Metabolite Identification of HIV-1 Capsid Modulators PF74 and 11L in Human Liver Microsomes

PF74 and 11L, as potent modulators of the HIV-1 capsid protein, have been demonstrated to act at both early and late stages in the HIV-1 life cycle. However, their clearance is high in human liver microsomes (HLMs). The main goal of this study was to clarify the metabolism of PF74 and 11L in HLMs, and provide guidance for future structural optimization. To accomplish this, the phase-I metabolites of PF74 and 11L, resulting from in vitro incubation with HLMs, were investigated via ultra-performance liquid chromatography–ultraviolet–high-resolution mass spectrometry (UPLC–UV–HRMS). The results show that 17 phase-I metabolites were putatively annotated for PF74, whereas 16 phase-I metabolites were found for 11L. The main metabolic pathways of PF74 in HLMs were oxidation and demethylation, and the secondary metabolic pathway was hydrolysis; thus, the di-oxidation and demethylation products (M7, M9, M11, and M14) were found to be major metabolites of PF74 in HLMs. In comparison, the main metabolic pathways of 11L in HLMs were oxidation, demethylation, dehydrogenation, and oxidative deamination, with M6′, M11′, M15′, and M16′ as the main metabolites. We suggest that the indole ring and N-methyl group of PF74, and the aniline group, benzene ring R1′, N-methyl, and methoxy group of 11L, were the main metabolic soft spots. Therefore, our research illuminates structural optimization options in seeking improved HIV-1 CA modulators.


Introduction
Human immunodeficiency virus type 1 (HIV-1) remains a worldwide healthcare concern despite the effective development of combination antiretroviral therapy (cART) [1,2]. Existing cART is projected to last a lifetime, and the virus will ultimately choose variants that are resistant to current treatment classes [3,4]. This necessitates the development of innovative medications with diverse resistance profiles and new targets. The multifunctional HIV-1 capsid protein (CA) represents an emerging and highly appealing target in HIV-1 medication research. CA plays essential roles in multiple events during viral replication, such as reverse transcription, nuclear entry, integration site distribution, assembly, and maturation [5][6][7]. Therefore, CA-targeting small molecules could provide antiviral characteristics in both the early and late stages. By far, the most intriguing CA-targeting compound is PF74; however, further clinical development of PF74 is mainly impeded by its inferior metabolic stability [8,9]. Additionally, our previous efforts involved replacing the indole moiety with benzenesulfonamide moiety, and we obtained compound 11L with increased antiviral activity, but only modestly better microsomal stability [10] (Figure 1). PF74 and 11L could affect the morphology and assembly process of viral particles through multiple effects to achieve the purpose of inhibiting viral replication, providing low stoichiometric loading advantages [10,11]. Theoretically, a small molecule could regulate Figure 1. The chemical structures of PF74 and 11L [10]. EC50 is the concentration of the compound required to achieve 50% protection of TZM-bl cells against HIV-1-induced cytopathic effect, deter mined in at least triplicate against HIV-1 in TZM-bl cells; t1/2 is the half-life; and CLint is the intrinsi clearance.
Metabolite profiling studies of new chemical entities (NCEs) are commonly per formed in the pharmaceutical industry to aid in medication development [12,13]. These investigations not only provide information regarding the metabolic fate of NCEs, they also assist in the identification of metabolic soft spots in the parent molecule [14]. Eluci dating the metabolic profile may allow for metabolic fortification during drug develop ment efforts, and it is also significant for assessing overall drug safety and efficacy [15,16] Ultra-performance liquid chromatography-ultraviolet-high-resolution mass spectrome try (UPLC-UV-HRMS) is a vital and effective method for the research of drug metabolism and pharmacokinetics, and it is widely utilized to characterize the structural properties o NCEs and their metabolites [17][18][19][20]. UPLC has a powerful separation capacity, making i one of the most useful tools currently available for separating complicated component [21][22][23]. The relative content of different metabolites can be determined according to the peak area of UV, which is helpful to preliminarily determine the main metabolites [18] HRMS possesses the qualities of high speed, sensitivity, and selectivity, and it has been extensively employed for online structural analysis and quantitative detection of the com ponents [22,24].
In this study, we investigated the phase-I metabolites and metabolic pathways o PF74 and 11L in human liver microsomes (HLMs). PF74 and 11L were incubated in the coexistence system of HLMs and NADPH for 60 min at 37 °C. The incubated samples were evaluated by UPLC-UV-HRMS. The relative content of each metabolite was determined according to the UV peak area. The structures of the metabolites were elucidated based on their MS spectra, tandem MS (MS/MS) spectra, fragmentation patterns, and compari son of the primary and secondary mass spectrometry signals with the parent drug. Fi nally, the metabolic pathways of PF74 and 11L were proposed based on metabolite data In the in vitro study, a positive control was used to monitor the incubation. As described in the Supplementary Materials, 7-ethoxycoumarin (7-EC, 10 μM) was utilized as a posi tive control for phase-I metabolism to assure that proper incubation conditions were  [10]. EC 50 is the concentration of the compound required to achieve 50% protection of TZM-bl cells against HIV-1-induced cytopathic effect, determined in at least triplicate against HIV-1 in TZM-bl cells; t 1/2 is the half-life; and CL int is the intrinsic clearance.
Metabolite profiling studies of new chemical entities (NCEs) are commonly performed in the pharmaceutical industry to aid in medication development [12,13]. These investigations not only provide information regarding the metabolic fate of NCEs, they also assist in the identification of metabolic soft spots in the parent molecule [14]. Elucidating the metabolic profile may allow for metabolic fortification during drug development efforts, and it is also significant for assessing overall drug safety and efficacy [15,16]. Ultra-performance liquid chromatography-ultraviolet-high-resolution mass spectrometry (UPLC-UV-HRMS) is a vital and effective method for the research of drug metabolism and pharmacokinetics, and it is widely utilized to characterize the structural properties of NCEs and their metabolites [17][18][19][20]. UPLC has a powerful separation capacity, making it one of the most useful tools currently available for separating complicated components [21][22][23]. The relative content of different metabolites can be determined according to the peak area of UV, which is helpful to preliminarily determine the main metabolites [18]. HRMS possesses the qualities of high speed, sensitivity, and selectivity, and it has been extensively employed for online structural analysis and quantitative detection of the components [22,24].
In this study, we investigated the phase-I metabolites and metabolic pathways of PF74 and 11L in human liver microsomes (HLMs). PF74 and 11L were incubated in the coexistence system of HLMs and NADPH for 60 min at 37 • C. The incubated samples were evaluated by UPLC-UV-HRMS. The relative content of each metabolite was determined according to the UV peak area. The structures of the metabolites were elucidated based on their MS spectra, tandem MS (MS/MS) spectra, fragmentation patterns, and comparison of the primary and secondary mass spectrometry signals with the parent drug. Finally, the metabolic pathways of PF74 and 11L were proposed based on metabolite data. In the in vitro study, a positive control was used to monitor the incubation. As described in the Supplementary Materials, 7-ethoxycoumarin (7-EC, 10 µM) was utilized as a positive control for phase-I metabolism to assure that proper incubation conditions were maintained. De-ethylated metabolites were observed in the 7-EC positive control, indicating phase-I metabolic activity in liver microsome incubation system.

Sample Preparation
To examine phase-I metabolites in vitro, microsomal incubation was performed with 100 mM phosphate buffer saline (pH 7.4) containing PF74 or 11L (10 µM), HLM (1.0 mg/mL), NADPH (1.0 mM), and MgCl 2 (3.0 mM), and the reaction volume was 400 µL. Furthermore, 7-EC (10 µM) was utilized as a positive control for phase-I metabolism to guarantee that proper incubation conditions were maintained. Incubation without samples was used as a blank control. After preincubation for 5 min at 37 • C, the reactions were started by adding NADPH. After incubation for 60 min at 37 • C, the reactions were quenched by adding 800 µL of acetonitrile solution containing 0.1% formic acid. Afterward, the supernatants were obtained under centrifugation at 13,000 rpm for 10 min, and were then dried under nitrogen gas and the residues redissolved in 200 µL water/acetonitrile solution (v:v = 10:3). Aliquots were taken at 0 and 60 min, then, the sample (10 µL) was injected into the UPLC-UV-HRMS system for metabolite profiling and identification.

Chromatographic and Mass Spectrometric Methods of PF74
The UPLC-UV-HRMS analysis was carried out using an ACQUITY UPLC system hyphenated to a Thermo Q-Exactive HF tandem mass spectrometer equipped with an electrospray ionization interface that operated in positive ion mode.
Mass Spectrometric Method: The electrospray ionization source parameters were set as follows: capillary temperature, 375 • C; spray voltage, 3.5 kV; sheath gas flow rate, 40 L/h; auxiliary gas flow rate, 15 L/h; auxiliary gas heater temperature, 300 • C; S-Lens voltage, 55 V. The data were collected in the m/z range of 140-1400 Da (full mass scan) and 200-2000 Da (MS/MS scan) in centroid mode. The collision energy was 10/15 V. The mass resolutions for full mass scans and MS/MS scans were chosen to be above 60,000 and 15,000/30,000, respectively. Collision-induced dissociation (CID) was utilized to attain the MS/MS fragment of PF74 and its metabolites.

Chromatographic and Mass Spectrometric Methods of 11L
The UPLC-UV-HRMS analysis was carried out using an ACQUITY UPLC system hyphenated to a Thermo Q-Exactive HF tandem mass spectrometer equipped with an electrospray ionization interface that operated in positive or negative ion mode.
Mass Spectrometric Method: The electrospray ionization source parameters were set as follows: capillary temperature, 375 • C; spray voltage, 3.

Data Processing
The acquired data sets were captured and analyzed by Thermo Xcaliber 4.2 workstation (Thermo Scientific, Bremen, Germany). The possible element composition was speculated by accurate molecular mass, and the data were processed by mass spectrometry fragmentation information. The molecular formulae of parent ions and fragment ions were predicted using a molecular formula prediction module, and the structures of metabolites, in vitro, of PF74 and 11L were identified. The maximum mass errors between the measured and the theoretical value were limited to 5 ppm. It is worth noting that after incubation at 37 • C for 60 min, PF74 was almost completely metabolized in HLMs. A total of 17 phase-I metabolites were discovered in the HLMs, and their spectra were compared to PF74 for structural confirmation (Figures 2 and 3). According to the UV peak area, M7, M9, M11, and M14 were preliminarily identified as the main metabolites ( Figure 2 and Table 1).      The semiquantitative data of UV (254-310 nm) peak area are shown in the table: P, parent; ND, not detected; +, the signal was detected in UPLC-HRMS, but the UV signal was too weak to integrate the peak area at 254-310 nm; #, peak area is the value after deducting blank matrix; *, the metabolite peaks overlapped, and the UV peak area was calculated and allocated according to the ratio of the mass spectrum peak area; NA, not applicable. Relative abundance was calculated by comparing the peak areas of a single metabolite to the sum of the peak areas of all given metabolites. Relative abundance = (peak area of a single metabolite/total peak area of metabolites) × 100%.

Identification of PF74 Metabolites and Proposed Metabolic Pathways in HLMs
The first step in identifying the structures of metabolites was to examine the MS/MS fragmentation pattern of PF74. The MS/MS fragmentation pattern of PF74 was studied in positive mode. A protonated molecular ion [M + H] + of PF74 at m/z 426.2151 was found in the full-scan mass spectrum. The protonated molecules then produced a series of characteristic fragment ions at m/z 320.1472, 319.1440, 291.1489, 263.1539, 214.1732, 172.0762, 144.0806, 120.0811 and 57.5965. PF74 was eluted at 9.91 min under the experimental conditions. The MS/MS product ion spectrum and predominant fragmentation patterns of PF74 were depicted in Figure 4.

Metabolite Profiling of PF74 in HLMs
A list of all the putative metabolites of PF74 with the proposed type of biotransformation and elemental composition, as well as the retention time, relative abundance, UV peak area, accurate mass of the protonated molecule and mass error, were summarized in Tables 1 and 2. UPLC-MS/MS analysis of unchanged PF74 and its 17 metabolites yielded informative and prominent product ions for structural characterization ( Figure S2). . It was 171 u lower than that of PF74, and produced secondary fragment ions at m/z 120.08, indicating that it might be the amide bond adjacent to the indole ring hydrolyzed to the amino (−171 u) product ( Figure S2A).  13, 323.14, 190.09, and 120.08, indicating that benzene ring R 2 did not undergo oxidation. In accordance with the secondary fragment ion at m/z 257.13, it is speculated that benzene ring R 1 underwent mono-oxidation and Ndemethylation ( Figure S2B,J). Additionally, the secondary fragment ions at m/z 206.08 and 136.08 of M3 suggest that the mono-oxidation of benzene ring R 2 had occurred ( Figure S2C). Metabolite M6 had secondary fragment ions at m/z 188.07 and 170.06, indicating that the indole ring was mono-oxidized, and the secondary fragment ions at m/z 367.13, 339.13, 222.08, and 152.07 indicate that the benzene ring R 2 was di-oxidized ( Figure S2F). Secondary fragment ions at m/z 202.05 and 186.05 suggest that the indole ring of metabolite M8 underwent oxidation ( Figure S2H). In accordance with the secondary fragment ions at m/z 367.13 and 339.13 of M12, the oxidation of the indole ring and benzene ring R 2 is also speculated ( Figure S2L).

Tri-oxidation Metabolites (M4 and M5)
Metabolites M4 and M5 possessed the same theoretical protonated molecular ion [M + H] + at m/z 474.2024 (C 27 H 27 N 3 O 5 ), and were detected at 6.68 and 7.05 min, respectively. They were 48 u higher than that of PF74; therefore, they could be the tri-oxidation products. The secondary fragment ion at m/z 456.19 was formed when they lost one molecule of water. The metabolite M4 had secondary fragment ions at m/z 188.07 and 170.06, demonstrating that the indole ring was mono-oxidized. The secondary fragment ions at m/z 222.08 and 152.07 show that the benzene ring R 2 was di-oxidized, and m/z 108.08 show that the benzene ring R 1 was not oxidized ( Figure S2D). The secondary fragment ions at m/z 202.05 and 174.06 in the metabolite M5 indicate that the indole ring was tri-oxidized, and the secondary fragment ions at m/z 120.08 and 108.08 indicate that the benzene rings R 1 and R 2 were not oxidized ( Figure S2E). , which indicates that they were di-oxidation (+32 u) and demethylation (−14 u) products. The metabolites M7 and M13 had secondary fragment ions at m/z 188.07 and 170.06, Implying that the indole ring was mono-oxidized. Secondary fragment ions at m/z 120.08 indicate that benzene ring R 2 was not oxidized, while secondary fragment ions at m/z 257.13 suggest that benzene ring R 1 was mono-oxidized and N-demethylated ( Figure S2G , which was 32 u higher than that of PF74, meaning that it might be a di-oxidation (+32 u) product of PF74. Secondary fragment ions at m/z 204.07 and 162.05 indicate that the indole ring was oxidized, and m/z 190.09 and 108.08 indicate that the benzene rings R 1 and R 2 were not oxidized. Additionally, m/z 440.20, 333.12, and 305.13 were a series of fragments formed by oxidative dehydration ( Figure S2I).

Mono-oxidation Metabolite (M15)
Metabolite M15 with a protonated ion [M + H] + at m/z 442.2134 (C 27 H 27 N 3 O 3 ), was eluted at 8.75 min. It was 16 u higher than that of PF74, showing that there was a monooxidation product. The secondary fragment ion at m/z 188.07 determines that the indole ring was mono-oxidized, and m/z 424.20, 317.13, 289.13, and 170.06 were a series of fragments formed by oxidative dehydration ( Figure S2O).

Mono-oxidation and Demethylation Metabolite (M17)
Metabolite M17, 2 u higher than that of PF74, possessed a protonated molecule ion [M + H] + at m/z 428.1969 (C 26 H 25 N 3 O 3 ) with the retention time of 9.37 min, suggesting that M17 was the product of mono-oxidation (+16 u) and demethylation (−14 u). In addition, m/z 188.07 leads us to speculate that the indole ring was mono-oxidized, and m/z 410.19, 317.18, 289.13, and 170.06 were a series of fragments formed by oxidative dehydration ( Figure S2Q).

Proposed Metabolic Pathways of PF74
In this investigation, a total of 17 metabolites were found in HLMs of PF74. The proposed metabolic pathways of PF74 are exhibited in Figure 5. There was a series of metabolic reactions containing mono-oxidation, di-oxidation, tri-oxidation, mono-oxidation and demethylation, di-oxidation and demethylation, tri-oxidation and demethylation, amide hydrolysis, and so on. Metabolites M7, M11, M14 (di-oxidation and N-demethylation products), and M9 (di-oxidation product) were the four most abundant metabolites, so the main metabolic pathways of PF74 in HLMs were di-oxidation and N-demethylation, which also suggests that the indole ring, benzene ring R 1 , and N-methyl group were the main metabolic soft spots. In a previous study by Wang et al. [25], upon substituting the electron-rich indole ring of PF74 with a less electron-rich benzamide group, a preferred compound with similar submicromolar potency and much longer (51-fold) half-life was reported. Additionally, by replacing the benzene ring R 1 with the electron-deficient benzothiazole moiety, we found a compound that exhibited markedly improved metabolic stability in HLMs, with a half-life 109-fold that of PF74 [26]. Furthermore, Sahani et al. [27] reported a PF74-like analog with 39 times enhanced metabolic stability over PF74, which led to increased resistance to N-methylation conceived via building the phenylalanine carboxamide into a pyridine ring. These results are consistent with our current findings, wherein we have identified the phase-I oxidative and N-demethylated metabolites as major metabolites of PF74 in HLMs. and demethylation, di-oxidation and demethylation, tri-oxidation and demethylation, amide hydrolysis, and so on. Metabolites M7, M11, M14 (di-oxidation and N-demethylation products), and M9 (di-oxidation product) were the four most abundant metabolites, so the main metabolic pathways of PF74 in HLMs were di-oxidation and N-demethylation, which also suggests that the indole ring, benzene ring R1, and N-methyl group were the main metabolic soft spots. In a previous study by Wang et al. [25], upon substituting the electron-rich indole ring of PF74 with a less electron-rich benzamide group, a preferred compound with similar submicromolar potency and much longer (51-fold) half-life was reported. Additionally, by replacing the benzene ring R1 with the electron-deficient benzothiazole moiety, we found a compound that exhibited markedly improved metabolic stability in HLMs, with a half-life 109-fold that of PF74 [26]. Furthermore, Sahani et al. [27] reported a PF74-like analog with 39 times enhanced metabolic stability over PF74, which led to increased resistance to N-methylation conceived via building the phenylalanine carboxamide into a pyridine ring. These results are consistent with our current findings, wherein we have identified the phase-I oxidative and N-demethylated metabolites as major metabolites of PF74 in HLMs.  Figure 6 shows the UPLC-UV chromatogram of 11L in HLMs at the detection wavelength of 240-320 nm, and Figure 7 show the XIC of 11L in HLMs. Metabolites were tentatively identified by comparing the UPLC-UV and UPLC-HRMS spectra and retention  Figure 6 shows the UPLC-UV chromatogram of 11L in HLMs at the detection wavelength of 240-320 nm, and Figure 7 show the XIC of 11L in HLMs. Metabolites were tentatively identified by comparing the UPLC-UV and UPLC-HRMS spectra and retention times of potential metabolites to the parent compound 11L. After incubation at 37 • C for 60 min, the relative abundance of 11L in HLMs was 7.11%, which explains why 11L (t 1/2 = 4.1 min) had a slightly longer half-life than PF74 (t 1/2 = 1.3 min). A total of 16 metabolites were discovered in HLMs, and their spectra were compared with those of 11L for structural confirmation (Figures 7 and 8). According to the UV peak area, M6 , M11 , M15 , and M16 are preliminarily identified as the main metabolites ( Figure 6 and Table 3).  211.5220, and 138.0914, and the deprotonated molecules then produced a series of characteristic fragment ions at m/z 441.1228, 417.1234, 311.0818, 294.0552, 254.0602, 228.0443,  186.0554, 156.0116, and 92.0495. Moreover, 11L was eluted at 10.69 min under the experimental conditions. The MS/MS product ion spectrum and predominant fragmentation patterns of 11L are displayed in Figure 8.   The semiquantitative data of UV (240-320 nm) peak area are shown in the table; P, parent; *, the measured values of M9 , M10 , and M13 (m/z) were obtained using the negative mode data; #, peak area is the value after deducting blank matrix; @, M3 , M4 , M14 -M16 were separated by cyano columns, M7 and M8 were separated by phenyl columns; NA, not applicable. Relative abundance was calculated by comparing the peak areas of a single metabolite to the sum of the peak areas of all given metabolites. Relative abundance = (peak area of a single metabolite/total peak area of metabolites) × 100%.

Metabolite Profiling of 11L in HLMs
A list of all the putative metabolites of 11L with the proposed type of biotransformation and elemental composition, as well as the retention time, relative abundance, UV peak area, accurate mass of the protonated (or deprotonated) molecule, and mass error, are summarized in Tables 3 and 4. UPLC-MS/MS analysis of unchanged 11L and its 16 metabolites yielded informative and prominent product ions for structural characterization ( Figure S3).  (C 29 H 33 N 5 O 9 S), which was 48 u higher than that of 11L, meaning that it might be a tri-oxidation (+48 u) product of 11L. The secondary fragment ion at m/z 284.07 suggests that the benzene ring R 1 had not been oxidized, whereas m/z 138.09 is evidence of the di-oxidation of benzene ring R 2 ( Figure S3A).

Di-oxidation Metabolites (M2 , M6 , M13 )
Metabolites M2 and M6 were observed at retention times of 8.49 and 8.82 min, respectively, with the same experimental protonated molecular ion [M + H] + at m/z 612.2123 (C 29 H 33 N 5 O 8 S). The two metabolites were 32 u higher than 11L, suggesting they were the di-oxidation products. The secondary fragment ions at m/z 268.08 and 138.09 of the metabolite M2 indicate that benzenesulfonamide piperazinone and benzene ring R 1 did not undergo oxidation, and m/z 475.13 and 296.07 indicate that benzene ring R 2 underwent di-oxidation ( Figure S3B). The secondary fragment ion at m/z 172.01 of metabolite M6 demonstrates that aniline was mono-oxidized, and m/z 475.13 and 312.07 indicate that benzene ring R 2 was mono-oxidized ( Figure S3F).
Furthermore, M13 was detected at a retention time of 10.06 min with the experimental deprotonated ion [M − H] − at m/z 610.1976 (C 29 H 33 N 5 O 8 S), which was 32 u higher than that of 11L, meaning that it might be a di-oxidation (+32 u) product of 11L. The secondary fragment ion at m/z 172.01 of metabolite M13 shows that aniline was mono-oxidized, and m/z 270.06 and 172.01 show that piperazinone was mono-oxidized ( Figure S3M).

Demethylation Metabolite (M14 )
Metabolite M14 , 14 u lower than that of 11L, presented an experimental protonated ion [M + H] + at m/z 566.2068 (C 28 H 31 N 5 O 6 S) with the retention time of 10.29 min, suggesting that M14 was the product of demethylation (−14 u). The secondary fragment ions at m/z 443.14, 296.07, 268.08, and 156.01 indicate that only the demethylation reaction occurred in M14 ( Figure S3N (C 29 H 32 N 4 O 8 S). It was 17u higher than that of 11L, showing that it was a product of oxidative deamination (+16 u and −15 u) and mono-oxidation (+16 u). The m/z 313.05 and 285.05 indicate that mono-oxidation and oxidative deamination of benzenesulfonamide piperazinone. The m/z 579.21 was the secondary fragment ion obtained by oxidative dehydration (Figure S3O).

Proposed Metabolic Pathways of 11L
On the basis of the MS/MS spectra and fragmentation patterns, the metabolic pathways of 11L are accordingly proposed, as shown in Figure 9. The metabolic reactions are summarized as follows: mono-oxidation, di-oxidation, tri-oxidation, demethylation, mono-oxidation and demethylation, mono-oxidation and oxidative deamination, monooxidation and dehydrogenation, Ndemethylation and O-demethylation. Metabolite M6 (di-oxidation product), M11 (mono-oxidation and demethylation product), M15 (monooxidation and oxidative deamination product), and M16 (mono-oxidation product) were the four metabolites with the highest relative abundance. Therefore, oxidation, demethylation, and oxidative deamination were the main metabolic pathways of 11L in HLMs, which also indicates that the aniline group, benzene ring R1 , N-methyl, and methoxy group of 11L were the main metabolic soft spots. It has been reported that the introduction of chlorine and fluorine atoms can block metabolically labile sites and improve the metabolic stability of compounds [28]. Recently, Sahani et al. [29] found that the substitution of a methoxy group on benzene ring R1 and an amino group on aniline via chlorine atoms could slightly improve the metabolic stability of 11L-like analogs. Notably, lenacapavir, a long-acting capsid modulator currently in clinical phase III, contains a core structure similar to PF74 and 11L [30]. However, its left wing introduced a fluorine-rich tetrahydrocyclopenta-pyrazole ring, and replaced the benzene ring R1 (R1 ) with an indazole ring, as well as introducing the pyridine group to avoid N-methylation [31]. The incorporation of electron-withdrawing groups (halogens and sulfonyls), metabolically stable ring systems (cyclopentane and pyrazoles), and rigidifying elements might be the reasons for its high metabolic stability.

Conclusions
The primary goal of metabolite screening in early drug development is to detect met abolic soft spots that result in poor metabolic stability. This information can direct syn thetic chemistry efforts through the identification of compounds with desirable pharmacokinetic characteristics. In this study, phase-I metabolites, and possible metabolic path ways of PF74 and 11L in HLMs, were identified for the first time. According to the ioni zation and fragmentation rules of ESI-MS, the metabolites were identified by UPLC-UV-HRMS data based on the characteristic fragments and characteristic neutral loss of PF74 and 11L. For PF74, the oxidation of the indole ring and N-demethylation of the paren compound were the main transformations. For 11L, oxidation of the aniline group and benzene ring R1′, demethylation, dehydrogenation, and oxidative deamination metabo lites were the relatively most intense. Therefore, it may be an effective method to improve the metabolic stability of compounds by substituting electron-deficient groups for elec tron-rich indole ring and aniline groups, and substituting benzo-aromatic heterocyclic rings for benzo-methoxy groups to overcome the defects of oxidation and demethylation In summary, this study's findings of metabolic transformation information will open up prospects for structural optimization, leading to the identification of novel, more metabolically stable HIV-1 CA modulators.

Conclusions
The primary goal of metabolite screening in early drug development is to detect metabolic soft spots that result in poor metabolic stability. This information can direct synthetic chemistry efforts through the identification of compounds with desirable pharmacokinetic characteristics. In this study, phase-I metabolites, and possible metabolic pathways of PF74 and 11L in HLMs, were identified for the first time. According to the ionization and fragmentation rules of ESI-MS, the metabolites were identified by UPLC-UV-HRMS data based on the characteristic fragments and characteristic neutral loss of PF74 and 11L. For PF74, the oxidation of the indole ring and N-demethylation of the parent compound were the main transformations. For 11L, oxidation of the aniline group and benzene ring R1 , demethylation, dehydrogenation, and oxidative deamination metabolites were the relatively most intense. Therefore, it may be an effective method to improve the metabolic stability of compounds by substituting electron-deficient groups for electron-rich indole ring and aniline groups, and substituting benzo-aromatic heterocyclic rings for benzo-methoxy groups to overcome the defects of oxidation and demethylation. In summary, this study's findings of metabolic transformation information will open up prospects for structural optimization, leading to the identification of novel, more metabolically stable HIV-1 CA modulators.