HRP20000110A2 - Optically pure camptothecin analogues, optically pure synthesis intermediate and method for preparing the same - Google Patents

Description

Camptothecin is a natural compound first isolated from the leaves and bark of a Chinese plant called Camptotheca acuminata (see Wall et al. J. Amer. Chem. Soc. 88:3888 (1966)). Camptothecin is a pentacyclic compound composed of an indolizino[1,2-b]quinoline fragment joined to a "-hydroxyacetone" by means of six bonds. The carbon at position 20, which carries the α-hydroxy group, is asymmetric and gives the molecule rotational power. The natural form of camptothecin has an absolute "S" configuration as far as carbon 20 is concerned and corresponds to the following formula:

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Camptothecin has anti-proliferative activity on several cancerous cell lines, including human colon, lung and breast tumor cell lines (Suffnes, M et al: The Alkaloids Chemistry and Pharmacology, Bross A., ed., Vol. 25, p. 73 (Academic Press, 1985)). It has been suggested that the anti-proliferative activity of camptothecin is related to the inhibitory activity of DNA topoisomerase I.

There are indications that for in vivo and in vitro activation of camptothecin there is an absolute need for α-hydroxylactone (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)). More recently, however, the patentee has perfected a new class of camptothecin analogs in which the ß-hydroxylacetone replaces the natural α-hydroxylacetone of camptothecin (see patent application WO 97/00876).

The field of this invention represents a new process for the preparation of enantiomerically pure synthetic intermediates as well as enantiomerically pure camptothecin analogues.

First, the field of the invention is represented by new analogs of camtothecin which are different from any known compound and are characterized by the corresponding formulas (I) and (II) presented below

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or characterized by being different salts of a compound of formula (II) for example as formula (III) presented below

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A key intermediate in the synthesis of this type of optically pure compounds is the product of general formula M presented below

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in which R represents a linear or branched alkyl radical containing 1 to 10 carbon atoms. It is preferred that R represents an alkyl radical.

Compounds from formulas (I) and (II) can be prepared in the following way:

- compound formula

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is combined individually with one or the other of the compounds represented in formulas N1 or N2, presented below:

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to obtain a compound of formula O1 or a compound of formula O2:

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- compound O1 is then cyclized, resulting in the compound of formula (I); by cyclization of the compound O2, a compound of formula (II) is formed, which, after conversion into a salt, gives a compound of formula (III).

The formation of compounds O1 and O2 starting with a compound of the general formula M in which R represents an ethyl radical while N1 and N2 are obtained by a treatment known to those skilled in the art as the Mitsunobu reaction (see 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 by final treatment with a phosphine, for example triphenylphosphine and an azodicarboxylate derivative, for example diethyl or diisopropyl azodicarboxylate in an aprotic solvent such as, for example, tetrahydrofuran or N,N -dimethylformamide. Cyclization of compounds O1 and O2 to give compounds of formula (I) or (II) is best carried out in the presence of a catalyst containing palladium (for example palladium diacetate) under basic conditions (facilitated for example by means of alkyl acetate optionally mixed with a change agent phase, such as, for example, tetrabutylammonium bromide), in an aprotic solvent such as acetonitrile or N,N-dimethylformamide at a temperature ranging between 50°C and 120°C (R. Grigg et al., Tetrahedron 46, page 4003 (1990)).

The invention also provides, as a new industrial product, a compound of the general formula M as indicated above. This product can be used for the production of medicines.

The compound of formula M was synthesized according to a new process that is part of this invention and includes the following successive stages:

- racemic t-butyl ester presented below

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(for its preparation see in particular patent application WO 97/00876) is treated with trifluoroacetic acid for 18 h at ambient temperature in order to produce the corresponding carboxylic acid;

- after that, the quinidine salt of the acid obtained immediately before was heated in isopropyl alcohol at a temperature higher than 30°C, preferably at 50°C, before the reaction mixture was left to cool at ambient temperature in order to crystallize the salt of one of the enantiomers of the above-mentioned acid while the salt of the other enantiomer, whose anion is represented below, remains in solution:

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- a solution in isopropyl alcohol of the salt of the enantiomer that did not crystallize was concentrated and treated with hydrochloric acid and mixed, resulting in a compound of the general formula A presented below

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- the compound of general formula A was then contacted with palladium on wet carbon, after which ammonium formate or formic acid was added to the mixture to produce the debenzylated product of the general formula presented below

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- then the compound of the general formula B is cyclized by means of the action of dicyclohexylcarbodiimide to obtain the lactone compound of the general formula C presented below

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- finally, the -OCH3 group of the lactone compound of the general formula C is converted into a carbonyl by means of sodium iodide and trimethylsilyl chloride in order to obtain the compound of the general formula M presented below

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As for the process described above, the reaction leading from the compound of the general formula A to the compound of the general formula B is preferably carried out in methanol and by heating the reaction mixture to about 40°C after the addition of ammonium formate. The cyclization of the compound of the general formula B to obtain the compound C can be carried out in THF, preferably at a temperature of about 50°C, while the reaction leading from the compound of the general formula C to the compound of the general formula M is preferably carried out at ambient temperature using acetonitrile as solvent.

In certain cases when R represents an ethyl group, the compound of formula M is synthesized according to a process that contains the following stages:

- racemic t-butyl ester presented below

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- (for its preparation see in particular patent application WO 97/00876) it was treated with trifluoroacetic acid for 18 hours at ambient temperature in order to obtain the corresponding carboxylic acid;

- the quinidine salt of 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxypentanioic acid was heated in isopropyl alcohol at a temperature higher than 30°C, preferably at about 50°C, before the reaction the mixture is cooled to ambient temperature to crystallize the salt of the (+) enantiomer of 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid, while the salt of the (-) isomer, whose anion is presented below, remains in solution

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- a solution in isopropyl alcohol of the (-) enantiomer salt of 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid was concentrated and treated with hydrochloric acid, after which it was mixed to form the compound of formula A' presented below

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- compound A' was then brought into contact with palladium on wet carbon after which ammonium formate or formic acid was added to the mixture to give the debenzylated product B' presented below

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- after that, the compound of formula B' is cyclized with the help of the action of dicyclohexylcarbodiimide, in order to obtain the lactone compound of formula C' presented below

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- finally, the -OCH3 group of the lactose compound of formula C' was converted into carbonyl with the help of sodium iodide and trimethylsulfyl chloride to obtain (+)-5-ethyl-5-hydroxy-1,3,4,5,8,9 -hexahydrooxepino[3,4-c]pyridine-3,9-dione (or (+)-EHHOPD) presented below

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A compound of formula N1 can be obtained starting with an aniline of formula P1 presented below

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according to the following process: the aniline of formula P1 is N-acetylated by treatment with an acetylating agent such as, for example, acetic anhydride. The thus obtained acetanilide was treated at a temperature between 50°C and 100°C, preferably at 75°C, with the aid of a reagent known to those skilled in the art as Vilsmeyer's reagent (obtained by the action of phosphoryl oxychloride on N ,N-dimethylformamide at a temperature ranging between 0°C and 100°C) to give the corresponding 2-chloro-3-quinolinecarbaldehyde (for examples see Meth-Cohn et al. J.Chem. Soc., Perkin Trans. I p.1520 (1981); Meth-Cohn et al. J. Chem. Soc., Perkin Trans. I p. 2509 (1981); and Nakasimhan et al. J. Am. Chem. Soc., 122. p. 4431 (1990)). The compound 2-chloro-6,7-difluoro-3-quinolinecarbaldehyde can be readily reduced to the corresponding 2-chloro-6,7-difluoro-3-quinolinemethanol of the formula N1 under the usual conditions known to those skilled in the art as treatment in an alcoholic solvent (for example methanol) using sodium borohydrate at a temperature between 0°C and 40°C.

The compound of formula N2 can be obtained according to the following process: aniline of formula P2 presented below:

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it is ortho-acetylated by reaction with chloroacetonitrile in the presence of boron trichloride and other Lewis acids such as aluminum trichloride, titanium tetrachloride or diethylaluminum chloride in an aprotic solvent followed by hydrolysis (see Sugasawa T. et al. J. Am. Chem. Soc 100 p. 4842 (1978)). The intermediate thus obtained is then treated with ethylmalonyl chloride in an aprotic solvent such as acetonitrile in the presence of a base such as triethylamine, followed by treatment with an alkaline alcoholate, for example, sodium methylate in methanol to give ethyl 7-chloro- 4-chloromethyl-6-methyl-2-oxo-1,2-dihydro-3-quinolinecarboxylate. This compound was converted to ethyl 2,7-dihydro-4-chloromethyl-6-methyl-3-quinolinecarboxylate by treatment with phosphoryl oxychloride. After that, nucleophilic substitution was carried out by treatment with 4-methylpiperidine. The ethyl carboxylate function is then reduced with diisobutylaluminum hydride in an aprotic solvent such as dichloromethane to give the compound of formula N2. The order in which the last two steps are performed can be changed as needed.

Analogs of compounds of intermediates of type N1 and N2 are described in the literature, especially in the application PCT 95/05427.

The compound of formula (II) can be converted into the form of pharmaceutically acceptable salts using conventional procedures. Acceptable salts include, by way of example and in a non-limiting sense, addition salts with inorganic acids such as chlorides, sulfates, phosphates, diphosphates, hydrobromides and nitrates or with organic acids such as acetates, malates, fumarates, tartrates, succinates, citrates, lactates, methane sulfonates, p-toluenesulfonates, pamoates, salicylates, oxalates and stearates. For other examples of pharmaceutically acceptable salts, see "Pharmaceutical Salts", J. Pharm. Sci. 66:1 (1977).

The compounds of the present invention possess useful pharmaceutical properties. Thus, the compounds of the presented invention have an inhibitory effect on topoisomerases I and/or II and anti-tumor activity. The current state of the art suggests that the compounds of the present invention possess anti-parasitic and/or anti-viral activity. In this sense, the compounds of the presented invention can be used for various therapeutic purposes.

An illustration of the pharmaceutical characteristics of the compounds of the present invention will be presented in the experimental section.

The compounds can inhibit topoisomerases, for example of type I and/or II, in a patient, for example in a mammal such as a human, by treating such patient with a therapeutically active amount of a compound of formula (I) or formula (II), or with a pharmaceutically acceptable salts of the compound of formula (II) or also any mixture of the aforementioned compounds.

The compounds of the presented invention possess anti-tumor activity. These compounds can be used for the treatment of tumors, for example tumors that express topoisomerases, in a patient by treating said patient with a therapeutically active amount of a compound of formula (I) or of formula (II), or with a pharmaceutically acceptable salt of a compound of formula (II), or also using a mixture of the aforementioned compounds. Examples of tumors or other types of cancer include various types of cancer of the esophagus, stomach, small intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, cervix, lining of the uterus, ovary, prostate, testicle, bladder, kidney , liver, pancreas, bone, connective tissue, skin, eyes, brain and central nervous system, also thyroid cancer, leukemia, Hodgkin's disease, lymphomas other than those associated with Hodgkin's disease, multiple myeloma and others.

Such compounds can also be used for the treatment of parasitic infections in a way that they inhibit blood flagella (for example in infections like trypanosomia or leishmaniasis) or in a way that they inhibit plasmodia (as in the example of malaria), but also the treatment of viral infections and viral diseases.

These characteristics make the products of formulas (I) and (II) suitable for pharmaceutical use. The subject of the presented invention is also, as drugs, the products of formulas (I) and (II) described above, but also addition salts of pharmaceutically acceptable mineral or organic acids of products of formula (II), as well as, for example, the salts of formula (III) described above , or also any mixture of the compounds mentioned so far. Similarly, the invention relates to pharmaceutical compositions that contain at least one of the drugs described above as an active ingredient.

Accordingly, the invention relates to pharmaceutical compositions containing the compound of the presented invention or one addition salt with a pharmaceutically acceptable acid, in combination with a pharmaceutically acceptable carrier according to the chosen method of administration (for example oral, intravenous, intraperitoneal, intramuscular, through the skin or subcutaneously). The pharmaceutical composition (for example therapeutic) can be in the form of solid, liquid, liposomal or lipid micelles.

The pharmaceutical composition may be in the form of a solid, such as powder, pill, granule, tablet, liposome, gelatin capsule or suppository. Pills, tablets or gelatin capsules may be coated with a substance capable of protecting the composition from the action of gastric acid or enzymes in the patient's stomach for a significant period of time to ensure that this composition passes into a non-digestible form later in the small intestine. The compound may also be administered locally, for example, at the same location as the tumor. The compound may also be administered by a sustained-release method (for example, a sustained-release composition or an infusion pump). A suitable solid carrier may be, for example, calcium phosphate, magnesium stearate, magnesium carbonate, talc, various sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine and wax. Pharmaceutical compositions containing a compound according to the present invention may also be presented in liquid form such as, for example, solutions, emulsions, suspensions or sustained release preparations. 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 various proportions in water.

The subject of this invention is also the use of products of formulas (I) and (II) as defined above, or addition salts with pharmaceutically acceptable mineral or organic acids of products of formula (II), such as for example the salt of formula (III), described before, or also a mixture of the above-mentioned compounds, for the preparation of drugs that can inhibit topoisomerases, more precisely topoisomerases of type I or II, drugs that can be used in the treatment of tumors, drugs that can be used in the treatment of parasitic infections (inhibition of invaders), as well as medicines that can be used to treat viral infections or viral diseases.

The dose of the compound according to the presented invention intended for the treatment of diseases or disorders mentioned above varies according to the method of administration, age and body weight of the patient being treated in the same way as the condition of the latter and will be finally determined based on the advice of a doctor or veterinarian. The amount determined in accordance with the recommendations of the attending physician or veterinarian is referred to herein as the “effective therapeutic amount”.

Unless otherwise indicated, all technical and scientific terms herein have the same meaning as that which is customary and understood by a person of ordinary skill in the field to which the invention belongs. Similarly, all publications, patent applications, all patents and all other references mentioned herein are presented in such a way as to constitute references themselves.

The following examples are presented to illustrate the above procedure and should in no way be construed as limiting the scope of the present invention.

EXPERIMENTAL PART:

Example 1: (+)-5-Ethyl-5-hydroxy-1,3,4,5,8,9-hexahydrooxepino[3,4-c]pyridine-3,9-dione [(+)-EHHOPD]

1.a. Quinidine salt of 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid:

Tertbutyl 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoate (40g; 100 mmol) was treated with trifluoroacetic acid (150 ml), and the reaction mixture was stirred for 18 hours at at a temperature of 20°C. After evaporation of trifluoroacetic acid, methylene chloride (200 ml) was poured in, and saturated sodium bicarbonate solution was added to pH = 7.5-8. After decantation, the aqueous phase was washed with 100 ml of methylene chloride. The pH value of the aqueous phase was then adjusted to 1 by the addition of a 6 N hydrochloric acid solution. After that, the product was extracted from the aqueous phase using methylene chloride (2 times per 200 ml). The solution was dried over magnesium sulfate and concentrated. The thus obtained 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid (31.1g; 90 mmol), was dissolved in isopropyl alcohol (30 ml), treated with quinidine solution (29, 2g; 90 mmol) in isopropyl alcohol (30 ml) at a temperature of 50°C with stirring until complete dissolution. After that, the reaction mixture was allowed to drop to 40°C, stirring was stopped and the temperature was lowered to 20°C. The mixture was left at a temperature of 0°C without stirring and kept at that temperature for a period of 16 hours. After that, the temperature was raised to 20°C, and stirring was continued until crystallization. Such a mixture was dissolved in isopropyl alcohol and then filtered. The precipitate was then washed with isopropyl alcohol. The salt of the (+) enantiomer precipitates (m = 26.6g), while the salt of the (-) enantiomer remains in the isopropyl alcohol solution. In this way, a filtrate was obtained which was concentrated to produce oil (34g) which can be used in the next phase without additional purification.

The products were analyzed by HPLC on a 5: CHIRAL AGP column (10 cm x 4 mm), eluted with a buffer mixture of isopropyl alcohol/water/phosphate in the ratio 30/920/50, pH = 6.5 at a flow rate of 1 ,2 ml/min, and UV detection at 280 nm. The residual times obtained are 6.4 minutes for the (-) enantiomer and 2.8 minutes for the (+) enantiomer. The ratio (-) enantiomer / (+) enantiomer is 83/17.

1.b. (-)-3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid

The solution of the quinidine salt (-) enantiomer of 3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid in isopropyl alcohol (Phase 1.a) is concentrated. The concentrate was dissolved in 270 ml of methylene chloride and 270 ml of 1 N hydrochloric acid solution. The reaction mixture was stirred for 16 hours at a temperature of 20°C. After decantation, the organic phase was concentrated, the concentrate was dissolved in methanol so that it could be used in the next phase.

13.5 g of product (87% yield) of (-) enantiomer / (+) enantiomer in the ratio 85/15 were obtained.

The lag time in HPLC (same protocol as in phase 1.a.) is:

- (-) enantiomer: 6.4 minutes

- (+) enantiomer: 2.8 minutes

1. c. (+)-5-Ethyl-5-hydroxy-1,3,4,5,8,9-hexahydrooxepino[3,4-c]pyridine-3,9-dione:

(-)-3-(3-benzyloxymethyl-2-methoxy-4-pyridyl)-3-hydroxy-pentanoic acid (13.5 g; 39 mmol; Phase 1.b) was added to 87 ml of methanol solution. The solution was poured under nitrogen onto 10% palladium on wet carbon at 50% (27.7 g; 13 mmol). The reaction mixture was stirred for about 5 min, after which it was poured into a solution of ammonium formate (11.5 g; 183 mmol) in 135 ml of methanol. The reaction mixture was stirred for the next 30 min allowing the temperature to rise to 40°C at which temperature the solution was heated for 30 min. The mixture was then filtered through a Clarcel plate after which it was concentrated. 40 ml of toluene was added which was later evaporated; the operation was repeated to eliminate the methanol. The residue thus obtained was dissolved in 45 ml of THF. Then a solution of dicyclohexylcarbodiimide (7.180 g; 34.5 mmol) in 20 ml THF was added to the mixture. The reaction mixture was heated to 50°C for 1 hour. The mixture was stirred at a temperature of 20°C, after which the dicyclohexylurea was filtered. The filtrate was concentrated to dryness. The residue was added to 46 ml of acetonitrile solution, after which 6.0 g (40.5 mmol) of sodium iodide and 5.13 ml (40.5 mmol) of trimethylsilyl chloride were added. The reaction mixture was kept at ambient temperature for 5 hours under stirring conditions. After that, 28 ml of acetonitrile and 5.6 ml of water were added. The thus obtained precipitate was filtered, after which it was dissolved in 1 ml of water, and the pH was adjusted to 7.5 by adding ammonium hydroxide solution. The obtained solid was filtered and dried. Thus, a mass of 4.2 g of the final product was obtained with a yield of 34% and a ratio of (+) enantiomer / (-) enantiomer of 88.4/11.6.

The HPLC analysis was carried out in a Chiralcel OD column of 25 cm x 4.6 mm, the eluates used were heptane 600 and ethanol 400, the flow rate was 1 ml/min 210 nm. The resulting lag times are:

- (+) enantiomer: 7.1 minutes

- (-) enantiomer: 9 minutes.

The product was dissolved in acetone (40 ml), after which water (150 ml) was added. The reaction was allowed to precipitate, after which 3 g of product was obtained with a (+) enantiomer / (-) enantiomer ratio of 99.4/0.6.

NMR 1H (250 MHz, DMSO D 6 ): 0.8 (t, 3H, CH 3 -CH 2 ); 1.65 (m, 2H, CH2-CH3); 3.00-3.35 (q, 1H+1H, -CH2-C=O); 5.3 (q, 2H, CH2-O); 5.7 (s, -OH); 6.35 (d, aromatic 1H); 7.3 (d, aromatic 1H); 11.7 (s, N-H).

Example 2: (+)-5-ethyl-9,10-difluoro-5-hydroxy-4,5,13,15-tetrahydro-1H,3H oxepino[3',4':6,7]indolizino[1, 2-b]quinoline-3,15-dione

2.a. N-(3,4-difluorophenyl)acetamide:

A mixture of 3,4-difluoroaniline (50 ml; 0.5 mol) and triethylamine (70 ml; 0.5 mol) in dichloromethane (1.5 L) was cooled using a cold bath. Acetic anhydride (71.5 ml; 0.75 mol) was added dropwise, after which the reaction mixture was stirred for 1 hour at ambient temperature. The resulting solution was washed individually with water, 10% sodium bicarbonate solution and saturated with salt water. The organic part was dried over sulfate and concentrated under reduced pressure. The residue was suspended in pentane, filtered and dried under reduced pressure to give the title product (78 g; 91% yield) as an off-white solid (M.P. 126-127.5°C).

NMR 1H (DMSO): 2.15 (s, 3H); 7.10-7.65 (m, 2H); 7.65-8.10 (m, 1H); 10.30 (broad signal, 1H).

2.b. 2-chloro-6,7-difluoro-3-quinoline-3-carbaldehyde:

The general procedure described by Meth-Chon et al., J. Chem., was used. Soc. Perkin Trans. And, in 1981, 1520 and 2509 were used.

The product of Phase 2.a (32 g; 220 mmol) was added to Vilsmeyer's reagent dropwise under an argon atmosphere of phosphorus oxychloride (103 ml; 1.1 mol) in anhydrous DMF (34 ml; 440 mmol) cooled in an ice bath and stirred for 30 minutes before the reaction temperature was allowed to equalize to ambient. The resulting mixture was stirred at a temperature of 70°C for 16 hours. After returning the reaction to ambient temperature, the product was added dropwise into a mixture of water and ice (400 ml) and stirred for 2 hours. The thus obtained precipitate was filtered and washed with water, after which it was dried to obtain the above-mentioned product (9 g; 18% yield) in the form of a yellow solid (M.p. 226.5-229°C).

NMR 1H (DMSO): 8.17 (dd, 1H); 8.39 (dd, 1H); 8.97 (d, 1H); 10.34 (d, 1H).

IR (KBr): 888, 1061, 1262, 1507, 1691 cm-1.

2. c. 2-chloro-6,7-difluoro-3-quinolylmethanol:

A suspension of the product from Phase 2.b (9 g; 39 mmol) in methanol (400 ml) was treated with sodium borohydride (2 g; 53 mmol) at ambient temperature for half an hour. Excess borohydride was destroyed by treatment with acetic acid (2 ml). Volatile substances were removed under reduced pressure. The residue was dissolved in ethyl acetate (500 ml), and the mixture thus obtained was washed separately with a dilute solution of sodium bicarbonate, water and salt saturated with water, after which it was dried over magnesium sulfate and concentrated under reduced pressure. The residue was then recrystallized from 1,2-dichloroethane to give the above product (8 g; 80% yield) as a yellow-gray solid (mp 166.5-167°C).

NMR 1H (DMSO): 4.67 (d, 2H); 5.80 (t, 1H); 8.01 (dd, 1H); 8.22 (dd, 1H); 8.48 (s, 1H).

IR (KBr): 871, 1038, 1253, 1513 cm-1.

2. d. (+)-8-(2-chloro-6,7-difluoro-3-quinolinemethanol)-5-ethyl-5-hydroxy-1,3,4,5,8,9-hexahydrooxepino[3,4-c] pyridine-3,9-dione:

Under conditions of ambient temperature and argon atmosphere, a solution of (+)-EHHOPD (1.58 g; 7.08 mmol; Phase 1.c.), the product of Phase 2.c (1.62 g; 7.06 mmol) and tributylphosphine (1.91 ml; 7.87 mmol) in anhydrous DMF (30 ml) was added dropwise diethyl azodicarboxylate (1.24 ml; 7.87 mmol). The resulting mixture was then stirred for 16 hours. After that, the reaction mixture was evaporated to dryness under reduced pressure. The residue was purified by chromatography on a silica column (eluent: ethyl acetate). The resulting solid was then dissolved in diethyl ether, filtered and dried to give the above product (1.56 g; 51% yield) as an off-white solid (m.p. 196°C).

NMR 1H (DMSO): 0.84 (t, 3H); 1.74 (m, 2H); 3.02 (d, 1H); 3.34 (d, 1H); 5.29 (s, 2H); 5.31 (dd, 2H); 5.75 (s, 1H); 6.51 (d, 1H); 7.80 (d, 1H); 8.03 (dd, 1H); 8.07 (s, 1H); 8.17 (dd, 1H).

IR (KBr): 875, 1057, 1360, 1507, 1574, 1647, 1749 cm-1.

2.e. (+)-5-ethyl-9,10-difluoro-5-hydroxy-4,5,13,15,-tetrahydro-1H,3H-oxepino[3',4':6,7]indolizino[1,2 -b]quinoline-3,15-dione:

A mixture of products from Phase 2.d (1.53 g; 3.52 mmol; Phase 2.d.), tetrabutylammonium bromide (1.25 g; 3.87 mmol), potassium acetate (520 mg; 5.28 mmol), triphenylphosphine (180 mg; 0.70 mmol) and palladium (II) acetate (79 mg; 0.35 mmol) were mixed under the conditions of an agricultural atmosphere in anhydrous acetonitrile and heated to reflux for 22 hours. After the reaction mixture returns to ambient temperature, before chromatography on a silicon column (eluent: CH2Cl2/MeOH mixture in the ratio 98/2), concentration is carried out under reduced pressure conditions. The above-mentioned product is then formed (960 mg; yield of 68%; purity determined by HPLC: 97.1%). This product was now dissolved in anhydrous CH2Cl2 (100 mL) and stirring was continued for a period of 24 hours, after which filtration and drying under reduced pressure was resorted to to give the purified above product (850 mg; 61% yield) ; purity determined by HPLC: 99.6 %) in the form of a white solid.

NMR 1H (DMSO): 0.87 (t, 3H); 1.85 (m, 2H); 3.08 (d, 1H); 3.44 (d, 1H); 5.26 (s, 2H); 5.39 (d, 2H); 5.52 (d, 2H); 5.99 (broad signal, 1H); 7.39 (s, 1H); 8.15 (dd, 1H); 8.23 (dd, 1H); 8.68 (s, 1H).

IR (KBr): 871, 1261, 1512, 1579, 1654, 1746 cm-1.

Example 3: (+)1-[9-chloro-5-ethyl-5-hydroxy-10-methyl-3,15-dioxo-4,5,13,15-tetrahydro-1H,3H-oxepino[3',4 ':6,7]indolizino[1,2-b]quinolin-12-ylmethyl]-4-methyl-hexahydropyridinium chloride

3.a. 1-(2-amino-4-chloro-5-methylphenyl)-2-chloro-ethanone:

3-Chloro-4-methylaniline (44.4 ml; 0.366 mol) in 1,2-dichloroethane (440 ml) under argon atmosphere was cooled in an ice bath. The following substances were added dropwise to that mixture in the specified order: boron trichloride (1 M in heptane; 400 ml; 0.4 mol), chloroacetonitrile (28 ml; 0.44 mol) and diethylaluminum chloride (1 M in heptane ; 400 ml; 0.4 mol). During the addition of the mentioned substances, the temperature was maintained below 20°C. The mixture thus obtained was then heated at reflux for 3 hours, after which the temperature was reduced to a value of 10°C. Hydrolysis of the reaction mixture was carried out carefully using 2 N hydrochloric acid (240 ml) and the mixture was heated under reflux for 1 hour. Water (1 l) and ethyl acetate (1 l) were added, and the resulting mixture was stirred for about 15 min before separating the phases. The aqueous phase was re-extracted with ethyl acetate (200 ml) and the combined organic phases were washed with water (500 ml). After drying over magnesium sulfate, the organic phase is concentrated. The residue was dissolved in petroleum ether (the fraction has a boiling point of 45-60°C; 150 ml), and the resulting mixture was left to stand at a temperature of 4°C for 16 hours. The resulting precipitate was collected by filtration, washed with petroleum ether and dried under reduced pressure to give the above product (25 g; 31% yield). T.t. 129-130°C.

NMR 1H (DMSO): 2.20 (s, 3H); 4.98 (s, 2H); 6.90 (s, 1H); 7.15 (broad signal, 2H); 7.70 (s, 1H).

IR (KBr): 871, 1018, 1183, 1225, 1270, 1533, 1577, 1619, 1662 cm-1.

3.b. Ethyl 7-chloro-4-chloromethyl-6-methyl-2-oxo-1,2-dihydro-3-quinolinecarboxylate:

The product of Phase 3.a (25 g; 0.11 mol) and triethylamine (30.6 ml; 0.22 mol) were mixed together with acetonitrile (520 ml). Ethylmalonyl chloride (28.1 ml; 0.22 mol) was added under ambient temperature and argon atmosphere. The resulting mixture was stirred for a period of 3 hours. Sodium ethanolate (prepared by dissolving 3 g, i.e. 0.13 mol, of sodium in 140 ml of absolute ethanol) was added dropwise, and the resulting mixture was stirred at ambient temperature for a period of 16 hours. The precipitate was collected by filtration, washed individually with ethanol, water, and ethanol and ether. It was then dried under reduced pressure and at a temperature of 70°C over phosphorus pentoxide to obtain the above product (28.6 g; 83% yield) in the form of a whitish powder.

NMR 1H (DMSO): 1.30 (t, 3H); 2.40 (s, 3H); 4.35 (q, 2H); 4.85 (s, 2H); 7.41 (s, 1H); 7.91 (s, 1H); 12.15 (broad signal, 1H).

IR (KBr): 879, 1108, 1250, 1288, 1483, 1664, 1721 cm-1.

3. c. Ethyl 2,7-dichloro-4-chloromethyl-6-methyl-3-quinolinecarboxylate:

The product of Phase 3.b (28.4 g; 90 mmol) was heated for 4 hours at reflux in phosphorus oxychloride (400 ml). The resulting mixture was concentrated under reduced pressure (20 mm Hg) at a temperature of 80°C. The residue was dissolved in diisopropylether (400 ml). The resulting precipitate was collected by filtration, washed with ether and petroleum ether, and then dried under reduced pressure to obtain the above-mentioned product (25.4 g; 85% yield) in the form of a whitish powder (m.p. 126-127°C ).

NMR 1H (DMSO): 1.37 (t, 3H); 2.58 (s, 3H); 4.49 (q, 2H); 5.14 (s, 2H); 8.16 (s, 1H); 8.35 (s, 1H).

IR (KBr): 874, 1006, 1163, 1243, 1278, 1577, 1723 cm-1.

3. d. 2,7-dichloro-4-chloromethyl-6-methyl-3-quinolylmethanol:

The product from Phase 3.c (25.2 g; 76.5 mmol) was mixed with dichloroethane (630 ml) under argon atmosphere. Diisobutylaluminum hydride (1 M in dichloromethane; 307 ml; 307 mmol) was added dropwise while the reaction mixture was stirred, and the temperature was maintained below 20°C. After that, the reaction mixture was stirred at ambient temperature for 3 hours, after which it was poured into an aqueous solution of potassium tartrate (concentrated to 20% by weight; 1.5 l). The resulting emulsion was stirred vigorously for 1 hour, filtered through celite, after which the two phases were separated. The aqueous phase was extracted with ethyl acetate (200 ml) and the combined organic phases were washed with aqueous sodium chloride solution (concentrated to 20% by weight; 500 ml). The obtained organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was dissolved in diethyl ether (50 ml), and the resulting precipitate was collected by filtration. Drying under reduced pressure gave the above-mentioned product (18.3 g; 93% yield) in the form of a whitish powder (m.p. 169-170°C).

NMR 1H (DMSO): 2.57 (t, 3H); 4.84 (s, 2H); 5.36 (s, 2H); 8.06 (s, 1H); 8.27 (s, 1H).

IR (KBr): 870, 1022, 1102, 1304, 1482, 1567 cm-1.

3.e. 2,7-dichloro-6-methyl-4-(4-methylpiperidinomethyl)-3-quinolylmethyl:

A solution of the product from Step 3.d (16.2 g; 55.7 mmol) in THF (70 ml) was treated with a solution of 4-methylpiperidine (23 ml; 195 mmol). The resulting mixture was stirred at ambient temperature for 2 hours. Water (200 ml) and dichloroethane (200 ml) were added. The organic phase was washed with an aqueous solution of sodium chloride (concentrated to 20% by weight; 100 ml), dried over magnesium sulfate and concentrated under reduced pressure. Crystallization of the residue from diethyl ether gave the above-mentioned product (18.3 g; 93% yield) in the form of a white crystalline solid (M.p. 170-171.5°C).

NMR 1H (CDCl 3 ): 0.88 (d, 3H); 1.17 (m, 2H); 1.42 (m, 1H); 1.60 (m, 2H); 2.19 (t, 2H); 2.56 (s, 3H); 2.82 (d, 2H); 4.02 (s, 2H); 4.93 (s, 2H); 6.36 (broad signal, 1H); 7.95 (s, 1H); 8.02 (s, 1H).

IR (KBr): 971, 1013, 1105, 1293, 1479, 1559 cm-1.

3.f. (+)-8-[2,7-dichloro-6-methyl-4-(4-methylpiperidinomethyl)-3-quinolylmethyl]-5-ethyl-5-hydroxy-1,3,4,5,8,9

- hexahydrooxepino[3,4-c]pyridin-3,9-dione:

A suspension of (+)-EHHOPD (obtained in Phase 1.c.; 1.56 g; 7.0 mmol) in anhydrous dioxane (70 ml) was treated separately with the product of Phase 3.e. (2.47 g; 7.0 mmol), triphenylphosphine (2.02 g; 7.7 mmol) and diisopropyl azodicarboxylate (1.07 ml; 10.5 mmol) under argon atmosphere. The mixture was stirred at ambient temperature for 16 hours. After that, under reduced pressure, the volatiles were evaporated from the mixture. The residue was purified by chromatography on a silicon column (eluent: ethyl acetate). The resulting solid was dissolved in diethyl ether, filtered and dried to give the above product (1.96 g; 50% yield) as an off-white solid (m.p. 182°C).

NMR 1H (DMSO): 0.89 (m, 8H); 1.23 (m, 1H); 1.41 (t, 2H); 1.64 (m, 2H); 2.09 (q, 2H); 2.59 (m, 5H); 3.15 (dd, 2H); 4.06 (dd, 2H); 5.31 (dd, 2H); 5.35 (dd, 2H); 5.75 (s, 1H); 6.29 (d, 1H); 7.17 (d, 1H); 8.06 (s, 1H); 8.46 (s, 1H).

IR (KBr): 878, 1053, 1275, 1474, 1572, 1648, 1747 cm-1.

3rd Mr. (+)-9-chloro-5-ethyl-5-hydroxy-10-methyl-12-(4-methylpiperidinomethyl)-4,5,13,15-tetrahydro-1H,3H-oxepino[3',4': 6,7] indolizino[1,2-c]quinoline-3,15-dione:

A mixture of the products from Phase 3.f (3.80 g; 6.80 mmol), tetrabutylammonium bromide (2.42 g; 7.5 mmol), potassium acetate (1.00 g; 10.2 mmol), triphenylphosphine (890 mg; 3.4 mmol) and palladium (II) acetate (220 mg; 0.68 mmol) were mixed under argon atmosphere in anhydrous acetonitrile (85 mg) under reflux for 24 hours. After cooling at ambient temperature, the resulting precipitate was collected by filtration and washed separately with acetonitrile, water, acetone and diethyl ether to obtain, after drying under reduced pressure, the above-mentioned product (2.5 g; 70 % yield) in the form of a whitish powder.

NMR 1H (DMSO): 0.86 (m, 6H); 1.12 (q, 2H); 1.36 (m, 1H); 1.56 (d, 2H); 1.84 (q, 2H); 2.12 (t, 2H); 2.56 (s, 3H); 2.83 (dd, 2H); 3.26 (dd, 2H); 4.03 (dd, 2H); 5.28 (dd, 2H); 5.45 (dd, 2H); 6.04 (s, 1H); 7.34 (s, 1H); 8.14 (s, 1H); 8.38 (s, 1H).

IR (KBr): 870, 1058, 1208, 1280, 1477, 1593, 1655, 1749 cm-1.

3. h. (+)1-[(5R)-9-chloro-5-ethyl-5-hydroxy-10-methyl-3,15-dioxo-4,5,13,15-tetrahydro-1H,3H-oxepino[3' ,4':6,7]indolizino[1,2-c]quinolin-12-ylmethyl]-4-methyl-hexahydropyridinium chloride:

The mixture of the product from Phase 3.g (2.3 g; 7.7 mmol) and absolute ethanol (300 ml) was subjected to ultrasound for 2 min. The resulting milky suspension was stirred and treated with hydrochloric acid (1 N solution; 13.2 ml; 13.2 mmol) to give a light yellow solution which, on standing, formed a gel-like precipitate. The precipitate was collected by Bηchner filtration and washed individually with ethanol and ether, then dried under reduced pressure to give the above product (2.1 g; 85% yield).

NMR 1H (DMSO): 0.87 (m, 6H); 1.59 (m, 5H); 1.84 (q, 2H); 2.64 (s, 3H); 3.28 (dd, 2H); 3.45 (s, 2H); 4.93 (s, 2H); 5.47 (dd, 2H); 5.61 (s, 2H); 6.04 (broad signal, 1H); 7.41 (s, 1H); 8.28 (s, 1H); 8.63 (s, 1H); 10.30 (broad signal, 1H).

IR (KBr): 1043, 1212, 1479, 1585, 1655, 1751 cm-1.

PHARMACOLOGICAL STUDY OF THE PRODUCT OF THIS INVENTION

Cell proliferation assay.

Five tumor cell lines were used in this study: SW620 (human colon adenocarcinoma), OVCAR-5 (human ovarian adenocarcinoma), PC-3 and DU 145 (human prostate cell line) and NCI-H69 (human lung adenocarcinoma). These lines were obtained from the NCI/Frederick Cancer Research and Development Center (Frederich, MD). They were cultivated on a complete medium containing RMPI-1640 medium enriched with 10% fetal bovine serum and 2 mM L-Glutamine. The cell lines were incubated at a temperature of 37°C in a humid atmosphere containing 5% CO2. Adhered cells were separated by treatment with a solution containing 0.25% trypsin and 0.2% EDTA (Worthington Biochemical Corp., Freehold, NJ) for 5 min at a temperature of 37°C. Cell counts were performed using a Coulter Z1 counter (Coulter Corp., Hialeah, FL). Cell viability was determined by staining them with propidium iodide, after which the cells were counted with an EPICS Elite type flow cytometer (Coulter).

The compounds from Examples 2 and 3 that were tested were dissolved at a concentration of 5 mM in a solution of N;N-dimethylacetamine (DMA, Aldrich). Appropriate dilutions were prepared with the culture medium. The tested final molar concentrations are: 1×10-6, 2×10-7, 4×10-8, 8×10-9, 1.6×10-9, 3.2×10-10, 6.4× 10-11, 1.28×10-11, 2.56×10-12 and 5.12×10-13. Each concentration was tested in nine wells. Testing for the influence of DMA was performed for all cell lines. The result of these checks is that at the maximum concentration used (0.02%), DMA has no effect. Doxorubicin in concentrations of 1×10-7 M and 2×10-7 M was used as a positive control.

Cells were seeded at a concentration of 5×10 3 cells per well in a 96-well microplate (Costar Corporation, Cambridge, MA). The cells were incubated for a period of 24 h at a temperature of 37°C to repeat the cell multiplication. The compounds from Examples 2 and 3 to be tested were added in the aforementioned concentrations, and the cells were incubated at a temperature of 37°C in a humid atmosphere with 5% CO2 for a period of 3 days for adherent cells (SW620, OVCAR-5, PC- 3 and DU 145) and from 5 days for cells in suspension (NCI-H69).

Adherent cells were tested by the SRB method (described in L.V. Rubenstein, R.H. Shoemaker, K.D. Paull, R.M. Simon, S. Tosini, P. Skehan, D.A. Scudiero, A. Monks, and M.R. Boyd “Comparison of in vitro anticancer-drug-screening data generated with tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines", J. Nat. Cancer Inst., 82:1113-1118, 1990). After incubation for a period of 3 days, the supernatant was removed, and then 200:l RPMI-1640 without fetal bovine serum was added. The cells were fixed with the addition of 50:1 50% trichloroacetic acid (the final concentration of trichloroacetic acid is 10%) and were incubated at a temperature of 4°C for 1 hour. The basins were washed 5 times with water, and then painted with 50 l of a solution of 0.4% sulforhodamine B (SRB, Sigma) in 1% acetic acid at ambient temperature for 10 min. The dye was dissolved using 100:l 10 mM TRIS buffer pH 10 for 5 minutes with stirring, and the microplates were analyzed by spectrophotometry at 570 nm.

Cells in suspension were tested by the XTT procedure (described in D.A. Scudero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger, M.J. Currens, D. Seniff and M.R. Boyd: “Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines", Cancer Research 48:4827-4833, 1988). After incubation in the presence of the compounds from Examples 2 and 3 being tested, XTT [sodium salt of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, (Sigma)] and phenazine methosulfate (PMS, Sigma) in saline phosphate buffer solution were added to the culture, and the cells were incubated for 4 hours at a temperature of 37°C in an atmosphere containing 5% CO2. The final concentrations of XTT and PMS are 50 and 0.38 g per pool. Formazan production was stopped by the addition of 10:1 10% sodium dodecyl sulfate (Sigma), and the absorbance was analyzed by spectrophotometry at 450 nm with a filter reference of 600-650 nm.

The results:

The molar concentrations of compounds from Examples 2 and 3 that inhibit cell proliferation by 50% are listed in the following table:

[image]

Claims (8)

1. A compound having one of formulas (I) or (II) presented below [image] or to represent various salts of the compound of formula (II) such as for example the compound of formula (III) presented below [image] 2. A novel compound, characterized in that it is described by the general formula M presented below [image] in which R represents an ethyl radical. 3. Procedure for obtaining the product of the general formula M presented below, [image] in which R represents the ethyl radical, characterized by the fact that it is composed of the following separate phases: - racemic t-butyl ester presented below [image] it was treated with trifluoroacetic acid for 18 hours at room temperature to obtain the corresponding carboxylic acid; - after that, the quinidine salt of the previously obtained acid was heated in isopropyl alcohol at a temperature higher than 30°C, preferably around 50°C, before the reaction mixture was allowed to cool to room temperature in order for the salt of one of the enantiomers of the above-mentioned acid to crystallize while the salt of the other enantiomer, whose anion is represented below, remains in solution [image] - a solution in isopropyl alcohol of the salt of the enantiomer that did not crystallize was concentrated and treated with hydrochloric acid, and then stirred to form the compound of general formula A presented below [image] - the compound of general formula A was then contacted with palladium on wet carbon, ammonium formate or formic acid was added to the mixture to give the debenzylated product of general formula B presented below [image] - then the compound of the general formula B is crystallized by means of the action of dicyclohexylcarbodiimide to obtain the lactone compound of the general formula C presented below [image] - finally, the -OCH3 group of the lactone compound of the general formula C is converted into a carbonyl by the action of sodium iodide and trimethylsilyl to obtain the compound of the general formula M presented below. [image] . 4. As a drug, a product characterized in that its formula is one of the formulas (I) or (II) presented below [image] or to represent an addition salt with a pharmaceutically acceptable mineral or organic acid of a product of formula (II) as for example in formula (III) presented below: [image] or also any mixture of the latter. 5. Pharmaceutical composition, characterized in that it contains at least one of the compounds indicated in claim 4 as an active ingredient. 6. Use of the compound according to claim 1, characterized in that the compound is used in the preparation of antitumor drugs. 7. Use of the compound according to claim 1, characterized in that the compound is used for the preparation of antiviral drugs. 8. Use of the compound according to claim 1, characterized in that the compound is used for the preparation of antiparasitic drugs.