Synthesis of 3-phenylpyrazolo[4,3-b]pyridines via a convenient synthesis of 4-amino-3-arylpyrazoles and SAR of corticotropin-releasing factor receptor type-1 antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00621-8

3-Phenylpyrazolo[4,3-b]pyridines were synthesized via a cyclization of 4-amino-3-phenylpyrazoles 11-13 with ethyl acetoacetate. These compounds were found to be potent CRF₁ antagonists. The 2-alkylpyrazolo[4,3-b]pyridines were more polar but le

Full text of DOI:10.1016/S0960-894X(03)00621-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3367–3370
Synthesis of 3-Phenylpyrazolo[4,3-b]pyridines Via a Convenient
Synthesis of 4-Amino-3-arylpyrazoles and SAR of Corticotropin-
Releasing Factor Receptor Type-1 Antagonists
Keith Wilcoxen, Charles Q. Huang, James R. McCarthy,
Dimitri E. Grigoriadis and Chen Chen*
Department of Medicinal Chemistry and Department of Pharmacology, Neurocrine Biosciences, Inc.,
10555 Science Center Drive, San Diego, CA 92121, USA
Received 28 January 2003; accepted 14 April 2003
Abstract—3-Phenylpyrazolo[4,3-b]pyridines were synthesized via a cyclization of 4-amino-3-phenylpyrazoles 11–13 with ethyl
acetoacetate. These compounds were found to be potent CRF1 antagonists. The 2-alkylpyrazolo[4,3-b]pyridines were more polar
but less active than the corresponding 1-alkyl-isomers.
# 2003 Elsevier Ltd. All rights reserved.
Corticotropin releasing factor (CRF), a 41-amino acid
peptide produced in hypothalamus and extracts its
function by acting on the CRF receptors in the pituitary
gland, is a major modulator of the body’s responses to
stress. Clinical evidences suggest that over-stimulation
of CRF may result in several neuropsychiatric diseases
including depression, anxiety and stress related dis-
orders. Ever since the discovery of the first nonpeptide
CRF1 antagonist CP-154,526 (1) in 1996,1 many differ-
ent classes of small molecules were reported as potent
CRF1 antagonists.2 In the efforts to search for small-
molecule CRF1 receptor antagonists for treatment of
anxiety/depression and other related diseases, we3 and
others4 have synthesized a series of pyrazolo[1,5-a]pyr-
imidines. Although these derivatives possess very potent
receptor binding affinity and functional antagonism,
like many other classes of CRF1 antagonists, this series
of compounds such as 2a are very lipophilic and have
poor water-solubility. CP-154,526 has extremely high
volume of distribution in rats, resulting in a very long
half-life,5 that may imply potential accumulation in the
body and thus may cause unexpected toxicity. We have
been able to optimize the pyrazolo[1,5-a]pyrimidine
series by incorporating a basic aminopyridine group
represented by compound 2b (NBI 30775).6 We also
reported recently7 a series of 4-aminoquinolines such as
3, which was designed and optimized based on the high
basicity of this core (4-aminoquinoline has a pKa value
of 9.1).8 Recently, a series of pyrazolo[4,3-d]pyrimidines
4 and 5 was reported as potent CRF1 antagonists with
low nanomolar binding affinity.9 Again this series of
derivatives may suffer from high lipophilicity and poor
water solubility. Here, we report the synthesis and
initial SAR study of a series of 3-phenylpyrazolo[4,3-
b]pyridines as CRF1 antagonists (Fig. 1).
The rationale for the design of this series of compounds
is as follow. Since the benzene-fused 4-aminopyridine
(pKa=9.17),10 that is 4-aminoquinoline (pKa=9.1),8 is
much
more
basic
than
4-aminoquinazoline
(pKa=5.73),11 the pyrazolo-fused 4-aminopyridine
structure (pyrazolo[4,3-b]pyridine) would be more basic
(estimated pKa ꢀ7.8)12 than the corresponding ring-
fused pyrimidines such as pyrrolo[2,3-b]pyrimidine (1),
pyrazolo[1,5-a]pyrimidine (2) and pyrazolo[4,3-d]pyr-
imidine (4). However, the chemistry of this bicyclic het-
erocycle remains unexplored due to a lack of efficient
synthetic routes to the key intermediate 4-amino-3-
arylpyrazoles 11–13 (Schemes 1 and 2). There are few
reports on the synthesis of 4-amino-3-arylpyrazoles in
the literature.13ꢁ15 For example, a five-step synthesis is
reported via the cyclization of ethyl 3-benzoyl-3-nitroso-
2-oxopropionate with hydrazine or methylhydrazine,
followed by saponification and decarboxylation, but the
*Corresponding author. Tel.: +1-858-658-7600; fax: +1-858-658-
7601; e-mail: cchen@neurocrine.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00621-8

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K. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3367–3370
Scheme 3. Reagents and conditions: (a) ethyl acetoacetate, benzene;
(b) Ph2O, heat; (c) POCl3, reflux; (d) R1R2NH, heat.
could be easily purified by ether trituration to pre-
cipitate the products (Scheme 1). Reaction of 7 with 2.5
equivof hydrazine in ethanol at room temperature for 1
h followed by refluxing the mixture for 2 h gave
4-amino-3-phenylpyrazoles 11 in 70–85% yield (two
steps from 6). Similarly, cyclization of 7 with alkylhy-
drazine afforded 4-amino-3-arylpyrazoles 12 and 13, in
good yields, as a mixture of regio-isomers.
Figure 1. Some bicyclic small-molecule CRF1 antagonists.
It is worth noting that 4-aminopyrazoles 11–13 are
labile compounds and they turn to dark color when
exposed to air. We discovered that the use of alkylhy-
drazine hydrochloride or sulfate salts avoided the for-
mation of the des-phthaloyl compounds even with a
large excess of hydrazine reagent or longer reaction
time. Thus, cyclization of hydrazine hydrochloride with
7 in refluxing aqueous ethanol (EtOH–H2O 10:1) for
2–16 h gave the 3-aryl-4-phthaloylaminopyrazoles 8 in
about 85% yield, which was deprotected with hydrazine
in refluxing ethanol to afford the 4-amino-3-arylpyr-
azoles 11 (Scheme 1). A similar method was used to
prepared compounds 12 and 13. Thus, cyclization of 7
with methylhydrazine sulfate gave a mixture of 9 and 10
in a ratio of about 2:1. These two regio-isomers were
easily separated by chromatography on silica gel.
Deprotection of the phthaloyl group of compound 9 or
10 proceeded in high yields (>90%) with two equiva-
lents of hydrazine free base in refluxing ethanol for an
hour (Scheme 2). The assignment of the two isomeric
structures of 9/10, and 12/13 are based on proton NMR
analyses and confirmed by NOE NMR experiments.17
Scheme 1. Reagents and conditions: (a) sodium phthalimide, DMF;
.
(b) DMFDMA, reflux; (c) NH2NH2 HCl, aq EtOH, reflux;
(d) NH2NH2, EtOH, reflux; (e) NH2NH2, EtOH, rt, 1 h, then reflux.
We applied the aminopyrazoles 11, 12, and 13 to syn-
thesize pyrazolo[3,4-b]pyridines 17, 18, and 19, which is
outlined in Scheme 3 and illustrated as follows.
4-Amino-3-(2,4-dichlorophenyl)-1H-pyrazole (11a) was
.
Scheme 2. Reagents and conditions: (a) RNHNH2 H2SO4, aq EtOH,
reflux, then, separation; (b) NH2NH2, EtOH, reflux.
overall yield is low (less than 1%).9 Here, we report the
synthesis of a series of pyrazolo[4,3-b]pyridines via an
efficient synthesis of 4-aminopyrazoles. The initial SAR
of the 1-alkyl- and 2-alkyl-pyrazolo[4,3-b]pyridines as
CRF1 receptor antagonists will also be discussed.
obtained
from
readily
available
a,20,40-tri-
chloroacetophenone in 65% yield. Compound 11a was
heated for two h with ethyl acetoacetate (2 equiv), in the
presence of catalytic amount of toluenesulfonic acid, in
benzene with a Dean–Stark water trap to remove water.
The crude enamine 14a was used directly, after concen-
tration in vacuo, for cyclization. Thus, 14a was added
into a hot phenyl ether solution at 240 ꢂC for 10 min to
give the pyrazolo[3,4-b]pyridone 15a as a brown solid.
Conversion of the pyridone 15a to the corresponding
chloropyrazolo[3,4-b]pyridine 16a was accomplished by
refluxing compound 15a in POCl3 for 2 h. The amine
replacement reaction proceeded in an excess amount of
Reaction of a-phthaloylaminoacetophenone derivatives
6, obtained from a-bromo- or a-chloro-acetophenones
and potassium phthalimide in DMF or ethanol,16 with
N,N-dimethylformamide dimethyl acetal (DMFDMA)
proceeded smoothly at reflux (with or without DMF as
solvent) to give the crude enamines 7 in quantitative
yields after concentration in vacuo. The enamines 7

K. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3367–3370
Table 2. SAR of the substituents on the 3-phenyl group
3369
primary or secondary amine, promoted by toluene-
sulfonic acid at 160 ꢂC in a sealed reacti-vial for 6 h, to
give the desired pyrazolo[3,4-b]pyridine 17a. Finally,
alkylation of 17 with alkyl halide in the presence of
sodium hydride in DMF gave two isomers 18 and 19,
which were separated by chromatography on silica gel
(Scheme 4). Their isomeric structures were assigned
based on proton NMR analyses as described before and
confirmed by NOE NMR experiments. This synthetic
route was not optimized but is very efficient for the
rapid generation of 18 and 19 analogues from readily
available a-chloro- or a-bromoacetophenones. Alter-
natively, compounds 18 and 19 were also prepared from
the corresponding 1-alkyl-4-aminopyrazoles 12 and 13,
respectively, according to a similar procedure described
above.
Compd
R
X
R1NR2
Ki (nM)
19d
19j
19k
19l
19m
19n
19o
19p
Me
2,4-Cl2
2,4-Cl2
4-Cl
Pr2N
BuNCH2CH2PhOMe-4
Pr2N
4.4ꢃ2.0
80
81.5ꢃ25.5
H
Pr2N
Bu2N
Bu2N
c-PrCH2NPr
Bu2N
230
27.5ꢃ7.5
70
2,4,6-Me3
2-Cl-4-MeO
2-Cl-4-Me
2-Cl-4-Me
Et
32.0ꢃ4.0
59
The desired compounds 17, 18, and 19 were assayed on
the binding affinities towards the human CRF1 receptor
stably express on HEK273 cells,18 and the results are
summarized in Table 1 and Table 2. The 1H-pyr-
azolo[4,3-b]pyridines 17a–c showed only moderate
binding affinity (Ki 26–60 nM), and they were much less
active than the corresponding pyrazolo[1,5-a]pyrimidine
analogues such as 2a. One explanation for the decrease
in binding affinity could be that the polar nature of the
NH functionality of 17a may be harmful to receptor
binding. The missing methyl group corresponding to the
2-position of pyrazolo[1,5-a]pyrimidine 2a (Ki=1 nM)
could also account for the 10-fold loss in activity. The
2-alkyl-pyrazolo[4,3-b]pyridines 19, especially the
2-methyl analogue 19d which had the same geometry as
2a, were much more polar than the corresponding
1-alkyl-isomers 18 and pyrazolo[1,5-a]pyrimidine 2 on
silica gel. Thus, methylation (19b) at the 2-position of
17b (Ki=26 nM) only slightly increased binding affinity,
whereas ethylation (19f) hardly changed the binding
(19f vs 17b). Alkylation of 17c with propyl or isopropyl
group (19h and 19i) also had minimal impact on the
binding affinity. The best compound from this subseries,
the dipropylamino derivative 19d, had a Ki value of 4.4
nM, which is comparable to the corresponding pyr-
azolo[4,3-d]pyrimidine analogue 5 (Ki=3 nM), but less
active than the pyrazolo[1,5-a]pyrimidine 2a (Ki=1.3
nM).
Scheme 4. Reagents and conditions: (a) RX, NaH, DMF, rt.
The 1-alkyl pyrrazolo[4,3-b]pyridine derivatives 18,
although they were less polar than their corresponding
isomers 19, were still much more polar on silica gel than
Table 1. Comparison of binding affinity between 1-alkyl and 2-
alkylpyrazolo[4,3-b]pyridines
the pyrazolo[1,5-a]pyrimidines
2
and exhibited
increased binding affinity. Thus, 18b and 18c were 15-
and 30-fold, respectively, more active than 17b and
17c; and 18a with a Ki value of 0.62 nM was 65-fold
better than 17a. The dipropylamino derivative 18d
(Ki=1.8 nM) had comparable activity with the corre-
sponding 1-methylpyrazolo[4,3-d]pyrimidine 4 (Ki=1
nM) and pyrazolo[1,5-a]pyrimidine 2a (Ki=1.3 nM).
The ethyl derivatives 18e, 18f and 18g were slightly less
active (<3-fold) than the corresponding methyl analo-
gues 18a–c. Interestingly, the bulky isopropyl com-
pound 18i (Ki=16 nM) was only 25-fold less active than
the optimal methyl analogue 18a, but the longer propyl
derivative 18h (Ki=180 nM) lost almost 300-fold activ-
ity (Table 1).
Compd
R
H
R1NR2
Ki (nM)
17a
17b
17c
18a
18b
18c
18d
18e
18f
18g
18h
18i
19a
19b
19c
19d
19e
19f
19g
19h
19i
Bu2N
c-PrCH2NPr
BuNEt
Bu2N
c-PrCH2NPr
BuNEt
Pr2N
40
26
60
Me
Et
0.62ꢃ0.35
1.7ꢃ0.7
2.0ꢃ0.2
3.3ꢃ1.5
2.1ꢃ0.2
1.7ꢃ0.7
4.2ꢃ0.7
180
16.1ꢃ1.0
32.5ꢃ5.7
11.0ꢃ4.8
35.0ꢃ5.0
4.4ꢃ2.0
32.8ꢃ4.0
46
Bu2N
c-PrCH2NPr
BuNEt
Bu2N
Bu2N
Bu2N
c-PrCH2NPr
BuNEt
Pr2N
Pr
i-Pr
Me
The effects of the substitution on the 3-phenyl group of
the 2-methylpyrazolo[4,3-b]pyridines 19 were also
investigated and the results are summarized in Table 2.
Similar to the pyrazolo[1,5-a]pyrimidine series, the
2,4-dichlorophenyl is the most favored group at this
position. In the current series, loss of the 4-chlorine of
the 2,4-dichlorophenyl group of 19d resulted in almost
20-fold decrease in binding affinity (19k, Ki=81 nM).
Et
Bu2N
c-PrCH2NPr
BuNEt
Bu2N
32
64
37
Pr
i-Pr
Bu2N

3370
K. Wilcoxen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3367–3370
The un-substituted phenyl analogue 19l had a Ki value
of 230 nM, much less active than 19d. The 2,4,6-tri-
methylphenyl derivative 19m (Ki=28 nM) was similarly
active as 19a, but the 2-chloro-4-methoxyphenyl was
not able to mimic the 2,4-chlorophenyl group and the
resulting compound 19n (Ki=70 nM) was 3-fold less
active than 19a. Other than 2,4,6-trimethylphenyl ring,
the 2-chloro-4-methylphenyl group was a reasonably
good replacement of the 2,4-dichlorophenyl ring (19o
and 19b, Ki=22 and 11 nM, respectively).
Schmidt, A. W.; Seeger, T.; Seymour, P.; Tingley, F. D., III;
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Wing, L.; Grigoriadis, D. E.; De Souza, E. B.; McCarthy, J. R.
Book of Abstracts, 217th American Chemical Society National
Meeting, Anaheim, CA, USA, March 21–25, 1999; MEDI
002.
4. (a) Wustrow, D. J.; Capiris, T.; Rubin, R.; Knobelsdorf,
J. A.; Akunne, H.; Davis, M. D.; MacKenzie, R.; Pugsley,
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Chem. Lett. 1998, 8, 2067. (b) Gilligan, P. J.; Baldauf, C.;
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Trainor, G.; Robertson, D. W.; Hartig, P. Bioorg. Med. Chem.
2000, 8, 181.
In general, the 1-alkyl-pyrazolo[4,3-b]pyridines 18 were
much more active than the corresponding 2-alkyl-iso-
mers 19, and the best compound from isomers 19 had a
Ki value of 4.4 nM (19d) on binding to the CRF1
receptor. The high polarity, which may contribute to the
lower binding affinity, is certainly an advantage for
designing CRF1 antagonists with desirable pharmaco-
kinetic profile and eventually in vivo efficacy.
5. Keller, C.; Bruelisauer, A.; Lemaire, M.; Enz, A. Drug
Metab. Dispos. 2002, 30, 173.
6. Chen, C.; Wilcoxen, K.; Bozigian, H.; Chen, T.; Cha, M.;
McCarthy, J. R.; Huang, C. H.; Haddach, M.; Zhu, Y. F.;
Murphy, B.; De Souza, E. B.; Grigoriadis, D. E. Book of
Abstracts, 221st ACS National Meeting, San Diego, April 1–5,
2001; MEDI 214.
7. Huang, C. Q.; Haddach, M.; Chen, C.; Wilcoxen, K. M.;
Xie, Y.; Grigoriadis, D. E.; McCarthy, J. R. Book of
Abstracts, 215th American Chemical Society National Meet-
ing, March 15, 1999, Anaheim, CA, USA. MEDI 002.
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12. The calculated pKa value obtained from ACD/Labs 6.00
software, 2002, Advanced Chemistry Development, Inc. The
calculated pKa values were 9.26 for 4-aminopyridine and 9.0
for 4-aminoquinoline, respectively.
13. Tarzia, G.; Panzone, G.; Depaoli, A.; Schiati, P.; Selva, D.
Farmaco Ed. Sci. 1984, 39, 618.
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Selective compounds from this series were further tested
for functional antagonism on the CRF1 receptor. Thus,
in a CRF-stimulated c-AMP production assay, com-
pounds 18a, 18b, and 18c inhibited c-AMP accumula-
tion with low nanomolar IC50 values (15.8, 5.0, and 16
nM, respectively), while compounds 19d was less active
(IC50=365 nM). All compounds were examined for
activity in a CRF2-receptor binding assay as previously
described19 and none of the listed compounds showed a
does-dependent inhibition of binding and none had a
greater than 40% inhibition at a concentration of 10
mM. These data demonstrate these compounds are
selective CRF1 antagonists.
In conclusion, condensation of a phthaloylaminoaceto-
phenone derivatives 6 with N,N-dimethylformamide
dimethyl acetal gave the enamines 7, which was cyclized
with hydrazine to give the pivotal intermediate 4-amino-
3-arylpyrazoles 10, 11, and 12. Application of this key
intermediate towards the synthesis of pyrazolo[4,3-
b]pyridines 17–19 resulted in the discovery of a class of
very potent and relatively more polar (most likely more
basic) CRF1 receptor antagonists. Isomers 19, which
were more polar on silica gel chromatography, were less
active than the corresponding 18 subseries. Isomers 18
possessed a basic core structure (calculated pKa value
for 18d was 10.4, ACD) and displayed excellent
potency. The optimization of 18 will be reported in the
following paper.
16. (a) Sheehan, J. C.; Bolhofer, W. A. J. Am. Chem. Soc.
1950, 72, 2786. (b) Gabriel, S. Chem. Ber. 1908, 41, 1132.
17. Chen, C.; Wilcoxen, K.; McCarthy, J. R. Tetrahedron
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18. Chen, C.; Dagnino, R., Jr.; De Souza, E. B.; Grigoriadis,
D. E.; Huang, C. Q.; Kim, K. I.; Liu, Z.; Moran, T.; Webb,
T. R.; Whitten, J. P.; Xie, Y. F.; McCarthy, J. R. J. Med.
Chem. 1996, 39, 4358.
Acknowledgements
We like to thank Mr. Zhengyu Liu for technical sup-
ports of this work.
References and Notes
19. For a CRF2 binding assay see: Grigoriadis, D. E.; Liu,
X. J.; Vaughn, J.; Palmer, S. F.; True, C. D.; Vale, W. W.;
Ling, N.; De Souza, E. B. Mol. Pharmacol. 1996, 50, 679.
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