Rutaecarpine derivatives synthesized via skeletal reorganization alleviate inflammation-associated oxidative damage by inhibiting the MAPK/NF-κB signaling pathway

Abstract

In recent years, skeleton reorganization based on bioactive natural products has emerged as a novel alternative strategy to the classical approach, mainly focusing on the peripheral modification of the inherent natural skeleton. Such reorganizations not only afford structurally unique molecules but also provide unanticipated bioactivities compared with the unaltered natural precursors. Herein, by rebuilding the inherent rigid skeleton of cardioprotective rutaecarpine (RUT), thirty-three structural derivatives were designed and synthesized, with 5Ci being the most representative example, which exhibited superior protective effects against inflammation-induced ROS accumulation and cellular damage compared with the clinically used anti-inflammation drug indomethacin.

In recent years, skeleton reorganization based on bioactive natural products has emerged as a novel alternative strategy to the classical approach, mainly focusing on the peripheral modification of the inherent natural skeleton.

1. Introduction

Inflammation is the normal defensive physiological response of the immune system to invasive microorganisms or physical injury. However, if it is not controlled, the immune system will instead attack the normal tissues.^1–3 The mechanism of inflammation is complex and regulated by various pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), nitric oxide (NO), interleukin-6 (IL-6) and interleukin-1β (IL-1β), which eventually produce a large amount of ROS, leading to DNA damage and subsequent cell death.^4–7

Natural products, which are characterized by their unique molecular structures, have continued to attract the attention of chemical and biological scientists for developing new chemical entities with great biological significances.^8–12 Although approximately 50% of FDA-approved new drugs released during the past four decades were natural product-based, only 3.8% of them were unaltered natural products, whereas the synthetic compounds derived from natural metabolites or natural pharmacophores accounted for the majority.¹³ Over the past few decades, the structural optimization of natural products has highly relied on a classical peripheral modification strategy to achieve multi-functionalization based on the inherent natural product framework,^14–17 but increasing reports in recent years have demonstrated the great advantages of natural product skeletal reorganization. Such reorganizations not only result in diverse chemical backbones and ring systems but also provide broader bioactivities than the original natural compounds.^18–21

Rutaecarpine (RUT) is a highly rigid pentacyclic alkaloid, which is isolated from one of the most famous traditional Chinese herbs, Evodia rutaecarpa.²² While most of the pharmacological studies have focused on the significant cardiovascular protective effects of RUT and its peripheral modification derivatives, increasing evidences indicate that using RUT as a starting material for further skeletal reorganization may lead to the discovery of a series of novel RUT frameworks with promising and unanticipated bioactivities. In 2012, Huang's group achieved a breakthrough in developing acetylcholinesterase inhibitors through strategic cleavage of the ring C coupled with structural modifications to the ring E of RUT.²³ Later, the same research group implemented an optimized strategy in 2015 by introducing structural modifications to both B and D rings, followed by ring C cleavage, ultimately obtaining novel hypolipidemic agents capable of inhibiting triglyceride (TG) accumulation.²⁴ In addition, Si et al. reported the identification of a series of ABCA1 up-regulators via successive opening of rings C, D, and B.²⁵

In the past few years, our group has conducted systematic research studies on the discovery of structurally unique and biologically significant molecules based on natural sources.^26–32 As an ongoing project, RUT was selected as a starting material for further skeletal reorganization. Consequently, thirty-three novel RUT derivatives were designed and synthesized via ring cleavage of the inherent B and C rings. Notably, these new chemical entities showed good anti-inflammatory effects against LPS-stimulated RAW 264.7 macrophages. The best compound 5Ci possessed two-fold potency compared with the unmodified natural RUT. Further mode-of-action studies evidenced that 5Ci alleviated the inflammation-induced oxidative damage by inhibiting the MAPK/NF-κB signaling pathway. Herein, we report the design and synthesis of RUT derivatives, along with their protective effects against inflammation-induced cellular damage.

2. Results and discussion

2.1. Chemistry

Structurally, RUT features three main biological fragments, namely an aromatic ring A, an amino group in ring B, and a quinazolinone moiety (rings C and D), but the inherent pentacyclic skeleton is highly rigid. Therefore, we decided to rebuild the ring system of RUT but retain the major pharmacophores. Previous investigation by Li's group revealed that the ring C cleavage of RUT may provide significant inhibition of AChE.²³ Inspired by this discovery, we attempted to approach a new RUT-derived skeleton mimicking a B,C-seco scaffold.

As shown in Scheme 1, the target compounds were readily accomplished in 3 steps starting from isatoic anhydride (1) and benzylamine derivatives or phenylhydrazine derivatives (2). First, 1 reacted with 2 using water as a solvent at room temperature to provide the intermediate 3. Second, 3 was refluxed in excess diethyl oxalate to afford quinazolinone 4 in high yields. Finally, 4 reacted with amines in ethanol to furnish the target compounds 5. All products were characterized by NMR and HRMS (see ESI† for more details).

Scheme 1. Synthesis of compounds 5Aa–5Fb.

2.2. Biological evaluation

2.2.1. Impact on RAW 264.7 macrophage cell viability

Before the anti-inflammation evaluation, we first assessed the effects of all RUT derivatives on the RAW 264.7 cell viability to exclude those with severe cytotoxicity with a cell viability cutoff of 65% at 100 μM. As shown in Table 1 and Fig. 1, RUT derivatives with an R group of p-chlorobenzyl (i.e., 5Ax series) and p-bromobenzyl (i.e., 5Bx series) exhibited significant cell inhibitory effects against RAW 264.7 cells, except for those with tetrahydropyrrole as an R² group (i.e., 5Af and 5Bf). A 5Cx series, which bear an R group of arylamino, featured only weak cytotoxicity, with 5Cn being the only exception. In addition, 5Eb showed a cell viability of 110.86 ± 0.08% at 100 μM. Thus, 5Af, 5Bf, 5Ca–5Cm and 5Eb were selected for further study.

Table 1. Effects of RUT derivatives (100 μM) on the viability of RAW 264.7 cells.

Cmpd.	X, Ar	n, R2	Cell viability rate [%]	Cmpd.	X, Ar	n, R2	Cell viability rate [%]
5Aa	CH2, p-chlorophenyl	2, N(CH3)2	8.87 ± 0.01	5Ce	NH, phenyl	3, NH2	99.78 ± 0.09
5Ab	CH2, p-chlorophenyl	3, N(CH3)2	9.98 ± 0.01	5Cf	NH, phenyl	4, NH2	67.63 ± 0.07
5Ac	CH2, p-chlorophenyl	2, N(C2H5)2	11.09 ± 0.01	5Cg	NH, phenyl	3, pyrrolidinyl	88.69 ± 0.09
5Ad	CH2, p-chlorophenyl	3, N(C2H5)2	7.77 ± 0.01	5Ch	NH, phenyl	1, benzenyl	96.86 ± 1.31
5Af	CH2, p-chlorophenyl	4, NH2	110.87 ± 0.07	5Ci	NH, phenyl	1, 4-toluenyl	100.89 ± 1.05
5Ag	CH2, p-chlorophenyl	3, pyrrolidinyl	11.09 ± 0.02	5Cj	NH, phenyl	1, 3-toluenyl	94.30 ± 2.82
5Ba	CH2, p-bromophenyl	2, N(CH3)2	9.98 ± 0.01	5Ck	NH, phenyl	1, 2-toluenyl	92.37 ± 2.78
5BB	CH2, p-bromophenyl	3, N(CH3)2	8.87 ± 0.01	5Cl	NH, phenyl	2, benzenyl	104.58 ± 5.29
5Bc	CH2, p-bromophenyl	2, N(C2H5)2	11.09 ± 0.01	5Cm	NH, phenyl	4, CH3	97.04 ± 2.81
5Bd	CH2, p-bromophenyl	3, N(C2H5)2	7.76 ± 0.01	5Cn	NH, phenyl	5, CH3	29.93 ± 0.03
5Be	CH2, p-bromophenyl	3, NH2	8.87 ± 0.01	5Da	NH, p-chlorophenyl	2, N(CH3)2	25.50 ± 0.02
5Bf	CH2, p-bromophenyl	4, NH2	66.52 ± 0.03	5Db	NH, p-chlorophenyl	3, N(CH3)2	28.82 ± 0.08
5Bg	CH2, p-bromophenyl	3, pyrrolidinyl	9.98 ± 0.01	5Ea	NH, p-fluorophenyl	2, N(CH3)2	11.09 ± 0.06
5Ca	NH, phenyl	2, N(CH3)2	66.58 ± 0.10	5Eb	NH, p-fluorophenyl	3, N(CH3)2	110.86 ± 0.08
5Cb	NH, phenyl	3, N(CH3)2	66.52 ± 0.07	5Fa	NH, p-methylphenyl	2, N(CH3)2	32.15 ± 0.01
5Cc	NH, phenyl	2, N(C2H5)2	99.78 ± 0.33	5Fb	NH, p-methylphenyl	3, N(CH3)2	63.19 ± 0.06
5Cd	NH, phenyl	3, N(C2H5)2	77.61 ± 0.06	IDMCa			77.61 ± 0.07

^aIDMC, indomethacin.

Fig. 1. Effect of compounds 5 on cell viability against RAW 264.7 cells.

2.2.2. Inhibition of NO production in LPS-induced RAW 264.7 cells

Abnormal overproduction of NO has been associated with the pathogenesis of inflammatory disorders and was identified as a classical indicator during the screening for anti-inflammatory molecules.^33,34 To evaluate whether the synthesized RUT derivatives could serve as potential anti-inflammatory agents, the selected compounds were subjected to the LPS-induced RAW 264.7 cells to detect their inhibitory effects on NO production. For this detection, we chose to examine the compounds at a concentration of 50 μM. As shown in Table 2, LPS stimulation significantly induced the production of NO in RAW 264.7 cells compared with the untreated control, which was inhibited after treatment with the RUT derivatives. None of the compounds displayed significant anti-proliferation activities against RAW 264.7 cells at the tested concentrations. Compared with the clinically used anti-inflammation drug indomethacin (IDMC, with NO inhibition of 57.87%), some of the synthetic RUT derivatives exhibited stronger inhibition, as exemplified by 5Ci (NO inhibition of 76.40%), followed by 5Cl (NO inhibition of 70.73%), 5Ca (NO inhibition of 67.88%), and 5Cc (NO inhibition of 66.90%). Moreover, the best compound 5Ci showed two-fold potency compared with the natural RUT (NO inhibition of 44.05%), further demonstrating the significance of skeletal rearrangement in natural product structural modifications.

Table 2. Effects of RUT derivatives (50 μM) on cell viability and NO inhibition against LPS-induced RAW 264.7 cells.

Cmpd.	Cell viability rate [%]	NO inhibition rate [%]		Cmpd.	Cell viability rate [%]	NO inhibition rate [%]
5Af	103.34 ± 2.54	47.37 ± 4.75		5Ch	94.64 ± 1.22	44.98 ± 2.21
5Bf	103.34 ± 5.67	50.73 ± 1.89		5Ci	97.48 ± 2.54	76.40 ± 5.41
5Ca	89.32 ± 4.68	67.88 ± 0.93		5Cj	97.92 ± 1.22	65.07 ± 1.66
5Cb	87.67 ± 7.93	56.35 ± 6.97		5Ck	89.23 ± 7.26	54.40 ± 2.10
5Cc	87.43 ± 4.23	66.90 ± 2.78		5Cl	104.87 ± 5.75	70.73 ± 3.02
5Cd	99.82 ± 10.21	50.66 ± 2.61		5Cm	107.75 ± 2.83	50.86 ± 3.70
5Ce	92.89 ± 8.56	53.92 ± 2.94		5Eb	102.77 ± 2.75	63.18 ± 7.90
5Cf	101.98 ± 3.89	55.04 ± 2.22		IDMCa	98.43 ± 4.76	57.87 ± 1.12
5Cg	82.73 ± 3.65	45.03 ± 3.22		RUTb	101.76 ± 8.89	44.05 ± 4.02

^aIDMC, Indomethacin.

^bRUT.

A schematic illustration of the structure–activity relationship of these molecules is shown in Fig. S100.† Structurally, these synthetic RUT derivatives feature a quinazolinone core, with different substitutions at N-3 (highlighted in red, part A) and 2-carboxamide (highlighted in blue, part B), of which the former mainly affects the cytotoxicity and NO inhibition potency, while the latter is responsible for the NO inhibitory effect of the molecules (Fig. S100†).

2.2.3. Compound 5Ci inhibited LPS-induced production of inflammatory mediators in RAW 264.7 cells

To further evaluate the anti-inflammation efficiency of 5Ci, we evaluated the potency of 5Ci at different concentrations. NO and PGE2 are key inflammatory mediators that play significant roles in the diverse inflammatory diseases, which are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively.^35,36 As shown in Fig. 2A and 3C, compound 5Ci dose-dependently reduced the release of NO and PGE2 from RAW 264.7 cells induced by LPS even at a concentration of 12.5 μM. In accordance with this observation, 5Ci dose-dependently suppressed the LPS-induced upregulation of iNOS and COX-2 (Fig. 2D–F). Therefore, compound 5Ci was selected for further mechanism investigation.

Fig. 2. Effects of compound 5Ci on the cells viability (A), NO release (B), PGE2 release (C), and expression levels of iNOS and COX-2 (D–F) in LPS-induced RAW 264.7 cells. Before treatment with LPS (1 μg mL⁻¹) for 24 h, cells were pretreated with compound 5Ci at different concentrations for 1 h. Statistical significance compared to the LPS group is indicated. Results are expressed as mean ± SD, n = 3, ### p < 0.001, vs. 0 μM. *p < 0.05, **p < 0.01, ***p < 0.001, vs. LPS alone.

Fig. 3. Compound 5Ci inhibited LPS-induced inflammatory responses in the MAPK signaling pathway. Cells were pretreated with different concentrations of 5Ci (12.5, 25, or 50 μM) for 1 h, and then exposed to 1 μg mL⁻¹ of LPS for 24 h. Results are expressed as mean ± SD, n = 3, ##p < 0.01, ###p < 0.001, ####p < 0.0001, vs. 0 μM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, vs. LPS alone.

2.2.4. Compound 5Ci inhibited LPS-induced MAPK activation in RAW 264.7 cells

MAPK signaling can be activated by different inflammatory stimuli, and it plays an important regulatory role in inflammation and a series of cellular processes such as cell differentiation, cell growth, and apoptosis.³⁷ It has been shown that LPS-induced activation of MAPK induces the phosphorylation of ERK, JNK, and p38, which eventually leads to oxidative stress and tissue damage.^38–40 To evaluate whether the MAPK signaling was blocked by 5Ci after LPS stimulation, Western blot assay was performed. As expected, the treatment of RAW 264.7 cells with LPS led to significant phosphorylation of ERK, JNK, and p38, while 5Ci diminished the LPS-induced phosphorylation of ERK, JNK, and p38 without affecting its protein expression (Fig. 3), indicating that 5Ci could be regarded as a MAPK inhibitor.

2.2.5. Compound 5Ci inhibited LPS-induced NF-κB activation in RAW 264.7 cells

The nuclear factor-κB (NF-κB) signaling pathway is a downstream target of MAPK signaling.⁴¹ In inactivated status, cytoplasmic NF-κB binds with inhibitor-κ binding protein (IκB) α. After stimulation by LPS and inflammatory mediators, IκBα is phosphorylated and degraded, which leads to the release and phosphorylation of NF-κB. The phosphorylated NF-κB will then translocate from the cytoplasm to the nucleus, which then initiates the transcription of target genes of inflammatory factors. Therefore, the ability to inhibit NF-κB signaling is considered to be one of the most important features for identifying anti-inflammatory agents.⁴² Based on the significant inhibitory effects of 5Ci on MAPK signaling, we evaluated whether 5Ci could also affect the downstream NF-κB pathway. As shown in Fig. 4, LPS significantly increased the phosphorylation of IκB and NF-κB p65 subunits, which were antagonized by 5Ci in a dose-dependent manner.

Fig. 4. Compound 5Ci inhibited LPS-induced inflammatory responses in the NF-κB signaling pathway. Cells were pretreated with different concentrations of 5Ci (12.5, 25, or 50 μM) for 1 h, and then exposed to 1 μg mL⁻¹ of LPS for 24 h. Results are expressed as mean ± SD, n = 3, ##p < 0.01, ###p < 0.001, vs. 0 μM. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. LPS alone.

2.2.6. Compound 5Ci down-regulated the mRNA levels of IL-1β, IL-6, and TNF-α

As one of the most highly expressed transcription factors in macrophages, NF-κB is responsible for the transcription of many inflammatory factors such as IL-1β, IL-6, and TNF-α.⁴³ To assess the alterations in inflammatory factors following the inhibition of LPS-induced MAPK/NF-κB activation by 5Ci, we employed qPCR to measure the mRNA levels of IL-1β, IL-6, and TNF-α. As expected, 5Ci dose-dependently reduced the mRNA levels of inflammatory factors stimulated by LPS (Fig. 5), indicating that 5Ci could effectively alleviate LPS-induced inflammatory responses as a result of inhibiting the NF-κB signaling pathway.

Fig. 5. IL-1β (A), IL-6 (B) or TNF-α (C) levels in the cells determined using qPCR assays. Cells were pretreated with different concentrations of 5Ci (12.5, 25, or 50 μM) for 1 h, and then exposed to 1 μg mL⁻¹ of LPS for 24 h. The results are expressed as mean ± SD, n = 3, #p < 0.05, ##p < 0.01, vs. 0 μM. *p < 0.05, **p < 0.01, vs. LPS alone.

2.2.7. Compound 5Ci attenuates LPS-induced intracellular ROS accumulation and inflammatory response

In response to inflammatory stimulation, cells take up excessive oxygen, which subsequently triggers a “respiratory burst” via ROS accumulation, and eventually results in DNA damage.^44,45 As shown in Fig. 6, pre-treatment of LPS significantly induced the ROS accumulation of RAW 264.7 cells, which was attenuated by 5Ci. Moreover, compared with IDMC, the clinically used anti-inflammation drug, 5Ci showed significant greater potency. Meanwhile, 5Ci antagonized the LPS-induced DNA damage of RAW 264.7 cells. These findings indicated that 5Ci can relieve the inflammation-associated oxidative damage.

Fig. 6. Effect of 5Ci on LPS-induced ROS generation (A) and expression of γ-H2AX (B and C) in RAW 264.7 cells. Cells were co-treated with 1 μg mL⁻¹ LPS for 1 h in the presence or absence of 25 μM compound. Images were captured using a Bio-Tek Cytation5 imaging reader with 20× objective.

2.2.8. Compound 5Ci binds to HK2 to inhibit the activation of the MAPK signaling pathway and downstream cascade reactions

Finally, to predict the potential anti-inflammatory targets of compound 5Ci, we used the PharmMapper database to construct its putative target profile.⁴⁶ Among the identified results, HK2 (hexokinase 2) emerged as the top-ranked human protein target. Since HK2 mediates the phosphorylation of p38,⁴⁷ the binding of 5Ci to HK2 may inhibit the activation of the MAPK signaling pathway and its downstream cascade reactions. Subsequently, the molecular interactions between 5Ci and HK2 were revealed through molecular docking. The molecular docking study showed that 5Ci could strongly bind to HK2 via hydrogen bonding with Thr620, Glu708, Asn735, and Glu742, as well as a series of π–cation and π–alkyl interactions (Fig. 7). These findings position HK2 as a potential target for 5Ci to inhibit the activation of the MAPK signaling pathway and downstream cascade reactions.

Fig. 7. Binding mode of compound 5Ci with the pocket of HK2 in 3D and 2D.

3. Conclusion

In conclusion, thirty-three RUT derivatives were designed and synthesized via natural product skeleton reorganization, which allowed the identification of a series of anti-inflammatory compounds with novel natural product-derived frameworks. The best compound 5Ci possesses superior anti-inflammation efficiency to the clinically used anti-inflammation drug and the unmodified natural RUT. Moreover, the mechanistic study indicated that 5Ci significantly inhibited the activation of the MAPK/NF-κB signaling pathways, and therefore, exhibited promising protective effects against inflammation-induced oxidative stress and DNA damage. Finally, HK2 was proposed to be the potential target of 5Civia virtual screening using the PharmMapper database combined with molecular docking.

Natural products and their derivatives are important sources for anti-inflammatory drug discovery. The most well-known examples include dexamethasone, a steroid analogue, and aspirin, a non-steroidal anti-inflammatory drug (NSAID) derived from natural salicylic acid via acetylation. Compared with the classical peripheral functionalization of inherent natural scaffolds, skeleton reorganization allows the rapid de novo construction of a natural product-derived skeleton by using simple and commercially available fragments, which lead to a novel framework and broader bioactivity. While RUT and its peripheral functionalization derivatives are known for their cardioprotective effects, previous studies have demonstrated the great potential of skeleton reorganization of RUT to build a novel scaffold with unexpected bioactivities, but most of these studies focused on the ring C cleavage. Herein, we report the cleavage of rings B and C of RUT and their significance in alleviating inflammation responses. Taken together, our research not only provided a novel structural framework for developing anti-inflammatory agents, but also verified the advantages of skeleton reorganization in natural product structural optimization.

4. Experimental section

4.1. General

All commercially available solvents and chemicals were used as received without further purification unless otherwise indicated. ¹H and ¹³C NMR spectra were recorded using a Bruker AV-400 instrument with chemical shifts reported in ppm. Deuterated chloroform (CDCl₃), deuterated dimethyl sulfoxide (DMSO-d₆) and deuterated methanol (CD₃OD) were used as the solvents and TMS as the internal standard. Melting points were recorded using an X-4B apparatus without correction. The high-resolution mass spectra (HRMS) were recorded using an Agilent 6545 Q-TOF LC/MS. ¹H and ¹³C NMR and HRMS spectra of compounds 5 are available in the Supporting Information.

4.2. Chemistry

4.2.1. Synthesis of compound series 3 (taking compound 3A as an example)

Compound 1 (3.06 mmol, 500 mg) was dissolved in 100 mL of H₂O, Compound 2 (para-chlorobenzylamine, 3.06 mmol, 434 mg) was added and the mixture was stirred at room temperature for 12 h until the reaction completed (monitored by TLC). The reaction mixture was cooled to room temperature, filtered, and concentrated in vacuo. The filter cake was collected to compound 3A as a white solid. Yield: 702 mg (88%).

4.2.2. Synthesis of compound series 4 (taking compound 4A as an example)

Compound 3A (2.69 mmol, 700 mg) was dissolved in 4 mL of diethyl oxalate (26.9 mmol), and the mixture was heated and refluxed for 4 h under stirring until the reaction completed (monitored by TLC). The reaction mixture was cooled to room temperature, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with ethyl acetate/petroleum ether (V_DCM: V_MeOH = 1:  50) to afford 4A as a white solid. Yield: 916 mg (99%).

4.2.3. General procedure for target compound series 5Aa–5Fb

Compound 4 (0.31 mmol, 106 mg) was dissolved in 5 mL of ethanol, amine derivatives (typically, 1.5 mmol L⁻¹) were added and the mixture was heated and refluxed for 1 h under stirring until the reaction completed (monitored by TLC). Then the solvent was removed in vacuo and the residue was purified by column chromatography over silica gel eluting with dichloromethane/methanol to provide 5Aa–5Fb. The structures of all these molecules were established by comprehensive analysis of their physicochemical data as well as MS and NMR evidence. The purity of these compounds was demonstrated to be >95% by NMR spectroscopy.

3-(4-Chlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Aa)

White solid, 88 mg, 74% yield, m.p. 174–177 °C. ¹H NMR (400 MHz, CDCl₃) δ 12.64 (s, 1H), 8.59 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.50–7.42 (m, 1H), 7.26 (s, 2H), 7.13–7.06 (m, 1H), 6.82 (s, 1H), 4.58 (d, J = 5.8 Hz, 2H), 3.47–3.39 (m, 2H), 2.50 (t, J = 6.1 Hz, 2H), 2.27 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 168.0, 159.9, 158.3, 138.0, 136.4, 133.4, 132.6, 129.3, 128.8, 126.8, 123.9, 121.4, 121.1, 57.4, 45.1, 43.2, 37.2. HRMS (ESI) m/z calcd for C₂₀H₂₂N₄O₂Cl [M + H]⁺ 385.1426, found 385.1418.

3-(4-Chlorobenzyl)-N-(3-(dimethylamino)propyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Ab)

White solid, 83.8 mg, 36% yield, m.p. 130–131 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.58 (d, J = 8.3 Hz, 1H), 7.77–7.71 (m, 1H), 7.55–7.49 (m, 1H), 7.37–7.30 (m, 4H), 7.24–7.18 (m, 1H), 4.55 (s, 2H), 3.36 (t, J = 6.9 Hz, 2H), 2.55–2.47 (m, 2H), 2.35 (s, 6H), 1.86–1.76 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 167.3, 159.0, 156.5, 136.0, 135.7, 131.0, 130.3, 127.3, 126.7, 126.1, 122.3, 120.7, 119.0, 55.0, 42.2, 40.7, 36.0, 24.6. HRMS (ESI) m/z calcd for C₂₁H₂₄N₄O₂Cl [M + H]⁺ 399.1570, found 399.1588.

3-(4-Chlorobenzyl)-N-(2-(diethylamino)ethyl)-4-oxo-3,4-dihydroquinazoline-2-carbox-amide (5Ac)

White solid, 103.5 mg, 43% yield, m.p. 152–154 °C. ¹H NMR (500 MHz, CDCl₃) δ 12.66 (s, 1H), 8.65–8.61 (m, 1H), 7.95 (s, 1H), 7.56–7.47 (m, 2H), 7.32–7.29 (m, 2H), 7.15–7.10 (m, 2H), 6.64 (s, 1H), 4.61 (d, J = 5.8 Hz, 2H), 3.41 (q, J = 6.0 Hz, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.57 (q, J = 7.1 Hz, 4H), 1.04 (t, J = 7.1 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 167.9, 159.7, 158.4, 138.1, 136.3, 133.5, 132.6, 129.4, 128.9, 126.7, 123.9, 121.4, 121.2, 51.2, 46.9, 43.3, 37.4, 11.8. HRMS (ESI) m/z calcd for C₂₂H₂₆N₄O₂Cl [M + H]⁺ 413.1739, found 413.1751.

3-(4-Chlorobenzyl)-N-(3-(diethylamino)propyl)-4-oxo-3,4-dihydroquinazoline-2-carbox-amide (5Ad)

White solid, 120.2 mg, 49% yield, m.p. 92–94 °C. ¹H NMR (500 MHz, CDCl₃) δ 12.60 (s, 1H), 8.87 (s, 1H), 8.62–8.58 (m, 1H), 7.55–7.51 (m, 1H), 7.50–7.44 (m, 1H), 7.30–7.28 (m, 2H), 7.13–7.07 (m, 1H), 6.81–6.66 (m, 1H), 4.60 (d, J = 5.8 Hz, 2H), 3.47–3.42 (m, 2H), 2.60–2.54 (m, 6H), 1.78–1.69 (m, 2H), 1.08 (t, J = 7.1 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 167.9, 159.8, 158.5, 138.1, 136.4, 133.4, 132.5, 129.4, 128.8, 126.8, 123.8, 121.4, 121.2, 51.9, 46.8, 43.2, 39.8, 25.4, 11.3. HRMS (ESI) m/z calcd for C₂₃H₂₈N₄O₂Cl [M + H]⁺ 427.1895, found 427.1891.

N-(4-Aminobutyl)-3-(4-chlorobenzyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Af)

White solid, 107 mg, 48% yield, m.p. 219–221 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.58 (d, J = 7.6 Hz, 1H), 7.77–7.71 (m, 1H), 7.56–7.50 (m, 1H), 7.38–7.28 (m, 4H), 7.25–7.18 (m, 1H)), 4.55 (s, 2H), 3.34 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H), 1.69–1.52 (m, 4H). ¹³C NMR (125 MHz, CD₃OD) δ 177.2, 169.0, 167.9, 147.7, 147.1, 141.7, 140.9, 138.6, 137.8, 137.7, 133.2, 130.7, 129.6, 51.4, 49.9, 38.5, 38.2, 35.5. HRMS (ESI) m/z calcd for C₂₀H₂₂N₄O₂Cl [M + H]⁺ 385.1426, found 385.1415.

3-(4-Chlorobenzyl)-4-oxo-N-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydroquinazoline-2-carboxamide (5Ag)

White solid, 185 mg, 75% yield, m.p. 115–117 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.60–8.55 (m, 1H), 7.76–7.72 (m, 1H), 7.56–7.50 (m, 1H), 7.39–7.30 (m, 4H), 7.25–7.20 (m, 1H), 4.56 (s, 2H), 3.37 (t, J = 6.8 Hz, 2H), 2.70–2.57 (m, 6H), 1.90–1.80 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 169.3, 160.8, 158.5, 137.9, 137.6, 133.0, 132.2, 129.3, 128.6, 128.0, 124.3, 122.7, 120.9, 54.0, 53.9, 42.6, 38.3, 27.9, 23.2. HRMS (ESI) m/z calcd for C₂₃H₂₆N₄O₂Cl [M + H]⁺ 425.1739, found 425.1725.

3-(4-Bromobenzyl)-N-(2-(dimethylamino)ethyl)-4-oxo-3,4-dihydroquinazoline-2-carbox-amide (5Ba)

White solid, 205 mg, 92% yield, m.p. 184–186 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.58 (d, J = 8.4 Hz, 1H), 7.78–7.69 (m, 1H), 7.57–7.51 (m, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.25–7.19 (m, 1H), 4.54 (s, 2H), 3.47 (t, J = 6.6 Hz, 2H), 2.57 (t, J = 6.6 Hz, 2H), 2.32 (s, 6H). ¹³C NMR (125 MHz, CD₃OD) δ 170.3, 161.9, 159.4, 139.4, 138.6, 133.2, 132.6, 130.6, 129.0, 125.3, 123.7, 122.0, 121.9, 58.9, 45.4, 43.7, 38.2. HRMS (ESI) m/z calcd for C₂₀H₂₂N₄O₂Br [M + H]⁺ 429.0921, found 429.0927.

3-(4-Bromobenzyl)-N-(3-(dimethylamino)propyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Bb)

White solid, 185 mg, 75% yield, m.p. 128–130 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.58 (d, J = 8.3 Hz, 1H), 7.76–7.70 (m, 1H), 7.55–7.50 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 7.6 Hz, 1H), 4.53 (s, 2H), 3.35 (t, J = 6.9 Hz, 2H), 2.49–2.42 (m, 2H), 2.31 (s, 6H), 1.82–1.75 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 170.3, 161.8, 159.5, 139.4, 138.7, 133.2, 132.6, 130.6, 129.0, 125.2, 123.6, 121.9, 121.8, 58.0, 45.2, 43.7, 39.0, 27.6. HRMS (ESI) m/z calcd for C₂₁H₂₄N₄O₂Br [M + H]⁺ 443.1077, found 443.1074.

3-(4-Bromobenzyl)-N-(2-(diethylamino)ethyl)-4-oxo-3,4-dihydroquinazoline-2-carbox-amide (5Bc)

White solid, 212 mg, 89% yield, m.p. 157–159 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.61–8.53 (m, 1H), 7.77–7.71 (m, 1H), 7.57–7.50 (m, 1H), 7.50–7.43 (m, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.25–7.17 (m, 1H), 4.53 (s, 2H), 3.43 (t, J = 7.0 Hz, 2H), 2.74–2.58 (m, 6H), 1.08 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 170.3, 161.8, 159.4, 139.4, 138.7, 133.2, 132.6, 130.6, 129.1, 125.3, 123.7, 122.0, 121.9, 52.2, 48.1, 43.7, 38.0, 11.6. HRMS (ESI) m/z calcd for C₂₂H₂₆N₄O₂Br [M + H]⁺ 457.1234, found 457.1215.

3-(4-Bromobenzyl)-N-(3-(diethylamino)propyl)-4-oxo-3,4-dihydroquinazoline-2-carbox-amide (5Bd)

White solid, 220 mg, 90% yield, m.p. 99–101 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.60–8.54 (m, 1H), 7.76–7.70 (m, 1H), 7.56–7.50 (m, 1H), 7.49–7.44 (m, 2H), 7.33–7.27 (m, 2H), 7.24–7.18 (m, 1H), 4.53 (s, 2H), 3.35 (t, J = 6.8 Hz, 2H), 2.65–2.54 (m, 6H), 1.81–1.72 (m, 2H), 1.07 (t, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 170.3, 161.8, 159.5, 139.4, 138.7, 133.2, 132.6, 130.6, 129.1, 125.3, 123.6, 122.0, 121.9, 51.5, 47.7, 43.7, 39.5, 26.6, 11.2. HRMS (ESI) m/z calcd for C₂₃H₂₈N₄O₂Br [M + H]⁺ 471.1390, found 471.1395.

N-(3-Aminopropyl)-3-(4-bromobenzyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Be)

White solid, 201 mg, 93% yield, m.p. 200–201 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.61–8.53 (m, 1H), 7.77–7.69 (m, 1H), 7.57–7.50 (m, 1H), 7.50–7.42 (m, 2H), 7.34–7.26 (m, 2H), 7.25–7.17 (m, 1H), 4.54 (s, 2H), 3.39 (t, J = 6.8 Hz, 2H), 2.71 (t, J = 7.0 Hz, 2H), 1.81–1.69 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 170.3, 162.0, 159.4, 139.4, 138.7, 133.3, 132.6, 130.6, 129.1, 125.3, 123.6, 121.9, 43.7, 39.5, 38.0, 32.6. HRMS (ESI) m/z calcd for C₁₉H₂₀N₄O₂Br [M + H]⁺ 415.0764, found 415.0758.

N-(4-Aminobutyl)-3-(4-bromobenzyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Bf)

White solid, 178 mg, 80% yield, m.p. 180–182 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.60–8.55 (m, 1H), 7.76–7.71 (m, 1H), 7.55–7.50 (m, 1H), 7.49–7.44 (m, 2H), 7.29 (d, J = 8.5 Hz, 2H), 7.24–7.19 (m, 1H), 4.54 (s, 2H), 3.33 (t, J = 6.3 Hz, 2H), 2.72–2.66 (m, 2H), 1.66–1.58 (m, 2H), 1.55–1.49 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 170.3, 161.8, 159.6, 139.4, 138.9, 133.2, 132.6, 130.6, 129.0, 125.3, 123.7, 122.0, 121.9, 43.7, 41.9, 40.5, 30.5, 27.6. HRMS (ESI) m/z calcd for C₂₀H₂₂N₄O₂Br [M + H]⁺ 429.0921, found 429.0916.

3-(4-Bromobenzyl)-4-oxo-N-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydroquinazoline-2-carboxamide (5Bg)

White solid, 229 mg, 94% yield, m.p. 106–108 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.61–8.54 (m, 1H), 7.76–7.72 (m, 1H), 7.56–7.50 (m, 1H), 7.49–7.43 (m, 2H), 7.32–7.26 (m, 2H), 7.24–7.17 (m, 1H), 4.53 (s, 2H), 3.37 (t, J = 6.8 Hz, 2H), 2.69–2.57 (m, 6H), 1.88–1.77 (m, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 170.3, 161.8, 159.5, 139.4, 138.7, 133.2, 132.6, 130.6, 129.1, 125.3, 123.6, 121.9, 55.0, 54.9, 43.7, 39.2, 28.9, 24.2. HRMS (ESI) m/z calcd for C₂₃H₂₆N₄O₂Br [M + H]⁺ 469.1234, found 469.1234.

N-(2-(Dimethylamino)ethyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Ca)

White solid, 103 mg, 45% yield, m.p. 120–121 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.25–8.18 (m, 1H), 7.93–7.86 (m, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.63–7.57 (m, 1H), 7.24–7.16 (m, 2H), 6.89 (t, J = 7.4 Hz, 1H), 6.79–6.73 (m, 2H), 3.43 (s, 2H), 2.48–2.39 (m, 2H), 2.24 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 163.5, 162.0, 154.2, 148.0, 136.4, 130.1, 129.2, 128.8, 127.8, 123.6, 122.5, 114.9, 58.5, 45.3, 38.0. HRMS (ESI) m/z calcd for C₁₉H₂₂N₅O₂ [M + H]⁺ 352.1768, found 352.1765.

N-(3-(Dimethylamino)propyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Cb)

White solid, 118 mg, 66% yield, m.p. 126–127 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.24–8.19 (m, 1H), 7.93–7.87 (m, 1H), 7.81–7.77 (m, 1H), 7.63–7.58 (m, 1H), 7.24–7.18 (m, 2H), 6.94–6.87 (m, 1H), 6.82–6.77 (m, 2H), 3.40 (s, 2H), 2.40–2.31 (m, 2H), 2.19 (s, 6H), 1.75–1.64 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 162.8, 160.9, 153.4, 147.2, 147.0, 135.4, 129.2, 128.2, 127.8, 126.8, 122.6, 121.6, 113.2, 56.5, 44.0, 37.3, 26.5. HRMS (ESI) m/z calcd for C₂₀H₂₄N₅O₂ [M + H]⁺ 366.1925, found 366.1918.

N-(2-(Diethylamino)ethyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Cc)

White solid, 193 mg, 98% yield, m.p. 82–84 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.23–8.19 (m, 1H), 7.93–7.87 (m, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.64–7.58 (m, 1H), 7.24–7.16 (m, 1H), 6.90 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 7.7 Hz, 2H), 3.42 (s, 2H), 2.61–2.52 (m, 1H), 1.02 (t, J = 7.2 Hz, 6H). ¹³C NMR (125 MHz, CD₃OD) δ 163.5, 162.0, 154.2, 148.0, 147.9, 136.4, 130.1, 129.3, 128.8, 127.8, 123.6, 122.5, 114.9, 52.0, 48.1, 37.7, 11.4. HRMS (ESI) m/z calcd for C₂₁H₂₆N₅O₂ [M + H]⁺ 380.2081, found 380.2072.

N-(3-(Diethylamino)propyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Cd)

Yellow liquid, 182 mg, 94% yield. ¹H NMR (500 MHz, CD₃OD) δ 8.26–8.19 (m, 1H), 7.96–7.89 (m, 1H), 7.83–7.79 (m, 1H), 7.66–7.61 (m, 1H), 7.27–7.19 (m, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.86–6.80 (m, 2H), 3.52–3.36 (m, 2H), 2.70–2.58 (m, 6H), 1.80–1.70 (m, 2H), 1.07 (t, J = 7.2 Hz, 6H). ¹³C NMR (125 MHz, CD₃OD) δ 162.9, 160.9, 153.3, 147.2, 146.9, 135.4, 129.2, 128.2, 127.8, 126.8, 122.6, 121.7, 114.0, 50.2, 46.8, 37.4, 25.4, 9.6. HRMS (ESI) m/z calcd for C₂₂H₂₈N₅O₂ [M + H]⁺ 394.2238, found 394.2235.

N-(3-Aminopropyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Ce)

White solid, 201 mg, 93% yield, m.p. 82–84 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.26–8.16 (m, 1H), 7.94–7.85 (m, 1H), 7.82–7.77 (m, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.21 (t, J = 8.0 Hz, 2H), 6.90 (t, J = 7.4 Hz, 1H), 6.78 (d, J = 7.8 Hz, 2H), 3.44–3.33 (m, 21H), 2.59 (t, J = 7.0 Hz, 2H), 1.71–1.57 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 160.9, 159.0, 151.5, 145.2, 145.0, 133.5, 127.2, 126.3, 125.8, 124.8, 120.7, 119.6, 111.9, 36.4, 34.6, 29.6. HRMS (ESI) m/z calcd for C₁₈H₂₀N₅O₂ [M + H]⁺ 338.1612, found 338.1607.

N-(4-Aminobutyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Cf)

White solid, 218 mg, 96% yield, m.p. 204–206 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.25–8.15 (m, 1H), 7.94–7.87 (m, 1H), 7.82–7.76 (m, 1H), 7.65–7.57 (m, 1H), 7.26–7.17 (m, 2H), 6.93–6.87 (m, 1H), 6.82–6.75 (m, 2H), 3.42–3.31 (m, 2H), 2.51 (t, J = 7.1 Hz, 2H), 1.53–1.44 (m, 2H), 1.41–1.32 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 162.2, 160.6, 153.2, 146.8, 146.6, 135.0, 128.7, 127.8, 127.4, 126.4, 122.2, 121.1, 113.4, 40.6, 38.6, 29.2, 26.0. HRMS (ESI) m/z calcd for C₁₉H₂₂N₅O₂ [M + H]⁺ 352.1768, found 352.1764.

4-Oxo-3-(phenylamino)-N-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydroquinazoline-2-carbox-amide (5Cg)

White solid, 249 mg, 98% yield, m.p. 109–111 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.23 (d, J = 7.7 Hz, 1H), 7.91 (t, J = 7.2 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.9 Hz, 2H), 6.92 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 7.9 Hz, 2H), 3.54–3.34 (m, 2H), 3.50–3.39 (m, 6H), 1.83–1.71 (m, 6H). ¹³C NMR (125 MHz, CD₃OD) δ 162.3, 160.6, 153.1, 146.8, 146.6, 135.0, 128.8, 127.8, 127.4, 126.4, 122.2, 121.2, 113.5, 53.4, 53.3, 37.3, 27.6, 22.7. HRMS (ESI) m/z calcd for C₂₂H₂₆N₅O₂ [M + H]⁺ 392.2081, found 392.2079.

N-Benzyl-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Ch)

White solid, 236 mg, 82% yield, m.p. 104–106 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.35 (t, J = 6.1 Hz, 1H), 9.28 (s, 1H), 8.16 (dd, J = 8.0, 1.5 Hz, 1H), 7.98–7.90 (m, 1H), 7.86–7.79 (m, 1H), 7.67–7.59 (m, 1H), 7.25–7.12 (m, 7H), 6.88–6.82 (m, 1H), 6.82–6.74 (m, 2H), 4.55–4.29 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.2, 159.6, 153.6, 147.1, 146.5, 138.2, 135.2, 128.7, 128.2, 127.8, 127.6, 126.9, 126.7, 126.4, 122.0, 120.4, 113.4, 41.7. HRMS (ESI) m/z calcd for C₂₂H₁₉N₄O₂ [M + H]⁺ 371.1503 found 371.1494.

N-(4-Methylbenzyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Ci)

White solid, 275 mg, 92% yield, m.p. 108–110 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.32 (dd, J = 8.0, 1.3 Hz, 1H), 8.06 (s, 1H), 7.83–7.77 (m, 1H), 7.75–7.70 (m, 1H), 7.60–7.54 (m, 1H), 7.40 (t, J = 5.4 Hz, 1H), 7.25–7.19 (m, 2H), 7.08–7.02 (m, 2H), 7.01–6.94 (m, 3H), 6.76–6.70 (m, 2H), 4.46 (d, J = 5.8 Hz, 2H), 2.31 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 160.5, 160.4, 148.5, 146.1, 145.2, 137.4, 135.0, 133.6, 129.4, 129.3, 128.6, 128.0, 127.6, 127.5, 122.5, 122.4, 114.6, 43.5, 21.1. HRMS (ESI) m/z calcd for C₂₃H₂₁N₄O₂ [M + H]⁺ 385.1659 found 385.1646.

N-(3-Methylbenzyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Cj)

White solid, 257 mg, 86% yield, m.p. 174–175 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.33 (t, J = 6.1 Hz, 1H), 9.26 (s, 1H), 8.19–8.12 (m, 1H), 7.97–7.90 (m, 1H), 7.85–7.78 (m, 1H), 7.66–7.59 (m, 1H), 7.22–7.15 (m, 2H), 7.09 (t, J = 7.4 Hz, 1H), 7.05–6.96 (m, 3H), 6.88–6.78 (m, 3H), 4.52–4.25 (m, 2H), 2.17 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.2, 159.6, 153.6, 147.2, 146.5, 138.1, 137.3, 135.2, 128.7, 128.1, 127.8, 127.6, 127.4, 127.4, 126.4, 124.0, 122.0, 120.5, 113.6, 41.7, 20.9. HRMS (ESI) m/z calcd for C₂₃H₂₀N₄NaO₂ [M + Na]⁺ 407.1478 found 407.1476.

N-(2-Methylbenzyl)-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Ck)

White solid, 186 mg, 81% yield, m.p. 174–175 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.26 (s, 1H), 9.22 (t, J = 5.9 Hz, 1H), 8.16 (J = 1.5 Hz, 1H), 7.97–7.89 (m, 1H), 7.85–7.78 (m, 1H), 7.66–7.58 (m, 1H), 7.21–7.14 (m, 3H), 7.14–7.09 (m, 2H), 7.01–6.94 (m, 1H), 6.87–6.82 (m, 1H), 6.81–6.75 (m, 2H), 4.47–4.28 (m, 2H), 2.22 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.1, 159.6, 153.6, 147.1, 146.5, 135.8, 135.5, 135.2, 129.7, 128.7, 127.8, 127.6, 127.2, 126.9, 126.4, 125.7, 122.0, 120.4, 113.5, 40.1, 18.5. HRMS (ESI) m/z calcd for C₂₃H₂₁N₄O₂ [M + H]⁺ 385.1659 found 385.1644.

4-Oxo-N-phenethyl-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Cl)

White solid, 220 mg, 96% yield, m.p. 177–178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.15 (s, 1H), 8.95 (t, J = 5.7 Hz, 1H), 8.14 (dd, J = 7.9, 1.5 Hz, 1H), 7.96–7.89 (m, 1H), 7.84–7.78 (m, 1H), 7.65–7.58 (m, 1H), 7.27–7.21 (m, 2H), 7.20–7.14 (m, 5H), 6.88–6.82 (m, 1H), 6.81–6.75 (m, 2H), 3.37–3.26 (m, 2H), 2.63 (q, J = 6.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.6, 160.1, 154.0, 147.7, 147.0, 139.5, 135.7, 129.2, 129.1, 128.8, 128.3, 128.1, 126.9, 126.6, 122.5, 121.0, 114.2, 40.8, 35.2. HRMS (ESI) m/z calcd for C₂₃H₂₁N₄O₂ [M + H]⁺ 385.1659 found 385.1659.

4-Oxo-N-pentyl-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Cm)

White solid, 202 mg, 88% yield, m.p. 69–71 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.36–8.27 (m, 1H), 7.83–7.79 (m, 1H), 7.78–7.73 (m, 1H), 7.59–7.54 (m, 1H), 7.23–7.18 (m, 2H), 7.17–7.12 (m, 1H), 6.97–6.91 (m, 1H), 6.76–6.70 (m, 2H), 3.31 (q, J = 6.8 Hz, 2H), 1.49–1.42 (m, 2H), 1.28–1.21 (m, 2H), 1.20–1.45 (m, 2H), 0.83 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 160.6, 160.4, 148.7, 146.1, 145.3, 134.9, 129.2, 128.5, 127.9, 127.5, 122.5, 122.4, 114.6, 39.9, 28.8, 28.7, 22.2, 13.9. HRMS (ESI) m/z calcd for C₂₀H₂₃N₄O₂ [M + H]⁺ 351.1816 found 351.1810.

N-Hexyl-4-oxo-3-(phenylamino)-3,4-dihydroquinazoline-2-carboxamide (5Cn)

White solid, 288 mg, 82% yield, m.p. 113–114 °C. ¹H NMR (500 MHz, CD₃OD) δ 8.24–8.18 (m, 1H), 7.92–7.85 (m, 1H), 7.82–7.76 (m, 1H), 7.63–7.56 (m, 1H), 7.22–7.16 (m, 2H), 6.93–6.84 (m, 1H), 6.81–6.75 (m, 2H), 3.31–3.16 (m, 2H), 1.47–1.41 (m, 2H), 1.27–1.16 (m, 6H), 0.86 (t, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 162.2, 160.6, 153.2, 146.7, 146.6, 135.0, 128.7, 127.8, 127.4, 126.3, 122.2, 121.1, 113.5, 39.0, 31.3, 28.7, 26.1, 22.2, 13.0. HRMS (ESI) m/z calcd for C₂₁H₂₅N₄O₂ [M + H]⁺ 365.1972, found 365.1972.

3-((4-Chlorophenyl)amino)-N-(2-(dimethylamino)ethyl)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Da)

White solid, 169 mg, 99% yield, m.p. 178–180 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.25–8.19 (m, 1H), 7.94–7.86 (m, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.65–7.58 (m, 1H), 7.22–7.16 (m, 2H), 6.80–6.73 (m, 2H), 3.45 (s, 2H), 2.50 (t, J = 6.7 Hz, 2H), 2.29 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 163.5, 161.8, 154.0, 147.8, 147.0, 136.5, 129.9, 129.3, 128.8, 127.8, 127.2, 123.6, 116.4, 58.5, 45.3, 37.9. HRMS (ESI) m/z calcd for C₁₉H₂₁N₅O₂Cl [M + H]⁺ 386.1378, found 386.1380.

3-((4-Chlorophenyl)amino)-N-(3-(dimethylamino)propyl)-4-oxo-3,4-dihydro-quinazoline-2-carboxamide (5Db)

White solid, 157 mg, 90% yield, m.p. 113–115 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.25–8.19 (m, 1H), 7.94–7.87 (m, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.64–7.58 (m, 1H), 7.23–7.16 (m, 2H), 6.81–6.74 (m, 2H), 3.48–3.32 (m, 1H), 2.33–2.25 (m, 2H), 2.18 (s, 6H), 1.73–1.63 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 163.6, 161.8, 154.3, 147.9, 147.1, 136.5, 130.0, 129.3, 128.8, 127.8, 127.3, 123.6, 116.4, 57.7, 45.2, 38.5, 27.7. HRMS (ESI) m/z calcd for C₂₀H₂₃ClN₅O₂ [M + H]⁺ 400.1535, found 400.1533.

N-(2-(Dimethylamino)ethyl)-3-((4-fluorophenyl)amino)-4-oxo-3,4-dihydroquinazoline-2-carboxamide (5Ea)

White solid, 160 mg, 94% yield, m.p. 100–102 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.23–7.57 (m, 1H), 7.93–7.87 (m, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.64–8.19 (m, 1H), 6.98–6.92 (m, 2H), 6.83–6.77 (m, 2H), 3.47 (t, J = 6.7 Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 2.32 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 163.6, 162.0, 159.5 (d, J = 236.5 Hz), 154.1, 147.9, 144.35 (d, J = 2.3 Hz), 136.4, 129.0 (d, J = 50.1 Hz), 127.8, 123.6, 116.6 (d, J = 7.8 Hz), 116.5, 116.3, 58.5, 45.2, 37.8. HRMS (ESI) m/z calcd for C₁₉H₂₁FN₅O₂ [M + H]⁺ 370.1674, found 370.1685.

N-(3-(Dimethylamino)propyl)-3-((4-fluorophenyl)amino)-4-oxo-3,4-dihydro-quinazoline-2-carboxamide (5Eb)

Yellow liquid, 167 mg, 95% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.24–8.18 (m, 1H), 7.93–7.86 (m, 1H), 7.82–7.75 (m, 1H), 7.64–7.57 (m, 1H), 6.99–6.91 (m, 2H), 6.86–6.78 (m, 2H), 3.41–3.32 (m, 2H), 2.31–2.23 (m, 2H), 2.15 (s, 6H), 1.73–1.63 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 163.7, 161.9, 159.5 (d, J = 236.4 Hz), 154.4, 148.0, 144.50 (d, J = 2.3 Hz), 136.4, 129.0 (d, J = 42.5 Hz), 127.7, 123.6, 116.6, 116.5 (d, J = 15.2 Hz), 57.8, 45.3, 38.6, 27.8. HRMS (ESI) m/z calcd for C₂₀H₂₃FN₅O₂ [M + H]⁺ 384.1830, found 384.1816.

N-(2-(Dimethylamino)ethyl)-4-oxo-3-(p-tolylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Fa)

White solid, 172 mg, 98% yield, m.p. 108–110 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.25–8.17 (m, 1H), 7.92–7.86 (m, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.63–7.56 (m, 1H), 7.01 (d, J = 8.1 Hz, 2H), 6.70–6.63 (m, 2H), 3.46–3.39 (m, 2H), 2.40 (t, J = 6.8 Hz, 2H), 2.23 (s, 3H), 2.21 (s, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 163.6, 162.1, 154.2, 147.9, 145.6, 136.4, 132.0, 130.5, 129.2, 128.8, 127.8123.6, 115.1, 58.5, 45.4, 38.1, 20.6. HRMS (ESI) m/z calcd for C₂₀H₂₄N₅O₂ [M + H]⁺ 366.1925, found 366.1918.

N-(3-(Dimethylamino)propyl)-4-oxo-3-(p-tolylamino)-3,4-dihydroquinazoline-2-carbox-amide (5Fb)

White solid, 122 mg, 70% yield, m.p. 111–113 °C. ¹H NMR (400 MHz, DMSO−d₆) δ 9.02 (s, 1H), 8.83 (t, J = 5.7 Hz, 1H), 8.16–8.08 (m, 1H), 7.96–7.88 (m, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.64–7.56 (m, 1H), 6.96 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 3.30–3.10 (m, 2H), 2.30 (t, J = 7.2 Hz, 2H), 2.18 (s, 3H), 2.16 (s, 6H), 1.62–1.49 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.1, 159.6, 153.7, 146.5, 144.8, 135.1, 129.2, 129.1, 127.7, 127.6, 126.3, 122.0, 113.7, 55.6, 44.2, 36.4, 25.9, 20.1. HRMS (ESI) m/z calcd for C₂₁H₂₆N₅O₂ [M + H]⁺ 380.2081, found 380.2087.

4.3. Biological assay methods

4.3.1. Cell culture

RAW 264.7 cells (Procell Life Science & Technology Co., Ltd., China) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), penicillin (100 U mL⁻¹) and streptomycin (100 μg mL⁻¹), at 37 °C in a humidified incubator containing 5% CO₂.

4.3.2. MTT assay for cell viability

The cytotoxic effects were analyzed by an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. RAW 264.7 cells were seeded in 96-well plates (2.0 × 10⁴ cells per well) and treated with a final concentration of 100 μM with the compounds. After treating for 48 h, an MTT (Solarbio, IM0280) solution was added to each well, and the cells were further incubated at 37 °C for 4 h. The reaction was stopped by adding dimethyl sulfoxide (DMSO), and the optical density (OD) was recorded at 570 nm using a microplate reader (CYTATION5, Bio-Tek, USA). Cell viability was calculated using the following formula: cell viability (%) = (A_570e/A_570c) × 100%, A_570e and A_570c represent the absorbance values from the experimental and control groups, respectively. Each test was repeated at least three times.

4.3.3. Measurement of nitric oxide (NO) production

Nitric oxide production in the cell supernatant was evaluated spectrophotometrically by measuring the amount of nitrite produced, which is the oxidative product of nitric oxide. RAW 264.7 cells were cultured in 96-well plates (2 × 10⁴ cells per well) for one night. The cells were pretreated with different concentrations of compounds for 1 h, and then induced with or without LPS (1 μg mL⁻¹) for 24 h. Equal amounts of cell supernatant (50 μL) were mixed with 50 μL Griess reagent I and II (Beyotime, S0023, China). The nitrite production was determined by measuring the optical density using a microplate reader (CYTATION5, Bio-Tek, USA) at 540 nm and the sodium nitrite standard curve was used to determine nitrite concentrations in the supernatant. Three experiments were conducted independently.

4.3.4. Western blot analysis

Protein levels were determined by the standard Western blot. RAW 264.7 cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells per well. The attached cells were exposed to different concentrations of 5Ci for 1 h, followed by LPS treatment (1 μg mL⁻¹) for 24 h. Then cells were harvested and lysed with a RIPA lysis buffer for 30 min, and then centrifuged at 12 000 rpm at 4 °C for 15 min to extract the total proteins. The protein was separated by using SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010, USA). The PVDF membranes were blocked with 5% BSA and incubated with primary antibodies (GAPDH (Affinity, AF7021, 1 : 1000); γ-H2AX (Beyotime, AF5836, 1 : 500); p-ERK (Affinity, AF1015, 1 : 1000); ERK (Abcam, ab184699, 1 : 10000); p-JNK (Abcam, ab124956, 1 : 5000); JNK (Abcam, ab199380, 1 : 2500); p-p38 (Abcam, ab195049, 1 : 1000); p38 (Abcam, ab170099, 1 : 2500); p-IκB (Abcam, ab133462, 1 : 10000), p-p65 (Abcam, ab76302, 1 : 1000), p65 (Abcam, ab32536, 1 : 1000); COX-2 (Abcam, ab179800, 1 : 5000); iNOS (Abcam, ab178945, 1 : 1000)) at 4 °C overnight, and GAPDH was used as the internal standard. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 2 h. Finally, the intensity of protein bands was detected using an ECL Western blot system (Tanon 5200). The relative optical density value for protein expression was analyzed using the ImageJ software.

4.3.5. RNA extraction and qPCR

RAW 264.7 cells were pretreated with 5Ci of different concentrations for 1 h, and then induced with or without LPS (1 μg mL⁻¹) for 24 h. The cells were collected and total RNA of the cells was isolated using a Trizol reagent (Invitrogen, Grand Island, NY, USA, REF 15596026). cDNA was synthesized from the isolated RNA (1000 ng) using MonScript™ RTlll All-in-One Mix with dsDNase (Monnad, China, REF: MR05101M) according to the manufacturer's instructions. Real-time PCR was conducted using a MonAmp™ SYBR® Green qPCR Mix (Low ROX) (Monnad, China, REF: MQ10201S), according to the manufacturer's instructions. The PCR cycle was 5 min at 95 °C, 40 cycles 10 s at 95 °C, 34 s at 60 °C, 30 s at 72 °C and stopped by heating at 95 °C for 14 min, finally holding at 4 °C. The comparative CT method was used to determine the relative expression normalized by different inflammatory factors.

The sequences of the primers are listed as follows:

GAPDH-F: 5′-GGTTGTCTCCTGCGATTCA-3′;

GAPDH-R: 5′-TGG CCAGGGTTTCTTACTCC-3′;

IL-6-F: 5′-TGGGACTGATGCTGGTGACA-3′;

IL-6-R: 5′-ACAGGTCTGTTGGGAGTGGT-3′;

IL-1β-F: 5′-ATCTCGCAGCAGCACATCAA-3′;

IL-1β-R: 5′-AGTTCAGGAACAGTTGCCAT-3′;

TNF-α-F: 5′-GCGACGTGGAACTGGCAGAAG-3′;

TNF-α-R: 5′-ACAAGCAGGAATGAGAAGAGG-3′.

GAPDH was used as an internal standard.

4.3.6. Measurement of reactive oxygen species (ROS) generation

RAW 264.7 macrophage cells were plated in 6-well plates at a density of 3 × 10⁵ cells per well for one night. Different concentrations of 5Ci were pretreated for 1 h before exposure to 1 μg mL⁻¹ LPS. After 24 h, cells were washed with PBS, fixed with 4% paraformaldehyde and stained with DCFH-DA. The ROS level were analyzed by fluorescence microscopy (CYTATION5, Bio Tek, USA).

4.3.7. Network pharmacology analysis

The putative direct target of 5Ci was predicted using the PharmMapper database (https://lilab-ecust.cn/pharmmapper/index.html) according to the instructions.⁴⁶ The complete screening results were archived in a publicly accessible result repository and can be queried at: https://lilab-ecust.cn/pharmmapper/results/250219084945.html. Job ID: 250219084945.

Discovery Studio (vision 2017R2, BIOVIA, USA) was employed to carry on molecular docking. The crystal structure of HK2 (PDB: 5HG1) was downloaded from the Protein Data Bank (https://www.rcsb.org). The structure was preprocessed (removing water molecules and heteroatoms). The CHARMm force field was applied, and the protein was defined as the receptor. The 3D structure of 5Ci was energy-minimized and docked into HK2 using the CDOCKER protocol, respectively.

Conflicts of interest

There is no conflict of interest to declare.

Data availability

The data supporting this article have been included as part of the Supplementary Information.