MD4349C1 - N-(3-methoxyphenyl)-2-(pyridine-2-ylmethylene)-hydrazinecarbothioamide compound - inhibitor of human melanoma MeW-164 cell proliferation - Google Patents
N-(3-methoxyphenyl)-2-(pyridine-2-ylmethylene)-hydrazinecarbothioamide compound - inhibitor of human melanoma MeW-164 cell proliferation Download PDFInfo
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- MD4349C1 MD4349C1 MDA20140062A MD20140062A MD4349C1 MD 4349 C1 MD4349 C1 MD 4349C1 MD A20140062 A MDA20140062 A MD A20140062A MD 20140062 A MD20140062 A MD 20140062A MD 4349 C1 MD4349 C1 MD 4349C1
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- 201000001441 melanoma Diseases 0.000 title claims abstract description 26
- -1 N-(3-methoxyphenyl)-2-(pyridine-2-ylmethylene)-hydrazinecarbothioamide compound Chemical class 0.000 title claims abstract description 16
- 230000004663 cell proliferation Effects 0.000 title abstract description 3
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- 150000001875 compounds Chemical class 0.000 claims description 9
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Pyridine Compounds (AREA)
Abstract
Description
Invenţia se referă la chimie şi medicină, şi anume la un compus organic din clasa tioamidelor care poate găsi aplicare în medicină în calitate de preparat citostatic la profilaxia şi tratamentul melanomului uman. The invention relates to chemistry and medicine, namely to an organic compound from the thioamide class that can find application in medicine as a cytostatic preparation for the prophylaxis and treatment of human melanoma.
Melanomul uman - unul dintre cele mai periculoase tipuri de cancer uman, o tumoare malignă care se dezvoltă din melanocite, celule producătoare de pigment. Aceste celule sub acţiunea razelor ultraviolete produc colorantul - melanina. Acestea sunt, de asemenea, găsite în cantităţi mari în negi şi aluniţe. Degenerarea melanocitelor apare ca rezultat al mai multor factori: radiaţiile ultraviolete, traume mecanice, arsuri termice sau chimice etc. Melanomul uman este mai periculos decât alte tipuri de cancer de piele, deoarece metastazează repede şi se răspândeşte la alte organe prin vasele sanguine şi ganglionii limfatici. Human melanoma - one of the most dangerous types of human cancer, a malignant tumor that develops from melanocytes, pigment-producing cells. These cells under the action of ultraviolet rays produce the dye - melanin. They are also found in large amounts in warts and moles. Degeneration of melanocytes occurs as a result of several factors: ultraviolet radiation, mechanical trauma, thermal or chemical burns, etc. Human melanoma is more dangerous than other types of skin cancer because it metastasizes quickly and spreads to other organs through blood vessels and lymph nodes.
În practica medicală pentru profilaxia şi tratamentul melanomului uman se foloseşte pe larg doxorubicina - unul din antibioticele din grupa antraciclinelor [1], care are următoarea formulă: In medical practice, doxorubicin is widely used for the prophylaxis and treatment of human melanoma - one of the anthracycline antibiotics [1], which has the following formula:
Mecanismul acţiunii doxorubicinei este bazat pe intercalarea celulelor ADN. Ea posedă un spectru larg de acţiune citostatică şi se aplică în cazul cancerului glandei mamare, prostatei, sarcomului ţesuturilor moi, sarcomului osteogen, tumorii lui Young, cancerului pulmonar, limfosarcomului, cancerului ovarian, cancerului pavimentos de diversă localizare, cancerului vezicii urinare, tumorii lui Williams, cancerului glandei tiroide, melanomului şi diverselor leucoze. The mechanism of action of doxorubicin is based on the intercalation of DNA cells. It possesses a broad spectrum of cytostatic action and is applied in the case of breast cancer, prostate, soft tissue sarcoma, osteogenic sarcoma, Young's tumor, lung cancer, lymphosarcoma, ovarian cancer, squamous cell carcinoma of various locations, bladder cancer, tumor of Williams, thyroid gland cancer, melanoma and various leukemias.
Dezavantajele doxorubicinei constau în faptul că întrebuinţarea ei este limitată, deoarece nu posedă o activitate anticancerigenă înaltă (concentraţia de inhibare semimaximală (IC50) alcătuieşte 4,4…7,5 µmol/L), precum şi în efectele secundare, pe care le cauzează: în procesul tratamentului cu acest preparat se pot dezvolta cardiomiopatia, dureri în regiunea cardiacă, dereglarea ritmului inimii, insuficienţa cardiacă, hipotensiunea. The disadvantages of doxorubicin consist in the fact that its use is limited, because it does not possess a high anticancer activity (the semi-maximal inhibition concentration (IC50) is 4.4...7.5 µmol/L), as well as in the side effects, which it causes: in the process of treatment with this preparation, cardiomyopathy, pain in the heart region, irregular heart rhythm, heart failure, hypotension may develop.
Anterior, compuşii organici din clasa tioamidelor sau derivaţii lor nu se foloseau în calitate de inhibitori ai melanomului uman (analogul structural lipseşte). Previously, organic compounds from the class of thioamides or their derivatives were not used as inhibitors of human melanoma (the structural analogue is missing).
Problema pe care o rezolvă invenţia constă în extinderea arsenalului de inhibitori ai proliferării celulelor MeW-164 ale melanomului uman cu activitate citostatică înaltă. The problem that the invention solves consists in expanding the arsenal of inhibitors of the proliferation of human melanoma MeW-164 cells with high cytostatic activity.
Esenţa invenţiei constă în sinteza compusului N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă cu formula: The essence of the invention consists in the synthesis of the compound N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide with the formula:
, ,
care manifestă proprietatea de inhibare a proliferării celulelor MeW-164 ale melanomului uman. which exhibits the property of inhibiting the proliferation of human melanoma MeW-164 cells.
Compusul dat, proprietăţile lui şi procedeul de obţinere nu sunt descrise în literatură. The given compound, its properties and the method of obtaining it are not described in the literature.
Rezultatul tehnic al invenţiei constă în stabilirea la compusul revendicat a activităţii anticancerigene, care depăşeşte de 8,8…15 ori concentraţia de inhibare semimaximală (IC50) a doxorubicinei. The technical result of the invention consists in establishing the claimed compound's anticancer activity, which exceeds by 8.8...15 times the semi-maximal inhibition concentration (IC50) of doxorubicin.
Rezultatul tehnic al invenţiei este condiţionat de faptul că pentru prima dată în calitate de inhibitor al celulelor MeW-164 ale melanomului uman se propune N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă, care conţine o combinare nouă de legături chimice deja cunoscute. The technical result of the invention is conditioned by the fact that for the first time N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide is proposed as an inhibitor of MeW-164 human melanoma cells, which contains a combination nine chemical bonds already known.
Compusul revendicat se obţine conform următoarei scheme : The claimed compound is obtained according to the following scheme:
Tiosemicarbazona revendicată se sintetizează prin condensarea 3-metoxi-feniltiosemicarbazidei cu 2-formilpiridina la încalzire într-un amestec de dimetilformamidă-etanol (1:3). Sinteza 3-metoxifeniltiosemicarbazidei iniţiale a fost efectuată după metodicile descrise în literatură (Kalinowski D.S., Sharpe P.C., Islam M., Liao Y.T., Lovejoy D.B., Kumar N., Bernhardt P.V., Richardson D. R. Design, synthesis and characterization of novel iron chelators: structure-activity relationships of the 2-benzoylpyridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent antitumor agents. Journal of Medicinal Chemistry, 2007, v. 50, p.3716-3729). Puritatea compusului revendicat a fost confirmată cromatografic prin analiza elementală şi spectrală (IR, 1H-RMN şi 13C-RMN). The claimed thiosemicarbazone is synthesized by the condensation of 3-methoxy-phenylthiosemicarbazide with 2-formylpyridine under heating in a mixture of dimethylformamide-ethanol (1:3). The synthesis of the initial 3-methoxyphenylthiosemicarbazide was carried out according to the methods described in the literature (Kalinowski D.S., Sharpe P.C., Islam M., Liao Y.T., Lovejoy D.B., Kumar N., Bernhardt P.V., Richardson D.R. Design, synthesis and characterization of novel iron chelators: structure -activity relationships of the 2-benzoylpyridine thiosemicarbazone series and their 3-nitrobenzoyl analogues as potent antitumor agents. Journal of Medicinal Chemistry, 2007, v. 50, p.3716-3729). The purity of the claimed compound was confirmed chromatographically by elemental and spectral analysis (IR, 1H-NMR and 13C-NMR).
Exemplu de obţinere a N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidei Example of obtaining N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide
La soluţia obţinută din 0,22 g (2 mmol) 2-formilpiridină în 15 mL etanol se adaugă 0,39 g (2 mmol) 3-metoxifeniltiosemicarbazidă, dizolvată în 5 mL dimetilformamidă, apoi amestecul se încălzeşte pe baie de apă 1 oră. După răcire produsul final se filtrează, se spală pe filtru cu etanol şi se usucă în aer. Se obţin 0,42 g (74%) de produs final. Compoziţia substanţei a fost stabilită în baza rezultatelor analizei elementelor. To the solution obtained from 0.22 g (2 mmol) of 2-formylpyridine in 15 mL of ethanol, add 0.39 g (2 mmol) of 3-methoxyphenylthiosemicarbazide, dissolved in 5 mL of dimethylformamide, then the mixture is heated on a water bath for 1 hour. After cooling, the final product is filtered, washed on the filter with ethanol and dried in air. 0.42 g (74%) of the final product is obtained. The composition of the substance was established based on the results of elemental analysis.
Determinat, %: C - 28,59; H - 4,69; N - 19,31; S - 11,14. Determined, %: C - 28.59; H - 4.69; N - 19.31; S - 11.14.
Calculat pentru compusul C14H15N4OS , %: C - 28,72; H - 4,93; N - 19,57; S - 11,20. Calculated for the compound C14H15N4OS, %: C - 28.72; H - 4.93; N - 19.57; S - 11.20.
P. t. = 142...144°C. P. t. = 142...144°C.
Spectrul 1H-RMN (DMSO, d6), δ, ppm: 3,79 (-CH3), 8,27 (-HC=N-), 6,83...8,55 (-C6H4-, -C5H4N), 1H-NMR spectrum (DMSO, d6), δ, ppm: 3.79 (-CH3), 8.27 (-HC=N-), 6.83...8.55 (-C6H4-, -C5H4N) ,
Spectrul 13C-RMN (DMSO, d6), δ, ppm: 177,20 (C=S); 141,53 (HC=N); 111,51...158,59 (-C6H4-, -C5H4N); 55,57 (CH3). 13C-NMR spectrum (DMSO, d6), δ, ppm: 177.20 (C=S); 141.53 (HC=N); 111.51...158.59 (-C6H4-, -C5H4N); 55.57 (CH 3 ).
Spectrul IR (fig. 1), cm-1: ν(NH) 3246, 3139, 3006, 2986, 2832; ν(C=N) 1608; δ(C-N) 1198, 1153; ν(C=S) 1107; ν(C-N) 970, 948. IR spectrum (fig. 1), cm-1: ν(NH) 3246, 3139, 3006, 2986, 2832; ν(C=N) 1608; δ(C-N) 1198, 1153; ν(C=S) 1107; ν(C-N) 970, 948.
Invenţia se explică cu ajutorul desenelor din fig. 1-6, care reprezintă: The invention is explained with the help of the drawings in fig. 1-6, which represent:
- fig 1, spectrul IR N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidei (domeniul 4000…650 cm-1), - fig 1, the IR spectrum of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide (range 4000...650 cm-1),
- fig 2, microfotografia celulelor melanomului uman MeW-164 cu adaos de 10 µmol de N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă, este inhibată proliferarea a 82% de celule, - fig 2, photomicrograph of MeW-164 human melanoma cells with the addition of 10 µmol of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide, the proliferation of 82% of cells is inhibited,
- fig 3, microfotografia celulelor melanomului uman MeW-164 cu adaos de 1 µmol de N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă, este inhibată proliferarea a 56% de celule, - fig 3, photomicrograph of MeW-164 human melanoma cells with the addition of 1 µmol of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide, the proliferation of 56% of cells is inhibited,
- fig 4, microfotografia celulelor melanomului uman MeW-164 cu adaos de 0,1 µmol de N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă, este inhibată proliferarea a 42% de celule, - fig 4, photomicrograph of MeW-164 human melanoma cells with the addition of 0.1 µmol of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide, the proliferation of 42% of cells is inhibited,
- fig. 5, microfotografia celulelor melanomului uman MeW-164 fără N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă (control), - fig. 5, photomicrograph of MeW-164 human melanoma cells without N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide (control),
- fig 6, IC50 N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidei este egală cu 0,5 µmol, eroarea determinării alcătuieşte ± 5,5%. - fig 6, the IC50 of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazine carbotioamide is equal to 0.5 µmol, the error of determination is ± 5.5%.
Esenţa invenţiei poate fi confirmată prin studierea proprietăţilor anticancerigene ale N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidei folosind metoda MTT. Această metodă este bazată pe capacitatea enzimelor mitocondriale de celule vii de a reduce sarea de tetrazoliu (MTT) (bromură de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazoliu) până la MTT-formazan. Numai celulele vii reduc substratul de MTT galben într-un formazan albastru închis. Celulele moarte şi mediul de cultură nu posedă astfel de abilităţi. Numărul de celule viabile este direct proporţional cu cantitatea de formazan redus, care poate fi determinat spectrofotometric după dizolvarea într-un solvent organic. Pentru experiment au fost utilizate celulele de melanom uman MeW-164 (pasaj 45). Ele au fost derivate de la metastazele melanomului, pe cale chirurgicală de la pacienţii Centrului Oncologic din Varşovia. Celule de melanom uman au fost puse în suspensie 90% EAGLE (MEM) (Biomed-Lublin), care conţine L-glutamină şi ser bovin 10% (FBS, Invitrogen), în baloane de cultură (Cellstar) şi incubate la 37°C, 5% CO2. The essence of the invention can be confirmed by studying the anticancer properties of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazine carbotioamide using the MTT method. This method is based on the ability of living cell mitochondrial enzymes to reduce tetrazolium salt (MTT) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to MTT-formazan. Only living cells reduce the yellow MTT substrate to a dark blue formazan. Dead cells and culture medium do not possess such abilities. The number of viable cells is directly proportional to the amount of reduced formazan, which can be determined spectrophotometrically after dissolution in an organic solvent. Human melanoma cells MeW-164 (passage 45) were used for the experiment. They were derived from melanoma metastases, surgically from patients of the Oncological Center in Warsaw. Human melanoma cells were suspended in 90% EAGLE (MEM) (Biomed-Lublin), containing L-glutamine and 10% bovine serum (FBS, Invitrogen), in culture flasks (Cellstar) and incubated at 37°C , 5% CO2.
Pentru a separa celulele se utilizeaza tripsină (EDTA 200 mg/L, Lonza, Belgia). Suspensia de celule a fost centrifugată (MPW 370 centrifugă) la 1000 rot/min timp de 5 min, apoi supernatantul a fost îndepărtat, fără a agita depozitul celular. Diluţii suplimentare au fost preparate în mediul de cultură celulară (90% RPMI 1640, conţinând 10% FBS ser bovin, Invitrogen) 5x104 celule pe mL. Trypsin (EDTA 200 mg/L, Lonza, Belgium) is used to separate the cells. The cell suspension was centrifuged (MPW 370 centrifuge) at 1000 rpm for 5 min, then the supernatant was removed without shaking the cell deposit. Further dilutions were prepared in cell culture medium (90% RPMI 1640, containing 10% bovine serum FBS, Invitrogen) 5x104 cells per mL.
Pe plăci cu 96 de godeuri pentru microtitrare s-a adăugat fracţia de celule (5x103 celule/100 pL) şi s-a incubat (SANYO incubator cu CO2) timp de 2 ore. După aceasta s-au adăugat 10 µL de probe experimentale în trei concentraţii: 10, 1 şi 0,1 µmol (fiecare concentraţie a fost testată de trei ori). După incubare timp de 24 ore la 37°C, 5% CO2, s-au adăugat 10 mL de reactiv MTT (ATCC) în fiecare godeu al plăcilor şi din nou au fost întoarse în incubator pentru culturi celulare pentru alte 24 de ore. Celulele au fost examinate periodic sub un microscop inversat (Olympus CK40), pentru depistarea momentului apariţiei cristalelor violete de formazan. Pentru a dizolva cristalele în toate godeurile, inclusiv cel de control, a fost adăugat solvent organic (reactiv ATCC). Apoi probele cercetate au fost lăsate într-un loc întunecat, timp de 2…4 ore la temperatura camerei. Determinarea cantităţii de celule de melanom uman MeW-164 a fost efectuată spectrofotometric (spectrofotometru Hamilton HR 7000) cu maximum de absorbţie la 540 nm. The cell fraction (5x103 cells/100 µL) was added to 96-well microtiter plates and incubated (SANYO CO2 incubator) for 2 hours. After that, 10 µL of experimental samples were added in three concentrations: 10, 1 and 0.1 µmol (each concentration was tested three times). After incubation for 24 hours at 37°C, 5% CO2, 10 mL of MTT reagent (ATCC) was added to each well of the plates and again returned to the cell culture incubator for another 24 hours. The cells were periodically examined under an inverted microscope (Olympus CK40), to detect the moment of the appearance of the purple formazan crystals. Organic solvent (ATCC reagent) was added to dissolve the crystals in all wells, including the control. Then the researched samples were left in a dark place for 2...4 hours at room temperature. The determination of the amount of MeW-164 human melanoma cells was performed spectrophotometrically (Hamilton HR 7000 spectrophotometer) with maximum absorption at 540 nm.
Datele experimentale obţinute (fig. 2-5) sunt prezentate în tabel, din care se observă că N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamida inhibă proliferarea celulelor MeW-164 ale melanomului uman în limitele concentraţiilor 0,1…10 µmol/L. La concentraţia 10 µmol/L ea inhibă 82%, la 1 µmol/L - 56% de celule MeW-164 ale melanomului uman, iar la concentraţia de 0,1 µmol/L - 42%. Concentraţia de inhibare semimaximală (IC50), care caracterizează eficacitatea compusului, la N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidă alcătuieşte 0,5 µmol/L (fig.6) şi de 8,8…15 ori depăşeşte IC50 corespunzătoare a doxorubicinei. The experimental data obtained (fig. 2-5) are presented in the table, from which it can be seen that N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbothioamide inhibits the proliferation of human melanoma MeW-164 cells within the limits concentrations 0.1...10 µmol/L. At the concentration of 10 µmol/L it inhibits 82%, at 1 µmol/L - 56% of human melanoma MeW-164 cells, and at the concentration of 0.1 µmol/L - 42%. The semi-maximal inhibition concentration (IC50), which characterizes the effectiveness of the compound, to N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazine carbotioamide is 0.5 µmol/L (fig. 6) and 8.8 ...15 times exceeds the corresponding IC50 of doxorubicin.
Tabel Table
Partea celulelor MeW-164 ale melanomului uman inhibate, % Proportion of human melanoma MeW-164 cells inhibited, %
Concentraţie (µmol/L) Proliferarea celulelor (%) Partea celulelor inhibate (%) Eroarea experimentului (%) IC50 (µmol/L) Numărul experimentului Rezultat mediu I II III Controlul 71 67 65 68 32 2,8 0,5 0,1 48 63 61 58 42 8,0 1 40 48 45 44 56 4,0 10 11 25 18 18 82 7,0 Concentration (µmol/L) Cell Proliferation (%) Fraction of Inhibited Cells (%) Experiment Error (%) IC50 (µmol/L) Experiment Number Mean Result I II III Control 71 67 65 68 32 2.8 0.5 0.1 48 63 61 58 42 8.0 1 40 48 45 44 56 4.0 10 11 25 18 18 82 7.0
Proprietăţile depistate ale N-(3-metoxifenil)-2-(piridin-2-ilmetilen)-hidrazincarbotioamidei prezintă interes pentru medicină din punct de vedere al extinderii arsenalului de inhibitori ai proliferării celulelor MeW-164 ale melanomului uman. The detected properties of N-(3-methoxyphenyl)-2-(pyridin-2-ylmethylene)-hydrazinecarbotioamide are of interest for medicine from the point of view of expanding the arsenal of inhibitors of proliferation of human melanoma MeW-164 cells.
1. Anuszewska E., Chlopkiewicz B., Gruber B., Marczewska J., Priebe W., Skurzak H. Estimation of DNA damage and cytotoxicity of anthracycline analogs in human melanoma cells on early and late passages. Acta Poloniae Pharmaceutica - Drug Research, 2006, vol. 63, nr. 4, p. 321-324 1. Anuszewska E., Chlopkiewicz B., Gruber B., Marczewska J., Priebe W., Skurzak H. Estimation of DNA damage and cytotoxicity of anthracycline analogs in human melanoma cells on early and late passages. Acta Poloniae Pharmaceutica - Drug Research, 2006, vol. 63, no. 4, pp. 321-324
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