Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

Abstract

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.

TRPV1, the target of capsaicin action, has emerged as a promising therapeutic target for the treatment of neuropathic pain and a range of other conditions, reflecting the central role of this nociceptor in the function of C-fiber sensory neurons.^1–3 Building on the demonstration of a receptor for capsaicin,⁴ its cloning as TRPV1,⁵ and insights arising from its structural analysis by cryo-EM^6,7 and homology modeling,^8,9 dramatic strides have been made in understanding the structure activity relations for antagonist binding to TRPV1.¹⁰ These advances have been coupled with an appreciation of the complexity of TRPV1 regulation, such as the finding that different antagonists may be differentially effective against different classes of TRPV1 activators such as capsaicin, low pH, or elevated temperature.¹¹ Such differences, in term, may translate into different physiological responses of animals or humans upon treatment with different TRPV1 antagonists.^12,13

Over the past several years we have described an extensive series of N-(pyridin-3-ylmethyl) 2-(3-fluoro-4-(methylsulfon amido) phenyl)propanamides that showed potent and stereospecific human TRPV1 (hTRPV1) antagonism for multiple activators (Fig. 1).^14–24 The pharmacophore of the antagonistic template (Template I) can be divided into the A, B and C-regions, analogous to the corresponding designation of regions for the agonist capsaicin. The structure activity relationships (SAR) of the template have been investigated in most detail for the pyridine C-region, in which a variety of functional groups including the amino, oxy, thio, alkyl, aryl and sulfonamido groups were incorporated at the 2-position of pyridine (R₂) along with substitution at the 6-position (R₁) with a trifluoromethyl^14–18,22 or tert-butyl group²⁴ as well as with the pyridine core modified by its isomers¹⁹ or by a phenyl group.^21,24 In these studies, a number of compounds demonstrated highly potent antagonism toward TRPV1 activators including capsaicin (CAP), N-arachidonoyl dopamine (NADA), low pH, and heat (45 °C) and their antagonism were stereospecific to the S-configuration. Consistent with the in vitro mechanism of action of the compounds as hTRPV1 antagonists, in vivo studies of selected potent antagonists indicated that these compounds blocked capsaicin-induced hypothermia and that they produced strong antiallodynic effects in neuropathic pain models. Docking studies using our established hTRPV1 homology model¹⁴ indicated that the 6-trifluoromethyl group and the 2-substituent in the pyridine C-region made hydrophobic interactions with pockets composed of Leu547/Thr550 and Met514/Leu515, respectively, that were critical for their potent antagonism.^14–18

Fig. 1.

Design of pyrazole C-region TRPV1 antagonists.

As part of our continuing effort to optimize TRPV1 antagonists as clinical candidates for neuropathic pain, we herein have investigated a series of pyrazole C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides (Template II) as a new antagonistic template. Since the pyrazole is a weak base and has a pyridine-like nitrogen (N2) with a lone electron pair, it serves as a bioisostere of pyridine. Furthermore, we anticipated that 1,3-substituents, R₁ and R₂, on pyrazole are better positioned to interact with the two hydrophobic pockets identified by the previous modeling than do the 2,6-substituents of pyridine.

In this paper, we synthesized a series of 1-substituted 3-(trifluoromethyl/t-butyl)pyrazole C-region derivatives and evaluated their antagonism toward activation of hTRPV1 by capsaicin. With selected potent antagonists in the series, we characterized in detail their in vitro activities and mode of action in vivo. Finally, we carried out a docking study using our hTRPV1 homology model to identify their mode of binding to the receptor.

N₁-Substituted 3-t-butyl-1H-pyrazole amines for the C-region were synthesized by one of two methods. In Method A, 4,4-dimethyl-3-oxopentanenitrile was condensed with different hydrazines to provide N₁-substituted 5-amino-3-t-butylpyrazoles whose amine was converted to the corresponding aminomethyl group in 3 steps to give the t-butylpyrazole C-region amine (Scheme 1). In Method B, N-Boc (3-t-butyl-pyrazol-5-yl)methanamine as a key intermediate for coupling with the corresponding boronic acids was prepared from 3,3-dimethylbutan-2-one in 6 steps. A Chan-Lam coupling reaction between pyrazole 1H-amine and aryl boronic acid followed by deprotection provided the t-butylpyrazole C-region amine (Scheme 2). N₁-Substituted 3-trifluoromethyl-1H-pyrazole amines for the C-region were synthesized from ethyl trifluoroacetic acid in 5 steps using different hydrazines (Scheme 3). A library of synthesized pyrazole C-region amines were coupled with 2-(3-fluoro-4-(methylsulfonamido)phenyl) propionic acids¹⁴ to provide the final compounds (Scheme 4).

Scheme 1.

Synthesis of 3-t-butyl pyrazole C-region (Method A). Reagents and conditions: (a) R-NHNH₂·HCl, EtOH:H₂O (1:1), reflux, overnight, 44–94%; (b) t-BuONO, CuI, CH₃CN, 0 °C to 65 °C; (c) Zn(CN)₂, Pd(PPh₃)₄, DMF, reflux, overnight; (d) LiAlH₄, THF, 0 °C to rt, 1 h, 12–26% (3 steps yield).

Scheme 2.

Synthesis of 3-t-butyl pyrazole C-region (Method B). Reagents and conditions: (a) LiHMDS, diethyloxalate, THF, −78 °C to rt, overnight, 97%; (b) NH₂-NH₂°2HCl, TEA, EtOH, rt, 2 h, 77%; (c) NaOH, MeOH:H₂O (5:1), rt, 2 h, 70%; (d) SOCl₂, MC, rt, overnight; then NH₄OH, MC, 0 °C, 1 h, 80%; (e) LiAlH₄, THF, 0 °C to rt, 3 h; (f) Boc₂O, MC, rt, 1 h, 50%; (g) Ar-B(OH)₂, Cu(OAc)₂, pyridine, MC, rt, 2 d; (h) TFA, MC, rt, overnight.

Scheme 3.

Synthesis of 3-trifluoromethyl pyrazole C-region (Method C). Reagents and conditions: (a) LiAlH₄, Et₂O, −78 °C, 2 h; (b) R-NHNH₂, EtOH, reflux, 5 h, (15: 90%, 16: 67%); (c) NCS, DMF, rt, (15: 48%, 16: 26%); (d) 2-chloroacylonitrile, TEA, toluene, 80 °C, 20 h, (15: 44%, 16: 80%); (e) LiAlH₄, THF, 0 °C to rt, 3 h, (15: 64%, 16: 62%).

Scheme 4.

General synthesis of 2-(3-fluoro-4-(methylsulfonamido)phenyl) propanamide analogues Reagents and conditions: (a) EDC, HOBt, DMF, room temperature, 12 h.

The synthesized compounds were evaluated in vitro for TRPV1 antagonism as measured by inhibition of activation by capsaicin (100 nM). The assays were conducted using a fluorometric imaging plate reader (FLIPR) with hTRPV1 heterologously expressed in Chinese hamster ovary (CHO) cells.¹⁴ The results are summarized in Tables 1–3.

Table 1.

In vitro hTRPV1 antagonistic activities for 3-t-butylpyrazole derivatives.

WE: weakly active, NE: not effective.

Table 3.

In vitro hTRPV1 antagonistic activities for α-substituted acetamide derivatives.

WE: weakly active (17: 46%, 18: 12%, 19: 49% inhibition% at 5 μM).

First, we investigated the SAR of the N₁-substituent in the 3-t-butylpyrazole C-region (Table 1). The unsubstituted derivative (1) was found to be inactive, suggesting that the two hydrophobic groups were indispensable for activity by providing needed interactions with the receptor pockets as previously characterized for the pyridine C-region.^14–24 The incorporation of an alkyl group (2–4) enhanced the antagonism progressively as the size increased. The rigidity of alkyl groups further increased the potency (3 vs 5, 4 vs 6). Most phenyl derivatives (7–14) showed potent antagonism. Among them, the 3-chlorophenyl derivative (13) displayed excellent antagonism with a K_i(CAP) = 0.3 nM and its S-stereoisomer (13S) exhibited a stereospecific activity with a K_i(CAP) = 0.1 nM. The 4-fluoro-3-chlorophenyl derivatives (14, 14S) also showed high potency like those of the 3-chlorophenyl derivatives. The analysis of 4-substituted phenyl derivatives indicated that whereas the chloro group provided promising antagonism, electron-donating (9) and bulky (11, 12) groups led to the dramatic reduction in activity.

Next, we explored the SAR of N₁-substituents in the 3-trifluoromethylpyrazole C-region (Table 2). Since 4- or 3-chlorophenyl derivatives in the 3-t-butylphenyl series provided the most promising activity, their surrogates were examined. 4-Chlorophenyl (15) and 3-chlorophenyl (16) derivatives displayed highly potent antagonism with K_i(CAP) = 0.4 and 0.3 nM, respectively, in line with those of the 3-t-butylpyrazole series. In addition, the S-configuration was confirmed as the active configuration, as dramatically shown by isomers 15S, 16S with K_i(CAP) = 0.3 and 0.1 nM, versus isomers 15R, 16R with K_i(CAP) = 31.7 and 26.5 nM.

Table 2.

In vitro hTRPV1 antagonistic activities for 3-trifluoromethylpyrazole derivatives.

Finally, we investigated α-substituted acetamide B-region derivatives of the potent antagonist 13 (Table 3) because some of these B-region derivatives previously had provided high potency.²⁰ Unfortunately, they all displayed weak antagonism.

Detailed in vitro activities of 13S and 16S, the most potent antagonists in this series, were investigated for four different TRPV1 activators, viz. capsaicin, N-arachidonoyl dopamine (NADA), pH and heat (45 °C), and compared to the activity of the previous lead GRT12360 (Table 4). Both 13S and 16S showed excellent antagonism of hTRPV1 activation by capsaicin and NADA comparable to that of GRT12360. However, 13S exhibited poor antagonism toward pH and heat, in contrast to 16S and GRT12360, indicating that the size of the hydrophobic group at the 3-position in the pyrazole C-region affected the selectivity of antagonism for different activators.

Table 4.

Antagonistic activities of 13S and16S for multiple activators in hTRPV1 and rTRPV1.

Activators, parameter	13S	16S	GRT12360b
hTRPV1
CAP (f)Ki (nM)	0.1	0.1	0.2
NADA (f)Ki (nM)	0.04	0.1	0.01
pH, IC50 (nM)	WE	8.0	6.3
heat 45 °C, IC50 (nM)	WE	8.1	0.8
rTRPV1
CAP (f)Ki (nM)	0.1	0.1
Anti-hypothermia	95%a at 3 mg/kg, po	85%a at 3 mg/kg, po

^aInhibition percent to hypothermic response by 3 mg/kg ip capsaicin.

^bCompound 49S in Ref. 9.

Both antagonists proved to be highly potent antagonists of capsaicin action in vivo against rat TRPV1 (rTRPV1). Consistent with its in vitro mechanism of action (K_i(CAP) = 0.1 nM in r/hTRPV1), they were able to block the acute hypothermic response to capsaicin (3 mg/kg, ip). The oral administration of 3 mg/kg 13S and 16S almost completely antagonized the effect of capsaicin on body temperature, with 95% and 85% inhibition, respectively, of the decrease in body temperature induced by capsaicin.

In order to investigate the binding interactions of 13S and 16S, flexible docking studies were carried out using our hTRPV1 model¹⁴ constructed on the basis of our rTRPV1 model.⁸ 13S and 16S share with the previously reported GRT12360 (Fig. 2A)¹⁴ the identical A,B-region structure of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide but are distinguished by different C-regions. Compound 16S has a 3-(trifluoromethyl)pyrazole ring and a chlorobenzene ring in the C-region and showed a different binding mode in the C-region compared to GRT12360. As shown in Fig. 2B, the sulfonaminobenzyl group in the A-region occupied the deep bottom hole and participated in the hydrophobic interactions with Val508, Tyr511, Leu515, Ile564, Tyr565, and Ile569. In addition, the fluorine atom in the A-region engaged in hydrogen bonding with Lys571 while the NH of the sulfonamide group formed a hydrogen bond with Ile564. The amide group in B-region was able to form a hydrogen bond with Tyr511 and contributed to the proper positioning of the C-region for the hydrophobic interactions. In the C-region, the 3-(trifluoromethyl)pyrazole ring formed hydrophobic interactions with Tyr554 and with Phe587 and Leu588 of the adjacent monomer. Instead of the 3-(trifluoromethyl)pyrazole ring, the chlorobenzene ring oriented towards the upper hydrophobic region composed of Leu547, Thr550 and was involved in hydrophobic interactions with Phe587 and Phe591 in the adjacent monomer, which might have caused the flipped positioning of the two rings in the C-region.

Fig. 2.

Binding modes of GRT12360 and 16S in the hTRPV1 model.25 (Top) 2-D representation of the binding interactions between GRT12360 (A) and 16S (B) with hTRPV1. Hydrogen bonding interactions are drawn in blue dashed line arrows, and hydrophobic interactions are displayed with curved patches. (Bottom) The Fast Connolly surface of hTRPV1 and the van der Waals surface of GRT12360 and 16S. Using MOLCAD, the hTRPV1 molecular surface was created and the surface with the lipophilic potential property is presented. For clarity, the surface of hTRPV1 is Z-clipped and that of the ligands are colored individually by magenta and purple.

On the other hand, 13S, which has the t-butyl pyrazole and chlorobenzene rings in the C-region, exhibited interactions similar to those of GRT12360.¹⁴ As shown in Fig. 3A, the 3-fluoro-4-methylsulfonamidophenyl group in the A-region bound well to the deep bottom area of the binding site, which consisted of Val508, Tyr511, Leu515, Tyr555, Ile564, Tyr565, and Ile569, forming hydrophobic interactions. Additionally, the NH of the sulfonamide group engaged in hydrogen bonding with Ser512. The amide group in the B-region made a hydrogen bond with the OH of Tyr511 and assisted the appropriate positioning of the C-region. The t-butyl pyrazole ring in the C-region extended toward the hydrophobic pocket composed of Leu515, Leu518, and Leu547, along with Phe587 from the adjacent monomer. Moreover, the chlorobenzene ring in the C-region participated in the hydrophobic interactions with Tyr511, Met514, and Leu515. Additionally, 13S presented another comparable binding mode by flipping the two rings in the C-region as shown in Fig. 3B. The 3-fluoro-4-methylsulfonamidophenyl group in the A-region fitted in the deep bottom region and showed hydrophobic interactions with Val508, Tyr511, Ile564, Tyr565, and Ile569. The NH of the amide group in the B-region formed a hydrogen bond with Asn551. Moreover, the t-butyl pyrazole ring in the C-region participated in hydrophobic interactions with Tyr511, Met514, and Leu515 as did the 4-methylpiperidine ring in GRT12360. The chlorobenzene ring approached the upper hydrophobic area, which included Leu518, Leu547, Thr550 and Phe587 from the adjacent monomer as did the 6-trifluoromethylpyridine ring in GRT12360.

Fig. 3.

Binding modes of 13S in the hTRPV1 model.25 (Top) 2-D illustrations of the two binding modes of 13S with hTRPV1. Hydrogen bonding interactions are shown in blue dashed line arrows and hydrophobic interactions are presented with curved patches. (Bottom) The Fast Connolly surface representation of hTRPV1 and the van der Waals surface representation of the ligand 13S. MOLCAD was used to generate the molecular surface of hTRPV1 and the surface is displayed with the lipophilic potential property. For clarity, the surface of hTRPV1 is Z-clipped and that of 13S is in magenta color.

In summary, the structure activity relationship of 3-(t-butyl) pyrazole and 3-(trifluoromethyl)pyrazole C-region derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl) propanamides as antagonists for hTRPV1 was investigated. The 3-chlorophenyl group at the 1-position of pyrazole was the optimal hydrophobic group for antagonism and the activity was stereospecific for the S-configuration, providing the exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Whereas 16S showed full antagonism to all activators as did the previous lead, 13S exhibited antagonism selective for capsaicin and NADA but not low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1 and blocked the hypothermic effect of capsaicin in vivo, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that they displayed different binding interactions and that the mode of 13S was more like that of GRT12360.