Synthesis and oral efficacy of a 4-(butylethylamino)pyrrolo[2,3- d]pyrimidine: A centrally active corticotropin-releasing factor1 receptor antagonist

Article abstract of DOI:10.1021/jm960861b

The syntheses of a centrally active nonpeptide CRF₁ receptor antagonist 2, butylethyl[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl]amine (CP-154,526), and its analogs 11-14 and [³H]-2 are reported. The in v

Full text of DOI:10.1021/jm960861b

J . Med. Chem. 1997, 40, 1749-1754
1749
Notes
Syn th esis a n d Or a l Effica cy of a 4-(Bu tyleth yla m in o)p yr r olo[2,3-d ]p yr im id in e:
A Cen tr a lly Active Cor ticotr op in -Relea sin g F a ctor 1 Recep tor An ta gon ist
Yuhpyng L. Chen,* Robert S. Mansbach, Steven M. Winter, Elizabeth Brooks, J udith Collins,
Michael L. Corman, Audrey R. Dunaiskis, W. Stephen Faraci, Randall J . Gallaschun, Anne Schmidt, and
David W. Schulz
Medicinal Chemistry and Neuroscience Departments, Central Research Division, Pfizer Inc., Groton, Connecticut 06340
Received December 23, 1996X
The syntheses of a centrally active nonpeptide CRF1 receptor antagonist 2, butylethyl[2,5-
dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amine (CP-154,526), and its
analogs 11-14 and [3H]-2 are reported. The in vitro CRF1 receptor binding affinity in the
series 2, the pharmacokinetic properties of 2 in rats, and the anxiolytic-like effects of orally
administered 2 are presented.
In tr od u ction
resulted in a more basic pyrrolo[2,3-d]pyrimidine se-
ries13 with increased solubility at pH 2.0. Subsequent
research on structure-activity relationships in the
pyrrolo[2,3-d]pyrimidine series focused on improving
potency, brain penetrability, and oral bioavailability,
leading to the discovery of compound 2, which is
centrally and orally active. As described in our previous
publication,14 compound 2 has a Ki of 2.7 nM against
Corticotropin-releasing factor (CRF), a 41-amino acid
neuropeptide hormone, coordinates the overall response
of the body to stress through the release of adrenocor-
ticotropic hormone (ACTH). CRF has been shown to
mediate stress-induced changes in the autonomic ner-
vous system, neuroendocrine functions, and behavior.1
Receptors for CRF are distributed throughout the
central and peripheral nervous systems. Receptor
subtypes CRF1 and CRF2 have been cloned and ex-
pressed from mouse, rat, and human.2-6 Clinical data
indicate that patients with depression and posttrau-
matic stress disorder show significantly elevated con-
centrations of CRF in cerebrospinal fluid when com-
pared to normal controls.7 In addition, patients with
depression, anxiety, anorexia nervosa, and posttrau-
matic stress disorder showed blunted ACTH response
to intravenous CRF,8 indicating their CRF receptors
may be down-regulated possibly due to chronic hyper-
secretion of CRF. A CRF antagonist may represent a
novel agent for the treatment of anxiety, depression, and
stress-related diseases. Several peptide antagonists
[
125I]Tyr0-o-CRF (ovine CRF) binding to the CRF1 recep-
tor and >10 µM against [125I]Tyr0-sauvagine binding to
the CRF2 receptor. It blocks CRF-induced elevation of
ACTH and antagonizes CRF-induced excitation of locus
coeruleus neurons. In addition 2 demonstrates efficacy
in anxiolytic14,15 and antidepressant16 models after
intraperitoneal (ip) administration, indicating the thera-
peutic potential of a CRF1 receptor antagonist. Herein
we report the syntheses of 2 and its derivatives 11-14
and [3H]-2. The in vitro CRF1 receptor binding affinity
of analogs in the series 2, the pharmacokinetic proper-
ties of 2, and the anxiolytic-like effects of orally admin-
istered 2 are presented.
9
including R-helical CRF9-41 have been discovered and
studied extensively. However, a peptide antagonist
would not be expected to penetrate the blood-brain
barrier, thereby limiting its clinical utility. Intensive
research has been focused on seeking nonpeptide CRF
receptor antagonists. Several nonpeptide CRF1 receptor
antagonists were published very recently.10 Herein we
describe the discovery of a centrally active nonpeptide
CRF1 receptor antagonist 2, butylethyl[2,5-dimethyl-7-
(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-
amine (CP-154,526), and its oral efficacy in an animal
model of anxiety.
Ch em istr y
An initial weak pyrazole lead (1)11 was identified from
a high-throughput CRF receptor binding screen. After
substantial structural modifications, a structurally dis-
tinct pyrazolo[3,4-d]pyrimidine series12 was identified.
Replacement of the pyrazolo ring with a pyrrolo ring
Synthesis of 2 is illustrated in Scheme 1. Knoevena-
gel condensation of malononitrile and excess acetone in
refluxing toluene or benzene gave 317 in 70% yield after
distillation. Bromination of 3 was attempted under
various conditions. All reaction conditions tried pro-
vided the desired monobromo 418 as a major component
contaminated with undesired dibromo derivative 5 and
starting material. The best conditions use 1-1.3 equiv
* Corresponding author: phone, 860-441-4208; fax, 860-441-4111;
e-mail, yuhpyng_l_chen@groton.pfizer.com.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00861-8 CCC: $14.00 © 1997 American Chemical Society

1750 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
Sch em e 1
Ta ble 1. Inhibition of Binding of 70 pM [125I]Tyr0-o-CRF to Rat Brain Membranes
compd
R1
R2
pIC50 (mean ( SEM)a
IC50 (nM)
2 (CP-154,526)
n-Bu
Et
Et
H
H
H
8.26 ( 0.02
8.12 ( 0.08
6.94 ( 0.16
6.21 ( 0.08
<5
5.5
7.6
110
11
12
13
14
CH2-CHdCH-CH3
n-Bu
Et
620
H
>10000
a
Geometric means are of at least three experimental determinations.
of N-bromosuccinimide in the presence of a catalytic
amount of benzoyl peroxide in chloroform. Reaction of
4 (76% pure) with trimethylaniline at room temperature
afforded the desired aminopyrrole intermediate 6 as a
purple solid that was purified through silica gel column
chromatography to give a 48% yield of tan crystals, mp
138-139 °C. Acetylation of aminopyrrole 6 followed by
acid hydrolysis and cyclization in 85% phosphoric acid
afforded 4-hydroxypyrrolopyrimidine derivative 8 in
81% overall yield. Reaction of 8 with phosphorous
oxychloride at reflux gave the corresponding 4-chloro
derivative 9 in 79% yield. Coupling 9 with n-butylethy-
lamine in dimethyl sulfoxide at 130 °C gave 2 in 88%
yield as a tan solid, mp 132-137 °C. The corresponding
HCl salt was prepared as white crystals, mp 169-170
°C . The corresponding analogs 11-14 were prepared
in a similar manner.
Biologica l Resu lts
Table 1 lists the in vitro CRF1 binding affinity in rat
cortex against radioligand [125I]Tyr0-o-CRF by com-
pounds 2 and 11-14. As shown, both 4-disubstituted
amino analogs 2 and 11 showed potent CRF1 binding
affinity. The monosubstituted amino analogs 12 and
13 had reduced potency, and the amino analog 14 is
inactive. Compound 2 was selected for anxiolytic-like
activity studies after oral administration in rats.
Previously we reported the activity of 2 in fear-
potentiated startle after ip administration.13 In this
report we present efficacy data after oral administra-
tion. The paradigm for the fear-potentiated startle
experiments is similar to that described previously. In
the training session, rats received light paired with
electric shock. In this way, the light acquired condi-
tioned aversive properties. Three days later, the acous-
tic startle session was performed in the presence and
absence of light without electric shock. At the oral dose
of 17.8 mg/kg, compound 2 significantly attenuated the
enhancement of startle amplitude induced by the light-
[2H]CP-154,526 was synthesized by reduction of the
precursor 11 with deuterium in the presence of 10%
Pd/C in ethyl acetate at atmospheric pressure. [3H]CP-
154,526 was prepared in a similar way with 60 Ci/mmol
specific activity.

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1751
F igu r e 1. Effect of compound 2 (17.8 mg/kg, po, 60 min prior
to testing) on potentiated startle in rats. Animals were exposed
to 108 dB[A] acoustic stimuli, some of which were presented
in darkness and others in the presence of a conditioned
stimulus (electric light, 25 W) formerly paired with brief
electric shock. Data are expressed as the absolute difference
in startle amplitude between light-paired and light-unpaired
startle trials, over 5 blocks of 10 trials each. The initial block
consisted of light-unpaired trials only and was presented to
eliminate the rapid phase of startle habituation. Controls (no
shock history/vehicle) received previous exposure to light
without the accompanying shock. Compound 2 significantly
reduced fear-potentiated startle without affecting baseline
startle magnitude (n ) 12): (O) no shock history/vehicle, (b)
vehicle, and (2) compound 2.
F igu r e 2. Mean plasma concentrations of compound 2 in
Sprague-Dawley rats administered a bolus intravenous dose
at 5 mg/kg ([, solid line) or an oral dose at 10 mg/kg (2, dotted
line). Data represent mean ( SD from four animals.
Exp er im en ta l Section
conditioned stimulus to that of vehicle controls as shown
in Figure 1.
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. High-field
1H NMR spectra were recorded on a Varian XL-300, Bruker
AM 250, or Bruker AM 300 instrument. Elemental analyses
were carried out by Mr. J . W. Greene, Analytical Department,
Pfizer Inc., or by Schwarzkopf Microanalytical, Woodside, NY.
Isop r op ylid en em a lon on itr ile (3). A mixture of malono-
nitrile (90 g, 1.362 mol), acetone (400 mL), sodium acetate (25
g, 0.3 mol), acetic acid (18 mL), and benzene (400 mL) was
heated at reflux using a Dean-Stark trap for 4 h. The reaction
mixture was concentrated to dryness. The reaction was
quenched with water and the residue extracted with ethyl
acetate. The organic layer was separated, dried, and concen-
trated to give a light yellow oil, which was distilled at 70 °C/1
mmHg to give 101.34 g (70.1%) of the title compound: 1H NMR
(CDCl3) δ 2.24 (s, 6H).
P h a r m a cok in etics in Ra t. The pharmacokinetics
of 2 were determined in male Sprague-Dawley rats
following an intravenous (iv) dose of 5 mg/kg (n ) 4)
and an oral (po) dose of 10 mg/kg (n ) 4). Following iv
administration, drug concentrations declined over time
in a biphasic manner (Figure 1). The pharmacokinetics
of 2 were characterized by a high plasma clearance (CL
82 mL/min/kg) and a large volume of distribution (Vdss
6.7 L/kg), resulting in an elimination half-life of 1.5 h.
Following oral administration in DMSO/emulphor/water
(5:5:90, v/v/v) the mean maximal plasma concentration
(Cmax) of drug was 367 ng/mL and occurred at 0.5-1 h
following dose administration (Figure 2). Oral bioavail-
ability of 2 at this dose was estimated to be 37%.
Assuming that blood concentrations are similar to those
measured in plasma, CL exceeds hepatic blood flow. An
oral bioavailability of 37% suggests that hepatic extrac-
tion of 2 was e63% when administered orally at 10 mg/
kg in DMSO/emulphor/water (5:5:90, v/v/v).
2-(2-Br om o-1-m eth yleth ylid en e)m a lon on itr ile (4).
A
mixture of isopropylidenemalononitrile (30.000 g, 282.68
mmol), N-bromosuccinimide (65.410 g, 367.48 mmol), and
benzoyl peroxide (0.600 g, 2.48 mmol) in 90 mL of chloroform
was heated at reflux for 15 h. The reaction mixture was
allowed to cool and filtered. The collected solid was washed
with chloroform. The filtrate was concentrated to dryness to
give a brown oil which showed a mixture of 4, 5, and 3 with a
ratio of 67%, 15%, and 18%, respectively. The product was
fractionated: bp 95-98 °C at 0.6 mmHg, to give 40.21 g (77%)
of light oil which showed a mixture of 4, 5, and 3 with a ratio
of 76%, 12%, and 12%, respectively, according to the H NMR
spectrum. The material was used directly for the next reaction
step: 1H NMR (CDCl3) δ 4.38 (s, 0.12 × 4H), 4.10 (s, 0.76 ×
2H), 2.40 (s, 0.76 × 3H), 2.24 (s, 0.12 × 6H).
Con clu sion s
After substantial structural modifications of the
initial pyrazole lead 1 aimed at improving potency,
pharmacokinetics, and physicochemical properties, we
have discovered a centrally and orally active CRF1
antagonist (2). Compound 2 not only shows potent
CRF1 binding affinity but also demonstrates oral efficacy
in animals. An orally bioavailable CRF1 receptor an-
tagonist, such as 2, may be a therapeutic alternative to
current antidepressant and anxiolytic drugs. Details
of structure-activity relationships will be reported later.
2-Am in o-4-m eth yl-1-(2,4,6-tr im eth ylph en yl)-1H-pyr r ole-
3-ca r bon itr ile (6). A mixture of 2-(2-bromo-1-methyleth-
ylidene)malononitrile (76% pure, 20.000 g, 91.24 mmol) and
2,4,6-trimethylaniline (12.330 g, 91.24 mmol) in 40 mL of
2-propanol was stirred at room temperature for 15 h. The
reaction mixture was concentrated to dryness and diluted with

1752 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
chloroform and water. The chloroform layer was neutralized
with dilute sodium hydroxide, washed with brine, separated,
dried, and concentrated to give 33.000 g of a dark brown oily
solid. The solid was purified through silica gel column
chromatography using chloroform as eluent to give 9.35 g
(47.5%) of 6 as an orange-yellow solid. The solid was recrys-
tallized from ethanol to give beige crystals: mp 138-139 °C;
1H NMR (CDCl3) δ 6.95 (s, 2H), 5.8 (s, 1H), 3.75 (brs, 2H),
2.35 (s, 3H), 2.15 (s, 3H), 2.0 (s, 6H). Anal. Calcd for
C15H17N3: C, 75.28; H, 7.16; N, 17.56. Found: C, 74.85; H,
6.92; N, 17.59.
N-[3-Cya n o-4-m eth yl-1-(2,4,6-tr im eth ylp h en yl)-1H-p yr -
r ol-2-yl]a ceta m id e (7). A mixture of 6 (3.200 g, 12.54 mmol)
and acetic anhydride (1.410 g, 13.82 mmol) in 3 mL of acetic
acid was refluxed for 45 min. The reaction mixture was
concentrated to dryness. The residue was treated with ice
water, neutralized, and extracted with ethyl acetate. The
organic layer was washed with brine, dried, and concentrated
to give 3.71 g of 7 as a brown foam. The brown foam was used
directly for the next reaction step. A small portion of the crude
material was purified through column chromatography to give
a pink glassy foam: 1H NMR (CDCl3) δ 6.9 (s, 2H), 6.8 (brs,
1H, NH), 6.2 (s, 1H), 2.32 (s, 3H), 2.2 (s, 3H), 1.95 (s, 6H).
Anal. Calcd. for C17H19N3O: C, 72.57; H, 6.81; N, 14.93.
Found: C, 72.79; H, 6.64; N, 14.98.
2,5-Dim eth yl-7-(2,4,6-tr im eth ylp h en yl)-7H-p yr r olo[2,3-
d ]p yr im id in -4-ol (8). A suspension of the crude compound
of 7 (9.820 g, 34.90 mmol) in 17 mL of 85% phosphoric acid
was immersed in an oil bath preheated to 130 °C for 30 min.
The reaction mixture was cooled, poured onto ice water and
extracted with chloroform. The organic layer was washed with
brine, dried, filtered, and concentrated to give 7.97 g (81.1%)
of 8 as a brown solid. The solid was used directly for the next
reaction step. The solid was recrystallized from a mixture of
ethanol and water to give brown crystals: 1H NMR (CDCl3) δ
6.95 (s, 2H), 6.42 (s, 1H), 2.45 (s, 3H), 2.41 (s, 3H), 2.32 (s,
3H), 1.92 (s, 6H). Anal. Calcd for C17H19N3O: C, 72.57; H,
6.81; N, 14.93. Found: C, 72.24; H, 7.01; N, 14.89.
4-Ch lor o-2,5-d im et h yl-7-(2,4,6-t r im et h ylp h en yl)-7H -
p yr r olo[2,3-d ]p yr im id in e (9). A mixture of 2,5-dimethyl-
7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-ol (7.800
g, 27.72 mmol) and POCl3 (10 mL) was heated at reflux for
2.5 h and cooled. The reaction mixture was poured into ice-
water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated to dryness to give 9 as a tan solid which was
purified via silica gel column chromatography using chloroform
as eluent to give 6.600 g (79.4%) of 9 as an off-white solid:
mp 108-110 °C; 1H NMR (CDCl3) δ 7.00 (s, 2H), 6.78 (s, 1H),
2.65 (s, 3H), 2.50 (s, 3H), 2.35 (s, 3H), 1.90 (s, 6H). Anal. Calcd
for C17H18ClN3: C, 68.11; H, 6.05; H, 14.02. Found: C, 67.85;
H, 5.90; N, 13.75.
crotyl bromide (3.000 g, 22.22 mmol) in 15 mL of dry methylene
chloride was added ethylamine (30 mL). The reaction mixture
was allowed to warm to room temperature for 2 h. Excess
ethylamine was evaporated to give 3.2 g of a yellow oil. The
oil was fractionated to give 1.32 g (60%) of 10 as a clear
1
liquid: bp 110-120 °C; H NMR (CDCl3) δ 5.4-5.6 (m, 2H),
3.1-3.3 (m, 2H), 2.6 (m, 2H), 1.6-1.7 (m, 3H), 1.0-1.2 (m, 3H).
Bu t-2-en yl[2,5-d im eth yl-7-(2,4,6-tr im eth ylp h en yl)-7H-
p yr r olo[2,3-d ]p yr im id in -4-yl]eth yla m in e (11). A mixture
of 9 (0.500 g, 1.67 mmol) and N-(2-butenyl)ethylamine (1.320
g, 13.36 mmol) in 5 mL of DMSO was heated at 130 °C for 2
h. The reaction was quenched with water and the mixture
extracted with ethyl acetate. The organic layer was washed
with brine, dried, filtered, and concentrated to give 0.850 g of
brown oil. The oil was purified via silica gel column chroma-
tography using 1% methanol in chloroform as eluent to give
0.540 g (90%) of a mixture of E and Z isomers 11 as a colorless
oil: 1H NMR (CDCl3) δ 6.95 (s, 2H), 6.58 (s, 1H), 5.5-5.8 (m,
2H), 4.25 (d, 0.2 × 2H), 4.15 (d, 0.8 × 2H), 3.6 (m, 2H), 2.5 (s,
3H), 2.4 (s, 3H), 2.3 (s, 3H), 1.9 (s, 6H), 1.7 (d, 3H), 1.2 (t, 3H).
The corresponding HCl salt was prepared to give white
crystals after recrystallization from ethyl acetate: mp 157-
1
159 °C; H NMR (CDCl3) δ 7.00 (s, 2H), 6.76 (s, 1H), 5.8-6.0
(m, 1H), 5.4-5.6 (m, 1H), 4.4-4.6 (m, 2H), 3.9-4.1 (m, 2H),
2.86 (s, 3H), 2.43 (s, 3H), 2.35 (s, 3H), 1.89 (s, 6H), 1.75 (m,
3H), 1.32 (m, 3H); IR (KBr) 2917, 1599, 1490 cm-1
. Anal. Calcd
for C23H30N4‚HCl: C, 69.24; H, 7.83; N, 14.04. Found: C,
68.97; H, 7.74; N, 13.97.
The following compounds 12-14 were prepared from com-
pound 9 and an appropriate amine by the same procedure as
described for 2. Compounds 13 and 14 were heated in a high-
pressure reactor to 110 °C at 150 and 250 psi, respectively.
Bu t yl[2,5-d im et h yl-7-(2,4,6-t r im et h ylp h en yl)-7H-p yr -
r olo[2,3-d ]p yr im id in -4-yl]a m in e (12). A 99% yield of brown
solid was obtained. The brown solid was recrystallized from
petroleum ether. A 40% yield of white crystals was collected
from the first crop, and a 55% yield of crude product was
obtained from the concentration of the filtrate: mp 114-116
1
°C; H NMR (CDCl3) δ 6.93 (s, 2H), 6.39 (s, 1H), 4.90 (t, 1H),
3.57 (q, 2H), 2.45 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3H), 1.90 (s,
6H), 1.62 (m, 2H), 1.41 (m, 2H), 0.98 (t, 3H); IR (KBr) 3648,
2918, 2858, 1591, 1577 cm-1. Anal. Calcd for C21H28N4: C,
74.96; H, 8.39; N, 16.65. Found: C, 75.04; H, 8.70; N, 16.50.
E t h yl[2,5-d im et h yl-7-(2,4,6-t r im et h ylp h en yl)-7H-p yr -
r olo[2,3-d ]p yr im id in -4-yl]a m in e (13). A 88% yield of light
brown solid was obtained. The light brown solid was recrys-
tallized from petroleum ether. A 44% yield of tan crystals was
collected from the first crop: mp 153-156 °C; 1H NMR (CDCl3)
δ 6.93 (s, 2H), 6.40 (s, 1H), 4.90 (t, 1H), 3.62 (q, 2H), 2.46 (s,
3H), 2.44 (s, 3H), 2.30 (s, 3H), 1.90 (s, 6H), 1.30 (t, 3H); IR
(KBr) 3670, 3458, 2917, 1592, 1578 cm-1. Anal. Calcd for
C19H24N4: C, 73.99; H, 7.84; N, 18.17. Found: C, 74.29; H,
7.95; N, 17.84.
Bu t yl[2,5-d im et h yl-7-(2,4,6-t r im et h ylp h en yl)-7H-p yr -
r olo[2,3-d ]p yr im id in -4-yl]eth yla m in e (2). A mixture of 9
(6.600 g, 22.01 mmol) and N-butylethylamine (11.130 g, 110.05
mmol) in 27 mL of DMSO was heated at 130 °C for 3 h. The
reaction was quenched with water and the mixture extracted
with ethyl acetate. The organic layer was washed with brine,
dried, filtered, and concentrated to give 9.1 g of brown oil. The
oil was purified via silica gel column chromatography using
2% methanol in chloroform as eluent to give 7.040 g (88%) of
2 as an oil, which crystallized out as a tan solid upon
standing: mp 132-137 °C; 1H NMR (CDCl3) δ 6.95 (s, 2H),
6.55 (s, 1H), 3.66 (q, 2H), 3.58 (t, 2H), 2.46 (s, 3H), 2.40 (s,
3H), 2.30 (s, 3H), 1.92 (s, 6H), 1.60-1.70 (m, 2H), 1.35 (m, 2H),
1.23 (t, 3H), 0.90 (t, 3H).
2,5-Dim eth yl-7-(2,4,6-tr im eth ylp h en yl)-7H-p yr r olo[2,3-
d ]p yr im id in -4-yla m in e (14). A 63% yield of golden solid was
obtained after silica gel column chromatography using 5%
MeOH in chloroform as eluent: mp 208-210 °C; 1H NMR
(CDCl3) δ 6.95 (s, 2H), 6.51 (s, 1H), 5.12 (s, 2H), 2.46 (s, 6H),
2.32 (s, 3H), 1.91 (s, 6H); IR (KBr) 3517, 3405, 2916, 1609,
1592 cm-1. Anal. Calcd for C17H20N4: C, 72.83; H, 7.19; N,
19.98. Found: C, 72.47; H, 6.94; N, 19.58.
[2H ]Bu t yl[2,5-d im et h yl-7-(2,4,6-t r im et h ylp h en yl)-7H -
p yr r olo[2,3-d ]p yr im id in -4-yl]eth yla m in e. A mixture of 11
(50 mg, 0.138 mmol) and 50 mg of 10% Pd/C in 10 mL of ethyl
acetate was hydrogenated using deuterium gas under atomo-
spheric pressure for 2 h. The mixture was filtered through
Celite and concentrated to give 50 mg (98%) of a clear oil. The
oil was purified via silica gel column chromatography using
1% methanol in chloroform as eluent to give 38 mg (75%) of
[2H]-2 as a colorless oil: 1H NMR (CDCl3) δ 6.96 (s, 2H), 6.56
(s, 1H), 3.67 (q, 2H), 3.59 (d, 2H), 2.47 (s, 3H), 2.42 (s, 3H),
2.33 (s, 3H), 1.92 (s, 6H), 1.60-1.70 (m, 1H), 1.35 (m, 1H), 1.23
(t, 3H), 0.90 (t, 3H).
The corresponding HCl salt was prepared by adding 1.5
equiv of 1 M HCl in methanol followed by evaporation of
solvent to give a white solid. The solid was recrystallized from
1
ethyl acetate: mp 169-170 C; H NMR (CDCl3) δ 14.45 (brs,
1H), 7.09 (s, 2H), 6.78 (s, 1H), 3.80-3.94 (m, 4H), 2.85 (s, 3H),
2.40 (s, 3H), 2.30 (s, 3H), 1.90 (s, 6H), 1.70 (m, 2H), 1.40 (m,
2H), 1.32 (t, 3H), 0.92 (t, 3H); IR (KBr) 2922, 2868, 1609, 1489
cm-1
. Anal. Calcd for C23H32N4‚HCl: C, 68.89; H, 8.30; N,
The above procedure was used for the synthesis of [3H]-2,
and the radiolabeled synthesis was performed by Chemsyn
Science Laboratory.
13.97. Found: C, 69.23; H, 8.34; N, 14.04.
N-(2-Bu ten yl)eth yla m in e (10). To a -78 °C solution of

Notes
Biologica l Meth od s. Recep tor Bin d in g Stu d ies. Rats
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1753
dynamic range of the assay used for these samples was 5-500
ng/mL. Plasma samples with concentrations of drug above 500
ng/mL were diluted appropriately with control plasma and
reanalyzed.
were sacrificed and their brains removed. The cortex was
dissected and homogenized in 50 mM Tris HCl buffer (pH 7.4)
using a Brinkman Polytron Model PT3000 (setting 15 000 rpm,
15 s). The homogenate was centrifuged for 10 min at 40000g.
The pellet was resuspended in fresh ice-cold 50 mM Tris HCl
buffer using the Polytron and recentrifuged for 10 min at
40000g. The final pellet was resuspended in 20 mM pipera-
zine-N,N′-bis[2-ethanesulfonic acid], 10 mM MgCl2, 2 mM
EGTA, 0.015% bacitracin, and aprotinin (100 kallikrein units/
mL) (pH 7.0). Incubations were initiated by the addition of
tissue homogenate (100 µL) to tubes containing [125I]o-CRF (70
pM) and buffer with 0.04% bovine serum albumin and either
vehicle or compound in a final volume of 200 µL. Nonspecific
binding was defined as the radioactivity remaining in the
presence of 1 µM rat/human CRF. After a 120 min incubation
at room temperature, assay samples were centrifuged at 1000g
for 10 min. The supernatant was discarded, and 100 µL of
ice-cold assay buffer (same as tissue buffer described above
plus 0.04% bovine serum albumin) was added to each assay
sample. After another centrifugation, assay samples were
filtered onto LKB Betaplate filtermats, presoaked in 0.2%
polyethylenimine, using a Skatron cell harvester and ice-cold
50 mM Tris buffer (pH 7.2). Radioactivity was quantified by
liquid scintillation counting (Betaplate, LKB/Wallac).
Pharmacokinetic parameters were calculated using the
program PK_PARAM. The concentration of drug at time t )
0 following iv dose administration was determined by regres-
sion of the data using data points from the distribution phase
of the apparent biexponential elimination. Area under the
) and AUC(0
plasma concentration-time curves (AUC(0 - tlast
- inf)) were calculated by the linear trapezoidal method.
Clearance (CL) was calculated as dose/AUC(0 - inf), and the
apparent volume of distribution at steady state was calculated
as CL × mean residence time (MRT), where MRT equaled area
under the momment curve (AUMC)/AUC. The terminal
elimination rate constant (Kel) was determined by unweighted
linear regression. The oral bioavailability (F) of 2 was
estimated by comparing the AUCs following oral and iv
administration and normalizing for dose.
Ack n ow led gm en t. We are grateful to Dr. J on
Bordner for the X-ray structure analyses and Chemsyn
Science Laboratories, Lenexa, KS, for conducting the
radiolabeled synthesis.
F ea r -P oten tia ted Sta r tle. One week after delivery, rats
were exposed to a startle response session consisting of twenty
120 dB[A], 40 ms acoustic startle stimuli, interspersed by 15
s of 68 dB[A] background noise. The results of this initial
baseline matching test were used to assign rats to experimen-
tal groups with approximately equal startle reactivity. One
day later, subjects were exposed to a classical conditioning
procedure in which illumination of an incandescent light (25
W a.c.) was paired with presentation of a scrambled foot shock
(1.2 mA). The shock occurred during the final 500 ms of the
3.2 s light presentation. Twenty such pairings were presented,
2 min apart, in darkened startle chambers. One group of
animals, “no shock”, was exposed to the light presentations
without the accompanying shocks. On the test day, conducted
3 days later, rats were exposed to a startle session in which
some of the 108 dB stimuli were preceded by a 3.2 s light
presentation and others were not. An initial block of 11 “noise-
alone” startle stimuli were presented to eliminate the rapid
phase of startle habituation. These trials were not included
in the data analysis. Compound 2 was administered po 60
min prior to the acoustic startle session. For each subject, the
difference between light-paired and -unpaired startle re-
sponses was calculated. This difference score represents the
magnitude of potentiated startle. Also analyzed was the effect
of drug on baseline (unpaired) startle, as a measure of
nonspecificity.
Refer en ces
(1) (a) Vale, W.; Spiess, J .; Rivier, C.; Rivier, J . Characterization of
a 41 residue ovine hypothalamic peptide that stimulates secre-
tion of corticotropin and beta endorphin. Science 1981, 213,
1394-1397. (b) Owens, M. J .; Nemeroff, C. B. Physiology and
pharmacology of corticotropin-releasing factor. Pharmacol. Rev.
1991, 43, 425-473. (c) Black, P. H. Psychoneuroimmunology:
brain and immunity. Sci. Am. Sci. Med. 1996, 16-25.
(2) Chen, R.; Lewis, K. A.; Perrin, M. H.; Vale, W. W. Expression
cloning of a human corticotropin-releasing-factor receptor. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 8967-8971.
(3) Perrin, M. H.; Donaldson, C. J .; Chen, R.; Lewis, K. A.; Vale,
W. W. Cloning and functional expression of a rat brain corti-
cotropin releasing factor (CRF) receptor. Endocrinology 1993,
133, 3058-3061.
(4) Perrin, M.; Donaldson, C.; Chen, R.; Blount, A.; Berggren, T.;
Bilezikjian, L.; Sawchenko, P.; Vale, W. Identification of a second
corticotropin-releasing factor receptor gene and characterization
of a cDNA expressed in heart. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 2969-2973.
(5) Liaw, C. W.; Lovenberg, T. W.; Barry, G.; Oltersdorf, T.;
Grigoriadis, D. E.; De Souza, E. B. Cloning and characterization
of the human corticotropin-releasing factor-2 receptor comple-
mentary deoxyribonucleic acid. Endocrinology 1996, 137, 72-
77.
(6) Chalmers, D. T.; Lovenberg, T. W.; Grigoriadis, D. E.; Behan,
D. P.; De Souza, E. B. Corticotrophin-releasing factor recep-
tors: from molecular biology to drug design. Trends Pharmacol.
Sci. 1996, 17, 166-172.
P h a r m a cok in etic Stu d ies in Ra t. Male Sprague-Daw-
ley rats (300-350 g) were surgically prepared with indwelling
jugular vein canulae for the intravenous administration of 2
and the sampling of blood. Surgery was performed under
pentobarbital anesthesia (65 mg/kg, ip) 1 day before dosing.
Two groups of rats (n ) 4) received either a bolus iv (5 mg/kg)
or po (10 mg/kg) dose of 2. For iv dosing, the compound was
prepared in dimethyl sulfoxide/emulphor/saline (5:5:90, v/v/
v) and administered as a 5 mg/mL solution at 1 mL/kg. For
oral dosing the compound was administered as a 2 mg/mL
solution in dimethyl sulfoxide/emulphor/water (5:5:90, v/v/v)
at 5 mL/kg. The dosing solutions were prepared immediately
prior to administration. Serial blood samples (0.5 mL) were
collected from the indwelling cannula at 0.08, 0.17, 0.25, 0.50,
1, 2, 3, 4, 6, and 24 h following iv dose administration into
heparinized syringes. Sampling time points following the oral
dose were 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h. The blood was
immediately centrifuged and the plasma transferred to clean
Eppendorf tubes and stored at -20 °C prior to analysis.
The concentration of 2 in plasma samples was determined
by HPLC using a Zorbax RX-C8 5 µm (15 cm × 4.6 mm id)
analytical column. The flow rate of the mobile phase, aceto-
nitrile:0.05 M phosphate buffer, pH 4.7 (70/30, v/v), was 1.5
mL/min. The column effluent was monitored by UV absorption
at 300 nm, and the limit of detection was 5 ng/mL. The
(7) (a) Nemeroff, C. B.; Widerlov, E.; Bissette, G.; Wallens, H.,;
Karlsson, I.; Eklund, K.; Kilts, C. D.; Loosen, P. T.; Vale, W.
Elevated concentrations of CSF corticotropin-releasing factor-
like immunoreactivity in depressed patients. Science 1984, 226,
1342-1344. (b) Darnell, A.; Bremner, J . D.; Licinio, J .; Krystal,
J .; Nemeroff, C. B.; Owens, M.; Erdos, J .; Charnev, D. S. CSF
levels of corticotropin releasing factor in chronic post-traumatic
stress disorder. Soc. Neurosci. Abstr. 1994, 20, 17.
(8) (a) Holsboer, F.; Von Bardeleben, U.; Gerken, A.; Stalla, G. K.;
Muller, O. A. Blunted corticotropin and normal cortisol response
to human corticotropin-releasing-factor in depression. N. Engl.
J . Med. 1984, 311, 1127. (b) Taylor, A. L.; Fishman, L. M.
Corticotropin-releasing hormone. N. Engl. J . Med. 1988, 319,
213-222.
(9) Rivier, J .; Rivier, C.; Vale, W. Synthetic competitive antagonists
of corticotropin-releasing factor: effect on ACTH secretion in the
rat. Science 1984, 224, 889-891.
(10) (a) Chen, Y. L. Pyridine, pyrimidine, purinones, pyrrolopyrimi-
dinones and pyrrolopyridinones as corticotropin releasing factor
antagonists. International Publication Number WO 9533750-
A1. The disclosed pyrimidine series in this patent was later
published by another group: Chen, C.; Dagnino, R.; De Souza,
E. B.; Grigoriasdis, D. E.; Huang, C. Q.; Kim, K.; 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) Whitten, J . P.; Xie, Y. F.;
Erickson, P. E.; Webb, T. R.; De Souza, E. B.; Grigoriadis, D.
E.; McCarthy, J . R. Rapid microscale synthesis, a new method

1754 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Notes
for lead optimization using robotics and solution phase chem-
istry: application to the synthesis and optimization of corti-
cotropin-releasing factor1 receptor antagonists. J . Med. Chem.
1996, 39, 4354-4357.
(15) Lundkvist, J .; Chai, Z.; Teheranian, R.; Hasanvan, H.; Bartfai,
T.; J enck, F.; Widmer, U.; Moreau, J . A non peptide corticotropin
releasing factor receptor antagonist attenuates fever and exhibits
anxiolytic-like activity. Eur. J . Pharamcol. 1996, 309, 195-200.
(16) Mansbach, R. S.; Winston, E. N.; Chen, Y. L. Antidepressant-
like effects of CP-154,526, a selective CRF1 receptor antagonist.
Eur. J . Pharmacol. 1997, 323, 21-26.
(11) Faraci, W. S.; Welch, W. M., J r. Pyrazoles and pyrazolopyrim-
idines having corticotropin-releasing factor antagonist activity.
International Publication Number WO 9413643-A1.
(12) Chen, Y. L. Pyrazolopyrimidines as CRF antagonists. Interna-
tional Publication Number WO 9413677-A1.
(17) Prout, F. S. Amino acid catalysis of the Knoevenagel reaction.
(13) Chen, Y. L. Pyrrolopyrimidines as CRF antagonists.International
Publication Number WO 9413676-A1.
J . Org. Chem. 1953, 18, 928-933.
(18) Gewald, K. 2-Amino-thiophene aus R-oxo-mercaptanen und
methyleneaktiven nitrilen. Chem. Ber. 1965, 98, 3571-3577.
(14) Schulz, W. D.; Mansbach, R. S.; Sprouse, J .; Braselton, J . P.;
Collins, J .; Corman, M.; Dunaiskis, A.; Faraci, S.; Schmidt, A.;
Seeger, T.; Seymour, P.; Tingley, F. D., III; Winston, E. N.; Chen,
Y. L.; Heym, J . CP-154,526: a potent and selective nonpeptide
antagonist of corticotropin releasing factor receptors. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 10477-10482.
J M960861B