Abstract:
The present invention relates to novel colchicine derivatives having antiproliferative, antineoplastic, antiinflammatory and muscle relaxant activities; said derivatives include novel colchine nitrogen amides for use either as such or after derivatization of the hydroxyl at C 3  of the aromatic ring and at C 10  of the tropolone ring. These novel compounds have a cytotoxicity on human tumoral cell lines comparable with colchicine but, in comparison with the latter, they are much more active on cells resistant to the usual antiblastics. The compounds can be included in pharmaceutical formulations useful for the intravenous, oral and topical administrations.

Description:
TECHNICAL FIELD 
     The present invention relates to novel colchicine derivatives having antiproliferative, antineoplastic, antiinflammatory and muscle relaxant activities, the methods for the preparation thereof and the pharmaceutical formulations containing them. 
     BACKGROUND ARTS 
     Colchicine is a known pseudo-alkaloid widely used for a very long time in therapy for the treatment of gout, a pathology on which it acts very quickly and specifically, even though it should be used for short times due to its toxicity. A colchicine derivative, namely thiocolchicoside, is widely used to treat contractures and in inflammatory conditions on skeletal muscles. In addition, colchicine is a very potent antiblastic agent, which acts block the formation of the mitotic spindle during cell division; this latter aspect has been investigated thoroughly for any antineoplastic activity and a great deal of colchicine derivatives have been prepared for this purpose. Colchicine as such and a number of its derivatives could not be used clinically due to their high toxicity, and therefore their unacceptable risk/benefit ratio. Only one colchicine derivative, demecolcine, is used in some degree in oncology for the treatment of certain leukemia forms. It would thus be desirable to have compounds which possess higher activity, lower toxicity and higher therapeutical index. More specifically, in the antineoplastic field, researches have been focused on the search for products having, besides a normal cytotoxicity, a cytotoxicity aimed at cell lines resistant to the known, usual antiblastic medicaments. 
     SUMMARY OF THE INVENTION 
     The derivatives of the present invention have the formula I: ##STR1## wherein R is a methoxyl or thiomethyl group; R 1  is hydroxy; a B-D-glucopyranosyloxy residue; a B-D-glucopyranosyloxy residue ketalized at the hydroxyls 4&#39; and 6&#39; with an aliphatic or aromatic aldehyde; a 6-deoxygalactopyranosyloxy residue; an acyloxy group of C 16  to C 22  polyunsaturated fatty acids; straight, branched or cyclic O-alkyl C 1  -C 6 , saturated or unsaturated; and R 2  is a C 1  -C 6  haloalkyl group. 
     Particularly preferred compounds of formula I are those in which R 1  is a methoxy group, a B-D-glucopyranyloxy residue optionally ketalized at the 4&#39; and 6&#39; hydroxyls with an aromatic or aliphatic aldehydes, for example, 2- or 3-thienal or a ximenoyloxy group. 
     R 2  is preferably trifluoromethyl, pentafluoroethyl or heptafluoropropyl. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Compounds I are prepared starting from the natural compounds colchicine or thiocolchicine (Formula I, R 1  =--OCH 3 , R 2  =CH 3 , R=--OCH 3  or --SCH 3 , respectively) or from the corresponding derivatives thereof glucosylated at the hydroxyl at the 3- position or also from the N-formyl-N-deacetyl-derivatives thereof. 
     The hydrolysis of these natural compounds with aqueous solutions of strong mineral acids makes it possible to obtain selectively, changing the temperature and the reaction time, the corresponding N-deacetyl and 3-demethyl-N-deacetyl derivatives which can then be subjected to conventional reactions of N-acylation and alkylation or acylation at the hydroxyl at the 3-position. 
     In the case of thiocolchicine, the hydrolysis with hydrohalogen acids or, more preferably, with sulfuric acid (20% H 2  SO 4  --120 h) allows one to obtain N-deacetylthiocolchicine and 3-demethyl-N-deacetylthiocolchicine in essentially quantitative yields. 
     The compounds of the invention have a remarkable antitumour activity. 
     The table shows the antimitotic activity of the compounds of the invention on a cultured breast tumour explant, compared with colchicine and Taxol. 
     
                       TABLE______________________________________In vitro cytotoxic activity of some thiocolchicinederivatives.   IC.sub.50 (nM)     MCF7-ADR  MCF-7Compounds (resistant)               (human breast)                           MCF7-ADR/MCF7______________________________________Colchicine     112 ± 4.2               4.4 ± 0.3                           25.45Compound I     26 ± 2.3               6.2 ± 0.4                           4.2Compound II     11 ± 1.9               5.0 ± 0.2                           2.2Compound III      7 ± 0.4               4.1 ± 0.3                           1.7Compound IV     31 ± 1.9               3.2 ± 0.2                           9.7Taxol     360 ± 7.8               6.1 ± 0.3                           59.01______________________________________ 
    
     This table evidences that the compound of the invention have significant advantages on the resistant cell lines, which are nowadays considered the main target for cytotoxic medicaments. 
     Moreover, the products according to the present invention have antiinflammatory and muscle relaxant activities and they can be incorporated in pharmaceutical formulations useful for the administration of the medicament for the indicated pathology. Formulations for the intravenous, oral, transdermal, epicutaneous administrations can conveniently be prepared. Such formulations are generally known to one of ordinary skill in the art and need not be described in great detail here. 
     Among the excipients useful to prepare said formulations, natural and synthetic phospholipids proved to be particularly useful for preparing liposomial forms for the parenteral, intravenous and/or topical routes. The same formulations proved to be useful in the topical treatment of cutaneous epitheliomas and in cutaneous hyperproliferative conditions, such as psoriasis. In the specific antineoplastic field, besides the phospholipids which allow the administration of the medicament in the liposomial form, some surfactants such as polyethoxylated castor oils, or polysorbates acting synergistically with the active ingredient, turned out to be particularly useful. Preferably the active principle is micronized until the compound is dissolved in water. In oncology, the products are used at dosages from 1 to 100 mg/m 2 . 
    
    
     EXAMPLES 
     The following examples further illustrate the invention. 
     Example I 
     Preparation of N-deacetyl-N-pentafluoro-propionyl-thiocolchicine. (Compound I; R=--SCH 3  R 1  =--OCH 3  R 2  =--CF 2  --CF 3 ) 
     20 g of thiocolchicine are dissolved in 300 ml of 20% sulfuric acid and heated under nitrogen atmosphere for 36 h at 100° C.; the reaction mixture is alkalinized to pH 8 to separate 15 g of N-deacetyl-thiocolchicine. 
     This product is dissolved in acetone and, in the presence of anhydrous Na 2  CO 3 , it is reacted with 1.5 equivalents of perfluoropropionic anhydride under strong stirring; after 2 h the reaction mixture is filtered and the solvent is evaporated off. The oily residue is taken up with methanol, from which N-deacetyl-N-pentafluoropropionylthiocolchicine is separated by crystallization. 
     Example II 
     Preparation of N-deacetyl-N-pentafluoropropionyl-3-O-ximenoyl-thiocolchicine. (Compound II; R=--SCH 3  R 1  =--O-Ximenoyl R 2  =--CF 2  --CF 3 ) 
     20 g of thiocolchicoside are dissolved in 300 ml of 20% sulfuric acid and the whole is heated under nitrogen atmosphere 36 h at 100° C.; from the reaction mixture 12 g of N-deacetyl-3-O-demethylthiocolchicine separate. 
     This product is dissolved in acetone and, in the presence of anhydrous Na 2  CO 3 , is reacted with 3 equivalents of perfluoropropionic anhydride under strong stirring; after 2h the reaction mixture is filtered and the solvent and the reactive excess are removed under vacuum. The residue consisting of N-deacetyl-N-pentafluoropropionyl-3-O-demethyl-3-O-pentafluoropropionate is taken up with methanol containing NH 4  Cl, checking the hydrolysis of the phenol ester by thin layer chromatography (toluene/ethyl acetate 1:1); the solvent is evaporated to dryness under vacuum and the residue is dissolved in acetone, filtering off the insolubles. The acetone solution is concentrated to dryness and the residue is taken up with 100 ml of pyridine; this solution is cooled at 0° C. and added with 2 eq. of ximeninic acid chloride under strong stirring. The reaction mixture is left to stand overnight and then poured onto 500 g of ice. The formed aqueous suspension is extracted for three times with 500 ml of methylene chloride. The organic phase is washed with water, then with a hydrochloric acid diluted solution and again with water. The phase organic is dried over Na 2  SO 4  and concentrated to dryness. The residue is crystallized from an ethyl acetate/isopropyl ether mixture, to obtain 27 g of N-deacetyl-N-pentafluoropropionyl-3-O-ximenoyl-thiocolchicine. 
     Example III 
     Preparation of N-deacetyl-N-pentafluoropropionyl-3-O-demethyl-3-O-cyclopentenyl-thiocolchicine. (Compound III; R=--SCH 3  R 1  =--O-cyclopentenyl R 2  =--CF 2  --CF 3 ) 
     20 g of thiocolchicoside are dissolved in 300 ml of 20% sulfuric acid and the mixture is heated under nitrogen atmosphere for 36 h at 100° C.; 12 g of N-deacetyl-3-O-demethylthiocolchicine separate from the reaction mixture. 
     This product is dissolved in acetone in the presence of anhydrous Na 2  CO 3  and reacted with 3 equivalents of pentafluoropropionic anhydride under strong stirring; after 2 h the reaction mixture is filtered and the solvent and reactive excess are removed under vacuum. The residue consisting of N-deacetyl-N-pentafluoro-propionyl-3-O-demethyl-3-O-pentafluoropropionate is taken up with methanol containing NH 4  Cl, checking the hydrolysis of the phenol ester by thin layer chromatography (toluene/ethyl acetate 1:1); the solvent is evaporated to dryness under vacuum and the residue is dissolved in acetone filtering off the insolubles. The acetone solution is added with Na 2  CO 3  and 5 equivalents of cyclopentenyl bromide with respect to starting product. The reaction is stirred for 6 h checking the alkylation by thin layer chromatography. When the reaction is complete, salts are filtered off and the solvent is distilled under vacuum. The residue is chromatographed on a silica gel column using ethyl acetate as eluent. The fractions containing the desired product are collected, solvent is removed and the product is crystallized from acetone/hexane. 9.2 g of N-deacetyl-N-pentafluoropropionyl-3-O-cyclopentenyl-thiocolchicine are obtained. 
     Example IV 
     Preparation of N-deacetyl-N-heptafluorobutyroyl-thiocolchicine. (Compound IV; R=--SCH 3  R 1  =--OCH 3  R 2  =--CF 2  --CF 2  --CF 3 ) 
     10 g of N-deacetylthiocolchicine are dissolved in 150 ml of anhydrous acetone in the presence of Na 2  CO 3  and treated at room temperature with 1.5 eq. of heptafluorobutyroyl anhydride. Na 2  CO 3  and the solvent are removed and the residue is purified with isopropyl ether to give 12.5 g of N-deacetyl-heptafluoro-butyroylthiocolchicine. 
     Example V 
     Preparation of N-deacetyl-N-pentafluoropropionyl-3-O-isopropyl-thiocolchicine. (R=--SCH 3  R 1  =--O-isopropyl R 2  =--CF 2  --CF 3 ) 
     For the preparation of this derivative, the procedures of example III are repeated, using isopropyl bromide as the reagent. After purification of the crude reaction product on silica gel and crystallization, 7.6 g of N-deacetyl-N-pentafluoropropionyl-3-O-isopropyl-thiocolchicine are obtained, having spectroscopic characteristics in agreement with the desired molecule. 
     Examples VI, VII and VIII illustrate typical pharmaceutical formulations of the derivatives of the invention 
     
         ______________________________________Example VI - Preparation of tablets containing compound______________________________________(I):Compound I                  20     mgLactose                     47     mgMicrocrystalline cellulose  20     mgCrosslinked carboxymethylcellulose sodium                       5      mgColloidal silica            1      mgTalc                        1      mgMagnesium stearte           1      mgExample VII - Preparation of a liposome cream containingcompound (II):Compound II                 0.2    gPhosphatidylcholine         20     gCholesterol                 0.5    gButylhydroxytoluene         0.01   gEthanol 95%                 8      gDisodium edetate            0.15   gImidazolidinyl urea         0.3    gSodium dehydroacetate       0.2    gHydroxyethylcellulose       2      g(Natrosol 250 HHX-Aqualon)Distilled water             67.75  gExample VIII - Preparation of an injectable solutioncontaining compound (IV):Compound IV                 50     mgPEG-660 12-hydroxystearate  2,500  mgPropylene glycole           1,000  mgAlcohol q.s.                5      ml______________________________________ 
    
     These formulations are prepared in a conventional manner which is within the abilities of the skilled artisan. These formulations can conveniently be administered to a subject orally (Example VI), topically (Example VII) or by injection (Example VIII) to achieve the beneficial advantages described previously.