Optimising metabolic stability in lipophilic chemical space: The identification of a metabolically stable pyrazolopyrimidine CRF-1 receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.09.016

Balancing potency and metabolic stability in a target which favours lipophilic ligands is a considerable challenge. Here we describe two strategies employed to achieve this balance in a series of pyrazolopyrimidine CRF antagonists: moderation of lipophili

Full text of DOI:10.1016/j.bmcl.2009.09.016

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6144–6147
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimising metabolic stability in lipophilic chemical space: The identification
of a metabolically stable pyrazolopyrimidine CRF-1 receptor antagonist
*
Duncan C. Miller , Wolfgang Klute, Andrew Calabrese , Alan D. Brown
Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2009
Revised 3 September 2009
Accepted 4 September 2009
Available online 11 September 2009
Balancing potency and metabolic stability in a target which favours lipophilic ligands is a considerable
challenge. Here we describe two strategies employed to achieve this balance in a series of pyrazolopyr-
imidine CRF antagonists: moderation of lipophilicity, and incorporation of a metabolically stable lipo-
philic group.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antagonist
Corticotrophin releasing factor 1
Metabolic stability
G-protein coupled receptor
Corticotrophin Releasing Factor (CRF) is a 41 amino acid peptide
which is the primary modulator of the HPA (hypothalamic–pitui-
tary–adrenal) axis. CRF is the endogenous ligand for two receptors,
CRF-1 and CRF-2, both of which belong to the class B subfamily of
G-Protein Coupled Receptors (GPCRs).1 CRF-1 antagonists have
historically been pursued primarily as potential treatments for
anxiety and depression.2 However reports have also implicated
CRF in female sexual behaviour.3 We hypothesised that an antago-
nist of the CRF-1 receptor may have utility in the treatment of
female sexual dysfunction.
pound was still highly lipophilic with poor in vitro metabolic sta-
bility in human liver microsomes.13
SAR for CRF-1 potency in chemotypes containing a bicyclic core
has been well described in the literature.4
N
H
HN
N
O
N
N
Several different classes of small molecule CRF-1 antagonists
have been reported in the literature.4 Representative examples
are shown in Figure 1. While there are examples of CRF-1 antago-
nists which have been studied in the clinic including R1219195 (3),
Pexacerfont6 (4) and Emicerfont7 (5), attempts to identify CRF-1
antagonists suitable for clinical development have often been ham-
pered by the apparent intolerance of the small molecule binding
site to polar functionality, generally resulting in high lipophilicity
for reported small molecule antagonists.8 This high lipophilicity re-
duces the chance of achieving a suitable pharmacokinetic profile.9
The lead compound for our studies was selected from a review
of substrate from previous CRF-1 antagonist programs at Pfizer.
Compound 610, a close analogue of the extensively studied pyrrol-
opyrimidine CRF-1 antagonist Antalarmin,11 was selected as a
promising lead due to its high potency12 and relatively heteroatom
rich, polar pyrazolopyrimidine core (Fig. 2). However this com-
N
N
N
OMe
N
OMe
OMe
2
1 DMP-904
NH
O
N
N
NH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NMe2
OMe
OMe
* Corresponding author. Tel.: +44 1304 640026.
5 Emicerfont
3 R121919
4 Pexacerfont
E-mail address: duncan.miller@pfizer.com (D.C. Miller).
Celgene, 4550 Towne Centre Court, San Diego, CA 92121, USA.
Figure 1. Representative examples of reported CRF-1 antagonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.016

D. C. Miller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6144–6147
6145
Table 1
In vitro functional potency, microsomal stability and clogP for compounds 7–15
N
4
3
N
1
5
MeO
N
6
2
N
N
7
N
N
N
N
N
Ar
6 CRF1 Ki 9nM clogP 6.75
HLM Clint >320 l/min/mg
Compd
Ar
Ki (nM)
HLM
172
RLM
23
clog P
Figure 2. Structure and in vitro functional potency, microsomal stability and clog P
for Compound 6.
7
13
4.8
OMe
OMe
In an attempt to identify a novel CRF-1 antagonist with good
pharmacokinetic parameters we chose to screen all analogues for
stability in human and rat liver microsomes in parallel with gener-
ating CRF-1 activity data, enabling us to track SAR and physico-
chemical trends for microsomal stability alongside those for
CRF-1 activity.
Our initial design strategy targeted reducing lipophilicity with
the aim of improving the metabolic stability of this template
through incorporation of more polar substituents at the 1- and 4-
positions.
We chose to first explore SAR at the 1-substituent while keep-
ing the 4-substituent constant. For these studies we selected N-
propyl-N-methoxyethylamine as as a less lipophilic replacement
of the N-ethyl-N-butylamine substituent at the 4-position of the
pyrazolopyrimidine template as it has previously been reported
as retaining good CRF-1 potency when incorporated into this posi-
tion in several bicyclic CRF-1 antagonist templates4 (Table 1).
Introduction of a 2-methyl-4-methoxyphenyl substituent into
the 1-position of 6,5 bicyclic systems has been widely reported to
give good CRF-1 potency.4 Its introduction into the pyrazolopyrimi-
dine template to give 7 did retain good CRF-1 activity and also
encouraging microsomal stability in rat. However stability in human
microsomes was poor.14 Substituting the phenyl ring with other po-
lar substituents (8–11) resulted in reduced potency which broadly
tracked with the reduction in lipophilicity. Despite this reduction
in lipophilicity only 11 showed a significant improvement in both
rat and human microsomal stability. Incorporation of substituted
pyridines with precedented activity in other 6,5 templates4,6 either
failed to improve microsomal stability (12–14) or in the case of 15
improvedstabilityinbothratandhumanmicrosomesattheexpense
of a significant reduction in CRF-1 activity.
These results suggested that further tuning the lipophilicity of
the template through introduction of polarity in the 1-substituent
was unlikely to lead to potent compounds with sufficient micro-
somal stability.
Our attention thus turned to exploring SAR at the 4-position.
We elected to explore SAR at this position with the 2-methyl-4-
methoxyphenyl substituent in place with the aim of identifying a
substituent at the 4-position which would improve human micro-
somal stability while retaining the encouraging potency and rat
stability of compound 7 (Table 2).
Reducing lipophilicity with both tertiary (16–24) and secondary
amines (25–30) uniformly resulted in a significant reduction in po-
tency. Secondary amines were generally more stable than tertiary
amines with similar lipophilicity (e.g., 18 vs 27 and 28).
At this point it appeared that the strategy of reducing lipophil-
icity through modifications to the 1- and 4-substituents would not
deliver a compound with the desired balance of properties. As an
alternative strategy two higher lipophilicity analogues, 31 and
OMe
8
9
74
101
632
275
86
147
151
17
90
4.4
4.1
3.1
3.5
<11
166
22
CN
CN
CN
10
11
CONH2
12
13
14
15
43
17
117
218
160
38
163
159
206
13
4.1
4.0
4.1
2.8
N
OMe
OMe
N
OMe
183
577
N
NMe2
N
CONH2

6146
D. C. Miller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6144–6147
Table 2
Table 3
In vitro functional potency, microsomal stability and clog P for compounds 7 and 16–
Comparison of pairs of compounds differing only at the 3-position (R4)
32
R2R3N
R2R3N
R4
N
N
N
N
N
N
N
N
OMe
OMe
NR2R3
R4
Ki (nM)
HLM
RLM
clog P
Compd
NR2R3
Ki (nM)
HLM
RLM
clog P
Compd
7
16
17
18
19
N-(nPr)(CH2)2OMe
N-(Et)(CH2)2OMe
N(CH2CH2OMe)2
N(nPr)(CH2CH2OH)
N(nPr)CH2CONMe2
13
117
193
129
1409
172
117
66
80
18
23
104
47
117
22
4.8
4.3
3.8
4.1
4.2
7
33
25
34
32
35
N(nPr)(CH2CH2OMe)
N(nPr)(CH2CH2OMe)
NCH(Et)2
NCH(Et)2
NHCH(cPr)2
Me
H
Me
H
Me
H
13
132
42
85
35
172
16
64
15
<12
<11
23
387
72
246
24
4.8
4.6
5.4
5.1
5.2
5.0
NHCH(cPr)2
32
25
Pr
Pr
Pr
O
N
20
21
22
23
996
84
34
51
29
96
40
66
58
67
3.3
4.2
4.2
4.4
N
N
3-methyl group was much less severe (25 vs 34 and 32 vs 35). We
believe that this observation may be due to the differences in confor-
mational preference between secondary and tertiary amines at this
position. Secondary amines can adopt a coplanar conformation with
the lone pair of the exocyclic sp2 nitrogen in conjugation with the pi
system of the bicyclic heterocycle (Fig. 3, compound 34). In this con-
formation the branched alkyl groups are able to occupy the proposed
binding pockets in the CRF-1 receptor above and below the plane of
thecore. Thepresenceorabsenceofthe3-methylgroupis unlikelyto
affect this conformation resulting in minimal potency difference.
Due to steric clash with the 3-methyl group the preferred conforma-
tion for tertiary amines is the non-conjugated form (Fig. 3, com-
pound 7) which again allows the alkyl groups to occupy the
proposed binding pockets. Substitution of the 3-methyl group for a
hydrogenatom reducesthis steric clash allowingthe exocyclicnitro-
gen to access a low energy coplanar conjugated conformation (Fig. 3,
compound 33).
N
N
N
O
N
N
124
400
O
N
Pr
Pr
N
N
N
N
N
24
2500
196
85
4.4
N
25
26
27
28
29
30
31
32
(S)-NHCH(CH2OMe)Et
(R)-NHCH(CH2OMe)Et
(S)-NHCH(nPr)CH2OH
(R)-NHCH(nPr)CH2OH
(S)-NHCH(CH2OH)Et
(R)-NHCH(CH2OH)Et
NHCH(Et)2
135
210
76
574
620
1130
42
52
37
23
10
<7
<7
64
<12
105
53
41
36
27
23
72
24
4.3
4.3
4.1
4.1
3.6
3.6
5.4
5.2
The use of the C,C-dicyclopropylmethylamine substituent pres-
ent in our most promising compounds (32 and 35) has previously
been reported in CRF-1 antagonists.15 In the reported triazole tem-
plate this group suffered from significant acid instability. However,
in accelerated solution stability studies 35 showed good stability
NHCH(cPr)2
35
32, were prepared lacking the potentially metabolically vulnerable
methoxy and hydroxyl groups present in the earlier secondary
amino analogues. These compounds both retained good CRF-1 po-
tency. However despite being effectively isolipophilic they had
markedly different microsomal stability profiles with 32 exhibiting
much greater microsomal stability in both rat and human systems
than 31. The microsomal stability of 32 was also superior to the
more polar analogues prepared in this series.
With the relatively lipophilic C,C-dicyclopropylmethylamine
identified as giving the best balance of CRF-1 potency and micro-
somal stability at the 4-position our attention now turned to iden-
tifying other areas of the template where we might be able to
moderate lipophilicity. Analysis of the reported bicyclic CRF-1
antagonists revealed that the methyl at the 6-position is generally
required for CRF-1 activity, whereas templates lacking the methyl
at the 3-position often retain CRF-1 activity. In an attempt to fur-
ther reduce the lipophilicity analogues with the 3-methyl deleted
were prepared (Table 3).
Figure 3. Minimised conformations of 34, 7 and 33.
When the 4-substituent was a tertiary amine deletion of the 3-
methyl group resulted in an increase in human microsomal stabil-
ity, albeit at the expense of a 10-fold reduction in CRF-1 activity
(33 vs 7).
Intriguingly, when the 4-substituent was a secondary rather than
a tertiary amine, the reduction in potency for deletion of the
Table 4
In vivo rat pharmacokinetic parameters for compound 35 iv dose 0.1 mg/kg, po dose
5 mg/kg
Compd
Cl (ml/min/kg)
27
Vd (L/kg)
Fu
T1/2 (h)
F (%)
35
7
0.02
1.9
43

D. C. Miller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6144–6147
6147
R1
N
N
Ar
References and notes
R1
H2NOC
i
iii
iv
NC
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R1
ii
CN
H2N
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R2R3N
R1
Cl
N
v
N
N
N
N
N
Ar
N
N
Ar
Scheme 1. Reagents: (i) ArNHNH2, MeOH, Et3N; (ii) NaOH, EtOH, H2O; (iii) NaOEt,
EtOAc, EtOH; (iv) POCl3, dimethylaniline, CH3CN; (v) iPrNEt2, R2R3NH, CH3CN.
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across the pH range, with only limited degradation at pH1.2 (51%
main band remaining after 14 days at 50 °C). Discussions with col-
leagues in our pharmaceutical sciences department confirmed that
this level of stability should not cause any issues in the develop-
ment of this compound.
Compound 35 was progressed into a rat pharmacokinetic study
to determine how in vitro metabolic stability would translate
in vivo (Table 4). Compound 35 exhibited an encouraging pharma-
cokinetic profile with moderate volume of distribution and clear-
ance suitable for further pre-clinical investigation.
Compounds were conveniently prepared from substituted aryl
hydrazines by the method described in Scheme 1. Condensation
of the hydrazine with the suitably substituted ethoxymethylidene-
malononitrile yielded the cyanopyrazole intermediate, which was
hydrolysed to the amide under basic conditions. Cyclisation to
the pyrazolopyrimidinone was followed by chlorination to give
the chloropyrimidine derivative, which was subsequently dis-
placed with an amine under basic conditions to give target com-
pounds such as 35.
In summary, introduction of the C,C-dicyclopropylmethylamine
group was instrumental in combining metabolic stability with
CRF-1 potency in this pyrazolopyrimidine template. This suggests
that in targets which poorly tolerate polar functionality, identify-
ing functional groups which display much higher metabolic stabil-
ity than would be expected from their lipophilicity can be an
effective strategy. Compound 35 has been identified as having an
improved balance of potency and in vitro metabolic stability rela-
tive to compound 6, and a pharmacokinetic profile suitable to sup-
port further progression of this compound. Further studies will be
reported in due course.
7. Di Fabio, R.; St-Denis, Y.; Sabbatini, F. M.; Andreotti, D.; Arban, R.; Bernasconi,
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12. CRF-1 potency is expressed as functional activity measured using CHO cells
(Cell Sciences SNB0000377) expressing recombinant human CRF-1 receptor
grown in DMEM:F12 (1:1) media containing 10% (V/V) Foetal Bovine Serum
(PAA), 400 lg/ml Geneticin (GIBCO-BRL 10131-027) and 1% (V/V) Glutamax in
a cell incubator at 37 °C, 5% CO2 to 70% confluence. FAC hCRF (10 nM) was
added with test compound to 10,000 cells/well in Phosphate Buffered Saline
containing 500 lM FAC IBMX. The functional response was measured using
DiscoveRx HitHunter cAMP II Assay kit (Amersham Biosciences—90-0034-03).
Each compound was tested multiple times using a 0.5 log serial dilution dose–
response with a top final assay concentration of 20 lM. The % response of the
test compound at different test doses were then fitted to a 4-parameter logistic
curve to determine the compound IC50. Ki values were determined from the
IC50 using the Cheng and Prussoff relationship and the EC50 for the agonist
dose–response curve which was determined on the same day.
13. Human liver microsome (HLM) and rat liver microsome (RLM) figures are
reported as intrinsic clearances (Clint) with units of lg/ml/mg of protein. This is
a measure of oxidative (Phase I) metabolic liability. Unpublished in-house
Pfizer experience suggests Clint figures of <30 are generally have a good chance
of translating to moderate to low in vivo clearance, and Clint figures of >70 are
likely to translate to rapid in vivo clearance.
Acknowledgements
14. The difference between human and rat microsomal stability in this case is
surprising. From unpublished in-house experience in these assays rat liver
microsomes generally show higher turn-over across chemotypes than human
liver microsomes. The high turnover in rat microsomes for this compound
suggests a possible atypical species difference in Cyp mediated metabolism for
compound 7.
15. Lowe, R. F.; Nelson, J.; Trunghau, N. D.; Crowe, P. D.; Pahuja, A.; McCarthy, J. R.;
Grigoriadis, D. E.; Conlon, P.; Saunders, J.; Chen, C.; Szabo, T.; Ta Kung, C.;
Bozigian, H. J. Med. Chem. 2005, 48, 1540.
We acknowledge Denise Harding, Laia Malet and Adam Sten-
nett for compound synthesis, Margaret Jackson, Nick Edmunds, Jo
Mulgrew, Graham Baker, David Winpenny, Pauline Carnell and Ra-
chel Russell for biological data and Joanne Phipps for ADME
studies.