Design, Synthesis, Antiviral Evaluation, and SAR Studies of New 1-(Phenylsulfonyl)-1H-Pyrazol−4-yl-Methylaniline Derivatives

A series of N-((3-phenyl-1-(phenylsulfonyl)-1H-pyrazol-4-yl)methyl)anilines 7a-p and 8a-l, structurally related to previously synthesized and tested (N-(1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines (1a-v), were designed and synthesized. The new derivatives were evaluated in cell-based assays for their cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses of public health significance. Generally, the tested compounds did not display cytotoxicity toward the cell lines used. The majority of derivatives 7a-p were able to interfered with YFV and RSV replication in the micromolar range showing a marked improvement in potency and selectivity with respect to the reference inhibitors 6-azauridine and ribavirin, respectively. The introduction of a p-methoxy substituent on the phenylsulfonyl group (compounds 8a-l) completely abolished the anti-RSV activity and reduced or eliminated the potency against YFV. On the contrary, several p-methoxy analogs were able to interfere with BVDV replication with a comparable (8b, 8c, 8g, and 8k) or better (8a and 8f) potency than the reference inhibitor, ribavirin. Compound 7e, selected for time of addition experiments on BHK-21 cell cultures infected with YFV, achieved the highest reduction of virus titer when added 2 h post infection and maintained up to 4 h post infection.

The genera Flavivirus and Hepacivirus include several human pathogenic viruses of global medical importance. Within the approximately 70 species of the genus Flavivirus, Yellow Fever Virus (YFV), West-Nile Virus (WNV), Dengue Virus (DENV), Japanese Encephalitis Virus (JEV), Tick-Borne Encephalitis Virus (TBEV), and Zika virus (ZIKV) are arthropod-borne emerging or reemerging pathogens. Flavivirus infections can result in diseases ranging from a "flu-like" illness with fever and general malaise, to sever and potentially fatal disease including hemorrhagic fever, jaundice, seizures, or fatal encephalitis (Sips et al., 2012;Holbrook, 2017). The Hepacivirus genus includes only the Hepatitis C Virus (HCV), a major cause of human hepatitis, worldwide. HCV can cause both acute and chronic infection. Acute HCV infection is usually asymptomatic and often undiagnosed. However, more than 60% of infected person will develop chronic HCV infection with risk of liver cirrhosis or hepatocellular carcinoma. Recently, effective, safer and well-tolerated new anti-HCV drugs have been developed (Li and De Clercq, 2017;Zajac et al., 2019). On the contrary, no effective antiviral therapy is currently available to fight Flavivirus infections. Although human vaccines are available for YFV, JEV, TBEV, and recently DENV, their use is lacking in many areas and outbreaks of Flavivirus infections still occur, with a significant mortality rate (Deen, 2016;Collins and Metz, 2017). Therefore, the development of effective drugs for the treatment of Flavivirus infections is urgently needed.
Viruses belonging to the Pestivirus genus comprise animal pathogens producing heavy economic losses for the livestock industry. The type specie Bovine Viral Diarrhea Virus (BVDV) together with Border Disease Virus (BDV) of sheep and Classical Swine Fever Virus (CSFV) are responsible of a range of clinical manifestations including respiratory problems, chronic wasting disease, immunosuppression leading to a higher susceptibility to secondary infections, abortion and teratogenicity. Despite the morbidity and even mortality caused by Pestivirus infections, no approved antiviral therapy is currently available (Yeşilbag et al., 2017).
In this paper, we report the design and synthesis of novel 1-(phenylsulfonyl)-1H-pyrazol-4-yl-methylaniline derivatives (7a-p and 8a-l), and their evaluation in cell-based assays for cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses.

Antiviral Tests
All the new synthesized pyrazole derivatives (7a-p and 8a-l) and reference inhibitors were initially tested in cell based assays for their cytotoxicity and antiviral activity against YFV and BVDV, representative of the Flavivirus and the Pestivirus genus, respectively, within the Flaviviridae family ( Table 2). Their efficacy was also initially evaluated against RSV, a single-stranded, negative RNA virus (ssRNA − ) belonging to Paramyxoviridae family ( Table 2). The previously studied N-((1,3-diphenyl-1Hpyrazol-4-yl)methyl)anilines and the new derivatives able to inhibit YFV and/or BVDV replication were also evaluated against two additional pathogenic viruses belonging to the Flavivirus genus, DENV-2 and WNV (Tables 1, 2). All the new compounds were further assayed against representative members of a large panel of virus families. Among ssRNA + viruses, a retrovirus (Human Immunodeficiency Virus type-1, HIV-1), and two Picornaviruses (Coxsackie Virus type-5, CVB-5, and Poliovirus type-1, Sabin strain, Sb-1) were also considered. Among ssRNA − viruses, in addition to RSV, a Rhabdoviridae family member (Vesicular Stomatitis Virus, VSV) was selected. Among doublestranded RNA (dsRNA) viruses, a reovirus (Reo-1, Reoviridae) was included. Finally, among DNA viruses, Herpes Simplex Virus type-1 (HSV-1, Herpesviridae), and Vaccinia Virus (VV, Poxviridae) were involved ( Table 3).
Moreover, despite the presence of a p-methoxy substituent on the phenylsulfonyl group was required for a potent antiflavivirus activity in SID compounds (Johnston et al., 2007;Sidique et al., 2009), the new p-methoxy phenylsulfonyl analogs 8a-l were totally inactive or less potent YFV inhibitors than the corresponding unsubstituted compounds 7e-p. Conversely, the introduction of a p-methoxy substituent generally converted the inactive or poor effective unsubstituted analogs 7e-p in the more potent anti-BVDV agents 8a-l. In particular, 8a was the best anti-BVDV compound among the new derivatives (EC 50 = 5.6 µM, SI > 17.9) showing almost a 3-fold improvement in potency and a 5-fold improvement in selectivity with respect to the reference inhibitor, ribavirin (EC 50 = 16.0 µM, SI = 3.4) ( Table 2).

Time of Addition Studies
Due to its potency against YFV (EC 50 = 3.6 µM) and low cytotoxicity against BHK-21 cell cultures (CC 50 > 100 µM), compound 7e were selected for time of addition studies. In order to determine the possible step(s) in YFV replication cycle inhibited by the derivative 7e, time of addition experiments were performed on infected BHK-21 cells under a YFV single cycle conditions. 6-Azauridine, a nucleoside analog inhibitor of the orotidine monophosphate (OMP) decarboxylase, was used as reference inhibitor. This enzyme is essential in the biosynthesis of pyrimidine nucleotides, as it converts the OMP in uridine monophosphate (UMP) (Neyts et al., 1996).  Figure 2 showed that both pyrazole 7e and 6-azauridine reduced the YFV titer when present in the pre-treatment or during the 2 h of infection. However, 6azauridine lost its effectiveness when added after the infection, while 7e retained its inhibitory activity when added at any time p.i. In particular, the highest reduction of virus yield was observed when 7e was added 2 h post infection and maintained up to 4 h post infection. These data suggest that 7e interferes with different steps of YFV replication starting from the virus binding to host cell membrane but also covering some subsequent phase of the virus replication. However, further studies are necessary to identify the antiviral target(s) of this pyrazole derivative.

CONCLUSIONS
In order to improve the anti-Flaviviridae activity of previously studied (N-(1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines (1av), a series of N-((3-phenyl-1-(phenylsulfonyl)-1H-pyrazol-4yl)methyl)anilines (7a-p and 8a-l) was designed, synthesized and assayed against a large panel of viruses belonging to Flaviviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae, Retroviridae, Herpesviridae, and Poxviridae families. SAR studies showed that the analogs unsubstituted in R 1 (7a-p) were generally potent and high selective YFV inhibitors (EC 50 ranging from 3.6 to 11.5 µM and SI ranging from >27.8 to >8.7). The analogs 7e-p also interfered with RSV replication in the micromolar concentrations (EC 50 ranging from 8.5 µM to 24.0 µM) providing an improvement in potency and selectivity with respect to the reference inhibitor ribavirin, the first drug licensed for the treatment of RSV infection. On the contrary, the introduction of a methoxy group in R 1 resulted in compounds that preferentially affected BVDV replication. Among these            Compound concentration required to reduce the viability of mock-infected VERO76 (monkey normal kidney) monolayers by 50% after contact with the cells for 2 days for Sb-1 and VSV, and 3 days for CVB-5, VV and HSV-1. Frontiers in Chemistry | www.frontiersin.org derivatives, 8a was the most potent and selective inhibitor of BVDV replication (EC 50 = 5.6 µM, SI > 17.9).
In conclusion the results of this work allowed the selection of a new generation of hits for the development of anti-YFV, -BVDV, and -RSV agents.

Chemistry
Solvents and reagents were purchased from Sigma-Aldrich or Alfa Aesar and were used without further purification. The progress of reactions was routinely checked by thinlayer chromatography (TLC). TLC was performed on silica gel or aluminum oxide fluorescent coated plates (Fluka, DC-Alufolien Kieselgel or aluminum oxide F 254 ). Melting points were determined on a Stuar Scientific SMP1 apparatus and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded on a Bruker AM-400 spectrometer in CDCl 3 or DMSO-d 6 , and chemical shifts were reported in ppm (δ) (Supplementary Figures S1-S31).
General Procedure for the Synthesis of the 3-Phenyl-1H-Pyrazol-4-Carbaldeydes (3a-d) To a solution of the suitable (E)-2-(1-phenylethylidene) hydrazinecarboxamides 2a-d (12 mmol) in dry DMF (9 ml) cooled in ice bath, POCl 3 (2 ml) was added dropwise. The mixture was stirred in ice bath for 30 min and then heated at 65 • C for 6 h. After cooling and stirring at room temperature for 18 h, the mixture was diluted with water and ice, neutralized with NaOH 2N and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and evaporated to dryness. The residue was purified by crystallization from CHCl 3 /n-Hexane. Intermediate 3c was purified by column chromatography on silica gel eluting with a mixture of AcOEt and n-Hexane 1:1.