Article pubs.acs.org/jmc
Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor Agonists with in Vivo Efficacy in a Mouse Model of Multiple Sclerosis Ying Shi,†,∇ Yan-Hui Duan,‡,∇ Yue-Yang Ji,† Zhi-Long Wang,§ Yan-Ran Wu,† Hendra Gunosewoyo,∥ Xiao-Yu Xie,⊥ Jian-Zhong Chen,⊥ Fan Yang,† Jing Li,§ Jie Tang,# Xin Xie,*,‡,§ and Li-Fang Yu*,† †
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China ‡ Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-Based Bio-Medicine, Collaborative Innovation Center for Brain Science, School of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China § CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China ∥ School of Pharmacy, Faculty of Health Sciences, Curtin University, Bentley, Perth, WA 6102, Australia ⊥ College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China # Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China S Supporting Information *
ABSTRACT: Selective CB2 agonists represent an attractive therapeutic strategy for the treatment of a variety of diseases without psychiatric side effects mediated by the CB1 receptor. We carried out a rational optimization of a black market designer drug SDB-001 that led to the identification of potent and selective CB2 agonists. A 7-methoxy or 7-methylthio substitution at the 3-amidoalkylindoles resulted in potent CB2 antagonists (27 or 28, IC50 = 16−28 nM). Replacement of the amidoalkyls from 3-position to the 2-position of the indole ring dramatically increased the agonist selectivity on the CB2 over CB1 receptor. Particularly, compound 57 displayed a potent agonist activity on the CB2 receptor (EC50 = 114−142 nM) without observable agonist or antagonist activity on the CB1 receptor. Furthermore, 57 significantly alleviated the clinical symptoms and protected the murine central nervous system from immune damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.
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INTRODUCTION The endocannabinoid system consists of at least two cannabinoid (CB) receptors, namely, the CB1 and CB2 receptors.1 Cloned in the early 1990s, these receptors belong to the class A family of G-protein-coupled receptors (GPCRs).2−4 Both receptors are coupled to the Gi/o proteins and subsequently inhibit the activity of adenylyl cyclase (AC), thus reducing the intracellular cyclic adenosine monophosphate (cAMP) production.5 Stimulation of the CB1 receptor results in blockade of the N-type and P/Q-type calcium channels while activating the inwardly rectifying K+ channels.6,7 The CB1 receptor consists of 472 amino acids and is one of the most abundant GPCR in the brain, especially in the hippocampus, cortex, basal ganglia, and cerebellum.8 It is expressed mainly in the presynaptic terminals where it modulates the release of various neurotransmitters.7,9 Stimulation of the CB1 receptor leads to modulation of feeding behavior, implicating an © 2017 American Chemical Society
important role for CB1 receptor in obesity and metabolic disorders.10,11 Rimonabant (1, SR141716A, Sanofi-Aventis), an inverse agonist/antagonist of the CB1 receptor, was approved in 2006 in Europe for the treatment of obesity.12 However, it was withdrawn 2 years later due to its severe central nervous system (CNS) related side effects including depression, anxiety, and suicidal thoughts.13 The crystal structure of human CB1 receptor14 was very recently resolved in a complex with the analogue of compound 1 (Figure 1), 4-(4-(1-(2,4-dichlorophenyl)-4-methyl-3-(piperidin-1-ylcarbamoyl)-1H-pyrazol-5yl)phenyl)but-3-yn-1-yl nitrate (AM6538),15 providing crucial insights into both the mechanistic studies and drug development in the field of CB receptor. Received: May 17, 2017 Published: July 20, 2017 7067
DOI: 10.1021/acs.jmedchem.7b00724 J. Med. Chem. 2017, 60, 7067−7083
Journal of Medicinal Chemistry
Article
Figure 1. Selected CB receptor ligands.
On the other hand, the CB2 receptor is primarily located in the peripheral immune cells such as splenocytes and leukocytes, with significantly lower expression levels in the neurons.1 The CB2 receptor signaling typically involves the activation of mitogen-activated protein kinases (MAPKs) and JUN Nterminal kinases (JNKs), as well as a transient increase in the intracellular calcium levels, resulting in complex physiological functions.16 The human CB2 receptor consists of 360 amino acids sharing 44% homology with CB1 at the amino acid level and 68% homology within the transmembrane regions.1 The crystal structure of the human CB2 receptor has not yet been resolved to date. Of particular interest, the stimulation of CB2 receptor was found to modulate the chemotactic profile of human monocytes via downregulation of the chemokine receptors and inhibition of interferon γ (IFNγ) induced intercellular adhesion molecule 1 (ICAM-1) expression.17 The CB2 receptor is responsible for many aspects of the CBs’ immunomodulatory and anti-inflammatory effects, and therefore CB2 receptor agonists have been suggested as a viable therapeutic option for conditions such as pain, osteoporosis, and multiple sclerosis (MS).18−21
MS is a neuroinflammatory disorder characterized by damages in the myelin sheath which leads to reduced nerve conductivity and the clinical symptoms of MS. The majority of MS patients initially have a relapsing−remitting MS and eventually progressing to a secondary stage of MS where loss of neurological function manifests. There is no cure for MS to date, and the main goal of currently available treatment is to relieve the symptoms such as spasticity and pain. In clinical settings, cannabinoids exhibit beneficial effects on relieving the symptoms of MS. Nabiximols (Sativex, GW Pharmaceuticals) is a combination of botanical extracts containing approximately 1:1 tetrahydrocannabinol (2, THC)/cannabidiol (3, CBD) and is marketed in more than 25 countries worldwide for the treatment of MS. The metabolite of compound 2, (6aR,10aR)1-hydroxy-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydro-6H-benzo[c]chromene-9-carboxylic acid (JBT-101, ajulemic acid, Corbus Pharmaceuticals), was found to selectively bind to the CB2 receptor on immune cells and fibroblasts,22 eventually granted an FDA fast-track development status for the treatment of systemic sclerosis in 2015. Recent studies have also shown that potent CB2 agonists that are devoid of CB1 activity could offer therapeutic benefits for 7068
DOI: 10.1021/acs.jmedchem.7b00724 J. Med. Chem. 2017, 60, 7067−7083
Journal of Medicinal Chemistry
Article
Scheme 1a
Reagents and conditions: (a) NaH, DMF, 0 °C; 4-(2-bromoethyl)morpholine, 100 °C; (b) (i) (CF3CO)2O, DMF, 0 °C to rt; (ii) NaOH, H2O, reflux; (c) (COCl)2, DMF (cat.), CH2Cl2, rt; amantadine, Et3N, CH2Cl2, rt; (d) TBSCl, imidazole, CH2Cl2, rt; (e) 30, NaH, DMF, 0−100 °C; (f) TBAF, THF, rt; (g) ma-chloroperoxybenzoic acid, CH2Cl2, −40 °C; (h) CH3I, NaH, DMF, 0 °C to rt.
a
of synthetic CB receptor ligands. WIN55212-2 (8) represents a nonselective CB agonist (Ki,CB1 = 1.9 nM, Ki,CB2 = 0.3 nM) that has been widely used as pharmacological tool.31 Selective CB2 agonists were developed based on an indole scaffold, such as aminoalkylindoles JWH-015 (9) and AM1241 (10).32−35 Dual function compounds with CB1 receptor antagonist and CB2 receptor agonist activities were also developed via the optimization of one common component of K2/spice naphthalen-1-yl-(1-butylindol-3-yl)methanone (JWH-073).36,37 SDB-001 (11) and AB-001 (12) belong to the 3-amidoalkylindole and 3-acylalkylindole class of cannabimimetics, identified in Japan and Ireland as a “designer drug” in 2012 and 2010, respectively.38,39 Pharmacological assessments reveal that compounds 11−13 were potent CB agonists, with similar EC50 values for both CB1 (35−40 nM) and CB2 (29−89 nM) receptors.40 Compound 11 induced hypothermia and heart rate in rats at 1 mg/kg, while compounds 12 and 13 only produced a weak cannabimimetic activity in rat up to 30 mg/kg. Although the linker moiety between the adamantane and the indole ring was not important for potent in vitro binding, an amide linker was crucial for the in vivo activities, as demonstrated by the significantly lower activity of the adamantyl ketone 12.40 On the basis of these observations, we selected 3-amidoalkylindole 11 as a starting point for the development of selective CB2 receptor ligands with minimal CNS-related adverse effects.
alleviating the symptoms of MS, although the exact mechanisms of action remain to be further established.23,24 In preclinical studies, the beneficial effects of cannabinoids have been reported in different animal models of MS including experimental autoimmune encephalomyelitis (EAE), chronic relapsing experimental allergic encephalomyelitis (CREAE), and Theiler’s murine encephalomyelitis virus induced demyelinating disease (TMEV-IDD).25 Chromenopyrazole (4), a selective and potent CB2 receptor agonist, was found to dampen neuroinflammation in the TMEV-IDD mouse model by reducing microglial activation.26 Administration of a selective CB2 receptor agonist quinoline-2,4(1H,3H)-dione (5) reduced the clinical scores in the mouse model of EAE, as shown by the decreased leukocyte infiltration and demyelination in the CNS.27 The bicyclic sesquiterpene (−)-β-caryophyllene (6) was previously shown to exhibit anti-inflammatory and analgesic effects in mouse models.28 Very recently, this compound was reported to prevent the progression of clinical symptoms and neuroinflammation in the mouse model of EAE.29 To date, numerous synthetic ligands for both CB1 and CB2 receptors have been investigated in both academia and pharma industry settings such as the widely used tool compound CP55940 (7).30 In particular, indole-containing compounds have been recognized as a popular scaffold for the investigation 7069
DOI: 10.1021/acs.jmedchem.7b00724 J. Med. Chem. 2017, 60, 7067−7083
Journal of Medicinal Chemistry
Article
Scheme 2a
a
Reagents and conditions: (a) NaOH, H2O, reflux; (b) (i) (COCl)2, DMF (cat.), CH2Cl2, rt; (ii) adamantan-1-amine or aniline, Et3N, CH2Cl2, rt; (c) NaH, R1Br or R1Cl, DMF, 0−100 °C; (d) aqueous KOH, reflux; (e) SelectFluor, NaHCO3, THF, rt; (f) (3-bromopropoxy)(tertbutyl)dimethylsilane, 37 or 38, NaH, DMF, 100 °C; (g) TBAF, THF, rt; (h) Dess−Martin periodinane, CH2Cl2, rt; (i) DAST, CCl4, rt; (j) (i) (Boc)2O, CH2Cl2, rt; (ii) TsCl, Et3N, CH2Cl2; (k) 43, NaH, DMF; (l) HCl in EtOAc, rt; (m) 1H-pyrazole-1-carboxamidine hydrochloride, MeOH, 40 °C.
Treatment with trifluoroacetic anhydride41 followed by basic hydrolysis gave carboxylic acid 15. Subsequent reaction with oxalyl chloride and amantadine afforded the final product 16. Alternatively, compound 16 could also be synthesized via route B: amidation followed by N-alkylation. Compounds 11, 25−28, 32−36 were synthesized in a similar manner to compound 16 starting from indole or various substituted indoles with appropriate bromides and amines. The sulfinyl analog 29 was prepared via oxidation of the thiomethyl compound 28 using m-chloroperoxybenzoic acid. tert-Butyldimethylsilyl (TBS) protection of commercially available 4-chlorobutan-1-ol and 5-chloropentan-1-ol yielded silyl ethers 37 and 38, respectively,
Herein we report our efforts on the investigation of 2- and 3amidoalkylindoles that led to the identification of potent and highly selective CB2 receptor ligands with beneficial effects in the mouse EAE model of MS.
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RESULTS AND DISCUSSION Chemistry. The 3-amidoalkylindoles 11, 16, 25−36, 39−41 were synthesized starting from commercially available substituted indoles utilizing the synthetic routes shown in Scheme 1. Indole was first N-alkylated with 4-(2-bromoethyl)morpholine in dimethylformamide (DMF) using sodium hydride as a base to form morpholine indole 14 (route A). 7070
DOI: 10.1021/acs.jmedchem.7b00724 J. Med. Chem. 2017, 60, 7067−7083
Journal of Medicinal Chemistry
Article
Table 1. Agonist and Antagonist Activities of 3-Amidoalkylindoles in CHO Cells Expressing Human CB1 or CB2 Receptor by Calcium Mobilization Assaysa agonism
antagonism
CB1
CB2
CB1
CB2
compd
EC50, μM
Emax, %
EC50, μM
Emax, %
IC50, μM
IC50, μM
11 16 30 25 26 27 28 29 32 31 33 34 35 36 39 40 41 7
0.039 ± 0.004 3.3 ± 0.1 NAb NAb NAb NAb NAb NAb 6.7 ± 2.0 NAb 0.084 ± 0.015 NAb NAb 0.63 ± 0.11 13 ± 0.1 4.1 ± 1.3 NAb 0.022 ± 0.005
68.3 ± 11.7 75.0 ± 7.6 NDc NDc NDc NDc NDc NDc 63.3 ± 1.7 NDc 133 ± 12 NDc NDc 105 ± 3 58.3 ± 1.7 76.7 ± 9.3 NDc 100 ± 15
0.059 ± 0.020 0.059 ± 0.005 0.63 ± 0.24 NAb NAb NAb NAb NAb NAb 0.50 ± 0.05 0.038 ± 0.006 0.41 ± 0.05 0.18 ± 0.03 0.031 ± 0.003 0.16 ± 0.04 0.062 ± 0.009 0.082 ± 0.016 0.085 ± 0.009
66.3 ± 4.4 56.7 ± 12.0 75.0 ± 2.9 NDc NDc NDc NDc NDc NAb 60.0 ± 2.9 76.7 ± 6.0 66.3 ± 1.7 73.3 ± 1.7 70.0 ± 5.0 68.3 ± 1.7 63.3 ± 1.7 70.0 ± 2.9 100 ± 6
NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb
NAb NAb NAb NAb NAb 0.016 ± 0.001 0.028 ± 0.005 NAb NAb NAb NAb NAb NAb NAb NAb NAb NAb
See Experimental Section. Data represent mean values ± SEM of eight-point experiments each performed in triplicates from three independent experiments. In the agonist mode, compound 7 was used as a positive control. In the antagonist mode, cells were preincubated with either the test compounds or compound 1 as a positive control for 10 min, followed by addition of agonist 7 at 100 nM. bNA: not active, defined as
