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Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents

Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine... Acta Pharm. 60 (2010) 311­323 10.2478/v10007-010-0030-y Original research paper AMEEN ALI ABU-HASHEM1 MOHAMED FATHY EL-SHEHRY2* FARID ABD-ELRAHEEM BADRIA3 1 Photochemistry Department (Heterocyclic Unit), National Research Center, 12622 Dokki Giza, Egypt Green Chemistry Department National Research Center 12622 Dokki, Giza, Egypt Department of Pharmacognosy Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt 2-Amino-5-acetyl-4-methyl-thiophene-3-carboxylic acid ethyl ester (1) and 5-acetyl-2-amino-4-methylthiophene-3-carbohydrazide (2) were synthesized and used as starting materials for the synthesis of new series of 1-(5-amino-4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3a), 1-(5-amino-4-(4-chloro-3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3b), 1-(4-methyl-2-amino-5-acetylthiophene-3-carbonyl)pyrazolidine-3,5-dione (4), (Z)-N'-(4 -methyl-2-amino-5-acetylthiophene-3-carbonyl) formohydrazonic acid (5a), (Z)-ethyl-N'-4-methyl-2-amino-5-acetylthiophene-3-carbonylformo hydrazonate (5b), 6-acetyl-3-amino-2,5-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (8), 5-methyl-3-amino-2-mercapto-6-acetylthieno [2,3-d]pyrimidin-4(3H)-one (10) and 5-methyl-6-acetyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (12) as potential antioxidant and antitumor agents. Pharmacological tests showed that compounds 6a, 6b, 8, 10 and 12 exhibited significant antitumor and antioxidant activity. Keywords: thienopyrazole, thieno[2,3-d]pyrimidines, thiophenecarbohydrazide, antioxidant activity, bleomycin-dependent DNA damage, antitumor activity Accepted July 16, 2010 The ultimate position of substituted 2-aminothiophenes in the field of drug design and synthesis of pharmaceuticals comes from their advantageous properties. The thiophene ring is bioisosteric replacement for the phenyl group broadly present in active drugs; the thiophene core exists in many natural and synthetic pharmaceuticals (1, 2). In the present work, ethyl-2-aminothiophene-3-carboxylic was expressed as a synthone for many new condensed heterocyclic systems viz. thieno[2,3-d]pyrimidine, thienylthiourea, thienylacetamide, thienylcarbohydrazide. Fused and polyfunctional substituted thiophene play an important role, especially when linked to a pyrimidine ring; thienopyri- * Correspondence; e-mail: moh_elshehry2000@yahoo.com A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. midine derivatives possess pharmacological properties as A1 adenosine receptor agonists (3, 4), phosphodiesterase inhibitors and many receptor antagonists (5, 6). Various thieno [2,3-d] pyrimidine derivatives show pronounced antitumor (7) and radioprotective activities (8), acts as immunomodulators (9) and were used for prophylaxis and therapy of cerebral ischemia (10), malaria (11), tuberculosis (12), Alzheimer's disease (13), Parkinson's disease (14), and other diseases (15, 16). Design and synthesis of the novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents were explored. EXPERIMENTAL All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Japan). Elemental analyses were performed at Vario EL (Elementar, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University. The IR spectra (KBr disk) were recorded using a Perkin-Elmer 1650 spectrometer (USA). 1H NMR spectra were determined using Jeol 270 MHz and Jeol JMS-AX 500 MHz (Jeol, Japan) with internal standard MeSi4. Mass spectra were recorded on an EI Ms-QP 1000 EX (Shimadzu) at 70 eV. Pharmacological evaluations were done in pharmacological unit, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt. DNA (calf thymus type 1), bleomycin sulfate, butylated hydroxyanisole (BHA), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA) and ascorbic acid were obtained from Sigma (USA). 2,2'-Azo-bis-(2-amidinopropane) dihydrochloride (AAPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) were purchased from Wako Co. (USA). Syntheses Ethyl-4-methyl-2-amino-5-acetylthiophene-3-carboxylate (1). ­ A mixture of acetylacetone (0.01 mol), ethyl cyanoacetate (0.01 mol), sulfur (0.01 mol) and diethylamine (0.01 mol) was heated at 70 °C under stirring in absolute ethanol (20 mL) for 4 h, then the mixture was left for 24 h at 0 °C. The solid formed was collected by filtration, washed with ethanol (20 mL), dried and crystallized from absolute ethanol. 5-Acetyl-2-amino-4-methylthiophene-3-carbohydrazide (2). ­ A suspension of dry compound 1 (0.01 mol) and hydrazine hydrate (5 mL) and (30 mL) absolute ethanol was stirred under gentle reflux. The solid dissolved within 10 min with copious evolution of hydrogen sulfide to form a clear solution. After 30 min, the solid product started separating out; heating was continued for 8 h. The reaction mixture was then allowed to cool to room temperature. The solid was filtered off, washed with ethanol, dried and crystallized from dioxane. 1-(5-Amino-4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3a) and 1-(5-amino-4-(4-chloro-3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3b). ­ A mixture of compound 2 (0.01 mol) and 2,4-diketone, namely, pentan-2,4-dione A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. or 3-chloropentan-2,4-dione (0.01 mol) in absolute ethanol (30 mL), was stirred under reflux for 12 h. The reaction mixture was allowed to cool to 0 °C for 24 h, the solid precipitate was filtered off, dried and crystallized from ethanol to produce 3a or 3b in high yields. 1-(4-Methyl-2-amino-5-acetylthiophene-3-carbonyl)pyrazolidine-3,5-dione (4). ­ A solution of compound 2 (0.01 mol) and freshly distilled diethylmalonate (0.01 mol) in sodium ethoxide was heated under reflux with stirring for 5 h. The solvent was evaporated under reduced pressure and the crude product was acidified with 10 % hydrochloric acid. The solid formed was filtered off, washed with cold water, and crystallized from ethanol. (Z)-N'-(4-methyl-2-amino-5-acetylthiophene-3-carbonyl) formohydrazonic acid (5a). ­ A mixture of compound 2 (0.01 mol) and formic acid (10 mL) was heated under reflux for 6 h. The reaction mixture was allowed to cool to room temperature and poured onto water (100 mL). The solid formed was collected by filtration, dried, and crystallized from benzene. (Z)-ethyl-N'-4-methyl-2-amino-5-acetylthiophene-3-carbonylformo hydrazonate (5b). ­ A mixture of compound 2 (0.01 mol) and triethylorthoformate (20 mL) was heated under reflux with stirring for 2 h. The reaction mixture was filtered hot and the filtrate was left to cool to room temperature. The solid formed was filtered off, dried and crystallized from ethanol. Synthesis of 1-(5-amino-3-methyl-4-(1,3,4-oxadiazol-2-yl) thiophen-2-yl) ethanone (6a) and 1-(5-amino-3-methyl-4-(1,3,4-oxathiadiazol-2-yl) thiophen-2-yl) ethanone (6b). ­ A mixture of compounds 5a or 5b (0.005 mol) and phosphorus pentoxide (2 g) or phosphorus pentasulfide (4 g) in dry xylene (30 mL) was heated under reflux for 6­12 h. The solid separated upon cooling was filtered off, crystallized from ethanol to produce 6a and 6b, respectively. Ethyl-2-acetamido-4-methyl-5-acetylthiophene-3-carboxylate (7). ­ A mixture of compound 1 (0.01 mol) and acetic anhydride (30 mL) was heated under reflux for 3 h. The reaction mixture was allowed to cool to room temperature and the solid formed was collected by filtration, dried, and crystallized from ethanol. 6-Acetyl-3-amino-2,5-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (8). ­ A mixture of 7 (0.01 mol) and hydrazine hydrate (10 mL) in ethanol (30 mL) was heated under reflux for 2 h. The reaction mixture was allowed to cool to room temperature and poured into water (100 mL). The solid formed was collected by filtration, dried and crystallized from ethanol as yellow powder. Ethyl-4-methyl-5-acetyl-2-(methylthiocarbonothioylamino) thiophene-3-carboxylate (9). ­ To a vigorously stirred solution of 1 (0.02 mol) in dimethylsulfoxide DMSO (10 mL) at room temperature, carbon disulfide (0.026 mol) and aqueous sodium hydroxide were added simultaneously over 30 min; stirring was continued for further 30 min. Dimethylsulfate (0.02 mol) was added dropwise to the reaction mixture under stirring at 5­10 °C. It was stirred for another 2 h and poured into ice-water. The solid obtained was filtered off, dried and recrystallized from ethanol. A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. 5-Methyl-3-amino-2-mercapto-6-acetylthieno[2,3-d]pyrimidin-4(3H)-one (10). ­ Method A: A solution of 9 (0.01 mol) in ethanol (30 mL) and hydrazine hydrate (0.01 mol) was added and refluxed on a water bath until the methyl mercaptan evolution ceased after 8 h. After cooling, the solid obtained was filtered off, dried and recrystallized from ethanol/ acetone mixture. Method B: A solution of 2 (0.01 mol) in ethanol (30 mL) and aqueous potassium hydroxide (0.01 mol) was refluxed for 30 min. After cooling to room temperature, carbon disulfide (1 mL) was added, refluxed for 2-3 hours and poured into ice-water. The solid obtained was filtered off, dried and recrystallized from ethanol. Ethyl-4-methyl-5-acetyl-2-thioureidothiophene-3-carboxylate (11). ­ A mixture of 1 (0.01 mol) and 10 % HCl (10 mL) was refluxed with potassium thiocyanate (0.015 mol) for 4 h. The reaction mixture was allowed to cool to room temperature. The solid formed was collected by filtration, washed with water, dried and crystallized from dioxane. 5-Methyl-6-acetyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (12). ­ A solution of 11 (0.01 mol) in ethanolic sodium ethoxide (0.23 g of sodium metal in 30 mL ethanol) was stirred under reflux for 6 h. After cooling, the reaction mixture was neutralized with cool 10 % HCl and the solid formed was collected by filtration, washed with water, dried and then crystallized from dimethylformamide. Pharmacological screening Ehrlich cells. ­ Ehrlich cells (Ehrlich ascites carcinoma, EAC) were derived from ascetic fluid from diseased mice (the cells were purchased from the National Cancer institute, Cairo, Egypt). Antioxidant activity screening via erythrocyte hemolysis. ­ The blood was obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes were separated from plasma and the buffy coat was washed three times with 10 volumes of 0.15 mol L­1 NaCl. During the last wash, the erythrocytes were centrifuged at 2500 rpm for 10 min to obtain a constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system (17). A 10 % suspension of erythrocytes in phosphate buffered saline pH 7.4 (PBS) was added to the same volume of 200 mmol L­1 AAPH solution in PBS containing samples to be tested (50 mL of 2 mmol L­1 solution). The reaction mixture was shaken gently while being incubated at 37 °C for 2 h. The reaction mixture was then removed, diluted with eight volumes of PBS and centrifuged at 1500´g for 10 min. The absorbance of the supernatant was read at 540 nm. Similarly, the reaction mixture was treated with 8 volumes of distilled water to achieve complete hemolysis, and the absorbance of the supernatant obtained after centrifugation was measured at 540 nm. The data for hemolysis percentage was expressed as mean ± standard deviation. L-ascorbic acid was used as a positive control. Antioxidant activity screening via ABTS method. ­ For each of the investigated compounds, 2 mL of ABTS solution (60 mmol L­1) was added to 3 mL MnO2 solution (g = 25 mg mL­1), all prepared in aqueous phosphate buffer (pH 7, 0.1 mol L­1). The mixture was shaken, centrifuged, filtered and the absorbance of the resulting green-blue solution (ABTS radical solution) at 734 nm was adjusted to approx. 0.5. Then, 50 mL of 2 mmol L­1) solution of the test compound in spectroscopic grade MeOH/phosphate buffer (1:1) A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. was added. The absorbance was measured and the reduction in color intensity was expressed as inhibition percentage. L-ascorbic acid was used as standard antioxidant (positive control). Blank sample was run without ABTS and using MeOH/phosphate buffer (1:1) instead of a sample. Negative control was run with ABTS and MeOH/phosphate buffer (1:1) instead of test compound (18­20). Bleomycin-dependent DNA damage. ­ The assay was done according to Aeschlach (21) with minor modifications. The reaction mixture (0.5 mL) contained calf thymus DNA (0.5 mg mL­1), bleomycin sulfate (0.05 mg mL­1), MgCl2 (5 mmol L­1), FeCl3 (50 mmol L­1) and the sample to be tested (50 mL of 2 mmol L­1 solution). L-ascorbic acid was used as a positive control. The mixture was incubated at 37 °C for 1 h. The reaction was terminated by addition 0.05 mL EDTA (0.1 mol L­1). The color was developed by adding 0.5 mL TBA (1 %, m/V) and 0.5 mL HCl (25 %, V/V) followed by heating at 37 °C for 15 min. After centrifugation, the extent of DNA damage was measured by the increase in absorbance at 532 nm. Antitumor activity using Ehrlich ascites in vitro assay. ­ Tested compounds were prepared in a concentration of 25 mg mL­1 in DMSO. Ascites fluid from the peritoneal cavity of a diseased mouse (containing Ehrlich cells) was aseptically aspirated. The cells were grown partly floating and partly attached in a suspension culture in RPMI 1640 medium, supplemented with 10 % fetal bovine serum. They were maintained at 37 °C in a humidified atmosphere with 5 % CO2 for 2 h. The viability of the cells was determined by microscopical examination using a hemocytometer and using trypan blue stain (stains only dead cells) (22). RESULTS AND DISCUSSION Chemistry First, aminothiophene (1) was prepared by the reaction of acetylacetone with ethylcyanoacetate and sulfur in the presence of diethylamine following the Gewald procedure (23). 1H NMR spectrum of 1 showed, beside the two signals of ester moiety, a new broad singlet at d 6.66 ppm characteristic of the amino group. Moreover, the mass spectra showed an ion peak m/z 227 [M+], which was in accord with the calculated molecular mass for C10H13NOS. Hydrazide 2 was achieved after refluxing compound 1 with hydrazine hydrate in ethanol. 1H NMR spectrum of 2 showed the absence of the two signals of ester moiety detected in parent 1 along with other protons at their expected locations. Hydrazide 2 reacted with 2,4-diketones, namely, pentane-2,4-dione or 3-chloropentane-2,4-dione to afford 3a and 3b, respectively. The 1H NMR spectrum of 3a revealed new signals at d 2.10 ppm assignable to 2 CH3 groups, d 7.96 ppm for pyrazole proton and d 12.51 ppm for NH2 (as D2O exchangeable). Also, hydrazide 2 reacted with diethylmalonate affording pyrazolidine-3,5-dione derivative 4. The 1H NMR spectrum showed the absence of the hydrazo signal and the presence of new signals at d 2.78 ppm for CH2 group and d 12.32 ppm corresponding to (as a D2O exchangeable NH proton) along with other protons detected in parent 4. A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. O O C 2 H5 O + O N HN(C2H5)2 S R` R S 1 COOC2H5 NH2 CS2 R` R S 10 O N N H NH2 S R R` S 3: a b H3 C N N NH2 X CH3 N 2 H4 H3COC H3COC X R` R S 2 HCOOH or CH(OEt)3 R R` S CONHNH2 NH2 KOH COOEt COOEt C2H5ONa R` R S 4 O N HN NH2 X= H X= Cl CONHN CHOR P 2O 5 NH2 or P2S5 R` R S N X NH2 1-6: R = CH3 R` = COCH3 5: a R = H b R = C 2 H5 6: a X = O b X=S Scheme 1 Treatment of 2 with formic acid or triethylorthoformate afforded 5a and 5b, respectively, in fairly good yields. 1H NMR spectrum of 5a showed signals at d 8.44 ppm for CH proton, and d 11.55 ppm (as a D2O exchangeable OH proton), while the 1H NMR spectrum of 5b showed new signals at d 1.31, 4.32 ppm for CH2CH3 moiety and d 6.67 ppm for CH proton. Moreover, compounds 5a and 5b undergo cyclization when treated with P2O5 and P2S5, to yield thienyloxadiazole (6a) and thienylthiadiazole (6b) derivatives, respectively. The 1H NMR spectrum of 6a and 6b showed signals at d 7.12 ppm characteristic of the oxadiazole proton of 6a and d 7.85 ppm for thiadiazole proton of 6b beside the other expected signals characteristic of methyl, acetyl and amino groups. Hydrazide 2 reacted with carbon disulphide in ethanolic potassium hydroxide to afford thienopyrimidinone derivative (10). 1H NMR spectrum showed signals at d 6.12 ppm for NH2 and d 7.44 ppm for NH proton (as D2O exchangeable) beside the signals characteristic of acetyl and methyl groups; moreover, the mass spectrum revealed an ion peak at m/z 255 [M+], which was in accord with the calculated molecular mass C9H9N3O2S2 (Scheme 1). Compound 1, when treated with acetic anhydride, afforded 2-thienylacetamide derivative (7). Moreover, when compound 7 was allowed to react with hydrazine hydrate, it yielded aminothienopyrimidinone derivative (8). 1H NMR of 8 revealed signals at d 1.97 ppm for CH3 and 7.33 ppm (D2O exchangeable NH2 protons) with a lack of peaks attributed to the ester protons detected in the parent compound 7. When compound 1 was stirred with a mixture of carbon disulphide, sodium hydroxide in dimethylsulphoxide and dimethylsulphate, according to the Alagarsamy proce316 A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table I. Physical and analytical data of newly synthesized compounds Microanalytical analysis Calcd./found (%) C 52.85 52.82 45.06 45.10 56.30 56.32 50.08 50.12 46.97 46.95 44.80 44.83 49.06 49.08 48.42 48.40 45.17 45.14 53.52 53.50 54.28 54.25 45.40 45.43 42.34 42.30 46.14 46.10 44.98 44.95 H 5.77 5.71 5.20 5.15 5.45 5.40 4.53 4.50 3.94 3.90 4.60 4.62 5.61 5.60 4.06 4.03 3.79 3.75 5.61 5.60 5.01 5.03 4.76 4.72 3.55 3.50 4.93 4.90 3.36 3.30 N 6.16 6.15 19.70 19.70 15.15 15.10 13.48 13.45 14.94 14.90 17.42 17.40 15.60 15.63 18.82 18.80 17.56 17.53 5.20 5.23 18.99 18.95 4.41 4.40 16.46 16.42 9.78 9.75 11.66 11.62 Compd. No. 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 Yield (%) M.p. (°C) Molecular formula (Mr) C10H13NO3S (227.28) C8H11N3O2S (213.26) C13H15N3O2S (277.34) C13H14ClN3O2S (311.79) C11H11N3O4S (281.29) C9H11N3O3S (241.27) C11H15N3O3S (269.32) C9H9N3O2S (223.25) C9H9N3OS2 (239.32) C12H15NO4S (269.32) C10H11N3OS (221.28) C12H15NO3S3 (317.45) C9H9N3O2S2 (255.32) C11H14N2O3S2 (286.37) C9H8N2O2S2 (240.30) 160­162 110­112 140­142 290­292 137­139 123­125 131­133 138­140 oil 146­148 273­275 198­200 330­332 201­203 239­241 dure (24), it afforded 9. 1H NMR spectrum of 9 showed the absence of the amino signal and appearance of new signals at d 3.85 ppm for SCH3 and d 11.72 ppm (as a D2O exchangeable NH proton). It is worth mentioning that compound 9 cyclized with hydrazine hydrate to yield 10. Finally, the reaction of compound 1 with potassium thiocyanate afforded the formation of 2-thioureidothiophene derivative (11). Moreover, when compound 11 was refluxed in sodium ethoxide, it underwent self-cyclization to yield 2-thioxothienopyrimidi- A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. H3C H3COC S COOC2H5 (CH3)2SO4 NH S S Na H3C H3COC S 9 COOC2H5 NH2NH2 NH S SCH3 10 CS 2 /NaOH H3C H3COC S 11 S C2H5ONa H3C H3COC S 12 O NH N H S H3C H3COC S 8 COOC2H5 NH NH2 H3C H3COC S 7 COOC2H5 NH O CH3 KSCN HCl Ac2O NH2NH2 O N N CH3 NH2 Scheme 2 none derivative (12). The 1H NMR of 12 revealed signals at d 11.90, 12.29 ppm (as D2O exchangeable 2 NH protons) with a lack of peaks attributed to the ester protons detected in the parent compound 11 (Scheme 2). Antioxidant activity All compounds were tested for antioxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and the rate of erythrocyte hemolysis. Pro-oxidant activities of the formed compounds were assayed via their effects on bleomycin-induced DNA damage. Compounds 10 and 12 manifested potent antioxidative activity in the lipid peroxidation assay but no inhibitory activity in the hemolysis assay. On the other hand, compounds 3a, 4, 5a, 5b, 6a, 6b, 8, 10 and 12 exhibited significant antioxidant activity and protected the DNA from damage (Table III). Antitumor activity using the in vitro Ehrlich used ascites assay The newly synthesized compounds were screened for their antitumor activity. Viability of the cells used in control experiments exceeded 95 %. Compound 6a proved to have the highest cytotoxic activity (83.1 %) followed by compounds 8, 10, 12 (79.9­77.2 %) and 11 (67.9 %). The other tested compounds showed very weak activity (45.1­4.9 %) (Table IV). A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table II. Mass, IR and 1H NMR spectral data of newly synthesized compounds Compd. Mass m/z No. (abundance, %) 1 227 [M+] (35) 213 [M+] (100) 277 [M+] (100) 311 [M+] (40) 281 [M+] (25) 241 [M+] (45) IR (KBr) (n, cm­1) 3425 (NH2) 1700, 1720 (2CO) 3420, 3355 (NH, NH2) 1700, 1690 (2CO) 3410 (NH2) 1705, 1685 (2CO) 3415 (NH2) 1706, 1689 (2CO) 3410 (NH) 1700, 1690, 1685, 1679 (4CO) 3420 (NH) 1710, 1685 (2CO) 1H NMR (DMSO-d6), d (ppm) 3a 3b 4 1.29 (t, 3H, CH3), 2.24 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 4.29 (q, 2H, CH2), 6.66 (br.s, 2H, NH2, D2O exchangeable) 2.25 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 6.12 (br.s, 2H, NH2, D2O exchangeable), 7.33 (br.s, 2H, NH2, D2O exchangeable), 8.16 (s, 1H, NH, D2O exchangeable) 2.10 (s, 6H, 2 CH3), 2.23 (s, 3H, CH3), 2.57 (s, 3H, CO CH3), 7.96 (s, 1H, pyrazole proton), 12.51 (br.s, 2H, NH2, D2O exchangeable) 2.07 (s, 6H, 2 CH3), 2.31 (s, 3H, CH3), 2.56 (s, 3H, COCH3), 12.53 (br.s, 2H, NH2, D2O exchangeable) 2.24 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 2.78 (s, 2H, CH2), 9.35 (br.s, 2H, NH2, D2O exchangeable), 12.32 (s, 1H, NH, D2O exchangeable) 2.25 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 8.44 (s, 1H, azomethine CH proton), 8.59 (br.s, 2H, NH2, D2O exchangeable), 11.31 (s, 1H, NH, D2O exchangeable), 11.55 (s, 1H, OH, D2O exchangeable) 1.31 (t, 3H, CH3), 2.26 (s, 3H, CH3), 2.60 (s, 3H, COCH3), 4.32 (q, 2H, CH2), 6.67 (s, 1H, azomethine CH proton), 8.49 (br.s, 2H, NH2, D2O exchangeable), 11.10 (s, 1H, NH, D2O exchangeable) 2.24 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 7.12 (s, 1H, oxadiazole proton), 8.16 (br.s, 2H, NH2, D2O exchangeable) 2.27 (s, 3H, CH3), 2.60 (s, 3H, COCH3), 7.85 (s, 1H, thiadiazole proton), 8.90 (br.s, 2H, NH2, D2O exchangeable) 1.28 (t, 3H, CH3), 2.24 (s, 3H, CH3) 2.44 (s, 3H, COCH3), 2.59 (s, 3H, COCH3), 4.31 (q, 2H, CH2), 11.10 (s, 1H, NH, D2O exchangeable) 1.97 (s,3H, CH3), 2.23 (s, 3H, CH3), 2.58 (s, 3H, COCH3) 7.33 (br.s, 2H, NH2, D2O exchangeable) 1.28 (t, 3H, CH3), 2.25 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 3.85 (s, 3H, SCH3) 4.28 (q, 2H, CH2), 11.72 (s, 1H, NH, D2O exchangeable) 2.10 (s, 3H, CH3), 3.21 (s, 3H, COCH3), 6.12 (br.s, 2H, NH2, D2O exchangeable), 7.44 (s, 1H, NH, D2O exchangeable) 1.29 (t, 3H, CH3), 2.24 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 4.32 (q, 2H, CH2), 6.84 (br.s, 2H, NH2, D2O exchangeable), 10.97 (s, 1H, NH, D2O exchangeable) 2.23 (s, 3H, CH3), 2.95 (s, 3H, COCH3), 11.90 (s, 1H, NH, D2O exchangeable), 12.29 (s, 1H, NH, D2O exchangeable) 5a 5b 269 [M+] (40) 223 [M+] (71) 239 [M+] (100) 269 [M+] (69) 237 [M+] (92) 317 [M+] (64) 255 [M+] (88) 286 [M+] (72) 240 [M+] (83) 3410 (NH) 1700, 1680, (2CO) 3395 (NH2) 1700 (CO) 3398 (NH2) 1705 (CO) 3420 (NH) 1720, 1690, 1685 (3CO) 3415 (NH2) 1705, 1680 (2CO) 3300 (NH) 1705, 1725 (2CO) 3250 (NH2) 1700, 1685 (2CO) 3380, 3260 (NH, NH2) 1700, 1720 (2CO) 3390, 3385 (2NH) 1705, 1680 (2CO) 6a 6b A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table III. Antioxidant activity of the prepared compounds Compd. No.a L-ascorbic acid 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 ABTS inhibition (%) 88.6 20.2 24.3 70.8 44.4 65.9 60.9 55.3 80.4 79.2 50.3 72.9 48.2 88.6 35.6 83.3 Erythrocyte hemolysis (%) 0.9 7.5 6.4 1.7 4.8 1.9 2.5 2.8 1.1 1.4 3.5 1.5 3.7 0.9 5.6 1.0 For testing 50 mL of 2 mmol L­1 solution in 1 mL methanol/phosphate buffer (1:1, V/V) was used. Table IV. Ehrlich in vitro assay Compd. No.a 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 5-Florouracilb a b Dead cells (%) 4.9 45.1 12.0 6.9 11.4 7.9 15.0 83.1 36.0 4.9 79.5 42.9 79.9 67.9 77.2 99.5 1 mg mL­1 in DMSO/RPMI-1640 (1:10) 25 mg mL­1 in DMSO/RPMI-1640 (1:10) A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. CONCLUSIONS In summary, an interesting thiophene derivatives with substituted diazole, thiadiazole, pyrazole and pyrimidine moieties were synthesized and evaluated for their antioxidant and antitumor activities. i) Novel thiophene derivatives wit substituted diazole, thiadiazole, pyrazole and pyrimidine moieties possess a potential as antioxidant and antitum agents. ii) Thienopyrimidinone derivatives were found to be of high antioxidant activity, while thienoxathiazol and thienoxadiazole showed moderate activity compared to thienopyrazole derivatives. iii) Thienoxadiazole showed the highest cytotoxic activity, higher than thienopyrimidinone and thienylthiourea derivatives. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Pharmaceutica de Gruyter

Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents

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Acta Pharm. 60 (2010) 311­323 10.2478/v10007-010-0030-y Original research paper AMEEN ALI ABU-HASHEM1 MOHAMED FATHY EL-SHEHRY2* FARID ABD-ELRAHEEM BADRIA3 1 Photochemistry Department (Heterocyclic Unit), National Research Center, 12622 Dokki Giza, Egypt Green Chemistry Department National Research Center 12622 Dokki, Giza, Egypt Department of Pharmacognosy Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt 2-Amino-5-acetyl-4-methyl-thiophene-3-carboxylic acid ethyl ester (1) and 5-acetyl-2-amino-4-methylthiophene-3-carbohydrazide (2) were synthesized and used as starting materials for the synthesis of new series of 1-(5-amino-4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3a), 1-(5-amino-4-(4-chloro-3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3b), 1-(4-methyl-2-amino-5-acetylthiophene-3-carbonyl)pyrazolidine-3,5-dione (4), (Z)-N'-(4 -methyl-2-amino-5-acetylthiophene-3-carbonyl) formohydrazonic acid (5a), (Z)-ethyl-N'-4-methyl-2-amino-5-acetylthiophene-3-carbonylformo hydrazonate (5b), 6-acetyl-3-amino-2,5-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (8), 5-methyl-3-amino-2-mercapto-6-acetylthieno [2,3-d]pyrimidin-4(3H)-one (10) and 5-methyl-6-acetyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (12) as potential antioxidant and antitumor agents. Pharmacological tests showed that compounds 6a, 6b, 8, 10 and 12 exhibited significant antitumor and antioxidant activity. Keywords: thienopyrazole, thieno[2,3-d]pyrimidines, thiophenecarbohydrazide, antioxidant activity, bleomycin-dependent DNA damage, antitumor activity Accepted July 16, 2010 The ultimate position of substituted 2-aminothiophenes in the field of drug design and synthesis of pharmaceuticals comes from their advantageous properties. The thiophene ring is bioisosteric replacement for the phenyl group broadly present in active drugs; the thiophene core exists in many natural and synthetic pharmaceuticals (1, 2). In the present work, ethyl-2-aminothiophene-3-carboxylic was expressed as a synthone for many new condensed heterocyclic systems viz. thieno[2,3-d]pyrimidine, thienylthiourea, thienylacetamide, thienylcarbohydrazide. Fused and polyfunctional substituted thiophene play an important role, especially when linked to a pyrimidine ring; thienopyri- * Correspondence; e-mail: moh_elshehry2000@yahoo.com A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. midine derivatives possess pharmacological properties as A1 adenosine receptor agonists (3, 4), phosphodiesterase inhibitors and many receptor antagonists (5, 6). Various thieno [2,3-d] pyrimidine derivatives show pronounced antitumor (7) and radioprotective activities (8), acts as immunomodulators (9) and were used for prophylaxis and therapy of cerebral ischemia (10), malaria (11), tuberculosis (12), Alzheimer's disease (13), Parkinson's disease (14), and other diseases (15, 16). Design and synthesis of the novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents were explored. EXPERIMENTAL All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus (Shimadzu, Japan). Elemental analyses were performed at Vario EL (Elementar, Germany). Microanalytical data were processed in the microanalytical center, Faculty of Science, Cairo University. The IR spectra (KBr disk) were recorded using a Perkin-Elmer 1650 spectrometer (USA). 1H NMR spectra were determined using Jeol 270 MHz and Jeol JMS-AX 500 MHz (Jeol, Japan) with internal standard MeSi4. Mass spectra were recorded on an EI Ms-QP 1000 EX (Shimadzu) at 70 eV. Pharmacological evaluations were done in pharmacological unit, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Egypt. DNA (calf thymus type 1), bleomycin sulfate, butylated hydroxyanisole (BHA), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA) and ascorbic acid were obtained from Sigma (USA). 2,2'-Azo-bis-(2-amidinopropane) dihydrochloride (AAPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) were purchased from Wako Co. (USA). Syntheses Ethyl-4-methyl-2-amino-5-acetylthiophene-3-carboxylate (1). ­ A mixture of acetylacetone (0.01 mol), ethyl cyanoacetate (0.01 mol), sulfur (0.01 mol) and diethylamine (0.01 mol) was heated at 70 °C under stirring in absolute ethanol (20 mL) for 4 h, then the mixture was left for 24 h at 0 °C. The solid formed was collected by filtration, washed with ethanol (20 mL), dried and crystallized from absolute ethanol. 5-Acetyl-2-amino-4-methylthiophene-3-carbohydrazide (2). ­ A suspension of dry compound 1 (0.01 mol) and hydrazine hydrate (5 mL) and (30 mL) absolute ethanol was stirred under gentle reflux. The solid dissolved within 10 min with copious evolution of hydrogen sulfide to form a clear solution. After 30 min, the solid product started separating out; heating was continued for 8 h. The reaction mixture was then allowed to cool to room temperature. The solid was filtered off, washed with ethanol, dried and crystallized from dioxane. 1-(5-Amino-4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3a) and 1-(5-amino-4-(4-chloro-3,5-dimethyl-1H-pyrazole-1-carbonyl)-3-methylthiophen-2-yl) ethanone (3b). ­ A mixture of compound 2 (0.01 mol) and 2,4-diketone, namely, pentan-2,4-dione A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. or 3-chloropentan-2,4-dione (0.01 mol) in absolute ethanol (30 mL), was stirred under reflux for 12 h. The reaction mixture was allowed to cool to 0 °C for 24 h, the solid precipitate was filtered off, dried and crystallized from ethanol to produce 3a or 3b in high yields. 1-(4-Methyl-2-amino-5-acetylthiophene-3-carbonyl)pyrazolidine-3,5-dione (4). ­ A solution of compound 2 (0.01 mol) and freshly distilled diethylmalonate (0.01 mol) in sodium ethoxide was heated under reflux with stirring for 5 h. The solvent was evaporated under reduced pressure and the crude product was acidified with 10 % hydrochloric acid. The solid formed was filtered off, washed with cold water, and crystallized from ethanol. (Z)-N'-(4-methyl-2-amino-5-acetylthiophene-3-carbonyl) formohydrazonic acid (5a). ­ A mixture of compound 2 (0.01 mol) and formic acid (10 mL) was heated under reflux for 6 h. The reaction mixture was allowed to cool to room temperature and poured onto water (100 mL). The solid formed was collected by filtration, dried, and crystallized from benzene. (Z)-ethyl-N'-4-methyl-2-amino-5-acetylthiophene-3-carbonylformo hydrazonate (5b). ­ A mixture of compound 2 (0.01 mol) and triethylorthoformate (20 mL) was heated under reflux with stirring for 2 h. The reaction mixture was filtered hot and the filtrate was left to cool to room temperature. The solid formed was filtered off, dried and crystallized from ethanol. Synthesis of 1-(5-amino-3-methyl-4-(1,3,4-oxadiazol-2-yl) thiophen-2-yl) ethanone (6a) and 1-(5-amino-3-methyl-4-(1,3,4-oxathiadiazol-2-yl) thiophen-2-yl) ethanone (6b). ­ A mixture of compounds 5a or 5b (0.005 mol) and phosphorus pentoxide (2 g) or phosphorus pentasulfide (4 g) in dry xylene (30 mL) was heated under reflux for 6­12 h. The solid separated upon cooling was filtered off, crystallized from ethanol to produce 6a and 6b, respectively. Ethyl-2-acetamido-4-methyl-5-acetylthiophene-3-carboxylate (7). ­ A mixture of compound 1 (0.01 mol) and acetic anhydride (30 mL) was heated under reflux for 3 h. The reaction mixture was allowed to cool to room temperature and the solid formed was collected by filtration, dried, and crystallized from ethanol. 6-Acetyl-3-amino-2,5-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (8). ­ A mixture of 7 (0.01 mol) and hydrazine hydrate (10 mL) in ethanol (30 mL) was heated under reflux for 2 h. The reaction mixture was allowed to cool to room temperature and poured into water (100 mL). The solid formed was collected by filtration, dried and crystallized from ethanol as yellow powder. Ethyl-4-methyl-5-acetyl-2-(methylthiocarbonothioylamino) thiophene-3-carboxylate (9). ­ To a vigorously stirred solution of 1 (0.02 mol) in dimethylsulfoxide DMSO (10 mL) at room temperature, carbon disulfide (0.026 mol) and aqueous sodium hydroxide were added simultaneously over 30 min; stirring was continued for further 30 min. Dimethylsulfate (0.02 mol) was added dropwise to the reaction mixture under stirring at 5­10 °C. It was stirred for another 2 h and poured into ice-water. The solid obtained was filtered off, dried and recrystallized from ethanol. A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. 5-Methyl-3-amino-2-mercapto-6-acetylthieno[2,3-d]pyrimidin-4(3H)-one (10). ­ Method A: A solution of 9 (0.01 mol) in ethanol (30 mL) and hydrazine hydrate (0.01 mol) was added and refluxed on a water bath until the methyl mercaptan evolution ceased after 8 h. After cooling, the solid obtained was filtered off, dried and recrystallized from ethanol/ acetone mixture. Method B: A solution of 2 (0.01 mol) in ethanol (30 mL) and aqueous potassium hydroxide (0.01 mol) was refluxed for 30 min. After cooling to room temperature, carbon disulfide (1 mL) was added, refluxed for 2-3 hours and poured into ice-water. The solid obtained was filtered off, dried and recrystallized from ethanol. Ethyl-4-methyl-5-acetyl-2-thioureidothiophene-3-carboxylate (11). ­ A mixture of 1 (0.01 mol) and 10 % HCl (10 mL) was refluxed with potassium thiocyanate (0.015 mol) for 4 h. The reaction mixture was allowed to cool to room temperature. The solid formed was collected by filtration, washed with water, dried and crystallized from dioxane. 5-Methyl-6-acetyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (12). ­ A solution of 11 (0.01 mol) in ethanolic sodium ethoxide (0.23 g of sodium metal in 30 mL ethanol) was stirred under reflux for 6 h. After cooling, the reaction mixture was neutralized with cool 10 % HCl and the solid formed was collected by filtration, washed with water, dried and then crystallized from dimethylformamide. Pharmacological screening Ehrlich cells. ­ Ehrlich cells (Ehrlich ascites carcinoma, EAC) were derived from ascetic fluid from diseased mice (the cells were purchased from the National Cancer institute, Cairo, Egypt). Antioxidant activity screening via erythrocyte hemolysis. ­ The blood was obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes were separated from plasma and the buffy coat was washed three times with 10 volumes of 0.15 mol L­1 NaCl. During the last wash, the erythrocytes were centrifuged at 2500 rpm for 10 min to obtain a constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system (17). A 10 % suspension of erythrocytes in phosphate buffered saline pH 7.4 (PBS) was added to the same volume of 200 mmol L­1 AAPH solution in PBS containing samples to be tested (50 mL of 2 mmol L­1 solution). The reaction mixture was shaken gently while being incubated at 37 °C for 2 h. The reaction mixture was then removed, diluted with eight volumes of PBS and centrifuged at 1500´g for 10 min. The absorbance of the supernatant was read at 540 nm. Similarly, the reaction mixture was treated with 8 volumes of distilled water to achieve complete hemolysis, and the absorbance of the supernatant obtained after centrifugation was measured at 540 nm. The data for hemolysis percentage was expressed as mean ± standard deviation. L-ascorbic acid was used as a positive control. Antioxidant activity screening via ABTS method. ­ For each of the investigated compounds, 2 mL of ABTS solution (60 mmol L­1) was added to 3 mL MnO2 solution (g = 25 mg mL­1), all prepared in aqueous phosphate buffer (pH 7, 0.1 mol L­1). The mixture was shaken, centrifuged, filtered and the absorbance of the resulting green-blue solution (ABTS radical solution) at 734 nm was adjusted to approx. 0.5. Then, 50 mL of 2 mmol L­1) solution of the test compound in spectroscopic grade MeOH/phosphate buffer (1:1) A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. was added. The absorbance was measured and the reduction in color intensity was expressed as inhibition percentage. L-ascorbic acid was used as standard antioxidant (positive control). Blank sample was run without ABTS and using MeOH/phosphate buffer (1:1) instead of a sample. Negative control was run with ABTS and MeOH/phosphate buffer (1:1) instead of test compound (18­20). Bleomycin-dependent DNA damage. ­ The assay was done according to Aeschlach (21) with minor modifications. The reaction mixture (0.5 mL) contained calf thymus DNA (0.5 mg mL­1), bleomycin sulfate (0.05 mg mL­1), MgCl2 (5 mmol L­1), FeCl3 (50 mmol L­1) and the sample to be tested (50 mL of 2 mmol L­1 solution). L-ascorbic acid was used as a positive control. The mixture was incubated at 37 °C for 1 h. The reaction was terminated by addition 0.05 mL EDTA (0.1 mol L­1). The color was developed by adding 0.5 mL TBA (1 %, m/V) and 0.5 mL HCl (25 %, V/V) followed by heating at 37 °C for 15 min. After centrifugation, the extent of DNA damage was measured by the increase in absorbance at 532 nm. Antitumor activity using Ehrlich ascites in vitro assay. ­ Tested compounds were prepared in a concentration of 25 mg mL­1 in DMSO. Ascites fluid from the peritoneal cavity of a diseased mouse (containing Ehrlich cells) was aseptically aspirated. The cells were grown partly floating and partly attached in a suspension culture in RPMI 1640 medium, supplemented with 10 % fetal bovine serum. They were maintained at 37 °C in a humidified atmosphere with 5 % CO2 for 2 h. The viability of the cells was determined by microscopical examination using a hemocytometer and using trypan blue stain (stains only dead cells) (22). RESULTS AND DISCUSSION Chemistry First, aminothiophene (1) was prepared by the reaction of acetylacetone with ethylcyanoacetate and sulfur in the presence of diethylamine following the Gewald procedure (23). 1H NMR spectrum of 1 showed, beside the two signals of ester moiety, a new broad singlet at d 6.66 ppm characteristic of the amino group. Moreover, the mass spectra showed an ion peak m/z 227 [M+], which was in accord with the calculated molecular mass for C10H13NOS. Hydrazide 2 was achieved after refluxing compound 1 with hydrazine hydrate in ethanol. 1H NMR spectrum of 2 showed the absence of the two signals of ester moiety detected in parent 1 along with other protons at their expected locations. Hydrazide 2 reacted with 2,4-diketones, namely, pentane-2,4-dione or 3-chloropentane-2,4-dione to afford 3a and 3b, respectively. The 1H NMR spectrum of 3a revealed new signals at d 2.10 ppm assignable to 2 CH3 groups, d 7.96 ppm for pyrazole proton and d 12.51 ppm for NH2 (as D2O exchangeable). Also, hydrazide 2 reacted with diethylmalonate affording pyrazolidine-3,5-dione derivative 4. The 1H NMR spectrum showed the absence of the hydrazo signal and the presence of new signals at d 2.78 ppm for CH2 group and d 12.32 ppm corresponding to (as a D2O exchangeable NH proton) along with other protons detected in parent 4. A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. O O C 2 H5 O + O N HN(C2H5)2 S R` R S 1 COOC2H5 NH2 CS2 R` R S 10 O N N H NH2 S R R` S 3: a b H3 C N N NH2 X CH3 N 2 H4 H3COC H3COC X R` R S 2 HCOOH or CH(OEt)3 R R` S CONHNH2 NH2 KOH COOEt COOEt C2H5ONa R` R S 4 O N HN NH2 X= H X= Cl CONHN CHOR P 2O 5 NH2 or P2S5 R` R S N X NH2 1-6: R = CH3 R` = COCH3 5: a R = H b R = C 2 H5 6: a X = O b X=S Scheme 1 Treatment of 2 with formic acid or triethylorthoformate afforded 5a and 5b, respectively, in fairly good yields. 1H NMR spectrum of 5a showed signals at d 8.44 ppm for CH proton, and d 11.55 ppm (as a D2O exchangeable OH proton), while the 1H NMR spectrum of 5b showed new signals at d 1.31, 4.32 ppm for CH2CH3 moiety and d 6.67 ppm for CH proton. Moreover, compounds 5a and 5b undergo cyclization when treated with P2O5 and P2S5, to yield thienyloxadiazole (6a) and thienylthiadiazole (6b) derivatives, respectively. The 1H NMR spectrum of 6a and 6b showed signals at d 7.12 ppm characteristic of the oxadiazole proton of 6a and d 7.85 ppm for thiadiazole proton of 6b beside the other expected signals characteristic of methyl, acetyl and amino groups. Hydrazide 2 reacted with carbon disulphide in ethanolic potassium hydroxide to afford thienopyrimidinone derivative (10). 1H NMR spectrum showed signals at d 6.12 ppm for NH2 and d 7.44 ppm for NH proton (as D2O exchangeable) beside the signals characteristic of acetyl and methyl groups; moreover, the mass spectrum revealed an ion peak at m/z 255 [M+], which was in accord with the calculated molecular mass C9H9N3O2S2 (Scheme 1). Compound 1, when treated with acetic anhydride, afforded 2-thienylacetamide derivative (7). Moreover, when compound 7 was allowed to react with hydrazine hydrate, it yielded aminothienopyrimidinone derivative (8). 1H NMR of 8 revealed signals at d 1.97 ppm for CH3 and 7.33 ppm (D2O exchangeable NH2 protons) with a lack of peaks attributed to the ester protons detected in the parent compound 7. When compound 1 was stirred with a mixture of carbon disulphide, sodium hydroxide in dimethylsulphoxide and dimethylsulphate, according to the Alagarsamy proce316 A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table I. Physical and analytical data of newly synthesized compounds Microanalytical analysis Calcd./found (%) C 52.85 52.82 45.06 45.10 56.30 56.32 50.08 50.12 46.97 46.95 44.80 44.83 49.06 49.08 48.42 48.40 45.17 45.14 53.52 53.50 54.28 54.25 45.40 45.43 42.34 42.30 46.14 46.10 44.98 44.95 H 5.77 5.71 5.20 5.15 5.45 5.40 4.53 4.50 3.94 3.90 4.60 4.62 5.61 5.60 4.06 4.03 3.79 3.75 5.61 5.60 5.01 5.03 4.76 4.72 3.55 3.50 4.93 4.90 3.36 3.30 N 6.16 6.15 19.70 19.70 15.15 15.10 13.48 13.45 14.94 14.90 17.42 17.40 15.60 15.63 18.82 18.80 17.56 17.53 5.20 5.23 18.99 18.95 4.41 4.40 16.46 16.42 9.78 9.75 11.66 11.62 Compd. No. 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 Yield (%) M.p. (°C) Molecular formula (Mr) C10H13NO3S (227.28) C8H11N3O2S (213.26) C13H15N3O2S (277.34) C13H14ClN3O2S (311.79) C11H11N3O4S (281.29) C9H11N3O3S (241.27) C11H15N3O3S (269.32) C9H9N3O2S (223.25) C9H9N3OS2 (239.32) C12H15NO4S (269.32) C10H11N3OS (221.28) C12H15NO3S3 (317.45) C9H9N3O2S2 (255.32) C11H14N2O3S2 (286.37) C9H8N2O2S2 (240.30) 160­162 110­112 140­142 290­292 137­139 123­125 131­133 138­140 oil 146­148 273­275 198­200 330­332 201­203 239­241 dure (24), it afforded 9. 1H NMR spectrum of 9 showed the absence of the amino signal and appearance of new signals at d 3.85 ppm for SCH3 and d 11.72 ppm (as a D2O exchangeable NH proton). It is worth mentioning that compound 9 cyclized with hydrazine hydrate to yield 10. Finally, the reaction of compound 1 with potassium thiocyanate afforded the formation of 2-thioureidothiophene derivative (11). Moreover, when compound 11 was refluxed in sodium ethoxide, it underwent self-cyclization to yield 2-thioxothienopyrimidi- A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. H3C H3COC S COOC2H5 (CH3)2SO4 NH S S Na H3C H3COC S 9 COOC2H5 NH2NH2 NH S SCH3 10 CS 2 /NaOH H3C H3COC S 11 S C2H5ONa H3C H3COC S 12 O NH N H S H3C H3COC S 8 COOC2H5 NH NH2 H3C H3COC S 7 COOC2H5 NH O CH3 KSCN HCl Ac2O NH2NH2 O N N CH3 NH2 Scheme 2 none derivative (12). The 1H NMR of 12 revealed signals at d 11.90, 12.29 ppm (as D2O exchangeable 2 NH protons) with a lack of peaks attributed to the ester protons detected in the parent compound 11 (Scheme 2). Antioxidant activity All compounds were tested for antioxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and the rate of erythrocyte hemolysis. Pro-oxidant activities of the formed compounds were assayed via their effects on bleomycin-induced DNA damage. Compounds 10 and 12 manifested potent antioxidative activity in the lipid peroxidation assay but no inhibitory activity in the hemolysis assay. On the other hand, compounds 3a, 4, 5a, 5b, 6a, 6b, 8, 10 and 12 exhibited significant antioxidant activity and protected the DNA from damage (Table III). Antitumor activity using the in vitro Ehrlich used ascites assay The newly synthesized compounds were screened for their antitumor activity. Viability of the cells used in control experiments exceeded 95 %. Compound 6a proved to have the highest cytotoxic activity (83.1 %) followed by compounds 8, 10, 12 (79.9­77.2 %) and 11 (67.9 %). The other tested compounds showed very weak activity (45.1­4.9 %) (Table IV). A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table II. Mass, IR and 1H NMR spectral data of newly synthesized compounds Compd. Mass m/z No. (abundance, %) 1 227 [M+] (35) 213 [M+] (100) 277 [M+] (100) 311 [M+] (40) 281 [M+] (25) 241 [M+] (45) IR (KBr) (n, cm­1) 3425 (NH2) 1700, 1720 (2CO) 3420, 3355 (NH, NH2) 1700, 1690 (2CO) 3410 (NH2) 1705, 1685 (2CO) 3415 (NH2) 1706, 1689 (2CO) 3410 (NH) 1700, 1690, 1685, 1679 (4CO) 3420 (NH) 1710, 1685 (2CO) 1H NMR (DMSO-d6), d (ppm) 3a 3b 4 1.29 (t, 3H, CH3), 2.24 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 4.29 (q, 2H, CH2), 6.66 (br.s, 2H, NH2, D2O exchangeable) 2.25 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 6.12 (br.s, 2H, NH2, D2O exchangeable), 7.33 (br.s, 2H, NH2, D2O exchangeable), 8.16 (s, 1H, NH, D2O exchangeable) 2.10 (s, 6H, 2 CH3), 2.23 (s, 3H, CH3), 2.57 (s, 3H, CO CH3), 7.96 (s, 1H, pyrazole proton), 12.51 (br.s, 2H, NH2, D2O exchangeable) 2.07 (s, 6H, 2 CH3), 2.31 (s, 3H, CH3), 2.56 (s, 3H, COCH3), 12.53 (br.s, 2H, NH2, D2O exchangeable) 2.24 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 2.78 (s, 2H, CH2), 9.35 (br.s, 2H, NH2, D2O exchangeable), 12.32 (s, 1H, NH, D2O exchangeable) 2.25 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 8.44 (s, 1H, azomethine CH proton), 8.59 (br.s, 2H, NH2, D2O exchangeable), 11.31 (s, 1H, NH, D2O exchangeable), 11.55 (s, 1H, OH, D2O exchangeable) 1.31 (t, 3H, CH3), 2.26 (s, 3H, CH3), 2.60 (s, 3H, COCH3), 4.32 (q, 2H, CH2), 6.67 (s, 1H, azomethine CH proton), 8.49 (br.s, 2H, NH2, D2O exchangeable), 11.10 (s, 1H, NH, D2O exchangeable) 2.24 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 7.12 (s, 1H, oxadiazole proton), 8.16 (br.s, 2H, NH2, D2O exchangeable) 2.27 (s, 3H, CH3), 2.60 (s, 3H, COCH3), 7.85 (s, 1H, thiadiazole proton), 8.90 (br.s, 2H, NH2, D2O exchangeable) 1.28 (t, 3H, CH3), 2.24 (s, 3H, CH3) 2.44 (s, 3H, COCH3), 2.59 (s, 3H, COCH3), 4.31 (q, 2H, CH2), 11.10 (s, 1H, NH, D2O exchangeable) 1.97 (s,3H, CH3), 2.23 (s, 3H, CH3), 2.58 (s, 3H, COCH3) 7.33 (br.s, 2H, NH2, D2O exchangeable) 1.28 (t, 3H, CH3), 2.25 (s, 3H, CH3), 2.58 (s, 3H, COCH3), 3.85 (s, 3H, SCH3) 4.28 (q, 2H, CH2), 11.72 (s, 1H, NH, D2O exchangeable) 2.10 (s, 3H, CH3), 3.21 (s, 3H, COCH3), 6.12 (br.s, 2H, NH2, D2O exchangeable), 7.44 (s, 1H, NH, D2O exchangeable) 1.29 (t, 3H, CH3), 2.24 (s, 3H, CH3), 2.59 (s, 3H, COCH3), 4.32 (q, 2H, CH2), 6.84 (br.s, 2H, NH2, D2O exchangeable), 10.97 (s, 1H, NH, D2O exchangeable) 2.23 (s, 3H, CH3), 2.95 (s, 3H, COCH3), 11.90 (s, 1H, NH, D2O exchangeable), 12.29 (s, 1H, NH, D2O exchangeable) 5a 5b 269 [M+] (40) 223 [M+] (71) 239 [M+] (100) 269 [M+] (69) 237 [M+] (92) 317 [M+] (64) 255 [M+] (88) 286 [M+] (72) 240 [M+] (83) 3410 (NH) 1700, 1680, (2CO) 3395 (NH2) 1700 (CO) 3398 (NH2) 1705 (CO) 3420 (NH) 1720, 1690, 1685 (3CO) 3415 (NH2) 1705, 1680 (2CO) 3300 (NH) 1705, 1725 (2CO) 3250 (NH2) 1700, 1685 (2CO) 3380, 3260 (NH, NH2) 1700, 1720 (2CO) 3390, 3385 (2NH) 1705, 1680 (2CO) 6a 6b A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. Table III. Antioxidant activity of the prepared compounds Compd. No.a L-ascorbic acid 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 ABTS inhibition (%) 88.6 20.2 24.3 70.8 44.4 65.9 60.9 55.3 80.4 79.2 50.3 72.9 48.2 88.6 35.6 83.3 Erythrocyte hemolysis (%) 0.9 7.5 6.4 1.7 4.8 1.9 2.5 2.8 1.1 1.4 3.5 1.5 3.7 0.9 5.6 1.0 For testing 50 mL of 2 mmol L­1 solution in 1 mL methanol/phosphate buffer (1:1, V/V) was used. Table IV. Ehrlich in vitro assay Compd. No.a 1 2 3a 3b 4 5a 5b 6a 6b 7 8 9 10 11 12 5-Florouracilb a b Dead cells (%) 4.9 45.1 12.0 6.9 11.4 7.9 15.0 83.1 36.0 4.9 79.5 42.9 79.9 67.9 77.2 99.5 1 mg mL­1 in DMSO/RPMI-1640 (1:10) 25 mg mL­1 in DMSO/RPMI-1640 (1:10) A. A. Abu-Hashem et al.: Design and synthesis of novel thiophenecarbohydrazide, thienopyrazole and thienopyrimidine derivatives as antioxidant and antitumor agents, Acta Pharm. 60 (2010) 311­323. CONCLUSIONS In summary, an interesting thiophene derivatives with substituted diazole, thiadiazole, pyrazole and pyrimidine moieties were synthesized and evaluated for their antioxidant and antitumor activities. i) Novel thiophene derivatives wit substituted diazole, thiadiazole, pyrazole and pyrimidine moieties possess a potential as antioxidant and antitum agents. ii) Thienopyrimidinone derivatives were found to be of high antioxidant activity, while thienoxathiazol and thienoxadiazole showed moderate activity compared to thienopyrazole derivatives. iii) Thienoxadiazole showed the highest cytotoxic activity, higher than thienopyrimidinone and thienylthiourea derivatives.

Journal

Acta Pharmaceutica – de Gruyter

Published: Sep 1, 2010

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