EP2125684A2 - Colchicine derivatives, process for their preparation and use in the medical field - Google Patents

Colchicine derivatives, process for their preparation and use in the medical field

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EP2125684A2
EP2125684A2 EP08720244A EP08720244A EP2125684A2 EP 2125684 A2 EP2125684 A2 EP 2125684A2 EP 08720244 A EP08720244 A EP 08720244A EP 08720244 A EP08720244 A EP 08720244A EP 2125684 A2 EP2125684 A2 EP 2125684A2
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colchicine
och
sch
cells
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French (fr)
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Francesco Fringuelli
Oriana Piermatti
Massimo Pinzani
Ferdinando Pizzo
Angelo Vedovelli
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    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/20—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to carbon atoms of rings other than six-membered aromatic rings of the carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/32—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a ring other than a six-membered aromatic ring
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    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/22—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and doubly-bound oxygen atoms bound to the same carbon skeleton
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C403/06—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by singly-bound oxygen atoms
    • C07C403/10—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by singly-bound oxygen atoms by etherified hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
    • C07C403/20—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by carboxyl groups or halides, anhydrides, or (thio)esters thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/24—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with monohydroxylic compounds
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/84—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring
    • C07C69/88—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring with esterified carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/96—Esters of carbonic or haloformic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
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    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
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    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/30—Ortho- or ortho- and peri-condensed systems containing three rings containing seven-membered rings
    • C07C2603/34—Benzoheptalenes; Hydrogenated benzoheptalenes

Definitions

  • the present invention concerns colchicine derived compounds, process for preparation and uses thereof in medical field.
  • the present invention concerns colchicine derived compounds to be used advantageously in antifibrotic therapy of chronic hepatic diseases.
  • Chronic hepatic, sclerosis developing diseases comprise a set of affections with infective, toxic/metabolic, genetic and autoimmune etiology.
  • Italy countries of the Mediterranean Basin and generally in Europe these diseases are very important in terms of morbidity and mortality.
  • infectious forms it is estimated that approximately 10% of the Italian population can be disease carrier and an incidence peak of chronic hepatopathy terminal forms is actually foreseen from 2010 to 2020.
  • HSCs hepatic active fibrogenesis perisinusoidal stellate cells constitute one of the main cell types responsible of the production of extracellular matrix, with a predominant synthesis of collagen I and III.
  • Stellate cells HSCs; known as hepatic stellate cells, fat-storing cells, or fat cells
  • HSCs are localised at level of Disse space in intimate contact both with liver and endothelial sinusoid cells.
  • HSCs are subjected to an activation and phenotypic modulation process resulting in so-called myofibroblast-like phenotype.
  • HSCs constitute the main retinoid reservoir in adult organism. This resulting from their selective uptake ability of retinol contained in the diet and retinol metabolization in retinol esters, which represent the main storage form as characteristic lipid drops in HSC cytoplasm. During the HSC activation process a progressive loss of intracellular retinoid deposits is observed. It has been assumed that the loss of the physiological intracellular retinoid content has an impact on some aspects of HSC activation and in particular on the proliferating characteristics of activated phenotype.
  • the main problem resulting from drugs suitable to affect the fibrogenic and inflammatory liver response is the lack of specificity of their action, both at organ and cellular type levels, from which various type collateral effects result. Such adverse reactions impose dosage limitations seriously compromising the effectiveness of the therapy.
  • the drugs acting as inhibitors or modulators of the main ways of intracellular signal transduction as a result of the occupation of membrane receptors by growth factors and cytokines acting in a pro-fibrogenic sense are not provided with any cellular specificity and can therefore result in highly counter-productive effects within the hepatic regenerating process.
  • Colchicine main alkaloid of Colchicum autumnalis and Glorious superba, most known and more extensively studied drug suitable to interfere with microtubules, fundamental components of the cytoskeleton .
  • the intimate action mechanism of the colchicine is based on virtually irreversible bond formed with cytosol free tubulin molecules, which, through localization in a critical region for the polymerization with other tubulin sub-units, prevents the assemblage to form microtubules. The result is the dissolution of cellular microtubules, since such structures exist as result of the perennial equilibrium between the association of new tubulin sub-units and the dissociation of integrated ones.
  • colchicine in hepatic fibrosis is based on the following experimental evidences: (a) colchicine is suitable to inhibit the extracellular secretion of collagen by cultured liver fibroblasts osteoblasts and chondrocytes, resulting in intracellular accumulation of collagen and "feedback inhibition" of synthesis thereof; (b) colchicine stimulates the extracellular secretion and expression of several proteinases, including collagenase, elastase, and gelatinase, in different cultured mesenchymal cell types; (c) in liver stellate cell cultures, the colchicine suppresses the proliferation and platelet-derived growth factor (PDGF) induced cell motility.
  • PDGF platelet-derived growth factor
  • colchicine derivative retinoyldeacetylcolchicine compound 2a in table.
  • the compound has been synthesized by Brossi et al. without any relationship with the antifibrotic use or liver chronic diseases but only in the context of studies concerning the relationships between colchicine structure and tubulin binding thereof.
  • the object was to study the in vitro effect of large molecular dimension substitution (vitamin A) in the C7 position of colchicine B ring in relation of the binding kinetics with tubulin.
  • retinoyldeacetyilcolchicine shown in the study a reduced binding affinity with tubulin, resulting in presumption of its reduced or absent biological activity 2 .
  • the invention is suitable to provide compounds able to selectively deliver antifibrotic drugs to the liver and specifically stellate cells, ideal system in order to assure effective and sure antifibrotic therapy for the patients with chronic hepatic diseases.
  • This targeting type confers to standard doses of an antifibrotic, conventional or experimental drug: a) dramatic increase of the therapeutic effectiveness, through the concentration in the organ and target cells; b) attenuation to the virtual cancellation of the adverse effects, due to the drastic reduction of the distribution in other districts.
  • the invention is based on the retinol peculiar metabolic pathway, which, after the absorption at intestinal level, is carried from kilomicrons into the liver where it stored in ester form within the stellate cells. Consequently the colchicine and derived compounds conjugation with retinol (or other retinoids) can allow their vehiculation preferentially at hepatic level (I order targeting) and, inside of the organ, specifically at level of the hepatic stellate cells (Il order targeting), the main cellular effector of the hepatic fibrogenesis. It is therefore a specific object of the present invention colchicine derived compounds having the general formula (I):
  • the group R is selected from OCH 3 , SCH 3 or SCH 2 Ph, X is selected from NH or O, Ri is selected from 14
  • R 1 is H when X is NH and R is SCH 2 Ph, or when X is O and R is SCH 3 or SCH 2 Ph; with provision that when Ri is X is NH then R is different than OCH 3 .
  • colchicine is (aR, 7S)-colchicine, more preferably (-)- (aR, 7S)-colchicine.
  • the compounds according to the invention are preferably selected from compounds having the following formulas:
  • a pharmaceutical composition comprising one or more compounds as above defined as active principle together with one or more pharmaceutically acceptable excipients or co-adjuvants represent a further object of the present invention.
  • the invention concerns the use of compounds and composition as above defined for the preparation of a medicament to be used in the antifibrotic therapy of the chronic hepatic diseases.
  • a further object of the present invention is represented by retinol derived compounds having the following general formula (II):
  • R 2 is selected from
  • Compounds according to formula (II) can be advantageously used as antifibrotic drugs wherein retinol acts as carrier.
  • the invention regards a pharmaceutical composition comprising one or more compounds of formula (II) as active principle together with one or more pharmaceutically acceptable excipients or co- adiuvants.
  • Said compounds and composition can advantageously be used for the prevention and treatment of hepatic diseases like hepatic fibrosis and in the antifibrotic therapy of the chronic hepatic diseases.
  • the present invention regards moreover the use of retinol like antifibrotic drug carrier.
  • FIGURE 1 shows the effect of two doses of colchicine on the synthesis of proto-collagen type I in stellate hepatic human cells.
  • FIGURE 2 shows the effect of two doses of the (-)-11b compound on the synthesis of proto-collagen type I in stellate hepatic human cells.
  • FIGURE 3 shows the effect of two doses of (+)-11b compound on the synthesis of proto-collagen type I in stellate hepatic human cells.
  • Example 1 Preparation of colchicine derivatives according to the present invention (Table 1).
  • (-)-Deacetylcolchicines 1a and 1b are prepared according to the literature 1 .
  • (-) -Deacetyl-benzyltiocolchicine 1c is a new product and we have prepared the same by hydrolysis of benzyltiocolchicinele with HCI in refluxing MeOH.
  • Retinoyl derivatives 2b and 2c are new products and have been prepared by reaction of deacetylcolchicines 1b-c with retinoic acid in CH2CI2 in the presence of diciclohexyl carbodiimide (DCC) and 4- pyrrolidin-pyridine (PPY) according to an already described procedure for (-)-N-retinoyl-deacetylcolchicine 2a.2
  • (2C) 1 30.0, 33.0, 34.2, 36.6, 36.8, 39.5, 51.8, 56.0, 61.4, 61.8, 107.2, 120.7, 125.6, 127.2, 127.6, 128.0 (2C), 128.7 (2C), 129.0, 129.1 , 129.7,
  • Amides 3a-c have been prepared by means of reaction of the deacetylcolchicine 1a-c with 3-carboxy cumarinic acid in CH 2 CI 2 in the presence of dicyclohexylcarbodiimide (DCC) and 4-pyrrolidin pyridine (PPY).
  • DCC dicyclohexylcarbodiimide
  • PPY 4-pyrrolidin pyridine
  • COLCHICOLS are obtained from corresponding deacetylcolchicines 1a-b, respectively, according to the literature.1c,3.
  • Racemic COLCHICOLO 4c is a new product and has been prepared by reaction of deacetylcolchicine 1c with 4-formyl-1-methylpiridinium benzene sulphonatel (FMB), at ambient temperature for 1 ,5 h. Obtained imine then is treated in situ with 1 ,8-diazabicyclo [5.4.0] undec-7-ene (DBU) and then with oxalic acid yielding correspondent colchicone which is reduced with
  • (+) and (-)-COLCH ICOLI 4a-c are obtained for the first time by re-crystallisation of menthyl derivatives 5a-c, obtained by treatment of the racemic mixture with (-)-menthyl-chloro formiate.
  • the use of ethyl acetate as re-crystallisation solvent yields (+)-(aS, 7R)-menthyl- derivative (+)-5a-c, with approximately 40% yield.
  • MeOH/CHCI 3 as re-crystallisation solvent provides (-)-(aR, 7S)-menthyl-derivative (-)-5a- c with approximately 40% yield.
  • (+)-5b or (-)-5b (1 ,8 mmol) in methanol KOH 0,5 M (25 ml) is stirred at 80 0 C for 30 min. After work-up the crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/methanol 50:45:5). (+)-(aS, 7R)-thioCOLCHICOLO (+)-4b ([ ⁇ ] 25 D +189° (c. 0.3, MeOH)), and (-)-(aR, 7S)-thioCOLCHICOLO (-)-4b ([ ⁇ ] 25 D -188° (c. 0.3, MeOH)), are obtained with 95% yield.
  • (+) and (-)-retinoyl derivatives 6a-c are prepared by reaction of (+) and (-)-COLCH ICOLI 4a-c with retinoic acid in CH 2 CI 2 in the presence of diciclohexyl carbodiimide (DCC) and 4-pyrrolidin pyridine (PPY).
  • DCC diciclohexyl carbodiimide
  • PPY 4-pyrrolidin pyridine
  • Various esters having 7-9 (a-c) structures have been prepared by reaction of (+) and (-)-COLCH ICOLI 4a-c with appropriated carboxylic acid (hexanoic, dodecanoic and hexadecanoic acids, 3-carboxy cumarinic and acetyl salicilic acids) in CH 2 CI 2 in the presence of DCC and PPY.
  • (+) and (-)-ester derivatives 7-9 are obtained with 70-75% yield.
  • (+) or (-)-COLCHICOLI lithium salts 4a-c obtained by treatment with lithium diisopropylamide (LDA), react with trans-1 ,4-dibromo-2-butene to yield corresponding (+)- and (-)-butenyl bromides 10a-c.
  • LDA lithium diisopropylamide
  • the reaction of 4-bromo-butenyl derivatives with retinol lithium salts yields (+)-and (-)- retinyl ethers 11a-c.
  • (+)-and (-)-butenyl bromides 10a-c are obtained with 40% yield.
  • the crude product is purified by column chromatography (silica gel, diethyl ether/petroleum ether 85:15). (+)-and (-)-retinyl ethers 11a-c are obtained with 30% yield.
  • (+) and (-)-COLCH ICOLI 4a-c lithium salts with a set of alkyl bromides (1-bromo-nonane, 1-bromo-dodecane, 1-bromo- esadodecane, genaryl bromide and famesyl bromide) provides corresponding (+)- and (-)-alkyl ethers 12-14 (a-c).
  • Example 2 Study about increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the proliferation and migration of human stellate cells both under control conditions and after stimulation with 10 ng/ml of PDGF-BB.
  • MATERIALS AND METHODS Isolation and culture of human liver stellate cells.
  • Human HSCs have been isolated from surgical sections (10-20 g) of not transplant usable normal human liver. Shortly, after collagenase and pronase combined digestion of surgical slices HSCs have been separated from other non parenchymal liver cells by means of ultra- centrifugation on stractan gradient (Cellsep TM isotonic solution, Larex Inc, St. Paul, MN, USA).
  • the cells have been cultured in plastic flasks (Falcon, Becton Dickinson, Lincoln, New Jersey, USA) in Iscove's modified Dulbecco's medium, supplemented with 0.6 U/ml of insulin, glutamine (2 mM), non essential amino acids (0,1 mM), sodium pyruvate (1 mM), antimycotic and antibacterial solution (all from GIBCO BRL Laboratories, Grand Island, New York, USA), and calf foetal serum 20% (v/v) (Imperial Laboratories, Andover, UK).
  • the cells have been cultured in atmosphere consisting of humidified air with 95% O 2 and 5% CO 2 , at constant temperature of 37 °C. Just isolated and in successively sub-cultured cells have been characterised as previously described. Described experiments have been carried out using HSCs between the third and fifth serial passage, at 1 :3 distribution ratio. Experiments have been carried out using three different cell lines.
  • DNA synthesis has been evaluated using the method involving incorporation of [methyl- 3 H]-thymidine in cellular material precipitated with trichloroacetic acid.
  • cells have been cultured in 24-well plates, at density of 2x10 4 cells/well, with complete culture medium containing 20% of calf foetal serum (FBS).
  • FBS calf foetal serum
  • the cells At confluency (approximately 1x10 5 cells/well), the cells have been made quiescent with the incubation in serum and insulin free medium (serum-free/insulin-free: SFIF) for 48 hours. Then the cells have been incubated with or without agonists, at doses and conditions indicated, for 20 hours.
  • the cells have been then incubated for 4 hours with 1.0 ⁇ Ci/ml of [methyl- 3 H]-thymidine (6,7 Ci/mM). At the end of pulsing period incorporated [methyl- 3 H]-thymidine has been measured as reported in literature. The number of cells has been estimated in three different wells for every plate and the final result of the experiment has been expressed as cpm/10 5 cells. Analysis of cell migration The cell migration has been analysed as described in literature.
  • the experiments have been carried out using Boyden chamber technique, equipped with 8 ⁇ m pore polycarbonate filters.
  • the filters have been pre-treated with type I human collagen (20 ⁇ g /ml) for 30 min at 37 0 C and subsequently inserted between the upper and lower chamber HSCs, cultured at confluence, have been incubated in SFIF for 48 hours and then have been treated for 10 min with increasing concentrations of NO donors. Then the cells have been re-suspended by means of mild trypsinization (0,05% trypsin/EDTA) and an aliquot of the cell suspension (210 ⁇ l), corresponding to approximately 4x10 4 cells, has been inserted in the upper chamber and incubated at 37 0 C for 6 hours.
  • (+)-6b Mean 174 168 274 180 1599 1065* * 1294 1167 SD 28 11 91 82 245 17 169 81
  • (+)-11b Mean 440 153* 201 268* 2737 634 ** 2138** 2068 * * SD 294 18 206 58 453 282 433 460
  • (+)-13b Mean 791 638 722 772 4098 1139 ** 3284** 3466 * SD 125 84 22 117 525 271 259 268
  • (+)-6b Mean 7 6 5 17 5** 4 ** 3** SD 1 0 1 2 2 2 1
  • (+)-13b Mean 1 1 1 1 6 2 * * 3** 2** SD 1 1 1 O O J C 1 1 0 1 1
  • Example 3 Sfudy about increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the synthesis of type I protocollagen in human stellate hepatic cells
  • Hepatic human stellate cells have been grown at 70-80% confluence and then incubated with serum free medium (SFIF) for 24 hours.
  • the culture medium have been replaced with serum-free fresh medium and the cells have been treated with test compounds (colchicines derivatives) in absence or presence of transforming growth factor- ⁇ 1 (TGF ⁇ -1 , 1 ng/ml), used as standard stimulus for the synthesis of collagen and other components of extracellular matrix.
  • TGF ⁇ -1 transforming growth factor- ⁇ -1 , 1 ng/ml
  • SFIF un serum free medium
  • TGF ⁇ -1 Transforming Growth Factor- ⁇ 1
  • TGF ⁇ -1 Transforming Growth Factor- ⁇ 1
  • concentration of type I pro-collagen has been determined by means of EIA method, using commercially available Procollagen Type C- peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
  • TGF ⁇ -1 1 ng/ml
  • concentration of type I pro-collagen has been determined by means of EIA method, using commercially available
  • Procollagen Type C-peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
  • TGF ⁇ -1 Transforming Growth Factor- ⁇ 1
  • TGF ⁇ -1 Transforming Growth Factor- ⁇ 1
  • concentration of type I pro-collagen has been determined by means of EIA method, using commercially available Procollagen Type C-peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
  • Example 4 Chemical stability tests of (-)-11b product
  • the (-)-11b product proves to be highly sensitive to the presence of oxidant agents, as atmospheric oxygen, which result in the destruction of conjugated double bond system of retinyl group generating various decomposition products.
  • sample stability was checked by HPLC analysis using a VISFER SILICA 5 ⁇ , 4,6 mm x 25 cm column; Eluent: ethyl acetate/n- hexane 75:25; flow rate 1 ml/min.
  • Table 4 shows decomposition percentage (%) of (-)-11b product stored in different storage conditions after 10, 30 and 60 days (g).

Abstract

The present invention concerns colchicine derived compounds, a process for their preparation and use thereof in medical field. In particular, the invention concerns the synthesis of colchicine conjugates and derivatives with a carrier molecule aimed to the antifibrotic treatment of chronic hepatic diseases.

Description

COLCHICINE DERIVATIVES, PROCESS FOR PREPARATION THEREOF AND RELATED USES IN MEDICAL FIELD
The present invention concerns colchicine derived compounds, process for preparation and uses thereof in medical field. In particular, the present invention concerns colchicine derived compounds to be used advantageously in antifibrotic therapy of chronic hepatic diseases.
Chronic hepatic, sclerosis developing diseases comprise a set of affections with infective, toxic/metabolic, genetic and autoimmune etiology. In Italy, Countries of the Mediterranean Basin and generally in Europe these diseases are very important in terms of morbidity and mortality. Particularly, as to infectious forms, it is estimated that approximately 10% of the Italian population can be disease carrier and an incidence peak of chronic hepatopathy terminal forms is actually foreseen from 2010 to 2020. It is to be pointed out that, notwithstanding the incidence of the chronic sclerogenous hepatopathies is lower than other sclerogenous pathologies, as for example cardiovascular diseases, their incidence in various age ranges is more widely distributed, many patients being affected by invalidating steps of the disease in relatively younger age ranges and wherein the production pressure is maximum. Since liver transplant is currently the only real effective therapeutic treatment, it is apparent that this situation results in a remarkable social and economic damages. Although remarkable efforts carried out under this aspect during the last few years, currently do not exist an effective treatment that can be defined surely proposable in anti-fibrogenic sense. The introduction of such type of treatment associated with therapies specifically aimed to treat the main cause of the chronic damage would constitute a sure strategic advantage within a combined therapy of several aspects of the chronic epatopathies. Below cellular bases of the hepatic fibrosis will be briefly described.
During the process of hepatic active fibrogenesis perisinusoidal stellate cells constitute one of the main cell types responsible of the production of extracellular matrix, with a predominant synthesis of collagen I and III. Stellate cells (HSCs; known as hepatic stellate cells, fat-storing cells, or fat cells) are localised at level of Disse space in intimate contact both with liver and endothelial sinusoid cells. In conditions of chronic hepatic damage HSCs are subjected to an activation and phenotypic modulation process resulting in so-called myofibroblast-like phenotype. This transition, detectable both in chronic damaged hepatic tissue and in vitro after extended " culture on plastic material, is characterised by a remarkable cell proliferation, cell migration, increase of the synthesis and secretion of extracellular fibrillary matrix (MEC) (particularly type I and III collagen), and relevant increase of their contractile abilities. Activated HSCs are able, at least in early activation steps, to carry out a degradation and remodelling of produced MEC. However this characteristic is gradually lost by perpetuating the activation process, while proliferative, synthetic and contractile characteristics are progressively emphasised. It is therefore possible to assert that hepatic fibrosis is practically the result of disequilibrium between fibrogenesis and matrix degradation processes.
HSCs constitute the main retinoid reservoir in adult organism. This resulting from their selective uptake ability of retinol contained in the diet and retinol metabolization in retinol esters, which represent the main storage form as characteristic lipid drops in HSC cytoplasm. During the HSC activation process a progressive loss of intracellular retinoid deposits is observed. It has been assumed that the loss of the physiological intracellular retinoid content has an impact on some aspects of HSC activation and in particular on the proliferating characteristics of activated phenotype. In support of this possibility it is the consideration of a reduction of cell proliferation, both in basal conditions and after mitogenic stimulation, after incubation with retinol or retinoic acid and successive, at least partial, restoration of intracellular retinoid deposits. In the last decade, significant advancement in the identification and development of new antifibrotic agents, mainly thanks to a better understanding of the cellular and molecular mechanisms leading to a progressive liver cirrhosis, has been realised. Currently, only few agents reached the possibility of a clinical experimentation, and an effective treatment of the hepatic cirrhosis is not still realisable. In general terms, the main problem resulting from drugs suitable to affect the fibrogenic and inflammatory liver response is the lack of specificity of their action, both at organ and cellular type levels, from which various type collateral effects result. Such adverse reactions impose dosage limitations seriously compromising the effectiveness of the therapy. On the other hand, the drugs acting as inhibitors or modulators of the main ways of intracellular signal transduction as a result of the occupation of membrane receptors by growth factors and cytokines acting in a pro-fibrogenic sense are not provided with any cellular specificity and can therefore result in highly counter-productive effects within the hepatic regenerating process. Colchicine, main alkaloid of Colchicum autumnalis and Glorious superba, most known and more extensively studied drug suitable to interfere with microtubules, fundamental components of the cytoskeleton . The intimate action mechanism of the colchicine is based on virtually irreversible bond formed with cytosol free tubulin molecules, which, through localization in a critical region for the polymerization with other tubulin sub-units, prevents the assemblage to form microtubules. The result is the dissolution of cellular microtubules, since such structures exist as result of the perennial equilibrium between the association of new tubulin sub-units and the dissociation of integrated ones. From this the inhibition of microtubulin-dependent cellular functions results, including mitosis and extracellular secretion of various macromolecules. As a whole, this explains the variety and the range of pharmacological effects that colchicine can exert on the organism as a result of its anti-microtubular action. A wide body of experimental evidences, accumulated in the course of last thirty years, pointed out that colchicine can exert a variety of potentially therapeutically exploitable pharmacological activities in liver damaged patients. The latter can artificially be distinguished in two classes: antifibrotic and anti-inflammatory, on one hand and cyto- protective on the other one. The reason for the use of colchicine in hepatic fibrosis is based on the following experimental evidences: (a) colchicine is suitable to inhibit the extracellular secretion of collagen by cultured liver fibroblasts osteoblasts and chondrocytes, resulting in intracellular accumulation of collagen and "feedback inhibition" of synthesis thereof; (b) colchicine stimulates the extracellular secretion and expression of several proteinases, including collagenase, elastase, and gelatinase, in different cultured mesenchymal cell types; (c) in liver stellate cell cultures, the colchicine suppresses the proliferation and platelet-derived growth factor (PDGF) induced cell motility.
In the light of above, the clinical experimentations in the humans aimed to estimate the effectiveness of colchicine in several types of hepatic fibrosis (alcoholic and not alcoholic) resulted globally in disappointing results. This contrasts with the antifibrotic effectiveness that colchicines - at high doses - proved in various experimental models of hepatic cirrhosis. In fact when administered in the animal laboratory at high dosages, the substance proved to be able not only to prevent, but also regress, experimental cirrhosis. This discrepancy is easy explicable considering that high doses used in test animal and having effective anti- fibrosis and anti-cirrhosis activity are not usable in humans due to significant and also serious side-effects resulting from drug activity in extra-hepatic districts. With respect to this some pharmacokinetic considerations suggest indirectly the order of magnitude of potential advantages deriving from a selective colchicine targeting for the liver. Distribution studies have shown that high drug percentages are concentrated normally in kidney, spleen, intestine, and circulating leukocytes, in addition to the liver. Moreover, assuming a uniform distribution of the drug in the different liver cellular types, it is obvious that a preponderant colchicine portion is dispersed in hepatocytes accounting of approximately 80% of the parenchyma volume, while only a minimal fraction is internalized by stellate cells, contributing only by 1 ,4% of the normal parenchyma volume. It is difficult to speculate that at colchicine dosage of 1 mg daily, a fraction of this type can influence significantly the "activation" process of the stellate cells which is the base of liver fibrosis.
It is known colchicine derivative retinoyldeacetylcolchicine (compound 2a in table).2 The compound has been synthesized by Brossi et al. without any relationship with the antifibrotic use or liver chronic diseases but only in the context of studies concerning the relationships between colchicine structure and tubulin binding thereof. The object was to study the in vitro effect of large molecular dimension substitution (vitamin A) in the C7 position of colchicine B ring in relation of the binding kinetics with tubulin. In comparison to colchicine, retinoyldeacetyilcolchicine shown in the study a reduced binding affinity with tubulin, resulting in presumption of its reduced or absent biological activity2.
In the light of above it therefore apparent the need to provide new compounds suitable to overcome the disadvantages of up to now known therapies. The authors of the present invention have now found new compounds with increased therapeutic effectiveness and reduced toxicity aimed to antifibrotic treatment of the chronic fibrogenic hepatic diseases. Moreover, the invention is suitable to provide compounds able to selectively deliver antifibrotic drugs to the liver and specifically stellate cells, ideal system in order to assure effective and sure antifibrotic therapy for the patients with chronic hepatic diseases. This targeting type confers to standard doses of an antifibrotic, conventional or experimental drug: a) dramatic increase of the therapeutic effectiveness, through the concentration in the organ and target cells; b) attenuation to the virtual cancellation of the adverse effects, due to the drastic reduction of the distribution in other districts. The invention is based on the retinol peculiar metabolic pathway, which, after the absorption at intestinal level, is carried from kilomicrons into the liver where it stored in ester form within the stellate cells. Consequently the colchicine and derived compounds conjugation with retinol (or other retinoids) can allow their vehiculation preferentially at hepatic level (I order targeting) and, inside of the organ, specifically at level of the hepatic stellate cells (Il order targeting), the main cellular effector of the hepatic fibrogenesis. It is therefore a specific object of the present invention colchicine derived compounds having the general formula (I):
with (aR, 7S), (aS, 7R); {aR, 7S) or (aS, 7R) configuration, the group R is selected from OCH3, SCH3 or SCH2Ph, X is selected from NH or O, Ri is selected from 14
,Br
— (CH2)nCH3 wherein n is 8, 11 or 15
and R1 is H when X is NH and R is SCH2Ph, or when X is O and R is SCH3 or SCH2Ph; with provision that when Ri is X is NH then R is different than OCH3.
Preferably colchicine is (aR, 7S)-colchicine, more preferably (-)- (aR, 7S)-colchicine.
The compounds according to the invention are preferably selected from compounds having the following formulas:
Above described compounds can advantageously be used in medical field.
A pharmaceutical composition comprising one or more compounds as above defined as active principle together with one or more pharmaceutically acceptable excipients or co-adjuvants represent a further object of the present invention.
Moreover, " the invention concerns the use of compounds and composition as above defined for the preparation of a medicament to be used in the antifibrotic therapy of the chronic hepatic diseases.
A further object of the present invention is represented by retinol derived compounds having the following general formula (II):
wherein R2 is selected from
with (aR, 7S), (aS, 7R); (aR, 7S) or (aS, 7R) configuration, where the group R is selected from OCH3, SCH3 or SCH2Ph, with the provision that when R2 is (aR, 7S) configuration, then R is different than OCH3.
Compounds according to formula (II) can be advantageously used as antifibrotic drugs wherein retinol acts as carrier. Moreover, the invention regards a pharmaceutical composition comprising one or more compounds of formula (II) as active principle together with one or more pharmaceutically acceptable excipients or co- adiuvants. Said compounds and composition can advantageously be used for the prevention and treatment of hepatic diseases like hepatic fibrosis and in the antifibrotic therapy of the chronic hepatic diseases.
The present invention regards moreover the use of retinol like antifibrotic drug carrier.
The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:
FIGURE 1 shows the effect of two doses of colchicine on the synthesis of proto-collagen type I in stellate hepatic human cells.
FIGURE 2 shows the effect of two doses of the (-)-11b compound on the synthesis of proto-collagen type I in stellate hepatic human cells.
FIGURE 3 shows the effect of two doses of (+)-11b compound on the synthesis of proto-collagen type I in stellate hepatic human cells.
Example 1 : Preparation of colchicine derivatives according to the present invention (Table 1).
Table 1
Preparation of (-)-N-Retinoyl-Deacetyl-Thiomethylcolchicine (2b) and (-)-N-Retinoyl-Deacetyl-Benzyltiocolchicine (2c)
(-)-Deacetylcolchicines 1a and 1b are prepared according to the literature1. (-) -Deacetyl-benzyltiocolchicine 1c is a new product and we have prepared the same by hydrolysis of benzyltiocolchicinele with HCI in refluxing MeOH.
Retinoyl derivatives 2b and 2c are new products and have been prepared by reaction of deacetylcolchicines 1b-c with retinoic acid in CH2CI2 in the presence of diciclohexyl carbodiimide (DCC) and 4- pyrrolidin-pyridine (PPY) according to an already described procedure for (-)-N-retinoyl-deacetylcolchicine 2a.2
Procedure: A mixture consisting of deacetylcolchicine 1b or 1c (1.4 mmol), retinoic acid (0.5 g, 1.65 mmol), DCC (0.5 g, 2.4 mmol), PPY (0.05 g, 0.34 mmol) in CH2C^ (15 ml) is stirred at room temperature for 20 h. The reaction mixture is filtered to eliminate dicyclohexylurea (DCU). The solution is washed with water, anhydrified over sodium sulphate and concentrated at reduced pressure. The crude product is purified by silica gel column chromatography using diethyl ether/petroleum ether 90:10 mixture as eluent. 2b, c retinoyl derivatives are obtained with 80-85% yields.
(-)-N-Retinoyl-Deacetyl- Thiomethylcolchicine (2b) m.p.: 208-211°C
[α] 25D -88.7° (c. 0.32; CHCI3) 1H-NMR (CDCI3, 400 MHz) δ: 1.00 (s, 6H, 2CH3-I1), 1.40-1.65
(m, 4H, CH2-2\ CH2-3'), 1.69 (s, 3H, CH3-5'), 1.92 (s, 3H, CH3-9'), 2.00 (m,
2H, CH2-4'), 2.22 (s, 3H, CH3-13'), 2.42 (s, 3H, SCH3-IO), 2.2-2.6 (m, 4H,
CH2-5, CH2-6), 3.71 (S, 3H, OCH3-I), 3.8 (s, 3H, OCH3-2), 3.94 (s, 3H,
OCH3-3), 4.73 (m, 1 H, H-7), 5.84 (s, 1 H, H-14'), 6.05-6.25 (m, 4H, H-7',H-
δ'.H-IO'.H-^1), 6.52 (s, 1H, H-4), 6.80 (dd, 1 H, H-111, J=14.8, 11.7 Hz),
7.06 (d, 1 H, H-11 , J=10.4 Hz), 7.30 (d, 1 H, H-12, J=10.4 Hz), 7.44 (s, 1 H,
H-8)
13C-NMR (CDCI3, 400 MHz) ppm: 12.7, 13.4, 15.1 , 19.2, 21.7,
28.9 (2C), 30.0, 33.0, 33.9, 36.6, 39.5, 51.8, 56.0, 61.3, 61.8, 107.2,
120.9, 125.7, 126.5, 127.8, 128.7, 129.6, 129.7, 129.8, 134.5, 134.7, 135.9, 137.4, 137.6, 138.36, 138.39, 141.5, 149.4, 151.1 , 151.6, 153.5, 156.8, 158.1 , 166.5, 182.3
(-)-N-Retinoyl-\Deacetyl-Benzylthiocolchicine (2c) m.p.: it decomposes at 144-146 0C
[α] 25 D -29.0° (c. 0.49; CHCI3) 1H-NMR (CDCI3, 400 MHz) δ: 1.03 (s, 6H, 2CH3-I'), 1.44-1.66
(m, 4H, CH2-2',CH2-3'), 1.72 (s, 3H, CH3-5'), 1.96 (s, 3H1 CH3-9'), 2.02 (m,
2H1 CH2-4'), 2.25 (s, 3H1 CH3-13'), 2.2-2.6 (m, 4H, CH2-5, CH2-6), 3.70 (s,
3H1 OCH3-I), 3.90 (s, 3H, OCH3-2), 3.95 (s, 3H, OCH3-3), 4.14 (s, 2H1
CH2Ph), 4.72 (m, 1H, H-7), 5.77 (s, 1H1 H-14'), 6.05-6.35 (m, 4H, H-7\ H-
8', H-10', H-12'), 6.53 (s, 1 H, H-4), 6.90 (dd, 1 H, H-11', J=14.4, 11.3 Hz),
7.1-7.5 (m, 8H, H-8, H-11 , H-12, Ph)
13C-NMR (CDCI3, 400 MHz) ppm: 12.8, 13.4, 19.2, 21.7, 28.9
(2C)1 30.0, 33.0, 34.2, 36.6, 36.8, 39.5, 51.8, 56.0, 61.4, 61.8, 107.2, 120.7, 125.6, 127.2, 127.6, 128.0 (2C), 128.7 (2C), 129.0, 129.1 , 129.7,
129.8, 129.9, 134.4, 134.7, 134.8, 135.7, 137.4, 137.7, 138.6, 138.8,
141.5, 149.6, 151.2, 151.4, 153.5, 156.7, 166.4, 182.4
Preparation of (-)-N- (Cumarin-3-carbonyl)-Deacetyl-Colchicine (3a), (-)-N- (Cumarin-3-carbonyl)-Deacetyl-Thiomethylcolchicine (3b) and (-)-N- (Cumarin-3-carbonyl)-Deacetyl-Benzylthiocolchicine (3c)
Amides 3a-c have been prepared by means of reaction of the deacetylcolchicine 1a-c with 3-carboxy cumarinic acid in CH2CI2 in the presence of dicyclohexylcarbodiimide (DCC) and 4-pyrrolidin pyridine (PPY). Procedure: A mixture consisting of deacetylcolchicine 1a, 1b or
1c (1.4 mmol), 3-carboxy cumarinic acid (0.31 g, 1.65 mmol), DCC (0.5 g, 2.4 mmol), PPY (0.05 g, 0.34 mmol) in CH2CI2 (15 ml) is stirred at room temperature for 20 h. The reaction mixture is filtered in order to eliminate dicyclohexylurea (DCU). The solution is washed with water, anhydrified over sodium sulphate and concentrated at reduced pressure. The crude product is purified by silica gel column chromatography using diethyl ether/petroleum ether 90:10 mixture as eluent. Derivatives 3a-c are obtained with 80-85% yields.
(-)-N- (Cumarin-3-carbonyl)-Deacetyl-Colchicine (3a) m.p. 151-156 °C [α] 25D -74.52° (c. 0.5; CHCI3)
1HNMR (CDCI3, 200 MHz) δ: 2.40-2.67 (m, 4H, CH2-5, CH2-6),
3.76 (s, 3H, OMe-10), 3.97 (s, 3H, OMe-1), 4.01 (s, 3H1 OMe-2), 4.03 (s, 3H, OMe-3), 4.81 (m, 1H, H-7), 6.63 (s, 1 H, H-4), 6.86 (d, 1 H, J=10.8Hz,
H-11), 7.32 (S1 1 H, H-6'), 7.33-7.74 (m, 5H, H-8, H-12, H-4', H-5', H-7'),
8.80 (s, 1 H. H-3');
13C-NMR (CDCI3, 400 MHz) ppm: 29.4, 36.3, 52.6, 55.7, 56.0, 61.1 , 107.0, 111.7, 116.3, 117.4, 118.0, 125.1 , 125.4, 129.5, 130.7, 133.9, 134.1 , 134.7, 135.5, 141.2, 148.3, 149.8, 150.8, 153.1 , 154.1 , 160.7, 161.0, 163.7, 179.0.
Preparation of optically active COLCHICOLO (4a), Thiomethyl- COLCHICOLO (4b) and Benzylthhio-COLCHICOLO (4c)
4a-b COLCHICOLS are obtained from corresponding deacetylcolchicines 1a-b, respectively, according to the literature.1c,3.
Racemic COLCHICOLO 4c is a new product and has been prepared by reaction of deacetylcolchicine 1c with 4-formyl-1-methylpiridinium benzene sulphonatel (FMB), at ambient temperature for 1 ,5 h. Obtained imine then is treated in situ with 1 ,8-diazabicyclo [5.4.0] undec-7-ene (DBU) and then with oxalic acid yielding correspondent colchicone which is reduced with
NaBH4 in methanol to yield racemic alcohol (±)-4c.
The separation of (+) and (-)-COLCH ICOLI 4a-c is obtained for the first time by re-crystallisation of menthyl derivatives 5a-c, obtained by treatment of the racemic mixture with (-)-menthyl-chloro formiate. The use of ethyl acetate as re-crystallisation solvent yields (+)-(aS, 7R)-menthyl- derivative (+)-5a-c, with approximately 40% yield. The use of MeOH/CHCI3 as re-crystallisation solvent provides (-)-(aR, 7S)-menthyl-derivative (-)-5a- c with approximately 40% yield.
The hydrolysis of menthyl-derivatives in KOH 0.5 M methanol solution provides optically pure (+) and (-)-COLCHICOLI 4a-c with 95%yields. The procedure for the separation of (±)-thioCOLCHICOLO 4b is below reported.
Procedure: to) To a solution of (±J-thioCOLCHICOLO 4b (6 g,
16.0 mmol) in anhydrous piridine (30 ml), (-)-menthyl-chloroformiate (6.8 ml, 32 mmol) is added and the mixture is stirred at room temp for 1 ,5 h.
After work-up the crude product is purified by silica gel column using diethyl ethe/chloroform/petroleum ether 20:50:30 mixture. Menthyl- derivative 5b is obtained with 94% yield. b) Separation of the diastereoisomers: Diatereoisomers (±)-5b have been separated by re-crystallisation. The use of ethyl acetate as re- crystallisation solvent provides diastereoisomer (+)-5b (α] 25 D +185.5° (c.
0.3 CHCI3)), with 44% yield. The use of MeOH/CHCI3 as re-crystallisation solvent provides (-)-5b ([α] 25 D -269.5° (c. 0.3 CHCI3)) with 40% yield.
[α]25D +185.5° (c. 0.33 CHCI3)
1H-NMR (CDCI3, 400 MHz) δ: 0.69 (d, 3H, CH3-5\ J= 6.9 Hz),
0.85 (d, 3H, CH3-CH-CH3, J=6.8 Hz), 0.86 (d, 3H1 CH3-CH-CH3, J= 6.2
Hz), 0.75-1.12 (m, 3H), 1.37 (m, 2H), 1.62 (m, 2H), 1.80-2.10 (m, 3H), 2.42
(s, 3H, SCH3), 2.35-2.60 (m, 3H), 3.62 (s, 3H, OCH3-I), 3.89 (s, 3H,
OCH3-2), 3.91 (s, 3H, OCH3-3), 4.38 (m, 1 H, H-1'), 5.16 (m, 1 H, H-7), 6.55
(s, 1H1 H-4), 7.02 (d, 1H, H-11 , J=10.3 Hz), 7.23 (d, 1H, H-12, J= 10.3 Hz)1
7.30 (s, 1H, H-8)
13C-NMR (CDCI3 400 MHz) ppm: 15.1 , 16.1 , 20.6, 21.8, 23.1 ,
25.8, 29.2, 31.3, 33.9, 35.8, 40.4, 46.8, 55.9, 61.2, 61.3, 75.9, 78.9, 107.3,
124.9, 125.8, 127.6, 134.2, 134.5, 136.0, 141.5, 147.9, 151.0, 153.5,
153.6, 158.6, 182.2
Menthyl derivative (-)-5b
[(X]25D -269.5° (c. 0.31 CHCI3)
1H-NMR (CDCI3, 400 MHz) δ: 0.69 (d, 3H, CH3-5', J= 6.9 Hz),
0.84 (d, 3H, CH3-CH-CH3, J=6.5 Hz), 0.89 (d, 3H, CH3-CH-CH3, J= 7.0
Hz), 0.75-1.05 (m,3H), 1.38 (m, 2H), 1.63 (m, 2H), 1.90-2.06 (m, 3H), 2.41
(s, 3H, SCH3), 2.35-2.60 (m, 3H), 3.62 (s, 3H, OCH3-I), 3.89 (s, 3H, OCH3-2), 3.91 (s, 3H, OCH3-3), 4.37 (m, 1 H, H-T), 5.15 (m, 1 H1 H-7), 6.54
(s, 1 H, H-4), 7.01 (d; 1 H, H-11 , J=10.4 Hz), 7.22 (d, 1 H, H-12, J= 10.4
Hz), 7.28 (s, 1 H, H-8)
13C-NMR (CDCI3) ppm: 15.0, 16.0, 20.6, 21.8, 23.2, 26.1 , 29.2,
31.2, 33.9, 35.7, 40.5, 46.9, 55.9, 61.2, 61.3, 75.9, 79.0, 107.3, 124.9,
125.8, 127.5, 134.2, 134.4, 135.9, 141.5, 147.8, 151.0, 153.6, 158.6,
181.9 c) Hydrolysis: A solution of (+)-5b or (-)-5b (1 ,8 mmol) in methanol KOH 0,5 M (25 ml) is stirred at 80 0C for 30 min. After work-up the crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/methanol 50:45:5). (+)-(aS, 7R)-thioCOLCHICOLO (+)-4b ([α] 25 D +189° (c. 0.3, MeOH)), and (-)-(aR, 7S)-thioCOLCHICOLO (-)-4b ([α] 25D -188° (c. 0.3, MeOH)), are obtained with 95% yield.
Preparation of (+) and (-)-7-O-Retinoyl-Deacetamido-colchicine (6a), 7-O-Retinoyl- Deacetamido-thiomethyl-colchicine (6b), and 7-0- Retinoyl-Deacetamido-benzylthiocolchicine (6c).
(+) and (-)-retinoyl derivatives 6a-c are prepared by reaction of (+) and (-)-COLCH ICOLI 4a-c with retinoic acid in CH2CI2 in the presence of diciclohexyl carbodiimide (DCC) and 4-pyrrolidin pyridine (PPY). Procedure: A mixture of (+) or (-J-COLCHICOLI (4a, 4b or 4c)
(1.4 mmol), retinoic acid (0.7 g, 2.3 mmol), DCC (0.58 g, 2.8 mmol), PPY (0.062 g, 0.42 mmol) in CH2CI2 (15 ml) is stirred at room temperature for 3 h. The reaction mixture is filtered in order to eliminate DCU; the filtered solution is washed with water, anhydrified over sodium sulphate and concentrated at reduced pressure. The crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/petroleum ether 10:50:40). (+) and (-)-retinoyl derivatives 6a-c are obtained with 70-75% yields.
(-)-7-O-Retinoyl- Deacetamido-thiomethylcolchicine (-)-6b [α] 25 D -68.9° (c. 0.37 CHCI3) m.p.: 113-1170C 1H-NMR (CDCI3, 400 MHz) δ: 1.02 (s, 6H1 2CH3-I'), 1.45-1.75
(m, 4H, CH2-13', CH2-14'), 1.71 (s, 3H, CH3-H'), 1.98 (s, 3H, CH3-7'), 1.9-
2.1 (m, 2H1 CH2-5), 2.25 (s, 3H, CH3-3'), 2.3-2.6 (m, 2H, CH2-5), 2.42 (s,
3H, S-CH3), 3.66 (s, 3H, OCH3-I), 3.90 (s, 3H, OCH3-2), 3.93 (s, 3H,
OCH3-3), 5.37 (m, 1 H, H-7), 5.87 (s, 1 H1 H-2'), 1.1-6.3 (m, 4H1 H^'.H-β'.H-
8',H-9'), 6.54 (s, 1 H, H-4), 6.99 (dd, 1H1 H-5', J=14.8, 11.6 Hz), 7.03 (d,
1H, H-11, J=10.3 Hz), 7.25 (d, 1H, H-12, J=10.3 Hz), 7.33 (s, 1H, H-8).
13C-NMR (CDCI3. 400 MHz) ppm: 12.8, 13.9, 15.1 , 19.1 , 21.7,
28.9 (2C)1 29.4, 33.0, 34.2, 36.0, 39.5, 56.0, 61.2, 61.4, 71.9, 107.4, 117.4, 125.1 , 126.2, 128.2, 128.8, 129.4, 130.0, 131.6, 134.5, 134.6,
134.7, 136.7, 137.1 , 137.6, 140.0, 141.5, 148.9, 151.0, 153.6, 154.5,
158.5, 165.4, 182.3
Preparation of various esters of (+) and (-)-COLCHICOLO (4a), ThiomethylCOLCH ICOLO (4b) and BenzylthioCOLCH ICOLO (4c). Various esters having 7-9 (a-c) structures have been prepared by reaction of (+) and (-)-COLCH ICOLI 4a-c with appropriated carboxylic acid (hexanoic, dodecanoic and hexadecanoic acids, 3-carboxy cumarinic and acetyl salicilic acids) in CH2CI2 in the presence of DCC and PPY.
Procedure: A mixture of (+) or (-)-COLCHICOLO (4a, 4b or 4c) (1.4 mmol), carboxylic acid (2.3 mmol), DCC (0.58 g, 2.8 mmol), PPY
(0.062 g, 0.42 mmol) in CH2CI2 (15 ml) is stirred at room temperature for 3 h. The reaction mixture is filtered in order to eliminate DCU; the filtered solution is washed with water, anhydrified over sodium sulphate and concentrated at reduced pressure. The crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/petroleum ether
10:50:40). (+) and (-)-ester derivatives 7-9 are obtained with 70-75% yield.
7-(cumarin-3-carboxylate)-Deacetamido-benzylthiocolchicine (8c) mp 116-118 0C; 1H-NMR (CDCI3, 400 MHz) δ: 2.18-2.61 (m, 4H1 CH2-5, CH2-6), 3.67 (s, 3H, OCH3-I), 3.92 (s, 3H, OCH3-2), 3.94 (s, 3H, OCH3-3), 4.14 (s, 1 H1 SCH2Ph), 5.56 (rn, 1 H, H-7), 6.57 (s, 1H, H-4), 7.2-7.7 (m, 12H, Ar1 H- 8, H-11 , H-12), 8.60 (s, 1 H, H-4"); 13C-NMR (CDCI3. 400 MHz) ppm: 28.9, 35.5, 36.2, 55.8, 61.0,
61.2, 73.9, 107.3, 116.2, 116.5, 117.4, 124.4, 124.7, 126.8, 127.3, 127.8, 128.4 (2C), 128.7 (2C), 129.6, 133.9, 134.4, 134.5, 134.6, 136.5, 141.2, 147.7, 149.0, 150.8, 153.4, 154.6, 156.0, 157.0, 161.0, 181.8.
Preparation of (+)-and (-)-7- {[(2'E)-4' (Retinyloxy) but-2'-enil] oxy}-deacetamidocolchicine (11a), 7- {[(2E)-4 (Retinylossi) but-2-enil] oxy}- deacetamido-thiomethylcolchicine (11b) and 7- {[(2E)-4 (Retinylossi) but-2- enil] oxy}-deacetamido-benzylthiocolchicine (11c).
(+) or (-)-COLCHICOLI lithium salts 4a-c obtained by treatment with lithium diisopropylamide (LDA), react with trans-1 ,4-dibromo-2-butene to yield corresponding (+)- and (-)-butenyl bromides 10a-c. The reaction of 4-bromo-butenyl derivatives with retinol lithium salts yields (+)-and (-)- retinyl ethers 11a-c.
Procedure: a) To a solution of (+) or (-J-COLCHICOLO (4a, 4b or 4c) (2,7 mmol) in THF (20 ml) cooled at -20 0C a solution of LDA (4 mmol) in THF (6 ml) is added. The resulting mixture is stirred at 0 0C for 30 min and then at room temperature for 1 h. A solution of 1 ,4-dibromo-2- butene (5,4 mmol) in DMF (15 ml) at room temperature is added and the solution stirred for 15 h. After work-up the crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/petroleum ether 50:40:10). (+)-and (-)-butenyl bromides 10a-c are obtained with 40% yield. b) To a solution of LDA (2,5 mmol) in 4 ml of Et2O, cooled at -20 0C, a solution of retinol (2,5 mmol) in 4 ml of Et2O is slowly added. The mixture is stirred at room temperature for 30 minutes and a solution of butenyl bromide 10 (1 mmol) in DMF is added and the solution stirred at room temperature for 15 h. After work-up the crude product is purified by column chromatography (silica gel, diethyl ether/petroleum ether 85:15). (+)-and (-)-retinyl ethers 11a-c are obtained with 30% yield.
(-)-7-{[(2E)-4 (Retinyloxy) but-2-enil] oxyj-deacetamido- thiomethylcolchicine (-)-11b [CX]25D -106.2° (c. 0.65 CHCI3)
1H-NMR (CDCI3, 400 MHz) δ: 1.00 (s, 6H, 2CH3-15"), 1.40-1.65
(m, 4H, CH2-13", CH2-14"), 1.70 (s, 3H, CH3-H"), 1.81 (s, 3H, CH3-7"),
1.85 (m, 1H, CHH-6), 1.94 (s, 1 H, CH3-3"), 2.00 (m, 2H, CH2-12"), 2.3-2.5
(m, 3H, CHH-6, CH2-5), 2.43 (s, 3H1 S-CH3), 3.61 (s, 3H, OCH3-I), 3.67
(m, 1 H1 CHH-1'), 3.85-4.0 (m, 4H1 CHH-1', CH2-4\ H-7), 3.91 (s, 6H1
OCH3-2, OCH3-3), 4.06 (d, 2H, CH2-I", J = 6.7 Hz), 5.58 (t, 1H, H-2", J =
6.7 Hz), 5.72 (m, 2H, H-21, H-3'), 6.05-6.30 (m, 4H, H-4",H-6",H-8",H-9"),
6.53 (S, 1 H, H-4), 6.57 (dd, 1 H, H-5", J=15.1 , 11.2 Hz), 7.03 (d, 1 H, H-11 ,
J=10.3 Hz), 7.20 (d, 1H, H-12, J=10.3 Hz), 7.50 (s, 1H, H-8).
13C-NMR (CDCI3 400 MHz) ppm: 12.7, 12.8, 15.1 , 19.2, 21.7, 28.9 (2C), 29.6, 33.0, 34.2, 37.6, 39.6, 56.0, 61.0, 61.2, 66.6, 69.6, 69.8, 78.8, 107.2, 124.8, 124.9, 125.8, 126.5, 127.7, 128.7, 129.0, 129.2, 129.8, 130.1 , 134.4, 135.1 , 135.9, 136.4, 137.2, 137.4, 137.0, 137.8, 141.2, 149.2, 150.7, 153.5, 158.5, 182.4.
Preparation of (+)- and (-)-7-(Alkyl-oxy)-deacetamidocolchicine (12-14a), 7-(Alkyl-oxy)-deacetamidothiomethylcolchicine (12-14b) and 7- (Alkyl-oxy)-deacetamido benzylthio-colchicine (12-14c).
The reaction of (+) and (-)-COLCH ICOLI 4a-c lithium salts with a set of alkyl bromides (1-bromo-nonane, 1-bromo-dodecane, 1-bromo- esadodecane, genaryl bromide and famesyl bromide) provides corresponding (+)- and (-)-alkyl ethers 12-14 (a-c).
Procedure: To a solution of (+) or (-J-COLCHICOLO (4a, 4b or
4c) (2.7 mmol) in THF (20 ml) cooled at -20 °C a solution of LDA (4 mmol) in THF (6 ml) is added. Resulting mixture is then stirred with magnetic bar at 0 °C for 30 minutes and 1 h at room temperature. A solution of alkyl bromide (4 mmol) in DMF (15 ml) is added and the solution is stirred at room temperature for 15 h. After work-up the crude product is purified by column chromatography (silica gel, diethyl ether/chloroform/petroleum ether 50:401:10). (+)-and (-)-alkyl ethers 12-14 (a-c) are obtained with 40% yield. (-)-7- (Geranyl-oxy)-deacetamidothiomethylcolchicine (-)-13b
[(X]25 D - 221.8° (c. 0.3 CHCI3)
1H-NMR (CDCI3, 400 MHz) δ: 1.50 (s, 3H, CH3-7'), 1.55 (s, 3H,
CH3-7'), 1.63 (S, 3H, CH3-3'), 1.77-2.05 (m, 5H, CHH-6, CH2-4',), 2.25-2.5
(m, 3H1 CHH-6, CH2-5), 2.42 (s, 3H, S-CH3), 3.63 (s, 3H, OCH3-I), 3.67
(m, 1H, CHH-1'), 3.85-4.0 (m, 4H, CHH-1 ', H-7), 3.89 (s, 6H, OCH3-2,
OCH3-3), 5.00 (t, 1 H, H-6\ J = 6.9 Hz), 5.28 (t, 1 H, H-2', J = 6.3 Hz), 6.53
(s, 1H, H-4), 7.02 (d, 1H, H-11 , J=10.3 Hz), 7.18 (d, 1H, H-12, J=10.3 Hz),
7.53 (s, 1 H, H-8).
13C-NMR (CDCI3 400 MHz) ppm: 15.1 , 16.4, 17.6, 25.6, 26.2,
29.6, 37.7, 39.5, 55.9, 60.9, 61.1 , 66.2, 78.5, 107.1 , 120.2, 123.8, 124.9,
125.8, 129.1 , 131.5, 134.3, 135.2, 137.3, 140.6, 141.1 , 149.4, 150.6,
153.4, 158.4, 182.4.
Example 2: Study about increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the proliferation and migration of human stellate cells both under control conditions and after stimulation with 10 ng/ml of PDGF-BB. MATERIALS AND METHODS Isolation and culture of human liver stellate cells. Human HSCs have been isolated from surgical sections (10-20 g) of not transplant usable normal human liver. Shortly, after collagenase and pronase combined digestion of surgical slices HSCs have been separated from other non parenchymal liver cells by means of ultra- centrifugation on stractan gradient (Cellsep ™ isotonic solution, Larex Inc, St. Paul, MN, USA). The cells have been cultured in plastic flasks (Falcon, Becton Dickinson, Lincoln, New Jersey, USA) in Iscove's modified Dulbecco's medium, supplemented with 0.6 U/ml of insulin, glutamine (2 mM), non essential amino acids (0,1 mM), sodium pyruvate (1 mM), antimycotic and antibacterial solution (all from GIBCO BRL Laboratories, Grand Island, New York, USA), and calf foetal serum 20% (v/v) (Imperial Laboratories, Andover, UK). The cells have been cultured in atmosphere consisting of humidified air with 95% O2 and 5% CO2, at constant temperature of 37 °C. Just isolated and in successively sub-cultured cells have been characterised as previously described. Described experiments have been carried out using HSCs between the third and fifth serial passage, at 1 :3 distribution ratio. Experiments have been carried out using three different cell lines.
Determination of DNA synthesis DNA synthesis has been evaluated using the method involving incorporation of [methyl-3H]-thymidine in cellular material precipitated with trichloroacetic acid. Shortly, cells have been cultured in 24-well plates, at density of 2x104 cells/well, with complete culture medium containing 20% of calf foetal serum (FBS). At confluency (approximately 1x105 cells/well), the cells have been made quiescent with the incubation in serum and insulin free medium (serum-free/insulin-free: SFIF) for 48 hours. Then the cells have been incubated with or without agonists, at doses and conditions indicated, for 20 hours. The cells have been then incubated for 4 hours with 1.0 μCi/ml of [methyl-3H]-thymidine (6,7 Ci/mM). At the end of pulsing period incorporated [methyl-3H]-thymidine has been measured as reported in literature. The number of cells has been estimated in three different wells for every plate and the final result of the experiment has been expressed as cpm/105 cells. Analysis of cell migration The cell migration has been analysed as described in literature.
Shortly, the experiments have been carried out using Boyden chamber technique, equipped with 8 μm pore polycarbonate filters. The filters have been pre-treated with type I human collagen (20 μg /ml) for 30 min at 37 0C and subsequently inserted between the upper and lower chamber HSCs, cultured at confluence, have been incubated in SFIF for 48 hours and then have been treated for 10 min with increasing concentrations of NO donors. Then the cells have been re-suspended by means of mild trypsinization (0,05% trypsin/EDTA) and an aliquot of the cell suspension (210 μl), corresponding to approximately 4x104 cells, has been inserted in the upper chamber and incubated at 37 0C for 6 hours. In the lower room SFIF (control) or PDGF-BB (10 ng/ml) with or without the same NO donor concentration as employed during the pre-incubation period were present. After methanol fixation at 96% and Harris hematoxylin staining, the cells migrated onto the filter lower side have been quantified as number of cells in 10 high-power fields (HPF). All the experiments have been carried out in triplicate. Each triplicate analysis has been repeated twice, in different experimental sessions, and with various HSC preparations. Possible cytotoxic effects have been checked using trypan blue exclusion test. This test has constantly indicated a 90 % cell viability. Data statistical analysis Results from experiments reported in Tables 2 and 3 are expressed as mean + standard deviation and refer to 18 determinations for each experimental point (n=18). The statistical analysis was carried out by variance analysis (ANOVA) and when F value was significant by Duncan Test. Experiments
In a first group of experiments (Table 2) the increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the proliferation (evaluated by tritiated thymidine incorporation in cell DNA) of human stellate cells both under control conditions and after stimulation with 10 ng/ml of PDGF-BB have been evaluated. In a second group of experiments (Table 3) the increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the migration of human stellate cells both under control conditions and after stimulation with 10 ng/ml of PDGF-BB have been evaluated.. The evaluation of test compounds by means of the aforesaid experimental design allows a rapid evaluation of antifibrogenic potentiality of a large number of compounds. For all the compounds listed in Tables 2 and 3 a toxic effect has been accurately excluded.
Cell proliferation (TABLE 2): As expected the PDGF-BB stimulation in all the experiments induced a significant increase of the cell proliferation in comparison to control (C). Co-incubation with three decreasing colchicine molar concentrations (10"6, 10~7, 10~8 M) at all the concentrations induced a remarkable reduction of the proliferation. All the retinol-colchicine compounds listed in Table 1 at least at one of tested molar concentrations (1O-6, 10~7, 10"8 M) induced a significant reduction of the cell proliferation. Moreover compounds 1a, 3a, 4c, (-)-6b, (+)-6b, (-)- 11b, (+)-11b, (-)-13b, (+)-13b (yellow coloured in the table) induced a significant inhibition of PDGF-BB induced proliferation in a dose- dependent way. A minor part of tested compounds resulted in a significant reduction of the basal proliferation (compared to the Control). Cell migration (TABLE 3): As expected the PDGF-BB stimulation in all the experiments induced a significant increase of the cell migration in comparison to control (C). Co-incubation with three decreasing colchicine molar concentrations (10"6, 10'7, 10~8 M) at all the concentrations induced a remarkable reduction of the proliferation. All the retinol-colchicine compounds listed in Table 2 at least at one of tested molar concentrations (1C)"6, 10"7, 10~8 M) induced a significant reduction of the cell migration. Moreover compounds 6b, 11b, 13b, (-)-6b, (+)-6b, (-)- 11b, (+)-11b, (-)-13b, (+)-13b (yellow coloured in the table) induced a significant inhibition of PDGF-BB induced proliferation in a dose- dependent way. A minor part of tested compounds resulted in a significant reduction of the basal proliferation (compared to the Control). TABLE 2
Effects on cell proliferation (*= P<0.05 or higher significance level vs. Control) (** = P<0.05 or higher significance level vs. PDGF-BB)
3c Mean 76 95 94 94 2480 683** 2200 2827 SD 6 32 8 14 181 77 1225 100
4c Mean 480 304* 284* 572 3251 649** 1468** 1816** SD 90 64 103 64 732 148 92 119
6a Mean 1018 817 1189 1004 3740 916** 4071 4326 SD 60 189 128 252 1177 80 201 236
6b Mean 754 726 881 987 10484 1823** 7652** 11987 SD 58 98 91 187 623 489 565 2156
8c, Mean 581 367 457 524 1809 356** 1601 1589 SD 619 392 635 417 1705 484 2146 2371
11b Mean 482 184* 351 460 1808 173** 1034** 1641 SD 55 86 112 137 286 73 206 123
13b Mean 402 301* 298* 395 2604 501** 1625** 2620 SD 21 16 52 32 40 151 300 280
B-6b Mean 340 157* 104* 330 981 134** 452** 517** SD 143 14 19 68 90 14 48 93
(+)-6b Mean 174 168 274 180 1599 1065** 1294 1167 SD 28 11 91 82 245 17 169 81
(-)-11b Mean 726 148* 272* 338* 2332 266** 1218** 1733** SD 44 9 15 145 131 57 255 130
(+)-11b Mean 440 153* 201 268* 2737 634** 2138** 2068** SD 294 18 206 58 453 282 433 460
(-)-13b Mean 748 655 758 747 3630 846** 1366** 2792** SD 152 71 40 41 897 243 59 452
(+)-13b Mean 791 638 722 772 4098 1139** 3284** 3466* SD 125 84 22 117 525 271 259 268
TABLE 3
Effects on cell migration
(*= P<0.05 or higher significance level vs. Control) (** = p<0.05 or higher significance level vs. PDGF-BB)
13b Mean 42 43 62 38 94 41** 68** 67** SD 5 5 6 4 2 1 3 1
(-)-6b Mean 7 8 6 7 17 0** 2** 4** SD 1 " 1 0 1 2 0 1 1
(+)-6b Mean 7 6 5 17 5** 4** 3** SD 1 0 1 2 2 2 1
(-)-11b Mean 30 5* 15* 29 98 -14** 68** 64** SD 5 1 2 6 8 2 3 8
(+)-11b Mean 16 19 16 19 43 19** 25** 28** SD 5 4 5 3 6 5 5 6
(-)-13b Mean 3 4 7 4 32 2** 20** 31** SD 1 1 2 1 3 3 2 5
(+)-13b Mean 1 1 1 1 6 2** 3** 2** SD 1 1 1 O OJ C 1 1 0 1 1
Example 3: Sfudy about increasing dose effects of colchicine and various retinol-colchicine conjugate inventive compounds on the synthesis of type I protocollagen in human stellate hepatic cells Hepatic human stellate cells have been grown at 70-80% confluence and then incubated with serum free medium (SFIF) for 24 hours. The culture medium have been replaced with serum-free fresh medium and the cells have been treated with test compounds (colchicines derivatives) in absence or presence of transforming growth factor-β1 (TGF β-1 , 1 ng/ml), used as standard stimulus for the synthesis of collagen and other components of extracellular matrix. After 24 hours of incubation the supernatant has been withdrawn and frozen at -20 0C. The secretion of collagen has been determined by means of EIA method, using commercially available Procollagen Type C-peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
Effect of two doses of colchicine on the synthesis of type I protocollagen in stellate hepatic human cells (see figure 1)
Cells grown at 70-80%confluence have been then incubated un serum free medium (SFIF) for 24 hours. Culture medium has been replaced with SFIF fresh medium and the cells treated with colchicine in absence or presence of Transforming Growth Factor-β1 (TGF β-1 , 1 ng/ml), used as standard stimulus for the synthesis of type I protocollagen. After 24 hours of incubation the supernatant has been withdrawn and the concentration of type I pro-collagen has been determined by means of EIA method, using commercially available Procollagen Type C- peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
* P<0.05 or higher significance value (Student T test). Both the colchicine doses induced a statistically significant reduction of TGF β-1 induced type I pro-collagen synthesis.
Effect of two doses of (-)-11b compound on the synthesis of type I protocollagen in stellate hepatic human cells (see figure 2)
Cells grown at 70-80% confluence have been then incubated un serum free medium (SFIF) for 24 hours. Culture medium has been replaced with SFIF fresh medium and the cells treated with (-)-11 b compound in absence or presence of Transforming Growth Factor-β1
(TGF β-1 , 1 ng/ml), used as standard stimulus for the synthesis of type I pro-collagen. After 24 hours of incubation the supernatant has been withdrawn and the concentration of type I pro-collagen has been determined by means of EIA method, using commercially available
Procollagen Type C-peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
* P<0.05 or higher significance value (Student T test). Both the (-)-11b compound doses induced a statistically significant reduction of TGF β-1 induced type I pro-collagen synthesis. Effect of two doses of (+)-11b compound on the synthesis of type I protocollagen in stellate hepatic human cells (see figure 3)
Cells grown at 70-80% confluence have been then incubated in serum free medium (SFIF) for 24 hours. Culture medium has been replaced with SFIF fresh medium and the cells treated with (+)-11b compound in absence or presence of Transforming Growth Factor-β1 (TGF β-1 , 1 ng/ml), used as standard stimulus for the synthesis of type I pro-collagen. After 24 hours of incubation the supernatant has been withdrawn and the concentration of type I pro-collagen has been determined by means of EIA method, using commercially available Procollagen Type C-peptide EIA kit (Takara Bio Inc., Otsu, Shiga, Japan).
* P<0.05 or higher significance value (Student T test). Only the 10'7 M dose of (+)-11b compound induced a statistically significant reduction of TGF β-1 induced type I pro-collagen synthesis.
Example 4: Chemical stability tests of (-)-11b product The (-)-11b product proves to be highly sensitive to the presence of oxidant agents, as atmospheric oxygen, which result in the destruction of conjugated double bond system of retinyl group generating various decomposition products.
The chemical stability of (-)-11b product, both as solid and triolein (glyceryl trioleate)or methanol solution at 200C or room temperature; in nitrogen or air atmosphere and presence or absence of light, has been evaluated.
The sample stability was checked by HPLC analysis using a VISFER SILICA 5 μ, 4,6 mm x 25 cm column; Eluent: ethyl acetate/n- hexane 75:25; flow rate 1 ml/min.
Disappearance of (-)-11b product at λ =326 nm and appearance of decomposition products at λ = 380 nm wavelength are monitored.
Table 4 shows decomposition percentage (%) of (-)-11b product stored in different storage conditions after 10, 30 and 60 days (g).
Table 4
a A 10 ml aliquot is diluted in 1ml of ethyl acetate and then analysed by HPLC. b The solution is used as it is for HPLC analysis. cThe light protection is provided by aluminum foil shield. d The light protection is provided by amber borosilicate vials.
As it can be observed from the data reported in table 4, in order to obtain a good chemical stability, the solid product must be stored in nitrogen atmosphere and therefore in absence of oxygen and day-light protected. At a temperature of -20 0C complete stability is assured. Amber borosilicate containers are not sufficient the light protection; the use of aluminium foil as container covering assures a complete protection. The product is better conserved in solution (tests 5-7) wherein the solvent provides a good protection from atmospheric oxygen. Particularly in glyceryl trioleate at -20 0C, in nitrogen atmosphere and absence of light a complete chemical stability is obtained.
Bibliographical references
1. (a) Lebeau, L.; Ducray, P.; Mioskowski, C. Synthetic Comm. 1997,
27, 293-296. (b) Danieli, B.; Lesma, G.; Passarella, D.; Prosperi, D.;
Silvani, A. HeIv. Chim Acta 1999, 82, 1502-1508. (c) Shi, Q.;
Verdier-Pinard, P.; Brossi, A.; Hamel, E.; McPhail, A. T.; Lee, K.-H.
J. Med. Chem. 1997, 40, 961-966. (d) Shi, Q.; Verdier-Pinard, P.;
Brossi, A.; Hamel, E.; Lee, K.-H. Biorg. Med. Chem. 1997, 5, 2277-
2282. (e) Velluz, L.; Muller G. Bull. Soc. Chim. Fr. 1954, 194-200
2. Brossi, A.; Sharma, P. N.; Atwell, L.; Jacobson, A. E.; lorio, M. A.;
Molinari, M.; Chignell, C- F. J. Med. Chem. 1983, 26, 1365-1369.
3. (a) Bombardelli. E.; PCT lnt Appl 1997, WO9747577. (b) AI-TeI, T.
H.; Abu Zarga, M. H., Sabri, S. S.; Freyer, A. J.; Shamma, M.
Journal of Natural Product, 1990, 53, 623-629.

Claims

CLAIMS 1. Ccolchicine derived compounds having the general formula
(I):
with (aR, 7S), (aS, 7R); (aR, 7S) or (aS, 7R) configuration, the group R is selected from OCH3, SCH3 or SCH2Ph, X is selected from NH or O, Ri is selected from
wherein n is 4, 10 or 14
,Br
—(CH2)nCH3 wherein n is 8, 11 or 15
and Ri is H when X is NH and R is SCH2Ph, or when X is O and R is SCH3 or SCH2Ph; with provision that when R1 is
X is NH then R is different than OCH3.
2. Compounds according to claim 1 selected from compounds havin the followin formulas:
3. Compounds as defined according to each of claims 1-3 to be used in medical field..
4. Pharmaceutical composition comprising one or more compounds as defined according to each of preceding claims as active principle together with one or more pharmaceutically acceptable excipients or co-adjuvants.
5. Use of compounds and composition as defined according to each of preceding claims for the preparation of a medicament to be used in the antifibrotic therapy of the chronic hepatic diseases.
6. Retinol derived compounds having the following general formula (II):
wherein R2 is selected from
with (aR, 7S), (aS, 7R); (aR, 7S) or (aS, 7R) configuration, wherein the group R is selected from OCH3, SCH3 or SCH2Ph, with the provision that when R2 is
ith {aR, 7S) configuration, then R is different than OCH3.
7. Use of compounds as defined according to claim 6 for the preparation of a medicament for the antifibrotic therapy wherein retinol acts as carrier.
8. Pharmaceutical composition comprising one or more compounds as defined according to claim 6 as active principle together with one or more pharmaceutically acceptable excipients or co-adiuvants.
9. Use of compounds and composition as defined according to claims 6 and 8 for the preparation of a medicament for the prevention and treatment of hepatic diseases.
10. Use according to claim 9 wherein the hepatic disease is hepatic fibrosis.
11. Use of the retinol as carrier for antifibrotic drugs.
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