# Kinase Inhibitors of Novel Pyridopyrimidinone Candidates: Synthesis andIn Vitro Anticancer Properties

Authors: Nagy M. Khalifa; Mohamed A. Al-Omar; Hamad M. Alkahtani; Ahmed H. Bakheit
Journal: Journal of Chemistry
 (2019)

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## Abstract

A new class of pyridopyrimidinone compounds containing different nitrogenous heterocycles were synthesized starting from the key precursor 2-hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one via condensation with series of aromatic aldehydes and cyclization using different reagents as ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, and ammonium isothiocyanate. The bioassay results showed compound 6 to be highly effective towards three human cancer cell lines (HepG2, PC-3, and HCT-116) in vitro with promising activity values (IC50: 0.5 μM) relative to the standard doxorubicin (IC50: 0.6 μM). Kinase inhibitory evaluation of compound 6 displays hopeful inhibitory action against BRAF V600E, EGFR, and PDGFRβ at100 μM. The molecular docking studies supported the initial kinase assay.


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## Body

## 1. Introduction

Cancer is a great public health issue characterized by an uncontrolled increase of cancer cells through cell division and the cells undergo modification by their DNA, leading to death [1–3]. Due to drug resistance and the serious effects of treatment by chemotherapy, the use of available chemotherapeutics is often limited [4]. The combination of chemotherapies with several targets increases selectivity, reduces the resistance, and lowers toxicity towards infected and noninfected cells. Heterocycles have emerged as strong scaffolds for numerous biological considerations [5] and represent a significant part in the designing and detection of novel pharmacologically active entities [6]. The pyridopyrimidine compounds are a group of fused heterocycles that possess various pharmacological applications as antitumor, topoisomerase I inhibitor, adenosine kinase inhibitor, growth regulator, antihepatitis C virus, antiinflammatory, antileishmanial, antiviral, antimicrobial, anticonvulsant, antimycobacterial, CNS depressant, antihypertensive, antiallergic, diuretic, tyrosine kinase inhibitor, and calcium channel antagonist [7–16]. Among them, pyrido[2,3-d]pyrimidin-4-ones (A–C) were found to lower cell proliferation in various cancer cell lines through inhibition of various kinases, e.g., TKs, PI3K, and CDK4/6 (Figure 1) [17–19]. In continuation of our earlier studies that involved synthesis of different other substituted pyridopyrimidine compounds [20–22] and based on the structural features of pyrido[2,3-d]pyrimidine, this study is designed to synthesize various groups containing different substituents in the phenyl ring at position 5 of the parent compound pyrido[2,3-d]pyrimidinone to further improve the SAR-relationship for their cytotoxicity and also for their inhibitory activity against TKs, CDK4/6, and PI3K enzymes.Figure 1
Reported and suggested pyridopyrimidines integrated with kinase inhibitors and anticancer properties.


## 2. Materials and Methods

### 2.1. General Information

Electrothermal apparatus with open capillary tubes was used in measuring melting points. Shimadzu 435 IR spectrophotometer was used in measuring IR spectra (KBr). Varian Mercury VX-300 NMR spectrophotometer was used in determining NMR spectra using DMSO-d6 as a solvent and TMS as internal standard. The proposed structures were within ±0.4% of the theoretical values in microanalyses data. Shimadzu GC/MS-QP 2010 plus spectrometer was used in recording mass spectra.


### 2.2. Chemistry

#### 2.2.1. 5-Phenyl-7-(pyridin-3-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (3)

In dry DMF (20 mL), equimolar quantities (0.01 mol) of 6-amino-2,3-dihydro-2-thioxopyrimidin-4(1H)-one 1 and α, β-unsaturated ketone 2 were refluxed for 10 h (monitored with TLC). The residue created was gathered and purified from DMF. Yield: 67%; mp: >300°C; IR (KBr, νmax, cm−1): 3327, 3234 (2NH), 1668 (CO), 1178 (CS); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.65 (m, 10H, Ar), 12.26, 13.01 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 108.94, 113.21, 123.87, 127.19, 129.45, 134.59, 135.01, 137.66, 147.82, 149.65, 151.14, 153.02, 153.72, 159.97, 178.01; MS: [m/z, 332 (M+)]; Anal. Calcd for: C18H12N4OS (332.38): C, 65.04; H, 3.64; N, 16.86% Found: C; 64.92, H, 3.57; N, 16.71.


#### 2.2.2. 2-Hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (4)

In dry ethyl alcohol (30 mL), (0.003 mol) of 2-thioxo derivative3 and (0.005 mol) of hydrazine hydrate 99% was heated for 12 h. The precipitate created was purified in DMF. Yield: 72%; mp: 286–287°C; IR (KBr, νmax, cm−1): 3446 (NH2), 3362, 3205 (2NH), 1671 (C=O); 1H NMR (δ, ppm, DMSO-d6): 4.80 (s, 2H, NH2), 7.21–8.73 (m, 10H, Ar), 11.38, 12.76, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 118.74, 121.02, 123.90, 126.38, 129.17, 130.01, 134.57, 135.01, 137.21, 147.83, 149.96, 151.93, 152.47, 153.12, 159.97, 162.41; MS: [m/z, 330 (M+)]; Anal. Calcd for: C18H14N6O (330.34): C, 65.44; H, 4.27; N, 25.44% Found: C; 65.29, H, 4.12; N, 25.34.


#### 2.2.3. Synthetic Method for Derivatives (5a–g)

In glacial acetic acid (10 mL), equimolar amounts of (0.001 mol) of hydrazinyl derivative4 and several aromatic aldehydes, benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-tolylaldehyde, 4-nitrobenzaldehyde, 4-methoxy benzaldehyde, or 4-N,N-dimethylamino benzaldehyde were refluxed for 5–8 h. After cooling and pouring into crushed ice, the precipitate obtained was purified in DMF/H2O.2-[2-Benzylidenehydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5a
). Yield: 69%; mp: 336°C; IR (KBr, νmax, cm−1): 3379, 3210 (2NH), 1667 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.20–8.66 (m, 16H, Ar + =CH), 10.87, 12.68 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.28, 119.97, 123.64, 127.80, 128.17, 128.63, 129.10, 130.03, 131.14, 132.99, 134.13, 134.49, 139.11, 143.54, 147.68, 149.98, 151.73, 152.71, 153.69, 161.34, 162.15; MS: [m/z, 418 (M+)]; Anal. Calcd for: C25H18N6O (418.45): C, 71.76; H, 4.34; N, 20.08% Found: C, 71.63; H, 4.17; N, 29.95.2-[2-(4-Fluorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5b
). Yield: 62%; mp: 317°C; IR (KBr, νmax, cm−1): 3365, 3217 (2NH), 1663 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.15–8.70 (m, 14H, Ar + =CH), 10.78, 12.29 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 115.79, 116.25, 119.85, 123.65, 128.13, 128.34, 128.76, 129.16, 130.24, 134.18, 134.59, 140.02, 143.28, 149.55, 150.48, 151.63, 152.69, 153.74, 161.27, 162.18, 164.72; MS: [m/z, 436 (M+)]; Anal. Calcd for: C25H17FN6O (436.44): C, 68.80; H, 3.93; N, 19.26% Found: C, 68.64; H, 3.79; N, 19.08.2-[2-(4-Chlorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5c
). Yield: 53%; mp: 345°C; IR (KBr, νmax, cm−1): 3381, 3195 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.72 (m, 14H, Ar + =-CH), 10.75, 12.36, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.34, 119.36, 123.60, 128.12, 128.45, 128.84, 129.04, 130.42, 131.56, 134.21, 134.68, 137.13, 140.15, 143.36, 149.47, 150.36, 151.24, 152.72, 153.61, 161.15, 162.19; MS: [m/z, 452 (M+)]; Anal. Calcd for: C25H17ClN6O (452.9): C, 66.30; H, 3.78; N, 18.56% Found: C, 66.12; H, 3.64; N, 18.39.2-[2-(4-Methylbenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5d
). Yield: 71%; mp: 321°C; IR (KBr, νmax, cm−1): 3348, 3212 (2NH), 1668 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.40 (s, 3H, CH3), 7.20–8.73 (m, 14H, Ar + =CH), 11.05, 12.45, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 24.10, 116.87, 119.82, 123.17, 128.05, 128.33, 128.96, 129.15, 130.36, 134.32, 134.59, 140.18, 141.82, 143.62, 149.38, 151.03, 151.46, 152.91, 153.73, 161.22, 162.31; MS: [m/z, 432 (M+)]; Anal. Calcd for: C26H20N6O (432.48): C, 72.21; H, 4.66; N, 19.43% Found: C, 72.04; H, 4.52; N, 19.28.2-[2-(4-Nitrobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5e
). Yield: 58%; mp: 312°C; IR (KBr, νmax, cm−1): 3365, 3202 (2NH), 1664 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.21–8.66 (m, 14H, Ar + =CH), 10.82, 12.35 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 119.87, 121.17, 123.47, 128.02, 128.66, 129.34, 130.03, 134.45, 134.62, 139.76, 140.16, 143.29, 149.35, 150.29, 151.04, 151.87, 153.04, 153.74, 161.13, 162.25; MS: [m/z, 463 (M+)]; Anal. Calcd for: C25H17N7O3 (463.45): C, 64.79; H, 3.70; N, 21.16% Found: C, 64.63; H, 3.56; N, 20.98.2-[2-(4-Methoxybenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5f
). Yield: 73%; mp: 295–296°C; IR (KBr, νmax, cm−1): 3376, 3198 (2NH), 1665 (CO); 1H NMR (δ, ppm, DMSO-d6): 3.81 (s, 3H, OCH3), 7.01–8.58 (m, 14H, Ar + =CH), 11.23, 12.56 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 56.19, 115.03, 116.89, 119.77, 123.59, 126.09, 128.14, 128.70, 129.15, 130.03, 134.26, 134.71, 140.12, 143.86, 149.27, 151.02, 151.78, 152.42, 153.84, 161.19, 162.34, 165.01; MS: [m/z, 448 (M+)]; Anal. Calcd for: C26H20N6O2 (448.48): C, 69.63; H, 4.49; N, 18.74% Found: C, 69.47; H, 4.31; N, 18.59.2-[2-(4-Dimethylaminobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5g
). Yield: 75%; mp: 302°C; IR (KBr, νmax, cm−1): 3401, 3234 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.67 (s, 6H, 2CH3), 7.20–8.68 (m, 14H, Ar + =CH), 11.24, 12.58, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 42.03, 115.63, 116.85, 119.47, 121.17, 123.57, 128.16, 128.77, 129.16, 130.08, 134.27, 134.45, 140.01, 143.67, 148.99, 151.02, 151.87, 152.04, 152.71, 153.14, 161.17, 162.23; MS: [m/z, 461 (M+)]; Anal. Calcd for: C27H23N7O (461.52): C, 70.27; H, 5.02; N, 21.24% Found: C, 70.12; H, 4.96; N, 21.08.


#### 2.2.4. Synthetic Method for Derivatives (6–9)

In glacial acetic acid (15 mL), equimolar amounts (0.001 mol) of hydrazinyl derivative4 and different reagents, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocanate were heated for 3–6 h. The mixture was allowed to cool, poured into ice, and the residue obtained was purified in AcOH.2-(3-Methyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
6
). Yield: 45%; mp: 302°C; IR (KBr, νmax, cm−1): 3368, 3214 (2NH), 1725, 1660 (2CO); 1H NMR (δ, ppm, DMSO-d6): 1.87 (s, 3H, CH3), 7.21–8.65 (m, 11H, Ar + CH-pyrazolone), 10.47, 12.80 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 21.57, 106.48, 116.99, 119.91, 123.69, 128.11, 128.48, 129.10, 134.20, 134.58, 140.03, 149.13, 151.01, 151.96, 152.67, 152.84, 153.22, 161.38, 162.45, 172.46; MS: [m/z, 396 (M+)]; Anal. Calcd for: C22H16N6O2 (396.4): C, 66.66; H, 4.07; N, 21.20% Found: C, 66.52; H, 3.94; N, 21.06.2-(3-Amino-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
7
). Yield: 60%; mp: 310°C; IR (KBr, νmax, cm−1): 3431 (NH2), 3346, 3196 (2NH), 1730, 1667 (2CO); 1H NMR (δ, ppm, DMSO-d6): 4.58 (s, 2H, NH2), 7.24–8.78 (m, 11H, Ar + CH-pyrazolone), 11.53, 12.39 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 106.75, 116.85, 119.65, 123.16, 128.05, 128.36, 129.18, 134.42, 134.81, 140.08, 149.01, 150.37, 151.90, 152.65, 153.12, 161.09, 163.14, 171.82; MS: [m/z, 397 (M+)]; Anal. Calcd for: C21H15N7O2 (397.39): C, 63.47; H, 3.80; N, 24.67% Found: C, 63.31; H, 3.65; N, 24.58.1-[4-Oxo-5-phenyl-7-(pyridin-3-yl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]pyrazolidine-3,5-dione (
8
). Yield: 39%; mp: 349°C; IR (KBr, νmax, cm−1): 3420, 3210 (2NH), 1705, 1682, 1667 (3CO); 1H NMR (δ, ppm, DMSO-d6): 2.98 (s, 2H, CH2), 7.22–8.71 (m, 10H, Ar), 11.84, 12.59 (2s, 2H, 2-NH); 13C NMR (δ, ppm, DMSO-d6): 90.21, 116.93, 119.73, 123.34, 127.91, 128.41, 129.30, 134.59, 134.66, 140.03, 149.28, 151.13, 151.86, 152.74, 153.21, 161.02, 163.18, 167.49, 170.56; MS: [m/z, 398 (M+)]; Anal. Calcd for: C21H14N66O3 (398.37): C, 63.31; H, 3.54; N, 21.10% Found: C, 63.17; H, 3.39; N, 20.91.3-Amino-6-phenyl-8-(pyridin-3-yl)pyrido[2,3-d]triazolo[4,3-a]pyrimidin-5(1H)-one (
9
). Yield: 63%; mp: 347°C; IR (KBr, νmax, cm−1): 3460 (NH2), 3317 (NH), 1669 (CO); 1H NMR (δ, ppm, DMSO-d6): 5.60 (s, 2H, NH2), 7.23–8.76 (m, 10H, Ar), 12.55 (s, 1H, NH); 13C NMR (δ, ppm, DMSO-d6): 117.30, 119.58, 123.79, 127.98, 129.01, 129.57, 134.25, 134.76, 139.53, 148.95, 151.06, 151.90, 152.64, 153.42, 155.47, 160.37, 161.77; MS: [m/z, 355 (M+)]; Anal. Calcd for: C19H13N7O (355.35): C, 64.22; H, 3.69; N, 27.59% Found: C, 64.06; H, 3.54; N, 27.43.


## 2.1. General Information

Electrothermal apparatus with open capillary tubes was used in measuring melting points. Shimadzu 435 IR spectrophotometer was used in measuring IR spectra (KBr). Varian Mercury VX-300 NMR spectrophotometer was used in determining NMR spectra using DMSO-d6 as a solvent and TMS as internal standard. The proposed structures were within ±0.4% of the theoretical values in microanalyses data. Shimadzu GC/MS-QP 2010 plus spectrometer was used in recording mass spectra.


## 2.2. Chemistry

### 2.2.1. 5-Phenyl-7-(pyridin-3-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (3)

In dry DMF (20 mL), equimolar quantities (0.01 mol) of 6-amino-2,3-dihydro-2-thioxopyrimidin-4(1H)-one 1 and α, β-unsaturated ketone 2 were refluxed for 10 h (monitored with TLC). The residue created was gathered and purified from DMF. Yield: 67%; mp: >300°C; IR (KBr, νmax, cm−1): 3327, 3234 (2NH), 1668 (CO), 1178 (CS); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.65 (m, 10H, Ar), 12.26, 13.01 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 108.94, 113.21, 123.87, 127.19, 129.45, 134.59, 135.01, 137.66, 147.82, 149.65, 151.14, 153.02, 153.72, 159.97, 178.01; MS: [m/z, 332 (M+)]; Anal. Calcd for: C18H12N4OS (332.38): C, 65.04; H, 3.64; N, 16.86% Found: C; 64.92, H, 3.57; N, 16.71.


### 2.2.2. 2-Hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (4)

In dry ethyl alcohol (30 mL), (0.003 mol) of 2-thioxo derivative3 and (0.005 mol) of hydrazine hydrate 99% was heated for 12 h. The precipitate created was purified in DMF. Yield: 72%; mp: 286–287°C; IR (KBr, νmax, cm−1): 3446 (NH2), 3362, 3205 (2NH), 1671 (C=O); 1H NMR (δ, ppm, DMSO-d6): 4.80 (s, 2H, NH2), 7.21–8.73 (m, 10H, Ar), 11.38, 12.76, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 118.74, 121.02, 123.90, 126.38, 129.17, 130.01, 134.57, 135.01, 137.21, 147.83, 149.96, 151.93, 152.47, 153.12, 159.97, 162.41; MS: [m/z, 330 (M+)]; Anal. Calcd for: C18H14N6O (330.34): C, 65.44; H, 4.27; N, 25.44% Found: C; 65.29, H, 4.12; N, 25.34.


### 2.2.3. Synthetic Method for Derivatives (5a–g)

In glacial acetic acid (10 mL), equimolar amounts of (0.001 mol) of hydrazinyl derivative4 and several aromatic aldehydes, benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-tolylaldehyde, 4-nitrobenzaldehyde, 4-methoxy benzaldehyde, or 4-N,N-dimethylamino benzaldehyde were refluxed for 5–8 h. After cooling and pouring into crushed ice, the precipitate obtained was purified in DMF/H2O.2-[2-Benzylidenehydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5a
). Yield: 69%; mp: 336°C; IR (KBr, νmax, cm−1): 3379, 3210 (2NH), 1667 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.20–8.66 (m, 16H, Ar + =CH), 10.87, 12.68 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.28, 119.97, 123.64, 127.80, 128.17, 128.63, 129.10, 130.03, 131.14, 132.99, 134.13, 134.49, 139.11, 143.54, 147.68, 149.98, 151.73, 152.71, 153.69, 161.34, 162.15; MS: [m/z, 418 (M+)]; Anal. Calcd for: C25H18N6O (418.45): C, 71.76; H, 4.34; N, 20.08% Found: C, 71.63; H, 4.17; N, 29.95.2-[2-(4-Fluorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5b
). Yield: 62%; mp: 317°C; IR (KBr, νmax, cm−1): 3365, 3217 (2NH), 1663 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.15–8.70 (m, 14H, Ar + =CH), 10.78, 12.29 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 115.79, 116.25, 119.85, 123.65, 128.13, 128.34, 128.76, 129.16, 130.24, 134.18, 134.59, 140.02, 143.28, 149.55, 150.48, 151.63, 152.69, 153.74, 161.27, 162.18, 164.72; MS: [m/z, 436 (M+)]; Anal. Calcd for: C25H17FN6O (436.44): C, 68.80; H, 3.93; N, 19.26% Found: C, 68.64; H, 3.79; N, 19.08.2-[2-(4-Chlorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5c
). Yield: 53%; mp: 345°C; IR (KBr, νmax, cm−1): 3381, 3195 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.72 (m, 14H, Ar + =-CH), 10.75, 12.36, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.34, 119.36, 123.60, 128.12, 128.45, 128.84, 129.04, 130.42, 131.56, 134.21, 134.68, 137.13, 140.15, 143.36, 149.47, 150.36, 151.24, 152.72, 153.61, 161.15, 162.19; MS: [m/z, 452 (M+)]; Anal. Calcd for: C25H17ClN6O (452.9): C, 66.30; H, 3.78; N, 18.56% Found: C, 66.12; H, 3.64; N, 18.39.2-[2-(4-Methylbenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5d
). Yield: 71%; mp: 321°C; IR (KBr, νmax, cm−1): 3348, 3212 (2NH), 1668 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.40 (s, 3H, CH3), 7.20–8.73 (m, 14H, Ar + =CH), 11.05, 12.45, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 24.10, 116.87, 119.82, 123.17, 128.05, 128.33, 128.96, 129.15, 130.36, 134.32, 134.59, 140.18, 141.82, 143.62, 149.38, 151.03, 151.46, 152.91, 153.73, 161.22, 162.31; MS: [m/z, 432 (M+)]; Anal. Calcd for: C26H20N6O (432.48): C, 72.21; H, 4.66; N, 19.43% Found: C, 72.04; H, 4.52; N, 19.28.2-[2-(4-Nitrobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5e
). Yield: 58%; mp: 312°C; IR (KBr, νmax, cm−1): 3365, 3202 (2NH), 1664 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.21–8.66 (m, 14H, Ar + =CH), 10.82, 12.35 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 119.87, 121.17, 123.47, 128.02, 128.66, 129.34, 130.03, 134.45, 134.62, 139.76, 140.16, 143.29, 149.35, 150.29, 151.04, 151.87, 153.04, 153.74, 161.13, 162.25; MS: [m/z, 463 (M+)]; Anal. Calcd for: C25H17N7O3 (463.45): C, 64.79; H, 3.70; N, 21.16% Found: C, 64.63; H, 3.56; N, 20.98.2-[2-(4-Methoxybenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5f
). Yield: 73%; mp: 295–296°C; IR (KBr, νmax, cm−1): 3376, 3198 (2NH), 1665 (CO); 1H NMR (δ, ppm, DMSO-d6): 3.81 (s, 3H, OCH3), 7.01–8.58 (m, 14H, Ar + =CH), 11.23, 12.56 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 56.19, 115.03, 116.89, 119.77, 123.59, 126.09, 128.14, 128.70, 129.15, 130.03, 134.26, 134.71, 140.12, 143.86, 149.27, 151.02, 151.78, 152.42, 153.84, 161.19, 162.34, 165.01; MS: [m/z, 448 (M+)]; Anal. Calcd for: C26H20N6O2 (448.48): C, 69.63; H, 4.49; N, 18.74% Found: C, 69.47; H, 4.31; N, 18.59.2-[2-(4-Dimethylaminobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5g
). Yield: 75%; mp: 302°C; IR (KBr, νmax, cm−1): 3401, 3234 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.67 (s, 6H, 2CH3), 7.20–8.68 (m, 14H, Ar + =CH), 11.24, 12.58, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 42.03, 115.63, 116.85, 119.47, 121.17, 123.57, 128.16, 128.77, 129.16, 130.08, 134.27, 134.45, 140.01, 143.67, 148.99, 151.02, 151.87, 152.04, 152.71, 153.14, 161.17, 162.23; MS: [m/z, 461 (M+)]; Anal. Calcd for: C27H23N7O (461.52): C, 70.27; H, 5.02; N, 21.24% Found: C, 70.12; H, 4.96; N, 21.08.


### 2.2.4. Synthetic Method for Derivatives (6–9)

In glacial acetic acid (15 mL), equimolar amounts (0.001 mol) of hydrazinyl derivative4 and different reagents, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocanate were heated for 3–6 h. The mixture was allowed to cool, poured into ice, and the residue obtained was purified in AcOH.2-(3-Methyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
6
). Yield: 45%; mp: 302°C; IR (KBr, νmax, cm−1): 3368, 3214 (2NH), 1725, 1660 (2CO); 1H NMR (δ, ppm, DMSO-d6): 1.87 (s, 3H, CH3), 7.21–8.65 (m, 11H, Ar + CH-pyrazolone), 10.47, 12.80 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 21.57, 106.48, 116.99, 119.91, 123.69, 128.11, 128.48, 129.10, 134.20, 134.58, 140.03, 149.13, 151.01, 151.96, 152.67, 152.84, 153.22, 161.38, 162.45, 172.46; MS: [m/z, 396 (M+)]; Anal. Calcd for: C22H16N6O2 (396.4): C, 66.66; H, 4.07; N, 21.20% Found: C, 66.52; H, 3.94; N, 21.06.2-(3-Amino-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
7
). Yield: 60%; mp: 310°C; IR (KBr, νmax, cm−1): 3431 (NH2), 3346, 3196 (2NH), 1730, 1667 (2CO); 1H NMR (δ, ppm, DMSO-d6): 4.58 (s, 2H, NH2), 7.24–8.78 (m, 11H, Ar + CH-pyrazolone), 11.53, 12.39 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 106.75, 116.85, 119.65, 123.16, 128.05, 128.36, 129.18, 134.42, 134.81, 140.08, 149.01, 150.37, 151.90, 152.65, 153.12, 161.09, 163.14, 171.82; MS: [m/z, 397 (M+)]; Anal. Calcd for: C21H15N7O2 (397.39): C, 63.47; H, 3.80; N, 24.67% Found: C, 63.31; H, 3.65; N, 24.58.1-[4-Oxo-5-phenyl-7-(pyridin-3-yl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]pyrazolidine-3,5-dione (
8
). Yield: 39%; mp: 349°C; IR (KBr, νmax, cm−1): 3420, 3210 (2NH), 1705, 1682, 1667 (3CO); 1H NMR (δ, ppm, DMSO-d6): 2.98 (s, 2H, CH2), 7.22–8.71 (m, 10H, Ar), 11.84, 12.59 (2s, 2H, 2-NH); 13C NMR (δ, ppm, DMSO-d6): 90.21, 116.93, 119.73, 123.34, 127.91, 128.41, 129.30, 134.59, 134.66, 140.03, 149.28, 151.13, 151.86, 152.74, 153.21, 161.02, 163.18, 167.49, 170.56; MS: [m/z, 398 (M+)]; Anal. Calcd for: C21H14N66O3 (398.37): C, 63.31; H, 3.54; N, 21.10% Found: C, 63.17; H, 3.39; N, 20.91.3-Amino-6-phenyl-8-(pyridin-3-yl)pyrido[2,3-d]triazolo[4,3-a]pyrimidin-5(1H)-one (
9
). Yield: 63%; mp: 347°C; IR (KBr, νmax, cm−1): 3460 (NH2), 3317 (NH), 1669 (CO); 1H NMR (δ, ppm, DMSO-d6): 5.60 (s, 2H, NH2), 7.23–8.76 (m, 10H, Ar), 12.55 (s, 1H, NH); 13C NMR (δ, ppm, DMSO-d6): 117.30, 119.58, 123.79, 127.98, 129.01, 129.57, 134.25, 134.76, 139.53, 148.95, 151.06, 151.90, 152.64, 153.42, 155.47, 160.37, 161.77; MS: [m/z, 355 (M+)]; Anal. Calcd for: C19H13N7O (355.35): C, 64.22; H, 3.69; N, 27.59% Found: C, 64.06; H, 3.54; N, 27.43.


## 2.2.1. 5-Phenyl-7-(pyridin-3-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (3)

In dry DMF (20 mL), equimolar quantities (0.01 mol) of 6-amino-2,3-dihydro-2-thioxopyrimidin-4(1H)-one 1 and α, β-unsaturated ketone 2 were refluxed for 10 h (monitored with TLC). The residue created was gathered and purified from DMF. Yield: 67%; mp: >300°C; IR (KBr, νmax, cm−1): 3327, 3234 (2NH), 1668 (CO), 1178 (CS); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.65 (m, 10H, Ar), 12.26, 13.01 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 108.94, 113.21, 123.87, 127.19, 129.45, 134.59, 135.01, 137.66, 147.82, 149.65, 151.14, 153.02, 153.72, 159.97, 178.01; MS: [m/z, 332 (M+)]; Anal. Calcd for: C18H12N4OS (332.38): C, 65.04; H, 3.64; N, 16.86% Found: C; 64.92, H, 3.57; N, 16.71.


## 2.2.2. 2-Hydrazinyl-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (4)

In dry ethyl alcohol (30 mL), (0.003 mol) of 2-thioxo derivative3 and (0.005 mol) of hydrazine hydrate 99% was heated for 12 h. The precipitate created was purified in DMF. Yield: 72%; mp: 286–287°C; IR (KBr, νmax, cm−1): 3446 (NH2), 3362, 3205 (2NH), 1671 (C=O); 1H NMR (δ, ppm, DMSO-d6): 4.80 (s, 2H, NH2), 7.21–8.73 (m, 10H, Ar), 11.38, 12.76, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 118.74, 121.02, 123.90, 126.38, 129.17, 130.01, 134.57, 135.01, 137.21, 147.83, 149.96, 151.93, 152.47, 153.12, 159.97, 162.41; MS: [m/z, 330 (M+)]; Anal. Calcd for: C18H14N6O (330.34): C, 65.44; H, 4.27; N, 25.44% Found: C; 65.29, H, 4.12; N, 25.34.


## 2.2.3. Synthetic Method for Derivatives (5a–g)

In glacial acetic acid (10 mL), equimolar amounts of (0.001 mol) of hydrazinyl derivative4 and several aromatic aldehydes, benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-tolylaldehyde, 4-nitrobenzaldehyde, 4-methoxy benzaldehyde, or 4-N,N-dimethylamino benzaldehyde were refluxed for 5–8 h. After cooling and pouring into crushed ice, the precipitate obtained was purified in DMF/H2O.2-[2-Benzylidenehydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5a
). Yield: 69%; mp: 336°C; IR (KBr, νmax, cm−1): 3379, 3210 (2NH), 1667 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.20–8.66 (m, 16H, Ar + =CH), 10.87, 12.68 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.28, 119.97, 123.64, 127.80, 128.17, 128.63, 129.10, 130.03, 131.14, 132.99, 134.13, 134.49, 139.11, 143.54, 147.68, 149.98, 151.73, 152.71, 153.69, 161.34, 162.15; MS: [m/z, 418 (M+)]; Anal. Calcd for: C25H18N6O (418.45): C, 71.76; H, 4.34; N, 20.08% Found: C, 71.63; H, 4.17; N, 29.95.2-[2-(4-Fluorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5b
). Yield: 62%; mp: 317°C; IR (KBr, νmax, cm−1): 3365, 3217 (2NH), 1663 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.15–8.70 (m, 14H, Ar + =CH), 10.78, 12.29 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 115.79, 116.25, 119.85, 123.65, 128.13, 128.34, 128.76, 129.16, 130.24, 134.18, 134.59, 140.02, 143.28, 149.55, 150.48, 151.63, 152.69, 153.74, 161.27, 162.18, 164.72; MS: [m/z, 436 (M+)]; Anal. Calcd for: C25H17FN6O (436.44): C, 68.80; H, 3.93; N, 19.26% Found: C, 68.64; H, 3.79; N, 19.08.2-[2-(4-Chlorobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5c
). Yield: 53%; mp: 345°C; IR (KBr, νmax, cm−1): 3381, 3195 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.23–8.72 (m, 14H, Ar + =-CH), 10.75, 12.36, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 116.34, 119.36, 123.60, 128.12, 128.45, 128.84, 129.04, 130.42, 131.56, 134.21, 134.68, 137.13, 140.15, 143.36, 149.47, 150.36, 151.24, 152.72, 153.61, 161.15, 162.19; MS: [m/z, 452 (M+)]; Anal. Calcd for: C25H17ClN6O (452.9): C, 66.30; H, 3.78; N, 18.56% Found: C, 66.12; H, 3.64; N, 18.39.2-[2-(4-Methylbenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5d
). Yield: 71%; mp: 321°C; IR (KBr, νmax, cm−1): 3348, 3212 (2NH), 1668 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.40 (s, 3H, CH3), 7.20–8.73 (m, 14H, Ar + =CH), 11.05, 12.45, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 24.10, 116.87, 119.82, 123.17, 128.05, 128.33, 128.96, 129.15, 130.36, 134.32, 134.59, 140.18, 141.82, 143.62, 149.38, 151.03, 151.46, 152.91, 153.73, 161.22, 162.31; MS: [m/z, 432 (M+)]; Anal. Calcd for: C26H20N6O (432.48): C, 72.21; H, 4.66; N, 19.43% Found: C, 72.04; H, 4.52; N, 19.28.2-[2-(4-Nitrobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5e
). Yield: 58%; mp: 312°C; IR (KBr, νmax, cm−1): 3365, 3202 (2NH), 1664 (CO); 1H NMR (δ, ppm, DMSO-d6): 7.21–8.66 (m, 14H, Ar + =CH), 10.82, 12.35 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 119.87, 121.17, 123.47, 128.02, 128.66, 129.34, 130.03, 134.45, 134.62, 139.76, 140.16, 143.29, 149.35, 150.29, 151.04, 151.87, 153.04, 153.74, 161.13, 162.25; MS: [m/z, 463 (M+)]; Anal. Calcd for: C25H17N7O3 (463.45): C, 64.79; H, 3.70; N, 21.16% Found: C, 64.63; H, 3.56; N, 20.98.2-[2-(4-Methoxybenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (
5f
). Yield: 73%; mp: 295–296°C; IR (KBr, νmax, cm−1): 3376, 3198 (2NH), 1665 (CO); 1H NMR (δ, ppm, DMSO-d6): 3.81 (s, 3H, OCH3), 7.01–8.58 (m, 14H, Ar + =CH), 11.23, 12.56 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 56.19, 115.03, 116.89, 119.77, 123.59, 126.09, 128.14, 128.70, 129.15, 130.03, 134.26, 134.71, 140.12, 143.86, 149.27, 151.02, 151.78, 152.42, 153.84, 161.19, 162.34, 165.01; MS: [m/z, 448 (M+)]; Anal. Calcd for: C26H20N6O2 (448.48): C, 69.63; H, 4.49; N, 18.74% Found: C, 69.47; H, 4.31; N, 18.59.2-[2-(4-Dimethylaminobenzylidene)hydrazinyl]-5-phenyl-7-(pyridin-3-yl)pyrido[2,3-d]-pyrimidin-4(3H)-one (
5g
). Yield: 75%; mp: 302°C; IR (KBr, νmax, cm−1): 3401, 3234 (2NH), 1660 (CO); 1H NMR (δ, ppm, DMSO-d6): 2.67 (s, 6H, 2CH3), 7.20–8.68 (m, 14H, Ar + =CH), 11.24, 12.58, (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 42.03, 115.63, 116.85, 119.47, 121.17, 123.57, 128.16, 128.77, 129.16, 130.08, 134.27, 134.45, 140.01, 143.67, 148.99, 151.02, 151.87, 152.04, 152.71, 153.14, 161.17, 162.23; MS: [m/z, 461 (M+)]; Anal. Calcd for: C27H23N7O (461.52): C, 70.27; H, 5.02; N, 21.24% Found: C, 70.12; H, 4.96; N, 21.08.


## 2.2.4. Synthetic Method for Derivatives (6–9)

In glacial acetic acid (15 mL), equimolar amounts (0.001 mol) of hydrazinyl derivative4 and different reagents, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocanate were heated for 3–6 h. The mixture was allowed to cool, poured into ice, and the residue obtained was purified in AcOH.2-(3-Methyl-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
6
). Yield: 45%; mp: 302°C; IR (KBr, νmax, cm−1): 3368, 3214 (2NH), 1725, 1660 (2CO); 1H NMR (δ, ppm, DMSO-d6): 1.87 (s, 3H, CH3), 7.21–8.65 (m, 11H, Ar + CH-pyrazolone), 10.47, 12.80 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 21.57, 106.48, 116.99, 119.91, 123.69, 128.11, 128.48, 129.10, 134.20, 134.58, 140.03, 149.13, 151.01, 151.96, 152.67, 152.84, 153.22, 161.38, 162.45, 172.46; MS: [m/z, 396 (M+)]; Anal. Calcd for: C22H16N6O2 (396.4): C, 66.66; H, 4.07; N, 21.20% Found: C, 66.52; H, 3.94; N, 21.06.2-(3-Amino-5-oxo-2,5-dihydro-1H-pyrazol-1-yl)-5-phenyl-7-(pyridin-3-yl)pyrido-[2,3-d]-pyrimidin-4(3H)-one (
7
). Yield: 60%; mp: 310°C; IR (KBr, νmax, cm−1): 3431 (NH2), 3346, 3196 (2NH), 1730, 1667 (2CO); 1H NMR (δ, ppm, DMSO-d6): 4.58 (s, 2H, NH2), 7.24–8.78 (m, 11H, Ar + CH-pyrazolone), 11.53, 12.39 (2s, 2H, 2NH); 13C NMR (δ, ppm, DMSO-d6): 106.75, 116.85, 119.65, 123.16, 128.05, 128.36, 129.18, 134.42, 134.81, 140.08, 149.01, 150.37, 151.90, 152.65, 153.12, 161.09, 163.14, 171.82; MS: [m/z, 397 (M+)]; Anal. Calcd for: C21H15N7O2 (397.39): C, 63.47; H, 3.80; N, 24.67% Found: C, 63.31; H, 3.65; N, 24.58.1-[4-Oxo-5-phenyl-7-(pyridin-3-yl)-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]pyrazolidine-3,5-dione (
8
). Yield: 39%; mp: 349°C; IR (KBr, νmax, cm−1): 3420, 3210 (2NH), 1705, 1682, 1667 (3CO); 1H NMR (δ, ppm, DMSO-d6): 2.98 (s, 2H, CH2), 7.22–8.71 (m, 10H, Ar), 11.84, 12.59 (2s, 2H, 2-NH); 13C NMR (δ, ppm, DMSO-d6): 90.21, 116.93, 119.73, 123.34, 127.91, 128.41, 129.30, 134.59, 134.66, 140.03, 149.28, 151.13, 151.86, 152.74, 153.21, 161.02, 163.18, 167.49, 170.56; MS: [m/z, 398 (M+)]; Anal. Calcd for: C21H14N66O3 (398.37): C, 63.31; H, 3.54; N, 21.10% Found: C, 63.17; H, 3.39; N, 20.91.3-Amino-6-phenyl-8-(pyridin-3-yl)pyrido[2,3-d]triazolo[4,3-a]pyrimidin-5(1H)-one (
9
). Yield: 63%; mp: 347°C; IR (KBr, νmax, cm−1): 3460 (NH2), 3317 (NH), 1669 (CO); 1H NMR (δ, ppm, DMSO-d6): 5.60 (s, 2H, NH2), 7.23–8.76 (m, 10H, Ar), 12.55 (s, 1H, NH); 13C NMR (δ, ppm, DMSO-d6): 117.30, 119.58, 123.79, 127.98, 129.01, 129.57, 134.25, 134.76, 139.53, 148.95, 151.06, 151.90, 152.64, 153.42, 155.47, 160.37, 161.77; MS: [m/z, 355 (M+)]; Anal. Calcd for: C19H13N7O (355.35): C, 64.22; H, 3.69; N, 27.59% Found: C, 64.06; H, 3.54; N, 27.43.


## 3. Results and Discussion

### 3.1. Chemistry

A group of substituted pyridopyrimidines were obtained via treatment of starting1 with α,β-unsaturated ketone 2 in dry DMF to afford 2-thioxo derivative 3. IR spectrum of 3 revealed four strong bands at 3327, 3234, 1668, and 1178 cm−1 due to 2NH, CO, and CS functions, respectively. In the same time, 1HNMR spectrum displayed two singlets at δ 12.26 and 13.01 ppm assignable for 2NH protons. Also, 13CNMR spectrum showed signal at 178.01 ppm for C=S carbon. Treatment of hydrazine hydrate with thioxo derivative 3 afforded the corresponding 2-hydrazinyl derivative 4. IR spectrum of 4 revealed four peaks at 3446, 3362, 3205, and 1671 cm−1 attributed to amino, two amides, and carbonyl functions, respectively. Moreover, 1H NMR spectrum confirmed the presence of amino and amide protons by existence of three singlet peaks at δ 4.80, 11.38, and 12.76 ppm. The MS revealed [M+] at m/z 330 agreed with the MF C18H14N6O. Compound 4 was allowed to react with different aromatic aldehydes to afford 2-arylidene derivatives 5a–g. Compounds 5a–g were confirmed on the basis of their IR spectra and showed strong peaks around the regions 3401–3348, 3234–3195, and 1668–1660 cm−1 corresponding to two amide and carbonyl functions. Furthermore, the methine functions were proved by presence of singlet peaks in the aromatic region in 1H NMR spectrum and signals around 143 ppm in 13H NMR spectrum.Treatment of the 2-hydrazinyl intermediate4 with active methylene, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocyanate afforded the corresponding 5-substituted pyrazolones and triazolopyrimidines 6–9 (Scheme 1). The new pyrazolone ring linked to the pyridopyrimidine backbone in compound 6 was proved with the appearance of strong bands at 3214 and 1725 cm−1 referred to NH and CO functions of the pyrazole ring in IR spectrum and existence of two singlet peaks at δ 1.87 and 10.87 ppm due to methyl attached to the pyrazole ring and -NH protons. The molecular formula C18H14N6O of 6 was confirmed by the presence of molecular ion peak [M+] at m/z 330 in MS.Scheme 1
Synthetic pathway for pyridopyrimidines (3–9).IR spectrum of compound7 confirmed attachment of the new 3-aminopyrazolone ring to the original backbone by the presence of broad bands at 3431, 3196, and 1730 cm−1 due to -NH2, -NH, and CO functions of the new moiety, respectively, besides other bands at 3346 and 1667 cm−1 for NH and CO functions of pyridopyrimidine. Also, 1H NMR spectrum proved the presence of the new aminopyrazolone ring by the existence of two singlet peaks at δ 4.58 and 11.53 ppm corresponding to amino and amide protons. The appearance of molecular ion peak [M+] at m/z 397 proved the molecular formula C21H15N7O2 of 7 in MS.On the contrary, the new pyrazolidine-3,5-dione ring in compound8 was proved by the existence of strong bands at 3210, 1705, and 1682 cm−1 referred to NH and 2 CO functions in the IR spectrum. The methylene group of the new ring appeared as a singlet peak at δ 2.98 ppm in 1H NMR spectrum and signal at 90.21 ppm in 13H NMR spectrum. MS gave a [M+]-ion peak at m/z 398 equivalents to the molecular formula C21H14N66O3.Finally, the new 3-aminotriazole ring fused with pyridopyrimidine moiety in9 was proved in the IR spectra by the existence of bands at 3460 and 3317 cm−1 referred to NH2 and NH functions besides other band at 1669 cm−1 due to carbonyl of pyridopyrimidine moiety. 1H NMR spectrum displayed two singlets at 5.60 and 12.55 attributed to NH2 and NH protons of the new fused ring. 13C NMR spectrum displayed the carbons at their expected regions, and MS gave a [M+]-ion peak at m/z 355 equivalents to the molecular formula of 9.


### 3.2. Biology

#### 3.2.1. In Vitro Cytotoxic Screening against HepG2, PC-3, and HCT-116 Cell Lines

Anticancer evaluation of the newly obtained products represented in Figure2 was screened against three human cancer cell lines (HepG2, PC3, or HCT-116) [23]. The tested compounds that displayed inhibitory effect more than 90% referring doxorubicin as a standard drug (IC50 0.6 μM) were chosen for IC50 examination (concentrations required for 50% inhibition of cell viability). The in vitro screening of compounds 3–9 at 100 μM (Figure 3) exhibited remarkable anticancer activities, and compound 6 showed promising potency against hepatic cancer cell line (anti-HepG2) with (IC50 = 0.5 μM).Figure 2
Percentage of growth inhibition activity against cancer cell lines at 100μM dose.Figure 3
IC50 of the tested compounds against cancer cell lines.


#### 3.2.2. Structure-Activity Relationships

The cytotoxic screening results revealed that thioxo precursor3 displayed poor anticancer activity against all cancer cell lines. Upon converting the thioxo group in 3 to hydrazide in 4, antihepatic cancer (HepG2) effect was greatly increased as a result of the presence of the hydrophilic electron-rich nature in compound 4 which causes the electron factor to give a positive impact on the antiproliferative properties. Attachment of the pyrazole ring as a substituent at position 2 of the backbone moiety as in compounds 6, 7, and 8 afforded the highest potency of anticancer activity. Compound 6 that carried 5-methyl-3-oxopyrazole exhibited the highest activity against all the tested cell lines. The activity was reduced and shifted toward the PC-3 cell line after replacement of the 5-methyl-3-oxopyrazole nucleus at 6 by 3-amino-5-oxopyrazol in 7. Upon replacing the mentioned pyrazole moiety in 6 by 3,5-dioxopyrazole in 8, or by fused triazolo[4,3-a]pyrimidine in 9, the activity profile was changed.


#### 3.2.3. Kinase Inhibition Screening

According to the data of cytotoxic assay, the highly potent derivative6 was chosen for in vitro inhibition assessment against a list of different protein, AKT1, AKT2, BRAF (V600E), CDK2/cyclin A1, CHK1, EGFR, PDGFRβ, and c-RAF kinases at 100 μM utilized the radiometric or ADP-Glo assay procedure. Three of the tested kinases (BRAF V600E, EGFR, and PDGFRβ) were highly inhibited by more than 90% with the highest inhibition recorded with EGFR at 97%. On the contrary, compound 6 appeared to partially activate the c-RAF kinase with a rise in counts of 37% over the control substrate rates (Table 1, Figure 4).Table 1
Percentage of kinase inhibition of derivative6 at 100 μM.
Kinase
Compound 6
% Inhibition
AKT1
−79
AKT2
−85
BRAF (V600E)
−91
CDK2/cyclin A1
−78
CHK1
−6
EGFR
−97
PDGFRβ
−94
c-RAF
37Figure 4
% of kinase inhibition of target molecule6.


#### 3.2.4. Molecular Docking Study

Molecular docking was used to analyze the supposed binding mode of the designed compound with CDK6 and EGFR to better understand the mechanism of inhibition. For the docking studies, the crystal structure of the complex CDK6 and EGFR has been chosen (PDB code 5L2I [24] and PDB code 4HJO [25]). The most active compound 6 in the current study was docked into the CDK6 and EGFR kinase’s putative active site. For the receptor preparation, the Molecular Operating Environment software package MOE® 2015 [26] has been used by means of the removal of water molecules and the addition of hydrogen atoms. MOE has also been used for the graphic structure of ligands 3D and then saved on data lists after minimizing structure and geometries energy. For each receptor, the pockets were then used to dock ligands after setting London dG to scoring function and GBVI/WSA dG to re scoring function. Therefore, the scoring and RMSD (root-mean-square deviation) values for the best conformation of each ligand with different receptors are shown in Table 2, as well as 2D and 3D figures of each selected conformation are shown in Figures 3 and 4. Docking simulation of compound 6 into kinase domain of CDK6 and EGFR postulated the pivotal function of both the backbone moiety and the side chain substituent (Figures 5 and 6).Table 2
Molecular simulation results for ACV/PNV interactions with HAS.
Ligand
Receptor
Amino acid residues
Interaction type
Distance (Å)
Total binding energy (kcal·mol−1)
RMSD
Compd.6
EGFR
LEU 694
pi-H
3.47
−6.156
1.878
(4HJO)
MET 769
H-acceptor
3.86
CDK6
VAL 101
H-acceptor
3.52
−6.942
1.251
LYS 147
H-acceptor
3.09
(5L2I)
VAL 27
pi-H
4.35Figure 5
The interaction between compound6 and CDK6 kinase protein (PDB code: 5L2I), presented by MOE 2015: (a, b) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (c) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (d) 2D diagrams of compound 6 (green) and padlbocyclib (red) were overlapping.Figure 6
The interaction between compound6 and EGFR kinase protein (PDB code: 4HJO), presented by MOE 2015: (a, d) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (b) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (c) 2D diagrams of compound 6 (green) and erlotinib (red) were overlapping.


## 3.1. Chemistry

A group of substituted pyridopyrimidines were obtained via treatment of starting1 with α,β-unsaturated ketone 2 in dry DMF to afford 2-thioxo derivative 3. IR spectrum of 3 revealed four strong bands at 3327, 3234, 1668, and 1178 cm−1 due to 2NH, CO, and CS functions, respectively. In the same time, 1HNMR spectrum displayed two singlets at δ 12.26 and 13.01 ppm assignable for 2NH protons. Also, 13CNMR spectrum showed signal at 178.01 ppm for C=S carbon. Treatment of hydrazine hydrate with thioxo derivative 3 afforded the corresponding 2-hydrazinyl derivative 4. IR spectrum of 4 revealed four peaks at 3446, 3362, 3205, and 1671 cm−1 attributed to amino, two amides, and carbonyl functions, respectively. Moreover, 1H NMR spectrum confirmed the presence of amino and amide protons by existence of three singlet peaks at δ 4.80, 11.38, and 12.76 ppm. The MS revealed [M+] at m/z 330 agreed with the MF C18H14N6O. Compound 4 was allowed to react with different aromatic aldehydes to afford 2-arylidene derivatives 5a–g. Compounds 5a–g were confirmed on the basis of their IR spectra and showed strong peaks around the regions 3401–3348, 3234–3195, and 1668–1660 cm−1 corresponding to two amide and carbonyl functions. Furthermore, the methine functions were proved by presence of singlet peaks in the aromatic region in 1H NMR spectrum and signals around 143 ppm in 13H NMR spectrum.Treatment of the 2-hydrazinyl intermediate4 with active methylene, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocyanate afforded the corresponding 5-substituted pyrazolones and triazolopyrimidines 6–9 (Scheme 1). The new pyrazolone ring linked to the pyridopyrimidine backbone in compound 6 was proved with the appearance of strong bands at 3214 and 1725 cm−1 referred to NH and CO functions of the pyrazole ring in IR spectrum and existence of two singlet peaks at δ 1.87 and 10.87 ppm due to methyl attached to the pyrazole ring and -NH protons. The molecular formula C18H14N6O of 6 was confirmed by the presence of molecular ion peak [M+] at m/z 330 in MS.Scheme 1
Synthetic pathway for pyridopyrimidines (3–9).IR spectrum of compound7 confirmed attachment of the new 3-aminopyrazolone ring to the original backbone by the presence of broad bands at 3431, 3196, and 1730 cm−1 due to -NH2, -NH, and CO functions of the new moiety, respectively, besides other bands at 3346 and 1667 cm−1 for NH and CO functions of pyridopyrimidine. Also, 1H NMR spectrum proved the presence of the new aminopyrazolone ring by the existence of two singlet peaks at δ 4.58 and 11.53 ppm corresponding to amino and amide protons. The appearance of molecular ion peak [M+] at m/z 397 proved the molecular formula C21H15N7O2 of 7 in MS.On the contrary, the new pyrazolidine-3,5-dione ring in compound8 was proved by the existence of strong bands at 3210, 1705, and 1682 cm−1 referred to NH and 2 CO functions in the IR spectrum. The methylene group of the new ring appeared as a singlet peak at δ 2.98 ppm in 1H NMR spectrum and signal at 90.21 ppm in 13H NMR spectrum. MS gave a [M+]-ion peak at m/z 398 equivalents to the molecular formula C21H14N66O3.Finally, the new 3-aminotriazole ring fused with pyridopyrimidine moiety in9 was proved in the IR spectra by the existence of bands at 3460 and 3317 cm−1 referred to NH2 and NH functions besides other band at 1669 cm−1 due to carbonyl of pyridopyrimidine moiety. 1H NMR spectrum displayed two singlets at 5.60 and 12.55 attributed to NH2 and NH protons of the new fused ring. 13C NMR spectrum displayed the carbons at their expected regions, and MS gave a [M+]-ion peak at m/z 355 equivalents to the molecular formula of 9.


## 3.2. Biology

### 3.2.1. In Vitro Cytotoxic Screening against HepG2, PC-3, and HCT-116 Cell Lines

Anticancer evaluation of the newly obtained products represented in Figure2 was screened against three human cancer cell lines (HepG2, PC3, or HCT-116) [23]. The tested compounds that displayed inhibitory effect more than 90% referring doxorubicin as a standard drug (IC50 0.6 μM) were chosen for IC50 examination (concentrations required for 50% inhibition of cell viability). The in vitro screening of compounds 3–9 at 100 μM (Figure 3) exhibited remarkable anticancer activities, and compound 6 showed promising potency against hepatic cancer cell line (anti-HepG2) with (IC50 = 0.5 μM).Figure 2
Percentage of growth inhibition activity against cancer cell lines at 100μM dose.Figure 3
IC50 of the tested compounds against cancer cell lines.


### 3.2.2. Structure-Activity Relationships

The cytotoxic screening results revealed that thioxo precursor3 displayed poor anticancer activity against all cancer cell lines. Upon converting the thioxo group in 3 to hydrazide in 4, antihepatic cancer (HepG2) effect was greatly increased as a result of the presence of the hydrophilic electron-rich nature in compound 4 which causes the electron factor to give a positive impact on the antiproliferative properties. Attachment of the pyrazole ring as a substituent at position 2 of the backbone moiety as in compounds 6, 7, and 8 afforded the highest potency of anticancer activity. Compound 6 that carried 5-methyl-3-oxopyrazole exhibited the highest activity against all the tested cell lines. The activity was reduced and shifted toward the PC-3 cell line after replacement of the 5-methyl-3-oxopyrazole nucleus at 6 by 3-amino-5-oxopyrazol in 7. Upon replacing the mentioned pyrazole moiety in 6 by 3,5-dioxopyrazole in 8, or by fused triazolo[4,3-a]pyrimidine in 9, the activity profile was changed.


### 3.2.3. Kinase Inhibition Screening

According to the data of cytotoxic assay, the highly potent derivative6 was chosen for in vitro inhibition assessment against a list of different protein, AKT1, AKT2, BRAF (V600E), CDK2/cyclin A1, CHK1, EGFR, PDGFRβ, and c-RAF kinases at 100 μM utilized the radiometric or ADP-Glo assay procedure. Three of the tested kinases (BRAF V600E, EGFR, and PDGFRβ) were highly inhibited by more than 90% with the highest inhibition recorded with EGFR at 97%. On the contrary, compound 6 appeared to partially activate the c-RAF kinase with a rise in counts of 37% over the control substrate rates (Table 1, Figure 4).Table 1
Percentage of kinase inhibition of derivative6 at 100 μM.
Kinase
Compound 6
% Inhibition
AKT1
−79
AKT2
−85
BRAF (V600E)
−91
CDK2/cyclin A1
−78
CHK1
−6
EGFR
−97
PDGFRβ
−94
c-RAF
37Figure 4
% of kinase inhibition of target molecule6.


### 3.2.4. Molecular Docking Study

Molecular docking was used to analyze the supposed binding mode of the designed compound with CDK6 and EGFR to better understand the mechanism of inhibition. For the docking studies, the crystal structure of the complex CDK6 and EGFR has been chosen (PDB code 5L2I [24] and PDB code 4HJO [25]). The most active compound 6 in the current study was docked into the CDK6 and EGFR kinase’s putative active site. For the receptor preparation, the Molecular Operating Environment software package MOE® 2015 [26] has been used by means of the removal of water molecules and the addition of hydrogen atoms. MOE has also been used for the graphic structure of ligands 3D and then saved on data lists after minimizing structure and geometries energy. For each receptor, the pockets were then used to dock ligands after setting London dG to scoring function and GBVI/WSA dG to re scoring function. Therefore, the scoring and RMSD (root-mean-square deviation) values for the best conformation of each ligand with different receptors are shown in Table 2, as well as 2D and 3D figures of each selected conformation are shown in Figures 3 and 4. Docking simulation of compound 6 into kinase domain of CDK6 and EGFR postulated the pivotal function of both the backbone moiety and the side chain substituent (Figures 5 and 6).Table 2
Molecular simulation results for ACV/PNV interactions with HAS.
Ligand
Receptor
Amino acid residues
Interaction type
Distance (Å)
Total binding energy (kcal·mol−1)
RMSD
Compd.6
EGFR
LEU 694
pi-H
3.47
−6.156
1.878
(4HJO)
MET 769
H-acceptor
3.86
CDK6
VAL 101
H-acceptor
3.52
−6.942
1.251
LYS 147
H-acceptor
3.09
(5L2I)
VAL 27
pi-H
4.35Figure 5
The interaction between compound6 and CDK6 kinase protein (PDB code: 5L2I), presented by MOE 2015: (a, b) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (c) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (d) 2D diagrams of compound 6 (green) and padlbocyclib (red) were overlapping.Figure 6
The interaction between compound6 and EGFR kinase protein (PDB code: 4HJO), presented by MOE 2015: (a, d) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (b) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (c) 2D diagrams of compound 6 (green) and erlotinib (red) were overlapping.


## 3.2.1. In Vitro Cytotoxic Screening against HepG2, PC-3, and HCT-116 Cell Lines

Anticancer evaluation of the newly obtained products represented in Figure2 was screened against three human cancer cell lines (HepG2, PC3, or HCT-116) [23]. The tested compounds that displayed inhibitory effect more than 90% referring doxorubicin as a standard drug (IC50 0.6 μM) were chosen for IC50 examination (concentrations required for 50% inhibition of cell viability). The in vitro screening of compounds 3–9 at 100 μM (Figure 3) exhibited remarkable anticancer activities, and compound 6 showed promising potency against hepatic cancer cell line (anti-HepG2) with (IC50 = 0.5 μM).Figure 2
Percentage of growth inhibition activity against cancer cell lines at 100μM dose.Figure 3
IC50 of the tested compounds against cancer cell lines.


## 3.2.2. Structure-Activity Relationships

The cytotoxic screening results revealed that thioxo precursor3 displayed poor anticancer activity against all cancer cell lines. Upon converting the thioxo group in 3 to hydrazide in 4, antihepatic cancer (HepG2) effect was greatly increased as a result of the presence of the hydrophilic electron-rich nature in compound 4 which causes the electron factor to give a positive impact on the antiproliferative properties. Attachment of the pyrazole ring as a substituent at position 2 of the backbone moiety as in compounds 6, 7, and 8 afforded the highest potency of anticancer activity. Compound 6 that carried 5-methyl-3-oxopyrazole exhibited the highest activity against all the tested cell lines. The activity was reduced and shifted toward the PC-3 cell line after replacement of the 5-methyl-3-oxopyrazole nucleus at 6 by 3-amino-5-oxopyrazol in 7. Upon replacing the mentioned pyrazole moiety in 6 by 3,5-dioxopyrazole in 8, or by fused triazolo[4,3-a]pyrimidine in 9, the activity profile was changed.


## 3.2.3. Kinase Inhibition Screening

According to the data of cytotoxic assay, the highly potent derivative6 was chosen for in vitro inhibition assessment against a list of different protein, AKT1, AKT2, BRAF (V600E), CDK2/cyclin A1, CHK1, EGFR, PDGFRβ, and c-RAF kinases at 100 μM utilized the radiometric or ADP-Glo assay procedure. Three of the tested kinases (BRAF V600E, EGFR, and PDGFRβ) were highly inhibited by more than 90% with the highest inhibition recorded with EGFR at 97%. On the contrary, compound 6 appeared to partially activate the c-RAF kinase with a rise in counts of 37% over the control substrate rates (Table 1, Figure 4).Table 1
Percentage of kinase inhibition of derivative6 at 100 μM.
Kinase
Compound 6
% Inhibition
AKT1
−79
AKT2
−85
BRAF (V600E)
−91
CDK2/cyclin A1
−78
CHK1
−6
EGFR
−97
PDGFRβ
−94
c-RAF
37Figure 4
% of kinase inhibition of target molecule6.


## 3.2.4. Molecular Docking Study

Molecular docking was used to analyze the supposed binding mode of the designed compound with CDK6 and EGFR to better understand the mechanism of inhibition. For the docking studies, the crystal structure of the complex CDK6 and EGFR has been chosen (PDB code 5L2I [24] and PDB code 4HJO [25]). The most active compound 6 in the current study was docked into the CDK6 and EGFR kinase’s putative active site. For the receptor preparation, the Molecular Operating Environment software package MOE® 2015 [26] has been used by means of the removal of water molecules and the addition of hydrogen atoms. MOE has also been used for the graphic structure of ligands 3D and then saved on data lists after minimizing structure and geometries energy. For each receptor, the pockets were then used to dock ligands after setting London dG to scoring function and GBVI/WSA dG to re scoring function. Therefore, the scoring and RMSD (root-mean-square deviation) values for the best conformation of each ligand with different receptors are shown in Table 2, as well as 2D and 3D figures of each selected conformation are shown in Figures 3 and 4. Docking simulation of compound 6 into kinase domain of CDK6 and EGFR postulated the pivotal function of both the backbone moiety and the side chain substituent (Figures 5 and 6).Table 2
Molecular simulation results for ACV/PNV interactions with HAS.
Ligand
Receptor
Amino acid residues
Interaction type
Distance (Å)
Total binding energy (kcal·mol−1)
RMSD
Compd.6
EGFR
LEU 694
pi-H
3.47
−6.156
1.878
(4HJO)
MET 769
H-acceptor
3.86
CDK6
VAL 101
H-acceptor
3.52
−6.942
1.251
LYS 147
H-acceptor
3.09
(5L2I)
VAL 27
pi-H
4.35Figure 5
The interaction between compound6 and CDK6 kinase protein (PDB code: 5L2I), presented by MOE 2015: (a, b) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (c) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (d) 2D diagrams of compound 6 (green) and padlbocyclib (red) were overlapping.Figure 6
The interaction between compound6 and EGFR kinase protein (PDB code: 4HJO), presented by MOE 2015: (a, d) 3D compound 6 binding geometry (yellow sticks) in the CDK6 binding site cavity. (b) 2D interaction diagram of compound 6 with the CDK6 binding site cavity. (c) 2D diagrams of compound 6 (green) and erlotinib (red) were overlapping.


## 4. Conclusions

A library of substituted pyridopyrimidines3–9 were designed and screened for their cytotoxicity. There are potent growth inhibitory effects against hepatic, prostate, and colon cancer cells lines, in comparison with doxorubicin as positive control. Regarding HepG2 cell line, compound 6 showed the greatest inhibitory activities against hepatic cancer (HepG2) with inhibition percent (IC50 = 0.5 μM) more potent than doxorubicin (IC50 = 0.6 μM). A molecular docking study of compound 6 into the ATP binding site of EGFR exhibited identical binding as erlotinib.



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*Source: 2635219-2019-03-20.xml*