Spirocyclic delta opioid receptor agonists for the treatment of pain: Discovery of N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4′-piperidine]-4- yl) benzamide (ADL5747)

J. Med. Chem. 2009, 52, 5685–5702 5685

DOI: 10.1021/jm900773n

Spirocyclic Delta Opioid Receptor Agonists for the Treatment of Pain: Discovery of

N,N-Diethyl-3-hydroxy-4-(spiro[chromene-2,4⁰-piperidine]-4-yl) Benzamide (ADL5747)

Bertrand Le Bourdonnec,*^,†Rolf T. Windh,^‡Lara K. Leister,^†Q. Jean Zhou,^†Christopher W. Ajello,^†Minghua Gu,^{†,^}

Guo-Hua Chu,^†Paul A. Tuthill,^†William M. Barker,^†Michael Koblish,^‡Daniel D. Wiant,^‡Thomas M. Graczyk,^‡

Serge Belanger,^‡Joel A. Cassel,^‡Marina S. Feschenko,^‡Bernice L. Brogdon,^§Steven A. Smith,^§,#Michael J. Derelanko,

Steve Kutz, Patrick J. Little,^‡Robert N. DeHaven,^‡Diane L. DeHaven-Hudkins,^‡and Roland E. Dolle^†

§

^†Departments of Chemistry and ^‡Pharmacology and DMPK and Preclinical Safety Assessment, Adolor Corporation,

700 Pennsylvania Drive, Exton, Pennsylvania 19341. ^{^}GlaxoSmithKline R&D, PA. ^#UCB Pharma SA, Belgium.

Received June 1, 2009

Selective, nonpeptidic δ opioid receptor agonists have been the subject of great interest as potential

novel analgesic agents. The discoveries of BW373U86 (1) and SNC80 (2) contributed to the rapid

expansion of research in this field. However, poor drug-like properties and low therapeutic indices have

prevented clinical evaluation of these agents. Doses of 1 and 2 similar to those required for analgesic

activity produce convulsions in rodents and nonhuman primates. Recently, we described a novel series

of potent, selective, and orally bioavailable delta opioid receptor agonists. The lead derivative,

ADL5859 (4), is currently in phase II proof-of-concept studies for the management of pain. Further

structure activity relationship exploration has led to the discovery of ADL5747 (36), which is

approximately 50-fold more potent than 4 in an animal model of inflammatory pain. On the basis of

its favorable efficacy, safety, and pharmacokinetic profile, 36 was selected as a clinical candidate for the

treatment of pain.

Introduction

neuropathic pain,^14-16and efficacy in visceral pain models

has also been reported.^17-19Efficacy of DOR agonists in

inflammatory pain models is more similar to that of nonster-

oidal anti-inflammatory drugs (NSAIDs) than of morphine in

that DOR agonists reverse the hyperalgesia associated with

inflammation but do not inhibit acute pain. Aside from pain,

DOR agonists also have potential application in several

therapeutic indications, including affective disorder,^20,21or-

gan protection,²²and neurodegenerative diseases.^23,24Delta

opioid agonists may possess potential clinical benefits

compared with the μ opioid agonists currently used for pain

relief, including reduced respiratory depression,^25,26constipa-

tion,^14,27physical dependence,^28-30and abuse liability.^26,31

Delta opioid receptor research received a major boost when

The biological effects of endogenous opioid peptides are

mediated through three classes of naloxone-sensitive opioid

receptors: mu (μ), kappa (κ), and delta (δ). All three opioid

receptors are localized throughout the central nervous system

(CNS^a) andperiphery, and allare associated withpain relief in

animal models.¹Whereas μ opioid receptor agonists such as

morphine still serve as the gold standards for managing a wide

range of pain conditions, their pronounced side effects, in-

cluding sedation, respiratory depression, and constipation,

limit their utility. The δ opioid receptor (DOR) is an attractive

target for the development of new drugs to control pain. Delta

opioid receptors in rodents and primates are localized in

cerebral cortex, striatum, amygdala, brainstem nuclei asso-

ciated with pain processing, spinal cord, and dorsal root

ganglia.^2-11The localization pattern of DORs is consistent

with a role in pain regulation and may also support the

proposed roles for DORs in modulation of affective disorders

and striatal neurodegeneration. Delta opioid receptor ago-

nists are active in animal models of inflammatory^12-14and

1

³²(Chart 1), a prototypic small molecule nonpeptidic DOR

agonist, was disclosed by Burroughs Wellcome in 1993.

Subsequent DOR agonist research centered around the

structure-activity relationships (SAR) of this compound

series,^33-35and led to the discovery of 2,³⁶the optically pure

enantiomer of the methyl ether analogue of 1. Receptor

binding and in vitro bioassays have revealed a high degree

of selectivity for δ over μ and κ opioid receptors for this

compound.^36,37

However, further development of 1 and 2 has not been

pursued because they both elicit convulsions in animals at

doses similar to those required for analgesic activity.³⁸

The DOR agonist induced convulsions consist of a single

mild, brief (10-15 s) convulsive event, usually occurring

within minutes of drug administration, and are followed by

a brief period of catalepsy before the animals return to

apparently normal behavior.³⁸Sensitivity to the convulsive

effects of these agonists in mice varies with genetic strain,³⁹

*To whom correspondence should be addressed. Phone: 1-484-595-

1061. Fax: 1-484-595-1551. E-mail: blebourdonnec@adolor.com.

^aAbbreviations: SAR, structure-activity relationship; CYP, cyto-

chrome P450; DDI, drug-drug interaction; hERG, human ether-a-

go-go related gene; FCA, Freund’s Complete Adjuvant; AUC, area

under the curve; CNS: central nervous system; EEG: electroencephalo-

gram; MEST, maximum electroshock seizure threshold; DMPK, drug

metabolism and pharmacokinetic; NSAID: nonsteroidal anti-inflam-

matory drug; TBTU, O-benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethyluro-

nium tetrafluoroborate; DOR: δ opioid receptor; SEM: standard error

of the mean; CHO, Chinese hamster ovary; MAMC, 7-methoxy-4-

aminomethyl-coumarin; HAMC, 7-hydroxy-4-aminomethyl-coumarin;

PPT, paw pressure thresholds; HEK, human embryonic kidney.

r

2009 American Chemical Society

Published on Web 08/20/2009

pubs.acs.org/jmc

5686 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Chart 1. Structures of 1-4

Le Bourdonnec et al.

and convulsions have also been reported in rats⁴⁰and nonhu-

manprimates,^26,41,42ofteninthe same doserange as analgesia.

The convulsions induced by 1 and 2 can be blocked with DOR

antagonists in all species tested,^26,39-42and the absence of

these convulsions in mice lacking the DOR clearly demon-

strates the requirement for DOR activation in this effect.³⁹

However, continued interest in DOR agonist research has

been encouraged by the knowledge that, in contrast to

compounds 1, 2, and structurally related analogues, DOR

peptide agonists do not generally produce convulsions in

rodents.^43-45Recent studies with DOR agonists of different

chemical classes also demonstrate that not all small molecule

DOR agonists produce the behavioral activation and convul-

sions that are characteristic of 1 and 2. SB-235863 (3)¹⁴does

not produce seizures at doses up to 70 mg/kg po and fails to

decrease maximum electroshock seizure threshold (MEST) or

potentiate metrazol-induced seizures.¹⁴Therefore, it appears

that whereas the δ receptor is required for behavioral activa-

tion and induction of convulsions by compounds 1, 2, and

structurally related analogues, activation of the DOR does

not necessarily result in these behaviors. The reasons for

differences in behavioral effects among DOR agonists are

unclear. We reported previously the discovery of ADL5859

(4), a potent and highly selective full agonist at the DOR that

is structurally distinct from other chemical classes of δ ago-

nists.⁴⁶Importantly, 4 does not produce compound 2-like

behaviors, including convulsions, increased locomotor activ-

ity, or stereotypicactivity, at oraldosesupto1000mg/kg. This

agent is in phase II clinical trials as an analgesic. Herein, we

describe our continued efforts in this area and report the

discovery of ADL5747 (36), a small molecule DOR agonist

currently undergoing phase I safety evaluation in normal

healthy volunteers.

Scheme 2. Condensation of the enol triflate 39a with

4-(methoxycarbonyl)phenylboronic acid under standard Su-

zuki coupling conditions provided the methyl ester 41, which

was hydrolyzed to the corresponding carboxylic acid deriva-

tive 42 under basic conditions. Coupling of 42 with various

amines using O-benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethyluro-

nium tetrafluoroborate (TBTU) as coupling agent afforded

the primary, secondary, and tertiary carboxamide deriva-

tives 43a-k. Treatment of the N-Boc derivatives 43a-k with

anhydrous hydrochloric acid in methanol provided the final

compounds 6-16. The 3,4-dihydroisoquinolin-1(2H)-one de-

rivative 17, constrained analogue of 5, was prepared accord-

ing to Scheme 3. Alkylation of 6-methoxy-3,4-dihydroiso-

quinolin-1(2H)-one 44³⁵with ethyl iodide in tetrahydrofuran

in the presence of sodium hydride afforded the methyl

ether 45, which was converted to the phenolic derivative 46

by treatment with boron tribromide. Condensation of 46 with

trifluoromethanesulfonic anhydride in dichloromethane in

the presence of pyridine gave the triflate derivative 47. Con-

version of the enol triflate 39a to the boronate derivative 48

was achieved in good yield in N,N-dimethylformamide in

the presence of bis(pinacolato)diborane, dichloro[1,1⁰-bis(di-

phenylphosphino)ferrocene]palladium(II) dichloromethane

adduct, and potassium acetate. Couplingof 48withthe triflate

derivative 47 under Suzuki coupling conditions afforded

the N-Boc derivative49, whichwas converted to thefinalcom-

pound 17 under acidic conditions. The preparation of com-

pounds 22-24 is described in Scheme 4. The spiro[chromene-

2,4⁰-piperidine] derivative 5 was converted to the spiro-

[chroman-2,4⁰-piperidine] analogue 22 (racemic mixture) by

hydrogenation in methanol in the presence of Pearlman’s

catalyst. The enantiomers 23 and 24, derived from 22, were

separated by chiral HPLC. Condensation of 23 with (1S)-(þ)-

10-camphorsulfonyl chloride (used as chiral derivatization

agent) in dichloromethane in the presence of triethylamine

provided the chiral sulfonamide derivative 50. The absolute

configuration of 50 was determined by X-ray crystallography

(Chart 2),⁴⁷thereby establishing, by inference, the absolute

configuration of enantiomers 23 and 24. Compounds 25-27

and 29-35 were prepared according to Scheme 5. The aryl

bromides 52a-d,f-j were obtained by coupling of their

corresponding carboxylic acid derivatives with N,N-diethyla-

mine using TBTU as coupling agent. Treatment of 5-bromo-

2-iodopyrimidine 53 with n-butyllithium in hexane/toluene

followed by addition of carbon dioxide provided 5-bromo-

pyrimidine-2-carboxylic acid (54). Treatment of 54 with ox-

alyl chloride in dichloromethane gave the corresponding acyl

chloride derivative, which reacted with diethylamine in tetra-

hydrofuran to yield the amide 52e. Coupling of the aryl

bromides 52a-j with the boronate derivative 48 in ethylene

glycol dimethyl ether in the presence of tetrakis(triphenyl-

phosphine)palladium(0), lithium chloride, and an aqueous

Chemistry

The examples listed in Tables 1-4 were prepared according

to Schemes 1-7. The synthesis of compounds 5⁴⁶and 18-21

is outlined in Scheme 1. Condensation of the ketone deriva-

tives 37a-e with 2-hydroxyacetophenone in refluxing metha-

nol in the presence of pyrrolidine provided the spirocyclic

ketones 38a-e, which were converted to the corresponding

enol triflate derivatives 39a-e using N-phenylbis(trifluoro-

methanesulfonamide) as triflating agent. Suzuki type cou-

pling of the enol triflate derivatives 39a-e with 4-(N,N-

diethylaminocarbonyl)phenylboronic acid in ethylene glycol

dimethyl ether in the presence of tetrakis(triphenylphos-

phine)palladium(0), lithium chloride, and an aqueous solu-

tion of sodium carbonate providedcompounds21 and 40a-d.

The N-Boc derivatives 40a-d were then converted to the

target compounds 5, and 18-20, respectively, under acidic

conditions. The synthesis of compounds 6-16 is described in

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5687

Table 1. In Vitro Profile of Compounds 5-17 at Opioid Receptors (δ, μ, and κ), hERG, and CYP2D6

^aSee detailed pharmacological methods in the Experimental Section. ^bInhibition of hERG channel currents in voltage-clamped HEK293 cells stably

expressing hERG potassium channels; IC₅₀values are geometric means from at least three separate determinations. ^cFluorescence-based assay; see

detailed methods in the Experimental Section. ^dSee structure in Scheme 3.

solution of sodium carbonate yielded the N-Boc protected

spirocyclic piperidine derivatives 51a-j, which were con-

verted to the final compounds 25-27 and 29-35, respectively,

under acidic conditions. The synthesis of compound 28 is

outlined in Scheme 6. Coupling of the carboxylic acid 55

with N,N-diethylamine in the presence of TBTU afforded the

bromide 56, which was converted to the boronate 57

inN,N-dimethylformamideinthe presence of bis(pinacolato)-

diborane, potassium acetate, and 1,1⁰-bis(diphenylphosphino)-

ferrocene palladium(II) chloride complex with dichloro-

methane. Suzuki coupling of the enol triflate 39a with 57 in

ethylene glycol dimethyl ether in the presence of palladium on

carbon, lithium chloride, and an aqueous solution of sodium

carbonate afforded compound 58, which was converted to the

final product (compound 28) under acidic conditions. The

medicinal chemistry route used to prepare compound 36 is

shown in Scheme 7. Compound 60 was prepared in two steps

from carboxylic acid 59, i.e., esterification of 59 using acetyl

chloride in methanol, followed by conversion of the resulting

phenolic intermediate to the methoxymethyl (MOM) ether

derivative 60 using chloro(methoxy)methane as alkylating

agent. Hydrolysis of ester 60 under basic conditions gave the

carboxylic acid 61, which was coupled with N,N-diethylamine in

the presence of TBTU to afford the corresponding carboxamide

derivative 62. Suzuki coupling of the boronate 48 with 62 in

ethylene glycol dimethyl ether in the presence of palladium on

5688 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

Table 2. In Vitro Profile of Compounds 18-21 at Opioid Receptors (δ, μ, and κ), hERG, and CYP2D6

^aSee detailed pharmacological methods in the Experimental Section. ^bInhibition of hERG channel currents in voltage-clamped HEK293 cells stably

expressing hERG potassium channels; IC₅₀values are geometric means from at least three separate determinations. ^cFluorescence-based assay; see

detailed methods in the Experimental Section. ^dnd: not determined.

Table 3. In Vitro Profile of Compounds 22-24 at Opioid Receptors (δ, μ, and κ), hERG, and CYP2D6

K_i(δ)

EC₅₀(δ)

%inh at

10 μM (μ)^a

%inh at

10 μM (κ)^a

IC₅₀(hERG)

(nM)^b

IC₅₀(CYP2D6)

(nM)^c

compd

configuration

(nM)^a

22

23

24

(()

R-(-)

S-(þ)

1.6 (1.0-2.6)

0.93 (0.57-1.5)

29 (18-47)

21 (11-42)

16 (10-25)

420 (310-560)

19 ( 6%

33 ( 7%

18 ( 4%

20 ( 2%

25 ( 3%

24 ( 5%

5094

2543

6200

7100

3100

18471

^aSee detailed pharmacological methods in the Experimental Section. ^bInhibition of hERG channel currents in voltage-clamped HEK293 cells stably

c

expressing hERG potassium channels; IC₅₀values are geometric means from at least three separate determinations. Fluorescence-based assay; see

detailed methods in the Experimental Section.

carbon, lithium chloride, and an aqueous solution of sodium

carbonate under microwave irradiation afforded compound 63,

which was converted to 36 under acidic conditions.

metabolizing enzyme cytochrome P450 2D6 (CYP2D6) in

vitro (IC₅₀=4300 nM). CYP2D6 is a polymorphic member of

the P450 superfamily, which is absent in 5-9% of the

Caucasian population, resulting in a deficiency in drug oxida-

tion known as debrisoquine/sparteine polymorphism.⁴⁹

A

Results and Discussion

number of drugs have been clinically implicated in major

drug-drug interactions (DDI) via CYP2D6 inhibition.^50-52

As a result, it is prudent to minimize potential issues related to

CYP2D6-mediated DDIs at an early step of the drug dis-

covery process. The objective of the lead optimization

program was to identify orally active, potent, and selective

DOR agonists [K_i(δ)<10 nM; K_i(μ), K_i(κ)>1000ꢀK_i(δ)]

displaying reduced hERG and CYP2D6 inhibitory activities

(IC₅₀>10000 nM) when compared to 5. The derivatives 5-36

were tested for their affinities toward the cloned human δ, μ,

and κ opioid receptors as measured by their abilities to

displace specific [³H]diprenorphine binding. Selected com-

pounds were also evaluated for their in vitro functional

Compound 5, the lead molecule in this series, was pre-

viously identified as a potent DOR agonist, displaying ex-

cellent selectivity (>1000-fold) for δ over μ and κ opioid

receptors.⁴⁶Off-target profiling of 5 revealed that this com-

pound weakly inhibited the human ether-a-go-go-related gene

(hERG) potassium channel (IC₅₀=7900 nM).⁴⁶Blocking the

hERG K^þchannel could raise serious cardiovascular issues.

The hERG channel is responsible for the I_Krcurrent, a critical

component in ventricular repolarization. Its blockade can

lead to increased QT_cinterval and potentially ventricular

arrhythmias.⁴⁸Additional studies also demonstrated that

compound 5 moderately inhibited the activity of the drug

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5689

Table 4. In Vitro Profile of Compounds 25-36 at Opioid Receptors (δ, μ, and κ), hERG, and CYP2D6

^aSee detailed pharmacological methods in the Experimental Section. ^bInhibition of hERG channel currents in voltage-clamped HEK293 cells stably

expressing hERG potassium channels; IC₅₀values are geometric means from at least three separate determinations. ^cFluorescence-based assay; see

detailed methods in the Experimental Section.

agonist potencies to stimulate binding of guanosine 5⁰-

O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) to DOR-contain-

ing cell membrane preparations. All compounds tested in this

[³⁵S]GTPγS functional assay behaved as full agonists at the

DORs, with maximal stimulation similar to the reference

DOR agonist 1. The compounds displaying potent affinity

at the DOR were also evaluated for their inhibitory activity

toward CYP2D6 and the hERG K^þchannel. The N,N-

diethylcarboxamide moiety of 5 was previously shown to be

a key element for potent affinity at the DOR.⁴⁶On the basis of

this result, the SAR at the amide position of 5 was explored

further. As indicated in Table 1, conversion of the N,N-

diethylbenzamide group of 5 to an N-ethylbenzamide moiety

(compound 6) resulted in only a slight decrease (3-fold) in

DOR binding affinity. However, this subtle structural mod-

ification led to a 47-fold increase in the CYP2D6 inhibitory

activity. Replacement of the two ethyl groups of 5 with

hydrogen atoms (compound 7) was accompanied by a ∼10-

fold reduction in the binding affinity toward DOR. Similarly,

the methyl and isobutyl carboxamide derivatives 8 and 9

displayed significantly weaker DOR binding affinity than 5.

The N,N-dimethylcarboxamide derivative 11, despite its

reduced DOR affinity when compared to 5, displayed

an improved hERG profile (IC₅₀= 23070 nM). However,

the CYP2D6 inhibitory profile of 11 was still less than

desirable (IC₅₀=2800 nM). Cyclization of the ethyl substi-

tuent of 5 into pyrrolidino-, piperidino-, or morpholino-

groups (compounds 12-14, respectively) resulted in reduced

binding affinities at the DOR. Comparison of the DOR

affinity of 5 (K_i=1.8 nM) and its constrained analogue 17

5690 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Scheme 1. Synthesis of 5, 18-21^a

Le Bourdonnec et al.

^aReagents and conditions: (a) 2-hydroxyacetophenone, pyrrolidine, MeOH; (b) C₆H₅N(SO₂CF₃)₂, LiHMDS, THF; (c) 4-(N,N-diethylaminocar-

bonyl)phenylboronic acid, Pd[P(C₆H₅)₃)₄, LiCl, aq Na₂CO₃, DME; (d) anhyd HCl, (C₂H₅)₂O, CH₂Cl₂.

Scheme 2. Synthesis of 6-16^a

^aReagents and conditions: (a) 4-(methoxycarbonyl)phenylboronic acid, Pd[P(C₆H₅)₃]₄, LiCl, aq Na₂CO₃, DME; (b) LiOH, THF, H₂O; (c) R¹R²NH,

TBTU, iPr₂EtN, CH₃CN; (d) anhyd HCl, (C₂H₅)₂O, MeOH.

Scheme 3. Synthesis of 17^a

^aReagents and conditions: (a) NaH, C₂H₅I, THF; (b) BBr₃, CH₂Cl₂; (c) (CF₃SO₂)O, pyridine, CH₂Cl₂; (d) bis(pinacolato)diborane, Pd-

(Cl₂)dppf CH₂Cl₂, KOAc, CH₂Cl₂, DMF; (e) 47, Pd(Cl₂)dppf CH₂Cl₂, KOAc, DMF; (f) anhyd HCl, (C₂H₅)₂O, CH₂Cl₂.

3

(K_i=8.1 nM) also indicated that the free rotation of the amide

bond is necessary for optimal binding affinity at the DOR.

Among the series of N,N-disubstituted derivatives, the iso-

indolin-2-yl carboxamide 15 showed the highest affinity at the

DOR (K_i=0.53 nM). However, compound 15 did not meet in

vitro advancement criteria for hERG and CYP2D6 inhibitory

activity (hERG: IC₅₀=3501 nM; CYP2D6: IC₅₀=1800 nM).

Wenextinvestigated the SAR atthe piperidine moietyof5. As

shown in Table 2, the azepane derivative 18 bound to the

DOR with the same affinity as 5. However, despite its

improved hERG profile when compared to 5, compound 18

still suffered from moderate inhibitory activity at CYP2D6

(IC₅₀= 2600 nM). Replacement of the 4,4⁰-disubstituted

piperidine template of 5 with a 3,3⁰-disubstituted pyrrolidine

(compound 19) ora3,3⁰-disubstituted piperidine (compound 20)

moieties resulted in a significant decrease (10-fold and

30-fold, respectively) in the binding affinity toward the

DOR. We previously reported that the NH functionality of 5,

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5691

Scheme 4. Synthesis of 22-24^a

^aReagents and conditions: (a) Pd(OH)₂, H₂, MeOH; (b) chiral separation; (c) (1S,4R)-7,7-dimethly1-2-oxobicyclo[2,2,1]heptan-1-yl)methane-

sulfonyl chloride, Et₃N₃, CH₂Cl₂.

Scheme 5. Synthesis of 25-27 and 29-35^a

^aReagents and conditions: (a) Et₂NH, TBTU, iPr₂EtN, CH₃CN; (b) (i) nBuLi, CO₂, hexane, toluene, (ii) AcOH; (c) (i) ClCOCOCl, CH₂Cl₂, (ii) Et₂-

NH, THF; (d) RBr (52a-j), Pd[P(C₆H₅)₃]₄, LiCl, Na₂CO₃, DME; (e) anhyd HCl, (C₂H₅)₂O, CH₂Cl₂.

Scheme 6. Synthesis of 28^a

^aReagents and conditions: (a) (i) ClCOCOCl, CH₂Cl₂, (ii) Et₂NH, Et₃N, CH₂Cl₂; (b) bis(pinacolato)diborane, Pd(Cl₂)dppf CH₂Cl₂, KOAc, DMF;

(c) 39a, 10% Pd/C, LiCl, Na₂CO₃, DME; (d) anhyd HCl, dioxane, CH₂Cl₂

3

protonated at physiological pH, is of crucial importance for

optimal affinity at the DOR.⁴⁶This positively charged nitrogen

is presumably involved in electrostatic interaction with an

anionic residue of the DOR. On the basis of this hypothesis,

5692 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Scheme 7. Synthesis of 36^a

Le Bourdonnec et al.

^aReagents and conditions: (a) (i) MeOH, CH₃COCl, (ii) CH₃OCH₂Cl, iPr₂EtN, CH₂Cl₂; (b) LiOH, acetone, THF, H₂O; (c) Et₂NH, iPr₂EtN, TBTU,

CH₃CN; (d) 48, 10% Pd/C, LiCl, Na₂CO₃, DME; (e) anhyd HCl, dioxane, MeOH.

Chart 2. X-ray Structure of 50 Showing Labeling of the Non-

hydrogen Atoms

derivative 25 was identified as a potent and selective DOR

agonist. However, 25 displayed increased hERG and CYP2D6

inhibitory activities when compared to 5. Replacement of the

thiophene group of 25 with a furan moiety (compound 26) led

to a 13-fold decrease in the binding affinity toward the DOR.

Substitution of the pendant phenyl ring of 5 with a pyridine

template (compound 28) resulted in a significant reduction

(5- to 7-fold) in the hERG and CYP2D6 inhibitory activities,

attributed to a decrease in the overall lipophilicity (5: clogP=

3.15; 28: clogP=2.05). Importantly, 28 retained potent affinity

(K_i= 2.5 nM) and high selectivity for the DOR. The pyridine

derivative 29, a regioisomeric analogue of 28, also displayed

weak inhibitory activity at hERG and CYP2D6 while main-

taining good DOR affinity and selectivity. The favorable in

vitro profile of compound 30 also demonstrated that the N,N-

diethylpyrimidine-2-carboxamide was an effective replace-

ment of the N,N-diethylbenzamide group. The effect of sub-

stitution of the pendant phenyl ring of 5 was also studied

(compounds 31-36). As illustrated in Table 4, substitution of

the phenyl ring of the N,N-diethylbenzamide moiety of 5 with

a polar hydroxyl group (compounds 33 and 36) was beneficial

to decrease the hERG and CYP2D6 inhibitory activities while

maintaining potent affinity at the DOR and good selectivity

for δ versus μ and κ. As anticipated, replacement of the

hydroxyl group of 33 and 36 with more lipophilic methyl or

fluorine substituent (compounds 31, 32, 34, 35) led to a

significant increase in the hERG inhibitory activity. Having

thoroughly investigated the in vitro SAR of this spirocyclic

class of DOR agonists, several compounds were evaluated for

analgesic action in vivo. In particular, compounds 28 and 36

were tested in the rat Freund’s Complete Adjuvant (FCA)

mechanical hyperalgesia assay used as a model of inflamma-

tory pain.^53,54At the screening dose of 3 mg/kg po, com-

pound 28 produced only 11% reversal of hyperalgesia in the

inflamed paw (the paw pressure threshold of the inflamed paw

returned to that of the uninflamed paw). In contrast, under

the same assay conditions, compound 36 when dosed orally at

3 mg/kg produced 148% reversal of hyperalgesia in the

inflamed paw. The reason for the difference of in vivo effi-

cacy of compounds 28 and 36 is unknown. The oral potency

of 36 in the FCA assay (ED₅₀=0.03 mg/kg) was greater than

that of 4 (ED₅₀= 1.4 mg/kg)⁴⁶(Figure 1). The level of

it was not too surprising to note that the replacement of the

piperidine nitrogen of 5 with an oxygen atom, as in 21, led to a

dramatic decrease (770-fold) in the DOR binding affinity. As

shown in Table 3, the spiro[chroman-2,4⁰-piperidine] deriva-

tive 22, saturated analogue of 5, displayed potent affinity and

agonist activity at the DOR (K_i=1.6 nM; EC₅₀=21 nM).

Because 22 is racemic, the synthesis of enantiomers 23 and 24

was undertaken to see if additional potency at the DOR and

selectivity versus hERG and CYP2D6 were to be gained. As

indicated in Table 3, the R-isomer 23 was superior to the

S-isomer 24 in terms of binding affinity and agonist potency

at the DOR. Unfortunately, the R-isomer 23 was also a more

potent hERG inhibitor than the S-isomer 24 and the racemic 22.

On the basis of its unfavorable hERG profile, compound 23 was

not profiled further. We then studied the effect of replacing the

phenyl group of the N,N-diethylbenzamide moiety of 5, with

various heterocyclic systems (compounds 25-30, Table 4). In

particular, the medicinal chemistry strategy consisted in redu-

cing the overall lipophilicity of the spirocyclic derivatives in an

attempt to decrease simultaneously hERG and CYP2D6 in-

hibitory activities while maintaining good affinity and selecti-

vity for the DOR. As indicated in Table 4, the thiophene

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5693

Figure 1. Dose response effect of the antihyperalgesic activity

produced by 4 and 36 in the rat FCA assay. Values on the graph

represent the mean and standard error of the mean (SEM) using n =

8-24 (*: significantly different from appropriate vehicle group, p <

0.05).

Figure 2. Antihyperalgesia produced by 4, 36, indomethacin, and

morphine in the rat FCA assay. Values on the chart represent the

mean and standard error of the mean (SEM) using n = 8-11.

antihyperalgesia observed following 3 mg/kg po of 36 was

similar to that produced by 3 mg/kg po of 4 and 30 mg/kg sc of

the nonselective cyclooxygenase inhibitor indomethacin

(Figure 2). The antihyperalgesic efficacy of 36 was less than

that of morphine (Figure 2), which increased paw pressure

thresholds in both the inflamed and the uninflamed paw.

Additional studies showed that the antihyperalgesia produced

by 3 mg/kg po of 36 was prevented by sc pretreatment with 0.3

mg/kg of the DOR antagonist naltrindole (Figure 3), thus

demonstrating that the antihyperalgesic activity of 36 is

mediated through activation of DORs.

Compound 36 was also evaluated across a broad in vitro

selectivity panel that included adrenergic, muscarinic, and

nicotinic cholinergic, dopaminergic, peptidergic, and sero-

tonergic receptors, various ion channels, and enzymes.

These studies demonstrated that 36 was a selective DOR

agonist as it did not inhibit ligand binding to over 100

nonopioid receptors, channels, and enzymes at a concen-

tration of 10 μM.

Figure 3. Antagonism by naltrindole of the antihyperalgesia pro-

duced by 3 mg/kg po of compound 36 in the rat FCA assay. Values

on the graph represent the mean and standard error of the mean

(SEM) using n = 15-16 (*: significantly different from vehicle-36

treatment group, p<0.05).

Table 5. Pharmacokinetics of 36 in Male Sprague-Dawley Rats and

Male Beagle Dogs after iv and po Administration^a

iv

po

The pharmacokinetics of 36 after iv and po administration

to male rats are summarized in Table 5. Similar to the rat

pharmacokinetics of4, 36is a highclearance compound witha

large volume of distribution. The elimination of 36 was

moderate with a half-life of 5.3 h after a single iv dose of

2.5mg/kg. Followingivadministration, the systemic clearance

of 36 approximated rat hepatic blood flow, i.e., 3.3 L/h/kg.⁵⁵

After a 10 mg/kg po dose, maximum plasma concentrations

were reached within 1.3 h postdose. The oral bioavailability of

36 in rat was 37.6%. Table 5 also summarizes the pharmacoki-

netics of 36 in male beagle dogs after single iv and po doses of

1 and 3 mg/kg, respectively.

pharmacokinetic

species

rat

dog

rat

dog

dose (mg/kg)

CLs (L/h/kg)

2.5

3.3 ( 0.5

18.1 ( 3.7 5.1 ( 0.8

5.3 ( 0.1 12.2 ( 0.4

780 ( 125 2,637 ( 426 1152 ( 84 4203 ( 346

1.0

10

3.0

0.38 ( 0.06

V

_dss(L/kg)

t_1/2(h)^b

4.9 ( 0.2

7.7 ( 0.8

AUC_0-¥

(ng h/mL)

3

_max(h)

T

1.3 ( 0.6

246 ( 55

37.6 ( 2.7 54.5 ( 13.0

0.4 ( 0.1

852 ( 46

C_max(ng/mL)

F (%)

^aValues represent the mean ( standard deviation of 3 animals.

^bExpressed as harmonic mean.

Unlike the moderate half-life elimination of 4 (5.1 h), the

half-life of 36 in dog was long, 12.2 h, after a 1 mg/kg iv dose.

After a single 3 mg/kg oral dose (solution gavage) of 36,

maximum plasma concentrations were reached between 15

and 30 min postdose and ranged from 808 to 900 ng/mL. The

volume of distribution, 5.1 L/kg, was greater than total body

water (0.6L/kg in dog),⁵⁵indicatingtissue binding. Compared

with hepatic blood flow, approximately 1.9 L/h/kg in dog,⁵⁵

the systemic plasma clearance was low, i.e., 0.38 L/h/kg. The

oral bioavailability of 36 in dog was 54.5%.

The plasma protein binding of 36 was species-dependent,

with the least degree of binding in human plasma. The overall

mean free fraction in rat, dog, and human plasma was 21.5%,

28.9%, and 36.5%, respectively. Compound 36 was moder-

ately metabolized by rat and dog hepatic microsomes, with

59.7% and 63.9% remaining after a 30 min incubation,

respectively. Little metabolism was observed in human liver

microsomes (94.5% remaining after 30 min). Compound 36

(1 μM) was metabolized in vitro by human P450 isozymes

CYP3A4, CYP2C19, and CYP2D6, with 22.5, 78.4, and

63.2% remaining after a 1 h incubation, respectively. Com-

pound 36 showed very little inhibitory activity toward drug-

metabolizing CYP enzymes (CYP1A2: 0% inhibition at

10 μM; CYP2C19: 12.8% inhibition at 10 μM; CYP2C9:

11.4% inhibition at 10 μM; CYP2D6: 12.5% inhibition at

10 μM; CYP3A4: 18% inhibition at 10 μM). Additional

experiments were conducted to study the disposition of 36 in

plasma and brain microdialysates of freely moving rats after

5694 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

oral dosing. The concentrations of 36 were determined in

plasma of rats for approximately 6 h following a 30 mg/kg po

dose of 36. The corresponding concentrations in the brain

were determined by analysis of microdialysates from probes

placed into the raphe magnus. The area under the curve

(AUC) of compound 36 (total concentration) in plasma was

(TLC) was performed on silica gel 6F glass-backed plates

(250 μm) from Analtech and visualized by UV 254 irradiation

and iodine. Flash chromatography was conducted using the

ISCO CombiFlash with RediSep silica gel cartridges (4 g, 12 g,

40 g, 120 g). Chromatographic elution solvent systems

are reported as volume:volume ratios. All ¹H NMR spectra

were recorded at ambient temperature on a Bruker 400 MHz

spectrometer. They are reported in ppm on the δ scale from

TMS. LC-MS data were obtained using a Thermo-Finnigan

Surveyor HPLC and a Thermo-Finnigan AQA MS using either

positive or negative electrospray ionization. Program (positive):

solvent A, 10 mM ammonium acetate, pH 4.5, 1% acetonitrile;

solvent B, acetonitrile; column: Michrom Bioresources Magic

C18 Macro Bullet, detector (PDA λ=220-300 nm; gradient:

96%A-100%B in 3.2 min, hold 100%B for 0.4 min). Program

(negative): solvent A, 1 mM ammonium acetate, pH 4.5,

1% acetonitrile; solvent B, acetonitrile; column, Michrom

Bioresources Magic C18 Macro Bullet, detector (PDA λ =

220-300 nm; gradient: 96%A-100%B in 3.2 min, hold 100%

B for 0.4 min). Elemental analyses were performed by Atlantic

Microlabs, Norcross, GA and are within (0.4% of theoretical

values.

4611 ng h/mL. On the basis of the plasma protein binding of

36 determined in vitro (see above), the AUC of compound 36

3

(free concentration) in plasma was calculated to be 990 ng

h/mL. The AUC of 36 in the raphe magnus was 105 ng h/mL,

3

which indicates that 36 was able to penetrate the CNS. The

levels of 36 in the CNS were approximately 10% of the

corresponding free concentration of drug in plasma.

Compound 36 was also evaluated in vitro for the potential

to inhibit four other major cardiac ion channels related to

potassium, sodium, and calcium current generation

[I_to(rKv4.3), hKvLQT1/h min K, hHNa, and I_Ca,L]. The

studies were conducted on human embryonic kidney (HEK)

cells transfected with cDNAs for the ion channels of interest.

Compound 36 showed relatively little inhibition of these ion

channel currents. At 100 μM, the following average inhibition

was observed: hKvLQT1/h min K, 30.5%; hHNa, 10.2%;

I_Ca,L, 29.5%; I_to(rKv4.3), 10.3%. A bacterial mutagenicity

assay (Ames study) conducted with 36 indicated that this

compound was not likely to be mutagenic. A chromosomal

aberration study was also performed in human peripheral

blood lymphocytes. In this study, it was concluded that36 was

negative for the induction of structural and numerical chro-

mosome aberrations. To determine whether compound 36

produced sedation or behavioral activation in rats, the effect

of oral administration of 30, 100, or 300 mg/kg po of 36 on the

spontaneous locomotor activity of rats was assessed in loco-

motor activity chambers. No significant effect on horizontal

or vertical activity was observed after any dose (data not

shown). In contrast, sc administration of 1 mg/kg of 2

significantly increased horizontal and vertical locomotor

activity. The effects of 36 on EEG were investigated following

iv administration. In a telemetered rat preparation, 36 failed

to produce seizures or preseizure waveforms following bolus

administration of 10 and 30 mg/kg. No overt side effects of

compound 36 were identified. These data highlight a vastly

improved CNS safety margin for compound 36 compared

with 2.

N,N-Diethyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(5). A 2 N solution of hydrochloric acid in diethyl ether (34.6 mL,

69.24 mmol, 5.5 equiv) was added dropwise to a cooled (0 ꢀC)

solution of 40a (6.00 g, 12.59 mmol, 1.0 equiv) in anhydrous

dichloromethane (70 mL). The mixture was warmed to room

temperature, and stirring was continued for an additional 10 h.

Diethyl ether (100 mL) was added to the solution, and the

resulting precipitate was collected by filtration and washed with

diethyl ether. Yield: 99%. ¹H NMR (DMSO-d₆) δ 9.06 (m, 2H),

7.43 (s, 4H), 7.27 (t, 1H), 7.00 (m, 3H), 5.95 (s, 1H), 3.45 (m,

2H), 3.23 (m, 6H), 2.00 (m, 4H), 1.12 (m, 6H). LCMS (ESI): m/z

377.4 (M þ H^þ). HPLC purity: 100%. Anal. (C₂₄H₂₈N₂O₂

3

1HCl) C, H, N.

N-Ethyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(6). Compound 6 was synthesized in a similar manner to 7

using ethylamine in step c and 43a in step d. ¹H NMR

(DMSO-d₆) δ 8.50 (m, 1H), 7.90 (d, 2H), 7.40 (d, 2H), 7.20

(m, 1H), 6.90 (m, 3H), 5.85 (s, 1H), 3.30 (m, 2H), 2.90 (m, 2H),

2.70 (m, 2H), 1.85-1.70 (m, 4H), 1.10 (t, 3H). LCMS (ESI):

m/z 349.2 (M þ H^þ). HPLC purity: 99.8%. Anal. (C₂₂H₂₄

-

N₂O₂1HCl ¹/₄H₂O) C, H, N.

3

4-(Spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide (7). A 2 N

solution of hydrochloric acid in diethyl ether (0.12 mL, 0.24

mmol, 5.5 equiv) was added dropwise to a cooled (0 ꢀC) solution

of 43b (18 mg, 0.04 mmol, 1.0 equiv) in anhydrous methanol

(5 mL). The mixture was stirred overnight at room temperature

and then concentrated under reduced pressure. The crude

product was triturated with ethyl acetate. The resulting pre-

cipitate was collected by filtration. Yield: 70%. ¹H NMR

(DMSO-d₆) δ 8.99 (m, 2H), 8.06 (m, 1H), 7.95 (m, 2H), 7.46

(m, 3H), 7.27 (m, 1H), 7.06 (m, 1H), 6.96 (m, 2H), 5.95 (s, 1H),

3.24 (m, 4H), 2.08 (m, 4H). LCMS (ESI): m/z 321.1 (M þ H^þ).

HPLC purity: 95.6%.

Conclusion

In summary, further SAR exploration in this novel series of

DOR agonists led to the discovery of 36, a potent and selective

DOR agonist displaying a preclinical profile appropriate for

clinical evaluation. Increasing the polarity of the lead com-

pound 5 was beneficial to further decrease the hERG and

CYP2D6 liabilities while keeping potent affinity and selectiv-

ity for the DOR. Compound 36 demonstrated robust and

potent activity in a rodent assay of inflammatory pain follow-

ing oral administration. Indeed, 36 was approximately 50-fold

more potent than 4 in the rat FCA assay of inflammatory pain

after oral dosing. Moreover, preclinical studies suggested that

36 shared the wide safety margin observed previously for 4.

Compound 36 has been advanced to phase I safety evaluation

conducted in normal healthy volunteers.

N-Methyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(8). Compound 8 was synthesized in a similar manner to 7 using

methylamine in step c and 43c in step d. (DMSO-d₆) δ 9.05 (m,

2H), 8.55 (m, 1H), 7.92 (m, 2H), 7.41 (m, 2H), 7.26 (m, 1H), 7.06

(m, 1H), 6.95 (m, 2H), 5.95 (s, 1H), 3.20 (m, 4H), 2.81 (m, 3H),

2.08 (m, 4H). LCMS (ESI): m/z 335.2 (M þ H^þ). HPLC purity:

96.4%.

N-Isobutyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(9). Compound 9 was synthesized in a similar manner to 7 using

isobutylamine in step c and 43d in step d. ¹H NMR (CDCl₃) δ

9.75 (brs, 1H), 9.31 (brs, 1H), 7.81 (d, 2H), 7.39 (d, 2H), 7.21 (m,

1H), 6.98 (m, 2H), 6.90 (m, 1H), 6.25 (m, 1H), 5.56 (s, 1H), 3.46

(m, 2H), 3.33 (m, 4H), 2.30 (m, 2H), 2.12 (m, 2H), 1.94 (m, 1H),

1.04 (d, 6H). LCMS (ESI): m/z 377.2 (M þ H^þ). HPLC purity:

99.5%.

Experimental Section

1. Chemistry. General. All chemicals were reagent grade and

used without further purification. Thin-layer chromatography

Article

N,N-Diisopropyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benz-

amide (10). Compound 10 was synthesized in a similar manner to

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5695

1.35-1.07 (m, 6H). LCMS (ESI): m/z 391.2 (MþH^þ). HPLC

purity: 99%. Anal. (C₂₅H₃₀N₂O₂1HCl) C, H, N.

3

1

7 using N,N-diisopropylamine in step c and 43e in step d. H

N,N-Diethyl-4-(spiro[chromene-2,3⁰-pyrrolidine]-4-yl)benz-

amide (19). Compound 19 was synthesized in a similar manner

to 5 using 37c in step a, 38c in step b, 39c in step c, and 40c in

step d. ¹H NMR (CDCl₃) δ 10.20 (m, 2H), 7.40 (m, 4H), 7.22

(m, 1H), 7.04 (m, 2H), 6.91 (m, 1H), 5.66 (s, 1H), 3.85-3.50

(m, 5H), 3.31 (m, 3H), 2.60 (m, 1H), 2.13 (m, 1H), 1.27 (m,

3H), 1.16 (m, 3H). LCMS (ESI): m/z 363.2 (M þ H^þ). HPLC

purity: 99%.

NMR (DMSO-d₆) δ 8.98 (m, 2H), 7.39 (dd, 4H), 7.24 (m, 1H),

6.95 (m, 3H), 5.91 (s, 1H), 3.66 (brs, 2H), 3.22 (m, 4H), 2.10 (m,

4H), 1.30 (m, 12H). LCMS (ESI): m/z 405.3 (M þ H^þ). HPLC

purity: 99%. Anal. (C₂₆H₃₂N₂O₂1HCl ¹/₂H₂O) C, H, N.

3

N,N-Dimethyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(11). Compound 11 was synthesized in a similar manner to 7 using

N,N-dimethylamine in step c and 43f in step d. ¹H NMR (DMSO-

d₆) δ 9.08 (m, 2H), 7.42 (m, 4H), 7.24 (m, 1H), 7.00 (m, 3H), 5.91 (s,

1H), 3.25 (m, 4H), 2.96 (m, 6H), 2.07 (m, 4H). LCMS (ESI): m/z

349.1 (M þ H^þ). HPLC purity: 98.3%.

N,N-Diethyl-4-(spiro[chromene-2,3⁰-piperidine]-4-yl)benz-

amide (20). Compound 20 was synthesized in a similar

manner to 5 using 37d in step a, 38d in step b, 39d in step c,

1

Pyrrolidin-1-yl(4-(spiro[chromene-2,4⁰-piperidine]-4-yl)phe-

nyl)methanone (12). Compound 12 was synthesized in a simi-

lar manner to 7 using pyrrolidine in step c and 43g in step d. ¹H

NMR (DMSO-d₆) δ 8.91 (m, 2H), 7.58 (d, 2H), 7.41 (d, 2H),

7.25 (m, 1H), 7.00 (m, 3H), 5.92 (s, 1H), 3.49 (m, 2H), 3.41 (m,

2H), 3.24 (m, 4H), 2.09 (m, 2H), 2.00 (m, 2H), 1.84 (m, 4H).

LCMS (ESI): m/z 375.1 (M þ H^þ). HPLC purity: 95.3%.

Piperidin-1-yl(4-(spiro[chromene-2,4⁰-piperidine]-4-yl)phe-

nyl)methanone (13). Compound 13 was synthesized in a

similar manner to 7 using piperidine in step c and 43h in

and 40d in step d. H NMR (400 MHz, CDCl₃) δ 10.33 (m,

1H), 9.21 (m, 1H), 7.39 (m, 5H), 7.21 (m, 1H), 6.98 (m, 1H),

6.87 (m, 1H), 5.50 (s, 1H), 3.55 (m, 4H), 3.34 (m, 2H), 2.93 (m,

2H), 2.44 (m, 1H), 2.33 (m, 1H), 1.83 (m, 1H), 1.70 (m, 1H),

1.26 (m, 3H), 1.16 (m, 3H). LCMS (ESI): m/z 377.0 (M þ H^þ).

HPLC purity: 99%.

N,N-Diethyl-4-(2⁰,3⁰,5⁰,6⁰-tetrahydrospiro[chromene-2,4⁰-pyran]-

4-yl)benzamide (21). Compound 21 was synthesized in a similar

manner to 40a using 37e in step a, 38e in step b, and 39e in step c. ¹H

NMR (DMSO-d₆) δ 7.42 (d, 2H), 7.38 (d, 2H), 7.19 (m, 1H), 6.97

(m, 2H), 6.86 (m, 1H), 5.62 (s, 1H), 3.96(m, 2H), 3.79(m, 2H), 3.57

(brs, 2H), 3.32 (brs, 2H), 2.03 (d, 2H), 1.84 (m, 2H), 1.21 (brd, 6H).

LCMS (ESI): m/z 378.2 (M þ H^þ). HPLC purity: 97.2%.

N,N-Diethyl-4-(spiro[chroman-2,4⁰-piperidine]-4-yl)benzamide

(22). A solution of 5 (0.66 g, 1.75 mmol, 1.0 equiv) in anhydrous

methanol (13 mL) was hydrogenated at atmospheric pressure in

the presence of palladium hydroxide (0.120 g, 0.09 mmol, 0.05

equiv) for 10 h. The mixture was then filtered through celite. The

filtrate was concentrated and was hydrogenated at atmospheric

pressure in the presence of palladium hydroxide (0.120 g) for an

additional 10 h. The mixture was filtered through celite, and the

filtrate was concentrated to dryness under reduced pressure. To a

cold (0 ꢀC) solution of the resulting oil in anhydrous dichlor-

omethane was added dropwise a 2 N solution of anhydrous

hydrochloric acid in diethyl ether (5 mL). The mixture was then

stirred for 1 h at room temperature and concentrated under

reduced pressure. Diethyl ether was added. The resulting pre-

cipitate was collected by filtration and washed with diethyl ether

and ethyl acetate. Yield: 63%. ¹H NMR (DMSO-d₆) δ 9.15 (m,

2H), 7.30 (m, 4H), 7.10 (m, 1H), 6.90 (m, 1H), 6.75 (m, 1H), 6.60

(m, 1H), 4.20 (m, 1H), 3.40 (m, 3H), 3.20 (m, 4H), 3.00 (m, 1H),

2.15 (m, 1H), 1.95 (m, 5H), 1.05 (m, 6H). LCMS (ESI): m/z

1

step d. H NMR (DMSO-d₆) δ 8.90 (m, 2H), 7.44 (m, 4H),

7.26 (m, 1H), 7.00 (m, 3H), 5.91 (s, 1H), 3.59 (m, 2H), 3.21 (m,

6H), 2.09 (m, 2H), 1.99 (m, 2H), 1.55 (m, 6H). LCMS (ESI):

m/z 389.1 (M þ H^þ). HPLC purity: 96.9%.

Morpholino(4-(spiro[chromene-2,4⁰-piperidine]-4-yl)phenyl)-

methanone (14). Compound 14 was synthesized in a similar

manner to 7 using morpholine in step c and 43i in step d. H

1

NMR (DMSO-d₆) δ 8.91 (m, 2H), 7.46 (m, 4H), 7.26 (m, 1H),

7.01 (m, 3H), 5.94 (s, 1H), 3.61 (m, 6H), 3.35 (m, 2H), 3.21 (m,

4H), 2.09 (m, 2H), 1.98 (m, 2H). LCMS (ESI): m/z 391.1 (Mþ

H^þ). HPLC purity: 98.0%.

Isoindolin-2-yl(4-(spiro[chromene-2,4⁰-piperidine]-4-yl)phe-

nyl)methanone (15). Compound 15 was synthesized in a

similar manner to 7 using isoindoline in step c and 43j in

step d. ¹H NMR (DMSO-d₆) δ 8.90 (m, 2H), 7.70 (d, 2H), 7.50

(d, 2H), 7.40 (m, 1H), 7.30 (m, 4H), 7.00 (m, 3H), 5.95 (s, 1H),

4.90 (s, 2H), 4.80 (s, 2H), 3.30 (brm, 4H), 2.05 (m, 4H). LCMS

(ESI): m/z 423.1 (M þ H^þ). HPLC purity: 96.1%. Anal.

(C₂₈H₂₆N₂O₂1HCl 1H₂O) C, H, N.

3

N-Benzyl-N-methyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)-

benzamide (16). Compound 16 was synthesized in a similar

manner to 7 using N-methyl-1-phenylmethanamine in step c

1

379.1 (M þ H^þ). HPLC purity: 99.3%. Anal. (C₂₄H₃₀N₂O₂

and 43k in step d. H NMR (DMSO-d₆) δ 8.88 (m, 2H), 7.40

3

1HCl ³/₄H₂O) C, H, N.

(brm, 10H), 7.00 (m, 3H), 5.94 (s, 1H), 4.70 (m, 1H), 4.52 (m,

1H), 3.21 (m, 4H), 2.88 (m, 3H), 2.02 (m, 4H). LCMS (ESI): m/z

425.2 (M þ H^þ). HPLC purity: 95.5%. Anal. (C₂₈H₂₈N₂-

O₂1HCl ³/₅H₂O) C, H, N.

3

(S)-N,N-Diethyl-4-(spiro[chroman-2,4⁰-piperidine]-4-yl)benz-

amide (23). The enantiomers derived from 22 (racemic mixture)

(10 g, 24.10 mmol, 1.0 equiv) were separated by chiral HPLC:

Column: Chiralpak AD-H, 4.6 mm ꢀ 250 mm, 5 μ, Chiral

Technologies PN no. 19325; column temperature: room tem-

perature; detection: UV photo diode array, 200-300 nm, extract

at 275 nm; injection volume: 40 μL of 2 mg/mL sample in EtOH/

MeOH (80:20); flow: 1 mL/min; mobile phase: 85% solution A,

15% solution B; solution A: 0.1% diisopropylethylamine in

hexane (HPLC grade); solution B: 80% ethanol, 20% methanol

(both HPLC grade); run time: 25 min.; HPLC: Waters Alliance

2695 (system dwell volume is ∼350 μL); detector: Waters 996

(resolution: 4.8 nm, scan rate: 1 Hz). Yield: 40%. ¹H NMR

(DMSO-d₆) δ 9.12 (m, 2H), 7.28 (m, 4H), 7.14 (m, 1H), 6.90 (d,

1H), 6.79 (m, 1H), 6.63 (d, 1H), 4.25 (m, 1H), 3.44 (m, 3H),

3.24 (m, 4H), 2.96 (m, 1H), 2.18 (m, 1H), 1.97 (m, 5H), 1.10 (m,

6H). LCMS (ESI): m/z 379.4 (M þ H^þ). HPLC purity: 99.1%.

Anal. (C₂₄H₃₀N₂O₂1HCl ¹/₄H₂O) C, H, N. Chiral HPLC

3

2-Ethyl-6-(spiro[chromene-2,4⁰-piperidine]-4-yl)-3,4-dihydroi-

soquinolin-1(2H)-one (17). To a solution of 49 (0.150 g, 0.316

mmol, 1.0 equiv) in anhydrous methylene chloride (5 mL) at

0 ꢀC under a nitrogen atmosphere was added a 1 N solution of

anhydrous hydrochloric acid in diethyl ether (1.26 mL, 1.26

mmol, 4.0 equiv). The reaction was warmed to room tempera-

ture and stirred for 4 days at room temperature. Diethyl ether

was added (5 mL), and the resulting precipitate was collected by

1

filtration. Yield: 27%. H NMR (DMSO-d₆) δ 8.80 (brs, 2H),

7.92 (d, 1H), 7.29 (m, 3H), 7.05 (d, 1H), 6.97 (m, 2H), 5.94 (s,

1H), 3.54 (m, 4H), 3.23 (brm, 4H), 3.00 (t, 2H), 2.08 (brm,

2H), 1.97 (brm, 2H), 1.13 (t, 3H). LCMS (ESI): m/z 375.3 (M þ

H^þ). HPLC purity: 99.4%. Anal. (C₂₄H₂₆N₂O₂1HCl 1H₂O)

3

C, H, N.

N,N-Diethyl-4-(spiro[azepane-4,2⁰-chromene]-4⁰-yl)benzamide

(18). Compound 18 was synthesized in a similar manner to 5

using 37b in step a, 38b in step b, 39b in step c, and 40b in step d.

¹H NMR (CDCl₃) δ 9.76 (m, 2H), 7.41 (m, 2H), 7.36 (m, 2H),

7.20 (m, 1H), 7.00 (dd, 1H), 6.97 (dd, 1H), 6.88 (m, 1H), 5.63 (s,

1H), 3.68-3.23 (m, 8H), 2.50-2.23 (m, 4H), 2.02-1.82 (m, 2H),

3

method: t_R=11.914 min. (ee=100%). [R]_D²⁵=-63.59 (c. 0.01,

MeOH).

(R)-N,N-Diethyl-4-(spiro[chroman-2,4⁰-piperidine]-4-yl)benz-

amide (24). The enantiomers derived from 22 (racemic mixture)

(10 g, 24.10 mmol, 1.0 equiv) were separated by chiral HPLC:

5696 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

Column: Chiralpak AD-H, 4.6 mmꢀ250 mm, 5 μ, Chiral

Technologies PN no. 19325; column temperature: room tem-

perature; detection: UV photo diode array, 200-300 nm, extract

at 275 nm; injection volume: 40 μL of 2 mg/mL sample in EtOH/

MeOH (80:20); flow: 1 mL/min; mobile phase: 85% solution A,

15% solution B. Solution A: 0.1% diisopropylethylamine in

hexane (HPLC grade); solution B: 80% ethanol, 20% methanol

(both HPLC grade); run time: 25 min. HPLC: Waters Alliance

2695 (system dwell volume is ∼350 μL); detector: Waters 996

(resolution: 4.8 nm, scan rate: 1 Hz). Yield: 40%. ¹H NMR

(DMSO-d₆) δ 9.10 (m, 2H), 7.28 (m, 4H), 7.14 (m, 1H), 6.90 (d,

1H), 6.80 (m, 1H), 6.63 (d, 1H), 4.25 (m, 1H), 3.42 (m, 3H), 3.24

(m, 4H), 2.97 (m, 1H), 2.20 (m, 1H), 1.97 (m, 5H), 1.10 (m, 6H).

LCMS (ESI): m/z 379.4 (M þ H^þ). HPLC purity: 99.3%. Anal.

(C₂₄H₃₀N₂O₂1HCl ¹/₄H₂O) C, H, N. Chiral HPLC method:

the following exceptions: Step a did not require heating. Step d used

52d and the reaction was refluxed overnight; the crude product was

washed with water instead of a 0.5 N aqueous solution of hydro-

chloric acid. Step e went to completion without being resubjected to

anhydrous hydrochloric acid and the product was isolated as the

hydrochloride salt. ¹H NMR (DMSO-d₆) δ 8.94 (brm, 2H), 8.64 (s,

1H), 7.92 (dd, 1H), 7.65 (d, 1H), 7.29 (m, 2H), 7.05 (d, 1H), 6.96 (t,

1H), 6.22 (s, 1H), 3.48 (m, 2H), 3.24 (brm, 6H), 2.05 (brm, 4H), 1.14

(brd, 6H). LCMS (ESI): m/z 378.4 (M þ H^þ). HPLC purity: 99%.

Anal. (C₂₃H₂₇N₃O₂1HCl ⁴/₃H₂O) C, H, N.

3

N,N-Diethyl-5-(spiro[chromene-2,4⁰-piperidine]-4-yl)pyrimidine-

2-carboxamide (30). Compound 30 was synthesized in a similar

manner to 33 with the following exceptions: Step a was replaced

with steps b and c. Step d used 52e and the reaction was refluxed

overnight; the crude product was washed with water instead of a

0.5 N aqueous solution of hydrochloric acid. Step e went to

completion without being resubjected to anhydrous hydrochloric

3

t_R=8.64 min. (ee=97%). [R]_D²⁵=þ58.40 (c. 0.01, MeOH).

N,N-Diethyl-5-(spiro[chromene-2,4⁰-piperidine]-4-yl)thiophene-

2-carboxamide (25). Compound 25 was synthesized in a similar

manner to 33 with the following exceptions: step a did not require

heating. Step d used 52a and the reaction was refluxed overnight;

the crude product was washed with water instead of a 0.5 N

aqueous solution of hydrochloric acid. Step e went to completion

without being resubjected to anhydrous hydrochloric acid and

the final product was isolated as the hydrochloride salt. ¹H NMR

(DMSO-d₆) δ 9.07 (brs, 2H), 7.41 (d, 1H), 7.37 (d, 1H), 7.31 (t,

1H), 7.22 (d, 1H), 7.07 (d, 1H), 7.02 (t, 1H), 6.12 (s, 1H), 3.50

(brm, 4H), 3.21 (brm, 4H0, 2.03 (brm, 4H), 1.18 (brt, 6H). LCMS

(ESI): m/z 383.3 (M þ H^þ). HPLC purity: 99%. Anal.

1

acid, and the product was isolated as the hydrochloride salt. H

NMR (DMSO-d₆) δ 8.81 (m, 2H), 7.18 (m, 1H), 6.92 (m, 2H), 6.85

(m, 1H), 6.06 (s, 0.8H), 6.04 (s, 0.2H), 3.41 (q, 2H), 3.06 (q, 2H),

2.86 (m, 2H), 2.76 (m, 2H), 1.73 (brm, 4H), 1.10 (t, 3H), 1.00 (t,

3H). LCMS (ESI): m/z 379.3 (M þ H^þ). HPLC purity: 99.1%.

N,N-Diethyl-2-methyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)-

benzamide (31). Compound 31 was synthesized in a similar

manner to 33 with the following exceptions: Step a did not require

heating. Step d used 52f and the reaction was refluxed overnight;

the crude product was washed with water instead of a 0.5 N

aqueous solution of hydrochloric acid. Step e went to completion

without being resubjected to anhydrous hydrochloric acid and

(C₂₂H₂₆N₂O₂S 1HCl) C, H, N.

3

1

N,N-Diethyl-5-(spiro[chromene-2,4⁰-piperidine]-4-yl)furan-

2-carboxamide (26). Compound 26 was synthesized in a

similar manner to 33 with the following exceptions: Step a

did not require heating. Step d used 52b, and the reaction was

refluxed overnight; the crude product was washed with water

instead of a 0.5 N aqueous solution of hydrochloric acid. Step

e went to completion without being resubjected to anhydrous

hydrochloric acid, and the product was isolated as the

the product was isolated as the hydrochloride salt. H NMR

(CDCl₃) δ 9.76 (brs, 1H), 9.63 (brs, 1H), 7.20 (m, 4H), 7.05 (dd,

1H), 6.93 (m, 2H), 5.60 (s, 1H), 3.76 (brs, 2H), 3.42 (brm, 4H),

3.18(q, 2H), 2.32(s, 3H), 2.21 (brm, 4H), 1.28(t, 3H), 1.08 (t, 3H).

LCMS (ESI): m/z 391.0 (M þ H^þ). HPLC purity: 100%. Anal.

(C₂₅H₃₀N₂O₂1HCl) C, H, N.

3

N,N-Diethyl-2-fluoro-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)-

benzamide (32). Compound 32 was synthesized in a similar

manner to 33 with the following exceptions: Step a did not

require heating. Step d used 52g and the reaction was refluxed

overnight; the crude product was washed with water instead of a

0.5 N aqueous solution of hydrochloric acid. Step e went to

completion without being resubjected to anhydrous hydrochlo-

ric acid and the product was isolated as the hydrochloride salt.

¹H NMR (DMSO-d₆) δ 8.85 (brs, 2H), 7.43 (t, 1H), 7.35 (d, 1H),

7.27 (m, 2H), 7.04 (m, 2H), 6.97 (m, 1H), 6.03 (s, 1H), 3.48 (q,

2H), 3.22 (brm, 6H), 2.04 (brm, 4H), 1.16 (t, 3H), 1.04 (t, 3H).

LCMS (ESI): m/z 395.0 (M þ Hþ). HPLC purity: 100%. Anal.

(C₂₄H₂₇FN₂O₂1HCl ¹/₄H₂O) C, H, N.

1

hydrochloride salt. H NMR (400 MHz, DMSO-d₆) δ 9.05

(brs, 2H), 7.52 (d, 1H), 7.32 (t, 1H), 7.07 (brm, 3H), 6.91 (d,

1H), 6.26 (s, 1H), 3.50 (brs, 4H), 3.20 (brm, 4H), 2.05 (brm,

4H), 1.17 (brs, 6H). LCMS (ESI): m/z 367.3 (M þ H^þ). HPLC

purity: 99%.

N,N-Diethyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)thiophene-

2-carboxamide (27). Compound 27 was synthesized in a similar

manner to 33 with the following exceptions: Step a did not require

heating. Step d used 52c and the reaction was refluxed overnight;

the crude product was washed with water instead of a 0.5 N

aqueous solution of hydrochloric acid. Step e went to completion

without being resubjected to anhydrous hydrochloric acid, and

3

N,N-Diethyl-2-hydroxy-4-(spiro[chromene-2,4⁰-piperidine]-

4-yl)benzamide (33). To a solution of 51h (0.20 g, 0.406 mmol, 1.0

equiv) in methylene chloride (2 mL) was added a 1 N solution of

anhydrous hydrochloric acid in diethyl ether (10 mL, 10 mmol, 25

equiv). The mixture was stirred at room temperature for 16 h. The

mixture was concentrated under reduced pressure and treated with

diethyl ether. The resulting precipitate was collected by filtration.

By LC/MS some starting material remained, so the precipitate was

treated with an excess of a 4 N solution of anhydrous hydrochloric

acid in dioxane. This mixture was stirred at room temperature for

16 h. The mixture was concentrated under reduced pressure and

the crude product was purified by column chromatography

(eluent: methylene chloride/methanol mixtures of increasing

1

the product was isolated as the hydrochloride salt. H NMR

(DMSO-d₆) δ 9.01 (brs, 2H), 7.80 (s, 1H), 7.41 (s, 1H), 7.27 (t,

1H), 7.19 (d, 1H), 7.04 (d, 1H), 6.99 (t, 1H), 6.04 (s, 1H), 3.48

(brm, 4H), 3.21 (brm, 4H), 2.02 (brm, 4H), 1.16 (brt, 6H). LCMS

(ESI): m/z 383.4 (M þ H^þ). HPLC purity: 98.9%.

N,N-Diethyl-5-(spiro[chromene-2,4⁰-piperidine]-4-yl)picolinamide

(28). To a cold (0 ꢀC) solution of 58 (2 g, 4.18 mmol, 1.0 equiv) in

anhydrous dichloromethane (20 mL) was slowly added a 4.0 M

solution of hydrogen chloride in dioxane (5.2 mL, 20.8 mmol, 5.0

equiv). The reaction mixture was stirred at room temperature for 10

h and then concentrated under reduced pressure. The resulting

foamy solids were soaked in diethyl ether to give the fine powders,

which were collected by filtration and washed sequentially with

ethyl acetate and diethyl ether. Yield: 95%. ¹H NMR (DMSO-d₆) δ

8.99 (m, 2H), 8.60 (d, 1H), 7.90 (dd, 1H), 7.61 (d, 1H), 7.29 (m, 1H),

7.06 (d, 1H), 6.98 (m, 2H), 6.09 (s, 1H), 3.47 (q, 2H), 3.35-3.13 (m,

6H), 2.06 (m, 4H), 1.17 (t, 3H), 1.11 (t, 3H). LCMS (ESI): m/z

1

polarity). Yield: 66%. H NMR (DMSO-d₆) δ 9.91 (brs, 1H),

9.08 (brs, 2H), 7.26 (m, 1H), 7.13 (d, 1H), 7.04 (m, 2H), 6.95 (m,

1H), 6.84 (m, 2H), 5.87 (s, 1H), 3.66 (brs, 4H), 3.20 (brm, 4H), 2.05

(brm, 4H), 1.08 (brd, 6H). LCMS (ESI): m/z 393.4 (M þ H^þ).

HPLC purity: 99%. Anal. (C₂₄H₂₈N₂O₃1HCl ³/₂H₂O) C, H, N.

3

378.4 (M þ H^þ). HPLC purity: 99%. Anal. (C₂₃H₂₇N₃O₂

N,N-Diethyl-6-(spiro[chromene-2,4⁰-piperidine]-4-yl)nicotinamide

(29). Compound 29 was synthesized in a similar manner to 33 with

N,N-Diethyl-3-methyl-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)-

benzamide (34). Compound 34 was synthesized in a similar

manner to 33 with the following exceptions: Step a did not require

heating. Step d used 52i and the reaction was refluxed overnight;

3

2HCl ¹/₂H₂O) C, H, N.

3

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5697

the crude product was washed with water instead of a 0.5 N

aqueous solution of hydrochloric acid. Step e went to completion

without being resubjected to anhydrous hydrochloric acid and

tert-Butyl 4-(4-(Diethylcarbamoyl)phenyl)spiro[chromene-2,4⁰-

piperidine]-1⁰-carboxylate (40a). To a solution of 39a (15 g, 33.37

mmol, 1.0 equiv) in dimethoxyethane (100 mL) was added

sequentially a 2 N aqueous solution of sodium carbonate

(50.06 mL, 100.12 mmol, 3.0 equiv), lithium chloride (4.24 g,

100.12 mmol, 3.0 equiv), 4-(N,N-diethylaminocarbonyl)phenyl-

boronic acid (8.12 g, 36.71 mmol, 1.1 equiv), and tetrakis-

(triphenylphosphine)palladium(0) (0.77 g, 0.67 mmol, 0.02

equiv). The mixture was refluxed for 10 h under nitrogen.

The mixture was then cooled to room temperature, and water

(250 mL) was added. The mixture was extracted with ethyl

acetate. The organic layer was further washed with brine and

dried over sodium sulfate. The crude product was purified by

column chromatography (eluent: hexane/ethyl acetate mixtures

of increasing polarity). Yield: 73%. ¹H NMR (CDCl₃) δ 7.35 (m,

4H), 7.15 (t, 1H), 7.00-6.80 (m, 3H), 5.55 (s, 1H), 3.85 (m, 2H),

3.55 (m, 2H), 3.30 (m, 4H), 2.00 (m, 2H), 1.65 (m, 2H), 1.40 (s,

9H); 1.20 (m, 6H). LCMS (ESI): m/z 477.2 (M þ H^þ).

1

the product was isolated as the hydrochloride salt. H NMR

(CDCl₃) δ 9.78 (brs, 1H), 9.62 (brs, 1H), 7.22 (m, 3H), 7.13 (d,

1H), 6.92 (d, 1H), 6.84 (t, 1H), 6.63 (dd, 1H), 5.48 (s, 1H), 3.42

(brm, 8H), 2.36 (brm, 2H), 2.21 (m, 2H), 2.13 (s, 3H), 1.21 (brd,

6H). LCMS (ESI): m/z 391.0 (M þ H^þ). HPLC purity: 100%.

Anal. (C₂₅H₃₀N₂O₂1HCl) C, H, N.

3

N,N-Diethyl-3-fluoro-4-(spiro[chromene-2,4⁰-piperidine]-4-yl)-

benzamide (35). Compound 35 was synthesized in a similar

manner to 33 with the following exceptions: Step a did not

require heating. Step d used 52j and the reaction was refluxed

overnight; the crude product was washed with water instead of a

0.5 N aqueous solution of hydrochloric acid. Step e went to

completion without being resubjected to anhydrous hydrochlo-

ric acid and the product was isolated as the hydrochloride salt.

¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (brs, 2H), 7.29 (m, 3H),

7.13 (s, 1H), 7.05 (d, 1H), 6.98 (m, 2H), 6.01 (s, 1H), 3.43 (brm,

2H), 3.23 (brm, 6H), 2.04 (brm, 4H), 1.10 (brd, 6H). LCMS

(ESI): m/z 395.0 (M þ H^þ). HPLC purity: 99%. Anal. (C₂₄H₂₇-

FN₂O₂1HCl ¹/₄H₂O) C, H, N.

tert-Butyl 4-(4-(Methoxycarbonyl)phenyl)spiro[chromene-2,4⁰-

piperidine]-1⁰-carboxylate (41). To a solution of 39a (7.80 g, 17.35

mmol, 1.0 equiv) in dimethoxyethane (75 mL) was added

sequentially a 2 N aqueous solution of sodium carbonate

(26.03 mL, 52.06 mmol, 3.0 equiv), lithium chloride (2.21 g,

52.06 mmol, 3.0 equiv), 4-(methoxycarbonyl)phenylboronic acid

(3.44 g, 19.09 mmol, 1.1 equiv), and tetrakis(triphenylphos-

phine)palladium(0) (0.40 g, 0.35 mmol, 0.02 equiv). The mixture

was refluxed overnight under nitrogen. The mixture was then

cooled to room temperature, and water (250 mL) was added. The

mixture was extracted with ethyl acetate. The organic layer was

further washed with brine and dried over sodium sulfate. The

crude product was purified by column chromatography (eluent:

hexane/ethyl acetate mixtures of increasing polarity). Yield:

3

N,N-Diethyl-3-hydroxy-4-(spiro[chromene-2,4⁰-piperidine]-

4-yl)benzamide (36). To a solution of 63 (0.647 g, 1.21 mmol, 1

equiv) in methanol (3 mL) was added an excess of a 4 N

solution of anhydrous hydrochloric acid in dioxane (20 mL).

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

mixture was concentrated under reduced pressure and treated

with a mixture of methylene chloride (15 mL) and ethyl

acetate (25 mL). The resulting precipitate was collected by

filtration and dried under vacuum. Yield: 77%. ¹H NMR

(DMSO-d₆) δ 9.75 (s, 1H), 8.84 (brm, 2H), 7.16 (m, 2H), 6.96

(d, 1H), 6.84 (m, 3H), 6.72 (d, 1H), 5.78 (s, 1H), 3.42 (brs, 2H),

3.22 (brs, 6H), 2.10 (brm, 2H), 1.96 (brm, 2H), 1.12 (brs,

6H). LCMS (ESI): m/z 393.3 (M þ H^þ). HPLC purity: 99.3%.

Anal. (C₂₄H₂₈N₂O₃1HCl ¹/₄H₂O) C, H, N.

1

64%. H NMR (DMSO-d₆) δ 8.02 (d, 2H), 7.49 (d, 2H), 7.23

(m, 1H), 6.99 (d, 1H), 6.92 (m, 2H), 5.92 (s, 1H), 3.88 (s, 3H), 3.70

(m, 2H), 3.27 (m, 2H), 1.89 (m, 2H), 1.71 (m, 2H), 1.42 (s, 9H).

LCMS (ESI): m/z 436.0 (M þ H^þ).

3

4-(1⁰-(tert-Butoxycarbonyl)spiro[chromene-2,4⁰-piperidine]-

4-yl)benzoic Acid (42). A solution of 41 (4.71 g, 10.81 mmol,

1.0 equiv) in tetrahydrofuran (30 mL) at 0 ꢀC under nitrogen

was added dropwise to a solution of lithium hydroxide

monohydrate (0.54 g, 12.98 mmol, 1.2 equiv) in water (30

mL). The mixture was stirred overnight at room temperature.

The mixture was then concentrated under reduced pressure.

Water was added to the residue. The mixture was then

acidified to pH 2 using concentrated hydrochloric acid. The

resulting precipitate was collected by filtration, and the crude

product was used for the next step without further purifica-

tion. Yield: 98%. ¹H NMR (DMSO-d₆) δ 13.03 (brs, 1H), 8.01

(d, 2H), 7.47 (d, 2H), 7.23 (m, 1H), 6.98 (d, 1H), 6.92 (m, 2H),

5.91 (s, 1H), 3.70 (m, 2H), 3.28 (m, 2H), 1.86 (m, 2H), 1.72 (m,

2H), 1.42 (s, 9H). LCMS (ESI): m/z 420.1 (M - H^-).

tert-Butyl 4-Oxospiro[chroman-2,4⁰-piperidine]-1⁰-carboxylate

(38a). Pyrrolidine (42 mL, 0.5 mol, 2.0 equiv) was added drop-

wise at room temperature to a solution of 37a (49.8 g, 0.249 mol,

1.0 equiv) and 2-hydroxyacetophenone (34 g, 0.249 mol, 1.0

equiv) in anhydrous methanol (400 mL). The solution was

refluxed overnight and then concentrated under reduced pres-

sure. Diethyl ether (500 mL) was added. The organic mixture was

washed with a 1 N aqueous solution of hydrochloric acid, a 1 N

aqueous solution of sodium hydroxide, brine, and dried over

sodium sulfate. Hexane (300 mL) was added to the mixture. The

resulting precipitate was collected by filtration, washed with

hexane, and used for the next step without further purification.

Yield: 72%. ¹H NMR (CDCl₃) δ 7.86 (d, 1H), 7.50 (t, 1H), 7.00

(m, 2H), 3.87 (m, 2H), 3.22 (m, 2H), 2.72 (s, 2H), 2.05 (d, 2H),

1.61 (m, 2H), 1.46 (s, 9H). LCMS (ESI): m/z 318.0 (M þ H^þ).

tert-Butyl 4-(Trifluoromethylsulfonyloxy)spiro[chromene-2,4⁰-

piperidine]-1⁰-carboxylate (39a). To a solution of 38a (25 g, 0.078

mol, 1.0 equiv) in tetrahydrofuran (250 mL) at -78 ꢀC under

nitrogen was added dropwise a 1 N solution of lithium bis-

(trimethylsilyl)amide in tetrahydrofuran (94.5 mL, 0.095 mol, 1.2

equiv). The mixture was stirred for 1 h at -78 ꢀC. A solution of

N-phenylbis(trifluoromethanesulfonamide) (33.8 g, 0.095 mol,

1.2 equiv) in tetrahydrofuran (150 mL) was added dropwise. The

mixture was warmed slowly to room temperature, and stirring

was continued for a further 12 h. The mixture was then poured

into ice water, and the two phases were separated. The organic

phase was washed with a 1 N aqueous solution of hydrochloric

acid, a 1 N aqueous solution of sodium hydroxide, brine, and

dried over sodium sulfate. The crude product was purified by

column chromatography (eluent: hexane/ethyl acetate mixtures

of increasing polarity). Yield: 70%. ¹H NMR (DMSO-d₆) δ

7.45-7.20 (m, 2H), 7.00 (m, 2H), 6.15 (s, 1H), 3.70 (m, 2H), 3.20

(m, 2H), 1.90 (m, 2H), 1.75 (m, 2H), 1.40 (s, 9H). LCMS (ESI):

m/z 450.1 (M þ H^þ).

tert-Butyl 4-(4-Carbamoylphenyl)spiro[chromene-2,4⁰-piperidine]-

1⁰-carboxylate (43b). O-Benzotriazol-1-yl-N,N,N⁰,N⁰-tetramethy-

luronium tetrafluoroborate (150.8 mg, 0.47 mmol, 1.1 equiv) was

added to a cooled (0 ꢀC) solution of 42 (180.0 mg, 0.43 mmol, 1.0

equiv), ammonium chloride (50.3 mg, 0.94 mmol, 2.2 equiv), and N,

N-diisopropylethylamine (0.25 mL, 0.94 mmol, 2.2 equiv) in acet-

onitrile (5 mL). The solution was stirred overnight at room tem-

perature and then concentrated under reduced pressure. Ethyl

acetate (10 mL) and a saturated aqueous solution of sodium

bicarbonate (10 mL) were added to the crude product, and the

mixture was stirred for 20 min at room temperature. The phases

were separated and the organic layer was separated, washed with a

saturated aqueous solution of sodium bicarbonate, brine, dried over

sodium sulfate, and filtered. The organics were concentrated under

reduced pressure, and the crude product was purified by column

chromatography (eluent: hexane/ethyl acetate mixtures of increas-

ing polarity). Yield: 10%. LCMS (ESI): m/z 421.2 (MþH^þ).

2-Ethyl-6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (45).

To a suspension of NaH (0.81 g, 33.86 mmol, 6.0 equiv) in

5698 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

tetrahydrofuran (30 mL) under a nitrogen atmosphere was

added dropwise a solution of 44 (1.0 g, 5.64 mmol, 1.0 equiv)

in tetrahydrofuran (15 mL). To this mixture was added

dropwise ethyliodide (2.28 mL, 28.22 mmol, 5.0 equiv), and

stirring was continued for 16 h at room temperature. A thick

precipitate formed; therefore, additional amounts of tetra-

hydrofuran (15 mL) and ethyliodide (1.0 mL, 12.4 mmol, 2.2

equiv) were added and stirring was continued for an addi-

tional 24 h at room temperature. The reaction was quenched

by addition of a 1 N aqueous solution of hydrochloric acid

followed by ethyl acetate and water. The layers were sepa-

rated. The organic phase was dried over sodium sulfate,

filtered, and concentrated under reduced pressure. The crude

product was purified by column chromatography (eluent:

hexane/ethyl acetate mixtures of increasing polarity). Yield:

83%. ¹H NMR (CDCl₃) δ 8.03 (d, 1H), 6.84 (dd, 1H), 6.65 (d,

1H), 3.84 (s, 3H), 3.61 (q, 2H), 3.53 (t, 2H), 2.95 (t, 2H), 1.21

(t, 3H). LCMS (ESI): m/z 206.1 (MþH^þ).

ing polarity). Yield: 96%. ¹HNMR(CDCl₃) δ7.71 (d, 1H), 7.11 (t,

1H), 6.90 (t, 1H), 6.83 (d, 1H), 6.28 (s, 1H), 3.84 (brs, 2H), 3.27

(brm, 2H), 1.96 (d, 2H), 1.60 (m, 2H), 1.34 (s, 9H), 1.26 (s, 12H).

LCMS (ESI): m/z 428.0 (M þ H^þ).

tert-Butyl 4-(2-Ethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-

spiro[chromene-2,4⁰-piperidine]-1⁰-carboxylate (49). To a solution

of 47 (0.100 g, 0.309 mmol, 1.0 equiv) in N,N-dimethylformamide

(5 mL) under a nitrogen atmosphere was added 48 (0.145 g, 0.340

mmol, 1.1 equiv), potassium acetate (0.091 g, 0.928 mmol, 3.0

equiv) and 1,1⁰-bis(diphenylphosphino)ferrocene palladium(II)/

dichloromethane complex (0.005 g, 0.006 mmol, 0.02 equiv). The

reaction was stirred at 65 ꢀC for 16 h. The mixture was cooled to

room temperature. Water was added, and the mixture was

extracted with ethyl acetate. The organic phase was dried over

sodium sulfate, filtered, and concentrated under reduced pressure.

The crude product was purified by column chromatography

(eluent: hexane/ethyl acetate mixtures of increasing polarity).

1

Yield: 45%. H NMR (CDCl₃) δ 8.11 (d, 1H), 7.31 (dd, 1H),

2-Ethyl-6-hydroxy-3,4-dihydroisoquinolin-1(2H)-one (46). To

a solution of 45 (0.96 g, 4.68 mmol, 1.0 equiv) in anhydrous

methylene chloride (30 mL) at -78 ꢀC under a nitrogen atmo-

sphere was added dropwise a 1 N solution of boron tribromide

in methylene chloride (9.35 mL, 9.35 mmol, 2.0 equiv). The

reaction was warmed to room temperature, and stirring was

continued for 16 h at room temperature. The mixture was cooled

in an ice bath, quenched with methanol (10 mL), and concen-

trated under reduced pressure. The crude mixture was dissolved

in ethyl acetate, and the solution was washed with a 1 N aqueous

solution of hydrochloric acid and brine. The organic phase was

dried over sodium sulfate, filtered, and concentrated under

reduced pressure. The crude solid was triturated in ethyl acet-

ate/hexane (1:1). The precipitate was collected by filtration.

7.19 (m, 1H), 7.15 (s, 1H), 6.96 (m, 2H), 6.86 (m, 1H), 5.58 (s, 1H),

3.86 (brm, 2H), 3.65 (q, 2H), 3.59 (t, 2H), 3.34 (m, 2H), 3.01 (t,

2H), 2.05 (m, 2H), 1.67 (m, 2H), 1.48 (s, 9H), 1.26 (t, 3H). LCMS

(ESI): m/z 475.3 (M þ H^þ).

4-((S)-1⁰-(((1S,4R)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)-

methylsulfonyl)spiro[chroman-2,4⁰-piperidine]-4-yl)-N,N-diethyl-

benzamide (50). ((1S,4R)-7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-

1-yl)methanesulfonyl chloride (0.45 g, 1.78 mmol, 1.1 equiv) was

added at 0 ꢀC to a solution of 24 (0.67 g, 1.61 mmol, 1 equiv) and

triethylamine (0.74 mL, 5.33 mmol, 3.3 equiv) in dichloromethane

(6 mL). The reaction was warmed to room temperature and stirred

overnight at room temperature. The mixture was washed with a

saturated aqueous solution of sodium bicarbonate and brine, dried

over sodium sulfate, filtered, and concentrated under reduced

pressure. The crude product was purified by column chromatog-

raphy (eluent: hexane/ethyl acetate mixtures of increasing polarity).

Yield: 64%. ¹H NMR(DMSO-d₆) δ7.30 (m, 4H), 7.11 (t, 1H), 6.90

(d, 1H), 6.77 (t, 1H), 6.61 (d, 1H), 4.23 (m, 1H), 3.39 (brm, 9H), 2.93

(d, 1H), 2.37(m, 2H), 2.24(m, 1H), 2.06 (m, 2H), 1.93 (m, 6H), 1.53

(m, 1H), 1.41 (m, 1H), 1.10 (m, 6H), 1.03 (s, 3H), 0.83 (s, 3H).

1

Yield: 74%. H NMR (CDCl₃) δ 7.89 (d, 1H), 6.82 (dd, 1H),

6.68 (d, 1H), 3.63 (q, 2H), 3.54 (t, 2H), 2.91 (t, 2H), 1.22 (t, 3H).

LCMS (ESI): m/z 192.1 (M þ H^þ).

2-Ethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl Trifluorometha-

nesulfonate (47). To a solution of 46 (0.38 g, 1.99 mmol, 1.0 equiv)

and pyridine (0.32 mL, 3.98 mmol, 2.0 equiv) in methylene chloride

(10 mL) at 0 ꢀC under a nitrogen atmosphere was added trifluor-

omethanesulfonic anhydride (0.40 mL, 2.38 mmol, 1.2 equiv). The

reaction was warmed to room temperature, and stirring was

continued for 2 h at room temperature. Methylene chloride was

added to the mixture, which was washed with a 1 N aqueous

solution of hydrochloric acid and with a 1 N aqueous solution of

sodium hydroxide. The organic phase was dried over sodium

sulfate, filtered, and concentrated under reduced pressure. The

crude product was purified by column chromatography (eluent:

hexane/ethyl acetate, 1:1). Yield: 45%.

LCMS (ESI): m/z 593.4 (M þ H^þ). Anal. (C₃₃H₄₄N₂O₅S ¹/₄H₂O)

3

C, H, N.

tert-Butyl 4-(4-(Diethylcarbamoyl)-3-hydroxyphenyl)spiro[chro-

mene-2,4⁰-piperidine]-1⁰-carboxylate (51h). To a solution of 52h

(0.30 g, 1.11 mmol, 1.0 equiv) in dimethoxyethane (10 mL) was

added sequentially a 2 N aqueous solution of sodium carbonate

(1.66 mL, 3.32 mmol, 3.0 equiv), lithium chloride (0.141 g, 3.32

mmol, 3.0 equiv), 48 (0.57 g, 1.33 mmol, 1.2 equiv), and tetrakis-

(triphenylphosphine)palladium(0) (0.128 g, 0.11 mmol, 0.1 equiv).

The reaction was conducted under microwave conditions (A: 25 to

170 ꢀC for 10 min; B: 170 ꢀC for 10 min). The crude mixture was

dissolved in ethyl acetate. The mixture was washed with a 0.5 N

aqueous solution of hydrochloric acid, brine, and dried over

magnesium sulfate. The mixture was filtered and the filtrate was

concentrated under reduced pressure. The crude product was

purified by column chromatography (eluent: hexane/ethyl acetate

mixtures of increasing polarity). Yield: 37%. ¹H NMR (CDCl₃) δ

9.94 (s, 1H), 7.29 (d, 1H), 7.18 (m, 1H), 7.06 (dd, 1H), 7.00 (d, 1H),

6.94 (d, 1H), 6.85 (m, 2H), 5.59 (s, 1H), 3.85 (brs, 2H), 3.55 (q, 4H),

3.34 (brs, 2H), 2.04 (brm, 2H), 1.66 (m, 2H), 1.48 (s, 9H), 1.30 (t,

6H). LCMS (ESI): m/z 493.2 (M þ H^þ).

¹H NMR (CDCl₃) δ 8.18 (d, 1H), 7.23 (dd, 1H), 7.11 (d, 1H),

3.62 (m, 4H), 3.04 (t, 2H), 1.23 (t, 3H). CDCl₃) δ 7.89 (d, 1H),

6.82 (dd, 1H), 6.68 (d, 1H), 3.63 (q, 2H), 3.54 (t, 2H), 2.91 (t, 2H),

1.22 (t, 3H). LCMS (ESI): m/z 324.1 (M þ H^þ).

tert-Butyl 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)spiro-

[chromene-2,4⁰-piperidine]-1⁰-carboxylate (48). To a solution of

bis(pinacolato)diboron (14.7 g, 57.8 mmol, 2.0 equiv) in N,N-

dimethylformamide (200 mL) at room temperature under a nitro-

gen atmosphere was added 1,1⁰-bis(diphenylphosphino)ferrocene

palladium(II) chloride complex with dichloromethane (710 mg,

0.867 mmol, 0.03 equiv) followed by addition of potassium acetate

(8.58 g, 86.7 mmol, 3.0 equiv.) The mixture was heated to 80 ꢀC,

followed by dropwise addition of a solution of 39a (13.0 g, 28.9

mmol, 1.0 equiv) in N,N-dimethylformamide (100 mL). After the

addition was complete, the reaction mixture was heated at 80 ꢀC

for an additional 16 h. The solvent was evaporated under vacuum,

and the residue was added to a 1 N aqueous solution of hydro-

chloric acid. The aqueous residue was extracted with ethyl acetate.

The organic extracts were washed with brine, dried over sodium

sulfate, filtered, and concentrated under reduced pressure to afford

a brown semisolid. The crude product was purified by column

chromatography (eluent: hexane/ethyl acetate mixtures of increas-

5-Bromo-N,N-diethylpyrimidine-2-carboxamide (52e). To a

solution of 54 (0.055 g, 0.27 mmol, 1.0 equiv) in methylene

chloride (5 mL) was added oxalyl chloride (0.050 mL, 0.58

mmol, 2.1 equiv). The mixture was refluxed for 1 h and

concentrated under reduced pressure to give the crude acyl

chloride. To a solution of the crude acyl chloride (0.060 g, 0.27

mmol, 1.0 equiv) in tetrahydrofuran (2.5 mL) was added N,N-

diethylamine (0.11 mL, 1.06 mmol, 4.0 equiv). The mixture was

stirred for 16 h and then diluted with ethyl acetate. The organic

mixture was washed with water, with a saturated aqueous

solution of sodium bicarbonate, a 1 N aqueous solution of

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5699

hydrochloric acid, and brine. The organic mixture was dried

over sodium sulfate, filtered, concentrated under reduced

pressure, and the crude product was used for the next step

without further purification. Note: the product was isolated

with a 17% impurity corresponding to N,N-diethyl-2-iodo-

pyrimidine-5-carboxamide. Yield: 86%. ¹H NMR (CDCl₃)

δ 8.82 (s, 2H), 3.56 (q, 2H), 3.20 (q, 2H), 1.28 (t, 3H), 1.18

(t, 3H).

4-Bromo-N,N-diethyl-2-hydroxybenzamide (52h). To a mix-

ture of diethylamine (0.85 g, 11.58 mmol, 2.5 equiv), O-benzo-

triazol-1-yl-N,N,N⁰,N⁰-tetramethyluronium tetrafluoroborate

(TBTU) (1.93 g, 6.02 mmol, 1.3 equiv) and N,N-diisopropy-

lethylamine (1.25 g, 9.72 mmol, 2.1 equiv) in acetonitrile (50 mL)

at 0 ꢀC was added dropwise a solution of 4-bromo-2-hydro-

xybenzoic acid (1.0 g, 4.63 mmol, 1.0 equiv) in acetonitrile

(10 mL). The mixture was warmed to room temperature and

stirred for 48 h at room temperature. An additional portion of

TBTU (1.04 g, 3.24 mmol, 0.7 equiv) was added to the mixture,

which was heated at 60 ꢀC for 5 h. The mixture was concentrated

under reduced pressure, and the residue was dissolved in ethyl

acetate. The mixture was washed with water and brine, dried

over magnesium sulfate, and filtered. The solution was concen-

trated under reduced pressure. The crude product was purified

by column chromatography (eluent: hexane/ethyl acetate mix-

tures of increasing polarity). Yield: 63%. ¹H NMR (CDCl₃) δ

10.08 (s, 1H), 7.17 (d, 1H), 7.12 (d, 1H), 6.98 (dd, 1H), 3.50 (q,

4H), 1.27 (t, 6H). LCMS (ESI): m/z 270.1 (M - H^-).

5-Bromopyrimidine-2-carboxylic Acid (54). To a mixture of a

2.5 M solution of n-butyl lithium in hexanes (0.84 mL, 2.1 mmol,

1.05 equiv) and toluene (4 mL) at -78 ꢀC was added a solution

of 53 (0.57 g, 2.0 mmol, 1.0 equiv) in toluene (2 mL). The

reaction was stirred for 1 h at -78 ꢀC. The reaction was

quenched with freshly crushed dry ice. The mixture was warmed

slowly to room temperature and was stirred for 2 h at room

temperature. The mixture was concentrated under reduced

pressure, and the resulting solid was treated with acetic acid.

The solid was collected by filtration, dried under vacuum, and

used for the next step without further purification. Yield: 62%.

¹H NMR (CD₃OD) δ 8.90 (s, 2H).

5-Bromo-N,N-diethylpicolinamide (56). To a suspension of 55

(808 mg, 3.01 mmol, 1.0 equiv) in dry dichloromethane (5 mL)

was added oxalyl chloride (0.34 mL, 3.96 mmol, 1.3 equiv)

followed by 2 drops of N,N-dimethylformamide. The reaction

mixture was heated under reflux for 1 h. After cooling to room

temperature, the mixture was concentrated under reduced pres-

sure to provide the crude acyl chloride. To a suspension of the

crude acyl chloride (as of 3.01 mmol, 1.0 equiv) in dry tetra-

hydrofuran (5 mL) was added N,N-diethylamine (1.56 mL,

15.08 mmol, 5.0 equiv) dropwise. The reaction mixture was

stirred at room temperature for 2 h. Ethyl acetate (20 mL) was

added, and the mixture was washed with water (20 mL),

saturated aqueous sodium bicarbonate (30 mL), 1 N aqueous

hydrochloric acid (20 mL), and brine. The organics were dried

over sodium sulfate, filtered, and concentrated under reduced

pressure to give a red-brown crystalline solid. Yield: 88% over

two steps. ¹H NMR (CDCl₃) δ 8.64 (d, 1H), 7.91 (dd, 1H), 7.53

(d, 1H), 3.56 (q, 2H), 3.39 (q, 2H), 1.27 (t, 3H), 1.17 (t, 3H).

LCMS (ESI): m/z 257.2 (M þ H^þ).

concentrated under reduced pressure to give a dark-brown oil,

which solidified to needles. The crude product was triturated with

hexane. The resulting solid was collected by filtration. Yield: 52%.

¹H NMR (CDCl₃) δ 8.92 (d, 1H), 8.14 (dd, 1H), 7.53 (d, 1H), 3.55

(q, 2H), 3.32 (q, 2H), 1.36 (s, 12H), 1.27 (t, 3H), 1.12 (t, 3H).

tert-Butyl 4-(6-(Diethylcarbamoyl)pyridin-3-yl)spiro[chromene-

2,4⁰-piperidine]-1⁰-carboxylate (58). To a solution of 39a (1.48 g,

3.29 mmol, 1.0 equiv) in dimethoxyethane (20 mL) under nitrogen

was added sequentially a 2 N aqueous solution of sodium carbo-

nate (4.94 mL, 9.87 mmol, 3.0 equiv), lithium chloride (0.42 g, 9.87

mmol, 3.0 equiv), palladium (70 mg, 10 wt. % (dry basis) on

activated carbon, 0.033 mmol, 0.01 equiv), and 57 (1.0 g, 3.29

mmol, 1.0 equiv). The mixture was heated under reflux for 10 h.

Dichloromethane (200 mL) was added to dilute the reaction

mixture, and palladium(0) on carbon was filtered off on a celite

pad. The filtrate was washed with brine, dried over sodium sulfate,

filtered, and concentrated under reduced pressure. The crude

product was purified by column chromatography (eluent: hex-

ane/ethyl acetate mixtures of increasing polarity). Yield: 76%. ¹H

NMR (CDCl₃) δ 8.56 (dd, 1H), 7.75 (dd, 1H), 7.64 (dd, 1H), 7.22

(m, 1H), 6.99-6.85 (m, 3H), 5.62 (s, 1H), 3.88 (m, 2H), 3.59 (q,

2H), 3.45 (q, 2H), 3.34 (m, 2H), 2.06 (m, 2H), 1.69 (m, 2H), 1.48 (s,

9H), 1.29 (t, 3H), 1.20 (t, 3H). LCMS (ESI): m/z 478.0 (M þ H^þ).

Methyl 4-Iodo-3-(methoxymethoxy)benzoate (60). To an

acidic methanolic solution, which was prepared by drop-

wise addition of acetyl chloride (25 mL) to anhydrous methanol

(500 mL), was added 59 (171.61 g, 0.65 mol). The mixture was

heated to reflux for 30 h. The reaction mixture was allowed to

cool to room temperature and was concentrated under reduced

pressure. The residue was diluted in ethyl acetate (750 mL),

washed with a saturated sodium bicarbonate solution (750 mL),

water (750 mL), brine (750 mL), and dried over sodium sulfate.

The solution was filtered, and the filtrate was concentrated

under reduced pressure. The crude product was dried under

vacuum. Yield: 94%. ¹H NMR (CDCl₃) δ 7.74 (d, 1H), 7.64 (d,

1H), 7.31 (dd, 1H), 5.80 (s, 1H), 3.90 (s, 3H). To a solution of the

crude ester (169.4 g, 0.61 mol, 1.0 equiv) and N,N-diisopropy-

lethylamine (318.8 mL, 1.83 mol, 3.0 equiv) in methylene

chloride (750 mL) at room temperature under nitrogen was

added dropwise chloromethyl methyl ether (136.7 mL, 1.80 mol,

2.95 equiv). The mixture was heated under reflux for 22 h and

then allowed to cool to room temperature. The mixture was

concentrated to half the volume under reduced pressure and

washed with a 1 N hydrochloric acid solution (2 ꢀ 1 L) and brine

(1 L). The organic layer was dried over sodium sulfate, filtered,

and the filtrate was concentrated under reduced pressure. The

crude product was used for the next step without further

purification. Yield: 95%. ¹H NMR (CDCl₃) δ 7.83 (d, 1H),

7.65 (d, 1H), 7.39 (dd, 1H), 5.30 (s, 2H), 3.90 (s, 3H), 3.52 (s, 3H).

4-Iodo-3-(methoxymethoxy)benzoic Acid (61). A solution of

60 (186.66 g, 0.58 mol, 1.0 equiv) in acetone (400 mL) was added

to a solution of lithium hydroxide monohydrate (97.32 g, 2.32

mol, 4.0 equiv) in a 1:1 tetrahydrofuran/water solution (900

mL). The mixture was stirred at room temperature for 16 h. The

mixture was reduced to half of its volume under reduced

pressure and acidified with a 6 N aqueous solution of hydro-

chloric acid (∼300 mL). The crude mixture was extracted with

ethyl acetate (750 mL). The organic layer was washed with brine,

dried over sodium sulfate, and filtered. The filtrate was con-

centrated under reduced pressure to afford the acid 61 as a light-

yellow crystalline solid, which was used for the next step without

N,N-Diethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pico-

linamide (57). To a solution of bis(pinacolato)diboron (2.18 g,

8.6 mmol, 1.2 equiv) in N,N-dimethylformamide (10 mL) at

0 ꢀC was added potassium acetate (2.3 g, 23.4 mmol, 3.0 equiv),

1,1⁰-bis(diphenylphosphino)ferrocene palladium(II) chloride com-

plex with dichloromethane (171 mg, 0.23 mmol, 0.03 equiv). The

reaction mixture was heated at 80 ꢀC at which point a solution of 56

(2.0 g, 7.8 mmol, 1.0 equiv) in N,N-dimethylformamide (10 mL)

was added dropwise. The reaction mixture was stirred at 80 ꢀC for

an additional 10 h. Ethyl acetate (75 mL) and water (50 mL) were

added, and the two phases were separated. The organic phase was

washed with brine (50 mL), dried over sodium sulfate, filtered, and

1

further purification. Yield: 99%. H NMR (CDCl₃) δ 7.89 (d,

1H), 7.73 (d, 1H), 7.47 (dd, 1H), 5.30 (s, 2H), 3.50 (s, 3H).

N,N-Diethyl-4-iodo-3-(methoxymethoxy)benzamide (62). To

a mixture of 61 (104.74 g, 0.34 mol, 1.0 equiv) and TBTU (125.0 g,

0.39 mol, 1.15 equiv) in acetonitrile (3.75 L) at 0 ꢀC was added

diethylamine (40.5 mL, 0.39 mol, 1.15 equiv) and N,N-diisopro-

pylethylamine (125.4 mL, 0.72 mol, 2.1 equiv). The mixture was

warmed to room temperature, stirred for 18 h at room tempera-

ture, and concentrated under reduced pressure. The crude mixture

5700 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

was dissolved in ethyl acetate (1 L). The mixture was washed with

a saturated aqueous solution of sodium bicarbonate (800 mL),

dried over sodium sulfate, and filtered. The filtrate was concen-

trated under reduced pressure. The crude product was purified by

column chromatography (first eluting with a 20% ethyl acetate/

hexane mixture then 40% ethyl acetate/hexane). Yield: 97%. ¹H

NMR (CDCl₃) δ 7.80 (d, 1H), 7.08 (d, 1H), 6.75 (dd, 1H), 5.23 (s,

2H), 3.50 (s, 3H), 3.62-3.07 (brm, 4H), 1.33-1.02 (brm, 6H).

tert-Butyl 4-(4-(Diethylcarbamoyl)-2-(methoxymethoxy)phenyl)-

spiro[chromene-2,4⁰-piperidine]-1⁰-carboxylate (63). To a solution of

62 (1.02 g, 2.82 mmol, 1.0 equiv) in dimethoxyethane (DME)

(20 mL) was added sequentially a 2-N aqueous solution of sodium

carbonate (4.23 mL, 8.46 mmol, 3.0 equiv), lithium chloride (0.359 g,

8.46 mmol, 3.0 equiv), 48 (1.44 g, 3.38 mmol, 1.2 equiv), and

palladium on carbon (10%, 50% water) (0.038 g, 0.007 mmol,

0.0025 equiv). The coupling reaction was conducted under micro-

wave conditions (A: 25-170 ꢀC for 10 min; B: 170 ꢀC for 7 min).

The mixture was dissolved in ethyl acetate, washed with water, and

dried over sodium sulfate. The mixture was filtered, and the filtrate

was concentrated under reduced pressure. The crude product was

purified by column chromatography (eluent: hexane/ethyl acetate

mixtures of increasing polarity). Yield: 50%. ¹H NMR (400 MHz,

CDCl₃) δ 7.21 (m, 2H), 7.13 (m, 1H), 7.06 (dd, 1H), 6.90 (d, 1H),

6.76 (m, 2H), 5.53 (s, 1H), 5.04 (s, 2H), 3.87 (brs, 2H), 3.55 (brs,

2H), 3.34 (brs, 4H), 3.30 (s, 3H), 2.08 (brm, 2H), 1.67 (brm, 2H),

1.48 (s, 9H), 1.24 (brm, 6H). LCMS (ESI): m/z 537.3 (M þ H^þ).

2. Biological Methods. Radioligand Binding Assays. Mem-

brane preparations from Chinese hamster ovary (CHO) cells

stably expressing human δ, μ, or κ opioid receptors were

prepared as described previously.⁵⁶The assay buffer used is

composed of 50 mM tris(hydroxymethyl) aminomethane HCl,

pH 7.8, 1.0 mM ethylene glycol bis(β-aminoethylether)-N,N,N⁰,

N⁰-tetraacetic acid (EGTA free acid), 5.0 mM MgCl₂, 10 mg/L

leupeptin, 10 mg/L pepstatin A, 200 mg/L bacitracin, and

0.5 mg/L aprotinin. After dilution in assay buffer and homo-

genization in a Polytron homogenizer (Brinkmann, Westbury,

NY) for 30 s at a setting of 1, membrane proteins (10-80 μg) in

250 μL of assay buffer were added to mixtures containing test

compound and [³H]diprenorphine (0.5-1.0 nM, 25000-50000

dpm) in 250 μL of assay buffer in 96-well deep-well polystyrene

titer plates (Beckman) and incubated at room temperature for

60 min. Reactions were terminated by vacuum filtration with a

Brandel MPXR-96T harvester through GF/B filters that had

been pretreated with a solution of 0.5% polyethylenimine and

0.1% bovine serum albumin for at least 1 h. The filters were

washed four times with 1.0 mL each of ice-cold 50 mM Tris-HCl,

pH 7.8, and 30 μL of Microscint-20 (PerkinElmer Life and

Analytical Sciences, Shelton, CT) was added to each filter.

Radioactivity on the filters was determined by scintillation

spectrometry in a PerkinElmer TopCount. [³H]Diprenorphine

with a specific activity of 50 Ci/mmol was purchased from

PerkinElmer Life and Analytical Sciences, Inc. (Shelton, CT).

The K_Dvalues for [³H]diprenorphine binding were 0.33 nM for

the κ and μ opioid receptors and 0.26 nM for the DOR. Receptor

expression levels, determined as B_maxvalues from Scatchard

analyses, were 4400, 4700, and 2100 fmol/mg of protein for the κ,

μ, and DORs, respectively. Preliminary experiments were per-

formed to show that no specific binding was lost during the wash

of the filters, that binding achieved equilibrium within the

incubation time and remained at equilibrium for at least an

additional 60 min, and that binding was linear with regard to

protein concentration. Nonspecific binding, determined in the

presence of 10 μM unlabeled naloxone, was less than 10% of

total binding. Protein was quantified by the method of Brad-

ford.⁵⁷The data from competition experiments were fit by

nonlinear regression analysis using GraphPad Prism 4.02 for

Windows (GraphPad Software, San Diego, CA) using the four-

parameter equation for one-site competition, and K_ivalues were

subsequently calculated from EC₅₀values by the Cheng-Prus-

off equation.

Receptor-Mediated [³⁵S]GTPγS Binding. Receptor-mediated

[³⁵S]GTPγS binding was performed by modifications of the

methods of Selley and collaborators⁵⁸and Traynor and Na-

horski.⁵⁹Assays were carried out in 96-well FlashPlates (Perkin-

Elmer Life and Analytical Sciences, Shelton, CT). Membranes

prepared from CHO cells expressing the human DOR (50-100 μg

of protein) were added to assay mixtures containing agonist,

approximately 100000 dpm (100 pM) [³⁵S]GTPγS, 3.0 μM

GDP, 75 mM NaCl, 15 mM MgCl₂, 1.0 mM EGTA, 1.1 mM

dithiothreitol, 10 mg/L leupeptin, 10 mg/L pepstatin A, 200 mg/L

bacitracin, and 0.5 mg/L aprotinin in 50 mM Tris-HCl buffer,

pH 7.8. After incubation at room temperature for 1 h, the plates

were sealed and centrifuged at 800g in a swinging bucket rotor

for 5 min and bound radioactivity was determined with a

TopCount microplate scintillation counter (PerkinElmer). Ago-

nist potencies were assessed by measuring stimulation of

[³⁵S]GTPγS binding by a series of concentrations of agonist,

using 1 as a reference agonist. The concentration to give half-

maximal stimulation (EC₅₀) was determined by nonlinear re-

gression using GraphPad Prism 4.02 for Windows (GraphPad

Software, San Diego, CA).

Fluorescence-Based CYP2D6 Inhibition Assay. CYP2D6 ac-

tivity was measured in microsomes containing human recombi-

nant CYP2D6 (BD Biosciences) using 7-methoxy-4-amino-

methyl-coumarin (MAMC) as substrate.⁶⁰Conversion of

MAMC to 7-hydroxy-4-aminomethyl-coumarin (HAMC)

was measured using a PerkinElmer Fusion with a 390 nm

excitation filter and a 460 nm emission filter. IC₅₀values are

geometric means computed from at least three separate deter-

minations.

Freund’s Complete Adjuvant (FCA)-Induced Mechanical Hy-

peralgesia in Rats. For the assay, the methods of DeHaven-

Hudkins and collaborators were used to determine mechanical

hyperalgesia in rats 24 h after intraplantar administration of

150 μL Freund’s Complete Adjuvant (FCA).⁵³To determine

paw pressure thresholds (PPTs), the rats were lightly restrained

in a gauze wrap and pressure was applied to the dorsal surface of

the inflamed and uninflamed paw with a conical piston using a

pressure analgesia apparatus (Stoelting Instruments, Wood

Dale, IL). The PPT was defined as the amount of force (in

grams) required to elicit an escape response using a cutoff value

of 250 g. PPTs were determined before and at specified times

after drug treatment. The antihyperalgesia value was calculated

for each rat using the following formula: % AH = 100-

(posttreatment PPT_(inflamed)- baseline PPT_(inflamed))/(baseline

PPT_(uninflamed)- baseline PPT_(inflamed)). The dose required to

produce a half-maximal effect (ED₅₀) was determined by non-

linear regression using GraphPad Prism 4.02 for Windows

(GraphPad Software, San Diego, CA), which was also used

for statistical analyses. The level of significance was set at p<

0.05. To determine the ED₅₀value for the dose-response

experiments, a mean % AH was determined for each dose and

the data were analyzed using nonlinear regression analysis of a

fitted sigmoidal dose-response curve. The minimum value was

set at 0, and the maximum value was constrained at a value that

did not exceed the mean % AH ( the standard deviation of the

mean for the highest value. One-way analysis of variance

(ANOVA) with Tukey’s posthoc test was used to determine

group differences in the dose-response and time course experi-

ments. Two-way ANOVA with Bonferroni’s posthoc test was

used to determine differences due to pretreatment and treatment

in the antagonism experiment. The level of significance was set

at p < 0.05. GraphPad Prism 4.02 for Windows (GraphPad

Software, San Diego, CA) was used for curve fitting and for all

statistical analyses.

Acknowledgment. This research was sponsored by Adolor

Corporation. All authors are currently or were employees of

Adolor Corporation at the time this work was conducted. We

thank Glenn P. A. Yap from the University of Delaware

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5701

(Department of Chemistry and Biochemistry) for providing

crystallographic data of compound 50. We also thank Pfizer

Inc. for their careful review of the manuscript. Administrative

assistance was provided by UBC Scientific Solutions and

funded by Adolor Corp. and Pfizer Inc.

(18) Harada, Y.; Nishioka, K.; Kitahata, L. M.; Nakatani, K.; Collins,

J. G. Contrasting actions of intrathecal U50,488H, morphine, or

[^D-Pen², D-Pen⁵] enkephalin or intravenous U50,488H on the

visceromotor response to colorectal distension in the rat. Anesthe-

siology 1995, 83, 336–343.

(19) Danzebrink, R. M.; Green, S. A.; Gebhart, G. F. Spinal mu and

delta, but not kappa, opioid-receptor agonists attenuate responses

to noxious colorectal distension in the rat. Pain 1995, 63, 39–47.

(20) Broom, D. C.; Jutkiewicz, E. M.; Rice, K. C.; Traynor, J. R.;

Woods, J. H. Behavioral effects of δ-opioid receptor agonists:

potential antidepressants? Jpn. J. Pharmacol. 2002, 90, 1–6.

(21) Jutkiewicz, E. M. The antidepressant-like effects of delta-opioid

receptor agonists. Mol. Interventions 2006, 6, 162–169.

Supporting Information Available: Table of crystallographic

data for compound 50. Table of elemental analyses for com-

pounds 5-36. This material is available free of charge via the

Internet at http://pubs.acs.org.

(22) Gross, G. J. Role of opioids in acute and delayed preconditioning.

References

J. Mol. Cell. Cardiol. 2003, 35, 709–718.

(23) Henry, B.; Fox, S. H.; Crossman, A. R.; Brotchie, J. M. Mu- and

delta-opioid receptor antagonists reduce levodopa-induced dyski-

nesia in the MPTP-lesioned primate model of Parkinson’s disease.

Exp. Neurol. 2001, 171, 139–146.

(24) Hill, M. P.; Hille, C. J.; Brotchie, J. M. Delta-opioid receptor

agonists as a therapeutic approach in Parkinson’s disease. Drug

News Perspect. 2000, 13, 261–268.

(25) Szeto, H. H.; Soong, Y.; Wu, D.; Olariu, N.; Kett, A.; Kim, H.;

Clapp, J. F. Respiratory depression after intravenous administra-

tion of delta-selective opioid peptide analogs. Peptides 1999, 20,

101–105.

(26) Negus, S. S.; Gatch, M. B.; Mello, N. K.; Zhang, X.; Rice, K.

Behavioral effects of the delta-selective opioid agonist SNC80 and

related compounds in rhesus monkeys. J. Pharmacol. Exp. Ther.

1998, 286, 362–375.

(27) Sheldon, R. J.; Riviere, J. M.; Malarchik, M. E.; Mosberg, I.;

Burks, T. F.; Porreca, F. Opioid regulation of mucosal ion trans-

port in the mouse isolated jejunum. J. Pharmacol. Exp. Ther. 1990,

253, 144–151.

(28) Cowan, A.; Zhu, X. Z.; Mosberg, H. I.; Omnaas, J. R.; Porreca, F.

Direct dependence studies in rats with agents selective for different

types of opioid receptor. J. Pharmacol. Exp. Ther. 1988, 246, 950–

955.

(29) Lee, P. H.; McNutt, R. W.; Chang, K. J. A nonpeptidic delta opioid

receptor agonist, BW373U86, attenuates the development and

expression of morphine abstinence precipitated by naloxone in

rat. J. Pharmacol. Exp. Ther. 1993, 267, 883–887.

(1) Aldrich, J. V. Analgesics. In Burger’s Medicinal Chemistry and

Drug Discovery; Wolff, M. E., Ed.; John Wiley & Sons: New York,

1996; Vol. 3, pp 321-341.

(2) Gouarderes, C.; Tellez, S.; Tafani, J. A.; Zajac, J. M. Quantitative

autoradiographic mapping of delta-opioid receptors in the rat

central nervous system using [¹²⁵I][D-Ala²]deltorphin-I. Synapse

1993, 13, 231–240.

(3) Traynor, J. R.; Hunter, J. C.; Rodrigues, R. E.; Hill, R. G.; Hughes,

J. Delta-opioid receptor binding sites in rodent spinal cord. Br. J.

Pharmacol. 1990, 100, 319–323.

(4) Quirion, R.; Zajac, J. M.; Morgat, J. L.; Roques, B. P. Autoradio-

graphic distribution of mu and delta opiate receptors in rat brain

using highly selective ligands. Life Sci. 1983, 33, 227–230.

(5) Hiller, J. M.; Fan, L. Q.; Simon, E. J. Autoradiographic compar-

ison of [³H]DPDPE and [³H]DSLET binding: evidence for distinct

delta 1 and delta 2 opioid receptor populations in rat brain. Brain

Res. 1996, 719, 85–95.

(6) Tempel, A.; Zukin, R. S. Neuroanatomical patterns of the μ, δ, and

K opioid receptors of rat brain as determined by quantitative in

vitro autoradiography. Neurobiology 1987, 84, 4308–4312.

(7) Pradhan, A. A. A.; Clarke, P. B. S. Comparison between δ-opioid

receptor functional response and autoradiographic labeling in rat

brain and spinal cord. J. Comp. Neurol. 2005, 481, 416–426.

(8) Mansour, A.; Fox, C. A.; Burke, S.; Meng, F.; Thompson, R. C.;

Akil, H.; Watson, S. J. Mu, delta, and kappa opioid receptor

mRNA expression in the rat CNS: an in situ hybridization study.

J. Comp. Neurol. 1994, 350, 412–438.

(30) Brandt, M. R.; Furness, M. S.; Rice, K. C.; Fischer, B. D.; Negus,

S. S. Studies of tolerance and dependence with the δ-opioid agonist

SNC80 in rhesus monkeys responding under a schedule of food

presentation. J. Pharmacol. Exp. Ther. 2001, 299, 629–637.

(31) Negus, S. S.; Butelman, R.; Chang, K.-J.; DeCosta, B.; Winger, G.;

Woods, J. H. Behavioral effects of the systemically active delta

opioid agonist BW373U86 in rhesus monkeys. J. Pharmacol. Exp.

Ther. 1994, 270, 1025–1034.

(32) Chang, K. J.; Rigdon, G. C.; Howard, J. L.; McNutt, R. W. A

novel, potent and selective nonpeptidic delta opioid receptor

agonist BW373U86. J. Pharmacol. Exp. Ther. 1993, 267, 852–857.

(33) Calderon, S. N.; Coop, A. SNC-80 and related δ opioid agonists.

Curr. Pharm. Des. 2004, 10, 733–742.

(34) Wei, Z.-Y.; Brown, W.; Takasaki, B.; Plobeck, N.; Delorme, D.;

Zhou, F.; Yang, H.; Jones, P.; Gawell, L.; Gagnon, H.; Schmidt,

R.; Yue, S.-Y.; Walpole, C.; Payza, K.; St-Onge, S.; Labarre, M.;

Godbout, C.; Jakob, A.; Butterworth, J.; Kamassah, A.; Morin, P.-

E.; Projean, D.; Ducharme, J.; Roberts, E. N,N-Diethyl-

4-(phenylpiperidin-4-ylidenemethyl)benzamide: a novel, excep-

tionally selective, potent δ opioid receptor agonist with oral

bioavailability and its analogues. J. Med. Chem. 2000, 43, 3895–

3905.

(35) Plobeck, N.; Delorme, D.; Wei, Z.-Y.; Yang, H.; Zhou, F.;

Schwarz, P.; Gawell, L.; Gagnon, H.; Pelcman, B.; Schmidt, R.;

Yue, S.-Y.; Walpole, C.; Brown, W.; Zhou, E.; Labarre, M.; Payza,

K.; St-Onge, S.; Kamassah, A.; Morin, P.-E.; Projean, D.; Duch-

arme, J.; Roberts, E. New diarylmethylpiperazines as potent and

selective nonpeptidic δ opioid receptor agonists with increased in

vitro metabolic stability. J. Med. Chem. 2000, 43, 3878–3894.

(36) Calderon, S. N.;Rothman, R. B.;Porreca, F.; Flippen-Anderson, J. L.;

McNutt, R. W.; Xu, H.; Smith, L. E.; Bilsky, E. J.; Davis, P.; Rice, K.

C. Probes for narcotic receptor mediated phenomena. 19. Synthesis of

(þ)-4-[(rR)-r-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-meth-

oxybenzyl]-N,N-diethylbenzamide (SNC 80): a highly selective, non-

peptide delta opioid receptor agonist. J. Med. Chem. 1994, 37, 2125–

2128.

(9) George, S. R.; Zastawny, R. L.; Briones-Urbina, R.; Cheng, R.;

Nguyen, T.; Heiber, M.; Kouvelas, A.; Chan, A. S.; O’Dowd, B. F.

Distinct distributions of mu, delta and kappa opioid receptor

mRNA in rat brain. Biochem. Biophys. Res. Commun. 1994, 205,

1438–1444.

(10) Bausch, S. B.; Patterson, T. A.; Appleyard, S. M.; Chavkin, C.

Immunocytochemical localization of delta opioid receptors in

mouse brain. J. Chem. Neuroanat. 1995, 8, 175–189.

(11) Cahill, C. M.; McClellan, K. A.; Morinville, A.; Hoffert, C.;

Hubatsch, D.; O’Donnell, D.; Beaudet, A. Immunohistochemical

distribution of delta opioid receptors in the rat central nervous

system: evidence for somatodendritic labeling and antigen-specific

cellular compartmentalization. J. Comp. Neurol. 2001, 440, 65–84.

(12) Fraser, G. L.; Gaudreau, G.-A.; Clarke, P. B. S.; Menard, D. P.;

Perkins, M. N. Antihyperalgesic effects of δ opioid agonists in a rat

model of chronic inflammation. Br. J. Pharmacol. 2000, 129, 1668–

1672.

(13) Brandt, M. R.; Furness, M. S.; Mello, N. K.; Rice, K. C.; Negus, S.

S. Antinociceptive effects of δ-opioid agonists in rhesus monkeys:

effects on chemically induced thermal hypersensitivity. J. Pharma-

col. Exp. Ther. 2001, 296, 939–946.

(14) Petrillo, P.; Angelici, O.; Bingham, S.; Ficalora, G.; Garnier, M.;

Zaratin, P. F.; Petrone, G.; Pozzi, O.; Sbacchi, M.; Stean, T. O.; Upton,

N.; Dondio, G. M.; Scheideler, M. A. Evidence for a selective role in

the δ-opioid agonist [8R-(4βS*,8ar,8aβ,12bβ)]7,10-dimethyl-1-meth-

oxy-11-(2-methylpropyl)oxycarbonyl 5,6,7,8,12,12β-hexahydro-(9H)-

4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochlor-

ide (SB-235863) in blocking hyperalgesia associated with inflamma-

tory and neuropathic pain responses. J. Pharmacol. Exp. Ther. 2003,

307, 1079–1089.

(15) Desmeules, J. A.; Kayser, V.; Guilbaud, G. Selective opioid

receptor agonists modulate mechanical allodynia in an animal

model of neuropathic pain. Pain 1993, 53, 277–285.

(16) Mika, J.; Przewlocki, R.; Przewlocka, B. The role of delta-opioid

receptor subtypes in neuropathic pain. Eur. J. Pharmacol. 2001,

415, 31–37.

(17) Craft, R. M.; Henley, S. R.; Haaseth, R. C.; Hruby, V. J.; Porreca,

F. Opioid antinociception in a rat model of visceral pain: systemic

versus local drug administration. J. Pharmacol. Exp. Ther. 1995,

275, 1535–1542.

(37) Calderon, S. N.; Rice, K. C.; Rothman, R. B.; Porreca, F.; Flippen-

Anderson, J. L.; Kayakiri, H.; Xu, H.; Becketts, K.; Smith, L. E.;

Bilsky, E. J.; Davis, P.; Horvath, R. Probes for narcotic receptor

mediated phenomena. 23. Synthesis, opioid receptor binding, and

5702 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18

Le Bourdonnec et al.

bioassay of the highly selective delta agonist (þ)-4-[(rR)-alpha-

((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,

N-diethylbenzamide (SNC 80) and related novel nonpeptide delta

opioid receptor ligands. J. Med. Chem. 1997, 40, 695–704.

(38) Comer, S. D.; Hoenicke, E. M.; Sable, A. I.; McNutt, R. W.;

Chang, K. J.; De Costa, B. R.; Mosberg, H. I.; Woods, J. H.

Convulsive effects of systemic administration of the delta opioid

agonist BW373U86 in mice. J. Pharmacol. Exp. Ther. 1993, 267,

888–895.

(39) Broom, D. C.; Nitsche, J. F.; Pintar, J. E.; Rice, K. C.; Woods, J.

H.; Traynor, J. R. Comparison of receptor mechanisms and

efficacy requirements for δ-agonist-induced convulsive activity

and antinociception in mice. J. Pharmacol. Exp. Ther. 2002, 303,

723–729.

CB2 1EZ, UK, (fax: þ44 1223 336033 or e-mail: deposit@ccdc.cam.

ac.uk).

(48) Pearlstein, R.; Vaz, R.; Rampe, D. Understanding the structure-

-activity relationship of the human ether-a-go-go-related gene

cardiac K^þchannel. A model for bad behavior. J. Med. Chem.

2003, 46, 2017–2022.

(49) Ioannides, C. Cytochromes P450: Role in the Metabolism and

Toxicity of Drugs and Other Xenobiotics; Royal Society of Chem-

istry: Cambridge, UK, 2008.

(50) Spina, E.; Scordo, M. G. Clinically significant drug interactions

with antidepressants in the elderly. Drugs Aging 2002, 19, 299–320.

(51) Hemeryck, A.; Belpaire, F. M. Selective serotonin reuptake in-

hibitors and cytochrome P-450 mediated drug-drug interactions:

an update. Curr. Drug Metab. 2002, 3, 13–37.

(40) Broom, D. C.; Jutkiewicz, E. M.; Folk, J. E.; Traynor, J. R.; Rice,

K. C.; Woods, J. H. Convulsant activity of a non-peptidic delta-

opioid receptor agonist is not required for its antidepressant-like

effects in Sprague-Dawley rats. Psychopharmacology 2002, 164,

42–48.

(41) Dykstra, L. A.; Schoenbaum, G. M.; Yarbrough, J.; McNutt, R.;

Chang, K.-J. A novel delta opioid agonist, BW373U86, in squirrel

monkeys responding under a schedule of shock titration. J. Phar-

macol. Exp. Ther. 1993, 267, 875–882.

(52) Sproule, B. A.; Naranjo, C. A.; Bremner, K. E.; Hassan, P. C.

Selective serotonin reuptake inhibitors and CNS drug interactions.

A critical review of the evidence. Clin. Pharmacokinet. 1997, 33,

454–471.

(53) DeHaven-Hudkins, D. L.; Burgos, L. C.; Cassel, J. A.; Daubert, J.

D.; DeHaven, R. N.; Mansson, E.; Nagasaka, H.; Yu, G.; Yaksh,

T. Loperamide (ADL 2-1294), an opioid antihyperalgesic agent

with peripheral selectivity. J. Pharmacol. Exp. Ther. 1999, 289,

494–502.

(42) Pakarinen, E. D.; Woods, J. H.; Moerschbaecher, J. M. Repeated

acquisition of behavioral chains in squirrel monkeys: comparisons

of a mu, kappa and delta opioid agonist. J. Pharmacol. Exp. Ther.

1995, 272, 552–559.

(43) Tortella, F. C.; Echevarria, E.; Robles, L.; Mosberg, H. I.; Hola-

day, J. W. Anticonvulsant effects of mu (DAGO) and delta

(DPDPE) enkephalins in rats. Peptides 1998, 9, 1177–1181.

(44) Vergura, R.; Balboni, G.; Spagnolo, B.; Gavioli, E.; Lambert, D.

G.; McDonald, J.; Trapella, C.; Lazarus, L. H.; Regoli, D.;

Guerrini, R.; Salvadori, S.; Calo, G. Anxiolytic- and antidepres-

sant-like activities of H-Dmt-Tic-NH-CH(CH₂-COOH)-Bid

(UFP-512), a novel selective delta opioid receptor agonist. Peptides

2008, 29, 93–103.

(45) Tortella, F. C.; Robles, L.; Mosbeg, H. L.; Holaday, J. W.

Electroencephalographic assessment of the role of δ receptors in

opioid peptide-induced seizures. Neuropeptides 1984, 5, 213–216.

(46) Le Bourdonnec, B.; Windh, R. T.; Ajello, C. W.; Leister, L. K.; Gu,

M.; Chu, G.-H.; Tuthill, P. A.; Barker, W. M.; Koblish, M.; Wiant,

D. D.; Graczyk, T. M.; Belanger, S.; Cassel, J. A.; Feschenko, M.

S.; Brogdon, B. L.; Smith, S. A.; Christ, D. D.; Derelanko, M. J.;

Kutz, S.; Little, P. J.; DeHaven, R. N.; DeHaven-Hudkins, D. L.;

Dolle, R. E. Potent, orally bioavailable delta opioid receptor

agonists for the treatment of pain: Discovery of N,N-diethyl-4-

(5-hydroxyspiro[chromene-2,4⁰-piperidine]-4-yl)benzamide

(ADL5859). J. Med. Chem. 2008, 51, 5893–5896.

(54) All housing conditions and experimental procedures were per-

formed in accordance with the “Guide for the Care and Use of

Laboratory Animals”. Experimental procedures followed the ethi-

cal guidelines of the IASP and were approved by the Adolor

Corporation Institutional Animal Care and Use Committee.

(55) Davies, B.; Morris, T. Physiological parameters in laboratory

animals and humans. Pharm. Res. 1993, 10, 1093–1095.

(56) Schlechtingen, G.; Zhang, L.; Maycock, A.; DeHaven, R. N.;

Daubert, J. D.; Cassel, J.; Chung, N. N.; Schiller, P. W.; Goodman,

M. [Pro³]Dyn A(1-11)-NH₂: a dynorphin analogue with high

selectivity for the κ opioid receptor. J. Med. Chem. 2000, 43,

2698–2702.

(57) Bradford, M. M. A rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of pro-

tein-dye binding. Anal. Biochem. 1976, 72, 248–254.

(58) Selley, D. E.; Sim, L. J.; Xiao, R.; Liu, Q.; Childers, S. R. μ-Opioid

receptor-stimulated guanosine-5⁰-O-(γthio)-triphosphate binding

in rat thalamus and cultured cell lines: signal transduction mechan-

isms underlying agonist efficacy. Mol. Pharmacol. 1997, 51, 87–96.

(59) Traynor, J. R.; Nahorski, S. R. Modulation by μ-opioid agonists of

guanosine-5⁰O-(3-[35S]thio)triphosphate binding to membranes

from human neuroblastoma SH-SY5Y Cells. Mol. Pharmacol.

1995, 47, 848–854.

(60) Chauret, N.; Dobbs, B.; Lackman, R. L.; Bateman, K.; Nicoll-

Griffith, D. A.; Stresser, D. M.; Ackermann, J. M.; Turner, S. D.;

Miller, V. P.; Crespi, C. L. The use of 3-[2-(N,N-diethyl-

N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin

(AMMC) as a specific CYP2D6 probe in human liver microsomes.

Drug Metab. Dispos. 2001, 29, 1196–1200.

(47) Crystallographic data for compound 50 has been deposited with the

Cambridge Crystallographic Data Centre as supplementary pub-

lication No. CCDC 719968. Copies of the data can be obtained, free

of charge, on application to CCDC, 12 Union Road, Cambridge

Article Doi

Spirocyclic delta opioid receptor agonists for the treatment of pain: Discovery of N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4′-piperidine]-4- yl) benzamide (ADL5747)

DOI: 10.1021/jm900773n

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm900773n

Full text of DOI:10.1021/jm900773n

Products guided by the article

R&D Labs maybe for 1013334-48-9

Relevant to this article

Hot Product