Discovery and pharmacological characterization of N-[2-({2-[(2S)-2- cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a] pyrimidine-6-carboxamide hydrochloride (anagliptin hydrochloride salt) as a potent and selective DPP-IV inhibitor

Article abstract of DOI:10.1016/j.bmc.2011.09.043

In the course of our program for discovery of novel DPP-IV inhibitors, a series of pyrazolo[1,5-a]pyrimidines were found to be novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2- methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (4a) and described its pharmacological profiles.

Full text of DOI:10.1016/j.bmc.2011.09.043

Bioorganic & Medicinal Chemistry 19 (2011) 7221–7227
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and pharmacological characterization of N-[2-({2-[(2S)-2-cyanopyrr-
olidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyri-
midine-6-carboxamide hydrochloride (anagliptin hydrochloride salt) as a
potent and selective DPP-IV inhibitor
Noriyasu Kato a, , Mitsuru Oka a, Takayo Murase a, Masahiro Yoshida b, Masao Sakairi a,
⇑
Satoko Yamashita a, Yoshika Yasuda a, Aya Yoshikawa b, Yuuji Hayashi a, Mitsuhiro Makino a,
Motohiro Takeda a, Yakufu Mirensha c, Takuji Kakigami b
a Central Research Laboratory, Sanwa Kagaku Kenkyusho, Co. Ltd, 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan
b Sanwa Kagaku Kenkyusho, Co. Ltd, 35 Higashisotobori-cho, Higashi-ku, Nagoya 461-8631, Japan
c Xinjiang Medical University, Urumqi 830011, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of our program for discovery of novel DPP-IV inhibitors, a series of pyrazolo[1,5-a]pyrimi-
dines were found to be novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-
Received 11 August 2011
Revised 22 September 2011
Accepted 23 September 2011
Available online 2 October 2011
oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide
(4a) and described its pharmacological profiles.
hydrochloride
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dipeptidyl peptidase IV
DPP-IV inhibitor
Pyrazolo[1,5-a]pyrimidine
Anagliptin
Glucagon-like peptide-1
Type 2 diabetes mellitus
1. Introduction
infusion,5 and based on accumulating clinical studies, greater than
2-fold enhancement of circulating levels of active GLP-1 is known
to result from inhibition of 80% or more of the plasma DPP-IV activ-
ity.6 Consequently, optimal glycemic control requires continuous
high-level exposure to DPP-IV inhibitors.
Glucagon-like peptide-1 (GLP-1), a 30-amino acid peptide hor-
mone, is secreted by intestinal L-cells in response to food ingestion
and stimulates insulin secretion from b-cells in a glucose-depen-
dent manner.1 GLP-1 is also known to have multiple actions such
as suppression of glucagon secretion, inhibition of gastric empty-
ing and induction of satiety.1,2 Based on these findings, GLP-1 has
been considered to be an attractive target for the therapy of type
2 diabetes mellitus (T2DM). However, GLP-1 is rapidly degraded
into inactive GLP-1 by a serine protease, dipeptidyl peptidase IV
(DPP-IV), which fueled the development of biologically stable
GLP-1 analogs.3 Therefore, inhibitors of DPP-IV capable of increas-
ing the circulating concentration of active GLP-1, have now
emerged as promising treatments for T2DM.4 In addition, it was
demonstrated in a clinical study of diabetic patients receiving ac-
tive GLP-1 infusion that a 24-h infusion of active GLP-1 resulted
in a more marked improvement in glycemic control than a 16-h
Following approval of sitagliptin (Januvia)7 in the US in 2006,
vildagliptin,8 saxagliptin9 and alogliptin10 have been launched or
have been under late stage clinical development ( Fig. 1). Their
emerging medical need was also exemplified by the fact that the
combined sales of Januvia and Janumet11 (sitagliptin/metformin)
have been growing at double-digit rates in all regions and ac-
counted for an 8% market share 3 years after their introduction into
the US and Asian oral anti-diabetic markets.12 Their rapid and wide
acceptance is thought to be due to a low incidence of hypoglycemia
and absence of body weight gain as well as excellent tolerability.
DPP-IV inhibitors on the market are structurally diverse and there-
fore the expected growth in prescriptions for DPP-IV inhibitors
would underscore compound-specific differences in efficacy and
side effects. Thus, there seem to be opportunities for development
of new chemical entities that have unique pharmacological profiles
and/or different pharmacokinetic properties, which include the
metabolic pathways.13
⇑
Corresponding author. Tel.: +81 594 72 6221; fax: +81 594 82 0072.
E-mail address: n_katoh@mb4.skk-net.com (N. Kato).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.043

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N. Kato et al. / Bioorg. Med. Chem. 19 (2011) 7221–7227
Table 1
OH
DPP inhibitory activities of 4a
HO
N
O
O
CN
N
H
N
N
H
H2N
CN
O
R
N
H
N
O
CN
Me Me
HCl
Vildagliptin
Saxagliptin
4
NH2
Compound
R
IC50
F3C
N
4
N
N
F
F
N
N
CN
DPP-IV
(nM)
DPP-8
(nM)
DPP-9
(nM)
N
O
NH2
N
F
O
N
Me
O
Me
Me
a
3.8
13
68
76
60
64
N
N
N
N
Alogliptin
Sitagliptin
N
b
Figure 1. Some gliptins.
Me
N
Me
Me
N
N
NC
c
d
e
f
32
1.8
15
11
80
>100
58
81
45
66
30
53
21
28
N
N
O
Me Me
CF3
N
N
N
N
H
O
Me
1
OH
N
O
O
CN
H
N
N
>100
43
N
N
N
N
H
Me Me
Me
N
N
N
N
Anagliptin
Me
N
Me
Me
Figure 2. Anagliptin and its prototype 1.
N
N
g
h
i
>100
12
N
R2
Me
N
CO2H
N
Me
N
28
110
N
N
R3
N
O
CN
H
N
Me
R1
3
H2N
N
N
Me
Me Me
2 HCl
Me
CDI TEA, THF
N
45
N
2
Me
R2
N
O
O
CN
a
H
N
IC50 values are the average of at least two independent experiments.
N
N
H
N
Me Me
R3
and safety. Herein, we would like to report the synthesis and phar-
macological profiles of novel and potent DPP-IV inhibitors, the
hydrochloride salts of N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-
oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrim-
idine-6-carboxamide (anagliptin)15 and related compounds (Fig. 2).
HCl
N
R1
4
f: R1 = Ph, R2 = H, R3 = Me
g: R1 = CMe3, R2 = R3 = Me
h: R1 = Ph, R2 = R3 = Me
i: R1 = R2 = R3 = Me
a: R1 = Me, R2 = R3 = H
b: R1 = Me, R2 = H, R3 = Me
c: R1 = Me, R2 = H, R3 = CF3
d: R1 = Me, R2 = H, R3 = OH
2. Chemistry
e: R1 = CMe3, R2 =H, R3 = Me
A series of pyrazolo[1,5-a]pyrimidine derivatives were prepared
as described in Scheme 1. 2-Amino-2-methylpropylamine was
selectively protected with di-tert-butyl dicarbonate and coupled
with (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile. After depro-
tection of the Boc group, compound 2 was WSC-coupled with var-
ious pyrazolo[1,5-a]pyrimidinecarboxylic acids 3a–h to yield the
desired compounds 4a–h. All unavailable acids were prepared by
methods described in the literature.14
Scheme 1. Synthesis of compounds 4.
As part of our effort to discover novel DPP-IV inhibitors, we
previously reported that SARs of isoindoline derivatives led to the
discovery of 2-[3-[2-{(2S)-cyanopyrrolidin-1-yl}-2-oxoethylami-
no]-3-methyl-1-oxobutyl]-5-methyl-1,3-dihydroisoindole 1 as a
highly selective, potent DPP-IV inhibitor.14 Unfortunately, in vivo
studies showed that an active metabolite of 1 had a very high inhib-
itory potency toward DPP-IV and we abandoned further develop-
ment of the compounds in this series. Further studies showed
that pyrazolo[1,5-a]pyrimidine functioned as a bioisostere of the
isoindoline moiety that imparted improved metabolic stability
3. Results and discussion
As described previously,14 compounds 4 were evaluated in vitro
for inhibition of human recombinant DPP-IV and were also

N. Kato et al. / Bioorg. Med. Chem. 19 (2011) 7221–7227
7223
Table 2
Selected PK parameters of 4a and 4b in rats and dogs
c
c
Compound
Species
CLtot (l/h/kg)
Vdss (l/h/kg)
Cmax (ng/ml)
tmax (h)
t1/2 (h)
AUC (ng/h/ml)
BA (%)
iv
po
309 (62)
261
747 (68)
282
4aa
4bb
Rat
Dog
Rat
2.00
0.65
2.23
0.62
0.68
0.83
1.61
0.75
0.8 (2.3)
1.5
0.25 (0.25)
1.0
1.9
1.0
1.9
1.3
1160
824
1111
6390
23
100
27
Dog
98
a
Compound 4a dose in rats, 1 mg/kg, iv (n = 3); 10 mg/kg, po (n = 3). 4a dose in dogs, 0.2 mg/kg, iv (n = 3); 0.5 mg/kg, po (n = 2).
Compound 4b dose in rats, 3 mg/kg, iv (n = 1); 10 mg/kg, po (n = 1). 4b dose in dogs, 0.2 mg/kg, iv (n = 2); 0.5 mg/kg, po (n = 2). CLtot, plasma clearance; Vdss, steady-state
b
volume of distribution; Cmax, maximal concentration; tmax, time of maximal concentration; t1/2, terminal half-life; AUC, area under the curve of plasma concentration; BA, oral
bioavailability. Data are given as the mean value for the indicated compound (compd; 4a or 4b), species (rat or dog), dose (see above), and route of administration (iv or po).
c
Values in parentheses were obtained at a 4a or 4b dose of 3 mg/kg (4a, n = 3; 4b, n = 1).
are also presented because the importance of selectivity over DPP-
8/9 has been frequently mentioned in animal toxicity studies.4 Our
parent compound in this series, the 2-methyl pyrazolopyrimidine
analogues 4a, had DPP-IV inhibitory activity at an IC50 of 3.8 nM
with more than 10,000-fold selectivity over DPP-8/9.16 Substitu-
tion at the 7-position of the fused ring (e.g., 4b–d) was well toler-
ated and the hydroxyl analogue 4d was the most potent with an
IC50 of 1.8 nM and excellent selectivity over DPP-8/9. Like the 7-
substituents, replacement of the methyl group with bulkier alkyl
groups such as the tert-butyl or phenyl group yielded compounds
4e and 4f, which had similar DPP-IV inhibitory potencies compared
with 4b. On the other hand, introduction of a methyl group at the
5-position (e.g., 4g–i) resulted in slight to significant loss of the
inhibitory activity relative to the corresponding unsubstituted ana-
logues 4b, 4e and 4f.
Table 3
Serum protein binding of 4a and 4b in different species
Compd
4a
Species
Protein binding (%)
20 hg/mla 2000 hg/mla
Rat
Dog
Human
Rat
Dog
77.3
64.8
29.2
22.1
22.5
18.9
77.1
51.8
26.4
17.9
19.9
21.6
4b
Human
a
Final compound concentration. Each value represents the mean of three
experiments.
screened for selectivity over dipeptidyl peptidase 8 and 9 (DPP-8/
9) by a fluorescence assay using glycyl-proline 4-methylcouma-
ryl-7-amide (Gly-Pro-MCA). Structure–activity relationships for
compounds in this series are summarized in Table 1. DPP-8/9 data
Eventually, taking their selectivity into account, compounds 4a,
4b and 4d were chosen for further investigation. However, early
pharmacokinetic (PK) studies showed compound 4d to have a very
vehicle
3mg/kg
10 mg/kg
120
100
80
60
40
20
0
-30
0
30
60
90
120
time (min)
vehicle
3mg/kg
10 mg/kg
8000
7800
160
7600
7400
7200
7000
*
135
*
**
110
85
**
**
6800
6600
6400
*
6200
6000
60
-30
0
30
60
90
120
vehicle
3
10
time (min)
4b (mg/kg)
Figure 3. Effect of 4b on plasma DPP-IV activity (upper graph) and plasma glucose concentration (lower left) after oGTT in rats; control group received vehicle after oGTT.
Glucose AUC (lower right) was determined between 0 and 60 min. Asterisks in lower plots indicate significance at p <0.05 versus control (⁄) and p <0.01 versus control (⁄⁄),
respectively, by Dunnett’s test.

7224
N. Kato et al. / Bioorg. Med. Chem. 19 (2011) 7221–7227
vehicle
0.3mg/kg
1 mg/kg
3 mg/kg
100
80
60
40
20
0
-30
0
30
60
90
120
time (min)
8000
7500
7000
vehicle
0.3mg/kg
1 mg/kg
3 mg/kg
160
135
*
6500
6000
*
110
85
*
5500
5000
60
-30
0
30
60
90
120
vehicle
0.3
1
3
time (min)
4a (mg/kg)
Figure 4. Effect of 4a on plasma DPP-IV activity (upper graph) and plasma glucose concentration (lower left) after oGTT in rats; control group received vehicle after oGTT.
Glucose AUC (lower right) was determined between 0 and 60 min. In lower plots, asterisk (⁄) indicates significance (p <0.05) versus control by Dunnett’s test.
low oral bioavailability of 4.1% due to poor systemic exposure at a
dose of 5 mg/kg po in Sprague–Dawley rats and then characteriza-
tion of 4d was discontinued. Selected PK parameters of 4a and 4b
are listed in Table 2. These two compounds had a low clearance
(4a, 0.65 L/h/kg; 4b, 0.62 L/h/kg) and very high oral bioavailability
(4a, 100%; 4b, 98%) in beagle dogs, while showing a relatively high
clearance (4a, 2.00 L/h/kg; 4b, 2.23 L/h/kg) and moderate oral bio-
availability in rats. As has been recently reported,17 high levels of
plasma protein binding of the inhibitor are considered a cause
for the disconnection between the observed in vivo efficacy and
the measured in vitro potency. To see if this would be the case with
our compounds, their plasma protein binding was measured Table
3. The measured binding activity was reasonably acceptable across
species. Remarkably, both compounds showed the lowest protein
binding (ꢀ20%) in human plasma and were expected to be suitable
for animal scale-up studies.
90% inhibition throughout the study. The inhibitory effect was also
dose-dependent, with a dose of only 0.1 mg/kg yielding 30% inhibi-
tion. Likewise, a significant reduction in the AUC was observed at
the 1 mg/kg dose. In addition, increased insulin levels at 10 min
post-challenge strongly suggested preservation of active GLP-1
(data not shown). The results showed a strong correlation between
in vivo levels of plasma DPP-IV inhibition and in vivo efficacy, and
confirmed superiority of 4a over 4b in improvement of glucose
tolerance.18
Besides evaluation of its potential for anti-diabetics, compound
4a was found to show an excellent preclinical safety profile; it did
not act as inhibitor or inducer of major liver metabolic enzymes
such as CYP3A4, CYP2C19, CYP2C9, CYP1A2, and CYP2D6 (IC50
>10
tion of 50
nel (E-4031, >90% inhibition at 0.1
l
M) or CYP3A4, CYP2C8, CYP2C9, and CYP1A2 at a concentra-
M. Compound 4a showed no binding to the hERG chan-
M, IC50 >500 M in patch
l
l
l
Given the preliminary PK data, we examined the potencies of 4a
and 4b in oral glucose tolerance tests (oGTT). Fasted male Wistar/ST
rats received orally administered vehicle, or compound 4a or 4b at
different dosages. Half an hour after treatment (t = 0), oral glucose
challenges (1 g/kg) were conducted. Plasma DPP-IV activities and
plasma glucose levels were then monitored at various intervals
over a 2 h period. Selected data are shown in Figures 3 and 4. At a
dose of 10 mg/kg, 4b caused approximately 90% inhibition of plas-
ma DPP-IV activity within 30 min, and the high levels of inhibition
were sustained throughout the study. The inhibitory effect was
dose-dependent, with a 3 mg/kg dose yielding approximately 60%
inhibition. Hence, reduction of the rise in blood glucose in response
to glucose challenge paralleled DPP-IV inhibition, and a significant
reduction of AUC0–60 min was observed at the 10 mg/kg dose. More
strikingly, administration of compound 4a at a dose of 3 mg/kg gave
95% inhibition of plasma DPP-IV activity and produced greater than
clamp using HEK 293 cells). 4a was negative in the Ames mutage-
nicity test. It was also tested against a panel of 62 receptor binding/
ion channel assays at a concentration of 10 lM, and did not show
specific binding of greater than 20% in any of the 62 assays under
control conditions.19 Briefly, preliminary studies on the excretion
pathway were conducted (Table 4). In bile duct-cannulated rats,
30% and 40% of 4a were excreted unchanged from the urine in
the 24 h period after iv administration at doses of 1 and 5 mg/kg,
respectively.20 Biliary excretion was also an important elimination
pathway, with recovery of ꢀ13% to 14% of the doses in bile. Simi-
larly, in dogs dosed at 1 mg/kg iv, 32% and 24% of 4a were excreted
unchanged from urine and feces, respectively, in the 24 h period
after drug administration. Parent compound 4a was primarily
eliminated into urine and biliary excretion was more significant
in dogs than in rats. The approximately 50% recoveries of 4a in
these species were consistent with the data on in vitro metabolism,

N. Kato et al. / Bioorg. Med. Chem. 19 (2011) 7221–7227
7225
Table 4
reduced pressure. The residues were subjected to column chroma-
tography (eluting solvent; dichloromethane/methanol, 50:1) to
yield a free base of 4b (690 mg, 33%). 4 N hydrochloric acid/1,4-
dioxane (0.50 ml) was added at 10 °C to a solution of the resulting
compound (690 mg) in 1,4-dioxane (5.0 ml) and stirred for 10 min.
Crystals were precipitated by adding ether and were then collected
by filtration. The crystals were dried under reduced pressure to
give 4b (670 mg, 90%) as yellow crystals.
Excertion of 4a after iv administration and metabolic stability
Excretion
Metabolic activity
Recovery of 4a (% dose)a
CL0int (l/h/kg)
Species
Rat
Dose (mg/kg)
Urine
Bile
Feces
1
5
1
30
41
32
13
14
ND
ND
ND
24
Rat
Dog
Monkey
Human
1.4
0.1
0.5
0.3
Dog
1H NMR: d 1.37 (6H, s), 2.05–2.31 (4H, m), 2.47 (3H, s), 2.87 (3H,
s), 3.30–3.80 (4H, m), 4.10–4.30 (2H, m), 4.84–4.86 (1H, m), 6.60
(1H, s), 8.68 (1H, s), 8.93–8.97 (3H, m); MS m/z 398 (M+H)+; IR
(ATR) 2974, 2348, 2138, 1981, 1647, 1604, 1523, 1433, 1308,
1173, 422 cmꢁ1; HRMS calcd for C20H28N7O2: 398.2299. Found:
398.2305.
a
Excretion of 4a in bile duct-cannulated rats (n = 2) and dogs (n = 3) after iv
administration. ND; not determined.
wherein the stability of 4a in rodent and human liver microsomes
was moderate to excellent (Table 4). Taken together, the data
suggest that compound 4a has a well-balanced elimination route.
5.1.2. (S)-2-Methylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid
{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-2-methylpropyl}
amide hydrochloride (4a)
4. Conclusions
1H NMR: d 1.36 (6H, s), 2.00–2.30 (4H, m), 2.50 (3H, s), 3.30–
3.80 (4H, m), 4.10–4.30 (2H, m), 4.80 (1H, m), 6.63 (1H, s), 8.80–
8.90 (3H, m), 9.50 (1H, s); MS m/z 384 (M+H)+; Anal. Calcd for
In the course of our program for discovery of novel DPP-IV
inhibitors, a series of pyrazolo[1,5-a]pyrimidines were found to be
novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrr-
olidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo
[1,5-a]pyrimidine-6-carboxamide hydrochloride (4a) as a potent
and selective DPP-IV inhibitor and have demonstrated several
unique pharmacological profiles. On the basis of the data presented
above, anagliptin, a free base of 4a, was chosen for further evaluation
as an option for DPP-IV inhibitors. Currently, late stage development
is under way.
C
19H25N7O2ꢂHCl: C, 59.51; H, 6.57; N, 25.57. Found: C, 59.91; H,
6.35; N, 25.71; IR (ATR) 3064, 2074, 2029, 1646, 1532, 1406,
1332, 1297, 1176, 665, 472, 413 cmꢁ1
HRMS calcd for
19H26N7O2: 384.2142. Found: 384.2147.
;
C
5.1.3. (S)-2-Methyl-7-trifluoromethylpyrazolo[1,5-a]pyrim
idine-6-carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoeth
ylamino]-2-methylpropyl}amide hydrochloride (4c)
1H NMR: d 1.37 (6H, s), 2.02–2.10 (2H, m), 2.19–2.24 (2H, m),
2.53 (3H, s), 3.49–3.62 (3H, m), 3.72 (1H, m), 4.13–4.16 (2H, m),
4.86 (1H, dd, J = 4.4, 6.9 Hz), 6.94 (1H, s), 9.00 (2H, br s), 9.09
(1H, t, J = 6.2 Hz), 9.77 (1H, s); MS m/z 452 (M+H)+.
5. Experimental
5.1. Compound synthesis
General: All commercially available reagents and solvents were
used without further purification. All reactions were carried out
using oven-dried flasks or glassware, and the mixtures were stirred
with magnetic stirring bars and concentrated using a standard ro-
tary evaporator, unless otherwise noted. Procedures for prepara-
tion of all intermediates 2 and 3a–h were described previously14a
and can be found in the supplementary data. The 1H NMR spectra
were recorded by a JEOL JNM-ECP400 spectrometer operating at
400 MHz in DMSO-d6 at 25 °C with tetramethylsilane as the inter-
nal standard. The data are reported as follows: chemical shift in
ppm (d), integration, multiplicity (s = singlet, d = doublet, t = trip-
let, q = quartet, br = broad singlet, m = multiplet), and coupling
constant (Hz). LC/MS spectra were determined on a Waters
ZMD2000 equipped with a Waters 2690 injector and a PDA detec-
tor operating at 210–400 nm and interfaced with a Micromass
ZMD mass spectrometer. Infrared spectra (IR) were recorded on a
JASCO FT/IR-4200 spectrometer. High-resolution mass spectra
(HRMS) were recorded on a Thermo. LTQ Orbitrap.
5.1.4. (S)-2-Methyl-7-hydroxypyrazolo[1,5-a]pyrimidine-6-
carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-
2-methylpropyl}amide hydrochloride (4d)
1H NMR: d 1.22 (6H, s), 2.05–2.27 (4H, m), 2.28 (3H, s), 3.48–
3.53 (4H, m), 3.65 (1H, m), 3.79–3.89 (2H, m), 4.79–4.82 (1H, m),
5.97 (1H, s), 8.47 (1H, s), 9.65 (1H, br t); MS m/z 400 (M+H)+; IR
(ATR) 3746, 3277, 2353, 2239, 2182, 2042, 1637, 1527, 1418,
1305, 1180, 792, 469, 418 cmꢁ1; HRMS calcd for C19H26N7O3:
400.2091. Found: 400.2096.
5.1.5. (S)-2-tert-Butyl-7-methylpyrazolo[1,5-a]pyrimidine-6-
carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-
2-methylpropyl}amide hydrochloride (4e)
1H NMR: d 1.36 (6H, s), 1.38 (9H, s), 1.97–2.08 (2H, m), 2.19–
2.25 (2H, m), 2.88 (3H, s), 3.51–3.58 (3H, m), 3.73 (1H, m), 4.12–
4.17 (2H, m), 4.87 (1H, dd, J = 4.4, 7.0 Hz), 6.73 (1H, s), 8.68 (1H,
s), 8.91 (1H, t, J = 6.2 Hz), 8.95 (2H, br s); MS m/z 440 (M+H)+; IR
(ATR) 2961, 2361, 2325, 2135, 2035, 2007, 1661, 1603, 1523,
1434, 1311, 1215, 474, 418 cmꢁ1; HRMS calcd for C23H34N7O2:
440.2768. Found: 440.2770.
5.1.1. Representative procedure for compound 4; (S)-2,7-
Dimethylpyrazolo[1,5-a]pyrimidine-6-carboxylic acid
{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-2-methylpropyl}
amide hydrochloride (4b)
5.1.6. (S)-2-Phenyl-7-methylpyrazolo[1,5-a]pyrimidine-6-
carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-
2-methylpropyl}amide hydrochloride (4f)
N,N’-Carbonyl diimidazole (930 mg) was added to a solution of
3b (1.00 g) in tetrahydrofuran (30 ml), and the mixture was stirred
at room temperature for 4 h. The reaction mixture was slowly
added dropwise to 2 (1.56 g) in an ice-cooled solution of triethyl-
amine (3.6 ml) in tetrahydrofuran (30 ml). The mixture was re-
turned to room temperature and stirred overnight. The reaction
mixture was concentrated under reduced pressure, and then
dichloromethane was added to the residues. Insoluble materials
were removed by filtration and the filtrate was concentrated under
1H NMR: d 1.39 (6H, s), 2.03–2.11 (2H, m), 2.19–2.25 (2H, m),
2.96 (3H, s), 3.54–3.69 (4H, m), 4.11–4.23 (2H, m), 4.88 (1H, dd,
J = 4.0, 7.0 Hz), 7.36 (1H, s), 7.47 (1H, t, J = 7.3 Hz), 7.52 (2H, dd,
J = 7.3, 7.7 Hz), 8.10 (2H, d, J = 7.7 Hz), 8.77 (1H, s), 9.01–9.06
(3H, m); MS m/z 460 (M+H)+; IR (ATR) 2979, 2329, 2116, 1994,
1660, 1604, 1579, 1524, 1460, 1436, 1400, 1314, 1260, 1174,
768, 693, 630, 419, 409 cmꢁ1
460.2455. Found: 460.2455.
; HRMS calcd for C25H30N7O2:

7226
N. Kato et al. / Bioorg. Med. Chem. 19 (2011) 7221–7227
5.1.7. (S)-2-tert-Butyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-
6-carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-
(0.5 mg/kg, n = 2). Following a 1-week washout period, a single
intravenous dose of 4a or 4b (0.2 mg/kg, n = 2) was given. Approx-
imately 1.5 ml of blood was collected from the vein with a heparin-
ized syringe at the following times: 0.083, 0.167, 0.25, 0.5, 1, 2, 3, 5,
7, 9, and 24 h for iv doses; 0.25, 0.5, 1, 2, 3, 5, 7, 9, and 24 h for oral
doses. Plasma was obtained by centrifugation at 4 °C and stored at
ꢁ70 °C until analysis. For excretion studies, three dogs were dosed
intravenously with 4a (1 mg/kg), and then urine was collected
from the dogs at 0–7 and 7–24 h intervals. Fecal and cage wash
samples were collected at 24 h post-dose.
oxoethylamino]-2-methylpropyl}amide hydrochloride (4g)
1H NMR: d 1.36 (15H, s), 2.01–2.10 (2H, m), 2.19–2.24 (2H, m),
2.47 (3H, s), 2.66 (3H, s), 3.51–3.71 (4H, m), 4.11–4.19 (2H, m), 4.87
(1H, dd, J = 4.4, 6.6 Hz), 6.52 (1H, s), 8.80 (1H, t, J = 6.3 Hz), 9.08
(2H, br s); MS m/z 454 (M+H)+; IR (ATR) 2964, 2361, 2333, 2070,
2010, 1656, 1539, 1432, 1281, 489 cmꢁ1
24H36N7O2: 454.2925. Found: 454.2926.
; HRMS calcd for
C
5.1.8. (S)-2-Phenyl-5,7-dimethylpyrazolo[1,5-a]pyrimidine-6-
carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-
2-methylpropyl}amide hydrochloride (4h)
5.2.3. Plasma pharmacokinetic analysis
Noncompartmental analysis was used to determine the phar-
1H NMR: d 1.38 (6H, s), 2.02–2.10 (2H, m), 2.19–2.25 (2H, m),
2.52 (3H, s), 2.75 (3H, s), 3.53–3.76 (4H, m), 4.11 (1H, dd, J = 6.6,
16.5 Hz), 4.18 (1H, dd, J = 5.9, 16.5 Hz), 4.87 (1H, dd, J = 4.4,
7.0 Hz), 7.18 (1H,s), 7.44 (1H, t, J = 7.3 Hz), 7.51 (2H, dd, J = 7.0,
7.3 Hz), 8.07 (2H, d, J = 7.0 Hz), 8.94 (1H, t, J = 6.0 Hz), 9.10 (2H,
br s); MS m/z 474 (M+H)+; IR (ATR) 2985, 2332, 2164, 1656,
1614, 1518, 1438, 1276, 1194, 769, 695, 445, 410 cmꢁ1; HRMS
calcd for C26H32N7O2: 474.2612. Found: 474.2612.
macokinetics of 4a and 4b. Maximal plasma concentration (Cmax)
and time of maximal plasma concentration (tmax) were assessed
by visual inspection. The terminal elimination half-life was calcu-
lated using the relationship 0.693/k, where k is the elimination rate
constant. The AUC0-t was calculated through the last measurable
time point t by the trapezoidal rule. From the intravenous plasma
concentration data, plasma clearance and volume of distribution of
4a and 4b were determined. BA was calculated according to the
following equation:
5.1.9. (S)-2,5,7-Trimethylpyrazolo[1,5-a]pyrimidine-6-
carboxylic acid{2-[(2-cyanopyrrolidin-1-yl)-2-oxoethylamino]-
2-methylpropyl}amide hydrochloride (4i)
BAð%Þ ¼ ðAUCpo=DpoÞ=ðAUCiv=DivÞ ꢃ 100
where AUCpo is the AUC after oral dosing; AUCiv, the AUC after
intravenous dosing; Dpo, the oral dose; and Div, the intravenous
dose.
1H NMR: d 1.37 (6H, s), 1.98–2.09 (2H, m), 2.18–2.27 (2H, m),
2.43 (3H, s), 2.47 (3H, s), 2.66 (3H, s), 3.52–3.63 (1H, m), 3.62
(2H, d, J = 6.2 Hz), 3.71–3.76 (1H, m), 4.10–4.21 (2H, m), 4.86
(1H, dd, J = 4.4, 7.0 Hz), 6.44 (1H, s), 8.92 (1H, br t), 9.12 (2H, br
s); MS m/z 412 (M+H)+; IR (ATR) 2981, 2344, 2117, 1998, 1656,
1536, 1433, 1322, 1277, 1173, 731, 553, 434, 418 cmꢁ1; HRMS
calcd for C21H30N7O2: 412.2455. Found: 412.2455.
5.2.4. In vitro protein binding
Ultrafiltration units (VIVASPINÒ, Sartorius) were used in protein
binding studies. Rat serum, dog serum and human serum contain-
ing drug concentrations of 20 and 2000 ng/ml were centrifuged for
0.5 h at 37 °C incubation. Following centrifugation, the amount of
drug in the supernatant was determined by LC–MS/MS. The frac-
tion of bound to protein was calculated from the concentrations
in the spiked sample and the supernatant.
5.2. Biological evaluation
General: Inhibition of DPP-IV and -8/9 activity and metabolic
stability were determined as described previously14b and can be
found in the supplementary data. All animal experiments were ap-
proved by the Animal Ethics Committee of the Sanwa kagaku
kenkyusho.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.043.
5.2.1. Pharmacokinetic studies in rats
Male Sprague–Dawley rats (6–7 weeks old/160–180 g) were
acclimated for at least 3 days before use. Food and water were sup-
plied ad libitum at all times throughout the experiment. Com-
pounds 4a or 4b were administered intravenously (iv) via the
femoral vein (4a, 1 mg/kg, n = 3; 4b, 3 mg/kg, n = 1) and by oral
(po) gavage at a dose of 10 mg/kg in 5% gum arabic solution (4a;
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concentrations of the two compounds available for DPP-IV inhibition (i.e., free
fraction of concentration) were estimated. From the data listed in Tables 2 and
3, free fraction concentrations of 4a and 4b at the 3 mpk dose are calculated to
be 14 and 54 nM at tmax, respectively, which suggest systemic exposures were
sufficient for high levels (ꢀ90%) of in vivo inhibition in both cases. Our attempt
at the rationalization failed, but it became evident that in vivo potency of 4a
was consistent with its in vitro potency.
13. (a) At least, in case of renal or hepatic impairment, there seems to be possibility
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Novartis AG PRESS RELEASE 2007 November 6. (c) No drug–drug interctions
with gliptins having been reported, caution is recommeded in some cases, see;
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067509: Chem.Abstr. 141: 190794.; (b) Kato, N.; Oka, M.; Murase, T.; Yoshida,
M.; Sakairi, M.; Yakufu, M.; Kawamura, S.; Yasuda, Y.; Yoshikawa, A.; Hayashi,
Y.; Shirai, M.; Mizuno, Y.; Takeuchi, M.; Makino, M.; Takeda, M.; Kakigami, T.
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15. (a) Sunami, Y.; Yoshioka, N.; Hayashi, I.; Ishida, T.; Presented at the American
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Ishida, T.; Matsumoto, Y.; Presented at the American Diabetes Association 67th
Scientific Session, Chicago, IL, Jun. 2–6, 2007, Abstract 1492-P.; (c) Furuta, S.;
Goto, M.; Furuta, Y.; Yasuda, Y.; Nakaya, K.; Tamura, M.; Uesugi, N.; Kawamura,
19. http://www.ricerca.com/.
20. Pharmacokinetic studies with [14C]compound 4a in rats and dogs indicated
that most (89–98%) of excretion occurred within 24 h. The detail will be
reported elsewhere.