Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 7

Article pubs.acs.org/jmc

Investigations on the 4‑Quinolone-3-carboxylic Acid Motif. 7. Synthesis and Pharmacological Evaluation of 4‑Quinolone-3carboxamides and 4‑Hydroxy-2-quinolone-3-carboxamides as High Affinity Cannabinoid Receptor 2 (CB2R) Ligands with Improved Aqueous Solubility Claudia Mugnaini,*,† Antonella Brizzi,† Alessia Ligresti,‡ Marco Allarà,‡ Stefania Lamponi,† Federica Vacondio,§ Claudia Silva,§ Marco Mor,§ Vincenzo Di Marzo,‡ and Federico Corelli*,† †

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy § Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy ‡

S Supporting Information *

ABSTRACT: 4-Quinolone-3-carboxamide derivatives have long been recognized as potent and selective cannabinoid type-2 receptor (CB2R) ligands. With the aim to improve their physicochemical properties, basically aqueous solubility, two different approaches were followed, entailing the substitution of the alkyl chain with a basic replacement or scaffold modification to 4-hydroxy-2-quinolone structure. According to the first approach, compound 6d was obtained, showing slightly reduced receptor affinity (Ki = 60 nM) compared to the lead compound 4 (0.8 nM) but greatly enhanced solubility (400−3400 times depending on the pH of the medium). On the other hand, shifting from 4-quinolone to 4-hydroxy-2-quinolone structure enabled the discovery of a novel class of CB2R ligands, such as 7b and 7c, characterized by Ki < 1 nM and selectivity index [SI = Ki(CB1R)/Ki(CB2R)] > 1300. At pH 7.4, compound 7c resulted by 100-fold more soluble than 4.

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INTRODUCTION Cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R) regulate a variety of physiological functions, including neuronal development, neuromodulatory processes, energy metabolism, as well as cardiovascular, respiratory, and reproductive functions, hormone release and action, bone formation, and cellular functions such as cell architecture, proliferation, motility, adhesion, and apoptosis.1 Therefore, compounds able to activate or block these receptors are expected to show interesting pharmacological properties and potentially produce therapeutically useful effects.2 The exciting and intensive research activity carried out during the last decades at both academic and industrial level has led to unveiling a wide number of cannabimimetic chemical scaffolds and to produce an ample variety of compounds able to target either CB1R or CB2R with very high affinity and selectivity.3 Nevertheless, only a few drugs in this field have been introduced so far into the market, that is, rimonabant, which was approved as an antiobesity drug in Europe but subsequently withdrawn for safety reasons, a standardized extract of Cannabis for symptomatic treatment of multiple sclerosis, and dronabinol and nabilone for treating chemotherapy-induced nausea and vomiting. As a result, the development of cannabinoid ligands is viewed by pharmaceut© XXXX American Chemical Society

ical companies as a very risky and likely unsuccessful activity. While a high attrition rate in drug research and development can be due in principle to a number of factors, in the case of cannabinoids, and particularly for CB2R-targeting compounds, inadequate drug-likeness may be a major issue. Using CB2R agonists/inverse agonists in vivo is challenging, as these compounds are inherently very lipophilic4 and usually show not optimal pharmacokinetic profile, being characterized by high binding to plasma proteins, long half-life, low bioavailability, and off-target effects.5 Structural optimization of CB2R ligands aimed at balancing lipophilicity (necessary to reach the target receptor) and aqueous solubility (necessary to ensure acceptable bioavailability) is a very challenging endeavor.6 The development of tailored system for delivery and specific targeting of these molecules, whenever applicable, is a strategy that can be pursued to overcome their limitations in terms of physicochemical and pharmacokinetic properties.7 In recent years, we have been investigating a large family of 4-quinolone-3-carboxamide derivatives endowed with high CB2R affinity and selectivity over CB1R and characterized by different functional profiles.8 Our structure−activity relationReceived: October 6, 2015

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DOI: 10.1021/acs.jmedchem.5b01559 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Chart 1. Schematic Summary of Relevant SAR for 4-Quinolone-3-carboxamides

Chart 2. Design of 4-Hydroxy-2-quinolone-3-carboxamides as Cannabinoid Ligands

ships (SAR) studies on this class of cannobinoid ligands suggested that the quinolone-3-carboxamide scaffold can be optimized through selected chemical modifications in order to obtain either CB1R or CB2R ligands with improved affinity and/or selectivity (Chart 1). In particular, the introduction of substituents into the 6 or 7 position of the quinolone ring in the prototypical compound 1 mainly affects receptor selectivity and receptor affinity, respectively, although the functional profile of the compounds is also modulated, as compound 2, for instance, is a full agonist at CB2R whereas compound 3 acts as an inverse agonist at the same receptor.9 However, along with an excellent in vitro profile, these compounds mostly showed ClogP values higher than 5 and low aqueous solubility. Herein, we describe our efforts, starting from prototypical structures 2 and 4 for obtaining new quinolone-3-carboxamides with improved physicochemical characteristics, mainly aqueous solubility, while retaining CB2R affinity and selectivity. The design of these compounds relied on the following approaches: (i) The replacement of the hydrophobic n-pentyl chain at N1 position with more polar chains eventually bearing basic groups able to enhance aqueous solubility in an acidic medium. We have already applied a similar strategy to a series of 4-quinolone-3-carboxamides bearing a fenchyl group as the amide substituent and demonstrated that this approach is compatible with cannabimimetic activity, although the selectivity profile of these componds was not satisfactory.8d In the present study, we came back to the adamantylamide derivatives of 4quinolones, which, based on previous results,8a,b are expected to retain better CB1R/CB2R selectivity. In addition, although the hydrophobic substituent constant for the adamantyl group (πadamantyl) has been estimated as

high as 3.1, the multidimensional value of this group in drug design is well recognized.10 (ii) The modification of the 4-quinolones into 4-hydroxy-2quinolones characterized as slightly acidic compounds with pKa values typically in the range 4.2−5.011 and hence expected to confer higher aqueous solubility in a basic medium. We8c and others12 have previously shown that the replacement of the 4-quinolone scaffold with the isomeric 2-quinolone ring is not detrimental for cannabinoid activity. As a step forward (Chart 2), we reasoned that the insertion of a further substituent (an OH group) at the 4-position of 2-quinolones should be tolerated in terms of steric hindrance while providing the new compounds with different electronic properties. We have reported previously two compounds of similar structure, but no evaluation of their solubility was performed nor rationalization of structure−affinity/ selectivity properties was possible.8c Therefore, because of the promising features of 4-hydroxy-2-quinolones as a novel class of cannabinoid ligands, compounds showing limited variations of the amide residue and aromatic substitution pattern compared to 2 and 4 were also considered. In accordance with these approaches, new cannabinoid ligands, represented by general structures 6−8 (Chart 3), were synthesized and evaluated for their ability to bind to human CB1R and CB2R. Calculated and experimental solubilities of the most promising compounds are also reported.

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RESULTS AND DISCUSSION Chemistry. The synthesis of quinolone derivatives 6a−h was accomplished starting from quinolone-3-carboxylic acids 10a,8a 10b,8d and 10c (Scheme 1). Amidation with 1B



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Scheme 2a

Chart 3. Structure of New Quinolone Derivatives 6−9

Reagents and conditions: (i) AcCl, AlCl3, 90 °C, 6 h; (ii) methyltriphenylphosphonium bromide, BuLi, THF, 0 °C to reflux, 1 h; (iii) H2, 10% Pd/C, MeOH, rt, 1 atm, 2 h; (iv) 6 N HCl, reflux, 4 h; (v) EMME, 120 °C, 1 h; (vi) Ph2O, reflux, 1 h; (vii) 6 N HCl/EtOH (1:1), reflux, 24 h. a

according to le Gould−Jacobs procedure, and the condensation product 17 was cyclized by heating in diphenyl ether to afford the quinolone derivative 18. Finally, hydrolysis with hydrochloric acid gave the expected acid 10c. 4-Hydroxy-2-quinolone derivatives 7a−d were synthesized as depicted in Scheme 3. While the 4-hydroxy-2-quinolone derivative 19a was prepared according to literature,14 the analogues 19b−d were synthesized from amides 12, 15, and commercially available N-[4-(propan-2-yl)phenyl]pentanamide 20. Reduction with lithium aluminun hydride (LAH) afforded the corresponding N-pentylanilines 21b−d, which in turn were reacted with triethyl methanetricarboxylate under microwave irradiation to provide the ethyl 4-hydroxy-2-quinolone-3carboxylate 19b−d. The final amides 7a−d were easily obtained in good yield by heating 19a−d with 1-aminoadamantane in toluene. For the preparation of analogous compounds 7e−h, bearing different substituents at N1/N3 positions, a similar synthetic approach was followed, although the N1-alkylation reaction gave better results when it was performed on amido derivatives instead that ester derivatives (Scheme 4). Accordingly, 2215 and 2316 were first converted by heating in the presence of the

aminoadamantane gave the intermediates 11a−c, which were then converted into the final compounds 6a−h by alkylation at N1 position in the presence of potassium carbonate and sodium iodide using the appropriate alkylating agents, i.e., 2(dimethylamino)ethyl chloride hydrochloride (6a,d,g), 2methoxyethoxymethyl chloride (6b,e), 4-(2-chloroethyl)thiomorpholine 1,1-dioxide hydrochloride (6c,f) (that was prepared according to a literature procedure13) and n-pentyl iodide (6h). The last compound was prepared to be used as a reference compound in pharmacological and solubility assays. The starting acid 10c, which had never been reported before, was synthesized through a seven-step procedure as described in Scheme 2. Commercially available N-(3-fluorophenyl)pentanamide 12 was subjected to Friedel−Crafts acylation to yield the corresponding methyl ketone 13. This reaction required heating at 90−100 °C in the absence of solvent to give the expected ketone in 37% yield. Wittig reaction with methyltriphenylphosphonium bromide and butyllithium gave the olefine derivative 14, which was then reduced (H2, Pd/C) to the saturated intermediate 15 in quantitative yield. Acidic hydrolysis led to 3-fluoro-4-isopropylaniline 16, which was condensed with diethyl ethoxymethylenemalonate (EMME) Scheme 1a

Reagents and conditions: (i) 1-aminoadamantane, HBTU, HOBt, DIPEA, DMF, rt, 3−5 h; (ii) appropriate alkyl halide, K2CO3, KI, DMF, 90 °C, 1−2 h.

a

C



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Scheme 3a

favored by performing the reaction under microwave irradiation in a basic medium. Interestingly enough, although compounds 24 and 25a,b due to the presence of reactive lactam and enol functionalities could behave in principle as ambident nucleophiles, thereby giving rise to either N- or O-alkylation products, we only isolated the N1-substituted compounds. While the reason for the high regioselectivity of the alkylation reaction under the employed experimental conditions was not investigated, the structure of the alkylated products 26a−d could be unequivocally assigned based on spectroscopic data and alternative synthesis. Thus, the 1H NMR spectrum of compounds 26a−d shows a singlet at δ > 17 ppm that is diagnostic for a free C4-hydroxy group engaged in a strong intramolecular hydrogen bond with the coplanar carbonyl of the amide, whose NH proton is in turn involved in hydrogen bonding to the lactam CO.11b Furthermore, the structure of compounds 7e−h could be confidently confirmed by comparison of their spectral data with those of 7a−d, obtained through a different synthetic route starting from N-alkylated precursors (Scheme 3). The 2-quinolone derivative 8 was best prepared starting from 19b (Scheme 5) that was converted into the corresponding triflate 27 by reaction with N-phenyl-bis(trifluoromethanesulfonimide). Reduction of triflate 27 with triisopropylsilane and palladium catalyst gave the 2-quinolone derivative 28, which was hydrolyzed to the acid 29. Amidation of 29, according to the procedure used for the preparation of compounds 11a−c, provided the expected compound 8.

a

Reagents and conditions: (i) LAH, THF, reflux, 2 h; (ii) triethyl methanetricarboxylate, microwaves, 225 °C, 1 h; (iii) 1-aminoadamantane, toluene, reflux, 2−3 h.

appropriate amine into the corresponding amides 24 and 25a,b, respectively, and these, in turn, were subjected to alkylation at N1-position with 4-acetoxybutyl iodide or 1-iodopentane to give intermediate compounds 26a−d. Finally, 26a was hydrolyzed with NaOH to provide compound 7e, while the bromo derivatives 26b−d were transformed into 7f−h by Suzuki−Miyaura coupling using the appropriate heteroarylboronic acid with concurrent deprotection of the N1 side chain Scheme 4a

a Reagents and conditions: (i) 1-aminoadamantane, toluene, reflux, 2−3 h; (ii) appropriate alkyl halide, K2CO3, KI, DMF, 90 °C, 1−2 h; (iii) 10% NaOH, reflux, 2 h; (iv) appropriate heteroarylboronic acid, Pd(OAc)2, PPh3, 1 M Na2CO3, EtOH, 1,2-dimethoxyethane, microwaves, 150 °C, 5 min.

D



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Scheme 5a

showed no affinity for CB1R and modest affinity for CB2R, with Ki(CB2) values ranging from 146 to 1931 nM. Therefore, the replacement of the N1 pentyl chain of 2 with more hydrophilic chains caused the total loss of affinity for CB1R and a >20-fold decrease of CB2R affinity, with derivative 6a, bearing the 2-(dimethylamino)ethyl chain, showing the highest CB2R affinity. Conversely, the same structural modifications of the lead compound 4 led to compounds 6d−f, which still exhibit CB2R affinity in the nanomolar range and, as for compound 6e, significant receptor selectivity as well. Within this series, the one presenting the 4-thiomorpholine 1,1-dioxide-2-ethyl chain (6f) resulted the least active derivative, while no significant difference in receptor affinity was seen between compounds bearing the 2-(dimethylamino)ethyl (6d) and the 2-methoxyethoxymethyl (6e) side chain. On the basis of these findings, 6g was synthesized with the aim of evaluating the effect of the 2-dimethylaminoethyl chain on a 4-quinolone scaffold substituted with both the 6-isopropyl and 7-fluoro groups. The binding affinity values of 6g, compared to those of 6h, possessing the N1 pentyl chain, demonstrate that the possibility does exist to insert solubilizing moieties in the 4-quinolone-3carboxamide scaffold while retaining CB2R affinity and selectivity. Actually, 6g proved to be the most interesting compounds among the 4-quinolones 6a−g, showing Ki(CB2) = 11.4 nM and Ki(CB1) = 717 nM (SI = 63). In vitro pharmacological evaluation of 4-hydroxy-2-quinolone-3-carboxamide derivatives led to the identification of very potent and selective CB2R ligands, with compounds 7a−c eliciting (sub)nanomolar CB2R affinity and high selectivity (SI in the range 2349−112). Interestingly enough, the insertion of the 6-isopropyl (7b) or 7-fluoro (7c) substituent into the basic scaffold of 7a did not affect CB2R affinity but decreased by more than 10 times the ability to bind to the CB1R, thereby enhancing significantly receptor selectivity. A general reduction of activity was observed when both those substituents were present, like in compound 7d, which showed no CB1R affinity and Ki(CB2) = 66.4 nM, that is 70−120 times higher than compounds 7a−c. By comparing 7b and 7c with 2 and 4, respectively, it can be seen that shifting from the 4-quinolone to the 4-hydroxy-2-quinolone scaffold can greatly improve receptor selectivity by decreasing affinity for CB1R while preserving, or even slightly enhancing, activity at CB2R. Nevertheless, this is not a general rule, as compound 7d is a 9-fold less potent CB2R ligand than the corresponding 4quinolone analogue 6h. The partial saturation of the aromatic ring of 7a gave derivative 9, which is a very potent, though unselective, cannabinoid ligand. Thus, reduction of flatness and π-system extension of the molecule seems to selectively increase its binding capacity to CB1R. On the other hand, the comparison between 7b and 8 demonstrates that also the removal of the 4hydroxy group favors binding to CB1R because 8 is as potent as 7b at CB2R but far less selective. Concurrent replacement of N1-pentyl chain and adamantyl residue with less lipophilic counterparts was carried out with the aim of improving the physicochemical properties of the compounds. However, these structural modifications of 4hydroxy-2-quinolone-3-carboxamides negatively affected the pharmacodynamic profile of the compounds. With the exception of 7e, which, though less potent than 7a still retained receptor affinity, derivatives 7f,g appeared basically devoid of activity. Notably, this drop of receptor affinity must be mainly attributed to the 4-hydroxybutyl chain because the

a

Reagents and conditions: (i) PhN(SO2CF3)2, K2CO3, DMF, rt, 5 h; (ii) (i-Pr)3SiH, PdCl2(PPh3)2, DMF, 85 °C, 24 h; (iii) 10% NaOH, reflux, 3 h; (iv) 1-aminoadamantane, HBTU, HOBt, DIPEA, DMF, rt, 3 h.

The synthesis of the partially hydrogenated analogue 9 (Scheme 6) started from ethyl cyclohexanone-2-carboxylate, Scheme 6a

a Reagents and conditions: (i) amylamine, 45 °C, 5 h; (ii) methyl malonyl chloride, Et3N, dichloromethane, 0 °C to rt, 24 h; (iii) MeONa, MeOH, reflux, 30 min; (iv) 1-aminoadamantane, toluene, reflux, 3 h.

which was reacted with amylamine to afford the enaminoester 30. Acylation with methyl malonyl chloride yielded the intermediate 31 that was cyclized to 32 through MeONacatalyzed Dieckmann condensation. The last compound was finally converted into the amide 9 by heating with 1aminoadamantane. The described synthetic sequence did not allow the characterization of all of the oily intermediates, which proved to be chemically unstable, but it gave access to 9 in a very easy and fast way, with an overall yield of 12% over four steps (corresponding to an average yield of approximately 60% for every step). Effect on Cannabinoid Receptors and SAR. The binding affinities (Ki values) of compounds 6a−h, 7a−h, 8, and 9 for human recombinant CB1R and CB2R are reported in Table 1. The tested compounds were evaluated in parallel with lead compounds 2 and 4 as well as 33 (SR144528)17 and rimonabant 18 as reference CB2R and CB1R ligands, respectively, as previously described.19 As to 4-quinolone derivatives, compounds 6a−c, bearing the 6-isopropyl group, E



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Table 1. CB1R and CB2R Affinity Values for Compounds 6−9a,b

F



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Table 1. continued

G



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Table 1. continued

a

Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM). bFor both receptor binding assays, the new compounds were tested using membranes from HEK cells transfected with either the CB1R or CB2R and [3H]-(−)-cis-3-[2-hydroxy4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP-55,940). cCB1R: human cannabinoid type 1 receptor. dCB2R: human cannabinoid type 2 receptor. eSI: selectivity index for CB2R, calculated as Ki(CB1R)/Ki(CB2R) ratio. fKi: “Equilibrium dissociation constant”, that is, the concentration of the competing ligand that will bind to half the binding sites at equilibrium in the absence of radioligand or other competitors. gCB2 reference compound. hCB1 reference compound. iThe binding affinities of reference compounds were evaluated in parallel with test compounds under the same conditions.

analogue 7h, where the N1-pentyl chain was present, retrieved activity at both cannabinoid receptors. As a result, we may infer that chemical modulation of the N1 position of 4-hydroxy-2quinolones by the insertion of the 4-hydroxybutyl chain can help decrese lipophilicity to more acceptable levels but, differently from 4-quinolone analogues,8d could deeply influence receptor affinity as well. Compounds 7a−c were taken as representatives of 4hydroxy-2-quinolone ligands and subjected to further in vitro pharmacological evaluation to assess their fuctional activity. As

shown in Figure 1, all compounds acted as inverse agonists as they enhanced the NKH477-induced production of cAMP. Although the compounds exhibited less than 50% of the efficacy of the high efficacy inverse agonist, AM630, the general rank of potency observed in the binding affinity (IC50 values of 2.0, 3.7, and 3.5 nM, respectively, for 7a, 7b, and 7c) correlated with what we observed in the functional activity (EC50 values of 2.4, 2.6, and 17.9 nM, respectively, for 7a, 7b, and 7c). In Vitro Cytotoxicity. Cytotoxicity assay was performed to establish the effects of selected compounds on cell viability in H



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Figure 1. Concentration−response curves of compounds 7a−c in a cAMP-based functional assay. (A) Curves show the effect of increasing concentrations of ligands on NKH477-induced cAMP levels in stable CHO cells expressing the human recombinant CB2 receptor. (B) Effect of AM630 as reference inverse agonist is also shown.

Table 2. Cytotoxicity Values of Selected Compounds 6−9a IC50 (μM)

6a

6d

6h

7a

7b

7c

7d

8

9

2

4

5

15

9

74

13

19

13

92

15

14

74

a

Cell viability (fibroblasts NIH3T3) measured by the neutral red uptake (NRU) test. All compounds were tested at increasing concentrations ranging from 2 to 300 μM. Each concentration was tested in six replicate. The standard deviation is less than 5%.

Table 3. Calculated and Experimental Solubility of Selected Compounds 6a,d,g, 7a−c, 9, and Reference Compounds 2 and 4 compd

MW

CLogPa

kinetic solubility pH 1.0 (μg/mL)

SDb

kinetic solubility pH 7.4 (μg/mL)

SDb

kinetic solubility pH 7.4 Log S

calculated solubility ACD/labsc Log S

calculated solubility ALOGPSd Log S

6a 6d 6g 7a 7b 7c 9 2 4

435.61 411.52 453.60 408.54 450.62 426.53 412.57 434.62 410.53

4.56 3.30 4.72 5.87 7.30 6.06 6.06 6.56 5.29

31.5 170.2 35.5 0.23 0.020 0.17 0.10 0.21 0.05

0.5 2.8 0.6 0.06 0.005 0.05 0.04 0.02 0.01

4.5 5.6 12.1 1.4 1.9 1.5 1.3 1.0 0.014

0.5 0.5 0.8 0.1 0.2 0.2 0.2 0.1 0.004

−4.99 −4.87 −4.57 −5.47 −5.38 −5.45 −5.50 −5.64 −7.47

−4.3 −4.01 −4.23 −4.47 −5.14 −4.77 −4.44 −6.04 −5.66

−4.93 −4.48 −5.1 −4.99 −5.62 −5.21 −4.41 −6.15 −5.73

a

Calculated with ChemDraw Ultra 8.0. bStandard deviations (n = 3−5). cACD calculated solubility values (ACD/Laboratories I-Lab 2.0: ilab. acdlabs.com). dALOGPS 2.1 calculated solubility values (http://www.vcclab.org).

comparable or higher than those of reference compounds 2 and 4 and in good agreement with calculated Log S values, particularly those obtained using ALOGPS software. To allow an easier comparison among the test compounds, Table 3 also shows solubility expressed as μg/mL at pH 1.0 and 7.4. As expected, 4-quinolones 6a,d,g, bearing the 2(dimethylamino)ethyl chain, were more soluble than lead compounds 2 and 4 at physiological pH and much more soluble at pH 1.0. In particular, compound 6a showed a solubility (μg/mL) 150 times (pH 1.0) and 25 times (pH 7.4) higher than its analogue 2. A more marked effect of the protonatable side chain was seen for the fluorinated couple of compounds 6d and 4, as the solubility of 6d was enhanced by 3400 (pH 1.0) and 280 (pH 7.4) times compared to 4. However, compound 6g, possessing both the isopropyl and fluoro substituents, although still much more soluble than 4, showed yet the same solubility values as 6a. It appeared clear that the fluoro substituent is irrelevant for solubility, whereas the apolar and lipophilic isopropyl group, which exerts a favorable effect in terms of pharmacodynamic profile, still is responsible for depressing aqueous solubility. Going on to 4-hydroxy-2-quinolone derivatives 7a−c and 9, their solubility was comparable to that of compound 2 but much better than that of 4 at both pH values. The test compounds exhibited higher solubility at pH 7.4 than at acidic pH, but the difference is approximately only 1 order of

vitro in comparison with reference compounds 2 and 4. The IC50 values on NIH3T3 cell line are reported in Table 2. These data demonstrated different but quite low cytotoxic potential for all the tested compounds toward NIH3T3. Therefore, structural modifications performed on prototypes 2 and 4 did not impact negatively on the cytotoxicity profile of the new compounds. Nevertheless, the 4-quinolone compounds 6a and 6d, bearing the (2-dimethylamino)ethyl chain at N1 position, were slightly more cytotoxic than the corresponding analogues 2 and 4 possessing the N1-pentyl moiety. On the other hand, among the 4-hydroxy-2-quinolone derivatives 7a−c, compound 7a, having no substituents on the aromatic ring, proved to elicit the lowest cytotoxicity. In Vitro Physicochemical Characterization. Compounds 6a,d,g, 7a−c, and 9 were selected for additional physicochemical profiling. To this end, ClogP and aqueous kinetic solubility were determined and the results are reported in Table 3. The ClogP values vary in the range 3.30−6.56, and in many cases exceed 5. In general, the 6-isopropyl derivatives are more lipophilic than the fluorinated ones (compare 6a and 6d), and the 4-quinolone structures are less lipophilic than their 4hydroxy-2-quinolone counterparts (compare 7c and 4). Appropriate Log S for a drug candidate should be between −4 and −6, while log values < −6 indicate low solubility.20 Experimental Log S values at physiological pH for the new compounds were between −4.57 and −5.50, which is I



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Microwave irradiations were conducted using a CEM Discover synthesis unit (CEM Corporation, Matthews, NC). Elemental analyses were performed on a PerkinElmer PE 2004 elemental analyzer, and the data for C, H, and N are within 0.4% of the theoretical values. The chemical purity of the target compounds was determined using the following conditions: an Agilent 1100 series LC/MSD with a Lichrocart 125-4 Lichrospher 100 RP-18 (4.6 mm × 100 mm, 5 μm) reversed phase column. 100-fold increase in aqueous solubility at pH 7.4 despite the concurrent increase of CLogP. Considering that 7c displays a very interesting pharmacological profile, with the same CB2R affinity as 4 and better selectivity (see Table 1), it may represent a significant step forward in the search of new cannabinoid ligands endowed with appropriate physicochemical properties.

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CONCLUSION Two sets of quinolone-3-carboxamide derivatives were designed as cannabinoid ligands possessing hopefully better solubility in order to overcome one of the typical shortcomings of cannabimimetic agents. As a first approach, the insertion of polar/basic chains at N1 position of 4-quinolone-3-carboxamides was considered. The new synthesized compounds proved to be somewhat less potent than reference compounds 2 and 4 but showed increased aqueous solubility, not only in an acidic solution but also under physiological conditions. Within this first set of derivatives, the 7-fluorinated compounds resulted as being more promising than the 6-isopropyl analogues. In the second approach, a deeper modification of the quinolone scaffold was taken into account, leading to 4hydroxy-2-quinolone derivatives, which were expected to undergo better solvation due to their possible conversion into anions at pH 7.4. In this case, the advantage in terms of solubility was less relevant, because most of the compounds 7− 9 proved to be as soluble as 2, although far more soluble compared to the other lead compound 4. Nevertheless, the new 4-hydroxy-2-quinolone chemotype worked very well from the pharmacological viewpoint, leading to the identification of compounds 7a−c and 9 as a novel class of subnanomolar CB2R ligands with high receptor selectivity, although only slightly improved physicochemical profile. Further work should be directed to increase binding affinity of soluble compounds 6d,g and to improve solubility of most potent compounds 7a−c.

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EXPERIMENTAL SECTION

Chemistry. General Methods. Reagents were purchased from commercial suppliers and used without further purification. Anhydrous reactions were run under (a positive pressure) dry N2. Merck silica gel 60 was used for flash chromatography (23−400 mesh). IR spectra were recorded on a PerkinElmer BX FT-IR system using CHCl3 as the solvent or a Nujol dispersion. 1H NMR and 13C NMR were recorded at 200 and 50 MHz, respectively, on a Bruker AC200F spectrometer or at 400 and 100 MHz on a Bruker Advance DPX400. Chemical shifts are reported relative to tetramethylsilane at 0.00 ppm. Mass spectral (MS) data were obtained using Agilent 1100 LC/MSD VL system (G1946C) with a 0.4 mL/min flow rate using a binary solvent system of 95:5 methanol/water. UV detection was monitored at 254 nm. Mass spectra were acquired either in positive or in negative mode scanning over the mass range of 105−1500. Melting points were determined on a Gallenkamp apparatus and are uncorrected. J



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crystals; mp 237 °C (dec). 1H NMR (400 MHz, DMSO-d6): δ 9.72 (s, 1H), 8.67 (s, 1H), 8.30−8.28 (m, 1H), 7.73 (d, J = 12.9 Hz, 1H), 7.31−7.26 (m, 1H), 4.43 (s, 2H), 2.91 (s, 8H), 2.78 (s, 2H), 1.97 (s, 9H), 1.58 (s, 6H). MS (ESI): m/z 502 [M + H]+. N-(Adamantan-1-yl)-1-[2-(dimethylamino)ethyl]-7-fluoro-6(propan-2-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (6g). Prepared from 11c and 2-(dimethylamino)ethyl chloride hydrochloride according to the procedure described for 6a. Reaction time: 8 h. Purified by flash column chromatography on silica gel eluting with dichloromethane/methanol (95:5); yield, 32%; pale-yellow solid; mp 166−167 °C. 1H NMR (400 MHz, CDCl3): δ 9.90 (s, 1H), 8.70 (s, 1H), 8.44 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 11.7 Hz, 1H), 4.22 (t, J = 6.7 Hz, 2H), 3.34−3.27 (m, 1H), 2.73 (t, J = 6.7 Hz, 2H), 2.31 (s, 6H), 2.17−2.11 (m, 9H), 1.76−1.68 (m, 6H), 1.32 (d, J = 6.9 Hz, 6H). MS (ESI): m/z 454 [M + H]+, 476 [M + Na]+. N-(Adamantan-1-yl)-7-fluoro-6-(propan-2-yl)-4-oxo-1-(1-pentyl)1,4-dihydroquinoline-3-carboxamide (6h). Prepared from 11c and 1iodopentane according to the procedure described for 6a. Reaction time: 6 h. Purified by flash column chromatography on silica gel eluting with dichloromethane; yield, 78%; white solid; mp 160−161 °C. 1H NMR (400 MHz, CDCl3): δ 9.84 (s, 1H), 8.62 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 11.6 Hz, 1H), 4.06 (t, J = 7.4 Hz, 2H), 3.27−3.20 (m, 1H), 2.10−2.03 (m, 9H), 1.82−1.78 (m, 2H), 1.69− 1.61 (m, 6H), 1.30 (d, 4H), 1.25 (d, J = 6.8 Hz, 6H), 0.84 (t, J = 6.8 Hz, 3H). MS (ESI): m/z 453 [M + H]+. N-(Adamantan-1-yl)-4-hydroxy-2-oxo-1-(1-pentyl)-1,4-dihydroquinoline-3-carboxamide (7a). A solution of ester 19a (303 mg, 1 mmol) and 1-aminoadamantane (362 mg, 2 mmol) in toluene (20 mL) was refluxed for 2−3 h while removing by azeotropic distillation the ethanol formed during the reaction. After cooling, the solution was washed with 1 N HCl and brine. The solid obtained after evaporation of the solvent was purified by flash column chromatography on silica gel eluting with dichloromethane; yield, quantitative; beige solid; mp 152−154 °C. 1H NMR (400 MHz, CDCl3): δ 17.49 (s, 1H), 10.24 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.58 (m, 1H), 7.20 (m, 2H), 4.14 (m, 2H), 2.08 (m, 9H), 1.66 (m, 6H), 1.42 (m, 6H), 0.87 (t, J = 6.5 Hz, 3H). MS (ESI): m/z 409 [M + H]+. N-(Adamantan-1-yl)-4-hydroxy-2-oxo-1-(1-pentyl)-6-(propan-2yl)-1,4-dihydroquinoline-3-carboxamide (7b). Prepared from 19b and 1-aminoadamantane as described for 7a and purified by flash column chromatography on silica gel eluting with petroleum ether/ AcOEt (7:1); yield, 70%; yellow solid; mp 129−131 °C. 1H NMR (400 MHz, CDCl3): δ 17.49 (s, 1H), 10.38 (s, 1H), 8.07 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 4.21 (t, J = 8.2 Hz, 2H), 3.07−3.00 (m, 1H), 2.20 (s, 7H), 2.15 (s, 3H), 1.79−1.65 (m, 10H), 1.44 (m, 4H), 1.32 (d, J = 8.1 Hz, 6H), 0.96 (t, J = 8.2 Hz, 3H). MS (ESI): m/z 473 [M + Na]+. N-(Adamantan-1-yl)-7-fluoro-4-hydroxy-2-oxo-1-(1-pentyl)-1,4dihydroquinoline-3-carboxamide (7c). Prepared from 19c and 1aminoadamantane as described for 7a and purified by trituration with diethyl ether; yield, 65%; white solid; mp 160−161 °C. 1H NMR (400 MHz, CDCl3): δ 17.62 (s, 1H), 10.11 (s, 1H) 8.16−8.12 (m, 1H), 6.92−6.88 (m, 2H), 4.06 (t, J = 7.9 Hz, 2H), 2.09−2.06 (m, 9H), 1.69−1.50 (m, 8H), 1.35−1.33 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H). MS (ESI): m/z 449 [M + Na]+. N-(Adamantan-1-yl)-7-fluoro-4-hydroxy-2-oxo-1-(1-pentyl)-6(propan-2-yl)-1,4-dihydroquinoline-3-carboxamide (7d). Prepared from 19d and 1-aminoadamantane as described for 7a and purified by flash column chromatography on silica gel eluting with dichloromethane/petroleum ether (2:1); yield, 52%; white solid; mp 127−128 °C. 1H NMR (400 MHz, CDCl3): δ 17.50 (s, 1H), 10.17 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 12.5 Hz, 1H), 4.03 (m, 2H), 3.21− 3.13 (m, 1H), 2.09−2.05 (m, 8H), 1.69−1.61 (m, 9H), 1.35−1.34 (m, 4H), 1.24 (d, J = 6.9 Hz, 6H), 0.88−0.84 (m, 3H). MS (ESI): m/z 491 [M + Na]+. N-(Cyclohexyl)methyl-4-hydroxy-1-(4-hydroxy-1-butyl)-2-oxo1,2-dihydroquinoline-3-carboxamide (7e). A suspension of ester 26a (414 mg, 1 mmol) in EtOH (4 mL) and 6 N HCl (4 mL) was heated at 100 °C for 18 h. After cooling to room temperature the solvent was evaporated. The residue was filtered and washed with water, then with

diethyl ether and petroleum ether, to give 7e which was recrystallized from ethanol; yield, 95%; white solid; mp 75−77 °C. 1H NMR (400 MHz, CDCl3): δ 17.29 (s, 1H), 10.33 (m, 1H), 8.23−8.21 (m, 1H), 7.68−7.64 (m, 1H), 7.38−7.36 (m, 1H), 7.29−7.26 (m, 1H), 4.29 (t, J = 7.2 Hz, 2H), 3.76 (t, J = 6.0 Hz, 2H), 3.29 (d, J = 6.2 Hz, 2H), 2.08−1.61 (m, 9H), 1.26−1.16 (m, 4H), 1.06−0.85 (m, 3H). MS (ESI): m/z 371 [M − H]−. N-(4-Fluorobenzyl)-6-(furan-2-yl)-4-hydroxy-1-(4-hydroxybutyl)2-oxo-1,2-dihydro quinoline-3-carboxamide (7f). To a solution of the aryl bromide 26b (505 mg, 1 mmol) in 1,2-dimethoxyethane (4 mL), palladium(II) diacetate (22.4 mg, 0.1 mmol), triphenylphosphine (78.7 mg, 0.3 mmol), 2-furanylboronic acid (168 mg, 1.5 mmol), EtOH (1 mL), and 1 M Na2CO3 (2 mL) were added and the reaction mixture was irradiated with microwaves at 150 °C for 5 min. The mixture was then filtered through a plug of Celite, the filtrate was diluted with AcOEt, washed with brine, dried over anhydrous Na2SO4 and evaporated to dryness. The solid residue was purified by flash column chromatography using dichloromethane/methanol (99:1) as eluent; yield, 90%; white solid; mp 147−149 °C. 1H NMR (400 MHz, CDCl3): δ 16.99 (s, 1H), 10.66 (t, J = 5.5 Hz, 1H), 8.44 (d, J = 1.6 Hz, 1H), 7.95−7.93 (m, 1H), 7.50 (m, 1H), 7.38−7.33 (m, 3H), 7.06− 7.01 (m, 2H), 6.71 (d, J = 3.2 Hz, 1H), 6.51−6.50 (m, 1H), 4.60 (d, J = 5.8 Hz, 2H), 4.27 (t, J = 7.4 Hz, 2H), 3.75 (t, J = 6.1 Hz, 2H), 1.87− 1.80 (m, 3H), 1.74−1.67 (m, 2H). MS (ESI): m/z 449 [M−H]−. 4-Hydroxy-1-(4-hydroxybutyl)-N-(4-methoxybenzyl)-2-oxo-6-(thiophen-2-yl)-1,2-dihydro quinoline-3-carboxamide (7g). Prepared from 26c and 2-thienylboronic acid as described for the preparation of 7f. It was purified by flash column chromatography using dichloromethane/methanol (99:1) as eluent; yield, 40%; colorless oil. 1H NMR (200 MHz, CDCl3): δ 17.10 (s, 1H), 10.59 (m, 1H), 8.24−8.20 (m, 1H), 7.80−7.62 (m, 1H), 7.46−7.26 (m, 6H), 6.86 (d, J = 8.4 Hz, 2H), 4.55 (d, J = 5.8 Hz, 2H), 4.26 (t, J = 8.0 Hz, 2H), 3.78 (s, 3H), 3.77 (t, J = 5.8 Hz, 2H), 2.03−1.71 (m, 5H). MS (ESI): m/z 477 [M − H]−. 4-Hydroxy-N-(4-methoxybenzyl)-1-(1-pentyl)-2-oxo-6-(thiophen2-yl)-1,2-dihydro quinoline-3-carboxamide (7h). Prepared from 26d and 2-thienylboronic acid as described for the preparation of 7f. It was purified by flash column chromatography using dichloromethane/ methanol (99:1) as eluent; yield, 25%; colorless oil. 1H NMR (200 MHz, CDCl3): δ 17.02 (s, 1H), 10.64 (m, 1H), 8.45−8.19 (m, 1H), 7.69−7.61 (m, 1H), 7.46−7.26 (m, 6H), 6.87 (d, J = 8.0 Hz, 2H), 4.55 (d, J = 5.8 Hz, 2H), 4.18 (t, J = 7.6 Hz, 2H), 3.88 (s, 3H), 2.00−1.23 (m, 6H), 0.9 (t, J = 6.0 Hz, 3H). MS (ESI): m/z 477 [M + H]+. N-(Adamantan-1-yl)-6-(propan-2-yl)-1-(1-propyl)-2-oxo-1,2-dihydroquinoline-3-carboxamide (8). To a solution of 29 (300 mg, 1 mmol) in dry DMF (5 mL) O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 760 mg, 2 mmol) was added followed by 1-hydroxybenzotriazole hydrate (HOBt, 153 mg, 1 mmol), diisopropylethylamine (DIPEA, 260 μL, 1.5 mmol), and 1aminoadamantane (181 mg, 1.2 mmol). The mixture was stirred under N2 atmosphere at room temperature for 30 min, and DIPEA (260 μL, 1.5 mmol) was added again. The reaction mixture was stirred at room temperature for 5 h, poured into water, and extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was purified by flash chromatography on silica gel (eluent: petroleum ether/AcOEt, 7:1) to give 8; yield, 70%; yellowish solid; mp 122−124 °C. 1H NMR (400 MHz, CDCl3): δ 9.67 (s, 1H), 8.69 (s, 1H), 7.42− 7.40 (m, 2H), 7.19 (d, J = 8.0 Hz, 1H), 4.16 (t, J = 8.0 Hz, 2H), 2.91− 2.84 (m, 1H), 2.06−1.98 (m, 9H), 1.64−1.59 (m, 8H), 1.31−1.28 (m, 4H), 1.17 (d, J = 8.0 Hz, 6H), 0.81 (t, J = 8.0 Hz, 3H). MS (ESI): m/z 435 [M + H]+. N-(Adamantan-1-yl)-4-hydroxy-2-oxo-1-(1-pentyl)-1,2,4,5,6,7hexahydroquinoline-3-carboxamide (9). A mixture of ethyl cyclohexanone-2-carboxylate (5.6 mL, 0.035 mol) and amylamine (6 mL, 0.052 mol) was heated at 45 °C for 5 h, then cooled to rt and diluted with n-hexane (10 mL). The reaction mixture was poured into a separatory funnel and left to stand overnight. The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated. The oily enaminoester 30 so obtained (3.3 g, 0.013 mol) was dissolved in K



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N-[3-Fluoro-4-(propen-2-yl)phenyl]pentanamide (14). To a suspension of methyltriphenylphosphonium bromide (5.19 g, 0.014 mol) in dry THF (40 mL), cooled to 0 °C under a nitrogen atmosphere, 1.6 N BuLi in hexane (9.0 mL, 0.014 mol) was added dropwise under stirring. After 1 h, a solution of 13 (0.69 g, 2.9 mmol) in dry THF (20 mL) was slowly added and the reaction mixture was gradually heated to room temperature and then refluxed for 1 h. After cooling, the mixture was poured into water, neutralized by adding 1 N HCl, and extracted with AcOEt. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and evaporated. The residue was purified by flash column chromatography on silica gel eluting with petroleum ether/AcOEt (2:1) to give 14; yield, 95%; yellow solid; mp 71−72 °C. 1H NMR (400 MHz, CDCl3): δ 7.40−7.35 (m, 2H), 7.19− 7.13 (m, 1H), 7.04 (d, J = 8.4 Hz, 1H), 5.15 (s, 1H), 5.12 (s, 1H), 2.28 (t, J = 7.4 Hz, 2H), 2.04 (s, 3H), 1.67−1.59 (m, 2H), 1.37−1.28 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). M/S (ESI): m/z 258 [M + Na]+. N-[3-Fluoro-4-(propan-2-yl)phenyl]pentanamide (15). A solution of 14 (1.45 g, 6.11 mmol) in methanol (80 mL) was hydrogenated over 10% Pd/C for 1 h at room temperature. Filtration on Celite and evaporation of methanol yielded the expected compound 15 as a pure orange solid; yield, quantitative; mp 47−48 °C. 1H NMR (400 MHz, CDCl3): δ 7.60 (s, 1H), 7.31 (d, J = 12.3 Hz, 1H), 7.07−7.04 (m, 2H), 3.13−3.06 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 1.65−1.59 (m, 2H), 1.35−1.25 (m, 2H), 1.14 (d, J = 6.9 Hz, 6H), 0.84 (t, J = 7.2 Hz, 3H). M/S (ESI): m/z 238 [M + H]+, 260 [M + Na]+. 3-Fluoro-4-(propan-2-yl)aniline (16). A solution of 15 (1.14 g, 4.8 mmol) in 6 N HCl (50 mL) was refluxed for 4 h, then cooled to room temperature, made basic (pH 10) by adding 1 N NaOH, and extracted with diethyl ether. Usual workup of the organic phase afforded 16 as a dark-orange oil, which was used in the next step without further purification; yield, 93%. 1H NMR (400 MHz, CDCl3): δ 7.27 (s, 1H), 7.00 (t, J = 8.2 Hz, J = 8.5 Hz, 1H), 6.44−6.41 (m, 1H), 6.38−6.35 (m, 2H), 3.15−3.08 (m, 1H), 1.21 (d, J = 6.9 Hz, 6H). MS (ESI): m/z 154 [M + H]+. Diethyl 2-[[[3-Fluoro-4-(propan-2-yl)phenyl]amino]methylene]malonate (17). A mixture of 16 (1.31 g, 0.01 mol) and diethyl ethoxymethylenemalonate (2.1 mL, 0.01 mol) was heated at 120 °C for 1 h. After cooling, the solid which formed was purified by flash column chromatography on silica gel eluting with petroleum ether/ AcOEt (1:1) to give 17; yield, 83%; orange oil. 1H NMR (400 MHz, CDCl3): δ 8.42 (d, J = 13.6 Hz, 1H), 7.21 (t, J = 6.6 Hz, J = 16.6 Hz, 1H), 6.85−6.77 (m, 2H), 4.28 (q, J = 7.0 Hz, J = 7.1 Hz, 2H), 4.22 (q, J = 7.3 Hz, J = 7.0 Hz, 2H), 3.91 (s, 1H), 3.22−3.12 (m, 1H), 1.35 (t, J = 7.1 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H), 1.27 (d, J = 10.8 Hz, 6H). MS (ESI): m/z 346 [M + Na]+. Ethyl 4-Hydroxy-2-oxo-1-(1-pentyl)-6-(propan-2-yl)-1,2-dihydroquinoline-3-carboxylate (19b). A mixture of 21b (205 mg, 1 mmol) and triethyl methanetricarboxylate (930 mg, 4 mmol) was irradiated with microwaves at 225 °C for 1 h (3 × 20 min). After cooling, the mixture was purified by flash column chromatography on silica gel eluting with petroleum ether/AcOEt (9:1 to 7:1); yield, 40%; orange oil. 1H NMR (400 MHz, CDCl3): δ 13.81 (s, 1H), 8.03 (s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 4.51 (q, J = 7.3 Hz, 2H), 4.19 (t, J = 8.2 Hz, 2H), 3.05−2.98 (m, 1H), 1.73−1.68 (m, 4H), 1.49 (t, J = 7.1 Hz, 3H), 1.46−1.38 (m, 2H), 1.33 (d, J = 12.4 Hz, 6H), 0.93 (t, J = 7.2 Hz, 3H). MS (ESI): m/z 346 [M + H]+. Ethyl 7-Fluoro-4-hydroxy-2-oxo-1-(1-pentyl)-1,2-dihydroquinoline-3-carboxylate (19c). Prepared from 21c following the same procedure described for 21b and purified by flash column chromatography on silica gel eluting with petroleum ether/Et2O (7:2); yield, 66%; yellow solid; mp 95−96 °C. 1H NMR (400 MHz, CDCl3): δ 13.77 (s, 1H), 8.13−8.09 (m, 1H), 6.91−6.86 (m, 2H), 4.43 (q, J = 7.1 Hz, 2H), 4.06 (t, J = 7.9 Hz, 2H), 1.64−1.59 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.38−1.31 (m, 4H), 0.87−0.83 (m, 3H). MS (ESI): m/z 322 [M + H]+. Ethyl 7-Fluoro-4-hydroxy-2-oxo-1-(1-pentyl)-6-(propan-2-yl)-1,2dihydroquinoline-3-carboxylate (19d). Prepared from 21d following the same procedure described for 21b. Purification by flash column chromatography on silica gel eluting with petroleum ether/Et2O (7:1) and then AcOEt/petroleum ether (3:1) gave a brown oil still

dry dichloromethane (35 mL), treated with triethylamine (4.52 mL, 0.032 mol), and cooled to 0−5 °C before adding dropwise methyl malonyl chloride (2.98 mL, 0.027 mol). After stirring at rt for 12 h, the solution was washed with water, then brine, and evaporated to give the amido derivative 31 (4 g, 0.011 mol) as an orange oil. This was added to NaOMe (0.016 mol) in dry MeOH (15 mL) and refluxed for 30 min. After cooling, the solution was made acidic (pH ≤ 2) with 1 N HCl and extracted with dichloromethane. The organic layer was washed with water (3 × 15 mL), then brine, and evaporated. The oily residue 32 (800 mg, 2.73 mmol) was dissolved in toluene (50 mL), and 1-aminoadamantane (824 mg, 5.46 mmol) was added. The solution was refluxed for 2−3 h, while removing azeotropically the ethanol that formed, then evaporated to provide a residue, which was purified by flash column chromatography on silica gel eluting with dichloromethane to give pure 9 as a white solid. Overall yield, 12%; mp 155−156 °C. 1H NMR (400 MHz, CDCl3): δ 16.41 (s, 1H) 10.27 (s, 1H), 3.87−3.83 (m, 2H), 2.59 (t, J = 6.0 Hz, 2H), 2.43 (t, J = 6.0 Hz, 2H) 2.09−2.05 (m, 9H)1.78−1.77 (m, 2H), 1.70−1.57 (m, 10H), 1.32 (s, 4H), 0.89−0.86 (m, 3H). MS (ESI): m/z 413 [M + H]+, 435 [M + Na]+. 7-Fluoro-4-oxo-6-(propan-2-yl)-1,4-dihydroquinoline-3-carboxylic Acid (10c). A solution of 17 (3.23 g, 0.01 mol) in diphenyl ether (25 mL) was refluxed for 1 h. After cooling, petroleum ether was added and the precipitate which formed was filtered and washed thoroughly with diethyl ether to give 18. This compound was suspended in ethanol (30 mL) and 6 N HCl (30 mL), and the resulting mixture was refluxed for 24 h. On cooling, a precipitate was obtained that was filtered and washed successively with water, ethanol, and diethyl ether. Purification by flash column chromatography on silica gel eluting with dichloromethane/methanol (95:5) gave 10c; yield, 37% (overall); white solid; mp > 270 °C. 1H NMR: spectrum was not registered due to the very low solubility of 10c in the common solvents. MS (ESI): m/z 248 [M − H]−. N-(Adamantan-1-yl)-6-(propan-2-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (11a). For spectroscopic and analytical data of this compound, see ref 8a. N-(Adamantan-1-yl)-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (11b). Prepared from 10b according to the procedure described for the preparation of 8. Purified by flash chromatography on silica gel (eluent: AcOEt/petroleum ether, 4:1); yield, 60%; white solid; mp > 270 °C. 1H NMR (400 MHz, CDCl3): δ 12.05 (s, 1H), 10.28 (s, 1H), 8.75 (s, 1 H), 8.42 (dd, J = 9.0 Hz, J = 6.0 Hz, 1H), 7.30−7.27 (m, 1H), 7.20−7.15 (m, 1H), 2.20−2.07 (m, 9H), 1.73 (s, 6H). MS (ESI): m/z 341 [M + H]+. N-(Adamantan-1-yl)-7-fluoro-6-(propan-2-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (11c). Prepared from 10c according to the procedure described for the preparation of 8. Purified by flash chromatography on silica gel (eluent: dichloromethane/methanol, 95:5); yield, 42%; beige solid; mp > 270 °C. 1H NMR (400 MHz, CDCl3): δ 11.81 (br s, 1H), 10.33 (s, 1H), 8.76 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 3.34−3.27 (m, 1H), 2.19−2.10 (m, 9H), 1.71 (s, 6H), 1.32 (d, J = 6.9 Hz, 6H). MS (ESI): m/z 381 [M−H]−, 405 [M + Na]+. N-(4-Acetyl-3-fluorophenyl)pentanamide (13). Acetyl chloride (1.45 mL, 0.02 mol) was slowly added (caution: very exothermic reaction!) to a mixture of 12 (2.0 g, 0.01 mol) and finely ground AlCl3 (4.0 g, 0.03 mol) kept under a nitrogen atmosphere and vigorous magnetic stirring. The reaction mixture was then gradually heated to 90 °C and maintained at this temperature for 6 h. After cooling, ice water and concd HCl (7 mL) were slowly added to the mixture (caution: exothermic reaction!). Extraction of the mixture with dichloromethane followed by usual workup of the organic phase gave a residue, which was purified by flash column chromatography on silica gel eluting with petroleum ether/AcOEt (2:1). Evaporation of the eluates provided a solid that was washed with diethyl ether to afford pure 13; yield, 37%; yellow solid; mp 70−73 °C. 1H NMR (400 MHz, CDCl3): δ 8.28 (s, 1H), 7.74−7.64 (m, 2H), 7.06 (d, J = 8.6 Hz, 1H), 2.52 (d, J = 4.9 Hz, 3H), 2.31 (t, J = 7.5 Hz, 2H), 1.64−1.57 (m, 2H), 1.33−1.24 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). M/S (ESI): m/z 260 [M + Na]+. L



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J2 = 1.8 Hz, 1H), 7.36−7.33 (m, 2H), 7.20 (d, J = 9.1 Hz, 1H), 7.07− 7.02 (m, 2H), 4.61 (d, J = 5.9 Hz, 2H), 4.24 (m, 2H), 4.14−4.10 (m, 2H), 2.05 (s, 3H), 1.77−1.76 (m, 4H). MS (ESI): m/z 503 [M−H]−. 1-(4-Acetoxy-1-butyl)-6-bromo-4-hydroxy-N-(4-methoxybenzyl)2-oxo-1,2-dihydroquinoline-3-carboxamide (26c). Prepared from 25b and 4-acetoxybutyl iodide according to the procedure described for the preparation of 6a. Purified by recrystallization from DMF; yield, 67%; white solid; mp 112−114 °C. 1H NMR (200 MHz, CDCl3): δ 17.18 (s, 1H), 10.51 (t, J = 5.7 Hz, 1H), 8.31 (d, J = 2.6 Hz, 1H), 9.69 (dd, J1 = 9.4 Hz, J2 = 2.6 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 9.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 4.54 (d, J = 5.7 Hz, 2H), 4.30−4.40 (m, 4H), 3.78 (s, 3H), 2.02 (s, 3H), 1.77−1.72 (m, 4H). MS (ESI): m/z 515 [M−H]−. 6-Bromo-4-hydroxy-N-(4-methoxybenzyl)-2-oxo-1-(n-pentyl)1,2-dihydroquinoline-3-carboxamide (26d). Prepared from 25b and 1-iodopentane according to the procedure described for the preparation of 6a. Purified by flash column chromatography on silica gel eluting with dichloromethane/methanol (98:2); yield, 70%; yellow oil. 1H NMR (400 MHz, CDCl3): δ 17.24 (s, 1H), 10.57 (m, 1H), 8.31 (d, J = 2.0 Hz, 1H), 7.60 (dd, J1 = 8.7 Hz, J2 = 1.7 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.7 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 4.50 (d, J = 5.8 Hz, 2H), 4.18 (t, J = 7.6 Hz, 2H), 3.88 (s, 3H), 1.70− 1.62 (m, 2H), 1.39 (m, 4H), 0.9 (t, J = 6.0 Hz, 3H). MS (ESI): m/z 473 [M + H]+. Ethyl 1-(1-Pentyl)-6-(propan-2-yl)-2-oxo-4-[[(trifluoromethane)sulfonyl]oxy]-1,2-dihydroquinoline-3-carboxylate (27). Potassium carbonate (91 mg, 0.66 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (107 mg, 0.3 mmol) were added to a solution of 19b (70 mg, 0.2 mmol) in dry DMF (1.5 mL), and the resulting mixture was stirred under a nitrogen atmosphere at rt for 24 h. The reaction was diluted with saturated solution of ammonium chloride and extracted with AcOEt. The organic layer was washed with water, then brine, and dried over anhydrous sodium sulfate. Evaporation of solvent gave 27 as a brown oil (homogeneous by TLC: SiO2-petroleum ether/AcOEt, 7:1), which was used directly in the following step; yield, 74%. MS (ESI): m/z 478 [M + H]+. Ethyl 1-(1-Pentyl)-6-(propan-2-yl)-2-oxo-1,2-dihydroquinoline-3carboxylate (28). Triisopropylsilane (25 mg, 0.16 mmol) was added to a mixture of 27 (70 mg, 0.15 mmol), PdCl2(PPh3)2 (10 mg, 0.015 mmol), and triethylamine (46 mg, 0.45 mmol) in dry DMF (1 mL). After heating at 85 °C for 24 h under nitrogen, the reaction mixture was cooled to rt and diluted with AcOEt and water. The organic layer was washed with water, then with brine, dried, and evaporated. The oily residue was purified by flash column chromatography on silica gel eluting with petroleum ether/AcOEt (7:1 to 3:1) to afford pure 28; yield, 98%; yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.39 (s, 1H), 7.53 (d, J = 12.2 Hz, 1H), 7.48 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 4.40 (q, J = 8.1 Hz, 2H), 4.27 (t, J = 8.3 Hz, 2H), 3.03−2.97 (m, 1H), 1.59−1.46 (m, 6H), 1.40 (t, J = 8.2 Hz, 3H), 1.29 (d, J = 4.2 Hz, 6H), 0.91 (t, J = 8.1 Hz, 3H). MS (ESI): m/z 330 [M + H]+. 1-(1-Pentyl)-6-(propan-2-yl)-2-oxo-1,2-dihydroquinoline-3-carboxylic Acid (29). A suspension of the ester derivative 28 (650 mg, 2.0 mmol) in 10% NaOH (30 mL) was refluxed for 3 h. After cooling, the mixture was washed with AcOEt, then the aqueous phase was brought to pH 1−2 with 6 N HCl. Extraction of the mixture with AcOEt and usual workup of the organic extract afforded an oily residue which was used directly in the next step. Competition Binding Assay. Membranes from HEK-293 cells overexpressing the respective human recombinant CB1 receptor (Bmax = 2.5 pmol/mg protein) and human recombinant CB2 receptor (Bmax = 4.7 pmol/mg protein) were incubated with [3H]-CP-55,940 (0.14 nM/Kd = 0.18 nM and 0.084 nM/Kd = 0.31 nM, respectively, for CB1 and CB2 receptor) as the high affinity ligand. Competition curves were performed by displacing [3H]-CP-55,940 with increasing concentration of compounds (0.1 nM to 10 μM). Nonspecific binding was defined by 10 μM of WIN55,212-2 as the heterologous competitor (Ki values 9.2 and 2.1 nM, respectively, for CB1 and CB2 receptor). All compounds were tested following the procedure described by the manufacturer (PerkinElmer, Italy). Displacement curves were generated by incubating compounds with [3H]-CP-55,940 for 90

containing traces of triethyl methanetricarboxylate that was used directly in the next step; yield, approximately 65%. MS (ESI): m/z 362 [M − H]−. N-(1-Pentyl)-4-(propan-2-yl)aniline (21b). A solution of 20 (1.31 g, 6 mmol) in dry THF (15 mL) was added dropwise under a nitrogen atmosphere to an ice-cooled suspension of LAH (0.3 g, 8 mmol) in 15 mL of the same solvent. After heating at reflux for 2 h, the reaction mixture was cooled, quenched with 10% NaOH and water, and extracted with AcOEt. The organic phase was washed with brine, dried, and evaporated to leave an oily residue, which was purified by flash column chromatography on silica gel eluting with petroleum ether/AcOEt (7:1); yield, 98%; orange oil. 1H NMR (200 MHz, CDCl3): δ 7.40 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 8.2 Hz, 2H), 3.60 (br s, 1H), 2.87−2.81 (m, 1H), 2.31 (t, J = 8.1 Hz, 2H), 1.75−1.60 (m, 2H), 1.43−1.35 (m, 2H), 1.31−1.26 (m, 2H), 1.22 (d, J = 4.2 Hz, 6H), 0.91 (t, J = 6.2 Hz, 3H). MS (ESI): m/z 206 [M + H]+. N-(1-Pentyl)-3-fluoroaniline (21c). Prepared from 12 according to the procedure described for the preparation of 21b and used in the next step without further purification; yield, 98%; orange oil. 1H NMR (400 MHz, CDCl3): δ 7.09−7.03 (m, 1H), 6.37−6.32 (m, 2H), 6.27 (dd, J = 11.7 Hz, J = 1.7 Hz, 1H), 3.65 (s, 1H), 3.04 (t, J = 7.2 Hz, 2H), 1.58 (t, J = 6.9 Hz, 2H), 1.36−1.35 (m, 4H), 0.93−0.92 (m, 3H). MS (ESI): m/z 182 [M + H]+. N-(1-Pentyl)-3-fluoro-4-(propan-2-yl)aniline (21d). Prepared from 15 according to the procedure described for the preparation of 21b and used in the next step without further purification; yield, 98%; orange oil. 1H NMR (400 MHz, CDCl3): δ 6.99−6.95 (m, 1H), 6.30 (d, J = 8.4 Hz, 1H), 6.24 (d, J = 13.0 Hz, 1H), 3.55 (br s, 1H), 3.12− 3.07 (m, 1H), 3.02 (t, J = 7.1 Hz, 2H), 1.58−1.55 (m, 2H), 1.34 (m, 4H), 1.20−1.18 (m, 6H), 0.90−0.89 (m, 3H). MS (ESI): m/z 224 [M + H]+. N-(Cyclohexyl)methyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3carboxamide (24). Prepared from 22 and aminomethylcyclohexane as described for 7a and purified by trituration with diethyl ether; yield, 81%; white solid; mp 227−229 °C. 1H NMR (400 MHz, DMSO-d6): δ 17.32 (s, 1H), 11.78 (s, 1H), 10.35−10.34 (m, 1H), 7.95−7.93 (m, 1H), 7.67−7.63 (m, 1H), 7.36−7.21 (m, 1H), 7.27−7.23 (m, 1H), 3.21 (t, J = 6.4 Hz, 2H), 1.68−1.52 (m, 6H), 1.25−1.07 (m, 3H), 1.00−0.92 (m, 2H). MS (ESI): m/z 299 [M − H]−. 6-Bromo-N-(4-fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamide (25a). Prepared from 23 and 4-fluorobenzylamine as described for 7a and purified by trituration with diethyl ether; yield, 85%; white solid; mp > 280 °C. 1H NMR (400 MHz, DMSOd6): δ 17.15 (s, 1H), 11.96 (s, 1H), 10.57 (m, 1H), 7.99−7.79 (m, 2H), 7.42−7.38 (m, 2H), 7.30−7.28 (m, 1H), 7.21−7.14 (m, 2H), 4.56 (d, J = 5.6 Hz, 2H). MS (ESI): m/z 389 [M − H]−. 6-Bromo-4-hydroxy-N-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxamide (25b). Prepared from 23 and 4-methoxybenzylamine as described for 7a and purified by recrystallization from DMF; yield, 76%; white solid; mp 253−255 °C. 1H NMR (200 MHz, DMSO-d6): δ 17.20 (s, 1H), 11.95 (s, 1H), 10.45 (t, J = 5.5 Hz, 1H), 7.99 (d, J = 1.5 Hz, 1H), 7.77 (dd, J1 = 8.8 Hz, J2 = 1.5 Hz, 1H), 7.29− 7.25 (m, 3H), 6.96 (d, J = 8.5 Hz, 2H), 4.48 (d, J = 5.5 Hz, 2H), 3.70 (s, 3H). MS (ESI): m/z 401 [M − H]−. 1-(4-Acetoxy-1-butyl)-N-(cyclohexyl)methyl-4-hydroxy-2-oxo-1,2dihydroquinoline-3-carboxamide (26a). Prepared from 24 and 4acetoxybutyl iodide according to the procedure described for the preparation of 6a. Purified by flash column chromatography on silica gel eluting with dichloromethane/methanol (98:2); yield, 40%; white solid; mp 76−78 °C. 1H NMR (400 MHz, CDCl3): δ 17.32 (s, 1H), 10.35 (m, 1H), 8.25−8.23 (m, 1H), 7.69−7.65 (m, 1H), 7.34−7.27 (m, 2H), 4.29 (m, 2H), 4.15−4.10 (m, 2H), 3.30 (t, J = 6.4 Hz, 2H), 2.06 (s, 3H), 1.84−4.61 (m, 9H), 1.32−1.17 (m, 4H), 1.04 (t, J = 12.0 Hz, 2H). MS (ESI): m/z 413 [M−H]−. 1-(4-Acetoxy-1-butyl)-6-bromo-N-(4-fluorobenzyl)-4-hydroxy-2oxo-1,2-dihydroquinoline-3-carboxamide (26b). Prepared from 25a and 4-acetoxybutyl iodide according to the procedure described for the preparation of 6a. Purified by recrystallization from DMF; yield, 58%; white solid; mp 134−136 °C. 1H NMR (400 MHz, CDCl3): δ 17.05 (s, 1H), 10.60 (m, 1H), 8.34 (d, J = 2.4 Hz, 1H), 7.75 (dd, J1 = 8.8 Hz, M



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min at 30 °C. Ki values were calculated by applying the Cheng− Prusoff equation to the IC50 values (obtained by GraphPad) for the displacement of the bound radioligand by increasing concentrations of the test compound. Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM), average SEM < 5%. Functional Activity at CB2 Receptors in Vitro. The cAMP Hunter assay enzyme fragment complementation chemiluminescent detection kit was used to characterize the functional activity in CB2 receptor-expressing cell lines. Gi-coupled cAMP modulation was measured following the manufacturer’s protocol (DiscoveRx, Fremont, CA). Briefly, CHO-K1 cells overexpressing the human CB2 receptor were plated into a 96-well plate (30000 cells/well) and incubated overnight at 37 °C, 5% CO2. Media was aspirated and replaced with 30 μL of assay buffer. Cells were treated with 15 μL of 3× dose−response solutions of samples prepared in the presence of cell assay buffer containing a 3× of 25 μM NKH477 solution (a water-soluble analogue of Forskolin) to stimulate adenylate cyclase and enhance basal cAMP levels. Following stimulation, cell lysis and cAMP detection were performed as per the manufacturer’s protocol. Luminescence measurements were measured using a GloMax Multi Detection System (Promega, Italy). Data are reported as mean ± SEM of two independent experiments conducted in triplicate and were normalized by considering the NKH477 stimulus alone as 100% of the response. The percentage of response was calculated using the following formula: % response = 100% × (1 − (RLU of test sample − RLU of NKH477 positive control)/(RLU of vehicle − RLU of NKH477 positive control). The data were analyzed using PRISM software (GraphPad Software Inc., San Diego, CA). Cytotoxicity Assay. Materials. Dulbecco’s Modified Eagle’s Medium (DMEM) and Eagle’s Minimum Essential Medium (EMEM), trypsin solution, and all the solvents used for cell culture were purchased from Lonza (Switzerland). Mouse immortalized fibroblasts NIH3T3 were purchased from American Type Culture Collection (USA). Cell Cultures and Cytotoxicity Assay. NIH3T3 were utilized for cytotoxicity experiments. NIH3T3 were maintained in DMEM at 37 °C in a humidified atmosphere containing 5% CO2. The culture media were supplemented with 10% fetal calf serum (FCS), 1% L-glutamine− penicillin−streptomycin solution, and 1% MEM nonessential amino acid solution. Once at confluence, cells were washed with PBS 0.1 M, taken up with trypsin−EDTA solution, and then centrifuged at 1000 rpm for 5 min. The pellet was resuspended in medium solution (dilution 1:15), and cells were seeded. After 24 h of incubation, the test compounds, solubilized in DMSO, were added to the cells. Concentrations ranging from 5 to 200 μM were tested. Each concentration was tested in six replicate. Cell viability after 24 h of incubation with the different compounds was evaluated by Neutral Red uptake (Sigma-Aldrich, Switzerland) by the procedure previously reported.21 Briefly, the following solutions were prepared in order to determine the percentage of viable cells:

order to protect them from light. After 5 min from the plate shaker removal, the absorbance was read at 540 nm by a UV/visible spectrophotometer (Lambda 25, PerkinElmer). Determination of Kinetic Solubility. Kinetic solubility values at pH 1.0 and pH 7.4 for reference compounds 2, 4, and for compounds 6a,d,g, 7a−c, and 9 were determined starting from freshly prepared 10 mM DMSO stock solutions. In a 96-well plate, 2 mL of each stock solution were added to 198 mL of (i) 0.1 M HCl, pH 1.0, or (ii) 10 mM PBS, pH 7.4, both adjusted to 0.15 M ionic strength by KCl addition. The plate was stirred (250 rpm, 2 h, room temperature). At the end of the stirring time, samples were centrifuged (1000g, 3 min, 20 °C) to separate undissolved compound and an aliquot of the supernatant was further diluted with MeOH and injected in the HPLC-UV system for quantification. Calibration curves for each compound were built in MeOH. Gradient elution was chosen for compound separation employing a Phenomenex Jupiter C18 column (150 mm × 4.6 mm; 5 μm particle size; Phenomenex Corporation, USA). Eluent A, water; eluent B, acetonitrile both additioned of 0.1% v/v HCOOH. A typical gradient was as follows: t = 0 min; A, 20%; B, 80%; t = 10 min; A, 5%; B, 95%; t = 12 min; A, 5%; B, 95%; t = 12.5 min; A, 20%; B, 80%, followed by a 3 min reconditioning time. Each compound was monitored at its relative absorbance maximum. HPLC flow was 1 mL/ min. A Shimadzu HPLC-UV gradient system (Shimadzu Corporation, Kyoto, Japan), consisting of two Shimadzu LC-10ADvp solvent delivery modules, a 10 mL Rheodyne sample injector (Rheodyne LLC, Rohnert Park, USA), and a SPD-10Avp UV−vis detector, interfaced with the software PeakSimple 2.83, was employed for data acquisition and analysis.

1. Neutral Red (NR) stock solution: 0.33 g NR dye powder in 100 mL of sterile H2O 2. NR medium: 1.0 mL NR stock solution (99.0 routine culture medium prewarmed to 37 °C) 3. NR desorb solution: 1% glacial acetic acid solution + 50% ethanol + 49% H2O At the end of the incubation the routine culture medium was removed from each well, and cells were carefully rinsed with 1 mL of prewarmed D-PBS. Multiwells were then gently blotted with paper towels. Then 1.0 mL of NR Medium was added to each well and further incubated at 37 °C, 95% humidity, 5.0% CO2 for 3 h. The cells were checked during the NR incubation for NR crystal formation. After incubation, the NR medium was removed and cells were carefully rinsed with 1 mL of prewarmed D-PBS. Then the PBS was decanted and blotted from the wells and exactly 1 mL of NR Desorb solution was added to each sample. Multiwells were then put on a shaker for 20−45 min to extract NR from the cells and form a homogeneous solution. During this step, the samples were covered in

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01559. 13 C NMR data for compounds 6a−h, 7a−h, 8, 9, and 28; elemental analyses data for compounds 6a−h, 7a−f, 8, 9, 24, 25a,b, and 26a−c (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*For C.M.: phone, + 39 0577 234318; fax, +39 0577 234333; E-mail, [email protected]. *For F.C.: phone, +39 0577 234308; fax, +39 0577 234333; Email, [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Alessandro Rabbito, Endocannabinoid Research Group, for technical help in functional assays. C.M., A.B., S.L., and F.C. thank the University of Siena for providing the facilities for the execution of this work and “Progetti FISM 2011 (2011/R/3)” for financial support. A.L., M.A., and V.D.M. thank Italian CNR for financial support. F.V., C.S., and M.M. acknowledge the support from the University of Parma.

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ABBREVIATIONS USED CB1R, cannabinoid type-1 receptor; CB2R, cannabinoid type-2 receptor; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s Modified Eagle’s Medium; EMEM, Eagle’s Minimum Essential Medium; EMME, diethyl ethoxymethylenemalonate; FCS, fetal calf serum; HBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyN



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luronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; LAH, lithium aluminum hydride; SI, selectivity index

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Pertwee, R. G.; Di Marzo, V. Structure-affinity relationships and pharmacological characterization of new alkyl-resorcinol cannabinoid receptor ligands: identification of a dual cannabinoid receptor/TRPA1 channel agonist. Bioorg. Med. Chem. 2014, 22, 4770−4783.

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