AP612A

AP612A - Oxathiolanes, method for their preparation and pharmaceutical compositions containing same.

Info

Publication number: AP612A
Application number: APAP/P/1996/000784A
Authority: AP
Inventors: Anne-Sophie Charbet-Faury
Original assignee: Laboratoire Laphal
Priority date: 1994-05-24
Filing date: 1996-01-23
Publication date: 1997-09-05
Also published as: CZ21996A3; HU219299B; WO1995032200A1; NZ287478A; HUT75112A; CA2167930A1; OA10258A; FI960316A0; FR2720397B1; KR960703903A; PL312693A1; NO306299B1; EP0733053A1; CN1130906A; US5708000A; CN1080264C; AU2620995A; RU2142462C1; PL181713B1; NO960272L

Abstract

The present invention pertains to the field of organic chemistry, and particularly therapeutic chemistry. The invention provides cis-form 5-cytosinyl-1 1,3-oxathiolanes of general formula i, wherein r is an acyl or aralkoyl radical, derivatized from a nitrogen monocyclic or bicyclic heterocycle, and the hydroxymethyl group in position 2 is in cis position in relation to the plane defined by positions 2 and 5. The compounds of general formula i are useful as active ingredients in pharmaceutical compositions, particularly with antiviral activity.

Description

OXATHIOLANES, METHOD FOR THEIR PREPARATION AND

PHARMAC EUTICAL COMPOSITIONS CONTAINING SAME

This invention relates to the field of organic chemistry and more precisely to that of medicinal chemistry

More particularly it has as a subject matter 2’,3’-didesoxynucleosides substituted in position 2 with an oxathiolane ring

Specifically this invention has as a subject matter the 5-(citosinyl-1) 1,3-oxathiolanes of cis configuration (2R-5S) or (2S-5R) having the general formula :

which may be represented with one out the two spatial representations :

*βΖ00/96 /d/dV wherein R is an acyl radical or an aralkyl radical, derived from a monocyclic or bicyclic nitrogenous heteroring and the hydroxymethyl group in position 2 is in cis position relating to the plane determined by the two positions 2 and 5

Among the various definitions taken by the substituent R, it may be differentiated

a) the nicotinic deris aloes where the heteroring is a pyridinic structure and the ac\I moietv is in position 2. ? or 4. basing the formula la

co_ ( la ) (X)

AP. Ο Ο 6 1 2 wherein X is a hydrogen, a halogen atom, a nitro group, a lower alkoxy or a trilluoromethyl radical and n is a integer from I to 3

b) the dihydropyridic derivatives wherein the heteroring is a 1,4-dihydropyridimc structure and the acyl radical is in position 2, 3 or 4, having the formula lb

wherein R| is an alkyl radical having from I to 10 carbon atoms and X and n are defined as previously

c) the quaternized nicotinic derivatives wherein the nitrogen atom bears an alkyl substituent and the acyl radical is in position 2, 3, or 4, having the general formula Ic

(X)n (Ic) wherein A is an inorganic or organic anion and Ri, X and n are defined as previously

d) the quinoleinic derivatives wherein the heteroring is a bicyclic structure, having the general formula Id

AP/P/ 9 6 / 0 0 7 8 4

wherein the carbons I radical may be in position 2, 3, or 4,

X represents a hydrogen, a halogen, a trifluoromethyi radical, a lower alkoxy or a nitro group and n' represents an integer from I to 6

e) the dihvdroquinoleines of general formula le

O

(le) *1 wherein R_t and X are defined as previously and n' is an integer from I to 6

f) the quaternized quinoleins of general formula If

O

(ID wherein Ri, X and n’ are defined as previously the acyl radical is present in position 2, 3, or 4, and A is an inorganic or organic anion

g) the (dihydropyridyl) alkyl derivatives of general formula lg ^iX’n

N (lg) *1 wherein R_b X andjn are defined as previously and m is an integer from 1 to 6 as well as the acid addition salts thereof with a mineral or organic acid.

The compounds of formula I may be resolved in their optically-active isomers. It may thus be obtained the isomer (+) and the isomer (-)

The retroviral infections are the cause of severe infections, particularly that of acquired immunodeficiency syndrom (AIDS) which is a viral infection often lethal and to a lesser degree hepatitis.Several compounds of nucleosidic or hetero-nucleosidic types are presently clinically utilized for the treatment of these retroviral infections. The former ones are the derivatives of AZT (3’-azido-2’,3’-didesoxythymidine) (Proc Natl. Acad. Sci. 82 . 7096-7100, 1985), ddC (2’,3’-didesoxycytidine) (Proc Natl Acad Sci 86, 1911-15, 1986), d4T (2’,3’-didehydro-3’desoxythymidine) (Biochem, Biophys, Res Comm. 142. 128-34, 1987), ddl (2’,3’didesoxyinosine) (Antiviral, Chem Chemother 2, 221, 1991), BCH-189 or 3TC (2',3 -dideoxy 3’-thiacytidine) disclosed in the european patent application 90301335 7 and the followings The limited number of the anti-retroviral compounds available to the practioner ant the limited efficacy suggested the search of new compounds, the therapeutic virtues of w hich would be increased and the side-effects will be decreased

AP/P/ 96/00784

Some derivatives of 2-(( I-hydroxy inethx I) 1.3-o\atltiolan-5yl) cytosin and namely the esters of the hydroxymethyl function (european patent application 0 382 526) or the halouenated derivatives in position 5' of the pyrimidine nng are already known (R F SCHI\ \/1 et al

Antimicrob Agents and Chemotherapy 5o ( I'>'•’2) 2423-2431)

AP. Ο Ο 6 1 2

The compounds of this invention possess the ability to inhibit the replication of human retroviruses, particularly VIII and hepatites B (HBV) The compounds derived from BCH-189 or TC-3, include in their structure a 1,4-dihydro I-methyl 3-quinoleylcarbonyl grouping bound to the exocyclic amino group of cytidine This particular structural entity gave in experimental pharmacology, the ability to increase and to facilitate the permeation of the hemato-encephalic barrier (Pharmacol Ther 19, 337-396, 1983 - Methods Enzymol I 12, 381-396, 1985 - Drug Des Del, I, 51-64, (1986) - J Med Chem 31, 244-249, 1988 - J Med Chem 32. 17821788, 1989 - J Med Chem 32, 1774-1781, 1989)

The chemical synthesis of these compounds has been carried out using classical experimental protocols The performance of these novel heteronucleosides requires as starting material, the compound 2’,3’-didesoxy 3 '-thiacytidine for which several methods of synthesis have been reported in the chemical litterature (J Org Chem 56, 6503, 1991 - J Org Chem 57, 2217. 1992 - Tet. Lett. 33, 4625, 1992 - Nucleosides and Nucleotides J_2, 225, 1993) Starting from this derivative, the synthesis of the compounds which are the subject matter of this invention, and that of the intermediate compounds, which are related thereto, are also described in this application.

This invention also relates to a process for producing the compounds of general formula I which consists in that a 5-(cytosinyI-1) 1.3-oxathiolane cis is submitted to the action of a functional derivative of a carboxylic acid of formula ROH wherein R is an acyl moiety derived from an aromatic or dihydroaromatic, mono- or bicyclic nitrogenous heteroring or to the action of an aralkylated active derivative of formula R’Z wherein R’ is an aralkyl radical deriving from an aromatic or dihydroaromatic, mono- or bicyclic nitrogenous heteroring and Z is an easily split, labile radical.

In a more precise manner, the functional derivative of acid ROH is an halogenide, an anhydride, a mixed anhydride obtained from a carbodiimide or a reactive ester such a phenolester.

In the case of an arylakylated derivative, Z is a halogen atom or an alkyl- or arylsulphonyl radical.

This invention has also as a subject matter the pharmaceutical compositions with anti-viral action characterized in that they include as an active ingredient at least one compound of general formula I, or an addition salt thereof with a mineral or organic acid, in admixture or in association with an inert, non toxic, pharmaceutically-compatible carrier or vehicle, notably suitable for the topic or systemic use

In these compositions the contents in active ingredient of general formula I ranges from 0.1 mg to 100 mg per unit dosage as a function of the way of administration

EXAMPLE 1

Isomers of cis 2-(diphenvltcrthuts Isilv loxvmethyl)-5 [N'*-(3”-pyridinylcarbouyl) extosiuI’-yll-l ,3-oxathiolanc_L

At room temperature under nitrogen stream, they are added to a mixture of diehloiomethane (7 ml) and dimethyl formatnide (2 ml) leq of nicotinic acid (27 mg, 0.21 mmol). 1.1 eq ot BOP (93mg. 0,23 mmol). 1.1 eq of HOBT (31 mg, 0,23 mmol). I eq of ^ς tertbutyldiphcnylsilyl-2’,3 -didesoxy- I'-tluaeytidin (39 mg, 0,23 mmol) and 4 eq of DIE.A *8200/96 /d/dV

AP . Ο Ο 6 1 2 (146μΙ, 0,84 mmol) The mixture is stirred for a night at room temperature , washed with a 5% citric acid solution (10 ml) then with an 5 % aqueous solution of sodium bicarbonate (IO ml) The resulting mixture is extracted with ethyl acetate (3x10 ml), dried on SO4 Na 2 , and evaporated The residue is purified on silica gel plate 48mg of pure compound are obtained ('H| NMR δ (CDCI3) = 1 1 (s.9H.tBu). 3 1-36 (m.2H.CH₂-O). 3.74.2 (rn.2H.C4H2).

25 (t.1H.C5-H). 3 1-3.6 (m.2H.CH₂-O). 5 5 (d. 1 H.Cg'-H), 6 35 (q.1H,C₂-H). 7 4-7 8 (m. 1 H.aromatique); 8.0 (d,1H,Cg’-H); 8 3 (d.1H.nicotinyl), 8 8 (d,1H,nicotinyl). 8.8 (d.IH.nicotinyl); 9.1 (d.1H.nicotinyl).

EXAMPLE II

Isomers of Cis 2-(hydroxymethyl)-5 (N⁴-(3”-pyridinylcarbonyl)cytosin-r-yl]-I,3oxathiolan 2 /- The compound 1 (25mg, 0,05 mmol) is dissolved in 3ml anhydrous tetrahydrofuran. To this solution 3 eq (135 μΙ, 0,15 mmol) of tetrabutylammonium fluoride are added. The solution is mixed at room temperature for 3 hours. After evaporation of the solvent, the resulting product is chromatographied on preparative silica plates (eluent : toluene/CHjOH 15 %). 13 mg of desired compound are obtained.

[’H] NMR δ (CDCI3) = 3.1-3.6 (m.2H.CH₂-O). 3 7-4.2 (m.2H.C₄H₂). 5 25 (t.lH.CsH). 5 5 (d.H.Cg'-H). 6.35 (q.1H.C₂-H). 7 4-7 8 (m. 1 H.aromatique). 8 0 (d. 1 H.Cg‘-H).

3 (d 1H.nicotinyl); 8.8 (d. 1H.nicotinyl). 9 1 (s. 1 H.nicotinyl)

EXAMPLE III

Isomers of Cis 2-(tertbutyl-diphenyI-silyIoxymethyl)-5-{N'⁴-(3”-(tosyl) cytosin-l’-yl]-l,3 oxathiolane 4

To a solution of compound 5 (56 mg, 0,12 mmol) in pyridine (3 ml) they are added at 60°C under nitrogen atmospher, 2 eq of p.toluene sulphonic acid chloride (46 mg, 0,24 mmol). After 20 hours stirring at room temperature, the mixture is evaporated off, washed with a 5 % citric acid solution (10 ml) then extracted with ethyl acetate (3x10 ml). The united organic phases are dried on Na₂SO4 and evaporated. 48 mg of the desired compound are isolated ['HJNMR : (CDCI?) = 11 (s,9H,tbu) ; 2,45 (s,3H.CHj tosyl) , 3 15-3 55 (m, 2H,C₂-CH₂-0);

85-4 2 (m,2H,C4H₂) , 5 25 (t,IH,C₅-H) . 6 3 (t.lH,C_;-H) , 7 3-7 7 (m,l4H,ArH) ; 8.05 (d,IH,C₆-H) ,

EXAMPLE IV

AP/P/ 96/00784

Isomers of cis 2-(diphenyltertbutyl silyIoxymclhyl)-5-(N4(3”-pyridinylcarbonyl)cytosinl’-yl| 1,3-oxathiolanc 3

To a solution of derivative 4 (47 nig, 0.075 mmol) in lutidine (2ml) under nitrogen atmospher at OOV. they arc added 6 eq (46 μΙ 0,45 mmol) of pyridinylniethylainine Heating is kept for 4S hours under stirring Alter cooling the mixture is evaporated, washed with a 5 °o citric acid solution (10 ml) then extracted with ethyl acetate (3x10 ml) and dried on Na.-S O₄ After

AP. Ο Ο 6 1 2 evaporation the residue is purifyicd by chromatography on silica gel plate (eluant

EtOAC/MeOH, I0/I)

EXAMPLE V

Isomers of cis 2(tertbutyldiphenylsilyloxy met hyl )-5-(cytosin-l’-yl)-l,3-oxath iolane 5

A solution of 2’, 3'-didesoxy 3’-thiacytidine (I05 mg, 0,45 mmol) in pyridine (6 ml) is treated under nitrogen atmospher with diphenyltertbutylsilyl chloride (140 pi, 0,5 mmol). The reaction mixture is stirred for 24 hours at room temperature After concentration under vaccuum, they are added 30 ml water and it is extracted with ethyl acetate (3 x 20 ml) The united organic phases are then dried on Na₂SO₄ and concentrated to dryness to give the compound 5 (210 mg, quantitative yield) in the form of a white solid

Rf(AcOEt/MeOH2/l) = 0,62 {’HJNMR(CDCb) δ : 1,1 (s,9H,tbu), 3,1-3,6 (m,2H,C₂-CH₂-O) , 3,7-4,2 (m,2H,C₄-H₂) ; 5,25 (t.lH.Cs-H) ; 5,5 (d,H,C₅-H) ; 6,35 (q,lH,C₂-H) 7,4-7,8 (m,10H,2Ph), 8,0 (d.lH.Ce-H).

C

EXAMPLE VI

Isomers of cis 2-(hydroxymethyl)-5-(N4-(l”-methyl-3-pyridinyIcarbonyl)cytosm -l’-yl]1,3-oxathiolane 6 as the iodide.

Under nitrogen atmospher to 1 eq. (46 mg, 0,14 mmol) of compound 2 dissolved in 4 mJ anhydrous acetonitrile, they are added 9 eq. (1,25 mmol, 77 μΐ) methyl iodide. The resulting solution is heated to 50°C for 48 hours. After evaporation of the solvants, the residue is purified by flash chromatography with as eluant BuOH/H20/acetic acid (5:2,5:2,5). A yellow solid is obtained (55 mg).

['H]NMR : (DMSO dj δ = 3.25 (t,2H,CH₂-4); 3.90 (dd,2h,C₂-CH₂); 4,45 (s,3H,N⁺CH₃); 5.25 (t.1H.CH-5); 6.30 (t.1H.CH-2); 6.92 (d.1H.CH-6'); 7.30 (d.lH.nicotinyl); 7.85 (d.lH.nicotinyl); 8.05 (d,1H,CH-5·); 8.20 (d.lH.nicotinyl); 7.85 (d.lH.nicotinyl);

¢-, 9.01 (d.lH.nicotinyl).

Mass spectrum (FAB*)349(M-1)*

EXAMPLE VII

Isomers of cis 2-(hydroxymethyl)-5-(N⁴-( r’-niethyl)-l”,4”-dihydro-3”-pyridinyI carbonyl)cytosin-l’-yl|-l,3-oxathiolane 7

In 3 ml of degased solution of methanol containing 10 °o water, they are dissolved 30 mg (0.06 mM)of compound 6 To this solution they are added I 5 mg of sodium bicarbonate and 60 mg sodium dithionite The reaction mixture is stirred under nitrogen for 3 hours The solution becomes orange The solvent is evaporated then the salts are resuspended in a minimal amount ot’methanol One filters The filtrate is purified bv pieparative chromatography on thin layer (I mm) (eluant Toluene/methanol 1 I) to give 6 mg of a\ellow white solid

Mass spectrum (FAB )349 (Μ-1)

AP. Ο Ο 6 1 2

EXAMPLE VIII

Isomers of cis 2-hydroxymethyl 5-|N⁴-3”-quinolinylcarbonyl) cytosin-1'-yl| 1,3oxathiolanc 8

To 2 ml anhydrous dimethylformamide they are added 74,4 mg (0,43 mmol) quinolinyl -3carboxylic acid then 96 mg (0,47 mmol) 1,3-dicyclohexyl carbodiimide and 63,4 mg (0,47 mmol) hydroxybenzotriazole hydrate The mixture is stirred for 1 hour at room temperature under nitrogen A white precipitate of dicyclohexylurea appears and 100 mg (0,43 mmol) 2,3’didesoxy 3’-thiacytidine are then added The mixture is stirred over night Dimethyl formamide is thereafter evaporated under reduced pressure and the residue is hydrolysed with a saturated aqueous solution of NaCl (10 ml) The residue is extracted two times with ethyl acetate (2x10 ml) The organic phases are combined, dried on magnesium sulphate and filtered The solvent is evaporated The product is purified by (flash) chromatography (eluant ethyl acetate/methanol 98 2) to give 78 mg of a white solid (0,2 mmole) i .e a yield of 46,5 %

MP- 116-118°C IR = 1656 cm’¹ (carbonyl of the amide function) [’H1NMR : (DMSO d₆) δ = 11,75 (s, 1 H,NH) ; 9,40 (d, 1 H,CH-2”), 9,1 5 (d, 1 H,CH-4”),

8,65 (d, 1H.CH-5’); 8.20 (m.2H.CH-8 et -CH-5), 8.0 (rn.1H.CH-6’·); 7,80 (rn.1H.CH7); 7,45 (d.1H.CH-6’); 6,35 (t.1H.CH-2); 5,55 (tjH.CH-5); 3,98 (t.2H.C₂-CH₂-4) [¹³C]NMR : (DMSO d₆ ) 5 = 37.14 (C-4); 61,95 (CH₂0-C2); 87.28 (C-2). 88,29 (C5); 95,85 (C-5'), 122,21 (C-3); 126,28 (C-6); 127,71 (C-5), 129.63 (C-10); 129.87 (C-Γ); 132,08 (C-8^H); 137,65 (C-4^M); 138,89 (C-6’); 148,91 (C-9), 149.29 (C-2),

163,16 (C-2*); 164,51 (C-4')

Mass spectrum : (Fab) 385 (M+l)⁺ , 769 (2M+1)⁺

EXAMPLE IX

Isomers of cis 2-hydroxymethyl 5-[N⁴-(l”-methyl 3”-quinolinylcarbonyl)cytosin-l’yl|13-oxathiolane 9 as the iodide.

A solution of compound 8 (78 mg i.e. 0,20 mmol) in 4 ml acetonitrile is prepared To this 112 <<

μΙ (1,8 mmol) methyl iodide are added and the mixture is heated to 40°C under nitrogen for 24 hours. The solvent is eliminated and the crude residue is then dissolved in the minimal amount of acetonitrile It is a new precipitated by adding ether. An orange solid is thus obtained (60 mg i e a yield of 55 ° o)

MP η 156-1 59°C (’H|NMR 1CD3OD) ? = 3 32 (t 2H CH₂-4) 3.99 (dd 2H C₂-CH₂) .150₍s3H’N·

CH ,) 5.15(1 IHCH?i 6 0 (d 1H CH-6). 6.33 (t. 1H CH-2) 8 1 3 (ni 1 H CH ⁷ ) S 39 (mlHCH-61 8 63 jnCHCH-S) 8.72 (dJH.CH-5) 9 ⁷9 to HU'H -Γ) 9 95 (d I H CH 3 1 /96/00784

Mass spectrum (l .ih ) 3^l,9(M-l] , 799 [2(M-1)]

AP.00612

EXAMPLE X

Isomers of cis 2-hydroxymcthyl 5-(N⁴-(l”-mcthyl 1”, 4”-dihydro 3”-quinolinylcarbonyl) cytosin-l’yl|- 1,3-oxathiolane 10

To a solution of 30 mg (0,057 mmol) of compound 9 in 3 ml degased aqueous methanol (3 ml methanol containing 10 % water), 15 mg of sodium bicarbonate and 60 mg sodium dithionite are added The whole mixture is stirred for one hour under nitrogen The solvent is evaporated and the crude residue is purified by preparative chromatography (eluent ethyl acetate/methanol I I) Finally 10 mg of a vitreous yellow solid are obtained, i e a yield of 46% (¹H) NMR (D2O) δ = 3.24 (m,3+2H.NCH3+CH2-43.48 (m.2H.CH2-4); 3.93 (rn.2K.C2H2). 5.29 (t.1H.CH-5); 6.96 (d.1 H.CH-6'). 7.13 (m.2H.CH-6+CH-7). 7.27 (m.2H.CH-5+CH-8). 7.45 (d.1H.CH-2); 8.23 (d.1H.CH-5 )

Mass spectrum (Fab ) 399 (M-l)’

EXAMPLE XI

Isomers of cis Z-hydroxymethyl-S-IN^l-ia.a.a-trifluoro-m-toluidinoJnicotinyO-cytosinl’ylj-l,3-oxathiolane 11

To a solution of 2’,3’-didesoxy 3’-thiacytidine (50 mg, 0,2 mmol) in 5 mJ anhydrous DMF they are added 1,1 eq. BOP (93mg 0,23 mmol), 1 eq. niflumic acid (2-a,a,a-trifluoro-m-toluidino nicotinic acid) and 4 eq. DEEA (146 μζ 0,84 mmol). The mixture is stirred overnight at room temperature, washed with a 5 % citric acid solution (10 ml) then with a 5 % aqueous solution of sodium bicarbonate (10 ml). The resulting mixture is extracted with ethyl acetate (3x10ml) dried on sodium sulphate, then evaporated.

The residue is purified by chromatography on a column of silica gel (eluent : ethyl acetate/toluene 1 9) It is obtained a white solid (49 mg, yield : 48 %)

AP/P.' 96/00784

TEST OF EVALUTATION OF THE ANTIVIRAL PROPERTIES

ANTI-HIV TESTS - Experimental protocol

a) Generalities

Apparatus an ultricentrifugal of Beckman type TL100 is used Counting of the radio labelled particles is performed on a Hewlett Packard apparatus Tri Carb Model 1600 The syncitia are observed with an invert microscope Labovert

Composition of the Iv sis butler NTE :

Trisbv drew inethvlaininomethane 10 mM

CINa 100 111M

IDEA I mM

AP . 0 0 6 1 2

b) Evaluation tests of the antiviral properties in cell cultures

The evaluation of the antiviral action is based on the study of cytopathogenic effect of HIV-l Virus on the cell line MT4 The cell line MT4 has as an origin T cells isolated from a patient, transformed by the TLHV-I virus The cytopathogenic effect of HIV-l virus is shown by the formation of multinucleated giant cells called «syncithia >> visible under the microscope This effect of HIV-l is observed 4 to 5 days after infection. It is followed with the death of cells

The cytopathogenic effect is directly correlated to the infection of the cells by the virus, to its intracellular replication and to the expression of viral antigens by the cells An inhibition of this effect thus corresponds to an inhibition of the multiplication of HIV-l virus

The MT4 cells are maintained at 3.10⁵ cells/ml in a RPMI 1640 medium : 10 % of decomplemented fetal calf serum for 30 mn at 56°C (hormons, serum growth factors . ), 1 % glutamine, 1 % penicillin streptomycin and 2 pg/ml Polyren which promotes the viral adhesion to the cells.

The action of the treatment by the anti-viral agent is continuous. In fact this effect is present before during and after the viral infection. Successive dilutions are performed in the 10 % fetal calf serum to be able to cultivate the MT4 cells for 8 days and to observe the formation of syncithias.

MT4 TEST

Before infection : 3xl0⁶ cells/100pl are distributed in a microtiter plate with 96 wells and centrifugated for 3 nun. at 2000 rpm (rounds per minute). The pellet is then pre-incubated for 1 hour at 37°C, with 100pl of various concentrations of the anti-viral agent to be tested.

Infection : The infection is achieved in microwells while adding 100 TC1TJ (infection units) of HIV-l virus (this titer of HIV-l virus is determined as to induce the formation of syncithias in 4 or 5 days). The anti-viral agent is always present at the time of infection and dilution of the virus at 100 TCIU.

C'

After infection : After incubation for 1 hour at 37°C, the MT4 cells are washed three times with RPMI 1640 medium and cultured on account of 3.10⁵ cells per milliliter of each of the concentration of the compounds to be tested on plates of 24 wells. When passing at day D3 the MT4 cells are diluted to 1/3. The concentration of the anti-viral agent is maintained Each day occurrence of syncithia is observed under the microscope to detect any optional delay with respect to the controls HIV-l At day Dg the determination of reverse transcriptase is carried out. When the cells are not infected thus it is a protection by the tested anti-viral agent

Determination of reverse transcriptase

Reverse transcriptase is a RNA dependant DNA polymerase. This enzyme allows the replication of retroviruses Thank to it, the viral RNA transcribed into DNA, is incorporated in the cell genome Proviral DNA is transcribed by the cell enzymes into a viral RNA For the determination of the reverse transcriptase activity, one concentrates 100 times 1 ml of supernatant of culture, by ultra centrifugation for 5 min at 95000 rpm The pellet of virus obtained after ultracentriftigation is resuspended into 10 μΙ of buffer NTE + TRITON 0,1 % (polyoxyethylene ether). The latter is used to lysate the virus and to release the reverse

AP/P/ 9 6 / 0 0 7 8 4

AP.00612 transcriptase In vitro it is the reverse transcriptase activity which is evidenced. This enzyme uses a synthetic matrix poly A possessing a primer oligo dT having from 12 to 18 residues The radio labelled substrate of this reaction is [³H]dTTP (radio active thymidin triphosphate at ImCi/ml) The novelly formed tritiated macromolecules are precipitated by trichloroacetic acid and separated from free [³H] TTP by filtration The enzymatic activity of the reverse transcriptase is measured through the radioactivity incorporated in the complexes poly-rA/poly -dT This radiolabelling is determined in a particle counter after addition of a scintillation liquid acting as an amplifyier.

ΑΝΤΙ-HBV TEST - EXPERIMENTAL PROTOCOL

Among the several methodologies used for evaluating the activity of compounds on Hepatitis B virus (HBV), the pattern of Hepatitis B virus of the duck (Duck HBV) has been utilized (J Med Virology (1990) 31 82-89 ; Viral Hepatitis and Liver disease (1988) 506-509 ; Hepatology (1989) J_0_186-191). The material of culture to test « in vitro» the anti HBV activity of the compounds is made of duck hepatocytes infected by the duck Hepatitis B virus (DHBV) (Antimicrob. Agents Chemother. (1989) 33 336-339 ; J.Med. Virology 40 , 59-64 , Antivir Res. (1993) 21. 155-171 ; Antimicrob. Agents Chemother. (1993) 37 1539-1542). Indeed the Hepatitis B virus of the duck is recognized as being very close to the human hepatitis B virus (Viral Hepatitis and Liver Disease (1988) 526-529. Antiviral Research (1987) 8_ 189-199) Under standardized conditions (Virology (1989) 171 564-572) the cultures of duck hepatocytes allow the complete replication of DHBV The antiviral activity of the tested compounds has been measured as a function of the amount of viral DNA produced during 10 days of culture of the infected hepatocytes.

Experimental Protocol

A young male duck 3 weeks old, affected by DHBV, is used for the production of hepatocytes. The duck is killed under anesthesia and the isolation of hepatocytes is carried out using the methodology of Guillouzou (Research in isolated and cultured hepatocytes, J. Libbey Eurotex Ltd / INSERM, 1986). The cells are cultured in the Leibowitz’s medium and 5 pg/ml bovine insuline, 7x10'⁵ mole cortisone hemisuccinate and 1,5% dimethylsulfoxide (DMSO) are added thereto. The density of the cells is 8xl0⁶ cells, they are sprayed in culture boxes of 100x20 mm The tested compounds are added to the culture after the spread out of the cells. The medium is renewed every day, during 10 days. The production of viral DNA of the duck hepatitis B virus in the cells surpematant is determined by means of hybridation method “Dot blot” according to Fourel and al. (Viral Hepatitis and Liver Disease, 1988, 506-509 , Hepatology, 1989, 10. 186-191). Inhibition is expressed as a percentage of the DNA amount present on the supernatant of cells with regard to a culture infected by DHBV untreated with a nucleosidic derivative.

V1ROLOG1CAL RESULTS

HIV Tests

The compounds the synthesis of which has been described in this patent application and the related structures given in the previous prior art, have an affect on the inhibition of the replication of BRU HIV-1 virus (BRU beeing the viral strain experienced) in the case where the infected cells are of the type MT4

In the table I they have been described the results of the tests Column 1 provides the names of the most representative studied compounds and in column 2 they are reported the so-called *8/00/96 /d/dV

AP . Ο Ο 6 1 2

IC » values (concentration of the component giving a 50% inhibition of the replication of HIV in the infected MT4 cells, measured at day D7 after infection) In observing and counting the number of formed syncitia in relation with that counted in the case of infected MT4 cells but untreated with antiviral compound, the value IC» is determined.

DBHV Tests

The most representative described compounds have been tested for their effect on the replication of the duck virus HBV (DHBV) along the protocol exposed in the experimental part of this document.

In the table II the results of the tests are described

AP/P/ 9 6 / 0 0 7 8 4

AP.00612

ABREVAT1ONS

AcOEt: ethyl acetate DNA: desoxyribonucleic acid PTSA para-toluene sulfonic acid RNA: ribonucleic acid

AZT: 3’-azido-2’-3’-dideoxy-thymidine or Zidovudine

BOP: benzotriazolyoxytrisdimethyl-aminophosphonium hexafluorophosphate

TLC: chromatography on thin-layer

DCC: N,N’-dicyclohexylcarbodiimide DCU: N,N’-dicyclohexyluree DIEA: Ν,Ν-diisopropyl N-ethylamine DMF: Ν,Ν-dimethylformamide DHBV: duck hepatitis B virus FAB⁺: fast positiv atomic bombardment ³HdTTP: thymidine triphosphate labelled with a radioactivity of lmCi/ml HOBT: 1-hydroxybenzotriazole IR: infra-red

MP: melting point NMR: nuclear magnetic resonance AIDS: acquired immuno deficiency syndrom MS: mass spectrum

Q TBAF: tetrabutyldiphenylsilyle fluoride

TBDPSCI tetrabutyldiphenylsilyle chloride

3-TC: (-)-(2R,3S)-2-hydroxymethyl-5-(cytosine-l’-yl)-l,3-oxathiolane or Lamivudine

TCIU: infection units

THF: tetrahydrofuran

RPM: round per minute

TsCl: tosyl chloride

UV: ultra-violet

HIV human immunodeficiency virus

HBV: hepatitis B virus

HLTV human lymphotropic T cells virus * 8 L 0 0 / 9 6 /d/dV

AP. Ο Ο 6 1 2

Table I

Effect of the nucleosides of this invention on the inhibition of the replication of HIV I virus, expressed with the sum of IC50

Compounds n°	COMPOUNDS	IC so
9		> 100 μΜ
io		1-10 μΜ
8		10 μΜ
6

AP/P/ 9 6 / 0 0 7 8 4

AP.00612

Compounds n°	COMPOUNDS	1C»
7
2		0.1-1 μΜ
U		< 100 μΜ
BCH-189 (reference product)		1 μΜ

AP/P/ 9 6 / 0 0 7 8 4

AP .Ο Ο 6 1 2

Table ΪΪ

Effect of the nucleosides of this invention on the inhibition of the replication of DHBV virus

Compounds n°	COMPOUNDS	IC 50
BCH-189 (reference compound)		1± 0.5 μΜ
2		2± 0.5 μΜ
8		2 μΜ
9		2 μΜ

tr 8 L 0 0 / 9 6 /d/dV

AP.00612

Compounds n°	COMPOUNDS	IC 50
1θ		2 μΜ
6		under testing
7		under testing

* 8 L 0 0 / 9 6 /d/dV

Claims

WHAT IS CLAIMED IS

1. The 5-(cytosinyl-1)1, 3-oxathiolanes of cis configuration (2R-5S) or (2S-5R) having the general formula I wherein R is an acyl radical or an aralkyl radical, derived from a monocyclic or bicyclic nitrogenous heteroring and the hydroxymethyl group in position 2 is in cis position relating to the plane determined by the two positions 2 and 5.
2. The 5-(cytosinyl-1) 1, 3-oxathiolanes according to claim 1, of 2R-5S configuration, having the general formula :

*8/00/96 /d/dV

AP.ΟΟ612
3. The 5-(cytosinyl-1) 1, 3-oxathiolanes, according to claim I, of (2S-5R) configuration, having the general formula
4. The nicotinic compounds, according to any of claims 1 to 3, wherein R has the formula la:

(X) co_ (la)

AP/P/ 9 6 / 0 0 7 8 4 wherein X is an hydrogen, a halogen atom, a nitro group, a lower alkoxy or a trifluoromethyl radical and n is an integer from 1 to 3 and the acyl radical is in position 2, 3 or 4
5. The dihydropyridic compounds, according to any of claims 1 to 3, wherein R has the formula rib)

AP.00612 wherein Rl is an alkyl radical having from I to 10 carbon atoms

X and n are defined as previously and the acyl radical is in position 2, 3 or 4
6. The quaternized nicotinic compounds according to any of claims 1 to 3, wherein R has the general formula lc wherein A is an inorganic or organic anion Rl, X and n are defined as previously and the acyl radical is in position 2, 3 or 4.
7. The quinoleinic compounds, according to any of claims 1 to 3, wherein R has the general formula Id

AP/P/ 9 6 / 0 0 7 8 4 wherein the carbonyl radical is in position 2, 3 or 4

X represents a hydrogen, a halogen, a trifluoromethyl radical, a lower alkoxy or a nitro group and n’ represents an integer from 1 to 6
8. The dihydroquinoleinyl compounds according to any of claims 1 to 3, wherein R has the general formula le

AP.OO612 ^(XG

(fe) *1 wherein Rl and X are defined as previously and n’ is an integer from 1 to 6
9. The quaternized quinoleinyl compounds according to any of claims 1 to 3, wherein R has the general formula If

I

R wherein Rl, X and n’ are defined as previously the acyl radical is present in position 2, 3 or 4 and A is an inorganic or organic anion lO.The (dihydropyridyl alkyl) compounds according to any of claims I to 3, wherein R has the general formula lg >5 wherein RI, X and n are defined as previously and m is an integer from 1 to 6

AP/P/ 9 6 / 0 0 7 8 4
11 The addition salts with a mineral or organic acid, of the compounds according to any of claims 1 to 10
12 The optically-active isomers of compounds according to any of claims I to 11.

AP. Ο Ο 6 1 2
13 A compound according to claim I or to claim 11 or to claim 12 i e the cis 2-hydroxymethyl

5-[N -(3”-pyridyl carbonyl) cytosin 1 ’-yl] 1,3-oxathiolane or one of its addition salts with a mineral or organic acid, in a racemic form or in an optically-active form
14. A process for producing the compounds according to any of claims 1 to 13 which consists in that a 5-(cytosinyl-l)l,3 oxathiolane cis is submitted to the action of a functional derivative of a carboxylic acid of formula ROH wherein R is an acyl moiety derived from an aromatic or dihydroaromatic, mono- or bicyclic nitrogenous heteroring or to the action of an aralkylated active derivative of formula R’Z wherein R’ is an aralkyl radical deriving from an aromatic or dihydroaromatic, mono- or bicyclic nitrogenous heteroring, and Z is an easily split, labile radical.
15. The pharmaceutical compositions with anti-viral action, characterized in that they include as an active ingredient at least one compound of general formula I y 8 L 0 0 / 9 6 /d'dV c

wherein R is an acyl radical or an aralkyl radical, derived from an aromatic or dihydroaromatic, monocyclic or bicyclic nitrogenous heteroring and the hydroxymethyl group in position 2 is in cis position relating to the plane determined by the two positions 2 and 5 or an addition salt with an acid thereof, in a racemic form or in an optically active form in admixture or in association with an inert, non toxic, pharmaceutically-compatible carrier