Discovery, Crystallographic Studies, and Mechanistic Investigations of Novel Phenylalanine Derivatives Bearing a Quinazolin-4-one Scaffold as Potent HIV Capsid Modulators

Abstract

Optimization of compound 11L led to the identification of novel HIV capsid modulators, quinazolin-4-one-bearing phenylalanine derivatives, displaying potent antiviral activities against both HIV-1 and HIV-2. Notably, derivatives 12a2 and 21a2 showed significant improvements, with 2.5-fold over 11L and 7.3-fold over PF74 for HIV-1, and approximately 40-fold over PF74 for HIV-2. The X-ray co-crystal structures confirmed the multiple pocket occupation of 12a2 and 21a2 in the binding site. Mechanistic studies revealed a dual-stage inhibition profile, where the compounds disrupted capsid-host factor interactions at the early stage and promoted capsid misassembly at the late stage. Remarkably, 12a2 and 21a2 significantly promoted capsid misassembly, outperforming 11L, PF74, and LEN. The substitution of easily metabolized amide bond with quinolin-4-one marginally enhanced the stability of 12a2 in human liver microsomes compared to controls. Overall, 12a2 and 21a2 highlight their potential as potent HIV capsid modulators, paving the way for future advancements in anti-HIV drug design.

Graphical Abstract

1. INTRODUCTION

Acquired immune deficiency syndrome, also known as AIDS, is a contagious illness that stems from the human-acquired immune deficiency virus (HIV), posing significant threats to human health and social development.¹ Based on genotypic differences, HIV can be classified into two primary subtypes: HIV-1 and HIV-2. HIV-1 is the globally predominant strain noted for its high infectivity and pathogenicity.^2,3 HIV-2 primarily exists in West Africa and countries with socioeconomic ties to the region.^4,5 Currently, it is estimated that approximately 2 million individuals worldwide who are infected with HIV-2, accounting for about 5% of the global HIV burden.⁶ Furthermore, the escalating incidence of HIV-2 infections across diverse geographical locations requires vigilant monitoring and robust response. Though combination antiretroviral therapy (cART) has shown substantial efficacy against HIV infection, hurdles such as the rise of multi-drug-resistant HIV strains, long-term toxicity, limited accessibility in economically disadvantaged regions, and barriers to treatment adherence impede its broad application.^7-9 Thus, exploring novel anti-HIV medications to diversify and bolster the available treatment options is crucial.

The HIV-1 capsid (CA) protein has garnered considerable interest as a promising target due to its pivotal roles in both structural and regulatory aspects.¹⁰ In the early stage, productive HIV-1 infection entails numerous CA-mediated post-entry events up to the completion of the integration, including uncoating, reverse transcription, nuclear entry, and selection of the integration sites.^11,12 These events often involve the interaction of CA with host factors like cleavage and polyadenylation specific factor 6 (CPSF6), nucleoporins 153 and 358 (NUP153, NUP358), cyclophilin A (CypA), and Sec24C.^13-16 These interactions have the added advantage of shielding the capsid from the innate immune system.¹⁷ In the late stage, individual CA monomers assemble into pentamers and hexamers that form through capsid–capsid interactions, creating the fullerene capsid cone that forms the inner shell of the retrovirus.¹⁸ A mature HIV CA comprises approximately 250 hexamers and 12 pentamers. This assembled structure is crucial for viral infectivity;^19,20 hence, small molecules targeting CA can potentially disrupt multiple virus replication processes.

The CA monomer is comprised of two main helical domains: the N-terminal domain (NTD, residues 1–145) and the C-terminal domain (CTD, residues 150–231), connected by a flexible linker.²¹ Vulnerable regions within this structure have been identified for HIV-1 CA modulation, the PF74 binding pocket being one of significance.²² PF74 binds to the interface formed between NTD and the adjacent CTD within a CA hexamer, its binding site also accommodating CPSF6 and NUP153, which are required for nuclear entry and integration.²³ Despite PF74’s unique antiviral profiles, its poor antiviral activity (EC₅₀ = 0.87 μM, MT-4 cells) and metabolic instability have hindered further studies.²⁴ Lenacapavir (LEN, Figure 1), a multi-round iterative modification of PF74 by Gilead Science, has picomolar-level anti-HIV activity (EC₅₀ = 105 pM, MT-4 cells) and excellent metabolic stability and is the first FDA-approved HIV-1 CA inhibitor.^25,26 However, resistant strains insensitive to LEN have emerged during clinical trials and in vitro screening, and its complex synthesis and high administration costs have limited its clinical application.^27,28 Therefore, the challenge remains to discover new structural types of HIV-1 CA modulators that can be seamlessly integrated into current cART regimens.

Figure 1.

(A) Chemical structures and anti-HIV-1 activities of PF74 and LEN. (B) Crystal structure of PF74 (PDB code: 4XFZ) bound at the dimeric interface of a CA hexamer. (C) Crystal structures of PF74 and LEN (PDB code: 6V2F) bound at the dimeric interface of two CA monomers. (D) Crystallographic overlay of PF74 and LEN. The figures were generated in PyMOL (www.pymol.org).

Previously, we attempted to enhance the antiviral activity and metabolic stability of PF74 by replacing the indole moiety with a benzenesulfonamide moiety, resulting in compound 11L.²⁴ This molecule exhibited improved activity but only marginal enhancements in microsomal stability. While a significant step forward in the continued development of HIV-1 CA modulators, room for optimization remains. Assessment of the 11L-CA complex structure (PDB code: 8F22) reveals ample space that can be exploited in the subsequent modification of 11L. According to the structural characteristics of 11L and the distribution of amino acids around it, the ligand binding sites can be divided into three regions: regions I–III (Figure 2). Region I (within the black dotted line, hotspot residues include Gln67, Lys70, Glu71, Tyr169, Thr186, Arg173, and Gln176) is located within the NTD-CTD interface region between adjacent subunits. Contacts within this region significantly contribute to the improved activity of 11L compared to PF74. We will continue to maintain this privileged structure in the new series. Region II (blue dashed line with hot spot residues such as Gln50, Thr54, and Ser41) is located at the NTD-NTD interface of adjacent subunits, and region III (red dashed line with hot spot residues such as Asn74, Thr107, and Tyr130) is located on the surface of the NTD. We also used a conformational restriction strategy to enhance the metabolic stability of 11L, cyclizing the quickly metabolized amide bond to achieve a quinazolin-4-one structure and introducing substituents (R₂) targeting the NTD-NTD interface.²⁹ Finally, apart from the methoxy group, which proves to feature more favorable antiviral activity,³⁰ we also introduced 4-sulfonylmorpholine and 4-sulfonyl-thiomorpholine-1,1-dioxide groups (R₃) in region III to explore more interactions. Overall, based on the co-crystal structure of 11L in complex with the CA hexamer, this study combined privileged structure repositioning and conformational restriction strategies with a multi-site binding strategy to target hot spot residues in regions I–III to optimize the lead compound 11L. This holistic approach aimed at disrupting the interactions between CA protein subunit interfaces, thereby altering CA assembly dynamics and blocking virus replication.

Figure 2.

Design strategy of novel phenylalanine derivatives bearing quinazolin-4-one scaffold as HIV CA modulators (PDB code: 8F22). The figure was generated in PyMOL (www.pymol.org).

All synthesized compounds were evaluated for their anti-HIV activity and cytotoxicity using the MTT method in MT-4 cells infected with WT HIV-1 strain (III_B) and HIV-2 strain (ROD). The results enabled us to establish a structure–activity relationship (SAR). Moreover, we conducted surface plasmon resonance (SPR) assays and crystallographic studies to validate the target and binding modes, and performed single-round infection (SRI) assays to investigate the action stages of representative compounds. We further explored the early and late-stage mechanisms of action through RT inhibition assays, SPR-based competition assays with CPSF6, in vitro CA assembly assays, enzyme-linked immunosorbent assay (ELISA)-based quantification of p24 content assays. Finally, we performed preliminary drug-likeness evaluation, including metabolic stability predicted in silico and assessed in human liver microsome (HLM) and human plasma in vitro, as well as pharmacokinetic (PK) profiles in vivo, for some promising compounds.

2. CHEMISTRY

Intermediate 4 was synthesized following the procedures outlined in Scheme 1, commercially available 4-nitrobenzene-sulfonyl chloride (1) reacted with piperazin-2-one in dichloromethane (DCM) under the action of triethylamine (TEA) through acylation reaction to get intermediate 2; Then, in the presence of sodium hydride (NaH), a nucleophilic substitution (S_N2) reaction took place between intermediate 2 and methyl bromoacetate, leading to the production of intermediate 3. Finally, intermediate 3 underwent a LiOH-mediated hydrolysis for conversion into intermediate 4.

Scheme 1. Preparation of Intermediate 4 ^a.

^aReagents and conditions: (i) piperazin-2-one, TEA, DCM, 0 °C to r.t. Yield: 90%; (ii) methyl bromoacetate, NaH, THF, 0 °C to r.t. Yield: 85%; (iii) LiOH, THF: H₂O = 1:1, r.t. Yield: 88%.

The synthesis of target compounds 11a1–11e1, 11a2–11e2, 12a1–12c1, 12e1, 12a2–12c2, and 12e2 were accomplished through a pathway described in Scheme 2. Starting with the corresponding substituted 2-nitro-benzoyl chloride (5a–5e), a well-established strategy involving the presence of TEA was employed to react it with the p-anisidine, resulting in the formation of intermediates 6a–6e. Next, the amino intermediates 7a–7e were prepared via the nitro reduction catalyzed by 10% palladium carbon from intermediates 6a–6e. Subsequently, coupling of N-Boc-l-phenylalanine or N-Boc-l-3,5-difluorophenylalanine with intermediates 7a–7e in DCM afforded intermediates 8a1–8e1 and 8a2–8e2. Then, intermediates 9a1–9e1 and 9a2–9e2 were obtained by cyclization reaction under the action of N,O-bis(trimethylsilyl)acetamide (BSA), N,N-diisopropylethylamine (DIEA), and 4-dimethylaminopyridine (DMAP) with acetonitrile as solvent. The Boc protecting group was removed using trifluoroacetic acid (TFA) to produce intermediates 10a1–10e1 and 10a2–10e2, which were used for the subsequent amide coupling with intermediate 4 to produce target compounds 11a1–11e1 and 11a2–11e2. Finally, the target compounds 12a1–12c1, 12e1, 12a2–12c2, and 12e2 were obtained by hydrogenation reduction reaction of 11a1–11e1 and 11a2–11e2 in DCM under the catalysis of 10% palladium carbon.

Scheme 2. Preparation of Target Compounds 11a1–11e1,11a2–11e2, 12a1–12c1, 12e1, 12a2–12c2, and 12e2^a.

^aReagents and conditions: (i) p-anisidine, TEA, DCM, 0 °C to r.t. Yield: 75%–81%; (ii) H₂, 10%Pd·C, DCM, r.t. Yield: 66%–72%; (iii) N-Boc-l-phenylalanine or N-Boc-l-3,5-difluorophenylalanine, 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), DIEA, DCM, 0 °C to r.t. Yield: 68%–81%; (iv) BSA, DIEA, DMAP, CH₃CN, 80 °C, reflux. Yield: 58%–72%; (v) TFA, DCM, r.t. Yield: 65%–77%; (vi) intermediate 4, HATU, DIEA, DCM, 0 °C to r.t. Yield: 46%–66%; (vii) H₂, 10%Pd·C, DCM, r.t. Yield: 29%–68%.

The synthetic route for the preparation of target compounds 20a1–20b1, 20a2–20b2, 21a1–21b1, and 21a2–21b2 is illustrated in Scheme 3. Treating the commercially available 4-nitrobenzenesulfonyl chloride (1) with morpholine or thiomorpholine-1,1-dioxide in TEA and DCM to give intermediates 13a–13b, followed by the nitro reduction, afforded the free amines 14a–14b. Intermediates 17a1–17b1 and 17a2–17b2 were obtained through acylation, reduction, and condensation reactions involving intermediates 14a–14b. Then the key intermediates 18a1–18b1 and 18a2–18b2 were generated by a cyclization reaction under the action of BSA, DIEA, and DMAP with acetonitrile as solvent. After removing the Boc protecting group from intermediates 18a1–18b1 and 18a2–18b2 using TFA, the target compounds 20a1–20b1 and 20a2–20b2 were obtained by amide condensation reaction with intermediate 4. Finally, the target compounds 21a1–21b1 and 21a2–21b2 were synthesized via a hydrogenation reduction reaction of 20a1–20b1 and 20a2–20b2 in DCM under 10% palladium carbon catalysis.

Scheme 3. Preparation of Target Compounds 20a1–20b1, 20a2–20b2, 21a1-21b1, and 21a2-21b2^a.

^aReagents and conditions: (i) morpholine or thiomorpholine-1,1-dioxide, TEA, DCM, 0 °C to r.t. Yield: 78%–79%; (ii) H₂, 10%Pd·C, DCM, r.t. Yield: 70%–72%; (iii) 2-nitrobenzoyl chloride, TEA, DCM, 0 °C to r.t. Yield: 69%–73%; (iv) H₂, 10%Pd·C, DCM, r.t. Yield: 75%–77%; (v) N-Boc-l-phenylalanine or N-Boc-l-3,5-difluorophenylalanine, HATU, DIEA, DCM, 0 °C to r.t. Yield: 72%–78%; (vi) BSA, DIEA, DMAP, CH₃CN, 80 °C, reflux. Yield: 69%–77%; (vii) TFA, DCM, r.t. Yield: 59%–66%; (viii) intermediate 4, HATU, DIEA, DCM, 0 °C to r.t. Yield: 48%–67%; (ix) H₂, 10%Pd·C, DCM, r.t. Yield: 32%–38%.

3. RESULTS AND DISCUSSION

3.1. In Vitro Anti-HIV Assays and Structure–Activity Relationship (SAR) Analysis.

The newly synthesized compounds in this work were evaluated for their antiviral activities and cytotoxicities using MT-4 cells infected by HIV-1 III_B and HIV-2 ROD. PF74 and 11L were utilized as controls in this assay. We quantified the biological evaluation results in terms of EC₅₀ values (anti-HIV activities), CC₅₀ values (cytotoxicity), and SI values (selectivity index, the ratio of CC₅₀ to EC₅₀).

3.1.1. SAR Analysis of the Newly Synthesized Compounds against HIV-1.

As depicted in Table 1, the majority of the tested compounds displayed remarkable activity in inhibiting HIV-1 III_B strain replication with submicromolar to low micromolar levels, of which five compounds surpassed the activity of 11L (EC₅₀ = 0.28 μM), and 12 compounds outperformed PF74 (EC₅₀ = 0.80 μM). Compounds 12a2 and 21a2 were the most potent HIV-1 CA modulators with EC₅₀ values of 0.11 μM. This significantly improved potency over the control compounds, specifically 2.5-fold over 11L and 7.3-fold over PF74. All tested compounds also exhibited high selectivity indices, indicating a desirable balance between potency and cytotoxicity.

Table 1.

Anti-HIV Activity against HIV-1 III_B and HIV-2 ROD Strains and Cytotoxicity of the Target Compounds 11a1–11e1, 11a2–11e2, 12a1–12c1, 12e1, 12a2–12c2, 12e2, 20a1, 21a2, 20b1, and 21b2 in MT-4 Cells

Compd	R1	R2	R3	R4	EC50a(μM)		Ratiob	CC50c(μM)	SId
					IIIB	ROD			IIIB	ROD
11a1	H	H	OCH3	NO2	1.51 ± 0.42	0.57 ± 0.07	2.65	>179.41	>119.05	>312.50
11b1	F	H	OCH3	NO2	1.99 ± 0.08	0.56 ± 0.14	3.55	>174.89	>88.03	>312.50
11c1	Cl	H	OCH3	NO2	2.16 ± 0.26	0.42 ± 0.04	5.14	>170.96	>79.11	>403.23
11d1	Br	H	OCH3	NO2	2.40 ± 0.81	0.36 ± 0.03	6.67	>161.16	>67.20	>446.43
11e1	SO2CH3	H	OCH3	NO2	2.77 ± 0.50	0.32 ± 0.03	8.66	>161.33	>58.14	>500.00
11a2	H	F	OCH3	NO2	0.53 ± 0.15	0.41 ± 0.04	1.29	>170.60	>320.51	>416.67
11b2	F	F	OCH3	NO2	0.49 ± 0.08	0.35 ± 0.03	1.40	>166.51	>337.84	>480.77
11c2	Cl	F	OCH3	NO2	0.59 ± 0.14	0.40 ± 0.10	1.48	>162.94	>277.78	>403.23
11d2	Br	F	OCH3	NO2	0.99 ± 0.25	0.59 ± 0.20	1.68	>154.01	>156.25	>260.42
11e2	SO2CH3	F	OCH3	NO2	1.84 ± 0.25	0.41 ± 0.05	4.49	>154.17	>83.89	>378.79
12a1	H	H	OCH3	NH2	0.64 ± 0.18	0.15 ± 0.04	4.27	>187.48	>290.70	>1250.00
12b1	F	H	OCH3	NH2	0.82 ± 0.15	0.15 ± 0.03	5.47	>182.55	>223.21	>1250.00
12c1	Cl	H	OCH3	NH2	0.61 ± 0.11	0.10 ± 0.02	6.10	>178.27	>290.70	>1736.11
12e1	SO2CH3	H	OCH3	NH2	3.84 ± 0.71	0.35 ± 0.03	10.97	>167.82	>43.71	>480.77
12a2	H	F	OCH3	NH2	0.11 ± 0.03	0.10 ± 0.01	1.10	>177.88	>1666.67	>1736.11
12b2	F	F	OCH3	NH2	0.31 ± 0.14	0.12 ± 0.03	2.58	>173.44	>568.18	>1488.10
12c2	Cl	F	OCH3	NH2	0.14 ± 0.03	0.08 ± 0.01	1.75	>169.57	>1250.00	>2083.33
12e2	SO2CH3	F	OCH3	NH2	0.68 ± 0.08	0.15 ± 0.03	4.53	>160.09	>235.85	>1041.67
20a1	H	H		NO2	1.21 ± 0.65	0.37 ± 0.09	3.27	>153.21	>126.26	>416.67
20a2	H	F		NO2	0.16 ± 0.09	0.18 ± 0.05	0.89	>146.74	>892.86	>833.33
21a1	H	H		NH2	0.15 ± 0.08	0.10 ± 0.01	1.50	>159.06	>1041.67	>1562.50
21a2	H	F		NH2	0.11 ± 0.05	0.08 ± 0.01	1.38	>152.09	>1329.79	>1923.08
20b1	H	H		NO2	12.62 ± 18.68	0.73 ± 0.16	17.29	>144.69	>11.47	>198.41
20b2	H	F		NO2	3.04 ± 1.41	1.97 ± 0.67	1.54	>138.90	>45.62	>70.62
21b1	H	H		NH2	1.98 ± 0.96	0.54 ± 0.13	3.67	>149.89	>75.76	>277.78
21b2	H	F		NH2	1.51 ± 1.34	0.60 ± 0.15	2.52	>143.69	>95.42	>240.38
11L	-	-	-	-	0.28 ± 0.09	0.03 ± 0.01	9.33	197.91 ± 6.85	717.00	6748.24
PF74	-	-	-	-	0.80 ± 0.09	3.78 ± 0.38	0.24	>293.75	>367.65	>77.64

^aEC₅₀: the concentration of the compound required to achieve 50% protection of MT-4 cells against HIV-induced cytotoxicity effect, determined in at least triplicate against HIV in MT-4 cells

^bRatio: III_B(EC₅₀)/ROD(EC₅₀)

^cCC₅₀: the concentration of the compound required to reduce the viability of uninfected cells by 50%, determined in at least triplicate against HIV in MT-4 cells; values were averaged from at least three independent experiments

^dSI: selectivity index, the ratio of CC₅₀/EC₅₀.

In assessing SARs, we noted several trends. For example, the R₁ position preferred other substitutes over the SO₂CH₃ group, as exemplified by 11e1–11e2 and 12e1–12e2. The presence of NO₂ in the R₄ position favored the F-substituted and unsubstituted compounds for anti-HIV-1 activity, and Cl− substituted (12c1–12c2) R₁ was found to maintain the same potency level with unmodified counterpart (12a1–12a2) when with NH₂ in R₄. The anti-HIV-1 activity of R₂ with F substitution was superior to that of unsubstituted R₂, consistent with previously reported data.³¹ This observation could be attributed to the fact that the 3,5-difluorophenyl substituent was embedded within a hydrophobic subpocket surrounded by Leu56, Val59, Met66, Leu69, Lys70, and Ile73.³² The introduction of F might enhance interactions with the surrounding hydrophobic amino acids, resulting in improved antiviral activity. Among the compounds where R₃ was replaced, 20a1–20a2 and 21a1–21a2 (EC₅₀ values ranging from 0.11 to 1.21 μM), substituted at R₃ with 4-sulfonylmorpholine, were found to improve anti-HIV-1 activities compared with methoxy-substituted (11a1–11a2 and 12a1–12a2, EC₅₀ values ranging from 0.11 to 1.51 μM) and 4-sulfonylthiomorpholine-1,1-dioxide-substituted (20b1–20b2 and 21b1–21b2, EC₅₀ values ranging from 1.51 to 12.62 μM), which might be due to the introduction of 4-sulfonylmorpholine group resulting in increased interactions with surrounding amino acid residues such as Asn74. These findings indicated that appropriate expansion of the volume of R₃ substituent positively impacts the enhancement of the compounds’ antiviral activity. Finally, the influence of R₄ substituents was investigated, and most NH₂-bearing compounds were notably more active than their NO₂ analogs.

3.1.2. SAR Analysis of the Newly Synthesized Compounds against HIV-2.

Intriguingly, all tested compounds also demonstrated strong anti-HIV-2 potency with EC₅₀ values ranging from 0.08 to 1.97 μM. Two compounds, 12c2 and 21a2, were equipotent with 11L and showed a 47-fold increase in potency compared to PF74. When R₂ = H or F, R₃ = OCH₃, and R₄ = NO₂, there was no statistical difference in R₁ substituted by SO₂CH₃, Br, Cl, F or unsubstituted. When R₂ = H or F, R₃ = OCH₃, R₄ = NH₂, the antiviral activity of the analogs with SO₂CH₃ at R₁ decreased in comparison with unsubstituted and F- or Cl-substituted R₁, as exemplified by 12e1 (EC₅₀ = 0.35 μM) vs 12a1–12c1 (EC₅₀ values ranging from 0.10 to 0.15 μM), 12e2 (EC₅₀ = 0.15 μM) vs 12a2–12c2 (EC₅₀ values ranging from 0.08 to 0.12 μM). Notably, no statistically significant difference was found when R₂ was H or F, as exemplified by 11a1–11e1 (EC₅₀ values ranging from 0.32 to 0.57 μM) vs 11a2–11e2 (EC₅₀ values ranging from 0.35 to 0.59 μM), 12a1–12c1 (EC₅₀ values ranging from 0.10 to 0.15 μM) vs 12a2–12c2 (EC₅₀ values ranging from 0.08 to 0.12 μM). From the R₃-substituted derivatives, the order of potency was as follows: 4-sulfonylmorpholine (20a1–21a2, EC₅₀ values ranging from 0.08 to 0.37 μM) > OCH₃ (11a1–11a2, 12a1–12a2, EC₅₀ values ranging from 0.10 to 0.57 μM) > 4-sulfonyl thiomorpholine-1,1-dioxide (20b1–21b2, EC₅₀ values ranging from 0.54 to 1.97 μM). Finally, R₄ substituted with an NH₂ was favorable for antiviral activity. The effect of R₃ and R₄ substituents on the anti-HIV-2 activity was consistent with the anti-HIV-1 activity of the compounds.

3.1.3. Effects of Different Structural Types on the Activity and Selectivity of Anti-HIV-1/HIV-2.

As mentioned above, the newly synthesized compounds investigated in this study demonstrated potent activities against both HIV-1 and HIV-2. Exploring the effects of different structural types on the activity and selectivity of anti-HIV-1/HIV-2 could lay the foundation for developing a new generation of potent HIV CA modulators, as well as selective CA modulators for HIV-1 or HIV-2. In our previous work, a homology model of HIV-2 CA hexamer has been successfully constructed, and the sequence of CA of HIV-2 is highly homologous to HIV-1 CA, a significant difference in the binding site is that Lys70 in the HIV-1 CA corresponds to Arg70 in the HIV-2 CA (Figure 3A,B).³³ Herein, we have analyzed recent results to develop a deeper insight into the SARs of anti-HIV potency and the structure–selectivity relationships (SERs) of anti-HIV-1/HIV-2 potency (Figure 3C, Tables S1-S8).

Figure 3.

(A) Binding site of HIV-1 CA hexamer (PDB code: 4XFZ). (B) Binding site of HIV-2 CA hexamer model (obtained by homology modeling³³). The figures were generated in PyMOL (www.pymol.org). (C) Scaffolds I–VIII (sorted by publication time). The sector diagram represents the selectivity of compounds against HIV-1 and HIV-2, and different colors represent different ranges of ratio [(III_B(EC₅₀)/ROD(EC₅₀)] (Blue: Ratio = [<1], Orange: Ratio = [1–3]; Green: Ratio = [3–10]; Red: Ratio = [>10]).

1. The sulfonyl group in scaffolds I^24,34 and VIII (this work) can play a role in stabilizing the conformation, leading to a “U”-shaped conformation of the compounds (PDB codes: 8F22, 8TOV, 8TQP). This facilitates hydrogen bond interactions between Lys70/Arg70 and the carbonyl group on the piperazinone or the carbonyl group on the amide bond, which could explain why these two scaffolds display high anti-HIV-1 and anti-HIV-2 activity. However, arginine is more basic and has more polar hydrogens than lysine, resulting in a stronger hydrogen bond formation profile, resulting in these compounds prefer suppressing HIV-2 over HIV-1.

2. While scaffolds II³⁰ and VII^31,33 also contained the piperazinone structure, the sulfonyl group replaced by the acyl group might disrupt the active conformation of the compounds, leading to a significant reduction of their anti-HIV activity.

3. The anti-HIV-2 activities of scaffold III³⁵ were significantly superior to their anti-HIV-1 activities, making them selective HIV-2 CA modulators. This might be attributed to the fact that the benzene ring can simultaneously form cation-π interactions with Arg70 and Arg173, thereby exhibiting better anti-HIV-2 activity. In comparison to scaffold III, the orientation of the carbonyl on the 2-pyridone moiety in scaffold V³⁶ might be unfavorable for interaction with Arg70, leading to a significant decrease in its anti-HIV-2 activity.

4. Scaffold IV³⁷ is a series of compounds obtained through the Ugi four-component reaction with significant structural differences resulting in substantial variations in their ratio values. This also underscores the critical role of structural changes in the interface region for the selectivity of compounds against HIV-1/HIV-2.

5. Scaffold VI³⁸ retains the indole structure of the lead compound PF74 and maintains similar anti-HIV activity, making them selective HIV-1 CA modulators.

Taking these observations together, we could find that the primary reason for differences in anti-HIV activity among compounds of different structural types might lie in the variations in the 70th amino acid at the binding site. Therefore, the sulfonyl-piperazinone structure (scaffolds I and VIII) capable of forming hydrogen bonds with Arg70 or groups capable of engaging in cation-π interactions with it (scaffold III) exhibit stronger anti-HIV-2 activity. These findings also guide the future development of selective HIV-1 of HIV-2 CA modulators.

In this study, compounds 12a2 and 21a2 displayed excellent anti-HIV potency while maintaining relatively low levels of cytotoxicity. These compounds, characterized by their unique structural features, hold great promise as lead compounds and warrant further study.

3.2. 12a2 and 21a2 Interact with HIV-1 CA As Determined by SPR.

To further explore the interactions of these promising compounds with the HIV-1 CA protein, we performed SPR analysis using the two most potent compounds, 12a2 and 21a2. We employed both the monomeric and hexameric forms of CA in these experiments, utilizing a previously established protocol.³³ PF74 and 11L were used as controls. Table 2 and Figures 4 and 5 show that both compounds directly bound with CA’s monomeric and hexameric forms. PF74 demonstrated rapid association and dissociation rates with the monomeric form of CA and slow dissociation rates with the hexameric form (K_D (monomer) = 0.969 μM, K_D (hexamer) = 0.047 μM). 11L, in contrast, exhibited the fastest association and dissociation rates but with a greater preference for HIV-1 CA monomers (K_D (monomer) = 0.849 μM, K_D (hexamer) = 1.626 μM).

Table 2.

SPR Results of 12a2, 21a2, 11L, and PF74 Binding to Monomeric and Hexameric HIV-1 CA NL4.3 Proteins

	Monomer			Hexamer
Compd	koff (s−1)	Residence Time (min)	KD (μM)	koff (s−1)	Residence Time (min)	KD (μM)	Ratioa
12a2	0.082 ± 0.011	0.203	0.838 ± 0.170	0.052 ± 0.005	0.321	0.561 ± 0.031	1.490
21a2	0.040 ± 0.007	0.417	0.244 ± 0.037	0.012 ± 0.000	1.389	0.199 ± 0.019	1.230
11L	13.400 ± 10.200	0.001	0.849 ± 0.540	2.040 ± 0.871	0.008	1.626 ± 0.175	0.520
PF74	1.580 ± 0.628	0.011	0.969 ± 0.447	0.036 ± 0.007	0.463	0.047 ± 0.001	20.620

^aRatio = K_D^Monomer/K_D^Hexamer. This value indicates how much the inhibitors prefer the monomer or hexamer. All values represent the average response from three replicates. Errors represent standard deviation (SD).

Figure 4.

SPR sensorgrams of 12a2 and 21a2 binding to monomeric or disulfide-stabilized hexameric HIV-1 CA NL4.3 proteins, respectively, with PF74 and 11L as controls. Experiments were performed in triplicate.

Figure 5.

SPR isotherms of 12a2 and 21a2 binding to monomeric or disulfide-stabilized hexameric HIV-1 CA NL4.3 proteins, with PF74 and 11L as controls. Isotherms are an average of three replicates with error bars representing standard deviation (SD).

In contrast to PF74 and 11L, compounds 12a2 and 21a2 demonstrated fast association and slow dissociation with both oligomeric forms of CA. Importantly, their dissociation rates were slower for CA hexamers (12a2: k_off (hexamer) = 0.052 s⁻¹, 21a2: k_off (hexamer) = 0.012 s⁻¹), suggesting a preference for this form. Further analysis revealed that the antiviral activity of 12a2 was similar to 21a2. Still, the affinities of 21a2 (K_D (monomer) = 0.244 μM, K_D (hexamer) = 0.199 μM) for both CA forms were approximately three times higher than those of 12a2 (K_D (monomer) = 0.838 μM, K_D (hexamer) = 0.561 μM). These findings suggest that introducing the 4-sulfonylmorpholine group in 21a2 might decrease its membrane permeability and intracellular stability compared to 12a2. Notably, when compared to 11L, the introduction of quinazolin-4-one in 12a2 led to a 2-fold increase in affinity toward the CA hexamer, a finding consistent with its potent anti-HIV-1 activity. This supports our hypothesis that the newly introduced quinazolin-4-one group can enhance interactions with surrounding amino acids and validates the rationality of our design strategy.

In summary, the SPR analysis validated the strong interactions between the newly developed compounds 12a2 and 21a2 and the HIV-1 CA protein and provided crucial insights into their preferential binding forms. These findings underscore these compounds’ potential as potent antiviral agents and warrant further investigations into their mechanism of action.

3.3. Molecular Insights into Binding Mechanisms: Crystallographic Studies of 12a2 and 21a2 in Complex with HIV-1 CA Hexamer.

To gain insights into the binding mode of the most potent HIV CA modulators of the present series, we co-crystallized 12a2 and 21a2 with a disulfide-linked HIV-1 CA hexamer and determined the complex structures by X-ray crystallography.

As shown in Figure 6A,B, compounds 12a2 and 21a2 occupy the same NTD-CTD interface as PF74. This is located between two adjacent CA protomers, denoted as A (in gray) and B (in cyan). For both compounds, a network of hydrogen bonds was observed, encompassing the following interactions (i) the carbonyl of the quinazolin-4-one core interacts with the side chain of Thr107B; (ii) the side chain of Asn57B forms interactions with both the ring-nitrogen atom of the quinazolin-4-one core and the amide N–H of 12a2 and 21a2; (iii) the sulfonyl group of 21a2 in region III interacts with the side chain of Asn74B. The 3,5-difluorophenyl substituent was embedded within a hydrophobic subpocket surrounded by Leu56B, Val59B, Met66B, Leu69B, Lys70B, and Ile73B. One fluorine is located surrounded by Leu69B, Lys70B, and Ile73B, making van der Waals (vdW) interactions. The other is positioned to interact with Met66B and forms a hydrogen bond with a coordinated water (Figure S1). The side chain of Lys70B was involved in a hydrogen bond with the carbonyl group of piperazinone (12a2) or the carbonyl group on the amide bond (21a2).

Figure 6.

X-ray co-crystal structures of 12a2 (green, A) and 21a2 (purple, B) in complex with HIV-1 CA disulfide-linked hexamer. Hydrogen bonds are indicated as yellow dashed lines. (C) Crystallographic overlay of 12a2 (green), 11L (yellow), and PF74 (orange). (D) Crystallographic overlay of 21a2 (purple), 11L (yellow), and PF74 (orange). The figures were generated in PyMOL (www.pymol.org).

Furthermore, the p-aminobenzenesulfonyl moiety extended into the NTD-CTD interface, establishing multiple hydrogen bonds with key surrounding amino acids. Specifically, the sulfonyl appeared to engage in a hydrogen bond with the side chain of Lys182A, while the amino group formed hydrogen bonds with the side chain of Asn183A (12a2) and Thr186A (12a2 and 21a2). These extensive binding interactions likely account for the high potency observed in this series.

Comparing the X-ray crystallographic structures of 12a2 and 21a2 with those of 11L and PF74 (Figure 6C,D) showed that the phenylalanine-glycine (FG) skeleton maintains the original binding mode. However, these new compounds displaying higher activity occupy multiple pockets in the binding site. This is particularly the case for 21a2, with the morpholine group located in shallow depression bounds by the side chain of Thr107B, Asn74B, and Ser102B. The base of this depression is hydrophobic in nature and is not accessed by either PF74 or LEN. Comparison with the structure of CPSF6 bound shows this same pocket to be occupied by Val314 of the CPSF6 peptide. This provides compelling evidence for the success of the “multi-site binding” strategy.

In summary, the crystallographic data elaborate upon the strong and specific binding interactions that 12a2 and 21a2 form with the HIV-1 CA protein. These molecular insights confirm their multi-site binding capabilities and offer a robust scientific foundation for their observed high potency in antiviral assays.

3.4. 12a2 and 21a2 Exhibit Both Early- and Late-Stage Inhibition Activity.

Following our observations of the interactions between our lead compounds 12a2 and 21a2 and the HIV-1 CA protein, we then aimed to assess their effectiveness in inhibiting different stages of the HIV-1 lifecycle, given the critical role of CA in both the early and late stages of the viral replication process. For this purpose, we conducted the SRI assay, which can efficiently distinguish between the early and late stages of the HIV-1 life cycle.³⁴

As shown in Table 3 and Figure 7, compounds 12a2 and 21a2 demonstrated inhibitory effects on both early and late stages of HIV-1 replication, with a slight improvement over PF74 and 11L potency at the late stage. While the antiviral activities of 12a2 and 21a2 surpassed those of PF74 and 11L, their impact on infection rates during the early stages of replication was comparable to the controls. This indicates that the superior antiviral performance of 12a2 and especially 21a2 over the control compounds likely derives from their more effective reduction of infection rates during the late stages of viral replication. This agrees with enhanced monomeric CA binding over control as judged by our SPR results.

Table 3.

Results of the Single-Round Infection Assay at Early and Late Stages

		% Infectiona
Compd	Concentration (μM)	Early Stage	Late Stage
12a2	1	0.092 ± 0.013	0.019 ± 0.002
21a2	1	0.109 ± 0.046	0.017 ± 0.003
11L	1	0.092 ± 0.027	0.807 ± 0.750
PF74	1	0.070 ± 0.007	4.185 ± 0.216
DMSO	–	100.0 ± 30.99	100.0 ± 18.40

^aInfections are an average of three replicates with error bars indicating standard error of the mean (SEM).

Figure 7.

Results of the single-round infection assay. Effect of compounds at 1 μM at (A) early stage and (B) late stage. Infections are an average of three replicates with error bars indicating the SEM.

In addition, these results consistent that our new compounds bind to multiple sites within the CA protein’s binding pocket, facilitating a wider range of interactions with the hexameric interface (post-late stage, during maturation and hexamer formation). This may result in stabilizing the assembled CA or accelerating the misassembly of CA at the late stage of infection, interfering with the typical replication process of the virus and enhancing its antiviral potency.

Our SRI assay results confirmed the broad-spectrum inhibitory effects of 12a2 and 21a2 at both early and late stages of the HIV-1 life cycle. These findings strengthen our understanding of these compounds as potential versatile antiviral agents and provide additional validation for their promising therapeutic profiles.

3.5. In Vitro Reverse Transcriptase Inhibition Assay.

Building on the evidence that 12a2 and 21a2 inhibit both early and late stages of the HIV-1 life cycle, it is crucial to elucidate whether this antiviral activity was specifically due to their interaction with HIV-1 CA. Antiviral activity in the early stages of HIV-1 replication can occur by disrupting many processes. The most common process that can be disrupted in the early stages is the action of reverse transcriptase (RT). Therefore, to demonstrate that the antiviral action of our compounds was due to direct action on HIV-1 CA, as indicated by their direct interaction with CA, and not through off-target action against RT, we performed an in vitro reverse transcriptase inhibition assay on representative compounds 12a2 and 21a2.

As illustrated in Figure 8, it is evident that the control RT inhibitor TMC-278 (at 100 nM) inhibits RT (approximately 50% inhibition compared to the RT control), whereas 12a2 and 21a2 did not to any appreciable level (about 80% compared to RT control at 900 nM). Consequently, the early-stage inhibition in the antiviral assay is most likely due to direct action on the capsid protein and not by RT inhibition.

Figure 8.

Effect of compounds 12a2 and 21a2 on RT activity in vitro, TMC278 was used as a positive control. The experiment was performed in triplicate, with box size representing the standard error of the mean (SEM) and whiskers the confidence interval at 95%. TMC-278 was used at 100 nM, while 12a2 and 21a2 were used at 900 nM. RT control represents the RT vehicle control only.

3.6. Interference with Host Factor CPSF6 Potentially Augments the Antiviral Potency of 12a2 and 21a2.

Previous studies have demonstrated that PF74 binds to the same interprotomer site in the hexamer as host factors, such as CPSF6, and competes with CPSF6 to modulate nuclear entry.^13,23 To investigate whether the newly synthesized compounds could also bind to this pocket and compete for the binding site with CPSF6, we performed an SPR-based competition assay with CPSF6 using the most potent compounds, 12a2 and 21a2.

As depicted in Table 4 and Figure 9, 12a2, 21a2, and PF74 impeded the binding of the CPSF6 peptides, suggesting a shared binding site. We conducted a crystallographic overlay of 12a2 and 21a2 with CPSF6 to further validate this result. From Figure 10, it can be observed that not only the FG motif of 12a2 and 21a2 could fit well with CPSF6, but also the benzenesulfonamide piperazinone group extending toward the CTD interface and the quinolin-4-one group targeting the NTD interface maintained a consistent conformation with CPSF6. Furthermore, 12a2 and 21a2 could effectively inhibit the binding of CPSF6 to CA hexamer with IC₅₀ values of 189.2 and 163.3 nM, respectively, demonstrating that competing with CA-host factor CPSF6 might be a contributing factor in its antiviral potency during the early stage of viral infection.

Table 4.

Results of CPSF6 Peptide Competition Assay Based on SPR Experiments with 12a2, 21a2, and PF74

Compd	IC50 (nM)a
12a2	189.2 ± 89.8
21a2	163.3 ± 47.1
PF74	26.6 ± 25.5

^aAll values represent the average response from three replicates.

Figure 9.

SPR-based CPSF6 peptide competition experiments with 12a2 and 21a2 utilizing hexameric HIV-1 CA NL4.3 protein.

Figure 10.

Crystallographic overlay of 12a2 (green, A) and 21a2 (purple, B) with CPSF6 (pink, PDB code: 4WYM) peptide. The figures were generated in PyMOL (www.pymol.org).

These results establish another facet of the antiviral mechanisms of 12a2 and 21a2, illuminating their ability to interfere with host–virus interactions involving CPSF6. This multi-pronged mechanism augments their antiviral potency and further solidifies their promise as effective therapeutic agents against HIV-1.

3.7. 12a2 and 21a2 Promote CA Assembly In Vitro.

Having established that compounds 12a2 and 21a2 can exert antiviral effects on HIV-1 during both the early and late stages of replication, we turned our attention to examining their impact on the assembly process of HIV-1 CA during the late stage. To explore their mechanism of action at the late stage of viral replication, we conducted an in vitro CA self-assembly assay through fluorescence resonance energy transfer (FRET).³⁹ We used PF74, 11L, and LEN as controls. Additionally, we included inositol hexakisphosphate (IP6) as a control, given its known capacity to accelerate the in vitro assembly of HIV-1 CA.⁴⁰

Figure 11 displays the results of this experiment, with the fluorescence intensity change over time. This change stems from the hydrolysis of the fluorescent primer tqON by an endonuclease, with the resulting fluorescence signal proportional to the quantity of unassembled CA-NC particles. Our results reveal that compounds 12a2 and 21a2, at a concentration of 50 μM, display a remarkable capability to enhance CA assembly, surpassing the reference compounds PF74, 11L, and the approved drug LEN. Furthermore, compared to IP6, 12a2 and 21a2 demonstrate a more pronounced effect in promoting CA assembly. Increased assembly could lead to abnormal CA structures and subsequent maturation defects.

Figure 11.

FRET-based in vitro assembly of HIV-1 CA-NC in the presence of 50 μM compound concentration. Error bars represent the standard deviation (SD) of three replicates.

These findings suggest that compounds 12a2 and 21a2 exert their antiviral activity in the late stages of HIV-1 replication by significantly facilitating the assembly of the CA protein. This action aligns with their observed high activity during the late stage, including initial CA monomer binding.

3.8. ELISA-Based Quantification of Capsid (p24) Content.

Having demonstrated that 12a2 and 21a2 could promote the assembly of CA during the late stage of viral replication, our subsequent objective was to examine the effect of these compounds on p24 content using an ELISA assay. We quantified the amount of virus (CA/p24) produced in the presence of compounds at 10× over EC₅₀ or DMSO. CA/p24 was captured on an ELISA plate coated with anti-p24 antibodies and compared to a virus produced in the presence of PF74 and 11L. As depicted in Figure 12, the p24 content of the produced virus in the presence of 12a2 and 21a2 did not change significantly, but it does increase CA assembly (Figure 11) and, therefore, might cause abnormal CA/Cone shapes, rendering them incompatible with infection.

Figure 12.

Effect of compounds 12a2 and 21a2 on p24 content of produced pseudoviruses. PF74 and 11L are used as controls.

3.9. Computational Assessment and Experimental Validation of Metabolic Stability.

3.9.1. In Silico Predicted Metabolic Stability of Representative Compounds.

Considering the significant drawback of PF74 related to its metabolic stability, one of our primary objectives in designing these compounds was to enhance their metabolic half-life. Therefore, we next sought to investigate in silico whether or not our compounds had improved predicted metabolic stability over 11L and PF74. We employed a computational analysis first demonstrated to be an accurate indicator of metabolic stability by the Cocklin group.^41-43 We utilize the P450 module in StarDrop 7 (Optibrium, Ltd., Cambridge, UK) to predict each compound’s major metabolizing cytochrome P450 isoforms using the WhichP450 model. Subsequently, we predict affinity to that isoform using the HYDE function in SeeSAR (BioSolveIT Gmbh, Germany).^44,45 The results of this analysis are shown in Figure 13.

Figure 13.

Computational prediction of metabolic stability. (A) Prediction of the major metabolizing CYP isoforms. All compounds are predicted to be metabolized primarily by the 3A4 isoform. (B) Overall composite site lability (CSL) score and number of labile sites within the compound (for metabolism). A lower CSL score indicates a more stable molecule. The prediction was achieved using the StarDrop (version 7) P450 module. (C) Predicted affinity of docked 12a2, 21a2, 11L, and PF74 to cytochrome P450 3A4 (PDB ID: 4D78). The lower boundary for predicted 3A4 affinity utilized the HYdrogen bond and dehydration scoring function (HYDE) implemented in SeeSAR12.1.

All compounds are predicted to be primarily metabolized by the CYP3A4 isoform (Figure 13A). We next investigated the predicted metabolic lability of these compounds by the CY3A4 isoform by comparing the overall composite site lability (CSL) score and the number of labile sites. The CSL score between our compounds and 11L is not significantly different; however, the number of labile sites indicated increased metabolic stability for 11L and PF74 (Figure 13B). In addition to the CSL score and number of labile sites, which assume that all compounds bind with similar affinity to the CYP3A4 isoform, other factors, such as compound reduction rate and actual binding affinity to the CYP3A4 isoform, can influence metabolic stability. Furthermore, intrinsic compound properties such as size and lipophilicity can infer affinity. We, therefore, performed predictive binding affinity calculations using the HYdrogen bond and DEhydration (HYDE) energy scoring function in SeeSAR 12.1 (BioSolveIT Gmbh, Germany)⁴⁶ using the structure of the human CYPA4 bound to an inhibitor (PDB code: 4D78).⁴⁷ The HYDE scoring function in SeeSAR provides a range of affinities, including an upper and lower limit. We used the lower limit as the affinity predictor to compare 12a2, 21a2, 11L, and PF74 (Figure 13C), which resulted in an affinity of 216.7 μM for 12a2, 558.4 μM for 21a2, 41 nM for 11L, and 35 nM for PF74. Combining the results from these predictions (CSL scores, labile sites, and predicted CYP3A4 affinity), this analysis indicates that compounds 12a2 and 21a2 have greater metabolic stability than 11L and PF74, primarily due to the significantly lower CYP3A4 affinity.

3.9.2. Experimental Validation of Metabolic Stability in Human Liver Microsomes and Human Plasma.

Equipped with the computational predictions, we next performed metabolic stability assays of compounds 12a2 and 21a2 in HLMs and human plasma. The reference compounds PF74 and 11L were employed for comparative analysis.

The metabolic stabilities of 12a2 and 21a2 in HLMs were assessed using testosterone, diclofenac, and propafenone as control compounds due to their rapid clearance rate (CL) in HLMs. Table 5 shows that the intrinsic CLs of 12a2 in microsomes and liver were 143.4 and 129.0 mL/min/kg, respectively, which resulted in improved stability with a half-life (T_1/2) of 9.7 min, showing a slight increase compared to 11L (T_1/2 = 4.0 min), and a nearly 14-fold increase compared to PF74 (T_1/2 = 0.7 min). The CLs of 21a2 was also reduced compared to PF74 and 11L, with CL_int(mic) and CL_int(liver) values of 312.7 and 281.5 mL/min/kg, respectively. We next examined the stability of 12a2 and 21a2 in human plasma. As depicted in Figure 14, 109.9% of 12a2 and 101.6% of 21a2 maintained intact after incubation for 120 min at 37 °C. On the contrary, PF74 was quickly metabolized, retaining only 85.2% of its initial amount at 120 min. In conclusion, 12a2 and 21a2 exhibited relatively high stability in HLMs and human plasma, aligning with our initial design of blocking the easily metabolizable amide bond by introducing quinolin-4-one. Meanwhile, our experimentally derived metabolic stability evaluation agrees with the previously described computational prediction approach.

Table 5.

Metabolic Stability in Human Liver Microsomes

	HLM (Final concentration of 0.5 mg protein/mL)
Sample	R 2a	T1/2(min)b	CLint(mic)c (μL/min/mg)	CLint(liver)d (mL/min/kg)	% Remaining (T = 60 min)	% Remaining (NCFe = 60 min)
12a2	0.9670	9.7	143.4	129.0	1.1%	107.3%
21a2	0.9668	4.4	312.7	281.5	0.2%	94.4%
11L	0.9852	4.0	343.6	309.3	0.0%	113.3%
PF74	1.0000	0.7	2114.6	1903.1	0.0%	106.1%
Testosterone	0.9791	13.2	104.8	94.3	4.8%	95.8%
Diclofenac	0.9995	4.5	310.7	279.7	0.0%	86.4%
Propafenone	0.9316	6.3	219.1	197.2	0.0%	103.5%

^aR² is the linear regression’s correlation coefficient for determining the kinetic constant.

^bT_1/2 is the half-life, and CL_int(mic) is the intrinsic clearance.

^cCL_int(mic) = (0.693/half-life)/mg microsome protein per mL.

^dCL_int(liver) = CL_int(mic) × mg microsomal protein/g liver weight × g liver weight/kg body weight.

^eNCF: no cofactor. No NADPH regenerating system was added to the NCF sample (replaced by buffer) during the 60 min incubation. A non-NADPH dependent reaction occurs if the remaining amount is less than 60%.

Figure 14.

Result summary of the human plasma stability assay. Experiments were performed in triplicate. % remaining = 100 × (PAR at appointed incubation time/PAR at time T₀). PAR is the peak area ratio of a test compound to the internal standard. Accuracy should be within 80–120% of the indicated value.

3.10. In Vivo Pharmacokinetic Study.

After demonstrating potent antiviral activity against both HIV-1 and HIV-2 and excellent performance in various in vitro evaluations, compound 21a2 was selected for in vivo PK analysis. We examined its PK profiles using a Sprague–Dawley (SD) rat model, following both intravenous (i.v.) and oral (p.o.) administration. The results are summarized in Table 6.

Table 6.

PK Profile of Compound 21a2^a

Parameter	Units	21a2 (i.v.)b	21a2 (p.o.)c
T 1/2	h	0.514 ± 0.176	2.02 ± NA
T max	h	0.083	0.333 ± 0.144
C max	ng/mL	1650 ± 361	6.58 ± 8.24
C 0	ng/mL	2985 ± 545	–
AUC0–t	h·ng/mL	572 ± 151	10.80 ± 17.4
AUC0–„	h·ng/mL	574 ± 151	37.3 ± NA
V z	mL/kg	2579 ± 707	–
CL	mL/h/kg	3633 ± 860	–
MRT0–„	h	0.284 ± 0.0468	3.03 ± NA

^aPK parameter (mean ± SD, n = 3).

^bDosed intravenously at 2 mg/kg.

^cDosed orally at 20 mg/kg.

After administering a single 2 mg/kg intravenous dose of 21a2, we obtained a mean clearance rate (CL) and half-life (T_1/2) of 3.6 L/h/kg and 0.514 h, respectively. When delivered orally at a dose of 20 mg/kg, compound 21a2 was rapidly absorbed, achieving a time to maximum concentration (T_max) of 0.333 h. The compound exhibited a moderate half-life of 2.02 h, while its mean residence time (MRT), the average length of time the drug stays in the body, was calculated to be 3.03 h.

LEN is reported to be a long-acting agent with a significantly longer oral half-life (T_1/2 ≈ 10–12 days)²⁵ and subcutaneous half-life (T_1/2 ≈ 8–12 weeks)⁴⁸ compared to 21a2. From a clinical perspective, long-acting agents offer potential solutions to overcome various adherence obstacles associated with daily pill usage. However, the utilization of long-acting agents raises inherent concerns, including the possibility of irreversible long-term adverse effects upon administration and an extended PK tail. These factors have implications for safety, tolerability, risk of drug resistance development, as well as persistent drug–drug interactions even after discontinuation of active dosing.⁴⁹ Therefore, developing drugs with appropriate half-life remains imperative in light of the aforementioned reasons.

Combining these in vivo PK studies with our in vitro and computational analyses, we can conclude that 21a2 exhibits remarkable antiviral potency and maintains excellent PK profiles, further solidifying its potential as a promising antiretroviral agent.

4. CONCLUSIONS

HIV remains a significant global health concern, underscoring the necessity for novel classes of therapeutic agents that employ unique mechanisms of action to address the limitations of existing treatments. The HIV-1 capsid protein, integral to the viral life cycle, has recently emerged as a promising therapeutic target. In this study, we designed, synthesized, and identified 26 novel phenylalanine derivatives bearing a quinazolin-4-one scaffold as HIV-1 CA modulators.

The results from our in vitro anti-HIV assays indicated that a majority of the synthesized compounds demonstrated excellent antiretroviral efficacy against both HIV-1 III_B and HIV-2 ROD strains while maintaining low cytotoxicity levels. The most potent analogs, 12a2 and 21a2, exhibited markedly superior anti-HIV-1 potency—2.5-fold higher than 11L and 7.3-fold higher than PF74—with notable safety indexes exceeding 1666 and 1329, respectively. Also, the derivatives demonstrated impressive activity against HIV-2, with EC₅₀ values between 0.08 and 1.97 μM, surpassing PF74 (EC₅₀ = 3.78 μM) significantly. Therefore, to explore the effects of different structural types on the activity and selectivity of anti-HIV-1/HIV-2, we have reviewed recently published articles and provided a more comprehensive summary of the SARs of anti-HIV and the SERs of anti-HIV-1/HIV-2. The results suggested that the transition from Lys70 of the ligand binding site of HIV-1 CA to Arg70 of HIV-2 CA might be the main reason for the selectivity of the compounds to anti-HIV-1 and anti-HIV-2. The higher significance of HIV-1 compared to HIV-2 for the human population necessitated the selection of 12a2 and 21a2, which exhibit the most potent anti-HIV activity, as representatives for further investigation into their mechanism of action against HIV-1.

SPR binding assays confirmed that 12a2 and 21a2 could directly bind to monomeric and hexameric HIV-1 CA with nanomolar affinities. X-ray co-crystal structures further validated the binding site and substantiated our design concepts, revealing that 12a2 and 21a2 occupied multiple binding pockets within the CA hexamer, which improved our understanding of the HIV-1 CA structure and its interaction potential with CA modulators. These valuable insights contribute significantly to the design of future HIV CA modulators. SRI assays revealed that these compounds disrupt the HIV-1 life cycle at early and late stages. Further, exploration through in vitro reverse transcriptase inhibition assays and SPR-based competition assays with CPSF6 provided insight into the early-stage mechanism of action of 12a2 and 21a2. Notably, both compounds compete with CPSF6 and possibly interfere with nuclear import during the early stage of viral replication, contributing to their antiviral activity. In the late stage, our CA assembly studies and the quantification of p24 content suggested that these compounds might accelerate HIV-1 CA misassembly.

We also aimed to tackle a key drawback associated with metabolic instability. Employing computational methods^23,31,33,36 and subsequent experimental validations, we assessed the metabolic stability of 12a2 and 21a2 in human liver microsomes and plasma. The moderate stability displayed by these compounds provides a valuable starting point for further PK optimization. To obtain a toxicity profile for the representative compounds, we computationally evaluated the genotoxicity and hepatotoxicity of compounds 12a2 and 21a2 using a knowledge-based algorithm within the Derek Nexus module in StarDrop V7 (Optibrium, Ltd., Cambridge, UK). The presence of aniline groups may account for the predicted carcinogenicity, phototoxicity, and hepatic and thyroid toxicity (Figure S2), but it also offers valuable guidance for our subsequent structural modifications.

In conclusion, this multidisciplinary study offers a compelling set of lead compounds—particularly 12a2 and 21a2—for advancing novel anti-HIV therapies. These leads demonstrate exceptional in vitro and in vivo properties and align well with our initial objectives of targeted mechanism-based intervention. Future efforts will focus on fine-tuning these promising leads in terms of their metabolic stability, safety, and PK profiles.

5. EXPERIMENTAL SECTION

5.1. Chemistry.

All new compounds’ melting points were determined on a micro melting point apparatus. ¹H NMR and ¹³C NMR spectra were obtained in DMSO-d₆ on Bruker AV-400 or Bruker AV-600 MHz spectrometer using tetramethylsilane (TMS) as the internal reference. Chemical shifts were reported in δ values (ppm), and J values were expressed in hertz (Hz). Thin-layer chromatography (TLC) was performed for monitoring reactions or purifying products on silica gel GF254 and HUANGHAI_HSGF254, 0.15–0.2 mm, respectively. Spots were visualized with iodine vapor or by irradiation with UV light (λ = 254 nm, or λ = 365 nm). Mass spectra (MS) were carried out on an LC Autosampler Device: Standard G1313A instrument. Flash column chromatography was performed on a column packed with Silica Gel 60 (200–300 mesh). Solvents were of reagent grade and, if needed, were purified and dried by standard methods. Rotary evaporators were involved in the concentration of the reaction solutions under reduced pressure. The solvents of DCM, acetonitrile, methanol, etc., were obtained from Sinopharm Chemical Reagent Co., Ltd. (SCRC), which were of AR grade. The key reactants, including 4-nitrobenzenesulfonyl chloride (CAS Registry No. 98-74-8), piperazin-2-one (CAS Registry No. 5625-67-2), methyl bromoacetate (CAS Registry No. 96-32-2), etc. were purchased from Shanghai Haohong Scientific Co., Ltd. The purity of target compounds was evaluated on a Shimadzu HPLC system. HPLC conditions were as follows: Agilent ZORBAX, SB-C18 column (250 mm × 4.6 mm × 5 μm); isocratic elution method: mobile phase A: methanol (80%); mobile phase B: water (20%); flow rate: 1.0 mL/min; wavelength: 254 nm, temperature: 30 °C, injection volume: 10 μL. All tested target compounds possessed purities of >95%.

5.1.1. 4-((4-Nitrophenyl)sulfonyl)piperazin-2-one (2).

Under an ice bath, the piperazin-2-one (1.00 g, 9.99 mmol, 1 equiv), TEA (2.77 mL, 19.98 mmol, 2 equiv), and 4-nitrobenzenesulfenyl chloride (2.66 g, 11.99 mmol, 1.2 equiv) were successively dissolved in DCM (20 mL). The resulting mixture was then stirred at room temperature (monitored by TLC). Then, the reaction mixture was filtered under reduced pressure, and the filter cake was washed with DCM. Finally, intermediate 2 was obtained after drying the filter cake. White solid, yield 90%. ¹H NMR (600 MHz, DMSO-d₆) δ 8.45–8.40 (m, 2H, Ph-H), 8.09–8.06 (m, 3H, Ph-H, NH), 3.59 (s, 2H, CH₂), 3.28 (dd, J = 6.5, 4.3 Hz, 2H, CH₂), 3.19 (td, J = 5.4, 4.8, 2.5 Hz, 2H, CH₂). ESI-MS: m/z 285.81 [M+H]⁺. C₁₀H₁₄N₃O₅S [285.04].

5.1.2. Methyl 2-(4-((4-Nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetate (3).

Intermediate 2 (2.50 g, 8.76 mmol, 1 equiv) and NaH (0.63 g, 26.28 mmol, 3 equiv) were mixed in THF (20 mL) and stirred in an ice bath for 30 min. Then methyl bromoacetate (1.61 g, 10.51 mmol, 1.2 equiv) was added to the above solution, and the reaction system was stirred at room temperature for an additional 2 h (monitored by TLC). Then, the reaction mixture was filtered under reduced pressure, and the filter cake was washed with ethyl acetate and water. Finally, intermediate 3 was obtained after drying the filter cake. White solid, yield 85%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (d, J = 8.8 Hz, 2H, Ph-H), 8.11 (d, J = 8.8 Hz, 2H, Ph-H), 4.09 (s, 2H, CH₂), 3.75 (s, 2H, CH₂), 3.62 (s, 3H, OCH₃), 3.42 (s, 4H, CH₂ × 2). ESI-MS: m/z 357.83 [M+H]⁺, 379.94 [M+Na]⁺. C₁₃H₁₅N₃O₇S [357.06].

5.1.3. 2-(4-((4-Nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetic Acid (4).

Intermediate 3 (2.50 g, 7.00 mmol, 1 equiv) was dissolved in a mixture of 10 mL THF and 10 mL water. Then, LiOH (0.50 g, 21.00 mmol, 3 equiv) was slowly added to the above solution and the mixture was stirred at room temperature (monitored by TLC). The resulting mixture solution was acidized to pH 2–3 with 1 N HCl. Then, the mixture was filtered under reduced pressure, and the filter cake was washed with water. Finally, intermediate 4 was obtained after drying the filter cake. White solid, yield 88%. ¹H NMR (600 MHz, DMSO-d₆) δ 12.77 (s, 1H, COOH), 8.44–8.40 (m, 2H, Ph-H), 8.10–8.06 (m, 2H, Ph-H), 3.95 (s, 2H, COCH₂), 3.72 (s, 2H, CH₂), 3.40 (dd, J = 7.0, 4.4 Hz, 2H, CH₂), 3.37 (dd, J = 6.9, 4.6 Hz, 2H, CH₂). ESI-MS: m/z 343.98 [M+H]⁺, 366.01 [M+Na]⁺. C₁₃H₁₅N₃O₇S [343.31].

5.1.4. General Procedure for the Synthesis of 6a–6e.

Under an ice bath, 4-methoxy-N-methylaniline (1 equiv), TEA (2 equiv), and the corresponding substituted 2-nitrobenzoyl chloride (5a–5e, 1.2 equiv) were successively dissolved in DCM (20 mL). The resulting mixture was then stirred at room temperature (monitored by TLC). Then the reaction mixture was extracted with DCM (20 mL), and the combined organic phase was washed with a saturated NaCl solution (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the corresponding crude products, which were purified by recrystallization (ethyl acetate:petroleum ether = 1:10) to afford intermediates 6a–6e.

N-(4-Methoxyphenyl)-2-nitrobenzamide (6a).

White solid, yield: 80%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 1H, NH), 8.13 (d, J = 8.2 Hz, 1H, Ph-H), 7.90–7.83 (m, 1H, Ph-H), 7.82–7.71 (m, 2H, Ph-H), 7.64–7.54 (m, 2H, Ph-H), 7.02–6.90 (m, 2H, Ph-H), 3.75 (s, 3H, OCH₃). ESI-MS: m/z 295.09 [M+Na]⁺, 271.51 [M−H]⁻; C₁₄H₁₂N₂O₄ [272.08].

4-Fluoro-N-(4-methoxyphenyl)-2-nitrobenzamide (6b).

White solid, yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.54 (s, 1H, NH), 8.10 (dd, J = 8.7, 2.5 Hz, 1H, Ph-H), 7.87 (dd, J = 8.5, 5.6 Hz, 1H, Ph-H), 7.77 (td, J = 8.3, 2.5 Hz, 1H, Ph-H), 7.64–7.53 (m, 2H, Ph-H), 7.02–6.91 (m, 2H, Ph-H), 3.75 (s, 3H, OCH₃). ESI-MS: 313.43 [M+Na]⁺, 289.32 [M−H]⁻; C₁₄H₁₁FN₂O₄ [290.07].

4-Chloro-N-(4-methoxyphenyl)-2-nitrobenzamide (6c).

White solid, yield: 76%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 1H, NH), 8.25 (d, J = 2.0 Hz, 1H, Ph-H), 7.96 (dd, J = 8.2, 2.1 Hz, 1H, Ph-H), 7.82 (d, J = 8.2 Hz, 1H, Ph-H), 7.56 (d, J = 9.0 Hz, 2H, Ph-H), 7.02–6.90 (m, 2H, Ph-H), 3.75 (s, 3H, OCH₃). ESI-MS: 329.08 [M+Na]⁺, 305.04 [M−H]⁻; C₁₄H₁₁ClN₂O₄ [306.04].

4-Bromo-N-(4-methoxyphenyl)-2-nitrobenzamide (6d).

White solid, yield: 81%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 1H, NH), 8.36 (d, J = 1.8 Hz, 1H, Ph-H), 8.09 (dd, J = 8.2, 1.9 Hz, 1H, Ph-H), 7.74 (d, J = 8.2 Hz, 1H, Ph-H), 7.56 (d, J = 9.0 Hz, 2H, Ph-H), 6.94 (d, J = 9.0 Hz, 2H, Ph-H), 3.75 (s, 3H, OCH₃). ESI-MS: 373.74 [M+Na]⁺, 375.51 [M+Na+2]⁺; C₁₄H₁₁BrN₂O₄ [349.99].

N-(4-Methoxyphenyl)-4-(methylsulfonyl)-2-nitrobenzamide (6e).

White solid, yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.67 (s, 1H, NH), 8.61 (d, J = 1.5 Hz, 1H, Ph-H), 8.39 (dd, J = 7.9, 1.6 Hz, 1H, Ph-H), 8.07 (d, J = 8.0 Hz, 1H, Ph-H), 7.56 (d, J = 9.0 Hz, 2H, Ph-H), 6.96 (d, J = 9.0 Hz, 2H, Ph-H), 3.75 (s, 3H, OCH₃), 3.41 (s, 3H, SO₂CH₃). ESI-MS: 351.4 [M+H]⁺, 373.2 [M+Na]⁺; C₁₅H₁₄N₂O₆S [350.06].

5.1.5. General Procedure for the Synthesis of 7a–7e.

Intermediates 6a–6e and 10% Pd–C (10% w/w) were dissolved in DCM (20 mL), and the solution was degassed and stirred at room temperature overnight in the atmosphere of hydrogen. The mixture was filtered and concentrated, and the resulting residue was purified by recrystallization (ethyl acetate:petroleum ether = 1:15) to provide the intermediates 7a–7e.

2-Amino-N-(4-methoxyphenyl)benzamide (7a).

White solid, yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H, NH), 7.60 (d, J = 8.0 Hz, 3H, Ph-H), 7.18 (t, J = 7.5 Hz, 1H, Ph-H), 6.90 (d, J = 8.2 Hz, 2H, Ph-H), 6.74 (d, J = 8.2 Hz, 1H, Ph-H), 6.58 (t, J = 7.4 Hz, 1H, Ph-H), 6.30 (s, 2H, NH₂), 3.74 (s, 3H, OCH₃). ESI-MS: 243.20 [M+H]⁺; C₁₄H₁₄N₂O₂ [242.11].

2-Amino-4-fluoro-N-(4-methoxyphenyl)benzamide (7b).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H, NH), 7.68 (dd, J = 8.7, 6.7 Hz, 1H, Ph-H), 7.57 (d, J = 9.0 Hz, 2H, Ph-H), 6.90 (d, J = 9.0 Hz, 2H, Ph-H), 6.65 (s, 2H, NH₂), 6.50 (dt, J = 11.9, 3.1 Hz, 1H, Ph-H), 6.38 (td, J = 8.6, 2.6 Hz, 1H, Ph-H), 3.74 (s, 3H, OCH₃). ESI-MS: 261.49 [M+H]⁺, 283.88 [M+Na]⁺, 259.59 [M−H]⁻; C₁₄H₁₃FN₂O₂ [260.10].

2-Amino-4-chloro-N-(4-methoxyphenyl)benzamide (7c).

White solid, yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.86 (s, 1H, NH), 7.68 (dd, J = 8.8, 6.7 Hz, 1H, Ph-H), 7.63–7.54 (m, 2H, Ph-H), 6.96–6.87 (m, 2H, Ph-H), 6.65 (s, 2H, NH₂), 6.50 (dd, J = 11.9, 2.6 Hz, 1H, Ph-H), 6.38 (td, J = 8.6, 2.6 Hz, 1H, Ph-H), 3.74 (s, 3H, OCH₃). ESI-MS: 299.66 [M+Na]⁺, 275.43 [M−H]⁻; C₁₄H₁₃ClN₂O₂ [276.07].

2-Amino-4-bromo-N-(4-methoxyphenyl)benzamide (7d).

White solid, yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 9.92 (s, 1H, NH), 7.56 (dd, J = 12.6, 8.8 Hz, 3H, Ph-H), 6.96 (d, J = 1.9 Hz, 1H, Ph-H), 6.90 (d, J = 9.0 Hz, 2H, Ph-H), 6.72 (dd, J = 8.4, 1.9 Hz, 1H, Ph-H), 6.55 (s, 2H, NH₂), 3.74 (s, 3H, OCH₃). ESI-MS: 320.92 [M+H]⁺, 319.18 [M−H]⁻; C₁₄H₁₃BrN₂O₂ [320.02].

2-Amino-N-(4-methoxyphenyl)-4-(methylsulfonyl)benzamide (7e).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.12 (s, 1H, NH), 7.79 (d, J = 8.2 Hz, 1H, Ph-H), 7.61 (d, J = 9.0 Hz, 2H, Ph-H), 7.30 (d, J = 1.6 Hz, 1H, Ph-H), 7.06 (dd, J = 8.2, 1.6 Hz, 1H, Ph-H), 6.92 (d, J = 9.0 Hz, 2H, Ph-H), 6.65 (s, 2H, NH₂), 3.74 (s, 3H, OCH₃), 3.18 (s, 3H, SO₂CH₃). ESI-MS: 319.20 [M−H]⁻; C₁₄H₁₃BrN₂O₂ [320.02].

5.1.6. General Procedure for the Synthesis of 8a1–8e1 and 8a2–8e2.

(tert-Butoxycarbonyl)-l-phenylalanine or (tert-butoxycarbonyl)-3,5-difluoro-l-phenylalanine (1.2 equiv) and HATU (1.5 equiv) was mixed in 30 mL of DCM and stirred in an ice bath for 30 min. Subsequently, DIEA (2 equiv) and intermediates 7a–7e (1 equiv) were added to the mixture and then stirred at room temperature for another 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed with 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:8) to afford the intermediates 8a1–8e1 and 8a2–8e2.

tert-Butyl (S)-(1-((2-((4-Methoxyphenyl)carbamoyl)phenyl)-amino)-1-oxo-3-phenylpropan-2-yl)carbamate (8a1).

White solid, yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.36 (s, 1H, NH), 10.36 (s, 1H, NH), 8.48 (d, J = 8.3 Hz, 1H, NH), 7.84 (d, J = 7.7 Hz, 1H, Ph-H), 7.65 (d, J = 8.8 Hz, 2H, Ph-H), 7.54 (d, J = 8.2 Hz, 1H, Ph-H), 7.26 (d, J = 6.4 Hz, 5H, Ph-H), 7.22 (d, J = 7.9 Hz, 1H, Ph-H), 7.20–7.14 (m, 1H, Ph-H), 6.92 (d, J = 8.9 Hz, 2H, Ph-H), 4.22–4.14 (m, 1H, CH), 3.74 (s, 3H, OCH₃), 3.20 (dd, J = 13.7, 3.7 Hz, 1H, CH), 2.84 (dd, J = 13.7, 11.0 Hz, 1H, CH), 1.25 (s, 9H, (CH₃)₃). ESI-MS: 490.2 [M+H]⁺; C₂₈H₃₁N₃O₅ [489.23].

tert-Butyl (S)-(1-((5-Fluoro-2-((4-methoxyphenyl)carbamoyl)-phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (8b1).

White solid, yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.72 (s, 1H, NH), 10.37 (s, 1H, NH), 8.38 (dd, J = 12.0, 2.3 Hz, 1H, NH), 8.01–7.90 (m, 1H, Ph-H), 7.63 (d, J = 8.9 Hz, 2H, Ph-H), 7.55 (d, J = 7.8 Hz, 1H, Ph-H), 7.31–7.22 (m, 4H, Ph-H), 7.18 (dq, J = 8.1, 5.6, 4.2 Hz, 1H, Ph-H), 7.10 (td, J = 8.5, 2.5 Hz, 1H, Ph-H), 6.92 (d, J = 9.0 Hz, 2H, Ph-H), 4.19 (ddd, J = 11.2, 8.0, 4.0 Hz, 1H, CH), 3.74 (s, 3H, OCH₃), 3.20 (dd, J = 13.9, 3.9 Hz, 1H, CH), CH, 2.85 (dd, J = 13.7, 10.9 Hz, 1H, CH), 1.25 (s, 9H, (CH₃)₃). ESI-MS: 508.38 [M+H]⁺, 530.40 [M+Na]⁺, 506.68 [M−H]⁻; C₂₈H₃₀FN₃O₅ [507.22].

tert-Butyl (S)-(1-((5-Chloro-2-((4-methoxyphenyl)carbamoyl)-phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (8c1).

White solid, yield: 81%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.55 (s, 1H, NH), 10.42 (s, 1H, NH), 8.61 (d, J = 1.7 Hz, 1H, NH), 7.90 (d, J = 8.5 Hz, 1H, Ph-H), 7.64 (d, J = 8.9 Hz, 2H, Ph-H), 7.53 (d, J = 7.7 Hz, 1H, Ph-H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H, Ph-H), 7.30–7.22 (m, 4H, Ph-H), 7.19 (qd, J = 5.1, 3.6, 2.5 Hz, 1H, Ph-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-H), 4.27–4.16 (m, 1H, CH), 3.75 (s, 3H, OCH₃), 3.20 (dd, J = 13.9, 4.0 Hz, 1H, CH), 2.85 (dd, J = 13.8, 10.8 Hz, 1H, CH), 1.25 (s, 9H, (CH₃)₃). ESI-MS: 524.31 [M+H]⁺, 546.82 [M+Na]⁺, 522.87 [M−H]⁻; C₂₈H₃₀ClN₃O₅ [523.19].

tert-Butyl (S)-(1-((5-Bromo-2-((4-methoxyphenyl)carbamoyl)-phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (8d1).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.50 (s, 1H, NH), 10.42 (s, 1H, NH), 8.74 (d, J = 1.6 Hz, 1H, NH), 7.81 (d, J = 8.4 Hz, 1H, Ph-H), 7.63 (d, J = 8.9 Hz, 2H, Ph-H), 7.53 (d, J = 7.7 Hz, 1H, Ph-H), 7.46 (dd, J = 8.4, 1.9 Hz, 1H, Ph-H), 7.32–7.22 (m, 4H, Ph-H), 7.18 (qd, J = 4.9, 3.5, 2.5 Hz, 1H, Ph-H), 6.92 (d, J = 9.0 Hz, 2H, Ph-H), 4.19 (ddt, J = 11.2, 7.7, 3.6 Hz, 1H, Ph-H), 3.74 (s, 3H, OCH₃), 3.18 (dd, J = 13.8, 4.0 Hz, 1H, CH), 2.84 (dd, J = 13.9, 10.9 Hz, 1H, CH), 1.24 (s, 9H, (CH₃)₃). ESI-MS: 567.85 [M+H]⁺, 569.82 [M+H+2]⁺; C₂₈H₃₀BrN₃O₅ [567.14].

tert-Butyl (S)-(1-((2-((4-Methoxyphenyl)carbamoyl)-5-(methylsulfonyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-carbamate (8e1).

White solid, yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.28 (s, 1H, NH), 10.58 (s, 1H, NH), 8.98 (s, 1H, NH), 8.06 (d, J = 8.2 Hz, 1H, Ph-H), 7.78 (d, J = 8.2 Hz, 1H, Ph-H), 7.66 (d, J = 8.7 Hz, 2H, Ph-H), 7.53 (d, J = 7.8 Hz, 1H, Ph-H), 7.26 (d, J = 6.0 Hz, 4H, Ph-H), 7.20–7.13 (m, 1H, Ph-H), 6.93 (d, J = 8.8 Hz, 2H, Ph-H), 4.23 (td, J = 9.3, 7.6, 4.3 Hz, 1H, CH), 3.75 (s, 3H, OCH₃), 3.28 (s, 3H, SO₂CH₃), 3.20 (dd, J = 14.0, 3.8 Hz, 1H, CH), 2.88–2.79 (m, 1H, CH), 1.25 (s, 9H, (CH₃)₃). ESI-MS: 590.19 [M+Na]⁺, 566.49 [M−H]⁻; C₂₉H₃₃N₃O₇S [567.20].

tert-Butyl (S)-(3-(3,5-Difluorophenyl)-1-((2-((4-methoxyphenyl)-carbamoyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (8a2).

White solid, yield: 77%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.39 (s, 1H, NH), 10.37 (s, 1H, NH), 8.46 (d, J = 8.2 Hz, 1H, NH), 7.84 (d, J = 7.7 Hz, 1H, Ph-H), 7.66 (d, J = 8.4 Hz, 2H, Ph-H), 7.54 (dt, J = 12.8, 6.5 Hz, 2H, Ph-H), 7.24 (t, J = 7.5 Hz, 1H, Ph-H), 7.03 (t, J = 9.5 Hz, 3H, Ph-H), 6.91 (d, J = 8.5 Hz, 2H, Ph-H), 4.34–4.18 (m, 1H, CH), 3.74 (s, 3H, OCH₃), 3.14 (dd, J = 7.0, 4.2 Hz, 1H, CH), 2.88–2.78 (m, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 526.3 [M+H]⁺; C₂₈H₂₉F₂N₃O₅ [525.21].

tert-Butyl (S)-(3-(3,5-Difluorophenyl)-1-((5-fluoro-2-((4-methoxyphenyl)carbamoyl)phenyl)amino)-1-oxopropan-2-yl)-carbamate (8b2).

White solid, yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.77 (s, 1H, NH), 10.40 (s, 1H, NH), 8.37 (dd, J = 12.0, 2.4 Hz, 1H, NH), 8.06–7.91 (m, 1H, Ph-H), 7.62 (dd, J = 13.3, 8.6 Hz, 3H, Ph-H), 7.12 (td, J = 8.4, 2.6 Hz, 1H, Ph-H), 7.08–6.98 (m, 3H, Ph-H), 6.92 (d, J = 9.0 Hz, 2H, Ph-H), 4.28 (ddd, J = 11.7, 8.5, 4.0 Hz, 1H, CH), 3.75 (s, 3H, OCH₃), 3.27 (dd, J = 13.9, 3.9 Hz, 1H, CH), 2.84 (dd, J = 13.6, 11.2 Hz, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 566.16 [M+Na]⁺, 542.38 [M−H]⁻; C₂₈H₂₈F₃N₃O₅ [543.20].

tert-Butyl (S)-(1-((5-Chloro-2-((4-methoxyphenyl)carbamoyl)-phenyl)amino)-3-(3,5-difluorophenyl)-1-oxopropan-2-yl)-carbamate (8c2).

White solid, yield: 76%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.58 (s, 1H, NH), 10.43 (s, 1H, NH), 8.58 (d, J = 1.7 Hz, 1H, NH), 7.90 (d, J = 8.5 Hz, 1H, Ph-H), 7.64 (d, J = 8.9 Hz, 2H, Ph-H), 7.57 (d, J = 8.1 Hz, 1H, Ph-H), 7.33 (dd, J = 8.4, 2.1 Hz, 1H, Ph-H), 7.01 (d, J = 9.1 Hz, 3H, Ph-H), 6.91 (d, J = 9.0 Hz, 2H, Ph-H), 4.28 (ddd, J = 11.5, 8.3, 4.1 Hz, 1H, CH), 3.74 (s, 3H, OCH₃), 3.26 (dd, J = 13.9, 4.0 Hz, 1H, CH), 3.07 (dd, J = 13.7, 4.4 Hz, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 582.39 [M+Na]⁺; C₂₈H₂₈ClF₂N₃O₅ [559.17].

tert-Butyl (S)-(1-((5-Bromo-2-((4-methoxyphenyl)carbamoyl)-phenyl)amino)-3-(3,5-difluorophenyl)-1-oxopropan-2-yl)-carbamate (8d2).

White solid, yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.54 (s, 1H, NH), 10.44 (s, 1H, NH), 8.82–8.67 (m, 1H, NH), 7.82 (d, J = 8.5 Hz, 1H, Ph-H), 7.63 (d, J = 8.9 Hz, 2H, Ph-H), 7.57 (d, J = 8.2 Hz, 1H, Ph-H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H, Ph-H), 7.03 (dt, J = 11.8, 4.9 Hz, 3H, Ph-H), 6.91 (d, J = 9.0 Hz, 2H, Ph-H), 4.28 (ddd, J = 11.1, 8.2, 3.8 Hz, 1H, CH), 3.74 (s, 3H, OCH₃), 3.25 (dd, J = 13.7, 3.8 Hz, 1H, CH), 2.83 (dd, J = 13.6, 11.3 Hz, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 626.16 [M+Na]⁺, 602.18 [M−H]⁻; C₂₈H₂₈BrF₂N₃O₅ [603.12].

tert-Butyl (S)-(3-(3,5-Difluorophenyl)-1-((2-((4-methoxyphenyl)-carbamoyl)-5-(methylsulfonyl)phenyl)amino)-1-oxopropan-2-yl)-carbamate (8e2).

White solid, yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.31 (s, 1H, NH), 10.59 (s, 1H, NH), 8.96 (s, 1H, NH), 8.06 (d, J = 8.3 Hz, 1H, Ph-H), 7.83–7.76 (m, 1H, Ph-H), 7.66 (d, J = 8.9 Hz, 2H, Ph-H), 7.56 (d, J = 8.2 Hz, 1H, Ph-H), 7.03 (td, J = 11.4, 10.4, 4.5 Hz, 3H, Ph-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-H), 4.37–4.28 (m, 1H, CH), 3.75 (s, 3H, OCH₃), 3.28 (s, 3H, SO₂CH₃), 3.23 (dd, J = 9.7, 3.4 Hz, 1H, CH), 2.88–2.79 (m, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 626.12 [M+Na]⁺, 602.49 [M−H]⁻; C₂₉H₃₁F₂N₃O₇S [603.19].

5.1.7. General Procedure for the Synthesis of 9a1–9e1 and 9a2–9e2.

The key intermediates 8a1–8e1 and 8a2–8e2 (1 equiv), DIEA (2 equiv), and DMAP (1 equiv) were dissolved in the solution of acetonitrile (30 mL), and then BSA (10 equiv) was added dropwise to the mixture solution. The reaction system was stirred at 80 °C for 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed by 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:3) to afford intermediates 9a1–9e1 and 9a2–9e2.

tert-Butyl (S)-(1-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (9a1).

White solid, yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (d, J = 7.8 Hz, 1H, NH), 7.89 (t, J = 7.6 Hz, 1H, Ph-H), 7.73 (d, J = 8.1 Hz, 1H, Ph-H), 7.57 (t, J = 7.5 Hz, 1H, Ph-H), 7.47 (d, J = 7.9 Hz, 1H, Ph-H), 7.34 (dd, J = 5.6, 2.9 Hz, 2H, Ph-H), 7.22–7.12 (m, 5H, Ph-H), 6.83 (d, J = 7.4 Hz, 2H, Ph-H), 4.44–4.33 (m, 1H, CH), 3.88 (s, 3H, OCH₃), 3.03 (dd, J = 13.6, 3.1 Hz, 1H, CH), 2.79 (dd, J = 13.6, 10.2 Hz, 1H, CH), 1.28 (s, 9H, (CH₃)₃). ESI-MS: 494.61 [M+Na]⁺; 470.18 [M−H]⁻; C₂₈H₂₉N₃O₄ [471.22].

tert-Butyl (S)-(1-(7-Fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (9b1).

White solid, yield: 65%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20 (dd, J = 8.3, 6.4 Hz, 1H, NH), 7.45 (td, J = 11.8, 10.8, 5.1 Hz, 3H, Ph-H), 7.39–7.31 (m, 2H, Ph-H), 7.22–7.12 (m, 5H, Ph-H), 6.88–6.79 (m, 2H, Ph-H), 4.44–4.31 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.02 (dd, J = 13.7, 3.2 Hz, 1H, CH), 2.78 (dd, J = 13.7, 10.2 Hz, 1H, CH), 1.27 (s, 9H, (CH₃)₃). ESI-MS: 490.45 [M+H]⁺, 512.64 [M+Na]⁺, 488.29 [M−H]⁻; C₂₈H₂₈FN₃O₄ [489.21].

tert-Butyl (S)-(1-(7-Chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (9c1).

White solid, yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (d, J = 8.5 Hz, 1H, NH), 7.72 (d, J = 1.7 Hz, 1H, Ph-H), 7.61 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.46 (d, J = 7.8 Hz, 1H, Ph-H), 7.38–7.29 (m, 2H, Ph-H), 7.22–7.12 (m, 5H, Ph-H), 6.88–6.80 (m, 2H, Ph-H), 4.45–4.32 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.02 (dd, J = 13.7, 3.3 Hz, 1H, CH), 2.77 (dd, J = 13.6, 10.1 Hz, 1H, CH), 1.28 (s, 9H, (CH₃)₃). ESI-MS: 506.34 [M+H]⁺, 528.46 [M+Na]⁺; C₂₈H₂₈ClN₃O₄ [505.18].

tert-Butyl (S)-(1-(7-Bromo-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (9d1).

White solid, yield: 58%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J = 8.5 Hz, 1H, NH), 7.90–7.85 (m, 1H, Ph-H), 7.78–7.70 (m, 1H, Ph-H), 7.45 (d, J = 7.8 Hz, 1H, Ph-H), 7.32 (t, J = 7.4 Hz, 2H, Ph-H), 7.23–7.10 (m, 5H, Ph-H), 6.83 (d, J = 7.3 Hz, 2H, Ph-H), 4.45–4.32 (m, 1H, CH), 3.86 (s, 3H, OCH₃), 3.02 (dd, J = 13.6, 3.3 Hz, 1H, CH), 2.76 (dd, J = 13.7, 10.1 Hz, 1H, CH), 1.28 (s, 9H, (CH₃)₃). ESI-MS: 550.18 [M+H]⁺, 552.20 [M+H+2]⁺; C₂₈H₂₈BrN₃O₄ [549.13].

tert-Butyl (S)-(1-(3-(4-Methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (9e1).

White solid, yield: 59%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (d, J = 8.3 Hz, 1H, NH), 8.21–8.14 (m, 1H, Ph-H), 8.04 (dd, J = 8.3, 1.4 Hz, 1H, Ph-H), 7.48 (d, J = 8.0 Hz, 1H, Ph-H), 7.35 (t, J = 7.3 Hz, 2H, Ph-H), 7.18 (dd, J = 12.3, 7.9 Hz, 5H, Ph-H), 6.84 (d, J = 7.0 Hz, 2H, Ph-H), 4.48–4.36 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.39 (s, 3H, SO₂CH₃), 3.10–3.01 (m, 1H, CH), 2.78 (dd, J = 13.6, 10.1 Hz, 1H, CH), 1.28 (s, 9H, (CH₃)₃). ESI-MS: 549.92 [M+H]⁺, 548.11 [M−H]⁻; C₂₉H₃₁N₃O₆S [549.19].

tert-Butyl (S)-(2-(3,5-Difluorophenyl)-1-(3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)carbamate (9a2).

White solid, yield: 62%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.15 (d, J = 7.8 Hz, 1H, NH), 7.90 (t, J = 7.6 Hz, 1H, Ph-H), 7.74 (t, J = 8.3 Hz, 1H, Ph-H), 7.61–7.51 (m, 2H, Ph-H), 7.47 (d, J = 8.2 Hz, 1H, Ph-H), 7.37–7.29 (m, 1H, Ph-H), 7.21 (d, J = 8.9 Hz, 3H, Ph-H), 6.48 (d, J = 6.8 Hz, 2H, Ph-H), 4.43–4.32 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.08–2.99 (m, 1H, CH), 2.84 (dd, J = 13.7, 10.6 Hz, 1H, CH), 1.27 (s, 9H, (CH₃)₃). ESI-MS: 508.29 [M+H]⁺, 530.53 [M+Na]⁺; C₂₈H₂₇F₂N₃O₄ [507.20].

tert-Butyl (S)-(2-(3,5-Difluorophenyl)-1-(7-fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)carbamate (9b2).

White solid, yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.27–8.17 (m, 1H, NH), 7.56–7.51 (m, 1H, Ph-H), 7.46 (q, J = 8.4, 7.9 Hz, 3H, Ph-H), 7.37–7.31 (m, 1H, Ph-H), 7.20 (d, J = 8.9 Hz, 2H, Ph-H), 7.08–6.97 (m, 1H, Ph-H), 6.49 (t, J = 9.3 Hz, 2H, Ph-H), 4.43–4.32 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.08–2.99 (m, 1H, CH), 2.82 (dd, J = 13.7, 10.7 Hz, 1H, CH), 1.27 (s, 9H, (CH₃)₃). ESI-MS: 526.22 [M+H]⁺; C₂₈H₂₆F₃N₃O₄ [525.19].

tert-Butyl (S)-(1-(7-Chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(3,5-difluorophenyl)ethyl)carbamate (9c2).

White solid, yield: 63%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (d, J = 8.5 Hz, 1H, NH), 7.73 (d, J = 1.7 Hz, 1H, Ph-H), 7.62 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.56–7.49 (m, 1H, Ph-H), 7.47 (d, J = 8.2 Hz, 1H, Ph-H), 7.38–7.28 (m, 1H, Ph-H), 7.20 (d, J = 8.8 Hz, 2H, Ph-H), 7.01 (t, J = 9.4 Hz, 1H, Ph-H), 6.48 (d, J = 6.8 Hz, 2H, Ph-H), 4.44–4.31 (m, 1H, CH), 3.86 (s, 3H, OCH₃), 3.09–2.98 (m, 1H, CH), 2.81 (dd, J = 13.7, 10.6 Hz, 1H, CH), 1.27 (s, 9H, (CH₃)₃). ESI-MS: 542.71 [M+H]⁺, 564.28 [M+Na]⁺, 540.69 [M−H]⁻; C₂₈H₂₆ClF₂N₃O₄ [541.16].

tert-Butyl (S)-(1-(7-Bromo-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(3,5-difluorophenyl)ethyl)carbamate (9d2).

White solid, yield: 61%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (d, J = 8.5 Hz, 1H, NH), 7.88 (d, J = 1.4 Hz, 1H, Ph-H), 7.78–7.72 (m, 1H, Ph-H), 7.55–7.49 (m, 1H, Ph-H), 7.46 (d, J = 8.2 Hz, 1H, Ph-H), 7.36–7.30 (m, 1H, Ph-H), 7.20 (d, J = 8.8 Hz, 2H, Ph-H), 7.01 (t, J = 9.4 Hz, 1H, Ph-H), 6.48 (d, J = 6.8 Hz, 2H, Ph-H), 4.43–4.32 (m, 1H, CH), 3.86 (s, 3H, OCH₃), 3.08–2.99 (m, 1H, CH), 2.81 (dd, J = 13.7, 10.6 Hz, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 586.22 [M+H]⁺, 588.32 [M+H+2]⁺; C₂₈H₂₆BrF₂N₃O₄ [585.11].

tert-Butyl (S)-(2-(3,5-Difluorophenyl)-1-(3-(4-methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-carbamate (9e2).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (d, J = 8.3 Hz, 1H, NH), 8.23–8.14 (m, 1H, Ph-H), 8.09–8.02 (m, 1H, Ph-H), 7.58–7.51 (m, 1H, Ph-H), 7.47 (d, J = 8.3 Hz, 1H, Ph-H), 7.39–7.31 (m, 1H, Ph-H), 7.28–7.17 (m, 2H, Ph-H), 7.02 (t, J = 9.3 Hz, 1H, Ph-H), 6.50 (d, J = 7.0 Hz, 2H, Ph-H), 4.48–4.37 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.38 (s, 3H, SO₂CH₃), 3.11–3.02 (m, 1H, CH), 2.83 (dd, J = 13.8, 10.5 Hz, 1H, CH), 1.26 (s, 9H, (CH₃)₃). ESI-MS: 586.08 [M+H]⁺, 584.47 [M−H]⁻; C₂₉H₂₉F₂N₃O₆S [585.17].

5.1.8. General Procedure for the Synthesis of 10a1–10e1 and 10a2–10e2.

TFA was added dropwise to intermediates 9a1–9e1 and 9a2–9e2 in 30 mL of DCM and stirred at room temperature. Then, the resulting mixture solution was alkalized to pH ~7 with a saturated sodium bicarbonate solution and then extracted with DCM (40 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the intermediates 10a1–10e1 and 10a2–10e2.

(S)-2-(1-Amino-2-phenylethyl)-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10a1).

White solid, yield: 77%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (s, 2H, Ph-H), 8.23–8.16 (m, 1H, Ph-H), 8.02–7.95 (m, 1H, Ph-H), 7.84 (d, J = 8.0 Hz, 1H, Ph-H), 7.71–7.63 (m, 1H, Ph-H), 7.36 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.31–7.24 (m, 3H, Ph-H), 7.19 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.03 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 6.81 (dd, J = 6.4, 2.8 Hz, 2H, NH₂), 6.75 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 4.07–4.01 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.27 (dd, J = 13.9, 5.7 Hz, 1H, CH), 2.98 (dd, J = 13.9, 7.9 Hz, 1H, CH). ESI-MS: 372.50 [M+H]⁺; C₂₃H₂₁N₃O₂ [371.16].

(S)-2-(1-Amino-2-phenylethyl)-7-fluoro-3-(4-methoxyphenyl)-quinazolin-4(3H)-one (10b1).

White solid, yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (dd, J = 8.8, 6.3 Hz, 1H, Ph-H), 7.55 (dd, J = 10.0, 2.4 Hz, 1H, Ph-H), 7.40 (ddd, J = 17.4, 8.7, 2.5 Hz, 2H, Ph-H), 7.17 (q, J = 6.0 Hz, 3H, Ph-H), 7.11 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.03 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 6.89–6.83 (m, 2H, Ph-H), 6.80 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 3.84 (s, 3H, OCH₃), 3.57 (dd, J = 7.7, 5.8 Hz, 1H, CH), 3.06 (dd, J = 13.2, 5.7 Hz, 1H, CH), 2.63 (dd, J = 13.2, 7.9 Hz, 1H, CH), 2.04 (s, 2H, NH₂). ESI-MS: 390.54 [M+H]⁺, 388.41 [M−H]⁻; C₂₃H₂₀FN₃O₂ [389.15].

(S)-2-(1-Amino-2-phenylethyl)-7-chloro-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10c1).

White solid, yield: 65%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (d, J = 8.5 Hz, 1H, Ph-H), 7.82 (d, J = 1.9 Hz, 1H, Ph-H), 7.57 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.38 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.17 (q, J = 6.1 Hz, 3H, Ph-H), 7.10 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.02 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 6.92–6.83 (m, 2H, Ph-H), 6.78 (dd, J = 8.7, 2.5 Hz, 1H, Ph-H), 3.84 (s, 3H, OCH₃), 3.61–3.54 (m, 1H, CH), 3.06 (dd, J = 13.1, 5.8 Hz, 1H, CH), 2.63 (dd, J = 13.2, 7.7 Hz, 1H, CH), 1.96 (s, 2H, NH₂). ESI-MS: 406.73 [M+H]⁺, 428.42 [M+Na]⁺; C₂₃H₂₀ClN₃O₂ [405.12].

(S)-2-(1-Amino-2-phenylethyl)-7-bromo-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10d1).

White solid, yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, J = 8.5 Hz, 1H, Ph-H), 7.98 (d, J = 1.8 Hz, 1H, Ph-H), 7.71 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.38 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.17 (q, J = 5.9 Hz, 3H, Ph-H), 7.10 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.02 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 6.88–6.83 (m, 2H, Ph-H), 6.78 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 3.84 (s, 3H, OCH₃), 3.59–3.54 (m, 1H, CH), 3.05 (dd, J = 13.2, 5.8 Hz, 1H, CH), 2.65–2.60 (m, 1H, CH), 1.99 (s, 2H, NH₂). ESI-MS: 450.37 [M+H]⁺; C₂₃H₂₀BrN₃O₂ [449.07].

(S)-2-(1-Amino-2-phenylethyl)-3-(4-methoxyphenyl)-7-(methylsulfonyl)quinazolin-4(3H)-one (10e1).

White solid, yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.34 (d, J = 8.3 Hz, 1H, Ph-H), 8.26 (d, J = 1.6 Hz, 1H, Ph-H), 8.01 (dd, J = 8.3, 1.8 Hz, 1H, Ph-H), 7.41 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.17 (q, J = 6.7, 6.3 Hz, 3H, Ph-H), 7.12 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.04 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 6.91–6.85 (m, 2H, Ph-H), 6.83 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 3.85 (s, 3H, OCH₃), 3.62–3.56 (m, 1H, CH), 3.40 (s, 3H, SO₂CH₃), 3.08 (dd, J = 13.2, 5.6 Hz, 1H, CH), 2.65 (dd, J = 13.2, 7.8 Hz, 1H, CH), 2.00 (s, 2H, NH₂). ESI-MS: 450.25 [M+H]⁺; C₂₄H₂₃N₃O₄S [449.14].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10a2).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 2H, Ph-H), 8.24–8.18 m, 1H, Ph-H), 8.04–7.93 (m, 1H, Ph-H), 7.80 (d, J = 8.0 Hz, 1H, Ph-H), 7.72–7.63 (m, 1H, Ph-H), 7.40 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.33 (dd, J = 8.7, 2.6 Hz, 1H, Ph-H), 7.24 (dd, J = 8.7, 2.9 Hz, 1H, Ph-H), 7.21–7.10 (m, 2H, Ph-H), 6.51–6.38 (m, 2H, NH₂), 4.10–4.03 (m, 1H, CH), 3.87 (s, 3H, OCH₃), 3.24 (dd, J = 14.5, 3.8 Hz, 1H, CH), 2.96 (dd, J = 14.5, 9.0 Hz, 1H, CH). ESI-MS: 408.41 [M+H]⁺; C₂₃H₁₉F₂N₃O₂ [407.14].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-7-fluoro-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10b2).

White solid, yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19 (dd, J = 8.8, 6.3 Hz, 1H, Ph-H), 7.53 (dd, J = 10.0, 2.5 Hz, 1H, Ph-H), 7.47–7.39 (m, 2H, Ph-H), 7.23 (dd, J = 8.7, 2.5 Hz, 1H, Ph-H), 7.19–7.10 (m, 2H, Ph-H), 7.00 (tt, J = 9.4, 2.3 Hz, 1H, Ph-H), 6.63–6.50 (m, 2H, Ph-H), 3.85 (s, 3H, OCH₃), 3.56 (dd, J = 8.8, 4.3 Hz, 1H, CH), 3.02 (dd, J = 13.4, 4.2 Hz, 1H, CH), 2.64 (dd, J = 13.4, 8.8 Hz, 1H, CH), 2.01 (s, 2H, NH₂). ESI-MS: 426.65 [M+H]⁺, 424.41 [M−H]⁻; C₂₃H₁₈F₃N₃O₂ [425.14].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-7-chloro-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10c2).

White solid, yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12 (d, J = 8.5 Hz, 1H, Ph-H), 7.81 (d, J = 1.9 Hz, 1H, Ph-H), 7.59 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.42 (dd, J = 8.4, 2.3 Hz, 1H, Ph-H), 7.22 (dd, J = 8.4, 2.5 Hz, 1H, Ph-H), 7.18–7.09 (m, 2H, Ph-H), 7.00 (ddd, J = 11.7, 5.8, 2.2 Hz, 1H, Ph-H), 6.55 (d, J = 6.6 Hz, 2H, Ph-H), 3.85 (s, 3H, OCH₃), 3.56 (dd, J = 8.6, 4.3 Hz, 1H, CH), 3.02 (dd, J = 13.4, 4.3 Hz, 1H, CH), 2.63 (dd, J = 13.4, 8.7 Hz, 1H, CH), 2.03 (s, 2H, NH₂). ESI-MS: 464.38 [M+Na]⁺; C₂₃H₁₈ClF₂N₃O₂ [441.11].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-7-bromo-3-(4-methoxyphenyl)quinazolin-4(3H)-one (10d2).

White solid, yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (d, J = 8.5 Hz, 1H, Ph-H), 7.94 (d, J = 1.7 Hz, 1H, Ph-H), 7.75 (dd, J = 8.5, 1.8 Hz, 1H, Ph-H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H, Ph-H), 7.23 (dd, J = 8.6, 2.5 Hz, 1H, Ph-H), 7.15 (ddd, J = 17.4, 8.7, 2.8 Hz, 2H, Ph-H), 7.04 (dt, J = 11.5, 5.7 Hz, 1H, Ph-H), 6.52 (d, J = 6.6 Hz, 2H, Ph-H), 3.85 (s, 3H, OCH₃), 3.71 (dd, J = 8.6, 4.2 Hz, 1H, CH), 3.08 (dd, J = 13.7, 4.2 Hz, 1H, CH), 2.73 (dd, J = 13.7, 8.8 Hz, 1H, CH). ESI-MS: 486.30 [M+H]⁺; C₂₃H₁₈BrF₂N₃O₂ [485.06].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-3-(4-methoxyphenyl)-7-(methylsulfonyl)quinazolin-4(3H)-one (10e2).

White solid, yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.36 (d, J = 8.3 Hz, 1H, Ph-H), 8.24 (d, J = 1.5 Hz, 1H, Ph-H), 8.02 (dd, J = 8.3, 1.8 Hz, 1H, Ph-H), 7.44 (dd, J = 8.8, 2.5 Hz, 1H, Ph-H), 7.26 (dd, J = 8.8, 2.5 Hz, 1H, Ph-H), 7.20–7.12 (m, 2H, Ph-H), 7.00 (tt, J = 9.5, 2.3 Hz, 1H, Ph-H), 6.61–6.53 (m, 2H, Ph-H), 3.86 (s, 3H, OCH₃), 3.59 (dd, J = 8.7, 4.2 Hz, 1H, Ph-H), 3.39 (s, 3H, SO₂CH₃), 3.04 (dd, J = 13.5, 4.2 Hz, 1H, CH), 2.65 (dd, J = 13.5, 8.8 Hz, 1H, CH), 2.04 (s, 2H, NH₂). ESI-MS: 486.23 [M+H]⁺, 484.30 [M−H]⁻; C₂₄H₂₁F₂N₃O₄S [485.12].

5.1.9. General Procedure for the Synthesis of 11a1–11e1 and 11a2–11e2.

Intermediate 4 (1.2 equiv) and HATU (1.5 equiv) were mixed in 30 mL of DCM and stirred in an ice bath for 30 min. Subsequently, DIEA (2 equiv) and intermediates 10a1–10e1 and 10a2–10e2 (1 equiv) were added to the mixture and then stirred at room temperature for another 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed with 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:1) to afford the target compounds 11a1–11e1 and 11a2–11e2.

(S)-N-(1-(3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11a1).

White solid, yield: 65%. Mp: 213–214 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (d, J = 7.6 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.17–8.11 (m, 1H, Ph-H), 8.07 (d, J = 8.8 Hz, 2H, Ph-H), 7.94–7.86 (m, 1H, Ph-H), 7.74 (d, J = 8.1 Hz, 1H, Ph-H), 7.57 (t, J = 7.3 Hz, 1H, Ph-H), 7.30 (dd, J = 7.8, 3.6 Hz, 1H, Ph-H), 7.25–7.07 (m, 6H, Ph-H), 6.90–6.82 (m, 2H, Ph-H), 4.63 (td, J = 9.1, 4.5 Hz, 1H, CH), 3.97–3.88 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.70–3.59 (m, 2H, CH₂), 3.31–3.24 (m, 2H, CH₂), 3.24–3.7 (m, 2H, CH₂), 3.14 (dd, J = 13.7, 4.3 Hz, 1H, CH), 2.79 (dd, J = 13.7, 9.5 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.63 (C═O), 163.46 (C═O), 161.88 (C═O), 160.09, 157.69, 150.76, 147.36, 141.03, 137.96, 135.28, 130.55, 130.33, 129.67, 129.32, 129.02, 128.64, 127.52, 127.35, 127.00, 126.96, 125.29, 121.22, 115.15, 56.02, 53.46, 48.69, 48.48, 46.93, 43.07, 38.76. HRMS calcd for m/z C₃₅H₃₂N₆O₈S [M+H]⁺ 697.2080, found 697.2075 [M+H]⁺. HPLC purity: 98.71%.

(S)-N-(1-(7-Fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11b1).

White solid, yield: 58%. Mp: 209–210 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (d, J = 7.5 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.23–8.16 (m, 1H, Ph-H), 8.07 (d, J = 8.8 Hz, 2H, Ph-H), 7.44 (t, J = 8.3 Hz, 2H, Ph-H), 7.34–7.28 (m, 1H, Ph-H), 7.24–7.20 (m, 1H, Ph-H), 7.20–7.09 (m, 5H, Ph-H), 6.90–6.82 (m, 2H, Ph-H), 4.67–4.57 (m, 1H, CH), 3.97–3.88 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.69–3.59 (m, 2H, CH₂), 3.28 (dt, J = 10.2, 5.4 Hz, 2H, CH₂), 3.24–3.17 (m, 2H, CH₂), 3.17–3.09 (m, 1H, CH), 2.79 (dd, J = 13.8, 9.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.74 (C═O), 166.40 (d, ¹J_CF = 252.3 Hz), 163.46 (C═O), 161.20 (C═O), 160.15, 159.36, 150.75, 149.51 (d, ³J_CF = 13.4 Hz), 140.92, 137.85, 130.52, 130.28, 129.68, 129.30, 128.76, 128.67, 127.01, 125.30, 118.29, 118.28, 116.11 (d, ²J_CF = 23.5 Hz), 115.17, 112.38 (d, ²J_CF = 21.6 Hz), 56.01, 53.61, 48.69, 48.45, 46.95, 43.06, 38.57. HRMS calcd for m/z C₃₅H₃₁FN₆O₈S [M+H]⁺ 715.1981, found 715.1981 [M+H]⁺. HPLC purity: 98.10%.

(S)-N-(1-(7-Chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11c1).

White solid, yield: 52%. Mp: 194–195 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (d, J = 7.5 Hz, 1H, NH), 8.43 (d, J = 8.9 Hz, 2H, Ph-H), 8.13 (d, J = 8.5 Hz, 1H, Ph-H), 8.07 (d, J = 8.9 Hz, 2H, Ph-H), 7.72 (d, J = 2.0 Hz, 1H, Ph-H), 7.61 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.35–7.28 (m, 1H, Ph-H), 7.24–7.07 (m, 6H, Ph-H), 6.90–6.82 (m, 2H, Ph-H), 4.61 (td, J = 9.2, 4.5 Hz, 1H, CH), 3.98–3.88 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.65 (d, J = 2.2 Hz, 2H, CH₂), 3.31–3.25 (m, 2H, CH₂), 3.25–3.16 (m, 2H, CH₂), 3.13 (dd, J = 13.8, 4.3 Hz, 1H, CH), 2.78 (dd, J = 13.8, 9.5 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.75 (C═O), 163.47 (C═O), 161.32 (C═O), 160.17, 159.41, 150.75, 148.47, 140.96, 139.87, 137.83, 130.49, 130.24, 129.68, 129.31, 129.18, 128.71, 128.68, 127.82, 127.01, 126.39, 125.30, 120.09, 115.18, 56.02, 53.62, 48.69, 48.46, 46.99, 43.06, 38.56. HRMS calcd for m/z C₃₅H₃₁ClN₆O₈S [M+H]⁺ 731.1685, found 731.1686 [M+H]⁺. HPLC purity: 98.56%.

(S)-N-(1-(7-Bromo-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11d1).

White solid, yield: 55%. Mp: 160–161 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (d, J = 7.5 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.06 (dd, J = 11.1, 8.7 Hz, 3H, Ph-H), 7.88 (d, J = 1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 8.5, 1.8 Hz, 1H, Ph-H), 7.30 (dd, J = 9.0, 2.3 Hz, 1H, Ph-H), 7.22–7.07 (m, 6H, Ph-H), 6.89–6.82 (m, 2H, Ph-H), 4.61 (td, J = 9.2, 4.5 Hz, 1H, CH), 3.97–3.87 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.65 (d, J = 1.9 Hz, 2H, CH₂), 3.32–3.25 (m, 2H, CH₂), 3.25–3.16 (m, 2H, CH₂), 3.12 (dd, J = 13.8, 4.2 Hz, 1H, CH), 2.78 (dd, J = 13.8, 9.5 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.75 (C═O), 163.47 (C═O), 161.46 (C═O), 160.17, 159.35, 150.75, 148.52, 140.96, 137.83, 130.58, 130.48, 130.23, 129.68, 129.49, 129.31, 129.16, 128.82, 128.71, 128.68, 127.02, 125.30, 120.37, 115.18, 56.02, 53.61, 48.69, 48.45, 47.00, 43.06, 38.57. HRMS calcd for m/z C₃₅H₃₁BrN₆O₈S [M+H]⁺ 775.1180, [M+H+2]⁺ 777.1160, found 775.1179, [M+H]⁺, 777.1165 [M+H+2]⁺. HPLC purity: 98.96%.

(S)-N-(1-(3-(4-Methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)-sulfonyl)-2-oxopiperazin-1-yl)acetamide (11e1).

White solid, yield: 62%. Mp: 185–186 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (d, J = 7.6 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.36 (d, J = 8.3 Hz, 1H, Ph-H), 8.17 (d, J = 1.4 Hz, 1H, Ph-H), 8.11–8.03 (m, 3H, Ph-H), 7.38–7.30 (m, 1H, Ph-H), 7.28–7.09 (m, 6H, Ph-H), 6.93–6.81 (m, 2H, Ph-H), 4.65 (td, J = 9.2, 4.4 Hz, 1H, CH), 3.98–3.89 (m, 2H, CH₂), 3.86 (s, 3H, OCH₃), 3.65 (s, 2H, CH₂), 3.39 (s, 3H, SO₂CH₃), 3.31–3.25 (m, 2H, CH₂), 3.20 (q, J = 8.9, 6.8 Hz, 2H, CH₂), 3.17–3.10 (m, 1H, CH), 2.80 (dd, J = 13.9, 9.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.76 (C═O), 163.47 (C═O), 161.22 (C═O), 160.26, 159.82, 150.75, 147.40, 146.64, 140.92, 137.76, 130.44, 130.19, 129.68, 129.29, 129.01, 128.71, 128.58, 126.12, 125.30, 124.60, 115.25, 56.04, 53.54, 48.69, 48.44, 46.97, 43.62, 43.05, 38.66. HRMS calcd for m/z C₃₆H₃₄N₆O₁₀S₂ [M+H]⁺ 775.1851, found 775.1851, [M+H]⁺. HPLC purity: 95.54%.

(S)-N-(2-(3,5-Difluorophenyl)-1-(3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11a2).

White solid, yield: 46%. Mp: 198–200 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75 (d, J = 7.8 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.18–8.12 (m, 1H, Ph-H), 8.07 (d, J = 8.8 Hz, 2H, Ph-H), 7.94–7.87 (m, 1H, Ph-H), 7.73 (d, J = 8.0 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz, 1H, Ph-H), 7.44 (dd, J = 9.0, 2.4 Hz, 1H, Ph-H), 7.31 (dd, J = 9.0, 2.4 Hz, 1H, Ph-H), 7.21–7.12 (m, 2H, Ph-H), 7.02 (ddd, J = 11.5, 5.7, 2.1 Hz, 1H, Ph-H), 6.51 (d, J = 6.4 Hz, 2H, Ph-H), 4.66–4.57 (m, 1H, CH), 3.96–3.88 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.71–3.58 (m, 2H, CH₂), 3.33–3.26 (m, 2H, CH₂), 3.26–3.17 (m, 2H, CH₂), 3.14 (dd, J = 13.9, 3.4 Hz, 1H, CH), 2.83 (dd, J = 13.9, 10.0 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.73 (C═O), 163.47 (C═O), 162.48 (dd, ¹J_CF = 246.0, ¹³J_CF = 13.5 Hz), 161.89 (C═O), 160.19, 157.02, 150.75, 147.22, 142.56 (t, ³J_CF = 9.1 Hz) 140.86, 135.34, 130.59, 130.43, 129.68, 128.98, 127.66, 127.37, 127.02, 125.31, 121.29, 115.33, 115.17, 112.38 (dd, ²J_CF = 24.9 Hz, ⁴J_CF = 5.5 Hz), 102.60 (t, ²J_CF = 25.8 Hz). 56.00, 53.10, 48.67, 48.51, 47.04, 43.06, 38.01. HRMS calcd for m/z C₃₅H₃₀F₂N₆O₈S [M+H]⁺ 733.1881, found 733.1884 [M+H]⁺. HPLC purity: 96.51%.

(S)-N-(2-(3,5-Difluorophenyl)-1-(7-fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11b2).

White solid, yield: 51%. Mp: 195–196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (d, J = 7.7 Hz, 1H, NH), 8.43 (d, J = 8.9 Hz, 2H, Ph-H), 8.21 (dd, J = 9.6, 6.2 Hz, 1H, Ph-H), 8.12–8.04 (m, 2H, Ph-H), 7.45 (ddd, J = 8.4, 6.3, 2.5 Hz, 3H, Ph-H), 7.32 (dd, J = 9.0, 2.5 Hz, 1H, Ph-H), 7.23–7.12 (m, 2H, Ph-H), 7.02 (tt, J = 9.4, 2.2 Hz, 1H, Ph-H), 6.57–6.47 (m, 2H, Ph-H), 4.69–4.56 (m, 1H, CH), 3.96–3.88 (m, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.71–3.59 (m, 2H, CH₂), 3.33–3.26 (m, 2H, CH₂), 3.27–3.17 (m, 2H, CH₂), 3.14 (dd, J = 13.9, 3.4 Hz, 1H, CH), 2.83 (dd, J = 13.9, 10.0 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.80 (C═O), 166.39 (d, ¹J_CF = 252.0 Hz), 163.49 (C═O), 162.56 (dd, ¹J_CF = 245.6, ³J_CF = 13.0 Hz), 161.20 (C═O), 160.27, 158.61, 150.75, 149.38 (d, ³J_CF = 13.1 Hz), 142.45 (t, ³J_CF = 9.4 Hz), 140.93, 130.54, 130.39, 130.34, 130.23, 129.67, 128.74, 125.29, 118.39, 118.38, 116.20 (d, ²J_CF = 23.7 Hz), 115.36, 115.20, 112.39 (dd, ²J_CF = 24.8, ⁴J_CF = 6.7 Hz), 102.63 (t, ²J_CF = 25.0 Hz), 56.01, 53.21, 48.68, 48.52, 43.07, 38.71. HRMS calcd for m/z C₃₅H₂₉F₃N₆O₈S [M+H]⁺ 751.1792, found 751.1790 [M+H]⁺. HPLC purity: 98.32%.

(S)-N-(1-(7-Chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11c2).

White solid, yield: 49%. Mp: 212–214 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (d, J = 7.7 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.14 (d, J = 8.5 Hz, 1H, Ph-H), 8.07 (d, J = 8.8 Hz, 2H, Ph-H), 7.72 (d, J = 1.9 Hz, 1H, Ph-H), 7.62 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.47–7.41 (m, 1H, Ph-H), 7.31 (dd, J = 7.8, 3.5 Hz, 1H, Ph-H), 7.21–7.11 (m, 2H, Ph-H), 7.02 (t, J = 9.4 Hz, 1H, Ph-H), 6.51 (d, J = 6.5 Hz, 2H, Ph-H), 4.65–4.55 (m, 1H, CH), 3.90 (d, J = 4.5 Hz, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.71–3.59 (m, 2H, CH₂), 3.32–3.26 (m, 2H, CH₂), 3.26–3.17 (m, 2H, CH₂), 3.13 (dd, J = 13.9, 3.3 Hz, 1H, CH), 2.82 (dd, J = 13.9, 9.9 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.82 (C═O), 163.49 (C═O), 162.43 (d, ¹J_CF = 246.0, ³J_CF = 13.6 Hz), 161.33 (C═O), 160.28, 158.67, 150.76, 148.34, 142.44 (t, ³J_CF= 9.2 Hz), 140.94, 139.87, 130.51, 130.36, 129.67, 129.19, 128.69, 127.92, 126.42, 125.30, 120.20, 115.38, 115.21, 112.40 (d, ²J_CF = 25.0, ⁴J_CF= 6.3 Hz), 102.64 (t, ²J_CF = 25.8 Hz), 56.02, 53.23, 48.68, 48.52, 47.09, 43.08, 37.84. HRMS calcd for m/z C₃₅H₂₉ClF₂N₆O₈S [M+H]⁺ 767.1497, found 767.1494 [M+H]⁺. HPLC purity: 98.07%.

(S)-N-(1-(7-Bromo-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11d2).

White solid, yield: 53%. Mp: >240 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (d, J = 7.6 Hz, 1H, NH), 8.43 (d, J = 8.7 Hz, 2H, Ph-H), 8.15–8.01 (m, 3H, Ph-H), 7.93–7.84 (m, 1H, Ph-H), 7.80–7.71 (m, 1H, Ph-H), 7.49–7.39 (m, 1H, Ph-H), 7.37–7.27 (m, 1H, Ph-H), 7.15 (t, J = 6.6 Hz, 2H, Ph-H), 7.02 (t, J = 9.3 Hz, 1H, Ph-H), 6.51 (d, J = 6.6 Hz, 2H, Ph-H), 4.66–4.54 (m, 1H, NH), 3.90 (d, J = 3.1 Hz, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.71–3.58 (m, 2H, CH₂), 3.32–3.26 (m, 2H, CH₂), 3.23 (dd, J = 12.2, 5.8 Hz, 2H, CH₂), 3.17–3.08 (m, 1H, CH), 2.81 (dd, J = 13.7, 10.0 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.82 (C═O), 163.48 (C═O), 162.49 (dd, ¹J_CF = 245.9, ³J_CF = 13.4 Hz), 161.47 (C═O), 160.28, 158.61, 150.76, 148.39, 142.44 (t, ³J_CF = 8.8 Hz), 140.95, 130.68, 130.50, 130.34, 129.67, 129.52, 129.16, 128.81, 128.69, 125.30, 120.48, 115.38, 115.21, 112.40 (d, ²J_CF = 24.4, ⁴J_CF = 5.5 Hz), 102.64 (t, ²J_CF = 25.8 Hz), 56.02, 53.23, 48.68, 48.52, 47.10, 43.08, 37.85. HRMS calcd for m/z C₃₅H₂₉BrF₂N₆O₈S [M+H]⁺ 811.0992, [M+2+H]⁺ 813.0971, found 811.0991 [M+H]⁺, 813.0978 [M+2+H]⁺. HPLC purity: 96.93%.

(S)-N-(2-(3,5-Difluorophenyl)-1-(3-(4-methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (11e2).

White solid, yield: 66%. Mp: 198–199 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (d, J = 7.7 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.37 (d, J = 8.3 Hz, 1H, Ph-H), 8.17 (d, J = 1.4 Hz, 1H, Ph-H), 8.12–8.03 (m, 3H, Ph-H), 7.51–7.42 (m, 1H, Ph-H), 7.34 (dd, J = 8.0, 3.4 Hz, 1H, Ph-H), 7.25–7.13 (m, 2H, Ph-H), 7.08–6.98 (m, 1H, Ph-H), 6.52 (d, J = 6.5 Hz, 2H, Ph-H), 4.73–4.59 (m, 1H, CH), 3.94 (d, J = 16.9 Hz, 2H, CH₂), 3.86 (s, 3H, OCH₃), 3.71–3.59 (m, 2H, CH₂), 3.38 (s, 3H, SO₂CH₃), 3.29 (dd, J = 8.8, 3.7 Hz, 2H, CH₂), 3.23 (dq, J = 12.5, 6.7, 5.7 Hz, 2H, CH₂), 3.15 (dd, J = 14.0, 3.3 Hz, 1H, CH), 2.84 (dd, J = 14.2, 10.2 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.83 (C═O), 163.51 (C═O), 162.50 (dd, ¹J_CF = 245.9, ³J_CF = 13.5 Hz), 161.25 (C═O), 160.37, 159.04, 150.75, 147.28, 146.61, 142.35 (t, ³J_CF = 9.5 Hz), 140.86, 130.46, 130.29, 129.67, 129.02, 128.55, 126.14, 125.31, 124.73, 115.44, 115.29, 112.38 (dd, ²J_CF = 24.8 ⁴J_CF = 6.6 Hz), 102.67 (t, ²J_CF = 25.4 Hz), 56.03, 53.16, 48.67, 48.50, 47.08, 43.62, 43.05, 37.92. HRMS calcd for m/z C₃₆H₃₂F₂N₆O₁₀S₂ [M+H]⁺ 811.1662, found 811.1664 [M+H]⁺. HPLC purity: 98.90%.

5.1.10. General Procedure for the Synthesis of 12a1–12c1, 12e1, 12a2–12c2, and 12e2.

Compounds 11a1–11c1, 11e1, 11a2–11c2, and 11e2 (150 mg) and 10% Pd·C (10% w/w, 15 mg) were dissolved in DCM (10 mL), and the solution was degassed and stirred at room temperature overnight in the atmosphere of hydrogen. The mixture was filtered and concentrated, and the resulting residue was purified by recrystallization (ethyl acetate) or preparative thin-layer chromatography (methanol:DCM = 1:30) to provide the target compounds 12a1–12c1, 12e1, 12a2–12c2, and 12e2.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (12a1).

White solid, yield: 68%. Mp: 201–202 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (d, J = 7.6 Hz, 1H, NH), 8.18–8.11 (m, 1H, Ph-H), 7.94–7.86 (m, 1H, Ph-H), 7.73 (d, J = 8.1 Hz, 1H, Ph-H), 7.57 (t, J = 7.5 Hz, 1H, Ph-H), 7.40 (d, J = 8.7 Hz, 2H, Ph-H), 7.32 (dd, J = 8.5, 2.5 Hz, 1H, Ph-H), 7.22 (dd, J = 8.5 2.5 Hz, 1H, Ph-H), 7.19–7.14 (m, 3H, Ph-H), 7.14–7.08 (m, 2H, Ph-H), 6.92–6.82 (m, 2H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.18 (s, 2H, NH₂), 4.64 (td, J = 9.3, 4.4 Hz, 1H, CH), 3.89 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.45–3.36 (m, 2H, CH₂), 3.17 (ddd, J = 14.2, 7.4, 3.5 Hz, 2H, CH₂), 3.13–3.02 (m, 2H, CH₂), 3.02–2.94 (m, 1H, CH), 2.80 (dd, J = 13.8, 9.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.73 (C═O), 164.00 (C═O), 161.89 (C═O), 160.09, 157.78, 154.17, 147.36, 137.97, 135.30, 130.56, 130.40, 130.30, 129.32, 129.03, 128.64, 127.52, 127.34, 127.01, 126.97, 121.23, 118.58, 115.17, 115.14, 113.32, 56.01, 53.46, 49.30, 48.45, 46.94, 43.33, 38.69. HRMS calcd for m/z C₃₅H₃₄N₆O₆S [M+H]⁺ 667.2333, found 667.2335 [M+H]⁺. HPLC purity: 99.20%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(7-fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (12b1).

White solid, yield: 35%. Mp: 188–189 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (d, J = 7.6 Hz, 1H, NH), 8.26–8.16 (m, 1H, Ph-H), 7.45 (d, J = 9.1 Hz, 2H, Ph-H), 7.43–7.37 (m, 2H, Ph-H), 7.33 (dd, J = 8.6, 2.5 Hz, 1H, Ph-H), 7.25–7.07 (m, 6H, Ph-H), 6.94–6.82 (m, 2H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.17 (s, 2H, NH₂), 4.63 (td, J = 9.4, 4.4 Hz, 1H, CH), 3.89 (s, 2H, CH₂), 3.85 (s, H, OCH₃), 3.46–3.34 (m, 2H, CH₂), 3.18 (ddd, J = 19.9, 7.9, 4.3 Hz, 2H, CH₂), 3.13–3.03 (m, 2H, CH₂), 3.03–2.94 (m, 1H, CH), 2.80 (dd, J = 13.8, 9.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.83 (C═O), 166.40 (d, ¹J_CF = 251.9 Hz), 164.05 (C═O), 161.23 (C═O), 160.16, 159.38, 154.16, 149.51 (d, ³J_CF = 13.2 Hz), 140.92, 137.84, 130.50, 130.29, 129.30, 128.76, 128.66, 127.02, 118.56, 118.29, 116.11 (d, ²J_CF = 23.8 Hz), 115.20, 115.17, 113.34, 112.39 (d, ²J_CF = 21.5 Hz), 56.01, 53.60, 49.28, 48.44, 46.96, 43.32, 38.53. HRMS calcd for m/z C₃₅H₃₃FN₆O₆S [M+H]⁺ 685.2239, found 685.2237 [M+H]⁺. HPLC purity: 99.15%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(7-chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (12c1).

White solid, yield: 33%. Mp: >240 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (d, J = 7.5 Hz, 1H, NH), 8.06 (d, J = 8.5 Hz, 1H, Ph-H), 7.65 (d, J = 1.9 Hz, 1H, Ph-H), 7.54 (dd, J = 8.5, 2.0 Hz, 1H, Ph-H), 7.33 (d, J = 8.7 Hz, 2H, Ph-H), 7.25 (dd, J = 8.6, 2.5 Hz, 1H, Ph-H), 7.19–6.99 (m, 6H, Ph-H), 6.86–6.74 (m, 2H, Ph-H), 6.60 (d, J = 8.7 Hz, 2H, Ph-H), 6.10 (s, 2H, NH₂), 4.62–4.50 (m, 1H, CH), 3.82 (s, 2H, CH₂), 3.77 (s, 3H, OCH₃), 3.31 (d, J = 7.6 Hz, 2H, CH₂), 3.16–3.05 (m, 2H, CH₂), 3.00 (dt, J = 17.2, 4.3 Hz, 2H, CH₂), 2.95–2.87 (m, 1H, CH), 2.72 (dd, J = 13.7, 9.5 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.84 (C–O), 164.01 (C═O), 161.34 (C═O), 160.17, 159.48, 154.18, 148.48, 139.87, 137.84, 130.48, 130.32, 130.29, 129.31, 129.19, 128.71, 128.67, 127.82, 127.02, 126.38, 120.09, 118.52, 115.21, 115.18, 113.31, 56.02, 53.62, 49.29, 48.42, 46.97, 43.32, 38.48. HRMS calcd for m/z C₃₅H₃₃ClN₆O₆S [M+H]⁺ 701.1944, found 701.1943 [M+H]⁺. HPLC purity: 97.78%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(3-(4-methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (12e1).

White solid, yield: 30%. Mp: 193–194 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J = 7.6 Hz, 1H, NH), 8.36 (d, J = 8.3 Hz, 1H, Ph-H), 8.20–8.15 (m, 1H, Ph-H), 8.05 (dd, J = 8.3, 1.5 Hz, 1H, Ph-H), 7.40 (d, J = 8.6 Hz, 2H, Ph-H), 7.35 (dd, J = 8.6, 2.4 Hz, 1H, Ph-H), 7.22 (dd, J = 8.5, 2.4 Hz, 1H, Ph-H), 7.20–7.08 (m, 5H, Ph-H), 6.91–6.82 (m, 2H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.16 (s, 2H, NH₂), 4.66 (td, J = 9.2, 4.4 Hz, 1H, CH), 3.90 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.38 (s, 5H, SO₂CH₃, CH₂), 3.24–3.14 (m, 2H, CH₂), 3.14–3.04 (m, 2H, CH₂), 3.04–2.96 (m, 1H, CH), 2.81 (dd, J = 13.8, 9.6 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.86 (C═O), 164.01 (C═O), 161.23 (C═O), 160.26, 159.89, 154.18, 147.41, 146.65, 137.78, 130.42, 130.31, 129.29, 129.00, 128.70, 128.59, 127.06, 126.12, 124.61, 124.59, 118.50, 115.28, 115.24, 113.31, 56.03, 53.55, 49.30, 48.41, 46.98, 43.63, 43.30, 38.58. HRMS calcd for m/z C₃₆H₃₆N₆O₈S₂ [M+H]⁺ 745.2109, found 745.2104 [M+H]⁺. HPLC purity: 95.21%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(2-(3,5-difluorophenyl)-1-(3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)acetamide (12a2).

White solid, yield: 45%. Mp: 185–186 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (d, J = 7.8 Hz, 1H, NH), 8.14 (d, J = 7.8 Hz, 1H, Ph-H), 7.90 (t, J = 7.6 Hz, 1H, Ph-H), 7.72 (d, J = 8.1 Hz, 1H, Ph-H), 7.58 (t, J = 7.5 Hz, 1H, Ph-H), 7.47–7.42 (m, 1H, Ph-H), 7.40 (d, J = 8.6 Hz, 2H, Ph-H), 7.37–7.30 (m, 1H, Ph-H), 7.22–7.12 (m, 2H, Ph-H), 7.02 (t, J = 9.4 Hz, 1H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.50 (d, J = 6.6 Hz, 2H, Ph-H), 6.19 (s, 2H, NH₂), 4.68–4.58 (m, 1H, CH), 3.89 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.46–3.36 (m, 2H, CH₂), 3.23 (ddd, J = 10.9, 6.7, 4.1 Hz, 1H, CH), 3.14 (dt, J = 16.6, 3.8 Hz, 2H, CH₂), 3.09–2.96 (m, 2H, CH₂), 2.85 (dd, J = 13.8, 10.1 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.81 (C═O), 164.02 (C═O), 162.48 (dd, ¹J_CF = 246.1, ³J_CF = 13.3 Hz), 161.88 (C═O), 160.20, 157.09, 154.18, 147.23, 142.60 (t, ³J_CF = 9.3 Hz), 135.33, 130.59, 130.51, 130.30, 128.99, 127.64, 127.35, 127.02, 121.31, 118.49, 115.32, 115.20, 113.31, 112.39 (dd, ²J_CF = 24.2, ⁴J_CF = 5.8 Hz), 102.63 (t, ²J_CF = 25.9 Hz), 56.00, 53.12, 49.29, 48.47, 47.04, 43.32, 37.95. HRMS calcd for m/z C₃₅H₃₂F₂N₆O₆S [M+H]⁺ 703.2145, found 703.2147 [M+H]⁺. HPLC purity: 99.73%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(2-(3,5-difluorophenyl)-1-(7-fluoro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)acetamide (12b2).

White solid, yield: 36%. Mp: 192–193 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.73 (d, J = 7.7 Hz, 1H, NH), 8.21 (dd, J = 9.6, 6.2 Hz, 1H, Ph-H), 7.49–7.42 (m, 3H, Ph-H), 7.40 (d, J = 8.7 Hz, 2H, Ph-H), 7.37–7.31 (m, 1H, Ph-H), 7.24–7.12 (m, 2H, Ph-H), 7.03 (dt, J = 11.4, 5.7 Hz, 1H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.51 (d, J = 6.5 Hz, 2H, Ph-H), 6.17 (s, 2H, NH₂), 4.68–4.58 (m, 1H, CH), 3.89 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.48–3.37 (m, 2H, CH₂), 3.21 (ddt, J = 20.7, 11.3, 4.9 Hz, 2H, CH₂), 3.15–3.06 (m, 2H, CH₂), 3.02 (ddd, J = 11.3, 6.7, 3.9 Hz, 1H, CH), 2.85 (dd, J = 13.9, 10.0 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.89 (C═O), 166.39 (d, ¹J_CF = 251.8 Hz), 164.04 (C═O), 162.56 (dd, ¹J_CF = 245.7, ³J_CF = 13.4 Hz), 161.21 (C═O), 160.27, 158.68, 154.17, 149.39 (d, ³J_CF = 13.2 Hz). 142.49 (t, ³J_CF = 9.2 Hz), 130.52, 130.48, 130.34, 130.29, 130.23, 128.75, 118.53, 118.41, 118.39, 116.19 (d, ²J_CF = 23.5 Hz), 115.36, 113.32, 112.38 (dd, ²J_CF = 25.0, ⁴J_CF = 6.4 Hz), 102.63 (t, ²J_CF = 26.0 Hz), 56.01, 53.25, 49.28, 48.46, 47.06, 43.32, 38.71. HRMS calcd for m/z C₃₅H₃₁F₃N₆O₆S [M+H]⁺ 721.2051, found 721.2056 [M+H]⁺. HPLC purity: 95.44%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(7-chloro-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-(3,5-difluorophenyl)ethyl)acetamide (12c2).

White solid, yield: 40%. Mp: 196–197 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (d, J = 7.8 Hz, 1H, NH), 8.14 (d, J = 7.6 Hz, 1H, Ph-H), 7.96–7.85 (m, 1H, Ph-H), 7.71 (d, J = 8.1 Hz, 1H, Ph-H), 7.58 (t, J = 7.5 Hz, 1H, Ph-H), 7.47–7.37 (m, 3H, Ph-H), 7.36–7.30 (m, 1H, Ph-H), 7.15 (d, J = 8.9 Hz, 2H, Ph-H), 7.01 (t, J = 9.4 Hz, 1H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.50 (d, J = 6.5 Hz, 2H, Ph-H), 6.17 (s, 2H, NH₂), 4.70–4.58 (m, 1H, CH), 3.88 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.47–3.36 (m, 2H, CH₂), 3.26–3.14 (m, 2H, CH₂), 3.14–3.05 (m, 2H, CH₂), 3.05–2.96 (m, 1H, CH), 2.85 (dd, J = 13.8, 10.0 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.90 (C═O), 163.94 (C═O), 162.43 (d, ¹J_CF = 246.0, ³J_CF = 13.4 Hz), 161.34 (C═O), 160.29, 158.72, 156.44, 148.35, 142.47 (t, ³J_CF = 9.3 Hz), 139.88, 130.50, 130.43, 129.70, 129.19, 128.69, 127.92, 126.42, 122.34, 120.20, 115.37, 115.24, 112.39 (d, ²J_CF = 24.3, ⁴J_CF = 6.6 Hz), 111.84, 102.63 (t, ²J_CF = 26.7 Hz), 56.02, 53.26, 49.20, 48.49, 47.11, 43.30, 37.77. HRMS calcd for m/z C₃₅H₃₁ClF₂N₆O₆S [M+H]⁺ 737.1755, found 737.1751 [M+H]⁺. HPLC purity: 97.74%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(2-(3,5-difluorophenyl)-1-(3-(4-methoxyphenyl)-7-(methylsulfonyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)acetamide (12e2).

White solid, yield: 29%. Mp: 193–194 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.71 (d, J = 7.7 Hz, 1H, NH), 8.38 (d, J = 8.3 Hz, 1H, Ph-H), 8.16 (d, J = 1.5 Hz, 1H, Ph-H), 8.06 (dd, J = 8.3, 1.6 Hz, 1H, Ph-H), 7.46 (dd, J = 8.9, 2.4 Hz, 1H, Ph-H), 7.44–7.33 (m, 3H, Ph-H), 7.24–7.14 (m, 2H, Ph-H), 7.03 (td, J = 9.4, 2.1 Hz, 1H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.51 (d, J = 6.5 Hz, 2H, Ph-H), 6.17 (s, 2H, NH₂), 4.72–4.61 (m, 1H, CH), 3.91 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃), 3.43 (d, J = 16.2 Hz, 2H, CH₂), 3.38 (s, 3H, SO₂CH₃), 3.20 (dq, J = 14.7, 5.5, 4.1 Hz, 2H, CH₂), 3.15–3.06 (m, 2H, CH₂), 3.03 (dd, J = 11.7, 4.5 Hz, 1H, CH), 2.85 (dd, J = 14.0, 10.1 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.93 (C═O), 164.06 (C═O), 162.58 (dd, ¹J_CF = 246.3 Hz, ³J_CF = 13.6 Hz), 161.24 (C═O), 160.38, 159.12, 154.17, 147.30, 146.65, 142.41 (t, J = 9.6 Hz), 140.85, 130.45, 130.30, 129.30, 129.01, 128.57, 126.12, 124.75, 118.52, 115.44, 115.31, 113.32, 112.38 (dd, ²J_CF = 24.8 Hz, ⁴J_CF = 6.2 Hz), 102.67 (t, ²J_CF = 25.4 Hz), 56.03, 53.20, 49.29, 48.45, 47.08, 43.65, 43.31, 37.83. HRMS calcd for m/z C₃₆H₃₄F₂N₆O₈S₂ [M+H]⁺ 781.1920, found 781.1923 [M+H]⁺. HPLC purity: 97.16%.

5.1.11. General Procedure for the Synthesis of 13a and 13b.

Under an ice bath, morpholine or thiomorpholine-1,1-dioxide (1 equiv), TEA (2 equiv), and 4-nitrobenzenesulfonyl chloride (1, 1.2 equiv) were successively dissolved in DCM (20 mL). The resulting mixture was then stirred at room temperature (monitored by TLC). Then the reaction mixture was extracted with DCM (20 mL), and the combined organic phase was washed with a saturated NaCl solution (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the corresponding crude products, which were purified by recrystallization (DCM:petroleum ether = 1:15) to afford intermediates 13a and 13b.

4-((4-Nitrophenyl)sulfonyl)morpholine (13a).

White solid, yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.50–8.44 (m, 2H, Ph-H), 8.05–7.99 (m, 2H, Ph-H), 3.69–3.61 (m, 4H, CH₂ × 2), 3.00–2.92 (m, 4H, CH₂ × 2). ESI-MS: 273.06 [M+H]⁺; C₁₀H₁₂N₂O₅S [272.05].

4-((4-Nitrophenyl)sulfonyl)thiomorpholine 1,1-Dioxide (13b).

White solid, yield: 79%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.49–8.43 (m, 2H, Ph-H), 8.15–8.07 (m, 2H, Ph-H), 3.59–3.51 (m, 4H, CH₂ × 2), 3.32–3.25 (m, 4H, CH₂ × 2). ESI-MS: 319.32 [M−H]⁻; C₁₀H₁₂N₂O₆S₂ [320.01].

5.1.12. General Procedure for the Synthesis of 14a and 14b.

Intermediates 13a, 13b and 10% Pd·C (10% w/w) were dissolved in DCM (20 mL), and the solution was degassed and stirred at room temperature overnight in the atmosphere of hydrogen. The mixture was filtered and concentrated, and the resulting residue was purified by recrystallization (ethyl acetate) to provide intermediates 14a and 14b.

4-(Morpholinosulfonyl)aniline (14a).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J = 8.6 Hz, 2H, Ph-H), 6.66 (d, J = 8.6 Hz, 2H, Ph-H), 6.12 (s, 2H, NH₂), 3.67–3.57 (m, 4H, CH₂ × 2), 2.82–2.72 (m, 4H, CH₂ × 2). ESI-MS: 243.1 [M+H]⁺, 265.1 [M+Na]⁺; C₁₀H₁₄N₂O₃S [242.07].

4-((4-Aminophenyl)sulfonyl)thiomorpholine 1,1-Dioxide (14b).

White solid, yield: 70%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.42 (d, J = 8.7 Hz, 2H, Ph-H), 6.68 (d, J = 8.7 Hz, 2H, Ph-H), 6.19 (s, 2H, NH₂), 3.39 (d, J = 6.8 Hz, 4H, CH₂ × 2), 3.26–3.18 (m, 4H, CH₂ × 2). ESI-MS: 313.82 [M+Na]⁺; 289.35 [M−H]⁻; C₁₀H₁₄N₂O₄S₂ [290.04].

5.1.13. General Procedure for the Synthesis of 15a and 15b.

Under an ice bath, the key intermediates 14a, 14b (1 equiv), TEA (2 equiv), and 2-nitrobenzoyl chloride (1.2 equiv) were successively dissolved in DCM (20 mL). The resulting mixture was then stirred at room temperature (monitored by TLC). Then the reaction mixture was extracted with DCM (20 mL), and the combined organic phase was washed with a saturated NaCl solution (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give the corresponding crude products, which were purified by recrystallization (ethyl acetate:petroleum ether = 1:10) to afford intermediates 15a and 15b.

N-(4-(Morpholinosulfonyl)phenyl)-2-nitrobenzamide (15a).

White solid, yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.13 (s, 1H, NH), 8.20 (d, J = 8.1 Hz, 1H, Ph-H), 7.93 (dd, J = 14.3, 8.1 Hz, 3H, Ph-H), 7.85–7.79 (m, 2H, Ph-H), 7.79–7.73 (m, 2H, Ph-H), 3.70–3.61 (m, 4H, CH₂ × 2), 2.93–2.83 (m, 4H, CH₂ × 2). ESI-MS: 390.61 [M−H]⁻; C₁₇H₁₇N₃O₆S [391.08].

N-(4-((1,1-Dioxidothiomorpholino)sulfonyl)phenyl)-2-nitrobenzamide (15b).

White solid, yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.17 (s, 1H, NH), 8.20 (d, J = 8.0 Hz, 1H, Ph-H), 7.95 (d, J = 8.8 Hz, 2H, Ph-H), 7.91 (d, J = 7.4 Hz, 1H, Ph-H), 7.83 (dd, J = 10.8, 8.7 Hz, 4H, Ph-H), 3.45 (s, 4H, CH₂ × 2), 3.32–3.21 (m, 4H, CH₂ × 2). ESI-MS: 462.62 [M+Na]⁺; C₁₇H₁₇N₃O₇S₂ [439.05].

5.1.14. General Procedure for the Synthesis of 16a and 16b.

Intermediates 15a, 15b and 10% Pd×C (10% w/w) were dissolved in DCM (20 mL), and the solution was degassed and stirred at room temperature overnight in the atmosphere of hydrogen. The mixture was filtered and concentrated, and the resulting residue was purified by recrystallization (ethyl acetate:petroleum ether = 1:10) to provide the intermediates 16a and 16b.

2-Amino-N-(4-(morpholinosulfonyl)phenyl)benzamide (16a).

White solid, yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.42 (s, 1H, NH), 8.02 (d, J = 8.8 Hz, 2H, Ph-H), 7.71 (d, J = 8.8 Hz, 2H, Ph-H), 7.68–7.63 (m, 1H, Ph-H), 7.28–7.20 (m, 1H, Ph-H), 6.78 (d, J = 7.8 Hz, 1H, Ph-H), 6.65–6.58 (m, 1H, Ph-H), 6.39 (s, 2H, NH₂), 3.69–3.60 (m, 4H, CH₂ × 2), 2.91–2.82 (m, 4H, CH₂ × 2). ESI-MS: 362.52 [M+H]⁺, 384.89 [M+Na]⁺; 360.83 [M−H]⁻; C₁₇H₁₉N₃O₄S [361.11].

2-Amino-N-(4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)benzamide (16b).

White solid, yield: 77%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.42 (s, 1H, NH), 8.02 (d, J = 8.9 Hz, 2H, Ph-H), 7.79 (d, J = 8.8 Hz, 2H, Ph-H), 7.71–7.62 (m, 1H, Ph-H), 7.28–7.19 (m, 1H, Ph-H), 6.78 (d, J = 7.7 Hz, 1H, Ph-H), 6.66–6.57 (m, 1H, Ph-H), 6.38 (s, 2H, NH₂), 3.44 (s, 4H, CH₂ × 2), 3.28 (q, J = 6.1, 4.7 Hz, 4H, CH₂ × 2). ESI-MS: 410.01 [M+H]⁺, 432.61 [M+Na]⁺, 408.54 [M−H]⁻; C₁₇H₁₉N₃O₅S₂ [409.08].

5.1.15. General Procedure for the Synthesis of 17a1–17b1 and 17a2–17b2.

(tert-Butoxycarbonyl)-l-phenylalanine or (tert-butoxycarbonyl)-3,5-difluoro-l-phenylalanine (1.2 equiv) and HATU (1.5 equiv) were mixed in 30 mL of DCM and stirred in an ice bath for 30 min. Subsequently, DIEA (2 equiv) and intermediates 16a, 16b (1 equiv) were added to the mixture and then stirred at room temperature for another 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed with 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:8) to afford the intermediates 17a1–17b1 and 17a2–17b2.

tert-Butyl (S)-(1-((2-((4-(Morpholinosulfonyl)phenyl)carbamoyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (17a1).

White solid, yield: 72%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (d, J = 18.7 Hz, 2H, NH), 8.35 (d, J = 8.3 Hz, 1H, NH), 8.00 (d, J = 8.8 Hz, 2H, Ph-H), 7.83 (d, J = 7.7 Hz, 1H, Ph-H), 7.72 (d, J = 8.7 Hz, 2H, Ph-H), 7.60 (t, J = 7.7 Hz, 1H, Ph-H), 7.47 (d, J = 7.7 Hz, 1H, Ph-H), 7.27 (q, J = 6.7, 5.9 Hz, 5H, Ph-H), 7.21–7.11 (m, 1H, Ph-H), 4.19 (ddd, J = 11.2, 8.1, 4.1 Hz, 1H, CH), 3.68–3.59 (m, 4H, CH₂ × 2), 3.17 (dd, J = 13.9, 3.7 Hz, 1H, CH), 2.84 (s, 4H, CH₂ × 2), 2.82–2.77 (m, 1H, CH), 1.22 (s, 9H, (CH₃)₃). ESI-MS: 609.36 [M+H]⁺, 631.62 [M+Na]⁺; C₃₁H₃₆N₄O₇S [608.23].

tert-Butyl (S)-(1-((2-((4-((1,1-Dioxidothiomorpholino)sulfonyl)phenyl)carbamoyl)phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (17b1).

White solid, yield: 78%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 2H, NH), 8.36 (d, J = 8.3 Hz, 1H, NH), 8.02 (d, J = 8.9 Hz, 2H, Ph-H), 7.82 (t, J = 9.2 Hz, 3H, Ph-H), 7.60 (t, J = 7.5 Hz, 1H, Ph-H), 7.49 (d, J = 7.8 Hz, 1H, Ph-H), 7.31–7.22 (m, 5H, Ph-H), 7.21–7.13 (m, 1H, Ph-H), 4.26–4.14 (m, 1H, CH), 3.42 (s, 4H, CH₂ × 2), 3.32–3.26 (m, 4H, CH₂ × 2), 3.18 (dd, J = 13.8, 3.9 Hz, 1H, CH), 2.83 (dd, J = 13.7, 10.8 Hz, 1H, CH), 1.22 (s, 9H, (CH₃)₃). ESI-MS: 679.46 [M+Na]⁺, 655.56 [M−H]⁻; C₃₁H₃₆N₄O₈S₂ [656.20].

tert-Butyl (S)-(3-(3,5-Difluorophenyl)-1-((2-((4-(morpholinosulfonyl)phenyl)carbamoyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (17a2).

White solid, yield: 76%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (d, J = 24.6 Hz, 2H, NH), 8.33 (d, J = 8.3 Hz, 1H, NH), 8.00 (d, J = 8.8 Hz, 2H, Ph-H), 7.84 (d, J = 7.4 Hz, 1H, Ph-H), 7.72 (d, J = 8.8 Hz, 2H, Ph-H), 7.64–7.57 (m, 1H, Ph-H), 7.52 (d, J = 8.2 Hz, 1H, Ph-H), 7.29 (t, J = 7.6 Hz, 1H, Ph-H), 7.03 (dd, J = 14.8, 5.5 Hz, 3H, Ph-H), 4.32–4.19 (m, 1H, CH), 3.73–3.56 (m, 4H, CH₂ × 2), 3.23 (dd, J = 13.7, 3.7 Hz, 1H, CH), 2.88–2.80 (m, 4H, CH₂ × 2), 2.82–2.75 (m, 1H), 1.23 (s, 9H, (CH₃)₃). ESI-MS: 667.41 [M+Na]⁺; C₃₁H₃₄F₂N₄O₇S [644.21].

tert-Butyl (S)-(3-(3,5-Difluorophenyl)-1-((2-((4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)carbamoyl)phenyl)-amino)-1-oxopropan-2-yl)carbamate (17b2).

White solid, yield: 74%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (d, J = 9.8 Hz, 2H, NH), 8.34 (d, J = 8.3 Hz, 1H, NH), 8.02 (d, J = 8.7 Hz, 2H, Ph-H), 7.82 (dd, J = 14.4, 8.3 Hz, 3H, Ph-H), 7.60 (t, J = 7.6 Hz, 1H, Ph-H), 7.52 (d, J = 8.1 Hz, 1H, Ph-H), 7.28 (t, J = 7.6 Hz, 1H, Ph-H), 7.01 (d, J = 8.6 Hz, 3H, Ph-H), 4.27 (td, J = 9.8, 8.4, 3.8 Hz, 1H, CH), 3.41 (s, 4H, CH₂ × 2), 3.31–3.26 (m, 4H, CH₂ × 2), 3.26–3.19 (m, 1H, CH), 2.89–2.78 (m, 1H, CH), 1.24 (s, 9H, (CH₃)₃). ESI-MS: 715.15 [M+Na]⁺, 691.50 [M−H]⁻; C₃₁H₃₄F₂N₄O₈S₂ [692.18].

5.1.16. General Procedure for the Synthesis of 18a1–18b1 and 18a2–18b2.

The key intermediates 17a1–17b1 and 17a2–17b2 (1 equiv), DIEA (2 equiv), and DMAP (1 equiv) were dissolved in the solution of acetonitrile (30 mL), and then BSA (10 equiv) was added dropwise to the mixture solution. The reaction system was stirred at 80 °C for 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed with 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:3) to afford intermediates 18a1–18b1 and 18a2–18b2.

tert-Butyl (S)-(1-(3-(4-(Morpholinosulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)carbamate (18a1).

White solid, yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20–8.14 (m, 1H, NH), 8.07–7.99 (m, 2H, Ph-H), 7.94 (dd, J = 11.0, 4.2 Hz, 1H, Ph-H), 7.83 (d, J = 8.4 Hz, 1H, Ph-H), 7.80–7.74 (m, 2H, Ph-H), 7.65–7.55 (m, 2H, Ph-H), 7.17 (dq, J = 14.1, 6.9 Hz, 3H, Ph-H), 6.82 (d, J = 6.8 Hz, 2H, Ph-H), 4.35–4.24 (m, 1H, CH), 3.81–3.68 (m, 4H, CH₂ × 2), 3.08–3.03 (m, 1H, CH), 3.03–2.95 (m, 4H, CH₂ × 2), 2.84 (dd, J = 13.8, 10.3 Hz, 1H, CH), 1.28 (s, 9H, (CH₃)₃). ESI-MS: 591.52 [M+H]⁺, 613.49 [M+Na]⁺; 589.74 [M−H]⁻; C₃₁H₃₄N₄O₆S [590.22].

tert-Butyl (S)-(1-(3-(4-((1,1-Dioxidothiomorpholino)sulfonyl)-phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-carbamate (18b1).

White solid, yield: 77%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (d, J = 7.9 Hz, 1H, NH), 8.13–8.06 (m, 2H, Ph-H), 7.96–7.90 (m, 1H, Ph-H), 7.76 (ddd, J = 11.1, 8.5, 5.2 Hz, 3H, Ph-H), 7.64–7.56 (m, 2H, Ph-H), 7.17 (dt, J = 23.5, 7.1 Hz, 3H, Ph-H), 6.82 (d, J = 7.1 Hz, 2H, Ph-H), 4.28 (td, J = 9.8, 4.0 Hz, 1H, CH), 3.61 (s, 4H, CH₂ × 2), 3.48 (s, 4H, CH₂ × 2), 3.07 (dd, J = 13.9, 3.6 Hz, 1H, CH), 2.84 (dd, J = 13.8, 10.0 Hz, 1H, CH), 1.27 (s, 9H, (CH₃)₃). ESI-MS: 639.35 [M+H]⁺, 661.39 [M+Na]⁺, 637.55 [M−H]⁻; C₃₁H₃₄N₄O₇S₂ [638.19].

tert-Butyl (S)-(2-(3,5-Difluorophenyl)-1-(3-(4-(morpholinosulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-ethyl)carbamate (18a2).

White solid, yield: 75%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20–8.13 (m, 1H, NH), 7.99 (ddd, J = 15.6, 8.4, 1.9 Hz, 2H, Ph-H), 7.95–7.88 (m, 2H, Ph-H), 7.78 (d, J = 8.0 Hz, 1H, Ph-H), 7.70 (dd, J = 8.2, 1.8 Hz, 1H, Ph-H), 7.61 (t, J = 7.5 Hz, 1H, Ph-H), 7.51 (d, J = 8.4 Hz, 1H, Ph-H), 7.10–6.97 (m, 1H, Ph-H), 6.64 (d, J = 6.3 Hz, 2H, Ph-H), 4.29 (td, J = 10.3, 3.5 Hz, 1H, CH), 3.66 (t, J = 4.4 Hz, 4H, CH₂ × 2), 3.15 (dd, J = 13.7, 3.3 Hz, 1H, CH), 2.99–2.94 (m, 4H, CH₂ × 2), 2.95–2.89 (m, 1H, CH), 1.23 (s, 9H, (CH₃)₃). ESI-MS: 627.44 [M+H]⁺, 649.31 [M+Na]⁺; 625.41 [M−H]⁻; C₃₁H₃₂F₂N₄O₆S [626.20].

tert-Butyl (S)-(2-(3,5-Difluorophenyl)-1-(3-(4-((1,1-dioxidothio-morpholino) sulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-ethyl)carbamate (18b2)

White solid, yield: 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.15 (d, J = 7.2 Hz, 1H, NH), 8.11–8.02 (m, 2H, Ph-H), 7.96–7.85 (m, 2H, Ph-H), 7.79 (t, J = 7.5 Hz, 1H, Ph-H), 7.68 (dd, J = 6.2, 2.2 Hz, 1H, Ph-H), 7.61 (t, J = 7.5 Hz, 1H, Ph-H), 7.46 (d, J = 8.4 Hz, 1H, Ph-H), 7.00 (t, J = 9.3 Hz, 1H, Ph-H), 6.62 (d, J = 6.4 Hz, 2H, Ph-H), 4.31 (td, J = 9.8, 3.7 Hz, 1H, CH), 3.58 (s, 4H, CH₂ × 2), 3.27 (s, 4H, CH₂ × 2), 3.15 (dd, J = 13.8, 3.3 Hz, 1H, CH), 2.95 (dd, J = 13.7, 10.3 Hz, 1H, CH), 1.22 (s, 9H, (CH₃)₃). ESI-MS: 675.38 [M+H]⁺, 697.34 [M+Na]⁺, 673.77 [M−H]⁻; C₃₁H₃₂F₂N₄O₇S₂ [674.17].

5.1.17. General Procedure for the Synthesis of 19a1–19b1 and 19a2–19b2.

TFA was added dropwise to intermediates 18a1–18b1 and 18a2–18b2 in 30 mL of DCM and stirred at room temperature for 3 h. Then, the resulting mixture solution was alkalized to pH ~7 with a saturated sodium bicarbonate solution and then extracted with DCM (20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the intermediates 19a1–19b1 and 19a2–19b2.

(S)-2-(1-Amino-2-phenylethyl)-3-(4-(morpholinosulfonyl)-phenyl)quinazolin-4(3H)-one (19a1).

White solid, yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.20–8.12 (m, 1H, Ph-H), 7.97 (td, J = 7.1, 6.0, 1.8 Hz, 1H, Ph-H), 7.94–7.89 (m, 2H, Ph-H), 7.89–7.78 (m, 2H, Ph-H), 7.64–7.56 (m, 1H, Ph-H), 7.43 (dd, J = 8.3, 2.0 Hz, 1H, Ph-H), 7.17 (ddt, J = 14.1, 10.2, 4.7 Hz, 3H, Ph-H), 6.88–6.77 (m, 2H, Ph-H), 3.84–3.66 (m, 4H, CH₂ × 2), 3.47 (dd, J = 8.5, 4.8 Hz, 1H, CH), 3.05 (dd, J = 13.4, 4.7 Hz, 1H, CH), 3.02–2.88 (m, 4H, CH₂ × 2), 2.60 (dd, J = 13.4, 8.6 Hz, 1H, CH), 2.07 (s, 2H, NH₂). ESI-MS: 491.53 [M+H]⁺; C₂₆H₂₆N₄O₄S [490.17].

(S)-2-(1-Amino-2-phenylethyl)-3-(4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)quinazolin-4(3H)-one (19b1).

White solid, yield: 63%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.17–8.11 (m, 1H, Ph-H), 8.05 (dd, J = 8.3, 2.1 Hz, 1H, Ph-H), 7.98–7.89 (m, 2H, Ph-H), 7.88–7.78 (m, 2H, Ph-H), 7.62–7.56 (m, 1H, Ph-H), 7.26–7.15 (m, 4H, Ph-H), 6.82 (d, J = 6.7 Hz, 2H, Ph-H), 3.57 (s, 4H, CH₂ × 2), 3.50–3.45 (m, 1H, CH), 3.35 (d, J = 4.9 Hz, 4H, CH₂ × 2), 3.07 (dd, J = 13.3, 5.6 Hz, 1H, CH), 2.66 (dd, J = 13.3, 7.9 Hz, 1H, CH), 2.33 (s, 2H, NH₂). ESI-MS: 539.16 [M+H]⁺; C₂₆H₂₆N₄O₅S₂ [538.13].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-3-(4-(morpholinosulfonyl)phenyl)quinazolin-4(3H)-one (19a2).

White solid, yield: 62%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.19–8.13 (m, 1H, Ph-H), 7.96 (ddd, J = 7.1, 4.9, 1.7 Hz, 2H, Ph-H), 7.94–7.89 (m, 1H, Ph-H), 7.84 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.79 (d, J = 8.1 Hz, 1H, Ph-H), 7.66 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.63–7.56 (m, 1H, Ph-H), 7.00 (tt, J = 9.4, 2.2 Hz, 1H, Ph-H), 6.64 (d, J = 6.5 Hz, 2H, Ph-H), 3.75–3.61 (m, 4H, CH₂ × 2), 3.45 (dd, J = 8.6, 4.4 Hz, 1H, CH), 3.07 (dd, J = 13.6, 4.3 Hz, 1H, CH), 3.01–2.90 (m, 4H, CH₂ × 2), 2.73 (dd, J = 13.7, 8.8 Hz, 1H, CH), 2.08 (s, 2H, NH₂). ESI-MS: 527.89 [M+H]⁺; C₂₆H₂₄F₂N₄O₄S [526.15].

(S)-2-(1-Amino-2-(3,5-difluorophenyl)ethyl)-3-(4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)quinazolin-4(3H)-one (19b2).

White solid, yield: 59%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18–8.12 (m, 1H, Ph-H), 8.08–8.01 (m, 2H, Ph-H), 7.95–7.89 (m, 1H, Ph-H), 7.88–7.82 (m, 1H, Ph-H), 7.78 (d, J = 8.0 Hz, 1H, Ph-H), 7.64–7.56 (m, 2H, Ph-H), 6.99 (tt, J = 9.3, 2.0 Hz, 1H, Ph-H), 6.68–6.57 (m, 2H, Ph-H), 3.57 (s, 4H, CH₂ × 2), 3.45 (dd, J = 8.3, 4.8 Hz, 1H, CH), 3.25 (d, J = 10.6 Hz, 4H, CH₂ × 2), 3.07 (dd, J = 13.5, 4.7 Hz, 1H, CH), 2.73 (dd, J = 13.4, 8.4 Hz, 1H, CH), 2.18 (s, 2H, NH₂). ESI-MS: 575.14 [M+H]⁺; C₂₆H₂₄F₂N₄O₅S₂ [574.12].

5.1.18. General Procedure for the Synthesis of 20a1–20b1 and 20a2–20b2.

Intermediate 4 (1.2 equiv) and HATU (1.5 equiv) were mixed in 30 mL of DCM and stirred in an ice bath for 30 min. Subsequently, DIEA (2 equiv) and intermediates 19a1–19b1 and 19a2–19b2 (1 equiv) were added to the mixture and then stirred at room temperature for another 5 h (monitored by TLC). The resulting mixture was evaporated under reduced pressure, and the residue was initially washed with 1 N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic layer was washed with saturated sodium bicarbonate (3 × 20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford the corresponding crude product, which was purified by flash column chromatography (ethyl acetate:petroleum ether = 1:1) to afford the target compounds 20a1–20b1 and 20a2–20b2.

(S)-N-(1-(3-(4-(Morpholinosulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)-sulfonyl)-2-oxopiperazin-1-yl)acetamide (20a1).

White solid, yield: 56%. Mp: 211–212 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (d, J = 7.8 Hz, 1H, NH), 8.43 (d, J = 8.9 Hz, 2H, Ph-H), 8.19–8.12 (m, 1H, Ph-H), 8.07 (d, J = 8.9 Hz, 2H, Ph-H), 8.02–7.90 (m, 3H, Ph-H), 7.78 (d, J = 8.0 Hz, 1H, Ph-H), 7.75–7.66 (m, 2H, Ph-H), 7.66–7.58 (m, 1H, Ph-H), 7.25–7.11 (m, 3H, Ph-H), 6.87 (d, J = 6.7 Hz, 2H, Ph-H), 4.54 (td, J = 8.8, 4.9 Hz, 1H, CH), 3.97–3.80 (m, 2H, CH₂), 3.69 (t, J = 4.3 Hz, 4H, CH₂ × 2), 3.63 (d, J = 4.3 Hz, 2H, CH₂), 3.32–3.24 (m, 2H, CH₂), 3.21 (dd, J = 11.7, 8.6 Hz, 2H, CH₂), 3.18–3.11 (m, 1H, CH), 2.97 (t, J = 7.3 Hz, 4H, CH₂ × 2), 2.84 (dd, J = 13.9, 9.3 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.74 (C═O), 163.47 (C═O), 161.58 (C═O), 156.07, 150.78, 147.23, 141.00, 140.83, 137.87, 135.78, 135.62, 130.86, 130.84, 129.72, 129.66, 129.35, 129.21, 128.73, 127.90, 127.55, 127.07, 126.99, 125.30, 121.11, 65.83, 53.31, 48.70, 48.48, 47.02, 46.45, 43.07, 38.43. HRMS calcd for m/z C₃₈H₃₇N₇O₁₀S₂ [M+H]⁺ 816.2116, found 816.2118 [M+H]⁺. HPLC purity: 99.41%.

(S)-N-(1-(3-(4-((1,1-Dioxidothiomorpholino)sulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (20b1).

White solid, yield: 67%. Mp: 219–220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.77 (d, J = 7.7 Hz, 1H, NH), 8.44 (d, J = 8.8 Hz, 2H, Ph-H), 8.18–8.13 (m, 1H, Ph-H), 8.08 (d, J = 8.8 Hz, 2H, Ph-H), 8.05–7.98 (m, 2H, Ph-H), 7.98–7.91 (m, 1H, Ph-H), 7.79 (d, J = 8.1 Hz, 1H, Ph-H), 7.73–7.68 (m, 1H, Ph-H), 7.65–7.58 (m, 2H, Ph-H), 7.19 (dt, J = 15.0, 7.0 Hz, 3H, Ph-H), 6.88 (d, J = 7.0 Hz, 2H, Ph-H), 4.54 (td, J = 8.4, 5.4 Hz, 1H, CH), 3.98–3.78 (m, 2H, CH₂), 3.67 (d, J = 16.6 Hz, 2H, CH₂), 3.58 (s, 4H, CH₂ × 2), 3.42–3.37 (m, 2H, CH₂), 3.34–3.30 (m, 4H, CH₂ × 2), 3.25 (td, J = 9.6, 5.6 Hz, 2H, CH₂), 3.18 (dd, J = 12.0, 5.6 Hz, 1H, CH), 2.85 (dd, J = 13.9, 9.1 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.64 (C═O), 163.49 (C═O), 161.57 (C═O), 156.04, 150.78, 147.21, 141.10, 140.75, 137.76, 137.70, 135.65, 131.14, 131.03, 129.71, 129.25, 129.16, 128.88, 128.80, 127.93, 127.56, 127.06, 126.98, 125.33, 121.07, 53.22, 50.93, 48.70, 48.42, 46.98, 45.49, 43.06, 38.54. HRMS calcd for m/z C₃₈H₃₇N₇O₁₁S₃ [M+H]⁺ 864.1786, found 864.1782 [M+H]⁺. HPLC purity: 96.84%.

(S)-N-(2-(3,5-Difluorophenyl)-1-(3-(4-(morpholinosulfonyl)-phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (20a2).

White solid, yield: 48%. Mp: 209–210 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (d, J = 8.1 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.19–8.13 (m, 1H, Ph-H), 8.07 (d, J = 8.8 Hz, 2H, Ph-H), 7.95 (dt, J = 8.2, 4.2 Hz, 2H, Ph-H), 7.88 (dd, J = 8.3, 2.0 Hz, 1H, Ph-H), 7.83–7.75 (m, 2H, Ph-H), 7.70–7.58 (m, 2H, Ph-H), 7.08–6.96 (m, 1H, Ph-H), 6.68 (d, J = 6.4 Hz, 2H, Ph-H), 4.56 (td, J = 8.5, 5.1 Hz, 1H, CH), 3.95–3.74 (m, 2H, CH₂), 3.74–3.57 (m, 6H, CH₂ × 3), 3.31–3.28 (m, 1H, CH), 3.28–3.24 (m, 2H, CH₂), 3.21 (dd, J = 11.5, 5.3 Hz, 2H, CH₂), 2.96 (dt, J = 12.0, 4.6 Hz, 4H, CH₂ × 2), 2.92–2.87 (m, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.44 (C═O), 163.47 (C═O), 162.50 (dd, ¹J_CF = 245.9, ³J_CF = 13.2 Hz), 161.62 (C═O), 155.14, 150.79, 147.05, 142.46 (t, ³J_CF = 9.3 Hz), 140.86, 140.71, 135.99, 135.65, 131.07, 130.49, 129.73, 129.52, 129.38, 128.06, 127.61, 126.97, 125.31, 121.20, 112.56 (dd, ²J_CF = 24.6, ⁴J_CF = 6.6 Hz), 102.59 (t, ²J_CF = 25.4 Hz), 65.82, 52.43, 48.68, 48.54, 47.13, 46.31, 43.07, 38.02. HRMS calcd for m/z C₃₈H₃₅F₂N₇O₁₀S₂ [M+H]⁺ 852.1928, found 852.1925 [M+H]⁺. HPLC purity: 98.09%.

(S)-N-(2-(3,5-Difluorophenyl)-1-(3-(4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)-4-oxo-3,4-dihydroquin-azolin-2-yl)ethyl)-2-(4-((4-nitrophenyl)sulfonyl)-2-oxopiperazin-1-yl)acetamide (20b2).

White solid, yield: 58%. Mp: 215–216 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 8.1 Hz, 1H, NH), 8.43 (d, J = 8.8 Hz, 2H, Ph-H), 8.15 (d, J = 7.9 Hz, 1H, Ph-H), 8.12–8.01 (m, 3H, Ph-H), 8.00–7.89 (m, 2H, Ph-H), 7.84–7.74 (m, 2H, Ph-H), 7.68–7.59 (m, 2H, Ph-H), 7.01 (dt, J = 11.5, 5.7 Hz, 1H, Ph-H), 6.67 (d, J = 6.4 Hz, 2H, Ph-H), 4.56 (td, J = 8.5, 5.2 Hz, 1H, CH), 3.94–3.71 (m, 2H, CH₂), 3.70–3.59 (m, 2H, CH₂), 3.56 (s, 4H, CH₂ × 2), 3.40–3.32 (m, 2H, CH₂), 3.31–3.24 (m, 6H, CH₂ × 3), 3.22–3.17 (m, 1H, CH), 2.98–2.88 (m, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.35 (C═O), 163.51 (C═O), 162.48 (dd, ¹J_CF = 246.0, ³J_CF = 13.3 Hz), 161.62 (C═O), 155.01, 150.80, 147.00, 142.42 (t, ²J_CF = 9.3 Hz), 140.98, 140.66, 137.78, 135.68, 131.37, 130.73, 129.71, 129.00, 128.94, 128.09, 127.63, 126.96, 125.34, 121.17, 112.62 (dd, ²J_CF = 24.6, ⁴J_CF = 6.4 Hz), 102.60 (t, ²J_CF = 25.5 Hz), 52.40, 50.93, 48.67, 48.44, 47.04, 45.34, 43.05, 37.97. HRMS calcd for m/z C₃₈H₃₅F₂N₇O₁₁S₃ [M+H]⁺ 900.1597, found 900.1600 [M+H]⁺. HPLC purity: 97.88%.

5.1.19. General Procedure for the Synthesis of 21a1–21b1 and 21a2–21b2.

Compounds 20a1–20b1 and 20a2–20b2 (150 mg) and 10% Pd·C (10% w/w, 15 mg) were dissolved in DCM (10 mL), and the solution was degassed and stirred at room temperature overnight in the atmosphere of hydrogen. The mixture was filtered and concentrated, and the resulting residue was purified by recrystallization (ethyl acetate) or prep-TLC (methanol:DCM = 1:30) to provide the target compounds 21a1–21b1 and 21a2–21b2.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(3-(4-(morpholinosulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (21a1).

White solid, yield: 35%. Mp: 202–203 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.78 (d, J = 7.7 Hz, 1H, NH), 8.23–8.12 (m, 1H, Ph-H), 8.02–7.90 (m, 3H, Ph-H), 7.78 (d, J = 8.1 Hz, 1H, Ph-H), 7.76–7.71 (m, 1H, Ph-H), 7.71–7.66 (m, 1H, Ph-H), 7.62 (t, J = 7.5 Hz, 1H, Ph-H), 7.41 (d, J = 8.7 Hz, 2H, Ph-H), 7.17 (dq, J = 14.2, 6.9 Hz, 3H, Ph-H), 6.87 (d, J = 6.8 Hz, 2H, Ph-H), 6.67 (d, J = 8.7 Hz, 2H, Ph-H), 6.19 (s, 2H, NH₂), 4.55 (td, J = 8.7, 5.0 Hz, 1H, CH), 3.97–3.81 (m, 2H, CH₂), 3.69 (t, J = 4.3 Hz, 4H, CH₂ × 2), 3.47–3.36 (m, 2H, CH₂), 3.21 (tt, J = 9.2, 4.2 Hz, 2H, CH₂), 3.16–3.04 (m, 2H, CH₂), 2.97 (t, J = 8.4 Hz, 5H, CH₂ × 2, CH), 2.85 (dd, J = 13.9, 9.4 Hz, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.83 (C═O), 164.04 (C═O), 161.60 (C═O), 156.15, 154.19, 147.25, 141.00, 137.88, 135.76, 135.63, 130.91, 130.85, 130.31, 129.73, 129.33, 129.20, 128.73, 127.89, 127.54, 127.08, 126.99, 121.11, 118.46, 113.33, 65.83, 53.32, 49.31, 48.49, 47.05, 46.47, 43.32, 38.37. HRMS calcd for m/z C₃₈H₃₉N₇O₈S₂ [M+H]⁺ 786.2374, found 786.2372 [M+H]⁺. HPLC purity: 98.93%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(1-(3-(4-((1,1-dioxidothiomorpholino)sulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-phenylethyl)acetamide (21b1).

White solid, yield: 37%. Mp: 206–208 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (d, J = 7.7 Hz, 1H, NH), 8.15 (d, J = 7.1 Hz, 1H, Ph-H), 8.02 (d, J = 8.6 Hz, 2H, Ph-H), 7.97–7.89 (m, 1H, Ph-H), 7.77 (d, J = 8.1 Hz, 1H, Ph-H), 7.75–7.69 (m, 1H, Ph-H), 7.66–7.56 (m, 2H, Ph-H), 7.40 (d, J = 8.7 Hz, 2H, Ph-H), 7.17 (dq, J = 14.2, 7.0 Hz, 3H, Ph-H), 6.86 (d, J = 7.0 Hz, 2H, Ph-H), 6.66 (d, J = 8.7 Hz, 2H, Ph-H), 6.17 (s, 2H, NH₂), 4.53 (td, J = 8.5, 5.4 Hz, 1H, CH), 3.93–3.79 (m, 2H, CH₂), 3.58 (s, 4H, CH₂ × 2), 3.47–3.36 (m, 2H, CH₂), 3.35 (s, 4H, CH₂ × 2), 3.21 (tt, J = 10.8, 4.7 Hz, 2H, CH₂), 3.10 (ddd, J = 16.5, 9.6, 5.2 Hz, 2H, CH₂), 2.95 (dq, J = 11.3, 5.3, 4.0 Hz, 1H, CH), 2.90–2.81 (m, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.80 (C═O), 164.13 (C═O), 161.61 (C═O), 156.11, 154.16, 147.20, 141.08, 137.70, 137.67, 135.70, 131.19, 131.04, 130.32, 129.22, 128.81, 127.94, 127.55, 127.10, 126.98, 121.02, 118.39, 113.35, 53.26, 50.91, 49.27, 48.49, 47.05, 45.51, 43.29, 38.48. HRMS calcd for m/z C₃₈H₃₉N₇O₉S₃ [M+H]⁺ 834.2044, found 834.2047 [M+H]⁺. HPLC purity: 99.68%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(2-(3,5-difluorophenyl)-1-(3-(4-(morpholinosulfonyl)phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)acetamide (21a2).

White solid, yield: 38%. Mp: 198–199 °C. ¹H NMR (400 MHz, DMSO-d₆) δ (d, J = 8.1 Hz, 1H, NH), 8.22–8.12 (m, 1H, Ph-H), 8.01–7.86 (m, 3H, Ph-H), 7.85–7.75 (m, 2H, Ph-H), 7.70–7.58 (m, 2H, Ph-H), 7.40 (d, J = 8.7 Hz, 2H, Ph-H), 7.02 (t, J = 9.4 Hz, 1H, Ph-H), 6.77–6.61 (m, 4H, Ph-H), 6.19 (s, 2H, NH), 4.56 (td, J = 8.5, 5.1 Hz, 1H, CH), 3.94–3.74 (m, 2H, CH₂), 3.64 (t, J = 4.4 Hz, 4H, CH₂ × 2), 3.44 (d, J = 16.1 Hz, 2H, CH₂), 3.32–3.20 (m, 2H, CH₂), 3.20–3.06 (m, 2H, CH₂), 3.05–2.84 (m, 6H, CH₂ × 2, CH× 2). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.53 (C═O), 164.07 (C═O), 162.51 (dd, ¹J_CF = 246.1, ³J_CF = 13.4 Hz), 161.63 (C═O), 155.20, 154.20, 147.06, 142.48 (t, ³J_CF = 9.3 Hz), 140.87, 136.04, 135.64, 131.13, 130.48, 130.32, 129.57, 129.34, 128.04, 127.61, 126.97, 121.21, 118.42, 113.33, 112.56 (dd, ²J_CF = 24.7, ⁴J_CF = 6.8 Hz), 102.60 (t, ²J_CF = 25.5 Hz), 65.83, 52.45, 49.30, 48.57, 47.18, 46.31, 43.32, 37.96. HRMS calcd for m/z C₃₈H₃₇F₂N₇O₈S₂ [M+H]⁺ 822.2186, found 822.2186 [M+H]⁺. HPLC purity: 99.63%.

(S)-2-(4-((4-Aminophenyl)sulfonyl)-2-oxopiperazin-1-yl)-N-(2-(3,5-difluorophenyl)-1-(3-(4-((1,1-dioxidothiomorpholino)sulfonyl)-phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)acetamide (21b2).

White solid, yield: 32%. Mp: 186–187 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 8.0 Hz, 1H, NH), 8.22–8.12 (m, 1H, Ph-H), 8.05 (dd, J = 8.3, 2.0 Hz, 1H, Ph-H), 7.99 (dd, J = 8.3, 2.0 Hz, 1H, Ph-H), 7.97–7.90 (m, 1H, Ph-H), 7.78 (d, J = 8.1 Hz, 2H, Ph-H), 7.72 (dd, J = 5.7, 3.2 Hz, 1H, Ph-H), 7.70–7.57 (m, 2H, Ph-H), 7.40 (d, J = 8.7 Hz, 2H, Ph-H), 7.08–6.95 (m, 1H, Ph-H), 6.66 (d, J = 8.7 Hz, 3H, Ph-H), 6.17 (s, 2H, NH₂), 4.56 (td, J = 8.5, 5.1 Hz, 1H, CH), 3.90–3.73 (m, 2H, CH₂), 3.57 (s, 4H, CH₂ × 2), 3.48–3.35 (m, 2H, CH₂), 3.30 (d, J = 5.2 Hz, 4H, CH₂ × 2), 3.23 (dd, J = 12.2, 5.5 Hz, 2H, CH₂), 3.13 (ddd, J = 11.5, 7.9, 4.7 Hz, 2H, CH₂), 3.04–2.95 (m, 1H, CH), 2.95–2.88 (m, 1H, CH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.53 (C═O), 164.15 (C═O), 162.48 (dd, ¹J_CF = 246.0, ³J_CF = 13.4 Hz), 161.64 (C═O), 155.12, 154.17, 147.01, 142.41(t, ³J_CF = 9.2 Hz), 140.98, 137.84, 135.70, 132.02, 131.42, 130.75, 130.32, 129.05, 128.08, 127.62, 126.97, 121.15, 118.39, 113.35, 112.59 (dd, ²J_CF = 24.8, ⁴J_CF = 6.5 Hz).102.61 (t, ²J_CF = 25.9 Hz), 52.48, 50.95, 49.25, 48.56, 47.16, 45.35, 43.28, 37.90. HRMS calcd for m/z C₃₈H₃₇F₂N₇O₉S₃ [M+H]⁺ 870.1856, found 870.1852 [M+H]⁺. HPLC purity: 99.70%.

5.2. In Vitro Anti-HIV Assay in MT-4 Cells.

The protocol for in vitro anti-HIV assay in MT-4 cells is provided in the Supporting Information.

5.3. Determination of the Mechanism of Action of Representative Compounds.

The protocols for determining the mechanism of action of representative compounds are provided in the Supporting Information, including the following assays: surface plasmon resonance (SPR) assay; single-round infection (SRI) assay; in vitro reverse transcriptase inhibition assay; SPR-based competition assay with CPSF6; in vitro capsid assembly assay; and ELISA-based quantification of capsid (p24) content.

5.4. Crystallization, Data Collection, and Processing.

The protocol for crystallization is provided in the Supporting Information.

5.5. Computational Assessment of Metabolic Stability and Toxicity.

The protocols for computational assessment of metabolic stability and toxicity are provided in the Supporting Information.

5.6. Metabolic Stability in Human Liver Microsomes and Human Plasma.

The protocols for metabolic stability in human liver microsomes and human plasma are provided in the Supporting Information.

5.7. In Vivo Pharmacokinetic Study.

The protocol for in vivo PK study is provided in the Supporting Information.

ABBREVIATIONS USED

AIDS

acquired immunodeficiency syndrome

BSA

N,O-bis(trimethylsilyl)acetamide

Boc

tert-butyloxycarbonyl

CA

capsid

cART

combination antiretroviral therapy

CL

clearance rate

CPSF6

cleavage and polyadenylation specific factor 6

CSL

composite site lability

CTD

C-terminal domain

CypA

cyclophilin A

CC₅₀

50% cytotoxicity concentration

¹³C NMR

carbon nuclear magnetic resonance

DCM

dichloromethane

DIEA

N,N-diisopropylethylamine

DMAP

4-dimethylaminopyridine

EC₅₀

effective concentration causing 50% inhibition of viral cytopathogenicity

ELISA

enzyme-linked immunosorbent assay

FG

phenylalanineglycine

FRET

fluorescence resonance energy transfer

HATU

2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HIV

human immunodeficiency virus

HLM

human liver microsome

¹H NMR

proton nuclear magnetic resonance

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

HYDE

HYdrogen bond and Dehydration

IP6

inositol hexakisphosphate

LEN

Lenacapavir

mp

melting point

MRT

mean residence time

MS

mass spectrometry

NTD

N-terminal domain

NUP153, NUP358

nucleoporins 153 and 358

PK

pharmacokinetic

RT

reverse transcriptase

SAR

structure–activity relationship

SER

structure–selectivity relationship

SD

Sprague–Dawley

SI

selectivity index

SPR

surface plasmon resonance

SRI

single-round infection

TEA

triethylamine

TFA

trifluoroacetic acid

T _1/2

half-time

TLC

thin-layer chromatography

T _max

time to maximum concentration

TMS

tetramethylsilane