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  • 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
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  • 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).

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