2-substituted-5-substituted-1,3-oxathiolanes with antiviral properties

Methods and compositions for preventing or treating human immunodeficiency virus (HIV) infections characterized by 2-substituted-5-substituted-1,3-oxathiolanes.

TECHNICAL FIELD OF INVENTION 
The present invention relates to novel substituted 1,3-oxathiolane cyclic 
compounds having pharmacological activity, to processes for and 
intermediates of use in their preparation, to pharmaceutical compositions 
containing them, and to the use of these compounds in the antiviral 
treatment of mammals. 
BACKGROUND ART 
Retroviral infections are a serious cause of disease, most notably, the 
acquired immunodeficiency syndrome (AIDS). The human immunodeficiency 
virus (HIV) has been recognized as the etiologic agent of AIDS and 
compounds having an inhibitory effect against HIV multiplication have been 
actively sought. 
Mitsuya et al., "3'-Azido-3'-deoxythymidine (BW A509U): An antiviral agent 
that inhibits the infectivity and cytopathic effect of human 
T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro", 
Proc. Natl. Acad. Sci. U.S.A., 82, pp. 7096-7100 (1985), refers to a 
compound of formula (A) (3'-azido-2'3'-dideoxythymidine), commonly 
referred to as AZT. This compound is said to be useful in providing some 
protection for AIDS carriers against the cytopathogenic effect of 
immunodeficiency virus (HIV). 
##STR1## 
Mitsuya et al., "Inhibition of the in vitro infectivity and cytopathic 
effect of human T-lymphotrophic virus type III/lymphadenopathy-associated 
virus (HTLV-III/LAV) by 2'3'-dideoxynucleosides", Proc. Natl. Acad. Sci. 
U.S.A., 86, pp. 1911-15 (1986), have also referred to a group of 2',3'- 
dideoxynucleosides shown in Formula (B) which are said to possess 
protective activity against HIV-induced cytopathogenicity. 
##STR2## 
Balzarini et al., "Patent and selective anti-HTLV-III/LAV activity of 
2',3'-dideoxycytidinene, the 2',3'-unsaturated derivative of 
2',3'-dideoxycytidine", Biochem. Biophys. Res. Comm., 140, pp. 735-42 
(1986), refer to an unsaturated analogue of these 
nucleosides--2'3'-dideoxy-cytidine, shown in Formula (C)--as being 
characterized by antiretroviral activity. 
##STR3## 
Baba et al., "Both 2',3'-dideoxythimidine and its 2',3'-unsaturated 
derivative (2',3'-dideoxythymidinene) are potent and selective inhibitors 
of human immunodeficiency virus replication in vitro", Biochem. Biophys. 
Res. Comm., 142, pp. 128-34 (1987), refer to the 2',3'-unsaturated 
analogue shown in Formula (D) of 2',3'-dideoxythymidine. This analogue is 
purported to be a potent selective inhibitor of HIV replication. 
##STR4## 
Analogues of AZT known as 3'-azido-Z', 3'-dideoxyuridine shown in Formula 
(E), where bromine or iodine have been said to have an inhibitory activity 
against Moloney murine leukemia in T. S. Lin et al., "Synthesis and 
antiviral activity of various 3'-azido, 3' amino, 2',3'-unsaturated and 
2',3'-dideoxy analogues of pyrimidine, deoxyribonucleosides against 
retroviruses", J. Med. Chem., 30, pp. 440-41 (1987). 
##STR5## 
Finally, the 3'-fluoro analogues of 2',3'-dideoxycytidine shown in Formula 
(F) and of 2',3'-dideoxythymidine shown in Formula (G) are referred to in 
Herdewijn et al., "3'-Substituted 2',3'-dideoxynucleoside analogues as 
potential anti-HIV(HTLV-III/LAV) agents", J. Med. Chem., 30, pp. 1270-78 
(1987), as having potent antiretroviral activity. 
##STR6## 
The most potent anti-HIV compounds thus far reported are 
2',3'-dideoxynucleosides, more particularly, 2',3'-dideoxy cytidine 
(ddCyd) and 3'-azido-2',3'-dideoxythymidine (AzddThd or AZT). These 
compounds are also active against other kinds of retroviruses such as the 
Moloney murine leukemia virus. Because of the increasing incidence and the 
life-threatening characteristics of AIDS, efforts are being expended to 
discover and develop new nontoxic and potent inhibitors of HIV and 
blockers of its infectivity. It is therefore an object of the present 
invention to provide effective anti-HIV compounds of low toxicity and a 
synthesis of such new compounds that is readily feasible. 
DISCLOSURE OF INVENTION 
A structurally distinct class of compounds known as 
2-substituted-5-substituted-1,3-oxathiolanes has now been discovered and 
found to have antiretroviral activity. In particular, these compounds have 
been found to act as non-toxic inhibitors of the replication of HIV-1 in 
T-lymphocytes over prolonged periods of time. Accordingly, the present 
invention provides a compound of Formula (I): 
##STR7## 
R.sub.1 may be selected from the group consisting of hydrogen, an acyl 
group having from 1 to 16 carbon atoms, preferably, benzoyl or a benzoyl 
substituted in any position by at least one halogen (bromine, chlorine, 
fluorine, or iodine), C.sub.1-6 alkyl, C.sub.1-6 alkoxy, nitro or 
trifluoromethyl groups R.sub.2 is a heterocyclic radical selected from: 
##STR8## 
R.sub.3 and R.sub.4 are independently selected from the group of hydrogen 
or C.sub.1-6 alkyl groups, 
R.sub.5 is selected from the group of hydrogen, C.sub.1-6 alkyl, bromine, 
chlorine, fluorine, or iodine, and 
X and Y are independently selected from the group of bromine, chlorine, 
fluorine, iodine, amino or hydroxy groups. Of particular interest are the 
following compounds of formula (I): 
(a) R.sub.1 is hydrogen and R.sub.2 is cytosin-1'-yl; 
(b) R.sub.1 is hydrogen and R.sub.2 is thymin-1'-yl; 
(c) R.sub.1 is hydrogen and R.sub.2 is uracil-1'-yl; 
(d) R.sub.1 is hydrogen and R.sub.2 is adenosin-9'-yl; 
(e) R.sub.1 is hydrogen and R.sub.2 is inosin-9'-yl; 
(f) R.sub.1 is hydrogen and R.sub.2 is 5-aza-cytosin-1'-yl; 
(g) R.sub.1 is hydrogen and R.sub.2 is 2'-amino-purin-9'-yl; 
(h) R.sub.1 is hydrogen and R.sub.2 is 2'-amino-6'-chloro-purin-9'-yl; 
(i) R.sub.1 is hydrogen and R.sub.2 is 2',6'-diamino-purin-9'-yl; and 
(j) R.sub.1 is hydrogen and R.sub.2 is guanin-9'-yl. 
Also included in this invention are the oxidized forms of Formula (I) as 
shown in Formulas (II) and (III). 
##STR9## 
There are two asymmetric carbons noted by asterisks in the Formulas (I), 
(II) and (III) which lead to two racemic forms (.+-.) and therefore, four 
optical isomers. These racemates differ in the relative configurations of 
the 2- and 5- substituents which can either assume the cis- or trans- 
configurations. The use of the graphic representation of Formulas (I), 
(II) and (III) is meant to include the dl- or racemic mixtures as well as 
their separate d-and l- isomers. The sulfoxide derivative of Formula (III) 
exists in two additional racemic forms as shown in Formulas (IIIa) and 
(IIIb) which differ in the configuration of the oxide oxygen atom relative 
to the 2,5-substituents. Accordingly, Formula (III) includes both forms as 
well as mixtures of them. 
##STR10## 
The present invention also provides a process for the preparation of 
compounds having Formulas (I), (II) and (III). This process, in which 
R.sub.2 is cytosin-1'-yl is illustrated in SCHEME I. The various steps 
involved in the synthesis as illustrated in Scheme 1 may be briefly 
described as follows: 
Step 1: Commercial bromoacetaldehyde diethyl acetal (or an equivalent halo 
alkyl acetal) is treated in boiling DMF with an excess of potassium 
thiobenzoate to give the benzoylthio acetal of Formula V. 
Step 2: The benzoyl group of Formula V is hydrolyzed with sodium hydroxide 
in an aqueous organic solvent to give the known mercaptoacetal shown in 
Formula VI (G. Hesse and I. Jorder, "Mercaptoacetaldehyde and dioxy-1, 
4-dithiane", Chem. Ber., 85, pp. 924-32 (1952)). 
Step 3. Glycerol 1-monobenzoate prepared according to the literature (E. G. 
Hallonquist and H. Hibbert, "Studies on reactions relating to 
carbohydrates and polysaccharides. Part XLIV: Synthesis of isomeric 
bicyclic acetal ethers", Can. J. Research, 8, pp. 129-36 (1933)), is 
oxidized with sodium metaperiodate to give the known 
benzoyloxyacetaldehyde of Formula VII (C. D. Hurd and E. M. Filiachione, 
"A new approach to the synthesis of aldehyde sugars", J. Am. Chem. Soc., 
61, pp. 1156-59 (1939)). 
Step 4. The aldehyde of Formula VII is then condensed with mercaptoacetal 
of Formula VI in a compatible organic solvent, such as toluene, containing 
a catalytic amount of a strong acid to give the novel intermediate shown 
in Formula VIII. 
Step 5. The 1,3-oxathiolane of Formula VIII is then reacted with cytosine 
previously silylated with hexamethyldisilazane in a compatible solvent 
using a Lewis acid or trimethylsilyl triflate to give intermediate of 
Formula IX. 
Step 6. The amine function of the compound shown in Formula IX is 
acetylated with acetic anhydride to yield the intermediate of Formula X as 
cis- and trans-isomers. 
Step 7: The cis- and trans- isomers of Formula X are treated with 
methanolic ammonia to obtain the desired product shown in Formula XI as 
cis- and trans- isomers. 
Step 8: The preceding isomers of Formula XI are treated with an oxidizing 
agent which may be a suitable peracid in a compatible solvent to give the 
5-oxide (sulfoxide) of Formula XII. 
This synthesis is applicable to any nucleoside base analogue as would be 
obvious to those skilled in the art of nucleoside chemistry. Other 
analogues defined by Formulas (I), (II), (IIIa) and (IIIb) may be obtained 
similarly from intermediate VII by using the appropriate heterocyclic 
compound in place of cytosine in Step 5. In Step 4, other esters of 
hydroxyacetaldehyde such as aliphatic acyl or substituted aroyl groups can 
be used following the same sequence of steps eventually leading to XI and 
XII respectively. 
A preferred embodiment of the present invention is the compounds having 
Formulas (I) and (III) 
##STR11## 
wherein R.sub.1 is selected from the group comprising H, CH.sub.3 CO--, 
CH.sub.3 (CH.sub.2).sub.2 CO--, CH.sub.3 (CH.sub.2).sub.10 CO--, CH.sub.3 
(CH.sub.2).sub.12 CO--, and CH.sub.3 (CH.sub.2).sub.14 CO-- or aroyl or 
substituted aroyl residues such as C.sub.6 H.sub.5 CO-- and its ortho, 
meta and para substituted derivatives comprising halogens (bromine, 
chlorine, fluorine or iodine), C.sub.1-6 alkyls, methoxy, C.sub.1-6 
alkoxy, nitro, trifluoromethyl groups; and R.sub.2 is selected from the 
group comprising cytosin-1'-yl, adenosin-9'-yl, thymin-1'-yl, 
guanosin-9'-yl, uracil-1'-yl, inosin -9'-yl, and analogous heterocycles 
such as 5-aza-cytosin-1'-yl, 5'-ethyluracil-1'-yl, 
N.sub.4,5'-dimethylcytosin-1'-yl, 5'-fluorouracil-1'-yl, 5'- 
iodouracil-1'-yl, or other related heterocycles well known to those 
familiar with nucleoside analogue chemistry and biochemistry. 
A more preferred embodiment is the compounds of Formulas XI, XII, and XIII 
##STR12## 
wherein R.sub.2 comprises adenosin-9'-yl, thymin-1'-yl, inosin-9'-yl, 
uracil-1'-yl and 5-aza-cytosin-1'-yl and where the sulfur atom may be in 
the sulfoxide form. 
In vitro testing was conducted on the compounds designated herein as cis-XI 
and trans-XI to determine their inhibitory properties. The results are 
shown in Tables 1 and 2. The concenrations reported are ug/ml in the 
incubation media which affect the susceptibility of a continuous line of 
T-cells developed at the Lady Davis Institute for Medical Research 
(Montreal) by Dr. Mark A. Wainberg toward infection by HIV-1 following a 
protocol similar to that of H. Mitsuya and S. Broder, "Inhibition of the 
in vitro infectivity and cytopathic effect of human T-lymphotropic virus 
type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 
2'3'-dideoxynucleosides", Proc. Natl. Acad. Sci. USA, 83, pp. 1911-15 
(1986). Protection of the cell line from infection was monitored by 
staining with monoclonal antibodies against viral proteins in the standard 
manner (Table 1). In all experiments, comparisons were made with the drug 
AZT as the control. In order to confirm the results, the drug effects were 
monitored by measuring reverse transcriptase (RT) activity in the U-937 
line of human monocytic cells as assayed in the usual manner with 
tritiated thymidine triphosphate (TTP) (Table 2). Finally, the drug 
effects on cell viability as measured by the well-know cytolytic effects 
of HIV-1 on the MT-4 cell line was evaluated in the accepted manner (Table 
1). 
It is apparent from the results of Table 1 that compound cis-XI exerts 
potent inhibitory activity against HIV-1, surpassing the potency of AZT in 
some respects. In some assays, significant inhibitory activity was also 
observed with the trans- isomer of XI. As is usually observed with 
compounds of type XI, the cis-isomer especially is expected to demonstrate 
in vivo activity as an inhibitor of retroviruses. It may also be used in 
combination with other antiviral agents at reduced doses owing to the 
possibility of synergistic effects. 
Toxicity 
No toxic effects were observed in the above tests. 
TABLE 1 
______________________________________ 
Inhibition of HIV-1 product by 
cis-XI in MT-4 cells 
______________________________________ 
(a) Viable Counts 
Time in Culture 
Cell Viability % 
(Days) No Drug 2 ug/ml AZT 2 ug/ml cis-XI 
______________________________________ 
3 86.7 95.0 94.0 
6 6.47 88.6 87.4 
______________________________________ 
(b) P-24 immunofluorescence 
Time in Culture 
Immunofluorescent Cells 
(Days) No Drug 2 ug/ml AZT 2 ug/ml cis-XI 
______________________________________ 
3 5.9 1.0 1.0 
6 99 1.0 7.6 
______________________________________ 
(c) Reverse transcriptase assay 
6 339.0 1.748 2.301 
Time in Culture 
RT Activity (CPM .times. 1000)/ml 
(Days) No Drug 2 ug/ml AZT 2 ug/ml cis-XI 
______________________________________ 
3 36.43 1.564 2.381 
6 339.0 1.748 2.301 
______________________________________ 
TABLE 2 
______________________________________ 
Inhibition of HIV-1 production by 
cis-XI in H-9 cells 
Reverse transcriptase assay 
Time in Culture 
RT Activity (CPM .times. 1000)/ml 
(Days) No Drug 2 ug/ml AZT 2 ug/ml cis-XI 
______________________________________ 
5 9.117 3.346 3.077 
8 438.5 3.414 5.853 
11 2550 2.918 3.560 
14 2002 8.320 2.872 
17 584.5 2.997 2.399 
21 365.2 3.111 2.907 
25 436.4 15.88 4.020 
29 92.38 32.08 3.756 
33 111.1 612.2 3.803 
37 32.28 878.2 4.193 
41 384.4 994.0 4.515 
45 33.64 32.91 3.441 
______________________________________ 
Also in accordance with the present invention, there is provided a 
pharmaceutical composition for administration to persons infected with the 
AIDS virus or other infectious agent which comprises a therapeutically 
effective amount of the 2-substituted-5-substituted-1,3-oxathiolane of 
Formula I, II, or III. Depending upon the route of administration, which 
could normally be either oral or parenteral, the compounds may be in the 
form of a solid, semi-solid, liquid, oil, or ingestible capsule and may 
either be present as the original compound or in the form of a 
pharmaceutically acceptable salt in association with or without an 
appropriate pharmaceutical carrier. 
Also within the scope of the present invention is a method for treating 
AIDS-infected persons by administering a therapeutically effective amount 
of 2-substituted-5-substituted-1,3-oxathiolane of Formula I, II or III or 
any combination of a 2-substituted-5-substituted-1,3-oxathiolane of 
Formula I, II or III with any other drug where such combination is 
therapeutically advantageous. The therapeutically antiviral effective 
amount of the compounds to be used in accordance with this invention to 
provide prophylaxis and treatment for individuals infected with, or at 
risk of being infected with HIV, can be determined by methods known in the 
art.