Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors

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  • l-3-yl)methylene)-quinucli-nity ligands for CB1 and CB2

    Lisa K. Brents b, Benjamin M. Ford b,

    r Medical Sciences, Little Rock, AR 72205, USAsas for Medical Sciences, Little Rock, AR 72205, USA

    indolequinuclidinone (IQD) analogs has been identied as a novel class of can-

    subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R = H, R = F) and 13 (R = COOCH3, R = R = H)exhibited high afnity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower

    C, Figmariju

    cannabinoid-specic receptors which have been characterized

    (CB2R) are known to be expressed in other tissues,4,5 predomi-nantly in cells of the immune system.6 Interestingly, many typesof cancer cells express relatively high densities of CB1 and CB2receptors, and most recent studies report that cannabinoids canreduce tumor growth and progression in several animal modelsof cancer.79 There are three main structural classes of cannabinoidligands. These are the classical cannabinoid analogues of

    Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.Figure 1. High afnity cannabinoid receptor ligands.

    Bioorganic & Medicinal Chemistry Letters 23 (2013) 20192021

    Contents lists available at SciVerse ScienceDirect

    nal Chemistry Letters

    .e lsevier .com/ locate/bmclE-mail address: pacrooks@uams.edu (P.A. Crooks).from mammalian tissues.2,3 CB1 receptors (CB1R) are abundantlyexpressed in the CNS. These receptors are also expressed in periph-eral nerve terminals and in many extra-neuronal sites. CB1R acti-vation is responsible for most of the pharmacological effects ofcannabinoids in the nervous system. In contrast, CB2 receptors0960-894X/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.02.025of potential therapeutic effects, including anti-convulsive, anti-emetic, analgesic, lowering of intraocular pressure and immunemodulation.1 However, undesirable central nervous system (CNS)effects and potential addictive properties of D9-THC has limitedits medical use, and raised the necessity for identifying the recep-tors responsible for cannabinoid activity in order to develop drugswith fewer adverse side-effects. CB1 and CB2 are the two majorAvailable online 14 February 2013

    Keywords:N-Benzyl indolequinuclidinoneN-Benzyl indolequinuclidinolN-Benzyl indolequinuclidin-3-one oximeCannabinoid ligandsCB1 receptorsCB2 receptors

    D9-Tetrahydrocannabinol (D9-THprimary psychoactive constituent ofafnities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highestselectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-timesgreater selectivity for CB2R over CB1R. These ndings are signicant for future drug development, givenrecent reports demonstrating benecial use of cannabinoid ligands in a wide variety of human diseasestates including drug abuse, depression, schizophrenia, inammation, chronic pain, obesity, osteoporosisand cancer.

    2013 Elsevier Ltd. All rights reserved.

    . 1), the well-knownana, exhibits a varietyReceived 3 January 2013Accepted 1 February 2013

    nabinoid ligands. The afnity and selectivity of these IQDs for the two established cannabinoid receptor2 1 1 2Evaluation of (Z)-2-((1-benzyl-1H-indodin-3-one analogues as novel, high afcannabinoid receptors

    Nikhil Reddy Madadi a, Narsimha Reddy Penthala a,Paul L. Prather b, Peter A. Crooks a,aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas fobDepartment of Pharmacology and Toxicology, College of Medicine, University of Arkan

    a r t i c l e i n f o

    Article history:

    a b s t r a c t

    A small library of N-benzyl

    Bioorganic & Medici

    journal homepage: www

  • D9-THC, the non-classical bicyclic and tricyclic cannabinoid ana-logues of D9-THC, the aminoalkylindoles (e.g., WIN 55212-2,Fig. 1)10 and other diverse heterocyclic structures such as the pyr-azoles (e.g., SR141716A, Fig. 1)1113 and indolopyridones (e.g.,IPDN, Fig. 1).14 In the evaluation of synthetic or non-classical can-nabinoids, the amino alkylindole WIN 55212-2 was reported as apotent non-selective cannabinoid receptor agonist with Ki valuesof 1.9 nM for CB1R and 0.3 nM for CB2R.14

    In contrast, the pyrazole compound SR141716A was reported toselectively bind to CB1R where it acts as an antagonist/inverse ago-nist without producing cannabimimetic activity in vivo.12,13 Theindolopyridone analogue IPDN exhibits higher afnity for CB2R(Ki = 1.0 nM) than for CB1R (Ki = 16 nM).14

    The indole nucleus has played an important role in the activityof the cannabinoid compound WIN 55212-2, and indolopyridone(IPDN) analogues and other analogs that incorporate a variety ofdifferent bicyclic ring systems as subunits have emerged as potentcannabinoid ligands.14,15 These observations prompted us toundertake a more detailed investigation into the structureactivityrelationships of various indole analogs that incorporate a quinuc-lidinone bicyclic ring system as a structural subunit. The presentstudy focuses on the synthesis and evaluation of some (Z)-2-((1-

    (PTC) conditions using triethylbenzylammonium chloride in 50%w/v aqueous NaOH solution in dichloromethane.16 Reduction ofcompounds 7 and 9 with NaBH4 in methanol afforded thecorresponding (Z)-()-2-(N-benzylindol-3-yl methylene)-quinucl-idin-3-ol derivatives 14 and 15 in 8288% yield.16 Reaction of com-pounds 7 and 9with hydroxyl amine hydrochloride in the presenceof sodium acetate/methanol afforded the (2Z,3E)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one oximes 16 and 17.17

    Conrmation of the structure and purity of these analogs wasobtained from 1H and 13C NMR spectroscopic analysis.18 Thegeometry of the double bond in these molecules was establishedfrom X-ray crystallographic data.17,19,20

    Membrane homogenates of either whole mouse brain (50 lg)(CB1R) or Chinese hamster ovary (CHO)-hCB2 cells (25 lg)(CB2R) were incubated for 90 min at room temperature with0.2 nM [3H]CP-55, 940, 5 mM MgCl2, and either increasing IQDconcentrations (10 fM10 lM), 1 lM WIN 55212-2 (to denenon-specic binding) or vehicle (to determine total binding). Eachligand concentration was examined in triplicate, in a nal volumeof 1 mL of buffer containing 50 mM Tris, 0.05% bovine serum albu-min (BSA) and 0.1% ethanol/0.1% dimethyl sulfoxide vehicle. Reac-tions were terminated by rapid vacuum ltration through

    2020 N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 20192021benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues(IQD) as novel potent ligands for CB1 and CB2 cannabinoidreceptors.

    The synthetic strategy for the preparation of IQDs 717 is sum-marized in Scheme 1. The appropriate 1-acetylindole-3-carboxal-dehyde (1 and 2) was prepared by N-acetylation of thecorresponding indole-3-carboxaldehyde with acetic anhydride/ tri-ethylamine in dichloromethane under reux conditions. Aldol con-densation of the appropriate 1-acetylindole-3-carboxaldehydewith quinuclidin-3-one hydrochloride in the presence of lithiumdiisopropylamide in tetrahydrofuran at 78 C afforded the (Z)-2-(N-acetylindol-3-ylmethylene)quinuclidin-3-one derivative (3and 4). Subsequent N-deacetylation in reuxing 1 N aqueous NaOHsolution afforded the appropriate (Z)-2-(1H-indol-3-ylmethylene)-quinuclidin-3-ones (5 and 6).

    The (Z)-2-(N-benzylindol-3-ylmethylene)-quinuclidin-3-onederivatives (713) were prepared in 8187% yield by treatmentof the (Z)-2-(1H-indol-3-yl methylene)quinuclidin-3-ones 5 and 6with a variety of benzyl halides under phase-transfer catalyticScheme 1. Synthesis of indole quinuclidine analogs (IQDS) (717). Reagents and condi78 C; (c) 1 N NaOH (aq), reux; (d) substituted benzyl halide, 50% NaOH (aq), triethhydrochloride, NaOAc, methanol, rt.Whatman GF/B glass ber lters, followed by ve washes withice-cold buffer (50 mM Tris, 0.05% BSA). Filters were immediatelyplaced into 7 mL scintillation vials to which 4 mL of scintillationuid was added. Bound radioactivity was determined afterovernight incubation at room temperature by liquid scintillationspectrophotometry. Specic binding was expressed as total minusnon-specic binding and graphed for each data point as a percent-age of specic binding occurring in the absence of any competitor.The afnity of IQDs for CB1R and CB2Rs was derived by employingthe ChengPrusoff equation21 to convert the observed IC50 valuesto Ki values, from 3 to 4 separate competition receptor bindingcurves for each ligand.

    All the synthesized IQDs were evaluated against the two can-nabinoid receptor subtypes, CB1 and CB2. Compounds 79, 11and 13 displayed high afnity (

  • abuse, depression, schizophrenia, inammation, chronic pain,obesity, osteoporosis and cancer.22


    We are grateful to NCI/NIH (Grant number CA 140409) and tothe Arkansas Research Alliance (ARA) for nancial support

    References and notes

    Table 1Ki values for (Z)-2-(N-benzylindol-3-ylmethylene) quinuclidin-3-ones (713) at CB1and CB2 receptors

    Compound Ki (nM)

    CB1 CB2

    7 135 44.1 11.7 5.558 9.23 0.64 1.33 0.459 31.7 9.25 20.3 7.43

    10 629 201 333 15711 55.3 8.89 114 36.212 953 162 588 23713 85.7 15.9 2.50 0.49

    N. R. Madadi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 20192021 2021compared to CB1R. Compound 13, which contains a COOCH3 moi-ety at the indolic 6-position, was a potent CB2 receptor ligand andexhibited 34-fold selectivity for CB2R (Ki = 2.50 nM) when com-pared to CB1R (Ki = 85.7 nM) (Table 1). The N-4-uorobenzyl ana-logue 8 displays high afnity for both CB1 (Ki = 9.23 nM) and CB2receptors (Ki = 1.33 nM), but was less selective for the CB2 receptorthan compound 13.

    The (Z)-()-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ols(14 and 15) and (2Z,3E)-2-((1-benzyl-1H-indol-3-yl) methylene)-quinuclidin-3-one oximes (16 and 17) exhibited lower afnity forboth CB1 and CB2 receptors when compared to their parent com-pounds (7 and 9) and were generally less selective. The overallafnities of quinuclidin-3-ol and quinuclidin-3-one oxime deriva-tives for CB1R and CB2R were reduced when compared to thoseof the corresponding quinuclidin-3-one derivatives (Table 2).

    Table 2Effects of simple and p-chloro substituted N-benzyl indole quinuclidin-3-ol analogs(14 and 15) the quinuclidin-3-one oxime analogs (16 and 17) on binding afnity

    Compound Ki (nM)

    CB1 CB2

    14 540 64.2 55.3 3.1015 85.3 42.5 113 2.0316 357 52.2 68.6 7.2717 95.7 2.03 107 8.75In summary N-benzylindolequinuclidinone (IQD) analogs havebeen identied as a novel class of cannabinoid ligands. These IQDswere evaluated against the two cannabinoid receptor subtypes,CB1 and CB2. Compounds 8 and 13 exhibited high afnity forCB2 receptors with Ki values of 1.33 and 2.50 nM, respectively,and had lower afnities for the CB1 receptor (Ki values of 9.23and 85.7 nM, respectively). Compound 13 had the highest selectiv-ity of all the compounds examined, and represents a potent can-nabinoid ligand with 34-times greater selectivity for CB2R overCB1R. These ndings are signicant for future drug development,given recent reports demonstrating benecial use of cannabinoidligands in a wide variety of human disease states including drug1. (a) Porter, A. C.; Felder, C. C. Pharmacol. Ther. 2001, 90, 45; (b) Williamson, E.M.; Evans, F. J. Drugs 2000, 60, 1303; (c) Hollister, L. E. Pharmacol. Rev. 1986, 38,1.

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    18. Analytical data and yields for the most active compounds: (8): 1H NMR (300 MHz,CDCl3): d 2.012.03 (m, 4H), 2.62 (p, J = 3.0 Hz, 1H), 2.923.02 (m, 2H), 3.083.18 (m, 2H), 5.34 (s, 2H), 6.967.25 (m, 7H), 7.46 (s, 1H), 7.877.90 (m, 1H),8.36 (s, 1H); 13C NMR (75 MHz, CDCl3): d 26.4, 40.5, 47.5, 50.0, 110.1, 111.4,115.6, 118.8, 121.1, 121.8, 122.8, 124.3, 127.7, 128.27, 130.7, 134.3, 135.5,135.8, 135.9, 185.2; yield: 87%: (13): 1H NMR (300 MHz, CDCl3): d 1.982.04(m, 4H), 2.622.63 (p, J = 2.7 Hz, 1H), 2.913.01 (m, 2H), 3.083.17 (m, 2H),3.90 (s, 3H, OCH3), 5.44 (s, 2H), 7.107.13 (m, 2H), 7.287.43 (m, 3H), 7.43 (s,1H), 7.817.89 (m, 2H), 8.03 (s, 1H), 8.51 (s, 1H); 13C NMR (75 MHz, CDCl3): d26.3, 40.4, 47.5, 50.6, 52.0, 110.6, 112.4, 117.1, 118.7, 122.0, 124.4, 126.5, 127.9,128.9, 132.2, 135.5, 136.2, 136.9, 141.3, 167.7, 205.3; yield: 91%.

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    Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptorsAcknowledgementsReferences and notes


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