AU738104B2

AU738104B2 - Oligo-thiophenes useful as antimetastatic agents, a preparation thereof and pharmaceutical compositions containing them

Info

Publication number: AU738104B2
Application number: AU88079/98A
Authority: AU
Inventors: Gianpiero De Cillis; Roberto Di Domenico; Bernhard Konig; Gerd Zimmermann
Original assignee: Roche Diagnostics GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1997-07-31
Filing date: 1998-07-28
Publication date: 2001-09-06
Anticipated expiration: 2018-07-28
Also published as: EP1000054A1; WO1999006393A1; JP2001512116A; CN1269797A; BR9811577A; AR020308A1; TR200000266T2; CO4970718A1; AU8807998A; CA2299045A1; KR20010022500A; MA26527A1; HRP980420A2; ZA986800B

Description

WO 99/06393 PCT/EP98/04705 OLIGO-THIOPHENES USEFUL AS ANTIMETASTATIC AGENTS, A PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM The present invention concerns oligo-thiophenes, optionally linked to natural or synthetic aminoacids.

These compounds have been found to possess uPA-uPAR antagonist activity and can be employed as antitumor and/or antimetastatic agents.

BACKGROUND OF THE INVENTION The serine proteases uPA (urokinase-type plasminogen activator) catalyzes the activation of plasminogen to plasmin which is involved in a variety of physiological and pathological processes. uPA is a multi-domain protein having a catalytic chain (armno-acids 144-411) and an amino-terminal fragment aa 1-143) comprised of a growth factor-like domain (aa 4-43) and a kringle domain (aa 47-135). uPA is a multifunctional protein involved in tissue proteolysis, cellular migration, cellular proliferation and growth factor activation. uPA is released from cells as a virtually inactive pro-enzyme, pro-uPA. The activation of the single-chain pro-uPA by plasmin (leading to the active two-chain form) is regulated by tight control mechanisms which are not completely understood yet. Most of the uPA activities are confined to the cell surface and the pericellular environment. This is accomplished by binding to a specific, highaffinity receptor on the cell surface (uPAR). Both forms of uPA bind to uPAR with similar affinity. The binding interaction is mediated by the growth factor-like domain Rabbani et al.J. Biol. Chem., 267, 14151-56, 1992].

The uPA receptor is a three domain glycoprotein where each triplicated motif comprizes a cysteine rich consensus sequence of approximately 90 amino acids Plough et al., J Biol. Chem., 268, 17539-46, 1993]. uPAR is anchored to cell membrane by a glycosylphosphatidylinositol moiety (GPI anchor). uPAR binds uPA with KD values between and 10" 9 M, depending on the experimental system. The major determinants for uPA binding are located in the N-terminal domain 1. uPAR can be cleaved by uPA and plasmin, liberating a water soluble domain 1 and by the action of phospholipase C, three domains uPAR can be released from the cell surface. This latter form of uPAR is also water soluble because the GPI-anchor is missing.

The inhibition of uPA dependent phenomena can principally be approached in two ways, either by direct inhibition of the proteolytic activity or by inhibition of uPA receptor WO 99/06393 PCT/EP98/04705 2 binding. The latter strategy has the potential of achieving greater specificity since inhibition might be localized to the pericellular environment.

A bacteriophage display technique and protein engineering have recently been used to discover peptidic and species-specific uPAR antagonists [Goodson et al., PNAS, 91, 7129, 1994; Stratton-Thomas et al., Prot. Eng., 5, 463-470, 1995, respectively].

The present invention concerns oligo-thiophenes with potent antagonistic activity.

Many bisthiophene and terthiophene derivatives seem to display interesting biological properties [Kagan, J. et al., J. Org. Chem., 48, 4317-20, 1983 and references cited therein]. Most notably they are toxic to nematodes and this effect can be greatly enhanced by the presence of ultraviolet light. The most carefully scrutinized of these compounds is c-terthienyl, a plant-derived natural product first recognized as phototoxin in the 1970s. This compound showed photoenhanced activity against nematodes, microorganisms, algae, human erythrocytes, insect larvae and eggs, in addition to generating skin pigmentation, acting as herbicide and as a seed germination inhibitor.

Photoactive antiviral and cytotoxic activities were also reported Cooper et al., Bioorg. Chem., 13, 362-374, 1985 and references cited therein; Rawls et al., Chem.

Engin. News, 21-23, 1986; Evans et al., J. Am. Chem. Soc., 112, 2694-2701, 1990; Kyo et al., Plant Cell Rep., 9, 393-397, 1990; Hudson et al., Planta Med, 59, 447-450, 1993; Hudson et al., Chemosphere, 19, 1329-1343, 1989]. A structureactivity relationship study was announced for the fourteen isomeric unsubstituted terhiophenes [Jayasuriya et al., Heterocycles, 24, 2261-2264 and 2901-2904, 1986].

The use of the thiophene ring is largely widespread in the different fields of therapeutic agents, while oligothiophenes are only rarely mentioned as pharmacological agents [Press et al., The Chemistry of Heterocycles Compounds, vol. 44, pan four, chapter III, edited by Salo Gronowitz, 396-502, 1989].

The uPA/uPAR system has been shown to be implicated in a variety of invasive biological processes such as tumor metastasis, trophoblast implantation, inflammation and angiogenesis. Therefore, uPAR antagonists should be able to block tumor invasiveness, metastasis and angiogenesis. Formulations containing uPAR antagonists represent novel therapeutic treatments for a number of highly invasive and metastazising cancers where uPA and uPAR have been found to be consistently present at the invasive foci of the tumor [Dano et al., Proteolysis and Protein Turnover, eds. Barret Bond, Portlan Press, 1994. London] breast, lung, colon, ovarian cancers). In patients with WO-99/06393 PCT/EP98/04705 3 breast cancer and non-small cell lung cancer increased levels of uPAR in plasma have been detected. Therefore, the amount of soluble uPAR appears to reflect the degree of proteolysis in the tumor and this might be closely related to patient prognosis. Both uPA and uPAR levels in tumor tissue are prognostic factors in may types of cancers.

S In addition to cancer, other diseases mediated by cell-surface activity of uPA are addressed by uPAR antagonists. Inhibitors of plasmin generation by receptor bound uPA therefore have mechanism-based tumoristatic, anti-invasive, anti-metastatic, antiangiogenic, anti-arthritic, anti-inflammatory, anti-osteoporotic, anti-retinopathic and contraceptive activities. These compounds are applied preferentially via the oral route, but also by i.v. or i.m. injections, nasal sprays or any other conventionally used application.

DESCRIPTION OF THE INVENTION The present invention concerns oligo-thiophenes of the general formula

B

wherein: A is a -CH 2

-CH

2

CH

2 -CO- or -CH=CH-CO- group; B is a 2-thienyl or a phenyl group, which is substituted by an R group, or it is a natural or synthetic armno acid, linked to the A group via its N-terminus; or A-B is a -CH 2

CH

2 -NH-A.A.-group. wherein the A.A.-group is a natural or synthetic u0 amino acid residue, linked to the CH 2

CH

2 -NH-group via its carboxy-terminus; R is hydrogen or a chlorine, bromine, iodine, fluorine, (C 1

-C

4 )alkyl, (Ci- C4)alkylene- COOR', (C 1 -C4)alkylene-NH 2 (CI-C4)alkvlene-NR'2 or (CI- C4)alkylene-NHCOR' group; R' is hydrogen or a (Ci-C4)alkyl group, isomers thereof, and salts thereof with pharmaceutically acceptable acids and bases.

A natural amino acid denotes one of the 20 a-amino acids which are the monomer units for polypeptides, for example Glycine, alanine, valine, leucine isoleucine, phenylalanine, tryptophan, proline, serine, threonine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, histidine, cysteine or methionine.

A synthetic amino acid consists of a compound comprising an amino group together with a carboxy group linked to the c-carbon atom.

Pharmaceuically acceptable salts of compounds of the present invention include physiologically acceptable acid addition salts for example hydrochlorides, S hydrobromides, sulphates, methane sulphonates, p-toluensulphonates, phosphates, aceates citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.

Preferred compounds are those in which B is an amino acid or A-B is a group of formula

-CH

2 CHr2NH-A.A Other preferred compounds are those in which B is a 2-thienyl group.

Particularly preferred compounds are those in which the amminoacid is triptophae.

Advantageously at least one embodiment of the present invention provides a process for the preparation of the compounds of formula Advantageously at least one embodiment of the present invention provides the use of the compounds of formula for the treatment of the diseases in which an inhibitor of the •uPAR can be effective, in particular tumor and tumor metastasis or invasiveness, as well as pharmaceutical compositions containing a pharmacologically effective amount of one or more compounds of formula in admixture with pharmaceutically suitable additives.

PREPARATION OF THE COMPUNRISOF TMEONVE Nr The compounds of formula in which B is a natural or synthetic amino acid (hereinafter referred to as can be prepared starring from the intermediate of formula (II): S0 T T (II) which is first transformed into the corresponding acyl chloride derivative and then it is reacted via Friedel-Craft reaction with the intermediate of formula (III): S -COOR" wherein A' is a -CH 2

-CH

2

CH

2 or -CH=CH- group and R" is a (Ci-C 4 )alkyl group, in S the presence of a Lewis acid.

WO 99/06393 PCT/EP98/04705 The transformation of the intermediate (II) into its acyl chloride is preferentially performed with thionyl chloride, which is used also as the solvent, at temperature between room temperature and the boiling temperature of the solvent.

The Friedel-Craft reaction is performed in an inert solvent and at a temperature ranging from -5 0 C and room temperature, using preferentially SnCL as the Lewis acid.

The intermediate (IV) so obtained: s IV) S A

COOR"

0 is hydrolized into the corresponding acid of formula S A 0 0

H

Suitable reaction conditions comprise the use of a base, preferentially an alkaline metal hydroxide, in water, an alcohol or mixtures thereof, at a temperature ranging from 0OC to room temperature.

The carboxylic group of intermediate is then activated, for example with carbonyldiimidazole to give the imidazolyl derivative, and subsequently reacted, S preferentially in an inert solvent and at a temperature between 0°C and 50 0 C. with an amino acid of formula to give the intermediates of formula (VI): S S

(VI)

S CONH-A.A.-COOR"

O

0 which are then converted into the corresponding compounds of formula via hydrolysis of the carboxylic ester -COOR", preferentially in acidic conditions.

If in the above process the intermediate of formula (III) is replaced by an intermediate of formula s CHCHNH-COCF,

I')

S WO-99/06393 PCT/EP98/04705 6 the compounds of formula in which A-B is a -CH 2

CH

2 -NH-A.A.-group can be prepared. In this case, the -COCF 3 protecting group should be removed after the Friedel- Craft reaction and the intermediate so obtained should be condensed with a N-BOCamino acid, previously activated through its carboxvi group. The compounds of formula are finally obtained after removal of the BOC protecting group.

The compounds of formula in which B is a 2-thienyl or a phenyl group, which is substituted by an R group having the above meanings, can be prepared by Friedel-Craft reaction of the intermediate of formula activated in its carboxylic functionality as described above, with an intermediate of formula (VII): sB wherein A has the meanings shown above and B' is a 2-thienyl or a phenvi group, which is substituted by an R group as shown above.

The intermediates of formula (II) can be obtained by reaction of a bis-thiophene, substituted on one ring with the R substituents, with carbon dioxide and a strong base such as butyl lithium, at a temperature between -50°C and -20°C. The starting products bis-thiophenes are commercially available or can be prepared according to procedures well known to one skilled in the art.

The intermediates of formula (VII):

\VI)

wherein A is -CH 2 -CO- or -CH=CH-CO- can be obtained starting from the commercially available intermediates of formula (VIII):.

A" 0

VIII)

H

wherein A" is -CH 2 or -CH=CH-, via transformation of the carboxylic functionality into its corresponding acyl chloride and subsequent Friedel-Craft reaction with a compound of formula B-H, wherein B-H is a thiophene or phenyi group substituted with an R group as shown above.

The intermediates of formula (VII) in which A is -CH 2

CH

2 -CO- can be obtained from the corresponding intermediates with A -CH=CH-CO- via hydrogenation of the C=C double bond in the presence of a suitable catalyst, such as (PPh 3 3 RhCI.

WO 99/06393 PCT/EP98/04705 7 Analogously, the intermediates of formula (III) in which A' is -CH 2

CH

2 can be obtained from the corresponding C=C carrying intermediates via catalytic hydrogenation.

BIOLOGICAL ACTIVITY The compounds of the invention were tested (ELISA test) as inhibitors of human urokinase (uPA) binding to its specific receptor uPAR mAk (BIO-R4), according to the procedure described in Biol. Chem. Hoppe-Seyler, 376, 587-94 (1995) by Rettenberger et al..

The assays are performed in Microtiterplates (96 wells). The following solutions are used: washing buffer: PBS-buffer (without Mg 2 and Ca 2 0.05% Tween incubation buffer 1% skimmed milk powder in PBS-buffer (without Mg2+ and Ca 2 BIO-R4 solution: 50 ng/well (0.5 Tg/ml 100 TI/well) in IP: uPAR solution: 3 ng/well (30 ng/ml; 100 Tl/well) in PBS-buffer (without Mg 2 and Ca2,); blocking solution: 1% skimmed milk powder in washing buffer (dissolved at 37 0

C);

uPA solution: 0.25 ng/well (5 ng/ml; 50 TI/well) in IP.

Detection solutions (per microtiterplate): 6 ml (100 mM Tris-Cl pH 7.2 0.15% Tween 80) 1.5 ml (10 Tg) Plasminogen in :0 aqua bidest: 6 ml (100 mM Tris-Cl pH 7.2 -0.15% Tween 80) 15 mi (7.5 mg) chromozyme PL in aqua bidest.

The detection solution must be continously stirred.

Testing substances: the testing substances are dissolved in DMSO. They are used in the test system with a highest concentration of 100 Tg/ml. The solutions are prepared using

PBS.

Three controls are performed: a) positive control: using 2% DMSO in PBS; b) negative control: assay without receptor; c) inhibition control: 1) inhibition (IC 9 s at 0.25 mg/ml) with dextranesulfate (MW 500.000); 2) inhibition (IC 9 0 at 1 Tg/ml) with inactivated uPA (175 Tg/ml).

Incubation is done as follows: WO 99/06393 PCT/EP98/04705 8 Each well is incubated by 100 TI of BIO-R4 (c=0.5 Tg/ml) for 1 hour at room temperature under shaking. After washing three times with the washing buffer, each well is incubated for 1 hour (37°C) with 200 Tl/well blocking solution. After triple washing each well is incubated for 1 hour at room temperature under shaking with 100 T1/well uPAR (c=30 ngiml), then the wells are washed again three times with the washing buffer.

The testing substance solution and the control solution, respectively, are added T/well) and are incubated for 30 minutes at room temperature under shaking. An additional 50 TI ofuPA solution (c=2.5 ng/ml) are added. After 1 hour at room temperature a triple washing is performed.

For detection, the following procedure is used: Incubation with 50 Tl each of detection solution and at room temperature. After minutes a yellow colour will be risible (the positive control reads an extinction of I after 45-60 minutes). The detection is performed at 405 nm (reference is 490 nm) using a Dynatech MR 7000 ELISA reader. To obtain the percentage of inhibition the following formula is used (E stands for extinction): Inhibition 100 100 x Enc. cont Epo.. cotr Encg. conal The data for some representative compounds of the invention are reported in table I.

Table I BIO-R4 Assay Inhibition of uPA binding to the specific uPAR receptor (BIO-R4) expressed as ICso (mM) WO 99/06393 PCT/EP98/04705 9 0 0 S S s /s 5 1.9

F

0S 3 0.7 The invention concerns pharmaceutical agents containing one or more compounds of formula In order to produce pharmaceutical agents, the compounds of formula are mixed in a known manner with suitable pharmaceutical carrier substances. aromatics, flavouring and dyes and are formed for example into tablets or coated tablets or they are suspended or dissolved in water or an oil such as e.g. olive oil with addition of appropriate auxiliary substances.

The substance of the general formula can be administered orally or parenterally in a liquid or solid form. Water is preferably used as the medium which contains the stabilizing agents, solubilizers and/or buffers which are usually used for injection solutions. Such additives are for example tartrate or borate buffers, ethanol, dimethylsulfoxide, complexing agents (such as ethvlendiaminotetraacetic acid), high molucuiar polymers (such as liquid polyethylene oxide) for the regulation of the viscosity or polyethylene derivatives of sorbitol anhydrides.

Solid carrier substances are e.g. starch, lactose, mannitol, methylcellulose. talcum, highly dispersed silicic acid, higher molecular fatty acids (such as stearic acid), gelatin, agaragar, calcium phosphate, magnesium stearate, animal and vegetable fats or solid high molecular polymers (such as polyethylene glycols). Suitable formulations for the oral route can if desired contain flavourings and sweeteners.

The administered dose depends on the age, the health and the weight of the patient, the extent of the disease, the type of treatments which are possibly being carried out concurrently, the frequency of the treatment and the type of the desired effect. The daily dose of the active compound is usually 0.1 to 50 mg/kg body weight. Normally 0.5 to and preferably 1 to 20 mg/kg/day in one or several applications per day are effective in order to obtain the desired results.

WO 99/06393 PCT/EP98/04705 The invention is further illustrated by the following examples.

Preparation 1 Synthesis of bis-thiophene-2-carboxylic acid g ofbis-thiophene are dissolved in 300 ml of tetrahydrofuran (THF) and the solution is cooled at under a nitrogen atmosphere. 10.8 ml of 10 M buthyl lithium in hexane are cautiously dropped, then the red solution is kept at -10°C for 1 hour and it is poured into a flask containing 500 g of dry ice (solid carbon dioxide). The yellow suspension dissolves in I hour, then the resulting solution is poured into 1000 mi of 2 N sodium hydroxide. After 30 minutes the organic phase is separated off and the solvent is concentrated to a little volume. The residue is added with 2 N sodium hydroxide and water and washed with hexane, then the aqueous phase is acidified to pH 2 with 37% hydrochloric acid, kept for 1 hour at room temperature, and the solid is filtered and washed with 50 mi of water. After drying it in an oven at 70 0 C, 17.8 g of the product are obtained, m.p. 171-174°C Preparation 2 Synthesis of 2-r3-(2-thienvl)acrvlovl]thiophene A solution of 5 g of 3-(2-thienyl)acrylic acid in 30 ml of thionyl chloride is heated at 0 C for I hour 30 minutes, then the unreacted thionyl chloride is eliminated under reduced pressure. The residue is added with 20 ml of heptane and dried twice, then it is dissolved in 50 ml of methylene chloride, cooled at 0°C and added dropwise with a o0 solution of 2.43 g of thiophene in 5 ml of methylene chloride. Keeping the temperature at o0C. a solution of 6.74 mi of SnCL in 12 ml of methylene chloride is then dropped. The reaction mixture is kept at room temperature for 2 hours, then it is poured into 200 ml of 2 N hydrochloric acid. The aqueous phase is separated and extracted with 100 ml of methylene chloride. The organic phase is dried over sodium sulfate and evaporated to dryness and the residue is purified by silica gel chromatography (eluent hexane/ethyi acetate obtaining, after recrystallization from hexane, 4.79 g of the product, m.p.

105-107 0

C.

Preparation 3 Synthesis of 2 3 2 -thienvi)nropanovllthiophene A solution of 4.64 g of 2 3 2 -thienyl)acryloyl]thiophene in 50 ml of dry methylene chloride and 0.97 g of tris(triphenyiphosphine)rhodium chloride is hydrogenated at room temperature overnight. The reaction mixture is then concentrated to dryness and purified by silica gel chromatography (eluent hexane/ethyl acetate 20:1) to give 4.3 g of the product as an oil.

WO-99/06393 PCT/EP98/04705 11 Preparation 4 Synthesis of 2-[2-((2.2'-bisthien-5'-vl)carbonvl)thien-5-vlacetic acid ethyl ester A solution of bis-thiophene-2-carboxyiic acid (3 g; preparation 1) in 20 ml ofthionyl chloride is heated at 50°C for 1 hour 30 minutes, then the unreacted thionyl chloride is evaporated off and the residue is treated three times with 10 ml of heptane and evaporated. The residue is dissolved in methylene chloride and cooled at 0°C, then it is added dropwise with a solution of 2-(thien-2-yl)acetic acid, ethyl ester (2.19 g) in 5 mi of methylene chloride. A solution of SnC1 4 (3 ml) in 10 ml of methylene chloride is subsequently added. The reaction mixture is kept at room temperature for 2 hours, then it is poured into 200 ml of 2 N hydrochloric acid. The aqueous phase is extracted with methylene chloride (3x150 ml) and the organic extracts are collected, dried over sodium sulfate and concentrated to dryness.The residue (8 g) is purified by silica gel chromatography (eluent hexaneiethyl acetate 8:1) to give 3.62 of the product.

Preparation 5 Synthesis of 2-[2-((2.2'-bisthien-5'-vl)carbonvl)thien-5-vliacetic acid A suspension of 3.62 g of 2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetic acid, ethyl ester, in 60 ml of ethanol and 5 ml of water is cooled at 0°C and added with 11.35 ml of 2 N sodium hydroxide. The mixture is kept at room temperature overnight, then it is concentrated to a little volume. After acidification, the product crystallizes, which is then separated by filtration and dried under vacuum at 40*C. 2.4 g of the product are recovered.

Preparation 6 4 -3-(2-thienvl)propanoll- I-fluorobenzene g of 2 2 -thienylidene)-4-fluoroacetophenone are dissolved in 50 ml of dry methylene chloride and added with 0.97 g of tris(triphenylphosphine)rhodium chloride. The reaction is hydrogenated for 8 hours (about 470 ml of hydrogen are reacted), then it is concentrated to dryness and purified by silica gel chromatography (eluant hexane/ethyl acetate 20 to give 4.85 g of the product.

Preparation 7 2-[2-(trifluoroacetamido- l-ethvl]thiophene A solution of 2-(2-thienyl)ethylamine (10 g) in 100 ml of tetrahydrofuran is cooled to 0 C then it is added dropwise with 9.05 ml of ethyl trifluoroacetate. After 4 hours under stirring at room temperature the reaction mixture is concentrated to dryness to give 18 g of an oil which is treated with 18 ml of ethyl ether and saturated with 90 ml of hexane.

After cooling for 2 hours 12.15 g of the product are collected by filtration, m.p. 47 0

C.

WO 99/06393 PCT/EP98/04705 12 Preparation 8 2-[2-((2.2'-bisthien-5'-vl)carbonvl)thien-5-vll-Ntrifluoroacetvlethvlamine A solution of bis-thiophene-2-carboxylic acid (3.4 g; preparation 1) in 25 ml of thionyl chloride is heated at 50 0 C for 2 hours. The reaction mixture is concentrated to dryness, then it is redissolved in heptane and concentrated again to dryness. This work up is repeated, then the residue is dissolved in methylene chloride, cooled at 0°C and added dropwise with a solution of 3.3 g of 2-[2-(trifluoroacetamido)- -ethyl]thiophene (preparation 7) in 20 ml of methylene chloride and subsequently with a solution of SnC14 (3.8 ml) in 3.8 ml of methylene chloride. The reaction mixture is kept under stirring at room temperature for 2 hours, then it is poured into 200 ml of 2 N hydrochloric acid and extracted with tetrahydrofuran (4 x 100 ml). The organic extracts are pooled, dried over sodium sulfate and the solvent is evaporated under reduced pressure. 18 g of a black oil are obtained, which is purified by silica gel chromatography (eluant hexane/tetrahydrofuran 2 After recrystallization from-hexane (100 ml) and drying under vacuum at 50 0 C, 4.3 g of the product are obtained, m.p. 168-170 0

C.

Preparation 9 -2-[2-((2.2'-bisthien-5'-vl)carbonyl)thien-5-yl]ethylamine To a mixture of 2-[2-((2,2'-bisthien-5'-yi)carbonyl)thien-5-yl]-Ntrifluoroacetylethylamine (10 g; preparation 8) in 800 ml of methanol, cooled at 0°C and kept under stirring, 60.17 ml of 2 N sodium hydroxide are added and the reaction mixture is heated at 50 0 C overnight. The mixture is then concentrated to a little volume, then it is added with water (500 ml) and extracted with chloroform (4 x 200 ml). The organic extracts are pooled, dried over sodium sulfate and concentrated to dryness. After crystallization from a ethyl acetate (16 ml)/hexane (16 ml) mixture, 5.1 g of the product are obtained, m.p. 102-104 0

C.

Example 1 Synthesis of N-[2-[2-((2.2'-bisthien-5'-vl)carbonvl)thien-5vllacetvlltriptophane. ethyl ester A suspension of 2.34 g of 2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetic acid (preparation 5) and 1.74 g of carbonyldiimidazole in 150 ml of dry THF is kept at 40 0

C

for 1 hour. The mixture is cooled to 20 0 C and it is added dropwise with a solution of 2.45 g of triptophane ethyl ester in 10 ml of dry THF. After 2 hours the reaction mixture is concentrated to dryness, then it is dissolved in ethyl acetate, washed with water and extracted with 200 ml of chloroform. The organic phase is concentrated to dryness to WO-99/06393 PCT/EP98/04705 13 give 7 g of a residue which is purified by silica gel chromatography (eluent hexane/ethyl acetate from 4:1 to 2.79 g of the product are obtained.

Example 2 Synthesis of N-[2-r2-((2.2'-bisthien-5'-yl)carbonvl)thien-5vllacetvyltriptophane A solution of 2.2 g of N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5yl]acetyl]triptophane. ethyl ester (example 1) in 320 ml of ethanol and 10 ml of water is cooled at 0°C, then it is added dropwise with 4.43 ml of 2 N sodium hydroxide. After 4 hours at room temperature, the mixture is concentrated to a little volume, added with water, washed with ethyl acetate and acidified to pH 2. The aqueous phase is extracted with ethyl acetate, the organic extracts is dried over sodium sulfate, concentrated to dryness and the residue is purified by silica gel chromatography (eluent chloroformimethanollacetic acid 9:1:0.25) to give 1.16 g of the product, m.p. 166- 168 0

C.

Example 3 Synthesis of 2-[3-f2-((2.2'-bisthien-5'-vl)carbonvl)thien-5vllDropanovl]thiophene A solution of 1 g of bis-thiophene-2-carboxylic acid (preparation 1) in 5 ml of thionyl chloride is heated at 50*C for 1 hour 30 minutes, then the unreacted thionyl chloride is evaporated off and the residue is added with heptane and again evaporated. The residue is dissolved in methylene chloride, cooled at o0C and added dropwise with a solution of 2 3 -(2-thienyl)propanoyl]thiophene (1 g; preparation 3) in 3 mi of dry methylene chloride. A solution of 1.05 ml of SnCL in 3 mi of dry methylene chloride is then dropped, keeping the temperature at 0°C. The reaction mixture is kept at room temperature for 1 hour, then it is poured into 100 mi of 2 N hydrochloric acid, extracted with ethyl acetate (3x100 ml), dried over sodium sulfate and concentrated to dryness.

The residue (3 g) is purified by silica gel chromatography (eluent hexaneiethyl acetate 5:1) to give 1.02 g of the product, m.p. 124-126 0

C.

Example 4 Synthesis of 2-r3-r2-((2,2'-bisthien-5'-vl)carbonvl)thien-5vlacryloylithiophene A solution of 0.24 g of bis-thiophene-2-carboxylic acid (preparation 1) in 3 ml of thionyl chloride is heated at 50°C for 1 hour 30 minutes, then the unreacted thionyl chloride is evaporated off and the residue is added with heptane and again evaporated. The residue is dissolved in methylene chloride, cooled at 0°C and added dropwise with a solution of 2-[3-(2-thienyl)acryioyl]thiophene (0.3 g; preparation 2) in 3 ml of dry methylene WO-99/06393 PCT/EP98/04705 14 chloride. A solution of 0.31 ml of SnCl in 3 ml of dry methylene chloride is then dropped. keeping the temperature at 0 0 C. The reaction mixture is kept at room temperature for 4 hour, then further 0.3 ml of SnC, are added and the mixture is kept at room temperature overnight. The reaction mixture is poured into 200 mi of 2 N hydrochloric acid, extracted with ethyl acetate (3x50 mi), dried over sodium sulfate and concentrated to dryness. The residue (1.3 g) is purified by silica gel chromatography (eluent hexane/ethyl acetate 5:1, then pure ethyl acetate) to give 0.37 g of the product.

Example 5 Synthesis of 2-((2.2'-bisthien-5 '-vi)carbonvl)thien-5-vlpropanovl-4fluorobenzene 1.06 g of bis-thiophene-2-carboxylic acid (preparation 1) in 10 mi of thionvl chloride are heated at 50 0 C for 1 hour 30 minutes, then the mixture is concentrated to dryness. The residue is treated twice with 20 ml of heptane, finally with 30 mi of methylene chloride, cooled to 0 0 C and added dropwise with a solution of 4- 3-(2-thienyl)propanoyl]- 1fluorobenzene (1.08 g; preparation 6) in 5 mi of methylene chloride. Keeping the temperature at about 0 0 C, a solution of SnCl (1.07 mi) in 5 ml of methylene chloride is added dropwise, then the mixture is stirred at room temperature for 2 hours. The reaction mixture is poured into 150 ml of 2 N hydrochloric acid, then it is extracted with ethyl acetate and the organic phase is concentrated to dryness. The residue is purified by silica gel chromatography (eluant methylene chloride), to give, after crystallization from ethyl acetate/hexane mixture, 1 g of the product, m.p. 138-140 0

C.

Example 6 According to the procedures described in the previous preparations and examples, the following oligothiophene derivatives are obtained: N-[2-[2-((2,2'-bisthien-5'-yl N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetv]alanine; N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetv]phenvlalanine; N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetyl]tyrosine; N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetyl]leucine; N-f2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetyl]lysine; N-(2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetyl]serine; N-[2-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]acetyl]isoleucine; 2-[3-[2-((2,2'-bisthien-5'-yl)carbonyl)thien-5-yl]propanoyl]4-fluorobenzene; 2-(3-[2-((2,2'-bisthien-5'-yl)carbonyvl)thien-5-yvi]propanoyi]-4-chlorobenzene: WO -99/06393 PCT/EP98/04705 2-[3 -bisthien-5 '-vl)carbonvl)thien-5-yllpropanovl]-2-bromobenzene; 2-fj3-[2.((2,2'-bshe- 2-[3 '-bisthien-5 '.Yl)carbonyl)thien-S-yl~propanov].4-(armnomethyl)benzene; 2-[3 -bisthien-5 '-yl)carbonyl)thien-S-yl]propanov]-3- -bisthien-5 '-vl)carbonyl)thien-S-ilpropanovi]--(ten-butyl)benzene; 2-j3 '-bisthien-5 -v)carbonvl)thien-5-yl]acryloyl.4-fluoroberizene; 2-[3 -bisthien-5 '-vl)carbonvl)thien-5-vl~acryioyl]A4-chlorobenene; 2-[3 -bisthien-5 '-yl)carbony)thien--yl]acryloyl]-2-bromobenene; 2-[3 2' -bisthien-5 '-yl)carbonyi)thien-S-yl]acryloyl]-4(.carboxrmethyl)benzene; 2-[3 '-bisthien-5 '-yl)carbony)thien-5-ylacryioyl].4(axninomethyl)benzene; 243 '-bisthien-5 '-vl)carbonvi)thien-5-vl]acrvovl]-'3dimethvlamiunomethyi)benzene.

'-bisthien-S '-yl)carbonyl)thien-5-ylacrvioy1i]-2(tert-butv)benzene.

1Example 7 4-r2-r2-((2.2 '-bisthien-5 '-vl)carbonvl)thien-5-vlethlamjinocarbonll.2.

(tert-butoxvcarbonvaminojbutanoic acid A mixture of glutamxnic acid N-BOC 88 g) and 1,1 '-carbonyldiimidazole 63 g) in mld of tetrahydrofuran is kept under stirring at room temperature for 2 hours, then a solution of 2 -[2-((2,2'-bisthien.5 '-yl)carbonyl)thien-5-y]ethvamine (1.11 g; preparation 9) in 40 ml of tetrahydrofuran is added dropwise and the reaction mixture is heated at overnieht. The solvent is evaporated under reduced pressure and the residue is dissolved in ethyl acetate and washed with a saturated aqueous solution of potassium hydrogensulfate. After separation by filtration of the solid which separates (starting product), the organic phase is concentrated to dryness. The residue t2.5 is purified by silica gel chromatography (eluant 1) chloroformjmethanol 15 2. 2) chloroformn/methanol 9 to give 0.42 g of the product.

Example 8 4-r2-2-((22 '-bisthien-5 '-yl)carbonvl)thien-5-vlethyiamianocarbonvll-2aminobutanoic acid A suspension of '-bisthien-5 '-yl)carbony)thien-5-yl]ethylaminocarbonyl]-2- (tert-butoxycarbonylamjno)butanoic acid (0.28 g; example 7) in 28 ml of methylene chloride is added with 0.39 ml of trifluoroacetic acid. The resultinia dark solution is kept under stirring at room temperature overnight, then it is concentrated to dryness. The residue is redissolved in absolute ethanol 15 ml) then it is concentrated again to dryness and this work up is repeated another time. The residue (0.3 g) is suspended in ml of hexane, stirred for I hour at room temperature, filtered and dried under vacuum at 0.2 g of the product are obtained.

'H-NN4R in d6-DMSQ D 2 0: 1.9 ppm (mn, 2H); 2.18 ppmn (mn, 2H); 3.05 ppm (mn, 211); 3.36 ppmn (br in. 2H); 3.7 ppm 111); 7.1 ppm (mn, 2H); 7.5 ppm (mn, 3H1); 7.9 ppm (in, 2H); 8.61 ppmn (br t, 11H).

ExAMie 9 A-ccording to the methods described in examples 7 and 8, starting from the suitable amino acids, the following oligo-thiophenes are obtained: 2 -[2-((2,2'-bisthien-5 -bisthien-5 -bisthien-5 '-yl)carbonvi)tien-5-1vIlethyiamiinojserinanjce: f 2 -[2-((2.2'-bisthien-5'-yi f2-[2-((2..z'-bisthien-5 135 '-bisthien-5 The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims

3. Oligo-thiophenes according to claim 2. wherein A-B is a group of formula 20 -CH2CH?-NH-A.A.-.
4. Qligo-ckiiophenes according to claims I to 3, wherein the armino acid is triptophane. Oigo-trhiophenes according to claim 1, wherein B is a 2-thienyi group. STR4 7 S
6. Oligo-tkiiophenes according to claim 1, selected from: -bisthien-5 '-bisthien-5 2 3 -(2-((2,2'-bisthien-5 -bisthien-S '-vl)carbonyl)tbien-5vl]propanov].4fluorb~enzne -N-[2-[2-((2,2'-bisthien5 '-vi)carbonyi)thien-S-yilacewll~glycine, -bisthiien-5' -bisthfien-5 -bistkiien-5 -bistkiien-5' N-(2-(2-((2,2'-biszjiien-5' -bistkiien-5 '-yl)carbonyl)thien-5-vI]propanoyiJ-4-fluorobenzene; 2 3 2 -((2,2'-bisthien-5'-y)carbony)thien-5-yl]propanoyJ..4.chjorobenzee -bisthien-5'-Yi)carbonyi)thien-5..yi]propanoylJ-2bromobenzene; 2 3 2 -((2,2'-bishien-5'-y)carbony)tiien.5.yi]propanoi].44.carboxymethyi)Ieimw; -biszhien-5 '-vI)carbonvl)thien-.5-vl]propanovi-4- aninomethvi)beuizme;e -bisthien-5 -l)carbonyi)thien-S-vl]propanoyil-3)- (dimethylaminomethyi)benzene-, -bisxhiei-5' -yI)carbonyl)thien-5-yI]propanoyi-24 tert-butyl)benzene; 3[2(22-itin5ylaroythe--larly]4furbzee 2 3 2 2 2 '-bisthien-5'-Yl)carbonyi)thien-5-yl]acryioyi]-4-lorobenzene; -2-3-2-((2,2'-bisthien-5' -yl)carbonyl)thien-5-yl~acryloyl]-2-1,roniobenzene; -bisthien- -yl)carbonyl)thien-5.yl]acrvloyl]-44.carboxyrnethyl)benzene; 20 -bisthien-5' -vl)carbony)then-5-yl]acrvioylp-4(aminomethv)benzene -bisthien-5' -v~I)carbonvi)thien-5-vI~acriovlj-3 (dimethvlaminomethyl)benzene: -bisthien-5' -Yl)carbonvi)thien-5-yI~acrvloyII-'2-(tert-butvl)benzene. 25 7. Pharmaceutical compositions containing a pharmacologically effective amount of one or more compounds of claims I to 6 in adznixture with pharmaceutically acceptable excipients.
8. The use of compounds of claims I to 6 for the preparation of a medicamnent having antitumor and antimetastatic activity.
9. Compounds of claims I to 6 for use as inhibitors of uPA binding to the specific uPAR \receptor. P:\OPERKbmX\88079-98 sp.doc-0307m1 -19- A method of treating diseases involving tumors, or tumor metastasis or invasiveness comprising administering a compound of any one of claims 1 to 6 or a pharmaceutical composition of claim 7.
11. An oligo-thiophene compound of general formula according to claim 1, substantially as hereinbefore described with reference to the Examples.
12. A pharmaceutical composition according to claim 7, substantially as hereinbefore described with reference to the Examples. DATED this 16th day of July, 2001 Roche Diagnostics GmbH By DAVIES COLLISON CAVE Patent Attorneys for the Applicants *o *o*