S. F. Neelamkavil et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4563–4565
4565
Table 5
a preferred group in a wide array of compounds. We believe that
these compounds which are more potent and have increased solu-
bility compared to our initial series have the potential for good
pharmacokinetics and in vivo activity in 11b-HSD1 models. Further
optimization of this series and SAR of structurally related com-
pounds will be reported in due course.
11b-HSD1 inhibition for 4-methoxyazepane sulfonamides
N
SO2
MeO
R
a
a
Compounds
R
11b-HSD1 hIC50 (nM)
11b-HSD1 mIC50 (nM)
Acknowledgements
30
31
32
33
Me
H
Cyclopropyl
Ph
3
7
43
57
165
328
210
We thank Drs. John Piwinski, Michael Graziano and Catherine
Strader for helpful discussions. We thank Dr. Pradip Das for obtain-
ing analytical data. We also thank Dr. Charles McNemar, Ashwin
Ranchod and Dr. William Windsor for solubility measurements.
110
a
15
hIC50 = human IC50, mIC50 = mouse IC50
.
References and notes
Table 6
11b-HSD1 inhibition for 4-methoxy-4-methylazepane sulfonamides
1. Brian, R. W. Eur. J. Endocrinol. 2007, 157, 545.
2. Chrousos, G. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6329.
N
SO2
R
MeO
3. Boyle, C. D.; Kowalski, T. J.; Zhang, L. Annu. Rep. Med. Chem. 2006, 41, 127.
4. Morton, N. M.; Paterson, J. M.; Masuzaki, H.; Holmes, M. C.; Staels, B.; Fievet, C.;
Walker, B. R.; Flier, J. S.; Mullins, J. J.; Seckl, J. R. Diabetes 2004, 53, 931.
5. Barf, T. Mini-Rev. Med. Chem. 2004, 4, 897.
a
a
Compounds
R
11b-HSD1 hIC50 (nM) 11b-HSD1 mIC50 (nM)
30
4-t-Bu–Ph
3
57
6. Seckl, J. R. Curr. Opin. Pharm. 2004, 4, 597.
7. Kotelevtsev, Y.; Holmes, M. C.; Burchell, A.; Houston, P. M.; Schmoll, D.;
Jamieson, P. M.; Best, R.; Brown, R.; Edwards, C. R. W.; Seckl, J. R.; Mullins, J. J.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14924.
8. Putignano, P.; Giraldi, F. P.; Cavagnini, F. J. Endocrinol. Invest. 2004, 27, 969.
9. Boyle, C. D. Curr. Opin. Drug Discovery Dev. 2008, 11, 495.
10. Webster, S. P.; Pallin, T. D. Expert Opin. Ther. Pat. 2007, 17, 1407.
11. Richards, S.; Sorensen, B.; Jae, H. S.; Winn, M.; Chen, Y.; Wang, J.; Fung, S.;
Monzon, K.; Frevert, E. U.; Jacobson, P.; Sham, H.; Link, J. T. Bioorg. Med. Chem.
Lett. 2006, 16, 6241. and references cited therein.
MeO
HO
34
20
229
35
113
656
a
15
hIC50 = human IC50, mIC50 = mouse IC50
.
12. Finney, Z. G.; Riley, T. N. J. Med. Chem. 1980, 23, 895.
13. Lyles-Eggleston, M.; Altundas, R.; Xia, J.; Sikazwe, D. M. N.; Fan, P.; Yang, Q.; Li,
S.; Zhang, W.; Zhu, X.; Schmidt, A. W.; Vanase-Frawley, M.; Shrihkande, A.;
Villalobos, A.; Borne, R. F.; Ablordeppey, S. Y. J. Med. Chem. 2004, 47, 497.
14. Isomers 9 and 10 were separated by silica gel chromatography with 40%
hexanes/ethyl acetate as the eluent system.
end, we introduced different alkyl and aryl groups at the 4-position
of the azepane ring.
We were very pleased to identify compound 27 which had a hu-
man 11b-HSD1 IC50 of 5 nM (Table 4).17 We quickly realized that
there was only a very small window for the R group in this struc-
ture, as simple H (29) or a bulkier phenyl group (23) resulted in
significant loss in potency. A small change from methyl (27) to
cyclopropyl (28) led to a twofold loss in potency.
To further examine the contribution from the hydroxy group to
potency, we alkylated the tertiary alcohol at the 4-position with
methyl iodide and several R groups were again explored. As shown
in Table 5 the SAR data showed that the free (27) as well as meth-
ylated (30) tertiary hydroxy groups had comparable potency,
which seems to indicate the absence of a hydrogen bond donation
to binding. It is also interesting to note that methylating the sec-
ondary hydroxyl compound 29 resulted in compound 31 which
had a significantly increased potency.
We were pleased that our SAR efforts at the 4-position resulted
in the discovery of a compound 30 which was ꢀ35 times more po-
tent than our initial lead 1. Importantly the compounds shown in
Tables 4 and 5 also were 10–20 times more soluble than our earlier
compounds (Tables 1 and 2) based on kinetic solubility measure-
ments.18 Finally, to incorporate additional solubility elements on
30, we introduced hydroxy groups at the t-butyl site and synthe-
sized compounds 34 and 35. However this resulted in a significant
loss in potency as shown in Table 6.
In conclusion, SAR studies of several novel azepane sulfona-
mides have resulted in the identification of several potent inhibi-
tors of 11b-HSD1. The most potent compound 30 has a human
IC50 of 3 nM. p-(t-Butyl)-phenylsulfonamide has been found to be
15. The following are the conditions used for the 11b-HSD1 binding assays: (a)
Preparation of 11b-HSD1 membranes. Human 11b-HSD1 with N-terminal myc
tag was expressed in Sf9 cells using baculovirus Bac-to-Bac expression system
(Invitrogen) according to manufacturer’s instructions. Cells were harvested
three days after infection and washed in PBS before frozen. To make
membranes, the cells were resuspended in buffer A (20 mM Tris–HCl, pH 7.4,
100 mM NaCl, 2 mM EDTA, 2 mM EGTA and CompleteTM protease inhibitor
tablets (Roche Molecular Biochemicals), and lysed in a nitrogen bomb at
900 psi. The cell lysate was centrifuged at 600g for 10 min to remove nuclei
and large cell debris. The supernatant was centrifuged at 100,000g for 1 h. The
membrane pellet was resuspended in buffer A, flash-frozen in liquid nitrogen
and stored at À70 °C before use. (b) Measurement of 11b-HSD1 activity. 11b-
HSD1 enzymatic activity was measured in a 50
NaPO4 pH 7.5, 0.1 mM MgCl2, 3 mM NADPH (prepared fresh daily), 125 nM 3H-
cortisone (American Radiochemicals) and 0.5 g membrane. The reaction was
incubated at room temperature for 1 h before it was stopped by addition of
50 M buffer containing 20 mM NaPO4 pH 7.5, 30 M 18b-glycyrrhetinic acid,
g/ml monoclonal anti-cortisol antibody (Biosource) and 2 mg/ml anti-
ll reaction containing 20 mM
l
l
l
l
1
mouse antibody coated scintillation proximity assay (SPA) beads (Amersham
Bioscience). The mixture was incubated at room temperature for 2 h with
vigorous shaking and analyzed on TopCount scintillation counter.
16. The lower activity in the mouse assay as compared to human makes it
challenging to predict human efficacy based on a rodent in vivo model.
17. Representative compounds 27 and 31 showed <10% inhibition in an 11b-HSD2
assay at a concentration of 50
selective inhibitors of 11b-HSD1.
18. Kinetic solubility of selected compounds: (12 = 10
31 = 100 M). The nephelometric (light scattering) method was used to
lM implying that these compounds are highly
lM, 27 = 200 lM, 23 = 100 lM,
l
determine the kinetic solubility of compounds. The test compound (1.0 mg)
was dissolved in DMSO at 25 mM. A serial dilution into DMSO was performed
and 3 ll of the compound in DMSO at various concentrations was added to the
buffer (10 mM phosphate, pH 7.4). Presence of precipitate was detected by
nephelometry. Solubility was defined as the highest concentration of material
that did not scatter light. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Delivery Rev. 2001, 46, 3.