CA2079796A1 - Nucleoside derivatives - Google Patents
Nucleoside derivativesInfo
- Publication number
- CA2079796A1 CA2079796A1 CA002079796A CA2079796A CA2079796A1 CA 2079796 A1 CA2079796 A1 CA 2079796A1 CA 002079796 A CA002079796 A CA 002079796A CA 2079796 A CA2079796 A CA 2079796A CA 2079796 A1 CA2079796 A1 CA 2079796A1
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- CA
- Canada
- Prior art keywords
- group
- groups
- formula
- compounds
- hydrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
Abstract
Nucleoside compounds of formula (I): Y1O-G-X wherein Y1 is a hydrogen atom or an acyl or acyloxymethyl group; G is the residue of the glycone moiety of the nucleoside; and X is a purine or pyrimidine base or an ester, amide or acyloxyalkyl derivative thereof. The compounds have antiviral activity.
Description
2~797~n NUCLEOSIDE DERIVATIVES
This invention relates to antiviral compounds and more particularly to esters, ethers and amides of nucleoside derivatives which are active against human immunodeficiency virus (HIV), the retrovirus which causes the disease AIDS, or other viruses such as herpes simplex virus (~ISV).
Since the recognition of AIDS as a new clinical entity in 1981 nearly five hundred thousand cases of the disease have probably been diagnosed, while the number of HIV infected persons is estimated-to be between 5 ~~~ million and 10 million.
AIDS is fatal, more than 50% of all diagnosed cases having ended in death. HIV and AIDS are today and will remain a worldwide health problem for many years to come.
Clinical symptoms are weight loss, chronic diarrhoea, persisting fever and opportunistic infections due to loss of T-cells, thus upsetting the overall balance of the immune system. The patient loses his/her ability to combat otherwise insignificant infections.
Many substances interfering with replication ha~e been tried, e.g. 3'-azido - 3l-deoxythymidine (AZT), 2',3'-dideoxyadenosine, 3'-fluoroarabinosyladenine, 2',3'-dideoxycytidine, ~'-chloro-2l3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxy - 2',3'-didehydrothymidine, 3'-azido- 2',3',-dideoxyuridine, 3'-azido - 2',3'-dieoxy-5-ethyl-uridine, 1-(2'-deoxy-2'-fluoro-~-D-arabinofuranosyl)-5-ethyluracil, 2,6-diamino-9-(3'-azido-2',3'-dideoxy-~-D-ervthropentofuranosyl)purine, suramin, Evans Blue, fushsin acid, 5-chloro-3'-fluoro-2',3'-dideoxy-uridine, hypericin, I-aurothioglucose, carbovir, dextran sulfate, int~rferon alpha, monoclonal antibodies against the HIV
envelope, peptide T, phosphonoformate (foscarnet), ; ' ` ' ' ~' ~ , ' ; `' :
- ~:
.. ..
-: .: . : :.~ . .:., .
. : : .. .
. ~
WO91/1~98 PCT/EP91/00639 207 ~rl9 ~ 2 phosphorothioate oligodeoxynucleotides, protease inhibitors, ribavirin and soluble CD4 receptor.
European Patent Application No. 0196185A, for instanc~, describes pharmaceutical compositions containing AZT, a known compound which has shown great promise in the treatment of AIDS and AIDS- related complex. It is believed that A2T works by inhibiting reverse transcriptase.
We have now found that acylation or alkylation of oxygen atoms in the glycone (sugar moiety) or in the purine or pyrimidine ring and~or acylation or alkylation of exocylic or endocyclic nitrogen atoms present in the purine or pyrimidine ring can give signiflcant advantages in terms of uptake, overall activity and site 15 of action. Our PCT Application W088/07532 describes certain esters and amides of this type carrying acyl groups at the 5' position or on nitrogens in the aglycone moiety nitrogens; the present invention extends this principle to a wider range of related compounds.
Thus according to one feature of the invention we provide nucleosides of khe general formula ylo - G - X ~ (I) ~wherein G is the residue of the glycone moiety of the nucleoside, Y is a hydrogen atom or a physiologioally acceptable group of the formula Rl(o)nCo(oCR2R3)m-where n is O or 1, m is O or 1 and Rl is an optionally su~stituted alkyl or aryl group or an N-~C1_7 alkyI)~1,4-dihydropyridin- 3-yl group or, where n is 0, a hydrogen atom;
R2 and R3 are independently hydrogen atoms or lower alkyl groups or R2 and R3 together are an alkylidene group; and .. . ...... . . . .
.,. ,::
.
WO91/1~98 PCT/EP91/00639 3 2~7~79S
X is a group selected from O OY N~Y
Y~ ~ R5 O N N
I
(A) (B) (C) NY4 Nr4 N~ y4 o~3 ~ N ~ N ~J~
( E ) ¦ ~ F ) .~
~ 3) (where the groups y2~ y3 and Y4 are as defined for and may be the same as or different from yl or each other, R4 is a hydrogen atom or a group -NY3Y4, where Y3 and Y4 have the above meanings and R5 is a hydrogen or halogen atom or a lower alkyl or trifluoromethyl group, :. . :
'~
, , : , . . . - . .
:: - : :: ~ . ~ .
:.. , ., :
WO91/tS498 PCT/EP9t/00639 2 ~ r~
with the following provisos a) at least one of the groups yll y2~ y3 and Y4 is other than hydrogen, (b) when all of those groups y2~ y3 and Y4 which are present are hydrogen or all of those groups y2~ y3 and R5 which are present in formulae I(C), I(F) and I(G) are hydrogen and Y4 is RlCO, then yl is a group Rl~o)n.Co.(ocR2R3)m in which n and/or m is 1, (c) the glycone group - G - is not a 2',3'-dideoxyribosyl group or such a group having 3'~
fluorine or 3'-azido substituent nor a 2',3'-dehydro-dideoxyribosyl group) _ and/or salts thereof.
In general, compounds in which m and/or n is 1 are preferred, that is the group Rl(o)n.Co.(ocR2R3)- is other than a simple acyl group R CO-.
Compounds in which X is a group of formula (D), (E), (G), (H), (I) or (J) wherein the groups y2 and/or Y are other than hydrogen are of particular interest.
It will be appreciated that s;ome of the groups X, for example those in which y2 is a hydrogen atom, are tautomers of other of the groups Y and exist in equilibrium with them The glycone moiety G will normally be of the formula with the group yl in the 5'-position and the group X in the l'-position. The remaining positions may be substituted, for example, by one or more halogen atoms such as chlorine or, more particularly fluorine; or by hydroxyl, protected hydroxy or a7ido groups. There may be a bond joining the 2'- and 3'-positions to form a 2',3'-didehydro glycone. The sterochemistry of the glycone moiety may be that of any pentose but will generally be that of ribose or arabinose.
- ~
W091/1549~ PCT/~P91/&0639 2 ~ 797~i~
Preferred glycone moieties include the 2,3-dideoxy-2-halo-pentofuranosyl group, for example the 2-chloro-and 2-fluoro-analogues, especially when the group has the threo configuration:
Y ~0~, Another preferred moiety is the 2,2-difluoro-2-deoxy-pentofuranosyl group I X
y 1~
~( r50 F
where Ys is a hydrogen atom or an acyl or acyloxy-alkyl yroup as defined above for y1. It is knnwn that 2,2-difluoro-2-deoxy-nucleosides are active against herpes simplex virus (HSV) and accordingly the compounds according to the invention having this glycone moiety will ~ind application in treatment of herpes infections.
Protected hydroxy groups will in general be groups of the ~ormula ylo_ where yl has the above meaning.
According to a further feature of the invention we provide for the use of compounds of formula (I) as hereinbefore de~ined, and/or salts thereo~, in the manufacture of a composition for the treatment or prophylaxis of virus infections, in particular neurotropic viruses and especially retroviruses such as HIV. Such compositions also form part of the invention.
The group Rl is preferably an alkyl group containing 1-20 carbon atoms which may be straight or branched, or an aryl group which may contain 6 to 20 carbon atoms and may be mono- or poly-cyclic.
Substituents which may be present on the alkyl groups include aryl groups preferably having 6-lO carbon atoms .: ~
-- :~ . ~ ., :
. : .: .,. . ~. :: .
WO 91/15498 PCl/~:P91/00639 2~7~79~;
(as in aralkyl groupings), hydroxy, alkoxy and carboxy groups. Aryl groups include 5- or 6-membered heterocyclic aryl groups having one or more heteroatoms selected from O, N and S, such as furyl, imidazolyl, pyrrolyl, pyridinyl and thienyl groups. Substituents which may be present on aryl groups include alkyl groups, e.g. having 1-6 carbon atoms, hydroxy and carboxy groups. Examples of such groups include methyl, ethyl, propyl, t-butyl, pentyl, stearyl, palmityl, carboxyethyl and benzyl groups.
The lower alkyl groups R2, R3 and R5 preferably contain 1-6 carbon atoms. However, R2 preferably represents a hydrogen atom. R is preferably a hydrogen atom or more preferably a methyl group. Where R5 is a halogen atom it may be a fluorine, chlorine, bromine or iodine atom. However, R5 is preferably a hydrogen or chlorine atom or a methyl group. ~hen R2 and R3 together form an alkyidene group this suitably contai~s 1-6 car~on atoms.
Where Rl in any oE the groups yl~ y2~ y3 or Y4 is an N-alkyl-1,4-dihydropyridin-3-yl group the alkyl group is preferably methyl.
It will be noted that the compounds of the invention may carry more than one of the groups yl~ y2 Y3 and Y4. In the compounds of formula (I) D,E, I and J, it is preferred that m in the group Y4 is O (zero).
Groups Y are preferably of the ~ormula Rl CO-, RlCO.O.CR2R3 or Rl.o.co.O.CR2R --The salts of the compounds of formula (I) may be acid addition salts with organic or inorganic acids, forinstance hydrochloric or phosphoric acid or methanesulphonic acid, ethane disulphonic acid, 2-naphthylsulphonic ~cid, pivalic acid and pamoic acid.
Antiviral counter-ions such as phosphonoformate or suramin may also be used. Organic or inorganic base salts may be formed with acidic groups present in the molecule; suitable counter-ions include alkali metal .;
, .
!
WO91t15498 PCT/EP91/00639 7 2~79 l ~
ions such as sodium and potassium ions, divalent ions such as calcium and zinc ions and organic ions such as tetraalkylammonium and choline or ions derived from meglumine or ethylenediamine. Salts according to the invention may be formed by reaction of the compound of formula (I) with an appropriate acid or base.
The compositions according to the invention may be used in the treatment and/or prophylaxis of virus infections, in particular HIV infections, and such a method forms a further feature of the invention. They may be formulated in conventional manner by admixture of one or more compounds of formula (I) as defined above with excipients and/or carriers.
-~ ~ While the compounds of formula (I) may themselves ba inhibitors of reverse transcriptase when the 5'-hydroxy group is free, it is possible that they are cohverted ln vivo to the desa~yl or desalkyl nucleosides. Nevertheless the substitution at the respective O- and N- atoms gives surprising advantages in terms of uptake and sustained activity. The compounds of formula (I) are more :Lipophilic than the parent compounds and this permits rapid and efficient absorption from the gastro-intestinal tract;lthe absorption rate ma~ be optimised by careful choice of the substituent group to give the desired balance of lipophilicity and hydrophilicity. The lipophilic nature of the compunds of formula (I~ also gives the molecules the ability to penetrate the cell membranes more easily and leads to higher intracellular concentrations, giving an improved dose/effect ratio. The steady hydrolysis of the compounds ensures a sustained concentration of ~he active compound in the cell and thereby permits longer intervals between doses, overcoming a significant drawback of the prior art compounds. Finally, the compnunds according to the invention can penetrate the blood-brain barrier and thus permit treatment of the neurological disorders which have been observed to be .
.,.. , , . :
,:: :: :, , . :
- : . , , :
:. ' '. :~ ., WO91/l54g8 PCT/EP91/00639 2~7~79~; 8 related to the presence of neurotropic viruses, e.g.
retroviruses such as HIV, and lentiviruses (Yarchoan et al, The Lancet, January 17, 1987, page 132~. This is a significant advantage compared to the corresponding unsubstituted compounds or other antiviral compounds and is not referred to anywhere in the prior art. Attempts have been made to treat these neurological disorders with AZT but with limited success.
The invention thus further provides a method of treatment of neurological disorders caused by neurotropic viruses wherein an effective dose of a compound of formula (I) or a salt thereof is administered to a patient suffering from such a disorder.
Compounds of formula (I) may be prepared in any convenient way, for example, by reaction of a compound of formula (II) ylo - G - XB (II) [wherein yl is as hereinbefore dei.ined and XB is as hereinbefore defined for X except that any of the groups yl~ y2~ y3 and Y4 may each additionally represent a protecting group, with the proviso that at least one of yl~ y2/ y3 and Y4 is a hydrogen atom] with a reagent serving to introduce a group Rl(O)~CO.(OCR2R )m as defined above followed where required by removal of any protecting groups and/or unwanted substituents so introduced.
It should be noted that where, in the starting material, more than one of yl~ y2~ y3 and Y4 is hydrogen, multiple reactions may occur.
WAere it is desired to ensure that acylation or alkylation is effected while one or more groups yl~ y2~
Y3 and Y4 remain as hydrogen atoms, it may be desirable to protect the latter first, to form a compound of formula (I) in which one or more of ylr y2, y3 and Y4 r ~ ' WO91/tS498 PCT/EP9t/00639 2~797~S
are protecting groups, these being removed after introduction of the desired acyl or ether group. Such protecting groups may, in fact, be conventional N- or O-protecting groups including groups RlOCO- which may be selectively removed in the presence of the group(s) intended to remain. Thus, for example, an N-benzyloxycarbonyl may be used to protect an exocylic amino group and if the group which is intended to remain is not one which is removable by reduction, for example a straight chain alkoxycarbonyl group, the N-benzyloxycarbonyl group can readily be removed selectively using hydrogen and a noble metal catalyst such as palladium. Trisubstituted silyl groups may also be used as protecting groups, especially for the 5'-oxygen atom, and include trialkylsilyl e.g.
trimethylsilyl, dimethyl- t-butylsilyl, and thexyldimethyl silyl groups. Where it is desired that the reagent introduces a group Rl(o)n.Co.(oCR2R3)m- only into the purine or pyrimidine base then it will be convenient to protect all of the hydroxyl groups present in the glycone, if any; adjacent hydroxyl groups can be protected with a bidentate protecting group such as the 1,1,3,3-tetraisopropyldisilox-1,3-diyl group.
In general, where more than one of yl~ y2~ y3 and Y4 are hydrogen, and a mixture of compounds is produced, the individual components may readily be separated, for example by chromatography.
Where 5'-0-monoalkylation is to be ef~ected in 2',3'-dideoxy derivatives ti.e. introduction of a group yl in which m is 1) it is especially effective to form a dianion of the nucleoside (e.g. by reacting with sodium hydride) and to react this with one equivalent of the alkylating agent~ Monoalkylation of a hydroxy group other than the 5'-hydroxy group in the sugar moiety is carried in a similar fashion using a 5'-protected nucleoside. It is of course, still possible to use protected forms of the nucleoside, for example by . . , , ~:
. ~ :: , . ,.: : : .,: ~ : ., :- ; .; :
WO91/1~98 PCT/EP91/00639 2~979~;
acylation of a nucleophilic nitrogen atom before salt - formation with sodium hydride.
Suitable acylating agents for use in the reaction have the formula Ac-L where L is a leaving group. When the acyl group Ac- is derived from a carboxylic acid, i.e. is of formula Rl-CO-, then suitable acylating agents include the acid halides and acid anhydrides advantageously in the presence of a base; when the acyl group is derived from a carbonic acid, i.e. is of formula Rl.O.CO-, then acylating agents include the haloformate esters and reactive carbonic acid diesters.
In such reagents, the halogen may, for example, be chlorine or bromine. The base for use in the reaction ~ with the acid halide or anhydride may, for example, be a heterocyclic ~ase such as pyridine or 4-dimethylamino-pyridine. The latter increases the speed of the reaction and may be used advantageously with pyridine.
The reaction will normally be carri.ed out in the presence of an inert solvent e.g. a substituted amide solvent such as dimethylformamide, dimethyl~ acetamide or a halogenated hydrocarbon such als dichloromethane.
In general, we have found that: using acid anhydrides as acylating agents to i.ntroduce a group R1CO, O-acylation in the glycone takes place more readily than N-acylation, whereas using acid halides, N-acylation or even N-diacylation predominates.
However, M-acyl groups R1CO- may be removed selectively, for example by reaction with a phenol such as p-methyl-phenol. Where multiple substitution is to be effected, a stronger base such as sodium hydride may be advantageous.
Suitable acyloxyalkylating agents for use in the invention will in general be of the formula RlCo.o.CR2R3L or Rlo.Co.o.CR2R3L, where L is a leaving group. Thus, the group L may for example, be a halogen atom such as a chlorine or bromine atom or a hydrocarbon-sulphonyloxy group such as a tosyloxy or WO91/1~98 PCT/EP91/00639 2~7~
mesyloxy group.
The alkylation reaction will normally be effected in the presence of a base, conveniently an inorganic carbonate such as potassium carbonate or an alkali metal hydride such as sodium hydride. Bases as used for acylation may also be useful.
The starting compounds of formula (II) wherein yl~
y2~ y3 and Y4 are all hydrogen atoms are well described in the literature (see for example the literature references cited in the introduction hereto). Starting compounds wherein one or more O~ yll y2~ y3 and Y4 are other than hydrogen may be prepared by preliminary reactions as described above.
The following literature references are of particular interest in describing the preparation of nucleoside starting materials:
2'-Fluoro-2'-deoxyarabinofuranosylpyrimidine nucleosides:
K. A. Watanabe, U. Reichman, K. Hirota, C. Lopez, J.J.
Fox J. Med. Chem. 22 (1979)21.
5-Substituted (2-Deoxy-2-halogeno--~-D-arabinofuranosyl)cytosines and -uracils. (2-halogeno =
F, Cl, Br ara, and 2-F ribo):
K. A. Watanabe, T.-L-Su, R. S. Klein, C. K. Chu, A.
Matsuda, M. W. Chun, C. Lopez, J. J. Fox J. Med.~Chem.
26 (1983) 152~
1-(2-Deoxy-2-fluoro-~-D-arabinofuranosyl)-5-ethyl(or methyl)uracil:
M. M. Mansuri, I. Ghazzouli, M. S. Chen, H. G. Howell, P. R. Brodfuehrer, D. A. Benigni, J. C. Martin J. Med.
Chem.30 (198~)86~.
Perbenzoylated 1-(2-fluoro-2-deoxy-~-D-arabino-furanosyl)cytosine:
... : :, :
" , . .:
: :: ; ~. ~ ~ . . .
WO91/1~98 PCT/EP91/00639 2~9~9~ 12 C. H. Tann, P. H. Brodfeuhrer, S. P. Brundidge, C.
Sapino. H. G. Howell J. Orq. Chem. 50 (1985)3644.
9-(2-Deoxy-2-~-fluoroarabinofuranosyl)guanine (2'-ara-fluoroquanosine):
A. D. Borthwick, S. Butt, K. Biggadi~e, A. M. Exall, S.
M. Roberts, P. M. Yods, B. E. Kirk, B. R. Booth, J. M.
Cameron, S. W. Cox, C. L. P. Marr, M. D. Shill J. Chem.
Soc. Chem. Commun. (1988)656.
1-(2-Fluoro-2,3-dideoxy-~-D-erythiro-pentofuranosyl)-thymine and l-(2-Fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)-thymine A. V. Aerschot, P. Herdewijn, J. Balzarini, R. Pauwels, E. De Clercq J.Med.Chem.32 (1989)1743.
9-(2-Chloro-2,3-dideoxy-~-D-threo-pentofuranosyl)adenine and its bromo analogue:
P. Herdewijn, J. Balzarini, M. Baba, R. Pauwels, A. V.
Aerschot, G. Janssen, E. De Clercq J. Med. Chem.
31(1988)2040.
1-(2,3-Dideoxy-2-fluoro-~-D-arabinofuranosyl)-cytosine:
EP-A-292033 and EP-A-349928.
The pharmaceutical compositions according to the invention may be ~ormulated conventionally by means well known in the art, and may be administered by any convenient route, for instance orally, rectally, vaginailly, intraveneously or intramuscularly. Examples of suitable formulations include tablets and capsules, aqueous formulations for intravenous injection and oil-based formulations for intramuscular injection.
Suitable dosages will lie in the range 0.1 to 100mg per kilogram of bodyweight per 24 hour period. The compositions according to the invention may also contain ` other active antivirals for instance acyclovir, :;:
" ~
; . - ~ , ~ .
~ .
;`
WO91/tS4g8 PCT/EP9ltO0639 2~7~79 ~
phosphonoformate, suramin, Evans Blue, interferons or AZT.
The invention is illustxated by the following Examples.
. . _ . . .
, - . . .. .. . . . ...
2~797~
Example 1 1-(2-Fluoro-2,3-dideoxy-~-D-threo~Pentofuranosyl)-3-~ivaloyloxymethvl-thvmine 1-(2-Fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)thymine (O.2 mmol) and imidazole (O.5 mmol) are dissolved in DMF
(0.5 mmol). Thexy,ldimethylsilyl chloride (0.25 mmol) is added, and the reaction mixture is stirred at ambient temperature for 24 hours. The solvent is removed at reduced pressure, chloroform (15 ml) added to the residue, washed with water (5ml x 2) and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 1-(2-Fluoro-5-0-thexyldimethYlsily1-2,3-dideoxv-~-D-threo-pentofuranosvl~-thymine.
The product thus prepared (0.1 mmol) and potassium carbonate (0.12 mmol) are added to DMF (lml), the mixture stirred for 1.5 hours at ambient temperature, cooled to O-C, chlorom2thyl pivalate (0.12 mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish 1-(2-Fluoro-5-O-thexvl-dimethylsilyl-2,3-dideoxy-~-D-threo-pentofuranosyl)-3-Pivaloylox~neth~l-th~mine The silyl group is removed by dissolution of the product -~
thus obtained (1 mmol~ in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved in chloroform (lOml), washsd with water (2ml), dried (MgSO4), evaporated and the residue purified by preparative chromatography on silica gel plates using diethyl ether. The product is extracted from the main band by chloroform-methanol.
. . .. .
.
. ~
WO91/1~98 PCT/EP91/00639 Exam~le_2 5-Chloro-3-~-(EthYloxvcarbonYloxy~ethyl-1-(2-fluoro-5-o-Propion~1-2~3-dideox~ D-threo-pentofuranosvl)uracil 5-Chloro-1 (2-fluoro~2,3-dideoxy-~-D~threo-pentofuranosyl)uracil (0.2mmol) and 4-N,N-dimethylaminopyridine (0.25mmol) are dissolved in pyridine (3ml), the solution cooled to O'C, propionic anhydride (0.3mmol) added, the mixture stirred at ambient temperature for 24 hours, the solvent evaporated at reduced pressure, toluene added, the mixture reevaporated at reduced pressure, and the residue chromatographed on silica using chloroform and subsequently chloroform-methanol. The product obtained is 5-Chloro-1-(2-fluoro-5-O-~ropion~1-2,3-dideoxv-a-D-t eo-Pentofuranosyl)uracil 5-Chloro-1-(2-fluoro-5-0-propionyl-2,3-dideoxy-~-D-threo-pantofuranosyl)uracil (0.2mmol~ and potassium carbonate (0.25mmol) are suspended in DMF (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to O-C, l-chlloroethyl ethyl carbonake (0.25 mmol) added, the mixture stirred at 0C
for 30 minutes, at ambient temperature for 2 hours, at 60'C for 24 hours, and the solvent evaporated at reduced pressure. The produ~t is purified by chromatography of the residue on silica gel using ethyl acetate-hexane.
Example 3 1-(2-Fluoro-5-pivalovloxvmethvl-2.3-dideoxy-B-D-threo-pentofuranosyl~t~y~ine A mixture of 1-(2-fluoro-2,3-dideoxy-~-D-threo-- 35 pentofuranosyl)thymine (0.1 mmol) and sodium hydride (0.2 mmol) in DMF ~1.5ml) is stirred at O C for 1.5 hours, chloromethyl pivalate (O.llmmol) added, the ' :, . :
:
.~ .. .. : . , 2V7~ 79 ~j` 16 mixture stirred for 1 hour at ambient temperature, acetic acid (lmmol) added, the solvent evapor~ted at reduced pressure, and the residue chromatographed on silica gel. The product is eluted with chloroform-methanol.
Example 4 N4-BenzYloxvcarbonvl-1-(2-fluoro-5-0-pivaloyloxymethyl-2.3-dideoxy-B-D-threo-pentofuranosvl)cytosine 1-(2-Fluoro-2,3-dideoxy-~-D-theo-pentofuranosyl)cytosine (0.2mmol) is dissolved in a mixture of pyridine (0.5 ml) and~DMF (0.5 ml), the solution cooled to O~C,benzyl chloroformate (0.5 mmol) and 4-N.N-dimethylaminopyridine (0.2 mmol) added, the mixture stirred at ambient temperature for 12 hours, water (4 ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel. The product,N~-enz~loxycarbonyl-1-¢2-fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)cytosine is eluted with chloro~orm:ethanol (99:1) A mixture of N4-benzyloxycarbonyl~ (2-fluoro-2,3- .
dideoxy-~-D-threo-pentofuranosyl)cytosine (O.lmmol) and sodium hydride (0.21mmol) in DMF (2ml) is stirred at ambient temperature for 1.5 hours, the mixture cooled to -50 C, chloromethyl pivalate (0.11 mmol) added, the mixture stirxed at -50 C for 4 hours, saturated ammonium chloride solution (lml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica. The product is eluted with chloroform-methanol.
WO91/15498 PCT/~P91/0~63g 17 207979~
Example 5 1-~2-Fluoro-5-O-pivaloyloxymeth~1-2,3-dideoxy-~-D-threo-pentofuranosyl)cvtosine N4-Benzyloxycarbonyl-1-(2-~luoro-5-O-pivaloyloxymethyl-2,3-dideoxy-~-D-threo-pentofuranosyl)cytosine (1.0 mmol) is added to a suspension of 5~ palladium on charcoal (8mg) in ethanol (4ml). The hydrogenolysis is run at atmospheric presure using a Brown apparatus where the hydrogen gas is generated in a controlled manner by the addition of 3N HCl to a solution of sodium hydride in a . . .
separate compartment. The reaction is run at ambient temperature and is monitored by TLC in order to ensure that overreduction in the heterocyclic ring does not occur. The reaction time is about 1 hour. The mixture is then filtered through a thin bed of Celite, the filtrate evaporated and the product purified by chromatography on silica gel using c:hloroform-ethanol (9: 1) -Example 6 25 N6-Benzvloxvcarbon~1-9-(2-fluoro 5-O-PivalovloxymethYl-2,3-dideoxy-~-D hreo-pentofuranos~l)adenine A solution of 9-(2-fluoro-~,3-dideoxy-~-D-threo-pentofuranosyl)adenine (O.2 mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) in pyridine (4ml) is cooled to 0C, benzyl chloro~ormate (0.4mmol) added., the mixture stirred under nitro~en at room temperature for 24 hours, the same amounts of N,N-dimethylpyridine and benzyl chloroformate added, ~nd the stirring continued 3S for 48 hours. The solvent is removed at reduced pressurP, the residue chromatographed on silica using chloroform:methanol (99-1 which is gradually changed to - ~
,: : ;: . ' ,. :. :
;: .. . ,, '.: , , ;
. .
WOgl/15498 PCT/EP91/00639 2~79i79 ~ 18 9:1). Evaporation gives the product N6-senzyloxycarbonyl-s-(2-fluoro-2 3-dideoxy-B-D-threo-pentofuranosyl)_denine The product (0.15mmol) thus obtained is dissolved in DMF
(3ml), sodium hydride (80% in oil, 0.33 mmol) added, the mixture stirred at room temperature for 1 hour, cooled to -50C and chloromethyl pivalate (0.16mmol) added, the mixture stirred at room temperature for 4 hours before the reaction is stoped by addition of acetic acid (2mmol). The mixture is evaporated to dryness at reduced pressure and the product isolated by chromatography on silica gel using chloroform:methanol ( 9 9 1 ~
Example 7 9-(2-Fluoro-5~0-~ivalovloxvmethYl-2,~-dideoxv-~-~-threo-pentofuranosyl~adenine N6-~enzyloxycarbonyl-9-(2-fluoro-5-O-pivaloyloxymethyl-~,3-dideoxy-~-D-threo-pentofuranosyl)adenine (0.1 mmol) is added to a suspension of 5% palladium on charcoal (8mg) in ethanol (4ml). The hydrogenolysis is run at atmospheric pressure using a Brown apparatus where the hydrogen gas is generated in a controlled manner by the addition of 3N HCl to a solution of sodium hydride in a separate compartment. The reaction is run at ambient temperature and is monitored by TLC in order to ensure that overreduction in the heterocyclic ring does not occur. The reaction time is about 1 hour. The mixture is then filtered through a thin bed of Celite, the filtrate evaporated and the product purified by chromatography on silica gel using chloroform-ethanol (9:1).
- : : . , , .. : ' .
' 19 2~7~795 Example 8 2' 2'-Difluoro-3-pivaloyloxymethylthymidine 2l,2'-Difluorothymidine (0.2 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.2mmol) are added to pyridine (2ml), the reaction mixture stirred at ambient temperature for 8 hours, the solvent removed at reduced pressure, chlorofoxm (15 ml) added to the residue, washed with aqueous bicarbonate and with water, and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 2'.2'-difluoro-3' 5'-0-(1.1 3 3-tetraiso~ropyldisilox-1 3-diyl)thymidine.
The product thus prepared (0.1 mmol) and potassium carbonate (0.12 mmol) are added to DMF (lml), the mixture stirred for 1.5 hours at aI~ient temperature, cooled to O~C, chloromethyl pivalate (0.12 mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish 2'.2'-di1Eluoro-3' 5'-O-(1.1.3.3-tetraisopro~yldisilox-1.3-diyl)-3-pivaloyloxymethylthvmidine.
The silyl group is removed by dissol~tion of the product thus obtained (lmmol) in THF~lml~ and adding 0.25 M
solution o~ tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved in chloroform (lOml), washed with water (2ml), dried (MgSO4~, evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
:
.~
-.: :
W091/15498 PCT/EP9ltO0639 2 ~ ~ 9 7 ~ ~
Example 9 3-~-(Ethvloxycarbonyloxv)ethvl-2'.2'-difluorothymidine S
2',2'-Difluoro-3',5'-0-tl,1,3,3-tetraisopropyldisilox-1,3-diyl)thymidine (o.2mmol) and potassium carbonate (0.25 mmol) are suspended in DMF (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to 0C, 1-chloroethyl ethyl carbonate (0.25 mmol) added, the mixture stirred at 0C for 30 minutes, at ambien~ temperature for 2 hours, at 60C for 24 hours, and the solvent evaporated at reduced pressure. The product 3-~-(Ethyloxvcarbonvloxv)ethvl-2',2'-difluoro-3' 5'-0-(111,3.3-tetraisopro~yldisilox-1,3-diyl)thymidine is purified by chromatography on silica gel using ethyl acetate-hexane.
The silyl group is removed by dissolution of the product thus obtained (lmmol) in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, t:he residue dissolved in chloroform (10 ml), washed with water (2ml), dried (MgSO4), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
Example 10 N4-Benzyloxvcarbonyl-2'r2'-difluoro-2'-deoxycytidine 2',2'-difluoro-2'-deoxycytidine (0.2mmol) is dissolved in a mixture of pyridine (0.5ml) and DMF (0.5ml), the solution cooled to 0C, benzyl chloroformate (0.5 mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) added, the mixture stirred at ambient temperature for 12 hours, -,: - .' ., . : ' : ~
:
21 2 ~ 9 ~
water (4ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel.
The product is eluated with chloroform:ethanol (99:1) S Exam~le 11 1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)-3-~ivaloyloxvmethvlthymine 1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)thymine (0.2mmol) and 1,3-dichloro-1,1,3,3-tetr~isopropyldisiloxane (0.2mmol) are added to pyridine (2ml), the reaction mixture stirred at ambient temperature for 8 hours, the solvent removed at reduced pressure, chloroform (15ml) added to the residue, washed with aqueous bicarbonate and with water, and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 1- r 2-fluoro-2-deoxy-~-D-arabinofuranosyl-3,5-0-(111~3,3-tetraisopropyldisilox-1,3-diyl)]thymine.
The product thus prepared (O.lmmol) and potassium carbonate (0.12mmol) are added to IDMF (lml), the mixture stirred for 1.5 hours at ambient temperature, cooled to 0C, chloromethylpivalate (0.12mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish l-~2-f_uoro-2-deoxy-~-D-arabinofuranosyl-3 5-0-(1 1.3~ -tetraisopro~yldis_lox-lL3~diyl)1-3-ivaloyloxymethvlthymine.
The silyl group is removed by dissolution of the product thus obtained (lmmol) in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved . ::: , . .
.. ..
.. - : ,; , ,: , - , , ,: ,~ . :
W09l/l5498 PCT/EP9t/00639 ~79~ 22 in chloroform (lOml), washed with water (2ml), dried (MgS04), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
Example 12 3-~-fEthylox~carbonvloxv~ethyl-1-(2-fluoro-2-deoxv-~-D-arabinofuranosyl)thymine l-[2-Fluoro~2-deoxy-~-D-arabinofuranosyl-5-0-(1,1,3,3-tetraisopropyldisilox-1,3-diyl)]thymine (0.2mmol) and potassium carbonate ~0.25mmol) are suspended in DMF _ (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to 0C, l-chloroethyl éthyl carbonate (0.25mmol) added, the mixture stirred at OoC for 30 minutes, at ambient temperature for 2 hours, at 60C for 24 hours, and the solvent evaporated at reduced pressure. The product 3-~-(ethyloxY-carbonYloxy)ethyl-1-~(2-fluoro-2-d,eoxy-~-D-arabinofuranosyl-3.5-0-(1,1.3,3)-tetraisop~opyldisilox-1,3-diyl)~thvmine is purified by chromatography on silica gel using ethyl acetate-hexane.
The silyl group is removed by dissolution of the product thus obtained (lmmol` in THF (lml) and adding 0.25M
solution of tetrabutylammonium fluoride in T~F (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved - 30 in chloroform (lOml), washed with water (2ml1, dried (MgS04), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
23 2~7~S
Examele 13 1-(2-Fluoro-2-deoxv-3-~ivaloyloxvmethyl-~-D-arabinofuranosvl)thvmine A mixture of 4-anisylchlorodiphenylmethane (1.05mmol) and 1-(2-fluoro-2-deoxy-~-D-arabinofuran~syl)thymine (lmmol) in dry pyridine is stirred at room temperature for 4 hours, the reaction stopped by addition of a few drops of methanol, the solution evaporated at reduced pressure, the residue extracted into chloroform, the chloroform solution washed and dried (MgS04), the solvent evaporated, and the crude product, l-(2-fluoro-2-deoxy-5-0-monomethox~tritvl-~-D-arabinofuranosyl)thvmine purified by chromatography on silica using chloroform:MeOH 98:2.
mixture of 1-(2-fluoro-2~deoxy-5--0-monomethoxytrityl-~0 ~-D-arabinofuranosyl~thymine (0.2~nol), sodium hydride 0.42mmol; 80% in oil) in DMF (4ml) is stirred at oc for l hour, chloromethyl pivalate (0.2:lmmol) added, the mixture stirred at O-C for 5 hours. The reaction is stopped by addition of 1 M aqueous ammonium chloride (lOml). The mixture is extracted with diethyl ether, the ether extracts ~ashed (saturated NaCl), dried, evaporated and the product fractionaked by silica gel chromatography. The product 1-(2-~luoro-2-deoxy-5-0-monomethoxytrityl-3-pivalo~loxymethyl-~-D-arabinofuranos~llthvmine is eluted bychloroform:methanol (99:1).
The product thus obtained is detritylated by heating a solution in 80% acetic acid at 60C for 15 minutes, the solution evaporated at reduced pressure, and the desired product isolated by chromatography on silica gel using chloroform:ethanol 9505.
.
.
t ' . ~ :
' . -.. ' : :
.. . . . .
2~7979b 24 Example 14 .
N4-IsobutYlox~carbonyl 1-12-fluoro-2-deoxy-B-D-arabinofuranosvl)-5-iodocytosine S
1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)-5-iodocytosine (0.2mmol) is dissolved in a mixture of pyridine (O.Sml~
and DMF (0.5ml), the solution cooled to OC, isobutyl chloroformate (0.5mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) added, the mixture stirred at ambient temperature for 12 hours, water (4ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel. The product is eluted with chloroform:ethanol (98:2). . :
:
,:
' : . ~: ' ' , .
, .
WO ~l/IS4~8 PCl/EP91/00639 2~7979$
N6-Ethvlox~carbonYI-N6-Pivalovlox~vmethvl-9-(2-fluoro-2,3-dideoxy-B-arabino-furanosyl~adenine a) 9-(2-Fluoro-S-O-thex~ldimethYlsilYI-2-deoxY-B~D-arabinofurarlos~ adenine:
Thexyldimethylsilyl chloride (840 mg, 4.7 mmol) was added to a solution of 9-(2-deoxy-2-fluoro-~-D-arabinofulanosyl)adenine (320 mg, 1.28 mmol) and imidazole (650 mg, 9.55 mmol) in pyridine (8 ml) and the mixture stirred at R.T. for 30 rnin. The reaction was stopped by addition to ice-water ~30 ml), the mixture stirred for 10 min and extracted with chloroform (2 x 30 ml). The washed and dried (~ gSO4) chloroform solution was cvaporated and the residue subjected to flash chromatography on silica gel using CHC13: EtOH (9 :1); yield 236 mg (47%). IH NM~ (200 MHz, CDCl3): ~ 0.23 (Me2Si), 0.85 (thexyl), 1.7 (IH), 3.9 (2H, m, H5 ), 4.05 (lH, m), 4.65 (lH, m, H3-), 5.1 (lH, m, H2 ), 5.80 (NH2), 6.50 (lH, dd, Hl ), 8.10 (IH, d, H8), 8.32 (lH, s, H2). --~~- ~~ ~~: ~
b) 9-(3-O-Phenoxvthiocarbonvl-5-O-thexYldimethvlsilvl-2-deoxv-2-fluoro-~-D-arabin~
furanosv!)adenine:
solution of 9{'7-fluoro-S-O-thexyldimcthylsilyl-2-~deoxy-2-fluoro-~-D-arabino-fu~zmosyl)adenine (136 mg, 0.35 mmol), 4-dimethylaminopyridine (95 mg, 0.82 mmol) and phenoxyt}uocarbonyl chloride (58 ~1) in acetonitrile (S ml) was stirred at R.T. under N2 for 16 h. The solventwas then removed at reduced pressure and the residue purified by flash chromatography using CHCI3: Et()H (9: 1), yield I l6 mg (63%). IH NMR
(200 MHz, CDC13): ~ 0.21 (MeSi), 0.89 (thex.), 1.6 (IH), 4.0 (CH2-S ), 4.3 (H4 ), 5.4 (H2 ), 5.90 (NH2), 6.0 (H3'), 6.5 (Hl ), 7.1-7.5 (Ph), 8.13 (d, H8), 8.40 (s, H2).
c) 9-(2-Fluoro-5~0-thexv!dimethvlsilvl-~3-dideoxv-B-D-arabinofi~ranosv!)-adenine:
Nitrogen was bubbled for 15 min through a solution of 9-(2-fluoro-S-O-thexyldimethylsilyl-2-deoxy-~B-D-arabinofuranosyl)adenine (116 mg, 0.22 mmol), tributylstannane (70 111) and a~obisisobutyronitrile (7 mg) in ~oluene (6 ml). The mixture was stirred at 7s C under N2 for 3 h and the solvent evaporated. The product was isolated by nash chromatography on silica gel using CHCI3: EtOH (9: 1); yield 82 mg (98%). IH NMR (200 MHz, CDCI3): o 0.22 (Me2Si), 0.90 (thexyl), 1.5 (IH), 2.4 (CH2-3 ), 3.8 (CH~- S ), 4.3 (H4 ), 5.3 (H2 ), S.90 (NH2), 6.3 (Hl ), 8.20 (d, H8), 8.35 (s, H2) d) N6--Ethy!oxvcarbonvl-9-~2-fluoro-5-O-thexvldimetvlsilvl-2.3-dideoxv-B-D-arabi;lo-fu~nosvl)adenine:
A solution of 9-(2-fluoro-S-O-thexyldimethylsilyl-2,3-dideoxy-~-D-arabino-furanosyl)-adenine (50 mg, 0.13 mmol) in dichlorome~hane (l.S ml) was added dropwise to a SU~3STITIJTE SHFET
' ~
: i, . ~ !
WO 91/15498 PC~/EP91/OOS39 2~7~79~ -26- --solution of N-metllylimidazole ( 0.5 ml) and ethyl chloroformate ~70 ml, 0.75 mmol) in diehloromethane ~1.5 ml). Thc mixture w~ stirred at r~om temperature ~or 20 h, the solvent evaporated and the residue subjected to flash chromatography using CHCI3:
EtOH (9:1); yield 37 mg (64%). lH-NMR (200 MHz, CDC13): o 0.15 ( Me2Si), 0.9 (thex) 1.40 and 4.30 (EtO), 1.5 (IH), 2.5 (CH2-3-), 3.8 (CH2-S ), 5.3 (H2 ), 6.4(Hl ), 8 30 (d, H8), 8.65 (NH), 8.75 (s, H2). r e) _6-Ethv!oxvcar~onv!-N6-pivaloYloxvmethyl-9-(2-fluor~S-O-thexvldimethvlsilYI-2.3-dideoxy-1~-D-arabinofilranosvl!adenine:
Chloromethyl pivalate (30 mg, 0.20 mmol) was added to a mixture of N6-ethyloxycarbonyl-9-(2-fluoro-5-O-thexyldimethylsilyl-2,3-dideoxy-,B-D-arabinofurano-syl)adenine (30 mg, 0.067 mmol) and potassium t-butoxide (24 mg, 0.20 mmol) in dry DMF (2 ml) under nitrogen atmosphere. The mixture was stirred at R.T. for 7 h, evaporated, the residue subjected to nash chromatography using CHCI3:Et2O:EtOH
(5:4:1); yield 28 mg (76 %j. ~H-NMR (200 MHz, CDCI3): o 0.15 (Me2Si), û.85 (thex), 1.30 (piv), 1.40 and 4.33 (OEt), 1.6 (IH), 2.5 (CH2-3-), 3.8 (CH2-5 ), 5.3 (H2 ), 6.05 (OCH2N), 6.5 (Hl ), 8.35 (H8), 8.79(s, H2).
f) N6-Eth~loxvcarbonYI-N6-~ivaloYl~xvmethvl-9-(2-fluoro-2 3-dideox~ -D-arabino-fu~n~svl)adenine:
A solution of tetrabutylarnmonium fluoride (0.5 M, S0 111) in d y TH~ was added dropwise to a solution of N6-ethyloxycarbonyt-N6-pivaloyloxymethyl-9-(2-fluoro-S-O-thexyldimethylsilyl-2,3-dideoxy-,B-D-arabinofuranosyl) adenine (18 mg, 0.032 mmol) in dry T~ ( Iml). The mixture was sti~red under nitrogen atmospher~ at R.T. for 30 min.
Saturated aqueous ammonium cl~loride solution (1 ml) was added, the mixture w~s stirred for S min and evaporated. The product was isolated by flash chromatography using chlorofonn:Et20:ethanot (5:4:1); yisld 60Yo. IH NMR (200 MHz, CDCI3): ~ 1.3 (piY), 1.4 and 4.3 (OEt), 6.1 (OCH2N), 8.3 (H8), 8.8 (H2).
2'.2 -Difluoro-3-pivalovloxvme~v!thvrnidine a) 3 .5-Bis -O-(thexv!dimethylsilvl)-2-deoxv-2 2-difluoro- 1 -oxoribose:
Thexyldimethylsilyl chloride (1.00 ml, 5.1 mol) was added to a solution of 2-deoxy-2,2-difluoro-I-oxoribose (2.5 mmol) and imid~7Ole (10 mmol) in d~y DMF (15 ml). The mixture was stirred at room temperature for 4 h before the solvent was removed under reduced pressure. The product was purified by flash-chromatography on silica gel using chloroform:ethanol (8:1); yield 80%. IH-NMR (200 MHz, CDCI3) o 0.1, 0.9 and 1.6 (2x thexSiMe2), 3.8-4.05 (CEI2), 4.3 (H3), 4.5-4.7 (H4).
$UBSTITUTE: SHE:ET
- : . .
-27- 2~7~
b) 3,5-Bis-O-(thexyldimethvlsilyl)-l-O-(methanesulphonvl)-2-deoxy 2,2-difluoro-n~ose:
The above lactone was rcduced to the corresponding ribose by diisobutylaluminum hydride and mesylated by methanesulphonyl chloride essentially as described for the corresponding t-butylsilyl analogue by Hertel et. al. (J. Org. Chem. 53 ( 1988) 2406.).
lH-NMR (200 MHz, CDCI3): ~ 0.1, 0.9 and 1.6 (2 x thexSiMe2), 3.1 (MeOSO2-), 3.7-3.9 (CH2), 4.2-4.3 (H3), 4.3-4.6(H4), 5.8-5.9 ~Hl).
c) 3'.5' Bis-O-tthexvldimethvlsilvl)-2'~2'-difluo~o~h~nidine: ' A mixture of 3,5-bis-O-(thexyldimethysilyl)-l-O-methanesulphonyl-2-deoxy-2,2-difluororibose (3.0 g) and 2,4-bis-O-(trimethylsilyl)thymine ( 2.5 g) in dry dichloromcthane ( 100 ml) ~ogether with (trifluoromethanesulphonyloxy)trimethylsilane (0.6 g) W'15 strirred at room temperature for 3 days before the reaction was stopped by the addition of methanol. The solvent was evaporated and the residue subjected to flash chsomatography on silica gel using light petroleum:CH2CI2:EtOAc ( 10:10 1) and finally EtOAc:hexane (7:3). The product was mainly the ,B-anomer with some of the a-anomer.
IH-NMR (200 MHz, CDC13): o 0.1, 0.9 and 1.6 (2 x thexSiMe2), 1.92 (S-M[e), 3.7-4.0 (CH2), 4.2 (H3'); 4.5 (H4'), 6.3 (Hl'), 7.24 and 7.16 (Hfi), 9.05 and 9.1 (NH).
d) 2'.2'- Dilluoro-3-pivalo~lo,~mcthv!thvmidine:
Potassium carbonate (7 mmol) was added to a solution of 3',5'-bis-O-(thexyldimethylsilyl)-2',2'-difluorothymidine (6 mmol) in Idry DMF (20 ml), the mixture stirred at 60C for I h, cooled to 0C, chloromethyl pivalate (10 mmol) added and the mixture stirred at 60C for 2 h. Water was added and the mixture freeze-dried and the residue subjected to flash chromatography on silica gel using EtOAc:hexane (5:7). The product, a colourless oily material, was dissolved in dry T~IF (20 ml) and a solution of anhydrous tetrabutylarnmonium fluoride in dry THF (0.2 ~ml, 15 ml) added dropwise with stirring. llle mix~ure was stirred at room temperature for 35 min, the solvent evaporated, water added, the mixture extracled Witll chloroform, ~he chloroforrn solution evaporated and the residue subjected to flash chromatography on silica gel using CHCI3:
MeOH (10:1). lH-NMR (200 MHz, CDC13): ~ 1.2 (piv), 1.98 (S-Me), 3.8-4.1 (5'-CH23, 4.3-4.6 (H3', H4'), 5.97 (OCH2N), 6.1-6.3 ~HI'), 7.2-7.3 (H6).
~7 3--(Ethyloxvcarbonvlox~)ethyl-2' .2 '-difluorothymidine Potassium carbonate (0.25 mmol) was added to a solution of 3' ,5' -bis-O-(thexyldimethylsilyl)-2',2'-difluorothymidine S0.2 mmol) in DMF l4 ml), the mixture was stirred at room temperature for l.S hours, cooled to 0C, I-chloroe~hyl ethyl carbonate (0.25 mmol) added and the mixture stirred at 40C for 2 days before the ~B~B5TITWTI~: SHE:ET
' "' .
; ', ~.:
WO 91/15498 PCr/EP91/00639 2~79h -28- ~
solvent was removed at reduced pressure. llle residue was dissolved in hexane: EtOAc (7:53 ,d the filtrate subjected to flash cromatography using the above eluant. The product thus obtained was dissolved in dry TH~ (S ml) and a solution of anhydrous tetrabutylammoium fluoride (0.6 mmol) in l~IF ( 8 ml) added. The mixture was stirred at room temperature for 35 min and the solvent evaporated. The residue was ext~acted with chloroform (20 ml) washed and dried (MgSO4), the solvent evaporated and theproduct purified by flash chroma~ography on silica gel using CHCI3:MeOH (1:1) . IH-NMR (200 M~, CDCI3): ~ 1.2 and 4.1 (OEt), 1.7 td, MeCH), 7.0 (OCHN).
Exam~le 1 8 N4-Ethvloxvcarbon~ (2-deoxv-2-fluoro-B-D-arabinofuranos~l)cYtosine Sodium hydridc (60% in oil; 4.4 mg, 0.18 mmol) was added to a solution of 1-(2-deoxy-2-fluoro-~-D-arabinofuranosyl)cy~osine--(17 mg, 0.07 mmol) i DMF (6 ml) at0C, and the mixture was stirred at room temperature under nitrogen for 1 h before a-chloroethyl ethyl carbonafe (I0 yl, 0.07 mmol) was added. The resu~tant mixture was stirred overnight beforc the reaction was stoppcd by the addition of saturafed aqueous ammonium chloride (6 ml). The solvents were removed by reduced pressure and the residue subjected t~ flash chromatography on silica gel using ethyl acetate:ethanol ( 1:1);
yield 10 mg (40 %). lH NMR (200 M~; CDCl3): o 1.26 and 4.19 (EtO), 3.6 t2H, m, H-S ), 3.9 (lH, m, H-4 ), 4.28 (lH, m, H-2 ), 5 2 and 4.95 (lH, m, H-2 ), 5.92 (IH, s; NH), 6.12 (IH, dd, H-l ), 7.10 (lH, d, H-S), 8.1~5 (IH, d, H-6).
. ~ 1
This invention relates to antiviral compounds and more particularly to esters, ethers and amides of nucleoside derivatives which are active against human immunodeficiency virus (HIV), the retrovirus which causes the disease AIDS, or other viruses such as herpes simplex virus (~ISV).
Since the recognition of AIDS as a new clinical entity in 1981 nearly five hundred thousand cases of the disease have probably been diagnosed, while the number of HIV infected persons is estimated-to be between 5 ~~~ million and 10 million.
AIDS is fatal, more than 50% of all diagnosed cases having ended in death. HIV and AIDS are today and will remain a worldwide health problem for many years to come.
Clinical symptoms are weight loss, chronic diarrhoea, persisting fever and opportunistic infections due to loss of T-cells, thus upsetting the overall balance of the immune system. The patient loses his/her ability to combat otherwise insignificant infections.
Many substances interfering with replication ha~e been tried, e.g. 3'-azido - 3l-deoxythymidine (AZT), 2',3'-dideoxyadenosine, 3'-fluoroarabinosyladenine, 2',3'-dideoxycytidine, ~'-chloro-2l3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxy - 2',3'-didehydrothymidine, 3'-azido- 2',3',-dideoxyuridine, 3'-azido - 2',3'-dieoxy-5-ethyl-uridine, 1-(2'-deoxy-2'-fluoro-~-D-arabinofuranosyl)-5-ethyluracil, 2,6-diamino-9-(3'-azido-2',3'-dideoxy-~-D-ervthropentofuranosyl)purine, suramin, Evans Blue, fushsin acid, 5-chloro-3'-fluoro-2',3'-dideoxy-uridine, hypericin, I-aurothioglucose, carbovir, dextran sulfate, int~rferon alpha, monoclonal antibodies against the HIV
envelope, peptide T, phosphonoformate (foscarnet), ; ' ` ' ' ~' ~ , ' ; `' :
- ~:
.. ..
-: .: . : :.~ . .:., .
. : : .. .
. ~
WO91/1~98 PCT/EP91/00639 207 ~rl9 ~ 2 phosphorothioate oligodeoxynucleotides, protease inhibitors, ribavirin and soluble CD4 receptor.
European Patent Application No. 0196185A, for instanc~, describes pharmaceutical compositions containing AZT, a known compound which has shown great promise in the treatment of AIDS and AIDS- related complex. It is believed that A2T works by inhibiting reverse transcriptase.
We have now found that acylation or alkylation of oxygen atoms in the glycone (sugar moiety) or in the purine or pyrimidine ring and~or acylation or alkylation of exocylic or endocyclic nitrogen atoms present in the purine or pyrimidine ring can give signiflcant advantages in terms of uptake, overall activity and site 15 of action. Our PCT Application W088/07532 describes certain esters and amides of this type carrying acyl groups at the 5' position or on nitrogens in the aglycone moiety nitrogens; the present invention extends this principle to a wider range of related compounds.
Thus according to one feature of the invention we provide nucleosides of khe general formula ylo - G - X ~ (I) ~wherein G is the residue of the glycone moiety of the nucleoside, Y is a hydrogen atom or a physiologioally acceptable group of the formula Rl(o)nCo(oCR2R3)m-where n is O or 1, m is O or 1 and Rl is an optionally su~stituted alkyl or aryl group or an N-~C1_7 alkyI)~1,4-dihydropyridin- 3-yl group or, where n is 0, a hydrogen atom;
R2 and R3 are independently hydrogen atoms or lower alkyl groups or R2 and R3 together are an alkylidene group; and .. . ...... . . . .
.,. ,::
.
WO91/1~98 PCT/EP91/00639 3 2~7~79S
X is a group selected from O OY N~Y
Y~ ~ R5 O N N
I
(A) (B) (C) NY4 Nr4 N~ y4 o~3 ~ N ~ N ~J~
( E ) ¦ ~ F ) .~
~ 3) (where the groups y2~ y3 and Y4 are as defined for and may be the same as or different from yl or each other, R4 is a hydrogen atom or a group -NY3Y4, where Y3 and Y4 have the above meanings and R5 is a hydrogen or halogen atom or a lower alkyl or trifluoromethyl group, :. . :
'~
, , : , . . . - . .
:: - : :: ~ . ~ .
:.. , ., :
WO91/tS498 PCT/EP9t/00639 2 ~ r~
with the following provisos a) at least one of the groups yll y2~ y3 and Y4 is other than hydrogen, (b) when all of those groups y2~ y3 and Y4 which are present are hydrogen or all of those groups y2~ y3 and R5 which are present in formulae I(C), I(F) and I(G) are hydrogen and Y4 is RlCO, then yl is a group Rl~o)n.Co.(ocR2R3)m in which n and/or m is 1, (c) the glycone group - G - is not a 2',3'-dideoxyribosyl group or such a group having 3'~
fluorine or 3'-azido substituent nor a 2',3'-dehydro-dideoxyribosyl group) _ and/or salts thereof.
In general, compounds in which m and/or n is 1 are preferred, that is the group Rl(o)n.Co.(ocR2R3)- is other than a simple acyl group R CO-.
Compounds in which X is a group of formula (D), (E), (G), (H), (I) or (J) wherein the groups y2 and/or Y are other than hydrogen are of particular interest.
It will be appreciated that s;ome of the groups X, for example those in which y2 is a hydrogen atom, are tautomers of other of the groups Y and exist in equilibrium with them The glycone moiety G will normally be of the formula with the group yl in the 5'-position and the group X in the l'-position. The remaining positions may be substituted, for example, by one or more halogen atoms such as chlorine or, more particularly fluorine; or by hydroxyl, protected hydroxy or a7ido groups. There may be a bond joining the 2'- and 3'-positions to form a 2',3'-didehydro glycone. The sterochemistry of the glycone moiety may be that of any pentose but will generally be that of ribose or arabinose.
- ~
W091/1549~ PCT/~P91/&0639 2 ~ 797~i~
Preferred glycone moieties include the 2,3-dideoxy-2-halo-pentofuranosyl group, for example the 2-chloro-and 2-fluoro-analogues, especially when the group has the threo configuration:
Y ~0~, Another preferred moiety is the 2,2-difluoro-2-deoxy-pentofuranosyl group I X
y 1~
~( r50 F
where Ys is a hydrogen atom or an acyl or acyloxy-alkyl yroup as defined above for y1. It is knnwn that 2,2-difluoro-2-deoxy-nucleosides are active against herpes simplex virus (HSV) and accordingly the compounds according to the invention having this glycone moiety will ~ind application in treatment of herpes infections.
Protected hydroxy groups will in general be groups of the ~ormula ylo_ where yl has the above meaning.
According to a further feature of the invention we provide for the use of compounds of formula (I) as hereinbefore de~ined, and/or salts thereo~, in the manufacture of a composition for the treatment or prophylaxis of virus infections, in particular neurotropic viruses and especially retroviruses such as HIV. Such compositions also form part of the invention.
The group Rl is preferably an alkyl group containing 1-20 carbon atoms which may be straight or branched, or an aryl group which may contain 6 to 20 carbon atoms and may be mono- or poly-cyclic.
Substituents which may be present on the alkyl groups include aryl groups preferably having 6-lO carbon atoms .: ~
-- :~ . ~ ., :
. : .: .,. . ~. :: .
WO 91/15498 PCl/~:P91/00639 2~7~79~;
(as in aralkyl groupings), hydroxy, alkoxy and carboxy groups. Aryl groups include 5- or 6-membered heterocyclic aryl groups having one or more heteroatoms selected from O, N and S, such as furyl, imidazolyl, pyrrolyl, pyridinyl and thienyl groups. Substituents which may be present on aryl groups include alkyl groups, e.g. having 1-6 carbon atoms, hydroxy and carboxy groups. Examples of such groups include methyl, ethyl, propyl, t-butyl, pentyl, stearyl, palmityl, carboxyethyl and benzyl groups.
The lower alkyl groups R2, R3 and R5 preferably contain 1-6 carbon atoms. However, R2 preferably represents a hydrogen atom. R is preferably a hydrogen atom or more preferably a methyl group. Where R5 is a halogen atom it may be a fluorine, chlorine, bromine or iodine atom. However, R5 is preferably a hydrogen or chlorine atom or a methyl group. ~hen R2 and R3 together form an alkyidene group this suitably contai~s 1-6 car~on atoms.
Where Rl in any oE the groups yl~ y2~ y3 or Y4 is an N-alkyl-1,4-dihydropyridin-3-yl group the alkyl group is preferably methyl.
It will be noted that the compounds of the invention may carry more than one of the groups yl~ y2 Y3 and Y4. In the compounds of formula (I) D,E, I and J, it is preferred that m in the group Y4 is O (zero).
Groups Y are preferably of the ~ormula Rl CO-, RlCO.O.CR2R3 or Rl.o.co.O.CR2R --The salts of the compounds of formula (I) may be acid addition salts with organic or inorganic acids, forinstance hydrochloric or phosphoric acid or methanesulphonic acid, ethane disulphonic acid, 2-naphthylsulphonic ~cid, pivalic acid and pamoic acid.
Antiviral counter-ions such as phosphonoformate or suramin may also be used. Organic or inorganic base salts may be formed with acidic groups present in the molecule; suitable counter-ions include alkali metal .;
, .
!
WO91t15498 PCT/EP91/00639 7 2~79 l ~
ions such as sodium and potassium ions, divalent ions such as calcium and zinc ions and organic ions such as tetraalkylammonium and choline or ions derived from meglumine or ethylenediamine. Salts according to the invention may be formed by reaction of the compound of formula (I) with an appropriate acid or base.
The compositions according to the invention may be used in the treatment and/or prophylaxis of virus infections, in particular HIV infections, and such a method forms a further feature of the invention. They may be formulated in conventional manner by admixture of one or more compounds of formula (I) as defined above with excipients and/or carriers.
-~ ~ While the compounds of formula (I) may themselves ba inhibitors of reverse transcriptase when the 5'-hydroxy group is free, it is possible that they are cohverted ln vivo to the desa~yl or desalkyl nucleosides. Nevertheless the substitution at the respective O- and N- atoms gives surprising advantages in terms of uptake and sustained activity. The compounds of formula (I) are more :Lipophilic than the parent compounds and this permits rapid and efficient absorption from the gastro-intestinal tract;lthe absorption rate ma~ be optimised by careful choice of the substituent group to give the desired balance of lipophilicity and hydrophilicity. The lipophilic nature of the compunds of formula (I~ also gives the molecules the ability to penetrate the cell membranes more easily and leads to higher intracellular concentrations, giving an improved dose/effect ratio. The steady hydrolysis of the compounds ensures a sustained concentration of ~he active compound in the cell and thereby permits longer intervals between doses, overcoming a significant drawback of the prior art compounds. Finally, the compnunds according to the invention can penetrate the blood-brain barrier and thus permit treatment of the neurological disorders which have been observed to be .
.,.. , , . :
,:: :: :, , . :
- : . , , :
:. ' '. :~ ., WO91/l54g8 PCT/EP91/00639 2~7~79~; 8 related to the presence of neurotropic viruses, e.g.
retroviruses such as HIV, and lentiviruses (Yarchoan et al, The Lancet, January 17, 1987, page 132~. This is a significant advantage compared to the corresponding unsubstituted compounds or other antiviral compounds and is not referred to anywhere in the prior art. Attempts have been made to treat these neurological disorders with AZT but with limited success.
The invention thus further provides a method of treatment of neurological disorders caused by neurotropic viruses wherein an effective dose of a compound of formula (I) or a salt thereof is administered to a patient suffering from such a disorder.
Compounds of formula (I) may be prepared in any convenient way, for example, by reaction of a compound of formula (II) ylo - G - XB (II) [wherein yl is as hereinbefore dei.ined and XB is as hereinbefore defined for X except that any of the groups yl~ y2~ y3 and Y4 may each additionally represent a protecting group, with the proviso that at least one of yl~ y2/ y3 and Y4 is a hydrogen atom] with a reagent serving to introduce a group Rl(O)~CO.(OCR2R )m as defined above followed where required by removal of any protecting groups and/or unwanted substituents so introduced.
It should be noted that where, in the starting material, more than one of yl~ y2~ y3 and Y4 is hydrogen, multiple reactions may occur.
WAere it is desired to ensure that acylation or alkylation is effected while one or more groups yl~ y2~
Y3 and Y4 remain as hydrogen atoms, it may be desirable to protect the latter first, to form a compound of formula (I) in which one or more of ylr y2, y3 and Y4 r ~ ' WO91/tS498 PCT/EP9t/00639 2~797~S
are protecting groups, these being removed after introduction of the desired acyl or ether group. Such protecting groups may, in fact, be conventional N- or O-protecting groups including groups RlOCO- which may be selectively removed in the presence of the group(s) intended to remain. Thus, for example, an N-benzyloxycarbonyl may be used to protect an exocylic amino group and if the group which is intended to remain is not one which is removable by reduction, for example a straight chain alkoxycarbonyl group, the N-benzyloxycarbonyl group can readily be removed selectively using hydrogen and a noble metal catalyst such as palladium. Trisubstituted silyl groups may also be used as protecting groups, especially for the 5'-oxygen atom, and include trialkylsilyl e.g.
trimethylsilyl, dimethyl- t-butylsilyl, and thexyldimethyl silyl groups. Where it is desired that the reagent introduces a group Rl(o)n.Co.(oCR2R3)m- only into the purine or pyrimidine base then it will be convenient to protect all of the hydroxyl groups present in the glycone, if any; adjacent hydroxyl groups can be protected with a bidentate protecting group such as the 1,1,3,3-tetraisopropyldisilox-1,3-diyl group.
In general, where more than one of yl~ y2~ y3 and Y4 are hydrogen, and a mixture of compounds is produced, the individual components may readily be separated, for example by chromatography.
Where 5'-0-monoalkylation is to be ef~ected in 2',3'-dideoxy derivatives ti.e. introduction of a group yl in which m is 1) it is especially effective to form a dianion of the nucleoside (e.g. by reacting with sodium hydride) and to react this with one equivalent of the alkylating agent~ Monoalkylation of a hydroxy group other than the 5'-hydroxy group in the sugar moiety is carried in a similar fashion using a 5'-protected nucleoside. It is of course, still possible to use protected forms of the nucleoside, for example by . . , , ~:
. ~ :: , . ,.: : : .,: ~ : ., :- ; .; :
WO91/1~98 PCT/EP91/00639 2~979~;
acylation of a nucleophilic nitrogen atom before salt - formation with sodium hydride.
Suitable acylating agents for use in the reaction have the formula Ac-L where L is a leaving group. When the acyl group Ac- is derived from a carboxylic acid, i.e. is of formula Rl-CO-, then suitable acylating agents include the acid halides and acid anhydrides advantageously in the presence of a base; when the acyl group is derived from a carbonic acid, i.e. is of formula Rl.O.CO-, then acylating agents include the haloformate esters and reactive carbonic acid diesters.
In such reagents, the halogen may, for example, be chlorine or bromine. The base for use in the reaction ~ with the acid halide or anhydride may, for example, be a heterocyclic ~ase such as pyridine or 4-dimethylamino-pyridine. The latter increases the speed of the reaction and may be used advantageously with pyridine.
The reaction will normally be carri.ed out in the presence of an inert solvent e.g. a substituted amide solvent such as dimethylformamide, dimethyl~ acetamide or a halogenated hydrocarbon such als dichloromethane.
In general, we have found that: using acid anhydrides as acylating agents to i.ntroduce a group R1CO, O-acylation in the glycone takes place more readily than N-acylation, whereas using acid halides, N-acylation or even N-diacylation predominates.
However, M-acyl groups R1CO- may be removed selectively, for example by reaction with a phenol such as p-methyl-phenol. Where multiple substitution is to be effected, a stronger base such as sodium hydride may be advantageous.
Suitable acyloxyalkylating agents for use in the invention will in general be of the formula RlCo.o.CR2R3L or Rlo.Co.o.CR2R3L, where L is a leaving group. Thus, the group L may for example, be a halogen atom such as a chlorine or bromine atom or a hydrocarbon-sulphonyloxy group such as a tosyloxy or WO91/1~98 PCT/EP91/00639 2~7~
mesyloxy group.
The alkylation reaction will normally be effected in the presence of a base, conveniently an inorganic carbonate such as potassium carbonate or an alkali metal hydride such as sodium hydride. Bases as used for acylation may also be useful.
The starting compounds of formula (II) wherein yl~
y2~ y3 and Y4 are all hydrogen atoms are well described in the literature (see for example the literature references cited in the introduction hereto). Starting compounds wherein one or more O~ yll y2~ y3 and Y4 are other than hydrogen may be prepared by preliminary reactions as described above.
The following literature references are of particular interest in describing the preparation of nucleoside starting materials:
2'-Fluoro-2'-deoxyarabinofuranosylpyrimidine nucleosides:
K. A. Watanabe, U. Reichman, K. Hirota, C. Lopez, J.J.
Fox J. Med. Chem. 22 (1979)21.
5-Substituted (2-Deoxy-2-halogeno--~-D-arabinofuranosyl)cytosines and -uracils. (2-halogeno =
F, Cl, Br ara, and 2-F ribo):
K. A. Watanabe, T.-L-Su, R. S. Klein, C. K. Chu, A.
Matsuda, M. W. Chun, C. Lopez, J. J. Fox J. Med.~Chem.
26 (1983) 152~
1-(2-Deoxy-2-fluoro-~-D-arabinofuranosyl)-5-ethyl(or methyl)uracil:
M. M. Mansuri, I. Ghazzouli, M. S. Chen, H. G. Howell, P. R. Brodfuehrer, D. A. Benigni, J. C. Martin J. Med.
Chem.30 (198~)86~.
Perbenzoylated 1-(2-fluoro-2-deoxy-~-D-arabino-furanosyl)cytosine:
... : :, :
" , . .:
: :: ; ~. ~ ~ . . .
WO91/1~98 PCT/EP91/00639 2~9~9~ 12 C. H. Tann, P. H. Brodfeuhrer, S. P. Brundidge, C.
Sapino. H. G. Howell J. Orq. Chem. 50 (1985)3644.
9-(2-Deoxy-2-~-fluoroarabinofuranosyl)guanine (2'-ara-fluoroquanosine):
A. D. Borthwick, S. Butt, K. Biggadi~e, A. M. Exall, S.
M. Roberts, P. M. Yods, B. E. Kirk, B. R. Booth, J. M.
Cameron, S. W. Cox, C. L. P. Marr, M. D. Shill J. Chem.
Soc. Chem. Commun. (1988)656.
1-(2-Fluoro-2,3-dideoxy-~-D-erythiro-pentofuranosyl)-thymine and l-(2-Fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)-thymine A. V. Aerschot, P. Herdewijn, J. Balzarini, R. Pauwels, E. De Clercq J.Med.Chem.32 (1989)1743.
9-(2-Chloro-2,3-dideoxy-~-D-threo-pentofuranosyl)adenine and its bromo analogue:
P. Herdewijn, J. Balzarini, M. Baba, R. Pauwels, A. V.
Aerschot, G. Janssen, E. De Clercq J. Med. Chem.
31(1988)2040.
1-(2,3-Dideoxy-2-fluoro-~-D-arabinofuranosyl)-cytosine:
EP-A-292033 and EP-A-349928.
The pharmaceutical compositions according to the invention may be ~ormulated conventionally by means well known in the art, and may be administered by any convenient route, for instance orally, rectally, vaginailly, intraveneously or intramuscularly. Examples of suitable formulations include tablets and capsules, aqueous formulations for intravenous injection and oil-based formulations for intramuscular injection.
Suitable dosages will lie in the range 0.1 to 100mg per kilogram of bodyweight per 24 hour period. The compositions according to the invention may also contain ` other active antivirals for instance acyclovir, :;:
" ~
; . - ~ , ~ .
~ .
;`
WO91/tS4g8 PCT/EP9ltO0639 2~7~79 ~
phosphonoformate, suramin, Evans Blue, interferons or AZT.
The invention is illustxated by the following Examples.
. . _ . . .
, - . . .. .. . . . ...
2~797~
Example 1 1-(2-Fluoro-2,3-dideoxy-~-D-threo~Pentofuranosyl)-3-~ivaloyloxymethvl-thvmine 1-(2-Fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)thymine (O.2 mmol) and imidazole (O.5 mmol) are dissolved in DMF
(0.5 mmol). Thexy,ldimethylsilyl chloride (0.25 mmol) is added, and the reaction mixture is stirred at ambient temperature for 24 hours. The solvent is removed at reduced pressure, chloroform (15 ml) added to the residue, washed with water (5ml x 2) and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 1-(2-Fluoro-5-0-thexyldimethYlsily1-2,3-dideoxv-~-D-threo-pentofuranosvl~-thymine.
The product thus prepared (0.1 mmol) and potassium carbonate (0.12 mmol) are added to DMF (lml), the mixture stirred for 1.5 hours at ambient temperature, cooled to O-C, chlorom2thyl pivalate (0.12 mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish 1-(2-Fluoro-5-O-thexvl-dimethylsilyl-2,3-dideoxy-~-D-threo-pentofuranosyl)-3-Pivaloylox~neth~l-th~mine The silyl group is removed by dissolution of the product -~
thus obtained (1 mmol~ in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved in chloroform (lOml), washsd with water (2ml), dried (MgSO4), evaporated and the residue purified by preparative chromatography on silica gel plates using diethyl ether. The product is extracted from the main band by chloroform-methanol.
. . .. .
.
. ~
WO91/1~98 PCT/EP91/00639 Exam~le_2 5-Chloro-3-~-(EthYloxvcarbonYloxy~ethyl-1-(2-fluoro-5-o-Propion~1-2~3-dideox~ D-threo-pentofuranosvl)uracil 5-Chloro-1 (2-fluoro~2,3-dideoxy-~-D~threo-pentofuranosyl)uracil (0.2mmol) and 4-N,N-dimethylaminopyridine (0.25mmol) are dissolved in pyridine (3ml), the solution cooled to O'C, propionic anhydride (0.3mmol) added, the mixture stirred at ambient temperature for 24 hours, the solvent evaporated at reduced pressure, toluene added, the mixture reevaporated at reduced pressure, and the residue chromatographed on silica using chloroform and subsequently chloroform-methanol. The product obtained is 5-Chloro-1-(2-fluoro-5-O-~ropion~1-2,3-dideoxv-a-D-t eo-Pentofuranosyl)uracil 5-Chloro-1-(2-fluoro-5-0-propionyl-2,3-dideoxy-~-D-threo-pantofuranosyl)uracil (0.2mmol~ and potassium carbonate (0.25mmol) are suspended in DMF (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to O-C, l-chlloroethyl ethyl carbonake (0.25 mmol) added, the mixture stirred at 0C
for 30 minutes, at ambient temperature for 2 hours, at 60'C for 24 hours, and the solvent evaporated at reduced pressure. The produ~t is purified by chromatography of the residue on silica gel using ethyl acetate-hexane.
Example 3 1-(2-Fluoro-5-pivalovloxvmethvl-2.3-dideoxy-B-D-threo-pentofuranosyl~t~y~ine A mixture of 1-(2-fluoro-2,3-dideoxy-~-D-threo-- 35 pentofuranosyl)thymine (0.1 mmol) and sodium hydride (0.2 mmol) in DMF ~1.5ml) is stirred at O C for 1.5 hours, chloromethyl pivalate (O.llmmol) added, the ' :, . :
:
.~ .. .. : . , 2V7~ 79 ~j` 16 mixture stirred for 1 hour at ambient temperature, acetic acid (lmmol) added, the solvent evapor~ted at reduced pressure, and the residue chromatographed on silica gel. The product is eluted with chloroform-methanol.
Example 4 N4-BenzYloxvcarbonvl-1-(2-fluoro-5-0-pivaloyloxymethyl-2.3-dideoxy-B-D-threo-pentofuranosvl)cytosine 1-(2-Fluoro-2,3-dideoxy-~-D-theo-pentofuranosyl)cytosine (0.2mmol) is dissolved in a mixture of pyridine (0.5 ml) and~DMF (0.5 ml), the solution cooled to O~C,benzyl chloroformate (0.5 mmol) and 4-N.N-dimethylaminopyridine (0.2 mmol) added, the mixture stirred at ambient temperature for 12 hours, water (4 ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel. The product,N~-enz~loxycarbonyl-1-¢2-fluoro-2,3-dideoxy-~-D-threo-pentofuranosyl)cytosine is eluted with chloro~orm:ethanol (99:1) A mixture of N4-benzyloxycarbonyl~ (2-fluoro-2,3- .
dideoxy-~-D-threo-pentofuranosyl)cytosine (O.lmmol) and sodium hydride (0.21mmol) in DMF (2ml) is stirred at ambient temperature for 1.5 hours, the mixture cooled to -50 C, chloromethyl pivalate (0.11 mmol) added, the mixture stirxed at -50 C for 4 hours, saturated ammonium chloride solution (lml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica. The product is eluted with chloroform-methanol.
WO91/15498 PCT/~P91/0~63g 17 207979~
Example 5 1-~2-Fluoro-5-O-pivaloyloxymeth~1-2,3-dideoxy-~-D-threo-pentofuranosyl)cvtosine N4-Benzyloxycarbonyl-1-(2-~luoro-5-O-pivaloyloxymethyl-2,3-dideoxy-~-D-threo-pentofuranosyl)cytosine (1.0 mmol) is added to a suspension of 5~ palladium on charcoal (8mg) in ethanol (4ml). The hydrogenolysis is run at atmospheric presure using a Brown apparatus where the hydrogen gas is generated in a controlled manner by the addition of 3N HCl to a solution of sodium hydride in a . . .
separate compartment. The reaction is run at ambient temperature and is monitored by TLC in order to ensure that overreduction in the heterocyclic ring does not occur. The reaction time is about 1 hour. The mixture is then filtered through a thin bed of Celite, the filtrate evaporated and the product purified by chromatography on silica gel using c:hloroform-ethanol (9: 1) -Example 6 25 N6-Benzvloxvcarbon~1-9-(2-fluoro 5-O-PivalovloxymethYl-2,3-dideoxy-~-D hreo-pentofuranos~l)adenine A solution of 9-(2-fluoro-~,3-dideoxy-~-D-threo-pentofuranosyl)adenine (O.2 mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) in pyridine (4ml) is cooled to 0C, benzyl chloro~ormate (0.4mmol) added., the mixture stirred under nitro~en at room temperature for 24 hours, the same amounts of N,N-dimethylpyridine and benzyl chloroformate added, ~nd the stirring continued 3S for 48 hours. The solvent is removed at reduced pressurP, the residue chromatographed on silica using chloroform:methanol (99-1 which is gradually changed to - ~
,: : ;: . ' ,. :. :
;: .. . ,, '.: , , ;
. .
WOgl/15498 PCT/EP91/00639 2~79i79 ~ 18 9:1). Evaporation gives the product N6-senzyloxycarbonyl-s-(2-fluoro-2 3-dideoxy-B-D-threo-pentofuranosyl)_denine The product (0.15mmol) thus obtained is dissolved in DMF
(3ml), sodium hydride (80% in oil, 0.33 mmol) added, the mixture stirred at room temperature for 1 hour, cooled to -50C and chloromethyl pivalate (0.16mmol) added, the mixture stirred at room temperature for 4 hours before the reaction is stoped by addition of acetic acid (2mmol). The mixture is evaporated to dryness at reduced pressure and the product isolated by chromatography on silica gel using chloroform:methanol ( 9 9 1 ~
Example 7 9-(2-Fluoro-5~0-~ivalovloxvmethYl-2,~-dideoxv-~-~-threo-pentofuranosyl~adenine N6-~enzyloxycarbonyl-9-(2-fluoro-5-O-pivaloyloxymethyl-~,3-dideoxy-~-D-threo-pentofuranosyl)adenine (0.1 mmol) is added to a suspension of 5% palladium on charcoal (8mg) in ethanol (4ml). The hydrogenolysis is run at atmospheric pressure using a Brown apparatus where the hydrogen gas is generated in a controlled manner by the addition of 3N HCl to a solution of sodium hydride in a separate compartment. The reaction is run at ambient temperature and is monitored by TLC in order to ensure that overreduction in the heterocyclic ring does not occur. The reaction time is about 1 hour. The mixture is then filtered through a thin bed of Celite, the filtrate evaporated and the product purified by chromatography on silica gel using chloroform-ethanol (9:1).
- : : . , , .. : ' .
' 19 2~7~795 Example 8 2' 2'-Difluoro-3-pivaloyloxymethylthymidine 2l,2'-Difluorothymidine (0.2 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.2mmol) are added to pyridine (2ml), the reaction mixture stirred at ambient temperature for 8 hours, the solvent removed at reduced pressure, chlorofoxm (15 ml) added to the residue, washed with aqueous bicarbonate and with water, and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 2'.2'-difluoro-3' 5'-0-(1.1 3 3-tetraiso~ropyldisilox-1 3-diyl)thymidine.
The product thus prepared (0.1 mmol) and potassium carbonate (0.12 mmol) are added to DMF (lml), the mixture stirred for 1.5 hours at aI~ient temperature, cooled to O~C, chloromethyl pivalate (0.12 mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish 2'.2'-di1Eluoro-3' 5'-O-(1.1.3.3-tetraisopro~yldisilox-1.3-diyl)-3-pivaloyloxymethylthvmidine.
The silyl group is removed by dissol~tion of the product thus obtained (lmmol) in THF~lml~ and adding 0.25 M
solution o~ tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved in chloroform (lOml), washed with water (2ml), dried (MgSO4~, evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
:
.~
-.: :
W091/15498 PCT/EP9ltO0639 2 ~ ~ 9 7 ~ ~
Example 9 3-~-(Ethvloxycarbonyloxv)ethvl-2'.2'-difluorothymidine S
2',2'-Difluoro-3',5'-0-tl,1,3,3-tetraisopropyldisilox-1,3-diyl)thymidine (o.2mmol) and potassium carbonate (0.25 mmol) are suspended in DMF (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to 0C, 1-chloroethyl ethyl carbonate (0.25 mmol) added, the mixture stirred at 0C for 30 minutes, at ambien~ temperature for 2 hours, at 60C for 24 hours, and the solvent evaporated at reduced pressure. The product 3-~-(Ethyloxvcarbonvloxv)ethvl-2',2'-difluoro-3' 5'-0-(111,3.3-tetraisopro~yldisilox-1,3-diyl)thymidine is purified by chromatography on silica gel using ethyl acetate-hexane.
The silyl group is removed by dissolution of the product thus obtained (lmmol) in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, t:he residue dissolved in chloroform (10 ml), washed with water (2ml), dried (MgSO4), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
Example 10 N4-Benzyloxvcarbonyl-2'r2'-difluoro-2'-deoxycytidine 2',2'-difluoro-2'-deoxycytidine (0.2mmol) is dissolved in a mixture of pyridine (0.5ml) and DMF (0.5ml), the solution cooled to 0C, benzyl chloroformate (0.5 mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) added, the mixture stirred at ambient temperature for 12 hours, -,: - .' ., . : ' : ~
:
21 2 ~ 9 ~
water (4ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel.
The product is eluated with chloroform:ethanol (99:1) S Exam~le 11 1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)-3-~ivaloyloxvmethvlthymine 1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)thymine (0.2mmol) and 1,3-dichloro-1,1,3,3-tetr~isopropyldisiloxane (0.2mmol) are added to pyridine (2ml), the reaction mixture stirred at ambient temperature for 8 hours, the solvent removed at reduced pressure, chloroform (15ml) added to the residue, washed with aqueous bicarbonate and with water, and the dried (MgSO4) solution evaporated. The residue is chromatographed on silica gel using ethyl acetate-hexane to furnish 1- r 2-fluoro-2-deoxy-~-D-arabinofuranosyl-3,5-0-(111~3,3-tetraisopropyldisilox-1,3-diyl)]thymine.
The product thus prepared (O.lmmol) and potassium carbonate (0.12mmol) are added to IDMF (lml), the mixture stirred for 1.5 hours at ambient temperature, cooled to 0C, chloromethylpivalate (0.12mmol) added, the mixture stirred at ambient temperature for 18 hours, the solvent evaporated at reduced pressure, and the residue chromatographed on silica gel using ethyl acetate-hexane to furnish l-~2-f_uoro-2-deoxy-~-D-arabinofuranosyl-3 5-0-(1 1.3~ -tetraisopro~yldis_lox-lL3~diyl)1-3-ivaloyloxymethvlthymine.
The silyl group is removed by dissolution of the product thus obtained (lmmol) in THF (lml) and adding 0.25 M
solution of tetrabutylammonium fluoride in THF (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved . ::: , . .
.. ..
.. - : ,; , ,: , - , , ,: ,~ . :
W09l/l5498 PCT/EP9t/00639 ~79~ 22 in chloroform (lOml), washed with water (2ml), dried (MgS04), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
Example 12 3-~-fEthylox~carbonvloxv~ethyl-1-(2-fluoro-2-deoxv-~-D-arabinofuranosyl)thymine l-[2-Fluoro~2-deoxy-~-D-arabinofuranosyl-5-0-(1,1,3,3-tetraisopropyldisilox-1,3-diyl)]thymine (0.2mmol) and potassium carbonate ~0.25mmol) are suspended in DMF _ (2ml), the mixture stirred at ambient temperature under nitrogen for 1.5 hours, cooled to 0C, l-chloroethyl éthyl carbonate (0.25mmol) added, the mixture stirred at OoC for 30 minutes, at ambient temperature for 2 hours, at 60C for 24 hours, and the solvent evaporated at reduced pressure. The product 3-~-(ethyloxY-carbonYloxy)ethyl-1-~(2-fluoro-2-d,eoxy-~-D-arabinofuranosyl-3.5-0-(1,1.3,3)-tetraisop~opyldisilox-1,3-diyl)~thvmine is purified by chromatography on silica gel using ethyl acetate-hexane.
The silyl group is removed by dissolution of the product thus obtained (lmmol` in THF (lml) and adding 0.25M
solution of tetrabutylammonium fluoride in T~F (lml).
The mixture is stirred at ambient temperature for 30 minutes, the solvent evaporated, the residue dissolved - 30 in chloroform (lOml), washed with water (2ml1, dried (MgS04), evaporated, and the product purified by chromatography on silica gel using chloroform:methanol 95:5.
23 2~7~S
Examele 13 1-(2-Fluoro-2-deoxv-3-~ivaloyloxvmethyl-~-D-arabinofuranosvl)thvmine A mixture of 4-anisylchlorodiphenylmethane (1.05mmol) and 1-(2-fluoro-2-deoxy-~-D-arabinofuran~syl)thymine (lmmol) in dry pyridine is stirred at room temperature for 4 hours, the reaction stopped by addition of a few drops of methanol, the solution evaporated at reduced pressure, the residue extracted into chloroform, the chloroform solution washed and dried (MgS04), the solvent evaporated, and the crude product, l-(2-fluoro-2-deoxy-5-0-monomethox~tritvl-~-D-arabinofuranosyl)thvmine purified by chromatography on silica using chloroform:MeOH 98:2.
mixture of 1-(2-fluoro-2~deoxy-5--0-monomethoxytrityl-~0 ~-D-arabinofuranosyl~thymine (0.2~nol), sodium hydride 0.42mmol; 80% in oil) in DMF (4ml) is stirred at oc for l hour, chloromethyl pivalate (0.2:lmmol) added, the mixture stirred at O-C for 5 hours. The reaction is stopped by addition of 1 M aqueous ammonium chloride (lOml). The mixture is extracted with diethyl ether, the ether extracts ~ashed (saturated NaCl), dried, evaporated and the product fractionaked by silica gel chromatography. The product 1-(2-~luoro-2-deoxy-5-0-monomethoxytrityl-3-pivalo~loxymethyl-~-D-arabinofuranos~llthvmine is eluted bychloroform:methanol (99:1).
The product thus obtained is detritylated by heating a solution in 80% acetic acid at 60C for 15 minutes, the solution evaporated at reduced pressure, and the desired product isolated by chromatography on silica gel using chloroform:ethanol 9505.
.
.
t ' . ~ :
' . -.. ' : :
.. . . . .
2~7979b 24 Example 14 .
N4-IsobutYlox~carbonyl 1-12-fluoro-2-deoxy-B-D-arabinofuranosvl)-5-iodocytosine S
1-(2-Fluoro-2-deoxy-~-D-arabinofuranosyl)-5-iodocytosine (0.2mmol) is dissolved in a mixture of pyridine (O.Sml~
and DMF (0.5ml), the solution cooled to OC, isobutyl chloroformate (0.5mmol) and 4-N,N-dimethylaminopyridine (0.2mmol) added, the mixture stirred at ambient temperature for 12 hours, water (4ml) added, the mixture evaporated at reduced pressure, and the residue chromatographed on silica gel. The product is eluted with chloroform:ethanol (98:2). . :
:
,:
' : . ~: ' ' , .
, .
WO ~l/IS4~8 PCl/EP91/00639 2~7979$
N6-Ethvlox~carbonYI-N6-Pivalovlox~vmethvl-9-(2-fluoro-2,3-dideoxy-B-arabino-furanosyl~adenine a) 9-(2-Fluoro-S-O-thex~ldimethYlsilYI-2-deoxY-B~D-arabinofurarlos~ adenine:
Thexyldimethylsilyl chloride (840 mg, 4.7 mmol) was added to a solution of 9-(2-deoxy-2-fluoro-~-D-arabinofulanosyl)adenine (320 mg, 1.28 mmol) and imidazole (650 mg, 9.55 mmol) in pyridine (8 ml) and the mixture stirred at R.T. for 30 rnin. The reaction was stopped by addition to ice-water ~30 ml), the mixture stirred for 10 min and extracted with chloroform (2 x 30 ml). The washed and dried (~ gSO4) chloroform solution was cvaporated and the residue subjected to flash chromatography on silica gel using CHC13: EtOH (9 :1); yield 236 mg (47%). IH NM~ (200 MHz, CDCl3): ~ 0.23 (Me2Si), 0.85 (thexyl), 1.7 (IH), 3.9 (2H, m, H5 ), 4.05 (lH, m), 4.65 (lH, m, H3-), 5.1 (lH, m, H2 ), 5.80 (NH2), 6.50 (lH, dd, Hl ), 8.10 (IH, d, H8), 8.32 (lH, s, H2). --~~- ~~ ~~: ~
b) 9-(3-O-Phenoxvthiocarbonvl-5-O-thexYldimethvlsilvl-2-deoxv-2-fluoro-~-D-arabin~
furanosv!)adenine:
solution of 9{'7-fluoro-S-O-thexyldimcthylsilyl-2-~deoxy-2-fluoro-~-D-arabino-fu~zmosyl)adenine (136 mg, 0.35 mmol), 4-dimethylaminopyridine (95 mg, 0.82 mmol) and phenoxyt}uocarbonyl chloride (58 ~1) in acetonitrile (S ml) was stirred at R.T. under N2 for 16 h. The solventwas then removed at reduced pressure and the residue purified by flash chromatography using CHCI3: Et()H (9: 1), yield I l6 mg (63%). IH NMR
(200 MHz, CDC13): ~ 0.21 (MeSi), 0.89 (thex.), 1.6 (IH), 4.0 (CH2-S ), 4.3 (H4 ), 5.4 (H2 ), 5.90 (NH2), 6.0 (H3'), 6.5 (Hl ), 7.1-7.5 (Ph), 8.13 (d, H8), 8.40 (s, H2).
c) 9-(2-Fluoro-5~0-thexv!dimethvlsilvl-~3-dideoxv-B-D-arabinofi~ranosv!)-adenine:
Nitrogen was bubbled for 15 min through a solution of 9-(2-fluoro-S-O-thexyldimethylsilyl-2-deoxy-~B-D-arabinofuranosyl)adenine (116 mg, 0.22 mmol), tributylstannane (70 111) and a~obisisobutyronitrile (7 mg) in ~oluene (6 ml). The mixture was stirred at 7s C under N2 for 3 h and the solvent evaporated. The product was isolated by nash chromatography on silica gel using CHCI3: EtOH (9: 1); yield 82 mg (98%). IH NMR (200 MHz, CDCI3): o 0.22 (Me2Si), 0.90 (thexyl), 1.5 (IH), 2.4 (CH2-3 ), 3.8 (CH~- S ), 4.3 (H4 ), 5.3 (H2 ), S.90 (NH2), 6.3 (Hl ), 8.20 (d, H8), 8.35 (s, H2) d) N6--Ethy!oxvcarbonvl-9-~2-fluoro-5-O-thexvldimetvlsilvl-2.3-dideoxv-B-D-arabi;lo-fu~nosvl)adenine:
A solution of 9-(2-fluoro-S-O-thexyldimethylsilyl-2,3-dideoxy-~-D-arabino-furanosyl)-adenine (50 mg, 0.13 mmol) in dichlorome~hane (l.S ml) was added dropwise to a SU~3STITIJTE SHFET
' ~
: i, . ~ !
WO 91/15498 PC~/EP91/OOS39 2~7~79~ -26- --solution of N-metllylimidazole ( 0.5 ml) and ethyl chloroformate ~70 ml, 0.75 mmol) in diehloromethane ~1.5 ml). Thc mixture w~ stirred at r~om temperature ~or 20 h, the solvent evaporated and the residue subjected to flash chromatography using CHCI3:
EtOH (9:1); yield 37 mg (64%). lH-NMR (200 MHz, CDC13): o 0.15 ( Me2Si), 0.9 (thex) 1.40 and 4.30 (EtO), 1.5 (IH), 2.5 (CH2-3-), 3.8 (CH2-S ), 5.3 (H2 ), 6.4(Hl ), 8 30 (d, H8), 8.65 (NH), 8.75 (s, H2). r e) _6-Ethv!oxvcar~onv!-N6-pivaloYloxvmethyl-9-(2-fluor~S-O-thexvldimethvlsilYI-2.3-dideoxy-1~-D-arabinofilranosvl!adenine:
Chloromethyl pivalate (30 mg, 0.20 mmol) was added to a mixture of N6-ethyloxycarbonyl-9-(2-fluoro-5-O-thexyldimethylsilyl-2,3-dideoxy-,B-D-arabinofurano-syl)adenine (30 mg, 0.067 mmol) and potassium t-butoxide (24 mg, 0.20 mmol) in dry DMF (2 ml) under nitrogen atmosphere. The mixture was stirred at R.T. for 7 h, evaporated, the residue subjected to nash chromatography using CHCI3:Et2O:EtOH
(5:4:1); yield 28 mg (76 %j. ~H-NMR (200 MHz, CDCI3): o 0.15 (Me2Si), û.85 (thex), 1.30 (piv), 1.40 and 4.33 (OEt), 1.6 (IH), 2.5 (CH2-3-), 3.8 (CH2-5 ), 5.3 (H2 ), 6.05 (OCH2N), 6.5 (Hl ), 8.35 (H8), 8.79(s, H2).
f) N6-Eth~loxvcarbonYI-N6-~ivaloYl~xvmethvl-9-(2-fluoro-2 3-dideox~ -D-arabino-fu~n~svl)adenine:
A solution of tetrabutylarnmonium fluoride (0.5 M, S0 111) in d y TH~ was added dropwise to a solution of N6-ethyloxycarbonyt-N6-pivaloyloxymethyl-9-(2-fluoro-S-O-thexyldimethylsilyl-2,3-dideoxy-,B-D-arabinofuranosyl) adenine (18 mg, 0.032 mmol) in dry T~ ( Iml). The mixture was sti~red under nitrogen atmospher~ at R.T. for 30 min.
Saturated aqueous ammonium cl~loride solution (1 ml) was added, the mixture w~s stirred for S min and evaporated. The product was isolated by flash chromatography using chlorofonn:Et20:ethanot (5:4:1); yisld 60Yo. IH NMR (200 MHz, CDCI3): ~ 1.3 (piY), 1.4 and 4.3 (OEt), 6.1 (OCH2N), 8.3 (H8), 8.8 (H2).
2'.2 -Difluoro-3-pivalovloxvme~v!thvrnidine a) 3 .5-Bis -O-(thexv!dimethylsilvl)-2-deoxv-2 2-difluoro- 1 -oxoribose:
Thexyldimethylsilyl chloride (1.00 ml, 5.1 mol) was added to a solution of 2-deoxy-2,2-difluoro-I-oxoribose (2.5 mmol) and imid~7Ole (10 mmol) in d~y DMF (15 ml). The mixture was stirred at room temperature for 4 h before the solvent was removed under reduced pressure. The product was purified by flash-chromatography on silica gel using chloroform:ethanol (8:1); yield 80%. IH-NMR (200 MHz, CDCI3) o 0.1, 0.9 and 1.6 (2x thexSiMe2), 3.8-4.05 (CEI2), 4.3 (H3), 4.5-4.7 (H4).
$UBSTITUTE: SHE:ET
- : . .
-27- 2~7~
b) 3,5-Bis-O-(thexyldimethvlsilyl)-l-O-(methanesulphonvl)-2-deoxy 2,2-difluoro-n~ose:
The above lactone was rcduced to the corresponding ribose by diisobutylaluminum hydride and mesylated by methanesulphonyl chloride essentially as described for the corresponding t-butylsilyl analogue by Hertel et. al. (J. Org. Chem. 53 ( 1988) 2406.).
lH-NMR (200 MHz, CDCI3): ~ 0.1, 0.9 and 1.6 (2 x thexSiMe2), 3.1 (MeOSO2-), 3.7-3.9 (CH2), 4.2-4.3 (H3), 4.3-4.6(H4), 5.8-5.9 ~Hl).
c) 3'.5' Bis-O-tthexvldimethvlsilvl)-2'~2'-difluo~o~h~nidine: ' A mixture of 3,5-bis-O-(thexyldimethysilyl)-l-O-methanesulphonyl-2-deoxy-2,2-difluororibose (3.0 g) and 2,4-bis-O-(trimethylsilyl)thymine ( 2.5 g) in dry dichloromcthane ( 100 ml) ~ogether with (trifluoromethanesulphonyloxy)trimethylsilane (0.6 g) W'15 strirred at room temperature for 3 days before the reaction was stopped by the addition of methanol. The solvent was evaporated and the residue subjected to flash chsomatography on silica gel using light petroleum:CH2CI2:EtOAc ( 10:10 1) and finally EtOAc:hexane (7:3). The product was mainly the ,B-anomer with some of the a-anomer.
IH-NMR (200 MHz, CDC13): o 0.1, 0.9 and 1.6 (2 x thexSiMe2), 1.92 (S-M[e), 3.7-4.0 (CH2), 4.2 (H3'); 4.5 (H4'), 6.3 (Hl'), 7.24 and 7.16 (Hfi), 9.05 and 9.1 (NH).
d) 2'.2'- Dilluoro-3-pivalo~lo,~mcthv!thvmidine:
Potassium carbonate (7 mmol) was added to a solution of 3',5'-bis-O-(thexyldimethylsilyl)-2',2'-difluorothymidine (6 mmol) in Idry DMF (20 ml), the mixture stirred at 60C for I h, cooled to 0C, chloromethyl pivalate (10 mmol) added and the mixture stirred at 60C for 2 h. Water was added and the mixture freeze-dried and the residue subjected to flash chromatography on silica gel using EtOAc:hexane (5:7). The product, a colourless oily material, was dissolved in dry T~IF (20 ml) and a solution of anhydrous tetrabutylarnmonium fluoride in dry THF (0.2 ~ml, 15 ml) added dropwise with stirring. llle mix~ure was stirred at room temperature for 35 min, the solvent evaporated, water added, the mixture extracled Witll chloroform, ~he chloroforrn solution evaporated and the residue subjected to flash chromatography on silica gel using CHCI3:
MeOH (10:1). lH-NMR (200 MHz, CDC13): ~ 1.2 (piv), 1.98 (S-Me), 3.8-4.1 (5'-CH23, 4.3-4.6 (H3', H4'), 5.97 (OCH2N), 6.1-6.3 ~HI'), 7.2-7.3 (H6).
~7 3--(Ethyloxvcarbonvlox~)ethyl-2' .2 '-difluorothymidine Potassium carbonate (0.25 mmol) was added to a solution of 3' ,5' -bis-O-(thexyldimethylsilyl)-2',2'-difluorothymidine S0.2 mmol) in DMF l4 ml), the mixture was stirred at room temperature for l.S hours, cooled to 0C, I-chloroe~hyl ethyl carbonate (0.25 mmol) added and the mixture stirred at 40C for 2 days before the ~B~B5TITWTI~: SHE:ET
' "' .
; ', ~.:
WO 91/15498 PCr/EP91/00639 2~79h -28- ~
solvent was removed at reduced pressure. llle residue was dissolved in hexane: EtOAc (7:53 ,d the filtrate subjected to flash cromatography using the above eluant. The product thus obtained was dissolved in dry TH~ (S ml) and a solution of anhydrous tetrabutylammoium fluoride (0.6 mmol) in l~IF ( 8 ml) added. The mixture was stirred at room temperature for 35 min and the solvent evaporated. The residue was ext~acted with chloroform (20 ml) washed and dried (MgSO4), the solvent evaporated and theproduct purified by flash chroma~ography on silica gel using CHCI3:MeOH (1:1) . IH-NMR (200 M~, CDCI3): ~ 1.2 and 4.1 (OEt), 1.7 td, MeCH), 7.0 (OCHN).
Exam~le 1 8 N4-Ethvloxvcarbon~ (2-deoxv-2-fluoro-B-D-arabinofuranos~l)cYtosine Sodium hydridc (60% in oil; 4.4 mg, 0.18 mmol) was added to a solution of 1-(2-deoxy-2-fluoro-~-D-arabinofuranosyl)cy~osine--(17 mg, 0.07 mmol) i DMF (6 ml) at0C, and the mixture was stirred at room temperature under nitrogen for 1 h before a-chloroethyl ethyl carbonafe (I0 yl, 0.07 mmol) was added. The resu~tant mixture was stirred overnight beforc the reaction was stoppcd by the addition of saturafed aqueous ammonium chloride (6 ml). The solvents were removed by reduced pressure and the residue subjected t~ flash chromatography on silica gel using ethyl acetate:ethanol ( 1:1);
yield 10 mg (40 %). lH NMR (200 M~; CDCl3): o 1.26 and 4.19 (EtO), 3.6 t2H, m, H-S ), 3.9 (lH, m, H-4 ), 4.28 (lH, m, H-2 ), 5 2 and 4.95 (lH, m, H-2 ), 5.92 (IH, s; NH), 6.12 (IH, dd, H-l ), 7.10 (lH, d, H-S), 8.1~5 (IH, d, H-6).
. ~ 1
Claims (8)
1. Nucleoside compounds of the general formula Y1O - G - X (I) (wherein G is the residue of the glycone moiety of the nucleoside, y1 is a hydrogen atom or a physiologically acceptable group of the formula R1(O)nCO(OCR2R3)m-where n is 0 or 1, m is 0 or 1 and R1 is an optionally substituted alkyl or aryl group or an N-(C1-7 alkyl)-1,4-dihydropyridin- 3-yl group or, where n is 0, a hydrogen atom;
R2 and R3 are independently hydrogen atoms or lower alkyl groups or R2 and R3 together are an alkylidene group; and X is a group selected from (A) (B) (C) (D) (E) (F) (G) (H) (I) (J) (where the groups Y2, Y3 and Y4 are as defined for Y1 and may be the same as or different from Y1 or each other, R4 is a hydrogen atom or a group -NY3Y4, where Y3 and Y4 have the above meanings and R5 is a hydrogen or halogen atom or a lower alkyl or trifluoromethyl group, with the following provisos (a) at least one of the groups Y1, Y2, Y3 and Y4 is other than hydrogen, (b) when all of those groups Y2, Y3 and Y4 which are present are hydrogen or all of those groups Y2, Y3 and R5 which are present in formulae I(C), I(F) and I(G) are hydrogen and Y4 is R1CO, then Y1 is a group R1(O)n.CO.(OCR2R3)m in which n and/or m is 1, (c) the glycone group - G - is not a 2',3'-dideoxyribosyl group or such a group having 3'-fluorine or 3'-azido substituent nor a 2',3'-dehydro-dideoxyribosyl group) and/or salts thereof.
R2 and R3 are independently hydrogen atoms or lower alkyl groups or R2 and R3 together are an alkylidene group; and X is a group selected from (A) (B) (C) (D) (E) (F) (G) (H) (I) (J) (where the groups Y2, Y3 and Y4 are as defined for Y1 and may be the same as or different from Y1 or each other, R4 is a hydrogen atom or a group -NY3Y4, where Y3 and Y4 have the above meanings and R5 is a hydrogen or halogen atom or a lower alkyl or trifluoromethyl group, with the following provisos (a) at least one of the groups Y1, Y2, Y3 and Y4 is other than hydrogen, (b) when all of those groups Y2, Y3 and Y4 which are present are hydrogen or all of those groups Y2, Y3 and R5 which are present in formulae I(C), I(F) and I(G) are hydrogen and Y4 is R1CO, then Y1 is a group R1(O)n.CO.(OCR2R3)m in which n and/or m is 1, (c) the glycone group - G - is not a 2',3'-dideoxyribosyl group or such a group having 3'-fluorine or 3'-azido substituent nor a 2',3'-dehydro-dideoxyribosyl group) and/or salts thereof.
2. Compounds of formula (I) as claimed in claim 1 wherein R1 is selected from optionally substituted C1-20 alkyl groups and C6-20 aryl groups.
3. Compounds of formula (I) as claimed in claim 1 wherein m represents 1 in at least one of the groups Y1, Y2, Y3 and Y4; R2 is a hydrogen atom; and R3 is a hydrogen atom or a methyl group.
4. Compounds of formula (I) as claimed in claim 1 wherein the glycone moiety is a 2,3-dideoxy-3-halo-pentofuranosyl group.
5. Compounds of formula (I) as claimed in claim 1 wherein the glycone moiety is a 2,2-difluoro-2-deoxy-pentofuranosyl group.
6. A pharmaceutical composition comprising as active ingredient one or more compounds of formula (I) as defined in any preceding claim and/or a non-toxic salt thereof, together with a pharmaceutical carrier or excipient.
7. A process for the preparation of a compound of formula (I) as defined in any of claims 1 to 5, which comprises reaction of a compound of formula (II) Y1O - G - XB (II) [wherein Y1 is as defined in claim 1 and XB is as defined in claim 1 for X except that any of the groups Y1, Y2, Y3 and Y4 may each additionally represent a protecting group, with the proviso that at least one of Y1, Y2, Y3 and Y4 is a hydrogen atom] with a reagent serving to introduce a group R1(O)nCO.(OCR2R3)m as defined in claim 1 followed where required by removal of any protecting groups and/or unwanted substituents so introduced.
8. Use of compounds of formula (I) as defined in any of claims 1 to 5, and/or salts thereof, in the manufacture of a medicament for the treatment or prophylaxis of virus infections.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9007566.4 | 1990-04-04 | ||
| GB9007650.6 | 1990-04-04 | ||
| GB909007650A GB9007650D0 (en) | 1990-04-04 | 1990-04-04 | Nucleoside derivatives |
| GB909007566A GB9007566D0 (en) | 1990-04-04 | 1990-04-04 | Nucleoside derivatives |
| GB909007651A GB9007651D0 (en) | 1990-04-04 | 1990-04-04 | Nucleoside derivatives |
| GB9007651.4 | 1990-04-04 | ||
| PCT/EP1991/000639 WO1991015498A2 (en) | 1990-04-04 | 1991-04-04 | Nucleoside derivatives |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2079796A1 true CA2079796A1 (en) | 1991-10-05 |
Family
ID=27265029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002079796A Abandoned CA2079796A1 (en) | 1990-04-04 | 1991-04-04 | Nucleoside derivatives |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0523110A1 (en) |
| JP (1) | JPH05505815A (en) |
| AU (1) | AU7558491A (en) |
| CA (1) | CA2079796A1 (en) |
| OA (1) | OA09677A (en) |
| WO (1) | WO1991015498A2 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU636678B2 (en) * | 1989-11-06 | 1993-05-06 | Nycomed Imaging As | Nucleoside derivatives |
| UA41261C2 (en) * | 1992-06-22 | 2001-09-17 | Елі Ліллі Енд Компані | METHOD OF OBTAINING BETA-ANOMER-ENRICHED NUCLEOSIDES |
| YU43193A (en) * | 1992-06-22 | 1997-01-08 | Eli Lilly And Company | 2'-DEOXY-2 ', 2'-DIFLUORO (4-SUBSTITUTED) PYRIMIDINE NUCLEOSIDS OF ANTIVIRUS AND ANTICANCEROGENIC ACTIVITY AND INTERMEDIATES |
| JPH0692987A (en) * | 1992-09-17 | 1994-04-05 | Tanabe Seiyaku Co Ltd | Uridine derivative and its production |
| JPH11217396A (en) * | 1998-01-30 | 1999-08-10 | Ajinomoto Co Inc | Production of nucleoside derivative |
| US6326507B1 (en) | 1998-06-19 | 2001-12-04 | Trustees Of Dartmouth College | Therapeutic compounds and methods of use |
| US7435755B2 (en) | 2000-11-28 | 2008-10-14 | The Trustees Of Dartmouth College | CDDO-compounds and combination therapies thereof |
| US7176237B2 (en) | 2002-01-15 | 2007-02-13 | The Trustees Of Dartmouth College | Tricyclic-bis-enone derivatives and methods of use thereof |
| EP1567169A4 (en) * | 2002-11-04 | 2009-10-21 | Xenoport Inc | Gemcitabine prodrugs, pharmaceutical compositions and uses thereof |
| CN1693309A (en) * | 2005-04-18 | 2005-11-09 | 成都正开生物科技发展有限公司 | N4 (substituted oxycarbonyl) 2',2'-bifluoro-2'-deoxycytidine derivate and application thereof |
| US8921340B2 (en) | 2006-11-17 | 2014-12-30 | Trustees Of Dartmouth College | Methods for using synthetic triterpenoids in the treatment of bone or cartilage diseases or conditions |
| EP2094651A1 (en) | 2006-11-17 | 2009-09-02 | Trustees Of Dartmouth College | Synthesis and biological activities of new tricyclic-bis-enones (tbes) |
| WO2008064132A2 (en) | 2006-11-17 | 2008-05-29 | Trustees Of Dartmouth College | Synthetic triterpenoids and tricyclic-bis-enones for use in stimulating bone and cartilage growth |
| RS58486B1 (en) | 2008-01-11 | 2019-04-30 | Reata Pharmaceuticals Inc | Synthetic triterpenoids and methods of use in the treatment of disease |
| BRPI0911105B1 (en) | 2008-04-18 | 2022-11-08 | Reata Pharmaceuticals, Inc | COMPOUNDS CONTAINING AN ANTI-INFLAMMATORY PHARMACOPHORE, PHARMACEUTICAL COMPOSITION, AS WELL AS THEIR USES |
| JP5588966B2 (en) | 2008-04-18 | 2014-09-10 | リアタ ファーマシューティカルズ インコーポレイテッド | Antioxidant inflammation modulator: C-17 homologated oleanolic acid derivative |
| BRPI0911208B1 (en) | 2008-04-18 | 2021-05-25 | Reata Pharmaceuticals, Inc | INFLAMMATORY ANTIOXIDANT MODULATOR COMPOUNDS, THEIR USE, AND PHARMACEUTICAL COMPOSITION |
| WO2009146216A2 (en) | 2008-04-18 | 2009-12-03 | Reata Pharmaceuticals. Inc. | Antioxidant inflammation modulators: novel derivatives of oleanolic acid |
| HUE041221T2 (en) | 2008-04-18 | 2019-05-28 | Reata Pharmaceuticals Inc | Antioxidant inflammation modulators: oleanolic acid derivatives with amino and other modifications at c-17 |
| PT2833905T (en) | 2012-04-04 | 2018-08-06 | Halozyme Inc | Combination therapy with hyaluronidase and a tumor-targeted taxane |
| US8921419B2 (en) | 2012-05-08 | 2014-12-30 | Trustees Of Dartmouth College | Triterpenoids and compositions containing the same |
| WO2016078397A1 (en) * | 2014-11-17 | 2016-05-26 | 常州方圆制药有限公司 | New type of cytidine derivative and application thereof |
| WO2016078160A1 (en) * | 2014-11-17 | 2016-05-26 | 常州方圆制药有限公司 | Cytidine derivative and application thereof |
| US20190351031A1 (en) | 2018-05-16 | 2019-11-21 | Halozyme, Inc. | Methods of selecting subjects for combination cancer therapy with a polymer-conjugated soluble ph20 |
| GR1009958B (en) * | 2019-04-24 | 2021-03-18 | ΕΝΟΡΑΣΙΣ ΑΝΩΝΥΜΗ ΕΜΠΟΡΙΚΗ ΕΤΑΙΡΕΙΑ ΦΑΡΜΑΚΕΥΤΙΚΩΝ ΚΑΙ ΙΑΤΡΙΚΩΝ ΥΛΙΚΩΝ ΚΑΙ ΜΗΧΑΝΗΜΑΤΩΝ με δ.τ. "ΕΝΟΡΑΣΙΣ Α.Ε." | Gemcitabine derivatives and methods of forming gemcotabine derivatives |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3651045A (en) * | 1968-10-21 | 1972-03-21 | Parke Davis & Co | 9-(beta-d-arabinofuranosyl)adenine esters and methods for their production |
| AU542271B2 (en) * | 1980-06-23 | 1985-02-14 | Regents Of The University Of Minnesota | Adenine nucleosides |
| DE3100478A1 (en) * | 1981-01-09 | 1982-08-12 | Dr. Thilo & Co GmbH, 8021 Sauerlach | 5'ESTERS OF PYRIMIDINE NUCLEOSIDES WITH ANTIVIRAL EFFECTIVENESS, METHOD FOR THE PRODUCTION AND MEDICINAL PRODUCTS THEREOF |
| US4495180A (en) * | 1982-06-21 | 1985-01-22 | Merck & Co., Inc. | Prodrugs of Ara-A an antiviral agent |
| EP0104857A1 (en) * | 1982-09-28 | 1984-04-04 | Beecham Group Plc | Deoxyuridine compounds, methods for preparing them and their use in medicine |
| EP0151189B1 (en) * | 1983-07-20 | 1990-01-31 | Teijin Limited | Antineoplastic agent |
| ATE190064T1 (en) * | 1985-09-17 | 2000-03-15 | Wellcome Found | COMBINATION THERAPEUTIC NUCLEOSIDES WITH OTHER THERAPEUTICALLY EFFECTIVE COMPONENTS. |
| US4751221A (en) * | 1985-10-18 | 1988-06-14 | Sloan-Kettering Institute For Cancer Research | 2-fluoro-arabinofuranosyl purine nucleosides |
| US4780452A (en) * | 1986-09-08 | 1988-10-25 | Burroughs Wellcome Co. | F-substituted-3-β-D-ribofuranosyl-3H-imidazo[4,5-b]pyridines and pharmaceutical compositions thereof |
| AU613026B2 (en) * | 1987-03-24 | 1991-07-25 | Nycomed As | 2',3' dideoxyribofuranoxide derivatives |
| CA1312599C (en) * | 1988-02-16 | 1993-01-12 | Larry Wayne Hertel | 2',3'dideoxy-2',2'difluoronucleosides |
| JPH02152976A (en) * | 1988-05-06 | 1990-06-12 | Bristol Myers Co | Prodrug of 2',3'-didehydro-2',3'-dideoxynucleoside |
| US4900828A (en) * | 1988-05-12 | 1990-02-13 | Hoffmann-Laroche Inc. | Intermediate compounds and an improved procedure for the synthesis of 2',3'-dideoxycytidine |
| EP0346108A3 (en) * | 1988-06-09 | 1991-04-24 | The Wellcome Foundation Limited | Anti-infective nucleosides |
| EP0355031A3 (en) * | 1988-08-17 | 1990-12-27 | MATTHES, Eckart, Dr. | Substituted pyrimidine nucleosides, process for their preparation and pharmaceutical compounds containing them |
| GB8823319D0 (en) * | 1988-10-05 | 1988-11-09 | Nycomed As | Chemical compounds |
| GB8823320D0 (en) * | 1988-10-05 | 1988-11-09 | Nycomed As | Chemical compounds |
| GB8920534D0 (en) * | 1989-09-11 | 1989-10-25 | Wellcome Found | Antiviral compounds |
-
1991
- 1991-04-04 WO PCT/EP1991/000639 patent/WO1991015498A2/en not_active Application Discontinuation
- 1991-04-04 AU AU75584/91A patent/AU7558491A/en not_active Abandoned
- 1991-04-04 CA CA002079796A patent/CA2079796A1/en not_active Abandoned
- 1991-04-04 EP EP91906957A patent/EP0523110A1/en not_active Withdrawn
- 1991-04-04 JP JP91506841A patent/JPH05505815A/en active Pending
-
1992
- 1992-10-02 OA OA60286A patent/OA09677A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPH05505815A (en) | 1993-08-26 |
| WO1991015498A2 (en) | 1991-10-17 |
| WO1991015498A3 (en) | 1992-06-11 |
| EP0523110A1 (en) | 1993-01-20 |
| OA09677A (en) | 1993-05-15 |
| AU7558491A (en) | 1991-10-30 |
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