Azoles and bis-Azoles: Synthesis and Biological Evaluation as Antimicrobial and Anti-cancer Agents

Abstract

Novel hydrazonoyl halides 3, 5 and bis-hydrazonoyl halides 7, 9 were synthesized. The synthetic utility of bis-hydrazonoyl halide 7 was explored to prepare novel bis-azole 13 with antipyrine moiety. On the other hand, [1,3,4]thiadiazol-2(3H)-ylidene 17 and thiazol-2(3H)-ylidene 21 derivatives, with antipyrine moiety, were prepared from the reaction of 3-mercapto-3-(phenylamino)acrylamide derivative 10 with N-phenyl benzenecarbohydrazonoyl chloride (14) and 3-(2-bromoacetyl)-2H-chromen-2-one (18), respectively. The structures of the isolated products were confirmed by spectral data (IR, ¹H-NMR, ¹³C-NMR, MS) and elemental analyses. The anti-cancer activitiy of the synthesized products against the colon carcinoma (HCT) cell line was determined and the results revealed promising activity of compound 3. In addition, the antimicrobial activity of some selected products was evaluated. The results proclaimed that compounds 9, 17, and 21 have high antibacterial activity against Gram-positive bacteria (SA, BS) and Gram-negative bacteria (PA, EC).

Development of heterocycles with antimicrobial and antitumor activities represents one of the most important researches in therapeutical chemistry.¹⁾ Azoles are an important class of heterocycles with broad spectrum of biological activities such as antimicrobial,^2–4) anticancer,⁵⁾ anti-inflammatory,⁶⁾ and antivirus.⁷⁾ Hydrazonoyl halides⁸⁾ are proved to be useful precursors for synthesis of numerous bioactive azoles such as, pyrazoles,^9,10) thiazoles,^11,12) 1,3,4-thiadiazoles,^13,14) spiropyrazoles,^15,16) and spiro[1,2,4]thiadiazoles.¹⁷⁾ Also, representative examples of bis-azoles exhibit anti-cancer activity.¹⁸⁾ bis-Hydrazonoyl halides are considered to be versatile synthons for construction of bis-azoles. 1,3-Dipolar cycloaddition of bis-nitrilimines [prepared in situ via dehydrohalogenation of bis-hydrazonoyl halides under basic conditions] with activated C=C bond gave the respective bis-pyrazoles.¹⁹⁾ Moreover, treatment of carbonothioic dihydrazide with bis-hydrazonoyl halides afforded bis-[1,3,4]thiadiazoles.²⁰⁾ As a part of our research interest towards developing new routes for the synthesis of a variety of heterocyclic systems with promising biological and pharmacological activities,^21–24) In the present work, we demonstrated a strategy for the synthesis of novel hydrazonoyl halides, bis-hydrazonoyl halides, azoles, and bis-azoles bearing antipyrine moiety and investigated their antimicrobial and anticancer potential activities.

Results and Discussion

Chemistry

The usual method for the synthesis of hydrazonoyl and bis-hydrazonoyl halides employs a Japp–Klingemann reaction²⁵⁾ via cleavage of the acetyl group(s). Thus, coupling of 3-chloro-2,4-pentanedione (1) with diazonium chlorides of 4-aminoantipyrine 2, 4-(N-pyrimidin-2-ylsulfamoyl)aniline 4, and benzidine 6 in ethanol, in the presence of sodium acetate afforded Nʹ-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-oxo-propanhydrazonoyl chloride (3), Nʹ-[4-(N-pyrimidin-2-ylsulfamoyl)phenyl]-2-oxopropanehydrazonoyl chloride (5), and Nʹ,Nʺ-(biphenyl-4,4ʹ-diyl)bis(2-oxopropanehydrazonoyl chloride) (7), respectively (Chart 1). Analytical and spectroscopic data were consistent with the final products 3, 5, and 7 (see Experimental).

Chart 1

Hydrazones can be formulated in different possible tautomeric structures, namely keto-hydrazone (A), azo-enol (B), and CH-azo tautomer (C) (Fig. 1). The ¹H-NMR spectra of the studied compounds showed signal in the region of δ=13.72–14.42 ppm assignable to hydrazone proton (–CH=N–NH–).^26–28) Signal at δ=5.27²⁹⁾ ppm, which is characteristic for the CH protons of CH-azo form, was not detected. Also, IR spectra did not reveal broad signal at 3434 cm⁻¹, which is characteristic for OH of azo-enol form.³⁰⁾ These spectroscopic analysis allows to rule out the azo-enol form (B) and CH-azo form (C) (Fig. 1).

Fig. 1. Possible Tautomeric Structures of Hydrazones

Keto-hydrazone tautomer (A) can be existed in two geometric structures (E and Z). IR spectra of compounds 3, 5, and 7 showed a shift of the CO bond stretching to lower wave number due to both conjugation with C=N and formation of a hydrogen bond with the NH group. Furthermore, the downfield shift of hydrazone proton signal in ¹H-NMR is characteristic for the formation of an intramolecular hydrogen bond of the NH proton for structure (A). These results confirm E-form.³¹⁾

Condensation of Nʹ,Nʺ-(biphenyl-4,4ʹ-diyl)bis(2-oxo-propanehydrazonoyl chloride) (7) with 4-aminoantipyrine (8) in ethanol in the presence of catalytic amount of glacial acetic acid, under reflux, gave novel bis-hydrazonoyl halide, namely as, Nʹ,Nʺ-(biphenyl-4,4ʹ-diyl)bis[2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)propanehydrazonoyl chloride] (9) (Chart 2).

Chart 2

The IR spectrum of the isolated product showed absorption bands at ν=3336, 1656 cm⁻¹ characteristic for NH and carbonyl of amide group, respectively. Its ¹H-NMR spectrum showed three singlet signals at δ 2.22, 2.42, 3.02 ppm due to three methyl protons, and another singlet signal (D₂O-exchangeable) at δ 13.94 ppm assignable to NH.

Treatment of Nʹ,Nʺ-(biphenyl-4,4ʹ-diyl)bis(2-oxopropanehydrazonoyl chloride) (7) (1 mol) with 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-mercapto-3-(phenylamino)acrylamide (10) (2 mol) in ethanol, in the presence of catalytic amount of triethylamine, furnished 2,2ʹ-[3,3ʹ-(biphenyl-4,4ʹ-diyl)bis(5-acetyl-1,3,4-thiadiazole-3(3H)-yl-2(3H)-ylidene)]bis[2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide] (13) (Chart 3).

Chart 3

The reaction proceeds via S-alkylation,³²⁾ with removal of two moles of hydrogen chloride, to give bis(S-alkylated) intermediate 11. Intramolecular Michael³³⁾ type addition of 11, under the employed reaction conditions, gives bis-cycloadduct 12. Elimination of two moles of aniline from 12 gives the final product 13 (cf. Chart 3). The characterization of the isolated product 13 is based on its spectral data (IR, ¹H-NMR, MS) and elemental analysis. The IR spectra exhibited absorption bands at ν 3428 (NH), 2193 (C≡N), 1653, 1637 (2C=O) cm⁻¹. The ¹H-NMR spectrum revealed singlet peak at δ 2.51 ppm attributable to the acetyl group and another singlet peak at δ 11.30 ppm (D₂O exchangeable) assignable to NH group.

Next, our study was extended to synthesis new azoles with antipyrine moiety. Thus, refluxing of 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-mercapto-3-(phenylamino)acrylamide (10) with N-phenyl benzenecarbohydrazonoyl chloride (14) in ethanol, in the presence of catalytic amount of triethylamine, furnished one isolable product (as tested by TLC), which identified as, 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)acetamide (17) (Chart 4).

Chart 4

The IR spectrum of the isolated product 17, revealed absorption bands at ν=1630, 1668, 2193, and 3384 cm⁻¹ due to two carbonyl groups, nitrile and NH functions, respectively. Its ¹H-NMR spectrum showed two singlet signals at δ 2.15 and 3.07 ppm due to two methyl protons, and another singlet signal (D₂O-exchangeable) at δ 8.33 ppm due to NH, in addition to aromatic multiplets in the region δ 7.32–7.86 ppm.

On the other hand, reaction of compound 10 with 3-(2-bromoacetyl)-2H-chromen-2-one (18) under the employed condition gave the respective 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-[4-(2-oxo-2H-chromen-3-yl)-3-phenylthiazol-2(3H)-ylidene)]acetamide (21) in agreement with literature^34,35) for the reaction of 2-cyano-N-aryl-3-(phenylamino)acrylamides with α-haloketones (Chart 5). The reaction proceeds via nucleophilic displacement of hydrogen bromide by thiol group to give S-alkylated intermediate 19, which underwent nucleophilic addition of (PhNH) group to carbonyl group of chromen-2-one ring to give the respective intermediate 20. Dehydration of the latter intermediate gave thiazole derivative 21 as the final product (cf. Chart 5).

Chart 5

Pharmacology. Anti-cancer Activity

The anti-cancer activity of the synthesized compounds 3, 5, 7, 9, 13, 17 and 21 was determined against the colon carcinoma (HCT) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and vinblastine was used as a reference drug. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC₅₀) was determined. Cytotoxic activity was expressed as the mean IC₅₀ of three independent experiments (Table 1).

Table 1. Viability Values and IC₅₀ of Compounds 3, 5, 7, 9, 13, 17, and 21 against HCT-116 Cell Line

Sample number	Sample concentration (µg/mL) viability %							IC50 (µg)
	50	25	12.50	6.25	3.125	1.56	0.00
Vinblastine	23.08	27.35	43.59	53.85	69.23	82.54	100.00	9.8
3	29.14	36.96	56.70	80.94	97.86	100.00	100.00	18.6
5	25.62	38.06	68.72	87.90	98.54	100.00	100.00	20.4
7	33.94	48.76	72.88	87.65	96.98	100.00	100.00	24.0
9	67.40	80.26	91.32	98.44	100.00	100.00	100.00	>50.0
13	34.54	60.42	78.60	90.22	97.68	100.00	100.00	36.2
17	54.28	76.07	90.68	98.54	100.00	100.00	100.00	>50.0
21	34.22	58.13	76.15	90.10	96.61	100.00	100.00	33.2

Table 2. Antimicrobial Activity (µg/mL) of Tested Samples (5, 9, 13, 17, and 21) against Tested Microorganisms

Sample/Tested organism	5	9	13	17	21	Standard (30 µg/mL)
Fungi						Itraconazole	Clotrimazole
Aspergillus fumigatus (AF)	15.2±0.08	14.4±0.5	12.6±0.05	12.4±0.5	15.2±0.06	28±0.05	26±0.1
Geotrichum candidum (GC)	18.4±0.5	18.4±0.1	14.7±0.01	15.4±0.1	18.4±0.04	27±0.1	23±0.3
Candida albicans (CA)	15.4±0.1	17.4±0.09	9.0±0.05	15.8±0.1	11.4±0.1	26±0.02	18±0.1
Syncephalastrum racemosum (SR)	NA	NA	NA	NA	NA	22±0.09	20±0.2
Gram-positive bacteria						Penicillin G	Streptomycin
Staphylococcus aureus (SA)	23.2±0.01	18.9±0.03	10.4±0.03	23.6±0.01	20.3±0.2	29.48±0.82	25±0.2
Bacillis subtilis (BS)	24.3±0.03	20.1±0.03	13.7±0.2	22.4±0.03	21.6±0.4	32.56±0.56	29±0.4
Gram-negative bacteria						Penicillin G	Streptomycin
Pseudomonas aeruginosa (PA)	NA	18.1±0.02	NA	20.1±0.02	19.6±0.1	28.32±0.10	24±0.1
Escherichia coli (EC)	9.4±0.5	18.3±0.06	NA	16.4±0.07	21.7±0.3	33.56±0.78	25±0.3

*NA: No activity, data are expressed in the form of mean±S.D.

The results revealed that compound 3, with antipyrine moiety, has promising anticancer activity against colon carcinoma (HCT) while compounds arylazo 5, bis-arylazo 7, bis-thiadiazoline 13, and thiazoline 21 have moderate activities. On the other hand, compounds 9 and 17 have poor inhibitory activities.

Antimicrobial Evaluation

The newly synthesized compounds (5, 9, 13, 17, and 21) were evaluated in vitro for antibacterial activity against Staphylococcus aureus (SA) and Bacillis subtilis (BS) as examples of Gram-positive bacteria and Pseudomonas aeruginosa (PA) and Escherichia coli (EC) as examples of Gram-negative bacteria. They were also evaluated in vitro for their antifungal activity against Aspergillus fumigatus (AF), Geotrichum candidum (GC), Candida albicans (CA) and Syncephalastrum racemosum (SR) fungal strains. Inhibition zone diameter (IZD) in mm was used as criterion for the antimicrobial activity using agar diffusion well method. Penicillin G and Streptomycin were used as reference drugs for antibacterial activity and Itraconazole, Clotrimazole were used as reference drugs for antifungal activity. The antimicrobial activity result is presented in Table 2.

From the data given by Table 2 we concluded that most of the tested compounds displayed variable degrees of antibacterial activity against Gram-positive bacteria, Gram-negative bacteria strains and also against fungal strains in comparison to the standard in each case which revealed that these compounds are biologically active. Compound 5 exhibited high degree of antibacterial activity against Gram-positive bacteria (SA) and (BS). Compounds 9, 17 and 21 have high degree of antibacterial activity against Gram-positive bacteria (SA, BS), Gram-negative bacteria (EC) and exhibited high inhibition effect against (PA) which emerged as one of the most problematic Gram-negative pathogens.³⁶⁾ All the tested compounds exhibited moderate antifungal activity against (AF, GC, CA) and no inhibition of growth against (SR). The structure antimicrobial activity relationship of the synthesized compounds revealed that the maximum activity was attained with compounds 9, 17 and 21, having antipyrine moiety.

Conclusion

Novel series of azoles and bis-azoles, bearing antipyrine moiety were synthesized and evaluated for their anti-cancer and antimicrobial activities. Most of the newly synthesized products revealed moderate anticancer activity against the colon carcinoma (HCT) cell line. Also they have promising activity against Gram-positive and Gram-positive bacteria.

Experimental

Chemistry. General

All melting points were measured on a Gallenkamp melting point apparatus (Weiss-Gallenkamp, London, U.K.). The infrared spectra were recorded in potassium bromide disks on a pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers (Pye Unicam Ltd., Cambridge, England and Shimadzu, Tokyo, Japan, respectively). The NMR spectra were recorded on a BRUKER VX-500 NMR spectrometer (Varian, Palo Alto, CA, U.S.A.). ¹H spectra were run at 500 MHz and ¹³C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d₆). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer (Shimadzu) at 70 eV. Elemental analyses were carried out at the Micro-analytical Center of Cairo University, Giza, Egypt. The antimicrobial and cytotoxic evaluation of some selected examples was carried out in the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt. 2-Cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-mercapto-3-(phenylamino)acrylamide (10),³⁷⁾ N-phenyl benzenecarbohydrazonoyl chloride (14),³⁸⁾ and 3-(2-bromoacetyl)-2H-chromen-2-one (18)^39,40) were prepared as described in literature.

Synthesis of Novel Hydrazonoyl Chlorides. General Procedure

To a cold solution of 3-chloro-2,4-pentanedione (1) (0.134 g, 1 mmol) in EtOH (50 mL), buffered with sodium acetate trihydrate (3 g), was added the diazonium chloride [prepared by diazotizing the appropriate arylamine (4-aminoantipyrine) or 4-amino-N-(pyrimidin-2-yl)benzenesulfonamide, (1 mmol of each), or benzidine (0.5 mmol) dissolved in concentrated hydrochloric acid (2 mL) with sodium nitrite solution (0.07 g, 1 mmol) in water (2 mL)]. The addition was carried out portion-wise with stirring at 0–5°C over a period of 30 min. After complete addition, the reaction mixture was stirred for further 4 h, at room temperature, then kept in an ice chest for 12 h, and finally diluted with water. The precipitated solid was collected by filtration, washed with water, dried and finally recrystallized from EtOH/N,N-dimethylformamide (DMF) to afford the corresponding hydrazonoyl chlorides 3, 5 and 7, respectively.

Nʹ-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-oxopropanehydrazonoyl Chloride (3)

Yellow needles, (0.25 g, 81%), mp 189–191°C; IR (KBr) ν 3310 (NH), 1685, 1647 (2C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.34 (s, 3H, CH₃), 2.47 (s, 3H, COCH₃), 3.16 (s, 3H, N-CH₃), 7.39–7.57 (m, 5H, Ar-H), 14.42 (s, 1H, D₂O-exchangeable, NH) ppm; ¹³C-NMR (DMSO-d₆) δ 11.2, 26.7, 31.1, 112.3, 124.4, 127.2, 129.3, 133.1, 134.0, 144.2, 158.1, 195.6 ppm; MS, m/z (%) 306 (M⁺, 14), 263 (20), 77 (100). Anal. Calcd for C₁₄H₁₅ClN₄O₂ (306.75): C, 54.82; H, 4.93; N, 18.26. Found: C, 54.87; H, 4.78; N, 18.33%.

Nʹ-[(4-(N-Pyrimidin-2-ylsulfamoyl)phenyl)]-2-oxopropanehydrazonoyl Chloride (5)

Yellow crystals, (0.28 g, 79%), mp 228–230°C; IR (KBr) ν 3309, 3245 (2NH), 1685 (C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.53 (s, 3H, COCH₃), 7.28 (br, 3H, pyrimidine-H), 7.58 (d, 2H, J=10 Hz, Ar-H), 7.81 (d, 2H, J=10 Hz, Ar-H), 10.95 (s, 1H, D₂O-exchangeable, NH), 13.95 (s, 1H, D₂O-exchangeable, NH) ppm; ¹³C-NMR (DMSO-d₆) δ 35.8, 113.8, 114.3, 124.7, 126.8, 127.1, 127.3, 137.7, 145.2, 188.1 ppm. Anal. Calcd for C₁₃H₁₂ClN₅O₃S (353.78): C, 44.13; H, 3.42; N, 19.80; S, 9.06. Found: C, 44.17; H, 3.29; N, 19.77; S, 9.18%.

Nʹ,Nʺ-(Biphenyl-4,4ʹ-diyl)bis(2-oxopropanehydrazonoyl Chloride) (7)

Yellow crystals, (0.31 g, 79%), mp 295–297°C; IR (KBr) ν 3252 (NH), 1676 (C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.52 (s, 6H, 2CH₃), 7.54 (d, 4H, J=5 Hz, Ar-H), 7.67 (d, 4H, J=5 Hz, Ar-H), 13.72 (s, 2H, D₂O-exchangeable, 2NH) ppm; MS, m/z (%) 393 (M⁺+2, 18.5), 392 (M⁺+1, 55.4), 391 (M⁺, 20.8), 286 (100), 186 (70). Anal. Calcd for C₁₈H₁₆Cl₂N₄O₂ (391.25): C, 55.26; H, 4.12; N, 14.32. Found: C, 55.17; H, 4.28; N, 14.40%.

Synthesis of Nʹ,Nʺ-(Biphenyl-4,4ʹ-diyl)bis[2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)-propanehydrazonoyl Chloride] (9)

To an ethanolic solution of hydrazonoyl chloride 7 (0.391 g, 1 mmol) and 4-aminoantipyrine (8) (0.406 g, 2 mmol) was added a few drops of acetic acid and the reaction mixture was refluxed for 4 h. The solvent was evaporated under reduced pressure and the precipitated solid was collected by filtration, washed with EtOH and finally purified by recrystallization from DMF/EtOH to afford brown crystals of 9 in 66%, mp 240–242°C; IR (KBr) ν 3336 (NH), 1656 (C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.22 (s, 6H, 2CH₃), 2.42 (s, 6H, 2CH₃), 3.02 (s, 6H, 2N-CH₃), 6.94–7.54 (m, 18H, Ar-H), 13.94 (s, 2H, D₂O-exchangeable, 2NH) ppm; Anal. Calcd for C₄₀H₃₈Cl₂N₁₀O₂ (761.70): C, 63.07; H, 5.03; N, 18.39. Found: C, 63.18; H, 5.15; N, 18.31%.

Synthesis of 2,2ʹ-[3,3ʹ-(Biphenyl-4,4ʹ-diyl)bis(5-acetyl-1,3,4-thiadiazole-3(3H)-yl-2(3H)-ylidene)]bis[2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide] (13)

To a solution of the compound 10 (0.81 g, 2 mmol) in EtOH (20 mL), the bis-hydrazonoyl chloride 7 (0.391 g, 1 mmol) was added. Triethylamine (0.2 mmol) was added dropwise and the reaction mixture was refluxed for 6 h then allowed to cool. The formed solid was filtered off, washed with EtOH, and recrystallized from DMF/EtOH to afford 13, 68%, mp>300°C; IR (KBr) ν 3428 (NH), 2193 (C≡N), 1653, 1637 (2C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 1.57 (s, 6H, 2CH₃), 2.51 (s, 6H, 2CH₃), 3.08 (s, 6H, 2N-CH₃), 6.77–7.65 (m, 18H, Ar–H), 11.3 (s, 2H, D₂O-exchangeable, 2NH) ppm; MS, m/z (%) 943 (M⁺, 2), 876 (10), 230 (20), 152 (12), 77 (100). Anal. Calcd for C₄₈H₃₈N₁₂O₆S₂ (943.02): C, 61.13; H, 4.06; N, 17.82; S, 6.80. Found: C, 61.04; H, 4.18; N, 17.77; S, 6.64%.

Reactions of 2-Cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-mercapto-3-(phenylamino)acrylamide (10) with N-Phenyl Benzenecarbohydrazonoyl Chloride (14) or 3-(2-Bromoacetyl)-2H-chromen-2-one (18). General Procedure

To a solution of the compound 10 (1 mmol) in EtOH (20 mL), the hydrazonoyl chloride 14 or 3-(2-bromoacetyl)-2H-chromen-2-one (18) (1 mmol) were added. Triethylamine (0.2 mmol) was added dropwise and the reaction mixture was refluxed for 6 h then allowed to cool. The formed solid was filtered off, washed with EtOH, and recrystallized from DMF/EtOH to afford the corresponding thiadiazole 17 or thiazole derivative 21.

2-Cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)acetamide (17)

Yellow crystals (0.35 g, 69%), mp 233°C; IR (KBr) ν 3384 (NH), 2193 (C≡N), 1668, 1630 (2 C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.15 (s, 3H, CH₃), 3.07 (s, 3H, N-CH₃), 7.32–7.86 (m, 15H, Ar-H), 8.33 (s, 1H, D₂O-exchangeable, NH) ppm; ¹³C-NMR (DMSO-d₆) δ 10.8, 36.0, 65.5, 108.1, 114.9, 123.3, 126.1, 126.5, 127.9, 128.2, 129.1, 129.3, 129.5, 130.7, 131.6, 135.2, 138.5, 153.2, 156.4, 162.0, 163.1, 164.7 ppm; MS, m/z (%) 506 (M⁺, 10), 305 (16), 277 (70), 230 (12), 187 (12), 77 (70), 56 (100). Anal. Calcd for C₂₈H₂₂N₆O₂S (506.58): C, 66.39; H, 4.38; N, 16.59; S, 6.33. Found: C, 66.46; H, 4.18; N, 16.49; S, 6.19%.

2-Cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-(4-(2-oxo-2H-chromen-3-yl)-3-phenylthiazol-2(3H)-ylidene)acetamide (21)

Yellow powder (0.35 g, 61%), mp 260°C; IR (KBr) ν 3443 (NH), 2197 (C≡N), 1684, 1653, 1636 (3 C=O) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 2.10 (s, 3H, CH₃), 3.20 (s, 3H, N-CH₃), 7.23–7.65 (m, 16H, Ar-H), 9.50 (s, 1H, D₂O-exchangeable, NH) ppm; MS, m/z (%) 575 (M⁺+2, 33) 573 (M⁺, 10), 386 (36), 202 (32), 77 (100). Anal. Calcd for C₃₂H₂₃N₅O₄S (573.62): C, 67.00; H, 4.04; N, 12.21; S, 5.59. Found: C, 67.06; H, 4.15; N, 12.29; S, 5.48%.

Pharmacology. Cytotoxic Activity

Evaluation of anticancer activity of compounds 3, 5, 7, 9, 13, 17, and 21 was performed at the Regional Center for Mycology and Biotechnology (Al-Azhar University, Cairo). The tested compounds were evaluated for cytotoxicity against the colon carcinoma (HCT) cell line in comparison with vinblastine as a standard drug. Different concentrations of the tested compounds were added to the cell monolayer of tumor. A 48 h continuous newly synthesized compound exposure is used to estimate all availability or growth. The concentration needed to inhibit the proliferation of HCT by 50% (IC₅₀) was determined by the standard MTT assay according to Vijayan et al.⁴¹⁾ using Crystal violet stain (1%). Cells were seeded in 96-well plate at a cell concentration 1×10⁴ cells per well in 100 µL of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers, flat-bottomed microtiter plates using a multichannel pipette. The microtiter plates were incubated at 37°C in a humidified incubator with 5% CO₂ for 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without test sample with DMSO. After incubation of the cells for 24 h at 37°C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125, and 1.56 µg) were added, and the incubation was continued for 48 h and the viable cells yield was determined by a colorimetric method. After the end of incubation period, media were aspirated and the crystal violet solution was added to each well for at least 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates was measured after gently shaken on Microplate reader, using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. Tests were performed in triplicate.

Agar Diffusion Well Method to Determine the Antimicrobial Activity

The microorganism inoculums were uniformly spread using sterile cotton swab on a sterile Petri dish Malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred micro-liter of each sample was added to each well (10 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24–48 h at 37°C (for bacteria) and at 28°C (for fungi). After incubation, the microorganism’s growth was observed. Inhibition of the bacterial and fungal growth were measured in mm. Tests were performed in triplicate.⁴²⁾

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