Synthesis and SAR of azolopyrimidines as potent and selective dipeptidyl peptidase-4 (DPP4) inhibitors for type 2 diabetes

Article abstract of DOI:10.1016/j.bmcl.2010.06.063

Several pyrazolo-, triazolo-, and imidazolopyrimidines were synthesized and evaluated as inhibitors of DPP4. Of these three classes of compounds, the imidazolopyrimidines displayed the greatest potency and demonstrated excellent selectivity over the other

Full text of DOI:10.1016/j.bmcl.2010.06.063

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4395–4398
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of azolopyrimidines as potent and selective dipeptidyl
peptidase-4 (DPP4) inhibitors for type 2 diabetes
a,
b
c
c
a
Robert P. Brigance a, , Wei Meng , Aberra Fura , Thomas Harrity , Aiying Wang , Robert Zahler ,
*
*
Mark S. Kirby c, Lawrence G. Hamann a
a Department of Discovery Chemistry, Bristol-Myers Squibb, Research and Development, PO Box 5400, Princeton, NJ 08543-5400, United States
b Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Research and Development, PO Box 5400, Princeton, NJ 08543-5400, United States
c Department of Metabolic Diseases, Bristol-Myers Squibb, Research and Development, PO Box 5400, Princeton, NJ 08543-5400, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Several pyrazolo-, triazolo-, and imidazolopyrimidines were synthesized and evaluated as inhibitors of
DPP4. Of these three classes of compounds, the imidazolopyrimidines displayed the greatest potency
and demonstrated excellent selectivity over the other dipeptidyl peptidases. SAR evaluation for these
scaffolds was described as they may represent potential treatments for type 2 diabetes.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 1 May 2010
Revised 8 June 2010
Accepted 10 June 2010
Available online 15 June 2010
Keywords:
DPP4
Azolopyrimidines
GLP-1
SAR
Diabetes mellitus is a chronic disease and a major healthcare
problem which is estimated to affect more than 171 million people
worldwide.1 It is further estimated that by the year 2030,2 there
will be over 366 million cases of diabetes, of which more than
90% will be type 2.3 Recently, DPP4 inhibition has shown promise
as a novel approach to treating type 2 diabetes.4 DPP4 is a ubiqui-
tous serine protease which belongs to a broad class of dipeptidyl
peptidases (DPP’s). Although the physiological functions of these
other DPP’s are not fully understood, the function of DPP4 has been
well documented in the literature.5 DPP4 specifically cleaves a pro-
line or aniline at the penultimate position of the N-terminus of a
variety of small peptides.6 When food is ingested, the intestinal
L-cells release a potent incretin hormone known as glucagon-like
peptide-1 (GLP-1). This 30-mer peptide is responsible for increases
in insulin secretion and b-cell function, and decreases in glucagon
secretion.7 These beneficial antidiabetic responses mediated by
GLP-1 make it a highly attractive direct or indirect target for drug de-
sign. Upon its release, the active GLP-1 (7–36) is rapidly degraded by
DPP4 (t1/2 <1 min) to provide the inactive form of GLP-1 (9–36).8
Inhibiting DPP4 has been shown in animals and clinically to effec-
tively increase the half-life of active GLP-1 and regulate plasma
glucose.9
To date, sitagliptin (1),10 saxagliptin (2),11 and vildagliptin
(3, approved in Europe, Brazil, and Mexico)12 have all obtained reg-
ulatory approval while alogliptin (4),13 linagliptin (5),14 and dutog-
liptin (6)15 have successfully advanced to late stage clinical trials
(Fig. 1). Compounds 2 and 3 belong to a potent class of
a-amino
acid derived DPP4 inhibitors which are dipeptide mimics, while 1
is a b-amino acid derived inhibitor.
Our continued interest in DPP4 inhibition as a therapy for type 2
diabetes prompted the investigation into other novel small mole-
cule DPP4 inhibitor manifolds that were both potent and selective
over the other DPP’s. This investigation led to the discovery of the
pyrazolopyrimidines, a class of compounds under investigation in
F
F
O
NH2
HO
N
N
N
N
HO
N
H
H2N
N
CN
N
F
O
CN
O
CF3
vildagliptin, 3
saxagliptin, 2
sitagliptin, 1
O
N
O
O
N
N
N
N
N
N
N
O
N
N
NH2
NH2
CN
alogliptin, 4
linagliptin, 5
* Corresponding authors. Tel.: +1 609 818 4968 (W.M.).
E-mail addresses: robert.brigance@bms.com (R.P. Brigance), wei.meng@bms.com
(W. Meng).
Figure 1. Structures of selected DPP4 inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.063

4396
R. P. Brigance et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4395–4398
Table 1
another in-house program. This class of compounds possessed a
basic amine functionality that was bound to a rigid system, a motif
sharing commonalities with some other non-amino acid derived
DPP4 inhibitors such as linagliptin and dutogliptin.16
Binding affinities for pyrazolopyrimidines 13–24
Compd
R1
R2
DPP4 Ki
(nM)
DPP8 Ki
M)
DPP9 Ki
(lM)
(
l
13
14
15
16
17
18
19
20
21
22
23
24
H
Ph
H
CO2Et
H
H
H
H
H
H
Me
H
Ph
H
2-Me-Ph
2-Cl-Ph
2-Furanyl
4-Cl-Ph
4-MeO-Ph
4-Me-Ph
3-Me-Ph
3-Cl-Ph
28 ( 2.5)a
>30
>30
>30
>30
7.0
6.8
>30
6.3
>30
>30
>20
>30
5.8
Our SAR started from pyrazolopyrimidine containing com-
pounds. Various substituents on the pyrazole ring which include
alkyl, aryl, substituted aryl and carboxylic ester groups were used
to explore the substitution pattern in the region. The synthesis of
pyrazolopyrimidines 13–24 was achieved by condensing the
appropriate 3-aminopyrazole 9 (obtained commercially or pre-
pared according to the literature procedures17) with 2,4-dichloro-
benzaldehyde 7 and methyl acetoacetate 8 in a three component,
one pot reaction to give the desired dihydropyrimidine intermedi-
ate 10 (Scheme 1). Formation of 10 for compound 16 required a
two-step process in which t-butyl acetoacetate was condensed with
7 to give the desired Knoevenagel product, which was then con-
densed with 4-carboethoxy-2-aminopyrrazole 9. Compound 10
was then oxidized to the desired pyrazolopyrimidine core 11 by
DDQ. The carboalkoxy group of 11 was reduced to the corresponding
hydroxymethyl 12 using DIBAL directly or by a three-step process in
which the ester was hydrolyzed to the corresponding acid with LiOH
(TFA for compound 16), converted to a mixed anhydride using ethyl
chloroformate, and reduced to the desired alcohol with NaBH4. The
resultant alcohol was then converted to the corresponding azide via
a chloride intermediate, and reduced by PPh3 to the desired pyrazol-
opyrimidine 13–24.
All inhibitors were tested in vitro against purified human DPP4
using H-Gly-Pro-pNA as a substrate and measuring the level of
p-nitroaniline production at 405 nm over 15 min.18 Inhibition of
the related proteases DPP8 and DPP9 was also assessed (Table 1).
This class of compounds possessed three desirable characteristics:
favorable DPP4 activity, selectivity over DPP8 and DPP9, and ample
opportunity for further optimization around the pyrazole ring. The
majority of the compounds in this series exhibited good potency
against DPP4, though only weak DPP8/DPP9 inhibition (most
141 ( 24)a
22
73
50
20
43
33
30
28
75
163
4.5
>30
>30
3.1
>10
7.8
5.5
>30
12.2
14.6
H
H
5.3
a
Values represent the mean of multiple determinations, standard deviation is
shown in parentheses.
tion was somewhat less favored than para-substitution on the R2
phenyl (comparing 20–24 and 22–23).
Expanded investigation into other fused heterobicyclic systems
led us to examine triazolopyrimidines as DPP4 inhibitors. Except
for compounds 32 and 40, triazolopyrimidines were prepared by
one of two similar methods. The synthesis of 2-alkyl or 2-aryl
substituted triazolopyrimidines 29–31 began with a same three
component, one pot reaction in which the appropriate 3-substi-
tuted 5-amino-1H-[1,2,4]-triazole 25 was condensed with 7 and
8, followed with DDQ oxidation, to give triazolopyrimidine 26
(Scheme 2). Compound 26 (R = C) was then converted to the de-
sired 5-aminomethyl triazolopyrimidine using the same methods
employed in the pyrazolopyrimidine series. For 2-amino substi-
tuted triazolopyrimidines 33–39, a common intermediate 27 pro-
vided a means for rapid analog generation and SAR evaluation,
beginning with 3,5-diamino-1H-[1,2,4]-triazole 25. To this end,
26 (R = NH2) was converted to the corresponding 2-bromotriazolo-
pyrimidine 27 by a Sandmeyer reaction, of which the bromide
could be readily replaced with appropriate amines by nucleophilic
substitution to form amine 28. Compound 28 was further con-
verted to the desired final products 33–39 using the same method-
ology as previously described. Compound 32 was generated by first
Boc protecting amine 31 with di-tert-butyl dicarbonate and Et3N in
THF, followed by hydrolysis of the ester using aqueous LiOH. The
final product 32 was then obtained by standard EDAC/HOAT cou-
pling of the resultant carboxylic acid with aniline to give the corre-
sponding amide, which was then deprotected with TFA. Compound
40 was generated by deprotection of compound 39 with TFA in
CH2Cl2.
Ki’s >30 lM) was observed. Additionally, all of these inhibitors
occupied a narrow potency range, indicating tolerability for a wide
variety of substituents around the pyrazole ring. Initially, com-
pounds 13–16 were synthesized to determine the more favorable
substitution pattern about the pyrazole ring (R1 vs R2). The SAR
from this small group of compounds clearly indicated that substit-
uents at R2 showed >6-fold greater activity than those at R1
(14 = 141 nM and 15 = 22 nM). Once this was established, the
remaining analogs (17–18 and 20–24) focused to probe the elec-
tronics and their substitution pattern on a phenyl group at the R2
position of the pyrazole ring. It was observed that meta-substitu-
N
R1
H
N
N
N
N
N
H
N
N
R
R
N
NH2
c-h
Cl
N
N
a, b
R2
H
CO2Me
Cl
NH2
N
O
O
N
CHO
N
N
(R = C)
a
N
Cl
CO2Me
Cl
+
N
+
R2
R
25
OMe
H2N
Cl
26
29-31
R1
i
7
8
9
10
(R = NH2)
Cl
Cl
N
Cl
N
R1
R1
N
N
R1
N
N
N
2
N
N
2
N
R
R
Br
j
c-h
N
NH2
R2
c
N
N
N
R2
d-f
R2
CO2Me
Cl
CO2Me
Cl
N
OH
N
NH2
Cl
N
N
(R = N)
N
N
Cl
CO2Me
Cl
b
Cl
28
33-39
27
11
12
13-24
Cl
Cl
Cl
Cl
Cl
Cl
Scheme 2. Reagents and conditions: (a) 7, 8, piperidine, THF, 70 °C; (b) DDQ,
CH2Cl2, 50–61% over two steps; (c) LiOH, THF, H2O, 50 °C; (d) ClCO2Et, Et3N, THF;
(e) NaBH4, THF, 30–60% over three steps; (f) MsCl, CH2Cl2, Et3N; (g) NaN3, DMF,
50 °C; (h) PPh3, THF, H2O, 50 °C, 39–50% over three steps; (i) t-BuONO, CuBr2,
CH3CN, 65 °C, 75–85%; (j) amine, dioxane, 75 °C, 51–95%.
Scheme 1. Reagents and conditions: (a) piperidine, heptane, THF, 70 °C; (b) DDQ,
CH2Cl2, 50–100% over two steps; (c) DIBAL, THF or LiOH, THF, H2O, 50 °C; ClCO2Et,
Et3N, THF; NaBH4, THF, 38–100%; (d) MsCl, CH2Cl2, Et3N; (e) NaN3, DMF, 50 °C;
(f) PPh3, THF, H2O, 50 °C, 26–65% over three steps.

R. P. Brigance et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4395–4398
4397
Table 2
Table 3
Binding affinities for triazolopyrimidines 29–40
Binding affinities for imidazolopyrimidines 44–52
Compd
R
DPP4 Ki
(nM)
DPP8 Ki
M)
DPP9 Ki
M)
Compd
R
DPP4 Ki
(nM)
DPP8 Ki
M)
DPP9 Ki
(lM)
(
l
(
l
(
l
29
30
31
32
33
34
35
36
37
38
39
40
Me
Ph
COOMe
CONHPh
Et2N
Me(Et)N
MeOCH2CH2(Me)N
Pyrrolidine
Morpholine
Thiomorpholine
N-Boc-piperazine
Piperazine
64
67
137
93
73
50
18
50
31
29
>30
>30
>30
13.3
>30
>30
>30
>10
nd
>30
>30
>30
>10
>30
>30
>30
>30
>30
>30
>30
>30
44
45
46
47
48
49
50
51
52
H
Ph
12 ( 3)a
6
18
24
9
22
38
>30
>30
5.3
7.2
4.7
2.7
3.4
1.3
7.8
>30
>30
9.8
5.9
4.8
5.6
5.5
2.1
>10
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
4-Me-Ph
3-MeO-Ph
4-MeO-Ph
2-MeO-Ph
5.6
13 ( 12)a
>30
>30
>30
a
Values represent the mean of multiple determinations, standard deviation is
shown in parentheses.
442
106
nd, not determined.
Table 4
Comparison of properties of azolopyrimidines 15, 30, and 45
These compounds, like the pyrazolopyrimidines, tolerated sig-
nificant substituent diversity on the fused five-membered hetero-
cycle, and retained DPP4 activity and DPP8/DPP9 selectivity
(Table 2). The degree of DPP4 inhibition in this series was depen-
dent on the nature of the substituent, with the most active com-
pounds bearing cyclic or straight-chain secondary amines that
contain either an oxygen or a sulfur (35 = 18 nM, 37 = 31 nM, and
38 = 29 nM). Other substitution of triazole displayed slightly weak-
er activity as observed for 29, 30, 33, 34, and 36 (50–73 nM). DPP4
activity appeared to diminish when the substituent was changed to
a carboalkoxy group or a secondary cyclic amine containing a sec-
ond nitrogen (31 = 137 nM, 39 = 442 nM, and 40 = 106 nM). Addi-
Compd
15
30
45
DPP4 Ki (nM)
22
>30
>30
95
95
35
1
12
3
6
67
6
DPP8 Ki (
DPP9 Ki (
l
l
M)
M)
>30
>30
85
67
17
nd
nd
nd
nd
>30
>30
74
58
26
1
>40
13
19
hERG (% @ 10
Na+ (% @ 10
L-type Ca++ (% @ 10
l
M)
M)
l
l
M)
CYP3A4 IC50
CYP2C9 IC50
(
(
l
M)
l
M)
CYP2C19 IC50
CYP1A2 IC50
(
l
M)
(
l
M)
nd, not determined.
tionally,
a direct comparison of DPP4 inhibition between
compounds 13 (Ki = 28 nM) and 29 (Ki = 64 nM), and compounds
15 (Ki = 22 nM) and 30 (Ki = 67 nM) indicated that comparable
compounds in the pyrazolopyrimidine series were 2–3-fold more
active than those in the triazolopyrimidine series.
son of DPP4 activity and DPP8/DPP9 selectivity among the three
respective azolopyrimidine series. Compounds 46–52 were gener-
ated to assess the SAR associated with the aryl substitution pat-
tern. All of these compounds were active against DPP4 and
displayed wide DPP8/DPP9 (200- to >2000-fold) selectivity. The
degree of DPP4 activity, however, exhibited more of a regiochem-
ical dependence (steric) than type of substitution (electronic), in
that ortho- and para-substituted aryls (46, 48, 51, and 52) were
preferred over meta-substituted aryls (47 and 50).
A direct comparison of the three phenyl substituted azolopyr-
imidines 15, 30, and 45 shows that of the three fused heterobicy-
clic cores, the imidazolopyrimidine core possessed the most
favorable attributes (Table 4). While all three cores showed excel-
lent selectivity over DPP8 and DPP9, imidazolopyrimidine com-
pound 45 was the most potent against DPP4, and also displayed
improved hERG, Na ion channel, and CYP liabilities than the other
two heterobicyclic systems.
Similarly, imidazolopyrimidines were prepared and evaluated
as DPP4 inhibitors. The chemistry utilized to prepare these analogs
was similar to that employed in the pyrazolopyrimidine and triaz-
olopyrimidine syntheses (Scheme 3). Formation of dihydropyrimi-
dine 43, however, required two steps. Compound 8 was first
condensed with 7 to give the desired Knoevenagel product 41,
which in turn, was condensed with the appropriate 2-aminoimi-
dazole 42 obtained commercially or prepared according to the lit-
erature.19 The dihydropyrimidine 43 was then converted to the
desired 5-aminomethyl imidazolopyrimidines (44–52) using the
same methods as described earlier.
Several compounds in this series were quite potent against
DPP4 and most had no significant DPP8/DPP9 inhibition
(Table 3). Compound 45 was synthesized to allow direct compari-
Based on its overall profile, the in vivo activity of compound 45
was assessed using an oral glucose tolerance test (oGTT) in ob/ob
mice, dosing at 3 and 10 l
mol/kg 1 h prior to a glucose challenge.20
O
O
Cl
Blood samples were taken and analyzed for insulin 15 min post
oGTT. Relative to the vehicle treated mice, the animals receiving
O
O
R
OMe
CHO
N
a
NH2
42
+
+
OMe
N
H
the 3
l
mol/kg dose had a mean increase in plasma insulin of 51%
mol/kg dose had a mean increase in
Cl
7
8
41
Cl
Cl
and those receiving the 10
l
H
N
plasma insulin of 74%. The pursuit of potent small molecule
DPP4 inhibitors that are selective over DPP8 and DPP9 led to the
discovery of pyrazolo-, triazolo-, and imidazolopyrimidine-based
inhibitors. These novel leads represent potential scaffolds for the
design of clinically useful DPP4 inhibitors. With potency and selec-
tivity already achieved, further exploration of SAR around the
periphery of the imidazolopyrimidine core was targeted in order
to further minimize CYP, hERG, and ion channel liabilities of the
current compounds in this class. The results of these explorations
will be reported in due course.
N
N
N
R
R
N
NH2
N
CO2Me
Cl
b
c-g
Cl
43
44-52
Cl
Cl
Scheme 3. Reagents and conditions: (a) piperidine, IPA, AcOH, 70%; (b) DMF, 70 °C;
(c) DDQ, CH2Cl2, 26–49% over two steps; (d) DIBAL, THF, 38–53%; (e) MsCl, CH2Cl2,
Et3N, 87–100%; (f) NaN3, DMF, 50 °C, 79–100%; (g) PPh3, THF, H2O, 50 °C, 56–72%.

4398
R. P. Brigance et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4395–4398
13. Feng, J.; Zhang, Z.; Wallace, M. B.; Stafford, J. A.; Kaldor, S. W.; Kassel, D. B.;
References and notes
Navre, M.; Shi, L.; Skene, R. J.; Asakawa, T.; Takeuchi, K.; Xu, R.; Webb, D. R.;
Gwaltney, S. L. J. Med. Chem. 2007, 50, 2297.
14. Eckhardt, M.; Langkopf, E.; Mark, M.; Tadayyon, M.; Thomas, L.; Nar, H.;
Pfrengle, W.; Guth, B.; Lotz, R.; Sieger, P.; Fuchs, H.; Himmelsbach, F. J. Med.
Chem. 2007, 50, 6450.
15. Pattzi, H. M. R.; Pitale, S.; Alpizar, M.; Bennett, C.; O’Farrell, A. M.; Li, J.;
Cherrington, J. M.; Guler, H.-P.PHX1149-PROT202 Study Group Diabetes. Obes.
Metab. 2010, 12, 348.
16. For other small molecular DPP-IV inhibitors, see: Havale, S. H.; Pal, M. Bioorg.
Med. Chem. 2009, 17, 1783. and references cited therein.
1. Diabetes Facts and Figures. International Diabetes Foundation, October 6, 2009.
http://www.idf.org/Facts_and_Figures.; (b) Wild, S.; Roglic, C.; Green, A.;
Sicree, R.; King, H. Diabetes 2004, 27, 1047.
2. For latest information, please visit the website of World Health Organization at
http://www.who.int/diabetes/facts/world_figures/en/.
3. Ross, S. A.; Gulve, E. A.; Wang, M. Chem. Rev. 2004, 104, 1255.
4. Rizzo, M.; Rizvi, A. A.; Spinas, G. A.; Rini, G. B.; Berneis, K. Expert Opin. Invest.
Drugs 2009, 18, 1495.
5. (a) Holst, J. J.; Deacon, C. J. Diabetes 1998, 47, 1663; (b) Lankas, G. R.; Leiting, B.;
Roy, R. S.; Eiermann, G. J.; Beconi, M. G.; Biftu, T.; Chan, C. C.; Edmonson, S.;
Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.; Lyons, K. A.; Ok, H. O.; Patel, R. A.;
Petrov, A. N.; Pryor, K. A.; Qian, X.; Reigle, L.; Woods, A.; Wu, J. K.; Zaller, D.;
Zhang, X.; Zhu, L.; Weber, A. E.; Thornberry, N. A. Diabetes 2005, 54, 2988.
6. Ahrén, B.; Hughes, T. E. Endocrinology 2005, 146, 2055.
7. (a) Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. Lancet 2002, 359, 824; (b)
Nauck, M. A.; Wollschlaeger, D.; Werner, J.; Holst, J. J.; Orskov, C.; Creutzfeldt,
W.; Williams, B. Diabetologia 1996, 39, 1546.
8. Hansen, L.; Deacon, C. F.; Orskov, C.; Holst, J. J. Endocrinology 1999, 140, 5356.
9. Ahrén, B. Expert Opin. Invest. Drugs 2006, 15, 431.
10. Kim, D.; Wang, L.; Beconi, M.; Fisher, M. H.; He, H.; Hickey, G. J.; Kowalchick, J.
E.; Leiting, B.; Lyons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.; Petrov, A.;
Scapin, G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt, M. J.; Zhang, B. B.; Zhu, L.;
Thornberry, N. A.; Weber, A. E. J. Med. Chem. 2005, 48, 141.
11. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson,
J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S. P.; Abboa-Offei, B.;
Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.;
Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med. Chem.
2005, 48, 5025.
17. Settepani, J. A.; Stokes, J. B. J. Org. Chem. 1968, 33, 2606.
18. Magnin, D. R.; Robl, J. A.; Sulsky, R. B.; Augeri, D. J.; Huang, Y.; Simpkins, L. M.;
Taunk, P. C.; Betebenner, D. A.; Robertson, J. G.; Abboa-Offei, B. E.; Wang, A.;
Cap, M.; Xin, L.; Tao, L.; Sitkoff, D. F.; Malley, M. F.; Gougoutas, J. Z.; Khanna, A.;
Huang, Q.; Han, S. P.; Parker, R. A.; Hamann, L. G. J. Med. Chem. 2004, 47, 2587.
19. Little, T. L.; Webber, S. E. J. Org. Chem. 1994, 59, 7299.
20. Oral glucose tolerance test in ob/ob mice. Male 13–14 weeks old ob/ob mice
(Jackson Labs) were maintained under constant temperature and humidity
conditions, a 12 h:12 h light–dark cycle, and had free access to a 10% fat rodent
diet (D1245B Research Diets) and tap water. After an overnight fasting period,
animals were dosed orally with vehicle (water) or DPP4 inhibitor (3 and
10
lmol/kg) at À60 min. Two blood samples were collected at À60 and 0 min
by tail bleed for glucose and insulin determinations. Glucose (2 g/kg) was then
administered orally (at 0 min). Additional blood samples were collected at 15,
30, 60, 90, and 120 min for glucose and insulin determinations. Blood samples
were collected into EDTA containing tubes (Sarstedt). Plasma insulin was
assayed using a mouse insulin ELISA kit (ALPCO Diagnostics). Data represent
the mean of 12–24 mice/group. All procedures were performed according to
BMS-IACUC guidelines.
12. Villhauer, E.; Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.; Dunning, B. F.; Prasad,
K.; Mangold, B. L.; Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46, 2774.