Surrogating and redirection of pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a novel class of potent and selective DPP-4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.01.006

The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in vitro DPP-4 inhibitory activity (IC₅₀: 80 nM), while maintaining other key cellular parameters such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on docking analysis and structure-based drug design knowledge resulted in d1. Compound d1 has nearly 2-fold increase of inhibitory activity (IC₅₀: 49 nM) and over 1000-fold selectivity against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose excursion by 34% at the dose of 10 mg/kg in diabetic mice. Herein we report the optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4 inhibitors.

Full text of DOI:10.1016/j.bmc.2018.01.006

Accepted Manuscript
Surrogating and redirection of Pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a nov-
el class of potent and selective DPP-4 Inhibitors
Xinxian Deng, Jian Shen, Hui Zhu, Jia Xiao, Ran Sun, Fangzhou Xie, Celine
Lam, Juntao Wang, Yixue Qiao, Mojdeh S. Tavallaie, Yang Hu, Yi Du, Jianqi
Li, Lei Fu, Faqin Jiang
PII:
S0968-0896(17)32286-1
https://doi.org/10.1016/j.bmc.2018.01.006
BMC 14160
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 November 2017
9 January 2018
10 January 2018
Please cite this article as: Deng, X., Shen, J., Zhu, H., Xiao, J., Sun, R., Xie, F., Lam, C., Wang, J., Qiao, Y., Tavallaie,
M.S., Hu, Y., Du, Y., Li, J., Fu, L., Jiang, F., Surrogating and redirection of Pyrazolo[1,5-a]pyrimidin-7(4H)-one
core, a novel class of potent and selective DPP-4 Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.01.006
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Surrogating and redirection of Pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a
novel class of potent and selective DPP-4 Inhibitors
Xinxian Denga,b, Jian Shena,c, Hui Zhud, Jia Xiaoa, Ran Suna, Fangzhou Xiea, Celine Lama, Juntao
Wanga, Yixue Qiaoa, Mojdeh S. Tavallaiea, Yang Hua, Yi Due,, Jianqi Li b*, Lei Fua* and Faqin
Jianga*
a. School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dongchuan Rd. Minhang District,
Shanghai, 200240, China.
b. China State Institute of Pharmaceutical Industry, No. 285 Gebaini Rd. Pudong District
Shanghai, 201203, China.
c. Viva Biotech Ltd. (Shanghai), No. 334 Aidisheng Rd., Pudong District Shanghai, 201203,
China.
d. Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong
University School of Medicine, No. 369 Zhizaoju Road, Huangpu District, Shanghai, 200011,
China
e. Xinhua hospital affiliated to Shanghai Jiao Tong University，School of Medicine, No. 1665
Kongjiang Rd., Yangpu District, Shanghai, 200092, China.
Abstract
The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-
4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in
vitro DPP-4 inhibitory activity (IC50: 80 nM), whilst maintaining other key cellular parameters
such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2
based on docking analysis and structure-based drug design knowledge resulted in d1. Compound
d1 has nearly 2-fold increase of inhibitory activity (IC50: 49 nM) and over 1000-fold selectivity
against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively
reduce glucose excursion by 34% at the dose of 10 mg/kg in diabetic mice. Herein we report the
optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-
one DPP-4 inhibitors.
Key words
DPP-4 inhibitor; pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives; structure-based drug design;
molecular docking; anti-diabetic.
*Corresponding authors. Tel./fax: +86 21 3420 4787 (Faqin Jiang.), +86 21 3420 4791 (Lei Fu.). E-mail addresses:
lijianqb@126.com (Jianqi Li), jfq2008@sjtu.edu.cn (Faqin Jiang), leifu@sjtu.edu.cn (Lei Fu)
1

1. Introduction
Type 2 diabetes is one of the greatest impending global health concerns. To date 415 million
people live with diabetes worldwide, and an estimated 193 million people have undiagnosed
diabetes. Type 2 diabetes accounts for more than 90% of diabetes patients1. Progressing efforts
regarding risk factors for type 2 diabetes and some prevention programs have been proved to be
resultful. But the incidence and widespread occurrence of the disease continues to rise globally
and more than two-fold increase is expected over next 20 years. Among all discovered paths, the
incretin pathway is emerging as a promising target for diabetes treatment2. It facilitates a
communication channel between the intestine and the endocrine pancreas and accounts for 50-
70% of the total ꢀ cell derived insulin secretion in response to oral glucose ingestion2. In fact,
incretin-receptor activation is mainly occurred by glucagon like peptide-1 (GLP-1), one of the key
hormones of this mechanism. GLP-1 stimulates the secretion of insulin after oral glucose
absorption and as a result normalizes the blood glucose level3. On the other side, incretin is
inactivated by the enzyme dipeptidyl peptidase (DPP-4) through catalyzing the hydrolization of
GLP-14. Therefore, therapeutic agents based on potentiation of incretin action provide new
physiologically based approaches for the treatment of type 2 diabetes. For instance, the
development of degradation-resistant GLP-1–receptor agonists or DPP-4 inhibitors would lead to
increase plasma concentration of active GLP-15. On recent advances in drug discovery, a variety
of DPP-4 inhibitors with diverse structural features has been created as anti-diabetic drugs.
Unfortunately, the currently available DPP-4 inhibitors, as shown in Figure 1, are inevitably
associated with several side effects such as nasopharyngitis, headache, nausea, hypersensitivity,
skin reactions and pancreatitis. These side effects are caused due to the off-target effect: the
inhibition of other DPP subtypes such as DPP-8 and DPP-9. To avoid those side effects, it’s
necessary to design advanced inhibitors with high activity for DPP-4 and great selectivity against
other DPP subtypes as well.
Figure 1. Key features of DPP-4 inhibitors.
Pyrazolo[1,5-a]pyrimidin derivatives are wildly used in pharmaceutical industries6-8 and academic
researches9-13 as lead compounds for the discovery of potential medical agents. However, they
were never utilized as anti-diabetic candidates. The current study outlined the new approach
towards creating a new series of DPP-4 inhibitors, with a focus on rational drug design and
2

structural optimization to pursue high in vitro activity, high selectivity and low cytotoxicity. To
earn these benefits, we executed two strategies to generate new inhibitors as outlined in Figure 2.
Firstly, we aimed to generate new class ligands by applying a novel core structure. By analyzing
the structural components of the DPP-4 inhibitors, we found that the two key building blocks
should be standing adjacent to each other with a distance of 1-2 atoms14, 15. It suggests that the
positions of the substitutions are important for activity. The skeleton of pyrazolo[1,5-a]pyrimidin-
7(4H)-one perfectly meets the demand of such structural requirement, where substitutions can be
attached to multiple contiguous positions. The ꢁ-ꢁ stacking between the core structure and Y547
would hopefully stabilize the overall conformation16. We then constructed 4, 5-substituted
pyrazolo[1,5-a]pyrimidin-7(4H)-one analogs and discovered a novel series of DPP-4 inhibitors.
Secondly, we study the nature of key building blocks. A hydrophobic or aromatic ring is needed to
occupy the S1 pocket of DPP-417-20. The salt bridge between the ligand and E205/E206 residues
plays a significant role for the generation of inhibition activity16, 21. Based on such findings, we
designed compounds with a substituted benzyl group on 4’ position for the S1 occupation; and a
cyclic amine group on 5’ position for salt bridge networking. This approach could yeild a unique
series of DPP-4 inhibitors.
Figure 2. Design of novel DPP-4 inhibitors.
We herein report the design, synthesis and evaluation of biological activity of these newly
designed compounds. We also highlight the key interactions between functional groups of
synthesized compounds and DPP-4 active site residues for rational drug design.
2. Results and discussion
2.1 Chemistry
The synthesis of the target compounds is depicted in the following schemes. First of all,
intermediate 3 (Scheme 1) was synthesized from two key intermediates. Intermediate 1 was
obtained by the condensation and cyclization of 1H-pyrazol-5-amine and diethyl malonate.
Chlorination of 1 with POCl3 with N, N-dimethyl-aniline as the base, yielded the key product
intermediate 2. Compound 3 was produced via the hydrolyzation of 2 in 1 N NaOH aqueous
3

solution.
Starting from intermediate 3, the syntheses of b series ligands were carried out following a
synthetic route depicted below (Scheme 2). Intermediate 4 was synthesized by reacting 3 with
corresponding benzyl halides under basic conditions, where Cs2CO3 or K2CO3 or DIPEA was
used. The final compounds were converted from intermediate 4 where 2’ Cl was substituted with
two different amines followed by deprotection.
Compound 6 was made by the combination of 2-(bromomethyl)benzonitrile and diethyl malonate
in EtOH using EtONa as the base (Scheme 3). A cyclization reaction was carried out to yield
compound 7 which was chloridized by POCl3 to form compound 8. Compound d1 was obtained
by a substitution reaction on compound 8 with (R)-tert-butyl piperidin-3-yl carbamate at the final
step.
Scheme 1: i. EtONa EtOH, reflux; ii. POCl3, N,N-dimethyl-aniline, 100 oC; iii 1 N NaOH, 90 oC.
Scheme 2: i. Cs2CO3 or K2CO3 or DIPEA, DMF, 90 oC; ii. DIPEA, DMF, 90 oC; iii. TFA, DCM.
EtOOC
COOEt
NC
O
i
ii
H2N
Br
CN
N
EtOOC
COOEt
+
+
N
NH
N
Bu3N
CN
O
N
H
6
7
NH2
iii
iv
NC
Cl
N
CN
N
N
N
N
N
N
Cl
Cl
8
d1
Scheme 3. i. EtONa, EtOH, r.t.; ii. Bu3N, 180 oC; iii. POCl3, 110 oC; iv. (R)-tert-butyl piperidin-3-
yl carbamate, TEA, THF, 60 oC; TFA, DCM.
4

2.2 Biological evaluation and preliminary SAR analysis
Table 1. DPP-4 IC50 values of b series compounds.
B
Structure features: A1
A2
No.
Structure
DPP-4
IC50 (ꢂM)
A
R
b1
A1
A2
A1
A2
A1
A2
A1
A2
A1
A1
A1
A2
2-CN
2-CN
H
1.3 0.45
0.08 0.005
46.5 5.5
2.5 0.4
b2
b3
b4
H
b5
4-Me
4-Me
4-NO2
4-NO2
4-COOEt
4-COOH
2-F
2.0 0.4
b6
0.4 0.1
b7
11.5 4.5
17.2 2.1
10.4 0.9
59.9 4.4
21.5 1.3
1.2 0.6
b8
b9
b10
b11
b12
Alogliptin
2-NO2
0.004 0.001
Values are means of three experiments. Data are represented as mean SD (n = 3)
Table 2. IC50 values of d series compounds
D
No.
d1
DPP-4 IC50 (ꢂM)
0.049 0.004
Values are means of three experiments. Data are represented as mean SD (n = 3)
The substituted benzyl group is supposed to stretch into the S1 pocket. We tried different
functional groups in order to verify the impact brought by the substitutions on the benzyl ring. We
found that placing a cyano group at ortho position could create the best inhibitory activity against
5

DPP-4 (Table 1, b1 and b2). Cyano group in the S1 pocket has a typical effect improving the
DPP-4 inhibitory activity in many successful DPP-4 inhibitors, such as alogliptin and vildagliptin.
The same effect can also be obtained in our ligands. We explored the binding conformation of our
ligands in the DPP-4 active site by molecular docking. In our docking model, the cyano group
forms a H-bond with residue R125. This H-bond can be observed in the crystal structure 2NOC as
well. We consider it as the reason why the cyano group can bring a tipical effect to the DPP-4
ligands.
(R)-piperidin-3-amine is one of the structural features in a variety of DPP-4 inhibitors21-25
.
Therefore we introduced the (R)-piperidin-3-amine to our compounds as a key component to
generate the DPP-4 inhibitory activity. However, (R)-piperidin-3-amine was not the only choice at
the same position26-28. For further evaluation of the amine group, piperazine was used as the
substitution. Surprisingly, such a transformation brought significant activity improvement to the
ligand. Among all of our synthetic compounds, those with the piperazine substitution have better
DPP-4 IC50 value than their counterparts with (R)-piperidin-3-amine substitution (b2 vs b1, b4 vs
b3, b6 vs b5 and b8 vs b7). A maximum of 18 times (b4 vs b3) improvement was obtained with
explained optimization. The most potent compound in b series is b2, which has DPP-4 IC50 of 80
nM.
We performed molecule docking to study the binding of b1 and b2 in the protein in order to
explain the activity differences. As is depicted in Figure 4A and 4B, compound b1 and b2 lie in
the binding site where S1 pocket is occupied by the substituted benzyl ring. The amine of the two
ligands forms H-bond interactions with E205 and E206. The one difference between the two
ligands’ binding is the number of the H-bonds. Compound b1 forms an H-bond with E205 3.0
away. On the other hand, b2 forms two H-bonds between the two amines and the two important
acidic amino acids E205 and E206. The distances are 3.4 Å and 3.5 Å, respectively. The two
molecules also forms H-bond between the –CN group and R125. Superimposition of the b1 and
the ligand in the crystal structure (PBD code: 2NOC) shows that they have similar interactions
with the enzyme. What distinguishes them is the position and spatial configuration of the core
structure. The pyrazolo[1,5-a]pyrimidin-7(4H)-one core of our compounds tends to stretch
towards the enzyme while the ligand (IC50 =13 nM) in co-crystal structure goes to another
direction with larger space. We reasonably assumed that the reversal of the direction of the core
structure may gain us a better inhibitory activity.
To generate a core-redirected ligand, we designed to switch the two substitutions’ position. We put
the benzonitrile group to 6’ position and amine group to 7’ position. Then we could obtain a ligand
with a core reversal. Under the circumstances we created and synthesized compound d1 to test the
hypothesis (Table 2). The substitutions’ positions swap could change the binding conformation of
the core. Indeed, d1 showed an improved inhibition activity against DPP-4 with IC50 of 49 nM
which is nearly 2 times better than b2 and over 22 times stronger than b1. In DPP4-d1 docking
model (Figure 4D), we found a redirection of the core structure in the binding pocket. The cyano
6

substituted benzyn ring of d1 occupied the S1 pocket. The salt bridge network between the (R)-
piperidin-3-amine and E205 and E206 can also be observed. The R125 residue, however, forms a
cation-ꢁ interaction with the core structure to further stabilize the conformation. These unique
binding conformation along with the redirection of the core of d1 boosts its inhibition activity.
We chose those with IC50 value less than 5 ꢂM to perform the selectivity assay against DPP-8 and
DPP-9 (Table 3). Results show that they all have good selectivity against the DPP-4 homologues
proteins. Compound b2 and d1 have selectivity over DPP-8 and DPP-9 about 1000 times.
Table 3. DPP-8 and DPP-9 inhibitory activities of the selected compounds.
No.
b1
DPP-8 IC50 (ꢂM)
>100
DPP-8/DPP-4
>100
DPP-9 IC50 (ꢂM)
>100
DPP-9/DPP-4
>100
b2
>100
>1000
>40
>100
>1000
>40
b4
>100
>100
b5
>100
>50
>100
>50
b6
>100
>200
>100
>200
b12
d1
>100
>100
>100
>100
>100
>1000
>100
>1000
Values are means of three experiments.
2.3 Cytotoxicity assay
We performed MTT assay to determine the effects of compounds b2 and d1 on cell growth rate
and viability. As is shown (Figure 3), b2 and d1 present a dose dependent manner in cell viability.
As is demonstrated in the chart, cell viability of b2 at 100 ꢂM is more than 60%. In comparison
d1 shows a dramatic decrease viable cells at the same dose. The low cytotoxicity of d1 at the dose
of 20 ꢂM or less is confident. Overall compound b2 clearly shows a significant low cytotoxicity
while compared to d1.
7

100
80
60
40
20
0
b2
d1
.
.
.
.
.
.
1
5
0
0
0
0
1
2
5
0
1
Concentration (µM)
Figure 3. Inhibitory effect of compound b2 and d1 on the viability of HepG2 cells after 48 hrs is
evaluated by MTT test. Each value was presented as mean SD, n=3.
2.4 Molecular docking study
Interactions between ligands and DPP-4 (PDB code: 2ONC) are explained by molecular modeling
using GOLD Suite v5.0.1. (CCDC, Cambridge, U.K., 2010). Before docking was run, parameters
were specifically addressed. The binding site was defined to include all residues within a 15.0 Å
radius of the E205 Cꢃ carbon atom. An H-bond was restricted between the amine of the ligand
((R)-piperidin-3-amine or piperazine) and E205 of the enzyme. After the docking runs were
completed, 20 conformations were produced for each ligand and GoldScore was used as scoring
function. Other parameters were set as standard default. High-scoring complexes were inspected
visually for selecting the most reasonable solution.
A
B
8

D
C
Figure 4. Docking of b1 and b2 to the DPP-4 model; A. compound b1 is colored in green, the
protein and the residues are colored in white; B. b2 is colored in cyan, the protein and the residues
are colored in white; C. Superimpose of the docking results to the crystal structure 2NOC, the
protein is depicted with surface, b1 is colored in green, d1 is colored in gold, ligand in the crystal
is colored in orange; D. compound d1 is colored in gold, the protein and the residues are colored in
white.
2.5 IPGTT assay
The in vivo hypoglycemic effects of compounds b2 was evaluated in an intraperitoneal injection
glucose tolerance test (IPGTT) using diabetic male C57BL/6 mice. Single intraperitoneal injection
(IP) administration of b2 to male BALB/c mice 30 min prior to an IPGTT reduced plasma glucose
excursion in a dose-dependent manner from 1 mg/kg to 10 mg/kg. The results are shown in Table
4 and Figure 5. Intraperitoneal injection of b2 at a dose of 10 mg/kg significantly reduced plasma
glucose levels in male BALB/c mice by 34%, which transcended the effect of metformin (20%
reduction, Figure 5) at the same dose.
The dose-dependent effects of compound b2 in the IPGTT are shown in Figure 5. The results
indicate that compound b2 produced a dose-dependent improvement of glucose tolerance in
diabetic male BALB/c mice after injection and its minimum effective dose was 1 mg/kg (Figure
5B).
Therefore, the potent and selective DPP-4 inhibitor b2 is expected to provide a potential
therapeutic efficacy for the treatment of type 2 diabetes.
Table 4. IPGTT assays of compound b2 in diabetic mice.
Blood glucose (mM)
AUC
0 h
15.6 2.4
0.5 h
33.6 2.3
1 h
25.0 1.2
1.5 h
23.9 2.9
2 h
23.9 0.7
Control
b2
51.2 3.9
9

1 mg/kg
5 mg/kg
10 mg/kg
Metformin
10 mg/kg
12.1 0.3
13.2 0.2
11.6 0.2
27.3 1.2
21.9 1.2
20.2 0.4
21.9 2.0
20.8 2.0
18.6 1.2
18.1 1.9
17.8 1.9
15.6 1.6
14.1 2.3
14.8 2.3
14.3 3.5
38.7 3.4
37.3 3.1
33.7 2.5
12.9 2.4
23.4 2.5
21.3 1.5
19.4 4.6
21.4 5.7
40.7 6.3
Data are represented as mean SD (n = 4)
B
A
Figure 5. A. Effects of compound b2 in various concentrations and 10 mg/kg metformin on
glucose levels after an intraperitoneal injection glucose tolerance test in diabetic BALB/c male
mice. Compound b2 or saline (control) was administered 30 min prior to an injected dextrose
challenge (2 g/kg). B. The glucose AUC was determined from 0-120 min. Percent reduction
values for each treatment were generated from the AUC data normalized to the saline-challenged
controls.
3. Conclusion
We have constructed a series of potent and greatly selective DPP-4 inhibitors with pyrazolo[1,5-
a]pyrimidin-7(4H)-one core surrogates. The modification of the core led us to b2 which has IC50
of 80 nM and > 1000 fold selectivity over DPP-8 and DPP-9. We utilized docking program to
study the interactions between the ligand and the enzyme. We discovered that in our model the
substituted benzyl ring occupies the S1 pocket, and the amine substitution forms H-bond
interactions with the key residue E205 and E206. Through the comparison of our model to the co-
crystal structure 2NOC, this hypothesis came up that the core structure might have an impact on
the ligand activity. Therefore we designed and synthesized d1 to redirect the core structure. As a
result of this act the performance of the ligand improved immensely. Compound d1 has DPP-4
IC50 of 49 nM and more than 1000 fold selectivity over DPP8 and DPP-9. Although d1 has DPP-4
IC50 result greater than b2, yet b2 is considerably less cytotoxic. Further in vivo IPGTT assay
further indicated that compound b2 exert positive impact on the diabetic male mice. To sum up,
we have identified a series of new derivatives as well potent DPP-4 inhibitors. Compounds b2 and
d1 possess good DPP-4 inhibitory activity, remarkable selectivity over other DPP subtypes such as
DPP-8 and DPP-9, low cytotoxicity, and excellent in vitro and in vivo efficacy. It produced a
10

potential therapeutic efficacy for the treatment of type 2 diabetes.
4. Experimental Section
Chemistry
General Chemistry Procedure: All references to EA are diethyl ether; brine refers to a saturated
aqueous solution of NaCl. Unless otherwise indicated, all temperatures are expressed in °C
(degrees Centigrade). All reactions conducted under an inert atmosphere at room temperature
unless otherwise noted. 1H NMR spectra were recorded on an Agilent 400 MHz NMR. 13C NMR
spectra were recorded on an Agilent 100 MHz NMR. Chemical shifts are expressed in parts per
million (ppm). Coupling constants are in units of Hertz (Hz). Splitting patterns describe apparent
multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and
br (broad). High-resolution mass spectra (HRMS) and compound purity data were acquired on an
Agilent 6200 TOF LC/MS system, UV detector (220 and 254 nm).
4.1. General procedure A:
To a solution of 5-chloropyrazolo[1,5-a]pyrimidin-7(4H)-one (1 eq) in DMF was added 2 eq. of
Cs2CO3 or K2CO3 or DIPEA and the corresponding (bromomethyl)benzene analog. The reaction
was heated to 90 oC overnight. After the completion of the reaction, EA and water were added, the
organic layer was washed with water 3 times. Collect the organic layer, remove the solvent to
obtain the crude product. The crude product was purified by F.C.C. to obtain the corresponding
product.
4.1.1. 4-benzyl-5-chloropyrazolo[1,5-a]pyrimidin-7(4H)-one (4a): This compound is synthesized
1
according to general procedure A (yield: 68.56%); H NMR (400 MHz, CHLOROFORM-d) δ
7.66 (d, J = 1.00 Hz, 1H), 7.33 - 7.48 (m, 5H), 6.36 (d, J = 3.52 Hz, 1H), 6.07 (s, 1H), 5.97 (s,
2H); HRMS (ESI): calcd for C13H11ClN3O [M + H]+ 260.0591; found 260.0587.
4.1.2. 2-((5-chloro-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-yl)methyl)benzonitrile (4b): This
1
compound is synthesized according to general procedure A (yield: 59.56%); H NMR (400 MHz,
CHLOROFORM-d) δ 8.03 (d, J = 3.52 Hz, 1H), 7.70 (d, J = 7.83 Hz, 1H), 7.54 - 7.65 (m, 2H),
7.37 - 7.51 (m, 1H), 6.42 (d, J = 3.52 Hz, 1H), 6.22 (s, 2H), 6.15 (s, 1H); HRMS (ESI): calcd for
C14H10ClN4O [M + H]+ 285.0543; found 285.0567.
4.1.3. 4-((5-chloro-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-yl)methyl)benzonitrile (4c): This
1
compound is synthesized according to general procedure A (yield: 59.56%); H NMR (400 MHz,
CHLOROFORM-d) δ 7.75 (d, J = 3.13 Hz, 1H), 7.59 (d, J = 7.83 Hz, 2H), 7.21 - 7.26 (m, 2H),
6.40 (d, J = 3.13 Hz, 1H), 6.02 (s, 1H), 5.98 (s, 2H); HRMS (ESI): calcd for C14H10ClN4O [M +
H]+ 285.0543; found 285.0567.
11

4.1.4. ethyl 4-((5-chloro-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-yl)methyl)benzoate (4d): This
1
compound is synthesized according to general procedure A (yield: 51.11%); H NMR (400 MHz,
CHLOROFORM-d) δ 7.53 (d, J = 7.83 Hz, 2H), 7.30 - 7.40 (m, 2H), 7.16 (s, 1H), 7.00 (d, J =
1.00 Hz, 1H), 6.21 (s, 1H), 5.42 (s, 2H), 4.38 (q, J = 7.00 Hz, 2H), 1.40 (t, J = 1.00 Hz, 3H);
HRMS (ESI): calcd for C16H15ClN3O3 [M + H]+ 332.0802; found 332.0811.
4.1.5. 5-chloro-4-(2-fluorobenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (4e): This compound is
synthesized according to general procedure A (yield: 61.07%); 1H NMR (400 MHz,
CHLOROFORM-d) δ 7.78 (br. d, J = 1.00 Hz, 1H), 7.41 - 7.56 (m, 1H), 7.33 (d, J = 6.65 Hz,
1H), 6.97 - 7.19 (m, 2H), 6.35 (d, J = 3.13 Hz, 1H), 6.10 (s, 1H), 6.05 (s, 2H); HRMS (ESI): calcd
for C13H10ClFN3O [M + H]+ 278.0496; found 278.0482.
4.1.6. 5-chloro-4-(4-nitrobenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (4f): This compound is
synthesized according to general procedure A (yield: 45.91%); 1H NMR (400 MHz,
CHLOROFORM-d) δ 8.20 (d, J = 8.61 Hz, 2H), 7.79 (d, J = 3.52 Hz, 1H), 7.36 (d, J = 8.61 Hz,
2H), 6.47 (d, J = 3.13 Hz, 1H), 6.08 (br. s., 1H), 6.07 (s, 2H); HRMS (ESI): calcd for
C13H10ClN4O3 [M + H]+ 305.0441; found 305.0451.
4.1.7. 5-chloro-4-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (4g): 1H NMR (400 MHz,
CHLOROFORM-d) δ 7.65 (d, J = 3.13 Hz, 1H), 7.10 - 7.18 (m, 4H), 6.35 (d, J = 3.13 Hz, 1H),
6.08 (s, 1H), 5.92 (s, 2H), 2.33 (s, 3H); HRMS (ESI): calcd for C14H13ClN3O [M + H]+
274.0747; found 274.0755.
4.1.8. 5-chloro-4-(2-nitrobenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (4h): This compound is
synthesized according to general procedure A (yield: 66.28%); 1H NMR (400 MHz,
CHLOROFORM-d) δ 8.13 (d, J = 7.83 Hz, 1H), 7.81 (d, J = 3.52 Hz, 2H), 7.59 (t, J = 7.43 Hz,
1H), 7.52 (t, J = 7.83 Hz, 1H), 7.00 (d, J = 7.43 Hz, 1H), 6.45 (s, 2H), 6.04 (s, 1H); HRMS (ESI):
calcd for C13H10ClN4O3 [M + H]+ 305.0441; found 305.0433.
4.2. General procedure B.
To a solution of 4-benzyl-5-chloropyrazolo[1,5-a]pyrimidin-7(4H)-one analogs, piperazine or (R)-
o
tert-butyl piperidin-3-ylcarbamate and DIPEA in DMF was heated to 90 C overnight. After the
completion of the reaction, EA was added to the reaction mixture and washed with water ×3. The
organic layer was collected. Removed the solvent to obtain the crude product. The crude was
purified by F.C.C. to obtain the desired product.
4.2.1.
(R)-tert-butyl
(1-(4-(2-cyanobenzyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-
12

yl)piperidin-3-yl)carbamate (5a): This compound is synthesized according to general procedure B
(yield: 31.74%); 1H NMR (400 MHz, CHLOROFORM-d) δ 8.03 (d, J = 3.52 Hz, 1H), 7.70 (d, J
= 7.83 Hz, 1H), 7.54 - 7.65 (m, 2H), 7.37 - 7.51 (m, 1H), 6.42 (d, J = 3.52 Hz, 1H), 6.22 (s, 2H),
6.15 (s, 1H), 3.64 (d, J = 10.62 Hz, 2H), 3.25 (d, J = 12.51 Hz, 2H), 1.91 - 2.15 (m, 2H), 1.66 -
1.79 (m, 2H), 1.50 - 1.61 (m, 1H), 1.45 (br. s., 9H); HRMS (ESI): calcd for C24H29N6O3 [M +
H]+ 449.2301; found 449.2364.
4.2.2. (R)-tert-butyl (1-(4-benzyl-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-
yl)carbamate (5b): This compound is synthesized according to general procedure B (yield:
30.88%); 1H NMR (400 MHz, CHLOROFORM-d) δ 8.00 (s, 1H), 7.50 (d, J = 3.13 Hz, 1H), 7.28
- 7.39 (m, 2H), 7.17 - 7.27 (m, 2H), 6.07 (br. d, J = 1.00 Hz, 1H), 5.80 (s, 2H), 5.33 (s, 1H), 4.54 -
4.78 (m, 2H), 3.49 - 3.93 (m, 2H), 2.96 (s, 2H), 1.81 - 2.14 (m, 2H), 1.70 (d, J = 10.96 Hz, 1H),
1.45 (br. s., 9H); HRMS (ESI): calcd for C23H30N5O3 [M + H]+ 424.2349; found 424.2361.
4.2.3.
(R)-ethyl
4-((5-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-7-oxopyrazolo[1,5-
a]pyrimidin-4(7H)-yl)methyl)benzoate (5c): This compound is synthesized according to general
1
procedure B (yield: 55.79%); H NMR (400 MHz, CHLOROFORM-d) δ 7.53 (d, J = 7.83 Hz,
2H), 7.30 - 7.40 (m, 2H), 7.16 (s, 1H), 7.00 (d, J = 1.00 Hz, 1H), 6.21 (s, 1H), 5.42 (s, 2H), 4.55
(br. s., 1H), 4.33 - 4.47 (m, 2H), 3.02 - 3.73 (m, 6H), 1.97 (br. s., 1H), 1.59 (s, 9H), 1.40 (t, J =
1.00 Hz, 3H), 1.26 (br. s., 1H); HRMS (ESI): calcd for C26H34N5O5 [M + H]+ 496.2560; found
496.2483.
4.2.4.
(R)-tert-butyl
(1-(4-(2-fluorobenzyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-
yl)piperidin-3-yl)carbamate (5d): This compound is synthesized according to general procedure B
(yield: 55.79%); 1H NMR (400 MHz, CHLOROFORM-d) δ 7.78 (br. d, J = 1.00 Hz, 1H), 7.41 -
7.56 (m, 1H), 7.33 (d, J = 6.65 Hz, 1H), 6.97 - 7.19 (m, 2H), 6.35 (d, J = 3.13 Hz, 1H), 6.10 (s,
1H), 6.05 (s, 2H), 4.09 - 4.27 (m, 1H), 3.92 (d, J = 6.10 Hz, 2H), 3.58 (br. s., 1H), 3.25 - 3.50 (m,
1H), 2.28 (br. s., 2H), 2.14 (br. s., 1H), 1.44 (m, 9H), 1.22 - 1.31 (m, 1H); HRMS (ESI): calcd for
C23H29FN5O3 [M + H]+ 442.2254; found 442.2311.
4.2.5.
(R)-tert-butyl
(1-(4-(4-methylbenzyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-
yl)piperidin-3-yl)carbamate (5e): This compound is synthesized according to general procedure B
(yield: 14.38%); 1H NMR (400 MHz, CHLOROFORM-d) δ 7.47 (d, J = 2.74 Hz, 1H), 7.05 - 7.21
(m, 4H), 6.06 (d, J = 4.30 Hz, 1H), 5.75 (s, 2H), 5.33 (s, 1H), 4.58 (br. s., 1H), 3.64 (d, J = 10.96
Hz, 2H), 3.18 - 3.47 (m, 2H), 2.31 (s, 3H), 1.96 (br. s., 1H), 1.64 - 1.92 (m, 2H), 1.45 (br. s., 9H),
1.22 - 1.31 (m, 1H); HRMS (ESI): calcd for C24H32N5O3 [M + H]+ 438.2505; found 438.2606
4.2.6.
(R)-tert-butyl
(1-(4-(4-nitrobenzyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-
13

yl)piperidin-3-yl)carbamate (5f): This compound is synthesized according to general procedure B
(yield: 54.38%); 1H NMR (400 MHz, CHLOROFORM-d) δ 8.20 (d, J = 8.61 Hz, 2H), 7.79 (d, J =
3.52 Hz, 1H), 7.36 (d, J = 8.61 Hz, 2H), 6.47 (d, J = 3.13 Hz, 1H), 6.08 (br. s., 1H), 6.07 (s, 2H),
3.81 (br. s., 1H), 3.65 (d, J = 10.17 Hz, 2H), 3.48 (br. s., 2H), 3.25 (br. s., 2H), 1.91 - 2.17 (m,
2H), 1.41 - 1.46 (m, 9H); HRMS (ESI): calcd for C23H29N6O5 [M + H]+ 469.2199; found
469.2239.
4.3. General procedure C.
To a solution of compound 8 in DCM was treated with TFA in a ratio of 1/10 v/v. The reaction
was monitored with TLC. After the completion of the reaction, the solvent was removed by
vacuum and the crude was purified by F.C.C. to obtain the desired product.
4.3.1.
(R)-2-((5-(3-aminopiperidin-1-yl)-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-
yl)methyl)benzonitrile (b1): This compound is synthesized according to general procedure C
(yield: 61.15%); m.p. = 138-140 oC; 1H NMR (400 MHz, CHLOROFORM-d) δ 8.03 (d, J = 3.52
Hz, 1H), 7.70 (d, J = 7.83 Hz, 1H), 7.54 - 7.65 (m, 2H), 7.37 - 7.51 (m, 1H), 6.42 (d, J = 3.52 Hz,
1H), 6.22 (s, 2H), 6.15 (s, 1H), 3.64 (d, J = 10.62 Hz, 2H), 3.25 (d, J = 12.51 Hz, 2H), 1.91 - 2.15
(m, 2H), 1.66 - 1.79 (m, 2H), 1.50 - 1.61 (m, 1H); 13C NMR (CHLOROFORM-d) δ: 161.7, 160.5,
157.8, 140.9, 139.8, 137.6, 133.1, 130.4, 129.1, 117.6, 111.1, 98.9, 80.9, 52.9, 45.4, 45.0, 44.1,
39.7, 29.6; HRMS (ESI): calcd for C19H21N6O [M + H]+ 349.1777; found 349.1792.
4.3.2. 2-((7-oxo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidin-4(7H)-yl)methyl)benzonitrile (b2):
This compound is synthesized according to general procedure B (yield: 5.88%); m.p. = 102-104
oC; 1H NMR (400 MHz, CHLOROFORM-d) δ 8.12 (d, J = 1.00 Hz, 1H), 7.85 (br. s., 1H), 7.64
(d, J = 6.65 Hz, 2H), 7.55 (t, J = 7.24 Hz, 1H), 6.13 (br. s., 1H), 6.07 (s, 2H), 5.39 (d, J = 1.00 Hz,
1H), 3.57 - 3.66 (m, 4H), 3.41 - 3.51 (m, 4H); 13C NMR (101 MHz, CHLOROFORM-d) δ 160.9,
158.8, 141.7, 139.8, 137.6, 133.4, 133.1, 130.4, 129.1, 117.4, 111.7, 98.9, 80.9, 52.9, 45.0, 44.1,
39.7, 28.3; HRMS (ESI): calcd for C18H19N6O [M + H]+ 335.1620; found 335.1615.
4.3.3. (R)-5-(3-aminopiperidin-1-yl)-4-benzylpyrazolo[1,5-a]pyrimidin-7(4H)-one (b3): This
o
compound is synthesized according to general procedure C (yield: 22.70%); m.p. = 110-112 C;
1H NMR (400 MHz, CHLOROFORM-d) δ 7.44 (d, J = 3.52 Hz, 1H), 7.27 - 7.33 (m, 2H), 7.14 -
7.24 (m, 3H), 6.01 (d, J = 3.13 Hz, 1H), 5.75 (s, 2H), 5.43 (s, 1H), 4.16 (d, J = 12.52 Hz, 1H),
3.91 (d, J = 12.91 Hz, 1H), 3.72 (q, J = 6.78 Hz, 1H), 3.29 - 3.41 (m, 1H), 3.12 - 3.29 (m, 2H),
2.08 (br. s., 1H), 1.69 - 1.83 (m, 2H); 13C NMR (101 MHz, CHLOROFORM-d) δ 161.7, 159.0,
152.9, 139.4, 134.4, 128.8, 128.4, 128.0, 98.0, 80.4, 55.7, 48.8, 47.2, 44.6, 29.4, 22.7; HRMS
(ESI): calcd for C18H22N5O [M + H]+ 324.1824; found 324.1983.
14

4.3.4. 4-benzyl-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (b4): This compound is
o
1
synthesized according to general procedure B (yield: 31.59%); m.p. = 100-102 C; H NMR (400
MHz, CHLOROFORM-d) δ 8.12 (s, 1H), 7.53 (d, J = 3.13 Hz, 1H), 7.27 - 7.33 (m, 4H), 6.10 (d,
J = 3.52 Hz, 1H), 5.84 (s, 2H), 5.31 (s, 1H), 3.60 (t, J = 1.00 Hz, 4H), 3.44 (t, J = 4.89 Hz, 4H);
13C NMR (101 MHz, CHLOROFORM-d) δ 160.8, 158.8, 153.2, 139.2, 134.2, 128.9, 128.6,
128.3, 98.2, 80.9, 55.8, 45.4, 45.1, 44.2, 39.8; HRMS (ESI): calcd for C17H20N5O [M + H]+
310.1668; found 310.1664.
4.3.5.
(R)-5-(3-aminopiperidin-1-yl)-4-(4-methylbenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one
(b5): This compound is synthesized according to general procedure C (yield: 59.65%); m.p. =
132-134 oC; 1H NMR (400 MHz, CHLOROFORM-d) δ 7.39 (br. d, J = 1.00 Hz, 1H), 6.99 - 7.18
(m, 4H), 5.99 (br. s., 1H), 5.62 - 5.79 (m, 2H), 5.50 (br. d, J = 1.00 Hz, 1H), 4.09 - 4.27 (m, 2H),
3.93 (d, J = 6.13 Hz, 1H), 3.58 (br. s., 1H), 3.25 - 3.50 (m, 1H), 3.13 (d, J = 6.65 Hz, 2H), 2.28
(br. s., 3H), 2.14 (br. s., 1H), 1.53 (br. s., 1H); 13C NMR (101 MHz, CHLOROFORM-d) δ 161.7,
152.9, 139.3, 138.4, 131.3, 129.6, 128.2, 97.9, 80.6, 77.3, 55.6, 47.2, 45.8, 44.5, 28.6, 22.6, 21.1;
HRMS (ESI): calcd for C19H24N5O [M + H]+ 338.1981; found 338.2091.
4.3.6. 4-(4-methylbenzyl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (b6): This
o
compound is synthesized according to general procedure B (yield: 19.47%); m.p. = 112-114 C;
1H NMR (400 MHz, CHLOROFORM-d) δ 7.49 (d, J = 3.13 Hz, 1H), 7.06 - 7.18 (m, 4H), 6.06 (d,
J = 3.52 Hz, 1H), 5.76 (s, 2H), 5.29 (s, 1H), 3.51 - 3.66 (m, 2H), 3.32 - 3.42 (m, 2H), 2.87 (td, J =
4.94, 17.51 Hz, 4H), 2.30 (s, 3H); 13C NMR (101 MHz, CHLOROFORM-d) δ 160.1, 146.9,
138.4, 130.5, 128.8, 128.7, 127.7, 127.6, 97.5, 79.6, 54.8, 46.0, 45.0, 40.4, 28.9, 20.3; HRMS
(ESI): calcd for C18H22N5O [M + H]+ 324.1824; found 324.1844.
4.3.7. (R)-5-(3-aminopiperidin-1-yl)-4-(4-nitrobenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (b7):
This compound is synthesized according to general procedure C (yield: 59.65%); m.p. = 124-126
oC; 1H NMR (400 MHz, CHLOROFORM-d) δ 8.20 (d, J = 8.61 Hz, 2H), 7.79 (d, J = 3.52 Hz,
1H), 7.36 (d, J = 8.61 Hz, 2H), 6.47 (d, J = 3.13 Hz, 1H), 6.08 (br. s., 1H), 6.07 (s, 2H), 3.81 (br.
s., 1H), 3.65 (d, J = 10.17 Hz, 2H), 3.48 (br. s., 2H), 3.25 (br. s., 2H), 1.91 - 2.17 (m, 2H); 13C
NMR (CHLOROFORM-d) δ: 160.9, 160.8, 148.6, 141.7, 133.7, 131.1, 128.6, 128.1, 124.0, 123.6,
46.0, 45.3, 44.9, 40.5, 39.4, 30.0; HRMS (ESI): calcd for C18H21N6O3 [M + H]+ 369.1675;
found 369.1692.
4.3.8. 4-(4-nitrobenzyl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (b8): This
o
compound is synthesized according to general procedure B (yield: 19.78%); m.p. = 114-116 C;
1H NMR (400 MHz, CHLOROFORM-d) δ 8.33 (d, J = 8.20 Hz, 1H), 8.17 (d, J = 8.40 Hz, 1H),
8.08 - 8.14 (m, 2H), 8.00 (d, J = 8.61 Hz, 1H), 7.93 (s, 1H), 7.60 (d, J = 8.22 Hz, 1H), 3.66 - 3.71
15

(m, 2H), 3.59 - 3.66 (m, 2H), 3.57 (s, 1H), 3.48 - 3.54 (m, 2H), 3.44 (s, 1H), 3.35 - 3.42 (m, 2H);
13C NMR (101 MHz, CHLOROFORM-d) δ 160.9, 160.8, 148.6, 141.7, 133.7, 131.1, 128.6,
128.1, 124.0, 123.6, 46.0, 44.9, 40.5, 39.4, 29.6; HRMS (ESI): calcd for C17H19N6O3 [M + H]+
355.1519; found 355.1576.
4.3.9.
(R)-ethyl
4-((5-(3-aminopiperidin-1-yl)-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-
yl)methyl)benzoate (b9): This compound is synthesized according to general procedure C (yield:
o
1
22.72%); m.p. = 134-136 C; H NMR (400 MHz, DMSO-d6) δ 7.83 - 8.01 (m, 4H), 7.56 (d, J =
7.83 Hz, 1H), 7.35 - 7.50 (m, 2H), 5.07 - 5.36 (m, 2H), 4.66 - 4.93 (m, 2H), 4.28 (t, J = 7.04 Hz,
2H), 4.10 (d, J = 4.70 Hz, 1H), 3.93 (s, 2H), 1.87 - 2.01 (m, 2H), 1.23 - 1.31 (m, 3H), 0.75 - 0.86
(m, 2H); 13C NMR (101 MHz, CHLOROFORM-d) δ 166.1, 162.9, 132.6, 130.4, 130.1, 130.0,
128.9, 128.8, 127.8, 127.7, 126.6, 61.2, 57.0, 29.8, 29.7, 29.6, 29.4, 29.3, 14.2; HRMS (ESI):
calcd for C21H26N5O3 [M + H]+ 396.2036; found 396.2108.
4.3.10.
(R)-4-((5-(3-aminopiperidin-1-yl)-7-oxopyrazolo[1,5-a]pyrimidin-4(7H)-
yl)methyl)benzoic acid (b10): To a solution of b9 in EtOH was added 2 eq. NaOH and heated to
reflux overnight. After the completion of the reaction, EtOH was removed by vacuum. The crude
o
1
product was acidified by AcOH and purified by F.C.C. (yield: 20.70%); m.p. = 136-138 C; H
NMR (400 MHz, CHLOROFORM-d) δ 8.04 (br. d, J = 1.00 Hz, 1H), 7.70 (d, J = 7.43 Hz, 1H),
7.59 (br. s., 2H), 7.39 - 7.53 (m, 1H), 6.42 (br. d, J = 1.00 Hz, 1H), 6.22 (s, 2H), 6.14 (s, 1H), 2.36
(s, 2H), 2.25 (br. s., 2H), 2.03 (br. s., 4H), 0.80 - 0.93 (m, 1H); 13C NMR (101 MHz,
CHLOROFORM-d) δ 169.3, 163.1, 143.2, 131.4, 130.8, 131.0, 128.9, 128.8, 128.0, 127.7, 126.6,
92.2, 57.0, 53.5, 50.4, 38.5, 22.3; HRMS (ESI): calcd for C19H22N5O3 [M + H]+ 368.1723;
found 368.1822.
4.3.11.
(R)-5-(3-aminopiperidin-1-yl)-4-(2-fluorobenzyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one
(b11): This compound is synthesized according to general procedure C (yield: 32.33%); m.p. =
138-140 oC; 1H NMR (400 MHz, CHLOROFORM-d) δ 7.78 (br. d, J = 1.00 Hz, 1H), 7.41 - 7.56
(m, 1H), 7.33 (d, J = 6.65 Hz, 1H), 6.97 - 7.19 (m, 2H), 6.35 (d, J = 3.13 Hz, 1H), 6.10 (s, 1H),
6.05 (s, 2H), 4.09 - 4.27 (m, 1H), 3.94 (d, J = 6.10 Hz, 2H), 3.58 (br. s., 1H), 3.25 - 3.50 (m, 1H),
2.28 (br. s., 2H), 2.14 (br. s., 1H), 1.22 - 1.31 (m, 1H); 13C NMR (101 MHz, CHLOROFORM-d) δ
161.5, 161.2, 139.2, 139.1, 133.9, 128.9, 127.2, 125.6, 120.2, 119.1, 117.2, 114.3, 34.9, 28.7, 28.3,
26.2, 21.7, 13.1；HRMS (ESI): calcd for C18H21FN5O [M + H]+ 342.1730; found 342.2113.
4.3.12. 4-(2-nitrobenzyl)-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (b12): This
o
compound is synthesized according to general procedure C (yield: 32.33%); m.p. = 106-108 C;
1H NMR (400 MHz, CHLOROFORM-d) δ 8.13 (d, J = 7.83 Hz, 1H), 7.81 (d, J = 3.52 Hz, 1H),
7.59 (t, J = 7.43 Hz, 1H), 7.52 (t, J = 7.83 Hz, 2H), 7.00 (d, J = 7.43 Hz, 1H), 6.45 (s, 2H), 6.04 (s,
16

1H), 3.64 - 3.71 (m, 2H), 3.52 - 3.64 (m, 4H), 3.36 - 3.47 (m, 2H); 13C NMR (101 MHz,
CHLOROFORM-d) δ 160.0, 140.8, 133.6, 132.6, 130.1, 129.3, 126.9, 124.0, 123.5, 123.0, 118.1,
115.3, 30.4, 29.2, 28.7, 21.7, 13.1; HRMS (ESI): calcd for C17H19N6O3 [M + H]+ 354.1440;
found 354.1476.
4.4.1. Diethyl 2-(2-cyanobenzyl)malonate (6): To a solution of 1 g (6.24 mM) diethyl malonate
and 1.22 g (6.24 mM) 2-(bromomethyl)benzonitrile in EtOH was added 0.85 g (12.5 mM) EtONa
at ice bath. The reaction mixture was stirred at ambient temperature overnight and monitored by
TLC. After the completion of the reaction, EtOH was removed by vacuum. Then EA and 1N HCl
solution were added to the mixture. The organic layer was washed with water 3 times and
concentrated under vacuum. The crude product was purified by F.C.C. to obtain the title product.
1H NMR (CHLOROFORM-d) δ: 7.50 - 7.64 (m, 4H), 4.65 (s, 2H), 4.21 (q, J = 7.02 Hz, 4H), 3.37
(s, 1H), 1.29 (t, J = 7.21 Hz, 6H).
4.4.2.
2-((5,7-dioxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-6-yl)methyl)benzonitrile
tributylamine salt (7): 1 g (3.63 mM) diethyl 2-(2-cyanobenzyl)malonate and 0.3 g (3.63 mM) 1H-
o
pyrazol-3-amine was added 1.35 g (7.26 mM) tributylamine and heated to 170 C for 6 h. It was
cooled to room temperature and excess tri-n-butylamine decanted away from the solid product.
The latter was triturated overnight with isohexane, filtered and dried under vacuum to give the
title compound. 1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, J = 1.00 Hz, 1H), 7.93 - 8.00 (m, 1H),
7.60 - 7.84 (m, 4H), 6.45 - 6.56 (m, 1H), 5.28 - 5.36 (m, 2H), 2.83 - 3.05 (m, 6H), 1.40 - 1.56 (m,
6H), 1.17 - 1.31 (m, 6H), 0.68 - 0.90 (m, 9H).
4.4.3. 2-((5,7-dichloropyrazolo[1,5-a]pyrimidin-6-yl)methyl)benzonitrile (8): Under nitrogen gas
atmosphere, phosphorous oxychloride 30 (160 mL, 1.72 mol) and dimethylaniline (16 mL, 132
mmol) was added successively to the 2-((5,7-dioxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-6-
yl)methyl)benzonitrile tributylamine salt (16 g, 97 mmol). The mixture was heated at 110° C. for 4
hours then excess POC13 was removed under vacuum. The residue was made basic with 3N
NaOH solution (pH=9-10) and extracted with ethyl acetate (×3). The combined organic layers
were dried over anhydrous Na2SO4, filtered, and con-centrated under vacuum. The residue was
purified by silica gel chromatography (100percent DCM) to provide 15.8 grams of the solid
1
yellow product, (81% yield). H NMR (400 MHz, DMSO-d6) δ 8.26 (d, 1H), 7.77 (d, 1H), 7.66
(m, 2H), 7.52 (m, 2H), 5.34 (s, 2H).
4.4.4.
(R)-2-((7-(3-aminopiperidin-1-yl)-5-chloropyrazolo[1,5-a]pyrimidin-6-yl)methyl)
benzonitrile (d1): To a solution of of 2-((5,7-dichloropyrazolo[1,5-a]pyrimidin-6-yl)methyl)
benzonitrile in THF was add (R)-tert-butyl piperidin-3-ylcarbamate and TEA and stirred at
ambient temperature overnight. The reaction was monitored by TLC. After the completion of the
17

reaction, the solvent was removed by vacuum and the crude product was purified by F.C.C. to
obtain (R)-tert-butyl (1-(5-chloro-6-(2-cyanobenzyl)pyrazolo[1,5-a]pyrimidin-7-yl)piperidin-3-
yl)carbamate. The product was put into DCM:TFA=20:1 solution and stirred at ambient
temperature for 1h. After the completion of the reaction, DCM and 1N NaOH was added to the
mixture, the organic layer was washed with water. Collect the organic layer and removed the
solvent with vacuum. The crude product was purified by F.C.C. to obtain the title product (10% in
o
1
two steps). m.p. = 138-140 C; H NMR (400 MHz, CHLOROFORM-d) δ 8.01 - 8.25 (m, 1H),
7.80 - 7.93 (m, 1H), 7.42 - 7.60 (m, 2H), 7.19 - 7.25 (m, 2H), 5.56 (m, 2H), 3.69 - 4.22 (m, 2H),
3.54 (d, J = 10.56 Hz, 1H), 2.83 (t, J = 7.43 Hz, 1H), 2.09 - 2.45 (m, 2H), 1.62 - 1.97 (m, 2H),
0.90 - 1.04 (m, 1H); 13C NMR (CHLOROFORM-d) δ: 161.5, 160.6, 158.8, 138.4, 137.6, 132.2,
131.4, 129.9, 126.4, 115.8, 113.3, 97.9, 83.5, 51.1, 46.7, 45.0, 44.1, 39.7, 29.6; HRMS (ESI):
calcd for C19H20ClN6 [M + H]+ 367.1438; found 367.1440.
4.5. IC50 assays for DPP-4, DPP-8 and DPP-9
Solutions of test compounds in varying concentrations (ꢄ10 mM final concentration) were
prepared in dimethyl sulfoxide (DMSO) and then diluted into assay buffer comprising: 20 mM
Tris, pH 7.4; 20 mM KCl; and 0.1 mg/mL BSA. Human DPP-4 (0.1 nM final concentration) was
added to the dilutions and pre-incubated for 10 minutes at ambient temperature before the reaction
was initiated with A-P-7-amido-4-trifluoromethylcoumarin (AP-AFC; 10 ꢂM final concentration).
The total volume of the reaction mixture was 100 ꢂL depending on assay formats used (96 well
plates). The reaction was followed kinetically (excitation ꢅ= 400 nm; emission ꢅ= 505 nm) for 5-
10 minutes or an end-point was measured after 10 minutes. Inhibition constants (IC50) were
calculated from the enzyme progress curves using standard mathematical models16.
Recombinant human DPP-8 and DPP-9 (X-Y biotechnology, Shanghai, China) were diluted to a
final volume of 50 ꢂL in assay buffer (100 mM Tris-HCl, 100 mM NaCl, pH 7.8) and added to 96-
well flat-bottom microtiter plates, followed by the addition of 10 ꢂL inhibitor and 40 ꢂL substrate
(H-Gly-Pro-AMC, GL Biochem(Shanghai)Ltd., final concentration in the assay, 303 ꢂM). The
plates were incubated at 37oC for 10 min. after incubation, fluorescence was measured using
microplate reader (excitation 380 nm/ emission 460 nm)29.
4.6. Cell cytotoxicity MTT assay
This experiment was carried out on a 96-well plate. HepG2 cells in 100 ꢂL DMEM were
incubated with the negative control (1 ꢂL of DMSO) or the inhibitors (dissolved in DMSO) at
doses ranging from 1 μM to 100 μM (final concentration) for 48 hrs. Then 20 ꢂL of 5 mg/ml MTT
was added to the mixture followed by a 6 h-incubation. After the incubation, the upper clear
solution was carefully removed followed by the addition of 100 ꢂL of DMSO. The plate was
shaked gently for 1 min so that complete dissolution was achieved. The absorbance of the solution
at 490 nm was measured using the microplate spectrophotometer system (Thermo Scientific
18

Varioskan Flash). Cell viablility was calculated based on the OD values.
Acknowledgments
We thank the Medicine and Engineering interdisciplinary Research Fund of Shanghai Jiao Tong
University (YG2015QN03, YG2014MS10 and YG2017MS77), National Natural Science
Foundation of China (81202397) and Shanghai Natural Science Fund (12ZR1415400) for the
financial support. We thank Professor Yongxiang Wang’s Laboratory (Shanghai Jiao Tong
University) for the biological support of this research.
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Surrogating and redirection of Pyrazolo[1,5-a]pyrimidin-7(4H)-one core, a
novel class of well potent and selective DPP-4 Inhibitors
Xinxian Denga,b, Jian Shena,c, Hui Zhud, Jia Xiaoa, Ran Suna, Celine Lama, Juntao Wanga, Yixue
Qiaoa, Mojdeh S. Tavallaiea, Yang Hua, Yi Due, Jianqi Li b*, Faqin Jianga* and Lei Fua†
The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-
4 inhibitors, led to a potent and selective inhibitor compound b2 with remarkable in vitro DPP-4
inhibitory action (IC50: 80 nM), whilst maintaining other key cellular parameters such as high
selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on
docking analysis and structure-based drug design knowledge resulted in d1 with nearly 2-fold
increase of inhibitory activity (IC50: 49 nM) and over 1000-fold selectivity against DPP-8 and
DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose
excursion by 34% at the dose of 10 mg/kg in diabetic mice. Herein we report the optimization and
design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4
inhibitors.
*Corresponding authors. Tel./fax: +86 21 3420 4787 (Faqin Jiang.), +86 21 3420 4791 (Lei Fu.). E-mail addresses:
lijianqb@126.com (Jianqi Li), jfq2008@sjtu.edu.cn (Faqin Jiang), leifu@sjtu.edu.cn (Lei Fu)
21