Design, synthesis and biological evaluation of novel 1,2,4-triazolo and 1,2,4-triazino[4,3- a ]quinoxalines as potential anticancer and antimicrobial agents

Doaa A. E. Issa; Nargues S. Habib; Abeer E. Abdel Wahab

doi:10.1039/C4MD00257A

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DOI: 10.1039/C4MD00257A (Concise Article) Med. Chem. Commun., 2015, 6, 202-211

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Design, synthesis and biological evaluation of novel 1,2,4-triazolo and 1,2,4-triazino[4,3-a]quinoxalines as potential anticancer and antimicrobial agents†

Doaa A. E. Issa *^ab, Nargues S. Habib ^a and Abeer E. Abdel Wahab ^c
^aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, 21521, Alexandria, Egypt
^bDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, 115020, Beirut, Lebanon. E-mail: d.issa@bau.edu.lb
^cGenetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Application, Borg El-Arab, 21934, Alexandria, Egypt

Received 16th June 2014 , Accepted 26th September 2014

First published on 29th September 2014

Abstract

In an effort to find new leads as anticancer or antimicrobial agents, the present work deals with the synthesis of some novel 1-substituted 1,2,4-triazolo[4,3-a]quinoxalines 7, 9a,b, and 14–19 and 1,2,4-triazino[4,3-a]quinoxalines 10a–c as well as 2-[5-amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline 13. These were synthesized using the key intermediate 3-benzyl-2-hydrazinoquinoxaline 6 with various reagents. Ten compounds, namely 7, 9a, 10b, 11, and 13–18 were chosen by the National Cancer Institute of Bethesda (NCI) for evaluation of their anticancer activity. The results indicated that 9a was the most active and was further evaluated for in vitro five dose assay against 60 human cell lines. It was proven to possess the highest broad spectrum anticancer activity. It showed particular effectiveness towards leukemia SR, non-small cell lung cancer HOP-92, NCI-H460, colon cancer HCT-116, HCT-15, CNS cancer U251, melanoma LOX IMVI, renal cancer A498, prostate cancer PC-3, and breast cancer MDA-MB-468 cell lines (GI₅₀ = 3.91, 3.45, 3.49, 3.21, 1.96, 5.18, 3.69, 1.80, 5.19, and 5.55 μM, respectively). All new compounds were screened for their antimicrobial activity and were very active against P. aeruginosa. Compounds 10a and 16 were twice as active as ampicillin against P. aeruginosa. Five compounds, 9a, b, 10b, 13, and 14 were equipotent to ampicillin against P. aeruginosa. In addition, compound 16 showed a broad spectrum antimicrobial activity. Furthermore, compound 9a showed dual activity as an anticancer and antimicrobial agent.

1. Introduction

The quinoxaline pharmacophore, being isosteric to purine antimetabolites, developed an appealing platform for the discovery of active chemotherapeutic agents. Several anticancer drugs containing a quinoxaline ring have been reported along with their pharmacological data, activity against solid tumours and clinical trials studies.¹ The antineoplastic antibiotic quinoxaline, Triostatin A, (Fig. 1) showed considerable interest. It is characterized by cross-linked octapeptide rings bearing two quinoxalines and is stabilized at its centre by a cysteine pair (disulfhydryl covalent bonds). The two quinoxaline rings represent the planar aromatic ring structure which is a major requirement for intercalation.² Besides the two antineoplastic quinoxalines topoisomerase II poisons, XK469 ((±)2-[4-(7-chloro-2-quinoxalinyloxy)phenoxyl]propanoic acid) and CQS (5-chloro quinoxalin-2-yl)-(4-aminobenzene sulfonamide) (Fig. 1) showed difference in DNA site specificity of topoisomerase II poisoning. This may be caused by differences in their geometry, side chains or electronic structure.³ In addition, XK469 induced apoptosis of human ovarian cancer cell line PA1.⁴ Several efforts have been made to search for new quinoxaline anticancer agents; thus a series of 2-alkylcarbonyl and 2-benzoyl-3-trifluromethylquinoxaline-1,4-di-N-oxide derivatives was found to inhibit the growth of Leukemia cell lines.⁵


	Fig. 1 Some quinoxaline derivatives having anticancer activity.

Recently, a series of 5,7-diamino-3-phenyl-2-benzylamino and 5,7-diamino-3-phenyl-2-benzyloxy, substituted quinoxalines of the general formula (A) (Fig. 1) has been synthesized. Among them two compounds showed promising anticancer activity.⁶ 4-Substituted anilino-1,2,4-triazolo[4,3-a]quinoxalines of the general formula (B) (Fig. 1) exhibited prominent cytotoxicity against mock-infected M.T-4 cells.⁷ Whereas, 3-benzoyl-2-piperazinylquinoxalines of the general formula (C) (Fig. 1) showed anticancer activity against melanoma, renal and colon cancer.⁸ Quinoxalines are currently recognized to display good affinity to the ATP-binding site of the c-kit tyrosine protein. Their activity as antitumor agents may be due to their ability to inhibit protein tyrosine kinases.⁹

During our ongoing research program on quinoxaline derivatives,^10–13 we were able to synthesize new lead structures. In particular: 1-(3-methoxyphenyl)-4-phenyl-1,2,4-triazolo[4,3-a]quinoxaline (D), 1-(substituted methyl)-4-phenyl-1,2,4-triazolo[4,3-a]quinoxalines (E), 2-(4-substituted phenyl)-5-phenyl-1H-1,2,4-triazino[4,3-a]quinoxalines (F),¹² 1-(N-arylcarbamoylmethyl)-3-phenylquinoxaline-2(1H)-ones (G),¹³ and 2-(N-arylcarbamoylmethylsulfonyl)-3-phenylquinoxaline (H) (Fig. 1).¹⁴ The antimicrobial activity of the triazolo and triazinoquinoxalines is well documented.^{10,11,13–15}

Lately the incorporation of 1,2,4-triazolo fused heterocycles in anti-proliferative and anti-microbial drug design projects revealed a very interesting scaffold to find new potential agents.^16,17 These findings prompted us to continue our investigations on quinoxalines having dual activity as anticancer and antimicrobial agents and to synthesize 1-substituted 4-benzyl-1,2,4-triazolo[4,3-a]quinoxalines having various pharmacophoric groups at the 1-position (7, 9a, b, and 16–19); also a series of 2-aryl-5-benzyl-1H-1,2,4-triazino[4,3-a]quinoxalines 10a–c were synthesized besides 2-[5-amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline 13 and 4-benzyl-1,2,4-triazolo[4,3-a]quinoxaline-1(2H)-one 15.

2. Results and discussion

2.1. Chemistry

Target 1,2,4-triazolo[4,3-a]quinoxalines 7, 9a, b, 14–19 and 1,2,4-triazino[4,3-a]quinoxalines 10a–c as well as 2-[5-amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline 13 were synthesized by the reactions depicted in Schemes 1–3.


	Scheme 1 Synthesis of key intermediate 6. Reagents and conditions: (i) (CH₃CO)₂O·H₂O, rt, 2 h, 91%; (ii) C₆H₅CHO, (CH₃CO)₂O, CH₃CO₂Na, 100 °C, 2 h, 86%; (iii) o-C₆H₄(NH₂)₂, CH₃CH₂OH, reflux, 5 h, 79%; (iv) POCl₃, 100 °C, 2 h, 75%; (v) NH₂NH₂·H₂O, CH₃CH₂OH, reflux, 2 h, 92%.


	Scheme 2 Synthesis of the target compounds 7–13. Reagents and conditions: (i) (CH₃CO)₂O or 2-acetylbuterolactone, dry xylene, reflux, 2–3 h, 55–77%; (ii) 2-Cl or 4-NO₂C₆H₄COOH, POCl₃, 100 °C, 2 h, 92–95%; (iii) 4-RC₆H₄COCH₂Br, dry dioxane, reflux, 1 h, 64–73%; (iv) CH₃COCH₂COOC₂H₅, 160–170 °C, 1 h, 69%; (v) 4-ClC₆H₄COCH₂CN, C₂H₅OH, CH₃COOH, reflux, 2 h, 97%.


	Scheme 3 Synthesis of the target compounds 14–19. Reagents and conditions: (i) HCOOH, reflux, 3 h, 57%; (ii) ClCOOC₂H₅, C₂H₅OH, reflux, 3 h, 69%; (iii) succinic anhydride, gl AcOH, reflux, 5 h, 57%; (iv) phthalic anhydride, gl AcOH, reflux, 5 h, 59%; (v) (COOC₂H₅)₂, dry xylene, reflux, 3 h, 68%; (vi) CH₂(COOC₂H₅)₂, 160–170 °C, 1 h, 87%.

Preparation of the hydrazino key intermediate 6 was accomplished according to the sequence of reactions of Scheme 1. The azlactone of α-acetamidocinnamic acid 3 has been prepared according to the literature.^18,19 Its condensation with o-phenylenediamine achieved ring closure to quinoxalinone 4 in good yield, the product was identical to that previously described by Moffitt and Schultz obtained from the hydrolysis and decarboxylation of 3-(α-carboxamido)benzyl-2(1H)-quinoxalinone.²⁰ Earlier, 3-benzyl-1H-quinoxalin-2-one 4 has been differently prepared from o-phenylenediamine and phenylpyruvic acid.⁸ Treatment of 4 with phosphorus oxychloride gave the chloroquinoxaline derivative 5. Replacement of the chlorine atom of 5 with the hydrazine moiety resulted in the key intermediate 6.

In the present work we studied the reaction of 6 with a variety of carbonyl compounds. The synthetic routes adopted to obtain the newly synthesized compounds are depicted in Schemes 2 and 3. Treatment of 6 with 2-acetylbutyrolactone failed to afford the corresponding 2-(pyrazol-1-yl)-3-benzylquinoxaline derivative 8 as reported for the preparation of related compounds.²¹ Instead, 4-benzyl-1-methyl-1,2,4-triazolo[4,3-a]quinoxaline 7 was obtained. The structure of 7 was confirmed by IR and ¹H-NMR spectra and chemically through its parallel synthesis from 6 and acetic anhydride.¹⁵ Cyclization of 6 using aromatic acids and phosphorus oxychloride yielded 1-aryl-4-benzyl-1,2,4-triazolo[4,3-a]quinoxaline 9a,b where 9a is reported to be differently prepared.²² The 1,2,4-triazino[4,3-a]quinoxalines 10a–c were obtained through the reaction of 6 with phenacyl bromides. Fusion of a mixture of 6 and ethyl acetoacetate failed to afford 2-[5-hydroxy-3-methylpyrazol-1-yl]-3-benzylquinoxaline 12 as previously reported for analogous compounds.¹¹ It yielded ethyl 3-[(3-benzylquinoxalin-2-yl)hydrazono]butyrate 11. Reacting 6 with 4-chlorophenyl-ω-cyanoacetophenone yielded 2-[5-amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline 13.

Compounds 14 and 15 were obtained by the reaction of 6 with formic acid and ethyl chloroformate, respectively. Compound 14 was previously reported to be synthesized from 2-benzyl-3-(2-methylenehydrazinyl) quinoxaline by its pyrolysis in dimethylformamide or its acylation with acetic anhydride in pyridine.²² Compound 15 was reported to be synthesized through the reaction of 2-chIoro-3-benzylquinoxaline with semicarbazide hydrochloride.¹⁵ Compounds 16 and 17 were obtained by cyclization of 6 using succinic or phthalic anhydride, respectively, in glacial acetic acid. Furthermore reacting 6 with diethyl oxalate or diethyl malonate in boiling dry xylene gave compounds 18 and 19 respectively as described for the synthesis of analogous compounds.¹³

2.2. Biological evaluation

2.2.1. Preliminary in vitro anticancer screening. Out of the newly synthesized compounds, ten candidates, namely: 7, 9a, 10b, 11, and 13–18 were selected by the National Cancer Institute (NCI) Bethesda-Maryland, USA, to be evaluated for their in vitro antitumor activity through the in vitro disease-oriented human cells screening panel assay. An effective one-dose assay has been added to the NCI-60 cell screen in order to increase compound throughput and reduce the data-turnaround time to suppliers while maintaining efficient identification of active compounds.^23,24 All compounds submitted to the NCI-60 cell screen are now tested initially at a single high dose (10 μM) in the full NCI-60 cell panel including leukemia, non-small cell lung, colon, CNS melanoma, ovarian, renal, prostate, and breast cancer cell lines. Only compounds which satisfy pre-determined threshold inhibition criteria would proceed to the five-dose screen. The threshold inhibition criteria for proceeding to the five-dose screen were designed to efficiently capture compounds with anti-proliferative activity, and are based on careful analysis of historical Development Therapeutic Program (DTP) screening data. Data are reported as a mean graph of the percent growth of treated cells, and presented as percentage growth inhibition (GI%) caused by the test compounds (Table 1). Moreover, three response parameters (GI₅₀, TGI, and LC₅₀) were calculated for each cell line for compound 9a (Table 2). The GI₅₀ value corresponds to the compound concentration causing a 50% decrease in net cell growth. The TGI value is the compound concentration resulting in total growth inhibition and the LC₅₀ value is the compound concentration causing a net 50% loss of initial cells at the end of the incubation period (48 h). Subpanel and fullpanel mean-graph midpoint values (MG-MID) for certain agents are the average of individual real and default GI₅₀, TGI, or LC₅₀ values of all cell lines in subpanel and fullpanel, respectively.²⁵

Table 1 In vitro percentage growth inhibition (GI%) caused by the test compounds against some selected tumor cell lines at the single dose assay^a

Cpd	NSC no.	Panel	Subpanel tumor cell lines (% growth inhibitory activity)
a The data obtained from NCI in vitro disease-oriented human tumor cell screen at 10 μM concentration.
7	761 669	Non-small cell lung cancer	HOP-92 (17.65); NCI-H226 (15.11)
		CNS cancer	SNB-75 (21.94)
		Ovarian cancer	NCI/ADR-RES (12.55)
9a	761 676	Leukemia	HL-60(TB) (46.36); K-562 (78.62); MOLT-4 (60.41); RPMI-8226 (63.81); SR (84.89)
		Non-small cell lung cancer	EKVX (48.06); HOP-92 (50.11); NCI-H460 (84.44)
		Colon cancer	HCT-116 (65.87); HCT-15 (55.37); KM12 (51.07)
		CNS cancer	SF-268 (47.55); U251 (57.23)
		Melanoma	LOX IMVI (60.62)
		Renal cancer	A498 (61.07)
		Prostate cancer	PC-3 (60.87)
10b	761 674	Leukemia	HL-60(TB) (74.81); K-562 (69.87); RPMI-8226 (50.59); SR (74.57)
		Non-small cell lung cancer	NCI-H522 (61.57)
		Colon cancer	HCT-116 (46.86)
		Renal cancer	CAKI-1 (44.63)
11	761 673	Renal cancer	A498 (17.75); UO-31 (15.85)
13	761 675	Non-small cell lung cancer	A549/ATCC (21.96); EKVX (21.36); NCI-H226 (15.73); NCI-H23 (18.49)
		Colon cancer	HCT-116 (27.18); HCT-15 (25.56); HT29 (30.32)
		CNS cancer	SNB-75 (15.58)
		Renal cancer	CAKI-1 (19.3); RXF 393 (21.29)
		Breast cancer	MDA-MB-231/ATCC (24.88)
14	761 678	Non-small cell lung cancer	NCI-H226 (15.47)
		CNS cancer	SNB-75 (23.49)
		Renal cancer	A498 (38.71); UO-31 (22.68)
		Prostate cancer	PC-3 (14.24)
15	761 677	Renal cancer	A498 (28.58); UO-31 (35.5)
16	761 671	Non-small cell lung cancer	HOP-92 (18.87)
		CNS cancer	SNB-75 (17.71)
		Renal cancer	UO-31 (12.22)
17	761 670	Non-small cell lung cancer	NCI-H522 (14.55)
		CNS cancer	SF-268 (14.1); SNB-75 (24.41)
		Ovarian cancer	OVCAR-3 (20.22)
		Renal cancer	UO-31 (18.01)
		Prostate cancer	PC-3 (43.92)
18	761 672	CNS cancer	SNB-75 (10.34)
18	761 672	Renal cancer	UO-31 (15.06)

Table 2 Growth inhibitory action (GI₅₀) of some selected in vitro tumor cell lines (μM)^a for compound 9a (NCS 761676)

Panel	Subpanel cell lines (cytotoxicity GI₅₀ μM)
a Data obtained from NCI in vitro disease-oriented human cell screen.
Leukemia	CCRF-CEM (7.11); HL-60(TB) (32.5); K-562 (5.55); MOLT-4 (9.04); RPMI-8226 (5.64); SR (3.91)
Non-small cell lung cancer	A549/ATCC (8.77); EKVX (5.96); HOP-62 (12.4); HOP-92 (3.45); NCI-H226 (9.96); NCI-H322M (53.9); NCI-H460 (3.49); NCI-H522 (53.9)
Colon cancer	COLO 205 (8.77); HCC-2998 (42.8); HCT-116 (3.21); HCT-15 (1.96); HT29 (9.15); KM12 (9.48); SW-620 (43.4)
CNS cancer	SF-295 (12.3); SF-539 (24.2); SNB-19 (27.8); SNB-75 (6.12); U251 (5.18)
Melanoma	LOX IMVI (3.69); MALME-3M (23.8); M14 (36.7); SK-MEL-2 (55.8); SK-MEL-5 (5.90); UACC-257 (34.3); UACC-62 (7.96)
Ovarian cancer	IGROV1 (27.7); OVCAR-3 (21.7); OVCAR-4 (8.01); OVCAR-8 (7.21); NCI/ADR-RES (7.04); SK-OV-3 (23.9)
Renal cancer	786-0 (10.4); A498 (1.80); ACHN (34.1); CAKI-1 (9.71); RXF 393 (8.45); TK-10 (37.4); UO-31 (25.0)
Prostate cancer	PC-3 (5.19); DU-145 (44.2)
Breast cancer	MCF7 (22.2); MDA-MB-231/ATCC (6.24); HS 578T (9.66); BT-549 (6.72); T-47D (6.82); MDA-MB-468 (5.55)

As revealed from Table 1 showing the percentage growth inhibition (GI%) caused by the test compounds, compound 9a, having the 2-chlorophenyl moiety at the 1-position of the triazoloquinoxaline, was the most active compound. It showed a broad spectrum anticancer activity against most cell lines, namely Leukemia HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR cell lines with a growth inhibition of 46.36%, 78.62%, 60.41%, 63.81% and 84.89%, respectively. Compound 9a was remarkably active against non-small cell lung cancer EKVX, HOP-92 and NCI-H460 cell lines with a growth inhibition of 48.06%, 50.11% and 84.44%, respectively. The colon cancer HCT-116, HCT-15 and KM12 cell lines showed a growth inhibition of 65.87%, 55.37%, and 51.07%, respectively. In addition CNS cancer SF-268 and U251 cell lines were reasonably inhibited by compound 9a with a growth inhibition of 47.55% and 57.23%, respectively. Also compound 9a manifested appreciable activity against melanoma LOX IMVI, renal cancer A498 and prostate cancer PC-3 cell lines with a growth inhibition of 60.62%, 61.07% and 60.87%, respectively.

Replacement of 2-chlorophenyl with 2-carboxyphenyl at the 1-position of the triazoloquinoxaline decreased the activity where compound 17 showed only activity against CNS cancer SNB-75, ovarian cancer OVCAR-3 and prostate cancer PC-3 cell lines with a growth inhibition of 24.41%, 20.22% and 43.92%, respectively. The unsubstituted triazoloquinoxaline 14 displayed moderate activity against CNS cancer SNB-75, renal cancer A498 and UO-31 cell lines with a growth inhibition of 23.49%, 38.71% and 22.68% respectively. Compound 15 having a carbonyl group at the 1-position of the triazoloquinoxaline was fairly active against renal cancer A498 and UO-31 cell lines with 28.58% and 35.5% growth inhibition, respectively. The 1-methyltriazoloquinoxaline 7 showed weak activity against the CNS cancer SNB-75 cell line with a growth inhibition of 21.94%. Conversely the triazinoquinoxaline 10b having 4-chloro substitution demonstrated a significant growth inhibition of 74.81%, 69.87%, 50.59%, 74.57%, 61.57%, 46.86% and 44.63% against leukemia HL-60(TB), K-562, RPMI-8226, SR, non-small cell lung cancer NCI-H522, colon cancer HCT-116 and renal cancer CAKI-1 cell lines, respectively.

Furthermore the pyrazolylquinoxaline 13 showed some activity against non-small cell lung cancer, colon cancer, renal cancer and breast cancer specifically non-small cell lung cancer A549/ATCC, EKVX, colon cancer HCT-116, HCT-15, HT29, renal cancer RXF 393 and breast cancer MDA-MB-231/ATCC cell lines with a growth inhibition of 21.96%, 21.36%, 27.18%, 25.56%, 30.32%, 21.29% and 24.88%, respectively.

Only compound 9a fulfilled the requirements of selection for five-dose assay. Further interpretation of the five-dose screening data for compound 9a (Table 2) revealed that it was the most active lead in this study with a broad spectrum anticancer activity against most of the tested subpanel tumor cell lines with particular effectiveness against leukemia CCRF-CEM K-562, MOLT-4, RPMI-8226 and SR (GI₅₀ = 7.11, 5.55, 9.04, 5.64 and 3.91 μM respectively). The compound also showed activity against non-small cell lung cancer A549/ATCC, EKVX, HOP-92, NCI-H226 and NCI-H460 (GI₅₀ = 8.77, 5.96, 3.45, 9.96 and 3.49 μM, respectively), while TGI against HOP-92 was 41.8 μM. It also illustrated significant activity against colon cancer COLO 205, HCT-116, HCT-15, HT29 and KM12 (GI₅₀ = 8.77, 3.21, 1.96, 9.15 and 9.48 μM, respectively). The compound displayed moderate activity against CNS Cancer SNB-75 and U251 (GI₅₀ = 6.12 and 5.18 μM respectively), while TGI against SNB-75 was 43.6 μM. Its GI₅₀ against Melanoma LOX IMVI, SK-MEL-5 and UACC-62 was equal to 3.69, 5.90 and 7.96 μM, respectively. Compound 9a demonstrated moderate activity against ovarian cancer OVCAR-4, OVCAR-8 and NCI/ADR-RES (GI₅₀ = 8.01, 7.21 and 7.04 μM, respectively). The compound revealed promising activity against renal cancer A498, CAKI-1, RXF 393, prostate cancer PC-3, breast cancer MDA-MB-231/ATCC, HS 578T, BT-549, T-47D and MDA-MB-468 (GI₅₀ = 1.80, 9.71, 8.45, 5.19, 6.24, 9.66, 6.72, 6.82 and 5.55 μM, respectively). The dose response curve of compound 9a is illustrated in Fig. 2.


	Fig. 2 Dose response curve of compound 9a against the nine tested subpanels.

The ratio obtained by dividing the compound fullpanel MG-MID (μM) by its individual subpanel MG-MID (μM) is considered as a measure of compound selectivity (Table 3). Ratios between 3 and 6 refer to moderate selectivity, ratios >6 indicate high selectivity toward the corresponding cell line, while compounds meeting neither of these criteria are rated non-selective.²³ Accordingly, compound 9a was proven to be non-selective.

Table 3 The MG-MID (GI₅₀, μM) and the selectivity ratio of compound 9a (NSC 761676)

MG-MID^a	Subpanel tumor cell lines^b GI₅₀ MG-MID (μM) (SI)^c
MG-MID^a	I	II	III	IV	V	VI	VII	VIII	IX
a GI₅₀: full panel mean-graph midpoint (μM). b I: leukemia; II: non-small cell lung cancer; III: colon cancer; IV: CNS cancer; V: melanoma; VI: ovarian cancer; VII: renal cancer; VIII: prostate cancer; IX: breast cancer. c SI: selectivity index.
16.80	10.62 (1.58)	18.98 (0.89)	16.97 (0.99)	15.12 (1.11)	24.02 (0.70)	15.93 (1.05)	18.12 (0.93)	24.69 (0.68)	9.53 (1.76)

2.2.2. Antimicrobial screening. All newly synthesized compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus and Bacillus subtilis as Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Candida albicans. Their inhibition zones (IZ) using the cup-diffusion technique were measured.²⁶ Further evaluation was carried out to determine their minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the twofold serial dilution method.²⁷ Ampicillin was used as standard antibacterial while clotrimazole was used as the antifungal reference. Dimethylsulfoxide (DMSO) was used as blank and showed no antimicrobial activity.

As revealed from Tables 4 and 5, regarding the antimicrobial activity against S. aureus, the tested compounds showed weak activity IZ (12–17 mm). Only compound 16 showed one fifth the activity of ampicillin. However, the tested compound demonstrated better activity against B. subtilis IZ (12–16 mm). Compound 16 was the most active compound in this respect. It was equipotent to ampicillin (MIC = 12.5 μg mL⁻¹). Compounds 9a and 10a exhibited half the potency of ampicillin (MIC = 25 μg mL⁻¹). The inhibition zone of the tested compounds against P. aeruginosa was 11–14 mm, two compounds 10a and 16 were double as active as ampicillin (MIC = 25 μg mL⁻¹), and five compounds 9a, b, 10b, 13, and 14 were equipotent to ampicillin (MIC = 50 μg mL⁻¹). The tested compounds displayed activity against E. coli; the inhibition zones ranging 12–16 mm, only compound 16 was nearly equipotent to ampicillin (MIC = 12.5 μg mL⁻¹). On the other hand, the inhibition zone against C. albicans was 12–17 mm. Compound 10a showed nearly half the potency of clotrimazole and compounds 9b, 10b, 17, 18, and 19 showed one fifth the activity of clotrimazole.

Table 4 The inhibition zones (IZ) in mm diameter of the tested compounds

Cpd	S. aureus	B. subtilis	P. aeruginosa	E. coli	C. albicans
7	12	13	11	12	14
9a	12	12	14	10	16
9b	14	12	12	14	15
10a	13	14	12	16	14
10b	15	16	14	15	15
10c	12	12	12	14	14
11	15	12	12	14	14
13	14	15	12	16	16
14	17	12	14	12	17
15	16	16	12	10	14
16	13	12	12	15	16
17	14	16	12	15	14
18	14	13	10	12	15
19	14	14	14	15	12

Table 5 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the tested compounds in μg mL⁻¹

Cpd	S. aureus		B. subtilis		P. aeruginosa		E. coli		C. albicans
Cpd	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
7	50	100	50	100	100	100	50	50	100	200
9a	100	200	25	50	50	100	25	50	100	200
9b	50	100	100	100	50	50	100	100	25	50
10a	50	50	25	50	25	50	50	50	12.5	25
10b	50	100	100	200	50	100	50	50	25	25
10c	50	50	50	50	100	100	50	50	50	50
11	100	100	50	50	100	100	50	100	50	100
13	100	100	100	100	50	50	100	100	50	50
14	50	100	50	50	50	100	25	25	100	200
15	100	200	100	200	100	100	100	100	50	100
16	25	50	12.5	25	25	25	12.5	25	50	100
17	100	200	100	100	100	100	25	50	25	50
18	50	50	50	100	100	100	50	50	25	50
19	50	100	100	100	100	100	100	100	25	25
Ampicillin	5		12.5		50		10
Clotrimazole									5

It can be concluded that the tested compounds were weakly active against S. aureus and C. albicans. Compounds 10a and 16 were double as active as ampicillin against P. aeruginosa and five compounds 9a, b, 10b, 13, and 14 had similar activity to ampicillin, i.e. the tested compounds were very active against P. aeruginosa. The tested compounds showed medium activity against B. subtilis and E. coli. It is worth mentioning that compound 16 possesses a broad spectrum antimicrobial activity.

3. Conclusions

Preliminary in vitro anticancer screening revealed that compound 9a was the most active. It was proven to possess the highest broad spectrum anticancer activity after its further evaluation for in vitro five dose assay against 60 human cell lines. It showed particular effectiveness towards leukemia SR, non-small cell lung cancer HOP-92, NCI-H460, colon cancer HCT-116, HCT-15, CNS cancer U251, melanoma LOX IMVI, renal cancer A498, prostate cancer PC-3, and breast cancer MDA-MB-468 cell lines (GI₅₀ = 3.91, 3.45, 3.49, 3.21, 1.96, 5.18, 3.69, 1.80, 5.19, and 5.55 μM, respectively). From the antimicrobial screening it was found that the most active compounds were 10a and 16. They showed twice the activity of ampicillin against P. aeruginosa. Moreover five compounds, namely 9a, b, 10b, 13 and 14 were equipotent to ampicillin against P. aeruginosa.

In conclusion, the compound 4-benzyl-1-(2-chlorophenyl)-1,2,4-triazolo[4,3-a]quinoxaline 9a proved to possess dual effects as a broad spectrum anticancer and antimicrobial agent against P. aeruginosa.

4. Experimental section

4.1. Chemistry

All reagents and solvents were purchased from commercial suppliers and were dried and purified when necessary by standard techniques. All melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus and are uncorrected. The IR spectra were recorded using KBr discs on a Perkin-Elmer 1430 spectrophotometer. ¹H-NMR (δ ppm) spectra were recorded on a JNM-LA 400 FT NMR system (400 MHz) and on a Jeol (500 MHz) spectrometer (both JEOL, Tokyo, Japan). ¹³C-NMR spectra were run on Jeol spectrometer using TMS as the internal standard and DMSO-d₆ as a solvent. Mass spectra were run on a Finnigan mass spectrometer model SSQ/7000 (70 eV). The microanalyses were performed at the Microanalytical Laboratory, National Research Center, Cairo, Egypt and the data were within ±0.4% of the theoretical values. Following up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel aluminum sheets (Type 60 GF254, Merck, Darmstadt, Germany) and the spots were detected by exposure to a UV-lamp at λ 254 nm for few seconds.

4.1.1. 4-Benzyl-1-methyl-1,2,4-triazolo[4,3-a]quinoxaline (7).
Method A. The title compound was prepared by refluxing a solution of 3-benzyl-2-hydrazinoquinoxaline 6 (0.5 g, 2 mmol) in acetic anhydride (2.5 mL) for 2 h. The reaction mixture was poured onto ice water under stirring and the solid obtained was collected by filtration, washed with water and recrystallized from ethanol as yellowish needles (0.42 g, 76.6%), m.p. 182–183 °C; reported m.p.176–178 °C.¹⁵
Method B. A mixture of 6 (0.5 g, 2 mmol) and 2-acetylbutyrolactone (0.28 g, 2.2 mmol) in dry xylene (5 mL) was refluxed for 3 h. The reaction mixture was cooled; the obtained crystalline product was filtered, dried, and recrystallized from ethanol to yield the desired compound (0.3 g, 54.8%). The products obtained from method A and B were identical in IR (KBr, cm⁻¹): 1677 (C [double bond, length as m-dash]

N); 1605, 1495 (C [double bond, length as m-dash]

C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 3.08 (s, 3H, CH₃), 4.54 (s, 2H, CH₂–C₆H₅), 7.20 (t, 1H, J = 7.4 Hz, C₆H₅–C₄–H), 7.31 (t, 2H, J = 7.4 Hz, C₆H₅–C_3,5–H), 7.46 (d, 2H, J = 7.4 Hz, C₆H₅–C_2,6–H), 7.59–7.78 (m, 2H, triazoloquinox. C₇,₈–H), 8.04 (ddd, 1H, J = 7.2, 4.65, 2.1 Hz, triazoloquinox. C₆–H), 8.30 (dd, 1H, J = 9, 4.5 Hz, triazoloquinox. C9–H). Anal. calcd for C₁₇H₁₄N₄ (274.32): C, 74.43; H, 5.14; N, 20.42. Found: C, 74.29; H, 5.27; N, 20.74.

4.1.2. 4-Benzyl-1-(substituted phenyl)-1,2,4-triazolo[4,3-a]quinoxalines (9). A mixture of 6 (0.5 g, 2 mmol) and the appropriate substituted benzoic acid (2 mmol) in POCl₃ (2 mL) was refluxed for 2 h in an oil bath at 100 °C. The reaction mixture was cooled to room temperature, poured onto crushed ice and neutralized with sodium bicarbonate solution. The resulting solid was filtered, dried and recrystallized from ethanol.
4.1.2.1. 4-Benzyl-1-(2-chlorophenyl)-1,2,4-triazolo[4,3-a]quinoxaline (9a). Reddish-orange crystals (0.7 g, 94.5%), m.p. 200–201 °C; reported m.p. 194–195 °C.²² IR (KBr, cm⁻¹): 1643 (C [double bond, length as m-dash]

N); 1597, 1489 (C [double bond, length as m-dash]

C); 767 (C–Cl). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.61, 4.70 (2d, each 1H, J = 14.1 Hz, CH₂–C₆H₅), 7.10 (d, 1H, J = 7.5 Hz, C₆H₅–C₂–H), 7.26 (d, 1H, J = 7.5 Hz, C₆H₅–C₆–H), 7.34 (t, 2H, J = 7.5 Hz, C₆H₅–C_3,5–H), 7.49–7.55 (m, 3H, C₆H₅–C₄–H and chlorophenyl C_4,5–H), 7.62–7.71 (m, 2H, triazoloquinox. C_7,8–H), 7.78–7.86 (m, 3H, chlorophenyl C_3,6–H and triazoloquinox. C₆–H), 8.09 (dd, 1H, J = 8.1, 1.5 Hz triazoloquinox. C₉–H). ¹³C-NMR (500 MHz, DMSO-d₆, δ ppm): 42.3 (CH₂–C₆H₅), 73.2 (triazoloquinox. C₁), 114.6 (triazoloquinox. C₉), 115 (triazoloquinox. C₇), 125 (triazoloquinox. C₆), 126.7 (benz. C₁), 128.0, 128.1, 129.0, 129.1, 129.7, 129.9, 130.1, 132.4, 133.2, 133.8, 135.7, 136.5, 153.6, 209.6. Anal. calcd for C₂₂H₁₅ClN₄ (370.84): C, 71.25; H, 4.08; N, 15.11. Found: C, 71.42; H, 4.04; N, 14.86.
4.1.2.2. 4-Benzyl-1-(4-nitrophenyl)-1,2,4-triazolo[4,3-a]quinoxaline (9b). Brownish clusters of needles (0.7 g, 91.9%), m.p. 212–213 °C. IR (KBr, cm⁻¹): 1630 (sh C [double bond, length as m-dash]

N); 1601 (C [double bond, length as m-dash]

C); 1527, 1348 (NO₂). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.64 (s, 2H, CH₂–C₆H₅), 7.23 (t, 1H, J = 7.65 Hz, C₆H₅–C₄–H), 7.33 (t, 2H, J = 7.65 Hz, C₆H₅–C_3,5–H), 7.36 (d, 1H, J = 7.65 Hz, C₆H₅–C₂–H), 7.47 (d, 1H, J = 7.65 Hz, C₆H₅–C₆–H), 7.51–7.60 (m, 2H, triazoloquinox. C_7,8–H), 7.64 (dd, 1H, J = 7.8, 1.2 Hz, triazoloquinox. C₆–H), 8.1 (dd, 1H, J = 8.6, 1.5 Hz triazoloquinox. C₉–H), 8.12 (d, 2H, J = 8.7 Hz, nitrophenyl C_2,6–H), 8.51 (d, 2H, J = 8.7 Hz, nitrophenyl C_3,5–H). Mass spectrum m/z (%): 381 (M⁺˙) (100), 380 (98), 334 (25), 233 (61), 232 (95), 205 (25), 102 (35), 91(87), 77 (25), 65 (45). Anal. calcd for C₂₂H₁₅N₅O₂ (381.39): C, 69.28; H, 3.96; N, 8.39. Found: C, 68.99; H, 4.09; N, 8.21.

4.1.3. General procedure for the synthesis of 2-aryl-5-benzyl-1H-[1,2,4]triazino[4,3-a]quinoxalines (10a–c). To a solution of 6 (0.5 g, 2 mmol) in dry dioxane (10 mL), the appropriate phenacyl bromide (2 mmol) was added. The reaction mixture was heated under reflux; while a reddish precipitate separated out during the first 5 minutes. The reflux was continued for 1 h, the reaction mixture was cooled, and the product was filtered, dried, and recrystallized from the proper solvent.
4.1.3.1. 5-Benzyl-2-phenyl-1H-[1,2,4]triazino[4,3-a]quinoxaline (10a). Yellow clusters of needles (0.51 g, 72.9%), m.p. 239–241 °C (ethanol/water). IR (KBr, cm⁻¹): 3430 (NH); 1635 (C [double bond, length as m-dash]

N); 1592 (C [double bond, length as m-dash]

C); 1546 (δNH). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.49 (s, 2H, CH₂–C₆H₅), 5.60 (s, 2H, triazinoquinox. C₁–H₂), 7.27–7.08 (m, 10H, Ar–H), 8.02–8.08 (m, 2H, triazinoquinox. C_8,9–H), 8.17 (dd, 1H, J = 7.8, 2 Hz, triazinoquinox. C₇–H), 8.35 (d, 1H, J = 6.3 Hz, triazinoquinox. C₁₀–H). Mass spectrum m/z (%): 351 (12), 350 (M⁺˙) (50), 349 (24), 247 (86), 246 (72), 219 (79), 218 (32), 116 (18), 103 (67), 102 (28), 91 (68), 77 (100), 65 (34), 51 (43). Anal. calcd for C₂₃H₁₈N₄ (350.42): C, 78.83; H, 5.18; N, 15.99. Found: C, 78.67; H, 4.96; N, 16.15.
4.1.3.2. 5-Benzyl-2-(4-chlorophenyl)-1H-[1,2,4]triazino[4,3-a]quinoxaline (10b). Orange-yellow fine needles (0.49 g, 63.7%), m.p. 244–245 °C (Ethanol). IR (KBr, cm⁻¹): 3431 (NH); 1632 (C [double bond, length as m-dash]

N); 1592 (C [double bond, length as m-dash]

C); 1541 (δNH); 830 (C–Cl). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.49 (s, 2H, CH₂–C₆H₅), 5.58 (s, 2H, triazinoquinox. C₁–H₂), 7.29–7.40 (m, 5H, CH₂–C₆H₅), 7.67–7.86 (m, 2H, triazinoquinox. C_8,9–H), 7.72 (d, 2H, J = 8.9 Hz, chlorophenyl C_2,6–H), 8.05 (dd, 1H, J = 8.25, 7.8 Hz triazinoquinox. C₇–H), 8.19 (d, 2H, J = 8.9 Hz, chlorophenyl C_3,5–H), 8.34 (d, 1H, J = 8.4 Hz, triazinoquinox. C₁₀–H). ¹³C-NMR (500 MHz, DMSO-d₆, δ ppm): 38.9 (CH₂–C₆H₅), 62.3 (triazinoquinox. C₁), 114.2 (triazinoquinox. C₁₀), 117.8 (triazinoquinox. C₈), 122.9 (triazinoquinox. C₇), 125.2 (phenyl C₄), 127.1 (chlorophenyl C₄), 127.8 (phenyl C_3,5), 128.5 (chlorophenyl C_3,5), 129.0 (phenyl C_2,6), 129.3 (chlorophenyl C_2,6), 130.4, 132.5, 134.8, 136.2, 137.1, 138.2, 138.9, 147.0, 164.5, 172.5, 177.5. Anal. calcd for C₂₃H₁₇ClN₄ (384.87): C, 71.78; H, 4.45; N, 14.56. Found: C, 72.06; H, 4.32; N, 14.68.
4.1.3.3. 5-Benzyl-2-(4-bromophenyl)-1H-[1,2,4]triazino[4,3-a]quinoxaline (10c). Yellowish crystals (0.58 g, 67.6%), m.p. 248–249 °C (Ethanol). IR (KBr, cm⁻¹): 3430 (NH); 1631 (C [double bond, length as m-dash]

N); 1587 (C [double bond, length as m-dash]

C); 1539 (δNH); 762 (C–Br). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.49 (s, 2H, CH₂–C₆H₅), 5.57 (s, 2H, triazinoquinox. C₁–H₂), 7.31–7.42 (m, 5H, CH₂–C₆H₅), 7.77–8.05 (m, 2H, triazinoquinox. C_8,9–H), 7.86 (d, 2H, J = 8.3 Hz, bromophenyl C_2,6–H), 8.08–8.14 (m, 1H, triazinoquinox. C₇–H), 8.10 (d, 2H, J = 8.3 Hz, bromophenyl C_3,5–H), 8.33 (d, 1H, J = 8.4 Hz, triazinoquinox. C₁₀–H). Anal. calcd for C₂₃H₁₇BrN₄ (429.32): C, 64.35; H, 3.99; N, 13.05. Found: C, 64.17; H, 3.72; N, 13.26.

4.1.4. Ethyl 3-[(3-benzylquinoxalin-2-yl)hydrazono]butyrate (11). A mixture of 6 (0.5 g, 2 mmol) and ethyl acetoacetate (0.29 g, 2.2 mmol) was heated for 1 h in an oil bath at 160–170 °C, triturated with petroleum ether (60–80 °C), filtered dried and recrystallized from ethanol/water. This compound was obtained as white crystals (0.5 g, 69.1%), m.p. 115–116 °C. IR (KBr, cm⁻¹): 1720 (C [double bond, length as m-dash]

O ester); 1630 (C [double bond, length as m-dash]

N); 1599, 1510 (C [double bond, length as m-dash]

C); 1287, 1193, 1090 (C–O–C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 1.43 (t, 3H, J = 6.6 Hz, CH₂CH₃), 2.50 (s, 3H, N [double bond, length as m-dash]

C–CH₃), 3.34 (s, 2H, CH₂), 4.57 (q, 2H, J = 6.6 Hz, CH₂CH₃), 4.65 (s, 2H, CH₂–C₆H₅), 7.22 (t, 1H, J = 7.4 Hz, C₆H₅–C₄–H), 7.30 (t, 2H, J = 7.4 Hz, C₆H₅–C_3,5–H), 7.47 (d, 2H, J = 7.4 Hz, C₆H₅–C_2,6–H), 7.74–7.78 (m, 2H, quinox. C_6,7–H), 8.09 (ddd, 1H, J = 7.4, 3.8, 2.1 Hz, quinox. C₈–H), 8.75 (ddd, 1H, J = 7.5, 3.8, 2.1 Hz quinox. C₅–H). ¹³C-NMR (500 MHz, δ ppm): 13.80 (2 × CH₃), 40 (CH₂–C₆H₅), 63.11(COOCH₂CH₃), 118.9 (quinox-C₆), 124.85 (quinox-C₈), 126.69 (phenyl-C₄), 128.43 (phenyl-C_3,5), 128.59 (quinox-C₅), 129.03 (quinox-C₇), 129.21 (phenyl-C_2,6), 129.61 (phenyl-C₁), 136.3 (quinox-C_8a), 136.59 (quinox-C_4a), 142.52 (quinox-C₃), 144.53 (quinox-C₂), 153.23 (N [double bond, length as m-dash]

C), 158.61(COOCH₂CH₃). Anal. calcd for C₂₁H₂₂N₄O₂ (362.17): C, 69.59; H, 6.12; N, 15.46. Found: C, 69.28; H, 6.20; N, 15.29.

4.1.5. 2-[5-Amino-3-(4-chlorophenyl)pyrazol-1-yl]-3-benzylquinoxaline (13). To a solution of 6 (0.5 g, 2 mmol) in ethanol (8 mL) and acetic acid (2 mL), 4-chlorophenyl-ω-cyanoacetophenone (2 mmol) was added. The reaction mixture was heated under reflux for 2 h, cooled, and the separated product was filtered, dried, and recrystallized from acetonitrile as white-greyish crystals (0.8 g, 97.2%), m.p. 174–175 °C. IR (KBr, cm⁻¹): 3462, 3368 (NH₂); 1609, 1578, 1559 (C [double bond, length as m-dash]

N, C

C); 761 (C–Cl). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.75 (s, 2H, CH₂–C₆H₅), 5.94 (s, 1H, pyrazole C₄–H), 6.13 (s, 2H, NH₂, D₂O exchangeable), 7.06–7.17 (m, 5H, C₆H₅), 7.48 (d, 2H, J = 8.1 Hz, chlorophenyl C_2,6–H), 7.80–7.88 (m, 3H, quinox. C_5,6,7–H), 7.81 (d, 2H, J = 8.1 Hz, chlorophenyl C_3,5–H), 8.11 (dd, 1H, J = 8.4, 3.6 Hz, quinox. C₈–H). Mass spectrum m/z (%): 412 (36), 411 (M⁺˙) (100), 334 (10), 320 (15), 273 (50), 227 (19), 218 (21), 197 (15), 138 (15), 116 (25), 102 (16), 91(44). Anal. calcd for C₂₄H₁₈ClN₅ (411.89): C, 69.99; H, 4.40; N, 17.00. Found: C, 70.24; H, 4.38; N, 17.16.

4.1.6. 4-Benzyl-1,2,4-triazolo[4,3-a]quinoxaline (14). A solution of 6 (0.5 g, 2 mmol) in formic acid (3 mL) was refluxed for 3 h. After cooling, the reaction mixture was poured onto ice water with stirring and the precipitated solid was collected by filtration, washed with water and recrystallized from ethanol as yellow fine needles (0.3 g, 57.3%), m.p. 240–241 °C; reported m.p. >320 °C.²² IR (KBr, cm⁻¹): 1681 (C [double bond, length as m-dash]

N); 1539 (C [double bond, length as m-dash]

C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 4.60 (s, 2H, CH₂–C₆H₅), 7.60 (t, 2H, J = 7.35 Hz, C₆H₅–C_3,5–H), 7.78 (ddd, 2H, J = 7.35, 7.3, 1.5 Hz, C₆H₅–C_2,6–H), 7.95 (t, 1H, J = 7.35 Hz, C₆H₅–C₄–H), 8.07–8.10 (m, 2H, triazoloquinox. C_7,8–H), 8.17 (dd, 1H, J = 8.4, 1.5 Hz, triazoloquinox. C₆–H), 8.54 (dd, 1H, J = 8.4, 1.5 Hz triazoloquinox. C₉–H), 10.27 (s, 1H, triazoloquinox. C₁–H). Anal. calcd for C₁₆H₁₂N₄ (260.30): C, 73.83; H, 4.65; N, 21.52. Found: C, 73.80; H, 4.59; N, 21.67.

4.1.7. 4-Benzyl-1,2-dihydro-1,2,4-triazolo[4,3-a]quinoxalin-1-one (15). Ethyl chloroformate (0.22 g, 2 moles) was added drop wise under stirring to a solution of 6 (0.5 g, 2 mmol) in absolute ethanol (10 mL). The reaction mixture was refluxed for 3 h. After cooling the separated crystals were filtered, dried, and recrystallized from ethanol as yellowish white crystals (0.38 g, 69.1%), m.p. 262–264 °C; reported m.p. 256–258 °C.¹⁵ IR (KBr, cm⁻¹): 3272, 3248, 3199, 3184 (NH); 1682 (C [double bond, length as m-dash]

O); 1658 (C [double bond, length as m-dash]

N); 1629 (C [double bond, length as m-dash]

C); 1450, 1372 (C–N lactam). Anal. calcd for C₁₆H₁₁N₄O, (275.29): C, 69.81; H, 4.03; N, 20.35. Found: C, 69.63; H, 4.16; N, 20.09.

4.1.8. 3-(4-Benzyl-1,2,4-triazolo[4,3-a]quinoxalin-1-yl)propanoic acid (16). A mixture of 6 (0.5 g, 2 mmol) and succinic anhydride (0.18 g, 2 mmol) in glacial acetic acid (5 mL) was heated under reflux for 5 h. The reaction mixture was cooled to ambient temperature. The obtained crystalline product was filtered, dried, and recrystallized from ethanol as yellow fine needles (0.38 g, 57.2%), m.p. 256–258 °C. IR (KBr, cm⁻¹): 3500–2543 (br OH); 1693 (C [double bond, length as m-dash]

O); 1665 (C [double bond, length as m-dash]

N). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 1.05 (t, 2H, J = 6.6 Hz, CH₂, 3-propanoic), 3.38 (t, 2H, J = 6.6 Hz, CH₂, 2-propanoic), 4.41, 4.51 (2d, 2H, J = 14 Hz, CH₂–C₆H₅), 7.35–7.42 (m, 3H, C₆H₅–C_2,4,6–H), 7.44 (t, 1H, J = 7.2 Hz, triazoloquinox. C₈–H), 7.57 (t, 2H, J = 7.8 Hz, C₆H₅–C_3,5–H), 7.66 (ddd, 1H, J = 10, 7.4, 1.5 Hz triazoloquinox. C₇–H), 7.83 (dd, 1H, J = 8.1, 1.2 Hz, triazoloquinox. C₆–H), 7.97 (d, 1H, J = 7.2 Hz, triazoloquinox. C₉–H), 12.87 (s, 1H, OH, D₂O exchangeable). Anal. calcd for C₁₉H₁₆N₄O₂ (332.36): C, 68.66; H, 4.85; N, 16.86. Found: C, 68.50; H, 4.93; N, 17.04.

4.1.9. 2-(4-Benzyl-1,2,4-triazolo[4,3-a]quinoxalin-1-yl)benzoic acid (17). This compound was prepared analogous to 16 from 6 (0.5 g, 2 mmol) and phthalic anhydride (0.3 g, 2 mmol) in glacial acetic acid (5 mL). The obtained crystalline product was filtered, dried, and recrystallized from ethanol/water as white crystalline flakes (0.45 g, 59.2%), m.p. 223–224 °C. IR (KBr, cm⁻¹): 3500–2919 (br-OH); 1717 (C [double bond, length as m-dash]

O); 1689 (C [double bond, length as m-dash]

N); 1521 (C [double bond, length as m-dash]

C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 5.60 (s, 2H, CH₂–C₆H₅), 7.37–8.10 (m, 11H, Ar–H, triazoloquinox. C₇,₈–H), 8.08 (dd, 1H, J = 6.6, 5.1 Hz, triazoloquinox. C₆–H), 8.14 (ddd, 1H, J = 9.6, 6.9, 1.8 Hz, triazoloquinox. C₉–H), 10.74 (s, 1H, OH, D₂O exchangeable). Mass spectrum m/z (%): 382 (M⁺˙ + 2), 380 (M⁺˙) (5), 247 (20), 235 (10), 219 (13), 206 (31), 205 (100), 102 (35), 90 (10), 77 (47). Anal. calcd for C₂₃H₁₆N₄O₂ (380.41): C, 72.62; H, 4.24; N, 14.73. Found: C, 72.47; H, 4.09; N, 14.61.

4.1.10. 1-Ethoxycarbonyl-4-benzyl-1,2,4-triazolo[4,3-a]quinoxaline (18). A mixture of 6 (0.5 g, 2 mmol) and diethyl oxalate (0.32 g, 2.2 mmol) in dry xylene (5 mL) was refluxed for 3 h. The reaction mixture was cooled; the crystalline formed product was filtered, dried, and recrystallized from ethanol to yield 18 (0.45 g, 67.8%), m.p. 117–118 °C. IR (KBr, cm⁻¹): 1720 (C [double bond, length as m-dash]

O ester); 1599, 1510 (C [double bond, length as m-dash]

N,C

C); 1278, 1193, 1091 (C–O–C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 1.42 (t, 3H, J = 7.4 Hz, CH₂CH₃), 4.58 (q, 2H, J = 7.4 Hz, CH₂CH₃), 4.65 (s, 2H, CH₂–C₆H₅), 7.22 (t, 1H, J = 7.33 Hz, C₆H₅–C₄–H), 7.30 (t, 2H, J = 7.33 Hz, C₆H₅–C_3,5–H), 7.46 (d, 2H, J = 7.33 Hz, C₆H₅–C_2,6–H), 7.74–7.79 (m, 2H, triazoloquinox. C_7,8–H), 8.10 (ddd, 1H, J = 7.5, 3.8, 2.4 Hz, triazoloquinox. C₆–H), 8.76 (ddd, 1H, J = 7.5, 3.8, 2.4 Hz triazoloquinox. C₉–H). Anal. calcd for C₁₉H₁₆N₄O₂ (332.36): C, 68.66; H, 4.85; N, 16.86. Found: C, 68.82; H, 4.68; N, 16.63.

4.1.11. 1-Ethoxycarbonylmethyl-4-benzyl-1,2,4-triazolo[4,3-a]quinoxaline (19). A mixture of 6 (0.5 g, 2 mmol) and diethyl malonate (0.35 g, 2.2 mmol) was heated for 1 h in an oil bath at 160–170 °C. After cooling, the product was triturated with petr. ether (60–80 °C), filtered dried and recrystallized from DMF as pure white crystals (0.6 g, 86.7%), m.p. above 300 °C. IR (KBr, cm⁻¹): 1719 (C [double bond, length as m-dash]

O ester); 1630 (C [double bond, length as m-dash]

N); 1606, 1516, 1489 (C [double bond, length as m-dash]

C); 1241, 1071 (C–O–C). ¹H-NMR (500 MHz, DMSO-d₆, δ ppm): 1.44 (t, 3H, J = 7.4 Hz, CH₂CH₃), 4.53 (s, 2H, CH₂–C₆H₅), 5.12 (q, 2H, J = 7.4 Hz, CH₂CH₃), 5.83 (s, 2H, CH₂–CO), 7.11 (t, 1H, J = 7.33 Hz, C₆H₅–C₄–H), 7.17 (t, 2H, J = 7.33 Hz, C₆H₅–C_3,5–H), 7.42 (d, 2H, J = 7.33 Hz, C₆H₅–C_2,6–H), 7.45–7.63 (m, 2H, triazoloquinox. C_7,8–H), 8.04 (d, 1H, J = 7.5 Hz, triazoloquinox. C₆–H), 8.18 (d, 1H, J = 7.5 Hz triazoloquinox. C₉–H). Anal. calcd for C₂₀H₁₈N₄O₂ (346.39): C, 69.35; H, 5.24; N, 16.17. Found: C, 69.42; H, 5.28; N, 16.02.

4.2. Biological evaluation methodology

4.2.1. Anticancer screening. Ten of the prepared compounds were selected by the National Cancer Institute (NCI) Bethesda-Maryland (USA) and tested for their in vitro anticancer activity against 60 human tumor cell lines, derived from nine clinically isolated types of cancer (leukemia, lung, brain, melanoma, colon, ovarian, renal, prostate and breast). These cell lines were incubated with one concentration (10 μM) for each tested compound. Only compounds which satisfy pre-determined threshold-inhibition criteria were tested at five tenfold dilutions (0.01 to 100 μM). A 48 h continuous drug-exposure protocol was used, and a sulforhodamine B (SRB) protein assay was employed to estimate the cell viability or growth.^23,24 The results are presented in Tables 1–3.

4.2.2. Antimicrobial screening.
4.2.2.1. Inhibition-zone measurements. All synthesized compounds were evaluated for their antimicrobial activity by the agar cup diffusion technique using a 1 mg mL⁻¹ solution in DMSO.²⁶ The test organisms were Staphylococcus aureus (DSM 1104) and Bacillus subtilis (ATCC 6633) as Gram-positive bacteria; Escherichia coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145) as Gram-negative bacteria. Candida albicans (DSM 70014) was also used as a representative for fungi. Each 100 mL of sterile molten agar (at 45 °C) received 1 mL of 6 h-broth culture and then the seeded agar was poured into sterile Petri dishes. Cups (8 mm in diameter) were cut in the agar. Each cup received 0.1 mL of the 1 mg mL⁻¹ solution of the test compounds. The plates were then incubated at 37 °C for 24 h or, in case of C. albicans, for 48 h. A control using DMSO without the test compound was included for each organism. Ampicillin was used as the standard antibacterial, while clotrimazole was used as the antifungal reference. The resulting inhibition zones are recorded (Table 4).
4.2.2.2. Minimal inhibitory concentration (MIC) measurement. The minimal inhibitory concentrations (MIC) of the most active compounds were measured using the twofold serial broth dilution method.²⁷ The test organisms were grown in their suitable broth: 24 h for bacteria and 48 h for fungi at 37 °C. Two fold serial dilutions of solutions of the test compounds were prepared using 200, 100, 50, 25, and 12.5 μg mL⁻¹. The tubes were then inoculated with the test organisms; each 5 mL received 0.1 mL of the above inoculum and were incubated at 37 °C for 48 h. Then, the tubes were observed for the presence or absence of microbial growth. The MIC values of the prepared compounds are listed in Table 5.
4.2.2.3. Minimal bactericidal concentration (MBC) measurement. MIC tests were always extended to measure the MBC as follows: A loop-full from the tube not showing visible growth (MIC) was spread over a quarter of Müller–Hinton agar plate. After 18 h of incubation, the plates were examined for growth. Again, the tube containing the lowest concentration of the test compound that failed to yield growth on subculture plates was judged to contain the MBC of that compound for the respective test organism (Table 5).

Acknowledgements

The authors are grateful to the staff of the department of Health and Human Services, National Cancer Institute, Bethesda, Maryland, USA for carrying out the anticancer screening of the selected synthesized compounds.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4md00257a