INTRODUCTION:

The biological significance of heterocyclic molecules has interesting synthetic objectives in drug design and discovery for several years. New pyrido-pyrimidines are a category of heterocyclic compounds that have advantageous benefits in the treatment of many disorders. Several pyridopyrimidine derivatives¹ have demonstrated potent anticancer properties by targeting key cellular pathways such as tyrosine kinase inhibition, DNA intercalation, and apoptosis induction. Their ability to interfere with cancer cell proliferation, migration, and survival has positioned them as promising candidates for therapeutic development.

Recent studies have explored the structure-activity relationships of pyridopyrimidines, leading to the synthesis of novel derivatives with enhanced selectivity and efficacy against various cancer cell lines. Given their promising biological profile, further research is warranted to optimize pyridopyrimidine-based anticancer agents² for clinical application. Many pyridine derivatives were reported to have anti-bacterial³, Anti-Alzheimer⁴, anti-platelet⁵ and anti-cancer activities⁶. The general structure of the parent compound was shown in Figure 1.

Figure 1: General structure of Pyridopyrimidine derivative

MATERIALS AND METHODS:

All chemicals, including standard drugs and solvents were obtained from Sigma-Aldrich, HiMedia, India and used without further purification with the exception of liquid aldehydes which were purified prior to use using standard procedures. The melting points of all compounds were measured using the VEEGO VMP-D Digital melting point apparatus. The FTIR spectra were obtained using JASCO FTIR 4100 series and are reported in cm^-1. TMS was used as an internal standard to measure signals from ¹H NMR and ¹³C NMR spectra on a BRUKER-II 400 (400 MHz NMR, ¹³C NMR 100 MHz) spectrophotometer. To test the purity of the compounds, pre-coated TLC plates were used, and spots were visualised using iodine vapours and ultraviolet rays. Elemental analyses were performed using a CHN-VarioElico Micro elemental analyzer. The anti-cancer activity studies were estimated using commercially available test kits (Sigma-Aldrich).

PROCEDURE:

Synthesis of 2-Thioxopyrimidine (3)

A mixture of urea (1; 0.12 mol), ethyl 2-cyanoacetate (2; 0.1 mol) were refluxed in ethanol for 10 hours with stirring in the presence of sodium ethanoate. The reaction mixture was kept at room temperature for overnight. The crude precipitate resulted in was filtered, washed with cold ethanol and dried to produce the desired product (3).

Synthesis of chalcone (6)

Synthesis of Chalcones was carried out by using Aldol condensation. A mixture of Acetophenone (4; 0.1mol; 5ml) and Benzaldehyde (5; 0.1mol; 5ml) were taken in round bottom flask with stirring in the presence of base NaOH as a catalyst. The reaction mixture was kept at 0-20℃ temperature for 12 hours. The progress of the reaction was monitored byTLC. The reactant mixture was acidified with HCl in an ice bath and the solid was then filtered and crystallized by ethanol.

Synthesis of 2,3-dihydro-2-thioxo-5,7- diarylpyrido [2,3-d]pyrimidin-4(1H)-one (7a-g)

2-Thioxopyrimidine 3 (1.43 g, 0.01 mol) and of ά-β-unsaturated ketones (6, 0.01 mol) in dry DMF solution (20 ml) was refluxed for 18–20 h and progress of the reaction was monitored by TLC. The solid mass created on cooling was filtered and crystallized from (DMF) to give the 2,3-dihydro-2-thioxo-5,7- diarylpyrido[2,3-d]pyrimidin-4(1H)-one (7).

Synthesis of 2-hydrazinyl -5,7- diarylpyrido[2,3-d]pyrimidin-4(3H)-one (8a–g)

2,3-dihydro-2-thioxo-5,7- diarylpyrido[2,3-d]pyrimidin-4(1H)-ones derivatives (7a-p; 0.004 mol) and hydrazine reagent (3 ml, 0.006 mol) was refluxed in absolute ethyl alcohol (20 ml) for 10–15 h. On cooling, the residue created was filtered and purified from (DMF).

Anti-cancer activity

The anticancer activity of new pyrimidine derivatives which have been found for their wide range of biological properties, including their anticancer qualities, is assessed using the in-vitro MTT test. The anticancer activity against three cell lines—MCF-7 (breast cancer), HeLa (cervical carcinoma), and MDA-MB-231 (breast cancer)—in vitro was evaluated for the synthesized compounds to assess their biological efficacy. The previously described MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) test was used for the evaluation. First, 96-well microtiter plates were filled with 1,105 cells per millilitre, and the plates were incubated for the entire night in minimally needed media that was enhanced with foetal bovine serum. Dimethyl sulfoxide (DMSO) was used to dissolve the compounds, resulting in a final concentration of 0.1M. The samples were then successively combined with the whole medium to yield test concentrations of 0.001, 0.01, 0.1, 1.0, and 10 micromolar (uM). For 96 hours, the MCF-7 breast cancer cells were cultivated in a 96-well plate and exposed to different doses of the test chemicals at 37°C. A 5% CO₂ concentration was used to control the environment's pH. The cells were then incubated for an additional four hours after being exposed to MTT reagent. Carefully, the medium and MTT solutions above each well were eliminated. A deep blue formazan substance was generated by the cells and dissolved in 100 milliliters of DMSO. A 96-well plate reader was used to detect absorbance at a particular wavelength of 570 nm in order to assess the vitality of the cells. Plotting the percentage inhibitions versus the concentrations required to obtain the IC50 values was done using the methods that was supplied.²³

OD Control- OD treated

% Inhibition = ------------------------------- X 100

OD Control

RESULTS AND DISCUSSION:

Pyrimidine is a particularly helpful framework for developing anticancer drugs. Its molecules have demonstrated remarkable effectiveness in a number of ways, such as the inhibition of angiogenesis, the disruption of cell migration, the activation of cell cycle arrest, the control of nuclear receptor responsiveness, and the regulation of apoptosis-induced cell growth. Pyrimidine derivatives have been shown to be effective against a wide range of cancer cell lines, including those from colorectal, lung, breast, colon, and renal malignancies. The synthetic route of Pyrido-pyrimidine quinazoline derivatives was shown in Scheme 1. The IR spectra, NMR spectra and the Mass spectra of compound 8e and 8g were shown in Figure 2 and Figure 3 respectively. The spectral interpretation was summarized in Table 1.

Scheme 1: Synthetic route of Pyridopyrimidine quinazoline derivatives

IR Spectrum

¹HNMR Spectrum

Mass Spectrum

Figure 2: Representative spectra of (8e): 2-(4-bromophenyl)-4-(3,4-difluorophenyl)-9-fluoro-12a,13-dihydro-5H-pyrido[2',3':4,5]-pyrimido[2,1-b]-quinazoline-5,7(12H)-dione

IR Spectrum

¹HNMR Spectrum

Mass Spectrum

Figure 3: Representative spectra of (8g): 8-bromo-2-(3-chlorophenyl)-4-(2,4-difluorophenyl)-12a,13-dihydro-5H pyrido[2',3':4,5]pyrimido[2,1-b]quinazoline-5,7(12H)-dione

Table 1: Characterisation of Pyridopyrimidine derivatives

Compound code	Yield (%)	Melting Point, ℃	TLC Rf	IR Spectral study	¹HNMR Spectral study (DMSO) 400MHz CDCl3
8a	56	160-162	0.9	3103cm^-1 Ar (C-H) 2922cm^-1 Ali(C-H) 3443cm^-1Amide(N-H) 1590cm ^-1Ar(c=c) 724 cm ^-1(C-F) 1184 cm ^-1(c-o)	δ 8.51 (s, 1H, amide) δ 7.77-7.81 (m, 2H, Ar-H) δ 7.40-7.47 (m,2H, Ar-H) δ 7.63-7.68 (m, 2H, Ar-H) δ 7.95-8.00 (d, 1H, Ar-H) δ 7.89-7.91 (d, 1H, Ar-H) δ 2.63 (s, 3H, hydrazinyl)
8b	68	190-192	0.5	3451cm^-1 (NH, Amide) 3065cm^-1 Ar (C-H) 2952 cm^-1Ali (C-H) 1725 cm^-1 (C=O) 1525cm^-1 Ar (C=C) 728cm^-1 (C-F)	δ 8.54 (s, 1H, amide) δ 7.92-7.96 (d, 1H, Ar-H) δ 7.82-7.84 (d, 1H, Ar-H) δ 7.70-7.75 (m, 2H, Ar-H) δ 7.62-7.68 (m, 2H, Ar-H) δ 2.68 (s, 3H, hydrazinyl)
8c	85	135-137	0.8	3451 cm^-1 (NH, amide) 3083 cm^-1Ar (C-H) 2958 cm^-1(C-H) 1598 cm^-1Ar (C=C) 720 cm^-1 ( C-F)	δ 8.56 (s, 1H, amide) δ 7.93-7.95 (d,1HAr-H) δ 7.80-7.82 (d, 1H,Ar-H) δ 7.74-7.79 (m, 3H, Ar-H) δ 2.69 (s, 3H, hydrazinyl)
8d	60	158-160	0.4	3401 cm^-1 (NH, amide), 3091cm^-1(C-H,Ar), 2968cm^-1(C-H,Ali), 1728cm^-1(C=O) 1590cm^-1 (C=C,Ar) 1180 cm^-1 (C-O) 760 cm^-1 ( C-Cl)	δ 8.58 (s, 1H, amide) δ 7.92-7.94 (d, 1H,Ar-H) δ 7.82-7.84 (d, 1H,Ar-H) δ 7.75-7.79 (m, 3H, Ar-H) δ 7.62-7.68 (m, 2H, Ar-H) δ 2.65 (s, 3H, hydrazinyl)
8e	74	148-150	0.5	3440 cm^-1 (NH,amide) 3068 cm^-1 (C-H, Ar), 2924 cm^-1 (C-H, Ali), 1726 cm^-1 (C=O), 1592 cm^-1 (C=C, Ar), 1124 cm^-1 (C-O), 728 cm^-1 ( C-F).	δ 8.59 (s, 1H, amide), δ 7.92-7.94 (d, 1H,Ar-H) δ 7.85-7.87 (d, 1H, Ar-H), δ7.73-7.78 (m, 2H, ArH), δ7.62-7.68 (m, 2H, Ar-H), δ 7.40-7.45 (m,2H, Ar-H), δ 2.60 (s, 3H, hydrazinyl)
8f	54	140-142	0.8	3401 cm^-1 (NH, amide), 3060 cm^-1 (C-H, Ar), 2952 cm^-1 (C-H, Ali), 1720 cm^-1 (C=O), 1520 cm^-1(C=C, Ar), 1260 cm^-1 (C-O) 628 cm^-1( C-Br)	δ 8.59 (s, 1H, amide) δ 7.96-7.98 (d, 1H, Ar-H) δ 7.86-7.88 (d, 1H, Ar-H), δ 7.75-7.79 (m, 2H, Ar-H), δ 7.66-7.69 (m, 2H, Ar-H), δ 7.40-7.44 (m,2H, Ar-H), δ 2.62 (s, 3H, hydrazinyl)
8g	54	180-182	0.6	3420 cm^-1 (NH, amide) 3061 cm^-1 (C-H, Ar), 2929 cm^-1(C-H, Ali), 1726 cm^-1 (C=O), 1592 cm^-1(C=C, Ar), 1124 cm^-1 (C-O), 728 cm^-1(C-F).	δ 8.65 (s, 1H, amide), δ 7.90-7.92 (d, 1H,Ar-H) δ 7.86-7.88 (d, 1H Ar-H), δ 7.73-7.78 (m, 2H, Ar-H), δ 7.62-7.68 (m, 2H, Ar-H), δ 7.40-7.45 (m,2H, Ar-H), δ 2.62 (s, 3H, hydrazinyl);

Table 2: Anti-cancer activity of new substituted 2,4-diphenyl-12a,13-dihydro-5H-pyrido [2',3':4,5] pyrimido [2,1-b] quinazoline-5,7(12H)-dione (8a-g)

Compound code	R	R₁	R₂	MDA-MB-231 IC₅₀(μM)	HELa IC₅₀(μM)	MCF-7 IC₅₀(μM)
8a	4-F	2,4 di flouro	H	1.12±0.03	1.50±0.12	1.95±0.12
8b	4-F	2,4 di bromo	4-Cl	3.10±0.20	3.30±0.10	3.90±0.15
8c	4-F	4-NO₂	5-Br	3.48±0.10	3.51±0.23	3.86±0.13
8d	4-F	3-Cl	5- Cl	5.34±0.42	5.52±0.12	6.05±0.13
8e	4-Br	2,4 di flouro	4-F	2.02±0.14	2.15±0.18	2.20±0.20
8f	4-Br	2,4 di bromo	H	2.82±0.13	2.96±0.23	3.10±0.12
8g	3-Cl	2,4 di flouro	5-Br	1.89±0.18	1.98±0.02	1.78±0.12
	Cisplatin		1.14±0.05	0.95±0.02	1.02±0.22	16

CONCLUSIONS:

New Substituted 2,4-diphenyl-12a,13-dihydro-5H-pyrido[2',3':4,5]pyrimido[2,1-b]quinazoline-5,7(12H)-dione (8a-g) were synthesized, characterized and evaluated for their anti-cancer activity. The corresponding pyrido [2, 3-d] pyrimidine derivatives (8a-g) were also synthesized from starting materials. In the MTT assay, the synthesized compounds showed low to moderate reducing activity. However, the majority of the compounds presented potent anti-cancer activity, with the most potent being pyrimidine derivatives. The compounds 8a containing pyrido[2,3-d]-pyrimidine fused with 2,4-difluorophenyl and flouro phenyl substituted moiety showed potent cytotoxic activity at low concentration with IC₅₀value 1.12±0.03μM, 1.50±0.12μM, 1.95±0.12μM and compound 8g containing 8-bromo-2-(3-chlorophenyl)-4-(2,4-difluorophenyl)-12a,13-dihydro-5H-pyrido[2',3':4,5]pyrimido[2,1-b]quinazoline-5,7(12H)-dione showed good anti-cancer activity 1.89±0.18μM, 1.98±0.02μM, 1.78±0.12μM against breast cancer cell line (MDA-MB-231), cervical carcinoma cell line (HeLa), breast cancer cell line (MCF-7). Thus, they could be further investigated as multifunctional molecules.

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Received on 08.11.2024 Revised on 20.01.2025

Accepted on 28.03.2025 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1725-1730.

DOI: 10.52711/0974-360X.2025.00247

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