Discovery of N -(1-ethylpropyl)-[3-methoxy-5-(2-methoxy-4- trifluoromethoxyphenyl)-6-methyl-pyrazin-2-yl]amine 59 (NGD 98-2): An orally active corticotropin releasing factor-1 (CRF-1) receptor antagonist

ARTICLE

pubs.acs.org/jmc

Discovery of N-(1-Ethylpropyl)-[3-methoxy-5-(2-methoxy-4-

trifluoromethoxyphenyl)-6-methyl-pyrazin-2-yl]amine 59 (NGD

98ꢀ2): An Orally Active Corticotropin Releasing Factor-1 (CRF-1)

Receptor Antagonist

Kevin J. Hodgetts,* Ping Ge, Taeyoung Yoon, Stꢀephane De Lombaert, Robbin Brodbeck,

Michael Gulianello, Andrzej Kieltyka, Raymond F. Horvath, John H. Kehne, James E. Krause,

George D. Maynard, Diane Hoﬀman, Younglim Lee, Laurence Fung, and Dario Doller

Neurogen Corporation, 35 Northeast Industrial Road, Branford, Connecticut 06405, United States

S Supporting Information

b

ABSTRACT: The design, synthesis, and structureꢀactivity

relationships of a novel series of pyrazines, acting as cortico-

tropin releasing factor-1 (CRF-1) receptor antagonists, are

described. Synthetic methodologies were developed to prepare

a number of substituted pyrazine cores utilizing regioselective

halogenation and chemoselective derivatization. Noteworthy,

an eﬃcient 5-step synthesis was developed for the lead com-

pound 59 (NGD 98ꢀ2), which required no chromatography.

Compound 59 was characterized as an orally bioavailable, brain

penetrant, and highly selective CRF-1 receptor antagonist. Occupancy of rat brain CRF-1 receptors was quantiﬁed using ex vivo

receptor occupancy assays, using both brain tissue homogenates as well as brain slices receptor autoradiography. Behaviorally, oral

administration of 59 signiﬁcantly antagonized CRF-induced locomotor activity at doses as low as 10 mg/kg and dose-dependently

reduced the restraint stress-induced ACTH increases.

’ INTRODUCTION

replacement of the hydrophobic top-chain with a more polar

group (e.g., 4¹⁰and 5¹¹) or substitution of the lipophilic pendant

phenyl ring by a heterocycle (e.g., the pyridines 6¹²and 7¹³and

the thiazole 8¹⁴).

Corticotropin releasing factor (CRF), a 41 amino acid peptide

originally isolated by Vale in 1981 from ovine brain extract, is the

prime regulator of the hypothalamic-pituitary-adrenal (HPA)

stress response.¹CRF exerts its biological functions through

activation of its receptors, CRF-1 and CRF-2, both of which

belong to the class B subfamily of G-protein coupled receptors.²

While the pharmacological beneﬁts of blocking the CRF-2 recep-

tor remain uncertain, evidence from preclinical animal models³

and early clinical studies⁴suggests that antagonism of the CRF-1

receptor has the potential to produce therapeutically useful

anxiolytic and antidepressant eﬀects. The ﬁrst small molecule

CRF-1 receptor antagonists disclosed in the late 1990s, and

subsequently well characterized, include 1 (CP-154,5264),⁵2

(NBI-27914),⁶and 3 (SSR125543A)⁷(Chart 1). These early

analogues were very potent in vitro and demonstrated eﬃcacy in

animal models. However, they are highly lipophilic (cLogP> 7)

and poorly water-soluble. Clinical development of compounds of

this nature, especially as CNS drugs, has been hindered by issues

including unattractive pharmacokinetics, extensive tissue accumu-

lation, and undesirably long elimination half-lives.

A number of CRF-1 receptor antagonists have been reported

to have entered clinical trials for depression and anxiety related

disorders.¹⁵Notably, 7 (R121919) demonstrated eﬃcacy in

treating patients with depression in a small open-label phase

IIa clinical trial, although further development was halted due to

hepatotoxicity issues.¹⁶In a placebo-controlled clinical study,

designed speciﬁcally to evaluate whether subchronic treatment

with 9 (NBI-34041, Chart 2)¹⁷would decrease the stress

hormone response following a psychological stressor, there was

improved resistance to psychological stress by reducing stress

hormone secretion.¹⁸This provided additional clinical support

to the CRF-1 receptor antagonism hypothesis. However, in

a double-blind, placebo-controlled clinical trial for the treat-

ment of major depressive disorder, 10 (CP-316311)¹⁹was not

eﬃcacious.²⁰Likewise, 6 (BMS-562086) also was not eﬃcacious

in generalized anxiety disorder.^12bWhile the clinical results to

date generally have been disappointing, a complete evaluation

requires consideration of a number of factors, including the

In the ensuing years, eﬀorts have focused on reducing the

lipophilicity of these molecules⁸to values considered more

suitable for a CNS drug (cLogP values between 2 and 5).⁹

Successful approaches to lowering lipophilicity have included the

Received: March 28, 2011

Published: May 27, 2011

r

2011 American Chemical Society

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Chart 1. Examples of CRF-1 Receptor Antagonists

potential need to target clinical subpopulations with demon-

strated CRF-1 receptor hyperactivation in stress response

pathways.^15a,bA number of additional compounds,²¹including

11 (CP-376395),^21a5 (BMS-561388),¹¹and GSK 561679,^21b

have been reported to enter clinical trials. The results of these

trials and additional clinical studies, in particular, those with

structurally diverse CRF-1 receptor antagonists, may further

deﬁne the role of CRF in various human illnesses.

Structural features common to most CRF-1 receptor antago-

nists include a core central heterocycle that contains a critical

hydrogen bond acceptor; an out-of-plane pendant aromatic ring;

and a top side-chain ﬁlling a largely hydrophobic area of the

receptor. These have previously been classiﬁed into two topol-

ogies based on distinct structural features.^8cIn the ﬁrst topology,

which contains the majority of compounds to have reached

advanced preclinical development or clinical stage, the hydrogen

bond acceptor and the pendant aryl substituent are separated by

two atoms (e.g., 1), while in the second topology, a single

intervening atom is present (e.g., 3).

Given the relatively limited patentable chemical space within

topology I, we set out to examine the possibility of identifying

and characterizing novel CRF-1 receptor antagonists from the

relatively under-explored topology II template. We have pre-

viously reported that repositioning of the pendant aryl group, the

hydrogen bond acceptor, and the 2-methyl group of the quino-

line 12²²(topology I) led to the identiﬁcation of the novel

isoquinoline 13, which formally belongs to the topology II

template class (Chart 3).²³In an eﬀort to increase hydrophilicity

and improve general pharmacokinetic properties, the bicyclic

isoquinoline was replaced with a less lipophilic, monocyclic

pyrimidine core. Identiﬁed compounds with good in vitro

potencies (e.g., 14) were optimized.²⁴However, despite lower

lipophilicity and increased solubility relative to isoquinoline 13,

very poor oral exposures in rat rendered this series of compounds

unsuitable for further evaluation. The low oral exposures were

attributed, in part, to metabolic liabilities (based on their poor in

vitro microsomal stability), which proved diﬃcult to overcome

within the pyrimidine series. To circumvent this issue, we

investigated other monocyclic heterocycles as replacements of

the pyrimidine core. In this paper, we describe the synthesis and

evaluation of a series of pyrazine analogues (generic structure 15)

as CRF-1 receptor antagonists. A related series of pyrazine

analogues from the topology II class of CRF-1 receptor antago-

nists were also recently described.²⁵The most potent analogue

was 16 with a top-chain derived from cis-1-amino-2-indanol and

the ether linked 4-methylpyridine.

’ CHEMISTRY

The general syntheses of the substituted 3,6-diethyl pyrazines

described in this study are outlined in Scheme 1. Buchwald

coupling of the known 2-chloro-3,6-diethylpyrazine 17²⁶with

1-ethylpropylamine produced amine 18a. Bromination of 18a

with N-bromosuccinimide (NBS) yielded the 5-bromopyrazine

18b. The introduction of diﬀerent aryl groups at the 5-position in

the ﬁnal step was accomplished by Suzuki coupling of 18b with

substituted aryl boronic acids and aﬀorded good yields of the

aryl-pyrazines 19aꢀm. Alternatively, the top-chain could be

introduced in the ﬁnal step. To this end, the aryl ring was ﬁrst

introduced by Suzuki coupling of 17 and 2,4-dichlorophenyl-

boronic acid, which gave 20 in good yield. Direct chlorination of

20 was not possible, but formation of the N-oxide and treatment

with phosphorus oxychloride gave a moderate yield of 21. The

2-chloropyrazine 21 proved to be an excellent intermediate for

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Chart 2. Additional CRF-1 Receptor Antagonists Reported to Have Reached Clinical Trials

Chart 3. Evolution of Topology II CRF-1 Receptor Antagonists

the introduction of a variety of top-chains with diﬀerent linking

atoms in the ﬁnal step. For example, (i) Buchwald amination gave

the N-substituted analogues 22aꢀg; (ii) reaction with the

alkoxide of 3-pentanol gave the ether 23; (iii) addition of the

anion of butan-2-thiol gave the S-linked analogue 24; and (iv)

Suzuki coupling with the borane derived from the hydroboration

of 3-methylenepentane with 9-borabicyclo[3.3.1]nonane (9-BBN)

gave the C-linked analogue 25.

For analogues that keep the 6-ethyl group constant, the

synthesis of compounds with diﬀerent groups at the 3-position

is described in Scheme 2. Commercially available 2,6-dichloro-

pyrazine 26 proved to be an excellent starting material in this

regard. Thermal reaction of 26 with 1-ethylpropylamine gave

only the monosubstituted pyrazine 27 in 84% yield. Optimal

conditions to introduce the 6-ethyl group proved to be a

Kumada,²⁷NiCl₂(dppp)-catalyzed Grignard reaction, which

gave minimal reduction of the starting material and produced

the desired 6-ethylpyrazine 28 in 87% yield. Treatment of 28

with NBS gave predominantly bromination at the 5-position, and

the 5-bromopyrazine 29 was isolated in 70% yield following

chromatography. The aryl ring was introduced by Suzuki cou-

pling of 29 and 2,4-dichlorophenylboronic acid, which gave 30 in

96% yield. With the 2-, 5-, and 6-substituents in place, a number

of groups could be introduced at the 3-position. Halogenation at

the 3-position gave the corresponding 3-ﬂuoro 31, 3-chloro 32,

3-iodo 33, and 3-bromo 34 analogues. The 3-bromopyrazine 34

was used to introduce either an ether substituent at the 3-position

(e.g., 35 and 36) or an alky group (e.g., 37 and 38). In a similar

manner, the 3,6-dimethylpyrazine analogue 39 was prepared

from commercially available 2-chloro-3,6-dimethylpyrazine.

The synthesis of the 3-ethyl-6-methylpyrazine core is shown in

Scheme 3. Bromination of the 2-aminopyrazine 27 aﬀorded

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Scheme 1. General Synthesis of Substituted 3,6-Diethyl Pyrazines^a

^aReaction conditions: (i) 1-ethylpropylamine, Pd₂(dba)₃, BINAP, Na^tOBu, PhMe, 80 °C (71%); (ii) NBS, CHCl₃, 0 °C to room temperature (83%);

(iii) ArB(OH)₂, 2 M K₂CO₃, Pd(PPh₃)₄, PhMe, 85 °C (83%); (iv) 2,4-dichlorophenylboronic acid, 2 M K₂CO₃, Pd(PPh₃)₄, PhMe, 90 °C (98%);

(v) MCPBA, CH₂Cl₂, 0 °C; (vi) OPCl₃, 90 °C (50% over two steps); (vii) HNR¹R², Pd₂(dba)₃, P^tBu₃, K^tOBu, PhMe, 80 °C (57ꢀ79%);

(viii) 3-pentanol, NaH, NMP, 70 °C (77%); (ix) butan-2-thiol, NaH, THF, reﬂux (51%); (x) 3-methylenepentane, 9-BBN, THF, reﬂux, then

Pd(PPh₃)₄, NaOH, H₂O, 50 °C (37%).

5-bromopyrazine 40 in 77% yield. Suzuki coupling of 40 was

selective for the 5-bromo position and gave the pyrazine 41 in 94%

isolated yield. A second bromination gave the 3-bromo-6-chloro-

pyrazine 42. Suzuki coupling of 42 with ethylboronic acid gave a

49% isolated yield of the desired product 43. LCMS andTLC (thin

layer chromatography) analyses of the crude reaction mixture also

indicated the presence of unreacted starting material 42 and the

debrominated product 41. Again, optimal conditions to introduce

the 6-alkyl group proved to be a Kumada coupling reaction, which

minimized formation of reduction side-product and produced the

desired 6-methylpyrazine 44 in 58% isolated yield.

with sodium methoxide gave 49, and bromination with NBS

produced a mixture of the two momobromides 50 and 51b

accompanied by the dibromide 51a, which were separated by

ﬂash chromatography. Nickel-catalyzed reaction of 50 with ethyl

magnesium bromide gave a mixture of the 3-ethylpyrazine 52 and

the reduction product 49, which were separated by chromatog-

raphy in 64% and 29% isolated yields, respectively. Bromination

of 52 gave 53 and Suzuki coupling gave the ﬁnal target 54.

Compounds 55ꢀ65 described in the study were prepared

using chemistry analogous to that outlined in the above schemes.

The 6-chloropyrazine 41 also proved to be a useful inter-

mediate in the synthesis of the 3,6-dimethoxypyrazine 46,

3-methyl-6-methoxypyrazine 47, and the isomeric 3-methoxy-

6-methylpyrazine 48 (Scheme 4). Treatment of 41 with sodium

methoxide installed the 6-methoxy group, and subsequent

bromination with NBS gave 3-bromopyrazine 45. Introduction

of a second methoxy group was possible (sodium methoxide in

NMP at 80 °C for three days) and gave the target 46 in 65% yield.

A Kumada reaction of 45 with methyl magnesium bromide gave a

47% yield of the 3-methyl-6-methoxypyrazine 47. The 3-meth-

oxy-6-methylpyrazine 48 was obtained by reversing the order of

reactions from the 6-chloropyrazine 41 (Kumada nickel-cata-

lyzed Grignard reaction, NBS bromination, and reaction with

sodium methoxide).

’ RESULTS AND DISCUSSION

The compounds described in this study are listed in Tables 1,

2, 3, and 4. The aﬃnity of these compounds for the CRF-1

receptor was determined by using a modiﬁed version of the assay

described by Grigoriadis and De Souza²⁸and by examining their

competition with ¹²⁵I-sauvagine at membrane preparations of

CRF-1 receptors endogenously expressed in IMR-32 human

neuroblastoma cells. The eﬀects of changes to the top-chain are

summarized in Table 1. The 2,4-dichlorophenyl and the 3,6-

diethylpyrazine core were kept unchanged to allow comparison

among compounds in the table. Small, secondary amines were

investigated ﬁrst, with both the isopentyl amine 19a and the

dicyclopropyl analogue 22a having excellent aﬃnity at the CRF-1

receptor (hK_i= 4.1 and 3.7 nM, respectively). However, the

lipophilicity of both compounds was high (cLogP > 7). Intro-

duction of methoxy ether substituents into the top-chain of a

A route to 3-ethyl-6-methoxy substituted analogues, that

allowed the introduction of the 5-aryl group in the ﬁnal step,

was developed (Scheme 5). Reaction of the 6-chloropyrazine 27

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Scheme 2. Representative Synthesis of 3-Substituted-6-Alkyl Pyrazines^a

^aReaction conditions: (i) 1-ethylpropylamine, PrOH, Et₃N, 98 °C (84%); (ii) EtMgBr, THF, NiCl₂(dppp), 10ꢀ15 °C (87%); (iii) NBS, CHCl₃, 0 °C

(70%); (iv) 2,4-dichlorophenylboronic acid, 1 M Na₂CO₃, Pd(PPh₃)₄, DME, 90 °C (96%); (v) SelectFluor, CHCl₃, 0 °C (63%); (vi) NCS, CHCl₃,

0 °C (74%); (vii) NIS, CHCl₃, 0 °C (70%); (viii) NBS, CHCl₃, 0 °C (93%); (ix) NaOR³, R³OH, NMP, 75 °C (49ꢀ85%); (x) R³MgBr, THF,

NiCl₂(dppp), room temperature (65ꢀ76%).

Scheme 3. Representative Synthesis of 3-Ethyl-6-Methyl Pyrazines^a

^aReaction conditions: (i) NBS, CHCl₃, 0 °C (77%); (ii) 2,4-dichlorophenylboronic acid, 1 M Na₂CO₃, Pd(PPh₃)₄, DME, 90 °C (94%); (iii) NBS,

CHCl₃, 0 °C (86%); (iv) EtB(OH)₂, 1 M Na₂CO₃, Pd(PPh₃)₄, DME, 90 °C (49%); (v) MeMgBr, THF, NiCl₂(dppp), 10ꢀ15 °C (58%).

topology I template (e.g., pyrazolotriazine 4) reduced lipophili-

city, while maintaining CRF-1 receptor aﬃnity. Unfortunately, in

the pyrazine series (e.g., 22b), this modiﬁcation resulted in a

considerable loss in aﬃnity (hK_i= 224 nM). Furthermore,

incorporation of a basic amine into the top-chain (e.g., 22c)

was not tolerated. It is also interesting to note that the isopentyl

amine top-chain only gave moderately active compounds

in the topology I pyrazine series previously described by Corbett

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Scheme 4. Synthesis of Various Methoxypyrazines^a

^aReaction conditions: (i) NaOMe, MeOH, NMP, 75 °C; (ii) NBS, CHCl₃, 0 °C (73% over two steps); (iii) NaOMe, MeOH, NMP, 75 °C (65%);

(iv) MeMgBr, THF, NiCl₂(dppp), 10ꢀ15 °C (47%).

Scheme 5. Representative Synthesis of 3-Alkyl-6-Substituted Pyrazines^a

^aReaction conditions: (i) NaOMe, MeOH, NMP, 50 °C (92%); (ii) NBS, CHCl₃, 0 °C (29%); (iii) EtMgBr, THF, NiCl₂(dppp), 0 °C (64%); (iv) NBS,

CHCl₃, 0 °C (98%); (v) 2,4-dichlorophenylboronic acid, 2 M K₂CO₃, Pd(PPh₃)₄, DME, 75 °C (99%).

(hK_i> 100nM); in that series, top-chains derived from cis-1-

amino-2-indanol were preferred (e.g., 16, K_i= 11 nM).²⁵Un-

fortunately, the analogous cis-1-amino-2-indanol derived analo-

gues were not made in the present study and so could not be

compared. In terms of stability, the quinoline²²and pyrimidine²³

templates were plagued by short microsomal half-lives and poor

oral exposures in rat. Despite its high lipophilicity, isopentyl

amine 19a had an encouraging stability in human microsomes

quinoline or pyrimidine series, particularly in human microsomes.

Turnover in rat microsomes for the series was much higher than

in human microsomes, although, in vivo, the compounds tested

generally had lower clearance and longer half-lives than might

have been predicted from microsomal data.

In the pyrimidine template, tertiary alkyl amine top-chains

were the preferred substituent in terms of CRF-1 receptor

activity. However, the pyrazine series was also diﬀerent in this

regard, too. For example, the tertiary amines 22d and 22e were

4ꢀ5-fold less active than the isopentyl amine 19a. Not surpris-

ingly, addition of ethers (22f) or alkyl amine (22g) to the top-

side chain did not improve the aﬃnity. Changing the linking

atom (X) from nitrogen to oxygen (23) or carbon (25) brought

about a 10-fold loss in aﬃnity relative to 19a, while the sulfur

analogue (24, sec-butyl top-chain) was considerably less active.

The 3-pentyl amine top-chain and 3,6-diethylpyrazine core

were kept constant as the 2-aryl group was modiﬁed (Table 2).

(T_1/2= 17 min) and modest stability in rat microsomes (T_1/2

=

4.2 min). In an in vivo rat PK study on 19a, its short microsomal

half-life did not translate to either a short half-life or high clearance

(Table 5). Compound 19a had low clearance (16 mL/min/kg),

compared to 65 mL/min/kg for rat hepatic blood ﬂow, and a

moderate volume of distribution of 6.5 L/kg and a T_1/2of 4.9 h. In

addition, 19a had an oral bioavailability of 83% and high brain

penetration (brain to plasma ratio of 2.5). In the pyrazine series, in

general, microsomal stability was better than in the corresponding

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Table 1. Eﬀects of the Top-Chain and Linking Atom (X) on CRF-1 Receptor Binding

microsomal T_1/2(min)

no

X

R¹

R²

CH(Et)₂

CH(^cPr)₂

cLogP

h

r

hCRF-1 K_i(nM)^a

19a

22a

22b

22c

22d

22e

22f

22g

23

N

H

7.4

7.3

5.3

6.1

7.6

7.7

5.7

6.9

7.6

7.8

7.7

17

11

9

4.2

2.5

4.1 ( 1.4

3.7 ( 1.2

224 ( 56

489 ( 202

26 ( 9

N

O

S

CH₂(CH₂OMe)₂

CH(Me)CH₂NMe₂

7

<1

4.2

c

CH₂Pr

8

ⁿPr

CH₂Pr

12

2

19.2 ( 4

128 ( 37

1538 ( 582

46 ( 27

c

CH₂CH₂OMe

CH₂CH₂NMe₂

CH(Et)₂

<1

ⁿPr

5

-

10

17

13

2.5

24

-

CH(CH₃)CH₂CH₃

4.1

2.5

401 ( 126

68 ( 14

25

CH

H

CH(Et)₂

^aUnless otherwise stated, all values are the mean ( SEM of at least three separate experiments.

Table 2. Eﬀects of the 5-Aryl Substituent on CRF-1 Receptor Binding

microsomal T_1/2(min)

no

R⁵

Cl

R⁶

Cl

R⁷

X

Y

ClogP

h

r

hCRF-1 K_i(nM)^a

19a

19b

19c

19d

19e

19f

H

CH

N

CH

N

7.4

5.8

6.9

6.3

6.6

6.2

7.3

5.6

6.9

7.3

6.1

5.6

5.3

17

8

4.2

2.5

4.1 ( 1.4

7.3 ( 1.9

7.2 ( 3.4

7.8 ( 4.3

4.2 ( 1.7

23.7 ( 3.8

5.6 ( 0.9

1181 ( 476

4.1 ( 0.8

4.4 ( 2.1

7.6 ( 2.4

11.6 ( 3.8

17.2 ( 6.7

OMe

OH

OMe

OCF₃

OEt

H

13

nt

11

18

16

12

7

H

nt

OiPr

Me

H

<1

nt

H

19g

19h

19i

CF₃

H

OMe

Me

H

5.9

OCF₃

CF₃

H

4.1

4.3

19j

H

19k

19l

H

NMe₂

H

<1

2.5

<1

OMe

N

9

19m

H

N

7

^aUnless otherwise stated, all values are the mean ( SEM of at least three separate experiments; nt = not tested.

For optimal CRF-1 receptor binding, previous studies²⁹sug-

gested that the aryl ring lies out of the plane of the core

heterocycle, and that an ortho-substituent is generally required

to enforce this conformation. Our earlier results with topology II

templates were in accord with this model, and, for these reasons,

analogues were focused on the 2,4-disubstitution pattern. The

2,4-dimethoxyphenyl analogue 19b had similar aﬃnity to the 2,4-

dichloro analogue 19a, but it was less stable in human micro-

somes. The ﬁve analogues 19cꢀ19g retained the 2-OMe group

as the 4-position was varied. The 4-OCF₃19c, 4-OEt 19d, and

4-OⁱPr 19e had very similar aﬃnities and stability in human

microsomes. The 4-Me analogue 19f was approximately 3-fold

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Table 3. Eﬀects of Variations to the R³and R⁴Groups on

CRF-1 Receptor Binding

achieved. The 3-methoxy analogue 35 had comparable activity to

the parent 19a. However, increasing the size to ethoxy 36 resulted

in a substantial loss in activity. Increasing the size of the substituent

to butyl 38 resulted in an almost complete loss in activity, and

contracting the 3-ethyl group to3-methyl 37led toa 10-fold loss in

activity. Reversing the R³and R⁴substituents, however, (R³= Et,

R⁴= Me, 44) produced a signiﬁcant boost in aﬃnity (compare 44

with 37), although the size of the R³group could not be reduced

further without a loss in aﬃnity (R³= Me, 39). The analogue 48

(R³= OMe) had both excellent aﬃnity and microsomal stability.

In the ﬁnal three examples, R⁴= OMe was kept constant as the R³

group was varied. The R³= OMe and Et analogues 46 and 54 had

similar activity (hK_i= 8.4 and 8.0 nM) although 54 was more

stable in microsomes. Modiﬁcations to the R³and R⁴groups also

brought about some interesting changes in lipophilicity. Surpris-

ingly, the change in R³from Et to OMe had no eﬀect on ClogP

(compare 19a to 35), whereas the change in R⁴from Et to OMe

resulted in the expected reduction of ClogP from 7.4 to 7.1

(compare 19a to 54). Presumably, in compound 35, an internal

hydrogen bond between the NH and 3-OMe masks the change in

polarity. The same unexpected eﬀect was observed between the

methyl analogues 48 (ClogP = 6.8) and 47 (ClogP = 6.6).

The 3-methoxy-6-ethyl-, 3-methoxy-6-methyl-, and 3-ethyl-6-

methoxy-pyrazine cores (analogues 35, 48, and 54) were identi-

ﬁed as having a good balance between CRF-1 receptor aﬃnity

and lipophilicity, and a number of compounds with diﬀerent

5-aryl groups were prepared with these cores (Table 4). Com-

pounds from all three diﬀerent cores were identiﬁed with

excellent aﬃnity at the human CRF-1 receptor and low turnover

in human microsomes (55ꢀ65). The most potent compound

was the 3-methoxy-6-methyl-pyrazine 59 (hK_i= 1.0 nM), which

was also stable in both human and rat microsomes.

microsomal T_1/2(min)

no R³

R⁴ClogP

h

r

hCRF-1 K_i(nM)^a

19a Et

Et

7.4

6.4

6.6

7.2

7.3

7.4

7.9

8.5

6.9

6.4

6.8

17

28

73

10

nt

4.2

4.1 ( 1.4

545 ( 78

167 ( 26

71 ( 12

30

31

H

F

Et

nt

21

<1

nt

32 Cl

33

I

12 ( 2.1

5.9 ( 0.6

151 ( 27

3657 ( 437

54 ( 7.6

8.6 ( 1.4

27 ( 4.0

4.3 ( 0.8

8.4 ( 1.1

14.4 ( 3.0

8.0 ( 2.2

35 OMe Et

36 OEt Et

38 ⁿBu Et

37 Me Et

35

120

22

27

18

29

nt

74

4.2

11

4.2

3.2

nt

2.5

17

11

44 Et

Me

39 Me Me

48 OMe Me

46 OMe OMe 6.6

47 Me OMe 6.6

16

56

42

54 Et

OMe 7.1

^aUnless otherwise stated, all values are the mean ( SEM of at least three

separate experiments; nt = not tested.

Compound 59 was selected for further evaluation. An im-

proved synthesis, capable of generating multigram quantities, was

developed as outlined in Scheme 6. The Kumada reaction proved

to be the method of choice for the introduction of the methyl

group, and an 87% yield of 66 resulted from the reaction of 27

and methylmagnesium bromide. Bromination of 66 with 2 equiv

of bromine gave the corresponding dibromide, which was used

without puriﬁcation in the next step. Treatment of the dibromide

with sodium methoxide was selective and gave the 3-methoxy-5-

bromopyrazine 67 in an excellent 85% yield. Finally, Suzuki

coupling of 67 with 2-methoxy-4-triﬂuoromethoxyphenylboronic

acid gave an 89% yield of 59. The 5-step sequence, from commer-

cially available 2,6-dichloropyrazine 26, could be performed con-

veniently on a 500 g scale, required no chromatography, and

resulted in an overall yield of 46% of compound 59.

The functional activity of 59 was assessed in whole IMR-32

cells. In this system, the CRF peptide dose-dependently stimu-

lated the production of 3⁰-5⁰-cyclic adenosine monophosphate

(cAMP) (EC₅₀= 0.5 ( 0.1 nM). Under the same conditions,

compound 59 had no eﬀect on cAMP production at concentra-

tions as high as 10 μM. Compound 59, however, dose-depen-

dently antagonized the cAMP production stimulated by a 3.0 nM

concentration of CRF with an IC₅₀of 93 ( 23 nM. These data

conﬁrm that compound 59 is a functional antagonist at the CRF-

1 receptor. Related analogues tested from the series (data not

shown) were also conﬁrmed to be functional antagonists at the

CRF-1 receptor.

less active than the 4-OMe analogue 19b, but switching from Me

to CF₃(19g) improved the receptor aﬃnity. Demethylation of the

ortho methoxy group of 19b gave the 2-phenol 19h and a dramatic

loss in aﬃnity (hK_i= 1181 nM). This loss in aﬃnity can be

attributed to an intramolecular H-bond interaction between the

2-OH and the pyrazine nitrogen, enforcing a more planar orienta-

tion between the core and the aryl ring. Other changes to the

2-position (e.g., 19i (R⁵= OCF₃) and 19j (R⁵= CF₃)) resulted in

compounds of similar aﬃnity to 19b. Our SAR ﬁndings at the

2-position are in contrast to the O- and N-aryl linked pyrazine

series described by Corbett; in that series, 2-substitution on the

aryl ring was not well tolerated. In the O- and N-aryl linked

pyrazine series, compounds presumably adopted an alternative

binding mode and ether-linked 4-methylpyridines were preferred

(e.g., 16, K_i= 11 nM).²⁵Previous approaches to lowering the

lipophilicity of CRF-1 receptor antagonists have included re-

placement of the aryl ring by a more polar heterocycle (e.g., 7,

Figure 1). Indeed, the dimethylaminopyridine 19k, the dimethoxy

pyridine 19l, and the pyrimidine 19m had similar activity, lipo-

philicity, and microsomal stability as 19b.

Variations to the 3- and 6-pyrazine substituents were next

examined, and the data are summarized in Table 3. In examples

30 to 38, the R⁴group was kept constant (R⁴= Et) and the R³

group was varied. Deletion of the R³substituent (30, R = H)

resulted in a dramatic loss in aﬃnity compared to the diethyl

analogue 19a. Introduction of a halogen substituent improved

aﬃnity as the size of the substituent increased from ﬂuoro 31, to

chloro 32, to iodo 33, although aﬃnities under 10 nM were not

The 3,6-diethylpyrazine 19a had an encouraging pharmaco-

kinetic proﬁle in rat. Compound 59 also demonstrated encoura-

ging oral and high brain exposure (Table 5). Following oral

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Table 4. Examples of Compounds with 3-Methoxy-6-ethyl-, 3-Methoxy-6-methyl- and 3-Ethyl-6-methoxy-pyrazine Cores

microsomal T_1/2(min)

no

R³

Et

R⁴

Et

R⁵

Cl

R⁶

ClogP

h

r

hCRF-1 K_i(nM)^a

19a

55

56

57

58

59

60

61

62

63

64

65

Cl

7.4

5.7

5.5

7.3

7.2

6.3

6.2

6.7

5.4

6.5

6.9

17

120

73

4.2

4.1 ( 1.4

1.9 ( 0.2

4.4 ( 0.9

2.2 ( 0.3

1.4 ( 0.2

1.0 ( 0.4

1.3 ( 1.0

4.6 ( 0.9

5.9 ( 0.7

6.1 ( 1.1

1.1 ( 0.3

2.5 ( 0.8

OMe

Et

OMe

120

Et

OMe

CF₃

Cl

CF₃

23

120

47

Et

OMe

120

52

Et

OCHF₂

OCF₃

CF₃

Me

OMe

Cl

120

48

OCHF₂

OEt

Cl

47

OMe

Cl

OCF₃

OMe

36

Et

120

nt

Et

Cl

OCHF₂

17

^aUnless otherwise stated, all values are the mean ( SEM of at least three separate experiments; nt = not tested.

Table 5. Rat Pharmacokinetic Data for 19a and 59^a

no

iv/po dose (mg/kg)

C_max(ng/mL)

AUC_{0ꢀ24 h}(ng.h/mL)

T_max(h)

T_1/2(h)

CL (mL/min/kg)

V_ss(L/kg)

F (%)

B/P

19a

59

3/5

627

456

4309

4499

1.3

4

4.9

8.4

16

12

6.5

7.8

83

45

2.5

5.4

1/10

^aCompounds were dosed in 2% Vitamin E TPGS (tocopheryl polyethylene glycol 1000 succinate).

Figure 1. Rat cortical homogenate CRF-1 receptor binding. Left panel: compound 59 dose-dependently displaces the binding of [¹²⁵I]-sauvagine from

rat CRF-1 receptor (rat cortical membrane homogenate) with a K_i= 9.8 ( 1.7 nM.

administration, 59 also had higher brain penetration (brain to

plasma ratio greater than 5) but lower oral exposure (compounds

19a and 59 had similar AUC_{0ꢀ24 h}, but 59 was at twice the dose).

In a subsequent assessment, plasma protein binding compound

59 was determined to be 99.2% bound to human plasma protein.

In order to quantitatively evaluate the rat cortical CRF-1

receptor occupancy of compounds following oral dosing,

ex vivo binding methods were utilized. Prior to the ex vivo

determinations, a control study using [¹²⁵I]-sauvagine, in which

multiple concentrations of test compound were directly added to

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Scheme 6. Optimized Synthesis of 59^a

^aReaction conditions: (i) MeMgCl, THF, NiCl₂(dppp), room temperature (87%); (ii) Br₂, HOAc (84%); (iii) NaOMe, MeOH, 65 °C (85%);

(iv) 2-methoxy-4-triﬂuoromethoxyphenylboronic acid, 2 M K₂CO₃, Pd(PPh₃)₄, PhMe, 85 °C (89%).

Table 6. Ex Vivo Data for 59 and 7

no

hCRF-1 K_i(nM)

rat cortex K_i(nM)

ex vivo ID₅₀(mg/kg)

ex vivo IC₅₀(ng/mL)

59

7

1.0 ( 0.4

8.7 ( 1.6

9.8 ( 1.7

3.8 ( 1.1

2.0

7.2

71

74

rat cortical membranes, was performed. In the case of 59, this

exogenous addition experiment generated a K_i= 9.8 ( 1.7 nM

(Figure 1, left-hand panel). Quantitative determination of the

CRF-1 receptor occupancy of 59, following oral administration

(1, 3, and 10 mg/kg), was determined by assessing the amount of

speciﬁc [¹²⁵I]-sauvagine bound and comparing that to the

amount speciﬁcally bound in the vehicle-treated rats

(theoretical 100% occupancy). Doseꢀresponse studies were

used to determine the dose of 59 required for 50% receptor

occupancy (ex vivo ID₅₀) at a time point of 3 h; the time

empirically determined from earlier ex vivo receptor binding

studies, with 59, to be the time of maximum receptor occupancy.

The ex vivo ID₅₀of 59 was determined to be 2.0 ( 0.2 mg/kg

(Figure 1, right-hand panel). In satellite cohorts, plasma samples

were taken 3 h after dosing to determine compound levels at each

of the three doses (1.0, 3.0, and 10.0 mg/kg); this allowed the

plasma concentration that was associated with 50% occupancy of

brain CRF-1 receptors (ex vivo IC₅₀) to be calculated. From these

data, the ex vivo IC₅₀of 59 was determined to be 71 ng/mL.

Compound 7, which had demonstrated eﬃcacy in a small open-

label phase IIa clinical trial,¹⁶was also characterized in the ex vivo

receptor occupancy assay. The proﬁles of 59 and 7 are compared

in Table 6. In IMR-32 human neuroblastoma cells, 59 was

approximately 9-fold more active than 7; in rat cortex, however,

the rank order of aﬃnities was reversed, and 7 was 2ꢀ3-fold more

active than 59. Despite the lower aﬃnity in rat cortex, a dose of

2 mg/kg of 59 was required to achieve 50% receptor occupancy at

3 h, whereas a dose of 7.2 mg/kg of 7 was required for the same

receptor occupancy. The lower required dose of 59 presumably

reﬂects a higher brain exposure relative to 7. The plasma con-

centrations associated with 50% occupancy of brain CRF-1

receptors for 7 and 59 weresimilar, 74 and 71 ng/mL, respectively.

Ex vivo receptor autoradiographic analysis of CRF receptors in

the rodent brain was performed. One micromolar oCRF is not

selective for the CRF-1 over the CRF-2 receptor subtype

(Figure 2 top-left), while [¹²⁵I]sauvagine labels both the CRF-1

and the CRF-2 receptor subtypes (Figure 2 top-right). The

receptors, ion channels, and 6 enzymes (see Supporting Infor-

mation for full list) and did not show inhibition greater than 16%

in any assay. In addition, no signiﬁcant hERG (human ether-ꢁa-

go-go related gene) activity was recorded in a whole cell (Cos7)

electrophysiological assay (17% inhibition @ 3 μM). On the

basis of the high aﬃnity and functional antagonism, excellent

oral, and high brain exposure, occupancy of brain CRF-1

receptors, as quantiﬁed by ex vivo receptor binding autoradio-

graphy and selectivity proﬁle, the pyrazine 59 was further

evaluated in several in vivo pharmacology models.

ICV administration of CRF causes a robust increase in

locomotor activity relative to vehicle (Figure 3). The large

increase in movement time elicited by 0.3 μg CRF (compare

TPGS vs TPGS/CRF) was signiﬁcantly reduced by a 3 h

pretreatment with compound 59 at both 10 and 30 mg/kg PO.

To rule out potential sedative eﬀects of 59 as an explanation

for the reduction of CRF-induced locomotor activity, the eﬀects

of 59 on spontaneous locomotor activity and rearing in rats that

were not habituated to the testing environment were measured.

Compound 59 had no eﬀect on horizontal (movement time) or

vertical activity at any of the doses (3, 10, and 30 mg/kg) tested.

In a second rodent model, a 10 min period of restraint causes a

marked elevation in plasma ACTH levels (compare veh/no

stress vs veh/stress, Figure 4). This eﬀect was dose-dependently

antagonized by pretreatment with 59, and a signiﬁcant

attenuation of plasma ACTH levels was demonstrated at

the 10 mg/kg dose.

The CRF-1 receptor antagonist 7 (1ꢀ30 mg/kg, po, 1 h

pretreatment) was run as a positive control in the mechanistic

in vivo paradigms to demonstrate their sensitivity for detecting

CRF-1 receptor antagonism. As expected from published

literature,³⁰7 produced dose-dependent reductions of icv CRF-

enhanced locomotor activity and restraint stress-induced stimula-

tion of ACTH release (minimal eﬀective doses of 3 mg/kg and

10 mg/kg, respectively). Compound 7 produced no eﬀect on spon-

taneous locomotor activity at the minimal eﬀective doses in these

two assays, though a slight reduction was found at the highest dose

of 30 mg/kg. Taken together, these results with 7 are consistent

with the conclusion that the eﬀects of 59 in the two mechanistic

assays are attributable to CRF-1 receptor antagonism.

[

¹²⁵I]sauvagine was dose-dependently displaced with increasing

doses of 59 (middle and bottom panels) with the pattern of

labeling indicating selectivity for CRF-1 over CRF-2 receptors.

Due to aqueous solubility limitations, the pyrazine 59 was

tested at a concentration of 4 μM in a selectivity panel of 69

In mass balance studies in rat with [¹⁴C]-59, poor mass

recoveries were observed and extensive accumulation of

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Figure 4. Antagonism of stress-induced ACTH release by compound

59. Dosed in 2% vitamin E TPGS. * = signiﬁcantly diﬀerent from

Vehicle/No Stress; þ = signiﬁcantly diﬀerent from Veh/Stress.

number of metabolites, further preclinical development of 59 was

terminated.

Figure 2. CRF-1 receptor occupancy, following oral dosing of 59 to rats

at 0.3, 1.0, 3.0, and 10 mg/kg, measured using [¹²⁵I]sauvagine

autoradiography.

’ CONCLUSION

The design, synthesis, and structureꢀactivity relationships

of a novel series of pyrazines as corticotropin releasing factor

(CRF-1) receptor antagonists were described. The pyrazine core

proved superior to the earlier quinoline and pyrimidine tem-

plates; in particular, in terms of increased metabolic stability and

oral and brain exposure. New methods were developed to

synthesize a number of substituted pyrazine cores utilizing

regioselective halogenation and chemoselective derivatization.

Compound 59 was extensively proﬁled and displayed a high

aﬃnity for both the human and rat CRF-1 receptor (K_i= 1.0 and

9.8 nM, respectively), and it also was a functional antagonist vs

CRF-mediated cAMP accumulation (IC₅₀= 93 nM) in human

neuroblastoma cells. Oral administration to rats resulted in good

systemic and high brain exposure. Occupancy of brain CRF-1

receptors was quantiﬁed using ex vivo receptor binding in brain

membranes and brain slice receptor autoradiography. Fifty

percent brain receptor occupancy was achieved at oral doses of

2.0 mg/kg in both paradigms, with the pattern of labeling

indicating selectivity for CRF-1 over CRF-2 receptors. The

in vivo pharmacological characteristics were evaluated in tests

of acute functional activity following ICV CRF application and in

behavioral/biochemical models of stress. Following ICV admin-

istration of CRF to male rats, a wide variety of well-characterized

behavioral, biochemical, and autonomic events occurred. Oral

administration of compound 59 signiﬁcantly antagonized CRF-

induced alterations of locomotor activity with eﬀective doses as

low as 10 mg/kg. Rats undergoing a 10 min session of physical

restraint demonstrated marked increases in hypothalamic pitui-

tary adrenal axis activation, as evidenced by increased plasma

ACTH levels. Compound 59, administered orally, signiﬁcantly

reduced the elevated levels of ACTH. The long terminal half-life

and potential to accumulation in adipose tissues raised a potential

Figure 3. Antagonism of 0.3 μg ICV CRF-elicited increase in

locomotor activity by 59. Dosed in 2% vitamin E TPGS. * = signiﬁ-

cantly diﬀerent from TPGS; þ = signiﬁcantly diﬀerent from TPGS/

CRF.

radioactivity in adipose tissues was determined. Subsequent

metabolite identiﬁcation in rat also indicated a high number of

metabolites that included oxidations of the isopentyl group,

dealkylation of the isopentyl group, and demethylation of either

or both methoxy groups. On the basis of the long terminal half-

life, accumulation in adipose tissues, and potential to form a large

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safety issue for chronic dosing in human, and further develop-

ment of 59 was halted. Our eﬀorts to overcome these issues with

other novel topology II compounds, in particular, those directed

toward increased hydrophilicity and a lower propensity to

partition in adipose tissue will be reported in due course.

5-Bromo-[N-(1-ethyl)propyl]-3,6-diethylpyrazine-2-amine

(18b). A solution of N-(1-ethyl)propyl-3,6-diethylpyrazine-2-amine

(18a) (13.29 g, 60 mmol) in chloroform (200 mL) was cooled to 0 °C

and NBS (0.72 g, 4.1 mmol) was added in portions. The mixture was

allowed to warm to room temperature and stirred for 2 h. The mixture

was concentrated, triturated with hexane, filtered, and washed with

hexane. The combined filtrates were concentrated and purified by flash

chromatography (elution with hexane/ethyl acetate 19:1) to give the title

’ EXPERIMENTAL SECTION

1

compound 18b (15.0 g, 83%). MS 301 (Mþ1), H NMR (CDCl₃)

δ 0.90 (t, 6H), 1.23 (t, 3H), 1.27 (t, 3H), 1.49 (m, 2H), 1.63 (m, 2H),

2.55 (q, 2H) 2.74 (q, 2H), 4.06 (brs, 2H).

General. Melting points were determined using a capillary melting

1

apparatus and were uncorrected. H NMR spectra were recorded in

3,6-Diethyl-N-(1-ethylpropyl)-5-(2,4-dichlorophenyl)pyrazin-

2-amine (19a). To a solution of 5-bromo-[N-(1-ethyl)propyl]-

deuteriochloroform (unless otherwise noted) with tetramethylsilane as

the internal standard at 400 MHz. Coupling constants (J values) are

quoted to the nearest 0.5 Hz. ¹³C NMR spectra were recorded in

deuteriochloroform (unless otherwise noted) at 100 MHz. Mass spectra

were recorded on a VG 70SE magnetic sector mass spectrometer. For

selected compounds, elemental analyses were obtained on the appro-

priate salt and were within 0.4% of theoretical C, H, and N. Analytical

HPLC, used to determine the purity of all biologically tested com-

pounds, was conducted using two different methods; all tested com-

pounds had purity >95% by both methods. Method 1: Analyses were

performed using a 2790 HT-Alliance HPLC system (Waters Corpora-

tion, Milford, MA), a Waters 996 Diode Array Detector interfaced

to a Waters Quattro Micro Mass Spectrometer. Data acquired using

MassLynx 4.0 software. HPLC Conditions: column: 4.6 ꢁ 150 mm,

Waters XBridge C18, 5 μm; column temperature: 30 °C; UV: 254 nm;

scan rate: 10 points/s; flow rate: 1.0 mL/min; injection volume: 20 μL;

mobile phase A: water with 0.1% formic acid; mobile phase B: methanol;

gradient: 10% B (0ꢀ1 min), 10ꢀ100% B (1ꢀ12 min), 100% B (12ꢀ19

min), 10% B (19.1 min); run time: 22 min. MS Conditions: Electrospray

in positive ion mode, cone voltage 35 V, scan range 100ꢀ750 amu.

Method 2: Analyses were performed using an Acquity Ultra Perfor-

mance Liquid Chromatography (UPLC) system (Waters Corporation,

Milford, MA) and a Waters Acquity PDA Detector interfaced to a

Waters ZQ Mass Spectrometer. Data was acquired using MassLynx 4.1

software. HPLC Conditions: column: 2.0 ꢁ 50 mm, Phenomenex Luna

C18(2), 2.5 μm; column temperature: 30 °C; UV: 254 nm; scan rate: 10

points/s; flow rate: 0.6 mL/min; injection volume: 5 μL; mobile phase

A: 95% water, 5% acetonitrile with 0.1% ammonium hydroxide; mobile

phase B: 5% water, 95% acetonitrile with 0.1% ammonium hydroxide;

gradient: 5% B (0ꢀ2 min), 5ꢀ100% B (2ꢀ7 min), 100% B (7ꢀ9.5

min), 5% B (9.51 min); run time: 10 min. MS Conditions: electrospray

in positive ion mode; cone voltage 35 V; and scan range 100ꢀ750 amu.

Organic solutions were dried using anhydrous magnesium sulfate and

concentrated by rotary evaporation. Analytical thin layer chromatogra-

phy (TLC) was carried out on Camlab Polygram SIL G/UV₂₅₄plates.

Unless otherwise stated, preparative column chromatography was

carried out on 60H silica gel (Merck 9385). Compositions of solvent

mixtures are quoted as ratios of volume. Known compounds gave

spectral and analytical data consistent with literature values.

3,6-diethylpyrazine-2-amine (18b) (300 mg, 1.0 mmol) and tetrakis-

(triphenylphosphine)palladium(0) (46 mg, 0.04 mmol) in toluene

(8 mL) at room temperature under a nitrogen atmosphere was added

2,4-dichlorobenzeneboronic acid (229 mg, 1.2 mmol) and aqueous

potassium carbonate (2.0 M, 1.5 mL). The mixture was heated at

85 °C for 1.5 h, diluted with 0.1 N sodium hydroxide (10 mL), and

extracted with 1:1 hexaneꢀether (2 ꢁ 10 mL). The combined extracts

were dried (Na₂SO₄), filtered, concentrated, and purified by flash

chromatography (elution with hexane/ethyl acetate 3:1) to give the

title compound 19a (304 mg, 83%). MS 366 (Mþ1), ¹H NMR (CDCl₃)

δ 0.96 (t, 6H), 1.14 (t, 3H), 1.28 (t, 3H), 1.57 (m, 2H), 1.67 (m, 2H),

2.43 (m, 2H), 2.64 (q, 2H), 4.12 (m, 2H), 7.25 (d, 1H), 7.30 (dd, 1H),

7.46 (d, 1H). HPLC (method 1): 96.2% purity. HPLC (method 2):

97.3% purity. To a solution of 19a (183 mg, 0.5 mmol) in ethyl acetate

(4 mL) at room temperature was added dropwise 1 M hydrochloric acid

in ether (0.6 mL, 0.6 mmol). After stirring at room temperature for 1 h,

the resulting white solid was collected by filtration, washed with ether

(5 mL), and dried in a vacuum oven (145 mg). Mp 136ꢀ137 °C. Anal.

Calcd for C₁₉H₂₆Cl₃N₃: C, 56.66; H, 6.51; N, 10.43. Found: C, 57.01; H,

6.68; N, 10.44.

Following the method described for the synthesis of 19a, the following

compounds were prepared from 5-bromo-[N-(1-ethyl)propyl]-3,6-

diethylpyrazine-2-amine (18b) and the appropriate boronic acid:

3,6-Diethyl-N-(1-ethylpropyl)-5-(2,4-dimethoxyphenyl)pyrazin-

2-amine (19b). MS 358 (Mþ1), ¹H NMR (CDCl₃) δ 0.95 (t, 6H), 1.15

(t, 3H), 1.25 (t, 3H), 1.6(m, 4H), 2.45 (q, 2H), 2.65(q, 2H), 3.75(s, 3H),

3.85 (s, 3H), 4.1 (br, 2H), 6.5 (s, 1H), 6.55 (d, 1H), 7.2 (d, 1H). HPLC

(method 1): 95.2% purity. HPLC (method 2): 96.3% purity. The HCl salt

of 19b had MP 127ꢀ128 °C. Anal. Calcd for C₂₁H₃₂ClN₃O₂: C, 64.02;

H, 8.19; N, 10.67. Found: C, 64.31; H, 8.33; N, 10.29.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-methoxy-4-trifluoromethoxy-

phenyl)pyrazin-2-amine (19c). MS 412 (Mþ1), ¹H NMR (CDCl₃) δ

0.96 (t, 6H), 1.15 (t, 3H), 1.28 (t, 3H), 1.57 (m, 2H), 1.67 (m, 2H), 2.42

(m, 2H), 2.66 (q, 2H), 3.78 (s, 3H), 4.10 (br, 2H), 6.77 (s, 1H), 6.89 (d,

1H), 7.27 (d, 1H). HPLC (method 1): 96.0% purity. HPLC (method 2):

96.8% purity.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-methoxy-4-ethoxyphenyl)pyrazin-

2-amine (19d). MS 372 (Mþ1), ¹H NMR (CDCl₃) δ 0.95 (t, 6H), 1.14

(t, 3H), 1.28 (t, 3H), 1.43 (t, 3H), 1.55 (m, 2H), 1.67 (m, 2H), 2.46 (q,

2H), 2.65 (q, 2H), 3.74 (s, 3H), 4.07 (q, 2H), 4.10 (m, 2H), 6.51 (d, 1H),

6.54 (dd, 1H), 7.15 (d, 1H). HPLC (method 1): 98.9% purity. HPLC

(method 2): 97.4% purity.

N-(1-Ethyl)propyl-3,6-diethylpyrazine-2-amine (18a). To a

mixture of 2-chloro-3,6-diethylpyrazine (17) (14.5 g, 85 mmol), BINAP

(3.11 g, 5 mmol), and tris(dibenzylideneacetone)dipalladium(0) (1.5 g,

1.7 mmol) in toluene (350 mL) under nitrogen was added 1-ethylpro-

pylamine (15 mL) followed by sodium tert-butoxide in THF (1M,

127 mL, 127 mmol). The mixture was heated at 80 °C for 2.5 h, cooled

to room temperature, diluted with aqueous ammonium chloride

(250 mL), and extracted with 1:1 hexane/ether (2 ꢁ 250 mL). The

combined extracts were dried (Na₂SO₄), filtered, concentrated, and

purified by flash chromatography (elution with hexane/ethyl acetate

10:1 to 4:1 gradient) to give the title compound 18a (13.3 g, 71%). MS

3,6-Diethyl-N-(1-ethylpropyl)-5-[2-methoxy-4-(2-propoxy)phenyl]-

1

pyrazin-2-amine (19e). MS 386 (Mþ1), H NMR (CDCl₃) δ 0.95

(t, 6H), 1.14 (t, 3H), 1.28 (t, 3H), 1.33 (d, 6H), 1.55 (m, 2H), 1.67

(m, 2H), 2.46 (q, 2H), 2.65 (q, 2H), 3.74 (s, 3H), 4.02 (d, 1H), 4.08

(m, 1H), 4.59 (m, 1H), 6.51 (d, 1H), 6.54 (dd, 1H), 7.15 (d, 1H). HPLC

(method 1): 99.0% purity. HPLC (method 2): 98.2% purity.

1

222 (Mþ1), H NMR (CDCl₃) δ 0.91 (t, 6H), 1.24 (t, 3H), 1.29

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-methoxy-4-methylphenyl)pyrazin-

1

(t, 3H), 1.50 (m, 2H), 1.64 (m, 2H), 2.59 (m, 4H), 4.06 (m, 2H), 7.56

(s, 1H).

2-amine (19f). MS 342 (Mþ1), H NMR (CDCl₃) δ 0.95 (t, 6H),

1.14 (t, 3H), 1.28 (t, 3H), 1.55 (m, 2H), 1.67 (m, 2H), 2.38 (s, 3H),

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2.46 (m, 2H), 2.64 (q, 2H), 3.75 (s, 3H), 4.10 (m, 2H), 6.74 (s,1H), 6.82

(d, 1H), 7.14 (d, 1H). HPLC(method1):95.0%purity. HPLC(method2):

96.3% purity.

to return to room temperature and stirred overnight. Sodium hydroxide

(1 M, 25 mL) was added, and the mixture extracted with further

dichloromethane (2 ꢁ 50 mL). The combined extracts were washed

with brine (50 mL), dried (Na₂SO₄), and evaporated. Phosphorus

oxychloride (20 mL) was added to the residue, and the mixture heated

at 90 °C for 4 h. The excess reagent was evaporated, the residue

partitioned between ethyl acetate (50 mL) and saturated aqueous

NaHCO₃(20 mL), and the mixture extracted with further ethyl acetate

(2 ꢁ 50 mL). The combined organic extracts were washed with brine

(50 mL), dried (Na₂SO₄), and evaporated. The residue was purified by

flash chromatography (elution with hexane/ether 19:1) and gave the

title compound 21 (1.6 g, 50%). MS 316 (Mþ1), ¹H NMR (CDCl₃) δ

1.20 (t, 3H), 1.32 (t, 3H), 2.60 (brs, 2H), 2.99 (q, 2H), 7.22 (d, 1H),

7.38 (d, 1H), 7.54 (s, 1H).

3,6-Diethyl-N-(dicyclopropylmethyl)-5-(2,4-dichlorophe-

nyl)pyrazin-2-amine (22a). To a solution of 2-chloro-5-(2,4-

dichlorophenyl)-3,6-diethylpyrazine (21) (296 mg, 0.94 mmol) and

tris(dibenzylideneacetone)dipalladium(0) (11 mg) in toluene (10 mL)

under nitrogen was added a 0.2 M solution of tri-tert-butylphosphine in

toluene (0.1 mL). After 15 min at room temperature, dicyclopropyl-

methylamine (125 mg, 1.0 mmol), and potassium tert-butoxide (1.0 M

in THF, 1.4 mL, 1.4 mmol) were added, and the reaction mixture heated

at 80 °C for 4 h. The mixture was cooled to room temperature, diluted

with ether, washed with aqueous ammonium chloride solution, dried

(Na₂SO₄) and evaporated. The residue was purified by flash chroma-

tography (elution with hexane/ethyl acetate 3:1) and give the title

compound 22a (288 mg, 76%). MS 391 (Mþ1), ¹H NMR (CDCl₃) δ

0.5 (m, 8H), 1.03 (m, 2H), 1.1 (t, 3H), 1.3 (t, 3H), 2.4 (b, 2H), 2.66 (m,

2H), 3.5 (m, 1H), 4.25 (d, 1H), 7.25 (m, 2H), 7.46 (s, 1H). HPLC

(method 1): 95.4% purity. HPLC (method 2): 96.0% purity.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-methoxy-4-trifluoromethylphenyl)-

1

pyrazin-2-amine (19g). MS 396 (Mþ1), H NMR (CDCl₃) δ 0.96

(t, 6H), 1.15 (t, 3H), 1.28 (t, 3H), 1.57 (m, 2H), 1.67 (m, 2H), 2.42

(m, 2H), 2.63 (q, 2H), 3.82 (s, 3H), 4.10 (m, 2H), 7.14 (s, 1H), 7.28

(d, 1H), 7.38 (d, 1H). HPLC(method1):97.7%purity. HPLC(method2):

96.3% purity.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-hydroxy-4-methoxyphenyl)-

1

pyrazin-2-amine (19h). MS 344 (Mþ1), H NMR (CDCl₃) δ 0.95

(t, 6H), 1.30 (t, 3H), 1.37 (t, 3H), 1.55 (m, 2H), 1.67 (m, 2H), 2.64

(q, 2H), 2.86 (q, 2H), 3.82 (s, 3H), 4.15 (m, 2H), 6.48 (dd, 1H), 6.59

(d, 1H), 7.31 (d, 1H). HPLC(method1):95.4%purity. HPLC(method2):

95.2% purity.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-trifluoromethoxy-4-methoxy-

phenyl)pyrazin-2-amine (19i). MS 412 (Mþ1), ¹H NMR (CDCl₃) δ

0.96 (t, 6H), 1.15 (t, 3H), 1.27 (t, 3H), 1.57 (m, 2H), 1.67 (m, 2H), 2.45

(q, 2H), 2.63 (q, 2H), 3.85 (s, 3H), 4.10 (br, 2H), 6.86 (brs, 1H), 6.89

(dd, 1H), 7.29 (d, 1H). HPLC (method 1): 97.7% purity. HPLC

(method 2): 95.5% purity. The HCl salt of 19i had Mp 154 °C. Anal.

Calcd for C₂₁H₂₉ClF₃N₃O₂: C, 56.31; H, 6.53; N, 9.38. Found: C,

56.45; H, 6.80; N, 9.09.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2-trifluoromethyl-4-methoxyphenyl)-

1

pyrazin-2-amine (19j). MS 396 (Mþ1), H NMR (CDCl₃) δ 0.96

(t, 6H), 1.11 (t, 3H), 1.24 (t, 3H), 1.57 (m, 2H), 1.67 (m, 2H), 2.34

(q, 2H), 2.63 (q, 2H), 3.87 (s, 3H), 4.10 (m, 2H), 7.07 (dd, 1H), 7.24

(d, 1H), 7.25 (s, 1H). HPLC (method 1): 95.9% purity. HPLC (method 2):

96.2% purity.

[N-(1-Ethyl)propyl]-5-[(2-dimethylamino-4-methyl)pyridin-5-yl]-

3,6-diethylpyrazine-2-amine (19k). MS 356 (Mþ1), ¹H NMR

(CDCl₃, 300 MHz) δ 0.96 (t, 6H), 1.14 (t, 3H), 1.28 (t, 3H), 1.57

(m, 2H), 1.67 (m, 2H), 2.12 (s, 3H), 2.48 (m, 2H), 2.64 (q, 2H), 3.12

(s, 6H), 4.10 (m, 2H), 6.43 (s, 1H), 7.89 (s, 1H). HPLC (method 1):

98.9% purity. HPLC (method 2): 96.0% purity.

3,6-Diethyl-N-(1-ethylpropyl)-5-(2,6-dimethoxypyridin-3-yl)-

pyrazin-2-amine (19l). MS 359 (Mþ1), ¹H NMR (CDCl₃, 300 MHz)

δ 0.97 (t, 6H), 1.15 (t, 3H), 1.28 (t, 3H), 1.6 (m, 4H), 2.46 (q, 2H), 2.65

(q, 2H), 3.91 (s, 3H), 3.94 (s, 3H), 4.07 (m, 2H), 6.40 (d, 1H), 7.50

(d, 1H). HPLC (method 1): 96.0% purity. HPLC (method 2): 96.2%

purity.

5-(2,4-Dimethoxypyrimidin-5-yl)-3,6-diethyl-N-(1-ethylpropyl)-

pyrazin-2-amine (19m). MS 360 (Mþ1), ¹H NMR (CDCl₃, 300 MHz)

δ 0.94 (t, 6H), 1.16 (t, 3H), 1.28 (t, 3H), 1.55 (m, 2H), 1.78 (m, 2H),

2.42 (q, 2H), 2.62 (q, 2H), 3.94 (s, 3H), 4.01 (s, 3H), 4.12 (m, 2H),

8.2 (s, 1H). HPLC (method 1): 99.4% purity. HPLC (method 2): 98.3%

purity.

3-(2,4-Dichlorophenyl)-2,5-diethylpyrazine (20). To a mix-

ture of 2-chloro-3,6-diethylpyrazine (17) (5.1 g, 30 mmol) and tetrakis-

(triphenylphosphine)palladium(0) (1.0 g, 0.86 mmol) in toluene

(150 mL) at room temperature under a nitrogen atmosphere was added

2,4-dichlorobenzeneboronic acid (5.74 g, 30 mmol) and aqueous

potassium carbonate (2.0 M, 15 mL). The mixture was heated at

85 °C for 4 h, then diluted with 0.1 N sodium hydroxide and extracted

with 1:1 hexane/ether (2 ꢁ 100 mL). The combined extracts were dried

(Na₂SO₄), filtered, concentrated, and purified by flash chromatography

(elution with hexane/ether 3:1) to give the title compound 20 (8.2 g,

98%). MS 281 (Mþ1), ¹H NMR (CDCl₃) δ 1.19 (t, 3H), 1.36 (t, 3H),

2.62 (brs, 2H), 2.84 (q, 2H), 7.25 (d, 1H), 7.38 (d, 1H), 7.50 (s, 1H),

8.42 (s, 1H).

Following the method described above, the following compounds

were prepared from 2-chloro-5-(2,4-dichlorophenyl)-3,6-diethylpyra-

zine (21) and the appropriate amine:

3,6-Diethyl-N-[2-methoxy-1-(methoxymethyl)ethyl]-5-(2,4-dichloro-

1

phenyl)pyrazin-2-amine (22b). MS 399 (Mþ1), H NMR (CDCl₃)

δ 1.14 (t, 3H), 1.29 (t, 3H), 2.43 (b, 2H), 2.66 (m, 2H), 3.42 (s, 6H),

3.56 (m, 2H), 3.68 (m, 2H), 4.55 (m, 1H), 4.86 (d, 1H), 7.3 (m, 2H),

7.48 (m, 1H). HPLC (method 1): 98.9% purity. HPLC (method 2):

97.7% purity.

N²-[3,6-Diethyl-5(ꢀ2,4-dichlorophenyl)pyrazin-2-yl]-N¹,N¹-dimethyl-

propane-1,2-diamine (22c). MS 382 (Mþ1), ¹H NMR (CDCl₃,

300 MHz) δ 1.16 (t, 3H), 1.28 (t, 3H), 1.35 (d, 3H), 2.25 (m, 2H),

2.29 (s, 6H), 2.5 (m, 2H), 2.68 (m, 2H), 4.06 (m, 1H), 5.15 (b, 1H), 7.28

(m, 2H), 7.47 (s, 1H). HPLC (method 1): 96.4% purity. HPLC

(method 2): 95.5% purity.

N,N-Bis(cyclopropylmethyl)-5-(2,4-dichlorophenyl)-3,6-diethylpyr-

azin-2-amine (22d). MS 404 (Mþ1), ¹H NMR (CDCl₃, 300 MHz) δ

0.5 (m, 8H), 1.03 (m, 2H), 1.1 (t, 3H), 1.3 (t, 3H), 2.4 (b, 2H), 2.66 (m,

2H), 3.5 (m, 1H), 4.25 (d, 1H), 7.25 (m, 2H), 7.46 (s, 1H). HPLC

(method 1): 99.7% purity. HPLC (method 2): 96.8% purity.

3,6-Diethyl-N-propyl-N-(cyclopropylmethyl)-5-(2,4-dichlorophenyl)-

1

pyrazin-2-amine (22e). MS 393 (Mþ1), H NMR (CDCl₃) δ 0.12

(m, 2H), 0.48 (m, 2H), 0.91 (t, 3H), 1.05 (m, 1H), 1.15 (m, 3H), 1.3

(m, 3H), 1.6 (m, 2H), 2.5 (b, 2H), 2.83 (m, 2H), 3.16 (m, 2H), 3.34

(m, 2H), 7.34 (s, 2H), 7.49 (s, 1H). HPLC (method 1): 96.4% purity.

HPLC (method 2): 99.1% purity.

3,6-Diethyl-N,N-(2-methoxyethyl)-5-(2,4-dichlorophenyl)pyrazin-

2-amine (22f). MS 413 (Mþ1), ¹H NMR (CDCl₃) δ 1.13 (t, 3H), 1.24

(t, 3H), 2.5 (b, 2H), 2.84 (q, 2H), 3.33 (s, 6H), 3.6 (m, 8H), 7.31

(m, 2H), 7.49 (s, 1H). HPLC (method 1): 99.0% purity. HPLC

(method 2): 96.9% purity.

2-Chloro-5-(2,4-dichlorophenyl)-3,6-diethylpyrazine (21).

To a solution of 3-(2,4-dichlorophenyl)-2,5-diethylpyrazine (20) (2.8 g,

10 mmol) in dichloromethane (60 mL) at 0 °C was added 3-chlor-

operbenzoic acid (∼77%, 3.44 g, ∼15 mmol). The solution was allowed

3,6-Diethyl-N-propyl-N-(2-dimethylaminoethyl)-5-(2,4-dichloro-

1

phenyl)pyrazin-2-amine (22g). MS 410 (Mþ1), H NMR (CDCl₃,

300 MHz) δ 0.90 (t, 3H), 1.05 (t, 3H), 1.25 (t, 3H), 1.4 (m, 2H), 1.6 (m,

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2H), 2.30 (s, 6H), 2.52 (m, 2H), 2.80 (m, 2H), 3.26 (m, 2H), 3.5 (b,

2H), 7.32 (m, 2H), 7.50 (s, 1H). HPLC (method 1): 97.4% purity.

HPLC (method 2): 96.3% purity.

cooled to 0 °C in an ice bath, and quenched by the dropwise addition of

methanol (2 mL) and then aqueous acetic acid (10% solution v/v,

38 mL). The mixture was stirred until homogeneous and extracted with

toluene (3 ꢁ 100 mL). The combined extracts were washed with water

(100 mL), aqueous sodium hydroxide (0.2 M, 100 mL), and brine

(100 mL). The combined extracts were dried (Na₂SO₄), evaporated,

and distilled (125ꢀ130 °C @ 1 mmHg) to afford the title compound 28

(5.3 g, 87%). MS 194 (Mþ1), ¹H NMR (CDCl₃) δ 0.93 (t, 6H), 1.25 (t,

3H), 1.52 (m, 2H), 1.62 (m, 2H), 2.60 (q, 2H), 3.62 (m, 1H), 4.31 (brs,

1H), 7.66 (s, 1H), 7.68 (s, 1H).

5-Bromo-N-(1-ethyl)propyl-6-ethylpyrazine-2-amine (29).

To a solution of N-(1-ethyl)propyl-6-ethylpyrazine-2-amine 28 (31.9 g,

0.165 mol) in chloroform (450 mL) at 0 °C was added NBS in portions

(29.3 g, 0.165 mol). The reaction mixture was stirred at room tempera-

ture for 1 h and evaporated. The residue was redissolved in ethyl acetate

(500 mL) and washed with saturated aqueous NaHCO₃(500 mL)and

brine (500 mL), dried (Na₂SO₄), and evaporated. The residue was

purified by flash chromatography (elution with hexane/diethyl ether 10:1

then5:1) and gave the title compound 29 (31.3 g, 70%). MS 273 (Mþ1),

¹H NMR (CDCl₃) δ 0.94 (t, 6H), 1.23 (t, 3H), 1.49 (m, 2H), 1.63 (m,

2H), 2.77 (q, 2H), 3.61 (m, 1H), 4.38 (brs, 1H), 7.42.

3,6-Diethyl-2-(1-ethylpropoxy)-5-(2,4-dichlorophenyl)-

pyrazine (23). To a suspension of sodium hydride (60% in mineral oil,

40 mg, 1.0 mmol) in DMF (0.5 mL) under a nitrogen atmosphere was

added 3-pentanol (88 mg, 1.0 mmol), and the mixture was stirred at

room temperature for 15 min. To the resulting solution was added

a solution of 2-chloro-5-(2,4-dichlorophenyl)-3,6-diethylpyrazine (21)

(63 mg, 0.2 mmol) in N-methylpyrrolidinone (1 mL). The mixture was

heated at 70 °C for 2 h, cooled, diluted with aqueous ammonium

chloride solution (10 mL), and extracted with ether (3 ꢁ 10 mL). The

combined organics were dried (Na₂SO₄) and evaporated. The residue

was purified by flash chromatography (elution with hexane/ether 19:1)

and give the title compound 23 (57 mg, 77%). MS 367 (Mþ1),

¹H NMR (CDCl₃) δ 0.99 (t, 6H), 1.16 (t, 3H), 1.24 (t, 3H), 1.75

(m, 4H), 2.5 (br, 2H), 2.82 (m, 2H), 5.13 (m, 1H), 7.26 (d, 1H), 7.32 (d,

1H), 7.49 (s, 1H). HPLC (method 1): 95.2% purity. HPLC (method 2):

96.1% purity.

2-sec-Butylsulfanyl-5-(2,4-dichloro-phenyl)-3,6-diethyl-

pyrazine (24). Sodium hydride (60% in mineral oil, 60 mg, 1.5 mmol)

was added to a solution of butane-2-thiol (170 μL, 1.5 mmol) in THF

(5 mL). After 10 min, 2-chloro-5-(2,4-dichlorophenyl)-3,6-diethylpyr-

azine (21) (104 mg, 0.33 mmol) in THF (1 mL) was added dropwise,

and the mixture heated at reflux for 16 h. The mixture was diluted with

aqueous ammonium chloride solution (10 mL) and extracted ether (3 ꢁ

10 mL). The combined organics were dried (Na₂SO₄) and evaporated.

The residue was purified by preparative thin layer chromatography

(elution with hexane) and furnished the title compound 24 (62 mg,

51%). MS 369 (Mþ1), ¹H NMR (CDCl₃) δ 0.98 (t, 3H,), 1.12 (t, 3H),

1.20 (t, 3H), 1.36 (t, 3H), 1.6ꢀ1.8 (m, 4H), 2.58 (br, 2H), 2.72 (q, 2H),

3.92 (sext, 1H), 7.18 (d, 1H), 7.19 (d, 1H), 7.26 (dd, 1H). HPLC

(method 1): 96.2% purity. HPLC (method 2): 97.1% purity.

5-(2,4-Dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]pyrazine-

2-amine (30). To a solution of 5-bromo-N-(1-ethyl)propyl-6-ethyl-

pyrazine-2-amine (29) (3.61 g, 13.24 mmol) and tetrakis(triphenyl-

phosphine)palladium(0) (760 mg, 0.66 mmol) in DME (80 mL) at

room temperature under a nitrogen atmosphere was added 2,4-dichloro-

benzeneboronic acid (2.86 g, 15 mmol) and an aqueous solution of

sodium carbonate (1.0 M, 30 mL). The mixture was heated at 90 °C

for 12 h, diluted with 0.1 N sodium hydroxide (100 mL), and

extracted with hexane/ether (1:1, 2 ꢁ 150 mL). The combined

extracts were dried (Na₂SO₄), filtered, concentrated, and purified by

flash chromatography (elution with hexane/ethyl acetate 9:1) to give

1

the title compound 30 (4.29 g, 96%). MS 338 (Mþ1), H NMR

(CDCl₃) δ 0.97 (t, 6H), 1.13 (t, 3H), 1.56 (m, 2H), 1.65 (m, 2H),

2.45 (m, 2H), 3.72 (m, 1H), 4.45 (d, 1H), 7.25 (d, 1H), 7.30 (dd, 1H),

7.48 (d, 1H), 7.74 (s, 1H). HPLC (method 1): 95.2% purity. HPLC

(method 2): 96.8% purity.

2-(2,4-Dichlorophenyl)-3,6-diethyl-5-(2-ethylbutyl)-

pyrazine (25). To a solution of 3-methylenepentane (252 mg, 3.0

mmol) in THF (20 mL) was added a solution of 9-BBN in THF (0.5 M,

6.0 mL, 3.0 mmol). The mixture was heated at reflux, under a nitrogen

atmosphere for 12 h, and cooled. To the solution was added 2-chloro-

5-(2,4-dichlorophenyl)-3,6-diethylpyrazine (21) (316 mg, 1.0 mmol),

tetrakis(triphenylphosphine) palladium(0) (115 mg, 0.1 mmol) and

sodium hydroxide (1.0 M, 3.0 mL, 3.0 mmol). The mixture was heated at

50 °C for 24 h and cooled. Hydrogen peroxide (30%, 1.0 mL) was added,

the solution stirred for 1 h, and the reaction mixture extracted with

ether (3 ꢁ 25 mL). The combined extracts were dried (Na₂SO₄) and

evaporated. The residue was purified by flash chromatography (elution

with hexane/diethyl ether 10:1) and gave the title compound 25

3-Fluoro-6-ethyl-N-(1-ethylpropyl)-5-(2,4-dichlorophenyl)-

pyrazin-2-amine (31). To a solution of 5-(2,4-dichlorophenyl)-

6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (30) (338 mg, 1 mmol)

in chloroform (5 mL) at 0 °C was added in portions SelectFluor

(708 mg, 2 mmol). The reaction mixture was stirred at room tempera-

ture for 4 h and evaporated. The residue was dissolved in dichloro-

methane (20 mL), washed with saturated aqueous NaHCO₃(25 mL)

and brine (20 mL), dried (Na₂SO₄), and evaporated. The residue was

purified by flash chromatography (elution with hexane/ether 9:1) and

gave the title compound 31 (224 mg, 63%). MS 356 (M þ 1), ¹H NMR

(CDCl₃) δ 0.96 (t, 6H), 1.13 (t, 3H), 1.66 (m, 4H), 2.42 (m, 2H), 4.05

(m, 1H), 4.60 (d, 1H), 7.24 (d, 1H), 7.30 (dd, 1H), 7.48 (d, 1H). HPLC

(method 1): 96.0% purity. HPLC (method 2): 95.8% purity.

1

(135 mg, 37%). MS 365 (Mþ1), H NMR (CDCl₃) δ 0.90 (t, 6H),

1.18 (t, 3H), 1.24 (t, 3H), 1.42 (m, 4H), 1.82 (m, 1H), 2.59 (brs, 2H),

2.82 (m, 4H), 7.26 (d, 1H), 7.38 (d, 1H), 7.56 (s, 1H). HPLC (method

1): 97.2% purity. HPLC (method 2): 99.1% purity.

N-(1-Ethyl)propyl-6-chloropyrazine-2-amine (27). A solu-

tion of 2,6-dichloropyrazine (26) (690 g, 4.63 mol), 1-ethylpropylamine

(810 mL), and triethylamine (970 mL) in propanol (2 L) was heated at

98 °C for 12 h. The mixture was concentrated, diluted with water (1 L),

and extracted with toluene (2 ꢁ 2 L). The combined extracts were dried

(Na₂SO₄) and evaporated to give the title compound 27 (780 g, 84%).

MS 200 (Mþ1), ¹H NMR (CDCl₃) δ 0.92 (t, 6H), 1.46 (m, 2H), 1.63

(m, 2H), 3.65 (m, 1H), 4.48 (brs, 1H), 7.74 (s, 1H), 7.76 (s, 1H).

N-(1-Ethyl)propyl-6-ethylpyrazine-2-amine (28). A solution

of N-(1-ethyl)propyl-6-chloropyrazine-2-amine (27) (6.25 g, 31.4

mmol) and NiCl₂(dppp) (170 mg, 0.31 mmol) in THF (50 mL) was

cooled to 10ꢀ15 °C and ethyl magnesium bromide (3 M, 24 mL, 72

mmol) was added at a rate so as to maintain the internal temperature at

10ꢀ15 °C. After the addition, the mixture was stirred for a further 6 h,

3-Chloro-6-ethyl-N-(1-ethylpropyl)-5-(2,4-dichlorophenyl)-

pyrazin-2-amine (32). To a solution of 5-(2,4-dichlorophenyl)-

6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (30) (34 mg, 0.1 mmol)

in chloroform (1 mL) at 0 °C was added in portions N-chlorosuccinimide

(20 mg, 0.15 mmol). The reaction mixture was stirred at room

temperature for 2 h. Dichloromethane (5 mL) was added, and the

solution was washed with saturated aqueous NaHCO₃(10 mL) and

brine (10 mL), dried (Na₂SO₄), and evaporated. The residue was

purified by preparative TLC (elution with hexane/ethyl acetate, 4:1)

and gave the title compound 32 (28 mg, 74%). MS 373 (M þ 1), ¹H

NMR (CDCl₃) δ 0.97 (t, 6H), 1.14 (t, 3H), 1.55 (m, 2H), 1.70 (m, 2H),

2.46 (m, 2H), 4.05 (m, 1H), 4.93 (d, 1H), 7.24 (d, 1H), 7.30 (dd, 1H),

7.47 (d, 1H). HPLC (method 1): 98.3% purity. HPLC (method 2):

99.7% purity.

4200

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3-Iodo-6-ethyl-N-(1-ethylpropyl)-5-(2,4-dichlorophenyl)-

pyrazin-2-amine (33). To a solution of 5-(2,4-dichlorophenyl)-

6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (30) (51 mg, 0.15 mmol)

in chloroform (1 mL) at 0 °C was added in portions N-iodosuccinimide

(56 mg, 0.25 mmol). The reaction mixture was stirred at room

temperature for 2 h. Dichloromethane (5 mL) was added, and the

solution was washed with saturated aqueous NaHCO₃(10 mL) and

brine (10 mL), dried (Na₂SO₄), and evaporated. The residue was

purified by preparative TLC (elution with hexane/ethyl acetate, 5:1)

to gave the title compound 33 (49 mg, 70%). MS 465 (M þ 1), ¹H NMR

(CDCl₃) δ 0.97 (t, 6H), 1.14 (t, 3H), 1.55 (m, 2H), 1.68 (m, 2H), 2.43

(m, 2H), 4.00 (m, 1H), 4.95 (d, 1H), 7.24 (d, 1H), 7.30 (dd, 1H), 7.46

(d, 1H). HPLC (method 1): 95.5% purity. HPLC (method 2): 96.0%

purity.

3-Bromo-5-(2,4-dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]-

pyrazine-2-amine (34). To a solution of 5-(2,4-dichlorophenyl)-

6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (30) (3.38 g, 10 mmol)

in chloroform (50 mL) at 0 °C was added NBS in portions (1.8 g,

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

2 h, and solvents were evaporated under reduced pressure. The residue

was dissolved in ethyl acetate (150 mL) and washed with saturated

aqueous NaHCO₃(100 mL) and brine (100 mL), dried (Na₂SO₄), and

evaporated. The residue was purified by flash chromatography (elution

with hexane/ether 10:1) and gave the title compound 34 (3.87 g, 93%).

MS 416 (M þ 1), ¹H NMR (CDCl₃) δ 0.97 (t, 6H), 1.14 (t, 3H), 1.56

(m, 2H), 1.65 (m, 2H), 2.45 (m, 2H), 4.02 (m, 1H), 5.00 (d, 1H), 7.25

(d, 1H), 7.30 (dd, 1H), 7.46 (d, 1H).

5-(2,4-Dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]-3-

methoxypyrazine-2-amine (35). To a solution of 3-bromo-5-

(2,4-dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (34)

(4.16 g, 10 mmol) in NMP (20 mL) was added 25% (w/v) sodium

methoxide in methanol (50 mL). The mixture was heated at 75 °C

overnight, cooled, diluted with water, and extracted with 20% EtOAc in

hexane (2 ꢁ 200 mL). The combined extracts were washed with water,

dried (Na₂SO₄), and evaporated. The residue was purified by flash

chromatography (elution with hexane/ether 4:1) and gave the title

compound 35 (3.12 g, 85%). MS 368 (M þ 1), ¹H NMR (CDCl₃) δ

0.97 (t, 6H), 1.12 (t, 3H), 1.56 (m, 2H), 1.65 (m, 2H), 2.40 (q, 2H), 3.92

(s, 3H), 4.04 (m, 1H), 4.82 (d, 1H), 7.28 (m, 2H), 7.48 (d, 1H). HPLC

(method 1): 96.7% purity. HPLC (method 2): 95.9% purity. The HCl

salt of 35 had Mp 158 °C. Anal. Calcd for C₁₈H₂₄ClN₃O: C, 53.41; H,

5.98; N, 10.38. Found: C, 53.22; H, 6.33; N, 10.69.

5-(2,4-Dichlorophenyl)-3-ethoxy-6-ethyl-[N-(1-ethyl)-

propyl]pyrazine-2-amine (36). To a solution of 3-bromo-5-

(2,4-dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (34)

(104 mg, 0.25 mmol) in NMP (1 mL) was added sodium ethoxide (68

mg, 1.0 mmol) in ethanol (2 mL). The mixture was heated at 75 °C

overnight, cooled, diluted with water, and extracted with EtOAc (2 ꢁ

10 mL). The combined extracts were washed with water, dried

(Na₂SO₄), and evaporated. The residue was purified by flash chroma-

tography (elution with hexane/ether 5:1) and gave the title compound

36 (47 mg, 49%). MS 383 (M þ 1), ¹H NMR (CDCl₃) δ 0.95 (t, 6H),

1.10 (t, 3H), 1.37 (t, 3H), 1.55 (m, 2H), 1.68 (m, 2H), 2.36 (m, 2H),

4.05 (m, 1H), 4.33 (q, 2H), 4.81 (d, 1H), 7.24 (d, 1H), 7.26 (dd, 1H),

7.46 (d, 1H). HPLC (method 1): 95.7% purity. HPLC (method 2):

95.2% purity.

extracted with ethyl acetate (2 ꢁ 10 mL), and the combined extracts

washed with water (10 mL), aqueous sodium hydroxide (10 mL), and

brine (10 mL). The residue was purified by flash chromatography

(elution with hexane/ethyl acetate 10:1) and gave the title compound

37 (268 mg, 76%). MS 352 (M þ 1), ¹H NMR (CDCl₃) δ 0.97 (t, 6H),

1.12 (t, 3H), 1.56 (m, 2H), 1.65 (m, 2H), 2.35 (s, 3H), 2.42 (m, 2H),

4.04 (m, 2H), 7.25 (d, 1H), 7.29 (dd, 1H), 7.46 (d, 1H). HPLC (method

1): 98.1% purity. HPLC (method 2): 96.8% purity.

5-(2,4-Dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]-3-

butylpyrazine-2-amine (38). A solution of 3-bromo-5-(2,4-

dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl] pyrazine-2-amine (34)

(208 mg, 0.50 mmol) and NiCl₂(dppp) (16 mg, 0.03 mmol) in THF

(5 mL) was cooled to 10ꢀ15 °C and butyl magnesium chloride (1M,

1.0 mL, 1.0 mmol) was added dropwise. After the addition, the mixture

was stirred for a further 2 h, and additional butyl magnesium chloride

(1 M, 0.5 mL, 0.5 mmol) was added. The mixture was stirred for a further

2 h, cooled to 0 °C, and quenched by the dropwise addition of dilute

aqueous acetic acid (10 mL). The mixture was extracted with ethyl

acetate (2 ꢁ 15 mL), and the combined extracts washed with water

(20 mL), aqueous sodium hydroxide (20 mL), and brine (20 mL). The

residue was purified by flash chromatography (elution with hexane/

ethyl acetate 19:1) and gave the title compound 38 (128 mg, 65%). MS

394 (M þ 1), ¹H NMR (CDCl₃) δ 0.96 (m, 9H), 1.14 (t, 3H), 1.42 (m,

2H), 1.56 (m, 3H), 1.67 (m, 3H), 2.41 (brd, 2H), 2.61 (t, 2H), 4.14 (m,

2H), 7.26 (m, 2H), 7.46 (d, 1H). HPLC (method 1): 95.7% purity.

HPLC (method 2): 96.4% purity.

[N-(1-Ethyl)propyl]-5-(2,4-dichlorophenyl)-3,6-dimethyl-

pyrazine-2-amine (39). Starting from commercially available

2-chloro-3,6-dimethylpyrazine, and following the methods described

above, the title compound 39 was prepared. MS 339 (M þ 1), ¹H NMR

(CDCl₃) δ 1.0 (t, 6H), 1.6 (m, 4H), 2.2 (s, 3H), 2.4 (s, 3H), 4.05 (br,

2H), 7.25 (d, 1H), 7.3 (d, 1H), 7.45 (s, 1H). HPLC (method 1): 98.1%

purity. HPLC (method 2): 96.3% purity.

5-Bromo-6-chloro-[N-(1-ethyl)propyl]pyrazine-2-amine (40).

To 2-(3-pentylamino)-6-chloropyrazine (27) (4.09 g, 20.48 mmol) in

chloroform (80 mL) at 0 °C was added NBS (3.65 g, 20.48 mmol) in

portions. The mixture was stirred at 0 °C for 30 min, poured into

saturated aqueous NaHCO₃(100 mL), and extracted with further

dichloromethane (2 ꢁ 125 mL). The combined extracts were washed

with water (150 mL) and brine (150 mL), dried (sodium sulfate), and

evaporated. The residue was purified by flash chromatography (hexane/

ethyl acetate 19:1) and gave the title compound 40 (4.37 g; 77%).

MS 279 (M þ 1), ¹H NMR (CDCl₃) δ 0.92 (t, 6H), 1.47 (m, 2H), 1.63

(m,2H),3.61(m,1H),4.50(m,1H),7.54(s,1H).Otherfractionscontained

3-bromo-6-chloro-[N-(1-ethyl)propyl]pyrazine-2-amine as a minor product

(0.53 g, 9%). MS 279 (M þ 1), ¹H NMR (CDCl₃) δ 0.94 (t, 6H), 1.53

(m, 2H), 1.66 (m, 2H), 3.98 (m, 1H), 5.08 (m, 1H), 7.52 (s, 1H).

6-Chloro-5-(2,4-dichlorophenyl)-[N-(1-ethyl)propyl]-

pyrazine-2-amine (41). To a solution of 5-bromo-6-chloro-[N-(1-

ethyl)propyl]pyrazine-2-amine (40) (2.63 g, 9.44 mmol) and tetrakis-

(triphenylphosphine)palladium(0) (273 mg, 0.23 mmol) in DME

(100 mL) at room temperature under a nitrogen atmosphere was added

2,4-dichlorobenzeneboronic acid (1.90 g, 10 mmol) and an aqueous

solution of sodium carbonate (1.0 M, 20 mL). The mixture was heated

at 90 °C for 14 h, diluted with 0.1 N sodium hydroxide (100 mL),

and extracted with hexane/ether (1:1, 3 ꢁ 150 mL). The combined

extracts were dried (Na₂SO₄), filtered, concentrated, and purified by

flash chromatography (elution with hexane/ethyl acetate 19:1 and then

10:1) to give the title compound 41 (3.55 g, 94%). MS 345 (M þ 1),

¹H NMR (CDCl₃) δ 0.96 (t, 6H), 1.53 (m, 2H), 1.68 (m, 2H), 3.74

(m, 1H), 4.65 (d, 1H), 7.32 (m, 2H), 7.49 (s, 1H), 7.82 (s, 1H).

3-Bromo-6-chloro-5-(2,4-dichlorophenyl)-[N-(1-ethyl)-

propyl]pyrazine-2-amine (42). To a solution of 6-chloro-5-(2,4-

dichlorophenyl)-[N-(1-ethyl)propyl]pyrazine-2-amine 41 (550 mg,

5-(2,4-Dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl]-3-

methylpyrazine-2-amine (37). A solution of 3-bromo-5-(2,4-

dichlorophenyl)-6-ethyl-[N-(1-ethyl)propyl] pyrazine-2-amine (34)

(415 mg, 1.0 mmol) and NiCl₂(dppp) (27 mg, 0.05 mmol) in THF

(5 mL) was cooled to 10ꢀ15 °C and methyl magnesium bromide (1 M,

2.0 mL, 2.0 mmol) was added dropwise. After the addition, the mixture

was stirred for a further 2 h, cooled to 0 °C, and quenched by the

dropwise addition of dilute aqueous acetic acid (5 mL). The mixture was

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1.6 mmol) in chloroform (5 mL) at 0 °C was added in portions NBS

(315 mg, 1.76 mmol). The reaction mixture was stirred at room

temperature for 1 h, diluted with dichloromethane (20 mL), washed

with saturated aqueous NaHCO₃(10 mL) and brine (10 mL), dried

(Na₂SO₄), and evaporated. The residue was purified by flash chroma-

tography (elution with hexane/ether 24:1) and gave the title compound

42 (586 mg, 86%). MS 424 (M þ 1), ¹H NMR (CDCl₃) δ 0.97 (t, 6H),

1.58 (m, 2H), 1.70 (m, 2H), 4.00 (m, 1H), 5.20 (d, 1H), 7.30 (s, 2H),

7.50 (s, 1H).

diluted with water (10 mL) and extracted with ethyl acetate

(3 ꢁ 20 mL). The combined extracts were washed with water

(3 ꢁ 25 mL) and brine (25 mL), dried (Na₂SO₄), and evaporated.

The residue was purified by flash chromatography (elution with

hexane/ethyl acetate 29:1) and gave the title compound 46 (192 mg, 65%).

MS 371 (M þ 1), ¹H NMR (CDCl₃) δ 0.97 (t, 6H), 1.56 (m, 2H),

1.68 (m, 2H), 3.86 (s, 3H), 3.94 (s, 3H), 3.95(m, 1H), 4.82 (d, 1H),

7.27 (dd, 1H), 7.40 (d, 1H), 7.44 (d, 1H). HPLC (method 1): 95.7%

purity. HPLC (method 2): 97.0% purity.

6-Chloro-5-(2,4-dichlorophenyl)-3-ethyl-[N-(1-ethyl)pro-

pyl]pyrazine-2-amine (43). To a solution of 3-bromo-6-chloro-

5-(2,4-dichlorophenyl)-[N-(1-ethyl)propyl]pyrazine-2-amine 42 (423

mg, 1.0 mmol) and tetrakis(triphenylphosphine)palladium(0) (27 mg,

0.02 mmol) in DME (10 mL) at room temperature under a nitrogen

atmosphere was added ethane boronic acid (370 mg, 5 mmol) and an

aqueous solution of sodium carbonate (1.0 M, 10 mL). The mixture was

heated at 90 °C for 14 h and additional ethane boronic acid (370 mg,

5 mmol) was added. The mixture was heated at 90 ̊C for 28 h, diluted

with 0.1 N sodium hydroxide (100 mL), and extracted with hexaneꢀ

ether (1:1, 3 ꢁ 25 mL). The combined extracts were dried (Na₂SO₄),

filtered, concentrated, and purified by flash chromatography (elution

with hexane/ethyl acetate 25:1) to give the title compound 43 (183 mg,

49%). MS 373 (M þ 1), ¹H NMR (CDCl₃) δ 0.96 (t, 6H), 1.30 (t, 3H),

1.56 (m, 2H), 1.70 (m, 2H), 2.65 (t, 2H), 4.08 (m, 1H), 4.35 (d, 1H),

7.32 (d, 1H), 7.33 (s, H), 7.48 (d, 1H).

3-Methyl-5-(2,4-dichlorophenyl)-[N-(1-ethyl)propyl]-6-

methoxypyrazine-2-amine (47). A solution of 3-bromo-5-(2,4-

dichlorophenyl)-[N-(1-ethyl)propyl]-6-methoxypyrazine-2-amine (45)

(100 mg, 0.24 mmol) and NiCl₂(dppp) (9 mg, 0.015 mmol) in THF

(4 mL) was cooled to 10ꢀ15 °C and methyl magnesium bromide (1M,

0.6 mL, 0.6 mmol) was added dropwise. After the addition, the mixture

was stirred for a further 12 h, cooled to 0 °C, and quenched by the

dropwise addition of aqueous acetic acid (3 mL). The mixture was

extracted with ethyl acetate (3 ꢁ 10 mL), and the combined extracts

washed with water (10 mL), aqueous sodium hydroxide (1 M, 10 mL),

and brine (15 mL). The combined extracts were dried (Na₂SO₄) and

evaporated, and the residue purified by preparative TLC (elution with

hexane/ethyl acetate, 4:1), to give the title compound 47 (40 mg, 47%).

MS 355 (M þ 1), ¹H NMR (CDCl₃) δ 0.98 (t, 6H), 1.59 (m, 2H), 1.68

(m, 2H), 2.36 (s, 3H), 3.85 (s, 3H), 4.04 (m, 1H), 4.09(m, 1H), 7.26

(dd, 1H), 7.36 (d, 1H), 7.42 (d, 1H). HPLC (method 1): 98.2% purity.

HPLC (method 2): 96.3% purity.

5-(2,4-Dichlorophenyl)-3-ethyl-6-methyl-[N-(1-ethyl)-

propyl]pyrazine-2-amine (44). A solution of 6-chloro-5-(2,4-

dichlorophenyl)-3-ethyl-[N-(1-ethyl)propyl]pyrazine-2-amine (43)

(180 mg, 0.48 mmol) and NiCl₂(dppp) (17 mg, 0.03 mmol) in THF

(5 mL) was cooled to 10ꢀ15 °C and methyl magnesium bromide

(1 M, 1 mL, 1.0 mmol) was added dropwise. After the addition, the

mixture was stirred for a further 16 h, cooled to 0 °C, and quenched

by the dropwise addition of aqueous acetic acid (5 mL). The mixture

was extracted with ethyl acetate (3 ꢁ 10 mL) and the combined

extracts washed with water (10 mL), aqueous sodium hydroxide

(1 M, 10 mL), and brine (15 mL). The combined extracts were dried

(Na₂SO₄), evaporated, and the residue purified by preparative TLC

(elution with hexane/ethyl acetate, 4:1) to give the title compound

44 (98 mg, 58%). MS 353 (M þ 1), ¹H NMR (CDCl₃) δ 0.95 (t, 6H),

1.28 (t, 3H), 1.54 (m, 2H), 1.67 (m, 2H), 2.20 (s, 3H), 2.65 (q, 2H),

4.13 (m, 2H), 7.27 (d, 1H), 7.31 (d, 1H), 7.48 (s, 1H). HPLC

(method 1): 97.1% purity. HPLC (method 2): 98.3% purity.

3-Bromo-5-(2,4-dichlorophenyl)-[N-(1-ethyl)propyl]-6-

methoxypyrazine-2-amine (45). To a solution of 6-chloro-5-(2,4-

dichlorophenyl)-[N-(1-ethyl)propyl]pyrazine-2-amine (41) (2.12 g,

5 mmol) in NMP (15 mL) was added sodium methoxide in methanol

(1 M, 15 mL, 15 mmol). The mixture was heated at 75 °C for 24 h,

cooled, diluted with water (100 mL), and extracted with 50% EtOAc in

hexane (2 ꢁ 100 mL). The combined extracts were washed with water,

dried (Na₂SO₄), and evaporated. The residue was redissolved in chloro-

form (50 mL) and NBS (1.07 g, 6 mmol) was added in portions. The

mixture was stirred for 4 h, diluted with dichloromethane (100 mL),

washed with saturated aqueous NaHCO₃(150 mL) and brine (100 mL),

dried (Na₂SO₄), and evaporated. The residue was purified by flash

chromatography (elution with hexane/ether 49:1) and gave the title

compound 45 (1.53 g, 73%). MS 419 (M þ 1), ¹H NMR (CDCl₃) δ

0.97 (t, 6H), 1.60 (m, 2H), 1.70 (m, 2H), 3.89 (s, 3H), 3.92 (m, 1H),

4.98 (d, 1H), 7.27 (dd, 1H), 7.34 (d, 1H), 7.44 (d, 1H).

5-(2,4-Dichlorophenyl)-[N-(1-ethyl)propyl]-3-methoxy-6-

methylpyrazine-2-amine (48). Following analogueous procedures,

1

the title compound 48 was prepared. MS 355 (M þ 1), H NMR

(CDCl₃) δ 0.95 (t, 6H), 1.6 (m, 4H), 2.15 (s, 3H), 3.9 (s, 3H), 4.05 (m,

1H), 4.8 (br d, 1H), 7.3 (s, 2H), 7.45 (s, 1H). HPLC (method 1): 97.7%

purity. HPLC (method 2): 96.4% purity. The HCl salt of 48 had Mp

143ꢀ145 °C. Anal. Calcd for C₁₇H₂₁Cl₂N₃O: C, 57.63; H, 5.97; N,

11.86. Found: C, 57.31; H, 6.28; N, 11.60.

N-(1-Ethyl)propyl-6-methoxypyrazine-2-amine (49). To a

solution of 2-(3-pentylamino)-6-chloropyrazine (27) (20.0 g, 100

mmol) in DMF (150 mL) at room temperature was added sodium

methoxide (13.5 g, 250 mmol). The resulting solution was heated at

75 °C for 20 h, evaporated, poured into water (250 mL), and extracted

with ethyl acetate (3 ꢁ 200 mL). The combined extracts were dried,

washed with water (5 ꢁ 200 mL) and brine (250 mL) (Na₂SO₄), and

evaporated to afford the title compound 49 (18 g, 92%). MS 196 (M þ

1), ¹H NMR (CDCl₃) δ 0.94 (t, 6H), 1.50 (m, 2H), 1.64 (m, 2H), 3.62

(m, 1H), 3.83 (s, 3H), 4.24 (m, 1H), 7.40 (s, 1H), 7.42 (s, 1H).

3-Bromo-[N-(1-ethyl)propyl]-6-methoxypyrazine-2-amine (50).

To a solution of N-(1-ethyl)propyl-6-methoxypyrazine-2-amine (49)

(25 g, 128 mmol) in chloroform (400 mL) at 0 °C was added NBS (23 g,

129 mmol) in portions. The mixture was allowed to warm to room

temperature and stirred for 2 h. The mixture was diluted with chloro-

form (200 mL), washed with saturated NaHCO₃(250 mL), water

(250 mL), and brine (250 mL), dried (Na₂SO₄), and evaporated. The

residue was purified by flash chromatography (elution with dichloro-

methane/hexane 2:1 gradient to 1:1 and then hexane/ether 1:1). The

first fractions contained 3,5-dibromo-[N-(1-ethyl)propyl]-6-methoxy-

1

pyrazine-2-amine (51a) (9.9 g, 22%). MS 354 (M þ 1), H NMR

(CDCl₃) δ 0.93 (t, 6H), 1.58 (m, 2H), 1.65 (m, 2H), 3.84 (m, 1H),

3.98 (s, 3H), 4.82 (brd, 1H). Later fractions contained the desired

3-bromo-[N-(1-ethyl)propyl]-6-methoxypyrazine-2-amine (51b) (10.2 g,

1

5-(2,4-Dichlorophenyl)-[N-(1-ethyl)propyl]-3,6-dimethoxy-

pyrazine-2-amine (46). To a solution of 3-bromo-5-(2,4-

dichlorophenyl)-[N-(1-ethyl)propyl]-6-methoxypyrazine-2-amine (45)

(333 mg, 0.8 mmol) in 1-methyl-2-pyrrolidinone (5 mL) was added

sodium methoxide (1 M, 3.0 mL, 3.0 mmol). The resulting mixture

was then heated to 80 °C for three days. The mixture was then

29%). MS 275 (M þ 1), H NMR (CDCl₃) δ 0.93 (t, 6H), 1.56

(m, 2H), 1.66 (m, 2H), 3.87 (s, 3H), 3.92 (m, 1H), 4.85 (d, 1H), 7.18

(s, 1H). Final fractions contained 5-bromo-[N-(1-ethyl)propyl]-6-methoxy-

pyrazine-2-amine (50) (7.4 g, 21%). MS 275 (M þ 1), ¹HNMR(CDCl₃) δ

0.94 (t, 6H), 1.48 (m, 2H), 1.62 (m, 2H), 3.60 (m, 1H), 3.92 (s, 3H), 4.24

(d, 1H), 7.20 (s, 1H).

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3-Ethyl-[N-(1-ethyl)propyl]-6-methoxypyrazine-2-amine (52).

To a stirred solution of 3-bromo-[N-(1-ethyl)propyl]-6-methoxy-

pyrazine-2-amine (50) (19.6 g, 71 mmol) in THF (250 mL) at 0 °C,

and under a nitrogen atmosphere, was added [1,3-bis(diphenylpho-

sphino)propane]dichloronickel(II) (2.2 g, 4 mmol). After 10 min, ethyl-

magnesium bromide (2.0 M in THF, 100 mL, 200 mmol) was added

dropwise, and the mixture was allowed to return to room temperature.

After 2 h, aqueous ammonium chloride (200 mL) was added dropwise, and

the mixture extracted with ether (3 ꢁ 200 mL). The combined extracts

were dried (Na₂SO₄) and evaporated. The residue was purified by flash

chromatography (elution with hexane/ether 4:1) and gave the title

163ꢀ165 °C. Anal. Calcd for C₂₀H₂₇ClF₃N₃O: C, 55.36; H, 6.27; N,

9.68. Found: C, 64.23; H, 8.09; N, 9.88.

5-(2-Chloro-4-(difluoromethoxy)phenyl)-6-ethyl-3-methoxy-

1

N-(pentan-3-yl)pyrazin-2-amine (58). MS 401 (M þ 1), H NMR

(CDCl₃) δ 0.95 (t, 6H), 1.11 (t, 3H), 1.51ꢀ1.73 (m, 4H), 2.38 (q, 2H),

3.90 (s, 3H), 4.00ꢀ4.06 (m, 1H), 4.81 (d, 1H), 6.54 (t, 1H), 7.07 (dd,

1H), 7.25 (d, 1H), 7.32 (d, 1H). HPLC (method 1): 97.2% purity.

HPLC (method 2): 96.4% purity.

3-Methoxy-5-(2-methoxy-4-(trifluoromethyl)phenyl)-6-methyl-

1

N-(pentan-3-yl)pyrazin-2-amine (60). MS 384 (M þ 1), H NMR

(CDCl₃) δ 0.94 (t, 6H), 1.52ꢀ1.56 (m, 2H), 1.62ꢀ1.71 (m, 2H), 2.13

(s, 3H), 3.85 (s, 3H), 3.92 (s, 3H), 4.01ꢀ4.07 (m, 1H), 4.78 (d, 1H),

7.15 (s, 1H), 7.28 (d, 1H), 7.41 (d, 1H). HPLC (method 1): 96.6%

purity. HPLC (method 2): 97.2% purity. The HCl salt of 60 had Mp

149ꢀ151 °C. Anal. Calcd for C₁₉H₂₅ClF₃N₃O₂: C, 54.35; H, 6.00; N,

10.01. Found: C, 54.72; H, 5.76; N, 9.83.

1

compound 52 (10.7 g, 64%). MS 237 (M þ 1), H NMR (CDCl₃) δ

0.91 (t, 6H), 1.27 (t, 3H), 1.52 (m, 2H), 1.63 (m, 2H), 2.57 (q, 2H), 3.88

(s, 3H), 4.01 (m, 2H), 7.38 (s, 1H). Laterfractionscontainedthereduction

product N-(1-ethyl)propyl-6-methoxypyrazine-2-amine 49 (4 g, 29%).

3-Ethyl-[N-(1-ethyl)propyl]-5-bromo-6-methoxypyrazine-

2-amine (53). A solution of 3-ethyl-[N-(1-ethyl)propyl]-6-methoxy-

pyrazine-2-amine (52) (9.0 g, 38 mmol) in chloroform (150 mL) was

cooled to 0 °C, and NBS (6.8 g, 38 mmol) was added in portions. After

the addition, the mixture was stirred for 1 h and allowed to warm to room

temperature. The mixture was diluted with chloroform (200 mL),

washed with saturated NaHCO₃(200 mL), water (200 mL), and brine

(250 mL), dried (Na₂SO₄), and evaporated. The residue was purified by

flash chromatography (elution with hexane/ether 9:1) and gave the title

5-(2-Chloro-4-(difluoromethoxy)phenyl)-3-methoxy-6-methyl-

1

N-(pentan-3-yl)pyrazin-2-amine (61). MS 387 (M þ 1), H NMR

(CDCl₃) δ 0.95 (t, 6H), 1.49ꢀ1.72 (m, 4H), 2.14 (s, 3H), 3.92 (s, 3H),

4.01ꢀ4.07 (m, 1H), 4.82 (d, 1H), 6.54 (t, 1H), 7.08 (dd, 1H), 7.25 (d,

1H), 7.33 (d, 1H). HPLC (method 1): 99.4% purity. HPLC (method 2):

98.8% purity.

5-(2-Chloro-4-ethoxyphenyl)-3-methoxy-6-methyl-N-(pentan-3-yl)-

1

pyrazin-2-amine (62). MS 365 (M þ 1), H NMR (CDCl₃) δ 0.95

1

(t, 6H), 1.43 (t, 3H), 1.50ꢀ1.58 (m, 2H), 1.62ꢀ1.71 (m, 2H), 2.14

(s, 3H), 3.92 (s, 3H), 4.02ꢀ4.08 (m, 1H), 4.76 (d, 1H), 6.84 (dd, 1H),

6.98 (d, 1H), 7.22 (d, 1H). HPLC (method 1): 96.9% purity. HPLC

(method 2): 97.4% purity.

compound 53 (11.2 g, 98%). MS 303 (M þ 1), H NMR (CDCl₃)

δ 0.91 (t, 6H), 1.23 (t, 3H), 1.52 (m, 2H), 1.65 (m, 2H), 2.57 (q, 2H),

3.88 (m, 1H), 3.92 (s, 3H), 4.05 (m, 2H).

5-(2,4-Dichlorophenyl)-[N-(1-ethyl)propyl]-3-ethyl-6-

methoxypyrazine-2-amine (54). To a solution of 3-ethyl-[N-(1-

ethyl)propyl]-5-bromo-6-methoxypyrazine-2-amine (53) (101 mg;

0.33 mmol) and tetrakis(triphenylphosphine)palladium(0) (20 mg,

0.017 mmol, 5 mol %) in DME (3 mL) under a nitrogen atmosphere

was added 2,4-dichlorobenzeneboronic acid (95 mg, 0.5 mmol) and

aqueous potassium carbonate (1.0 M, 0.75 mL). The mixture was heated

at 75 °C for 15 h, diluted with water, and extracted with ethyl acetate

(2 ꢁ 10 mL). The combined extracts were dried (Na₂SO₄) and

evaporated. The residue was purified by flash chromatography

(elution with hexane/ethyl acetate 19:1) and gave the title compound

54 (121 mg, 99%). MS 368 (M þ 1), ¹H NMR (CDCl₃) δ 0.97 (t, 6H),

1.27 (t, 3H), 1.56 (m, 2H), 1.70 (m, 2H), 2.62 (q, 2H), 3.85 (s, 3H), 4.00

(m, 1H), 4.18 (d, 1H), 7.28 (d, 1H), 7.38 (d, 1H), 7.44 (s, 1H). HPLC

(method 1): 98.7% purity. HPLC (method 2): 99.1% purity.

Following the methods described above, the following compounds

were prepared:

5-(2-Ethyl-4-methoxyphenyl)-6-ethyl-[N-(1-ethyl)propyl]-3-methoxy-

pyrazine-2-amine (55). MS 358 (M þ 1), ¹H NMR (CDCl₃) δ 0.97

(t, 6H), 1.12 (m, 6H), 1.62 (m, 4H), 2.38 (q, 2H), 2.54 (q, 2H), 3.83

(s, 3H), 3.91 (s, 3H), 4.02 (m, 1H), 4.75 (d, 1H), 6.78 (dd, 2H), 6.86

(s, 1H), 7.12 (d, 1H). HPLC (method 1): 98.2% purity. HPLC (method 2):

96.9% purity. The HCl salt of 55 had Mp 138ꢀ139 °C. Anal. Calcd for

C₂₁H₃₂ClN₃O₂: C, 64.02; H, 8.19; N, 10.67. Found: C, 63.92; H, 8.02;

N, 10.77.

5-(2-Methoxy-4-trifluoromethoxyphenyl)-[N-(1-ethyl)propyl]-3-ethyl-

6-methoxypyrazine-2-amine (63). MS 414 (M þ 1), ¹H NMR

(CDCl₃) δ 0.96 (t, 6H), 1.25 (t, 3H), 1.59 (m, 2H), 1.69 (m, 2H),

2.62 (q, 2H), 3.80 (s, 3H), 3.87 (s, 3H), 4.00 (m, 1H), 4.10 (d, 1H), 6.80

(s, 1H), 6.89 (d, 1H), 7.39 (d, 1H). HPLC (method 1): 98.4% purity.

HPLC (method 2): 99.1% purity.

5-(2-Chloro-4-methoxyphenyl)-3-ethyl-6-methoxy-N-(pentan-3-yl)-

1

pyrazin-2-amine (64). MS 365 (M þ 1), H NMR (CDCl₃) δ 0.97

(t, 6H), 1.27 (t, 3H), 1.52ꢀ1.74 (m, 4H), 2.63 (q, 2H), 3.81 (s, 3H),

3.87 (s, 3H), 3.98ꢀ4.02 (m, 1H), 4.09 (d, 1H), 6.85 (dd, 1H), 6.98

(d, 1H), 7.33 (d, 1H). HPLC(method1):95.9%purity. HPLC(method2):

96.3% purity. The HCl salt of 64 had Mp 152ꢀ153 °C. Anal. Calcd for

C₁₉H₂₆ClN₃O₂: C, 62.71; H, 7.20; N, 11.55. Found: C, 62.99; H, 7.54;

N, 11.41.

5-(2-Chloro-4-(difluoromethoxy)phenyl)-3-ethyl-6-methoxy-

1

N-(pentan-3-yl)pyrazin-2-amine (65). MS 401 (M þ 1), H NMR

(CDCl₃) δ 0.97 (t, 6H), 1.25 (t, 3H), 1.49ꢀ1.61 (m, 2H), 1.63ꢀ1.77

(m, 2H), 2.63 (q, 2H), 3.88 (s, 3H), 3.95ꢀ4.04 (m, 1H), 4.19 (d, 1H),

6.50 (t, 1H), 7.07 (dd, 1H), 7.22 (d, 1H), 7.43 (d, 1H). HPLC (method

1): 98.7% purity. HPLC (method 2): 99.1% purity.

N-(1-Ethyl)propyl-6-methylpyrazine-2-amine (66). A solu-

tion of 2-(3-pentylamino)-6-chloropyrazine (27) (625 g, 3.14 mol) and

NiCl₂(dppp) (17 g, 31.4 mmol) in THF (5 L) was cooled to 10ꢀ15 °C

and methyl magnesium bromide (3 M, 2.4 L, 7.2 mol) was added at a rate

so as to maintain the internal temperature at 10ꢀ15 °C. After the

addition, the mixture was stirred for a further 6 h, cooled to 0 °C in an ice

bath, quenched by the dropwise addition of methanol (190 mL), and then

aqueous acetic acid (10% solution v/v, 3.8 L). The mixture was stirred until

homogeneous and extracted with toluene (2 ꢁ 2 L). The combined extracts

were washed with water (3 L), aqueous sodium hydroxide (0.2 M, 3 L) and

brine (3 L). The combined extracts were dried (Na₂SO₄), evaporated, and

distilled (121ꢀ123 °C at 1 mmHg) to afford the title compound 66 (530 g,

87%). MS 180 (M þ 1), ¹H NMR (CDCl₃) δ 0.93 (t, 6H), 1.47 (m, 2H),

1.62 (m, 2H), 2.34 (s, 3H), 3.59 (m, 1H), 4.32 (m, 1H).

5-(2-Methoxy-4-trifluoromethylphenyl)-6-ethyl-[N-(1-ethyl)propyl]-

3-methoxypyrazine-2-amine (56). MS 398 (M þ 1), ¹H NMR

(CDCl₃) δ 0.95 (t, 6H), 1.13 (t, 3H), 1.57 (m, 2H), 1.68 (m, 2H),

2.37 (q, 2H), 3.84 (s, 3H), 3.92 (s, 3H), 4.04 (m, 1H), 4.77 (d, 1H), 7.15

(s, 1H), 7.28 (d, 1H), 7.39 (d, 1H). HPLC (method 1): 97.7% purity.

HPLC (method 2): 96.0% purity.

6-Ethyl-3-methoxy-5-(4-methoxy-2-(trifluoromethyl)phenyl)-

1

N-(pentan-3-yl)pyrazin-2-amine (57). MS 399 (M þ 1), H NMR

(CDCl₃) δ 0.95 (t, 6H), 1.09 (t, 3H), 1.52ꢀ1.62 (m, 2H), 1.64ꢀ1.71

(m, 2H), 2.33 (q, 2H), 3.88 (s, 6H), 4.01ꢀ4.06 (m, 1H), 4.75 (d, 1H),

7.07 (dd, 1H), 7.26ꢀ7.29 (br, 2H). HPLC (method 1): 99.7% purity.

HPLC (method 2): 99.8% purity. The HCl salt of 57 had Mp

3-Methoxy-[N-(1-ethyl)propyl]-5-bromo-6-methylpyrazine-

2-amine (67). N-(1-Ethyl)propyl-6-methylpyrazine-2-amine (66)

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Journal of Medicinal Chemistry

ARTICLE

(570 g, 3 mol) was dissolved in glacial acetic acid (3 L) and cooled to

0 °C. Bromine (307 mL, 6 mol) was added at a rate such that the

temperature of the reaction remained at room temperature or below.

After the addition, the mixture was stirred for a further 1 h and water

(4 L) and toluene (4 L) were added. The toluene layer was washed with

water (3 L), aqueous sodium hydroxide (0.3 M, 3 L), aqueous Na₂SO₃

(0.1 M, 3 L), and water (3 L), and concentrated to give 3,5-dibromo-

N-(1-ethyl)propyl-6-methylpyrazine-2-amine (852 g, 84%), MS 337

(M þ 1), which was used without further purification. 3,5-Dibromo-

N-(1-ethyl)propyl-6-ethylpyrazine-2-amine (564 g, 1.67 mol) was dis-

solved in methanol (1 L) and sodium methoxide (574 mL, 25 wt % in

methanol) added dropwise. The mixture was heated to 65 °C for 16 h,

cooled to 0 °C, and glacial acetic acid (53 mL) added. The mixture was

concentrated and partitioned between water (1.5 L) and toluene (2.5 L).

The toluene layer was collected, washed with water (1 L) and concen-

trated. The residue was purified by distillation (112ꢀ115 °C at

0.7 mmHg) and gave the title compound 67 (408 g, 85%). MS 289

(M þ 1), ¹H NMR (CDCl₃) δ 0.89 (t, 6H), 1.46 (m, 2H), 1.61 (m, 2H),

2.38 (s, 3H), 3.94 (s, 3H), 4.62 (m, 1H).

5-(2-Methoxy-4-trifluoromethoxyphenyl)-[N-(1-ethyl)-

propyl]-3-methoxy-6-methylpyrazine-2-amine (59). 3-Methoxy-

[N-(1-ethyl)propyl]-5-bromo-6-methylpyrazine-2-amine (67) (380 g,

1.32 mol) was dissolved in toluene (2.5 L). 2-Methoxy-4-trifluoro-

methoxy-phenyl-boronic acid (342 g, 1.45 mol) and aqueous potassium

carbonate solution (2 M, 1.3 L) were added, and the stirred solution was

degassed with argon. Tetrakis(triphenylphosphine)palladium(0) (30 g,

25 mmol) was added, and the solution was heated at 85 °C for 12 h.

The mixture was filtered through Celite, and the toluene layer washed with

water (3 ꢁ 1 L) and concentrated. The residue was purified by distillation

(157ꢀ158 °C at 0.6 mmHg) and gave the title compound 59 (476 g, 90%)

as a clear viscous oil that crystallized on standing (Mp 45ꢀ47 °C). MS 400

(M þ 1), ¹H NMR (CDCl₃) δ 0.96 (t, 6H), 1.56 (m, 2H), 1.64 (m, 2H),

2.12 (s, 3H), 3.81 (s, 3H), 3.93 (s, 3H), 4.04 (m, 1H), 4.78 (d, 1H), 6.80

(s, 1H), 6.98 (d, 1H), 7.32 (d, 1H). HPLC (method 1): 98.1% purity.

HPLC (method 2): 97.3% purity. Compound 59 (465.5 g, 1.16 mol) in

ethyl acetate (2.32 L) was heated to 50ꢀ55 °C and tert-butyl methyl ether

(2.3 L) added. The resulting solution was stirred for 0.5 h and toluene-

sulfonic acid monohydrate (230 g, 1.2 mol) was added in portions. The

solution was stirred for 10 min at 50ꢀ55 °C and cooled in an ice bath for

2 h. The resulting solid was collected by filtration, washed with tert-butyl

methyl ether (3 ꢁ 1 L) and dried in a vacuum oven at 85ꢀ90 °C for 2 h.

The product was white and crystalline (612.5 g, 89.2%). Mp 162ꢀ4 °C.

Anal. Calcd for C₂₆H₃₂F₃N₃O₆S: C, 54.63; H, 5.64; N, 7.35. Found: C,

54.58; H, 5.81; N, 7.09.

Present Addresses

Galenea, 300 Technology Square, Cambridge, MA 02139, USA.

’ ABBREVIATIONS:

CRF, corticotropin releasing factor; HPA, hypothalamic-pituitary-

adrenal; ACTH, adrenocorticotropic hormone; CNS, central

nervous system;hERG, human ether-ꢁa-go-go related gene;

TPGS, tocopheryl polyethylene glycol 1000 succinate; TLC,

thin layer chromatography; NBS, N-bromosuccinimide; 9-BBN,

9-borabicyclo[3.3.1]nonane; cAMP, 3⁰-5⁰-cyclic adenosine

monophosphate

’ REFERENCES

(1) Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41-

residue ovine hypothalamic peptide that stimulates secretion of cortico-

tropin and β-endorphin. Science 1981, 213, 1394–1397.

(2) Spiess, J.; Dautzenberg, F. M.; Sydow, S.; Hauger, R. L.;

Ruhmann, A.; Blank, T.; Radulovic, J. Molecular properties of the

CRF receptor. Trends Endocrinol. Metab. 1998, 9, 140–145.

(3) (a) Chalmers, D. T.; Lovenberg, T. W.; Grigoriadis, D. E.;

Behan, D. P.; De Souza, E. B. Corticotropin-releasing factor receptors:

from molecular biology to drug design. Trends Pharmacol. Sci. 1996,

17, 166–172. (b) Lovenberg, T. W.; Liaw, C. W.; Grigoriadis, D. E.;

Clevenger, W.; Chalmers, D. T.; De Souza, E. B.; Oltersdorf, T. Cloning

and characterization of a functionally distinct corticotropin-releasing

factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. U.S.A. 1995,

92, 836–840.

(4) (a) Holsboer, F.; Ising, M. Central CRH system in depression

and anxiety - Evidence from clinical studies with CRH1 receptor

antagonists. Eur. J. Pharmacol. 2008, 583, 350–357. (b) Ising, M.;

Zimmermann, U. S.; Kuenzel, H. E.; Uhr, M.; Foster, A. C.; Learned-

Coughlin, S. M.; Holsboer, F.; Grigoriadis, D. E. High-Aﬃnity CRF1

receptor antagonist NBI-34041: preclinical and clinical data suggest

safety and eﬃcacy in attenuating elevated stress response. Neuropsycho-

pharmacology 2007, 32, 1941–1949. (c) Zoumakis, E.; Rice, K. C.; Gold,

P. W.; Chrousos, G. P. Potential uses of corticotropin-releasing hormone

antagonists. Ann. N.Y. Acad. Sci. 2006, 1083, 239–251. (d) Holsboer, F. J.

The rationale for corticotropin-releasing hormone receptor (CRH-R)

antagonists to treat depression and anxiety. Psychiatr. Res. 1999,

33, 181–214. (e) Bale, T. L.; Vale, W. V. CRF and CRF receptors: Role

in stress responsivity and other behaviors. Annu. Rev. Pharmacol. Toxicol.

2004, 44, 525–557.

(5) (a) Chen, Y. L.; Mansbach, R. S.; Winter, S. M.; Brooks, E.;

Collins, J.; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.; Gallaschun,

R. J.; Schmidt, A.; Schulz, D. W. Synthesis and oral eﬃcacy of a

4-(butylethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active corti-

cotropin-releasing factor1 receptor antagonist. J. Med. Chem. 1997,

40, 1749–1754. (b) Schultz, W. D.; Mansbach, R. S.; Sprouse, J.;

Braselton, J. P.; Collins, J.; Corman, M.; Tingley, F. D., III; Winston,

E. N.; Chen, Y. L.; Heym, J. CP-154,526: a potent and selective non

peptide antagonist of corticotropin releasing factor receptors. Proc. Natl.

Acad. Sci. U.S.A. 1996, 93, 10477–10482.

(6) Baram, T. Z.; Chalmers, D. T.; Chen, C.; Koutsoukos, Y.; De

Souza, E. B. The CRF1 receptor mediates the excitatory actions of

corticotropin releasing factor (CRF) in the developing rat brain: in vivo

evidence using a novel, selective, non-peptide CRF receptor antagonist.

Brain Res. 1997, 770, 89–95.

’ ASSOCIATED CONTENT

S

Supporting Information. Procedures for the radioligand

b

binding assay, IMR 32 whole cell functional assay, ex vivo

receptor occupancy assay, ICV CRF-induced locomotor activity,

restraint-stress-induced ACTH release, spontaneous locomotor

activity, statistical analyses of behavioral data and Cerep selec-

tivity proﬁle for compound 59. Data for reference compound 7,

in the icv CRF-enhanced locomotor activity, restraint stress-

enhanced ACTH release and spontaneous locomotor activity, is

also included. This material is available free of charge via the

Internet at http://pubs.acs.org.

(7) Griebel, G.; Simiand, J.; Steinberg, R.; Jung, M.; Gully, D.; Roger,

P.; Geslin, M.; Scatton, B.; Maﬀrand, J. ꢀP.; Soubrie, P. 4-(2-Chloro-4-

methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-ﬂuoro-4-methyl-

phenyl)ethyl]5-methyl-N-(2-propynyl)-1, 3-thiazol-2-amine hydro-

chloride (SSR125543A), a potent and selective corticotrophin-releasing

factor(1) receptor antagonist. II. Characterization in rodent models

of stress-related disorders. J. Pharmacol. Exp. Ther. 2002, 301,

333–345.

’ AUTHOR INFORMATION

Corresponding Author

*Tel.: 617-374-1010. Fax: 617-374-1320. E-mail: khodgetts@

galenea.com.

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(8) For reviews see:(a) Chen, C. Recent advances in small molecule

antagonists of the corticotropin- releasing factor type-1 receptor-focus

on pharmacology and pharmacokinetics. Curr. Med. Chem. 2006,

13, 1261–1282. (b) Gilligan, P. J.; Li, Y.-W. Corticotropin-releasing

factor antagonists: recent advances and exciting prospects for the

treatment of human diseases. Curr. Opin. Drug Discovery Dev. 2004,

7, 487–497. (c) Kehne, J.; De Lombaert, S. Non-peptidic CRF1 receptor

antagonists for the treatment of anxiety, depression and stress disorders.

Current Drug Targets ꢀ CNS & Neurological Disorders 2002, 1, 467–493.

(d) Gilligan, P. J.; Robertson, D. W.; Zaczek, R. Corticotropin releasing

factor (CRF) receptor modulators: progress and opportunities for new

therapeutic agents. J. Med. Chem. 2000, 43, 1641–1660.

(9) (a) Lipinski, C. A. Drew University Medical Chemistry Special

Topics Course, July 1999; (b) Hitchcock, S. A. Bloodꢀbrain barrier

permeability considerations for CNS-targeted compound library design.

Curr. Opin. Chem. Biol. 2008, 12, 318–323. (c) Pajouhesh, H.; Lenz,

G. R. Medicinal chemical properties of successful central nervous system

drugs. NeuroRx 2005, 2, 541–553.

(10) (a) He, L.; Gilligan, P. J.; Zaczek, R.; Fitzgerald, L. W.; McElroy,

J.; Shen, H. S.; Saye, J. A.; Kalin, N. H.; Shelton, S.; Christ, D.; Trainor,

G.; Hartig, P. 4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,

4-dichlorophenyl)pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bio-

available CRF(1) receptor antagonist. J. Med. Chem. 2000, 43, 449–

456. (b) Li, Y.-W.; Hill, G.; Wong, H.; Kelly, N.; Ward, K.; Pierdomenico,

M.; Ren, S.; Gilligan, P. J.; Grossman, S.; Trainor, G.; Taub, R.; McElroy,

J.; Zaczek, R. Receptor occupancy of non-peptide corticotropin-releasing

factor 1 antagonist DMP696: correlation with drug exposure and anxiolytic

eﬃcacy. J. Pharmacol. Exp. Ther. 2003, 305, 86–96.

(11) Gilligan, P. J.; He, L.; Clarke, T.; Tivitmahaisoon, P.; Lelas, S.;

Li, Y.-W.; Heman, K.; Fitzgerald, L.; Miller, K.; Zhang, G.; Marshall, A.;

Krouse, C.; McElroy, J.; Ward, K.; Shen, H.; Wong, H.; Grossman, S.;

Nemeth, G.; Zaczek, R.; Arneric, S. P.; Hartig, P.; Robertson, D. W.;

Trainor, G. 8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: selec-

tive and centrally active corticotropin-releasing factor receptor-1

(CRF1) antagonists. J. Med. Chem. 2009, 52, 3073–3083.

Therapeutic Strategies 2009, 5, 161–168.(c) Dzierba, C. D.; Hartz, R. A.;

Bronson, J. J.Recent advances in corticotropin-releasing factor receptor

antagonists. In Annual Reports in Medicinal Chemistry, Macor, J. E., Ed.;

Academic: San Diego, 2008; Vol. 43, pp 1ꢀ23. (d) Paez-Pereda, M.;

Hausch, F.; Holsboer, F. Corticotropin releasing factor receptor antago-

nists for major depressive disorder. Expert Opinion on Investigational

Drugs 2011, 20, 519–535.

(16) (a) Zobel, A. W.; Nickel, T.; Kunzel, H. E.; Ackl, N.; Sonntag,

A.; Ising, M.; Holsboer, F. Eﬀects of the high-aﬃnity corticotropin-

releasing hormone receptor 1 antagonist R121919 in major depression:

the ﬁrst 20 patients treated. J. Psych. Res. 2000, 34, 171. (b) Kunzel, H. E.;

Zobel, A. W.; Nickel, T.; Ackl, N.; Uhr, M.; Sonntag, A.; Ising, M.;

Holsboer, F. Treatment of depression with the CRH-1-receptor antago-

nist R121919: endocrine changes and side eﬀects. J. Psych. Res. 2003,

37, 525–533.

(17) Guo, Z.; Tellew, J. E.; Gross, R. S.; Dyck, B.; Grey, J.; Haddach,

M.; Kiankarimi, M.; Lanier, M.; Li, B.-F.; Luo, Z.; McCarthy, J. R.;

Moorjani, M.; Saunders, J.; Sullivan, R.; Zhang, X.; Zamani-Kord, S.;

Grigoriadis, D. E.; Crowe, P. D.; Chen, T. K.; Williams, J. P. Design and

synthesis of tricyclic imidazo[4,5-b]pyridin-2-ones as corticotropin-

releasing factor-1 antagonists. J. Med. Chem. 2005, 48, 5104–5107.

(18) Ising, M.; Zimmermann, U. S.; Kunzel, H. E.; Uhr, M.; Foster,

A. C.; Learned-Coughlin, S. M.; Holsboer, F.; Grigoriadis, D. E. High-

aﬃnity CRF (1) receptor antagonist NBI-34041: preclinical and clinical

data suggest safety and eﬃcacy in attenuating elevated stress response.

Neuropsychopharmacology 2007, 32, 1941–1949.

(19) Chen, Y. L.; Braselton, J.; Forman, J.; Gallaschun, R. J.;

Mansbach, R.; Schmidt, A. W.; Seeger, T. F.; Sprouse, J. F.; Tingley,

F. D., III; Winston, E.; Schulz, D. W. Synthesis and SAR of 2-aryloxy-4-

alkoxy-pyridines as potent orally active corticotropin releasing factor 1

receptor antagonists. J. Med. Chem. 2008, 51, 1377–1384.

(20) Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger,

T. A. 6-week randomized, placebo-controlled trial of CP-316,311

(a selective CRH1antagonist) in the treatment of major depression.

Amer. J. Psychiatry 2008, 165, 617–620.

(12) (a) Gilligan, P. J.; Clarke, T.; He, L.; Lelas, S.; Li, Y.-W.; Heman,

K.; Fitzgerald, L.; Miller, K.; Zhang, G.; Marshall, A.; Krouse, C.;

McElroy, J. F.; Ward, K.; Zeller, K.; Wong, H.; Bai, S.; Saye, J.; Grossman,

S.; Zaczek, R.; Arneric, S. P.; Hartig, P.; Robertson, D.; Trainor, G.

Synthesis and structure-activity relationships of 8-(pyrid-3-yl)pyrazolo-

[1,5-a]-1,3,5-triazines: potent, orally bioavailable corticotropin releasing

factor receptor-1 (CRF1) antagonists. J. Med. Chem. 2009, 52, 3084–

3092. (b) Coric, V.; Feldman, H. H.; Oren, D. A.; Shekhar, A.; Pultz, J.;

Dockens, R. C.; Wu, X.; Gentile, K. A.; Huang, S. P.; Emison, E.;

Delmonte, T.; D’Souza, B. B.; Zimbroﬀ, D. L.; Grebb, J. A.; Goddard,

A. W.; Stock, E. G. Multicenter, randomized, double-blind, active

comparator and placebo-controlled trial of a corticotropin-releasing

factor receptor-1 antagonist in generalized anxiety disorder. Depression

and Anxiety 2010, 27, 417–425.

(13) Chen, C.; Wilcoxen, K. M.; Huang, C. Q.; Xie, Y. F.; McCarthy,

J. R.; Webb, T. R.; Zhu, Y.-F.; Saunders, J.; Liu, X. J.; Chen, T. K.;

Bozigian, H.; Grigoriadis, D. E. Design of 2,5-dimethyl-3-(6-dimethyl-4-

methylpyridin-3-yl)-7-dipropylaminopyrazolo[1,5-a]pyrimidine (NBI

30775/R121919) and structure-activity relationships of a series of

potent and orally active corticotropin-releasing factor receptor antago-

nists. J. Med. Chem. 2004, 47, 4787–4798.

(21) (a) Chen, Y. L.; Obach, R. S.; Braselton, J.; Corman, M. L.;

Forman, J.; Freeman, J.; Gallaschun, R. J.; Mansbach, R.; Schmidt, A. W.;

Sprouse, J. S.; Tingley, F. D., III; Winston, E.; Schulz, D. W. 2-Aryloxy-4-

alkylaminopyridines: discovery of novel corticotrophin releasing factor 1

antagonists. J. Med. Chem. 2008, 51, 1385–1392. (b) Tellew, J. E.; Lanier,

M.; Moorjani, M.; Lin, E.; Luo, Z.; Slee, D. H.; Zhang, X.; Hoare, S. R. J.;

Grigoriadis, D. E.; St Denis, Y.; Di Fabio, R.; Di Modugno, E.; Saunders,

J.; Williams, J. P. Discovery of NBI-77860/GSK561679, a potent

corticotropin-releasing factor (CRF1) receptor antagonist with im-

proved pharmacokinetic properties. Bioorg. Med. Chem. Lett. 2010,

20, 7259–7264.

(22) Huang, C. Q.; Wilcoxen, K. M.; McCarthy, J. R.; Haddach, M.;

Webb, T. R.; Gu, J.; Xie, Y. F.; Grigoriadis, D.; Chen., C. Synthesis and

SAR of 8-arylquinolines as potent corticotropin-releasing factor1 (CRF1)

receptor antagonists. Bioorg. Med. Chem. Lett. 2003, 13, 3375–3379.

(23) Yoon, T.; De Lombaert, S.; Brodbeck, R.; Gulianello, M.;

Chandrasekhar, J.; Horvath, R. F.; Ge, P.; Kershaw, M. T.; Krause,

J. E.; Kehne, J.; Hoﬀman, D.; Doller, D.; Hodgetts, K. J. The design,

synthesis and structure-activity relationships of 1-aryl-4-aminoalkyliso-

quinolines. A novel series of CRF-1 receptor antagonists. Bioorg. Med.

Chem. Lett. 2008, 18, 891–896.

(14) Gehlert, D. R.; Cippitelli, A.; Thorsell, A.; Le, Anh, D.;

Hipskind, P. A.; Hamdouchi, C.; Lu, J.; Hembre, E. J.; Cramer, J.; Song,

M.; McKinzie, D.; Morin, M.; Ciccocioppo, R.; Heilig, M. 3-(4-Chloro-

2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo-

[1,2-b]pyridazine: A novel brain-penetrant, orally available corticotropin-

releasing factor receptor 1 antagonist with eﬃcacy in animal models of

alcoholism. J. Neurosci. 2007, 27, 2718–2726.

(15) (a) Kehne, J. H.; Cain, C. K. Therapeutic utility of non-peptidic

CRF1 receptor antagonists in anxiety, depression, and stress-related

disorders: evidence from animal models. Pharmacology & Therapeutics

2010, 128, 460–487. (b) Kehne, J. H.; Maynard, G. D. CRF1 receptor

antagonists: treatment of stress-related disorders. Drug Discovery Today:

(24) (a) Hodgetts, K. J.; Yoon, T.; Huang, J.; Gulianello, M.;

Kieltyka, A.; Primus, R.; Brodbeck, R.; De Lombaert, S.; Doller, D. 2-

Aryl-3-alkyl-5-dialkylaminopyrimidin-4-ones as novel CRF-1 receptor

antagonists. Bioorg. Med. Chem. Lett. 2003, 13, 2497–2500. (b) Yoon, T.;

De Lombaert, S.; Brodbeck, R.; Gulianello, M.; Krause, J. E.; Hutchison,

A.; Horvath, R. F.; Ge, P.; Kehne, J.; Hoﬀman, D.; Chandrasekhar, J.;

Doller, D.; Hodgetts, K. J. 2-Arylpyrimidines: Novel CRF-1 receptor

antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 4486–4490.

(25) Corbett, J. W.; Rauckhorst, M. R.; Qian, F.; Hoﬀman, R. L.;

Knauer, C. S.; Fitzgerald, L. W. Heteroatom-linked indanylpyrazines are

corticotropin releasing factor type-1 receptor antagonists. Bioorg. Med.

Chem. Lett. 2007, 17, 6250–6256.

4205

dx.doi.org/10.1021/jm200365y |J. Med. Chem. 2011, 54, 4187–4206

Journal of Medicinal Chemistry

ARTICLE

(26) Sato, N.; Matsuura, T. Studies on pyrazines. Part 32. Synthesis

of trisubstituted and tetrasubstituted pyrazines as ant pheromones.

J. Chem Soc., Perkin Trans. 1 1996, 19, 2345–2350.

(27) (a) Tamao, K.; Sumitani, K.; Kiso, Y.; Zembayashi, M.; Fujioka,

A.; Kodama, S.; Nakajima, I.; Minato, A.; Kumada, M. Nickel-phosphine

complex-catalyzed Grignard coupling. I. Cross-coupling of alkyl, aryl,

and alkenyl Grignard reagents with aryl and alkenyl halides: General

scope and limitations. Bull. Chem. Soc. Jpn. 1976, 49, 1958–1969. (b)

Tamao, K.; Kodama, S.; Nakajima, I.; Kumada, M.; Minato, A.; Suzuki,

K. Nickel-phosphine complex-catalyzed Grignard coupling II. Grignard

coupling of heterocyclic compounds. Tetrahedron 1982, 38, 3347–3354.

(28) Grigoriadis, D. E.; De Souza, E. B. Biochemical, pharmacolo-

gical, and autoradiographic methods to study corticotropin-releasing

factor receptors. Methods Neurosci. 1991, 5, 510–538.

(29) (a) Chen, C.; Dagnino, R., Jr.; De Souza, E. B.; Grigoriadis,

D. E.; Huang, C. Q.; Kim, K.-I.; Liu, Z.; Moran, T.; Webb, T. R.; Whitten,

J. P.; Xie, Y. F.; McCarthy, J. R. Design and synthesis of a series of non-

peptide high-aﬃnity human corticotropin-releasing factor1 receptor

antagonists. J. Med. Chem. 1996, 39, 4358–4360. (b) Hodge, C. N.;

Aldrich, P. E.; Wasserman, Z. R.; Fernandez, C. H.; Nemeth, G. A.;

Arvanitis, A.; Cheeseman, R. S.; Chorvat, R. J.; Ciganek, E.; Christos,

T. E.; Gilligan, P. J.; Krenitsky, P.; Scholﬁeld, E.; Strucely, P. Cortico-

tropin-releasing hormone receptor antagonists: framework design and

synthesis guided by ligand conformational studies. J. Med. Chem. 1999,

42, 819–832.

(30) Heinrichs, S. C.; De Souza, E. B.; Schulteis, G.; Lapsansky, J. L.;

Grigoriadis, D. E. Brain penetrance, receptor occupancy and antistress

in vivo eﬃcacy of a small molecule corticotropin releasing factor type I

receptor selective antagonist. Neuropsychopharmacology 2002, 27,

194–202.

4206

dx.doi.org/10.1021/jm200365y |J. Med. Chem. 2011, 54, 4187–4206

Article Doi

Discovery of N -(1-ethylpropyl)-[3-methoxy-5-(2-methoxy-4- trifluoromethoxyphenyl)-6-methyl-pyrazin-2-yl]amine 59 (NGD 98-2): An orally active corticotropin releasing factor-1 (CRF-1) receptor antagonist

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