WO2005092873A2

WO2005092873A2 - Thiadiazoles for treatment of cancer or neurological disease

Info

Publication number: WO2005092873A2
Application number: PCT/PL2005/000019
Authority: WO
Inventors: Joanna Matysiak; Andrzej Niewiadomy; Wojciech Rzeski
Original assignee: Uniwersytet Marii Curie Sklodowskiej; Akademia Rolnicza
Priority date: 2004-03-26
Filing date: 2005-03-25
Publication date: 2005-10-06
Also published as: PL204750B1; PL366644A1; WO2005092873A3

Abstract

The invention discloses a new compounds as per general formula (I): where -R stands for: alkyl, alkenyl, aryl, substituted aryl (with chlorine, alkyl, alkoxyl, -CF3, CN, -NO2), phenoxyphenyl, or morpholinoalkyl. The new compounds are applicable as antineoplastic agents, and additionally, one of them is particularly applicable in treatment of neurological diseases. The method of obtaining the new compounds was also disclosed.

Description

NEW COMPOUND S, THERAPEUTIC USAGE OF NEW COMPOUND S, AND METHOD OF THEIR DERIVATION

The invention concerns new chemical compounds, their therapeutic usage and the method of obtaining the same.

PRI OR ART 2-Aminothiadiazole and its derivatives are well known compounds. They display multidirectional biological activity, and their antineoplastic and antimycotic properties seem particularly significant. A limited level of activity of the initial system was observed against the following neoplastic lines: S91 melanoma (melanosarcoma), 8110 glioblastoma and 6C3HED lymphosarcoma. Methyl- and acethyl- aminoderivatives display lowered antiproliferative properties. Their antineoplastic action was confirmed against breast adenoid carcinoma, sarcoma 180, and leukaemia PI 534 in mice. The compounds were also active against leukaemia L1210 and 8174 in mice (MM. Ciotti, S.R. Humphreys, J.M. Venditti, N.O. Kaplan, A Goldin, Cancer. Res. 20,1195, 1960). Whereas in the case of the phenyl derivative, no activity in respect to the above lines was observed (D.L. Hill, Cancer. Chemother. Pharmacol. 4, 215-220, 1980). Significant activity of 2,2'-(methylenodiamino)bis-l,3,4-thidiazole was observed against leukaemia LI 210, 6C3HED lymphosarcoma, C1498 meloblastic leukaemia, sarcoma 180, B16 melanoma (melanosarcoma), and to a lesser extent against hepatomas (T. Matsumoto, K. Ootsu, Y. Okada, Cancer Chemother. Rep. 58, 331, 1975). Substitution at C-5 heterocyclic ring usually leads to weakening antineoplastic properties. In the case of 5-hydroxy-2-aminothiadiazole, activity against melanoma and glioblastoma was observed, while the 5-tiolo- and 5-chloroderivatives were inactive against the same (D.L. Hill, Cancer. Chemother. Pharmacol. 4,215-220, 1980).

Clinical application of 2-aminothiadiazole is complicated by hyperuricemia and stomach pains, which necessitates simultaneous action preventing these effects. The compound was, however, allowed to the II stage of clinical tests. The research included patients suffering from lung cancer (J.A. Stewart, C.C. Ackerly, C.F. Myers, R.A. Newman, I.H. Krakoff, Cancer Chemother. Pharmacol. 16, 287-291, 1986), kidney cancer (P.J. Elson, L.K. Kvols, S.E. Vogl, D.J. Glover, R.G. Hahn, D.L. Trump, P.P. Carbone, J.D. Earle, T.E. Davis, Invest. New Drugs 6, 97-103, 1988), aidvanced colon neoplasm (R.F. Asbury, A. Kramar, D.G. Haller, Am. J. Klin. Oncol. 10,380-382, 1987), advanced ovarian neoplasm (R.F. Asbury, J. Wilson, J.A. Blessing, H . Buchsbaum, P.J. DiSaia, Am. J. Klin. Oncol. 9, 334- 336, 1986), mesodermal tumor of the body of the uterus (R.F. Asbury, J.A. Blessing, D. Moore, Am. J. Klin. Oncol. 19, 400-402, 1996) and advanced neoplasm of the large intestine (G.Y. Locker, L. Kilton, J.D. Khandeker, T.E. Lad, R.H. Knop, K. Albain, R. Blough, S. French, A.B. Benson, Invest. New Drugs, 12, 299-301, 1994). Most commonly, the results of clinical tests did not provide the basis for further research due to the lack of sufficiently positive reaction or the occurrence of various side effects. A wide spectrum of "in vitro" activity of N-substituted 5-amino-l,3,4-thiadiazole-2- sulphonamide was recently observed during tests of compounds on a number of various human neoplasm cells. A significant influence of the type of N-substitution was also noted, both qualitative and quantitative, upon the antiproliferative effect (C.T. Supuran, A. Scozzafava, Eur. J. Med. Chem. 35, 867-874, 2000). The complex connections of metal cations and aminothiadiazole have an impact on the antineoplastic characteristics. For iron ions(II) and III with 5-substituted-l,3,4-thiadiazole. antiproliferative properties were observed against the cell line P388 (lymphatic leukaemia in mice). (L. Mishra, M.K. Said, H. Itokawa, K. Takeya, Bioorg. Med. Chem. 3, 1241-1245, 1995). There are a number of methods of 2-amino-5-substituted 1,3,4-thiadiazoles synthesis. Most commonly, it involves acylation of thiosemicarbazides with acidic chlorides, and cyclization of the intermediary product, acetylothiosemicabazide. Dehydrating substances are used, such as sulphuric acid, phosphorus oxychloride, and benzoic or acetic acid chloride. However, the use of such substances is not always possible, and their presence in the reaction environment significantly lowers efficiency and complicates separation. Originating from thiosemocarbazide, in the reaction of 5-nitrofurfurilidene diacetate,

2-amino-5-(5-nitro-2-furyl)-l,3,4-thiadiazole was obtained, as described in the publication by A. Fourmadi, A. Asadipour, M. Mirzaei, J. Karimi, S. Emami, _L Farmaco, 57, 2002, 765-769. tiosemikarbazyd

HCl,NH₄Fe(S0₄)₂ -12 H₂0

2-amino-5 substituted 1,3,4-thiadiazoles were derived in a single-stage reaction from arylthio-, arylsulfonyloacetic or -propionic acid in the reaction with thiosemicarbazides in the environment of polyphosphoric acid. Instead of acids, appropriate nitriles were also used, as described in the publication by S.M. Golovlyov, Y.A. Moskvichev, E.M.A. Alov, D.B. Kobylinsky, N.N. Ermolaev, Chemistry of Heterocyclic Compounds, 37 (9), 2001, 1102-1106.

H₂Ν ICSΝHR' + RCO2H

H₂ΝΝHCSΝHR' + RCN

The publication by A. Zerzouf, A. Keita, M. Salem, E.M. Essassi, M.L. Roumestant, P. Niallefront, Organic and Organometallic Synthesis, t.2, series II, 1999, 435-439 describes condensation of 2-phenylotioacyl-l,3-benzothiazole with nitrilimines which leads to substitution at position 2,2,3,5.

X=C1, Br: R'=C₆H₅, CO₂Et, p-CH₃C₆H₄. Thiadiazoles were derived from hydrazides synthesized from acidic chlorides and phenylhydrazine. Hydrazides were chlorinated with PC1₅, leading to the derivation of hydrazonoyl chlorides. Exposition of the products to potassium thiocyanate in methanol leads to cyclization to a suitable thiadiazole, as described in the publication by B.H. Lee, F.E. Dutton, M.F. Clothier, J.W. Bowman, J.P. Davis, S.S. Johnson, E.M. Thomas, M.R. Zantelle, E.W. Zinser, J.C. McGuire, D.P. Thompson, T.G. Geary, Bioorganic and Medicinal Chemistry Letters 9, 1999, 1727-1732. The method utilizing aldehyde instead of acidic chloride can also be used. In the reaction with phenylhydrazine hydrazone is derived. Its bromation leads to a suitable hydrazonoyl bromide, which is cyclized with the use of potassium thiocyanate.

90 Narious substituted thiadiazoles are also obtained via reactions of regioselective oxidative cyclization or photocyclization from thiosemicarbazones. Furthermore, a Polish patent application No. PL330263A1 describes a new thioacylating reagent sulfinyl-bis(2,4-dihydroxythiobenzoyl), therein referred to as SBKT.

95 DISCLOUSURE OF THE INVENTION New compound as per general formula (I):

where R stands for: alkyl, alkenyl, aryl, substituted aryl (with chlorine, alkyl, alkoxyl, -CF₃, -CN, -NO₂), phenoxyphenyl, or morpholinoalkyl.

100 The following compounds are of particular interest: • 2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-l,3,4-thiadiazole as per formula (IN) therein referred to as FABT, • 2-phenylamino-5-(2,4-dihydroxyphenyl)-l,3,4-thiadiazole; • 2-metylamino-5 -(2,4-dihydroxyphenyl)- 1 , 3 ,4-thiadiazole;

105 • 2-tert-butylamino-5-(2,4-dihydroxyphenyl)-l,3,4-thiadiazole; • 2-(4-phenoxyfenylamino)-5-(2,4-dihydroxyphenyl)-l,3,4-tiadiazole . The new compounds are applicable as antineoplastic agents, and the compound as per formula (IN) is particularly suitable for the treatment of neurological diseases, including epilepsy, Parkinson's disease, and Alzheimer's disease. 110 The method of obtaining 2-aryl(alkyl, alkenyl)amino-5-(2,4-dihydroxyphenyl)-l,3,4- thiadiazole as per general formula (I), where R stands for: alkyl, alkenyl, aryl, substituted aryl (with chlorine, alkyl, alkoxyl, -CF₃, -CΝ, -ΝO₂), phenoxyphenyl, morphinoalkyl is based on a reaction of thiosemicarbazide (general formula 2), where R has the above mentioned meaning, with a thioacylating agent -sulfinyl-bis(2,4-dihydroxythiobenzoyl) as 115 per formula (III), which is performed in elevated temperature in organic solvent. Whereas the method of obtaining a positive compound as per formula (IN) is based on the utilization of 4-fluorophenyl-3 -thiosemicarbazide as thiosemicarbazide. The reactions has positive effects when conducted in the ebullition temperature of the organic solvent. 120 Methyl alcohol is a suitable organic solvent. Specific substituted 2,4-dihydroxyphenyl derivatives constitute a new group of heterocyclic compounds derived from linear systems. The use of the SBKT reagent of proper oxidizing potential ensures the reaction with organic alkalis, and additionally, selective endocyclization. 125 The value of the calculated minimum energies for both isomers indicate limited probability of annular structures (Y) formation. After the reaction of SE with the thiosemicarbazide, the further course of the process depends on the capacity for linear structures stabilization by thiol transition and oxidative bonding of H₂S by surplus electrophilic reagent.

130

Example I New compounds as per general formula (I):

135 where:

Where in compounds No. la - lh R-. stands for:

140 Example H. 2-phenylamino-5-(2,4-dihydroxyphenyl)-l,3,4-thiadiazole (compound la) 0.02 mol of SBKT and 0.01 mol of 4-phenyl-3 -thiosemicarbazide were transferred into 50 ml of methanol and heated until ebullition for 3h. The postreactive mixture was filtrated while hot, and the filtrate condensed until dry. The secreted product of the reaction was 145 rinsed with water and crystallized from methanol (40ml). t.t. 238-240°C. Elemental analysis for the formula:

(285.41) % N calculated: 14.71; found: 14.90 LSIMS (NaOAc) Molecular ion clusters: [M+Na⁺]=308.0 m/z and [M+H]⁺=286.0 m/z (main peak) 150 [ΗJ MR, DMSO-d₆ (IMS), δppm: 10.93; 10.38; 9.92 IR (cm-¹): 3367, 3251, 3198, 3060, 1623, 1601, 1576, 1531, 1501, 1456, 1434, 1410, 1325, 1240, 1218, 1184, 1111, 986, 968, 875, 841, 803.

155 Example m. 2-methylamino-5-(2,4-dihydroxyphenyl)- 1 ,3,4-thiadiazole (compound 4) 0.02 mol of SBKT and 0.01 mol of 4-methyl-3 -thiosemicarbazide were transferred into 50 ml of methanol and heated until ebullition for 3h. The postreactive mixture was filtered while hot, and the filtrate condensed until dry. The secreted product of the reaction was 160 rinsed with water and crystallized from water-diluted (5:3.5) methanol (85 ml), t.t. 224- 226°C (R) Elemental analysis for the formula: C.H9N3O2S (223.25) % N calculated: 18.82; found: 18.39 EI-MS: 165 M^+* (223 m/e) main peak, fragmentations (m/e): 194, 137, 121, 109, 94, 88, 74, 69, 52 [Η]NMR, DMSO-dβ (IMS), δppm: 10.90; 9.95; 9.80; 2.40 IR Ccmr¹): 3387, 1631, 1559, 1477, 1411, 1326, 1270, 1228, 1182, 1137, 1088, 1045, 986, 966, 841. Example IN. 2-(4-fluorophenylamino)-5-(2,4-dihydroksyphenyl) - 1,3,4- thiadiazole (compound lh) 0.02 mol of SBKT and 0.01 mol of 4-(-4-fluorophenyl)-3-thiosemicarbazide were 175 transferred into 50 ml of methanol and heated until ebullition (3h). The postreactive mixture was filtered while hot, and the filtrate was exposed to 100 ml of water. The secreted compound was filtered, rinsed with water and dried. It was crystallized from methanol (70ml). t.t. 279-280°C Analytical characteristics 180 Elemental analysis for the formula: Cι₄Hι₀FΝ₃O₂S (303.34) % N calculated: 13.84; found: 13.76 EI-MS: M^+' (303 m/z), main peak, fragmentations (m/z): 168, 141, 136, 109, 94, 83, 66, 52. [Η]NMR, (DMSO-d₆ , IMS), δ pm): 10.85; 10.27; 9.92 185 IR (cnr¹): 3400, 3259, 3218, 3166, 1629, 1590, 1510, 1476, 1449, 1411, 1325, 1252, 1231, 1183, 1134, 1113, 1052, 1013, 987, 967, 875, 825.

Example V. 2-tert-butyloamino-5-(2,4-dihydroksyphenyl)-l,3,4-tiadiazole (compound 6)

190 0.0075 mol of SBKT and 0.01 mol of 4-tert-butylo-3 -thiosemicarbazide (Lancaster) were transferred into 50 ml methanol and heated until ebullition (3h). The secreted compound was crystallized from methanol (40 ml). 1. 1. 109-111°C. Elemental analysis for the formula:

(265.34): . %N calculated: 15.84 found: 15.61

195 EI-MS : M^+' 265 (m/e), fragmentations (m/e): 250, 209 (100), 136, 74, 57. [Η] NMR (DMSO-d₆, TMS): δppm: 9.475, 1.445-1.399 (9H, CH₃, m). IR (cnr¹): 3433, 3308 (OH+NH), 2920, 2854, 1735, 1665, 1506, 1439, 1368, 1267, 1219, 1163, 1093, 806.

200 Example VI. 2-(4-phenoksyphenylamino)-5-(2,4-dihydroksyphenyl)-l,3,4-tiadiazole (compound 7) 0.0075 mol of SBKT and 0.01 mol of 4-(4-phenoksyphenyl)-3-thiosemikarbazide (Aldrich) transferred into 50 ml of metanol and heated until ebullition (3h). The postreactive mixture 205 was filtered while hot, and the filtrate was exposed to 100 ml of water. The secreted compound was filtered, rinsed with water and dried. It was crystallized from methanol (70ml). t.t.87°C. Elemental analysis for the formula: C20H15N3O3S (377.42) %N . calculated: 11.13 found: 10.88

210 EI-MS : M^+' 377 (m/e), main peak, fragmentations (m/e): 302, 284, 242, 227, 210, 185, 165, 150, 135, 121, 94, 77, 51. [Η] NMR (DMSO-dβ, TMS): δppm: 10.863 (H, 2-COH, s), 10.263 (H, 4-COH, s), 9.897 (H, NH, s). 215 TR (cm-^!): 3198, 3307, 2918, 2851, 1735, 1665, 1583, 1501, 1436, 1465, 1330, 1270, 1220, 1162 C-OH, 1091, 1021, 999, 958, 927, 883.

Comparative example I. Refers to the compound described in Example IN.

220 Neuron culture Neuron cultures were set up from 18-day-old rat fetuses' brains. The brain tissue was dissociated into separate cells with 0.25% solution of trypsin-EDTA. The cell suspension of the density 5 x 10⁵ cell/ml was poured onto poly-L-lysine coated, 96-hole microplates in the quantity of 100μl/hole, with the use of Neurobasal medium + 2% B-27

225 supplement (Life Technologies) with additional 1% antibiotic/antisemiotic solution (Life Technologies). The neurons were cultured for 14 days in the temperature of 37°C and atmosphere of 95% air and 5% CO₂. The culture medium was replaced every 3 days. Neuron identification was performed immunocytochemically by dying the cells towards the . characteristic NSE marker (neuron specific enolase).

230 Determining neuron vitality Neuron vitality was examined on a 14-day culture. In order to determine the neuroprotective effect the study examined the influence of FABT on vitality of neurons under trophic stress (b-27 supplement was removed from the medium) and the influence of stimulating amino acids: glutamate, N-methyl-D-aspartic acid (NMDA) and a-amino-3-

235 hydroxy-5-methyl-4-isoxazolopropionic acid (AMP A). Neuron vitality was determined via the MTT method. MTT Method (with the "Cell proliferation kit III", Boehringer Manheim) The method was developed in order to determine cell proliferation and vitality for

240 the purpose of research on substances with cytotoxic and antiproliferative properties. In metabolically active cells, yellow tertazolium salt MTT is reduced to blue formazan by mitochondrial dehydrogenases. Formazan crystals are insoluble in water, thus they accumulate in cells and in order to dissolve them an organic detergent has to be used, which penetrates membranes simultaneously dissolving the dye. The SDS-HC1 buffer of pH 7.4 is

245 used for that purpose. The concentration of the released dye is measured quantitatively in a 96-hole microplates reader with the wavelength of 570 nm. The intensity of the dye is directly proportional to the number of living cells. MTT solution in PBS in a concentration of 5 mg/ml, was added by 15 μl per each hole on a plastic plate. The plates were incubated for 3 hours in the temperature of 37°C,

250 after that time 100 μl of SDS-HC1 buffer were added to each hole and the plates were left for the night in the temperature of 37°C. The results were read the following day with an E- max reader (Molecular Devices Corporation, Menlo Park, CA, USA). Determining neuroprόtective activity The study of the influence of FABT on neuron cultures in concentrations of 10, 25 and

255 50μM showed a significant statistical increase in cell vitality. In the concentration of 50μM the strongest effect was observed, amounting to 385. (fig. 1) In order to show the neuroprotective properties of the compound, the neuron cultures were exposed to neurodegenerating agents. It was observed that in the concentration of 25μM the compound weakens the action of such neurotoxic agents as trophic stress (serum

260 deprivation - SD) and stimulating amino acids (glutamate, NMDA and AMP A) (fig. 2).

Comparative example π. The compounds derived in Examples II, V and VI were tested for antiproliferative activity. Line A549 cells (lung cancer) were cultured on DMEM:F-12 HAM (2:1) medium. 265 To the culture medium 10% fetal bovine serum (FBS) was added, along with 100 j/ml of penicillin and 100 μG/ml of streptomycin. The culture medium was provided by Sigma company (Sigma, St. Louis, MO, USA). The fetal bovine serum (FBS) was provided by Life Technologies (Life Technologies, Karlsruhe, Germany). The remaining reagents were provided by Sigma company. Initial solutions of lOmM and lOOmM in DMSO were used. Working solutions were prepared by proper dilution of initial solutions in the culture medium. Preparation of cell cultures Cells stored in liquid nitrogen in a tissue bank were thawed in the temperature of 37°C and placed in plastic bottles containing appropriate culture medium. The cells were cultured in the temperature of 37°C in an incubator with a 5% flow of CO₂. After cell multiplication the liquid was poured out, the cells rinsed with PBS (without calcium or magnesium ions) and exposed to 0.25% solution of trypsin + EDTA in order to obtain the required cell suspension for the experiment. Determining the antiproliferative activity of the studied compounds in a cell culture. The prepared cell suspension was poured into a flat-bottom 96-hole microplate (provided by NUNC, Roskilde, Denmark) in culture medium of the density: M0⁴ cell/ml (A549), and volume of μl 100 per hole. After cell adhesion (24h) the liquid was carefully drawn off. Next, the examined substances in various concentration were added in liquid form, with the addition of 10% FBS (100 μl per hole). The microplate cultures were incubated for 96h in the temperature of 37°C and in the atmosphere of 95% air and 5% CO₂. The antiproliferative activity of the examined compounds was determined via the MTT method. The activity of the compounds was determined against human lung cancer cells

(A549) in the concentration of 10 and 50 μM. The results are presented in the table.

(H) (m)

(I) (Y)

295

(IV)

300

NH₂NHCSNH- o.

(V)

Claims

305 CLAIM S

1. New compound as per general formula (I):

310 where R stands for: alkyl, alkenyl, aryl, substituted aryl (with chlorine, alkyl, alkoxyl, -CF₃, -CN, -NO₂), phenoxyphenyl, or morpholinoalkyl.

2. The compound as claimed in claim 1, characterized by the fact, that it is constituted by 2-(4-fluorophenylamino)-5-(2,4-dihydroxyphenyl)-l,3,4-thiadiazole as per formula (IV).

315 3. The use of the compound as per formula (IV) for treatment of neurological diseases.

4. The use of the compound as per formula (IV) for treatment of epilepsy, Parkinson's disease or Alzheimer's disease.

5. The use of 2-phenylamiono-5(2,4-dihydroxyphenyl)-l,3,4-thiadiazole as an antineoplastic agent.

320 6. The use of 2-tert-butylamino-5-(2,4-dihydroksyphenyl)-l,3,4-tiadiazole as an antineoplastic agent.

7. The use of2-(4-phenoxyphenylamino)-5-(2,4-dihydroksyphenyl)-l,3,4-tiadiazole as an antineoplastic agent.

8. The method of obtaining 2-aryl(alkyl, alkenyl)amino-5-(2,4-dihydroxyphenyl)-l,3,4- 325 thiadiazoles as per general formula (I), where R stands for: alkyl, alkenyl, aryl, substituted aryl (with chlorine, alkyl, alkoxyl, -CF₃, -CN, -NO₂), phenoxyphenyl, or morpholinoalkyl, characterized by the reaction of thiosemicarbazyd as per general formula (II), where R has the above mentioned meaning, with a thioacylating agent -sulfinyl-bis(2,4- dihydroxythiobenzoyl) as per formula (III), which is performed in elevated temperature in 330 organic solvent.

9. The method of obtaining 2-(4-fluorophenylamino)-5-(2,4-dihydroxyphenyl)-l,3,4- thiadiazole as per general formula (IV), claimed in claim 8, characterized by the fact, that 4- fluorophenyl-3 -thiosemicarbazide as per general formula (V) is used as thiosemicarbazide.

10. The method claimed in claim 8 or 9, characterized by the use of methyl alcohol as the 335 organic solvent.

11. The method claimed in claim 8 or 9 characterized by the fact, that the reaction in conducted in the ebullition temperature of the organic solvent.