NO324973B1 - Camptothecin compounds and their use and preparation, drug and pharmaceutical composition. - Google Patents

Description

The present invention relates to camptothecin compounds as well as their use and production, drug and pharmaceutical composition.

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 indolizinope ,2-b]quinoline fragment (rings A, B, C and D) fused to a six-membered α-hydroxylactone (ring E). Carbon at position 20 bearing the α-hydroxy group is asymmetric and exhibits a rotatory force of The natural form of camptothecin has an absolute "S" configuration at carbon 20 and corresponds to the following formula:

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

Camptothecin and certain analogues thereof are not water-soluble, and this makes their administration by the parenteral route difficult. Water-soluble derivatives of camptothecin have been prepared in which rings A and B carry salt-forming substituents Qrnf. for example US 4,981,968, US 5,049,668, EP 540,099). These products have shown an antitumoral activity which was reduced with respect to that of the non-hydro-soluble derivatives. Other water-soluble derivatives of camptothecin have also been prepared where the hydroxyl group in position 20 is esterified with an acid that carries a salt-forming residue such as, for example, glycine (cf. US Patent No. 4,943,579 and PCT No. WO 96/02546). These derivatives are constructed by a person skilled in the art under the name "prodrug forms" in that they are not biologically active in themselves, but only after a first metabolizing phase when administered to the patient. The prodrug forms of α-hydroxylactone analogues of camptothecin have shown a good anti-tumoural effectiveness in animals and clinically, but are accompanied by harmful side effects such as the occurrence of severe diarrhea which may endanger the patient's life. It is therefore necessary to develop water-soluble analogues of camptothecin which are more effective and which can be better tolerated.

It has further been indicated that α-hydroxylactone has an absolute requirement for both in vivo and in vitro activity of camptothecins (Camptothecins: 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)). On the other hand, the applicant has discovered that β-hydroxylactones with 7 members have a biological activity that is comparable to or higher than α-hydroxylactones (unpublished PCT Application No. FR 96/00980). The present invention relates to new derivatives from this class of analogues of camptothecin, where a β-hydroxylactone with 7 members replaces the natural α-hydroxylactone of camptothecin. By p-hydroxylactone is meant a lactone comprising an additional carbon atom between the carbon of carboxyl and the a-carbon which carries the hydroxyl in a-hydroxylactone.

Two solutions were chosen to increase the water solubility of camptothecin analogues: the first consists in grafting an oxazine onto the ring of the molecule, and the second construction of the prodrug form by acetylation of the hydroxy function to p-hydroxylactone.

From this new class of camptothecin analogues, the compounds according to the present invention are either analogues modified by fixing an oxazine ring on carbon atoms 10 and 11 or prodrug forms where a p-hydroxylactone replaces the natural a-hydroxylactone of camptothecin. Compounds according to the present invention are therefore camptothecin analog p-hydroxylactones on an oxazine ring or hydrosoluble prodrugs have been grafted and provide a powerful biological activity that is unexpected in light of the state of the art.

A particular object according to the present invention is a compound characterized in that said compound has formula (I):

in racemic or enantiomeric form or any combination of these forms, wherein

R 1 represents the ethyl group;

R 2 represents H or halogen;

R3 represents H, lower alkyl, halogen or OR6 where R6 represents H,

lower alkyl or lower arylalkyl;

R 4 represents H or -(CH 2 )mNR 6 R 7 , where R 6 and R 7 represent, independently,

H or lower alkyl;

R 5 represents H, lower alkyl or -(CH 2 )n[N«X], substituted or unsubstituted and [N«X] represents piperidyl, morpholinyl, piperazinyl or indolyl group and said substituent is a lower alkyl;

or R3 and R4 form an oxazine ring optionally substituted on the nitro atom with

an Rg residue representing a lower alkyl;

Rp represents H or an easily cleavable group of formula -C(O)-(Ai)-NH2 where

A-\ represents (CH 2 ) m or a branched lower alkylene radical;

m is a number between 0 and 6;

n is 1 or 2; and

it is understood that when Rp is a hydrogen atom, R3 and R4 together form an optionally substituted oxazine ring,

and when the designation lower is associated with alkyl or alkylene, it means no more than 6 carbon atoms;

or a pharmaceutically acceptable salt thereof.

As used herein, the term lower with reference to alkyl, alkylthio and alkoxy groups denotes linear or branched saturated aliphatic hydrocarbon groups containing 1 to 6 carbon atoms, such as, for example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, methylthio, ethylthio , methoxy and ethoxy. With reference to alkenyl or alkynyl groups, the term denotes lower groups containing 2 to 6 carbon atoms and one or more double or triple bonds, such as, for example, vinyl, alkyl I, isopropenyl, pentenyl, hexanyl, ethynyl propenyl, propynyl and butynyl groups. The term cycloalkyl indicates a ring with 3 to 7 carbon atoms, such as, for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl groups. The term aryl denotes a mono-di- or tricyclic hydrocarbon compound with at least one aromatic ring, each ring containing a maximum of 7 members, such as, for example, phenyl, naphthyl, anthracyl, biphenyl or indenyl. The term halogen indicates chlorine, bromine, iodine or fluorine. The residues corresponding to the terms lower haloalkyl, lower cyanoalkyl, lower nitroalkyl, lower amidoalkyl, lower hydrazinoalkyl, lower azidoalkyl, lower arylalkyl, lower hydroxyalkyl, lower alkoxy-lower alkyl, lower alkylthio lower alkyl and lower alkyl-lower sulfonylalkyl are substituted, respectively, with one to three halogen, cyano, nitro, amido, hydrazino, azido, aryl, hydroxy, lower alkoxy, lower alkylthio or lower sulfonylalkyl groups. Lower alkylamino residue may contain one or two lower alkyl groups and represent, for example, NHCH3, NHChtøCHs, N(CH3)2 or N(CH3)(CH2CH3). The term free, esterified or salt-formed hydroxy indicates OH, OCOR26. OR27 groups and alcoholate salt.

The compounds according to the present invention have two possible enantiomeric forms, ie under "R" and "S" configurations. The present invention encompasses two enantiomeric forms and any combination of these forms, including "RS" racemic mixtures. In an attempt to simplify the preparation, when no specific configuration is indicated in the structural formulas it should be understood that two enantiomeric forms and their mixtures are represented.

Examples of substituted camptocins used as starting compounds can be found

in US Patents Nos. 4,473,692, 4,604,463, 4,894,956, 5,162,532, 5,395,939, 5,315,007, 5,264,579, 5,258,516, 5,254,690, 5,212,317 and 5,341,745, US PCT Application No. 91/ 08028, US94/06451, US90/05172, US92/04611, US93/10987, US91/09598, EP94/03058 and EP95/00393 and European Patent Applications Nos. 325 247, 495 432, 321 122 and 540 099.

The present invention further relates to a process for the preparation of compounds of formula la similar to the products of formula I where R3 and R4 form an oxazine ring according to any one of claims 1, 2 or 4, characterized in that a B-hydroxylactone compound of a general formula D where R 3 is a hydroxyl radical, R 4 is H, and R 1 and R 2 have the meaning indicated above is treated with primary amino, under Mannich's conditions, to obtain a B-hydroxylactone compound of general formula la

where R1, R2, R5 and R9 have the meanings given above.

This process consists in heating the starting compound in the presence of a primary amine such as benzylamine, formaldehyde in an acidic solvent such as acetic acid or propionic acid at a temperature of 30°C to 80°C for a period of 0.5 to 5 hours. Alternatively, a suspension of the starting compound in acetic acid with a tri-A/-substituted hexahydrotriazine such as hexahydro-1,3,5-trimethyltriazine, 1,3,5-triethylhexahydrotriazine or 1,3,5-tribenzylhexahydrotriazine can be heated at a temperature of 30°C to 80°C for a period of 0.5 to 5 hours.

The present invention further relates to a method for producing compounds of formula Ib corresponding to the products of formula I where Rp is not a hydrogen atom, according to any one of claims 1, 2, 3 or 5, characterized in that

the compound of general formula D

or let

is acylated with a derivative of the -C(0)-Ai-NH2 residue as defined in claim 1, to produce the B-hydroxylactone compound of general formula I wherein Rp is different from H.

In the above process, the R 2 , R 3 , R 4 and R 5 groups can be protected if necessary according to standard methods of protection (Green, T., Protective Groups in Organic Synthesis 10-86 (John Wiley & Sons 1981)). A protecting group commonly used for the amines is tert-butyloxycarbonyl (BOC). The acylation reactions are then carried out as described above, then protecting groups are cleaved, for example by treatment with trifluoroacetic acid in the case of BOC, to produce the compound of general formula (I). The use of protecting groups is known to those skilled in the art (for other examples, reference may be made to Green, T., Protective Groups in Organic Synthesis, John Wiley & Sons, 1981).

The compounds of formula D are prepared as follows:

- a compound of the general formula M

is coupled to 2-halo-3-quinoline-methanol of the general formula N where Pt2, R3, R4 and R5 are as defined above and X represents a halogen atom, to produce the compound of formula O - then the compound of the general formula becomes O cyclized to obtain the compound of general formula D as defined above.

In the above-mentioned method, R-| t The R 2 , R 3 and R 4 groups are protected if necessary according to standard procedures for protection (Green. T., Protective Groups in Organic Synthesis 10-86 (John Wiley & Sons 1981)). The formation of compound O starting from the compounds of the general formulas M and N is carried out by a procedure known to those skilled in the art under the name Mitsunobu's reaction (reference is made to Mitsunobu, O. et al. Synthesis, p,1 (1981)) . The hydroxyl function of compound N is replaced by a nucleophile such as compound M or a deprotonated derivative of the latter, by treatment with a phosphine, for example triphenylphosphine and an azodicarboxylate derivative, for example diethyl azodicarboxylate, in an aprotic solvent such as tetrahydrofuran or A/,A/-Dimethylformamide. Cyclization of compound O is preferably carried out in the presence of a palladium catalyst (for example palladium diacetate) under basic conditions (provided for example by an alkaline acetate optionally combined with a phase transfer agent, such as tetrabutylammonium bromide), in an aprotic solvent such as acetonitrile or /V,/V-dimethylformamide, at a temperature consisting of between 50°C and 120°C (R. Grigg et al., Tetrahedron 46, page 4003 (1990)).

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. Salts formed from bases such as sodium or potassium hydroxide also form part of the scope of 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 present invention further relates to a drug, characterized in that it comprises a compound according to any of the preceding claims or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition is further described, characterized in that it comprises, as active ingredient, at least one of the compounds according to any one of claims 1 to 5.

The invention further relates to the use of a compound according to any one of claims 1 to 5 for the production of antitumor drugs.

The compounds according to the present invention consequently have useful pharmacological properties. The compounds according to the present invention have an inhibitory effect on topoisomerase I and/or II and an anti-tumoral activity. The state of the art suggests that the compounds according to the invention have an anti-parasitic and/or anti-viral activity. The compounds according to the present invention can also be used in various therapeutic applications.

Below, in the experimental part, an illustration of the pharmacological properties of the compounds according to the invention is shown.

The compounds can inhibit topoisomerase, for example type I and/or II, in a patient, for example a mammal such as a human, by administering to the patient a therapeutically effective amount of a compound of formula (I) or (II).

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 (II). Examples of tumors or cancers include cancer of the fallopian tube, stomach, intestine, rectum, oral cavity, pharynx, throat, lung, intestine, breast, cervix uteri, corpus endometrium, ovaries, prostate, testes, bladder, kidneys, liver, pancreas, bones, 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.

They can also be used for the treatment of parasitic infections by inhibiting haemoflagellates (for example in trypanosomiasis or leishmania infections) or by inhibiting plasmodia (also for example in malaria), but also the treatment of viral infections and diseases.

These properties make the products with formulas (I) or (II) suitable for pharmaceutical use.

The present invention therefore relates to pharmaceutical compositions. These may be in combination with a pharmaceutically acceptable carrier according to the chosen method of administration (for example oral, intravenous, intra-peritoneal, intramuscular, trans-dermal or sub-cutaneous). The pharmaceutical composition (for example therapeutic) can be in solid, liquid, liposome or lipidic micelle form.

The pharmaceutical composition may be in solid form, for example powder, pills, granules, tablets, liposomes, gelatin capsules or suppositories. the pill,

the tablet or gelatin capsule may be converted into a compound capable of protecting the composition from the action of gastric acid or enzymes in the stomach of the individual for a sufficient period of time to enable this composition to pass in an undigested form into the small intestine of the latter. The compound can also be administered locally, for example, in 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 can be, for example, calcium phosphate, magnesium stearate, magnesium carbonate, talc, sugar compounds, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine and wax. Pharmaceutical compositions containing a compound according to the invention can also be presented in liquid form such as 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 invention intended for the treatment of 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 will be decided by the attending physician or veterinarian. Such an amount determined by the attending physician or veterinarian is referred to herein as "effective therapeutic amount".

The following examples are presented to illustrate the above procedures.

EXPERIMENTAL PART

Preparation 1: 5-ethyl-4,5-dihydro-1H-oxepino [3\4':6,7]-indolizine [1,2-b]quinoline

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

1.a. 4-ethyl-3,4-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14(4H,12H)-one

Sodium borohydride (14 g, 370 mmol) is added portionwise to a suspension of (S)-(+)-camptocin (14 g, 40 mmol, which can be obtained from various commercial sources such as Aldrich Chemical Co. (Milwaukee, Wl)), in methanol (750 mL) and the resulting mixture is gently warmed to 55°C to obtain a clear solution which is then stirred for 16 hours at ambient temperature. The solvent is then evaporated under reduced pressure, the residue is taken up in water (250 ml), neutralized by the addition of acetic acid (21 ml) and left to stand for 2 hours at 4°C. The resulting suspension is filtered and washed successively with cold water, acetone and diethyl ether, which enables the desired product to be obtained, after drying under reduced pressure, in the form of a white solid m.p. 280°C.

1.b. 8-formyloxymethyl-7-propionylindolizino [1,2-b]quinolin-9 (11 H )-one

A solution of sodium metaperiodate (14 g, 65 mmol) in water (140 mL) is added dropwise to a suspension of 4-ethyl-3,4-dihydroxy-1H-pyrano[3',4':6,7]indolizine [1,2-b]quinolin-14(4H,12H)-one (13.4 g, 38 mmol) in glacial acetic acid (720 mL) and the resulting solution is stirred for one hour at ambient temperature. The reaction mixture is then poured into an ice/water mixture (650 ml) and the resulting suspension is then stirred for half an hour, then filtered and washed successively with water, isopropyl alcohol and diethyl ether, enabling the desired product (11.5 g ) is obtained, after drying under reduced pressure, in the form of a pale yellow solid m.p. > 200°C (d).

1.c. tert-butyl B-ethyl-B-hydroxy-B-(8-hydroxymethyl-9-oxo (11 H )-indolizino-[1,2-5]quinolin-7-yl)-propionate

A suspension of zinc (6.5 g, 100 mmol) stirred with a magnetic stirrer in anhydrous diethyl ether (50 mL) under argon is activated by dropwise addition of chlorotrimethylsilane (0.75 mL, 5.7 mmol). Stirring is continued for 15 minutes at ambient temperature, then the reaction medium is heated to reflux. The heating bath is then removed and tert-butyl bromoacetate (15 mL, 100 mmol) is added dropwise at a rate that ensures reflux is achieved. The external heating is switched on again and heating continues for one hour. The resulting ethereal solution of Reformatsky's reagent is allowed to cool to ambient temperature, then transferred using a cannula to a suspension of 8-formyloxymethyl-7-propionylindolizino[1,2-b]quinoline-9(11H)- or\ (1 .6 g, 4.7 mmol) in anhydrous tetrahydrofuran (40 mL) under argon. The reaction mixture is stirred under reflux for one hour, then allowed to cool to ambient temperature and the reaction is stopped by the addition of saturated ammonium chloride (100 ml) and the extraction is carried out with chloroform (3 x 100 ml). The combined chloroform-containing extracts are dried over sodium sulfate, evaporated and the residue is purified by chromatography on a silica gel column (1-2% MeOH/CH2Cl2), which enables 0.64 g of the desired product (31%) to be obtained as a pale yellow solid, m.p. 146-149°C.

NMR-1H (CDCl3 ): 0.93 (t, 3H); 1.37 (s, 9H); 1.99 (m, 2H); 2.97 (dd, 2H); 3.5 (see 1H); 5.10 (s, 2H); 5.24 (s, 2H); 7.40 (s, 1H); 7.59 (t, 1H); 7.83 (t, 1H); 7.90 (d, 1H); 8.20 (d, 1H); 8.34 (p, 1H).

NMR-C 13 (CDCl 3 ): 8.18; 27.90; 34.59; 45.34; 49.91; 58.55; 77.39; 82.42; 100.52; 127.67; 127.97; 128.10; 128.64; 129.44; 129.79; 130.42; 130.99; 142.86; 148.69; 152.75; 155.16; 162.38; 172.24.

IR(KBr):764; 1016; 1157; 1580; 1651; 1726.

1.d. 5-ethyl-4,5-dihydro-5-hydroxy-1H-oxepino[3',4,:6,7]-indolizine[1,2-b]quinoline-3,15 (4H,13W)-dione

tert-butyl B-ethyl-B-hydroxy-B-(8-hydroxymethyl-9-oxo(11 H )-indolizino-[1,2-b]quinolin-7-yl)-propionate (1.45 g, 3, 32 mmol) is dissolved in anhydrous dichloromethane (25 ml) and treated with a saturated solution of hydrogen chloride in dichloromethane (100 ml). The resulting mixture is kept at -20°C for 16 hours. The precipitate is filtered, washed with methanol and dried under reduced pressure, which enables 662 mg (55%) of the desired product to be obtained as a yellow solid, m.p. > 300°C.

NMR-1H (DMSO): 0.90 (t, 3H); 1.20 (q, 2H); 3.27 (dd, 2H); 5.29 (s, 2H); 5.49 (dd, 2H); 7.42 (s, 1H); 7.73 (t, 1H); 7.90 (t, 1H); 8.16 (t, 2H); 8.71 (p, 1H).

NMR-C 13 (DMSO): 8.45; 36.48; 42.54; 50.68; 61.44; 73.34; 99.78; 122.71; 127.83; 128.15; 128.75; 129.08; 130.07; 130.61; 131.81; 144.66; 148.04; 152.80; 155.91; 159.26; 172.08.

IR (KBr): 761; 1127; 1204; 1285; 1580; 1653; 1757.

Preparation 2: cleavage of 5-ethyl-4,5-dihydro-5-hydroxy-1 W -oxepino

[3',4':6,7]indolizine [1,2-6]quinoline-3,15 (4H,13H)-dione

A mixture of B-ethyl-B-hydroxy-(8-hydroxymethylindolizino-[1,2-b]quinolin-9-(11H-on-7-yl)-propionic acid (19.5 g, 51 mmol) and L -(-)-α-methylbenzylamine (12.12 g, 100 mmol) in absolute ethanol (1 L) is heated to boiling, followed by filtration while hot and allowed to stand for 68 h. The precipitate is filtered and washed with ethanol and ether to produce 9.8 g of a white solid.Analysis by high pressure liquid chromatography on chiral stationary phase ("Chiral HPLC" on Chiral-AGP column (Chromtech, Stockholm, Sweden) 100 x 4 mm, eluent 2% acetonitrile in 10 mM phosphate buffer at pH 6.9, peaks during elution at 4.5 and 7.5 min) show two peaks integrating respectively 24% and 76% of the total area of two peaks. The solid is taken up in 93 % ethanol (350 mL) under reflux, then allowed to stand for 48 h. The precipitate is filtered off, then washed with ethanol and ether to obtain 4.8 g of a white solid which produces two peaks integrating 9% and 91% of that tot ale the area of two peaks using chiral HPLC. The solid is taken up in 50% ethanol (48 ml) under reflux, then allowed to rest for 48 hours. The precipitate is filtered out, then washed with ethanol and ether to produce 2.7 g of a white solid which produces two peaks integrating 3% and 97% respectively of the total area of two peaks using chiral HPLC. The solid is taken up in 50% ethanol (22 ml) under reflux, then allowed to rest for 48 hours. The precipitate is filtered out, then washed with ethanol and ether to produce 1.6 g of a white solid which produces two peaks integrating 1% and 99% of the total area respectively into two peaks using chiral HPLC. The resulting salt, diastereoisomerically enriched, is taken up in distilled water (20 mL), treated with acetic acid (0.35 mL, 6.4 mmol) for 15 minutes. The precipitate obtained is filtered out, washed with water, with acetone and with ether, then dried under vacuum at 80°C to obtain 1.1 g of a white solid. The latter is taken up in absolute ethanol (55 ml) and concentrated hydrochloric acid (11.5 N, 11 ml) is added to this to obtain a yellow solution which is kept under agitation at ambient temperature for 68 hours. The precipitate thus obtained is filtered out and washed with water, with ethanol and with ether, then dried under vacuum at 80°C to obtain 770 mg of 5-ethyl-4,5-dihydro-5-hydroxy-1H-oxepinde [3,,4':6,7]-indolizine [1,2-5]quinoline-3,15 (4H,13H)-dione which is enantiomerically enriched. Analysis by chiral HPLC (Chiral-AGP column, eluted with a 2 to 5% gradient of acetonitrile in 10 mM phosphate buffer at pH 6.9, peaks during elution at 15 and 20 minutes) and shows an enantiomeric excess of 98%. The method described above is carried out again by replacing L-(-)-rj-methylbenzylamine with D-(+)-□-methylbenzylamine. In this way, other enantiomers of 5-ethyl-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]-indolizine [1,2-5]quinoline-3,15 { AH, 13/^)-dione obtained.

Preparation 3: 5,12-diethyl-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]-indolizino[1,2-5]quinoline-3,15 (4H ,13H)-dione

This compound is prepared in a similar manner to Example 1, except that in step 1.a.., 7-ethyl camptocin (Sawada et al., Chem. Pharm. Bull. 39:2574 (1991)) is used instead of camptocin. The desired compound is obtained in the form of a pale yellow solid, m.p. > 270°C.

NMR-1H (DMSO): 0.92 (t, 3H); 1.39 (t, 3H); 1.93 (q, 2H); 3.08 (d, 2H); 3.25 (q, 2H); 3.51 (d, 2H); 5.32 (s, 2H); 5.52 (dd, 2H); 7.42 (s, 1H); 7.76 (t, 1H); 7.89 (t, 1H);

8.18 (d, 1H); 8.32 (d, 1H).

NMR-C 13 (DMSO): 8.46; 14,15; 22.42; 36.50; 42.54; 49.95; 61.45; 73.35; 99.68; 122.61; 124.27; 126.76; 127.70; 128.27; 129.92; 130.18; 145.17; 145.82; 148.57; 152.15; 155.89; 159.26; 172.08.

Preparation 4: 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3\4':6,7]indolizino[1,2-5]quinoline-3,15 (4H,13W)-dione

4. a. 2-Ethyl-2-(2-methoxy-4-pyridyl)-1,3-dioxolane

The water is azeotropically distilled (overnight) with a Dean Stark apparatus from a mixture of 2-chloro-4-propionylpyridine (10 g, 59 mmol) obtained as in Lamattina, J.L. J. Heterocyclic Chem. 20, p. 553 (1983), ethylene glycol (20 ml) and p-toluenesulfonic acid (250 mg) in toluene (150 ml). The solvent is then eliminated under reduced pressure, the acid is neutralized with saturated aqueous sodium bicarbonate

(100 ml) and the product is extracted with ether. The combined ethereal extracts are washed with brine, dried over sodium sulfate and evaporated, yielding 13.3 g (96%) of carbonyl-protected crude product which is heated to reflux

with 3 equivalents of sodium methoxide in acetonitrile until the reaction is complete (examined by thin-layer chromatography: SiO2, tert-butyl methyl oxide/hexane (TBMO/HX) 50/50). Acetonitrile solution is then filtered and evaporated. The residue is taken up in ether, washed with water and with brine, dried over sodium sulfate and evaporated, producing a brown oil which is distilled (70-75°C, 0.04 mbar); 10.7 g (total yield 81%) of product (F) is collected in the form of a transparent oil.

4.b. 2-Ethyl-2-(3-hydroxymethyl-2-methoxy-4-pyridyl)-1,3-dioxolane tert-butyllithium (1.7 M in pentane, 100 mL, 170 mmol) is added dropwise using a cannula to a solution of the bromomesitylene (13 mL, 85 mmol) in anhydrous tetrahydrofuran (300 mL) at -78°C and under argon. The resulting white precipitate is stirred at -78°C for one hour, then 2-ethyl-2-(2-methoxy-4-pyridyl)-1,3-dioxolane (10 g, 44.8 mmol) is added and the reaction mixture is stirred for 15 minutes at -78°C, for one hour at 0°C and for one hour at ambient temperature. After further cooling to -78°C, anhydrous N,N-dimethylformamide (100 mmol) is added and the reaction mixture is warmed up to ambient temperature and then stirred for 16 hours, with analysis by thin layer chromatography (S1O2, TBMO/HX: 50/50) showing complete consumption of the output connection. The reaction is quenched with saturated ammonium chloride and the reaction mixture is extracted with diethyl ether (200 mL, 50 mL, 50 mL). The combined extracts are dried over sodium sulfate and evaporated, and this produces a yellow oil which is purified by column chromatography (SiO 2 , TBMO/HX: 0/100 to 5/95 to elute mestylene derivatives then 20/80 to 50/50 to elute the product) to obtain the intermediate aldehyde (7 g). The aldehyde is dissolved in methanol (100 mL) and treated with sodium borohydride (5 g, 132 mmol) and the resulting mixture is stirred until complete consumption of the intermediate aldehyde (about 1 hour) with analytical control by thin layer chromatography. The solvent is then evaporated, the residue taken up in ether, washed with water and with brine, dried and the solvent is evaporated. Column chromatography (SiO 2 , TBMO/HX: 10/90 to 50/50) the residue produces 7 g (overall yield 62%) of product (G) in the form of a yellow oil.

4. c. 2-(3-Benzyloxymethyl-2-methoxy-4-pyridyl)-2-ethyl-1,3-dioxolane

A solution of 2-ethyl-2-(3-hydroxymethyl-2-methoxy-4-pyridyl)-1,3-dioxolane (7 g,

30 mmol) and benzyl chloride (5 mL, 45 mmol) in anhydrous tetrahydrofuran (50 mL) are added dropwise to a suspension of sodium hydride (80% in mineral oil, 1.85 g, 61 mmol) in

anhydrous tetrahydrofuran (100 ml) and the reaction mixture is refluxed for 16 hours. The reaction mixture is then cooled to ambient temperature, the reaction quenched with water (50 ml) and the reaction mixture concentrated under reduced pressure. The residue is dissolved in diethyl ether (150 ml) and washed with water and with brine, dried and evaporated. Purification by column chromatography (SiO 2 , TBMO/HX: 5/95 to 20/80) produced the product protected by benzyl (H), 9 g, (87%) as a clear oil. 4th d. 1 -(3-Benzyloxymethyl-2-methoxy-4-pyridyl)-propan-1 -one 2-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-2-ethyl-1,3-dioxolane (9 g, 27 mmol) is treated with trifluoroacetic acid (10 ml) and water (5 ml) at a bath temperature of 120°C for 3 hours. The reaction mixture is concentrated under reduced pressure and remaining traces of acids are neutralized by the addition of saturated aqueous sodium bicarbonate. Extraction is carried out with ether followed by column chromatography (S1O2, TBMO/HX: 10/90) producing 5.5 g (70%) of product (I). 4.e. tert-butyl □-ethyl-D-hydroxy-D-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-propionate

tert-butyl bromoacetate (13 mL, 80 mmol) is added dropwise to a zinc suspension (5.3 g, 80 mmol) activated with 6N HCl over 10 seconds, then washed successively with water until a neutral pH is obtained, with acetone and with diethyl ether ) in anhydrous form

tetrahydrofuran (60 mL) under reflux. The reaction medium is kept under reflux for a further 10 minutes after the addition is finished. Then a solution of 1-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-propan-1-one (5.8 g, 20 mmol) in anhydrous tetrahydrofuran (20 mL) is added and the reaction mixture is stirred under reflux in a further hour. The reaction is quenched at 0°C with saturated aqueous ammonium chloride (100 mL) and the reaction mixture is extracted with diethyl ether. The combined extracts are dried over sodium sulfate and evaporated to yield a yellow oil which is purified by column chromatography (SiO 2 , TBMO/HX: 5/95 to 10/90) to obtain tert-butyl ester (J) (7 g, 95 %) in the form of a transparent oil.

4.f. tert-butyl B-ethyl-B-hydroxy-B-(3-hydroxymethyl-2-methoxy-4-pyridyl)-propionate

tert-butyl B-ethyl-B-hydroxy-B-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-propionate (1 g, 2.5 mmol) is subjected to hydrogenolysis at atmospheric pressure and at ambient temperature using 5% palladium on carbon as catalyst (50 mg) and absolute ethanol as solvent (10 ml). When the reaction is

terminated (6 hours), the catalyst is separated by filtration and the solvent is evaporated, this gives 0.7 g (90%) of product (K) with a sufficient purity for a subsequent use for synthesis.

4th g. 5-ethyl-1,5-dihydro-5-hydroxy-9-methoxy-oxepino[3,4-c]pyridin-3(4H)-one

tert-butyl B-ethyl-B-hydroxy-B-(3-hydroxymethyl-2-methoxy-4-pyridyl)-propionate (8.8 g,

28 mmol) is treated with trifluoroacetic acid (30 ml) for 3 hours at ambient temperature. Volatile components are evaporated and the residue is purified by column chromatography (SiO 2 , CH 2 Cl 2 /MeOH: 100/0 to 98/2), which produces a clear oil which, after treatment with toluene, yields 5.9 g of product (L)

(89%) in the form of white crystals, m.p. 97-98°C.

4th h. 5-ethyl-1,5-dihydro-5-hydroxy-oxepino[3,4-c]pyridine-3,9(4H,8H)-dione 5-ethyl-1,5-dihydro-5-hydroxy-9- methoxy-oxepino[3,4-c]pyridin-3(4H)-one (0.5 g, 2.1 mmol) is heated under reflux for 9 h in 1 N hydrochloric acid (20 mL). The reaction mixture is concentrated under reduced pressure and the residue is again dried by the addition and evaporation of toluene twice, then left overnight under reduced pressure in the presence of phosphorus pentoxide. The resulting oil is dissolved in anhydrous acetonitrile (5 mL) and stirred under argon for 24 hours. The precipitate is filtered off and dried, this produces 0.23 g (49%) of a white solid (M), m.p. 118-119°C.

4.i. 2-chloro-6,7-difluoro-3-quinoline-methanol

The method described by Meth-Cohn and collaborators, J. Chem. Soc. Perkin Trans. IN,

pp. 1520 (1981); Meth-Cohn, J. Chem. Soc. Perkin Trans. I, p. 2509 (1981) is used. 3,4-Difluoroacetanilide (38 g, 22 mmol) is added to Vilsmeyer's reagent obtained by dropwise addition of phosphoryloxychloride (103 mL, 1.1 mol) to anhydrous dimethylformamide (34 mL, 44 mmol), cooled with a water/ice bath and stirred for 0.5 hours under an argon atmosphere. The resulting mixture is heated at 70°C for 16 hours. After cooling to ambient temperature, the reaction mixture is added to a mixture of ice and water (400 ml) which is kept under agitation for 2 hours, then filtered and washed successively with water, with ethanol and with ether to produce 9 g of 2-chloro -6,7-difluoroquinoline-3-carbaldehyde as a yellow solid, m.p. 222-224°C.

This intermediate is treated with sodium borohydride (2 g, 52 mmol) in methanol

(400 ml) at ambient temperature for 0.5 hours, then excess regens is destroyed by the addition of acetic acid (2 ml). The solvent is eliminated under reduced pressure, the residue is dissolved in ethyl acetate and washed successively with dilute sodium bicarbonate, with water and with a saturated aqueous solution of sodium

chloride. The organic phase is dried over sodium sulfate, filtered and concentrated. The resulting solid is recrystallized from 1,2-dichloroethane to produce 8 g of 2-chloro-6,7-difluoro-3-quinoline-methanol as a beige solid.

4th j. 5-ethyl-8-(2-chloro-6,7-difluoro-3-quinolinemethyl)-1,5-dihydro-5-hydroxy-oxepino[3,4-c]pyridine-3,9(4H,8rV) -dion

Diethyl azodicarboxylate (570 L, 3.6 mmol) is added dropwise over 5 minutes to a solution of 5-ethyl-1,5-dihydro-5-hydroxy-oxepino[3,4-c]pyridine-3,9(4H, 8H)-dione (400 mg, 1.79 mmol), the compound obtained in previous step 4.i. (770 mg, 2.23 mmol) and triphenylphosphine (934 mg, 3.58 mmol) in anhydrous N,N-dimethylformamide (45 mL) and the resulting mixture is stirred under argon at ambient temperature for 16 h. The reaction mixture is then concentrated under reduced pressure and the residue is dissolved in ether (100 mL). The resulting solution is washed with brine (4 x 50 mL), dried over sodium sulfate and evaporated. The residue is purified by column chromatography (SiO 2 , CH 2 Cl 2 /MeOH: 99/1 to 98/2), this produces 650 mg (66%) of product (O) as a white solid, m.p. 165-167°C. 4.k. 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-1 H- oxepino[3',4':6,7]indolizino[1,2-b]quinoline-3,15 (4H,13H)-dione 5-ethyl-8-(2-chloro-6,7-difluoro-3-quinolinemethyl)-1,5-dihydro-5-hydroxy-oxepino[3,4-c]pyridine-3,9(4H,8H) -dione (600 mg, 1.1 mmol), tetrabutylammonium bromide (352 mg, 1.1 mmol), sodium acetate (359 mg, 4.4 mmol) and palladium llacetate (98 mg, 0.43 mmol) are dissolved in anhydrous acetonitrile (40 ml) and heated at 90°C under argon for 16 hours. After cooling down to ambient temperature, a white precipitate is separated from the reddish solution. This precipitate is filtered out and dried under reduced pressure. The crude product is suspended in water, filtered and dried under reduced pressure over phosphorus pentoxide, this produces 250 mg of the desired compound in the form of a beige solid, m.p. > 250°C.

NMR-1H (DMSO): 0.91 (t, 3H); 1.87 (m, 2H); 3.08 (d, 1H); 3.51 (d, 1H); 4.45 (s, 4H); 5.19 (s, 2H); 5.47 (dd, 2H); 6.02 (see 1H); 7.33 (s, 1H); 7.54 (s, 1H); 7.55 (s, 1H);

8.43 (p, 1H).

NMR-C 13 (DMSO): 8.43; 36.47; 42.54; 50.52; 61.43; 64.43 (2C); 73.31; 99.07; 112.27; 113.14; 122.00; 124.24; 128.18; 129.74; 144.59; 145.01; 145.33; 147.63; 150.88; 155.88; 159.23; 172.07.

Preparation 5: 5-ethyl-4,5-dihydro-5,10-dihydroxy-1N-oxepino[3,,4,:6,7]indolizino[1,2-b]quinoline-3,15(4H> 13rl)-dione 10-benzyloxy-5-ethyl-4,5-dihydro-5-hydroxy-1H-oxepino[3',4,:6,7]-indolizino [1, 2-b]-quinoline-3 ,15 (4H,13H)-dione (370 mg, 0.79 mmol) is treated with hydrogen at atmospheric pressure and at ambient temperature using 10% palladium on carbon as catalyst (60 mg) and trifluoroacetic acid as solvent (15 mL) . When the reaction is terminated (16 hours), dichloromethane (50 ml) and methanol (50 ml) are added to the reaction mixture, the catalyst is filtered out and volatile components are evaporated under reduced pressure, and this enables the crude desired product to be obtained containing traces of trifluoroacetic acid. These traces are eliminated by co-distillation with 1,4-dioxane. The product is obtained in the form of an orange solid, m.p. 150°C (d), with a sufficient purity for a subsequent synthesis application. NMR-1H (DMSO): 0.89 (t, 3H); 1.85 (q, 2H); 3.02 (d, 1H); 3.45 (d, 1H); 5.19 (s, 2H); 5.37 (d, 1H); 5.50 (d, 1H); 5.98 (see 1H); 7.26 (p 1H); 7.31 (s, 1H); 7.40 (d, 1H); 8.00 (d, 1H); 8.42 (s, 1H); 10.32 (p, 1H).

NMR-C 13 (DMSO): 8.47; 36.50; 42.61; 50.57; 61.46; 73.35; 98.84; 109.02; 121.83; 123.18; 129.50; 129.85; 130.12; 130.80; 143.39; 145.10; 149.69; 155.97; 156.82; 159.30; 172.11.

Preparation 6: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-1 H -oxepino

[3<l>,4,:6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione

This compound is obtained from 3-fluoro-4-methoxyaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C. NMR-1H (DMSO): 0.89 (t, 3H); 1.85 (q, 2H); 3.08 (d, 1H); 3.49 (d, 1H); 4.00 (p, 3H); 5.25 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.00 (p, 1H); 7.32 (s, 1H); 7.72 (d, 1H); 7.91 (d, 1H); 8.58 (s, 1H).

NMR-C 13 (DMSO): 8.43; 36.48; 42.51; 50.68; 56.60; 61.42; 73.29; 99.25; 108.68; 113.52; 122.23; 126.33; 129.99; 130.30; 143.79; 144.70; 148.42; 151.18; 153.19; 155.81; 159.20; 172.06.

IR (KBr): 1259; 1503; 1602; 1737.

Preparation 7: 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

This compound is obtained from 3-chloro-4-methoxyaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C. NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 2.55 (s, 3H); 3.07 (d, 1H); 3.45 (d, 1H); 5.25 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.05 (s, 1H); 7.39 (s, 1H); 8.10 (s, 1H); 8.20 (s, 1H); 8.60 (p, 1H).

NMR-C 13 (DMSO): 8.43; 20,20; 36.47; 42.49; 50.67; 61.41; 73.28; 99.87; 122.82; 126.98; 127.99; 129.60; 130.53; 131.08; 135.64; 136.56; 144.39; 147.11; 153.10; 155.85; 159.18; 172.03.

IR (KBr): 1208; 1479; 1606; 1656; 1724.

Preparation 8: 8-ethyl-2,3,8,9-tetrahydro-8-hydroxy-10H,12H-[1,4]dioxino [ 2, 3- g]

oxepino [3',4':6,7] indolizino [1,2-b]quinoline-10,13 (15H)-dione This compound is obtained from 3,4-ethylenedioxyaniline according to the method illustrated in steps 4i, 4j and 4k according to Preparation 4. Yellow solid, m.p. > 250°C. NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.47 (d, 1H); 5.25 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.05 (s, 1H); 7.39 (s, 1H); 8.15 (q, 1H); 8.25 (q, 1H);

8.68 (p, 1H).

NMR-C 13 (DMSO): 8.41; 36.45; 42.48; 50.68; 61.40; 73.25; 99.92; 114.44; 115.42; 115.58; 122.96; 125.52; 130.56; 131.46; 144.21; 145.25; 142.36; 153.41; 155.85; 159.15; 172.00.

IR (KBr): 1266; 1512; 1581; 1618; 1751.

Preparation 9: 7-ethyl-7,8-dihydro-7-hydroxy-9H,11H-[1,3]dioxolo [4,5-g]

oxepino [3',4':6,7] indolizino [1,2-b]quinoline-9,12 (14W)-dione This compound is obtained from 3,4-methylenedioxyaniline according to the method illustrated in steps 4i, 4j and 4k according to Preparation 4. Cream solid, m.p. > 250°C. NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 5.20 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.00 (p, 1H); 6.30 (s, 2H); 7.30 (s, 1H); 7.49 (d, 2H); 8.45 (p.1H).

NMR-C 13 (DMSO): 8.43; 36.49; 42.56; 50.58; 61.42; 73.31; 98.87; 102.75; 103.33; 104.92; 121.76; 125.74; 128.59; 130.33; 145.08; 146.69; 148.78; 150.19; 151.49; 155.90; 159.24; 172.08.

IR (KBr): 1248; 1459; 1606; 1731.

Preparation 10: 9-chloro-5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino

[3\4':6,7] indolizino [1,2-b]quinoline-3,15 (4H,13H)-dione

This compound is obtained from 3-chloro-4-methoxyaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. White solid, m.p. > 250°C. NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 4.01 (s, 3H); 5.22 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.02 (s, 1H); 7.31 (s, 1H); 7.68 (s, 1H); 8.20 (s, 1H); 8.55 (p, 1H).

NMR-C 13 (DMSO): 8.22; 36.27; 42.30; 50.48; 56.69; 61.23; 73.08; 99.16; 107.44; 122.16; 127.12; 128.12; 129.25; 130.02; 130.53; 143.29; 144.37; 151.12; 153.29; 155.71; 158.98; 171.84.

IR (KBr): 1056; 1256; 1483; 1592; 1657; 1747.

Preparation 11: 5-ethyl-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino

[3',4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione

This compound is obtained from 4-methoxyaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 3.95 (s, 3H); 5.28 (s, 2H); 5.40 (d, 1H); 5.51 (d, 1H); 6.00 (p, 1H); 7.38 (s, 1H); 7.51 (d, 2H); 8.07 (d, 1H); 8.55 (p, 1H).

NMR-C 13 (DMSO): 8.45; 36.48; 42.51; 50.64; 55.92; 61.42; 73.33; 99.01; 106.49; 122.02; 123.19; 129.59; 130.20; 130.43; 144.17; 144.94; 150.40; 155.92; 158.31; 159.26; 172.07.

IR (KBr): 1251; 1604; 1655; 1735.

Preparation 12: 9,11-dichloro-5-ethyl-4,5-dihydro-5-hydroxy-1 H -oxepino [3',4':6,7]

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

This compound is obtained from 3,5-dichloroaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 5.30 (s, 2H); 5.41 (d, 1H); 5.55 (d, 1H); 6.08 (s, 1H); 7.41 (s, 1H); 8.05 (s, 1H); 8.21 (s, 1H); 8.91 (p, 1H).

NMR-C 13 (DMSO): 8.39; 36.45; 42.51; 51.03; 61.39; 73.25; 100.62; 123.55; 124.63; 127.60; 128.08; 128.56; 132.06; 132.19; 134.53; 143.77; 148.80; 154.88; 155.82; 159.13; 171.98.

IR (KBr): 1064; 1275; 1586; 1651; 1743.

Preparation 13: 5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methyl-1 H -oxepino

[3\4':6,7] indolizino [1,2-6]quinoline-3,15 (4H,13H)-dione

This compound is obtained from 3-fluoro-4-methylaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C. NMR-1H (DMSO): 0.89 (t, 3H); 1.85 (q, 2H); 2.49 (s, 3H); 3.08 (d, 1H); 3.49 (d, 1H); 5.21 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.05 (s, 1H); 7.39 (s, 1H); 7.87 (d, 1H); 8.05 (d, 1H); 8.61 (s, 1H).

NMR-C 13 (DMSO): 8.40; 15.14; 36.45; 42.52; 50.60; 61.41; 73.28; 99.71; 112.00; 122.66; 125.38; 127.66; 129.59; 130.28; 144.49; 147.88; 152.88; 155.85; 159.18; 162.25; 172.02.

IR (KBr): 1054; 1580; 1651; 1760.

Preparation 14: 5-ethyl-10-fluoro-4,5-dihydro-5-hydroxy-1 H -oxepino [3',4':6,7]

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

This compound is obtained from 4-fluoroaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. White solid, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 5.29 (s, 2H); 5.39 (d, 1H); 5.55 (d, 1H); 6.30 (s, 1H); 7.39 (s, 1H); 7.80 (q, 1H); 7.99 (q, 1H); 8.23 (q, 1H); 8.68 (s, 1H).

NMR-C 13 (DMSO): 8.40; 36.46; 42.48; 50.66; 61.41; 73.31; 99.68; 111.83; 122.75; 128.93; 130.93; 131.22; 131.93; 144.46; 145.27; 152.60; 155.89; 159.21; 172.04. IR (KBr): 1209; 1589; 1659; 1739.

Preparation 15:10-chloro-5-ethyl-4,5-dihydro-5-hydroxy-1 H -oxepino [3<I>,4':6,7]

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

This compound is obtained from 4-chloroaniline according to the method illustrated by steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.47 (d, 1H); 5.25 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.05 (s, 1H); 7.39 (s, 1H); 7.89 (d, 1H); 8.19 (d, 1H); 8.29 (s, 1H); 8.67 (s, 1H).

NMR-C 13 (DMSO): 8.40; 36.46; 42.47; 50.70; 61.42; 73.31; 100.00; 122.96; 127.31; 127.42; 128.87; 131.11; 132.12; 144.34; 146.53; 153.38; 155.88; 159.20; 172.04. IR (KBr): 1069; 1483; 1606; 1741.

Preparation 16: 9-chloro-5-ethyl-10-fluoro-4,5-dihydro-5-hydroxy-1 H -oxepino

[3\4':6,7] indolizino [1,2-fa]quinoline-3,15 (4H,13H)-dione

This compound is obtained from 4-chloro-3-fluoroaniline according to the method illustrated in steps 4i, 4j and 4k of Preparation 4. Yellow solid, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.07 (d, 1H); 3.45 (d, 1H); 5.25 (s, 2H); 5.39 (d, 1H); 5.51 (d, 1H); 6.05 (s, 1H); 7.40 (s, 1H); 8.20 (d, 1H); 8.40 (d, 1H); 8.68 (p, 1H).

NMR-C 13 (DMSO): 8.38; 36.47; 42.58; 50.71; 61.40; 73.26; 99.99; 113.59; 123.09; 124.28; 127.74; 130.64; 131.31; 144.13; 145.08; 153.57; 154.13; 155.84; 156.61; 159.14; 172.00.

IR (KBr): 1488; 1583; 1655; 1743.

Preparation 17: 5,12-diethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino[3\4':6,7]indolizino[1,2-b]quinoline -3,15 (4H,13H)-dione 17.a. 5-fluoro-4-methoxy-2-propionylaniline

(This product is obtained according to Sugasawa T; Toyoda T; Adachi M; Sasakura K, J. Am. Chem. Soc., 100 (1978), pp. 4842-4852). Boron trichloride (1M i

heptane, 156 mL, 156 mmol) is added dropwise under an argon atmosphere at 0°C to a solution of 3-fluoro-4-methoxyaniline (20 g, 142 mmol) in anhydrous dichloromethane (200 mL). The pink suspension thus obtained is kept under agitation for 5 minutes, then propionitrile (33 ml, 420 mmol) is added dropwise followed by aluminum trichloride (20.8 g, 156 mmol) in small portions. The reaction medium is heated under reflux for 3 hours, cooled to 0°C, hydrolyzed by careful addition of 2N hydrochloric acid (100 ml), then heated under reflux for 45 minutes. After cooling to 0°C, a precipitate is obtained, filtered out, washed with dichloromethane, then taken up in water (300 ml). The aqueous phase is basified to an alkaline pH, extracted with dichloromethane then ethyl acetate. The organic phase is dried (MgSO 4 ) then evaporated to produce a crude product which is purified by column chromatography (SiO 2 , AcOEt/Hpt: 1/99 to 20/80). 15.3 g of a yellow solid is obtained.

NMR-1H (CDCl3): 1.20 (t, 3H); 2.92 (q, 2H); 3.83 (s, 3H); 6.2 (s, 2H); 6.40 (d, 2H); 7.32 (d, 2H).

IR (KBr): 857; 1148; 1240; 1561; 1583; 1662.

17.b. Ethyl 4-ethyl-7-fluoro-2-hydroxy-6-methoxy-3-quinolinecarboxylate A solution of ethylmalonyl chloride (12.9 mL, 100 mmol) in anhydrous acetonitrile (30 mL) is added dropwise, under argon and at 0 °C to a solution of 5-fluoro-4-methoxy-2-propionylaniline (15.3 g, 77.5 mmol) and triethylamine (13.9 mL, 100 mmol) in anhydrous acetonitrile (110 mL). The reaction medium is allowed to return to ambient temperature, a solution of sodium ethylate (obtained from 1.8 g, 78 mmol sodium in 80 ml ethanol) is cannulated dropwise and under argon, then the reaction medium is left under agitation for 12 hours at ambient temperature. The reaction mixture is poured into ice-cooled water (100 ml) and the agitation is carried out for two hours, then the precipitate is filtered off and washed with water, with ethanol and with ether. 19.4 g of a white solid is obtained.

NMR-1H (DMSO): 1.25 (m, 6H); 2.78 (q, 2H); 3.92 (s, 3H); 4.30 (q, 2H); 7.15 (d, 2H); 7.40 (d, 2H); 11.93 (p, 1H).

IR (KBr): 786; 1083; 1410; 1521; 1644; 1725. 17th c. Ethyl 2-chloro-4-ethyl-7-fluoro-6-methoxy-3-quinolinecarboxylate A suspension of ethyl 4-ethyl-7-fluoro-2-hydroxy-6-methoxy-3-quinolinecarboxylate (19.4 g, 0.066 mol ) in phosphoryl chloride (243 mL) is heated at reflux for 6 h. Phosphoryl chloride is distilled out. The reaction mixture is decanted into ice-cooled water, then taken up in dichloromethane for solubilization. The organic phase is washed with water, then with a saturated solution of sodium chloride. The organic phase is dried over magnesium sulfate and the solvent is evaporated. The residue is suspended in ether and unconverted starting compound (4 g) is filtered off. The filtrate is evaporated and the residue is purified by column chromatography (SiO 2 , AcOEt/Hpt: 5/95 to 20/80). 10.9 g of a white solid is obtained.

NMR-1H (DMSO): 1.30 (t, 3H); 1.39 (t, 3H); 3.08 (q, 2H); 4.09 (s, 3H); 4.49 (q, 2H); 7.64 (d, 2H); 7.86 (d, 2H).

IR (KBr): 865; 1016; 1082; 1190; 1224; 1253; 1272; 1508; 1571; 1732.

17th 2-chloro-4-ethyl-7-fluoro-6-methoxy-3-quinolinemethanol

A solution of ethyl 2-chloro-4-ethyl-7-fluoro-6-methoxy-3-quinolinecarboxylate (10.8 g, 35 mmol) in anhydrous dichloromethane (200 mL) is treated dropwise at ambient temperature under an inert atmosphere with diisobutylaluminum hydride (1M in dichloromethane, 65 mL, 65 mmol), then heated at 40°C for 4 hours. After cooling to 0°C, a 20% aqueous solution of Rochelle salt (105 mL) and dichloromethane (200 mL) is added carefully and the reaction mixture is kept under agitation for 1 hour, followed by decantation and washing three times with water. The organic phase is dried over magnesium sulfate and the solvent is evaporated. The residue is purified by column chromatography (SiO 2 , AcOEt/Hpt: 5/95 to 50/50). 6 g of a white solid is obtained.

NMR-1H (DMSO): 1.28 (t, 3H); 3.25 (q, 2H); 4.04 (s, 3H); 4.77 (d, 2H); 5.27 (t, 1H); 7.55 (d, 2H); 7.73 (d, 2H).

IR (KBr): 840; 864; 1023; 1232; 1267; 1317; 1444; 1511; 1569.

17.e. 5,12-diethyl-9-fluoro-4,5-dihydro-5-hydroxy-10-methoxy-1H-oxepino[3',4':6,7]indolizino[1,2-b]quinoline -3,15 (4H,13H)-dione 2-chloro-4-ethyl-7-fluoro-6-methoxy-3-quinolinemethanol is coupled with compound (M) as described in step 4.j. according to Preparation 4. The resulting coupled product is cyclized according to the method described in step 4.k. A yellow solid is obtained, m.p. > 275°C.

NMR-1H (CF3COOD): 1.07 (m, 3H); 1.62 (m, 3H); 2.27 (m, 2H); 3.44 (d, 1H);

3.54 (m, 2H); 3.91 (d, 1H); 4.25 (s, 3H); 5.60 (d, 1H); 5.74 (s, 2H); 5.98 (d, 1H); 7.85 (m, 1H); 8.16 (m, 1H); 8.31 (p, 1H).

NMR-C 13 (CF 3 COOD): 9.03; 14.20; 26.68; 38.77; 43.98; 53.79; 58.27; 64.73; 77.93; 106.85; 109.24; 110.15; 128.99; 129.20; 131.61; 137.32; 141.23; 144.13; 154.79; 158.32; 160.25; 160.81; 179.30.

IR (KBr): 1013; 1068; 1265; 1466; 1514; 1601; 1655; 1748.

Preparation 18: 5-ethyl-4,5-dihydro-5-hydroxy-12-methyl-1H-oxepino [3\4':6,7]

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

The method described in examples 17.b., 17.c and 17.d. is applied to 2-acetylaniline to produce 2-chloro-4-methyl-3-quinolinemethanol. The latter is coupled to compound (M) as described in step 4.j according to Preparation 4. The resulting coupled product is cyclized according to the method described in step 4.k. A yellow solid is obtained, m.p. > 260°C.

NMR 1H (DMSO): 0.87 (t, 3H); 1.87 (q, 2H); 2.78 (s, 3H); 2.80 (d, 1H); 3.55 (d, 1H); 5.27 (s, 2H); 5.42 (d, 1H); 5.52 (d, 1H); 6.04 (s, 1H); 7.39 (s, 1H); 7.75 (t, 1H);

7.88 (t, 1H); 8.13 (d, 1H); 8.25 (d, 1H).

NMR-C 13 (DMSO): 8.23; 36.26; 42.36; 62.00; 73.11; 78.65; 79.13; 79.25; 99.52; 122.36; 124.30; 127.67; 129.54; 129.55; 129.56; 140.11; 145.06; 148.07; 152.00; 155.79; 159.09; 171.89.

IR (KBr): 1649; 1751; 3404.

Preparation 19: 10-benzyloxy-5-ethyl-9-fluoro-4,5-dihydro-5-hydroxy-1H-oxepino[3',4':6,7]indolizino[1,2-6]quinoline- 3,15 (4H,13H)-dione The method exemplified in step 4.i. is applied to 3-fluoro-4-methoxy-acetanilide to produce 2-chloro-7-fluoro-6-methoxy-quinoline-3-carbaldehyde which is treated with an excess of boron tribromide in dichloromethane at ambient temperature for 24 hours. 2-Chloro-7-fluoro-6-hydroxy-quinoline-3-carbaldehyde is obtained and O-benzylated in dimethylformamide in the presence of benzyl bromide and potassium carbonate to produce 6-benzyl-oxy-2-chloro-7-fluoro-quinoline- 3-carbaldehyde which is reduced with sodium borohydride in methanol to produce the corresponding quinoline methanol. The latter is coupled with compound (M) as described in step 4.j according to Preparation 4. The resulting coupled product is cyclized according to the method described in step 4.k. A yellow solid is obtained, m.p. > 275°C.

NMR-1H (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).

Preparation 20: 5-ethyl-9-fluoro-4,5-dihydro-5,10-dihydroxy-1 H -oxepino

[3',4':6,7]indolizino[1,2-6]quinoline-3,15(4H,13H)-dione

The compound of Preparation 19 (0.79 mmol) dissolved in trifluoroacetic acid (15 ml) is treated with hydrogen using 10% palladium on carbon (60 mg). A yellow solid is obtained, m.p. > 275°C.

NMR-1H (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).

The preparations indicated above will form the basis for illustrating the invention in the examples that follow.

EXAMPLE 1:

5-ethyl-9,10-difluoro-4,5-dihydro-5-(2-amino-1-oxoethoxy)-1 H -oxepino [ 3' A' :6, 7] indolizino [1, 2- b ] quinoline-3,15 (4H,13H)-dione

a. 5-Ethyl-9,10-difluoro-4,5-dihydro-5-(2-(f-butyloxycarbonyl-amino)-1-oxoethoxy)-1H-oxepino [3',4':6,7 ] indolizino [1,2-6] quinoline-3,15 (4H,13H)-dione, hydrochloride

A mixture of 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-1 H -oxepino [3',4':6,7]

indolizino[1,2-b]quinoline-3,15(4H,13H)-dione (200 mg, 0.526 mmol, obtained according to Preparation 4), A/-Boc-glycine (185 mg, 1.051 mmol) and a catalytic amount of 4-dimethylaminopyridine (20 mg) in anhydrous pyridine (10 mL) is treated at 0°C and under argon with dicyclohexylcarbodiimide (239 mg, 1.16 mmol), then stirred at ambient temperature for 48 h. The volatiles are driven off under vacuum and the residue chromatographed (SiO 2 , 1% methanol in chloroform) to produce the desired intermediate (40 mg, 14%), a yellow solid.

NMR-1H (CDCl3): 1.20 (t, 3H); 1.38 (s, 9H); 1.40-1.70 (m, 2H); 3.10 (d, 1H); 4.00 (d, 2H); 4.30 (d, 1H); 5.00 (h, 1H); 5.20 (s, 2H); 5.30-5.90 (dd, 2H); 7.20 (s, 1H); 7.50-8.10 (m, 2H); 8.30 (p, 1H).

b. 5-ethyl-9,10-difluoro-4,5-dihydro-5-(2-amino-1-oxoethoxy)-1H-oxepino

[3',4':6,7] indolizino [1,2-b] quinoline-3,15 (4H,13H)-dione, hydrochloride

The intermediate obtained above (40 mg, 0.072 mmol) in solution in dichloromethane (10 mL) is kept at 0°C and dioxane saturated with hydrogen chloride (8 mL) is added dropwise. Yellow suspension which is formed is subjected to agitation for 2 hours, then the volatiles are driven off under vacuum. The residue, taken up in water (5 ml), is washed with dichloromethane (3 x 30 ml). The aqueous phase is frozen and lyophilized to produce the expected salt, a hygroscopic yellow solid (20 mg, 50%).

NMR-1H (CDCl3 ): 1.00 (t, 3H); 2.15 (m, 1H); 2.30 (m, 1H); 3.60 (d, 1H); 3.90 (d, 1H); 4.15 (s, 2H); 5.10 (s, 2H); 5.40 (d, 1H); 5.70 (d, 2H); 7.40 (s, 1H); 7.80 (m, 2H); 8.50 (p, 1H).

EXAMPLE 2:

5-ethyl-9,10-difluoro-4,5-dihydro-5-(2-amino-1-oxopropoxy)-1H-oxepino [3',4':6,7] indolizino [1,2- b] quinoline-3,15 (4H,13H)-dione

The method according to example 1 is applied to 5-ethyl-9,10-difluoro-4,5-dihydro-5-hydroxy-1H-oxepino [3',4':6,7] indolizino [1,2-b] quinoline-3,15 (4H,13H)-dione using /V-Boc-b-alanine instead of A/-Boc-glycine, then the Boe protector of the intermediate thus obtained is cleaved by treatment with trifluoroacetic acid in dichloromethane. The volatile substances are evaporated under vacuum and the residue is taken up in dichloromethane. The resulting solution is washed with dilute bicarbonate, dried and evaporated. A yellow solid is obtained.

When applying the method of Examples 1 and 2 to other compounds, similar results are obtained. In this way, a whole class of campocin analogues becomes available in "prodrug" form.

EXAMPLE 3: 1,8-diethyl-8,9-dihydro-8-hydroxy-2H,10H,12H-[1,3]oxazino[5,6-r] oxepino [3\4':6,7]indolizino [1,2-b]quinoline-10,13(15H)-dione

A suspension of 5-ethyl-4,5-dihydro-5,10-dihydroxy-1H-oxepino[^'Æ^indolizino[1,2-b]quinoline-3,15(4H,13H)-dione (84 mg obtained according to Preparation 5) in acetic acid (2.5 ml) is treated with 1,3,5-triethylhexahydrotriazine (0.5 ml). The reaction mixture is stirred at 70°C for 30 minutes, then evaporated under vacuum. The residue is taken up in ethanol, filtered and washed with ether. A solid is obtained, m.p. > 275°C.

NMR-1H (DMSO): 0.87 (t, 3H); 1.50 (t, 3H); 1.85 (q, 2H); 2.77 (q, 2H); 3.05 (d, 1H); 3.47 (d, 1H); 4.37 (s, 2H); 5.00 (p, 2H); 5.22 (s, 2H); 5.45 (dd, 2H); 6.00 (p, 1H); 7.34 (s, 1H); 7.36 (d, 1H); 7.93 (d, 1H); 8.53 (p, 1H).

NMR-C 13 (DMSO): 8.46; 13.48; 36.46; 42.49; 45.49; 46.44; 50.75; 61.43; 73.33; 82.06; 99.02; 112.90; 122.00; 122.98; 125.42; 127.04; 129.04; 130.20; 144.09; 144.97; 149.87; 152.92; 155.98; 172.07.

IR (KBr): 1045; 1215; 1502; 1604; 1657, 1722.

EXAMPLE 4: 8-ethyl-8,9-dihydro-8-hydroxy-1-methyl-2H,10H,12H-[1,3]oxazino[5,6-f] oxepino [3',4':6, 7] indolizino [1,2-b] quinoline-10,13 (15H)-dione

A suspension of 5-ethyl-4,5-dihydro-5,10-dihydroxy-1H-oxepino[^'^TJindolizino[1,2-b]quinoline-3,15(4H,13H)-dione (200 mg obtained according to Preparation 5) in acetic acid (5 ml) is treated with hexahydro-1,3,5-trimethyltriazine (110 mg). The reaction mixture is stirred at 70°C for 30 minutes, then evaporated under vacuum. The residue is taken up in ethanol, filtered and washed with ether. A solid is obtained, m.p. > 275°C.

NMR-1H (DMSO): 0.87 (t, 3H); 1.85 (q, 2H); 3.04 (d, 1H); 3.48 (d, 1H); 4.33 (s, 2H); 4.93 (s, 2H); 5.28 (s, 2H); 5.45 (dd, 2H); 6.01 (s, 1H); 7.35 (s, 1H); 7.38 (d, 1H); 7.94 (d, 1H); 8.49 (p, 1H).

NMR-C 13 (DMSO): 8.46; 36.43; 37.85; 42.55; 48.68; 50.79; 61.43; 73.35; 83.82; 99.04; 112.49; 122.04; 123.00; 125.46; 127.14; 129.07; 130.27; 144.99; 149.95; 152.46; 155.99; 172.09

IR (KBr): 1047; 1058; 1219; 1246; 1295, 1439; 1504; 1604, 1655, 1735.

EXAMPLE 5: 8-ethyl-8,9-dihydro-8-hydroxy-1-benzyl-2H,10H,12H-[1,3]oxazino[5,6-r] oxepino [3\4':6,7 ] indolizino [1,2-b] quinoline-10,13 (15H)-dione

A suspension of 5-ethyl-4,5-dihydro-5,10-dihydroxy-1H-oxepino[3',4':6,7]indolizino[1,2-b]quinoline-3,15 (4H, 13H)-dione (200 mg obtained according to Preparation 5) in acetic acid (5 ml) is treated with 1,3,5-tribenzylhexahydrotriazine (285 mg). The reaction mixture is stirred at 70°C for 30 minutes, then evaporated under vacuum. The residue is taken up in ethanol, filtered and washed with ether. A solid is obtained, m.p. > 275°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.05 (d, 1H); 3.47 (d, 1H); 3.96 (s, 2H); 4.33 (s, 2H); 5.04 (s, 2H); 5.17 (s, 2H); 5.44 (dd, 2H); 6.01 (s, 1H); 7.38 (m, 6H); 7.42 (d, 1H); 7.97 (d, 1H); 8.42 (p, 1H).

NMR-C 13 (DMSO): 8.42; 19.96; 36.45; 42.51; 46.36; 50.78; 55.38; 61.39; 73.31; 99.00; 112.55; 122.01; 123.08; 125.38; 127.09; 127.47; 128.70; 129.14; 130.35; 128.40; 139.19; 144.18; 149.99: 152.84; 155.92; 159.24; 172.05.

IR (KBr): 1056; 1205; 1225; 1248; 1504; 1535; 1599; 1655; 1726.

EXAMPLE 6: 8-ethyl-8,9-dihydro-4-fluoro-8-hydroxy-1-benzyl-2H,10H,12H-[1,3]oxazino[5,6-f] oxepino [3', 4':6,7]indolizino[1,2-b]quinoline-10,13(15H)-dione A suspension of 5-ethyl-9-fluoro-4,5-dihydro-5,10-dihydroxy-1 H -oxepino[3',4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione (200 mg obtained according to Preparation 20) in acetic acid (5 ml) is treated with 1,3,5-tribenzylhexahydrotriazine (285 mg). The reaction mixture is stirred at 70°C for 30 minutes, then evaporated under vacuum. The residue is taken up in ethanol, filtered and washed with ether. A solid is obtained, m.p. > 250°C.

NMR-1H (DMSO): 0.85 (t, 3H); 1.85 (q, 2H); 3.05 (d, 1H); 3.48 (d, 1H); 3.95 (s, 2H); 4.45 (s, 2H); 5.20 (s, 4H); 5.45 (dd, 2H); 6.05 (s, 1H); 7.40 (s, 7H); 7.90 (d, 1H); 8.45 (p, 1H).

IR (KBr): 1248; 1451; 15001; 1598; 1657; 1727.

PHARMACOLOGICAL STUDY OF THE PRODUCTS ACCORDING TO THE INVENTION

Relaxation activity assay of DNA induced by topoisomerase 1.

All reactions are carried out 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 tetraacetic acid (EDTA), 30 µg/ 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 are initially dissolved in dimethylsulfoxide (DMSO) or in water for hydro-soluble compounds, other dilutions being carried out with distilled water. The final concentration of DMSO does not exceed 1% (v/v). The reaction is initiated by the addition of one unit of purified calf thymus DNA topoisomerase 1 (Life Technologies/Gibco-BRL, Paisley, United Kingdom) so that the reaction is completed within 15 minutes at 37° C. The reactions are stopped by the addition of 3 µl of a mixture containing 1% dodecyl sodium sulfate in 1%, 20 mM EDTA and 500 µg/ml K proteinase (Boehringer Mannheim, Meylan, France). After a further incubation period of 30 min at 37°C, 2 µl of an application buffer containing 10 mM of Na 2 HPO 4 , 0.3% bromophenol blue in 16% Ficoll is added to samples electrophoresed in 1.2% agarose gels 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 chlorquine. Gels are labeled with 2 µg/ml ethidium bromide, photographed under UV light at 312 nm with a charge-coupled device (ccd) camera, and fluorescence intensity is measured using a bioProfil bead-forming analyzer (Vilber Lourmat, Lyon, France) with an image for to determine the percentage of relaxed DNA.

In each experiment, supercoiled plasmid DNA is incubated alone or with topoisomerase 1. The reaction is completed within 15 minutes. For each compound to be tested or controlled (carrier alone is designated control), superspun plasmid DNA is incubated in the presence of the maximum concentration selected for the experiment of the compound to be tested or control without enzyme or in the presence of the compound to be tested, in concentrations varying from 1 uM to 200 uM or control in the presence of enzymes. As indicated in Table I, Examples 3 to 6 inhibit the relaxation activity obtained by topoisomerase 1 in a concentration-dependent manner.

Claims (10)

1. Compound, characterized in that said compound has formula (I): in racemic or enantiomeric form or any combination of these forms, wherein Rt represents the ethyl group; R 2 represents H or halogen; R3 represents H, lower alkyl, halogen or OR6 where R6 represents H, lower alkyl or lower arylalkyl; R 4 represents H or -(CH 2 )mNR 6 R 7 , where R 6 and R 7 represent, independently, H or lower alkyl; R 5 represents H, lower alkyl or -(CH 2 ) n [N«X], substituted or unsubstituted and [N*X] represents piperidyl, morpholinyl, piperazinyl or indolyl group and said substituent is a lower alkyl; or R 3 and R 4 form an oxazine ring optionally substituted on the nitro atom with an R 9 residue representing a lower alkyl; Rp represents H or an easily cleavable group of formula -C(0)-(A1)-NH2 where Ai represents (CH 2 ) m or a branched lower alkylene radical; m is a number between 0 and 6; n is 1 or 2; and it is understood that when Rp is a hydrogen atom, R3 and R4 together form an optionally substituted oxazine ring, and when the designation lower is associated with alkyl or alkylene, it means no more than 6 carbon atoms; or a pharmaceutically acceptable salt thereof. 2. Compound according to claim 1, characterized in that R3 and R4 form an optionally substituted oxazine ring; or a pharmaceutically acceptable salt thereof. 3. Compound according to claim 1, characterized in that Rp is an easily cleavable group; or a pharmaceutically acceptable salt thereof. 4. Compound according to claim 2, characterized in that said compound is selected from compounds corresponding to the following formulas: 1,8-diethyl-8,9-dihydro-8-hydroxy-2H,10H,12H-[1,3]oxazino [S^-rJoksepinop^eyjindolizinoIl ,2-Z>]quinoline-10,13(15H)-dione; 8-ethyl-8,9-dihydro-8-hydroxy-1-methyl-2H,10H,12H-[1,3]oxasino [5,6-/]oxepino[3',4':6,7]indolizino[1,2-b]quinoline-10,13(15HJ-dione; 8-Ethyl-8,9-dihydro-8-hydroxy-1-benzyl-2H, 10H, 12H-[1,3]oxazin[5,6-/] oxepino[3',4':6,7]indolizino[1,2-b]quinoline-10,13(15H)-dione; 8-ethyl-8,9-dihydro-4-fluoro-8-hydroxy-1-benzyl-2H,10H,12H-[1,3]oxazine 5,6-r]oxepino[3',4':6,7]indolizino[1,2-b]quinoline-10,13[15HJ-dione; or a pharmaceutically acceptable salt thereof. 5. Compound according to claim 3, characterized in that said compound is selected from compounds corresponding to the following formulas: 5-ethyl-9,10-difluoro-4,5-dihydro-5-(2-amino-1-oxoethyl)-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-(2-amino-1-oxopropoxy)-1H-oxepino [3',4':6,7]indolizino[1,2-b]quinoline-3,15(4H,13H)-dione; or a pharmaceutically acceptable salt thereof. 6. Medicine, characterized in that it comprises a compound according to any one of the preceding claims or a pharmaceutically acceptable salt thereof. 7. Pharmaceutical composition, characterized in that it comprises, as active ingredient, at least one of the compounds according to any one of claims 1 to 5. 8. Use of a compound according to any one of claims 1 to 5 for the production of antitumor drugs. 9. Process for the preparation of compounds of formula la similar to the products of formula I where R3 and R4 form an oxazine ring according to any one of claims 1, 2 or 4, characterized in that a B-hydroxylactone compound of general formula D where R3 is a hydroxyl radical, R4 is H, and R, and R2 has the meaning indicated above is treated with primary amino, under Mannich's conditions, to obtain a B-hydroxylactone compound of general formula la where R 1 , R 2 , R 5 and R 9 have the meanings given above. 10. Process for the preparation of compounds of formula Ib corresponding to the products of formula I where Rp is not a hydrogen atom, according to any one of claims 1, 2, 3 or 5, characterized in that the compound of general formula D or let is acylated with a derivative of the -C(0)-ArNH2 residue as defined in claim 1, to produce the B-hydroxylactone compound of general formula I wherein Rp is different from H.