The development and SAR of pyrrolidine carboxamide 11β-HSD1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.03.032

The design and development of a series of highly selective pyrrolidine carboxamide 11β-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11β-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11β-HSD1 selective inhibitor 42.

Full text of DOI:10.1016/j.bmcl.2010.03.032

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2897–2902
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The development and SAR of pyrrolidine carboxamide 11b-HSD1 inhibitors
a
a
a
a
a
Hengmiao Cheng a, , Jacqui Hoffman , Phuong Le , Sajiv K. Nair , Stephan Cripps , Jean Matthews ,
Christopher Smith a, Michele Yang a, Stan Kupchinsky a, Klaus Dress a, Martin Edwards a, Bridget Cole b,
Evan Walters b, Christine Loh b, Jacques Ermolieff a, Andrea Fanjul a, Ganesh B. Bhat a, Jocelyn Herrera a,
Tom Pauly a, Natilie Hosea a, Genevieve Paderes a, Paul Rejto a
*
a Pfizer Global Research & Development, La Jolla Labs, 10770 Science Center Drive, San Diego, CA, 92121, United States
b Pfizer Global Research & Development, Research Technology Center, 620 memorial Drive, Cambridge, MA 02139, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and development of a series of highly selective pyrrolidine carboxamide 11b-HSD1 inhibitors
are described. These compounds including PF-877423 demonstrated potent in vitro activity against both
human and mouse 11b-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone
to cortisol. Structure guided optimization effort yielded potent and stable 11b-HSD1 selective inhibitor 42.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 February 2010
Revised 4 March 2010
Accepted 5 March 2010
Available online 10 March 2010
Keywords:
11b-HSD1
Inhibitor
Diabetes
Pyrrolidine carboxamide
PF-877423
11b-HSD1 catalyzes the intracrine conversion of inactive corti-
sone to the active glucocorticoid agonist cortisol.1 The 11b-HSD1
knockout mouse is protected from hyperglycemia associated with
stress or obesity through reduced hepatic expression of phosphoe-
nol-pyruvate carboxy kinase (PEPCK), which controls the rate lim-
iting step of gluconeogenesis, as well as other glucose mobilizing
enzymes such as glucose-6-phosphatase (G6Pase).2 Positive effects
may also occur outside of the liver via improved b-cell function3
and lipid modulation through adipose tissue.4 In addition, trans-
genic mice that over-express 11b-HSD2, the enzyme that catalyzes
the conversion of the active glucocorticoid cortisol back to inactive
cortisone, have improved insulin sensitivity and glucose tolerance
and are protected from weight gain on a high-fat diet.5 Because
11b-HSD1 is highly expressed in the liver relative to other tissues,6
inhibition of 11b-HSD1 activity provides an opportunity to reduce
glucocorticoid levels specifically in the liver and splanchnic circu-
lation.7 Hence, inhibition of 11b-HSD1 activity in the liver is an
attractive approach to treat diabetes.
report the development of
a
series of amides such as PF-
-prolinamide, that demon-
87742311, N-adamantan-2-yl-1-ethyl-
D
strated high selectivity, potent in vitro activity against both human
and mouse enzymes, and good in vivo activity inhibiting the con-
version of cortisone to cortisol.
Cell based high throughput screening (HTS) was carried out to
search for non-steroid 11b-HSD1 inhibitors, the adamantine car-
boxamide 5 was identified as a lead with Ki of 175 nM against
human enzyme12 and cell IC50 of 114 nM13 (Fig. 2). Compound 5
also demonstrated very good selectivity against 11b-HSD2, with
O
N
N
N
N
S
O
H3C
O
S
N
N
N
Cl
H
1, BVT.2733
2, Merck 544
Several classes of selective, non steroid 11b-HSD1 inhibitors
have been published (Fig. 1). These include sulfonamides such as
BVT.2733,8 triazoles such as the Merck compound 544,9 and ada-
mantine amides from Abbott with generic structure 3.10 Here we
R3
R2
O
H
N
H
N
R4
N
N
R1
R5
O
3, Abbott amide
Figure 1. Non-steroid inhibitors of 11b-HSD1.
4, PF-877423
* Corresponding author. Tel.: +1 858 622 3208; fax: +1 858 526 4151.
E-mail address: henry.cheng@pfizer.com (H. Cheng).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.032

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H. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2897–2902
Ki greater than 10
l
M. However, compound 5 did not show
braries. Compound 6 was identified via this approach. Compound 6
demonstrated comparable in potency as 5 in the cell based assay
but was 3 units lower in clog P. Effort continued by combining
structural aspects of 5 and 6 to produce chimeric compounds of
measurable activity against murine enzyme at 100 nM. Initially a
combinatorial chemistry approach was taken to further expand
the SAR knowledge around 5 by investigating a series of amide li-
N
O
H
H
N
N
make a ring
hybrid design
5
N
N
O
O
7
4, PF-877423
N
N
O
N
O
6
Figure 2. Discovery of PF-877423.
Figure 3. Crystal structure of guinea pig 11b-HSD1 with PF-877423. Resolution is 1.84 Å.
H
N
a
NH2
b
HO
N
N
Boc
+
Boc
O
O
8
H
N
c or d
H
N
N
N
R1
H
O
O
10
9
Scheme 1. Reagents and conditions: (a) HATU, Et3N, CH2Cl2; or EDC, HOBt, Et3N, CH2Cl2; (b) TFA; (c) R1-X, Et3N, CH2Cl2; (d) R0CHO, NaBH(OAc)3, THF.

H. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2897–2902
2899
Table 1
5 and 6. A 10-fold increase in potency was achieved following this
strategy as exemplified by 7 (Table 1).
Summarized data for compounds 4–7
Significant chemical space around 7 had already been explored
via our combinatorial chemistry approach thus a new design strat-
egy was investigated—‘make a ring, break a ring’. These efforts
identified 4, PF-877423, which gave moderate metabolism in
HLM (Clint 44 mL/min/Kg), good potency in the cell and potent
inhibition of the mouse 11b-HSD1 enzyme. PF-877423 is selective
H
N
N
R1
O
Compound
Kiapp (nM)
IC50 (nM)
clog P
Human
Mouse
over 11b-HSD2, with Ki greater than 10 lM.
PF-877423 is highly ligand efficient with ligand efficiency (LE)
of 0.57 based on the cell IC50. The cocrystal structure of PF-
877423 with guinea pig 11b-HSD1 enzyme was determined to
understand how it binds in the enzyme active site (Fig. 3).
5
6
7
4
175
15
3.1
1.4
6% @100 nM
1.75% @100 nM
261
290
2.2
4.58
1.63
2.99
4.22
0.63
4.2
Table 2
H
N
N
R1
O
Compound
R1
Kiapp (nM) or % inhibition @100 nM
HEK IC50 (nM)
clog P
HLM Clint (ml/min/kg)
Human
1.3
Mouse
4
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Et
Me
Pr
0.63
5.4
1
9.4
1
ND
ND
3.5
35.2
3.6
6.3
ND
170
ND
40
4.15
22
1.2
4.22
3.69
4.75
4.53
5.67
5.15
5.11
6.23
4.48
4.46
6.12
4.84
4.84
3.12
3.91
43.9
22.2
93.3
ND
75.3
79.3
85.8
62.8
ND
ND
ND
73.2
ND
ND
2.4
1.4
1.6
ND
ND
ND
2.4
1
1
2
ND
1
3.6
1
i-Pr
3.5
Cyclopentylmethyl
Isobutyl
Cyclobutylmethyl
Cyclohexylmethyl
CF3CH2
0.03
0.03
0.03
0.32
0.32
0.32
0.32
0.03
0.32
20
CF3CH2CH2
p-Cl-Benzyl
p-CN-Benzyl
m-CN-Benzyl
Hydroxyethyl
2-Pyridyl
5
79.2
O
25
1
16
10
3.83
20.3
OH
26
27
ND
ND
17
48
0.34
0.03
4.58
5.14
86.2
64.6
OH
H
N
H
N
NH2
b
a
HO
O2NO
O
O
29
28
30
H
NH2
N
c
N
d
HO
HO
Boc
HO
N
Boc
+
O
O
32
31
H
N
H
N
N
H
N
f or g
e
R1
HO
HO
O
O
33
34
Scheme 2. Reagents and conditions: (a) HNO3, room temperature, 63%; (b): 6 N HCl, water, reflux, 60%; (c) chromatography separation of the two isomers; (d) HATU, Et3N,
CH2Cl2; or EDC, HOBt, Et3N, CH2Cl2; (e) TFA; (f) R1-X, Et3N, CH2Cl2; (g) R0CHO, NaBH(OAc)3, THF.

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H. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2897–2902
PF-877423 interacts with NADP and several active site residues,
most notably Ser408, Tyr469, Leu364, Ile418, Leu 455, Tyr469
and Tyr415. The adamantyl of PF-877423 packs against the nico-
tinamide ring of the NADP and the pyrrolidine ring is stacked
with the ring of Tyr415. Most of the interactions are hydrophobic
in nature with the exception of a hydrogen bond between the
amide oxygen of PF-877423 and the side chain of Ser408.
Structure analysis indicated that there is space to accommodate
a variety of side chains off the pyrrolidine nitrogen. In order to ex-
plore the SAR, as illustrated in Scheme 1, different substituents
were placed on the pyrrolidine nitrogen by either SN2 displace-
ment or reductive amination. Listed in Table 2 are in vitro bio-
chemical data such as human and mouse enzyme Kiapp and
cellular IC50, clog P and human liver microsomal clearance. The
in vitro cell potency is correlated to clog P of the compounds. For
example, when the R1 group is smaller alkyl groups as in com-
pounds 4, 11, 12, and 13, these compounds exhibited cell IC50
greater than 1 nM. When the R1 group is larger groups as in com-
pounds 14 through 22, these compounds exhibited higher clog P
and demonstrated cell IC50 less than 1 nM. The data from the table
also shows that human liver microsome intrinsic clearance is in-
versely correlated to clog P. Interestingly, compound 25 with lower
clog P demonstrated good in vitro activity and metabolic stability.
In vitro metabolic identification studies with PF-877423 indi-
cated that the adamantly ring and the pyrrolidine ring were oxidized
by HLM. In order to reduce the clearance by either block the meta-
bolic soft spots or lower clog P by introducing polar groups such as
hydroxyl group, the cocrystal structure of PF-877423 with the pro-
tein was further investigated. Around the pyrrolidine ring, there is
space to introduce small functional groups such as hydroxyl and
methyl. Introduction of a hydroxyl group to the adamantly ring is
also viable since this hydroxyl group can occupy the space next to
Table 3
Compound
Structure
Kiapp (nM) or % inhibition @100 nM
HEK IC50 (nM)
clog P
HLM Clint (ml/min/kg)
Human
Mouse
OH
H
N
35
1.4
23
34
3.02
ND
N
O
O
O
O
O
OH
H
N
36
37
1
3.3
12
0.8
3.94
4.5
ND
72
N
OH
H
N
1
0.173
N
OH
H
N
38
39
40
41
1
129
ND
ND
3.8
1
4.15
4.3
ND
ND
84.2
65
N
OH
H
N
1
0.03
30
N
F
OH
H
N
2.6
2.66
3.98
N
O
O
H
H
H
N
1
19.4
N
O
H
N
N
42
43
ND
ND
8.4
ND
23
1.09
2.02
11.8
60.2
HO
HO
O
O
H
N
N
31.3

H. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2897–2902
2901
25
20
15
10
5
50
40
30
20
10
0
100%
0
*52%
1
3
10
30
10
*46%
PF-00877423, mpk
*23%
*20%
*18%
Vehicle
1
3
10
30
100
PF-00877423 (mpk) p.o.
Figure 4. In vivo inhibition of 11b-HSD1 activity in normal C57Bl/6 mice by PF-877423.
2390; (c) Edwards, C. R. W.; Stewart, P. M.; Burt, D.; Brett, L.; McIntyre, M. A.;
Sutanto, W. S.; De Kloet, E. R.; Monder, C. Lancet 1988, 2, 986; (d) Funder, J. W.;
Pearce, P. T.; Smith, R.; Smith, A. I. Science 1988, 242, 583.
the phosphate group of NADP. Subsequently, the hydroxyproline
derivatives were prepared following similar synthetic methods as
outlined in Scheme 1 using the commercially available N-Boc-
hydroxyproline. The cyclopropane fused proline derivative 39 was
also prepared following the same synthetic route. The synthesis of
the hydroxyl-adamantyl ring is described in Scheme 2.15
The data for the hydroxylated amide derivatives is summarized
in Table 3. Introduction of hydroxyl group to the proline core low-
ered the clog P. Compounds with lipophilic substituents such as
compounds 35–41 demonstrated good in vitro potency. However,
their metabolic stability was not improved. The in vitro clearance
for the cyclopropane fused proline derivative 41 is also high. Inter-
estingly, the hydroxyl adanmantyl compound 42, which has cell
IC50 of 23 nM, has significantly lower clog P and is metabolically
stable in the in vitro human liver microsomal assay. Replacement
of the ethyl by isobutyl increased clog P by one unit as in com-
pound 43. This compound showed increased in vitro potency, but
high in vitro clearance.
In order to establish the correlation between in vitro activity
and in vivo inhibition, PF-877423 was progressed to the in vivo
biomarker assay methodology using exogenous substrate cortisone
conversion to cortisol in normal mice.14
As shown in Figure 4, oral treatment of normal mice with 1–
100 mg/kg PF-877423 dose dependently reduced the generation
of cortisol thirty minutes following compound administration with
a maximum inhibition of 82% in animals treated with 100 mpk. As
shown in the figure inset, the calculated dose to inhibit 50% 11b-
HSD1 in vivo activity was 5.0 mpk of PF-877423.
2. Kotelevtsev, Y.; Holmes, M. C.; Burchell, A.; Houston, P. M.; Schmoll, D.;
Jamieson, P.; Best, R.; Brown, R.; Edwards, C. R. W.; Seckl, J. R.; Mullins, J. J. Proc.
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Science 2001, 294, 2166.
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54, 1023.
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Rizza, R. Diabetes 2004, 53, 2051.
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Vosatka, A.; LiGrand, C. B.; Mundt, S. S.; Robbins, M. A.; Schaeffer, J. M.;
Slossberg, L. H.; Szymonifka, M. J.; Thieringer, R.; Wright, S. D.; Balkovec, J. M.
Bioorg. Med. Chem. Lett. 2005, 15, 4359.
10. (a) Rohde, J. J.; Pliuschev, M. A.; Wodka, D.; Sorensen, B. K.; Yong, H.; Madar, D.
J.; Shuai, Q.; Patel, J. R.; Jae, H.; Sarris, K. A.; Wang, J.; Fung, S.; Monzon, K.;
Chiou, W.; Pan, L.; Deng, X.; Chovan, L. E.; Longenecker, K. L.; Judge, R. A.; Qin,
W.; Wagaw, S. H.; Engstrom, K. M.; Kerdesky, F. A. J.; Stolarik, D. F.; Imade, H.
M.; Marsh, K. C.; Beno, D. W. A.; Fey, T. A.; Droz, B. A.; Brune, M.; Camp, H.;
Sham, H. L.; Frevert, E. U.; Jocobson, P.; Link, J. T. J. Med Chem. 2007, 50, 149; (b)
Patel, J. R.; Shuai, Q.; Dinges, J.; Winn, M.; Pliushchev, M.; Fung, S.; Monzon, K.;
Cjiou, W.; Wang, J.; Pan, L.; Wagaw, S.; Engstrom, K.; Kerdesky, F. A.;
Longenecker, K.; Judge, R.; Qin, W.; Imade, H. M.; Stolarik, D.; Beno, D. W. A.;
Brune, M.; Chovan, L. E.; Sham, H. L.; Jacobson, P.; Link, J. T. Bioorg. Med. Chem.
Lett. 2007, 17, 750.
11. Cheng, H.; Smith, C. R.; Wang, Y.; Parrott, T. J.; Dress, K. R.; Nair, S. K.; Hoffman,
J. E.; Le, P. T. Q.; Kupchinsky, S. W.; Yang, Y.; Cripps, S. J.; Huang, B. PCT Int.
Appl. WO 2005108359, 2005. The preparation, 1H NMR and MS data for the
compounds that are discussed in this article are described in this patent.
12. The enzyme assay is described in Ref. 11.
13. HEK293-11b-HSD1/GRE-Luciferase cell-based assay: EC50 determination/
inhibition of 11b-HSD1 enzyme activity was measured using human kidney
HEK293 stable transfected cells, over-expressing human 11b-HSD1, and a
reporter plasmid containing DNA sequences for specific recognition of
glucocorticoid-activated glucocorticoid receptors (GRE). These sequences
were fused to a luciferase reporter gene (Luc) allowing for quantification of
11b-HSD1 enzyme modulation. Cortisol (but not cortisone) binds and activates
glucocorticoid receptors (GR), which will result in activation of luciferase and
Additional studies with PF-877423 indicated that blocking adi-
pogenesis with selective 11b-HSD1 inhibitor may represent a novel
approach to treat obesity in patients with MS.16
In summary, we have discovered a series of pyrrolidine carbox-
amides with good selectivity over 11b-HSD2 and potent in vitro
activity against both human and mouse 11b-HSD1 enzymes. PF-
877423 was progressed to in vivo biomarker conversion assay,
and demonstrated in vivo activity. Introduction of the hydroxyl
group to the adamantly ring afforded compound 42 that is stable
in the in vitro human liver microsomal assay.
production of light (assay readout).
A compound with the capability of
inhibiting 11b-HSD1 will reduce the luciferase signal, compare to cortisone
control (enzyme substrate). Cells were plated in 384 well flat bottom white
polystyrene TC-treated microplates, at 20,000 cell/well at a volume of 40 lL/
well, in serum-free DME Medium. Plates were incubated at 37 °C, 5% CO2
overnight before addition of inhibitor compounds. Different concentrations of
Accession code: The coordinates for PF-877423 with Guinea Pig
11b hydroxysteroid dehydrogenase have been deposited, and the
PDB ID code is 3LZ6.
inhibitor compounds were added in 10% (v/v) dimethylsulfoxide (5
followed by addition of 3 M cortisone (5 L/well), and cells were incubated at
37 °C (5% CO2) for six hours. At the end of the incubation, 25 L/well SteadyLite
lL/well),
l
l
l
References and notes
HTS were added and plates were incubated 10 min at room temp on shaker.
Plates were then read on Top Count using 384HSD1 program. The concentration
of inhibitor compound causing 50% inhibition of light signal was determined via
1. (a) Amelung, D.; Hubener, H. J.; Roka, L.; Meyerheim, G. J. Clin. Endocrinol.
Metab. 1953, 13, 1125; (b) Lakshmi, V.; Monder, C. Endocrinology 1988, 123,

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H. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2897–2902
a custom made Excel Macro. All results were compared to 100% activation
using ELISA assay kit (Cat# 1850, Alpha Diagnostic International, San Antonio,
TX). Background value from no cortisone treated mice sample was subtracted
from all cortisone treated samples.
control, that is, cells treated only with cortisone (no inhibitors added).
14. Normal mice were used to develop an acute mechanistic biomarker model. For
this mechanistic biomarker study, normal 8–10 weeks old C57Bl mice (Charles
River Laboratories, Wilmington, MA) were dosed orally with vehicle (0.5%
methyl cellulose) or with 1–100 mg/kg of PF 00877423. Thirty minutes after
the vehicle or drug treatment, mice were dosed orally with cortisone (Cat#
C2755, Sigma-Aldrich, St Louis, MO, 10 mg/kg in 0.5% methyl cellulose). Plasma
samples were collected 60 min later. Plasma cortisol levels were measured
15. The authors would like to thank Wuxi Apptec Co. for the preparation of
intermediate compound 30.17
16. Bujalska, I. J.; Gathercole, L. L.; Tomlinson, J. W.; Darimont, C.; Ermolieff, J.;
Fanjul, A. N.; Rejto, P. A.; Stewart, P. M. J. Endocrinol. 2008, 197, 297.
17. Geluk, H. W. Synthesis 1972, 374.