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. Author manuscript; available in PMC: 2012 Aug 16.
Published in final edited form as: Bioorg Med Chem Lett. 2010 Nov 5;21(1):299–302. doi: 10.1016/j.bmcl.2010.11.012

Receptor Activity and Conformational Analysis of 5’-Halogenated Resiniferatoxin Analogues as TRPV1 Ligands

Kwang Su Lim a, Dong Wook Kang a, Yong Soo Kim a, Myeong Seop Kim a, Seul-Gi Park b, Sun Choi b, Larry V Pearce c, Peter M Blumberg c, Jeewoo Lee a,*
PMCID: PMC3420360  NIHMSID: NIHMS388384  PMID: 21111618

Abstract

A series of 5’-halogenated resiniferatoxin analogues have been investigated in order to examine the effect of halogenation in the A-region on their binding and the functional pattern of agonism/antagonism for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Halogenation at the 5-position in the A-region of RTX and of 4-amino RTX shifted the agonism of parent compounds toward antagonism. The extent of antagonism was greater as the size of the halogen increased (I > Br > Cl > F) while the binding affinities were similar, as previously observed for our potent agonists. In this series, 5-bromo-4-amino RTX (39) showed very potent antagonism with Ki(ant) = 2.81 nM, which was thus 4.5-fold more potent than 5’-iodo RTX, previously reported as a potent TRPV1 antagonist. Molecular modeling analyses with selected agonists and the corresponding halogenated antagonists revealed a striking conformational difference. The 3-methoxy of the A-region in the agonists remained free to interact with the receptor whereas in the case of the antagonists, the compounds assumed a bent conformation, permitting the 3-methoxy to instead form an internal hydrogen bond with the C4-hydroxyl of the diterpene.

Keywords: TRPV1 agonist, TRPV1 antagonist, partial agonist, halogenation, resiniferatoxin, molecular modeling

Resiniferatoxin (RTX, 1),1–2 isolated from Euphorbia resinifera, is an extremely potent irritant tricyclic diterpene which structurally falls within the daphnane subclass of phorbol-related diterpenes but is set apart by its homovanillyl ester group at C-20.3,4 RTX has proven to function pharmacologically as an ultrapotent agonist for the transient receptor potential vanilloid 1 (TRPV1) channel, displaying 103- to 104-fold greater potency than the prototypic agonist capsaicin.5

RTX was found to evoke large inward currents in dorsal root ganglion (DRG) neurons and TRPV1 transfected cell lines.6 The actions of RTX are mediated by binding, with picomolar affinity, directly to the capsaicin-binding site on the TRPV1 receptor.7 Whereas capsaicin under normal conditions produces only short term desensitization of TRPV1 mediated responses, the apparent desensitization to RTX can be of very long duration, lasting for weeks.8,9 RTX is being developed as a potent desensitizing agent for neurons in the treatment of urinary urge incontinence and the pain associated with diabetic neuropathy, as well as for cancer pain.10,11

Structure-activity relationships for RTX derivatives have been investigated mostly through partial modifications starting from RTX or ROPA (resiniferonol orthophenylacetate) based on the three structural regions including the A-region (4-hydroxy-3-methoxyphenyl), B-region (C20 ester), and C-region (diterpene).12–21 In the SAR of RTX, it should be noted that a divergence in potencies between ligand binding and agonism for TRPV1 ligands is frequently observed15 and may reflect the distinction between the properties of the small fraction of the receptor at the cell surface and the predominant proportion in internal membranes.22

Previous SAR indicated that although most modifications of RTX reduced binding affinity and agonism, several have proven to be of considerable interest interesting (Figure 1). 5-Iodination in the A-region of RTX shifted the activity from agonism to antagonism providing a potent antagonist, 5-iodo RTX (2), for both rat and human TRPV1.16,17 Surprisingly, the carbonate surrogate (3) in the B-region of 5-iodo RTX showed excellent agonism rather than antagonism and its potency was 4-fold higher than RTX with a one-digit picomolar value reported for hTRPV1 expressed in HEK cells.21 Substitution of an isosteric methanesulfonamide on RTX provided a metabolically stable and potent agonist (4), which was 2.5-fold more potent than RTX with an EC50 = 0.106 nM for rat TRPV1 heterologously expressed in CHO cells.20 The results demonstrated that halogenation and isosteric substitution of the phenolic group in the A-region appear to be key modifications influencing the functional activity and potency toward TRPV1.

Figure 1.

Figure 1

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In this paper, we have investigated the effect of halogenation in the A-region of RTX and the isosteric 4-amino surrogate in a systematic manner and we describe the synthesis and receptor activity, as well as conformational analysis for explaining the observed functional differences.

The halogenated RTX analogues (31–39) were prepared starting from commercially available ROPA (resiniferonol-9,13,14-orthophenylacetate) by the condensation with the corresponding 2-phenyl acetic acids (A-region) using EDC (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) coupling reagent, followed by appropriate O-deprotection if needed (Scheme 1).

Scheme 1.

Scheme 1

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Reagents and conditions: (a) R-CO2H, EDC, CH2Cl2, 70–90%; (b) CF3CO2H-CH2Cl2 (1:2) for MOM deprotection, 60–96%

The syntheses of the A-region are represented in Schemes 2–6. The 5-fluoro-4-hydroxyphenyl A-region was prepared from 2-fluoro-6-methoxyphenol employing regioselective formylation (Scheme 2) and the 5-chloro and 5-bromo-4-hydroxyphenyl A-regions were synthesized starting from vanillin and homovanillic acid using electrophilic addition of halogens, respectively (Schemes 2 and 3). The 3,5-dibromophenyl A-region was synthesized from 1,3-dibromo-5-fluorobenzene by nucleophilic substitution of ethyl cyanoacetate followed by decarboxylation (Scheme 4). The 5-fluoro-4-amino A-region was prepared from 1,3-difluoro-2-nitrobenzene employing the reactions of Makosza’s vicarious nucleophilic substitution23 and selective nucleophilic substitution of the methoxy group (Scheme 5). The 4-amino, 5-chloro and 5-bromo-4-amino A-regions were synthesized from (3-hydroxyphenyl)acetic acid by nitration followed by halogenation of the 4-aminophenyl (Scheme 6).

Scheme 2.

Scheme 2

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Reagents and conditions: (a) i) formaldehyde, diethylamine, EtOH, reflux, ii) CH3I, CH2Cl2, rt, 18 h, iii) HMTA, HOAc-H2O, 120 °C, 2 h, 37% for 3 steps; (b) NCS, NaH, THF, 49%; (c) MOMCl, DIPEA, CH3CN, 68% for X=F, 87% for X=Cl; (d) LAH, THF, 98% for X=F and Cl; (e) CBr4, PPh3, benzene, 48% for X=F, 32% for X=Cl; (f) X=F: NaCN, DMF, 80 °C, 93%, X=Cl: KCN, 18-crown-6, CH3CN, 85%; (g) NaOH, THF-H2O, reflux, 99% for X=F and Cl

Scheme 6.

Scheme 6

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Reagents and conditions: (a) cat. H2SO4, EtOH, 98%; (b) HNO3, AcOH, 29%; (c) K2CO3, CH3I, acetone, 71%; (d) 10% Pd/C, H2, THF-EtOH, 97%; (e) oxone, NaCl or NaBr, acetone-H2O, 30%; (f) LiOH, THF-H2O, 60–90%

Scheme 3.

Scheme 3

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Reagents and conditions: (a) cat. H2SO4, EtOH, 99%; (b) oxone, NaBr, acetone-H2O, 56%; (c) NaOH, THF-H2O, 100%; (d) Ac2O, cat. H2SO4, CH2Cl2, 95%

Scheme 4.

Scheme 4

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Reagents and conditions: (a) ethyl cyanoacetate, NaH, DMF, 25%; (b) NaOH, H2O 100%

Scheme 5.

Scheme 5

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Reagents and conditions: (a) t-BuOK, ethyl chloroformate, NMP, 22%; (b) NaOMe, MeOH, 55%; (c) Pd/C, H2, MeOH, 69%; (d) LiOH, THF-H2O, 82%

The halogenated resiniferatoxin analogues were evaluated for their binding affinities (expressed as Ki values) using a competitive binding assay with [3H]RTX and for their agonism/antagonism using a functional 45Ca2+ uptake assay (expressed as EC50 and Ki(ant) values) for rat TRPV1 heterologously expressed in Chinese hamster ovary (CHO) cells.24 In this system, resiniferatoxin displayed Ki = 0.043 nM and EC50 = 0.27 nM.20 The receptor binding and functional activities of the halogenated RTX analogues are listed in Table 1.

Table 1.

Potencies of RTX analogues for binding to rat TRPV1 and for inducing calcium influx in CHO/TRPV1 cells

graphic file with name nihms388384t1.jpg
R3 R4 R5 RTX1
binding
(Ki =nM)		 Ago1,2
(EC50 = nM)		 Ant1,3
(Ki =nM)
1 OCH3 OH H 0.043 0.27 NE
2 OCH3 OH I 0.61 ± 0.08 NE 12.2 ± 4.0
31 OCH3 OH F 0.26 ± 0.04 1.88 ± 0.24 NE
32 OCH3 OH Cl 1.03 ± 0.14 5.7 ± 1.7 NE
33 OCH3 OH Br 0.17 ± 0.05 (19%) 7.2 ± 1.6
34 OCH3 OAc Br 0.30 ± 0.02 (37%) (30%)
35 Br H Br 110 ± 36 1070 ± 110 NE
36 OCH3 NH2 H 0.13 ± 0.03 0.46 ± 0.10 NE
37 OCH3 NH2 F 0.70 ± 0.16 1.14 ± 0.23 NE
38 OCH3 NH2 Cl 0.85 ± 0.02 (34%) (56%)
39 OCH3 NH2 Br 1.99 ± 0.27 NE 2.81 ± 0.26
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1

Values represent the mean ± SEM of three or more independent experiments for Ki or EC50.

2

Values in parentheses represent the mean % of agonism relative to capsaicin.

3

Values in parentheses represent the mean % of antagonism of the capsaicin stimulated 45Ca2+ uptake response.

NE, no effect

Starting with RTX, 5-halogenation on the A-region progressively shifted the agonism toward antagonism as the size of halogen increased. 5-Fluoronation and 5-chlorination yielded the full agonists 31 and 32, which were 9- and 21-fold less potent, respectively, than RTX for their functional activity. 5-Bromination caused a partial shift in function, providing the partial antagonist 33 with Ki (ant) = 7.2 nM and 19% residual agonism. Finally, 5-iodination (I-RTX) afforded the full antagonist 2 with Ki (ant) = 12.2 nM under the same assay conditions. The 5-halogenated RTX analogues (31–33) bound to TRPV1 with lower affinity than did RTX (1) but showed little difference as the size of the halogen increased, with a range of Ki = 0.17 to 1.03 nM. Compound 34, the O-acetyl analogue of 33, displayed modestly weaker binding affinity and antagonism compared to 33. The 3,5-dibromo analogue 35 was a full agonist, albeit with weaker binding affinity and agonist potency.

Next, the halogenated 4-amino RTX analogues were explored as bioisosteres of the corresponding 4-phenolic agonists. The parent 4-amino RTX (36) showed high affinity binding and potent full agonism. The values of Ki = 0.13 nM and EC50 = 0.46 nM were only 2–3 fold less potent than RTX. As was the case with the 5-halogenated RTX derivatives, 5-halogenation on the A-region in 4-amino RTX also shifted the agonism toward antagonism progressively as the size of the halogen increased. 5-Fluoronation produced full agonist 37, which was 2.5-fold less potent than the parent 36, as found in the RTX series. However, 5-chlorination already started to shift the agonism of 36 toward antagonism to afford the partial antagonist 38 and 5-bromination yielded the full antagonist 39. Of particular note, compound 39 displayed high potency as an antagonist with Ki (ant) = 2.81 nM, which was 4-fold better than I-RTX under the same assay conditions. As found in the RTX series, the binding affinities of the 5-halogenated 4-amino RTX analogues (37–39) were weaker than that of 4-amino RTX (36) and little difference was examined as the size of the halogen increased, with a range of Ki = 0.7 to 1.99 nM. Unfortunately, the 5-iodo 4-amino analogue could not be investigated due to synthetic difficulty.

In order to investigate the structural basis for the difference in the functional activities of the TRPV1 agonists and their halogenated analogues as antagonists, we performed conformational analysis of RTX (1) and 36 (KMS-112) as agonists and I-RTX (2) and 39 (DWK-1427) as antagonists using the SYBYL Grid Search method.25 The resulting lowest energy conformers are as shown in Figure 2 in which overlays of RTX vs I-RTX and 36 vs 39 are represented in Figure 2A and Figure 2B, respectively.

Figure 2.

Figure 2

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The lowest energy conformers of (A) RTX and I-RTX, and (B) 36 and 39. Carbon atoms are shown in green for RTX, white for I-RTX, purple for 36 and pink for 39. Agonists RTX and 36 are depicted as ball-and-stick. Antagonists I-RTX and 39 are depicted as capped stick. Hydrogen bonds are displayed in black dashed lines, and non-polar hydrogens are undisplayed for clarity.

In their lowest energy conformers, the antagonists 2 and 39 showed bent conformations in which the methoxy group appeared to form an intramolecular hydrogen bond with the C-4 hydroxyl group of the diterpene core. In contrast, the agonists did not show this intramolecular hydrogen-bond in their lowest conformers and instead allowed the A-region to remain free to interact with residues in the binding site. The analysis indicates that the 5-halogen promoted bending of the B-region to permit this internal hydrogen-bond formation between 3-methoxy of the A-region and C4-OH of the diterpene and the preference for this conformation was higher as the size of the halogen increased. A prediction is thus that the binding of this bent conformation, unlike the more extended conformation of RTX agonists, should fail to induce the shift in the conformation of the tetrameric TRPV1 channel associated with channel opening. This model thus suggests novel strategies for antagonist design.

In conclusion, we have systematically modified the aromatic A-region of RTX and its 4-amino surrogate by halogenation at the 5-position in order to explore the role of halogens in the reversal of activity from agonism to antagonism. 5-Halogenation converted the agonists to partial or full antagonists, and the extent of antagonism reflected the order of I > Br > Cl > F. Antagonism was further favored in derivatives of the 4-amino RTX surrogate compared to derivatives of RTX itself. Of particular note, the 5-bromo 4-amino RTX analogue (39) was a potent, full antagonist with Ki (ant) = 2.81 nM, which was 4.5-fold more potent than I-RTX (2) under our conditions. Molecular modeling of selected agonists and antagonists demonstrated that the 3-methoxy of the A-region in agonists remained free to interact with the receptor for agonism, whereas a 5-halogen in the antagonists favored a bent B-region, allowing the 3-methoxy to form an intramolecular hydrogen bond with the C4-hydroxyl of the diterpene.

Acknowledgments

This research was supported by Grant R11-2007-107-02001-0 from the National Research Foundation of Korea (NRF), the National Core Research Center (NCRC) program (R15-2006-020) of MEST and NRF through the Center for Cell Signaling & Drug Discovery Research at Ewha Womans University (to S. Choi), and by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, National Cancer Institute. We thank numerous research fellows for some of the biological analyses.

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