1991, 113, 9377-9379)
but racemization or loss of selectivity become a serious limitation
(Jin et al. Tetrahedron Asymmetry 1993, 4, 2111-2114).
SUMMARY OF THE INVENTION
[0012] According to an aspect of the present invention, there is
provided a process for producing a .beta.-nucleoside analogue
compound of formula (III): 2
[0013] and salts thereof, wherein R.sub.1 is a hydroxyl protecting
group; and R.sub.2 is a purine or pyrimidine base or an analogue
thereof, the process comprising glycosylating said purine or
pyrimidine base at a temperature below about -10.degree. C., with
an intermediate of formula (II): 3
[0014] wherein L is halogen.
[0015] Subsequent to glycosylation, the compound of formula (III)
may then undergo deprotection of the hydroxyl protecting group
R.sub.1 to give a 1,3-dioxolane nucleoside analogue of formula (I)
4
[0016] wherein R.sub.2 is as previously defined.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a novel method for producing
dioxolane nucleoside analogues by coupling sugar precursors
carrying a C-2 protected hydroxymethyl group with purine or
pyrimidine nucleobases in high yield and selectivity in favour of
the desired .beta.-isomers.
[0018] A <<nucleoside >> is defined as any compound
which consists of a purine or pyrimidine base or analogue or
derivative thereof, linked to a pentose sugar.
[0019] A <<nucleoside analogue or derivative>> as used
hereinafter is a compound containing a 1,3-dioxolane linked to a
purine or pyrimidine base or analog thereof which may be modified
in any of the following or combinations of the following ways: base
modifications, such as addition of a substituent (e.g.
5-fluorocytosine) or replacement of one group by an isosteric group
(e.g. 7-deazaadenine); sugar modifications, such as substitution of
hydroxyl groups by any substituent or alteration of the site of
attachment of the sugar to the base (e.g. pyrimidine bases usually
attached to the sugar at the N-1 site may be, for example, attached
at the N-3 or C-6 site and purines usually attached at the N-9 site
may be, for example, attached at N-7.
[0020] A purine or pyrimidine base means a purine or pyrimidine
base found in naturally occurring nucleosides. An analogue thereof
is a base which mimics such naturally occurring bases in that its
structure (the kinds of atoms and their arrangement) is similar to
the naturally occurring bases but may either possess additional or
lack certain of the functional properties of the naturally
occurring bases. Such analogues include those derived by
replacement of a CH moiety by a nitrogen atom, (e.g.
5-azapyrimidines, such as 5-azacytosine) or conversely (e.g.,
7-deazapurines, such as 7-deazaadenine or 7-deazaguanine) or both
(e.g., 7-deaza, 8-azapurines). By derivatives of such bases or
analogues are meant those bases wherein ring substituent are either
incorporated, removed, or modified by conventional substituents
known in the art, e.g. halogen, hydroxyl, amino, C.sub.1-6 alkyl.
Such purine or pyrimidine bases, analogs and derivatives are well
known to those of skill in the art.
[0021] R.sub.1 is a hydroxyl protecting group. Suitable protecting
groups include those described in detail in Protective Groups in
Organic Synthesis, Green, John, J. Wiley and Sons, New York (1981).
Preferred hydroxyl protecting groups include ester forming groups
such as C.sub.1-6 acyl i.e. formyl, acetyl, substituted acetyl,
propionyl, butanoyl, pivalamido, 2-chloroacetyl; aryl substituted
C.sub.1-6 acyl i.e. benzoyl, substituted benzoyl; C.sub.1-6
alkoxycarbonyl i.e. methoxycarbonyl; aryloxycarbonyl i.e.
phenoxycarbonyl. Other preferred hydroxyl protecting groups include
ether forming groups such as C.sub.1-6 alkyl i.e. methyl, t-butyl;
aryl C.sub.1-6 alkyl i.e. benzyl, diphenylmethyl any of which is
optionally substituted i.e. with halogen. Particularly preferred
hydroxyl protecting groups are t-butoxycarbonyl, benzoyl and benzyl
each optionally substituted with halogen. In a more particularly
preferred embodiment the R.sub.1 hydroxyl protecting group is
benzyl.
[0022] In a preferred embodiment, R.sub.2 is selected from the
group consisting of 5
[0023] wherein
[0024] R.sub.3 is selected from the group consisting of hydrogen,
C.sub.1-6 alkyl and C.sub.1-6 acyl groups;
[0025] R.sub.4 and R.sub.5 are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, bromine, chlorine,
fluorine, and iodine;
[0026] R.sub.6 is selected from the group of hydrogen, halogen,
cyano, carboxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxycarbonyl,
C.sub.1-6 acyl, C.sub.1-6 acyloxy, carbamoyl, and thiocarbamoyl;
and
[0027] X and Y are independently selected from the group of
hydrogen, bromine, chlorine, fluorine, iodine, amino, and hydroxyl
groups.
[0028] In a particularly preferred embodiment R.sub.2 is 6
[0029] wherein R.sub.3 and R.sub.4 are as previously defined.
[0030] In a particularly preferred embodiment R.sub.2 is cytosine
or an analogue or derivative thereof. Most preferably R.sub.2 is
cytosine, N-acetylcytosine or N-acetyl-5-fluorocytosine.
[0031] In preferred embodiments R.sub.3 is H. In another preferred
embodiment R.sub.3 is C.sub.1-4 acyl such as acetyl.
[0032] In preferred embodiments R.sub.4 and R.sub.5 are
independently selected from hydrogen, C.sub.1-4 alkyl such as
methyl or ethyl and halogen such as F, Cl, I or Br. In particularly
preferred embodiments R.sub.4 and R.sub.5 are hydrogen. In another
particularly preferred embodiment R.sub.4 and R.sub.5 are F.
[0033] In preferred embodiments R.sub.6 is selected from hydrogen,
halogen, carboxy and C.sub.1-4 alkyl. In particularly preferred
embodiments R.sub.6 is H, F or Cl and most preferably H.
[0034] In preferred embodiments X and Y are independently selected
from the group of H, F or Cl. In a particularly preferred
embodiment X and Y are hydrogen.
[0035] L is selected from the group consisting of fluoro, bromo,
chloro and iodo.
[0036] In a particularly preferred embodiment L is an iodo group.
In this instance, leaving group (L) may be prepared by displacement
of another leaving group (L') i.e. acetoxy with Lewis acids
containing an iodo moiety. Preferably such Lewis acids have the
formula (IV): 7
[0037] wherein R.sub.3, R.sub.4 and R.sub.5 are independently
selected from the group consisting of hydrogen; C.sub.1-20 alkyl
(e.g. methyl, ethyl, ethyl, t-butyl), optionally substituted by
halogens (F, Cl, Br, I), C.sub.6-20 alkoxy (e.g., methoxy) or
C.sub.6-20 aryloxy (e.g., phenoxy); C.sub.7-20 aralkyl (e.g.,
benzyl), optionally substituted by halogen, C.sub.1-20 alkyl or
C.sub.1-20 alkoxy (e.g., p-methoxybenzyl); C.sub.6-20 aryl (e.g.,
phenyl), optionally substituted by halogens, C.sub.1-20 alkyl or
C.sub.1-20 alkoxy; trialkylsilyl; fluoro; bromo; chloro and iodo;
and R.sub.6 is selected from the group consisting of halogen (F,
Cl, Br, I) preferably I (iodo);
[0038] L' is a leaving group capable of being displaced by an iodo
leaving group using a Lewis acid of formula (IV). Suitable leaving
groups L' include acyloxy; alkoxy; alkoxycarbonyl; amido; azido;
isocyanato; substituted or unsubstituted, saturated or unsaturated
thiolates; substituted or unsubstituted, saturated or unsaturated
seleno, seleninyl or selenonyl compounds; --OR wherein R is a
substituted or unsubstituted, saturated or unsaturated alkyl group;
a substituted or unsubstituted, aliphatic or aromatic acyl group; a
substituted or unsubstituted, saturated or unsaturated alkoxy or
aryloxy carbonyl group, substituted or unsubstituted sulphonyl
imidazolide; substituted or unsubstituted, aliphatic or aromatic
amino carbonyl group; substituted or unsubstituted alkyl imidiate
group; substituted or unsubstituted, saturated or unsaturated
phosphonate; and substituted or unsubstituted, aliphatic or
aromatic sulphinyl or sulphonyl group. In a preferred embodiment L'
is acetoxy.
[0039] In a preferred embodiment, the present invention provides a
stereoselective process for producing .beta.-nucleoside analogues
of formula (III), and salt or ester thereof, by glycosylation of
the purine or pyrimidine base or analogue or derivative thereof,
with an intermediate of formula (II) as defined previously under
low temperature conditions. Preferably, the glycosylation reaction
takes place at temperatures below -10.degree. C. i.e. about -10 to
-100.degree. C. and more preferably below -20.degree. C. In a most
preferred embodiment the glycosylation reaction occurs between
about -20 to -78.degree. C.
[0040] The intermediate of formula II is reacted with a silylated
purine or pyrimidine base, conveniently in a suitable organic
solvent such as a hydrocarbon, for example, toluene, a halogenated
hydrocarbon such as dichloromethane (DCM), a nitrite, such as
acetonitrile, an amide such as dimethylformamide, an ester, such as
ethyl acetate, an ether such as tetrahydrofuran, or a mixture
thereof, at low temperatures, such as -40.degree. C. to -78.degree.
C. Silylated purine or pyrimidine bases or analogues and
derivatives thereof may be prepared as described in WO92/20669, the
teaching of which is incorporated herein by reference. Such
silylating agents are 1,1,1,3,3,3-hexamethyldisilazane,
trimethylsilyl triflate, t-butyldimethylsilyl triflate or
trimethylsilyl chloride, with acid or base catalyst, as
appropriate. The preferred silylating agent is
1,1,1,3,3,3,-hexamethyldisilazane.
[0041] To form the compound of formula (I), appropriate
deprotecting conditions include methanolic or ethanolic ammonia or
a base such as potassium carbonate in an appropriate solvent such
as methanol or tetrahydrofuran for N-4 deacetytion.
[0042] Transfer deacetylation hydrogenolysis with a hydrogen donor
such as cyclohexene or ammonium formate in the presence of a
catalyst such as palladium oxide over charcoal are appropriate for
the removal of the 5'-aryl group.
[0043] It will be appreciated that the intermediate of formula (II)
is constituted by intermediates IIa and IIb: 8
[0044] It will be further appreciated that, if the glycosylation
step is carried out using equimolar amounts of intermediates IIa
and IIb, a racemic mixture of .beta.-nucleosides of formula I is
obtained.
[0045] It will be apparent to those of skill in the art that
separation of the resulting diastereomic mixture, for example after
the coupling reaction between compounds of formula II and a
silylated base, can be achieved by chromatography on silica gel or
crystallization in an appropriate solvent (see, for example: J.
Jacques et al. Enantiomers, Racemates and Resolutions, pp 251-369,
John Wiley and Sons, New York 1981).
[0046] However, it is preferred that glycosylation is effected
using an optically pure compound of either formula IIa or IIb,
thereby producing the desired nucleoside analog in high optical
purity.
[0047] The compounds of formula IIa or IIb exist as mixture of two
diastereomers epimeric at the C-4 centre. We have now found that a
single diastereomer, as well as any mixture of the diastereomers
comprising the compounds of formula IIa, react with silylated bases
to produce .beta.-L nucleosides in high optical purity. The base at
C-4 having the cis-stereochemistry relative to the hydroxymethyl
moiety at C-2. The rate of the reaction of the two diastereomers of
formula IIa with silylated bases may however, be different. Similar
findings exist for the intermediates of formula IIb for the
synthesis of .beta.-D nucleosides.
[0048] In a preferred embodiment, the present invention provides a
step for producing anomeric iodides of formula II by reacting known
anomeric 2S-benzyloxymethyl-1,3-dioxolane-4S and -4R acetoxy
derivatives of formula (V) with iodotrimethylsilane or diiodosilane
at low temperatures (-78.degree. C.) prior to glycosylation with
silylated pyrimidine or purine base or analogue or derivative
thereof (Scheme 1). 9
[0049] Reagents and conditions:
[0050] i) 10
[0051] Toluene TSHO/80%/2.7:1.0 cis/trans;
[0052] ii) MeOH/LiOH;
[0053] iii) Column separation;
[0054] iv) Pb(OAc).sub.4/MeCN/Py/2h/RT/80%; and
[0055] v) TMSI or SiH.sub.2I.sub.2/CH.sub.2Cl.sub.2/-78.degree.
C.
[0056] Suitable methods for producing the anomeric acetoxy
intermediate (VI) will be readily apparent to those skilled in the
art and include oxidative degradation of benzyloxymethylacetals
derived from L-ascorbic acid (Belleau et al. Tetrahedron Lett.
1992, 33, 6949-6952) or D-mannitol (Evans et al. Tetrahedron
Asymmetry 1993, 4, 2319-2322).
[0057] We have also found that the known
2S-benzyloxymethyl-1,3-dioxolane-- 4S-carboxyclic acid (V) can be
generated in preference to its 2S,4R isomer by reacting
commercially available 2,2-dimethyl-1,3-dioxolane-4S-carboxyl- ic
acid with a protected derivative of hydroxyacetaldehyde, such as
benzyloxyacetaldehyde, under acidic conditions.
[0058] In the diastereoselective process of this invention, there
is also provided the following intermediates:
[0059] 2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and
2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane;
[0060] .beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-N-4-acetyl-cytidine;
[0061] .beta.-L-5'-Benzyloxy-2'-deoxy-3'-oxacytidine;
[0062]
.beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine;
and
[0063] .beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluorocytidine.
EXAMPLE 1a
2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and
2S-Benzyloxymethyl-4S-iodo-1,- 3 dioxolane (Compound #1)
[0064] 11
[0065] A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3
dioxolane and 2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2
ratio (6 g; 23.8 mmol) was dried by azeotropic distillation with
toluene in vacuo. After removal of toluene, the residual oil was
dissolved in dry dichloromethane (60 ml) and iodotrimethylsilane
(3.55 ml; 1.05 eq) was added at -78.degree. C., under vigorous
stirring. The dry-ice/acetone bath was removed after addition and
the mixture was allowed to warm up to room temperature (15 min.).
The .sup.1H NMR indicated the formation of
2S-benzyloxymethyl-4R-iodo-1,3-dioxolane and
2S-benzyloxymethyl-4S-iodo-1- ,3 dioxolane.
[0066] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.3.65-4.25 (2H,m);
4.50-4.75 (4H,m) 5.40-5.55 (1H, overlapping triplets); 6.60-6.85
(1H, d of d); 7.20-7.32 (5H,m).
EXAMPLE 1b
2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and
2S-Benzyloxymethyl-4S-iodo-1,- 3 dioxolane (Compound #1)
[0067] 12
[0068] A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3
dioxolane and 2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2
ratio (6 g; 23.8 mmol) was dried by azeotropic distillation with
toluene in vacuo. After removal of toluene, the residual oil was
dissolved in dry dichloromethane (60 ml) and diiodosilane (2.4 ml;
1.05 eq) was added at -78.degree. C., under vigorous stirring. The
dry-ice/acetone bath was removed after addition and the mixture was
allowed to warm up to room temperature (15 min.). The .sup.1H NMR
indicated the formation of 2S-benzyloxymethyl-4R-i-
odo-1,3-dioxolane and 2S-benzyloxymethyl-4S-iodo-1,3 dioxolane.
[0069] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.3.65-4.25 (2H,m);
4.50-4.75 (4H,m) 5.40-5.55 (1H, overlapping triplets); 6.60-6.85
(1H, d of d); 7.20-7.32 (5H,m).
EXAMPLE 2
.beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-N-4-acetyl-cytidine (Compound
#2)
[0070] 13
[0071] The previously prepared iodo intermediate (example 1) in
dichloromethane, was cooled down to -78.degree. C. Persylilated
N-acetyl cytosine (1.1 eq) formed by reflux in
1,1,1,3,3,3-hexamethyl disilazane (HMDS) and ammonium sulphate
followed by evaporation of HMDS was dissolved in 30 ml of
dichloromethane and was added to the iodo intermediate. The
reaction mixture was maintained at -78.degree. C. for 1.5 hours
then poured onto aqueous sodium bicarbonate and extracted with
dichloromethane (2.times.25 ml). The organic phase was dried over
sodium sulphate, the solid was removed by filtration and the
solvent was evaporated in vacuo to produce 8.1 g of a crude
mixture. Based on .sup.1H NMR analysis, the
.beta.-L-5'-benzyl-2'-deoxy-3'-oxacytidine and its .alpha.-L isomer
were formed in a ratio of 5:1 respectively. This crude mixture was
separated by chromatography on silica-gel (5% MeOH in EtOAc) to
generate the pure .beta.-L (cis) isomer (4.48 g). Alternatively,
recrystallization of the mixture from ethanol produces 4.92 g of
pure .beta. isomer and 3.18 g of a mixture of .beta. and
.alpha.-isomers in a ratio of 1:1.
[0072] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.2.20 (3H,S,Ac);
3.87 (2H,m,H-5'), 4.25 (2H,m,H-2'); 4.65 (2H,dd,OCH.sub.2Ph); 5.18
(1H,t,H-4'); 6.23 (1H,m,H-1'); 7.12 (1H,d,H-5); 7.30-7.50
(5H,m,Ph); 8.45 (2H,m,NH+H-6).
EXAMPLE 3
.beta.-L-5'-Benzyloxy-2'-deoxy-3'-oxacytidine (Compound #3)
[0073] 14
[0074] The protected .beta.-L isomer (4.4 g) of example 2 was
suspended in saturated methanolic ammonia (250 ml) and stirred at
room temperature for 18 hours in a closed-vessel. The solvents were
then removed in vacuo to afford the deacetylated nucleoside in pure
form.
[0075] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.3.85 (2H,m,H-5');
4.20 (2H,m,H-2'); 4.65 (2H,dd,OCH.sub.2Ph); 5.18 (1H,t,H-4'); 5.43
(1H,d,H-5); 5.50-5.90 (2H,br.S,NH.sub.2); 6.28 (1H,m,H-1');
7.35-7.45 (5H,m,Ph); 7.95 (1H,d,H-6).
EXAMPLE 4
.beta.-L-2'-deoxy-3'-oxacytidine (Compound #4)
[0076] 15
[0077] .beta.-L-5'-Benzyl-2'-deoxy-3'-oxacytidine from the previous
example, was dissolved in EtOH (200 ml) followed by addition of
cyclohexene (6 ml) and palladium oxide (0.8 g). The reaction
mixture was refluxed for 7 hours then it was cooled and filtered to
remove solids. The solvents were removed from the filtrate by
vacuum distillation. The crude product was purified by flash
chromatography on silica-gel (5% MeOH in EtOAc) to yield a white
solid (2.33 g; 86% overall yield,
.alpha..sub.D.sup.22=-46.7.degree. (c=0.285; MeOH)
m.p.=192-194.degree. C.
[0078] .sup.1H NMR (300 MHz, DMSO- d.sub.6) .delta.3.63
(2H,dd,H-5'); 4.06 (2H,m,H-2'); 4.92 (1H,t,H-4'); 5.14 (1H,t,OH);
5.70 (1H,d,H-5); 6.16 (2H,dd,H-1'); 7.11-7.20 (2H,brS,NH.sub.2);
7.80 (1H,d,H-6) .sup.13C NMR (75 MHz, DMSO- d.sub.6) .delta.59.5
(C-2'); 70.72 (C-5'); 81.34 (C-4'); 93.49 (C-1'); 104.49 (C-5);
140.35 (C-4); 156.12 (C-6); 165.43 (C-2).
EXAMPLE 5
.beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine
(Compound #5)
[0079] 16
[0080] The previously prepared iodo derivatives (example 1) in
dichloromethane, was cooled down to -78.degree. C. Persylilated
N-acetyl-5-fluorocytosine (1.05 eq) formed by reflux in
1,1,1,3,3,3-hexamethyldisilazane (HMDS) and ammonium sulphate
followed by evaporation of HMDS was dissolved in 20 ml of
dichloromethane (DCM) and was added to the iodo intermediate. The
reaction mixture was maintained at -78.degree. C. for 1.5 hours
then poured onto aqueous sodium bicarbonate and extracted with
dichloromethane (2.times.25 ml). The organic phase was dried over
sodium sulphate, the solid was removed by filtration and the
solvent was evaporated in vacuo to produce 8.1 g of a crude
mixture. Based on .sup.1H NMR analysis, the
.beta.-L-5'-benzyl-2'-d- eoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine
and its .alpha.-L isomer were formed in a ratio of 5:1
respectively. This crude mixture was separated by chromatography on
silica-gel (5% MeOH in EtOAc) to generate the pure .beta.-L (cis)
isomer (4.48 g). Alternatively, recrystallization of the mixture
from ethanol produces 4.92 g of pure .beta. isomer and 3.18 g of a
mixture of .beta. and .alpha.-isomers in a ratio of 1:1.
[0081] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.2.20 (3H,S,Ac);
3.87 (2H,m,H-5'), 4.25 (2H,m,H-2'); 4.65 (2H,dd,OCH.sub.2Ph); 5.18
(1H,t,H-4'); 6.23 (1H,m,H-1'); 7.12 (1H,d,H-5); 7.30-7.50
(5H,m,Ph); 8.45 (2H,m,NH+H-6).
EXAMPLE 6
.beta.-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluorocytidine (Compound
#6)
[0082] 17
[0083] The crude mixture from previous step (example 5) was
suspended in methanolic ammonia (100 ml) and stirred for 18 hours
at room temperature in a closed reaction vessel. The solvents were
removed in vacuo to afford the deacetylated mixture which was
separated by flash chromatography on silica gel (2% to 3% MeOH in
EtOAc) to yield 1.21 g pure .beta. isomer (yield 45% with respect
to this isomer).
EXAMPLE 7
.beta.-L-2'-deoxy-3'-oxa-5-fluorocytidine (Compound #7)
[0084] 18
[0085] The deacetylated pure .beta.-L isomer (900 mg; 2.8 mmol)
prepared as described in example 6 was dissolved in EtOH (40 ml)
followed by addition of cyclohexene (3 ml) and palladium oxide
catalyst (180 mg). The reaction was refluxed for 24 hours and the
catalyst was removed by filtration. The solvents were removed from
the filtrate by vacuum distillation. The crude product was purified
by flash chromatography on silica-gel (5% to 7% MeOH in EtOAc) to
yield a white solid (530 mg; 82% yield).
(.alpha..sup.22.sub.D)=-44.18.degree. (c=0.98; MeOH).
[0086] .sup.1H NMR (300 MHz, DMSO-d.sub.6); .delta.3.62-3.71
(2H,m,H-5'); 4.03-4.13 (2H;m,H-2'); 4.91 (1H,t,H-4'); 5.32
(1H,t,OH); 6.11 (1H;t;H-1'); 7.53-7.79 (2H,b,NH.sub.2); 8.16
(1H;d,H-6); .sup.13C NMR (75 MHz, DMSO-d.sub.6); .delta.59.34
(C-2'); 70.68 (C-5'); 80.78 (C-4'); 104.53-(C-1'); 124.90, 125.22
(C-4); 134.33, 136.73 (C-5); 153.04 (C-2); 156.96, 157.09
(C-6).
EXAMPLE 8
Isomeric Purity Determination of .beta.-L-2'-deoxy-3'-oxacytidine
Nucleoside Analogues
[0087] The determination of the isomeric purity (.beta.-L versus
.alpha.-L and .beta.-L versus .beta.-D isomers) was determined on a
Waters HPLC system consisting of a 600 controller pump for solvent
delivery, 486 uv detector, 412 WISP auto sampler and a 740 Waters
integrator module. An analytical chiral reverse phase cyclobond I
RSP column (Astec, 4.6.times.250 mm i.d.) was used and packed by
the manufacturer with .beta.-cyclodextrin derivatized with
R'S-hydroxypropyl ether. The mobile phase consisted of acetonitrile
(A) and water containing 0.05% triethylamine (B) with the pH
adjusted to 7.05 by glacial acetic acid. The column was operated
under isocratic conditions at 0.degree. C. using a mixture of 5% A
and 95% B. Such conditions are modifications of those reported in
DiMarco et al. (J. Chromatography, 1993, 645, 107-114). The flow
rate was 0.22 ml/min and the pressure was maintained at 648-660
psi. Detection of nucleosides was monitored by uv absorption at 215
and 265 nm. Samples of .beta.-D isomer and racemic compounds were
prepared as reported (Belleau et al. Tetrahedron Lett 1992, 33,
6948-6952) and used for internal references and co-injection. Under
these conditions the isomeric purity of compound #4 produced
according to example 4 was >99% and that of compound #7
according to example 7, was >96%.
[0088] The isomeric purity of dioxolane nucleosides having been
prepared according to the general scheme 2, under varying
conditions i.e. temperature and Lewis acid is represented in table
1 below. Those prepared at temperatures above -10.degree. C.
exhibited reduced stereoselectivity. 19
1TABLE 1 Base Lewis acid Temperature(.degree. c.) Cis:trans
5F-N(Ac)-cytosine TMSI a:-78 b:-78 8:1 5F-N(Ac)-cytosine
SiH.sub.2I.sub.2 a:-78 b:-78 7:2 N(Ac)-cytosine TMSI a:-78 b:-78
5:1 note: all reactions in DCM solvent and bases silylated with
HMDS.
* * * * *