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The replacement of amide and alkene groups in a biological setting with the 1,2,3-triazole group led to the discovery of compounds with a unique vanilloid/cannabinoid mixed profile. For example, the natural amides (see picture, above) and their triazole mimics (below) exhibit similar agonistic (X = H) or antagonistic (X = I) activity towards the TRPV1 receptor; however, only the triazole derivatives also show cannabinomimetic activity.

Starting from ROPA (2), analogues of RTX (1a) modified on the acyl side chain were prepared and evaluated for vanilloid activity in HEK-293 cells over-expressing the human recombinant TRPV1. The ROPA motif provided an enhancement of potency sufficient to expand the range of vanillyl surrogates to structural elements (e.g., an unsubstituted phenyl ring) that afford inactive analogues in compounds from the capsaicin series.

Replacement of the benzylamide motif of synthetic capsaicin (nonivamide, 1c) with a tetrazole moiety was detrimental for TRPV1 binding, but unexpectedly generated a potent and non-electrophilic TRPA1 agonist (4a). Spurred by this observation and by the relatively small number of non-covalent TRPA1 ligands reported so far, the benzylamide-to-tetrazole swap was investigated in the more lipophilic and powerful vanilloids olvanil (1d), rinvanil (1e), and phenylacetylrinvanil (1f). In all cases, the replacement was detrimental for TRPV1 binding, but a clear modulation of TRPA1 activity was observed. These observations show that the capsaicinoid pharmacophore displays orthogonal structure-activity relationships for TRPV1 and TRPA1 binding, and suggest the possibility of obtaining compounds with dual TRPV1/TRPA1 modulatory properties by exploration of the chemical space around the capsaicin motif. (C) 2015 Elsevier Ltd. All rights reserved.

Aromatic iodination ortho to the phenolic hydroxyl reverts the activity of the ultrapotent vanilloid agonist resiniferatoxin (RTX, 1a), generating the ultrapotent antagonist 5'-iodoRTX (1b). To better understand the role of iodine in this remarkable switch of activity, a systematic investigation on the halogenation of vanillamides, a class of compounds structurally simpler than resiniferonoids, was carried out. The results showed that (a) the antagonistic activity depends on the site of halogenation and is maximal at C-6', (b) iodine is more efficient than chlorine and bromine at reverting the agonistic activity, and (c) iodine-carbon exchange decreases antagonist activity. Iodine-induced reversal of vanilloid activity was also observed in vanillamides more powerful than capsaicin, but a poor correlation was found between agonistic and antagonistic potencies, suggesting that differences exist in the way vanillamides and their 6'-iodo derivatives bind to TRPV1.' '

The shift in nutritional sciences from survival and safety to the promotion of well-being has led to systematic investigations on the biological activity of natural products of dietary origin, questioning the assumption that food plants contain little if any secondary metabolites apart those revealed by our senses and responsible for their colour, taste, and flavour. With 25% of the human population consuming chilli pepper every day, capsaicin is the most important pharmacological agent we get from our diet, and the study of its pungency set in motion a multidisciplinary investigation that ultimately led to the discovery of vanilloid receptors (TRPVs), a class of ion channels involved in thermo-, chemo-, and mechanosensation, and whose malfunctioning is implicated in neurogenic inflammation and a host of other pathological conditions. A series of studies centred on the modification of capsaicin will be described, focusing on a) the preparation of a library of unnatural natural capsaicinoids and the identification of leads with the lipophilic C-moiety amenable to structure-activity study, and b) the reversal of the biological activity of capsaicin from a TRPV1 agonist into an antagonist by modification of its vanillyl moiety.

Olvanil ( N -9- Z -octadecenoyl-vanillamide) is an agonist of transient receptor potential vanilloid type 1 (TRPV1) channels that lack the pungency of capsaicin and was developed as an oral analgesic. Vanillamides are unmatched in terms of structural simplicity, straightforward synthesis, and safety compared with the more powerful TRPV1 agonists, like the structurally complex phorboid compound resiniferatoxin. We have modified the fatty acyl chain of olvanil to obtain ultra-potent analogs. The insertion of a hydroxyl group at C-12 yielded a compound named rinvanil, after ricinoleic acid, significantly less potent than olvanil (EC 50 = 6 versus 0.7 nM), but more versatile in terms of structural modifications because of the presence of an additional functional group. Acetylation and phenylacetylation of rinvanil re-established and dramatically enhanced, respectively, its potency at hTRPV1. With a two-digit picomolar EC 50 (90 pM), phenylacetylrinvanil (PhAR, IDN5890) is the most potent vanillamide ever described with potency comparable with that of resiniferatoxin (EC 50 , 11 pM). Benzoyl- and phenylpropionylrinvanil were as potent and less potent than PhAR, respectively, whereas configurational inversion to ent -PhAR and cyclopropanation (but not hydrogenation or epoxidation) of the double bond were tolerated. Finally, iodination of the aromatic hydroxyl caused a dramatic switch in functional activity, generating compounds that behaved as TRPV1 antagonists rather than agonists. Since the potency of PhAR was maintained in rat dorsal root ganglion neurons and, particularly, in the rat urinary bladder, this compound was investigated in an in vivo rat model of urinary incontinence and proved as effective as resiniferatoxin at reducing bladder detrusor overactivity.

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.

Background: Capsiate, the non-pungent ester isoster of capsaicin, and its dihydroderivative are the major capsaicinoids of sweet peppers. The remarkable difference between the sensory properties of capsaicin vs capsiate is solely due to the way the vanillyl and the acyl moieties of this basic structural motif are linked, via an amide bond in capsaicin-type compounds and via an ester bond in capsiate-type compounds. Aim of the study: Since capsaicin induces apoptosis in tumoral cells by a vanilloid receptor type 1(VR1)-independent pathway, we examined the effects of capsiates derived from sweet peppers in the ROS generation and induction of apoptosis in tumoral cells and if these are mediated independently from VR1. Methods: We have developed an expeditious synthesis of capsiates based on the esterification of vanillol with the Mitsunobu protocol. Capsiate-induction of apoptosis, generation of reactive oxygen species and disruption of the mitochondria transmembrane potential in tumoral cell lines were measured by flow cytometry. Chemopreventive activity was studied in a two-stage mouse skin carcinogenesis assay. Results: Capsiates induce apoptosis that was preceded by an increase in the production of reactive oxygen species and by a subsequent loss of mitochondria transmembrane potential (ΔΨm). These properties were retained in simplified synthetic analogues of natural capsiates, one of which (nor-dihydrocapsiate) showed powerful chemopreventive activity. Conclusions: These results suggest that capsiates and related synthetic analogues target a variety of pathways involved in cancer development and inflammation, and have considerable potential for dietary health benefits as well as for pharmaceutical development.

A simple and general synthesis of vanillamides was developed and employed to screen acids from the fatty and isoprenoid pools for new acyl templates of biological relevance as capsaicin analogues. Potent activation of the human vanilloid receptor 1 (VR1) was observed for the vanillamides of certain polyfunctional acids from both pools, showing that the vanilloid activity of capsaicinoids can be substantially improved by introducing polar groups and/or unsaturations on the acyl moiety. The activity of the unsaturated analogues was maintained or even increased by cyclopropanation, while omega dimerization led to a substantial increase of activity. Because of the wide structural diversity of the library of compounds screened, these observations could not be translated into a single framework of structure-activity relationships. Nevertheless, a series of new highly active leads was identified, validating the pharmacological potential of the unnatural combination of natural building blocks to provide new bioactive compounds.

The side chain benzylic methylene is a critical element for the vanilloid activity of resiniferatoxin (2a, RTX), and introduction of branching, oxygen functions, or isosteric substitution at this center proved detrimental, with a decrease of potency of 2-3 orders of magnitude compared to the natural product. Conversely, only a modest erosion of activity was observed upon a-methylation and alpha-methylenation of the side chain. Surprisingly, introduction of an iodine atom in the guaiacyl moiety of the oxygen isoster 2h led to an unexpected and remarkable (> 1000-fold) increase of potency, affording 2i, a compound that outperforms RTX in terms of vanilloid agonism and represents the first one-digit picomolar ligand of a TRP channel discovered to date. (C) 2009 Elsevier Ltd. All rights reserved.

The introduction of an iodine atom on the vanillyl moiety of nonivamide causes a switch in the vanilloid activity (TRPV1 antagonism versus TRPV1 desensitization) and nullifies the aversive properties of capsaicinoids. In the present study we investigated the effect of iodination in the vanillyl moiety on the antioxidant activity of capsaicinoids. To this aim, we have compared the protective effects of nonivamide and three iododerivatives, 2-iodononivamide (2IN), 5-iodononivamide (5IN), and 6-iodononivamide (6IN) in a series of in vitro models of lipid oxidation, namely the autoxidation and the FeCl(3)-mediated oxidation of linoleic acid at 37 degrees C and the thermal (140 degrees C), solvent-free oxidation of cholesterol. All tested compounds could protect linoleic acid and cholesterol against oxidative degradation, the order of potency being: nonivamide>5IN>6IN approximately 2IN. Our results show that, in general, the introduction of an iodine atom on the vanillyl moiety of nonivamide causes a decrease in the antioxidant activity, and this effect is sensitive to the position of iodine on the aromatic ring, with 5IN substantially retaining the efficacy of nonivamide to protect linoleic and cholesterol against free radical attack. Moreover, the pre-treatment with 5IN, at noncytotoxic concentrations, significantly preserved LDL from Cu(2+)-induced oxidative damage at 37 degrees C for 2h, inhibiting the reduction of polyunsaturated fatty acids and cholesterol and the increase of their oxidative products. The results of the present work suggest that 5IN exerts useful antioxidant properties in different in vitro systems of lipid peroxidation. This, coupled to its lacks of pungency and TRPV1 inhibiting properties, qualifies this phenolic compound as an attractive candidate for further investigations in vivo.

Synthetic N-acylvanillamines were designed and developed as metabolically stable compounds with pharmacological potential in analgesia and inflammation because of their interaction with cannabinoid receptors and the vanilloid receptor (TRPV1). Here, we show that arvanil inhibits early events in T-cell receptor (TCR)-mediated T-cell activation, such as calcium mobilization and nuclear factor of activated T-cell activation, and in late events in TCR-mediated activation, such as interleukin (IL)-2 gene transcription, IL-2R expression, and cell-cycle progression. Arvanil also prevents tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activation by direct inhibition of IkappaBalpha degradation, NF-kappaB binding to DNA, and NF-kappaB-dependent transcription. Aromatic iodination meta to the phenolic hydroxyl (on the 6'-carbon atom) converts arvanil and olvanil from TRPV1 agonists into antagonists. However, this structural modification did not affect the immunosuppressive and proapoptotic activity of these compounds. In summary, we described here novel activities of arvanil on T-cell functions and the development of two novel inhibitors of neurogenic inflammation (6'-I-olvanil and 6'-I-arvanil) endowed with a unique combination of TRPV1 antagonistic-, immunosuppressive-, and NF-kappaB-inhibitory properties. Our findings provide new mechanistic insights into the biological activities of N-alkylvanillamines and should foster the synthesis of improved analogs amenable to pharmaceutical development as analgesic and anti-inflammatory agents.

Capsiate, the non-pungent ester isoster of capsaicin, and its dihydroderivative are the major capsaicinoids of sweet peppers. The remarkable difference between the sensory properties of capsaicin vs capsiate is solely due to the way the vanillyl and the acyl moieties of this basic structural motif are linked, via an amide bond in capsaicin-type compounds and via an ester bond in capsiate-type compounds.Since capsaicin induces apoptosis in tumoral cells by a vanilloid receptor type 1(VR1)-independent pathway, we examined the effects of capsiates derived from sweet peppers in the ROS generation and induction of apoptosis in tumoral cells and if these are mediated independently from VR1.We have developed an expeditious synthesis of capsiates based on the esterification of vanillol with the Mitsunobu protocol. Capsiate-induction of apoptosis, generation of reactive oxygen species and disruption of the mitochondria transmembrane potential in tumoral cell lines were measured by flow cytometry. Chemopreventive activity was studied in a two-stage mouse skin carcinogenesis assay.Capsiates induce apoptosis that was preceded by an increase in the production of reactive oxygen species and by a subsequent loss of mitochondria transmembrane potential (DeltaPsi(m)). These properties were retained in simplified synthetic analogues of natural capsiates, one of which (nor-dihydrocapsiate) showed powerful chemopreventive activity.These results suggest that capsiates and related synthetic analogues target a variety of pathways involved in cancer development and inflammation, and have considerable potential for dietary health benefits as well as for pharmaceutical development.

Capsiate and its dihydroderivatives are the major capsaicinoids of sweet pepper. These new capsaicinoids do not activate the vanilloid receptor type 1 (VR1) but they share with capsaicin (CPS)some biological activities mediated in a VR1-independent fashion. In this study we show that CPS and nordihydrocapsiate (CPT) inhibit early and late events in T cell activation, including CD69, CD25 and ICAM-1 cell surface expression, progression to the S phase of the cell cycle and proliferation in response to TCR and CD28 co-engagement. Moreover, both CPS and CPT inhibit NF-kappaB activation in response to different agents including TNF-alpha. CPS itself does not affect the DNA-binding ability of NF-kappaB but it prevents IkappaB kinase activation and IkappaBalpha degradation in a dose-dependent manner, without inhibiting the activation of the mitogen-activated protein kinases, p38, extracellular regulated kinase and c-Jun N-terminal protein kinase. Moreover, intraperitoneal pretreatment with CPT prevented mice from lethal septic shock induced by lipopolysaccharide. In a second model of inflammation CPT pretreatment greatly reduced the extensive damage in the glandular epithelium observed in the bowel of DSS-treated mice. Taken together, these results suggest that CPT and related synthetic analogues target specific pathways involved in inflammation, and hold considerable potential for dietary health benefits as well as for pharmaceutical development.

The cover picture showsEuphorbia resinifera Berg and its constituent resiniferatoxin (RTX), a phorboid that acts as an ultrapotent biological analogue of capsaicin, the hot principle of chili pepper and the archetypal vanilloid. Ever since its isolation, interest within the organic community for phorbol has been high on account of its challenging structure, puzzling reactivity, and remarkable bioactivity. Diacyl derivatives of phorbol can act as ultrapotent analogues of the endolipid sn-1,2-diacylglycerol (DAG) and show powerful tumour-promoting properties. The more recent discovery that certain non-tumour-promoting phorbol derivatives and phorbol-related compounds can activate vanilloid receptors of the TRPV1 and TRPV4 type has made phorbol very interesting also for neurosciences. The article by G. Appendino et al. on p. 3413 ff. describes the synthesis of hybrid phorbol-resiniferatoxin of interest for the study of vanilloid receptors, which discloses some new and unpredictable aspects of the chemistry of phorbol. (Photo courtesy by Prof. Mauro Ballero, University of Cagliari)

Capsiate and its dihydroderivatives are the major capsaicinoids of sweet pepper. These new capsaicinoids do not activate the vanilloid receptor type 1 (VR1) but they share with capsaicin (CPS)some biological activities mediated in a VR1-independent fashion. In this study we show that CPS and nordihydrocapsiate (CPT) inhibit early and late events in T cell activation, including CD69, CD25 and ICAM-1 cell surface expression, progression to the S phase of the cell cycle and proliferation in response to TCR and CD28 co-engagement. Moreover, both CPS and CPT inhibit NF-kappaB activation in response to different agents including TNF-alpha. CPS itself does not affect the DNA-binding ability of NF-kappaB but it prevents IkappaB kinase activation and IkappaBalpha degradation in a dose-dependent manner, without inhibiting the activation of the mitogen-activated protein kinases, p38, extracellular regulated kinase and c-Jun N-terminal protein kinase. Moreover, intraperitoneal pretreatment with CPT prevented mice from lethal septic shock induced by lipopolysaccharide. In a second model of inflammation CPT pretreatment greatly reduced the extensive damage in the glandular epithelium observed in the bowel of DSS-treated mice. Taken together, these results suggest that CPT and related synthetic analogues target specific pathways involved in inflammation, and hold considerable potential for dietary health benefits as well as for pharmaceutical development.