Identification of 3-Oxindole Derivatives as Small Molecule HIV-1 Inhibitors Targeting Tat-Mediated Viral Transcription

The heterocyclic indole structure has been shown to be one of the most promising scaffolds, offering various medicinal advantages from its wide range of biological activity. Nonetheless, the significance of 3-oxindole has been less known. In this study, a series of novel 3-oxindole-2-carboxylates were synthesized and their antiviral activity against human immunodeficiency virus-1 (HIV-1) infection was evaluated. Among these, methyl (E)-2-(3-chloroallyl)-4,6-dimethyl-one (6f) exhibited the most potent inhibitory effect on HIV-1 infection, with a half-maximal inhibitory concentration (IC50) of 0.4578 μM but without severe cytotoxicity (selectivity index (SI) = 111.37). The inhibitory effect of these compounds on HIV-1 infection was concordant with their inhibitory effect on the viral replication cycle. Mode-of-action studies have shown that these prominent derivatives specifically inhibited the Tat-mediated viral transcription on the HIV-1 LTR promoter instead of reverse transcription or integration. Overall, our findings indicate that 3-oxindole derivatives could be useful as a potent scaffold for the development of a new class of anti-HIV-1 agents.


Introduction
Acquired immune deficiency syndrome (AIDS) is a major global problem threatening public health. The condition is caused by human immunodeficiency virus type-1 (HIV-1) infection [1]. Currently, six classes of drugs have been developed to treat individuals infected with HIV-1, and a combination of at least two classes of drugs, called combination anti-retroviral therapy (cART), are used to suppress viral replication efficiently [2]. Nonetheless, these treatments allow persistent HIV-1 infection and are frequently inadequate with regard to the emergence and transmission of drug-resistant mutations [3][4][5]. Therefore, global efforts to develop new classes of drugs that inhibit the HIV-1 life cycle are ongoing in medicinal research.
Heterocyclic chemical structures, acting as highly functionalized scaffolds, have been widely adopted in medicinal and pharmaceutical research. In this context, 2-oxindole (also referred to as 2-indolinone or oxindole) comprises a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring connecting a carbonyl group at C2 position. The structure is found widely in plants and mammals where it plays an important role in the maintenance of physiological homeostasis. Several derivatives of 2-oxindole obtained from natural sources are used in traditional medicinal practices, with   The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl The effects of compounds (including AZT) on HIV-1 infection and cell viability were determined in TZM-bl cells treated at a concentration of 3 μM with infection of HIV-1 NL4-3 at an MOI of 1.

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1NL4-3 at a final concentration of 3 μM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl

Evaluation of Anti-HIV-1 Activity
To explore whether the synthetic 3-oxindole-2-carboxylates are capable of inhibiting HIV-1 infection, all 11 3-oxindole derivatives were used to treat TZM-bl cells with infection of HIV-1 NL4-3 at a final concentration of 3 µM, and the inhibitory effect on HIV-1 infectivity was compared with that of azidothymidine (AZT), a marketed inhibitor of HIV-1 infection, as a control. Among the tested substances, compound 6f, which has a 3-chloroallyl group at the 2-position and 4,6-dimethyl substituents of 3-oxindole-2-carboxylates, exhibited a nearly complete inhibitory effect on HIV-1 infectivity without cytotoxicity. The compound 6g, which has a 3,3-dichloroallyl group at the 2-position, also showed a notable inhibitory effect on HIV-1 infection. Additionally, compound bearing 4-methyl (6a) with the 3-chloroallyl group at the 2-position and compounds that have the 3-chloroallyl group at the 3-position of 3-oxodindoline-2-carboxylates (7a and 7b) showed a slight inhibitory effect of approximately 20% ( Figure 1A and Table 1).
To determine the potency of the 3-oxindole-2-carboxylates on anti-HIV-1 activity, we analyzed their dose response with 1:2 serially diluted compounds at an initial concentration of 25 µM, which is relevant to medicinal application. The most significant activity, as shown in Table 2, was demonstrated by compound 6f, with a half-maximal inhibitory concentration (IC 50 ) value of 0.4578 µM. The compound 6g also showed potent activity with an IC 50 value of 1.276 µM and higher toxicity compared with 6f. The compound 6a exhibited a moderate inhibitory effect on HIV-1 infection, with an IC 50 value of 13.685 µM ( Figure 1B and Table 2). Of the compounds that showed slight activity ( Figure 1A), 7a and 7b gradually inhibited HIV-1 infectivity in a dose-dependent manner up to a concentration of 25 µM. While these inhibition ratios reached almost 50%, 6h did not show further increased inhibitory effect even at concentrations higher than 1.56 µM (Supplementary Materials Figure S1A). To determine the potency of the 3-oxindole-2-carboxylates on anti-HIV-1 activity, we analyzed their dose response with 1:2 serially diluted compounds at an initial concentration of 25 μM, which is relevant to medicinal application. The most significant activity, as shown in Table 2, was demonstrated by compound 6f, with a half-maximal inhibitory concentration (IC50) value of 0.4578 μM. The compound 6g also showed potent activity with an IC50 value of 1.276 μM and higher toxicity compared with 6f. The compound 6a exhibited a moderate inhibitory effect on HIV-1 infection, with an IC50 value of 13.685 μM ( Figure 1B and Table 2). Of the compounds that showed slight activity ( Figure 1A), 7a and 7b gradually inhibited HIV-1 infectivity in a dose-dependent manner up to a concentration of 25 μM. While these inhibition ratios reached almost 50%, 6h did not show further increased inhibitory effect even at concentrations higher than 1.56 μM (Supplementary Materials Figure S1A). The inhibitory effects on inhibition of HIV-1 infection and cell viability were determined in TZM-bl cells with infection of HIV-1 NL4-3 at an MOI of 1. a IC50: half-maximal inhibitory concentration. b CC50: concentration that reduces cell viability by 50%. c SI: selectivity index, the ratio of IC50 to CC50. Cell viability was assessed using a resazurin-based cell viability reagent in parallel with infectivity assay. EFV, AZT and seliciclib were used as the experimental control, and the data are presented as the mean ± SD (n = 3). The CC 50 (concentration that reduces cell viability by 50%) value of these compounds could not be determined at the concentration tested because of the low toxicity of these compounds (Table 2, and Figure 1B). Thus, the CC 50 values of the most promising compounds (6a, 6f, and 6g) were determined through serial dilution of the compounds starting from 100 µM. Based on the determined CC 50 values, the selectivity indices (SIs, the ratio of IC 50 to CC 50 ) of compounds 6a, 6f, and 6g were determined as 6.38, 111.37, and 34.19, respectively. As controls, the IC 50 values of efavirenz (EFV) and AZT were determined to be <0.05 µM and 0.079 µM, and SIs were >683.86 and >1265.5, respectively, similar to that reported in previous studies (Table 2) [14,15]. The other compounds showed no effect on either viral infectivity or cytotoxicity (data not shown). These data demonstrate that the 3-oxindole scaffold has potent intrinsic activity against HIV-1 infection. The compounds showing prominent activity have several similar structural features. All have the 3-chloroallyl group at the 2-position of the 3-oxindole; the compounds substituted with propyl (6i) or allyl (6h) at the 2-position exhibited decreased anti-HIV-1 activity. Moreover, 4,6-dimethyl substitution on 3-oxindole-2-carboxylate (6f and 6g) might be critical for anti-HIV-1 activity because either no substitution (6d) or monosubstitution with a methoxy (6b) or methyl (6e) at C5 exhibited reduced activities.
To verify the inhibitory effect of prominent 3-oxindole-2-carboxylates on the overall HIV-1 life cycle, HIV-1 replication assays were performed with A3.01 T cell lines (Figure 2A) and peripheral blood mononuclear cells (PBMCs) ( Figure 2B) infected with CXCR4 tropic HIV-1 NL4-3 in the presence of the test compounds. The compound 6f exhibited nearcomplete abrogation of viral replication at a concentration of 1.5 µM, 6g exhibited half inhibition of viral replication and the other compounds (6a, 6b, 6h, and 6i) had no inhibition of viral replication at the concentration tested without cytotoxicity, all of which was consistent with the inhibitory effects of the compounds on viral infectivity. Moreover, prominent compounds 6f and 6g inhibited replication of the CCR5 tropic HIV-1 Ad8 strain in the PBMCs similar to that shown in the CXCR4 tropic HIV-1 NL4-3 strain at the same concentration ( Figure 2C). To further address the mode of action linked to anti-HIV-1 activity of prominent 3- To further address the mode of action linked to anti-HIV-1 activity of prominent 3-oxindole derivatives, a HIV-1 reverse transcriptase (RTase) assay and an integrase assay were performed initially. RTase inhibitors (EFV and AZT) and integrase inhibitors (dolutegravir: DOL and raltegravir: RAL) inhibited the activities of RTase and integrase, respectively, but our compounds did not inhibit either of the two HIV-1 enzymatic activities ( Figure 3A,B). To examine whether the compounds inhibit HIV-1 transcription induced by the viral transcriptional factor Tat, a Tat-mediated transcription assay was performed in bl-DTR cells, which express simultaneously Tat-induced F-Luc and R-Luc upon doxycycline treatment (see 3. Materials and Methods), in the presence of our compounds. As shown in Figure 3C, Tat-induced transcriptional activity (F-Luc) was significantly reduced by treatment with our lead compounds without a decrease in R-Luc activity. Seliciclib and triptolide also exhibited specific inhibition of Tat-induced HIV-1 transcription as controls ( Figure 3C). Thus, we conducted a concentration response assay of the prominent derivatives to evaluate their activity against Tat-mediated HIV-1 transcription. Notably, compound 6f inhibited the Tat-induced transcriptional activity completely at a concentration of approximately 6 μM, with an IC50 value of 1.2 μM. In fact, the inhibitory effect was stronger than that shown by well-known Tat inhibitor seliciclib (IC50 value of 2.41 μM). The application of compounds 6a and 6g resulted in IC50 values of 9.53 μM and 2.85 μM, respectively ( Figure 4 and Table 3). Compounds that showed no inhibitory effect on HIV-1 infectivity did not inhibit Tat-induced HIV-1 transcription, as exemplified by 6d. Their Tat inhibitory effects closely corresponded to their activities against HIV-1 infection. These data revealed that derivatives bearing a 3-oxindole backbone inhibited Tat-induced viral transcription, which led to their inhibitory effects on HIV-1 infection. As shown in Figure 4, the increase in R-Luc activity, in phase with a decrease in F-Luc activity, might have been because of an excess of common factor(s) for transcription caused by the suppression of F-Luc expression as described previously [16,17], while the decrease in R-Luc activity in high dosages (12.5 μM and 25 μM) of compounds 6g and seliciclib might be To examine whether the compounds inhibit HIV-1 transcription induced by the viral transcriptional factor Tat, a Tat-mediated transcription assay was performed in bl-DTR cells, which express simultaneously Tat-induced F-Luc and R-Luc upon doxycycline treatment (see 3. Materials and Methods), in the presence of our compounds. As shown in Figure 3C, Tat-induced transcriptional activity (F-Luc) was significantly reduced by treatment with our lead compounds without a decrease in R-Luc activity. Seliciclib and triptolide also exhibited specific inhibition of Tat-induced HIV-1 transcription as controls ( Figure 3C). Thus, we conducted a concentration response assay of the prominent derivatives to evaluate their activity against Tat-mediated HIV-1 transcription. Notably, compound 6f inhibited the Tat-induced transcriptional activity completely at a concentration of approximately 6 µM, with an IC 50 value of 1.2 µM. In fact, the inhibitory effect was stronger than that shown by well-known Tat inhibitor seliciclib (IC 50 value of 2.41 µM). The application of compounds 6a and 6g resulted in IC 50 values of 9.53 µM and 2.85 µM, respectively ( Figure 4 and Table 3). Compounds that showed no inhibitory effect on HIV-1 infectivity did not inhibit Tat-induced HIV-1 transcription, as exemplified by 6d. Their Tat inhibitory effects closely corresponded to their activities against HIV-1 infection. These data revealed that derivatives bearing a 3-oxindole backbone inhibited Tat-induced viral transcription, which led to their inhibitory effects on HIV-1 infection. As shown in Figure 4, the increase in R-Luc activity, in phase with a decrease in F-Luc activity, might have been because of an excess of common factor(s) for transcription caused by the suppression of F-Luc expression as described previously [16,17], while the decrease in R-Luc activity in high dosages (12.5 µM and 25 µM) of compounds 6g and seliciclib might be relevant to cellular toxicity and/or off-target effects as described in the report in developing the cellular system ( Figure 1B) [18].   The compounds were assessed in bl-DTR cells using a concentration-response test. a IC50: half-maximal inhibitory concentration Current HIV-1 drugs target the specific viral replication steps of entry, reverse transcription, integration, and protease-mediated viral maturation. However, these antiretroviral treatments often lead to the emergence of drug-resistant mutations and adverse effects, as well as reservoirs of cells infected with latent HIV-1 that are not eradicated by current treatments [19]. Therefore, efforts are needed to develop new HIV-1 drugs to control the spread of HIV/AIDS.
In this study, a series of compounds containing a heterocyclic 3-oxindole core were synthesized and screened for inhibition of HIV-1 infection. The 3-oxindole core is structurally similar to indoles, which have occupied a salient place in medicinal chemistry because of their diverse biological activities as to anticancer, antimicrobial, antioxidation, and anti-HIV-1 effects. In particular, extensive investigations on the indole scaffold has led to the development of potent RTase inhibitors including derivatives of EFV, AZT, and  Current HIV-1 drugs target the specific viral replication steps of entry, reverse transcription, integration, and protease-mediated viral maturation. However, these antiretroviral treatments often lead to the emergence of drug-resistant mutations and adverse effects, as well as reservoirs of cells infected with latent HIV-1 that are not eradicated by current treatments [19]. Therefore, efforts are needed to develop new HIV-1 drugs to control the spread of HIV/AIDS.
In this study, a series of compounds containing a heterocyclic 3-oxindole core were synthesized and screened for inhibition of HIV-1 infection. The 3-oxindole core is structurally similar to indoles, which have occupied a salient place in medicinal chemistry because of their diverse biological activities as to anticancer, antimicrobial, antioxidation, and anti-HIV-1 effects. In particular, extensive investigations on the indole scaffold has led to the development of potent RTase inhibitors including derivatives of EFV, AZT, and nevirapine (NVP) [20,21], inhibitors of HIV-1 attachment to host membrane as exemplified by the BMS-663068 [22], integrase strand-transfer inhibitors (INSTIs) such as fluorobenzylindole derivatives [23], and inhibitors of HIV-1 transcription such as aristolactam [17] and 18BIOder [24]. Furthermore, 2-oxindole derivatives were also suggested to act as inhibitors of RTase [25]. In spite of the structural similarity of 3-oxindole to indole/ 2-oxindole, the potency of 3-oxindole in the developmental field of anti-HIV-1 agents has not been explored. In this study, we demonstrate for the first time that the 3-oxindole scaffold can provide an ability to inhibit HIV-1 infection. Although the anti-HIV-1 activity of 3-oxindole-2-carboxylates was facilitated by substituting 3-chloroallyl and 3,3-dichloroallyl at the 2-position with 4,6-dimethyl substituents (6f and 6g, respectively), the SAR study using more diverse substitutions on 3-oxindole-carboxylates may be further required to improve their potency against HIV-1 infection and reduce the toxicity. The mode of action of the lead compounds against HIV-1 infection has been revealed to inhibit the Tat-mediated viral transcription rather than RTase inhibition shown in several indole/2-oxindole derivatives against HIV-1. In addition, we conducted several biological experiments to determine whether these compounds inhibit Tat activity directly. However, these prominent compounds (6f and 6g) did not inhibit the cellular expression level of Tat protein, the nuclear localization of Tat, the interaction between Tat and cyclin T1 which is an essential component of super elongation complex (SEC) to facilitate viral transcription, and the association of Tat with TAR RNA, which is a step critical for the SEC to access viral promoter LTR. These results might indicate that these 3-oxindole derivatives do exert for Tat inhibition indirectly (manuscript in preparation) [26]. Details of the mechanism based on inhibition of Tat-mediated transcription remain to be elucidated in a further study. Less active compounds (7a and 7b) exhibited gradual inhibition of HIV-1 infectivity in a dose-dependent manner but do not affect the Tat-mediated viral transcription (Supplementary Materials Figure S1B). These data suggest that the 3-oxindole scaffold may have multifaceted modes of action to inhibit HIV-1 infection.

General Chemical Information
All reagents used were of the highest grade commercially available and used as purchased from Aldrich, TCI, Alfa Aesar, or Combi block. Thin-layer chromatography was carried out using Merck Kiesel gel 60 F 254 plates. Chromatographic purification was carried out using silica gel (Merck, 70-230 mesh). The purity of the synthesized compounds was ascertained by proton nuclear magnetic resonance ( 1 H NMR) and 13 C NMR spectroscopy, measured with a Bruker Avance 300-MHz spectrometer with tetramethylsilane as an internal standard. Chemical shifts in the NMR data are given as a chemical shift (δ) in ppm, multiplicity (if applicable), coupling constants (J) in Hz (if applicable), and integration (if applicable). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). MS spectra were collected with a Waters' LC-MS system (ZMD-1) and used to confirm ≥95% purity of each compound. The column used was an L-column 2 ODS (3.0 × 50 mm I.D., CERI, Saitama, Japan) with a temperature of 40 • C and a flow rate of 1.2 mL/min. Mobile phase A was 0.05% TFA in ultrapure water, and mobile phase B was 0.05% TFA in acetonitrile, which was increased linearly from 5% to 90% over 2 min and was 90% over the next 1.5 min, after which the column was equilibrated to 5% for 0.5 min. High-resolution mass spectroscopy (HRMS) was carried out at Takeda Analytical Laboratories, Ltd., without optimizing the yields.
A solution of compound 3 (1.88 g, 6 mmol) in CH 3 OH (20 mL) and H 2 O (10 mL) at 25 • C was treated with Dowex™ 50 (6g), and the reaction mixture was stirred for 24 h. The mixture was diluted with ethyl acetate (20 mL) and filtered to remove the resin. The filtrate was washed with brine (20 mL), dried with MgSO 4 , and concentrated in vacuo. The mixture was also purified by silica gel flash column chromatography (hexane to EtOAc/ hexane = 1:5) to provide compound 4 (420 mg, 22%) as a yellow solid; mp 180 • C; R f = 0.2 (EtOAc/ hexane = 1:5 A solution of compound 4 (400 mg, 1.3 mmol) in CH 2 Cl 2 (10 mL) was treated with trifluoroacetic acid (1.4 mL) at 0 • C under N 2 , and the reaction mixture was stirred at room temperature for 24 h. NaOH (2 M) solution was added until the pH reached up to 11. The mixture was extracted with ethyl acetate (10 mL), distilled water (10 mL), and brine (10 mL).

Assay for HIV-1 Infectivity
TZM-bl cells (also referred as JC53BL-13) expressing human CD4, CXCR4, and CCR5 in order to facilitate HIV-1 infection, which contain the firefly luciferase (F-Luc) gene under the control of HIV-1 long terminal repeat (LTR) promoter was used to determine inhibitory effect of the compounds on HIV-1 infection, as described previously with minor modifications [27,28]. In brief, 1 × 10 4 cells were cultured in 96-well plates for 24 h and then treated with compounds at a final concentration of 3 µM ( Figure 1A) or with 1:2 serially diluted compounds at concentrations ranging from 0 to 25 µM (dose-response assay; Figure 1B and Table 2). Without replacement of a culture medium, the cells were then infected with the HIV-1 NL4-3 strain at a multiplicity of infection (MOI) of 1 to determine the exact inhibitory effect of compounds on a single round infection. After 48 h, the inhibitory effect of the compounds was determined using a Bright Glo luciferase assay kit (Promega). The infectivity data are presented as a percentage relative to the DMSO control (vehicle). The inhibitory effects of the compounds on viral replication were determined with some minor modifications, as described previously [17,29]. In brief, 5 × 10 4 cells/ well of A3.01 cells were infected with HIV-1 NL4-3 at an MOI of 0.1 with the spinoculation at 300 g for 2 h at 25 • C. PBMCs were pre-activated with phytohemagglutinin M (PHA-M) for 3 days. The pre-activated 4 × 10 5 of PBMCs were washed with fresh media to remove excess PHA-M and then infected with HIV-1 NL4-3 or HIV-1 AD8 as that of A3.01 infection. After infection, the infectious media were replaced with fresh media (supplemented with international 10 units (IU)/mL of IL-2 for PBMCs containing 1.5 µM of compounds. At 72 h after infection, the inhibitory effect of the compounds on viral replication was determined by measuring the amount of p24, an HIV capsid protein, using an HIV-1 p24 ALPHAL-ISA™ kit (PerkinElmer, Waltham, MA, USA). Cell viability was determined by using the resazurin-based PrestoBlue™ cell viability reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. Tat-Mediated HIV-1 Transcription bl-DTR (TZM-bl-derived dual Tat reporter) cells derived from TZM-bl cells by insertion of two doxycycline-inducible lentiviral vector-expressing flag-tagged tat and Renillaluciferase (R-Luc) genes were used to determine simultaneously both activities of Tatmediated HIV-1 transcription and general cellular transcription, as described previously [18]. Determination of the inhibitory effect of the compounds on Tat-mediated transcription was performed as described previously. In brief, 1 × 10 4 of bl-DTR cells cultured in 96-well plates were treated with compounds at a final concentration of 3 µM ( Figure 3C) or with 1:2 serially diluted compounds at concentrations ranging from 0 to 25 µM (Figure 4 and Table 3). Subsequently, doxycycline was treated at a final concentration of 50 ng/mL to induce Tatmediated F-Luc and R-Luc. At 24 h after treatment, the activities of F-Luc and R-Luc were determined by using a Dual-Glo™ luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's protocols. The data are presented as a percentage relative to the DMSO control (vehicle) in the presence of doxycycline, and the experiment was performed in triplicate.

Assay for HIV-1 Reverse Transcription (RT) Activity
Activity of HIV-1 reverse transcription was determined, as described previously [17], with minor modifications. In brief, after treatment with DNase I, 2 × 10 6 of A3.01 cells were treated with the indicated compounds prior to infection with HIV-1 NL4-3 (at an MOI ratio of 1). Sixteen hours after infection, cytosolic DNA from the cells was isolated and the levels of reverse transcription (RT) products were determined by a quantitative PCR (qPCR). The primers for RT products were 5 -GGTCCAAAATGCGAACCCAG (forward) and 5 -TCTTGCTTTATGGCCGGGTC (reverse). To determine the relative levels of the RT products, rRNA from the lysed cells were analyzed with one-step quantitative RT-PCR using the following primer sets for 18S rRNA: 5 -GTAACCCGTTGAACCCCATT (forward) and 5 -CCATCCAATCGGTAGTAGGG (reverse). The relative level of each RT product was analyzed by using the delta-delta CT method, as described previously.

Assay for HIV-1 Integrase Activity
The integrase activity was determined using an HIV-1 integrase assay kit (XpressBio, Frederick, MD, USA), according to the manufacturer's protocols. In brief, a 96-well plate coated with donor substrate (DS) DNA was blocked with blocking buffer for 30 min at 37 • C. One hundred µL of HIV-1 integrase diluted with 1:300 onto reaction buffer was added to the plate, and the plate was then incubated for 30 min at 37 • C. Subsequently, 50 µL of compounds were added to the plate at final concentrations of 10 and 20 µM. At 5 min after addition of compounds, 50 µL of target substrate DNA diluted at 1:100 was added to the plate and incubated for 30 min at 37 • C. The plate was washed and then incubated with 100 µL of horse radish peroxidase antibody solution for 30 min at 37 • C. Integrase activity was assessed by measuring the peroxidase activity reacted with 3,3 ,5,5 -tetramethylbenzidine substrate at optical density 450 nM. The experiment was performed in triplicate.

Statistical Analysis
All data are expressed as the mean ± SD (n = 3), and the data were compared using a student's t-test, with * p < 0.05, ** p < 0.01 considered as being statistically significant. All statistical analyses were performed using the Prism software (v.5.0; GraphPad Software, San Diego, CA, USA).

Conclusions
A series of 3-oxindole-carboxylates were synthesized and their anti-HIV-1 activities were tested using TZM-bl cells with infection of HIV-1. The compound 6f showed the most prominent inhibitory effect on HIV-1 infection (IC 50 = 0.4578 µM and SI = 111.37), and 6a and 6g were moderately active (6a; IC 50 = 13.685 µM and SI = 6.38, 6g; IC 50 = 1.276 µM and SI = 34.19). Anti-HIV-1 activity of these compounds was validated based on the viral replication in CD4+ T cell lines and primary PBMCs infected with either X4 tropic or R5 tropic HIV-1. Anti-HIV-1 activity of these compounds was closely associated with the specific inhibition of Tat-mediated viral transcription. Together, our results demonstrate that some derivatives bearing the 3-oxindole core can be regarded as promising compounds for the development of novel anti-HIV-1 agents.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.