Synthesis of pyrido-annelated [1,2,4,5]tetrazines, [1,2,4]triazepine, and [1,2,4,5]tetrazepines for anticancer, DFT, and molecular docking studies

Abstract

In this strategy, we attempt to design various novel nitrogen-rich heterocycles in one molecule. Green, simple, and efficient aza-annulations of an active, versatile building block, 1-amino-4-methyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbonitrile (1), with different bifunctional reagents were developed under solvent-free conditions, resulting in the bridgehead tetrazines and azepines (triazepine and tetrazepines). Pyrido[1,2,4,5]tetrazines have been synthesized through two pathways; [3 + 3]- and [5 + 1]-annulations. In addition, pyrido-azepines have been developed by applying [4 + 3]-and [5 + 2]-annulations. This protocol establishes an efficient technique for synthesizing essential biological derivatives of 1,2,4,5-tetrazines, 1,2,4-triazepines, and 1,2,4,5-tetrazepine, tolerating a diverse variety of functionalities without the need for catalysis and fast reaction rates in high yields. The National Cancer Institute (NCI, Bethesda, USA) examined twelve compounds produced at a single high dosage (10⁻⁵ M). Compounds 4, 8, and 9 were discovered to have potent anticancer action against certain cancer cell types. To explain NCI results, the density of states was calculated to conduct a better description of the FMOs. The molecular electrostatic potential maps were created to explain a molecule's chemical reactivity. In silico ADME experiments were performed to better understand their pharmacokinetic characteristics. Finally, the molecular docking investigations on Janus Kinase-2 (PDB ID: 4P7E) were carried out to study the binding mechanism, binding affinity, and non-bonding contacts.

Introduction

Heterocyclic compounds containing the pyridine¹ nucleus show a wide range of interesting biological effects, such as anticancer^2,3,4, antioxidant^5,6, antimicrobial^2,6,7, and anti-viral⁸. Furthermore, it has been demonstrated that 1,2,4-triazepines have a wide spectrum of therapeutic and biological effects^9,10,11.

Moreover, 1,2,4,5-tetrazines are a family of heterocyclic compounds having a diverse variety of biological actions, including anticonvulsant, anti-inflammatory, and antibacterial properties¹². Many studies have shown that several 1,2,4,5-tetrazine compounds exhibit anticancer activity^13,14,15.

Tetrazepines are seven-membered azaheterocyclic molecules with valuable medical and pesticide applications¹⁶. Tetrazepine derivatives interest chemists and pharmacists, but there are only a few ways to synthesize them. Because of the importance of tetrazepines in pharmaceuticals and their wide variety of uses¹⁷, great emphasis has lately been dedicated to the synthesis of novel tetrazepines as well as new methodologies.

In light of the above findings, following the pot, atom, and step economy (PASE)¹⁸ concept aims to reduce the number of steps required to synthesize various fused heterocyclic structures as the vessel must be operated economically in a single reaction vessel without any of the requirement for workup or transitional species separation. We provide a simple method for synthesizing several bioactive nitrogen heterocycles regarding the idea of 'solvent-free', including fused pyrido-1,2,4-triazepines, 1,2,4,5-tetrazines, and 1,2,4,5-tetrazepines.

Computational chemistry is a prominent method for investigating the biological characteristics of recently produced compounds. Density functional theory (DFT) is a quantitative quantum mechanical modelling approach used to explore the thermal and electrical stability of the produced compounds in this work. This study's determined parameters include dipole moment, the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), and the subtraction (HOMO–LUMO) gap energies. The molecular electrostatic potential surfaces (MEPs) were computed using Gauss View to measure their chemical and thermal characteristics.

Molecular docking is a bioinformatics modelling technique that deals with the interaction of two or more molecules to produce a stable adduct. The primary goal of molecular docking is to anticipate the possible binding geometries of produced molecules with a target protein in a three-dimensional structure. As many small ligands with JAK2 inhibitory activity produced therapeutic effects, and visualization of the compounds' greatest binding mode into Janus Kinase-2 (PDB ID: 4P7E) than other proteins, using the computer software Molecular Operating Environment (MOE), the more active compounds were investigated in silico to highlight their likely binding energy and interaction patterns with the active site of JAK2 (PDB ID: 4P7E). The more potent compounds were then screened for drug-likeness using Lipinski's rule of five and ADME characteristics.

Experimental

Chemistry

All melting points were measured and uncorrected in open glass capillaries using an Electrothermal LA 9000 SERIS digital melting point instrument. Bruker high-performance digital FT-NMR spectrometer Avance III was used to scan the ¹H,¹³C NMR spectra at [(600, 400) & 213] MHz in deuterated dimethyl sulfoxide and chloroform (DMSO-d₆ & CDCl₃) as a solvent. Mass spectra were acquired at 70 eV using a Schimadzu GC/MS-QP-5050A mass spectrometer et al.-Azhar University's regional center for mycology and biotechnology.

General procedure for the synthesis of compounds (2, 3, 5–7)

Equivalent amounts of 1¹⁹ (2.25 g, 10 mmol) and each of [cyanoguanidine (0.84 g, 10 mmol), chloroacetone (0.92 g, 10 mmol), anthranilic acid (1.37 g, 10 mmol), hydrazinecarbonitrile (0.58 mL, 10 mmol), or thiosemicarbazide (0.91 g, 10 mmol)] were fused at 150–170 °C for 3 h. The fused mass was allowed to cool, triturated with ethanol, and then the precipitate was filtered, washed with ethanol, and dried.

N-(8-Cyano-7-methyl-5-phenyl-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyanamide (2)

Yellow crystals are obtained, crystalized from EtOH/DMF (40/60%); yield (79) %, mp.: 228–230 °C; FT‐IR (KBr, ν, cm⁻¹): 3190(NH), 3003(CH-Ar), 2980 (CH-aliph.), 2230, 2150 (2(C≡N), 1608(C=N); ¹H NMR (400 MHz, DMSO‐d₆) δ(ppm): 2.51 (s, 3H, CH₃), 3.80(br., 1H, NH–C≡N, exchangeable with D₂O), 7.29–7.56 (m, 5H, Ar–H), 8.09 (s, 1H, CH-pyridine). ¹³C NMR (213 MHz, CDCl₃) δ(ppm): 29.24(CH₃),111.23 (pyridine C–C≡N), 118.93 (C≡N),125.11 (CH-pyridine), 128.87, 129.73, 138.00 (phenyl C), 146.18 (2C=N), 156.37 (pyridine C–CH₃), 170.61 (pyridine C–Ph). MS (m/z, %): 274.08 (M^+·, 31); 272.08 (M^+·–2H, 29); 141.84 (100). Anal. Calcd. for C₁₅H₁₀N₆ (274.29); C, 65.68; H, 3.67; N, 30.64. Found C, 65.75; H, 3.77; N, 30.76.

4-Methyl-2-oxo-1-((2-oxopropyl)amino)-6-phenyl-1,2-dihydropyridine-3-carbonitrile (3)

Orange crystals are obtained, crystallized from EtOH; yield (85) %, mp.: 115–118 °C; FT‐IR (KBr, ν, cm⁻¹): 3400 (OH tautomer), 3250(NH), 3000, 2890 (CH), 2220 (C≡N), 1650, 1630 (2C=O), 1590 (C=N); ¹H NMR: (400 MHz, DMSO‐d₆) δ(ppm): 2.24, 2.29 (2s, 6H, CH₃ & CH₃-tautomer); 2.19 (2s, 6H, COCH₃ & COCH₃—tautomer); 3.80 (s, 2H, CH₂); 4.17, 4.28 (2s, 2H, NH & NH—tautomer, exchangeable with D₂O); 5.05(s, 1H, =CH(tautomer)); 6.50, 6.55 (2s, 2H, CH-pyridine & CH-tautomer); 7.30–7.73 (m, 10H, Ar–H), 10.51 (s, 1H, OH(tautomer), exchangeable with D₂O). MS (m/z, %): 281.46(M⁺, 16); 138.67(100). Anal. Calcd. for C₁₆H₁₅N₃O₂ (281.32); C, 68.31; H, 5.37; N, 14.94. Found C, 68.39; H, 5.42; N, 15.05.

3,9-Dimethyl-2,7-diphenyl-2,5-dihydropyrido[1,2-b][1,2,4,5]tetrazepine-10-carbonitrile (4)

Equivalent amounts of 3 (2.81 g, 10 mmol) and phenylhydrazine (1.08 mL, 10 mmol) were fused at 150–170 °C for 2.5 h. The resultant solid was triturated with ethanol, filtered, washed by ethanol, dried, and crystallized from EtOH.

Brown crystals are obtained; yield (72) %, mp.: 241–243 °C; FT‐IR (KBr, ν, cm⁻¹): 3170 (NH), 3020 (CH-Ar), 2870 (CH-aliph.), 2150 (C≡N); 1600 (C=N); ¹H NMR (400 MHz, DMSO‐d₆) δ(ppm): 2.22, 2.24 (2s, 6H, 2CH₃), 2.65(s, 1H, NH-tetrazepine, exchangeable with D₂O), 4.18 (s,1H, CH-tetrazepine), 7.25–7.58 (m, 10H, Ar–H), 7.73 (s, 1H, CH-pyridine). ¹³C NMR (213 MHz, CDCl₃) δ (ppm): 15.62 (CH₃), 21.20 (CH₃), 95.07(CH-pyridine), 104.75 (CH-tetrazepine),118.81 (C≡N), 119.06, 119.24, 122.15, 122.37, 126.07, 126.25, 129.00, 130.15, 131.73 (phenyl C). MS (m/z, %): 353.66 (M^+·, 9); 352.56(M^+·–H, 23); 328.22(100). Anal. Calcd. for C₂₂H₁₉N₅ (353.43); C, 74.77; H, 5.42; N, 19.82. Found, C, 74.80; H, 5.48; N, 19.78.

2-Methyl-7-oxo-4-phenyl-6,7-dihydrobenzo[e]pyrido[1,2-b][1,2,4]triazepine-1-carbonitrile (5)

Brown powder are obtained, crystallized from EtOH; yield (78) %, mp.: 189–191 °C; FT‐IR (KBr, ν, cm⁻¹): 3420 (NH/OH), 3005 (CH–Ar), 2890 (CH–aliph.), 2226 (C≡N), 1625 (C=O); 1605 (C=N); ¹H NMR: (400 MHz, CDCl₃) δ(ppm): 2.20 (s, 3H, CH₃), 7.17–7.85 (m, 9H, Ar–H), 7.40 (s, 1H, CH–pyridine), 10.24(s, 1H, NH, exchangeable with D2O). ¹³C NMR (213 MHz, DMSO-d₆) δ (ppm): 24.31 (CH₃), 95.63 (CH–pyridine), 117.98 (C≡N), 122.03, 122.36, 124.75, 128.50, 128.68, 129.42, 138.62, 141.79, 143.40 (phenyl C), 148.17 (C=N), 162.56 (C=O). MS (m/z, %): 323.51(M^+·–3H, 9); 94.24 (100). Anal. Calcd. for C₂₀H₁₄N₄O (326.36); calc. C, 73.61; H, 4.32; N, 17.17. Found C, 73.64; H, 4.30; N, 17.15.

3-Amino-8-methyl-6-phenyl-4H-pyrido[1,2-b][1,2,4,5]tetrazine-9-carbonitrile (6)

VYellow crystals are obtained, crystallized from EtOH; yield (67) %, mp.: 187–189 °C; FT‐IR (KBr, ν, cm⁻¹): 3380, 3250, 3120 (NH₂, NH), 3010 (CH–Ar), 2950 (CH–aliph.), 2227 (C≡N), 1611 (C=N). Anal. Calcd. for C₁₄H₁₂N₆ (264.29); C, 63.62; H, 4.58; N, 31.80. Found C, 63.72; H, 4.52; N, 31.73.

3-Amino-8-methyl-6-phenyl-2H-pyrido[1,2-b][1,2,4,5]tetrazine-9-carbonitrile (7)

Yellow crystals are obtained, crystallized from EtOH; yield (89) %, mp.: 202–204 °C; FT‐IR (KBr, ν, cm⁻¹): 3300, 3220 (NH₂, NH), 3000 (CH-Ar), 2900 (CH-aliph.), 2170 (C≡N), 1610 (C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ (ppm): 2.25 (s, 6H, CH₃, CH₃(tautomer)), 6.43(s, 2H, NH₂, exchangeable with D₂O), 7.25–7.40 (m, 5H, Ar–H), 7.73, 7.75 (2 s, 2H, CH-pyridine & CH-pyridine (tautomer)), 8.07 (s, 1H, NH-tetrazine (tautomer), exchangeable with D₂O), 12.38, 12.60 (2s, 2H, =NH, NH-tetrazine, exchangeable with D₂O). ¹³C NMR (213 MHz, DMSO‐d₆) δ(ppm): 15.34, 15.63 (CH₃, CH₃ (tautomer)), 89.35, 94.23 (CH–pyridine, CH–pyridine (tautomer)), 117.36 (C≡N), 120.68, 121.49, 125.36, 129.28, 139.00, 140.40 (phenyl C). MS (m/z, %): 264.80 (M^+·, 9); 262.47 (M^+·–2H, 25); 50.27 (100). Anal. Calcd. for C₁₄H₁₂N₆ (264.29); C, 63.62; H, 4.58; N, 31.80. Found C, 63.72; H, 4.52; N, 31.89.

General procedure for the synthesis of compounds (8–10)

Equivalent amounts of 7 (2.64 g, 10 mmol) and each of [(diethylmalonate (1.58 g, 10 mmol), phenacyl bromide (1.97 g, 10 mmol) or diethyl oxalate (1.47 mL, 10 mmol)] were fused at 150–180 °C for 3 h. The produced mass was allowed to cool, triturated with ethanol, and then the precipitate was filtered, washed with ethanol, dried, and crystallized from EtOH.

8-Methyl-2,4-dioxo-10-phenyl-1,2,3,4-tetrahydropyrido[1,2-b]pyrimido[1,2-e][1,2,4,5]tetrazine-7-carbonitrile (8)

Orange crystals are obtained; yield (78) %, mp.: 250–252 °C; FT‐IR (KBr, ν, cm⁻¹): 3420 (OH tautomer), 3380 (NH), 3000 (CH-Ar), 2970(CH-aliph.), 2225 (C≡N), 1750, 1670 (2C=O), 1600 (C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ(ppm): 2.14 (s, 3H, CH₃), 3.38 (s, 2H, CH₂-pyrimidindione), 7.16–7.45 (m, 5H, Ar–H), 8.05, 8.07 (2s, 2H, CH-pyridine & CH-pyridine (tautomer), 8.33, 8.36 (2s, 2H, NH-pyrimidindione & NH-pyrimidindione (tautomer), exchangeable with D₂O), 12.08, 12.09 (br.,2H,2OH(tautomer), exchangeable with D₂O). ¹³C NMR (213 MHz, DMSO‐d₆) δ (ppm): 17.13 (CH₃), 43.47 (CH₂), 88.05 (CH-pyridine), 118.42(C≡N), 121.01, 124.89, 125.45, 129.34, 138.59, 139.41 (phenyl C), 160.74, 163.55 (2C=O). MS (m/z, %): 332.05 (M^+·, 25); 309.49(100). Anal. Calcd. for C₁₇H₁₂N₆O2 (332.32); C, 61.44; H, 3.64; N, 25.29. Found, C, 61.35; H, 3.52; N, 25.21.

7-Methyl-2,9-diphenyl-3H-imidazo[1,2-b]pyrido[1,2-e][1,2,4,5]tetrazine-6-carbonitrile (9)

Orange crystals are obtained; yield (85) %, mp.: 258–260 °C; FT‐IR (KBr, ν, cm⁻¹): 3100 (CH-Ar), 2970 (CH-aliph.), 2215 (C≡N), 1609(C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ(ppm): 2.08 (s, 3H, CH₃), 4.59 (s, 2H, CH₂-imidazole), 7.28–7.76 (m, 10H, Ar–H), 7.40 (s,1H, CH-pyridine). ¹³C NMR (213 MHz, CDCl₃) δ(ppm): 22.94(CH₃), 52.29 (CH₂-imidazole), 99.54 (CH-pyridine), 119.40 (C≡N), 120.82, 121.15, 125.88, 126.60, 128.84, 129.41, 137.42, 138.37 (phenyl C). MS (m/z, %): 364.39 (M^+·, 28); 198.96 (100). Anal. Calcd. for C₂₂H₁₆N₆ (364.41); C, 72.51; H, 4.43; N, 23.06. Found C, 72.45; H, 4.49; N, 23.02.

7-Methyl-2,3-dioxo-9-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrido[1,2-e][1, 2, 4, 5] tetrazine-6-carbonitrile (10)

Orange crystals are obtained; yield (74) %, mp.: 143–145 °C; FT‐IR (KBr, ν, cm⁻¹): 3300 (NH), 3000 (CH-Ar), 2870 (CH-aliph.), 2220 (C≡N), 1750, 1650 (2C=O), 1600(C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ (ppm): 2.25 (s, 3H, CH₃), 7.25–7.73 (m, 5H, Ar–H), 7.75 (s, 1H, CH-pyridine), 8.63 (s, 1H, NH-imidazole, exchangeable with D₂O). ¹³C NMR (213 MHz, DMSO‐d₆) δ (ppm): 20.81 (CH₃), 87.75 (CH-pyridine), 116.70 (C≡N), 121.36, 125.12, 129.33, 137.56, 138.40 (phenyl C), 175.17, 175.70 (2C = O). MS (m/z, %): 318.44 (M^+·, 23); 248.85 (100). Anal. Calcd. for C₁₆H₁₀N₆O₂ (318.30); C, 60.38; H, 3.17; N, 26.40. Found C, 60.46; H, 3.25; N, 26.49.

1-Amino-2-hydrazono-4-methyl-6-phenyl-1,2-dihydropyridine-3-carbonitrile (11)

Equivalent amounts of 1¹⁹ (2.25 g, 10 mmol) and hydrazine hydrate (0.4 mL, 10 mmol) were fused at 120–140 °C for 3 h. The reaction mixture was allowed to cool, triturated with ethanol, and then the precipitate was filtered, washed with ethanol, dried, and crystallized from EtOH.

Orange powder are obtained; yield (84) %, mp.: 176–178 °C; FT‐IR (KBr, ν, cm⁻¹): 3352, 3315(NH₂), 3003 (CH-Ar), 2990 (CH-aliph.), 2250 (C≡N), 1608 (C=N). ¹H NMR (600 MHz, DMSO‐d₆) δ (ppm): 2.11 (s, 3H, CH₃), 4.50(s,2H, NH_2, exchangeable with D₂O), 5.49 (s, 2H, =N–NH₂, exchangeable with D₂O), 7.28 (s, 1H, CH-pyridine), 6.99–7.29 (m, 5H, Ar–H). ¹³C NMR (213 MHz, DMSO‐d₆) δ (ppm): 19.01(CH₃), 90.34(CH-pyridine), 118.11(CN), 120.44, 123.72, 125.07, 130.07, 134.61 (phenyl C), 146.49 (C=N). MS (m/z, %): 239.72(M^+·, 18); 71.42(100). Anal. Calcd. for C₁₃H₁₃N₅ (239.12); C, 65.25; H, 5.48; N, 29.27. Found C, 65.20; H, 5.53; N, 29.34.

General procedure for the synthesis of compounds (12–16)

Equivalent amounts of 11 (2.39 g, 10 mmol) and each of [phenylisothiocyanate (1.35 mL, 10 mmol), chloroacetone (0.92 mL, 10 mmol), phenacyl bromide (1.97 g, 10 mmol), diethyl oxalate (1.47 mL, 10 mmol) or 3,4-dimethoxybenzaldehyde (1.66 g, 10 mmol)] were fused at 140–160 °C for 2 h. The fused mass was allowed to cool, triturated with ethanol, and then the precipitate was filtered, washed with ethanol, and dried.

8-Methyl-6-phenyl-3-(phenylamino)-4H-pyrido[1,2-b][1,2,4,5]tetrazine-9-carbonitrile (12)

Brown crystals are obtained, crystallized from EtOH/DMF (70–30%); yield (71) %, mp.: 223–225 °C; FT‐IR (KBr, ν, cm⁻¹): 3420(NH), 3010(CH-Ar), 2980 (CH-aliph.), 2230 (C≡N), 1619 (C=N); ¹H NMR: (600 MHz, CDCl₃) δ (ppm): 2.45 (s, 3H, CH₃), 4.03 (s, 1H, NH tetrazine), 6.77–7.49 (m, 10H, CH-aromatic), 7.82 (s, 1H, CH-pyridine), 7.86 (s, 1H, NH–Ph), ¹³C NMR (213 MHz, DMSO‐d₆) δ (ppm): 24.31(CH₃), 95.63, 96.4 (2CH-pyridine), 117.84, 117.99 (2C≡N), 118.27, 118.80, 122.03, 122.36, 124.75, 128.50, 129.42, 138.62, 141.17(phenyl C), 152.56, 154.74 (2 (C=N)-tetrazine). MS (m/z, %): 340.16(M^+·, 13); 339.17(M^+·–H, 43); 148.56(100). Anal. Calcd. for C₂₀H₁₆N₆ (340.14); C, 70.57; H, 4.74; N, 24.69. Found C, 70.63; H, 4.82; N, 24.75.

3,9-Dimethyl-7-phenyl-4,5-dihydropyrido[1,2-b][1,2,4,5]tetrazepine-10-carbonitrile (13)

Yellow crystals are obtained, crystallized from EtOH; yield (83) %, mp.: 210–212 °C; FT‐IR (KBr, ν, cm⁻¹): 3350(NH), 3009(CH-Ar), 2950(CH-aliph.), 2225 (C≡N), 1590 (C=N). ¹H NMR(600 MHz, CDCl₃) δ (ppm): 2.20 (s, 6H, 2CH₃)_, 3.44 (s, 2H, CH_2-tetrazepine), 4.20 (s, 1H, NH-tetrazepine, exchangeable with D₂O), 7.18–7.40 (m, 5H, Ar–H), 7.85(s, 1H, CH-pyridine), ¹³C NMR (213 MHz, DMSO‐d₆) δ(ppm): 21.36, 22.40 (2CH₃), 65.41 (CH₂-tetrazepine), 87.72 (CH-pyridine), 114.20 (CH-tetrazepine), 118.20 (C≡N), 120.68, 121.48, 125.36, 129.28, 139.00 (phenyl C), 152.56, 154.74 (2 (C=N)-tetrazepine). MS (m/z, %): 277.47(M^+·, 34); 65.36 (100). Anal. Calcd. for C₁₆H₁₅N₅ (277.33); C, 69.29; H, 5.45; N, 25.25. Found C, 69.35; H, 5.38; N, 25.18.

9-Methyl-3,7-diphenyl-2,5-dihydropyrido[1,2-b][1,2,4,5]tetrazepine-10-carbonitrile (14)

Orange powder is obtained, crystallized from EtOH; yield (86) %, mp.: 230–232 °C; FT‐IR (KBr, ν, cm⁻¹): 3330 (NH), 3010 (CH-Ar), 2960 (CH-aliph.), 2220 (C≡N), 1605 (C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ(ppm): 2.23 (s, 3H, CH₃), 4.51(s, 2H, CH & NH-tetrazepine), 7.25–7.70 (m,10H, Ar–H), 7.76 (s, 1H, CH-pyridine), 8.10 (s, 1H, NH-tetrazepine). ¹³C NMR (213 MHz, DMSO‐d₆) δ(ppm): 24.21(CH₃), 86.41 (CH-pyridine), 104.85 (CH-tetrazepine), 117.84 (C≡N), 122.03, 122.36, 124.75, 128.57, 128.68, 129.42, 138.62 (phenyl C), 149.17 (C=N-tetrazepine). MS (m/z, %): 339.67 (M^+·, 4); 336.80 (M^+·–3H, 21); 78.29 (100). Anal. Calcd. for C₂₁H₁₇N₅ (339.40); C, 74.32; H, 5.05; N, 20.63. Found C, 74.29; H, 5.01; N, 20.69.

9-Methyl-3,4-dioxo-7-phenyl-2,3,4,5-tetrahydropyrido[1,2-b][1,2,4,5]tetrazepine-10-carbonitrile (15)

Faint yellow crystals are obtained, crystallized from EtOH; yield (86) %, mp.: 200–202 °C; FT‐IR (KBr, ν, cm⁻¹): 3450, 3335 (2NH), 3009 (CH-Ar), 2890 (CH-aliph.), 2220 (C≡N), 1650, 1630 (2C=O), 1600 (C=N). ¹H NMR (400 MHz, DMSO‐d₆) δ (ppm): 2.12 (s, 3H, CH₃), 7.70 (s, 1H, CH-pyridine) 7.64–7.76 (m, 5H, Ar–H), 8.44, 8.56 (2s, 2H, 2NH-tetrazepine. ¹³C NMR (213 MHz, DMSO‐d₆) δ (ppm): 21.53 (CH₃), 82.71 (CH_-pyridine), 118.22 (C≡N), 120.51, 123.74, 124.17, 127.68, 129.24, 129.41 (phenyl C), 147.74 (C=N-tetrazepine), 151.08, 152.51 (2C=O). MS (m/z, %): 293.06 (M^+·, 20); 55.68 (100). Anal.Calcd. for C₁₅H₁₁N₅O₂ (293.29); C, 61.43; H, 3.78; N, 23.88. Found, C, 61.38; H, 3.72; N, 23.80.

1-((3,4-Dimethoxybenzylidene)amino)-2-hydrazono-4-methyl-6-phenyl-1,2-dihydro- pyridine-3-carbonitrile (16)

Orange crystals are obtained; crystallized from EtOH; yield (88) %, mp.: 246–248 °C; FT‐IR (KBr, ν, cm⁻¹): 3450, 3340 (NH₂), 3010 (CH-Ar), 2950 (CH-aliph.), 2225 (C≡N), 1600(C=N). ¹H NMR (600 MHz, DMSO‐d₆) δ (ppm): 2.15 (s, 3H, CH₃), 3.44 (s, 3H, OCH₃), 3.70 (s, 3H, OCH₃), 5.36 (s, 2H, NH₂, exchangeable with D₂O), 7.07–7.42 (m, 8H, Ar–H), 7.40 (s, 1H, CH-pyridine), 7.69 (s, 1H, CH=N–). Anal. Calcd. for C₂₂H₂₁N₅O₂ (387.44); C, 68.20; H, 5.46; N, 18.08. Found, C, 68.14; H, 5.54; N, 18.02.

Biological activities

NCI anticancer activity screening

The preliminary anticancer screening for novel fused pyridine derivatives (2–16) was performed in the Developmental Therapy Program of the National Cancer Institute (NCI) in the United States. In accordance with the NCI, Bethesda, Drug Evaluation Branch procedure (http://dtp.nci.nih.gov), the twelve derivatives 2, 4, 5, 7–10, and 12–16 were chosen and tested for initial in vitro one dose anticancer testing at 10^–5 M concentration against full NCI 60 cell line screens indicating nine various types of cancer comprising renal cancer, leukaemia, melanoma, prostate cancer, non-small cell lung cancer, ovarian cancer, CNS cancer, breast cancer. The results of each tested compound (2, 4, 5, 7–10, and 12–16) were provided as a mean chart of the growth percentage of the treated cells as compared to the control, which reveals both inhibitory values (between 0 and 100) and toxicity values (less than 0). The COMPARE tool was used to examine the single-dose evaluation findings of all chosen compounds against sixty cancer cell lines. As previously stated²⁰, every molecule supplied for the NCI-60 Cell screen is examined at a single high dosage (10⁻⁵ M).

Protein preparation and molecular docking work

The docking investigation was carried out using the computer software Molecular Operating Environment (MOE) version 2015.10²¹, Chemical Computing Group Inc., Montreal, Quebec, Canada. The docking technique was carried out as previously reported²². The 3-dimensional (3D) structure of Janus Kinase (Jak2) with PDB ID: 4P7E was obtained through the link (https://www.rcsb.org/structure/4P7E) on the Protein Data Bank site. Using the program's default parameters, the co-crystallized ligand was re-docked in its original protein structure. Interactions of the amino acids, affinities by bond strength, and hydrogen bond lengths were illustrated in Table 1.

Table 1 In vitro anticancer screening results of compounds 2, 4, 5, (7–10) and (12–16) against sixty human tumour cell lines with single dose assay (10^–5 M concentration). Data was provided as cell growth inhibition percentage.

Designing and optimization of more potent fused pyridine derivatives

Molecular orbital and electrostatic characteristics are frequently calculated using quantum mechanical techniques. The Gaussian 09 software program was used to complete the calculations, which used density functional theory (DFT)²³. DFT with Beck's (B)²⁴ three-parameter hybrid models and Lee, Yang, and Parr's (LYP)²⁵ association function under 6-31G (d,p) basis set was used to optimize and forecast the molecular orbital features of the more active fused pyridine derivatives (4, 8, and 9). At the same level of theory, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) were counted as frontier molecular orbital characteristics. The HOMO–LUMO energy gap was computed for each of the more powerful derivatives.

In silico physicochemical properties, drug-likeness, and pharmacokinetics profiles of most active compounds 4, 8, and 9 compared to the reference 2HB

Drug development is incomplete unless the pharmacokinetic parameters of drug candidates are evaluated during the primary screening stage because some drug candidates do not act therapeutically due to insufficient pharmacokinetic characteristics. This process may result in better hits and less late-stage abrasion of drug candidates. To identify the physicochemical properties, drug-likeness, and ADME profile, "absorption, distribution, metabolism, and excretion" were calculated hypothetically based on SwissADME online software [www.SwissADME.ch].

Discussion of the results

Chemistry

As reported, the cyclocondensation of 2-cyanoacetohydrazide with benzoyl acetone in a slightly alkaline medium afforded the dihydropyridine-3-carbonitrile (1)¹⁹ as the primary entry material for the synthesis of bridgehead pyrido[1,2,4,5]tetrazine and pyrido-azepines with one ring joint nitrogen atom.

The treatment of building block 1¹⁹ with cyanoguanidine yielded the fused triazolopyridine 2 in good yield, rather than the predictable 2,4-diamino-9-methyl-7-phenylpyrido[1,2-b][1,2,4,6]tetrazepine-10-carbonitrile (Fig. 1). Compound 2 is produced due to the elimination of the water molecule, followed by intramolecular cyclization by removing the NH₃ molecule. The appearance of an NH proton signal at 3.80 ppm exchangeable with D₂O in the ¹H-NMR spectrum, and a new carbon signal corresponding to the cyano group at 118.93 ppm in the ¹³C NMR spectrum, affirmed the structure of compound 2.

Figure 1

Synthesis of compounds 2–5.

Additionally, substrate 1¹⁹ was alkylated with chloroacetone, furnishing the open-chain product 3, which was further subjected to cyclocondensation upon fusion with phenylhydrazine to form the cyclized [1,2,4,5]tetrazepine analogue 4. The structures of these compounds have been verified by their spectral data (See the Supplementary file).

The fused heterocyclic system (6:7:6) with a single ring connection atom of nitrogen was synthesized through the fusion of the key compound 1¹⁹ with the binucleophilic anthranilic acid at 140–160 °C, affording the biologically active benzo[e]pyrido[1,2,4]triazepine derivative 5 (Fig. 1).

Figure 2 depicts the synthesis of pyrido[1,2,4,5]tetrazines via two pathways: [3 + 3]-and [5 + 1]-annulations. The first pathway was developed under solvent-free conditions via [3 + 3] cyclocondensation of 1¹⁹ and the binucleophilic thiosemicarbazide. The reaction gave the corresponding 3-aminopyrido[1,2-b][1,2,4,5]tetrazine 7 in 89% yields. The reaction mechanism of compound 7 was hypothesized to proceed via the elimination of both H₂O and H₂S molecules as follows: initial condensation of the carbonyl functionality of compound 1 with the primary amine group of the thiosemicarbazide to give an intermediate by removal of a water molecule, followed by ring closure via removal of a hydrogen sulphide molecule to give the target compound 7, which can be tautomerized to its tautomeric form, as illustrated in Fig. 2. The ¹H-NMR spectrum of compound 7 recorded two singlet signals at 6.43 and 12.60 ppm exchangeable with D₂O, attributed to amine and tetrazine-NH, respectively. It is important to note that the fusion of substrate 1¹⁹ with a different reactive reagent, hydrazinecarbonitrile, produced the tautomer of compound 7 through a different mechanism and resulted in 3-imino-2H-pyrido[1,2-b][1,2,4,5]tetrazine 6 in a 67% yield (Fig. 2). A reaction mechanism is postulated with an initial nucleophilic attack on the carbonyl functional group of compound 1 by the amino nitrogen of the hydrazinecarbonitrile, providing an intermediate by the elimination of a water molecule. The intermediate then undergoes cyclization by attacking the primary amino group on the nitrile carbon, leading to the formation of compound 6.

Figure 2

Synthesis of compounds 6–10.

The presence of a primary amine functionality in the ortho position to tetrazine-NH opens the way for subsequent chemical modification of tetrazines. Thus, tetrazine derivative 7 was a good substrate for synthesizing a wide range of five- and six-membered heterocycles with high yields via [3 + 2]-and [3 + 3]-cyclocondensation reactions with 1,2- and 1,3-synthons, respectively. The fused heterocyclic system (6:6:6) with two nitrogen ring connections was obtained by fusing compound 7 with a 1,3-dicarbonyl compound, such as diethyl malonate, resulting in the respective dihydropyrimidinone derivative 8. The formation of the pyrimidine ring was confirmed through ¹³C-NMR with signals at δ 43.47, 160.74, and 163.55 ppm assignable for CH₂-pyrimidine and two amidic carbonyl carbons, respectively (Fig. 2). While tricyclic imidazo[1,2-b]pyrido[1,2-e][1,2,4,5]tetrazine analogues 9 and 10 were formed through the treatment of compound 7 with active methylene compound as phenacyl bromide and 1,2-di electrophilic diethyl oxalate; respectively. Compounds 9 and 10’s structures were determined using spectroscopic data (see the Supplementary file).

Another important starting point was hydrazone derivative 11, formed by hydrazinolysis of the substrate 1¹⁹ with an equimolar amount of hydrazine hydrate in the absence of solvent (Fig. 3). The latter compound was affirmed by the appearance of a new deuterium oxide exchangeable singlet in ¹H NMR at δ 5.49 ppm due to =N–NH₂ protons. In addition, the ¹³C NMR spectrum assured the absence of a carbonyl signal as well as the existence of a signal at δ 146.49 ppm correlated to C = N. Compound 11 was utilized as another way for the preparation of [1, 2, 4, 5]tetrazine and derivatives of [1, 2, 4, 5]tetrazepine via [5 + 1]- and [5 + 2]-annulation, respectively. The fusion of hydrazone derivative 11 with one carbon compound as phenylisothiocyanate was the second pathway for synthesizing pyrido[1,2,4,5]tetrazine 12 via [5 + 1]-annulation. The mechanism of compound 12 was expected to take place first via Michael's addition of the primary amine of 11 to the electron-deficient double bond in PhNCS, generating a Michael-type open-chain adduct (thiocarbamoyl intermediate) that underwent a cyclocondensation reaction with H₂S removal. Compound 12 displayed characteristic ¹H-NMR signals at δ 7.86 ppm and 4.03 ppm corresponding to NH-phenyl and NH-tetrazine, respectively, whereas the ¹³C NMR demonstrated signals at 152.56 and 154.74 ppm due to C=N-tetrazine carbons. On the other hand, the catalyst- and solvent-free [5 + 2]-cyclocondensation was applied to synthesize pyrido[1,2-b][1,2,4,5]tetrazepine derivatives 13 and 14 by fusing the key starting material 11 with α-halo carbonyl compounds, namely chloroacetone and phenacyl bromide, respectively at 140–160 °C. Additionally, 2,3,4,5-tetrahydropyrido[1,2-b][1,2,4,5]tetrazepine-3,4-dione 15 was easily obtained in high yield by treating hydrazone derivative 11 with diethyl oxalate. ¹H-NMR demonstrated new signals at 8.44 and 8.56 ppm for two tetrazepine-NH protons, respectively, whereas ¹³C-NMRR displayed distinguishable signals at 151.08 and 152.51 ppm for two C=O carbons. Finally, Schiff base 16 can be formed through the condensation of compound 11 with 3,4-dimethoxybenzaldehyde. The ¹H-NMR spectrum of 16 showed a singlet signal at 7.69 ppm due to the (CH=N–) proton.

Figure 3

Synthesis of compounds 11–16.

Biological activity

NCI screening of anticancer activity

Preliminary single high dose screening at 10^–5 M concentration

Twelve synthesized derivatives were tested for in vitro anticancer activity; some showed modest activity in numerous cancer cell lines, while others showed impact against further than one cancer cell line. Table 1 shows the most sensitive cell lines' growth inhibition percentages (GI %).

More specifically, compounds (4, 5, 7, 8, 10, 12, and 16) showed moderate activity with a mean GI% range (of 21.02–36.25) % against several forms of cancers, including Leukemia (RPMI-8226), non-small cell lung cancer (HOP-62 & NCI-H522), Melanoma (UACC-62), Renal cancer (UO-31), Brest cancer (T-47D), Prostate cancer (PC-3), and Ovarian Cancer (SK-OV-3) (Fig. 4).

Figure 4

Sunburst chart represents GI% higher than 15% for compounds 4, 5, 7, 8, 10, 12, and 16 against cancer cell lines of the nine tumour panels.

Among all tested compounds, 9 demonstrated strong anticancer cell line efficacy against Leukemia-RPMI-8226 (GI% = 51.58%) and Colon cancer-HCT116 (GI% = 77.94%). On the other hand, compound 9 inhibited various cell lines in a mild to moderate manner with a mean GI% range (of 8.07–36.84) % (Fig. 5a).

Figure 5

Radial bar chart represents (a): GI% of compound 9 against cancer cell lines of the nine tumour panels. (b): GI% of compounds (2, 13, 14, and 15) against cancer cell lines of the nine tumour panels.

Compounds 2 and 13–15 were ineffective against the bulk of the subpanels of cancer cell lines. They revealed a weak effective anticancer efficacy against certain cell lines, such as Non-small cell lung cancer (HOP-62 & NCI-H522) and Renal cancer (UO-31 & TK-10) with GI% range (8.76–16.88)% (Fig. 5b). The screening findings revealed that 4, 8, and 9 exhibited the maximum activity in some of the cell lines in the current study.

Analysis of molecular docking

The conjugates (4, 8, and 9) exceeded all other produced fused pyridine derivatives in terms of biological activity against various cancer cell lines. To examine their binding mechanism and non-bonding effects, we did a molecular docking versus Janus Kinase (Jak2) (PDB: 4P7E). The docking process was first confirmed by re-docking the co-crystallized ligand 2HB (N-(5–4)[(1,1-dioxidothiomorpholin-4-yl)methyl]phenyl[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropane carboxamide) at the enzyme's active sites. 2HB has an energy score (S) =  − 7.45 kcal/mol and showed one H-bond as Lys 882 with SO₂ group and three arene-H interactions with Gly 856, Ser 936, and Leu 855 residues (Fig. 6).

Figure 6

2D& 3D interaction of 2HB in the active site of Janus kinase Jak2 (PDB ID: 4P7E). Hydrogen bonds are displayed in cyan & H-pi-bonds are in dark pink.

Table 2 revealed that compounds 4 and 9 have an energy score (S) =  − 7.24 and − 7.25 kcal/mol, respectively, close to that of the 2HB ligand. Compound 4 can establish two H-bonds as Leu 932 with CN group and one hydrogen bond between the CH₃ group and the residue Met 929. On the other hand, Leu 855 showed three H-arene contacts with the benzene and tetrazepine ring of compound 4, also the pyridine nucleus produced four arene-H interactions with Val 863 and Leu 983 amino acids (Fig. 7). Compound 8 had an energy score (S) =  − 6.56 kcal/mol, two H-bonds were observed between CN group and the residue Leu 932 and one H-bond formed between Leu 855 and N of tetrazine ring, also, Leu 855 showed two H-arene interaction with tetrazine ring. In addition, the pyridine ring of 8 was oriented in close contact with Leu 983 and Val 863, forming four arene-H interactions (Fig. 8). Finally, compound 9 established one hydrogen bond as Val 911 with CN group and eight arene-H contacts as benzene ring with Leu 855, pyridine ring with (Ser 936, Leu 855 & Val 863) and tetrazine ring with (Val 863 & Leu 983) (Fig. 9).

Table 2 Docking results of compounds 4, 8, 9, and 2HB ligand inside Janus Kinase Jak2 (PDB ID: 4P7E) active spots.

Figure 7

2D& 3D interaction of 4 in the active site of Janus kinase Jak2 (PDB ID: 4P7E). Hydrogen bonds are displayed in cyan & H-pi-bonds are in dark pink.

Figure 8

2D& 3D interaction of 8 in the active site of Janus kinase Jak2 (PDB ID: 4P7E). Hydrogen bonds are displayed in cyan & H-pi-bonds are in dark pink.

Figure 9

2D& 3D interaction of 9 in the active site of Janus kinase Jak2 (PDB ID: 4P7E). Hydrogen bonds are displayed in cyan & H-pi-bonds are in dark pink.

Analysis of frontier molecular orbitals

The most fundamental orbitals in molecules are the frontier molecular orbitals, which are used to determine kinetic stability and chemical reactivity. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are the names given to the Frontier molecular orbitals. Figure 10 shows that the HOMO orbitals are less localized than the LUMO orbitals. Electronic absorption is the transition from the ground state to the first excited state and is best defined by the excitation of an electron from HOMO to LUMO²⁶. Kinetic stability rises when the HOMO–LUMO gap widens. As a result, moving electrons from the stable state HOMO to the excited state LUMO needs more energy. For compounds 4, 8, and 9, the majority of the HOMO is placed mainly on the pyridine ring and the fused triazepine or triazine rings, with a little contribution from the phenyl ring connected to triazepine nitrogen atom as in compound 4. Compounds 4 and 8 have LUMOs localized on the pyridine ring, nitrogen atoms of the fused ring, and the phenyl ring attached to pyridine. In contrast, compound 9 has LUMOs localized on the pyridine ring, 4-phenyl imidazole moiety, and nitrogen atoms of fused triazine. Table 3 displays orbital energy and dipole moment values calculated for the selected compounds (4, 8, 9) and 2HB. The dipole moments of the more potent conjugates and 2HB were investigated, revealing that the compounds have large dipole moments in the order 8 > 2HB < 4 > 9. Although, compounds 4, 8, and 9 have an energy gap of 0.1052, 0.1153, and 0.0875 eV, respectively, which are smaller than that of 2HB (0.1594 eV) (Fig. 10). As a result, these chemicals (4, 8 and 9) may enable higher interaction with high dipole moment species, particularly in biological systems.

Figure 10

Molecular orbital distribution plots of HOMO and LUMO for compounds 4, 8, 9, and 2HB.

Table 3 DFT parameters calculated for the synthesized compounds (4, 8, 9) & 2HB.

The electrostatic potential of molecules (MEP)

The molecular electrostatic potential (MEP) aids in interpreting the biological recognition phase and hydrogen bonding connections²⁷. The MEP map of compounds (4, 8, 9) and 2HB was generated using the B3LYP with the basis set 6-31G optimized outputs (Fig. 11). MEP was computed to identify the reactive functional groups for electrophilic and nucleophilic reactions in terms of colour grading of the optimized structure of compounds (4, 8, 9) and 2HB (Fig. 12). The red colour represents the maximum negative area, which suggests a desirable site for electrophilic attacks, the blue colour represents the largest positive area, which indicates a favourable site for nucleophilic attacks, and the green colour represents zero potential regions.

Figure 11

Chemical and optimized structure of compounds (4, 8, 9) and 2HB. Optimized with DFT-B3LYP/6-31G.

Figure 12

MEP map of compounds (4, 8, 9) and 2HB.

In silico ADME studies

Physicochemical properties and drug-likeness

A bioinformatics study was performed on the most active compounds 4, 8, and 9 to predict their physicochemical and drug-likeness properties. The physicochemical properties for oral bioavailability, which include (size: MW between 150 and 500 g/mol, lipophilicity: Log P between − 0.7 and + 5.0, polarity: TPSA between 20 and 140 Å, flexibility: no more than 10 rotatable bonds, and solubility: log S not higher than 6) demonstrated that all examined compounds have good physicochemical properties similar to the reference 2HB (Table 4). Furthermore, compounds should meet the following criteria under the Lipinski rule of five, which indicates that drugs have good absorption and bioavailability: M.wt. ≤ 500, log P ≤ 5, HBD ≤ 5, and HBA ≤ 10, as shown in Table 4. The examined compounds 4, 8, and 9 had good values for all of the rule principles with potential drug-like properties, indicating that these compounds may meet cell membrane permeability and bioavailability requirements.

Table 4 In silico physicochemical properties and drug-likeness of compounds 4, 8, and 9, as well as the reference 2HB.

The ADME profile

Further on, the SwissADME Web tool was used to investigate the pharmacokinetics of compounds 4, 8, and 9, as well as the reference 2HB. According to the Boiled-egg model²⁸, all compounds had high gastrointestinal absorption (GI), indicating their ability to easily absorb through the intestinal wall. Compounds 4 and 9 were also shown to cross the blood–brain barrier (BBB), which may be useful in anticancer drugs targeting the CNS. In contrast, compound 8 was distinguished by a lack of BBB permeability, indicating the absence of CNS drawbacks like the reference 2HB (Table 5). Furthermore, compounds 4, 8, and 9 were shown not to bind to P-glycoproteins, in contrast to the reference 2HB.

Table 5 In silico pharmacokinetic study of compounds 4, 8, and 9 as well as the reference 2HB.

Moreover, the compounds' interactions with cytochromes P450 (CYP) in the liver, which is the main factor for drug elimination via metabolic biotransformation, have been investigated. Compounds 4 and 9 are indicated to inhibit four and three of the five major isoforms of hepatic cytochrome P-450 (CYP), respectively, and should be administered at time intervals when other drugs are prescribed to avoid potential drug interactions, similar to the reference 2HB, which is indicated to inhibit both CYP2C9 and CYP3A4. On the other hand, compound 8 is expected to inhibit none of the cytochrome P-450 (CYP) isoforms in the liver, making it safe to use together with other drugs (Table 5).

Conclusion

The fusion technique was applied for synthesizing several novel bioactive nitrogen-rich heterocycles, including pyrido-annelated [1, 2, 4, 5]tetrazines, [1, 2, 4]triazepine, and [1, 2, 4, 5]tetrazepines, with yields ranging from 67 to 89%. Additionally, molecules 4, 8, and 9 had the highest anticancer effect against many cancer cell lines. Furthermore, compounds 4, 8, and 9 have low HOMO–LUMO energy gaps, so that they may become more reactive than the 2HB ligand. Moreover, the docked complex of analogues 4, 8, and 9 with 4P7E had a higher binding affinity. The MEP study findings revealed the most negative and positive surface areas of the examined conjugates, allowing us to predict potential hydrogen bonding sites. Finally, In silico investigations of the compounds produced encouraging results, including strong GI absorption, good oral bioavailability, and perfect physicochemical features, indicating their potential as attractive medicinal targets “Supplementary Information”.