NO326464B1 - Analogues of camptotecin, its use, and drug - Google Patents

Description

The present invention relates to new analogues of camptothecin, their use, and medicine.

Camptothecin is a natural compound first isolated from the leaves and bark of the Chinese plant camptotheca acuminata (see Wall et al. J. Amer. Chem. Soc. 88:3888 (1966)). Camptothecin is a pentacyclic compound consisting of an indolizino[1,2-b]quinoline fragment fused to a six-membered oc-hydroxy-lactone. The carbon in position 20 which carries the oc-hydroxy group is asymmetric and exhibits a rotating force on the molecule. The natural form of camptothecin has an absolute "S" configuration with respect to carbon 20 and corresponds to the following formula:

Camptothecin has an anti-proliferative activity in several cancerous cell lines, including cell lines from human tumors of the intestine, lungs and breast (Suffness, M et al: The Alkaloids Chemistry and Pharmacology, Brass A., ed., Vol. 25, p. 73 (Academic Press, 1985)). It has been proposed that the anti-proliferative activity of camptothecin is related to its inhibitory activity on DNA topoisomerase I.

It has been indicated that α-hydroxylactone has an absolute requirement both for in vivo and

in vitro activity of campotothecin (Camptotecins: New Anticancer Agents, Putmesil, M et al, ed., p. 27 (CRC Press, 1995); Wall M. et al, Cancer Res. 55:753 (1995); Hertzberg et al , J. Med. Chem. 32:715 (1982) and Crow et al, J. Med. Chem. 35:4160 (1992)). The present invention relates to a new class of compounds of camptothecin, where a p-hydroxylactone replaces the natural a-hydroxylactone of camptothecin. The compounds according to the present invention have a powerful biological activity which is unexpected with regard to the state of the art.

A purpose according to the invention is therefore new analogues of camptothecin which differ from all known derivatives of camptothecin in that they contain (3-hydroxy-lactone (or its open hydroxycarboxyl form) instead of α-hydroxylactone (or its open hydroxycarboxyl form); or a pharmaceutically acceptable salt of one of the latter. By derivative of camptothecin is meant a compound having the same structural skeleton as that of camptothecin (ie, an indolizino[1,2-b]quinoline fragment fused to a six-membered α-hydroxylactone), with or without other chemical substitutions on the skeletal structure. Various derivatives of camptothecin are well known to those skilled in the art, as described below. By (3-hydroxy-lactone is meant a lactone containing an additional carbon atom between the carbon of the carboxyl and the a-carbon bearing the hydroxyl group of α-hydroxylactone.

The present invention therefore relates to a compound, characterized in that it is selected from the compounds:

5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(1,2,5,6-tetrahydopyridinomethyl-

1 H-oxepino[3',4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione hydrochloride 5-ethyl-9,10-difluoro-4,5-dihydro -5-hydroxy-12-(4-methyl piperidinomethyl)-1 H- oxepino[3',4':6,7]indolizino[1,2-b] quinoline-3,15(4H,13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5 -hydroxy-12-pyrrolidinomethyl-1H-oxepino- [3,,4,:6,7]indolizino[1,2-d]quinoline-3,15(4H,13H)-dione

5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methyl piperazinomethyl-1 H -oxepino[3',4':6,7]indolizino[1,2-b ] quinoline-3,15(4H,13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-piperidinomethyl-1H-oxepino-[3,,4': 6,7]indolizino[1,2-ef]quinoline-3,15(4H,13W)-dione

5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-dimethylamino-methyl-1 /-/oxepino(3',4,:6,7]indolizino[1,2-d ]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-morpholino methyl-1H-oxepino[3',4< l>:6,7]indolizino[1,2-6]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12 -(4-methylpiperazinomethyl)-1H-oxepino(3,,4<l>:6,7]indolizino[1)2-£>]quinoline-3,15(4H,13H)-dione 12-benzylpiperazinomethyl-9 -chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino[3',4':6,7]indolizino[1,2-fc]quinoline-3,15( 4H,13H)-dione 12-(4-benzylpiperazinomethyl)-9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino[3,,4,:6,7] indolizino[1I2-b]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-piperidinomethyl-1H-oxepino-( 3',4':6,7]indolizino[1,2-/?]quinoline-3,15(4H,13H)-dione

12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]indolizino[1,2-£)] quinoline-3,15(4H,13H)-dione 12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino[3', 4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12 -dimethylaminomethyl-1 /-/ oxepino[3,,4,:6,7]indolizino[1,2-t>]quinoline-3,15(4H,13H)-dione 5-ethyl-12-diethylaminomethyl-9-fluoro-4,5-dihydro -5-hydroxy-10-methyl-1 /-/ oxepino[3,,4,:6I7]indolizino[1,2-/?]quinoline-3,15(4HI13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl -12-(4-methyl piperidinomethyl)-1H-oxepino[3',4:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9- fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-pyrrolidinomethyl-1 H- oxepino[3,14':6,7]indolizino[1,2-d]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10 -methyl-12-(1,2,5,6-tetrahydro- pyridinomethyl)-1 H-oxepino[3\4^67]indolizino[1,2-/?]quinoline-3 J 5(4H, 13H)-dione 12-diisobutylaminomethyl-5-ethyl-9-fluoro-4,5 -dihydro-5-hydroxy-10-methyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy- 10-methoxy-12-(4-methylpiperazinomethyl)- 1H-oxepino[3',4<l>:6,7]indolizino[1,2-6]quinoline-3,15(4H)13H)-dione 5-ethyl-9-fluoro-4,5-dihydro- 5-hydroxy-10-methoxy-12-piperidino methyl-1 /-/ oxepino[3,,4<l>:6,7]indolizino[1,2-b]quinoline-3,15(4HI13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy- 10-methoxy-12-dimethylaminomethyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-t>]quinoline-3,15(4H, 13W)-dione

9-ch1 oro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidinomethyl-1 /-/oxepino[3'I4,:6)7]indolizino[1,2-^] quinoline-3,15(4H,13H)-dione hydrochloride 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(1,2,5,6-tetrahydropyridino-methyl)-1H-oxepino[ 3,)4,:6,7]indolizino[1I2-/?]quinoline-3,15(4H13H)-dione hydrochloride

5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methylpiperidinomethyl)-1H-oxepino[3',4':6,7]indolizino[1,2-d]quinoline -3,15(4H,13H)-dione 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methylpiperazinomethyl)-1H-oxepino[3\4^67]indolizino[ 1,2-fc]quinoline-3,15(4H 13H)-dione 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-pyrrolidinomethyl-1H-oxepino-[3,,4, :6>7]indolizino[1,2-6]quinoline-3I15(4H113H)-dione

12-(4-benzylpiperazinomethyl)-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino[3,14'I:6,7]indolizino[1I2-/3]quinoline- 3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-(-4-methylpiperidino methyl)-1H-oxepino[3,,4 ,:6,7]indolizino[1,2-6]quinoline-3115(4H,13H)-dione;

10-benzyloxy-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]-indolizino[1,2-fc]quinoline-3, 15(4H,13H)-dione;

5-ethyl-9-fluoro-4,5-dihydro-5,10-dihydroxy-1H-oxepino[3',4':6,7]indolizino

[1,2-b]quinoline-3,15(4H,13H)-dione;

or a pharmaceutically acceptable salt thereof.

There is further described a compound according to claim 1, characterized in that it is selected from the compounds: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methyl piperidinomethyl)-1 H- oxepino[3',4,:6,7]indolizino[1,2-d]quinoline-3,15(4H,13H)-dione; 5-ethyl-12-diethylaminomethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-f}]quinoline-3,15(4H,13H)-dione; 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methyl piperidiiromethyl)- 1H-oxepino[3',4<1>:6,7]indolizino[1,2-£»]quinoline-3,15(4H,13H)-dione; 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-pyrrolidinomethyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-d]quinoline-3,15(4H,13W)-dione; 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidinomethyl-1 /-/ oxepino[3',4':6,7]indolizino[1,2-£?]quinoline-3,15(4 H -/,13 H )-dione hydrochloride; 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methyl piperidinomethyl)-1 H- oxepino[3',4,:6,7]indolizino[1,2-6]quinoline-3115(4H,13H)-dione; 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methylpiperidino methyl)-1 H- oxepino[3',4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione;

or a pharmaceutically acceptable salt thereof.

The invention further relates to medication, characterized in that it contains a compound according to any of the preceding claims or a pharmaceutically acceptable salt thereof.

The present invention also describes the use of a compound according to any one of claims 1 to 2 for the production of anti-tumoral drugs.

As observed for camptothecin, the carbon atom bearing the hydroxy function in the β-hydroxylactone or β-hydroxycarboxylate group of the compounds is asymmetric. Accordingly, the compounds according to the present invention have two possible enantiomeric forms, i.e. the "R" and the "S" configuration. The present invention encompasses two enantiomeric forms and any combination of these forms, including "RS" racemic mixtures. In an attempt to simplify matters, when no specific configuration is indicated in the structural formula, it should be understood that the two enantiomeric forms and their mixtures are represented.

Certain compounds according to the invention can be prepared in the form of pharmaceutically acceptable salts according to usual methods. Acceptable salts include, by way of example and without limitation, addition salts with inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide and nitrate or with organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate , methanesulfonate, p-toluenesulfonate, pamoate, salicylate, oxalate and stearate. The salts formed from bases such as sodium or potassium hydroxide also form part of the range of applications according to the present invention, when these can be used. For other examples of pharmaceutically acceptable salts, see "Pharmaceutical Salts", J. Pharm. Pollock. 66:1 (1977).

The compounds according to the present invention possess useful pharmacological properties. The compounds according to the present invention can be used in various therapeutic applications.

An illustration of the pharmacological properties of the compound according to the invention will emerge from the experimental part.

The compounds according to the invention also have an anti-tumoral activity. They can be used for the treatment of tumors, for example tumors expressing a topoisomerase, in a patient by administering to the latter a therapeutically effective amount of a compound of formula (I) or of formula (II). Examples of tumors or cancers include cancer of the esophagus, stomach, intestine, rectum, oral cavity, trachea, larynx, lung, intestine, breast, cervix uteri, corpus endometrium, ovary, prostate, testicles, bladder, kidney, liver, pancreas, bone , connective tissue, skin, eyes, brain and central nervous system, as well as thyroid cancer, leukaemia, Hodgkin's disease, lymphomas other than those related to Hodgkin's, multiple myeloma and others.

The invention therefore relates to pharmaceutical drugs containing a compound according to the invention or an addition salt with a pharmaceutically acceptable acid thereof, in combination with a pharmaceutically acceptable carrier according to the chosen administration method (for example oral, intravenous, intraperitoneal, intramuscular, transdermal or subcutaneous ). The pharmaceutical drug (eg therapeutic) can be in the form of a solid, liquid, liposome or lipid micelle.

Pharmaceutical drug can be in solid form, for example powders, pills, granules, tablets, liposomes, gelatin capsules or suppositories. The pill, tablet or gelatin capsule may be surrounded by a compound capable of protecting the composition from the action of gastric acid or enzymes in the stomach of the subject for a sufficient period of time to enable this composition to pass in an undigested form into the small intestine of the subject . The compound can also be administered locally, for example, at the same location as the tumor. The compound may also be administered according to a sustained release method (eg, a sustained release composition or an infusion pump). Suitable solid carriers may, for example, be calcium phosphate, magnesium stearate, magnesium carbonate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine and waxes. Pharmaceutical compositions containing a compound according to the invention can also be presented in liquid form such as, for example, solutions, emulsions, suspensions or a formulation with sustained release. Suitable liquid carriers are, for example, water, organic solvents such as glycerol or glycols such as polyethylene glycol, as well as mixtures thereof, in varying proportions, in water.

The object of the present invention is also the use of the compounds indicated above for the production of drugs intended for the treatment of tumors.

The dose of a compound according to the present invention for the treatment of the diseases or disorders mentioned above varies according to the method of administration, age and body weight of the individual as well as the condition of the latter and it will be definitively decided by the attending physician or veterinarian. Such an amount determined by the attending physician or veterinarian is stated herein as "effective therapeutic amount".

An illustration of the pharmacological properties of the compounds according to the invention can be found in the experimental section.

The compounds according to the invention also have an anti-tumoral activity. They can be used for the treatment of tumors, for example tumors expressing a topoisomerase, in a patient by administering to the latter a therapeutically effective amount of a compound of formula (I) or of formula (II). Examples of tumors or cancers include cancer of the oesophagus, stomach, intestine, rectum, oral cavity, trachea, larynx, lung, small intestine, breast, cervix uteri, corpus endometrium, ovaries, prostate, testes, bladder, kidney, liver, pancreas, bones , connective tissue, skin, the eyes, brain and central nervous system, as well as thyroid cancer, leukaemia, Hodgkin's disease, lymphomas other than those related to Hodgkin, multiple myeloma and others.

These properties make the compounds suitable for pharmaceutical use.

The pharmaceutical drug may be in solid form, for example, powders, pills, granules, tablets, liposomes, gelatin capsules or suppositories. The pill, tablet or gelatin capsule may be coated with a compound capable of protecting the composition from the action of gastric acid or enzymes in the stomach of the subject for a sufficient period of time to enable this composition to pass in an undigested form into the small intestine of the individual. The compound may also be administered locally, for example, at the location of the tumor. The compound may also be administered according to a sustained release method (eg, a sustained release composition or an infusion pump). Suitable solid carriers may be, for example, calcium phosphate, magnesium stearate, magnesium carbonate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine and waxes. The pharmaceutical drugs containing a compound according to the invention can also be presented in liquid form such as, for example, solutions, emulsions, suspensions or a formulation with sustained release. Suitable liquid carriers can be, for example, water, organic solvents such as glycerol or glycols such as polyethylene glycol, as well as mixtures thereof, in varying proportions, in water.

The dose of a compound according to the present invention intended for the treatment of diseases or disorders mentioned above varies according to the administration method, age and body weight of the individual as well as the condition of the latter and it will be finally decided by the attending physician or veterinarian. Such an amount determined by the attending physician or veterinarian is stated herein as "effective therapeutic amount".

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are usually used by specialists in the field to which the invention belongs.

The following examples are presented to illustrate the above procedures.

EXPERIMENTAL PART

Example 1: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(1,2,5,6-tetrahydropyridinomethyl-1N-oxepino[3',4':6,7 ]-indolizino[1,2-b]

quinoline-3,15 (4H, 13W)-dione hydrochloride

3,4-difluoroaniline was used to produce ethyl 2-chloro-4-chloromethyl-6,7-difluoro-3-quinolinecarboxylate, and treated using 1,2,5,6-tetrahydropyridine and then reduced to equivalent to quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. The free base thus obtained was suspended in absolute ethanol (50 ml/mmol) then treated with ethanolic hydrogen chloride (2.5N, 5 eq.). Initially a yellow solution was formed, then a precipitate which was collected by filtration after concentration to 40% of the original volume and washed with ether. A light orange solid was obtained, m.p. 264°C.

NMR-1H (DMSO): 0.87 (t, 3H); 1.85 (q, 2H); 2.26-2.30 (m, 1H); 2.50 (m, 1H); 3.09 (d, 1H); 3.40 (m, 2H); 3.48 (d, 1H); 3.87 (m, 2H); 5.05 (m, 2H); 5.48 (q, 2H); 5.65 (m, 2H); 5.89 (m, 1H); 7.42 (s, 1H); 8.24-8.30 (m, 1H); 8.76-8.82 (m, 1H); 10.86 (p, 1H). NMR-C 13 (DMSO): 8.44; 22.36; 36.5; 42.7; 48.71; 50.30; 51.49; 61.42; 73.23; 100.16; 112.64; 112.83; 116.05; 120.26; 123.31; 125.29; 125.40; 131.17; 133.97; 144.15; 146.26; 146.37; 148.74; 150.52; 151.23; 153.20; 153.53; 155.99; 159.04; 172.02.

IR (KBr): 662; 1064; 1268; 1452; 1523; 1598; 1652; 1743; 2936; 3027; 3418.

Example 2: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methyl piperidinomethyl)-1H-oxepino-3',4':6,7indolizino1,2-bquinoline -3,15(4H,13W)-dione

3,4-Difluoro-aniline was used to produce ethyl 2-chloro-4-chloromethyl-6,7-difluoro-3-quinoline carboxylate and treated using 4-methyl-piperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A beige solid was obtained, m.p. > 250°C.

NMR-1H (DMSO): 0.9 (m, 6H); 1.1 (m, 2H); 1.4 (m, 1H); 1.55 (d, 2H); 1.85 (q, 2H); 2.1 (t, 2H); 2.85 (m, 2H); 3.25 (dd, 2H); 4 (p, 2H); 5.3 (s, 2H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.35 (s, 1H); 8.15 (dd, 1H); 8.45 (dd, 1H).

IR (KBr): 1454; 1518; 1608; 1658; 1733; 2804; 2926; 3311.

Suspension of the above free base in absolute ethanol (50 ml/mmol) followed by treatment with ethanolic hydrogen chloride (2.5 N, 5 eq.) enables the corresponding hydrochloride to be obtained. Initially, a yellow solution is formed, then a precipitate which was collected by filtration after concentration to 40% of the original volume, then washed with ether. A pale orange solid was obtained, m.p. > 250°C. NMR-1H (DMSO): 0.85 (m, 6H); 1.7 (m, 5H); 1.85 (q, 2H); 3.15 (s, 1H); 3.25 (dd, 2H); 3.3 (m, 2H); 4.9 (s, 2H); 5.45 (dd, 2H); 5.6 (s, 2H); 6.1 (s, 1H); 7.4 (s, 1H); 8.25 (dd, 1H); 8.75 (dd, 1H); 10.35 (p, 1H).

IR (KBr): 1270; 1455; 1523; 1606; 1653; 1742; 2943; 3419.

Example 3: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-pyrrolidinomethyl-1H-oxepino[3',4,:6,7]indolizino[1,2-b] quinoline 3,15(4H,13H)-dione 3,4-difluoroaniline was used to produce ethyl 2-chloro-4-chloromethyl-6,7-difluoro-3-quinolinecarboxylate which was treated using pyrrolidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A beige solid was obtained, m.p.

> 250°C.

NMR-1 H (DMSO): 0.85 (t, 3H); 1.7 (s, 4H); 1.85 (q, 2H); 2.55 (s, 4H); 3.25 (dd, 2H); 4.15 (d, 2H); 5.35 (s, 2H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.35 (s, 1H); 8.15 (dd, 1H); 8.45 (dd, 1H).

IR (KBr): 1455; 1518; 1605; 1657; 1731; 2801; 2970; 3422.

Suspension of the above-mentioned free base in absolute ethanol (50 ml/mmol) followed by treatment with ethanolic hydrogen chloride (2.5 N, 5 eq.) enables the corresponding hydrochloride to be obtained. Initially a yellow solution is formed, then a precipitate which was collected by filtration after concentration to 40% in original volume, then washed with ether. A light orange solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.85 (t, 3H); 1.9 (m, 4H); 2.1 (s, 2H); 3.25 (dd, 2H); 3.3 (m, 2H); 3.55 (m, 2H); 5.05 (s, 2H); 5.45 (dd, 2H); 5.6 (s, 2H); 6.1 (s, 1H); 7.4 (s, 1H); 8.3 (dd, 1H); 8.75 (dd, 1H); 10.75 (p, 1H).

IR (KBr): 1454; 1522; 1603; 1653; 1743; 2970; 3394.

Example 4: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methyl piperazinomethyl-1H-oxepino3',4':6,7indolizino1,2-toquinoline-3, 15(4H,13H)-dione

3,4-difluoroaniline was used to produce ethyl 2-chloro-4-chloromethyl-6,7-difluoro-3-quinolinecarboxylate which was treated and then reduced to the equivalent

quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C. NMR-1 H (CDCl 3 + CD 3 OD): 0.99 (t, 3H); 2.00 (q, 2H); 2.32 (s, 3H); 3.24 (d, 1H); 3.37 (s, 1H); 3.42 (d, 1H); 4.04 (s, 2H); 5.37 (s, 2H); 5.43 (d, 1H); 5.64 (d, 1H); 7.56 (s, 1H); 7.84 (dd, 1H); 8.22 (dd, 1H).

NMR-C 13 (CDCl 3 + CD 3 OD): 7.87; 36.11; 42.16; 45.33; 52.67; 54.52; 56.47; 61.97; 73.26; 101.17; 110.81; 115.49; 122.93; 128.63; 139.83; 144.28; 146.40; 149.27; 151.27; 151.64; 152.31; 153.82; 156.50; 159.71; 172.56.

IR (KBr): 1607; 1656; 1732; 2795; 3411.

Example 5: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy 12-piperidinomethyl-

1 H-oxepino[3\4':6,7]indolizino[1,2-/>]quinoline 3,15(4H,13H)-dione 3,4-difluoroaniline was used to produce ethyl 2-chloro -4-chloromethyl-6,7-difluoro-3-quinolinecarboxylate which was treated using piperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light green solid was obtained, m.p. 266-268°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.42-1.49 (m, 6H); 1.85 (q, 2H); 2.47 (m, 4H); 3.06 (d, 1H); 3.48 (d, 1H); 4.00 (q, 2H); 5.31 (s, 2H); 5.46 (dd, 2H); 6.04 (s, 1H); 7.37 (s, 1H); 8.14 (m, 1H); 8.46 (m, 1H).

NMR-C 13 (DMSO): 8.43; 24.01; 25.8; 36.52; 42.56; 50.60; 54.29; 56.91; 61.41; 73.30; 99.81; 111.86; 115.67; 122.94; 130.10; 140.66; 144.49; 146.12; 153.18; 155.86; 159.14; 172.03.

IR (KBr): 1258; 1452; 1517; 1607; 1661; 1731; 2950; 3480.

Example 6: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-dimethylamino-methyl-1H-oxepino[3a,4:6,7]indolizino[1,2-b] quinoline-3,15(4H,13H)-dione

3,4-Difluoroaniline was used to produce ethyl 2-chloro-4-chloromethyl-6,7-difluoro-3-quinoline carboxylate which was treated using dimethylamine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light beige solid was obtained, m.p. > 270°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 2.25 (s, 6H); 3.08 (d, 1H); 3.47 (d, 1H); 3.95 (q, 2H); 5.28 (s, 2H); 5.46 (dd, 2H); 6.06 (s, 1H); 7.37 (s, 1H); 8.14 (s, 1H); 8.42 (p, 1H).

NMR-C 13 (DMSO): 8.42; 14.06; 33.36; 45.44; 50.57; 61.40; 65.14; 72.05; 72.93; 73.30; 99.82; 99.95; 115.78; 115.85; 122.96; 125.01; 130.08; 140.56; 144.54; 146.16; 155.86; 159.19; 172.03.

IR (KBr): 1516; 1613; 1654; 1731; 3450.

Example 7: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-morpholino methyl-1H-oxepino[3,,4,:6,7indollzino[1,2-to ]quinoline-3,15(4H,13W)-dione

3-Chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinoline carboxylate which was treated using morpholine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p.

> 300°C.

NMR-1 H (DMSO): 0.87 (t, 3H); 1.84 (q, 2H); 2.50 (s, 4H); 2.58 (s, 3H); 3.07 (d, 1H); 3.46 (d, 1H); 3.57 (s, 4H); 4.08 (dd, 2H); 5.30 (s, 2H); 5.51 (dd, 2H); 6.06 (s, 1H); 7.35 (s, 1H); 8.15 (p, 1H): 8.41 (p, 1H).

NMR-C 13 (DMSO): 8.42; 20.57; 36.51; 42.55; 50.76; 53.46; 55.86; 61.42; 66.42; 73.29; 99.73; 122.78; 128.40; 130.10; 135.31; 136.26; 139.36 144.61; 147.79; 152.81; 155.86; 159.16; 172.04.

IR (KBr): 1613; 1657; 1736; 3432.

Example 8: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methylpiperazinomethyl)-1H-oxepino[3',4':6,7indolizino [1, 2-b]

quinoline-3,15(4H,13H)-dione

3-Chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinoline carboxylate which was treated and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. 262-268°C. NMR-1 H (DMSO): 0.87 (t, 3H); 1.86 (q, 2H); 2.15 (s, 3H); 2.20-260 (m, 8H); 2.60 (s, 3H); 3.05 (d, 1H); 3.49 (d, 1H); 4.09 (dd, 2H); 5.32 (s, 2H); 5.50 (dd, 2H); 6.05 (s, 1H); 7.37 (s, 1H); 8.21 (s, 1H); 8.43 (p, 1H).

NMR-C 13 (DMSO): 8.42; 20.56; 36.50; 42.55; 45.91; 50.81; 53.00; 54.94; 55.65; 61.43; 73.29; 79.36; 99.69; 122.75; 126.32; 128.37; 129.84; 135.25; 136.23; 139.87; 144.57; 147.75; 152.76; 155.87; 159.15; 172.04.

IR (KBr): 1607; 1658; 1733; 3424.

Example 9: 12-benzylmethylaminomethyl-9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino[3',4,:6,7]indolizino[1,2- b]quinoline-3,15(4H,13H)-dione

3-Chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinoline carboxylate which was treated using N-methylbenzylamine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. 275-278°C.

NMR-1 H (DMSO): 0.88 (t, 3H); 1.85 (m, 2H); 2.13 (s, 3H); 2.55 (s, 3H); 3.10 (d, 1H); 3.50 (d, 1H); 3.67 (s, 2H); 4.05 (dd, 2H); 5.30 (s, 2H); 5.39-5.57 (dd, 2H); 6.05 (s, 1H); 7.36 (m, 6H); 8.15 (s, 1H); 8.31 (p, 1H).

NMR-C 13 (DMSO): 9.10; 21.15; 37.20; 42.86; 43.23; 51.32; 55.78; 62.10; 62.88; 73.99; 80.05; 100.44; 123.47; 126.99; 127.32; 128.09; 129.17; 129.96; 130.86; 135.75; 136.84; 139.51; 140.67; 145.38; 148.54; 153.50; 156.54; 159.85: 172.73.

IR (KBr): 1609; 1655; 1729; 3395.

Example 10: 12-(4-benzylpiperazinomethyl)-9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-1H oxepino[3',4':6,7]indolizino[1 ,2-b)quinoline-3,15(4H,13H)-dione

3-Chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinoline carboxylate which was treated using N-benzylpiperazine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A beige solid was obtained, m.p. 244-249°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.83 (m, 2H); 2.38-2.60 (m, 8H); 2.57 (s, 3H); 3.08 (d, 1H); 3.46 (s .2H); 4.08 (m, 2H); 5.30 (s, 2H); 5.51 (dd, 2H); 6.05 (s, 1H); 7.30 (m, 6H); 8.16 (s. 1H); 8.40 (p, 1H).

NMR-C 13 (DMSO): 9.10; 21.23; 37.19; 43.21; 51.48; 53.54; 53.80; 56.35; 62.09; 62.84; 73.97; 97.67; 100.39; 123.45; 127.05; 127.75; 129.02 129.63; 130.61; 135.95; 136.93; 139.14; 140.52; 145.27; 148.45; 153.47; 156.52; 159.83; 172.72.

IR (KBr): 1567; 1587; 1652; 1748; 3422.

Example 11: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-piperidinomethyl-1H-oxepino[3\4':6,7)indolizino[1,2-b ]quinoline-3,15(4H,13H)-dione

3-Chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinoline carboxylate which was treated using piperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M).

The resulting coupled product was cyclized. A yellow solid was obtained, m.p. 255°C (dec.).

NMR-1 H (DMSO): 0.86 (t, 3H); 1.50 (m, 6H); 1.84 (m, 2H); 2.50 (m, 4H); 2.58 (s, 3H); 3.05 (d, 1H); 3.45 (d, 1H); 4.04 (m, 2H); 5.32 (s, 2H); 5.51 (dd, 2H); 6.10 (s, 1H); 7.37 (s, 1H); 8.20 (s, 1H); 8.42 (p, 1H).

NMR-C 13 (DMSO): 9.11; 21.24; 24.70; 26.50; 37.20; 43.23; 51.43; 55.10; 57.21; 62.09; 73.99; 98.05; 100.38; 123.44; 127.10; 129.12; 130.59; 135.89; 136.91; 140.99; 145.31; 148.50; 153.52; 156.51; 159.85; 172.73.

IR (KBr): 1601; 1654; 1728; 3436.

Example 12: 12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]indolizino(1,2 -b]quinoline-3,15(4H,13H)-dione

4-Fluoroaniline was used to produce ethyl 2-chloro-4-chloromethyl-6-fluoro-3-quinolinecarboxylate which was treated using N-benzylpiperazine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A white solid was obtained, m.p. 262°C.

NMR-1 H (DMSO): 0.87 (t, 3H); 1.85 (q, 2H); 2.37 (s, 4H); 2.37 (s, 4H); 3.07 (d, 1H); 3.45 (s, 2H); 3.47 (d, 1H); 4.08 (q, 2H); 5.32 (s, 2H); 5.46 (dd, 2H); 6.03 (s, 1H); 7.35 (m, 5H); 7.38 (s, 1H); 7.77 (m, 1H); 8.20 (m, 2H).

NMR-C 13 (DMSO): 8.41; 36.49; 42.53; 50.65; 52.82; 53.03; 55.95; 61.41; 62.14; 72.3; 99.55; 109.31; 120.14; 120.40; 122.70; 127.05; 128.32 128.55; 128.96; 130.40; 138.42; 139.65; 144.66; 145.83; 152.15; 155.89; 159.15; 161.57; 172.02. IR (KBr): 740; 834; 1071; 1193; 1220; 1288; 1360; 1451; 1516; 1592; 1655; 1749; 2813; 2950; 3434.

Example 13: 12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino(3',4,:6,7]indolizino1, 2-6]quinoline-3,15(4H,13H)-dione

3-Fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinoline carboxylate which was treated using N-benzylpiperazine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light beige solid was obtained, m.p. 259°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 2.38 (m, 4H); 2.50 (s, 4H) 3.06 (d, 1H); 3.36 (s, 3H); 3.46 (s, 2H); 3.47 (d, 1H); 4.07 (q, 2H); 5.29 (s, 2H); 5.46 (dd, 2H); 6.02 (s, 1H); 7.23-7.35 (m, 6H); 7.8 (d, 1H); 8.35 (d, 1H).

NMR-C 13 (DMSO): 8.40; 15.45; 36.47; 42.54; 50.7; 52.84; 53.13; 55.81; 61.4; 62.14; 73.29; 99.57; 112.45; 122.61; 124.73; 127.05; 128.32; 128.96; 138.45; 139.81; 1444.68; 152.63; 155.85; 159.15; 172.02.

IR(KBr): 1013; 1069; 1169; 1241; 1266; 1475; 1577; 1594; 1655; 1744.

Example 14: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-dimethylaminomethyl-1H oxepino[3l,4':6,7]indolizino(1,2-b] quinoline-3,15(4H,13H)-dione

3-Fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinoline carboxylate which was treated using dimethylamine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light beige solid was obtained, m.p. 184-190°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 2.26 (s, 6H); 2.5 (s, 3H); 3.05 (d, 1H); 3.48 (d, 1H); 3.98 (q, 2H); 5.28 (s, 2H); 5.46 (dd, 2H); 6.06 (s, 1H); 7.37 (s, 1H); 7.84 (d, 1H); 8.35 (d, 1H).

NMR-C 13 (DMSO): 8.45; 15.50; 36.52; 45.59; 50.62; 57.36; 61.43; 73.33, 99.66; 112.29; 112.50; 122.67; 124.71; 126.99; 127.20; 127.44; 129.08; 140.16; 144.80; 148.82; 152.71; 155.89; 159.22; 160.75; 172.07.

IR (KBr): 1448; 1595; 1653; 1749; 2950; 3438.

Example 15: 5-ethyl-12-diethylaminomethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino3\4':6,7indolizino[1,2-b]quinoline3,15 (4H,13H)-dione

3-Fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinolinecarboxylate which was treated using diethylamine and then reduced to the corresponding quinolinemethanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light beige solid was obtained, m.p. > 270°C.

NMR-1 H (DMSO): 0.87 (t, 3H); 1.04 (t, 6H); 1.86 (q, 2H); 2.50 (q, 2H); 2.54 (s, 3H); 2.56 (q, 2H); 3.08 (d, 1H); 3.48 (d, 1H); 4.11 (q, 2H); 5.25 (s, 2H); 5.46 (dd, 2H); 6.05 (p. 1H); 7.35 (s, 1H); 7.80 (d, 1H); 8.36 (d, 1H).

NMR-C 13 (DMSO): 8.45; 11.68; 11.78; 15.43; 15.57; 36.5; 42.5; 46.68; 46.83; 46.99; 50.77; 51.85; 52.08; 61.44; 73.30; 99.60; 112.18; 112.36; 122.6; 124.6; 126.9; 127.1; 128.8; 141.45; 144.6; 148.6; 148.7; 152.65 155.9; 159.1; 160.7; 163.2; 172.1. IR (KBr): 1217; 1295; 1448; 1463; 1507; 1609; 1660; 1725; 2971; 3559. Suspension of the above free base in absolute ethanol (50 ml/mmol) followed by treatment with ethanolic hydrogen chloride (2.5 N, 5 eq.) enables the corresponding hydrochloride to be obtained. Initially a yellow solution is formed, then a precipitate which was collected by filtration after concentration to 40% in original volume, then washed with ether. A pale yellow solid was obtained, m.p. 269-272°C.

NMR-1 H (DMSO): 0.87 (t, 3H); 1.34 (m, 1H); 1.86 (q, 2H); 2.56 (s, 3H); 3.07 (d, 1H); 3.19 (m, 2H); 3.39 (m, 2H); 3.49 (d, 1H); 4.97 (m, 2H); 5.41 (d, 1H); 5.54 (d, 1H); 5.58 (s, 2H); 6.08 (s, 1H); 7.42 (s, 1H); 7.96 (d, 1H); 8.43 (d, 1H); 10.38 (p, 1H). IR (KBr): 1039; 1070; 1226; 1282; 1509; 1654; 1724; 1744; 2921; 3409; 3489.

Example 16: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methyl piperidinomethyl)-1H oxepino[3',4l:6,7]indolizino1,2 - b] quinoline-3,15(4H,13H)-dione

3-fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinoline carboxylate which was treated using 4-methylpiperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 1.00-0.80 (complex, 6H); 1.12 (q, 1H); 1.37 (s, 1H); 1.57 (d, 3H); 1.85 (q, 2H); 2.13 (t, 2H); 2.82 (s, 1H); 2.85 (s, 1H); 3.05 (d, 1H); 3.25 (s, 3H); 3.48 (d, 1H); 4.04 (q, 2H); 5.28 (s, 2H); 5.39 (d, 1H); 5.52 (d, 1H); 6.03 (s, 1H); 7.36 (s, 1H); 7.82 (d, 1H); 8.40 (d, 1H).

NMR-C 13 (DMSO): 0.29; 8.43; 13.68; 15.48; 19.40; 21.93; 23.23; 30.39; 34.20; 36.52; 42.55; 50.67; 53.84; 56.29; 57.67; 61.40; 73.32; 99.59; 112.49; 122.62; 124.80; 127.18; 129.10; 140.31; 144.58; 148.64; 152.69; 155.84; 159.19; 172.05. IR (KBr): 1597; 1653; 1747; 3446.

Example 17: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-pyrrolidinomethyl-1H-oxepino(3',4':6,7)indolizino[1,2- b) quinoline-3,15(4H,13H)-dione

3-fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinoline carboxylate which was treated using pyrrolidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.72 (s, 4H); 1.85 (q, 2H); 2.57 (s, 4H); 3.05 (d, 1H); 3.28 (s, 3H); 3.48 (d, 1H); 4.18 (q, 2H); 5.28 (s, 2H); 5.39 (d, 1H); 5.52 (d, 1H); 6.03 (s, 1H); 7.36 (s, 1H); 7.82 (d, 1H); 8.35 (d, 1H).

NMR-C 13 (DMSO): 0.37; 8.47; 15.57; 23.48; 36.53; 42.61; 50.61; 53.45; 54.09; 61.42; 73.33; 99.59; 112.37; 122.64; 124.51; 127.00; 127.25; 128.63; 140.65; 144.77; 148.65; 152.73; 155.87; 159.20; 162.00; 167.00; 172.07.

IR (Kflr): 1608; 1656; 1729; 3400.

Example 18: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-(1,2,5,6-tetrahydropyridinomethyl)-1H-oxepino(3',4': 6,7]indolizino [1,2-b]quinoline-3,15(4H,13H)-dione

3-Fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinolinecarboxylate which was treated using 1,2,5,6-tetrahydropyridine and then reduced to equivalent to quinoline methanol. The latter bee connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 2.08 (s, 2H); 3.03 (s, 2H); 3.05 (d, 1H); 3.28 (s, 3H); 3.48 (d, 1H); 4.12 (d, 1H); 5.28 (s, 2H); 5.39 (d, 1H); 5.52 (d, 1H); 5.64 (d, 1H); 6.03 (s, 1H); 7.36 (s, 1H); 7.83 (d, 1H); 8.36 (d, 1H).

NMR-C 13 (DMSO): 8.45; 15.54; 25.84; 36.54; 42.55; 49.78; 50.68; 52.52; 55.81; 61.42; 73.33; 99.62; 112.53; 122.66; 124.78; 125.03; 127.09; 127.19; 131.73; 139.98; 144.76; 148.79; 152.73; 155.86; 159.19; 160.76; 163.25; 172.07.

IR (KBr): 1605; 1656; 1733; 3451.

Example 19: 12-diisobutylaminomethyl-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1H-oxepino[3',4':6,7]indolizino[1,2 -b] quinoline-3,15(4H,13W)-dione

3-fluoro-4-methylaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methyl-3-quinoline carboxylate which was treated using diisobutylamine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.75 (d. 12 H); 0.87 (t, 3H); 1.83 (m, 4H); 2.15 (d, 1H); 2.48 (s, 3H); 3.06 (d, 1H); 3.47 (d, 1H); 4.01 (q, 2H); 5.28 (s, 2H); 5.39 (d, 1H); 5.53 (d, 1H); 6.03 (s, 1H); 7.37 (s, 1H); 7.83 (d, 1H); 8.49 (d, 1H).

NMR-C 13 (DMSO): 9.09; 16,14; 21.73; 26.57; 26.70; 37.15; 43.14; 51.05; 55.49; 62.08; 64.74; 73.98; 100.42; 113.03; 123.38; 125.58; 127.12; 127.32; 128.59;

130.27; 141.32; 145.51; 149.38; 149.51; 153.20; 156.62; 159.86; 161.31; 163.79; 172.72.

IR (KBr): 1599; 1656; 1747; 2796; 3448.

Example 20: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methyl piperazinomethyl)-1N-oxepino[3',4':6,7]indolizino 1,2-b]quinoline-3,15(4H,13H)-dione

3-Fluoro-4-methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methoxy-3-quinoline carboxylate which was treated and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light yellow solid was obtained, m.p. 274°C. NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 2.15 (s, 3H); 2.31 (m, 4H); 2.47 (m, 4H); 3.06 (d, 1H); 3.47 (d, 1H); 4.05 (m, 2H); 4.05 (s, 3H); 5.28 (s, 2H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.35 (s, 1H); 7.87 (d, 1H); 7.94 (d, 1H).

NMR-C 13 (DMSO): 8.44; 36.53; 45.58; 45.95; 50.68; 52.86; 55.07; 56.20; 56.47; 61.45; 73.32; 99.19; 105.90; 113.74; 113.91; 122.22; 125.60; 129.46; 138.83; 144.51; 144.62; 144.94; 147.85; 147.98; 150.96; 152.82; 155.34; 155.96; 159.19; 172.09.

IR (KBr): 1270; 1515; 1594; 1648; 1747; 2950; 3438.

Example 21: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidino methyl-1^oxepino[3\4':6,7]indolizino1,2-b]quinoline -3,15(4H,13H)-dione

3-Fluoro-4-methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-7-fluoro-6-methoxy-3-quinoline carboxylate which was treated using piperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A light green solid was obtained, m.p. > 275°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.42-1.50 (m, 6H); 1.84 (q, 2H); 2.50 (m, 4H); 3.05 (d, 1H); 3.48 (d, 1H); 4.03 (s, 2H); 4.05 (s, 3H); 5.30 (s, 2H); 5.45 (dd, 2H); 6.02 (s, 1H); 7.35 (s, 1H); 7.9 (d, 1H) 7.99 (d, 1H).

NMR-C 13 (DMSO): 8.44; 24.07; 25.9; 36.54; 42.57; 50.60; 54.26; 56.40; 57.11; 61.42; 73.33; 99.17; 105.97; 113.75; 113.92; 122.21; 125.66; 129.46; 139.23; 144.54; 144.98; 147.94; 151.0; 152.82; 155.34; 155.89; 159.20; 172.07. IR (KBr): 860; 1057; 1270; 1514; 1656; 1748; 2857; 2932; 3397.

Suspension of the above-mentioned free base in absolute ethanol (50 ml/mmol) followed by treatment with ethanolic hydrogen chloride (2.5 N, 5 eq.) enables the corresponding hydrochloride to be obtained. Initially a yellow solution is formed, then a precipitate which was collected by filtration after concentration to 40% in original volume, then washed with ether. A light yellow solid was obtained, m.p. 264°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.42 (m, 1H); 1.70-1.85 (m, 7H); 3.06 (d, 1H); 3.33 (m, 4H); 3.47 (m, 1H); 4.19 (s, 3H); 5.00 (p, 2H); 5.40 (d, 1H); 5.54 (d, 1H); 5.61 (s, 2H); 6.02 (s, 1H); 7.37 (s, 1H); 7.95-8.04 (m, 2H); 10.46(s, 1H).

NMR-C 13 (DMSO): 9.12; 22.11; 22.91; 37.63; 43.20; 52.27; 53.20; 54.00; 54.75; 57.91; 58.15; 62.12; 62.78; 73.97; 100.06; 106.96; 107.14; 114.80; 123.20; 126.58; 130.48; 134.14; 145.33; 145.48; 149.49; 149.62; 151.76; 153.84; 156.36; 156.69; 159.76; 172.73.

IR (KBr): 1010; 1072; 1240; 1271; 1469; 1511; 1574; 1598; 1648; 1734; 2525; 2944; 3430; 3507.

Example 22: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-dimethylaminomethyl-1H-oxepino[3',4':6,7]indolizino 1,2-b] quinoline-3,15(4H,13H)-dione

3-Chloro-4-methoxyaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methoxy-3-quinoline carboxylate which was treated using dimethylamine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.84 (q, 2H); 2.29 (s, 6H); 3.06 (d, 1H); 3.42 (d, 1H); 3.98 (q, 2H); 4.05 (s, 3H); 5.27 (s, 2H); 5.45 (s, 2H); 5.95 (s, 1H); 7.32 (s, 1H); 7.82 (s, 1H); 8.19 (p.1H).

NMR-C 13 (DMSO): 8.41; 36.50; 42.55; 45.58; 50.62; 56.70; 57.42; 61.42; 73.29; 99.28; 104.66; 122.34; 126.92; 127.55; 129.89; 130.04; 139.19 144.20; 144.81; 151.08; 153.15; 155.91; 159.18; 172.04.

IR (KBr): 1048; 1242; 1482; 1611; 1659; 1730; 3301; 3417.

Example 23: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidinomethyl-1H-oxepino[3',4,:6,7]indolizino[1, 2- b quinoline-3,15(4H,13H)-dione hydrochloride

3-Chloro-4-methoxyaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methoxy-3-quinoline carboxylate which was treated using piperidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. The free base thus obtained was suspended in absolute ethanol (50 ml/mmol) then treated

with ethanolic hydrogen chloride (2.5 N, 5 eq.)- Initially a yellow solution was formed, then a precipitate which was collected by filtration after concentration to 40% in original volume and washed with ether. An orange solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.43 (q, 1H); 1.70 (d, 1H); 1.76 (m, 2H); 1.86 (m, 4H); 3.07 (d, 1H); 3.28 (m, 2H); 3.47 (m, 3H); 4.20 (s, 3H); 5.00 (q, 2H); 5.41 (d, 1H); 5.54 (d, 1H); 5.62 (s, 1H); 6.10 (s, 1H); 7.36 (s, 1H); 7.88 (s, 1H); 8.31 (p, 1H). NMR-C 13 (CF 3 COOD): 8.44; 22.11; 24.79; 38.27; 43.51; 54.28; 56.01; 58.51; 58.75; 64.23; 77.59; 104.22; 110.49; 124.68; 129.44; 131.91; 136.61; 140.01; 141.33; 144.72; 158.25; 161.10; 161.89; 178.85.

IR (KBr): 1079; 1288; 1488; 1562; 1578; 1648; 1747; 2936; 3406.

Example 24: 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(1,2,5,6-tetrahydropyridinomethyl)-1H-oxepino3',4':6,7indolizino

(1,2-b]quinoline-3,15(4H,13H)-dione hydrochloride

4-Methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-6-methoxy-3-quinoline carboxylate which was treated using 1,2,5,6-tetrahydropyridine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. The free base thus obtained was suspended in absolute ethanol (50 ml/mmol) then treated with ethanolic hydrogen chloride (2.5 N, 5 eq.). Initially a yellow solution was formed, then a precipitate which was collected by filtration after concentration to 40% in original volume and washed with ether. A yellow solid was obtained, m.p.

>250°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.87 (q, 2H); 2.32 (m, 1H); 3.07 (d, 1H); 3.48 (m, 3H); 3.89 (m, 8H); 4.06 (s, 3H); 5.08 (m, 2H); 5.40 (d, 1H); 5.54 (d, 1H); 5.63 (q, 2H); 5.67 (d, 2H); 5.93 (d, 2H); 7.37 (s, 1H); 7.59 (q, 1H); 7.79 (d, 1H); 8.14 (d, 1H); 10.80 (p, 1H).

NMR-C 13 (DMSO): 8.47; 25.97; 36.40; 42.55; 49.75; 50.25; 50.61; 52.36; 56.05; 61.44; 73.36; 98.95; 103.74; 121.99; 122.29; 124.98; 125.50; 128.84; 129.84; 131.18; 138.47; 144.63; 145.18; 150.01; 155.93; 159.24; 172.10.

IR (KBr): 827; 1065; 1228; 1289; 1592; 1653; 1746; 2363; 3373.

Example 25: 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methyl piperidinomethyl)-1H-oxepino[3\4':6,7)indolizino[1,2-b ]quinoline-3,15(4H,13H)-dione

4-Methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-6-methoxy-3-quinolinecarboxylate which was treated using 4-methyl-piperidine and

then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (CF 3 COOD): 1.17 (m, 6H); 1.62 (m, 2H); 1.89 (s, 1H); 2.07 (q, 2H); 2.25 (m, 2H); 3.54 (m, 3H); 3.89 (d, 1H); 4.02 (s, 2H); 4.19 (s, 3H); 7.94 (s, 1H); 8.10 (m, 1H); 8.29 (s, 1H); 8.50 (m, 1H).

NMR-C 13 (CF 3 COOD): 8.43; 13.79; 17.43; 20.89; 30.01; 32.85; 38.26; 43.50; 54.13; 56.09; 57.87; 58.27; 64.22; 77.57; 107.37; 110.56; 125.75 129.36; 129.42; 132.78; 136.04; 136.65; 139.91; 140.38; 144.31; 158.30; 161.94; 164.90; 178.84. IR (KBr): 825; 1056; 1230; 1260; 1516; 1641; 1655; 1736; 2921; 3395.

Example 26: 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methyl piperazinomethyl)-1H-oxepino[3',4':6,7indolizino[1,2-b ]quinoline-3,15(4H,13H)-dione

4-Methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-6-methoxy-3-quinoline carboxylate which was treated and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. 215-219X. NMR-1 H (DMSO): 0.85 (t, 3H); 1.85 (m, 2H); 2.15 (s, 3H); 2.35 (m, 4H); 2.5 (m, 4H); 3.25 (dd, 2H); 3.95 (s, 3H); 4.05 (s, 2H); 5.3 (s, 2H); 5.45 (dd, 2H); 6 (p, 1H); 7.3 (s, 1H); 7.5 (d, 1H); 7.7 (s, 1H); 8.05 (d, 1H).

NMR-C 13 (DMSO): 9.12; 14.36; 20.08; 23.93; 46.61; 51.35; 53.58; 55.71; 56.34; 56.73; 58.37; 62.11; 74.03; 99.62; 104.49; 122.66; 123.11; 129.54; 130.53; 131.82; 139.05; 145.3; 145.86; 150.67; 156.62; 158.71; 159.91; 172.77.

IR (KBr): 1590; 1624; 1655; 1744; 2801; 2935; 3423.

Example 27: 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-pyrrolidinomethyl-

1 H-oxepino3\4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 4-methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl -6-methoxy-3-quinoline carboxylate which was treated using pyrrolidine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p.

> 250°C.

NMR-1 H (DMSO): 0.85 (t, 3H); 1.7 (s, 4H); 1.85 (q, 2H); 2.55 (s, 4H); 3.25 (dd, 2H); 3.9 (s, 3H); 4.15 (s, 2H); 5.25 (s, 2H); 5.45 (dd, 2H); 6 (p, 1H); 7.35 (s, 1H); 7.5 (d, 1H); 7.7 (s, 1H); 8.05 (d, 1H).

NMR-C 13 (DMSO): 9.68; 24.74; 51.8; 54.71; 55.25; 56.3; 56.87; 62.3; 62.64; 74.5; 100.14; 104.8; 104.92; 123.19; 123.45; 129.79; 130.49; 132.32; 132.50; 140.5; 145.83; 146.4; 151.27; 157.15; 159.25; 160.45; 173.3.

IR (KBr): 1255; 1516; 1535; 1613; 1655; 1735; 3438; 3762; 3830.

Example 28: 12-(4-benzylpiperazinomethyl)-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino[3',4':6,7]indolizino[1,2- bquinoline-3,15(4H,13H)-dione

4-Methoxyaniline was used to produce ethyl 2-chloro-4-chloromethyl-6-methoxy-3-quinoline carboxylate which was treated using N-benzyl-piperazine and then reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A beige solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.45 (s, 2H); 2.4 (m, 4H); 2.55 (m, 4H); 3.25 (dd, 2H); 3.45 (s, 2H); 3.95 (s, 3H); 4.05 (s, 2H); 5.3 (s, 2H); 5.45 (dd, 2H); 6 (p, 1H); 7.3 (m, 6H); 7.5 (d, 1H); 7.75 (s, 1H); 8 (d, 1H).

NMR-C 13 (DMSO): 7.38; 49.56; 51.89; 54.46; 54.82; 54.98; 55.1; 60.1; 60.35; 61.11; 72.26; 97.86; 102.6; 102.76;;120.9; 121; 121.2; 121.4; 126; 127.25; 127.77; 127.88; 128.76; 130.13; 130.2; 137.25; 137.36; 143.53 144.08; 148.86; 156.86; 156.95; 158.15; 171.02.

IR (KBr): 1235; 1259; 1517; 1586; 1614; 1654; 1747; 2927; 3450; 3762; 3848.

Suspension of the above free base in absolute ethanol (50 ml/mmol) followed by treatment with ethanolic hydrogen chloride (2.5 N, 5 eq.) enables the corresponding hydrochloride to be obtained. Initially a yellow solution is formed, then a precipitate which was collected by filtration after concentration to 40% in original volume, then washed with ether. A yellow solid was obtained, m.p. > 250°C.

NMR-1 H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 2.5 (s, 2H); 2.65 (m, 2H); 3 (m, 2H); 3.2 (m, 2H); 3.35 (dd, 2H); 3.35 (s, 2H); 3.95 (s, 3H); 4.15 (s, 2H); 4.3 (s, 2H); 5.3 (s, 2H); 5.45 (dd, 2H); 7.3 (s, 1H); 7.4 (s, 2H); 7.55 (m, 2H); 7.7 (s, 1H); 8.05 (d, 1H); 10.45 (p, 1H). IR (KBr): 1207; 1233; 1439; 1449; 1458; 1508; 1610; 1620; 1655; 1727; 3398.

Example 29: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methylpiperidinomethyl)-1H-oxepino3',4<*>:6,7indolizino[1, 2-b]

quinoline-3,15(4H,13H)-dione

3-chloro-4-methylaniline was used to produce ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinolinecarboxylate which was treated using piperidine and then

reduced to the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p.

> 275X.

NMR-1 H (DMSO): 0.86 (m, 6H); 1.15 (m, 2H); 1.37 (m, 1H); 1.60 (m, 2H); 1.80 (m, 2H); 2.10 (m, 2H); 2.60 (s, 3H); 2.80 (m, 2H); 3.05 (d, 1H); 3.48 (d, 1H); 4.02 (s, 2H); 5.30 (s, 2H); 5.45 (dd, 2H); 6.02 (s, 1H); 7.40 (s, 1H); 8.20 (s, 1H); 8.40 (p, 1H). NMR-C 13 (DMSO): 9.10; 21.28; 22.61; 31.07; 34.89; 37.18; 43.22; 54.53 56.83; 62.10; 73.94; 80.06; 100.43; 123.41; 127.08; 129.11; 130.58; 135.88; 136.89; 141.00; 145.28; 148.49; 153.51; 156.60; 159.85; 172.77; 174.05.

IR (KBr): 1605; 1657; 1734; 3342.

Example 30: 10-benzyloxy-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino

3\4':6,7]indolizino 1,2-to]quinoline-3,15(4H,13H)-dione 3-fluoro-4-methoxy-acetanilide was used to produce ethyl 2-chloro-7-fluoro- 6-Methoxy-quinoline-3-carbaldehyde which was treated with an excess of boron tribromide in dichloromethane at ambient temperature for 24 hours. 2-chloro-7-fluoro-6-hydroxy-quinoline-3-carbaldehyde was obtained, was O-benzylated in dimethylformamide in the presence of benzyl bromide and in potassium carbonate to produce

6-benzyloxy-2-chloro-7-fluoro-quinoline-3-carbaldehyde, which was reduced with sodium borohydride in methanol to produce the corresponding quinoline methanol. The latter was connected to compound (M). The resulting coupled product was cyclized. A yellow solid was obtained, m.p. > 275°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 3.05 (d, 1H); 5.25 (s, 2H); 5.37 (s, 2H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.4-7.6 (m, 5H); 7.88 (d, 1H); 7.95 (d, 1H); 8.56 (p, 1H).

Example 31: 5-ethyl-9-fluoro-4,5-dihydro-5,10-d i hydroxy-1 H -oxepino [3\4':6,7]

indolizino [1,2-6]quinoline-3,15(4H,13H)-dione

The hydrogenolysis procedure was applied to the compound of Example 30. A yellow solid was obtained, m.p. > 275°C.

NMR-1 H (DMSO): 0.86 (t, 3H); 1.85 (q, 2H); 3.05 (d, 1H); 5.25 (s, 2H); 5.37 (s, 2H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.8 (d, 1H); 7.90 (d, 1H); 8.56 (p, 1H).

PHARMACOLOGICAL STUDY OF THE PRODUCTS

1. Relaxation activity assay of DNA induced by topoisomerase 1.

All reactions were performed in a 20 µl reaction buffer consisting of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.5 mM dithiothreitol, 10 mM MgCl2, 0.1 mM ethyldiamine tetra-acetic acid (EDTA), 30 ug/ml bovine serum albumin and 300 ng superspun pUC19 (Pharmacia Biotech, Orsay, France) with or without the compounds to be tested at defined concentrations. All the compounds to be tested were initially dissolved in dimethyl sulfoxide (DMSO) at 50 mM, other dilutions were made with distilled water. The final concentration of DMSO does not exceed 1%

(v/v). The reaction was initiated by the addition of one unit of DNA topoisomerase 1 in purified calf thymus (Gibco-BRL, Paisley, United Kingdom) and was carried out for 15 minutes at 37°C. The reactions were stopped by the addition of 3 µl of a mixture containing 1% dodecyl sodium sulfate at 1%, 20 mM in EDTA and 500 µg/ml K proteinase (Boehringer Mannheim, Meylan, France). After a further incubation period of 30 minutes at 37°C, 2 µl of an application buffer containing 10 mM Na 2 HPO 4 , 0.3% bromophenol blue and 16% Ficoll were added to samples which were subjected to electrophoresis in agarose gels at 1.2% at 1 V/cm for 20 hours in a buffer containing 36 mM Tris-HCl at pH 7.8, 30 mM Na 2 HPO 4 , 1 mM EDTA and 2 µg/ml chloroquine. Gels were labeled with 2 µg/ml ethidium bromide, photographed under UV light at 312 nm with a camera and the fluorescence intensity was measured with a bioProfil camera (Vilber Lourmat, Lyon, France) with an image to determine the percentage of relaxed DNA. Each experiment was performed at least three times in duplicate.

In each experiment, supercoiled plasmid DNA was incubated alone or with topoisomerase 1. The reaction was completed within 15 minutes. In each compound to be tested or in the control, supercoiled plasmid DNA was incubated in the presence of 500 pM of compound to be tested with enzyme or without enzyme plus the compound to be tested, at the concentrations of 10 pM, 100 uM,

200 uM and 500 pM.

2. Test for cell proliferation

a. Eight tumoral cell lines are used in this study: L1210 (mouse lymphocytic leukemia), HCT15 and LOVO (human intestinal adenocarcinoma cell lines), A549 (human lung carcinoma), A172, U373 and U87 (human glioblastoma). All these lines were obtained from the American Type Culture Collection (ATCC),

Rockville, Md. L1210 cell cultures in suspension are grown in Dulbecco's modified Eagle's medium (DMEM) (BioWhitaker, Verviers, Belgium) together with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. HT29 cells are grown in monolayer cultures in a McCoy 5a medium (Gibco, Paisley, United Kingdom) together with 10% heat-inactivated fetal calf serum plus 2 mM glutamine and 50 µg/ml gentamycin. The other cells are cultured in Earle's modified essential medium (EMEM; Gibco, Paisley, United Kingdom) together with 5% heat-inactivated fetal calf serum, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. All cell lines are grown at 37°C in a humidified atmosphere containing 95% air and 5% CO2.

Inhibition of tumor cell line proliferation is determined using an MTT test. 1500 L1210 cells in a culture medium (according to the needs of the cell medium) are seeded in a well of a microwell dish (tissue culture level: 96 wells, flat-bottomed) 24 hours before treatment with the compounds to be tested. In these dose-response studies, the cells are incubated with each of the compounds to be tested or their corresponding dissolution media (controls) for 48 hours over a total concentration range of 1.10"<10> to 1.10"4 M. All compounds are dissolved just before use in dimethyl sulfoxide (DMSO) at a concentration of 50 mM. Other diluents of the drugs are carried out in culture medium. The final concentration of DMSO never exceeds 0.2% (v/v). As the controls, the solutions of drugs are replaced with the solubilizer which is successively diluted in the same manner as the compounds to be tested.

After the incubation period, the labeling reagent MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazol blue, Sigma M 565, Sigma, St Louis, MO) is added to a final concentration of 0, 3 mg/ml to each well. The cells are incubated for 4 hours at 37°C in a humidified atmosphere. This stage enables the mitochondrial dehydrogenase of living cells to convert the yellow tetrazolium salt MTT into crimson formazan crystals. The supernatant part is eliminated and the formazan crystals that are formed are dissolved with DMSO. Resulting colored solution is quantified by absorbance at 570 nm using a multi-cuvette scanning spectrophotometer. Data regarding proliferation are expressed as a percentage of live cells in treated wells, divided by live cells in the controls. Each point represents the mean of three independent experiments, each experiment representing six determinations.

For all the other cell lines (HCT15, LOVO, A549, A172, U373, U87), 1000 to 2000 cells are seeded into the well of a micro-well dish 24 hours before the medical treatment. They are incubated with each of the compounds to be tested or their corresponding dissolution media (controls) for 72 hours over a final concentration range of 1.10-<10> to 1.10"6 M.

The results were expressed as percentages of proliferation calculated from the optical density (OD) of the cells treated with a drug divided by the OD of the control cells (the cells treated with DMSO).

Nine tumoral cell lines are used in this study: PC3, DU 145 (human prostate cell lines), MCF7 and MCF7-ADR (breast lines, the symbol "ADR" is used to indicate that the line has been made adriamycin resistant), A427 (human lung adenocarcinoma), HT29 (human intestinal adenocarcinoma cell line), T24s, T24r (human bladder cell line, T24r's are resistant to adriamycin, and others). PC3, DU145 and A427 lines have been obtained from the American Type Culture Collection (ATCC, Rockville, Md). The MCF7 and MCF7-ADR cells were donated by Dr Jacques Soudon (Pharmacell, Paris, France). The T24s and T24r cells were donated by Dr Robert Kiss (Free University in Brussels, Belgium). HT29 cells were grown in monolayer cultures in a 4.5 g/l DMEM medium (Gibco, Paisley, United Kingdom) supplemented with 10% heat-inactivated fetal calf serum plus 2 mM glutamine and 50 g/ml gentamycin (Gibco, Paisley, United Kingdom) . The other cells are cultured in an Earle's modified essential medium DMEM at 4.5 g/l (Gibco, Paisley, United Kingdom) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine (Gibco, Paisley, United Kingdom), 50 U /ml penicillin and 50 g/ml streptomycin (BioWhitaker, Verviers, Belgium). All cell lines are grown at 37°C in a humidified atmosphere containing 95% air and 5% CO2.

Inhibition of tumor cell line proliferation is determined using a WST1 colorimetry assay. 500 to 4000 cells in a culture medium (according to the needs of the cell medium) were seeded into a well of a microwell dish (96 wells, flat bottom) 24 hours before treatment with the compounds to be tested. In these concentration-response studies, the cells are incubated with each of the compounds to be tested or their corresponding solvent (control) for 72 hours over a final concentration range of 1x10-13 to 1x10-5 M. All compounds are dissolved in dimethyl sulfoxide ( DMSO) or in water to water-soluble compounds. The following dilutions of the compounds according to the present invention were carried out in a culture medium so that the final concentration of DMSO, when it is part of the composition of the carrier, is always 0.1% (v/v). As the controls, solutions of the compounds are replaced with the solvent which is successively diluted in the same manner as the compounds to be tested.

After incubation, the labeling reagent WST1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2/j-5tetrazolio-1,3-benzene) (Boehringer, Mannheim, Germany) is added to a final concentration of 9% to each well. The cells are incubated for 2 to 4 hours at 37°C in a humidified atmosphere. This stage enables the mitochondrial dehydrogenase of living cells to convert the orange tetrazolium salt WST1 into crimson formazan crystals. The resulting colored solution is quantified by a dual beam reading (450 and 690 nm) using a multi-cuvette scanning spectrophotometer.

The results are expressed in the form of a concentration table, expressed in mol/litre, including the 50% inhibitory concentration (IC50). They are shown in tables III A) and UIB). Examples where the number is followed by an "s" correspond to salts of the compound. Cpt, Adr and Tpt are abbreviations for camptothecin, adriamycin and topotecan respectively.

Claims (4)

1. Compound, characterized in that it is selected from the compounds: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(1,2,5,6-tetrahydopyridinomethyl- 1H-oxepino[3,,4':6,7]indolizino[1,2-D]quinoline-3,15(4H,13H)-dione hydrochloride 5-ethyl-9,10-difluoro-4,5-dihydro- 5-hydroxy-12-(4-methyl piperidinomethyl)-1 H- oxepino^^ÆJjindolizinotl^-b] quinoline-3,15(4H,13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-pyrrolidinomethyl-1H-oxepino- ^'^'le.yiindolizinoll ,2-o]quinoline-3,15(4H, 13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methylpiperazinomethyl -1 H- oxepino[3,,4':6,7]indolizino[1, 2- b ] quinoline-3,15(4H, 13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5- hydroxy-12-piperidinomethyl-1H-oxepino- [3',4':6,7]indolizino[1,2-o]quinoline-3,15(4H,13H)-dione 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy -12-dimethylamino-methyl-1/-/- oxepino(3',4,:6,7]indolizino[1,2-6]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy- 10-methyl-12-morpholino methyl-1 H- oxepino[3',4':6,7]indolizino[1,2-/>]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy -10-methyl-12-(4-methylpiperazinomethyl)-1H- oxepino(3',4,:6,7]indolizino[1,2-£>]quinoline-3,15(4H,13H)-dione 12-benzylpiperazinomethyl-9-chloro-5-ethyl-4,5-dihydro -5-hydroxy-10-methyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 12-(4-benzylpiperazinomethyl)-9-chloro-5-ethyl-4, 5-dihydro-5-hydroxy-10-methyl-1H-oxepino^^-^.^indolizinotl ,2-o]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4, 5-dihydro-5-hydroxy-10-methyl-12-piperidinomethyl-1H-oxepino- (3',4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4,5 -dihydro-5-hydroxy-1 H- oxepino[3',4,:6,7]indolizino[1,2-t>]quinoline-3,15(4H,13H)-dione 12-(4-benzylpiperazinomethyl)-5-ethyl-9-fluoro-4 ,5-dihydro-5-hydroxy-10-methyl-1H-oxepinoI^^VSJlindolizinotl ,2-o]quinoline-3,15(4H, 13H)-dione 5-ethyl-9-fluoro-4,5-dihydro -5-hydroxy-10-methyl-12-dimethylaminomethyl-1 H- oxepino[3,,4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-12-diethylaminomethyl-9-fluoro-4,5-dihydro- 5-hydroxy-10-methyl-1 H- oxepino[3',4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy- 10-methyl-12-(4-methylpiperidinomethyl)-1H- oxepino[3',4:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10 -methyl-12-pyrrolidinomethyl-1 H- oxepino[3,,4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro -5-hydroxy-10-methyl-12-(1,2,5,6-tetrahydro- pyridinomethyl)-1H-oxepino[3\4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 12-diisobutylaminomethyl-5-ethyl-9-fluoro-4 ,5-dihydro-5-hydroxy-10-methyl-1 H- oxepino^.^ejlindolizinoll ,2-b]quinoline-3,15(4H, 13H)-dione 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methylpiperazinomethyl )-1H-oxepino[3,,4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-9-fluoro-4,5-dihydro -5-hydroxy-10-methoxy-12-piperidino methyl-1H- oxepino[3,l4,:6l7]indolizino[1I2-b]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12 -dimethylaminomethyl-1 H- oxepino^^veyjindolizinoll ,2-b]quinoline-3,15(4H,13H)-dione 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidinomethyl-1H- oxepino^^^yiindolizinoll ,2-b]quinoline-3,15(4H, 13H)-dione hydrochloride 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(1,2,5,6 -tetrahydropyridino- methyl)-1H-oxepino[3,,4':6,7]indolizino[1,2-b]quinoline-3,15(4H13H)-dione hydrochloride 5-ethyl-4,5-dihydro-5- hydroxy-10-methoxy-12-(4-methylpiperidinomethyl)-1 H- oxepino[3,,4,:6)7]indolizino[1l2-b]quinoline-3,15(4H,13H)-dione 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12- (4-methylpiperazinomethyl)-1 H- oxepino[3,,4,:6,7]indolizino[1,2-b]quinoline-3,15(4/-/ 13H)-dione 5-ethyl-4,5-dihydro-5-hydroxy-10- methoxy-12-pyrrolidinomethyl-1H-oxepino- [3,,4,:6,7]indolizino[1,2-b]quinoline-3l15(4H,13H)-dione 12-(4-benzylpiperazinomethyl)-5-ethyl-4,5-dihydro-5-hydroxy -10-methoxy-1 H- oxepino[3,,4',:6,7]indolizino[1,2-b]quinoline-3,15(4H,13/-/)-dione 9-chloro-5-ethyl-4,5-dihydro- 5-Hydroxy-10-methyl-12-(-4-methylpiperidinomethyl)-1H-oxepino[3,,4,:6,7]indolizino[1,2-b]quinoline-3115(4H113H)-dione; 10- benzyloxy-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3',4,:6,7]- indolizino[1,2-b]quinoline-3115(4H,13H)-dione; 5-ethyl-9-fluoro-4,5-dihydro-5,10-dihydroxy-1 /-/-oxepino[3',4':6,7]indolizino [1,2-b]quinoline-3,15(4H, 13ft)-dione; or a pharmaceutically acceptable salt thereof. 2. Compound according to claim 1, characterized in that it is selected from the compounds: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-12-(4-methylpiperidinonethyl)-1H- oxepino[3,4':6,7]indolizino[1,2nb]quinoline-3,15(4H,13H)-dione; 5-ethyl-12-diethylaminomethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1 H- oxepino[3,,4,:6,7]indolizino[1,2-ft]quinoline-3,15(4H,13H)-dione; 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methylpiperidinomethyl)-1 H- oxepino[3',4,:6,7]indolizino[1,2-t>]quinoline-3,15(4H,13H)-dione; 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-12-pyrrolidinomethyl-1 H- oxepino[3,14,:6,7]indolizino[1,2-ft]quinoline-3,15(4H,13H)-dione; 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-piperidinomethyl-1 H- oxepino[3,,4,:6,7]indolizino[1,2-6]quinoline-3115(4H,13H)-dione hydrochloride; 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-12-(4-methylpiperidinomethyl)-1 H- oxepino[3,,4,:6,7]indolizino[1,2-/]quinoline-3,15(4H,13H)-dione; 9-Chloro-5-ethyl 1-4,5-dihydro-5-hydroxy-10-methyl-12-(4-methylpiperidinomethyl)-1H- oxepino[3,4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione; or a pharmaceutically acceptable salt thereof. 3. Medicine, characterized in that it contains a compound according to any of the preceding claims or a pharmaceutically acceptable salt thereof. 4. Use of a compound according to any one of claims 1 to 2 for the production of anti-tumoral drugs.