Coumarin–furo[2,3-d]pyrimidone hybrid molecules targeting human liver cancer cells: synthesis, anticancer effect, EGFR inhibition and molecular docking studies

Abstract

The design, synthesis and investigation of antitumor activities of some coumarin–furo[2,3-d]pyrimidone hybrid molecules are reported. In vitro, HepG2 cells were used to investigate the cytotoxicity of 6a–n and 10a–n. The results demonstrated that coupling a furopyrimidone scaffold with coumarin through a hydrazide linker can effectively improve their synergistic anticancer activity. The coumarin–furo[2,3-d]pyrimidone combination 10a exhibited significant inhibitory activity against HepG2 cells with IC₅₀ = 7.72 ± 1.56 μM, which is better than those of gefitinib and sorafenib. It is worth mentioning that the coumarin–furo[2,3-d]pyrimidone combination 10a showed excellent inhibition of the EGFR enzymatic activity with IC₅₀ = 1.53 μM and 90% inhibition at 10 μM concentration. In silico investigation predicts the possibility of direct binding between the new coumarin–furo[2,3-d]pyrimidone hybrid molecules and the EGFR. The results suggest that coumarin–furo[2,3-d]pyrimidone hybrid molecules are potential antitumor agents targeting human liver cancer cells.

Coupling a furopyrimidone scaffold with coumarin through a hydrazide linker can effectively improve their synergistic anticancer activity targeting HepG2.

Introduction

As the third leading cause of cancer death worldwide, liver cancer is a serious threat to human health and life. Hepatocellular carcinoma (HCC) accounts for about 85–90% of all liver malignancies.¹ EGFR overexpression is notable in many types of human cancers and is responsible for poor prognosis. In particular, overexpression of EGFR has been found in 40–70% of HCC cases.² EGFR overexpression and mutation represent pivotal therapeutic targets for HCC. Inhibition of aberrant EGFR signaling can effectively impede tumor cell proliferation and metastasis, and induce apoptosis. Accumulating evidence suggests that natural products such as piperine, curcumin and oleocanthal are potential drug candidates for prevention and treatment of liver cancer.^3,4 The low systemic toxicity and fewer side effects of natural compounds provide a direction for the development of liver cancer drugs. Coumarin derivatives, as a typical class of natural compounds with highly privileged fused heterocyclic molecules, showed different pharmacological properties such as anticancer, anti-HIV, antioxidant, anti-inflammatory, anticoagulant, and antimicrobial activities and fewer harmful effects against normal cells.^5–9 Many reported anticancer mechanisms including interactions with a variety of enzymes and receptors such as steroid sulfatase, stress activated protein kinase, topoisomerase, gyrase enzymes, p38MAPK, estrogen receptor were found.^10–12 The inhibitory activity of coumarin derivatives against HCC and EGFR has been extensively documented in the literature.^13–15 Moreover, the clinical applications of their certain members, such as irosustat and esculetin, have been reported extensively, implying that coumarin is a highly privileged pharmacophore for the development of novel anticancer drugs.

Molecular hybridization integrates two or more pharmacophores into a single molecular scaffold through fusion or ligation to obtain a better molecular structure, with the potential to enhance the bioaffinity of the target system, improve activity, reduce side effects, overcome the drug resistance and generate new biological properties.^16,17 The EGFR inhibitory activity of fused pyrimidine scaffolds such as quinazoline, thienopyrimidine and pyrrolopyrimidine has been extensively investigated with compounds like erlotinib, gefitinib, ribociclib, olmutinib, PKI-166, and AEE-788 (Fig. 1).^18–20 However, the exploration of furopyrimidine scaffolds remains limited. The representative compound DBPR112 (Fig. 1), featuring a furopyrimidine scaffold, has successfully completed phase I clinical trials and exhibits significant potential to emerge as a 4th-generation EGFR-targeted small molecule inhibitor.²¹ Monia Hossam et al. reported furo[2,3-d]pyrimidine derivative II (Fig. 2) has excellent anticancer activity as a dual inhibitor of EGFR/HER2 tyrosine kinase.²² Furo[2,3-d]pyrimidine derivatives I and III (Fig. 2) have shown prominent VEGFR-2 inhibitory activity.^23–26 Our previous studies have previously reported a diverse range of fused pyrimidine scaffolds, some of which exhibit promising anticancer activity and potent EGFR inhibitory effects.^27–30

Fig. 1. Representative EGFR inhibitors.

Fig. 2. Design of furopyrimidone–coumarin combinations by employing the scaffold hopping and molecular hybridization strategies.

The hybridization of coumarin and fused pyrimidine scaffolds might afford new potent anticancer agents. A variety of coumarin-based hybrid molecules have been found to be more potent than coumarin, such as coumarin–isatin hybrids,³¹ coumarin–quinone hybrids,³² coumarin–indole hybrids,³³ and coumarin–benzimidazole hybrids,³⁴ indicating hybridization is a useful tool in the discovery of novel anticancer agents.³⁵ When different fusion heterocyclic scaffolds are combined with a promising medicinal chemistry linker, a synergistic effect of both fused heterocyclic scaffolds is obtained in the target system.^36,37 A hydrazide linker that provides NH and C O groups to include hydrogen-bond acceptor and donor sites and to perform an interaction with a wide range of amino-acid residues, as well as its ability to chelate catalytic zinc ion of metalloenzymes, can play a crucial role in cancer treatment.^38,39 Various hydrazide derivatives have been established as potential drugs such as isoniazid, iproniazide, benserazide and aldoxorubicin. The biological application of hydrazides and their structure–activity relationship in drugs were comprehensively studied.⁴⁰ Not only the pharmacokinetic and safety properties but also the toxicities and adverse effects of hydrazide-based compounds are well known.⁴¹ Therefore, given the diverse effects of hydrazide compounds as drug candidates in various diseases studied earlier, this unique scaffold may be utilized to design novel anticancer agents.

In this context, we designed a series of furopyrimidone–coumarin combinations by fusion heterocyclic scaffold hopping and molecular hybridization strategies (Fig. 2) and previously reported that some substituted coumarin–furopyrimidone derivatives exhibited multi-targeted inhibitory effects and selective inhibition of HepG cell lines. In this work, we linked new furopyrimidone moieties to substituted coumarin through a hydrazide linker and these agents showed excellent anticancer activities. The results of computational studies revealed that the substituents at the C-2 position of the furo[2,3-d]pyrimidinone could enhance the bioactivity and the butoxy present in the coumarin structure did not exert a significant impact on the activity.⁴²

In continuation of our studies to develop new cytotoxic candidates based on the naturally derived coumarin scaffold and fused heterocyclic scaffold, we intended to use the same substitution pattern on the furopyrimidone nucleus (C-2, N-3) as done in our previously published work. In the presented study, we reported the design and synthesis of new hybrid derivatives carrying furo[2,3-d]pyrimidone derivatives conjugated with non-substituted coumarin via a hydrazide linker as potential EGFR inhibitors aiming to improve their synergistic anticancer activity.⁴³ The in vitro anticancer activity of the target compounds against HepG2 cell lines was screened by 24 h drug exposure test using fluorouracil and gefitinib as the control. The molecular docking of compound 10a with the highest activity was studied to find out the possible binding mode with the EGFR binding site, and its binding stability was evaluated, and the relationship between its physicochemical properties and inhibition was discussed.

Results and discussion

2.1. Rationale and design

Amongst the attractive therapeutic targets and dominant strategies for cancer, epidermal growth factor receptor (EGFR) signalling plays an important role in the progression of tumors. At present, well-defined EGFR inhibitors share essential pharmacophoric features encompassing: (i) a central heteroaromatic unit that fits the adenine binding site through a H-bond with a methionine amino acid residue (Met793); (ii) a terminal hydrophobic unit interacting with the hydrophobic region I; (iii) oxygen and nitrogen in an amide linker as the H-bond acceptor–donor pair to link the hinge-binding central moiety with the terminal fragment that occupies the hydrophobic region I; (iv) a terminal lipophilic unit that is directly linked to the hydrophobic region II.⁴⁴

The primary objective of this study was to identify novel high-potency EGFR inhibitors based on furopyrimidone–coumarin hybrid scaffolds. This was inspired by our previous work on furo-, thieno- and pyrrolopyrimidines. As both fused pyrimidine and coumarin scaffolds have been applied for construction of highly potent EGFR antagonists and the coumarin–furo[2,3-d]pyrimidone hybrid scaffolds have the characteristics of well-defined EGFR inhibitors described above, we hope that the coumarin–furo[2,3-d]pyrimidone hybrid molecules can be potential antitumor agents (Fig. 3).

Fig. 3. Proposed hypothetic pharmacophoric features of the designed furopyrimidone–coumarin compounds for EGFR inhibitors. The coumarin scaffold is presented in blue. The hydrazide linker is presented in blue and the substituted aromatic moiety of furo[2,3-d]pyrimidone occupying the allosteric hydrophobic pocket is presented in red.

2.2. Chemistry

The synthetic routes are outlined in Scheme 1. The synthesis of target hybride molecules 2-alkylamino-6-methyl-N′-(coumarin-3-carbonyl)-3-aryl-3,4-dihydrofuro[2,3-d]pyrimidone-5-carbohydrazides (10a–h) was performed by the reaction of 2-alkylamino-6-methyl-3-aryl-3,4-dihydrofuro[2,3-d]pyrimidone-5-carbohydrazides (6a–h) with coumarin-3-carbonyl chloride (9). Firstly, iminophosphorane 3 was prepared according to our previous paper.²⁷ Then, this compound was reacted with aromatic isocyanates in dichloromethane to obtain corresponding carbodiimede derivatives (4a–c). Next, compounds (4a–c) were reacted with amines in ethanol to synthesize ethyl 2-alkylamino-6-methyl-3-aryl-3,4-dihydrofuro[2,3-d]pyrimidone-5-carboxylate derivatives (5a–n). Ester derivatives of compounds (6a–n) were prepared by the reaction with hydrazine hydrate in the presence of sodium ethoxide. The synthesis of second intermediate coumarin-3-carbonyl chloride (9) was performed in three steps, in the light of the literature methods.⁴⁵ First, coumarin-3-carboxylic acid ethyl ester (8) was prepared by the reaction of salicylaldehyde 7 with ethyl acetoacetate in ethanol with morpholine as a catalyst. Then, compound 8 was hydrolyzed in base solution, the coumarin-3-carboxylic acid reacted with SOCl₂ to prepare coumarin-3-carbonyl chloride (9). The target hydrazide linked coumarin–furopyrimidone hybrid molecules (10a–h) were obtained by the reaction of intermediate 9 and compounds 6a–n.

Scheme 1. Synthesis of target compounds 10a–h. Reagents and conditions: (i) ethyl cyanoacetate, NEt₃, 0 °C; (ii) PPh₃, C₂Cl₆, NEt₃, 0 °C; (iii) ArNCO, CH₂Cl₂, 0–5 °C; (iv) (a) CH₂Cl₂, HY, 3 h, r.t.; (b) NaOEt, ethanol, r.t.; (v) NH₂NH₂, ethanol, 60–80 °C; (vi) ethyl acetoacetate, ethanol, 60 °C; (vii) KOH, ethanol, H₂O, 70 °C; and (viii) CH₂Cl₂, NEt₃, 0 °C.

The spectral results of the target compounds 10a–n correspond to the proposed structures. The ¹HNMR and ¹³C NMR spectra of compound 10j–n have not been obtained due to their insolubility, but their mass spectrometry analysis was correct. For example, the ¹HNMR spectrum of 10g shows two NH signals of the hydrazide linker at 11.08 and 12.77 ppm and an NH signal of the C-2 position in furo[2,3-d]pyrimidone was observed at about 6.50 ppm. The NCH₂ signal was found at about 3.23 ppm. The signals of CH₃ at 0.84–0.88 ppm were found as triplets. The signals of CH₂ at1.18–1.50 and 3.21–3.24 ppm and Ar–H at 7.39–8.80 ppm were found as multiplets. In the ¹³C NMR spectra, C–O signals were found at about 165.05 and 160.76 ppm as two hydrazides, and coumarin C-2 and furo[2,3-d]pyrimidone C-4 were observed at 160.62 and 159.42 ppm. In addition, the MS spectrum of 10g shows a strong molecular ion peak at m/z 526.17 with 100% abundance (Fig. S46†).

2.3. Biological evaluation

The cytotoxicity of the furopyrimidone compounds 6a–n and furopyrimidone–coumarin combinations 10a–n was examined against the HepG2 cell line in comparison to fluorouracil, sorafenib and gefitinib using CCK8 assay. The cytotoxic activities of compounds are expressed by the median growth inhibitory concentration (IC₅₀) as shown in Table 1. The results revealed that most of the target compounds have good activity against hepatocellular carcinoma cells. Except for compound 10f, the activity of the furopyrimidone–coumarin combinations was significantly higher than that of the corresponding furopyrimidone compounds, indicating that coumarin could significantly improve its antitumor activity by linking to furan [2,3-d] pyrimidone scaffolds through hydrazine–hydrazine junctions. In addition, the results indicated that it is feasible to splice the furopyrimidone scaffold with coumarin to improve their synergistic anticancer activity. Compounds 10a, 10d and 10g were found to be potent and selective anticancer agents against HepG2 cells with IC₅₀ values of 7.72 ± 1.56, 7.89 ± 0.79 and 8.74 ± 0.96 μM, respectively, versus 16.10 ± 0.94 μM for the reference gefitinib. For the furan[2,3-d]pyrimidone compounds 6a–n, the activity of the compounds with diethylamine and dipropylamino substituents at the Y site is superior to those of other amino compounds, where compounds 6d, 6e and 6f were better than 6a–6c and 6g–6l. The results showed that the cytotoxic activity of the furan[2,3-d]pyrimidone compounds is related to the different substitutions at the Y site, and the activity of diethylamine substituents was stronger than those of propylamine, n-butylamine and morpholino substituents. For the furopyrimidone–coumarin compounds with Y-site diethylamine substituents 10d, 10e and 10f, the activity of the compound 10d (IC₅₀ = 7.89 ± 0.79 μM) with an R group as phenyl is significantly stronger than those of compounds 10f (IC₅₀ > 100 μM) and 10e (IC₅₀ = 26.47 ± 0.85 μM) with R groups as p-phenylmethyl and m-phenylmethyl. For the furopyrimidone–coumarin compounds with Y-site propylamine substituents 10a, 10b and 10c, the activity of the compound 10a (IC₅₀ = 7.72 ± 1.56 μM) with an R group as phenyl is significantly stronger than those of compounds 10c (IC₅₀ = 21.82 ± 1.52 μM) and 10b (IC₅₀ = 24.31 ± 1.64 μM) with R groups as p-phenylmethyl and m-phenylmethyl. The results showed that the cytotoxic activity of the target furopyrimidone–coumarin compounds is related to the different substitutions at the R site, and the activity of the phenyl group was stronger than those of p-phenylmethyl and m-phenylmethyl groups.

Cytotoxicity (IC₅₀) of the tested compounds on the HepG2 cell line.

Compd.	R	Y	IC50 (μM)	Compound	R	Y	IC50 (μM)
6a	H	Propylamino	69.30 ± 0.83	10a	H	Propylamino	7.72 ± 1.56
6b	m-Me	Propylamino	58.91 ± 1.57	10b	m-Me	Propylamino	24.31 ± 1.64
6c	p-Me	Propylamino	84.09 ± 0.63	10c	p-Me	Propylamino	21.82 ± 1.52
6d	H	Diethylamino	30.72 ± 1.09	10d	H	Diethylamino	7.89 ± 0.79
6e	m-Me	Diethylamino	36.53 ± 1.70	10e	m-Me	Diethylamino	26.47 ± 0.85
6f	p-Me	Diethylamino	17.20 ± 1.20	10f	p-Me	Diethylamino	>100
6g	H	Butylamino	>100	10g	H	Butylamino	8.74 ± 0.96
6h	m-Me	Butylamino	73.90 ± 1.81	10h	m-Me	Butylamino	12.97 ± 1.74
6i	p-Me	Butylamino	>100	10i	p-Me	Butylamino	14.52 ± 1.11
6j	H	Morpholino	45.09 ± 1.26	10j	H	Morpholino	24.20 ± 0.96
6k	m-Me	Morpholino	40.82 ± 1.01	10k	m-Me	Morpholino	26.81 ± 1.09
6l	p-Me	Morpholino	55.89 ± 0.96	10l	p-Me	Morpholino	40.00 ± 1.34
6m	H	Dipropylamino	17.04 ± 1.31	10m	H	Dipropylamino	14.33 ± 1.47
6n	p-Me	Dipropylamino	8.53 ± 1.15	10n	p-Me	Dipropylamino	22.15 ± 0.68
5-FU			23.45 ± 0.65	5-FU			23.45 ± 0.65
Sorafenib			12.33 ± 1.62	Sorafenib			12.33 ± 1.62
Gefitinib			16.10 ± 0.94	Gefitinib			16.10 ± 0.94

2.4. EGFR enzyme inhibition activity

Based on the biological cytotoxicity study, compounds 6d–f, 10d–f, 10a, and 10g were initially screened for EGFR kinase activity inhibition at a single dose of 10 μM concentration (Table 2). At 10 μM concentration, compounds 10a, 10d, and 10g have demonstrated potent EGFR inhibition with 90%, 80% and 82%. Intermediate compounds 6d–f showed poor EGFR inhibitory activity with 76%, 31%, and 46%. However, the target compounds 10d–f carrying intermediate furo[2,3-d]pyrimidone derivatives 6d–f conjugated with coumarin via the hydrazide linker exhibited EGFR inhibitory activity of 80%, 62% and 58%, respectively.

Percent inhibition of EGFR enzymatic activity at 10 μM and IC₅₀ values of promising compounds.

Comp. no	EGFR	EGFR
	Inhibition (%)	IC50 (μM)
6d	7̲6̲	32.36
6e	31	—
6f	46	—
10a	9̲0̲	1.53
10d	8̲0̲	3.01
10e	62	—
10f	58	—
10g	8̲2̲	3.09
5-Fu	—	ND
Sorafenib	—	1.83

Promising candidates, which exhibited EGFR inhibition percentage above 75% at 10 μM concentration (6d, 10a, 10d and 10g), were further evaluated for inhibition against EGFR kinase using ELISA assay with sorafenib and 5-fluorouracil as the reference drugs. Compound 10a exhibited potent EGFR inhibition activity with IC₅₀ = 1.53 μM comparable to the reference drug sorafenib (IC₅₀ = 1.83 μM). Compound 10a was 2-fold more active than 10d and 10g in the EGFR kinase assays, respectively. In addition, the furopyrimidone–coumarin hybrid compound 10d has smaller IC₅₀ values than intermediate compound 6d. As a result, the newly synthesized furopyrimidone–coumarin hybrid compounds were found to be good EGFR inhibitors and could inhibit EGFR in a dose-dependent manner. The in vitro EGFR kinase activity assay and HepG2 cytotoxicity assay showed that furopyrimidone–coumarin hybrid compound 10a is an excellent EGFR inhibitor, which may interfere with the biological process of tumor cells by inhibiting EGFR kinase.

2.5. Effects of transwell migration against the HepG2 cell layer

To further confirm the antitumor activity of compounds 6f and 10a targeting human liver cancer cells, transwell migration assay was used to detect the inhibitory effect of compounds 6f and 10a against HepG2 cell layer development. The assay was designed for HepG2 cells to proceed upward from the bottom well to the upper transwell insert cup containing the test reagent. The results are depicted in Fig. 4. As shown in Fig. 4, both furo[2,3-d]pyrimidone hydrazide hybrid derivative 6f and furo[2,3-d]pyrimidone–coumarin combination 10a can significantly inhibit HepG2 cell migration and invasion. Furopyrimidone–coumarin combination 10a showed better inhibition activity than intermediate 6f. These studies could provide potential antitumor agents of furo[2,3-d]pyrimidone–coumarin combinations against human liver cancer cells.

Fig. 4. Transwell migration effects of compounds 6f and 10a against the HepG2 cell layer.

2.6. Molecular simulation results

As we all know, EGFR is one of the members of the epidermal growth factor receptor (HER) family, which plays an important role in the physiological processes such as cell growth, proliferation and differentiation. The loss of function of protein tyrosine kinases such as EGFR or abnormal activity of key factors in its related signaling pathways or cell localization will cause the occurrence of tumors. Studies have shown that EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of cell apoptosis. Therefore, EGFR is the main target of antitumor drug research. To further clarify the structure–activity relationship between the furopyrimidone–coumarin combinations and EGFR, the molecular docking simulation studies of compound 6d and 10a were carried out using SYBYL 7.0 software. The results of docking are presented in Fig. 5, indicating that both 6f and 10a bind to ATP binding domains of EGFR, and 10a can enter deeper into the catalytic pocket of ATP binding sites, and its structure is located near the DFG-motif (aspartate–phenylalanine–glycine module). The DFG motif plays an important role in regulating kinase activity, and 10a may exhibit strong kinase inhibitory activity through interaction with the DFG motif. The furo[2,3-d]pyrimidone compound 6f formed three hydrogen bond interactions with T790, A743 and D855. The furopyrimidone–coumarin combination 10a formed four hydrogen bond interactions with T790, K745 and D855. In addition, the π–π interaction with F723 was also observed. From the perspective of binding mode, the space between the position of the benzene ring of 10a and EGFR is limited. If a substituent group is introduced into the benzene ring, there will be steric hindrance between small molecules and EGFR, which will reduce the activity. Therefore, the furopyrimidone–coumarin hybrid scaffolds 10a–h with substituents on the benzene ring display lower activity than the molecule without substituents on the benzene ring. Molecular simulation results showed that the furopyrimidone–coumarin hybrid derivatives are potential antitumor agents with potential EGFR inhibition.

Fig. 5. Docking pose of the target compounds: (a) 6f to the active site of EGFR (DFG-motif) and (b) 10a to the active site of EGFR (DFG-motif).

Experimental

3.1. Chemistry

All the furo[2,3-d]pyrimidone derivatives studied were synthesized from the corresponding carbonyl compounds, according to Scheme 1. The first three steps of chemical synthesis were described by our research group previously.⁴⁶ All melting points were uncorrected and measured using uncorrected X-4 digital melting point apparatus. The nuclear magnetic resonance NMR spectra were determined utilizing a Varian Mercury VRX-400 spectrometer with CDCl₃ and DMSO-d₆ as solvents and TMS as the internal standard. The chemical shift values (δ) are expressed in ppm, and the coupling constants (J) are expressed in Hz. The mass spectra were recorded on a Waters XEVO G2-XS mass spectrometer.

A solution of the corresponding ethyl 3,4-dihydro-6-methyl-2-amino-4-oxo-3-aryl furo[2,3-d]pyrimidine-5-carboxylates 5a–5n (ref. 28) (5 mmol) and hydrazine hydrate (10 mmol) in anhydrous CH₃CH₂OH (25 mL) was stirred for 30 h at 60 °C, and then the solvent was concentrated under reduced pressure to precipitate and after filtration 3,4-dihydro-6-methyl-4-oxo-2-amino-3-aryl-furo[2,3-d]pyrimidine-5-carbohydrazides 6a–6n were obtained, which were used directly without further purification.

3,4-Dihydro-6-methyl-4-oxo-3-phenyl-2-(n-propylamino)furo[2,3-d]pyrimidine-5-carbohydrazide (6a)

White solid (yield, 65%). M.p.: 234–236 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.79 (t, J = 4.0 Hz, 3H, CH₃), 1.45–1.51 (m, 2H, CH₂), 2.63 (s, 3H, CH₃), 3.16–3.19 (m, 2H, NCH₂), 4.42–4.38 (m 2H, NH₂), 6.43 (t, J = 4.0 Hz, 1H, NH), 7.36–7.63 (m, 5H, ArH), 10.85 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 164.96, 161.11, 160.13, 152.55, 134.29, 130.21, 129.04, 109.99, 92.90, 56.00, 43.05, 21.60, 18.51, 12.96, 11.05; anal. calcd for C₁₇H₁₉N₅O₂ (341.15); ESI-MS (70 eV) m/z (%): 342.17 (M + 1, 100).

3,4-Dihydro-6-methyl-4-oxo-2-(n-propylamino)-3-m-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6b)

White solid (yield, 71%). M.p.: 259–261 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.79 (t, J = 8.0 Hz, 3H, CH₃), 1.45–1.51 (m, 2H, CH₂), 2.39 (s, 3H, CH₃), 2.63 (s, 3H, CH₃), 3.15–3.22 (m, 2H, NCH₂), 4.43 (s, 2H, NH₂), 6.42–6.44 (m, 1H, NH), 7.13–7.5 (m, 4H, ArH), 10.87 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 164.95, 161.09, 160.11, 152.58, 152.53, 139.73, 134.15, 130.25, 129.99, 129.38, 125.87, 109.99, 92.88, 43.03, 21.62, 20.84, 12.97, 11.06; anal. calcd for C₁₈H₂₁N₅O₃ (355.16); ESI-MS (70 eV) m/z (%): 356.17 (M + 1, 100).

3,4-Dihydro-6-methyl-4-oxo-2-(n-propylamino)-3-p-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6c)

White solid (yield, 69%). M.p.: 260–263 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.80 (t, J = 8.0 Hz, 3H, CH₃), 1.46–1.51 (m, 2H, CH₂), 2.43 (s, 3H, CH₃), 2.65 (s, 3H, CH₃), 3.18 (t, J = 8.0 Hz, 2H, NCH₂), 4.44 (s, 2H, NH₂), 6.45 (s, 1H, NH), 7.23–7.42 (m, 4H, ArH), 10.88 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 165.45, 161.63, 160.70, 153.22, 153.00, 139.52, 132.11, 131.25, 129.24, 110.52, 93.38, 43.52, 22.11, 21.36, 13.48, 11.58; anal. calcd for C₁₈H₂₁N₅O₃ (355.16); ESI-MS (70 eV) m/z (%): 356.15 (M + 1, 100).

2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6d)

White solid (yield, 68%). M.p.: >280 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.74 (t, J = 8.0 Hz, 6H, 2 × CH₃), 2.68 (s, 3H, CH₃), 3.07–3.12 (m, 4H, 2 × NCH₂), 4.49 (d, J = 4.0 Hz, 2H, NH₂), 7.43–7.57 (m, 5H, ArH), 10.84 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.94, 161.53, 161.36, 156.82, 154.64, 138.13, 129.69, 129.52, 128.95, 110.63, 96.30, 45.36, 13.61, 12.52; anal. calcd for C₁₈H₂₁N₅O₃ (355.16); ESI-MS (70 eV) m/z (%): 356.16 (M + 1, 100).

2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-m-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6e)

White solid (yield, 68%). M.p.: 262–264 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.75 (t, J = 8.0 Hz, 6H, 2 × CH₃), 2.38 (s, 3H, CH₃), 2.68 (s, 3H, CH₃), 3.08–3.14(m, 4H, 2 × CH₂), 4.49 (s, 2H, NH₂), 7.2–7.45 (m, 4H, ArH), 10.84 (s, 1H, NH); ¹³C NM R (100 MHz, DMSO-d₆) δ = 163.95, 161.55, 161.36, 156.75, 154.58, 139.08, 138.04, 130.03, 129.48, 129.32, 126.62, 110.62, 96.16, 45.32, 21.22, 13.60, 12.52; anal. calcd for C₁₉H₂₃N₅O₃ (369.18); ESI-MS (70 eV) m/z (100%): 370.12 (M + 1, 100).

2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-p-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6f)

White solid (yield, 74%). M.p.: 234–236 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.76 (t, J = 8.0 Hz, 6H, 2 × CH₃), 2.39 (s, 3H, CH₃), 2.68 (s, 3H, CH₃), 3.10 (q, 4H, J = 8.0 Hz, 2 × NCH₂), 4.48 (d, J = 4.0 Hz, 2H, NH₂), 7.28–7.36 (m, 4H, ArH), 10.85 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.91, 161.59, 161.39, 156.86, 154.58, 138.46, 135.49, 129.97, 129.31, 110.61, 96.22, 45.38, 21.19, 13.60, 12.62; anal. calcd for C₁₉H₂₃N₅O₃ (369.18); ESI-MS (70 eV) m/z (%): 370.22 (M + 1, 100).

2-(n-Butylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6g)

White solid (yield, 66%). M.p.: 211–213 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.85 (t, J = 8.0 Hz, 3H, CH₃), 1.21 (s, 2H, CH₂), 1.43–1.48 (m, 2H, CH₂), 2.64 (s, 3H, CH₃), 3.19–3.24 (m, 2H, NCH₂), 4.43 (d, J = 4.0 Hz, 2H, NH₂), 6.42 (t, J = 4.0 Hz, 1H, NH), 7.2–7.62 (m, 5H, ArH), 10.86 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 164.99, 161.09, 160.16, 152.88, 152.54, 134.29, 130.21, 129.06, 110.03, 92.90, 41.14, 30.57, 19.38, 13.72, 12.99; anal. calcd for C₁₈H₂₁N₅O₃ (355.16); ESI-MS (70 eV) m/z (%): 354.18 (M − 1, 100).

2-(n-Butylamino)-3,4-dihydro-6-methyl-4-oxo-3-m-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6h)

White solid (yield, 73%). M.p.: 230–232 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.86 (t, J = 8.0 Hz, 3H, CH₃), 1.19–1.23 (m, 2H, CH₂), 1.45–1.46 (m, 2H, CH₂), 2.38 (s, 3H, CH₃), 2.64 (s, 3H, CH₃), 3.22 (t, J = 8.0 Hz, 2H, NCH₂), 4.42 (s, 2H, NH₂), 6.41–6.43 (m, 1H, NH), 7.13–7.50 (m, 4H, ArH), 10.87 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 165.44, 161.57, 160.60, 153.04, 153.02, 140.21, 134.63, 130.74, 130.48, 129.88, 126.36, 110.49, 93.35, 41.62, 31.06, 21.34, 19.88, 14.20, 13.47; anal. calcd for C₁₉H₂₃N₅O₃ (369.18); ESI-MS (70 eV) m/z (%): 368.13 (M − 1, 100).

2-(n-Butylamino)-3,4-dihydro-6-methyl-4-oxo-3-p-tolylfuro[2,3-d]pyrimidine-5-carbohydrazide (6i)

White solid (yield, 65%). M.p.: 235–237 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.85 (t, J = 8.0 Hz, 3H, CH₃), 1.19–1.22 (m, 2H, CH₂), 1.44–1.46 (m, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.63 (s, 3H, CH₃), 3.19–3.23 (m, 2H, NCH₂), 4.42 (d, J = 4.0 Hz, 2H, NH₂), 6.42 (t, J = 4.0 Hz, 1H, NH), 7.21–7.41 (m, 4H, ArH), 10.87 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 164.94, 161.13, 160.19, 152.68, 152.48, 138.99, 131.59, 130.73, 128.73, 110.01, 92.85, 41.09, 30.56, 20.84, 19.36, 13.71, 12.97; anal. calcd for C₁₉H₂₃N₅O₃ (369.18); ESI-MS (70 eV) m/z (%): 370.17 (M + 1, 100).

3,4-Dihydro-6-methyl-2-morpholino-4-oxo-3-phenylfuro[2,3-d]pyrimidine-5-carbohydrazide (6j)

White solid (yield, 75%). M.p.: 266–268 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 2.74 (s, 3H, CH₃), 3.10 (s, 4H, 2 × NCH₂), 3.36–3.38 (m, 4H, 2 × OCH₂), 4.55 (s, 2H, NH₂), 7.53–7.60 (m, 5H, ArH), 10.85 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.07, 160.78, 160.6, 156.03, 154.67, 136.77, 128.92, 128.83, 128.59, 110.19, 96.97, 65.02, 48.78, 13.17; anal. calcd for C₁₈H₁₉N₅O₄ (369.14); ESI-MS (70 eV) m/z (%): 368.17 (M − 1, 100).

3,4-Dihydro-6-methyl-2-morpholino-4-oxo-3-m-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6k)

White solid (yield, 66%). M.p.: 261–263 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 2.38 (s, 3H, CH₃), 2.74 (s, 3H, CH₃), 3.06 (s, 4H, 2 × NCH₂), 3.33–3.34 (m, 4H, 2 × OCH₂), 4.49 (s, 2H, NH₂), 7.26–7.43 (m, 4H, ArH), 10.81 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.04, 160.76, 160.58, 155.92, 154.60, 138.45, 136.64, 129.14, 128.72, 125.71, 110.16, 96.80, 65.05, 48.72, 20.76, 13.15; anal. calcd for C₁₉H₂₁N₅O₄ (383.16); ESI-MS (70 eV) m/z (%): 384.17 (M + 1, 100).

3,4-Dihydro-6-methyl-2-morpholino-4-oxo-3-p-tolylfuro[2,3-d]pyrimidine-5-carbohydrazide (6l)

White solid (yield, 66%). M.p.: 270–272 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 2.38 (s, 3H, CH₃), 2.68 (s, 3H, CH₃), 3.04–3.05(m, 4H, 2 × NCH₂), 3.37 (m, 4H, 2 × OCH₂), 4.49 (s, 2H, NH₂), 7.35–7.6 (m, 4H, ArH), 10.82 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.03, 160.80, 160.68, 156.09, 154.60, 138.07, 134.16, 129.38, 128.49, 110.18, 96.87, 65.05, 48.75, 20.74, 13.17; anal. calcd for C₁₉H₂₁N₅O₄ (383.16); ESI-MS (70 eV) m/z (%): 384.13 (M + 1, 100).

2-(Dipropylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenyl furo[2,3-d]pyrimidine-5-carbohydrazide (6m)

White solid (yield, 72%). M.p.: 203–205 °C; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.65 (t, J = 8.0 Hz, 6H, 2 × CH₃), 1.17 (m, 4H, 2 × CH₂), 2.67 (s, 3H, CH₃), 2.96–2.98(m, 4H, 2 × NCH₂), 4.48 (d, J = 4.0 Hz, 2H, NH₂), 7.41–7.57 (m, 5H, ArH), 10.81 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.43, 160.86, 156.27, 154.14, 137.47, 129.08, 129.00, 128.54, 121.95, 110.14, 95.72, 53.63, 20.08, 13.10, 11.07; anal. calcd for C₂₀H₂₅N₅O₂ (384.20); ESI-MS (70 eV) m/z (%): 385.20 (M + 1, 100).

2-(Dipropylamino)-3,4-dihydro-6-methyl-4-oxo-3-p-tolyl-furo[2,3-d]pyrimidine-5-carbohydrazide (6n)

White solid (yield, 70%). M.p.: 203–205 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.67 (t, J = 8.0 Hz, 6H, 2 × CH₃), 1.20 (q, J = 4.0 Hz, 4H, 2 × CH₂), 2.40 (s, 3H, CH₃), 2.68 (s, 3H, CH₃), 2.98 (m, 4H, 2 × NCH₂), 4.48 (s, 2H, NH₂), 7.28–7.37 (m, 4H, ArH), 10.84 (s, 1H, NH); ¹³C NMR (100 MHz, DMSO-d₆) δ = 163.40, 161.04, 156.31, 154.08, 138.08, 134.82, 129.43, 128.69, 110.11, 95.61, 52.57, 20.69, 20.16, 13.09, 11.07; anal. calcd for C₂₁H₂₇N₅O₃ (397.21); ESI-MS (70 eV) m/z (%): 398.18 (M + 1, 100).

To a solution of coumarin formyl chloride 9 (4 mmol) in dichloromethane (15 mL), the respective furopyrimidones 6a–n (1 equiv., 4 mmol) were added. Triethylamine (5 mmol, 7.2 mL) was slowly dropped in the mixture in an ice bath, and then the mixture was stirred for 6–8 h at room temperature. The precipitated solid was collected by filtration, washed with ethanol and recrystallized with EtOH : H₂O (V : V = 2 : 1) to give the title compounds (10a–n).

2-oxo-N′-[2-(n-Propylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenylfuro[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10a)

Yellow solid (yield, 78%). M.p.: 228.3–230.6 °C; ¹H NMR (400 MHz, CDCl₃) δ: 0.79 (d, J = 8.0 Hz, 3H, CH₃), 1.41–1.45 (m, 4H, 2 × CH₂), 2.67 (s, 3H, CH₃), 4.10 (t, J = 12.0 Hz, 2H, NCH₂), 6.52 (s, 1H, NH), 7.09–7.89 (m, 8H, ArH), 11.06 (d, J = 4.0 Hz, 1H, NH), 12.79 (d, J = 4.0 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ: 165.03, 160.47, 160.19, 156.21, 154.33, 152.77, 147.97, 134.16, 131.63, 130.28, 129.10, 114.02, 113.19, 111.84, 108.86, 100.70, 92.63, 68.41, 56.01, 43.11, 30.37, 21.61, 18.60, 18.54, 13.60, 11.07; anal. calcd for C₂₇H₂₃N₅O₆ (513.1648); ESI-MS (70 eV) m/z (%): 514.1723 (M + 1, 100).

2-oxo-N′-[2-(n-Propylamino)-3,4-dihydro-6-methyl-4-oxo-3-m-tolyl-furo[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10b)

Yellow solid (yield, 80%). M.p.: >280 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.80 (t, J = 8.0 Hz, 3H, CH₃), 1.47–1.52 (m, 2H, CH₂), 2.40 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 3.17–3.24 (m, 2H, CH₂), 6.52 (s, 1H, NH), 7.18–8.80 (m, 9H, ArH), 11.10 (s, 1H, NH), 12.80 (s, 1H, NH); ¹³C NMR (400 MHz, DMSO-d₆) δ: 13.11, 13.71, 19.39, 20.88, 30.55, 41.19, 45.39, 92.57, 108.85, 116.24, 117.77, 118.25, 125.23, 125.91, 129.43, 130.06, 130.31, 130.37, 134.00, 134.40, 136.77, 139.81, 147.65, 152.77, 153.87, 154.42, 159.95, 165.06; anal. calcd for C₂₈H₂₅N₅O₆ (527.1805); HRESI-MS m/z (%): 528.1884 (M + 1, 100).

2-oxo-N′-[2-(Propylamino)-3,4-dihydro-6-methyl-4-oxo-3-p-tolyl-furo[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10c)

Yellow solid (yield, 81%). M.p.: >280 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.84 (t, J = 8.0 Hz, 3H, CH₃), 1.53–1.59 (m, 2H, CH₂), 2.40 (s, 3H, CH₃), 2.75 (s, 3H, CH₃), 3.28–3.30 (m, 2H, CH₂), 6.71 (s, 1H, NH), 7.33–8.92 (m, 9H, ArH), 11.26 (s, 1H, NH), 12.96 (s, 1H, NH); ¹³C NMR (400 MHz, DMSO-d₆ and pyridine-d₅) δ: 10.96, 13.03, 20.73, 21.63, 43.09, 92.76, 109.01, 116.15, 117.75, 118.24, 125.12, 128.72, 130.23, 130.77, 131.52, 134.28, 139.13, 147.73, 152.92, 153.88, 154.32, 157.10, 158.19, 159.99, 160.33, 165.04; anal. calcd for C₂₈H₂₅N₅O₆ (527.1805); HRESI-MS m/z (%): 528.1882 (M, 100).

2-oxo-N′-[2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenyl-furo[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10d)

Yellow solid (yield, 78%). M.p.: >280 °C; ¹H NMR (400 MHz, DMSO-d₆ and pyridine-d₅) δ: 0.84 (t, J = 8.0 Hz, 3H, CH₃), 1.53–1.59 (m, 2H, CH₂), 2.40 (s, 3H, CH₃), 2.75 (s, 3H, CH₃), 3.28–3.30 (m, 2H, CH₂), 6.71 (s, 1H, NH), 7.33–8.92 (m, 9H, ArH), 11.26 (s, 1H, NH), 12.96 (s, 1H, NH); ¹³C NMR (400 MHz, DMSO-d₆ and pyridine-d₅) δ: 10.96, 13.03, 43.09, 92.76, 109.01, 116.15, 117.75, 118.24, 125.12, 128.72, 130.23, 130.77, 131.52, 134.28, 139.13, 147.73, 152.92, 153.88, 154.32, 157.10, 158.19, 159.99, 160.33, 165.04; anal. calcd for C₂₈H₂₅N₅O₆ (527.18); ESI-MS (70 eV) m/z (%): 527.19 (M, 100).

2-oxo-N′-[2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-m-tolyl-furo[2,3-d] pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10e)

Yellow solid (yield, 75%). M.p.: 236.6–225.4 °C; ¹H NMR (400 MHz, CDCl₃) δ: 0.86 (d, J = 8.0 Hz, 6H, 2 × CH₃), 1.51 (d, J = 8.0 Hz, 2H, CH₂), 2.41 (s, 3H, CH₃), 2.79 (s, 3H, CH₃), 3.16 (t, J = 16.0 Hz, 4H, 2 × NCH₂), 6.84–8.02 (m, 7H, ArH), 11.05 (d, J = 8.0 Hz, 1H, NH), 12.73 (d, J = 4.0 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ: 164.67, 164.8, 163.80, 161.8, 160.6, 157.19, 156.85, 156.6, 148.60, 139.49, 137.51, 130.93, 129.48, 129.4, 129.31129.20, 126.01, 114.47, 113.47, 112.16, 109.75, 100.83, 96.74, 45.49, 21.32, 13.76, 12.38; anal. calcd for C₂₉H₂₇N₅O₆ (541.20); ESI-MS (70 eV) m/z (%): 541.21 (M, 100).

2-oxo-N′-[2-(Diethylamino)-3,4-dihydro-6-methyl-4-oxo-3-p-tolyl-furo[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10f)

Yellow solid (yield, 80%). M.p.: >280 °C; ¹H NMR (400 MHz, C₅H₅N + CHCl₃) δ: 0.86 (t, J = 8.0 Hz, 3H, CH₃), 1.20–1.48 (m, 4H, 2 × CH₂), 2.40 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 3.22–3.29 (m, 2H, CH₂), 6.50 (s, 1H, NH), 7.17–8.80 (m, 9H, ArH), 11.09 (s, 1H, NH), 12.80 (s, 1H, NH); anal. calcd for C₂₉H₂₇N₅O₆ (541.20); ESI-MS (70 eV) m/z (%): 541.25 (M, 100).

2-oxo-N′-[2-(n-Butylamino)-3,4-dihydro-6-methyl-4-oxo-3-phenyl-furo[2,3-d]pyrimidine-5-carbonyl]-2H-chromene-3-carbohydrazide (10g)

Yellow solid (yield, 83%). M.p.: 271–273 °C; ¹H NMR (400 MHz, DMSO-d₆) δ: 0.86 (t, J = 8.0 Hz, 3H, CH₃), 1.18–1.50 (m, 4H, 2 × CH₂), 2.68 (s, 3H, CH₃), 3.21–3.24 (m, 2H,CH₂), 6.50 (s, 1H, NH), 7.39–8.80 (m, 10H, ArH), 11.08 (s, 1H, NH), 12.77 (s, 1H, NH). ¹³C NMR (400 MHz, DMSO-d₆) δ: 13.43(13.51), 19.78, 30.90, 41.77 (41.91), 94.42, 109.25, 116.63, 116.93, 118.34, 125.39, 128.71, 129.92, 130.34, 130.81, 133.77, 134.50, 148.96, 150.46, 152.47, 154.41, 156.38, 157.82, 159.42, 160.62, 160.76, 165.05; anal. calcd for C₂₈H₂₅N₅O₆ (527.1805); HRESI-MS m/z (%): 528.1888 (M + 1, 100).

2-(Butylamino)-6-methyl-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-(m-tolyl)-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10h)

Yellow solid (yield, 81%). M.p.: 240.1–241.7 °C; ¹H NMR (400 MHz, CDCl₃) δ: 0.86 (d, J = 8.0 Hz, 3H, CH₃), 1.23 (s, 2H, CH₂), 1.46 (d, J = 8.0 Hz, 4H, 2 × CH₂), 2.42 (s, 3H, CH₃), 2.78 (s, 3H, CH₃), 3.24 (t, J = 16.0 Hz, 2H, NCH₂), 6.51 (s, 1H, NH), 6.52–7.91 (m, 7H, ArH), 11.06 (d, J = 4.0 Hz, 1H, NH), 12.81 (d, J = 4.0 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ: 165.54, 164.62, 160.99, 160.69, 158.27, 156.73, 154.82, 153.26, 148.49, 140.31, 134.51, 132.16, 130.87, 130.57, 129.95, 126.43, 114.55, 113.76, 112.36, 109.36, 101.22, 99.99, 93.08, 41.71, 31.06, 21.39, 19.91, 14.11, 13.61; anal. calcd for C₂₉H₂₇N₅O₆ (541.1961); HRESI-MS m/z (%): 542.2040 (M, 100).

2-(Butylamino)-6-methyl-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-(p-tolyl)-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10i)

Yellow solid (yield, 82%). M.p.: >280 °C; ¹H NMR (400 MHz, CDCl₃-d₆) δ: 0.85 (t, J = 8.0 Hz, 3H, CH₃), 1.06 (s, 2H, CH₂), 1.45 (m, 2H, CH₂), 2.42 (s, 3H, CH₃), 2.67 (s, 3H, CH₃), 3.28 (d, J = 8.0 Hz, 2H, CH₂), 6.5 (s, 1H, NH), 7.27–7.99 (m, 8H, ArH), 8.79 (s, 1H, CH), 11.10 (s, 1H, NH), 12.82 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃-d₆) δ: 165.08, 160.26, 158.04, 157.04, 154.43, 153.87, 152.78, 147.67, 134.40, 134.12, 130.27, 129.09, 125.23, 118.25, 117.75, 116.24, 108.87, 99.49, 92.60, 14.20, 30.54, 19.38, 13.71, 13.11; anal. calcd for C₂₉H₂₇N₅O₆ (541.1961); HRESI-MS m/z (%): 542.2043 (M + 1, 100).

6-Methyl-2-morpholino-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-phenyl-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10j)

Yellow solid (yield, 90%). M.p.: >280 °C; anal. calcd for C₂₉H₂₇N₅O₆ (541.16); ESI-MS (70 eV) m/z (%): 541.21 (M, 100).

6-Methyl-2-morpholino-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-(m-tolyl)-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10k)

Yellow solid (yield, 90%). M.p.: >280 °C; anal. calcd for C₂₉H₂₇N₅O₆ (555.17); ESI-MS (70 eV) m/z (%): 555.19 (M, 100).

6-Methyl-2-morpholino-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-(p-tolyl)-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10l)

Yellow solid (yield, 87%). M.p.: >280 °C; anal. calcd for C₂₉H₂₇N₅O₆ (555.17); ESI-MS (70 eV) m/z (%): 555.19 (M, 100).

2-(Dipropylamino)-6-methyl-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-phenyl-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10m)

Yellow solid (yield, 80%). M.p.: >280 °C; anal. calcd for C₂₉H₂₇N₅O₆ (555.21); ESI-MS (70 eV) m/z (%): 556.15 (M + 1, 100).

2-(Dipropylamino)-6-methyl-4-oxo-N′-(2-oxo-2H-chromene-3-carbonyl)-3-(p-tolyl)-3,4-dihydrofuro[2,3-d]pyrimidine-5-carbohydrazide (10n)

Yellow solid (yield, 84%). M.p.: >280 °C; anal. calcd for C₂₉H₂₇N₅O₆ (569.22); ESI-MS (70 eV) m/z (%): 569.21 (M, 100).

3.2. Bioassays of cell cytotoxicity

Tests were performed on HepG2 cells to determine whether the target compounds were cytotoxic. The cell lines were obtained from the Institute of Biomedical Research (Hubei University of Medicine). CCK8 assays were used to evaluate the cytotoxic activities of the tested compounds after 48 h of exposure. The cells were cultured in RPMI 1640. All media were supplemented with 10% fetal bovine serum (FBS) and 100 units per mL penicillin and 100 mg mL⁻¹ streptomycin at 37 °C in a 5% CO₂ incubator. A 96-well plate with 1.0 × 10⁴ cells per well was seeded at 37 °C under 5% CO₂ for 48 h. A variety of compounds were then added to the cells after they had been incubated for 24 h. Incubation of 20 mL of MTT solution at 5 mg mL⁻¹ for 4 h was followed by treatment with the drug for 24 h. In each well, 100 mL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan. Cell viability in percent was calculated as (A₅₇₀ of treated samples/A₅₇₀ of untreated samples) × 100%.

3.3. In vitro EGFR inhibition assays

In human liver cancer cell line HepG2, the most promising cytotoxic compounds 6d, 6e, 6f, 10a, 10d, 10f and 10g were tested for their effect on EGFR. Cells in culture medium were treated with 20 μl of the compounds dissolved in DMSO, and then incubated for 24 h at 37 °C in 5% CO₂. After harvesting the cells, the homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is used to determine the level of human EGFR. A monoclonal antibody for EGFR was precoated onto 96-well plates. In addition to the test samples, a biotinylated detection polyclonal antibody from goat specific for EGFR was added, followed by PBS washing. An avidin–biotin peroxidase complex was added, and the unbound conjugates were washed away with PBS. HRP substrate TMB was used to visualize HRP enzymatic reactions. After adding an acidic stop solution, TMB was catalyzed by HRP to produce a yellow color. The density of the yellow color is proportional to the amount of human EGFR in the plate. The chroma of the samples and the concentration of the human EGFR were positively correlated, and the optical density was determined at 450 nm. Based on duplicate determinations from the standard curve, the level of human EGFR was calculated (pg mL⁻¹). Percent inhibition was calculated in comparison to control untreated cells.

In vitro inhibition of human EGFR was evaluated using ELISA kits according to manufacturer's instructions (Ray Biotech) for compounds 6a, 10a, and 10g. An antibody specific for human EGFR is coated on 96-well plates for this assay. After adding samples and standards, the wells were incubated overnight at 4 °C with gentle shaking. After washing the wells, the biotinylated antibody was added and incubated for one hour at room temperature. Incubation at room temperature for 45 minutes was performed after washing away unbound biotinylated antibodies. Following washing, the TMB substrate solution was added and incubated for 30 minutes. The color developed was in proportion to the amount of bound EGFR. The stop solution was added and 450 nm intensity was measured. Compounds treated with control incubations were compared to calculate the percent inhibition. Based on the concentration inhibition response curve, the concentration of the test compound causing 50% inhibition (IC₅₀) was compared with sorafenib, a standard EGFR inhibitor.

3.4. Transwell migration assay

Transwell migration assay was used to detect the migration ability against HepG2 cells. The treated HepG2 cells were diluted to 10 × 10⁵ cells per mL with serum-free DMEM medium, and 200 μL cell suspensions were added to the upper transwell chamber, and 650 μL DMEM containing 10% FBS was added to the bottom chamber, respectively. The upper chamber was carefully immersed in the lower chamber liquid with sterile forceps; the 12-well plate with the transwell chamber was incubated in a 37 °C incubator for 24 h. Then, drugs were not added to the control group, and the experimental group was given different concentrations of 6f (0, 5.42, 16.26, and 27.10 μmol L⁻¹) and 10a (0, 3.90, 11.70, and 19.50 μmol L⁻¹) treated HepG2 for 24 h. The liquid was removed from the upper chamber and placed in a hole containing 600 μL PBS three times. After crystal violet staining, the cells were fixed with paraformaldehyde, and observed under an electron microscope, photographed, and statistically analyzed.

3.5. Molecular docking

SYBYL 7.0 was used to prepare the three-dimensional (3D) structure of compounds 6f and 10a. The 2000 steepest descent minimization and 2000 conjugate gradient minimization were performed. A crystal structure of wild-type (WT) EGFR (PDB ID: 5UGB)⁴⁷ in complex with an irreversible inhibitor was downloaded from the Protein Data Bank (https://www.rcsb.org/).⁴⁸ The inhibitor is removed from the complex structure and used to define the active site. Hydrogen was added to EGFR using Discovery Studio 4.0. 6f and 10a were connected to the activity centre using GOLD 3.0.⁴⁹ The radius of the active site is set to 10 Å by using the genetic algorithm (GA) for 300 runs. The highest level conformation was selected as the representative.

Conclusions

A series of furo[2,3-d]pyrimidone derivatives linked to coumarin via a hydrazide linker were designed, synthesized and evaluated for their in vitro EGFR inhibitory activity as well as their anti-proliferative activity against the HepG2 cell line. Most of the furopyrimidone–coumarin combinations 10a–n exhibited better potent HepG2 inhibition than furo[2,3-d]pyrimidone derivatives 6a–n. Compound 10a exhibited significant inhibitory activity against HepG2 cells with IC₅₀ = 7.72 ± 1.56 μM, which are better than fluorouracil, sorafenib and gefitinib. The excellent inhibition of EGFR enzymatic activity with IC₅₀ = 1.53 μM and 90% inhibition at 10 μM concentration is also noteworthy. To further confirm the antitumor activity of compound 10a targeting human liver cancer cells, transwell migration assay was used to detect the inhibitory effect of compound 10a against HepG2 cell layer development. The results demonstrated that compound 10a is a potential antitumor agent targeting HepG2. Molecular docking studies revealed the ability of the coumarin–furo[2,3-d]pyrimidone hybrid molecules to form a network of key interactions, known to be essential for wild-type EGFR inhibitors. The findings demonstrate the potent enhancement of coumarin–furo[2,3-d]pyrimidone hybrid molecules in synergistic anticancer activity, suggesting their potential as synergistic EGFR inhibitors and promising anti-tumor drugs targeting human liver cancer cells, warranting further investigation.

Author contributions

Tianshuai Wang was involved in conceptualization and design of the work; Tianshuai Wang and Yumeng Gao were involved in the acquisition, analysis, and interpretation of data; Fengxu Wu and Lun Luo were involved in the creation of new software used in the work; Tianshuai Wang was involved in writing – original draft preparation; Junkai Ma and Yanggen Hu were involved in writing – review and editing.

Conflicts of interest

There are no conflicts to declare.