Identification of 2-(4-(Phenylsulfonyl)piperazine-1-yl)pyrimidine Analogues as Novel Inhibitors of Chikungunya Virus

The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, and it is the causative agent of chikungunya fever (CHIKF). Although it has re-emerged as an epidemic threat, so far there are neither vaccines nor pharmacotherapy available to prevent or treat an infection. Herein, we describe the synthesis and structure–activity relationship studies of a class of novel small molecule inhibitors against CHIKV and the discovery of a new potent inhibitor (compound 6a). The starting point of the optimization process was N-ethyl-6-methyl-2-(4-(4-fluorophenylsulfonyl)piperazine-1-yl)pyrimidine-4-amine (1) with an EC50 of 8.68 μM, a CC50 of 122 μM, and therefore a resulting selectivity index (SI) of 14.2. The optimized compound 6a, however, displays a much lower micromolar antiviral activity (EC50 value of 3.95 μM), considerably better cytotoxic liability (CC50 value of 260 μM) and consequently an improved SI of greater than 61. Therefore, we report the identification of a promising novel compound class that has the potential for further development of antiviral drugs against the CHIKV.

S3 effective concentration (EC90) are the concentrations of compound that inhibit virus replication by 50% and 90%, respectively. The overall antimetabolic effect of the compounds is shown as Cytostatic/Cytotoxic Concentration (CC50). The CC50 is the calculated concentration of compound that causes a 50% adverse effect on non-infected host cells (incorporates cytotoxic, cytostatic, and antimetabolic effect). All assay conditions producing an antiviral effect exceeding 50% were checked microscopically for minor signs of CPE or adverse effects on the host cell (i.e. altered cell morphology). A compound was only considered to elicit a selective antiviral effect on virus replication when, following microscopic quality control, at least at one concentration of the compound, no CPE nor any adverse effect is observed (image resembling untreated, uninfected cells). Multiple, independent experiments were performed.

Instrumental and General Analytical Methods
All reagents and solvents (of analytical and HPLC grades) were purchased from Sigma-Aldrich or other commercial suppliers like Fluka and Alfa Aesar and were used without further purification. Reactions that were moisture-sensitive were performed using anhydrous solvents and under argon atmosphere. EtOH, THF and DCM were dried before use. Disposable needles and syringes (B| Braun Inject and B| Braun Sterican®) were applied to transfer the solvents into the reaction flask. The solvents were removed under reduced pressure using the Heildolph Laborota 4000 efficient evaporator. Analytical thin-layer chromatography (TLC) was carried out on Polygram® SIL G/UV254 plates, layer 0.2 mm silica gel with fluorescent indicator. The spots were visualised by UV light (254 and/or 366nm) with UV light Vilmer Lourmat VL-6L. A part of the syntheses was performed in Cem Discover to obtain microwave conditions. Compounds were purified, performing a flash chromatography on a glass column using Merck silica gel (40−60 mesh). The solvent mixtures for chromatography are always referred to as a vol/vol ratio, and the melting points were determined on Cambridge Instruments. NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer (UltraShield) using a 5 mm switchable probe (TCI Prodigy Kryo-probe head, 5 mm, triple resonance-inverse-detection probe head) with z-axis gradients and automatic tuning and matching accessory (Bruker BioSpin).
The resonance frequency for 1 H NMR was 500.13 MHz and for 13 C NMR 125.75 MHz. All measurements were performed for a solution in fully deuterated dimethylsulfoxide at 298 K.
Standard 1D and gradient-enhanced 2D experiments, like double quantum filtered (DQF) COSY, HSQC, and HMBC, were used as supplied by the manufacturer. Chemical shifts are referenced internally to the residual, non-deuterated solvent signal 1H (δ 2.50 ppm) and the carbon signal 13C (δ 39.50 ppm) of dimethylsulfoxide. 19 F NMR spectra were recorded on a Bruker Avance III 400 S5 NMR spectrometer (UltraShield) using a 5 mm switchable probe (BBFOPLUS, BB/19F -1H/D) with z-axis gradients and automatic tuning and matching accessory (Bruker BioSpin). The resonance frequency for 1 H NMR was 400.23 MHz and for 19 F NMR 376.55 MHz. All measurements were performed for a solution in fully deuterated dimethylsulfoxide at 298 K. 19 F NMR spectra (broadband decoupled for 1H) were measured as supplied by the manufacturer using absolute referencing via Ξ ratio. Chemical shifts are given in ppm and are reported relative to TMS and referenced to the residual proton signal of d6-DMSO (2.49 ppm). d6-DMSO with 0.03% TMS (V/V), purchased at Euriso-top®, was used as a solvent for the samples. The specified abbreviations were used to characterize the signals: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, and br = broad signal. The spectra were analysed by a computer using the software MestReNova 6 (Mestrelab research, 1994).
HRESIMS spectra were obtained on a maXis HD ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Samples were dissolved to 20 µg/mL in MeOH and directly infused into the ESI source at a flow rate of 3 µL/min with a syringe pump. The ESI ion source was operated as follows: capillary voltage: 0.9 to 4.0 kV (individually optimised), nebulizer: 0.4 bar (N2), dry gas flow: 4 L/min (N2), and dry temperature: 200 °C. Mass spectra were recorded in the range of m/z 50 -1550 in the positive-ion mode. The sum formulas were determined using Bruker Compass DataAnalysis 4.2 based on the mass accuracy (Δm/z ≤ 2 ppm) and isotopic pattern matching (SmartFormula algorithm).
The purity of the compounds was determined by HPLC on LC-2010A HT Liquid Chromatograph device (Shimadzu Corporation, Tokyo, Japan). The separation was carried out on an Acclaim 120 C18, 2.1 x 150 mm, 3 µm HPLC column (Thermo Fisher Scientific) using LC-MS-grade water with 0.1% FA and acetonitrile with 0.1% FA as mobile phase A and B, S6 respectively. The sample components were separated and eluted with a linear gradient from 5% to 95% B in 30 min, followed by an isocratic column cleaning and re-equilibration step. The flow rate was 0.1 mL/min, and the column oven temperature was set to 25 °C. The purity was determined from the UV chromatogram (254 nm) as the ratio of the peak area of the compound to the total peak area (i.e., the sum of the areas of all peaks that were not present in the solvent blank).
Based on the HPLC data, all final compounds are ≥95% pure.

General Procedures
General Procedure A: Amine alkylation reaction for the synthesis of pyrimidine amines 2a-2j.
A stirred solution of pyrimidine (1 equiv) in anhydrous ethanol under an inert atmosphere was cooled to 0 °C before amine (2 equiv) was added dropwise. The reaction mixture was brought to room temperature and then stirred for 48 hours. After evaporation of the solvent, the residue was dissolved in dichloromethane and washed twice with water and with brine. The organic layer was dried over sodium sulphate, and then evaporated to dryness. The crude product was purified by column chromatography (SiO2, eluent: hexane/EtOAc or DCM/MeOH) to afford the desired pyrimidine amines.
After the solvent was evaporated, the resulting crude product was dissolved in dichloromethane and washed twice with K2CO3 solution (10% in water) and with water. The organic layer was dried over sodium sulphate, filtered, and then evaporated to dryness. The crude was purified by column chromatography (SiO2, eluent: 80% dichloromethane/20% methanol), to obtain the pure product.

S7
General Procedure C: Boc-deprotection reaction of compounds 4a-4b and 4d-4m. Carboxylate (1 equiv) was dissolved in dry tetrahydrofurane and stirred at room temperature, then the hydrochloric acid solution (4.0 M in dioxane, 10 equiv) was added dropwise. The mixture was stirred for 24 hours and evaporated to dryness. The residuum was dissolved in dichloromethane, washed twice with an aqueous solution of potassium carbonate (10%) and with water. The organic phase was dried over sodium sulphate, and filtered to obtain the desired product.
General Procedure D: Sulfonacylation reaction of nitrogen heterocycles. The pyrimidine (1 equiv) was dissolved in DCM. Sulfonyl chloride (1 equiv) and triethylamine (1 equiv) as a base was added and stirred overnight at room temperature. The mixture was diluted in DCM and washed twice with H2O and with brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuum. The crude product was purified by recrystallization (DIPE/EtOAc; DIPE/EtOH or Hexane/EtOAc) or by column chromatography (SiO2, eluent: DCM/MeOH).
Compound 1 was prepared following general procedure D using 4a (