WO2011121629A1

WO2011121629A1 - Indolic derivatives and use thereof in medical field

Info

Publication number: WO2011121629A1
Application number: PCT/IT2011/000099
Authority: WO
Inventors: Romano Silvestri; Giuseppe La Regina; Ernest Hamel; Andrea Brancale; Patrizia Lavia; Ettore Novellino
Original assignee: Universita' Degli Studi Dl Roma "La Sapienza"; Consiglio Nazionale Delle Ricerche
Priority date: 2010-04-01
Filing date: 2011-04-01
Publication date: 2011-10-06
Also published as: ITRM20100151A1; IT1399130B1

Abstract

The present invention concerns indolic derivatives and use thereof in medical field. Particularly, the invention concerns 3-[(3',4',5'- trimethoxyphenyl)thio]-1 H-indole derivatives and bioisosteric derivatives thereof and their use for the treatment of tumours.

Description

INDOLIC DERIVATIVES AND USE THEREOF IN MEDICAL FIELD

It is known that microtubules are involved in several cell functions (for example cell mobility, intracellular transport and cell division or mitosis). As to cell division, i.e. the process underlying the neoplastic proliferation, microtubules are arranged in order to form a complex and highly dynamic structure, the mitotic spindle, providing for chromosome distribution within new formed cells. Since tumour cells present a mitosis frequency higher than not tumour ones, the mitotic spindle is considered for many years an important target for antitumor therapies (Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Past and future of the mitotic spindle as an oncology target. Current Opin. Pharmacol. 2001 , 1 , 370-377; Jordan, M. A.; Wilson, L. 2004. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 2004, 4, 253-265).

The major protein component occurring within microtubules is tubulin. Molecules suitable to interfere with microtubule assembling, both by inhibition of tubulin polymerization and blocking microtubule disassembling, prevent the mitosis completion and block the cells in metaphase stage. The microtubule block represents therefore a validated approach for antitumor therapy [Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibithion of the binding of colchicine to tubulin. Biochemistry 1989, 28, 6984-6991 ; Beckers, T.: Mahboodi, S. Natural, semisynthetic and synthetic microtubule inhibitors for cancer therapy Drugs Fut. 2003, 28, 767-785].

Various natural products proved to be successful in exerting antimitotic properties. Vinca alkaloids from Catharanthus roseus (named Vinca rosea) and colchicine from Colchicum autumnalis have been the first tubulin interfering agents to be discovered [Pohle, K.; Matthies, E.; Peters, J. E. On the tumor growth-inhibiting acthion of colchicine. Arch. Geschwulstforsh. 1965, 25, 17-20]. Vincristine, vinblastine and vindesine are used by intravenous route for the treatment of several human cancer forms including leukaemia, lymphomas, and various solid breast and lung tumours. Vinorelbine is widely used by intravenous route for treatment of advanced breast cancer and lung carcinomas. Vinca alkaloids induce neurotoxic effects and hyper-expression of drug resistance inducing glycoprotein-P.

Podophyllotoxin is obtained from Podophyllum peltatum and

Podophyllum emodi (pharmaceutical preparations obtained from extracts of plant referred as podophyllum), and forms a complex with tubulin thus preventing the formation of microtubules. Podophyllotoxin is the choice drug for treatment of genital warts, but it must be manipulated carefully due to toxicity thereof.

The term taxoid is used to mean paclitaxel and derivatives thereof, firstly obtained from plants of Taxus genus. Paclitaxel proved to be successful in exerting excellent activity against ovarian and breast tumours and, together with docetaxel, is under evaluation against various other tumour types. Taxoids suffer from the problem in that are not suitable to be administered orally and result in various side-effects. Further often said drugs result in multi-drug resistance development.

Combrestatin A-4 (CSA4) is a natural product obtained from Combretum caffrum. It is the most active structure of this family, and like phosphate pro-drug (more soluble in water) is under clinical study phase. CSA4 shares structural characteristics with colchicine and podophyllotoxin, and binds tubulin in the same binding site as colchicine.

In the light of above it is therefore apparent the need to provide for availability of active principles for tumour therapy suitable to overcome the side-disadvantages associated to already known molecules (Jackson, J. R.; Patrick D. R.; Dar, M. M,; Huang, P.S. Targeted anti-mitotic therapies: can we improve on tubulin agents? Nature Rev. Cancer 2007, 7, 107).

The indole nucleus occurs in several tubulin polymerization inhibitors [Brancale, A.; Silvestri, R. Indole, a core nucleus for potent inhibitors of tubulin polymerizathion. Medicinal Research Reviews 2007, 27, 209-238]. Taking into account that natural antimitotics display toxicity problems, drug-resistance and limited bioavailability, a large synthetic effort has been focused towards synthetic inhibitors. Examples of indolic inhibitors of tubulin polymerization are disclosed WO2006/084338, WO2004/099139, WO0119794, Flynn et al. (Journal of Medicinal Chemistry, vol. 45, 1 January 2002, pagg. 2670-2673). The authors of the present invention in addition, have synthesized arylindole derivatives exerting anti-tubulin activity (WO2006/041961). The study about structure-activity relationship (SAR) resulted in detection of aryl thioindole derivatives displaying powerful tubulin polymerization inhibition activity, active at nanomolar range concentration against MCF-7 tumour cells [De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C; Barbera, M. C; Brancale, A.; Wilcox, E.; Hamel, E.; Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of tubulin polymerizathion. J. Med. Chem. 2004, 47, 6120-6123; De Martino, G.; Edler, M. C; La Regina, G.; Coluccia, A.; Barbera, M. ^'C; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.; Hamel, E.; Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of tubulin polymerizathion. 2. Structure activity relathionships and molecular modeling studies. J. Med. Chem. 2006, 49, 947-954; La Regina, G.; Edler, M. C; Brancale, A.; Kandil, S.; Coluccia, A.; Piscitelli, F.; Hamel, E.; De Martino, G.; Matesanz, R.; Diaz, J. F.; Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico, M.; Silvestri, R. New arythioindoles inhibitors of tubulin polymerizathion. 3. Biological evaluathion, SAR and molecular modeling studies. J. Med. Chem. 2007, 50, 2865-2874; La Regina, G.; Sarkar T.; Bai, R.; Edler, M. C; Saletti, R.; Coluccia, A.; Piscitelli, F.; Minelli, L; Gatti, V.; Mazzoccoli, C; Palermo, V.; Mazzoni, C; Falcone, C; Scovassi, A. I.; Giansanti, V.; Campiglia, P.; Porta, A.; Maresca, B.; Hamel, E.; Brancale, A.; Novellino, E.; Silvestri, R. New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerizathion, cell growth inhibithion, and molecular modeling studies. J. Med. Chem. 2009, 52, 7512-7527].

The authors of the present invention have synthesised a new family of arylthioindole compounds and bioisosteric derivatives thereof displaying a powerful inhibiting activity, both in vitro and in alive, against tubulin polymerization and/or microtubule formation that are hyper- expressed/hyper-stimulated in tumour cells of several cancer forms. In addition, the compounds of the invention are suitable to induce tumour cell apoptosis.

The ester moiety at 2 position and arylthio group at 3 position on indole nucleus represented essential structural requirements for first generation arylthioindoles. However, the ester moiety can be hydrolyzed by ubiquitous esterase resulting in the formation of carboxylic acid derivatives that during in vivo experiments did not display activity.

In compounds according to the present invention, heterocyclic nuclei, as for example; pyrrole, thiophene, furan, imidazole, pyridine, have been substituted at 2 position as bioisosteric groups of the ester moiety. The sulfur atom of arylthio group can be oxidized to sulfoxide and sulfone resulting in formation of less active derivatives. Although arylthioindole structure sterically protects sulfur atom from oxidative activity of CYP450 enzymes and there are no experimental evidences of the in vivo oxidation of arylthioindoles, in order to prevent the potential inactivation, the sulfur atom has been also replaced with a carbonyl or methylene group.

It has been advantageously observed that, with respect to non- tumour cells, arylthioindoles demonstrate lower toxicity than paclitaxel.

The compounds of the invention have proved to be powerful inhibitors of tubulin polymerization and MCF-7 cell growth, being active in the lower micro- and nanomolar concentration range, respectively, with a displacement activity for [^3H] colchicine up to approximately 90%. The antitumor activity in addition proved to be extended to a panel of clinically interesting tumour cells (Hela, PC3, HT-29, A-549, A2780wt, A2780-CIS, OVCAR-3, OVCAR-8, NCI/ADR-RES), some of which are resistant to currently used antitumor drugs in therapy. Hela cell treatment with said compounds resulted in a complete stop of cell cycle in G2/M phase. Apoptotic cell death by means of not p-53-dependent mechanism has been selectively induced during the mitotic arrest, while for inter-phase cells the level of apoptosis remained low or negligible.

Based on obtained results, it is possible therefore to affirm that compounds of the invention display an higher therapeutic effectiveness than known compounds as to the elimination of actively proliferating tumour cells but not of not tumour cells.

It is therefore a specific object of the present invention 3-[(3',4',5'- trimethoxyphenyl)thio]-1 H-indole derivatives and bioisosteric derivatives thereof of formula (I):

wherein

Ri is selected from phenyl, heterocycle, benzo fused heterocycle, optionally substituted at one or more positions with the following same or different groups: alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C-i-C₆ alkoxy, C₂- C6 alkoxycarbonyl, halogen, -CN, -NH₂, -OH, -NO₂, or aromatic ring selected from phenyl, phenoxy, benzyl, benzyloxy, naphthyl, naphthyloxy, biphenyl, or heterocycle, said aromatic ring being optionally substituted at one or more positions with one or more of the following same or different groups selected from alkyl, C₂-C₆ alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C2-C6 alkoxycarbonyl, halogen, -CN, -IMH2, -OH, -NO₂;

R₂ is selected from the following groups, at one or more positions selected from 4, 5, 6 and/or 7 on indole nucleus, the groups being the same or different, hydrogen, alkyl, for example methyl or ethyl, C₂-C6 alkenyl, C₂-C6 alkynyl, C C6 alkoxy, for example methoxy or ethoxy, C₂- C6 alkoxycarbonyl, chlorine, bromine, fluorine, iodine, -CN, -NH₂, -OH, - NO₂, aromatic ring selected from phenoxy, benzyl, benzyloxy, naphthyl, naphthyloxy, biphenyl, or heterocycle, said aromatic ring optionally independently substituted at para, meta and/or orto positions, with from one to five same or different substituents selected from alkyl, C₂-C6 alkenyl, C₂-C6 alkynyl, C₁-C6 alkoxy, C₂-C₆ alkoxycarbonyl, halogen, -

X is selected from S, SO, SO₂, C=O, CHOH, CH₂, C=OC=O, C=OCH₂, CH₂C=O or CH₂CH₂, with the provision that when X is C=O, S, SO or SO2, R2 at position 6 is not methoxy.

According to a preferred embodiment the heterocycle is a 5 membered heterocycle selected from pyrrole, pyrazole, imidazole, furan, thiophene, oxazole, isooxazole, thiazole or dihydro- or tetrahydro- derivatives thereof or a 6 membered heterocycle selected from pyridine, pirimidine, piridazine, thiazine, oxazine, or dihydro-, tetrahydro-, or hexahydro-derivatives thereof. Benzo fused heterocycle can be selected from indole, benzofuran, benzothiophene, benzimidazole or partially hydrogenated derivatives thereof.

The alkyl group can be selected from C-1-C12 alkyl, (C3-C8 cycloalkyl)C₀-C6 alkyl.. Preferably the compounds according to the invention are those wherein X is S, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophene-2-yl, thiophene-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl and R₂ is selected from H, 5-halogen, for example chlorine, bromine or fluorine, 5-methoxy, for example the following compounds:

2-(1 H-Pyrryl-3-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]-1 H-indole (16) (RS3544);

2-(Thiophene-2-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]-1 H-indole (25) (RS3142);

5-Methoxy-2-(thiophene-2-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]- 1 H-indole (28) (RS3552);

2-(Pyridin-4-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]-1 H-indole (41 ) (RS3301 ).

The above reported compounds have the following formulae

16 25 28 41

(RS 3544) (RS 3142) (RS 3552) (RS 3301 )

Further preferred are compounds according to the invention wherein X is C=O, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophene-2-yl, thiophene-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl and R₂ is selected from H, 5-halogen, for example chlorine, bromine, fluorine, 5-methoxy, as for example [2- (Thiophene-2-yl)-1 H-indol-3-yl]-(3',4',5'-trimethoxyphenyl)metanone (26) (RS3223) having the following formula..

(RS 3223)

Other preferred compounds according to the invention are those wherein X is CH2, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophene-2-yl, thiophene-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl, and R₂ is selected from H, 5-halogen, for example chlorine, bromine, fluorine, 5-methoxy, as for example 2- (Thiophene-2-yl)-3-(3',4',5'-trimethoxybenzyl)-1 H-indole (27) (RS3250) having the following formula.

(RS 3250)

Further examples of compounds according to the present invention are those having the following groups R-i, R₂ and X:

The compounds as above defined for use in medical field are a further object of the present invention.

In addition the present invention concerns a pharmaceutical composition comprising or consisting of one or more compounds as above defined as an active principle, in combination with one or more pharmaceutically acceptable excipients and/or adjuvants.

The compounds or composition as above defined for the treatment of tumours, as for example, mammary carcinoma, cervix carcinoma, ovary tumour and other histological origin carcinomas are a further object of the present invention.

It is to be pointed out that the compounds displayed effectiveness against carcinoma cell lines lacking of p53 function, an important oncosuppressor not present or mutated in 50% of human tumours.

Various arylthioindole compounds (for example compounds 16, 25, 28 and 41) of the present invention are generally obtainable by microwave synthesis through reaction of suitable 2-heterocyclil-1 /-/- indoles with bis-(3,4,5-trimethoxyphenyl)disulfide in the presence of sodium hydride in DMF at 1 10°C (150 W) for 2 minutes (Scheme 1). [Bis-(3,4,5-trimethoxyphenyl)disulfide is prepared according to literature: De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C; Barbera, M. C; Brancale, A.; Wilcox, E.; Hamel, E.; Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of tubulin polymerizathion. J. Med. Chem. 2004, 47, 6120-6123].

ariltioindoli

(Es.: 16, 25, 28 e 41 )

(arylthioindoles)

(Ex. 16, 25, 28, 41) Scheme 1. Synthesis of arylthioindoles. ^a Reagents and reaction conditions: (i) NaH, DMF, 25°C, 10 min; (ii), closed vessel, 1 10°C, 150

W, Pmax = 250 PSI, 2 min.

Various arylmethanoneindole derivatives (for example compound

26) are obtainable by microwave assisted reaction through reaction of suitable 2-heterocyclil-1 /-/-indoles with 3,4,5-trimethoxybenzoyl chloride in the presence of anhydrous aluminiuml chloride in 1 ,2-dichloroethane at 1 10°C (150 W) for 2 minutes (Scheme 2).

arilmetanoneindoli

(Es.: 26)

(arylthioindoles Ex. 26) Scheme 2. Synthesis of arylmethanoneindoles. ^aReagents and reaction conditions: Anhydrous AICI3, 1 ,2-dichloroetane, closed vessel, 1 10°C, 150 W, Pmax = 250 PSI, 2 min.

Various arylmethyleneindole derivatives (as for example compound 27) are obtainable by reduction with sodium borohydride from corresponding ketones in hot ethanol (Scheme 3).

arilmetilenendoli

(Es.: 27)

(arylthioindoles Ex. 27) Scheme 3. Synthesis of arylmethyleneindole. ^aReagents and reaction conditions: NaBH₄ (10 eq), ethanol, reflux, 2,5 h.

2-heterocyclil-1 /-/-indole intermediates are obtainable as reported in Schemes 4-9. [2-(1/-/-Pyrrole-2-yl]-1H-indole is prepared according to literature: Hugon, B.; Pfeiffer, B.; Renard, P.; Prudhomme, M. Synthesis of isogranulatimide analogues possessing a pyrrole moiety instead of an imidazole heterocycle. Tetrahedron Lett. 2003, 44, 3927-3930].

2-Nitrophenylacetic acid chloride is treated with 1- (phenylsolfonil)-IH-pyrrole in the presence of anhydrous aluminium chloride to yield 2-[1-(phenylsolfonil)-1 /-/-pyrrol-3-yl]-1 /-/-indole. By reduction of nitro group using iron metal powder in glacial acetic acid by intramolecular cyclization of amino ketone intermediate 2-[1- (phenylsolfonil)-1 H-pyrrol-3-yl]-1 H-indole is obtained (Scheme 4).

Scheme 4. Synthesis of 2-[1-(phenylsolfonil)-1 H-pyrrol-3-yl]-1H-indole. ^a Reagents and reaction conditions: (i) oxalyl chloride, DMF cat., 1 ,2- dichloroethane, 0°C, under Ar, 30 min (ii) 1-(phenylsolfonil)-1 /-/-pyrrole, AICI₃, 0°C, Ar stream, 15 min, yield 10%; (b) Fe, AcOH, 60°C, 12 h.

By microwave reaction of 2-acetyl furan with phenyl hydrazine hydrochloride in the presence of anhydrous potassium acetate in open vessel at 100°C (150 W) for 5 minutes 1-[1-(furan-2-yl)ethyliden-1-yl]-2- phenylidrazina is obtained. Hydrazone is transformed to 2-(furan-2-yl)- 1 /-/-indole by heating in polyphosphoric acid (PPA) at 1 10°C (Scheme

Scheme 5. Synthesis of 2-(furan-2-yl)-1 /-/-indole. ^a Reagents and reaction conditions: (a) anhydrous CH₃COONa, open vessel, 100°C, 250 W, 5 min, PowerMAX; (b) PPA, 1 10°C, 1 h.

By microwave reaction of 2-iodo-1 H-indole with furan-3-boronic acid pinacol ester in the presence of palladium acetate (II) and potassium carbonate in closed vessel at 1 10°C (200 W) for 15 min 2- (furan-3-yl)-1 H-indole (Scheme 6) is obtained. 2-lodo-1 /-/-indole is prepared according to literature: Bergman, J.; Venemalm, L. Efficient synthesis of 2-chloro-, 2-bromine-, and 2-iodoindole. J. Org. Chem. 1992, 57, 2495-2497].

Scheme 6. Synthesis of 2-(furan-3-yl)-1 /-/-indole. ^a Reagents and reaction conditions: Pd(ll) acetate, potassium carbonate, 1-methyl-2- pyrrolidinone/water, closed vessel, 10°C, 200 W, Pmax = 250 PSI, 15 min. 2-Acetyl- and 3-acetylthiophene are brominated with bromine and treated with aniline and catalytic DMF under microwave irradiation in closed vessel at 100°C (150 W) for 1 min, to yield 2-(thiophene-2-yl)- 1H-indole and 2-(thiophene-3-yl)-1 /-/-indole, res ectively (Schema 7).

Scheme 7. Synthesis of 2-(thiophene-2-yl)-1H-indole and 2-(thiophene- 3-yl)- /-/-indole. ³ Reagents and reaction conditions: (a) bromine, dichloromethane, 25°C, 1 hour; (b) (i) aniline, 25°C, 3 h; (ii) DMF cat, closed vessel, 100°C, 150 W, Pmax = 250 PSI, 1 min.

By reaction of 5-methoxy-2-nitrobenzyl trimethylsilane with thiophene-2-carboxyaldehyde in the presence of tetrabutyi ammonium fluoride (TBAF) 2-(5-methoxy-2-nitrophenyl)-1 -(thiophene-2-yl)ethanol is obtained, the latter is oxidized to ketone using pyridinium chlorochromate. Reduction with stannous chloride of 2-(5-methoxy-2- nitrophenyl)-1-(thiophene-2-yl)ethan-1-one yields an amine intermediate that by intramolecular cyclization yields 5-methoxy-2-(thiophene-2-yl)- 1 H-indole (Scheme 8). [5-Methoxy-2-nitrobenzyl trimethylsilane is prepared according to literature: Bartoli, G.; Bosco, M.; Dalpozzo, R. Todesco. P. E. Functionalization of aromatic systems: a highly chemoselective synthesis of trimethylsilylmethylnitroarenes. J. Org. Chem. 1986, 51, 3694-3696].

Scheme 8. Synthesis of 5-methoxy-2-(thiophene-2-yl)-1H-indole. ^a Reagents and reactions conditions: (a) thiophene-2-carboxyaldehyde, TBAF, anhydrous THF, 25°C, under Ar, 15 min; (b) pyridinium chlorochromate, anhydrous dichloromethane, 25°C, 1.5 hours; (c) stannous chloride dihydrate, ethyl acetate, reflux, 3 h.

Pyrimidinil indoles are obtained by reaction of 2-acetyl-, 3-acetyl- or 4-acetylpyrimidine with phenyl hydrazine hydrochloride in the presence of anhydrous sodium acetate by microwave irradiation at 100°C, 250 W, per 5 min. Subsequent cyclization of hydrazone intermediates is carried out by heating at 110°C with PPA (Schema 9).

X = N, Y = Z = CH; X = CH, Y = N, Z = CH; X = Y = CH, Z = N. Scheme 9. Synthesis of 2-(pyrimidinil)-1H-indoles. ^a Reagents and reaction conditions: (a) phenyl hydrazine hydrochloride, anhydrous CH₃COONa, open vessel, 100°C, 250 W, PowerMAX, 5 min; (b) PPA, 110°C, 1 hour.

The present invention now will be described by an illustrative, but not limitative, way according to preferred embodiments thereof, with particular reference to enclosed drawings, wherein:

Figure 1 shows the possible binding mode of compound 15 (gray); on the left Thr179 residue is highlighted; on the right Cys249 residue is highlighted; DAMA-colchicine is in violet colour.

Figure 2 shows the possible binding mode of compound 25

(gray); on the left Thr179 residue is highlighted; on the right Cys249 residue is highlighted; DAMA-colchicine is in violet colour.

Figure 3 shows the possible binding mode of compound 28 (gray); on the left Thr179 residue is highlighted; on the right Cys249 residue is highlighted; DAMA-colchicine is in violet colour.

Example 1 : Synthesis of compounds according to the present invention Chemistry. Melting points are determined using Buchi 510 instrument and are not corrected. Infrared spectra (IR) have been obtained using Perkin-Elmer SpectrumOne spectrophotometer. Absorption band position is reported in cm^"1. Nuclear magnetic resonance spectra (¹H NMR) are recorded using Fourier Transform Bruker Advance 400 MHz spectrometer in reported solvent. Chemical shifts relative to tetramethyl silane are expressed in δ unit (ppm). Chromatographic columns are packed with silica 60 gel, or alumina 90 Merck. Thin layer chromatography (TLC) is carried out using on silica gel or alumina coated aluminium plates with a Fluka 254 nm fluorescence detector. Organic solutions are dried over anhydrous sodium sulfate. Solvent evaporation is carried out using Buchi R-210 Rotavapor equipped with BQchi V-850 vacuum control device and BQchi V-700 (approximately 5 mbar) and V-710 (approximately 2 mbar) pumps. The purity of compounds is determined by combustion. Elemental analysis yields ±0.4% of theoretical values, and compounds are >95% pure.

Example 1.1 : 2-(1H-Pyrryl-3-yl)-3-[(3',4',5'-trimethoxyphenyl) thio]-1H-indole (16) (RS3544).

2-[1-(Phenylsulfonil)-1H-pyrrol-3-yl]-1 H-indole (0.45 g, 0.0014 mole) is carefully added to a sodium hydride suspension (60% in mineral oil, 0.13 g, 0.0031 mol) in anhydrous DMF (2 ml_) under stirring. After 10 min, bis-(3,4,5-trimethoxyphenyl)disulfide (0.62 g, 0.0015 mole) is added. [Bis-(3,4,5-trimethoxyphenyl)disulfide is prepared according to literature: De Martino, G.; La Regina, G.; Coluccia, A.; Edler, M. C; Barbera, M. C; Brancale, A.; Wilcox, E.; Hamel, E.; Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of tubulin polymerizathion. J. Med. Chem. 2004, 47, 6120-6123] and the reaction mixture vessel is put in microwave device cavity (closed vessel mode, Pmax = 250 PSI) irradiating at 150 W. Reaction temperature is increased from 25°C to 1 10°C in 1 min and then maintained at this temperature for 2 min. After cooling the mixture is diluted with water and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the obtained residue is purified by column chromatography (alumina, chloroform as eluent) to give 16 with 10 % yield, melting point 200-204°C (after ethanol crystallization). ¹H NMR (DMSO-d₆): δ 3.52 (s, 6H), 3.56 (s, 3H), 6.33 (s, 2H), 6.75-6.77 (m, 1 H), 6.84-6.86 (m, 1 H), 7.00-7.04 (m, 1 H), 7.08-7.12 (m, 1 H), 7.39 (d, J = 8.6 Hz, 2H), 7.48- 7.50 (m, 1 H), 1 1.11 (br s, 1 H, it disappears after treatment with D₂0), 11.58 ppm (br s, 1 H, it disappears after treatment with D₂0). IR: □ 3358, 3390 cm^-1. Anal. (C21 H20N2O3S (380.46)) C, H, N, S.

Example 1.2: 2-(Thiophene-2-yl)-3-[(3',4',5'- trimethoxyphenyl) thio]-1H-indole (25) (RS3142).

It is obtained as compound 16 from 2-(thiophene-2-yl)-1H-indole. Yield 60%, melting point 195-198°C (after ethanol crystallization). ¹H NMR (DMSO-de): δ 3.53 (s, 6H), 3.56 (s, 3H), 6.35 (s, 2H), 7.09-7.13 (m, 1 H), 7.19-7.24 (m, 2H), 7.47-7.49 (m, 2H), 7.65 (dd, J = 1.1 and 5.0 Hz, 1 H), 7.77 (dd, J * 1.1 and 3.7 Hz, 1 H), 12.1 1 ppm (br s, 1 H, it disappears after treatment with D₂0). IR:v 3317 cm^"1. Anal. (C2i H₁₉N0₃S2 (397.51)) C, H, N, S.

Example 1.3: 5-Methoxy-2-(thiophene-2-yl)-3-[(3',4',5'- trimethoxyphenyl)thio]-1H-indole (28) (RS3552).

5-Methoxy-2-(thiophene-2-yl)-1 H-indole (0.19 g, 0.00083 mol) is added at 0"C to a sodium hydride suspension (60% in mineral oil, 0.05 g, 0.0012 mol) in anhydrous DMF (2 ml_). After 15 min bis-(3,4,5- trimethoxyphenyl)disulfide (0.36 g, 0.00091 mol) is added and the reaction mixture is heated at 1 10°C overnight under argon. After cooling the mixture is carefully diluted with water and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the obtained residue is purified by column chromatography (silica gel, ethyl acetate:r?-hexane 1 :2 as eluent) to give 28 (0.25 g, 70%), melting point 55-58°C (after ethanol crystallization). ¹H NMR (CDCI3), δ 3.67 (s, 6H), 3.78 (s, 3H), 3.84 (s, 3H), 6.39 (s, 2H), 6.92 (dd, J = 2,5 and 8.7 Hz, 1 H), 7 11-7.13 (m, 2H), 7.31 (dd, J = 0.5 and 8.7 Hz, H), 7.39 (dd, J = 1.1 and 5.1 Hz, 1 H), 7.52 (dd, J = 1.1 and 3.7 Hz, 1 H), 8.54 ppm (br s, 1 H, it disappears after treatment with D₂0). IR: v 3301 cm^-1. Anal. (C22H21NO4S2 (427.54)) C, H, N, S.

Example 1.4: 2-(Pyridin-4-yl)-3-[(3',4',5'-trimethoxyphenyl) thio]-1H-indole (41) (RS3301).

It is obtained as compound 16 from 2-(pyridin-4-yl)-1 /-/-indole. Yield 60%, melting point 215-218°C (after ethanol crystallization). ¹H NMR (DMSO-d₆): δ 3.53 (s, 6H), 3.57 (s, 3H), 6.32 (s, 2H), 7.14-7.18 (m, 1 H), 7.27-7.31 (m, 1 H), 7.53-7.57 (m, 2H), 7.92 (dd, J = 1.7 and 4.5 Hz, 2H), 8.71 (dd, J = 1.6 and 4.5 Hz, 2H), 12.36 ppm (br s, 1H, it disappears after treatment with D2O). IR: y 3320 cm^"1. Anal. (C22H20N2O3S (392.47)) C, H, N, S.

Example 1.5: [2-(Thiophene-2-yl)-1W-indole-3-yl]-(3',4',5'- trimethoxyphenyl)methanone (26) (RS3223).

A mixture of 2-(thiophene-2-yl)-1H-indole (0.25 g, 0.00125 mol), 3,4,5-trimethoxybenzoyl chloride (0.29 g, 0.00125 mol) and anhydrous aluminium chloride (0.17 g, 0.00125 mol) in anhydrous 1 ,2- dichloroethane (2 mL) is microwave irradiated (150 W, closed vessel mode, Pmax = 250 PSI), resulting in a temperature increase from 25°C to 110°C in 1 min and keeping for 2 min. After cooling the reaction is blocked by pouring in 1 N HCI acidified ground ice and extracted with chloroform. The organic layer is washed with water and dried. After solvent removal the obtained residue is purified by column chromatography (silica gel, ethyl acetate: n-hexane 3:7 as eluent) to give 26 (0.17 g, 34%), melting point 155-159°C (after ethanol crystallization). H NMR (DMSO-d₆): δ 3.75 (s, 6H), 3.88 (s, 3H), 6.96- 6.98 (m, 1 H), 7.08 (s, 2H), 7.20-7.24 (m, 2H), 7.30-7.34 (m, 2H), 7.45 (d, J = 8.0 Hz, 1 H), 7.80 (d, J = 7.8 Hz, 1H), 8.73 ppm (br s, 1H, it disappears after treatment with D₂0). IR: v 1570, 3214 cm^"1. Anal. (C₂₂H₁₉N0₄S (393.46)) C, H, N, S.

Example 1.6: 2-(Thiophene-2-yl)-3-(3',4',5'-trimethoxybenzyl)- 1H-indole (27) ( S3250).

A mixture of [2-(thiophene-2-yl)-1H-indol-3-yl]-(3',4',5'-trimethoxy phenyl)methanone (26) (0.1 g, 0.00025 mol) and sodium borohydride (0.1 g, 0.0025 mol) in ethanol (10 mL) is reflux heated for 2.5 h. After cooling the mixture is diluted wit water and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the obtained residue is purified by column chromatography (silica gel, ethyl acetate: n-hexane 3:7 as eluent) to give 26 (0.02 g, 21%), melting point 43-47°C (after ethanol crystallization). ¹H NMR (CDCI₃): δ 3.73 (s, 6H), 3.80 (s, 3H), 4.27 (s, 2H), 6.47 (s, 2H), 7.07-7.13 (m, 2H), 7.19- 7.23 (m, 2H), 7.35-7.39 (m, 2H), 7.49 (d, J = 7.9 Hz, 1 H), 8.17 ppm (br s, 1 H, it disappears after treatment with D2O). IR: v 3340 cm^"1. Anal. (C22H21 NO3S (379.47)) C, H, N, S.

Example 1.7: 2-(2-Nitrophenyl)-1-[1-(phenylsulfonil)-1H- pyrrol-3-yl]ethan-1-one.

Oxalyl chloride (2.46 g, 1.64 mL, 0.019 mol) and a catalytic amount of DMF are added to 2-nitrophenylacetic acid (3.52 g, 0.019 mol) in anhydrous 1 ,2-dichloroethane (88 mL) under argon. After 30 min, 1-(phenylsulfonil)-1H-pyrrole (4.04 g, 0.00019 mol) and anhydrous aluminium chloride (2.59 g, 0.0019 mol) and the reaction mixture is stirred at 0°C for 25 min. After cooling the reaction is blocked by pouring in 1 N HCI acidified ground ice and extracted with chloroform. The organic layer is washed with water and dried. After solvent removal the obtained residue is purified by column chromatography (silica gel, acetone:n-hexane 1 :2 as eluent) to give 2-(2-nitrophenyl)-1-[1- (phenylsulfonil)-1H-pyrrol-3-yl]ethan-1-one (0.7 g, 10%), melting point 135°C (after ethanol crystallization). ¹H NMR (DMSO-d₆): δ 4.45 (s, 2H), 6.41-6.43 (m, 1 H), 7.25-7.30 (m, 2H), 7.45-7.60 (m, 5H), 7.85-7.86 (m, 1 H), 7.90-7.92 (m, 2H), 8.06 ppm (dd, J = 1.2 and 8.18 Hz, 1 H). IR: v 1680 cm-¹. Anal. (C₁₈H₁₄N₂0₅S (370.38)) C, H, N, S.

Example 1.8: 2-[1-(Phenylsulfonil)-1H-pyrrol-3-yl]-1H-indole. A mixture of 2-(2-nitrophenyl)-1-[1-(phenylsulfonil)-1H-pyrrol-3- yl]ethan-1-one (0.25 g, 0.0007 mol) and iron metal powder (0.27 g, 0.0049 ga) in glacial acetic acid (8 ml_) is heated at 60°C overnight. After cooling the mixture is diluted wit water under stirring and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the solid residue is purified by column chromatography (silica gel, ethyl acetate: n-hexane 3:7 as eluent) to give 2-[1-(phenylsulfonil)-1/-/-pyrrol-3-yl]-1H-indole (0.7 g, 10%), melting point 180-185°C (after ethanol crystallization). H NMR (DMSO-d₆): δ 6.66 (δ, J = 1.4 Hz, 1 H), 6.83-6.85 (m, 1 H), 6.93-6.97 (m, H), 7.02- 7.07 (m, 1 H), 7.31 (d, J = 8.0 Hz, 1 H), 7.44-7.46 (m, 2H), 7.65-7.69 (m, 2H), 7.74-7.79 (m, 1 H), 7.84 (t, J = 1 ,9 Hz, 1 H), 7.98-8.00 (m, 2H), 11.30 ppm (br s,1 H, it disappears after treatment with D2O). IR: v 3407 cm^"1. Anal. (Ci₈H₁₄N₂02S (322.38)) C, H, N, S.

Example 1.9: 1-[1-(Furan-2-yl)ethyliden-1-yl]-2-phenyl hydrazine.

A mixture of phenyl hydrazine hydrochloride (3.91 g, 0.027 mol), 2-acetylthiophene (2.0 g, 1.82 imL, 0.018 mol) and anhydrous sodium acetate (4.9 g, 0.0.036 mol) in ethanol (25 ml_) is treated by microwave irradiation at 250 W in open vessel. The temperature is increased from 25°C to 100°C in 2 min and maintained for 5 min under stirring and then cooled. After cooled at room temperature the reaction mixture is filtered to give 1-[1-(furan-2-yl)ethyliden-1-yl]-2-phenylhydrazine as orange solid (2.9 g, 80%) that is stored at -20°C. ¹H NMR (CDCI3): δ 2.20 (s, 3H), 6.45-6.46 (m, 1 H), 6.63 (d, J = 3.3 Hz, 1 H), 6.89 (t, J = 6.8 Hz, 1 H, it disappears after treatment with D₂0), 7.16 (m, 2H), 7.27-7.31 (m, 3H), 7.46 ppm (m, 1 H). IR: v 1599, 1638, 3251 cm^"1. Anal. (C12H12N2O (200.24)) C, H, N.

Example 1.10: 2-(Furan-2-yl)-1H-indole.

1-[1-(Furan-2-yl)etiliden-1-yl]-2-phenylhydrazine (1.18 g, 0.006 mol) is portion wise added to PPA (10 g) pre-heated at 110°C and the reaction mixture is maintained at the same temperature for 1 hour. After reaction blocking by pouring in ground ice the mixture is stirred at 25°C overnight, then made basic using saturated potassium carbonate aqueous solution and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the solid residue is purified by column chromatography (alumina, acetone: n-hexane 1 :5 as eluent) to give 2-(furan-2-yl)-1 /-/-indole (0.6 g, 55%), melting point 123- 125°C (after ethanol crystallization). [2-(Furan-2-yl)-1 H-indole is prepared according to a different procedure, i.e.: Kraus, G. A.; Guo, H. One-Pot Synthesis of 2-substituted indoles from 2-aminobenzyl phosphonium salts. A formal total synthesis of arcyriacyanin A. Org. Lett. 2008, 10, 3061-3063, melting point 120-123°C].

Example 1.11 : 2-(Furan-3-yl)-1H-indole.

A mixture of 2-iodo-1 /-/-indole (0.25 g, 0.001 mol) [2-iodo-1 H- indole is prepared according to literature: Bergman, J.; Venemalm, L. Efficient synthesis of 2-chloro-, 2-bromo-, and 2-iodoindole. J. Org. Chem. 1992, 57, 2495-2497], furan-3-boronic acid pinacol ester (0.26 g, 0.00134 mol), palladium (II) acetate (10%, 0.03 g, 0.000134 mol) and potassium carbonate (0.18 g, 0.00134 mol) in 1-methyl-2-pyrrolidone (2.3 ml_) containing 0.18 mL of water is treated by microwave irradiation at 200 W in closed vessel at 110°C per 15 min (temperature ramp from 25°C to 1 10°C in 2 minutes). After cooling the mixture is diluted with water under stirring and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the solid residue is purified by column chromatography (silica gel, ethyl acetate: n- hexane 1 :5 as eluent) to give 2-(furan-3-yl)-1 /-/-indole (0.18 g, 95%), melting point 145-148°C (after ethanol crystallization). ¹H NMR (CDCI₃): 5 6.61-6.62 (m, 1 H), 6.70-6.71 (m, 1 H), 7.09-7.19 (m, 2H), 7.36 (dd, J = 0.87 and 8.0 Hz, 1 H), 7.50-7.52 (m, 1 H), 7.58-7.60 (m, 1 H), 7.75 (m, 1 H), 8.10 ppm (br s, 1 H, it disappears after treatment with D₂O). IR: v 3417 cm^"1. Anal. (C₁₂H₉NO (183.21)) C, H, N.

Example 1.12: 2-Bromo-1 -(thiophene-2-yl)ethan-1-one.

A solution of bromine (2.53 g, 0.82 mL, 0.0158 mol) in dichloromethane (8 mL) is drop wise added to a solution of 2- acethylthiophene (2.0 g, 1.71 ml_, 0.0158 mol) in same solvent (10 ml_). The reaction mixture is stirred at 25°C per 1 hour and then neutralized with sodium hydrogen carbonate saturated aqueous solution. The organic layer is washed with water and dried. After solvent removal the solid residue is purified by column chromatography (silica gel, ethyl acetate: n-hexane 5:95 as eluent) to give 2-bromo-1-(thiophen-2- yl)ethan-1-one as an oil (2.60 g, 80%). [Alternative preparation of 1- (thiophen-2-yl)ethan-1-one has been reported recently: Ostrowski, T.; Golankiewicz, B.; De Clercq, E.; Andrei, G.; Snoeck, R. Synthesis and anti-VZV activity of 6-heteroaryl derivatives of tricyclic acyclovir and 9- {[cis-1 \2'-bis(hydroxymethyl)cycloprop-1 '-yl]methyl}guanine analogues. Eur. J. Med. Chem. 2009, 44, 3313-3317].

Example 1.13: 2-(Thiophen-2-yl)-1H-indole.

A mixture of 2-bromo-1-(thiophen-2-yl)ethan-1-one (0.5 g, 0.0024 mol) and aniline (0.4 g, 0.4 ml_, 0.0041 mol) is maintained at 25°C under occasional stirring for 3 hours. After the addition of a catalytic amount of DMF, the reaction mixture is microwave irradiated at 150 W (closed vessel mode, Pmax = 250 PSI). The temperature is increased from 25°C to 100°C under stirring in 1 min, then the temperature is maintained for one further minute. After cooling the mixture is diluted with 1 N HCI and extracted with ethyl acetate. The organic layer is washed with water and dried. After solvent removal the solid residue is purified by column chromatography (silica gel, ethyl acetate: n-hexane 3:7 as eluent) to give 2-(thiophen-2-yl)-1H-indole (0.19 g, 40%), melting point 175-180°C (after cyclohexane crystallization). [2-(Thiophen-2-yl)- 1 /-/-indole is prepared according to a different procedure according to: Hudkins, R. L; Diebold, J. L; Marsh, F. D. Synthesis of 2-aryl- and 2- vinyl-1 /-/-indoles via palladium-catalyzed cross-coupling of aryl and vinyl halides with 1-carboxy-2-(tributylstannyl)indole. J. Org. Chem. 1995, 60, 6218-6220, melting point 167-168°C (da hexane)].

Example 1.14: 2-bromo-1-(thiophen-3-yl)ethan-1-one.

It is synthesised as for 2-bromo-1-(thiophen-2-yl)ethan-1-one from 3-acethylthiophene. Yield 57 %, melting point mp 60-65°C (after ethanol crystallization). ¹H NMR (CDCI₃): δ 4.34 (s, 2H), 7.35-737 (m, 1 H), 7.57 (dd, J = 1.3 and 5.1 Hz, 1 H), 8.17-8.18 ppm (m, 1 H). IR:v 1672 cm^"1. Anal. (C₆H₅BrOS (205.07)) C, H, N, S.

Example 1.15: 2-(Thiophen-3-yl)-1 /-/-indole.

It is synthesised as for 2-(thiophen-2-yl)-1 /-/-indole from 2-bromo- 1-(thiophen-3-yl)ethan-1-one. Yield 16%, melting point 210-215°C (after cyclohexane crystallization). [2-(Thiophen-3-yl)-1 /-/-indole has been prepared by a different procedure according to: Fang, Y.-Q.; Lautens, Mark. A highly selective tandem cross-coupling of gem-dihalo-olefins for a modular, efficient synthesis of highly functionalized indoles. J. Org. Chem. 2008, 73, 538-549, melting point 212-214°C].

Example 1.16: 2-(5-Methoxy-2-nitrophenyl)-1-(thiophen-2- yl)ethanol.

TBAF (1 M in THF, 0.83 ml_, 0.00083 mol) is drop wise added to a solution of 5-methoxy-2-nitrobenzyl trimethylsilane (0.2 g, 0.00083 mol) [5-methoxy-2-nitrobenzyl trimethylsilane is prepared according to literature: Bartoli, G.; Bosco, M.; Dalpozzo, R. Todesco. P. E. Functionalization of aromatic systems: a highly chemoselective synthesis of trimethylsilylmethylnitroarenes. J. Org. Chem. 1986, 51, 3694-3696] and thiophen-2-carboxyaldehyde (0.11 g, 0.092 ml_, 0.001 mol) in anhydrous THF (5 ml_) a 25°C under argon. After 15 min the mixture is treated with HCI al 37% (0.83 ml_). The mixture is extracted with diethyl ether, washed with water and dried. After solvent removal solid residue is purified by column chromatography (silica gel, chloroform as eluent) to give 2-(5-methoxy-2-nitrophenyl)-1-(thiophen-2- yhethanol (0.14 g, 61 %) as an oil. ¹H NMR (CDCI₃): δ 2.46 (d, J = 4.19 Hz, 1 H), 3.37 (dd, J = 8.4 and 13.3 Hz, 1 H), 3.55 (dd, J = 4.4 and 13.3 Hz, 1 H), 3.84 (s, 3H), 5.31-5.35 (m, 1 H), 6.77 (d, J = 2.8 Hz, 1 H), 6.86 (dd, J = 2.8 and 9.1 Hz, 1 H), 6.97-7.02 (m, 2H), 7.27-7.30 (m, 1 H), 8.08 ppm (d, J = 9.1 Hz, 1 H). IR: v 3289 cm^"1. Anal. (Ci₃Hi₃N0₄S (279.31)) C, H, N, S. Example 1.17: 2-(5-Methoxy-2-nitrophenyl)-1-(thiophen-2- yl)ethan-1-one.

A solution of 2-(5-methoxy-2-nitrophenyl)-1-(thiophen-2- yl)ethanol (0.74 g, 0.00265 mol) in dichloromethane (1.7 mL) is drop wise added to a suspension of pyridinium chlorochromate (0.87 g, 0.004 mol) in same solvent (6 mL). The reaction mixture is stirred at 25°C for 1.5 h and then filtered. Solid obtained after solvent evaporation is purified by column chromatography (silica gel, chloroform as eluent) to give 2-(5-methoxy-2-nitrophenyl)-1-(thiophen-2-yl)ethan-1-one (0.7 g, 56%), melting point 140-145°C (after ethanol crystallization). ¹H NMR (CDCI₃): δ 3.90 (s, 3H), 4.65 (s, 2H), 6.83 (d, J = 2,7 Hz, 1 H), 6.93 (dd, J = 2.8 and 9.1 Hz, 1 H), 7.14-7.20 (m, 1 H), 7.65-7.69 (m, 1 H), 7.84-7.92 (m, 1 H), 8.22 ppm (d, J = 9.2 Hz, 1 H). IR: v 1654 cm^"1. Anal. (C₁₃H₁₁N0₄S (277.04)) C, H, N, S.

Example 1.18: 5-Methoxy-2-(thiophen-2-yl)-1H-indole.

A mixture of 2-(5-methoxy-2-nitrophenyl)-1-(thiophen-2-yl)ethan- 1-one (1.07 g, 0.0039 mol) and tin (II) chloride bihydrate (4.98 g, 0.019 mol) in ethyl acetate has been heated at reflux for 3 hours. After cooling, the mixture is alkalinized at pH = 9 using potassium carbonate aqueous saturated solution, extracted with ethyl acetate, washed with water and dried. After solvent removal, solid residue is purified by column chromatography (silica gel, dichloromethane as eluent) to give 5-methoxy-2-(thiophen-2-yl)-1H-indole (0.19 g, 21 %), melting point 125°C (after ethanol crystallization). [5-Methoxy-2-(thiophen-2-yl)-1 H- indole has been prepared by a different procedure according to: Caubere, C; Caubere, P.; lanelli, S.; Nardelli, M.; Jamart-Gregoire, B. Aggregative activation and heterocyclic chemistry. I. Complex base promoted arynic cyclization of imines or enamino ketones; regiochemical synthesis of indoles. Tetrahedron 1994, 50, 11903- 1 1920, melting point 124°C].

Example1.19:4-[1-(2-Phenylhydrazone)ethyl]pyridine.

It is obtained as for 1-[1-(furan-2-yl)ethyliden-1-yl]-2-phenyl hydrazine from 4-acetylpyridine. Yield 90%, melting point 143-145°C (after ethanol crystallization). ¹H NMR (CDCI₃): δ 2.22 (s, 3H), 6.92-6.96 (m, 1 H), 7.20-7.34 (m, 4H), 7.58 (br s, 1 H, it disappears after treatment with D₂0), 7.66 (dd, J = 1.6 and 4.6 Hz, 2H), 8.59 ppm (dd, J = 1.6 and 4.6 Hz, 2H). IR: v 3225 cm^"1. Anal. (Ci₃Hi₃N₃ (21 1.26)) C, H, N. [4-[1-(2- Phenylhydrazone)ethyl]pyridine has been prepared by a different procedure according to: Hajipour, A. R.; Mohammadpoor-Baltork, I.; Bigdeli, M. A convenient and mild procedure for the synthesis of hydrazones and semicarbazones from aldehydes or ketones under solvent-free conditions. J. Chem. Res. S. 1999, 9, 570-571 , melting point 135-137°C].

Example 1.20: 2-(Pyridin-4-yl)-1H-indole.

It is obtained as for 2-(furan-2-yl)-1 /-/-indole from 4-[1-(2- phenylhydrazone)ethyl]pyridine. Yield 68%, melting point 210-215°C (after toluene crystallization). [2-(Pyridin-4-yl)- H-indole has been prepared by a different procedure according to: Barolo, S. M.; Lukach, A. E.; Rossi, R. A. Syntheses of 2-Substituted Indoles and Fused Indoles by Photostimulated Reactions of o-lodoanilines with Carbanions by the SRN1 Mechanism. J. Org. Chem. 2003, 68, 2807-2811 , melting point 204-206°C (ethanol)].

Example 1.21 : Elemental analysis of compounds 16, 25, 26, 27, 28 and 41.

Analysis % calculated/found

Compound C H N CI S

66.69 5.30 7.36 8.43

16

66.44 5.19 7.06 - 8.12

63.45 4.82 3.52 16.13

25

63.26 4.70 3.36 - 15.90

67.16 4.87 3.56 - 8.15

26

66.87 4.21 3.34 - 7.86

27 69.63 5.58 3.69 - 8.45 69.32 5.39 3.35 - 8.22

61.80 4.95 3.28 _ 15.00

28

61.51 4.86 2.95 - 14.74

67.33 5.14 7.14 8.17

41

67.11 4.98 6.82 - 7.87

Example 2: Inhibition activity studies on tubulin polymerization and MCF-7 (human breast carcinoma cells) cell growth by the compounds of the invention

Tubulin assembling.

The reaction mixtures contain monosodium glutamate 0,8 M (pH

6,6 with HCI 2M), tubulin 10 μΜ, and various drug concentrations. After pre-incubation at 30°C for 15 minutes, the sample is treated with ice, then GTP 0.4 mM is added. Turbidity development is recorded spectrophotometrically at 350 nm for 20 min at controlled temperature of 30°C [a detailed description of experimental procedures is literature reported according to a paper by Hamel E. co-inventor: Hamel, E. Evaluation of antimitotic agents by quantitative comparisons of their effects on the polymerization of purified tubulin, Cell Biochem. Biophys. 2003, 38, 1-21].

Colchicine binding assay.

Reaction mixtures containing tubulin 1.0 μΜ, [^3H] colchicine 5.0 μΜ and the inhibitor at 1 .0 or 5.0 μΜ are incubated for 10 min at 37°C [experimental details are reported by Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D.G.; Nambu, M.; White, J.D.; Falck, J.R.; Gerwick, W.H.; Day, B.W.; and Hamel, E. Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells, Mol. Pharmacol. 1998, 53, 62-76].

Inhibition of MCF7 cell growth.

The methodology for the determination of MCF-7 cell growth inhibition is described by Verdier-Pinard, P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D.G.; Nambu, M.; White, J.D.; Falck, J.R.; Gerwick, W.H.; Day, B.W.; and Hamel, E. Structure-activity analysis of the interacthion of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells, Mol. Pharmacol. 1998, 53, 62-76.

Molecular Modelling.

Molecular modelling studies are carried out using MacPro dual at 2.66 GHz Xeon with Ubuntu 8. Tubulin structures are obtained from PDB (http://www.rcsb.org/: PDB code 1 SA0) [Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M. Insight into tubulin regulathion from a complex with colchicine and a stathmin- like domain. Nature 2004, 428, 198-202]. Hydrogen atoms are added to the protein using MOE 2007.09 [Molecular Operating Environment (MOE 2007.09). Chemical Computing Group, Inc. Montreal, Quebec, Canada, http://www.chemcomp.com], and minimized maintaining the heavy atoms up to rmsd 0.05 kcal mole^"1 A^"1 gradient. Ligand structures are represented using MOE and minimized using MMFFP94x force field up to rmsd 0.05 kcal mole^"1 A^"1 gradien Docking simulations are carried out using FleX [FlexX 3.0; BioSolvelT GmbH: Sankt Augustin, Germany; http://www.biosolveit.de. Accessed on 1 1/12/2008] with MOE interface. Rmsd of trimetoxyphenyl group of each tested compound is calculated in comparison to DAMA-colchicine A ring [Code "fragment_rmsd.svl" obtained from SVL exchange website: SVL Exchange: An SVL Code Exchange Site for the MOE User Community; Chemical Computing Group, Inc.: Montreal, Canada, 2004; http://svl.chemcomp.com. Accessed on 11/12/2008]. Imagines are generated using Zodiac [Zodiac 0.5b; http://www.zeden.org. Accessed on 11/12/2008].

Table 1 shows the inhibition of tubulin polymerization and MCF-7 cell growth (not metastatic cell line of human breast carcinoma cells) by arylthioindoles and bioisosteric derivatives thereof 1-44 according to the invention. Table 1

inhibition of tubulin polymerization. inhibition of MCF-7 cell growth (human breast carcinoma cells). ^c Colchicine binding inhibition; tubulin =1 μΜ, [³H]colch. = 5 μΜ , inhibitor = 5 μΜ.

Compounds 45, 46 and 47 are already known and are not object of the present invention. Compound 45 has general formula (I) wherein Ri is H, f¾ is 5-Br and X is S [La Regina, G.; Edler, M. C; Brancale, A.; Kandil, S.; Coluccia, A.; Piscitelli, F.; Hamel, E.; De Martino, G.; Matesanz, R.; Diaz, J. F.; Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico, M.; Silvestri, R. New arythioindoles inhibitors of tubulin polymerizathion. 3. Biological evaluation, SAR and molecular modeling studies. J. Med. Chem. 2007, 50, 2865-2874]. Compound 46 is Combretastatin A-4 and compound 47 is colchicine.

For example, the activity of compounds 6, 25-28 and 41 is described in detail.

Inhibition of HeLa cell cycle

The results of flow citometry analysis (FACS) on cell cultures indicate that compounds 16, 25, 26 and 41 (100 nm) have inhibited consistently the cell cycle progression of HeLa cells (uterine cervix epithelium tumour cell line): after 24 hours of treatment, a proportion from 70% to 90% of cell population is stopped with a DNA content correspondent to phase G2/M, indicating an arrest at mitotic division level. Compounds 27 and 28 and reference arylthioindole 45 (previous reference 3) have not shown meaningful effect up 250 nM concentration.

Table 2

Effects on mitotic apparatus.

The effects on mitotic apparatus have been evaluated in cell HeLa culture by immunofluorescence microscopy (IF). Compounds 16, 25, 26 and 41 inhibited consistently the formation of mitotic spindle, by arresting the mitosis at initial stage. This block resulted in a meaningful increment of mitotic index, i.e. [mitotic cells/(mitotic cells + inter-phase cells)] ratio, in cultures treated with compounds 16, 25, 26 and 41 compared to controls. On the contrary, compounds 27 and 45, not active on HeLa cells have not shown mitotic accumulation, while 28 derivative has shown an intermediate level effect (35% of mitotic arrest)

Table 3

Effectively mitosis blocking compounds have evidenced three classes of phenotypes, correspondent to the progressive ability to inhibit tubulin polymerization. Class A phenotvpe: complete absence of tubulin polymerization, no microtubule formation, prometaphase cell arrest (compounds 25, 41 , CSA4 and vinblastine).

Class B phenotvpe: not assembled mitotic spindle, bud of microtubules in arrangement according to defined radial structures named ^'aster' but completely unable to lengthen in order to form spindle fibres (compound 26; 16 A-B class intermediate).

Class C phenotvpe:: microtubule occurrence, but partial de- polymerization resulting in formation of anomalous spindles (no compound reported in example).

Table 4

The concentration increase of compound 16 from 100 nM to 200 nM eliminated the formation of microtubule aster by changing phenotype B to phenotype A.

Reversibility.

The reversibility of the effects has been evaluated for compounds 26 and 41 after exposition of cells to 100 nM concentration for 24 hours, then carrying out the cultures in inhibitor free medium and analyzing the possible re-formation of mitotic structures by means of IF. After 30 minutes, cells treated with 26 have shown microtubule re-growth, indicating a rapidly reversible effect. Following the treatment with 41 , the re-growth has been slower: it was not detectable after 30 minutes, appearing evident after 3 hours, but not so effective as for compound 26. Table 5

Induction of apoptosis during the mitosis.

The compounds resulting in mitotic arrest have also induced a meaningful level of apoptosis in treated cell populations, as detected by FACS analysis (annexin V positive hypodiploid cells; Table 6), indicating a strong correlation between the two events. Table 6 shows the induction of apoptosis in treated cell culture.

Table 6

(1). Apoptotic cells are detected by ACS analysis as annexin V positive cells in the entire cell population

The apoptosis evaluation at level of whole cell population has studied using in vivo videorecording (Time-lapse recording): the compounds have been suitable to induce an absolute block of mitosis initiating cells and to activate the cell death as direct result of mitotic block. In fact, while not treated control cells have completed the cellular division in 1-2 hours, for compound exposed cell cultures, 100% mitosis entering cells have been arrested during all the videorecording time (5-6 hours) and, after an extended mitotic arrest, said cells activated a cell death pathway with an efficiency similar or higher than induced by vinblastine and combretastatin. Table 7 shows the apoptosis induction during the mitotic arrest in Time-lapse recording experiments (the cells has been individually video-recorded from mitosis start for 4-5 hours). Table 7

Cell death has been selectively induced during the mitotic arrest, while for interphase cells the apoptosis level remained low (for A class 3301 compounds) or negligible (for class B compounds) (table 8). This selectivity confirms the possible effectiveness of compounds as therapeutic agents for the elimination of actively proliferating tumour cells, but not of not tumour cells. Table 8 shows the resistance of interphase cells to apoptosis induction. The video-recording of interphase cells demonstrates that the cell death is selectively induced in mitosis blocked cells.

Table 8

Since HeLa cells are functional p53 free, obtained data indicates that the compounds are suitable to activate a p53 not dependent cell death pathway in mitosis blocked cells and therefore are potentially suitable for the treatment also of tumours wherein p53 is mutated or not present.

Molecular modelling studies evidence that compounds of this class bind tubulin in colchicine binding site in similar way as colchicine itself. Figures 1-3 show that these compounds form an hydrogen bond between the indolic NH and Thr179 residue, while trimethoxy phenyl group is located within hydrophobic pocket in proximate contact with Cys249 residue. Simulation obtained results are in agreement with experimental data and represent a valid structural hypothesis for the powerful antitubulinic activity' of this compound class.

Example 3: Study an inhibition activity for ovarian cell growth

A2780, OVCAR-3, A2780CIS cells are from European Collection of Cell Cultures (Salisbury, United Kingdom). The cells are incubated at 37°C in humid atmosphere containing 5% of carbon dioxide. For PCR semiquantitative transcription, total cellular RNA is obtained using a TRI-REAGEN solution (Molecular Research Center, Inc., Cincinnati, OH). PCR products are loaded on 1 % agarose gel and treated with ethidium bromide. Gel images are obtained using Cohu CCD camera and quantified with Phetrix 1 D.

Inhibition of ovarian cells

Potential selectivity of compounds against drug-resistant cells is evaluated analyzing the growth inhibition for an ovarian tumour model displaying high pharmacological sensitivity (A2780wt), cis-platinum resistant (A2780-CIS) counterpart thereof and a cell line derived from cis-platinum resistant patients (the OVCAR-3). Resistance indexes (Rl) are calculated dividing A2780wt IC50 values by cis-platinum resistant cell IC₅₀ values (RI-1 and RI-2 for A2780-CIS and OVCAR-3, respectively), compared with same cis-platinum.

For the model of cis-platinum acquired resistance (R1-I) compounds 16 and 26 show IC50 values lower than original cell line, with clear difference compared to cis-platinum displaying twenty fold higher values. This increase is not detectable for resistance endogenous model, because resistance values (RI-2) for OVCAR-3 cell line are essentially identical to cis-platinum. Table 9 shows the inhibition of cell growth for A2780w, A2780-CIS and OVCAR-3 ovarian tumour cells.

Table 9

Comp. A2780wt A2780-CIS OVCAR-3 Rl-1^a RI-2"

16 +++ +++ ++ + ++

26 +++ +++ ++ + ++

Cisplatin +++ + ++ +++ ++

Inhibition of ADR resistant cells

Compounds 16, 25 and 26 are uniformly more active against NCI/ADR-RES cells (a cell line hyper-expressing P glycoprotein (PGP) than original OVCAR-8 cells. On the contrary, vinorelbine (VRB), vinblastine (VBL) and paclitaxel (PTX) show typical multiresistance difference. Compounds 16, 25 and 26 are always better than vinorelbine, vinblastine and paclitaxel. Table 10 shows the inhibition of OVCAR-8 and NCI/ADR-RES cells by 16, 25 and 26 compounds compared to VRB, VBL, PTX.

Table 10

Comp. OVCAR-8b NCI/ADR-RES ^c

16 ++ +++

25 ++ +++

26 ++ +++

VRB + +

VBL ++ ++

PTX +++ + Inhibition of not transformed cells

A recurrent tumour drug problem is wide toxicity against normal cells. The use of not transformed cells serves in order the selectivity of possible anti-tumour agents against normal cells to be evaluated. Compound 16 shows some anti-proliferative activity against MCF-7 cells higher than not-transformed cell lines, with effects lower than PTX, and higher than combretastatin A4. Table 11 shows the inhibition of HOSMAC, A10, PtK2 and HUVEC cell lines by 16, 2, 5, 11 compounds and PTX.

Table 1

IC₅₀ ± SD (nM)

Compd MCF-7 HAOSMC^a A10^b PtK2° HUVEC"

16 ++ +++ +++ +++ ++

CSA4 ++ + + + +

PTX + + +++ +++ ++

Claims

1) Compounds derived from 3-[(3\4\5'-trimethoxyphenyl)thio]-1 H- indole and bioisosteric derivatives thereof of formula (I):

wherein

Ri is selected from phenyl, heterocycle, benzo fused heterocycle, optionally substituted at one or more positions with the following same or different groups: alkyl, C2-C6 alkenyl, C₂-C6 alkynyl, C1-C6 alkoxy, C2- C6 alkoxycarbonyl, halogen, -CN, -NH₂, -OH, -NO2, or aromatic ring selected from phenyl, phenoxy, benzyl, benzyloxy, naphthyl, naphthyloxy, biphenyl, or heterocycle, said aromatic ring being optionally substituted at one or more positions with one or more of the following same or different groups selected from alkyl, C2-C6 alkenyl, C₂-C6 alkynyl, C1-C6 alkoxy, C2-C6 alkoxycarbonyl, halogen, -CN, -NH2, -OH, -NO2;

R₂ is selected from the following groups, at one or more positions selected from 4, 5, 6 and/or 7 on indole nucleus, the groups being the same or different, hydrogen, alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C2-C6 alkoxycarbonyl, chlorine, bromine, fluorine, iodine, -CN, - NH₂, -OH, -NO2, aromatic ring selected from phenoxy, benzyl, benzyloxy, naphthyl, naphthyloxy, biphenyl, or heterocycle, said aromatic ring optionally independently substituted at para, meta and/or orto, with from one to five same or different substituents selected from alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ alkoxy, C₂-C₆ alkoxycarbonyl, halogen, -CN, -NH₂, -OH, -NO₂;

X is selected from S, SO, SO₂, C=O, CHOH, CH₂, C=OC=O, C=OCH₂, CH₂C=O or CH₂CH₂, with the provision that when X is C=O, S, SO or SO₂, R₂ at position 6 is not methoxy.

2) Compounds according to claim 1 , wherein the heterocycle is a 5 membered heterocycle selected from pyrrole, pyrazole, imidazole, furan, thiophene, oxazole, isooxazole, thiazole or dihydro- or tetrahydro- derivatives thereof or a 6 membered heterocycle selected from pyridine, pirimidine, piridazine, thiazine, oxazine, or dihydro-, tetrahydro-, or hexahydro-derivatives thereof.

3) Compounds according to claim 1, wherein benzo fused heterocycle is selected from indole, benzofuran, benzothiophene, benzimidazole or partially hydrogenated derivatives thereof.

4) Compounds according to anyone of claims from 1 to 3, wherein the alkyl group is selected from Ci-Ci₂ alkyl, (C3-C8 cycloalkyl)Co-C6 alkyl

5) Compounds according to anyone of preceding claims wherein X is S, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4- yl and R₂ is selected from H, 5-halogen, 5-methoxy.

6) Compounds according to claim 5, wherein said compounds are as below:

2-(Thiophen-2-yl)-3-[(3',4^,,5'-trimethoxyphenyl)thio]-1 H-indole (25) (RS3142);

5-Methoxy-2-(thiophen-2-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]- 1 H-indole (28) (RS3552); 2-(Pyridin-4-yl)-3-[(3',4',5'-trimethoxyphenyl)thio]-1 H-indole (41 ) (RS3301).

7) Compound according to anyone of claims from 1 to 4, wherein X is C=O, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophen-2- yl, thiophen-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl and R₂ is selected from H, 5-halogen, 5-methoxy.

8) Compound according to claim 7 which is [2- (thiophen-2-yl)- 1 H-indol-3-yl] - (3\ 4', 5' - trimethoxyphenyl) methanone (26) (RS3223).

9) Compound according to anyone of claims from 1 to 4 wherein X is CH₂, Ri is selected from 1 H-pyrrol-2-yl, 1 H-pyrrol-3-yl, thiophen-2- yl, thiophen-3-yl, furan-2-yl, furan-3-yl, pyridin-2-yl, pyridin-3-yl or pyridin-4-yl, and R₂ is selected from H, 5-halogen, 5-methoxy.

10) Compound according to claim 9 which is 2- (Thiophen-2-yl) - 3- (3", 4', 5' - trimethoxybenzyl)-1 H-indole (27) (RS3250).

1 1) Compounds according to each of preceding claims said compounds having the following R-i, R₂ and X groups:

35 5-Br S

36 5-OMe S

37 H S

38 5-CI S

39 5-Br S

40 H S

42 5-CI S

43 H S

44 H S

12) Compounds as defined in anyone of claims from 1 to 11 for use in medical field.

13) Pharmaceutical composition comprising or consisting of one or more compounds as defined according to anyone of claims from 1 to 11 as active principle in combination with or more pharmaceutically acceptable excipients and/or adjuvants.

14) Compounds as defined in anyone of claims from 1 to 11 or pharmaceutical composition as defined in claim 13 to be used for the treatment of tumours.

15) Compounds or composition according to claim 14, wherein the tumours are selected from the group consisting of mammary carcinoma, cervix carcinoma or ovarian tumour.