New 1,2,3-triazole/1,2,4-triazole hybrids linked to oxime moiety as nitric oxide donor selective COX-2, aromatase, B-RAF^V600E and EGFR inhibitors celecoxib analogs: design, synthesis, anti-inflammatory/anti-proliferative activities, apoptosis and molecular modeling study

Figures & data

Figure 1. Structure of selective oestrogen receptor blocker Tamoxifen (1), Steroidal aromatase inhibitors Exmestane (2), nonsteroidal aromatase inhibitor letrozole (4), anastrozole (5) Kinase inhibitors, Vemurafenib (6), Encorafenib (7), Non selective NSAIDs, Indomethacin (8), selective COX-2 inhibitors celecoxib (9), and 1,2,4 triazoles with anti-cancer activities (10) and anti-cancer 1,2,4 triazoles (10), (11), and B-RAF^V600E inhibitor (12).

Figure 2. structural hyperdization of designed compounds from COX-2 inhibitors celecoxib (9) and anti-cancer 1,2,4 triazoles (10), (11), B-RAF^V600E inhibitors (12a&b).

Table 1. Cell cycle analysis of compounds 19c, 19f, 19h, 19 l and control for breast MCF7 cells.

Cell cycle phase	%G0-G1	%S	%G2-M
19c	37.88	35.14	26.98
19f	69.07	26.89	4.04
19h	45.44	28.17	26.39
19l	63.69	23.42	12.62
Cont. MCF7	59.04	29.83	11.13

Table 2. Apoptosis of compounds 19c, 19f, 19h, 19 l and control against breast MCF7 cells.

	Total	Early	Late	Necrosis
19c	41.55	21.85	12.36	7.34
19f	39.18	24.15	12.61	2.42
19h	42.21	22.54	14.91	4.76
19l	44.76	12.91	24.74	7.11
Cont. MCF7	1.88	0.41	0.27	1.2

Scheme 1. Reagents and conditions: (a) ethanol 95% reflux 4h., (b) alcoholic NaOH reflux 4h., (c) acetonitrile, triethylamine, reflux 10h., (d) acetonitrile, hydroxylamine hydrochloride, sod. acetate, reflux, 10h.

Table 3. In vitro COX-1 and COX-2 inhibitory activity of triazole derivatives 19a-l and reference drug celecoxib.

Compound	COX-1 IC50 (μM)a ± S.E.	COX-2 IC50 (μM)^a± S.E.	COX-2 S.I.b
19a	4.84 ± 0.020	0.343 ± 0.070	14.11
19b	4.42 ± 0.021	0.485 ± 0.048	9.11
19c	10.70 ± 0.025	0.309 ± 0.041	34.62
19d	4.63 ± 0.023	0.437 ± 0.037	10.59
19e	4.39 ± 0.033	0.442 ± 0.058	9.93
19f	6.08 ± 0.016	0.329 ± 0.049	18.48
19g	4.76 ± 0.034	0.394 ± 0.031	12.08
19h	8.60 ± 0.027	0.309 ± 0.071	27.83
19i	4.60 ± 0.048	0.363 ± 0.061	12.67
19j	3.43 ± 0.026	0.397 ± 0.025	8.63
19k	4.76 ± 0.025	0.377 ± 0.040	12.62
19l	12.00 ± 0.027	0.243 ± 0.011	49.38
Celecoxib	8.78 ± 0.030	0.416 ± 0.010	21.10

The bold values indicates the highest S.I. of the most potent compounds.

^aThe concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme.

^bThe in vitro COX-2 selectivity index (COX-1/COX-2).

Figure 3. Cell viability % of compounds 19a-l and reference compound single dose (10 μM) against MCF-7, HEP-38, HCT-116 and A549 cancer cell line.

Figure 4. Compounds 19c, 19f, 19h, 19 l effect on DNA-ploidy flow cytometric analysis of breast MCF7 cells compared to negative control.

Figure 5. The percentage of Annexin-V-FITC-positive staining in breast MCF7 cells when treated with compounds 19c, 19f, 19h, 19 l compared to sorafenib and negative control.

Figure 6. IC₅₀% values of compounds 19c, 19f, 19h, 19 l compared to letrozole on aromatase against breast MCF7 cells.

Figure 7. IC₅₀ values of compounds 19c, 19f, 19h, 19 l compared to encorafenib on EGFR.

Table 4. In vitro MCF-7, HEP-3B, HCT-116, A549 and F180 inhibitory activity of triazole derivatives 19c,e,f,h,j-l and reference drugs doxorubicin, sorafenib, 5-F.U. and tamoxifen..

Compound	MCF-7 (μM)^a	HEP-3B (μM)^a	HCT-116 (μM)^a	A549 (μM)^a	F180 (μM)^a
19c	14	N.D.	N.D.	4.5	15.4
19e	N.D.	N.D.	4.8	N.D.	N.D.
19f	16	14	N.D.	3	10.4
19h	15	N.D.	N.D.	4	11.3
19j	N.D.	4.5	9.9	N.D.	N.D.
19k	N.D.	12	13.7	N.D.	N.D.
19l	9	12	8	3.3	16.5
Doxorubicin	0.3	—	—	—	4.28
Sorafenib	—	3.5	—	—	—
5-F.U.	—	—	4.8	6	---
Tamoxifen^78	27.9	—	—	—	—

^aThe concentration of test compound produce 50% inhibition of MCF-7, HEP-3B, HCT-116, A549 and F180 cell lines, the result is the mean of six values.

Figure 8. IC₅₀ values of compounds 19c, 19f, 19h, 19 l compared to that of encorafenib on B-RAF^V600E.

Table 5. % of NO release of the synthesised compounds.

Compound No.	% NO released
19a	1.52
19b	1.66
19c	3.18
19d	0.73
19e	1.76
19f	2.94
19g	2.14
19h	2.87
19i	2.55
19j	0.90
19k	0.88
19l	3.36

Figure 9. (A) Visual representation (3D) of celecoxib docked with 3LN1 active site (B) Visual representation (3D) of compound 19c docked with 3LN1active site, (C) Visual representation (3D) of compound 19f docked with 3LN1 active site (D) Visual representation (3D) of compound 19h docked with 3LN1 active site (E) Visual representation (3D) of compound 19 l docked with 3LN1 active site.

Table 6. Docking data of triazoles compounds 19a-l and celecoxib with COX-2 active site (3LN1).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of bonds	Functional group
19a	−7.3	Pro 500 (2.61), Val509 (3.5)	H-bond	SO2NH2
19b	−6.3	Val509 (3.6), Ser516 (3.8)	H-bond	SO2NH2
19c	−8.1	Leu338(2.6), Val509 (2.7), Pro500 (2.5),	H-bond, hydrophobic	SO2NH2, OCH3
19d	−6.7	Ser516 (3.6), Trp343 (2.8)	H-bond	SO2NH2
19e	−7.9	Leu517 (2.4), Thr79 (2.7)	H-bond	SO2NH2
19f	−8.9	Pro500 (3.9), Val509 (2.7)	H-bond, hydrophobic	SO2NH2, NO2
19g	−7.6	Gly340 (2.8), Val509 (3.7)	H-bond	SO2NH2
19h	−8.5	Pro500 (2.3), Val509 (2.6)	H-bond, hydrophobic	SO2NH2, OCH3
19i	−7.5	Phe504 (2.1), Ser516 (2.9)	H-bond	SO2NH2
19j	−7.2	Leu338 (2.6), Gly340 (3.9)	H-bond	SO2NH2
19k	−6.2	Val102 (2.9), pro500 (2.3)	H-bond	SO2NH2
19l	−8.2	Arg499(2.4)	H-bond	SO2NH2, NO2
Celecoxib	−9.6	Ser339 (2.7), Gln178 (2.1), Arg499 (2.9).	H-bond	SO2NH2

Figure 10. (ِA) Visual representation (2D) of compound 19c docked with 1M17 active site, (B) Visual representation (2D) of compound 19f docked with 1M17 active site (C) Visual representation (2D) of compound 19h docked with 1M17 active site (D) Visual representation (2D) of compound 19 l docked with 1M17 active site (E)Visual representation (2D) of encorafenib docked with 1M17 active site (F) Visual representation (2D) of 4-anilinoquinazline docked with 1M17 active site.

Table 7. Docking data of triazoles 19a-l and encorafenib with EGFR enzyme (1M17).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19a	−8.3	Pro770 (3.8), Met769 (3.7), Lys721 (4.1), Thr830 (3.2), Asp831 (2.9)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19b	−8.4	Asp831 (2.9), Pro770(3.1), Lys721 (3.9).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19c	−8.7	Asp830 (4.2), Met769 (3.5), Lys721(3.7), Thr830 (3.9).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19d	−8.3	Asp831 (3.7), Leu694 (3.9), Lys721(2.8).	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19e	−8.2	Lys721(3.8), Met769 (3.7), Val702 (3.6)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19f	−8.9	Met742 (7.2), Asp831(3.1), Lys721 (3.4), Lys773 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19g	−8.1	Pro770 (3.2), Thr830 (3.7), Asp831 (4.1), Met769 (3.9), Lys721(3.5)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19h	−8.8	Met769v(3.8), vCys773v(3.7), Lys721(3.9), Leu694 (3.7), Gly772 (4.1)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19i	−8.5	Thr830 (4.1), Asp831(3.9), Lys692 (4.0), Lys721 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19j	−8.3	Asp831(3.7), Met769 (3.8), His781(3.7), Asp831(3.8), Lys721(3.9)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19k	−8.1	Asp831(3.8), Pro770 (3.2), Met742 (3.9), Lys721 (3.9), Cys773 (3.2)	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
19l	−8.8	Met742 (3.7), Cys773 (3.1), Lys721(3.5), pro770	H-bond π … π stacking	SO2NH2 1,2,3triazole 1,2,4triazole
encorafenib	−7.5	Lys721(2.3), Met769 (2.8), Arg857 (2.8)	H-bond π … π stacking	SO2NH- pyrazole
4-anilinoquinazoline	−8.5	Lys721(2.1), Meth761(2.6)	H-bond π … π stacking	Pyrimidine moiety Phenyl moiety

Figure 11. (A) Visual representation (3D & 2D) of letrozole docked with aromatase enzyme 3EQM active site (B) Visual representation (3D & 2D) of compound 19c docked with aromatase enzyme 3EQM active site, (C) Visual representation (3D & 2D) of compound 19f docked with aromatase enzyme 3EQM active site (D) Visual representation (3D & 2D) of compound 19h docked with aromatase enzyme 3EQM active site (E) Visual representation (3D & 2D) of compound 19 l docked with aromatase enzyme 3EQM active site.

Table 8. Docking data of triazoles 19a-l and letrozole with aromatase enzyme (3EQM).

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19a	−10.8	Met447 (3.1), Met311(3.2), Arg115 (3.4), Cys437 (3.6)	H-bond π … π stacking	SO2NH2 1,2,4triazole 1,2,3 triazole
19b	−10.9	Met374 (3.9), Leu372 (3.8), Leu477 (3.7)	H-bond π … π stacking	SO2NH2, 1,2,4 triazole 1,2,3 triazole
19c	−11.3	Met374 (4.1), Ile133 (4.0), Ala438 (4.2)	H-bond π … π stacking	SO2NH2- 1,2,3 triazole 1,2,4triazole
19d	−10.1	Met311(3.8), Ile132 (3.9), Trp141 (3.5), Arg145 (3.5), Arg115 (3.6), Ala348(3.7)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19e	−8.6	Val369 (3.7), Arg115 (3.6), Arg435 (3.8), Ala306 (3.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19f	−8.0	Met374 (3.5), Leu372 (3.8), Ala307 (3.9)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19g	−10.1	Met311 (3.7), Leu372 (3.5), Met374 (3.4), Phe430 (3.4)	H-bond π … π stacking	SO2NH2 1,2,4 triazole 1,2,3 triazole
19h	−10.2	Met303 (2.8), Met446 (2.9), Ala307 (2.7), Val370 (2.6)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
19i	−7.7	Met447 (3.1), Ser314 (3.2), Leu372 (2.9)	H-bond π … π stacking	SO2NH2 1,2,4 triazole 1,2,3 triazole
19j	−6.5	Val370 (3.5), 369 (3.6), Met303(3.8), Ala438 (3.8)	H-bond π … π stacking	SO2NH2-1,2,3 triazole 1,2,4triazole
19k	−7.5	Met303 (3.8), Ala307 (3.7), Arg375 (3.7), Leu372 (3.9)	H-bond π … π stacking	SO2NH2-1,2,4 triazole 1,2,3 triazole
19l	−8.8	Met311 (2.4), Met446(2.6), Arg115 (2.7), Leu372 (2.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole 1,2,4triazole
letrozole	−7.1	Arg115 (2.9), Arg435(2.8)	H-bond π … π stacking	CN group 1,2,3 triazole

Figure 12. (A) Visual representation (2D) of compound 19c docked with enzyme B-RAF^V600E (PDB entry 1UWJ) active site, (B) Visual representation (2D) of compound 19f docked with B-RAF^V600E (C) Visual representation (2D) of compound 19h docked with B-RAF^V600E (D) Visual representation (2D) of compound 19 l docked with B-RAF^V600E (E)Visual representation (2D) of encorafenib docked with B-RAF^V600E (F) Visual representation (2D) of Sorafenib docked with B-RAF^V600E.

Table 9. Docking data of the most active triazoles 19c,f,h,l and encorafenib kinase enzyme B-RAF^V600E (PDB, ID: 1UWJ) active site.

Compounds	Docking Score (Kcal/mol)	Amino acid residue (distance A^o)	Types of interaction	Functional group
19c	−8.3	Glu500 (2.9), Asp593 (3.0), Ser535 (2.9), Asp593 (3.3), Val470 (4.1)	H-bond π … π stacking	SO2NH2 1,2,3triazole Oxime moiety
19f	−7.7	Lys482 (2.3), Phe594 (2.9), Asp593 (2.7)	H-bond π … π stacking	1,2,3 triazole 1,2,4triazole
19h	−8.0	Phe594 (2.7), Lys482 (2.8), Asp593 (2.8)	H-bond π … π stacking	SO2NH2 1,2,3 triazole
19l	−7.4	Asp593 (2.9), Glu500 (2.9), Phe594 (2.8), Lys482 (2.7)	H-bond π … π stacking	SO2NH2 1,2,3triazole Oxime moiety
Encorafenib	−7.4	Asp593 (2.9), Glu500 (3.1), Val470 (2.7).	H-bond π … π stacking	Amide moiety Pyrimidine moiety
Sorafenib	−7.8	Glu500(2.9), Cys531(2.7), Asp593(2.9)	H-bond π … π stacking	Thiourea moiety Pyrimidine moiety

Abdellatif KRA, Belal A, Omar HA. Design, synthesis and biological evaluation of novel triaryl (Z)-olefins as tamoxifen analogues. Bioorg Med Chem Lett. 2013;23(17):4960–4963.

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