Discovery and optimization of a novel series of N-arylamide oxadiazoles as potent, highly selective and orally bioavailable cannabinoid receptor 2 (CB 2) agonists

J. Med. Chem. 2008, 51, 5019–5034

5019

Discovery and Optimization of a Novel Series of N-Arylamide Oxadiazoles as Potent, Highly

Selective and Orally Bioavailable Cannabinoid Receptor 2 (CB₂) Agonists

Yuan Cheng,*^,†Brian K. Albrecht,^§James Brown,^†John L. Buchanan,^§William H. Buckner,^§Erin F. DiMauro,^§

Renee Emkey, Robert T. Fremeau, Jr.,^|Jean-Christophe Harmange,^§Beth J. Hoffman,^|Liyue Huang,^#Ming Huang,^|

Josie Han Lee, Fen-Fen Lin,³Matthew W. Martin,^§Hung Q. Nguyen,³Vinod F. Patel,^§Susan A. Tomlinson,^§

Ryan D. White,^§Xiaoyang Xia,^‡and Stephen A. Hitchcock^†

Chemistry Research and DiscoVery, Department of Molecular Structure, Department of Neuroscience, Department of Protein Science, Amgen

Inc., One Amgen Center DriVe, Thousand Oaks, California 91320, Chemistry Research and DiscoVery, Department of HTS and Molecular

Pharmocology, Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1 Kendall Square Building 1000,

Cambridge, Massachusetts 02139

ReceiVed April 22, 2008

The CB₂receptor is an attractive therapeutic target for analgesic and anti-inﬂammatory agents. Herein we

describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB₂agonist

leads were developed. Initial hit 7 was identiﬁed from a cannabinoid target-biased library generated by

virtual screening of sample collections using a pharmacophore model in combination with a series of

physicochemical ﬁlters. 7 was demonstrated to be a selective CB₂agonist (CB₂EC₅₀) 93 nM, E_max

)

98%, CB₁EC₅₀> 10 µM). However, this compound exhibited poor solubility and relatively high clearance

in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route

toward improving potency, physicochemical properties, and solubility. This effort resulted in identiﬁcation

of 63 that is a potent and selective agonist at CB₂(EC₅₀) 2 nM, E_max) 110%) with excellent pharmacokinetic

properties.

Introduction

Two distinct receptors have been identiﬁed that mediate the

biologic effect of plant-derived, synthetic, and endogenous

cannabinoids, the CB₁¹and CB₂²receptors. These receptors are

composed of seven transmembrane proteins that couple to

inhibitory G_i/G_oproteins and thus inhibit adenylate cyclase and

activate MAP kinases. The CB₁and CB₂receptors share 44%

amino acid sequence identity and exhibit different pharmaco-

logic proﬁles and distinct tissue expression patterns.²The CB₁

receptor is highly expressed in the central nervous system

(CNS),^awhere it is primarily localized on axons and nerve

terminals.³Activation of presynaptic CB₁receptors inhibits

* To whom correspondence should be addressed. Phone: 805-447-3543.

Fax: 805-480-3016. E-mail:yuanc@amgen.com.

†

Chemistry Research and Discovery, Amgen Inc., Thousand Oaks, CA.

Department of Molecular Structure, Amgen Inc., Thousand Oaks, CA.

Chemistry Research and Discovery, Amgen Inc., Cambridge, MA.

Department of Neuroscience, Amgen Inc., Thousand Oaks, CA.

‡

§

|

Department of HTS and Molecular Pharmocology, Amgen Inc.,

Cambridge, MA.

#

Department of Pharmacokinetics and Drug Metabolism, Amgen Inc.,

Cambridge, MA.

3

Department of Protein Science, Amgen Inc., Thousand Oaks, CA.

^aAbbreviations: CB₂, cannabinoid receptor 2; CB₁, cannabinoid receptor

1; SAR, structure-activity relationship; FLAME, fexibly align molecules;

MAP kinases, mitogen-activated protein kinases; CNS, central nervous

system; ∆⁹-THC, ∆⁹-tetrahydrocannabinol; HLM, human liver microsomes;

RLM, rat liver microsomes; F, bioavailability; CL, in vivo clearance; AUC,

the area under the curve; C_max, the maximum concentration of a compound

observed after its administration; SIF, simulated intestinal ﬂuid solubility;

ACD-SC, available chemicals directory-screening compounds; PSA, polar

surface area; DMF, N,N-dimethylformamide; DMA, N,N-dimethylacetamide;

EtOAc, ethyl acetate; NMP, N-methylpyrrolidone, EDC, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride; HOBt, 1-hydroxylben-

zotrizole; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium

hexaﬂuorophosphate methanaminium; DMSO, dimethyl sulfoxide; PS-

carbodiimide, N-cyclohexylcarbodiimide-N′-propyloxymethylpolystyrene;

MP-carbonate, macroporous triethylammonium methylpolystyrene carbon-

ate; Ph₃P, triphenylphosphine; cAMP, cyclic adenosine monophosphate;

GTP, guanosine-5′-triphosphate.

Figure 1. Structure of 1 (∆⁹-THC), 2 (CP-55,940),¹¹3 (Cannabinor),¹²

4 (GW842166X),¹³5 (A-796260),¹⁴and 6.

neurotransmitter release and produces a characteristic tetrad of

behavioral effects including hypomotility, hypothermia, cata-

lepsy, and acute analgesia. It is likely that ∆⁹-tetrahydrocan-

nabinol (∆⁹-THC, 1, Figure 1), the active ingredient in marijuana

produces most, if not all of its psychoactive behavioral effects

through action of the CB₁receptor.⁴In contrast, the CB₂receptor

is primarily expressed by immune cells and tissues.⁵Activation

of the CB₂receptor is thought to mediate the immunosuppressive

effects of cannabinoids. It has been recently reported that CB₂

10.1021/jm800463f CCC: $40.75

2008 American Chemical Society

Published on Web 08/05/2008

5020 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Cheng et al.

Scheme 1. Synthesis of Oadiazole Aide Drivatives^a

Figure 2. Initial lead structure as CB2 agonist.

^aReagents and conditions: (a) NH₂OH, MeOH, reﬂux; (b) succinimide,

DMF, 120 °C; (c) EDC, CH₂Cl₂, R²NH₂, 0-25 °C; or thionyl chloride,

then R²NH₂, N,N-diisopropylethylamine, CH₂Cl₂.

is also found within the CNS.⁶Given the predominant peripheral

distribution of the CB₂receptor, we hypothesized that selective

activation of CB₂should be devoid of undesired psychoactive

effects that are believed to be mediated through central CB₁.

The role of CB₁and CB₂in the transduction and perception

of pain is supported by an emerging body of evidence.⁷The

role of CB₁receptors in pain response is more thoroughly

understood than that of CB₂, however CB₁agonists typically

illicit problematic psychotropic effects and are consequently

hampered by the potential for abuse.^3,4Since its identiﬁcation

and cloning in 1993, understanding of the pharmacology

associated with the CB₂receptor subtype continues to evolve.

The expression pattern of CB₂receptors on cells and in tissues

that modulate the immune system renders CB₂an attractive

potential therapeutic target for analgesic and anti-inﬂammatory

agents. Recent studies indicate that the CB₂receptor could be

involved in the modulation of inﬂammation,⁸neuropathic, and

inﬂammatory pain.⁹Multiple CB₂agonists of different structural

classes have been reported to reduce inﬂammatory and neuro-

pathic pain. In addition, CB₂agonists have been reported to

elicit anti-inﬂammatory effects and to promote functional

recovery in animal models of multiple sclerosis.¹⁰These factors,

as well as the need for safe and effective anti-inﬂammatory

agents and analgesics, make the CB₂receptor an attractive target.

The development of highly selective CB₂agonists with desirable

physical and pharmacokinetic properties is necessary for both

understanding the biological role of the CB₂receptors and

evaluation of their therapeutic potential.

In recent years, there has been growing interest from a number

of groups focused on developing selective CB₂agonists.⁹

However, only a few compounds are known in the literature to

be selective for the CB₂receptor (3-6, Figure 1).^9,11-15For

example, compound 6 was recently disclosed to be potent (CB₂

K_i) 13 nM) and selective (CB₁K_i) 3500 nM).¹⁵However

there are even fewer examples of CB₂agonists that have

acceptable oral bioavailability (4, formulation dependent F )

25-60% in rats and dogs; 6, F ) 52% in rats).^13,15Cannabinoid

ligands are typically highly lipophilic, often leading to low

aqueous solubility and poor physicochemical properties. There-

fore, design of selective cannabinoid agonists that have good

oral bioavailability and retain high afﬁnity remains a major

challenge.

a full CB₂agonist (CB₂EC₅₀) 93 nM, E_max) 98%) with

high selectivity over the CB₁receptor (CB₁EC₅₀> 10 µM).

Full agonist activity was deﬁned as E_maxequal to 100% efﬁcacy

of 2, a known agonist at CB₁and CB₂receptor.¹¹Broader

selectivity of compound 7 for the CB₂receptor was further

demonstrated by absence of activity when screened against a

panel of 141 receptors/enzymes/ion channels. Compound 7

showed moderate microsomal stability (HLM ) 274 µL/min/

mg and RLM ) 198 µL/min/mg) and moderate in vivo clearance

(CL ) 1.2 L/h/kg, iv dose of 0.5 mg/kg in rats).

However, compound 7 suffered from poor solubility (simu-

lated intestinal ﬂuid solubility, SIF ) 2 µg/mL) and low and

variable oral bioavailability (F ) 13-24% in rats). Overall the

results indicated that compound 7 represented a promising

proﬁle as a lead compound. We therefore carried out SAR

studies on this compound, particularly focused on improving

solubility and physicochemical properties. Herein we describe

the discovery and systematic SAR investigation on the oxadia-

zole structural class that has revealed CB₂agonists that exhibit

subnanomolar activity and excellent oral bioavailability.

Chemistry. The representative synthesis of oxadiazole amide

analogues is shown in Scheme 1. Commercially available or

readily prepared nitriles 8 were treated with hydroxylamine in

methanol to give amide oxime 9, which upon heating with

succinimide in DMF gave oxadiazole acid 10.¹⁶The acid was

subjected to standard coupling under standard conditions with

commercially available or readily made amines to afford formal

target 11.

Speciﬁcally, 2-cyano-4-ﬂuoro-phenyl 14 and 2-cyano-4-

pyridyl 17 derivatives were prepared as illustrated in Scheme

2. Protection of the carboxylic acid functionality was necessary

to allow convertion of the bromide to the correspondent nitrile

by heating with copper(I) cyanide under microwave conditions.

Hydrolysis to acids (13 and 16) and coupling with the

appropriate amines gave 14 and 17.

As part of our SAR investigation, we replaced the amide

linker with ether and amine linkers using the sequence described

in Scheme 3. Reduction of the ester 18 to alcohol 19 and

Mitsunobu reaction or converting the alcohol to bromide

followed by displacement with the appropriate alcohols afforded

the ether-linked analogues 20. Alternatively, half-reduction of

ester 18 could also provide aldehyde 21, which underwent

reductive amination with various amines to produce oxadiazole

amine analogues 22.

Our efforts to identify effective CB₂agonists have revealed

a novel class of aryl oxadiazoles that are potent and highly

selective. Our program began with the generation of a phar-

macophore model, based on known cannabinoid ligands, which

was used to conduct a virtual screen of our in-house and

commercial compound databases. This method led to a can-

nabinoid target-biased library, which was then screened in both

CB₁and CB₂binding assays. Hits showing IC₅₀< 10 µM at

CB₂and >10-fold selectivity over CB₁were evaluated for their

agonist activity in functional assays. Those that demonstrated

selective CB₂functional agonist activity were selected for their

physicochemical and pharmacokinetic proﬁles. This line of

investigation led to identiﬁcation of oxadiazole 7 (Figure 2) as

Intermediate nicotinonitrile 26, used in the preparation of 56

and 60, was synthesized as described in Scheme 4. Displacement

of the chloride in 23 with triﬂuoroethanol gave intermediate

24. Subsequent conversion of bromide in 24 to the nitrile group

followed by selective reduction of the nitro group to amine

yielded 26.

Intermediates 6-chloroquinolin-3-amine for 47 and 63, 6-tri-

ﬂuoromethoxyquinolin-3-amine for 64, and 6-chloro-1,8-naph-

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5021

Scheme 2. Synthesis of 2-Cyano-4-ﬂuoro-phenyl and 2-Cyano-4-pyridyl Derivatives^a

^aReagents and conditions: (a) trimethylsilyldiazomethane, toluene/methanol (9:1), rt; (b) KCN, CuCl, DMA, 130 °C; (c) LiOH, methanol or DME, rt; (d)

EDC, CH₂Cl₂, RNH₂, 0-25 °C. (e) CuCN, DMF, microwave, 180 °C.

Scheme 3. Synthesis of Oxadiazole Ether and Amine Analogues^a

^aReagents and conditions: (a) diisobutylaluminum hydride, dichloromethane, -78-0 °C; (b) R¹OH, Ph₃P, diisopropyl azodicarboxylate, benzene, 25 °C,

or Br₂, THF, then R¹OH, K₂CO₃, DMF, 60 °C; (c) diisobutylaluminum hydride, dichloromethane, -78 °C; (d) titanium isopropoxide, R²NH₂, NaBH₄.

4(1H)-on-3-carboxamide 28,¹⁸and indolopyridone 29,¹⁹(Figure

Scheme 4. Synthesis of 5-Amino-2-(2,2,2-triﬂuoroethoxy)nicoti-

3) using an in-house developed software (FLAME, ﬂexibly align

molecules).²⁰These ligands have high afﬁnity for the CB₂

receptor but limited selectivity over CB₁. The FLAME program

aligns three molecules by ﬁrst ﬁnding maximum common

pharmacophores among them using a generic algorithm. The

resulting alignments were then subjected to simultaneous

optimizations of their internal energies and given an alignment

score. Figure 3 shows the 3-feature pharmacophore model

generated by the three cannabinoid ligands. The best alignment

was then used to search the in-house sample collection (363416

compounds) and commercial compound databases (preﬁltered

ACD-SC database by MRL, 778296 compounds). Top 1000

scored compounds from each database search were extracted

and were further reﬁned by applying physicochemical ﬁlters,

such as calculated logP (ClogP) < 5, polar surface area (PSA)

< 110 Å², number of rotatable bonds e10, and hydrogen bond

donors e2. This exercise led to a cannabinoid target-biased

library with 1092 compounds. After screening this library in

CB₁and CB₂binding assays, 74 compounds met the criteria of

CB₂binding afﬁnity less than 10 µM with greater than 10-fold

selectivity over the CB₁receptor. These compounds were next

tested in GTP-Eu binding functional assays for CB₂and CB₁

receptors, respectively. Eighteen compounds were conﬁrmed as

full CB₂agonists with EC₅₀values less than 1 µM and greater

than 25-fold selectivity over the CB₁receptor. The structural

diversity of the resulting hits compared to the three original

ligands used the build the pharmacophore model, demonstrates

the utility of the FLAME model to generate novel starting points.

Figure 4 shows the alignments of compound 7 and the

pharmacophore model.

nonitrile^a

^aReagents and conditions: (a) Sodium hydride, 2,2,2-triﬂuoroethanol,

0-25 °C; (b) copper(I) cyanide, N-methylpyrrolidone, microwave, 195 °C;

(c) iron, acetic acid, H₂O, ethanol, reﬂux.

thyridin-3-amine for 48 were prepared by cyclization of

methazonic acid with correspondent 2-amino-5-chlorobenzal-

dehyde, 2-amino-5-(triﬂuoromethoxy)benzaldehyde, and 2-amino-

5-chloropyridine-3-carboxaldehyde, respectively, under acidic

conditions. Intermediate 6-chloroquinolin-3-ol for 67 was

synthesized by diazotizing 6-chloroquinolin-3-amine with so-

dium nitrite, and the diazonium compound was decomposed to

6-chloroquinolin-3-ol (see Experimental Section). Compounds

(30-36, Table 1) with alternative heterocycles were prepared

using known procedures to form the heterocycle cores (see

Experimental Section).

Results and Discussion

FLAME Model and Screening. The cannabinoid pharma-

cophore model was designed by employing three known

cannabinoid ligands, 27 (WIN 55,212-2),¹⁷1,8-naphthyridin-

5022 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Cheng et al.

Figure 4. Mapping of compound 7 to the pharmacophore model. F1

in magenta, aromatic; F2 in green, hydrophobic; F3 in yellow, hydrogen

bond acceptor.

receptor but also displayed excellent selectivity over CB₁. This

lead identiﬁcation process demonstrated that combining phar-

macophore model screening and physicochemical ﬁlters could

provide a powerful and efﬁcient tool for generating high quality

leads.

Pharmacology. The CB receptor activity and selectivity of

the compounds reported in this paper were evaluated in two

functional assays, a GTP binding assay and a cAMP assay.

Membranes prepared from cells expressing either human CB₁

or CB₂receptor were used in the GTP binding assay. This is a

proximal assay that measures activation of G-protein coupled

receptors by the ability of the associated G_R-protein to bind GTP.

Compound 2 (Figure 1) was used to deﬁne 100% efﬁcacy

(Figure 5).

Compounds that exhibited full agonism with good potency

in the GTP binding assay were progressed for evaluation in the

cell-based cAMP assay. This assay monitors a distal signaling

event that emanates from the CB₁and CB₂receptors. Activation

of CB₁and CB₂receptors results in inhibition of adenylate

cyclase. Compounds were assessed for their ability to inhibit

forskolin-induced increase in intracellular cAMP. Species dif-

ference in agonist activity was also investigated in this assay

by using cells expressing human CB₂receptor, human CB₁

receptor, rat CB₂receptor, and rat CB₁receptor. Although the

CB₂receptor exhibits 80% sequence identity between human

and rodent,²¹the analogues of the oxadiazole class showed

consistent activity in both human and rat CB₂receptor (Tables

1-4).

SAR Studies. We ﬁrst examined the oxadiazole core of 7

by replacing it with a number of alternative heterocycles shown

in Table 1. Removing either one of the nitrogens of the

oxadiazole, giving isoxazole 30 and oxazole 31, resulted in a

drop in activity (over 2-fold and 17-fold, respectively) and a

decrease in efﬁcacy (E_max). Pyrazole 32 and methylated pyrazole

33 showed no CB₂agonist activity up to 10 µM, while triazole

34 also registered as a 0.5 µM partial agonist of CB₂. Although

the isomeric oxadiazole 35 and tetrazole 36 were similar and

displayed comparable potency and efﬁcacy to compound 7, none

had better potency than compound 7, suggesting that the

oxadiazole was optimal.

Figure 3. The pharmacophore model generated by canabinoid ligands,

27 in green, 28 in magenta, and 29 in orange. (A) Geometrical

relationships among the pharmacophore features. F1 in magenta,

aromatic; F2 in green, hydrophobic; F3 in yellow, hydrogen bond

acceptor. The distances (in Å) among the centers of the features are

labeled. (B) Mapping of 27, 28, and 29 to the pharmacophore model.

We conﬁrmed the structures of 18 lead compounds and

evaluated their physicochemical properties, solubility, melting

point, permeability, rat (RLM) and human (HLM) microsomal

stability, and broader selectivity proﬁle with pharmacology

screening. RLM and HLM were used as a ﬁlter to select

compounds for in vivo pharmacokinetic studies in rats. In vivo

clearance was determined by intravenous (iv) administration in

rats with compounds that had HLM/RLM less than 300 µL/

min/mg. This investigation allowed us to prioritize oxadiazole

7 that showed in vivo clearance less than 2 L/h/kg. Although

we derived compound 7 from three relatively nonselective CB₂

ligands, it not only showed good agonist activity on the CB₂

Next, we investigated modiﬁcation of the aryl group attached

to the oxadizole (Table 2). The SAR proved very sensitive in

this region of the molecule. Removing the 2-ﬂuoro substituent,

as with compound 37, caused a reduction in activity. Replace-

ment of the ﬂuorine atom with chlorine (38) increased the

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5023

Figure 5. Compounds 2 and 7 in the GTP binding assay for CB₂and CB₁.

potency over 18-fold without loss of efﬁcacy, but larger ortho

substituents such as isopropyl 39 were not well tolerated.

Moving the chlorine atom to the meta-position resulted in

compound 40 with a marked reduction in activity. However,

ﬂuoro substitution at the para-position 41 provided good

potency. Combining both ortho and para substituents proved

very productive, delivering compounds 42-44 that possessed

excellent CB₂activity with low nanomolar and picomolar CB₂

potency. Indeed, compound 43 exhibited an EC₅₀value of 89

pM in the GTP-Eu binding assay. Replacement of the phenyl

group by 4-pyridyl 45 was well tolerated. In addition, saturated

ring systems could also be tolerated. 4-Pyran 46, for example,

showed better potency than the corresponding phenyl analogue

37. Gratifyingly, the excellent potency and selectivity of

analogues 41-46 was consistent in the cAMP assay against

cells expressing rat or human CB₂receptors.

Table 1. Functional Activity for Oxadiazole Replacements

b

^aThe results are expressed as the means ( SEM for n ) 2-20 independent measurements and were calculated in Prism by use of a logistic ﬁt. E_max

is the maximal effect of the test compounds and expressed as a percentage of that value obtained with 2. ^cND ) not determined.

5024 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Table 2. Functional Activity for Right Hand Side Variant Analogues

Cheng et al.

^aThe results are expressed as the mean of at least two determinations ( SEM and were calculated in Prism by use of a logistic ﬁt. ^bE_maxis the maximal

effect of the test compounds and expressed as a percentage of that value obtained with 2. ^cND ) not determined.

Using optimal aryl oxadiazoles, attention shifted to exploring

the carbazole region of the lead molecule 7. With a mindset to

improve solubility and physicochemical properties of 7 by

lowering molecular weight and decreasing hydrophobicity and

the precedent for mutagenic potential among certain carbazole

containing compounds,²²we elected to search for carbazole

replacements. The results of this endeavor are shown in Table

3. This region of the molecule appears to be fairly accom-

modating, accepting a variety of aromatic ring systems such as

quinoline 47, naphthyridine 48, benzothiazole 49, and thiadiazole

50, all of which showed better potency and selectivity than 7.

In addition, they all demonstrated improved metabolic stability

in human and rat liver microsomes (HLM/RLM < 100 µL/

min/mg). However, these compounds 47-50 showed little

improvement in solubility and oral bioavailability relative to 7.

A further reduction in hydrophobicity relative to 7 via replace-

ment of the carbazole unit with a smaller isopropylphenyl group

51 demonstrated an impressive 10-fold increase in potency as

compared to the lead compound 7. Expanded SAR studies

around additional aryl groups showed that para-substituted

phenyl ring was important for CB₂potency. Adding a chlorine

atom at the ortho-position 53 led to 5-fold decrease in activity.

On the other hand, chlorine substitution at the meta-position

54 was equipotent to 52. To introduce polarity, we prepared

heteroaryl derivative 6-substituted pyridin-3-amines 55, 2-sub-

stituted pyrimidin-5-amines 57, and 5-substituted isoxazole-3-

amine 58. Although compounds 57 and 58 were potent, they

displayed CB₁agonism in the cAMP assay. Further investigation

yielded 2-triﬂuoroethoxy-3-cyano-pyridin-5-amine analogue 56,

which showed low nanomolar potency and good selectivity in

the GTP binding and functional cAMP experiments. Compound

56 is more soluble than compound 7 with SIF solubility of 24

µg/mL and demonstrated more favorable pharmacokinetic

properties in rat relative to compound 1 (Table 5). On oral

dosing 56, >30-fold higher levels in AUC and >40-fold C_max

compared to 7 were achieved at an equivalent dose. The oral

bioavailability of compound 56 (36%) was also superior to 7.

As our work progressed, hydrolysis of the amide bond was

observed as a major clearance pathway in the in vivo experi-

ments in rat. Acid 59 was the major circulating metabolite

(Figure 6). To suppress this metabolic pathway, we sought to

remedy this problem by preparing suitable nonamide-linked

analogues.

To this end, a number of amine-linked compounds were

examined as shown in Table 4. We found, in general, the amine-

linked monocyclic analogues gave lower activity as compared

to their amide counterparts (cf. 60 and 56, 61 and 58). The loss

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5025

Table 3. Functional Activity for Left Hand Side Variant Analogues

^aThe results are expressed as the mean of at least two determinations ( SEM and were calculated in Prism by use of a logistic ﬁt. ^bE_maxis the maximal

effect of the test compounds and expressed as a percentage of that value obtained with 2. ^cND ) not determined.

in potency was also observed with the benzothiazole analogues

such as compound 62.

and 49 µg/mL, respectively, an order of magnitude improved

over compound 7. Encouraged by these observations, we further

evaluated the pharmacokinetic proﬁle of several amine-linked

analogues in rats of which compound 63 showed excellent in

vivo properties (Table 5). The oral administration of 63 at a

dose 10 mg/kg (po) in rats resulted in high plasma exposure

(AUC at 43.1 µg h/mL), twice that of compound 56 and over

80-fold that of compound 7 at equivalent dose levels. Compound

63 also generated twice the C_maxof 56 and over 90-fold that of

7. The half-life of 63 was also improved over that of 56 and 7

(5.1, 3.1, and 1.6 h, respectively). More importantly, Compound

63 showed outstanding oral bioavailability (100%) in rats.

However, the amine-linked quinoline analogues exhibited a

comparable potency and selectivity proﬁle to corresponding

amide analogues (cf. 63 and 47). The 3-(triﬂuoromethox-

y)quinolin-6-amine 64 was found to be one of the most potent

analogues, with EC₅₀value at 15 pM in the GTP-Eu assay.

Methylation of the amine (66) caused a large drop in potency.

The amine-linked compounds also showed a signiﬁcant im-

provement in solubility relative to amide congeners. As an

example, SIF solubility of 63 and 64 was determined to be 87

A number of ether-linked and carbon-linked analogues were

also investigated. The ether-linked analogues, such as 67 and

68, showed modestly lower but still respectable potency

compared to amine-linked analogue 63 and amide-linked

analogue 57, respectively. However, solubility and the phar-

macokinetic proﬁle of these compounds were generally less

desirable than the amine-linked analogues. Carbon-linked

Figure 6. The acid metabolite of amide hydrolysis.

5026 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Table 4. Functional Activity for Non-Amide Analogues

Cheng et al.

^aThe results are expressed as the mean of at least two determinations ( SEM and were calculated in Prism by use of a logistic ﬁt. ^bE_maxis the maximal

effect of the test compounds and expressed as a percentage of that value obtained with 2. ^cND ) Not determined.

Table 5. Pharmacokinetics in Rats (dosed at 10 mpk)

(i.e., 56). Subsequently, we incorporated an amine linker in an

effort suppress plasma accumulation of an acid metabolite

resulting from amide hydrolysis. This study led to highly potent,

selective CB₂agonists, as exempliﬁed by 63, which displayed

an excellent pharmacokinetic proﬁle with 100% oral bioavail-

ability in rats.

compd

C_max(µg/mL)

AUC (µg h/mL)

F (%)

t_1/2(h)

7

56

63

0.1

3.4

8.4

0.46

21

43

13

36

1.6

3.1

5.1

<100

analogues generally suffered from weaker CB₂activity, such

Experimental Section

as compound 69 vs 65.

FLAME Model and Screening. The pharmacophore model was

designed using three known cannabinoid ligands selected from the

literature using FLAME following this procedure: CONCORD was

used to generate their three-dimensional structure. No further

modiﬁcation was made to their conformation. For each compound,

100 different conformations were generated using FLAME. They

were combined as the probe molecules. Each conformation of the

probe molecules was used as a static probe to align all target

molecules. In other words, multiple conformations of all molecules

were used as ﬁxed probes, one at a time, to align all other molecules.

For each resulting alignment, the total score (energies and alignment

scores of all molecules) was minimized with respect to the

coordinates of all atoms of all molecules. All alignments were saved

and sorted by the total scores for visual inspection. A reasonable

active conformation was derived from the alignment and used for

Summary

Targeted library screening of a cannabinoid agonist-biased

library and subsequent hit assessment has identiﬁed a novel class

of potent and selective CB₂agonists. SAR investigation of lead

compound 7 showed that the oxadiazole core was essential and

substitutions at the 2- and 4- position of the phenyl ring

appended to the oxadiazole were important for desired CB₂

potency. Subsequent optimization involving replacement of the

carbazole group revealed that a variety of aromatic groups,

especially simple ring systems, such as phenyl and pyridyl rings,

provided excellent CB₂potency. This effort led to compounds

with greatly improved physical properties and pharmacokinetics

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5027

subsequent database searching against in-house compound database

and commercial database. Top 1000 scored compounds from each

database search were kept and further ﬁltered by physicochemical

criteria (ClogP < 5, PSA < 110 Å, number of rotatable bonds e

10, and hydrogen bond donors e 2) to give 1092 compounds.

Preparation of rCB₁or rCB₂-Transfected CHO Cell Lines. One

day before transfection, approximately 3-4 × 10⁶AM1 cells,

derived from CHO-K1 cells, were plated in Dulbecco’s modiﬁed

Eagle’s medium containing 10% fetal bovine serum, 1× MEM

nonessential amino acids solution, 1× HT supplement, and 1×

penicillin-streptomycin-glutamate (Gibco/Invitrogen, Carlsbad, CA).

The cells were transfected with 5.6 µg of pcDNA3.1hyg-rCB₁or

5.6 µg of pcDNA3.1hyg-rCB₂using 16.8 µL FuGENE 6 transfec-

tion reagent following manufacturer’s protocol (Roche Applied

Science, Indianapolis, IN). One day later, the transfected cells were

split at 1:10 dilution and were placed under antibiotic selection in

the medium containing 250 µg/mL hygromycin B (Roche Applied

Science, Indianapolis, IN) and colonies were picked after two weeks

of antibiotic selection.

GTP-Europium Binding Assay. Two membrane preparations

(B_max: 1.7 pmol/mL for both) from Sf9 insect cells cotransfected

with CB₁or CB₂and Gai3 were assessed by monitoring binding

of a nonhydrolyzable GTP analogue, GTP-europium. Three µM of

2 was used to deﬁne 100% efﬁcacy. The procedure was modiﬁed

from a commercial kit (Perkin-Elmer, AD-0167) in that 25 µL of

buffer A (50 mM Hepes, 0.1% BSA) containing a certain

concentration (0.01 nM to 10 µM) of tested compound was added

to 125 µL of buffer B (50 mM Hepes, 0.1% BSA, 10 mM MgCl₂,

150 mM NaCl, 5 µM GDP, 10 nM GTP-europium, 0.0005%

saponin, and CB₁(4.5 µg/well) or CB₂(14 µg/well) membranes)

in a 96-well ﬁltration assay plate that came with the kit. The ﬁnal

DMSO concentration was 1%. The plate was incubated at room

temperature for 45 min, followed by ﬁltering and washing twice

with 300 µL of cold GTP wash buffer (supplied with the kit) on a

vacuum manifold device (Millipore, MAVM 096 0R). The plate

was then read in a ﬂuorescent plate reader (Perkin-Elmer Envision)

at 615 nm and data analyzed with ActivityBase.

Whole Cell cAMP Assay. Intracellular cAMP levels were

measured using the Lance cAMP kit from Perkin-Elmer (Waltham,

MA). CHO-K1 cells stably expressing human CB₁-receptor (Eu-

roscreen, Belgium), human CB₂-receptor (Euroscreen), rat CB₁-

receptor, or rat CB₂-receptor were used. Compound concentration

curves were prepared in DMSO at 100 times the desired ﬁnal

concentration in the assay. The compounds were diluted 50-fold

into assay buffer (HBSS/5 mM HEPES/0.1% BSA) containing 60

µM forskolin (EMD Biosciences/Calbiochem; San Diego, CA). The

compound/forskolin mixture was transferred to the assay plate (96-

well half area, white opaque plate; No. 3693 Corning), 12 µL/well.

Cells were harvested, pelletted at 1000 rpm for 5 min, and

resuspended in assay buffer to a density of 416667 cells/mL. The

Alexa-ﬂuor 647 labeled anti-cAMP antibody supplied with the

Lance cAMP kit was added to the cells at a dilution of 1:100. The

cells/antibody mixture was added to the assay plate (12 µL/well,

which is 5000 cells/well) containing compound/forskolin. The assay

plate was incubated at room temperature for 1 h. The detection

mix, which included biotinylated cAMP, was prepared per manu-

facturer’s instructions and was added to the assay plate (24 µL/

well) and the plate was incubated at room temperature for an

additional hour. The plate was read on the RUBYstar (BMG

LabTech, Durham, NC). The emission at 665 nm was used to plot

the concentration response curves of the compounds and calculate

their EC₅₀. A CB₁or a CB₂agonist results in inhibition of the

forskolin-induction production of intracellular cAMP, which results

in an increase in emission at 665 nm. Compound 2 was used to

deﬁne 100% efﬁcacy.

synthesizer from Personal Chemistry, Uppsala, Sweden. All ﬁnal

compounds were puriﬁed to >95% purity, as determined by LC/

MS obtained on an Agilent 1100 or HP 1100 spectrometer. Silica

gel chromatography was performed using either glass columns

packed with silica gel (230-400 mesh, EMD Chemicals, Gibb-

stown, NJ) or prepacked silica gel cartridges (Biotage or RediSep).

¹H NMR spectra were determined with a Bruker 300 MHz or DRX

400 MHz spectrometer. Chemical shifts are reported in parts per

million (ppm, δ units). Low-resolution mass spectral (MS) data

were determined on a Perkin-Elmer-SCIEX API 165 mass spec-

trometer using ES ionization modes (positive or negative).

3-(3-(2-Cyano-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid

(13). 3-(3-(2-Bromo-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic

acid (0.88 g, 2.8 mmol, prepared in the same manner as in 37) was

suspended in toluene/methanol (9:1) 30 mL. Trimethylsilyldiaz-

omethane (2 M in hexanes, 2.09 mL, 4.2 mmol) was added dropwise

with stirring until the solution remained pale yellow in color. The

solution was evaporated to dryness under reduced pressure. Dry

dimethylacetamide (20 mL) was added to dissolve the crude ester

followed by copper(I) chloride (0.102 g, 1 mmol) and potassium

cyanide (0.726 g, 11 mmol). The mixture was heated under nitrogen

at 130 °C for 14 h before cooling to room temperature. Saturated

ammonium chloride (20 mL), EtOAc (80 mL), and water (70 mL)

were added and the phases were mixed and separated. The organic

layer was then dried (MgSO₄), and the solvent was concentrated

in vacuo. The crude product was puriﬁed by column chromatog-

raphy on silica gel (eluting with 10-100% EtOAc/hexane) to give

methyl 3-(3-(2-cyano-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)pro-

panoate (0.104 g, 0.38 mmol, 14% yield). The solid was dissolved

in methanol (15 mL) and treated with a solution of lithium

hydroxide monohydrate (0.079 g, 1.9 mmol) in water (2 mL). The

mixture was stirred for 30 min. EtOAc (60 mL) and 5N HCl (20

mL) were added, and the phases were mixed and separated. The

organic layer was then dried (MgSO₄), and the solvent was

concentrated in vacuo to give 13. This material was used without

further puriﬁcation. MS (ESI, pos. ion) m/z: 262.0 (M + 1).

3-(3-(3-Cyanopyridin-4-yl)-1,2,4-oxadiazol-5-yl)propanoic acid

(16). 3-(3-(3-Bromopyridin-4-yl)-1,2,4-oxadiazol-5-yl)propanoic acid

(3.12 g, 10 mmol, prepared in the same manner as in 37) was

suspended in toluene/methanol (9:1) 70 mL. The solution was

cooled in a ice bath. Trimethylsilyldiazomethane (2 M in hexane,

5 mL, 10 mmol) was added dropwise until the solution remained

pale yellow in color. The solvent was removed, and the residue

was puriﬁed by column chromatograph on silica gel (eluting with

0-100% EtOAc/hexane). The resulting material (2.50 g, 8.01

mmol), copper(I) cyanide (2.87 g, 32.0 mmol) and DMF 30 mL

were combined and divided into two microwave tubes. The reaction

was heated at 180 °C for 10 min in microwave reactor. After cooling

to room temperature, the reaction mixture was poured into water

and extracted with EtOAc three times. The combined organic layers

were washed with water and brine, dried on Na₂SO₄, ﬁltered, and

concentrated. The residue was puriﬁed by column chromatography

on silica gel (eluting with 0-100% EtOAc/hexane). The resulting

material (1.23 g, 5 mmol) was dissoved in dimethoxyethane (25

mL) and 1 M lithium hydroxide aqueous solution (7 mL, 7 mmol)

was added. The mixture was stirred at room temperature for 16 h.

The solvent was evaporated and 1N HCl was added. The solution

was extracted with EtOAc. The combined organic layers were

washed with water and brine, dried on Na₂SO₄, ﬁltered, and

concentrated. The crude material was puriﬁed by column chroma-

tography on silica gel (eluting with 0-100% EtOAc/hexane) to

1

give 16 (0.68 g, 28% yield). H NMR (300 MHz, DMSO-d₆) δ

9.25 (s, 1H), 9.05 (d, J ) 5.02 Hz, 1H), 8.12 (d, J ) 5.52 Hz, 1H),

3.27 (t, J ) 6.78 Hz, 2H), 2.85 (t, J ) 6.78 Hz, 2H). MS (ESI,

pos. ion) m/z: 245.0 (M + 1).

Chemistry. Unless otherwise noted, all materials were obtained

from commercial suppliers and used without further puriﬁcation.

Anhydrous solvents were obtained from Aldrich or EM Science

and used directly. All reactions involving air- or moisture-sensitive

reagents were performed under a nitrogen or argon atmosphere.

All microwave assisted reactions were conducted with a Smith

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propan-

1-ol (19). Methyl 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-

5-yl)propanoate (0.501 g, 1.85 mmol, esteriﬁcation as in 13 from

the acid prepared in the same manner as in 37) was dissolved in

dry dichloromethane (40 mL) and cooled in a dry ice bath under

nitrogen. Diisobutylaluminum hydride (1.0 M in toluene, 4.0 mL,

5028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Cheng et al.

4.0 mmol) was added dropwise and the solution was allowed to

warm up to room temperature overnight. The reaction was quenched

by addition of 1N HCl (50 mL). Dichloromethane (50 mL) was

added, and the phases were mixed for 30 min, then separated and

the organic layer was dried with MgSO₄before evaporating to

dryness. The crude material was puriﬁed by column chromatography

on silica gel (eluting with 30-100% EtOAc/hexane) to give 19

(0.40 g, 84% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.92 (dd, J )

8.8, 6.1 Hz, 1H), 7.28 (dd, J ) 8.4, 2.5 Hz, 1H), 7.11 (m, 1H),

3.80 (t, J ) 6.0 Hz, 2H), 3.12 (t, J ) 7.3 Hz, 2H), 2.25 (br s, 1H),

2.13 (m, 2H). MS (ESI, pos. ion) m/z: 257.0 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanal (21).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid

(1.54 g, 5.7 mmol, prepared in the same manner as in 37 using

2-chloro-4-ﬂuorobenzonitrile) was dissolved in dichloromethane (80

mL) and methanol (10 mL) and treated with (trimethylsilyl)diaz-

omethane (2 M in hexanes, 4.3 mL, 8.5 mmol). The mixture was

stirred for 15 min, after which nitrogen evolution ceased and the

solution was evaporated to dryness under reduced pressure. The

crude ester was then dissolved in dry dichloromethane (80 mL)

and cooled to -78 °C under nitrogen. Diisobutylaluminum hydride

(1.0 M in toluene, 8.5 mL, 8.5 mmol) was added slowly, and the

solution was stirred for 60 min, then methanol (20 mL) was added

to quench the reaction. The mixture was added water and 1N HCl.

The phases were separated. The organic layer was dried (MgSO₄),

and the solvent was concentrated in vacuo. The crude material was

puriﬁed by column chromatography on silica gel (eluting with

0-100% EtOAc/hexane) to give 21 (0.88 g, 61% yield). ¹H NMR

(400 MHz, CD₃OD) δ 9.89 (s, 1H), 7.92 (dd, J ) 8.6, 6.4 Hz,

1H), 7.26 (dd, J ) 8.6, 2.4 Hz, 1H), 7.11 (dd, J ) 8.5, 2.5 Hz,

1H), 3.28 (t, J ) 6.1 Hz, 2H), 3.15 (t, J ) 6.2 Hz, 2H). MS (ESI,

pos. ion) m/z: 255.1 (M + 1).

(s, 1H), 4.77 (q, J ) 8.18 Hz, 2H), 3.66 (s br, 2H). MS (ESI, pos.

ion) m/z: 218.1 (M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-(2-ﬂuorophenyl)isoxazol-5-yl)

propanamide (30). 2-Fluorobenzaldehyde (2.0 mL, 19 mmol) was

dissolved in dry dichloromethane (50 mL) and treated with

hydroxylamine (50% in water, 1.16 mL, 19 mmol). MgSO₄(1.75

g) was added, and the reaction was stirred for 7 h. The mixture

was ﬁltered and evaporated to dryness to give an off-white solid.

The imine was dissolved in dry DMF (20 mL) under nitrogen and

treated with N-chlorosuccinimide (2.53 g, 19 mmol). The reaction

was stirred for 90 min, then poured into a separatory funnel

containing water (300 mL) and diethyl ether (100 mL). The phases

were mixed and separated and the organic layer was washed twice

with water (100 mL each). The organic layer was then dried

(MgSO₄), and the solvent was concentrated in vacuo to give

2-ﬂuorobenzoyl chloride oxime that was used without further

puriﬁcation.

Methyl 4-pentynoate (1.80 g, 16 mmol) was dissolved in THF

(20 mL) and added to the 2-ﬂuorobenzoyl chloride oxime. Tri-

ethylamine (2.6 mL, 19 mmol) was added, and the reaction was

stirred under nitrogen overnight. The reaction was evaporated to

dryness under reduced pressure. The crude residue was redissolved

in EtOAc (60 mL) and washed with 1N HCl (20 mL), water (20

mL), and 1N NaOH (20 mL). The organic layer was then dried

(MgSO₄), and the solvent was concentrated in vacuo to give methyl

3-(3-(2-ﬂuorophenyl)isoxazol-5-yl)propanoate.

The crude methyl 3-(3-(2-ﬂuorophenyl)isoxazol-5-yl)propanoate

was dissolved in methanol (30 mL) and treated with a solution of

lithium hydroxide monohydrate (0.98 g, 23 mmol) in water (5 mL).

The mixture was stirred for 3 h. EtOAc (80 mL) and water (80

mL) were added, and the phases were separated. The aqueous layer

was acidiﬁed with 5N HCl (20 mL) and extracted with EtOAc (90

mL). The organic layer was then dried (MgSO₄), and the solvent

was concentrated in vacuo to give 3-(3-(2-ﬂuorophenyl)isoxazol-

5-yl)propanoic acid (2.14 g, 48% yield), which was used without

further puriﬁcation.

3-Bromo-5-nitro-2-(2,2,2-triﬂuoroethoxy)pyridine (24). 2,2,2-

Triﬂuoroethanol (300 mL) was stirred at 0 °C and sodium hydride

(15 g, 379 mmol) was added by small portions carefully to the

solution. The mixture was then stirred at room temperature for 30

min. 3-Bromo-2-chloro-5-nitropyridine (10 g, 42 mmol) in triﬂuo-

roethanol was added, and the reaction was reﬂuxed overnight. The

solvent was removed, and the residue was taken up in water and

extracted with EtOAc. The combined organic layers were washed

with water and brine, dried over Na₂SO₄, and concentrated. The

product was puriﬁed by column chromatography on silica gel

(eluting with 0-100% EtOAc/hexane) to give 24 (11.2 g, 88%

General Procedure for Amide Coupling Method A. 3-(3-(2-

Fluorophenyl)isoxazol-5-yl)propanoic acid (0.50 g, 2.1 mmol) and

3-amino-9-ethylcarbazole (0.45 g, 2.1 mmol) were dissolved in

dichloromethane (10 mL). The reaction mixture was cooled in an

ice bath. A suspension of EDC (0.41 g, 2.1 mmol) in dichlo-

romethane (10 mL) was added slowly, and the reaction was stirred

for 15 min. The mixture was removed from the ice bath and stirred

for 2 h. The reaction was quenched by addition of water (50 mL)

and EtOAc (80 mL), and the phases were separated. The organic

layer was dried with MgSO₄, and the solvent was concentrated in

vacuo. The crude material was puriﬁed by column chromatography

on silica gel (eluting with 0-100% EtOAc/hexane) to give

1

yield). H NMR (300 MHz, CDCl₃) δ 9.00 (d, J ) 2.48 Hz, 1H),

8.70 (d, J ) 2.48 Hz, 1H), 4.92 (q, J ) 8.18 Hz, 2H).

5-Nitro-2-(2,2,2-triﬂuoroethoxy)nicotinonitrile (25). Compound

24 (4.8 g, 16 mmol) was divided into eight 20 mL microwave vials

and copper(I) cyanide (5.7 g, 64 mmol), divided into 8 portions,

was added to each vial. The vials were diluted with 20 mL NMP

and sealed. Each reaction vial was heated to 195 °C for 5 min.

The crude material was combined, diluted with water and EtOAc,

and ﬁltered through celite. The ﬁltrate was extracted with EtOAc

(4 × 50 mL). The combined organic fractions were washed with

water and brine, dried over Na₂SO₄, and concentrated. The crude

residue was puriﬁed by column chromatography on silica gel

(eluting with 15-50% EtOAc/hexane). The pure fractions were

combined and concentrated to give the product (2.4 g, 61%) as a

1

compound 30 (0.29 g, 31% yield). H NMR (400 MHz, CD₃OD)

δ 8.31 (d, J ) 1.9 Hz, 1H), 8.03 (d, J ) 7.9 Hz, 1H), 7.88 (dt, J

) 1.7, 7.5 Hz, 1H), 7.35-7.55 (m, 5H), 7.50-7.15 (m, 3H), 6.65

(d, J ) 3.2 Hz, 1H), 4.38 (q, J ) 7.2 Hz, 2H), 3.30 (t, J ) 7.4 Hz,

2H), 2.90 (t, J ) 7.4 Hz, 2H), 1.40 (t, J ) 7.2 Hz, 3H). MS (ESI,

pos. ion) m/z: 428.1 (M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-(2-ﬂuorophenyl)isoxazol-5-yl)

propanamide (31). Methyl succinimate (0.54 g, 4.1 mmol) and

2-bromo-1-(2-ﬂuorophenyl)ethanone (0.95 g, 2.1 mmol) were

dissolved in NMP (20 mL). The mixture was heated to 120 °C for

6 h, then allowed to cool to room temperature. The reaction was

diluted with EtOAc (80 mL) and water (70 mL) and the phases

were separated. The organic layer was dried (MgSO₄), and the

solvent was concentrated in vacuo. The crude material was puriﬁed

by column chromatography on silica gel (eluting with 0-100%

EtOAc/hexane) to give methyl 3-(4-(2-ﬂuorophenyl)oxazol-2-

yl)propanoate (0.35 g, 34% yield).

1

light yellow oil. H NMR (300 MHz, CDCl₃) δ 9.24 (d, J ) 2.78

Hz, 1H), 8.78 (d, J ) 2.63 Hz, 1H), 5.02 (q, J ) 8.04 Hz, 2H).

MS (ESI, pos. ion) m/z: 248.1 (M + 1).

5-Amino-2-(2,2,2-triﬂuoroethoxy)nicotinonitrile (26). Compound

25 (1.8 g, 7.4 mmol) was dissolved in ethanol 50 mL. Acetic acid

(5 mL) and water (1 mL) were added, followed by addition of iron

powder (0.82 g, 24 mmol). The reaction was reﬂuxed for 1 h. The

mixture was ﬁltered through a pad of celite and the solvent was

removed. The residue was taken in saturated aqueous NaHCO₃

solution and extracted with dichromethane. The organic layers were

combined and washed with water and brine, dried on Na₂SO₄,

ﬁltered, and concentrated under reduced pressure to give 26 (1.03

Hydrolysis of 3-(4-(2-ﬂuorophenyl)oxazol-2-yl)propanoate (in the

same manner as in 30) followed by amide coupling method A (in

the same manner as 30) gave compound 31 (0.16 g, 18% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.35-8.25 (m, 2H), 8.1 (dt, J )

1.5, 7.3 Hz, 1H), 7.95-8.05 (m, 2H), 7.05-7.55 (m, 7H), 4.31 (q,

J ) 7.0 Hz, 2H), 3.30 (t, J ) 6.9 Hz, 2H), 2.98 (t, J ) 6.9 Hz,

1

g, 65% yield). H NMR (300 MHz, CDCl₃) δ 7.80 (s, 1H), 7.27

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5029

2H), 1.7-1.5 (br s, 1H), 1.40 (t, J ) 7.1 Hz, 3H). MS (ESI, pos.

ion) m/z: 428.1 (M + 1).

chromatography on silica gel (eluting with 0-100% EtOAc/hexane)

to afford 34 as an off-white solid (0.079 g, 39% yield over two

steps). H NMR (400 MHz, acetone-d₆) δ 9.32 (s, 1H), 8.47 (d, J

1

N-(9-Ethyl-9H-carbazol-3-yl)-3-(5-(2-ﬂuorophenyl)-1H-pyrazol-

3-yl)propanamide (32). 2′-Fluoroacetophenone (2.0 mL, 16 mmol)

was dissolved in dry THF (60 mL) under nitrogen and cooled in

an ice bath. Lithium bis(trimethylsilyl)amide (1 M in THF, 16.2

mL, 16 mmol) was added slowly and the reaction stirred for 20

min. Succinic anhydride (1.32 mL, 16 mmol) was added in one

portion, and the reaction was stirred overnight. Water (50 mL),

1N HCl (30 mL), and ethyl acetate (70 mL) were added, and the

phases were separated. The organic was dried with MgSO₄and

concentrated to 10 mL of the crude solution of 6-(2-ﬂuorophenyl)-

4,6-dioxohexanoic acid under reduced pressure.

The 6-(2-ﬂuorophenyl)-4,6-dioxohexanoic acid solution (8 mmol)

was diluted with ethanol (20 mL) and treated with anhydrous

hydrazine (0.26 mL, 8.1 mmol). The reaction was stirred overnight.

Water (50 mL), 1N NaOH (10 mL), and EtOAc (50 mL) were

added, and the phases were separated. The aqueous layer was

acidiﬁed with 1N HCl (30 mL) and extracted with EtOAc (80 mL).

The organic layer was dried (MgSO₄), and the solvent was

concentrated in vacuo to give the crude material of 3-(5-(2-

ﬂuorophenyl)-1H-pyrazol-3-yl)propanoic acid. Using the crude acid

and following amide coupling method A (as in 30) gave 32 (0.18

g, 3.3% yield over three steps) as an off white solid. ¹H NMR

(400 MHz, CD₃OD) δ 8.29 (d, J ) 1.6 Hz, 1H), 8.01 (d, J ) 7.8

Hz, 1H), 7.79 (dd, J ) 8.6, 1.8 Hz, 1H), 7.50 (dd, J ) 8.6, 1.8 Hz,

1H), 7.38-7.47 (m, 3H), 7.28-7.36 (m, 1H), 7.10-7.25 (m, 3H),

6.62 (d, J ) 2.7 Hz, 1H), 4.39 (q, J ) 7.2 Hz, 2H), 3.15 (t, J ) 7.5

Hz, 2H), 2.82 (t, J ) 7.5 Hz, 2H), 1.39 (t, J ) 7.2 Hz, 3H). MS

(ESI, pos. ion) m/z: 427.1 (M + 1).

) 1.89 Hz, 1H), 8.33 (d, J ) 3.92 Hz, 1H), 8.24 (dt, J ) 7.71,

1.64 Hz, 1H), 8.07 (d, J ) 7.71 Hz, 1H), 7.60 (dd, J ) 8.65, 1.96

Hz, 1H), 7.51-7.55 (m, 1 H), 7.41-7.50 (m, 2H), 7.34-7.40 (m,

1H), 7.26 - 7.32 (m, 1H), 7.15 - 7.25 (m, 2H), 4.90 (t, J ) 6.63

Hz, 2H), 4.45 (q, J ) 7.20 Hz, 2H), 4.45 (q, J ) 7.20 Hz, 2H),

1.37 (t, J ) 7.14 Hz, 3H). MS (ESI, pos. ion) m/z: 428 (M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(5-(2-ﬂuorophenyl)-1,2,4-oxadia-

zol-3-yl)propanamide (35). A mixture of 3-cyanopropanoic acid (1.0

g, 10 mmol), HOBt (2.0 g, 17 mmol), and PS-carbodiimide (16 g,

20 mmol, Argonaut Technologies) in methylene chloride was stirred

at room temperature for 10 min, at which time 3-amino-9-

ethylcarbazole (2 g, 10 mmol) was added. The resulting mixture

was stirred overnight at room temperature. MP-carbonate (17 g,

50 mmol, Argonaut Technologies) was then added, and the reaction

mixture was stirred an additional 1 h. The reaction mixture was

ﬁltered and the resins washed with dichloromethane. The ﬁltrate

was concentrated in vacuo and the residue was triturated with

methanol and ﬁltered, affording 3-cyano-N-(9-ethyl-9H-carbazol-

3-yl)propanamide (1.44 g, 49% yield) as a yellow solid.

A ﬂask containing 3-cyano-N-(9-ethyl-9H-carbazol-3-yl)pro-

panamide (0.23 g, 0.79 mmol) was dissolved in methanol MeOH

(8 mL). Hydroxylamine hydrochloride (0.060 g, 0.87 mmol),

sodium bicarbonate (0.080 g, 0.95 mmol), and 0.62 mL of water

were added. The reaction mixture was reﬂuxed for 48 h. An

additional 80 mg of sodium bicarbonate and 60 mg of hydroxyl-

amine hydrochloride was added to the reaction, and reﬂux was

continued overnight. The reaction mixture was cooled to room

temperature, and the solvents were removed. To the crude reaction

product was added dichloromethane and water. The organic layers

were separated and washed with brine, dried over Na₂SO₄, ﬁltered,

and concentrated. The crude product was puriﬁed by column

chromatography on silica gel (eluting with 0-10% methanol/

dichloromethane) providing (Z)-4-amino-N-(9-ethyl-9H-carbazol-

3-yl)-4-(hydroxyimino)butanamide as a light purple solid (72 mg,

28% yield).

A vial containing (Z)-4-amino-N-(9-ethyl-9H-carbazol-3-yl)-4-

(hydroxyimino)butanamide (0.072 g, 0.22 mmol) was charged with

dichloromethane (1.7 mL). To the stirring solution was added

2-ﬂuorobenzoic acid (0.031 g, 0.22 mmol), HOBt (0.034 g, 0.22

mmol), EDC (0.043 g, 0.22 mmol), and N,N-diisopropylethylamine

(0.078 mL, 0.44 mmol). The reaction was stirred at room temper-

ature for 6 h then diluted with dichloromethane and transferred to

a separation funnel. 1N HCl was added to wash the organic layer.

The phases were separated, and the aqueous layer was extracted

with dichloromethane. The organic fractions were combined and

washed with saturated aqueous NaHCO₃and brine, dried over

Na₂SO₄, ﬁltered, and concentrated to afford a brown ﬁlm, (Z)-4-

(2-ﬂuorobenzoyloxyimino)-4-amino-N-(9-ethyl-9H-carbazol-3-yl)b-

utanamide (0.045 g, 46% yield), which was used without further

puriﬁcation.

N-(9-Ethyl-9H-carbazol-3-yl)-3-(5-(2-ﬂuorophenyl)-1-methyl-1H-

pyrazol-3-yl)propanamide (33). The 6-(2-ﬂuorophenyl)-4,6-dioxo-

hexanoic acid (8 mmol, prepared in the same manner as in 32)

was diluted with ethanol (20 mL) and treated with 1-methylhydra-

zine (0.47 mL, 8.9 mmol). The reaction was stirred overnight. The

reaction was diluted with water (50 mL), ethyl acetate (60 mL),

and 1N NaOH (20 mL). The phases were separated and the aqueous

layer was acidiﬁed with 5N HCl (10 mL) and extracted with ethyl

acetate (70 mL). The organic layer was then dried (MgSO₄), and

the solvent was concentrated in vacuo to give the crude 3-(5-(2-

ﬂuorophenyl)-1-methyl-1H-pyrazol-3-yl)propanoic acid. Using the

crude acid and following amide coupling method A (as in 30) gave

1

33 (0.047 g, 1.4% yield over three steps). H NMR (400 MHz,

CD₃Cl) δ 8.2-8.3 (m, 2H), 8.05 (d, J ) 7.7 Hz, 1H), 7.3-7.55

(m, 6H), 7.15-7.25 (m, 3H), 6.21 (s, 1H), 4.33 (q, J ) 7.3 Hz,

2H), 3.80 (s, 3H), 3.15 (t, J ) 7.0 Hz, 2H), 2.85 (t, J ) 7.1 Hz,

2H), 1.40 (t, J ) 7.3 Hz, 3H). MS (ESI, pos. ion) m/z: 441.1 (M

+ 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(4-(2-ﬂuorophenyl)-1H-1,2,3-

triazol-1-yl)propanamide (34). To a solution of 3-amino-9-ethyl-

carbazole (0.50 g, 2.38 mmol) and N,N-diisopropylethylamine (0.54

mL, 3.09 mmol) in DMF (10 mL) was added 3-chloropropionyl

chloride (0.25 mL, 2.62 mmol). The resulting mixture was allowed

to stir for 1 h, at which time sodium azide (0.42 mL, 11.9 mmol)

was added. The resulting mixture was heated to 60 °C overnight.

The reaction was cooled to room temperature and poured onto water

(100 mL) and extracted with ether (3 × 25 mL). The combined

organic layers were washed with brine (20 mL), dried over Na₂SO₄,

ﬁltered, and concentrated to afford 3-azido-N-(9-ethyl-9H-carbazol-

3-yl)propanamide, which was used without further puriﬁcation.

To a slurry of 1-ethynyl-2-ﬂuorobenzene (0.053 mL, 0.47 mmol)

and 3-azido-N-(9-ethyl-9H-carbazol-3-yl)propanamide (0.15 g, 0.47

mmol) in t-BuOH (0.5 mL) and water (0.5 mL) was added a freshly

prepared solution of sodium ascorbate (0.094 mL, 0.047 mmol)

and a freshly prepared solution of copper sulfate pentahydrate

(0.0094 mL, 0.0047 mmol). The mixture was allowed to stir at

room temperature. After 5 h, the reaction was diluted with water

(0.75 mL) and t-butanol (0.75 mL) and continued to stir overnight.

Then the mixture was diluted with water and ﬁltered. The solid

material was washed with water, then reconstituted in acetonitrile

and dichloromethane. The crude mixture was puriﬁed via ﬂash

A vacuum-dried ﬂask containing (Z)-4-(2-ﬂuorobenzoyloxy-

imino)-4-amino-N-(9-ethyl-9H-carbazol-3-yl)butanamide (0.045 g,

0.1 mmol) was added pyridine (1.0 mL). The reaction mixture was

heated to reﬂux overnight, then it was cooled and pyridine was

removed. The crude material was puriﬁed by column chromatog-

raphy on silica gel (eluting with 0-100% EtOAc/hexane). The

product-containing fractions were combined and puriﬁed by column

chromatography on silica gel (eluting 0-5% methanol/dichlo-

romethane). The impure fractions were combined and puriﬁed by

column chromatography on silica gel (eluting with 3:1 EtOAc/

hexanes followed by 1:1 EtOAc/hexanes) to afford additional

material. The combined product fractions were concentrated to

1

afford 35 (9.3 mg, 21.5% yield). H NMR (400 MHz, CDCl₃) δ

8.33 (s, 1H), 8.13 (t, J ) 6.82 Hz, 1H), 8.06 (d, J ) 7.83 Hz, 1H),

7.75 (s, 1H), 7.63-7.57 (m, 1H), 7.53 (d, J ) 7.20 Hz, 1H),

7.50-7.44 (m, 1H), 7.42-7.37 (m, 1H), 7.37-7.25 (m, 3H), 7.21

(t, J ) 7.33 Hz, 1H), 4.35 (q, J ) 7.16 Hz, 2H), 3.36 (t, J ) 7.26

5030 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Cheng et al.

Hz, 2H), 2.99 (t, J ) 7.26 Hz, 2H), 1.42 (t, J ) 7.20 Hz, 3H). MS

(ESI, pos. ion) m/z: 429.1 (M + 1).

7.07 Hz, 2H), 1.82 (t, J ) 7.14 Hz, 3H). MS (ESI, pos. ion) m/z:

411.1 (M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(5-(2-ﬂuorophenyl)-2H-tetrazol-

2-yl)propanamide (36). 2-Fluorobenzonitrile (0.80 mL, 7.4 mmol),

dibutyltin oxide (0.092 g, 0.37 mmol), and azidotrimethylsilane

(1.47 mL, 11 mmol) were added to a microwave reaction vessel

containing dimethoxyethane (2 mL). The reaction was heated to

150 °C in the microwave for 10 min, then cooled to room

temperature. The solution was transferred to a separatory funnel

and diluted with water (100 mL), 1N HCl (20 mL), and EtOAc

(80 mL). The phases were separated, and the organic layer was

dried with MgSO₄before evaporating to dryness under reduced

pressure to give 5-(2-ﬂuorophenyl)-2H-tetrazole, which was used

without puriﬁcation. The crude 5-(2-ﬂuorophenyl)-2H-tetrazole (7.4

mmol) was dissolved in dimethoxyethane (3 mL) and transferred

to a microwave reaction vial. Methyl 3-bromopropanoate (0.81 mL,

7.4 mmol) was added, and the vial was heated to 150 °C in a

microwave reactor for 10 min. The crude material was concentrated

and redissolved in methanol and treated with an aqueous solution

of lithium hydroxide (1M, 10 mL, 37 mmol). After stirring for 15

min, the mixture was acidiﬁed with 5N HCl (10 mL) and diluted

with water (100 mL) and EtOAc (100 mL). The phases were

separated, and the organic layer was dried with MgSO₄before

evaporating to dryness under reduced pressure. The crude material

was puriﬁed by column chromatography on silica gel (eluting with

0-10% methanol/dichromethane) to give 3-(5-(2-ﬂuorophenyl)-

2H-tetrazol-2-yl)propanoic acid (0.20 g, 12% yield).

3-(5-(2-Fluorophenyl)-2H-tetrazol-2-yl)propanoic acid (0.20 g,

0.86 mmol) and 3-amino-9-ethylcarbazole (0.20 g, 0.95 mmol)

dissolved in a minimal amount of dry DMF (2 mL) and treated

with HATU (0.36 g, 0.95 mmol). The reaction was stirred for 10

min. Water (60 mL) and EtOAc (60 mL) were added, and the phases

were separated. The organic layer was dried with MgSO₄and

evaporated to dryness under reduced pressure. The crude material

was puriﬁed by column chromatography on silica gel (eluting with

0-100% EtOAc/hexane) to give 36 (0.11 g, 30% yield). ¹H NMR

(400 MHz, CDCl₃) δ 8.24 (d, J ) 1.8 Hz, 1H), 8.13 (dt, J ) 7.5,

1.8 Hz, 1H), 8.02 (d, J ) 7.7 Hz, 1H), 7.72 (br s, 1H), 7.50-7.35

(m, 4H), 7.1-7.3 (m, 4H), 5.15 (t, J ) 6.9 Hz, 2H), 4.30 (q, J )

7.2 Hz, 2H), 3.25 (t, J ) 6.9 Hz, 2H), 1.38 (t, J ) 7.2 Hz, 3H).

MS (ESI, pos. ion) m/z: 428.9 (M + 1).

3-(3-(2-Chlorophenyl)-1,2,4-oxadiazol-5-yl)-N-(9-ethyl-9H-car-

bazol-3-yl)propanamide (38). Using 2-chlorobenzonitrile and fol-

lowing the procedure used to prepare 37 gave 38. H NMR (400

1

MHz, DMSO-d₆) δ 10.16 (s, 1H), 8.41 (s, 1H), 8.05 (d, J ) 7.73

Hz, 1H), 7.90 (dd, J ) 7.63, 1.66 Hz, 1H), 7.68 (dd, 1H), 7.49-7.63

(m, 5H), 7.44 (t, J ) 7.19 Hz, 1H), 7.17 (t, J ) 7.43 Hz, 1H), 4.42

(q, J ) 7.04 Hz, 2H), 3.32-3.44 (m, 2H), 3.01 (t, J ) 6.94 Hz,

2H), 1.30 (t, J ) 7.09 Hz, 3H). MS (ESI, pos. ion) m/z: 445.0 (M

+ 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-(2-isopropylphenyl)-1,2,4-oxa-

diazol-5-yl)propanamide (39). Using 2-isopropylbenzonitrile and

following the procedure in 37 to prepare 3-(3-(2-isopropylphenyl)-

1,2,4-oxadiazol-5-yl)propanoic acid. Then 39 was prepared by

amide coupling method A (as in 30). ¹H NMR (400 MHz, CDCl₃)

δ 8.18 (d, J ) 1.88 Hz, 1H), 7.93 (d, J ) 7.91 Hz, 1H), 7.68 (m,

2H), 7.37 (m, 6H), 7.20 (m, 2H), 7.11 (t, J ) 7.44 Hz, 1H), 4.24

(q, J ) 7.22 Hz, 2H), 3.58 (m, 1H), 3.35 (t. J ) 7.06 Hz, 2H),

2.94 (t, J ) 7.06 Hz, 2H), 1.31 (t, J ) 7.25 Hz, 3H), 1.15 (d, J )

6.78 Hz, 6H). MS (ESI, pos. ion) m/z: 452.8 (M + 1).

3-(3-(3-Chlorophenyl)-1,2,4-oxadiazol-5-yl)-N-(9-ethyl-9H-car-

bazol-3-yl)propanamide (40). Using 2-chlorobenzonitrile and fol-

1

lowing the procedure used to prepare 37 gave 40. H NMR (400

MHz, DMSO-d₆) δ 9.82 (s, 1H), 8.96 (s, 1H), 8.55-8.44 (m, 2H),

8.12-8.02 (m, 3H), 8.01-7.92 (m, 2H), 7.89 (t, J ) 7.39 Hz, 1H),

7.62 (t, J ) 7.45 Hz, 1H), 4.90 (q, J ) 7.03 Hz, 2H), 3.86 (t, J )

6.95 Hz, 2H), 3.86 (t, J ) 6.95 Hz, 2H), 3.86 (t, J ) 6.95 Hz, 2H).

MS (ESI, pos. ion) m/z: 445.1 (M + 1).

N-(9-ethyl-9H-carbazol-3-yl)-3-(3-(4-ﬂuorophenyl)-1,2,4-oxadia-

zol-5-yl)propanamide (41). Using 2-chlorobenzonitrile and follow-

ing the procedure used to prepare 37 gave 41. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.16 (s, 1H), 8.41 (t, 1H), 7.98-8.11 (m, 3H),

7.51-7.62 (m, 3H), 7.36-7.48 (m, 3H), 7.16 (t, 1H), 4.41 (q, J )

7.11 Hz, 2H), 3.33 (t, 2H), 3.01 (t, J ) 6.99 Hz, 2H), 1.29 (t, J )

7.14 Hz, 3H). MS (ESI, pos. ion) m/z: 429.1 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(9-ethyl-

9H-carbazol-3-yl)propanamide (42). Using 2-chloro-4-ﬂuoroben-

zonitrile and following the procedure used to prepare 37 gave 42.

¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J ) 1.64 Hz, 1H), 8.06 (d,

J ) 7.58 Hz, 1H), 7.94 (dd, J ) 8.65, 6.00 Hz, 1H), 7.59 (s, 1H),

7.48 (t, J ) 8.02 Hz, 2H), 7.33-7.43 (m, 2H), 7.28-7.31 (m, 2H),

7.06-7.14 (m, 1H), 3.47 (t, J ) 7.14 Hz, 2H), 3.05 (t, J ) 7.20

Hz, 2H), 1.43 (t, J ) 7.14 Hz, 3H). MS (ESI, pos. ion) m/z: 463.0

(M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-(4-ﬂuoro-2-methylphenyl)-

1,2,4-oxadiazol-5-yl)propanamide (43). Using 2-chlorobenzonitrile

and following the procedure used to prepare 37 gave 43. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 8.41 (s, 1H), 8.04 (d, J )

7.96 Hz, 1H), 7.97 (dd, J ) 8.53, 6.13 Hz, 1H), 7.57 (m, 3H), 7.44

(t, J ) 7.58 Hz, 1H), 7.30 (dd, J ) 10.0, 2.46 Hz, 1H), 7.20 (m,

2H), 4.42 (q, J ) 6.91 Hz, 2H), 3.34 (m, 2H), 3.00 (t, J ) 7.01

Hz, 2H), 2.56 (s, 3H), 1.30 (t, J ) 7.01 Hz, 3H). MS (ESI, pos.

ion) m/z: 443.0 (M + 1).

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-phenyl-1,2,4-oxadiazol-5-yl)

propanamide (37). To a mixture of sodium carbonate (1.0 g, 10

mmol) and hydroxylamine hydrochloride (1.0 g, 19 mmol) in

methanol/H₂O was added benzonitrile (2 mL, 19 mmol). The

mixture was heated to 70 °C for 1 h. The cooled reaction mixture

was concentrated, and the residue was taken up in dichloromethane.

The organic layer was washed with water and concentrated to give

(Z)-N′-hydroxybenzamidine (1.85 g, 70% yield), which was used

without further puriﬁcation.

A mixture of succinic anhydride (1.1 mL, 13.6 mmol) and (Z)-

N′-hydroxybenzamidine (1.85 g, 13.6 mmol) was heated to 120

°C for 30 min. The reaction was cooled to room temperature,

concentrated to dryness, and triturated with methanol. The white

solid was ﬁltered, washed with water, and dried under high vacuum

to give 3-(3-phenyl-1,2,4-oxadiazol-5-yl)propanoic acid (2.11 g,

71.2% yield), which was used without further puriﬁcation.

A mixture of 3-(3-phenyl-1,2,4-oxadiazol-5-yl)propanoic acid

(0.15 g, 0.69 mmol), HOBt (0.11 g, 0.78 mmol), and PS-

carbodiimide (0.72 g, 0.92 mmol) in dichloromethane was mixed

for 10 min, at which time 9-ethyl-9H-carbazol-3-amine (0.096 g,

0.46 mmol) was added. The resulting mixture was mixed for

overnight at room temperature. MP-carbonate (0.76 g, 2.29 mmol)

was then added, and the mixture was stirred for an additional hour.

The reaction mixture was ﬁltered and the resins washed with

dichloromethane. The ﬁltrate was concentrated down and the residue

triturated with methanol and ﬁltered to give 37 (93.2 mg, 50% yield)

3-(3-(2-Cyano-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(9-ethyl-

9H-carbazol-3-yl)propanamide (44). 44 was prepared by amide

1

coupling method A (as in 30) using 13 (90 mg, 43% yield). H

NMR (400 MHz, CDCl₃) δ 8.32 (d, J ) 1.88 Hz, 1H), 8.17 (dd, J

) 5.3, 8.8 Hz, 1H), 8.03 (d, J ) 7.7 Hz, 1H), 7.79 (br s, 1H),

7.30-7.55 (m, 6H), 7.18 (t, J ) 7.0 Hz, 1H), 4.32 (q, J ) 7.2 Hz,

2H), 3.46 (t, J ) 6.9 Hz, 2H), 3.05 (t, J ) 6.9 Hz, 2H), 1.39 (t, J

) 7.2 Hz, 3H). MS (ESI, pos. ion) m/z: 454.1 (M + 1).

3-(3-(3-Cyanopyridin-4-yl)-1,2,4-oxadiazol-5-yl)-N-(9-ethyl-9H-

carbazol-3-yl)propanamide (45). 45 was prepared by amide cou-

pling method A (as in 30) using 3-(3-(3-cyanopyridin-4-yl)-1,2,4-

1

oxadiazol-5-yl)propanoic acid 16 (142 mg, 78% yield). H NMR

1

as a off-white solid. H NMR (400 MHz, DMSO-d₆) δ 9.81 (s,

(300 MHz, DMSO-d₆) δ 10.16 (s, 1H), 9.25 (s, 1H), 9.05 (d, J )

5.12 Hz, 1H), 8.40 (s, 1H), 8.13 (d, J ) 5.12 Hz, 1H), 8.04 (d, J

) 7.60 Hz, 1H), 7.55 (m, 3H), 7.43 (t, J ) 7.67 Hz, 1H), 7.16 (t,

J ) 7.38 Hz, 1H), 4.41 (q, J ) 7.02 Hz, 2H), 3.41 (t, J ) 13.45

1H), 8.96 (d, J ) 1.89 Hz, 1H), 8.46-8.55 (m, 3H), 8.08 (dd, J )

8.78, 1.83 Hz, 1H), 7.84-8.02 (m, 6H), 7.61 (t, J ) 7.26 Hz, 1H),

4.89 (q, J ) 7.07 Hz, 2H), 3.84 (t, J ) 7.07 Hz, 2H), 3.55 (t, J )

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5031

Hz, 2H), 3.04 (t, J ) 13.74 Hz, 2H), 1.28 (t, J ) 6.94 Hz, 3H).

MS (ESI, pos. ion) m/z: 437.1 (M + 1).

MgSO₄and evaporated to dryness under reduced pressure to

give 6-chloro-1,8-naphthyridin-3-amine (0.18 g, 15% yield),

which was used without further puriﬁcation.

N-(9-Ethyl-9H-carbazol-3-yl)-3-(3-(tetrahydro-2H-pyran-4-yl)-

1,2,4-oxadiazol-5-yl)propanamide (46). Using tetrahydro-2H-pyran-

4-carbonitrile and following the procedure in 37 to prepare 3-(3-

(tetrahydro-2H-pyran-4-yl)-1,2,4-oxadiazol-5-yl)propanoic acid. Then

46 was prepared by amide coupling method A (as in 30). ¹H NMR

(300 MHz, CDCl₃) δ 8.27 (d, J ) 1.88 Hz, 1H), 8.04 (d, J ) 7.72

Hz, 1H), 7.72 (s, 1H), 7.40 (m, 4H), 7.20 (m, 1H), 4.33 (q, J )

7.22 Hz, 2H), 4.03 (m, 2H), 3.54 (m, 2H), 3.34 (d, J ) 7.06 Hz,

2H), 3.00 (m, 3H), 1.93 (m, 4H), 1.40 (t, J ) 7.25 Hz, 3H). MS

(ESI, pos. ion) m/z: 418.8 (M + 1).

Amide Coupling Method B. 3-(3-(2-Chloro-4-ﬂuorophenyl)-

1,2,4-oxadiazol-5-yl)propanoic acid (0.69 g, 2.5 mmol, as in 42)

was dissolved in thionyl chloride (5 mL) and stirred for 2 h.

The solution was evaporated to dryness under reduced pressure

to give a pale yellow oil. The crude acid chloride was dissolved

in dichloromethane (10 mL) and added to a solution of 6-chloro-

1,8-naphthyridin-3-amine (0.46 g, 2.5 mmol) and diisopropyl-

ethylamine (0.44 mL, 2.5 mmol) in dichloromethane (30 mL).

The reaction was stirred for 3 h, after which water (100 mL)

was added and the phases were separated. The organic layer

was dried with MgSO₄and evaporated to dryness under reduced

pressure. The crude material was puriﬁed using column chro-

matography on silica gel eluting with (0-10% methanol/

dichloromethane) to give enriched material. This material was

triturated with methanol (10 mL) and ﬁltered to 48 (0.15 g, 14%

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(6-chlo-

roquinolin-3-yl)propanamide (47). NaOH 10N (6.0 mL, 60 mmol)

was cooled in an ice bath and nitromethane (1.0 mL, 18.5 mmol)

was added dropwise. The reaction was stirred for 15 min, then

removed from the cold bath and stirred for another 20 min. The

solution was placed in a room temperature water bath, and

nitromethane (1.0 mL, 18.5 mmol) was added slowly with vigorous

stirring. The mixture was stirred for 25 min then poured into ice

water (100 mL) with added concentrated HCl (6 mL) and diethyl

ether (100 mL). The mixture was stirred until the ice melted, after

which the phases were separated and the organic layer was dried

with MgSO₄. The solution was ﬁltered and concentrated under

reduced pressure to give methazonic acid as a pale yellow solid

(1.88 g, 98% yield, this compound is thermally sensitive!).

Methazonic acid (1.9 g, 19 mmol) solution (10 mL aqueous)

was added to 5N HCl (10 mL) and ice (∼50 mL). A solution of

2-amino-5-chlorobenzaldehyde (1.1 g, 7.2 mmol) in a mixture of

ethanol (70 mL), water (60 mL), and 5N HCl (2 mL) was added

slowly with stirring. The mixture was heated to 50 °C for 60 h,

then concentrated under reduced pressure to ∼10 mL solution that

was cooled in an ice bath. The suspension was ﬁltered through a

fritted funnel and washed with water (10 mL) to give the

intermediate 3-nitro-6-chloroquinoline. This intermediate was then

suspended in isopropanol (50 mL) and water (5 mL) and heated to

50 °C. Saturated ammonium chloride (4 mL) and iron powder (4.03

g, 72 mmol) were added, and the mixture was stirred overnight.

The reaction mixture was cooled and ﬁltered through a pad of celite.

The ﬁltrate was evaporated to dryness under reduced pressure and

redissolved in a mix of EtOAc (100 mL), 1N NaOH (20 mL), and

water (80 mL). The phases were mixed and separated, and the

organic layer was dried with MgSO₄and evaporated to dryness

under reduced pressure. The crude material was puriﬁed by column

chromatography on silica gel (eluting with 0-100% EtOAc/hexane)

to give 6-chloroquinolin-3-amine (0.40 g, 31% yield).

47 was prepared by 6-chloroquinolin-3-amine and 3-(3-(2-chloro-

4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid (as in 42) using

amide coupling method A (as in 30). ¹H NMR (400 MHz, CD₃OD)

δ 8.79 (d, J ) 2.2 Hz, 1H), 8.70 (d, J ) 2.4 Hz, 1H), 7.90-8.0

(m, 2H), 7.79 (d, J ) 2.2 Hz, 1H), 7.56 (dd, J ) 2.3, 8.9 Hz, 1H),

7.29 (dd, J ) 8.4, 2.5 Hz, 1H), 7.12 (m, 1H), 3.43 (t, J ) 7.2 Hz,

2H), 3.09 (t, J ) 7.2 Hz, 2H). MS (ESI, pos. ion) m/z: 430.7 (M

+ 1).

N-(6-Chloro-1,8-naphthyridin-3-yl)-3-(3-(2-chloro-4-ﬂuorophe-

nyl)-1,2,4-oxadiazol-5-yl)propanamide (48). 2-Amino-5-chloropy-

ridine-3-carboxaldehyde (1.1 g, 7.0 mmol) was dissolved in a

mixture of water (10 mL) and concentrated HCl (8 mL) and

added to a solution of methazonic acid (1.9 g, 18 mmol, prepared

as in 47) in water (20 mL). The reaction was stirred overnight.

The resulting suspension was ﬁltered through a fritted funnel

and dried in vacuo. The intermediate 3-chloro-6-nitro-1,8-

naphthyridine (0.42 g, 2.0 mmol) was suspended in isopropanol

(40 mL) and methanol (10 mL) and treated with saturated

ammonium chloride (2 mL) and water (1 mL). Iron powder (1.1

g, 20 mmol) was added, and the mixture was heated to 60 °C

with stirring. After 1 h, the mixture was cooled and ﬁltered

through a pad of celite. The ﬁltrate was evaporated to dryness

under reduced pressure. The crude material was dissolved in

water (100 mL), 1N NaOH (20 mL), and EtOAc (100 mL), and

the phases were separated. The organic layer was dried with

1

yield). H NMR (400 MHz, CD₃OD) δ 10.88 (s, 1H), 9.04 (d,

J ) 2.1 Hz, 1H), 8.92 (s, J ) 2.1, 1H), 8.83 (d, J ) 2.3 Hz,

1H), 8.68 (d, J ) 2.1 Hz, 1H), 7.96 (dd, J ) 8.8, 6.3 Hz, 1H),

7.70 (dd, J ) 8.7, 2.1 Hz, 1H), 7.43 (dt, J ) 8.7, 1.9 Hz, 1H)

3.40 (t, J ) 6.4 Hz partially obscured by MeOD, 1H), 3.10 (t,

J ) 6.4 Hz, 2H). MS (ESI, pos. ion) m/z: 432.0 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(6-(trif-

luoromethoxy)benzo[d]thiazol-2-yl)propanamide (49). 49 was pre-

pared by riluzole and 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-

5-yl)propanoic acid (as in 42) using amide coupling method A (as

in 30, 88 mg, 33% yield). ¹H NMR (300 MHz, DMSO-d₆) δ 12.67

(s, 1H), 8.12 (d, J ) 1.51 Hz, 1H), 7.94 (dd, J ) 8.85, 6.22 Hz,

1H), 7.83 (d, J ) 8.85 Hz, 1H), 7.70 (dd, J ) 8.85, 2.64 Hz, 1H),

7.41 (m, 2H), 3.39 (t, J ) 6.78 Hz, 2H), 3.16 (t, J ) 6.97 Hz, 2H).

MS (ESI, pos. ion) m/z: 486.6 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(5-(pip-

eridin-1-yl)-1,3,4-thiadiazol-2-yl)propanamide (50). 50 was prepared

by 5-(piperidin-1-yl)-1,3,4-thiadiazol-2-amine and 3-(3-(2-chloro-

4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid (as in 42) using

amide coupling method B (as in 48, 141 mg, 44%). ¹H NMR (400

MHz, DMSO-d₆) δ 12.17 (s, 1H), 7.94 (t, 1H), 7.71 (d, J ) 8.97

Hz, 1H), 7.43 (t, 1H), 3.25 - 3.42 (m, 8H), 1.50-1.64 (m, 6H).

MS (ESI, pos. ion) m/z: 437.0 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(4-isopro-

pylphenyl)propanamide (51). 51 was prepared by 4-isopropylben-

zenamine and 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-

yl)propanoic acid (as in 42) using amide coupling method A (as in

30, 50 mg, 36% yield). ¹H NMR (300 MHz, CDCl₃) δ 7.90 (dd, J

) 8.67, 6.03 Hz, 1H), 7.46 (s, 1H), 7.40 (d, J ) 8.48 Hz, 2H),

7.28 (m, 1H), 7.17 (d, J ) 8.29 Hz, 2H), 7.09 (m, 1H), 3.40 (t, J

) 6.97 Hz, 2H), 2.97 (t, J ) 7.06 Hz, 2H), 2.87 (m, 1H), 1.22 (d,

J ) 6.97 Hz, 6H). MS (ESI, pos. ion) m/z: 387.8 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(4-(trif-

luoromethyl)phenyl)propanamide (52). 52 was prepared by 4-(tri-

ﬂuoromethyl)benzenamine and 3-(3-(2-chloro-4-ﬂuorophenyl)-

1,2,4-oxadiazol-5-yl)propanoic acid (as in 42) using amide coupling

method A (as in 30, 29 mg, 37% yield). ¹H NMR (300 MHz,

CDCl₃) δ 7.81 (dd, J ) 8.67, 6.03 Hz, 1H), 7.61 (s br, 1H), 7.53

(q, J ) 8.85 Hz, 4H), 7.21 (m, 1H), 7.02 (m, 1H), 3.34 (t, J )

6.88 Hz, 2H), 2.94 (t, J ) 6.88 Hz, 2H). MS (ESI, pos. ion) m/z:

413.7 (M + 1).

N-(2-Chloro-4-(triﬂuoromethyl)phenyl)-3-(3-(2-chloro-4-ﬂuo-

rophenyl)-1,2,4-oxadiazol-5-yl)propanamide (53). 53 was prepared

by 2-chloro-4-(triﬂuoromethyl)benzenamine and 3-(3-(2-chloro-

4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid (as in 42)

using amide coupling method A (as in 30, 58 mg, 29% yield).

¹H NMR (300 MHz, CDCl₃) δ 8.55 (d, J ) 8.61 Hz, 1H), 8.01

(s br, 1H), 7.91 (dd, J ) 8.80, 6.06 Hz, 1H), 7.64 (m, 1H), 7.52

(m, 1H), 7.28 (m, 1H), 7.10 (m, 1H), 3.43 (t, J ) 6.94 Hz, 2H),

3.12 (t, J ) 6.94 Hz, 2H). MS (ESI, pos. ion) m/z: 447.6 (M +

1).

N-(3-Chloro-4-(triﬂuoromethyl)phenyl)-3-(3-(2-chloro-4-ﬂuo-

5032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16

Cheng et al.

rophenyl)-1,2,4-oxadiazol-5-yl)propanamide (54). 54 was prepared

by 3-chloro-4-(triﬂuoromethyl)benzenamine and 3-(3-(2-chloro-

4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propanoic acid (as in 42)

using amide coupling method A (as in 30, 130 mg, 79% yield).

¹H NMR (300 MHz, CDCl₃) δ 7.88 (dd, J ) 8.67, 6.03 Hz,

1H), 7.84 (s br, 1H), 7.79 (d, J ) 1.70 Hz, 1H), 7.61 (d, J )

8.48 Hz, 1H), 7.46 (dd, J ) 8.57, 1.41 Hz, 1H), 7.29 (dd, J )

8.48, 2.45 Hz, 1H), 7.10 (m, 1H), 3.41 (t, J ) 6.88 Hz, 2H),

3.02 (t, J ) 6.78 Hz, 2H). MS (ESI, pos. ion) m/z: 447.6 (M +

1).

5.7 Hz, 1H), 7.38-7.49 (m, 1H), 7.71 (dd, J ) 9.0, 2.5 Hz, 1H),

7.97 (dd, J ) 8.7, 6.2 Hz, 1H). MS (ESI, pos. ion) m/z: 379 (M +

1).

6-Chloro-N-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)

propyl)benzo[d]thiazol-2-amine (62). 11 mg, 6.5% yield. ¹H NMR

(400 MHz, CD₃OD) δ 7.80-8.00 (m, 2H), 7.35-7.55 (m, 3H),

7.19 (dt, J ) 7.8, 2.5 Hz, 1H), 3.77 (t, J ) 6.6 Hz, 2H), 3.23 (t, J

) 7.0 Hz, 2H), 2.40 (m, 2H). MS (ESI, pos. ion) m/z: 423.0 (M +

1).

6-Chloro-N-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)

propyl)quinolin-3-amine (63). 63 was prepared using 6-chloro-

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(6-(trif-

luoromethyl)pyridin-3-yl)propanamide (55). 55 was prepared by

6-(triﬂuoromethyl)pyridin-3-amine and 3-(3-(2-chloro-4-ﬂuorophe-

nyl)-1,2,4-oxadiazol-5-yl)propanoic acid (as in 42) using amide

1

quinolin-3-amine (prepared as in 47); 1.2 g, 50% yield. H NMR

(400 MHz, CDCl₃) δ 8.21 (d, J ) 2.5 Hz, 1H), 7.74 (dd, J ) 8.8,

6.1 Hz, 1H), 7.63 (d, J ) 8.8 Hz, 1H), 7.40 (d, J ) 2.2 Hz, 1H),

7.10-7.20 (m, 2H), 6.98 (dt, J ) 2.1, 8.1 Hz, 1H), 6.81 (d, J )

2.5 Hz, 1H), 4.05 (br s, 1H), 3.23 (t, J ) 6.8 Hz, 2H), 3.01 (t, J )

7.2 Hz, 2H), 2.14 (m, 2H). MS (ESI, pos. ion) m/z: 417.1 (M + 1).

N-(3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propyl)-

6-(triﬂuoromethoxy)quinolin-3-amine (64). Butyl 2-formyl-4-(tri-

ﬂuoromethoxy)phenylcarbamate (1.28 g, 4.2 mmol) was dissolved

in dry THF (20 mL) and added to a solution of methazonic acid

(0.44 g, 4.2 mmol, prepared as in 47) in water (20 mL).

Concentrated HCl (2 mL) was added, and the mixture was heated

to 80 °C. After 2 h, the mixture was concentrated under reduced

pressure to about 20 mL that was diluted with water (50 mL) and

ﬁltered through a fritted funnel. The crude 3-nitro-6-(triﬂuo-

romethoxy)quinoline (0.52 g, 2.0 mmol) was dissolved in isopro-

panol (80 mL) and treated with saturated ammonium chloride (5

mL), water (5 mL), acetic acid (0.5 mL), and iron powder (0.57 g,

10 mmol). The reaction mixture was heated to 60 °C for 40 min.

After cooling, the mixture was ﬁltered through a pad of celite. The

ﬁltrate was evaporated to dryness under reduced pressure, and the

crude material was resuspended in ethyl acetate (100 mL) before

washing with 1N NaOH (20 mL) and water (60 mL). The organic

layer was dried with MgSO₄and evaporated to dryness under

reduced pressure. The crude material was puriﬁed by reverse-phase

HPLC to give 6-(triﬂuoromethoxy)quinolin-3-amine (0.38 g, 40%

yield) as a yellow solid.

1

coupling method B (as in 48, 95 mg, 21% yield). H NMR (400

MHz, DMSO-d₆) δ 10.76 (s, 1H), 8.86 (d, J ) 2.0 Hz, 1H), 8.30

(dd, J ) 8.2. 1.6 Hz, 1H), 7.95 (dd, J ) 8.8, 6.3 Hz, 1H), 7.86 (d,

J ) 8.8 Hz, 1H), 7.71 (dd, J ) 8.9, 2.5 Hz, 1H), 7.43 (m, 1H),

3.32 (m, 2H), 3.06 (t, J ) 7.0 Hz 2H). MS (ESI, pos. ion) m/z:

415.0 (M + 1).

3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)-N-(5-cyano-

6-(2,2,2-triﬂuoroethoxy)pyridin-3-yl)propanamide (56). 56 was

prepared by amide coupling method B (as in 48, 1.2 g, 68% yield)

using 26 and 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-

1

yl)propanoic acid (as in 42). H NMR (300 MHz, CDCl₃) δ 8.35

(m, 2H), 7.91 (s br, 1H), 7.88 (m, 1H), 7.26 (m, 1H), 7.12 (m,

1H), 4.85 (q, J ) 8.23 Hz, 2H), 3.42 (t, J ) 6.78 Hz, 2H), 3.02 (t,

J ) 6.78 Hz, 2H). MS (ESI, pos. ion) m/z: 469.7 (M + 1).

N-(2-tert-Butylpyrimidin-5-yl)-3-(3-(2-chloro-4-ﬂuorophenyl)-

1,2,4-oxadiazol-5-yl)propanamide (57). 57 was prepared by 2-tert-

butylpyrimidin-5-amine and 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-

oxadiazol-5-yl)propanoic acid (as in 42) using amide coupling

method B (as in 48, 127 mg, 75% yield). ¹H NMR (300 MHz,

CDCl₃) δ 8.87 (s, 2H), 7.88 (m, 2H), 7.28 (m, 1H), 7.11 (m, 1H),

3.42 (t, J ) 6.88 Hz, 2H), 3.03 (t, J ) 6.88 Hz, 2H), 1.39 (s, 9H).

MS (ESI, pos. ion) m/z: 403.8 (M + 1).

N-(5-tert-Butylisoxazol-3-yl)-3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-

oxadiazol-5-yl)propanamide (58). 58 was prepared by 5-tert-

butylisoxazol-3-amine and 3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-

oxadiazol-5-yl)propanoic acid (as in 42) using amide coupling

method B (as in 48, 123 mg, 37% yield). ¹H NMR (400 MHz,

DMSO-d₆) δ 11.13 (s, 1H), 7.95 (dd, J ) 8.84, 6.06 Hz, 1H), 7.71

(dd, J ) 8.91, 2.59 Hz, 1H), 7.47-7.40 (m, 1H), 6.56 (s, 1H),

3.29 (t, J ) 6.82 Hz, 2H), 2.97 (t, J ) 6.82 Hz, 2H), 1.27 (s, 9H).

MS (ESI, pos. ion) m/z: 393.1 (M + 1).

64 was prepared using 6-(triﬂuoromethoxy)quinolin-3-amine and

compound 21 following the procedure used to prepare 60 (27 mg,

1

30% yield). H NMR (300 MHz, CDCl₃) δ 8.41 (d, J ) 2.92 Hz,

1H), 7.92 (m, 2H), 7.40 (m, 1H), 7.30 (dd, J ) 8.48, 2.48 Hz, 1H),

7.24 (m, 1H), 7.10 (m, 1H), 7.00 (d, J ) 2.78 Hz, 1H), 4.36 (m,

1H), 3.44 (q, J ) 6.72 Hz, 2H), 3.15 (t, J ) 7.09 Hz, 2H), 2.31

(m, 2H). MS (ESI, pos. ion) m/z: 467.1 (M + 1).

N-(3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propyl)

quinolin-3-amine (65). 65 was prepared using quinolin-3-amine and

compound 21 following the procedure used to prepare 60 (30 mg,

40% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.09-2.22 (m, 2H),

3.19 (t, J ) 7.2 Hz, 2H), 3.24-3.31 (m, 2H), 6.44 (t, J ) 5.1 Hz,

1H), 7.07 (d, J ) 2.4 Hz, 1H), 7.31 (t, J ) 7.1 Hz, 1H), 7.35-7.45

(m, 2H), 7.63 (d, J ) 7.5 Hz, 1H), 7.71 (dd, J ) 8.8, 2.8 Hz, 1H),

7.76 (d, J ) 7.5 Hz, 1H), 7.94 (dd, J ) 8.6, 6.2 Hz, 1H), 8.45 (d,

J ) 2.5 Hz, 1H). MS (ESI, pos. ion) m/z: 383 (M + 1).

6-Chloro-N-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)

propyl)-N-methylquinolin-3-amine (66). Compound 63 (0.034 g,

0.08 mmol) was dissolved in dichloroethane (20 mL) and treated

with acetic acid (2 mL) and formaldehyde, 37% in water (0.80 mL,

11 mmol). The mixture was heated to 60 °C for 2 h, then sodium

triacetoxyborohydride (0.087 g, 0.4 mmol) was added and the

mixture stirred at room temperature for 40 min. The reaction was

evaporated to dryness and redissolved in acetic acid (5 mL). Another

portion of formaldehyde (37% in water, 0.80 mL, 11 mmol) was

added, and the mixture was stirred for another 45 min prior to

addition of sodium triacetoxyborohydride (0.088 g, 0.4 mmol). The

mixture was stirred for an additional 40 min then evaporated to

dryness under reduced pressure. 1N NaOH (20 mL) and ethyl

acetate (40 mL) were added, and the phases were mixed for 0.5 h.

The organic layer was dried with MgSO₄and evaporated to dryness

under reduced pressure. The crude material was puriﬁed by column

chromatography on silica gel (eluting with 30-100% EtOAc/

5-(3-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)propy-

lamino)-2-(2,2,2-triﬂuoroethoxy)nicotinonitrile (60). Compound 21

(0.080 g, 0.31 mmol) and compound 26 (0.068 g, 0.31 mmol) were

combined in dichloroethane (1 mL). The mixture was stirred at

room temperature. Titanium isopropoxide (1.4 mL, 4.7 mmol) was

added, and the mixture was stirred for 45 min. The resulting solution

was added to a stirring solution of sodium borohydride (0.066 mL,

1.9 mmol) in 5 mL of methanol. The reaction mixture was stirred

for 30 min, and then the solvent was removed. The resulting

material was taken in water and extracted with EtOAc three times.

The combined organic layers were washed with water and brine,

dried on sodium sulfate, ﬁltered, and concentrated. The resulting

residue was puriﬁed by column chromatography on silica gel eluting

with 0-10% methanol/dichromethane to give 60 (0.10 g, 70%

1

yield). H NMR (300 MHz, CDCl₃): δ 7.93 (dd, J ) 8.70, 6.07

Hz, 1H), 7.74 (d, J ) 2.92 Hz, 1H), 7.30 (dd, J ) 8.48, 2.48 Hz,

1H), 7.21 (d, J ) 2.92 Hz, 1H), 7.13 (m, 1H), 4.76 (q, J ) 8.33

Hz, 2H), 3.88 (s br, 1H), 3.31 (q, J ) 6.63 Hz, 2H), 3.12 (t, J )

7.02 Hz, 2H), 2.23 (m, 2H). MS (ESI, pos. ion) m/z: 456.0 (M +

1).

5-tert-Butyl-N-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-

yl)propyl)isoxazol-3-amine (61). 77 mg, 35% yield. ¹H NMR (400

MHz, DMSO-d₆) δ 1.21 (s, 9H), 1.96-2.10 (m, 2H), 3.08 (t, J )

7.5 Hz, 2H), 3.15 (q, J ) 6.6 Hz, 2H), 5.55 (s, 1H), 6.11 (t, J )

CB2 Cannabinoid Receptor Agonists

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5033

hexane) to give 66 (0.029 g, 81% yield) as a yellow oil. ¹H NMR

(400 MHz, CDCl₃) δ 8.67 (d, J ) 2.9 Hz, 1H), 7.85-8.00 (m,

2H), 7.56 (d, J ) 2.2 Hz, 1H), 7.36 (dd, J ) 8.9, 2.3, 1H), 7.30

(dd, J ) 8.5, 2.5 Hz, 1H), 7.10-7.20 (m, 2H), 3.67 (t, J ) 7.2 Hz,

2H), 3.12 (s, 3H), 3.07 (t, J ) 7.1 Hz, 2H), 2.20-2.35 (m, 2H).

MS (ESI, pos. ion) m/z: 431.1 (M + 1).

6-Chloro-3-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)

propoxy)quinoline (67). Compound 19 (0.33 g, 1.3 mmol) and

triphenylphosphine (0.39 g, 1.5 mmol) were dissolved in dry THF

(30 mL) prior to addition of bromine (0.085 mL, 1.6 mmol). The

reaction was stirred at room temperature for 6 h then concentrated

to dryness under reduced pressure. The crude was puriﬁed using

silica chromatography (eluting with 0-50% EtOAc/hexane) to give

5-(2-bromoethyl)-3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazole (0.27

g, 65% yield).

6-Chloroquinolin-3-amine (0.34 g, 1.9 mmol, prepared as in 47)

was dissolved in 2N H₂SO₄(40 mL) and cooled in an ice bath. A

solution of sodium nitrite (0.15 g, 2.1 mmol) in water (10 mL)

was added slowly and the mixture stirred for 30 min. The solution

was added to 70 °C 5N H₂SO₄(20 mL) and stirred at this

temperature for 20 min. The mixture was cooled to room temper-

ature, and EtOAc (100 mL) was added. Disodium hydrogen

phosphate was added to adjust the pH to 5, and the phases were

separated. The organic layer was dried with MgSO₄then evaporated

to dryness under reduced pressure to give 6-chloroquinolin-3-ol

that was used without puriﬁcation.

The crude 6-chloroquinolin-3-ol and 5-(3-bromopropyl)-3-(2-

chloro-4-ﬂuorophenyl)-1,2,4-oxadiazole (0.70 g, 2.2 mmol) were

dissolved in dry DMF (5 mL) and treated with K₂CO₃(0.45 g, 3.3

mmol). The reaction was heated to 60 °C for 20 min. Water (100

mL) and ethyl acetate (100 mL) were added. The phases were

separated and the organic layer was dried with MgSO₄before

evaporating to dryness under reduced pressure. The crude material

was puriﬁed by column chromatography on silica gel (eluting with

10-100% EtOAc/hexane) to give 67 (0.28 g, 30% yield). ¹H NMR

(400 MHz, CDCl₃) δ 8.62 (d, J ) 2.8 Hz, 1H), 7.87-8.00 (m,

2H), 7.67 (d, J ) 2.3 Hz, 1H), 7.49 (dd, J ) 9.0, 2.3 Hz, 1H),

7.25-7.32 (m, contains CHCl₃peak, 2H), 7.10 (dt, J ) 1.7, 7.1

Hz, 2H), 4.25 (t, J ) 5.8 Hz, 2H), 3.26 (t, J ) 7.3 Hz, 2H),

2.40-2.55 (m, 2H). MS (ESI, pos. ion) m/z: 418.1 (M + 1).

2-tert-Butyl-5-(3-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-

yl)propoxy)pyrimidine (68). Compound 19 (0.085 g, 0.33 mmol),

2-tert-butylpyrimidin-5-ol (0.050 g, 0.33 mmol), and triphenylphos-

phine (0.087 g, 0.33 mmol) were dissolved in benzene (10 mL)

and treated with diisopropyl azodicarboxylate (0.065 mL, 0.33

mmol). After stirring for 2 h, the reaction was evaporated to dryness

under reduced pressure and the crude material was puriﬁed by

column chromatography on silica gel (eluting with 5-100% EtOAc/

hexane). Further puriﬁcation using reverse phase HPLC gave 68

(0.037 g, 28% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 2H),

7.93 (dd, J ) 8.6, 6.1 Hz, 1H), 7.29 (dd, J ) 8.4, 2.6 Hz, 1H),

7.12 (dt, J ) 2.6, 8.8 Hz, 1H), 4.25 (t, J ) 5.9 Hz, 2H), 3.22 (t, J

) 7.3 Hz, 2H), 2.40-2.50 (m, 2H). MS (ESI, pos. ion) m/z: 391.1

(M + 1).

3-(4-(3-(2-Chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)butyl)quin-

oline (69). To a solution of (E)-2-chloro-4-ﬂuoro-N′-hydroxyben-

zamidine (4.4 g, 23 mmol, prepared in the same manner as in 37

using 2-chloro-4-ﬂuorobenzonitrile) in THF (100 mL) at 0 °C was

added pent-4-ynoyl chloride (3.26 g, 28 mmol) followed by

triethylamine (4.9 mL, 35 mmol). The white mixture was allowed

to warm to room temperature. After 1 h, the mixture was ﬁltered

and concentrated. The residue was taken up in EtOAc, washed with

water and brine, and dried with Na₂SO₄. After concentration in

vacuo, the tan oil was advanced without further puriﬁcation. A

solution of this material (6.2 g, 23.1 mmol) in toluene (100 mL)

was heated to reﬂux. After 16 h, the solvent was removed in vacuo

and the residue puriﬁed by column chromatography on silica gel

eluting with 5-40% EtOAc/hexane to afford 5-(but-3-ynyl)-3-(2-

chloro-4-ﬂuorophenyl)-1,2,4-oxadiazole (4.1 g, 70% yield) as a

white solid.

To a mixture of 5-(but-3-ynyl)-3-(2-chloro-4-ﬂuorophenyl)-1,2,4-

oxadiazole (0.15 g, 0.60 mmol), 3-bromoquinoline (0.10 mL, 0.78

mmol), copper(I) iodide (0.0057 g, 0.03 mmol), and dichloro-

bis(triphenylphosphine)palladium (II) (0.021 g, 0.03 mmol) was

added acetonitrile (4 mL) and triethylamine (1.67 mL, 12 mmol).

The mixture was heated to 80 °C in a sealed tube. After 16 h, the

solvent was removed in vacuo and the residue puriﬁed by column

chromatography on silica gel (eluting with 5-30% EtOAc/hexane)

to afford 3-(4-(3-(2-chloro-4-ﬂuorophenyl)-1,2,4-oxadiazol-5-yl)but-

1-ynyl)quinoline (0.12 g, 54% yield) as a light yellow solid. This

material (0.052 g, 0.14 mmol) and palladium on activated carbon

10% (0.015 g) in benzene was exposed to an atmosphere of H₂.

After 16 h, the mixture was ﬁltered over a silica plug and

concentrated in vacuo to afford 69 (0.040 g, 76% yield) as an off-

1

white solid. H NMR (400 MHz, DMSO-d₆) δ 8.81 (d, J ) 2.02

Hz, 1H), 8.16 (s, 1H), 7.88-8.00 (m, 3H), 7.66-7.72 (m, 2H),

7.54-7.60 (m, 1H), 7.38-7.45 (m, 1H), 3.10 (t, J ) 6.88 Hz, 2H),

2.87 (t, J ) 6.88 Hz, 2H), 1.76-1.92 (m, 4H). MS (ESI, pos. ion)

m/z: 382.1 (M + 1).

Supporting Information Available: Detailed description of

HPLC methods, HPLC purity analysis of all ﬁnal compounds, and

HPLC spectra of compound 56 and 63. This material is available

free of charge via the Internet at http://pubs.acs.org.

References

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JM800463F

Article Doi

Discovery and optimization of a novel series of N-arylamide oxadiazoles as potent, highly selective and orally bioavailable cannabinoid receptor 2 (CB 2) agonists

DOI: 10.1021/jm800463f

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