Kumar et al. BMC Chemistry(2024) 18:161
https://doi.org/10.1186/s13065-024-01228-w

BMC Chemistry

RESEARCH

Open Access

Synthesis and anticancer evaluation of diaryl
pyrido[2,3-d]pyrimidine /alkyl substituted
pyrido[2,3-d]pyrimidine derivatives as
thymidylate synthase inhibitors
Adarsh Kumar1, Nabeel Backer1†, Harshali Paliwal1†, Ankit Kumar Singh1, Tanushree Debbaraman2, Vikramjeet Singh3
and Pradeep Kumar1*
Abstract
Worldwide, colorectal cancer (CRC) is the third most common type of cancer and the second most common causeof cancer-related deaths. Thymidylate synthase (TS) is a crucial component of DNA biosynthesis and has drawninterest as an essential target for cancer treatment. In the current work, we have designed and synthesized twentyeight new diaryl-based pyrido[2,3-d]pyrimidine/alkyl-substituted pyrido[2,3-d]pyrimidine derivatives and evaluatedtheir anticancer activity against the HCT 116, MCF-7, Hep G2, and PC-3 cell lines cell lines. Additionally, we havecarried out TS inhibitory activity and in silico studies for compounds 1n and 2j. All the synthesized compounds
exhibited good anticancer activity, but among them, compounds 1n and 2j showed excellent anticancer activity,
having IC50 values of 1.98 ± 0.69, 2.18 ± 0.93, 4.04 ± 1.06, and 4.18 ± 1.87 µM; and 1.48 ± 0.86, 3.18 ± 0.79, 3.44 ± 1.51,
and 5.18 ± 1.85 µM, against the HCT 116, MCF-7, Hep G2, and PC-3 cell lines respectively with control raltitrexed(IC50 1.07 ± 1.08, 1.98 ± 0.72, 1.34 ± 1.0, and 3.09 ± 0.96 µM, respectively) and hTS inhibitory activity with IC50 values of
20.47 ± 1.09 and 13.48 ± 0.96 nM with control raltitrexed (IC50 14.95 ± 1.01 nM). Further, the mechanism of inhibition
was revealed by molecular docking, which showed the binding pattern of 1n and 2j to the catalytic site of TS withdocking scores of -10.6 and − 9.5 kcal/mol, respectively, with reference raltitrexed (-9.4 kcal/mol). Additionally, theassessment of physicochemical, biochemical, structural, and toxicological characteristics were also in the acceptable
range for these compounds. Based on the anticancer activity of compounds, SAR was also performed for lead
optimization.
Keywords Colorectal cancer, Pyrido[2,3-d]pyrimidine, Thymidylate synthase, Anticancer, In silico studies†

Nabeel Backer and Harshali Paliwal may be treated as second
author.
*Correspondence:
Pradeep Kumar
pradeepyadav27@gmail.com
1
Department of Pharmaceutical Sciences and Natural Products, Central
University of Punjab, Bathinda 151401, India
2
Rajiv Gandhi Centre for Diabetes & Endocrinology, J N Medical College &Hospital, Aligarh Muslim University, Aligarh 202002, UP, India
3
Department of Pharmaceutical Sciences, Guru Jambheshwar Universityof Science and Technology, Hisar 125001, HR, India© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Kumar et al. BMC Chemistry(2024) 18:161

Introduction
Numerous people around the world are impacted bycancer, which is a significant health concern [1–3]. Globally, colorectal cancer (CRC) is the second most common cause of cancer-related mortality and the third mostcommon cancer [4]. According to the Globocan report,
2022, 19,964,811 new cases of cancer have been reported
throughout the world; among them, 1,926,118 (9.6%)
were colorectal cancer cases and 904,000 colorectal cancer deaths. This amounts to nearly one in ten cancerrelated deaths and cases [5]. Elderly patients are typicallydiagnosed with CRC. The median age of CRC diagnosis
in the USA was 67 years from 2013 to 2017, while 68%
of new cases are anticipated to occur in people over 65
during 2020. The American Cancer Society estimated106,970 new cases of colon cancer and 46,050 new casesof rectal cancer in the United States for 2023 [6].
Human thymidylate synthase (hTS) is gaining attention in cancer chemotherapy because of its crucial function in DNA biosynthesis [7]. It is a rate-limiting enzyme
in the de novo production of 2-deoxythymidine-5-monophosphate, which is required for DNA synthesis [4].
Nitrogen-containing heterocyclic compounds, such as
β-lactam, quinoxaline, pyrazole, pyrrolobenzodiazepine,
pyrido[2,3-d]pyrimidines, quinoline, quinazoline, pyrimidine, benzimidazole, pyridine, carbazole, imidazole,
triazole, indole, and isatin, are widely recognized pharmacophores in medicinal chemistry, exhibiting a varietyof pharmacological actions. The effect of pyrido[2,3-d]pyrimidine derivatives on cancer has not received asmuch attention as those of the other nitrogen-containing
heterocyclic compounds mentioned [8]. Therefore, toexplore the role of pyrido[2,3-d]pyridine in cancer treatment, we have performed a detailed literature search andfound that eleven different cancer targets, including tyrosine kinase, extracellular regulated protein kinases - ABL
kinase, etc. have been reported worldwide, but not a single molecule explored on TS [1].
Therefore, we have designed and synthesized benzylated diaryl-based pyrido[2,3-d]pyrimidine /alkyl substituted pyrido[2,3-d]pyrimidine derivatives to explore
their hTS inhibitory potential. The designed compounds
were developed on the basis of reported pyrido[2,3-d]pyrimidine derivatives as anticancer agents [1]. The compound considered for the drug design was 2-(2-(4-Fluorobenzylidene)hydrazinyl)-5-phenyl-7-(thiophen-2-yl)
pyrido[2,3-d]pyrimidin-4(3 H)-one (I) (Fig. 1), reported
by Fares M. et al. [9]. The molecules of the present series
were designed by replacing thiophene with an aromaticring and substituting hydrazine with a carbonyl group (asthat of 5-FU), as it is a primary requirement of hTS inhibitors. Further, the free NH of reported molecules has
also been substituted with methyl (Fig. 1). The designed
compounds were docked into the catalytic pocket of hTS

Page 2 of 15(PDB code: 1HVY). The docking score and interactionsof designed compound 1n revealed the molecules bindsimilar to raltitrexed within the active site of hTS, and all
the designed compounds had better docking scores than
the reported inhibitors (I), 5-FU and raltitrexed (Table 1).

Materials and methods
General chemistry

We used the starting ingredients and solvents purchasedfrom commercial suppliers without further purification.
Silica gel 60 F254 was used for identification, and TLC
was utilized to monitor the reaction’s progress. Silica gel
60–120 and solvent ethyl acetate/CHCl3 and petroleum
ether were used for column chromatography. A 600 MHz
Jeol NMR spectrometer was used to record 1H NMR
and 13C NMR spectra in CDCl3/d6-DMSO, with TMS(δ = 0) as the internal standard. The splitting patterns
are singlet (s), doublet (d), triplet (t), and multiplet (m),and the chemical shifts are reported in δ ppm. The coupling constant values are expressed in hertz (Hz). Uncorrected melting points were determined using an open
glass capillary tube and a Stuart melting point instrument(SMP-30). GCMS spectra were recorded by SHIMADZUGCMS at 70 eV. UV Visualization of TLC was done bySpectroline Fluorescence Analysis Cabinet (Model
CM-10 A). FTIR of synthesized molecules was recorded
by FTIR 4X (Make-JASCO). HRMS spectrum of leadinhibitors 1n and 2j was recorded by Triple TOF 5600
(Make-SCIEX). HPLC of potent compounds was performed on Agilent 1260 Infinity II with column Eclipse
XDB-C18, having a particle size of 5 μm.
Synthetic procedure and spectral data
General procedure for the synthesis of intermediate (I)A round bottom flask charged with 100 ml of puremethanol containing 6.011 g of sodium ethoxide (1 eq.)
was used to dissolve 10 gm of cyanoacetic ester (1 eq).
After that, urea (1 eq) was added, and the mixture washeated over reflux for 10 h. The alcohol was subsequently
removed, and the remaining substance was dissolvedin water. By adding acetic acid, the 6-aminopyrimidine-2,4(1 H,3 H)-dione precipitated out, which was collected by filtering [10].
General procedure for the synthesis of substituted
chalcone (II)In a 100 ml round bottom flask, substituted benzaldehyde(1 eq) was dissolved in methanol and stirred for 10 min.
Added 20% (0.5 ml) aq. NaOH solution to the reaction
mixture. After 10 min, added, substituted acetophenone(1 eq) to the reaction mixture and stirred it for 4–6 h. The
progress of the reaction was monitored by a TLC plateand visualized under UV light. On the completion of theKumar et al. BMC Chemistry(2024) 18:161

Page 3 of 15

Fig. 1 Design of proposed compounds: (A) Design of basic pharmacophore based on reported pyrido[2,3-d]pyrimidine derivatives along with 5-fluorouracil; (B) Overlay of docked raltitrexed (blue) and structure of compound 1n (red); (C) 3D-interaction diagram of the compound 1n inside catalytic pocketof human thymidylate synthase (PDB: 1HVY)

Table 1 Dock score of reported pyrido[2,3-d]pyrimidine
derivative I and designed compound 2a, raltitrexed and 5-FU
Code
I
1n
5-FU
Ral

Docking Score (kcal/mol)-6.8 Kcal/mol-10.6 Kcal/mol-5.4 Kcal/mol-9.4 Kcal/mol

reaction, the reaction mixture was filtered, washed, andthen crystallized using methanol [11].
(E)-1-(4-methoxyphenyl)-3-(naphthalen-2-yl)prop-2-en-1one

Yield 90.1%; yellowish white powder; m.p.: 140–142 ºC; IR(cm− 1) νmax: 3055 (C-Har.), 2360 (C = C), 1650 (C = O); 1H
NMR (600 MHz, CDCl3) δ (ppm): 8.08 (d, J = 8.8 Hz, 2H,
H-2’, H-6’), 8.03 (d, J = 5.8 Hz, 2 H, H-3’, H-5’), 7.95 (s, 1H,H-1), 7.88–7.86 (m, 2 H, H-7, H-8), 7.85 (d, J = 1.2 Hz, 1H,H-3), 7.84 (d, J = 4.9 Hz, 1H, H-4) 7.81–7.78 (m, 2 H, H-5,
H-6), 7.66 (d, J = 15.6 Hz, 1H, H-β), 7.00 (d, J = 8.8 Hz, 1H,H-α), 3.90 (s, 3 H, H-7’); 13C NMR (150 MHz, CDCl3)

δ (ppm): 188.6 (C = O), 155.4 (C-4’), 144.1 (C-β), 143.5(C-2), 134.4 (C-10), 133.5 (C-1’), 130.9 (C-2’), 130.6 (C-1),
130.4 (C-5), 128.7 (C-7), 127.8 (C-6), 127.3 (C-3), 126.8(C-α), 125.8 (C-1), 123.8 (C-8), 122.1 (C-4), 113.9 (C-3’),
55.6 (C-5’); GCMS (EI) calculated for C20H16O2, 288.11[M]+; observed: 288.15.
General procedure for the synthesis of diaryl based
pyrido[2,3-d]pyrimidines 1(a-n)

In a 100 ml RBF, substituted chalcone (II, 1 Eq) was dissolved in pure methanol and stirred for 20 min. 40% aq
NaOH (0.8 ml) solution was added to the reaction mixture. After 15 min, the previously synthesized intermediate I (1 eq.) was put into the reaction mixture andrefluxed for 14–16 h. The progress of the reaction wasmonitored by a TLC plate and visualized under UV light.
On the completion of the reaction, the mixture was filtered, washed, and then crystallized using methanol. [11].

Kumar et al. BMC Chemistry(2024) 18:161

Page 4 of 15

5-(4-chlorophenyl)-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (a)Yield: 84.2%; yellow powder; m.p.: 158–160 ºC; IR(cm− 1) νmax: 3727 (NH), 3432 (NH), 3251 (C-Har.),2922 (C-Haliph.), 2360 (C = C), 1619 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.67 (s, 1H, H-1), 11.20(s, 1H, H-3), 8.11 (d, J = 8.4 Hz, 2H, H-2’, H-3’), 7.51 (s,1H, H-6), 7.46 (d, J = 4.0 Hz, 4 H, H-4’, H-5’, H-2’’, H3’’),7.34 (d, J = 8.5 Hz, 2 H, H-4’’, H-5’’), 2.38 (s, 3 H, H-7’);
13
C NMR (150 MHz, DMSO-d6) δ (ppm): 161.9 (C-4),
159.4 C-2), 154.0 (C-5), 152.8 (C-9), 150.7 (C-7), 141.1(C-1’’), 138.2 (C-1’), 134.4 (C-4’’), 133.3 (C-4’), 131.0(C-3’), 130.01 (C-3’’), 127.9 (C-2’’), 127.9 (C-2’), 117.9(C-6), 106.0 (C-10), 21.4 (C-5’); GCMS (EI) calculated for
C20H14ClN3O2, 363.80 [M]+; observed: 364 [M + 1].
5-(4-chloro-3-fluorophenyl)-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (b)Yield: 56.6%; yellowish brown; m.p.: 178–180 ºC; IR(cm− 1) νmax: 3729 (NH), 3600 (NH), 3100 (C-Har.),2919 (C-Haliph.), 2360 (C = C), 1716 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.61 (s, 1H, H-1), 11.15(s, 1H, H-3), 8.05 (s, 1H, H-6), 7.49 (s, 1H, H-3’’), 7.45 (d,J = 3.9 Hz, 1H, H-4’’), 7.43 (d, J = 1.8 Hz, 1H, H-2’’), 7.27
(d, J = 5.4 Hz, 2 H, H-2’, H-3’), 7.22 (d, J = 8.3 Hz, 2 H, H-4’,H-5’), 2.32 (s, 3 H, H-7’); 13C NMR (150 MHz, DMSOd6) δ (ppm): 161.7 (C-4), 159.7 C-2), 157.7 (C-5’’), 153.7(C-9), 151.5 (C-5), 150.5 (C-7), 141.2 (C-1’’), 140.4 (C-1’),
137.4(C-4’’), 134.3 (C-3’), 129.9 (C-2’), 127.8 (C-2’’),
127.6 (C-4’), 126.3 (C-6’’), 119.5 (C-3’’), 117.6 (C-6),
105.9 (C-10), 21.2 (C-5’); GCMS (EI) calculated for
C20H13ClFN3O2, 381.06 [M]+; observed: 381.
5-(4-(dimethylamino)phenyl)-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (c)2923 (C-Haliph.), 2360 (C = C), 1713 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.57 (s, 1H, H-1), 11.13(s, 1H, H-3), 8.10 (d, J = 6.2 Hz, 2H, H-2’, H-3’), 7.50 (s,1H, H-6), 7.34 (d, J = 3.6 Hz, 1H, H-2’’), 7.33 (d, J = 4.2 Hz,
1H, H-4’’), 7.06 (s, 1H, H-3’’), 6.98 (d, J = 2.0 Hz, 2 H, H-4’,H-5’), 3.82 (s, 3 H, H-8’’), 3.76 (s, 3 H, H-7’’), 2.37 (s, 3 H,H-7’); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 161.9(C-4), 159.1 (C-2), 154.2 (C-9), 154.0 (C-5), 150.7 (C-5’’),149.4 (C-6’’), 148.2 (C-7), 140.9 (C-1’’), 134.6 (C-1’),
131.7 (C-4’), 129.9 (C-3’), 127.8 (C-2’), 121.8 (C-2’’),118.1 (C-3’’), 113.8 (C-4’’), 111.3 (C-10), 106.1 (C-6), 56.1
(C-7’’), 56.0 (C-8’’), 21.4 (C-5’); GCMS (EI) calculated for
C22H19N3O4, 389.13 [M]+; observed: 389.
5-(2,6-dichlorophenyl)-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (e)Yield: 42.7%; golden brown; m.p.: 180–182 ºC; IR(cm− 1) νmax: 3729 (NH), 3600 (NH), 3171 (C-Har.),2921 (C-Haliph.), 2360 (C = C), 1675 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.84 (s, 1H, H-1), 11.32(s, 1H, H-3), 8.13 (d, J = 3.8 Hz, 1H, H-3’), 8.12 (s, 1H,
H-6), 7.63 (d, J = 3.5 Hz, 1H, H-2’), 7.56 (d, J = 3.3 Hz,
1H, H-4’’), 7.55 (d, J = 3.7 Hz, 1H, H-5’’), 7.45 (dd, J = 8.4,
3.2 Hz, 1H, H-6’’), 7.34 (d, J = 8.1 Hz, 2 H, H-4’, H-5’), 2.37
(s, 3 H, H-7’); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
161.9 (C-4), 159.4 (C-2), 154.0 (C-5), 152.8 (C-9), 150.7(C-7), 141.1 (C-1’’), 138.2 (C-1’), 134.4 (C-4’’), 133.3(C-4’), 131.0 (C-2’’), 130.0 (C-3’), 127.9 (C-3’’), 127.9(C-2’), 117.9 (C-10), 106.0 (C-6), 21.4 (C-5’); GCMS (EI)
calculated for C20H13Cl2N3O2, 397.03 [M]+; observed:
396 [M − 1].
5-(3-bromophenyl)-7-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (f)Yield: 55.6%; yellowish brown; m.p.: 172–174 C; IR(cm− 1) νmax: 3728 (NH), 3550 (NH), 3174 (C-Har.),2922 (C-Haliph.), 2360 (C = C), 1713 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.51 (s, 1H, H-1), 11.08(s, 1H, H-3), 8.08 (d, J = 10.4 Hz, 2H, H-2’, H-3’), 7.44 (s,1H, H-6), 7.33 (d, J = 2.5 Hz, 2 H, H-4’’, H-5’’), 6.79 (d,J = 9.0 Hz, 1H, H-3’’), 6.73 (d, J = 4.8 Hz, 2 H, H-4’, H-5’),
6.55 (d, J = 1.8 Hz, 1H, H-2’’), 2.98 (s, 6 H, H-7’’, H-8’’), 2.38
(s, 3 H, H-7’); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
162.1 (C-4), 158.8 (C-2), 154.7 (C-4’’), 140.8 (C-5), 134.7(C-9), 130.7 (C-3’), 129.9 (C-2’’), 129.5 (C-7), 128.6 (C-1’),
127.9 (C-1’’), 127.8 (C-2’), 127.3 (C-3’’), 117.9 (C-4’), 113.0(C-10), 105.6 (C-6), 29.5 (C-5’’), 21.6 (C-5’); GCMS (EI)
calculated for C22H20N4O2, 372.15 [M]+; observed: 372.

Yield: 62.7%; pale yellow; m.p.: 170–172 ºC; IR(cm− 1) νmax: 3729 (NH), 3600 (NH), 3164 (C-Har.),2924 (C-Haliph.), 2360 (C = C), 1685 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.65 (s, 1H, H-1), 11.18(s, 1H, H-3), 8.20 (d, J = 8.9 Hz, 2H, H-2’, H-3’), 7.62 (s,1H, H-6), 7.60 (d, J = 7.9 Hz, 1H, H-2’’), 7.50 (s, 1H, H-3’’),7.42 (d, J = 7.8 Hz, 1H, H-6’’), 7.37 (t, J = 7.7 Hz, 1H, H-4’’),7.07 (d, J = 8.9 Hz, 2 H, H-4’, H-5’), 3.84 (s, 3 H, H-7’); 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 162.0 (C-4), 161.8(C-2), 159.4 (C-6’), 153.6 (C-4’), 152.3 (C-5), 150.7 (C-9),
141.5 (C-7), 131.4 (C-1’’), 131.2 (C-3’’), 130.2 (C-4’’), 129.7(C-2’), 129.3 (C-1’), 127.9 (C-2’’), 121.1 (C-5’’), 117.4(C-10), 114.8 (C-3’), 105.3 (C-6), 55.8 (C-5’); GCMS (EI)
calculated for C20H14BrN3O3, 423.02 [M]+; observed:
423.00.

5-(3,4-dimethoxyphenyl)-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (d)5-(2-chlorophenyl)-7-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (g)ºYield: 53.0%; pale yellow; m.p.: 182–184 ºC; IR(cm− 1) νmax: 3728 (NH), 3600 (NH), 3100 (C-Har.),Yield: 69.3%; light orange; m.p.: 166–168 ºC; IR(cm− 1) νmax: 3700 (NH), 3600 (NH), 3178 (C-Har.),Kumar et al. BMC Chemistry(2024) 18:161

2839 (C-Haliph.), 2360 (C = C), 1695 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.71 (s, 1H, H-1), 11.20(s, 1H, H-3), 8.20 (d, J = 9.0 Hz, 2H, H-2’, H-3’), 7.51 (d,J = 1.7 Hz, 1H, H-2’’), 7.49 (s, 1H, H-6), 7.45–7.39 (m,2 H, H-4’’, H-6’’), 7.35 (d, J = 7.0, 2.2 Hz, 1H, H-5’’), 7.07
(d, J = 8.9 Hz, 2 H, H-4’, H-5’), 3.84 (s, 3 H, H-7’); 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 162.0 (C-4), 161.6(C-2), 159.7 (C-4’), 153.5 (C-5), 150.85 (C-9), 150.7 (C-7),
138.8 (C-1’’), 131.8 (C-3’’), 130.0 (C-2’), 129.8 (C-2’’),129.7 (C-6’’), 129.4 (C-5’’), 129.0 (C-4’’), 127.2(C-1’), 116.9(C-10), 114.8 (C-3’), 114.7 (C-6), 55.9 (C-5’); GCMS (EI)
calculated for C20H14ClN3O3, 379.07 [M]+; observed:379.10.
5-(4-bromophenyl)-7-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1(h)Yield: 58%; pale yellow; m.p.: 171–173 ºC; IR (cm− 1) νmax:
3720 (NH), 3600 (NH), 3177 (C-Har.), 2839 (C-Haliph.),2360 (C = C), 1698 (C = O); 1H NMR (600 MHz, DMSOd6) δ (ppm): 11.65 (s, 1H, H-1), 11.18 (s, 1H, H-3), 8.18
(d, J = 9.0 Hz, 2H, H-2’, H-3’), 7.60 (d, J = 8.4 Hz, 2 H,
H-4’’, H-5’’), 7.47 (s, 1H, H-6), 7.38 (d, J = 8.4 Hz, 2 H,
H-2’’, H-3’’), 7.07 (d, J = 9.0 Hz, 2 H, H-4’, H-5’), 3.84 (s,3 H, H-7’); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
161.9 (C-4, C-4’), 159.2 (C-2), 153.9 (C-5), 152.7 (C-9),
150.7 (C-7), 138.7 (C-4’’), 131.3 (C-2’’), 130.8 (C-2’), 129.6(C-3’’), 129.5 (C-1’’), 121.9 (C-1’), 117.3 (C-10), 114.7(C-3’), 105.4 (C-6), 55.9 (C-5’); GCMS (EI) calculated for
C20H14BrN3O3, 423.02 [M]+; observed: 423.00.
5-(4-chlorophenyl)-7-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (i)Yield: 67%; pale yellow; m.p.: 164–166 ºC; IR (cm− 1) νmax:
3729 (NH), 3600 (NH), 3200 (C-Har.), 2900 (C-Haliph.),2360 (C = C), 1702 (C = O); 1H NMR (600 MHz, DMSOd6) δ (ppm): 11.65 (s, 1H, H-1), 11.18 (s, 1H, H-3), 8.18 (d,J = 8.9 Hz, 2H, H-2’, H-3’), 7.47 (s, 1H, H-6), 7.46–143 (m,3 H, H-3’’, H-4’’, H-5’’), 7.43 (d, J = 2.4 Hz, 1H, H-2’’), 7.07
(d, J = 9.0 Hz, 2 H, H-4’, H-5’), 3.84 (s, 3 H, H-7’); 13C NMR(150 MHz, DMSO-d6) δ (ppm): 161.9 (C-4, C-4’), 159.2(C-2), 153.9 (C-5), 152.7 (C-9), 150.6 (C-7), 138.3 (C-4’’),133.2 (C-1’’), 131.0 (C-1’), 129.6 (C-2’’), 129.5 (C-2’), 127.8(C-3’’), 117.4 (C-10), 114.7 (C-3’), 105.5 (C-6), 55.9 (C-5’);
GCMS (EI) calculated for C20H14ClN3O3, 379.07 [M]+;observed: 379.10.
5-(4-(dimethylamino)phenyl)-7-(4-methoxyphenyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1(j)Yield: 72%; yellowish brown; m.p.: 158–160 ºC; IR(cm− 1) νmax: 3729 (NH), 3600 (NH), 3200 (C-Har.),2924 (C-Haliph.), 2360 (C = C), 1701 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.50 (s, 1H, H-1), 11.07(s, 1H, H-3), 8.16 (d, J = 9.0 Hz, 2H, H-2’, H-3’), 7.41 (s,1H, H-6), 7.32 (d, J = 8.8 Hz, 2 H, H-2’’, H-3’’), 7.07 (d,Page 5 of 15

J = 8.9 Hz, 2 H, H-4’, H-5’), 6.73 (d, J = 8.9 Hz, 2 H, H-4’’,H-5’’), 3.84 (s, 3 H, H-7’), 2.97 (s, 6 H, H-7’’, H-8’’); 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 162.1 (C-4), 161.7(C-2), 158.6 (C-4’), 154.6 (C-4’’), 154.2 (C-5), 150.9 (C-9),
150.7 (C-7), 130.6 (C-2’), 129.9 (C-2’’), 129.4 (C-1’’), 126.4(C-1’), 117.4 (C-10), 114.7 (C-3’), 111.4 (C-3’’), 105.1(C-6), 55.8 (C-5’), 40.6 (C-5’’); GCMS (EI) calculated for
C22H20N4O3, 388.42 [M]+; observed: 388.
5-(2,6-dimethoxyphenyl)-7-(4-methoxyphenyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1(k)Yield: 55%; creamish white; m.p.: 168–170 ºC; IR(cm− 1) νmax: 3700 (NH), 3600 (NH), 3167 (C-Har.),2834 (C-Haliph.), 2360 (C = C), 1682 (C = O); 1H NMR(600 MHz, DMSO-d6) δ (ppm): 11.57 (s, 1H, H-1), 11.08(s, 1H, H-3), 8.17 (d, J = 8.9 Hz, 2H, H-2’, H-3’), 7.44 (s,1H, H-6), 7.08 (d, J = 5.0 Hz, 2 H, H-4’, H-5’), 6.97–6.90(m, 2 H, H-4’’, H-5’’), 6.81 ( t, J = 2.6 Hz, 1H, H-6’’), 3.84
(s, 3 H, H-7’), 3.74 (s, 3 H, H-7’’), 3.60 (s, 3 H, H-8’’); 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 161.8 (C-4), 159.3(C-2), 153.3 (C-4’), 153.3 (C-2’’), 150.7 (C-5), 150.7 (C-9),
129.6 (C-2’), 129.5 (C-3’), 117.5 (C-7), 117.5 (C-1’), 115.5(C-10), 115.4 (C-1’’), 114.7 (C-3’’), 114.7(C-4’’), 114.1(C-6), 56.4 (C-5’’), 55.9 (C-5’); GCMS (EI) calculated for
C22H19N3O5, 405.13 [M]+; observed: 405.
5-(2,5-dimethoxyphenyl)-7-(4-methoxyphenyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1(l)Yield: 62%; pale yellow; m.p.: 168–170 ºC; IR (cm− 1) νmax:
3729 (NH), 3600 (NH), 3177 (C-Har.), 2921 (C-Haliph.),2360 (C = C), 1703 (C = O); 1H NMR (600 MHz, DMSOd6) δ (ppm): 11.53 (s, 1H, H-1), 11.05 (s, 1H, H-3), 8.15
(d, J = 8.9 Hz, 2H, H-2’, H-3’), 7.40 (s, 1H, H-6), 7.14 (d,J = 8.9 Hz, 1H, H-5’’), 7.06 (d, J = 8.9 Hz, 2 H, H-4’, H-5’),
6.59 (s, 1H, H-2’’), 6.57 (d, J = 4.4 Hz, 1H, H-6’’), 3.84 (s,3 H, H-7’), 3.82 (s, 3 H, H-7’’), 3.65 (s, 3 H, H-8’’); 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 161.8 (C-4), 161.7(C-2), 161.3 (C-4’), 159.2 (C-4’’), 158.1 (C-3’’), 153.3 (C-5),
150.8 (C-9), 150.7 (C-7), 132.0 (C-1’), 130.0 (C-1’’), 129.7(C-6’’), 129.5 (C-2’’), 129.2 (C-2’), 117.8 (C-5’’), 114.7(C-3’), 106.9 (C-6), 104.8 (C-10), 55.9 (C-7’’), 55.8 (C-8’’),55.8 (C-5’); GCMS (EI) calculated for C22H19N3O5, 405.13[M]+; observed: 405.
5-(4-methoxyphenyl)-7-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1(m)Yield: 63%; pale yellow; m.p.: 158–160 ºC; IR (cm− 1) νmax:
3729 (NH), 3600 (NH), 3164 (C-Har.), 2919 (C-Haliph.),2360 (C = C), 1615 (C = O); 1H NMR (600 MHz, DMSOd6) δ (ppm): 11.57 (s, 1H, H-1), 11.07 (s, 1H, H-3), 8.17
(d, J = 8.9 Hz, 2H, H-2’, H-3’), 8.13 (d, J = 9.0 Hz, 1H, H-2’’),
7.43 (s, 1H, H-6), 7.20 (d, J = 7.4, 1.8 Hz, 1H, H-3’’), 7.06 (d,J = 9.0 Hz, 2 H,, , H-4’, H-5’), 7.04 (d, J = 1.2 Hz, 1H, H-4’’),7.02 (d, J = 2.9 Hz, 1H, H-5’’), 3.84 (s, 3 H, H-7’), 3.66 (s,Kumar et al. BMC Chemistry(2024) 18:161

3 H, H-7’’); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
161.8 (C-4), 161.6 (C-2), 159.3 (C-5’), 156.8 (C-5’’), 153.3(C-5), 151.0 (C-9), 150.7 (C-7), 129.9 (C-1’), 129.6 (C-1’’),129.5 (C-2’), 129.3(C-3’’), 129.0 (C-2’’), 120.5 (C-4’), 117.5(C-4’’), 114.7 (C-3’), 111.1 (C-10), 106.8 (C-5), 55.9 (C-6’,
C-6’’); GCMS (EI) calculated for C21H17N3O4, 375.12[M]+; observed: 375.
7-(4-methoxyphenyl)-5-(naphthalen-2-yl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 1 (n)Yield: 70.1%; % Purity ≥ 98 (HPLC); light brown; m.p.:
176–178 ºC; IR (cm-1) νmax: 3700 (NH), 3600 (NH), 3176
(C-Har.), 2920 (C-Haliph.), 2360 (C = C), 1703 (C = O); 1H
NMR (600 MHz, DMSO-d6) δ (ppm): 11.62 (s, 1H, H-1),
11.15 (s, 1H, H-3), 8.18 (d, J = 8.9 Hz, 2H, H-2’, H-3’),
7.95–7.92 (m, 3 H, H-7’’, H-8’’, H-1’’), 7.87 (d, J = 8.5 Hz,
1H, H-5’’), 7.55 (d, J = 3.8 Hz, 2 H, H-4’, H-5’), 7.54 (s,1H, H-6), 7.51 (dd, J = 8.4, 1.8 Hz, 1H, H- H-6’’’), 7.04 (d,J = 8.9 Hz, 2 H, H-3’’, H-4’’), 3.85 (s, 3 H, H-7’); 13C NMR(150 MHz, DMSO-d6) δ (ppm): 161.9 (C-4), 161.9 (C-2),
159.3 (C-4’) 154.0 (C-5), 150.8 (C-9), 137.5 (C-7), 133.0(C-2’’), 129.7 (C-8’’), 129.5 (C-2’), 128.5 (C-6’’), 127.8(C-7’’), 127.1 (C-1’), 127.1 (C-1’’), 126.6 (C-4’’), 126.6(C-3’’), 126.5 (C-10), 117.7 (C-5’’), 114.6 (C-3’), 105.6(C-5), 55.7 (C-5’); GCMS (EI) calculated for C24H17N3O3,
395.12 [M]+; observed: 395.15; HRMS (TOF MS) calculated for C24H17N3O3, 396.1348 [M+H]; observed:
396.1357 [M + H].
General procedure for the synthesis of alkyl substituted
pyrido[2,3-d]pyrimidine derivatives 2 (a-n)

In a round bottom flask, potassium hydroxide (4 eq) in
acetonitrile 25 ml was stirred for 10–15 min. Diarylbased pyrido[2,3-d]pyrimidines 1(a-n) (1 eq) was added
to the solution, and then the reaction mixture was stirred
for 20–25 min at 0–4 ºC. Then methyl iodide (2.5 eq)[11] was added to the stirred mixture. The reaction wasmonitored by TLC. After the completion of the reaction (2–4 h), a workup was done using ethyl acetate andwater, and the final product was purified by column
chromatography.
5-(4-chlorophenyl)-1,3-dimethyl-7-(p-tolyl)pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (a)Yield: 86.1%; light brown; m.p.: 164–166 ºC; IR (cm− 1)
νmax: 3200 (C-Har.), 2924 (C-Haliph.), 2360 (C = C), 1710
(C = O); 1H NMR (600 MHz, CDCl3) δ (ppm): 7.97 (d,J = 8.3 Hz, 2H, H-2’, H-3’), 7.42 (d, J = 8.5 Hz, 2 H, H-2’’,H-3’’), 7.38 (d, J = 8.6 Hz, 2 H, H-4’’, H-5’’), 7.26–7.24 (m,2 H, H-4’, H-5’), 6.91 (s, 1H, H-6), 3.11 (s, 3 H, H-1), 2.63(s, 3 H, H-3), 2.40 (s, 3 H, H-7’); 13C NMR (150 MHz,CDCl3) δ (ppm): 169.3 (C-4), 157.5 (C-2), 156.6 (C-9),
147.4 (C-1’’), 139.4 (C-1’), 138.1 (C-4’’), 136.3 (C-4’),
134.8, 129.6 (C-3’), 129.3 (C-2’’), 129.1 (C-3’’), 126.9 (C-7),

Page 6 of 15

117.9, 110.9 (C-6), 108.8 (C-10), 28.3 (C-1), 26.5 (C-3),
21.4 (C-5’); GCMS (EI) calculated for C22H18ClN3O2,
391.10 [M]+; observed: 391.35.
5-(4-chloro-3-fluorophenyl)-1,3-dimethyl-7-(p-tolyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (b)Yield: 81.1%; brown; m.p.: 180 − 122 ºC; IR (cm-1) νmax:
3251 (C-Har.), 2922 (C-Haliph.), 2360 (C = C), 1619 (C = O);
1
H NMR (600 MHz, CDCl3) δ (ppm): 7.96 (d, J = 8.1 Hz,
2H, H-2’, H-3’), 7.45 (t, J = 7.8 Hz, 1H, H-4’’), 7.27 (d,J = 1.9 Hz, 1H, H-3’’), 7.16 (dd, J = 8.2, 1.5 Hz, 2 H, H-4’,H-5’), 6.88 (s, 1H, H-6), 6.46 (d, J = 4.5 Hz, 1H, H-2’’),
3.10 (s, 3 H, H-1), 2.65 (s, 3 H, H-3), 2.39 (s, 3 H, H-7’);
13
C NMR (150 MHz, CDCl3) δ (ppm): 169.0 (C-4), 159.0(C-2), 157.4 (C-5’’), 156.8 (C-9), 146.3 (C-5), 140.2 (C-7),
139.5 (C-1’’), 136.1 (C-1’), 131.0 (C-4’’), 129.4 (C-2’), 126.9(C-3’), 124.8 (C-4’), 121.4 (C-2’’), 116.5 (C-2’’), 116.3(C-3’’), 110.9 (C-6), 108.5 (C-10), 28.4 (C-1), 26.6 (C-3),
21.4 (C-5’); GCMS (EI) calculated for C22H17ClFN3O2,
409.09 [M]+; observed: 409.
5-(4-(dimethylamino)phenyl)-1,3-dimethyl-7-(p-tolyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (c)Yield: 87%; dark brown; m.p.: 176–178 ºC; IR (cm-1) νmax:
2972 (C-Haliph.), 2360 (C = C), 1698 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 8.03 (d, J = 8.2 Hz, 2H, H-4’,H-5’), 7.45 (s, 1H, H-6,), 7.30 (d, J = 8.7 Hz, 4 H, H-2’’,H-3’’, H-2’, H-3’), 6.79 (d, J = 8.7 Hz, 2 H, H-4’’, H-5’’),
3.86 (s, 3 H, H-1), 3.41 (s, 3 H, H-3), 3.03 (s, 6 H, H-7’’,H-8’’), 2.43 (s, 3 H, H-7’); 13C NMR (150 MHz, CDCl3) δ(ppm): 160.8 (C-4), 158.8 (C-2), 155.7 (C-4’’), 152.1 (C-9),
151.8 (C-5), 150.6 (C-7), 140.9 (C-1’), 134.9 (C-1’’), 129.7(C-3’), 129.6 (C-2’’), 127.4 (C-2’), 126.7 (C-4’), 118.4 (C-6),
111.3 (C-3’’), 106.2 (C-10), 40.4 (C-5’’), 30.2 (C-1), 28.5
(C-3), 21.5 (C-5’); GCMS (EI) calculated for C24H24N4O2,
400.18 [M]+; observed: 400.
5-(3,4-dimethoxyphenyl)-1,3-dimethyl-7-(p-tolyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (d)Yield: 86%; yellowish brown; m.p.: 184–186 ºC; IR (cm-1)
νmax: 3381 (= C-Har.), 2942 (C-Haliph.), 2360 (C = C), 1708
(C = O); 1H NMR (600 MHz, CDCl3) δ (ppm): 7.99 (d,J = 8.1 Hz, 2H, H-2’, H-3’), 7.24 (s, 1H, H-6), 7.03 (dd,J = 8.2, 2.0 Hz, 1H, H-2’’), 6.95 (d, J = 3.1 Hz, 2 H, H-4’,H-5’), 6.93 (d, J = 2.2 Hz, 1H, H-4’’), 6.68 (s, 1H, H-3’’),3.93 (s, 3 H, H-8’’), 3.90 (s, 3 H, H-7’’), 3.12 (s, 3 H, H-1),
2.63 (s, 3 H, H-3), 2.40 (s, 3 H, H-7’); 13C NMR (150 MHz,CDCl3) δ (ppm): 169.8 (C-4), 157.6 (C-2), 156.4 (C-6’’),149.4 (C-5’’), 149.2 (C-9), 148.7 (C-5), 139.2 (C-7), 136.5(C-1’), 132.3 (C-1’’), 129.3 (C-2’), 126.9 (C-4’), 120.6)(C-3’), 118.3 (C-2’’, 111.8 (C-3’’), 111.4 (C-6), 110.7 (C-4’’),109.1 (C-10), 56.0 (C-7’’, C-8’’), 28.3 (C-1), 26.6 (C-3), 21.4
(C-5’); GCMS (EI) calculated for C24H23N3O4, 417.16[M]+; observed: 417.

Kumar et al. BMC Chemistry(2024) 18:161

5-(2,6-dichlorophenyl)-1,3-dimethyl-7-(p-tolyl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (e)Yield: 79%; dark brown; m.p.: 188–190 ºC; IR (cm− 1) νmax:
2942 (C-Haliph.), 2360 (C = C), 1697 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 7.96 (d, J = 1.8 Hz, 2H, H-2’,
H-3’), 7.55 (d, J = 8.0 Hz, 1H, H-6’’), 7.29 (d, J = 3.1 Hz,
2 H, H-4’, H-5’), 7.25 (s, 1H, H-6), 7.07 (d, J = 8.0 Hz, 1H,
H-5’’), 6.91 (d, J = 7.9 Hz, 1H, H-4’’), 3.12 (s, 3 H, H-1),
2.95 (s, 3 H, H-3), 2.43 (s, 3 H, H-7’); 13C NMR (150 MHz,CDCl3) δ (ppm): 161.5 (C-4), 158.5 (C-2), 151.3 (C-9),
146.5 (C-5), 139.4 (C-7), 138.3 (C-1’), 135.6 (C-1’’),132.6 (C-4’), 129.7 (C-2’’), 128.4, (C-4’’) 128.1 (C-3’),
128.0 (C-6), 127.0 (C-3’’), 125.2 (C-2’), 108.1 (C-10), 31.0(C-1), 28.0 (C-3), 21.3 (C-5’); GCMS (EI) calculated for
C22H17Cl2N3O2, 425.06 [M]+; observed: 427.15 [M + 2].
5-(3-bromophenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (f)Yield: 82%; brown; m.p.: 178–180 ºC; IR (cm− 1) νmax: 3391(= C-Har.), 2945 (C-Haliph.), 2360 (C = C), 1697 (C = O);
1
H NMR (600 MHz, CDCl3) δ (ppm): 8.05 (d, J = 8.8 Hz,
2H, H-2’, H-3’), 7.63–7.62 (m, 2 H, H-4’’, H-6’’), 7.36 (d,J = 6.4 Hz, 1H, H-3’’), 7.31 (s, 1H, H-6), 6.97 (d, J = 8.8 Hz,
2 H, H-4’, H-5’), 6.87 (s, 1H, H-2’’), 3.86 (s, 3 H, H-7’),
3.11 (s, 3 H, H-1), 2.63 (s, 3 H, H-3); 13C NMR (150 MHz,CDCl3) δ (ppm): 160.8 (C-4), 157.5 (C-2), 156.3 (C-9),
147.2 (C-5), 141.9 (C-1’), 131.7 (C-1’’), 131.0 (C-4’), 130.3(C-2’’), 129.2 (C-3’), 128.4 (C-4’’), 127.1 (C-3’’), 122.9(C-7), 117.3 (C-6’’), 114.4 (C-5’’), 114.0 (C-2’), 110.3(C-10), 108.3 (C-6), 55.4 (C-5’), 28.3 (C-1), 26.5 (C-3);
GCMS (EI) calculated for C22H18BrN3O3, 451.05 [M]+;observed: 451.00.
5-(2-chlorophenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (g)Yield: 86%; yellowish brown; m.p.: 170–172 ºC; IR (cm− 1)
νmax: 2941 (C-Haliph.), 2360 (C = C), 1697 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 8.12 (d, J = 8.9 Hz, 2H, H-2’,
H-3’), 7.96 (d, J = 8.9 Hz, 1H, H-3’’), 7.50 (d, J = 9.1 Hz,
1H, H-5’’), 7.37 (s, 1H, H-6), 7.02 (d, J = 8.9 Hz, 2 H, H-4’,H-5’), 6.91 (d, J = 8.8 Hz, 2 H, H-4’’, H-6’’), 3.89 (s, 3 H,H-7’), 3.86 (s, 3 H, H-1), 3.38 (s, 3 H, H-3); 13C NMR(150 MHz, CDCl3) δ (ppm): 162.1 (C-4), 159.5 (C-2),
151.6 (C-9), 138.9 (C-5), 132.1 (C-1’), 130.5 (C-1’’), 129.4(C-4’), 129.2 (C-2’), 128.7 (C-3’’), 128.4 (C-2’’), 127.7(C-4’’), 127.0 (C-7), 126.6 (C-5’’), 116.9 (C-6’’), 114.4(C-3’), 113.8 (C-10), 109.1 (C-6), 55.5 (C-5’), 30.1 (C-1),
28.4 (C-3); GCMS (EI) calculated for C22H18ClN3O3,
407.85 [M]+; observed: 407.15.
5-(4-bromophenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (h)Yield: 88%; brown; m.p.: 175–177 ºC; IR (cm− 1) νmax:
2962 (C-Haliph.), 2360 (C = C), 1698 (C = O); 1H NMR

Page 7 of 15(600 MHz, CDCl3) δ (ppm): 8.11 (d, J = 8.9 Hz, 2H, H-2’,
H-3’), 8.04 (d, J = 8.8 Hz, 1H, H-2’’), 7.35 (s, 1H, H-6),
7.33–7.32 (m, 1H, H-3’’), 7.22 (d, J = 8.4 Hz, 2 H, H-4’’,H-5’’), 7.02 (d, J = 8.9 Hz, 2 H, H-4’, H-5’), 3.87 (s, 3 H,H-7’), 3.11 (s, 3 H, H-1), 2.63 (s, 3 H, H-3); 13C NMR(150 MHz, CDCl3) δ (ppm): 160.6 (C-4), 159.1 (C-2),
153.7 (C-9), 151.6 (C-5), 147.5 (C-4’), 138.6 (C-1’’), 132.1(C-1’’), 131.1 (C-2’’), 129.6 (C-2’), 129.2 (C-3’’), 128.4(C-4’’), 117.3 (C-7), 114.4 (C-3’), 114.0 (C-10), 108.3(C-6), 55.5 (C-5’), 30.2 (C-1), 28.5 (C-3); GCMS (EI) calculated for C22H18BrN3O3, 451.05 [M]+; observed: 453.00[M + 2].
5-(4-chlorophenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (i)Yield: 81%; yellowish brown; m.p.: 168–170 ºC; IR (cm− 1)
νmax: 2928 (C-Haliph.), 2360 (C = C), 1653 (C = O); 1H
NMR (600 MHz, CDCl3) δ (ppm): 8.11 (d, J = 9.2 Hz,
2H, H-2’, H-3’), 8.04 (d, J = 9.1 Hz, 2 H, H-2’’, H-3’’), 7.35
(s, 1H, H-6), 7.02 (d, J = 9.1 Hz, 2 H, H-4’’, H-5’’), 6.97 (d,J = 9.1 Hz, 2 H, H-4’, H-5’), 3.89 (s, 3 H, H-7’), 3.10 (s, 3 H,
H-1), 2.63 (s, 3 H, H-3); 13C NMR (150 MHz, CDCl3) δ(ppm): 162.1 (C-4), 159.1 (C-2), 156.3 (C-9), 153.7 (C-5),
147.5 (C-4’), 138.1 (C-1’), 130.5 (C-1’’), 129.6 (C-2’’),
129.2 (C-2’), 128.4 (C-3’’), 128.2 (C-4’’), 117.4 (C-7), 114.4(C-3’), 113.8 (C-10), 108.4 (C-6), 55.5 (C-5’), 30.2 (C-1),
28.3 (C-3); GCMS (EI) calculated for C22H18ClN3O3,
407.85 [M]+; observed: 407.15.
5-(4-(dimethylamino)phenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (j)Yield: 90%; % Purity ≥ 98 (HPLC); dark brown; m.p.:
162–164 ºC; IR (cm− 1) νmax: 2920 (C-Haliph.), 2360 (C = C),
1699 (C = O); 1H NMR (600 MHz, CDCl3) δ (ppm): 8.10
(d, J = 8.7 Hz, 2H, H-2’, H-3’), 7.40 (s, 1H, H-6), 7.29 (d,J = 8.6 Hz, 2 H, H-2’’, H-3’’), 7.00 (d, J = 8.7 Hz, 2 H, H-4’’,H-5’’), 6.78 (d, J = 8.6 Hz, 2 H, H-4’, H-5’), 3.88 (s, 3 H,H-7’), 3.85 (s, 3 H, H-1), 3.40 (s, 3 H, H-3), 3.02 (s, 6 H,
H-7’’, H-8’’); 13C NMR (150 MHz, CDCl3) δ (ppm): 161.7(C-4), 160.8 (C-2), 158.3 (C-9), 155.5 (C-5), 150.5 (C-4’),
130.3 (C-6’’), 130.1 (C-2’), 129.5 (C-2’’), 129.0 (C-7), 126.7(C-1’’), 117.9 (C-1’), 114.2 (C-3’), 113.7 (C-10), 111.2(C-3’’), 105.7 (C-6), 55.4 (C-5’), 40.3 (C-5’’), 29.7 (C-1),
28.4 (C-3); GCMS (EI) calculated for C24H24N4O3, 416.18[M]+; observed: 416.20; HRMS (TOF MS) calculated
for C24H24N4O3, 417.1926 [M+H]; observed: 417.1941[M + H].
5-(2,6-dimethoxyphenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (k)Yield: 82%; yellowish brown; m.p.: 171–173 ºC; IR (cm− 1)
νmax: 2940 (C-Haliph.), 2360 (C = C), 1697 (C = O); 1H
NMR (600 MHz, CDCl3) δ (ppm): 8.11 (d, J = 8.9 Hz,
2H, H-2’, H-3’), 8.04 (d, J = 8.8 Hz, 2 H, H-4’, H-5’), 7.39

Kumar et al. BMC Chemistry(2024) 18:161

Page 8 of 15(s, 1H, H-6), 7.00 (d, J = 2.3 Hz, 1H, H-5’’), 6.8–6.92 (m,2 H, H-4’’, H-6’’), 3.89 (s, 3 H, H-7’), 3.85 (s, 3 H, H-7’’),3.74 (s, 3 H, H-8’’), 3.68 (s, 3 H, H-1), 3.38 (s, 3 H, H-3);
13
C NMR (150 MHz, CDCl3) δ (ppm): 161.8 (C-4), 159.2(C-2), 157.4 (C-2’’), 153.9 (C-3’’), 151.2 (C-4’), 145.4 (C-9),
130.3 (C-5), 129.1 (C-7), 128.4 (C-2’), 127.8 (C-1’), 117.4(C-1’’), 115.0 (C-5’’), 114.3 (C-4’’), 113.9 (C-3’), 111.5(C-6’’), 110.5 (C-10), 109.4 (C-6), 55.8 (C-8’’), 55.5 (C-7’’),55.4 (C-5’), 30.0 (C-1), 28.3 (C-3); GCMS (EI) calculated
for C24H23N3O5, 433.46 [M]+; observed: 433.20.

2 H, H-3’’, H-4’’), 6.98 (d, J = 8.9 Hz, 2 H, H-4’, H-5’), 6.93
(s,, 1H, H-1’’), 3.86 (s, 3 H, H-7’), 3.15 (s, 3 H, H-1), 2.51(s, 3 H, H-3); 13C NMR (150 MHz, CDCl3) δ (ppm):
160.7 (C-4), 157.7 (C-2), 156.2 (C-4’), 148.9 (C-9), 137.4(C-5), 131.9 (C-1’), 130.5 (C-2’’), 129.2 (C-3’’), 128.4 (C-7),
128.3 (C-2’), 127.9 (C-1’’), 127.8 (C-9’’), 127.1 (C-10’’),126.8 (C-8’’), 126.4 (C-7’’), 126.2 (C-6’’), 125.1 (C-4’’),117.9 (C-5’’), 114.4 (C-10), 113.9 (C-3’), 109.0(C-6), 55.5
(C-5’), 30.2 (C-1), 28.4 (C-3); GCMS (EI) calculated for
C26H21N3O3, 423.15 [M]+; observed: 422.10 [M-1].

5-(2,5-dimethoxyphenyl)-7-(4-methoxyphenyl)-1,3dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (l)Biology

Yield: 81%; yellowish brown; m.p.: 172–174 ºC; IR (cm− 1)
νmax: 2941 (C-Haliph.), 2360 (C = C), 1692 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 8.04 (d, J = 2.0 Hz, 2H, H-2’,
H-3’), 7.94 (d, J = 8.9 Hz, 2 H, H-4’, H-5’), 7.22 (s, 1H,
H-6), 6.80 (s, 1H, H-3’’), 6.75 (d, J = 8.4 Hz, 1H, H-5’’), 6.58
(d, J = 2.3 Hz, 1H, H-6’’), 3.86 (s, 3 H, H-7’), 3.79 (s, 3 H,
H-7’’), 3.73 (s, 3 H, H-8’’), 3.38 (s, 3 H, H-1), 2.62 (s, 3 H,H-3); 13C NMR (150 MHz, CDCl3) δ (ppm): 160.4 (C-4),
157.5 (C-2), 155.8 (C-4’), 150.7 (C-4’’), 145.3 (C-2’’), 132.1(C-9), 130.6 (C-5), 130.2 (C-7), 129.0 (C-1’), 128.3 (C-2’),
127.9 (C-1’’), 121.6 (C-3’’), 117.9 (C-5’’), 114.0 (C-6’’),113.8 (C-3’), 110.9 (C-10), 109.8 (C-6), 55.6 (C-8’’), 55.5
(C-7’’), 55.3 (C-5’), 30.0 (C-1), 28.2 (C-3); GCMS (EI) calculated for C24H23N3O5, 433.46 [M]+; observed: 433.20.
5,7-bis(4-methoxyphenyl)-1,3-dimethylpyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (m)Yield: 79%; brown; m.p.: 162–164 ºC; IR (cm− 1) νmax:
2942 (C-Haliph.), 2360 (C = C), 1697 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 8.11 (d, J = 8.8 Hz, 2H, H-2’,
H-3’), 8.04 (d, J = 8.8 Hz, 2 H, H-2’’, H-3’’), 7.44–7.41 (m,1H, H-4’), 7.40 (s, 1H, H-6), 7.39–7.37 (m, 1H, H-5’), 7.28
(dd, J = 7.4, 1.6 Hz, 1H, H-4’’), 7.17 (dd, J = 7.3, 1.5 Hz, 1H,
H-5’’), 3.88 (s, 3 H, H-7’), 3.73 (s, 3 H, H-7’’), 3.11 (s, 3 H,
H-1), 2.58 (s, 3 H, H-3); 13C NMR (150 MHz, CDCl3) δ(ppm): 160.6 (C-4), 159.1 (C-2), 157.5 (C-6’), 155.9 (C-6’’),151.6 (C-9), 145.6 (C-5), 132.1 (C-1’’), 130.0 (C-1’), 129.7(C-7), 129.1 (C-2’), 121.3 (C-2’’), 120.5 (C-3’’), 117.6(C-4’’), 114.3 (C-3’), 111.0 (C-5’’), 110.5 (C-10), 109.6(C-6), 55.7 (C-5’), 55.4 (C-7’’), 30.0 (C-1), 28.3 (C-3);
GCMS (EI) calculated for C23H21N3O4, 403.43 [M]+;observed: 404.10 [M + 1].
7-(4-methoxyphenyl)-1,3-dimethyl-5-(naphthalen-2-yl)
pyrido[2,3-d]pyrimidine-2,4(1 H,3 H)-dione 2 (n)Yield: 88%; yellowish brown; m.p.: 181–183 ºC; IR (cm− 1)
νmax: 2928 (C-Haliph.), 2360 (C = C), 1653 (C = O); 1H NMR(600 MHz, CDCl3) δ (ppm): 8.14 (d, J = 8.9 Hz, 1H, H-8’’),8.09 (d, J = 8.9 Hz, 2 H, H-2’, H-3’), 8.00 (d, J = 9.0 Hz,
1H, H-5’’), 7.97 (s, 1H, H-6), 7.46 (dd, J = 8.4, 1.7 Hz, 1H,
H-6’’), 7.13 (d, J = 8.8 Hz, 1H, H-7’’), 7.02 (d, J = 8.9 Hz,

Cell culture and maintenance

Human colorectal cancer (HCT-116), breast cancer (MCF-7), prostate cancer (PC-3), and liver cancer(HepG2) cells were grown in DMEM media containing 10% Fetal Bovine Serum (FBS) and 1% penicillin andstreptomycin. It was maintained at 37 °C in an incubator, humidified with 5% CO2. At 70–80% confluency, theculture cells were trypsinized with 0.25% trypsin-EDTA(Invitrogen) and subcultured for further experimentation[12].
MTT assay

MTT reagent was used in the cytotoxicity assay to test
the drug’s sensitivity to the cancer cells (HCT-116, MCF7, PC-3, and HepG2). The reduction of yellow-colored
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) into insoluble, dark purple-colored formazan crystals served as the basis for this assay. The cells
were seeded in 96-well plates (1 × 104 cells/well) and kept
at 37 °C in an incubator with CO2 filtration for the entire
night. They were then treated for 24 to 48 h with varying concentrations (1–50 µM) of synthesized compounds
and raltitrexed.
Following the treatments, treated cells were incubatedfor 4 h at a final concentration of 0.5 mg/ml of MTT.
After the incubation period, each well received 200 µL of
acidified DMSO to dissolve the purple formazan crystals.
The cell culture medium was then carefully aspirated.
After putting the plates on a shaker for ten to twenty minutes at room temperature, a microplate reader was used
to measure the absorbance at 570 nm. Untreated cells
were employed as a control. Raltitrexed was employed
as a positive control. Three measurements of each were
made. The results were displayed as mean ± SEM [13, 14].
Thymidylate synthase inhibitory assay

According to the procedure described by Wahba et
al. [15] and Davisson et al. [16], an enzymatic assay forthymidylate synthase was performed. The hTS enzyme
was procured from Elabscience using the catalog. No
PKSH033115. Thymidylate synthase was assayed spectrophotometrically at 30 ºC and pH 7.4 in a mixtureKumar et al. BMC Chemistry(2024) 18:161

Page 9 of 15

containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R, S)tetrahydrofolate, 0.012 M formaldehyde, 0.02 M MgCl2,
0.001 M dUMP, 0.04 M Tris–HCl, and 0.00075 M
NaEDTA. The reaction was initiated by the addition of
30 nM hTS, yielding a change in absorbance at 340 nm
of 0.019/min in the absence of inhibitor. Inhibitory
concentration was then calculated using two inhibitor
concentrations (1n, 2j) [17]. The test compounds’ concentration–inhibition response curve was then created toascertain the median inhibitory concentration (IC50). The
acquired information was compared with raltitrexed.

predicts the properties of molecules that are relevant topharmaceuticals. Prepared molecules were imported
from the file for the analysis, and the job was instructed
to be executed. The pharmacokinetic characteristics
of ligands, including their molecular weight, acceptor
and donor hydrogen bonds, QPlogP(o/w), QPlogHERG,QPPMDCK, QPPCaco, QPlogBB, QPlogKp, Polar Surface Area (PSA), and percentage of oral absorption byhumans, have been assessed. The application of Lipinski’s
rule aids in the prediction of the fundamental idea andphysiological characteristics of inhibitors [7, 27].

In silico study
Molecular docking

Results and discussion
As depicted in scheme 1, 28 new diaryl-based pyrido[2,3d]pyrimidine/alkyl-substituted
pyrido[2,3-d]pyrimidine derivatives have been synthesized. Diaryl-based
pyrido[2,3-d]pyrimidine/alkyl-substituted pyrido[2,3-d]pyrimidine derivatives were produced by condensing
different substituted acetophenones and substituted
benzaldehydes using NaOH as a base, were reacted with
6-aminouracil (I) to obtain diaryl based pyrido[2,3-d]pyrimidine derivatives 1(a-n) and then subjected toalkylation with methyl iodide to afford alkylated diaryl
pyrido[2,3-d]pyrimidine derivatives 2(a-n). Synthesizedtarget compounds were characterized using proton andcarbon NMR, GCMS (EI), and melting point characteristics and purified using column chromatography.
The purity of potent compounds was further verified by
HPLC, results of which revealed that both compounds
1n and 2j had purity of more than 98% (SI). Alkylationof methyl at NH of pyrido[2,3-d]pyrimidine can be confirmed by two methyl singlet at 2 to 3.6 ppm in 1H NMR
and 20 to 32 ppm in 13C NMR. Further, both carbonyls ofalkyl-substituted pyrido[2,3-d]pyrimidine were detected
between 156 and 175 ppm in 13C NMR.

Molecular docking study was conducted using the Mac
operating system on an Apple I Mac operating Maestro
12.9 (Schrodinger 2022-23) [18]. Using PDB ID: 1HVY
(resolution: 1.90 Å), in silico studies were carried out
to ascertain the binding interaction of the potent compounds within the target protein’s active site. The main
steps in molecular docking studies are protein preparation and selection, grid creation [19], ligand preparation[20], docking [21], along with subsequent docking analysis [22]. Impurities and other software-reported flaws
were eliminated in order to optimize and minimize theprotein. The minimized protein was utilized in grid generation, where the chosen ligand served as the reference
and represented the drug’s binding sites in relation to thetarget. The LigPrep module developed compounds thatwere depicted in three dimensions [23]. For every input
structure, the molecules were exposed to an OPLS-2005
force field, which produced a single, low-energy 3D structure. Glide software was used to conduct docking studies[24]. It was completed with extra precision and involved
XP descriptor data. After that, energy-minimized poses
were scored in order to produce a Glide score and docking score. Further, the validation of the docking study wasdone by using raltitrexed (Fig. 2) [25, 26].
Physicochemical, biochemical, structural, and toxicological(ADME/T) studies

The Schrodinger suite’s QikProp module provides a quick
and easy way to estimate ligand absorption, distribution,
metabolism, and excretion properties. The ADME properties were measured using prepared ligands. It accurately

Biological assessment
In vitro cytotoxicity activity

All the synthesized compounds were assessed for theiranticancer properties against four different humancancer cell lines, including HCT 116 (colorectal cancer), MCF-7 (breast cancer), Hep G2 (liver cancer), andPC-3 (prostate cancer). Among all the synthesized compounds, compounds 1n and 2j exhibited excellent anticancer activity against all cancer cell line with IC50 values

Fig. 2 Overlay of experimental (red) and docked (blue) structures of raltitrexed inside catalytic pocket of human thymidylate synthase (PDB code IHVY)Kumar et al. BMC Chemistry(2024) 18:161

Page 10 of 15

Scheme 1 Synthesis of diaryl based pyrido[2,3-d]pyrimidine/ alkyl substituted pyrido[2,3-d]pyrimidine derivatives 1 & 2 (a-n)

1.98 ± 0.69, 2.18 ± 0.93, 4.04 ± 1.06, and 4.18 ± 1.87 µM;
and 1.48 ± 0.86, 3.18 ± 0.79, 3.44 ± 1.51, and 5.18 ± 1.85
µM, respectively with control raltitrexed (IC50 1.07 ± 1.08,
1.98 ± 0.72, 1.34 ± 1.0 and 3.09 ± 0.96 µM respectively).
Compound 1i and 2e also exhibited potent activity at
IC50 values of 2.98 ± 0.87, 3.59 ± 0.59, 3.81 ± 1.86, and5.24 ± 1.18 µM; and 4.89 ± 0.09, 6.41 ± 0.84, 7.49 ± 0.87,
9.89 ± 1.64 µM, respectively against respective cell lines.
Compound 1a showed good activity against HCT 116,
MCF-7, and Hep G2 but more than 10 µM against PC-3
cells. Surprisingly, compound 2k showed very potent
activity against HCT 116 (IC50 5.44 ± 1.18 µM), but it wasless potent against other cell lines. Further, compound 2n
showed potent inhibitory activity against HCT 116 (IC50
5.59 ± 0.93 µM) and MCF-7 (IC50 8.45 ± 1.06 µM) but lesspotent to Hep G2 and PC-3 cells. Four compounds (1i,1n, 2e, and 2j) had IC50 values lower than 5 µM against
HCT 116, and three compounds (1i, 1n, and 2j) against
MCF-7 and Hep G2 and 1n against PC-3. Compounds 1a,1c, 1f, 1 h, 1 m, 2a, 2f, 2i, 2k, and 2n were having IC50 values between 5 and 10 µM against HCT 116; compounds
1a, 1 h, 1 m, 2a, 2e and 2n against MCF-7; compounds 1a,
1 m, 2a, 2e, and 2 h against Hep G2; and compounds 1i,
2e, 2 g, 2 h and 2j against PC-3 (Table 2).
From the anticancer results, a structure-activity relationship relationship of synthesized compounds against
tested cell lines was depicted as follows:a. Pyrido[2,3-d]pyrimidine pharmacophore was
essential for anticancer activity, which is inaccordance with the findings of Kumar et al., 2023
[1].
b. Compounds 1n and 2j with electron-donatinggroups at position R and R1 showed potent activity
against all tested cell lines along with excellent TS
inhibitory activity with reference to raltitrexed,
which is in accordance with the findings of Malagu etal., 2009 [28].
c. Further, compound 2e with methyl groups at Npositions also had potent activity against all tested
cell lines. These results are in accordance with thefindings of Connolly et al., 1997 and Kumar et al.,2023 [1, 29].
d. Compounds substituted at the para position had thegood IC50 than those at ortho and meta-positions[30].
e. Compounds 1a, 1f, 1i, 2a, and 2e with electron
withdrawing group showed promising activity
against HCT 116 and MCF 7 cell lines concerning
raltitrexed.
f. Overall, compounds with methyl group at the Nposition of pyrido[2,3-d]pyrimidine derivatives (2a-n) had potent activity over compounds having NH(Fig. 3).

Kumar et al. BMC Chemistry(2024) 18:161

Page 11 of 15

Table 2 Antiproliferative activity of synthesized compounds 1 & 2 (a-n)
Cmp.

R1

IC50 µM ± SEMa, b
1a
4-CH3
1b
4-CH3
1c
4-CH3
1d
4-CH3
1e
4-CH3
1f
4-OCH31g
4-OCH31h
4-OCH31i
4-OCH31j
4-OCH31k
4-OCH31L
4-OCH31m
4-OCH31n4-OCH3
2a
4-CH3
2b
4-CH3
2c
4-CH3
2d
4-CH3
2e
4-CH3
2f
4-OCH32g
4-OCH32h
4-OCH32i
4-OCH32j
4-OCH32k
4-OCH32L
4-OCH32m
4-OCH32n4-OCH3Raltitrexed

R2

R3

R4

HCT 116(Colorectal cancer)

MCF-7(Breast cancer)

Hep G2(Liver cancer)

PC-3(Prostate cancer)

4-Chloro
3-F,4-Cl
4-dimethylamino
3,4-dimethoxy
2,6-dichloro
3-bromo
2-Chloro
4-bromo
4-Chloro
4-dimethylamino
2,6-dimethoxy
2,5-dimethoxy
4-methoxy
4-(naphthalen-2-yl
4-Chloro
3-F,4-Cl
4-dimethylamino
3,4-dimethoxy
2,6-dichloro
3-bromo
2-Chloro
4-bromo
4-Chloro
4-dimethylamino
2,6-dimethoxy
2,5-dimethoxy
4-methoxy
4-(naphthalen-2-yl

H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3

H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3

5.44 ± 0.81
11.21 ± 1.80
7.66 ± 0.70
18.99 ± 1.21
27.98 ± 1.33
9.77 ± 1.68
13.44 ± 0.41
7.88 ± 0.78
2.98 ± 0.87
13.69 ± 1.09
28.98 ± 1.61
11.75 ± 0.99
6.59 ± 0.81
1.98 ± 0.69
5.87 ± 1.37
21.35 ± 1.50
11.99 ± 0.73
20.78 ± 1.14
4.89 ± 0.09
7.88 ± 0.88
14.11 ± 1.80
10.77 ± 0.79
7.54 ± 1.11
1.48 ± 0.86
5.44 ± 1.18
11.11 ± 0.71
11.57 ± 1.26
5.59 ± 0.93
1.07 ± 1.08

9.67 ± 0.62
14.56 ± 1.31
10.11 ± 1.81
26.58 ± 0.95
38.97 ± 1.21
13.68 ± 0.69
17.46 ± 1.38
9.94 ± 1.41
3.59 ± 0.59
21.41 ± 1.07
32.32 ± 0.92
14.34 ± 1.91
8.38 ± 1.37
2.18 ± 0.93
7.49 ± 1.83
36.97 ± 1.01
15.76 ± 1.49
29.47 ± 0.76
6.41 ± 0.84
13.47 ± 1.19
21.48 ± 1.63
29.37 ± 0.95
14.38 ± 1.72
3.18 ± 0.79
11.31 ± 1.91
19.84 ± 1.33
18.57 ± 1.82
8.45 ± 1.06
1.98 ± 0.72

8.46 ± 1.87
15.68 ± 0.81
12.11 ± 1.21
34.66 ± 1.01
42.66 ± 1.67
16.21 ± 0.91
18.95 ± 0.67
11.25 ± 0.73
3.81 ± 1.86
19.32 ± 1.11
36.49 ± 1.42
13.11 ± 0.53
9.62 ± 1.87
4.04 ± 1.06
7.42 ± 0.61
38.78 ± 1.13
14.38 ± 0.71
32.46 ± 1.61
7.49 ± 0.87
12.38 ± 1.51
18.93 ± 1.88
8.49 ± 1.09
17.26 ± 1.15
3.44 ± 1.51
10.47 ± 0.86
16.38 ± 1.09
17.31 ± 1.71
11.39 ± 0.94
1.34 ± 1.01

13.57 ± 1.02
21.23 ± 1.41
17.45 ± 1.08
39.11 ± 0.87
48.87 ± 1.91
18.47 ± 1.63
28.43 ± 0.78
15.93 ± 1.11
5.24 ± 1.18
35.84 ± 0.74
41.54 ± 1.97
19.11 ± 1.29
12.31 ± 1.38
4.18 ± 1.87
11.67 ± 1.01
41.47 ± 1.85
21.52 ± 1.11
38.87 ± 1.72
9.89 ± 1.64
18.37 ± 0.97
6.79 ± 1.71
8.58 ± 1.41
13.52 ± 1.21
5.18 ± 1.85
18.58 ± 1.06
24.55 ± 1.91
26.23 ± 1.43
12.49 ± 1.04
3.09 ± 0.96

a- The assay was performed in triplicate, and data were compiled for 48 h incubation; b- Data were presented as means ± standard error mean of three independent
experiments

Fig. 3 Structure-activity relationship based on anticancer activity of synthesized compounds 1 &2 (a-n)

Kumar et al. BMC Chemistry(2024) 18:161

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Table 3 In vitro thymidylate synthase (TS) activity of compounds
1n and 2j, along with raltitrexed
Compound No.
1n2j
Raltitrexed

IC50 (nM) ± SEMa, b
20.47 ± 1.09
13.48 ± 0.96
14.95 ± 1.01

a- Assay was performed in triplicate; b- Data were presented as means ± standard
error mean of three independent experiments

In vitro thymidylate synthase (TS) inhibitory activity

The most potent compounds in the cytotoxic assay (1nand 2j) were further examined to ascertain their inhibitory effects against thymidylate synthase (TS) with theobjective of identifying these molecules’ likely mechanism of action as anticancer medications. Raltitrexed
was used as control for this activity and the results werereported as IC50 (Table 3). With IC50 concentrations of
20.47 ± 1.09 and 13.48 ± 0.96 nM, the tested compounds
1n and 2j demonstrated good inhibitory activity that wasin line with their proven in vitro anticancer effects with
control raltitrexed (IC50 14.95 ± 1.01 nM).
Evaluation of insilico study
Molecular docking analysis

Hydrophobic interactions and hydrogen bonds, which
are generally highlighted by protein-ligand interactions,are essential to predict the binding conformation ofligands with hTS. The binding conformations of synthesized compounds in the binding pocket of thymidylate
synthase have been analyzed.
To explore the possible binding mechanisms of compounds at the catalytic site, flexible docking of compounds with raltitrexed into the hTS active site (PDB:
IHVY) was done with the aid of Glide in Schrödinger.
The catalytic gatekeeper residue cys195 has been associated to the pyridogrido[2,3-d]pyrimidine core. Previous research [7], has shown that interactions occur
frequently and that cysteine sulfur is often found in thesame plane as the fundamental ring system (Fig. 4). The
docked ligands 1n and 2j exhibited polar interactions
with Gln, Ser, Thr, Asn, and Asn; hydrophobic interactions with Phe, Tyr, Ala, Met Ile, Trp, Leu, Ile, and Cys;
and charged interactions with Arg, Glu and Asp aminoacid residue (Table 4). When compared to raltitrexed, thepotent compounds 1n and 2j displayed superior docking
scores.
Analysis of physicochemical, biochemical, structural, andtoxicological characteristics (ADME/T) of compounds 1n
and 2jThe ADME/T parameters for the compounds 1n and 2jwith good activity have been determined. ADME is a crucial pharmacological measure to assess the bioavailability,
absorption, and other pharmacokinetic characteristics ofpotent compounds 1n and 2j. It was found that the pharmacokinetic properties of the potent compounds against
the thymidylate synthase were within the acceptable
range (Table 5).
Molecular weight was expressed as MW, and partition
coefficient QPlogP (o/w) ranged from − 2 to 6.5, and thepredicted brain/blood partition coefficient (QPlogBB)
was within the accepted range of -3.0 to -1.2. The predicted skin permeability range (QPlogKp) spans from − 8
to -1. For apparent gut-blood barrier permeability (QPP
Caco), values ≤ 25 suggest poor permeability, while values ≥ 500 indicate high permeability. The forecast forhuman serum albumin binding (QPlogKhsa) ranges from− 1.5 to 1.2. Moreover, the percent of human oral absorption (HA) is categorized as high if ≥ 80% and low if ≤ 25%.
Donor HB: The anticipated count of hydrogen bonddonors should not exceed 5. Acceptor HB: The projected
number of hydrogen bond acceptors should remain
below 10. Polar Surface Area (PSA) falls within the rangeof < 90 Å, indicating favorable, and > 140 Å, suggesting unfavorable. QPlogHERG: Predicted IC50 value forblockage of HERG K+ channels ≤ -5 good; QPPMDCK:
Predicted apparent MDCK cell permeability accepted
range ≤ 25 poor, ≥ 500 excellent.

Conclusion
In the present study, we designed and synthesized 28
new diaryl-based pyrido[2,3-d]pyrimidine/alkyl-substituted pyrido[2,3-d]pyrimidine derivatives as anticancer agents against colorectal, breast, liver, and prostatecancer cell lines with positive control raltitrexed. Tested
compounds 1j and 2n demonstrated strong TS inhibitory activity with IC50 of 20.47 ± 1.09 and 13.48 ± 0.96 nM
with control raltitrexed (IC50 14.95 nM). Compounds 1jand 2n showed potent anticancer activity with IC50 values of 1.98 ± 0.69, 2.18 ± 0.93, 4.04 ± 1.06, and 4.18 ± 1.87
µM; and 1.48 ± 0.86,3.18 ± 0.79, 3.44 ± 1.51, and 5.18 ± 1.85
µM, respectively with control raltitrexed (IC50 1.07 ± 1.08,
1.98 ± 0.72, 1.34 ± 1.0, and 3.09 ± 0.96 µM, respectively).
Potent compounds 1j and 2n showed good docking
scores of -10.6 and − 9.5 kcal/mol, respectively with reference raltitrexed (-9.4 kcal/mol), and are having polarinteractions with Gln, Ser, Thr, Asn, and Asn; hydrophobic interactions with Phe, Tyr, Ala, Met Ile, Trp, Leu, Ile,and Cys; and charged interactions with Arg, Glu and Asp
amino acid residue with catalytic amino acid Cys195.
Further, synthesized compounds 1j and 2n satisfied
Lipinski criteria, suggesting good drug-like qualities withgood oral bioavailability profile. Based on the aforementioned results, compounds 1j and 2n may be developed aspromising inhibitors of TS.

Kumar et al. BMC Chemistry(2024) 18:161

Page 13 of 15

Fig. 4 A, C, and E were 2D docking orientations of compounds 1n, 2j, and raltitrexed, respectively, in the active site of hTS, demonstrating binding andsignificant interactions with some of the key amino acid residues; B and D were the orientations of the diaryl based pyrido[2,3-d]pyrimidine core in aclose-up view of compounds 1n and 2j; F was the close-up view of raltitrexed inside catalytic pocket of hTS (PDB code IHVY)

Table 4 Interaction pattern of compounds 1n, and 2j along with raltitrexed
S.
No.

Cpd.

H-BondingHydrophobic

Polar

ChargedPi-cation1

1n

Tyr135

Thr51, Hie196,
Ser216, and Asn226

2j

Asn226

Asp49,
Arg50,Glu87,Asp218,
Hip256,
Glu87, Arg50, Glu87,
Asp218 and Hip256,

Asp49,Arg50,
Glu87,

2

Glu87,
Asp218

-9.5

3

Ral

Ser216Phe80, Ile108, Trp109, Tyr135, Leu192,
Cys195, Leu221, Phe225, Tyr258, Met311,
and Ala312
Phe80, Ile108, Trp109, Tyr135, Leu192,
Cys195, Leu221, Phe225, Tyr258, Met311,
and Ala312
Phe80, Ile108, Trp109, Leu192, Cys195,
Leu221, Phe225, Tyr258, and Met311

Glu87, Asp218,
Hip256

Glu87,
Asp218-9.4

Thr51, Asn112,
Hie196, Ser216,
Asn226 and Hie261
Hie196, Gln214,
Ser216, and Asn226

Docking
Score(Kcal/mol)-10.6

Kumar et al. BMC Chemistry197.150
208.194
0.173
-8.6
-8.2
-2.6

96
100
36

QPPMDCK
QPlogHERG%HA(2024) 18:161

Page 14 of 15

Abbreviations
CRC	Colorectal cancerhTS	Human thymidylate synthaseTS	Thymidylate synthaseRal	Raltitrexed
IC50	Concentration which inhibits 50% of the tested populationnM	Nano molarµM	Micro molar
THF	TetrahydrofolatedUMP	deoxyUridine monophosphatedTMP	deoxyThymidine monophosphate
Aq	AqueousEq	Equivalent
GCMS	Gas Chromatography-mass spectrometry

100.7
80.3
190.1
-2.38
-1.41
-6.51
-0.78
-1.12
-3.62

QPlogK
hsa
0.623
0.467
0.347
QPlogBB

QPP
Caco
309.1
369.6
0.321
QPlogP
o/w
3.82
4.67
2.4
Acceptor
HB
4.86
4.76
9.25

Author contributions
Adarsh Kumar, Nabeel Backer, and Harshali Paliwal synthesized the molecules,
Ankit Kumar Singh and Tanushree Debbaraman performed anticancer activity,
Vikramjeet Singh and Adarsh Kumar performed molecular docking studies,
Pradeep Kumar and Adarsh Kumar wrote the manuscript.
Funding
There was no specific funding for this research.
Data availability
Data is provided within the manuscript or supplementary information files.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.

2
0
3
1n
2j
Raltitrexed12
3

395.12416.18458.48

Donor HB

Acknowledgements
The authors are thankful to the CIL-Central University of Punjab and DST-FIST
for providing Infrastructural support for the completion of this study. The
authors are also thankful to DST SERB (CRG/2023/001580) for financial support.

Received: 29 February 2024 / Accepted: 13 June 2024

Compound

MWThe online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01228-w.
Supplementary Material 1

S. No

Table 5 Physicochemical, biochemical, structural, and toxicological characteristics (ADME/T) of compounds 1n and 2j

QPlogKp

PSA

Supplementary Information

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