Discovery of N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives as anti-HIV-1 agents

The discovery and development of novel inhibitors with activity against variants of human immunodeficiency virus type 1 (HIV-1) is pivotal for overcoming treatment failure. As our ongoing work on research of anti-HIV-1 inhibitors, 32 N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives were prepared by introduction of the hydrazone fragments on the N-arylsulfonyl-3-acylindolyl skeleton and preliminarily screened in vitro as HIV-1 inhibitors for the first time. Among of all the reported analogues, eight compounds exhibited significant anti-HIV-1 activity, especially N-(3-nitro)phenylsulfonyl-3acetylindole benzoyl hydrazone (18) and N-(3-nitro)phenylsulfonyl-3-acetyl-6-methylindole benzoyl hydrazone (23) displayed the most potent anti-HIV-1 activity with EC50 values of 0.26 and 0.31 μg/mL, and TI values of >769.23 and >645.16, respectively. It is noteworthy that introduction of R3 as the methyl group and R2 as the hydrogen group could result in more potent compounds. This suggested that introduction of R3 as the methyl group could be taken into account for further preparation of these kinds of compounds as anti-HIV-1 agents.


INTRODUCTION
Acquired immunodeficiency syndrome (AIDS) is mainly caused by human immunodeficiency virus type 1 (HIV-1) infection and remains one of the biggest medical barriers for human health, since it was first reported in 1981 (Gottlieb et al., 1981).The reverse transcriptase (RT) of the HIV-1 plays a significant role in the viral replication process, which makes it a pivotal target for anti-HIV-1 inhibitor discovery (Jonckheere, Anné, De Clercq, 2000;Yisma et al., 2014).Although numerous RT inhibitors, including primarily the nucleoside/ nucleotide RT inhibitors (NRTIs), e.g., zidovudine, and non-nucleoside RT inhibitors (NNRTIs), e.g., nevirapine, delavirdine and efavirenz, have been developed, like other anti-HIV inhibitors, effectiveness of now approved NRTIs and NNRTIs have been hampered because of the fast development of resistance (Boone, 2006;De Clercq, 2002;Sluis-Cremer, Wainberg, Schinazi, 2015;Yu et al., 2011).It is estimated that 36.9 million people (including 2.6 million children) were living with HIV infection in the year 2014 according to UNAIDS-2015 report, and 1.2 million people died due to HIV as well as related diseases (Chander et al., 2016).To circumvent this challenge, there is an urgent need to discover and develop safe, green, efficient, selective and novel anti-HIV inhibitors having significant potency against drugresistant RT viral strains as well as less toxicity (Chander et al., 2016;Huang et al., 2007;Polanski et al., 2006;Safakish et al., 2017).
Interestingly, all reactants dissolved well in anhydrous ethanol in the beginning of the reaction.As the reaction progressed, some insoluble species were gradually precipitated out, as well as at the end of the reaction, a large amount of solid accumulated in the bottom of the flask.As a result of this behavior, all the desired N-arylsulfonyl-3-acylindole benzoyl hydrazone analogs (1-32) could be obtained by filtration.This procedure imbues the synthetic methodology with green credentials.

Biological activities
As our ongoing work on research of anti-HIV-1 inhibitors, purified and characterized N-arylsulfonyl-3-acylindole benzoyl hydrazones 1-32 were screened in vitro for their inhibitory activity against HIV-1 replication in acutely infected C8166 cells, and 3′-azido-3′-deoxythymidine (AZT) was used as a positive control presented in Table I.
In order to elucidate the anti-HIV-1 activity of compounds 1-32 at a molecular basis and to reveal structural features critical for their anti-HIV-1 activity, a brief investigation of structure activity relationship (SAR) was determined, which revealed how the substituents on 1-32 were related to the anti-HIV-1 activity.In general, (1) R 3 = Me was more vital for the anti-HIV-1 activities than R 3 = H, Et, or n-pentyl.For example, when R 1 and R 2 were H, introduction of R 3 as the methyl group could lead to the pronounced compound (14 vs 30 and 31, EC 50 values of 0.52, 14.53 and 38.23 μg/mL, TI values of >384.61,>13.76 and >5.23, respectively; that is, the TI value of 14 was close to 28 times of that of 30 and was more than 73 times of that of 31).(2) When R 1 = H or 6-Me and R 2 = 3-NO 2 , compounds with R 3 = Me exhibited significant inhibitory potential against the HIV viral replication (18 vs 5, EC 50 values of 0.26 and 4.06 μg/mL, TI values of >769.23 and 6.67, respectively, especially the TI value of 18 was more than 115 times of that of 5; 23 vs 10, EC 50 values of 0.31 and 1.02 μg/mL, TI values of >645.16 and 116.33, respectively).(3) R 2 as the 3-nitro group also plays an important role in the activities against the HIV viral replication.For example, when R 3 = H and R 1 = 6-Me, the corresponding compound 10 usually displayed more potent anti-HIV-1 activity (TI = 116.23);When R 3 = Me and R 1 = H or 6-Me, the corresponding compounds 18 and 23 also exhibited the most remarkable anti-HIV-1 activity (TI >769.23 and >645.16,respectively).( 4) It is noteworthy that introduction of R 3 as the methyl group and R 2 as the hydrogen group could result in more potent compounds.For example, the EC 50 and TI values of compounds 14, 19, 24 and 27 were 0.52, 0.49, 0.67, 0.50 μg/mL, and >384.61,>408.16,>298.50, 249.86, respectively.This suggested that introduction of R 3 as the methyl group could be taken into account for further preparation of these kinds of compounds as anti-HIV-1 agents.

Cells and virus
The cell line (C8166) and the laboratory-derived virus (HIV-1 IIIB ) were obtained from the Medical Research Council, AIDS Reagent Project, London, UK.C8166 was maintainedin RPMI-1640 medium supplemented with 10% heat-inactivated newborn calf serum (Gibco, Grand Island, NY, USA).The cells used in all experiments were in log-phasegrowth.The 50% HIV-1 IIIB tissue culture infectious dose (TCID 50 ) in C8166 cells was determined and calculated by the Reed and Muench method.Virusstocks were stored in small aliquots at -70 o C.

MTT-based cytotoxicity assay
Cellular toxicity of compounds 1-32 on C8166 cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described previously.Briefly, cells were seeded in a 96well microtiter plate in the absence or presence of various concentrations of compounds in triplicate and incubated at 37 o C in a humid atmosphere of 5% CO 2 for 3 days.The supernatants were discarded and MTT reagent (5 mg/mL in PBS) was added to each well, then incubated for 4 h, after which 100 μL of 50% DMF-20% SDS was added.After the formazan was dissolved completely, the plates were read on a BioTekElx800 ELISA reader (BioTek, Winooski, VT, USA) at 595/630 nm.Thecytotoxic concentration that caused the reduction of viable C8166cells by 50% (CC 50 ) was determined from the dose-response curve.

Syncytia assay
In the presence of 100 μL of various concentrations of compounds 1-32, C8166 cells (4×10 5 /mL) were infected with HIV-1 IIIB at a multiplicity of infection (M.O.I) of 0.06.The final volume per well was 200 μL.Control assays were performed without the test compounds in HIV-1 IIIB infected and uninfected cultures.After 3 days of culture, the cytopathic effect (CPE) was measured by counting the number of syncytia.Percentage inhibition of syncytia formationwas calculated, and the 50% effective concentration (EC 50 ) was calculated.3′-Azido-3′-deoxythymidine (AZT; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control.The therapeutic index (TI) was calculated as CC 50 /EC 50 .

CONCLUSIONS
In the present study, 32 N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives were prepared and screened in vitro as HIV-1 inhibitors for the first time.

FIGURE 1 -
FIGURE 1 -Structures of currently approved NRTIs and NNRTIs by the U.S. FDA.
eAZT was used as a positive control.