The present invention relates to the use of 1,4- dihydropyridine-5-carboxylic acid derivatives comprising a coumarin group and pyridine-5-carboxylic acid derivatives comprising a coumarin group for the preparation of an anti-viral agent. The use of 1, 4-dihydropyridine-5-carboxylic acid ester derivatives comprising a coumarin group as anti-viral agents is known from the International patent application WO 01/14370 (Rephartox B. V.). The virus diseases may be those virus diseases caused by: 1 Adenovirus type 2 2 Coxsackie virus B4

3 Cytomegalovirus (AD-169 strain; Davis strain)

4 Herpes simplex virus 1 (KOS; F; Mclntyre; TK-B2006; TK-VMW1837; TK-Cheng C158/77; TK-Field C137/101)

5 Herpes simplex virus-2 (G; 196; Lyons) 6 HIV-I

7 HIV-2

8 Influenza virus A

9 Influenza virus B

10 Parainfluenza-3 virus 11 Polio virus-1

12 Reovirus

13 Respiratory syncytial virus

14 Semliki forest virus

15 Sindbis virus 16 Vaccinia virus

17 Varicella zoster virus (TK+ OKA strain; TK+ YS strain; TK- 07/1 strain; TK- YS/R strain;

18 Vesicular stomatitus virus.

The object of the present invention is to provide a new use. Thus, the present invention relates to the use of compounds comprising a coumarin group of the general formula (I) or (II)

wherein

R is hydrogen, CN, NO ₂ or a -COOR' -group, wherein R' represents a linear or branched (Ci_ ₆ ) alkyl group;

Rl has a meaning as defined for R4 or represents hydrogen, fluoro, chloro, bromo, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci-β) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, optionally esterified with a linear or branched (Ci_ _e ) alkanol, carbox- amide;

R2 represents OH, linear or branched (Ci_ ₆ ) alkoxy or has a meaning as defined for R4 with the exception of hydrogen;

R3, R3' represent independently of each other H or independently of each other have a meaning as defined for R4;

R4 represents H or a coumarinyl group of the formula III

[HI)

wherein

X represents a single bond, - (CH ₂ ) _n -O-, or - (CH ₂ ) _ra -Y-

(CH ₂ ) _k ~0, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and

Y represents phenylene, (C ₃ -β) cycloalkylene, or a carbon atom substituted with 1 or 2 (Ci-C ₂ ) alkyl groups ;

R5 represents hydrogen, linear or branched (C ₁ -^) alkyl, linear or branched (Ci_ ₆ ) alkoxy, halogen, nitro, hydroxyl, or amino; wherein alkyl, alkoxy, alkanol, benzyloxy, phenylene and cycloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, and nitro; with the provisio that at least 1 of the groups Rl, R2, R3, R3' and R4 is chosen from, optionally substituted, coumarin-4-yl and cou- marin-7-yl, as well as the N-oxide, stereo isomers and/or addition salts thereof with a pharmaceutically acceptable acid or base for the preparation of a drug for the prevention of and/or treatment of Severe Acute Respiratory Syndrome (SARS) .

It is noted that more than 1 Rl- or R5-groups may be present. If there are more than 1 Rl (or R5) groups, these may optionally be different. R5 may be present on any carbon atom of the backbone of the coumarin ring system. In the present application an optionally present alkyl group, alkanol group or alkoxy group, preferably has 1 or 2 carbon atoms. Alkanol is an alkylgroup substituted with an hydroxyl group.

In the present application the term cycloalkyl is understood to mean a cycloalkyl group having 3 to 8 carbon atoms. Halogen is understood to mean a halogen atom chosen from the group consisting of fluoro, chloro, bromo or iode, preferably fluoro or chloro.

SARS is caused by a virus named SARS-CoV (SARS associated corona virus) . The largest group of anti-viral compounds known in the art include nucleoside-analogues . The disadvantage is that virus strains are increasingly becoming resistant to these analogues. For this reason there is a strong need for new compounds having an anti-viral activity. It has been found that the compounds of the formulas (I) and (II) , which are not nucleoside-analogues, show excellent anti-viral activity against SARS. The importance of an effective medicine against SARS cannot be emphasized enough.

Literature also discloses other coumarine derivatives having anti-viral activity. For example, the International patent application WO92/18123 discloses coumarine derivatives, and as most related compounds alkoxy coumarine derivatives. These compounds are indicated to be suitable for inhibiting the replication of retrovirusses .

In "Synthesis of new dihydropyridine derivates" in Huaxue Yan- jiu Yu Yingyong, April 2002, 14_(2), p. 233-234, Deng et al disclose compounds containing ester bond coupled coumarine groups.

Preferred compounds are those wherein R4 is an optionally substituted coumarin-4-yl-group.

Furthermore, it is preferred that R2 and R3, R3' are chosen from an optionally substituted coumarin-4-yl-group and an optionally substituted coumarin-7-yl-group.

The present invention also relates to a pyridine-5-carboxylic acid ester derivative comprising a coumarine group, of the formula I

(i:

wherein

R represents hydrogen, CN, NO ₂ or a -COOR' -group, wherein R' is a linear or branched (Ci- ₆ ) alkyl group;

Rl has a meaning as defined for R4 or represents hydrogen, fluoro, chloro, bromo, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci_ ₆ ) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, an optionally esterified linear or branched (Ci_ ₆ ) alkanol, carboxamide;

R2 represents OH, linear or branched (Ci-ε) alkoxy or has a meaning as defined for R4 with the exception of hydrogen;

R3, R3' represent independently of each other H or independently of each other have a meaning as defined for R4;

R4 represents H or a coumarinyl group of the formula III

;iii;

wherein

X represents a single bond, - (CH ₂ ) _n -O-, or - (CH ₂ ) _ra -Y-

(CH ₂ ) _k ~0, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and

Y represents phenylene, (C ₃ _ ₈ ) cycloalkylene, or a carbon atom substituted with 1 or 2 (Ci-C ₂ ) alkyl groups ; R5 represents hydrogen, linear or branched (Ci-ε) alkyl, linear or branched (Ci_ ₆ ) alkoxy, halogen, nitro, hydroxyl, or amino; wherein alkyl, alkoxy, alkanol, benzyloxy, phenylene and cycloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, and nitro; with the provisio that at least 1 of the groups Rl, R2, R3, R3' and R4 is chosen from, optionally substituted, coumarin-4-yl and cou- marin-7-yl, as well as the N-oxide, stereo isomers and/or addition salts thereof with a pharmaceutically acceptable acid or base. If there is more than 1 Rl group, these may optionally be different.

The present invention also relates to a method for the preparation of a compound of the formula (I)

(i;

wherein

R represents hydrogen, CN, NO ₂ or a -COOR' -group, wherein R' is a linear or branched (Ci_ ₆ ) alkyl group;

Rl has a meaning as defined for R4 or represents hydrogen, fluoro, chloro, bromo, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci-β) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, an optionally esterified linear or branched (Cx-g) alkanol, carboxamide;

R2 represents OH, linear or branched (Ci-β) alkoxy or has a meaning as defined for R4 with the exception of hydrogen; R3, R3' represent independently of each other H or independently of each other have a meaning as defined for R4;

R4 represents H or a coumarinyl group of the formula III

(III)

wherein

X represents a single bond, -(CHa) _n -O-, or - (CH ₂ ) m~Y~ (CH ₂ J _k -O, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and

Y represents phenylene, (C ₃ _ ₈ ) cycloalkylene, or represents a carbon atom substituted with 1 or 2 (C ₁ -C ₂ ) alkyl groups;

R5 represents hydrogen, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci- ₆ ) alkoxy, halogen, nitro, hydroxyl, or amino; wherein alkyl, alkoxy, alkanol, benzyloxy, phenylene and cycloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, and nitro; with the provisio that at least 1 of the groups Rl, R2, R3, R3' and R4 is chosen from, optionally substituted, coumarin-4-yl and cou- marin-7-yl, by subjecting a compound of the formula (II),

wherein R, Rl, R2, R3, R3' and R4 are as defined before, to an oxidation yielding the coumarine derivative of the formula (I).

Suitable oxidizing agents are, for example, 3-chloroperbenzoic acid, hydrogen peroxide, silver oxide, hydroxy (tosyloxy) iodobenzene .

Compounds of the formula II can be prepared as disclosed in WO 01/14370.

In addition, the present invention relates to new coumarine derivatives of the formula (II'), a subset of the compounds of the formula II

( II '

wherein

R represents hydrogen, CN, NO ₂ or a -COOR' -group, wherein R' is a linear or branched (Cχ- ₆ ) alkyl group;

Rl has a meaning as defined for R4 or represents hydrogen, fluoro, chloro, bromo, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci_ ₆ ) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, optionally esterified with a linear or branched (Ci_ ₆ ) alkanol, carbox- amide;

R2 represents OH, linear or branched (Ci_ ₆ ) alkoxy or has a meaning as defined for R4 with the exception of hydrogen;

R3, R3' represent independently of each other H or independently of each other have a meaning as defined for R4;

R4 represents H or a coumarinyl group of the formula III

(III)

wherein

X represents a single bond, - (CH ₂ ) _n -0~, or - (CH ₂ ) _m -Y- (CH ₂ ) _k -0, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and

Y represents phenylene, (C ₃ _ ₈ ) cycloalkylene, or a carbon atom._ sυb.At.itjα.te.d..._with 1 or 2 (Ci-C ₂ ) alkyl groups;

R5 represents hydrogen, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci_ ₆ ) alkoxy, halogen, nitro, hydroxyl, or amino; wherein alkyl, alkoxy, alkanol, benzyloxy, phenylene and cycloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, and nitro; with the provisio that at least 1 of the groups Rl, R2 , R3, R3' and R4 is chosen from, optionally substituted, coumarin-4-yl and cou- marin-7-yl as well as the N-oxide, stereo isomers and/or addition salts thereof with a pharmaceutically acceptable acid or base, with the exception of those compounds of the formula (II) wherein R = CN, NO ₂ or a -COOR' -group, wherein R' is a (Ci-C ₆ ) alkyl group;

Rl = Rβ or R4; R2 = (Ci-C ₆ ) alkoxy or R4; R3 = H or R4; R4 = a coumarine group of the formula III;

R5 = H, (Ci-C ₆ ) alkyl, (Ci-C ₅ ) alkoxy, halogen, nitro, hydroxyl, or amino;

R6 = H, (Ci-C ₆ ) alkyl, (Ci-C ₆ ) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, optionally esterified with a (Ci-C ₆ ) alkanol, carboxamide, optionally substituted with 1-2 (Ci-C ₆ ) alkyl groups ;

X = a single bond, -(CH2J _n -O-, or - (CH2 J _1n -Y- (CH2) _k -0, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and Y = phenylene, cycloalkylene, or a carbon atom substituted with 1 or 2 (Ci-C ₂ ) -alkyl groups; wherein (Ci-C ₆ ) alkyl, (Ci-C ₆ ) alkoxy, (Ci-C ₆ ) alkanol, benzyloxy, phenylene and cycloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, in particular, chlσro or fluoro, and nitro; with the provisio that at least 1 of the groups Rl, R2 and R3 is the same as of R4,

as well as the N-oxide, stereo isomers and/or addition salts thereof with a pharmaceutically acceptable acid or base, wherein more than 1 Rl- and R6-groups may be present, the (Ci- C ₆ ) alkyl and (Ci-C ₆ ) alkoxy are linear or branched, the term cycloalkyl is understood to mean a cycloalkyl group having 3 to 8 carbon atoms, and halogen is understood to mean a halogen atom chosen from the group containing fluoro, chloro, bromo or iodo.

The disclaimer aims to exclude all compounds as disclosed in WO 01/14370 and in Dutch patent application NL1012886 on which it is based. The new compounds as claimed herein can be prepared using the method as disclosed in WO 01/14370 and NL1012886.

Finally, the present invention relates to a pharmaceutical composition comprising a compound according to the invention together with a pharmaceutically acceptable carrier or excipient. The present invention will now be elucidated on the basis of the following embodiments and in vivo experiments. Starting materials can either be obtained commercially or can be easily prepared by those skilled in the art analogous to known compounds. Known starting materials are disclosed in PCT WO 94/12488. The invention will now be elucidated on the basis of the following examples.

EXAMPLES

I) Preparation of compounds

Known and new compounds were prepared using Methods A to C as disclosed in the International patent application WO 01/14370, and Methods D and E, as described below.

Ia) Method D which includes the steps of a) subjecting 2, 6-dimethyldihydropyridine derivative of the formula

wherein

R represents hydrogen, CN, NO ₂ or a -COOR' -group, wherein R' is a linear or branched (Ci_ ₆ ) alkyl group;

Rl has a meaning as defined for R4 or represents hydrogen, fluoro, chloro, bromo, linear or branched (Cχ-6) alkyl, linear or branched (Ci_ ₆ ) alkoxy, benzyloxy, cyano, nitro, carboxylic acid, optionally esterified a linear or branched (Cχ-β) alkanol, carboxamide;

R2 represents OH, linear or branched (Ci-ε) alkoxy or has a meaning as defined for R4 with the exception of hydrogen;

R3, R3' represent hydrogen;

R4 represents H or a coumarinyl group of the formula III

:iii)

wherein

X represents a single bond, - (CH ₂ ) _n -0-, or - (CH ₂ ) _m -Y- (CH ₂ ) _k ~0, wherein n ranges from 1 to 6, m and k each are equal to or larger than 1 and the sum of m and k is smaller than or equal to 4; and

Y represents phenylene, (C ₃ _ ₈ ) cycloalkylene, or a carbon atom substituted with 1 or 2 (C ₁ -C ₂ ) alkyl groups ;

R5 represents hydrogen, linear or branched (Ci_ ₆ ) alkyl, linear or branched (Ci-β) alkoxy, halogen, nitro, hydroxyl, or amino; wherein alkyl, alkoxy, alkanol, benzyloxy, phenylene and cy- cloalkylene are optionally substituted with 1-3 groups chosen from hydroxyl, cyano, amino, halogen, and nitro;

to a bromination yielding 2-bromomethyl derivatives and bis-2- bromomethyl derivatives of the formulas

(IV) (V)

wherein R, Rl, R2 and R4 are as defined before b) subjecting the product of step a) to a reaction with a hy- droxycoumarine corresponding to the formula (VI)

(VI)

wherein X and R5 are as defined before,

yielding a compound of the formula II

( II )

wherein R, Rl, R2, R3, R3' and R4 are as defined before, with the provisio that at least 1 of R3 and R3' is an optionally substituted coumarinyl group.

Suitable bromination reagents for carrying out step a) are, for example, bromine, fosfortribromide, N-bromosuccinimide, pyridinium- bromideperbromide .

After step a) and/or b) a purification step may be carried out, for example, chromatography over silica or crystallisation, wherein the purified product can be used in the subsequent step. The method for the preparation of the 2-bromoethyl- dihydropyridine derivative, intermediate obtained after step a) , takes place analogous to that disclosed by D. Alker et al in: Tetrahedron Letter, 3JL' P- 1479-1482, 1990.

Solid pyridiniumbromideperbromide (15 mmol) was added portion- wise to a stirred ice-cold solution of a dihydropyridine (10 mmol) in CH ₂ Cl ₂ . After completing the addition, the mixture was stirred for another 15 minutes, washed with ice-cold 2 M HCl, dried over MgSO ₄ and concentrated by evaporation without heating.

The residue was dissolved in DMF together with 15 mmol of a hy- droxycoumarine corresponding to the formula (III) and 20 mmol of K ₂ CO ₃ . The mixture was heated for 6 hours at 60°C, cooled down to room temperature and poured into water. The precipitate was filtered off, dissolved in CH ₂ Cl ₂ and applied onto a silica gel column. Elution with ethyl acetate and crystallisation from ethanol yielded the 2 compounds (a 2, 6-bis- (coumarinyloxymethyl) 1, 4-dihydropyridine-3, 5-

carboxylic acid diethyl ester and a 2- (coumarinyloxymethyl) -1, 4- dihydropyridine-3, 5-carboxylic acid diethyl ester derivative).

Method D is visualized in the diagram of the sole figure, wherein 1: pyridiniumbromideperbromide in CH ₂ Cl ₂ , 0°C 2: hydroxycoumarine, K ₂ CO ₃ , DMF, 60°C

Ib) Method E

The method for the preparation of the pyridine derivatives ac- cording to the invention takes place analogous to that disclosed by

Kang-Hyeok Lee and Kwang-Youn Ko in Bull. Korean Chem. Soc, 2_3, p.

1505-1506, 2002. According to the invention a compound of the formula

(II) is used as a starting material.

A mixture of 10 mmol of a dihydropyridine of the formula (II) and 12 mmol of hydroxy (tosyloxy) iodobenzene as an oxidizing agent in dichloromethane was stirred for 5 minutes at room temperature and then washed using 1 M bicarbonate. The organic solution was dried over magnesium sulfate and concentrated by evaporation. The residue was purified using chromatography over silica (eluent: ethyl acetate or a mixture of ethyl acetate and hexane) .

Using the Methods A to E, starting from the suitable starting compounds, the following compounds were prepared (Table 1) . The mass

(M+H) of the prepared compounds was measured using ESI (electron spray impact) mass spectrometry via continue flow injection equipped with an LCQ Deca (Finnigan) .

Table 1

In Vitro activity

The biological activity was tested for anti-SARS activity by means of standard experiments by the Rega Instituut (Leuven, Belgium) . The results (anti-viral action and cytotoxicity) are shown in Table 2.

Table 2

IC50: Concentration of the substance to be tested at which the virus production is inhibited by 50 %.

CC50: Concentration of the substance to be tested required to reduce cell growth by 50%.