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Brazilian Journal of Pharmaceutical Sciences

On-line version ISSN 2175-9790

Braz. J. Pharm. Sci. vol.54 no.4 São Paulo  2018  Epub Apr 08, 2019

https://doi.org/10.1590/s2175-97902018000417543 

Article

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

Zhiping Che1 

Yuee Tian1 

Shengming Liu1 

Mei Hu1 

Genqiang Chen1  * 

1 Laboratory of Pharmaceutical Design & Synthesis, Department of Plant Protection, College of Forestry, Henan University of Science and Technology, Luoyang 471003, Henan Province, People′s Republic of China


ABSTRACT

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-3-acetylindole 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.

Keywords: Benzoyl hydrazone; Human immunodeficiency virus type-1; Inhibitor of virus replication; Anti-HIV-1 agent

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 drug-resistant RT viral strains as well as less toxicity (Chander et al., 2016; Huang et al., 2007; Polanski et al., 2006; Safakish et al., 2017).

Figure 1: Structures of currently approved NRTIs and NNRTIs by the U.S. FDA. 

Hydrazones are excellent candidates for the research of antiprotozoal agents (Carvalho et al., 2014; de Sá Alves, Barreiro, Fraga, 2009), multidentate ligands (Bessy Raj, Prathapachandra Kurup, Suresh, 2008), pesticidal agents (Che et al., 2013b; Guo et al., 2012) and gelatinase inhibitors (Yang et al., 2016). Meanwhile, N-arylsulfonylindoles or N-arylsulfonyl-3-acylindoles are excellent candidates for the study of anti-HIV-1 inhibitors (Che et al., 2016; Fan et al., 2009; Ran et al., 2010), and especially some N-arylsulfonyl-3-acetylindoles showed potent anti-HIV-1 activity. Nevertheless, the anti-HIV-1 activity of the N-arylsulfonyl-3-acylindole benzoyl hydrazones has not been previously published. Inspired by these previous observations, and the goal in this program is to discover new compounds with potent biological activity (Che et al., 2013a; Che et al., 2015; Che et al., 2016; Xu, Che, Wang, 2011), we report here the results of the anti-HIV-1 activity of 32 N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives for the first time.

Results and Discussion

Chemistry

32 N-Arylsulfonyl-3-acylindole benzoyl hydrazone analogs (1-32,Figure 2) were smoothly synthesized as shown in Scheme 1. N-Arylsulfonyl-3-acetylindoles (A) reacted with the corresponding benzoyl hydrazides (B) in the presence of AcOH at reflux, and compounds 1-32 were prepared, as well as characterized by satisfactory proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), electrospray ion trap mass spectrometry (ESI-TRAP-MS), high-resolution mass spectra (HR-MS), as well as melting point (Che et al., 2013b). Owing to the steric hindrance, the substituents on the CN bond of all compounds adopted a trans configuration (E configuration) (Carvalho et al., 2014; Che et al., 2013b; Lourenço et al., 2008).

Scheme 1: Synthetic route for the preparation of N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives 1-32

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.

Moreover, target compounds 1-32 were analysed by reverse phase high-performance liquid chromatography (RP-HPLC), and all compounds purity were >95%.

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.

Table I: Anti-HIV-1 activity of n-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives 1-32 in vitro a

Compd R1 R2 R3 CC50 b(μg/mL) EC50 c(μg/mL) TId
1 H 4-Me H 89.93 11.02 8.16
2 H 4-OMe H 192.02 3.02 63.58
3 H 4-NHAc H 18.31 1.71 10.70
4 H 4-Cl H 53.15 9.92 5.36
5 H 3-NO2 H 27.09 4.06 6.67
6 H 3-NO2, 4-Cl H 1.77 59.99 0.03
7 6-Me 4-Me H 41.74 8.25 5.06
8 6-Me 4-OMe H 74.86 3.66 20.45
9 6-Me 4-Cl H 0.68 3.64 0.19
10 6-Me 3-NO2 H 118.66 1.02 116.33
11 5-CN 4-Me H 123.68 3.13 39.51
12 5-CN 3-NO2 H 21.47 3.13 6.85
13 5-NO2 4-Me H 73.09 20.82 3.51
14 H H Me >200 0.52 >384.61
15 H 4-Me Me 8.79 0.93 9.45
16 H 4-OMe Me 1.30 0.16 8.13
17 H 4-Cl Me >200 125.16 >1.60
18 H 3-NO2 Me >200 0.26 >769.23
19 6-Me H Me >200 0.49 >408.16
20 6-Me 4-Me Me >200 1.65 >121.21
21 6-Me 4-OMe Me >200 12.37 >16.16
22 6-Me 4-Cl Me 96.71 2.51 38.53
23 6-Me 3-NO2 Me >200 0.31 >645.16
24 5-CN H Me >200 0.67 >298.50
25 5-CN 4-Me Me >200 59.99 >3.33
26 5-CN 4-Cl Me 19.76 11.72 1.68
27 5-NO2 H Me 124.93 0.50 249.86
28 5-NO2 4-Me Me >200 2.55 >78.43
29 5-NO2 4-Cl Me >200 7.27 >27.51
30 H H Et >200 14.53 >13.76
31 H H n-pentyl >200 38.23 >5.23
32 H 4-Me n-pentyl >200 52.23 >3.83
AZTe \ \ \ 1139.47 0.00324 351688.3

a Values are means of two separate experiments (the values exhibited standard deviation (SD) less than ±5% from mean).

b CC50 (50% cytotoxic concentration), concentration of drug that causes 50% reduction in total C8166 cell number.

c EC50 (50% effective concentration), concentration of drug that reduces syncytia formation by 50%.

d In vitro therapeutic index (CC50 value/EC50 value).

e AZT was used as a positive control.

The results of anti-HIV-1 viral replication assay revealed that, out of thirty-two evaluated compounds, two (18 and 23) showed significant anti-HIV-1 activity (EC50 values of 0.26 and 0.31 μg/mL, TI values of >769.23 and >645.16, respectively), six (10, 14, 19, 20, 24 and 27) showed moderate anti-HIV-1 activity (EC50 values of 1.02, 0.52, 0.49, 1.65, 0.67 and 0.50 μg/mL, TI values of 116.33, >384.61, >408.16, >121.21, >298.50 and >249.86, respectively), while the rest of tested compounds displayed weak anti-HIV-1 activity (EC50 values of 1.71 to >125.16 µg/mL, except 16, the EC50 value of 0.16 μg/mL, and TI values of 0.03 to 78.43).

Figure 2: Chemical structures of N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives 1-32

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) R3 = Me was more vital for the anti-HIV-1 activities than R3 = H, Et, or n-pentyl. For example, when R1 and R2 were H, introduction of R3 as the methyl group could lead to the pronounced compound (14 vs 30 and 31, EC50 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 R1 = H or 6-Me and R2= 3-NO2, compounds with R3 = Me exhibited significant inhibitory potential against the HIV viral replication (18 vs 5, EC50 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, EC50 values of 0.31 and 1.02 μg/mL, TI values of >645.16 and 116.33, respectively). (3) R2 as the 3-nitro group also plays an important role in the activities against the HIV viral replication. For example, when R3 = H and R1= 6-Me, the corresponding compound 10 usually displayed more potent anti-HIV-1 activity (TI = 116.23); When R3 = Me and R1 = 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 R3 as the methyl group and R2 as the hydrogen group could result in more potent compounds. For example, the EC50 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 R3 as the methyl group could be taken into account for further preparation of these kinds of compounds as anti-HIV-1 agents.

Experimental Section

Compound purity assessment

The purity of N-arylsulfonyl-3-acylindole benzoyl hydrazone derivatives 1-32 were recorded on a Shimadzu LC-15C liquid chromatograph [SPD-15C UV-vis spectrophotometric detector (190-700 nm)] using a flow rate of 1.0 mL/min (MeOH/H2O = 5/1) and a Hypersil ODS C18 column (5 μm, 4.6 × 150 mm) as the stationary phase, and all compounds purity were >95%.

Anti-HIV-1 activity assay

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 (TCID50) in C8166 cells was determined and calculated by the Reed and Muench method. Virusstocks were stored in small aliquots at -70oC.

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 96-well microtiter plate in the absence or presence of various concentrations of compounds in triplicate and incubated at 37 oC in a humid atmosphere of 5% CO2 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% (CC50) was determined from the dose-response curve.

Syncytia assay

In the presence of 100 μL of various concentrations of compounds 1-32, C8166 cells (4×105/mL) were infected with HIV-1IIIB 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-1IIIB 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 (EC50) 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 CC50/EC50.

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. Among the reported analogues, compounds 10, 14, 18-20, 23, 24 and 27 exhibited significant anti-HIV-1 activity with EC50 values of 1.02, 0.52, 0.26, 0.49, 1.65, 0.31, 0.67 and 0.50 μg/mL, and TI values of 116.33, >384.61, >769.23, >408.16, >121.21, >645.16, >298.50 and >249.86, respectively. Especially N-(3-nitro)phenylsulfonyl-3-acetylindole benzoyl hydrazone (18) and N-(3-nitro)phenylsulfonyl-3-acetyl-6-methylindole benzoyl hydrazone (23) displayed the highest anti-HIV-1 activity.

Acknowledgements

The present research was supported by the National Natural Science Foundation of China (Grant No. U1604105), the Doctoral Scientific Research Fund Project of Henan University of Science and Technology (Grant No. 09001763) and Henan Province Natural Science Foundation (Grant No. 162300410079). We would like to acknowledge the MRC AIDS Research Project and the NIH AIDS Research and Reference Reagent Program for providing cell lines and viruses.

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Received: September 03, 2017; Accepted: March 01, 2018

*Correspondence: G. Q. Chen. Laboratory of Pharmaceutical Design & Synthesis, Department of Plant Protection, College of Forestry, Henan University of Science and Technology, Luoyang 471003, Henan Province, People′s Republic of China. Tel./fax: +86-0379-69978303. genqiangchen@126.com.

Conflict of Interest

The authors have reported no conflict of interest.

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