Design, synthesis, in silico, anticancer evaluations of N-acylhydrazone derivatives as STAT3 inhibitors

ABSTRACT

Aim

N-acylhydrazone derivatives were synthesized, evaluated in-vitro and further subjected to cell cycle analysis, cell apoptosis, western bloting, and in-silico analysis targeting STAT3.

Materials and methods

A series of N-acylhydrazones (5a-5l) were synthesized and evaluated against MIA PaCa-2 cancer cells. 5 h and 5 l were further evaluated against PANC-1, MCF-7 and hTERT-HPNE cells. Additionally, cell cycle analysis, apoptosis, western blotting, docking, in-silico ADMET, molecular dynamics and MM-GBSA were done on 5l.

Results and conclusion

5 l and 5 h suppressed the growth of MIA PaCa-2 cancer cells and were very less cytotoxic to hTERT-HPNE cells. Additionally, the anticancer efficacy was confirmed against PANC-1 and MCF-7 for 5l and 5h. Further, 5l treatment induced G1/S arrest marked decrease in G2/M and displayed a significant increase in the apoptosis of MIA PaCa-2 cells. 5 l treatment significantly inhibited the phosphorylated level STAT3 and JAK1. In-silico studies confirm the binding affinity and stability and further synthetic modifications and biological investigations can be done to explore the medicinal potential of the derivatives in future.

GRAPHICAL ABSTRACT

1. Introduction

Cancer is a complex and lethal group of disease reported with alarming rate of incidences and mortalities all around the globe [1]. The multi-factorial interlinked ailments causing human malignancies are largely associated with overexpressed or mutationally modified kinases. Among all cancers, the mortality ratio is consistently high for pancreatic and breast cancer, demonstrating its poor diagnosis and treatment options. For the past 5 years as per global cancer data, more than 90% of cases with pancreatic carcinoma have less than 8 months survival. STAT3 and its downstream signaling cascades have been hyperactivated in about 70% of carcinomas including solid tumors and hematological malignancies [2,3]. Over-expressed STAT3 in cancer cells stimulates the progressive and continued upregulated transcription of growth factors and anti-apoptosis that imparts a fundamental role toward maintaining cell growth and survival. Moreover, STAT3 confers tumorigenesis by promoting tumor invasion, metastasis, and angiogenesis [3,4].

A number of drugs have been developed as target-based anticancer compounds for different kinases, but the unavailability of STAT3 targeted anticancer drugs, as well as the frequent association of deregulated STAT3 mediated signaling in different cancers notably necessitates developing STAT3 inhibitors as anticancer drugs [5,6]. STAT3 protein is characterized with six domains as the amino terminal domain, coiled coil domain, DNA binding domain, linker domain, SH2 and the carboxyl terminal transactivation domain [7,8].

Various STAT3 inhibitors under development (clinical trials) include WP1066, TTI-101, Napabucasin (BBI-608), Silibrin, SH-4–54, LYW-6, S3I–201, and Stattic. Derivatives of N-acylhydrazones has been the important and commons functional groups in medicinal chemistry. A number of potential acylhydrazone derivatives have been synthesized with significant anticancer activity using doxorubicin, cisplatin, colchicine and related compounds as reference drugs. (E)-N’-((6-(tert-butyl)-4-oxo-4 H-chromen-3-yl)methylene)nicotinohydrazide (Erasin) induced apoptosis in erlotinib-resistant neoplasm as a result of selective STAT3 inhibition. (E,E)-3-(5-chloro-2-nitrophenyl)-1-(2-chlorophenyl)-5-(2-((2-methylquinolin-8-yl)oxy) acety l)formazan (BT-1F) specifically induced apoptosis by inhibiting the STAT3 signal. The azo-hydrazone analog attenuated the solid tumor growth without inducing significant toxicity. Erasin and BT-1F comprising N-acylhdrazone moiety in common (Figure 1) are reported with STAT3 inhibition as anticancer molecules [9,10]. Thus, a series of NAH derivatives as small molecule inhibitors were synthesized and the anticancer evaluation was done, further targeting STAT3.

Figure 1.

Chemical structure of the compounds as STAT3 inhibitors (S31–201, napabucasin, WP-1066, TTI-101, SH-4–54, Stattic, Erasin and BT-1F).

2. Material and methods

2.1. Instruments and materials

All the chemicals were purchased from E. Merck, Sigma-Aldrich, Loba Chemie, TCI, and SD Fine Chemicals. ¹H NMR and ¹³C NMR analyses were carried out on a Bruker Neo Avance 500 spectrometer using tetramethylsilane (TMS) as an internal standard and DMSO-d₆ as a solvent. Mass spectra were obtained on an Agilent UHD-6450 mass spectrometer. IR spectra were obtained using Agilent Technologies FTIR spectrometer. Melting points were recorded in the Systonic S-9750 Microprocessor Melting Point Apparatus and are uncorrected. Thin-layer chromatography was performed on pre-coated silica gel plates and column chromatography was performed using silica gel 60 − 120 mesh.

The MIA PaCa-2 and PANC-1 Pancreatic carcinoma cells and MCF-7 breast carcinoma cells were procured from the American Type Culture Collection. All three cell lines were grown in ATCC-recommended medium, Dulbecco’s Modified Eagle Medium at 37°C under humidified conditions with 5% CO₂.

2.2. Experimental methods

2.2.1. Procedure for the synthesis of N-acylhydrazone derivatives (5a–5l)

Benzoyl chloride (0.2 g, 2.64 mmol) was added to the saturated solution of 2-aminoacetic acid (0.97 mL, 2.64 mmol) in potassium carbonate. The reaction mixture was acidified using hydrochloric acid after vigorous shaking followed by cooling in the ice bath [11]. The reaction progress was monitored using TLC. The solid crystals obtained were washed and recrystallized to get 2-benzamidoacetic acid (2). Excess methanol was added to benzamidoacetic acid (0.5 g, 2.8 mmol) and the reaction mixture was further refluxed for 4–6 h. Sulphuric acid was added drop-wise (constant stirring) to get the desired product [12]. The reaction progress was monitored using TLC. The solid product obtained after cooling was recrystallized to get methyl-2-benzamidoacetate (3).

Methyl-2-benzamidoacetate (1.93 g, 0.01 mol) was added to hydrazine hydrate (1.0 g, 0.02 mol) and was refluxed for 6 h using ethanol. Reaction progress was monitored using TLC. Excess ethanol was distilled off after the reaction, and the contents were poured into ice-cold water. The product obtained was filtered dried and recrystallized to get 2-benzamidoacetohydrazide (4). 2-Benzamidoacetohydrazide (4) (0.1 mol) was treated with different aldehydes (a-l) (0.1 mol), and the reaction mixture was refluxed for 4–6 h using ethanol. Glacial acetic acid was added in catalytic concentration. The reaction progress was monitored using TLC. After completion of the reaction, the reaction mixture was cooled and poured into ice-cold water. The product obtained was filtered and dried [13]. The dried product was purified using column chromatography (n-hexane:ethyl acetate) to get N-acylhydrazone derivatives (5a-5l). In most cases, NMR data revealed the presence of conformer mixtures that were not distinguishable by TLC, making it difficult to isolate the individual conformers. The characterization data is provided as supplementary material. Therefore, we proceeded with the biological evaluation using the isolated and purified compounds.

2.2.1.1. N-(2-oxo-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)ethyl)benzamide (5a)

Mol. formula: C₁₉H₂₁N₃O₅, yield: 50%, Off-white solid, m.p. 200–202°C, IR(v_max) 3383 (NH str.), 1700 (CO str.), 1652 (CN str.), 1238 (CO str. O-CH₃) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.52–11.48 (d, J = 20.3 Hz, 1H), 8.89–8.69 (dt, J = 11.5, 5.9 Hz, 1H), 8.17–7.90 (m, 3H), 7.57–7.48 (dt, J = 14.5, 7.2 Hz, 3H), 7.02–7.01 (d, J = 6.3 Hz, 2H), 4.44–3.98 (d, J = 5.9 Hz, 2H), 3.84–3.83 (d, J = 3.9 Hz, 6H), 3.70 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.2, 166.5, 166.5, 165.4, 153.1, 146.6, 143.1, 139.1, 138.9, 134.1, 133.8, 131.3, 131.2, 129.7, 129.5, 128.2, 127.2, 127.1, 104.2, 103.9, 60.0, 55.3, 55.8, 41.9, 40.7, 40.0, 39.9, 39.8, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₉H₂₂N₃O₅ 372.1554; found 372.1555.

2.2.1.2. N-(2-oxo-2-(2-(2,4,5-trimethoxybenzylidene)hydrazinyl)ethyl)benzamide (5b)

Mol. formula: C₁₉,H₂₁N₃O₅, yield: 67%, Off-white solid, m.p. 218–220°C, IR (v_max) 3391 (NH str.), 1968 (CO str.), 1586 (CN str.), 1212 (CO str. O-CH₃) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.38–11.32 (d, J = 9.0, 11.32 Hz, 1H), 8.85–8.67 (dt, J = 10.0, 10.0 Hz, 1H), 8.52–8.28 (d, J = 7.1, 6.45 Hz, 1H), 7.92–7.90 (d, J = 8.2 Hz, 2H), 7.55–7.47 (m, 3H), 7.36–7.31 (d, J = 7.09, 3.6 Hz, 1H), 6.73 (s, 1H), 4.44–4.43 (d, J = 5.9 Hz, 1H), 3.98–3.97 (d, J = 5.8 Hz, 1H), 3.84 (s, 6H), 3.75–3.73 (d, J = 12.9 Hz, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ 169.9, 166.6, 166.5, 165.1, 153.2, 153.0, 151.9, 151.8, 143.2, 142.4, 139.0, 134.1, 133.9, 131.3, 131.2, 128.2, 128.2, 127.3, 127.2, 113.4, 107.8, 107.7, 97.8, 97.8, 56.4, 56.4, 55.9, 55.8, 55.7, 55.7, 42.0, 40.8, 39.9, 39.9, 39.8, 39.7, 39.6, 39.5, 39.4, 39.3, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₉H₂₂N₃O₅ 372.1554; found 372.1556.

2.2.1.3. N-(2-(2-(4-Nitrobenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5c)

Mol. formula: C₁₆H₁₄N₄O₄, yield: 86%, Off-white solid, m.p. 223–225°C, IR (v_max) 3346 (NH str.), 1979 (CO str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.84–11.79 (d, J = 25.4 Hz, 1H), 8.92–9.73 (dt, J = 5.92, 5.9 Hz, 1H), 8.35–8.28 (m, 2H), 8.12–7.90 (m, 5H), 7.58–7.49 (dt, J = 31.9, 7.1 Hz, 3H), 4.48–4.47, 4.04–4.03 (dd, J = 5.9, 5.25 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.7, 166.7, 166.7, 166.2, 147.9, 147.7, 144.3, 141.1, 140.6, 140.4, 134.0, 133.7, 131.5, 131.4, 128.4, 127.9, 127.7, 127.3, 127.2, 124.0, 42.0, 40.8, 39.9, 39.8, 39.7, 39.6, 39.5, 39.5, 39.4, 39.3, 39.2, 39.09 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₆H₁₅N₄O₄ 327.1088; found 327.1083.

2.2.1.4. N-(2-(2-(3-Nitrobenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5d)

Mol. formula: C₁₆H₁₄N₄O₄, yield:94%, Off-white solid, m.p. 219–221°C, IR(v_max) 3342 (NH str.), 1808 (CO str.), 1521 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.79–11.75 (d, J = 22.2 Hz, 1H), 8.93–8.73 (dt, J = 6.5, 6.2 Hz, 1H), 8.53–8.50 (d, J = 14.1 Hz, 1H), 8.37–8.24 (m, 1H), 8.18–8.13 (m, 2H), 7.93–7.92 (d, J = 7.1 Hz, 2H), 7.76–7.72 (t, J = 8.0 Hz, 1H), 7.58–7.49 (dt, J = 7.6, 7.2 Hz, 3H), 4.49–4.48, 4.05–4.04 (dd, J = 5.72, 5.45 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.5, 166.6, 166.5, 166.0, 148.2, 148.1, 144.2, 141.1, 136.1, 135.9, 133.9, 133.7, 133.1, 132.7, 131.4, 131.3, 130.3, 128.3, 127.3, 127.1, 124.1, 123.9, 120.9, 120.9, 41.9, 40.7, 39.7, 39.6, 39.4, 39.2, 39.0; HRMS m/z: (M+H)⁺ calcd for C₁₆H₁₅N₄O₄ 327.1088; found 327.1090.

2.2.1.5. N-(2-(2-(2-Nitrobenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5e)

Mol. formula:C₁₆H₁₄N₄O₄, yield: 46%, Off-white solid, m.p. 154–156°C, IR(v_max) 3372 (NH str.), 1972 (CO str.), 1722 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.86–11.78 (d, J = 12.3, 9.02 Hz, 1H), 8.90–8.70 (dt, J = 5.9, 5.8 Hz, 1H), 8.64–8.37 (d, J = 5.3, 9.05 Hz, 1H), 8.10–8.03 (m, 2H), 7.91–7.90 (d, J = 7.0 Hz, 2H), 7.81–7.77 (q, J = 7.1 Hz, 1H), 7.68–7.64 (m, 1H), 7.57–7.48 (m, 3H), 4.42–4.40, 4.02–4.01 (dd, J = 5.9, 5.72 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.5, 166.5, 166.5, 166.0, 148.0, 147.9, 142.0, 138.7, 134.0, 133.7, 133.7, 133.4, 131.3, 131.2, 130.5, 130.4, 128.7, 128.3, 128.2, 128.0, 127.9, 127.2, 127.2, 127.1, 124.5, 124.4, 48.5, 41.9, 40.6, 40.0, 39.9, 39.9, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺calcd for C₁₆H₁₅N₄O₄ 327.1088; found 327.1086.

2.2.1.6. N-(2-(2-((1 H-Indol-3-yl)methylene)hydrazinyl)-2-oxoethyl)benzamide (5f)

Mol. formula: C₁₈,H₁₆N₄O₂, yield: 83%, Off-white solid, m.p. 224–226°C, IR(v_max) 3250 (NH str.), 1611 (CO str.), 1540 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 11.21–11.17 (d, J = 19.6 Hz, 1H), 8.87–8.69 (dt, J = 79.1, 5.9 Hz, 1H), 8.42–8.15 (m, 2H), 7.94–7.79 (m, 3H), 7.58–7.43 (m, 4H), 7.23–7.11 (m, 2H), 4.51–4.50, 4.01–4.0 (dd, J = 6.24, 6.12 Hz, 2H);¹³C NMR (126 MHz, DMSO-d₆) δ 169.4, 166.6, 166.5, 164.7, 143.8, 140.8, 137.0, 136.9, 134.1, 133.9, 131.3, 131.2, 130.3, 130.1, 128.3, 128.2, 127.3, 127.2, 124.2, 124.0, 122.6, 122.5, 121.8, 121.4, 120.5, 120.3, 111.8, 111.7, 111.5, 111.3, 42.0, 40.7, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₈H₁₇N₄O₂ 321.1346; found 321.1347.

2.2.1.7. N-(2-(2-(4-Hydroxybenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5g)

Mol. formula: C₁₆H₁₅N₃O₃, yield: 41%, Off-white solid, m.p. 221–223°C; ¹H NMR (500 MHz, DMSO-d₆) δ 11.30–11.29 (d, J = 5.9 Hz, 1H), 9.88 (s, 1H), 8.85–8.64 (dt, J = 6.0, 5.8 Hz, 1H), 8.13–7.90 (m, 3H), 7.57–7.48 (dq, J = 22.7, 7.1 Hz, 5H), 6.84–6.82 (d, J = 8.8 Hz, 2H), 4.40–4.39, 3.97–3.96 (dd, J = 5.77, 5.6 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 169.8, 166.4, 166.4, 165.1, 159.2, 159.1, 146.8, 143.6, 134.0, 133.8, 131.3, 131.2, 128.6, 128.4, 128.2, 127.2, 127.1, 125.1, 125.0, 115.6, 115.5, 41.8, 40.6, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M-H)⁺ calcd for C₁₆H₁₆N₃O₃ 298.1186; found 296.9455.

2.2.1.8. N-(2-(2-(4-Chlorobenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5h)

Mol. formula: C₁₆,H₁₄N₃O₂Cl, yield: 78%, Off-white solid, m.p. 207–209°C, IR (v_max) 3342 (NH str.), 1689 (CO str.), 1637 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.60–11.55 (d, J = 21.7 Hz, 1H), 8.89–8.69 (dt, J = 5.9, 5.45 Hz, 1H), 8.23–8.01 (d, J = 6.6, 4.43 Hz, 1H), 7.92–7.90 (d, J = 7.0 Hz, 2H), 7.75–7.72 (m, 2H), 7.58–7.49 (m, 5H), 4.44–4.43, 4.01–4.0 (dd, J = 6.09, 5.6 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.4, 166.7, 166.7, 165.8, 145.4, 142.2, 134.5, 134.3, 134.0, 133.8, 133.2, 133.0, 131.5, 131.4, 128.9, 128.7, 128.4, 128.4, 127.3, 127.2, 42.0, 40.8, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₆H₁₅N₃O₂Cl 316.0848; found 316.0848.

2.2.1.9. (2-oxo-2-(2-((1E,2E)-3-phenylallylidene)hydrazinyl)ethyl)benzamide (5i)

Mol. formula: C₁₈,H₁₇N₃O₂, yield: 77%, Off-white solid, m.p. 191–193°C, IR (v_max) 3339 (NH str.), 1689 (CO str.), 1644 (CN str.), 1518 (C = C str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.42 (s, 1H), 8.86–8.64 (dt, J = 5.7, 5.4 Hz, 1H), 8.03–7.84 (dd, J = 7.2, 5.9 Hz, 3H), 7.62–7.31 (m, 8H), 7.07–6.96 (m, 2H), 4.35–4.34, 4–3.99 (dd, J = 5.9, 5.8 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 169.9, 166.4, 166.4, 165.4, 148.5, 145.9, 138.7, 138.6, 135.8, 133.9, 133.7, 131.3, 131.2, 128.7, 128.7, 128.2, 127.2, 127.1, 126.9, 125.4, 125.0, 41.7, 40.5, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.2, 39.1, 39.0, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₈H₁₈N₃O₂ 308.1394; found 308.1396.

2.2.1.10. N-(2-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)-2-oxoethyl)benzamide (5j)

Mol. formula: C₁₈H₂₀N₄O₂, yield:  81%, Off-white solid, m.p. 201–203°C, IR(v_max) − 3312 (NH str.), 1678 (CO str.), 1555 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.21–11.19 (d, J = 7.4 Hz, 1H), 8.84–8.63 (dt, J = 6.12, 5.08 Hz, 1H), 8.09–7.88 (m, 3H), 7.57–7.48 (m, 5H), 6.75–6.73 (d, J = 9.0 Hz, 2H), 4.40–4.39, 3.97–3.96 (dd, J = 216.7, 5.9 Hz, 2H), 2.96 (s, 6H); ¹³C NMR (126 MHz, DMSO-d₆) δ 169.6, 166.5, 166.4, 164.9, 151.4, 151.3, 147.4, 144.2, 134.1, 133.8, 131.3, 131.2, 128.3, 128.2, 128.0, 127.3, 127.2, 121.4, 121.4, 111.7, 111.7, 41.9, 40.6, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₈H₂₁N₄O₂ 325.1659; found 325.1663.

2.2.1.11. N-(2-(2-(4-Methoxybenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5k)

Mol. formula:C₁₇H₁₇N₃O₃, yield:  71%, Off-white solid, m.p.145–147°C, IR(v_max) 3342 (NH str.), 1666 (CO str.), 1540 (CN str.), 1246 (CO str. O-CH₃) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.38 (s, 1H), 8.87–8.66 (dt, J = 5.94, 5.31 Hz, 1H), 8.19–7.91 (m, 3H), 7.66–7.48 (m, 5H), 7.02–7.0 (m, 2H), 4.43–4.42, 3.99–3.98 (dd, J = 217.2, 5.9 Hz, 2H), 3.80 (s, 3H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.0, 166.5, 166.5, 165.3, 160.7, 160.6, 146.5, 143.2, 134.0, 133.8, 131.3, 131.2, 128.5, 128.3, 128.2, 127.3, 127.2, 126.7, 126.6, 114.2, 114.2, 55.2, 41.9, 40.7, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.5, 39.4, 39.3, 39.2, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺ calcd for C₁₇H₁₈N₃O₃ 312.1343; found 312.1343.

2.2.1.12. N-(2-(2-(2-Hydroxybenzylidene)hydrazinyl)-2-oxoethyl)benzamide (5l)

Mol. formula:C₁₆,H₁₅N₃O₃, yield: 80%, Off-white solid, m.p. 229–231°C, IR (v_max) 3370 (NH str.), 1633 (CO str.), 1566 (CN str.) cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 11.76–10.09 (m, 2H), 8.92–6.87 (m, 11H), 4.43–4.42, 4.04–4.03 (dd, J = 5.93, 5.54 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 169.9, 166.6, 166.5, 165.5, 157.3, 156.3, 147.2, 141.1, 134.0, 133.7, 131.4, 131.3, 131.0, 129.3, 128.3, 127.3, 127.2, 126.4, 120.0, 119.4, 119.3, 118.6, 116.3, 116.1, 41.9, 40.7, 40.0, 39.9, 39.8, 39.7, 39.7, 39.6, 39.4, 39.3, 39.2, 39.1, 38.9; HRMS m/z: (M+H)⁺calcd for C₁₆H₁₆N₃O₃ 298.1186; found 298.1188.

2.2.2. Evaluation of anticancer activity using MTT assay

To determine the effect of the synthesized N-acylhydrazone derivatives on the proliferation of pancreatic (MIA PaCa-2, PANC-1) and breast (MCF-7) cancer cells, the methyl thiazolyl-diphenyl-tetrazolium bromide (MTT) assays were performed [14–16]. About 4,000 to 5,000 cells (MIA PaCa-2, PANC-1) were seeded in each well of the 96-well plate in quadruplets. After 24 h of incubation, the cells were treated with 0–100 μM concentration using serial dilutions of the synthesized derivatives (5a−5l). After 72 h of the treatment, the 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) was added into each well incubated to form Formazan crystals. These crystals were dissolved using DMSO, and absorbance was recorded at 570 nm using a spectrophotometer. The percentage of growth inhibition and IC₅₀ of each derivative was calculated using Graph-Pad.

2.2.3. Cell cycle arrest analysis

For cell cycle analysis, the 5 × 105MIA PaCa-2 cells were plated in 60 mm dishes and incubated with increasing doses of 10 µM and 100 µM of active derivative 5l for 48 h. DMSO solvent was used as the control. The cells were reaped, washed with cold 1X PBS, and fixed with 3 mL of chilled 75% ethanol. The fixed MIA PaCa-2 cells were washed again with 1X PBS, stained with 400–500 µl of propidium iodide staining solution (50 µg/ml PI, 0.1%Triton X-100 (v/v), 20 µg/ml DNase-free RNase) for 30 min [15,16]. Ten thousand events per sample were acquired using BD FACS ARIA III flow cytometer. The % of cells in Sub G1, G0/G1, S, and G2/M phases of the cell cycle were examined with FACS Diva software.

2.2.4. Apoptosis assay

To check the activity of derivatives on the apoptosis of MIA PaCa-2 cells, 5 × 105 cells were plated in 60 mm dishes and were incubated with 10 µM and 100 µM concentrations of 5 l for 48 h. Post-treatment, the cells were reaped and washed with 1X binding buffer. This was then followed by staining of each sample with 5µL Annexin V-FITC and 10 µL Propidium Iodide using Apoptosis Detection Kit [14,16]. The incubation was done at room temperature in dark conditions for 30 min. The samples were run and observed using BD FACS ARIA III flow cytometer instrument. A total of 10,000 events were captured for each sample which were further analyzed using the FACS Diva software.

2.2.5. Western blot analysis

To check the phosphorylation of JAK1 and STAT3 in MIA PaCa-2 and MCF-7 cells, we performed western blooting experiments as described earlier [4]. Briefly, the monolayer cells were washed with 1X PBS for three times and serum starved for 24 h in the serum-free medium. After 24 h, the fresh medium with 10% FBS was added to the DMSO and 5l treated plates for 20 min. The stimulated cells were harvested and lysed with Mammalian protein extraction buffer (Thermo-Fisher Scientific, USA) followed by a resolution of 30 µg of proteins on 10% SDS-PAGE and transferred on PVDF membrane (Millipore, Merck, USA). The PVDF membranes were blocked followed by incubation with p-JAK1, JAK, p-STAT3, STAT3, and GAPDH antibodies. Next, HRP-conjugated secondary antibody was used to visualize the protein signals using ECL (Thermo-Fisher Scientific, USA).

2.2.6. STAT3 preparation and active core determination

Human STAT3 protein (6NJS) X-ray crystallographic structure was retrieved from the RCSB pdb [17,18]. The protein was then subjected to AutoDock. To confirm the probity of STAT3, the ligand (co-crystallized) was isolated, and then water molecules and the unwanted chains or hetatoms were eliminated before protein preparation [19]. Addition of the nonpolar H atoms and Kollman charges was done [20]. The prepared structure was then exported to pdbqt format. The grid area [X = 10.2873, Y = 50.3617, Z = 6.1684] was prepared in the active positions, and binding pockets of STAT3 were visualized using Pymol and Biovia Discovery studio [21,22].

2.2.7. Ligand preparation

The structure in 2D was drawn in using Chemdraw and was subjected to energy minimization for converting to 3D structure. The minimization was done till the RMSD value of <.001 kcal/mol. Chem3D extreme v10.0 integral option was used for the minimized structure followed by importing the subjected molecule to AutoDock Tools. The instigation of gasteiger charges and merging of the H (nonpolar) was done, and the rotatable bonds were adjusted along with the addition of TORSDOF for calculating the change in free energy and were stored in the pdbqt format [23–25].

2.2.8. Molecular docking

It is an effective and competent adjunct for in-silico screening. It is helpful toward predicting and exploring the key interactions responsible for activity as well as the binding affinity of the designed molecule against the target. The docking analysis was done using PyRx (AutoDockVina) [26–28].

2.2.9. ADME screening and drug likeliness

Swiss-ADME (http://www.swissadme.ch) and ADMET 2.0 (https://admetmesh.scbdd.com/) servers were used for drug-likeness and ADME properties of the derivative [29–32]. Physicochemical properties, synthetic feasibility, GI absorption, solubility parameters, drug likeliness, metabolizing enzymes and toxicity parameters were taken into consideration while screening the selected derivative on ADME criteria.

2.2.10. Molecular dynamics simulation

To analyze the stability of interactions of the complex in physiological conditions, MD simulation of 100 ns was done [33–35]. Desmond was used in this study to simulate molecular dynamics. The specification of the simulation depends on study design, protein size, and the way of structure prediction. For dynamic simulations, the Schrodinger 2021.4 was used with the OPLS2.1, 3 and 3e force fields with the usual settings. The Desmond in-built workstations were used to create all of the topologies and simulation files. The salt content was maintained at 0.15 M sodium and chloride ions to mimic physiological conditions. Desmond was performed in an NPT setup for intervals of 70 to 100 ns at a temperature of 300 K and a pressure of 1.70 bar [36,37].

3. Results and discussion

3.1. Synthesis

Series of N-acylhydrazone derivatives was synthesized using benzoyl chloride and 2-aminoacetic acid as starting materials utilizing reported literature. The synthetic scheme for the target derivatives (5a-5l) comprising benzamide, amide linkage, Schiff bases, and the substituted aromatic aldehydes is depicted in Figure 2. Saturated solution of 2-aminoacetic acid in potassium carbonate was added to benzoyl chloride to give 2-benzamidoacetic acid (2). Methyl-2-benzamidoacetate (3) was prepared via Fischer esterification in the presence of methanol. Methyl-2-benzamidoacetate (3) upon reacting with hydrazine hydrate give 2-benzamidoacetohydrazide (4). NAH derivatives (5a-5l) were prepared using different substituted aromatic aldehydes via dehydration condensation reaction. IR, ¹H NMR, ¹³C NMR and mass spectrometry was done for the characterization of the synthesized derivatives.

Figure 2.

The synthetic route of target compound 5a−5l. Reagent and conditions: (a) K₂CO₃, NH₂CH₂COOH, HCl; (b) CH₃OH, H₂SO₄, 4 h, 60°C; (c) C₂H₅OH, NH₂NH₂.H₂O, 80 °C; (d) C₂H₅OH, CH₃COOH, 80 °C, Ar-CHO (−R), 6h.

3.2. In-vitro cytotoxic activity

To determine the effect of NAH derivatives (5a−5l) on the proliferation of pancreatic (MIA PaCa-2) cancer cells, the methyl thiazolyl-diphenyl-tetrazolium bromide (MTT) assays were performed. Initially, we evaluated the anticancer activity of the derivatives (5a-5l) using MIA PaCa-2 cells. The MIA PaCa-2 cells were treated with varying doses of these derivatives (1–100 μM) and DMSO as a solvent control for 72 h. Treatment of MIA PaCa-2 cells with derivatives (5a-5l) led to a significant reduction in the cellular proliferation of MIA PaCa-2 cells in a dose-dependent manner compared with DMSO solvent control (Figure 3(a)). The IC₅₀ of these derivatives ranged from approximately 52.46 to 398.75μM. Notably among all, 5h and 5l were found to be effective with IC₅₀ 61.21 μM, and 52.46 μM, respectively. The cytotoxicity activity data of NAH’s (5a-5l) is given in Table 1. Furthermore, we noticed that 5h and 5l does not produce significant cytotoxicity on the human normal pancreatic (hTERT-HPNE) cells suggesting the specific anticancer activity in pancreatic cancer cells (Figure 3(b)).

Figure 3.

Effect of NAH derivatives on the proliferation of normal pancreatic, pancreatic carcinoma and breast cancer cells. (a) Screening the NAH derivatives using MIA PaCa-2 cells. Doxorubicin was used as positive control for analyzing the anticancer activity of NAH derivatives. (b) Effect of 5h and 5l derivative on the growth of human normal pancreatic epithelial (hTERT-HPNE) cells. (c) Effect of 5h and 5l derivative on the growth of PANC-1 (pancreatic ductal adenocarcinoma) cells. (d) Effect of 5h and 5l derivative on the growth of MCF-7 (breast cancer) cells. All the experiments were performed thrice in biological triplicates. The results are presented as mean ±SD; n=3. p-value *<0.05, **<0.01, ***<0.001, two-tailed Student’s t-test.

Table 1.

Cytotoxicity results of the NAH derivatives (general structure) against MIA PaCa-2, PANC-1, and MCF-7 cancer cell lines.

Sr. No.	Derivative	R	MIA PaCa-2 (IC50)	PANC-1 (IC50)	MCF-7 (IC50)
01	5a		154.30	–	–
02	5b		145.77	–	–
03	5c		155.17		–
04	5d		131.45	–	–
05	5e		107.81	–	–
06	5f		144.85	–	–
07	5g		189.61	–	–
08	5h		61.21	110.91	201
09	5i		169.61	–	–
10	5j		272.71	–	–
11	5k		398.75	–	–
12	5l		52.46	63.47	58.79
13	Doxorubicin		4.32	0.9	0.25

*IC₅₀ represents half m aximal inhibitory concentration in µM.

Henceforth, the most active 5l derivative was selected for further analysis. Additionally, we evaluated the anticancer activity of the derivative 5l in PANC-1 and MCF-7 cell lines. Both cell lines were treated with increasing concentrations of 5l for 72 h. The treatment of the 5l resulted in a significant decrease in the cellular proliferation of PANC-1 and MCF-7 cells in a dose-dependent fashion as compared to DMSO with IC₅₀ of 63.47μM and 58.79μM in PANC-1 and MCF-7 cells, respectively (Figure 3(c,d)). Doxorubicin was used as a standard chemotherapy control (IC₅₀ 4.32 ± 1.06 µM (MIA PaCa-2 cells)) along with the synthesized derivatives. As expected, doxorubicin was found to be the most potent anticancer agent in both PANC-1 and MCF-7 cells. The IC₅₀ values of the derivatives are given in Table 1.

3.3. Cell cycle and apoptosis analysis

To check the mechanism linked with the growth inhibition of MIA PaCa-2 with increasing doses of derivative 5l, cell cycle and apoptosis experiments were performed. The flow cytometric analysis displayed significant arrest in the G1/S phase during the cell cycle followed by a profound decrease in the G2/M cell population with an accumulation of apoptotic cells in the Sub-G1 in the MIA PaCa-2 after 48 h treatment with 5l compared to DMSO control. Additionally, the annexin and propidium iodide staining data displayed a significant and simultaneous increase in both early and late apoptosis with increasing doses of 5l concentrations. The percentage of early apoptotic and late apopototic cell population significantly increased upon 5l treatment in a dose-dependent manner. Altogether, results indicated that NAH derivative 5l has promising anticancer activity by arresting cell cycle progression which resulted in increased cell apoptosis (Figure 4) as well as inhibited the phosphorylated levels of JAK1/STAT3 signaling (Figure 5(a)) and densitometry analysis revealed 5l treatment significantly inhibited the phosphorylation of JAK1 and STAT3 protein (Figure 5(b)).

Figure 4.

Treatment with 5l derivative induced cell cycle arrest and cell apoptosis. (a) the effect of NAH derivative 5l on the cell cycle analysis showed arrest in the G1/S phase. (b) Annexin-V and PI staining of of MIA PaCa-2 cells using flow cytometry analysis displayed increase early and late apoptosis. The results are presented as mean ±SD; n=3. p-value *<0.05, **<0.01, ***<0.001, two-tailed Student’s t-test.

Interestingly, the treatment of 5l in MIA PaCa-2 and MCF-7 cells displayed significant decrease in the phosphorylation of the JAK1 and STAT3 proteins. These results indicated that 5l treatment can suppressed the JAK1/STAT3 signaling pathway.

Figure 5.

Effect of 5l on the JAK1 and STAT3 signaling pathway. (a) the western blotting analysis of MIA PaCa-2 and MCF-7 cells treated with 5l showed marked reduction in phosphorylated JAK1 and STAT3 protein. (b) Densitometry analysis revealed 5l treatment significantly inhibited the phosphorylation of JAK1 and STAT3 protein. The results are presented as mean ±SD; n = 3. p-value ** < 0.01, *** < 0.001 (two-tailed Student’s t-test).

3.4. Molecular docking

To analyze the binding affinity and interaction of the derivative 5l in complex with STAT-3, the 3D structure was retrieved from protein data bank and was subjected for protein preparation. Molecular docking of the targeted complex demonstrated good binding affinities and interactions with the targeted human STAT3 core complex. Derivative 5l displayed the binding affinity of −7.0 kJ/mol and the binding region residue as SER: 611 (H bond) and GLU: 638 (salt bridge, attractive charge and C-H bond). Both of the interactions are also observed in the co-crystallized ligand. The representation of the binding interactions of 5 l and co-crystallized ligand are given in Figure S25.

3.5. Physicochemical properties and ADME profiling

The derivative 5l was further screened for ADME and drug likeliness analysis. The compound 5l was following the drug likeliness criteria including Lipinski criteria, partition coefficient and exhibit good physicochemical properties, good solubility, high GI absorption, and is not permeable to BBB. The compound on the criteria of toxicity analysis displayed no significant toxicity including carcinogenicity, ames, respiratory toxicity, skin sensitivity and genotoxicity. The physicochemical properties and the predicted ADME properties are given in Table 2.

Table 2.

Physicochemical properties and ADME profiling of derivative 5l.

Descriptors	Derivative 5l	Descriptors	Derivative 5l
Formula	C16H15N3O3	GI absorption	High
Molecular Weight	297.31	BBB permeant	No
Heavy atoms	22	Pgp Substrate	No
H bond Acceptors	4	CYP inhibitor	No
H bond donors	3	Bioavailability Score	0.55
Molar Refractivity	82.25	Carcinogenicity	0.294
Topological Polar Surface Area	90.79	Skin sensitivity	0.414
ClogP	1.81	Ames	0.203
IlogP	1.94	Respiratory	0.672
Lipinski Violations	0	Genotoxicity	0.0
PAINS alert	1

3.6. Molecular dynamics

The best derivative chosen as best fit was subjected to subsequent submission to a simulation investigation of molecular dynamics. Though dock interactions and binding affinity is a common perspective of the docking posture of chosen derivative in binding site areas of target proteins, dynamic investigations lead to calculating the derivatives with time and Newton’s classic formula of motions. For complex derivatives, the force field was administered for 100 ns at a steady rate. The RMSD and RMSF revealed the stable trajectories and the start run (100 ns). The RMSD values were presented on the Y-axis for the docked molecules, whereas the X-axis on right side represents ligand RMSD. Figure S26a depicts RMSD of STAT3 and 5l as a decisive system to corroborate the correctness of docking geometry. Protein RMSD exemplifies explained explorations regarding the structural conformation of STAT3 along the run whereas RMSD of 5l derivative suggests its stability within STAT3 core complex. Irregular fluctuation was observed initially and reaches stability after 20 ns. The protein RMSD maintains its stability between 20-80 ns and the average STAT3 value is 2.4 to 4.2Å throughout the run whereas minor fluctuations were observed within 3.6 Å and issupposed as the acceptable range for human STAT3 core complex. RMSD of ligand fit protein stable at 20–60 ns throughout the simulation within 1.8 to 3.6 Å. Initially, before 20 ns and after 60 ns, few peaks indicated the presence of rotatable bonds within the acceptable area. Throughout the simulation run, little or no significant changes clearly suggests the stable binding of the 5 l derivative within the active site of STAT3 core complex. Figure S26b shows the ligand RMSF value (Red color) and the protein RMSF value (Blue color). While many amino acid residues continue to have an RMSF value >3.2 Å, one significant peak initially and N and C terminal domains possess considerable fluctuations, suggesting the presence of loop areas. Protein loop sections holdup to 45% of the total STAT3, vary more than other 2° structures like α-helices and β-strands, and provide stiffness in protein structure. For most atoms in relation to protein, our ligand represents an RMSF value <2.0 Å.

The represented stacked bar chart given in Figure S26c depicts multiple interactions occurring between STAT3 and 5l derivative including H-bonds, hydrophobic interactions, and water bridges throughout the simulation run. PRO:471, LYS:573, and LYS:615 make hydrophobic interactions while ASP:334 and ASP:566 make hydrogen bonds. The water bridges are formed by HIS:332, ARG:335, and GLU:616 residues. The stabilization of Ligand RMSD suggests that the ligand molecule has found a favorable binding pose within the protein structure, and further significant conformational changes or movements are limited.

The structure activity relationship (SAR) of the derivatives suggests the impact of different substitutions on activity. The R₁ substituted derivatives were significantly more active than R₂ and R₃ substituted derivatives, respectively. Mono-substituted derivatives were more active as compared to the cyclic/tri-substituted or aliphatic substitutions, respectively. Electron donating groups displayed better activity than electron withdrawing groups at position R₁. Derivative 5l was significantly more active than derivative 5e. Electron withdrawing groups displayed better activity than electron donating groups at R_3. Derivatives 5h and 5c were also active (5 h > 5c), whereas derivatives 5g, 5j and 5k (5g > 5j > 5k) were least active. R₂ substituted derivative (5d) was moderately active. The structure activity relationship of the synthesized derivatives is represented in Figure S27. Among all, derivative 5l displayed significant anticancer activity, whereas 5g, 5j and 5k were found to be least active or inactive.

4. Conclusion

STAT3 proteins are reported to be overexpressed in different cancer types including pancreatic, and breast carcinoma. Thus, a series of N-acylhydrazone derivatives (5a-5l) were synthesized and evaluated against MIA PaCa-2. The active derivative 5l showed good anticancer potential. Next, 5l was further evaluated for anticancer evaluation against the pancreatic cancer cell line (PANC-1) and breast cancer carcinoma (MCF-7). Among all the synthesized derivatives, 5l was found to be the most active and was further evaluated to understand the mechanism of growth arrest in MIA PaCa-2 cells. The results of cell cycle analysis revealed that 5l is potent to arrest cells in the G1/S phase during cell cycle. This arrest led to the accumulation of apoptotic cells in the Sub-G1 phase which was confirmed by annexin V and PI staining. The annexin V and PI experiment demonstrated a marked increase in apoptosis in both the early and late phases in a dose-dependent fashion. Further, the molecular docking of the 5l derivative followed by molecular dynamics supported that 5l can bind with the human STAT3 core complex 1 receptor. Furthermore, our data displayed that 5l treatment markedly inhibited the phosphorylation of JAK1 and STAT3, suggesting the potential of 5l to suppressed the JAK1/STAT3 pathway. In-vitro evaluation and computational studies confirm the anticancer activity of 5l derivative as STAT3 inhibitor. Thus, the current findings uphold the significant potential of the synthesized derivatives for targeting cancer. Further synthetic modifications and biological investigations may provide better insights toward developing more potent derivatives with improved therapeutics.

Funding Statement

This paper was not funded.

Article highlights

• Over-expressed STAT3 in cancer cells stimulates the progressive and continued upregulated transcription of growth factors and anti-apoptosis that imparts a fundamental role towards maintaining cell growth and survival.

• A series of N acylhydrazone derivatives synthesized, characterized evalauted in vitro against MIA PaCa-2 cancer cells.

• Compounds 5h and 5l were further evaluated against PANC-1, MCF-7 and hTERT-HPNE cells and the anticancer efficacy was confirmed against PANC-1 and MCF-7 for 5l and 5h.

• Cell cycle analysis revealed 5l as potent molecule to arrest cells in the G1/S phase during cell cycle. The annexin V and PI experiment showed marked increase in apoptosis in both the early and late phases in a dose-dependent manner.

• Cpmpds 5l inhibited the phosphorylation of JAK1 and STAT3, suggesting the potential of 5l to suppress the JAK/STAT pathway.

• In silico investigations including molecular docking, ADMET and molecular dynamics supports the STAT3 inhibition and potential anticancer activity.

Author contributions

V.P. experimental, investigation, data curation, validation, writing – original draft. Y.R. and M. validation, analysis, writing – review and editing, A.S., B.S.; Biology, validation, analysis, writing – review and editing, D.K, M.K, M.G: Conceptualization, Investigation resources and writing – review and editing, D.K. supervision. All the authors approved the manuscript.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

Writing assistance

No writing assistance was utilized in the production of this manuscript

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2527604