ES2322534B1 - CIS DE PLATINO BICYCLE COMPLEXES (II) WITH ANTI-TARGET ACTIVITY. - Google Patents

Abstract

Bicyclic complexes of platinum (II) cis with anticancer activity.

The compounds of formula (I) where the line dashed represents a single or double bond; X is N, O, S, or - (CH 2) n -; n is an integer from 1 to 3; R_ {1} is a radical independently selected from (C 1 -C 4) alkoxy, phenoxy, phenyl (CH 2) r -O; R 2 and R '' 2 are a radical independently selected from hydrogen, hydroxyl, (C 1 -C 4) -alkoxy, phenoxy, phenyl (CH2) r -O, and O-Gp; r is an integer from 1 to 4; Gp is a group alcohol protector; the radicals being with rings optionally mono, di- or benzene tri-substituted in their benzene rings by (C 1 -C 4) -alkyl, (C 1 -C 4) -alkoxy and halogen; R 3 and R '' 3 are halogen or, taken together, they are a birradical selected from -OCO- (CH2) m -COO-, (C 3 -C 7) - cycloalkane-1,1-dicarboxylate, and (C 3 -C 7) cycloalkane-1,2-dicarboxylate; and m is an integer from 0 to 4, have shown to be active against several cancer cell lines This means that these compounds are Useful agents to treat various types of cancer.

Description

Bicyclic complexes of platinum (II) cis with anticancer activity.

The present invention relates to new bicyclic cis -platinum (II) complexes of 1,4-diamines having different degrees of functionalization, as well as to their preparation process and their use for the treatment and / or prevention of cancer.

Prior art

One of the most widely used anticancer drugs is cis- (diamine) dichloroplatin (II) or cisplatin, cis - [PtCl 2 (NH 3) 2]. The cytotoxic activity of this compound was first observed by Rosenberg in 1969. It is known that when cisplatin is administered to a patient this substance is distributed in the body and reaches the target cell without undergoing any structural modification. However, when cisplatin enters the cell, it is rapidly hydrolyzed to give rise to the cis - [PtCl (H2O) (NH3) 2] + species, because The concentration of chloride ions inside the cell (approximately 3 mM) is lower than the concentration of chloride ions in the extracellular medium (approximately 100 mM). The positively charged platinum adduct is a molecule with an increased electrophilia with respect to the corresponding neutral compound and reacts with nucleophiles present inside the cell such as the guanine nucleobase of the DNA, in the N-7 position, or the proteins with sulfur groups . Cisplatin can undergo a second hydrolysis inside the cell, which allows the drug to bind to DNA forming bifunctional cis adducts. Among the different modes of connection between DNA and cisplatin, intracatenary junctions through two neighboring guanine bases appear to be a key stage in the mechanism of action of this drug, since the distortion produced in the helical structure of the DNA can induce cell death by apoptosis. Thus, cisplatin is a bifunctional cytotoxic alkylating agent that reacts with DNA to produce inter and intracatenary bonds which alter the processes of replication and transcription of cellular DNA and cause cell cycle arrest in the G2 phase.

Currently, cisplatin is frequently used to treat some types of cancer, such as ovarian, lung or testicular cancer (cf. YP Ho et al ., Med. Res. Rev. 2003, vol. 23, p 633). However, cancer cells develop several mechanisms of drug resistance. Some examples of these mechanisms are: a) the inactivation of the drug through its interaction with the sulfur residues of proteins and peptides; b) decreased absorption of the drug by the cell or c) DNA repair mechanisms. In addition, the clinical use of cisplatin causes problems of nephro- and neuro-toxicity, which limits the dose of administration to patients. For the reasons mentioned above, it is of great interest to find new platinum (II) complexes that overcome the problems of cross resistance to cisplatin and are more efficient and less toxic than the drugs currently used in clinical oncology.

Explanation of the invention.

The inventors have found new agents antitumor agents with relevant cytotoxic activity. These compounds have been shown to be potent cancer cell inhibitors in front of several tumor cell lines, especially those corresponding to gynecological cancers, resistant and not resistant to cisplatin. It is worth mentioning that some of these new compounds are four to five times more active than the cisplatin and that are able to avoid the resistance developed by cancer cells against cisplatin.

Thus, a first aspect of the present invention is to provide a compound of formula (I), or its salts pharmaceutically acceptable, or its solvates, including any stereoisomer or mixture of stereoisomers,

one

where: the dashed line represents a single or double bond; X is a biradical that is select from the group consisting of N, O, S, and - (CH 2) n -; n is an integer from 1 to 3; R_ {1} is a radical independently selected from the group consisting of hydrogen, (C 1 -C 4) -alkoxy, phenoxy, phenyl- (CH2) r -O, and a radical derived from the aforementioned with rings benzene, through a mono-, di- or tri-substitution in its benzene rings; being the substituents a radical independently selected from halogen group, (C 1 -C 4) -alkyl, (C 1 -C 4) -alkoxy and halogen; R2 and R2 'are a radical independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4) -alkoxy, phenoxy, phenyl- (CH2) r -O, and O-Gp, and a radical derived from the above mentioned with benzene rings, through a mono-, di- or tri- substitution in its benzene rings; being the substituents a radical independently selected from the group formed by halogen, (C 1 -C 4) -alkyl, (C 1 -C 4) -alkoxy and halogen; r is an integer from 1 to 4; Gp is a protective group of alcohols; R 3 and R '3 are independently a halogen or alternatively R 3 and R '3 taken together are a birradical selected from the group consisting of -OCO- (CH 2) m -COO-, (C 3 - C7) - cycloalkane-1,1-dicarboxylate Y (C 3 -C 7) -cycloalkane-1,2-dicarboxylate; and m is an integer from 0 to Four.

The Gp can be any acetal such as an acetonide ie 1,1-methylmethylenedioxy; ethers, such as methyl, ethyl, isopropyl, phenyl or benzyl ethers or esters such as acetate, benzoate or oxalate. The protecting group can be introduced and removed by methods known in the state of the art (cf. TW Greene and PGM Wuts, Protective Groups in Organic Synthesis , third EditionWiley-Interscience, (1999)). The specific conditions depend on the protective group used.

The complexes form a seven-member metalacycle between a platinum (II) atom and a 1,4-diamine system, giving rise to cis -flat-plane complexes. The stereocenters present in the molecules may exist in the R or S configuration.

In a particular embodiment, the compounds of formula (I) have the formula (Ia), where R 1, R 2, R '2, R 3, R' 3 and X have the same meaning as You mentioned above.

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In a preferred embodiment, the compounds of formula (I), are those where X is O or a selected radical between methylene, ethylene and propylene.

In another preferred embodiment, the compounds of formula (I) are those where R 3 and R '3 are both chlorine or iodine

In another preferred embodiment, the compounds of formula (I) are those where R 3 and R '3 taken together they are a dicarboxylate of one of the following diacids: oxalic acid, malonic acid, succinic acid and acid cyclobutane-1,1-dicarboxylic.

In another preferred embodiment, the compounds of Formula (I) are those where R 1 is hydrogen or methoxy.

In another preferred embodiment, the compounds of formula (I) are those where R 2 and R '2 are independently selected from hydrogen, hydroxyl, methoxy, phenoxy, benzyloxy or an acetonide.

The most preferred compounds are those selected from those in the following list:

(one) cis - ((((3-aminomethyl-bicyclo [2.2.1] hept-5-en-2-yl) methylamine) dichloride) platinum (II);

(2) cis - (((3-aminomethylbicyclo [2.2.1] hept-2-yl) -methylamine) dichloride) platinum (II);

(3) cis - ((((3-aminomethyl-7-oxabicyclo [2.2.1 ] hept-5-en-2-yl) methylamine) dichloride) platinum (II);

(4) cis - ((((3-aminomethyl-7-oxabicyclo [2.2.1] hept-2-yl) methylamine) dichloride) platinum (II);

(5) cis - ((((3-aminomethyl-bicyclo [2.2.2] oct-5-en-2-yl) methylamine) dichloride) platinum (II); Y

(6) cis - ((((3-aminomethyl-bicyclo [2.2.2] oct-2-yl) methylamine) dichloride) platinum (II).

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The compounds of the present invention are can get from a series of 1,4-bicyclic diamines by the reaction of Diels-Alder between fumaronitrile and / or maleonitrile as dienophile and a suitable diene such as furan, methoxyfuran or other functionalized C-2 furans, cyclopentadiene, cyclohexadiene, cycloheptadiene or substituted dienes.

The introduction of the R 2 and R '2 groups is achieved by double link functionalization C5-C6, of the precursor cycduct, via dihydroxylation with RuO4, followed by derivatization of the diol resulting in the form of an ether (by S N 2 reactions with the corresponding haloalkanes), in the form of an acetal (for reactions with the corresponding ketone, eg acetone, in presence of an amount of catalyst of an acid) or in the form of an ester by reaction with the corresponding acid chloride or a acid anhydride in the presence of a base eg pyridine or Triethylamine as an acid scavenger.

The compounds of formula (I) where the line Discontinuous is a simple bond, they can be prepared from corresponding intermediate cycloducts by hydrogenation in presence of a catalyst, for example, Pd / C. The nitriles are they can reduce with LiAIH4 in an appropriate solvent such as diethyl ether or tetrahydrofuran (THF) to give diamines corresponding.

Finally the 1,4-diamines are they can react with K 2 PtCl 4, PtCl 2 or others Pt (II) reagents in an appropriate solvent, for example, in ethanol / water 1: 1 to give rise to the complexes of corresponding cis-platinum (cf. Fig. 1). Through this process the 1,4-diamines were coordinated to platinum giving rise to the corresponding platinum complexes with a yield close to 70%.

The antiproliferative activity has been evaluated and the structure-activity relationships of compounds of the present invention. Kinetics have been tested of the interaction with glutathione, which has shown the excellent resistance factors observed for the new complexes of platinum.

Cytotoxicity tests were performed. against cancerous, sensitive cell lines (A2780) and resistant (A2780cisR) to cisplatin. An excellent was observed cytotoxicity for most of these complexes, even much greater than that of cisplatin as a reference drug. Fits mention that all the new complexes evaluated have better  resistance factors that cisplatin against the cell line cancerous A2780cisR.

Thus, according to another aspect of the present invention a compound is provided as defined above for Use as an anti-carcinogen.

The present invention is also related with the use of the compounds of the present invention for the preparation of a medicament for therapeutic treatment and / or prophylactic cancer in a mammal, including humans. Preferably, the types of cancer against which the Compounds of the present invention may be especially assets are selected from those in the following group: cancer ovary, breast cancer, leukemia, lung cancer and cancer testicular

The compounds of the present invention are they can use in the same way as other therapeutic agents known. Thus, they can be used alone or in combination with others. suitable bioactive compounds.

The invention is also related to a method of treatment and / or prophylaxis of a mammal, including the human being, who suffers or is susceptible to cancer, in particular one of the cancers mentioned above, said method comprises administering to said patient an amount therapeutically effective of the compounds of the present invention, together with pharmaceutically excipients or carriers acceptable.

Another aspect of the present invention is related to a pharmaceutical composition comprising a therapeutically effective amount of the compounds herein invention, together with appropriate amounts of excipients or pharmaceutically acceptable carriers.

The chemotherapeutic treatment of cancer that derived from this invention is a new approach to therapy oncological and has the advantage of being useful for the treatment of Several types of cancer.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. The summary of this application is incorporated here as reference. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The The following examples are provided by way of illustration, and are not It is intended to be limiting of the present invention.

Brief description of the drawings

Fig. 1 summarizes the synthetic route for preparation of bicyclic complexes of cis platinum (II). In the Fig. 1 the synthetic route has been particularized for Y = CI as labile ligand.

Fig. 2 illustrates the results of the studies kinetics of the interaction of platinum complexes with glutathione % Platinum coupling (A) is represented (Pt) -glutation (GSH) versus time in hours.

Examples Example 1 Preparation of bicyclo [2.2.1] hept-5-en-2,3-dicarbonitrile (one)

Under anhydrous conditions, it dissolved fumaronitrile (1.0 g, 13.1 mmol) in absolute ethanol (15 mL) and he added, dropwise, cyclopentadiene (1.2 mL, 13.9 mmol). After if the addition was complete, the reaction mixture was cooled in an ice and water bath and, after 30 min of agitation, it reduced the volume of ethanol in half, under vacuum. The solution obtained at 4 ° C in the dark for 3 days, obtaining a white solid precipitate that was filtered (1.70 g). Yield = 73%, IR (KBr) 3080, 2990, 2950, 2244. EM (CI, NH 3,%) 145 (1, M + H +), 162 (88, M + NH 4 +) 163 (10, M + NH 4 + + H +).

Example 2 Preparation of 7-oxabicyclo [2.2.1] hept-5-en-2,3-dicarbonitrile (2)

Under anhydrous conditions, it dissolved fumaronitrile (1.0 g, 12.8 mmol) in furan (20 mL, 275 mmol). Be the mixture was heated to reflux and after 19 hours it was cooled and filtered to isolate the product (2) as a white solid. The waters mothers refluxed for 30 hours, isolating a new portion of the product (2) by filtration (total: 1.48 g). Yield = 81%. P.F. = 113-114 ° C (furan). IR (KBr) 3100, 2961, 2925, 2244, 1559. MS (CI, NH3,%) 102 (100, M-2 (CH-CN) + N 2 H 7 +), 103 (33, M-2 (CH-CN) + N 2 H 7 + + H +), 164 (9, M + NH 4 + + H +), 181 (56, M + N 2 H 7 +).

Example 3 Preparation of 1-methoxy-7-oxabicyclo [2.2.1] hept-5-en-2,3-dicarbonitrile (3a) and (3b)

Fumaronitrile (550 mg, 7 mmol) was dissolved in absolute ethanol (5 mL) and then 2-methoxyfuran was added at one time and under nitrogen. The reaction mixture was stirred for four days at room temperature and filtered to obtain 1.2 g of a white solid consisting of a mixture of diastereoisomers: (3a) (1 R *, 2 R *, 3 R *, 4 S *) ( 70%) and (3b) (1 R *, 2 S *, 3 S * 4 S *) (30%). The product (3b) was separated by fractional crystallization in a mixture of chloroform and ethanol. After purification by column chromatography of the mixture, the pure product (3a) was obtained. Suitable crystals of (3a) were also obtained for analysis by X-ray diffraction. Yield = 83%.

(3a): P. F. = 124-126 ° C (CHCl 3 / ethanol 6/4). IR (film) 3099, 2988, 2968, 2954, 2857, 2250, 1584, 1455, 1330, 1273, 1171, 1086, 1073, 1048, 984, 951, 932

(3b): IR (film) 3099, 2988, 2968, 2954, 2857, 2250, 1584, 1455, 1330, 1273, 1171, 1086, 1073, 1048, 984, 951, 932. MS (CI, NH3,%): 78 (3, M-O 5 H 6 O 2), 99 (83, M-C2 H-2CN), 177 (51, M + H <+>), 194 (100, M + NH4 <+>).

Example 4 Preparation of bicyclo [2.2.2loct-5-en-2,3-dicarbonitrile (4)

Under anhydrous conditions it dissolved fumaronitrile (1.0 g, 13.1 mmol) in absolute ethanol (10 mL) and added 1,3-cyclohexadiene (2.5 mL) dropwise. A after the addition was completed, the reaction mixture was stirred for 16  hours at room temperature, after which it was added, of only once, BF_ {3} .Et_ {2} O (5 µL). After 36 hours it evaporated the solvent in vacuo and the product was purified by chromatography column using hexane and acetate mixtures of ethyl of increasing polarity as eluents. The product (4) is isolated by elution with hexane-ethyl acetate 60:40 (1.31 g). Yield = 42%. IR (KBr) 3060, 2952, 2876, 2244. MS (CI, NH 3,%) 176 (92, M + NH 4 +), 193 (100, M + N 2 H 7 +).

Example 5 Preparation of bicyclo [3.2.2] non-8-en-6,7-dicarbonitrile (5)

In a Pyrex tube, fumaronitrile was dissolved (400 mg, 5.13 mmol) in anhydrous toluene (5 mL). In another flask it dissolved cycloheptadiene (500 mg, 5.10 mmol) in anhydrous toluene (2 mL) and was added via cannula on the previous solution of fumaronitrile Once the addition was complete, BF3.Et_ {2} was added (3 µL). The Pyrex tube was purged with nitrogen, closed hermetically and heated to 80 ° C. After several days the reaction stopped (control by chromatography). The solvent is evaporated in vacuo and the reaction crude was purified by column chromatography, using hexane / ethyl acetate mixtures of increasing polarity as a mobile phase. Compound (5) was isolated with hexane / ethyl acetate 80:20 (91 mg). Yield = 18%. IR (film) 3050, 2934, 2865, 2242, 1674, 1447, 1095, 816. MS (CI, NH3,%) 208 (27, M + N 2 H 7 +), 190 (100, M + NH 4 +).

Example 6 General procedure for hydrogenation of nitriles bicyclic

This procedure is exemplified for the substrate hydrogenation reaction (1) in order to obtain the bicycle [2.2.1] heptane-2,3-dicarbonitrile (6).

Under argon atmosphere, a solution was added of the substrate (1) (263 mg, 1.8 mmol) in THF (10 mL) over 60 mg of Pd / C (10% by mass) as catalyst. Another 12 mL of THF and the reaction flask was purged by five cycles of vacuum / hydrogen The system was vigorously stirred under atmosphere of hydrogen for 15 hours at room temperature. Then the reaction mixture was sonicated in a bath of ultrasound in order to desorber the product retained by the palladium and coal, subsequently proceeding to filtration at through Celite. The solvent was evaporated in vacuo, obtaining the product (6) in the form of white solid (189 mg). Yield = 99%. P.f. = 80-82 ° C (THF). IR (Film, v, cm -1): 2960-2885 (Csp3 -H, st), 2242 (C \ equivN, st), 1457, 1306, 841. MS (CI, NH3,%) 164 (55, M + NH4 <+>), 167 (66, M + N 2 H 7 -CH 2), 182 (100, M + N 2 H 7 + H +).

Example 7 Preparation of 7-oxabicyclo [2.2.1] heptane-2,3-dicarbonitrile (7)

Obtaining compound (7) from (2) it was carried out using H2 (Pd / C), following the procedure described in example 6. Yield = 67%. P.F. = 116-117 ° C (hexane / ethyl acetate 3/7). IR (film) 2998, 2962, 2927, 2889, 2246, 1468, 1177, 1038, 1017, 928, 905 843. MS (CI, NH3,%) 91 (46, M-CH 2 OH-CN), 100 (100, M-2CN + 4H), 104 (50, M-CN-H 2 O), 117 (M-CH 2 OH).

Example 8 Preparation of 1-methoxy-7-oxabicyclo [2.2.1] heptane-2,3-dicarbonitrile (8)

The reaction to obtain the diastereoisomers 8a (1 R *, 2 R *, 3 R *, 4 S *) and 8b (1 R *, 2 S *, 3 S * 4 S *) was carried out following the procedure described in example 6, starting from the precursor (3).

(8a): Yield = 99%. IR (film) 2962, 2856, 2366, 1465, 1320, 1264, 1189, 1150, 1032, 965. MS (CI, NH3,%) 178 (1, M +), 196 (100, M + NH 4 +)

(8b): Yield = 99%. P.F. = 114-117 ° C (hexane / ethyl acetate 75/25). GO (film) 2962, 2856, 2366, 1465, 1320, 1264, 1189, 1150, 1032, 965

Example 9 Preparation of bicyclo [2.2.2] octane-2,3-dicarbonitrile (9)

Obtaining (9) from (4) led to Perform following the procedure described in Example 6. Yield = 99%. P. F. = 171-173 ° C (THF). GO (film) 2952, 2875, 2246, 1457, 1347, 1301, 1248, 1026, 911. MS (CI, NH3,%) 178 (100, M + NH4 +).

Example 10 General procedure for reducing nitriles to diamines

This procedure is exemplified for the transformation of dinitrile (1) into 1,4-diamine (10) Thus, dinitrile (1) (963 mg, 6.7 mmol) was dissolved in ether diethyl (30 mL) and added dropwise to a suspension of LiAIH 4 (1.02 g, 26.8 mmol) in ether (10 mL). After two hours of stirring at room temperature, excess hydride is destroyed by careful addition of water (2 mL) drop by drop, followed by the addition of 30% sodium hydroxide (4 mL) and finally water (5 mL). The suspension was filtered and the solvent was removed at empty, getting the (3-aminomethyl-bicyclo [2.2.1] hept-5-en-2-yl) -methylamine (10) as a yellow oil (788 mg). Yield = 92%. IR (film) 3286, 3056, 2959, 2871, 1576. MS (CI, NH3,%) 134 (2, M-NH4 <+>), 153 (100, M + H <+>), 154 (10, M + 2H +).

Example 11 Preparation of (3-aminomethyl-7-oxa-bicyclo [2.2.1] hept-5-en-2-yl) -methylamine (eleven)

Obtaining (11) from (2) led to carried out following the procedure described in example 10, but using THF (3 mL for 1.4 mmol of substrate) as solvent. The diamine (11) was isolated by column chromatography (77 mg, eluting with EtOH / NH3 80:20). Yield = 36%. IR (film) 3349, 3320, 3200, 3100, 3075, 2990, 2921, 2880, 1574. EM (CI, NH 3,%) 155 (100, M + H +), 156 (12, M + 2H +), 157 (5, M + 3H <+>), 172 (4, M + NH4 <+>), 309 (5, 2M + H <+>).

Example 12 Preparation of (3-aminomethyl-bicyclo [2.2.2] oct-5-en-2-yl) -methylamine (12)

Obtaining (12) from (4) led to Perform following the procedure described in Example 10. Yield = 88%. IR (film) 3291, 3040, 2938, 2867, 1576. EM (CI, NH 3,%) 167 (100, M + H +).

Example 13 Preparation of (7-aminomethyl-bicyclo [3.2.2] non-8-en-6-yl) -methylamine (13)

Obtaining (13) from (5) led to Perform following the procedure described in Example 10. Yield = 47%. IR (film) 3284 (N-H, st), 3031, 2921, 1574, 1462, 1383, 1325. MS (CI, NH3,%) 181 (100, M + H +).

Example 14 Obtaining hydrogenated bicyclic diamines. Preparation from (3-aminomethyl-bicyclo [2.2.1] hept-2-yl) -methylamine (14)

Saturated amines were obtained by of the following methodologies (see FIG. 1): a) Reduction of corresponding saturated dinitriles (6) to (9), to obtain the diamines (14) to (17) following a similar synthetic procedure of example 6; b) Catalytic hydrogenation of diamines unsaturated (10), (11) or (12), to obtain the corresponding saturated diamines (14), (15) and (17), respectively, following a synthetic procedure similar to that described in example 6.

Obtaining the product (14): a) From substrate (10), Yield = 97%; b) From the substrate (6), Yield = 94%. IR (film) 3322, 2948, 2875, 1564. EM (CI, NH 3,%) 155 (100, M + H +), 172 (10, M + NH 4 +).

Example 15 Preparation of (3-aminomethyl-7-oxa-bicyclo [2.2.1] hept-2-yl) -methylamine (fifteen)

It was obtained by applying a procedure similar to of Example 6 from the substrate (11). Yield = 87%. Be obtained by applying a procedure analogous to that of Example 14 a starting from the substrate (7). Yield = 24%. IR (film) 3250, 3150 (N-H, st), 2965, 2921, 1655. MS (CI,%) 138 (13, M-NH 4 +) 157 (100, M + H +), 158 (11, M + 2H <+>), 167 (4).

Example 16 Preparation of 2,3-bis (aminomethyl) -1-methoxy-7-oxabicyclo [2.2.1] heptane, (diastereoisomers (16a) (1R *, 2R *, 3R *, 4S *) and (16b) (1 R *, 2S *, 3S *, 4S *)

It was obtained by applying a procedure similar to of Example 14 from (8a) and / or (8b). Performance = 42% (16a): IR (film) 3288, 2925, 1567, 1466, 1387, 1310, 1223, 1191, 1144, 1028, 978. MS (CI, NH3,%) 170 (56, M-NH 2), 187 (100, M + H +).

(16b): IR (film) 3288, 2925, 1567, 1466, 1387, 1310, 1223, 1191, 1144, 1028, 978. MS (CI, NH3,%) 170 (56, M-NH 2), 187 (100, M + H +).

Example 17 Preparation of (3-aminomethyl-bicyclo [2.2.2] octane-2-yl) -methylamine (17)

It was obtained by applying a procedure similar to of Example 6 from (12). It was obtained by applying a procedure analogous to that of Example 14 from (9). Yield = 93% Yield = 81%. IR (film): 3290, 2950, 2860, 1650, 1550. MS (CI, NH 3%) 169 (100, M + H +).

Example 18 Preparation of 5,6-dihydroxybicyclo [-2.2.1] -heptane-2,3-dicarbonitrile, (diastereoisomers (18a) (1S *, 2S *, 3S *, 4R *, 5R *, 6S *) and 18b (1S *, 2S *, 3S *, 44R *, 5S *, 6R *))

The substrate (1) (250 mg, 1.7 mmol) was dissolved in a mixture of acetone / ether 8: 2 (6 mL). This solution is added osmium tetraoxide (16 mg) in water (1 mL) and then 35% H2O2 (0.8 mL) at 0 ° C. The resulting solution is stirred in the dark for 6 hours. The reaction mixture is concentrated to dryness in vacuo to obtain a brown solid, which was subjected to a chromatography column using hexane mixtures and ethyl acetate of increasing polarity. The diol (18a) (183 mg) is eluted with hexane / ethyl acetate 3/7 and its diastereoisomer (18b) (38 mg) with hexane / ethyl acetate 35/65. Yield = 57%.

(18a): P.f. = 137-138 ° C (hexane / ethyl acetate 7/3). IR (film): 3423 (OR-H, st), 2983-2948 (Csp3 -H, st), 2248 (C?, St), 1268, 1083, 1026. 92: 3417 (OR-H, st), 2948 (Csp3-H, st), 2246 (C \ equivN, st), 1457, 1420, 1331, 1308, 1182, 1088, 1032, 1005, 957. MS (CI, NH3,%) 109 (23, M-2CN-OH), 178 (100, M).

(18b): IR (film): 3423 (OR-H, st), 2983-2948 (Csp3 -H, st), 2248 (C \ equivN, st), 1268, 1083, 1026. 92: 3417 (OR-H, st), 2948 (Csp3 -H, st), 2246 (C \ equivN, st), 1457, 1420, 1331, 1308, 1182, 1088, 1032, 1005, 957. MS (CI, NH3,%) 109 (23, M-2CN-OH), 178 (100, M).

Example 19 Preparation of (1S *, 2S *, 4R *, 6R *, 8S *, 9S *) - 4,4-dimethyl-3,5- dioxatricyclo [5.2.1.02.6] decane-8,9-dicarbonitrile (19)

To a solution of the diol (18a) (60 mg, 0.34 mmol) in anhydrous acetone (6 mL) were added 2,2-dimethoxypropane (1 mL, 8.2 mmol) and p- toluenesulfonic acid (10 mg). The mixture was heated at reflux for 3 hours and stirred at room temperature for an additional hour. After the reaction mixture was concentrated to dryness, it was dissolved with ethyl acetate (7 mL) and the resulting organic solution was washed with saturated aqueous sodium bicarbonate solution (2 mL) and saturated sodium chloride solution (2 mL) . The resulting organic phase was dried over anhydrous magnesium sulfate and concentrated to dryness, yielding 64 mg of (19). Yield = 87%. MP = 162-163 ° C (ethyl acetate). IR (film) 2991, 2952, 2937, 2913, 2246, 1465, 1382, 1272, 1208, 1169, 1158, 1075, 1050, 864. MS (CI, NH3,%) 190 (9, M-CH2) -CH 3 + H +), 203 (100, M-CH 3), 219 (6, M + H +), 236 (9, M + NH 4) +}).

Example 20 Preparation of (1S *, 2S *, 4R *, 6R *, 8S *, 9S *) - 8,9-bis (aminomethyl) -4,4-dimethyl-3,5-dioxatricyclo [5.2.1.02.6] decane, (twenty)

Obtaining compound (20) from (19) it was carried out following the procedure described in the example 10. Yield = 75%. IR (film) 3348, 3274, 3172, 2979, 2937, 1654, 1561, 1382, 1374, 1316, 1270, 1208, 1164, 1044. EM (CI, NH 3,%) 152 (5, M-C (CH 3) 2 -CH 2 NH 2), 183 (5, M-C (CH 3) 2), 227 (82, M + H +).

Example 21 Preparation of (1R *, 2R *, 3S *, 4S *, 5S *, 6S *) - 5,6-bis (aminomethyl) bicyclo [2.2.1] heptane-2,3-diol, (twenty-one)

Compound (20) (52 mg, 0.28 mmol) was dissolved in 2 M HCl (4 mL). The mixture was stirred for 80 min and added 3.5 M NaOH (3.5 mL) to neutralize the acidic medium. The mixture of reaction was concentrated to dryness and the resulting brown solid was dissolved in methanol and the resulting solution was filtered through alumina and celite, obtaining 287 mg of a solid that was purified by chromatography column using mixtures of ethanol and ammonia as eluents. The product (21) (7 mg) was eluted with ethanol / ammonia 75:25 and 70:30. Yield = 18%. IR (film) 33280 (N-H, OR-H, st), 2925-2840 (Csp3-H, st), 1684, 1654, 1576, 1561, 1507, 1459, 1322, 1082. MS (CI, NH3,%) 78 (100, M-2H2 O-2CH2NH2 -CH2), 90 (9, M-2H 2 O-2CH 2 NH 2), 104 (40, M-2 H 2 O-CH 2 NH 2 -CH 2), 119 (12, M-OH-H 2 O-2NH 2),  136 (16, M-2OH-NH2), 187 (9, M + H +).

Example 22 Preparation of cis - {[(3-aminomethyl-bicyclo [2.2.1] hept-5-en-2-yl) -methylamine] dichloro} platinum (II), (22)

A solution of diamine (10) (81 mg, 0.53 mmol) in ethanol (5 mL) to a solution of K 2 PtCl 4 (220 mg, 0.53 mmol) in water (5 mL). The reaction mixture was stirred. in the dark for 24 hours and subsequently leaked to through a sintered glass plate (size per 10-16 µm). The resulting yellow precipitate is washed successively with water, ethanol, acetone and dichloromethane and dried under vacuum to obtain 200 mg of the product (22) (0.48 mmol). Yield = 91%. P.f. = 240-242 ° C. IR (KBr) 3440, 3250, 3210, 3120 (N-H, st), 3075, 2957, 2873, 1601, 1454 cm -1. E.M. (MALDI-TOF, m / z): 345.7 (M-2CI-H). E. Anal .: N (6.70%), C (25.80%), H (3.82%). Calcd for C_ {9} H_ {16} N_ {Cl} {P2}: (6.70%), C (25.85%), H (3.86%).

Example 23 Preparation of cis - {[(3-aminomethyl-7-oxabicyclo [2.2.1] hept-5-en-2-yl) -methylamine] dichloro} platinum (II) (2. 3)

Following the same procedure as described in example 22, diamine (11) was transformed into the complex of platinum (23). Yield = 44%. P.F. = 230-232 ° C. IR (KBr) 3459, 3226, 3150 (N-H, st), 2950, 2886, 1593, 1456 cm -1. E.M. (FAB (+), m / z): 502 (M-2CI + NBA), 537 (M-CI + NBA). AND. Anal .: N (6.16%), C (23.15%), H (3.87%). Calcd for C_ {H} {14} ON_ {2} Cl2 {Pt}: N (6.66%), C (22.85%), H (3.33%).

Example 24 Preparation of cis - {[(3-aminomethyl-bicyclo [2.2.2] oct-5-en-2-yl) -methylamine] dichloro} platinum (II) (24)

Following the same procedure as described in example 22, the diamine (12) was transformed into the complex of platinum (24). Yield = 72%. P.F. = 245-247 ° C. IR (KBr) 3444, 3220, 3210 (N-H, st), 3130, 3050, 2940, 2873, 1597, 1456 cm -1. E.M. (MALDI-TOF, m / z): 359.9 (M-2CI-H). E. Anal .: N (6.13%), C (27.61%), H (4.57%). Calcd for C 10 H 18 N 2 Cl 2 Pt: N (6.48%), C (27.8%), H (4.16%).

Example 25 Preparation of cis - {(7-aminomethyl-bicyclo [3.2.21non-8-en-6-yl) -methylamine} dichloroplatin (II), (25)

Diamine (13) (38 mg, 0.21 mmol) was dissolved in absolute ethanol (2 mL). On the other hand, it dissolved K 2 PtCI 4 (89 mg, 0.21 mmol) in water (4 mL). This second solution was added dropwise to the first solution and the mixture  The reaction was maintained at room temperature for 5 h. Was formed a brown yellow precipitate that was filtered and washed, obtaining 57 mg of the product (25). Yield = 61%. P.F. = 247-249 ° C (water / ethanol). IR (KBr, v, cm -1): 3448, 3220, 3131, 3035, 2929, 2861, 1599, 1383, 1207, 1036. EM [FAB (+), NBA, m / z]: 489 (M-CI + DMSO + H +). AND. Anal. Found: C (32.19%), H (5.14%), N (6.20%). Calcd for C 11 H 20 Cl 2 N 2 Pt (446): C (29.60%), H (4.52%), N (6.28%).

Example 26 Preparation of cis - {[(3-aminomethyl-bicyclo [2.2.1] hept-2-yl) -methylamine] dichloro} platinum (II) (26)

Following the same procedure as described in example 22, the diamine (14) was transformed into the complex of platinum (26). Yield = 71%. P.F. = 179-182 ° C. IR (KBr) 3432, 3255, 3222, 3125 (N-H, st), 2948, 2875, 1603, 1452, 1383 cm -1. E.M. (MALDI-TOF, m / z): 414 (M-7H), 385 (M-CI), 348 (M-2CI-H), 345 (M-2CI-4H). E. Anal .: N (6.29%), C (25.93%), H (4.54%). Calcd for C 9 H 18 N 2 Cl 2 Pt: N (6.66%), C (25.71%), H (4.28%).

Example 27 Preparation of cis - {[(3-aminomethyl-7-oxabicyclo [2.2.1] hept-2-yl) -methylaminaldicloro} platinum (II) (27)

Following the same procedure as described in example 22, the diamine (15) was transformed into the complex of platinum (27). Yield = 24%. P.F. = 248-250 ° C. IR (KBr) 3492, 3270, 3226, 3150 (N-H, st), 2969, 2900, 1589, 1456 cm -1. E.M. (MALDI-TOF, m / z): 351 (M-2CI-H), 386 (M-CI). E. Anal .: N (6.31%), C (23.46%), H (4.25%). Calcd for C_ {H} {16} ON_ {2} Cl_ {2} Pt: N (6.63%), C (22.75%), H (3.79%).

Example 28 Preparation of cis - {[(3-aminomethyl-bicyclo [2.2.2] oct-2-yl) -methylamine] dichloro} platinum (II) (28)

Following the same procedure as described in example (22), diamine (17) was transformed into the complex of platinum (28). Yield = 74%. P.F. = 185-187 ° C. IR (KBr) 3444, 3270, 3222 (N-H, st), 3125, 2934, 2880, 1600, 1456 cm -1. E.M. (MALDI-TOF, m / z): 355.9 (M-2CI-7H). E. Anal .: N (6.16%), C (27.66%), H (5.05%). Calcd for C 10 H 20 N 2 Cl 2 Pt: N (6.45%), C (27.65%), H (4.61%).

Example 29 Preparation of {(1S *, 2S *, 4R *, 6R *, 8S *, 9S *) - [8,9-bis (aminomethyl) -4,4-dimethyl-3,5-dioxatricyclo [5.2.1.02.6] decane-dichloro platinum (II), (29)

Following the same procedure described in Example 22, the diamine (20) was transformed into a platinum complex (29). Yield = 704%. Brown solid, mp = 275 ° C (decomp.) IR (KBr, u, cm -1): 3504-3260-3220-3130 (NH, st), 2990-2956 (Csp3-H, st), 1654, 1602, 1507, 1459, 1382, 1268, 1210, 1046. MS (MALDI): 531 (100, M-Cl + DMSO), 575 (40, M-2CI + 2DMSO). HRMS (Electrospray): 513.0576 (91%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na35 {Cl} {194} Pt:
513.0577 Δ (mDa) = 0.1; 514.0598 (100%); calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na35 {Cl} {195} Pt: 514.0598 Δ (mDa) = 0.02; 515.0525 (43%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na35 {Cl} {37} Cl <{} 194} Pt: 515.0548 Δ (mDa) = 2.2; 515.0615 (55%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na35 {Cl} {37} Cl <{} 196} Pt: 515.0600 Δ (mDa) = 1.5; 516.0566 (62%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na35 {Cl} {37}
Cl 196 Pt: 516.0569 Δ (mDa) = 0.3; 517.0566 (35%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na <35> CI <37} CI < 196} Pt: 517.0570 Δ (mDa) = 0.4; 518.0532 (10%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Nal <37} Cl_ {2} <195} Pt: 518.0539 Δ (mDa) = 0.8; 519.0610 (11%), calcd. for <12> C_12 <1> H_ {22} <14> N2 {16} O2 {23} Na37 {Cl} {{}} {196} Pt: 519.0541 Δ (mDa) = 6.9. Anal. Found:% N = 5.07% C = 29.87% H = 4.91. Calcd .:% N = 5.69% C = 29.28% H = 4.50.

Example 30 Kinetic studies of the interaction of platinum complexes  with glutathione

One of the main resistance mechanisms developed by cancer cells is the increase in deactivation of drugs by increasing interaction of the platinum (II) complexes with the sulfur groups of biomolecules such as glutathione, which act as good nucleophiles, displacing chlorine atoms from the complex (or other labile ligands). Glutathione is a biomolecule capable of regulating the activity of some platinum drugs due to coordination of the metal atom to thiol groups.

To see if there was a difference between new complexes in their interaction with this biomolecule, it was evaluated the glutathione binding kinetics of this new series of compounds. Kinetics was determined using spectroscopy. ultraviolet measuring absorption changes at 260 nm (at this wavelength there is a link absorption Pt-thiol and also an absorption of the Pt atom isolated, however, this second phenomenon has a coefficient minor extinction). Aqueous solutions of platinum complexes they were mixed with freshly prepared glutathione solutions and reaction kinetics was measured over a period of 24 hours For each compound.

\hbox{tanto del  cisplatino como de los nuevos
complejos de platino se ilustran en la Fig. 2.}

The reactions of the platinum complexes with glutathione were monitored by UV spectroscopy, using an Ultraspec-2100-Pro UV / visible spectrophotometer with cuvettes or quartz cells of 1 cm optical path. The solutions of the platinum and glutathione compounds were prepared in Tris-HCI buffer. Suitable volumes of the complex and glutathione solutions were mixed to reach a final ratio of 1: 500 (platinum / glutathione complex), close to the conditions that would be reached in the cell, and under these conditions the samples were analyzed for determine the absorbance at 260 nm. As a blank a glutathione solution was used. The graphs of glutathione binding kinetics,

 \ hbox {both cisplatin and new
Platinum complexes are illustrated in Fig. 2.} 

The time values of semi-reaction (t 1/2) obtained for Different compounds are listed in Table 1. In this test, observed differences between the complexes evaluated in their interaction with glutathione. In addition, all compounds studied produced slower binding kinetics than the cisplatin

3

The complex that has an oxygenated bridge and a double bond (23), does not show any interaction with the glutathione This compound showed no increase in absorption relative to the reference target, even after Repeat the experiment carefully five times. So, in this particular case, it was so slow that it could not be measured by this technique due to the oxidation of glutathione over time.

The observed differences of the platinum complexes of the present invention with respect to cisplatin in the interaction with glutathione are very important because they are consistent with the resistance factors observed in the antiproliferative studies carried out against resistant cancer cell lines (see below). ). This fact is remarkable because one of the resistance mechanisms acquired by cancer cells is the increase in the inactivation of drugs by reaction with glutathione and metallothionine (A. Eastman et al .; Cancer Cells 1990, vol. 2, pp. 275-280; B. Koberle et al ., Curr. Biol . 1999, vol. 9 , pp. 273-6; X. Yang et al ., J. Cancer Res. Clin. Oncol . 2004, vol. 130, pp. 423-8).

Example 31 Cytotoxicity studies

The antiproliferative activity of complexes Platinum was studied against ovarian cancer lines human A2780 and A2780cisR, sensitive and resistant to cisplatin respectively. Work with cancer cell lines Cisplatin resistant was carried out because the cisplatin resistance is one of the main limitations of use of this drug in clinic. Therefore, the value of the factor of resistance (RF = IC 50 resistant cell line / IC 50 sensitive cell line) is an important value to evaluate the ability of the new compounds to overcome the resistance of Cisplatin cancer cells. The cancerous cell line studied resistance has an acquired resistance due to several factors: the decrease in cell transport, the increased repair of DNA lesions and increased of glutathione levels.

The growth inhibitory effect of the investigated compounds was evaluated in the pair of human ovarian cancer cell lines A2780 and A2780cisR (patent line originating from a patient not treated with cisplatin and derivative line resistant to cisplatin, respectively). Lines A2780 and A2780cisR were maintained at 37 ° C in an atmosphere containing 10% CO2 and humidified air and grown in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% heat-inactivated bovine fetal serum. , 2 mM glutamine, 10 µg / mL insulin, 0.5 µg / mL hydrocortisone, 2.5 µg / mL amphotericin B and 50 µg / mL gentamicin. All culture media and reagents were supplied by Euroclone (Paignton, UK). The growth inhibition effect of platinum compounds was evaluated by the sulforrodamine-B (SRB) assay. The cells were seeded in a 96-well microplate in 100 µL of the appropriate culture medium with a cell density of 10,000 cells per well. After sowing the microplates were incubated at 37 ° C for 24 hours before the addition of the compounds. After 24 hours, several samples of each cell line were fixed in situ with cold trichloroacetic acid (TCA), in order to obtain a measurement of the cell population at the time the compound was added. The platinum complexes evaluated were dissolved in the culture medium and diluted in a staggered manner until the desired final concentrations were reached. Different concentrations of the compounds in the wells were then added in quadruplicate. The plates were subsequently incubated at 37 ° C for 96 hours. The cells were fixed in situ by rapid addition of 50 µL of 50% cold trichloroacetic acid (mass / volume) until reaching the final concentration of 10% and then incubated for 1 hour at 4 ° C. The supernatant was discarded and the plates washed four times with water and dried with air. 0.4% SRB solution (100 µL) (mass / volume) in 1% acetic acid was added to each well and the plates were incubated for 30 minutes at room temperature. After staining, the unbound dye was removed by five washes with 1% acetic acid and the plates were air dried. The fixed dye was dissolved in TRIZMA [tris (hydroxymethyl) aminomethane] as a base and the absorbance was read in an automatic plate reader at 515 nm. The concentration of compound capable of inhibiting cell growth by 50% (IC 50 ± SD) was calculated from the dose-response semi-logarithmic plots. The results after 96 hours of incubation of the compounds with the cell lines are shown in Table 2 as IC 50 values.

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TABLE 2 Biological activity of compounds 23, 24, 26, 28 tested against cisplatin sensitive cell lines (A2780) and cisplatin resistant (A2780cisR). Cytotoxicity of the evaluated complexes related to cisplatin was estimated as the relationship: IC_ {50} (cisplatin) / IC 50 (complexes)

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In these series of complexes it was observed that compounds 23, 24 and 28 showed much better biological activity than cisplatin In fact, compounds 23 and 28 are 4.28 times more active than cisplatin and compound 24 is 2.5 times more active than this standard drug. Compound 26 has a similar activity It should be noted that all the compounds evaluated have lower resistance factors than cisplatin when were tested against cell lines resistant to it, showing that this structural family of complexes is capable of overcome the resistance to cisplatin developed by the line A2780cisR cancer cell.

All these results show that Bicyclic platinum complexes of formula (I) are strongly active against gynecological cancers and also to treat others types of cancer such as lung or testicular among others.

Claims (15)

1. Compound of formula (I), or a salt pharmaceutically acceptable thereof, or a solvate thereof, including any stereoisomer or mixture of stereoisomers, 5 where: the dashed line represents a simple link or double; X is a birradical that is selected from the group formed by N, O, S, and - (CH 2) n -; n is an integer from 1 to 3; R_ {1} is a radical selected from the group that consists of hydrogen, (C 1 -C 4) -alkoxy, phenoxy, phenyl- (CH2) r -O and a radical derived from the aforementioned with rings benzene, through a mono-, di- or tri-substitution in its benzene rings; being the substituents a radical independently selected from group formed by (C 1 -C 4) -alkyl, (C 1 -C 4) -alkoxy and halogen; R 2 and R '2 are a radical independently selected from the group formed by hydrogen, hydroxyl, (C 1 -C 4) -alkoxy, phenoxy, phenyl- (CH2) r -O, and O-Gp, and a radical derived from the above mentioned with benzene rings, through a mono-, di- or tri-substitution in its benzene rings; being the substituents a radical independently selected from group formed by (C 1 -C 4) -alkyl, (C 1 -C 4) -alkoxy and halogen; r is an integer from 1 to 4; Gp is an alcohol protecting group; R 3 and R '3 are independently a halogen or, alternatively, R 3 and R '3 taken together they are a birradical selected from the group consisting in -OCO- (CH2) m -COO-, (C 3 -C 7) - cycloalkane-1,1-dicarboxylate, Y (C 3 -C 7) -cycloalkane-1,2-dicarboxylate; Y m is an integer from 0 to 4. 2. Compound according to claim 1, which has the formula (Ia), 6 where R 1, R '2, R 3, R '3 and X have the same meaning as in the claim one. 3. Compound according to any of the claims 1-2, wherein X is O or a radical selected from the group consisting of methylene, ethylene and propylene 4. Compound according to any of the claims 1-3, wherein R 3 and R '3 are both chlorine or both iodine. 5. Compound according to any of the claims 1-3, wherein R 3 and R '3 taken together are a dicarboxylate of one of the following diacids: oxalic acid, malonic acid and acid cyclobutane-1,1-dicarboxylic. 6. Compound according to any of the claims 1-5, wherein R1 is hydrogen or methoxy. 7. Compound according to any of the claims 1-6, wherein R 2 and R '2 are independently selected from hydrogen, hydroxyl, methoxy, phenoxy, benzyloxy, or both radicals R2 and R2 'form an acetonide. 8. Compound according to any of the claims 1-7, which is selected from the following list: (one) cis - ((((3-aminomethyl-bicyclo [2.2.1] hept-5-en-2-yl) methylamine) dichloride) platinum (II); (2) cis - (((3-aminomethylbicyclo [2.2.1] hept-2-yl) -methylamine) dichloride) platinum (II); (3) cis - (((3-aminomethyl-7-oxabicyclo [2.2.1] hept-5-en-2-yl) methylamine) dichloride) platinum (II); (4) cis - ((((3-aminomethyl-7-oxabicyclo [2.2.1] hept-2-yl) methylamine) dichloride) platinum (II); (5) cis - ((((3-aminomethyl-bicyclo [2.2.2] oct-5-en-2-yl) methylamine) dichloride) platinum (II); Y (6) cis - ((((3-aminomethyl-bicyclo [2.2.2] oct-2-yl) methylamine) dichloride) platinum (II).

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9. Use of the compound defined in any of claims 1-8 for the preparation of a medicine for the treatment and / or prevention of cancer in a mammal including the human being. 10. Use according to claim 9, wherein the type of cancer is ovarian cancer. 11. Use according to claim 9, wherein the type Cancer is breast cancer. 12. Use according to claim 9, wherein the type Cancer is leukemia. 13. Use according to claim 9, wherein the type of cancer is lung cancer. 14. Use according to claim 9, wherein the type Cancer is testicular cancer. 15. Pharmaceutical composition comprising a therapeutically effective amount of any of the compounds defined in claims 1-8, together with sufficient amounts of excipients or carriers pharmaceutically acceptable.