Synthesis of 1-methyl-3-phenylpyrazolo[4,3-b]pyridines via a methylation of 4-phthalimino-3-phenylpyrazoles and optimization toward highly potent corticotropin-releasing factor type-1 antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00622-X

1-Methyl-3-phenylpyrazolo[4,3-b]pyridines were synthesized via a cyclization reaction of 1-methyl-4-amino-3-phenylpyrazoles 8 with ethyl acetoacetate. Optimization of this series of compounds resulted in CRF ₁ antagonists with subnanomolar binding affinity. Compounds bearing a polar group such as methoxy or hydroxy were also found to be very active.

Full text of DOI:10.1016/S0960-894X(03)00622-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3371–3374
Synthesis of 1-Methyl-3-phenylpyrazolo[4,3-b]pyridines Via a
Methylation of 4-Phthalimino-3-phenylpyrazoles and
Optimization toward Highly Potent Corticotropin-Releasing
Factor Type-1 Antagonists
Charles Q. Huang, Keith Wilcoxen, James R. McCarthy,
Mustaph Haddach, Dimitri 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—1-Methyl-3-phenylpyrazolo[4,3-b]pyridines were synthesized via a cyclization reaction of 1-methyl-4-amino-3-phe-
nylpyrazoles 8 with ethyl acetoacetate. Optimization of this series of compounds resulted in CRF1 antagonists with subnanomolar
binding affinity. Compounds bearing a polar group such as methoxy or hydroxy were also found to be very active.
# 2003 Elsevier Ltd. All rights reserved.
In the preceding paper1 we described the synthesis and
initial SAR studies of a series of 3-phenylpyrazolo[4,3-
b]pyridines (1, 2, and 3, Fig. 1) as corticotrophin-
releasing factor type-1 antagonists, on the assumption
that this series of compounds may be more polar than
3-phenylpyrazolo[1,5-a]pyrimidines which we developed
earlier and suffered, like many other known CRF1
antagonists, high lipophilicity. Because the pyrazolo-
fused 4-aminopyridine is similar to 4-aminoquinoline
(pKa=9.08),2 it may offer a core with high basicity
(estimated pKa>7.8),3 which is desirable for com-
pounds with better pharmacokinetic profile. Although
isomers 3 are more polar than 2, compounds from this
subseries were somewhat less active than 2. Because of
the lack of highly active compounds 3 from our initial
SAR, we turn our attention to the 1-alkylpyrazolo[4,3-
b]pyridines 2 in our efforts to discover higher hydro-
philic CRF1 antagonists. Here we report the detailed
SAR and optimization of physicochemical properties on
this subseries of compounds.
cyclization of enamines 4 with methylhydrazine sulfate
in a mixture of solvents (EtOH/H2O=10:1) at reflux
gave 1-methyl-3-phenyl-4-phthalimidopyrazoles 7 and
1-methyl-5-phenyl-4-phthalimidopyrazoles 6 as mix-
tures with a ratio ranging from 3:1 to 3:2. The two iso-
mers 6 and 7 were easily separated by chromatography
on silica gel. Alternatively, 4-phthalimidopyrazolo[4,3-
b]pyrimidines 5, which were obtained from the cycli-
zation of enamines 4 with hydrazine hydrochloride in
refluxing aqueous ethanol (EtOH–H2O, 10:1) in
85–95% yields, were alkylated with alkyl halide in dry
DMF promoted by sodium hydride to give the corre-
sponding alkyl analogues 7. This regio-selective proce-
dure avoided the formation of 6 as a by-product from
the alkylhydrazine cyclization. Compounds 7 were then
deprotected with two equivalents of hydrazine in
refluxing ethanol to give the 4-aminopyrazoles 8 which
were converted to the desired 1-alkyl-3-phenyl-7-chloro-
pyrazolo[4,3-b]pyridines 9 as described in Scheme 2.
Compounds 9 were subjected to a nucleophilic replace-
ment with various alkyl amines to give the desired
products 11. By using the above reaction, we also
introduced amines bearing a hetero-atoms. Thus reac-
tions of 9 with primary amines such as 3-methoxy-
propylamine under the similar conditions gave
compounds 10, which were alkylated with alkyl halide
in DMF to give the tertiary amine products 12.
The synthesis of 1-alkyl-3-phenylpyrazolo[4,3-b]pyridines
described in Scheme 1 is based on the new synthetic
method for 4-phthalimido-3-phenylpyrazoles 5.4 Thus,
*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)00622-X

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C. Q. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3371–3374
Table 1. Effects of some small lipophilic alkyl group
Compd
R1NR2
Ki (nM)
Figure 1. 1-Methyl- and 2-methyl-3-phenyl-7-aminopyrazolo[4,3-b]-
pyridines 2 and 3.
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
n-BuNH
n-PrNMe
n-PrNEt
c-PrCH2NEt
(n-Pr)2N
170
24Æ9
4.1Æ2.0
4.4Æ0.8
3.3Æ1.5
1.7Æ0.7
1.3Æ0.6
3.7Æ1.2
1.5Æ0.5
1.0Æ0.1
2.0Æ0.2
0.62Æ0.35
1.3Æ0.7
1.2Æ1.2
0.9Æ0.1
7.0Æ3
c-PrCH2NPr-n
c-PrCH2NBu-n
c-PrCH2NBu-i
c-PrCH2N(C5H11)-n
c-PrCH2N(C6H13)-n
n-BuNEt
n-Bu2N
n-(C5H11)NEt
n-(C5H11)NPr-n
n-(C5H11)NBu-n
(Et)2CHNBu
Table 2. Effects of seven-side chain with a polar group
.
Scheme 1. Reagents and conditions: (a) RNHNH2 H2SO4, EtOH–
.
H2O, reflux; (b) NH2NH2 HCl, EtOH–H2O, reflux; (c) RX, NaH,
DMF, rt; (d) NH2NH2, EtOH, reflux.
Compd
R1NR2
Ki (nM)
12a
12b
12c
12d
12e
12f
12g
12h
12i
MeOCH2CH2NMe
MeOCH2CH2NEt
MeOCH2CH2NPr-n
MeOCH2CH2NPr-i
MeOCH2CH2NBu-n
MeOCH2CH2NBu-i
MeOCH2CH2N(C5H11)-n
MeOCH2CH2N(C6H13)-n
(MeOCH2CH2)2N
MeOCH2CH2CH2NEt
MeOCH2CH2CH2NPr-n
MeOCH2CH2CH2NBu-n
MeSCH2CH2NBu-n
32Æ11
3.2Æ0.8
5.1Æ0.8
9.7Æ3.0
10.4Æ0.5
10.5Æ0.5
6.7Æ0.6
4.0Æ1.2
8.2Æ0.7
12.5Æ1.1
9.8Æ3.2
18Æ4
Scheme 2. Reagents and conditions: (a) (i) ethyl acetoacetate, ben-
zene, reflux; (ii) Ph2O, 260 ꢀC; (iii) POCl3, reflux; (b) R1R2NH, TsOH,
140 ꢀC; (c) R2X, NaH, DMF, rt.
12j
12k
12l
The binding affinity of the compounds prepared were
determined as CRF1 antagonists by using a binding
assay reported by Grigoriadis, and using [125I]-sauva-
gine from CRF1 receptor expressed in HEK293 cells.5
The effects of alkyl substituents at the 7-position of the
1-methyl-3-(2,4-dichlorophenyl)-5-methyl-7-aminopyr-
azolo[4,3-b]pyridines on the binding to the CRF1
receptor were summarized in Table 1 and 2.
12m
1.4Æ0.2
12n
12o
11.2Æ2.8
380
The mono-butylamino derivative 11a (Ki=170 nM) was
found to be only moderately active, but N-methyl-N-
propylamino analogue (11b, Ki=24 nM) was much
improved. Increasing the chain length from ethyl to
hexyl resulted in a gain in binding affinity (0.62–4.4
nM), and the dibutylamino (11l) was optimal with a
Ki value of 0.62 nM. Interestingly, this result was
slightly different from the pyrazolo[1,5-a]pyrimidines,
in which dipropylamine or N-butyl-N-ethylamine is
the optimal side chain.6 The requirement of a more
lipophilic group for optimal binding indicates that the
more basic pyrazolo[1,5-a]pyrimidine core needs to be
off-set with a more hydrophobic group. This hypo-
thesis was further supported by compounds 11m–o
(Ki’s=0.9-1.3 nM) that bear a highly lipophilic group
12p
12q
12r
12s
12t
12u
12v
4-PyCH2NBu-n
4-PyCH2CH2NBu-n
2-PyCH2CH2NBu-n
4-HOPhCH2NH
4-HOPhCH2NPr-n
HOCH2CH2NPr-n
HOCH2CH(n-Pr)NH
68Æ11
34Æ13
13Æ2
>10,000
13Æ8
4.4Æ0.9
62
such as N-butyl-N-pentylamine. Only the branched
2-pentyl derivative 11p was less active (Ki=7 nM).
Although compounds from this series had high binding
affinity, they were still very lipophilic (calculated logD

C. Q. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3371–3374
3373
value for 11e was 4.0, ACD software). In order to
reduce the lipophilicity of this series of compounds, a
reduction of logP/logD at least one log unit is desirable.
This means an introduction of a hydroxyl or at least a
methoxy group. The results from compounds bearing a
hetero-atom are summarized in Table 2. The N-methyl-
N-methoxyethylamino compound (12a, Ki=32 nM) had
comparable affinity to the N-methyl-N-propyamine 11b
(Ki=24 nM); similarly the N-ethyl-N-methoxy-
ethylamine 12b had a Ki value of 3.2 nM, close to the N-
ethyl-N-propyl analogue (11c, Ki=4.1 nM). Unfortu-
nately, increasing the length of this alkyl chain did not
further increase activity. Instead, a decrease in binding
affinity for the butyl or the isobutyl group (12e and 12f
Ki=10.4 and 10.5 nM, respectively) was observed. Sub-
stitution with the polar bis(methoxyethyl)amino group
resulted in compound 12i (Ki=8.2 nM) with less activ-
ity. N-Methoxypropyl-N-alkylamines (12j–l) were 2- to
4-fold less active (10–18 nM) than the 2-methox-
yethylamino analogues, and N-(tetrahydrofuranmethyl)-
N-butylamine (12n, Ki=11 nM) was also less active. As
we expected, 12o, which bears a basic pyrrolidine-
methylamino side chain, was much less active in binding
affinity (Ki=380 nM). Attempt to incorporate a less
basic pyridine moiety into the side chain also was not
very successful, and compounds 12p–r all had Ki values
of >10 nM. Although the N-methylthioethyl-N-butyl-
amine 12m was very potent (Ki=1.4 nM), this com-
pound might not be polar enough. However, the N-
(hydroxyethyl)-N-propylamine 12u had a Ki value of 4.4
nM, while the 1-hydroxy-2-pentylamine analogue (12v,
Ki=62 nM) was not very active. Another interesting
compound is 12t that had an acidic phenol moiety and
still had a Ki value of 13 nM, much better than its des-
propyl analogue 12s (inactive).
Table 4. CRF functional antagonistic activitya of selected compounds
Compd
Ki (nM)
IC50 (nM)
11f
11g
11k
11l
12b
12c
12i
1.7Æ0.7
1.3Æ0.6
2.0Æ0.2
0.62Æ0.35
3.2Æ0.8
5.1Æ0.8
8.2Æ0.7
4.4Æ0.9
16
3.0
16
5.0
15
36
38
18
12u
aInhibition of CRF-stimulated cAMP production.
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 11f, 11g, 11k, 11l, 12b, and 12u displayed low
nanomolar IC50 values while compounds 12c and 12i
were slightly less active (Table 4). These data seems to
be in agreement with the binding affinity which mea-
sures the interaction between the small molecule ligands
and the CRF1 receptor. All compounds were examined
for activity in a CRF2-receptor binding assay as pre-
viously described7 and none of the listed compounds
showed a does-dependent inhibition of binding and
none had a greater than 40% inhibition at a concen-
tration of 10 mM. These data demonstrate these com-
pounds are selective CRF1-selective antagonists.
In conclusion, we developed a regio-selective synthesis
of
1-alkyl-3-phenyl-7-aminopyrazolo[4,3-b]pyridines.
These compounds were tested as CRF1 antagonists and
many of them were highly potent. This core structure,
comparing with 4-aminoquinoline (pKa=9.1), should be
more basic than the previous pyrazolo[1,5-a]pyrimidine,
pyrazolo[4,3-d]pyrimidine and pyrrolo[2,3-d]pyrimidine,
therefore, more hydrophilic. Compound 2l with a dibu-
tylamino side chain had a Ki value of 0.62 nM, but was
still quite lipophilic (calculated values for pKa 10.4,
logP>6 and logD>4, ACD software). However, the
hydroxy and methoxy analogues 12b and 12u, despite of
slightly decreased binding affinity (Ki ꢁ4 nM) and
functional antagonistic activity (cAMP IC50 ꢁ15 nM),
possess desirable physicochemical properties (calculated
values for 12b: logP 4.3, logD 2.7; for 12u: logP 4.2,
logD 1.8, ACD software), which are required for good
PK profiles and in vivo efficacy. Results for further
studies of these compounds will be reported elsewhere in
due course.
We also examined the possible alternatives for the
3-(2,4-dichlorophenyl) group (Table 3). While the
2-chloro-4-methoxy- and 2,4,6-trimethylphenyl replace-
ments (11q and 11t) resulted in compounds with about
8-fold less active; 2-chloro-4-methyl, especially 2-chloro-
4,6-dimethylphenyl analogues (11r and 11s) had com-
parable binding affinity. On the other hand, the
4-chlorophenyl compound was significantly less active
(11u, Ki=160 nM) in binding affinity.
Table 3. Substitution effects on the 3-phenyl group
References and Notes
1. Wilcoxen, K.; Huang, C. Q.; McCarthy, J. R.; Grigoriadis,
D.; Chen, C. Bioorg. Med. Chem. Lett. See preceding paper.
doi: 10.1016/S0960-849X(03)00621-8.
2. (a) Brown, H. C. Determination of Organic Structure by Phy-
sical Methods; In Braude, E. A., Nachod, F. C. Eds; Academic:
New York, 1955. (b) Hawley, S. R.; Bray, P. G.; O’Neill, P. M.;
Park, B. K.; Ward, S. A. Biochem. Pharmacol. 1996, 52, 723.
3. ACD/Labs 6.00, 2002, Advanced Chemistry Development,
Inc.
Compd
X
Ki (nM)
11l
11q
11r
11s
11t
11u
2,4-Cl2
2-Cl-4-MeO
2-Cl-4-Me
2-Cl-4,6-Me2
2,4,6-Me3
4-Cl
0.62Æ0.35
4.8Æ0.9
2.8Æ0.9
2.1Æ0.7
5.1Æ0.2
340
4. Chen, C.; Wilcoxen, K.; McCarthy, J. R. Tetrahedron Lett.
1998, 39, 8229.

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C. Q. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3371–3374
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6. (a) Wustrow, D. J.; Capiris, T.; Rubin, R.; Knobelsdorf, J. A.;
Akunne, H.; Davis, M. D.; MacKenzie, R.; Pugsley, T. A.; Zoski,
K. T.; Heffner, T. G.; Wise, L. D. Bioorg. Med. Chem. Lett. 1998,
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D. W.; Hartig, P. Bioorg. Med. Chem. 2000, 8, 181.
7. 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.