Novel pyrazole–oxadiazole–chalcone/oxime hybrids as dual EGFR/VEGFR-2 inhibitors with promising anticancer potential: a comprehensive cytotoxicity evaluation, mechanistic insights and SAR analysis

Abstract

A novel series of pyrazole–1,3,4-oxadiazole hybrids incorporating chalcone/oxime scaffolds (10a–10c, 11a–11c, 12a–12i, 13a–13i, and 14a–14i) was synthesized and subjected to an detailed cytotoxicity profile against the NCI-60 human cancer cell line panel. Several derivatives demonstrated broad-spectrum growth inhibition, with compounds 10b, 11a, and 11b emerging as the most active candidates, exhibiting mean GI₅₀ values in the low micromolar range (4.36–16.4 µM). A deeper biological assessment revealed that these hybrids act as dual inhibitors of EGFR and VEGFR-2, with compound 11b showing the highest potency (IC₅₀ = 26.38 nM and 114.17 nM, respectively). Mechanistic studies further confirmed that 11b induced G2/M phase arrest and triggered apoptotic pathways in MCF-7 cells. The oxime-containing analogs exhibited enhanced nitric oxide (NO) release, a property associated with modulation of angiogenesis and increased susceptibility of cancer cells to apoptosis, contributing to their observed anticancer activity. Molecular docking and structure–activity relationship analysis clarified the binding interactions and substitution patterns governing activity, and both cytotoxicity and mechanistic analyses converged on compound 11b as the most promising lead of the series.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11030-025-11411-3.

Introduction

With an estimated 15 million deaths per year by 2030, cancer has become a significant global health crisis, posing a serious challenge for healthcare systems worldwide [1, 2]. Traditional anticancer drugs often suffer from limitations such as drug resistance, severe side effects, and lack of selectivity, which further complicate treatment outcomes [3]. As a result, innovative solutions have been explored to address this global health issue As a result, innovative solutions have been explored to address this global health issue [4]. One promising approach in this regard is the development of multitarget or smart hybrid molecules that combine two or more pharmacophores to target cancer [5, 6]. These rationalized hybrid compounds have attracted considerable interest in cancer treatment, as they have the potential to simultaneously inhibit multiple cancer pathways or targets [7–9].

Two promising protein kinases that could be of particular interest for further work are EGFR (Epidermal Growth Factor Receptor) and VEGF (vascular endothelial growth factor) [10, 11]. EGFR is a receptor tyrosine kinase that plays a crucial role in cell proliferation and survival, and its overexpression or mutations have been observed in various cancers, making it an attractive target for cancer therapy [12–14]. Furthermore, the EGFR mediated signaling pathway stimulates the vascular endothelial growth factor (VEGF) which is the main factor inducing tumor angiogenesis. Hence, after binding of VEGF to VEGFR-2 (vascular endothelial growth factor receptor-2) which is abundantly expressed in the endothelial cells of cancer [15], a series of signaling pathways responsible for tumor angiogenesis begins and results in promoting survival, proliferation, migration, and vascular permeability for cancer tissues [16]. From the foregoing, we can infer that the EGFR and VEGFR-2 pathways are closely related; VEGF expression is reduced when EGFR is blocked. Nevertheless, VEGFR-2 inhibition also increases the anticancer effect of EGFR blocking medications [17]. Therefore, targeting EGFR and VEGFR-2 through a dual inhibition is a promising protocol for cancer treatment.

Pyrazole is among the most important heterocyclic compounds incorporated in several drugs and utilized for innovation and discovery of new biologically active candidates that has been reported to exhibit a wide range of pharmacological activities, including anti-inflammatory, analgesic, antipyretic, anticonvulsant, antidepressant, antimicrobial, and antifungal properties [18–21]. pyrazole has gained attention in cancer research due to its potential as an anticancer agent [22, 23]. One of the mechanisms by which pyrazole exhibits its anticancer activity is through the inhibition of kinases, particularly EGFR and VEGFR-2 [24–26]. Pyrazole derivatives have demonstrated inhibitory effects on EGFR, preventing its activation and downstream signaling pathways involved in cancer cell growth and metastasis, and for instance, mavelertinib I is a potent EGFR inhibitor and represent promising candidate currently in clinical trials for treating EGFR-mutated cancers [27–30], and lazertinib II is an EGFR inhibitor that approved for treatment of non-small cell lung cancer [31]. Similarly, compound III showed potent antiproliferative activity against MCF-7 and B16-F10 cell lines with IC₅₀ values of 0.30 ± 0.04 µM and 0.44 ± 0.05 µM, respectively with potent inhibitory activity against EGFR with IC₅₀ = 0.21 ± 0.05 µM [32]. Moreover, pyrazole-based compounds have shown promising inhibitory effects on VEGFR-2, leading to the suppression of cancer cell proliferation and tumor growth [15, 33]. Moreover, compound IV (Fig. 1) showed potent cytotoxicity against all of the tested cancer cell lines, with IC₅₀ values ranging from 3.17 to 6.77 µM. Moreover, compound IV demonstrated c.

Fig. 1.

Reported pyrazole derivatives, as EGFR and VEGFR-2 inhibitors as a template

onsiderable in vitro inhibitory effects on VEGFR-2 with IC₅₀ value of 97 nM and displayed antiangiogenic properties in an in vivo model using transgenic zebrafish (Tg(flila: EGFP)) [34].

Furthermore, 1,3,4-oxadiazole moiety has displayed remarkable anticancer activity and significant activity against wide range of Kinases specially EGFR inhibition activity [35–39]. Compound V (Fig. 2) showed cytotoxic activity with IC₅₀ of 1.95 µM, 2.36 µM and 3.45 µM against K-562, Jurkat and KG-1a leukemia cell lines, respectively. Simultaneously inhibiting EGFR with IC₅₀ of 0.24 µM [35]. Likewise compound VI ( Fig. 2) demonstrated antiproliferation activity against four human cancer cell lines (A-549, MCF‐7, Panc‐1, and HT‐29) with IC₅₀ of 1.30, 0.80, 1.20, and 1.13 µM, respectively, and a noteworthy EGFR inhibition with IC₅₀ of 1.80 µM [37].

Fig. 2.

Reported EGFR and VEGFR-2 inhibitors containing chalcone and 1,3,4-oxadiazole moieties as a template

Moreover, chalcones are naturally occurring compounds present in various plant sources and have demonstrated potential as anticancer agents [40–45]. Studies indicates that chalcones act as a Michael acceptor and can inhibit kinases such as EGFR and VEGFR-2, which contributes to their anticancer properties [42, 46]. By targeting these kinases, chalcones can disrupt key signaling pathways involved in cancer cell growth and survival [47, 48]. Compounds VII (Fig. 2) was assayed for their antiproliferation activity against NCI cell line panel and showed remarkable activities, and were found to have a potency towards VEGFR-2 kinase comparable to that of sorafenib with a an IC₅₀ value of 0.11 µM [49].

Furthermore, oximes have emerged as promising nitric oxide (NO) donors with applications in the field of anticancer therapy due to their unique ability to release NO in a controlled manner [50–52]. This release mechanism can induce various cellular responses, including apoptosis in cancer cells, inhibition of tumor growth, and enhancement of the immune response against tumors [53]. By targeting specific pathways, such as those involved in angiogenesis and metastasis, oxime derivatives can disrupt the cancerous environment, making them effective adjuncts to traditional therapies [51]. Moreover, the ability of oximes to selectively deliver NO to tumor tissues while minimizing systemic toxicity further enhances their therapeutic potential [54, 55]. A series of nitric oxide (NO)-releasing quinolone-1,2,4-triazole/oxime hybrids were developed to target STAT3 in melanoma [53]. The most potent compound, VIII and (Fig. 3), bound to the STAT3-SH domain with IC₅₀ value 0.25 µM. They effectively inhibited STAT3 phosphorylation in BRAFV600E melanoma cells and prevented STAT3 nuclear translocation and DNA-binding activity. Furthermore, Fadaly, W. A., et al. [51] synthesized a series of pyrazole/triazole/oxime hybrids, which showed significant antiproliferative activity, particularly the sulphamoyl derivative IX (Fig. 3). This compound exhibited IC₅₀ value of 0.33 µM against the PC-3 cancer cell line, with selectivity ratios of 39.4-fold over F180 fibroblasts. Moreover, it also effectively inhibited p38MAPK with IC₅₀ of 0.58 µM, and VEGFR-2 with IC₅₀ of 0.54 µM. Another series of quinazoline-based compounds were designed and synthesized as VEGFR-2 inhibitors [52]. Among these, compound X (Fig. 3) demonstrated remarkable anti-cancer activity across three human cancer cell lines (HepG-2, MCF-7, and HCT-116). In addition, compound X exhibited strong VEGFR-2 inhibitory activity with an IC₅₀ of 2.5 µM.

Fig. 3.

Reported compounds with antiproliferative activity bearing oxime moiety as a template

In this study, the objective was to gather the benefits of combining pyrazole, with 1,3,4-oxadiazole scaffolds into hybrid molecules. The introduction of a chalcone moiety as a Michael acceptor group and incorporating of nitric oxide-releasing oxime group is expected to result in more potent and targeted anticancer candidates, with enhanced binding ability to the target sites (Fig. 4). Aiming that this strategy offers potential advantages such as reduced toxicity, decreased resistance, and enhanced efficacy compared to single scaffold compounds.

Fig. 4.

Design of novel pyrazole-1,3,4-oxadiazole hybrids as potential EGFR inhibitors

Results and discussion

Chemistry

The designed compounds 10a–10c, 11a-11c, 12a-12i, 13a-13i, and 14a-14i were prepared as outlined in Schemes 1, 2 and 3. Firstly, chalcones (1a-1i) were prepared via Claisen-Schmidt condensation of 4-aminoacetophenone and aromatic aldehydes [14], followed by acylation with chloroacetyl chloride to afford acylated chalcones (2a-2i) and similarly treating 4-aminoacetophenone to afford acylated 4-aminoacetophenone (3) [56, 57] as depicted in Scheme 1.

Scheme 1.

Synthesis of the intermediates 1–3

Scheme 2.

Synthesis of the target pyrazole-1,3,4-oxadiazole hybrids 10a-10c and 11a-11c

Reagents and conditions: (a) EtOH, 60% NaOH, Ice bath, stirring (b) DCM, TEA, ClCOCH₂Cl, stirring 4 h.

Next, hydrazone (4a-4c) was prepared by refluxing substituted acetophenone derivatives with phenyl hydrazine in ethanol and drops of acetic acid as a catalyst [58]. Furthermore, pyrazole-4-carbaldehyde (5a-5c) was then obtained through Vilsmeier-Haack reaction by reaction of POCl₃ with DMF, followed by addition of hydrazone (4a-4c) and heating [59]. Afterwards, oxidation of compound (5a-5c) using KMnO₄ and KOH in pyridine/water afforded pyrazole carboxylic acid (6a-6c) [60], which was then esterified with methanol and sulfuric acid to give ester (7a-7c) [61], which upon refluxing with hydrazine hydrate in ethanol produced pyrazole hydrazide (8a-8c) [62]. Furthermore, pyrazole hydrazide (8a-8c) was subsequently cyclized with CS₂ and KOH to form pyrazole-1,3,4-oxadiazole hybrids (9a-8c). Moreover, the target hybrids 10a–10c were obtained by the reaction of compounds (9a-9c) and (3) in the presence of triethylamine. Additionally, target hybrids 11a-11c were obtained by the reaction of compounds (10a-10c) with hydroxylammonium hydrochloride in the presence of pyridine as illustrated in Scheme 2.

Reagents and conditions: (c) AcOH, EtOH, reflux, 2 h; (d) DMF, POCl₃ (e) Pyridine, KMnO₄, stirring 3 h; (f) MeOH, H₂SO₄, reflux,8 h; (g) EtOH, Hydrazine hydrate 85% reflux, 8 h; (h) EtOH, NaOH, CS₂ reflux,6 h; (i) CH₃CN, TEA, stirring 4 h; (j) EtOH, NH₂OH.HCl, pyridine reflux, 4 h.

Finally, the target hybrids 12a-12i, 13a-13i, and 14a-14i were afforded by the reaction of compounds (9a-9c) and (2a-2i) in the presence of triethylamine as shown in Scheme 3. Structures of the synthesized compounds 9a-c, 10a-10c, 11a-11c, 12a-12i, 13a-13i, and 14a-14i were elucidated by FT-IR, ¹H NMR, ¹³C NMR, and Elemental analysis. ¹H NMR spectra of all compounds displayed their intended protons in the expected chemical shifts. Similarly, ¹³C NMR spectra of compounds showed the presence of the exact number of carbon atoms at their corresponding ppm. In addition, Elemental analysis showed matching of the calculated values and the found values.

Scheme 3.

Synthesis of the target pyrazole-oxadiazole-chalcones hybrids 12a-12i, 13a-13i, and 14a-14i

Reagents and conditions: (i) CH₃CN, TEA, stirring 4 h.

Biological evaluation

In vitro anticancer activity

Thirty-three newly synthesized target compounds were selected by the National Cancer Institute (NCI); potential pyrazole-oxadiazole hybrids 10a-10c, 11a-11c, 12a-12i, 13a-13i, and 14a-14i for their in vitro anticancer screening against full NCI’s disease-oriented 60 human cell lines. The tested cell lines are derived from nine tumor subpanels, including leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancer cell lines according to the reported protocol of the Drug Evaluation Branch, NCI, Bethesda (http://www.dtp.nci.nih.gov).

In vitro single-dose assay on full NCI − 60 cell lines

The selected hybrids 10a-10c, 11a-11c, 12a-12i, 13a-13i, and 14a-14i were tested at single concentration of 10 µM. Screening results were recorded as growth percent of the cells treated with the tested compound as compared to the untreated reference cells. Mean graph midpoint (MG-MID) (differential activity patterns, bar scale) was constructed for each cell line to facilitate visual scanning of data for potential NCI patterns of selectivity, with bars depicting the deviation of the individual tumor cell lines from the overall mean value of all growth percentages recorded for all cell lines tested. Bars pointed to the left (positive values) denote resistance where the growth is greater than the average, while bars pointed to the right (negative values) denote sensitivity where the growth is less than the average. Delta means the logarithm of the difference between the (MG-MID). The screening results were redisplayed as percentage growth inhibition (GI %), (Supporting information Tables 1–5).

Pyrazole-oxadiazole hybrids 10a-10c with 4-acetylphenyl group showed significant cytotoxicity with varied broadness in the spectrum of activity. The results of the ketone derivatives showed that compounds 10b and 10c displayed strong anticancer activity (growth inhibition % of 60.78% to 174.47%) against a number of 31, and 33 cancer cell lines, respectively. Moreover, compounds 10b and 10c showed mean growth inhibition percentages of 63.11, and 78.02%, respectively, and showed a moderate anticancer activity against the remaining cancer cell lines, as shown in (Supporting information Table 1). Furthermore, compound 10a showed moderate activity against most cancer cell lines except non-small cell lung cancer NCI-H522 cell line, showed significant growth inhibition percent of 122.26% and a mean growth inhibition percent of 30% (Fig. 5).

Fig. 5.

Mean GI % for in vitro tumor cell lines at 10 µM concentration for the most potent hybrids 10a-10c and 11a-11c

Changing the acetyl group into oxime in hybrids 11a, 11b, and 11c resulted in increased activity and broadness with growth inhibition ≥ 60.78% to 197.63% against almost all cancer cell lines, with mean growth inhibition percentage of 104.23%, 115.10% and 95.10%, respectively (Fig. 26). The antiproliferative activity of these oxime derivatives showed that compound 11b is the most active hybrid displaying very strong antiproliferative activity against almost all cancer cell lines, as shown in (Supporting information Table 1). This activity could be attributed to both oxime group introduction and the presence of fluorine substituent on the pyrazole phenyl ring.

On the other hand, pyrazole-oxadiazole-chalcone hybrids 12a-12i, 13a-13i, and 14a-i resulted in significant decrease in both activity and broadness compared with ketone, and oxime hybrids 10a-10c, and 11a-11c. The obtained results of hybrids 12a-12i, with unsubstituted phenyl ring on C₃ of pyrazole ring, revealed that most of the tested compounds showed potent inhibition on four cancer cell lines (Leukemia RPMI-8226, Colon Cancer HCT-116, Melanoma LOX IMVI, and Breast Cancer MCF7 cell lines). Compounds 12a and 12e were found to be the most active in these derivatives and they displayed a potent anticancer activity (growth inhibition ≥ 61.29% and up to 112.16%) against the four cancer cell lines that mentioned above. compound 12a exhibited inhibitory activity on leukemia (RPMI-8226; GI% = 94.94%), colon cancer (HCT-116; GI% = 65.39%), breast cancer (MCF7; GI% = 61.29%). compound 12e exhibited inhibitory to cytotoxic (complete cell death) activity on leukemia (RPMI-8226; GI% = 76.4%), Colon Cancer (HCT-116; GI% = 112.16%), melanoma (LOX IMVI; GI% = 63.42%), breast cancer (MCF7; GI% = 93.56%). Compound 12d showed significant selectivity against leukemia cell lines, while compound 12f showed selectivity against all CNS cancer cell lines except SF-268 cell line with GI% ranging from 34.96 up to 84.72, with remarkable cytotoxic activity on renal cancer cell line RXF 393 with GI % of 104.52. And finally compound 12i showed significant cytotoxic activity on melanoma cancer cell line LOX IMVI with GI % of 169.52. The remaining derivatives showed a moderate to weak anticancer activity against most of the used cancer cell lines (Supporting information Tables 2–3). The investigation of the effect of introducing a 4-fluoro substitution on the pyrazole phenyl ring in the activity of hybrids 13a-13i showed that the presence of the fluorine atom resulted in almost equal activity with the unsubstituted series with enhanced potency against few cell lines and these results can be attributed for the fact of the fluorine atom is a bio-isostere for the hydrogen and can play crucial role for additional binding with the target enzyme. Like the previous series most of the compounds showed potent inhibition on the same four cancer cell lines (RPMI-8226, HCT-116, LOX IMVI, and MCF7). Among this group of compounds, derivatives 13a and 13 g demonstrated the highest level of activity. compound 13a inhibited the growth of cancer cells by 70.03% and 72.66% against RPMI-8226 and HCT-116 respectively and compound 13 g inhibited the growth of cancer cells by 63.66% against RPMI-8226, 70.11% against HCT-116, and 65.36% against MCF7. Compound 12d showed significant selectivity against leukemia cell lines, while compound 12f displayed remarkable inhibition of the Leukemia cell line RPMI-8226, with a growth inhibition percentage (GI %) of 80.11. Additionally, compound 13i exhibited a significant cytotoxic activity against the melanoma cancer cell line LOX IMVI, with a GI % of 107.63. The remaining derivatives displayed moderate to weak anticancer activity against most of the tested cancer cell lines (Supporting information Tables 2–3).

Substitution of the pyrazole phenyl with 4-methoxy group generally resulted in decreased activity against most cell lines and even greater reduction in the activity where the chalcone contained an electron donating group. Among this series few compounds showed noteworthy activity as compound 14a which inhibited the growth of cancer cells by 57.03% and 71.48% against RPMI-8226 and HCT-116, respectively, while compounds 14e and 14f displayed remarkable inhibition of the Leukemia cell line RPMI-8226, with a growth inhibition percentage (GI %) of 80.37 and 75.09, respectively. The remaining derivatives displayed moderate to weak anticancer activity against most of the tested cancer cell lines (Table 4 and Supporting information Table 5).

Table 4.

Physicochemical characteristics, lipophilicity, water solubility, pharmacokinetics, drug likeness, medicinal chemistry, and toxicity parameters of compounds 10b, 11a, and 11b that we acquired from Swiss ADME and PkCSM server

Physicochemical properties		10b			11a			11b
Formula		C27H20FN5O3S			C27H22N6O3S			C27H21FN6O3S
Molecular weight		513.54 g/mol			510.57 g/mol			528.56 g/mol
Num. heavy atoms		37			37			38
Num. arom. heavy atoms		28			28			28
Fraction Csp3		0.07			0.07			0.07
Num. rotatable bonds		9			9			9
Num. H‐bond acceptors		7			7			8
Num. H‐bond donors		1			2			2
Molar refractivity		138.00			142.03			141.99
TPSA (topological polar surface area)		128.21 Å2			143.73 Å2			143.73 Å2
Log Po/w		4.49			4.18			4.50
Water solubility
Log S (ESOL)		-5.88			-5.95			-6.11
Solubility		6.84e-04 mg/ml; 1.33e-06 mol/l			5.71e-04 mg/ml; 1.12e-06 mol/l			4.09e-04 mg/ml ; 7.74e-07 mol/l
Class		Moderately soluble			Moderately soluble			Poorly soluble
Pharmacokinetics
GI absorption		Low			Low			Low
BBB permeant		No			No			No
P‐gp substrate		No			No			No
Log Kp (skin permeation)		-6.18 cm/s			-6.06 cm/s			-6.09 cm/s
Physicochemical properties
Druglikeness
Lipinski		Yes;	1	Violation	Yes;	1	Violation	Yes;	1	Violation
Veber		Yes			No	1	Violation	No	1	Violation
Muegge		Yes			Yes			Yes
Bioavailability	Score	0.55			0.55			0.55
Toxicity
AMES toxicity (Yes/No)		No			No			No
Max. tolerated dose (human) (log mg/ kg/day)		0.757			0.742			0.775
hERG I inhibitor (YES/NO)		No			No			No
hERG II inhibitor (Yes/No)		Yes			Yes			Yes
Oral rat acute toxicity (LD50) (mol/kg)		3.104			2.934			2.927
Oral rat chronic toxicity (LOAEL) (log mg/Kg bw/day)		-0.481			0.854			0.834
Hepatotoxicity (Yes/No)		Yes			Yes			Yes
Skin sensitization (Yes/No)		No			No			No
T. pyriformis toxicity (log μg/L)		0.285			0.285			0.285
Minnow toxicity (log mM)		-0.073			0.25			0.375

In vitro five-dose assay on full NCI − 60 cell panel

The pyrazole-oxadiazole hybrids 10b and 11a-11c were further selected by NCI upon their 1st screening results for advanced NCI full panel five dose assays at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 & 100 µM). The resulting data of the evaluated compounds was displayed with calculated three response parameters for each cell line GI₅₀ (growth inhibitory activity) value: compound concentration that causes 50% decrease in the net growth of cells, TGI (cytostatic activity) value: compound concentration that results in total growth inhibitions, and LC₅₀ (cytotoxic activity) value: compound concentration that causes net 50% loss of initial cells at the end of the incubation period of 48 h as illustrated in Supporting information Tables 6–9. Moreover, mean graph midpoint (MG-MID) values have been calculated giving an averaged activity parameter over subpanel and full panel cell lines for each tested compound [MID_a: the average sensitivity of all cell lines for the tested compound while MID_b: the average sensitivity of cell lines of a specific subpanel for the tested compound].

As displayed in Supporting information Table 6, The ketone hybrid 10b, exhibited very strong antiproliferative activity with GI₅₀ ≤ 10 µM against 49 cancer cell lines (GI₅₀ range from 1.71 to 9.52 µM) with outstanding activity against 26 cancer cell lines with GI₅₀ ≤ 5 µM and remarkable activity against 23 cancer cell lines with GI₅₀ ranging between 5.11 and 9.52 µM. Furthermore, hybrid 10b showed strong activity against 11 cancer line with GI₅₀ range from 10.4 to 15.8 µM. Additionally, hybrid 10b showed an average GI₅₀ (MID_a) of 6.39 µM, and TGI of 21.93 µM which is comparable with the reference drug gefitinib average GI₅₀ of 6.08 µM, and TGI of 22.25 µM. Moreover, hybrid 10b showed more potent LC₅₀ value of 47.55 µM compared with gefitinib LC₅₀ value of 52.59 µM indicating potent cytotoxic activity (Fig. 6).

Fig. 6.

Mean (MID_a) GI₅₀ and TGI of compounds 10a, and 11a-11c compared with Gefitinib

Regarding the sensitivity of various cell lines for hybrid 10b, it showed distinct pattern of homogenous anticancer activity on the individual cell lines. It exhibited remarkable growth inhibition GI₅₀ on entire panel of tumor cell lines with average sensitivity values (MID_b) ranged from 3.35 to 8.91 µM. The most sensitive subpanel for 10b was breast cancer with GI₅₀ (MID_b) = 3.35 µM, (Supporting information Table 7). For selectivity of 10b at GI₅₀ level for anti-proliferative activity and according to selectivity index (SI), hybrid 10b showed non-selective anti-proliferative activity as the anti-proliferative activity selectivity index ranged from 0.83 to 1.91, and its selectivity at TGI level for cytostatic activity, it has non-selective potency with selectivity index ranged from 0.65 to 1.30 (Supporting information Table 7).

Once again, as in one dose assay converting ketone function group to its corresponding oxime functionality increase both activity and broadness. Hybrids 11a, and 11b with oxime function group, were the most active compounds among all of the synthesized derivatives. Compound 11a exhibited very strong anti-proliferative activity with GI₅₀ ≤ 10 µM against 56 cancer cell lines (GI₅₀ range from 0.31 to 10.0 µM) with outstanding activity against 33 cancer cell lines with GI₅₀ ≤ 5 µM and remarkable activity against 23 cancer cell lines with GI₅₀ ranging between 5.15 and 10.0 µM. (Supporting information Table 8). Moreover, 11a showed strong antitumor activity against 3 cancer cell lines with GI₅₀ ≤ 20 µM (GI₅₀ range from 11.9 to 17.0 µM). Regarding the sensitivity of various cell lines for hybrid 11a, it showed distinct pattern of homogenous anticancer activity on the individual cell lines. It exhibited remarkable growth inhibition GI₅₀ on entire panel of tumor cell lines with average sensitivity (MID_a) of 4.88 µM which is more potent than the reference drug gefitinib (GI₅₀ = 6.08 µM) as illustrated in Fig. 6, and on subpanels of tumor cell with average sensitivity values (MID_b) ranged from 1.45 to 6.99 µM. The most sensitive subpanel for 11a was leukemia with GI₅₀ (MID_b) = 1.45 µM, (Supporting information Table 9). For selectivity of 11a at GI₅₀ level for anti-proliferative activity and according to selectivity index, hybrid 11a showed moderate selective anti-proliferative activity toward leukemia cell lines with selectivity index of 3.37, and the remaining subpanels selectivity index ranged from 0.70 to 1.29. compound 11a selectivity at TGI level for cytostatic activity, it showed moderate selective potency toward leukemia subpanel with selectivity index of 5.39, and the remaining subpanels selectivity index ranged from 0.60 to 1.36 (Supporting information Table 9).

Moreover, hybrid 11b, exhibited very strong antiproliferative activity against 57 cancer cell lines with GI₅₀ ≤ 10 µM (GI₅₀ range from 0.21 to 9.68 µM) with outstanding activity against 44 cancer cell lines with GI₅₀ ≤ 5 µM and remarkable activity against 13 cancer cell lines with GI₅₀ ranging between 5.24 and 9.68 µM. Moreover, 11a showed strong antiproliferative activity against 2 cancer cell lines (Non-Small Cell Lung Cancer NCI-H322M, and Ovarian cancer OVCAR-5 cell lines) with GI₅₀ of 13.4 and 15.9 µM respectively (Supporting information Table 8). Regarding the sensitivity of various cell lines for hybrid 11b, it exhibited remarkable growth inhibition GI₅₀ on entire panel of tumor cell lines with average sensitivity (MID_a) of 4.36 µM as illustrated in Fig. 6. Moreover, on subpanels of tumor cell it showed average sensitivity values (MID_b) ranged from 1.40 to 6.00 µM. The most sensitive subpanel for 11b were leukemia with GI₅₀ (MID_b) = 1.40 µM. Hybrid 11b showed moderate selectivity toward leukemia subpanel with selectivity index of 3.11, while the remaining subpanels selectivity index ranged from 0.73 to 1.35, and its selectivity at TGI level for cytostatic activity, exhibited moderate selectivity towards leukemia subpanel with selectivity index of 4.88 over the other subpanels with SI ranged from 0.71 to 1.33 (Supporting information Table 9). Notably, hybrids 11a and 11b showed potent cytotoxic activity towards the major of leukemia cells with LC₅₀ values ranged between 0.89 and 34.3 µM.

Finally, hybrid 11c, exhibited very strong anticancer activity with GI₅₀ ≤ 10 µM against 30 cancer cell lines (GI₅₀ range from 0.88 to 9.58 µM) and strong anticancer activity against 10 cancer line with GI₅₀ ≤ 20 µM (GI₅₀ range from 11.7 to 18.1 µM). Moreover, hybrid 11c displayed moderate to weak activity against the remaining cancer cell lines with (GI₅₀ values ranging from 20.7 to 97.9 µM) as presented in Supporting information Table 8. Ongoing through the details of the antiproliferative assay results hybrid 11c displayed outstanding activity against 16 cancer cell lines with GI₅₀ ≤ 5 µM and remarkable activity against 14 cancer cell lines with GI₅₀ ranging between 5.01 and 9.58 µM. Regarding the sensitivity of various cell lines for hybrid 11c, it showed moderate growth inhibition GI₅₀ on entire panel of tumor cell lines with average sensitivity (MID_a) of 16.4 µM (Fig. 6). Furthermore, it exhibited remarkable growth inhibition GI₅₀ on leukemia subpanel with average sensitivity (MID_b) of 2.77 µM, while showing strong to moderate sensitivity on the remaining subpanels of tumor cell with average sensitivity values (MID_b) ranged from 6.00 to 28.77 µM (Supporting information Table 9). For selectivity of 11c at GI₅₀ level for anti-proliferative activity and according to selectivity index, hybrid 11c showed moderate-selective anti-proliferative activity toward leukemia subpanel with SI of 5.92, over the other subpanels with SI ranged from 0.57 to 2.73, while its selectivity at TGI level for cytostatic activity, it has highly-selective potency toward leukemia cell lines with SI of 8.79 over the other subpanels with SI ranged from 0.55 to 1.32. Summing up, the results of the five-dose investigation for the oxime hybrids 11a, 11b and 11c, indicating a positive impact of the oxime moiety on the activity.

Comparing the resulting antitumor activity of the most potent hybrids 11a, and 11b with the reference drug gefitinib, it is obvious that hybrids 11a, and 11c for the unsubstituted and the fluorine substituted pyrazole-C₃ phenyl ring, exhibited average anti-proliferative activity (GI₅₀) more potent than gefitinib with GI₅₀ (MID_a) of 4.88 and 4.36 µM, respectively compared with gefitinib GI₅₀ (MID_a) of 6.08 µM and showed anti-proliferative activity (GI₅₀) more potent than gefitinib on 39, and 44 cancer cell lines, respectively, and the both hybrids were shared in displaying highly potent antitumor activity against leukemia (SR) cell line, Moreover, hybrids 11a and 11b showed cytostatic activity (TGI) more potent than gefitinib on 38, and 43 cancer cell lines, respectively, with (MID_a) for cytostatic activity on all cancer cell lines (TGI of 20.90, 15.8, and 22.25 µM for 11a, 11b, and gefitinib, respectively). Additionally, both hybrids 11a and 11b have potent cytotoxicity (LC₅₀) more than gefitinib on 29 cancer cell lines, and LC₅₀: (MID_a) on all cancer cell lines for hybrid 11a and 11b (57.70 and 42.29 µM, respectively) in comparison with gefitinib (LC₅₀; MID_a = 52.59 µM). Furthermore, hybrid 11b exhibited sub-micromolar cytotoxicity (cytotoxic effects) on leukemia (SR) cell line with (LC₅₀ = 0.89 µM) more potent than gefitinib (LC₅₀ = 18.58 µM) (Supporting information Tables 8 and 9).

In-vitro cytotoxic effects against normal cells WI-38

The cytotoxicity of hybrid 11b was evaluated on normal human WI-38 cells, which are derived from fibroblasts obtained from the lung tissue of a three-month pregnant aborted female embryo. The results indicated that compound 11b exhibited low cell toxicity, with an IC₅₀ of 145.5 µM, in contrast to the control drug gefitinib, which had an IC₅₀ of 43.2 µM. This suggests that hybrid 11b is comparatively safer than gefitinib against WI-38 normal human cell lines (Table 1).

Table 1.

Cytotoxic IC₅₀ of compounds 11b, and gefitinib on human normal cells WI-38 (n = 3)

Compound	Cytotoxicity IC50 / µM
	WI-38
11b	145.5 ± 4.7
Gefitinib	43.2 ± 1.8

Enzymatic inhibitory assay

Assay of EGFR and VEGFR-2

The most active newly synthesized hybrids (10b, 11a, and 11b) were selected for further evaluation of their inhibitory potential against both EGFR and VEGFR-2 enzymes (Table 2). These compounds demonstrated promising activity compared to the reference drugs, Gefitinib (for EGFR) and Sorafenib (for VEGFR-2), suggesting their potential as dual EGFR/VEGFR-2 inhibitors. Compound 11b exhibited the most potent inhibitory activity against both targets, with IC₅₀ values of 26.38 µM against EGFR and 114.17 µM against VEGFR-2. This compound showed superior EGFR inhibition compared to Gefitinib (IC₅₀ = 39.56 µM) and also a comparable VEGFR-2 IC₅₀ than Sorafenib (IC₅₀ = 108.74 µM), indicating a favorable profile. While compounds 10b (IC₅₀ = 51.63 µM for EGFR and 137.76 µM for VEGFR-2) and 11a (IC₅₀ = 32.47 µM for EGFR and 146.09 µM for VEGFR-2) also showed inhibitory activity against both EGFR and VEGFR-2, their potency was less pronounced than that of 11b. These results suggest that compound 11b, in particular, warrants further investigation as a potential dual EGFR/VEGFR-2 inhibitor.

Table 2.

In vitro EGFR, and VEGFR-2 inhibitory activity (IC₅₀ nM) of the most potent compounds 10b, 11a, and 11b (n = 3)

Compounds	EGFR	VEGFR-2
10b	50.97 ± 2.05	136.82 ± 6.92
11a	31.46 ± 1.30	149.03 ± 3.55
11b	26.94 ± 0.33	114.10 ± 2.12
Gefitinib	39.57 ± 3.52	–
Sorafenib	–	110.86 ± 5.06

Cell cycle analysis

CDKs inhibition is strongly associated with the induction of cell cycle arrest at the G2/M phase [63]. Therefore, to gain more insights into the potent cytotoxic activity of hybrid 11b, cell cycle analysis of MCF7 cells was performed via flow cytometry assay (Fig. 7), which clearly showed that hybrid 11b caused a significant G2/M phase arrest. After treating MCF7 cells with IC₅₀ of hybrid 11b (3.54 µM) for 48 h, there was an increase in the number of cells in the G2/M phase (53.20%) compared to the control (28.95%). These changes were concomitant with a noteworthy decrease in the G0/G1 (51.13%) compared to the control (67.92%), similarly S phases decreased (12.47%) compared with the control (14.39%), indicating that these S phase cells can’t progress to the next cell cycle stage.

Fig. 7.

Cell cycle analysis of control (a) and after treatment with hybrid 11b (b)

Apoptosis assay

To understand the mechanism of cell death, whether apoptosis or necrosis, induced by hybrid 11b. MCF7 cells were assessed using Annexin-V/FITC staining coupled with flow cytometry (Fig. 8). Hybrid 11b induced late apoptosis (programmed cell death) in MCF7 with an apoptosis percentage of 27.68% compared to untreated control cells (0.71%), suggesting hybrid 11b as an apoptotic inducer. In addition, necrotic cell death (non-programmed) (12.86%) was also observed after treatment with hybrid 11b, but less than apoptosis, indicating that apoptosis is the predominant cell death mechanism of hybrid 11b. Regarding cell cycle arrest and apoptosis findings, hybrid 11b exhibited excellent anticancer activity causing programmed cell death apoptosis induction and cell cycle arrest at the G2/M phase.

Fig. 8.

Percentage of apoptosis and necrosis of control cells (a), treated with hybrid 11b (b)

Evaluation of nitric oxide release

Due to the significant biological importance of nitric oxide (NO), quantifying NO released is an important criterion in nitric oxide donor research [1]. The synthesized compounds 11a-11c are considered NO donors, as they can release NO under certain physiological conditions. The spontaneous oxidation of NO under physiological conditions into nitrites was the basis for a colorimetric method for measuring NO in biological systems using the Griess diazotization reaction. The Griess reaction method is a simple and sensitive technique that was used to measure the nitric oxide release from the synthesized compounds 11a-11c. It was carried out in phosphate buffer, pH 7.4, in the presence of L-cysteine, using sodium nitrite solution (1–50 nmol/mL) as a standard to establish the standard sodium nitrite curve. The role of L-cysteine is to accelerate the NO release from the organic nitrate [64, 65]. The formed intense purple color was measured at ƛ-max 540 nm after 5 min intervals. The percentage of NO release is calculated from the following equation:

Results revealed that all the tested compounds (11a-11c) released NO with relatively similar %, and compound 11b exhibited the highest % of release (7.197%) (Table 3). Correlation between time and the % of NO released indicated a gradual increase in the % of NO till reach plateau (after about 25–35 min) followed by decreasing in it (Fig. 9).

Table 3.

Percentage of NO release of compounds (11a-11c)

Comp.	Percentage of NO release
	5	10	15	20	25	30	35	40	45	50
11a	1.765	3.333	3.529	3.529	3.725	4.118	4.314	4.706	4.510	4.118
11b	4.735	6.061	6.061	6.818	7.197	6.250	3.409	3.409	2.462	2.083
11c	4.259	5.185	5.370	6.111	6.111	6.111	5.741	4.815	4.815	4.259

Fig. 9.

Percentage of NO release of compounds (11a-11c)

Docking study

Auto Dock vina was used as molecular-docking tool in order to carry out the docking simulations. The conformations with the most favorable (least) free binding energy were selected for analyzing the interactions between the target receptor and ligands by Discovery Studio Visualizer and PyMOL. The ligands are represented in different color; H-bonds and the interacting residues are represented in ball and stick model representation. Reference drugs dinaciclib, gefitinib, hybrids 10b, 11a, and 11b, was built using ChemBioDraw Ultra 12.0, and finally the crystal structures of VEGFR-2 (PDB ID: 2OH4) and EGFR (PDB ID: 1M17) enzymes were downloaded from protein data bank.

Molecular Docking of VEGFR-2

Analysis of the docking results revealed that the docking studies were consistent with the VEGFR-2 assay. From the inspection of the docking results, the reference drug sorafenib against VEGFR-2 (PDB ID: 2OH4), as presented in (Fig. 10) was incorporated in the formation of five hydrogen bonds with Glu883 (2), Cys917 (2), and Asp1044 and three carbon hydrogen bonds with Glu915, His1024, and Cys1043. Moreover, it formed one halogen bond with Ile1042, also, it was engaged in the formation of many hydrophobic interactions such as: Pi Sigma with Leu1033 as well as one Pi-Anion interaction with Asp1044 and ten Pi alkyl interactions with Leu838, Val846, Ala864, Leu887, Ile890, Val896, Val897, Val914, Leu1017, and His1024.

Fig. 10.

3D (A)/2D (B) Binding modes/interactions of sorafenib into the active site of VEGFR-2 (PDB ID: 2OH4)

Furthermore, it was found that hybrid 10b against VEGFR-2 (PDB ID: 2OH4), as presented in (Fig. 11) was incorporated in the formation of two hydrogen bonds with Arg840 and Asp1062, one carbon hydrogen bond with Gly920, also, it was engaged in the formation of many hydrophobic interactions such as: three Pi-Cation with Arg1049 (2), and Lys1053, as well as one Pi-Sigma interactions with Leu1033 and one Pi-Pi stacked with Phe916 and five Pi-alkyl with Leu838, Ala864, Cys917, Leu1033 and Arg1049.

Fig. 11.

3D (A)/2D (B) Binding modes/interactions of 10b into the active site of VEGFR-2 (PDB ID: 2OH4)

Furthermore, it was found that hybrid 11a with oxime function group resulted in enhanced binding to the active site of VEGFR-2 (PDB ID: 2OH4), as presented in (Fig. 12) it was incorporated in the formation of two hydrogen bonds with Ala1048, and Arg1049, one carbon hydrogen bond with Gly1046 amino acid residue. Also, it was engaged in the formation of many hydrophobic interactions such as: four Pi-Cation with Lys866, Glu883, Asp1026, and Asp1044, and one Pi-Sigma interaction with Ala879 as well as five Pi-alkyl with Ala879, Leu880, Ile886, Phe1045, and Ala1048 amino acid residues.

Fig. 12.

3D (A)/2D (B) Binding modes/interactions of hybrid 11a into the active site of VEGFR-2 (PDB ID: 2OH4)

Similarly, it is clear that hybrid 11b can fit perfectly into the catalytic binding pocket of VEGFR-2 (PDB ID: 2OH4), demonstrating good uniformity between the in vitro VEGFR-2 screening and the in-silico prediction, forming three hydrogen bonds with Lys866, Glu883, and Cys917 amino acid residues, as well as three carbon hydrogen bonds with Leu838, Phe916, and Asn921, and one halogen bond with Phe919. Also, it was engaged in the formation of many hydrophobic interactions such as: two Pi-Cation interations with Arg1049 (2), eight Pi-alkyl with Leu838, Val846, Ala864, Val897, Val914, Leu1033, and Cys1043 (2) amino acid residues (Fig. 13).

Fig. 13.

3D (A)/2D (B) Binding modes/interactions of hybrid 11b into the active site of VEGFR-2 (PDB ID: 2OH4)

Molecular Docking of EGFR

Docking results against EGFR (PDB ID: 1M17), showed consistency with the in vitro enzymatic assay results with the results being illustrated in Figs. 14, 15, 16 and 17. From the inspection of the docking results, the reference drug gefitinib as presented in Fig. 14 was incorporated in the formation of two hydrogen bonds with Leu694, and Cys773 amino acid residues, three carbon hydrogen bonds with Arg817 and Asp831 (2), as well as several hydrophobic interactions including one Pi-Sigma with Leu694, and four Pi-Alkyl with Leu694, Lys721, Cys773 (2) amino acid residues.

Fig. 14.

3D (A)/2D (B) Binding modes/interactions of gefitinib into the active site of EGFR (PDB ID: 1M17)

Fig. 15.

3D (A)/2D (B) Binding modes/interactions of hybrid 10b into the active site of EGFR (PDB ID: 1M17)

Fig. 16.

3D (A)/2D (B) Binding modes/interactions of hybrid 11a into the active site of EGFR (PDB ID: 1M17)

Fig. 17.

3D (A)/2D (B) Binding modes/interactions of hybrid 11b into the active site of EGFR (PDB ID:1M17)

Furthermore, in agreement with the in-vitro enzymatic assay, hybrid 10b showed enhanced binding affinity toward the binding site of EGFR (PDB ID: 1M17), and formed three hydrogen bonds with Lys721, Met769, Asp831 amino acid residues, one carbon hydrogen bond with Gly772, one halogen bond with Gln767 as well as several hydrophobic interactions including one Pi-sulfur with Met742, one Pi-Cation with Lys721, and five Pi-Alkyl with Leu694, Val702 (2), Ala719, Leu820 amino acid residues (Fig. 15).

Similarly, hybrids 11a and 11b showed consistency with the in vitro enzymatic assay results as well, with significant increase in binding affinity with the active site of EGFR enzyme. For instance, compound 11a (Fig. 16) formed three hydrogen bonds with Ala719, Thr766, and Asp831 amino acid residues, and carbon hydrogen bond with Gly772 as well as several hydrophobic interactions including one Pi-Cation with Lys721, one Pi-Sigma with Leu694. Furthermore, it showed one Amide-Pi Stacked interaction with Phe771, one Alkyl with Leu820, and four Pi-Alkyl with Val702 (2), Met742, and Leu764 amino acid residues.

Finally, compound 11b showed outstanding affinity toward the active site of EGFR and formed seven hydrogen bonds with Ala719, Lys721, Thr766, Met769, Cys773 (2), and Asp831 amino acid residues, as well as several hydrophobic interactions including two Pi-Cation with Lys721, and one Pi-Sulfur with Cys773. Additionally, it showed six Pi-Alkyl with Leu694 (2), Val702, Met742, Leu764, and Leu820 amino acid residues (Fig. 17).

Finally, from the previously mentioned data the binding mode of the compounds with the active sites of the enzymes reveals a complex network of interactions. The two nitrogens of the oxadiazole ring engaged in multiple hydrogen bonds with EGFR, enhancing the compound’s affinity for this target. Additionally, the acetamide and oxime groups are involved in the formation of hydrogen bonds with all studied enzymes, contributing to their inhibitory activity. The 1,3,4-trisubstituted pyrazole moiety participated in numerous hydrophobic interactions with all molecular targets, stabilizing the compound-enzyme complexes. Notably, the fluorine atom forms both halogen and hydrogen bonds with EGFR and VEGFR-2, further strengthening the binding interactions.

Physicochemical, ADME, pharmacokinetic characteristics and drug-likeness forecast

The Swiss Institute of Bioinformatics (SIB) provides the purchase free simply entered Swiss ADME website, which is one of the greatest useful computational systems for providing a worldwide evaluation of the pharmacokinetics characterization and prospects of the drug-likeness feature of tiny scaffolds. To save time, money, and enhance the likelihood of success, this was done to make sure the newly synthesized scaffolds had promising physiological potency and pharmacokinetic features. The most effective antiproliferative hybrids (10b, 11a, and 11b) had strong hydrophilicity, and good BBB penetration, with projected log P_o/w values of 4.49, 4.18, and 4.50, respectively.

Based on drug-likeness criteria of the Lipinski and Veber rules, the oral bioavailability of the target compounds 10b, 11a, and 11b was assessed (Table 4). All compounds have an unsaturation property that is outside the colored zone (Fig. 18) with Fraction Csp3 value of 0.07 which indicate less hydrophobicity. Moreover, the solubility of the compounds in water is poorly soluble. Furthermore, the compound with the best profile, according to the toxicity parameters, is 11b, followed by 11a, then 10b. All the compounds belong to the category of non-hERG I inhibitors, non-AMES toxicity, and non-skin sensitizers. Furthermore, the results showed that compound 11b is not an inhibitor of CYP1A2 and CYP2D6, but it exhibited inhibitory activity against CYP2C19, CYP2C9, and CYP3A4.

Fig. 18.

The bioavailability radar chart for hybrids 10b, 11a, and 11b. The pink area represents the range of ideal property values. The red lines characterize hybrids 10b, 11a, and 11b, whose estimated properties are almost entirely comprised in the pink area, indicating their good oral bioavailability

Structure activity relationship (SAR)

The SAR analysis revealed that the nature of the substituent on the phenyl ring at position 3 of the pyrazole and the functional group attached to the phenylacetamide moiety significantly influenced anticancer activity. The ketone series (10a-10c) with acetyl group attached to the phenylacetamide moiety exhibited strong to moderate anticancer activity. Among these, the compound with a 4-methoxyphenyl substitution at position 3 of the pyrazole 10c demonstrated the highest activity. The order of activity was determined to be 4-methoxypheny > 4-fluorophenyl > unsubstituted phenyl. Moreover, conversion of the acetyl group to an oxime functionality in series 11a-11c resulted in a significant increase in activity. Furthermore, compound 11b with 4-fluorophenyl group was the most active, followed by the unsubstituted phenyl 11a and the 4-methoxyphenyl 11c. Lastly, the introduction of a chalcone group in hybrids 12a-12i, 13a-13i, and 14a-14i generally led to a decrease in activity compared to the ketone and oxime derivatives. However, the chalcones activity varied significantly based on the substitutions on both pyrazole C3 phenyl ring and chalcone distal phenyl ring. The pyrazole C3 phenyl ring substitutions followed the order: unsubstituted phenyl > 4-fluorophenyl > 4-methoxyphenyl. For the chalcone distal phenyl ring, electron-withdrawing groups increased activity compared to unsubstituted derivatives, while electron-donating groups decreased activity. The overall order of activity was: 4-fluoro < 4-bromo < 4-nitro < unsubstituted < 1-naphthyl < 4-methyl < 3,4,5-trimethoxy < 3,4-dimethoxy < 4-methoxy. The 3,4,5-trimethoxy group showed increased selectivity towards the leukemia subpanel, and the 1-naphthyl group was selective towards the melanoma LOX IMVI cell line as illustrated in SAR (Fig. 19).

Fig. 19.

SAR of the pyrazole-oxadiazole derivatives

Conclusion

In conclusion, this work focused on designing, synthesizing, and evaluating a novel series of pyrazole-1,3,4-oxadiazole hybrid compounds linked to chalcone as potential dual inhibitors of EGFR and VEGFR-2 enzymes for anticancer therapy. In-vitro screening revealed that compounds 10b, 11a, 11b, and 11c exhibited significant cytotoxic activity. Compound 10b showed a mean GI₅₀ of 6.39 µM, while 11a and 11b had mean GI₅₀ values of 4.88 µM and 4.36 µM, respectively. Compound 11c was effective with a mean GI₅₀ of 16.4 µM. Enzymatic assays confirmed the dual inhibitory activity, with compounds 10b, 11a, and 11b showing potent inhibition of EGFR and VEGFR-2. Notably, 11b induced G2/M phase arrest and apoptosis in MCF7 cells. Molecular modeling and NO release studies supported these findings, highlighting the therapeutic potential of these hybrids. Overall, compounds 10b, 11a, 11b, and 11c are promising anticancer leads for further development.

Experimental

Chemistry

Materials and methods

All chemicals used in the preparation of the intermediate and target compounds are of commercial grade, purchased from Merck, Fluka, and El-Nasr pharmaceutical chemicals companies. All chemicals are used without further purification. Solvents used are of commercial grade and purchased from Merck, and El-Nasr pharmaceutical chemicals companies. DMF was dried with anhydrous sodium sulphate and purified by distillation on rotary evaporator.

Melting points were determined on Stuart electro-thermal melting point apparatus and were uncorrected. NMR spectra were carried out using a Bruker Advance 400 MHz NMR spectrometer at Bani-Swef University-Faculty of pharmacy, using TMS as internal reference. Chemical shifts (δ) values are given in parts per million (ppm) relative to DMSO-d₆ (2.50 for proton and 39.50 for carbon) and coupling constants (J) in Hertz. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. The progress of reactions for preparation of compounds was monitored by thin-layer chromatography (TLC) using Merck 9385 pre-coated aluminum plate silica gel (Kieselgel 60) 5 × 20 cm plates with a layer thickness of 0.2 mm. The spots were detected by exposure to UV-lamp at λ = 254 nm. Elemental analyses were performed on Vario El Elementar CHN Elemental analyzer; organic microanalysis section, The Regional center for Mycology and Biotechnology, Naser City, Cairo, Egypt and the results were within ± 0.4% of the theoretical values.

General procedure for the synthesis of 5-(1-phenyl-3-substitutedphenyl-1 H-pyrazol-4-yl)-1,3,4-oxadiazole-2-thiol (9a-9c)

In an ice bath, an equimolar mixture of potassium hydroxide (0.561 g, 10 mmol) in 20 mL of ethanol was combined with the appropriate hydrazide (8a-8c) (10 mmol) and carbon disulfide (0.761 g, 10 mmol). The mixture was stirred for 10 min and then heated under reflux for 6 h, or until the evolution of hydrogen sulfide had nearly ceased. The reaction progress was monitored by TLC using a methylene chloride and methanol elution system (9:1 v/v). Once the reaction was deemed complete, ethanol was removed under vacuum. The resulting solid residue was dissolved in cold water, filtered, and acidified with diluted hydrochloric acid to yield the desired oxadiazoles (9a-9c). The product was then filtered, washed with water, dried, and crystallized from 90% aqueous ethanol.

5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-thiol (9a)

White crystals (yield, 2.8 g 88%); m.p. 225–227 °C; FT-IR; ν_max 2758 (SH); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 14.6 (s, 1H, NH, D₂O exchangeable), 9.3 (s, 1H, pyrazole C5–H), 8.03 (d, 2 H, J = 8.00 Hz Ar–H), 7.88–7.86 (m, 2 H, Ar-H), 7.59–7.48 (m, 2 H, Ar-H), 7.44–7.42 (m, 3 H, Ar-H), 7.41–7.37 (m, 1H, Ar-H); ¹³C-NMR (101 MHz, DMSO-d₆) δ (ppm): 177.19 (oxadiazole C₂), 156.68 (oxadiazole C₅), 150.92 (pyrazole C₃), 139.10 (pyrazole C₅), 131.81, 131.67, 130.15, 129.48, 129.17, 128.69, 127.99, 119.52, 105.49 (pyrazole C₄); Elemental analysis for C₁₇H₁₂N₄OS (320.37) (Calcd./Found): C, 63.73/63.91; H, 3.78/3.89; N, 17.49/17.65.

5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-thiol (9b)

Light brown crystals (yield, 2.73 g 81%); m.p. 229–230 °C, MW. 338.36; FT-IR; ν_max 2759 (SH); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ), 14.56 (s, 1H, NH, D₂O exchangeable), 9.31 (s, 1H, pyrazole C₅–H), 8.02 (d, 2 H, J = 8.00 Hz Ar–H), 7.93 (d, 2 H, J = 8.00 Hz Ar–H), 7.56–7.54 (m, 2 H, Ar-H), 7.43–7.40 (m, 1H, Ar-H), 7. 35-7.32 (m, 2 H,, Ar–H); ¹³C-NMR (101 MHz, DMSO-d₆) δ (ppm): 177.19 (oxadiazole C₂), 164.27, 161.82, 156.61 (oxadiazole C₅), 149.91 (pyrazole C₃), 139.00 (pyrazole C₅), 131.71, 131.49, 131.40, 130.14, 128.12, 128.01, 119.48, 115.74, 115.53, 105.48 (pyrazole C₄); Elemental analysis for C₁₇H₁₁FN₄OS (338.36) (Calcd./Found): C, 60.35/60.37; H, 3.28/3.30; N, 16.56/16.59.

5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-thiol (9c)

White crystals (yield, 2.55 g 73%); m.p. 209–210 °C; FT-IR; ν_max 2760 (SH); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 9.26 (s, 1H, pyrazole C₅–H), 8.01 (d, 2 H, J = 8.00 Hz Ar–H), 7.82 (d, 2 H, J = 8.00 Hz Ar–H), 7.60–7.5 (m, 2 H, Ar-H), 7.45–7.35 (m, 1H, Ar-H), 7. 04 (d, 2 H, J = 8.00 Hz, Ar–H), 3.83 (s, 3 H, OCH₃ ); ¹³C-NMR (101 MHz, DMSO-d₆) δ (ppm): 177.22 (oxadiazole C₂), 160.35, 156.82 (oxadiazole C₅), 150.71 (pyrazole C₃), 139.08 (pyrazole C₅), 131.57, 130.52, 130.14, 127.87, 123.97, 119.41, 114.12, 105.22 (pyrazole C₄), 55.70 (OCH₃) ; C₁₈H₁₄N₄O₂S (350.40) (Calcd./Found): C, 61.70/61.74; H, 4.03/4.05; N, 15.99/16.02.

General procedure for the synthesis of N-(4-acetylphenyl)-2-((5-(1-phenyl-3-substitutedphenyl-1 H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (10a-10c)

A mixture of the appropriate oxadiazole (9a-9c) (1 mmol), triethylamine (3 mmol), and compound 3 (0.211 g, 1 mmol) was stirred at room temperature in 20 ml of acetonitrile for 4 h. TLC was used to follow the reaction progression. The obtained product was filtered off and washed with water, dried, crystallized from methanol to afford compounds (10a-10c) in a good yeild.

N-(4-Acetylphenyl)-2-((5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (10a)

White crystals (0.193 g, 78% yield); m.p. 169–172 °C; FT-IR; ν_max 3350 (NH), 1675 (Ketone C = O), 1598 (amide C = O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.85 (s, 1H, CO-NH), 9.28 (s, 1H, pyrazole C₅–H), 7.99 (d, 2 H, J = 8.00 Hz, Ar–H), 7.95–7.88 (m, 4 H, Ar-H), 7.75–7.70 (m, 3 H, Ar-H), 7.62–7.55 (m, 2 H, Ar-H), 7.49–7.41 (m, 3 H, Ar-H), 4.36 (s, 2 H, CH₂) 2.52 (s, 3 H, CH₃) under DMSO peak; ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 196.95 (ketone C = O), 165.99 (amide C = O), 162.82 (oxadiazole C₂), 161.03 (oxadiazole C₅), 150.92 (pyrazole C₃), 143.41 (C- NH), 139.14 (pyrazole C₅), 132.60, 131.79, 131.68, 130.16, 129.97, 129.42, 129.05, 128.69, 127.97, 119.49, 118.99, 105.80 (pyrazole C₄), 37.32 (CH₂), 26.86 (CH₃); Elemental analysis for C₂₇H₂₁N₆O₃S (495.56) (Calcd./Found): C, 65.44/65.46; H, 4.27/4.28; N, 14.13/14.15; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 4.7 min; peak area, 94.6% at λ_max 254 nm.

N-(4-acetylphenyl)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (10b)

White crystals (0.156 g, 61% yield); m.p. 179–181 °C; FT-IR; ν_max 3346 (NH), 1679 (Ketone C = O), 1602 (amide C = O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.92 (s, 1H, CO-NH), 9.28 (s, 1H, pyrazole C₅–H), 8.02-7. 98 (m, 4 H, Ar–H), 7.93 (d, 2 H, J = 8.00 Hz, Ar-H), 7.76 (d, 2 H, J = 8.00 Hz, Ar-H), 7.58–7.54 (m, 2 H, Ar-H), 7.45–7.40 (m, 1H, Ar-H), 7.31 (m, 2 H, Ar-H), 4.36 (s, 2 H, CH₂) 2.52 (s, 3 H, CH₃) under DMSO peak; ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 196.95 (ketone C = O), 166.02 (amide C = O), 164.24, 162.80 (oxadiazole C₂), 161.80 (oxadiazole C₅), 160.98, 149.89 (pyrazole C₃), 143.40 (C- NH), 139.06 (pyrazole C₅), 132.60, 131.66, 131.35, 131.27, 130.13, 129.95, 128.25, 127.98, 119.46, 118.99, 115.72, 115.50, 105.77 (pyrazole C₄), 37.31(CH₂), 26.83 (CH₃); Elemental analysis for C₂₇H₂₀FN₅O₃S (513.55) (Calcd./Found): C, 63.15/63.18; H, 3.93/3.93; N, 13.64/13.66; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 3.7 min; peak area, 99.1% at λ_max 254 nm.

N-(4-acetylphenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (10c)

White crystals (0.176 g, 67% yield); m.p. 165–166 °C; FT-IR; ν_max 3308 (NH), 1676 (Ketone C = O), 1603 (amide C = O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.86 (s, 1H, CO-NH), 9.23 (s, 1H, pyrazole C₅–H), 7.99–7.88 (m, 6 H, Ar–H), 7.75 (d, 2 H, J = 8.00 Hz, Ar-H), 7.58–7.55 (m, 2 H, Ar-H), 7.44–7.40 (m, 1H, Ar-H), 7.03 (d, 2 H, J = 8.00 Hz, Ar-H), 4.37 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃) 2.52 (s, 3 H, CH₃) under DMSO peak; ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 196.94 (ketone C = O), 166.02 (amide C = O), 162.71 (oxadiazole C₂), 161.20 (oxadiazole C₅), 160.35, 150.72 (pyrazole C₃), 143.39 (C- NH), 139.15 (pyrazole C₅), 132.61, 131.51, 130.41, 130.11, 129.97, 127.82, 124.15, 119.38, 118.99, 114.13, 105.46 (pyrazole C₄), 55.67 (OCH₃), 37.34 (CH₂), 26.84 (CH₃); Elemental analysis for C₂₈H₂₃N₅O₄S (525.58) (Calcd./Found): C, 63.99/64.01; H, 4.41/4.42; N, 13.33/13.35; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 3.7 min; peak area, 99.1% at λ_max 254 nm.

General procedure for the synthesis of (E)-2-((5-(1-phenyl-3-substitutedphenyl-1 H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(1-(hydroxyimino)ethyl)phenyl)acetamide (11a-11c)

To a mixture of the desired ketones 10a-10c (0.3 mmol) and pyridine (0.079 g, 1.0 mmol) in absolute ethanol (30 ml) hydroxylamine hydrochloride (0.041 g, 0.6 mmol) added and refluxed for 4 h, and then left to cool. The formed precipitate was filtered off, washed with distilled water, dried, and crystallized from absolute ethanol affording the pure products 11a-11c in a good yield.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(1-(hydroxyimino)ethyl)phenyl)acetamide (11a)

White crystals (0.84 g, 55% yield); m.p. 191–193 °C; FT-IR; ν_max 3292 (NH), 1597 (amide C = O), 1533 (C = N); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.05 (s, 1H, C = N-OH), 10.68 (s, 1H, CO-NH), 9.30 (s, 1H, pyrazole C₅–H), 8.00 (d, 2 H, J = 8.00 Hz, Ar–H), 7.92 (d, 2 H, J = 8.00 Hz, Ar-H), 7.62 -7.60-7.55(m, 6 H, Ar-H), 7.49–7.42 (m, 4 H, Ar-H), 4.33 (s, 2 H, CH₂), 2.12 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 165.46 (amide C = O), 162.89 (oxadiazole C₂), 160.97(oxadiazole C₅), 152.84 (oxime C = N), 150.68 (pyrazole C₃), 139.50 (pyrazole C₅), 139.09, 132.65, 131.75, 131.68, 130.19, 129.45, 129.08, 128.71, 127.99, 126.56, 119.43, 119.32, 105.81 (pyrazole C₄), 37.21(CH₂), 11.83 (CH₃); Elemental analysis for C₂₇H₂₂N₆O₃S (510.57) (Calcd./Found): C, 63.52/63.55; H, 4.34/4.35; N, 16.46/16.48; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 3.3 min; peak area, 98.3% at λ_max 254 nm.

(E)-2-((5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(1-(hydroxyimino)ethyl)phenyl)acetamide (11b)

White crystals (0.97 g, 62% yield); m.p. 208–210 °C; FT-IR; ν_max 3311 (NH), 1599 (amide CO), 1516 (C = N); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.06 (s, 1H, C = N-OH), 10.96 (s, 1H, CO-NH), 9.28 (s, 1H, pyrazole C₅–H), 8.01–7.93 (m, 4 H, Ar–H), 7.67-7.57- (m, 6 H, Ar–H), 7.43–7.42 (m, 1H, Ar–H), 7.33–7.30 (m, 2 H, Ar–H), 4.39 (s, 2 H, CH₂), 2.12 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 165.57 (amide C = O), 164.22, 162.84 (oxadiazole C₂), 161.78 (oxadiazole C₅), 160.91, 152.88 (oxime C = N), 149.85 (pyrazole C₃), 139.51(pyrazole C₅), 138.99, 132.63, 131.67, 131.39, 131.31, 130.17, 128.19, 128.01, 126.52, 119.40, 119.33, 115.74, 115.33, 105.76 (pyrazole C₄), 37.11 (CH₂), 11.81(CH₃); Elemental analysis for C₂₇H₂₁FN₆O₃S (528.56) (Calcd./Found): C, 61.35/61.37; H, 4.00/4.02; N, 15.90/15.91; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 3.26 min; peak area, 98.9% at λ_max 254 nm.

(E)-N-(4-(1-(hydroxyimino)ethyl)phenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (11c).

White crystals (0.115 g, 71% yield); m.p. 186–187 °C; FT-IR; ν_max 3227 (NH), 1599 (amide CO), 1516 (C = N); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.06 (s, 1H, C = N-OH), 10.54 (s, 1H, CO-NH), 9.21 (s, 1H, pyrazole C₅–H), 7.96–7.88 (m, 4 H, Ar–H), 7.61–7.41 (m, 7 H, Ar-H), 7.12–6.99 (m, 2 H, Ar-H), 4.32 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃), 2.13 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 165.43 (amide C = O), 162.79 (oxadiazole C₂), 161.13 (oxadiazole C₅), 160.31, 152.80 (oxime C = N), 150.69 (pyrazole C₃), 139.45 (pyrazole C₅), 139.12, 132.68, 131.50, 130.43, 130.14, 127.83, 126.58, 124.09, 119.32, 114.11, 105.47 (pyrazole C₄), 55.66 (OCH₃), 37.28 (CH₂), 11.82 (CH₃); Elemental analysis for C₂₈H₂₄N₆O₄S (540.60) (Calcd./Found): C, 62.21/62.22; H, 4.48/4.50; N, 15.55/15.55; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 3.5 min; peak area, 97.2% at λ_max 254 nm.

General procedure for the synthesis of N-(4-cinnamoylphenyl)-2-((5-(1-phenyl-3-substitutedphenyl-1 H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (12a-12i, 13a-13i and 14a-14i)

A mixture of the desired oxadiazoles (9a-9c) (1 mmol), triethylamine (303 mg, 3 mmol) and the desired chalcone derivatives (2a-2i) (1 mmol) was stirred at room temperature in 20 ml of acetonitrile for 4 h. TLC was used to follow the reaction progression. The formed precipitate was filtered off, washed with water, dried, and crystallized from acetonitrile to afford compounds (12a-12i, 13a-13i, and 14a-14i).

(E) N-(4-Cinnamoylphenyl)-2-[(5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (12a).

White crystals (0.206 g, 71% yield); m.p. 230–232 °C; FT-IR; ν_max 3315 (NH), 1662 (Ketone CO), 1603 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.82 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.16 (d, 2 H, J = 8.00 Hz, Ar–H), 7.98–7.53 (m, 2 H, Ar-H), 7.88–7.86 (m, 5 H, Ar-H), 7.78 (d, 2 H, J = 8.00 Hz, Ar–H),7.72 (d, 1H, CH = CH J = 16.00 Hz, Ar-H), 7.58–7.54 (m, 2 H, Ar-H), 7.48–7.45 (m, 6 H, Ar-H), 7.41 (t, 1H, J = 8.00 Hz Ar-H), 4.34 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): δ 188.17 (ketone C = O), 166.05 (amide C = O), 162.86 (oxadiazole C₂), 161.02 (oxadiazole C₅), 150.96 (pyrazole C₃), 144.05 (Ar-C = C), 143.43, 139.05 (pyrazole C₅), 135.14, 133.18, 131.71, 131.53, 131.05, 130.45, 130.19, 129.47, 129.41, 129.26, 129.05, 128.71, 128.02, 122.36, 119.45 (CO-C = C), 119.15, 105.77 (pyrazole C₄), 37.27 (CH₂); Elemental analysis for C₃₄H₂₅N₅O₃S (583.67) (Calcd./Found): C, 69.97/69.85; H, 4.32/4.15; N, 12.00/12.23; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.48 min; peak area, 97.8% at λ_max 254 nm.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (12b)

Yellow crystals (0.206 g, 69% yield); m.p. 230–232 °C; FT-IR; ν_max 3312 (NH), 1647 (Ketone CO), 1597 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.77 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.14 (d, 2 H, J = 8.00 Hz, Ar–H), 7.96 (d, 2 H,, J = 8.00 Hz, Ar-H), 7.91–7.89 (m, 2 H, Ar-H), 7.81 (d, 2 H, J = 8.00 Hz Ar-H), 7.79–7.75 (m, 3 H, Ar-H), 7.68 (d, 1H, CH = CH J = 16.00 Hz), 7.56 (m, 2 H, Ar-H), 7.49–7.43 (m, 3 H, Ar-H), 7.40 (m, 1H, Ar-H), 7.01 (d, 2 H, J = 8.00 Hz, Ar–H), 4.34 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.97 (ketone C = O), 165.98 (amide C = O), 162.86 (oxadiazole C₂), 161.78, 161.03 (oxadiazole C₅), 150.94 (pyrazole C₃), 143.98 (Ar-C = C), 143.25, 139.09 (pyrazole C₅), 133.47, 131.75, 131.59, 131.17, 130.30, 130.18, 129.45, 129.06, 128.70, 127.99, 127.84, 119.87 (CO-C = C), 119.45, 119.09, 114.89, 105.79 (pyrazole C₄), 55.85 (OCH₃), 37.34 (CH₂); Elemental analysis for C₃₅H₂₇N₅O₅S (613.69) (Calcd./Found): C, 68.50/68.61; H, 4.43/4.68; N, 11.41/11.68; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.24 min; peak area, 96.8% at λ_max 254 nm.

(E)-N-(4-(3-(3,4-Dimethoxyphenyl)acryloyl)phenyl)-2-((5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (12c)

Yellow crystals (0.196 g, 61% yield); m.p. 185–187 °C; FT-IR; ν_max 3319 (NH), 1656 (Ketone CO), 1597 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.73 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.19–8.17 (m, 3 H, Ar–H), 8.00-7.91 (m, 2 H, Ar-H), 7.85–7.76 (m, 6 H, Ar-H), 7.60–7.37 (m, 7 H, Ar-H), 7.04–7.02 (m, 1H, Ar-H), 4.34 (s, 2 H, CH₂), 3.87 (s, 3 H, OCH₃), 3.83 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.97 (ketone C = O), 166.26, 165.94 (amide C = O), 165.34, 162.85 (oxadiazole C₂), 161.04 (oxadiazole C₅), 151.75, 150.95 (pyrazole C₃), 149.56, 144.44 (Ar-C = C), 143.26, 139.13 (pyrazole C₅), 133.52, 131.62, 130.29, 130.15, 129.42, 129.05, 128.69, 128.11, 127.97, 124.25, 120.05, 119.49 (CO-C = C), 119.08, 112.16, 111.38 (pyrazole C₄), 56.28 (OCH₃), 56.12 (OCH₃), 37.40 (CH₂); Elemental analysis for C₃₆H₂₉N₅O₅S (643.72) (Calcd./Found): C, 67.17/67.61; H, 4.54/4.62; N, 10.88/11.07; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 4.9 min; peak area, 96.9% at λ_max 254 nm.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (12d).

Pale yellow crystals (0.206 g, 70% yield); m.p. 110–112 °C; FT-IR; ν_max 3317 (NH), 1661 (Ketone CO), 1597 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.82 (s, 1H, CO-NH), 9.26 (s, 1H, pyrazole C₅–H), 8.18 (d, 2 H, J = 8.00 Hz, Ar–H), 7.98 (d, 2 H, J = 8.00 Hz, Ar-H), 7.91–7.89 (m, 2 H, Ar-H), 7.86 (s, 1H, Ar-H), 7.79 (d, 2 H, J = 8.00 Hz, Ar-H), 7.68 (d, 1H, CH = C, J = 16.00 Hz), 7.59–7.54 (m, 2 H, Ar-H), 7.50–7.45 (m, 3 H, Ar-H), 7.42 (m, 1H, Ar–H), 7.22 (s, 2 H, Ar-H), 4.35 (s, 2 H, CH₂), 3.87 (s, 6 H, OCH₃), 3.72 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.02 (ketone C = O), 166.01 (amide C = O), 162.87 (oxadiazole C₂), 161.03 (oxadiazole C₅), 153.58, 150.94 (pyrazole C₃), 144.50 (Ar-C = C), 143.41, 140.18, 139.09 (pyrazole C₅), 133.28, 131.75, 131.60, 130.76, 130.45, 130.18, 129.46, 129.06, 128.71, 128.00, 121.57, 119.46 (CO-C = C), 119.07, 106.96, 105.79 (pyrazole C₄), 60.62 (OCH₃), 56.62 (OCH₃), 37.34 (CH₂); Elemental analysis for C₃₇H₃₁N₅O₆S (673.74) (Calcd./Found): C, 65.96/65.82; H, 4.64/4.68; N, 10.39/10.61; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 4.9 min; peak area, 96.9% at λ_max 254 nm.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)acetamide (12e)

Light grey crystals (0.159 g, 53% yield); m.p. 207–209 °C; FT-IR; ν_max 3341 (NH), 1651 (Ketone CO), 1606 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.82 (s, 1H, CO-NH), 9.28 (s, 1H, pyrazole C₅–H), 8.19–8.17 (m, 2 H, Ar–H), 8.00-7.89 (m, 7 H, Ar-H), 7.84–7.72 (m, 4 H, Ar-H), 7.59–7.56 (m, 2 H, Ar-H), 7.49–7.42 (m, 3 H, Ar-H), 7.34–7.29 (m, 2 H, Ar–H), 4.37 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.97 (ketone C = O), 166.00 (amide C = O), 162.86 (oxadiazole C₂), 161.03 (oxadiazole C₅), 150.93 (pyrazole C₃), 143.48 (Ar-C = C), 142.72, 139.11 (pyrazole C₅), 133.16, 131.94, 131.77, 131.68, 130.47, 130.17, 129.44, 129.07, 128.70, 127.99, 127.98, 122.35, 119.46 (CO-C = C), 119.08, 116.50, 116.29, 105.80 (pyrazole C₄), 37.37 (CH₂); Elemental analysis for C₃₄H₂₄FN₅O₃S (601.66) (Calcd./Found): C, 67.87/68.05; H, 4.02/4.19; N, 11.64/11.87; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 7.1 min; peak area, 95.3% at λ_max 254 nm.

(E)-N-(4-(3-(4-Bromophenyl)acryloyl)phenyl)-2-((5-(1,3-diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (12f)

White crystals (0.208 g, 68% yield); m.p. 194–196 °C; FT-IR; ν_max 3314 (NH), 1652 (Ketone CO), 1601 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.34 (s, 1H, CO-NH), 9.38 (s, 1H, pyrazole C₅–H), 8.15 (d, 2 H, J = 8.00 Hz, Ar–H), 8.03–7.98 (d, 2 H, J = 8.00 Hz, Ar-H), 7.94–7.92 (m, 3 H, Ar-H), 7.87–7.83 (m, 4 H, Ar-H), 7.69–7.64 (m, 3 H, Ar-H), 7.59–7.56 (m, 2 H, Ar-H), 7.50–7.44 (m, 3 H, Ar-H), 7.44–7.39 (m, 1H, Ar-H), 4.44 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.87 (ketone C = O), 166.09 (amide C = O), 164.24, 162.84 (oxadiazole C₂), 160.97 (oxadiazole C₅), 149.93 (pyrazole C₃), 148.48, 143.75 (Ar-C = C), 141.69, 141.11, 139.01 (pyrazole C₅), 132.78, 131.57, 131.38, 131.30, 130.68, 130.24, 130.18, 128.04, 126.44, 124.40, 119.45 (CO-C = C), 119.14, 115.75, 115.53, 105.76 (pyrazole C₄), 37.32 (CH₂); Elemental analysis for C₃₄H₂₄BrN₅O₃S (662.56) (Calcd./Found): C, 61.64/61.85; H, 3.65/3.73; N, 10.57/10.84; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 5.3 min; peak area, 97.2% at λ_max 254 nm.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)acetamide (12 g)

Orange crystals (0.185 g, 59% yield); m.p. 190–192 °C; FT-IR; ν_max 3293 (NH), 1657 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.97 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.29 (d, 2 H, J = 8.00 Hz, Ar–H), 8.21–8.16 (m, 3 H, Ar-H), 8.15–8.10 (m, 2 H, Ar-H), 7.99 (d, 2 H, J = 8.00 Hz, Ar-H), 7.92–7.90 (m, 2 H, Ar-H), 7.83–7.78 (m, 3 H, Ar-H), 7.59–7.56 (m, 2 H, Ar-H), 7.55–7.45 (m, 3 H, Ar–H), 7.44–7.39 (m, 1H, Ar-H), 4.38 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.82 (ketone C = O), 166.11 (amide C = O), 162.83 (oxadiazole C₂), 161.03 (oxadiazole C₅), 150.90 (pyrazole C₃), 148.49, 143.84 (Ar-C = C), 141.75, 141.07, 139.10 (pyrazole C₅), 132.76, 131.77, 131.65, 130.68, 130.26, 130.16, 129.43, 129.07, 128.68, 127.96, 126.49, 124.39, 119.43 (CO-C = C), 119.11, 105.79 (pyrazole C₄), 37.36 (CH₂); Elemental analysis for C₃₄H₂₄N₆O₅S (628.66) (Calcd./Found): C, 64.96/65.12; H, 3.85/3.94; N, 13.37/13.60; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.6 min; peak area, 95.7% at λ_max 254 nm.

(E)-2-((5-(13-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(p-tolyl)acryloyl)phenyl)acetamide (12 h)

Yellow crystals (0.182 g, 61% yield); m.p. 197–199 °C; FT-IR; ν_max 3317 (NH), 1650 (Ketone CO), 1599 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.85 (s, 1H, CO-NH), 9.27 (s, 1H, pyrazole C₅–H), 8.15 (d, 2 H, J = 8.00 Hz, Ar–H), 7.98 (d, 2 H, J = 8.00 Hz, Ar–H), 7.92–7.85 (m, 3 H, Ar-H), 7.79–7.75 (m, 4 H,, Ar-H), 7.69 (d, 1H, CH = CH J = 16.00 Hz), 7.59–7.55(m, 2 H, Ar-H), 7.50–7.45 (m, 3 H, Ar-H), 7.44–7.39 (m, 1H, Ar-H), 7.28 (d, 2 H, J = 8.00 Hz, Ar–H), 4.36 (s, 2 H, CH₂), 2.35 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.08 (ketone C = O), 166.02 (amide C = O), 162.85 (oxadiazole C₂), 161.02 (oxadiazole C₅), 150.94 (pyrazole C₃), 144.08, 143.38 (Ar-C = C), 141.11, 139.08 (pyrazole C₅), 133.28, 132.47, 131.74, 131.60, 130.38, 130.18, 130.03, 129.46, 129.31, 129.06, 128.71, 128.00, 121.32, 119.46 (CO-C = C), 119.11, 105.79 (pyrazole C₄), 37.30 (CH₂), 21.54 (CH₃); Elemental analysis for C₃₅H₂₇N₅O₃S (597.69) (Calcd./Found): C, 70.33/70.19; H, 4.55/4.67; N, 11.72/11.98; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 8.2 min; peak area, 96.8% at λ_max 254 nm.

(E)-2-((5-(1,3-Diphenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(naphthalen-1-yl)acryloyl)phenyl)acetamide (12i)

Yellow crystals (0.260 g, 82% yield); m.p. 113–114 °C; FT-IR; ν_max 3313 (NH), 1651 (Ketone CO), 1604 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.86 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.55 (d, 1H, J = 16.00 Hz, Ar–H), 8.28 (d, 1H, J = 8.00 Hz, Ar–H), 8.24–8.10 (m, 3 H, Ar-H), 7.96–8.07 (m, 6 H, Ar-H), 7.90 (d, 2 H, J = 8.00 Hz), 7.81 (d, 2 H, J = 8.00 Hz, Ar-H), 7.68–7.55 (m, 5 H, Ar-H), 7.50–7.45 (m, 2 H, Ar-H), 7.42–7.39 (m, 1H, Ar-H), 4.36 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.05 (ketone C = O), 166.06 (amide C = O), 162.85 (oxadiazole C₂), 161.03 (oxadiazole C₅), 150.94 (pyrazole C₃), 143.54 (Ar-C = C), 139.92 (pyrazole C₅), 139.08, 133.84, 133.14, 131.84, 131.74, 131.66, 131.59, 131.24, 130.55, 130.18, 129.46, 129.27, 129.06, 128.70, 127.99, 127.73, 126.80, 126.19, 126.10, 124.91, 123.44, 119.45 (CO-C = C), 119.17, 105.79 (pyrazole C₄), 37.33 (CH₂); Elemental analysis for C₃₈H₂₇N₅O₃S (633.73) (Calcd./Found): C, 72.02/72.25; H, 4.29/4.37; N, 11.05/11.26; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 11.1 min; peak area, 96.7% at λ_max 254.

N-(4-Cinnamoylphenyl)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (13a)

White crystals (0.234 g, 78% yield); m.p. 240–242 °C; FT-IR; ν_max 3312 (NH), 1656 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.82 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.16 (d, 2 H, J = 8.00 Hz, Ar–H), 7.99–7.93 (m, 4 H, Ar-H), 7.89–7.86 (m, 3 H, Ar-H), 7.77 (d, 2 H, J = 8.00 Hz, Ar–H) 7.72 (d, 1H, CH = CH J = 16.00 Hz), 7.58–7.52 (m, 2 H, Ar-H), 7.47–7.46 (m, 3 H, Ar-H), 7.42–7.38 (m, 1H, Ar-H), 7.31 (m, 2 H, Ar-H), 4.34 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.15 (ketone C = O), 166.05 (amide C = O), 162.86 (oxadiazole C₂), 160.97 (oxadiazole C₅), 149.96 (pyrazole C₃), 144.04, 143.43 (Ar-C = C), 139.01 (pyrazole C₅), 135.16, 133.18, 131.57, 131.39, 131.31, 131.05, 130.45, 130.20, 129.42, 129.26, 128.06, 122.38, 119.47 (CO-C = C), 119.14, 115.77, 115.55, 105.77 (pyrazole C₄), 37.29 (CH₂); Elemental analysis for C₃₄H₂₄FN₅O₃S (601.66) (Calcd./Found): C, 67.87/67.91; H, 4.02/4.05; N, 11.64/11.67; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 7.96 min; peak area, 96.6% at λ_max 254.

(E)-2-((5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (13b)

white crystals (0.205 g, 65% yield); m.p. 220–222 °C; FT-IR; ν_max 3277 (NH), 1659 (Ketone CO), 1599 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.97 (s, 1H, CO-NH), 9.29 (s, 1H, pyrazole C₅–H), 8.14 (d, 2 H, J = 8.00 Hz, Ar–H), 8.01–7.97 (m, 4 H, Ar-H), 7.85–7.76 (m, 5 H, Ar-H), 7.69 (d, 1H, CH = CH J = 16.00 Hz), 7.77–7.55 (m, 2 H, Ar-H), 7.41 (m, 1H, Ar-H), 7.34–7.10 (m, 2 H, Ar-H), 7.02 (d, 2 H, J = 8.00 Hz, Ar–H), 4.38 (s, 2 H, CH₂), 3.82 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.95 (ketone C = O), 166 (amide C = O), 162.83 (oxadiazole C₂), 161.77 (oxadiazole C₅), 160.96, 149.91 (pyrazole C₃), 143.96 (Ar-C = C), 143.31, 139.04 (pyrazole C₅), 133.43, 131.67, 131.39, 131.31, 131.17, 130.27, 130.18, 128.24, 128.21, 128.02, 127.85, 119.89, 119.45 (CO-C = C), 119.07, 115.76, 115.54, 114.89, 105.79 (pyrazole C₄), 55.85 (OCH₃), 37.29 (CH₂); Elemental analysis for C₃₅H₂₆FN₅O₄S (631.68) (Calcd./Found): C, 66.55/66.58; H, 4.15/4.18; N, 11.09/11.12; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 7.12 min; peak area, 96.3% at λ_max 254.

(E)-N-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (13c)

Pale yellow crystals (0.244 g, 74% yield); m.p. 140–142 °C; FT-IR; ν_max 3298 (NH), 1656 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.84 (s, 1H, CO-NH), 9.26 (s, 1H, pyrazole C₅–H), 8.17 (d, 2 H, J = 8.00 Hz, Ar–H), 8.00-7.96 (m, 4 H, Ar-H), 7.83–7.78 (m, 3 H, Ar-H), 7.69 (d, 1H, CH = CH J = 16.00 Hz), 7.59–7.53 (m, 2 H, Ar-H), 7.528–7.520 (m, 1H, Ar-H), 7.43–7.39 (m, 1H, Ar-H), 7.38–7.36 (m, 1H, Ar-H), 7.33–7.29 (m, 2 H, Ar-H), 7.02 (d, 1H, J = 8.00 Hz, Ar–H), 4.36 (s, 2 H, CH₂), 3.86 (s, 3 H, OCH₃), 3.82 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.97 (ketone C = O), 166.00 (amide C = O), 164.24, 162.86 (oxadiazole C₂), 161.79 (oxadiazole C₅), 160.97, 151.69, 149.93 (pyrazole C₃), 149.49, 144.52, 143.26 (Ar-C = C), 139.02 (pyrazole C₅), 133.47, 131.59, 131.39, 131.30, 130.33, 130.17, 128.20, 128.03, 124.34, 119.91 (CO-C = C), 119.45, 119.06, 115.75, 115.53, 112.04, 111.16, 105.77 (pyrazole C₄), 56.21 (OCH₃), 56.06 (OCH₃), 37.32 (CH₂); Elemental analysis for C₃₆H₂₈FN₅O₅S (661.71) (Calcd./Found): C, 65.34/65.39; H, 4.27/4.30; N, 10.58/10.63; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 5.31 min; peak area, 97.6% at λ_max 254.

(E)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (13d)

Pale yellow crystals (0.200 g, 58% yield); m.p. 145–147 °C; FT-IR; ν_max 3301 (NH), 1656 (Ketone CO), 1597 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.46 (s, 1H, CO-NH), 9.40 (s, 1H, pyrazole C₅–H), 8.16 (d, 2 H, J = 8.00 Hz, Ar–H), 8.03–7.99 (m, 4 H, Ar-H), 7.86–7.93 (m, 3 H, Ar-H), 7.67 (d, 1H, CH = CH, J = 16.00 Hz), 7.59–7.55 (m, 2 H, Ar-H), 7.43–7.41 (m, 1H, Ar–H), 7.34–7.29 (m, 2 H, Ar–H), 7.25–7.21 (m, 2 H, Ar-H), 4.35 (s, 2 H, CH₂), 3.87 (s, 6 H, OCH₃), 3.72 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.04 (ketone C = O), 166.28 (amide C = O), 162.75 (oxadiazole C₂), 161.78 (oxadiazole C₅), 160.95, 153.57, 149.81 (pyrazole C₃), 144.42, 143.67 (Ar-C = C), 140.15, 139.03 (pyrazole C₅), 133.16, 131.85, 131.40, 131.31, 130.89, 130.79, 130.36, 130.15, 127.97, 121.63, 119.41 (CO-C = C), 119.23, 119.06, 115.74, 115.52, 107.15, 106.97, 105.77 (pyrazole C₄), 60.62 (OCH₃), 56.64 (OCH₃), 37.15 (CH₂); Elemental analysis for C₃₇H₃₀FN₅O₆S (691.73) (Calcd./Found): C, 64.25/64.28; H, 4.37/4.39; N, 10.12/10.14; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.37 min; peak area, 97.4% at λ_max 254.

(E)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)acetamide (13e)

Gray crystals (0.189 g, 61% yield); m.p. 240–243 °C; FT-IR; ν_max 3315 (NH), 1654 (Ketone CO), 1602 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.82 (s, 1H, CO-NH), 9.27 (s, 1H, pyrazole C₅–H), 8.17 (d, 2 H, J = 8.00 Hz, Ar–H), 8.01–7.95 (m, 6 H, Ar-H), 7.89 (d, 1H, CH = CH, J = 16.00 Hz), 7.78 (d, 2 H, J = 8.00 Hz, Ar-H), 7.72 (d, 1H, CH = CH, J = 16.00 Hz), 7.59–7.55 (m, 2 H, Ar-H), 7.43–7.41 (m, 1H, Ar-H), 7.34–7.28 (m, 4 H, Ar-H), 4.36 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.98 (ketone C = O), 166.02 (amide C = O), 162.85 (oxadiazole C₂), 162.59, 161.79 (oxadiazole C₅), 160.98, 149.92 (pyrazole C₃), 143.47 (Ar-C = C), 142.73, 139.04 (pyrazole C₅), 133.16, 131.92, 131.66, 131.61, 131.58, 131.39, 131.30, 130.46, 130.17, 128.25, 128.02, 122.32, 119.45 (CO-C = C), 119.08, 116.50, 116.28, 115.75, 115.54, 105.78 (pyrazole C₄), 37.36 (CH₂); Elemental analysis for C₃₄H₂₃F₂N₅O₃S (619.65) (Calcd./Found): C, 65.90/65.92; H, 3.74/3.77; N, 11.30/11.31; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.59 min; peak area, 98.0% at λ_max 254.

(E)-N-(4-(3-(4-bromophenyl)acryloyl)phenyl)-2-((5-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (13f)

White crystals (0.241 g, 71% yield); m.p. 210–212 °C; FT-IR; ν_max 3316 (NH), 1661 (Ketone CO), 1596 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.43 (s, 1H, CO-NH), 9.27 (s, 1H, pyrazole C₅–H), 8.14 (d, 2 H, J = 8.00 Hz, Ar–H), 8.03–7.93 (m, 4 H, Ar-H), 7.89–7.83 (m, 4 H, Ar-H), 7.69–7.64 (m, 3 H, Ar-H), 7.57–7.54 (m, 3 H, Ar-H), 7.43–7.41 (m, 1H, Ar-H), 7.33–7.29 (m, 2 H, Ar-H), 4.47 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.99 (ketone C = O), 166.33 (amide C = O), 164.21, 162.73 (oxadiazole C₂), 160.94 (oxadiazole C₅), 149.81 (pyrazole C₃), 142.53 (Ar-C = C), 138.99 (pyrazole C₅), 134.47, 132.92, 132.34, 131.82, 131.39, 131.30, 131.19, 130.41, 130.17, 128.19, 128.00, 124.30, 123.22, 119.40 (CO-C = C), 119.10, 115.74, 115.52, 105.75 (pyrazole C₄), 37.14 (CH₂); Elemental analysis for C₃₄H₂₃BrFN₅O₃S (680.55) (Calcd./Found): C, 60.01/60.03; H, 3.41/3.41; N, 10.29/10.31; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.21 min; peak area, 97.7% at λ_max 254.

(E)-2-((5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)acetamide (13g)

Pale yellow crystals (0.210 g, 65% yield); m.p. 210–212 °C; FT-IR; ν_max 3310 (NH), 1654 (Ketone CO), 1600 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.85 (s, 1H, CO-NH), 9.26 (s, 1H, pyrazole C₅–H), 8.28 (d, 2 H, J = 8.00 Hz, Ar–H), 8.20–8.14 (m, 4 H, Ar-H), 8.12 (d, 1H, CH = CH, J = 16.00 Hz), 8.00-7.96 (m, 4 H, Ar-H), 7.81–7.77 (m, 3 H, Ar-H), 7.58–7.55 (m, 2 H, Ar-H), 7.41 (m, 1H Ar-H), 7.33–7.29 (m, 2 H, Ar-H), 4.36 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.87 (ketone C = O), 166.09 (amide C = O), 164.24, 162.84 (oxadiazole C₂), 161.79 (oxadiazole C₅), 160.97, 149.93 (pyrazole C₃), 148.48, 143.75 (Ar-C = C), 141.69, 141.11, 139.01 (pyrazole C₅), 131.57, 131.38, 131.30, 130.68, 130.24, 130.18, 128.04, 126.44, 124.40, 119.45 (CO-C = C), 119.14, 115.75, 115.53, 105.76 (pyrazole C₄), 37.32 (CH₂); Elemental analysis for C₃₄H₂₃FN₆O₅S (680.55) (Calcd./Found): C, 63.15/63.18; H, 3.59/3.560; N, 13.00/13.02; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.47 min; peak area, 94.2% at λ_max 254.

(E)-2-((5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(p-tolyl)acryloyl)phenyl)acetamide (13h)

Orange crystals (0.175 g, 57% yield); m.p. 230–232 °C; FT-IR; ν_max 3328 (NH), 1658 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.88 (s, 1H, CO-NH), 9.30 (s, 1H, pyrazole C₅–H), 8.16 (d, 2 H, J = 8.00 Hz, Ar–H), 8.02–7.98 (m, 4 H, Ar-H), 7.88 (d, 1H, CH = C, J = 16.00 Hz), 8.00-7.96 (m, 4 H, Ar-H), 7.70 (d, 1H, CH = CH, J = 16.00 Hz), 7.59–7.55 (m, 2 H, Ar-H), 7.43–7.41 (m, 1H, Ar-H), 7.34–7.27 (m, 4 H, Ar-H), 4.38 (s, 2 H, CH₂), 2.36 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): δ 188.03 (ketone C = O), 166.02 (amide C = O), 162.84 (oxadiazole C₂), 160.97 (oxadiazole C₅), 149.92 (pyrazole C₃), 144.04, 143.40 (Ar-C = C), 141.07, 139.05 (pyrazole C₅), 133.28, 132.50, 131.65, 131.39, 131.31, 130.38, 130.17, 130.02, 129.32, 128.02, 121.34, 119.46 (CO-C = C), 119.08, 115.76, 115.54, 105.79 (pyrazole C₄), 37.34 (CH₂), 21.55 (CH₃); Elemental analysis for C₃₅H₂₆FN₅O₃S (615.68) (Calcd./Found): C, 68.28/68.30; H, 4.26/4.29; N, 11.38/11.41; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 8.97 min; peak area, 95.3% at λ_max 254.

(E)-2-((5-(3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(naphthalen-1-yl)acryloyl)phenyl)acetamide (13i)

Yellow crystals (0.253 g, 78% yield); m.p. 110–112 °C; FT-IR; ν_max 3313 (NH), 1651 (Ketone CO), 1603 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.93 (s, 1H, CO-NH), 9.29 (s, 1H, pyrazole C₅–H), 8.55 (d, 1H, CH = CH, J = 16.00 Hz, Ar–H), 8.29 (d, 1H, J = 8.00 Hz, Ar–H), 8.25–8.22 (m, 3 H, Ar-H), 8.07 (d, 1H, J = 8.00 Hz, Ar–H), 8.02–7.98 (m, 6 H, Ar-H), 7.81 (d, 2 H, J = 8.00 Hz Ar-H), 7.69–7.61 (m, 3 H, Ar-H), 7.59–7.55 (m, 2 H, Ar-H), 7.41 (m, 1H, Ar-H), 7.34–7.41 (m, 2 H, Ar-H), 4.38 (s, 2 H, CH₂); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.00 (ketone C = O), 166.07 (amide C = O), 164.24, 162.84 (oxadiazole C₂), 160.98 (oxadiazole C₅), 149.91 (pyrazole C₃), 143.57 (Ar-C = C), 139.89 (pyrazole C₅), 139.05, 133.85, 133.13, 131.88, 131.67, 131.39, 131.31, 131.22, 130.55, 130.17, 129.27, 128.01, 127.72, 126.79, 126.18, 126.10, 124.95, 123.46, 119.46 (CO-C = C), 119.15, 115.76, 115.55, 105.79 (pyrazole C₄), 37.35 (CH₂); Elemental analysis for C₃₈H₂₆FN₅O₃S (651.72) (Calcd./Found): C, 70.03/70.04; H, 4.02/4.03; N, 10.75/10.77; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 8.00 min; peak area, 94.7% at λ_max 254.

N-(4-Cinnamoylphenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (14a)

White crystals (0.202 g, 66% yield); m.p. 185–186 °C; FT-IR; ν_max 3312 (NH), 1664 (Ketone CO), 1600 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.83 (s, 1H, CO-NH), 9.21 (s, 1H, pyrazole C₅–H), 8.17 (d, 2 H, J = 8.00 Hz, Ar–H), 7.96–7.92 (m, 3 H, Ar-H), 7.92–7.84 (m, 4 H, Ar-H), 7.79 (d, 2 H, J = 8.00 Hz, Ar–H), 7.73 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.59–7.55 (m, 2 H, Ar-H), 7.50–7.43 (m, 3 H, Ar-H), 7.41–7.39 (m, 1H, Ar-H), 7.02 (d, 2 H, J = 8.00 Hz, Ar–H), 4.36 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.11 (ketone C = O), 166.06 (amide C = O), 162.75 (oxadiazole C₂), 161.19 (oxadiazole C₅), 160.33, 150.74 (pyrazole C₃), 144.01, 143.45 (Ar-C = C), 139.11 (pyrazole C₅), 135.19, 133.19, 131.47, 131.02, 130.46, 130.44, 130.15, 129.40, 129.27, 127.87, 124.09, 122.40, 119.36 (CO-C = C), 119.12, 114.12, 105.45 (pyrazole C₄), 55.66 (OCH₃), 37.33 (CH₂); Elemental analysis for C₃₅H₂₇N₅O₄S (613.69) (Calcd./Found): C, 68.50/68.62; H, 4.43/4.46; N, 11.41/11.43; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.22 min; peak area, 96.2% at λ_max 254.

(E)-2-((5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (14b)

Pale yellow crystals (0.225 g, 70% yield); m.p. 118–120 °C; FT-IR; ν_max 3314 (NH), 1653 (Ketone CO), 1599 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.81 (s, 1H, CO-NH), 9.23 (s, 1H, pyrazole C₅–H), 8.15 (d, 2 H, J = 8.00 Hz, Ar–H), 7.97 (d, 2 H, J = 8.00 Hz, Ar–H), 7.89–7.77 (m, 7 H, Ar-H), 7.70 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.58–7.55 (m, 2 H, Ar-H), 7.41 (m, 1H, Ar-H), 7.04–7.01 (m, 4 H, Ar–H), 4.36 (s, 2 H, CH₂), 3.83 (s, 3 H, OCH₃), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.93 (ketone C = O), 166.00 (amide C = O), 162.76 (oxadiazole C₂), 161.78 (oxadiazole C₅), 161.19, 160.33, 150.73 (pyrazole C₃), 143.96, 143.26 (Ar-C = C), 139.13 (pyrazole C₅), 133.47, 131.50, 131.17, 130.43, 130.31, 130.15, 127.87, 127.84, 124.11, 119.90 (CO-C = C), 119.36, 119.06, 114.88, 114.13, 105.47 (pyrazole C₄), 55.85 (OCH₃), 55.66 (OCH₃), 37.37(CH₂); Elemental analysis for C₃₆H₂₉N₅O₅S (643.72) (Calcd./Found): C, 67.17/67.18; H, 4.54/4.55; N, 10.88/10.90; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 5.84 min; peak area, 97.4% at λ_max 254.

(E)-N-(4-(3-(3,4-Dimethoxyphenyl)acryloyl)phenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide )14c)

Yellow crystals (0.215 g, 64% yield); m.p. 168–170 °C; FT-IR; ν_max 3319 (All chemicals used in the preparation NH), 1650 (Ketone CO), 1602 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.91 (s, 1H, CO-NH), 9.25 (s, 1H, pyrazole C₅–H), 8.17 (d, 2 H, J = 8.00 Hz, Ar–H), 7.97 (d, 2 H, J = 8.00 Hz, Ar–H), 7.88 (d, 2 H, J = 8.00 Hz, Ar–H), 7.83–7.76 (m, 3 H, Ar-H), 7.69 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.58–7.53 (m, 3 H, Ar-H), 7.42–7.36 (m, 2 H, Ar-H), 7.04–7.01 (m, 3 H, Ar–H), 4.38 (s, 2 H, CH₂), 3.87 (s, 3 H, OCH₃), 3.83 (s, 3 H, OCH₃), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.93 (ketone C = O), 166.05 (amide C = O), 162.75 (oxadiazole C₂), 161.19 (oxadiazole C₅), 160.33, 151.70, 150.72 (pyrazole C₃), 149.51, 144.49, 143.32 (Ar-C = C), 139.13 (pyrazole C₅), 133.46, 131.52, 130.44, 130.33, 130.14, 128.06, 127.84, 124.34, 124.11, 119.94 (CO-C = C), 119.35, 119.04, 114.12, 112.05, 111.20, 105.47 (pyrazole C₄), 56.23 (OCH₃), 56.08 (OCH₃), 55.66 (OCH₃), 37.33 (CH₂); Elemental analysis for C₃₇H₃₁N₅O₆S (673.74) (Calcd./Found): C, 65.96/65.97; H, 4.64/4.67; N, 10.39/10.42; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 4.78 min; peak area, 96.2% at λ_max 254.

(E)-2-((5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (14d)

White crystals (0.225 g, 70% yield); m.p. 130–132 °C; FT-IR; ν_max 3315 (NH), 1650 (Ketone CO), 1605 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.83 (s, 1H, CO-NH), 9.23 (s, 1H, pyrazole C₅–H), 8.18 (d, 2 H, J = 8.00 Hz, Ar–H), 7.97 (d, 2 H, J = 8.00 Hz, Ar–H), 7.90–7.86 (m, 3 H, Ar-H), 7.81 (d, 2 H, J = 8.00 Hz, Ar–H), 7.68 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.58–7.54 (m, 2 H, Ar-H), 7.43–7.41 (m, 1H, Ar-H), 7.22 (s, 2 H, Ar-H), 7.03 (d, 2 H, J = 8.00 Hz, Ar–H), 4.37 (s, 2 H, CH₂), 3.87 (s, 6 H, OCH₃), 3.81 (s, 3 H, OCH₃), 3.72 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): δ 188.04 (ketone C = O), 166.07 (amide C = O), 165.35, 162.76 (oxadiazole C₂), 161.19 (oxadiazole C₅), 160.33, 153.58, 150.74 (pyrazole C₃), 144.50, 143.41 (Ar-C = C), 140.17, 139.10 (pyrazole C₅), 133.28, 131.48, 130.76, 130.44, 130.16, 127.87, 124.08, 121.57, 119.36 (CO-C = C), 119.08, 114.12, 106.95, 105.45 (pyrazole C₄), 60.63 (OCH₃), 56.62 (OCH₃), 55.66 (OCH₃), 37.30 (CH₂); Elemental analysis for C₃₈H₃₃N₅O₇S (703.77) (Calcd./Found): C, 64.85/64.87; H, 4.73/4.75; N, 9.95/9.96; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 5.16 min; peak area, 95.4% at λ_max 254.

(E)-N-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (14e)

Gray crystals (0.233 g, 74% yield); m.p. 185–186 °C; FT-IR; ν_max 3315 (NH), 1657 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.13 (s, 1H, CO-NH), 9.29 (s, 1H, pyrazole C₅–H), 8.15 (d, 2 H, J = 8.00 Hz, Ar–H), 8.01–7.92 (m, 4 H, Ar-H), 7.92–7.79 (m, 6 H, Ar-H), 7.72 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.58–7.54 (m, 2 H,, Ar-H), 7.43–7.41 (m, 1H, Ar-H), 7.30 (m, 2 H, Ar-H), 7.03 (d, 1H, J = 8.00 Hz, Ar–H), 4.40 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.01 (ketone C = O), 166.19 (amide C = O), 162.70 (oxadiazole C₂), 161.18 (oxadiazole C₅), 160.32, 150.69 (pyrazole C₃), 143.59 (Ar-C = C), 142.71, 139.11 (pyrazole C₅), 133.10, 131.91, 131.59, 130.43, 130.14, 127.84, 124.10, 122.34, 122.32, 119.34 (CO-C = C), 119.09, 116.49, 116.28, 114.12, 105.46, 105.43 (pyrazole C₄), 55.66 (OCH₃), 37.24 (CH₂); Elemental analysis for C₃₅H₂₆FN₅O₄S (631.68) (Calcd./Found): C, 66.55/66.56; H, 4.15/4.18; N, 11.09/11.10; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.33 min; peak area, 97.1% at λ_max 254.

(E)-N-(4-(3-(4-Bromophenyl)acryloyl)phenyl)-2-((5-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)acetamide (14f)

White crystals (0.200 g, 62% yield); m.p. 185–186 °C; FT-IR; ν_max 3313 (NH), 1653 (Ketone CO), 1601 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.42 (s, 1H, CO-NH), 9.35 (s, 1H, pyrazole C₅–H), 8.15 (d, 2 H, J = 8.00 Hz, Ar–H), 8.01–7.94 (m, 3 H, Ar-H), 7.91–7.83 (m, 7 H, Ar-H), 7.70–7.64 (m, 3 H, Ar-H), 7.58–7.54 (m, 2 H, Ar-H), 7.42–7.40 (m, 1H, Ar-H), 7.03 (d, 1H, J = 8.00 Hz, Ar–H), 4.46 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.91 (ketone C = O), 162.66 (amide C = O), 161.16 (oxadiazole C₂), 160.30 (oxadiazole C₅), 150.61 (pyrazole C₃), 143.78 (Ar-C = C), 142.54, 139.10 (pyrazole C₅), 134.53, 132.91, 132.33, 131.72, 131.69, 131.22, 130.46, 130.14, 127.81, 124.33, 124.09, 123.24, 123.19, 119.30 (CO-C = C), 119.05, 114.10, 105.46 (pyrazole C₄), 55.66 (OCH₃), 37.16 (CH₂); Elemental analysis for C₃₅H₂₆BrN₅O₄S (692.59) (Calcd./Found): C, 60.70/60.72; H, 3.78/3.78; N, 10.11/10.12; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 6.46 min; peak area, 95.3% at λ_max 254.

(E)-2-((5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)acetamide (14g)

Orange crystals (0.168 g, 51% yield); m.p. 168–170 °C; FT-IR; ν_max 3313 (NH), 1660 (Ketone CO), 1597 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.99 (s, 1H, CO-NH), 9.25 (s, 1H, pyrazole C₅–H), 8.28 (d, 2 H, J = 8.00 Hz, Ar–H), 8.20–8.10 (m, 5 H, Ar-H), 7.98 (d, 2 H, J = 8.00 Hz, Ar–H), 7.88 (d, 2 H, J = 8.00 Hz, Ar–H), 7.83–7.77 (m, 3 H, Ar-H), 7.58–7.54 (m, 2 H, Ar-H), 7.42–7.41 (m, 1H, Ar-H), 7.03 (d, 2 H, J = 8.00 Hz, Ar–H), 4.39 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 187.84 (ketone C = O), 166.16 (amide C = O), 162.71 (oxadiazole C₂), 161.19 (oxadiazole C₅), 160.32, 150.70 (pyrazole C₃), 148.49, 143.83 (Ar-C = C), 141.74, 141.08, 139.12 (pyrazole C₅), 132.76, 131.53, 130.68, 130.43, 130.26, 130.14, 127.84, 126.48, 124.39, 124.10, 119.35 (CO-C = C), 119.11, 114.12, 105.45 (pyrazole C₄), 55.66 (OCH₃), 37.32 (CH₂); Elemental analysis for C₃₅H₂₆N₆O₆S (658.69) (Calcd./Found): C, 63.82/63.84; H, 3.98/3.99; N, 12.76/12.77; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 5.93 min; peak area, 96.0% at λ_max 254.

(E)-2-((5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(p-tolyl)acryloyl)phenyl)acetamide (14h)

Yellow crystals (0.247 g, 79% yield); m.p. 119–121 °C; FT-IR; ν_max 3313 (NH), 1651 (Ketone CO), 1604 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 11.17 (s, 1H, CO-NH), 9.32 (s, 1H, pyrazole C₅–H), 8.14 (d, 2 H, J = 8.00 Hz, Ar–H), 7.99 (d, 2 H, J = 8.00 Hz, Ar–H), 7.90 (d, 2 H, J = 8.00 Hz, Ar–H), 7.83 (d, 2 H, J = 8.00 Hz, Ar–H), 7.80–7.76 (m, 3 H, Ar-H), 7.70 (d, 1H, CH = CH J = 16.00 Hz Ar-H), 7.58–7.55 (m, 2 H, Ar-H), 7.42–7.41 (m, 1H, Ar-H), 7.27 (d, 2 H, J = 8.00 Hz, Ar–H), 7.03 (d, 2 H, J = 8.00 Hz, Ar–H), 4.42 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃), 2.36 (s, 3 H, CH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.04 (ketone C = O), 166.16 (amide C = O), 162.70 (oxadiazole C₂), 161.18 (oxadiazole C₅), 160.32, 150.68 (pyrazole C₃), 144.02, 143.51 (Ar-C = C), 141.06, 139.13 (pyrazole C₅), 133.23, 132.50, 131.60, 130.44, 130.34, 130.14, 130.01, 129.32, 127.83, 124.11, 121.35, 119.34 (CO-C = C), 119.08, 114.12, 105.47 (pyrazole C₄), 55.67 (OCH₃), 37.25 (CH₂), 21.55 (CH₃); Elemental analysis for C₃₆H₂₉N₅O₄S (627.72) (Calcd./Found): C, 68.88/68.90; H, 4.66/4.69; N, 11.16/11.18; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 7.83 min; peak area, 95.3% at λ_max 254.

(E)-2-((5-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-(3-(naphthalen-1-yl)acryloyl)phenyl)acetamide (14i)

Yellow crystals (0.209 g, 63% yield); m.p. 120–122 °C; FT-IR; ν_max 3312 (NH), 1656 (Ketone CO), 1598 (amide CO); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 10.83 (s, 1H, CO-NH), 9.22 (s, 1H, pyrazole C₅–H), 8.55 (d, 1H, CH = CH J = 16.00 Hz, Ar–H), 8.29 (d, 1H, J = 8.00 Hz, Ar–H), 8.23 (t, 3 H, J = 8.00 Hz, Ar-H), 8.07 (d, 1H, J = 8.00 Hz, Ar–H), 8.03–8.01 (m, 2 H, Ar-H), 7.97 (d, 2 H, J = 8.00 Hz Ar-H), 7.88 (d, 2 H, J = 8.00 Hz Ar-H), 7.81 (d, 2 H, J = 8.00 Hz Ar-H), 7.69–7.61 (m, 3 H, Ar-H), 7.59–7.55 (m, 2 H, Ar-H), 7.42-7,41 (m, 1H Ar-H), 7.03 (d, 2 H, J = 8.00 Hz Ar-H), 4.37 (s, 2 H, CH₂), 3.81 (s, 3 H, OCH₃); ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 188.02 (ketone C = O), 169.44, 166.07 (amide C = O), 160.33 (oxadiazole C₂), 160.12 (oxadiazole C₅), 150.74 (pyrazole C₃), 143.55 (Ar-C = C), 139.91 (pyrazole C₅), 139.12 133.85, 133.15, 131.86, 131.67, 131.50, 131.24, 130.57, 130.44, 130.16, 130.13, 129.27, 127.87, 127.73, 126.79, 126.19, 124.94, 124.31, 124.10, 123.46, 119.37 (CO-C = C), 119.16, 115.43, 114.13, 55.67 (OCH₃), 37.36 (CH₃); Elemental analysis for C₃₉H₂₉N₅O₄S (663.75) (Calcd./Found): C, 70.57/70.59; H, 4.40/4.41; N, 10.55/10.58; HPLC analysis: Mobile phase (dipotassium hydrogen phosphate buffer [pH 3.2]: ACN, 30:70 respectively) FR 1mL /min, retention time = 9.83 min; peak area, 94.8% at λ_max 254.

Biology

In vitro anticancer activity

In vitro one-dose assay on NCI 60 cancer cell lines

The thirty-three selected compounds (10a-10c, 11a-11c, 12a-12i, 13a-13i and 14a-14i) were primary evaluated for their in vitro anticancer activity at single dose concentration on a panel of about 60 cancer cell lines derived from nine different cancer types: lung, leukemia, colon, ovarian, melanoma, renal, prostate, CNS and breast cancers. The tested compounds seeded to the culture at a single at single concentration 10^{− 5} M, then incubated for 48 h. the end determinations were made with a protein binding dye, Sulforhodamine B (SRB). The percent growth was evaluated spectrophotometrically versus control (untreated with test agents). Compounds which exhibit significant growth inhibition (10b, 11a, 11b and 11c) are again evaluated against the 60 cell lines panel at five concentration levels, 10-fold dilutions each with the top dose being 10^{− 4} molar (100 μm) via solubilizing drug in dimethylsulphoxide. After drug addition, cells were incubated at at 37 °C, 5% CO₂, 95% air, and 100% relative humidity for 48 h then staining by SRB and the absorbance was evaluated spectrophotometrically by using an automated plate. The percent growth was calculated at different drug concentration levels and at different time. These methods of assay were performed according to the protocol of the Drug Evaluation Branch, National Cancer Institute (NCI), Bethesda, MA, USA. The methodology details of the assay is described on the web site of NCI [66] (https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm).

Cytotoxicity IC₅₀ on normal human cells WI-38

Cell lines were obtained from American Type Culture Collection; cells were cultivated using Dulbecco’s modified Eagle’s medium (DMEM(Invitrogen/Life Technologies)), and the cytotoxicity of the tested compounds was determined using MTT (3-[4,5- dimethylthiazole-2yl]-2,5-diphenyltetrazolium bromide) assay [67]. Cell viability is determined spectrophotometrically by counting the number of living cells after treating the cells with tested compounds compared to untreated control cell lines.

Enzymatic assay

VEGFR-2 Inhibition assay

Enzyme reactions were performed in 50 mM Tris-HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20 and 2 mM dithiothreitol, containing 10 µM ATP, 0.1 µg/ml biotinylated poly-GluTyr (4:1) and 0.1 nM of VEGFR-2 (Millipore, UK). Before the catalytic initiation with ATP, the tested compounds at final concentrations ranging from 0 to 300 µg/ml and enzyme were incubated for 5 min at room temperature. The reactions were quenched by the addition of 25 µl of 100 mM ethylenediaminetetraacetic acid, 10 µg/ml Alpha Screen streptavidin donor beads, and 10 µg/ml acceptor beads in 62.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.4, 250 mM NaCl, and 0.1% bovine serum albumin. The plate was incubated in the dark overnight and then read by ELISA Reader (PerkinElmer). Wells containing the substrate and the enzyme without compounds were used as reaction control. Wells containing biotinylated poly-GluTyr (4:1) and enzyme without ATP were used as basal control. Percent inhibition was calculated by the comparison of compounds treated to control incubations. The concentration of the test compound causing 50% inhibition (IC₅₀) was calculated from the concentration–inhibition response curve (triplicate determinations) and the data were compared with sorafenib (Sigma-Aldrich) as standard VEGFR-2 inhibitor.

EGFR inhibitory assay

A cell-free assay was used to investigate the mechanism of inhibition of EGFR kinase. Kit used for immune-assay was cloud clone SEA757Hu 96 Tests. 200 lM (EGFR) was used. From the following equation: E (%) = E max/(1 + [I]/ID50), where E (%) is the fraction of the enzyme activity measured in the presence of the inhibitor, E max is the activity in the absence of the inhibitor, [I] is the inhibitor concentration and ID₅₀ is the inhibitor concentration at which E (%) = 0.5 E max, a dose–response curve was generated. Mean values of two independent replicates for each experiment were used for the interpolation.

Cell cycle analysis

After treatment with test compounds for the specified duration, cells (105 cells) are collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Cells are resuspended in two milliliters of 60% ice-cold ethanol and incubated at 4 °C for 1 h for fixation. Fixed cells are washed twice again with PBS (pH 7.4) and re-suspended in 1 mL of PBS containing 50 µg/mL RNAase A and 10 µg/mL propidium iodide (PI). After 20 min of incubation in dark at 37 C, cells are analyzed for DNA contents using flow cytometry analysis using FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte™ flowcytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events are acquired. Cell cycle distribution is calculated using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA) [68].

Apoptosis assay

Apoptosis and necrosis cell populations are determined using Annexin V-FITC apoptosis detection kit (Abcam Inc., Cambridge Science Park, Cambridge, UK) coupled with 2fluorescent channels flowcytometry. After treatment with test compounds for the specifiedduration, cells (105 cells) are collected by trypsinization and washed twice with ice-coldPBS (pH 7.4). Then, cells are incubated in dark with 0.5 ml of Annexin V-FITC/PI solutionfor 30 min in dark at room temperature according to manufacturer protocol. After staining, cells are injected via ACEA Novocyte™ flowcytometer (ACEA Biosciences Inc., SanDiego, CA, USA) and analyzed for FITC and PI fluorescent signals using FL1 and FL2signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI).For each sample, 12,000 events are acquired and positive FITC and/or PI cells are quantifiedby quadrant analysis and calculated using ACEA NovoExpress™ software (ACEABiosciences Inc., San Diego, CA, USA) [69, 70].

Evaluation of nitric oxide release

A solution of the appropriate compound (20 µL of 10^{− 4} M in 100 mL DMSO) was added to 2 ml of 1:1 V/V mixture of 50 mM phosphate buffer (pH 7.4) and methanol containing (5 × 10-4 M) L-cysteine. After 1 h at 37Cº, 1 ml of the reaction mixture was treated with 250 µL of Griess reagent. After 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min., the absorbance was measured at 540 nm. Sodium nitrite standard solutions (10–50 nmol/ml) were used to construct the calibration curve [65]. Griess reagent was prepred as follows: [sulfanilamide (4 g), N-naphthylethylenediamine dihydrochloride (0.2 g), 85% phosphoric acid (10 ml) in distilled water (final volume: 100 ml)].

Docking methodology

AutoDock Vina v.1.2.0 was used for carrying out the molecular docking and Discovery Studio Visualizer was used for analyzing the interactions between the target receptor and ligands. For more details see supporting information.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Author Contribution: Conception and design: B, C, DExperimental work: A, DBiological studies: DDocking Study: D Validation: A,D Formal analysis: DInvestigation: B, C Resources: A Data interpretation: B, C, D Writing-original draft preparation: A, D Intellectual content writing-review and editing: B,C Visualization: DSupervision: C,D.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.