Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines

J . Med. Chem. 1999, 42, 833-848

833

Syn th esis, Cor ticotr op in -Relea sin g F a ctor Recep tor Bin d in g Affin ity, a n d

P h a r m a cok in etic P r op er ties of Tr ia zolo-, Im id a zo-, a n d P yr r olop yr im id in es a n d

-p yr id in es

Robert J . Chorvat,*^,†Rajagopal Bakthavatchalam,*^,†J ames P. Beck,^†Paul J . Gilligan,^†Richard G. Wilde,^†

Anthony J . Cocuzza,^†Frank W. Hobbs,^†Robert S. Cheeseman,^†Matthew Curry,^†J oseph P. Rescinito,^†

Paul Krenitsky,^†Dennis Chidester,^†J erry A. Yarem,^†J ohn D. Klaczkiewicz,^†C. Nicholas Hodge,^†

Paul E. Aldrich,^†Zelda R. Wasserman,^†Christine H. Fernandez,^†Robert Zaczek,^‡Lawrence W. Fitzgerald,^‡

Shiew-Mei Huang,^§,Helen L. Shen,^§Y. Nancy Wong,^§,∇Ben M. Chien,^§Check Y. Quon,^§and Argyrios Arvanitis^†

Departments of Chemical and Physical Sciences and of Biological Sciences, DuPont Pharmaceuticals Company,

Experimental Station, P.O. Box 80500, Wilmington, Delaware 19880-0500, and Drug Metabolism and Pharmacokinetic

Section, Stine-Haskell Research Center, Newark, Delaware 19714

Received October 1, 1998

The synthesis and CRF receptor binding affinities of several new series of N-aryltriazolo- and

-imidazopyrimidines and -pyridines are described. These cyclized systems were prepared from

appropriately substituted diaminopyrimidines or -pyridines by nitrous acid, orthoester, or acyl

halide treatment. Variations of amino (ether) pendants and aromatic substituents have defined

the structure-activity relationships of these series and resulted in the identification of a variety

of high-affinity agents (K_i’s < 10 nM). On the basis of this property and lipophilicity differences,

six of these compounds (4d ,i,n ,x, 8k , 9a ) were initially chosen for rat pharmacokinetic (PK)

studies. Good oral bioavailability, high plasma levels, and duration of four of these compounds

(4d ,i,n ,x) prompted further PK studies in the dog following both iv and oral routes of

administration. Results from this work indicated 4i,x had properties we believe necessary for

a potential therapeutic agent, and 4i¹has been selected for further pharmacological studies

that will be reported in due course.

In tr od u ction

and neurological diseases, including depression and

anxiety-related diseases, is considerable and compelling

and is reviewed in the preceding paper.¹¹

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

peptide, is the primary physiological regulator of the

hypothalamic-pituitary-adrenal (HPA) axis by its ac-

tion as a secretagogue of adrenocorticotropic hormone

(ACTH), â-endorphin, and other pro-opiomelanocortin

(POMC)-derived peptides from the anterior pituitary

gland.^2,3In addition to its endocrine role at the pituitary,

immunohistochemical localization of CRF has demon-

strated that the peptide has broad extrahypothalamic

distribution in the central nervous system and produces

a wide spectrum of autonomic, electrophysiological, and

behavioral effects consistent with a neurotransmitter

or neuromodulator role in the brain.^4-7Evidence dem-

onstrating that CRF may play a significant role in

integrating response of the immune system to physi-

ological, psychological, and immunological stressors also

exists.^8,9

A role for CRF has also been postulated in the etiology

of anxiety-related disorders. Centrally administered

CRF produces effects in animals, including increased

arousal in familiar and agitation to unfamiliar sur-

roundings, consistent with the manifestations of anxi-

ety.^12,13Interactions between both benzodiazepine and

non-benzodiazepine anxiolytics and CRF have been

demonstrated in a variety of behavioral anxiety mod-

els.^14,15Preliminary studies using the putative CRF

receptor antagonist R-helical ovine CRF_9-41in a variety

of behavioral paradigms show that the antagonist

produces “anxiolytic-like” effects that are qualitatively

similar to those of benzodiazepines.^12,16-18Neurochemi-

cal, endocrine, and receptor binding studies have all

demonstrated interactions between CRF and benzodi-

azepine anxiolytics providing further evidence for in-

volvement of CRF in these disorders. Chlordiazepoxide

has been shown to attenuate the “anxiogenic” effects of

CRF in both the conflict¹⁴and the acoustic startle tests¹⁹

in rats. The benzodiazepine receptor antagonist (Ro15-

1788), which was without behavioral activity alone in

the operant conflict test, reversed effects of CRF in a

dose-dependent manner; the benzodiazepine inverse

agonist (FG7142) enhanced the actions of CRF.²⁰

Preclinical studies in rats and nonhuman primates

provide some pharmacological support for the hypoth-

esis that hypersecretion of CRF may be involved in

symptoms seen in human depression and related dis-

orders.¹⁰Clinical evidence supporting the hypothesis

that CRF may indeed play a role in psychiatric disorders

^†Department of Chemical and Physical Sciences, DuPont Pharma-

ceuticals Co.

^‡Department of Biological Sciences, DuPont Pharmaceuticals Co.

^§Stine-Haskell Research Center.

The effects of centrally administered CRF are largely

independent of the activation of ACTH and corticoster-

oids,¹⁷and the primary event through which CRF

initiates its stressor responses is by interaction with

Current address: Food and Drug Administration, 1451 Rockville

Park, Rockville, MD 20852.

^∇Current address: Cephalon Inc., Brandywine Parkway, West

Chester, PA.

Published on Web 02/17/1999

834 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

cell-surface proteins on brain target cells. The recent

cloning of CRF₁and CRF₂receptor subtypes, also

reviewed in the previous paper,¹¹provides opportunity

to differentiate some of the intricacies of CRF function

by allowing study of its action as a hormone and a

neurotransmitter.²¹However, to fully comprehend the

physiological significance of these receptor subtypes and

their associated responses, subtype-selective compounds

must be identified.

Thus, the search for small-molecule receptor subtype-

specific ligands is being carried out to help clarify the

role of these various receptors in mediating physiological

response. More importantly, however, based on the

substantial body of pharmacological and clinical evi-

dence, there is high anticipation that a small-molecule

CRF receptor antagonist should indeed provide benefi-

cial therapy for affective disorders or anxiety-related

conditions due to CRF’s role in orchestrating the body’s

response to stress.

ylamino adduct 2, a key intermediate for the construc-

tion of the annelated heterocyclic systems. Cyclization

of 2 to triazolopyrimidines 3 was accomplished using

sodium nitrite in aqueous acid with methylene chloride

as a cosolvent that considerably improved the yield of

this conversion. Treatment of bicyclic chlorides 3 with

primary or secondary amines in protic solvents such as

ethanol in the presence of triethylamine produced the

aminated triazolopyrimidines 4. Triazolopyrimidine ring

desmethyl analogues 11 and 12 were prepared from

commercially available 5-amino-4,6-dichloropyrimidine

using the reactions described to prepare 4 from 2.

Alternatively, the diaminopyrimidine 2 was converted

to imidazopyrimidines 5 (R ) H) by heating with

triethyl orthoformate in acetic anhydride. When other

orthoesters were used under these reaction conditions,

cyclization to the 8-substituted imidazopyrimidine was

slow and displacement of the chloro group was observed.

In these cases treatment of 2 with excess orthoester in

CH₂Cl₂in the presence of acid at room temperature

cleanly afforded iminoether intermediates that ther-

mally cyclized in refluxing xylene to the imidazopyri-

midines 5 (R ) CH₃, n-Bu, Ph). In the case of the

8-(trifluoromethyl)imidazopyrimidine 8n , 2 was acy-

lated with trifluoroacetic anhydride to give amide 6 that

unfortunately underwent hydrolysis when thermal cy-

clization in xylene was attempted. The isolated pyrimi-

done amide was converted to 7 in reluxing EtOH in the

presence of Et₃N, and subsequent treatment with POCl₃

provided 5 (R ) CF₃). Condensation with appropriate

amines as described above yielded the aminated imi-

dazopyrimidines 8 (Ar ) 2-bromo-4-(1-methylethyl)-

phenyl, R ) H, CH₃, CF₃) (Table 1) or 8 (Ar ) 2-chloro-

4,6-dimethoxyphenyl, R ) H, CH₃, n-Bu, Ph) (Table 2).

While a considerable amount of steric bulk could be

accommodated as amino group substituents on the six-

membered heterocycle of the bicyclic systems, it was of

interest to determine whether chirality at this region

in space might influence binding affinity. Thus, in the

case where the two enantiomers of a chiral primary

amine adduct were studied to determine whether ster-

eochemistry at this site might influence binding affinity,

HPLC was used for isomer separation. A Chiralcel-OJ

column (95% MeOH/H₂O/0.1% TEA) of 4x afforded both

enantiomers, 4y,z, in greater than 98% ee.

Ch em istr y

Our initial chemistry effort in this area focused on

optimization of the binding affinity of a non-peptide

lead, identified by high-throughput screening of our in-

house compound library.¹¹After this lead identification,

our Computer Assisted Drug Design (CADD) group

determined conformational preferences and barriers to

rotation by semiempirical methods and established

global energy minima for certain of these compounds.²²

X-ray determinations and variable-temperature NMR

spectroscopy provided physical evidence to support these

calculated conformations. This work determined that a

cyclized version of these diarylamines, wherein the alkyl

group of the central nitrogen atom of i was rigidly held

in position through attachment with the heterocyclic

ring, should provide a constrained bicyclic ligand ii with

potential high affinity for this receptor. This prediction

Intermediates 3 and 5 were also used to prepare

alkoxy analogues of both of the above bicyclic systems.

Thus, by treating these chloro derivatives with alkoxide,

generated from the appropriate alcohol and sodium

hydride in aprotic solvent, aryl ether adducts 9 and 10,

respectively, were produced (Table 1).

was indeed validated by the binding affinities of the first

members of this series that were synthesized which

were comparable to some of the best members of the

noncyclized series as described in the accompanying

paper.²³A recent Pfizer report²⁴also described high CRF

receptor binding affinity for a similar bicyclic system

during the course of this work. We then proceeded to

optimize the activity of these bicyclics by preparing a

variety of related molecules for evaluation of their

binding affinity, as well as absorption, distribution, and

pharmacokinetic properties of selected agents that

might be considered for further evaluation as a potential

therapeutic.

Alter n a te Rou te to 4 a n d 8 (Sch em e 2). Attempted

incorporation of arylamines other than the 2-bromo-4-

isopropylaniline into these bicyclic systems using 1

proceeded with great difficulty. Typically we observed

little or no coupling products with the more-hindered

2,4,6-trisubstituted amines, as well as with other 2,4-

disubstituted anilines. In these cases we found that the

more chemically reactive dichloronitropyrimidine 13

was a necessary precursor to these bicyclic systems

(Scheme 2). Thus, using this nitro compound, monoad-

ducts 14 were produced in DMSO at room temperature

in high yield, albeit in most cases with hydrolysis of the

remaining chloro group, a transformation which may

Tr ia zolo- a n d Im id a zop yr im id in es 4 a n d 8, Re-

sp ectively (Sch em e 1). Treatment of dichloroami-

nopyrimidine 1²⁵with 2-bromo-4-isopropylaniline in

ethoxyethanol at elevated temperatures afforded diar-

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 835

Sch em e 1

have prevented bis-arylamine addition. Interestingly,

of all the arylamines used in this sequence (Table 2),

only collidylamine afforded 15 directly from 13 under

the mild reaction conditions. The hydrolysis products

14 were readily reconverted to the desired nitro chlo-

rides 15 using POCl₃; subsequent reduction of the nitro

group with Fe in acidic media provided 2.

Intermediate 15 was also routinely used for the

introduction of the dialkylamino substituent on the

pyrimidine ring. The resultant diaminonitropyrimidines

16 were then reduced with sodium dithionite in am-

monium hydroxide solution to produce the triaminopy-

rimidines 17, precursors to 4 or 8. Alternatively, dichlo-

ronitropyrimidine 13 was initially treated with secondary

amines in ethanol in the presence of triethylamine at

room temperature. In these cases there was generally

no evidence of hydrolysis product. Upon subsequent

treatment with arylamines in DMF, the nitro adduct

18 also provided 16.

In certain cases we also reduced nitro adduct 14 to

the diaminopyrimidone 19 which was then converted

to bicyclic 20 under the usual conditions. This tria-

zolopyrimidone readily chlorinated with POCl₃provid-

ing yet another route to the bicyclic chlorides 3. Thus,

with these various pathways we were able to prepare

an array of di- and trisubstituted aryl derivatives of

these bicyclic systems.

An analogue of 4 with a norvaline-type side chain was

also prepared by treatment of 13 with DL-norvaline

dimethylamide to give 18, which upon aniline condensa-

tion afforded the nitrodiamine 16 with the amide

pendant. Borane reduction at room temperature yielded

the reduced amide with the nitro group intact. Both of

the norvaline-like pendants were converted to triazolo-

pyrimidines 4h h ,ii after reduction with dithionite and

cyclization with nitrous acid.

Tr ia zolo- a n d Im id a zop yr id in es 25 a n d 26, Re-

sp ectively (Sch em e 3). 2,4,-Dichloro-6-methyl-3-ni-

tropyridine (21) readily prepared from commercially

available 4-hydroxy-6-methyl-3-nitro-2(1H)-pyridone and

POCl₃, was treated with a dialkylamine in refluxing

ethanol to give a 3:1 mixture of regioisomers, 22 and

22′. The position of the amine substituent was estab-

lished by NOE experiments where enhancement of the

butyl methylene protons adjacent to the nitrogen atom

of the butylethylamino group was observed for the major

isomer 22 upon irradiation of the ring proton. Coupling

of the major product 22 with substituted anilines at

elevated temperatures (120-140 °C) afforded adducts

23 which were reduced with Na₂S₂O₄to the correspond-

ing diaminopyridines 24. Cyclization of these com-

pounds with NaNO₂/HOAc or CH₃C(OEt)₃/HCl produced

triazolopyridines 25 or imidazopyridines 26 (Table 3).

In the case of the butylethylamino adduct, the minor

836 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

Sch em e 2

isomer 22′ was also isolated in sufficient quantity to

allow synthesis of the isomeric triazolopyridine 25′,

using the same route and conditions described for the

major isomer.

P yr r olop yr im id in es (Sch em e 4). Selected repre-

sentatives of this series 28 were synthesized from

intermediates 27 described in a recent Pfizer patent²⁶

by condensation with primary and secondary alkyl-

amines (Table 4).

activity relationships (SAR) in these series were on

systems with the 2-bromo-4-isopropylphenyl substituent

on the triazole or imidazole ring. Since the presence of

the pyrimidine methyl group, as in the case of the

anilinopyrimidines and -triazines,¹¹was also found to

be important for high binding affinity (cf. 4d and 11, K_i

) 7.5 and 287 nM, respectively; 4i and 12, 3.7 and 203

nM, respectively), our study maintained this group as

a constant. An early look at cyclic amines on the

pyrimidine ring of these sytems (4a ,b) also established

that comparable open-chain substituents in general

afforded compounds with better binding affinities. We

also found that in the case of these noncyclic secondary

Resu lts

SAR of Tr iazolo- an d Im idazopyr im idin es (Tables

1 a n d 2). Our initial investigations of the structure-

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 837

Sch em e 3

Sch em e 4

enantiomers (4x-z) all possessed comparable affinities

(<10 nM). Methylation of the secondary amine of one

of these compounds (4a a ) had an inconsequential effect

on binding. The elimination of chirality by incorporating

a symmetrical bis-ether pendant on the amine (4b b )

also did not diminish binding from the 10 nM level.

Replacement of the amine proton of this symmetrical

bis-ether with an alkyl group significantly affected

binding when this group was larger than methyl (4ee

vs 4d d , 21 vs 123 nM). The presence of a gem-dimethyl

group on the side-chain of the molecule with a single

ether substituent (4ee) also afforded a compound with

the poor binding affinity (ca. 200 nM) of the unsubsti-

tuted chain analogue (4o). Other variations in the

branched chain of these primary amine adducts in-

cluded benzyl (4ff), ester (4gg), amido (4h h ), and a

second amino functionality (4ii). The amide was the

poorest binder (235 nM), whereas the corresponding

amine had surprisingly good affinity (38.7 nM) in

contrast to these types of diamino pendant analogues

in the anilinopyrimidine and -triazine series.¹¹

amine adducts, the presence of unsymmetrical alkyl

substituents was preferable to identical chains. Using

the high affinity of the N-ethylbutylamino compound

4d as a template, we evaluated the effect of insertion

of oxygen atoms within this side chain on receptor

binding. Of these compounds bis(methoxyethyl)amino

compound 4i (3.7 nM) possessed binding affinity com-

parable to that of 4d and slightly better than that of 4f

(10.5 nM). Lengthening one of these ether chains (4j,

66.9 nM) or converting ethers to hydroxyl groups (4g,h ,

3460, 64 nM, respectively) diminished binding. When

one methoxyethyl chain was maintained on the amino

group, the other amino substituent could be varied

(allyl, cyclopropyl, benzyl) with little change in the high

binding (<10 nM) affinity (4l-n ). However, the 3-picolyl

analogue 4k and secondary amines (4o-q) displayed

poorer affinity.

We further explored primary amine adducts and

determined that when the substituent on the nitrogen

was a branched alkyl group, the K_ifor these compounds

was in most cases <10 nM (e.g., 4r ,t-4v). However,

when these alkyl chains were tied back into a ring

system (4s), the receptor affinity was considerably

weaker (>100 nM). An alcohol group on this branched

chain (4w ) slightly diminished binding (25 nM) from

the alkyl chains, whereas the ether homologue (4x) was

comparable to the alkyl system. The presence of a chiral

center in this latter compound allowed an evaluation

of the effect of the stereochemistry of this side chain on

binding affinity. In this case, however, racemate and

The imidazopyrimidines generally followed the same

SAR as the triazolopyrimidines for amine substituents

at this position of the pyrimidine ring. Compounds with

acyclic alkyl-chain substituents on the amine with or

without ether linkages (8a -c,e,f) possessed high bind-

ing affinity (ca. 10 nM); primary amine adducts also

showed comparable binding affinity to the triazolo

system. In the case of a molecule with a second amino

group on an alkyl chain of a secondary amino adduct

(8d ), the binding affinity (2665 nM) was considerably

weaker than that of the branched primary amino adduct

(4ii) of the triazolo analogue (38.7 nM). The branched

primary amine adducts (8h -k ) also possessed the high

affinity (<10 nM) of their analogues in the previous

series. The presence of a methyl or trifluoro group at

the 8-position of the imidazopyrimidine ring system (8l-

8n ) had little effect on binding affinity in these cases,

indicating this site of the molecule has some steric

tolerance. Replacement of the N atom linker of the

838 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

Ta ble 1. CRH Receptor Binding Affinities of 2-Bromo-4-isopropylphenyltriazolo- and -imidazopyrimidines

compd

Z

R₁

R₂

Q

% yield (method)^a

mp (°C)

formula

anal.^bK_i(nM)^c

4a

4b

4c

4d

11

4e

N

O

-CH₂CH₂CH₂CH₂CH₂-

N

CH

CMe

CCF₃

N

80 (A)

96 (A)

89 (A)

97 (A)

55 (A)

64 (A)

83 (A)

42 (A)

92 (A)

38 (A)

76 (A)

27 (A)

80 (A)

67 (A)

69 (A)

91 (A)

29 (A)

95 (A)

86 (A)

99 (A)

90 (A)

48 (A)

83 (A)

76 (A)

73 (A)

59 (A)

29 (A)

59 (A)

98 (A)

81 (A)

96 (A)

82 (A)

27 (A)

67 (A)

67 (D)

73 (D)

48 (B)

56 (B)

49 (B)

41 (B)

31 (B)

96 (B)

79 (B)

81 (B)

62 (B)

87 (B)

55 (B)

46 (B)

85 (B)

52 (B)

62 (A′)

39 (B′)

21 (A′)

29 (B′)

39 (A′)

11 (B′)

66 (A′)

63 (A′)

amorph solid C₁₉H₂₃BrN₆

C,H,N

46^d

194

25.3

7.5

287

10.5

32.5

3460

64.0

3.7

-CH₂CH₂CH₂CH₂-

147.5-148

92-93

84-86

oil

C₁₈H₂₁BrN₆

C₁₈H₂₃BrN₆

Et

n-Bu

(CH₂)₂OCH₃

n-Bu

C

₂₀H₂₇BrN₆

₁₉H₂₅BrN₆

oil

C₁₉H₂₅BrN₆O

C₂₂H₃₁BrN₆

4f

n-Bu

4g

4h

4i

12

4j

(CH₂)₂OH

70-72

120-122

93-94

oil

C₁₈H₂₃BrN₆O₂

C₁₉H₂₅BrN₆O₂

C₂₀H₂₇BrN₆O₂

C₁₉H₂₅BrN₆O₂

C₂₁H₂₉BrN₆O₂

C₂₃H₂₆BrN₇O

C₂₀H₂₅BrN₆O

C₂₁H₂₇BrN₆O

C₂₄H₂₇BrN₆O

C₁₇H₂₁BrN₆O

C₁₈H₂₃BrN₆O

(CH₂)₂OCH₃

(CH₂)₃OCH₃

n-Bu

(CH₂)₂OH

(CH₂)₂OCH₃

(CH₂)₃OCH₃

3-picolyl

allyl

cyclopropylmethyl

benzyl

H

203

66.9

48.5

4,3

4.6

5.0

242

291

69.6

5.2

120

2.8

3.1

4.5

25.3

6.1

7.5

4k

4l

oil

4m

4n

4o

4p

4q

4r

121-122

134-136

109-110

149-151

171-172

120.5-122

154-155

137-138

162-163

157-159

133-134

114-115

oil

156-158.5

oil

129.5-130

67-69

163-164

179-180 dec C₂₁H₃₀BrN₇H₂O C,H,N

oil

C

₁₈H₂₃BrN₆

C₁₉H₂₅BrN₆

₁₉H₂₃BrN₆

CH(Et)₂

c-pentyl

4s

4t

C

CH(Et)(CH₂)₂CH₃

CH(Et)(CH₂)₃CH₃

CH[(CH₂)₃CH₃]₂

CH(Et)CH₂OH

CH(Et)CH₂OCH₃

(+)-CH(Et)CH₂OCH₃

(-)-CH(Et)CH₂OCH₃

C₂₀H₂₇BrN₆

C₂₁H₂₉BrN₆

4u

4v

4w

4x

4y

4z

4a a

4bb

4cc

4d d

4ee

4ff

4gg

4h h

4ii

8a

C₁₈H₂₃BrN₆O

C₁₉H₂₅BrN₆O

C₂₀H₂₇BrN₆O

C₁₉H₂₅BrN₆O₂

C₂₀H₂₇BrN₆O₂

C₂₁H₂₉BrN₆O₂

C₁₉H₂₅BrN₆O

C₂₄H₂₇BrN₆O

C₁₉H₂₃BrN₆O₂

C₂₁H₂₈BrN₇O

d

e

9.4

(+)-CH(Et)CH₂OCH₃Me

11.2

10.3

21.3

123

195

36.7

19.4

235

38.7

12.4

4.3

9.2

2665

6.1

5.9

210

4.4

5.1

3.6

8.0

0.9

6.7

1.4

1.9

9.7

1.9

3.5

1.5

2.9

7.1

39.7

CH(CH₂OMe)₂

C(CH₃)₂(CH₂OMe)

CH(Bz)CH₂OCH₃

CH(Et)COOCH₃

CH(nPr)CON(CH₃)₂

CH(nPr)CH₂N(CH₃)₂

Et

H

CH₃

Et

H

Et

C

₁₉H₂₄BrN₅

₂₁H₂₈BrN₅

HRMS^f

C,H,N

HRMS

C,H,N

8b

8c

8d

8e

Et

n-Bu

(CH₂)₂OCH₃

(CH₂)₂N(CH₃)₂

n-Bu

oil

C₂₀H₂₆BrN₅O

C₂₁H₂₉BrN₆

oil

C

₂₃H₃₂BrN₅

8f

(CH₂)₂OCH₃

CH(Et)(CH₂)₂CH₃

CH(Et)(CH₂)₃CH₃

CH[(CH₂)₂CH₃]₂

CH(Et)(CH₂)₂OCH₃

Et

(CH₂)₂OCH₃

H

n-Bu

oil

125-126

amorph solid C₂₁H₂₈BrN₅

C₂₁H₂₈BrN₅O₂

C₁₈H₂₂BrN₅O

8g

8h

8i

8j

8k

8l

8m

8n

9a

10a

9b

10b

9c

10c

9d

9e

oil

C₂₂H₃₀BrN₅

C₂₄H₃₄BrN₅

C₂₀H₂₆BrN₅O

87-88

105-106

oil

C₂₂H₃₀BrN₅

(CH₂)₂OCH₃

Et

(CH₂)₂OCH₃

n-Bu

oil

C₂₂H₃₀BrN₅O₂

C₂₂H₂₇BrF₃N₅

C₁₉H₂₄BrN₅O₂

C₂₀H₂₅BrN₄O₂

C₁₉H₂₄BrN₅O

C₂₀H₂₅BrN₄O

C₂₁H₂₈BrN₅O

C₂₂H₂₉BrN₄O

C₁₉H₂₄BrN₅O₃

C₂₁H₂₈BrN₅O₃

69-70

75-76

oil

112-114

CH(Et)CH₂OMe

CH(Et)₂

CH

N

CH

N

CH

N

CH(Et)₂

CH(Et)(CH₂)₃Me

CH(CH₂OMe)₂

CH(CH₂OEt)₂

a

Methods are described in Schemes 1-4; yields reported are for the last step in the sequence. ^bAll compounds gave acceptable combustion

analysis results ((0.4%) unless otherwise indicated. ^cReceptor binding affinities are for the human cloned CRH recetor (hCRF₁) expressed

in 293EBNA cells. K_ivalues < 50 nM are the result of at least two separate test results; K_ivalues > 50 nM are single measurements.

d

Prepared from the enatiomerically pure chiral amine (+35.5°, c ) 0.2, CHCl₃). ^eIsolated from the racemate by HPLC using a chiral

column (-32.5°, c ) 0.2, CHCl₃). ^fHigh-resolution mass spectrometry.

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 839

Ta ble 2. CRH Receptor Binding Affinities of N-Aryltriazolo- and -imidazolopyrimidines

% yield

K_i

compd

R₂

R₄

R₆

Q

R₁

R₃

(method)^a

mp (°C)

formula

anal.^b(nM)^c

4i

Br

H

SMe

CH(Me)₂

H

N

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₂CH₂Me 83 (C)

CH₂CH₂OMe

CH₂CH₂CH₂Me 80 (C)

CH₂CH₂OMe

CH₂CH₂CH₂Me 83 (D)

CH₂CH₂CH₂Me 39 (D)

CH₂CH₂CH₂Me 89 (D)

CH₂CH₂CH₂Me 14 (D)

CH₂CH₂OMe

92 (A)

12^e

74 (D)

68 (D)

105-109S

142-143

oil

143-145

oil

124-126

141.5-143.5 C₂₀H₂₈BrN₇O₃

112.5-114

156-157.5

oil

C₂₀H₂₇N₆BrO₂

C₂₀H₂₈N₆O₂

C₂₁H₃₀N₆O₂S

C₂₁H₃₀N₆O₄S

C₁₉H₂₅BrN₆O₂‚HCl

C₁₉H₂₅BrN₆O₄

C,H,N

C,H,N 3918

C,H,N

C,H,N 1026

C,H,N

3.7

4jj

4k k

4ll

15.3

SO₂Me CH(Me)₂

4m m Br

OMe

Br

OMe

N(Me)₂

OMe

H

3.0

4n n

4oo

4p p

4qq

4r r

4ss

4tt

4u u

4vv

4w w Br

Br

Cl

Br

46 (D)

45 (C)

28.3

48.2

4.4

21.0

7.4

20.4

10.6

100

33.7

10.5

33.2

23.5

15.4

24.1

27.5

C₁₉H₂₅ClN₆O₂

C₁₉H₂₅ClN₆O₄

C₁₇H₂₀Br₂N₆O₂

C₁₇H₂₀Br₂N₆

C₁₈H₂₃BrN₆S

C₁₈H₂₃BrN₆SO₂

69 (C)

98 (D)

Br

SMe

SO₂Me

COMe

N(Me)₂

OCF₃

N(Me)₂

CH₃

Br

141-143

oil

CH₂CH₃

C₁₉H₂₃BrN₆O‚1/2H₂O C,H,N

H

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH(Et)CH₂OMe

CH(Et)₂

CH(Et)n-Bu

CH(Me)n-Pr

CH(n-Pr)₂

CH(Bn)CH₂OMe

CH(n-Pr)₂

CH₂CH₂OMe

CH₂CH₃

CH₂CH₂OMe

CH₂CH₃

95 (F,A) 108-109

55 (C)

88 (C)

C₁₉H₂₆BrN₇O₂

C₁₈H₂₀BrF₃N₆O₃

C₂₀H₂₆N₇F₃O₂

C₂₀H₂₆N₇F₃

C₂₀H₂₈N₆O₂

C₁₉H₂₇N₆

C,H,N

4xx

4yy

4zz

4a b

4a c

Br

oil

CF₃

CH₃

105-109

142-143

83.5-85

oil

59-60.5

oil

141.5-143.5 C₁₆H₂₂N₆O

oil

156-157.5

184.5-186.5 C₁₉H₂₆N₆

141.5-142.5 C₂₁H₃₀N₆

151-152

145-146.5

oil

128-129

oil

125.5-126

137-138

126.5-127

141.5-142

117-120

86.5-88.5

106.5-108.5 C₂₁H₂₈ClN₅O₂

oil

CH₂CH₂CH₂Me 91 (C)

CH₂CH₂OMe 98 (C)

CH₂CH₂CH₂Me 97 (C)

CH₂CH₂OMe 92 (E)

CH₂CH₂CH₂Me 86 (E)

CH₃

CH3

CH₃

H

4a d ^dCH₃

C₁₉H₂₇N₇O₂

C₁₉H₂₇N₇

416

65

4a e^dCH₃

4a f

4a g

4a h

4a i

4a j

4a k

4a l

4a m

4a n

8f

8o

8p

8q

8r

8s

8t

8u

8v

CH₃

Br

Cl

H

97 (C)

86 (C)

75 (C)

79 (C)

73 (C)

86 (C)

72 (C)

75 (C)

83 (C)

96 (B)

C,H,N 1204

CH₂Me

H

C₁₈H₂₆N₆O

C₁₉H₂₆N₆O

HRMS

C,H,N

HRMS

C,H,N

20.7

59.4

5.7

7.6

7.1

4.0

27.7

0.8

5.9

3.5

31.3

5.2

23.8

20.7

25.0

3.5

C₁₉H₂₆N₆

C₂₁H₃₀N₆

C₂₄H₂₈N₆O

C₁₈H₂₁Br₂N₆

C₂₁H₂₈BrN₅O₂

C₂₀H₂₆BrN₅O₂

C₂₀H₂₆BrN₅O₄

C₂₀H₂₆ClN₅O₂

C₂₁H₂₈ClN₅O₄

C₂₁H₂₉N₅O₂

C₂₁H₂₉N₅

CH(Me)₂

OMe

CH₃

H

CH

CH₂CH₂OMe

OMe

CH₃

OMe

CH₂CH₂CH₂Me 79 (C)

CH₂CH₂OMe 10 (D)

CH₂CH₂CH₂Me 94 (C)

Cl

CH₂CH₂CH₂OMe CH₂CH₂OMe

CH₂CH₂OMe

CH₂CH₃

82 (C)

86 (C)

CH₃

Cl

CH₂CH₂OMe

CH₃

CH₂CH₂CH₂Me 87 (C)

CH₂CH₂CH₂Me 97 (C)

CH₂CH₂CH₂Me 86 (C)

CH₂CH₂CH₂Me 99 (C)

OMe

CMe

C-n-Bu CH₂CH₃

CC₆H₅CH₂CH₃

CH₂CH₃

C₂₄H₃₄ClN₅O₂

C₂₆H₃₀ClN₅O₂

22

133

8w

d

^a-cSee Table 1 for explanation. 3-Pyridinyl rather than phenyl. ^eObtained as a side product from a stannylation reaction of the

2-bromo compound; details will be reported at a later date.

primary amine adducts with an oxygen resulted in a

series of ethers that also had high binding affinity (<10

nM), comparable to that of the nitrogen-linked ana-

logues, in either of these systems (9a -d , 10a -c).

Extension of the terminal ether groups (9e) did, how-

ever, adversely affect binding (39.7 nM), a result not

observed with 4v where all atoms of this extended chain

were carbon (4.5 nM).

The influence on binding affinity of substituents on

the phenyl ring and its replacement with an ap-

propriately substituted pyridine were also studied (Table

2). Since the bis(methoxyethyl)amino and the N-ethyl-

butylamino groups afforded high-affinity agents in the

2-bromo-4-isopropylphenyl series, the effect of changes

in phenyl substitution was primarily determined on

triazolo- and imidazopyrimidines with these two amino

groups. As in the case of the anilinopyrimidines and

-triazines,¹¹there was a need for at least one bulky

ortho-substituent on the phenyl ring for high binding

affinity (4i,jj, 4.5 and 3918 nM, respectively), conferring

orthogonality between the planes of the phenyl and

bicyclic ring systems.²³Replacement of bromo with

methylthio (4k k ) was without significant effect, whereas

the methyl sulfone (4ll) was a much weaker binder

(1026 nM). Compounds with a 2-bromo substituent and

a replacement for the 4-isopropyl group (4r r -w w )

tended to be slightly weaker binders than the isopropyl

analogue, with the electron-withdrawing acetyl (4vv)

and methylsulfonyl (4u u ) groups showing the greatest

decrease in binding affinity (33.7 and 100 nM, respec-

tively). This 4-position of the phenyl group seems to

favor the presence of an alkyl substituent as long as it

does not exceed certain steric requirements as delin-

eated in the previous paper. Other disubstituted phenyl

groups also afforded no improvement in the binding of

the bromoisopropyl pairing (4xx-zz), except when the

840 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

Ta ble 3. CRH Receptor Binding Affinities of N-Substituted Phenyltriazolo- and -imdazolopyridines

% yield

compd

R₁

R₂

R₃

Q

(method)^a

mp (°C)

formula

anal.^b

K_i(nM)^c

25a

26a

25b

26b

25c

25d

25e

25f

Et

n-Bu

(CH₂)₂OCH₃

n-Bu

Br

SMe

SO₂Me

SMe

N

CCH₃

N

CCH₃

N

70 (G)

68 (H)

45 (G)

90 (H)

81 (G)

68 (G)

74 (G)

68 (G)

70 (G)

168-171

oil

87-89.5

oil

151-153

oil

C₂₁H₂₈BrN₅‚HCl

C,H,N,Br

C,H,N

C,H,N,S

C,H,N

3.2

3.9

4.7

9.8

2.2

C₂₃H₃₁BrN₄‚HCl‚H₂O

C₂₁H₂₈BrN₅O₂‚HCl‚H₂O

C₂₃H₃₁BrN₄O₂‚HCl‚H₂O

(CH₂)₂OCH₃

Et

C

₂₂H₃₁N₅S

Et

n-Bu

C₂₂H₃₁N₅O₂S

C₂₂H₃₁N₅O₄S

83

4.7

476

>10000

(CH₂)₂OCH₃

Et

(CH₂)₂OCH₃

n-Bu

SO₂Me

Br

143-145

oil

25a ′

C₂₁H₂₈N₅Br

^a-cSee Table 1 for explanation.

2,4-dibromophenyl group was paired with the 4-heptyl-

amino adduct in the triazolopyrimidine series (4a n )

providing a compound with about 1 nM affinity.

the annelated pyrimidines, replacement of 2-bromo for

2-thiomethyl maintained this high affinity (25c,e),

whereas the corresponding sulfones diminished binding

20-100-fold (25d ,f). Surprisingly, 25a ′, the isomeric

pyridine analogue of 25a , showed no affinity for the CRF

receptor up to 10 µM, demonstrating the importance of

a nitrogen heteroatom at the 4-position of this bicyclic

system for optimal receptor interaction of these mol-

ecules.

SAR of P yr r olop yr im id in es (Ta ble 4). Selected

members of these compounds were synthesized to

determine the importance of the five-membered ring N

atoms on binding, in light of the profound effect ob-

served for the pyrimidine nitrogens (vide supra). In the

course of this study, a Pfizer patent on this series of

molecules²⁷disclosed their utility as CRF receptor

antagonists. The compounds we prepared are included

for comparison with the other bicyclic systems. This

series showed trends comparable to the other N-ethyl-

butylamino compounds with 28a ,d possessing high

binding (<2 nM), which in this case was slightly better

than the bis-ether analogue 28f (7 nM). The morpholinyl

derivative (28e) also showed the diminished binding

(201 nM) of other cyclic secondary amines found in the

earlier described series. The presence of an alcohol on

the side chain (28b) or an unbranched primary amine

adduct (28c) showed decreased binding affinity (about

30 nM) as was also seen in the other bicyclic series.

Certain compounds with a 2,4,6-trisubstituted phenyl

group were also prepared. In the case of the 2-halo-4,6-

dimethoxy compounds the butylethylamino compounds

(4m m ,p p ) were comparable in binding affinity to 4i and

5-9 times more potent the bis-ether analogues (4n n ,qq,

28 and 21 nM, respectively). Thus, substituents on the

phenyl ring do indeed influence the SAR of the bicyclic

system. Replacement of the 6-methoxy group with

dimethylamino also diminished affinity about 2-fold in

the bromo series. Compounds with a 2,4,6-trimeth-

ylphenyl group (4a b,a c,a f-a h ) tended to be poorer

binders by a factor of 2-fold, compared to 2-bromo-4-

isopropylphenyl compounds that possessed the same

amino substituents. However, when the 2,4,6-trimeth-

ylphenyl series was explored more fully, primary amino

adducts with branched alkyl chains (4a i-a l) had com-

parable binding affinity (<10 nM) to 4i; an amino group

with a 2-(1-methoxy-3-phenylpropyl) chain (4a m ) was

a weaker binder (28 nM). The 3-collidyl derivatives of

both the bis-ether and dialkylamino compounds (4ad,ae)

were not as potent (416 and 65 nM) as their mesityl

counterparts (4a b,a c) with K_i’s of about 25 nM. There

was no significant difference between the binding af-

finities of the trisubstituted phenyl compounds in the

triazolo and imidazo series (8o-t vs 4m m ,n n ,p p ,qq,a -

b,a c). A further look at the effect of substituents on the

carbon atom of the imidazo ring in the 2-chloro-4,6-

dimethoxyphenyl series indicated that methyl was again

equivalent (3.5 nM) to hydrogen (8u ), but a butyl chain

diminished binding by about 6-fold (8v) and a phenyl

group by about 40-fold (8w ).

Discu ssion a n d P h a r m a cok in etic Stu d ies

The evaluation of amino groups on the pyrimidine

ring established early on that acyclic alkyl groups were

preferred substituents for maximum CRF receptor

binding affinity in these bicyclic systems. The interrup-

tion of this alkyl chain with oxygen atoms generally

maintained this high afffinity while decreasing the

lipophilicity of these molecules. Lipohilicity has long

been recognized as an important property for enabling

molecules to penetrate the blood-brain barrier (BBB).

The identification of agents with appropriate hydrophobic/

hydrophilic balance has been shown to increase the

probability of finding potential centrally acting thera-

SAR of Tr ia zolo- a n d Im id a zop yr id in es (Ta ble

3). In the annelated pyridine series, representatives

with both the bis(methoxyethyl)amino and the N-

ethylbutylamino groups were prepared. In the cases

with the 2-bromo-4-isopropylphenyl substituent, tri-

azolo- and 8-methylimidazopyridines (25a ,b, 26a ,b)

possessed high affinity (<10 nM) that was comparable

to that of the pyrimidine analogues. As in the case of

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 841

Ta ble 4. CRH Receptor Binding Affinities of N-(2,4,6-Trimethylphenyl)pyrrolopyrimidines

% yield

compd

R₁

R₂

R₃

(method)^a

mp (°C)

formula

anal.^b

K_i(nM)^c

28a

28b

28c

28d

28e

28f

Et

H

n-Bu

HOCH₂CHEt

n-Bu

CH₃

H

CH₃

79 (I)

72 (I)

89 (I)

78 (I)

34 (I)

>325

C₂₄H₃₄N₄‚HCl

C₂₂H₃₀N₄O‚HCl

C₂₃H₃₀N₄

C₂₃H₃₂N₄

C₂₂H₂₈N₄O

C₂₄H₃₄N₄O₂

C,H,N

HRMS

0.84

27.3

199-200

219-220

171-172

157.5-15

oil

29.0^d

1.61^d

201^d

7.1^d

Et

n-Bu

-CH₂CH₂OCH₂CH₂-

(CH₂)₂OCH₃

d

^a-cSee Table 1 for explanation. Single test results.

peutic agents,^28,29the goal of this work. Indeed, when

the log P of certain of these molecules was calculated,³⁰

we routinely found values >6 suggesting that these

lipophilic agents might possess unacceptable absorption,

distribution, or pharmacokinetic properties. Thus, the

investigation of secondary amines in these series af-

forded a way of decreasing this property through

removal of one of these chains. However, it was found

in these cases that a branched chain alkyl group of five

or six carbon atoms was necessary for optimal binding

(4r ,t-w ) diminishing the advantage of removing one

of the alkyl chains; the ether analogues of these chains,

then, provided the means of reducing the hydrophobicity

of these substituents. In the case of 4x, a branched alkyl

ether chain analogue, enantiomers 4y,z were also

evaluated for binding affinity and found to be equivalent

suggesting that while a hydrophobic binding pocket with

definite steric limits was present at the site of interac-

tion, discrete spatial orientation of the group occupying

this location was not necessary. This same high binding

affinity was also observed in the pyrimidinyl ethers for

these branched chain analogues though no significant

lipophilicity advantage was attained (9a -c, 10a -c).

The introduction of various functionality on these side

chains that we felt might favorably affect lipophilicity

or solubility generally diminished binding affinity (4gg-

ii).

A number of di- and trisubstituted phenyl analogues

in the triazolopyrimidine series maintained the high

binding affinity of N-ethylbutylamino and bis(methoxy-

ethyl)amino compounds 4d ,i, respectively (Table 2). In

some cases where groups such as the methanesulfonyl

(4ll,u u ) were introduced to decrease lipophilicity, bind-

ing affinity was adversely affected; diminished binding

was also observed with an acetyl group (4vv). A larger

number of analogues were prepared in the 2,4,6-tri-

methylphenyl series of both the triazolo- and imida-

zopyrimidines, and in general the SAR followed that of

the 2-bromo-4-isopropylphenyl series. The trimethylated

pyridyl analogues 4a d ,a e both showed diminished af-

finity compared to their phenyl counterparts.

diminishing methanesulfonyl group had the same det-

rimental effect on binding as indicated earlier. While

two of the pyrrolopyrimidines (28a ,d ) were among the

compounds with the highest binding affinity in this

group, their high lipophilicity (calculated log P > 6) and

extreme insolubility made them difficult to work with

and evaluate in secondary pharmacological paradigms.

The in vivo activity of 28d as a selective CRF receptor

antagonist in the learned-helplessness procedure, a

putative model of depression, has recently been re-

ported.³¹The high doses (10-32 mg/kg) of this com-

pound needed to show activity via intraperitoneal

administration are suggestive of suboptimal pharma-

cokinetic properties.

Of the compounds in these various series, we selected

high-affinity representatives with various amine sub-

stituents and the 2-bromo-4-isopropylphenyl group on

the triazole or imidazole ring for further study. These

included a dialkylamino (4d ), a bis(alkoxyalkyl)amino

(4i), a methoxyethylbenzylamino (4n ), two racemic

methoxypentylamino (4x, 8k ), and a branched chain

alkoxyalkyl ether (9a ) compound. Our initial work³²

evaluated the pharmacokinetic (PK) parameters of these

compounds in rats after po dosing (1 mg/kg, methocel

suspension). We felt that compounds with good bioavail-

ability, blood plasma levels, and duration would provide

reliable indicators of the biological activity of this new

type of potential therapeutic agent. As shown in Table

5 several of these compounds (4d ,i,n ,x) have reasonably

good oral bioavailability (16-27%); moreover, maximum

plasma levels (C_max) 3-10 times the K_ivalue of the

compound and long plasma duration, where the plasma

concentration persisted above the K_ilevel of the com-

pound for 8-16 h, were also observed. Since these

compounds were expected to mediate their in vivo

responses through interaction with receptors in the

CNS, steady-state brain-to-plasma ratios for these

compounds were also determined in the rat. Of these

compounds, 4i,x showed a favorable brain-to-plasma

ratio of ca. 0.6.

Subsequently, we performed further in vivo evalua-

tions of the four compounds with good rat oral bioavail-

ability in the dog at 1 mg/kg by both iv (ethanol/PEG

The SAR of the active regioisomer in the triazolo- or

imidazopyridines was also comparable to the SAR of the

pyrimidine series. Introduction of the lipophilicity-

842 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

Ta ble 5. Summary of Pharmacokinetic Parameters of Selected

Compounds in Rats after 1 mg/kg po Dose

bioavailability and high plasma levels and duration of

four of these compounds (4d ,i,n ,x) prompted further

pharmacokinetic studies in the dog following both iv and

oral routes of administration. Results from this work

indicated 4i,x had properties we believe to be important

for a potential therapeutic agent, and 4i has been

selected for further pharmacological studies that will

be reported in subsequent manuscripts.

plasma plasma

b

K_i^a

C_max

AUCT^c

plasma

_max/K_i^eB/P^f

cLog

compd

(nM) (nM) (nM‚h) %F^d

C

P^g

4d (soln)^k7.5

4i (susp)^l3.7

4n (susp)^l5.8

4x (susp)^l6.1

8k (susp)^l8.0

9a (susp)^l1.9

49.2ⁱ

471ⁱ

26.5ⁱ

17^j

15.5ⁱ

21ⁱ

7^j

7ⁱ

8^j

2ⁱ

5ⁱ

2^j

4^j

ND^h7.4

30^j

98^j

0.6^m5.04

0.2^j.n6.23

0.6^o4.93

12.5ⁱ

30.7ⁱ

19.9^j

5.9^j

137ⁱ

146ⁱ

30^j

ND

4.85

5.20

21^j

6^j

Exp er im en ta l Section

a

Proton NMR spectra were obtained on VXR or Unity 300-

or 400-MHz instruments (Varian Instruments, Palo Alto, CA);

chemical shifts were recorded in ppm (δ) from an internal TMS

standard in deuteriochloroform or deuteriodimethyl sulfoxide

as specified. Coupling constants were measured in hertz (Hz).

Mass spectra were measured with a Hewlett-Packard 5988A

or a Finnigan MAT 8230 mass spectrometer with a particle

beam interface using NH₃for chemical ionization. High-

resolution mass spectra (HRMS) were obtained on a VG 70-

VSE instrument with NH₃as a carrier gas for chemical

ionization. Optical rotations were obtained on a Perkin-Elmer

241 polarimeter. Elemental analyses were performed by

Quantitative Technologies, Inc., Whitehouse, NJ . Solvents and

reagents were obtained from commercial vendors and used

without further purification unless otherwise indicated. Melt-

Receptor binding affinities are for the cloned CRF receptor

b

(hCRF₁) expressed in 293EBNA cells. Peak plasma level of

compound in nM. ^cPlasma area under the compound concentra-

tion-time curve. Oral bioavailability. ^eThe ratio of the peak

d

plasma concentration (nM) over the K_iof the compound. ^fBrain

concentration to plasma concentration. ^gCalculated log P of these

h

i

compounds. Not determined. Average of two determinations.

^jSingle determination. Compounds were administered in EtOH/

k

l

m

PG (20:80). Compound administered in methocel. Infusion of

n

0.67 mg/kg/h in EtOH/PG (20:80). Infusion of 0.025 and 0.25 mg/

kg/h in EtOH/PG/PEG/H2O (10:30:30:30). ^oInfusion of 0.033 and

0.33 mg/kg/h in EtOH/PG/PEG (20:40:40).

Ta ble 6. Pharmacokinetic Parameters of Selected Compounds

after 1 mg/kg iv^aAdministration in Dogs

d

compd

n

Cl^b(L/h/kg)

V_ss^c(L/kg)

t_1/2(h)

ing points were obtained on

apparatus and are uncorrected.

a Thomas-Hoover capillary

4d

4i

4n

4x

4

6

5

0.555 ( 0.130

0.478 ( 0.078

0.523 ( 0.098

0.443 ( 0.119

7.54 ( 2.68

15.55 ( 3.90

24.72 ( 14.79

10.10 ( 4.36

26.0 ( 10.5

33.2 ( 9.7

46.7 ( 26.3

19.0 ( 11.1

4-N-[2-Br om o-4-(1-m eth yleth yl)p h en yl]-6-ch lor o-2-m e-

th ylp yr im id in e-4,5-d ia m in e (2). 5-Amino-4,6-dichloro-2-me-

thylpyrimidine (3)²⁵(28.5 g, 0.16 mol) and 2-bromo-4-isopro-

pylaniline (34.24 g, 0.16 mol) in 2-ethoxyethanol (100 mL) were

refluxed at 135 °C for 30 h. After the reaction mixture was

cooled, the solvent was removed in vacuo, the residue taken

up into dichloromethane, and the organic phase washed with

water, dried over anhydrous magnesium sulfate, and filtered.

Solvent removal gave an oil that was purified by flash

chromatography (silica gel) using methanol/CH₂Cl₂(1:100) to

yield the desired product as a cream-colored solid (32.1 g,

56%): mp 144.5-146 °C.

Meth od A: Gen er a l P r oced u r es for th e Syn th esis of

2-Br om o-4-isop r op ylp h en yltr ia zolop yr im id in es 4. 3-[2-

Br om o-4-(1-m eth yleth yl)p h en yl]-7-ch lor o-5-m eth yl-3H-

1,2,3-tr ia zolo[4,5-d ]p yr im id in e (3). To 2 (12.5 g, 0.035 mol)

in dichloromethane (125 mL) and 50% aqueous acetic acid (125

mL) was added sodium nitrite (2.55 g, 0.037 mol) in water (10

mL) dropwise at room temperature. After addition, the reac-

tion was stirred for an additional 15 min. The organic layer

was then separated, washed with water, and dried with

anhydrous magnesium sulfate. Solvent removal gave a residue

that was purified by flash chromatography (silica gel) using

CH₂Cl₂to afford a light brown oil. Crystallization from 1:1

hexane/pentane (150 mL) yielded 3 as an off-white solid (12.1

g, 94%): mp 72-74 °C.

7-[Bis(2-m eth oxyeth yl)a m in o]-3-[2-br om o-4-(1-m eth yl-

et h yl)p h en yl]-5-m et h yl-3H -1,2,3-t r ia zolo[4,5-d ]p yr im i-

d in e (4i). To 3 (3.1 g, 8.45 mmol) in ethanol (50 mL) was added

bis(2-methoxyethyl)amine (1.35 g, 10.1 mmol), followed by

triethylamine (1.02 g, 10.1 mmol) and the reaction mixture

was refluxed for 3 h. Solvent removal in vacuo gave a residue

that was partitioned between ethyl acetate and water. The

organic layer was then washed with brine, dried with anhy-

drous magnesium sulfate, and stripped down to a pale yellow

liquid that was purified by a flash chromatography (silica gel)

using CH₂Cl₂. Recrystallization of the isolated product from

hexane gave 4i as a white crystalline solid (3.62 g, 92%): mp

93-94 °C; ¹H NMR (CDCl₃) δ 1.25 (d, 6H, CH(CH₃)₂), 2.58 (s,

3H, C-5 CH₃), 3.0 (m, 1H, CH(CH₃)₂), 3.39-3.4 (2s, 6H, 2

OCH₃), 3.7-3.85 (2t, 4H, 2 N-CH₂), 4.1-4.6 (2t, 4H, 2 -CH₂-

O-CH₃),7.4-7.6 (2m, 3H, Ar); MS (CI) M⁺) 463.2, M + 2 )

465.2.

a

iv formulation: ethanol/PEG 400/water (5:65:30) for 4d ,n ,x;

ethanol/PG/PEG 400/water (10:30:30:30) for 4i. ^bClearance. ^cVol-

d

ume of distribution at steady state. Elimination half-life.

400/H₂O) and po (methocel with or without Tween 80/

H₂O) routes of administration. In the iv study (Table

6) clearance, volume of distribution, and elimination

half-lives were determined. Of particular significance

in these measurements were the longer elimination half-

lives generally observed in the dog than in the rat. In

the oral dog study (Table 7) C_max, t_max(the time to reach

maximum plasma levels), area under the curve (AUCT)

(a measure of the duration of the parent molecule over

time), and bioavailability (%F) were determined. Of

these compounds 4i,x showed the best pharmacokinetic

profiles: high oral bioavailabilities (30% and 53%,

respectively) and plasma levels (307 and 546 nM,

respectively); rapid absorption to reach peak plasma

levels (0.67 and 0.5 h, respectively); and long elimination

half-lives (33 and 19 h, respectively). Thus, in this

species these two compounds again showed PK proper-

ties that were sought for a pharmacological agent with

therapeutic potential. We favored 4i for additional

evaluation since it possessed favorable in vivo pharma-

cological properties which along with that of other

related compounds will be reported in due course.

Con clu sion

We have described the synthesis and CRF receptor

binding affinities of several series of N-aryl polyazain-

doles. Variations of amino (ether) pendants and aro-

matic substituents have defined the structure-activity

relationships of these series and resulted in the iden-

tification of a variety of high-affinity agents. On the

basis of this property and lipophilicty differences, six

of these compounds (4d ,i,n ,x, 8k , 9a ) were initially

chosen for rat pharmacokinetic (pk) studies. Good oral

Meth od B: Gen er a l P r oced u r es for th e Syn th esis of

2-Br om o-4-isop r op ylp h en ylim id a zop yr im id in es 8 a n d

Oth er Ar yl 8-Su bstitu ted Im id a zop yr id in es. 9-[2-Br om o-

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 843

Ta ble 7. Pharmacokinetic Parameters of Selected Compounds after 1 mg/kg po Administration in Dogs

b

compd^a

n

C_max(nM)

T_max^c(h)

AUC^e(nM‚h)

%F^e

K_i(nM)^f

4d

4i

4n

4x

4

6

5

141.3 ( 55.1

307.6 ( 131.6

39.04 ( 15.49

545.8 ( 123.9

1.25 ( 0.5

0.67 ( 0.26

0.8 ( 0.3

824.3 ( 89.3

914.4 ( 212.7^g

365.7 ( 191.8

3144.9 ( 958.7

18.4 ( 7.1

30.7^h

7.3 ( 4.1

53.1 ( 10.5

5.0

4.7

5.8

7.3

0.50 ( 0.31

a

b

po formulation: 0.25% methocel (with 0.1% Tween 80 for 4d ,n ). Peak plasma concentration of compound after po administration.

^cTime to achieve maximum plasma levels. Area under the compound concentration-time curve. ^eOral bioavailability. ^fBinding affinity

constant. Value determined from 0 to 24 h. Dogs were given iv or po dose.

d

g

h

4-(1-m eth yleth yl)p h en yl]-6-ch lor o-2-m eth yl-9H-im id a zo-

[4,5-d ]p yr im id in e (5, R ) H). Compound 2 (12.2 g, 0.034

mol) in triethyl orthoformate (90 mL) and acetic anhydride

(90 mL) was heated at 120 °C for 5 h. The solvent was stripped

off in vacuo, the residue was partitioned between chloroform

and water, and the pH of the aqueous phase was adjusted to

8. After extracting with additional chloroform, the extracts

were washed with brine, dried with anhydrous magnesium

sulfate, and stripped down to a brown oil. The crude oil was

purified by flash chromatography (silica gel) using CH₂Cl₂,

followed by recrystallization from petroleum ether to give 5

(R ) H) as an off-white crystalline solid (4.9 g, 40%): mp 90-

pale yellow liquid. The crude residue was purified by a flash

chromatography (silica gel) using CH₂Cl₂to give 8b as a light

brown oil (0.73 g, 56%): ¹H NMR (CDCl₃) δ 1.0-1.2 (2t, 6H, 2

CH₃), 1.25 {d, 6H, CH(CH₃)₂}, 1.4-1.6 (2m, 4H, 2 CH₂) 2.58

(s, 3H, C-2 CH₃), 3.0 (m, 1H, CH(CH₃)₂), 3.5 (q, 2H, -N-CH₂-

CH₃), 4.0 (br, 2H, -N-CH₂), 7.25-7.4 (m, 2H, Ar), 7.6 (s, 1H,

Ar), 7.8 (s, 1H, C-8 CH); MS M⁺) 430.1, M + 2 ) 432.1

Meth od s A′ a n d B′: Gen er a l P r oced u r e for th e Syn -

th esis of 3-[2-Br om o-4-(1-m eth yleth yl)p h en yl]-7-a lk oxy-

5-m eth yl-3H-1,2,3-tr ia zolo[4,5-d ]- or -9H-im id a zo[4,5-d ]-

p yr im id in es 9 a n d 10. 3-[2-Br om o-4-(1-m eth yleth yl)p h en -

y l]-7-(1-m e t h o x y m e t h y lp r o p o x y )-5-m e t h y l-3H -1,2,3-

tr iazolo[4,5-d]pyr im idin e (9a). To 1-methoxy-2-butanol (0.26

g, 2.4 mmol) in toluene (20 mL) was added 60% NaH (mineral

oil) (0.12 g, 2.4 mmol), and the mixture was stirred at room

temperature for 10 min. Compound 3 (0.74 g, 2.0 mmol) was

then added, and the reaction mixture was refluxed for 1 h,

cooled to room temperature, and quenched with water (10 mL).

The organic layer was separated, washed with brine, dried

with anhydrous magnesium sulfate, and stripped down to a

pale yellow liquid that was purified by a flash chromatography

(silica gel) using CH₂Cl₂as an eluent to afford 9a as a colorless

oil (0.54 g, 62%): ¹H NMR (CDCl₃) δ 1.05 (t, 3H, CH₃), 1.35

(d, 6H, CH(CH₃)₂), 1.95 (q, 2H, CH₂), 2.78 (s, 3H, C-5 CH₃),

3.0 (m, 1H, CH(CH₃)₂), 3.4 (s, 3H, OCH₃), 3.6-3.8 (m, 2H,

O-CH₂), 5.85 (m, H, O-CH), 7.4 (m, 2H, Ar), 7.6 (s, 1H, Ar);

MS M⁺) 434.2, M + 2 ) 436.2

1

91 °C; H NMR (CDCl₃) δ 1.25 {d, 6H, CH(CH₃)₂}, 2.8 (s, 3H,

C-2 CH₃), 3.0 {m, 1H, CH(CH₃)₂}, 7.2 (m, 2H, Ar), 7.45 (s, 1H,

Ar), 8.18 (s, 1H, C-8 CH).

5 (R ) CH ₃, n -Bu , P h ; Ar ) 2-ch lor o-4,6-d im et h oxy-

p h en yl). To 1 mmol of 2 in 10 mL of CH₂Cl₂was added 10

mmol of ortho ester and 5 mmol of anhydrous HCl/dioxane

solution, and the reaction mixture was stirred for 1-2 h (R )

n-Bu, CH₃) on 30 h (R ) Ph) at room temperature. Partitioning

the reaction mixture with saturated NaHCO₃solution and

CHCl₃and the usual workup gave a residue that was taken

up into xylenes and refluxed for 13-16 h (R ) n-Bu, CH₃) or

26 h (R ) Ph). The solvent was removed in vacuo to give a

residue that was crystallized from ether (R ) CH₃) or hexane

(R ) n-Bu) or chromatographed (R ) Ph) to afford 5 (Ar )

2-chloro-4,6-dimethoxyphenyl; R ) CH₃, n-Bu, Ph).

4-[2-Br om o-4-(1-m et h ylet h yl)p h en yla m in o]-6-ch lor o-

2-m eth yl-5-(tr iflu or oa cetyla m in o)p yr im id in e (6). A solu-

tion of 2.5 g (7 mmol) of 2 in 15 mL of trifluoroacetic anhydride

was refluxed under nitrogen overnight. Solvent removal in

vacuo gave a homogeneous oil [TLC, silica: 1:50 MeOH/CH₂-

Cl₂; R_f(SM) 0.41; R_f(prod) 0.65] that was used without further

purification for the cyclization process.

6-Hydr oxy-9-[2-br om o-4-(1-m eth yleth yl)ph en yl]-2-m eth -

yl-9H-im id a zo[4,5-d ]p yr im id in e (7). The above residue was

taken up into 15 mL of p-xylene and reluxed overnight. Solvent

removal gave, after crytallization from EtOH, 2.4 g of white

solid that turned out to be the pyrimidone analogue of 6: mp

>265 °C; MS: M⁺433. To 1.65 g (0.0038 mol) of this

pyrimidone amide in 35 mL of EtOH was added 1.5 mL of

Et₃N, and the solution was refluxed overnight. The solvent

was removed in vacuo and the residue taken up into CH₂Cl₂,

washed with water, and dried. Solvent removal gave 1.55 g

(99%) of 7 which was converted to the chloride 5 without

further purification.

Con ver sion of 7 to 5 (R ) CF ₃). To 1.85 g (4.5 mmol) of 7

in 20 mL of POCl₃was added 0.8 g of N,N-diethylaniline

dropwise over a 10-min period, and the reaction mixture was

refluxed for 3 h. After solvent removal in vacuo, the brown

residue was treated with ice-water, and the mixture was

stirred for 1 h and then extracted with ether. The combined

extracts were washed with water and dried with MgSO₄.

Solvent removal gave a viscous residue that was chromato-

graphed (CH₂Cl₂) to afford 1.1 g of oil that crystallized upon

trituration with pentanes to give 1.05 g (54%) of white solid 5

(R ) CF₃): mp 132-133 °C.

6-(N-Eth ylbu tyla m in o)-9-[2-br om o-4-(1-m eth yleth yl)-

ph en yl]-2-m eth yl-9H-im idazo[4,5-d]pyr im idin e (8b). Com-

pound 5 (1.1 g, 3 mmol) in N-ethylbutylamine (5.0 g) was

heated at reflux for 1 h. The excess amine was removed under

vacuum, and the residue was partitioned between ethyl acetate

and water. The organic layer was washed with brine, dried

with anhydrous magnesium sulfate, and stripped down to a

Meth od C: Gen er a l P r oced u r es for th e Syn th esis of

15. 4-N-(2,4,6-Tr im eth ylp h en yl)-6-h yd r oxy-2-m eth yl-5-n i-

tr op yr im id in -4-a m in e (14, Ar ) 2,4,6-tr im eth ylp h en yl).

To 4,6-dichloro-2-methyl-5-nitropyrimidine (13) (12.58 g, 60.48

mmol) dissolved in DMSO (200 mL) was added 2,4,6-tri-

methylaniline (7.43 mL, 52.8 mmol) dropwise via syringe over

1 h. The reaction was stirred at room temperature for 18 h,

then poured onto water (1.6 L), and allowed to stir overnight.

The resultant precipitated pyrimidone was filtered and dried

to constant weight affording 12 (8.02 g, 51%) as a light yellow

1

solid: mp >225 °C; H NMR (CDCl₃) δ 12.23 (bs, 1H), 10.60

(s, 1H), 6.95 (s, 2H), 2.34 (s, 3H), 2.33 (s, 3H), 2.16 (s, 6H).

Anal. (C₁₄H₁₆N₄O₃) C, H, N.

In a similar manner the following compounds were prepared

in 40-80% yield: 14, Ar ) 2-bromo-4-(trifluoromethoxyl)-

phenyl; 14, Ar ) 2-(trifluoromethyl)-4-(dimethylamino)phenyl;

14, Ar ) 2-bromo-4,6-dimethoxyphenyl; 14, Ar ) 2-chloro-4,6-

dimethoxyphenyl; 14, Ar ) 2-bromo-4-(dimethylamino)-6-

methoxyphenyl.

4-N-(2,4,6-Tr im eth ylp h en yl)-6-ch lor o-2-m eth yl-5-n itr o-

pyr im idin -4-am in e (15, Ar ) 2,4,6-tr im eth ylph en yl). Prod-

uct 14 from part A (3.1 g, 11 mmol) was suspended in

phosphorus oxychloride (25 mL) and heated to just under

reflux for 1 h, to give a dark solution. The reaction was pipetted

slowly and cautiously onto 700 mL of ice/water, stirred 30 min

at room temperature, partitioned with methylene chloride (200

mL), and transferred to a separatory funnel. The aqueous layer

was separated and extracted with methylene chloride (3 × 50

mL). The combined organic extracts were dried over anhydrous

magnesium sulfate, filtered, and concentrated in vacuo to

constant weight to afford (3.18 g, 97%) 15 as a bright yellow

solid: mp 128-130 °C; ¹H NMR (CDCl₃) δ 8.79 (bs, 1H), 6.96

(s, 2H), 2.42 (s, 3H), 2.33 (s, 3H), 2.15 (s, 6H).

In a similar manner the following compounds were prepared

in 40-80% yield: 15, Ar ) 2-bromo-4-(trifluoromethoxy)-

phenyl; 15, Ar ) 2-(trifluoromethyl)-4-(dimethylamino)phenyl;

844 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

15, Ar ) 2-bromo-4,6-dimethoxyphenyl; 15, Ar ) 2-chloro-4,6-

dimethoxyphenyl; 15, Ar ) 2-bromo-4-(dimethylamino)-6-

methoxyphenyl.

residue that was partitioned between ethyl acetate and water.

The organic layer was then washed with brine, dried with

anhydrous magnesium sulfate, and stripped down to a residue

that was purified by a flash chromatography (silica gel, 1:50

MeOH/CH₂Cl₂) to afford 16 (R¹, R²) MeOCH₂CH₂; Ar )

2-bromo-4,6-dimethoxyphenyl) (4.03 g, 64%) as a yellow crys-

Gen er a l P r oced u r e for th e Con ver sion of 15 to 16. 4-N-

(2,4,6-Tr im eth ylph en yl)-6-(N-n -bu tyleth ylam in o)-2-m eth -

yl-5-n it r op yr im id in -4-a m in e (16, Ar ) 2,4,6-t r im et h yl-

p h en yl; R¹) Et, R²) n -Bu ). To the chloronitropyrimidine

15 (Ar ) 2,4,6-trimethylphenyl) (0.2 g, 0.7 mmol) in 5 mL of

acetone were added 87 mg (87 mmol) of Et₃N and 88 mg (88

mmol) of N-n-butylethylamine via syringe causing a precipitate

to form in the bright yellow-orange solution. After stirring at

room temperature for 1-2 h, the solvent was removed in vacuo

and the residue chromatographed (SiO₂, 10% EtOAc/hexanes)

to give 262 mg (98%) of bright yellow crystalline solid 16 (Ar

) 2,4,6-trimethylphenyl; R¹) Et, R²) n-Bu): mp 76-77.5

1

talline solid: mp 95-96 °C; H NMR (CDCl₃) d 2.15 (s, 3H,

CH₃), 3.4 (s, 6H, 2 O-CH₃'s), 3.6-3.75 (m, 8H, 4 CH₂'s), 3.8-

3.83 (2 s, 6H, 2 Ar-OCH₃’s), 6.45 (s, 1H, Ar-H), 6.8 (s, 1H,

Ar-H), 9.15 (s, 1H, NH); HRMS calcd for M + H (C₁₉H₂₇N₅O₆-

Br₁) 500.114470, found 500.114616.

In a similar manner 16 (R¹) Et, R²) Bu) was also prepared

in comparable yield. These intermediates were prepared with

the following aryl groups: 16, Ar ) 2-(methylthio)-4-isoprop-

ylphenyl; 16, Ar ) 2-(methylsulfonyl)-4-isopropylphenyl; 16,

Ar ) 2,4-dibromophenyl; 16, Ar ) 2-bromo-4-(methylthio)-

phenyl; 16, Ar ) 2-bromo-4-(methylsulfonyl)phenyl; 16, Ar )

2-bromo-4-acetylphenyl.

1

°C; H NMR (CDCl₃) δ 2.16 (s, 6H), 2.20 (s, 3H), 2.31 (s, 3H),

3.47 (m, 4H), 6.93 (s, 2H).

4-N-[2-Br om o-4-(d im et h yla m in o)p h en yl]-5-a m in o-6-

h yd r oxy-2-m eth ylp yr im id in -4-a m in e (19). To 14 (Ar )

2-bromo-4-(dimethylamino)phenyl) (3.8 g, 11.2 mmol) in 50 mL

of MeOH containing 220 mL of 1 N HCl solution was added

1.2 g of powdered Fe. After stirring the mixture at room

temperature overnight, the solution was then basified to pH

10 with 1 N NaOH solution and filtered through a cake of

Celite which was washed with several portions of EtOAc and

then MeOH. The layers were separated, and the aqueous

phase was extracted with additional EtOAc. The combined

extracts were washed with water, dried over MgSO₄, and

filtered. Solvent removal gave a residue that was chromato-

graphed (SiO₂, 10% MeOH/CHCl₃) to give 1.35 g (63%) of gray

solid 19: ¹H NMR (CD₃OD) δ 2.20 (s, 3H), 2.90 (s, 6H), 6.78

(dd, J ) 9.1, 2.9 Hz, 1H), 6.95 (d, J ) 2.9 Hz, 1H), 7.38 (d,

J +9.1 Hz, 1 Hz); MS M + 1 338.

3-[2-Br om o-4-(d im et h yla m in o)p h en yl]-7-h yd r oxy-5-

m eth yl-3H-1,2,3-tr iazolo[4,5-d]pyr im idin e (20). To 19 (1.35

g, 14 mmol) in 17 mL of HOAc and 11 mL of CH₂Cl₂was added

NaNO₂(4.3 mmol), and the reaction mixture was stirred at

room temperature for 4 h. Water was then added, the mixture

was extracted with several portions of CH₂Cl₂, and the

combined extracts were washed with water, dried over MgSO₄,

and filtered. Solvent removal gave a residue that was chro-

matographed (SiO₂, 10% MeOH/CHCl₃) to give 0.91 g (65%)

of brown solid 20: MS M + 1 349.

3-[2-Br om o-4-(dim eth ylam in o)ph en yl]-7-ch lor o-5-m eth -

yl-3H-1,2,3-tr ia zolo[4,5-d ]p yr im id in e (3, Ar ) 2-br om o-

4-(d im eth yla m in o)p h en yl). This intermediate was prepared

by the same procedure as is descibed for the conversion of 14

to 15. The crude product was purified by chromatography

(SiO₂, 10% MeOH/CHCl₃) to give the title compound (50%):

MS M + 1 367.

Meth od D: Gen er a l P r oced u r es for th e Syn th esis 17

fr om 13 via 18. 4-N-Bis(2-m et h oxyet h yl)-6-ch lor o-2-

m eth yl-5-n itr op yr im id in -4-a m in e (18, R¹, R²) MeOCH₂-

CH₂). To 13 (4.16 g, 20 mmol) in ethanol (50 mL) was added

triethylamine (2.02 g, 20.0 mmol) followed by dropwise addi-

tion of bis(2-methoxyethyl)amine (2.7 g, 20.0 mmol) in ethanol

(10.0 mL) over 30 min at room temperature. After stirring the

reaction mixture at room temperature for 1 additional h,

solvent removal in vacuo gave a residue that was partitioned

between ethyl acetate and water. The organic layer was

washed with brine, dried with anhydrous magnesium sulfate,

and stripped down to a residue that was purified by a flash

chromatography (silica gel, CH₂Cl₂) to afford 5.9 g (97%) of

an orange-yellow liquid (18, R¹, R²) MeOCH₂CH₂): ¹H NMR

(CDCl₃) δ 2.45 (s, 3H, C-2 CH₃), 3.35 (s, 6H, 2 -OCH₃’s), 3.55

(t, 4H, 2 -N-CH₂’s), 3.75 (t, 4H, 2 -O-CH₂’s).

4-N-Bis(2-m eth oxyeth yl)-6-N-(2-br om o-4,6-d im eth oxy-

p h en yl)-2-m eth ylp yr im id in e-4,5,6-tr ia m in e (17, R¹, R²

)

MeOCH₂CH₂; Ar ) 2-br om o-4,6-d im eth oxyp h en yl). Com-

pound 16 was reduced according to the method described in

the preparation of 24 from 23 (Scheme 3) to afford 17 as a

viscous oil (72%): ¹H NMR (CDCl₃) δ 2.35 (s, 3H, CH₃), 3.4 (s,

6H, 2 O-CH₃), 3.5-3.6 (2 t, 8H, 4 CH₂), 3.75-3.80 (2 s, 6H, 2

Ar-OCH₃), 6.25 (bs, 1H, NH), 6.45 (s, 1H, Ar-H), 6.75 (s, 1H,

Ar-H).

7-N-Bis(2-m et h oxyet h yl)-3-(2-b r om o-4,6-d im et h oxy-

phenyl)-5-methyl-3H-1,2,3-triazolo[4,5-d]pyrimidin-7-amine

(4n n ). Compound 17 was cyclized according to the method

used in the conversion of 2 to 3 to afford 4n n as a white

1

crystalline solid (46%): mp 124-126 °C; H NMR (CDCl₃) δ

2.55 (s, 3H, CH₃), 3.4 (s, 6H, 2 O-CH₃’s), 3.65 (s, 3H, Ar-

OCH₃), 3.75-3.85 (2 t, 4H, 2 CH₂'s), 3.9 (s, 3H, Ar-OCH₃),

4.1 (t, 2H, CH₂), 4.55 (t, 2H, CH₂), 6.55 (s, 1H, Ar-H), 6.85 (s,

1H, Ar-H); Mass M⁺) 481.1, M + 2 ) 483.1.

Syn t h esis of 4h h ,ii. ^DL-Nor va lin e Dim et h yla m id e. A

solution of N-CBz-^DL-norvaline (10.0 g, 39.8 mmol) in THF (100

mL) was treated with 1-hydroxybenzotriazole hydrate (6.45

g, 47.7 mmol) in portions over 30 min. Dimethylamine

hydrochloride (4.22 g, 51.7 mmol), triethylamine (8.00 mL, 57.4

mmol), and dicyclohexylcarbodiimide (9.84 g, 47.7 mmol) were

then added in sequence. The resulting mixture was allowed

to stir for 12 h at room temperature, then diluted with ethyl

acetate (100 mL), and filtered through Celite. The filtrate was

washed with water (150 mL) and brine (100 mL), and the

aqueous layers were back-extracted in sequence with ethyl

acetate (100 mL). The organic extracts were combined, dried

over MgSO₄, filtered, and evaporated. The residual oil was

separated by flash chromatography (silica gel, 30:70 ethyl

acetate/hexane) to afford N-CBz-^DL-norvaline dimethylamide

(7.52 g, 27.0 mmol, 68%): TLC R_f0.10 (30:70 ethyl acetate/

1

hexane); H NMR (CDCl₃) δ 7.36-7.27 (5H, m), 5.68 (1H, br

d, J ) 8.4 Hz), 5.09 (2H, s), 4.67 (1H, dt, J ) 8.2, 5.1 Hz), 3.08

(3H, s), 2.96 (3H, s), 1.71-1.30 (4H, m), 0.93 (3H, t, J ) 7.3

Hz); MS (ESI) m/e 281 (1), 280 (15), 279 (100).

A solution of N-CBz-^DL-norvaline dimethylamide (6.82 g,

24.5 mmol) in methanol (50 mL) was treated with 5% Pd on

carbon (1 g) and subjected to hydrogen atmosphere (50 psi) in

a Paar apparatus for 14 h. The mixture was filtered through

Celite and evaporated to afford the title product as an oil (2.72

g, 18.9 mmol, 77%): TLC R_f0.06 (30:70 ethyl acetate/hexane);

¹H NMR (CDCl₃) δ 3.77-3.72 (1H, m), 3.04 (3H, s), 2.98 (3H,

s), 2.53 (2H, br s), 1.62-1.32 (4H, m), 0.94 (3H, t, J ) 7.0 Hz);

MS (H₂O-GC/MS) m/e 145 (100).

4-N-[2-Br om o-4-(1-m et h ylet h yl)p h en yl]-6-[1-(N,N-d i-

methylcarboxamido)butylamino]-2-methyl-5-nitropyrimidin-

4-a m in e (16, Ar ) 2-br om o-4-(1-m eth yleth yl)p h en yl; R¹

In a similar manner 18 (R¹) Et, R²) Bu) was also prepared

in 80% yield. 4-N-Bis(2-m eth oxyeth yl)-6-N-(2-br om o-4,6-

d im et h oxyp h en yl)-2-m et h yl-5-n it r op yr im id in e-4,6-d i-

a m in e (16, R¹, R²) MeOCH₂CH₂; Ar ) 2-br om o-4,6-

d im eth oxyp h en yl). To 18 (R¹, R²) MeOCH₂CH₂) (3.85 g,

12.6 mmol) in anhydrous DMF (30.0 mL) was added 2-bromo-

4,6-dimethoxyaniline (3.07 g; 13.3 mmol), and the mixture was

heated at 60 °C for 6 days. Solvent removal in vacuo gave a

) H, R²

) DL-n or va lin e d im eth yla m id e). The general

methods used to convert 13 to 18 to 16 were employed (65%

over both steps): mp 139-141 °C; TLC R_f0.17 (30:70 ethyl

acetate/hexane); ¹H NMR (CDCl₃) δ 11.37 (1H, br s), 9.67 (1H,

br d, J ) 6.6 Hz), 8.20 (1H, d, J ) 8.4 Hz), 7.47 (1H, d, J ) 1.8

Hz), 7.20 (1H, dd, J ) 8.4, 1.8 Hz), 5.37 (1H, q, J ) 5.5 Hz),

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 845

3.21 (3H, s), 3.01 (3H, s), 2.89 (1H, heptet, J ) 7.0 Hz), 2.36

(3H, s), 1.90-1.79 (2H, m), 1.50-1.39 (2H, m), 1.26 (6H, d, J

) 7.0 Hz), 0.97 (3H, t, J ) 7.3 Hz); MS (ESI) m/e 496 (23), 495

(100), 494 (22), 493 (99).

(CDCl₃) δ 8.61 (1H, d, J ) 8.4 Hz), 8.20 (1H, br s), 7.48 (1H,

d, J ) 1.8 Hz), 7.26 (1H, dd, J ) 8.4, 1.8 Hz), 5.08-4.98 (1H,

m), 3.27 (1H, dd, J ) 12.6, 9.7 Hz), 2.91 (1H, heptet, J ) 7.0

Hz), 2.68 (3H, s), 2.67 (1H, dd, J ) 12.6 Hz), 2.22 (6H, s), 2.21-

2.11 (1H, m), 1.99-1.89 (1H, m), 1.29-1.19 (1H, m), 1.27 (6H,

d, J ) 7.0 Hz), 1.16-1.05 (1H, m), 0.88 (3H, t, J ) 7.1 Hz);

MS (NH₃-CI) m/e 464 (3), 463 (25), 462 (100), 461 (29), 460

(98).

Meth od E (Ar ) collid yl): Alter n a te Meth od for th e

Syn th esis of 4-Am in o-6-ch lor o-2-m eth yl-5-n itr op yr im -

id in es 15 (Ar ) collid yl). 3-Am in o-2,4,6-tr im eth ylp yr i-

d in e. 3-Nitro-2,4,6-trimethylpyridine (14.89 g, 89.70 mmol) in

methanol (250 mL) containing 10% palladium/carbon (1.5 g)

was hydrogenated at 55 psi for 2 h. The reaction mixture was

filtered through wet Celite, and the Celite filter rinsed with

methanol (5 × 30 mL). The filtrate was concentrated in vacuo

to dryness and the residue purified by chromatography (silica

gel; methylene chloride/methanol, 95/5) to 3-amino-2,4,6-

trimethylpyridine (12.42 g, 100%) as a viscous oil.

3-[2-Br om o-4-(1-m e t h yle t h yl)p h e n yl]-7-[1-(N ,N -d i-

methylcarboxamido)butylamino]-5-methyl-3H-1,2,3-triazolo-

[4,5-d ]p yr im id in e (4h h ). The general method used to convert

16 to 17 was employed to prepare 5-amino-4-[2-bromo-4-(1-

methylethyl)phenyl]-6-[1-(N,N-dimethylcarboxamido)butylami-

no]-2-methylpyrimidine (96%). The general method used to

convert 2 to 3 was used to prepare the title product (67%):

mp 163-164 °C (ether); TLC R_f0.16 (50:50 ethyl acetate/

hexane); ¹H NMR (CDCl₃) δ 7.64 (0.314H, d, J ) 1.8 Hz), 7.63

(0.686H, d, J ) 1.8 Hz), 7.41-7.30 (2H, m), 6.97 (0.686H, br

d, J ) 8.0 Hz), 6.66 (0.314H, br d, J ) 8 Hz), 6.20-6.12

(0.314H, m), 5.54-5.44 (0.686H, m), 3.27 (0.942H, s), 3.25

(2.058H, s), 3.02 (3H, s), 2.97 (1H, heptet, J ) 7 Hz), 2.57

(2.058H, s), 2.55 (0.942H, s), 1.99-1.78 (2H, m), 1.58-1.41 (2H,

m), 1.31 (1.884H, d, J ) 7 Hz), 1.30 (4.116H, d, J ) 7 Hz),

0.99 (2.058H, t, J ) 7 Hz), 0.97 (0.942H, t, J ) 7 Hz); MS

(NH₃-CI) m/e 478 (3), 477 (25), 476 (100), 475 (31), 474 (100).

4-N-(2,4,6-Tr im et h ylp yr id yl)-6-ch lor o-2-m et h yl-5-n i-

tr op yr im id in -4-a m in e (15, Ar ) 2,4,6-tr im eth ylp yr id yl).

To 4,6-dichloro-2-methyl-5-nitropyrimidine (13) (10.10 g, 48.60

mmol) in anhydrous tetrahydrofuran (200 mL) were added

triethylamine (6.8 mL, 48.60 mmol) and 3-amino-2,4,6-trim-

ethylpyridine (3.30 g, 24.3 mmol), and the reaction was stirred

for 72 h at room temperature. The solution was diluted with

water (1 L) and extracted with ethyl acetate (4 × 200 mL).

The combined organic extracts were dried over anhydrous

magnesium sulfate, filtered, and concentrated to dryness in

vacuo. Chromatography of the crude product (silica gel; ethyl

acetate/hexanes, 1/1) gave 15 (Ar ) collidyl, 4.8 g, 64%) as a

4-N-[2-Br om o-4-(1-m et h ylet h yl)p h en yl]-6-[1-(N,N-d i-

m eth ylam in o)-2-pen tylam in o]-2-m eth yl-5-n itr opyr im idin -

4-a m in e (16, Ar ) 2-br om o-4-(1-m eth yleth yl)p h en yl; R¹

) H, R²) 1-(N,N-d im eth yla m in o)-2-p en tyla m in o). A

solution of 4-[2-bromo-4-(1-methylethyl)phenyl]-6-[1-(N,N-dim-

ethylcarboxamido)butylamino]-2-methyl-5-nitropyrimidin-4-

amine (650 mg, 1.32 mmol) in THF (5 mL) was treated with a

solution of borane-THF complex (3.00 mL, 1.0 M, 3.0 mmol)

at ambient temperature. After being allowed to stir for 14 h,

the reaction mixture was delivered slowly to an equal volume

of 1 N aqueous NaHCO₃solution with stirring. The resulting

mixture was extracted with dichloromethane (2 × 50 mL), and

the extracts were combined, dried over Na₂SO₄, filtered, and

evaporated. The residue was separated by flash chromatog-

raphy (silica gel, 20:80 ethyl acetate/hexane) to afford first the

title product (390 mg, 0.813 mmol, 62%) and then recovered

unreacted starting material (180 mg, 28%). For the product

1

faint yellow solid: mp 134-136 °C; H NMR (CDCl₃) δ 8.78

(bs, 1H), 6.97 (s, 1H), 2.43 (s, 3H), 2.39 (s, 3H), 2.16 (s, 3H),

2.05 (s, 3H).

4-N-(2,4,6-Tr im eth ylp yr id yl)-6-ch lor o-2-m eth ylp yr im i-

d in e-4,5-d ia m in e (2, Ar ) collid yl). To 15 (Ar ) collidyl)

(4.8 g, 15.6 mmol) in acetic acid (6 mL) was added powdered

iron (4.36 g, 78.0 mmol), and the heterogeneous reaction was

stirred for 5 min at 0 °C, then refluxed for 3 h, cooled, and

filtered through Celite. The Celite pad was washed with ethyl

acetate (500 mL), and the dark filtrate was concentrated in

vacuo to near dryness. The residue was redissolved in ethyl

acetate/water, and the layers were separated. The aqueous

layer was extracted several times with ethyl acetate, and the

combined extracts were dried over anhydrous magnesium

sulfate, filtered, and concentrated in vacuo. Chromatography

of the crude product (silica gel; methylene chloride/methanol,

95/5) gave 2 (Ar ) collidyl) (3.1 g, 72%): ¹H NMR (CDCl₃) δ

6.94 (s, 1H), 6.26 (bs, 1H), 3.36 (bs, 2H), 2.52 (s, 3H), 2.40 (s,

3H), 2.34 (s, 3H), 2.16 (s, 3H).

2,4-Dich lor o-6-m eth yl-3-n itr op yr id in e (21). 4-Hydroxy-

6-methyl-3-nitropyridone (18.67 g, 0.11 mol) and diethylaniline

(19 mL, 0.12 mol) were heated at reflux in POCl₃(85 mL) for

3 h. After cooling the solution was poured into ice/water (800

mL), and after stirring for 2.5 h the mixture was extracted

with EtOAc (3 × 400 mL). The combined organic extracts were

washed with NaHCO₃(200 mL) and brine (200 mL), dried

(MgSO₄), and stripped in vacuo. The residue was dissolved in

EtOAc (100 mL) and passed through a glass funnel packed

with 1 in. of silica gel and 1 in. of Celite. The filtrate was

stripped in vacuo to give 21 which was used without further

purification: ¹H NMR (CDCl₃) δ 7.30 (s, 1H), 2.61 (s, 3H).

2-Ch lor o-4-(N-b u t yl-N-et h yla m in o)-6-m et h yl-3-n it r o-

p yr id in e (22, R¹) Et, R²) Bu ). 2,4-Dichloro-6-methyl-3-

nitropyridine (21) (1 g, 4.83 mmol), N-ethylbutylamine (0.75

mL, 5.55 mmol), and N,N-diisopropylethylamine (1 mL, 6

mmol) were stirred at 25 °C for 24 h and at reflux for 5 h. The

mixture was stripped in vacuo, and the residue was partitioned

between EtOAc (75 mL) and water (50 mL). The organic layer

was washed with water (30 mL) and brine (30 mL), dried

(MgSO₄), and stripped in vacuo. The residue was chromato-

graphed (silica gel; 10% EtOAc/hexanes) to give regiomer 22′

(270 mg, 21%): ¹H NMR (CDCl₃) δ 6.56 (s, 1H), 3.30-3.42 (m,

4H), 2.38 (s, 3H), 1.49-1.59 (m, 2H), 1.23-1.35 (m, 2H),1.15

1

(an oil): TLC R_f0.47 (30:70 ethyl acetate/hexane); H NMR

(CDCl₃) δ 11.39 (1H, br s), 9.27 (1H, br d, J ) 8.8 Hz), 8.24

(1H, d, J ) 8.4 Hz), 7.49 (1H, d, J ) 1.8 Hz), 7.22 (1H, dd, J

) 8.4, 1.8 Hz), 5.20-5.09 (1H, m), 3.10 (1H, dd, J ) 13.9, 2.6

Hz), 3.00 (1H, dd, J ) 13.9, 7.7 Hz), 2.90 (1H, heptet, J ) 7.0

Hz), 2.69 (3H, s), 2.65 (3H, s), 2.40 (3H, s), 1.79-1.50 (2H, m),

1.46-1.34 (2H, m), 1.26 (6H, d, J ) 7.0 Hz), 0.96 (3H, t, J )

7.3 Hz); MS (NH₃-CI) m/e 483 (3), 482 (25), 481 (100), 480 (29),

479 (99).

4-N-[2-Br om o-4-(1-m et h ylet h yl)p h en yl]-6-[1-(N,N-d i-

m eth yla m in o)-2-p en tyla m in o]-2-m eth ylp yr im id in e-4,5-

d ia m in e (17, Ar ) 2-br om o-4-(1-m eth yleth yl)p h en yl; R¹

) H , R ²) 1-(N,N-d im et h yla m in o)-2-p en t yla m in o). The

general procedure used to prepare 17 from 16 was employed

(100%): ¹H NMR (CDCl₃) δ 8.27 (1H, d, J ) 8.4 Hz), 7.49 (1H,

br s), 7.38 (1H, d, J ) 1.8 Hz), 7.16 (1H, dd, J ) 8.4, 1.8 Hz),

4.99 (1H, d, J ) 8.8 Hz), 4.73 (1H, br), 3.02 (2H, d, J ) 4.4

Hz), 2.84 (1H, heptet, J ) 6.6 Hz), 2.75 (3H, s), 2.62 (3H, s),

2.41 (3H, s), 2.15 (2H, s), 1.65-1.55 (2H, m), 1.42-1.32 (2H,

m), 1.23 (6H, d, J ) 6.6 Hz), 0.93 (3H, t, J ) 7.1 Hz); MS (NH₃-

CI) m/e 453 (3), 452 (25), 451 (100), 450 (27), 449 (99).

3-[2-Br om o-4-(1-m e t h yle t h yl)p h e n yl]-7-[1-(N ,N -d i-

methylamino)-2-pentylamino]-5-methyl-3H-1,2,3-triazolo[4,5-d]-

p yr im id in e (4ii). The general method used to convert 2 to 3

was employed and upon workup with chromatography afforded

first the title product (73%), followed by the isomeric product

7-[2-bromo-4-(1-methylethyl)phenyl]-3-[1-(N,N-dimethylamino)-

2-pentylamino]-5-methyl-3H-1,2,3-triazolo[4,5-d]pyrimidine

1

(13%). For 4ii: mp 179-180 °C dec; H NMR (CDCl₃) δ 7.66

(1H, d, J ) 1.5 Hz), 7.39 (1H, d, J ) 8.4 Hz), 7.36 (1H, dd, J

) 8.4, 1.5 Hz), 6.20 (1H, d, J ) 8 Hz), 5.11 (1H, br), 3.13 (2H,

d, J ) 5.5 Hz), 3.00 (1H, heptet, J ) 7.0 Hz), 2.76 (3H, s), 2.68

(3H, s), 2.60 (3H, s), 1.80-1.65 (2H, m), 1.52-1.42 (2H, m),

1.31 (6H, d, J ) 7.0 Hz), 0.97 (3H, t, J ) 7.3 Hz); MS (NH₃-

CI) m/e 464 (3), 463 (25), 462 (99), 461 (28), 460 (100). For the

isomer: TLC R_f0.05 (30:70 ethyl acetate/hexane); ¹H NMR

846 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5

Chorvat et al.

(t, 3H, J ) 7.3 Hz), 0.92 (t, 3H, J ) 7.0 Hz). And regiomer 22

(840 mg, 64%): ¹H NMR (CDCl₃) δ 6.52 (s, 1H), 3.28 (q, 2H, J

) 7.0), 3.18 (dd, 2H, J ₁) 7.3 Hz, J ₂) 8.1 Hz), 2.44 (s, 3H),

1.49-1.60 (m, 2H), 1.24-1.37 (m, 2H), 1.17 (t, 3H, J ) 7.3

Hz), 0.94 (t 3H, J ) 7.3 Hz). The major regioisomer was

characterized as the 4-adduct 22 by NOE NMR experiments.

3-Am in o-2-N-[2-br om o-4-(1-m eth yleth yl)ph en yl)am in o]-

4-(N,N-bis(m eth oxyeth yl)a m in o)-6-m eth ylp yr id in e (24b,

R

Synthesized by reducing the corresponding 3-nitro analogue

21 as described earlier and purified by chromatography (silica

gel; 20%EtOAc/hexanes) to give 24b (96%).

¹, R²) m eth oxyeth yl; Ar ) 4-(1-m eth yleth yl)p h en yl).

2-[N-(2-Br om o-4-(1-m et h ylet h yl)p h en yl)a m in o]-4-(N-

bu tyl-N-eth yla m in o)-6-m eth yl-3-n itr op yr id in e (23a ). 2-

Chloro-4-(N-butyl-N-ethylamino)-6-methyl-3-nitropyridine (22)

(R¹) Et, R²) Bu) (1.088 g, 4 mmol) and 2-bromo-4-

isopropylaniline (1.712 g, 8 mmol) were heated at 140 °C for

4.5 h. The cooled mixture was then partitioned between EtOAc

(11 mL) and 0.5 N NaOH solution (30 mL). The EtOAc solution

was washed with brine (30 mL), dried (MgSO₄), and stripped

in vacuo, and the residue was chromatographed (silica gel; 5%

EtOAc/hexanes) to give 23a (920 mg, 51%): ¹H NMR (CDCl₃)

δ 9.54 (s, 1 H), 8.33 (d, 1H, J ) 8.8 Hz), 7.41 (d, 1H, J ) 1.8

Hz), 7.14 (dd 1H, J ₁) 8.8 Hz, J ₂) 1.8 Hz), 6.24 (s, 1H), 3.18-

3.32 (m, 4H), 2.80-2.90 (m, 1H), 2.36 (s, 3H), 1.54-1.65 (m,

2H), 1.18-1.40 (m, 11H), 0.93 (t, 3H, J ) 7.0 Hz).

4-Isop r op yl-2-(th iom eth yl)a n ilin e In a dried flask, under

N₂a mixture of 2-iodo-4-isopropylaniline (6.0 g, 23.0 mmol),

sodium thiomethoxide (1.9 g, 26.4 mmol), and copper powder

(0.70 g, 11.0 mmol) in anhydrous DMF (50 mL) was refluxed

for l h. The cooled reaction mixture was filtered through Celite

and the filtrate partitioned between H₂O and EtOAc. The

aqueous layer was extracted with EtOAc (2×), and the

combined extracts were washed with H₂O (3×) followed by

brine and dried over MgSO₄. The filtered solution was con-

centrated in vacuo to give crude product (5.38 g.) that was

chromatographed (silica gel; 5% EtOAc/hexane) to give the

aniline as a brown liquid (2.10 g, 50%): ¹H NMR (CDCl₃) δ

7.22 (d, 1H, J ) 2.1 Hz), 6.97 (dd, 1H, J ₁) 8 Hz, J ₂) 2.2 Hz),

6.68 (d, 1H, J ) 8 Hz), 4.15 (br s, 2H), 2.75-2.82 (m, 1H),

2.36 (s, 3H), 1.20 (d, 6H, J ) 7 Hz).

2-Ch lor o-4-(N,N-bis(m eth oxyeth yl)a m in o)-6-m eth yl-3-

n it r op yr id in e (22, R¹, R²) b is(m et h oxyet h yl)). 2,4-

Dichloro-6-methyl-3-nitropyridine (4 g, 19.32 mmol) was treated

with bis(methoxyethyl)amine (3.5 mL, 23.66 mmol) in the

presence of N,N-diisopropylethylamine in ethanol (30 mL) at

25 °C for 60 h and at reflux for 7 h. The product was purified

by chromatography (silica gel; 20%EtOAc/hexanes followed by

40%EtOAc/hexanes) to give 22 (R¹, R²) methoxyethyl) (4 g,

68%).

2-[N-(2-Br om o-4-(1-m eth yleth yl)p h en yl)a m in o]-4-(N,N-

bis(m eth oxyeth yl)a m in o)-6-m eth yl-3-n itr op yr id in e (23b,

R¹, R²) m eth oxyeth yl, Ar ) 4-(1-m eth yleth yl)p h en yl).

Heating 2-chloro-4-(N,N-bis(methoxyethyl)amino)-6-methyl-3-

nitropyridine (1.87 g, 6.15 mmol) with 2-bromo-4-isopropyl-

aniline (2.63 g, 12.3 mmol) at 140 °C for 6 h afforded a residue

that was purified by chromatography (silica gel; 25%EtOAc/

hexanes) to give 23b (1.3 g, 44%).

3-Am in o-2-[N-(2-(t h iom et h yl)-4-(1-m et h ylet h yl)p h en -

yl)a m in o]-4-(N-b u t yl-N-et h yla m in o)-6-m et h ylp yr id in e

(24c). Purified by chromatography (silica gel; 10% EtOAc/CH₂-

Cl₂) to give 22c as an oil (62%).

3-Am in o-2-[N-(2-(t h iom et h yl)-4-(1-m et h ylet h yl)p h en -

yl)amino]-4-[N,N-bis(methoxyethyl)amino]-6-methylpyridine

(22e). Purified by chromatography (silica gel; 30% EtOAc/

hexane) to give 22e as an oil (68%).

6-(N-Eth ylbu tyla m in o)-9-[2-br om o-4-(1-m eth yleth yl)-

p h en yl]-2-m et h yl-9H -t r ia zolo[4,5-d ]p yr id in e

(25a )

(Meth od G). To a two-phase mixture containing 3-amino-2-

[N-(2-bromo-4-(1-methylethyl)phenyl)amino]-4-(N-butyl-N-eth-

yl amino)-6-methylpyridine (0.5 g, 1.19 mmol) dissolved in

CH₂Cl₂and 50% aqueous AcOH (4 mL) was added sodium

nitrite (87.5 mg, 1.27 mmol) in small portions. The reaction

mixture was stirred at 25 °C for 2 h and partitioned between

EtOAc (80 mL) and water (30 mL). The organic extract was

washed with brine (30 mL), dried (MgSO₄), and stripped in

vacuo. The residue was chromatographed on silica gel (10%

EtOAc/hexanes eluent) to give 25a (360 mg, 70%): ¹H NMR

(CDCl₃) δ 7.63 (d, 1H, J ) 1.5 Hz), 7.44 (d, 1H, J ) 8.1 Hz),

7.33 (dd, 1H, J ₁) 8.1 Hz, J ₂) 1.5 Hz), 6.10 (s, 1H), 3.8-4.0

(br m, 4 H), 2.90-3.05 (m, 1H), 2.49 (s, 3H), 1.70-1.82 (m,

2H), 1.40-1.55 (m, 2H), 1.35 (t, 3H, J ) 6.9 Hz), 1.30 (d, 6H,

J ) 7.0 Hz), 1.01 (t, 3H, J ) 7.0 Hz). The free base was

converted to the hydrochloride salt using anhydrous HCl in

ether.

6-(N-Eth ylbu tyla m in o)-9-[2-br om o-4-(1-m eth yleth yl)-

p h en yl]-2-m eth yl-9H-tr ia zolo[4,5-d ]p yr id in e (25a ′). The

title compound was prepared from pyridine regioisomer 22′

(Scheme 3, method G) via the methodology described for the

preparation of 25a to give 25a ′ in comparable yield as an

opaque oil: ¹H NMR (CDCl₃) δ 7.65 (s, 1H), 7.35 (s, 2H), 6.19

(s, 1H), 4.05 (m, 4H), 3.01 (quintet, 1H, J ) 7 Hz), 2.40 (s,

3H), 1.75 (m, 2H), 1.45 (m, 2H), 1.33 (t, 3H, J ) 7.3 Hz), 1.32

(d, 6H, J ) 7 Hz), 0.99 (t, 3H, J ) 7.3 Hz).

6-(N-Eth ylbu tyla m in o)-9-[2-(th iom eth yl)-4-(1-m eth yl-

eth yl)p h en yl]-2-m eth yl-9H-tr ia zolo[4,5-d ]p yr id in e (25c).

Purified by chromatography (silica gel; 20% EtOAc/hexane) to

give 25c as an oil (81% yield).

6-(N,N-Bis(m eth oxyeth yl)a m in o)-9-[2-(th iom eth yl)-4-

(1-m eth yleth yl)p h en yl]-2-m eth yl-9H-tr ia zolo[4,5-d ]p yr i-

d in e (25e). Purified by chromatography (silica gel; 30%

EtOAc/hexane) to give 25e as an oil (68% yield).

6-(N-Eth ylbu tyla m in o)-9-[2-(m eth ylsu lfon yl)-4-(1-m e-

t h ylet h yl)p h en yl]-2-m et h yl-9H -t r ia zolo[4,5-d ]p yr id in e

(25d ). To 25c (250 mg, 0.63 mmol) in methanol (5 mL) and

H₂O (2.5 mL) at room temperature was added sodium perio-

date (200 mg, 0.95 mmol), and the reaction mixture was stirred

overnight. After H₂O and EtOAc were added, the aqueous

phase extracted with additional EtOAc (2×). The combined

extracts were washed with H₂O followed by brine, dried over

MgSO₄, filtered, and concentrated in vacuo to give a foam (0.24

g) which was taken up into CH₂Cl₂(5 mL) and H₂O (5 mL).

Benzyltriethylammonium chloride (132 mg, 0.58 mmol) fol-

lowed by KMnO₄(275 mg, 1.74 mmol) were added, and the

reaction mixture was stirred at room temperature overnight.

After partitioning between H₂O and EtOAc, the layers were

separated and the aqueous layer was extracted with EtOAc

(2 × 10 mL). The combined extracts were washed with H₂O

followed by brine, dried over MgSO₄, filtered, and concentrated

in vacuo to give an oil (0.26 g) that was chromatographed

(silica gel; gradient from 20% to 30% EtOAc/hexane) to give

2-[N-(2-(Th iom eth yl)-4-(1-m eth yleth yl)p h en yl)a m in o]-

4-(N-bu tyl-N-eth yla m in o)-6-m eth yl-3-n itr opyr idin e (23c).

Using the method above, the title compound was produced as

an oil in 65% yield after purification by chromatography (silica

gel; 95% hexane/3%EtOAc/CH₂Cl₂).

3-Am in o-2-[N-(2-br om o-4-(1-m eth yleth yl)ph en yl)am in o]-

4-(N-b u t yl-N-et h yla m in o)-6-m et h ylp yr id in e (24a ). To

2-[N-(2-bromo-4-(1-methylethyl)phenyl)amino]-4-(N-butyl-N-ethyl-

amino)-6-methyl-3-nitropyridine (1.17 g, 2.6 mmol) in dioxane

(60 mL) and water (60 mL) containing concentrated NH₄OH

(2 mL) was added Na₂S₂O₄(3.63 g, 20.8 mmol), and the

mixture was stirred at 25 °C for 2 h. The reaction was

extracted with EtOAc (160 mL), and the organic extract was

washed with water (3 × 50 mL) and brine (50 mL), dried

(MgSO₄), and stripped in vacuo. The residue was chromato-

graphed (silica gel; 5% EtOAc/hexanes) to give 24a (960 mg,

88%): ¹H NMR (CDCl₃) δ 7.59 (d, 1H, J ) 8.4 Hz), 7.36 (d,

1H, J ) 2.2 Hz), 7.06 (dd, 1H, J ₁) 8 Hz, J ₂) 2.2 Hz), 6.54 (s,

1H), 6.51 (brs, 1H), 3.59 (brs, 2H), 2.94-3.06 (m, 4H), 2.77-

2.81 (m, 1H), 2.39 (s, 3H), 1.35-1.50 (m, 2H), 1.25-1.34 (m,

2H), 1.21 (d, 6H, J ) 7.0 Hz), 1.03 (t, 3H, J ) 7.3 Hz), 0.90 (t,

3H, J ) 7.3 Hz).

1

25d (0.17 g, 68%): mp 151-153 °C; H NMR (CDCl₃) δ 7.39

(d, 1H, J ) 8 Hz), 7.32 (d, 1H, J ) 1.8 Hz), 7.19 (dd, 1H, J ₁)

8 Hz, J ₂) 1.8 Hz), 6.09 (s, 1H), 3.9-4.0 (m, 2H), 3.8-3.9 (m,

2H), 2.95-3.04 (m, 1H), 2.49 (s, 3H), 2.38 (s, 3H), 1.7-1.8 (m,

Synthesis and CRF Receptor Binding Affinity

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 847

2H), 1.4-1.53 (m, 2H), 1.34 (t, 3H, J ) 7.0 Hz), 1.31 (d, 6H, J

) 6.6 Hz), 1.00 (t, 3H, J ) 7.3).

was resuspended to a protein concentration of 360 µg/mL to

be used in the assay.

Binding assays were performed in 96-well plates, each well

having a 300-µL capacity. To each well was added 50 µL of

test drug dilutions (final concentration of drugs ranged from

10^-10to 10^-5M), 100 µL of [¹²⁵I]o-CRF (final concentration 150

pM), and 150 µL of the cell homogenate described above. Plates

were then allowed to incubate at room temperature for 2 h

before filtering the incubate over GF/F filters (presoaked with

0.3% poly(ethylenimine)) using an appropriate cell harvester.

Filters were rinsed two times with ice-cold assay buffer before

removing individual filters and assessing them for radioactiv-

ity on a gamma counter.

Curves of the inhibition of [¹²⁵I]o-CRF binding to cell

membranes at various dilutions of test drug were analyzed

by the iterative curve-fitting program LIGAND, which provides

K_ivalues for inhibition which are then used to assess biological

activity.

2. P h a r m a cok in etic Stu d ies. Pharmacokinetic param-

eters were determined in rats after an oral dose of 1 mg/kg

prepared in a 0.25% methylcellulose suspension. At 30 min

and 1, 2, 4, 8, and 16 h after dosing, rats were sacrificed and

blood samples were collected into tubes containing EDTA. Dogs

(n ) 4) were given 1 mg/kg of compound iv in a cosolvent

vehicle or 1 mg/kg po in 0.25% methylcellulose suspension

(with 0.1% Tween 80 in some cases). Blood samples were

collected from jugular veins at predose, 5, 15, and 30 min, and

1, 2, 4, 8, 10, 12, 16, 24, 32, 48, 56, and 72 h after dosing.

Steady-state brain-to-plasma ratios in rats were determined

approximately 76 h after infusion. Blood and brain tissue were

collected at the end of the infusion. Plasma was separated by

centrifugation and stored frozen until analysis. Compounds

were extracted from plasma by simple liquid-liquid extraction.

LC/MS/MS analysis was performed on a Sciex (Thornhill,

Ontario) model APIIII triple quadrapole mass spectrometer

interfaced with a turbo ion spray ionization source. The liquid

chromatography consisted of a Perkin-Elmer series 200 solvent

delivery system (Norwalk, CT), a Perkin-Elmer ISS 200

autoinjector, and a Waters Symmetry octyl minibore column

(2.1 × 50 mm).

6-(N,N-Bis(m eth oxyeth yl)am in o)-9-[2-(m eth ylsu lfon yl)-

4-(1-m eth yleth yl)p h en yl]-2-m eth yl-9H-tr ia zolo[4,5-d ]p y-

r id in e (25f). Prepared using the same sequence as described

for 25d and purified by chromatography (silica gel; 50% EtOAc/

hexane) to give 25f as a white solid (68%).

6-(N-Eth ylbu tyla m in o)-9-[2-br om o-4-(1-m eth yleth yl)-

p h en yl]-2,8-d im et h yl-9H -im id a zo[4,5-d ]p yr id in e (26a )

(Meth od H). To a solution of 3-amino-2-[N-(2-bromo-4-(1-

methylethyl)phenyl)amino]-4-(N-butyl-N-ethylamino)-6-meth-

ylpyridine (460 mg, 1.1 mmol) in triethyl orthoacetate (5 mL)

was added 0.5 mL of concentrated HCl, and the reaction was

stirred at 25 °C for 2 h. The mixture was partitioned between

EtOAc (75 mL) and NaHCO₃(50 mL), and the organic extract

was washed with brine (30 mL), dried (MgSO₄), and stripped

in vacuo. The residue was chromatographed (silica gel; 20%

EtOAc/hexanes) to give 26a (330 mg, 68%): ¹H NMR (CDCl₃)

δ 7.59 (s, 1H), 7.32 (dd, 1H, J ₁) 8.4 Hz, J ₂) 1.8 Hz), 7.28 (d,

1H, J ) 8.4 Hz), 6.13 (s, 1H), 3.6-4.0 (m, 4H), 2.9-3.03 (m,

1H), 2.41 (s, 3H), 2.29 (s, 3H), 1.61-1.75 (m, 2H), 1.35-1.5

(m, 2H). The free base was converted to the hydrochloride salt

using anhydrous HCl in ether.

6-(N,N-Dim eth oxyeth yla m in o)-9-[2-br om o-4-(1-m eth yl-

eth yl)p h en yl]-2-m eth yl-9H-tr ia zolo[4,5-d ]p yr id in e (25b).

Synthesized by cyclization of the 3-aminopyridine 24 with

NaNO₂in AcOH. The product was purified by chromatography

(silica gel; 2% MeOH/CH₂Cl₂), followed by HCl salt formation

and crystallization fron EtOAc/ether/hexanes to give 25b

(45%): ¹H NMR (CDCl₃) δ 7.68 (s, 1H), 7.55 (d, 1H J ) 8.0

Hz), 7.45 (d, 1H J ) 8 Hz), 6.42 (s, 1H), 4.6-4.7 (m, 1H).

6-(N,N-Bis(m eth oxyeth yl)a m in o)-9-[2-br om o-4-(1-m e-

t h ylet h yl)p h en yl]-2,8-d im et h yl-9H -im id a zo[4,5-d ]p yr i-

d in e (26b ). Synthesized by cyclization of 3-amino-2-[N-(2-

bromo-4-(1-methylethyl)phenyl)amino]-4-(N,N-bis(methoxy-

ethyl)amino)-6-methylpyridine with triethyl orthoacetate un-

der HCl catalysis as described earlier. The product was

purified by chromatography (silica gel; 25%EtOAc/hexanes) to

1

give 26b (90%); H NMR (CDCl₃) δ 8.12 (d, 1H, J ) 2.2 Hz),

7.69 (dd, 1H, J ₁) 8 Hz, J ₂) 2.2 Hz), 7.55 (d, 1H, J ) 8 Hz),

6.23 (s, 1H), 4.1-4.22 (m, 4H), 3.75 (t, 4H J ) 5.5 Hz), 3.39 (s,

6H), 3.37 (s, 3H), 3.05-3.15 (m, 1H), 2.48 (s, 3H), 1.36 (d, 6H,

J ) 7.0 Hz).

Ack n ow led gm en t. The authors gratefully acknowl-

edge the expert technical assistance of Susan Keim,

Carol Krause, Anne Marshall, J ohn Patterson, David

Rominger, and Cindy Rominger in performing the

binding assays; Gregory Nemeth and Ernest Schubert

in acquiring NMR data; Karl Bloom, Carl Schwarz,

Michael Haas, and Robert Carney for providing mass

spectral data; and Michael Xia for compound synthesis.

Biology. 1. CRF -R1 Recep tor Bin d in g Assa y. The fol-

lowing is a description of the isolation of cell membranes

containing cloned human CRF-R1 receptors for use in the

standard binding assay as well as a description of the assay

itself.

Messenger RNA was isolated from human hippocampus.

The mRNA was reverse-transcribed using oligo (dt) 12-18,

and the coding region was amplified by PCR from start to stop

codons. The resulting PCR fragment was cloned into the

EcoRV site of pGEMV, from whence the insert was reclaimed

using XhoI + XbaI and cloned into the XhoI + XbaI sites of

vector pm3ar (which contains a CMV promoter, the SV40 ‘t′

splice and early poly(A) signals, an Epstein-Barr viral origin

of replication, and a hygromycin selectable marker). The

resulting expression vector, called phchCRFR, was transfected

in 293EBNA cells, and cells retaining the episome were

selected in the presence of 400 µM hygromycin. Cells surviving

4 weeks of selection in hygromycin were pooled, adapted to

growth in suspension, and used to generate membranes for

the binding assay described below. Individual aliquots con-

taining approximately 1 × 10⁸of the suspended cells were then

centrifuged to form a pellet and frozen.

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J M980224G

Article Doi

Synthesis, corticotropin-releasing factor receptor binding affinity, and pharmacokinetic properties of triazolo-, imidazo-, and pyrrolopyrimidines and -pyridines

DOI: 10.1021/jm980224g

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