Synthesis and Cytotoxic Activity Evaluation of Novel Imidazopyridine Carbohydrazide Derivatives

doi:10.21203/rs.3.rs-1837075/v1

Download PDF

Research Article

Synthesis and Cytotoxic Activity Evaluation of Novel Imidazopyridine Carbohydrazide Derivatives

https://doi.org/10.21203/rs.3.rs-1837075/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 06 Jan, 2024

Read the published version in BMC Chemistry →

You are reading this latest preprint version

Two series of novel imidazo[1,2-a]pyridine-2-carbohydrazide derivatives have been designed, synthesized and evaluated for cytotoxic activity. Target compounds were designed in two series: aryl hydrazone derivatives that were devoid of triazole moiety (7a-e) and aryl triazole group (11a-e). In vitro cytotoxicity screening was carried out using MTT assay against three human cancer cells including breast cancer (MCF-7), colon cancer (HT-29) and leukemia (K562) cell lines. Compound 7d bearing 4-bromophenyl pendant from aryl hyrazone series exhibited the highest cytotoxic potential with IC₅₀ values of 22.6 µM and 13.4 µM against MCF-7 and HT-29 cells, respectively. Cell cycle analysis revealed that 7d decreased the cells in the G2/M phase at 50 and 100 µM concentrations. Overall, 7d could serve as a suitable candidate for further modifications as a lead anticancer structure.

Imidazopyridine

3-triazole ring

carbohydrazide

cytotoxic activity

In spite of considerable advances in the treatment of cancer, cancer incidence and mortality are rapidly growing worldwide and remain a major health concern. According to the GLOBOCAN 2020 database, the number of new cancer cases is estimated roughly as 19.3 million patients in 2020. In this regard, one of the world’s top medical priorities should be the development of new drugs for the prevention and treatment of cancer [1].

The imidazopyridine scaffold possesses a broad range of activities including anxiolytic, antiulcer, anti-mycobacterial, antiviral, anti-inflammatory and anticancer properties [2–5]. Recently, during the investigation of imidazopyridine derivatives with anticancer properties, some 6-substituted imidazo[1,2-a] pyridine derivatives have been found to be effective against colon cancer cell lines (compound I, Fig. 1) [6]. Another effort on structural modification of these series containing pyrazol moiety showed the potential of these molecules as potent and selective p110a inhibitors (compound II, Fig. 1) [7]. On the other hand, 1,2,3-triazole-based structures have been previously reported as anti-cancer agents (Compound III, Fig. 1) [8–13]; Compound IV from triazolo linked nicotinamides exhibited promising antiproliferative activity and ability to inhibit tubulin polymerization. In this regard, the triazole linkage would presumably be favorable for potent and selective anticancer agents [14–16].

As a continuation of our interest in the design of novel anticancer agents [17–20], herein we report the design and synthesis of imidazopyridine carbohydrazide derivatives characterized by a imidazopyridine core and two different substituted pendants. This strategy yielded two different series: aryl hydrazone derivatives lacking triazole moiety (7a-e) and aryl triazole group (11a-e). Various aryl moieties were subjected to gradual structural alteration to investigate the importance of their contribution to the enhancement of cytotoxic potency. Afterwards, the synthesized compounds were evaluated for their cytotoxic potential against three human cancer cell lines MCF-7, HT-29 and K562 cells. Furthermore, the distribution of cancer cells incubated with the most potent compound 7d in different phases of cell cycle was studied in order to understand the mode of action of these compounds.

Figure. 1.

3.1. Chemistry

A series of novel imidazo[1,2-a]pyridine-2-carbohydrazide derivatives (7a-e, 11a-e) were synthesized in several steps as outlined in Scheme 1. The chemical reaction of aminopyridine 1 and ethyl 3-bromo-2-oxo propanoate 2 in refluxing ethanol led to synthesis of ethylimidazo[1,2-a]pyridine-2-carboxylate 3. In the second step, treatment of ester intermediate 3 with excess amount of hydrazine hydrate afforded carbohydrazide derivative 5 under refluxing condition. ¹HNMR spectrum of 5 represented two singlet peaks at 9.53 and 4.48 ppm which were assigned for a hydrazine group at position 2. In addition IR spectrum of 5 showed the appearance of the band at 1660 cm characteristic for carboxamide group stretch. Different substituted aldehydes 6a-e were reacted with intermediate 5 to afford the corresponding benzylidene imidazo[1,2-a]pyridine-2-carbohydrazides 7a-e [21]. In ¹HNMR spectrum of 7a-e, disappearance of the proton shift of NH₂ and presence of the singlet peaks (related to the protons N = CH) were obtained as the result of further formation of the imine bond in the corresponding products. Further reaction of 5 with propargyl bromide in the presence of K₂CO₃ in refluxing DMF resulted in the corresponding prop-2-yn-1-yl imidazo[1,2-a]pyridine-2-carbohydrazide 9. ¹HNMR spectrum of 9 presented two singlet peaks at 3.70 and 2.23 ppm which corresponded to a 2-propynyl group. The click reaction of 9 with different in situ-produced azides, catalyzed by CuSO₄.5H₂O in the presence of sodium ascorbate afforded the final product 11a-e. The ¹HNMR spectrums were consistent with the assigned structures of triazole, benzyl substituted pendant and all other aromatic protons at expected regions.

Scheme 1

3.2 Biological activity

The results of in vitro cytotoxic activity of imidazopyridine derivatives are summarized in Table 1. All synthesized compounds were evaluated for cytotoxic activity against three human cancer cell lines including MCF-7, HT-29 and K562 by employing MTT assay. Cisplatin and doxorubicin were also tested as standard chemotherapeutic reference agents.

Five compounds (9, 7c, 7d, 11a and 11d) exhibited weak to moderate cytotoxic activity with IC₅₀ values as low as 22.6 µM (MCF-7), 13.4 µM (HT-29) and 23.7 µM (K562). None of the compounds except derivative 9 showed any cytotoxicity against K562 cell lines up to the highest tested concentration of 100 µM. Among all evaluated derivatives, compound 7d containing bromide substitution showed the best cytotoxic activity with IC₅₀ values of 22.6 and 13.4 µM against MCF-7 and HT-29 cell lines, respectively. However introduction of triazole linkage between substituted phenyl and imidazopyridine in other bromide bearing compound 11d decreased the activity against HT-29 cell line with the IC₅₀ values of 83.9 µM and no activity was observed in the two other cell lines.

Assessment of the cytotoxic activity and preliminary structure–activities relationship (SAR) of tested compounds revealed that the lipophilicity might be an effective parameter. So that the mild change in activity was found between intermediate of the template structure of imidazopyridine-2-carbohydrazide 5 and propyn-1yl imidazopyridine-2-carbohydrazide 9 with increase in logP value from − 0.08 to 0.65. Considering less cytotoxic activity and logP of compound 7c bearing chlorine substitute (IC₅₀ values of 64.8 and 85.4 µM against MCF-7 and HT-29 cell lines, respectively) compared to 7d, it could be implied that the indispensable role of lipophilicity parameter on cytotoxic potential of these series of compounds. A linear dependency between lipophilicity and cytotoxicity could be observed so the more lipophilic compounds might have improved cytotoxic effect.

On the other hand, the in vitro screening results of the triazole bearing analogues revealed that compound 11a containing benzyl moiety with a logP value of 1.64 demonstrated superior cytotoxic activity with against both MCF-7 and HT-29 cells compared to 11d with a logP value of 2.0. Hence, it could be construed that in 11a-11e compounds, unlike 7a-7e series, higher lipophilicity is not necessarily associated with improved cytotoxicity.

Overall, it was observed that imidazopyridine derivatives (7a-7e) exhibited superior cytotoxicity compared to imidazopyridine triazole counterparts (11a-11e) against tested cell lines.

Furthermore, arrest of cancer cells at any phase of the cell cycle caused by certain anti-cancer agents prevents proliferation, and could be exploited as a therapeutic strategy. In order to explore the possible mechanisms of action of the most potent imidazopyridine derivative, the cell-cycle distribution of MCF-7 cells incubated with 7d was evaluated using propidium iodide (PI)-based assessment of cell cycle by flow cytometry after 48 h. The distribution of treated and control cells in different phases of cell cycle is presented in Table 2 and Fig. 2. The data obtained clearly indicated that this compound cased a dose dependent increase in G0/G1 phase and a concomitant decrease in G2/M phase. These data indicate that the compounds cause a prolongation or arrest in the G1 phase or probably increase the number of cells that leave the cycle and enter the G0 phase.

Table 2

Percent distribution of MCF-7 cells in phases of cell cycle in the presence or absence of 7d
Sample	Sub G1	G0/G1	S	G2/M
Control	0.84 ± 0.3	61.37 ± 1.4	11.65 ± 2.3	26.13 ± 2.9
7d (20 µM)	0.81 ± 0.58	64.23 ± 7.62	11.93 ± 5.74	23.27 ± 9.95
7d (50 µM)	0.81 ± 0.56	66.95 ± 8.41	11.14 ± 4.88	21.10 ± 8.79
7d (100 µM)	1.12 ± 0.92	67.18 ± 7.46	10.18 ± 5.31	21.52 ± 7.17

Some novel imidazopyridine derivatives have been designed and synthesized in two different groups: compounds bearing aryl hydrazine pendant (7a-7e) and aryl-triazole hydrazine bearing derivatives (11a-11e). We also investigated the role of 1,2,3-triazole linkage on the cytotoxic potential of the imidazopyridine derivatives against MCF-7, K562 and HT-29 cancer cells. The results demonstrated that triazole motif could not improve the cytotoxic potency of the tested compounds against any of the tested cell lines. Among them, compound 7d from imine series with para-bromine substitution was found to be the most potent analogue with IC₅₀ value of 22.6 and 13.4 µM against MCF-7 and HT-29 cell lines, respectively. In addition, among triazole-bearing derivatives, compound 11a showed moderate activity at IC₅₀ values of 40.3 and 61.2 µM against the mentioned cell lines, respectively. The results indicated that the potent analog of this scaffold, 7d with superior LogP value among all compounds could serve as a suitable candidate for further modification as a lead anti-cancer structure.

General procedure for the synthesis of compounds 7a-e and 11a-e

The synthetic route of compounds is displayed in Scheme 1. The condensation of 2-aminopyridine 1 with appropriate ethyl 3-bromo-2-oxo propanoate 2 resulted in the formation of ethyl imidazo[1,2-a]pyridine-2-carboxylate 3. The carbohydrazide derivative 5 was produced in the presence of excess amount of hydrazine hydrate. Compound 7a-e was prepared via the reaction of different aldehydes 6a-e and 5. Moreover, compound 5 was treated with propargyl bromide to provide prop-2-yn1yl imidazo[1,2-a]pyridine-2-carbohydrazide 9 which was further coupled with various benzyl azide intermediates 10a-e through click reactions to generate 11a‑e derivatives.

Synthesis of ethyl imidazo[1,2-a]pyridine-2-carboxylate (3)

2-Aminopyridine (1, 5 mmol) was first dissolved in ethanol (10 mL) and ethyl 3-bromo-2-oxo propanoate (2, 7.5 mmol,) was added gently to the mixture. The reaction mixture was stirred at 0°C for 1 hour. The reaction was completed after 24 hours; the progression of the reaction was regularly monitored by thin layer chromatography (TLC). After completion, the solvent was evaporated. The precipitate was filtered off and washed with diethylether, dried and recrystallized from ethanol to afford creamy white solid.

Yield: 90%, Rf = 0.70 (CHCl₃:MeOH 90:10); mp: 135-137 ^◦C, IR (KBr, cm^-1 ) υ max: 3086 (O-H), 1729 (Ester C=O); ¹HNMR (300MHz, Acetone-d6): δ (ppm) 8.42-8.40 (d, J=6.0 Hz, 1H, imidazopyridine-H5), 8.30 (s, 1H, imidazopyridine-H), 7.45-7.42 (d, J=9.0 Hz, 1H, imidazopyridine-H), 7.22-7.17 (t, J=6.0 Hz, 1H, imidazopyridine-H), 6.86-6.82 (t, J=6.0 Hz, 1H, imidazopyridine-H), 4.25-4.18 (q, 2H, COOCH₂CH₃), 1.25-1.20 (t, J=9.0, 3H, COOCH₂CH₃), MS (EI) m/z (%): 190 (M⁺, 28), 145 (34), 118 (100), 91 (19), 78 (40).

Synthesis of imidazo[1,2-a]pyridine-2-carbohydrazide (5)

Ethylimidazo[1,2-a]pyridine-2-carboxylate (3, 5 mmol) was reacted with excess amounts (3-4 mL) of hydrazine hydrate 4 for 3 hours under the reflux condition. After completion, the mixture was poured into ice water (50 mL), filtered and washed with cold water.

light creamy solid, Yield: 25%, Rf = 0.36 (CHCl₃:MeOH; 90:10), mp: 192-194 ^◦C. IR (KBr, cm ^-1) υ max : 3353 and 3187 (NH2), 1660 (Amide C=O), ¹HNMR (300MHz, CDCl₃): δ_H (ppm) 9.53 (s, 1H, CONH), 8.59-8.57 (d , J = 6.0 Hz, 1H, imidazopyridine-H), 8.36 (s, 1H, imidazopyridine-H), 7.60-7.57 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.36-7.30 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 6.99-6.95 (t, J = 6.0 Hz, 1H, imidazopyridine-H-6), 4.48 (s, 2H, CONHNH₂), MS (EI) m/z (%): 176 (M+, 100), 145 (100), 117 (45), 97 (34), 78 (39).

General procedure for synthesis of N´-benzylidene imidazo[1,2-a]pyridine-2-carbohydrazide derivatives (7a-e)

Title compounds 7a-e were synthesized by adding appropriate aromatic aldehydes (1 mmol) into the solution of imidazo[1,2-a]pyridine-2-carbohydrazide ( 5, 1 mmol) in ethanol and refluxed for 24 h. After completion of the reaction as indicated by TLC (chloroform methanol 95:5), the reaction mixture was allowed to cool in room temperature. The precipitated product was filtered and re-crystallized from methanol.

Synthesis of N´-benzylidene imidazo[1,2-a]pyridine-2-carbohydrazide (7a)

Yield: 53%, Rf = 0.40 (CHCl₃:MeOH 95:5), mp: 257-259^◦C, IR (KBr, cm ^-1) υ max: 3244 (Amide N-H), 1666 (Amide C=O).¹HNMR (300MHz, DMSO-d₆): δ_H (ppm) 11.95 (s, 1H, CONH), 8.62 (m, 2H, N=CH and imidazopyridine-H), 8.55 (s, 1H, imidazopyridine-H), 7.72- 7.70 (m, 2H, Ar-H), 7.66-7.63 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.47-7.36 (m, 4H, imidazopyridine-H and Ar-H), 7.05-7.00 (t, J = 6.0 Hz, 1H, imidazopyridine-H); ¹³CNMR (70 MHz, DMSO): d_C (ppm) 158.97, 148.49, 144.34, 138.74, 134.92, 130.49, 129.32 (2C), 128.24, 127.54 (2C), 127.34, 117.64, 116.37, 113.97.MS (EI) m/z (%): 263 (M+, 30), 161 (91), 143 (77), 118 (100), 97 (25), 78 (38).

Synthesis of N´-(4-fluorobenzylidene)imidazo[1,2-a]pyridine-2-carbohydrazide (7b)

Yield: 39%, Rf = 0.43 (CHCl₃:MeOH 95:5), mp: 234-236^◦C, IR (KBr, cm ^-1 ) υ max: 3302 (Amide N-H), 1669 (Amide C=O), ¹HNMR (300MHz, DMSO-d₆): δ_H (ppm) 11.95 (s, 1H, CONH), 8.63-8.61 (m, 2H, N=CH and imidazopyridine-H), 8.55 (s, 1H, imidazopyridine-H), 7.78- 7.74 (m, 2H, Ar-H), 7.66-7.63 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.42-7.36 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 7.33-7.27 (m, 2H, Ar-H), 7.05-7.00 (t, J = 6.0 Hz, 1H, imidazopyridine-H); ¹³CNMR (70 MHz, DMSO): d_C (ppm) 161.86, 159.04, 147.26, 144.41, 138.95, 131.57, 129.71, 129.60, 128.20, 127.14, 117.73 116.53, 116.38, 116.24, 113.86.MS (EI) m/z (%): 282 (M+, 28), 161 (100), 144 (100), 118 (100), 91 (28), 78 (50).

Synthesis of N´-(4-chlorobenzylidene)imidazo[1,2-a]pyridine-2-carbohydrazide (7c)

Yield: 44%, Rf = 0.44 (CHCl₃:MeOH 95:5), mp: 226-228^◦C, IR (KBr, cm ^-1 ) υ max: 3441 (Amide N-H), 1656 (Amide C=O), ¹HNMR (300MHz, DMSO-d₆): δH (ppm) 12.02 (s, 1H, CONH), 8.63-8.61 (m, 2H, N=CH, imidazopyridine-H-5), 8.55 (s, 1H, imidazopyridine-H-3), 7.74-7.71 (d, J = 9.0 Hz, 2H, Ar-H), 7.66-7.63 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.54-7.51 (d, J = 9.0 Hz, 2H, Ar-H), 7.42- 7.36 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 7.05-7.00 (t, J = 6 Hz, 1H, imidazopyridine-H), ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 157.30, 145.69, 143.29, 135.34, 131.16, 127.94 (3C), 125.85, 125.59, 116.94, 114.48, 112.95. MS (EI) m/z (%): 298 (M+, 27), 161 (100), 144 (100), 118 (100), 91 (30), 78 (50).

Synthesis of N´-(4-bromobenzylidene)imidazo[1,2-a]pyridine-2-carbohydrazide (7d)

Yield: 48%, Rf = 0.59 (CHCl3:MeOH 95:5), mp: 230-232^◦C, IR (KBr, cm ^-1 ) υ max: 3441 (Amide N-H), 1654 (Amide C=O),¹HNMR (300MHz, DMSO-d₆): δ_H (ppm) 12.03 (s, 1H, CONH), 8.63-8.61 (d, J = 6.0 Hz, 1H, imidazopyridine-H), 8.59 (s, 1H, N=CH) 8.56 (s, 1H, imidazopyridine-H), 7.66-7.63 (m, 5H, imidazopyridine-H and Ar-H), 7.42-7.37 (m, 1H, imidazopyridine-H-7), 7.05-7.00 (t, J = 6.0 Hz, 1H, imidazopyridine-H); ¹³CNMR (70 MHz, DMSO): d_C (ppm) 159.11 (CONH), 147.18, 144.42, 138.80, 134.24, 132.47, 132.31, 130.67, 129.37, 128.22, 127.21, 123.64, 117.73, 116.46, 113.90. MS (EI) m/z (%): 342 (M⁺, 14), 161 (100), 144 (77), 118 (99), 91 (33), 78 (34).

Synthesis of N´-(4-methylbenzylidene)imidazo[1,2-a]pyridine-2-carbohydrazide (7e)

Yield: 38%, Rf = 0.48 (CHCl3:MeOH 95:5), mp: 180-182^◦C, IR (KBr, cm ^-1 ) υ max: 3432 (Amide N-H), 1653 (Amide C=O)^{, 1}HNMR (300MHz, CDCl3): δ_H (ppm) 10.37 (s, 1H, CONH), 8.23 (s, 1H, imidazopyridine-H), 8.15 (s, 1H, N=CH), 8.12-8.09 (d, J = 9.0, 1H, imidazopyridine-H), 7.65-7.62 (d, J = 9.0 Hz, 2H, Ar-H), 7.53- 7.50 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.24-7.21 (t, overlap with CDCl3, 1H, imidazopyridine-H), 7.16-7.13 (d, J = 9.0 Hz, 2H, Ar-H), 6.84-6.80 (t, J = 6.0 Hz, 1H, imidazopyridine-H), 2.31 (s, 3H, CH₃);¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 158.11, 148.45, 144.15, 140.93, 138.06, 130.86, 129.41(2C), 127.86 (2C), 127.04, 126.72, 117.78, 115.55, 114.02, 21.58. MS (EI) m/z (%): 278 (M⁺, 25), 161 (84), 144 (71), 118 (100), 91 (21), 78 (43).

Synthesis of N´-(Prop-2-yn-1-yl) imidazo[1,2-a]pyridine-2-carbohydrazide (9)

A mixture of imidazo[1,2-a]pyridine-2-carbohydrazide (5 ,2 mmol), propargyl bromide (8, 2 mmol) and K₂CO₃ (2 mmol) in DMF was stirred at room temperature for 72 hours. The mixture was concentrated and extraction was performed three times with chloroform: water. The chloroform layer was separated, washed with saturated NaCl solution and dried over anhydrous Na₂SO₄and filtered then the solvent was removed under reduced pressure. The product was purified by column chromatography (ethylacetate: methanol 90:10). Yield: 22%, Rf = 0.85 (CHCl₃:MeOH 90:10), mp: 142-144 ^◦C. IR (KBr, cm ^-1 ) υ max = 3583 (NH), 3307 (amide N-H), 1711 (Amide C=O); ¹HNMR (300MHz, CDCl₃): δ_H (ppm) 8.77 (s, 1H, CONH), 8.10-8.08 (m, 2H, imidazopyridine-H), 7.53-7.50 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.23 (t, overlap with CDCl₃, 1H, imidazopyridine-H), 6.83-6.78 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 4.88 (s, 1H, CONHNH), 3.70 (s, 2H, CH₂), 2.23 (s, 1H, C CH ); MS (EI) m/z (%): 214 (M+, 14), 161 (100), 144 (99), 118 (80), 78 (79), 39 (27)[13, 14].

General procedure for synthesis of N´-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)imidazo [1,2a] pyridine-carbohydrazide derivatives (11a-e)

The mixture of benzyl bromide/ chloride reagent, NaN₃, triethylamine (2 drops), water (2 mL) and tertiary butanol (2 mL) was stirred and heated at 70 ºC for 1 hour. After completion, the formation of benzyl azide intermediate was confirmed by TLC (petroleum ether 100%), Then the relevant benzyl azide solution was added to a solution of N´-(Prop-2-yn-1-yl)imidazo[1,2-a]pyridine-2-carbohydrazide (9) in water (2 mL) and tertiary butanol (2 mL). The reaction mixture was stirred in the presence of catalytic amount of sodium ascorbate and CuSO₄.5H₂O (0.05 mmol) for a period of 4 to 7 days. After completion of the reaction, the mixture was extracted using water and chloroform mixture for three times. The combined organic layers were dried over saturated NaCl solution and Na₂SO₄. The product was purified by plate chromatography (ethylacetate: methanol, 90:10). At last the products were recrystallized using ethanol to get a pure compound [22].

Synthesis of N´-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-2-carbohydrazide (11a)

For this reaction, benzyl bromide (10a, 1.1 mmol) , NaN₃ (1 mmol) and compound 9 (0.8 mmol) were employed as starting materials. Yield: 21%, Rf = 0.24 (EtOAc:EtOH 90:10), mp: 150-152 ^◦C, IR (KBr, cm ^-1) υ max: 3584 (amine N-H), 3405 (amide N-H), 1711 (amide C=O).¹HNMR (300MHz, CDCl₃): δ_H (ppm) 8.31(s, 1H, CONH), 8.20 (s, 1H, imidazopyridine-H), 8.11 -8.08 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 8.03 (s, 1H, triazole-H), 7.67-7.60 (m, 1H, imidazopyridine-H), 7.54-7.51` (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.34-7.32 (m, 3H, Ar-H), 7.25-7.23 (m, 2H, Ar-H), 6.85-6.80 (t, J = 9.0 Hz, 2H, imidazopyridine-H), 5.49 (s, 2H, CH₂), 4.14 (s, 2H, CH₂), ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 161.06 , 144.48, 143.26 , 138.76,134.62, 129.00 (2C), 128.58, 128.01(2C),126.27, 125.93,123.63, 118.43, 114.51, 113.43, 54.12 , 51.39. MS (EI) m/z (%): 345 (M+, 24), 162 (57), 145 (86), 118 (100), 91 (57).

Synthesis of N´-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-carbohydrazide (11b)

For this reaction , 4-fluorobenzyl chloride (10b,1.65 mmol), NaN₃ (1.5 mmol) and compound 9 (1.2 mmol) were employed .Yield: 17%, Rf = 0.20 (EtOAc:EtOH 90:10), mp: 205-207 ^◦C, IR (KBr, cm ^-1) υ max: 3584 (amine N-H), 3306 (amide N-H), 1710 (amide C=O), ¹HNMR (300MHz, CDCl₃): δ_H (ppm) 8.47(s, 1H, CONH), 8.06-8.04 (d, J = 6.0 Hz, 1H, imidazopyridine-H-5), 8.00 (s, 1H, imidazopyridine-H), 7.62 (s, 1H, triazole-H), 7.50-7.47 (d, J = 9.0 Hz, 1H, imidazopyridine-H), 7.20-7.15 (t, overlap with CDCl₃, 1H, imidazopyridine-H), 7.13-7.08 (m, 2H, Ar-H), 6.90-6.84 (t, J = 9.0 Hz, 2H, Ar-H), 6.81-6.76 (t, J = 9.0 Hz, 1H, imidazopyridine-H) 5.37 (s, 2H, CH₂), 4.21 (s, 2H, CH₂); ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 160.85, 144.50, 144.13, 138.57, 130.61, 130.57, 129.88, 129.77,126.34, 126.12,123.02, 118.35, 116.04, 115.75, 114.69, 113.55, 53.33, 51.89. MS (EI) m/z (%): 365 (M+, 2), 258 (60), 213 (34), 191 (9), 162 (23), 145 (86), 118 (43). 109 (100), 90 (16).

Synthesis of N´-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-carbohydrazide (11c)

For this reaction, 4-chlorobenzyl chloride (10c, 1.1 mmol) NaN₃ (1 mmol), and compound 9 (0.8 mmol) were used. Yield: 23%, Rf = 0.22 (EtOAc:EtOH 90:10), mp: 193-195 ^◦C. ¹HNMR (300MHz, CDCl₃): δ_H (ppm) 8.44 (s, 1H, CONH), 8.06-8.04 (m, 2H, imidazopyridine-H), 7.97 (s, 1H, imidazopyridine-H), 7.63 (s, 1H, triazole-H), 7.50-7.47 (d, J = 9.0 Hz, 2H, Ar-H), 7.19-7.13 (t, overlap with CDCl3, 1H, imidazopyridine-H), 7.05-7.02 (d, J = 9.0 Hz, 2H, Ar-H), 6.80-6.75 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 5.36 (s, 2H, CH₂), 4.21 (s, 2H, CH₂), ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 160.57, 144.05, 143.56, 134.54, 133.21 (2C), 129.33 (2C), 129.17 (2C), 126.56, 123.55(2C), 117.98, 114.63, 113.84, 53.35, 51.78. MS (EI) m/z (%): 379 (M⁺, 21), 161 (42), 145 (71), 125 (46), 91 (21), 78 (36).

Synthesis of N´-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-carbohydrazide (11d)

For this reaction, 4-bromobenzyl bromide (10d ,1.65 mmol), NaN₃ (1.5 mmol), and compound 9 (1.2 mmol) were used.Yield: 17%, Rf = 0.32 (EtOAc:EtOH 90:10), mp: 186-188 ^◦C, ¹HNMR (300MHz, CDCl₃): δ_H (ppm) 8.45 (s, 1H, CONH), 8.07-8.05 (d, J = 6.0 Hz, 1H, imidazopyridine-H), 7.99 (s, 1H, imidazopyridine-H), 7.63 (s, 1H, triazole-H), 7.50-7.47 (d, J = 9.0 Hz, 2H, imidazopyridine-H), 7.31-7.28 (d, J = 9.0 Hz, 2H, Ar-H), 6.98-6.95 (d, J = 9.0 Hz, 2H, Ar-H), 6.81 -6.76 (t, J = 9.0 Hz, 1H, imidazopyridine-H), 5.35 (s, 2H, CH₂), 4.21 (s, 2H, CH₂); ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 160.71 (CONH), 144.03, 143.56, 137.78, 133.83, 133.77, 132.15, 132.08, 129.68, 129.62, 126.76, 123.76, 122.71, 117.86, 114.88, 113.91, 53.41, 51.86. MS (EI) m/z (%): 425 (M⁺, 3), 320 (35), 251 (10), 213 (45), 169 (61), 145 (100), 118 (52), 90 (37).

Synthesis of N´-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)imidazo[1,2-a]pyridine-carbohydrazide (11e)

For this reaction, 4-methylbenzyl chloride 10e (4.125 mmol, MW: 140.6 g/mol) was added to NaN₃ (3.75 mmol), and in the second part, the amount of compound 9 used was 3 mmol.Yield: 24%, Rf = 0.28 (EtOAc:EtOH 90:10), mp: 140-142 ^◦C, IR (KBr, cm ^-1 ) υ max: 3682 (Amine N-H), 3306 (Amide N-H), 3018 (Ar C-H), 1710 (Amide C=O), ¹HNMR (300MHz, CDCl3): δ_H (ppm) 8.51(s, 1H, CONH), 8.04 (s, 3H, Ar-H), 7.59-7.48 (brm, 2H, Ar-H), 7.13-7.02 (brm, overlap with CDCl3, 2H, Ar-H), 6.78 (s, 2H, Ar-H), 5.64 (m, 1H, Ar-H), 5.39-5.35 (brd, 2H, CH₂), 4.20 (s, 2H, CH₂), 2.23 (s, 3H, CH₃); ¹³CNMR (70 MHz, CDCl₃): d_C (ppm) 160.84, 144.50, 143.84, 138.59, 134.72, 131.61, 129.62, 128.95, 128.57, 128.03, 126.38, 126.09, 123.19, 118.36, 114.77, 113.54, 53.94, 51.78, 46.61. MS (EI) m/z (%): 361 (M⁺, 6), 347 (9), 213 (27), 162 (89), 145 (100), 1 18 (64), 105 (45), 91 (60).

Biological activity

Cytotoxicity assay

Assessment of cell viability after exposure of cancer cells to synthetic compounds was performed using MTT reduction assay. Cells were plated in 96-well microplates at densities of 30,000 cells/mL (100 μL in each well). The plates were incubated overnight at 37 °C and then 50 μL of the growth medium was replaced with the same amount of fresh growth medium containing 3-4 different concentrations of synthesized analogues at the final concentrations of 10-100 µM. Test compounds were initially dissolved in DMSO and then diluted in growth medium. The upper limit of DMSO concentration in each well was 0.5%. Fresh culture medium was added to the wells, followed by incubation for 72 hours. Afterwards, 80 μL of the solution in each well was substituted with the identical quantity of growth the medium comprising 0.5 mg/mL of MTT. Cells were subsequently incubated at 37°C for 4 hours and then 80 μL of the solution in each well was deleted. Introduction of DMSO (200 μL) to each well caused the formazan crystals formed inside the viable cells to dissolve. The process lasted 1.5 hours (1 hour incubation and 30 minute shaking). The optical absorbance of the final solution was quantified at the wavelength of 570 nm using a microplate reader. IC₅₀ values for all derivatives were calculated by the means of CurveExpert 1.34 software. (35) To prove the reliability of the results, we repeated each experiment 3 to 5 times [22, 23].

Cell Cycle Analysis:

The analysis of cells in different phases of the cell cycle and the sub-G1 phase were monitored using propidium iodide (PI)-based assessment of cell cycle by flow cytometry. MCF-7 cells were seeded in 12-well plates (1 × 10⁵ cells/well) and after being incubated overnight to allow cell attachment, were treated with different concentrations of 7d (20, 50, and 100 nM) for 48 h. At the end of the incubation, the cells in each well were collected and washed with PBS. Then, they were fixed with 70% ethanol overnight at -20 °C. After 24 h, the fixed cells were washed with PBS and subsequently stained with DNA staining solution (PI 20 μg/mL and RNase 200 μg/mL) at room temperature for 30 min in the dark. Ten thousand cells of each sample were analyzed using a FACS Calibur flow cytometer (BD Biosciences) and the percentage of the cells in sub-G1, G0/G1, S, and G2/M phases were calculated using CellQuest (BD, USA) software [24].

Ethical approval and consent to participate

Not applicable.

Consent for publication:

Not applicable.
Availability of data and materials

All data generated or analyzed during this study are included in its supplementary information files.

Competing interest:

The authors declare that they have no competing interests.

Funding:

This work was supported by a grant from Shiraz University of Medical Sciences (Grant numbers 13298 and 7426).

Author contributions

M.F. Synthesized the compounds and perpetrated of first draft of the manuscript, Z.H. perpetrated In silico studies and wrote the manuscript, M.E. Synthesized the compounds, M.M. Carried out the Cytotoxicity test, M.H.J. Synthesized the compounds, R.M., O.F., M.K. and N.E. Supervision of the research project, Investigation and designing the compounds and biological experiments. All authors read and approved the final manuscript.

Acknowledgements

This study was the part of the PharmD thesis of Maryam Firouzi.

Author Information

¹Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. ²Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021, 71(3):209-249.
Goel R, Luxami V, Paul K: Imidazo [1, 2-a] pyridines: Promising drug candidate for antitumor therapy. Curr Top Med Chem. 2016, 16(30):3590-3616.
Jose G, Kumara TS, Nagendrappa G, Sowmya H, Sriram D, Yogeeswari P, Sridevi JP, Row TNG, Hosamani AA, Ganapathy PS: Synthesis, molecular docking and anti-mycobacterial evaluation of new imidazo [1, 2-a] pyridine-2-carboxamide derivatives. Eur J Med Chem. 2015, 89:616-627.
Geronikaki A, Babaev E, Dearden J, Dehaen W, Filimonov D, Galaeva I, Krajneva V, Lagunin A, Macaev F, Molodavkin G: Design, synthesis, computational and biological evaluation of new anxiolytics. Bioorg. Med. Chem. 2004, 12(24):6559-6568.
Asif M: An Overview of Various Heterocyclic Imidazopyridine, Triazolopyridine and Quinoline Derivatives and Their Biological Significances. Mor J Chem. 2017, 5(2):317-324.
Dahan-Farkas N, Langley C, Rousseau AL, Yadav DB, Davids H, de Koning CB: 6-Substituted imidazo [1, 2-a] pyridines: Synthesis and biological activity against colon cancer cell lines HT-29 and Caco-2. Eur J Med Chem. 2011, 46(9):4573-4583.
Farag AM, Dawood KM, Kandeel ZE: Facile synthesis of novel polysubstituted thiopene and 1, 3,4-thiadiazole derivatives. Tetrahedron 1997, 53(1):161-166.
Kamal A, Rao AS, Vishnuvardhan M, Reddy TS, Swapna K, Bagul C, Reddy NS, Srinivasulu V: Synthesis of 2-anilinopyridyl–triazole conjugates as antimitotic agents. Org Biomol Chem. 2015, 13(17):4879-4895.
Reddy TS, Kulhari H, Reddy VG, Rao AS, Bansal V, Kamal A, Shukla R: Synthesis and biological evaluation of pyrazolo–triazole hybrids as cytotoxic and apoptosis inducing agents. Org Biomol Chem. 2015, 13(40) 10136-10149.
Nagavelli VR, Nukala SK, Narsimha S, Battula KS, Tangeda SJ, Reddy YN: Synthesis, characterization and biological evaluation of 7-substituted-4-((1-aryl-1H-1, 2, 3-triazol-4-yl) methyl)-2H-benzo [b][1, 4] oxazin-3 (4H)-ones as anticancer agents. Med Chem Res. 2016, 25(9):1781-1793.
Singh P, Raj R, Kumar V, Mahajan MP, Bedi P, Kaur T, Saxena A: 1, 2, 3-Triazole tethered β-lactam-chalcone bifunctional hybrids: synthesis and anticancer evaluation. European journal of medicinal chemistry 2012, 47:594-600.
Gregorić T, Sedić M, Grbčić P, Paravić AT, Pavelić SK, Cetina M, Vianello R, Raić-Malić S: Novel pyrimidine-2, 4-dione–1, 2, 3-triazole and furo [2, 3-d] pyrimidine-2-one–1, 2, 3-triazole hybrids as potential anti-cancer agents: Synthesis, computational and X-ray analysis and biological evaluation. Eur J Med Chem. 2017, 125:1247-1267.
Khazir J, Hyder I, Gayatri JL, Yandrati LP, Nalla N, Chasoo G, Mahajan A, Saxena A, Alam M, Qazi G: Design and synthesis of novel 1, 2, 3-triazole derivatives of coronopilin as anti-cancer compounds. Eur J Med Chem. 2014, 82:255-262.
Kumar A, Ahmad I, Chhikara BS, Tiwari R, Mandal D, Parang K: Synthesis of 3-phenylpyrazolopyrimidine-1, 2, 3-triazole conjugates and evaluation of their Src kinase inhibitory and anticancer activities. Bioorg. Med. Chem Lett. 2011, 21(5):1342-1346.
Kamal A, Reddy NS, Nayak VL, Bolla NR, Rao AS, Prasad B: Synthesis and evaluation of N-((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl) nicotinamides as potential anticancer agents that inhibit tubulin polymerization. Bioorg. Med. Chem. 2014, 22(13):3465-3477.
Lal K, Yadav P: Recent advancements in 1, 4-disubstituted 1H-1, 2, 3-triazoles as potential anticancer agents. Anti-Cancer Agents Med. Chem. 2018, 18(1):21-37.
Akbarzadeh T, Noushini S, Taban S, Mahdavi M, Khoshneviszadeh M, Saeedi M, Emami S, Eghtedari M, Sarrafi Y, Khoshneviszadeh M: Synthesis and cytotoxi ativity of novel poly-substituted imidazo [2, 1-c][1, 2, 4] triazin-6-amines. Mol Divers. 2015, 19(2):273-281.
Ranjbar S, Edraki N, Khoshneviszadeh M, Foroumadi A, Miri R, Khoshneviszadeh M: Design, synthesis, cytotoxicity evaluation and docking studies of 1, 2, 4-triazine derivatives bearing different arylidene-hydrazinyl moieties as potential mTOR inhibitors. Res Pharm Sci. 2018, 13(1):1.
Edraki N, Jamei MH, Haghighijoo Z, Kayani Z, Raufi E, Eskandari M, Firouzi M, Sadeghpour H, Miri R, Khoshneviszadeh M: Phenanthrotriazine Derivatives Containing Arylidine Hydrazone Moieties as Novel Potential c-Met Inhibitors with Anticancer Effect. Iran. J Pharm Sci. 2021, 20(3):516.
Damghani T, Hadaegh S, Pirhadi S, Sabet R, Khoshneviszadeh M, Edraki N: Design, synthesis, in vitro evaluation and molecular docking study of N'-Arylidene imidazo [1, 2-a] pyridine-2-carbohydrazide derivatives as novel tyrosinase inhibitors. J Mol Struct. 2020, 1222:128876.
Özdemir A, Turan-Zitouni G, Kaplancıklı ZA, İşcan G, Khan S, Demirci F: Synthesis and the selective antifungal activity of 5, 6, 7, 8-tetrahydroimidazo [1, 2-a] pyridine derivatives. Eur J Med Chem. 2010, 45(5):2080-2084.
Azizmohammadi M, Khoobi M, Ramazani A, Emami S, Zarrin A, Firuzi O, Miri R, Shafiee A: 2H-chromene derivatives bearing thiazolidine-2, 4-dione, rhodanine or hydantoin moieties as potential anticancer agents. Eur J Med Chem. 2013, 59:15-22.
van Meerloo J, Kaspers GJ, Cloos J: Cell sensitivity assays: the MTT assay. In: Cancer cell culture. Springer; 2011: 237-245.
Menezes JC, Edraki N, Kamat SP, Khoshneviszadeh M, Kayani Z, Mirzaei HH, Miri R, Erfani N, Nejati M, Cavaleiro JA: Long chain alkyl esters of hydroxycinnamic acids as promising anticancer agents: selective induction of apoptosis in cancer cells. J Agric Food Chem. 2017, 65(33):7228-7239.

Table 1 is available in the Supplementary Files section.

No competing interests reported.

Download PDF

Journal Publication

published 06 Jan, 2024

Read the published version in BMC Chemistry →

Editorial decision: Major revision
07 Feb, 2023
Reviews received at journal
27 Jan, 2023
Reviewers agreed at journal
30 Nov, 2022
Reviewers agreed at journal
26 Sep, 2022
Reviews received at journal
15 Sep, 2022
Reviewers agreed at journal
05 Sep, 2022
Reviewers invited by journal
01 Sep, 2022
Editor assigned by journal
31 Aug, 2022
Editor invited by journal
31 Aug, 2022
Submission checks completed at journal
31 Aug, 2022
First submitted to journal
07 Jul, 2022

You are reading this latest preprint version

Synthesis and Cytotoxic Activity Evaluation of Novel Imidazopyridine Carbohydrazide Derivatives

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results And Discussion

3.1. Chemistry

3.2 Biological activity

Conclusion

Experimental Methods

Declarations

References

Table 1

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1