Synthesis, antiproliferative, anti-tubulin activity, and

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Sep 28, 2017 - In the current study, new cis-restricted Combretastatin A4 analogues ... CA4 has a dual mechanism on tumors; the 1st mech- ... presence of a methoxy group in the para position of ring B are all ... On the other hand, in series 2, compounds 6a-6f showed ..... In conclusion, these two series of 1,2,4-triazole.

European Journal of Medicinal Chemistry 141 (2017) 293e305

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis, antiproliferative, anti-tubulin activity, and docking study of new 1,2,4-triazoles as potential combretastatin analogues Muhamad Mustafa a, b, Dalia Abdelhamid a, ElShimaa M.N. Abdelhafez a, Mahmoud A.A. Ibrahim c, Amira M. Gamal-Eldeen d, Omar M. Aly a, * a

Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia, Egypt Pharmaceutical Chemistry Department, Faculty of Pharmacy, Deraya University, Minia, Egypt Computational Chemistry Laboratory, Chemistry Department, Faculty of Science, Minia University, Minia, Egypt d Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 January 2017 Received in revised form 13 March 2017 Accepted 27 September 2017 Available online 28 September 2017

Combretastatin A4 (CA4) is a natural product characterized by a powerful inhibition of tubulin polymerization and a potential anticancer activity. However, therapeutic application of CA4 is substantially hindered due to geometric isomerization. In the current study, new cis-restricted Combretastatin A4 analogues containing 1,2,4-triazle in place of the olefinic bond were designed and synthesized. The synthesized compounds were evaluated for their in vitro antiproliferative activity in human hepatocellular carcinoma HepG2 and leukemia HL-60 cell lines using MTT assay. Moreover, fourteen compounds were selected and tested for their antiproliferative activity by the National Cancer Institute. Some of the tested compounds showed moderate activity against sixty cell lines. In vitro tubulin polymerization inhibitory activity was evaluated on HepG2 cells. The assay revealed that 6a showed a remarkable tubulin inhibition compared to CA4. Moreover, the cell cycle analysis revealed significant G2/M cell cycle arrest of the analogue 6c in HepG2 cells. Molecular docking combined with AMBER-based molecular mechanical minimization results showed several noncovalent interactions, including van der Waals and hydrogen-bonding with several amino acids within the colchicine binding site of b-subunit of tubulin. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Combretastatin A4 Antiproliferation 1,2,4-Triazole Tubulin Molecular docking

1. Introduction Combretastatin A4 (CA4) is a simple natural compound discovered three decades ago. It was isolated from the stem wood of the South African tree combretum caffrum [1e3]. CA4 showed structural similarity with most tubulin polymerization inhibitors resulting in a higher matching with the colchicine binding site of tubulin [4]. CA4 has a dual mechanism on tumors; the 1st mechanism is by binding to tubulin dimers and preventing microtubule polymerization leading tumor cells to apoptosis. The 2nd mechanism is by disrupting the VE-cadherin, which is responsible for cellcell adhesion, resulting in ischemic necrosis of tumor tissues [5e7]. Some water soluble prodrugs of CA4 were subjected to clinical trials such as phosphate disodium (CA-4P, fosbretabulin) and serinamido derivative (AVE-8062, ombrabulin), they have shown promising

* Corresponding author. Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt. E-mail addresses: [email protected], [email protected] (O.M. Aly). https://doi.org/10.1016/j.ejmech.2017.09.063 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

results on the anaplastic thyroid carcinoma [8e10] (Fig. 1). Unlike most anticancer drugs, CA4P showed less bone marrow toxicity and alopecia [11,12]. The structure-activity relationship (SAR) study of CA4 showed a number of structural features which are highly essential for its antiproliferative activity. The trimethoxy group in ring A, the cisoid configuration at the olefinic bridge, and the presence of a methoxy group in the para position of ring B are all fundamental for CA4 anticancer potency [13,14]. Several trials were performed through provision of substitutions and bioisosteric replacements on ring A and B aiming to find the optimum scaffold [15,16]. Moreover, it was revealed that the cis olefinic double bond is transformed into trans isomer in vivo leading to a significant loss in the inhibition activity [17]. Therefore, several studies were carried out to prevent cis-trans transformation of the olefinic bond by introducing hetero-aromatic rings such as isomeric triazole [18,19], tetrazole [20,21], thiadiazole [22], dithiole [23], imidazole [24], isoxazole [25] and oxadiazole [26]. In the present study, new cis restricted analogues of CA4 containing 1,2,4-triazole were synthesized working through two

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mechanical minimization of top potent compounds towards the colchicine binding site of b-subunit of tubulin was carried out. 2. Results and discussion 2.1. Chemistry The synthesis of the designed triazole carboxamide derivatives 5a-5s and 6a-6f (Table 1) is outlined in Scheme 1. 2-(3,4,5trimethoxybenzamido) acetic acid 1 was prepared in high yield through Schotten Baumann reaction by reacting glycine with 3,4,5trimethoxybenzoyl chloride in 10% NaOH [27]. Treatment of compound 1 with acetic anhydride afforded the oxazoline-2-one derivative 2. Coupling of the diazonuim salt 3 with the active methylene group of 2 in presence of anhydrous sodium acetate and acetic acid afforded the hydrazone derivative (compound 4). Based on Sawdey rearrangement [28]; treating compound 4 with primary aromatic amines in presence of glacial acetic acid and sodium acetate afforded the target compounds 5a-5s in 39e74% yield. While, treating 4 with benzyl amines in presence of methanol afforded the target compounds 6a-6f in 63e71% yield. The prepared compounds were identified by 1HNMR and 13 CNMR, while the purity of the newly prepared compounds was checked by elemental analysis.

Fig. 1. Chemical structures of combretastatin A4 and its analogues.

series; concerning series 1; through using ring A as 3,4,5trimethoxyphenyl moiety and ring B as 4-ethoxyphenyl moiety for compounds 5a-5j and 3-methoxyphenyl moiety for compounds 5k-5s, also an extension was afforded to the structure by adding a third ring (ring C, which is directly attached to a carboxamide group) with variation of substituents for the purpose of improvement of the binding with the colchicine binding site of tubulin (Table 1). On the other hand, in series 2, compounds 6a-6f showed an increase in ring C flexibility through inserting a benzyl group into the structure which may result in more possible interactions. The synthesized compounds were evaluated for their in vitro anticancer activity against two different types of cancer cells; human hepatocellular carcinoma HepG2 cell lines (hard tissue tumor) and Leukemia HL-60 cells (soft tissue tumor). In addition, fourteen of the synthesized compounds, including nine compounds of series 1 and five compounds of series 2, were selected and tested for their antiproliferative activity by National Cancer Institute (NCI). Moreover, cell cycle analysis was performed on 6c to evaluate its ability to induce apoptosis and arrest different phases of the cell cycle. In vitro tubulin polymerization inhibitory activity of six selected compounds (5a, 5b, 5k, 5q, 6a, and 6c) was evaluated in HepG2 cells. To reveal the binding features of the synthesized compounds, molecular docking followed by AMBER-based molecular

2.2. Biological investigation 2.2.1. Evaluation of in vitro antiproliferative activity against HepG2 and HL-60 cells The effect of the tested compounds was studied using MTT assay on the viability of two different human cancer cell lines after 48 h of incubation. The treatment of hepatocellular carcinoma HepG2 cells and leukemia HL-60 cells with gradual concentrations of the prepared compounds was carried out. According to the in vitro results, it was found that compounds 5q, 6a, 6b, 6c, 6e, and 6f exhibited IC50 values (1.38, 0.62, 1.90, 1.41, 1.67 and 0.90 mM, respectively) better than CA4 (2,41 mM) in HepG2 cells (Table 2). Hence, increasing flexibility of ring C showed a dramatic increase in the antiproliferative activity in HepG2 cells. Moreover, compounds 5h, 5k, 5o, 5p, and 5r displayed significant antiproliferative activity with IC50 values 10.82, 6.53, 8.51, 8.75, and 10.98 mM, respectively. Among the synthesized compounds; 6a exhibited the best antiproliferative activity against HL-60 cells with IC50 value of 0.60 mM which is close to CA4 with IC50 value of 0.56 mM. Moreover, compounds 6c and 5a showed promising activity with IC50 values of 0.95 and 1.28 mM, respectively (Table 2). cLog P values were calculated using ACD/ChemSketch software version D10E41 to

Table 1 Variable attachment points and substituents of the prepared compounds. Cpd

R1

R2

R3

R4

R5

R6

Cpd

R1

R2

R3

R4

R5

R6

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m

H H H H H H H H H H OCH3 OCH3 OCH3

OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 OC2H5 H H H

H H H H H H H F H OCH3 H H OCH3

H OCH3 OCH3 OCH3 H F F F H H H OCH3 H

H H OCH3 OCH3 OC2H5 H F F OCH3 H H H H

H H H OCH3 H F H H OH H H H H

5n 5o 5p 5q 5r 5s 6a 6b 6c 6d 6e 6f

OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 H H H OCH3 OCH3 OCH3

H H H H H H OC2H5 OC2H5 OC2H5 H H H

H H H H H F OCH3 H H CH3 OCH3 H

OCH3 OCH3 H F F F H CH3 F H H OCH3

OCH3 OCH3 OC2H5 H F F H H H H H H

H OCH3 H F H H H H F H H H

M. Mustafa et al. / European Journal of Medicinal Chemistry 141 (2017) 293e305

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Scheme 1. (Series 1&2): (I) NaNO2, HCl, 0e5  C; (II) AC2O, 60  C, 50min; (III) diazonium chloride of an appropriate amine 3, NaOAC, AcOH, 0e5  C; (IV) aromatic amine, NaOAc, AcOH, reflux, 2 h; (V) benzyl amine, methanol, reflux, 2 h.

Table 2 IC50 (mM) values for the tested compounds against HepG2 and HL-60 cell lines compared to CA4. IC50 (mM)

Cpd

5a 5b 5c 5d 5e 5f 5g 5h 5i 5k 5l 5m 5n

IC50 (mM)

Cpd

HepG2

HL-60

cLog P

41.82 36.84 32.81 37.84 29.75 36.48 31.84 10.82 13.65 6.53 15.29 29.89 32.54

1.28 16.60 4.30 >100 6.00 >100 17.10 10.89 >100 >100 15.29 29.89 14.21

5.85 6.11 5.96 6.01 6.33 6.86 6.69 7.13 5.23 5.32 5.32 5.21 5.43

5o 5p 5q 5r 5s 6a 6b 6c 6d 6e 6f CA4

HepG2

HL-60

cLog P

8.51 8.75 1.38 10.98 16.28 0.62 1.90 1.41 3.85 1.67 0.90 2.41

6.02 14.51 54.16 31.34 27.17 0.60 98.82 0.95 89.41 >100 >100 0.56

5.48 5.80 6.33 6.16 6.07 5.79 5.79 6.01 5.80 5.26 5.26 3.57

explore the impact of fluorine substitution on the antiproliferative activity of the synthesized compounds. cLog P values indicated that the fluorinated compounds, which exhibited moderate antiproliferative activity, showed higher lipophilicity (Table 2). This may be attributed to the formation of the non-polarizable C-F bond which may result in higher activity [29,30]. 2.2.2. Antiproliferative investigation against 60 cell lines at the NCI The synthesized compounds were submitted to National Cancer Institute (NCI), USA at www.dtp.nci.nih.gov. Fourteen of the synthesized compounds were selected and tested for their antiproliferative activity according to the drug evaluation branch by NCI which mainly depends on comparing the results of similar scaffolds tested previously in the NCI60 assay. The selected compounds were subjected to in vitro anticancer assay against tumor cells in a full panel of 60 cell lines derived from nine different cancer types (leukemia, lung, colon, CNS, melanoma, ovarian,

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against most cell lines; a complete cell death was recorded against leukemia MOLT-4, melanoma SK-MEL-5, and renal cancer UO-31. Also, it exhibited the best activity against leukemia HL-60, K-562, RPMI-8226, SR, lung cancer A549/ATCC, HOP-92, NCI-H460, NCIH522, colon cancer COLO205, HCT-116, HCT-15, KM12, CNS cancer SF-295, SNB-75, melanoma MDA-MB-435, ovarian cancer OVCAR-4, NCI/ADR-RES, renal cancer CAKI-1, A498, ACHN, prostate PC-3, and breast cancer MCF7, MDA-MB231/ATCC, BT-549, T-47D. However, compound 6b exhibited mild activity against most cancer cells. Compound 6c indicated the best activity against renal cancer UO-31, and exhibited moderate activity against prostate cancer PC3, and breast cancer MCF7, MDA-MB231/ATCC cells, while showing a remarkable activity against other cells. It was expected that presence of benzyl group will improve the activity as in compounds 6a and 6c. However, compounds containing a meta methoxy group on ring B (6d, 6e) did not exhibit remarkable antiproliferative activity in spite of having a more flexible ring C. From the obtained results we can deduce that presence of para ethoxy group on ring B enhanced the antiproliferative activity especially upon increasing the flexibility of ring C (as in compounds 6a and 6c), this extension increased the potency of the prepared compounds against most tested cells and they were better than the reference CA4 against HepG2 cells.

renal, prostate and breast cancers). The selected compounds were added at single concentration 105 M and the culture was incubated for 48 h. Growth inhibition percentages are illustrated by Tables 3 and 4. Concerning series 1 (Table 3): Compound 5a exhibited relatively high growth inhibitory activity against most cell lines. A relatively complete cell death 99.8% was recorded against Melanoma MDAMB-435 cells. Also, 5a showed the best growth inhibition activity against leukemia SR, Colon cancer HT29, and renal cancer A498, UO-31, whereas remarkable to moderate activity was observed against the other cell lines. Compound 5b showed the best activity against leukemia RPMI-8226. Also, 5b revealed moderate activity against leukemia CCRF-CEM, SR, and renal cancer UO-31, however, mild activity was observed against the other tested cells. Compound 5g exhibited the best activity against leukemia RPMI-8226. Also, moderate activity was observed against leukemia CCRF-CEM, ovarian cancer, OVCAR-8, and breast cancer T-47D cells. On the other hand compounds 5d, 5f, 5h, 5i, 5j, and 5k exhibited mild activity against most of the tested cell lines. Concerning series 2 (Table 4): there was an attempt to figure out the effect of increasing the flexibility of ring C on the biological activity by adding a benzyl group to the structure. Compound 6a achieved a remarkable cell growth inhibition

Table 3 Percentage growth inhibition (GI%) of in vitro subpanel tumor cell Lines of compounds 5a, 5b, 5d, and 5f-k. Subpanel tumor cell lines

Leukemia CCRF-CEM HL-60(TB) MOLT-4 RPMI-8226 SR Lung Cancer A549/ATCC HOP-92 NCI-H460 NCI-H522 Colon Cancer COLO 205 HCT-116 HCT-15 HT29 KM12 CNS Cancer SF-295 SNB-75 U251 Melanoma LOX IMVI M14 MDA-MB-435 SK-MEL-2 Ovarian Cancer OVCAR-3 OVCAR-8 NCI/ADR-RES Renal Cancer A498 UO-31 Prostate Cancer PC-3 Breast Cancer MCF7 MDA-MB-231/ATCC HS 578T T-47D MDA-MB-468 (N.T.) not tested.

% Growth Inhibition (GI%) 5a

5b

5d

5f

5g

5h

5k

5j

5i

49.22 67.15 57.07 52.56 83.00

48.13 12.48 37.54 72.10 47.43

33.54 1.84 32.51 44.28 59.27

15.91 35.91 36.08 30.64 38.16

55.90 10.96 49.99 87.65 36.05

N.T. 18.55 N.T. 12.81 13.52

10.47 5.70 25.12 32.32 31.48

6.00 26.8 23.54 31.78 34.27

0 0 0 0 6.06

58.63 54.00 72.06 77.20

31.80 32.76 20.72 42.14

13.13 30.08 7.90 34.09

26.18 32.23 5.32 38.44

38.00 32.96 22.12 38.85

14.28 15.13 0 28.53

5.96 31.88 2.43 29.68

22.66 13.21 16.62 30.75

7.57 2.10 0 34.47

77.92 78.80 64.60 86.96 77.49

33.37 32.73 25.72 21.71 28.84

19.66 24.20 17.88 15.44 18.16

10.09 4.77 22.14 11.02 14.13

36.30 48.77 36.25 25.37 43.32

10.01 26.61 35.71 12.18 4.04

0 3.32 2.79 0.76 7.18

20.30 27.30 19.15 26.00 3.83

0 8.66 12.03 20.07 3.42

65.98 64.86 63.90

31.51 31.91 21.47

4.11 34.39 14.94

15.43 41.98 18.00

39.47 25.60 29.65

7.04 31.70 1.80

0 28.79 6.80

15.24 33.36 5.99

8.09 44.53 6.41

44.25 61.98 99.81 62.29

19.24 16.30 13.20 13.11

10.97 8.51 20.37 14.36

13.42 2.45 8.98 7.06

33.56 29.77 29.67 21.63

6.04 6.14 10.01 15.86

9.39 0 5.03 4.04

7.77 26.17 13.10 18.13

0 0 9.57 2.02

56.79 38.37 54.31

21.34 46.91 22.51

13.08 26.03 14.23

6.38 15.42 18.34

38.95 57.75 32.53

8.40 6.64 11.42

2.23 21.3 13.56

4.38 9.65 14.74

0.13 0 6.88

89.72 62.31

21.16 49.10

32.69 47.39

43.97 46.48

31.11 57.38

11.28 48.54

0 43.95

16.21 40.43

0 38.37

55.12

41.04

29.76

30.07

52.91

28.39

28.05

34.53

10.04

63.02 56.12 60.36 60.56 57.49

16.52 44.89 26.79 41.54 6.58

16.82 49.5 34.42 48.63 17.98

21.03 35.70 24.46 44.09 26.41

21.18 36.91 51.88 58.27 1.41

20.52 26.4 19.34 34.34 9.76

9.08 34.21 13.17 11.22 0

22.64 55.51 18.50 49.81 55.51

8.42 0 0.26 37.79 15.55

M. Mustafa et al. / European Journal of Medicinal Chemistry 141 (2017) 293e305 Table 4 Percentage growth inhibition (GI%) of in vitro subpanel tumor cell Lines of compounds 6a-e. Subpanel tumor cell lines

Leukemia CCRF-CEM HL-60(TB) K-562 MOLT-4 RPMI-8226 SR Non-Small Cell Lung Cancer A549/ATCC EKVX HOP-92 NCI-H460 NCI-H522 Colon Cancer COLO 205 HCT-116 HCT-15 HT29 KM12 CNS Cancer SF-295 SF-539 SNB-19 SNB-75 U251 Melanoma LOX IMVI M14 MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-62 Ovarian Cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-8 NCI/ADR-RES Renal Cancer 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 Prostate Cancer PC-3 DU-145 Breast Cancer MCF7 MDA-MB-231/ATCC BT-549 T-47D

297

the lower right quadrant illustrating the early apoptotic cells which keep their membrane integrity indicated the ability of 6c to initiate apoptosis.

% Growth Inhibition (GI%) 6a

6b

6c

6d

6e

60.26 80.88 77.95 99.62 73.09 64.15

5.82 8.95 11.53 30.11 15.72 27.50

14.38 38.65 37.97 54.92 53.05 35.99

0 11 9.21 19.96 4.27 34.23

0 0 0 0 0 6.25

65.49 58.69 70.96 69.52 64.32

26.82 10.88 4.39 0 41.31

42.95 37.18 38.91 33.94 49.06

30.82 26.7 18.16 3.56 39.23

7.18 8.89 0 0 39.55

69.71 74.70 70.99 59.36 66.23

8.15 4.00 10.04 26.49 3.57

34.98 50.26 38.98 38.31 34.25

15.70 14.81 25.65 19.77 18.01

0 0 3.17 18.67 4.39

65.19 50.82 54.79 69.98 57.47

9.69 11.06 9.87 46.76 5.83

34.75 29.29 37.74 56.58 25.71

23.87 12.28 16.81 68.3 14.97

4.91 3.52 0 36.99 2.91

63.66 57.85 77.95 58.53 63.36 101.22 63.04

11.10 7.09 7.53 9.96 0 7.83 17.07

33.42 34.85 32.99 34.52 34.7 49.90 42.11

21.34 13.29 14.20 21.90 0 13.25 27.45

4.07 0.15 9.22 22.15 0 0 2.57

56.34 50.57 76.59 56.28 72.25

19.62 6.46 1.13 5.71 17.48

37.53 28.85 34.21 25.01 38.28

28.00 19.05 18.43 12.72 36.86

11.48 0 9.19 0 22.61

58.47 70.04 80.37 74.06 56.46 60.27 53.46 102.66

9.77 1.88 2.47 N.T. 7.47 3.46 12.22 55.67

34.07 35.63 45.57 55.60 31.42 32.23 20.14 74.17

24.02 4.13 9.47 N.T. 0 8.30 26.78 64.12

6.03 0 0 N.T. 0 5.19 19 40.88

69.98 57.47

46.76 5.83

56.58 25.71

68.3 14.97

36.99 2.91

77.92 82.21 66.14 82.96

15.87 23.67 7.36 30.24

55.66 56.85 31.26 55.85

17.86 26.49 13.82 43.64

1.27 6.96 0 24.20

2.2.3. Cell cycle analysis Cell cycle analysis was performed for compound 6c at different concentrations. The analysis indicated that HepG2 cells treated with compound 6c showed a significant growth arrest at the preG1 (G0) and G2/M phases compared to control cells, where the Sphase progression of HepG2 cells was substantially delayed (Fig. 2). The results of Annexin V/PI flow cytometry of HepG2 cells after treatment with IC50 value (1.41 mM) of 6c showed increasing of the percentage of the necrotic cells in late apoptosis to 14% (upper right quadrant of the cytogram) (Fig. 3). Hence, compound 6c showed a considerable ability to dissipate cell membrane integrity. Whereas

2.2.4. Evaluation of in vitro tubulin polymerization inhibitory activity Compounds 5a, 5b, 5k, 5q, 6a, and 6c were tested for their ability to inhibit tubulin polymerization inhibitory at their IC50 concentrations using ELISA assay for b-tubulin. The assay revealed that tubulin inhibition percentages were 42.7, 49.6, 62.3, 65.2, 85.6, 71.8, and 72.3 for compounds 5a, 5b, 5k, 5q, 6a, 6c and the reference CA4, respectively. Compound 6a showed the highest ability to inhibit tubulin polymerization (85.6%) compared to all the tested compounds and the reference CA4 (72.3%). Also, 6a showed the best IC50 value (0.62 mM). On the other hand, although compound 5a showed a significant antiproliferative activity, compound 5a showed a low inhibition percentage of 42.7. This may be attributed to that compound 5a has an off target mechanism. Compounds 5k, 5q, and 6c showed comparable percentages of inhibition to the standard CA4. It worth mentioning that tubulin polymerization inhibitory results were positively correlated with the obtained IC50 values in HepG2 cell lines (Fig. 4). 2.3. Molecular docking study Using AutoDock Vina software; molecular docking calculations were carried out on CA4 and the top potent compounds towards the colchicine binding site of b-subunit of tubulin. To assess AutoDock Vina performance, the co-crystallized ligand DAMAcolchicine was firstly docked and the binding mode was investigated (Fig. 5a). According to the predicted docking pose, Vina accurately reproduced the crystal structure observed binding mode of DAMA, forming an essential hydrogen bond with CYS241 with a bond length of 1.90 Å. As well, strong hydrophobic interactions with Leu255 and Leu248 amino acids were also observed. Molecular docking of CA4 and the top potent compounds 6a, 6c and 6f towards tubulin was then performed and the corresponding binding energies were calculated. It is worth mentioning that the binding energies calculated using the MM-GBSA approach were in a good agreement with the comparable experimental data, with a correlation coefficient R2 value of 0.93 (Table 5). According to the docked CA4 structure (Fig. 5b); CA4 forms two hydrogen bonds with CYS241 and Leu248 with a bond length of 2.22 and 1.71 Å, respectively. Further interactions, including van der Waals and hydrophobic interactions, were observed giving a total binding energy (DGMM/GBSA) value of -41.42 kcal/mol for CA4 with tubulin. For the synthesized potent compounds, the docked structures revealed that compounds 6a, 6c and 6f have the same binding features as CA4 and DAMA (Fig. 5c). According to the AMBER-based minimized docked structures (Fig. 6), compounds 6a, 6c and 6f form a hydrogen bond with the key CYS241 amino acid with a bond length of 2.08, 1.97 and 2.02 Å, respectively. As well, a moderate H … pi interaction was observed between ring C and terminal NH2 group of ASN285 residue. The highest potency of compound 6a may be attributed to the existence of ortho-methoxy group on ring C which forms a hydrogen bond with LYS352. The latter hydrogen bond is absent in compounds 6c and 6f. 3. Conclusion A group of new 1,2,4-triazole-3-carboxamide derivatives resembling cis-restricted CA4 analogues were synthesized and characterized by their spectral data. They were compromised in two series; 5a-5s and 6a-6f, these compounds showed promising to moderate anticancer activities. The obtained results revealed

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Fig. 2. Cell cycle analysis of HepG2 cells treated with Annexin PI at IC50 Concentration. A) Untreated cells, B) Treated cells.

that compound 6a which has more flexible ring C bearing omethoxy group showed IC50 value better than that of CA4 against HepG2 cells. 6a was capable of completely stopping the growth of three diverse cancer cell lines (MOLT-4, SK-MEL-5, and UO-31) in NCI60 assay. Also, compounds 5a, 6a, and 6c exhibited promising IC50 values against leukemia HL-60 along with promising antiproliferative activity against most cell lines. Most of compounds containing fluorine atoms on ring C exhibited potent antiproliferative activity in both NCI60 assay and against HepG2 cells which may be attributed to the enhanced lipophilicity. 6c was able to arrest G2/M cell cycle and 6a showed the highest ability to inhibit tubulin polymerization, whereas compound 5a may have an off target mechanism. The molecular docking results revealed that compounds 6a, 6c and 6f showed interaction energies and features comparable to CA4. In conclusion, these two series of 1,2,4-triazole derivatives could be taken in consideration as promising anticancer compounds acting through inhibition of tubulin polymerization. 4. Experimental 4.1. Chemistry Fig. 3. Contour diagram of Annexin V/PI Flow Cytometry.

Chemicals were purchased from Aldrich, Merck, Fluka,

Fig. 4. Tubulin percentage inhibition of the prepared compounds compared to CA4.

M. Mustafa et al. / European Journal of Medicinal Chemistry 141 (2017) 293e305

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Fig. 5. Docking pose of (a) DAMA-colchicine (predicted in cyan; crystal structure in pink), (b) Combretastatin A4, and (c) compounds 6a (in cyan), 6c (in pink) and 6f (in purple), with b-subunit of tubulin (PDB code: 1SA0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 5 Calculated binding energies (DG) (kcal/mol) and binding features for the top potent compounds, compared to CA4, with b-subunit of tubulin. IC50a (mM)

DGexp. (kcal/mol)

CA4

2.41

6a

Compound

DGcalc (kcal/mol)

Ligand-receptor interactions

Vina

MM/GBSA

Residue

Type

Length (Ǻ)

7.66

6.30

41.42

CYS241 Leu248

Hydrogen bond Hydrogen bond

2.22 1.71

0.62

8.46

7.80

54.23

CYS241 ASN258 LYS352

Hydrogen bond N-H$$$Pi Hydrogen bond

2.08 2.98 2.74

6c

1.41

7.98

8.10

48.83

CYS241 ASN258

Hydrogen bond N-H$$$Pi

1.97 3.12

6f

0.90

8.24

8.20

52.83

CYS241 ASN258

Hydrogen bond N-H$$$Pi

2.02 3.11

Correlation coefficient (R2) a

0.93

IC50 Values for the tested compounds against HepG2 cell line.

Fig. 6. AMBER-based minimized docked structure of (a) compound 6a (in cyan), (b) compound 6c (in pink) and compound 6f (in purple), with b-subunit of tubulin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Cambrian chemicals, and El-Nasr pharmaceutical chemicals companies, and used without further purification. Reactions were monitored by thin layer chromatography (TLC), using Merck9385 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 254 nm. Melting points were determined on Stuart electro thermal melting point apparatus and were uncorrected. NMR spectra were carried out using a Bruker Avance 400 MHz 1HNMR spectrometer and 100 MHz 13CNMR spectrometer (Baniswif, Egypt), using TMS as internal reference. Chemical shifts (d values are given in parts per million (ppm) relative to TMS CDCl3 (7.29 for 1HNMR and 76.9 ppm for 13CNMR) 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. Elemental analysis was performed on Vario El Elementar CHN

Elemental Analyzer; Organic Microanalysis Section, Cairo University, Giza, Egypt. 4.1.1. Synthesis of 4-[(Aryl)-hydrazono]-2-(3,4,5-trimethoxyphenyl)-4H-oxazol-5-one 4 Trimethoxyhippuric acid 1 (0.17 mol, 46.67 g) was heated with acetic anhydride (100 mL) at 60  C for 50 min or until a clear solution of 2 was obtained; the mixture was cooled to room temperature (solution A). Stirring appropriate amine (ex: 4ethoxyaniline) (0.13 mol), 5 N HCl (40 mL) and glacial acetic acid (40 mL) in an ice-salt bath 0e5  C, a solution of sodium nitrite (0.17 mol, 11.96 g) in water (20 mL) was added in a drop wise manner. The reaction mixture was left for 10 min then anhydrous sodium acetate (0.24 mol, 20 g) was added (solution B). Solution A was added to solution B in a drop wise manner at 0e10  C with

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continuing stirring for 2 h; the formed precipitate was filtered off and dried to afford light red solid with 70% yield. 4.1.2. General procedure for synthesis of 1-(Aryl)-5-(3,4,5trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamides 5a-5s To a mixture of compound 4 (3.99 g, 0.01 mol) and appropriate primary aromatic amine (0.01 mol) was added and the mixture was refluxed for 2 h in acetic acid (50 mL) in presence of anhydrous sodium acetate (1.5 g, 0.018 mol) for. The reaction mixture was cooled and poured into iced cold water (50 mL) while stirring. The formed precipitate was filtered off, dried and recrystallized from methanol. 4.1.2.1. 1-(4-Ethoxy-phenyl)-5-(3,4,5-trimethoxy-phenyl)-1H-[1,2,4] triazole-3-carboxylic acid phenylamide 5a. Yellowish brown crystals (5.49 g, 74% yield); mp 145e147  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.45 (t, 3H, J ¼ 6.97 Hz, O-CH2-CH3), 3.71 (s, 6H, 3,5-diOCH3), 3.88 (s, 3H, O-CH3), 4.08 (q, 2H, J ¼ 6.96 Hz, O-CH2), 6.79 (s, 2H, Ar-H), 6.98 (d, 2H, J ¼ 8.92 Hz, Ar-H), 7.17 (t, 1H, J ¼ 7.41 Hz, ArH), 7.35e7.41 (m, 4H, Ar-H), 7.78 (d, 2H, J ¼ 7.68 Hz, Ar-H), 9.03 (s, 1H, CO-NH); 13CNMR (100 MHz, CDCl3, d ppm): 14.13, 14.65, 29.69, 30.93, 31.92, 55.97, 56.07, 60.95, 64.00, 106.08, 106.33, 115.09, 119.92, 121.82, 124.61, 127.42, 129.11, 130.56, 137.46, 139.85, 15316, 154.91, 156.12, 156.84 and 159.75; Anal. Calcd for C26H26N4O5: C, 65.81; H, 5.52; N, 11.81. Found: C, 65.48; H, 5.36; N, 11.58. 4.1.2.2. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3-methoxyphenylamide 5b. Yellow crystals (2.30 g, 39% yield); mp 165e167  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.45 (t, 3H, J ¼ 6.97 Hz, OCH2-CH3), 3.71 (s, 6H, 3,5-diOCH3), 3.84 (s, 3H, O-CH3), 3.88 (s, 3H, O-CH3), 4.07 (q, 2H, J ¼ 6.97 Hz, O-CH2), 6.79 (s, 2H, Ar-H), 6.97 (d, 2H, J ¼ 8.94 Hz, Ar-H), 7.24e7.29 (m, 3H, Ar-H), 7.35 (d, 2H, J ¼ 8.92 Hz, Ar-H), 7.53 (s, 1H, Ar-H), 9.00 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 14.66, 55.34, 56.06, 60.96, 64.00, 105.59, 106.30, 110.52, 112.09, 115.08, 121.84, 127.23, 129.78, 130.56, 138.65, 139.82, 153.15, 154.90, 156.08, 156.84, 159.74 and 160.19. Anal. Calcd for C27H28N4O6: C, 64.27; H, 5.59; N, 11.10. Found: C, 64.13; H, 5.55; N, 11.31. 4.1.2.3. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3,4-dimethoxyphenylamide 5c. Yellow crystals (3.98 g, 64% yield); mp 176e178  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.44 (t, 3H, J ¼ 6.94 Hz, O-CH2-CH3), 3.70 (s, 6H, 3,5-di-OCH3), 3.87 (s, 3H, O-CH3), 3.88 (s, 3H, O-CH3), 3.92 (s, 3H, O-CH3), 4.07 (q, 2H, J ¼ 6.92 Hz, O-CH2), 6.78 (s, 2H, Ar-H), 6.86 (d, 1H, J ¼ 8.65 Hz, Ar-H), 6.96 (d, 2H, J ¼ 8.82 Hz, Ar-H), 7.17 (d, 1H, J ¼ 8.60 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.34 (d, 2 H, J ¼ 8.79 Hz, Ar-H), 8.94 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 14.65, 55.94, 56.05, 56.10, 60.95, 63.98, 104.71, 106.28, 111.32, 111.86, 115.07, 121.86, 127.20, 130.56, 131.08, 139.80, 146.03, 149.06, 153.14, 154.85, 156.17, 156.71, 159.71; Anal. Calcd for C28H30N4O7: C, 62.91; H, 5.66; N, 10.48. Found: C, 62.94; H, 5.52; N, 10.56. 4.1.2.4. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3,4,5-trimethoxyphenylamide 5d. Yellowish brown crystals (3.12 g, 48% yield); mp 175e177  C; 1 HNMR (400 MHz, CDCl3) (d ppm): 1.45 (t, 3H, J ¼ 6.96 Hz, O-CH2CH3), 3.70 (s, 6H, 3,5-di-OCH3), 3.84 (s, 3H, O-CH3), 3.88 (s, 9H, OCH3), 4.08 (q, 2H, J ¼ 6.95 Hz, O-CH2), 6.78 (s, 2H, Ar-H), 6.97 (d, 2H, J ¼ 8.88 Hz, Ar-H), 7.09 (s, 2H, Ar-H), 7.35 (d, 2H, J ¼ 8.87 Hz, Ar-H), 8.93 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 14.65, 56.07,56.15, 60.96, 60.99, 64.01, 97.57, 106.30, 115.10, 121.82, 127.17, 130.52, 133.55, 134.86, 139.87, 153.18, 153.38, 154.96, 156.06, 156.82, 159.76; Anal. Calcd for C29H32N4O8: C, 61.69; H, 5.71; N, 9.92. Found: C, 62.05; H, 5.39; N, 9.67.

4.1.2.5. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid (4-ethoxyphenyl)-amide 5e. White crystals (3.53 g, 69% yield); mp 200e202  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.39e1.46 (m, 6H, O-CH2-CH3), 3.70 (s, 6H, 3,5-di-OCH3), 3.87 (s, 3H, O-CH3), 4.02e4.09 (m, 4H, O-CH2), 6.78 (s, 2H, Ar-H), 6.90 (d, 2H, J ¼ 8.77 Hz, Ar-H), 6.96 (d, 2H, J ¼ 8.72 Hz, Ar-H),7.34 (d, 2H, J ¼ 8.69 Hz, Ar-H), 7.66 (d, 2H, J ¼ 8.75 Hz, Ar-H), 8.93 (s, 1H, CO-NH); 13C(100 MHz, CDCl3, d ppm): 14.64, 14.84, 56.04, 60.93, 63.67, 63.97, 106.30, 114.82, 115.05, 121.51, 121.88, 127.25, 130.53, 130.60, 139.78, 153.12, 154.79, 155.93, 156.23, 156.63, 159.69; Anal. Calcd for C28H30N4O6: C, 64.85; H, 5.83; N, 10.80 Found: C, 65.00; H, 5.81; N, 11.00. 4.1.2.6. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3,5-difluorophenylamide 5f. Yellowish brown crystals (3.23 g, 55% yield); mp 75e77  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.43 (t, 3H, J ¼ 6.96 Hz, O-CH2-CH3), 3.66 (s, 6H, 3,5di-OCH3), 3.85 (s, 3H, O-CH3), 4.05 (q, 2H, J ¼ 6.93 Hz, O-CH2), 6.74 (s, 2H, Ar-H), 6.89 (d, 1H, J ¼ 6.16 Hz, Ar-H), 6.95 (d, 2H, J ¼ 8.83 Hz, Ar-H),7.08 (d, 1H, J ¼ 8.81 Hz, Ar-H), 7.31 (d, 2H, J ¼ 8.81 Hz, Ar-H), 7.72 (t, 1H, J ¼ 6.34 Hz, Ar-H), 9.98 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 14.61, 42.46, 55.97, 60.90, 63.96, 102.84, 103.24, 103.36, 103.31, 103.36, 106.20, 110.27, 110.73, 115.06, 121.77, 127.21, 130.53, 139.76, 142.06, 153.08, 155.68, 159.33, 161.78, 164.38; Anal. Calcd for C26H24F2N4O5: C, 61.17; H, 4.74; N, 10.98. Found: C, 60.96; H, 5.05; N, 11.00. 4.1.2.7. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3,4-difluorophenylamide 5g. Pale Yellow crystals (3.62 g, 61% yield); mp 150e152  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.4 (t, 3H, J ¼ 6.43 Hz, O-CH2-CH3), 3.65 (s, 6H, 3,5di-OCH3), 3.84 (s, 3H, O-CH3), 4.03 (q, 2H, J ¼ 6.71 Hz, O-CH2), 6.72 (s, 2H, Ar-H), 6.93 (d, 2H, J ¼ 8.02 Hz, Ar-H), 7.09 (d, 1H, J ¼ 8.86 Hz, Ar-H), 7.29 (d, 2H, J ¼ 7.59 Hz, Ar-H),7.85 (t, 1H, J ¼ 7.32 Hz, Ar-H), 8.07 (d, 1H, J ¼ 8.37 Hz, Ar-H), 9.09 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 14.59, 55.97, 56.37, 60.88, 61.00, 63.96, 64.16, 105.52, 106.22, 109.48, 109.70, 115.55, 115.60,115.64, 121.59,126.73, 127.16, 130.38, 139.81, 151.16, 151.29, 153.08, 153.54, 154.94, 155.60, 156.92, 159.73; Anal. Calcd for C26H24F2N4O5: C, 61.71; H, 4.74; N, 10.98. Found: C, 60.88; H, 4.64; N, 10.94. 4.1.2.8. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 2,3,4-trifluorophenylamide 5h. Yellow crystals (3.92 g, 64% yield); mp 169e171  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.46 (t, 3H, J ¼ 6.95 Hz, O-CH2-CH3), 3.72 (s, 6H, 3,5di-OCH3), 3.89 (s, 3H, O-CH3), 4.09 (q, 2H, J ¼ 6.96 Hz, O-CH2), 6.80 (s, 2H, Ar-H), 7.00 (d, 2H, J ¼ 8.88 Hz, Ar-H), 7.04 (d, 1H, J ¼ 7.97 Hz, Ar-H), 7.37 (d, 2H, J ¼ 8.85 Hz, Ar-H), 8.28 (d, 1H, J ¼ 9.19 Hz, Ar-H), 9.16 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 14.65, 56.05, 56.47, 60.96, 64.02, 106.32, 111.75, 111.93, 11.96, 115.13, 115.75, 115.82, 115.86, 121.61, 127.25, 130.48, 139.95, 141.08, 153.16, 155.14, 155.47, 157.03, 159.85; Anal. Calcd for C26H23F3N4O5: C, 59.09; H, 4.39; N, 10.60. Found: C, 59.03; H, 4.44; N, 10.81. 4.1.2.9. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3-hydroxy-4-methoxyphenylamide 5i. Brown crystals (3.24 g, 54% yield); mp 228e230  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.45 (t, 3H, J ¼ 6.88 Hz, O-CH2-CH3), 3,70 (s, 6H, 3,5-di-OCH3), 3.87e3.89 (m, 6H, O-CH3), 4.07 (q, 2H, J ¼ 6.81 Hz, O-CH2), 5.96 (s, 1H, OH), 6.78 (s, 2H, Ar-H), 6.85 (d, 1H, J ¼ 8.51 Hz, Ar-H), 6.96 (d, 2H, J ¼ 8.49 Hz, Ar-H), 7.28e7.36 (m, 4H, Ar-H), 8.91 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm):14.66, 56.07, 56.20, 60.97, 64.00, 106.29, 107.42, 110.96, 111.82, 115.08, 121.89, 127.26, 130.61, 131.34, 139.80, 143.79, 145.86, 153.15, 154.82, 156.21, 156.63, 159.71; Anal. Calcd for C27H28N4O7: C, 62.30; H, 5.42;

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N, 10.76. Found: C, 62.61; H, 5.38; N, 10.61. 4.1.2.10. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 2-methoxyphenylamide 5j. White crystals (3.41 g, 58% yield); mp 193e195  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.40 (t, 3H, J ¼ 6.93 Hz, O-CH2-CH3), 3.67 (s, 6H, 3,5-diOCH3), 3.84 (s, 3H, O-CH3), 3.88 (s, 3H, O-CH3), 4,03 (q, 2H, J ¼ 6.92 Hz, O-CH2), 6.78 (s, 2H, Ar-H), 6.88 (d, 1H, J ¼ 7.98 Hz, Ar-H), 6.94 (d, 2H, J ¼ 8.78 Hz, Ar-H), 6.98e7.05 (m, 2H, Ar-H), 7.33 (d, 2H, J ¼ 8.78 Hz, Ar-H), 8.58 (d, 1H, J ¼ 7.86 Hz, Ar-H), 9.57 (s, 1H, CONH); 13CNMR(100 MHz, CDCl3, d ppm): 14.59, 55.74, 55.96, 60.82, 63.90, 105.99, 106.34, 109.97,114.97, 119.92, 120.92, 121.92, 124.16, 127.59, 130.58, 139.66, 148.22, 152.99, 154.82, 156.33, 156.71, 159.63; Anal. Calcd for C27H28N4O6: C, 64.27; H, 5.59; N, 11.10. Found: C, 64.28; H, 5.40; N, 11.36. 4.1.2.11. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid phenylamide 5k. Yellowish brown crystals (2.7 g, 58% yield); mp 138e140  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.72 (s, 6H, 3,5-di-OCH3), 3.84 (s, 3H, O-CH3), 3.90 (s, 3H, O-CH3), 6.81 (s, 2H, Ar-H), 6.99e7.06 (m, 3H, Ar-H), 7.19 (t, 1H, J ¼ 7.38 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.37e7.43 (m, 2H, Ar-H), 7.79 (d, 2H, J ¼ 8.00 Hz, Ar-H), 9.03 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 55.72, 56.09, 61.00, 106.35, 111.29, 115.80, 118.04, 119.92, 121.70, 124.68, 129.15, 130.23, 137.42, 138.80, 139.99, 153.20, 154.97, 156.32, 156.72, 160.43; Anal. Calcd for C25H26N4O5: C, 65.21; H, 5.25; N, 12.17. Found: C, 65.00; H, 5.30; N, 11.87. 4.1.2.12. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 3-methoxyphenylamide 5l. Yellow crystals (3.2 g, 54% yield); mp 147e149  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.73 (s, 6H, 3,5-di-OCH3), 3.84 (s, 3H, OCH3), 3.87 (s, 3H, O-CH3), 3.90 (s, 3H, O-CH3), 6.81 (s, 2H, Ar-H), 6.99e7.06 (m, 3H, Ar-H), 7.27e7.31 (m, 3H, Ar-H), 7.39 (t, 1H, J ¼ 8.05 Hz, Ar-H), 7.56 (s, 1H, Ar-H), 9.01 (s, 1H, CO-NH); DEPTQ(100 MHz, CDCl3, d ppm): 55.37, 55.72, 56.09, 61.00, 105.60, 106.34, 110.62, 111.27, 112.09, 115.81, 118.01, 121.70, 129.82, 130.23, 138.60, 138.80, 153.21, 154.98, 156.28, 156.73, 160.23, 160.43; Anal. Calcd for C26H26N4O6: C, 63.66; H, 5.34; N, 11.42. Found: C, 63.51; H, 5.31; N, 11.22. 4.1.2.13. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 2-methoxyphenylamide 5m. Yellow crystals (3.4 g, 58% yield); mp 131e134  C;1HNMR (400 MHz, CDCl3) d (ppm): 3.72 (s, 6H, 3,5-di-OCH3), 3.84 (s, 3H, OCH3), 3.90 (s, 3H, O-CH3), 3.96 (s, 3H, O-CH3), 6.83 (s, 2H, Ar-H), 6.96 (d, 1H, J ¼ 7.95 Hz, Ar-H), 7.00e7.07 (m, 3H, Ar-H), 7.13 (t, 1H, J ¼ 7.01 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.39 (t, 1H, J ¼ 8.03 Hz, Ar-H), 8.63 (d, 1H, J ¼ 9.40 Hz, Ar-H), 9.62 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 55.62, 55.83, 56.00, 106.45, 110.01, 111.40, 115.68, 118.14, 120.28, 121.19, 121.91, 124.27, 127.24, 130.18, 138.92, 148.33, 153.14, 154.95, 156.70, 160.39; Anal. Calcd for C26H26N4O6: C, 63.66; H, 5.34; N, 11.42. Found: C, 64.05; H, 5.65; N, 11.59. 4.1.2.14. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 3,4-dimethoxyphenylamide 5n. Light gray crystals (3.5 g, 56% yield); mp 141e144  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.70 (s, 6H, 3,5-di-OCH3), 3.81 (s, 3H, OCH3), 3.88 (m, 6H, O-CH3), 3.92 (s, 3H, O-CH3), 6.79 (s, 2H, Ar-H), 6.86 (d, 1H, J ¼ 8.64 Hz, Ar-H), 6.96e7.07 (m, 2H, Ar-H), 7.17 (d, 1H, J ¼ 8.60 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.37 (t, 1H, J ¼ 7.99 Hz, ArH), 7.57 (d, 1H, J ¼ 2.01 Hz, Ar-H), 8.95 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 55.70, 55.95, 56.06, 56.10, 60.98, 104.68, 106.29, 111.25, 111.29, 111.86, 115.72, 117.96, 121.70, 130.22,

301

131.02, 138.77, 139.92, 146.06, 149.05, 153.17, 154.91, 156.33, 156.60, 160.39; Anal. Calcd for C27H28N4O7: C, 62.30; H, 5.42; N, 10.76 Found: C, 62.21; H, 5.63; N, 10.99. 4.1.2.15. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 3,4,5-trimethoxyphenylamide 5o. Pale white crystals (3.3 g, 50% yield); mp 186e189  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.72 (s, 6H, 3,5-di-OCH3), 3.83 (s, 3H, OCH3), 3.86 (s, 3H, O-CH3), 3.89 (s, 3H, O-CH3), 3.91 (s, 6H, O-CH3), 6.80 (s, 2H, Ar-H), 6.98e7.06 (m, 2H, Ar-H), 7.10 (s, 2H, Ar-H), 7.28 (s, 1H, Ar-H), 7.39 (t, 1H, J ¼ 7.95 Hz, Ar-H), 8.93 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 55.71, 55.96, 61,00, 97.58, 106.34, 111.26, 115.74, 117.94, 121.68, 130.25, 133.50, 134.92, 138.76, 140.03, 153.22, 153.41, 155.02, 156.23, 156.71, 160.44; Anal. Calcd for C28H30N4O8: C, 61.08; H, 5.49; N, 10.18. Found: C, 61.15; H, 5.33; N, 10.05. 4.1.2.16. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 4-ethoxyphenylamide 5p. Pale white (3.6 g, 59% yield); mp 167e169  C; 1HNMR (400 MHz, CDCl3) d (ppm): 1.43 (t, 3H, J ¼ 6.97 Hz, O-CH2-CH3), 3.71 (s, 6H, 3,5-diOCH3), 3.82 (s, 3H, O-CH3), 3.89 (s, 3H, O-CH3), 4.05 (q, 2H, J ¼ 6.97, O-CH2), 6.80 (s, 2H, Ar-H), 6.92 (d, 2H, J ¼ 8.97 Hz, Ar-H), 6.97e7.05 (m, 2H, Ar-H), 7.28 (s, 1H, Ar-H), 7.38 (t, 1H, J ¼ 8.02 Hz, Ar-H), 7.68 (d, 2H, J ¼ 8.95 Hz, Ar-H), 8.94 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 14.85, 55.70, 56.07, 60.97, 63.69, 106.33, 111.30, 114.85, 115.74, 118.03, 121.54, 121.74, 130.19, 130.47, 138.82, 139.93, 153.16, 154.86, 155.98, 156.41, 156.53, 160.40; Anal. Calcd for C24H28N4O6: C, 64.27; H, 5.59; N, 11.10. Found: C, 64.08; H, 5.61; N, 11.08. 4.1.2.17. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 3,5-difluorophenylamide 5q. Dark Brown crystals (3.6 g, 60% yield); mp 104e107  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.69 (s, 6H, 3,5-di-OCH3), 3.81 (s, 3H, OCH3), 3.08 (s, 3H, O-CH3), 6.61 (t, 1H, J ¼ 8.70 Hz, Ar-H), 6.77 (s, 2H, Ar-H), 6.96 (d, 1H, J ¼ 8.01 Hz, Ar-H), 7.03 (d, 1H, J ¼ 7.90 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.35e7.40 (m, 3H, Ar-H), 9.16 (s, 1H, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 55.70, 56.05, 60.97, 99.63, 100.14, 103.15, 106.32, 111.34, 115.81, 117.96, 121.39, 130.29, 138.63, 153.18, 155.64, 156.87, 160.43, 161.89, 162.04, 164.49; Anal. Calcd for C25H22F2N4O5: C, 60.48; H, 4.47; N, 11.29. Found: C, 60.40; H, 4.22; N, 11.38. 4.1.2.18. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 3,4-difluorophenylamide 5r. Yellowish brown crystals (2.9 g, 49% yield); mp 141e144  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.70 (s, 6H, 3,5-di-OCH3), 3.82 (s, 3H, OCH3), 3.88 (s, 3H, O-CH3), 6.78 (s, 2H, Ar-H), 6.97e7.04 (m, 3H, Ar-H) 7.15 (d, 1H, J ¼ 7.80 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.33e7.40 (m, 2H, Ar-H), 9.04 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 55.94, 55.70, 56.05, 60.98, 106.31, 109.57, 109.79, 111.33, 115.53, 115.56, 115.59, 115.62, 115.78, 117.26, 117.43, 117.97, 121.48, 130.27, 138.68, 153.19, 155.07, 160.43; Anal. Calcd for C25H22F2N4O5: C, 60.48; H, 4.47; N, 11.29. Found: C, 60.37; H, 4.62; N, 11.62. 4.1.2.19. 1-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H[1,2,4]triazole-3-carboxylic acid 2,3,4-trifluorophenylamide 5s. Yellow crystals (3.4 g, 55% yield); mp 137e139  C; 1HNMR (400 MHz, CDCl3) d (ppm): 3.71 (s, 6H, 3,5-di-OCH3), 3.83 (s, 3H, OCH3), 3.89 (s, 3H, O-CH3), 6.82 (s, 2H, Ar-H), 6.99e7.08 (m, 4H, ArH), 7.28 (s, 1H, Ar-H), 7.41 (t, 1H, J ¼ 8.28 Hz, Ar-H), 9.17 (s, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 55.72, 56.08, 60.98, 106.41, 109.18, 111.39, 111.78, 115.87, 118.06, 121.42, 123.46, 130.29, 138.73, 140.16, 141.11, 143.57, 146.49, 148.95, 153.21, 155.64, 156.93,

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160.47; Anal. Calcd for C25H21F3N4O5: C, 58.37; H, 4.11; N, 10.89. Found: C, 58.33; H, 4.10; N, 10.94. 4.1.3. General procedure for synthesis of N-benzyl-1-(Aryl)-5(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 6a-6d A mixture of compound 4 (3.99 g, 0.01 mol) and appropriate benzylamine aromatic amine (0.01 mol) was refluxed for 2 h in absolute methanol (40 mL). Compounds were purified using flash column technique using CH2Cl2:MeOH (9:1) as an eluent. 4.1.3.1. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 2-methoxybenzylamide 6a. Orange oil (3.4 g, 65% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 1.43 (t, 3H, J ¼ 6.94 Hz), 3.66 (s, 6H, 3,5-di-OCH3), 3.85 (s, 3H, O-CH3), 3.86 (s, 3H, O-CH3), 4.05 (q, 2H, J ¼ 6.92 Hz), 4.70 (d, 2H, J ¼ 5.96 Hz, NHCH2), 6.74 (s, 2H, Ar-H), 6.87e6.95 (m, 4H, Ar-H), 7.24e7.31 (m, 3H, Ar-H), 7.37 (d, 1H, J ¼ 7.39 Hz, Ar-H), 7.67 (t, 1H, J ¼ 5.90 Hz, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 14.65, 38.97, 55.37, 56.01, 60.93, 63.97, 106.23, 110.32, 115.04, 120.62, 121.95, 125.76, 127.27, 128.98, 129.87, 130.63, 139.65, 153.05, 154.62, 156.14, 157.59, 158.98, 159.64; Anal. Calcd for C28H30N4O6: C, 64.85; H, 5.83; N, 10.80. Found: C, 64.68, H, 6.03; N, 10.72. 4.1.3.2. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3-methoxybenzylamide 6b. Dark orange oil (3.1 g, 59% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 1.45 (t, 3H, J ¼ 6.96 Hz, O-CH2-CH3), 3.68 (s, 6H, 3,5-di-OCH3), 3.81 (s, 3H, OCH3), 3.86 (s, 3H, O-CH3), 4.07 (q, 2H, J ¼ 6.97 Hz, O-CH2), 4.69 (d, 2H, J ¼ 5.99 Hz, NH-CH2), 6.75 (s, 2H, Ar-H), 6.85 (d, 1H, J ¼ 8.25 Hz, Ar-H), 6.95e6.99 (m, 3H, Ar-H), 7.26 (s, 1H, Ar-H), 7.28e7.30 (m, 1H, Ar-H), 7.33 (d, 2H, J ¼ 8.86 Hz, Ar-H), 7.55 (t, 1H, J ¼ 5.91 Hz, CONH); 13CNMR(100 MHz, CDCl3, d ppm): 14.64, 43.42, 55.29, 56.03, 60.94, 63.97, 106.23, 113.18, 113.63, 115.05, 120.34, 121.94, 127.26, 129.75, 130.66, 139.44, 139.73, 153.09, 154.09, 156.01, 159.09, 159.68, 159.91; Anal. Calcd for C28H30N4O6: C, 64.85; H, 5.53; N, 10.80. Found: C, 65.21, H, 5.80; N, 11.12. 4.1.3.3. 1-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3,5-difluorobenzylamide 6c. Orange oil (4.3 g, 71% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 1.45 (t, 3H, J ¼ 6.81 Hz, O-CH2-CH3), 2.68 (s, 6H, 3,5-di-OCH3), 3.87 (s, 3H, OCH3), 4.07 (q, 2H, J ¼ 6.97 Hz, O-CH2), 4.69 (d, 2H, J ¼ 6.94 Hz, NHCH2), 6.70e6.78 (m, 3H, Ar-H), 6.92 (m, 2H, Ar-H), 6.97 (d, 2H, J ¼ 8.94 Hz, Ar-H), 7.28 (s, 1H), 7.33 (d, 2H, J ¼ 8.93 Hz, Ar-H); 13 CNMR(100 MHz, CDCl3, d ppm): 14.64, 42.51, 56.02, 60.95, 63.99, 102.95, 106.23, 110.66, 115.09, 121.81, 127.23, 130.56, 139.80, 141.99, 153.12, 154.89, 155.69, 159.74, 161.98, 164.45; Anal. Calcd for C27H26F2N4O5: C, 61.83; H, 5.00; N, 10.68. Found: C, 62.00, H, 5.01; N, 11.05. 4.1.3.4. 1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 2-methylbenzylamide 6d. Dark red oil (3.2 g, 65% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 2.41 (s, 3H, CH3), 3.68 (s, 6H, O-CH3), 3.81 (s, 3H, 3,5-di-OCH3), 3,87 (s, 3H, OCH3), 4.73 (d, 2H, J ¼ 5.6 Hz, NH-CH2), 6.75 (s, 2H, Ar-H), 6.96 (d, 1H, J ¼ 8.78 Hz, Ar-H), 7.01e7.03 (m, 2H, Ar-H), 7.22e7.25 (m, 2H, J ¼ 6.34 Hz, Ar-H), 7.28 (s, 1H, Ar-H), 7.34e7.36 (m, 2H, Ar-H), 7.38(brs, 1H, CO-NH); 13CNMR(100 MHz, CDCl3, d ppm): 19.16, 41.74, 55.69, 56.05, 60.96, 106.28, 111.28, 115.73, 118.05, 121.77, 126.25, 127.98, 128.97, 130.19, 130.61, 135.38, 136.85, 138.85, 139.86, 153.12, 154.80, 156.14, 158.84, 160.39; Anal. Calcd for C27H28N4O5: C, 66.38; H, 5.78; N, 11.47. Found: C, 66.52, H, 5.84; N, 11.69. 4.1.3.5. 1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 2-methoxybenzylamide 6e. Dark red oil

(2.9 g, 71% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 3.69 (s, 6H, 3,5-di-OCH3), 3.81 (s, 3H, O-CH3), 3.87 (s, 3H, O-CH3), 3.89 (s, 3H, OCH3), 4.73 (d, 2H, J ¼ 5.95 Hz, NH-CH2), 6.78 (s, 2H, Ar-H), 6.91 (d, 1H, J ¼ 8.22 Hz, Ar-H), 6.96 (d, 1H, J ¼ 7.45 Hz, Ar-H), 7.01e7.03 (m, 2H, Ar-H), 7.27e7.31 (m, 2H, Ar-H), 7.35 (d, 1H, J ¼ 7.77 Hz, Ar-H), 7.40 (d, 1H, J ¼ 8.83 Hz, Ar-H), 7.76 (t, 1H, J ¼ 5.78 Hz, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 39.01, 55.39, 55.70, 56.07, 60.97, 106.38, 110.33, 111.33, 115.77, 118.07, 120.64, 121.45, 125.79, 128.99, 129.94, 130.20, 138.79, 140.00, 153.13, 154.50, 156.00, 157.63, 158.55, 160.40; Anal. Calcd for C27H28N4O6: C, 64.72; H, 5.59; N, 11.10. Found: C, 64.49, H, 5.67; N, 11.34. 4.1.3.6. 1-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-[1,2,4] triazole-3-carboxylic acid 3-methoxybenzylamide 6f. Dark red oil (2.6 g, 63% yield); 1HNMR (400 MHz, CDCl3) d (ppm): 3.69(s, 6H, 3,5-di-OCH3), 3.82e3.83(m, 6H, O-CH3), 3.88(s, 3H, O-CH3), 4.71(d, 2H, J ¼ 5.84 Hz, NH-CH2), 6.79(s, 2H, Ar-H), 6.86(d, 1H, J ¼ 8.27 Hz, Ar-H), 6.97e7.05(m, 5H, Ar-H), 7.29(t, 1H, J ¼ 7.87 Hz, Ar-H), 7.38(t, 1H, J ¼ 8.01 Hz, Ar-H), 7.78(t, 1H, J ¼ 6.34 Hz, CO-NH); 13 CNMR(100 MHz, CDCl3, d ppm): 43.52, 55.31, 55.72, 56.10, 60.98, 103.05, 106.39, 111.32, 113.23, 113.67, 115.91, 118.06, 120.39, 121.05, 129.77, 130.05, 138.67, 139.31, 140.16, 153.18, 154.52, 155.53, 159.92, 160.45; Anal. Calcd for C27H28N4O6: C, 64.72; H, 5.59; N, 11.10. Found: C, 64.48, H, 5.62; N, 11.32. 4.2. Biology 4.2.1. Cell culture Human hepatocarcinoma cell line (HepG2), and leukemia (HL60), which were purchased from ATCC, USA, were used to evaluate the cytotoxic effect of the tested samples. Cells were routinely cultured in DMEM (Dulbecco's Modified Eagle's Medium). Media was supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine, containing 100 units/ml penicillin G sodium, 100 units/ ml streptomycin sulphate, and 250 ng/mL amphotericin B. Cells were maintained at sub-confluency at 37  C in humidified air containing 5% CO2. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37  C. Cells were used when confluence had reached 75%. Tested samples were dissolved in dimethyl sulphoxide (DMSO), and then diluted thousand times in the assay to begin with the indicated concentration. All cell culture material was obtained from Cambrex BioScience (Copenhagen, Denmark). All chemicals were from Sigma/Aldrich, USA, except mentioned. All experiments were repeated three times, unless mentioned. 4.2.1.1. Anti-tumor activity. Cytotoxicity of tested samples was measured against each cell line using the MTT Cell Viability Assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and form a dark blue insoluble formazan crystals which is largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is directly proportional to the level of soluble formazan dark blue color. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm. Briefly, cells (0.5  105 cells/well), in serum-free media, were plated in a flat bottom 96-well microplate, and treated with 20 mL of serial concentrations of the tested samples for 48 h at 37  C, in a humidified 5% CO2 atmosphere. After incubation, media were removed and 40 mL MTT solution (5 mg/mL of MTT in 0.9% NaCl) in each well were added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 mL of acidified isopropanol/well and plate

M. Mustafa et al. / European Journal of Medicinal Chemistry 141 (2017) 293e305

was shacked at room temperature, followed by photometric determination of the absorbance at 570 nm using microplate ELISA reader. Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells compared with the vehicle control, with cytotoxicity indicated by

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