Synthesis and pharmacological evaluation of novel octahydro-1H-pyrido[1,2- a]pyrazine as μ-opioid receptor antagonists

7290

J. Med. Chem. 2006, 49, 7290-7306

Synthesis and Pharmacological Evaluation of Novel Octahydro-1H-pyrido[1,2-a]pyrazine as

µ-Opioid Receptor Antagonists

Bertrand Le Bourdonnec,*^,†Allan J. Goodman,^†,‡Thomas M. Graczyk,^§Serge Belanger,^§Pamela R. Seida,^†

Robert N. DeHaven,^§and Roland E. Dolle^†

Department of Chemistry and Pharmacology, Adolor Corporation, 700 PennsylVania DriVe, Exton, PennsylVania 19341

ReceiVed April 25, 2006

To better understand structural requirements for a µ ligand of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)-

piperidine class to interact with the µ opioid receptor, we have described in the previous article (Le

Bourdonnec, B. et al. J. Med. Chem. 2006, 25, 7278-7289) new, constrained analogues of the N-phenethyl

derivative 3. One of the active constrained analogues, compound 4, exhibited subnanomolar µ-opioid receptor

affinity (K_i) 0.62 nM) and potent µ-opioid antagonist activity (IC₅₀) 0.54 nM). On the basis of structure

4, a new series of µ-opioid receptor antagonists were designed. In these compounds the octahydroquinolizine

template of 4 was replaced by an octahydro-1H-pyrido[1,2-a]pyrazine scaffold. The new derivatives were

tested for their binding affinities and in vitro functional activity against the cloned human µ-, δ-, and κ-opioid

receptors. From this study, we identified compound 36, which displays high affinity toward the µ-opioid

receptor (K_i) 0.47 nM), potent µ in vitro antagonist activity (IC₅₀) 1.8 nM) and improved binding selectivity

profile µ/κ and µ/δ, when compared to 4.

Introduction

receptor affinity (K_i(µ) ) 0.62 nM) and potent µ-opioid

antagonist activity (IC₅₀(µ) ) 0.54 nM). This novel µ-opioid

receptor antagonist was prepared in 11 steps and 0.07% overall

yield from (+)-4(R)-(3-hydroxyphenyl)-3(R)-4-dimethyl-1-pip-

eridine.⁹The synthetic complexity of such derivatives prevented

structure-activity relationship (SAR) exploration at positions

6 and 7 of the octahydroquinolizine scaffold (Figure 1). On the

Among the opioid receptor antagonists family, naloxone (1a),

naltrexone (1b), and structurally related analogues have received

the most attention.¹The trans-3,4-dimethyl-4-(3-hydroxyphen-

yl)piperidines (2) class of opioid antagonists described for the

2

first time in 1978 by Zimmerman and collaborators has also

been widely investigated.^3-6A representative of this class, (+)-

N-phenethyl trans-3(R),4(R)-dimethyl-4-(3-hydroxyphenyl)pip-

eridine (3), has been previously reported to bind cloned human

opioid receptors with good affinity [K_i(µ) ) 1.9 nM; K_i(κ) )

17 nM; K_i(δ) ) 33 nM].^3,7

Due to the fact that the whole phenethyl side chain of 3 can

freely rotate relative to the piperidine ring, it is not possible to

identify the exact location of this binding site relative to the

rest of the molecule. To better understand structural requirements

for a ligand of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)-

piperidine class to interact with the µ-opioid receptor, in the

previous paper⁸we described new, constrained analogues of

the N-phenethyl derivative 3. One of the active constrained

analogues, compound 4, exhibited subnanomolar µ-opioid

Figure 1. X-ray structure of 62 showing labeling of the nonhydrogen

atoms. Displacement ellipsoids are at the 20% probability level.

basis of the structure of 4, we investigated the bioisosteric

replacement of the methylene group at position 6 of the

octahydroquinolizine scaffold by an NH or NR moiety. We now

wish to report the synthesis, opioid receptor binding properties,

and in vitro functional activity of this novel series of octahydro-

1H-pyrido[1,2-a]pyrazine derivatives.

Chemistry

* To whom correspondence should be addressed. Tel.: 1-484-595-1061.

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

^†Department of Chemistry.

The synthesis of the octahydro-1H-pyrido[1,2-a]pyrazine

derivatives 5-56 is shown in Schemes 1-6. The preparation

of compounds 5-7 is described in Scheme 1. The key step of

^‡Postdoctoral fellow, 2003-2004.

^§Department of Pharmacology.

Published on Web 11/10/2006

NoVel µ-Opioid Receptor Antagonists

Scheme 1. Synthesis of 5-7^a

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7291

^aReagents and conditions: (a) (CH₃)₃Si(CH₃)₂Cl, imidazole, DMAP, DMF, 86%; (b) s-BuLi, TMEDA, CO₂, Et₂O, 73%; (c) (S)-methyl 2-amino-2-

phenylacetate, TBTU, IPr₂NEt, CH₃CN, 82%; (d) 4 M HCl in dioxane, 95%; (e) toluene, 47%; (f) BH₃S(CH₃)₂, THF, 69%; (g) CH₃COCl, Et₃N, CH₂Cl₂

or CH₂O, NaCNBH₃, Et₃N, MeOH, THF, 53% (6), 76% (7).

the chemistry relied on the regio and stereoselective introduction

of a carboxylic acid functionality at the 6â-position of the N-tert-

butoxycarbonyl (N-Boc) piperidine derivative 58, prepared by

condensation of (3R,4R)-tert-butyl 4-(3-hydroxyphenyl)-3,4-

dimethylpiperidine-1-carboxylate (57)⁶with tert-butylchlorodi-

methylsilane in the presence of imidazole and 4-(dimethylamino)-

pyridine (DMAP). It has been previously reported that the N-Boc

group is an effective directing group for the R-lithiation of

piperidines.^10-12Treatment of 58 with sec-butyllithium in the

presence of N,N,N′,N′-tetramethylenediamine (TMEDA) af-

forded the lithiated intermediate which reacted with carbon

dioxide to provide the carboxylic acid 59 isolated in 86% yield.¹³

Condensation of 59 with (S)-methyl 2-amino-2-phenylacetate

in the presence of O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-

uronium tetrafluoroborate (TBTU) provided the amide 60, which

was converted to 61 under acidic conditions. Intramolecular

cyclization of 61, conducted in refluxing toluene, provided the

lactam derivative 62 in 47% isolated yield. The absolute regio-

and stereochemistry of 62 was determined by X-ray crystal-

lography (Figure 1).¹⁴This crystal structure established by

inference the absolute configuration of the synthetic precursors

59-61. Reduction of compound 62 with borane-dimethyl

sulfide complex provided the target compound 5. The acetamide

6 was obtained by treatment of 5 with acetyl chloride.

Condensation of 5 with formaldehyde under reductive amination

conditions afforded the N-methyl derivative 7.

The preparation of compounds 8-13 is described in Scheme

2. Coupling of 5 with benzaldehyde under reductive amination

conditions, using borane-pyridine complex (BAP) as reducing

agent, afforded the N-benzyl derivative 8, which was converted

to the triflate 63 by condensation with N-phenyltrifluoromethane-

sulfonimide. The derivative 9 was obtained by palladium-

catalyzed reduction of the triflate 63. Palladium-catalyzed

carbonylation of the triflate 63 provided the methyl ester 10,

which was hydrolyzed under basic conditions to give the

carboxylic acid 11. Coupling of 11 with ammonium chloride

in the presence of triethylamine and TBTU provided the primary

amide 12, which was converted to 13 by hydrogenation.

The compounds 14-19 were prepared from 59 according to

a reaction sequence (Scheme 3) similar to the one described

for the synthesis of 5. The intramolecular cyclization of

compounds 65 was performed in either toluene (65a,c,d) or

o-xylene (65e,f). Compound 66b was synthesized in quantitative

yield from 64a in a one-pot, two-step procedure.

The preparation of compounds 20-28 and 30-32 is described

in Scheme 4. Condensation of 15 with formaldehyde under

reductive amination conditions afforded the N-methyl derivative

20. Coupling of 15 with acetyl chloride, benzoyl chloride,

2-phenylacetyl chloride, or 3-phenylpropanoyl chloride provided

the amides 21-24, respectively. Reduction of 22-24 with

borane-dimethyl sulfide complex provided the target com-

pounds 26-28, respectively. Condensation of 15 with meth-

anesulfonyl chloride or phenylisocyanate, under standard con-

ditions, provided the sulfonamide 30 or the urea 32, respectively.

The O-benzyl derivative 67, prepared from 15 in three steps

(Boc protection, alkylation of the phenol hydroxyl group with

benzyl bromide, and acid-mediated N-Boc deprotection) was

used as starting material for the preparation of compounds 25

and 31. Condensation of 67 with potassium phenyltrifluoroborate

at room temperature, in the presence of triethylamine, molecular

sieves, and copper(II) acetate, provided the corresponding

N-phenyl derivative, which was converted to the target com-

pound 25 by hydrogenation. Coupling of 67 with benzenesulfo-

nyl chloride, followed by debenzylation of the sulfonamide

intermediate, provided compound 31. Compound 29 was

prepared in four steps from 59, using a chemical route (Scheme

5) similar to the one described for the synthesis of 5. Condensa-

tion of 15 with a selected range of aldehydes under reductive

amination conditions in trimethylorthoformate/methanol/acetic

acid using resin-supported cyano borohydride as reducing agent

afforded the corresponding derivatives 33-56. These library

compounds were purified by preparative HPLC. Representative

compounds (33, 36, 39, 54, and 56) were resynthesized

according to Scheme 6 to confirm the biological data obtained

for the library compounds.

Results and Discussion

The derivatives 1a,b, and 3-56 were tested for their affinities

toward the cloned human µ-, δ-, and κ-opioid receptors as

measured by their abilities to displace [³H]-diprenorphine from

7292 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Scheme 2. Synthesis of 8-13^a

Le Bourdonnec et al.

^aReagents and conditions: (a) C₆H₅CHO, BH₃‚pyridine, EtOH, 71%; (b) C₆H₅N(SO₂CF₃)₂, Et₃N, CH₂Cl₂, 72%; (c) Et₃N, HCO₂H, PPh₃, Pd(OAc)₂,

DMF, 57%; (d) Et₃N, Pd(OAc)₂, dppf, MeOH, CO, DMSO, 76%; (e) LiOH‚H₂O, H₂O, THF, MeOH, 100%; (f) NH₄Cl, Et₃N, TBTU, DMF, 91%; (g) Pd/C,

H₂, EtOH, 12%.

Scheme 3. Synthesis of 14-19^a

^aReagents and conditions: (a) H₂NCHRCO₂Me, TBTU, i-Pr₂NEt, CH₃CN, 83% (64a), 95% (64b), 81% (64c), 71% (64d), 82% (64e), 75% (64f); (b) 4

M HCl in dioxane, 84% (65a), 99% (65c), 77% (65d), 97% (65e), 100% (65f); (c) toluene (66a,c,d) or o-xylene (66e,f), 99% (66a), 38% (66c), 70% (66d),

37% (66e), 43% (66f); (d) (i) 4 M HCl in dioxane; (ii) Et₃N, 100%; (e) BH₃‚S(CH₃)₂, THF, 69% (14), 73% (15), 43% (16), 80% (17), 68% (18), 22% (19).

its specific binding sites. The µ antagonist potencies of the

compounds were assessed by their abilities to inhibit agonist

(loperamide)-stimulated guanosine 5′-O-(3-[³⁵S]thio)triphosphate

([³⁵S]GTPγS) binding to membranes containing µ-opioid recep-

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7293

Scheme 4. Synthesis of 20-28 and 30-32^a

^aReagents and conditions: (a) HCHO, NaCNBH₃, Et₃N, THF, EtOH, 4%; (b) ROCl, Et₃N, THF, 22% (21), 57% (22), 96% (23), 54% (24); (c) BH₃‚S(CH₃)₂,

THF, 100% (26), 42% (27), 45% (28); (d) ((CH₃)₃COCO)₂O, Et₃N, THF, 74%; (e) C₆H₅CH₂Br, K₂CO₃, DMF, 99%; (f) 2 M HCl in ether, MeOH, 100%;

(g) Et₃N, Cu(OAc)₂, CH₃C₆H₄BF₃K, Et₃N, CH₂Cl₂, 30%; (h) Pd/C, H₂, EtOH, 50% (25), 85% (31); (i) C₆H₅SO₂Cl, Et₃N, THF, 70%; (j) CH₃SO₂Cl, Et₃N,

THF, 15%; (k) C₆H₅NCO, Et₃N, CH₂Cl₂, 58%.

Scheme 5. Synthesis of 29^a

^aReagents and conditions: (a) C₆H₅NHCH₂CH₂CO₂CH₂CH₃, TBTU, i-Pr₂NEt, CH₃CN, 60%; (b) 4 M HCl in dioxane, 92%; (c) o-xylene, 25%; (d)

BH₃‚S(CH₃)₂, THF, 10%.

tors. As shown in Table 1, bioisosteric replacement of the

methylene group at position 6 of the octahydroquinolizine

scaffold of 4 by an NH functionality was successful. Indeed

the octahydro-1H-pyrido[1,2-a]pyrazine derivative 5, NH ana-

logue of 4, displayed high affinity toward the µ-opioid receptor

(K_i) 3.6 nM) and potent µ in vitro antagonist activity (IC₅₀)

1.1 nM). No µ agonist activity was detectable for compound 5

at concentrations up to 10 µM, demonstrating that this new

ligand was a pure µ antagonist. We then explored the effect of

substituting the NH functionality of 5 by various lipophilic

7294 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

Table 1. Opioid Receptor (µ, κ, and δ) Binding Data and in Vitro Antagonist Activity (µ) of Compounds 1a, 1b, and 3-13

K_i(µ)^a(nM)

or % inh.^b@

10 µM

K_i(κ)^a(nM)

or % inh.^b@

10 µM

K_i(δ)^a(nM)

or % inh.^b@

10 µM

IC₅₀(µ)^c

(nM)

compd

X

R

1a

1b

3

3.7 (3.1-4.5)

1.0 (0.78-1.3)

1.8 (0.76-4.4)

0.62 (0.41-0.81)

3.6 (2.2-6.0)

94 (55-160)

3.3 (1.9-5.9)

4.7 (0.85-26)

1600 (450-6200)

24% ( 2%

7.3 (5.0-10)

4.1 (1.8-9.1)

1.1 (0.3-2.0)

0.54 (0.32-2.0)

1.1 (0.52-2.2)

220 (120-390)

26 (17-42)

40 (20-79)

nd^e

9.2 (6.9-13)

4.4 (3.4-5.6)

17 (12-25)

9.0 (6.3-12)

18 (13-25)

620 (300-1300)

13 (3.4-47)

120 (78-180)

11% ( 3%

33 (27-41)

14 (9.8-20)

33 (19-57)

4^d

5^d

6

CH²

NH

NCOCH₃

NCH₃

NCH₂C₆H₅

NH

OH

H

CO₂CH₃

CO₂H

CONH₂

31 (21-45)

89 (68-120)

57% ( 1%

7^d

8^d

9

10

11

12

13^d

2300 (190-4700)

340 (68-1700)

26% ( 2%

nd^e

6% ( 4%

39% ( 5%

44% ( 2%

nd^e

9% ( 2%

460 (280-770)

11 (8.3-13)

2500 (1000-5700)

1100 (760-1700)

60 (37-99)

17 (9.3-30)

1.8 (0.81-4.1)

28 (14-56)

1.5 (1.0-2.3)

^aThe potencies of the compounds were determined by testing the ability of a range of concentrations of each compound to inhibit the binding of the

nonselective opioid antagonist, [³H]diprenorphine, to cloned human µ, κ, and δ opioid receptors, expressed in separate cell lines. K_ivalues are geometric

means and 95% confidence intervals computed from at least three separate determinations. ^bThe % inhibition of [³H]diprenorphine binding to the cloned

human µ-, κ-, and δ-opioid receptors using a concentration of the competitor of 10 µM. Mean ( S.E.M. ^cThe potencies of the antagonists were assessed

by their abilities to inhibit agonist (µ: loperamide; κ: U50, 488; δ: BW373U86) stimulated [³⁵S]GTPγS binding to membranes containing the respective

cloned human opioid receptor. ^dThe κ and δ antagonist potency of selected ligands. 4: IC₅₀(κ) ) 3.2 nM (0.81-13), IC₅₀(δ) ) 51 nM (45-58). 5: IC₅₀(κ)

) 3.2 nM (0.39-27), IC₅₀(δ) ) 98 nM (9.3-1000). 7: IC₅₀(κ) ) 2.4 nM (0.43-13), IC₅₀(δ) ) 580 nM (220-1500). 8: IC₅₀(κ) ) 99 nM (54-180),

IC₅₀(δ) ) 1300 nM (730-2400). 13: IC₅₀(κ) ) 2.3 nM (0.37-14), IC₅₀(δ) ) 110 nM (33-380). ^eNot determined.

Scheme 6. Synthesis of 33-56^a

Table 2. Opioid Receptor (µ, κ, and δ) Binding Data and in Vitro

Antagonist Activity (µ) of Compounds 5 and 14-19

^aReagents and conditions: (a) RCHO, PS-CNBH₃, TMOF, MeOH,

CH₃COOH; (b) RCHO, BH₃‚pyridine, EtOH, 39% (33), 62% (36), 38%

(39), 47% (54), 65% (56).

moieties. N-Acetylation of 5 resulted in a 20-fold decrease in µ

binding (6: K_i) 94 nM). In contrast, the N-methyl (7) and the

N-benzyl (8) derivatives displayed µ-opioid receptor binding

affinities similar to that of the precursor from which they were

derived. The phenolic hydroxy group of opiate-derived ligands

is important for biological activity.¹As expected, replacement

of the hydroxyl group of 8 with a hydrogen atom (compound

9) resulted in a significant (340-fold) decrease in µ binding.

The methyl ester 10 and its carboxylic acid analogue 11 were

also devoid of appreciable opioid receptor binding. We showed

previously in the trans-3,4-dimethyl-4-(3-hydroxyphenyl)pip-

eridine series of µ-opioid receptor antagonists that the CONH₂

group was an effective isostere of the phenolic OH moiety.⁷

As shown in Table 1, the carboxamide derivative 12 retained

good µ binding affinity (K_i) 17 nM). Furthermore, replacement

of the phenolic OH functionality of 5 with a CONH₂group

resulted in a 2-fold increase in the binding affinity toward the

µ-opioid receptor (13: K_i) 1.8 nM). The presence and spatial

orientation of the phenyl ring of the octahydroquinolizine

derivative 4 are of critical importance for good binding affinity

toward the µ-opioid receptor.⁸

^aSee Table 1, footnote a. ^bSee Table 1, footnote b. ^cSee Table 1,

footnote c. ^dThe κ and δ antagonist potency of selected ligands. 5: IC₅₀(κ)

) 3.2 nM (0.39-27), IC₅₀(δ) ) 98 nM (9.3-1000). 16: IC₅₀(κ) ) 16 nM

(4.1-64), IC₅₀(δ) ) 33 nM (3.6-290). 18: IC₅₀(κ) ) 1.2 nM (0.47-3.0),

IC₅₀(δ) ) 28 nM (18-44). ^eSee Table 1, footnote e.

14 demonstrated that the S-stereochemistry at the carbon atom

bearing the phenyl ring was highly preferred. Furthermore, the

binding data for compound 15, which differs from 5 by the

absence of the phenyl group, supported the necessity of this

lipophilic substituent for good µ-opioid receptor binding affinity.

Replacement of the phenyl moiety of 5 (K_i) 3.6 nM) with a

As shown in Table 2, comparison of the µ binding affinity

of 5 with the binding affinity of its diastereoisomeric analogue

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7295

Table 3. Opioid Receptor (µ, κ, and δ) Binding Data and in Vitro

Antagonist Activity (µ) of Compounds 15 and 20-32

Figure 2. Lowest-energy conformers of (a) set A and (b) set B. Side

chain coloring: 4, green; 5, blue; 22, teal; 26, aqua; 25, dark magenta;

29, orange; 31, magenta.

group of 21 to a benzoyl moiety (compound 22) resulted in a

250-fold increase in the affinity toward the µ receptor. This

result demonstrated that both the increased binding affinity and

potency of 22, when compared to 15, were directly related to

the N-benzoyl functionality. Introduction of a phenacetyl (23),

phenpropionyl (24), or phenyl (25) group in place of the benzoyl

functionality of 22 gave rise to compounds of lower binding

affinity (Table 3). The N-benzyl derivative 26 displayed

comparable µ-opioid affinity to the N-benzoyl analogue 22,

indicating that the pK_aof the nitrogen atom attaching the benzyl

or benzoyl functionalities does not have an effect on µ-opioid

binding affinity. To explore the size of the lipophilic pocket in

which the benzyl group of 26 interacts, we prepared the

N-phenethyl (compound 27) and N-phenpropyl (compound 28)

analogues of 26. These structural modifications led to a decrease

in the affinity toward the µ-opioid receptor, indicating that the

hydrophobic cavity in which the benzyl group of 26 interacts

is relatively small. Furthermore, extending the octahydro-1H-

pyrido[1,2-a]pyrazine scaffold of 26 to a decahydropyrido[1,2-

a][1,4]diazepine template (compound 29) resulted in a 130-fold

decrease in µ binding. Replacement of the benzyl group of 26

with a phenylsulfonamide (compound 31) also led to a decrease

in µ binding. These data showed that subtle structural modifica-

tions have an important impact on µ-opioid receptor binding.

In an attempt to rationalize these findings, we compared the

low-energy conformations of the potent µ ligands (set A: 4, K_i

) 0.62 nM; 5, K_i) 3.6 nM; 22, K_i) 2.0 nM; 26, K_i) 1.2

nM) with the low-energy conformations of weaker µ ligands

(set B: 25, K_i) 220 nM; 29, K_i) 160 nM; 31, K_i) 210 nM),

structurally related to 5, 22, and 26. To examine the confor-

mational behavior of the investigated ligands, a conformational

analysis was performed. All calculations were conducted using

the MOE software.¹⁵Stochastic conformational searches using

the default MMFF94x force field without solvation were

performed to identify the global minimum energy conformers

for the ligands of set A (4, 5, 22, and 26) and set B (25, 29,

and 31). Antagonist structures were aligned in MOE by

superposing the piperidine ring common to the central fused

bicyclic templates. As shown in Figure 2, the ligands of set A

(potent µ ligands) assume an extended conformation as their

lowest-energy conformer.

^aSee Table 1, footnote a. ^bSee Table 1, footnote b. ^cSee Table 1,

footnote c. ^dThe κ and δ antagonist potency of selected ligands. 22: IC₅₀(κ)

) 50 nM (7.2-350), IC₅₀(δ) ) 14 nM (3.4-58). 26: IC₅₀(κ) ) 8.4 nM

(2.3-31), IC₅₀(δ) ) 150 nM (120-200). 27: IC₅₀(κ) ) 20 nM (10-39),

IC₅₀(δ) ) 310 nM (98-1000). ^eSee Table 1, footnote e.

benzyl substituent (compound 16; K_i) 8.9 nM) or a cyclohexyl

group (compound 18; K_i) 2.7 nM) was well tolerated.

However, replacement of the phenyl ring of 5 with an isopropyl

moiety (compound 19) resulted in a 100-fold decrease in µ

binding, suggesting that the isopropyl group of 19 is of

insufficient size for an optimal lipophilic contact with the

µ-opioid receptor. On the basis of the structure of 4, we then

demonstrated that the bioisosteric replacement of the methylene

group at position 6 of the octahydroquinolizine scaffold by an

NH or NR moiety successfully led to a new series of potent

µ-opioid receptor antagonists. As expected from the limited SAR

obtained in the octahydroquinolizine series,⁸we further provided

evidence that the presence and proper orientation of a lipophilic

substituent (phenyl, benzyl, or cyclohexyl) connected to the

octahydro-1H-pyrido[1,2-a]pyrazine template are of primary

importance for good µ binding and antagonist activity. As

indicated previously, the absence of this key pharmacophoric

element in structure 15 is likely to explain the weak µ binding

affinity of this ligand. On the basis of this assumption, we

hypothesized that introduction of various lipophilic substituents

at the secondary amine functionality of 15 could lead to

compounds with improved µ-opioid receptor binding affinity.

As shown in Table 3, the N-methyl (compound 20) and

N-acetyl (compound 21) analogues of 15 displayed weak µ

binding affinity. However, as anticipated, changing the acetyl

In the low-energy conformations of the high-affinity ligands

(set A), the phenyl moieties of compounds 4 and 5 are restricted

to the downward region shown in Figure 2a. As expected, the

N-benzoyl and N-benzyl side chains of compounds 22 and 26

7296 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

Figure 3. Molecular volumes enclosing (a) high-potency µ antagonists (set A) and (b) lower-potency µ antagonists (set B). The overlay of the two

volumes is shown in Figure 4c. Lipophilic substituents in the lower green region, unique to set A, are essential for high µ binding affinity.

Table 4. Opioid Receptor (µ, κ, and δ) Binding Data and in Vitro

are more flexible, and compounds 22 and 26 have low-energy

Antagonist Activity (µ) of Compounds 26 and 33-43

conformations (0.6 and 0.1 kcal/mol above the lowest-energy

conformers, respectively) in which the phenyl moiety is

positioned to the side of the bicyclic template. In the lowest-

energy conformations of ligands of set B (weaker µ ligands),

the phenyl moiety is positioned to the side of the bicyclic

template in an orientation different to the one adopted for the

phenyl rings of the constrained analogues 4 and 5. There are

no conformers in which the phenyl group of 25 can reach the

putative hydrophobic pocket occupied by the phenyl groups in

the lowest-energy conformers of 4, 5, 22, and 26. Conformers

of 29 and 31 with the benzyl and phenyl sulfonamide side chains

extended like the low-energy conformers of 22 and 26 are 2.6

and 2.8 kcal/mol higher in energy, respectively, than their

lowest-energy conformers. This molecular modeling study

combined with SAR analysis led us to hypothesize that the

phenyl groups of 25, 29, and 31 are positioned in such an

orientation that they are less likely to interact efficiently with

the putative hydrophobic pocket of the µ receptor that is of

critical importance for ligand recognition. We also generated

the molecular volume maps for each set of ligands (Figure 3).

The molecular volume can be visualized using a Gaussian

approximation of the Connolly surface, representing the solvent

accessible surface area of the molecule.¹⁶The overlay of the

two volumes (Figure 3c) further highlighted the differences

between the placement of phenyl groups in the two sets of

antagonists. The weaker binding affinity of ligands of set B,

when compared with ligands of set A, could also be explained

by unfavorable steric interactions of the phenyl groups of 25,

29, and 31 with the µ-opioid receptor.

With the identification of 26 as a novel µ-opioid antagonist,

the SAR at the benzyl functionality was investigated. This

additional study was conducted to further characterize the

lipophilic pocket in which the benzyl group of 26 interacts. The

biological data obtained for the library compounds 33-56 are

summarized in Tables 4 and 5. Representative compounds (33,

^aSee Table 1, footnote a. ^bSee Table 1, footnote b. ^cSee Table 1,

36, 39, 54, and 56) were resynthesized according to Scheme 6

and further purified to confirm the binding data obtained for

the library compounds. The K_ivalues obtained for the purified

products were generally within 2-4-fold of the K_ivalues

obtained for the library compounds. Various substituents were

introduced at the 2-, 3-, and 4-position of the benzyl group of

26. Comparison of the in vitro profile of 33, 34, and 35

suggested that substitution at the 2- and 3-position of the benzyl

group is preferred. Hence, substitution in the para position might

hinder the locking of the aromatic moiety within the binding

pocket. Introduction of a chloro substituent at the 2-position of

footnote c. ^dThe κ and δ antagonist potency of selected ligands. 26: IC₅₀(κ)

) 8.4 nM (2.3-31), IC₅₀(δ) ) 150 nM (120-200). 33: IC₅₀(κ) ) 15 nM

(4.4-52), IC₅₀(δ) ) 62 nM (1.5-5800). 36: IC₅₀(κ) ) 10 nM (6.8-16),

IC₅₀(δ) ) 190 nM (27-1400). 39: IC₅₀(κ) ) 9.4 nM (3.9-23), IC₅₀(δ) )

570 nM (38-8600). ^eBiological data of purified library compound

expressed as the geometric mean, and 95% confidence intervals of at least

three separate determinations.

the benzyl group of 26 resulted in a slight increase in affinity

toward the µ receptor. Indeed, compound 36 displayed high

affinity toward the µ-opioid receptor (K_i) 0.47 nM) and potent

µ in vitro antagonist activity (IC₅₀) 1.8 nM). This compound

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7297

Table 5. Opioid Receptor (µ, κ, and δ) Binding Data and in Vitro

Antagonist Activity (µ) of Compounds 26 and 45-56

cyclohexylmethyl moiety. This result is consistent with a non

π-π-type hydrophobic interaction. Comparison of the µ binding

affinity of 5 (K_i) 3.6 nM) with the µ binding affinity of its

cyclohexyl analogue (18: K_i) 2.7 nM) conclusively supports

that statement.

Conclusion

There are multiple reasons for the use of bioisosterism to

design new drugs, including the necessity to improve pharma-

cological activity, gain selectivity for a determined receptor or

enzymatic isoform subtype, or optimize pharmacokinetics. In

this study, we explored the concept of bioisosterism to prepare

synthetically simplified analogues of the conformationally

constrained µ-opioid antagonist 4, thereby allowing us to

potentially expand SAR studies. The bioisosteric replacement

of the methylene group at position 6 of the octahydroquinolizine

scaffold of 4 (K_i(µ) ) 0.62 nM) by an NH functionality

(compound 5; K_i(µ) ) 3.6 nM) was well tolerated. In addition,

the new octahydro-1H-pyrido[1,2-a]pyrazine series offers several

advantages over the octahydroquinolizine series, including ease

of preparation, stereochemical control, and potential for scale-

up. Indeed, the preparation of compound 5 (7 steps; 15% overall

yield) was conducted in a more straightforward manner when

compared to the synthesis of 4 (11 steps; 0.07% yield). This

new series of octahydro-1H-pyrido[1,2-a]pyrazine provided

numerous ligands with good affinity toward the µ-opioid

receptor and potent µ in vitro antagonist activity. The SAR

studies indicated that the presence of a lipophilic group at

position 2 or 3 of the octahydro-1H-pyrido[1,2-a]pyrazine

template is essential for high µ-opioid receptor binding affinity.

This key lipophilic moiety, which can be an aromatic, het-

eroaromatic, or an aliphatic group, is thought to interact with

the µ receptor by hydrophobic contacts. The SAR showed that

the nature of the linker connecting this lipophilic moiety to the

fused bicyclic heterocyclic template has an important impact

on µ-opioid receptor binding affinity. Hence, some of the best

ligands incorporated an N-benzoyl (22), N-benzyl (26), or

N-cyclohexylmethyl (56) moieties. From this study, we identi-

fied compound 36, which displayed high affinity toward the

µ-opioid receptor (K_i) 0.47 nM) and potent µ in vitro

antagonist activity (IC₅₀) 1.8 nM). Furthermore, this compound

also showed an improved binding selectivity profile (µ/κ and

µ/δ) when compared to the selectivity profile of 4 from which

it evolved. This new series of octahydro-1H-pyrido[1,2-a]-

pyrazines has much latitude for structural manipulation. In

particular, variations of the substituents at position 2 and/or 3

of this template could be further explored to identify new

subtype selective compounds. The SAR data as well as modeling

studies obtained with these rigid structures provided insights

into the pharmacophoric features (hydroxyphenyl, piperidine

nitrogen, and lipophilic moieties) important for µ binding and

expression of antagonist activity. This information provides

useful tools for further refinement of receptor modeling and

docking studies in this class of µ antagonists.

^aSee Table 1, footnote a. ^bSee Table 1, footnote b. ^cSee Table 1,

footnote c. ^dThe κ and δ antagonist potency of selected ligands. 26: IC₅₀(κ)

) 8.4 nM (2.3-31), IC₅₀(δ) ) 150 nM (120-200); 54: IC₅₀(κ) ) 6.6 nM

(2.0-22), IC₅₀(δ) ) 340 nM (20-5800); 56: IC₅₀(κ) ) 36 nM (15-87),

IC₅₀(δ) ) 85 nM (9.4-770). ^eBiological data of purified library compound

expressed as the geometric mean of at least three separate determinations.

also displayed improved selectivity profile (µ/κ, 34-fold; µ/δ,

120-fold) when compared to the selectivity profile of 4 (µ/κ,

13-fold; µ/δ, 40-fold) from which it derived. As shown in Table

4, the phenolic derivative 39 was also found to be a potent

µ-opioid receptor antagonist. The in vitro profile of compounds

44 and 45 (Table 5) showed that introduction of additional

lipophilic functionalities at the 3-position of the benzyl group

of 26 resulted in a decrease in µ binding. This confirmed that

the size and depth of this lipohilic pocket might be relatively

small. However, replacement of the benzyl group of 26 by a

naphthalen-1-yl-methyl moiety (compound 47) was well toler-

ated. Replacing the phenyl ring of 26 by various heterocycles

(compounds 48-55) also provided ligands with good affinity

for the µ-opioid receptor. In particular, the thiophene derivative

54 bound to the µ-opioid receptor with high affinity (K_i) 1.4

nM) and was a potent µ antagonist in vitro (IC₅₀(µ) ) 0.74

nM). The interaction of these compounds with the hypothesized

lipophilic pocket may either be of π-π-type stacking with an

aromatic receptor moiety or simply hydrophobic in nature. The

biological data for compound 56 (K_i) 1.1 nM) demonstrated

that the benzyl group of 26 could be efficiently replaced by a

Experimental Section

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

used without further purification. Thin-layer chromatography (TLC)

was performed on silica gel 6F glass-backed plates (250 microns)

from Analtech and visualized by UV 254 irradiation and iodine.

Flash chromatography was conducted using the ISCO CombiFlash

with RediSep silica gel cartridges (4, 12, 40, and 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

7298 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

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 Biore-

sources Magic C18 Macro Bullet; detector, PDA λ ) 220-300

nm; gradient, 96% A-100% B in 3.2 min and 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 and hold 100% B

for 0.4 min. Mass spectra were obtained on a Finnigan 4000 or

VG707EHF spectrometer by the mass spectrometry laboratories at

the Department of Chemistry, University of Minnesota. Elemental

analyses were performed by Atlantic Microlabs, Norcross, GA and

are within (0.4% of theoretical values.

(3R,4R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

3,4-dimethylpiperidine-1-carboxylate (58). To a solution of 57

(72.69 g, 238 mmol) in N,N-dimethylformamide (500 mL) was

added imidazole (43.13 g, 309 mmol), DMAP (2.9 g, 23.8 mmol),

and tert-butyldimethylsilyl chloride (43.13 g, 286 mmol). The

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

reaction mixture was then poured into water and extracted with

hexanes. The combined organic extracts were washed successively

with water and brine solution, dried over sodium sulfate, and

concentrated under reduced pressure. The crude product was

purified by column chromatography (eluent, hexane/ethyl acetate

mixtures of increasing polarity). Yield 86% (colorless oil); ¹H NMR

(CDCl₃) δ 0.18 (s, 6H), 0.62 (d, J ) 6 Hz, 3H), 0.98 (s, 9H), 1.26

(s, 1H), 1.34 (s, 3H), 1.46 (s, 9H), 1.96 (m, 1H), 2.17 (m, 1H),

3.03 (m, 1H), 3.30 (m, 1H), 3.79 (m, 0.6H), 3.88 (m, 0.4H), 4.06

(m, 0.5H), 4.23 (m, 0.5H), 6.67 (dd, J ) 8 and 2 Hz, 1H), 6.73 (s,

1H), 6.84 (d, J ) 7 Hz, 1H), 7.16 (t, J ) 8 Hz, 1H); LCMS (ESI)

m/z 420 (M + H⁺).

Hz, 1H), 4.33 (dd, J ) 12 and 6 Hz, 1H), 5.59 (d, J ) 7 Hz, 1H),

6.68 (dd, J ) 8 and 2 Hz, 1H), 6.75 (t, J ) 2 Hz, 1H), 6.87 (d, J

) 8 Hz, 1H), 7.14 (t, J ) 8 Hz, 2H), 7.33 (m, 1H), 7.35 (m, 4H);

LCMS (ESI) m/z 611 (M + H⁺).

(S)-Methyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)-2-phenylacetate (61). To a solution

of 60 (2.16 g, 3.54 mmol) in methanol (75 mL) was added a 4 M

solution of anhydrous hydrogen chloride in dioxane (3.8 mL, 14.4

mmol). The mixture was heated to reflux for 2 h. The mixture was

concentrated under reduced pressure, and the residue was taken up

in ethyl acetate (75 mL). A saturated solution of sodium bicarbonate

was added to the mixture, which was stirred for 2 h at room

temperature. The layers were separated, and the organic layer was

washed with water and brine, dried over sodium sulfate, filtered,

and concentrated under reduced pressure. The crude product was

washed with hexanes and used for the next step without further

purification. Yield 95% (white solid); ¹H NMR (CDCl₃) δ 0.73 (d,

J ) 7 Hz, 3H), 1.28 (s, 4H), 1.92 (m, 1H), 2.03 (m, 1H), 2.81 (dd,

J ) 12 and 2 Hz, 1H), 3.29 (dd, J ) 12 and 3 Hz, 1H), 3.61 (dd,

J ) 11 and 4 Hz, 1H), 3.74 (s, 3H), 5.63 (d, J ) 13 Hz, 1H), 6.64

(dd, J ) 8 and 2 Hz, 1H), 6.72 (s, 1H), 6.79 (d, J ) 7 Hz, 1H),

7.15 (t, J ) 8 Hz, 1H), 7.38 (m, 5H), 7.92 (d, J ) 7 Hz, 1H);

LCMS (ESI) m/z 397 (M + H⁺).

(3S,7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-3-phenyl-

hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (62). A solution

of 61 (1.33 g, 3.36 mmol) in toluene (200 mL) was heated to reflux

for 60 h. The mixture was concentrated under reduced pressure.

The crude product was purified by column chromatography

(eluent: dichloromethane/methanol mixtures of increasing polarity).

Yield 47% (yellow solid); ¹H NMR (CDCl₃) δ 0.64 (d, J ) 7 Hz,

3H), 1.39 (s, 3H), 2.11 (m, 1H), 2.26 (t, J ) 13 Hz, 1H), 2.43 (dd,

J ) 14 and 2 Hz, 1H), 3.12 (dd, J ) 14 Hz and 3 Hz, 1H), 4.35

(dd, J ) 12 and 3 Hz, 1H), 4.41 (dd, J ) 14 and 2 Hz, 1H), 5.16

(s, 1H), 6.51 (s, 1H), 6.67 (dd, J ) 8 and 2 Hz, 1H), 6.73 (s, 1H),

6.79 (d, J ) 8 Hz, 2H), 7.19 (t, J ) 8 Hz, 1H), 7.39 (m, 5H);

LCMS (ESI) m/z 363 (M - H⁺).

(2R,4R,5R)-1-(tert-Butoxycarbonyl)-4-(3-(tert-butyldimethyl-

silyloxy)phenyl)-4,5-dimethylpiperidine-2-carboxylic acid (59).

An oven-dried flask under nitrogen atmosphere was charged with

a solution of 58 (5.73 g, 13.64 mmol) and ¢¢TMEDA in diethyl

ether (30 mL). The solution was cooled to -78 °C and sec-

butyllithium (1.4 M in cyclohexane, 14.6 mL, 20.46 mmol) was

added dropwise over 30 min. After stirring at -78 °C for 4.5 h,

carbon dioxide was bubbled through the solution, and the reaction

mixture was allowed to warm to room temperature overnight. The

mixture was poured into saturated ammonium chloride solution,

and the aqueous layer was extracted once with diethyl ether. The

ether extract was washed with water and brine, dried over sodium

sulfate, filtered, and concentrated under reduced pressure. The crude

product was purified by column chromatography (eluent, dichlo-

romethane/methanol/acetic acid mixtures of increasing polarity).

3-((3S,7R,8R,9RR)-7,8-Dimethyl-3-phenyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)phenol (5). To a solution of 62 (0.57 g,

1.57 mmol) in anhydrous tetrahydrofuran (10 mL) was added

borane-dimethyl sulfide complex (2 M solution in tetrahydrofuran,

4.7 mL, 9.4 mmol), and the reaction was heated to reflux under a

nitrogen atmosphere for 16 h. The mixture was then cooled to 0

°C. Methanol (20 mL) was added to the reaction mixture, which

was stirred at 0 °C for 1 h. A 2 M anhydrous solution of hydrogen

chloride in diethyl ether (5 mL) was then added to the reaction,

which was heated to reflux for 1 h. After cooling to room

temperature, an aqueous ammonium hydroxide solution (5 mL) was

added to the mixture, which was stirred for 10 min at room

temperature. The mixture was concentrated under reduced pressure.

The residue was dissolved in methanol and concentrated under

reduced pressure. This process was repeated three times. The crude

product was purified by column chromatography (eluent: dichlo-

romethane/methanol/ammonium hydroxide mixtures of increasing

1

Yield 73% (white solid); H NMR (CDCl₃) δ 0.19 (s, 6H), 0.69

(d, J ) 7 Hz, 3H), 0.98 (s, 9H), 1.37 (s, 3H), 1.46 (s, 9H), 2.01

(dd, J ) 14 and 6 Hz, 2H), 2.40 (t, J ) 12 Hz, 1H), 3.37 (dd, J )

14 and 8 Hz, 1H), 3.74 (m, 1H), 4.30 (dd, J ) 11 and 6 Hz, 1H),

6.67 (dd, J ) 8 and 2 Hz, 1H), 6.77 (s, 1H), 6.88 (d, J ) 7 Hz,

1H), 7.14 (t, J ) 8 Hz, 1H); LCMS (ESI) m/z 464 (M + H⁺).

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((S)-2-methoxy-2-oxo-1-phenylethylcarbamoyl)-4,5-dimeth-

ylpiperidine-1-carboxylate (60). To a stirred solution of 59 (2 g,

4.32 mmol) in acetonitrile (20 mL) under a nitrogen atmosphere

was added, sequentially, N,N-diisopropylethylamine (3 mL, 17.28

mmol), (S)-methyl 2-amino-2-phenylacetate (1.04 g, 5.18 mmol),

and ¢¢TBTU (2.08 g, 6.48 mmol). The reaction was stirred at room

temperature overnight, poured into saturated ammonium chloride

solution, and extracted with ethyl acetate. The organic extracts were

washed with saturated brine, 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 82% (white solid); ¹H NMR

(CDCl₃) δ 0.18 (s, 6H), 0.54 (d, J ) 6 Hz, 3H), 0.98 (s, 9H), 1.34

(s, 3H), 1.38 (s, 9H), 2.03 (m, 2H), 2.40 (t, J ) 13 Hz, 1H), 3.03

(dd, J ) 13 and 8 Hz, 1H), 3.74 (s, 3H), 3.92 (dd, J ) 11 and 8

1

polarity). Yield 69% (white solid); H NMR (CD₃OD) δ 0.79 (d,

J ) 7 Hz, 3H), 1.38 (s, 3H), 1.57 (d, J ) 13 Hz, 1H), 1.95 (t, J )

12 Hz, 1H), 2.06 (m, 1H), 2.30 (t, J ) 13 Hz, 1H), 2.49 (t, J ) 11

Hz, 1H), 2.57 (dd, J ) 12 and 2 Hz, 1H), 2.81 (m, 3H), 3.06 (dd,

J ) 13 and 2 Hz, 1H), 4.04 (dd, J ) 11 and 3 Hz, 1H), 6.59 (dd,

J ) 8 and 2 Hz, 1H), 6.72 (t, J ) 2 Hz, 1H), 6.76 (d, J ) 7 Hz,

1H), 7.11 (t, J ) 8 Hz, 1H), 7.30 (m, 1H), 7.36 (t, J ) 7 Hz, 2H),

7.41 (d, J ) 7 Hz, 2H); LCMS (ESI) m/z 337 (M + H⁺). Anal.

(C₂₂H₂₈N₂O‚0.33H₂O) C, H, N.

1-((3S,7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-3-phen-

yl-hexahydro-1H-pyrido[1,2-R]pyrazin-2(6H)-yl)ethanone (6).

To a solution of 5 (0.1 g, 0.30 mmol) in tetrahydrofuran (5 mL)

was added triethylamine (0.09 g, 0.90 mmol) and acetyl chloride

(0.05 mL, 0.65 mmol), and the reaction was stirred at room

temperature for 1 h. Aqueous 1 N solution of sodium hydroxide

(10 mL) was then added to the reaction mixture, which was stirred

for 1 h. The mixture was concentrated under reduced pressure. The

crude product was purified by column chromatography (eluent:

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7299

dichloromethane/methanol/ammonium hydroxide mixtures of in-

creasing polarity). Yield 53% (white solid); H NMR (CD₃OD) δ

(3S,7R,8R,9RR)-2-Benzyl-7,8-dimethyl-3,8-diphenyl-octahy-

dro-1H-pyrido[1,2-R]pyrazine (9). A solution of 63 (0.35 g, 0.63

mmol) in N,N-dimethylformamide (20 mL) under a nitrogen

atmosphere was treated with triethylamine (0.35 mL, 2.52 mmol),

formic acid (0.1 mL, 2.52 mmol), palladium(II) acetate (0.02 g,

0.09 mmol), and triphenylphosphine (0.03 g, 0.13 mmol), and the

mixture was heated to 60 °C for 18 h. After cooling to room

temperature, the reaction was treated with a 0.5 N aqueous solution

of hydrochloric acid (20 mL) and stirred for 30 min at room

temperature. The reaction mixture was poured into dichloromethane,

and the layers were separated. The organic extracts were washed

with water until the washings attained a pH of 7. The organic

extracts were then dried over sodium sulfate, filtered, and concen-

trated under reduced pressure. The crude product was purified by

column chromatography (eluent: hexane/ethyl acetate mixtures of

1

0.79 (d, J ) 7 Hz, 3H), 1.35 (s, 3H), 1.66 (d, J ) 12 Hz, 1H), 2.05

(m, 5H), 2.82 (dd, J ) 11 and 2 Hz, 1H), 3.09 (m, 1H), 3.61 (br

m, 1H), 3.81 (dd, J ) 14 and 6 Hz, 1H), 4.98 (dd, J ) 10 and 6

Hz, 1H), 6.60 (dd, J ) 8 and 2 Hz, 1H), 6.74 (s, 1H), 6.77 (d, J )

7 Hz, 1H), 7.12 (t, J ) 8 Hz, 1H), 7.26 (m, 3H), 7.33 (m, 2H);

LCMS (ESI) m/z 379 (M + H⁺). Anal. (C₂₄H₃₀N₂O₂‚0.2H₂O) C,

H, N.

3-((3S,7R,8R,9RR)-2,7,8-Trimethyl-3-phenyl-octahydro-1H-

pyrido[1,2-R]pyrazin-8-yl)phenol (7). To a solution of 5 (0.1 g,

0.30 mmol) in tetrahydrofuran (5 mL) and ethanol (5 mL) was

added triethylamine (0.067 g, 0.66 mmol) and formaldehyde (40%

aqueous solution; 0.05 mL, 0.60 mmol). After 10 min, sodium

cyanoborohydride (0.03 g, 0.36 mmol) was added to the mixture,

which was stirred at room temperature for 60 h. The mixture was

concentrated under reduced pressure. The crude product was

purified by column chromatography (eluent: dichloromethane/

methanol/ammonium hydroxide mixtures of increasing polarity).

Yield 76% (white solid); ¹H NMR (CD₃OD) δ 0.77 (d, J ) 7 Hz,

3H), 1.37 (s, 3H), 1.57 (d, J ) 13 Hz, 1H), 1.96 (t, J ) 13 Hz,

1H), 2.07 (s, 4H), 2.24 (t, J ) 11 Hz, 1H), 2.32 (t, J ) 11 Hz,

1H), 2.55 (m, 1H), 2.66 (m, 2H), 2.74 (m, 1H), 2.92 (d, J ) 10

Hz, 1H), 3.24 (d, J ) 9 Hz, 1H), 6.58 (dd, J ) 8 and 2 Hz, 1H),

6.72 (t, J ) 2 Hz, 1H), 6.76 (d, J ) 8 Hz, 1H), 7.11 (t, J ) 8 Hz,

1H), 7.29 (m, 1H), 7.36 (m, 4H); LCMS (ESI) m/z 351 (M + H⁺).

Anal. (C₂₃H₃₀N₂O‚0.4H₂O) C, H, N.

3-((3S,7R,8R,9RR)-2-Benzyl-7,8-dimethyl-3-phenyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (8). To a solution of 5 (1

g, 2.98 mmol) in ethanol (20 mL) was added benzaldehyde (0.95

g, 8.93 mmol), and the reaction mixture was stirred at room

temperature for 10 min. To this was then added BAP (0.82 g, 8.93

mmol), and the reaction mixture was stirred at room temperature

for 18 h. The mixture was concentrated under reduced pressure.

The residue was taken up in ethyl acetate. The mixture was washed

with a saturated aqueous sodium bicarbonate solution, water, and

brine, dried over magnesium sulfate, filtered, and concentrated under

reduced pressure. The crude product was purified by column

chromatography (eluent: dichloromethane/methanol/ammonium

hydroxide mixtures of increasing polarity). Yield 71% (white solid);

¹H NMR (CD₃OD) δ 0.76 (d, J ) 7 Hz, 3H), 1.31 (s, 3H), 1.38 (d,

J ) 13 Hz, 1H), 1.89 (t, J ) 13 Hz, 1H), 2.04 (m, 2H), 2.32 (t, J

) 11 Hz, 1H), 2.48 (m, 1H), 2.54 (dd, J ) 11 and 2 Hz, 1H), 2.69

(t, J ) 3 Hz, 1H), 2.72 (t, J ) 3 Hz, 1H), 2.79 (dd, J ) 11 and 2

Hz, 1H), 2.90 (d, J ) 13 Hz, 1H), 3.51 (dd, J ) 10 and 3 Hz, 1H),

3.74 (d, J ) 13 Hz, 1H), 6.55 (dd, J ) 7 and 1 Hz, 1H), 6.67 (t,

J ) 2 Hz, 1H), 6.71 (d, J ) 7 Hz, 1H), 7.08 (t, J ) 8 Hz, 1H),

7.21 (m, 1H), 7.28 (m, 5H), 7.37 (t, J ) 8 Hz, 2H), 7.53 (d, J )

6 Hz, 2H); LCMS (ESI) m/z 427 (M + H⁺). Anal. (C₂₉H₃₄N₂O) C,

H, N.

3-((3S,7R,8R,9RR)-2-Benzyl-7,8-dimethyl-3-phenyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenyl trifluoromethanesulfonate

(63). To a solution of 8 (0.9 g, 2.11 mmol) in dichloromethane (10

mL) at 0 °C under a nitrogen atmosphere was added N-phenyltri-

fluoromethane sulfonimide (0.83 g, 2.32 mmol) and triethylamine

(0.71 mL, 5.06 mmol). The reaction mixture was allowed to warm

to room temperature overnight and then concentrated under reduced

pressure. The residue was taken up in ethyl acetate. The organic

mixture was washed successively with brine, a 1 N aqueous solution

of sodium hydroxide, and brine, 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 72% (white solid); ¹H NMR

(CDCl₃) δ 0.75 (d, J ) 7 Hz, 3H), 1.33 (s, 3H), 1.44 (d, J ) 12

Hz, 1H), 1.57 (s, 1H), 1.88 (t, J ) 12 Hz, 1H), 2.05 (m, 1H), 2.32

(t, J ) 11 Hz, 1H), 2.44 (m, 1H), 2.51 (dd, J ) 12 and 2 Hz, 1H),

2.67 (dd, J ) 12 and 3 Hz, 1H), 2.76 (dd, J ) 11 and 3 Hz, 1H),

2.83 (dd, J ) 11 and 2 Hz, 1H), 2.88 (d, J ) 14 Hz, 1H), 3.51 (dd,

J ) 11 and 3 Hz, 1H), 3.82 (d, J ) 13 Hz, 1H), 7.09 (m, 2H), 7.24

(m, 4H), 7.30 (m, 4H), 7.37 (m, 3H), 7.53 (m, 1H); LCMS (ESI)

m/z 559 (M + H⁺)

1

increasing polarity). Yield 57% (white solid); H NMR (CD₃OD)

δ 0.72 (d, J ) 7 Hz, 3H), 1.33 (s, 3H), 1.43 (d, J ) 13 Hz, 1H),

1.93 (t, J ) 13 Hz, 1H), 2.08 (m, 2H), 2.31 (t, J ) 12 Hz, 1H),

2.48 (m, 1H), 2.54 (dd, J ) 12 and 2 Hz, 1H), 2.70 (dd, J ) 11

and 2 Hz, 2H), 2.81 (dd, J ) 11 and 2 Hz, 1H), 2.90 (d, J ) 13

Hz, 1H), 3.51 (dd, J ) 11 and 2 Hz, 1H), 3.74 (d, J ) 13 Hz, 1H),

7.12 (m, 1H), 7.26 (m, 10H), 7.37 (t, J ) 8 Hz, 2H), 7.52 (m, 2H);

LCMS (ESI) m/z 411 (M + H⁺). Anal. (C₂₉H₃₄N₂) C, H, N.

Methyl-3-((3S,7R,8R,9RR)-2-benzyl-7,8-dimethyl-3-phenyl-oc-

tahydro-1H-pyrido[1,2-R]pyrazin-8-yl)benzoate (10). To a solu-

tion of 63 (0.5 g, 0.90 mmol) in methanol (6 mL) and dimethyl

sulfoxide (8 mL) was added triethylamine (0.28 mL, 1.97 mmol).

Carbon monoxide was then bubbled through the solution for 5 min.

Palladium(II) acetate (0.02 g, 0.09 mmol) and 1,1′-bis(diphen-

ylphosphino)ferrocene (dppf; 0.1 g, 0.18 mmol) were added to the

mixture. Carbon monoxide was bubbled through the reaction

mixture for 15 min while the reaction was heated to 65 °C. The

reaction mixture was heated at 65 °C under an atmosphere of carbon

monoxide for 18 h. The reaction mixture was poured into water

and extracted with ethyl acetate. The organic extracts were washed

with water and saturated brine solution, 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 76% (white solid);

¹H NMR (CD₃OD) δ 0.72 (d, J ) 7 Hz, 3H), 1.36 (s, 3H), 1.50 (d,

J ) 13 Hz, 1H), 1.98 (t, J ) 13 Hz, 1H), 2.11 (m, 2H), 2.35 (t, J

) 11 Hz, 1H), 2.54 (m, 2H), 2.74 (m, 2H), 2.84 (dd, J ) 11 and

2 Hz, 1H), 2.92 (d, J ) 13 Hz, 1H), 3.52 (dd, J ) 10 and 3 Hz,

1H), 3.74 (d, J ) 13 Hz, 1H), 3.89 (s, 3H), 7.22 (m, 1H), 7.28 (m,

5H), 7.39 (m, 3H), 7.53 (d, J ) 8 Hz, 3H), 7.82 (dd, J ) 8 and 2

Hz, 1H), 7.90 (s, 1H); LCMS (ESI) m/z 469 (M + H⁺). Anal.

(C₃₁H₃₆N₂O₂‚0.33H₂O) C, H, N.

3-((3S,7R,8R,9RR)-2-Benzyl-7,8-dimethyl-3-phenyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)benzoic acid (11). A solution of 10

(0.29 g, 0.62 mmol) in tetrahydrofuran (4 mL) and water (2 mL)

was treated with lithium hydroxide monohydrate (0.08 g, 1.86

mmol) and methanol (10 mL), and the mixture was stirred at room

temperature for 2 days. The reaction mixture was neutralized to

pH ∼6-7 by the addition of a 1 N aqueous solution of hydrochloric

acid. The mixture was then concentrated under reduced pressure.

The residue was taken up in dichloromethane. The organic solution

was dried over sodium sulfate, filtered, and concentrated under

reduced pressure. The product was isolated without further purifica-

tion. Yield 100% (white solid); ¹H NMR (CD₃OD) δ 0.76 (d, J )

7 Hz, 3H), 1.42 (s, 3H), 1.69 (d, J ) 13 Hz, 1H), 2.14 (t, J ) 13

Hz, 1H), 2.27 (m, 1H), 2.34 (m, 1H), 2.80 (t, J ) 11 Hz, 1H), 2.88

(dd, J ) 13 and 2 Hz, 1H), 2.95 (dd, J ) 12 and 2 Hz, 1H), 3.02

(m, 3H), 3.11 (dd, J ) 12 and 3 Hz, 1H), 3.75 (m, 2H), 7.22 (m,

1H), 7.28 (m, 4H), 7.36 (m, 2H), 7.42 (m, 3H), 7.56 (d, J ) 7 Hz,

2H), 7.81 (d, J ) 8 Hz, 1H), 7.90 (s, 1H); LCMS (ESI) m/z 455

(M + H⁺). Anal. (C₃₀H₃₄N₂O₂‚2HCl‚3H₂O) C, H, N.

3-((3S,7R,8R,9RR)-2-Benzyl-7,8-dimethyl-3-phenyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)benzamide (12). To a stirred solu-

tion of 11 (0.22 g, 0.48 mmol) in N,N-dimethylformamide (10 mL)

were added, sequentially, triethylamine (0.15 mL, 1.06 mmol),

ammonium chloride (0.1 g, 2.40 mmol), and ¢¢TBTU (0.23 g, 0.72

7300 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

mmol). The reaction was stirred at room temperature for 4 h, poured

into saturated ammonium chloride solution, and extracted with ethyl

acetate. The organic extracts were washed with saturated brine, dried

over sodium sulfate, and concentrated under reduced pressure. The

crude product was purified by column chromatography (eluent:

dichloromethane/methanol/ammonium hydroxide mixtures of in-

J ) 8 and 3 Hz, 1H), 4.33 (dd, J ) 12 and 6 Hz, 1H), 4.84 (q, J

) 6 Hz, 1H), 6.66 (dd, J ) 8 and 2 Hz, 1H), 6.73 (t, J ) 2 Hz,

1H), 6.84 (d, J ) 8 Hz, 1H), 7.13 (m, 3H), 7.24 (m, 1H), 7.29 (m,

2H); LCMS (ESI) m/z 625 (M + H⁺).

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((R)-1-methoxy-1-oxo-3-phenylpropan-2-ylcarbamoyl)-4,5-

dimethylpiperidine-1-carboxylate (64d). Compound 64d was

synthesized in a manner similar to 64a, using (R)-methyl 2-amino-

1

creasing polarity). Yield 91% (white solid); H NMR (CD₃OD) δ

0.74 (d, J ) 7 Hz, 3H), 1.39 (s, 3H), 1.64 (d, J ) 13 Hz, 1H), 2.06

(t, J ) 13 Hz, 1H), 2.22 (m, 2H), 2.59 (t, J ) 12 Hz, 1H), 2.76 (m,

2H), 2.89 (m, 1H), 2.95 (m, 3H), 3.61 (dd, J ) 11 and 3 Hz, 1H),

3.75 (d, J ) 13 Hz, 1H), 7.22 (m, 1H), 7.28 (m, 6H), 7.40 (t, J )

8 Hz, 3H), 7.47 (d, J ) 8 Hz, 1H), 7.54 (m, 2H), 7.68 (m, 2H),

7.778 (s, 1H); LCMS (ESI) m/z 454 (M + H⁺); HRMS for

C₃₀H₃₅N₃O (M, 453.2780 [M + H]) calcd, 454.2853; found,

454.2853.

1

3-phenylpropanoate. Yield 71% (white foam); H NMR (CDCl₃)

δ 0.19 (s, 6H), 0.43 (d, J ) 7 Hz, 3H), 0.99 (s, 9H), 1.30 (s, 3H),

1.44 (s, 9H), 1.98 (dd, J ) 14 and 6 Hz, 2H), 2.14 (m, 1H), 2.79

(m, 1H), 3.05 (dd, J ) 14 and 6 Hz, 1H), 3.22 (dd, J ) 14 and 6

Hz, 1H), 3.74 (s, 3H), 3.82 (dd, J ) 12 and 4 Hz, 1H), 4.25 (q, J

) 6 Hz, 1H), 4.92 (m, 1H), 6.55 (m, 1H), 6.68 (m, 1H), 6.72 (t, J

) 2 Hz, 1H), 6.81 (d, J ) 8 Hz, 1H), 7.12 (m, 1H), 7.16 (t, J )

8 Hz, 2H), 7.24 (m, 3H); LCMS (ESI) m/z 625 (M + H⁺).

3-((3S,7R,8R,9RR)-7,8-Dimethyl-3-phenyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)benzamide (13). To a solution of 12 (0.1

g, 0.22 mmol) in ethanol (20 mL) was added 10% palladium on

charcoal (0.01 g), and the mixture was stirred at room temperature

under a hydrogen atmosphere for 16 h. The mixture was then

filtered through Celite. The Celite was washed with ethanol, and

the filtrate was evaporated under reduced pressure. The crude

product was purified by column chromatography (eluent: dichlo-

romethane/methanol/ammonium hydroxide mixtures of increasing

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((S)-3-cyclohexyl-1-methoxy-1-oxopropan-2-ylcarbamoyl)-4,5-

dimethylpiperidine-1-carboxylate (64e). Compound 64e was

synthesized in a manner similar to 64a, using (S)-methyl 2-amino-

1

2-cyclohexyl acetate. Yield 82% (white foam); H NMR (CDCl₃)

δ 0.18 (s, 6H), 0.50 (d, J ) 7 Hz, 3H), 0.98 (s, 9H), 1.04-1.16

(m, 2H), 1.19-1.28 (m, 2H), 1.34 (s, 3H), 1.50 (s, 9H), 1.59 (s,

2H), 1.60-1.69 (m, 2H), 1.70-1.84 (m, 3H), 2.00 (m, 2H), 2.34

(t, J ) 13 Hz, 1H), 2.92 (dt, J ) 14 and 4 Hz, 1H), 3.73 (s, 3H),

3.96 (br s, 1H), 4.38 (dd, J ) 11 and 6 Hz, 1H), 4.54 (dd, J ) 9

and 5 Hz, 1H), 6.67 (dd, J ) 8 and 2 Hz, 1H), 6.74 (t, J ) 2 Hz,

1H), 6.86 (dd, J ) 9 and 1 Hz, 1H), 7.13 (t, J ) 8 Hz, 1H); LCMS

(ESI) m/z 617 (M + H⁺).

1

polarity). Yield 12% (white solid); H NMR (CD₃OD) δ 0.77 (d,

J ) 7 Hz, 3H), 1.43 (s, 3H), 1.67 (d, J ) 12 Hz, 1H), 2.02 (t, J )

12 Hz, 1H), 2.18 (m, 1H), 2.24 (t, J ) 11 Hz, 1H), 2.46 (m, 1H),

2.58 (dd, J ) 11 and 2 Hz, 1H), 2.79 (m, 3H), 3.04 (dd, J ) 12

and 3 Hz, 1H), 3.97 (dd, J ) 11 and 3 Hz, 1H), 7.27 (m, 1H), 7.33

(t, J ) 8 Hz, 2H), 7.42(m, 3H), 7.52 (d, J ) 8 Hz, 1H), 7.70 (dd,

J ) 8 and 2 Hz, 1H), 7.83 (s, 1H); LCMS (ESI) m/z 364 (M +

H⁺). Anal. (C₂₃H₂₉N₃O‚0.33H₂O) C, H, N.

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((S)-1-methoxy-4-methyl-1-oxopentan-2-ylcarbamoyl)-4,5-di-

methylpiperidine-1-carboxylate (64f). Compound 64f was syn-

thesized in a manner similar to 64a, using (S)-methyl 2-amino-3-

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((R)-2-methoxy-2-oxo-1-phenylethylcarbamoyl)-4,5-dimeth-

ylpiperidine-1-carboxylate (64a). To a stirred solution of 59 (2

g, 4.32 mmol) in acetonitrile (20 mL) under a nitrogen atmosphere

were added, sequentially, N,N-diisopropylethylamine (3 mL, 17.28

mmol), (R)-methyl 2-amino-2-phenylacetate (1.04 g, 5.18 mmol),

and ¢¢TBTU (2.08 g, 6.48 mmol). The reaction mixture was stirred

at room temperature overnight, poured into a saturated aqueous

solution of ammonium chloride, and extracted with ethyl acetate.

The organic extracts were washed with saturated brine, dried over

sodium sulfate, and concentrated under reduced pressure. The crude

product was purified by column chromatography (eluent: hexane/

ethyl acetate mixtures of increasing polarity). Yield 83% (white

1

methylbutanoate. Yield 75% (white foam); H NMR (CDCl₃) δ

0.18 (s, 6H), 0.50 (d, J ) 7 Hz, 3H), 0.90 (d, J ) 7 Hz, 3H), 0.98

(s, 12H), 1.34 (s, 3H), 1.49 (s, 9H), 1.61 (br s, 1H), 2.05 (m, 2H),

2.19 (m, 1H), 2.36 (t, J ) 13 Hz, 1H), 2.93 (dd, J ) 14 and 11 Hz,

1H), 3.74 (s, 3H), 4.40 (dd, J ) 12 and 6 Hz, 1H), 4.56 (dd, J )

9 and 5 Hz, 1H), 6.67 (dd, J ) 8 and 2 Hz, 1H), 6.75 (t, J ) 2 Hz,

1H), 6.87 (d, J ) 8 Hz, 1H), 7.13 (m, 1H); LCMS (ESI) m/z 577

(M + H⁺).

(R)-Methyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)-2-phenylacetate (65a). To a solution

of 64a (2.18 g, 3.49 mmol) in methanol (100 mL) was added a 2

M solution of hydrogen chloride in diethyl ether (7.2 mL, 14.4

mmol), and the mixture was heated to reflux for 2 h. The solvents

were removed under vacuum, and the residue was taken up in ethyl

acetate (75 mL). A saturated aqueous solution of sodium bicarbonate

(100 mL) was then added to the mixture, which was stirred for 2

h at room temperature. The layers were separated, and the organic

layer was washed with water and brine, dried over sodium sulfate,

filtered, and concentrated under reduced pressure. The product was

washed with hexanes and used in the next step without further

purification. Yield 84% (white solid); ¹H NMR (CDCl₃) δ 0.69 (d,

J ) 7 Hz, 3H), 1.26 (m, 1H), 1.32 (s, 3H), 1.95 (m, 4H), 2.83 (dd,

J ) 12 and 2 Hz, 1H), 3.32 (dd, J ) 12 and 3 Hz, 1H), 3.68 (dd,

J ) 13 and 2 Hz, 1H), 3.73 (s, 3H), 5.60 (d, J ) 8 Hz, 1H), 6.64

(dd, J ) 8 and 2 Hz, 1H), 6.68 (s, 1H), 6.76 (d, J ) 8 Hz, 1H),

7.15 (t, J ) 8 Hz, 1H), 7.37 (m, 5H), 7.80 (d, J ) 7 Hz, 1H);

LCMS (ESI) m/z 397 (M + H⁺).

(S)-Methyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)-3-phenylpropanoate (65c). Com-

pound 65c was synthesized in a manner similar to 65a, using 64c

as starting material. Yield 99% (white solid); ¹H NMR (CDCl₃) δ

0.67 (d, J ) 7 Hz, 3H), 1.28 (s, 3H), 1.92 (m, 3H), 2.75 (dd, J )

13 and 2 Hz, 1H), 3.08 (d, J ) 13 Hz, 0.5H), 3.11 (d, J ) 6 Hz,

0.5H), 3.21 (d, J ) 6 Hz, 1H), 3.27 (m, 2H), 3.55 (dd, J ) 11 and

6 Hz, 1H), 3.74 (s, 3H), 4.88 (q, J ) 6 Hz, 1H), 6.64 (dd, J ) 8

and 2 Hz, 1H), 6.70 (s, 1H), 6.77 (d, J ) 7 Hz, 1H), 7.15 (m, 3H),

7.21 (m, 2H), 7.31 (m, 3H); LCMS (ESI) m/z 411 (M + H⁺).

1

foam); H NMR (CDCl₃) δ 0.17(s, 6H), 0.47 (d, J ) 7 Hz, 3H),

0.97 (s, 9H), 1.34 (s, 3H), 1.46 (s, 9H), 2.00 (m, 2H), 2.37 (t, J )

12 Hz, 1H), 2.94 (dd, J ) 14 and 9 Hz, 1H), 3.73 (s, 3H), 3.90 (m,

1H), 4.37 (dd, J ) 12 and 6 Hz, 1H), 5.57 (d, J ) 7 Hz, 1H), 6.66

(dd, J ) 8 and 2 Hz, 1H), 6.73 (s, 1H), 6.85 (d, J ) 8 Hz, 1H),

7.12 (t, J ) 8 Hz, 1H), 7.35 (m, 5H); LCMS (ESI) m/z 611 (M +

H⁺).

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-(2-methoxy-2-oxoethylcarbamoyl)-4,5-dimethylpiperidine-1-

carboxylate (64b). Compound 64b was synthesized in a manner

similar to 64a, using methyl 2-aminoacetate. Yield 95% (yellow

oil); ¹H NMR (CDCl₃) δ 0.18 (s, 6H), 0.53 (d, J ) 6 Hz 3H), 0.98

(s, 9H), 1.35 (s, 3H), 1.48 (s, 9H), 2.39 (t, J ) 13 Hz, 1H), 3.04

(dd, J ) 14 and 9 Hz, 1H), 3.76 (s, 3H), 3.89 (dd, J ) 14 and 6

Hz, 1H), 4.05 (dd, J ) 10 and 5 Hz, 2H), 4.35 (dd, J ) 11 and 6

Hz, 1H), 6.60 (br s, 1H), 6.67 (dd, J ) 8 and 2 Hz, 1H), 6.76 (t,

J ) 2 Hz, 1H), 6.87 (dd, J ) 8 and 1 Hz, 1H), 7.14 (t, J ) 8 Hz,

3H); LCMS (ESI) m/z 535 (M + H⁺).

(2R,4R,5R)-tert-Butyl-4-(3-(tert-butyldimethylsilyloxy)phenyl)-

2-((S)-1-methoxy-1-oxo-3-phenylpropan-2-ylcarbamoyl)-4,5-

dimethylpiperidine-1-carboxylate (64c). Compound 64c was

synthesized in a manner similar to 64a using (S)-methyl 2-amino-

3-phenylpropanoate. Yield 81% (white solid); ¹H NMR (CDCl₃) δ

0.18 (s, 6H), 0.45 (d, J ) 7 Hz, 3H), 0.98 (s, 9H), 1.32 (s, 3H),

1.45 (s, 9H), 1.98 (m, 2H), 2.33 (t, J ) 13 Hz, 1H), 2.89 (dd, J )

14 and 9 Hz, 1H), 3.12 (t, J ) 6 Hz, 2H), 3.70 (s, 3H), 3.88 (dd,

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7301

Author: In the paragraph below, the starting material information

seems to be missing.

J ) 8 and 2 Hz, 2H), 7.31 (t, J ) 7 Hz, 1H), 7.36 (m, 2H); LCMS

(ESI) m/z 377 (M - H⁺).

(R)-Methyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)-3-phenylpropanoate (65d). Com-

pound 65d was synthesized in a manner similar to 65a, using as

starting material. Yield 77% (white solid); ¹H NMR (CDCl₃) δ 0.68

(d, J ) 7 Hz, 3H), 1.28 (s, 3H), 1.73 (m, 1H), 1.86 (m, 1H), 1.94

(m, 1H), 2.79 (dd, J ) 12 and 2 Hz, 1H), 3.07 (dd, J ) 14 and 8

Hz, 1H), 3.23 (dd, J ) 14 and 6 Hz, 1H), 3.30 (dd, J ) 13 and 3

Hz, 1H), 3.59 (dd, J ) 12 and 3 Hz, 1H), 3.75 (s, 3H), 4.96 (m,

1H), 6.66 (m, 2H), 6.74 (d, J ) 9 Hz, 1H), 7.17 (m, 2H), 7.27 (m,

4H), 7.38 (d, J ) 9 Hz, 1H); LCMS (ESI) m/z 411 (M + H⁺).

(S)-Methyl-3-cyclohexyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-

4,5-dimethylpiperidine-2-carboxamido)propanoate (65e). Com-

pound 65e was synthesized in a manner similar to 65a, using 64e

as starting material. Yield 97% (white foam); ¹H NMR (CDCl₃) δ

0.72 (d, J ) 7 Hz, 3H), 1.02-1.16 (m, 3H), 1.18-1.23 (m, 1H),

1.30 (s, 3H), 1.64 (m, 4H), 1.76 (m, 3H), 1.81-1.89 (m, 1H), 1.93

(m, 1H), 1.99 (m, 1H), 2.84 (dd, J ) 13 and 2 Hz, 1H), 3.32 (dd,

J ) 12 and 3 Hz, 1H), 3.63 (dd, J ) 10 and 6 Hz, 1H), 3.76 (s,

3H), 4.56 (dd, J ) 9 and 5 Hz, 1H), 6.65 (dd, J ) 7 and 2 Hz,

1H), 6.72 (t, J ) 2 Hz, 1H), 6.79 (d, J ) 8 Hz, 1H), 7.16 (t, J )

8 Hz, 1H), 7.44 (d, J ) 9 Hz, 1H); LCMS (ESI) m/z 403 (M +

H⁺).

(S)-Methyl-2-((2R,4R,5R)-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)-4-methylpentanoate (65f). Com-

pound 65f was synthesized in a manner similar to 65a, using 64f

as starting material. Yield 100% (yellow foam); ¹H NMR (CDCl₃)

δ 0.71 (d, J ) 7 Hz, 3H), 0.94 (d, J ) 7 Hz, 3H), 0.97 (d, J ) 7

Hz, 3H), 1.29 (s, 3H), 1.93 (m, 3H), 2.00 (d, J ) 8 Hz, 1H), 2.21

(m, 1H), 2.83 (dd, J ) 13 and 2 Hz, 1H), 3.32 (dd, J ) 12 and 3

Hz, 1H), 3.65 (t, J ) 8 Hz, 1H), 3.76 (s, 3H), 4.58 (dd, J ) 9 and

5 Hz, 1H), 6.65 (dd, J ) 8 and 2 Hz, 1H), 6.72 (t, J ) 2 Hz, 1H),

6.78 (d, J ) 8 Hz, 1H), 7.15 (t, J ) 8 Hz, 1H), 7.47 (d, J ) 9 Hz,

1H); LCMS (ESI) m/z 363 (M + H⁺).

(3R,7R,8R,9RR)-3-Benzyl-8-(3-hydroxyphenyl)-7,8-dimethyl-

hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (66d). Compound

66d was synthesized in a manner similar to 66a, using 65d as

1

starting material. Yield 70% (white solid); H NMR (CD₃OD) δ

0.17 (d, J ) 7 Hz, 3H), 0.98 (t, J ) 13 Hz, 1H), 1.30 (s, 3H), 1.80

(d, J ) 12 Hz, 1H), 1.93 (m, 1H), 3.01 (dd, J ) 14 and 5 Hz, 1H),

3.07 (dd, J ) 13 and 3 Hz, 1H), 3.30 (m, 1H), 4.07 (m, 1H), 4.24

(dd, J ) 13 and 2 Hz, 1H), 4.43 (t, J ) 4 Hz, 1H), 6.45 (m, 2H),

6.58 (dd, J ) 8 and 2 Hz, 1H), 7.07 (t, J ) 8 Hz, 1H), 7.22 (m,

5H); LCMS (ESI) m/z 377 (M - H⁺).

(3S,7R,8R,9RR)-3-Cyclohexyl-8-(3-hydroxyphenyl)-7,8-dimethyl-

hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (66e). Compound

66e was synthesized in a manner similar to 66a, using 65e as

starting material. The reaction was performed in o-xylene instead

of toluene. Yield 37% (yellow solid); ¹H NMR (DMSO-d₆) δ 0.49

(d, J ) 7 Hz, 3H), 1.00-1.29 (m, 6H), 1.34 (s, 3H), 1.45 (m, 1H),

1.53 (d, J ) 8 Hz, 1H), 1.62 (d, J ) 8 Hz, 1H), 1.72 (t, J ) 8 Hz,

1H), 1.84 (m, 1H), 1.96 (t, J ) 13 Hz, 1H), 2.10 (m, 1H), 2.50 (m,

1H), 3.12 (dd, J ) 14 and 3 Hz, 1H), 3.74 (t, J ) 2 Hz, 1H), 4.15

(dd, J ) 12 and 2 Hz, 1H), 4.24 (dd, J ) 13 and 2 Hz, 1H), 6.57

(dd, J ) 8 and 1 Hz, 1H), 6.65 (d, J ) 2 Hz, 1H), 6.69 (d, J ) 8

Hz, 1H), 7.11 (t, J ) 8 Hz, 1H), 8.24 (d, J ) 3 Hz, 1H), 9.24 (s,

1H); LCMS (ESI) m/z 369 (M - H⁺).

(3S,7R,8R,9RR)-8-(3-Hydroxyphenyl)-3-isopropyl-7,8-dimeth-

yl-hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (66f). Com-

pound 66f was synthesized in a manner similar to 66e, using 65f

1

as starting material. Yield 43% (yellow foam); H NMR (CDCl₃)

δ 0.72 (d, J ) 7 Hz, 3H), 0.94 (d, J ) 7 Hz, 3H), 0.97 (d, J ) 7

Hz, 3H), 1.32 (s, 3H), 1.99 (d, J ) 8 Hz, 2H), 2.32 (s, 0.5H), 2.37

(s, 0.5H), 2.45 (s, 1H), 2.83 (dd, J ) 14 and 3 Hz, 1H), 3.34 (m,

1H), 3.64 (m, 1H), 4.58 (dd, J ) 9 and 5 Hz, 1H), 6.66 (m, 1H),

6.72 (m, 1H), 6.80 (d, J ) 8 Hz, 1H), 7.11 (m, 1H); LCMS (ESI)

m/z 329 (M - H⁺).

3-((3R,7R,8R,9RR)-7,8-Dimethyl-3-phenyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)phenol (14). Compound 14 was synthe-

sized in a manner similar to 5, using 66a. Yield 69% (white solid);

¹H NMR (CD₃OD) δ 0.85 (d, J ) 7 Hz, 3H), 1.37 (s, 3H), 1.47 (d,

J ) 13 Hz, 1H), 1.59 (m, 1H), 1.88 (t, J ) 12 Hz, 1H), 2.08 (m,

1H), 2.48 (m, 1H), 2.58 (m, 3H), 2.74 (m, 2H), 3.11 (d, J ) 12

Hz, 1H), 3.57 (m, 1H), 4.07 (d, J ) 3 Hz, 1H), 6.58 (dd, J ) 8 and

2 Hz, 1H), 6.71 (s, 1H), 6.75 (d, J ) 8 Hz, 1H), 7.10 (t, J ) 8 Hz,

1H), 7.22 (t, J ) 8 Hz, 1H), 7.30 (t, J ) 8 Hz, 2H), 7.74 (t, J )

8 Hz, 2H); LCMS (ESI) m/z 337 (M + H⁺). Anal. (C₂₂H₂₈N₂O‚

0.75H₂O) C, H, N.

3-((7R,8R,9RR)-7,8-Dimethyl-octahydro-1H-pyrido[1,2-R]py-

razin-8-yl)phenol (15). Compound 15 was synthesized in a manner

similar to 5, using 66b as starting material. Yield 73% (white solid);

¹H NMR (CDCl₃) δ 0.79 (d, J ) 8 Hz, 3H), 1.36 (s, 3H), 1.46 (d,

J ) 12 Hz, 1H), 1.95 (t, J ) 12 Hz, 1H), 2.03 (br s, 1H), 2.17 (dt,

J ) 11 and 7 Hz, 1H), 2.25 (t, J ) 11 Hz, 1H), 2.52 (dd, J ) 11

and 2 Hz, 1H), 2.74 (m, 3H), 3.02 (m, 3H), 6.60 (s, 1H), 6.63 (dd,

J ) 9 and 1 Hz, 1H), 6.78 (d, J ) 9 Hz, 1H), 7.19 (t, J ) 8 Hz,

1H); LCMS (ESI) m/z 261 (M + H⁺); HRMS for C₁₆H₂₄N₂O (M,

260.1889 [M + H]) calcd, 261.1961; found, 261.1981.

(3R,7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-3-phenyl-

hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (66a). A solution

of 65a (1.28 g, 3.23 mmol) in toluene (100 mL) was heated to

reflux for 60 h. The mixture was concentrated under reduced

pressure. The crude product was purified by column chromatog-

raphy (eluent: dichloromethane/methanol mixtures of increasing

1

polarity). Yield 99% (white solid); H NMR (CDCl₃) δ 0.40 (d, J

) 7 Hz, 3H), 1.37 (s, 3H), 1.75 (br s, 1H), 2.09 (m, 1H), 2.24 (t,

J ) 13 Hz, 1H), 2.43 (dd, J ) 13 and 2 Hz, 1H), 3.11 (dd, J ) 13

and 3 Hz, 1H), 4.27 (dd, J ) 13 and 2 Hz, 1H), 4.38 (dd, J ) 13

and 2 Hz, 1H), 5.15 (s, 1H), 6.64 (dd, J ) 8 and 2 Hz, 1H), 6.71

(s, 1H), 6.75 (m, 2H), 7.16 (t, J ) 8 Hz, 1H), 7.37 (m, 4H); LCMS

(ESI) m/z 363 (M - H⁺).

(7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahydro-

6H-pyrido[1,2-R]pyrazine-1,4-dione (66b). To a solution of 64b

(1.55 g, 2.90 mmol) in methanol (20 mL) was added a 4 M

anhydrous solution of hydrogen chloride in dioxane (2.2 mL, 8.8

mmol), and the mixture was heated to reflux for 2 h. Triethylamine

(3.51 g, 34.8 mmol) was then added, and the mixture was heated

to reflux for 60 h. The reaction mixture was concentrated under

reduced pressure. The crude product was purified by column

chromatography (eluent: dichloromethane/methanol mixtures of

1

3-((3S,7R,8R,9RR)-3-Benzyl-7,8-dimethyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)phenol (16). Compound 16 was synthe-

sized in a manner similar to 5, using 66c as starting material. Yield

increasing polarity). Yield 100% (white solid); H NMR (CDCl₃)

δ 0.64 (d, J ) 6 Hz, 3H), 1.43 (s, 3H), 1.59 (s, 1H), 2.16 (m, 1H),

2.22 (t, J ) 13 Hz, 1H), 2.37 (m, 1H), 3.16 (dd, J ) 14 and 3 Hz,

1H), 3.49 (s, 1H), 4.23 (dd, J ) 14 and 3 Hz, 1H), 4.50 (dd, J )

14 and 3 Hz, 1H), 6.26 (br s, 1H), 6.70 (m, 2H), 6.81 (d, J ) 8 Hz,

1H), 7.20 (t, J ) 8 Hz, 1H); LCMS (ESI) m/z 287 (M - H⁺).

(3S,7R,8R,9RR)-3-Benzyl-8-(3-hydroxyphenyl)-7,8-dimethyl-

hexahydro-6H-pyrido[1,2-R]pyrazine-1,4-dione (66c). Compound

66c was synthesized in a manner similar to 66a, using 65c as

1

43% (white solid); H NMR (CD₃OD) δ 0.67 (d, J ) 7 Hz, 3H),

1.29 (s, 3H), 1.43 (d, J ) 8 Hz, 1H), 1.79 (t, J ) 12 Hz, 1H), 1.87

(t, J ) 11 Hz, 1H), 1.96 (m, 3H), 2.25 (dt, J ) 13 and 3 Hz, 1H),

2.40 (dd, J ) 12 and 2 Hz, 1H), 2.52 (d, J ) 12 Hz, 1H), 2.56 (m,

1H), 2.67 (m, 2H), 2.82 (dd, J ) 12 and 2 Hz, 1H), 3.03 (m, 1H),

6.52 (dd, J ) 8 and 2 Hz, 1H), 6.64 (t, J ) 2 Hz, 1H), 6.68 (d, J

) 8 Hz, 1H), 7.04 (t, J ) 8 Hz, 1H), 7.17 (d, J ) 7 Hz, 3H), 7.25

(t, J ) 7 Hz, 2H); LCMS (ESI) m/z 351 (M + H⁺). Anal.

(C₂₃H₃₀N₂O‚0.2H₂O) C, H, N.

1

starting material. Yield 38% (yellow solid); H NMR (CDCl₃) δ

0.59 (d, J ) 7 Hz, 3H), 1.21 (s, 3H), 2.00 (m, 1H), 2.16 (m, 1H),

2.87 (dd, J ) 14 and 3 Hz, 1H), 3.05 (d, J ) 11 Hz, 1H), 3.20 (t,

J ) 4 Hz, 2H), 4.38 (m, 2H), 6.31 (br s, 1H), 6.64 (m, 1H), 6.69

(m, 1H), 6.76 (d, J ) 8 Hz, 1H), 7.17 (t, J ) 8 Hz, 2H), 7.21 (dd,

3-((3R,7R,8R,9RR)-3-Benzyl-7,8-dimethyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)phenol (17). Compound 17 was synthe-

7302 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

sized in a manner similar to 5, using 66d as starting material. Yield

80% (white solid); H NMR (CD₃OD) δ 0.89 (d, J ) 7 Hz, 3H),

H⁺); HRMS for C₁₈H₂₆N₂O₂(M, 302.1994 [M + Na]) calcd,

325.1886; found, 325.1880.

1

1.33 (s, 3H), 1.56 (d, J ) 14 Hz, 1H), 2.06 (m, 2H), 2.29 (dd, J )

12 and 3 Hz, 1H), 2.38 (dd, J ) 12 and 2 Hz, 1H), 2.50 (m, 2H),

2.70 (dd, J ) 12 and 3 Hz, 1H), 2.92 (m, 2H), 3.10 (t, J ) 11 Hz,

1H), 3.38 (d, J ) 10 Hz, 2H), 6.60 (dd, J ) 8 and 2 Hz, 1H), 6.73

(t, J ) 2 Hz, 1H), 6.77 (d, J ) 8 Hz, 1H), 7.12 (t, J ) 8 Hz, 1H),

7.23 (d, J ) 7 Hz, 3H), 7.30 (t, J ) 8 Hz, 2H); LCMS (ESI) m/z

351 (M + H⁺); HRMS for C₂₃H₃₀N₂O (M, 350.2358 [M + H])

calcd, 351.2431; found, 351.2438.

((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahydro-

1H-pyrido[1,2-R]pyrazin-2(6H)-yl)(phenyl)methanone (22). Com-

pound 22 was synthesized in a fashion similar to compound 21,

using benzoyl chloride as starting material. Yield 57% (white solid);

¹H NMR (CD₃OD) δ 0.78 (br s, 3H), 1.29 (br s, 1.5H), 1.37 (br s,

1.5H), 1.63 (m, 0.5H), 1.82 (m, 0.5H), 1.99 (m, 0.5H), 2.06 (br s,

1H), 2.25-2.35 (m, 1.4H), 2.38 (m, 0.6H), 2.61 (m, 1H), 2.70 (m,

1H), 2.79 (dd, J ) 12 and 2 Hz, 1H), 2.86 (m, 0.5H), 3.06 (m,

1H), 3.37 (m, 1H), 3.59 (m, 1H), 4.60 (m, 1H), 6.58 (m, 1H), 6.67

(m, 1H), 6.77 (m, 1H), 7.11 (m, 1H), 7.44 (m, 2H), 7.48 (m, 3H);

LCMS (ESI) m/z 365 (M + H⁺); HRMS for C₂₃H₂₈N₂O₂(M,

364.2151 [M + H]) calcd, 365.2224; found, 365.2235.

1-((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahydro-

1H-pyrido[1,2-R]pyrazin-2(6H)-yl)-2-phenylethanone (23). Com-

pound 23 was synthesized in a fashion similar to compound 21,

using phenylacetyl chloride as starting material. Yield 96% (yellow

foam); ¹H NMR (CD₃OD) δ 0.72 (d, J ) 7 Hz, 1H), 0.75 (d, J )

7 Hz, 2H), 1.13 (s, 1.5H), 1.30 (s, 1.5H), 1.55 (d, J ) 14 Hz, 0.5H),

1.79 (m, 1H), 1.89 (d, J ) 11 Hz, 0.5H), 1.98 (m, 1.4H), 2.04 (m,

1H), 2.19 (m, 0.6H), 2.53 (m, 2H), 2.65 (m, 1.7H), 2.86 (m, 1.3H),

3.78 (d, J ) 15 Hz, 1.5H), 3.86 (d, J ) 15 Hz, 1H), 3.96 (dd, J )

13 and 2 Hz, 0.5H), 4.43 (dt, J ) 13 and 2 Hz, 0.5H), 4.49 (dd, J

) 13 and 2 Hz, 0.5H), 6.57 (d, J ) 8 Hz, 1H), 6.65 (s, 1H), 6.70

(s, 1H), 6.74 (d, J ) 8 Hz, 1H), 7.09 (m, 1H), 7.25 (m, 1H), 7.32

(m, 3H); LCMS (ESI) m/z 379 (M + H⁺). Anal. (C₂₄H₃₀N₂O₂‚

0.1H₂O) C, H, N.

3-((3S,7R,8R,9RR)-3-Cyclohexyl-7,8-dimethyl-octahydro-1H-

pyrido[1,2-R]pyrazin-8-yl)phenol (18). Compound 18 was syn-

thesized in a manner similar to 5, using 66e as starting material.

Yield 68% (white solid); ¹H NMR (CD₃OD) δ 0.76 (d, J ) 7 Hz,

3H), 1.12 (m, 2H), 1.25 (m, 2H), 1.34 (m, 4H), 1.47 (m, 1H), 1.56

(d, J ) 13 Hz, 1H), 1.70 (d, J ) 10 Hz, 1H), 1.84 (m, 5H), 2.09

(m, 2H), 2.46 (m, 1H), 2.59 (dd, J ) 11 and 2 Hz, 1H), 2.77 (m,

2H), 2.93 (m, 2H), 3.13 (dd, J ) 12 and 3 Hz, 1H), 6.59 (dd, J )

8 and 2 Hz, 1H), 6.73 (m, 2H), 7.10 (t, J ) 8 Hz, 1H); LCMS

(ESI) m/z 343 (M + H⁺). Anal. (C₂₂H₃₄N₂O‚1.5H₂O) C, H, N.

3-((3S,7R,8R,9RR)-3-Isopropyl-7,8-dimethyl-octahydro-1H-

pyrido[1,2-R]pyrazin-8-yl)phenol (19). Compound 19 was syn-

thesized in a manner similar to 5, using 66f as starting material.

Yield 22% (white solid); ¹H NMR (CD₃OD) δ 0.75 (d, J ) 7 Hz,

3H), 0.94 (d, J ) 7 Hz, 3H), 0.98 (d, J ) 7 Hz, 3H), 1.34 (s, 3H),

1.47 (d, J ) 13 Hz, 1H), 1.58 (sx, J ) 7 Hz, 1H), 1.83 (d, J ) 8

Hz, 1H), 1.88 (t, J ) 9 Hz, 1H), 2.03 (m, 1H), 2.26 (m, 1H), 2.55

(m, 3H), 2.72 (dd, J ) 12 and 3 Hz, 1H), 2.79 (dd, J ) 11 and 3

Hz, 1H), 2.88 (dd, J ) 12 and 3 Hz, 1H), 6.57 (dd, J ) 8 and 3

Hz, 1H), 6.70 (d, J ) 2 Hz, 1H), 6.73 (d, J ) 8 Hz, 1H), 7.09 (t,

J ) 8 Hz, 1H); LCMS (ESI) m/z 303 (M + H⁺). Anal. (C₁₉H₃₀N₂O)

C, H, N.

3-((7R,8R,9RR)-2,7,8-Trimethyl-octahydro-1H-pyrido[1,2-R]-

pyrazin-8-yl)phenol (20). To a solution of 15 (0.1 g, 0.39 mmol)

in tetrahydrofuran (5 mL) and ethanol (5 mL) was added triethyl-

amine (0.085 g, 0.85 mmol) and formaldehyde (40% aqueous

solution; 0.06 mL, 0.77 mmol). After 10 min, sodium cyanoboro-

hydride (0.03 g, 0.46 mmol) was added to the mixture, which was

stirred at room temperature for 2 h. The mixture was concentrated

under reduced pressure. The crude product was purified by column

chromatography (eluent: dichloromethane/methanol/ammonium

hydroxide mixtures of increasing polarity). Yield 4% (white solid);

¹H NMR (CD₃OD) δ 0.80 (d, J ) 7 Hz, 3H), 1.42 (s, 3H), 1.77 (d,

J ) 13 Hz, 1H), 2.10 (t, J ) 13 Hz, 1H), 2.26 (m, 1H), 2.94 (s,

3H), 3.00 (d, J ) 12 Hz, 2H), 3.15 (m, 2H), 3.59 (m, 2H), 6.63

(dd, J ) 8 and 2 Hz, 1H), 6.71 (t, J ) 2 Hz, 1H), 6.75 (d, J ) 8

Hz, 1H), 7.15 (t, J ) 8 Hz, 1H); LCMS (ESI) m/z 275 (M + H⁺);

HRMS for C₁₇H₂₆N₂O (M, 274.2045 [M + H]) calcd, 274.2118;

found, 275.2122.

1-((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahydro-

1H-pyrido[1,2-R]pyrazin-2(6H)-yl)-3-phenylpropan-1-one (24).

Compound 24 was synthesized in a fashion similar to compound

21, using phenylpropanoyl chloride as starting material. Yield 54%

1

(white solid); H NMR (CD₃OD) δ 0.75 (m, 3H), 1.28 (s, 1.4H),

1.31 (s, 1.6H), 1.43 (d, J ) 11 Hz, 0.5H), 1.53 (d, J ) 13 Hz,

0.5H), 1.85 (m, 2H), 2.03 (m, 2H), 2.47 (m, 2H), 2.64 (m, 3H),

2.77 (m, 2H), 2.93 (m, 2.5H), 3.17 (dt, J ) 13 and 3 Hz, 0.5H),

3.68 (d, J ) 13 Hz, 0.5H), 3.82 (d, J ) 13 Hz, 0.5H), 4.41 (dd, J

) 13 and 3 Hz, 0.5H), 4.48 (d, J ) 12 Hz, 0.5H), 6.59 (dd, J ) 8

and 1 Hz, 1H), 6.71 (m, 2H), 7.11 (m, 1H), 7.19 (m, 1H), 7.25 (m,

4H); LCMS (ESI) m/z 393 (M + H⁺). Anal. (C₂₅H₃₂N₂O₂‚0.25H₂O)

C, H, N.

(7R,8R,9RR)-8-(3-(Benzyloxy)phenyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazine (67). To a stirred solution of 15 (1 g,

3.85 mmol) in tetrahydrofuran (20 mL) under nitrogen at 0 °C was

added triethylamine (2.14 mL, 15.40 mmol) and di-tert-butyl

dicarbonate (1.84 g, 8.46 mmol). The solution was stirred at room

temperature for 18 h. The mixture was concentrated under reduced

pressure, and the residue was dissolved in ethyl acetate (20 mL).

The solution was then washed with a 0.5 M aqueous solution of

hydrochloric acid (2 × 25 mL) and dried over sodium sulfate. The

mixture was filtered, and the residue was concentrated under

reduced pressure. The crude product was purified by column

chromatography (eluent: hexane/ethyl acetate mixtures of increasing

polarity) to give (7R,8R,9RR)-tert-butyl 8-(3-hydroxyphenyl)-7,8-

dimethyl-hexahydro-1H-pyrido[1,2-R]pyrazine-2(6H)-carboxyl-

1-((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahydro-

1H-pyrido[1,2-R]pyrazin-2(6H)-yl)ethanone (21). A solution of

15 (0.02 g, 0.08 mmol) in tetrahydrofuran (2 mL) was treated with

triethylamine (0.03 g, 0.32 mmol) and acetyl chloride (0.01 g, 0.17

mmol), and the mixture was stirred overnight at room temperature.

A 1 N aqueous solution of sodium hydroxide (2 mL) was added to

the mixture, and stirring was continued at room temperature for an

additional 12 h. The mixture was poured into saturated ammonium

chloride solution and extracted with ethyl acetate. The organic

extracts were washed with water and brine, dried over sodium

sulfate, filtered, and concentrated under reduced pressure. The crude

product was purified by column chromatography (eluent: dichlo-

romethane/methanol/ammonium hydroxide mixtures of increasing

1

ate. Yield 74% (white solid); H NMR (CDCl₃) δ 0.73 (d, J ) 7

Hz, 3H), 1.35 (s, 3H), 1.48 (s, 9H), 1.52 (s, 1H), 1.66 (s, 1H), 1.97

(m, 2H), 2.19 (dt, J ) 12 and 4 Hz, 1H), 2.26 (m, 1H), 2.56 (dd,

J ) 12 and 2 Hz, 1H), 2.68 (m, 3H), 3.02 (br s, 1H), 4.00 (br s,

1H), 6.64 (dd, J ) 8 and 2 Hz, 1H), 6.74 (t, J ) 2 Hz, 1H), 6.80

(d, J ) 8 Hz 1H), 7.17 (t, J ) 8 Hz 1H); LCMS (ESI) m/z 361 (M

+ H⁺).

1

polarity). Yield 22% (white solid); H NMR (CD₃OD) δ 0.78 (d,

To a stirred solution of (7R,8R,9RR)-tert-butyl 8-(3-hydroxyphen-

yl)-7,8-dimethyl-hexahydro-1H-pyrido[1,2-R]pyrazine-2(6H)-car-

boxylate (1.03 g, 2.86 mmol) in N,N-dimethylformamide (10 mL)

was added benzyl bromide (0.41 mL, 3.43 mmol) and potassium

carbonate (1.18 g, 8.58 mmol), and the reaction mixture was stirred

at room temperature for 18 h. The reaction mixture was then poured

into water (20 mL) and extracted with hexanes. The combined

organics were washed with water and brine and dried over sodium

sulfate. The mixture was filtered, and the filtrate was concentrated

J ) 7 Hz, 3H), 1.33 (s, 1.5H), 1.35 (s, 1.5H), 1.59 (dd, J ) 12 and

2 Hz, 1H), 1.92 (t, J ) 13 Hz, 1H), 2.07 (m, 1H), 2.11 (s, 1.5H),

2.13 (s, 1.5H), 2.23 (m, 2H), 2.49 (t, J ) 12 Hz, 0.5H), 2.59 (d, J

) 12 Hz, 1H), 2.79 (m, 2.5H), 3.00 (t, J ) 12 Hz, 0.5H), 3.38 (m,

0.5H), 3.79 (dt, J ) 13 and 2 Hz, 0.5H), 3.86 (dt, J ) 13 and 2

Hz, 0.5H), 4.40 (dt, J ) 13 and 2 Hz), 4.48 (dd, J ) 13 and 2 Hz,

0.5 H), 6.59 (d, J ) 8 Hz, 1H), 6.72 (d, J ) 2 Hz, 1H), 6.76 (d, J

) 8 Hz, 1H), 7.12 (t, J ) 8 Hz, 1H); LCMS (ESI) m/z 303 (M +

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7303

under reduced pressure to give (7R,8R,9RR)-tert-butyl 8-(3-

(benzyloxy)phenyl)-7,8-dimethyl-hexahydro-1H-pyrido[1,2-R]pyra-

zine-2(6H)-carboxylate which was used for next step without further

purification. Yield 99% (white solid); ¹H NMR (CDCl₃) δ 0.75 (d,

J ) 7 Hz, 3H), 1.33 (s, 3H), 1.48 (s, 10H), 1.51 (s, 1H), 1.60 (s,

1H), 1.87 (t, J ) 12 Hz, 1H), 2.01 (m, 1H), 2.18 (m, 1H), 2.52

(dd, J ) 11 Hz and 2 Hz, 2H), 2.67 (m, 2H), 2.96 (br s, 1H), 3.98

(br s, 1H), 5.05 (s, 2H), 6.81 (dd, J ) 8 and 2 Hz, 1H), 6.89 (m,

2H), 7.23 (d, J ) 9 Hz 1H), 7.34 (m, 1H), 7.39 (t, J ) 8 Hz, 2H),

7.45 (d, J ) 9 Hz, 2H); LCMS (ESI) m/z 451 (M + H⁺).

in a manner similar to 5, using 22 as starting material. Yield 100%

(white solid); H NMR (CD₃OD) δ 0.73 (d, J ) 7 Hz, 3H), 1.32

1

(s, 3H), 1.41 (d, J ) 13 Hz, 1H), 1.65 (m, 1H), 1.84 (m, 2H), 2.00

(m, 1H), 2.33 (m, 1H), 2.43 (t, J ) 11 Hz, 1H), 2.56 (dd, J ) 11

and 2 Hz, 1H), 2.70 (m, 1H), 2.74 (m, 1H), 2.82 (dd, J ) 8 and 2

Hz, 1H), 3.56 (m, 2H), 3.58 (m, 1H), 6.56 (dd, J ) 8 and 2 Hz,

1H), 6.68 (m, 1H), 6.72 (d, J ) 8 Hz, 1H), 7.08 (t, J ) 8 Hz, 1H),

7.27 (m, 1H), 7.34 (m, 4H); LCMS (ESI) m/z 351 (M + H⁺);

HRMS for C₂₃H₃₀N₂O (M, 350.2358 [M + H]) calcd, 351.2446;

found, 351.2431.

To a stirred solution of (7R,8R,9RR)-tert-butyl 8-(3-(benzyloxy)-

phenyl)-7,8-dimethyl-hexahydro-1H-pyrido[1,2-R]pyrazine-2(6H)-

carboxylate (1.27 g, 2.82 mmol) in methanol (10 mL) was added

a 2 M anhydrous solution of hydrogen chloride in diethyl ether (6

mL), and the reaction mixture was stirred at room temperature for

18 h. The mixture was concentrated under reduced pressure. A

saturated sodium bicarbonate solution (20 mL) and ethyl acetate

(20 mL) were added to the residue, and the resultant mixture was

stirred at room temperature for 1 h. The layers were separated, and

the organic layer was washed with brine and dried over sodium

sulfate. The mixture was filtered, and the filtrate was concentrated

under reduced pressure to give 67, which was used without further

3-((7R,8R,9RR)-7,8-Dimethyl-2-phenethyl-octahydro-1H-py-

rido[1,2-R]pyrazin-8-yl)phenol (27). Compound 27 was synthe-

sized in a similar fashion as compound 5, using compound 23 as

1

starting material. Yield 42% (white solid); H NMR (CD₃OD) δ

0.76 (d, J ) 7 Hz, 3H), 1.36 (s, 3H), 1.53 (d, J ) 13 Hz, 1H), 1.65

(m, 0.5H), 1.93 (t, J ) 13 Hz, 1H), 1.98 (m, 0.5H), 2.07 (m, 1H),

2.16 (t, J ) 11 Hz, 1H), 2.44 (m, 2H), 2.61 (d, J ) 12 Hz, 1H),

2.71 (m, 2H), 2.79 (d, J ) 10 Hz, 1H), 2.87 (m, 2H), 3.00 (d, J )

9 Hz, 1H), 3.05 (d, J ) 9 Hz, 1H), 3.58 (m, 1H), 6.58 (dd, J ) 7

and 2 Hz, 1H), 6.70 (s, 1H), 6.74 (d, J ) 8 Hz, 1H), 7.10 (t, J )

8 Hz, 1H), 7.19 (m, 1H), 7.24 (m, 2H), 7.28 (m, 2H); LCMS (ESI)

m/z 365 (M + H⁺); HRMS for C₂₄H₃₂N₂O (M, 364.2515 [M +

H]) calcd, 365.2587; found, 365.2582.

1

purification. Yield 100% (yellow oil); H NMR (CDCl₃) δ 0.75

(d, J ) 7 Hz, 3H), 1.34 (s, 3H), 1.45 (d, J ) 13 Hz, 1H), 1.88 (t,

J ) 12 Hz, 1H), 2.00 (m, 1H), 2.27 (m, 2H), 2.48 (dd, J ) 12 and

2 Hz, 1H), 2.60 (t, J ) 11 Hz, 1H), 2.70 (m, 2H), 2.94 (m, 2H),

2.99 (m, 2H), 5.05 (s, 2H), 6.79 (dd, J ) 9 and 2 Hz, 1H), 6.87

(m, 2H), 7.23 (t, J ) 8 Hz 1H), 7.32 (m, 1H), 7.38 (t, J ) 8 Hz,

2H), 7.44 (m, 2H); LCMS (ESI) m/z 351 (M + H⁺)

3-((7R,8R,9RR)-7,8-Dimethyl-2-(3-phenylpropyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (28). Compound 28 was

synthesized in a similar fashion as compound 5, using compound

1

24 as starting material. Yield 45% (white solid); H NMR (CD₃-

OD) δ 0.75 (d, J ) 7 Hz, 3H), 1.34 (s, 3H), 1.50 (d, J ) 12 Hz,

1H), 1.87 (m, 3H), 1.97 (t, J ) 11 Hz, 1H), 2.05 (m, 1H), 2.32 (t,

J ) 12 Hz, 2H), 2.43 (m, 3H), 2.57 (dd, J ) 12 and 2 Hz, 1H),

2.65 (t, J ) 8 Hz, 2H), 2.72 (m, 2H), 2.84 (d, J ) 11 Hz, 1H),

2.91 (d, J ) 9 Hz, 1H), 6.59 (dd, J ) 8 and 2 Hz, 1H), 6.70 (t, J

) 2 Hz, 1H), 6.74 (d, J ) 8 Hz, 1H), 7.10 (t, J ) 8 Hz, 1H), 7.16

(m, 1H), 7.22 (m, 2H), 7.27 (m, 2H); LCMS (ESI) m/z 379 (M +

H⁺). Anal. (C₂₅H₃₄N₂O‚0.5H₂O) C, H, N.

3-((7R,8R,9RR)-7,8-Dimethyl-2-(methylsulfonyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (30). To a solution of 15

(0.1 g, 0.39 mmol) in methylene chloride (5 mL) was added

triethylamine (0.16 mL, 1.17 mmol) and methanesulfonyl chloride

(0.04 mL, 0.47 mmol). The mixture was stirred at room temperature

for 3 h. A 1 N aqueous solution of sodium hydroxide was added,

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

was concentrated in vacuo, and the residue was stirred with ethyl

acetate for 1 h. The mixture was filtered, and the solvents were

evaporated. The crude product was subjected to LC separation.

Yield 15% (white solid); ¹H NMR (CD₃OD) δ 0.77 (d, J ) 7 Hz,

3H), 1.35 (s, 3H), 1.57 (dt, J ) 13 and 2 Hz, 1H), 1.93 (t, J ) 13

Hz, 1H), 2.07 (m, 1H), 2.33 (dt, J ) 12 and 3 Hz, 1H), 2.42 (m,

1H), 2.61 (m, 2H), 2.80 (dt, J ) 12 and 2 Hz, 2H), 2.86 (s, 3H),

2.95 (dt, J ) 12 and 2 Hz, 1H), 3.55 (dt, J ) 12 and 2 Hz, 1H),

3.61 (dt, J ) 11 and 2 Hz, 1H), 6.58 (dd, J ) 8 and 2 Hz, 1H),

6.71 (t, J ) 2 Hz, 1H), 6.76 (d, J ) 7 Hz, 1H), 7.11 (t, J ) 8 Hz,

1H); LCMS (ESI) m/z 339 (M + H⁺). Anal. (C₁₇H₂₆N₂O₃S‚0.5H₂O)

C, H, N.

3-((7R,8R,9RR)-7,8-Dimethyl-2-(phenylsulfonyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (31). To a solution of 67

(0.5 g, 1.43 mmol) in tetrahydrofuran (10 mL) was added

triethylamine (0.6 mL, 4.26 mmol) and benzenesulfonyl chloride

(0.22 mL, 1.72 mmol). The mixture was stirred at room temperature

for 2 h. The mixture was poured into a saturated ammonium

chloride solution and extracted with ethyl acetate. The organic

extracts were washed with water and brine, 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) to give (7R,8R,9RR)-

8-(3-(benzyloxy)phenyl)-7,8-dimethyl-2-(phenylsulfonyl)-octahydro-

1H-pyrido[1,2-R]pyrazine. Yield 70% (white solid); ¹H NMR

(CDCl₃) δ 0.64 (d, J ) 7 Hz, 3H), 1.33 (s, 3H), 1.49 (dd, J ) 12

and 2 Hz, 1H), 1.78 (t, J ) 12 Hz, 1H), 1.99 (m, 1H), 2.09 (t, J )

11 Hz, 1H), 2.39 (m, 1H), 2.48 (m, 3H), 2.71 (m, 2H), 3.60 (dt, J

) 11 and 2 Hz, 1H), 3.69 (dt, J ) 8 and 2 Hz, 1H), 5.04 (s, 2H),

3-((7R,8R,9RR)-7,8-Dimethyl-2-phenyl-octahydro-1H-pyrido-

[1,2-R]pyrazin-8-yl)phenol (25). To a solution of 67 (0.5 g, 1.43

mmol) in dichloromethane (10 mL) was added potassium phenyl-

trifluoroborate (0.5 g, 2.71 mmol), triethylamine (0.4 mL, 2.86

mmol), and cupric acetate (0.2 g, 1.43 mmol). The mixture was

stirred at room temperature for 2 days. The mixture was poured

into water and extracted with dichloromethane. The organic extracts

were washed with water and brine, dried over sodium sulfate,

filtered, and concentrated under reduced pressure. The crude product

was purified by column chromatography (eluent: dichloromethane/

methanol/ammonium hydroxide mixtures of increasing polarity) to

give (7R,8R,9RR)-8-(3-(benzyloxy)phenyl)-7,8-dimethyl-2-phenyl-

octahydro-1H-pyrido[1,2-R]pyrazine. Yield 30% (yellow solid); ¹H

NMR (CDCl₃) δ 0.78 (d, J ) 7 Hz, 3H), 1.37 (s, 3H), 1.55 (t, J )

12 Hz, 1H), 2.02 (m, 2H), 2.44 (m, 1H), 2.49 (m, 1H), 2.60 (t, J

) 12 Hz, 1H), 2.75 (dd, J ) 11 and 3 Hz, 1H), 2.81 (dd, J ) 11

and 3 Hz, 1H), 2.94 (dt, J ) 12 and 3 Hz, 1H), 3.51 (dd, J ) 11

and 2 Hz, 1H), 3.57 (d, J ) 11 Hz, 1H), 5.06 (s, 2H), 6.81 (dd, J

) 8 and 2 Hz, 1H), 6.85 (d, J ) 8 Hz, 1H), 6.88 (d, J ) 5 Hz,

1H), 6.91 (s, 2H), 6.96 (d, J ) 7 Hz, 2H), 7.28 (m, 2H), 7.33 (d,

J ) 7 Hz, 1H), 7.39 (t, J ) 8 Hz, 2H), 7.46 (d, J ) 7 Hz, 2H);

LCMS (ESI) m/z 427 (M + H⁺).

To a solution of (7R,8R,9RR)-8-(3-(benzyloxy)phenyl)-7,8-

dimethyl-2-phenyl-octahydro-1H-pyrido[1,2-R]pyrazine (0.18 g,

0.42 mmol) in ethanol (20 mL) was added 10% palladium on

charcoal (0.02 g), and the mixture was stirred at room temperature

under a hydrogen atmosphere for 16 h. The mixture was then

filtered through Celite. The Celite was washed with ethanol, and

the filtrate was evaporated under reduced pressure. The crude

product was purified by column chromatography (eluent: dichlo-

romethane/methanol/ammonium hydroxide mixtures of increasing

1

polarity) to give 25. Yield 50% (white solid); H NMR (CD₃OD)

δ 0.79 (d, J ) 7 Hz, 3H), 1.37 (s, 3H), 1.61 (d, J ) 12 Hz, 1H),

1.99 (t, J ) 12 Hz, 1H), 2.08 (m, 1H), 2.44 (dt, J ) 11 and 2 Hz,

1H), 2.54 (m, 2H), 2.63 (dd, J ) 11 and 2 Hz, 1H), 2.77 (dd, J )

12 and 3 Hz, 1H), 2.84 (m, 1H), 2.90 (m, 1H), 3.55 (t, J ) 11 Hz,

2H), 6.58 (dd, J ) 8 and 2 Hz, 1H), 6.74 (m, 1H), 6.77 (d, J ) 8

Hz, 1H), 6.83 (t, J ) 7 Hz, 1H), 6.99 (d, J ) 8 Hz, 2H), 7.11 (t,

J ) 8 Hz, 1H), 7.23 (t, J ) 8 Hz, 2H); LCMS (ESI) m/z 337 (M

+ H⁺). Anal. (C₂₂H₂₈N₂O‚0.2H₂O) C, H, N.

3-((7R,8R,9RR)-2-Benzyl-7,8-dimethyl-octahydro-1H-pyrido-

[1,2-R]pyrazin-8-yl)phenol (26). Compound 26 was synthesized

7304 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

6.78 (m, 1H), 6.82 (m, 2H), 7.22 (t, J ) 8 Hz, 1H), 7.34 (m, 1H),

7.39 (t, J ) 8 Hz, 2H), 7.45 (d, J ) 8 Hz, 2H), 7.56 (m, 2H), 7.61

(m, 1H), 7.78 (d, J ) 8 Hz, 1H); LCMS (ESI) m/z 491 (M + H⁺).

To a solution of (7R,8R,9RR)-8-(3-(benzyloxy)phenyl)-7,8-

dimethyl-2-(phenylsulfonyl)-octahydro-1H-pyrido[1,2-R]pyrazine

(0.52 g, 1.06 mmol) in ethanol (20 mL) was added 10% palladium

on charcoal (0.05 g), and the mixture was stirred at room

temperature under a hydrogen atmosphere for 16 h. The mixture

was then filtered through Celite. The Celite was washed with

ethanol, and the filtrate was concentrated under reduced pressure.

The crude product was purified by column chromatography

(eluent: dichloromethane/methanol/ammonium hydroxide mixtures

of increasing polarity) to give 31. Yield 85% (white solid); ¹H NMR

(CD₃OD) δ 0.65 (d, J ) 7 Hz, 3H), 1.31 (s, 3H), 1.52 (d, J ) 12

Hz, 1H), 1.77 (t, J ) 12 Hz, 1H), 2.00 (m, 1H), 2.10 (t, J ) 11

Hz, 1H), 2.30 (dt, J ) 11 and 3 Hz, 1H), 2.37 (m, 1H), 2.43 (dd,

J ) 12 and 3 Hz, 1H), 2.52 (m, 2H), 3.60 (d, J ) 11 Hz, 1H), 3.65

(d, J ) 11 Hz, 1H), 6.57 (dd, J ) 8 and 3 Hz, 1H), 6.67 (s, 1H),

6.70 (d, J ) 7 Hz, 1H), 7.08 (t, J ) 8 Hz, 1H), 7.63 (m, 2H), 7.68

(m, 1H), 7.79 (d, J ) 8 Hz, 2H); LCMS (ESI) m/z 401 (M + H⁺).

Anal. (C₂₂H₂₈N₂O₃S) C, H, N.

2.02 (m, 2H), 2.68 (m, 2H), 3.12 (m, 0.5H), 3.46 (m, 1H), 3.55

(m, 1H), 3.63 (dd, J ) 14 and 4 Hz, 1H), 3.86 (dd, J ) 14 and 6

Hz, 1H), 4.22 (m, 0.5H), 4.54 (m, 1H), 4.63 (dd, J ) 12 and 3 Hz,

1H), 4.75 (d, J ) 14 Hz, 1H), 6.70 (m, 1H), 6.78 (m, 1H), 7.11

(m, 3H), 7.29 (m, 2H), 7.34 (m, 2H); LCMS (ESI) m/z 391 (M -

H⁺).

3-((8R,9R,10RR)-2-Benzyl-8,9-dimethyl-decahydropyrido[1,2-

R][1,4]diazepin-9-yl)phenol (29). Compound 29 was synthesized

in a fashion similar to compound 5, using compound 70 as starting

material. Yield 10% (white solid); ¹H NMR (CD₃OD) δ 0.71 (d, J

) 7 Hz, 3H), 1.27 (s, 3H), 1.58 (m, 0.5H), 1.84 (m, 2H), 1.95 (m,

2H), 2.60 (m, 4H), 2.73 (m, 2H), 2.81 (m, 2H), 2.88 (m, 1H), 3.56

(m, 0.5H) 3.69 (q, J ) 13 Hz, 2H), 6.65 (dd, J ) 8 and 2 Hz, 1H),

6.69 (s, 1H), 7.07 (t, J ) 8 Hz, 1H), 7.24 (m, 1H), 7.31 (t, J ) 8

Hz, 2H), 7.37 (m, 1H); LCMS (ESI) m/z 365 (M + H⁺). Anal.

(C₂₄H₃₂N₂O‚0.33H₂O) C, H, N.

3-((7R,8R,9RR)-7,8-Dimethyl-2-(2-methylbenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (33). To a 10 µM solution

of compound 15 (100 µL) in methanol/acetic acid (8:1) was added

tetramethyl orthoformate (TMOF; 100 µL) and a 12 µM solution

of 2-methylbenzaldehyde (100 µL) in methanol/acetic acid (8:1).

The reaction mixture was shaken for 16 h at room temperature. To

this mixture was added resin-bound cyanoborohydride, and shaking

was continued for 60 h. The reaction mixture was filtered through

a SCX-2 cartridge, and the resin was washed with methanol. The

product was eluted from the cartridge by washing with a 2 M

solution of ammonia in methanol. The product was purified by

liquid chromatographic methods. LCMS (ESI) m/z 365 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(3-methylbenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (34). Compound 34 was

synthesized in a fashion similar to compound 33, using 3-methyl-

benzaldehyde as starting material. LCMS (ESI) m/z 365 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(4-methylbenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (35). Compound 35 was

synthesized in a fashion similar to compound 33, using 4-methyl-

benzaldehyde as starting material. LCMS (ESI) m/z 365 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(2-chlorobenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (36). Compound 36 was

synthesized in a fashion similar to compound 33 using 2-chlo-

robenzaldehyde as starting material. LCMS (ESI) m/z 385 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(3-chlorobenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (37). Compound 35 was

synthesized in a fashion similar to compound 33 using 3-chlo-

robenzaldehyde as starting material. LCMS (ESI) m/z 385 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(4-chlorobenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (38). Compound 38 was

synthesized in a fashion similar to compound 33, using 4-chlo-

robenzaldehyde as starting material. LCMS (ESI) m/z 385 (M +

H⁺).

2-(((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahy-

dro-1H-pyrido[1,2-R]pyrazin-2(6H)-yl)methyl)phenol (39). Com-

pound 39 was synthesized in a fashion similar to compound 33,

using 2-hydroxybenzaldehyde as starting material. LCMS (ESI) m/z

367 (M + H⁺).

3-((7R,8R,9RR)-2-(3-Hydroxybenzyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (40). Compound 40 was

synthesized in a fashion similar to compound 33, using 3-hydroxy-

benzaldehyde as starting material. LCMS (ESI) m/z 367 (M + H⁺).

3-((7R,8R,9RR)-2-(4-Hydroxybenzyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (41). Compound 41 was

synthesized in a fashion similar to compound 33, using 4-hydroxy-

benzaldehyde as starting material. LCMS (ESI) m/z 367 (M + H⁺).

3-((7R,8R,9RR)-2-(4-(Dimethylamino)benzyl)-7,8-dimethyl-oc-

tahydro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (42). Compound

42 was synthesized in a fashion similar to compound 33, using

4-(dimethylamino)benzaldehyde as starting material. LCMS (ESI)

m/z 394 (M + H⁺).

(7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-N-phenyl-

hexahydro-1H-pyrido[1,2-R]pyrazine-2(6H)-carboxamide (32).

To a solution of 15 (0.1 g, 1.43 mmol) in methylene chloride (5

mL) was added triethylamine (0.11 mL, 0.76 mmol) and phenyl-

isocyanate (0.05 mL, 0.46 mmol). The mixture was stirred at room

temperature overnight. The mixture was concentrated in vacuo. The

crude product was purified by column chromatography (eluent:

dichloromethane/methanol/ammonium hydroxide mixtures of in-

1

creasing polarity) Yield 58% (white solid); H NMR (CD₃OD) δ

0.80 (d, J ) 7 Hz, 3H), 1.37 (s, 3H), 1.60 (d, J ) 13 Hz, 1H), 1.94

(t, J ) 13 Hz, 1H), 2.08 (m, 1H), 2.26 (dt, J ) 12 and 3 Hz, 1H),

2.34 (t, J ) 11 Hz, 1H), 2.62 (dd, J ) 11 and 2 Hz, 1H), 2.76 (m,

3H), 3.13 (m, 1H), 4.05 (dt, J ) 13 and 2 Hz, 1H), 4.09 (m, 1H),

6.60 (dd, J ) 8 and 2 Hz, 1H), 6.73 (t, J ) 2 Hz, 1H), 6.77 (d, J

) 8 Hz, 1H), 7.03 (t, J ) 8 Hz, 1H), 7.12 (t, J ) 8 Hz, 1H), 7.27

(t, J ) 8 Hz, 2H), 7.36 (dd, J ) 8 and 1 Hz, 2H); LCMS (ESI) m/z

380 (M + H⁺). Anal. (C₂₃H₂₉N₃O₂‚0.33H₂O) C, H, N.

(2R,4R,5R)-tert-Butyl-2-(benzyl(3-ethoxy-3-oxopropyl)car-

bamoyl)-4-(3-(tert-butyldimethylsilyloxy)phenyl)-4,5-dimethylpi-

peridine-1-carboxylate (68). Compound 68 was synthesized in a

fashion similar to compound 60, using ethyl 3-(benzylamino)-

1

propanoate. Yield 60% (yellow oil); H NMR (CDCl₃) δ 0.18 (s,

6H), 0.47 (m, 3H), 0.88 (m, 1H), 0.98 (s, 9H), 1.14 (s, 2H), 1.24

(m, 4H), 1.47 (s, 9H), 1.64 (s, 1H), 1.89 (m, 1H), 2.01 (m, 0.5H),

2.35 (m, 0.5H), 2.47 (m, 0.5H), 2.56 (m, 0.5H), 2.70 (t, J ) 7 Hz,

1H), 3.36 (m, 1H), 3.54 (m, 1H), 3.89 (m, 1H), 4.11 (m, 2H), 4.46

(m, 0.5H), 4.61 (m, 2H), 4.96 (m, 0.5H), 6.65 (m, 2H), 6.77 (m,

1H), 6.90 (m, 1H), 7.13 (m, 1H), 7.30 (m, 4H); LCMS (ESI) m/z

654 (M + H⁺).

Ethyl-3-((2R,4R,5R)-N-benzyl-4-(3-hydroxyphenyl)-4,5-dimeth-

ylpiperidine-2-carboxamido)propanoate (69). Compound 69 was

synthesized in a fashion similar to compound 61, using compound

68 as starting material. Yield 92% (white solid); ¹H NMR (CDCl₃)

δ 0.65 (d, J ) 7 Hz, 2H), 0.69 (d, J ) 7 Hz, 1H), 1.23 (m, 5.5H),

1.38 (m, 0.5H), 1.46 (m, 1H), 1.50 (m, 0.5H), 1.66 (d, J ) 13 Hz,

0.5H), 1.82 (m, 0.5H), 1.90 (m, 0.5H), 2.08 (m, 1H), 2.20 (t, J )

13 Hz, 0.5H), 2.47 (m, 0.5H), 2.59 (m, 1H), 2.67 (m, 1H), 2.81

(m, 0.5H), 2.86 (m, 1H), 3.27 (dd, J ) 14 and 3 Hz, 0.5H), 3.37

(dd, J ) 14 and 3 Hz, 0.5H), 3.62 (m, 1H), 3.71 (m, 1H), 3.93 (dd,

J ) 12 and 3 Hz, 0.5H), 4.11 (m, 2H), 4.63 (m, 2H), 6.66 (m, 2H),

6.75 (m, 1H), 7.14 (m, 1H), 7.20 (m, 2H), 7.28 (m, 3H); LCMS

(ESI) m/z 439 (M + H⁺).

(8R,9R,10RR)-2-Benzyl-9-(3-hydroxyphenyl)-8,9-dimethyl-

hexahydropyrido[1,2-R][1,4]diazepine-1,5(2H,7H)-dione (70). A

solution of 69 (2.58 g, 5.89 mmol) in o-xylene (200 mL) was heated

to reflux for 60 h. The mixture was concentrated under reduced

pressure. The crude product was purified by column chromatog-

raphy (eluent: dichloromethane/methanol mixtures of increasing

1

polarity). Yield 25% (yellow oil); H NMR (CDCl₃) δ 0.52 (d, J

Methyl 3-(((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-

hexahydro-1H-pyrido[1,2-R]pyrazin-2(6H)-yl)methyl)benzo-

) 7 Hz, 2H), 0.63 (d, J ) 7 Hz, 1H), 1.36 (s, 1H), 1.41 (s, 2H),

NoVel µ-Opioid Receptor Antagonists

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7305

ate (43). Compound 43 was synthesized in a fashion similar to

compound 33, using methyl 3-formylbenzoate as starting material.

LCMS (ESI) m/z 409 (M + H⁺).

methanol/ammonium hydroxide mixtures of increasing polarity).

Yield 39% (white solid); ¹H NMR (CD₃OD) δ 0.75 (d, J ) 7 Hz,

3H), 1.32 (s, 3H), 1.42 (d, J ) 13 Hz, 1H), 1.90 (t, J ) 12 Hz,

1H), 2.03 (m, 2H), 2.35 (m, 2H), 2.38 (s, 4H), 2.57 (d, J ) 11 Hz,

1H), 2.74 (m, 4H), 3.52 (s, 2H), 6.57 (dd, J ) 8 and 2 Hz, 1H),

6.69 (t, J ) 2 Hz, 1H), 6.73 (d, J ) 8 Hz, 1H), 7.09 (t, J ) 8 Hz,

1H), 7.15 (m, 3H), 7.25 (d, J ) 8 Hz, 2H); LCMS (ESI) m/z 365

(M + H⁺). Anal. (C₂₄H₃₂N₂O‚0.16H₂O) C, H, N.

3-((7R,8R,9RR)-7,8-Dimethyl-2-(3-phenoxybenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (44). Compound 44 was

synthesized in a fashion similar to compound 33, using 3-phenoxy-

benzaldehyde as starting material. LCMS (ESI) m/z 443 (M + H⁺).

3-((7R,8R,9RR)-2-(3-(Benzyloxy)benzyl)-7,8-dimethyl-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (45). Compound 45 was

synthesized in a fashion similar to compound 33, using 3-(benzyl-

oxy)benzaldehyde as starting material. LCMS (ESI) m/z 457 (M

+ H⁺).

3-((7R,8R,9RR)-2-(2-Chlorobenzyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (36). Compound 36 was

synthesized in a fashion similar to compound 33, using 2-chlo-

1

robenzaldehyde as starting material. Yield 62% (white solid); H

3-((7R,8R,9RR)-7,8-Dimethyl-2-(naphthalen-2-ylmethyl)-oc-

tahydro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (46). Compound

46 was synthesized in a fashion similar to compound 33, using

2-naphthaldehyde as starting material. LCMS (ESI) m/z 401 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(naphthalen-1-ylmethyl)-oc-

tahydro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (47). Compound

47 was synthesized in a fashion similar to compound 33, using

1-naphthaldehyde as starting material. LCMS (ESI) m/z 401 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(quinolin-4-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (48). Compound 48 was

synthesized in a fashion similar to compound 33, using quinoline-

4-carbaldehyde as starting material. LCMS (ESI) m/z 402 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(pyridin-2-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (49). Compound 49 was

synthesized in a fashion similar to compound 33, using picolinal-

dehyde as starting material. LCMS (ESI) m/z 352 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(pyridin-3-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (50). Compound 50 was

synthesized in a fashion similar to compound 33, using nicotinal-

dehyde as starting material. LCMS (ESI) m/z 352 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(pyridin-4-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (51). Compound 51 was

synthesized in a fashion similar to compound 33, using isonicoti-

naldehyde as starting material. LCMS (ESI) m/z 352 (M + H⁺).

3-((7R,8R,9RR)-2-(Furan-2-ylmethyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (52). Compound 52 was

synthesized in a fashion similar to compound 33, using furan-2-

carbaldehyde as starting material. LCMS (ESI) m/z 341 (M + H⁺).

3-((7R,8R,9RR)-2-(Furan-3-ylmethyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (53). Compound 53 was

synthesized in a fashion similar to compound 33, using furan-3-

carbaldehyde as starting material. LCMS (ESI) m/z 341 (M + H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(thiophen-2-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (54). Compound 54 was

synthesized in a fashion similar to compound 33, using thiophene-

2-carbaldehyde as starting material. LCMS (ESI) m/z 357 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(thiophen-3-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (55). Compound 55 was

synthesized in a fashion similar to compound 33, using thiophene-

3-carbaldehyde as starting material. LCMS (ESI) m/z 357 (M +

H⁺).

3-((7R,8R,9RR)-2-(Cyclohexylmethyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (56). Compound 56 was

synthesized in a fashion similar to compound 33, using cyclohex-

anecarbaldehyde as starting material. LCMS (ESI) m/z 357 (M +

H⁺).

3-((7R,8R,9RR)-7,8-Dimethyl-2-(2-methylbenzyl)-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (33). To a solution of 15

(0.1 g, 0.4 mmol) in ethanol (10 mL) under a nitrogen atmosphere

was added 2-methylbenzaldehyde (0.13 mL, 1.15 mmol), and the

reaction mixture was stirred at room temperature for 10 min. To

this was then added BAP (0.12 mL, 1.15 mmol), and the reaction

mixture was stirred at room temperature overnight. The reaction

mixture was concentrated under reduced pressure. The crude product

was purified by column chromatography (eluent: dichloromethane/

NMR (CD₃OD) δ 0.75 (d, J ) 7 Hz, 3H), 1.34 (s, 3H), 1.45 (d, J

) 13 Hz, 1H), 1.91 (t, J ) 12 Hz, 1H), 2.05 (m, 1H), 2.12 (t, J )

10 Hz, 1H), 2.45 (m, 2H), 2.60 (m, 1H), 2.79 (m, 5H), 3.69 (s,

2H), 6.57 (dd, J ) 8 and 2 Hz, 1H), 6.69 (s, 1H), 6.73 (d, J ) 7

Hz, 1H), 7.09 (t, J ) 7 Hz, 1H), 7.27 (m, 2H), 7.39 (dd, J ) 7 and

2 Hz, 1H), 7.51 (dd, J ) 8 and 2 Hz, 2H); LCMS (ESI) m/z 385

(M + H⁺). Anal. (C₂₃H₂₉ClN₂O‚0.33H₂O) C, H, N.

2-(((7R,8R,9RR)-8-(3-Hydroxyphenyl)-7,8-dimethyl-hexahy-

dro-1H-pyrido[1,2-R]pyrazin-2(6H)-yl)methyl)phenol (39). Com-

pound 39 was synthesized in a fashion similar to compound 33,

using 2-hydroxybenzaldehyde as starting material. Yield 38% (white

solid); ¹H NMR (CD₃OD) δ 0.75 (d, J ) 7 Hz, 3H), 1.34 (s, 3H),

1.47 (d, J ) 12 Hz, 1H), 1.90 (t, J ) 12 Hz, 1H), 2.06 (m, 2H),

2.34 (m, 1H), 2.41 (m, 2H), 2.58 (dd, J ) 12 and 2 Hz, 1H), 2.75

(dd, J ) 12 and 2 Hz, 2H), 2.84 (d, J ) 11 Hz, 1H), 2.90 (d, J )

11 Hz, 1H), 3.59 (m, 1H), 3.66 (m, 1H), 3.74 (s, 2H), 6.56 (dd, J

) 8 and 2 Hz, 1H), 6.68 (t, J ) 2 Hz, 1H), 6.74 (m, 2H), 6.78 (m,

1H), 7.09 (m, 3H); LCMS (ESI) m/z 367 (M + H⁺); HRMS for

C₂₃H₃₀N₂O₂(M, 366.2307 [M + H]) calcd, 367.2380; found,

367.2403.

3-((7R,8R,9RR)-7,8-Dimethyl-2-(thiophen-2-ylmethyl)-octahy-

dro-1H-pyrido[1,2-R]pyrazin-8-yl)phenol (54). Compound 54 was

synthesized in a fashion similar to compound 33, using thiophene-

1

2-carbaldehyde as starting material. Yield 47% (white solid); H

NMR (CD₃OD) δ 0.74 (d, J ) 7 Hz, 3H), 1.34 (s, 3H), 1.48 (m,

1H), 1.91 (t, J ) 12 Hz, 1H), 2.06 (br s, 2H), 2.40 (br s, 2H), 2.62

(m, 1H), 2.82 (m, 4H), 3.80 (s, 2H), 6.58 (dd, J ) 8 and 2 Hz,

1H), 6.69 (s, 1H), 6.72 (d, J ) 8 Hz, 1H), 6.98 (d, J ) 5 Hz, 1H),

7.01 (s, 1H), 7.10 (t, J ) 8 Hz, 1H), 7.34 (d, J ) 5 Hz, 1H); LCMS

(ESI) m/z 357 (M + H⁺). Anal. (C₂₁H₂₈N₂OS‚0.33H₂O) C, H, N.

3-((7R,8R,9RR)-2-(Cyclohexylmethyl)-7,8-dimethyl-octahydro-

1H-pyrido[1,2-R]pyrazin-8-yl)phenol (56). Compound 56 was

synthesized in a fashion similar to compound 33, using cyclohex-

anecarboxaldehyde as starting material. Yield 65% (white solid);

¹H NMR (CD₃OD) δ 0.75 (d, J ) 7 Hz, 3H), 0.91 (m, 2H), 1.25

(m, 3H), 1.35 (s, 3H), 1.47 (d, J ) 13 Hz, 1H), 1.57 (m, 1H), 1.89

(m, 3H), 2.05 (dd, J ) 13 Hz and 2 Hz, 1H), 2.21 (m, 3H), 2.36

(t, J ) 12 Hz, 1H), 2.45 (t, J ) 12 Hz, 1H), 2.58 (d, J ) 11 Hz,

1H), 2.71 (m, 2H), 2.75 (m, 1H), 2.84 (dd, J ) 12 and 2 Hz, 1H),

6.58 (dd, J ) 8 and 2 Hz, 1H), 6.70 (s, 1H), 6.74 (d, J ) 8 Hz,

1H), 7.10 (t, J ) 8 Hz, 1H); LCMS (ESI) m/z 357 (M + H⁺).

Anal. (C₂₃H₃₆N₂O‚0.25H₂O) C, H, N.

B. Biological Methods. Radioligand Binding Assays. Mem-

brane preparations from Chinese hamster ovary (CHO) cells stably

expressing human κ-, µ-, or δ-opioid receptors were prepared as

described previously.¹⁷

The assay buffer used is composed of 50

mM tris(hydroxymethyl) aminomethane HCl, pH 7.8, 1.0 mM

ethylene glycol bis(â-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

(EGTA-free acid), 5.0 mM MgCl

₂, 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 homogenization in a Polytron homo-

genizer (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, 25 000-50 000 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%

7306 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25

Le Bourdonnec et al.

(4) Mitch, C. H.; Leander, J. D.; Mendelsohn, L. G.; Shaw, W. N.; Wong,

D. T.; Cantrell, B. E.; Johnson, B. G.; Reel, J. K.; Snoddy, J. D.;

Takemori, A. E.; Zimmerman, D. M. 3,4-Dimethyl-4-(3-hydroxy-

phenyl)piperidines: Opioid antagonists with potent anorectant activ-

ity. J. Med. Chem. 1993, 36, 2842-2850.

(5) Zimmerman, D. M.; Gidda, J. S.; Cantrell, B. E.; Schoepp, D. D.;

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selective trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid

antagonist for the treatment of gastrointestinal motility disorders. J.

Med. Chem. 1994, 37, 2262-2265.

(6) Thomas, J. B.; Mascarella, S. W.; Rothman, R. B.; Partilla, J. S.;

Xu, H.; McCullough, K. B.; Dersch, C. M.; Cantrell, B. E.;

Zimmerman, D. M.; Carroll, F. I. Investigation of the N-substituent

conformation governing potency and µ receptor subtype selectivity

in (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid an-

tagonists. J. Med. Chem. 1998, 41, 1980-1990.

(7) Le Bourdonnec, B.; Belanger, S.; Cassel, J. A.; Stabley, G. J.;

DeHaven, R. N.; Dolle, R. E. trans-3,4-Dimethyl-4-(3-carboxami-

dophenyl)piperidines: A novel class of µ-selective opioid antagonists.

Bioorg. Med. Chem. Lett. 2003, 13, 4459-4462.

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of the bioactive conformation of the N-substituted trans-3,4-dimethyl-

4-(3-hydroxyphenyl)piperidine class of µ-opioid receptor antagonists.

J. Med. Chem. 2006, 25, 7278-7289.

(9) Mitch, C. H.; Zimmerman, D. M.; Snoddy, J. D.; Reel, J. K.; Cantrell,

B. E. Synthesis and absolute configuration of LY255582, a potent

opioid antagonist. J. Org. Chem. 1991, 56, 1660-1663.

(10) Cossy, J.; Belotti, D. Direct diastereoselective synthesis of (()-cis-

and (()-trans-4-methylpipecolic acid and derivatives. Tetrahedron

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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 (Packard

Instrument Company, Meriden, CT) was added to each filter.

Radioactivity on the filters was determined by scintillation spec-

trometry in a Packard TopCount.

[³H]Diprenorphine with a specific activity of 50 Ci/mmol was

purchased from Perkin-Elmer Life Sciences, Inc. (Boston, MA).

The K_Dvalues for [³H]diprenorphine binding were 0.33 nM for

the κ and µ receptors and 0.26 nM for the δ receptor. Receptor

expression levels, determined as B_maxvalues from Scatchard

analyses, were 4400, 4700, and 2100 fmol/mg of protein for the κ,

µ, and δ receptors, respectively. Preliminary experiments were

performed 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 Bradford.¹⁸

The data from competition experiments were fit by nonlinear

regression analysis with the program Prism (GraphPad Software,

Inc., San Diego, CA) using the four-parameter equation for one-

site competition, and K_ivalues were subsequently calculated from

EC₅₀values by the Cheng-Prusoff equation.

Receptor-Mediated [³⁵S]GTPγS Binding. Receptor-mediated

[³⁵S]GTPγS binding was performed by modifications of the

methods of Selley and co-workers¹⁹and Traynor and Nahorski.²⁰

Assays were carried out in 96-well FlashPlates (Perkin-Elmer Life

Sciences, Inc., Boston, MA). Membranes prepared from CHO cells

expressing the appropriate receptor (50-100 µg of protein) were

added to assay mixtures containing agonist with or without

antagonists, approximately 100 000 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 800 g in a swinging bucket rotor

for 5 min, and bound radioactivity was determined with a TopCount

microplate scintillation counter (Packard Instrument Co., Meriden,

CT).

(13) This material also contained, as an impurity (i.e., up to 20% dependent

upon reaction scale; percentage determined by ¹H NMR), an

additional carboxylic acid derivative, an isomer of 59. The mixture

was used for the next step without further purification.

(14) Crystallographic data for compound 62 has been deposited with the

Cambridge Crystallographic Data Centre as supplementary publica-

tion No. CCDC 604294. Copies of the data can be obtained, free of

charge, on application to CCDC, 12 Union Rd., Cambridge CB2 1EZ,

U.K., (fax: +44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).

(15) MOE 2004.03; Chemical Computing Group: 1010 Sherbrooke St.

West, Suite 910, Montreal, Quebec H3A 2R, Canada.

Antagonist activities were obtained by titration in the presence

of a concentration of loperamide (50 nM) that yielded 80% of its

maximal stimulation (EC₈₀), and the data were analyzed by

nonlinear regression fit using Prism. Potency was expressed as the

concentration of antagonist that achieved 50% of the maximum

inhibition of that antagonist.

(16) Connolly, M. L. Solvent-accessible surfaces of proteins and nucleic

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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.

(18) Bradford, M. M. A Rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of protein-

dye binding. Anal. Biochem. 1976, 72, 248-254.

(19) Selley, D. E.; Sim, L. J.; Xiao, R.; Liu, Q.; Childers, S. R. µ-Opioid

receptor-stimulated guanosine-5′-O-(γ-thio)-triphosphate binding in

rat thalamus and cultured cell lines: signal transduction mechanisms

underlying agonist efficacy. Mol. Pharmacol. 1997, 51, 87-96.

(20) Traynor, J. R.; Nahorski, S. R. Modulation by µ-opioid agonists of

guanosine-5′-O-(3-[³⁵S]thio)triphosphate binding to membranes from

human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 1995, 47,

848-854.

Supporting Information Available: Table of crystallographic

data for compound 62. Table of elemental analyses. This material

is available free of charge via the Internet at http://pubs.acs.org.

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JM0604878

Article Doi

Synthesis and pharmacological evaluation of novel octahydro-1H-pyrido[1,2- a]pyrazine as μ-opioid receptor antagonists

DOI: 10.1021/jm0604878

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