Synthesis, Anticancer Evaluation, Computer-Aided Docking Studies, and ADMET Prediction of 1,2,3-Triazolyl-Pyridine Hybrids as Human Aurora B Kinase Inhibitors.

A novel series of 1,2,3-triazolyl-pyridine hybrids were prepared through the reaction of the triazole derivative (1) with the appropriate aldehyde (2a-g) and malononitrile or ethyl cyanoacetate in the presence of ammonium acetate in refluxed acetic acid. The chemical composition of the products was established on the basis of spectral and elemental analyses. Aurora B kinase is a protein with diverse biological actions in controlling tumorigenesis by inhibiting apoptosis and promoting proliferation and metastasis, making it an emerging target for diseases such as hepatocellular carcinoma (HCC). Alteration in the target protein expression causes unequal distribution of genetic information, causing HCC. The new compounds were tested for their antihepatic cancer activity, and some of them had strong efficacy against human hepatoblastoma (HepG2) cell lines.

Introduction

Investigation of novel compounds which may be of use in designing new less toxic, selective, and potent anticancer agents is still the main challenge for medical chemists. It is being reported in the literature that compounds containing 1,2,3-triazole moiety are used extensively as precursors for preparation of molecules which have various pharmaceutical and biological applications, such as antimicrobial,[1−4] antitumor,[5,6] aromatase inhibitors, 5α-reductase inhibitors,[7] anti-inflammatory and antinociceptive agents,[8] LSD1inhibitors,[9] α-glycosidase inhibitors,[10] and HIV-1 protease inhibitors.[11] On the other hand, pyridine-based compounds are well validated for the synthesis of some new medical scaffolds that are showing anticancer, antifungal. and antibacterial activities, gastric H+/K+-ATPase inhibitors, and so forth.[12−20] Lavendamycin, streptonigrone, and streptonigrin are considered as good examples of drug possessing pyridine nuclei candidates, which are reported to be anticancer drugs. Many other pyridine analogues are screened for their cytotoxic activity against some human cancer cell lines to develop novel anticancer drugs. Also, it has been found that various pyridines, which are bioisosteres for α-terthiophene,[21] exhibit significant topoisomerase I (and/or) II inhibition activity. There are early reports on the ability of pyridines to bind with DNA/RNA[22] and to form metal complexes.[23] MCRs (multicomponent reactions) are potent processes used to facilitate the construction of druglike compounds, helping in drug discovery.[24,25] During the last decade, a hybridization approach was adopted to design 1,2,3-triazole-pyridine hybrids, and their synthetic and medicinal impacts were estimated.[26−29] Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer death worldwide.[30−32] Overexpression of Aurora B kinase occurred frequently during the process of hepatocarcinogenesis.[33,34] The investigation of the inhibition activity of Aurora B kinase on HepG2 cell invasion and migration and its role in hepatocellular carcinogenesis is well reported.[35,36] In the present work, in silico docking approach has been carried out to investigate docking interactions between triazolyl-pyridine hybrids and the target protein Aurora B kinase. Our results provide that the newly synthetic hybrid molecules could be targets for HCC treatment. Focusing on the aforementioned findings and in continuation of our aim to synthesize new pharmaceutically active compounds,[1,2,6,7,37−47] the aim of the present work is to design and synthesize a new series of 1,2,3-triazole-pyridine hybrids through MCRs, which are anticipated to be useful as anticancer candidates.

Results and Discussion

Chemistry

The key compound (1) was used for synthesis of a number of substituted nicotine analogues through a one-pot three-component reaction. For example, a series of novel substituted 2-amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)nicotine nitrile (3–9) was prepared via one-pot reaction of acetyl triazole (1) with different aldehydes (2a–g), malononitrile, and a catalytic amount of AcONH4 (Scheme ). The chemical compositions of newer synthesized compounds were elucidated by means of spectral and elemental analyses. The IR spectra of compounds (3–9) had shown in each case two absorption bands in the regions 3421–3208 cm–1, which was attributed to NH2 groups, and in 2192–2222 cm–1, which was attributed to CN groups. As a representative example of 3–9, the 1H NMR spectrum of compound (3) represented two signals at δ (6.98, 2H, NH2) and δ (8.25, 1H, pyridine-H5), along with the expected signals of compound (3). Moreover, the molecular weight determination was observed in the expected region, which confirmed the formation of compound (3).

Scheme 1

Synthesis of Substituted Nicotine Derivatives 3–9

For the formation of products (3–9), it was suggested that the reaction proceeded via the condensation reaction between the acetyl group of compound (1) with the appropriate of the aldehydes (2a–g) to afford the corresponding chalcones, which then reacted with AcONH4 to yield the corresponding imino derivatives, followed by tandem Michael addition of the active methylene group of (3–9) to give the nonisolable intermediates A. The latter underwent in situ auto-oxidation that was followed by tautomerization and formation of the final products (3–9) (Scheme ).

Scheme 2

Mechanism of the Synthesis of Nicotine Derivatives 3–9

Analogously, another series of nicotine derivatives (10–16) were prepared by using ethyl cyanoacetate in lieu of malononitrile. Thus, the reaction of acetyl triazole (1) with the appropriate of aldehydes (2a–g), ethyl cyanoacetate, and AcONH4 had yielded the corresponding products (10–16) (Scheme ). Chemical structures for the assigned compounds (10–16) were inferred from their elemental analysis and spectral data. Compound (10) was taken as a representative example. Its IR spectrum showed strong absorption bands at 3362 and 3280 cm–1 owing to the NH2 group, while the band at 1735 cm–1 was attributed to the carbonyl ester group. 1H NMR spectrum for (10) revealed a triplet signal at δ 1.02 for protons of CH3 of the ester group, a quartet signal at δ 4.13 for the two protons of CH2 of the ester group, singlet signals at δ 6.23 for the two protons of the NH2 group, and a singlet signal at δ 8.39 ppm for the proton of the pyridine ring.

Scheme 3

Synthesis of Substituted Nicotine Derivatives (10–16)

Pharmacology

As Aurora B kinase was found in hepatoblastoma (HepG2) cell lines;[35,36] HepG2 cells have been chosen as model to identify the expression effect of Aurora B kinase on the growth of hepatocellular cancer cells. The cytotoxic activities of the prepared derivatives (3–16) were screened against HepG2 and BALB/3T3 (murine fibroblast) using the standard drug doxorubicin with an IC50 value of 3.56 ± 0.46 μg/mL and the MTT assay. The results were used in plotting a dose response curve in which the concentrations of the tested samples required to kill half of the cell population (IC50) were determined. The cytotoxicity was expressed as the mean IC50 of three independent experiments as tabulated in Table .

Table 1

Antiproliferative Activity of New Derivatives toward HepG2 and Normal Cell Lines (BALAB/3T3)a

compound	HepG2 IC50 ± SD [μg/mL]	BALAB/3T3 IC50 ± SD [μg/mL]
doxorubicin	3.56 ± 0.46	3.21 ± 0.32
3	17.24 ± 0.83	Nd
4	2.34 ± 0.16	15.09 ± 0.23
5	Nd	Nd
6	0.64 ± 0.14	Nd
7	1.08 ± 0.94	Nd
8	9.65 ± 0.37	Nd
9	11.15 ± 0.01	Nd
10	23.24 ± 0.45	76.72 ± 0.24
11	16.66 ± 0.83	Nd
12	Nd	Nd
13	6.24 ± 0.71	61.24 ± 0.21
14	9.54 ± 0.05	Nd
15	11.34 ± 0.95	18.34 ± 1.81
16	13.24 ± 0.12	Nd

Compounds were tested in concentration from 100 to 0.1 μg/mL; Nd: no detectable activity in the used concentrations; *Concentration of DMSO: 1%

The data show that most of the compounds have promising anticancer activity against HepG2 compared with doxorubicin as the standard drug, and compounds 6 and 7 show excellent anticancer activities against HepG2 in comparison to the reference drug. Meanwhile, they show no toxicity on the normal cell line (BALAB/3T3). Accordingly, the newly synthetic compounds are promising molecules and can be used as antihepatic carcinoma candidates.

Computational Studies

In this study, the potential Aurora B kinase inhibitory activity of these novel hybrid compounds was theoretically investigated by using in silico molecular docking simulation. The 3D structure of the target (ID: 4AF3) obtained at a resolution of 2.75 Å has been used for the docking study. Before the docking process, the 3D model of the target protein was prepared by merging all nonpolar hydrogen atoms and removing water molecules. On the basis of various literature surveys,[48] the amino acid residues which represented the active site region are Arg81, Leu83, Phe88, Phe101, Lys106, Arg139, Tyr141, Tyr156, Ala157, Pro158, Lys164, Glu165, His198, Leu207, Lys215, and Arg248. The grid box was then set around the active pocket for specific docking. Computer-based docking study was achieved by means of PyRx software using Autodock vina. Among various confirmations for each docked compound, the confirmation with the lowest binding energy and good interactions with the target was selected. Table summarizes the binding characterizations between the target protein and synthesized compounds. According to the data tabulated, the 15 compounds interacted with the active site regions of Aurora B kinase through a network of noncovalent interactions. Figure represents the two-dimensional and three-dimensional schematic representations demonstrating the molecular interactions between the docked molecules 6 and 7 and the active pocket of protein. The remains of complexes are included in the Supporting Information as Figure S1. The starting compound 1 displayed a dock score of -7.2 kcal/mol with one H-bond, one π–π, and one π–sigma interaction at distances of 2.98, 4.66, and 3.65 Å, respectively. For a set of compounds (3–9) of docking scores in the range of −10.5 to −8.6 kcal/mol, the intermolecular interactions will clearly be discussed. Compound 3 of phenyl ring at position 4 of the pyridine moiety exhibited a binding energy of −9.1 kcal/mol and formed various π-interactions such as π–π, π–cation, and π–sigma contacts with Tyr156, Lys106, and Leu83 at 6.32, 6.22, and 3.77 Å, respectively. Substitution of electron-donating group -OMe (molecule 4) increases the docking energy to −9.9 kcal/mol.[49] The molecule interacted with the residues Tyr156, Tyr151, Lys106, and Leu83 through π-interactions. Replacing the phenyl ring on the pyridine moiety by thiophenyl/furfuryl rings as in compounds 5 and 9 will decrease the docking energy to −8.6 and −8.7 kcal/mol, respectively. For x = O in place of x = S, the docking scores remain the same. Derivative 5 formed one arene-cation interaction between the benzene ring and Lys106 at a distance of 5.31 Å beside forming arene-sigma contact between the triazole moiety and Leu207 at a distance of 3.78 Å, while analogue 9 exhibited one π–π and one π–cation interaction with Tyr156 and Lys106, respectively. Moreover, derivatives 6 (−10.5 kcal/mol) and 7 (−10.1 kcal/mol) with −Br (weak) and −NO2 (strong), respectively, as electron-withdrawing groups, showed various interactions with the target. Introducing deactivating groups (−Br and −NO2) on the phenyl ring decreases the docking score.[49] Monosubstitution on the benzene ring is preferred over disubstitution. Thus, the docking energy of compound 8 is decreased to −9.8 kcal/mol. Compound 8 displayed three arene-cation contacts between triazole and benzene rings and Arg248, beside forming one arene-sigma contact triazole moiety and His198. For the set of compounds 10–16 of docking scores in the range −10.2 to −8.3 kcal/mol, the intermolecular interactions will be presented a head. The docking energy for compound 10 is −8.8 kcal/mol. Molecule 10 interacted with the residues Lys106 and Leu83. π–Cation interface is seen with the positively charged amino on its side chain (H3N+) of Lys106 and pyridine ring, while Leu83 is involved in the π–sigma interaction with the triazole moiety. Molecule 11 with the activating group −OMe at the position-4 of the benzene ring increases the docking energy to -9.2 kcal/mol. The molecule exhibited π–cation and π–sigma interactions with the residues Lys106 and Leu83 at the distances of 6.08 and 3.93 Å, respectively. On the other hand, replacing the phenyl ring on the pyridine moiety as in derivatives 12 (with thiophenyl) and 16 (with furfuryl) decreases the docking energy to −8.3 and −8.5 kcal/mol, respectively. Analogues 13 and 14 with deactivating groups such as −Br (weak) and −NO2 (strong) showed docking scores of −10.2 and −9.5 kcal/mol, respectively. Finally, molecule 15 with disubstituent groups on the phenyl ring showed decreasing docking score (−8.9 kcal/mol) compared to the monosubstituent group on the phenyl ring as in molecule 11 (−9.2 kcal/mol). The in silico docking results revealed that all docked molecules exhibited the lowest binding energy with good affinity toward the binding site of Aurora B kinase. All compounds obeyed Lipinski’s rule of 5 (RO5) except compounds 6, 7, 11, 13, 14, and 15 as represented in Table ; their molecular masses are less than 500 Da except analogues 6, 7, 11, 13, 14, and 15; their polar solvent accessibilities are in the acceptable range except compounds 7, 13, and 14; their solubility is viewed good; their log p is <5 except for latter compounds, proposing that these compounds except 7, 13, and 14 are the most lipophilic with least water solubility; additionally, the hydrogen bond donating and accepting abilities are in the agreeable range. Furthermore, the values of blood brain barrier (BBB+) and % human intestinal absorption (HIA+) are in the permissible range, which indicate that they could be absorbed by human intestines. Further, they showed negative toxicity and negative carcinogenicity test. Based upon the information obtained from the in silico docking study and their ADME-Tox parameters, we suggested that the newly synthetic compounds can be more potent toward HCC inhibition.

Table 2

Energy-Based Intermolecular Interactions Between the Target and Docked Compoundsa

	B.E. (kcal/mol)	interactions	length (Å)
		H-bonds
1	–7.2	Lys106:NZ--compound 1	2.98
		π–π contact
		Tyr156--compound 1	4.66
		π–sigma contact
		Leu83:CD1--compound 1	3.65
		π–π contact
3	–9.1	Tyr156--compound 3	6.32
		π–cation contact
		Lys106:NZ--compound 3	6.22
		π–sigma contact
		Leu83:CB--compound 3	3.77
		π–cation contact
4	–9.9	Lys106:NZ--compound 4	6.11
		π–cation contact
5	–8.6	Lys106:NZ--compound 5	5.31
		π–sigma contact
		Leu207:CD2--compound 5	3.78
6	–10.5	H-bonds
		Tyr156:OH--compound 6	2.97
		Tyr156:OH--compound 6	3.20
		Pro158:O--compound 6	2.96
		π–cation contact
		Arg81:NH1--compound 6	4.73
		Arg81:NH2--compound 6	5.50
7	–10.1	H-bonds
		Glu165:N--compound 7	2.98
		Pro158:O--compound 7	2.41
		Tyr156:OH--compound 7	2.04
		π–cation contact
		Lys164:NZ--compound 7	6.30
		Lys106:NZ--compound 7	5.85
		Lys164:NZ--compound 7	5.51
8	–9.8	π–cation contact
		Arg248:NH1--compound 8	3.73
		Arg248:NH2--compound 8	4.99
		Arg248:NH2--compound 8	6.07
		π–sigma contact
		His198:CD2--compound 8	3.62
9	–8.7	π–π contact
		Tyr156--compound 9	6.23
		π–cation contact
		Lys106:NZ--compound 9	6.05
10	–8.8	π–cation contact
		Lys106:NZ--compound 10	6.08
		π–sigma contact
		Leu83:CB--compound 10	3.93
11	–9.2	H-bonds
		Tyr141:OH--compound 11	2.96
		π–π contact
		Phe101--compound 11	4.31
		π–cation contact
		Lys215:NZ--compound 11	4.83
		Arg139:NH1-compound 11	3.87
		Arg139:NH2-compound 11	6.08
12	–8.3	π–cation contact
		Lys106:NZ—compound 12	6.19
13	–10.2	H-bonds
		Tyr156:OH--compound 13	2.97
		π–π contact
		Phe88--compound 13	3.97
		π–cation contact
		Lys164:NZ--compound 13	6.36
14	–9.5	H-bonds
		Tyr156:OH--compound 14	2.74
		Glu165:N--compound 14	3.04
		Tyr156:OH--compound 14	2.21
		Ala157:O--compound 14	2.29
		π–π contact
		Phe88--compound 14	4.19
		π–cation contact
		Lys164:NZ--compound 14	6.33
		Lys164:NZ--compound 14	5.45
15	–8.9	π–π contact
		Phe88--compound 15	3.94
		π–cation contact
		Lys106:NZ--compound 15	4.78
16	–8.5	H-bonds
		Tyr156:OH--compound 16	2.88
		Pro158:O--compound 16	2.20
		Tyr156:OH--compound 16	2.28
		π–π contact
		Phe88--compound 16	4.08
		π–cation contact
		Arg81:NH1--compound 16	4.68
		Arg81:NH2--compound 16	6.96

B.E, estimated free binding energy.

Figure 1

(A) Two-dimensional and (B) three-dimensional representations showing interactions between compounds 6 and 7 and the active pocket of target. H-bonds are shown in green and blue dotted lines, while π-stackings are declared in blue lines.

Table 3

List of ADME and Physicochemical Properties of the Title Compounds 1–16a

	MW (g/mol)	BBB+	Caco2+	HIA+	log p	TPSA A2	HBA	HBD	N-rot.	N violations	volume A3	AMES toxicity	carcinogenicity
	180–500	–3 to 1.2	<25 poor >500 great	<25 poor >80 high	<5	≤140	2.0–20.0	0.0–6.0	≤10	<5		nontoxic	non carcinogenic
1	280.12	0.98	76.59	99.12	2.54	47.8	4	0	2	0	202.50	nontoxic	noncarcinogenic
3	431.30	0.96	56.08	98.32	3.94	93.5	6	1	3	0	333.76	nontoxic	noncarcinogenic
4	461.32	0.97	65.59	98.43	4.00	102.7	7	2	4	0	359.31	nontoxic	noncarcinogenic
5	437.33	0.97	58.20	97.02	3.72	93.4	6	2	3	0	324.47	nontoxic	noncarcinogenic
6	733.43	0.97	84.97	98.32	5.36	140.0	11	2	6	2	540.70	nontoxic	noncarcinogenic
7	699.53	0.97	86.51	96.57	4.70	187.8	14	2	7	2	546.15	nontoxic	noncarcinogenic
8	477.32	0.97	73.35	97.93	3.28	122.9	8	3	4	0	367.32	nontoxic	noncarcinogenic
9	421.26	0.97	58.35	97.52	3.08	106.6	7	2	3	0	315.33	nontoxic	noncarcinogenic
10	478.35	0.98	60.68	98.60	4.39	95.9	7	2	6	0	378.23	nontoxic	noncarcinogenic
11	508.38	0.98	66.97	98.66	4.45	105.2	8	2	7	1	403.78	nontoxic	noncarcinogenic
12	484.38	0.97	66.99	97.11	4.18	95.94	7	2	6	0	368.94	nontoxic	noncarcinogenic
13	780.49	0.98	86.02	98.60	5.82	144.5	12	2	9	3	585.17	nontoxic	noncarcinogenic
14	746.59	0.97	87.19	96.85	5.15	190.3	15	2	10	3	590.62	nontoxic	noncarcinogenic
15	524.38	0.97	75.53	98.26	3.73	125.4	9	3	7	1	411.80	nontoxic	noncarcinogenic
16	468.31	0.98	65.27	97.30	3.54	109.1	8	2	6	0	359.80	nontoxic	noncarcinogenic

MW, molecular weight; BBB+, blood–brain barrier; Caco2+, Caco-2 permeability; HIA+, % human intestinal absorption; log p, logarithm of partition coefficient between n-octanol and water; TPSA, topological polar surface area; HBA, number of hydrogen bond acceptors; HBD, number of hydrogen bond donors; n-rot., number of rotatable bonds.

Conclusions

Two series of biologically active 1,2,3-triazolyl-pyridine derivatives were synthesized using MCRs of the acetyltriazole analogue with the appropriate aldehydes, malononitrile, and/or ethyl cyanoacetate in the presence of excess ammonium acetate in refluxed acetic acid. The mechanism of the reaction was proposed, and the structure and chemical composition of the products were elucidated on bases based of elemental analyses and spectral data (IR, NMR, and MS). In addition, the novel 1,2,3-triazole-pyridine hybrids were tested for their potency as antitumor agents, and the obtained data revealed high potency for some of such compounds against hepatic cancer as compared with the reference drug. Hence, the output of the presented research holds a promising insight in the synthesis of novel compounds with significant biological activities. Also, based on the results of the computational studies, the newly synthetic hybrid derivatives could be considered as efficient drug candidates for further molecular development of HCC agents. Therefore, the research work presented herein may aid in compiling effective strategies for the drug design for HCC.

Experimental Section

Experimental Instrumentation

All melting points were determined on an electrothermal apparatus and were left uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. 1H NMR and 13C NMR spectra were recorded in (CD3)2SO solutions on a BRUKER 400 FT-NMR spectrometer, and chemical shifts were expressed in ppm units using TMS as an internal reference. Mass spectra were recorded on a GC–MS QP1000 EX Shimadzu spectrometer. Elemental analyses were carried out at the Microanalytical Center of Cairo University. 1-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)ethanone (1).[6,37,50]

Synthesis of Substituted Nicotine Derivatives 3–16

General Procedure

A mixture of 1-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl) ethanone (1) (1 mmol), the appropriate of aldehydes 2a–g (1 mmol), and malononitrile (1 mmol) or ethyl cyanoacetate (1 mmol) in glacial acetic acid (10 mL) containing ammonium acetate (0.616 gm, 8 mmol) were refluxed 6–8 h (monitored by TLC). After the completion of the reaction, the mixture was cooled and the precipitated products were filtered, washed with water, dried, and recrystallized from ethanol to give nicotine derivatives 3–16. Physical and spectral data of compounds 3–16 are listed below.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-phenylnicotinonitrile (3)

Brown solid, (80% yield), mp 168–170 °C; IR (KBr)νmax 3364, 3210 (NH2), 2192 (CN), 1620(C=N), 1600(C=C) cm–1; 1H NMR (DMSO-d6): δ 2.58 (s, 3H, CH3), 6.98 (s, 2H, NH2), 7.43–8.02 (m, 9H, Ar–H), 8.75 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 9.64, 85.6, 111.2, 113.7, 123.1, 127.4, 128.5, 129.2, 131.6, 133.5, 134.9, 142.8, 154.4, 158.6, 161.2; (MS m/z (%) 431 (M+, 15), 327 (47), 257 (19), 169 (28), 77 (56), 50 (15), 43 (100). Anal. Calcd for C21H15BrN6 (431): C, 58.48; H, 3.51; N, 19.49. Found: C, 58.46; H, 3.49; N, 19.45%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(4-methoxyphenyl)nicotine Nitrile (4)

Orange crystals, (72% yield), mp 179–181 °C; IR (KBr)νmax 3322, 3198 (NH2), 2222 (CN), 1630 (C=N), 1600(C=C) cm–1; 1H NMR (DMSO-d6): δ 2.58 (s, 3H, CH3), 3.95 (s, 3H, CH3), 6.93 (s, 2H, NH2), 7.12–7.98 (m, 8H, Ar–H), 8.34 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 10.05, 55.8, 85.9, 112.9, 114.8, 123.5, 128.1, 129.7, 131.6, 154.4, 161.9; MS m/z (%) 461 (M+, 10), 352 (27), 266 (12), 178(3), 165 (35), 77 (50), 65 (19), 43 (100). Anal. Calcd for C22H17BrN6O (461): C, 57.28; H, 3.71; N, 18.22. Found: C, 57.32; H, 3.67; N, 18.19%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(thiophen-2-yl)nicotinenitrile (5)

Brown crystals, (75% yield), mp 150–152 °C; IR (KBr)νmax 3370, 3211 (NH2), 2210 (CN), 1620 (C=N), 1590(C=N) cm–1; 1H NMR (DMSO-d6): δ 2.61 (s, 3H, CH3), 6.54 (s, 2H, NH2), 7.34–8.03 (m, 7H, Ar–H), 8.70 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 9.62, 85.9, 112.8, 123.1, 127.4, 128.6, 134.9, 138.2, 148.6, 159.7; MS m/z (%) 437(M+, 37), 415(20), 316 (100), 257 (19), 199 (23), 118 (50), 100 (28), 78 (72), 50 (15). Anal. Calcd for C19H14rN6S (437): C, 52.18; H, 3.00; N, 19.22. Found: C, 52.21; H, 2.95; N, 19.17%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinonitrile (6)

Beige crystals, (68% yield), mp 202–204 °C; IR (KBr)νmax 3367, 3212 (NH2), 2210 (CN), 1620 (C=C), 1600(C=N) cm–1; 1H NMR (DMSO-d6) δ 2.64 (s, 3H, CH3), 2.59 (s, 3H, CH3), 6.18 (s, 2H, NH2), 7.23–7.87 (m, 13H, Ar–H), 8.47 (s, 1H, pyrazole-H), 8.93 (s, 1H, Pyridine-H5); MS m/z (%) 735(M+2, 35), 733(M+, 39), 640(12), 590(9), 560(18), 511(22), 490(10), 461(3), 380 (17), 280 (29), 170 (11), 76 (12), 65 (100). Anal. Calcd for C33H24r2N11 (733): C, 54.04; H, 3.16; N, 21.01. Found: C, 54.08; H, 3.13; N, 20.97%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(3-(5-methyl-1-(3-nitro phenyl)-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinonitrile (7)

Yellow crystals, (73% yield), mp 192–194 °C; IR (KBr)νmax 3360, 3218 (NH2), 2210 (CN), 1620 (C=C), 1600(C=N) cm–1; 1H NMR (DMSO-d6): δ 2.61 (s, 3H, CH3), 2.58 (s, 3H, CH3), 6.81 (s, 2H, NH2), 7.45–8.24 (m, 13H, Ar–H), 8.51 (s,1H, pyrazole-H), 8.62 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 10.34, 12.5, 76.2, 113.2, 122.9, 123.7, 128.1, 133.5, 141.2, 142.3, 143.9, 147.9, 151.6, 159.6; MS m/z (%) 699 (M+, 100), 620(50), 542 (10), 498 (3), 496 (7), 483(30), 327 (47), 257 (19), 127 (27), 105 (48), (77 (14), 65 (11). Anal. Calcd for C33H24rN12O2 (699): C, 56.66; H, 3.31; N, 24.03. Found: C, 56.69; H, 3.27; N, 23.97%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(4-hydroxy-3-methoxyphenyl)nicotinonitrile (8)

Yellow crystals, (81% yield), mp 171–173 °C; IR (KBr)νmax 3650(OH), 3331, 3212 (NH2), 2220 (CN), 1615 (C=C), 1595(C=N) cm–1; 1H NMR (DMSO-d6): δ 2.48 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.18 (s, 1H, Ar–H), 6.57 (s, 2H, NH2), 7.23–7.87 (m, 6H, Ar–H), 8.18 (s, 1H, Pyridine-H5)10.02 (s, 1H, OH); MS m/z (%) 477 (M+, 90), 416 (80), 212 (10), 170 (27), 105 (48), 76 (63).

Anal. Calcd for C22H17BrN6O2 (477): C, 55.36; H, 3.59; N, 17.61. Found: C, 55.32; H, 3.54; N, 17.58%.

2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(furan-2-yl)nicotinonitrile (9)

Brown crystals, (62% yield), mp 152–154 °C; IR (KBr)νmax 3421, 3318 (NH2), 2213 (CN), 1625 (C=C), 1600(C=N)cm–1; 1H NMR (DMSO-d6): δ 2.50 (s, 3H, CH3), 6.65 (s, 2H, NH2), 7.18–7.90 (m, 7H, Ar–H), 8.18 (s, 1H, Pyridine-H5); MS m/z (%) 421 (M+, 12), 316 (60), 191 (55), 127 (51), 85 (47), 57 (100). Anal. Calcd for C19H14rN6O (421): C, 54.17; H, 3.11; N, 19.95. Found: C, 54.22; H, 3.05; N, 19.89%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-phenylnicotinate (10)

Yellow crystals, (67% yield), mp 163–165 °C; IR (KBr)νmax 3362, 3280 (NH2), 1735 (C=O ester) cm–1; 1H NMR (DMSO-d6): δ 1.02 (t, J = 7.2 Hz, 3H, CH3), 2.58 (s, 3H, CH3), 4.13 (q, J = 7.2 Hz, 2H, CH2), 6.23 (s, 2H, NH2), 7.15–7.92 (m, 9H, Ar–H), 8.39 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 9.6, 14.1, 60.9, 117.1, 123.0, 123.1, 123.5, 124.5, 128.2, 131.6, 133.5, 133.9, 142.6, 158.2, 160.9; MS m/z (%) 478 (M+, 12), 473(100), 458 (9), 410(5), 403(8), 389 (47), 314(14), 234 (19), 157 (28), 50 (10). Anal. Calcd for C23H20BrN5O2 (478): C, 57.75; H, 4.21; N, 14.64. Found: C, 59.82; H, 4.18; N, 14.61%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(4-methoxyphenyl)nicotinate (11)

Brown crystals, (62% yield), mp 152–154 °C; IR (KBr)νmax 3360, 3208 (NH2), 1735 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.23 (t, J = 7.2 Hz, 3H, CH3), 2.50 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.26 (q, J = 7.2 Hz, 2H, CH2), 6.25(s, 2H, NH2), 7.18–7.90 (m, 9H, Ar–H), 8.12 (s, 1H, Pyridine-H5); MS m/z (%) 508 (M+, 100), 503(62), 489(14), 465(5), 388(9), 308(3),227 (47), 211(19), 165 (28), 77 (56), 65 (15). Anal. Calcd for C24H22BrN5O3 (508.38): C, 56.70; H, 4.36; N, 13.78. Found: C, 56.65; H, 4.33; N, 13.73%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(thiophen-2-yl)nicotinate (12)

Yellow crystals, (81% yield), mp 170–172 °C; IR (KBr)νmax 3330,3280 (NH2), 1715 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.15 (t, J = 7.2 Hz, 3H, CH3), 2.58 (s, 3H, CH3), 4.26 (q, J = 7.2 Hz, 2H, CH2), 6.32 (s, 2H, NH2), 7.01–7.98 (m, 7H, Ar–H), 8.11 (s, 1H, Pyridine-H5); MS m/z (%) 494 (M+, 10), 465(25), 439(8), 375(6), 327 (17), 295(45), 257 (19), 177 (28), 77 (56), 65 (15), 50 (100). Anal. Calcd for C21H18BrN5O2S (484.37): C, 52.07; H, 3.75; N, 14.46. Found: C, 52.12; H, 3.72; N, 14.41%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(3-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinate (13)

Brown crystals, (87% yield), mp 179–181 °C; IR (KBr)νmax 3335, 3250 (NH2), 1735 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.22 (t, J = 7.2 Hz, 3H, CH3), 2.58 (s, 3H, CH3), 4.25 (q, J = 7.2 Hz, 2H, CH2), 6.52 (s, 2H, NH2), 7.12–7.89 (m, 13H, Ar–H), 8.42 (s,1H, pyrazole-H), 8.65 (s, 1H, Pyridine-H5); MS m/z (%) 780 (M+, 10), 690(15), 645(23), 620(8), 568(7), 553(4), 539(15), 507(52), 430(6), 360(8), 177(10), 65(100).Anal. Calcd for C35H28Br2N10O2 (780): C, 53.86; H, 3.62; N, 17.95. Found: C, 53.83; H, 3.59; N, 17.91%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(3-(5-methyl-1-(3-nitro phenyl)-1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)nicotinate (14)

Yellow crystals, (62% yield), mp 230–232 °C; IR (KBr)νmax 3291, 3218 (NH2), 1725 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.17 (t, J = 7.2 Hz, 3H, CH3), 2.58 (s, 3H, CH3), 4.22 (q, J = 7.2 Hz, 2H, CH2), 6.32 (s, 2H, NH2), 7.28–7.92 (m, 15H, Ar–H), 8.42 (s, 1H, pyrazole-H), 8.71 (s, 1H, Pyridine-H5); MS m/z (%) 746 (M+, 12), 710(30), 682(12), 640(5), 599 (90), 553(7), 473(6), 427(23), 312 (47), 257 (10), 176 (2), 56 (100). Anal. Calcd for C35H28BrN11O4 (746): C, 56.31; H, 3.78; N, 20.64. Found: C, 56.27; H, 3.74; N, 20.61%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(4-hydroxy-3-methoxy phenyl)nicotinate (15)

Beige crystals, (75% yield), mp 192–194 °C; IR (KBr)νmax 3360, 3218 (NH2), 1725 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.22 (t, J = 7.2 Hz, 3H, CH3), 2.61 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 4.32 (q, J = 7.2 Hz, 2H, CH2), 6.22 (s, 2H, NH2), 7.22–7.91 (m, 7H, Ar–H), 8.21 (s, 1H, Pyridine-H5), 9.81(a, 1H, OH); 13C NMR (DMSO-d6): δ 10.22, 14.5, 56.1, 60.9, 113.2, 115.2, 122.1, 123.6, 128.2, 133.7, 134.9, 141.2, 142.3, 143.9, 144.5, 147.9, 158.6; MS m/z (%) 524 (M+, 16), 517(10), 486(27), 406(36), 392(5), 321(47), 252(39), 179(41), 105(100), 57(83). Anal. Calcd for C24H22BrN5O4 (524.38): C, 54.97; H, 4.23; N, 13.36. Found: C, 54.92; H, 4.18; N, 13.33%.

Ethyl 2-Amino-6-(1-(4-bromophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)-4-(furan-2-yl)nicotinate (16)

Brown crystals, (82% yield), mp 185–187 °C; IR (KBr)νmax 3291, 3220 (CN), 1737 (C=O) cm–1; 1H NMR (DMSO-d6): δ 1.02 (t, J = 7.2 Hz, 3H, CH3), 2.59 (s, 3H, CH3), 4.41 (q, J = 7.2 Hz, 2H, CH2), 6.23 (s, 2H, NH2), 7.57–7.94 (m, 7H, Ar–H), 8.68 (s, 1H, Pyridine-H5); 13C NMR (DMSO-d6): δ 10.39, 14.2, 60.9, 107.4, 117.2, 122.9, 123.7, 128.1, 139.5, 146.3, 147.9, 151.6, 158.6; MS m/z (%) 468 (M+, 11), 452 (3), 441 (13), 429 (57), 322 (39), 180 (28), 105 (47), 259 (9), 77 (100), 50 (15), 43 (10). Anal. Calcd for C21H18BrN5O3 (468.31): C, 53.86; H, 3.87; N, 14.95. Found: C, 53.91; H, 3.82; N, 14.92%.

Antiproliferative Activity

The cytotoxic evaluation of the synthesized compounds was carried out at the Regional Centre for Mycology and Biotechnology at the Al-Azhar University, Cairo, Egypt according to the reported method. The details of this technique were described by Skehan et al.[51,52]

Computational Methods

To get more insight into the binding interactions between the hybrid molecules and the active site pocket of the target, in silico molecular docking studies are performed.[44,53,54] The chemical structures of the hybrids are drawn in cdx format using ChemDraw Ultra 0.8 and then converted to SDF format by using Open Babel 2.4.1 tool.[55] An in-house library of 15 triazolyl-pyridine hybrid molecules is generated. The X-ray crystal structure of the target protein Aurora B kinase is retrieved from the RCSB Protein Data Bank web server (www.rcsb.org/pdb/).[56] The stable confirmations of the target and molecules are obtained after energy minimization using CHARMm Force Field in Discovery Studio 3.5 Visualizer and Universal Force Field (UFF) in Open Babel GUI, respectively.[57,58] A grid with specific dimensions 25 Å × 25 Å × 25 Å is covered with the active site region of the target. All synthesized compounds are docked to the target protein using PyRx- Virtual screening tool version 0.8 through inbuilt Autodock vina,[59] and their binding energies are calculated. Nine confirmations of each compound are obtained from the docking protocol, and the confirmation with the best scored pose and with the lowest binding energy is selected for further study.[46,60,61] Accelrys discovery studio 3.5 (Accelrys Discovery Studio Visualizer Software 2010) is used to visualize the 2D and 3D representations of the intermolecular interactions between the target and newly synthetic compounds. ADME-Tox[62] (Absorption, Distribution, Metabolic, Excretion, and Toxicity) and physiochemical parameters of the newer compounds are predicted using admetSAR and Mol inspiration web-based tools to check whether they possess druglike properties or not.[63−66]