J. Dudash, Jr. et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4790–4793
Table 3. Fed blood glucose levels in ob/ob mice after compound administration
4793
Vehicle
Hours after compounds dosing:
2
0
1
4
8
339.5 37.4
334.0 35.9
357.4 37.5
320.3 36.5
319.3 21.5
306.4 12.1
305.4 23.8
349.1 40.6
375.3 15.8
351.3 36.3
356.0 28.9
338.4 31.5
341.1 28.8
313.3 29.1
362.5 38.7
383.3 33.5
222.9 17.3
224.9 22.6
328.4 50.7
325.9 47.4
7d
11c
11e
*P < 0.05, compared with vehicle treated group.
attach for several hours, followed by addition of [14C]glu-
cose for overnight incorporation into glycogen. The next
day, unincorporated radioactivity is removed and cells are
treated with test compound with or without .1 nM gluca-
gon for 2 h. Cellular glycogen is collected by potassium
hydroxide solubilization of the cells followed by ethanol
extraction and the radioactivity determined. Glycogen
breakdown is determined by comparing reduction of
radioactivity from cells at time 0 versus time 2 h and
inhibition of this breakdown by test compounds assessed.
13. Tests were conducted at Absorption Systems, Exton, PA.
Caco-2 monolayers were grown to confluence, dosed at
10 lM on the apical side (A-to-B) or basolateral side (B-
to-A), and incubated at 37 ꢁC. The permeability assay
buffer was HanksÕ balanced salt solution containing
10 mM HEPES and 15 mM glucose at a pH of 7.0–7.2.
Samples were assayed by LC/MS using electrospray
ionization. The apparent permeability, Papp, and percent
recovery were calculated using Papp = (dCr/dt) · Vr/
(A · C0) and Percent Recovery ¼ 100 ꢁ ððV r ꢁ CfrinalÞþ
ðV dꢁ CdfinalÞÞ=ðV d ꢁ C0Þ, where dCr/dt is the cumulative
concentration in the receiver compartment versus time in
M sꢂ1. Vr is the volume of the receiver compartment in
cm3. Vd is the volume of the donor compartment in cm3. A
is the area of the cell monolayer. C0 is the concentration of
the dosing solution in M. Cfrinal is the cumulative receiver
concentration in M at the end of the incubation period.
Cfdinal is the concentration of the donor in Mat the end of
the incubation period.
14. Tests were conducted at Absorption Systems, Exton, PA.
Incubation at 37 ꢁC with test compound at 5 lM and 1 mg
protein/mL human liver microsomal prep. Half life was
determined by measuring the percent parent compound
remaining. In this system, values above 30 min were
considered acceptable.
15. Tests were conducted at Absorption Systems, Exton, PA.
Equilibrium solubility was measured in pH 2.0 and pH 7.4
aqueous buffers. At least 1 mg of powder was combined
with 1 mL of buffer to make P1 mg/mL mixture. These
samples were shaken for P2 h and left to stand overnight
at room temperature. The samples were then filtered
through a 0.45-lm Nylon syringe filter that was first
saturated with the sample. The filtrate was sampled twice,
consecutively. All samples were assayed by LC/MS using
electrospray ionization.
References and notes
1. DeFronzo, R. A.; Bonadonna, R. C.; Ferrannini, E.
Diabetes Care 1992, 15, 318.
2. (a) Hellerstein, M. K.; Neese, R. A.; Linfoot, P.; Chris-
tiansen, M.; Turner, S.; Letscher, A. J. Clin. Invest. 1997,
100, 1305; (b) Pimenta, W.; Nurjhan, N.; Jansson, P. A.;
Stumvoli, M.; Gerich, J.; Korytkowski, M. Diabetologia
1994, 37, 697.
3. Ogawa, A. K.; Willoughby, C. A.; Bergeron, R.; Ells-
worth, K. P.; Geissler, W. M.; Myers, R. W.; Yao, J.;
Harris, G.; Chapman, K. T. Bioorg. Med. Chem. Lett.
2003, 13, 3405.
4. (a) DeFossa, E.; Kadereit, D.; Scoenafinger, K.; Klabun-
de, T.; Burger, H. J.; Herling, A.; Wendt, K. U.; Von
Roedern, E.; Enhsen, A.; Rieke-Zapp, J. WO 03/084922;
Chem. Abstr. 2003, 139, 323338; (b) DeFossa, E.; Kade-
reit, D.; Scoenafinger, K.; Klabunde, T.; Burger, H. J.;
Herling, A.; Wendt, K. U.; Von Roedern, E.; Enhsen, A.
WO 03/084923; Chem. Abstr. 2003, 139, 307602.
5. Hoover, D. J.; Lefkowitz-Snow, S.; Burgess-Henry, J. L.;
Martin, W. H.; Armento, S. J.; Stock, I. A.; McPherson,
R. K.; Genereux, P. E.; Gibbs, E. M.; Treadway, J. L.
J. Med. Chem. 1998, 41, 2934.
6. A three dimensional pharmacophore was derived from
a known GPa inhibitor, CP-91149. 3D searches were
carried out against a multiconformer version of the
corporate database using the Catalyst software avail-
pounds with desirable ADME (absorption, distribution,
metabolism, and excretion) properties were subsequent-
ly selected from the original Catalyst electronic hit list
by calculating ADME descriptors with the MOE
software (available from The Chemical Computing
Group, 1010 Sherbrooke Street West, Suite 910,
Montreal, Canada H#A 2R7). The final list of
compounds was tested and afforded compound 1 as
one of the confirmed hits.
7. Enzyme inhibitory activity was determined using glycogen
phosphorylase from rabbit muscle (Sigma P1261) by the
method of Martin et al. Martin, W. H.; Hoover, D. J.;
Armento, S. J.; Stock, I. A.; McPherson, R. K.; Danley,
D. F.; Stevenson, R. W.; Barrett, E. J.; Treadway, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1776.
8. All compounds provided satisfactory spectral data
(1H NMR, LCMS) and were homogeneous by TLC.
9. Lavieri, S.; Zoltewicz, J. A. J. Org. Chem. 2001, 66, 7227,
and references cited therein.
10. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett.
1977, 48, 4171.
11. Lumma, W. C.; Hartman, R. D.; Saari, W. S.; Engelhardt,
E. L.; Lotti, V. J.; Stone, C. A. J. Med. Chem. 1981, 24, 93.
12. The rat hepatocyte glycogenolysis assay was adapted from
the method of Ciaraldi, G. et al. Diabetes 1992, 41, 975.
Briefly, hepatocytes are obtained by in vivo collagenase
perfusion followed by ex vivo dispersion of rat liver.
Resultant cells are plated in 6 well plates and allowed to
16. Female C57BL/6J ob/ob mice (C57BL/KsJ-Lepob/ob Jack-
son Labs, Bar Harbor, ME) were at 6–7 weeks of age
when received. Upon arrival, they were quarantined for
5 days, housed two mice per cage and given access to
water and food ad libitum. Next, these mice were
grouped based on their blood glucose levels. They were
then given either vehicle (0.5% methycellulose), or
compound (suspended in 0.5% methycellulose) at
30 mg/kg body weight via oral gavage. Tail blood
samples were collected at 0, 1, 2, 4, and 8 h after
dosing to measure the blood glucose using a glucometer
(One Touch Ultra, Lifescan, Milpitas, CA).