Non-nucleoside Inhibitors of the Measles Virus RNA-dependent RNA Polymerase: Synthesis, Structure-Activity Relationships and Pharmacokinetics

Abstract

The measles virus (MeV), a member of the paramyxovirus family, is an important cause of pediatric morbidity and mortality worldwide. In an effort to provide therapeutic treatments for improved measles management, we previously identified a small, non-nucleoside organic inhibitor of the viral RNA-dependent RNA polymerase (RdRp) by means of high-throughput screening (HTS). Subsequent structure-activity relationship (SAR) studies around the corresponding pyrazole carboxamide scaffold led to the discovery of 2 (AS-136a), a first generation lead with low nanomolar potency against life MeV and attractive physical properties suitable for development. However, its poor water solubility and low oral bioavailability (F) in the rat suggested that the lead could benefit from further SAR studies to improve the biophysical characteristics of the compound. Optimization of in vitro potency and aqueous solubility led to the discovery of 2o (ERDRP-00519), a potent inhibitor of MeV (EC₅₀ = 60 nM) with aqueous solubility of approximately 60 μg/ml. The agent shows a 10-fold exposure (AUC/Cmax) increase in the rat model relative to 2, displays near dose proportionality in the range of 10 mg/kg to 50 mg/kg, and exhibits good oral bioavailability (F = 39%) in the rat. The significant solubility increase appears linked to the improved oral bioavailability.

INTRODUCTION

The paramyxoviruses family comprised non-segmented, negative strand RNA viruses that are primarily responsible for acute respiratory diseases. The family includes major human and animal pathogens such as measles virus (MeV), human parainfluenza virus (HPIV), mumps virus, respiratory syncytial virus (RSV) and the Newcastle disease virus. Despite the existence of an effective vaccine protecting against MeV infection, we have witnessed in the recent past an increasing number of cases particularly in the developed world.^1,2 For example, in the United States from January 1 through May 21 of 2011, 118 cases were reported across 23 states according to the CDC. Recently, in Ashland, Oregon, 25-30% of children entering kindergarten were unvaccinated.³ This has been attributed to elected exemption from vaccination on the basis of philosophical or religious beliefs. Vaccination rates in Europe in recent years have never fully recovered from a discredited 1998 British study linking the vaccine for measles, mumps and rubella to autism. At that time, parents, particularly in the U.K. abandoned the vaccine followed by precipitous drops in vaccination rates. For 2011, the World Health Organization reported 4,937 cases of measles between January and March in France alone, compared with 5,090 cases during all of 2010. The World Health Organization reports that as of October, there have been 26,000 measles cases, and nine deaths, in Europe since the start of 2011, rendering it the worst year for MeV activity in the Western World since 1996.⁴

Measles is not currently treatable by drug therapy. Ribavirin, a nucleoside based anti-viral agent, is the only small molecule drug approved for paramyxoviruses (RSV) therapy. ^{5, 6} However, efficacy is limited. To improve case management of severe measles and achieve rapid control of outbreaks through post-exposure prophylaxis, the development of an effective anti-measles drug is highly desirable.⁷ We previously reported the discovery of an MeV inhibitor targeting the viral RNA dependent RNA polymerase (RdRp) complex by means of a cell-based high-throughput screening (HTS).^8,9 Iterative optimization of a corresponding series of pyrazole carboxamides, exemplified by hit 1 (16677), led to the first-generation lead molecule 2 (AS-136a) (Figure 1).^10,11 The latter piperidine derivative exhibits superior in vitro cellular potency against MeV with nanomolar EC₅₀ concentrations. It was also subjected to a number of in vitro toxicity and metabolism assays. There, the compound was found to be non-mutagenic in a non-GLP in vitro bacterial reverse mutation (Ames) assay, and it did not block hERG channels at a concentration of 10 μM or below. Compound 2 shows moderate metabolic stability in mouse and human S9 fractions after one-hour incubation with 79% and 69% parent remaining, respectively. However, poor solubility and low rat plasma concentrations of 2 might hamper its in vivo efficacy. In an effort to improve pharmacological properties of 2, in particular water solubility, we initiated a structure activity relationship (SAR) study to identify a suitable solubilizing group. Earlier efforts had shown that the piperidine ring is amenable to chemical manipulation without adversely affecting activity. However, any changes to the central ring or the pyrazole group of 2 are detrimental to activity.¹¹ Consequently, the present study focuses on appending a solubilizing group to the piperidine ring or replacing it with either a substituted phenyl or an alicyclic group. This led to the identification of compound 2o (ERDRP-00519, Figure 1), which has significantly improved water solubility, while retaining high antiviral potency. The agent shows a 10-fold exposure (AUC/Cmax) increase in rat relative to 2 and displays near dose proportionality in the range of 10 mg/kg to 50 mg/kg. The significant solubility increase appears to contribute to the improvement in oral bioavailability. We describe herein the synthesis and a structure-activity relationship (SAR) strategy that led to the discovery of 2o as well as the pharmacokinetic comparison of first and second-generation lead candidates.

Figure 1.

Structures of hit and lead compounds.

CHEMISTRY

Synthesis of Substituted Piperidine Analogs

Our previous work showed that introduction of a piperidine moiety resulted in compounds that were about 10 times more active than the corresponding pyrrolidine analogs.¹⁰ Accordingly, linkers were installed at the 2-, 3- and 4-positions of the pyrrolidine ring to explore which position could best accommodate hydrophilic substituents while maintaining potency. Reaction of different amino alcohols (4a-c) with 4-nitrobenzene sulfonyl chloride (5) followed by formation of methoxymethyl (MOM) ethers and reduction of the nitro group afforded anilines 7a-c. Coupling of acid chloride 8, derived from 3-trifluoromethyl pyrazole using the method of Lahm,¹² with anilines 7a-c provided analogs 1a-c (Scheme 1). With preliminary data showing the 2-position of the piperidine to yield more active compounds compared to the 3- or 4-position (Table 1), additional analogs of the previously reported 2-piperidinemethanol compound 2a¹³ were prepared by a sequence similar to that depicted in Scheme 1.

Scheme 1.

Exploring the optimal substitution position on the piperidine ring^a

^aReagents and conditions: (a) Na₂CO₃, acetone; (b) MOMCl, i-Pr₂NEt, CH₂Cl₂; (c) SnCl₂. 2H₂O, EtOAc; (d) (8), Pyridine, CH₂Cl₂; (e) H2 (50 psi), Pd-C, MeOH.

Table 1.

MeV antiviral action (CPE) of the piperidine series of analogs, (EC₅₀).

Comp.	R	EC50 (μM)a (MV-Alaska) (CPE inhib.)	Comp.	R	EC50 (μM)a (MV-Alaska) (CPE inhib.)
2	−H	2.0	2h		3.7
1a		1.5	2i		6.7
1b		3.8	2j		2.7
1c		16.0	2k		2.7
2a		2.8	2l		8.3
2b		9.3	2m		3.1
2c		1.5	2n		4.6
2d		55.0	2o		2.5
2e		14.0	2p		25.0
2f		>150.0	2q		8.3
2g		6.8

^avalues represent averages of four experiments; highest concentration assessed 150 μM

Further analogs were prepared by PCC oxidation of 6a to obtain aldehyde 14, which was subjected to reductive amination with morpholine followed by the procedures illustrated in Schemes 2 to ultimately give analog 2b. Tosylation of 6a, reduction of the nitro group, coupling with acid chloride 8 and displacement of the tosylate with an azide furnished 2c. Reduction of the azide, dimethylation of the resultant amine or acylation resulted in compounds 2d-f. Further extension of the side chain including both saturated and unsaturated derivatives could be achieved from aldehyde 14. Horner-Wadsworth-Emmons olefination of 14 gave 12. Union of 12 with acid chloride 8 afforded analog 2g, which was then reduced with DIBAL-H to obtain analog 2h. Hydrogenation of 2g delivered the saturated analog 2i, which was converted to 2j by treatment with DIBAL-H (Scheme 2).

Scheme 2.

Synthesis of three-carbon substituents at the piperidine C-2 position^a

^aReagents and conditions: (a) PCC, CH₂Cl₂; (b) morpholine, NaBH(OAc)₃, CH₂Cl₂; (c)SnCl₂.2H₂O, CH₂Cl₂/MeOH; (d) 8, i-Pr₂NEt, CH₂Cl₂; (e) 4-Toluenesulfonyl chloride, CH₂Cl₂; (f) NaN₃, DMF, 120 °C; (g) H₂, Pd/C, MeOH; (h) AcCl, i-Pr₂NEt, CH₂Cl₂; (i) CH₃I, K₂CO₃, DMF; (j) ) t-BuOK, Et₂P(O)CH₂COOEt, THF/CH₂Cl₂; (k) SnCl₂.2H₂O, EtOAc; (l) DIBAL-H, THF;

Preparation of two-carbon side chain analogs was accomplished by utilizing 2-(2-piperidinyl) ethanol 9. Direct coupling of the latter with p-nitro-benzenesulfonyl chloride 5 gave low yields of the desired product due to further coupling of the product with the sulfonyl chloride. To circumvent this shortcoming, the NH- and OH- groups of 9 were protected using benzyl chloroformate¹⁴ and t-butyldimethylsilyl chloride (TBSCl), respectively. De-protection of the amine, coupling with 5 and reduction of the nitro group afforded aniline 11. Coupling of 11 with acid chloride 8 followed by cleavage of the silyl group furnished alcohol 2k which, when subjected to Swern oxidation and reductive amination with morpholine, gave 2n (Scheme 3). Chiral pure enantiomer 2o was then prepared similar to 2n starting from (S)-2-piperidine ethanol.

Scheme 3.

Introduction of a two-carbon tether at the piperidine C-2 position^a

^aReagents and conditions: (a) Na₂CO₃, BzOCOCl, H2O/acetone; (b) TBSCl, imidazole, DMF; (c) H₂, Pd/C, ethanol; (d) 5, i-Pr₂NEt, CH₂Cl₂; (e) H₂, Pd/C, ethanol, 40 psi; (f) 8, i-Pr₂NEt, CH₂Cl₂; (g) TBAF, THF; (h) (COCl)₂, DMSO, CH₂Cl₂; (i) morpholine, NaBH(OAc)₃, CH₂Cl₂.

We hypothesized that attaching an ethylene glycol moiety would give compounds with better aqueous solubility. Due to the instability of 6a under basic conditions, the synthesis of 2p was initiated by addition of a rhodium carbenoid across the hydroxylic bond^15,16 to form an ether bond. Thus, decomposition of ethyl diazoacetate in the presence of Rh₂OAc₄ generated a carbenoid that inserted into the OH bond to give 13. Reduction of the nitro group of 13 followed by coupling with 8 afforded analog 2p, which on hydrolysis of the ester and BOP/NaBH₄¹⁷ mediated reduction of the resultant carboxylic acid, provided 2q (Scheme 4).

Scheme 4.

Synthesis of O-alkylated analogs^a

^aReagents and conditions: (a) ethyl diazoacetate, Rh₂OAc₄, CH₂Cl₂; (b) H₂, Pd/C, MeOH; (c) 8, i-Pr₂NEt, CH₂Cl_2; (d) NaOH, THF/H₂O; (e) BOP, i-Pr₂NEt, THF, NaBH₄.

Synthesis of the Phenyl Series

Replacement of the piperidine ring with phenyl or substituted phenyl via the general route shown in Scheme 5 was also explored. Unsubstituted phenyl analog 3a was found to be as active as lead compound 2 triggering an SAR study of the series (Table 2). Coupling of 2-methoxylthiophenol 16a with 1-fluoro-4-nitrobenzene¹⁸ followed by oxidation of sulfur using MCPBA gave corresponding sulfone, which went through reduction of the nitro group and followed by coupling with acid chloride 8 furnished analog 3b. Demethylation of 3b with BBr₃ afforded phenol analog 3c, which on acylation gave analog 3d. Similarly, coupling of 2-bromothiophenol 16b with 1-fluoro-4-nitrobenzene obtained 17. To make additional analogs of the phenyl series, we envisioned utilizing bromide 17 to append substituents. However, attempts to lithiate bromide 17 using n-BuLi or t-BuLi were unfruitful resulting in decomposition of the bromide. Stille coupling offered an alternative. When 17 was treated with tributyl(vinyl)tin in the presence of Pd(PPh₃)₄, the desired coupling product 18 was obtained in 80% yield. Reduction of the nitro group followed by coupling with acid chloride 8 afforded analog 3e (Scheme 5 and Table 2). Subjecting olefin 18 to osmium tetroxide-mediated oxidative cleavage of the double bond gave aldehyde 19, a compound utilized in the synthesis of additional analogs. Reduction of the aldehyde, SnCl₂ reduction of the nitro group and protection of the alcohol as a silyl ether gave aniline 20. Coupling of 20 with acid chloride 8 followed by cleavage of the silyl group furnished analog 3f. Aldehyde 19 was also used for the synthesis of morpholine 3g by means of reductive-amination, followed by reduction of the nitro group and coupling with acid chloride 8 (Scheme 5).

Scheme 5.

Synthesis of the phenyl series^a

^aReagents and conditions: (a) 1-fluoro-4-nitrobenzene, Na₂CO₃, EtOH, 80°C; (b) m-CPBA, CH₂Cl₂; (c) tributyl(vinyl)tin, Pd(PPh₃)₄, THF, 80°C; (d)OsO₄, NaIO₄, THF/H₂O; (e) DIBAL-H, THF; (f) SnCl₂.2H₂O, CH₂Cl₂/MeOH; (g) TBSCl, imidazole, DMF; (h) 8, i-Pr₂NEt, CH₂Cl₂; (i) TBAF, THF ; (j) BBr₃, CH₂Cl₂; (k) CH₃COCl, THF; (l) morpholine, NaBH(OAc)₃, CH₂Cl₂.

Table 2.

MeV antiviral action (CPE) of the phenyl and acyclic series of analogs, (EC₅₀).

Comp.	R2	EC50 (μM)a (MV-Alaska) (CPE inhib.)	Comp.	R2	EC50 (μM)a (MV-Alaska) (CPE inhib.)
3a	-H	2.8	3e		> 50.0
3b	-OMe	3.1	3f		3.5
3c	-OH	4.5	3g		>75.0
3d	-OAc	4.5

^avalues represent averages of four experiments; highest concentration assessed 75 μM

Single Dose Antiviral Activity of Analogs of 2

In order to better understand the potency profile of compound 2 analogs, the most active analogs were subjected to a measles virus yield assay at a single concentration of 1.0 μM to generate data points for comparison with 2.

RESULTS AND DISCUSSION

The SAR data are summarized in Tables 1 and 2 for the piperidine and phenyl series, respectively. From previous experience, we have learned the necessity of preserving the structure of the phenyl, amide and fluorinated pyrazole units of the molecule in order to maintain anti-viral potency. Modification of either the 3-trifluoromethyl-pyrazole or the central phenyl ring in most cases leads to significant loss of activity.^10,11 All analogs listed in Tables 1 and 2 incorporate only variations on the left side of lead molecule 1. The MOM ether analogs (1a-c) demonstrate a trend whereby substitution at C-2 of the piperidine is favored. The 2-piperidine 1a is 2-fold more potent than the corresponding 3-piperidine, while the 4-substituted derivatives reduce activity by almost 10-fold (1a, 1b and 1c, Table 1). For compounds with a hydroxyl group, elongation of the pendant chain from one carbon to two does not adversely affect potency as exemplified by compounds 2a and 2k.

Further extension to three carbons leads to a decrease in activity by 3-fold (2j, Table 1). Introduction of basic amines led to significant reduction or complete loss of activity (2d and 2f, EC₅₀ = 55.0 and >150 μM, respectively). Replacement of the amino groups with a less basic morpholine (2b and 2n) restored good potency. Esters 2g and 2i were found to be 2-fold less active by comparison with the corresponding alcohols (2h and 2j, Table 1). There is a clear superiority of S-chirality over R- as demonstrated by the 3-fold loss of activity for 2l compared to 2m. For the phenyl series, analog 3a is as active as the lead compound in reducing virus-induced cytopathicity, and its activity is comparable to that of methoxy 3b and alcohol 3f (Table 3). However, the morpholine analog 3g loses activity completely, which stands in significant contrast to alterations in the piperidine series (2b and 2n). The previous SAR and that derived from the current three series of MeV-RdRp inhibitors suggests a highly hydrophobic environment on the target protein housing the left part of the molecules, strongly disfavoring hydrogen bonding. To explore whether poor aqueous solubility contributes to the low oral bioavailability that was observed with the existing lead 2, we measured the aqueous solubility for some of the more potent derivatives via nephylometry (buffer, pH = 7.4, Table 3). 2 and phenyl analog 3a show equally poor solubility with values at 15 μg/ml and 22 μg/ml, respectively. The alcohol analogs 2a and 2k both deliver improved solubility as expected with measured values at 61 and 62 μg/ml, respectively. Importantly, the morpholine analog 2n also furnishes similar solubility compared with the corresponding free alcohol derivative 2k. Compounds with moderate solubility (~60 μg/ml) and good potency (< 3.0 μM) in the CPE assay were advanced to assessment of virus yield reduction. The primary alcohol derivative 2k (EC₅₀ 2.7 μM, CPE assay; solubility 62 μg/ml) delivers an EC₅₀ of 100 nM in this assay (2k; Table 3). Optically pure analogs of compound 2k, 2l and 2m, both delivered slightly decreased potency (EC₅₀ 8.3 and 3.1 μM, respectively, CPE assay). Replacement of the hydroxyl group with morpholine led to racemate 2n with an EC₅₀ of 4.6 μM, while the corresponding optically pure analog 2o provided an EC₅₀ of 2.5 μM in the CPE assay, 60 nM in the virus yield reduction assay and solubility around 60 μg/ml (2o; Table 3).

Table 3.

Aqueous solubility, virus yields (EC₅₀) and toxicity (CC₅₀) for selected compounds.

	Solubility	EC50 (μM) (MV-Alaska)		CC50(μM)
Comp.	(μg/ml) Testa	CPE inhibit.b	virus titer reductionc	(MTT cytotox)d
2	<15	2.0	0.014	>75
2a	61	2.8	0.85	>75
2k	62	2.7	0.1	>75
2n	55	4.6	nd	>75
2o	60	2.5	0.06	>75
3a	22	2.8	0.09	>75
3b	<15	3.1	nd	>75
3c	67	4.5	nd	75
3f	46	3.5	nd	>75

^asolubility data generated through Nephelometer using standard procedure.

^bValues represent averages of four experiments; highest concentration assessed 75 μM, lowest concentration assessed 2.0 μM.

^cDetermined only when CPE inhibition-based EC50 concentration < 3.0 μM.

^dValues represent averages of at least three experiments; highest concentration assessed 75 μM.

Considering the advanced potencies of 2k and 2o in the virus yield reduction assay (EC₅₀ = 100 and 60 nM, respectively), we selected these two compounds for comparison with 2 in a pharmacokinetic (PK) study in Sprague-Dawley rats.

PHARMACOKINETIC PROFILES

Figure 3 shows oral pharmacokinetic parameters of compounds 2k and 2o in comparison with the first generation lead 2; a summary of the numerical PK analysis is provided in Table 4. Compound 2o shows a 10-fold exposure (with respect to both AUC and Cmax) increase in the rat model relative to 2 and displays good dose proportionality in the range of 10 mg/kg to 50 mg/kg. In contrast, the primary alcohol analog 2k reveals a good Cmax and AUC at 50 mg/kg dosing, but it generates poor plasma concentrations in rat and non- proportionality possibly due to high first-pass metabolism of the primary alcohol. On the basis of its high in vitro potency, good solubility and pharmacokinetic profile, the oral bioavailability of compound 2o was assessed. The compound was dosed at 2 mg/kg i.v. and 10 mg/kg p.o. in rat and exhibits good oral bioavailability (F = 39 %) (Figure S1 and Table S1). In the Caco-2 bi-directional permeability assay, both 2 and 2o showed high permeability with an efflux ratio of 1.1 and 2.6, respectively, which indicates that they are probably not a substrate for p-glycoprotein in humans. (Figure S2)^{19, 20} However, compound 2o proved to be less stable in human liver S9 fractions after one hour incubation. Only 24% of the parent remains as compared with 69% for compound 2.

Figure 3.

Time course of rat plasma concentration following p.o. dosing by oral gavage. Preliminary pharmacokinetic (PK) studies in the Sprague-Dawley rat compared 2 with compounds 2k and 2o following p.o. dosing by oral gavage at 10 mg/kg and 50 mg/kg in a PEG200/0.5% methylcellulose (10/90) vehicle (n=4/group)

Table 4.

PK Profile for Compounds 2, 2k and 2o

Comp.	oral dose (mg/kg)a	Tmax (hr)	Cmax (ng/mL)b	T½ (hr)b	AUC (0-t) (hr*ng/mL)b	AUC (0-∞) (hr*ng/mL)b
2	10	2.5	26.9	12.7	132	513
2	50	2.7	72.2	3.7	308	483
2k	10	1	19.8	0.8	56.3	56.8
2k	50	0.5	184	2.7	754	973
2o	10	1.1	195	2.2	683	818
2o	50	1.5	823	6.5	3521	7860

^aStudy in Sprague-Dawley rat dosed at 10 mg/kg and 50 mg/kg as a suspension in PEG200/0.5% methylcellulose (10/90) formulation, respectively. n = 4 animals per study.

^bEstimation of PK parameters by non-compartmental analysis of these data, which was accomplished using standard PK software (WinNonlin 5.3, Pharsight®).

Mechanism of Action of 2o

We previously demonstrated that compound 2 blocks MeV RdRp activity by targeting the viral polymerase (L) protein.¹¹ To test whether this mechanism of activity likewise extends to lead molecule 2o, a plasmid-based mini-replicon assay²¹ was employed to assess RdRp activity in the presence of 2o and 2, respectively. BSR-T7/5 cells were transfected with plasmid DNA encoding MeV-L, N, P and the firefly luciferse mini-genome reporter construct, and the cell were incubated in the presence of different inhibitor concentrations or vehicle for control. Relative luciferase activities in cell lysates were assessed 36 hours post-transfection and dose-response inhibition curves generated. For both compounds, we observed a dose-dependent inhibition of viral RdRp activity with virtually identical potency (Figure 4), supporting comparable mechanism of antiviral activity.

Figure 4.

Compounds 2o and 2 inhibit viral RdRp activity with equal potency. Values are expressed relative to vehicle-treated samples and represent averages of three experiments± SD.

SUMMARY

Modification and replacement of the piperidine moiety in the first-generation lead 2, derived from our MeV-RdRp inhibitor program has been investigated. An SAR study revealed that hydrophilicity in this molecular sector strongly influences antiviral activity. We identified compounds incorporating hydroxyl (2k) and morpholinyl (2o) moieties that furnish potencies within a 10-fold range of 2, but with much improved aqueous solubility and oral bioavailability. In the series that replaces piperidine with the phenyl group, the most promising compound was found to be 3a with antiviral activity around 90 nM in a virus yield reduction assay. Unfortunately, the solubility rates of 3a and 2 are equally low, which stands in strong contrast to analogs 2k and 2o. Accordingly, the latter were advanced to pharmacokinetic studies in the Sprague-Dawley rat model. Analog 2o displays a 10-fold exposure (AUC/Cmax) increase in this model relative to 2 and displays near dose proportionality in the range of 10 to 50 mg/kg. The Caco-2 permeability assessment demonstrated the high permeability of this class of molecule. This significant solubility increase might be a major determinant for the overall improvement in oral bioavailability. Compound 2o was therefore identified as a second-generation lead for further development towards a novel measles therapeutic.

EXPERIMENTAL SECTION

GENERAL

Unless otherwise noted, all materials were obtained from commercial suppliers and used without purification. Dry organic solvents (DriSolv) were purchased from EMD Chemicals and packaged under nitrogen in Sure Seal bottles. Reactions were monitored using thin-layer chromatography on 250 μm plates or using Agilent 1100 series LC/MS with UV detection at 254 nm and low resonance electrospray mode (ESI). Elemental analysis was done by Atlantic Microlab. Purification of title compounds was accomplished by liquid chromatography on a Biotage SP4 purification system with normal phase silica gel. ¹H NMR spectra were recorded on a Varian spectrometer (400 MHz) at ambient temperature. Chemical shifts are reported in ppm relative to CDCl₃ or CD₃OD and coupling constants (J) are reported in hertz (Hz). Solvents for NMR were deuteriochloroform (CDCl₃) (residual shifts: δ 7.26 for ¹H and δ 77.7 for ¹³C) and deuteriomethanol (CD₃OD) (residual shift: δ 3.31 for ¹H). The residual shifts were taken as internal references and reported in parts per million (ppm). Purities of all compounds were ≥ 95% determined by high performance liquid chromatography (HPLC) with UV detection at two wavelengths of 220 and 254 nM. Purities of key compounds were also confirmed by elemental analysis.

Typical Procedures for the Synthesis of 1-methyl-N-(4-(piperidin-1-ylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides (1a-c)

4-Amino-sulfonamide 7a-c (1.0 mmol) in dichloromethane (5 ml) and pyridine (0.1 ml) was treated with 1-methyl-3-trifluoromethyl-5-pyrazolecarbonyl chloride (8) at rt. Reaction was monitored by LC-MS till no more starting material was seen, then the mixture was poured into saturated aqueous NaHCO₃ (10 ml), extracted with CH₂Cl₂ (3× 10ml). The CH₂Cl₂ extracts were collected and dried over anhydrous Na₂SO₄. Products were purified by chromatography.

N-(4-((2-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (1a)

¹H NMR (CDCl₃, 400MHz): δ 8.17 (s, 1H), 7.74 - 7.79 (m, 2H), 7.64 - 7.69 (m, 2H), 7.06 (s, 1H), 4.51 (s, 2H), 4.19 - 4.28 (m, 4H), 3.76-3.68 (m, 1H), 3.54 - 3.65 (m, 2H), 3.27 (s, 3H), 3.03-2.94 (m, 1H), 1.76-1.70 (m, 1H), 1.42 - 1.60 (m, 4H), 1.20 - 1.37 (m, 1H). Anal. calcd for C₂₁H₂₉F₃N₄O₅S: C, 49.79; H, 5.77; N, 11.06. Found: C, 49.07; H, 5.06; N, 11.31.

N-(4-((3-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (1b)

¹H NMR (CDCl₃, 400MHz): δ 8.09 (s, 1H), 7.69 - 7.78 (m, 4H), 7.03 (s, 1H), 4.56 (s, 2H), 4.25 (s, 3H), 3.78 (d, J = 11.7 Hz, 2H), 3.30 - 3.38 (m, 5H), 2.27 (td, J = 2.3, 11.9 Hz, 2H), 1.72 - 1.83 (m, 2H), 1.50 (m, 1H), 1.29 - 1.42 (m, 2H); LC-MS (ESI) (LCT, 3 min) Rt 1.58 min; >95% purity at λ 254 and 210 nm, MS: m/z 491.5 [M+1].

N-(4-((4-((Methoxymethoxy)methyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (1c)

¹H NMR (CDCl₃, 400MHz): δ 7.97 (s, 1H), 7.71 - 7.77 (m, 4H), 7.01 (s, 1H), 4.56 (s, 2H), 4.26 (s, 3H), 3.79 (d, J = 11.3 Hz, 2H), 3.30 - 3.38 (m, 5H), 2.27 (td, J = 2.5, 11.8 Hz, 2H), 1.79 (d, J = 10.6 Hz, 2H), 1.45 - 1.56 (m, 1H), 1.35 (m, 2H). Anal. calcd for C₂₁H₂₉F₃N₄O₅S: C, 49.79; H, 5.77; N, 11.06. Found: C, 49.17; H, 5.09; N, 11.21.

Synthesis of N-(4-((2-(hydroxymethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2a)

A solution of (1-((4-nitrophenyl)sulfonyl)piperidin-2-yl)methanol 6a (90 mg, 0.3 mmol) in MeOH (10 ml) was treated with H₂ (50 Psi) for 4 h in the presence of Pd/C (32 mg, 0.03 mmol). The Pd/C residue was removed by filtration, followed by evaporation of the solvent. The crude product was purified by chromatography (Hexane/EtOAc) to obtain amine product as white solid 70 mg (Y=86%).

4-Amino-sulfonamide (70 mg, 0.25 mmol) in dichloromethane (5 ml) and pyridine (0.1 ml) was treated with 1-methyl-3-trifluoromethyl-5-pyrazolecarbonyl chloride (8) at rt. Reaction was monitored by LC-MS till no more starting material was seen, then the mixture was poured into saturated aqueous NaHCO₃ (10 ml), extracted with CH₂Cl₂ (3× 10ml). The CH₂Cl₂ extracts were collected and dried over anhydrous Na₂SO₄. Products were purified by chromatography (Hex/EtOAc) to obtain product 2a as light yellow solid (81 mg, 73%). ¹H NMR (400MHz, CDCl₃) δ 1.23-1.62 (6H, m), 2.20 (1H, m), 3.08 (1H, t, J=13.2 Hz), 3.53-3.59 (1H, m), 3.77 (1H, d, J=14.0Hz), 3.84 (1H, t, J=10.4Hz), 4.00-4.06 (1H, m), 4.26 (3H, s), 7.11 (1H, s), 7.74-7.81 (4H, m), 8.48 (1H, s). Anal. calcd for C₁₈H₂₁F₃N₄O₄S: C, 48.43; H, 4.74; N, 12.55. Found: C, 48.33 ; H, 4.84 ; N, 12.23.

General Procedure for the Synthesis of Morpholinyl Analog (2b, 2n and 2o)

To a solution of aldehyde (1.0 mmol) in CH₂Cl₂ (10 ml) was added morpholine (1.3 eq, 1.3 mmol) and NaBH(OAc)₃ (2.0 eq, 2.0 mmol) and the mixture was kept stirring at room temperature for 3h. NaHCO₃ (sat. aq) was added and the organic layer separated and washed with brine, dried over Na₂SO₄, filtered and concentrated. The product was purified by column to give morpholinyl analog.

1-Methyl-N-(4-((2-(morpholinomethyl)piperidin-1-yl)sulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2b)

¹H NMR (CDCl₃, 400MHz): δ 8.03 (s, 1H), 7.83 - 7.89 (m, 2H), 7.67 - 7.72 (m, 2H), 7.02 (s, 1H), 4.26 (s, 3H), 4.21 (br. s., 1H), 3.64 (m, 5H), 2.88 - 2.97 (m, 1H), 2.38 - 2.51 (m, 6H), 1.77 (m, 1H), 1.41 - 1.58 (m, 4H), 1.31(m, 1H). Anal. calcd for C₂₂H₂₈F₃N₅O₄S: C, 51.25; H, 5.47; N, 13.58. Found: C, 51.05; H, 5.45; N, 13.42.

N-(4-((2-(Azidomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2c)

¹H NMR (CDCl₃, 400MHz): δ 7.93 (s, 1H), 7.80 - 7.86 (m, 2H), 7.69 - 7.75 (m, 2H), 6.99 (s, 1H), 4.26 (s, 3H), 4.16 (m, 1H), 3.79 (d, J = 13.3 Hz, 1H), 3.51 (dd, J = 7.2, 12.3 Hz, 1H), 3.30 - 3.38 (m, 1H), 2.92 - 3.02 (m, 1H), 1.65 - 1.71 (m, 1H), 1.53 - 1.62 (m, 5H).

N-(4-((2-(Aminomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2d)

¹H NMR (CDCl₃, 400MHz): δ 8.27 (s, 1H), 7.77 - 7.83 (m, 2H), 7.68 - 7.75 (m, 2H), 7.03 (s, 1H), 4.25 (s, 3H), 3.87 - 3.96 (m, 1H), 3.77 (d, J = 11.0 Hz, 1H), 2.92 - 3.06 (m, 2H), 2.64 (dd, J = 5.7, 13.5 Hz, 1H), 1.28 - 1.60 (m, 6H). LC-MS (ESI) (LCT, 3 min) Rt 0.54 min; >95% purity at λ 254 and 210 nm, MS: m/z 446.0 [M+1].

N-(4-((2-(Acetamidomethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2e)

¹H NMR (CDCl₃, 400MHz): δ 9.31 (s, 1H), 7.83 - 7.90 (m, 2H), 7.76 - 7.82 (m, 2H), 7.22 (s, 1H), 6.08 (t, J = 5.5 Hz, 1H), 4.26 (s, 3H), 4.03 - 4.13 (m, 1H), 3.67 - 3.77 (m, 1H), 3.56 (ddd, J = 5.3, 10.9, 14.0 Hz, 1H), 3.20 - 3.28 (m, 1H), 3.02 - 3.11 (m, 1H), 2.0 (m, 3H), 1.38 - 1.53 (m, 4H), 1.20 - 1.34 (m, 1H). Anal. calcd for C₂₀H₂₅F₃N₄O₄S: C, 49.28; H, 4.96; N, 14.37. Found: C, 49.02; H, 4.98; N, 14.08.

(E)-Ethyl 3-(1-((4-(1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamido)phenyl)-sulfonyl)piperidin-2-yl)acrylate (2g)

¹H NMR (CDCl₃, 400MHz): δ 8.19 (s, 1H), 7.69 - 7.78 (m, 4H), 7.03 (s, 1H), 6.75 (dd, J = 4.0, 16.0 Hz, 1H), 5.89 (dd, J = 2.0, 16.0 Hz, 1H), 4.69 (br. s., 1H), 4.25 (s, 3H), 4.15 (q, J = 7.0 Hz, 2H), 3.67 (d, J = 12.9 Hz, 1H), 2.95 - 3.05 (m, 1H), 1.63 - 1.78 (m, 2 H), 1.56 (d, J = 11.0 Hz, 7 H), 1.32 - 1.47 (m, 7 H), 1.25 (t, J = 8.0 Hz, 3H). Anal. calcd for C₂₂H₂₅F₃N₄O₅S: C, 51.36; H, 4.90; N, 10.89. Found: C, 51.36; H, 4.90; N, 10.89. Found: C, 51.42; H, 4.90; N, 10.79.

(E)-N-(4-((2-(3-Hydroxyprop-1-en-1-yl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2h)

¹H NMR (CDCl₃, 400MHz): δ 8.32 (s, 1H), 7.63 - 7.77 (m, 4H), 7.05 - 7.10 (m, 1H), 5.63 - 5.72 (m, 1H), 5.52 - 5.61 (m, 1H), 4.54 (br. s., 1H), 4.24 (s, 3H), 3.95 - 4.08 (m, 2H), 3.64 (d, J = 12.5 Hz, 1H), 3.47 (d, J = 5.1 Hz, 1H), 2.91 - 3.02 (m, 1H), 1.81 (t, J = 5.9 Hz, 1H), 1.34 - 1.74 (m, 6H). Anal. calcd for C₂₀H₂₃F₃N₄O₄S: C, 50.84; H, 4.91; N, 11.86. Found: C, 50.57; H, 4.98; N, 11.63.

Ethyl 3-(1-((4-(1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamido)phenyl)-sulfonyl)piperidin-2-yl)propanoate (2i)

¹H NMR (CDCl₃, 400MHz): δ 8.34 (s, 1H), 7.66 - 7.83 (m, 4H), 7.07 (s, 1H), 4.25 (s, 3H), 4.02 (s, 1H), 3.73 (d, J = 14.5 Hz, 1H), 3.63 (m, 2H), 2.93 - 3.05 (m, 1H), 2.16 (s, 3H), 1.59 - 1.81 (m, 2H), 1.28 - 1.59 (m, 6H). LC-MS (ESI) (LCT, 3 min) Rt 2.11 min; >95% purity at λ 254 and 210 nm, MS: m/z 517.1 [M+1].

N-(4-((2-(3-Hydroxypropyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide (2j)

¹H NMR (CHLOROFORM-d, 400MHz): δ 8.26 (s, 1H), 7.67 - 7.79 (m, 4H), 7.06 (s, 1H), 4.25 (s, 3H), 4.11 (q, J = 7.0 Hz, 2H), 3.99 - 4.07 (m, 1H), 3.74 (d, J = 14.5 Hz, 1H), 2.96 - 3.07 (m, 1H), 2.36 (t, J = 7.4 Hz, 2H), 2.00 - 2.13 (m, 1H), 1.60 - 1.72 (m, 1H), 1.30 - 1.55 (m, 5H), 1.24 (t, J = 7.2 Hz, 3H), 1.01 - 1.17 (m, 1H). Anal. calcd for C₂₀H₂₅F₃N₄O₄S: C, 50.62; H, 5.31; N, 11.81. Found: C, 50.35; H, 5.28; N, 11.62.

N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2k)

¹H NMR (CDCl₃, 400MHz): δ 8.00 (s, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 6.99 (s, 1H), 4.26 (s, 3H), 4.17 - 4.25 (m, 1H), 3.90 (d, J = 14.1 Hz, 1H), 3.74 - 3.83 (m, 1H), 3.67 (d, J = 5.1 Hz, 1H), 2.97 - 3.06 (m, 1H), 2.84 (dd, J = 4.9, 8.4 Hz, 1H), 1.93 - 2.02 (m, 1H), 1.40 - 1.54 (m, 5H), 1.32-1.40 (M, 1H). Anal. calcd for C₁₉H₂₃F₃N₄O₄S: C, 49.56; H, 5.03; N, 12.17. Found: C, 49.36; H, 5.08; N, 11.98.

(R)-N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2l)

¹H NMR (CDCl₃, 400MHz): δ 8.34 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 7.07 (s, 1H), 4.27 (s, 3H), 4.22 - 4.19 (m, 1H), 3.91 (d, J = 14.4 Hz, 1H), 3.80 (t, J = 11.2 Hz, 1H), 3.67 (br, 1H), 3.06 – 2.04 (m, 2H), 2.03-1.95 (m, 1H), 1.57 - 1.41 (m, 4H), 1.28-1.21 (m, 2H). LC-MS (ESI) (LCT, 3 min) Rt 1.09 min; >95% purity at λ 254 and 210 nm, MS: m/z 461.2 [M+1].

(S)-N-(4-((2-(2-Hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2m)

¹H NMR (CDCl₃, 400MHz): δ 8.12 (m, 1H), 7.86 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.03 (s, 1H), 4.28 (s, 3H), 4.20 - 4.23 (m, 1H), 3.91 (d, J = 14.1 Hz, 1H), 3.81 (t, J = 11.6 Hz, 1H), 3.68 (m, 1H), 3.03 (t, J = 12.8 Hz, 1H), 2.88 (m, 1H), 2.05 – 1.96 (m, 1H), 1.58-1.26 (m, 5H), 1.13-1.08 (m, 1H). LC-MS (ESI) (LCT, 3 min) Rt 1.09 min; >95% purity at λ 254 and 210 nm, MS: m/z 461.2 [M+1]. Anal. calcd for C₁₉H₂₃F₃N₄O₄S: C, 49.56; H, 5.03; N, 12.17. Found: C, 49.50; H, 5.05; N, 11.95.

(S)-1-methyl-N-(4-((2-(2-morpholinoethyl)piperidin-1-yl)sulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamid (2o)

¹H NMR (CDCl₃, 400MHz): δ 8.18 (s, 1H), 7.74 - 7.80 (m, 2H), 7.65 - 7.72 (m, 2H), 7.05 (s, 1H), 4.25 (s, 3H), 4.04 - 4.11 (m, 1H), 3.76 (dd, J = 4.1, 14.3 Hz, 1H), 3.67 (t, J = 4.5 Hz, 1H), 2.97 - 3.08 (m, 1H), 2.23 - 2.45 (m, 6H), 1.78 - 1.90 (m, 1H), 1.55 - 1.66 (m, 1H), 1.30 - 1.53 (m, 5H). LC-MS (ESI) (LCT, 3 min) Rt 0.57 min; >95% purity at λ 254 and 210 nm, MS: m/z 530.2 [M+1]. Anal. calcd for C₂₃H₃₀F₃N₅O₄S.H₂O: C, 50.45; H, 5.89; N, 12.79. Found: C, 50.98; H, 5.72; N, 12.74.

Ethyl 2-((1-((4-(1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamido)phenyl) sulfonyl)piperidin-2-yl)methoxy)acetate (2p)

¹H NMR (CDCl₃, 400MHz): δ 8.14 (s, 1H), 7.77 - 7.83 (m, 2H), 7.65 - 7.70 (m, 2H), 7.02 - 7.06 (m, 1H), 4.25 (s, 3H), 4.14 - 4.23 (m, 3H), 3.99 (d, J = 3.1 Hz, 2H), 3.73 (d, J = 14.1 Hz, 1H), 3.60 - 3.67 (m, 2H), 2.96 - 3.06 (m, 1H), 1.77 (d, J = 12.9 Hz, 1H), 1.37 - 1.56 (m, 3H), 1.26 (t, J = 8.0 Hz, 3H). LC-MS (ESI) (LCT, 3 min) Rt 1.71 min; >95% purity at λ 254 and 210 nm, MS: m/z 533.2 [M+1].

N-(4-((2-((2-hydroxyethoxy)methyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (2q, JMN6-093)

¹H NMR (CDCl₃, 400MHz): δ 8.73 (s, 1H), 7.70 - 7.83 (m, 4H), 7.11 (s, 1H), 4.30 - 4.39 (m, 1H), 4.25 (s, 3H), 3.72 (t, J = 9.4 Hz, 2H), 3.58 - 3.68 (m, 2H), 3.45 - 3.54 (m, 2H), 3.40 (d, J = 10.6 Hz, 1H), 3.25 (br. s., 1H), 2.98 (td, J = 2.5, 13.2 Hz, 1H), 1.63 - 1.74 (m, 2H), 1.35 - 1.63 (m, 3H). Anal. calcd for C₂₀H₂₅F₃N₄O₅S: C, 48.97; H, 5.14; N, 11.42. Found: C, 48.94; H, 5.08; N, 11.26.

1-Methyl-N-(4-(phenylsulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3a)

¹H NMR (CDCl₃, 400MHz): δ 8.10 (s, 1H), 7.85 - 7.93 (m, 4H), 7.69 - 7.75 (m, 2H), 7.53 - 7.59 (m, 1H), 7.46 - 7.53 (m, 2H), 7.03 (s, 1H), 4.22 (s, 3H). Anal. calcd for C₁₈H₁₄F₃N₃O₃S: C, 52.81; H, 3.45; N, 10.26. Found: C, 52.31; H, 3.41; N, 9.95.

N-(4-((2-methoxyphenyl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3b)

¹H NMR (CDCl₃, 400MHz): δ 8.11 (dd, J = 1.8, 8.0 Hz, 1H), 8.04 (s, 1H), 7.90 - 7.95 (m, 2H), 7.66 - 7.71 (m, 2H), 7.51 - 7.57 (m, 1 H), 7.07 - 7.13 (m, 1 H), 7.02 (s, 1 H), 6.87 - 6.91 (m, 1 H), 4.24 (s, 3 H), 3.76 (s, 3H). LC-MS (ESI) (LCT, 3 min) Rt 1.11 min; >95% purity at λ 254 and 210 nm, MS: m/z 440.0 [M+1].

Synthesis of N-(4-((2-Hydroxyphenyl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3c)

To a solution of 3b (110.0 mg, 0.250 mmol) in CH₂Cl₂ (6.0 mL), was added BBr₃ (1.0 mL, 1.0 mmol) and the mixture stirred for an overnight. The reaction was cooled to 0 °C and NaHCO₃ solution (3.0 mL) slowly added. The reaction was allowed to warm to RT and CH₂Cl₂ (9.0 mL) and MeOH (1.0 mL) added. The organic layer was separated and washed with NaHCO₃, brine, dried over Na₂SO₄, filtered and concentrated. The product was purified by column (CH₂Cl₂/MeOH) and dried under vacuum to give 106.0 mg of a white solid in 96% yield. ¹H NMR (CDCl₃, 400MHz): δ 7.86 - 7.92 (m, 2 H), 7.77 - 7.82 (m, 2 H), 7.65 (dd, J = 1.6, 8.2 Hz, 1 H), 7.37 - 7.43 (m, 1H), 7.11 (s, 1H), 6.90 - 6.96 (m, 2H), 4.20 (s, 3H). Anal. calcd for C₁₈H₁₄F₃N₃O₄S: C, 50.82; H, 3.32; N, 9.88. Found: C, 50.67; H, 3.29; N, 9.61.

Synthesis of 2-((4-(1-Methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamido)phenyl)sulfonyl)phenylacetate (3d)

To a solution of 3c (52.0 mg, 0.122 mmol) in dimethylformamide (1.0 mL) was added K₂CO₃ (33.8 mg, 0.244 mmol) and acetic anhydride (0.023 mL, 0.244 mmol) and the mixture allowed to stir for an overnight. DMF was removed under vacuum and the residue purified by column (hexanes/ethylacetate) to give 43.4 mg of 3d as a white solid in 76% yield. ¹H NMR (CDCl₃, 400 MHz) δ 8.14 (dd, J = 1.76, 8.02 Hz, 1H), 7.87 - 7.93 (m, 3H), 7.70 - 7.76 (m, 2H), 7.58 - 7.63 (m, 1H), 7.41 (dt, J = 1.17, 7.83 Hz, 1H), 7.14 (dd, J = 0.98, 8.02 Hz, 1H), 6.96 (s, 1H), 4.24 (s, 3H), 2.32 (s, 3H). Anal. calcd for C₂₀H₁₆F₃N₃O₅S: C, 51.39; H, 3.45; N, 8.99. Found: C, 51.31; H, 3.32; N, 8.80.

1-Methyl-3-(trifluoromethyl)-N-(4-((2-vinylphenyl)sulfonyl)phenyl)-1H-pyrazole-5-carboxamide (3e)

¹H NMR (CDCl₃, 400 MHz) δ 8.12 - 8.17 (m, 1H), 8.08 (s, 1H), 7.76 - 7.83 (m, 2H), 7.65 - 7.71 (m, 2H), 7.51 - 7.59 (m, 2H), 7.41 - 7.50 (m, 2H), 7.02 (s, 1H), 5.52 (dd, J = 1.17, 17.22 Hz, 1H), 5.33 (dd, J = 0.78, 10.96 Hz, 1H), 4.22 (s, 3H). Anal. calcd for C₂₀H₁₆F₃N₃O₃S: C, 55.17; H, 3.70; N, 9.65. Found: C, 54.99; H, 3.60; N, 9.64.

N-(4-((2-(Hydroxymethyl)phenyl)sulfonyl)phenyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3f)

¹H NMR (CDCl₃, 400 MHz) δ 8.10 (dd, J = 1.17, 7.83 Hz, 1H), 8.00 (s, 1H), 7.84 - 7.90 (m, 2H), 7.71 - 7.77 (m, 2H), 7.59 - 7.65 (m, 1H), 7.48 - 7.57 (m, 2H), 6.98 (s, 1H), 4.73 (d, J = 6.26 Hz, 2H), 4.23 (s, 3H). Anal. calcd for C₁₉H₁₆F₃N₃O₄S: C, 51.93; H, 3.67; N, 9.56. Found: C, 52.01; H, 3.53; N, 9.40.

1-Methyl-N-(4-((2-(morpholinomethyl)phenyl)sulfonyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (3g)

¹H NMR (CDCl₃, 400MHz): δ 8.16 (dd, J = 1.2, 7.8 Hz, 1 H), 7.99 (s, 1H), 7.83 - 7.89 (m, 2H), 7.68 - 7.74 (m, 3H), 7.54 - 7.60 (m, 1H), 7.42 - 7.48 (m, 1H), 7.01 (s, 1H), 4.24 (s, 3H), 3.77 (s, 2H), 3.50 - 3.57 (m, 4H), 2.27 (m, 4H). Anal. calcd for C₂₃H₂₃F₃N₄O₄S: C, 54.32; H, 4.56; N, 11.02. Found: C, 54.36; H, 4.42; N, 10.85.

BIOLOGY

Antiviral assays and toxicity measurements were performed as described previously.²²

Figure 2.

Evaluation of compunds 2 and analogs against MV-Alaska. All compounds were tested at 1.0 μM. Compounds comparable in activity to 2 were further examined at a range of concentrations to generate dose-response curves.

ABBREVIATION USED

MeV

measles virus

RNA

ribonucleic acid

RdRp

RNA dependent RNA polymerase

HTS

high-throughput screening

HPIV

human parainfluenza virus

RSV

respiratory syncytial virus

EC₅₀

50% effective concentration

CC₅₀

50% cytotoxicity concentration

MOM

methoxymethyl

TBSCl

t-butyldimethylsilyl chloride

DIBALH

diisobutylaluminium hydride

MCPBA

meta-chloroperoxybenzoic acid

PK

pharmacokinetic