Discovery of quinazolin-4-one-based non-covalent inhibitors targeting the severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2 M^pro)

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a great threat to public health while various vaccines are available worldwide. Main protease (M^pro) has been validated as an effective anti-COVID-19 drug target. Using medicinal chemistry and rational drug design strategies, we identified a quinazolin-4-one series of nonpeptidic, noncovalent SARS-CoV-2 M^pro inhibitors based on baicalein, 5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one. In particular, compound C7 exhibits superior inhibitory activity against SARS-CoV-2 M^pro relative to baicalein (IC₅₀ = 0.085 ± 0.006 and 0.966 ± 0.065 μM, respectively), as well as improved physicochemical and drug metabolism and pharmacokinetics (DMPK) properties. In addition, C7 inhibits viral replication in SARS-CoV-2-infected Vero E6 cells more effectively than baicalein (EC₅₀ = 1.10 ± 0.12 and 5.15 ± 1.64 μM, respectively) with low cytotoxicity (CC₅₀ ＞ 50 μM). An X-ray co-crystal structure reveals a non-covalent mechanism of action, and a noncanonical binding mode not observed by baicalein. These results suggest that C7 represents a promising lead for development of more effective SARS-CoV-2 M^pro inhibitors and anti-COVID-19 drugs.

Graphical abstract

1. Introduction

The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has heavily impacted the global economy and threatened public health. According to the World Health Organization (WHO) report, there have been more than 645 million confirmed cases of COVID-19 worldwide, including more than 6.6 million deaths, as of 13 December 2022 [1]. Since the outbreak of COVID-19, enormous global efforts from academic and pharmaceutical companies have been made to discover preventive and therapeutic strategies to fight against this life-threatening disease [2,3]. To date, more than 20 effective SARS-CoV-2 vaccines have been made available globally, and play an important preventive role in controlling the COVID-19 pandemic. Nevertheless, a large number of people have not yet been vaccinated due to personal unwillingness or limited medicinal conditions; on the other hand, increasing numbers of breakthrough infections have been observed in convalescent and/or vaccinated populations, mainly due to decline in protective efficacy of vaccines with time since vaccination, or compromised effectiveness against the emerging omicron variants and subvariants [[4], [5], [6], [7], [8]]. Accordingly, the public are still at high risk of being infected, and effective antiviral drugs against SARS-CoV-2 and its emerging variants are still highly needed.

A large number of anti-COVID-19 drug targets have been reported, with SARS-CoV-2 main protease [M^pro; also known as 3C-like (3CL) protease or nonstructural protein 5 (nsp5)] capturing much attention. M^pro has been validated as an effective target for development of orally available small-molecule antiviral drugs [9,10]. M^pro cleaves viral polyproteins at 11 distinct sites to release functional non-structural proteins (nsps) that are essential for viral replication. Inhibition of M^pro is therefore able to block the viral replication and shut down the viral life cycle. Moreover, M^pro features a unique substrate specificity of glutamine (Gln) at the P1 position and no homologous human proteases have been known, and hence M^pro inhibitors are likely to cause no side-effects by interfering with host proteases. In contrast, M^pro are highly conserved among various coronaviruses, such as SARS-CoV-2, SARS-CoV and MERS, making it a promising target for development of broad-spectrum coronavirus antivirals. Notably, M^pro inhibitors should be effective against all the emerging variants of SARS-CoV-2, since the mutations occurring in spike proteins of variant strains cannot affect M^pro, and M^pro itself was shown to have an extremely low mutation rate in the emerging SARS-CoV-2 variants [[10], [11], [12]]. To date, a large number of SARS-CoV-2 M^pro inhibitors have been reported, and are mainly divided into two types: peptide-like covalent inhibitors represented by nirmatrelvir (PF-07321332, 1) [13], PF-00835231 (2) [14], PF-07304814 (3) [15], GC-376 (4) [16], MI-09 (5), MI-30 (6) [17] and compounds (7–9) [18,19], as well as nonpeptidic, noncovalent inhibitors represented by Ensitrelvir (S-217622, 10) [11], ML-188 (11), compound 12 [20], HL-3-68 (13) [21], baicalein (14) [22] and compounds 15–17 [[23], [24], [25]] (Fig. 1 ). Potential off-target effects, low membrane permeability and poor metabolic stability pose challenges for the extensive clinical application of peptidelike covalent inhibitors, notwithstanding the approval of Paxlovid (a combination of PF-07321332 and ritonavir) by the US Food and Drug Administration (FDA). The nonpeptidic, noncovalent M^pro inhibitor seems to be a more attractive modality. Ensitrelvir (S-217622) developed by Shionogi & Co., Ltd. showed robust antiviral potency against SARS-CoV-2 and its variants, excellent selectivity for M^pro over host proteases, as well as an outstanding DMPK profile in preclinical models [11]. Ensitrelvir (S-217622) exhibited promising single-agent antiviral potency and safety profiles in phase II/III clinical trials [26]. More recently, it was approved for clinical application by the Pharmaceuticals and Medical Device Agency (PMDA). But effective nonpeptidic, non-covalent M^pro inhibitors are very limited, and it is still necessary to search for more non-covalent M^pro inhibitors with diverse chemical scaffolds and improved properties.

Fig. 1.

Chemical structures of representative peptide-like covalent, and nonpeptidic, noncovalent SARS-CoV-2 M^pro inhibitors.

Here, we describe our discovery of quinazolin-4-one-based nonpeptidic, non-covalent inhibitors of SARS-CoV-2 M^pro that are derived from baicalein (14). Baicalein was the first reported nonpeptidic, noncovalent inhibitor of SARS-CoV-2 M^pro discovered by Shanghai Institute of Materia Medica, Chinese Academy of Sciences [22], and its good biochemical and cellular antiviral activity captured our interest. Nevertheless, the potency of baicalein requires further structural optimization and extensive structure activity relationship (SAR) studies. Moreover, the drug metabolism and pharmacokinetics (DMPK) properties and target specificity were largely unknown. In order to fill in gaps in this area and explore the potential of this series as a lead for the development of anti-COVID-19 drugs, we firstly used a scaffold hopping strategy turning baicalein's chromen-4-one core to the alternative privileged scaffold, quinazolin-4-one. In this way, we discovered the first quinazolin-4-one-based SARS-CoV-2 M^pro inhibitor, and further structural optimizations allowed us to produce quinazolin-4-one-based SARS-CoV-2 M^pro inhibitors that are superior to baicalein in terms of biochemical potency, cellular antiviral activity and DMPK profile. An X-ray cocrystal structure of D8 in SARS-CoV-2 M^pro revealed a ligand-induced conformation change, which allows the sec-butyl moiety of D8 to occupy a newly formed binding site between the canonical S1′ and S2 subpockets, suggesting a binding mode to M^pro of quinazolin-4-one-based SARS-CoV-2 M^pro inhibitors that is different from that of baicalein.

2. Results and discussion

2.1. Design of the quinazolin-4-one class of SARS-CoV-2 M^pro inhibitors

The active site of M^pro is composed of five subpockets, S4–S1′, which can accommodate substrate and inhibitor groups at positions P4–P1′, with a Cys145-His41 catalytic dyad. As summarized by researchers from Shionogi Pharmaceutical in a recent publication, the pharmacophore based on the known M^pro inhibitors generally includes: (i) a hydrogen acceptor that interacts with the side-chain NH of His163 in the S1 subpocket, (ii) a hydrogen acceptor that forms a hydrogen bond with the main-chain NH of Glu166, and (iii) a lipophilic group in the S2 subpocket [11]. As shown in Fig. 2 A, the cocrystal structure of baicalein (14) in SARS-CoV-2 M^pro (PDB code: 6M2N [22]) showed that baicalein follows this pharmacophore model. Specially, three phenolic hydroxyl groups in baicalein form a hydrogen-bond network with the side chains of Ser144/His163 and main chains of Leu141/Gly143 directly or indirectly through water molecules. The carbonyl group at C4 position forms a critical hydrogen bond with the Glu166. Accordingly, three phenolic hydroxyl groups and C4 carbonyl group are necessary for the potency, and therefore were retained in our design. The free phenyl ring (C ring) occupies the S2 subpocket with extensive hydrophobic interactions. The core chromen-4-one forms many key interactions contributing to baicalein's binding affinity with protein: (i) the A ring forming S–π and NH₂–π with the catalytic Cys145 and Asn142, respectively; (ii) the B ring π–π stacking with catalytic His41, and forming hydrophobic interactions with Met165. In addition, the chromen-4-one motif acts as a scaffold to assemble these necessary components. We envisioned that other aromatic heterocycles with similar structure could replace the chromen-4-one structure in baicalein. Because quinazolin-4-one and quinolin-4-one have been often seen in many natural products, biologically active compounds and clinically used drugs, together with well-established efficient synthesis and functionalization methodologies of these heterocycles, we firstly used a scaffold hopping strategy to replace the core chromen-4-one in baicalein with quinazolin-4-one or quinolin-4-one, respectively, in order to discover structurally novel non-covalent M^pro inhibitors. The resulting quinolin-4-one-based compound 18 had a complete loss of M^pro enzymatic inhibitory activity, while quinazolin-4-one-based compound 19 exhibited comparable inhibitory activity with that of baicalein. Therefore, we proceeded with 19 as a lead compound. A step-by-step optimization strategy and SAR studies of substituent groups at the C2 and N3 positions of quinazolin-4-one were conducted (Fig. 2B). Finally, the optimal substituents at the C2 and N3 positions were integrated, leading to a series of potent SARS-CoV-2 M^pro inhibitors (Fig. 2B).

Fig. 2.

Rational design of non-covalent SARS-CoV-2 M^pro inhibitors. (A) Binding pocket of baicalein in SARS-CoV-2 M^pro (PDB code 6M2N). (B) Detailed binding mode of baicalein complexed with M^pro. The protein is shown in gray cartoon, baicalein in blue sticks, and the selected residues in yellow sticks. Hydrogen bonds are indicated as gray dashes, and water molecule as red sphere. S–π, NH₂–π and π–π stacks are indicated as green dashes. (C) Step-by-step optimization strategy of non-covalent SARS-CoV-2 M^pro inhibitors starting from baicalein.

2.2. Chemistry

As shown in Scheme 1 , the quinolin-4-one derivative 18 was obtained by the base-mediated cyclization of N-(ketoaryl)amide (the Camps cyclization) according to the published literature [27]. Condensation of compound 20 and benzoyl chloride afforded benzamide (21). The Friedel-Crafts acylation of 21 using acetyl chloride in the presence of SnCl₄ gave the cyclization precursor (22) [28], which was then cyclized at 110 °C in the presence of KOH to generate compound 23 [27], and this was followed by BBr₃-mediated demethylation to yield the desired product (18).

Scheme 1.

Synthesis of compound 18. Reagents and conditions: (a) benzoyl chloride, TEA, THF, 0 °C to rt, 2 h, 91%; (b) acetyl chloride, SnCl₄, anhydrous DCM, 0 °C to rt, 5 h, 45%; (c) KOH, anhydrous 1,4-dioxane, reflux, 4 h, 35%; (d) BBr₃/DCM, DCM, −10 °C to rt, 24 h, 75%.

There are many efficient ways to construct quinazolin-4-one derivatives. We selected the appropriate synthetic method for each target compound based on the availability of raw materials. Compounds A1–A6, A8–A12, A14, A16, A18, A19, A21–A27 and 19 were obtained in two steps: (i) the reaction of key intermediate 24 [29] with various amidine hydrochlorides 25a–25w that are commercially available or easily synthesized in efficient mild copper-catalyzed conditions to yield compounds 26a–26w [30]; (ii) BBr₃-mediated demethylation of compounds 26a–26w (Scheme 2 ). A one-pot I₂-mediated oxidative cyclization of o-anthranilamide 27 [31] and various commercially available aldehydes 28a–28v [32] and subsequent demethylation were used to synthesize compounds A7, A13, A15, A17, A20 and compounds B1–B17 (Scheme 3 ). As depicted in Scheme 4 , the N ₃ substituted quinazolin-4-one derivatives C1–C15 were prepared by condensation of the prepared o-anthranilamides 30a–30o with benzaldehyde under similar reaction conditions with B series compounds followed by demethylation. Compounds D6–D9 were obtained by the oxidative cyclization approach that was used to access compounds in the B and C series in a good yield (Scheme 7), but compounds D1–D5 and D10–D12 could not be synthesized under the same conditions. D1–D4, D10 and D11 were successfully achieved by cyclization of N-acylanthranilic acids and amines in the presence of PCl₃ in a good yield (Scheme 5 ) [33]. However, N ₃-alkyl substituted compounds D5 and D12 could not be prepared with the synthetic method shown in Scheme 5, and these were obtained by a one-pot, two step cyclization: (i) the reaction of compound 36 with the prepared 2-phenylpropanoyl chloride or commercially available phenylacetyl chloride in the presence of triphenyl phosphite (TPP) and pyridine, and (ii) the substitution with isobutylamine [34], followed by demethylation (Scheme 6 ).

Scheme 2.

Synthesis of compounds A1–A6, A8–A12, A14, A16, A18, A19, A21–A27 and 19. Reagents and conditions: (a) CuI, CsCO₃, DMF, rt, overnight, 55%–90%; (b) BBr₃/DCM, DCM, −10 °C to rt, 24–36 h, 35%–64%.

Scheme 3.

Synthesis of compounds A7, A13, A15, A17, A20, B1–B17. Reagents and conditions: (a) I₂, EtOH, reflux, 3–5 h, 40%–80%; (b) BBr₃/DCM, DCM, −10 °C to rt, 24–36 h, 40%–63%.

Scheme 4.

Synthesis of compounds C1–C15. Reagents and conditions: (a) I₂, EtOH, reflux, 3–5 h, 45%–87%; (b) BBr₃/DCM, DCM, −10 °C to rt, 24–36 h, 35%–57%.

Scheme 7.

Synthesis of compounds D6–D9. Reagents and conditions: (a) I₂, EtOH, reflux, 5 h, 72%–88%; (b) BBr₃/DCM, DCM, −10 °C to rt, 24 h, 55%–56%.

Scheme 5.

Synthesis of compounds D1–D4, D10 and D11. Reagents and conditions: (a) PCl₃, 50 °C, 4–12 h, 70%–82%; (b) BBr₃/DCM, DCM, −10 °C to rt, 24–36 h, 45%–63%.

Scheme 6.

Synthesis of compounds D5 and D12. Reagents and conditions: (a) For 37a: (i) 2-phenylpropanoic acid (38), (COCl)₂, DCM, cat. DMF, 0 °C to rt, 2 h; (ii) triphenyl phosphite (TPP), Py, 0 °C–70 °C, 2 h; iii) isobutylamine (40), 70 °C, overnight, 40%; (b) For 37b: (i) phenylacetyl chloride (39), TPP, Py, 0 °C–70 °C, 2 h; (ii) isobutylamine (40), 70 °C, overnight, 45%; (c) BBr₃/DCM, DCM, −10 °C, 24 h, 50%–60%.

2.3. SAR studies of the quinazolin-4-one class of SARS-CoV-2 M^pro inhibitors

All target compounds were preliminarily evaluated for inhibitory activity against SARS-CoV-2 M^pro in fluorescence resonance energy transfer (FRET)-based enzymatic assays. As shown in Table 1 , quinolin-4-one-based compound 18 had a complete loss of enzymatic inhibitory activity against SARS-CoV-2 M^pro. To our delight, quinazolin-4-one-based compound 19 was only slightly less potent than baicalein (IC₅₀ = 1.372 ± 0.047 μM and 0.966 ± 0.065 μM, respectively). With the promising potency for compound 19 in hand, we firstly performed the SAR analysis of the substituents on the free phenyl group (C ring) in 19. Generally, the positions on the phenyl ring led to this order of potency: 2′-position > 3′-position > 4′-position (Table 1). In particular, compounds with fluorine (F), trifluoromethyl (CF₃), trifluoromethoxy (OCF₃) or tert-butyl at the C2′ position exhibited much more potency than the corresponding compounds with 3′ and/or 4′ substituents (A1 vs A2 and A3, A13 vs A14, A15 vs A16, A21 vs A22). The significant influence of the substituent position on activity can also be concluded from the difluorine-substituted compounds: 2′,3′-difluoro (A25, IC₅₀ = 1.130 ± 0.031 μM) > 2′,4′-difluoro (A26, IC₅₀ = 1.541 ± 0.042 μM) > 3′,4′-difluoro (A27, IC₅₀ = 2.716 ± 0.051 μM).

Table 1.

SAR exploration of the substituent groups on the C ring of quinazolin-4-one ^a.

Compd.	R1	IC50 (μM)
A1	2′-F	1.443 ± 0.060
A2	3′-F	5.132 ± 0.094
A3	4′-F	12.760 ± 0.067
A4	2′-Cl	0.435 ± 0.041
A5	3′-Cl	6.960 ± 0.076
A6	4′-Cl	7.706 ± 0.055
A7	2′-Br	0.554 ± 0.041
A8	3′-Br	8.017 ± 0.033
A9	4′-Br	9.797 ± 0.051
A10	2′-methyl	0.365 ± 0.033
A11	3′-methyl	2.277 ± 0.029
A12	4′-methyl	1.867 ± 0.034
A13	2′-CF3	1.481 ± 0.047
A14	3′-CF3	10.53 ± 0.050
A15	2′-OCF3	1.798 ± 0.061
A16	4′-OCF3	＞20
A17	2′-NO2	10.588 ± 0.040
A18	3′-NO2	11.169 ± 0.034
A19	4′-NO2	＞20
A20	3′-isopropyl	7.635 ± 0.058
A21	3′-tert-butyl	8.423 ± 0.031
A22	4′-tert-butyl	＞20
A23	2′-OH	18.285 ± 0.040
A24	4′-OH	＞20
A25	2′,3′-di-F	1.130 ± 0.031
A26	2′,4′-di-F	1.541 ± 0.042
A27	3,4′-di-F	2.716 ± 0.051
19	–	1.372 ± 0.047
baicalein (14)	–	0.966 ± 0.065

^aBaicalein was used as the positive control. Inhibitory activity against SARS-CoV-2 M^prowas determined with the FRET protease activity assay. Values represent a mean ± SD of at least three independent experiments.

The volume, polarity and electronegativity of the substituent groups also affect the potency significantly. Introduction of a bulky isopropyl (A20) and tert-butyl (A21 and A22), or a polar hydroxyl (A23 and A24) and a nitro group (A17–A19) resulted in a sharp decline in potency. It seemed that less bulky substituents with moderate-to-low electronegativity benefit the inhibitory activity against M^pro. A10 with 2′-methyl and A4 with 2′-chlorine were the two most active compounds among the A series, with IC₅₀ values of 0.365 ± 0.033 μM and 0.435 ± 0.04 μM, respectively. Adding a bromine (Br) atom at the C2′ position, whose van der Waals radius is slightly larger than that of chlorine (Cl) atom and whose electronegativity is lower than that of Cl, led to compound A7, which showed a slightly decreased potency relative to A4 (A7, IC₅₀ = 0.554 ± 0.041 μM), but was more active than 19 and baicalein. The introduction of more electronegative substituents at the C2′ position led to a poorer potency against M^pro, for example, F (IC₅₀ = 1.443 ± 0.060 μM) > CF₃ (IC₅₀ = 1.481 ± 0.047 μM) > nitro group (IC₅₀ = 10.588 ± 0.040 μM). Compound A7 containing an electron-donating trifluoromethoxy (OCF₃) group was slightly less potent than compound A13 with an electron-withdrawing CF₃ (IC₅₀ = 1.798 ± 0.061 μM for A7 vs 1.481 ± 0.047 μM for A13), which might be attributed to the larger volume of OCF₃ than that of CF₃, and the volume effect affected the potency more significantly. Together, these results suggested that the position, steric size, polarity and electronic property of substituents on the C ring integratedly affect the inhibitory activity against SARS-CoV-2 M^pro.

Next, we replaced the C ring with different types of groups in order to diversify the structures of quinazolin-4-one based M^pro inhibitors and search for more desirable substituents at the C2 position. As shown in the Table 2 , the replacement of phenyl (the C ring) with naphthyl and heteroaromatic rings, including thienyl, 3-pyridyl, 4-pyridyl and pyrazolyl, led to compounds B1–B5, and resulted in a remarkable decrease in potency against M^pro. Different cycloalkyl replacements afforded compounds B6–B8. Substituting phenyl with cyclopentyl (B7, IC₅₀ = 0.539 ± 0.061 μM) led to improved potency against M^pro over that of 19 (IC₅₀ = 1.372 ± 0.047 μM) and baicalein (14) (IC₅₀ = 0.966 ± 0.065 μM). B8 with a cyclohexyl (IC₅₀ = 1.370 ± 0.140 μM) was equipotent with 19, while B6 with cyclopropyl (IC₅₀ = 5.485 ± 0.791 μM) remarkably decreased the potency. The phenyl was replaced by different alkyl groups leading to compounds B9–B12. The introduction of isopropyl (B9) and tert-butyl (B10) resulted in a sharp drop of the potency. In contrast, B11 with sec-butyl (IC₅₀ = 0.385 ± 0.024 μM) and B12 with a bulkier tert-amyl (IC₅₀ = 0.970 ± 0.075 μM) were more potent than or comparable to 19, respectively. Subsequently, we explored the effects of styryl and benzyl groups at the C2 position on the inhibitory activity against M^pro. Compound B13 with a rigid styryl (IC₅₀ = 2.874 ± 0.030 μM) led to some loss of potency against M^pro compared to 19. The relatively flexible benzyl group in compound 14 improved the IC₅₀ value to 0.327 ± 0.052 μM. Small groups, such as methyl or ethyl, when introduced to the benzyl in 14 could further increase the activity, and the resulting compounds 15 and 16 exhibited the most potent inhibitory activity against M^pro among B series, with IC₅₀ values of 0.174 ± 0.038 μM and 0.210 ± 0.028 μM, respectively. Addition of a larger isopropyl group (compound 17) led to a slight loss of the potency of 14, with an IC₅₀ value of 0.390 ± 0.048 μM.

Table 2.

SAR exploration of substituent groups at the C2 position of quinazolin-4-one^a.

Compd	R2	IC50 (μM)
B1		＞20
B2		＞20
B3		15.19 ± 0.0405
B4		＞20
B5		3.565 ± 0.295
B6		5.485 ± 0.791
B7		0.539 ± 0.061
B8		1.370 ± 0.140
B9		4.943 ± 0.504
B10		4.086 ± 0.647
B11		0.385 ± 0.024
B12		0.970 ± 0.075
B13		2.874 ± 0.030
B14		0.327 ± 0.052
B15		0.174 ± 0.038
B16		0.210 ± 0.028
B17		0.390 ± 0.048
19		1.372 ± 0.047
baicalein (14)	–	0.966 ± 0.065

^aBaicalein was used as the positive control. Inhibitory activity against SARS-CoV-2 M^prowas determined by using the FRET protease activity assay. Values represent a mean ± SD of at least three independent experiments.

We tentatively kept R₂ as phenyl and explored the SAR at the N3 position (Table 3 ). Generally, C series compounds with different substituents at the N-3 position of quinazolin-4-one exhibited improved inhibitory potency against M^pro compared to compound 19 and baicalein (14), except for compounds C2 with the polar hydroxyethyl (IC₅₀ = 1.365 ± 0.062 μM), C3 with a bulky tert-butyl (0.949 ± 0.077 μM) and C14 with a biphenylyl (IC₅₀ = 1.476 ± 0.117 μM). For cycloalkyl replacements, cyclopentyl was the optimal substituent group, and the corresponding compound C5 showed significantly increased potency relative to 19, with an IC₅₀ value of 0.124 ± 0.014 μM (Table 3, Fig. S1). C1 with a less bulky sec-butyl substituent showed potency as good as C5 (Table 3, Fig. S1). The introduction of phenyl leading to compound C7 improved the IC₅₀ value to 0.085 ± 0.006 μM (Table 3, Fig. S1). Inspired by this result, different substituted phenyl groups were introduced to the N3 position. F, Br and hydroxyl substituents affording compounds C8–C11 retained the most of the potency of C7, showing IC₅₀ values of about 0.2 μM. Compound C12 containing a 3′-methyl-4′-fluorophenyl substituent was equipotent with C7 (IC₅₀ = 0.117 ± 0.016 μM, Table 3, Fig. S1). In addition, adding a 3-pyridyl or 4-pyrazolyl at the N3 position resulted in compounds C13 and C14, which had good inhibitory activity against M^pro (IC₅₀ = 0.212 ± 0.024 and 0.390 ± 0.003 μM, respectively).

Table 3.

SAR exploration of substituent groups at the N3 position of quinazolin-4-one^a.

Compd	R3	IC50 (μM)
C1		0.124 ± 0.018
C2		1.365 ± 0.062
C3		0.949 ± 0.077
C4		0.290 ± 0.028
C5		0.124 ± 0.016
C6		0.274 ± 0.022
C7		0.083 ± 0.006
C8		0.205 ± 0.033
C9		0.236 ± 0.018
C10		0.271 ± 0.018
C11		0.207 ± 0.024
C12		0.117 ± 0.016
C13		1.476 ± 0.117
C14		0.212 ± 0.024
C15		0.390 ± 0.003
19		1.372 ± 0.047
baicalein (14)	–	0.966 ± 0.065

^aBaicalein was used as the positive control. Inhibitory activity against SARS-CoV-2 M^prowas determined by using the FRET protease activity assay. Values represent a mean ± SD of at least three independent experiments.

D series compounds were derived from the combination of the desirable substitutions at the C2 and N3 positions that we have identified (Table 4 ). Unexpectedly, the combination of 2-methyl-benzyl that is the optimal substitution at the C2 position with phenyl and sec-butyl groups that were the favorable substituents at the N3 position resulted in decreased activity compared to the corresponding C series compounds. In particular, compounds D2 (IC₅₀ = 1.005 ± 0.136 μM) and D5 (IC₅₀ = 0.692 ± 0.119 μM) showed a sharp decrease in potency relative to the corresponding compounds C12 (IC₅₀ = 0.117 ± 0.016 μM) and C1 (IC₅₀ = 0.124 ± 0.018 μM). The possible reason for the decreased inhibitory activity was that C₂-2-methyl-benzyl clashed with N₃-phenyl or N₃-sec-butyl, leading one or both of them to be unable to smoothly enter into the corresponding M^pro protein subpockets. N₃-sec-butyl series compounds D6–D9 did not improve the potency compared to C7, with IC₅₀ values ranging from 0.100 to 0.284 μM. The combination of C₂-benzyl with N₃-phenyl resulted in compound D10 being equipotent with C7 (IC₅₀ = 0.103 ± 0.014 μM), while the combination of C₂-benzyl with 3-methyl-4-fluorobenzyl and sec-butyl at the N3 position led to some loss of the potency relative to C7.

Table 4.

SAR exploration of substituent groups at C-2 and N-3 positions of quinazolin-4-one^a.

Compd	R4	R5	IC50 (μM)
D1			0.477 ± 0.078
D2			1.005 ± 0.136
D3			0.290 ± 0.030
D4			0.386 ± 0.017
D5			0.692 ± 0.119
D6			0.107 ± 0.023
D7			0.239 ± 0.028
D8			0.100 ± 0.012
D9			0.284 ± 0.028
D10			0.131 ± 0.022
D11			0.502 ± 0.038
D12			0.466 ± 0.040
C7			0.085 ± 0.006

^aC7 was used as the positive control. Inhibitory activity against SARS-CoV-2 M^prowas determined with the FRET protease activity assay. Values represent a mean ± SD of at least three independent experiments.

2.4. Target validation and selectivity

To examine if the inhibitory activity against M^pro is associated with covalent modification of cysteine residues, we compared the inhibition of C5, C7, D6, D8 and D10 in the presence or absence of the reducing agent dithiothreitol (DTT) (Table S1). Addition of DTT guarantees accessible cysteine residues of a protein at their reduced state and will indicate whether an inhibitor acts by a false mechanism of redox-cycling [24,35]. There were no significant differences in the IC₅₀ values in the presence of DTT and in the absence of DTT.

One of the major limitations encountered by cysteine protease inhibitors is the target selectivity. Compound C7 was selected as an example of our target scaffold to be profiled with the selectivity over several common human proteases, including caspase 2, cathepsin L, thrombin, cathepsin B and cathepsin D. Compound C7 showed no apparent inhibitory activity against these host proteins at a concentration of 10 μM, suggesting that C7 had a good target specificity against M^pro over host proteases (Table S2).

2.5. In vitro DMPK profiling

As shown in Table S3, compounds C7 (Papp AB = 9.67 × 10⁻⁶ cm/s, Efflux radio (ER) = 0.44), D6 (Papp AB = 7.45 × 10⁻⁶ cm/s, ER = 0.40), and D8 (Papp AB = 7.83 × 10⁻⁶ cm/s, ER = 0.41) exhibited improved Madin-Darby canine kidney (MDCK) membrane permeability relative to baicalein (Papp AB = 6.07 × 10⁻⁶ cm/s, ER = 0.34). The kinetic solubility in phosphate buffer solution (PBS, pH = 7.4) of C7 and D6 was measured as 179.35 and 155.98 μM, respectively, which were superior to baicalein (112.18 μM). C7, D6, D8 and baicalein exhibited reasonable plasma protein binding (PPB) in human plasma (fraction unbound fu = 0.70%, 2.61%, 3.17% and 0.93%, respectively). Baicalein exhibited high intrinsic clearance in human liver microsome (HLM), with clearance rate (CL_int) of 333.05 μL/min/mg protein and half-time (T_1/2) of 4.16 min. C7 and D6 showed improved HLM stability relative to baicalein (CL_int = 108.68 and 68.34 μL/min/mg protein, respectively; T_1/2 = 12.75 and 20.28 min). The two compounds showed relatively high HLM metabolic stability in the presence of the cofactor NADPH alone, indicative of mainly undergoing phase II metabolism. With the presence of three phenolic hydroxyl groups in the structures, the above results were not unexpected. Despite the improvement relative to baicalein, the metabolic stability of this quinazolin-4-one series inhibitors requires further optimization.

2.6. Cellular cytotoxicity and antiviral activity

The antiviral activity of compounds C7, D6 and D8 was evaluated in SARS-CoV-2-infected Vero E6 cells. Prior to antiviral assays, the cytotoxicity of selected M^pro inhibitors against Vero E6 cells was evaluated by using cell counting kit-8 (CCK-8) assays. The test compounds exhibited no significant cytotoxicity at the highest tested concentration (50% cytotoxicity concentration, CC₅₀ > 50 μM, Table 5 and Fig. S2). The antiviral activity was determined in a yield reduction assay that assesses the inhibitory activity of the test compounds on the viral replication using quantitative real-time polymerase chain reaction (qRT-PCR), with baicalein as the positive control. As shown in Table 5 and Fig. S2, compounds C7 (EC₅₀ = 1.10 ± 0.12 μM), D6 (EC₅₀ = 2.87 ± 1.43 μM) and D8 (EC₅₀ = 2.11 ± 1.12 μM) showed better antiviral activity in Vero E6 cells than baicalein (EC₅₀ = 5.15 ± 1.64 μM).

Table 5.

Antiviral activity and cytotoxicity of selected compounds in Vero E6 cells.

Compd.	EC50 (μM)a	CC50 (μM)
C7	1.10 ± 0.12	＞50
D6	2.87 ± 1.43	＞50
D8	2.11 ± 1.16	＞50
baicalein	5.15 ± 2.46	＞50

^aInhibitory effect on viral replication induced by SARS-CoV-2 infection in Vero E6 cells (RT-qPCR assay). Values are expressed as mean ± SD from three independent experiments.

2.7. X-ray crystal structure of D8 in complex with SARS-CoV-2 M^pro

To further examine the binding mode of quinazolin-4-one inhibitors in SARS-CoV-2 M^pro, we resolved an X-ray co-crystal structure of D8 complexed with M^pro at a resolution of 2.2 Å (PDB code 8I4S, Fig. 3 A–3C). The X-ray data and refinement statistics are depicted in Table S4. The trihydroxyphenyl moiety of D8 sits in a similar position as in the baicalein/M^pro complex (Fig. 3B and E). Three phenolic hydroxyl groups of D8 form critical hydrogen bond interactions with the main chains of Gly143/Ser144/Gly145, and also form hydrogen bond interactions with the side chain of His163 via a buried water molecule (Fig. 3B). Distinct from the case of baicalein, the C4 carbonyl oxygen atom of D8 was a little aside from Glu166, and forms a hydrogen bond with the backbone NH of Glu166 with the aid of a buried water molecule (Fig. 3B). Unexpectedly, the 3′-methyl-4′-fluorophenyl at the N3 position occupies the S2 pocket, forming hydrophobic interactions with Gln189 and Met49, while the sec-butyl at the C2 position projects into a newly formed binding site that was observed in CCF0058981 analogue (12) complexed with M^pro (PDB code 7LMF), termed the S2_c pocket by Han et al. (Fig. 3D).²⁰ When D8 bound to M^pro, the flexible side chains of some amino acid residues of the protein, particularly Met49 and Gln189, exhibited a ligand-induced conformation change relative to most other inhibitor/M^pro structures (e.g., baicalein, nirmatrelvir and ensitrelvir), further rearranging the binding surface of subpockets S4 and S2 (Fig. 3D and S3). This allows the sec-butyl to occupy the S2c pocket, forming favorable hydrophobic interactions with Cys44, Thr45 and Ser46. As with baicalein, the phenyl ring of D8 with three hydroxyl groups is sandwiched between Cys145 and Asn142 by forming S–π and NH₂-π with Cys145 and Asn142, respectively (Fig. 3B). In addition, the middle ring π–π stacks with catalytic His41, also contributing to the binding affinity of D8 with protein (Fig. 3B).

Fig. 3.

(A) Overview of the co-crystal structure of D8 bound to SARS-CoV-2 M^pro (PDB code 8I4S). The protein is shown in cartoon, and domains I, II and III are colored light orange, magenta and violet, respectively. D8 is shown as spheres with carbons in cyan. (B) Detailed binding mode of compound D8 in complex with SARS-CoV-2 M^pro. The protein is shown in gray cartoon, D8 in cyan sticks, and the selected residues in yellow sticks. Hydrogen bonds are indicated as gray dashes, and the water molecule as a red sphere. S–π, NH₂–π and π–π stacks are indicated as green dashes. (C) Fo−Fc density map (contoured at 3.00σ) around D8 (cyan mesh). (D) Overlay of D8/SARS-CoV-2 M^pro (PDB code 8I4S, cyan) with 12/SARS-CoV-2 M^pro (PDB code 7LMF²⁰, pink) complexes, highlighting the residues Gln189 and Met49. (E) Overlay of D8/SARS-CoV-2 M^pro (cyan) with baicalein/SARS-CoV-2 M^pro (PDB code 6M2N²², blue) complexes, highlighting the residues Gln189 and Met49.

Although highly conserved among variants of SARS-CoV-2, some mutated amino acid residues in M^pro have been observed, such as K90R in Beta strain B.1.351, K90R and A193V in Beta B.1.351.2, L205V in Zeta P.2, and P132H in Omicron B.1.529 [36]. However, it is worth noting that these amino residues are more than 10 Å away from the binding site of D8 (Fig. S4), suggesting that D8 should be have inhibitory effects against M^pro from SARS-CoV-2 variants. The assays assessing the inhibitory activity of the inhibitors against M^pro from SARS-CoV-2 Omicron variant are currently in progress.

3. Conclusions

M^pro has been validated as an effective target for development of orally available small molecule anti-COVID19 drugs. In this study, we sought to use medicinal chemistry and rational drug design approaches to structurally modify the first reported nonpeptidic, noncovalent SARS-CoV-2 M^pro inhibitor, baicalein. These efforts led to a series of quinazolin-4-one-derived noncovalent inhibitors with nanomolar potencies against SARS-CoV-2 M^pro. In particular, an optimized compound, C7 exhibited superior inhibitory potency against M^pro relative to baicalein and is endowed with improved physicochemical and DMPK properties. Significantly, C7 also showed more potent antiviral activity than baicalein (EC₅₀ = 1.1 and 5.15 μM, respectively) in SARS-CoV-2-infected Vero E6 cells. Moreover, C7 exhibited relatively high selectivity over a panel of human proteases (IC₅₀ ＞ 10 μM) and low cytotoxicity against Vero E6 cells (CC₅₀ ＞ 50 μM). The co-crystal structure of another potent inhibitor D8 complexed with M^pro showed that the inhibitor noncovalently binds to the active site of M^pro, and occupies a newly formed S2c pocket that is not observed with baicalein and most other M^pro inhibitors, and can be further exploited for inhibitor design. Meanwhile, the S1 and S4 subpocket remain largely unoccupied by D8 and its analogues, leaving room for further improvement. While having improvements, the inhibitors still lack sufficient properties necessary to be profiled for in vivo antiviral efficacy in SARS-CoV-2-infected animal models. Further structural optimizations of this series of inhibitors are ongoing, and are focused on improving the DMPK properties, as well as further improving biochemical and cellular potencies. In addition, profiling the potency against M^pro from SARS-CoV-2 Omicron variant and other coronavirus is underway in our laboratory. Collectively, compound C7 represents a promising lead for further development of more effective M^pro inhibitors and antiviral drugs against SARS-CoV-2 infection.

4. Experimental

4.1. Chemistry

Reagents and solvents from commercial sources were used without further purification. The progress of all reactions was monitored by TLC using EtOAc/petroleum ether (PE) or dichloromethane (DCM)/MeOH as the solvent system, and spots were visualized by irradiation with UV light (254 nm) or by staining with phosphomolybdic acid. Flash chromatography was performed using silica gel (200−300 mesh). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance ARX-400, a Bruker Avance ARX-500 or a Bruker Avance ARX-600. Chemical shifts δ are reported in ppm, and multiplicity of signals are denoted as: br = broad, s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. The low resolution ESI-MS was recorded on Shimadzu GCMS-2010 instruments and the high resolution mass spectra (HRMS) on a Water Q-Tofmicro mass spectrometer. Anhydrous DCM and N,N-dimethylformamide (DMF) were freshly distilled from calcium hydride. Anhydrous tetrahydrofuran (THF) was freshly distilled over sodium using benzophenone as the indicator. All other solvents were reagent grade. All moisture sensitive reactions were carried out in flame dried flasks under an argon atmosphere. The chemical purity of the target compounds was analyzed by high performance liquid chromatography (HPLC) on an InertSustain C18 column (4.6 mm × 250 mm, 5 μm) under gradient 60–100% MeOH in water (with 0.1% TFA in each mobile phase), with a flow rate of 1.0 mL/min and peak detection at 254 nm. All the target compounds showed purity greater than 95%. The synthesis and compound information of the intermediates can be found in the Supporting Information.

4.1.1. 5,6,7-Trihydroxy-2-phenylquinolin-4(1H)-one(18)

• Step1: To the stirred solution of compound 20 (1.83 g, 10 mmol) in dry THF (25 mL) was added TEA (2 mL, 15 mmol) and benzoyl chloride (1.8 mL, 15 mmol) at 0 °C. Then the reaction mixture was stirred at room temperature (rt) for 2 h. The resulting mixture was concentrated and partitioned between water and DCM. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under the reduced pressure. The resulting residue triturated with n-hexane, filtered, washed with n-hexane, dried in vacuum to afford 21 as a white solid (2.61g, 91%), which was used directly in the next step without further purification.

• Step2: To the stirred solution of compound 21 in anhydrous DCM (30 mL) was successively dropwise added acetyl chloride (0.78 mL, 11 mmol) and SnCl₄ (2 mL, 20 mmol) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 4 h and then moved to room temperature for 1 h, after which the reaction mixture was poured into ice water. The aqueous portion was extracted with DCM (3 × 20 mL), and the combined organics were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica column chromatography and eluted with petroleum ether: EtOAc (20:1) to give compound 22 as a white solid (1.48 g, 45%). ¹H NMR (400 MHz, CDCl₃) δ 12.37 (s, 1H), 8.42 (s, 1H), 8.05–7.99 (m, 2H), 7.59–7.47 (m, 3H), 4.00 (s, 6H), 3.85 (s, 3H), 2.67 (s, 3H). m/z (ESI-MS): 330.2 [M + H ]⁺.

• Step3: To the stirred solution of compound 22 (1.32 g, 4.01 mmol) in anhydrous 1,4-dioxane (40 mL) was added NaOH (480 mg, 12 mmol), and the reaction mixture was heated to reflux under a nitrogen atmosphere for 4 h. Then, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. Next, the residue was treated with water and n-hexane and the resulting mixture was sonicated for approximately 2 min. The resulting suspension was adjusted to pH∼7 with 1 M HCl and filtered. The precipitate obtained was washed with n-hexane and dried under vacuum to afford compound 23 as a white solid (435 mg, 35%). ¹H NMR (400 MHz, CDCl₃) δ 8.07–8.01 (m, 2H), 7.96 (s, 1H), 7.54–7.45 (m, 3H), 7.02 (s, 1H), 4.21 (s, 3H), 4.05 (s, 3H), 3.95 (s, 3H). m/z (ESI-MS): 312.2 [M + H ]⁺.

• Step4: To the stirred suspension of compound 23 (200 mg, 0.64 mmol) in anhydrous DCM (1.5 mL) was dropwise added BBr₃/DCM (9.6 mL, 9.6 mmol, 1 M) at −10 °C under a nitrogen atmosphere. The resulting reaction mixture was stirred at −10 °C overnight and then moved to room temperature for 12 h. Then, the mixture was moved to −10 °C again, and was quenched by slowly adding ice methanol, after which the solvent was removed under reduced pressure. The resulting residue was triturated with water, filtered, washed with water and DCM, dried in vacuum to afford 18 as a pale yellow solid (130 mg, 75%). ¹H NMR (400 MHz, DMSO‑d ₆) δ 14.02 (s, 1H), 11.26 (s, 1H), 9.31 (s, 1H), 8.04–7.95 (m, 2H), 7.67–7.57 (m, 3H), 7.32 (s, 1H), 7.03 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.7, 156.1, 152.6, 139.2, 134.4, 132.2, 131.8, 131.5, 129.1, 128.5, 105.3, 101.1, 94.6. HRMS (ESI): m/z calcd for C₁₅H₁₀NO₄ [M − H]^–: 268.0615, found 268.0610. HPLC analysis: t_R = 10.216 min, 96.3%.

4.1.2. General procedure a for the preparation of A1–A6, A8 – A12, A14, A16, A18, A19, A21 – A27 and 19

Compound 24 (337 mg, 1 mmol) was dissolved in anhydrous DMF (6 mL), and compounds 25a–25w (1.5 mmol), CsCO₃ (652 mg, 2 mmol) and CuI (39 mg, 0.2 mmol) were added. The resulting mixture was stirred at room temperature under nitrogen atmosphere overnight. After completion of the reaction, excess saturated aqueous NH₄Cl was added and filtered. The precipitate obtained was washed with a large amount of water, dried in vacuum to afford trimethoxyquinazolinones 26a–26w.

To the stirred suspension of the obtained trimethoxyquinazolinone (0.5 mmol) in anhydrous DCM (1.5 mL) was dropwise added BBr₃/DCM (5 mL, 6 mmol, 1 M) at −10 °C under a nitrogen atmosphere. The resulting reaction mixture was stirred at −10 °C overnight and then moved to room temperature for 12–24 h. Then, the mixture was moved to −10 °C again, and was quenched by slowly adding ice methanol, after which the solvent was removed under the reduced pressure. The resulting residue was triturated with water, filtered, washed with water and DCM, dried in vacuum to afford target compounds A1–A6, A8–A12, A14, A16, A18, A19, A21–A27 and 19.

4.1.3. 5,6,7-Trihydroxy-2-phenylquinazolin-4(3H)-one (19)

The product was obtained as a offwhite solid (91 mg), yield 67%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.46 (s, 1H), 11.76 (s, 1H), 10.33 (s, 1H), 8.91 (s, 1H), 8.10 (d, J = 6.7 Hz, 3H), 7.59–7.49 (m, 3H), 6.65 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.4, 154.0, 149.4, 146.8, 142.1, 132.8, 131.1, 131.1, 128.6, 127.5, 103.2, 100.6. HRMS (ESI): m/z calcd for C₁₄H₁₁N₂O₄ [M + H]⁺: 271.0713, found 271.0717. HPLC analysis: t_R = 6.765min, 96.9%.

4.1.4. 2-(2-Fluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A1)

According to general procedure A, the product was obtained as a white solid (65 mg), yield 45%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.48 (s, 1H), 11.71 (s, 1H), 10.33 (s, 1H), 8.93 (s, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.63–7.56 (m, 1H), 7.41–7.31 (m, 2H), 6.63 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6, 159.5 (d, J = 250.2 Hz), 153.9, 146.8 (d, J = 6.8 Hz), 142.0, 132.6 (d, J = 8.5 Hz), 131.3, 131.0, 124.6 (d, J = 3.7 Hz), 122.2 (d, J = 13.1 Hz), 116.1 (d, J = 21.3 Hz), 103.2, 100.6. HRMS (ESI): m/z calcd for C₁₄H₁₀FN₂O₄ [M + H]⁺: 289.0619, found 289.0623. HPLC analysis: t_R = 6.825 min, 95.3%.

4.1.5. 2-(3-Fluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A2)

According to general procedure A, the product was obtained as a white solid (68 mg), yield 47%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.51 (br, 1H), 11.77 (s, 1H), 10.29 (s, 1H), 8.97 (s, 1H), 8.04–7.87 (m, 1H), 7.63–7.52 (m, 1H), 7.41 (td, J = 8.5, 2.6 Hz, 1H), 6.67 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.6, 162.1 (d, J = 243.5 Hz), 154.0, 148.4, 146.8, 141.4, 135.0 (d, J = 8.1 Hz), 131.4, 130.7 (d, J = 8.4 Hz), 123.7 (d, J = 2.7 Hz), 118.0 (d, J = 21.0 Hz), 114.3 (d, J = 23.8 Hz), 103.0, 101.0. HRMS (ESI): m/z calcd for C₁₄H₁₀FN₂O₄ [M + H]⁺: 289.0619, found 289.0622. HPLC analysis: t_R = 7.435 min, 95.5%.

4.1.6. 2-(4-Fluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A3)

According to general procedure A, the product was obtained as a white solid (56 mg), yield 39%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.80 (s, 1H), 10.33 (s, 1H), 8.89 (br, 1H), 8.25–8.08 (m, 2H), 7.44–7.29 (m, 2H), 6.64 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.7, 163.8 (d, J = 247.5 Hz), 153.9, 148.9, 146.8, 141.5, 131.0, 130.1 (d, J = 8.9 Hz), 129.2 (d, J = 2.9 Hz), 115.6 (d, J = 21.9 Hz), 102.7, 100.4. HRMS (ESI): m/z calcd for C₁₄H₁₀FN₂O₄ [M + H]⁺: 289.0619, found 289.0617. HPLC analysis: t_R = 8.683 min, 98.9%.

4.1.7. 2-(2-Chlorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one(A4)

According to general procedure A, the product was obtained as a white solid (90 mg), yield 59%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.56 (s, 1H), 11.73 (s, 1H), 10.35 (s, 1H), 8.93 (s, 1H), 7.65–7.52 (m, 3H), 7.48 (td, J = 7.4, 1.5 Hz, 1H), 6.60 (s, 1H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 165.5, 153.8, 149.1, 146.8, 141.7, 133.6, 131.6, 131.5, 131.2, 131.0, 129. 6, 127.2, 102.9, 100.7. HRMS (ESI): m/z calcd for C₁₄H₁₀ClN₂O₄ [M + H]⁺: 305.0324, found 305.0323. HPLC analysis: t_R = 6.920 min, 95.2%.

4.1.8. 2-(3-Chlorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A5)

According to general procedure A, the product was obtained as a white solid (94 mg), yield 62%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.50 (br, 1H), 11.73 (s, 1H), 10.35 (br, 1H), 8.98 (br, 1H), 8.16 (t, J = 1.9 Hz, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.69–7.48 (m, 2H), 6.67 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.5, 153.9, 148.2, 146.8, 141.7, 134.8, 133.4, 131.4, 130.8, 130.5, 127.3, 126.2, 103.4, 100.7. HRMS (ESI): m/z calcd for C₁₄H₁₀ClN₂O₄ [M + H]⁺: 305.0324, found 305.0323. HPLC analysis: t_R = 12.106 min, 96.2%.

4.1.9. 2-(4-Chlorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A6)

According to general procedure A, the product was obtained as a white solid (97 mg), yield 64%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.51 (s, 1H), 11.72 (s, 1H), 10.36 (s, 1H), 8.94 (s, 1H), 8.15–8.09 (m, 2H), 7.62–7.56 (m, 2H), 6.64 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.3, 154.0, 148.4, 146.8, 141.9, 135.9, 131.6, 131.3, 129.3, 128.7, 103.3, 100.6. HRMS (ESI): m/z calcd for C₁₄H₁₀ClN₂O₄ [M + H]⁺: 305.0324, found 305.0317. HPLC analysis: t_R = 9.457 min, 98.0%.

4.1.10. 2-(3-Bromophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A8)

According to general procedure A, the product was obtained as a white solid (104 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.54 (br, 1H), 11.78 (s, 1H), 10.37 (s, 1H), 8.96 (s, 1H), 8.29 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 6.66 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.6, 153.9, 148.3, 146.8, 141.3, 134.9, 133.7, 131.3, 130.7, 130.1, 126.5, 121.9, 102.9, 100.6. HRMS (ESI): m/z calcd for C₁₄H₁₀ ⁷⁹BrN₂O₄ [M + H]⁺: 348.9819, found 348.9823. HPLC analysis: t_R = 10.440 min, 95.4%.

4.1.11. 2-(4-Bromophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A9)

According to general procedure A, the product was obtained as a white solid (108 mg), yield 62%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.51 (s, 1H), 11.77 (s, 1H), 10.35 (s, 1H), 8.94 (s, 1H), 8.09–8.02 (m, 2H), 7.780–7.71 (m, 2H), 6.65 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.6, 153.9, 148.8, 146. 8, 141.5, 131.9, 131.6, 131.2, 129.5, 124.8, 102.8, 100.6. HRMS (ESI): m/z calcd for C₁₄H₈ ⁷⁹BrN₂O₄ [M − H]^–: 346.9673, found 346.9666. HPLC analysis: t_R = 11.866 min, 96.6%.

4.1.12. 5,6,7-Trihydroxy-2-(o-tolyl)quinazolin-4(3H)-one (A10)

According to general procedure A, the product was obtained as a white solid (50 mg), yield 35%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.37 (s, 1H), 11.78 (s, 1H), 10.28 (s, 1H), 8.87 (s, 1H), 7.47–7.38 (m, 2H), 7.35–7.28 (m, 2H), 6.58 (s, 1H), 2.35 (s, 3H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.3, 153.8, 151.7, 146.8, 141.3, 136.2, 134.0, 131.0, 130.5, 129.8, 129.2, 125.7, 102.8, 100.5, 19.6. HRMS (ESI): m/z calcd for C₁₅H₁₁N₂O₄ [M − H] ^–: 283.0724, found 283.0718. HPLC analysis: t_R = 9.143 min, 99.2%.

4.1.13. 5,6,7-Trihydroxy-2-(m-tolyl)quinazolin-4(3H)-one (A11)

According to general procedure A, the product was obtained as a white solid (57 mg), yield 40%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.84 (br, 1H), 10.36 (s, 1H), 8.90 (br, 1H), 7.93 (s, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.45–7.34 (m, 2H), 6.65 (s, 1H), 2.39 (s, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.8, 153.9, 150.1, 146.8, 141.5, 137.9, 132.5, 131.8, 131.0, 128.5, 128.1, 124.7, 102.6, 100.5, 21.0. HRMS (ESI): m/z calcd for C₁₅H₁₁N₂O₄ [M − H] ^–: 283.0724, found 283.0722. HPLC analysis: t_R = 7.412 min, 95.1%.

4.1.14. 5,6,7-Trihydroxy-2-(p-tolyl)quinazolin-4(3H)-one (A12)

According to general procedure A, the product was obtained as a white solid (61 mg), yield 43%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.38 (br, 1H), 11.82 (s, 1H), 10.31 (s, 1H), 8.87 (s, 1H), 8.05–7.98 (m, 2H), 7.36–7.30 (m, 2H), 6.62 (s, 1H), 2.38 (s, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.8, 154.4, 150.1, 147.3, 141.6, 131.3, 130.3, 129.6, 127.9, 102.7, 100.9, 21.42. HRMS (ESI): m/z calcd for C₁₅H₁₁N₂O₄ [M − H] ^–: 283.0724, found 283.0717. HPLC analysis: t_R = 5.061 min, 97.0%.

4.1.15. 5,6,7-Trihydroxy-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (A14)

According to general procedure A, the product was obtained as a white solid (68 mg), yield 40%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.68 (s, 1H), 11.72 (s, 1H), 10.38 (s, 1H), 8.99 (s, 1H), 8.46 (s, 1H), 8.41 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 6.68 (s, 1H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.3, 154.0, 148.0, 146.7, 141.8, 133.7, 131.53, 131.45, 129.8, 129.4 (q, J = 32.0 Hz), 127.5, 124.2, 124.0 (q, J = 272.3 Hz), 103.4, 100.7. HRMS (ESI): m/z calcd for C₁₅H₈F₃N₂O₄ [M − H] ^–: 337.0442, found 337.0436. HPLC analysis: t_R = 8.083 min, 97.2%.

4.1.16. 5,6,7-Trihydroxy-2-(4-(trifluoromethoxy)phenyl)quinazolin-4(3H)-one (A16)

According to general procedure A, the product was obtained as a yellow solid (76 mg), yield 43%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.78 (br, 1H), 10.35 (br, 1H), 8.27–8.18 (m, 2H), 7.56–7.49 (m, 2H), 6.66 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.5, 158.2, 153.9, 150.2, 148.6, 146.8, 141.4, 131.8, 131.2, 129.8, 128.3, 120.8, 120.0 (q, J = 257.4 Hz), 102.7, 100.5. HRMS (ESI): m/z calcd for C₁₅H₈F₃N₂O₅ [M − H] ^–: 353.0391, found 353.0381. HPLC analysis: t_R = 8.742 min, 96.3%.

4.1.17. 5,6,7-Trihydroxy-2-(3-nitrophenyl)quinazolin-4(3H)-one (A18)

According to general procedure A, the product was obtained as a yellow solid (71 mg), yield 45%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.76 (s, 1H), 11.71 (s, 1H), 10.42 (s, 1H), 9.02 (s, 1H), 8.94 (s, 1H), 8.53 (d, J = 7.8 Hz, 1H), 8.38 (dd, J = 8.2, 2.2 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 6.69 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 167.0, 154.4, 148.4, 147.8, 147.2, 141.9, 134.7, 134.2, 132.0, 130.7, 125.9, 122.8, 103.9, 101.2. HRMS (ESI): m/z calcd for C₁₄H₈N₃O₆ [M − H] ^–: 314.0419, found 314.0412. HPLC analysis: t_R = 11.254 min, 97.5%.

4.1.18. 5,6,7-Trihydroxy-2-(4-nitrophenyl)quinazolin-4(3H)-one (A19)

According to general procedure A, the product was obtained as a yellow solid (61 mg), yield 39%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.74 (s, 1H), 11.70 (s, 1H), 10.44 (s, 1H), 9.07 (s, 1H), 8.41–8.30 (s, 4H), 6.70 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.1, 153.9, 148.7, 147.6, 146.7, 141.6, 138.5, 131.8, 128.9, 123.6, 103.6, 100.7. HRMS (ESI): m/z calcd for C₁₄H₈N₃O₆ [M − H] ^–: 314.0419, found 314.0412. HPLC analysis: t_R = 8.392 min, 97.7%.

4.1.19. 2-(3-(tert-Butyl)phenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A21)

According to general procedure A, the product was obtained as a white solid (68 mg), yield 42%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.53 (br, 1H), 11.84 (s, 1H), 10.37 (s, 1H), 8.89 (s, 1H), 8.10 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 6.66 (s, 1H), 1.34 (s, 9H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.9, 153.9, 151.1, 150.0, 146.8, 141.4, 132.4, 131.0, 128.4, 128.1, 125.0, 124.3, 102.5, 100.5, 34.8, 31.1.

HRMS (ESI): m/z calcd for C₁₈H₁₇N₂O₄ [M − H] ^–: 325.1194, found 325.1189. HPLC analysis: t_R = 12.850 min, 95.2%.

4.1.20. 2-(4-(tert-Butyl)phenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A22)

According to general procedure A, the product was obtained as a white solid (67 mg), yield 41%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.38 (s, 1H), 11.80 (s, 1H), 10.29 (s, 1H), 8.85 (s, 1H), 8.10–8.01 (m, 2H), 7.56–7.51 (m, 2H), 6.63 (s, 1H), 1.32 (s, 9H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.3, 153.9, 149.6, 146.8, 142.2, 130. 9, 130.0, 128.6, 127.3, 125.4, 102.9, 100.5, 34.7, 30.9. HRMS (ESI): m/z calcd for C₁₈H₁₇N₂O₄ [M − H] ^–: 325.1194, found 325.1185. HPLC analysis: t_R = 10.744 min, 97.0%.

4.1.21. 5,6,7-Trihydroxy-2-(2-hydroxyphenyl)quinazolin-4(3H)-one (A23)

According to general procedure A, the product was obtained as a white solid (50 mg), yield 35%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 13.71 (br, 1H), 12.42 (br, 1H), 11.60 (s, 1H), 10.48 (s, 1H), 9.03 (s, 1H), 8.15 (dd, J = 8.1, 1.6 Hz, 1H), 7.47–7.37 (m, 1H), 7.01–6.92 (m, 2H), 6.63 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.4, 159.8, 154.2, 151.1, 147.0, 139.0, 133.4, 131.4, 127.4, 118.8, 117.8, 113.8, 101.8, 100.4. HRMS (ESI): m/z calcd for C₁₄H₉N₂O₅ [M − H] ^–: 285.0517, found 285.0509. HPLC analysis: t_R = 9.255 min, 95.4%.

4.1.22. 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)quinazolin-4(3H)-one (A24)

According to general procedure A, the product was obtained as a white solid (54 mg), yield 38%. ¹H NMR (300 MHz, DMSO‑d ₆) δ 12.27 (br, 1H), 11.85 (s, 1H), 10.27 (s, 1H), 10.13 (s, 1H), 8.81 (s, 1H), 8.06–7.93 (m, 2H), 6.93–6.82 (m, 2H), 6.58 (s, 1H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.4, 160.3, 154.0, 149.2, 146.8, 142.4, 130.5, 129.3, 123.3, 115.3, 102.5, 100.2. HRMS (ESI): m/z calcd for C₁₄H₉N₂O₅ [M − H] ^–: 285.0517, found 285.0514. HPLC analysis: t_R = 10.262 min, 96.1%.

4.1.23. 2-(2,3-Difluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A25)

According to general procedure A, the product was obtained as a white solid (77 mg), yield 50%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.60 (s, 1H), 11.67 (s, 1H), 10.40 (s, 1H), 9.01 (s, 1H), 7.69–7.60 (m, 1H), 7.58–7.51 (m, 1H), 7.40–7.32 (m, 1H), 6.64 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.0, 154.35, 151.17 (d, J = 12.5 Hz), 149.17 (dd, J = 13.2, 9.5 Hz), 147.20, 146.07 (d, J = 3.5 Hz), 142.26, 132.04, 126.60 (d, J = 3.5 Hz), 125.88–125.24 (m), 124.68 (d, J = 9.6 Hz), 119.93 (d, J = 17.3 Hz), 103.9, 101.1. HRMS (ESI): m/z calcd for C₁₄H₇F₂N₂O₄ [M − H] ^–: 305.0379, found 305.0369. HPLC analysis: t_R = 8.571 min, 96.1%.

4.1.24. 2-(2,4-Difluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A26)

According to general procedure A, the product was obtained as a white solid (84 mg), yield 55%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.52 (s, 1H), 11.69 (s, 1H), 10.36 (s, 1H), 8.97 (s, 1H), 7.83–7.76 (m, 1H), 7.50–7.42 (m, 1H), 7.25 (td, J = 8.5, 2.5 Hz, 1H), 6.62 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6, 163.5 (dd, J = 248.8, 11.3Hz), 160.0 (J = 253.2, 12.6 Hz), 153.9, 146.7, 146.0, 141.9, 132.6 (dd, J = 10.6, 3.7 Hz), 131.4, 119.0 (dd, J = 13.1, 3.5 Hz), 111.9 (dd, J = 22.1, 3.1 Hz), 104.6 (t, J = 26.0 Hz), 103.2, 100.6. HRMS (ESI): m/z calcd for C₁₄H₇F₂N₂O₄ [M − H] ^–: 305.0379, found 305.0369. HPLC analysis: t_R = 7.082 min, 95.4%.

4.1.25. 2-(3,4-Difluorophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A27)

According to general procedure A, the product was obtained as a white solid (77 mg), yield 50%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.53 (s, 1H), 11.69 (s, 1H), 10.38 (s, 1H), 8.97 (s, 1H), 8.20–8.13 (m, 1H), 8.04–7.97 (m, 1H), 7.67–7.57 (m, 1H), 6.65 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6 (d, J = 141.3 Hz), 153.9, 152.2 (d, J = 13.4 Hz), 150.2 (t, J = 12.7 Hz), 148.3 (d, J = 12.9 Hz), 147.3, 146.7, 141.7, 131.32, 130.2 (dd, J = 6.2, 3.4 Hz), 124.84, 117.79 (d, J = 17.7 Hz), 116.76 (d, J = 19.3 Hz), 103.3, 100.5. HRMS (ESI): m/z calcd for C₁₄H₇F₂N₂O₄ [M − H] ^–: 305.0379, found 305.0371. HPLC analysis: t_R = 7.863 min, 95.4%.

4.1.26. General procedure B for the preparation of compounds A7, A13, A15, A17, A20, B1–B17, C1–C15 and D6–D9

To the stirred solution of 27 or 30a–30p (1 mmol) in anhydrous EtOH (10 mL) was added I₂ (1.2 mmol) and the corresponding aldehyde (1.2 mmol). The resulting reaction mixture was heated to reflux and stirred for 3–5 h. After completion of the reaction, the reaction mixture was cooled to room temperature and was quenched with excess 5% aqueous Na₂S₂O₃. The aqueous portion was extracted with DCM (3 × 10 mL), and the combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica column chromatography (DCM/MeOH = 300:1 to 150:1) to give the corresponding quinazolinones 29a–29v, 32a–32o, and 41a–41d.

According to general procedure A, the demethylation of the above quinazolinones afforded the target compounds A7, A13, A15, A17, A20, B1–B17, C1–C15 and D6–D9.

4.1.27. 2-(2-Bromophenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (A7)

According to general procedure B, the product was obtained as a white solid (99 mg), yield 57%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.55 (s, 1H), 11.76 (s, 1H), 10.36 (s, 1H), 8.94 (s, 1H), 7.76 (dd, J = 7.7, 1.5 Hz, 1H), 7.60 (dd, J = 7.4, 2.1 Hz, 1H), 7.56–7.42 (m, 2H), 6.60 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.5, 153.8, 150.2, 146.8, 141.8, 135.7, 132.6, 131.6, 131.3, 130.9, 127.6, 121.1, 103.2, 100.7. HRMS (ESI): m/z calcd for C₁₄H₁₀ ⁷⁹BrN₂O₄ [M + H]⁺: 348.9819, found 348.9822. HPLC analysis: t_R = 9.626 min, 98.2%.

4.1.28. 5,6,7-Trihydroxy-2-(2-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (A13)

According to general procedure B, the product was obtained as a yellow solid (71 mg), yield 42%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.62 (s, 1H), 11.70 (s, 1H), 10.35 (s, 1H), 8.94 (s, 1H), 7.94–7.85 (m, 1H), 7.84–7.69 (m, 3H), 6.57 (s, 1H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 165.5, 153.9, 149.3, 147.0, 141.6, 132.9, 132.4, 131.3, 130.8, 130.4, 127.09 (q, J = 30.8 Hz), 126.41 (q, J = 4.7 Hz), 123.76 (q, J = 273 Hz), 103.0, 100.6. HRMS (ESI): m/z calcd for C₁₅H₈F₃N₂O₄ [M − H] ^–: 337.0442, found 337.0431. HPLC analysis: t_R = 7.223 min, 96.4%.

4.1.29. 5,6,7-Trihydroxy-2-(2-(trifluoromethoxy)phenyl)quinazolin-4(3H)-one (A15)

According to general procedure B, the product was obtained as a white solid (71 mg), yield 40%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.58 (s, 1H), 11.70 (s, 1H), 10.36 (s, 1H), 8.97 (s, 1H), 7.76 (dd, J = 7.6, 1.8 Hz, 1H), 7.68 (td, J = 7.8, 1.8 Hz, 1H), 7.58–7.50 (m, 2H), 6.62 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6, 153.9, 147.2, 146.7, 145.7, 141.9, 132.1, 131.4, 128.0, 127.6, 121.5, 120.0 (q, J = 257.4 Hz), 103.3, 100.5. HRMS (ESI): m/z calcd for C₁₅H₈F₃N₂O₅ [M − H] ^–: 353.0391, found 353.0377. HPLC analysis: t_R = 9.658 min, 95.0%.

4.1.30. 5,6,7-Trihydroxy-2-(2-nitrophenyl)quinazolin-4(3H)-one (A17)

According to general procedure B, the product was obtained as an orange solid (68 mg), yield 43%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.76 (s, 1H), 11.68 (s, 1H), 10.37 (s, 1H), 8.97 (s, 1H), 8.20–8.16 (m, 1H), 7.94–7.76 (m, 3H), 6.54 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.7, 153.9, 148.4, 147.6, 146.8, 141.9, 133.8, 131.6, 131.5, 131.4, 129.0, 124.5, 103.2, 100.6. HRMS (ESI): m/z calcd for C₁₄H₈N₃O₆ [M − H] ^–: 314.0419, found 314.0411. HPLC analysis: t_R = 6.699 min, 95.1%.

4.1.31. 5,6,7-Trihydroxy-2-(3-isopropylphenyl)quinazolin-4(3H)-one (A20)

According to general procedure B, the product was obtained as a white solid (72 mg), yield 46%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.46 (s, 1H), 11.83 (s, 1H), 10.30 (s, 1H), 8.87 (s, 1H), 7.98 (s, 1H), 7.94–7.89 (m, 1H), 7.44 (d, J = 4.8 Hz, 2H), 6.64 (s, 1H), 2.99 (hept, J = 6.8 Hz, 1H), 1.27 (s, 3H), 1.26 (s, 3H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.6, 153.9, 150.0, 148.8, 146.8, 141.8, 132.6, 131.0, 129.2, 128.6, 125.4, 125.2, 102.8, 100.5, 33.5, 23.8. HRMS (ESI): m/z calcd for C₁₇H₁₅N₂O₄ [M − H] ^–: 311.1037, found 311.1033. HPLC analysis: t_R = 13.162 min, 97.7%.

4.1.32. 5,6,7-Trihydroxy-2-(thiophen-2-yl)quinazolin-4(3H)-one (B1)

According to general procedure B, the product was obtained as a offwhite solid (83 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.59 (br, 1H), 11.75 (s, 1H), 10.33 (s, 1H), 8.90 (br, 1H), 8.16–8.11 (m, 1H), 7.85–7.78 (m, 1H), 7.24–7.17 (m, 1H), 6.54 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.3, 154.0, 147.00, 145.40, 141.5, 137.5, 131.5, 131.0, 128.7, 128.5, 102.4, 100.4. HRMS (ESI): m/z calcd for C₁₂H₇N₂O₄S [M − H] ^–: 275.0123, found 275.0132. HPLC analysis: t_R = 7.024 min, 96.7%.

4.1.33. 5,6,7-Trihydroxy-2-(pyridin-2-yl)quinazolin-4(3H)-one (B2)

According to general procedure B, the product was obtained as an orange solid (61 mg), yield 45%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.92 (s, 1H), 10.45 (s, 1H), 9.04 (s, 1H), 8.73 (d, J = 4.6 Hz, 1H), 8.36 (d, J = 7.8 Hz, 1H), 8.04 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 6.1 Hz, 1H), 6.74 (s, 1H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.1, 153.8, 148.9, 148.8, 148.0, 147.0, 140.7, 137.9, 131.6, 126.3, 122.0, 102.8, 101.3. HRMS (ESI): m/z calcd for C₁₃H₈N₃O₄ [M − H] ^–: 270.0520, found 270.0520. HPLC analysis: t_R = 5.840 min, 95.0%.

4.1.34. 5,6,7-Trihydroxy-2-(pyridin-4-yl)quinazolin-4(3H)-one (B3)

According to general procedure B, the product was obtained as an yellow solid (68 mg), yield 50%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.68 (s, 1H), 11.73 (s, 1H), 10.44 (s, 1H), 9.07 (s, 1H), 8.82–8.73 (m, 2H), 8.12–8.05 (m, 2H), 6.70 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.9, 153.9, 149.9, 149.8, 147.5, 146.7, 146.0, 141.3, 140.2, 131.8, 121.5, 103.6, 101.0. HRMS (ESI): m/z calcd for C₁₃H₈N₃O₄ [M − H] ^–: 270.0520, found 270.0519. HPLC analysis: t_R = 5.971 min, 96.7%.

4.1.35. 2-(1,3-Dimethyl-1H-pyrazol-5-yl)-5,6,7-trihydroxyquinazolin-4(3H)-one (B4)

According to general procedure B, the product was obtained as a yellow solid (75 mg), yield 52%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.34 (br, 1H), 11.73 (s, 1H), 10.38 (s, 1H), 8.97 (br, 1H), 6.89 (s, 1H), 6.62 (s, 1H), 4.10 (s, 3H), 2.18 (s, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.2, 153.9, 146.9, 145.7, 142.4, 141.1, 134.7, 131.4, 107.8, 103.0, 100.4, 39.6, 13.2. HRMS (ESI): m/z calcd for C₁₃H₁₁N₄O₄ [M − H] ^–: 287.0786, found 287.0777. HPLC analysis: t_R = 7.618 min, 96.5%.

4.1.36. 5,6,7-Trihydroxy-2-(naphthalen-2-yl)quinazolin-4(3H)-one (B5)

According to general procedure B, the product was obtained as a white solid (99 mg), yield 62%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.84 (s, 1H), 10.37 (s, 1H), 8.73 (s, 1H), 8.23 (dd, J = 8.6, 1.9 Hz, 1H), 8.08–7.97 (m, 3H), 7.67–7.56 (m, 2H), 6.70 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.6, 154.0, 150.0, 146.9, 141.2, 134.0, 132.3, 131.3, 129.7, 128.9, 128.2, 127.9, 127.8, 127.7, 127.0, 124.4, 102.5, 100.6. HRMS (ESI): m/z calcd for C₁₈H₁₁N₂O₄ [M − H] ^–: 319.0724, found 319.0721. HPLC analysis: t_R = 8.708 min, 97.4%.

4.1.37. 2-Cyclopropyl-5,6,7-trihydroxyquinazolin-4(3H)-one (B6)

According to general procedure B, the product was obtained as a white solid (49 mg), yield 42%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.34 (s, 1H), 11.71 (s, 1H), 10.15 (s, 1H), 8.65 (s, 1H), 6.35 (s, 1H), 1.92–1.84 (m, 1H), 1.06–0.93 (m, 4H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.0, 156.3, 153.8, 146.9, 141.8, 129.8, 102.0, 100.2, 13.4, 9.2. HRMS (ESI): m/z calcd for C₁₁H₉N₂O₄ [M − H] ^–: 233.0568, found 233.0560. HPLC analysis: t_R = 5.988 min, 95.3%.

4.1.38. 2-Cyclopentyl-5,6,7-trihydroxyquinazolin-4(3H)-one (B7)

According to general procedure B, the product was obtained as a white solid (49 mg), yield 48%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.06 (s, 1H), 11.75 (s, 1H), 10.19 (s, 1H), 8.71 (s, 1H), 6.46 (s, 1H), 3.00–2.90 (m, 1H), 1.99–1.89 (m, 2H), 1.88–1.77 (m, 2H), 1.76–1.65 (m, 2H), 1.63–1.52 (m, 2H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 165.9, 157.4, 153.7, 146.8, 142.2, 130.2, 102.6, 100.4, 43.7, 30.8, 25.3. HRMS (ESI): m/z calcd for C₁₃H₁₃N₂O₄ [M − H] ^–: 261.0881, found 261.0875. HPLC analysis: t_R = 8.081min, 95.7%.

4.1.39. 2-Cyclohexyl-5,6,7-trihydroxyquinazolin-4(3H)-one (B8)

According to general procedure B, the product was obtained as a white solid (49 mg), yield 53%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.01 (s, 1H), 11.75 (s, 1H), 10.19 (s, 1H), 8.71 (s, 1H), 6.47 (s, 1H), 2.53–2.45 (m, 1H), 1.91–1.82 (m, 2H), 1.80–1.72 (m, 2H), 1.66 (d, J = 10.6 Hz, 1H), 1.58–1.45 (m, 2H), 1.35–1.13 (m, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.8, 157.6, 153.7, 146.8, 142.3, 130.3, 102.1, 100.4, 42.6, 30.2, 25.5, 25.3. HRMS (ESI): m/z calcd for C₁₄H₁₅N₂O₄ [M − H] ^–: 275.1037, found 275.1034. HPLC analysis: t_R = 9.657 min, 96.3%.

4.1.40. 5,6,7-Trihydroxy-2-isopropylquinazolin-4(3H)-one (B9)

According to general procedure B, the product was obtained as a white solid (71 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.03 (s, 1H), 11.73 (s, 1H), 10.16 (s, 1H), 8.69 (s, 1H), 6.48 (s, 1H), 2.81 (hept, J = 6.8 Hz, 1H), 1.21 (d, J = 6.9 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.1, 158.7, 153.7, 146.9, 142.0, 130.3, 102.4, 100.4, 33.1, 20.4. HRMS (ESI): m/z calcd for C₁₁H₁₁N₂O₄ [M − H] ^–: 235. 0724, found 235.0721. HPLC analysis: t_R = 5.456 min, 96.1%.

4.1.41. 2-(tert-Butyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (B10)

According to general procedure B, the product was obtained as a white solid (70 mg), yield 56%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.79 (s, 1H), 10.22 (s, 1H), 8.75 (s, 1H), 6.51 (s, 1H), 1.29 (s, 9H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.5, 160.1, 153.8, 146.7, 141.4, 130.5, 102.5, 100.1, 37.1, 27.8. HRMS (ESI): m/z calcd for C₁₂H₁₃N₂O₄ [M − H] ^–: 249.0881, found 249.0873. HPLC analysis: t_R = 7.203 min, 95.2%.

4.1.42. 5,6,7-Trihydroxy-2-isobutylquinazolin-4(3H)-one (B11)

According to general procedure B, the product was obtained as a white solid (58 mg), yield 56%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.08 (s, 1H), 11.70 (s, 1H), 10.21 (s, 1H), 8.74 (s, 1H), 6.48 (s, 1H), 2.39 (d, J = 7.3 Hz, 2H), 2.11 (hept, J = 6.7 Hz, 1H), 0.90 (d, J = 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.1, 153.7, 146.8, 142.1, 130.3, 102.4, 100.4, 43.1, 27.0, 22.1. HRMS (ESI): m/z calcd for C₁₂H₁₃N₂O₄ [M − H] ^–: 249.0881, found 249.0879. HPLC analysis: t_R = 9.580 min, 98.8%.

4.1.43. 5,6,7-Trihydroxy-2-neopentylquinazolin-4(3H)-one (B12)

According to general procedure B, the product was obtained as a white solid (79 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.99 (s, 1H), 11.71 (s, 1H), 10.21 (s, 1H), 8.74 (s, 1H), 6.49 (s, 1H), 2.41 (s, 2H), 0.97 (s, 9H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.7, 153.7, 152.4, 146.8, 142.1, 130.3, 102.2, 100.1, 47.0, 31.9, 29.4. HRMS (ESI): m/z calcd for C₁₃H₁₅N₂O₄ [M − H] ^–: 263.1037, found 263.1030. HPLC analysis: t_R = 7.704 min, 95.1%.

4.1.44. (E)-5,6,7-Trihydroxy-2-styrylquinazolin-4(3H)-one (B13)

According to general procedure B, the product was obtained as a white solid (93 mg), yield 63%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.27 (s, 1H), 11.78 (s, 1H), 10.28 (s, 1H), 8.89 (s, 1H), 7.84 (d, J = 16.2 Hz, 1H), 7.69–7.60 (m, 2H), 7.51–7.37 (m, 3H), 6.93 (d, J = 16.2 Hz, 1H), 6.58 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6, 153.8, 148.4, 146.8, 142.1, 137.2, 135.1, 130.9, 129.5, 129.0, 127.5, 121.0, 102.8, 100.6. HRMS (ESI): m/z calcd for C₁₆H₁₁N₂O₄ [M − H] ^–: 295.0724, found 295.0720. HPLC analysis: t_R = 9.694 min, 95.3%.

4.1.45. 2-Benzyl-5,6,7-trihydroxyquinazolin-4(3H)-one (B14)

According to general procedure B, the product was obtained as a white solid (85 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.34 (s, 1H), 11.68 (s, 1H), 10.23 (s, 1H), 8.77 (s, 1H), 7.38–7.28 (m, 4H), 7.27–7.21 (m, 1H), 6.48 (s, 1H), 3.86 (s, 2H). ¹³C NMR (150 MHz, DMSO‑d ₆) δ 166.1, 153.9, 153.6, 146.8, 141.1, 136.4, 130.7, 128.8, 128.5, 126.9, 101.6, 100.2, 40.3. HRMS (ESI): m/z calcd for C₁₅H₁₁N₂O₄ [M − H] ^–: 283.0724, found 283.0716. HPLC analysis: t_R = 8.088 min, 95.0%.

4.1.46. 5,6,7-Trihydroxy-2-(1-phenylethyl)quinazolin-4(3H)-one (B15)

According to general procedure B, the product was obtained as a white solid (85 mg), yield 57%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.16 (s, 1H), 11.65 (s, 1H), 10.25 (s, 1H), 8.76 (s, 1H), 7.40–7.27 (m, 4H), 7.26–7.18 (m, 1H), 6.54 (s, 1H), 4.03 (q, J = 7.0 Hz, 1H), 1.55 (d, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.8, 155.9, 153.8, 146.8, 142.4, 142.0, 130.5, 128.5, 127.4, 126.8, 102.8, 100.4, 43.6, 19.3. HRMS (ESI): m/z calcd for C₁₆H₁₃N₂O₄ [M − H] ^–: 297. 0881, found 297. 0871. HPLC analysis: t_R = 7.318 min, 97.7%.

4.1.47. 5,6,7-Trihydroxy-2-(1-phenylpropyl)quinazolin-4(3H)-one (B16)

According to general procedure B, the product was obtained as a white solid (92 mg), yield 59%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.69 (br, 1H), 10.27 (br, 1H), 7.42–7.35 (m, 2H), 7.34–7.29 (m, 2H), 7.26–7.20 (m, 1H), 6.55 (s, 1H), 3.74 (t, J = 7.7 Hz, 1H), 2.27–2.15 (m, 1H), 1.96–1.84 (m, 1H), 0.83 (t, J = 7.3 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.1, 156.2, 154.5, 153.9, 146.8, 140.7, 130.7, 128.5, 127.9, 127.1, 101.8100.4, 51.2, 26.3, 12.2. HRMS (ESI): m/z calcd for C₁₇H₁₅N₂O₄ [M − H] ^–: 311.1037, found 311.1028. HPLC analysis: t_R = 7.867 min, 95.1%.

4.1.48. 5,6,7-Trihydroxy-2-(2-methyl-1-phenylpropyl)quinazolin-4(3H)-one (B17)

According to general procedure B, the product was obtained as a white solid (103 mg), yield 63%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.23 (s, 1H), 11.61 (s, 1H), 10.24 (s, 1H), 8.76 (s, 1H), 7.44 (d, J = 7.1 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.25–7.18 (m, 1H), 6.55 (s, 1H), 3.40 (d, J = 11.3 Hz, 1H), 2.67–2.53 (m, 1H), 0.93 (d, J = 6.4 Hz, 3H), 0.69 (d, J = 6.6 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.7, 155.4, 153.8, 146.7, 142.3, 140.3, 130.4, 128.33, 128.28, 127.0, 102.7, 100.2, 57.8, 30.9, 21.2, 20.5. HRMS (ESI): m/z calcd for C₁₈H₁₈N₂O₄Na [M + Na]⁺: 349. 1159, found 349. 1158. HPLC analysis: t_R = 9.494 min, 96.0%.

4.1.49. 5,6,7-Trihydroxy-3-isobutyl-2-phenylquinazolin-4(3H)-one (C1)

According to general procedure B, the product was obtained as a light yellow solid (77 mg), yield 47%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.83 (s, 1H), 10.37 (s, 1H), 8.95 (s, 1H), 7.61–7.56 (m, 2H), 7.55–7.47 (m, 3H), 6.57 (s, 1H), 3.83 (d, J = 7.4 Hz, 2H), 1.73 (hept, J = 6.9 Hz, 1H), 0.61 (d, J = 6.7 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.2, 153.9, 153.0, 146.4, 140.3, 135.2, 131.4, 129.5, 128.5, 128.3, 102.8, 100.4, 50.6, 27.3, 19.7. HRMS (ESI): m/z calcd for C₁₈H₁₉N₂O₄ [M + H] ⁺: 327.1339, found 327.1342. HPLC analysis: t_R = 14.369 min, 96.7%.

4.1.50. 5,6,7-Trihydroxy-3-(2-hydroxyethyl)-2-phenylquinazolin-4(3H)-one (C2)

According to general procedure B, the product was obtained as a white solid (71 mg), yield 45%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.80 (s, 1H), 10.33 (s, 1H), 8.93 (s, 1H), 7.62–7.55 (m, 2H), 7.54–7.47 (m, 3H), 6.55 (s, 1H), 4.83 (t, J = 5.7 Hz, 1H), 3.94 (t, J = 6.2 Hz, 2H), 3.48 (q, J = 5.9 Hz, 2H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 153.9, 153.4, 146.4, 140.5, 135.4, 131.3, 129.3, 128.6, 128.3, 102.6, 100.7, 57.8, 46.9. HRMS (ESI): m/z calcd for C₁₆H₁₅N₂O₅ [M + H] ⁺: 315. 0976, found 315. 0973. HPLC analysis: t_R = 11.691 min, 98.1%.

4.1.51. N-(tert-butyl)-2-(5,6,7-trihydroxy-4-oxo-2-phenylquinazolin-3(4H)-yl)acetamide (C3)

According to general procedure B, the product was obtained as a white solid (82 mg), yield 43%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.57 (s, 1H), 10.39 (s, 1H), 8.96 (br, 1H), 7.67 (s, 1H), 7.58–7.43 (m, 5H), 6.58 (s, 1H), 4.36 (s, 2H), 1.17 (s, 9H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.4, 164.7, 154.0, 153.1, 146.3, 140.4, 134.8, 131.4, 129.6, 128.3, 128.1, 102.8, 100.2, 50.3, 47.1, 28.3. HRMS (ESI): m/z calcd for C₂₀H₂₁N₃O₅Na [M + Na] ⁺: 406. 1373, found 406.1370. HPLC analysis: t_R = 12.294 min, 96.8%.

4.1.52. 3-Cyclopropyl-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C4)

According to general procedure B, the product was obtained as a white solid (71 mg), yield 46%.¹H NMR (400 MHz, DMSO‑d ₆) δ 11.88 (s, 1H), 10.26 (s, 1H), 8.88 (s, 1H), 7.84–7.66 (m, 2H), 7.56–7.41 (m, 3H), 6.55 (s, 1H), 3.17 (tt, J = 7.3, 4.1 Hz, 1H), 0.87–0.65 (m, 2H), 0.57–0.30 (m, 2H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.7, 153.9, 153.6, 146.4, 140.1, 136.0, 131.2, 129.3, 128.4, 127.9, 102.7, 100.4, 29.0, 10.7. HRMS (ESI): m/z calcd for C₁₇H₁₅N₂O₄ [M + H] ⁺: 311. 1026, found 311. 1023. HPLC analysis: t_R = 12.671 min, 98.4%.

4.1.53. 3-Cyclopentyl-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C5)

According to general procedure B, the product was obtained as a white solid (85 mg), yield 50%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.90 (s, 1H), 10.30 (s, 1H), 8.91 (s, 1H), 7.70–7.38 (m, 5H), 6.53 (s, 1H), 4.35–4.25 (m, 1H), 2.34–2.21 (m, 2H), 1.96–1.82 (m, 2H), 1.79–1.65 (m, 2H), 1.46–1.34 (m, 2H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.4, 153.8, 153.7, 146.5, 140.1, 135.9, 131.3, 129.5, 128.6, 127.8, 102.4, 101.4, 61.0, 28.7, 25.4. HRMS (ESI): m/z calcd for C₁₉H₁₉N₂O₄ [M + H] ⁺: 339.1339, found 339.1338. HPLC analysis: t_R = 11.188 min, 96.5%.

4.1.54. 3-Cyclohexyl-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C6)

According to general procedure B, the product was obtained as a white solid (97 mg), yield 55%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.98 (s, 1H), 10.30 (s, 1H), 8.91 (s, 1H), 7.60–7.48 (m, 5H), 6.52 (s, 1H), 3.71 (t, J = 12.1 Hz, 1H), 2.58–2.43 (m, 2H), 1.81–1.62 (m, 4H), 1.54–1.42 (m, 1H), 1.16–0.98 (m, 1H), 0.91–0.73 (m, 2H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 166.4, 154.2, 153.9, 147.0, 140.5, 136.4, 131.7, 129.9, 129.0, 127.9, 102.9, 101.9, 62.0, 28.8, 26.3, 25.1. HRMS (ESI): m/z calcd for C₂₀H₂₁N₂O₄ [M + H] ⁺: 353.1496, found 353.1503. HPLC analysis: t_R = 8.000 min, 98.4%.

4.1.55. 5,6,7-Trihydroxy-2,3-diphenylquinazolin-4(3H)-one (C7)

According to general procedure B, the product was obtained as a white solid (99 mg), yield 57%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.60 (s, 1H), 10.42 (s, 1H), 9.00 (s, 1H), 7.37–7.15 (m, 10H), 6.67 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.0, 154.1, 152.1, 146.8, 140.7, 136.9, 135.3, 131.5, 129.6, 129.0, 128.8, 128.6, 128.3, 127.5, 103.2, 100.4. HRMS (ESI): m/z calcd for C₂₀H₁₅N₂O₄ [M + H] ⁺: 347.1026, found 347.1025. HPLC analysis: t_R = 8.939 min, 98.9%.

4.1.56. 3-(2-Fluorophenyl)-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C8)

According to general procedure B, the product was obtained as a white solid (73 mg), yield 40%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.32 (s, 1H), 10.50 (s, 1H), 9.07 (s, 1H), 7.51 (td, J = 7.8, 1.7 Hz, 1H), 7.43–7.20 (m, 7H), 7.16 (td, J = 7.7, 1.4 Hz, 1H), 6.70 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.3, 157.0 (d, J = 248.1 Hz), 154.3, 151.7, 146.8, 140.4, 134.5, 131.7 (d, J = 24.6 Hz) 131.2 (d, J = 8.2 Hz), 129.3, 128.3, 127.7, 124.7 (d, J = 3.4 Hz), 124.5, 124.4, 115.8 (d, J = 19.6 Hz), 103.6, 99.9. HRMS (ESI): m/z calcd for C₂₀H₁₄FN₂O₄ [M + H] ⁺: 365.0932, found 365.0931. HPLC analysis: t_R = 7.934 min, 98.3%.

4.1.57. 3-(3-Fluorophenyl)-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C9)

According to general procedure B, the product was obtained as a white solid (73 mg), yield 40%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.50 (s, 1H), 10.44 (s, 1H), 9.03 (s, 1H), 7.45–7.07 (m, 9H), 6.67 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.8, 161.6 (d, J = 244.4 Hz), 154.1, 151.8, 146.8, 140.5, 138.4 (d, J = 10.6 Hz), 135.1, 131.6, 130.1 (d, J = 9.0 Hz), 128.9, 127.6, 126.1 (d, J = 3.1 Hz), 117.2 (d, J = 23.7 Hz), 115.4 (d, J = 20.8 Hz), 103.3, 100.3. HRMS (ESI): m/z calcd for C₂₀H₁₄FN₂O₄ [M + H] ⁺: 365.0932, found 365.0930. HPLC analysis: t_R = 7.986 min, 96.7%.

4.1.58. 5,6,7-Trihydroxy-3-(4-hydroxyphenyl)-2-phenylquinazolin-4(3H)-one (C10)

According to general procedure B, the product was obtained as a white solid (94 mg), yield 52%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.69 (s, 1H), 10.37 (s, 1H), 9.62 (s, 1H), 8.97 (s, 1H), 7.34–7.29 (m, 2H), 7.27–7.19 (m, 3H), 7.10–7.05 (m, 2H), 6.65–6.60 (m, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.3, 157.0, 154.0, 152.6, 146.8, 140.7, 135.6, 131.4, 130.5, 129.0, 128.7, 127.9, 127.5, 115.1, 103.1, 100.5. HRMS (ESI): m/z calcd for C₂₀H₁₅N₂O₅ [M + H] ⁺: 363.0976, found 363.0984. HPLC analysis: t_R = 7.195 min, 98.2%.

4.1.59. 3-(4-Bromo-2-fluorophenyl)-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one(C11)

According to general procedure B, the product was obtained as a white solid (93 mg), yield 42%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.23 (s, 1H), 10.57 (s, 1H), 9.13 (br, 1H), 7.66 (dd, J = 9.4, 2.1 Hz, 1H), 7.53 (t, J = 8.2 Hz, 1H), 7.42 (dd, J = 8.6, 2.1 Hz, 1H), 7.39–7.25 (m, 5H), 6.70 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.1, 156.9 (d, J = 253.0 Hz), 154.4, 151.4, 146.8, 140.3, 134.3, 133.2, 131.9, 129.5, 128.3, 128.0 (d, J = 3.4 Hz), 127.9, 124.2 (d, J = 13.1 Hz), 122.7 (d, J = 9.0 Hz), 119.4 (d, J = 23.2 Hz), 103.7, 99.8. HRMS (ESI): m/z calcd for C₂₀H₁₁ ⁷⁹BrFN₂O₄ [M − H]^–: 440.9892, found 440.9878. HPLC analysis: t_R = 8.670 min, 97.1%.

4.1.60. 3-(4-Fluoro-3-methylphenyl)-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C12)

According to general procedure B, the product was obtained as a white solid (108 mg), yield 57%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.57 (s, 1H), 10.42 (br, 1H), 9.01 (br, 1H), 7.38–7.29 (m, 3H), 7.31–7.13 (m, 4H), 7.06 (t, J = 9.1 Hz, 1H), 6.66 (s, 1H), 2.12 (d, J = 1.9 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.0, 159.8 (d, J = 244.8 Hz), 154.1, 152.0, 146.8, 140.5, 135.2, 132.69 (d, J = 3.4 Hz), 132.66, 132.61, 131.5, 128.9, 128.8 (d, J = 9.2 Hz), 127.5, 124.5 (d, J = 18.8 Hz), 115.0 (d, J = 23.7 Hz), 103.2, 100.3, 14.0 (d, J = 3.1 Hz). HRMS (ESI): m/z calcd for C₂₁H₁₆FN₂O₄ [M + H] ⁺: 379. 1089, found 379.1089. HPLC analysis: t_R = 8.177 min, 96.6%.

4.1.61. 3-([1,1′-biphenyl]-4-yl)-5,6,7-trihydroxy-2-phenylquinazolin-4(3H)-one (C13)

According to general procedure B, the product was obtained as a white solid (74 mg), yield 35%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.61 (s, 1H), 10.44 (s, 1H), 9.02 (s, 1H), 7.67–7.60 (m, 4H), 7.50–7.33 (m, 7H), 7.28–7.19 (m, 3H), 6.68 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 154.1, 152.0, 146.8, 140.7, 139.7, 138.8, 136.2, 135.3, 131.6, 130.1, 129.1, 129.0, 128.9, 127.8, 127.6, 126.7, 126.7, 126.6, 103.3, 100.4. HRMS (ESI): m/z calcd for C₂₆H₁₉N₂O₄ [M + H] ⁺: 423. 1340, found 423. 1345. HPLC analysis: t_R = 10.271 min, 97.1%.

4.1.62. 5,6,7-Trihydroxy-2-phenyl-3-(pyridin-3-yl)quinazolin-4(3H)-one (C14)

According to general procedure B, the product was obtained as a white solid (74 mg), yield 50%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.42 (s, 1H), 10.49 (s, 1H), 9.06 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H), 8.43 (dd, J = 4.8, 1.5 Hz, 1H), 7.88–7.83 (m, 1H), 7.44–7.30 (m, 3H), 7.29–7.19 (m, 3H), 6.69 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.9, 154.2, 151.7, 150.0, 149.0, 146.8, 140.6, 137.2, 134.9, 133.9, 131.7, 129.1, 129.0, 127.7, 123.5, 103.4, 100.2. HRMS (ESI): m/z calcd for C₁₉H₁₄N₃O₄ [M + H] ⁺: 348.0979, found 348.0974. HPLC analysis: t_R = 6.881min, 97.9%.

4.1.63. 5,6,7-Trihydroxy-2-phenyl-3-(1H-pyrazol-4-yl)quinazolin-4(3H)-one (C15)

According to general procedure B, the product was obtained as a white solid (82 mg), yield 49%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 12.86 (br, 1H), 11.62 (s, 1H), 10.44 (s, 1H), 9.00 (s, 1H), 7.69 (br, 1H), 7.42–7.35 (m, 2H), 7.32–7.23 (m, 3H), 6.64 (s, 1H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 154.1, 152.9, 146.8, 140.4, 135.4, 131.5, 128.9, 128.9, 127.6, 117.8, 103.2, 100.2. HRMS (ESI): m/z calcd for C₁₇H₁₁N₄O₄ [M − H] ^–: 335.0786, found 335.0779. HPLC analysis: t_R = 8.686 min, 95.4%.

4.1.64. 5,6,7-Trihydroxy-2-isobutyl-3-phenylquinazolin-4(3H)-one (D6)

According to general procedure B, the product was obtained as a white solid (98 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.53 (s, 1H), 10.27 (s, 1H), 8.82 (s, 1H), 7.62–7.48 (m, 3H), 7.47–7.39 (m, 2H), 6.57 (s, 1H), 2.16 (d, J = 6.9 Hz, 2H), 2.08–1.96 (m, 1H), 0.78 (d, J = 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 153.9, 152.7, 146.7, 140.6, 136.4, 130.8, 129.4, 129.0, 128.9, 102.6, 100.1, 43.2, 25.8, 22.2. HRMS (ESI): m/z calcd for C₁₈H₁₉N₂O₄ [M + H]⁺: 327.1339, found 327.1345. HPLC analysis: t_R = 8.174 min, 99.8%.

4.1.65. 3-(2-Fluorophenyl)-5,6,7-trihydroxy-2-isobutylquinazolin-4(3H)-one (D7)

According to general procedure B, the product was obtained as a white solid (107 mg), yield 62%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.26 (s, 1H), 10.43 (br, 1H), 7.71–7.58 (m, 2H), 7.54–7.46 (m, 1H), 7.42 (t, J = 7.4 Hz, 1H), 6.60 (s, 1H), 2.29–2.12 (m, 2H), 1.99 (hept, J = 6.7 Hz, 1H), 0.80 (dd, J = 8.2, 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 163.9, 157.3 (d, J = 248.5 Hz), 154.4, 153.1, 146.7, 145.9, 139.3, 132.0 (d, J = 7.9 Hz), 131.3 (d, J = 43.4 Hz), 125.5 (d, J = 3.6 Hz), 123.4 (d, J = 13.4 Hz), 116.5 (d, J = 19.4 Hz), 102.3, 99.6, 42.7, 25.9, 22.1 (d, J = 7.7 Hz). HRMS (ESI): m/z calcd for C₁₈H₁₇FN₂O₄ [M − H]^–: 343.1100, found 343.1090. HPLC analysis: t_R = 9.123 min, 96.3%.

4.1.66. 3-(4-Fluoro-3-methylphenyl)-5,6,7-trihydroxy-2-isobutylquinazolin-4(3H)-one (D8)

According to general procedure B, the product was obtained as a white solid (107 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.51 (s, 1H), 10.31 (s, 1H), 8.84 (s, 1H), 7.42–7.37 (m, 1H), 7.34–7.29 (m, 2H), 6.56 (s, 1H), 2.28 (d, J = 2.1 Hz, 3H), 2.17 (d, J = 6.3 Hz, 2H), 2.11–1.98 (m, 1H), 0.81 (dd, J = 6.6, 1.9 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 160.5 (d, J = 245.1 Hz), 154.0, 152.7, 146.7, 140.6, 132.2 (d, J = 3.2 Hz), 132.0 (d, J = 5.8 Hz), 130.9, 128.3 (d, J = 8.8 Hz), 125.7 (d, J = 18.7 Hz), 115.9 (d, J = 23.7 Hz), 102.6, 100.1, 43.1, 25.8, 22.3 (d, J = 5.4 Hz), 14.1 (d, J = 2.9 Hz). HRMS (ESI): m/z calcd for C₁₉H₁₈FN₂O₄ [M − H]^–: 357.1256, found 357.1252. HPLC analysis: t_R = 10.674 min, 95.2%.

4.1.67. 3-(4-Bromo-2-fluorophenyl)-5,6,7-trihydroxy-2-isobutylquinazolin-4(3H)-one (D9)

According to general procedure B, the product was obtained as a white solid (116 mg), yield 55%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.17 (s, 1H), 10.43 (br, 1H), 7.94–7.87 (m, 1H), 7.70–7.61 (m, 2H), 6.60 (s, 1H), 2.29–2.11 (m, 2H), 2.01 (hept, J = 6.6 Hz, 1H), 0.82 (dd, J = 7.8, 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.1, 157.2 (d, J = 253.0 Hz), 154.3, 152.0, 146.7, 140.2, 132.6, 131.3, 128.8 (d, J = 3.5 Hz), 123.3, 123.2 (d, J = 5.4 Hz), 120.1(d, J = 23.2 Hz), 102.9, 99.5, 42.8, 25.7, 22.1 (d, J = 2.7 Hz). HRMS (ESI): m/z calcd for C₁₈H₁₅ ⁷⁹BrFN₂O₄ [M − H]^–: 421.0205, found 421.0200. HPLC analysis: t_R = 10.166 min, 96.6%.

4.1.68. General procedure C for the preparation of D1–D4, D10 and D11

To a solution of compound 33a or 33b (1 mmol) in anhydrous acetonitrile (8 mL) was added the corresponding anilines (1.2 mmol) and PCl₃ (0.18 mL, 2 mmol) at 0 °C, and the resulting suspension was heated to 50 °C and stirred for 4–12 h. Then, the reaction was moved to 0 °C and was quenched by adding 1 N aqueous HCl solution. The aqueous portion was extracted with DCM (3 × 20 mL), and the combined organics were washed with 10% aqueous NaHCO₃ solution and brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica column chromatography (petroleum ether/EtOAc = 8:1 to 6:1) to give the corresponding quinazolinones 35a–35f.

According to general procedure A, the demethylation of the above quinazolinones afforded the target compounds D1–D4, D10 and D11.

4.1.69. 5,6,7-Trihydroxy-3-phenyl-2-(1-phenylethyl)quinazolin-4(3H)-one (D1)

According to general procedure C, the product was obtained as a white solid (118 mg), yield 63%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.49 (s, 1H), 10.39 (s, 1H), 8.92 (s, 1H), 7.57 (d, J = 4.5 Hz, 2H), 7.49–7.40 (m, 1H), 7.23–7.12 (m, 4H), 6.80 (dd, J = 6.6, 2.9 Hz, 2H), 6.68 (s, 1H), 6.54–6.48 (m, 1H), 3.81 (q, J = 6.8 Hz, 1H), 1.46 (d, J = 6.9 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.2, 155.0, 154.0, 146.7, 142.3, 140.3, 135.8, 131.2, 129.6, 129.4, 128.9, 128.7, 128.4, 127.1, 126.6, 103.1, 100.3, 43.2, 21.8.

HRMS (ESI): m/z calcd for C₂₂H₁₉N₂O₄ [M + H]⁺: 375. 1339, found 375. 1345. HPLC analysis: t_R = 9.099 min, 99.1%.

4.1.70. 3-(4-Fluoro-3-methylphenyl)-5,6,7-trihydroxy-2-(1-phenylethyl)quinazolin-4(3H)-one (D2)

According to general procedure C, the product was obtained as a white solid (106 mg), yield 52%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.46 (s, 1H), 10.40 (s, 1H), 8.92 (s, 1H), 7.54–7.48 (m, 0.4 H), 7.49–7.39 (m, 0.6 H), 7.32 (t, J = 9.0 Hz, 0.6 H), 7.23–7.11 (m, 3H), 6.94 (t, J = 9.1 Hz, 0.4H), 6.88–6.81 (m, 1H), 6.80–6.72 (m, 1H), 6.68 (d, J = 4.5 Hz, 1H), 6.36–6.28 (m, 0.4 H), 6.20–6.14 (m, 0.6H), 3.89–3.74 (m, 1H), 2.31 (s, 1.2 H), 1.91 (s, 1.9 H), 1.45 (dd, J = 6.9, 3.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.2, 161.6 (d, J = 5.0 Hz), 159.2 (d, J = 5.0 Hz), 154.9 (d, J = 8.7 Hz), 154.1 (d, J = 1.9 Hz), 146.7, 142.6, 142.4, 140.3 (d, J = 1.2 Hz), 133.1 (d, J = 6.0 Hz), 131.7 (d, J = 5.7 Hz), 131.5 (d, J = 3.3 Hz), 131.4 (d, J = 3.2 Hz), 131.2, 128.9 (d, J = 8.9 Hz), 128.4 (d, J = 4.7 Hz), 128.0 (d, J = 8.8 Hz), 127.1 (d, J = 6.9 Hz), 126.6 (d, J = 7.8 Hz), 125.5 (d, J = 18.7 Hz), 124.7 (d, J = 18.9 Hz), 115.7 (d, J = 23.9 Hz), 115.0 (d, J = 23.6 Hz), 103.2, 103.1, 100.2, 43.7, 43.3, 22.0, 21.9, 14.2 (d, J = 2.9 Hz), 13.8 (d, J = 3.1 Hz). HRMS (ESI): m/z calcd for C₂₃H₂₀FN₂O₄ [M + H]⁺: 407.1402, found 407.1395. HPLC analysis: t_R = 13.337 min, 96.5%.

4.1.71. 3-(2-Fluorophenyl)-5,6,7-trihydroxy-2-(1-phenylethyl)quinazolin-4(3H)-one (D3)

According to general procedure C, the product was obtained as a white solid (110 mg), yield 56%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.28 (s, 1H), 10.45 (s, 1H), 8.96 (s, 1H), 7.81–7.71 (m, 1H), 7.58–7.44 (m, 1H), 7.43–7.35 (m, 1H), 7.26–7.03 (m, 3H), 7.03–6.93 (m, 1H), 6.72 (s, 1H), 6.71–6.60 (m, 2H), 3.95 (q, J = 6.7 Hz, 1H), 1.47 (d, J = 6.8 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.4, 157.7 (d, J = 250.6 Hz), 154.5, 154.3, 146.7, 141.3, 140.2, 131.6 (d, J = 8.0 Hz), 131.5, 130.6, 128.5, 128.3, 126.8, 126.7, 125.0 (d, J = 3.5 Hz), 123.3 (d, J = 13.4 Hz), 116.1, 115.9, 103.4, 99.7, 43.5, 21.9. HRMS (ESI): m/z calcd for C₂₂H₁₈FN₂O₄ [M + H]⁺: 393. 1245, found 393. 1251. HPLC analysis: t_R = 8.657 min, 96.6%.

4.1.72. 3-(4-Bromo-2-fluorophenyl)-5,6,7-trihydroxy-2-(1-phenylethyl)quinazolin-4(3H)-one (D4)

According to general procedure C, the product was obtained as a white solid (141 mg), yield 60%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.18 (s, 1H), 10.48 (s, 1H), 8.98 (s, 1H), 7.77 (t, J = 8.2 Hz, 1H), 7.63 (dd, J = 8.5, 2.1 Hz, 1H), 7.32 (dd, J = 9.3, 2.1 Hz, 1H), 7.17–7.09 (m, 3H), 6.74–6.68 (m, 3H), 3.95 (q, J = 6.7 Hz, 1H), 1.47 (d, J = 6.8 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 164.1, 158.6, 156.6, 154.3, 154.1, 146.7, 141.2, 140.0, 132.1, 131.5, 128.4, 128.3 (d, J = 3.5 Hz), 126.8, 126.7, 123.1–122.9 (m), 119.3 (d, J = 23.2 Hz), 103.5, 99.6, 43.6, 21.9. HRMS (ESI): m/z calcd for C₂₂H₁₇ ⁷⁹BrFN₂O₄ [M + H]⁺: 471.0350, found 471.0352. HPLC analysis: t_R = 13.244 min, 99.4%.

4.1.73. 2-Benzyl-5,6,7-trihydroxy-3-phenylquinazolin-4(3H)-one (D10)

According to general procedure C, the product was obtained as a white solid (112 mg), yield 62%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.48 (s, 1H), 10.33 (s, 1H), 8.88 (s, 1H), 7.49–7.38 (m, 3H), 7.23–7.14 (m, 5H), 6.90–6.82 (m, 2H), 6.59 (s, 1H), 3.75 (s, 2H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.1, 154.0, 152.2, 146.7, 140.5, 136.0, 135.8, 131.2, 129.1, 129.0, 128.5, 128.2, 126.6, 102.8, 100.3, 41.3. HRMS (ESI): m/z calcd for C₂₁H₁₇N₂O₄ [M + H]⁺: 361.1183, found 361.1190. HPLC analysis: t_R = 8.737 min, 95.8%.

4.1.74. 2-Benzyl-3-(4-fluoro-3-methylphenyl)-5,6,7-trihydroxyquinazolin-4(3H)-one (D11)

According to general procedure C, the product was obtained as a white solid (88 mg), yield 45%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.44 (s, 1H), 10.35 (s, 1H), 8.89 (s, 1H), 7.23–7.16 (m, 4H), 7.14–7.08 (m, 1H), 6.94 (dd, J = 7.0, 2.6 Hz, 1H), 6.88–6.83 (m, 2H), 6.60 (s, 1H), 3.77 (q, J = 15.5 Hz, 2H), 2.12 (d, J = 1.8 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.6, 160.9 (d, J = 245.1 Hz), 154.5, 152.8, 147.2, 140.9, 136.2, 132.8 (d, J = 5.8 Hz), 132.1 (d, J = 3.2 Hz), 131.7, 128.9, 128.7 (d, J = 9.0 Hz), 128.6, 127.0, 125.6 (d, J = 18.8 Hz), 116.0 (d, J = 23.9 Hz), 103.3, 100.7, 42.0, 14.4 (d, J = 2.9 Hz). HRMS (ESI): m/z calcd for C₂₅H₁₈FN₂O₄ [M + H]⁺: 393.1245, found 393.1249. HPLC analysis: t_R = 12.068 min, 96.2%.

4.1.75. General procedure D for the preparation of D5 and D12

To the stirred solution of the prepared 2-phenylpropanoyl chloride or commercially available phenylacetyl chloride (1 mmol) in pyridine (5 mL) was added TPP (816 mg, 2.5 mmol) under the N₂ atmosphere at 0 °C, and the reaction mixture was heated to 70 °C and stirred for 2 h. Then the reaction mixture was cooled to room temperature, and was treated with isobutylamine (40) (0.12 mL, 1.2 mmol). Then the resulting mixture was heated to 70 °C and stirred overnight. After completion, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the residue. The residue was then diluted with DCM (50 mL), washed with 3 N aqueous HCl solution (1 × 20 mL) and brine (3 × 20 mL), and dried anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 6:1) to give the corresponding quinazolinones 37a, 37b.

According to general procedure A, the demethylation of the above quinazolinones afforded the target compounds D5, D12.

4.1.76. 5,6,7-Trihydroxy-3-isobutyl-2-(1-phenylethyl)quinazolin-4(3H)-one (D5)

According to general procedure D, the product was obtained as a white solid (97 mg), yield 55%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.75 (s, 1H), 10.30 (s, 1H), 8.83 (s, 1H), 7.36–7.20 (m, 5H), 6.61 (s, 1H), 3.80 (d, J = 15.5 Hz, 1H), 3.73 (d, J = 15.5 Hz, 1H), 4.02–3.93 (m, 1H), 3.50 (dd, J = 14.0, 7.1 Hz, 1H), 2.04 (hept, J = 6.9 Hz, 1H), 1.56 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.3, 155.1, 153.9, 146.3, 142.7, 139.9, 131.1, 128.9, 127.1, 126.9, 102.6, 100.0, 48.0, 42.3, 28.1, 22.5, 19.7, 19.6. HRMS (ESI): m/z calcd for C₂₀H₂₃N₂O₄ [M + H]⁺: 355.1652, found 355.1655. HPLC analysis: t_R = 11.131 min, 96.2%.

4.1.77. 2-Benzyl-5,6,7-trihydroxy-3-isobutylquinazolin-4(3H)-one (D12)

According to general procedure D, the product was obtained as a white solid (95 mg), yield 56%. ¹H NMR (400 MHz, DMSO‑d ₆) δ 11.74 (s, 1H), 10.30 (s, 1H), 8.84 (s, 1H), 7.36–7.30 (m, 2H), 7.28–7.22 (m, 3H), 6.52 (s, 1H), 4.18 (s, 2H), 3.77 (d, J = 7.5 Hz, 2H), 2.08 (hept, J = 7.0 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO‑d ₆) δ 165.3, 153.9, 152.6, 146.3, 140.3, 136.2, 131.0, 128.7, 128.4, 126.8, 102.3, 100.1, 49.0, 40.6, 27.8, 19.7. HRMS (ESI): m/z calcd for C₁₉H₂₁N₂O₄ [M + H]⁺: 341.1496, found 341.1495. HPLC analysis: t_R = 11.644 min, 97.8%.

4.2. SARS-CoV-2 M^pro protein expression and purification

The cDNA of SARS-CoV-2 M^pro (GeneBank: YP_009725301.1) was synthesized and cloned into pET-28a (+) vector with codon optimization by Sangon (Sangon Biotech, Shanghai, China). The SARS-CoV-2 M^pro construct contains protease autoprocessing site of M^pro (SAVLQ⬇SGFRK, arrow indicating the cleavage site) and PreScission site (SGVTFQ⬇GP, arrow indicating the cleavage site) in the N terminus and C terminus, respectively. The pET-28a(+)-SARS-CoV-2 M ^pro plasmid was then transformed into competent E.coli BL21 (DE3) cells and a single colony was picked, which was inoculated in 5 ml LB medium supplemented with 50 g/mL kanamycin and grown at 37 °C to an optical density at 600 nm of 0.8, and then induced using 0.5 mM isopropyl-β-d-thiogalactoside (IPTG). Induced cultures were grown at 25 °C for an additional 16 h. Cells were harvested via centrifugation at 10,000 rpm for 10 min and the precipitate were resuspended in a buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole) and homogenized with an ultrahigh-pressure cell disrupter at 4 °C. The insoluble material was removed by centrifugation at 18,000 rpm for 60 min. The protein was eluted by elution buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 300 mM imidazole) after incubation with Ni-resin and washed by buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole). After auto-cleaved and cleaved by PreScission protease at 4 °C overnight, native M^pro was generated. In order to obtain homogeneous and pure protein suitable for crystallization and enzymatic inhibition assays, the further purification using Q column was performed with A buffer (20 mM HEPES, pH 7.5, 50 mM NaCl) and B buffer (20 mM HEPES, pH 7.5, 1 M NaCl). Protein sample was concentrated into 10 mg/mL and stored at −80 °C.

4.3. SARS-CoV-2 M^pro enzymatic assays

The enzyme activity of SARS-CoV-2 M^pro was determined using a FRET assay, which was performed as previously reported [37]. The FRET assay uses fluorogenic peptide Dabcyl-KLSAVLQSGFRKM-Edans-NH₂ as the substrate, which was custom synthesized and obtained from GL Biochem Ltd (Shanghai, China). In brief, protease SARSCov-2 M^pro (600 nM at a final concentration) was mixed with compounds at the indicated concentration in 50 μL of assay buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 150 mM NaCl) and incubated at room temperature for 30 min. Then, the reaction was initiated by adding 50 μL of peptide substrate to the mixture (20 μM at a final concentration) and monitored at 340 nM (excitation)/490 nM (emission) with a Tecan's Spark multimode reader. The Vmax of reactions with compounds added at the indicated concentrations compared to the solvent control was calculated and used to generate IC₅₀ curves. For each compound, the half-maximal inhibitory concentration (IC₅₀) values against SARS-CoV-2 M^pro were measured at 10 different concentrations, and three independent experiments were performed. The data were analyzed using GraphPad Prism 8.4.3 software.

4.4. Cellular antiviral assays

A clinical isolate of SARS-CoV-2 (GenBank: MT121215.1) was propagated in Vero E6 cells and the viral titer was determined as 50% tissue culture infectious dose (TCID₅₀) per milliliter (mL) by CPE quantification. All the infection experiments were performed at biosafety level-3 (BLS-3) as previously reported [38]. Pre-seeded Vero E6 cells (5 × 10⁴ cells/well) were incubated with test compounds at the indicated concentrations at 37 °C in 5% CO₂ for 1 h. Then, 100 μL of the cell supernatant was removed, followed by addition of the prepared virus-drug mixture (100 μL) to the final virus titer of 100 TCID₅₀/well. After infection for 1 h, the supernatant medium was discarded, and the cells were washed twice with PBS and further cultured with maintenance medium at 37 °C in 5% CO₂ for 48 h. At 48 h postinfection, the cell supernatant was collected and lysed using TRIzol LS Reagent (Invitrogen, Carlsbad, USA). Subsequently, RNA was extracted using a PureLink™ RNA Mini Kit (Thermo Fisher, Waltham, USA). The viral RNA copy number was quantified by RT-qPCR using Verso SYBR Green 1-Step qRT-PCR Kit (Thermo Fisher, Waltham, USA) on CFX96™ Real-Time PCR System (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. The PCR primers targeting the N gene (nt608-706) of SARS-CoV-2 are as follows:

5′-GGGGAACTTCTCCTGCTAGAAT-3’;

5′-CAGACATTTTGCTCTCAAGCTG-3’.

4.5. SARS-CoV-2 M^pro protein crystallization and structure determination

8 mg/mL and 12 mg/mL SARS-CoV-2 M^pro in buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 150 mM NaCl) were incubated with 2 mM compound D8 at 4 °C for 18 h. Then, the mixture was centrifuged at 12,500 rpm for 10 min to remove the precipitate, and the supernatant was used for crystallization. All crystallization experiments were set up at 293 K using the hanging drop approach where 1 μL of each precipitant solution was mixed with 1 μL of protein solution. Crystals were observed after a few days of incubation in 12% PEG 20,000, 0.1 M MES monohydrate (pH 6.5), which were next immersed in the crystallization solution containing 15% glycerol, and were frozen in liquid nitrogen before diffraction. The diffraction data sets were collected at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) on beamline BL18U1. Using the molecular-replacement method, the structure was resolved with the Phenix program Phaser-MR [39,40], and the electron density could be clearly observed in the protein catalytic site. The eLBOW and LigandFit program in Phenix was used to fit the ligand D8 [41]. Next, adjustment of the structural model was performed in Coot, and structure refinement in Phenix was conducted with NCS restrains [42,43]. Finally, the MolProbity program in Phenix was used to check the quality of the resulting structure [44]. Data collection and structural refinement statistics are depicted in Table S3.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. Z Liu

Abbreviations

CCK-8

cell counting kit-8

COVID-19

Coronavirus Disease 2019

DTT

dithiothreitol

DMPK

drug metabolism and pharmacokinetics

FRET

fluorescence resonance energy transfer

HLM

human liver microsome

IPTG

isopropyl-β-d-thiogalactoside

M^pro

main protease

nsp

non-structural protein

PBS

phosphate buffer solution

PPB

plasma protein binding

SAR

structure activity relationship

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

TPP

triphenyl phosphite

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supporting data to this article can be found online.

Data availability

Data will be made available on request.