Halogenation of 4-Hydroxy/Amino-3-methoxyphenyl Acetamide TRPV1 Agonists Showed Enhanced Antagonism to Capsaicin
As an extension of our analysis of the effect of halogenation on thiourea TRPV1 agonists, we have now modified selected 4-hydroxy(or 4-amino)-3-methoxyphenyl acetamide TRPV1 agonists by 5- or 6-halogenation on the aromatic A-region and evaluated them for potency for TRPV1 binding and regulation and for their pattern of agonism / antagonism (efficacy). Halogenation shifted the functional activity at TRPV1 toward antagonism with a greater extent of antagonism as the size of the halogen increased (I > Br > Cl), as previously observed for the thiourea series. The extent of antagonism was greater for halogenation at the 5-position than at the 6-position, in contrast to SAR for the thiourea series. In this series, compounds 55 and 75 showed the most potent antagonism, with Ki(ant) = 2.77 and 2.19 nM, respectively, on rTRPV1 expressed in Chinese hamster ovary cells. The compounds were thus ca. 40-60 fold more potent than 6\xe2\x80\x99-iodononivamide.
1. Introduction<!>2.1. Chemistry<!>2.2. Biological Activity<!>3. Conclusion
<p>The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a molecular integrator of nociceptive stimuli expressed predominantly on unmyelinated pain-sensing nerve fibers (C-fibers) and small Aδfibers in the dorsal root, trigeminal, and nodose ganglia.1-3 TRPV1 is structurally a homotetramer and functions as a non-selective cation channel with high Ca2+ permeability. The receptor is activated not only by protons,4 heat,5 and endogenous substances such as anandamide6 and lipoxygenase products7 but also by natural vanilloids such as capsaicin (CAP)8 and resiniferatoxin (RTX)9 or indirectly by bradykinin10. Its activation by these agents leads to an increase in intracellular Ca2+ resulting in excitation of the primary sensory neurons. The functional blockade of this receptor, by antagonism or by desensitization subsequent to stimulation by agonists, promises considerable therapeutic utility targeting inflammatory and neuropathic pain, cystitis, and bladder hyperreflexia.11-15</p><p>It was previously reported that the halogenation of the aromatic A-ring of agonists shifted the agonism of the ligands toward antagonism. Two leading examples are 5-iodoresiniferatoxin (1)16 and 6-iodononivamide (2)17, iodinated products of the agonists RTX and nonivamide, which showed potent antagonism. These finding prompted us to investigate how halogenation on the aromatic A-region of our potent N-(4-hydroxy-3-methoxybenzyl)thiourea agonists (3, 4)18-20 would modulate their functional activity. For this series, we found that halogenation shifted the activity of the ligands from agonism toward antagonism and that the extent depended on both the size of the halogen and the position halogenated. Iodination conferred the most enhancement of antagonism, with lesser effects from bromination and chlorination in that order. 21 The extent of antagonism upon 6-halogenation was higher than that of the corresponding 5-halogenation. The pattern of SAR resembled that of the capsaicinoids rather than that of resiniferatoxin, although the lead structure was derived from a pharmacophore model of resiniferatoxin.</p><p>As a continuing part of our SAR analysis of the effect of halogenation, we have investigated the effect of halogenation on potent acetamide TRPV1 agonists. Here, we describe the structure activity relationships of 5 and 6-halogenated analogues of our lead agonists (5, 6)22 for TRPV1 binding and regulation and for their pattern of agonism / antagonism (efficacy).</p><!><p>The target halogenated acetamide compounds (41-81) were synthesized in general by the coupling of the carboxylic acids of halogenated A-region moieties with the corresponding C-region amines, followed by appropriate O-deprotection if needed as represented in Scheme 1. The syntheses of 5-halogenated 3-methoxy-4-hydroxy (or 4-acetoxy) and 6-halogenated 3-methoxy-4-hydroxy (or 4-acetoxy) A-regions were shown in Schemes 2 and 3, respectively, and the regioselectivity of halogenation was controlled by 4-hydroxy or 4-O-protected groups. The syntheses of 3-halo(or 3,5-dihalo)-4-hydroxy and 3-halo(or 3,5-dihalo)-4-amino A-regions were shown in Schemes 4 and 5 and selectivity was obtained by controlling the amount of halogenating reagent. 3-Methoxy-4-amino and 5-halo-3-methoxy-4-amino A-regions were synthesized by nitration of (3-hydroxyphenyl)acetic acid and halogenation of the 4-aminophenyl as represented in Scheme 6. 3-Methoxy-4-methylsulfonylamino and 6-bromo-3-methoxy-4-methylsulfonylamino A-regions were synthesized starting from the 3-methoxy-4-amino intermediate 31 as depicted in Scheme 7. Finally, the 3,5-dibromophenyl A-region was synthesized from 1,3-dibromo-5-fluorobenzene by nucleophilic substitution of ethyl cyanoacetate followed by decarboxylation as represented in Scheme 8.</p><!><p>The binding affinities and agonistic/antagonistic functional activities of the synthesized TRPV1 ligands were assessed in vitro by a binding competition assay with [3H]RTX and a functional 45Ca2+ uptake assay using rat TRPV1 heterologously expressed in Chinese hamster ovary (CHO) cells, as previously described.23 The results are summarized in Tables 1-3, together with the potencies of the parent agonists 5 and 6. In particularly, compound 6, N-[3-pivaloyloxy-2-(4-t-butylbenzyl)propyl]-2-(4-hydroxy-3-methoxyphenyl)acetamide, was previously reported as a simplified RTX analogue and was a potent high affinity TRPV1 agonist with Ki (binding) = 13.1 nM and EC50 (agonism) = 4.08 nM. 6'-Iodononivamide (2)17, previously reported as the most potent antagonist in a series of nonivamides, was also evaluated as a reference and displayed full antagonism with Ki (binding) = 1,320 nM and Ki (antagonism) = 127 nM, respectively.</p><p>The structure-activity relationship analysis for A-region halogenation of (4-hydroxy-3-methoxyphenyl)acetamide agonists used as an initial choice a simple 4-t-butylbenzyl C-region. The TRPV1 binding and functional activity of halogenated analogues of N-(4-t-butylbenzyl)-2-(4-hydroxy-3-methoxyphenyl)acetamide (5) are outlined in Table 1. Starting with agonist 5, 5-halogenation on the A-region progressively shifted the agonism toward antagonism as the size of halogen increased. For example, whereas 5-chlorination produced a partial antagonist 41 with 66% antagonism, 5-bromination gave an almost full antagonist 42 with only residual agonism, and 5-iodination afforded a full antagonist 43 with Ki (ant) = 17.2 nM. The 5-halogenated analogues (41-43) bound with higher affinity than did 6'-iodononivamide (2) and the parent compound (5), but showed little difference as the size of the halogen increased. In contrast, although 6-halogenation also shifted the agonism of 5 toward antagonism, the extent of the antagonism was less compared to 5-halogenation, full antagonism was not achieved even with the 6-iodo derivative (46), and the binding affinities of the 6-halogenated analogues (44-46) were weaker than those of the corresponding 5-halogenated analogues. A similar SAR pattern was observed with the 4-O-acetylated analogues (47-52) of 5- and 6-halogenated parent compounds.</p><p>The corresponding SAR of halogenated analogues possessing an N-(3-pivaloyloxy-2-benzylpropyl) C-region are described in Table 2. Similar to the SAR of the derivatives with an N-(4-t-butylbenzyl) C-region shown in Table 1, the halogenation of parent compound 6 converted the agonists to partial or full antagonists, and the extent of antagonism reflected the order of I > Br > Cl and 5-halogenation > 6-halogenation. Consequently, the 5- and 6-Iodo analogues (55, 58) were potent, full antagonists with Ki (ant) = 2.8 and 13.5 nM, respectively. Also, the 5-bromo compound (54) showed high affinity with a Ki = 9.4 nM and potent antagonism with a Ki (ant) = 8.77 nM.</p><p>A similar SAR pattern was again observed for the 4-O-acetylated analogues (59-64) of the halogenated parent compounds. Their extents of functional antagonism were similar to those of the corresponding 4-hydroxy compounds, and the binding affinities were comparable or weaker.</p><p>The SAR of halogenated analogues possessing an N-3-pivaloyloxy-2-(3,4-dimethylbenzyl)propyl C-region was similar to that of the 4-t-butylbenzyl series, although they variably showed weaker binding or less antagonism, emphasizing the contribution of the C-region to the pattern of functional response (Table 2).</p><p>Since compound 54 was found to be a potent high affinity TRPV1 antagonist, further optimization was conducted with its derivatives and the results are shown in Table 3. Removal of the 3-methoxy group in 54 led to partial agonist 69 with lower binding affinity. Incorporation of an additional halogen, providing 3,5-dichloro and -dibromo analogues, gave potent antagonists 70 and 71 with Ki (ant) = 4.26 and 10.7 nM, respectively. Deletion of the 4-hydroxyl group in 71 led to the reduction in both binding affinity and antagonist potency as expected, although complete antagonism was still retained.</p><p>Next, the halogenated 4-amino-3-methoxy analogues as biosteres of the corresponding 4-hydroxy-3-methoxy agonists were explored as shown in Table 3. The 4-amino surrogate (73) of lead agonist 5 was also a full agonist with Ki = 107 nM and EC50 = 22.9 nM. However its binding affinity and potency as an agonist were 8-fold and 5-fold less than for 5. The 5-halogenation of 73, providing 74 and 75, shifted the agonism to full and potent antagonism for both derivatives, whereas the 5-chloro derivative (41) of the corresponding 4-hydroxy analogue had been only a partial antagonist. Of particular note, the 5-bromo compound 75 display very potent antagonism with Ki (ant) = 3.4 nM. The 5-halogenated 4-amino compounds (76, 77) demonstrated mixed functional activity. However, incorporation of an additional halogen, giving 3,5-dihalo-4-amino analogues (78, 79), was able to convert the partial antagonism to full antagonism.</p><p>The halogenation of the 4-methylsulfonylamino-3-methoxy analogue (80), another biostere of the 4-hydroxy-3-methoxy agonist, also caused a shift to more antagonism. Thus, the 6-bromo analogue (81) had more antagonistic activity compared to 80.</p><p>Previously, halogenated resiniferatoxin (RTX) and capsaicinoid analogues were shown to have different structure-activity relationships for the reversal of activity from agonism to antagonism. In the RTX series, whereas 5-iodination produced full antagonism,16 6-iodination led to partial agonism with 50% efficacy.24 On the other hand, in the capsaicinoid series, although both 5- and 6-iodo derivatives behaved as powerful antagonists under the same assay conditions, the 6-iodo derivatives showed a greater extent of antagonism than did the corresponding 5-iodo analogues. We describe here that halogenation at the 5-position of our TRPV1 ligands possessing an amide B-region was more effective than halogenation at the 6-position to confer antagonism. This contrasts with the SAR we have previously reported for halogenated analogues with a thiourea containing B-region21. We conclude that halogenated analogues with an amide containing B-region behave more like RTX-related ligands, whereas those with a thiourea containing B-region behave more like capsaicinoid-type ligands.</p><!><p>We have systematically modified the aromatic A-region of potent acetamide agonists by 5- and 6-halogenation in order to explore the role of halogens in the reversal of activity from agonism to antagonism and we have analyzed their structure-activity relationships. In general, the halogenation of 4-hydroxy, 4-acetoxy, 4-amino and 4-methylsulfonylamino parent compounds shifted the activity of the ligands from agonism to antagonism and the extent of the shift depended on both the size of the halogen, the halogenated position, and number of halogens added. As was the case with the thiourea B-region analogues previously reported,21 the amide B-region analogues showed more antagonism as the size of the halogen increased, I > Br > Cl. However, in contrast to the results with the thiourea B-region analogues, the amide B-region analogues showed more antagonism upon 5-halogenation than upon 6-halogenation. Among the halogenated ligands synthesized, compounds 55 and 75 were found to be very potent full antagonists with Ki (antagonism) = 2.8 and 3.4 nM, being 45-fold and 37-fold more potent than 6'-iodononivamide, respectively. Our analysis indicated that the SAR of acetamide B-region agonists upon halogenation is similar to that of resiniferatoxin.</p>