Design, synthesis and biological evaluation of novel aminomethyl- piperidones based DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.03.009

A series of novel aminomethyl-piperidones were designed and evaluated as potential DPP-IV inhibitors. Optimized analogue 12v ((4S,5S)-5-(aminomethyl)-1- (2-(benzo[d][1,3]dioxol-5-yl)ethyl)-4-(2,5-difluorophenyl)piperidin-2-one) showed excellent in vitro p

Full text of DOI:10.1016/j.bmcl.2014.03.009

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1918–1922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel
aminomethyl-piperidones based DPP-IV inhibitors q
a,
b,
a
a
a
Pradip Jadav a,b, , Rajesh Bahekar , Shailesh R. Shah , Dipam Patel , Amit Joharapurkar , Mukul Jain ,
⇑
⇑
⇑
Kalapatapu V. V. M. Sairam a, Praveen Kumar Singh a
a Zydus Research Centre, Sarkhej-Bavla N.H. 8A Moraiya, Ahmedabad 382210, India
b Department of Chemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel aminomethyl-piperidones were designed and evaluated as potential DPP-IV inhibitors.
Optimized analogue 12v ((4S,5S)-5-(aminomethyl)-1-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-4-(2,5-difluo-
rophenyl)piperidin-2-one) showed excellent in vitro potency and selectivity for DPP-IV over other serine
proteases. The lead compound 12v showed potent and long acting antihyperglycemic effects (in vivo),
along with improved pharmacokinetic profile.
Received 20 December 2013
Revised 18 February 2014
Accepted 4 March 2014
Available online 12 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Aminomethyl-piperidones
DPP-IV inhibitors
Long-acting
Selective
Antidiabetic
The prevalence of Type 2 diabetes mellitus (T2DM) is rapidly
increasing (ꢀ371 million diabetic patients worldwide) and there
is a great need for new drug classes to ameliorate hyperglycemia,
while addressing additional accompanying elements of the meta-
bolic syndrome.1 Dipeptidyl peptidase-IV (DPP-IV) is a serine pro-
tease,2 responsible for the inactivation of glucagon-like peptide 1
(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP),
both of which enhance insulin secretion in a glucose-dependent
manner.3 DPP-IV inhibitors are a new class of oral medications,
which have been in use for 7 years, as a second-line therapy.
DPP-IV inhibitors are generally well tolerated, safe (low risk of
hypoglycemia) and weight neutral. There are currently eight glip-
tins approved worldwide with several more on the way.4
In recent years, a large variety of scaffolds being discovered as a
next generation DPP-IV inhibitors, particular efforts being made to
develop the long acting DPP-IV inhibitors.4,5 Two drugs (Omariglip-
tin and Trelagliptin) are currently under development for once-
weekly dosing to improve patients compliance.4 Structurally,
DPP-IV enzyme resembles with several other protease, so while
designing new class of DPP-IV inhibitors, it is essential to consider
selectivity of DPP-IV inhibitors over other serine protease, espe-
cially DPP-2, DPP-8 and DPP-9.6
Structurally distinct and rigid analogs of Sitagliptin (1), such as
N-aryl aminopiperidine (2), aminopiperidine-fused imidazoles (3)
and tetrahydropyran (4) derivatives were identified as a novel class
of DPP-IV inhibitors (Fig. 1).7–10 These newly discovered DPP-IV
inhibitors exhibit potent DPP-IV inhibitory activity, good off-target
selectivity and improved pharmacokinetic profiles. Earlier, we
reported peptidomimetics based long acting DPP-IV inhibitors.11
In continuation to our ongoing research on DPP-IV inhibitors, we
report herein design, synthesis and biological evaluation of novel
aminomethyl-piperidones (12a–v, 13a–e and 14a–e) based DPP-
IV inhibitors. Title compounds are designed based on the piperi-
done skeleton and we anticipated that the aminomethyl and the
amide groups of the piperidone ring might contribute for improved
pharmacokinetic and pharmacodynamic effects, along with the
potent and selective DPP-IV inhibitory activity.
As depicted in Scheme 1, synthesis of the aminomethyl-
piperidones based DPP-IV inhibitors (12a–v, 13a–e and 14a–e)
commenced with
a Horner–Wadsworth–Emmons reaction of
aldehydes (5a–c), followed by Michael addition, to get diester
(6a–c). Reduction of nitrile group of 6a–c by hydrogenation, using
Adam’s catalyst, followed by cyclization and ester regeneration
by trimethylsilyldiazomethane yielded piperidone-carboxylate
(7a–c), with >85% trans selectivity.12 Trans racemic mixture
[(3R,4S) and (3S,4R)] of (7a–c) were isolated in pure form by
q
ZRC communication no.: 459 (Part of PhD thesis work of Mr. P. Jadav).
Corresponding authors. Tel.: +91 2717 665555; fax: +91 2717 665355.
E-mail addresses: jadavpradeep37@rediffmail.com (P. Jadav), rajeshbahekar@
⇑
zyduscadila.com (R. Bahekar), shailesh-chem@msubaroda.ac.in (S.R. Shah).
http://dx.doi.org/10.1016/j.bmcl.2014.03.009
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

P. Jadav et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1918–1922
1919
F
F
F
F
F
F
NH2
F
F
NH2
NH2
O
N
N
N
N
N
F
N
N
N
N
N
CF3
(Sitagliptin; 1)
(2)
CF3
(3)
N
F
NH2
NH2
Ar
N
R
F
O
N
O
NH
N
(4)
12a-v, 13a-e & 14a-e
Figure 1. Structurally diverse small molecule based-DPP-IV inhibitors.
O
OCH3
NH
as n-hexane and 0.1% diethyl amine in EtOH (98:02)). Further, pro-
tection of primary amine of 10a–c with Boc-group and subsequent
oxidative removal of PMB group gave Boc-aminopiperidones (11a–
c). Various haloheterocycles/halo-aromatics of the interest were
coupled with 11a–c, by Goldberg reaction13 or by nucleophilic sub-
stitution, followed by Boc-deprotection to get the chiral pure
(4S,5S) aminomethyl-piperidones (12a–v, 13a–e and 14a–e).14 All
the test compounds obtained were purified by preparative HPLC
(yield 70–85%; HPLC purity >97% and chiral purity >97%ee) and
characterized by various spectroscopic techniques (13C NMR, 1H
NMR and ESI MS). Elemental analyses were determined within
0.04% of theoretical values (see Supplementary data for analytical
and spectral data).
O
OCH3
CN
Ar
a, b
c, d, e
Ar CHO
Ar
5a-c
O
OCH3
O
5a: Ar =2,5-difluoro phenyl
6a-c
7a-c
5b:
Ar =2,4,5-trifluoro phenyl
5c: Ar =2,4-dichloro phenyl
O
OH
NH2
N
N
j, k
h, i
Ar
Ar
f, g
O
Ar
N
N
PMB
PMB
PMB
O
O
O
8a-c
9a-c
10a-c
H
The in vitro DPP-IV inhibitory activity was determined in order
to establish the structure-activity relationship (SAR).15 Three sets
of the aminomethyl-piperidones (12a–v, 13a–e and 14a–e) were
prepared (Table 1). In the first set (Ar = 2,5-difluoro phenyl), 22
compounds (12a–v) were prepared by coupling 2,5-difluoro phe-
nyl-aminopiperidone (11a) with various halo-heterocycles/halo-
aromatics. In the second set (Ar = 2,4,5-trifluoro phenyl), 5 com-
pounds (13a–e) were prepared by replacing 2,5-difluoro phenyl
with 2,4,5-trifluoro phenyl, while in third set (Ar = 2,4-dichloro
phenyl), 5 compounds (14a–e) were prepared by replacing 2,5-di-
fluoro phenyl with 2,4-dichloro phenyl. All the test compounds
showed varying degrees of DPP-IV inhibitory activity (IC50),
depending on the nature of the substituents.
Within the first set (12a–v), test compounds showed diverse
DPP-IV inhibitory activity depending on the nature of substituents
on piperidone ring system. Compounds with electron withdrawing
groups (12b: –CN, 12c: –F and 12d: –CF3) at para-position of phenyl
ring system showed improved DPP-IV inhibitory activity, compared
to unsubstituted derivative (R = –Ph; 12a). Compounds with
electron donating groups (12e: –OMe and 12f: –SO2–Me) at para-
position of phenyl ring showed further improvement in in vitro
DPP-IV inhibitory activity. Replacement of phenyl ring system with
3-pyridyl (12g) and further substitutions with electron donating
(12h) and withdrawing (12i and 12j) groups at para-position
showed moderate DPP-IV inhibitory activity. Replacement of phe-
nyl ring system with quinoline (12m), triazolo[4,3-a]pyrazine
(12n), 2-methyl-pyrimido[1,2-b]pyridazinone (12o), benzyl (12k)
and further substitutions with electron withdrawing (12l) groups
at para-position showed moderate DPP-IV inhibitory activity.
Substitutions with ethylbenzene (12p), ethylpyridine (12q),
NH2
O
N
Ar
O
Ar
n, o
l, m
N
R
NH
O
O
12a-12v Ar = 2,5-difluoro phenyl
Ar = 2,4,5-trifluoro phenyl
14a-14e Ar = 2,4-dichloro phenyl
11a-c
13a-13e
Scheme 1. Synthesis of compounds 12a–v, 13a–e and 14a–e. Reagents and
conditions: (a) (Et2O)2POCH2COOMe, Na2CO3, EtOH; (b) NCCH2COOMe, NaOMe,
MeOH; (c) H2, PtO2, HCl, MeOH; (d) K2CO3, toluene/MeOH; (e) Me3SiCHN2, Et2O/
MeOH; (f) PMB-Br, NaHMDS, THF/DMF(4:1), ꢁ78 °C; (g) LiAlH4, THF, 0 °C; (h)
CH3SO2Cl, NEt3, DCM, 0 °C; (i) potassium phthalimide, DMF, 90 °C; (j) NH2–NH2,
EtOH, 25 °C; (k) chiral resolution:
D-tartaric acid, MeOH; (l) Boc2O, NEt3, THF/
H2O(3:2), 25 °C; (m) CAN, CH3CN/H2O(3:1), 25 °C; (n) R–X, CuI, N,N0-dimethyleth-
ylenediamine, toluene, reflux or R–X, NaH, DMF, 0 °C to 25 °C; (o) concd HCl/
EtOAc(1:3), ꢁ50 °C, 2 h, 0 °C, 1 h.
removing corresponding cis racemic mixture [(3R,4R) and (3S,4S)],
by column chromatography (mobile phase: 0–3% methanol in
DCM, using 100–200 mesh silicagel). Amide –NH protection of
trans racemic 7a–c with para-methoxy benzyl (PMB) group and
reduction of ester with lithium aluminium hydride (LiAlH4)
yielded trans racemic alcohol (8a–c). Subsequently, 8a–c were con-
verted to a good leaving group (methanesulfonate derivatives),
which upon treatment with potassium phthalimide via Gabriel
synthesis type reaction lead to the formation of trans racemic
phthalimido-piperidones (9a–c).
Hydrazinolysis of phthalimido group of 9a-c lead to the forma-
tion of trans racemic aminopiperidones (10a–c). trans racemic
10a–c was subjected for chiral resolution (10a–c was added to a
solution of
D
-tartaric acid (1.1 equiv
D
-tartaric acid, dissolved in
dimethylpyrazolo[1,5-a]-pyrimidine
(12r),
3-methyl-triazolo
100 ml methanol) and the mixture was stirred for 15 h at 25 °C,
solid precipitated was filtered off, washed with methanol
(200 ml) and dried to get enantiomerically pure (4S,5S) desired
piperidones (10a–c) as a tartrate salt, with >97% ee (chiral HPLC
analysis conditions: CHIRALCEL OD-H column, using mobile phase
[4,3-b]pyridazine (12s), 3-trifluoromethyl-triazolo[4,3-b]pyrida-
zine (12t) and 2-trifluoromethyl-triazolo[1,5-b]pyridazine (12u)
showed good DPP-IV inhibitory activity, while 12v (methylenedi-
oxy phenethyl) showed superior DPP-IV inhibitory activity
(IC50: 8.5 0.4 nM), compared to Sitagliptin (IC50: 18 2.4 nM).

1920
P. Jadav et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1918–1922
Table 1
In vitro DPP-IV inhibitory activity of aminomethyl-piperidones (12a–v, 13a–e and 14a–e).*
**
IC50 (nM)
**
IC50 (nM)
**
IC50 (nM)
Compd
R
Compd
R
Compd
R
12a
1436 12.3
12l
910 3.1
13a
157 4.1
119 1.0
N
CF3
N
N
N
12b
12c
378 1.4
382 4.5
12m
12n
1034 21.2
13b
13c
CN
N
N
N
N
CF3
N
N
N
N
1023 3.1
997 13.5
125 2.7
111 2.1
N
F
N
O
N
N
N
N
CF3
N
12d
342 3.3
12o
13d
N
CF3
O
O
12e
12f
217 8.6
193 8.4
12p
12q
119 4.2
84 2.6
13e
14a
19 5.1
O
197 4.2
N
N
S
O2
N
N
N
N
N
N
12g
12h
12i
1388 5.9
452 3.7
443 5.3
12r
12s
12t
77.6 1.2
122 3.2
79 0.2
14b
14c
14d
148 3.7
134 7.3
137 9.6
N
N
N
N
CF3
N
N
N
N
N
N
N
O
N
N
N
CF3
N
N
N
N
CF3
N
N
N
N
CF3
CN
N
O
O
CF3
12j
404 7.7
12u
12v
74 0.9
14e
43 3.2
N
O
O
12k
885 11.2
8.5 0.4
Sitagliptin
—
18 2.4
Bold IC50 values of 12v and sitagliptin represents most potent compounds in Table 1.
*
DPP-IV inhibitory activity determined by fluorescence-based assay; fluorescence measured using Spectra Max fluorometer (Molecular Devices, CA) by exciting at 380 nm
and emission at 460 nm. IC50 determined using Graph Pad prism software.
DPP-IV inhibitory activity represented as IC50 (nM), expressed as the mean SD (n = 3).
**
In the second set (13a–e, Ar = 2,4,5-trifluoro phenyl), all the five
compounds showed good activity, but compared to 2,5-difluoro
phenyl series (Set-1 analogs, 12q, 12r, 12t, 12u and 12v), in vitro
DPP-IV inhibition were found to be bit weaker, while in set three
(14a–e, Ar = 2,4-dichloro phenyl), in vitro DPP-IV inhibition were
found to be slight weaker than Set-1 and Set-2 corresponding ana-
logs. Thus the nature and position of halogen atom on aromatic
ring system contributed significantly towards in vitro DPP-IV
inhibition.
The in vitro selectivity over serine protease, especially DPP-2,
DPP-8 and DPP-9 was evaluated for 12V and it showed >5000-fold
selectivity over DPP-2 and >10,000-fold selectivity over DPP-8 and
DPP-9.15 To assess the CYP liabilities, 12v was subjected for CYP1A2,
CYP2C8, CYP2C9, CYP2D6, CYP2C19 and CYP3A4 inhibition studies
(@ 1, 10 and 100
was found to be devoid of CYP1A2, CYP2C8, CYP2C9, CYP2D6,
CYP2C19 and CYP3A4 inhibition up to 100
M concentrations.16
lM concentrations) and the test compound 12v
l
Detailed pharmacodynamic (PD) as well as pharmacokinetic
(PK) profiling of 12v was carried out. The in vivo antidiabetic activ-
ity of 12v and Sitagliptin (@ 3 mg/kg, p.o.) was evaluated in male
C57BL/6J mice, using OGTT (oral glucose tolerance test) protocol
and changes in serum glucose levels (AUC glucose up to 240 min;
mg/dL) was estimated (Fig. 2).17 Compound 12v showed good oral
antidiabetic activity (% decrease in AUC glucose 38.9 5.20), which
*P<0.05, Two-Way ANOVA followed by Bonferroni posttest, Mean SEM
Figure 2. In vivo antidiabetic activity of 12v and Sitagliptin in C57 mice.
was found to be better than Sitagliptin (% decrease in AUC glucose
17.9 4.58). In C57 mice, it was interesting to observe that 12v
showed suppression in the blood glucose at all the time points

P. Jadav et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1918–1922
1921
600
400
200
*
*
*
*
Vehicle Control, PO
Sitagliptin (3mg/kg, PO)
12v (3mg/kg, PO)
*
*
0.51
2
4
6
8
214
Hours post treatment
Drug
*P<0.05, Two-Way ANOVA followed by Bonferroni post test, M SEM
Figure 3. In vivo antidiabetic activity of 12v and Sitagliptin in db/db mice.
(15, 30, 60, 120 and 240 min) compared to vehicle control, while
Sitagliptin showed blood glucose reduction only at 30 and 60 min.
Further to understand the duration of action and effect of 12v
on post-prandial glucose excursion, single dose (@ 3 mg/kg, po)
antidiabetic activity of 12v and Sitagliptin was evaluated in fed-
db/db mice (hyperglycemic animals) for 24 h (Fig. 3). Under fed
condition, compared to vehicle control, 12v and Sitagliptin showed
good antidiabetic activity (% decrease in AUC glucose 38.29 12.13
and 20.80 11.06, respectively) up to 2 h. However, 12v showed
prolonged suppression of serum glucose levels (% decrease in
AUC glucose 20.62 7.05 for 12v and 1.48 11.84 for Sitagliptin,
up to 24 h).
Figure 4. Key interactions of compound 12v and Sitagliptin with active sites of
DPP-IV enzyme. Binding pose of compound 12v (Orange) and Sitagliptin (Maroon)
in the DPP-IV active site is indicated (Surface view: Blue), wherein both compounds
interact closely with key residues of site S1, S2 and S3.
(XP) Glide docking method (Fig. 4).20 The X-ray structure of the
DPP-IV enzyme (PDB ID: 2OQI) was obtained from the protein data
bank and the protein structure was prepared using protein prepa-
ration wizard module of Schrödinger. For docking study, the
ligands were geometrically optimized and prepared by using
ligprep module of Schrödinger.18 The overlay of binding poses of
12v (Orange) and Sitagliptin (Maroon) in the DPP-IV active site is
shown in Figure 4. As observed with Sitagliptin, 12v docks very
well into all the three sites (G-scores ꢁ11.81 (9/9) and ꢁ10.99
(9/9) for 12v and Sitagliptin respectively). Although, G-score of
12v and Sitagliptin are comparable, however, in vitro, DPP-IV
IC50 of 12v is half of that of Sitagliptin, which could be due to favor-
able interactions of 12v, in all the three binding pockets. Di-fluoro-
phenyl ring of 12v occupies S1 pocket. In S2 pocket, aminomethyl
groups of piperidone ring forms H-bonding with the side-chains
of Glu205 and Glu206 dyad, while methylenedioxy phenyl ring of
12v accommodates very well in S3 pocket, which together supports
excellent in vitro DPP-IV activity and selectivity of 12v over other
protease.
A comparative single dose (3 mg/kg iv or p.o.) PK profile of 12v
and Sitagliptin was evaluated in male C57BL/6J mice (n = 6) and
the various PK parameters (Tmax, T1/2, Cmax, AUC and %F) were re-
corded (Table 2).18 In PK study, 12v showed rapid Tmax, higher
AUC (ꢀtwofold compared to Sitagliptin), extended half-life (T1/2
:
>8 h) compared to Sitagliptin and good oral bioavailability (%F:
79.5%). Compound 12v showed extended half-life and higher
AUC, which could be due to its low clearance compared to Sitaglip-
tin (elimination rate constant (kel; hꢁ1), 0.12 0.02 for 12v and
0.84 0.14 for Sitagliptin). Thus improved pharmacokinetic profile
of compound 12v justifies its potent and prolonged antidiabetic
activity in C57 and db/db mice.
Interestingly, various gliptins, currently used in the clinic
(Sitagliptin, Vildagliptin, Saxagliptin, Alogliptin and Linagliptin),
exhibit short half-life thereby requires once or twice daily drug
administration.19 Further to regulate the pre- and post-prandial
blood glucose and thereby to control HbA1c, several long-acting
DPP-IV inhibitors (Omarigliptin and Trelagliptin) are under devel-
opments, as once-weekly drugs.4 Their clinical efficacy and side ef-
fects profile appear to be comparable with other gliptins in the
class, however, their infrequent dosing creates a niche and pro-
motes patients compliance.4 In this context, overall pre-clinical
profile of 12v demonstrated added advantages over currently prac-
ticed gliptins and appears to serve as long-acting DPP-IV inhibitors.
The molecular docking analysis of 12v and Sitagliptin, in the
binding pocket of DPP-IV was carried out using extra precision
In summary, we report discovery of novel aminomethyl-
piperidone derivatives as potent, selective and long acting DPP-IV
inhibitors for the treatment of T2DM. The lead compound 12v
((4S,5S)-5-(aminomethyl)-1-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-4-
(2,5-difluorophenyl)-piperidin-2-one) showed prolonged suppres-
sion of pre-and post-prandial blood glucose levels (in vivo), which
correlates with its extended PK profile.
Acknowledgment
We are grateful to the management of Zydus Group for encour-
agement and support.
Table 2
Pharmacokinetic study parametersa of 12v and Sitagliptin
Compd
Tmax (h)
Cmax
(l
g/ml)
T1/2 (h)
AUC (0-
a
) h
lg/ml
%Fb
12v
Sitagliptin
0.28 0.12
0.22 0.10
0.42 0.03
0.31 0.01
8.99 0.31
1.56 0.11
1.01 0.09
0.56 0.02
79.5
75.7
a
In male C57BL/6J mice (n = 6), compounds were administered orally (p.o.) at 3 mg/kg dose and plasma concentration was analyzed by LC–MS, values indicate mean SD.
Oral bioavailability (%F) was calculated wrt to iv AUC (12v: 1.27 0.08 and Sitagliptin: 0.74 0.09 h lg/ml) administered at 3 mg/kg dose, iv.
b

1922
P. Jadav et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1918–1922
DPP-IV, DPP-2, DPP-8 or DPP-9 enzymes in the presence of various
Supplementary data
concentrations of test compounds. Reaction was carried out at pH 7.8
(HEPES buffer 25 mM containing 1.0% BSA, 140 mM NaCl, 16 mM MgCl2, 2.8%
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03.
009.
DMSO) in a total volume of 100
was terminated with acetic acid (25
was measured using Spectra Max fluorometer (Molecular Devices, Sunnyvale
CA) by exciting at 380 nm and emission at 460 nm. The IC50 values were
determined for test compounds using Graph Pad prism software.
l
l at 25 °C for 30 min., in the dark. Reaction
ll of 25% solution). Activity (fluorescence)
16. Dierks, E. A.; Stams, K. R.; Lim, H. K.; Cornelius, G.; Zhang, H.; Ball, S. E. Drug
Metab. Dispos. 2001, 29, 23.
References and notes
17. (a) Chen, D.; Wang, M. Diabetes Obes. Metab. 2005, 7, 307; (b) Kim, J. G.; Baggio,
L. L.; Bridon, D. P.; Castaigne, J. P.; Robitaille, M. F.; Jette, L.; Benquet, C.;
Drucker, D. J. Diabetes 2003, 52, 751; (c) Study was conducted in male C57BL/6J
(using OGTT protocol) or without glucose load, in db/db mice (age 8–
12 weeks). All animal experiments were conducted according to the
internationally valid guidelines following approval by the ‘Zydus Research
Center Animal Ethical Committee’. Two days prior to the study, the animals
were randomized and divided into 2 groups (n = 6), based upon their fed
1. IDF Diabetes Atlas 6th addition update, 2013. Available from: http://
www.idf.org/worlddiabetesday/toolkit/gp/facts-figures.
2. Lambeir, A. M.; Durinx, C.; Scharpé, S.; Meester, I. Crit. Rev. Clin. Lab. Sci. 2003,
40, 209.
3. Kim, W.; Egan, J. M. Pharmacol. Rev. 2008, 60, 470.
4. Cahn, A.; Raz, I. Expert Opin. Emerg. Drugs 2013, 18, 245.
5. (a) Mulakayala, M.; Reddy, C. H. U.; Iqbal, J.; Pal, M. Tetrahedron 2010, 66, 4919;
(b) Mendieta, L.; Tarrago, T.; Giralt, E. Expert Opin. Ther. Pat. 2011, 21, 1693.
6. Kang, N. S.; Ahn, J. H.; Kim, S. S.; Chae, C. H.; Yoo, S. Bioorg. Med. Chem. Lett.
2007, 17, 3716.
glucose levels. Animals were left for
2 days under acclimatization and
maintained on standard diet. On the day of experiment, food was
a
withdrawn from all the cages, water was given ad-libitum and were kept for
overnight fasting. Briefly, in OGTT protocol (C57 mice) overnight fasted mice
were dosed orally (p.o.) with the test compounds (3 mg/kg), 0.5 h prior to the
oral glucose load, while in db/db mice, fed mice were dosed orally (p.o.) with
the test compounds (3 mg/kg) and the blood samples were collected at various
time points. Blood samples were centrifuged and the separated serum was
immediately subjected for the glucose estimation. The glucose estimation was
carried out with DPEC-GOD/POD method (Ranbaxy Fine Chemicals Limited,
Diagnostic division, India), using Spectramax-190, in 96-microwell plate
reader (Molecular devices Corporation, Sunnyvale, California). Mean values
of duplicate samples were calculated using Microsoft excel and the Graph Pad
Prism software (Ver 4.0) was used to plot an area under the curve (0–240 min
AUC). The AUC obtained from graphs were analyzed for two-way ANOVA,
followed by Bonferroni post test, using Graph Pad prism software.
7. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; 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.
8. Biftu, T.; Scapin, G.; Singh, S.; Feng, D.; Becker, J. W.; Eiermann, G.; Huaibing He,
H.; Lyons, K.; Patel, S.; Petrov, A.; Sinha-Roy, R.; Zhang, B.; Wu, J.; Zhang, X.;
Doss, G. A.; Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett. 2007, 17,
3384.
9. Edmondson, S. D.; Mastracchio, A.; Cox, J. M.; Eiermann, G. J.; He, H.; Lyons, K.
A.; Patel, R. A.; Patel, S. B.; Petrov, A.; Scapin, G.; Wu, J. K.; Xu, S.; Zhu, B.;
Thornberry, N. A.; Sinha-Roy, R.; Weber, A. E. Bioorg. Med. Chem. Lett. 2009, 19,
4097.
10. Biftu, T.; Qian, X.; Chen, P.; Feng, D.; Scapin, G.; Gao, Y. D.; Cox, J.; Sinha-Roy, R.;
Eiermann, G.; He, H.; Lyons, K.; Salituro, G.; Patel, S.; Petrov, A.; Xu, F.; Xu, S. S.;
Zhang, B.; Caldwell, C.; Wu, J. K.; Weber, A. E. Bioorg. Med. Chem. Lett. 2013, 23,
5361.
11. Jadav, P.; Bahekar, R.; Shah, S. R.; Patel, D.; Joharapurkar, A.; Kshirsagar, S.; Jain,
M.; Shaikh, M.; Sairam, K. V. V. M. Bioorg. Med. Chem. Lett. 2012, 22, 3516.
12. Moos, W. H.; Gless, R. D.; Rapoport, H. J. Org. Chem. 1981, 46, 5064.
13. Klapars, A.; Xiaohua Huang, X.; Stephen, L.; Buchwald, S. L. J. Am. Chem. Soc.
2002, 124, 7421.
14. (a) Cox, J. M.; Edmondson, S. D.; Harper, B.; Weber, A. E. WO 039325, 2006; PCT
Int. Appl. 2006.; (b) Biftu, T.; Chen, P.; Cox, J. M.; Weber, A. E. WO 056708, 2010;
PCT Int. Appl. WO 056708, 2010.
15. (a) Blackmon, D. L.; Watson, A. J.; Montrose, M. H. Anal. Biochem. 1992, 200,
352; (b) In vitro enzyme (DPP-IV, DPP-2, DPP-8 and DPP-9) inhibitory activity
was determined using fluorescence-based assay. The Gly-Pro-AMC was used as
a substrate (which is cleaved by the enzymes to release the fluorescent AMC)
and soluble human proteins (DPP-IV, DPP-2, DPP-8 and DPP-9 enzymes)
produced in a baculovirus expression system (Life Technologies) was used as
18. Briefly, for single dose PK study, test compounds were administered orally/iv
on a body weight basis (3 mg/kg) to overnight fasted male C57BL/6 J mice.
Serial blood samples were collected in microcentrifuge tubes containing EDTA
at pre-dose, 0.15, 0.3, 0.5, 0.75, 1, 2, 4, 6, 8, 24, 36 and 48 h post-dose after
compounds administration. Approximately 0.2 ml of blood was collected at
each time point and centrifuged at 4 °C. The obtained plasma was frozen,
stored at ꢁ70 °C and the concentrations of compounds in plasma were
determined by the LC–MS/MS (Shimadzu LC10AD, USA), using YMC
hydrosphere C18 (2.0 ꢂ 50 mm,
3
l
m) column (YMC Inc., USA). The
pharmacokinetic parameters, such as Tmax
,
t1/2 Cmax AUC and %F were
,
,
calculated using a non-compartmental model of WinNonlin software version
5.2.1.
19. (a) Baetta, R.; Corsini, A. Drugs 2011, 71, 1441; (b) Scheen, A. J. Expert Opin.
Pharmacother. 2012, 13, 81.
20. Schrodinger Suite 2010, Glide version 5.8, Prime version 2.2; Schrodinger, LLC:
New York, 2010.
the enzyme source. The H-Gly-Pro-AMC (200 lM) was incubated with either