Functionalization of β-Caryophyllene Generates Novel

May 5, 2014 - *E-mail: [email protected]., *E-mail: ... poorly understood intrinsic promiscuity may be exploited to generate novel pr...
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Functionalization of β‑Caryophyllene Generates Novel Polypharmacology in the Endocannabinoid System Andrea Chicca,†,∥ Diego Caprioglio,‡,∥ Alberto Minassi,‡ Vanessa Petrucci,† Giovanni Appendino,*,‡ Orazio Taglialatela-Scafati,§ and Jürg Gertsch*,† †

Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland ‡ Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale “Amedeo Avogadro”, Largo Donegani 2, 28100 Novara, Italy § Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy S Supporting Information *

ABSTRACT: The widespread dietary plant sesquiterpene hydrocarbon β-caryophyllene (1) is a CB2 cannabinoid receptor-specific agonist showing anti-inflammatory and analgesic effects in vivo. Structural insights into the pharmacophore of this hydrocarbon, which lacks functional groups other than double bonds, are missing. A structure− activity study provided evidence for the existence of a welldefined sesquiterpene hydrocarbon binding site in CB2 receptors, highlighting its exquisite sensitivity to modifications of the strained endocyclic double bond of 1. While most changes on this element were detrimental for activity, ringopening cross metathesis of 1 with ethyl acrylate followed by amide functionalization generated a series of new monocyclic amides (11a, 11b, 11c) that not only retained the CB2 receptor functional agonism of 1 but also reversibly inhibited fatty acid amide hydrolase (FAAH), the major endocannabinoid degrading enzyme, without affecting monoacylglycerol lipase (MAGL) and α,β hydrolases 6 and 12. Intriguingly, further modification of this monocyclic scaffold generated the FAAH- and endocannabinoid substrate-specific cyclooxygenase-2 (COX-2) dual inhibitors 11e and 11f, which are probes with a novel pharmacological profile. Our study shows that by removing the conformational constraints induced by the medium-sized ring and by introducing functional groups in the sesquiterpene hydrocarbon 1, a new scaffold with pronounced polypharmacological features within the endocannabinoid system could be generated. The structural and functional repertoire of cannabimimetics and their yet poorly understood intrinsic promiscuity may be exploited to generate novel probes and ultimately more effective drugs. β-Caryophyllene (β-humulene or (−)-trans-caryophyllene, 1), a major sesquiterpenoid constituent of cloves (Syzygium aromaticum L.) and of the essential oils of numerous plants, including hemp (Cannabis sativa L.), has played an important role in shaping our ideas on transannular interactions and the synthesis of medium-sized compounds.1 Its structure elucidation and synthesis have represented important milestones in isoprenoid chemistry,2,3 but the discovery that 1 is a pharmacologically selective agonist of the peripheral cannabinoid receptor (CB2) and an orally active cannabinoid present in many food plants4−6 has now also fostered interest in its biological activity. The absence of functional groups other than C−C double bonds makes olefins unusual lead structures, since their substantial reluctance to engage in polar interactions does not fit well with the lock-and-key model of receptor binding.7 Thus, despite their lipophilic nature, all known ligands of cannabinoid receptors show critical polarized bonds, variously expressed as an amide/ester group or an alcohol/phenolic © 2014 American Chemical Society

hydroxyl.8,9 On the other hand, the inactivity of closely related analogues of 1 (Figure 1), such as β-caryophyllene oxide (2) and α-caryophyllene (α-humulene or humulene, 3),4 suggests the existence of a specific sesquiterpene pharmacophore for CB2 receptor binding, identifying some critical elements of its cannabimimetic nature. This, the beneficial effects of 1 in different animal models of pain and chronic inflammation10−13 and, in general, the clinical potential of CB2 receptor modulation14−16 have provided a rationale for exploring the structure−activity relationships and functionalization of 1 and the possibility to obtain novel cannabimimetic agents targeting major receptor sites in the endocannabinoid system (ECS). Received: March 7, 2014 Accepted: May 5, 2014 Published: May 5, 2014 1499

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(Figure 1).17 While the latter was inactive in the CB2 binding assay, the mixture of 4 and 5 partially retained the CB2 binding property of the natural product at the tested concentration of 3 μM (Figure 2A). However, the difficulty to resolve this mixture into its two individual isomers and the extensive reorganization of the molecular connectivity suggested a more conservative approach, retaining the basic features of the natural products and carrying out point-like structural changes. In this context, the inactivity of 2 suggests that the endocyclic double bond is a critical element of the pharmacophore of 1, a view confirmed by the inactivity of its products of cyclopropanation (7a), bis-cyclopropanation (7b), and hydroboration/oxidation (8a),18 which were all inactive on CB1 and CB2 cannabinoid receptors at the tested concentration of 3 μM (Figure 2A). Moreover, no CB2 receptor binding activity was observed after conversion of the secondary alcohol 8a to its acetate, benzoate, or phenylacetate (8b−d, respectively), all acyl moieties known to increase the cannabinoid activity of lipophilic amides (Scheme 1, Figure 2A).19 The inclusion into medium-sized rings is known to induce strain in endocyclic trans double bonds,21,22 whose four substituents cannot attain planarity. By fostering polarizability,20,21 this property could be critical for the binding interaction of 1 with CB2 receptors. To provide further support for this view, we explored the replacement of the endocyclic double bond in 1 with a pair of

Figure 1. Known analogues of β-caryophyllene (1) (germacrane numbering of the parent ring system).



RESULTS AND DISCUSSION The chemistry of 1 has been extensively investigated, especially as regards skeletal rearrangements, but most of the classic chemistry of this compound affords mixtures of products difficult to separate.2 For instance, treatment of 1 with H2SO4 triggers a transannular cyclization that, after extensive cationic rearrangements, eventually provides a mixture of clovene (4), neoclovene (5), and caryolanol (β-caryophyllene alcohol, 6)

Figure 2. Cannabinoid receptor binding and FAAH inhibition. (A) Screening of all derivatives of 1 for binding to CB1 (white bars) and CB2 (gray bars) receptors at 3 μM. Displacement of radioligand by ≥50% was defined as significant binding. Results are expressed as % of [3H]CP55940 bound compared to vehicle. (B) Screening of all derivatives of 1 at 10 μM for FAAH inhibition in pig brain homogenate. (C) Concentration-dependent inhibition of FAAH activity by 11a, 11b, and 11e in pig brain homogenate. (D) Concentration-dependent inhibition of hFAAH activity by 11b. (E) Time-dependency of 11b and (F) reversibility and (G) competitiveness of inhibition of hFAAH activity by 11b at 3 μM. Data shown are mean values of at least 3 independent experiments each performed in triplicates ± SD. 1500

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and amidation in the presence of propylphosphonic anhydride (T3P)24 (Scheme 3).

Scheme 1. Modification of the Endocyclic Double Bond of 1a

Scheme 3. Modification of the Trishomo Seco-caryophyllene 9

a

Ac = acetate; Bz = benzoate; PhAC = phenylacetate.

polarized double bonds, exploiting the high reactivity of strained endocyclic double bonds in reactions of ring-opening cross metathesis (ROCM).22 While much less known than other forms of olefin metathesis, ROCM provides unique opportunities to exploit the chemical diversity of medium-sized isoprenoid olefins, complementarily differentiating the two olefinic carbons of an endocyclic double bonds in terms of electrophilicy and nucleophilicity. In the event, refluxing 1 with ethyl acrylate in the presence of the second-generation Hoveyda−Grubbs catalyst afforded as major product (75% yield) the trishomo seco-caryophyllene 9. The stereogenic double bond of 9 was generated exclusively in the Econfiguration (Scheme 2). Only traces (ca. 0.7%, mixture of

A significant potency was observed in the lipophilic amides 11a−c, with the 2-propenylamide 11b showing the most prominent binding to human CB2 receptors (Ki value = 680 ± 130 nM) with a 12-fold lower affinity to human CB1 receptors (Ki value = 8.64 ± 2.12 μM) (Table 1). Compared to 1, which shows a Ki value of 150−500 nM for CB2 receptors, depending on assay conditions,4 11b is only marginally less potent. The propyl (11a) and the 2-chloroethyl (11c) amides are slightly less potent CB2 receptor ligands but exhibited a reduction in their selectivity toward CB2 over CB1 receptors (2/3-fold) (Table 1). The presence of polar group as in the ethanolamide 11d, the vanillamide 11e, and the dopamide 11f could not improve the activity of 9 but rather skewed the receptor affinity from CB2 to CB1 receptors, as indicated by the clearly different binding property at the tested concentration of 3 μM for 11d (40% binding at CB1 vs 14% binding at CB2 compared to vehicle control) and 11e (37% binding at CB1 vs 25% binding at CB2 compared to vehicle control) (Figure 2A). This suggests substantially different structure−activity relationships of this scaffold compared to fatty acids amides. While no activity was observed in the reverse amides 13a−c, a significant increase of binding activity was obtained in the reverse ester, the acetate 14a, with CB2 receptor Ki values in the low micromolar range (data not shown). No significant binding was observed in its corresponding benzoate (14b) (data not shown). Notwithstanding the use of the least active regioisomer from the ROCM reaction of 1 (Scheme 3), a significant activity could be obtained by chemical modification of the carboxylate head. Taken together, these data suggest that the polarization of the endocyclic double bond is critical for binding of derivatives of 1 to CB2 receptors, since pyramidalization as in 2, 7a, and 7b is detrimental for activity. On the other hand, as discussed above, the opening of the macrocycle does not necessarily lead to an impairment of the CB2 receptor binding since the amides 11a− c showed a similar affinity at CB2 receptors as 1.4 The most potent ligands were also tested on CB2 receptor-mediated

Scheme 2. Ring-Opening Cross Metathesis of 1 and Ethyl Acrylate

cis and trans diastereomers) of its regioisomer 10 were obtained, in accordance with the chemoselective formation of the four-membered ruthena-cyclobutane intermediate resulting from the interaction of the bulky ruthenium ion with the least substituted carbon of the double bond.22 Remarkably, the trace reaction product 10 still showed a certain affinity and selectivity at CB2 receptors at the tested concentration of 3 μM, while the major reaction product 9 was only marginally active on both CB1 and CB2 receptors (Figure 2A). Binding activity could be resumed in 9 by chemical modification (vide infra), but the very different activity of 9 and 10 suggests a well-defined relationship between the polarized carbon−carbon bond on the lower rim of 1 (the exomethylene and the four-membered ring) and the polar elements in the upper rim of the molecule. Since some fatty acid amides, including the endogenous cannabinoid anandamide (AEA) and some plant-derived Nalkylamides, interact with different targets of the ECS,19,23 it was interesting to evaluate the effect of the replacement of the ester group of trishomo seco-caryophyllenes 9 and 10 with a series of amide functionalities. Due to the very limited availability of the active isomer 10, this issue was investigated in its less active isomer 9, which was more easily available. To this aim, compounds 11a−f were prepared by hydrolysis of 9 1501

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Table 1. Summary of the Pharmacological Effects of the Seco-caryophyllenes 11a−c,e,f on the Different Components of the ECSa Ki value (receptor binding)

EC50 value

IC50 value

compound

hCB1

hCB2

cAMP (CB2)

hFAAH

hCOX-2 (AA)

hCOX-2 (AEA)

AEA uptake

1 11a 11b 11c 11e 11f WIN55,212 JWH133 DuP-697 URB597 UCM707

>10 3.66 ± 1.21 8.64 ± 2.12 3.83 ± 0.34 >10 >10 0.0075 ± 0.002 nd nd nd nd

0.15−0.5± 1.01 ± 0.8 0.68 ± 0.1 1.43 ± 0.5 >10 6.5 ± 1.7 0.00045 ± 0.0001 0.013 ± 0.004 nd nd nd

1.9 ± 0.3± 9.4 ± 2.8 1.8 ± 1.2 8.9 ± 1.6 nd nd nd 0.032 ± 0.01 nd nd nd

>10 6.3 ± 1.5 2.7 ± 0.4 >10 7.1 ± 1.2 >10 nd nd nd 0.0012 ± 0.0003 nd

>50 >50 >50 >50 14.5 ± 2.1 8.9 ± 2.9 nd nd 0.068 ± 0.007 nd nd

>50 >50 >50 >50 2.2 ± 1.2** 0.8 ± 0.7** nd nd 0.071 ± 0.01 nd nd

>10 7.9 ± 0.6 0.76 ± 0.23 >10 2.4 ± 0.3 4.6 ± 0.5 nd nd nd 0.0008 ± 0.0001 1.8 ± 0.8

a Ki values for human CB1 and CB2 receptors and IC50 values for human FAAH and COX-2 are shown. The values were calculated from concentration-dependent curves each performed in triplicate. WIN55,212 was used as positive control for CB1 and CB2 receptor binding, JWH133 as positive control for CB2 receptor binding and cAMP inhibition, DuP-697 as positive control for COX-2 inhibition, URB597 as reference for FAAH inhibition, and UCM707 as positive control for AEA uptake inhibition Data shown are mean values (μM) of at least 3 independent experiments each performed in triplicate ± SD. **p < 0.01 COX-2 mediated oxygenation of AEA vs AA. ±Values from ref 4. nd = not determined

makes 11a−f suitable for the interaction with the catalytic pocket of this serine hydrolase. Remarkably, apart for the 2hydroxypropylamide derivative 11d, all of the other amides showed a weak inhibition of FAAH activity, with the 2propylamide (11a), 2-propenylamide (11b), and 2-(2-methoxy3-hydroxy-) phenyl amide (11e) being the most potent inhibitors. Among the different R-groups, smaller and/or more lipophilic substituents appear to have a better interaction with the enzyme. In order to confirm the FAAH inhibition observed in pig brain homogenate, 11a, 11b, and 11e were also tested by using human purified FAAH (hFAAH). As illustrated in Figure 2D and Table 1, all three compounds showed identical inhibition potencies in purified hFAAH as in the brain homogenates. In order to determine the mechanism of FAAH inhibition by the most potent FAAH inhibitor 11b, we assessed the time-dependency, reversibility, and competitive nature of the enzymatic inhibition. As shown in Figure 2E, 11b produced a rapid (1 min) and constant inhibition of hFAAH activity. Reversibility was tested by employing a rapid dilution assay. Under these conditions, the incubation of hFAAH with the covalent inhibitor URB597 (1 μM)28 resulted in the formation of an enzyme−inhibitor complex that was resistant to dilution of the assay mixture. By contrast, rapid dilution of the FAAH11b mixture resulted in a full recovery of the enzymatic activity already after 5 min from the dilution, indicating a reversible mechanism of FAAH inhibition by 11b (Figure 2F). In Figure 2G, it is shown that 11b significantly decreased the Michealis− Menten constant (Km) of the enzyme from 8.4 ± 0.8 μM to 11.9 ± 1.7 μM and 31.2 ± 3.5 μM for concentrations of 3 μM and 30 μM, respectively. On the contrary, the maximal catalytic velocity (Vmax) of hFAAH remained unchanged (Figure 2G and Supplemental Table 1). Taken together, these data suggest that 11b inhibits hFAAH activity in a rapid, reversible, and competitive manner. The series of compounds was further tested for 2-AG hydrolysis inhibition. Monoacylglcerol lipase (MAGL) is the principle enzyme involved in 2-AG degradation, accounting for about 60−80% of the total hydrolysis in CNS and periphery,29 with the residual activity being under control of other hydrolases. Among them, two α,β hydrolases (ABHD-6 and -12) have been recently identified to account for about 15−20%

activity using cAMP (Gαi) as functional readout. As shown in Table 1 (and Supplemental Figure 1), 11a, 11b, and 11c showed a concentration-dependent inhibition of the forskolininduced cAMP production in CHO-hCB2 cells, thus revealing their agonistic effects. While 11b and 11c fully inhibited the cAMP production at higher concentrations, 11a more potently but only partially inhibited cAMP production (about 80% total inhibition) (Supplemental Figure 1). All three compounds triggered the activation of the receptor with a similar potency as observed in the hCB2 receptor binding assays (Table 1). Interestingly, more hydrophilic (11d) and bulky substituents (11e and 11f) led to a complete loss of CB2 receptor binding affinity. Next, we explored whether the newly synthesized compounds may target protein members within the ECS, other than CB2 receptors. The concept that cannabimimetic agents may exert polypharmacology within the ECS network is becoming a new paradigm for rational drug design.25,26 To this aim, we evaluated the effects of the compounds on the major known components of the ECS. Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for endocannabinoid (i.e., AEA) hydrolysis, thus controlling the termination of its biological actions.27 Initially, all compounds were tested at 10 μM on FAAH activity in pig brain homogenates (Figure 2B). A few active compounds (defined by an inhibition of the enzymatic activity larger than 50% as compared to vehicle control) were identified and further characterized for a full concentrationdependent effect. As shown in Figure 2C, the propylamide (11a), 2-propenylamide (11b), and vanillamide (11e) analogues of 9 showed a concentration-dependent inhibition of FAAH activity (pig brain homogenates) with IC50 values of 6.9 ± 2.5 μM, 2.2 ± 0.9 μM, and 2.1 ± 1.1 μM, respectively. Importantly, 1 did not show any effect on hFAAH activity up to a concentration of 10 μM (Table 1). The opening of the macrocycle modifies the rigidity of the molecule, leading to higher rotational freedom and allowing different spatial conformations. Within the amides (11a−f), reverse amides (13a−c), and esters (9, 10, and 14a,b) only the amide derivatives showed FAAH inhibitory activity. The FAAH typically recognizes and hydrolyzes amide bonds (mainly ethanolamines) linked to hydrophobic chains, a property that 1502

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Figure 3. Inhibition of hCOX-2 activity. (A) Screening of all derivatives of 1 at 50 μM for hCOX-2-mediated oxygenation of arachidonic acid (AA). DuP-697 was used at 500 nM. The most potent compounds (B) 11e and (C) 11f were tested for concentration-dependent inhibition of hCOX-2mediated oxygenation of AA (dotted line) and AEA (solid line). Data shown are mean values of at least 3 independent experiments each performed in triplicate ± SD.

of the total 2-AG hydrolysis.30,31 We tested the effect of the derivatives of 1 on both MAGL and ABHDs by using different preparations (pig brain homogenate and BV-2 cell homogenate) as previously described.32 At the screening concentration of 10 μM none of the compounds exhibited a significant (more than 50%) enzymatic inhibition (Supplemental Figure 2). AEA and 2-AG can also undergo enzymatic degradation through alternative pathways such as COX-2-mediated oxygenation.33,34 Recently, several studies have suggested that prostaglandinsethanolamide (PG-EAs) and prostaglandins-glycerol ester (PGGEs), the COX-2 metabolites of AEA and 2-AG, respectively, are important physiological players. It has been shown that PGEAs and PG-GEs play an active role in the inflammatory process both in vitro and in vivo by acting at CB1/2 receptorindependent targets.35−37 We tested the compound library for inhibition of COX-2 activity using arachidonic acid at the screening concentration of 50 μM (Figure 3A). Most of the compounds were inactive, while 11e and 11f showed significant inhibition. A full inhibition curve for these compounds revealed a concentration-dependent inhibition of COX-2-mediated oxygenation of arachidonic acid with IC50 values of 14.5 ± 2.9 μM and 8.5 ± 2.2 μM for 11e and 11f, respectively (Figure 3B,C and Table 1). As mentioned above, COX-2 can also oxygenate ECs, and several molecules were described to inhibit selectively AEA and 2-AG oxygenation.33 Therefore, 11e and 11f were next tested for COX-2 inhibition using AEA as substrate. Noteworthy, both compounds showed significant higher potency in inhibiting AEA oxygenation compared to arachidonic acid. As shown in Figure 3B,C and Table 1, 11e and 11f were substrate-specific inhibitors of endocannabinoid oxygenation with a 7- and 10-fold lower IC50 value compared to arachidonic acid oxygenation, respectively. Another potentially important target in the ECS is the putative endocannabinoid

membrane transporter (EMT), which controls the bidirectional movement of AEA and 2-AG across the plasma membrane.38 We therefore tested our hit compounds (11a−c, 11e, and 11f) for inhibition of [3H]AEA uptake in U937 cells, as previously reported.38 11b showed the most potent inhibition of [3H]AEA cellular uptake with an EC50 value of 0.76 ± 0.23 μM (Table 1), while the other compounds showed only a weak or negligible inhibition of AEA uptake. As previously described, a FAAH inhibitor can lead to a strong reduction of [3H]AEA uptake in U937 cells.38 Since 11b is the most potent FAAH inhibitor (IC50 value of 1.8 ± 1.2 μM) its reduction of [3H]AEA uptake is likely due to the enzymatic inhibition rather than directly interfering with the EMT. Similarly, 11a and 11e, both of which showed inhibition of FAAH, also reduced the uptake of [3H]AEA in U937 cells. URB597, the potent and widely used FAAH inhibitor also exhibited this dual effect (Table 1). We next measured the levels of [3H]ethanolamine formed in the cells upon [3H]AEA administration. All compounds showed a reduction of [3H]ethanolamine formation at the same concentrations at which they exhibited the inhibition of [3H]AEA uptake (Supplemental Figure 3). Overall, these results confirmed that the FAAH inhibition measured in pig brain homogenate and purified enzyme takes place also in intact cells, controlling the level of AEA uptake as previously shown.38 In conclusion, by modification of 1 we were able to generate trishomo secocaryophyllene amides that retained potent and/or selective binding activity and agonism at CB2 receptors, as exemplified by 11b. Remarkably, moderately potent FAAH inhibitors (11a, 11b and 11e) and two endocannabinoid substrate-specific COX-2 inhibitors (11e and 11f) endowed with low micromolar to nanomolar potency could be identified. In particular, the CB2 agonists 11a and 11b exhibited a competitive and reversible 1503

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intestinal problems and potential cardiovascular side effects.41 Intriguingly, there is growing awareness that some popular NSAIDs exert a moderate FAAH inhibitory activity that can contribute to the overall anti-inflammatory effect of these drugs.41−43 Combination of substrate-specific inhibition of COX-2 and moderate, possibly reversible, FAAH inhibition may thus also overcome the therapeutic failure of the ultrapotent covalent and selective FAAH inhibitors. Recently, PF04457845, a clinical representative of this class of selective enzyme inhibitors, has failed to show any significant clinical anti-inflammatory and analgesic effects in patients suffering from knee osteoarthitis, despite a strong increase of the plasma levels of all major FAAH substrates. One reason for this clinical failure may be the intrinsic pharmacological mechanism of action of these compounds. PF04457845 leads to a complete and prolonged pharmacological ablation of FAAH activity, resulting in an “overflow“ of AEA that undergoes alternative degradation pathways. A dominant degradation pathway is the COX-2-mediated oxygenation, since this enzyme is upregulated in immune cells during inflammation. COX-2 oxygenation of AEA leads to the production of proinflammatory PG-EAs that do not show significant activity on CB1/2, FP, or EP receptors,36 as recently observed for PG-F2αEA in the spinal cord of mice with knee inflammation.37 PGF2α-EA binds to heterodimers between wild type FP receptors and an alternative FP splice variant,36 contributing to proinflammatory and nociceptive effects.37 Overall, a full and prolonged chemical knock-down of FAAH might determine a redirection of AEA degradation from hydrolysis to oxygenation, triggering the production of pro-inflammatory prostamides. This was, indeed, observed in tissues from FAAH knockout mice,44 and this mechanism may therefore counteract the antiinflammatory effects elicited by AEA and other NAEs via CB1/2 receptors, PPARs, and TRPV1 desensitization.45−48 Conclusion. Terpenes are generally regarded as poor lead structures for drug discovery despite their evolutionary importance in olfaction and plant−insect interactions.49 Nonetheless, the sesquiterpene β-caryophyllene (1) shows a remarkable pharmacological action on the endocannabinoid system (ECS) by acting as selective agonist at CB2 receptors with good to moderate binding activity and significant in vivo efficacy.4,10−12 In this article, we have investigated the impact of chemical modifications on the activity of 1, discovering that the opening and functionalization of the macrocycle generates molecules with novel polypharmacological features within the ECS. Pyramidalization of the endocyclic double bond of 1 by epoxidation or methylenation was detrimental for activity. Conversely, opening via metathesis with ethyl acrylate followed by amidation led to a potent and selective endocannabinoid substrate-specific COX-2 inhibitor (11f), as well as to three compounds exhibiting polypharmacology within the ECS. Thus, the propylamide 11a and the 2-propenylamide 11b showed CB2 receptor agonistic effect coupled with a reversible and competitive inhibition of FAAH, while the 2-(2-methoxy-3hydroxy-) phenyl amide (11e) lost binding properties to CB2 receptors but acted as a dual enzymatic inhibitor of FAAH and COX-2 in a substrate-specific manner. Interestingly, methylation of the phenolic hydroxyl of 11e generated a moderate FAAH inhibitor, while the more hydrophilic dihydroxy derivative 11f did not significantly affect FAAH activity, showing, however, a 3-fold higher potency for the substratespecific inhibition of COX-2. Overall, five derivatives of 1 with

inhibitory activity of FAAH, with IC50 values in the low micromolar range, indicative of a novel class of ECS modulating agents. In a therapeutic setting, this polypharmacological combination could lead to synergistic anti-inflammatory effects by directly modulating CB2 receptors and indirectly raising AEA levels as a consequence of the inhibition of its main degradation route (vide infra). While 2-AG is known to be a full agonist at CB2, AEA possesses only partial agonistic property at CB receptors, but unlike 2-AG it can activate several other targets, such as intracellular CB1 receptors, the membraneassociated TRPV1 channel, and nuclear PPARs. While covalent inhibitors lead to a strong and prolonged increase of the intracellular levels of AEA (and other N-acylethanolamines (NAEs) as well), this will eventually result in the COX-2mediated formation of pro-inflammatory and nociceptive prostamides.34,37 A moderate and reversible inhibition of FAAH could therefore be more effective than its irreversible and highly potent enzymatic blockage. Conversely, the competitive and reversible inhibition of FAAH is expected to induce only a transient and moderate increase of AEA and NAEs levels, with overall beneficial anti-inflammatory effects. Despite their functional similarities, both AEA and 2-AG are generally needed for the physiological modulation of the ECS. Compound 11b shows these features, being capable to directly activate CB2 receptors, directly mimicking the effects of 2-AG, and at the same time to inhibit FAAH, thus indirectly increasing the levels of AEA. The dual moderate FAAH and endocannabinoid substrate specific COX-2 inhibition showed by 11e is also remarkable and may be improved further by future synthetic attempts. This combination of effects offers the opportunity to pharmacologically modulate the ECS at two different levels. Simultaneous substrate-specific inhibition of COX-2 and FAAH might lead to synergistic anti-inflammatory effects because of the tight interplay between these two enzymes. During inflammation, the activity of the ECS is significantly increased with a higher production of ECs and a higher expression/ activity of the degrading enzymes. The more endocannabinoids are produced, the more endocannabinoids are hydrolyzed (mainly via FAAH and MAGL/ABHDs), eventually leading to an increased level of free arachidonic acid. Arachidonic acid is oxygenated by the up-regulated COX-2 expression/activity, leading to an amplification loop of the inflammatory process. The inhibition of 2-AG hydrolysis reduces the levels of free arachidonic acid, prostaglandins, and pro-inflammatory cytokines in the brain and in the periphery.39 This clearly links the release of arachidonic acid upon endocannabinoid hydrolysis with the COX-mediated production of prostaglandins, especially upon inflammation.39 On the same line of evidence, the inhibition of FAAH can hamper this inflammatory loop by reducing the amount of free arachidonic acid, but at the same time, it increases the levels of intact AEA that can undergo COX-2-mediated oxygenation. The increase of PGs-EA upon FAAH inhibition has been recently reported in JWF2 murine squamous carcinoma cell, a model of keratinocyte cancer with high endogenous levels of COX-2 expression.40 Therefore, agents simultaneously inhibiting FAAH and endocannabinoid oxygenation by COX-2 can hinder the production of both arachidonic acid-derived prostaglandins (indirectly) and AEAderived prostamides (directly). It has been cogently argued that this polypharmacological combination would provide the same degree of therapeutic effect by reducing the actual COXinhibitory component and hence the incidence of gastro1504

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Figure 4. Model of potential synergism caused by polypharmacophoric cannabimimetic agents. Trishomo seco-caryophyllene amides show novel polypharmacology illustrated by CB2 receptor activation and simultaneous inhibition of FAAH, as well as endocannabinoid substrate-specific inhibition of COX-2, while being inactive at MAGL and ABHDs. microtiter plate. Cells were incubated overnight at 37 °C with 5% CO2. Culture medium was removed and replaced by 40 μL of equilibration medium. Plates were incubated for 2 h at RT in the dark. Equilibration medium was prepared by diluting GloSensor cAMP Reagent stock solution to 5% v/v in CO2-independent medium (Invitrogen) plus 10% FBS. Tested compounds (11a, 11b, and 11c) at different concentrations (0.01−100 μM), JWH133 as positive control, or vehicle were diluted in 10 μL of assay medium containing 2.5 μM of forskolin and 250 μM of 3-isobutyl-1-methylxanthine (IBMX). Luminescence was monitored continuously by using a 1450 MicroBeta TriLux counter starting from immediately after the addition until 30 min after forskolin addition (keeping the plate at RT and in the dark). Basal levels of luminescence (cells plus IBMX and vehicle without forskolin) were subtracted from the measured values. Results were normalized by subtracting the residual cAMP production and expressed as % of vehicle control. FAAH Activity Assays. AEA hydrolysis was assessed by using two different systems, a cellular homogenate from fresh pig brains obtained from the slaughterhouse and purified human FAAH (hFAAH). The method was performed as previously reported for brain homogenate32 and adapted from literature for hFAAH.56 For details see Supporting Information. 2h-AG Activity Assay. [1,2,3-3H]-2-Oleoyl glycerol ([3H]-2-OG) hydrolysis was performed in pig brain homogenate as previously reported.32 For details see Supporting Information. ABHDs Activity Assays. ABHD-6 and -12 activity was assessed by using BV-2 cell homogenate adapted from Marrs et al.,31 as previously reported.32 For details see Supporting Information. hCOX-2 Activity. Inhibition of human recombinant cyclooxygenase-2 (hCOX-2) was assessed using an in house validated COX fluorescent inhibitor screening assay kit from Cayman Chemical Europe as recently described. 57 For details see Supporting Information. Determination of AEA uptake. The uptake of [ethanolamine-13H]AEA in intact cells was performed by using U937 cells as previously described.38 For details see Supporting Information. Data Analysis. Results are expressed as mean values ± SD for each examined group. Experiments were repeated at least three times in independent experiments each in triplicate. Statistical differences between treated and vehicle control groups were determined by Student’s t test for dependent samples. Differences between the

distinctly different pharmacology and polypharmacology for the ECS emerged from our study (Figure 4). These compounds could serve as probes to guide the de novo design of agents that synergistically target different components of the ECS, en route to an anti-inflammatory equivalent of the multikinase inhibitors used in cancer therapy.50 Moreover, novel probes differentially targeting this intricate network of lipid pathways may be useful to better understand endocannabinoid function.51 Polypharmacology is still a major challenge in drug discovery and, despite its potential to deliver more effective and less toxic agents, is still in short supply of specific examples that could inspire its rational design.52,53



METHODS

General Experimental Procedures for Chemical Synthesis. Gravity Column Chromatography (GCC): Merck Silica Gel 60 (70− 230 mesh). IR: Shimadzu DR 8001 spectrophotometer. NMR: Jeol Eclipse (300 and 75 MHz for 1H and 13C, respectively). For 1H NMR, CDCl3 as solvent and CHCl3 at δ = 7.25 as reference. For 13C NMR, CDCl3 as solvent and CDCl3 at δ = 77.0 as reference. Mass spectra were registered on a LTQ OrbitrapXL (Thermo Scientific) apparatus. Reactions were monitored by TLC on Merck 60 F254 (0.25 mm) plates that were visualized by UV inspection and/or staining with 5% H2SO4 in ethanol and heating. Organic phases were dried with Na2SO4 before evaporation. β-Caryophyllene was purchased from TCI. Compound Characterization. Detailed compound characterization is provided in Supporting Information. Radioligand Binding Assays on Cannabinoid Receptors. Receptor-binding experiments were performed with membrane preparations expressing hCB1 or hCB2 receptors as previously reported.32 For details see Supporting Information. Measurement of cAMP Accumulation. CHO-hCB2 cells were stably transfected with the pGloSensor 22-F plasmid (Promega) as previously described54 and cultured in complete F-12 medium supplemented with 400 μg/mL Geneticin (G418) and 200 μg/ml Hygromycin B. Since 2-AG can be present in large amounts in FBS,55 we used a batch of FBS low in 2-AG content as verified by LC−MS. Cells were detached and resuspended to a cell density of 3 × 105 cells/ mL in 100 μL of F-12 complete medium and seeded in a 96-well 1505

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analyzed samples were considered as significant if p < 0.05. Concentration-dependence curves and EC50 values were generated using GraphPad Prism 5.



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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http:/ pubs.acs.org



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We are grateful to MURST (Italy) for financial support (Project 2009RMW3Z5: Metodologie sintetiche per la generazione di diversità molecolare di rilevanza biologica) and the Swiss Science Foundation SNSF NCCR TransCure for financial support. We thank M. Rau for artwork.



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