Synthesis and Screening of New [1,3,4]Oxadiazole, [1,2,4]Triazole, and [1,2,4]Triazolo[4,3-b][1,2,4]triazole Derivatives as Potential Antitumor Agents on the Colon Carcinoma Cell Line (HCT-116).

New derivatives of [1,3,4]oxadiazole-2-thione and triazole-3-thione were synthesized through the cyclocondensation of dicarbonyl ester 2 with phenyl hydrazine followed by hydrazinolysis to give the corresponding hydrazide, which reacted with carbon disulfide or ammonium thiocyanate to afford [1,3,4]oxadiazole 5 or triazole-3-thione 7, respectively. Hydrazinolysis of compound 5 gave [1,2,4]triazole-3-thiol 9 which was treated with different aromatic aldehydes to obtain 10a-c. Mannich bases 11a-c were obtained from the reaction of Schiff bases 10a-c with morpholine and formaldehyde. Moreover, treatment of triazole-3-thione 7 with hydrazine was followed by cyclocondensation with diethyl oxalate, chloroacetic acid, or formic acid to give the corresponding [1,2,4]triazine-3,4-dione 14, [1,2,4]triazin-4-one 15, or [1,2,4]triazolo[4,3-b][1,2,4] triazole 16, respectively. Screening of some chosen synthesized compounds against the human colon carcinoma cancer cell lines showed that the compound [1,2,4]triazole-3-thiol 9 exhibiting cytotoxic activity was roughly equivalent to standard Vinblastine, while compounds 4, 7, 10, 11a, 14, and 16 exhibited moderate cytotoxic activity.

Introduction

As we mentioned in our recent articles, triazolo compounds and particular [1,2,4]triazolo derivatives are well known to have many physiological activities, such as anticancer.[1,2] Also, [1,2,4]triazole derivatives have been reported as antimicrobial, insecticidal, fungicidal, antidepressant, hypnotic, anti-inflammatory, antihypertensive, and plant growth regulator.[3−8] On the other hand, 1,3,4-oxadiazole has become an essential building unit for the creation of new drugs where a literature survey showed that a slight systemic change in 1,3,4-oxadiazole moiety structure could lead to qualitative as well as quantitative changes in 1,3,4-oxadiazole derivative activity, which convinced us to start synthesizing different new 1,3,4-oxadiazole derivatives in order to increase their activity and lower their toxicity. Over the last 2 decades, the synthesis of novel 1,3,4-oxadiazole derivatives and the investigation into their chemical properties and biological behavior have increased.[9−12] Also, the combination of oxadiazole and other heterocycles such as pyrrole and pyrazole rings in one compound led to several biological activities of these compounds.[13−15] We may therefore conclude that 1,3,4-oxadiazole is one of the main pharmacophores that plays an important role in pharmaceutical chemistry and has a broad range of important biological activities including anti-inflammatory,[16,17] anticancer,[18−21] antimalarial,[22] antibacterial,[23−26] anticonvulsant,[27] antitubercular,[28−30] anti-allergic agent,[31] analgesic,[32] and vasodilator.[33] Also, the survey showed that the presence of a morpholine ring on a heterocyclic system leads to an enhancement of pharmacological activities in most cases,[34−36] where the presence of morpholine in heterocyclic compounds could increase the solubility of these compounds in aqueous solutions because of the formation of a morpholine ammonium salt.[37] Also, Schiff bases derived from different heterocyclic compounds have a broad variety of biological activities.[38,39]

Results and Discussion

Chemistry

It is known that the reaction of heterocyclic acetyl derivatives with diethyl oxalate gives the corresponding alkyl-2,4-dioxobutanoate.[40] Therefore, the reaction of 3-acetyl-1-methylpyrrole 1 with diethyl oxalate in the presence of sodium ethoxide gave the corresponding ethyl 2,4-dioxobutanoate derivative 2, as shown in Scheme . The IR spectrum of compound 2 indicated a shoulder or a single broadened absorption band at 1733 cm–1 corresponding to the carbonyl groups, while its 1H NMR spectrum showed a triplet at δ 1.37, singlet at 3.42, quartet at 4.11, singlet at 4.67, and singlet at 7.33 ppm corresponding to methyl protons of the ethyl group, methyl protons of NCH3 and CH2 of the ethyl group, and protons of C3 and H2 of the pyrrolyl group, respectively. The cyclocondensation under reflux of dicarbonyl ester 2 with phenyl hydrazine in acetic acid gave the corresponding ethyl-5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazole-3-carboxylate 3. The IR spectrum of compound 3 showed disappearance of the shoulder absorption band of compound 2 with the appearance of a sharp band at 1724 cm–1 corresponding to the carbonyl ester, and also the 1H NMR spectrum indicated a singlet signal of the pyrazolyl proton at δ 7.83 ppm in addition to the signals of the ethyl, methyl, and the aromatic protons; see experimental part. 13C NMR of compound 3 confirmed the formation of a pyrazole ring by showing a signal at 162.24 ppm corresponding to (C=N). The reaction of hydrazine hydrate with ethyl-pyrazole-3-carboxylate 3 in ethanol under reflux gave the corresponding carboxylic acid hydrazide 4 in a good yield. The structure of the acid hydrazide 4 was confirmed by spectral data and elemental analyses, wherein the IR spectrum of compound 4 showed two characteristic bands at 3356 and 3189 cm–1 for NH and NH2 groups along with a band of amidic carbonyl at 1678 cm–1, while the 1H NMR spectrum indicated the disappearance of ethyl protons and appearance of two D2O exchangeable singlet peaks at 4.34 and 9.89 ppm for NH and NH2 groups, which confirmed the formation of acid hydrazide 4. Heterocyclization of acid hydrazide 4 with carbon disulfide in ethanolic potassium hydroxide solution afforded the novel substituted [1,3,4]oxadiazole-2-thione 5Scheme . Both IR and 1H NMR spectra showed the absence of NH2 signals and the presence of the NH signal due to thione–thiole tautomerism. 13C NMR of compound 5 indicated a characteristic peak at δ = 186.11 ppm corresponding to the C=S group, which suggested the presence of compound 5 on the thione form over the thiole form. The second pathway of the reaction of acid hydrazide 4 for the preparation of the [1,2,4]triazole-3-thione derivative starting from its reaction with ammonium thiocyanate in the presence of hydrochloric acid under reflux to give the corresponding thiosemicarbazide 6 which was refluxed in ethanolic potassium hydroxide afforded the corresponding triazole-3-thione 7. The IR spectrum of compound 6 showed moderately strong bands around (3265–3471) cm–1 characteristic of the NH and NH2 groups, along with a band at 1679 cm–1 for amidic carbonyl of thiosemicarbazide. Also, the 1H NMR spectrum of compound 6 indicated three peaks at 4.05, 4.66, and 9.14 ppm corresponding to NH2, CSNH, and CONH protons, respectively. The mass spectrum of compound 7 revealed the molecular ion peak at m/z 322 which is consistent with the chemical structure of the compound 7; also, both IR and 1H NMR spectra indicated the presence of 2 NH groups, along with a characteristic peak at 179.66 ppm in 1 3C NMR for the C=S group which suggested a thione form for compound 7. Hydrazinolysis of [1,3,4]oxadiazole-2-thione 5 was performed on the oxygen atom of oxadiazole to give 4-amino-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-4H-[1,2,4]triazole-3-thiol 9, and this did not occur on the thiol group to give {5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-[1,3,4]oxadiazol-2-yl}-hydrazine 8, as shown in Scheme . The formation of [1,2,4]triazole-3-thiol 9 was confirmed mainly through the elemental analysis data and the molecular ion peak at m/z 337, and this is in agreement with compound 9, not 8. Also, the IR spectrum of compound 9 showed only one band at 3256 cm–1 for the NH2 group, while the 1H NMR spectrum indicated characteristic signals at 4.85 and 12.95 ppm for NH2 and SH protons, respectively. Condensation of 4-amino[1,2,4]triazole-3-thiol 9 with different aromatic aldehydes gave the Schiff bases 10a–c. 1H NMR of compounds 10a–c showed singlet peaks resonating at (8.21–8.62) for the NH groups and at (8.72–8.94) ppm for the imine (N=CH) protons. Also, the appearance of a peak at 182.04 ppm at 13C NMR of compound 10a corresponding to C=S confirmed the thione form of these compounds.

Scheme 1

Scheme 2

Reaction of compounds 10a–c with morpholine and formaldehyde afforded the corresponding Mannich bases 11a–c. The 1H NMR of bases 11a–c showed disappearance of the NH proton signal and appearance of signals at (2.70–2.84) and (3.71–3.92) ppm for N–CH2 and O–CH2 of morpholine, respectively. Methylene protons of the morpholine ring appeared as a broad signal showing some splitting due to the rapid ring flipping which makes axial and equatorial protons of morpholine ring almost equivalent.[41] Also, the 1H NMR of compounds 11a–c indicated a signal at (4.81–4.92) belonging to the N–CH2–N protons.

In addition, 1 3C NMR for Mannich base 11a showed new carbon peaks related to the morpholine ring at 50.89 and 64.33 ppm. On the other hand, methylation of oxadiazole-2-thione 5 with methyl iodide in ethanolic potassium hydroxide yielded the methylthio derivative 12. Treatment of triazole-3-thione 7 with hydrazine hydrate gave 3-hydrazino[1,2,4]triazol derivative 13, as shown in Scheme .

Scheme 3

The structure of hydrazino compound 13 established using IR spectra which indicated a group of bands at (3371–3182) cm–1 for (2NH, NH2) groups, while 1H NMR showed three D2O exchangeable signals at 4.89, 5.76, and 9.13 ppm for NH2, NH of pyrrole, and NH of the hydeazino group, respectively. Heating of hydrazino[1,2,4]triazol 13 with diethyl oxalate under reflux produced [1,2,4]triazine-3,4-dione derivative 14. The IR spectra of compound 14 showed bands around (3362–3165) cm–1 characteristic of the 2 NH groups besides a broad band at 1711 ppm for the two carbonyl groups, while 1H NMR showed two D2O exchangeable signals at 5.88 and 10.73 ppm for 2 NH groups. Also, the cyclocondensation of hydrazinotriazol 13 with chloroacetic acid and/or formic acid gave [1,2,4]triazin-4-one derivative 15 and [1,2,4]triazolo[4,3-b][1,2,4] triazole derivative 16, respectively. IR spectra of 15 showed two bands for 2 NH groups at (3376–3181) and only one sharp band at 1686 cm–1 for the C=O group, while compound 16 showed only one band for at 3289 cm–1 for the NH group. The 1H NMR spectrum of compound 15 showed four singlet peaks at δ 3.55, 3.78, 5.61, and 6.09 ppm for NCH3, CH2, and 2 NH protons, respectively, while the 1H NMR spectrum of compound 16 indicated two characterized singlet peaks at 3.39 and 5.34 ppm for NCH3 and NH protons.

Anticancer Activity

Anticancer Activity Discussion

In this study, the synthesized compounds 4, 5, 7, 9, 10, 11a, 14, and 16 were evaluated for potential cytotoxicity according to the methods reported by Mossman,[42] Gangadevi, and Muthumary[43] for their anticancer activity against the human colon carcinoma cancer cell lines (HCT-116) using the Vinblastine drug as the standard. The data of anticancer activity were expressed as a cytotoxic effect of the synthesized compounds. The inhibitory activities against colon carcinoma cells (HCT-116) were calculated by dissolving the tested compounds in DMSO and diluted with saline to an appropriate volume using different concentrations of the samples (50, 25, 12.5, 6.25, 3.125, and 1.56 μg), and the cell viability (%) of the tested compounds was determined by the colorimetric method, as shown in Table . The relation between cell viability (%) and different concentrations of the tested compounds is plotted in order to obtain the survival curve for colon carcinoma cells (HCT-116), as shown in Figure . The response parameter, inhibitory concentration fifty (IC50) which corresponds to the concentration required for 50% inhabitation of cell viability, was calculated from Table . Screening of the tested compounds against human colon carcinoma cancer cell lines showed that the compound [1,2,4]triazole-3-thiol 9 exhibiting IC50 (4.16 μg) was roughly equivalent to the standard Vinblastine drug (3.34 μg) in cytotoxic activity, as shown in Table and Figure . Meanwhile, compounds 4, 7, 10, 11a, 14, and 16 exhibited moderate cytotoxic activity with IC50 in the range of (6.15–12.05) μg. Moreover, compound 5 showed weak cytotoxic activity with IC50 (19.17 μg). The data showed that compounds containing a polynitrogen ring such as compounds 4, 9, 11a, and 16 exhibit the highest cytotoxic activity and this activity increases when the polynitrogen ring is attached to the acyl hydrazide moiety (compound 4), thio bond (compound S 9 and 11a), and NH2 group (compound 9). Therefore, we recommended that we can use the synthesized compound, especially [1,2,4]triazole-3-thiol 9 and [1,2,4]triazolo[4,3-b][1,2,4] triazole derivative 16, in the formulation of antibiotics as drugs to increase the sensitivity of antibiotics that stimulate cancer treatment and cause apoptosis for the human colon carcinoma cancer cell.

Table 1

Evaluation of Cytotoxicity as a Function of Cell Viability of Some Chosen Compounds on Colon Carcinoma Cells (HCT-116)

	cell viability (%)
sample conc. (μg)	4	5	7	9	10	11a	14	16	vinblastine standard
50	18.17	30.14	27.54	14.12	24.11	19.12	22.84	15.33	13.31
25	23.33	43.03	38.22	16.22	34.15	25.75	30.93	18.03	15.65
12.5	33.11	54.50	48.12	22.09	45.07	36.65	40.76	29.56	20.11
6.25	57.18	79.64	73.98	44.05	69.33	59.90	66.77	49.87	41.76
3.125	68.85	86.32	80.08	53.21	82.33	73.89	79.33	60.98	50.22
1.56	85.23	97.86	95.32	68.15	93.13	88.23	90.77	78.94	63.43
0.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00

Figure 1

Inhibitory activities against colon carcinoma cells (HCT-116).

Table 2

IC50 (μg) of Some Chosen Compounds on Colon Carcinoma Cells (HCT-116)

compounds	4	5	7	9	10	11a	14	16	vinblastine standard
IC50	8.44	19.17	12.05	4.16	11.23	8.72	10.11	6.15	3.34

Figure 2

Comparison of IC50 (μg) for compounds 4, 5, 7, 9, 10, 11a, 14, 16, and Vinblastine.

In vitro Studies

Cell lines: Human colon carcinoma (HCT-116) cells were received from the American Type Culture set (ATCC, Rockville, MD, USA). The cells have been grown supplemented with 10% inactivated fetal calf serum and 50 μg/mL of gentamycin with RPMI-1640 medium. The cells were stored in a humid atmosphere with 5% CO2 and 37° C and subcultured 2 to 3 days a week. Cytotoxic assay of the tested compounds 4, 5, 7, 9, 10, 11a, 14, and 16: In a 96-well microtiter plate incubated for 24 h at 37° C in a humidified incubator with 5% CO2, monolayers of 10,000 cells adhered to the bottom of wells. The monolayers were then washed with sterile phosphate-buffered saline (0.01 M pH 7.2), and the cells were simultaneously processed in a fresh maintenance medium and incubated at 37° C with 100 μL of various dilutions from the tested compounds or Vinblastine drug as the standard. For each concentration of the tested compound, six wells were used while control cells were made in the absence of the tested compounds. The observation was performed under an inverted microscope every 24 h, and the number of (viable) surviving cells was counted using staining of cells with crystal violet followed by cell lysis using glacial acetic acid(33%) and recording the absorbance at 495 nm, taking into account that the absorption of the untreated cell is 100%.

The percentage of cell viability % was calculated from the following eq where ODt is the mean optical density of wells treated with the test compound and ODc is the mean optical density of untreated (control) cells. The 50% inhibitory concentration (IC50), which is the concentration required to cause toxic effect in 50% of inactivated cells, was estimated from graphic plots.

Conclusions

New derivatives of [1,3,4]oxadiazole, [1,2,4]triazol, [1,2,4]triazine-3,4-dione, [1,2,4]triazin-4-one, and [1,2,4]triazolo[4,3-b][1,2,4]triazole were designed and synthesized. The synthetic schemes of the synthesized compounds include the cyclocondensation of dicarbonyl ester 2 with phenyl hydrazine to give the 1H-pyrazole-3-carboxylate derivative 3, followed by hydrazinolysis of 3 to give the corresponding carboxylic acid hydrazide 4, which reacted with carbon disulfide or ammonium thiocyanate to afford [1,3,4]oxadiazole 5 or triazole-3-thione 7, respectively. Hydrazinolysis of [1,3,4]oxadiazole-2-thione 5 afforded the corresponding amino[1,2,4]triazole-3-thiol 9 which was treated with different aromatic aldehydes to obtain a new series of Schiff bases 10a–c. Mannich bases 11a–c were obtained from the reaction of Schiff bases 10a–ce with morpholine and formaldehyde. Moreover, treatment of triazole-3-thione 7 with hydrazine hydrate gave 3-hydrazino[1,2,4]triazol derivative 13, which underwent cyclocondensation with diethyl oxalate, chloroacetic acid, and formic acid to give the corresponding [1,2,4]triazine-3,4-dione 14, [1,2,4]triazin-4-one 15, and [1,2,4]triazolo[4,3-b][1,2,4]triazole 16, respectively. Screening of the tested compounds 4, 5, 7, 9, 10, 11a, 14, and 16 against the human colon carcinoma cancer cell lines showed that the compound [1,2,4]triazole-3-thiol 9 exhibiting cytotoxic activity was roughly equivalent to the standard Vinblastine drug, while compounds 4, 7, 10, 11a, 14, and 16 exhibited moderate cytotoxic activity. Therefore, we recommended that we can use the synthesized compounds [1,2,4]triazole-3-thiol 9 and [1,2,4]triazolo[4,3-b][1,2,4] triazole derivative 16 in the formulation of antibiotics as drugs to increase the sensitivity of antibiotics that stimulate cancer treatment and cause apoptosis for the human colon carcinoma cancer cell.

Experimental Section

Materials and Methods

Melting points for the prepared compounds are uncorrected and have been measured using MEL TEMP II equipment. The IR spectra (KBr) were recorded on a Perkin-Elmer FTIR spectrophotometer. On a Bruker spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR) in DMSO-d6 as solvent, the NMR spectrum, including 1H NMR and 13C NMR, was recorded using tetramethylsilane (TMS) as the internal reference standard. Chemical shift values are expressed in ppm and are abbreviated as follows: (s) for singlet, (d) for doublet, (t) for triplet, and (m) for multiplet signals. The NMR spectra were performed at Faculty of Science, Kafr Elsheikh University. At the Micro Analytical Center, El-azhr University, elemental microanalyses were performed. Mass spectra were run on a DI analysis Shimadzu QP-2010 plus mass spectrometer at the Micro Analytical Unit of Cairo University. In order to follow the progress of the chemical reaction and the purity of the compounds, a TLC analytical silica gel plate 60 F254 was used. The chemical reagents used in synthesis were purchased from Aldrich, Fluka, and Sigma.

Ethyl-4-(1-methyl-1H-pyrrol-3-yl)-2,4-dioxobutanoate 2

A solution of 3-acetyl-1-methylpyrrole (0.01 mol, 1.23 g) and sodium ethoxide (0.015 mol, 1.02 g) in 100 mL of ethanol was stirred for 30 min at room temperature gradually, and 0.01 mol (1.46 g) of diethyloxalate was added and stirred for 10 h under the same conditions. The reaction mixture was neutralized using (1:1) hydrochloric acid to give a pale yellow precipitate, then filtrated and washed with water, and recrystallized from ethanol to give compound 2. Yield (1.79 g, 80%); mp (91–93) °C; IR (KBr) νmax: 3023 (C–H aromatic), (C–H aliphatic) 2911, 1733 (C=O), 1637 cm–1 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 1.37 (t, 3H, J = 7.5 Hz, CH3), 3.42 (S, 3H, NCH3), 4.11 (q, 2H, J = 7.2 Hz, CH2), 4.67 (S, 2H, CH2), 6.50–6.89 (m, H-4 and H-5 of pyrrolyl), 7.33 ppm (s, H-2 of pyrrolyl). (m/z): 223 (M+, 73%). Anal. Calcd for C11H13NO4 (223.23): C, 59.19; H, 5.83; N, 6.28. Found: C, 59.11; H, 5.77; N, 6.31.

Ethyl-5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazole-3-carboxylate 3

A solution of phenylhydrazine (0.01 mol, 1.08 g) and ethyl ester 2 (0.01 mol, 2.23 g) in 15 mL of acetic acid was heated under reflux for 6 h. After cooling, the reaction mixture was poured into ice water and then left at room temperature for complete precipitation, then filtrated and washed with water, and recrystallized from ethanol to give compound 3. Yield (2.27 g, 77%); mp (108–110) °C; IR (KBr) νmax: 3047 (C–H aromatic), (C–H aliphatic) 2934, 1724 (C=O), 1653, 1629 cm–1 (C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 1.25 (t, 3H, J = 7.6 Hz, CH3), 3.51 (S, 3H, NCH3), 4.29 (q, 2H, J = 7.6 Hz, CH2), 6.65–7.65 (m, 8H, Ar–H), 7.83 ppm (S, H, pyrazolyl-H). 13C NMR (100 MHz, DMSO-d6): δ 16.21 (CH3), 42.24 (NCH3), 64.66 (CH2), 114.43, 119.54, 122.32, 125.02, 129.12, 131.44, 136.64, 138.90, 140.22 (12 C of aryl carbons), 162.24 (C=N), 176.37 ppm (C=O). (m/z): 294 (M+, 37%). Anal. Calcd for C17H17N3O2 (294.34): C, 69.07; H, 5.76; N, 14.22. Found: C, 68.94; H, 5.65; N, 14.20.

5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazole-3-carboxylic Acid Hydrazide 4

A solution of ethyl ester 3 (0.01 mol, 2.95 g) and 2 mL of hydrazine hydrate (99%) in 50 mL of ethanol was heated under reflux for 5 h. The reaction mixture was concentrated, filtrated, and recrystallized from ethanol to give the corresponding hydrazide 4. Yield (2.03 g, 73%); mp (142–144) °C; IR (KBr) νmax: 3356, 3189 (NH, NH2), 3051 (C–H aromatic), 2942 (C–H aliphatic), 1678(C=O), 1642, 1618 cm–1 (C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.42 (S, 3H, NCH3), 4.34 (s, 2H, NH2, D2O exchangeable), 6.78–7.95 (m, 9H, Ar–H), 9.89 ppm (s, 1H, NH, D2O exchangeable. 13C NMR (100 MHz, DMSO-d6): δ 39.83 (NCH3), 116.37, 118.33, 124.25, 127.11, 130.49, 134.09, 138.07, 140.77 (12 C of aryl carbons), 156.54 (C=N), 170.26 ppm (C=O). (m/z): 281 (M+, 26%). Anal. Calcd for C15H15N5O (281.31): C, 63.98; H, 5.32; N, 24.88. Found: C, 63.92; H, 5.35; N, 24.83.

5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-3H-[1,3,4]oxadiazole-2-thione 5

A mixture of hydrazide 4 (0.01 mol, 2.81 g) and carbon disulfide (1.5 mL) in 60 mL of 10% ethanolic potassium hydroxide was heated under reflux for 10 h, and then the reaction mixture was allowed to cool and poured into ice water. The reaction mixture was neutralized with 10% hydrochloric acid in order to provide a precipitate, and then the precipitate was filtrated and washed with water and recrystallized from ethanol/DMF to offer compound 5. Yield (2.43 g, 75%); mp (124–126) °C; IR (KBr) νmax: 3363, (NH), 3049 (C–H aromatic), 2977 (C–H aliphatic), 1642, 1618 cm–1 (C=N, C=C), 1238 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.56 (S, 3H, NCH3), 6.69–7.83 (m, 9H, Ar–H), 8.44 ppm (s, 1H, NH, D2O exchangeable. 13C NMR (100 MHz, DMSO-d6): δ 44.30 (NCH3), 114.99, 116.08, 121.22, 125.16, 127.49, 129.12, 133.34, 137.88, 139.63 (12 C of aryl carbons), 154.48, 159.55 (2 C=N), 186.11 ppm (C=S). (m/z): 323 (M+, 27%). Anal. Calcd for C16H13N5OS (323.37): C, 59.37; H, 4.02; N, 21.65. Found: C, 59.40; H, 4.00; N, 21.58.

1-(5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazoloyl)-3-thiosemicarbazide 6

A solution of carboxylic acid hydrazide 4 (0.01 mol, 2.81 g), ammonium thiocyanate (0.01 mol, 1.52 g), and 5 mL of hydrochloric acid in 50 mL of ethanol was heated under reflux for 6 h. After cooling, a white precipitate is formed, which is filtrated and recrystallized with DMF/ethanol to give the corresponding thiosemicarbazide 6. Yield (2.79 g, 82%); mp (173–175) °C; IR (KBr) νmax: 3471, 3285, 3265 (NH2, 2NH), 3012 (C–H aromatic), 2955 (C–H aliphatic), 1679 (C=O), 1631, 1606 cm–1 (C=N, C=C), 1176 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.39 (S, 3H, NCH3), 4.05 (s, 2H, NH2, D2O exchangeable), 4.66 (s, H, SNH, D2O exchangeable), 6.52–7.69 (m, 9H, Ar–H), 9.14 ppm (s, 1H, NH, D2O exchangeable). (m/z): 340(M+, 36%). Anal. Calcd for C16H16N6OS (340.40): C, 56.40; H, 4.70; N, 24.68. Found: C, 56.33; H, 4.64; N, 24.61.

5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,4-dihydro-[1,2,4]triazole-3-thione 7

A solution (0.005 mol, 1.70 g) of thiosemicarbazide 6 was refluxed in 30 mL of 10% ethanolic potassium hydroxide for 5 h. The reaction mixture was cooled and neutralized with 10% hydrochloric. The precipitate was filtrated, washed with water, and recrystallized from ethanol/DMF to give the corresponding triazole-3-thione 7. Yield (1.26 g, 78%); mp (193–195) °C; IR (KBr) νmax: 3363,3287 (2NH), 3011 (C–H aromatic), 2932 (C–H aliphatic), 1637, 1609 (C=N, C=C), 1187 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.61 (S, 3H, NCH3), 4.89 ppm (s, 1H, NH, D2O exchangeable, 6.81–8.02 (m, 9H, Ar–H), 8.84 ppm (s, 1H, NH, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6): δ 39.54 (NCH3), 111.45, 113.17, 120.66, 124.90, 126.37, 129.18, 133.77, 138.22, 139.10 (12 C of aryl carbons), 152.52, 154.06 (2 C=N), 179.66 ppm (C=S). (m/z): 322 (M+, 34%). Anal. Calcd for C16H14N6S (322.39): C, 59.56; H, 4.34; N, 26.06. Found: C, 59.52; H, 4.28; N, 26.13.

4-Amino-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-4H-[1,2,4]triazole-3-thiol 9

1 mL of hydrazine hydrate (99%) was added to a solution of 0.005 mol (1.66 g) of compound 5 in 40 mL of ethanol and then refluxed for 5 h. Drops of 10% potassium hydroxide was added in order to obtain a precipitate and then the precipitate is filtrated, acidified with drops of hydrochloric acid, washed with water, and recrystallized from ethanol to give the corresponding compound 9. Yield (1.02 g, 61%); mp (153–155) °C; IR (KBr) νmax: 3256 (NH2), 3069 (C–H aromatic), 2975 (C–H aliphatic), 1623, 1601 cm–1 (C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.37 (S, 3H, NCH3), 4.85 (s, 2H, NH2, D2O exchangeable), 6.89–8.07 (m, 9H, Ar–H), 12.95 ppm (s, 1H, SH). 13C NMR (100 MHz, DMSO-d6): δ 44.17 (NCH3), 120.88, 122.76, 127.53, 129.43, 135.77, 138.02, 139.65, 140.90 (12 C of aryl carbons), 158.22, 160.05, 163.41 ppm (3C=N). (m/z): 337 (M+, 56%). Anal. Calcd for C16H15N7S (337.40): C, 56.91; H, 4.45; N, 29.05. Found: C, 56.84; H, 4.39; N, 28.98.

General method for the preparation of 4-(Arylidene-amino)-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,4-dihydro-[1,2,4]triazole-3-thiones 10a–c

Triazole-3-thiol 9 (0.005 mol, 1.69 g) was fused with 0.01 mol of aromatic aldehydes in sand bath for 3 h at (160–170) °C. After cooling, the formed solid was recrystallized from DMF to give the corresponding [1,2,4]triazole-3-thiones 10a–c.

4-(Benzylidene-amino)-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,4-dihydro-[1,2,4]triazole-3-thione 10a

It was synthesized according to the previous general method: Yield (1.53 g, 72%); mp (182–184) °C; IR (KBr) νmax: 3042 (C–H aromatic), 2961 (C–H aliphatic), (1643–1630), 1609 cm–1 (3C=N, C=C), 1157 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.63 (S, 3H, NCH3), 6.56–7.93 (m, 14H, Ar–H), 8.21 (s, 1H, NH, D2O exchangeable), 8.78 ppm (s, 1H, ArCHN) . 13C NMR (100 MHz, DMSO-d6): δ 38.99 (NCH3), 124.68, 129.43, 131.61, 133.22, 134.08, 136.54, 137.44, 138.20, 142.49, 143.33, 144.55 (18 C of aryl carbons), 154.09, 158.66, 162.58 (3C=N), 182.04 ppm (C=S). (m/z): 425 (M+, 39%). Anal. Calcd for C23H19N7S (425.54): C, 64.86; H, 4.47; N, 23.03. Found: C, 64.79; H, 4.43; N, 22.94.

4-[(4-Chloro-benzylidene)-amino]-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,4-dihydro-[1,2,4]triazole-3-thione 10b

A solid of 10b was obtained after recrystallization according to the previous general method: Yield (1.79 g, 78%); mp (196–198) °C; IR (KBr) νmax: 3079 (C–H aromatic), 2988 (C–H aliphatic), (1638–1627), 1602 cm–1 (3C=N, C=C), 1138 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.41 (S, 3H, NCH3), 6.76–7.98 (m, 13H, Ar–H), 8.57 (s, 1H, NH, D2O exchangeable), 8.72 ppm (s, 1H, ArCHN). Anal. Calcd for C23H18N7SCl (459.95): C, 60.01; H, 3.91; N, 21.30. Found: C, 59.94; H, 3.85; N, 21.32.

5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-4-[(4-nitro-benzylidene)-amino]-2,4-dihydro-[1,2,4]triazole-3-thione 10c

It was synthesized according to the previous general method: Yield (1.91 g, 81%); mp (212–214) °C; IR (KBr) νmax: 3055 (C–H aromatic), 2949 (C–H aliphatic), (1643–1629), 1609 cm–1 (3C=N, C=C), 1185 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 3.64 (S, 3H, NCH3), 6.81–8.11 (m, 13H, Ar–H), 8.62 (s, 1H, NH, D2O exchangeable), 8.94 ppm (s, 1H, ArCHN). Anal. Calcd for C23H18N8O2S (470.51): C, 58.66; H, 3.83; N, 23.80. Found: C, 58.57; H, 3.80; N, 23.73.

General method for the preparation of 4-(Arylidene -amino)-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2-morpholin-4-ylmethyl-2,4-dihydro-[1,2,4]triazole-3-thione 11a–c

A solution of 0.003 mol of the 10a-c compounds in DMF was added to a solution of morpholine (0.03 mol, 2.61 g) and formaldehyde (0.03 mol, 0.9 g) in DMF. The reaction mixture was stirred for 12 h at room temperature. The precipitate was collected by filtration and recrystallized from ethanol after washing by water to obtain 11a–c compounds.

4-(Benzylidene-amino)-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2-morpholin-4-ylmethyl-2,4-dihydro-[1,2,4]triazole-3-thione 11a

According to the abovementioned method, compound 11a was obtained with the following data: Yield (1.18 g, 75%); mp (158–160) °C; IR (KBr) νmax: 3069 (C–H aromatic), 2943 (C–H aliphatic), (1637–1628), 1589 cm–1 (3C=N, C=C), 1181 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 2.84 (b, 4H, 2NCH2, morpholine), 3.38 (S, 3H, NCH3), 3.71 (b, 4H, 2OCH2, morpholine), 4.92 (S, 2H, NCH2N), 6.77–7.82 (m, 14H, Ar–H), 8.59 ppm (s, 1H, ArCHN). 13C NMR (100 MHz, DMSO-d6): δ 41.07 (NCH3), 50.89 (2NCH2, morpholine), 64.33 (2OCH2, morpholine), 69.85 (NCH2N), 123.05, 127.33, 129.76, 132.15, 134.17, 136.22, 138.64, 139.99, 143.49, 145.41, 147.79 (18 C of aryl carbons), 157.11, 160.23, 163.59 (3C=N), 173.42 ppm (C=S). (m/z): 524 (M+, 31%). Anal. Calcd for C28H28N8OS (524.64): C, 64.04; H, 5.43; N, 21.35. Found: C, 63.96; H, 4.41; N, 21.38.

4-[(4-Chloro-benzylidene)-amino]-5-[5-(1-methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2-morpholin-4-ylmethyl-2,4-dihydro-[1,2,4]triazole-3-thione 11b

After recrystallization according to the previous general method, compound 11b was obtained: Yield (1.31 g, 78%); mp (169–171) °C; IR (KBr) νmax: 3088 (C–H aromatic), 2971 (C–H aliphatic), (1640–1631), 1610 cm–1 (3C=N, C=C), 1196 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 2.70 (b, 4H, 2NCH2, morpholine), 3.47 (S, 3H, NCH3), 3.78 (b, 4H, 2OCH2, morpholine), 4.86 (S, 2H, NCH2N), 6.83–7.95 (m, 13H, Ar–H), 8.73 ppm (s, 1H, ArCHN). Anal. Calcd for C28H27N8OSCl (559.09): C, 60.10; H, 4.83; N, 20.03. Found: C, 60.03; H, 4.78; N, 19.95.

5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2-morpholin-4-ylmethyl-4-[(4-nitro-benzylidene)-amino]-2,4-dihydro-[1,2,4]triazole-3-thione 11c

According to the previous general method, compound 11c was obtained: Yield (1.37 g, 80%); mp (181–183) °C; IR (KBr) νmax: 3063 (C–H aromatic), 2962 (C–H aliphatic), (1643–1628), 1608 cm–1 (3C=N, C=C), 1161 cm–1 (C=S); 1H NMR (400 MHz, DMSO-d6): δ 2.74 (b, 4H, 2NCH2, morpholine), 3.54 (S, 3H, NCH3), 3.92 (b, 4H, 2OCH2, morpholine), 4.81 (S, 2H, NCH2N), 6.83–8.13 (m, 13H, Ar–H), 8.69 ppm (s, 1H, ArCHN). Anal. Calcd for C28H27N9O3S (569.64): C, 58.98; H, 4.74; N, 22.12. Found: C, 58.91; H, 4.75; N, 22.04.

2-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-5-methylsulfanyl-[1,3,4]oxadiazole 12

A solution of 0.005 mol (1.66 g) of oxadiazole-2-thione 5 with 0.30 mL of methyl iodide and 40 mL of 10% ethanolic potassium hydroxide was refluxed for 7 h. The reaction mixture was cooled and poured into ice water and neutralized with 10% hydrochloric. The precipitate was filtrated, washed with water, and recrystallized from ethanol/DMF to give oxadiazole 12. Yield (1.30 g, 77%); mp (106–108) °C; IR (KBr) νmax: 3033 (C–H aromatic), 2956 (C–H aliphatic), 1642,1635, 1618 cm–1 (2 C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.49(S, 3H, NCH3), 3.82(S, 3H, SCH3, 6.69–7.83 (m, 9H, Ar–H). (m/z): 337 (M+, 31%). Anal. Calcd for C17H15N5OS (337.40): C, 60.46; H, 4.45; N, 20.75. Found: C, 60.41; H, 4.42; N, 20.67.

3-Hydrazino-5-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-4H-[1,2,4]triazol 13

A mixture of 0.005 mol (1.61 g) of triazole-3-thione 7 and 8 mL of hydrazine hydrate in 20 mL DMF was heated under reflux for 8 h. The reaction mixture was allowed to cool and poured into ice water. After filtration, the crystallization took place from DMF/ethanol to obtain the corresponding [1,2,4]triazol 13. Yield (1.19 g, 74%); mp (221–223) °C; IR (KBr) νmax: (3371–3182) (2NH, NH2), 3034 (C–H aromatic), 2952 (C–H aliphatic), (1645–1632), 1612 cm–1 (3C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.42 (S, 3H, NCH3), 4.89 ppm (s, 2H, NH2, D2O exchangeable), 5.76 ppm (s, H, NH of pyrrole, D2O exchangeable) 6.88–8.11 (m, 9H, Ar–H), 9.13 ppm (s, 1H, NH, D2O exchangeable). (m/z): 320 (M+, 41%). Anal. Calcd for C16H16N8 (320.35): C, 59.93; H, 4.99; N, 34.96. Found: C, 59.86; H, 5.02; N, 34.89.

7-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,8-dihydro-[1,2,4]triazolo[5,1-c] [1,2,4]triazine-3,4-dione 14

A solution of 3-hydrazio-[1,2,4]triazol (0.003 mol, 0.96 g) 13 and 0.003 mol (0.44 g) of diethyloxalate in 30 mL of DMF was heated under reflux for 10 h. The reaction mixture was cooled and added to ice water, followed by filtration and recrystallization from DMF to obtain the triazine-3,4-dione 14. Yield (0.76 g, 68%); mp (252–254) °C; IR (KBr) νmax: (3362–3165) (2NH), 3031 (C–H aromatic), 2943 (C–H aliphatic), 1711 (b, 2C=O) (1633–1624), 1601 cm–1 (3C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.69 (S, 3H, NCH3), 5.88 (s, H, NH of pyrrole, D2O exchangeable),6.54–7.89 (m, 9H, Ar–H), 10.73 ppm (s, 1H, NHCO, D2O exchangeable). 13C NMR (100 MHz, DMSO-d6): δ 43.22 (NCH3), 113.23, 116.09, 122.54, 125.11, 127.43, 129.44, 132.09, 135.58, 138.61 (12 C of aryl carbons), (151.22–153.17) (3C=N), 168.84 ppm (C=O). (m/z): 374 (M+, 31%). Anal. Calcd for C18H14N8O2 (374.36): C, 57.70; H, 3.74; N, 29.92. Found: C, 57.74; H, 3.69; N, 29.87.

7-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-2,8-dihydro-3H-[1,2,4]triazolo [5,1-c][1,2,4]triazin-4-one 15

A mixture of 3-hydrazio-[1,2,4]triazol (0.003 mol, 0.96 g) 13 and 0.003 mol (0.29 g) of chloroacetic acid in 30 mL of DMF was refluxed for 5 h and then cooled, and the reaction mixture was poured into ice water, washed with water, dried, and recrystallized from DMF. Yield (0.81 g, 75%); mp (234–236) °C; IR (KBr) νmax: (3376–3181) (2NH), 3056 (C–H aromatic), 2963 (C–H aliphatic), 1686 (C=O), (1640–16220), 1604 cm–1 (3C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.55 (S, 3H, NCH3), 3.78 (S, 2H, CH2), 5.61, 6.09 (s,2 H, 2NH of, D2O exchangeable), 6.70–7.84 (m, 9H, Ar–H) .13C NMR (100 MHz, DMSO-d6): δ 41.19 (NCH3), 52.349 (CH2), 111.17, 114.23, 119.34, 123.22, 125.64, 128.48, 131.11, 134.76, 137.37 (12 C of aryl carbons), (149.83–151.23) (3C=N), 165.18 ppm (C=O). (m/z): 360 (M+, 31%). Anal. Calcd for C18H16N8O (360.37): C, 59.94; H, 4.44; N, 31.08. Found: C, 59.87; H, 4.45; N, 31.07.

6-[5-(1-Methyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-7H-[1,2,4]triazolo[4,3-b][1,2,4] Triazole 16

3-hydrazio-[1,2,4]triazol (0.003 mol, 0.96 g) 13 was refluxed in 20 mL of formic acid for 6 h, and then the mixture was cooled, poured into ice/water, stirred well, filtered, washed with water, dried, and recrystallized from DMF to give the compound 16. Yield (0.70 g, 71%); mp (211–213) °C; IR (KBr) νmax: 3289 (NH), 3061 (C–H aromatic), 2967 (C–H aliphatic), (1637–16,223), 1600 cm–1 (4C=N, C=C); 1H NMR (400 MHz, DMSO-d6): δ 3.39 (S, 3H, NCH3), 5.34 (s,1 H, NH of, D2O exchangeable), 6.61–7.75 (m, 10H, Ar–H),. (m/z): 330 (M+, 38%). Anal. Calcd for C17H14N8 (330.35): C, 61.75; H, 4.24; N, 33.90. Found: C, 61.77; H, 4.18; N, 33.85.