U.S. patent application number 11/182835 was filed with the patent office on 2005-11-10 for 1,3-oxathiolane nucleoside analogues.
Invention is credited to Coates, Jonathan Allan, Mutton, Ian Martin, Penn, Charles Richard, Storer, Richard, Williamson, Christopher.
Application Number | 20050250950 11/182835 |
Document ID | / |
Family ID | 10675345 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250950 |
Kind Code |
A1 |
Coates, Jonathan Allan ; et
al. |
November 10, 2005 |
1,3-oxathiolane nucleoside analogues
Abstract
(-)-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one,
its pharmaceutically acceptable derivatives, pharmaceutical
formulations thereof, methods for its preparation and its use as an
antiviral agent are described.
Inventors: |
Coates, Jonathan Allan;
(Greenford, GB) ; Mutton, Ian Martin; (Greenford,
GB) ; Penn, Charles Richard; (Greenfield, GB)
; Williamson, Christopher; (Greenford, GB) ;
Storer, Richard; (Greenford, GB) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
10675345 |
Appl. No.: |
11/182835 |
Filed: |
July 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11182835 |
Jul 18, 2005 |
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08460854 |
Jun 5, 1995 |
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08460854 |
Jun 5, 1995 |
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07835964 |
Feb 20, 1992 |
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6180639 |
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Current U.S.
Class: |
549/200 |
Current CPC
Class: |
A61P 31/18 20180101;
A61K 45/06 20130101; A61P 31/00 20180101; A61K 31/70 20130101; A61K
31/505 20130101; A61K 31/506 20130101; A61K 31/7068 20130101; C07D
411/04 20130101; A61P 37/02 20180101; A61P 37/04 20180101; A61P
31/12 20180101; A61K 31/7068 20130101; A61K 31/506 20130101; A61K
31/7068 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
549/200 |
International
Class: |
C07D 321/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 2, 1991 |
WO |
PCT/GB91/00706 |
May 2, 1990 |
GB |
9009861.7 |
Claims
1-18. (canceled)
19. A composition for use in treating an HIV infection in a
patient, said composition comprising an amount of a 2',3'-dideoxy
nucleoside compound effective to inhibit the growth or replication
of HIV in combination with a toxicity reducing effective amount of
the compound
(-)-(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimid-
in-2-one or a pharmaceutically acceptable salt thereof.
20. A composition for use in treating an HIV infection in a
patient, said composition comprising an amount of an anti-HIV
D-nucleoside compound effective to inhibit the growth or
replication of HIV in combination with a toxicity reducing
effective amount of the L-nucleoside compound
(-)-(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimid-
in-2-one or pharmaceutically acceptable salt thereof.
21. A method for reducing the toxicity associated with the
administration of a D-nucleoside compound in a patient, comprising
co-administering with said D-nucleoside compound a toxicity
reducing effective amount of (-)-(2R,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimi-
din-one or a pharmaceutically acceptable salt thereof.
22. A method for reducing the toxicity associated with
administration of a 2',3'-dideoxy nucleoside in a patient
comprising co-administering with said 2',3'-dideoxy nucleoside a
toxicity reducing effective amount of
(-)-(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimid-
in-2-one or a pharmaceutically acceptable salt thereof.
23. A composition for use in treating an HIV infection in a
patient, said composition comprising an amount of an anti-HIV
D-nucleoside compound effective to inhibit the growth or
replication of HIV in combination with a toxicity reducing
effective amount of the L-nucleoside compound
.beta.-L-2'-deoxy-3'-thiacytidine or a pharmaceutically acceptable
salt thereof.
24. A method for reducing toxicity associated with the
administration of a D-nucleoside compound in a patient, comprising
co-administering with said D-nucleoside compound a toxicity
reducing effective amount of, .beta.-L-2'-deoxy-3'-thiacytidine or
a pharmaceutically acceptable salt thereof.
25. A composition for use in treating an HIV infection in a
patient, said composition comprising an effective amount of a
2',3'-dideoxy nucleoside compound in combination with an effective
amount of the compound
(-)-(2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimid-
in-2-one or a pharmaceutically acceptable salt thereof.
26. A composition for use in treating an HIV infection in a
patient, said composition comprising an amount of an anti-HIV
D-nucleoside compound and an amount of the L-nucleoside compound
(-)-(2R,cis)-4-amino-1-(2-hydroxym-
ethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one or a
pharmaceutically acceptable salt thereof.
27. A composition for use in treating an HIV infection in a
patient, said composition comprising an amount of an anti-HIV
D-nucleoside compound in combination with an amount of the
L-nucleoside compound .beta.-L-2'-deoxy-3'-thiacytidine or a
pharmaceutically acceptable salt thereof.
Description
[0001] The present invention relates to nucleoside analogues and
their use in medicine. More specifically the invention is concerned
with 1,3-oxathiolane nucleoside analogues, pharmaceutial
formulations thereof and the use thereof in the treatment of viral
infections.
[0002] The compound of formula (I) 1
[0003] also known as BCH-189 or NGPB-21 has been described as
having antiviral activity in particular against the human
immunodeficiency viruses (HIV's), the causative agents of AIDS (5th
Anti-Aids Conference, Montreal, Canada 5th-9 Jun. 1989: Abstracts
T.C.O.1 and M.C.P. 63; European Patent Application Publication No.
0382562). The compound of formula (I) is a racemic mixture of the
two enantiomers of formulae (I-1) and (I-2):-- 2
[0004] and was described and tested in the form of its racemate.
The only compound currently approved for the treatment of
conditions caused by HIV is 3'-azido-3'-deoxythymidine (AZT,
zidovudine, BW 509U) However, this compound has a significant
side-effect liability and thus either cannot be employed or, once
employed, may have to be withdrawn in a significant number of
patients. There is in consequence a continuing need to provide
compounds which are effective against HIV but with a concommitant
significantly better therapeutic index.
[0005] We have now found that, surprisingly, enantiomers of the
compound of formula (I) are equipotent against HIV but that one of
the enantiomers (the (-)-enantiomer) has considerably lower
cytotoxicity than the other enantiomer. There is thus provided in a
first aspect of the invention the (-) (or laevorotatory) enantiomer
of the compound of formula (I) and pharmaceutically acceptable
derivatives thereof.
[0006] The (-) enantiomer has the chemical name
(-)-cis-4-Amino-1-(2-hydro-
xymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one (hereinafter
compound (A)). It has the absolute stereochemistry of the compound
of formula (I-1) which has the name (2R,
cis))-4-amino-1-(2-hydroxymethyl-1,3-oxathi-
olan-5-yl)-(1H)-pyrimidin-2-one.
[0007] Preferably Compound A is provided substantially free of the
corresponding (+)-enantiomer, that is to say no more than about 5%
w/w of the (+)-enantiomer, preferably no more than about 2%, in
particular less than about 1% w/w is present.
[0008] By "a pharmaceutically acceptable derivative" is meant any
pharmaceutically acceptable salt, ester, or salt of such ester, of
compound (A) or any other compound which, upon administration to
the recipient, is capable of providing (directly or indirectly)
compound (A) or an antivirally active metabolite or residue
thereof.
[0009] It will be appreciated by those skilled in the art that
compound (A) may be modified to provide pharmaceutically acceptable
derivatives thereof, at functional groups in both the base moiety
and at the hydroxymethyl group of the oxathiolane ring.
Modification at all such functional groups are included within the
scope of the invention. However of particular interest are
pharmaceutically acceptable derivatives obtained by modification of
the 2-hydroxymethyl group of the oxathiolane ring.
[0010] Preferred esters of compound (A) include the compounds in
which the hydrogen of the 2-hydroxymethyl group is replaced by an
acyl function 3
[0011] in which the non-carbonyl moiety R of the ester is selected
from hydrogen, straight or branched chain alkyl (e.g. methyl,
ethyl, n-propyl, t-butyl, n-butyl), alkoxyalkyl (e.g.
methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g.
phenoxymethyl), aryl (e.g. phenyl optionally substituted by
halogen, C.sub.1-4 alkyl or C.sub.1-4 alkoxy); sulphonate esters
such as alkyl- or aralkylsulphonyl (e.g. methanesulphonyl); amino
acid esters (e.g. L-valyl or L-isoleucyl) and mono-, di- or
tri-phosphate esters.
[0012] With regard to the above described esters, unless otherwise
specified, any alkyl moiety present advantageously contains 1 to 16
carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety
present in such esters advantageously comprises a phenyl group.
[0013] In particular the esters may be a C.sub.1-16alkyl ester, an
unsubstituted benzyl ester or a benzyl ester substituted by at
least one halogen (bromine, chlorine, fluorine or iodine),
C.sub.1-6alkyl, C.sub.1-6alkoxy, nitro or trifluoromethyl
groups.
[0014] Pharmaceutically acceptable salts of the compound (A)
include those derived from pharmaceutically acceptable inorganic
and organic acids and bases. Examples of suitable acids include
hydrochloric, hydrobromic, sulphuric, nitric, perchloric fumaric,
maleic, phosphoric, glycollic, lactic, salicylic, succinic,
toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic,
formic, benzoic, malonic, naphthalene-2-sulphonic and
benzenesulphonic acids. Other acids such as oxalic, while not in
themselves pharmaceutically acceptable, may be useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically acceptable acid addition salts.
[0015] Salts derived from appropriate bases include alkali metal
(e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and
NR.sub.4-- (where R is C.sub.1-4alkyl) salts.
[0016] References hereinafter to a compound according to the
invention include both the compound (A) and its pharmaceutically
acceptable derivatives.
[0017] The compounds of the invention either themselves possess
antiviral activity and/or are metabolizable to such compounds. In
particular these compounds are effective in inhibiting the
replication of retroviruses, including human retroviruses such as
human immunodeficiency viruses (HIV's), the causative agents of
AIDS.
[0018] There is thus provided as a further aspect of the invention
compound (A) or a pharmaceutically acceptable derivative thereof
for use as an active therapeutic agent in particular as an
antiviral agent, for example in the treatment of retroviral
infections.
[0019] In a further or alternative aspect there is provided a
method for the treatment of a viral infection, in particular an
infection caused by a retrovirus such as HIV, in a mammal
including-man comprising administration of an effective amount of
compound (A) or a pharmaceutially acceptable derivative
thereof.
[0020] There is also provided in a further or alternative aspect
use of compound (A) or a pharmaceutically acceptable derivative
thereof for the manufacture of a medicament for the treatment of a
viral infection.
[0021] The compounds of the invention are also useful in the
treatment of AIDS related conditions such as AIDS-related complex
(ARC), progressive generalised lymphadenopathy (PGL), AIDS-related
neurological conditions (such as dementia or tropical paraparesis),
anti-HIV antibody positive and HIV-positive conditions; Kaposi's
sarcoma, thrombocytopenia purpurea and associated opportunistic
infections for example Pneumocystis carinii.
[0022] The compounds of the invention are also useful in the
prevention of progression to clinical illness of individuals who
are anti-HIV antibody or HIV-antigen positive and in prophylaxis
following exposure to HIV.
[0023] The compound (A) or pharmaceutically acceptable derivatives
thereof may also be used for the prevention of viral contamination
of physiological fluids such as blood or semen in vitro.
[0024] It will be appreciated by those skilled in the art that
reference herein to treatment extends to prophylaxis as well as the
treatment of established infections or symptoms.
[0025] It will be further appreciated that the amount of a compound
of the invention required for use in treatment will vary not only
with the particular compound selected but also with the route of
administration, the nature of the condition being treated and the
age and condition of the patient and will be ultimately at the
discretion of the attendant physician or veterinarian. In general
however a suitable dose will be in the range of from about 0.1 to
about 750 mg/kg of bodyweight per day preferably in the range of
0.5 to 60 mg/kg/day, most preferably in the range of 1 to 20
mg/kg/day.
[0026] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example as two, three, four or more sub-doses per day.
[0027] The compound is conveniently administered in unit dosage
form; for example containing 10 to 1500 mg, conveniently 20 to 1000
mg, most conveniently 50 to 700 mg of active ingredient per unit
dosage form.
[0028] Ideally the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 1 to about 75 .mu.M, preferably about 2 to 50 .mu.M, most
preferably about 3 to about 30 .mu.M. This may be achieved, for
example, by the intravenous injection of a 0.1 to 5% solution of
the active ingredient, optionally in saline, or orally administered
as a bolus containing about 1 to about 100 mg of the active
ingredient. Desirable blood levels may be maintained by a
continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour
or by intermittent infusions containing about 0.4 to about 15 mg/kg
of the active ingredient.
[0029] While it is possible that, for use in therapy, a compound of
the invention may be administered as the raw chemical it is
preferable to present the active ingredient as a pharmaceutical
formulation.
[0030] The invention thus further provides a pharmaceutical
formulation comprising compound (A) or a pharmaceutically
acceptable derivative thereof together with one or more
pharmaceutically acceptable carriers therefor and, optionally,
other therapeutic and/or prophylactic ingredients. The carrier(s)
must be `acceptable` in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0031] Pharmaceutical formulations include those suitable for oral,
rectal, nasal, topical (including buccal and sub-lingual), vaginal
or parenteral (including intramuscular, sub-cutaneous and
intravenous) administration or in a form suitable for
administration by inhalation or insufflation. The formulations may,
where appropriate, be conveniently presented in discrete dosage
units and may be prepared by any of the methods well known in the
art of pharmacy. All methods include the step of bringing into
association the active compound with liquid carriers or finely
divided solid carriers or both and then, if necessary, shaping the
product into the desired formulation.
[0032] Pharmaceutical formulations suitable for oral administration
may conveniently be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
active ingredient; as a powder or granules; as a solution, a
suspension or as an emulsion. The active ingredient may also be
presented as a bolus, electuary or paste. Tablets and capsules for
oral administration may contain conventional excipients such as
binding agents, fillers, lubricants, disintegrants, or wetting
agents. The tablets may be coated according to methods well known
in the art. Oral liquid preparations may be in the form of, for
example, aqueous or oily suspensions, solutions, emulsions, syrups
or elixirs, or may be presented as a dry product for constitution
with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, emulsifying agents, non-aqueous vehicles (which may include
edible oils), or preservatives.
[0033] The compounds according to the invention may also be
formulated for parenteral administration (e.g. by injection, for
example bolus injection or continuous infusion) and may be
presented in unit dose form in ampoules, pre-filled syringes, small
volume infusion or in multi-dose containers with an added
preservative. The compositions may take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory agents such as suspending, stabilising and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilisation from solution, for constitution with a suitable
vehicle, e.g. sterile, pyrogen-free water, before use.
[0034] For topical administration to the epidermis the compounds
according to the invention may be formulated as ointments, creams
or lotions, or as a transdermal patch. Ointments and creams may,
for example, be formulated with an aqueous or oily base with the
addition of suitable thickening and/or gelling agents. Lotions may
be formulated with an aqueous or oily base and will in general also
contain one or more emulsifying agents, stabilising agents,
dispersing agents, suspending agents, thickening agents, or
colouring agents.
[0035] Formulations suitable for topical administration in the
mouth include lozenges comprising active ingredient in a flavoured
base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatin
and glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0036] Pharmaceutical formulations suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter and other materials commonly used in the art, and the
suppositories may be conveniently formed by admixture of the active
compound with the softened or melted carrier(s) followed by
chilling and shaping in moulds.
[0037] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
sprays containing in addition to the active ingredient such
carriers as are known in the art to be appropriate.
[0038] For intra-nasal administration the compounds of the
invention may be used as a liquid spray or dispersible powder or in
the form of drops.
[0039] Drops may be formulated with an aqueous or non-aqueous base
also comprising one more more dispersing agents, solubilising
agents or suspending agents. Liquid sprays are conveniently
delivered from pressurised packs.
[0040] For administration by inhalation the compounds according to
the invention are conveniently delivered from an insufflator,
nebuliser or a pressurised pack or other convenient means of
delivering an aerosol spray. Pressurised packs may comprise a
suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurised aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount.
[0041] Alternatively, for administration by inhalation or
insufflation, the compounds according to the invention may take the
form of a dry powder composition, for example a powder mix of the
compound and a suitable powder base such as lactose or starch. The
powder composition may be presented in unit dosage form in, for
example, capsules or cartridges or e.g. gelatin or blister packs
from which the powder may be administered with the aid of an
inhalator or insufflator.
[0042] When desired the above described formulations adapted to
give sustained release of the active ingredient may be
employed.
[0043] The pharmaceutical compositions according to the invention
may also contain other active ingredients such as antimicrobial
agents, or preservatives.
[0044] The compounds of the invention may also be used in
combination with other therapeutic agents for example other
antiinfective agents. In particular the compounds of the invention
may be employed together with known antiviral agents.
[0045] The invention thus provides, in a further aspect, a
combination comprising the compound (A) or a physiologically
acceptable derivative thereof together with another therapeutically
active agent, in particular an antiviral agent.
[0046] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation and
thus pharmaceutical formulations comprising a combination as
defined above together with a pharmaceutically acceptable carrier
therefor comprise a further aspect of the invention.
[0047] Suitable therapeutic agents for use in such-combinations
include acyclic nucleosides such as acyclovir or ganciclovir,
interferons such as .alpha., .beta. or .gamma.-interferon, renal
excretion inhibitors such as probenecid, nucleoside transport
inhibitors such as dipyridamole, 2',3'-dideoxynucleosides such as
AZT, 2',3'-dideoxycytidine, 2',3'-dideoxyadenosine,
2',3'-dideoxyinosine, 2',3'-dideoxythymidine,
2',3'-dideoxy-2',3'-didehydrothymidine and
2',3'-dideoxy-2',3'-didehydroc- ytidine, immunomodulators such as
interleukin II (IL2) and granulocyte macrophage colony stimulating
factor (GM-CSF), erythropoetin, ampligen, thymomodulin,
thymopentin, foscarnet, ribavirin and inhibitors of HIV binding to
CD4 receptors e.g. soluble CD4, CD4 fragments, CD4 hybrid
molecules, glycosylation inhibitors such as 2-deoxy-D-glucose,
castanospermine and 1-deoxynojirimycin.
[0048] The individual components of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical formulations.
[0049] When the compound (A) or a pharmaceutically acceptable
derivative thereof is used in combination with a second therapeutic
agent active against the same virus the dose of each compound may
be either the same as or differ from that when the compound is used
alone. Appropriate doses will be readily appreciated by those
skilled in the art.
[0050] The compound (A) and its pharmaceutically acceptable
derivatives may be prepared by any method known in the art for the
preparation of compounds of analogous structure, for example as
described in European Patent Application Publication No.
0382562.
[0051] It will be appreciated by those skilled in the art that for
certain of the methods described herein below the desired
stereochemistry of the compound (A) may be obtained either by
commencing with an optically pure starting material or by resolving
the racemic mixture at any convenient stage in the synthesis. In
the case of all the processes the optically pure desired product
may be obtained by resolution of the end product of each
reaction
[0052] In one such process (A) a 1,3-oxathiolane of formula (VIII)
4
[0053] wherein the anomeric group L is a displaceable group, is
reacted with an appropriate base. Suitable groups L include --OR
where R is an alkyl group, e.g. a C.sub.1-6alkyl group such as
methyl or R is an acyl group, e.g. a C.sub.1-6alkyl group such as
acetyl or halogen, for example iodine, bromine or chlorine.
[0054] The compound of formula VIII is conveniently reacted with
cytosine or an appropriate pyrimidine base precursor thereof
(previously silylated with a silylating agent such as
hexamethyldisilazane) in a compatible solvent such as methylene
chloride using a Lewis acid such as titanium tetrachloride, tin
(IV) compound such as SnCl.sub.4, or trimethylsilyltriflate.
[0055] The 1,3-oxathiolanes of formula (VIII) may be prepared for
example by reaction of an aldehyde of formula (VII) with a
mercaptoacetal of formula (VI) in a compatible organic solvent,
such as toluene in the presence of an acid catalyst for example a
Lewis acid such as zinc chloride.
HSCH.sub.2CH(OC.sub.2H.sub.5).sub.2 (VI)
C.sub.6H.sub.5CO.sub.2CH.sub.2CHO (VII)
[0056] The mercaptoacetals of formula (VI) may be prepared by
methods known in the art, for example G. Hesse and I. Jorder, Chem.
Ber 85, 924-932 (1952).
[0057] The aldehydes of formula (VII) may be prepared by methods
known in the art for example E. G. Halloquist and H. Hibbert, Can.
J. Research, 8, 129-136 (1933). Conveniently the crude aldehyde
(VII) may be purified by conversion to the crystalline bisulphite
addition adduct and subsequent reconversion to the free
aldehyde.
[0058] In a second process (B) the compound (A) is obtained by base
interconversion of a compound of formula (IX) 5
[0059] where B is a base convertible to cytosine. Such
interconversion may be effected either by simple chemical
transformation (e.g. the conversion of uracil base to cytosine) or
by an enzymatic conversion using a deoxyribosyl transferase. Such
methods and conditions for base interconversion are well known in
the art of nucleoside chemistry.
[0060] In a third process (C) a compound of formula (XI) 6
[0061] may be converted to the compound (A) by conversion of the
anomeric NH.sub.2 group to the cytosine base by methods well known
in the nucleoside chemistry art.
[0062] Many of the reactions described hereinabove have been
extensively reported in the context of nucleoside synthesis, for
example in Nucleoside Analogs--Chemistry, Biology and Medical
Applications, R. T. Walker et. al., eds, Plenum Press, New York
(1979) at pages 165-192; T. Ueda in Chemistry of Nucleosides and
Nucleotides, Vol I, L B Townsend ed., Plenum Press, New York (1988)
at pages 165-192 the disclosures of which are incorporated by
reference herein.
[0063] It will be appreciated that the above reactions may require
the use of, or conveniently may be applied to, starting materials
having protected functional groups, and deprotection might thus be
required as an intermediate or final step to yield the desired
compound. Protection and deprotection of functional groups may be
effected using conventional means. Thus, for example, amino groups
may be protected by a group selected from aralkyl (e.g. benzyl),
acyl, aryl (e.g. 2,4-dinitrophenyl) or silyl; subsequent removal of
the protecting group being effected when desired by hydrolysis or
hydrogenolysis as appropriate using standard conditions. Hydroxyl
groups may be protected using any conventional hydroxyl protecting
group, for example, as described in `Protective Groups in Organic
Chemistry`, Ed. J. F. W. McOmie (Plenum Press, 1973) or `Protective
Groups in Organic Synthesis` by Theodora W. Greene (John Wiley and
Sons, 1981). Examples of suitable hydroxyl protecting groups
include groups selected from alkyl (e.g. methyl, t-butyl or
methoxymethyl), aralkyl (e.g. benzyl, diphenylmethyl or
triphenylmethyl), heterocyclic groups such as tetrahydropyranyl,
acyl (e.g. acetyl or benzoyl) and silyl groups such as
trialkylsilyl (e.g. t-butyldimethylsilyl). The hydroxyl protecting
groups may be removed by conventional techniques. Thus, for
example, alkyl, silyl, acyl and heterocyclic groups may be removed
by solvolysis, e.g. by hydrolysis under acidic or basic conditions.
Aralkyl groups such as triphenylmethyl may similarly be removed by
solvolysis, e.g. by hydrolysis under acidic conditions. Aralkyl
groups such as benzyl may be cleaved for example by treatment with
BF.sub.3/etherate and acetic anhydride followed by removal of
acetate groups so formed at an appropriate stage in the synthesis.
Silyl groups may also conveniently be removed using a source of
fluoride ions such as tetra-n-butylammonium fluoride.
[0064] In the above processes compound (A) is generally obtained as
a mixture of the cis and trans isomers of which the cis isomer is
the compound of interest.
[0065] These isomers may be separated by physical means e.g.
chromatography on silica gel or by fractional crystallisation,
either directly or on a suitable derivatives thereof, eg acetates
(prepared for example with acetic anhydride) followed, after
separation, by conversion back to the parent product (eg by
deacetylation with methanolic ammonia).
[0066] Pharmaceutically acceptable salts of the compounds of the
invention may be prepared as described in U.S. Pat. No. 4,383,114,
the disclosure of which is incorporated by reference herein. Thus,
for example, when it is desired to prepare an acid addition salt of
compound (A) the product of any of the above procedures may be
converted into a salt by treatment of the resulting free base with
a suitable acid using conventional methods. Pharmaceutically
acceptable acid addition salts may be prepared by reacting the free
base with an appropriate acid optionally in the presence of a
suitable-solvent such as an ester (e.g. ethyl acetate) or an
alcohol (e.g. methanol, ethanol or isopropanol). Inorganic basic
salts may be prepared by reacting the parent compound with a
suitable base such as an alkoxide (e.g. sodium methoxide)
optionally in the presence of a solvent such as an alcohol (e.g.
methanol). Pharmaceutically acceptable salts may also be prepared
from other salts, including other pharmaceutically acceptable
salts, of the compound (A) using conventional methods.
[0067] Compound (A) may be converted into a pharmaceutically
acceptable phosphate or other ester by reaction with a
phosphorylating agent, such as POCl.sub.3, or a suitable
esterifying agent, such as an acid halide or anhydride, as
appropriate. An ester or salt of compound (A) may be converted to
the parent compound for example by hydrolysis.
[0068] Resolution of the final product, or an intermediate or
starting material therefor may be effected by any suitable method
known in the art: see for example `Stereochemistry of Carbon
Compounds` by E. L. Eliel (McGraw sill, 1962) and `Tables of
Resolving Agents` by S. H. Wilen.
[0069] Thus for example the compound (A) may obtained by chiral
HPLC using a suitable stationary phase for example acetylated
.beta.-cyclodextrin or cellulose triacetate and a suitable solvent
for example an alcohol such as ethanol or an aqueous solution of
for example triethyl ammonium acetate. Alternatively the compounds
may be resolved by enzyme mediated enantioselective catabolism with
a suitable enzyme such as cytidine deaminase or selective enzymatic
degradation of a suitable derivative using a 5'nucleotidase. When
resolution is effected enzymatically the enzyme may be employed
either in solution or, more conveniently, in immobilised form.
Enzymes may be immobilised by any method known in the art, for
example by adsorption onto a resin such as Eupergit C.
[0070] The invention will be further described by the following
examples which are not intended to limit the invention in any way.
All temperatures are in degrees Celcius.
[0071] Intermediate 1
5-Methoxy-1,3-oxathiolane-2-methanol, benzoate
[0072] A solution of zinc chloride (1.6 g) in hot methanol (15 ml)
was added to a stirred solution of mercaptoacetaldehyde, dimethyl
acetal (34.2 g) and benzoyloxy acetaldehyde (48.3 g) in toluene
(1300 ml) which was then heated to reflux under nitrogen for 50
min. The cooled mixture was concentrated, diluted with some
toluene, then filtered through Kiesulguhr. The combined filtrates
and toluene were washed with aqueous saturated sodium bicarbonate
solution (.times.2) and brine, dried (MgSO.sub.4) then evaporated
to an oil which was subjected to column chromatography on silica (2
kg, Merck 9385 eluted with chloroform to give the title product as
an oil (45.1 g) a mixture of anomers (ca 1:1); .sup.1H NMR
(DMSO-d.sub.6) 3.1-3.3 (4H), 3.42 (6H), 4.4-4.6 (4H), 5.41 (1H),
5.46 (1H), 5.54 (1H), 5.63 (1H), 7.46 (4H), 7.58 (2H), 8.07 (4H);
.gamma.max (CHBr.sub.3) 1717.6 cm.sup.-1.
[0073] Intermediate 2
(-)-cis-1-(2-Benzoyloxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-4-dion-
e
[0074] A mixture of finely ground uracil (9.62 g) hexamethyl
disilazane (50 ml) and ammonium sulphate (30 mg) was heated at
reflux under nitrogen until a clear solution was obtained. This was
cooled and then evaporated to a colourless oil, which was
dissolved, under nitrogen atmosphere, in acetonitrile (100 ml). The
solution was added to a stirred ice cooled solution of
5-methoxy-1,3-oxathiolane-2-methanol, benzoate (intermediate 1)
(19-43 g), in acetonitrile (600 ml) and trimethyl silyl
trifluoromethanesulphonate (14.7 ml) was added. The ice bath was
removed, and the solution was heated at reflux under nitrogen for
45 mins. After cooling and evaporation, the residue was purified by
column chromatography over 1 kg of silica gel (Merck 9385) eluting
with chloroform/methanol 9:1. Appropriate fractions were cooled and
evaporated to afford a crude residue. This was fractionally
crystallized from the minimum of hot methanol (c.1200 ml) to afford
the title compound (6.32 g) as white crystals. 1H NMR (d.sup.6DMSO)
.delta. 11.36 (1H, bs). 7.50-8.00 (6H, m), 6.20 (1H, t), 5.46 (2H,
m), 4.62 (2H, m), 3.48 (1H, m), 3.25 (1H, m).
[0075] Intermediate 3
(.+-.)-(cis)-4-Amino-1-(2-benzoyloxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrim-
idin-2-one
[0076] Method (a)
[0077] A suspension of cytosine (20.705 g) and ammonium sulphate
(few mgs) in hexamethyldisilazane (110 ml) was stirred and heated
at reflux for 21/2 h, under nitrogen. Solvent was removed by
evaporation, and the residual solid was dissolved in dry
acetonitrile (350 ml). This solution was transferred using flexible
needle techniques into a stirred, ice-chilled solution of
5-methoxy-1,3-oxathiolane-2-methanol, benzoate (Intermediate I)
(43.57 g) in acetonitrile (650 ml) under nitrogen. Trimethylsilyl
trifluoromethanesulphonate (33 ml) was added, the solution was
allowed to warm to ambient temperature (11/2 h) then heated to
reflux for an overnight period. The residue mixture was
concentrated, diluted with saturated aqueous sodium bicarbonate
solution (500 ml), then extracted with ethyl acetate (3.times.500
ml). The combined extracts were washed with water (2.times.250 ml)
and brine (250 ml) dried (MgSO.sub.4) then evaporated to a foam
which was subjected to column chromatography on silica (600 g,
merck 7734), eluted with ethyl acetate-methanol mixtures to give a
mixture of anomers (ca 1:1 31.59 g). The mixture was crystallised
from water (45 ml) and ethanol (9.0 ml) to give a solid (10.23 g)
which was recrystallised from ethanol (120 ml) and water (30 ml) to
give the title product as a white solid (9.26 g); .lambda.max
(MeOH) 229.4 mm (E.sup.1%.sub.1cm610); 272.4 mm
(E.sup.1%.sub.1cm293); .sup.1H NMR (DMSO d6) .delta. 3.14 (1H),
3.50 (1H), 4.07 (2H), 5.52 (1H), 5.66 (1H), 6.28 (1H), 7.22 (2H),
7.56 (2H), 7.72 (2H), 8.10 (2H).
[0078] Method (b)
[0079] Phosphorus oxychloride (7.0 ml) was added dropwise to a
stirred ice-cooled suspension of 1,2,4-triazole (11.65 g) in
acetonitrile (120 ml) then, keeping the internal temperature below
15.degree. C., triethylamine (22.7 ml) was added dropwise. After 10
min a solution of
(.+-.)-cis-1-(2-benzoyloxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2,4--
dione (Intermediate 2) (6.27 g) in acetonitrile (330 ml) was slowly
added. Stirring was then continued at room temperature overnight.
The mixture was cooled by means of an ice bath and triethylamine
(30 ml) was slowly added followed by water (21 ml). The resultant
solution was evaporated, and the residue was partitioned between
saturated sodium bicarbonate solution (400 ml) and chloroform
(3.times.200 ml). The combined chloroform extracts were dried and
magnesium sulphate, filtered and evaporated to give a crude residue
(9.7 g). The residue was dissolved in 1,4-dioxan (240 ml) and
concentrated aqueous ammonia solution (s.g 0.880, 50 ml) was added.
After 11/2 h the solution was evaporated and the residue dissolved
in methanol. This caused precipitation of a solid, which was
filtered off. The mother liquors were purified by column
chromatography over silica gel (Merck 9385, 600 g). Appropriate
fractions were pooled and evaporated to give the title compound as
a fawn solid (2.18 g), identical to that obtained by Method
(a).
EXAMPLE 1
(.+-.)-(cis)-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidi-
n-2-one
[0080] A suspension of
(cis)-4-amino-1-(2-benzoyloxymethyl-1,3-oxathiolan--
5-yl)-(1H)-pyrimidin-2-one (Intermediate 3) (8.19 g) and Amberlite
IRA-400 (OR) resin (8.24 g) in methanol (250 ml) was stirred and
heated to reflux for 11/4 h. Solids were removed by filtration then
washed with methanol. The combined filtrates were evaporated. The
residue was triturated with ethyl acetate (80 ml). The resulting
white solid was collected by filtration to give the title product
(5.09 g), .sup.1H NMR (DMSO-d.sub.6) 3.04 (1H), 3.40 (1H), 3.73
(2H), 5.16 (1H), 5.29 (1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81
(1H).
EXAMPLE 2
Chiral HPLC separation of the enantiomers of
(.+-.)-(cis)-4-Amino-1-(2-hyd-
roxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one
[0081] (A) The racemic product of Example 1 (25 mg) was subjected
to preparative HPLC under the following conditions:
[0082] Column: Merck Hibar cellulose triacetate, 250.times.10 mm,
10.mu.
[0083] Eluant: ethanol;
[0084] Flow: 3 ml/min;
[0085] Detection: uv, 270 nm;
[0086] Temperature: ambient.
[0087] Evaporation of the appropriate pooled fractions gave (2R,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one
(6.5 mg, e.e. ca 100%) and (2S,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxath-
iolan-5-yl)-(1H)-pyrimidin-2-one (3.6 mg, e.e. ca 90%).
[0088] (B) The racemic product of Example 1 (26 mg) was subjected
to preparative HPLC under the following conditions:
[0089] Column: ASTEC cyclobond I acetyl, 250.times.4.6 mm;
[0090] Eluant: 0.2% triethylammonium acetate (prepared by adding
glacial acetic acid to 0.2% triethylamine in water, to a final pH
7.2);
[0091] Flow: 2 ml/min;
[0092] Detection: uv, 300 nm
[0093] Temperature: ambient.
[0094] Evaporation of appropriate fractions gave crude (2R,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one
(25 mg) and crude (2S,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-y-
l)-(1H)-pyrimidin-2-one (0.7 mg). These fractions were separately
subjected to further preparative HPLC run under the following
conditions:
[0095] Column: ASTEC cyclobond I acetyl, 250.times.4.6 mm
[0096] Eluent: 15 mM ammonium acetate, pH 6.8;
[0097] Flow: 0.5 ml/min
[0098] Detection: uv, 300 nm;
[0099] Temperature: 5.degree..
[0100] Evaporation of the appropriate pooled fractions gave (2R,
Cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one
(5.0 mg, e.e ca. 91%) and (2S,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathi-
olan-5-yl)-(1)-pyrimidin-2-one (7.6 mg, e.e. ca 96%)
EXAMPLE 3
(-)-cis-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-o-
ne
(i)
(.+-.)-cis-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimi-
dinone, dihydrogenphosphate, ammonium salt
[0101] A cold (0.degree.), stirred suspension of
(.+-.)-cis-4-amino-1-(2-h-
ydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one (1.00 g)
(Example 1) in dry trimethylphosphate (20 ml) was treated with
phosphorous oxychloride (2.44 ml), and the mixture was stirred at
0.degree. for 35 mins then quenched in ice water (60 g). The cold
mixture was adjusted to pH 2.5 by the addition of aqueous N-sodium
hydroxide, then applied to a charcoal column (10 g, DARCO), which
was eluted with water, then ethanol-aqueous ammonia. Fractions
containing crude monophosphate were combined and concentrated. The
resulting solution was applied to a column containing 25 g of DEAE
Sephadex A-25 (HCO.sub.3-form). Elution was performed with a
gradient of water (120 ml) to 0.1 M-NH.sub.4HCO.sub.3 (240 ml),
then 0.2, 0.3 and 4M NH.sub.4HCO.sub.3 (120, 240, 400 mL
respectively). Appropriate fractions were combined and
concentrated. The residual solution was diluted with water (40 ml)
and freeze-dried to give the title product as a white solid (1.37
g); .lambda.max (pH6 buffer), 271.0 nm (E.sup.1%.sub.1 cm190);
.sup.1F. NMR (D20) .delta.3.23 (1H), 3.55 (1H), 4.0-4.2 (2H), 5.43
(1H), 6.07 (1H), 6.33 (1H), 8.08 (1H).
(ii) (2R,
cis)-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5yl)-(1H)-pyrimid-
in-2-one and (2S,
cis)-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H-
)-pyrimidin-2-one
[0102] 5'-Nucleotidase (from crotalus atrox venom) (EC 3.1.3.5] (60
mg at 17 units/mg) was added to a solution of
(.+-.)-cis-4-amino-(2-hydroxymeth-
yl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one,
6'-dihydrogenphosphate, ammonium salt (1.35 g) in buffer [30 ml,
prepared from glycine (526 mg) and magnesium chloride (190 mg) in
water (100 ml)] and the mixture was incubated at 37.degree. for 2.5
h. More enzyme (20 mg) was added, and incubation was continued for
a further 3.5 h. The resulting mixture was applied to a column of
DEAE Sephadex A-25 (HCO.sub.3-form) Elution was performed with
water (160 ml), then 0.1, 0.2, 0.3 and 0.4M NH.sub.4HCO.sub.3 (200
ml each). Appropriate fractions containing the first eluted
component were combined and evaporated, the residue was subjected
to column chromatography on silica (60 g, Merck 7734), eluted with
chloroform-methanol mixtures. Evaporation of the appropriate
fractions from methanol-ethyl acetate gave (2R,
Cis)-4-amino-1-(2-hydroxy-
methyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one as a white solid
(0.30 g)-[.alpha.]D.sup.21+137.degree.(c.1.04 MeOH); .sup.1H NMR
(DMSO) .delta. 3.04 (1H), 3.40 (1H), 3.73 (2H), 5.18 (1H), 5.29
(1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81 (1H)
[0103] Appropriate fractions from the Sephadex column, containing
the second eluted component, were combined and evaporated. The
residue was dissolved in water (30 ml), treated with alkaline
phosphatase (from Escherichia coli) [EC 3.1.3.1) (1.5 ml at 416
units/ml), then incubated at 37.degree. for one hour. Solvent was
removed by evaporation and the residue was subjected to column
chromatography on silica (60 g, Merck 7734), eluted with
chloroform-methanol mixtures. Evaporation of the appropriate
fractions from methanol-ethyl acetate gave (2S,
cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one
as a white solid (0.32 g); [.alpha.]D.sup.21-132.degree. (c.1.08,
MeOH); .sup.1H NMR (DMSO) .delta. 3.04 (1H), 3.40 (1H), 3.73 (2H),
5.18.degree. F.), 5.29 (1H), 5.73 (1H), 6.21 (1H), 7.19 (2H), 7.81
(1H).
EXAMPLE 4
(-)-cis-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)
pyrimidin-2-one
[0104] (i) Three 50 ml flasks of nutrient broth (Oxoid Ltd) were
innoculated with a loopful each of Escherichia coli (ATCC 23848)
scraped from a Nutrient Agar plate. The flasks were incubated
overnight at 37.degree. C. with shaking at 250 rev/min and then
each flask was used to innoculate 41 of CDD medium (glutamic acid,
3 g/l; MgSO.sub.4, 0.2 g/l: K.sub.2SO.sub.4, 2.5 g/l; NaCl, 2.3
g/l, Na.sub.2HPO.sub.42H.sub.2O, 1.1 g/l,
NaH.sub.2PO.sub.42H.sub.2O 0.6 g/l cytidine, 1.2 g/l) in a seven
litre fermenter. The cultures were fermented at 750 rev/min,
37.degree. C. with aeration at 41/min. After growth for 24 hrs the
cells were collected by centrifugation (5000 g, 30 minutes) to
yield 72 g wet weight. The cell pellet was resuspended in 300 ml of
20 mM Tris HCl buffer (pH 7.5) and disrupted by sonication
(4.times.45 seconds). The cell debris was removed by centrifugation
(30,000 g, 30 minutes) and the protein in the supernatant was
precipitated by addition of ammonium sulphate to 75% saturation.
The precipitate was collected by centrifugation (30,000 g. 30
minutes) and the pellet was resuspended in 25 ml of HEPES buffer
(100 mM, pH 7.0) containing ammonium sulphate (75% saturation).
Enzyme solution was prepared by centrifugation at 12,000 rpm for 30
mins. The supernatant was discarded and the pellet dissolved in
Tris HCl buffer (pH 7.0; 100 mM) to the original volume.
[0105] (ii) The product of Example 1 (115 mg was dissolved in water
(100 ml), and stirred. Enzyme solution (0.5 ml) was added, and the
mixture was maintained at a constant pH by the continual addition
of HCl (25 mM). The conversion was monitored by chiral HPLC, which
showed that the (+) enantiomer of the substrate was preferentially
deaminated. After 22 hr the (+) enantiomer of the substrate (RT
12.5 min) had been completely removed, and the solution was
adjusted to pH 10.5 by the addition of conc. sodium hydroxide.
[0106] The solution produced above was eluted through a column of
QAE Sephadex (A25; Pharmacia; 30.times.1.6 cm), pre-equilibrated to
pH11. The column was washed with water (200 ml) and then with HCl
(0.1M). Fractions (40 ml) were taken, and analysed by reversed
phase HPLC. Fractions 5-13, containing the unreacted (-) enantiomer
of the substrate, were combined and adjusted to pH 7.5 with HCl.
Fraction 47, containing deaminated product, was adjusted to pH7.5
with dil. NaOH. Analysis by chiral HPLC showed that this material
was a mixture, consisting of one enantiomer (RT 10.2 min) as the
major component with the other enantiomer (RT 8.5 min) as a minor
component (e.e ca 90%).
[0107] (iii) Stage (ii) above was repeated on a larger scale. The
compound of Example 1 (363 mg) in 250 ml of water was incubated
with enzyme solution (0.5 ml), prepared as in Stage (i). Further
aliquots (0.5 ml) of enzyme were added after 18 and 47 hrs. The
reaction mixture was stirred for 70 hr., then left standing for a
further 64 hr. Analysis by chiral hplc indicated that the (+)
enantiomer of the substrate had been completely deaminated, and the
resulting solution was adjusted to pH10.5 with NaOH.
[0108] The solution above was loaded onto the same QAE column, and
eluted as in stage (i). Fractions 2-6, containing a mixture of the
residual substrate and deaminated product, were bulked. Fractions
7-13, containing the residual substrate ((-) enantiomer), were
bulked and adjusted to pH7.5. Fractions 25-26, containing
deaminated product, were bulked and neutralised
[0109] Fractions 2-6 above were re-eluted through the same QAE
column. Fractions 3-11 from this second column contained unrected
substrate ((-) enantiomer). Fraction 70 contained the deaminated
product.
[0110] (iv) The resolved substrate fractions from stage (ii) and
(iii) were combined and adjusted to pH7.5. This solution was eluted
through a column of XAD-16 (40.times.2.4 cm), packed in water. The
column was washed with water, and then eluted with acetone: water
(1:4 v/v).
[0111] Fractions containing the (-) enantiomer of BCH 189 were
bulked and freeze-dried to give a white powder (190 mg).
[0112] The HPLC methods used above were as follows:--
[0113] 1. Reversed Phase analytical HPLC
1 Column Capital Cartridge Spherisorb ODS-2 (5 uM) 150 .times. 4.6
mm Eluant Ammonium dihydrogen phosphate (50 mM) + 5% MeCN Flow 1.5
ml/min Detection UV, 270 nm Retention Times BCH 189 5.5 min
deaminated BCH -189 8.1 min
[0114] 2. Chiral analytical HPLC
2 Column Cyclobond I Acetyl 250 .times. 4.6 mm Eluant 0.2%
Triethylammonium acetate (pH 7.2) Flow 1.0 ml/min Detection UV, 270
nm Retention Times BCH 189 11.0 and 12.5 min deaminated BCH-189 8.5
and 10.2 min (The bioconversion was followed by monitoring the loss
of the peak at 12.5 min., and accumulated of product at 10.2
min).
EXAMPLE 5
(-)-cis-4-Amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-o-
ne
[0115] A loopful of E. coli B cells (ATCC 32848) scraped from a
well grown nutrient agar plate was used to inoculate two Florence
flasks each containing 250 ml of nutrient broth. The culture was
incubated at 37.degree. with shaking (250 rev/min, 5 cm throw) for
18 hours. This was then used to inoculate 40 L of CDD-medium, with
cytidine, in a 70 L fermenter.
[0116] Conditions for the fermentation were as follows: 40 L/min
aeration, 750 rev/min stirring speed at a temperature of 37.degree.
C. Three Rushton impellers were fitted to the fermenter. The
fermentation was run for 18 hours before harvest using a Sharples
continuous centrifuge. The cell paste (150 g net weight) was frozen
at -20.degree. C. prior to cell breakage.
3 CDD Medium g/L L-Glutamic acid 3 MgSO.sub.4 0.2 K.sub.2SO.sub.4
2.5 NaCl 2.3 Na.sub.2HPO.sub.4 1.1 NaH.sub.2PO.sub.4 0.6
[0117] Made with distilled water. Sterilised at 121.degree. C. for
30 minutes. Cytidine (1.2 g/L) was filter sterilised and added
prior to inoculation.
[0118] The frozen cell paste (150 g) was thawed and suspended in
750 ml of 100 mM Hepes (N-(2-hydroxyethyl]piperazine-N'-[2-ethane
sulphonic acid]) buffer (pH 7.5) containing 1 mM
ethylenediaminetetraacetic acid (sodium salt) and 1 mM
dithiothreitol (disruption buffer). The cells were disrupted by
passing the suspension through a Manton-Gaulin homogeniser
operating at 7500 psi. This was carried out three times with
cooling of the suspension to approximately 5.degree. C. after each
pass through the homogeniser. The homogenate was clarified by
centrifugation (1400 g; 60 min). The cytidine deaminease activity
was adsorbed onto a Q-Sepharose column (490 mg bed volume)
pre-equilibrated with 50 mM Tris (hydroxymethyl)methylamine (pH
7.5) containing 1 mM sodium chloride. The pooled active fractions
(210 ml) were added onto a phenyl-Sepharose column (490 ml bed
volume), pre-equilibrated with the sample buffer containing 3.2M
ammonium sulphate. The bound enzyme was eluted by a gradient of
decreasing ammonium sulphate concentration. Fractions containing
the cytidine deaminase activity were pooled (695 ml) and the
partially purified enzyme was then precipitated with 80% ammonium
sulphate. After centrifugation (1400 g; 60 min) the pellet was
resuspended in 54 ml of supernatant from the above and stored at
4.degree. C.
[0119] 6.2 ml of this solution was centrifuged (18000 g, 90 min)
and the pellet was dissolved in 24 ml of 0.5M potassium phosphate
buffer (pH 7.5). The suspension was dialysed overnight against 1M
potassium phosphate buffer (pH 7.5). The retentate (20 ml) was then
diluted with an equal volume of distilled water. To 35 ml of this
solution dry Eupergit C beads were added (1 g) and the mixture was
allowed to stand at room temperature 150-300 hours (determined by
measuring residual cytidine deaminase activity in solution). The
immobilised enzyme was washed with 100 mM Tris/HCl buffer (pH 7.0)
containing 1 mM EDTA, 1 mM DTT, 0.5M NaCl and 500 ppm
p-hydroxybenzoic acid ethyl ester (storage buffer). The immobilised
enzyme (2.7 g wet weight) was stored in this buffer at 4.degree. C.
until required for biotransformation.
[0120] The product of Example 1 (3 g) was dissolved in 500 ml of
distilled water in a magnetically stirred 1 L flask. The
bioconversion was carried out at 32.degree. C. in a pH-stat. The pH
was maintained constant at 7.0 by addition of 1M acetic acid. 1 g
Wet weight of the immobilised enzyme beads was washed with
distilled water prior to start of the reaction. The conversion was
monitored by chiral-HPLC which showed that the (+) enantiomer of
the substrate was preferentially deaminated. At the end of the
reaction (72 h) the enzyme beads were filtered off and the filtrate
was used for isolation of the desired (-)-enantiomer.
[0121] The pH of the reaction mixture was adjusted to pH 10.5 using
ammonia solution (1M) and the solution applied to Duolite A113
super resin in the OH cycle (50 ml; 0.4 bed volumes per hour). The
uridine analogue adsorbed to the resin and the (-)-enantiomer
passed straight through. Any (-)-enantiomer still on the resin was
removed by washing with 0.04% ammonia solution (2 bed volumes; flow
rate 0.8 bed volumes/hour).
[0122] The pH of the spent solutions and washes (600 ml) was
adjusted to pH 7.0 with concentrated sulphuric acid and the
solution applied to XAD16 resin (50 ml; flow rate 1.4 bed volumes
per hour). The column was washed with distilled water (2.5 bed
volumes, flow rate 2 bed volumes per hour) and the (-)-enantiomer
eluted with acetone:water 1:3 (flow rate 1.5 bed volumes per
hour).
[0123] The bulk (-)-enantiomer containing fraction (4 bed volumes)
was concentrated on a Buchi evaporator to a small volume before
filtration through a No. 3 glass sinter. The filtered solution was
freeze dried to yield 1.2 g of title product identical to that
obtained in Example 4.
EXAMPLE 6
[0124] Tablet Formulations
[0125] A. The following formulation is prepared by wet granulation
of the ingredients with a solution of providone in water, drying
and screening, followed by addition of magnesium stearate and
compression.
4 mg/tablet (a) Active ingredient 250 (b) Lactose B.P. 210 (c)
Providone B.P. 15 (d) Sodium Starch Glycollate 20 (e) Magnesium
Stearate 5 500
[0126] B. The following formulation is prepared by direct
compression; the lactose is of the direct compression type.
5 mg/tablet Active Ingredient 250 Lactose 145 Avicel 100 Magnesium
Stearate 5 500
[0127] C. (Controlled Release Formulation). The formulation is
prepared by wet granulation of the ingredients (below) with a
solution of providone in water, drying and screening followed by
the addition of magnesium stearate and compression.
6 mg/tablet (a) Active ingredient 500 (b)
Hydroxypropylemethylcellulose 112 (Methocel K4M Premium) (c)
Lactose B.P. 53 (d) Providone B.P. 28 (e) Magnesium Stearate 7
700
EXAMPLE 7
[0128] Capsule Formulation
[0129] A capsule formulation is prepared by admixing the
ingredients below and filling into a two-part hard gelatin
capsule.
7 mg/capsule Active ingredient 125 Lactose 72.5 Avicel 50 Magnesium
Stearate 2.5 250
EXAMPLE 8
[0130] Injectable Formulation
[0131] Active ingredient 0.200 g
[0132] Sodium hydroxide solution, 0.1M q.s. to a pH of about
11.
[0133] Sterile water q.s. to 10 ml
[0134] The active ingredient is suspended in some of the water
(which may be warmed) and the pH adjusted to about 11 with a
solution of sodium hydroxide. The batch is then made up to volume
and filtered through a sterilizing grade membrane filter into a
sterile 10 ml glass vial and sealed with sterile closures and
overseals.
EXAMPLE 9
[0135]
8 mg/suppository Active ingredient 250 Hard Fat, B.P. 1770 2020
[0136] One-fifth of the hard fat is melted in a steam-jacketed pan
at 45.degree. C. maximum. The active ingredient is sifted through a
200 .mu.m sieve and added to the molten base with mixing, using a
high shear stirrer, until a smooth dispersion is achieved.
Maintaining the mixture at 45.degree. C., the remaining hard fat is
added to the suspension and stirred to ensure a homogenous mix. The
entire suspension is passed through a 250 .mu.m stainless steel
screen and, with continuous stirring, is allowed to cool to
40.degree. C. At a temperature of 38.degree. C. to 40.degree. C.,
2.02 g of the mixture is filled into suitable, 2 ml plastic molds.
The suppositories are allowed to cool to zoom temperature.
EXAMPLE 10
[0137] Biological Activity
[0138] Antiviral Activity
[0139] Antiviral activity of the compounds of Example 2 were
determined against three strains of HIV-1 and one strain of HIV-2
in the following cell lines.
[0140] JM cells, a semi-mature T-cell line derived from a patient
with lymphoblastic leukaemia, infected with HIV-1 strain GB8.
[0141] C8166 cells, a human T-lymphoblastoid cell line, infected
with HIV-1 strain RF.
[0142] MT-4 cells, a human T-cell leukaemia cell line, infected
with HIV-1 strain RF.
[0143] CEM cells, a human T-lymphoblastoid cell line, infected with
HIV-1 strains RF and U455, or HIV-2 strain ROD.
[0144] Antiviral activities in C8166, JM, and CEM cells were
determined by inhibition of syncytium formation (Tochikura et al
Virology, 164, 542-546) and in MT-4 cells by inhibition of formazan
conversion (Baba et al; (1987) Biochem Biophys Res Commun., 142,
128-134; Mossman (1983) J. Immun Meth; 65, 55-57). Antiviral
activities were also determined by analysing the amount of HIV p24
antigen synthesised in the presence and absence of enantiomers.
[0145] The results are shown in Tables 1 and 2 below:--
9 TABLE 1 50% Antiviral Activity (.mu.g/ml) Assay Formazan
inhibition of syncytium formation Cells MT-4 CEM CEM CEM JM C8166
Virus (HIV-1) HIV-1 RF HIV-2 ROD HIV-1 RF HIV-1 U455 HIV-1GB8 HIV-1
RF (+)-enantiomer 0.28 0.045 0.07 0.006 0.03 0.05 (-)-enantiomer
0.20 0.055 0.04 0.008 0.05 0.01
[0146]
10TABLE 2 Inhibition of HIV p24 Synthesis 50% Inhibition HIV p24
Synthesis (.mu.g/ml) Cells C8166 JM MT-4 Virus RF GB8 RF
(+)-enantiomer 0.021 0.033 0.0008 (-)-enantiomer 0.016 0.016
0.0004
[0147] B Cytoxicity
[0148] The cytotoxicites of the compounds of Example 2, the racemic
compound (BCH-189; Example 1) and a 50/50 mixture of the two
enantiomers were determined in five CD4 cell lines: H9, JM, CEM,
C8166 and U937.
[0149] Compounds for test were serially diluted from 100 .mu.g/ml
to 0.3 .mu.g/ml (final concentrations) in 96 well microtitre
plates. 3.6.times.10.sup.4 cells were inoculated into each well of
the plates including drug-free controls. After incubation at
37.degree. C. for 5 days, the viable cell count was determined by
removing a sample of cell suspension and counting trypan blue
excluding cells in a haemocytometer.
[0150] The results are shown in Table 3.
11TABLE 3 Cytotoxicity 50% Cytoxicity (.mu.g/ml) Compound CEM cells
JM cells H9 cells U937 cells C8166 cells (+)-enantiomer 1 1.5 2 4
35 (-)-enantiomer >100 30 >100 >100 >100 BCH-189 3 3.5
8 15 90 1:1 Mix of 2.5 ND* ND ND ND ND = Not Determined
* * * * *