Design and biological evaluation of 3-substituted quinazoline-2,4(1H,3H)-dione derivatives as dual c-Met/VEGFR-2-TK inhibitors

Abstract

The dual c-Met/vascular endothelial growth factor receptor 2 (VEGFR-2) TK inhibition is a good strategy to overcome therapeutic resistance to small molecules VEGFR-2 inhibitors. In this study, we designed 3-substituted quinazoline-2,4(1H,3H)-dione derivatives as dual c-Met/VEGFR-2 TK inhibitors. We introduced new synthetic methods for reported derivatives of 3-substituted quinazoline-2,4(1H,3H)-dione 2a–g, in addition to the preparation of some new derivatives namely, 3 and 4a–j. Three compounds namely, 2c, 4b, and 4e showed substantial amount of inhibition for both c-Met and VEGFR-2 TK (IC₅₀ range 0.052–0.084 µM). Both compounds 4b, 4e showed HB with highly conserved residue Asp1222 in the HB region of c-Met TK. For VEGFR-2 TK, compound 4b showed HB with a highly conserved residue Asp1046 in the HB region. Compound 4e showed HB with Glu885 and Asp1046. Moreover, in silico prediction of pharmacokinetic and physicochemical parameters of target compounds was carried out using SwissADME website. The quinazoline-2,4(1H,3H)-dione derivatives are promising antiproliferative candidates that require further optimisation.

Highlights

• New 3-substituted quinazoline-2,4(1H,3H)-dione derivatives were synthesised and characterised.

• Compounds 4b and 4e showed higher cytotoxic activity than cabozantinib against HCT-116 colorectal cell lines.

• Both compounds 4b and 4e showed less toxicity to WI38 normal cell line compared to HCT 116 colon cancer cell line.

• Compound 4b was superior to cabozantinib in VEGFR-2 inhibition while compound 2c was equipotent to cabozantinib.

• Compounds 4b and 4e showed remarkable c-Met inhibitory activity.

• Compounds 4b and 4e arrested cell cycle and induced significant levels of apoptosis.

• In silico ADME prediction revealed high oral bioavailability and enhanced water solubility of target compounds as compared to cabozantinib.

• Target compounds interacted with both c-Met and VEGFR-2 active site in similar way to cabozantinib.

Keywords:

• c-Met
• VEGFR-2
• colorectal cancer
• quinazoline-24(1H3H)-dione
• N-acylhydrazone

Introduction

Globally, cancer kills about 10 million people every year and its mortality rate will reach 16.3 million deaths in 2040¹. Conventional chemotherapeutic drugs can raise the survival rate of cancer patients but they have negative impact on patients’ quality of life because of their severe adverse effects². With growing knowledge of chemistry and function of cellular molecular targets, tyrosine kinase (TK) inhibitors become the main arm of chemotherapeutic arsenal³. Compared to conventional chemotherapy, TK inhibitors afford good anticancer activity with few and tolerable adverse effects².

The activation of c-Met TK receptor is achieved by binding of HGF/SF, that binding promotes different cellular signalling pathways and results finally in cell proliferation, motility, migration, and survival⁴. These cellular activities are fundamental in normal physiological processes like wound healing but can lead to tumorigenesis when these pathways are deregulated⁵. c-Met TK was found to be overexpressed or mutated in human tumours and it was related to poor prognosis⁴. Consequently, c-Met was considered as a potential molecular target for cancer therapy⁶.

Another TK is a vascular endothelial growth factor receptor 2 (VEGFR-2), which is expressed in endothelial cells, mediated by VEGF, and fundamentally regulates the angiogenesis of tumours^7,8. Albeit VEGF/VEGFR pathway is overexpressed in cancer cells and it is considered as important drug target, many antiangiogenic drugs suffering from humble efficacy⁹. Many neoplasms acquired therapeutic resistance against bevacizumab as well as small molecules inhibitors that target catalytic domain of VEGFR-2^10–13. There are several mechanisms beyond this resistance like expression of Tie2 by macrophage⁹, tumour hypoxia¹⁴, upregulation of SDF,1¹⁵ and overexpression of pERK¹⁶. It is found that HGF/c-Met pathway drives this resistance and dual inhibition of c-Met/VEGFR-2 was found to be effective against tumours that are resistant to pan-VEGFR inhibition^14,17. Single-target VEGFR-2 inhibitors does increase hypoxia which leads to c-Met mediated invasion and metastasis of cancer cells. On the contrary, the combination of VEGFR-2 and c-Met inhibitors, like sunitinib and PF-04217903 remarkably lowers tumour invasion and metastasis.¹⁸ Hence regarding drug resistance, multitarget TK inhibitors are more superior than single target inhibitors¹⁹.

Four structural requirements for dual c-Met/VEGFR-2 inhibition are heterocycle, linker, HB domain, and hydrophobic tail (Figure 1)¹⁹. Aza-heterocycle occupies adenine site in hinge region and forms from 0 to 3 HBs with highly conserved residues Met1160 and Cys919 in c-Met and VEGFR-2, respectively. Heterocycle ring can be surrogated by phenyl ring with HB groups like halogens and carboxamide (Figure 2)^18,20. Linker occupied the region adjacent to gatekeeper. It has a little role in protein binding, but it is important to fix other pharmacophoric elements in the corresponding binding sites⁸. It may be (hetero)aromatic or aliphatic chain (Figure 2)^{6,7,18,20–22}.

Figure 1. Pharmacophoric elements for dual inhibition of c-Met/VEGFR-2 TKs.

Figure 2. Four structural elements of reported dual c-Met/VEGFR-2 inhibitors.

HB domain can form HB with Asp1046 and Asp1222 of the DFG motif in c-Met and VEGFR-2, respectively. For c-Met inhibition, HB domain acts as HBA by forming two HB by two carbonyl oxygen with Asp1222 and Lys1110 while for VEGFR-2 inhibition, HB domain acts as dual HBA/HBD by forming two HB by carbonyl oxygen and amidic NH with Asp1046 and Glu885^8,23. HB domain can be dicarboxamide, 3-sulfonylacrylamide, (thio)urea, N-acyl(thio)urea, carbamoylmethyl carboxylate, N-acylhydrazone, and semicarbazone^{3,18,22,24–27}. It also can be incorporated in nitrogenous five or six membered ring system with carboxamide substituent like pyrazole, imidazole, 1,2,3-triazole, pyridine, pyrimidine, pyridazine, quinoline, cinnoline, and naphthyridine^{3,4,6,7,20,21,24,25,28–33}. Hydrophobic tail occupies the phenyl pocket in the allosteric site of TK and it may be phenyl, (non)aromatic heterocyclic, or alicyclic ring (Figure 2)^3,19,34.

Therefore, encouraged by the abovementioned rationale, we designed and synthesised a series of 3-substituted quinazoline-2,4(1H,3H)-dione analogues via a multi component reaction (Figure 3). The antiproliferative activity of target compounds was performed against c-Met/VEGFR-2 overexpressing HCT-116 colon cancer cell line. In addition, cytotoxicity of selected compounds against WI38 normal cells was carried out to assess safety profile of new series of compounds. In vitro screening of inhibitory activity against both c-Met and VEGFR-2 was carried out using cabozantinib as a positive control. Cell cycle analysis and apoptosis assay were carried out. Furthermore, the molecular docking studies were performed to investigate the interactions of the target molecules with c-Met and VEGFR-2 TKs. Finally, in silico pharmacokinetic profile and druglikeness of the synthesised molecules were investigated by SwissADME.

Figure 3. General structures of target compounds.

Results and discussion

Chemistry

The synthetic protocol adopted in this study is illustrated in Schemes 1 and 2. The azide 1 was first prepared by Younes et al. by treatment of a suspension of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetohydrazide in hydrochloric acid with a solution of sodium nitrite in water at 0–5 °C³⁵. Treatment of a cold solution of azide 1 with different primary and secondary amines afforded a new method to synthesise reported amides 2a–g^36–40. In these reactions, a low temperature (around 0 °C) is required to prevent decomposition of the acyl azide which releases nitrogen gas. The coupling step was performed under basic conditions. In our study, the amines were used as reactants as well as basic catalysts. For the synthesis of compound 2g, we used triethylamine as a catalyst. Another method was also used for the synthesis of compounds 2b, 2c, 2g in which it was observed that the coupling reaction can be performed under reflux as it is finished in a time less than that needed for decomposition of the azide. The reflux was carried out in dry benzene for compounds 2b and 2c, while dry pyridine was utilised as a solvent and basic catalyst for the synthesis of compound 2g (Figure 4)⁴¹.

Scheme 1. Synthesis of target compounds 2a–g.

Scheme 2. Synthesis of target compounds 3 and 4a–j; reagents and conditions: (a) N₂H₄·H₂O, EtOH, reflux. (b) Ar-CHO, piperidine, EtOH, reflux. (c) Cyclohexanone, EtOH, reflux. (d) Isatin, AcOH, reflux. (e) Acetylacetone, EtOH, reflux. (f) CS₂, pyridine, reflux. (g) PhNCS, EtOH, reflux. (h) NaOH.

Figure 4. Suggested mechanism of reaction of acyl azide with primary and secondary amines.

The reaction of azide 1 with glycine ethyl ester hydrochloride gave 2g. Then, treatment of compound 2g with hydrazine hydrate yielded hydrazide 3. IR spectrum of 3 showed characteristic absorption bands for NH₂ and NH’s at 3291 and 3175, and for C═O’s at 1704 and 1644. Besides, the ¹H NMR spectrum revealed the disappearance of the triplet and quartet signals related to the ethyl group of the starting ester 2g, and the appearance of new characteristic singlet signals for NH and NH₂ groups of the resulting hydrazide at δ 9.02 and 4.26 ppm, respectively. Compound 3 is a useful precursor in the synthesis of target compounds 4a–j.

Condensation of hydrazide 3 with various aromatic aldehydes namely, benzaldehyde, 4-nitrobenzaldehyde, anisaldehyde, and furfural afforded a series of hydrazone derivatives 4a–d, respectively in good yields. It is known that N-acylhydrazones may exist as E/Z geometrical isomers with respect to the imine C═N double bond, and cis/trans amide conformers due to rotation across the amide C(O)–NH single bond (Figure 5)^42,43. According to the literature, N-acylhydrazones derived from aryl and heteroaryl aldehydes tend to exist in the form of the less hindered E geometrical isomer both in the solid state and in DMSO solution; however, in less polar solvents, the Z isomer can be detected due to its stabilisation with intramolecular hydrogen bonds.^42–48

Figure 5. Isomers of N-acylhydrazones.

From the spectroscopic studies of the obtained hydrazones 4a–d, IR spectra showed characteristic stretching bands for NH’s in the range of 3249–3127 cm⁻¹, for C═O’s in the range of 1736–1644 cm⁻¹ and for C═N in the range of 1570–1518 cm⁻¹. The ¹H NMR spectra of these hydrazones exhibited two sets of signals for certain protons (NCH₂C(O)NH, α-CH₂, C(O)–NH–N, N═CH), which revealed their existence as an equilibrium mixture from cis/trans conformers of the E geometrical isomer. In addition to the abovementioned studies, this conclusion is supported by previous studies on similar hydrazones.^49,50 It was found that the cis-conformer predominates in DMSO solution.⁴² For α-CH₂ protons, the smaller upfield signal is assigned to the trans-conformer while the larger downfield signal to the cis-conformer. On the contrary, for N═CH proton the larger upfield signal is attributed to the cis-conformer while the smaller downfield signal to the trans-conformer. These data are in accordance with literature results.⁵¹ The signals of NCH₂C(O)NH and C(O)–NH–N protons of cis- and trans-conformers were recognised from the integration of their signals relative to those of α-CH₂ and N═CH protons (Table 1). The ratio of cis- to trans-conformers for every compound was calculated from the ratio of integrations of the paired peaks (see Supplementary Data). ¹³C NMR spectra also displayed duplicated signals for certain carbon atoms, which confirmed the presence of a mixture of conformational isomers.

Table 1. In vitro cytotoxicity of compounds 2a–g, 3, 4a–j, and cabozantinib against HCT116 cell line.

Compound	IC50 (µM)	Compound	IC50 (µM)
2a	15.11 ± 0.80	4b	0.734 ± 0.04
2b	16.02 ± 0.85	4c	31.82 ± 1.68
2c	4.692 ± 0.25	4d	10.83 ± 0.57
2d	47.52 ± 2.51	4e	1.408 ± 0.07
2e	37.99 ± 2.01	4f	44.1 ± 2.33
2f	5.401 ± 0.29	4g	3.697 ± 0.20
2g	12.5 ± 0.66	4h	6.989 ± 0.37
3	24.29 ± 1.28	4i	9.204 ± 0.49
4a	36.94 ± 1.95	4j	18.89 ± 1.00
Cabozantinib	16.350 ± 0.86

The reaction of carbohydrazide 3 with cyclohexanone afforded 4e. IR spectrum of 4e showed characteristic stretching bands for NH’s at 3256 and 3210 cm⁻¹, for C═O’s at 1738 and 1647 cm⁻¹ and for C═N at 1540 cm⁻¹. In ¹H NMR spectrum of hydrazone 4e in DMSO-d₆, the NHCH₂ appeared as two doublets with similar intensities at δ 3.84 and 4.15 ppm for CH₂ protons and two triplets with similar intensities at δ 8.32 and 8.51 ppm for NH proton. Furthermore, C(O)–NH–N proton appeared as two singlets at δ 10.14 and 10.46 ppm also with similar intensities. We might conclude that the hydrazone 4e solution in DMSO is present in the form of cis/trans amide conformers in 1:1 ratio due to rotation across the amide C(O)–NH single bond (Figure 6).

Figure 6. Cis/trans amide conformers of hydrazone 4e.

Refluxing of carbohydrazide 3 with isatin resulted in the formation of hydrazone 4f. The ¹H NMR spectrum of this hydrazone in DMSO-d₆ showed that some protons exhibit two sets of signals for each proton (NCH₂C(O)NH, α CH₂, C(O)–NH–N). The downfield shift of the two peaks related to the NH–N proton (δ 12.55 and 13.26 ppm) indicates the presence of intramolecular hydrogen bonding stabilising the Z geometrical isomer. Thus, it is more likely that compound 4f is found as an equilibrium mixture of cis/trans conformers of the Z geometrical isomer due to rotation across the amide CO–NH single bond, and the ratio of cis to trans conformers is 7:4, respectively.^52,53

The compound 4g was prepared via the Knorr pyrazole synthesis by treatment of hydrazide 3 with acetylacetone as a β-diketone. The ¹HNMR spectrum displayed singlet signals at δ 1.77, 1.96, and 6.40 ppm indicating the presence of two methyl groups and one methine group, beside the disappearance of NH and NH₂ signals related to the hydrazide 3. ¹³C NMR spectrum was also used to confirm the structure where the two CH₃ carbons appeared at 16.32 and 26.38 ppm.

The compound 4h was prepared by heating carbohydrazide 3 with carbon disulphide in pyridine. IR spectrum showed absorption bands at 3304 and 3214 attributed to NH’s, at 1714 and 1661 attributed to C═O’s, and at 1140 attributed to C═S. The ¹H NMR spectrum showed the disappearance of NH₂ signal of hydrazide 3 and the appearance of a new characteristic signal at δ 10.98 ppm related to NHCS proton of compound 4h.

The reaction of hydrazide 3 with phenyl isothiocyanate yielded 4i. IR spectrum showed bands for NH’s at 3444, 3285, and 3201 cm^–,¹ for C═O’s at 1735, 1704, and 1668 cm^–,¹ and for C═S at 1267 cm⁻¹. The ¹H NMR spectrum indicated a multiplet for aromatic protons (9H) at δ 7.06–7.95 ppm, besides the disappearance of NH₂ signal of hydrazide 3 and the appearance of new characteristic signals at δ 9.31, 9.74, and 10.21 ppm related to the new NH groups.

Heating the thiosemicarbazide 4i in NaOH solution under gentle reflux leads to the formation of 4j. IR spectrum showed bands for NH’s at 3272 and 3200 cm⁻¹, for C═O’s at 1718 and 1658 cm⁻¹, and for C═S at 1210 cm⁻¹. The ¹H NMR spectrum showed the disappearance of the signals of NH groups of the thiosemicarbazide and the appearance of a new characteristic signal at δ 13.86 ppm related to the NH proton of the triazole ring.

Biology

In vitro antiproliferative activity against HCT-116

The target compounds were evaluated for their anticancer activity against HCT-116 colon cancer cell line in which both c-Met and VEGFR-2 are overexpressed.^54,55 MTT assay was carried out to test the effect of different concentrations of the target compounds on HCT-116 cell line using DMSO as a negative control whereas cabozantinib was used as positive control. The half maximal inhibitory concentration (IC₅₀) was calculated for each compound (Table 1). Most target compounds exhibited significant anticancer activities. Amongst all compounds, compound 4b exhibited promising inhibition activity with IC₅₀ less than 1 µM. Eleven out of 18 compounds namely, 2a, 2b, 2c, 2f, 2g, 4b, 4d, 4e, 4g, 4h, and 4i displayed remarkable anticancer activity (IC₅₀=0.734–16.02 µM) and they were more superior than cabozantinib. On the other hand, compound 4j (IC₅₀=18.89 µM) showed comparable anticancer activities to cabozantinib. Compounds 2d, 2e, 3, 4a, 4c, and 4f showed noticeable anticancer activity (IC₅₀=24.29–47.52 µM).

In vitro toxicity against normal cells

To scrutinise the safety of target compounds on normal cells, compounds 4b and 4e were selected to evaluate their cytotoxic activity against WI38 normal cell line (Table 2). Both compounds showed less toxicity to WI38 normal cell line compared to HCT 116 colon cancer cell line. Compounds 4b and 4e exhibited cytotoxic activity against HCT-116 colon cancer cells 25 and 7.5 times more than WI38 normal cells, respectively.

Table 2. In vitro cytotoxicity of compounds 4b and 4e against WI38 normal cell line.

Compound	IC50 (µM)
4b	18.45 ± 1.09
4e	10.55 ± 0.62
Cabozantinib	44.71 ± 2.65

In vitro activity against c-Met and VEGFR-2 tyrosine kinases

As our rationale is to target c-Met/VEGFR-2 TKs, herein, we selected six 3-substituted quinazoline-2,4(1H,3H)-dione derivatives for evaluation of their inhibitory activity against both c-Met and VEGFR-2 enzymes.⁸ Cabozantinib was used as a positive control. The results in Table 3 showed that all scrutinised compounds have dual c-Met/VEGFR-2 inhibitory activity in the nanomolar range (IC₅₀ range 0.035–0.297 µM). Three compounds namely, 2c, 4b, and 4e showed substantial amount of inhibition for c-Met enzyme (IC₅₀ range 0.063–0.084 µM). Interestingly, to agree with the c-Met inhibition results, compounds 2c, 4b, and 4e were found to be the most potent agents against VEGFR-2 enzyme (IC₅₀ range 0.035–0.082 µM) (Figure 7). This correlation between the inhibitory activity of target compounds against c-Met and VEGFR-2 TKs is in consonance with the shared pharmacophoric requirements for inhibition of the two TKs. Compared to cabozantinib, all target compounds showed less inhibitory activity against c-Met enzyme. On the other hand, compound 4b (IC₅₀=0.035 µM) exhibited superior inhibition than cabozantinib against VEFGR-2. Additionally, compound 2c (IC₅₀=0.052 µM) was equipotent to cabozantinib.

Figure 7. Correlation of the inhibitory activity of c-Met enzyme and VEGFR-2 enzyme.

Table 3. Inhibitory activity of selected compounds against c-Met and VEGFR-2.

Compound	c-Met IC50 (μM)	VEGFR-2 IC50 (μM)
2c	0.084 ± 0.004	0.052 ± 0.002
2f	0.161 ± 0.007	0.210 ± 0.009
4b	0.063 ± 0.003	0.035 ± 0.002
4e	0.069 ± 0.003	0.082 ± 0.004
4g	0.271 ± 0.012	0.103 ± 0.005
4h	0.245 ± 0.011	0.297 ± 0.013
Cabozantinib	0.03 ± 0.002	0.059 ± 0.003

Apoptosis assay

Aiming to further study the mechanism of the target compounds, the cell cycle arrest experiment of 4b and 4e blocking the cell cycle of HCT-116 was carried out using Annexin V-FITC/PI staining (Table 4 and Figure 8).⁵⁶ HCT-116 colon cancer cells were treated with target compounds 4b, 4e, and cabozantinib at their IC₅₀ concentrations. Compared to the control, the percentage of HCT-116 cells in the S process increased to 51.07% and 46.89% after treatment with 4b and 4e, respectively.

Figure 8. Cell cycle analysis in HCT-116 colon cancer cell line treated with compounds 4b and 4e.

Table 4. Cell cycle analysis in HCT-116 colon cancer cell line treated with compounds 4b and 4e.

Compound	%G0–G1	%S	%G2/M
4b	39.59	51.07	9.34
4e	44.60	46.89	8.51
Cabozantinib	43.12	32.16	24.72
Control	47.41	39.28	13.31

The mechanism of HCT-116 apoptosis induced by compounds 4b, 4e, and cabozantinib was further scrutinised by annexin V/PI staining (Table 5).⁵⁶ Compared to the control group, the number of both early and late apoptotic cells of target compounds 4b and 4e was increased. The number of early apoptotic cells of 4b and 4e (15.28 and 26.76%, respectively) was higher than that of cabozantinib. In addition, the number of late apoptotic and death cells of 4b (26.41%) was higher than that of cabozantinib. It can be concluded that the synthesised compounds 4b and 4e can induce apoptosis of HCT-116 cells more efficiently than cabozantinib (Figures 9–11).

Figure 9. Apoptosis induction analysis using Annexin-V/propidium iodide (PI) staining assay.

Figure 10. Representative cytograms of apoptotic HCT-116 cells induced by 4b and 4e compared to cabozantinib for 24 h.

Figure 11. Representative cytograms of apoptotic HCT-116 cells induced by 4b and 4e compared to cabozantinib for 24 h.

Table 5. Apoptosis induction analysis for compounds 4b, 4e, and cabozantinib.

Compound	Apoptosis			%Necrosis
	%Total	%Early	%Late
4b	41.69	15.28	26.41	4.53
4e	38.78	26.76	12.02	2.28
Cabozantinib	32.13	12.52	19.61	3.63
Control HCT-116	0.52	0.33	0.19	1.33

In silico studies

Molecular modelling studies

Molecular docking of the target compounds was carried out in the active site of both c-Met (PDB: 3lq8) and VEGFR-2 (PDB: 4asd) TKs^8,19. Figure 12 represents docking styles of compounds 4b and 4e in the active site of c-Met TK. Compound 4b showed dual HB with highly conserved residue Asp1222 (2.64 and 2.84 Å) in the HB region as well as hydrophobic interactions with Met1131 and with Met1211 in the hinge region. Compound 4e showed HB with Asp1222 (2.88 Å) and Met1131 (3.42 Å) in addition to hydrophobic interactions with Met1131 and Met1211. On the other hand, docking poses of compounds 4b and 4e in the active site of VEGFR-2 TK are shown in Figure 13. Compound 4b showed HB with Cys1045 (3.73 Å) and with a highly conserved residue Asp1046 (3.13 Å) in the HB region. Compound 4e showed HB with Glu885 (H₂O 3.28 Å) and Asp1046 (3.20 Å).^8,56

Figure 12. Docking pose of compound 4b and 4e with c-Met TK (PDB: 3lq8).

Figure 13. Docking pose of compound 4b and 4e with VEGFR-2 TK (PDB: 4asd).

In silico prediction of physicochemical and pharmacokinetic properties

Many drug candidates failed to reach the clinic because of their improper pharmacokinetics and physicochemical properties. Consequently, in silico prediction of pharmacokinetic and physicochemical parameters of target compounds was carried out using SwissADME website (see Supplementary Data)⁸. SwissADME provides different reported filters to estimate drug likeness of tested compounds namely, Lipinski, Ghose, Veber, Egan, Muegge as well as bioavailability score. All target compounds showed zero or only one violation regarding all applied drug likeness filters. All target compounds have no Lipinski violation except compounds 4b and 4f which have only one violation just like cabozantinib. The Abbott Bioavailability Score of all tested compounds is equal to that of cabozantinib.

All target compounds have enhanced water solubility than cabozantinib. Like cabozantinib, most synthesised compounds were predicted to have high GI absorption (see Supplementary Data). Compared to cabozantinib, tested compounds were predicted not to be substrates for CYP2C19, CYP2C9, CYP2D6, and CYP3A4 that decrease the probability of drug–drug interactions.

Another tool to assess the oral bioavailability of small molecules is the Bioavailability Radar (Figure 14). It is a hexagon with specific physicochemical property at each vertex and the central pink region represents the optimum physicochemical space for good oral bioavailability. Compound 4e showed good fit to all six physicochemical parameters and it is predicted to have good oral bioavailability. Compound 4b exhibited deviation in both (Fraction Csp3 = 11) and (TPSA = 171.24 Å²) which expressed insaturation and polarity. In addition, cabozantinib showed deviation in both flexibility and insaturation as it has (10 rotatable bonds) and (Fraction Csp3 = 0.18).

Figure 14. Bioavailability Radar for 4b, 4e, and cabozantinib.

BOILED-Egg (The Brain Or IntestinaL EstimateD permeation) model is a robust method that carefully prognosticates both GI absorption and BBB accessibility by estimating both the lipophilicity (expressed in WLOGP) and polarity (expressed in TPSA) of target compounds (Figure 15). Like cabozantinib, compounds 2a–g, 4a, 4c–e, and 4g showed a high GI absorption while compounds 3, 4b, 4f, and 4h–j showed low gastrointestinal absorption. The high GI absorption of some synthesised compounds is due to reasonable balance between their lipophilicity (WLOGP –0.83 to 1.14) and their polarity (TPSA 75.17–138.56 Å²). Moreover, all tested compounds as well as cabozantinib do not pass BBB confirming their good CNS safety profile.

Figure 15. BOILED-Egg model of target compounds and cabozantinib.

Conclusions

The current study was aimed at identifying novel drug-like molecules as anticancer drug candidates using in silico and in vitro techniques. Novel series of 3-substituted quinazoline-2,4(1H,3H)-dione derivatives of quinazolinone scaffold were synthesised and their chemical structures were confirmed by elemental and spectroscopic analyses. New synthetic methods were introduced for reported derivatives of 3-substituted quinazoline-2,4(1H,3H)-dione 2a–g, in addition to the preparation of some new derivatives namely, 3 and 4a–j. Compounds 2c, 4b, and 4e showed remarkable dual inhibition for both c-Met and VEGFR-2 TKs. Designed compounds exhibited binding mode with target proteins like that of cabozantinib. Compounds 4b and 4e showed HB with highly conserved residue Asp1222 in the HB region of c-Met TK. For VEGFR-2 TK, compound 4b showed HB with a highly conserved residue Asp1046 in the HB region. Compound 4e showed HB with Glu885 and Asp1046. Moreover, in silico prediction of pharmacokinetic and physicochemical parameters of target compounds was carried out using SwissADME website. The quinazoline-2,4(1H,3H)-dione derivatives are promising antiproliferative candidates that require further optimisation.

Experimental

Chemistry

All reactions were followed by thin layer chromatography (TLC). Melting points were measured using an electrothermal melting apparatus and are uncorrected. IR spectra were recorded on a Shimadzu FT-IR 8101 PC infra-red spectrophotometer (Kyoto, Japan). ¹H and ¹³C NMR spectra were recorded on a BRUKER 400 MHz instrument (400.2 MHz for ¹H and 100.64 MHz for ¹³C). Chemical shifts are expressed in parts per million (ppm) units using tetramethylsilane (TMS) as an internal reference. Mass spectra were recorded on a Shimadzu GC-MS QP-2010 Plus EX mass spectrometer (Kyoto, Japan) at 70 eV. Elemental analyses were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt.

General procedure for synthesis of amides 2a–f

To a cold solution of azide 1 (0.49 g, 2 mmol) in ethyl acetate (15 ml), a solution of the corresponding amine derivative (2 mmol) in ethyl acetate (5 ml) was added dropwise with stirring. The reaction mixture was stirred at 0–5 °C for 6–14 h, then kept at room temperature overnight. The solid product was collected by filtration and crystallised from the appropriate solvent.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-phenylacetamide (2a)

Crystallisation from acetic acid yielded 0.52 g (88%) of compound 2a as white crystals; m.p. over 300 °C (reported 294–296 °C)³⁶. FT-IR (KBr, υ, cm⁻¹): 3204, 3136 (NH’s), 1737, 1636 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 4.72 (s, 2H, NCH₂), 7.06 (t, 1H, ArH), 7.25 (m, 2H, ArH_quinazoline), 7.32 (m, 2H, ArH), 7.57 (m, 2H, ArH), 7.71 (t, 1H, ArH_quinazoline), 7.96 (d, 1H, ArH_quinazoline), 10.31 (s, 1H, CONH), and 11.61 (s, 1H, quinazoline NH). MS m/z (%): 295 [M]⁺ (4.36%). Anal. Calcd. for C₁₆H₁₃N₃O₃: C, 65.08; H, 4.44; N, 14.23%. Found C, 65.16; H, 4.62; N, 14.12%.

N-Benzyl-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide (2b)

Crystallisation from acetic acid yielded 0.56 g (90.6%) of compound 2b as white crystals; m.p. 298–299 °C (reported 294–296 °C)³⁶. FT-IR (KBr, υ, cm⁻¹): 3199 (NH’s), 2954, 2874 (aliphatic CH’s), 1735, 1640 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 4.31 (d, J= 6 Hz, 2H, NHCH₂), 4.56 (s, 2H, NCH₂), 7.20–7.35 (m, 7H, ArH), 7.69 (t, J= 7.6 Hz, 1H, ArH_quinazoline), 7.96 (d, J= 7.6 Hz, 1H, ArH_quinazoline), 8.66 (t, J= 6 Hz, 1H, NHCH₂), and 11.54 (s, 1H, quinazoline NH). MS m/z (%): 309 [M]⁺ (9.03%) and 310 [M + 1]⁺ (2.06%). Anal. Calcd. for C₁₇H₁₅N₃O₃: C, 66.01; H, 4.89; N, 13.58%. Found C, 66.23; H, 5.05; N, 13.44%.

Another method for synthesis of compound 2b

A mixture of azide 1 (0.49 g, 2 mmol) and benzylamine (0.22 ml, 2 mmol) in dry benzene (15 ml) was refluxed for 4 h, then allowed to cool. The solid product was filtered off, dried, and crystallised from acetic acid to give 0.51 g (82.5%) of the target product 2b as white crystals.

N-Cyclohexyl-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide (2c)

Crystallisation from AcOH/H2O yielded 0.56 g (93%) of compound 2c as white crystals; m.p. over 300 °C (reported 308–310 °C)³⁶. FT-IR (KBr, υ, cm⁻¹): 3252, 3202 (NH’s), 2929, 2855 (aliphatic CH’s), 1734, 1642 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 1.08–1.75 (m, 10H, cyclohexyl ring 5CH₂), 3.52 (m, 1H, cyclohexyl ring CH), 4.46 (s, 2H, NCH₂), 7.20–7.25 (m, 2H, ArH), 7.68 (t, J= 7.6 Hz, 1H, ArH), 7.94 (d, J= 7.6 Hz, 1H, ArH), 8.01 (d, J= 7.6 Hz, 1H, NH), and 11.48 (s, 1H, quinazoline NH). MS m/z (%): 301 [M]⁺ (6.80%). Anal. Calcd. for C₁₆H₁₉N₃O₃: C, 63.77; H, 6.36; N, 13.94%. Found C, 63.95; H, 6.48; N, 14.02%.

Another method for synthesis of compound 2c

A mixture of azide 1 (0.49 g, 2 mmol) and cyclohexylamine (0.23 ml, 2 mmol) in dry benzene (15 ml) was refluxed for 5 h, then allowed to cool. The solid product was filtered off, dried, and crystallised from AcOH/H₂O to give 0.47 g (78%) of the target product as white crystals.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N,N-dimethylacetamide (2d)

The solid product was collected by filtration and dried to give 0.21 g (42.5%) of the desired product; m.p. 218–220 °C. FT-IR (KBr, υ, cm⁻¹): 3245 (NH), 1730, 1661 (C═O’s).¹H NMR (DMSOd₆), δ, ppm: 2.85, 3.08 (s, 6H, 2CH₃), 4.73 (s, 2H, NCH₂), 7.20–7.25 (m, 2H, ArH), 7.69 (t, J= 7.6 Hz, 1H, ArH), 7.93 (d, J= 6.8 Hz, 1H, ArH), and 11.54 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 35.56, 36.21, 41.94, 114.01, 115.66, 123.14, 127.86, 135.69, 139.87, 150.51, 162.22, and 166.35. MS m/z (%): 247 [M]⁺ (15.03%) and 248 [M + 1]⁺ (2.30%). Anal. Calcd. for C₁₂H₁₃N₃O₃: C, 58.29; H, 5.30; N, 17.00%. Found C, 58.11; H, 5.19; N, 16.91%³⁷.

3-(2-Oxo-2-(piperidin-1-yl)ethyl)quinazoline-2,4(1H,3H)-dione (2e)

Crystallisation from benzene yielded 0.39 g (68%) of compound 2e as white crystals; m.p. 204 °C. FT-IR (KBr, υ, cm⁻¹): 3193 (NH), 2934, 2857 (aliphatic CH’s), 1721, 1652 (C═O’s).¹H NMR (CDCl₃), δ, ppm: 1.61–1.70 (m, 6H, CH₂-3, 4 and 5), 3.53 and 3.62 (2t, 4H, CH₂-2 and 6), 4.90 (s, 2H, NCH₂), 6.97–7.04 (m, 2H, ArH), 7.35–7.40 (td, J= 7.6 Hz, 1H, ArH), 7.84–7.86 (dd, J= 8 Hz, 1H, ArH), and 10.38 (s, 1H, quinazoline NH). MS m/z (%): 287 [M]⁺ (27.44%), 288 [M + 1]⁺ (6.28%). Anal. Calcd. for C₁₅H₁₇N₃O₃: C, 62.71; H, 5.96; N, 14.63%. Found C, 62.87; H, 5.80; N, 14.56%³⁸.

3-(2-Morpholino-2-oxoethyl)quinazoline-2,4(1H,3H)-dione (2f)

Crystallisation from benzene/ethanol yielded 0.515 g (89%) of compound 2f as white crystals; m.p. 254–255 °C. FT-IR (KBr, υ, cm⁻¹): ∼3430 (NH), 2907, 2874 (aliphatic CH’s), 1715, 1650 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 3.44 (t, 4H, morpholine ring 2 N-linked CH₂), 3.64 (t, 4H, morpholine ring 2 O-linked CH₂), 4.77 (s, 2H, NCH₂), 7.21–7.25 (m, 2H, ArH), 7.69 (t, J= 7.6 Hz, 1H, ArH), 7.94 (d, J= 7.6 Hz, 1H, ArH), and 11.56 (s, 1H, quinazoline NH). MS m/z (%): 289 [M]⁺ (11.29%) and 290 [M + 1]⁺ (1.98%). Anal. Calcd. for C₁₄H₁₅N₃O₄: C, 58.13; H, 5.23; N, 14.53%. Found C, 58.03; H, 5.08; N, 14.45%³⁹.

Ethyl [2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetyl]glycinate (2g)

Method A

A solution of glycine ethyl ester hydrochloride (0.14 g, 1 mmol) in ethyl acetate (10 ml) containing 0.2 ml of Et₃N was added to a solution of azide 1 (0.245 g, 1 mmol) in ethyl acetate (10 ml). The mixture was stirred at 0–5 °C for 24 h, then kept at room temperature for another 24 h. The solvent was evaporated, and the residue was crystallised from benzene/ethanol to give the target product as white crystals; yield 0.15 g (49%); m.p. 228–230 °C (reported 223–225 °C)⁴⁰.

Method B

A mixture of azide 1 (0.245 g, 1 mmol) and glycine ethyl ester hydrochloride (0.14 g, 1 mmol) in dry pyridine (5 ml) was refluxed for 5 h. The reaction mixture was allowed to cool, then acidified with cold dilute hydrochloric acid (1:1). The resultant precipitate was collected by filtration, dried, and crystallised from benzene/ethanol to give the target product as white crystals; yield 0.235 g (77%). FT-IR (KBr, υ, cm⁻¹): ∼3255, 3205 (NH’s), ∼2945, 2885 (aliphatic CH’s), 1740, 1645 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 1.19 (t, J= 7.2 Hz, 3H, OCH₂CH₃), 3.86 (d, J= 6 Hz, 2H, NHCH₂), 4.10 (q, J= 7.2 Hz, 2H, OCH₂CH₃), 4.56 (s, 2H, NCH₂), 7.20–7.24 (m, 2H, ArH), 7.68 (t, J= 7.2 Hz, 1H, ArH), 7.94 (d, J= 8 Hz, 1H, ArH), 8.62 (t, J = 6 Hz, 1H, CONHCH₂), and 11.53 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 14.52, 41.13, 42.78, 60.93, 114.16, 115.66, 123.08, 127.88, 135.65, 139.96, 150.47, 162.25, 167.80, and 170.15. MS m/z (%): 305 [M]⁺ (6.86%) and 306 [M + 1]⁺ (1.30%). Anal. Calcd. for C₁₄H₁₅N₃O₅: C, 65.08; H, 4.95; N, 13.76%. Found C, 64.90; H, 5.09; N, 13.86%.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(2-hydrazineyl-2-oxo-ethyl)acetamide (3)

To a solution of ethyl [2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetyl]glycinate 2g (0.305 g, 1 mmol) in absolute ethanol (10 ml), 0.5 ml of hydrazine hydrate was added. The reaction mixture was refluxed for 5 h, then allowed to cool. The solid product was collected by filtration, washed with boiling ethanol and dried to yield 0.217 g (74.6%) of compound 3 as white powder; m.p. 296–298 °C. FT-IR (KBr, υ, cm⁻¹): 3291, 3175 (NH₂, NH’s), 2933, 2894 cm⁻¹ (aliphatic CH’s), 1704, 1644 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 3.70 (d, J= 6 Hz, 2H, NHCH₂), 4.26 (s, 2H, NH₂), 4.57 (s, 2H, NCH₂), 7.20–7.25 (m, 2H, ArH), 7.69 (t, J= 7.8 Hz, 1H, ArH), 7.94 (d, J= 7.4 Hz, 1H, ArH), 8.49 (t, J= 6 Hz, 1H, CONHCH₂), 9.02 (s, 1H, NHNH₂), and 11.55 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 41.31, 43.00, 114.18, 115.66, 123.10, 127.89, 135.65, 139.93, 150.52, 162.33, 167.63, and 168.49. MS m/z (%): 291 [M]⁺ (4.95%). Anal. Calcd. for C₁₂H₁₃N₅O₄: C, 49.48; H, 4.50; N, 24.04%. Found C, 49.35; H, 4.39; N, 23.94%.

General procedure for synthesis of arylidenes 4a–d

To a solution of hydrazide 7 (0.291 g, 1 mmol) in absolute ethanol (15 ml), aldehyde (1 mmol) and few drops of piperidine were added. The reaction mixture was refluxed for 9–14 h (determined by TLC), then allowed to cool. The formed precipitate was filtered off and crystallised from the appropriate solvent.

(E)-N-[2-(2-Benzylidenehydrazineyl)-2-oxoethyl]-2-(2,4-dioxo-1,4-dihy-droquinazolin-3(2H)-yl)acetamide (4a)

Crystallisation from DMF/ethanol afforded compound 4a as white powder; yield 0.36 g (95%); m.p. 325 °C. FT-IR (KBr, υ, cm⁻¹): 3252, 3202 (NH’s), 1735, 1690, 1644 (C═O’s), 1518 (C═N). ¹H NMR (DMSO-d₆), δ, ppm: 3.86, 4.30 (2d, 2H, NHCH₂), 4.61 (s, 2H, NCH₂), 7.21–7.25 (m, 2H, ArH), 7.42–7.44 (m, 3H, ArH), 7.68–7.69 (m, 3H, ArH), 7.95 (d, J= 7.6 Hz, 1H, ArH), 7.99, 8.22 (2s, 1H, N═CH), 8.45, 8.63 (2t, 1H, CONHCH₂), 11.39, 11.52 (2s, 1H, NH–N), and 11.52 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 41.92, 42.89, 114.21, 115.66, 123.02, 127.23, 127.52, 127.87, 129.25, 130.30, 130.51, 134.54, 135.56, 139.97, 143.97, 147.37, 150.52, 162.26, 165.66, 167.54, 167.80, and 170.56. MS m/z (%): 379 [M]⁺ (22.86%). Anal. Calcd. for C₁₉H₁₇N₅O₄: C, 60.15; H, 4.52; N, 18.46%. Found C, 60.04; H, 4.45; N, 18.42%.

(E)-2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[2-(2-[4-nitrobenz-ylidene]hydrazineyl)-2-oxoethyl]acetamide (4b)

Crystallisation from DMF/H₂O afforded compound 4b as yellowish white powder; yield 0.408 g (96%); m.p. over 300 °C. FT-IR (KBr, υ, cm⁻¹): 3187, 3137 (NH’s), 1706, 1669 (C═O’s), 1563 (C═N). ¹H NMR (DMSO-d₆), δ, ppm: 3.91, 4.34 (2s, 2H, NHCH₂), 4.60 (s, 2H, NCH₂), 7.21 (m, 2H, ArH), 7.67 (t, 1H, ArH), 7.93 (m, 3H, ArH), 8.24–8.27 (m, 2H, ArH), 8.06, 8.29 (2s, 1H, N═CH), 8.50, 8.67 (2brs, CONHCH₂), 11.52 (s, 1H, quinazoline NH), 11.73, 11.80 (2s, 1H, NH–N). ¹³C NMR (DMSO-d₆), δ, ppm: 41.96, 42.91, 114.20, 115.66, 123.01, 124.43, 127.86, 128.18, 128.44, 130.03, 135.55, 139.96, 140.84, 141.56, 144.87, 148.18, 148.35, 150.51, 162.26, 166.17, 167.61, 167.86, and 170.99. MS m/z (%): 424 [M]⁺ (26.86%). Anal. Calcd. for C₁₉H₁₆N₆O₆: C, 53.78; H, 3.80; N, 19.80%. Found C, 65.67; H, 4.67; N, 14.64%.

(E)-2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[2-(2-[4-methoxy-benzylidene]hydrazineyl)-2-oxoethyl]acetamide (4c)

Crystallisation from ethanol afforded compound 4c as white powder; yield 0.393 g (96%); m.p. 290–292 °C. FT-IR (KBr, υ, cm⁻¹): 3294, 3200 (NH’s), 1725, 1656 (C═O’s), 1570 (C═N). ¹H NMR (DMSO-d₆), δ, ppm: 3.80, 3.81 (2s, 3H, OCH₃), 3.84, 4.27 (2d, 2H, NHCH₂), 4.61 (s, 2H, NCH₂), 6.98–7.03 (m, 2H, ArH), 7.21–7.26 (m, 2H, ArH), 7.60–7.72 (m, 3H, ArH), 7.96 (d, 1H, ArH), 7.94, 8.16 (2s, 1H, N═CH), 8.41, 8.60 (2t, CONHCH₂), 11.22, 11.38 (2s, 1H, NH–N), and 11.52 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 41.84, 42.87, 43.02, 55.75, 114.17, 114.78, 115.66, 123.07, 123.12, 127.10, 127.15, 127.89, 128.83, 129.15, 135.62, 135.67, 139.96, 143.85, 147.19, 150.52, 150.61, 161.11, 161.31, 162.28, 162.39, 165.41, 167.53, 167.79, and 170.33. MS m/z (%): 409 [M]⁺ (13.29%). Anal. Calcd. for C₂₀H₁₉N₅O₅: C, 58.68; H, 4.68; N, 17.11%. Found C, 58.45; H, 4.53; N, 17.00%.

(E)-2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[2-(2-[furan-2-ylmethylene]hydrazineyl)-2-oxoethyl]acetamide (4d)

Crystallisation from DMF/ethanol afforded compound 4d as beige powder; yield 0.347 g (94%); m.p. over 300 °C. FT-IR (KBr, υ, cm⁻¹): 3200, 3127 (NH’s), 1736, 1688, 1644 (C═O’s), and 1526 (C═N). ¹H NMR (DMSO-d₆), δ, ppm: 3.85, 4.23 (2d, 2H, NHCH₂), 4.61 (s, 2H, NCH₂), 6.61–6.64 (dd, 1H, furan ring H_X), 6.88–6.93 (dd, 1H, furan ring H_M), 7.21–7.26 (m, 2H, ArH), 7.69 (t, 1H, ArH), 7.81–7.84 (dd, 1H, furan ring H_A), 7.95 (d, 1H, ArH), 7.88, 8.11 (2s, 1H, N═CH), 8.40, 8.61 (2t, CONHCH₂), 11.32, 11.46 (s, 1H, NH–N), and 11.51 (s, 1H, quinazoline NH). MS m/z (%): 369 [M]⁺ (42.26%). Anal. Calcd. for C₁₇H₁₅N₅O₅: C, 55.28; H, 4.09; N, 18.96%. Found C, 55.37; H, 4.06; N, 18.91%.

N-[2-(2-Cyclohexylidenehydrazineyl)-2-oxoethyl]-2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide (4e)

To a solution of compound 3 (0.58 g, 2 mmol) in ethanol (20 ml), cyclohexanone (0.21 ml, 2 mmol) was added, and the reaction mixture was refluxed for 10 h. After cooling, the solid product was filtered off and dried to afford pure compound 4e as a white powder without further purification. Yield 0.64 g (86%); m.p. 264–266 °C. FT-IR (KBr, υ, cm⁻¹): 3256, 3210 (NH’s), 2942, 2860 (aliphatic CH’s), 1738,1647 (C═O’s), 1540 (C═N). ¹H NMR (DMSO-d₆), δ, ppm: 1.62–1.76 (m, 6H, 3CH₂), 2.19–2.36 (m, 4H, 2CH₂), 3.84, 4.15 (2d, 2H, NHCH₂), 4.60 (s, 2H, NCH₂), 7.21–7.30 (m, 2H, ArH), 7.69 (t, 1H, ArH), 7.94 (d, 1H, ArH), 8.32, 8.51 (2t, 1H, CONHCH₂), 10.14, 10.46 (2s, 1H, NH), 11.51 (s, 1H, quinazoline NH). MS m/z (%): 371 [M]⁺ (23.14%). Anal. Calcd. for C₁₈H₂₁N₅O₄: C, 58.21; H, 5.70; N, 18.86%. Found C, 58.26; H, 5.72; N, 18.82%.

(Z)-2-[2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl]-N-[2-oxo-2-(2-(2-oxoindolin-3-ylidene)hydrazineyl)ethyl]acetamide (4f)

A mixture of hydrazide 3 (0.291 g, 1 mmol) and isatin (0.147 g, 1 mmol) in acetic acid (15) was heated under reflux for 8 h, then allowed to cool. The solid product was filtered off and recrystallised from acetic acid to give compound 4f as yellow crystals; yield 89.2%; m.p over 300 °C. FT-IR (KBr, υ, cm⁻¹): 3328, 3228 (NH’s), 1724, 1695, 1650 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 4.01, 4.44 (2s, 2H, NHCH₂), 4.64 (s, 2H, NCH₂), 6.94 (d, J= 8 Hz, 1H, ArH), 7.09 (t, J= 8 Hz, 1H, ArH), 7.21–7.25 (m, 2H, ArH_quinazoline), 7.38 (t, J= 8 Hz, 1H, ArH), 7.57 (d, J= 8 Hz, 1H, ArH), 7.69 (t, J= 8 Hz, 1H, ArH_quinazoline), 7.95 (d, J= 8 Hz, 1H, ArH_quinazoline), 8.60, 8.92 (2s, 1H, CONHCH₂), 11.30 (s, 1H, indolinone NH), 11.51 (s, 1H,quinazoline NH), 12.55, 13.26 (2s, 1H, NH–N). MS m/z (%): 420.60 [M]⁺ (17.99%).

N-[2-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-oxoethyl]-2-(2,4-dioxo-1,4-dihyd-roquinazolin-3(2H)-yl)acetamide (4g)

To a solution of compound 3 (0.58 g, 2 mmol) in ethanol (20 ml), acetyl acetone (0.2 ml, 2 mmol) was added in presence of few drops of piperidine, and the reaction mixture was refluxed for 16 h. After cooling, the solid product was filtered off and recrystallised from ethanol/benzene to afford compound 4g as white powder; yield 0.62 g (87.3%); m.p. 284–286 °C. FT-IR (KBr, υ, cm⁻¹): 3241, 3199 (NH’s), 1732, 1646 (C═O’s). ¹H NMR (DMSO-d₆), δ, ppm: 1.77 (s, 3H, CH₃), 1.96 (s, 3H, CH₃), 4.10 (d, J= 4 Hz, 2H, NHCH₂), 4.60 (s, 2H, NCH₂), 6.40 (s, 1H, CH), 7.20–7.24 (m, 2H, ArH), 7.68 (t, J= 8 Hz, 1H, ArH), 7.94 (d, J= 8 Hz, 1H, ArH), 8.32 (t, J= 4 Hz, 1H, CONHCH₂), and 11.49 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 16.32, 26.38, 42.19, 42.85, 52.37, 90.82, 114.13, 115.65, 123.08, 127.88, 135.61, 139.92, 150.50, 155.68, 162.26, 166.30, and 167.46. MS m/z (%): 355 [M]⁺ (23.14%). Anal. Calcd. for C₁₇H₁₇N₅O₄: C, 57.46; H, 4.82; N, 19.71%. Found C, 57.34; H, 4.74; N, 19.67%.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl]acetamide (4h)

To a solution of compound 3 (0.58 g, 2 mmol) in pyridine (15 ml), 1 ml of carbon disulphide was added. The reaction mixture was refluxed for 10 h, then allowed to cool, acidified with cold dilute hydrochloric acid (1:1), and the solid formed was collected by filtration and dried to obtain the target product as grey powder without further purification; yield 0.24 g (36%); m.p. over 300 °C. FT-IR (KBr, υ, cm⁻¹): 3304, 3214 (NH’s), 1714, 1661 (C═O’s), and 1140 (C═S). 1H NMR (DMSO-d₆), δ, ppm: 3.86 (d, 2H, NHCH₂), 4.59 (s, 2H, NCH2), 7.22–7.26 (m, 2H, ArH), 7.70 (t, 1H, ArH), 7.95 (d, 1H, ArH), 8.65 (t, 1H, CONHCH₂), 10.98 (br s, 1H, NHCS), and 11.56 (s, 1H, quinazoline NH). ¹³C NMR (DMSO-d₆), δ, ppm: 40.87, 42.89, 114.15, 115.71, 123.08, 127.86, 135.64, 139.97, 150.49, 162.29, 167.91, and 168.78. MS m/z (%): 333 [M]+ (20.79%). Anal. Calcd. for C₁₃H₁₁N₅O₄S: C, 46.84; H, 3.33; N, 21.01; S, 9.62%. Found C, 46.75; H, 3.28; N, 20.96; S, 9.66%.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[2-oxo-2-(2-[phenylcar-bamothioyl]hydrazineyl)ethyl]acetamide (4i)

To a solution of hydrazide 3 (0.58 g, 2 mmol) in absolute ethanol (20 ml), phenyl isothiocyanate (0.24 ml, 2 mmol) was added and the reaction mixture was refluxed for 15 h, then allowed to cool. The solid product was collected by filtration and crystallised from ethanol to afford compound 4i as white powder; yield 0.71 g (83.3%); m.p. 287–290 °C. FT-IR (KBr, υ, cm⁻¹): 3444, 3285, 3201 (NH’s), 1735, 17.0, 1668 (C═O’s), and 1267 (C═S). ¹H NMR (DMSO-d₆), δ, ppm: 3.82 (d, 2H, NHCH₂), 4.64 (s, 2H, NCH₂), 7.06–7.95 (m, 9H, ArH), 8.75 (br s, 1H, CONHCH₂), 9.31 (br s, 1H, NH), 9.74 (br s, 1H, NH), 10.21 (s, 1H, NH), and 11.56 (s, 1H, quinazoline NH). MS m/z (%): 426.56 [M]⁺ (60.16%). Anal. Calcd. for C₁₉H₁₈N₆O₄S: C, 53.51; H, 4.25; N, 19.71; S, 7.52%. Found C, 53.38; H, 4.18; N, 19.62; S, 7.61%.

2-(2,4-Dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-[(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]acetamide (4j)

The thiosemicarbazide 4i (0.426 g, 1 mmol) was suspended in 2% solution of NaOH (15 ml) and heated under gentle reflux for 6 h. after cooling, the solution was neutralised with dil. HCl. The resulting precipitate was filtered off, washed with distilled water, and recrystallised from ethanol to afford compound 4j as white crystals. Yield 0.29 g (71%); m.p. over 300 °C. FT-IR (KBr, υ, cm⁻¹): 3272, 3200 (NH’s), 1718, 1658 (C═O’s), and 1210 (C═S). ¹H NMR (DMSO-d₆), δ, ppm: 4.12 (d, J= 4 Hz, 2H, NHCH₂), 4.42 (s, 2H, NCH₂), 7.19–7.24 (m, 2H, ArH_quinazoline), 7.40–7.57 (m, 5H, ArH), 7.68 (t, J= 8 Hz, 1H, ArH_quinazoline), 7.94 (d, J= 8 Hz, 1H, ArH_quinazoline), 8.61 (t, J= 4 Hz, 1H, CONHCH2), 11.50 (s, 1H, quinazoline NH), and 13.86 (s, 1H, NHCS). MS m/z (%): 408 [M]⁺ (39.11%). Anal. Calcd. for C₁₉H₁₆N₆O₃S: C, 55.87; H, 3.95; N, 20.58; S, 7.85%. Found C, 55.74; H, 3.78; N, 20.52; S, 7.93%.

Biological evaluation

In vitro antiproliferative activity against normal and cancer cells

To scrutinise the cytotoxicity of the target compounds on colorectal cancer and normal cells, MTT assay was carried out against HCT-116 and WI38 cell lines, respectively.

In vitro activity against c-Met and VEGFR-2

Both c-Met and VEGFR-2 assay of the target compounds 2c, 2f, 4b, 4e, 4g, and 4h was performed by a well-known methodology of BPS KDR Assay Kit Catalog # 40325 (BPS Bioscience, San Diego, CA) (see Supplementary Data)⁸.

Cell cycle analysis and apoptosis assay

The effects of both 4b and 4e on progress of cell cycle and induction of apoptosis in HCT-166 cells were carried out using Annexin V-FITC Apoptosis Detection Kit (BioVision Research Products, Mountain View, CA) (see Supplementary Data)⁸.

In silico studies

Molecular modelling study

Molecular modelling of compounds 4b and 4e and 2D and 3D visualisation processes were carried out within c-Met and VEFGR-2 TK active sites using MOE 2014.0901 software. The co-crystal structures of c-Met (PDB: 3lq8) and VEGFR-2 (PDB: 4asd) proteins were retrieved from the Protein Data Bank (RCSB). At first, 4b and 4e were prepared with the standard protocol of MOE 2014. After that the structures of the two proteins were prepared by using the MOE structure preparation protocol. Validation of the molecular docking study at the active sites was carried out by redocking of the original ligands into the active sites. The RMSD values of the most stable poses were 1.0648 (foretinib) and 0.9797 (sorafenib) in c-Met and VEGFR-2 active sites, respectively. Then, compounds 4b and 4e were docked into the active sites using the Alpha Triangle placement method. Refinement was carried out using Forcefield and scored by London dG scoring system.

In silico prediction of physicochemical and pharmacokinetic properties

The Swiss Institute of Bioinformatics provides a freely available tools for prediction of pharmacokinetics and physicochemical properties of small molecules via URL: http://www.swissadme.ch/index.php#. Different filters were used to compute druglikeness as follows: Pfizer Lipinski (MW ≤ 500, MLOGP ≤ 4.15, N or O ≤ 10, NH or OH ≤ 5); Ghose (160 ≤ MW ≤ 480, −0.4 ≤ WLOGP ≤ 5.6, 40 ≤ MR ≤ 130, 20 ≤ atoms ≤ 70); Veber (rotatable bonds ≤ 10, TPSA ≤ 140); Egan (WLOGP ≤ 5.88, TPSA ≤ 131.6); Muegge (200 ≤ MW ≤ 600, −2 ≤ XLOGP ≤ 5, TPSA ≤ 150, rings ≤ 7, C > 4, heteroatoms > 1, rotatable bonds > 15, HBA ≤ 10, HBD ≤ 5); Abbott Bioavailability Score (probability of F > 10% in rat).⁵⁷ iLOG⁵⁸. BOILED Egg is a plot of TPSA on the X-axis versus WLOGP on the Y-axis. The white oval is the suitable TPSA/WLOGP value for the highest probability of GI absorption, and the yolk circle is the suitable TPSA/WLOGP value for the highest probability to BBB accessibility. The optimal values of physicochemical properties involved in Bioavailability Radar are as follows: size (150 g/mol < MV > 500 g/mol), polarity (20 Å²<TPSA > 130 Å²), lipophilicity (–0.7 < XLOGP3> +5.0), insolubility (0 < Log S (ESOL)>6), insaturation (0.25 < Fraction Csp3 > 1.0), and flexibility (rotatable bonds < 9)⁵⁹.

Disclosure statement

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Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.