New pyridopyrimidine derivatives as dual EGFR and CDK4/cyclin D1 inhibitors: synthesis, biological screening and molecular modeling

Abstract

Aim: A series of pyridopyrimidine derivatives 5–20 was designed, synthesized and examined for antitumor activity using four types of malignant cells.

Materials & methods: Cervical cancer (HeLa), hepatic cancer (HepG-2), breast cancer (MCF-7) and colon cancer (HCT-166) cells, as well as normal human lung fibroblast cells (WI-38) were used to determine the cytotoxicity.

Results: Pyrazol-1-yl pyridopyrimidine derivative 5 was found to be the most active compound against three malignant cells Hela, MCF-7 and HepG-2 with IC₅₀ values of 9.27, 7.69 and 5.91 μM, respectively, related to standard Doxorubicin. Moreover, compounds 5 and 10 showed good inhibition against cyclin dependent kinase (CDK4/cyclin D1) and epidermal growth factor (EGFR) enzymes.

Graphical Abstract

Plain language summary

Article highlights.

• Pyridopyrimidines derivatives (3–20) have been designed and synthesized through the different reaction conditions.

• The chemistry of all synthesized compounds were confirmed by the different spectroscopic techniques; IR, ¹H NMR, ¹³C NMR and mass spectroscopy.

• In vitro cytotoxicity assay has been conducted to evaluate the anti-cancer activities of pyridopyrimidines derivatives toward four types of cancer cell lines.

• The structure–activity relationship have shown that the cyclization of pyridopyrimidine ring with pyrazole ring and the hybridization with benzylidine resulted in producing derivatives with potent anticancer effects.

• Enzyme inhibition assay has been performed for compounds 5 and 10 which showed strong inhibition of EGFR and CDK4-cyclin D1, utilizing Erlotinib and Palbociclib, respectively as reference drugs.

• The molecular modeling calculations have been performed which went with the results of enzyme inhibition assay.

• Cell cycle arrest and apoptosis assay have been done for compounds 5 and 10 which showed arrest at G0/G1 phase and induced apoptosis.

• Real-time Reverse transcription polymerase chain reaction for Bax and Bcl2 has been conducted to compounds 5 and 10 to investigate their ability to act as pro-apoptotic or anti-apoptotic inducers.

• The Lipinski's rule has been calculated using the Molinspiration online program for all new synthesized compounds. Fortunately, compounds 5 and 10 have obeyed the Lipinski's rule, so they can be considered as bioavailable drugs.

1. Background

Cancer is one of the most life-threatening diseases [1]. The major problems of chemotherapeutics as an efficient anticancer remedy are drug resistance and sever side effects [2]. Tumor has genes that express pumps. These efflux pumps result in a drug accumulation outside the malignantly mutated cells [3]. Thus, the discovering new anticancer agents with less resistance and side effects has become an urgent matter. Pyridopyrimidine ring is a vital core with various bioactivities, especially anticancer activity due to great similarity between pyridopyrimidine moiety and DNA nucleus [4]. Also, pyridopyrimidines can inhibit several enzymes presented in cancer cells such as tyrosine kinase enzymes. Tyrosine kinase inhibitors are used to treat specific types of cancer [5]. Pyridopyrimidines are considered structurally similar to pyridopyrimidines. So, the importance of pyridopyrimidinone is closely related to pyridopyrimidine. There are several reported pyridopyrimidine derivatives I–IV that exhibit potent inhibitory activities against EGFR and cyclin-dependent kinase (CDK4/cyclin D1) (Figure 1) [6–9]. Therefore, the study aimed to design new substituted or fused pyridpyrimidine derivatives with potential EGFR and CDK4-cyclin D1 inhibition activity. Also, their in vitro cytotoxicity has been screened against four cancer cell lines; cervical cancer cell line (HeLa), hepatic cancer cell line (HepG-2), colon cancer cell line (HCT-166) and breast cancer cell line (MCF-7), as well as diploid human cell line composed of fibroblasts (WI-38). Further, cell cycle arrest and apoptosis induction assay have been investigated for the most potent derivatives.

Figure 1.

Reported pyridopyrimidine derivatives that suppress both EGFR and CDK4/Cyclin D1 enzymes. Compound I inhibited CDK4/cyclin D1, compound II inhibited both EGFR and CDK4/cyclin D1 and compound III, IV inhibited EGFR enzyme.

2. Rational & design

Gefitinib (V) has been reported as a marketed EGFR inhibitor drug [10]. Therefore, some structural modifications have been investigated to increase enzyme inhibition and binding affinity to enzyme pockets. In addition, decreasing severe cytotoxicity was considered another critical purpose.

As shown in Figure 2, based on the fact that nitrogen heterocyclic compounds are known for their higher anticancer activity [11], isosteric replacement of benzene ring with the pyridine nucleus was attempted to generate a hydrogen bond acceptor. Structural modifications at positions 2, 4, 5 and 7 were done as follows: substitution with phenyl moities at positions 5 and 7 were investigated to increase hydrophobic binding within enzyme pocket via van der Waals forces; replacement of phenylamino moiety at position 4 with carbonyl group to allow H-bond formation with receptor pocket; substitutions at position 2 with a hydrazinyl group were explored and this group was further subjected to another modification based on molecular hybridization theory as shown in Figure 2.

Figure 2.

Our designed pharmacophore compared with reported Gefitinib followed by structural modifications at position 2 designing the target compounds.

First, cyclization of hydrazinyl group into pyrazole or 1,2,4 triazole moieties has been done depending on previously reported antitumor compounds II,VI [7,12]. Also, a molecular hybridization technique has been adopted with other scaffolds like various benzaldehyde and acetamide derivatives (Figure 2).

3. Experimental

3.1. Chemistry

The Stuart equipment was used to record melting points. A Bruker spectrometer (MA, USA) was used to create ¹H NMR and ¹³C NMR using (CDCl₃) or (DMSO-d₆) as solvents. The device power was 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR at the NMR Unit, Faculty of pharmacy, University of Mansoura. Thermo Scientific GCMS model ISQ was used to perform mass spectrometry (MS) spectra at the Mycology and Biotechnology Regional Center. The high-resolution mass spectrometry (HRMS) was recorded on LC/Q-TOF, 6530 (Agilent Technologies, CA, USA) at Faculty of Pharmacy, Fayoum University. The Nicolet type FTIR spectrophotometer (4000-400 cm^-1) was used to obtain Infrared absorption spectra. The TLC technique was used to detect the completion of the reaction by employing Silica gel sheets from Merck (Darmstadt, Germany).

3.1.1. Procedure for the synthesis of 5-[(4-(dimethylamino)phenyl)]-7-(4-methylphenyl)2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (3)

6-Aminothiouracil (1) (1.43 g, 0.01 mol) and prepared chalcone (2) (0.01 mol) were reacted in DMF (20 ml) in the presence of triethylamine (1 ml) at 90°C for 24 h. The product was gathered by pouring it onto crushed ice. The collected precipitate (ppt) was recrystallized from aqueous methanol to give a brown solid, m.p = 245°C, yield = 46 %. IR (cm^-1): ν 3399 (NH), 1700 (C=O); ¹H NMR (CDCl₃): δ 9.57 (s, 1H, NH, exchangeable), 9.07 (s, 1H, NH, exchangeable), 7.99 (d, 2H, J = 8.4 Hz), 7.50 (s, 1H, pyridopyrimidine- C₆), 7.38 (d, 2H, J = 8.4 Hz), 7.34 (d, 2H, J = 8.4 Hz), 6.79 (d, 2H, J = 8.4 Hz), 3.07 (s, 6H, N(CH₃)₂), 2.35 (s, 3H, CH₃).

3.1.2. Procedure for the synthesis of 5-[2-hydrazinyl(4-(dimethylamino)phenyl)]-7-(4-methylphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (4)

Pyridopyrimidine-4one derivative 3 (1.5 gm, 0.004 mol) and hydrazine hydrate 99% (0.006 mol) were reacted in ethanol 100% (15 ml) at 70°C for 15 h. The formed ppt was filtered, and then recrystallized from petroleum ether/ethylactetate (5/1) as yellow precipitate, m.p = 125–127°C, yield = 56 %. IR (cm^-1): ν 3413 (NH), 3335,3198 (NH₂), 1664 (C=O); ¹H NMR (DMSO-d6): δ 8.07 (s, H, NH, exchangeable), 8.05 (s, 1H, NH, exchangeable), 7.31 (d, 4H, J = 7.6 Hz), 7.28 (s, 1H, pyridopyrimidine-C₆), 6.73 (d, 4H, J = 8 Hz), 2.97 (s, 6H, N(CH₃)₂), 2.38 (s, 3H, CH₃).

3.1.3. Procedure for the synthesis of 2-(3-amino-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-[4-(dimethylamino)phenyl]-7-(4-methylphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (5)

Hydrazinyl derivative 4 (3.86 gm, 10 mmol) in anhydrous acetic acid and ethylcyanoacetate (1.13 gm, 10 mmol) were reacted at 90°C for 24 h. The product was obtained by pouring it into ice-cooled water and then filtering. It was purified by column chromatograph with methylene chloride: methanol (20:1). Purified dark orange precipitate was obtained, m.p = 293–295°C, yield = 40 %. IR (cm^-1): ν 3393,30332 (NH,NH₂), 1692 (C=O), 1642 (N–C=O); ¹H NMR (DMSO-d6): δ 12.54 (s, 1H, NH, exchangeable), 8.19 (d, 1H, J = 8 Hz), 8.15 (d, 2H, J = 8 Hz), 7.90 (s, 1H, pyrazolone-C₄) 7.80 (s, 1H, pyridopyrimidine-C₆), 7.39–7.35, (m, 4H), 6.76 (d, 1H, J = 8 Hz), 3.00 (s, 6H, N(CH₃)₂), 2.97 (s, 2H, NH, exchangeable), 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 170.7, 169.9, 161.4, 156.2, 155.2, 154.0, 152.9, 151.2, 131.2, 130.2, 129.5 (2C), 128.1 (2C), 125.6, 122.7 (2C), 122.1, 117.0, 115.8 (2C), 80.8, 40.6 (2C), 29.4; MS: [m/z (%), 453 (13.52, M⁺), 243.77 (100)].

3.1.4. Procedure for the synthesis of 6-[4-(dimethylamino)phenyl]-8-(4-methylphenyl)]pyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (6)

Hydrazinyl derivative 4 (0.17 g, 0.001 mol) and dimethylformamide (10 ml) were reacted at 90 Cº for 24 h. The product was obtained after filtration and recrystallization from ethanol as purified brown precipitate, m.p = 168–170°C, yield = 40 %. IR (cm^-1): ν 3301 (NH), 1676 (C=O), ¹H NMR (DMSO-d6): δ 8.24–8.07 (m, 2H), 7.97 (s, 1H, pyridopyrimidine-C₆), 7.72 (s, 1H, triazole HC = N), 7.37–7.30 (m, 4H), 6.73–6.56 (m, 2H), 2.96 (s, 6H, N(CH₃)₂), 2.38 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 169.0, 156.0, 154.8, 153.9, 153.0, 139.2, 136.3, 135.9, 130.6, 129.9 (2C), 127.9 (2C), 125.0 (2C), 120.8, 112.3 (2C), 111.5, 40.6 (2C), 21.4, MS: [m/z (%), 396 (32.81, M⁺), 378 (100)]; ESI-MS: [m/z (%), 396.1697 (M+ H)⁺].

3.1.5. Procedure for the synthesis of 3-amino-6-[4-(dimethylamino)phenyl]-8-(4-methylphenyl)pyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (7)

Hydrazinyl derivative 4 (0.17 g, 2 mmol) and ammonium thiocyanate (2.38 g, 30 mmol) were reacted in anhydrous acetic acid at 90°C for 24 h. The product was collected by pouring onto ice-cooled water then filtered and dried. Purification was done by column chromatograph with methylene chloride: methanol (20:1) as orange solid, m.p = 349°C, yield = 76 %. IR (cm^-1): ν 3416 (NH), 3128,3054 (NH₂), 1660 (C=O); ¹H NMR (DMSO-d6): δ 12.46 (s, 1H, NH, exchangeable), 8.17 (d, 2H, J = 8.4 Hz), 7.82 (s, 1H, pyridopyrimidine-C₆), 7.40–7.36 (t, 4H), 6.76 (d, 2H, J = 8.4 Hz), 3.00 (s, 6H, N(CH₃)₂), 2.98 (s, 2H, NH₂, exchangeable), 2.40 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 173.0, 156.5, 148.5, 135.4, 130.1, 129.4 (2C), 128.0 (2C), 125.5 (2C), 121.0, 111.4 (2C), 109.5, 106.0, 103.4, 102.4, 88.9, 49.1 (2C), 16.0, MS [m/z (%), 411 (24.15, M⁺), 397 (100)]; ESI-MS: [m/z (%), 411.1887 (M+ H)⁺].

3.1.6. Procedure for the synthesis of 6-[(4-(dimethylamino)phenyl]-3-phenyl-8-(4-methylphenyl)pyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (8)

Hydrazinyl derivative 4 (0.37 g, 0.001 mol) and benzoyl chloride (0.21 gm, 0.0015 mol) were reacted in dry pyridine (10 ml) at 90°C for 20 h. The product was obtained by pouring it onto ice water, and then neutralizing the reaction mixture with diluted HCL and filtering. The purified product was obtained by column chromatograph using methylene chloride: methanol [20:1] to obtain a dark green solid, m.p = 163–165°C, yield = 85 %. IR (cm^-1): ν 3214 (NH), 1681 (C=O); ¹H NMR (DMSO-d6): δ 11.5 (s, 1H, NH, exchangeable), 8.015 (d, 4H, J = 8 Hz), 7.63–7.55 (m, 3H), 7.42 (s, 1H, pyridopyrimidine-C₆), 7.35–7.25 (m, 4H), 6.74 (d, 2H, J = 8 Hz), 2.98 (s, 6H, N(CH₃)₂), 2.38 (s, 3H, CH₃), ¹³C NMR (DMSO-d6): 175.8, 168.7, 156, 155.2, 154.4, 151.9, 150.8, 146.8, 140.3, 137.0, 132.5, 130.4, 129.8 (2C), 128.8 (2C), 128.7 (2C), 128.3 (2C), 127.7 (2C), 120.4, 117.5 (2C), 111.4, 40.6 (2C), 21.3; MS [m/z (%), 472 (43.54, M⁺), 269 (100)]; EIS-MS: [m/z (%), 472.2007 (M+ H)⁺].

3.1.7. General procedure for preparation of 2-(substituted benzylidene hydrazinyl)-5,7-diarylpyrido[2,3-d] pyrimidin-4(3H)-ones 9–15

Hydrazinyl derivative 4 (3.86 gm, 10 mmol) and different aromatic aldehydes (20 mmol) were condensed in ethanol 100% (10 ml) and 1 ml of anhydrous acetic acid at 70°C for 14 h. The product was obtained by pouring it onto iced-cold water then, filtering. Schiff bases (9–15) were purified by column chromatography with an elution system methylene chloride: methanol (20:1).

3.1.8. (E)-2-{2-[(4-(Dimethylamino)benzylidene]hydrazinyl}-5-[(4-(dimethylamino)phenyl]-7-(4-methylphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (9)

The aldehyde used was 4-aminodimethylbenzaldehyde and it produced orange solid, m.p = 336–337°C, yield = 97 %. IR (cm^-1): ν 3391 (NH), 3451 (NH), 1693 (C=O); ¹H NMR (DMSO-d6): δ 9.68 (s, 1H, NH, exchangeable), 8.51 (s, 1H, pyridopyrimidine-C₆), 8.16 (d, 2H, J = 8 Hz), 8.02 (s, 1H, CH=N), 7.76–7.65 (m, 4H), 7.37–7.34 (m, 2H), 6.81–6.76 (m, 4H), 3.00 (s, 12H, 2 N(CH₃)₂, 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 161.1, 155.2, 154.3, 153.4, 152.5, 137.2, 131.2, 130.4, 130.1, 130.0, 129.8, 129.7 (2C), 128.6 (4C), 127.7, 127.4, 122.9, 121.6, 117.0, 112.9 (2C), 111.6, 40.5 (2C), 40.1 (2C), 21.5; MS: [m/z (%), 517 (30.19, M⁺), 377 (100)].

3.1.9. (E)-5-{(4-(Dimethylamino)phenyl)-2-[2-(2-hydroxy-3-methoxybenzylidene)hydrazinyl]-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (10)

Using O-Vaniline gave the product as an orange solid, m.p = 246–248°C, yield = 95%. IR (cm^-1): ν 3500 (OH), 3202 (NH), 1685 (C=O); ¹H NMR (DMSO-d6): δ 8.51 (s,1H, pyridopyrimidine-C₆), 8.46 (s, 1H, CH=N), 8.17 (d, 2H, J = 8 Hz), 7.40–7.33 (m, 4H), 7.04–6.98 (m, 1H), 6.84–6.74 (m, 4H), 3.85 (s, 1H, OH, exchangeable), 3.83 (s, 3H, OCH₃), 2.99 (s, 6H, N(CH₃)₂), 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 161.2, 159.9, 154.4, 153.3, 150.7, 150.6, 148.3, 140.4, 140.2, 136.1, 130.5, 130.4 (2C), 129.8 (2C), 127.6 (2C), 119.3, 117.7 (2C), 115.8, 111.4 (2C), 108.0, 56.3, 40.6 (2C), 21.3; MS [m/z(%), 520 (17.90, M⁺), 99 (100)]; ESI-MS: [m/z (%), 520.2224 (M+ H)⁺].

3.1.10. (E)-2-{2-[(3,4-Dimethoxybenzylidene)hydrazinyl]-5-(4-(dimethylamino)phenyl)-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (11)

Using 3,4-dimethoxybenzaldehyde produced product as yellow solid, m.p = 282–284°C, yield = 90 %. IR (cm^-1): ν 3379 (NH), 1698 (C=O); ¹H NMR (DMSO-d6): δ 11.60 (s, 1H, NH, exchangeable), 11.02 (s, 1H, NH, exchangeable), 8.22 (s, 1H, CH=N), 8.17 (d, 2H, J = 8 Hz), 7.69 (s, 1H, pyridopyrimidine-C₆), 7.46–7.28 (m, 6H), 6.99 (d, 1H, J = 8 Hz), 6.75 (d, 2H, J = 8 Hz), 3.86 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 2.99 (s, 6H, N(CH₃)₂, 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 174.7, 164.3, 160.2, 155.5, 151.1 (2C), 149.8, 148.9, 145.5, 142.4, 137.5, 135.3, 132.7, 130.6, 129.8 (2C), 127.9 (2C), 125.0, 122.9 (2C), 111.4 (2C), 82.2, 74.7, 55.8, 40.5, 21.1; MS [m/z (%), 534 (30.71, M⁺), 303 (100)].

3.1.11. (E)-5-{(4-(Dimethylamino)phenyl)-2-[2-(3-nitrobenzylidene)hydrazinyl]-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (12)

Using 3-nitrobenzaldehyde gave the product as yellow precipitate, m.p = 305–307°C, yield = 85 %. IR (cm^-1): ν 3414 (NH), 1697 (C=O), 1530 and 1351 (NO₂); ¹H NMR (DMSO-d6): δ 11.78 (s, 1H, NH, exchangeable), 11.76 (s, 1H, NH, exchangeable), 8.86 (s, 1H, CH=N), 8.445 (d, 1H, J = 4 Hz), 8.31 (s, 1H, pyridopyrimidine-C₆), 8.26–8.17 (m, 2H), 8.12 (d, 1H, J = 8 Hz), 7.76–7.69 (m, 1H), 7.47–7.34 (m, 5H), 6.76 (d, 2H, J = 8 Hz), 3.00 (s, 6H, N(CH₃)₂), 2.1 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 186.1, 175.8, 167.0, 164.4, 163.4, 153.8, 150.2, 144.7, 136.1, 135.4, 131.8, 130.9 (2C), 127.8 (2C), 125.0, 124.2 (2C), 112.5, 111.3 (2C), 110.5, 78.6, 76.5, 48.6 (2C), 25.7; MS [m/z (%), 519 (48.38, M⁺), 307 (100)].

3.1.12. (E)-5-{(4-(Dimethylamino)phenyl)-2-[2-(4-hydroxybenzylidene)hydrazinyl]-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (13)

Using p-hydroxy benzaldehyde gave orange precipitate, m.p = 263–265°C, yield = 86 %. IR (cm^-1): ν 3381 (OH), 1688 (C=O); ¹H NMR (DMSO-d6): δ 10.09 (s, 1H, NH, exchangeable), 9.86 (s, 1H, OH, exchangeable), 8.57 (s, 1H, pyridopyrimidine-C₆), 8.17–8.09 (m, 2H), 8.06 (s, 1H, CH=N), 7.80 (d, 1H, J = 8 Hz), 7.70 (d, 1H, J = 8 Hz), 7.42–7.32 (m, 4H), 6.87 (d, 1H, J = 8 Hz), 6.81 (d, 1H, J = 8 Hz), 6.75 (d, 2H, J = 8 Hz), 2.99 (s, 6H, N(CH₃)₂), 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 173.1, 163.9, 157.6, 155.5, 154.2, 153.3 (2C), 138.2, 130.6 (2C), 129.8 (2C), 127.3(2C), 126.3, 123.2 (2C), 119.5, 118 116.6 (2C), 115.8, 112.2 (2C), 111.4, 89.5, 40.6 (2C), 16.2; MS [m/z (%), 490 (50.36, M+), 181 (100)].

3.1.13. (E)-2-{2-[(4-Bromobenzylidene)hydrazinyl)]-5-(4-(dimethylamino)phenyl)-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (14)

4-Bromobenzaldehyde gave yellow solid, m.p = 346–348°C, yield = 80 %. IR (cm^-1): ν 3415 (NH), 1661 (C=O); ¹H NMR (DMSO-d6): δ 11.49 (s, 1H, NH, exchangeable), 11.32 (s, 1H, NH, exchangeable), 9.70 (s, 1H, pyridopyrimidine-C₆), 8.13 (s, 1H, CH=N), 7.99–7.94 (m, 2H), 7.84 (d, 2H, J = 8 Hz), 7.72–7.62 (m, 8H), 2.20 (s, 6H, N(CH₃)₂), 1.96 (s, 3H,CH₃); ¹³C NMR (DMSO-d6): δ 163.9, 161.2, 159.5, 158.1, 155.7, 153.3, 152.1, 136.2, 133.4, 132.5, 131.8 (2C), 130.7, 129.7 (2C), 127.4 (4C), 125.4, 124.4 (2C), 121.0, 117.1 (2C), 89.5, 40.6 (2C), 16.2; MS [m/z (%), 535 (20.9, M ⁺ + 2) 533 (58.3, M⁺), 200 (100)].

3.1.14. (E)-2-{2-[(4-Chlorobenzylidene)hydrazinyl)]-5-(4-(dimethylamino)phenyl)-7-(4-methylphenyl)}pyrido[2,3-d]pyrimidin-4(3H)-one (15)

4-Chlorobenzaldehyde gave orange precipitate, m.p = 346–348°C, yield = 85 %. IR (cm^-1): ν 3415 (NH), 1659 (C=O); ¹H NMR (DMSO-d6): δ 8.29 (s, 1H, pyridopyrimidine-C₆), 8.19–8.03 (m, 4H), 7.52 (d, 2H, J = 8 Hz), 7.41 (s, 1H, CH=N), 7.38–7.33 (m, 3H), 6.75 (d, 3H, J = 8 Hz), 2.9 (s, 6H, N(CH₃)₂), 2.34 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 162.9, 161.1, 153.5, 152.0, 148.5, 146.2, 136.5, 135.0, 133.5, 133.0, 131.8, 130.8, 130.5 (2C), 130.1, 129.5 (2C), 127.7 (4C), 127.2 (2C), 126.7, 125.9, 119.8, 117.8, 111.6 (2C), 40.6 (2C), 29.4; MS [m/z (%), 511 (14.95, M⁺ +2), 509 (22.76, M⁺), 424 (100)].

3.1.15. General procedure for the synthesis of 3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)hydrazinyl)-N-(4-methyl phenyl) acetamide derivatives 17–20

Acetamide derivatives 16a–d were synthesized through the reaction of different aniline derivatives (0.2 gm, 10 mmol) and chloroacetylchloride (1.1 gm, 10 mmol) in acetone and catalytic amount of potassium carbonate.

Hydrazinyl derivative 4 (0.1gm, 2 mmol) and different synthesized acetamide derivatives (0.002 mol) in DMF (15 ml) and triethylamine at catalytic amount were reacted at 90°C for 4 h. The obtained product was poured onto cooled water then, it was filtered. Purification of desired products was done by applying column chromatograph with elution system methylene chloride: methanol [20:1].

3.1.16. 2-{2-[5-(4-(Dimethylamino)phenyl]-4-oxo-7-(4-methylphenyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)hydrazinyl}-N-(4-methylphenyl)acetamide (17)

4-Methyl phenyl acetamide derivative gave yellow precipitate, m.p = 296-298°C, yield = 55%. IR (cm^-1): ν 3426 (NH), 1596 (C=O), 1570 (C=O); ¹H NMR (DMSO-d6): δ 8.41 (s, 1H, pyridopyrimidine-C₆), 8.11 (d, 3H, J = 8 Hz), 7.37–7.32 (m, 6H), 6.74 (d, 3H, J = 8 Hz), 4.68 (s, 1H, NH, exchangeable), 3.95 (s, 2H, CH₂), 3.65 (s, 6H, N(CH₃)₂), 2.98 (s, 3H,CH₃), 2.39 (s, 3H, CH₃); ¹³C NMR (DMSO-d6): δ 175.2, 174.2, 169.8, 166.6, 164.5, 163.7, 151.7, 149.3, 147.4, 140.3, 137.6, 129.5 (4C), 128.1 (2C), 124.3 (4C), 119.7, 118.4 (2C), 73.2, 71.4, 60.7, 40.5 (2C), 25.7 (2C), MS [m/z (%), 533 (30.99, M⁺), 388 (100)].

3.1.17. 2-{2-[5-(4-(Dimethylamino)phenyl]-4-oxo-7-(4-methylphenyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)hydrazinyl}-N-(4-methoxyphenyl)acetamide (18)

p-Methoxyphenyl acetamide derivative gave orange precipitate, m.p = 168–170°C, yield = 50 %. IR (cm^-1): ν 3273 (NH), 1685 (C=O), 1608 (C=O); ¹H NMR (DMSO-d6): δ 10.25 (s, 1H, NH, exchangeable), 8.59 (s, 1H, pyridopyrimidine-C₆), 8.21 (d, 1H, J = 8 Hz), 8.12 (d, 1H, J = 8 Hz), 8.09 (s, 1H, NH, exchangeable), 7.80 (s, 1H, NH, exchangeable), 7.48 (d, 2H, J = 8 Hz), 7.40–7.26 (m, 4H), 6.90 (d, 2H, J = 8 Hz), 6.77–6.73 (t, 2H), 4.75 (s, 2H, CH₂), 3.72 (s, 6H, N(CH₃)₂), 3.52 (s, 3H, OCH₃), 2.98 (s, 3H, CH₃)); ¹³C NMR (DMSO-d6): δ 168.9, 160.1, 158, 155.5, 155.2, 153.6, 152.4, 151, 140.8, 132.5, 130.6, 130.0 (2C), 128.4 (2C), 127.9, 123.8 (2C), 121.8 (2C), 121.0, 119.5, 114.4 (2C), 111.9 (2C), 111.5, 55.6, 55.4, 40.5 (2C), 21.4, MS [m/z (%), 549 (19.58, M⁺), 212 (100)]; ESI-MS: [m/z (%), 549.2526 (M+ H)⁺].

3.1.18. 2-{2-[5-(4-(Dimethylamino)phenyl]-4-oxo-7-(4-methylphenyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)hydrazinyl}-N-phenyl acetamide (19)

N-Phenylacetamide derivative gave orange precipitate, m.p = 198°C, yield = 30 %. IR (cm^-1): ν 3327 (NH), 1689 (C=O), 1606 (C=O); ¹H NMR (CDCL₃): δ 8.02 (s, 1H, pyridopyrimidine-C6), 7.85 (d, 2H, J = 8 Hz, Ar-H), 7.19–6.72 (m, 8H, Ar-H), 6.71–6.65 (m, 3H, Ar-H), 3.67 (s, 2H, CH₂), 2.97 (s, 6H, (NCH₃)₂), 2.33 (s, 3H, CH₃); ¹³C NMR (CDCL₃): δ 174.7, 172.4, 155.7, 152.8, 129.8, 129.7, 129.6 (2C), 128.9 (2C), 128.7 (2C), 127.9, 127.6, 126.3 (2C), 124.7 (2C), 119.9, 111.2 (2C), 108.9, 108, 76.7, 40.2 (2C), 29.7, MS [m/z(%), 519 (16.35, M⁺), 347 (100)].

3.1.19. 2-{2-[5-(4-(Dimethylamino)phenyl]-4-oxo-7-(4-methylphenyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)hydrazinyl}-N-(4-fluorophenyl)acetamide (20)

4-Fluorophenylacetamide derivative gave brown precipitate, m.p = 230 ºC, yield = 45 %. IR (cm^-1): ν 3419 (NH), 1676 (C=O), 1611 (C=O); ¹H NMR (DMSO-d6): δ 10.43 (s, 1H, NH, exchangeable), 8.59 (s, 1H, pyridopyrimidine-C₆), 8.34–8.13 (m, 2H), 7.78 (s, 1H, NH, exchangeable), 7.58–7.16 (m, 8H), 6.74–6.56 (m, 2H), 4.77 (s, 2H, CH₂), 2.96 (s, 6H, N(CH₃)₂), 2.40 (s, 3H, CH₃), ¹³C NMR (DMSO-d6): δ 165.8, 162.9, 160.3, 154.9, 153.9, 152.1, 135.5, 132, 130.6, 130.0 (2C), 127.9 (2C), 123.4 (2C), 121.4, 120.6 (2C), 117.8, 115.9, 115.7 (2C), 111.6 (2C), 111.5, 66.9, 40.6 (2C), 21.4, MS [m/z (%), 537 (34.79, M⁺), 523 (100)].

3.2. Biological screening

3.2.1. In vitro cytotoxicity assay

MMT, DMSO, the medium RPMI-1640 (MI, US ) and Bovine Fetal serum (GIBCO, New York, UK) were used as reagents. Colorectal, colon cancer (HCT-116), cervix cancer (HeLa), breast cancer (MCF-7) and hepatic cancer (HepG-2) cell lines have been used to evaluate anticancer inhibition for all synthesized target compounds (3–20). As well, normal lung fibroblasts in humans (WI38) have been used to detect the toxicity of the compounds against normal cells. Vaccines and Biological Products Holding Company (VACSERA; Cairo, Egypt) provided the ATCC with the cell lines. MTT assay has been applied according the previously reported procedure [13,14] using Doxorubicin as a reference anticancer medication.

3.2.2. Kinase inhibition assay

3.2.2.1. EGFR inhibition assay

EGFR enzyme inhibition has been evaluated by using Elabscience Human EGFR ELISA Kit based on the reported method [15].

3.2.2.2. CDK/cyclinD1 inhibition assay

The CDK Assay Kit has been used to detect CDK4/cyclinD1 inhibition enzyme based on the previously described method [16].

3.2.3. Cell cycle arrest

The cell cycle arrest has been evaluated by using propidium iodide flow cytometry method according to the reported method [17].

3.2.4. Apoptotic assay

The apoptotic assay has been evaluated by utilizing a Detection Kit for Annexin V-FITC Apoptosis based on the reported procedure [17].

3.2.5. The evaluation of Bax & Bcl2 expression levels

The most active compounds 5 and 10 have been evaluated for their activity upon pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 using one-step real time-reverse transcription polymerase chain reaction (RT-PCR) Kit with SYBR Green according to the procedure [18].

3.2.6. Molecular modeling Investigation

Docking simulation and S score have been evaluated using Molecular Operating Environment (MOE) 2015.10. EGFR 3D x-ray structures [19,20] and CDK6 crystallized structure [21,22] have been obtained from the Protein Data Bank. Due to the similarity in structure between CDK4 and CDK6, we used CDK6 protein structure [23].

4. Results

4.1. Chemistry

Regarding to Figure 3, the synthesis of prop-2-en-1-one (intermediate chalcone) 2 was first done through the reaction of N-dimethyl amino benzaldehyde and 4-methyl acetophenone [24].

Figure 3.

Synthetic method of compounds (9–20) with different conditions: (A) DMF, TEA, 90°C, 24 h; (B) NH₂-NH₂.H₂O, EtOH, 70°C, 10–15 h; (C) Ethylcyanoacetate, anhydrous acetic acid, 90°C, 24 h; (D) DMF, 90°C, 24 h; (E) ammonuim thiocyanate, anhydrous acetic acid, 90°C, 24 h; (F) benzoyl chloride, dry pyridine, 90°C, 20 h.

Pyridopyrimidine 3 was synthesized through the reaction of 6-aminothiouracil and prepared prop-2-en-1-one 2 in dry DMF and a catalytic amount of triethylamine (TEA) according to the reported method [25]. In addition, triethyl hydrogen sulfate was used as green chemistry, which showed also higher yield and lower chemical hazards.

Nucleophilic substitution reaction of hydrazine hydrate with compound 3 yielded hydrazine derivative 4 (Figure 3), according to the previously reported procedure [26]. Hydrazinyl derivative 4 was allowed to condense with different reagents for example, ethylcyanoacetate, DMF, ammonium thiocyanate and benzoyl chloride under various reaction conditions [12,27].

First, condensation of hydrazinyl derivative 4 with ethylcyanoacetate occurred in anhydrous acetic acid to give the corresponding pyrazolone derivative 5.

Further, the reaction of hydrazinyl derivative 4 with DMF resulted in the formation of tricyclic pyridotriazolopyrimidine derivative 6.

Hydrazinyl derivative 4 was also allowed to react with ammonium isothiocyanate in anhydrous acetic acid resulting in 3-amino triazole derivative 7.

Further, hydrazinyl derivative 4 was reacted with benzoyl chloride in pyridine to give 3-phenyltriazole derivative 8.

Regarding Figure 4, hydrazinyl derivative 4 was reacted with benzaldehydes and yielded the corresponding schiff bases 9–15 [26,28,29].

Figure 4.

Synthetic method of compounds (9–20) with different conditions: (A) EtOH, anhydrous acetic acid, 70°C, 14 h; (B) DMF, TEA, 90°C, 4 h.

N-acetamide derivatives 16a–d were synthesized by reacting of various aniline derivatives and chloroacetylchloride in acetone and a catalytic amount of K₂CO₃ [30]. Then, hydrazinyl acetamide derivatives 17–20 were prepared by reacting N-acetamide derivatives 16a–d with hydrazinyl derivative 4 in DMF and catalytic amount of TEA (Figure 4) [31].

4.2. Biological screening

4.2.1. In vitro Cytotoxicity assay

Four cancerous cells were utilized to evaluate the anti-cancer properties of all 3–20 synthesized compounds namely, hepatic cancer (HepG-2), breast cancer (MCF-7), cervical cancer (HeLa) and colon cancer (HCT-166) cells, as well normal human lung fibroblast cells (WI-38). Doxorubicin was used as a reference medication in initial screening against cancer cell lines, with the doses of 100 μM. For the compounds 3–20 that were evaluated, inconsistent outcomes were recorded (Table 1).

Table 1.

Inhibition the growth of cells (IC₅₀) values of target compounds 3–20 against four malignant cells and normal human lung fibroblast cells (WI 38).

Comp. No.	IC50 (μM) for in vitro cytotoxicity
	WI-38	HeLa	HePG-2	HCT-116	MCF-7
3	62.34 ± 3.5	31.83 ± 1.9	15.47 ± 1.2	24.85 ± 1.8	13.44 ± 1.0
4	34.77 ± 2.4	4.28 ± 0.2	1.82 ± 0.1	6.24 ± 0.3	2.86 ± 0.1
5	25.20 ± 1.8	9.27 ± 0.6	5.91 ± 0.3	12.94 ± 0.9	7.69 ± 0.5
6	75.76 ± 3.8	35.44 ± 2.2	21.41 ± 1.5	28.67 ± 2.0	19.50 ± 1.4
7	18.01 ± 1.4	61.71 ± 3.6	47.46 ± 2.6	56.35 ± 3.2	63.83 ± 3.4
8	>100	82.75 ± 4.1	50.09 ± 2.8	79.33 ± 4.1	58.80 ± 3.0
9	30.17 ± 2.2	95.68 ± 5.1	69.43 ± 3.7	89.67 ± 4.5	74.89 ± 3.9
10	>100	23.26 ± 1.5	10.09 ± 0.8	16.14 ± 1.2	9.72 ± 0.8
11	86.85 ± 4.5	>100	71.38 ± 3.8	91.93 ± 4.8	78.32 ± 3.8
12	39.96 ± 2.5	>100	76.41 ± 3.9	>100	81.85 ± 4.1
13	52.76 ± 3.1	>100	84.96 ± 4.2	>100	92.35 ± 4.7
14	>100	77.56 ± 3.8	64.07 ± 3.3	72.63 ± 3.8	53.11 ± 2.8
15	27.63 ± 2.0	54.06 ± 3.2	42.86 ± 2.3	51.14 ± 2.9	45.98 ± 2.6
17	55.43 ± 3.3	73.34 ± 3.9	39.29 ± 2.4	65.48 ± 3.6	41.17 ± 2.4
18	48.68 ± 2.9	88.60 ± 4.4	67.72 ± 3.6	83.56 ± 4.3	70.35 ± 3.7
19	>100	46.60 ± 2.7	37.44 ± 2.2	44.56 ± 2.5	32.65 ± 2.1
20	87.28 ± 4.7	38.39 ± 2.3	29.73 ± 1.9	36.77 ± 2.2	26.10 ± 1.8
DOX	6.72 ± 0.5	5.57 ± 0.4	4.50 ± 0.2	5.23 ± 0.3	4.17 ± 0.2

Bolded values represent the most potent derivatives.

DOX: Doxorubicin; IC₅₀: The average ± standard deviation of five independent experiments.

4.2.2. Kinase inhibition assay

Compounds 4, 5 and 10 have shown promising inhibition toward malignant cell lines specifically, MCF-7 and HepG-2. Thus, the EGFR and CDK-cyclinD1 inhibition assay have been applied for these most potent antitumor compounds to investigate their manner of action.

4.2.2.1. Inhibition of the EGFR

Compounds 4, 5 and 10 have been evaluated for their EGFR enzyme inhibition and the results have been presented in Table 2.

Table 2.

EGFR and CDK4/cyclin D1 inhibition (IC₅₀) of compounds 4, 5 and 10 in HepG-2 and MCF-7 cell lines versus Erlotinib and Palbociclib, respectively.

Comp. No.	Cell line	EGFR IC50 (ng/ml)	CDK4/cyclinD1 IC50 (µg/ml)
4	HepG-2	96	0.172
5	HepG-2	3.114	0.152
	MCF-7	2.852
10	HepG-2	4.479	0.349
	MCF-7	3.382
Erlotinib/Palbociclib	HepG-2	2.247	0.034

4.2.2.2. Cyclin dependent kinase 4-cyclin D1 inhibition

Compounds 4, 5 and 10 have been evaluated for their direct inhibition of CDK4/cyclin D1 relative to reference Palbociclib, as shown in Table 2.

4.2.3. Cell cycle analysis

Compounds 5 and 10 have been exposed to cell cycle analysis in HepG-2 cell line. They have been chosen to investigate their effects on the growth of the cell cycle as presented in Figure 5 (Supplementary Table S1).

Figure 5.

Cell cycle arrest and apoptosis assay for pyridopyrimidines 5 and 10. (A) Flow cytometry chart of pyridopyrimidines 5 and 10 upon cell cycle progression of HepG-2 cell line compared with control HepG-2 cells. (B) Apoptotic effect induced by pyridopyrimidines 5 and 10 on HepG-2 cell lines using Annexin VIPI double staining technique compared with control HepG-2 cells.

PI: Propidium iodide.

4.2.4. Apoptotic assay

Using flow cytometry and Annexin V-FITC-treated cells, cellular apoptosis has been assessed for the most active compounds 5 and 10. The findings have been displayed in Figure 5 (Supplementary Table S1).

4.2.5. Real-time reverse transcription polymerase chain reaction for Bax & Bcl2

RT-PCR analysis of Bax and Bcl2 has been conducted for the most active compounds 5 and 10 to show their apoptotic mechanism. The results have been presented in the Supplementary Table S4.

4.2.6. Molecular Modeling

4.2.6.1. Molecular docking on EGFR & CDK4/cyclin D1 pockets

Kinase inhibition assay showed that compounds 5 and 10 had promising EGFR and CDK4/cyclin D1 inhibition. Thus, docking simulation has been done between EGFR and CDK4/cyclinD1 using CDK6 enzymes as macromolecule and our most potent synthesized compounds 5 and 10 as ligands. The Molecular Operating Environment (MOE) has automatically performed all docking calculations and interactions 2015.10.

As shown in Figure 6, compound 5 has bound with the EGFR backbone versus Erlotinib as a reference ligand (Supplementary Table S2).

Figure 6.

2D presentation of pyridopyrimidine 5 with EGFR and CDK6 binding site versus Erlotinib and ligand PD0332991, respectively as references. (A) Binding of pyridopyrimidine 5 with EGFR, compared to Erlotinib binding, (B) Binding of pyridopyrimidine 5 with CDK6 versus the ligand PD0332991 binding. 

Also, compound 10 has interacted with EGFR backbone (Supplementary Figures S1 & S2) (Supplementary Table S2).

As shown in Figure 6, compound 5 has interacted with CDK4/cyclin D1 pocket versus PD0332991 as a reference ligand. 3D presentation was presented in Supplementary Figure S3.

Also, compound 10 has bound with CDK4/cyclin D1 pocket (Supplementary Figures S1 & S3). The docking score of compound 10 was lower than that of the ligand (Supplementary Table S3).

5. Discussion

5.1. Chemistry

We aimed to synthesis the pyridopyrimidine nucleus then, prepare the anti-cancer pyridopyrimidine derivatives.

Compound 2 as intermediate was synthesized and confirmed through IR spectroscopy which showed the formation of the conjugated carbonyl group at 1645 cm^-1 and the disappearance of formyl proton (HC=O) at 2700 and 2800 cm^-1 which have proven the reaction completion.

Pyridopyrimidine 3 was confirmed through ¹H NMR spectrum which showed three singlet peaks at 7.54, 9.07 and 9.57 ppm, corresponding to pyridopyrimidine-C₆-H and 2 heteroatom-H (X-H) protons. In addition, two singlet peaks at 3.07 and 2.46 ppm were attributed to N(CH₃)₂ and CH₃ groups, respectively.

In the IR spectrum, compound 4 showed two peaks at 3335 and 3402 cm^-1 for (NH₂) group and a sharp peak at 1664 cm^-1 for the (C=O) group. Furthermore, ¹H NMR spectral data exhibited two singlet peaks at 8.07, and 8.05 ppm for 2NH protons.

Pyrazolone derivative 5 was confirmed through the ¹H NMR, IR and MS spectra. ¹H NMR showed a characteristic singlet peak at 7.91 ppm corresponding to C₄-H of pyrazolone. In addition, the IR spectrum showed bands at 3393, 30332 cm^-1 for (NH, NH₂) groups, a sharp band at 1692 cm^-1 for (C=O) group and a band at 1642 cm^-1 for (N–C=O) group. Further, the mass spectrum showed a peak of molecular ion at m/z 453, which agreed with molecular weight of the structure.

Tricyclic pyridotriazolopyrimidine derivative 6 was confirmed through ¹H NMR spectrum which showed a singlet peak at δ 8.24 ppm, due to the azomethine proton (CH=N) of triazole ring. In addition, the ¹³C NMR spectrum showed a characteristic signal of CH=N at 163.3 ppm. Moreover, EIS-MS spectrum showed (M + H)⁺ peak at m/z, 396.1697.

The structure of derivative 7 was confirmed through the ¹H NMR spectrum showed two singlet peaks at 12.46 and 2.98 ppm, corresponding to NH and NH₂ protons, respectively. Also, EIS-MS showed (M+ H)⁺ peak at m/z, 411.1887, which confirmed the exact molecular weight of compound.

¹H NMR of compound 8 showed singlet peak at 11.5 ppm, assigned to the proton of NH. Furthermore, there was an increase in the aromatic integration due to the phenyl ring at the third position of the 1,2,4 triazole ring. In addition, EIS-MS showed (M+ H)⁺ peak at m/z, 472.2007.

In HNMR spectrum, the singlet peak appeared at about 8.25 ppm for –N=CH proton for the compounds 9–15. ¹³C NMR spectrum also showed signals around 148.30 and 161.20 ppm, assigned for (–N=CH) and (C=O) carbons, respectively. Most compounds' mass spectra were in agreement with a molecular ion peak. For example, derivative 15 showed a peak of molecular ion [M⁺] at m/z 509 and [M ⁺ + 2], at m/z 511, which are indicative of the isotope of chlorine. Moreover, compound 10 showed (M + H)⁺ peak at m/z 520.2224 in EIS-MS.

N-acetamide derivatives 16a–d were confirmed through an IR spectrum that showed a sharp peak at 1670 cm^-1 for (C=O) of amide and a broad peak at 3295 cm^-1 for NH groups with the disappearance of two spikes of NH₂ group of starting material.

In ¹HNMR, the recently prepared compounds 17–20 showed a characteristic singlet peak at 4.3 ppm, indicating CH₂ protons. In addition, the increase in aromatic integration has been noticed because of the new phenyl ring. Also, ¹³C NMR spectrum showed signal around 61.2 ppm, attributed for CH₂ carbon. For example, compound 18 showed (M+ H)⁺ peak at m/z 549.2526 in EIS-MS.

5.2. Biological screening

5.2.1. In vitro Cytotoxicity assay

Among the tested compounds, derivatives 4, 5 and 10 showed remarkable activity toward HepG-2. Pyrazolylpyrido[2,3-d] pyrimidine 5 demonstrated extremely potent activity against HepG-2 (IC₅₀ = 5.9 μM) with equipotent activity relative to Doxorubicin. Additionally, compound 10 exhibited a highly effective anti-HepG-2 activity (IC₅₀ = 10.0 μM). While compounds 6, 15, 17, 19 and 20 showed moderate activity with IC₅₀ range of (21.41–47.46 μM). Other compounds 7, 8, 9, 11, 12, 13, 14, 18 showed weak activity against HepG-2 cell line.

Concerning the MCF-7 cell line, compounds 4, 5 and 10 demonstrated potent activity against MCF-7 (IC₅₀ = 2.86, 7.69 and 9.72 μM, respectively). Derivative 6 showed strong activity against MCF-7 (IC₅₀ = 19.50 μM). Compounds 15, 17, 20 revealed moderate activity against MCF-7 cell line.

Considering, cervical (HeLa) and colon (HCT-116) cancer cell lines, compound 4 demonstrated remarkably high efficacy against them (IC₅₀ 4.28 and 6.24 μM), respectively. Also, compound 5 showed strong activity toward both (IC₅₀ 9.27 and 12.94 μM, respectively). While most of the tested compounds showed weak activity, except for compounds 6, 10, 19 and 20 showed moderate activity.

Hydrazinyl derivative 4 exhibited the strongest activities against all tested cancer cell lines, IC₅₀ = 1.82, 2.86, 4.28 and 6.24 μM, corresponding to HepG-2, MCF-7 HeLa and HCT-116, respectively.

The cytotoxicity of the new pyridopyrimidines has been tested against normal human lung fibroblast cells (WI-38). All synthesized compounds showed low toxicity against normal cells compared with Doxorubicin which has IC₅₀ = 6.72 μM in WI-38 cell line. The results are shown in Table 1.

The promising potent compounds 5 and 10 had IC₅₀ = 25.20 and 100 μM, respectively against normal cell WI-38. Both promising two compounds indicated safety more than Doxorubicin.

5.2.2. Structure–activity relationship analysis

Fused pyridopyrimidine with pyrazole ring at position 2 (derivative 5) showed superior anti-tumor behavior toward HepG-2, MCF-7 and HeLa. This showed that fused pyrido[2,3-d] pyrimidine with pyrazole ring exhibited broad anti-cancer activity. Fused pyrido[2,3-d] pyrimidine compounds with unsubstituted triazole ring showed moderate anticancer activity, while substituted triazole with aryl or amino groups showed weak activity. In addition, pyridopyrimidine with open chain linker having different benzaldehyde derivatives at position 2 showed medium to low anticancer activity, except for derivative 10 which showed superior anti-cancer activity against HepG-2 and MCF-7. This may suggest that two distinct electron-donating groups (OH, OCH₃) on phenyl ring have improved the anti-cancer activity compared with electron withdrawing groups. Derivatives 12, 14 and 15 with electron withdrawing groups showed low anti-cancer activity.

Regarding pyridopyrimidine derivatives with open chain linkers having different phenyl acetamide moieties, moderate anticancer activity was observed except for derivative 17, which showed low anticancer activity.

Thioxo pyridopyrimidine precursor 3 showed low anti-cancer activity, while it is converted to hydrazide derivative 4 and it gives strong anticancer activity almost resembling standard drug. This may be due to many reasons such as:

1. Hydrazide derivatives were previously reported as biologically important scaffold which showed anticancer, anti-bacterial and other numerous of biological activities [32].

2. The nitrogen heterocyclic compounds are known for their higher anticancer activity [11].

3. Hydrazide derivative bound with enzyme pockets is more than thioxo derivative depending on molecular docking technique.

So, hydrazine moiety is important to improve anticancer activity.

5.2.3. Kinase inhibition assay

5.2.3.1. Inhibition of the epidermal growth factor receptor (EGFR)

Compound 4 inhibited EGFR enzyme at 96 ng/ml in the HepG-2 cell line, relative to reference Erlotinib.

Compound 5 showed EGFR enzyme inhibition in good value of 54.7% at 3.114 ng/ml in HepG-2 cell line and 29.8% at 2.852 ng/ml in MCF-7 cell line, relative to the standard Erlotinib.

Compound 10 has shown reasonable inhibition of EGFR enzyme in HepG-2 and MCF-7 cell lines that possess values 34.8 and 14.5%, respectively, compared with Erlotinib. So, compound 5 has indicated superior inhibition of EGFR enzyme in MCF-7 and HepG-2 malignant cells.

5.2.3.2. Cyclin dependent kinase 4-cyclin D1 inhibition

Compounds 4, 5 and 10 have shown direct inhibition of CDK4/cyclin D1 with high values of 92.6, 93.5 and 90.7%, respectively relative to reference Palbociclib.

5.2.4. Cell cycle analysis

The results have revealed a growth in the G0/G1 phase cells for both compounds 5 and 10. They could arrest HepG-2 cells in the G1 phase through the accumulation of cells by 55.13 and 49.51%, respectively, relative to the control. Further, both compounds 5 and 10 exhibited a reduction in the S phase cell accumulation (28.64 and 32.19%, respectively) as well as in G2/M phase (16.23 and 18.3%, respectively) compared with the control 44%. These findings prove that compounds 5 and 10 cause complete cell growth arrest at the G1 phase by inducing apoptosis at the preG1 phase.

5.2.5. Apoptosis assay

The results have shown that compounds 5 and 10 led to early apoptosis by 18.11 and 15.37%, respectively in HepG-2 cell lines. Moreover, they caused apoptosis at the late stage by 33.91 and 26.85%, respectively compared with control HepG-2 cells. So, compounds 5 and 10 have been valued as apoptotic inducers.

5.2.6. Real-time reverse transcription polymerase chain reaction for Bax & Bcl2

Compounds 5 and 10 have shown an increase in the expression levels of apoptotic protein (Bax), relative to control cells with fold change 2.65 and 5.1089, respectively. Also, compounds 5 and 10 have declined anti-apoptotic protein (Bcl2) with fold change 0.732 and 0.287, respectively. So, compounds 5 and 10 have been suggested to induce the intrinsic apoptosis pathway by increasing the level of pro-apoptotic Bax and decreasing the level of anti-apoptotic Bcl2.

5.2.7. Molecular modeling

Compound 5 bound with EGFR pocket through hydrogen bonding with Met769 through carbonyl group of pyrazolone ring. Also, it interacted with Thr766 presented in the pocket side chain through an amino group of pyrazolone ring via hydrogen bond. Hydrophobic interactions were also observed through aromatic part of pyrimidine ring with Gly772 and Leu694 amino acids. A binding score of compound 5 recorded a lower value of 6.80 kcal /mol than standard Erlotinib that recorded -5.35 kcal/mol. These findings went with EGFR assay result.

Erlotinib interacted through hydrogen bonding with the binding site backbone via Met769 occurred in compound 5 and Gln767 in binding site backbone formed hydrogen bond with Erlotinib.

Compound 10 has interacted through the hydroxyl group of benzylidene ring with Thr766, which was found in EGFR backbone through hydrogen bond interaction and hydrophobic interaction was observed with Cys773 through its pyridine ring.

Regarding compound 10, its binding with EGFR receptor has not occurred through the same amino acids, but it has formed hydrogen bonding with binding site backbone as in Erlotinib and its docking score has been found and was lower than Erlotinib with good EGFR inhibition assay result.

Compound 5 has interacted with CDK4/cyclin D1 pocket through hydrophobic interaction with Ile19, which resembles the ligand PD0332991, but compound 5 has interacted through low docking energy score. In addition, the free amino group of pyrazolone ring has interacted with Val101 and Asp102 through hydrogen bonding.

Ligand PD0332991, as reference was found to bind with CDK6 via hydrophobic interaction with Ile19 and hydrogen bond interaction with Val101 and Asp163. Also, the ligand was found to bind with CDK6 though hydrogen bonding with Thr107.

Compound 10 has bound with CDK4/cyclin D1 pocket via Asp104 and Asp163 amino acids through hydrophobic interaction unlike the ligand that has bound through hydrogen bond with Asp163, and it formed hydrophobic interaction with Ile19 as found in ligand. Compound 10 also interacted with Thr107 through hydrophobic interaction. The docking score of compound 10 was lower than that of the ligand.

5.2.8. Drug likeness prediction & ADME toxicity screening

Absorption, distribution, metabolism and excretion (ADME) properties have been evaluated for our newly synthesized compounds 5–20 using online Molinspiration property program [33]. This software uses Lipinski's rule of five, which evaluates the properties of tested compounds. According to this rule, the compound that has no more than one violation can be considered as orally active drug.

The results showed that compounds 5–7 can be used as bioavailable drugs as they do not exhibit any violation of Lipinski's rule. While compounds 8, 10 and 13 had only one violation so, the rule can be also applied to them. Compounds 9, 11, 12, 14, 15, 17–20 had clog p < 5 and more than one violation (Supplementary Table S5).

From these findings, the most biologically active compounds 5 and 10 have been considered as orally active drugs, according to the Lipinski's rule.

6. Conclusion

A new class of pyrido[2,3-d] pyrimidines has been designed, produced and evaluated for their antitumor activities toward four cancer cell lines, relative to Dox. The most active compounds 5 and 10 showed anticancer activity against HepG-2 and MCF-7. Also, compound 5 showed considerable HCT-116 and HeLa cancer cell line inhibition. Moreover, compounds 5 and 10 strongly inhibited EGFR in HepG-2 cancer cell line and CDK4-cyclin D1 utilizing Erlotinib and Palbociclib, respectively, as reference drugs. In addition, compounds 5 and 10 are assessed for cell cycle arrest and apoptosis. In addition, they showed good fitting to EGFR and CDK4-cyclin D1 pockets. Fortunately, compounds 5 and 10 have obeyed the Lipinski's rule, thus they can be considered bioavailable drugs.

Supplemental material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2366147

Financial disclosure

The authors have no 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.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity 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 disclosure

No writing assistance was utilized in the production of this manuscript.

Data availability declaration

It is simple to access the reference section, which includes works that complement the findings of this work.

The authors certify that the supplementary materials for this article are available, which support the study's conclusions.