Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel

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Targeting transient receptor potential vanilloid 1 (TRPV1) channel softly: the discovery of Passerini adducts as a topical treatment for inflammatory skin disorders Marta Serafini, Alessia Griglio, Silvio Aprile, Fabio Seiti, Cristina Travelli, Franco Pattarino, Giorgio Grosa, Giovanni Sorba, Armando A. Genazzani, Sara Gonzales-Rodriguez, Laura Butron, Isabel Devesa, Asia Fernández-Carvajal, Tracey Pirali, and Antonio Ferrer-Montiel J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00109 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Journal of Medicinal Chemistry

Targeting transient receptor potential vanilloid 1 (TRPV1) channel softly: the discovery of Passerini adducts as a topical treatment for inflammatory skin disorders Marta Serafini,† Alessia Griglio,† Silvio Aprile,† Fabio Seiti,† Cristina Travelli,† Franco Pattarino,† Giorgio Grosa,† Giovanni Sorba,† Armando A. Genazzani,† Sara GonzalezRodriguez,‡ Laura Butron,‡ Isabel Devesa, # Asia Fernandez-Carvajal,*, ‡ Tracey Pirali,*, † and Antonio Ferrer-Montiel ‡ AUTHOR ADDRESS: †

Department of Pharmaceutical Sciences, Università del Piemonte Orientale, Largo Donegani 2,

28100 Novara, Italy ‡

Instituto de Biología Molecular y Celular, Universitas Miguel Hernandez, Av de la Universidad

s/n, 03202 Elche, Spain #

AntalGenics, SL. Ed. Quorum III, Parque Científico UMH. Av de la Universidad s/n, 03202

Elche, Spain

ACS Paragon Plus Environment

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KEYWORDS: TRPV1 channel, capsaicin, Passerini multicomponent reaction, isocyanide, soft drug, skin disorders, skin inflammation, pruritus

ABSTRACT Despite being an old molecule, capsaicin is still a hot topic in scientific community and the development of new capsaicinoids is a promising pharmacological approach in the management of skin disorders related to inflammation and pruritus. Here we report the synthesis and the evaluation of capsaicin soft drugs that undergo deactivation by the hydrolyzing activity of skin esterases. The implanting of an ester group in the lipophilic moiety of capsaicinoids by the Passerini multicomponent reaction affords both agonists and antagonists that retain transient receptor potential vanilloid 1 channel (TRPV1) modulating activity and, at the same time, are susceptible to hydrolysis. The most promising antagonist identified shows in vivo antinociceptive activity on pruritus and hyperalgesia withouth producing hyperthermia, thus validating it as novel treatment for dermatological conditions that implicate TRPV1 channel dysfunction.

Introduction TRPV1 is arguably the best-characterized member of the transient receptor potential (TRP) family: it is a calcium permeable non-selective ion channel gated by noxious heat, extracellular protons and bioactive lipids. Its role in pain physiology and neurogenic inflammation is widely validated,1 but there is accumulating evidence that TRPV1 has additional functional roles away from sensory nerve activity. Indeed, different reports have unambiguously identified the presence of TRPV1 on numerous non-neuronal cell types, such as both epidermal and hair follicle keratinocytes, mast cells, dendritic cells and sebocytes.2 Particular attention has been


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recently given to the physiologic role of TRPV1 in keratinocytes, which has been related not only to differentiation, proliferation and homeostasis of the epidermal barrier, but also to cutaneous immunological function.3 By virtue of its dual “sensory” and “non-sensory” localization, TRPV1 plays a fundamental role in the pathogenesis of dermatological pathologies especially related to inflammation and pruritus.4 Early studies on TRPV1 modulators were centered on agonists, as they lead to the opening of TRPV1, followed by desensitization of the channel which is linked to their therapeutic potential. The most notorious clinical limitation of capsaicin and related vanilloids is the TRPV1-coupled acute excitation of the sensory C-afferents, which paradoxically results in a marked burning sensation and reduced patient compliance.5 Besides the algogenic side-effect, capsaicin is a very lipophilic, non-water-soluble compound and can reside in human skin relatively unchanged for a long period of time, leading to frequent erythema reactions depending on drug concentration in stratum corneum.6 Recently, it has been reported that chronic, long-term topical application of capsaicin increases skin carcinogenesis in mice treated with a tumor promoter.7 These results might imply that caution should be exercised when using capsaicin-containing topical applications in the presence of a tumor promoter, such as for example sunlight. Hence, existing research is trying to circumvent these side-effects with a focus on (i) increasing the local concentration of TRPV1 agonists either by site-specific injections or topical patches;8 (ii) developing agonists which cause only minor excitation, but hold strong desensitization power;9 (iii) using pore permeable capsaicinoids which target only hyperactive channels;10 (iv) developing photoswitchable agonists that are applied globally, but activated only locally under the control of light.11


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Besides agonists, a number of potent, small molecule antagonists have been developed.12 To date, at least fifteen compounds entered Phase 1 clinical trials and five of these agents have progressed into Phase 2 clinical trials, but none of them have advanced beyond, due to their undesirable side effects that led to the termination of several drug development programs.13 Yet, TRPV1 antagonists show worrisome adverse effects, largely arising from interference with the physiological function of TRPV1-expressing cells. The failure of the first TRPV1 antagonist, Amgen’s AMG-517, in Phase I clinical trial was due to marked hyperthermia and alerted the scientific community about the potential of hyperthermic risk associated with systemic absorption of this class of molecules.14 To circumvent the systemic side effects of these agents, a number of topical formulations are being developed, but this might not be enough. Indeed, similarly to capsaicin, long treatment with AMG-9810 (Chart 1), a typical antagonist, has been shown to promote tumor development through EGFR/Akt/mTOR signalling pathway in preclinical murine models15 and this evidence has warned about the topical use of TRPV1 antagonists. Very recently, this risk has been discharged by in vitro and in vivo experiments with respect to AMG-9810 (Chart 1), SB-705498 (Chart 1), PAC-14028 (Chart 1) and capsazepine (Chart 1)16 and the correlation between the topical use of antagonists and tumor development is still a subject for debate.Taken together, topical TRPV1 modulators, either alone or in conjunction with other agents, have the potential to alleviate a host of skin conditions associated with inflammation and/or pruritus, but have limitations in patient compliance and safety that drive the need for novel therapeutic strategies. Here we address this issue and report the synthesis and the evaluation of capsaicin soft drugs designed to undergo deactivation by the hydrolyzing activity of esterases. Soft drugs are active analogues of a lead compound that are deactivated in a predictable and controlled way


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(preferentially hydrolitically) after having exerted their biological activity. The desired activity is generally local, and the soft drug is applied or administered near the site of action. Therefore, they usually produce their therapeutic effect locally, but their distribution away from the site results in a metabolic deactivetion that prevents undesired systemic activity or toxicity. Examples for practical use of soft drugs are provided in the literature, covering the area of antimicrobials, anticholinergics, corticosteroids, ACE inhibitors,17 and have resulted in a number of already marketed drugs (e.g. esmolol, remifentanil). Here we report that the implanting of an ester group in the tail of capsaicinoids provides TRPV1 agonists and antagonists that retain the modulating activity, while being susceptible to deactivation by hydrolysis (Scheme 1). The most promising antagonist, compound 36, attenuates in vivo hyperalgesia and pruritus when administered systemically and locally, without affecting the thermal sensitivity nor body temperature. Thus, our capsaicin-based soft antagonists have the potential to be topically deactivated and to minimize both the side reactions associated to the systemic manipulation of TRPV1 and the burning effect of topical application of capsacin, pointing to a promising, costeffective and well-tolerated new strategy for treating those refractory cutaneous diseases where TRPV1 plays a pivotal role.

Results Chemistry Multicomponent reactions (MCRs)18 are those transformations where three or more substrates combine in one step to give a product that contains essential parts of all of them, allowing the generation of complex molecules in very few synthetic steps. Driven by high atom and step economy, MCRs have emerged over the last 15 years as perfectly suited tools for the synthesis of


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collections of drug-like compounds to be used in the realm of drug discovery.18a,19 Among MCRs, the Passerini reaction20 is less described in medicinal chemistry and the reason resides in the fact that the Passerini adduct contains an amide together with an ester moiety, the latter being hydrolytically unstable under in vivo conditions, and therefore precluding a systemic use of these compounds. Nevertheless, in some circumstances this problem can be turned into an opportunity and we have exploited this multicomponent reaction to generate soft drugs of capsaicin. The soft drug principles is based on bioactive molecules which undergo predictable metabolism to inactive metabolites, after exerting their therapeutic effect.17 Capsaicin accommodates in the TRPV1 intracellular vanilloid pocket in a “tail-up, head-down” configuration, where the aliphatic tail points towards the membrane face of TRPV1 and interacts with the channel through van der Waals forces, while the vanillyl moiety and the amide function face toward the cytoplasmic side of TRPV1 and form crucial hydrogen bonds with residues of S513, E571 and T551, together with other minor interactions.21 As a proof of principle, we inserted via Passerini reaction an ester moiety in the lipophilic tail of capsaicin and synthesized product 12, demonstrating that the activity on TRPV1 was retained, despite the change in terms of polarity and electronic properties. Moreover, we demonstrated that the corresponding metabolite 49 was devoid of activity on TRPV1. Proven the validity of our strategy, we then proceeded to synthesize two different series of modulators, the first as putative agonists and the second as putative antagonists of the TRPV1 channel. Indeed, simply changing the C-6´ substitution of vanillyl isocyanide (non-iodinated or iodinated), agonists and antagonists were easily accessed (Scheme 1). This is in accordance with similar observations done on resiniferatoxin,22 and on capsaicinoids,23 which demonstrated that the iodination at C-6´ causes a dramatic switch in functional activity, generating compounds that behave as TRPV1 antagonists


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rather than agonists. As depicted in Scheme 2, the preparation of the vanillyl isocyanides 3 and 4 was straightforward. The vanillylamine was formylated and subsequently tert-butyldimethyl silyl (TBDMS) protected at the phenolic group. The use of a protecting group was required for the success of the dehydrative reaction. The common formamide intermediate 54 was directly dehydrated or selectively iodinated at 6´ first and dehydrated thereafter. For the first series of compounds, a carboxylic acid 1, formalin 2 and the TBDMS-protected vanillyl isocyanide 3 reacted to form α-acyloxy vanillylamides 7-25 (Scheme 3). After completion of the reaction, the crude material was directly deprotected in the presence of tetra-nbutylammonium fluoride (TBAF) and acetic acid. The latter was necessary in order to avoid the partial hydrolysis of the ester moiety during the reaction and to increase the yield. Indeed, fluoride ion, because of its pronounced basicity, mediates the basic hydrolisis of the ester group displayed by the Passerini adduct, causing low yields of the final product in this two-step sequence. On the other hand, we have demonstrated that the addition of acetic acid during the deprotection buffers the reaction mixture and subsequently improves the yield of the reaction. In parallel, a second series of products (26-44) was prepared starting from 6´-iodovanillyl isocyanide 4 in a similar way (Scheme 3). A vast array of carboxylic acids 1 from the fatty pools was screened and the choice of the carboxylic partner in the Passerini reaction was inspired by the results described in the literature by modifying the acyl moiety of capsaicin.24 The only exception is represented by 1,1′,2-tris-norsqualene acid, as this substructure has never been reported in capsaicinoid modulators. Previous reports where capsaicinoids with a 3,4-cathecolic moiety retained TRPV1 activity25 prompted us to investigate whether the removal of the methyl group at 3´ position affected the


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agonism/antagonism on TRPV1 channel. To this aim, we prepared the two isocyanides 5 and 6 according to Scheme 4. After protection of the cathecolic moiety, the benzylamine was formylated and directly dehydrated to give the isocyanide 5, or alternatively, selectively iodinated at 6´ and dehydrated thereafter. With these isocyanides in our hands, two agonists and two antagonists were synthesized via Passerini reaction followed by deprotection (Scheme 5). Metabolites (49-52) deriving from the hydrolysis of the two series of compounds were prepared according to Scheme 6. Vanillyl hydroxyamides (49 and 50) were simply synthesized by basic hydrolysis of the Passerini products 12 and 31, while the metabolites displaying the cathecolic moiety needed a specifically tailored synthetic procedure. Indeed, all the attempts made to synthesize these products by saponification of the Passerini adducts failed, as the cathecolic function was prone to oxidation and reacted giving complex reaction mixtures. Therefore, we designed a synthetic procedure made of two steps. The first one is a truncated Passerini reaction where the 2hydroxymethyl benzoic acid furnishes a pseudo-molecule of water,26 avoiding the use of acidic promoters and allowing the retention of the protecting groups. The second reaction is the deprotection of the TBDMS moieties in the presence of TBAF. Thanks to this synthetic strategy we were able to access the metabolites 51 and 52 in high yields. The forty-two synthesized agonists and antagonists and the four hydroxyamide metabolites were then biologically evaluated. Screening of synthesized compounds by Ca2+-fluorimetry All compounds were tested at three different concentrations (100, 10 and 1 µM) on TRPV1 channels stably expressed in the human neuroblastoma SH-SY5Y cell line. The agonist-induced


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intracellular Ca2+ signals were measured using a fluorescent Ca2+ indicator, in the absence and in the presence of test compounds. Capsaicin was used as TRPV1 agonist and ruthenium red as TRPV1 antagonist. The EC50 and IC50 values were calculated for compounds displaying a percentage of activity higher than 50% of the reference compound at 1 µM (Table 1, Supporting Information for concentration-response curves). Regarding the non-iodinated compounds with a saturated and linear chain, compounds 7-10, 1214 and 16-19 show a bell-shaped activity/chain-length curve (Figure 1), with a plateau in correspondence of a number of acyl carbons of eight (13, 80.2%). Increasingly shorter (7-10, 12) or longer chains (14, 16-19) cause a progressive decrease in activity. The presence of E unsaturations in 10 (11; 32.0%) at 1 and 3 positions does not improve the activity of compound 10 (38.9%). In a similar way, the introduction of double bonds at 2 (E) and 6 positions, together with branching, in compound 13 (80.2%) slightly decreases the agonistic activity (15, 59.8%). On the other hand, the insertion of a double bond (Z) at position 8 (20) in the stearic substructure (19) significantly improves the TRPV1 agonism, while the simultaneous introduction of a hydroxyl group (R) at position 11 (21) causes a drop in activity, in contrast with reported observations done on olvanil.24a Esterification of such hydroxyl group with phenylacetic acid does not ameliorate the activity (22). The arachidonic substructure still allows for an activity on TRPV1 channel (24, 54.7%), while the longer erucic portion (23, 22.7%) decreases the agonism. Finally, squalene substructure displaying six unsaturations (25, 71.8%) significantly improves the activity. As the use of the squalene-incorporating capsaicinoids has never been reported, it is interesting to stress that the conjugate with 1,1′,2-tris-nor-squalene acid displayed a high potency on TRPV1 channel. This serendipitous discovery paves the way for further evaluations from a pharmaceutical standpoint as squalene derivatives are known to form self-


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assembled nanostructures and squalenoylation approach is finding applications in several contexts.27 Removal of the methyl at 3´ position on the aromatic ring did not affect the agonism, leading to two compounds (45, 46) which conserve an activity comparable to the corresponding methylated analogues (15, 25). Iodination of the 6´ carbon atom of the vanillyl moiety abolished the agonist activity, caused a reversal of vanilloid activity in all cases and led to the discovery of two potent antagonists (34, 36), with an IC50 value in the sub-micromolar range (0.09 µM; 0.93 µM). A good correlation was found between the potency of agonists and their corresponding 6´-iodo derivatives as antagonists, except for compounds 25 and 46, which show good activities in terms of TRPV1 activation in contrast to the lower potencies displayed by the antagonists (44 and 48, respectively) with the same acyl carbon chain. As for agonists, the optimal length of the side chain is eight acyl carbon atoms (32), while the unsaturation and branching displayed by geranic substructure increase the activity (34). A significant antagonism is also seen when lauric acid is used in the Passerini reaction (36), while, in contrast to the non-iodinated series, the removal of the methyl at 3´ position is detrimental for the activity (47, 48). Metabolic stability Considering calcium fluorimetry results, eleven compounds (8 agonists and 3 antagonists) were selected and evaluated in terms of hydrolytic stability (see Supporting Information for full experimental data and chromatographic methods).28 For this task, a combined approach based on HaCaT (a spontaneously immortalized male human keratinocyte cell line) homogenate, human plasma and liver S9 fraction was exploited, considering the relevance of these three systems in esterase activity. In particular, the hydrolytic


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stability was evaluated by HPLC-UV analysis monitoring the disappearance of substrate over incubation time with respect to the initial amount (Table 2) and assessing the presence of peaks related to the metabolites 49-51. Overall, all the tested compounds resulted rather stable toward HaCaT homogenate incubation. Only the agonists 17, 18 and 20 underwent a mild substrate depletion, with the residual substrate ranging from 83.8 to 92.6%. In human plasma incubations, all the tested TRPV1 agonists showed an overall good stability, being the compounds 17, 20, and 25 slightly hydrolysed with residual substrates after 20 minutes of 89.6, 92.5, and 78.1%, respectively. Conversely, the tested TRPV1 antagonist 32, which displays a short, saturated and linear chain, resulted largely unstable in plasma with a residual substrate of 32.1%, while the longer 36 is slowly hydrolized (82.0%) and the branched and unsaturated 34 is exceptionally stable (100.5%). In liver S9 fraction, all the compounds resulted less stable than in HaCaT homogenate and plasma. The most resistant compounds toward hepatic esterases resulted to be the squalene derivatives 25 and 46 (residual substrate 71.8 and 70.9%, respectively), whereas the long linear (20) and branched esters (15 and 34) resulted quite stable, being the residual substrate 53.7, 63.2, and 50.1%, respectively. The esters displaying linear alkyl chains containing up to twelve acyl carbon atoms (17, 32) were totally depleted within 15 minutes. Curiously, among the compounds displaying the geranic substructure, 45 underwent faster hydrolysis (residual substrate 2.2%) compared to 15 (63.2%) in S9 incubations. This suggests that for compounds featuring the geranic residue, the presence of the catecholic moiety might increase the reactivity toward liver esterases, as confirmed also for the iodinated analogues 47 (3.6%) and 34 (50.1%). According to both their potency in vitro and hydrolytic behaviour, the agonists 17 and 46 and the antagonists 34 and 36 were selected for further evaluation of their metabolic stability in both


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primary adult epidermal keratinocytes and dermal fibroblasts homogenates (Table 2). It should be noticed, that unlike what predicted, the hydrolytic behaviour in HaCaT was not as informative for the selection, and this was the main reason to perform further metabolic assays. As expected, 17 and the corresponding iodinated derivative 36 are significantly hydrolyzed, while 46 is slowly metabolized and 34 results to be hydrolitically stable. The significant difference in biotransformation capacity among the three skin cell lines is ascribable to the different expression and activity between immortalized and primary cell cultures.29 Furthermore, in order to evaluate the role of human carboxylesterases (hCEs) in the hydrolysis, the four selected compounds were incubated in the presence of recombinant hCE isoforms 1 and 2 (Table 3). Whereas nonspecific esterase activity has been demonstrated in human skin and cultured skin cells, investigations into the expression of specific CEs in skin were controversial in the literature.30 Indeed, both Zhu and Fu examined the expression of CEs in HaCaT cell line and detected CE2 mRNA, whereas the CE1 counterpart was undetectable,29,31 while a more recent proteomic analysis of human skin highlighted the presence of only CE1 isoform.32 It is known that the preferential substrates for hCE1 are compounds esterified by small alcohols, while those for hCE2 are compounds esterified by relatively large alcohols.33 Based on these considerations, our TRPV1 modulators are expected to be preferential substrate for the hCE2 isoform. Indeed, while the four TRPV1 modulators were substrate for both hCE1 and hCE2, the hCE2 in vitro clearance for compounds 34 and 36 is markedly higher if compared with the other isoform. Moreover, the results were consistent with the hydrolytic activity observed in human liver S9 incubations.

Indeed, linear alkanoates 17 and 36 showed the highest metabolic

clearance, whereas the branched and long alkanoates 34 and 46 were hydrolysed to a lesser extent.


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Despite their good in vitro potencies, 34 was discarded because of its marked hydrolytic stability which goes against the soft drug principle, while 46 was not selected for further investigations as the cathecolic function is theoretically prone to microsomal oxidation and formation of reactive ortho-quinone species. Indeed, albeit at low levels compared to hydrolysis we were able to demonstrate in vitro this behaviour by detection and characterization by mass spectrometry of the corresponding glutathione (GSH) hydroquinone adducts 46-GSH and 51-GSH (see Supporting Information). The propensity of the cathecolic function to form ortho-quinone species together with their unknown role in vivo is the reason why in our structure-activity relationship (SAR) study only four compounds (45-48) displaying the cahecolic moiety were synthesized, despite their significant activity on TRPV1. In vitro effects of compound 36 on TRPV1 mediated neuronal excitability As iodinated compounds are devoid of the initial aversive side-effects related to the opening of the TRPV1 channel, we focused our efforts on the antagonists and especially on 36, as it shows a good potency (IC50 = 930 nM), it features a controlled rate of hydrolysis in the skin, it displays the lauric acid substructure which is not susceptible to isomerization or oxidation side-reactions, it shows a good chemical stability34 and excellent synthetic accessibility. To study the potential effects of compound 36 on sensory neuronal excitability, planar multielectrode arrays technology (MEA) was used to monitor TRPV1-evoked electrical activity in rat dorsal root ganglion (DRGs) cultures with stablished neuronal networks. For this purpose, high-density DRGs neurons were cultured on arrays in close contact with electrodes. Capsaicin application promoted action potentials and consequent neuronal spiking detection (Figure 2A). As expected, a second pulse of capsaicin partly reduced electrical activity due to desensitization of TRPV1 channel (ratio P2/P1 mean spike frequency = 0.87 ± 0.04). As expected, compound 36


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significantly suppressed TRPV1-mediated neuronal excitability in a concentration-dependent manner (Figure 2B). At 10 µM, Compound 36 inhibited 43% capsaicin-evoked action potentials (ratio P2/P1 mean spike frequency = 0.53 ± 0.03; ****p

Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel

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