Long-acting peptidomimetics based DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.078

Pyrrolidine based peptidomimetics are reported as potent and selective DPP-IV inhibitors for the treatment of T2DM. Compounds 16c and 16d showed excellent in vitro potency and selectivity towards DPP-IV and the lead compound 16c showed sustained antihyper

Full text of DOI:10.1016/j.bmcl.2012.03.078

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3516–3521
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Long-acting peptidomimetics based DPP-IV inhibitors q
Pradip Jadav a,b, Rajesh Bahekar a, , Shailesh R. Shah b, , Dipam Patel a, Amit Joharapurkar a,
⇑
⇑
Samadhan Kshirsagar a, Mukul Jain a, Mubeen Shaikh a, Kalapatapu V.V.M. Sairam a
a Zydus Research Centre, Sarkhej-Bavla N.H. 8A Moraiya, Ahmedabad 382 210, India
b Department of Chemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrrolidine based peptidomimetics are reported as potent and selective DPP-IV inhibitors for the treat-
ment of T2DM. Compounds 16c and 16d showed excellent in vitro potency and selectivity towards
DPP-IV and the lead compound 16c showed sustained antihyperglycemic effects, along with improved
pharmacokinetic profile.
Received 9 December 2011
Revised 12 March 2012
Accepted 22 March 2012
Available online 28 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Peptidomimetic
DPP-IV inhibitors
Slow-binding
Selectivity
Antidiabetic
Long-acting
Dipeptidyl peptidase-IV (DPP-IV) is a serine protease, which
selectively cleaves first two amino acids of glucose-dependent
insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP-
1) thereby makes it inactive.1,2 Inhibition of DPP-IV activity ele-
vates endogenous GIP, GLP-1 and insulin levels thereby improve
glucose excursion. Thus, DPP-IV inhibitors hold great potential
for the treatment of type 2 diabetes mellitus (T2DM). 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-9.3
His740) and the S2 pocket involves key interactions with Glu205 and
Glu206 dyad. The S3 pocket (Ser209, Arg358 and Phe357) of DPP-IV dif-
fers a lot from other protease and it govern selectivity against DPP-
8 and 9.9
In T2DM patients, gliptins (Vildagliptin, Sitagliptin, Alogliptin
and Saxagliptin; Fig. 1) do not lower post-prandial glucose to
greater extent as monotherapy, but they are more effective in com-
bination.10 Gliptins demonstrated good oral bioavailability but due
to the rapid clearance, most of them exhibit shorter half-life, there-
by require repeated dosing. Thus, attempts are still underway to
develop long acting DPP-IV inhibitors, which could potentially pro-
vide sustained antidiabetic effect by reducing the post-prandial
glucose excursion and HbA1c (>1%), to anticipate cost and dosing
frequency. One of the approaches to develop long acting DPP-IV
inhibitor could be design of a cyanopyrrolidines based slow-bind-
ing inhibitors. The cyanopyrrolidines forms a reversible covalent
enzyme-inhibitor complex in which inhibitor bind and dissociates
slowly (two-step slow-binding inhibition). Consequently the
enzyme catalytic activity can be inhibited even after the free drug
has been cleared from the circulation, thereby cyanopyrrolidines
inhibits DPP-IV activity for longer duration despite their short
half-lives (Vildagliptin and Saxagliptin t1/2: ꢀ2–4 h).11 The NVP-
DPP728 represents first slow-binding cyanopyrrolidine-based
DPP-IV inhibitor and it showed good antidiabetic activity in clinical
trials, despite its short half-life (t1/2 ꢀ0.85 h).12 Recently RBx-0597,
has been reported as a potent, selective and slow-binding DPP-IV
inhibitor.13
DPP-IV inhibitors are classified as peptidomimetics (a- and b-ser-
ies) and non-peptidomimetics. In -series, pyrrolidine derivatives
a
have been widely explored and depending upon nature of substitu-
ents (Z; at the C2 position of pyrrolidine ring), it is divided into; irre-
versible (Z = diphenyl-phosphonate ester or O-acylhydroxamic acid)
and reversible (Z = boronic acid/nitrile) inhibitors.4 The NVP-
DPP728, Vildagliptin, Saxagliptin and Denagliptin represents ad-
vanced molecules in cyanopyrrolidine series.5 The b-series, such as
Sitagliptin and the non-peptidomimetic DPP-IV inhibitors, including
Alogliptin were developed through high-throughput screening
(HTS).6,7 The DPP-IV enzyme has three binding pockets/sites (S1, S2
and S3).8 The S1 pocket consists of catalytic triad (Ser630, Asn710 and
q
ZRC Communication no.: 389 (Part of PhD thesis work of Pradip Jadav).
⇑
Corresponding authors. Tel.: +91 2717 665555; fax: +91 2717 665355.
E-mail addresses: rajeshbahekar@zyduscadila.com (R. Bahekar), Shailesh-chem@
Considering clinical implication of long acting DPP-IV inhibitors
and to overcome the limitations of existing gliptins, we report
msubaroda.ac.in (S.R. Shah).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.078

P. Jadav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3516–3521
3517
F
NC
F
O
NH2
O
H
N
N
N
N
N
N
H
N
F
N
NC
CF3
MK-0431 (Sitagliptin; 2)
NVP-DPP728 (1)
O
OH
H3C
N
O
HO
H
NH2
N
O
N
N
N
N
H2N
NC
LAF-237 (Vildagliptin; 3)
CN
BMS-477118 (Saxagliptin; 4)
O
CN
SYR-322 (Alogliptin; 5)
Figure 1. Structurally diverse small molecule based-DPP-IV inhibitors.
design, synthesis and biological evaluation of pyrrolidine contain-
ing peptidomimetic based DPP-IV Inhibitors. The peptidomimetics
(11a–h, 12a–h, 15a–h, 16a–d and 17a,b) consist of substituted-
pyrrolidine ring attached to sterically hinder aromatic acid, with
suitable linkers/spacers.
fied by preparative HPLC (yield 70–85%; HPLC purity >99%) and
characterized by various spectroscopic technique (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).
Synthesis (Schemes 1 and 2) of peptidomimetics (11a–h, 12a–h,
15a–h, 16a–d and 17a,b) was carried out, using Fmoc-based solid
phase peptide synthesis (SPPS) approach, starting from commer-
cially available Rink-amide MBHA resin, preloaded with Fmoc-pro-
tected proline (6).14 Deprotection of 6 with piperidine (20% DMF)
and 1,3-diisopropycarbodiimide (DIC) coupling with Fmoc-pro-
The in vitro DPP-IV inhibitory activity was determined in order
to establish the structure–activity relationship (SAR).16 Two series
of the peptidomimetics (11a–h, 12a–h, 15a–h, 16a–d and 17a,b)
were prepared (Tables 1 and 2). In the first series (pyrrolidinecarb-
oxamides), 24 compounds (11a–h, 12a–h, 15a–h) were prepared
by linking proline with substituted benzoic acids, using suitable
spacers (set-1: b-Ala (11a–h); set-2: GABA (12a–h) and set-3:
PABA (15a–h)). In the second series (pyrrolidinecarbonitriles), six
compounds (16a–d and 17a,b) were prepared by replacing pyrroli-
dinecarboxamides with pyrrolidinecarbonitriles. All the test com-
pounds showed varying degrees of DPP-IV inhibitory activity
(IC50) depending on the nature of the substituents.
Within the first series (11a–h, 12a–h, 15a–h), the set-1 (11a–h)
containing b-alanine spacer attached to para-substituted benzam-
ides, showed diverse DPP-IV inhibitory activity depending on the
nature of substituents at the para-position. Compounds with elec-
tron donating groups (11b: –OMe and 11c: –OH) showed weak
tected b-Ala (b-alanine), GABA (c-amino butanoic acid) or PABA
(para-amino benzoic acid) provided the resin-bound Fmoc-pro-
tected dipeptides (7, 8 or 13). Deprotection of 7, 8 and 13 with
piperidine (20% DMF) and DIC coupling with substituted benzoic
acids gives resin-bound tripeptides (9a–h, 10a–h and 14a–h). Tri-
fluroacetic acid (TFA) mediated cleavage of resin-bound peptides (
9a–h, 10a–h and 14a–h) gives pyrrolidinecarboxamides (11a–h,
12a–h and 15a–h). Trifluroacetic anhydride (TFAA) mediated
dehydration of pyrrolidinecarboxamides (11d, 11f, 12d, 12f, 15d
and 15f) afforded title compounds as pyrrolidinecarbonitriles
(16a–d and 17a,b).15 All the test compounds obtained were puri-
NH2
R
R
N
N
b
H
c
H
N
O
O
N
N
O
O
FmocHN
n
O
n
O
n
O
O
11a-h (n= 2)
7 (n= 2)
8 (n= 3)
9a-h (n= 2)
12a-h (n= 3)
10a-h (n= 3)
a
N
O
Fmoc
NH2
6
N
N
N
O
O
O
c
b
O
O
O
O
O
Fmoc
N
H
N
H
N
H
13
R
14a-h
R
15a-h
9e-12e, 14e& 15e
; R= CN
9a-12a, 14a & 15a
; R= H
9f-12f, 14f & 15f; R= CF3
= Rink Amide MBHA Resin
9b-12b, 14b & 15b; R= OCH3
9c-12c, 14c & 15c; R= OH
9d-12d, 14d & 15d; R= F
9g-12g, 14g & 15g; R= CH3
9h-12h, 14h & 15h; R= Ph
Scheme 1. Synthesis of compounds 11a–h, 12a–h and 15a–h. aReagents and conditions: (a) (1) 20% piperidine in DMF; (2) Fmoc–NH–(CH2)n–COOH or Fmoc–PABA, HOBt,
DIC, DMF, N2; (b) (1) 20% piperidine in DMF; (2) substituted benzoic acids, HOBt, DIC, DMF, N2; (c) TFA/H2O/triisopropylsilane (95:2.5:2.5), 3 h.

3518
P. Jadav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3516–3521
NH2
R
R
CN
N
a
N
H
N
H
N
O
O
n
O
n
O
O
16a: R = CN, n = 2
16b: R = CF3, n = 2
16c: R = CN, n = 3
16d: R = CF3, n = 3
11d, 11f, 12d & 12f
NH2
CN
N
N
O
a
O
O
O
O
N
H
N
H
R
R
17a (R= CN)
17b (R= CF3)
15d & 15f
Scheme 2. Synthesis of compounds 16a–d and 17a,b. aReagents and conditions: (a) TFAA, CH2Cl2, 25 °C, 6 h.
Table 1
In vitro DPP-IV inhibitory activity*
NH2
N
NH2
O
R
N
O
O
H
N
O
O
n
N
H
O
R
11a-h (n= 2); 12a-h (n= 3)
15a-h
S. No.
R
DPP-IV inhibition**
S. No.
R
DPP-IV inhibition**
11a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
–H
320 29
890 21
863 18
93 9.3
31 2.5
28 1.7
715 27
107 19
298 19
869 43
843 26
74 11
12e
12f
–CN
–CF3
–CH3
–Ph
–H
–OCH3
–OH
–F
–CN
–CF3
–CH3
–Ph
26 3.1
19 2.3
694 14
104 16
311 24
879 22
863 13
100 8.5
34 7.6
31 8.3
723 21
116 13
–OCH3
–OH
–F
–CN
–CF3
–CH3
–Ph
12g
12h
15a
15b
15c
15d
15e
15f
–H
–OCH3
–OH
–F
15g
15h
*
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).
inhibitory activity relative to that of un-substituted (11a: –H),
whereas compounds with electron withdrawing groups (11d;
11e and 11f) showed good DPP-IV inhibitory activity. Among the
11d, 11e and 11f tested, 11e and 11f showed improved DPP-IV
inhibitory activity, which could be due to increase in the electro-
negativity at para-position of benzamide. Aliphatic substitution
at para-position (11g; –CH3) showed weak inhibitory activity,
while aromatic substitution at para-position (11h; –Ph) showed
moderate DPP-IV inhibitory activity. The second and third set of
compounds (12a–h, 15a–h) comprising GABA and PABA spacers
attached to para-substituted benzamides, showed similar trend
in DPP-IV inhibitory activity as observed with first set of com-
pounds, with respect to nature of para-substituents.
The first series was specifically designed to understand the role
of spacer and effect of para-substituents on benzamide. The SAR
study of first series reveals that the DPP-IV inhibitory activity of
test compounds drastically varies with para-substituents, whereas
alteration in spacers (aliphatic with two/three carbon chain-length
versus aromatic) do not exhibit significant change. In general, neu-
tral effect of spacers on inhibitory activity might be due to the flex-
ibility in S2 pocket and stapled orientation of Glu-dyad.
Substituents on para-position of benzamide altered inhibitory
activity to greater extent because in S3 pocket, para-substituents
play crucial role for its interaction with Ser209, Arg358 and Phe357
.
From first series, altogether in three different sets, 11e, 11f, 12e,
12f, 15e and 15f (para-nitrile/trifluoromethyl benzamide) were

P. Jadav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3516–3521
3519
Table 2
In vitro DPP-IV inhibitory activity and selectivity*
CN
N
R
CN
O
O
N
H
N
N
H
O
n
O
R
17a-b
16a-b (n= 2); 16c-d (n= 3)
S. No.
R
DPP-IV**
DPP2$
DPP8$
DPP9$
16a
16b
16c
16d
17a
17b
–CN
–CF3
–CN
–CF3
–CN
–CF3
10.3 1.9
13.2 2.3
2.3 0.9
3.8 0.5
11.6 1.6
14.3 2.5
7.2 1.3
3.2 0.5
—
—
—
—
—
—
>25,000
>25,000
—
>15,000
>15,000
—
>15,000
>15,000
—
—
—
—
NVP-DPP728#
Vildagliptin#
>25,000
—
>15,000
—
>15,000
—
*
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).
DPP2, DPP8 and DPP9 inhibitory activity represented as fold-selectivity wrt DPP-IV inhibitory activity.
Reported literature values for NVP-DPP728 and Vildagliptin are 7 1.7 and 2.7 0.1 respectively. (Ref. 5b,c).
$
#
identified as primary lead compounds. Further to study effect of ni-
trile group on pyrrolidine ring system, a second series (16a–d and
17a,b) was prepared by replacing pyrrolidinecarboxamides of first
series lead compounds with pyrrolidinecarbonitriles.
over DPP-8 and DPP-9, which was found to be comparable with
reference standard compounds (NVP-DDP728). Among all the com-
pounds tested, 16c and 16d were found most potent and selective,
hence subjected for pharmacodynamic (PD) as well as pharmacoki-
netic (PK) profiling.
As depicted in Table 2, all the six compounds (16a–d and 17a,b)
from second series showed potent inhibitory activity and was
found to be comparable with standard compounds (NVP-DPP728
and Vildagliptin).5 Compared to first series (pyrrolidinecarboxa-
mides), significant improvement in the inhibitory activity was
observed with second series (pyrrolidinecarbonitriles) of com-
pounds (16a–d and 17a,b), which could be due to the favorable
interactions of pyrrolidinecarbonitriles with the key residues of
S1 pocket. Among six compounds tested (second series), 16c and
16d were found to be equipotent as Vildagliptin.
The in vivo antidiabetic activity of 16c, 16d and NVP-DPP728 (@
20 mg/kg, po) was evaluated in male C57BL/6 J mice, using IPGTT
(intraperitoneal glucose tolerance test) protocol and changes in
serum glucose levels (AUC glucose up to 240 min; mg/dL) is re-
ported (Fig. 2).17 Compound 16c showed good oral antidiabetic
activity (% decrease in AUC glucose 54.9 3.86), whereas 16d and
NVP-DPP728 (positive control) showed moderate activity upon
oral administration (% decrease in AUC glucose 17.4 5.35 and
21.5 6.1, respectively). In C57 mice (IPGTT protocol), it was inter-
esting to observe that 16c showed suppression in the blood glucose
at all the time points (30, 60, 120 and 240 min) compared to vehi-
cle control, while 16d and NVP-DPP728 showed blood glucose
reduction only at 30 and 60 min.
The in vitro selectivity over serine protease (Table 2), especially
DPP-2, DPP-8 and DPP-9 was evaluated for most potent com-
pounds (16c and 16d).16 Compounds 16c and 16d showed
>25000-fold selectivity over DPP-2 and >15000-fold selectivity
Further to understand the duration of action and effect of test
compounds on post-prandial glucose excursion, single dose (@
Control
400
300
200
100
0
16d (20 mpk, po)
16c (20 mpk, po)
NVP-DPP728 (20mpk, po)
Control
700
600
500
400
300
200
100
0
16c (20 mg/kg) PO
NVPDPP-728 (20 mg/kg) PO
$
$
$
*
*
$
*
*
$
$
*
$
*
*
Compound
-1 0 0.51.0
0
50
100
150
200
250
Time (min)
2
4
6
24
Time (hr)
*P<0.05, $P<0.01, Two-Way ANOVA followed by Bonferroni post test, M SEM
*P<0.05, $P<0.01, Two-Way ANOVA followed by Bonferroni post test, M SEM
Figure 2. In vivo antidiabetic activity of 16c, 16d and NVP-DPP728 in C57 mice
(OGTT).
Figure 3. In vivo antidiabetic activity of 16c and NVP-DPP728 in db/db mice.

3520
P. Jadav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3516–3521
20 mg/kg, po) antidiabetic activity of 16c and NVP-DPP728 was
evaluated in fed-db/db mice (hyperglycemic animals) for 24 h
(Fig. 3). Under fed condition, compared to vehicle control, NVP-
DPP728 and 16c showed good antidiabetic activity (% decrease in
AUC glucose 31.4 8.7 and 33.5 7.4, respectively) up to 2 h. How-
ever, 16c showed sustained suppression in serum glucose levels for
>8 h (% decrease in AUC glucose, 14.9 6.3 for NVP-DPP728 and
30.8 6.2 for 16c, after 8 h).
A comparative single dose (20 mg/kg iv or po) PK profile of 16c,
16d and NVP-DPP728 was evaluated in male C57BL/6J mice (n = 6)
and the various PK parameters such as Tmax, T1/2, Cmax, AUC and %F
were recorded (Table 3).18 In PK study, all the test compounds
showed rapid Tmax, good Cmax and oral bioavailability (%F ꢀ63–
72%). Compound 16c showed higher AUC (>twofold compared to
16d and NVP-DPP728 1) and extended half-life (T1/2: >7 h com-
pared to 16d and NVP-DPP728). Compound 16c showed extended
half-life and higher AUC, which could be due to its low clearance
compared to 16d (elimination rate constant (kel; hÀ1), 0.11 0.03
for 16c and 0.82 0.18 for 16d). Thus improved pharmacokinetic
profile of compound 16c justifies its potent and sustained antidia-
betic activity in C57 and db/db mice.
The overlay of binding poses of 16c (Turquoise) and NVP-
DPP728 (Rose) in the DPP-IV active site is shown in Fig. 4. As ob-
served with NVP-DPP728, 16c docks very well into all the three
sites (G-scores À11.85 (9/9) and À10.16 (7/9) for 16c and NVP-
DPP728 respectively). Both NVP-DPP728 and 16c showed covalent
interaction of cyanopyrrolidine–CN with OH-group of side-chain of
Ser630 (S1 pocket) and H-bonding of amide-NH backbone with C@O
groups of side-chains of Glu205 and Glu206 dyad (S2 pocket), which
supports excellent in vitro DPP-IV selectivity of 16c over other pro-
tease. Especially, incorporation of GABA linkage (spacer) and para-
nitrile benzamide in 16c allows it to adopt new confirmation,
which favors strong H-bonding of benzamide with the NH of gua-
nidine side-chain of Arg358 and aromatic p–p stacking of para-ni-
trile phenyl ring with Phe357 in S3 pocket (Fig. 1; Supplementary
data). These additional interactions of 16c in S3 pocket justify its
3-fold potent DPP-IV inhibitory activity (in vitro) over NVP-
DPP728.
Kinetic study of DPP-IV inhibition by radiolabeled NVP-DPP728
established its slow-binding inhibition mechanism and nitrile
functionality play crucial role in the formation of the high-affinity
complex, via imidate intermediate.12 Due to unavailability of radi-
olabeled 16c, inhibitory kinetic of 16c was not evaluated. However,
docking studies supports involvement of key interactions of 16c
with all the three pockets, which apparently establish
slow-binding kinetic of 16c as cyanopyrrolidine class effect.
In summary, we report SPPS approach to discover peptidomi-
metic based cyanopyrrolidine derivatives as potent, selective and
long acting DPP-IV inhibitors for an effective treatment of T2DM.
The lead compound 16c showed sustained suppression of pre-
The molecular docking analysis of 16c and NVP-DPP728 was
carried out using extra precision (XP) Glide docking method
(Fig. 4).9,19 The crystal structure of the DPP-IV enzyme (PDB ID:
2I03) was obtained from the protein data bank and the protein
structure was prepared using protein preparation wizard module
of Schrödinger. For docking study, the ligands were minimized by
applying an OPLS-AA forcefield, using ligprep module of
Schrödinger.19
Table 3
Pharmacokinetic study parametersa of 16c, 16d and NVP-DPP728
Compd
Tmax (h)
Cmax
(lg/ml)
T1/2 (h)
AUC (0-a) h
lg/ml
F* (%)
16c
16d
NVP-DPP728
0.29 0.11
0.28 0.10
0.32 0.08
7.1 0.83
5.9 0.88
6.2 0.91
7.99 0.33
0.99 0.14
0.88 0.11
14.3 1.13
6.89 1.21
6.49 1.11
72.5
63.1
65
a
In male C57BL/6 J mice (n = 6), compounds were administered orally (po) at 20 mg/kg dose and plasma concentration was analyzed by LC–MS, values indicate Mean SD.
Oral bioavailability (%F) was calculated wrt to iv AUC (16c: 11.02 0.11; 16d: 10.92 0.12 & NVP-DPP728: 9.98 0.09 h g/ml) administered at 20 mg/kg dose, iv.
*
l
Figure 4. Key interactions of compound 16c and NVP-DPP728 with active sites of DPP-IV enzyme. Binding pose of compound 16c (Turquoise) and NVP-DPP728 (Rose) in the
DPP-IV active site is indicated (Surface view: Green), wherein both compounds interact closely with key residues of site S1, S2 and S3.

P. Jadav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3516–3521
3521
12. Hughes, T. E.; Mone, M. D.; Russell, M. E.; Weldon, S. C.; Villhauer, E. B.
Biochemistry 1999, 38, 11597.
13. Singh, S.; Sethi, S.; Khanna, V.; Benjamin, B.; Kant, R.; Sattigeri, J.; Bansal, V. S.;
Bhatnagar, P.; Davis, J. A. Eur. J. Pharmacol. 2011, 652, 157.
and post-prandial blood glucose levels (in vivo), which correlates
with its extended PK profile.
14. Sheppard, R. C.; Atherton, E. Solid Phase Peptide Synthesis- a practical approach.
I.R.L. Press publication, Oxford University Press.
Acknowledgments
15. North, M.; Pattnden, G. Tetrahedron 1990, 46, 8267.
We are grateful to the management of Zydus Group for encour-
agement and support.
16. (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
Supplementary data
the enzyme source. The H-Gly-Pro-AMC (200 lM) was incubated with either
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.078.
DPP-IV, DPP-2, DPP-8 or DPP-9 enzymes in the presence of various
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%
DMSO) in a total volume of 100
was terminated with acetic acid (25
l
l at 25 °C for 30 min., in the dark. Reaction
References and notes
ll of 25% solution). Activity (fluorescence)
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.
1. (a) Holst, J. J. Diabetologia 2006, 491, 253; (b) Baggio, L. L.; Drucker, D. J.
Gastroenterology 2007, 132, 2131; (c) Havale, S. H.; Pal, M. Bioorg. Med. Chem.
2009, 17, 1783.
2. (a) Heins, J.; Weiker, P.; Schonlein, C.; Born, I.; Hartrodt, B.; Neubert, K.; Tsuru,
D.; Barth, A. Biochim. Biophys. Acta 1988, 954, 161; (b) Hegen, M.; Niedobitek,
G.; Klein, C. E.; Stein, H.; Fleischer, B. J. Immunology 1990, 144, 2908; (c) Zhu, L.;
Tamvakopoulos, C.; Xie, D.; Dragovic, J.; Shen, X.; Fenyk-Melody, J. E.; Schmidt,
K.; Bagchi, A.; Griffin, P. R.; Thornberry, N. A.; Roy, R. S. J. Biol. Chem. 2003,
278(25), 22418.
3. Augustyns, K.; Veken, P. V.; Senten, K.; Haemers, A. Curr. Med. Chem. 2005, 12,
971.
4. Gupta, R.; Walunj, S. S.; Tokala, R. K.; Parsa, V. L.; Singh, S.; Pal, M. Curr. Drug
Targets 2009, 10, 71.
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 IPGTT 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
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 IPGTT protocol (C57 mice) overnight fasted mice
were dosed orally (po) with the test compounds (20 mg/kg), 0.5 h prior to the
intraperitoneal (ip) glucose load (1.5 g/kg), while in db/db mice, fed mice were
dosed orally (po) with the test compounds (20 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.
5. (a) Peters, J. Curr. Top. Med. Chem. 2007, 7, 579; (b) Villhauer, E. B.; Brinkman, J.
A.; Naderi, G. B.; Dunning, B. E.; Mangold, B. L.; Mone, M. D.; Russell, M. E.;
Weldon, S. C.; Hughes, T. E. J. Med. Chem. 2002, 45, 2362; (c) Villhauer, E. B.;
Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.; Dunning, B. E.; Prasad, K.; Mangold,
B. L.; Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46, 2774; (d) 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.; 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; (e) Haffner, C. D.; McDougald, D. L.; Randhawa, A. S.; Reister, S. M.;
Lenhard, J. M. PCT Int. Appl. WO2003/002531, 2003.; (f) Mulakayala, M.; Reddy,
C. H. U.; Iqbal, J.; Pal, M. Tetrahedron 2010, 66, 4919.
6. 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.
7. Feng, J.; Zhang, Z.; Wallace, M. B.; Stafford, J. A.; Kaldor, S. W.; Kassel, D. B.;
Navre, M.; Shi, L.; skene, R. J.; Asakawa, T.; Takeuchi, K.; Xu, R.; Webb, D. R.;
Gwaltney, S. L., II J. Med. Chem. 2007, 50, 2297; (b) Parsa, K. V. L.; Pal, M. Exp.
Opin. Drug. Disc. 2011, 6, 855.
18. Briefly, for single dose PK study, test compounds were administered orally/iv
on a body weight basis (20 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 and 30 h post-dose after
compounds administration. Approximately 0.3 ml of blood was collected at
each time point and centrifuged at 4 °C. The obtained plasma was frozen,
stored at À70 °C and the concentrations of compounds in plasma were
determined by the LC–MS/MS (Shimadzu LC10AD, USA), using YMC
8. (a) Oefner, C.; D’Arcy, A.; Sweeney, A. M.; Pierau, S.; Gardiner, R.; Dale, G. E. Acta
Crystallogr., Sect. D 2003, 59, 1206; (b) Engel, M.; Hoffmann, T.; Wagner, L.;
Wermann, M.; Heiser, U.; Kiefersauer, R.; Huber, R.; Bode, W.; Demuth, H.;
Brandstetter, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(9), 5063; (c) Rasmussen, H.
B.; Branner, S.; Wiberg, F. C.; Wagtmann, N. Nat. Struct. Biol. 2003, 10, 19.
9. (a) Kang, N. S.; Ahn, J. H.; Kim, S. S.; Chae, C. H.; Yoo, S. Bioorg. Med. Chem. Lett.
2007, 17, 3716; (b) Kuhn, B.; Hennig, M.; Mattei, P. Curr. Top. Med. Chem. 2007,
7, 609.
hydrosphere C18 (2.0 Â 50 mm,
3 lm) column (YMC Inc., USA). The
pharmacokinetic parameters were calculated using
model of WinNonlin software version 5.2.1.
a non-compartmental
19. (a) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177; (b)
Schrodinger Suite 2010, Glide version 5.6, Prime version 2.2; Schrodinger, LLC:
New York, 2010.
10. Bohannon, N. Postgrad. Med. 2009, 121, 40.
11. Deacon, C. F. Diabetes Obes. Metab. 2011, 13, 7. and referances cited therein.