Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa inhibitors with potent anticoagulant activity

Article abstract of DOI:10.1016/j.bmc.2017.03.055

A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl, and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC₅₀ (FXa) value of 0.14?μM and 98% inhibition rate, warranted further investigation as an FXa inhibitor.

Full text of DOI:10.1016/j.bmc.2017.03.055

Accepted Manuscript
Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone
derivatives as FXa inhibitors with potent anticoagulant activity
Xiaoqing Sun, Zexin Hong, Moyi Liu, Su Guo, Di Yang, Yong Wang, Tian Lan,
Linyu Gao, Hongxia Qi, Ping Gong, Yajing Liu
PII:
S0968-0896(17)30420-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.055
BMC 13653
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 February 2017
24 March 2017
26 March 2017
Please cite this article as: Sun, X., Hong, Z., Liu, M., Guo, S., Yang, D., Wang, Y., Lan, T., Gao, L., Qi, H., Gong,
P., Liu, Y., Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa
inhibitors with potent anticoagulant activity, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.03.055
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Design, synthesis, and biological activity of novel tetrahydropyrazolopyridone
derivatives as FXa inhibitors with potent anticoagulant activity
Xiaoqing Suna,b, Zexin Honga,b, Moyi Liua,b, Su Guoa,b, Di Yanga,b, Yong Wanga,b, Tian Lana,b,
Linyu Gaoa,b, Hongxia Qic, Ping Gonga,b, Yajing Liua,b,*
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry
of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
bDepartment of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, PR China
cShengjing Hospital of China Medical University, Shenyang 110004, PR China
Abstract
A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl,
and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and
evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed
moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further
examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b
were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC50
(FXa) value of 0.14 µM and 98% inhibition rate, warranted further investigation as an FXa
inhibitor.
Keywords: FXa; anticoagulant activity; synthesis; tetrahydropyrazolopyridone derivatives
1. Introduction
Anticoagulants are commonly used for the prevention and treatment of thromboembolic
disorders.1 Warfarin, heparin and its derivatives are traditional anticoagulants; these drugs are
effective and relatively safe when used properly. However, their short comings are
well-established, including drug-drug and drug-food interactions, parenteral administration, and
the need for frequent laboratory monitoring.2-3 Therefore, the development of safe and efficacious
oral anticoagulants for the prevention and treatment of a wide range of thrombotic diseases is
urgently needed.
Coagulation enzyme factor Xa (FXa), a serine protease involved in the coagulation cascade,
is the key enzyme at the convergence point of the intrinsic and extrinsic coagulation pathways.
FXa can specifically catalyze the conversion of prothrombin into thrombin and lead to the
production of fibrin. Accordingly, FXa is a promising target for antithrombotic drugs.4-9
Over the past few decades, several oral and selective FXa inhibitors, including rivaroxaban,
apixaban, and edoxaban, have been approved (Fig. 1). Table 1 contained a brief overview about
the ongoing approval processes for various clinical indications.3,10 Apixaban, a promising oral
anticoagulant in clinical development, also has some limitations. For example, it resulted in a
dose-related increase in bleeding, and the number of bleeding events in a trial for treatment of an
acute coronary syndrome resulted in the cessation of the trial.11-13 On the basis of the features of
apixaban, we endeavored to find alternative FXa inhibitors with good safety and potency.
Table1
*Corresponding author. Tel./fax: +86 24 43520217.
E-mail address: lyjpharm@126.com (Y. Liu).

Overview about the ongoing approval processes of the clinical indications of FXa inhibitors.
Prophylaxis of VTE
FXa
Prophylaxis of
strokec
Acute coronary
syndrome
1a
U.S.FDAd
2b
U.S.FDA
inhibitors
Pradaxa
EMAe
√
EMA
√
U.S.FDA
EMA
U.S.FDA
EMA
-
-
√
√
√
√
U.S.FDA
EMA
U.S.FDA
EMA
U.S.FDA
EMA
U.S.FDA
EMA
Rivaroxaban
Apixaban
-
√
√
√
√
EMA
-
√
√
√
EMA
-
U.S.FDA
EMA
U.S.FDA
U.S.FDA
EMA
U.S.FDA
-
-
√
√
√
√
U.S.FDA
EMA
U.S.FDA
EMA
U.S.FDA
EMA
U.S.FDA
EMA
-
Edoxaban
-
√
√
√
√
√
√
a Used following elective hip and knee replacement surgery.
b Used as treatment and secondary prophylaxis of VTE.
c Used as prophylaxis of stroke and systemic embolism in patients with nonvalvular atrial fibrillation.
d FDA, Food and Drug Administration.
e EMA, European Medicines Agency.
Cl
O
H2N
N
O
O
O
HN
O
N
NH
N
N
N
H
N
O
O
O
O
HN
S
O
N
S
N
N
Cl
N
O
O
NMe2
O
Rivaroxaban
Apixaban
Edoxaban
Fig. 1. Structures of oral FXa inhibitors.
In previous studies, we identified the X-ray structure of apixaban bound to FXa (Fig. 2) and
explored the structure–activity relationships (SARs).14-15 The tetrahydropyrazolopyridone scaffold
(P3 moiety) interacted with Gln192 and Gly216, and was necessary for the anticoagulant activity.
The p-methoxyl group was oriented in a planar manner relative to the phenyl P1 moiety, and the
electric effect of the substituents in the P1 moiety could influence activity. The X-ray structure
appeared to show the interaction of a carbonyl oxygen group of the pendant lactam (P4 moiety)
with a water molecule. Therefore, groups in the P4 moiety with moderate hydrophilicity were
beneficial to activity. The C-3 pyrazole carboxamido (P2 moiety) showed hydrogen bonding to
Glu146. Furthermore, the investigation of the C-3 pyrazole position revealed the breadth of
substitutions that were readily accommodated at the FXa active site.
P2
O
H2N
P3
N
N
N
O
O
O
N
Apixaban
P1
P4

Fig. 2. Structure of apixaban bound to FXa.14
In our previous study,15 it was observed that the 1,2,4-triazole compound 14a (PT = 0.5,
APTT = 0.8, IC50 = 0.15 µM) (Fig. 3) showed superior anticoagulant activity to apixaban (PT =
0.8, APTT = 0.9, IC50 = 0.23 µM). Consequently, we changed the position of the nitrogen atom on
the triazole and introduced a series of 1,3,4-triazole groups in the C-3 pyrazole position to
evaluate the anticoagulant activity. Furthermore, we noticed that compound 33a (Fig. 3), which
contained 1,2,3-triazolylmethyl in the C-3 pyrazole position, showed strong anticoagulant activity
with a Ki (FXa) value of 0.67 nM.14 Therefore, two series of triazolylmethyl derivatives,
1,2,3-triazolylmethyl and 1,2,4-triazolemethyl, were designed. In addition, a selection of tetrazole
compounds was also explored for the anticoagulant activity. Furthermore, to investigate the
potency of partially saturated heterocyclic compounds, a series of five-, six-, and seven-membered
nitrogenous heterocyclics was also incorporated into C-3 pyrazole position (Fig. 3).
Fig. 3. Structures of compounds 14a, 33a, and target compounds.
2. Chemistry
All target compounds listed in Fig. 3 were prepared from the key intermediate 11 shown in
Schemes 1-5.
2.1. Synthesis of ethyl 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetr
ahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate (11)
The key intermediate 11 was achieved using the convenient eleven-step procedure outlined in
Scheme 1, which was reported in detail in our previous study.15

Scheme 1. Reagents and conditions: (i) 5-chlorovaleryl chloride, TMA, THF, 0 °C, 30 min, rt., 6 h; (ii) K2CO3,
DMSO, 80 °C, 6 h; (iii) PCl5, CH2Cl2, reflux, 5 h; (iv) morpholine, 130 °C, 1.5 h; (v) NaNO2, HCl, H2O, 0 °C, 30
min; (vi) ethyl 2-chloro-3-oxobutanoate, sodium acetate, rt., 6 h; (vii) TEA, ethyl acetate, 80 °C, 6 h; (viii) TFA,
CH2Cl2, rt., 1 h; (ix) Fe powder, HCl, NH4Cl, EtOH/H2O, reflux, 2.5 h; (x) 5-chlorovaleryl chloride, THF, 0 °C, 30
min, rt., 6 h; (xi) NaH, DMF, 0 °C, 1 h.
2.2. Synthesis of target compounds 15a-15f and 24
The target compounds 15a-15f and 24 were synthesized as outlined in Scheme 2.16 The
aminolysis of intermediate 11 with formamide provided the carboxamido compound 12.17 Then,
the amino derivative 13 was obtained via Hofmann rearrangement of 12 under basic conditions in
the presence of iodobenzene diacetate,18 which was then converted to 14 by using DMF-DMA at
reflux. Subsequently, the target compounds 15a-15f were obtained via cyclization of compound 14
and different hydrazides. Using an alternative route, the tetrazole compound 24 was afforded by
cyclization from 13, triethyl orthoformate, and sodium azide.
Scheme 2. Reagents and conditions: (i) CH3ONa, HCONH2, DMF, 50 °C, 4 h; (ii) a: KOH, PhI(OAc)2, MeOH, rt.,
5 h; b: NaOH, dioxane, 100 °C, 6 h; (iii) DMF-DMA, CH2Cl2, reflux, 3 h; (iv) hydrazide, HOAc, 80 °C, 2 h; (v) a:
triethyl orthoformate, HOAc, 45 °C, 2 h; b: NaN3, 100 °C, 5 h.
2.3. Synthesis of target compounds 20a-20f and 26a-26b
The preparation of target compounds 20a-20f and 26a-26b is illustrated in Scheme 3. The
key intermediate 11 was converted into the chlorinated derivative 18 via hydrolysis, reduction, and

chlorination reactions. Then, the target compounds 20a-20f and 26a-26b were obtained via
substitution of 18 with intermediates 19a-19f and 25a-25b, respectively.14 The triazoles 19a-19f
were synthesized from different substituted benzamides through condensation and cyclization
reactions.19 While the compounds 25a-25b were prepared from different substituted
benzaldehydes through condensation, cyclization, and hydrolysis.20-21
Scheme 3. Reagents and conditions: (i) NaOH, MeOH, H2O, 60 °C, 1 h; (ii) a: TEA, THF, ethyl chloroformate,
30 °C, 4 h; b: sodium borohydride, 0 °C, 30 min; (iii) SOCl2, CH2Cl2, rt., 1 h; (iv) NaH, LiBr, DMF, 90 °C, 6 h; (v)
DMF-DMA, 120 °C, 2 h; (vi) HOAc, 80% hydrazine hydrate, 90 °C, 2 h; (vii) NaOAc, semicarbazide, MeOH, rt.,
2 h; (viii) Br2, HOAc, NaOAc, rt., 2 h; (ix) KOH, EtOH, reflux, 5 h; (x) HCl, reflux, 3 h.
2.4. Synthesis of target compounds 22a-22c and 23
As described in Scheme 4, the substitution of compound 18 with sodium azide obtained 21,
which was converted into final products 22a and 22b via cyclization with propiolic acid or
2-chloroacrylonitril in the presence of CuI.22-24 Subsequently, target compound 22c was afforded
from 22b via hydrolysis, and the benzotriazole compound 23 was obtained via substitution of 18
with benzotriazole directly.25
Scheme 4. Reagents and conditions: (i) NaN3, DMF, rt., 6 h; (ii) propiolic acid, CuI, CsCO3, DMF, 120 °C, 5 h; (iii)
2-chloroacrylonitril, CuI, 80 °C, 4 h; (iv) 30% H2O2, NaOH, EtOH, 50 °C, 2 h; (v) K2CO3, benzotriazole, DMF,
90 °C, 6 h.
2.5. Synthesis of target compounds 28a-28e
The synthesis of target compounds 28a-28e is summarized in Scheme 5.26 Compound 12 was
treated with trifluoroacetic anhydride in TEA to afford the nitrile compound 27, which was then
subjected to reaction with different diamines to obtain the target compounds 28a-28e, respectively.

Scheme 5. Reagents and conditions: (i) TEA, CH2Cl2, trifluoroacetic anhydride, 30 °C, 2 h; (ii) diamine, sulfur,
116 °C, 2 h.
3. Results and discussion
3.1. In vitro anticoagulant activity
The EC2X values for the anticoagulant activity were determined for all target compounds by
using human and rabbit plasma in vitro. The EC2X values were defined by the prothrombin time
(PT) and activated partial thromboplastin time (APTT). PT measures the effect of compounds on
the extrinsic pathway of coagulation, whereas APTT represents the effect on the intrinsic pathway.
The EC2x values were the average of at least three independent experiments. The effects of target
compounds on anticoagulant activity are illustrated in Table 2.
Table 2
Effects of all target compounds on anticoagulant activity.
EC2x(µM)a
Compd.
P2
PTb(human/rabbit)
4.9/5.7
APTTb(human/rabbit)
10.0/13.7
15a
1.2/1.7
0.5/0.9
4.1/5.9
7.6/8.4
7.2/8.0
2.8/3.0
0.7/1.2
15b
15c
15d
15e
15f
8.3/9.1
15.6/18.0
9.6/11.5
2.4/3.4
0.8/1.2
0.9/2.1
3.6/5.0
1.1/1.7
2.3/3.1
20a
20b
20c

1.9/3.5
1.6/1.9
2.0/3.2
2.4/2.1
3.6/5.0
2.3/2.1
4.3/5.2
3.7/3.9
20d
20e
20f
22a
0.7/0.9
1.4/2.1
1.0/1.5
3.1/5.2
22b
22c
25.2/29.4
4.2/5.7
31.6/39.1
7.3/8.2
23
24
11.7/12.4
27.1/29.8
26a
17.7/19.3
19.5/23.2
26b
6.2/7.5
8.8/9.6
11.8/12.5
10.5/12.0
14.6/17.1
13.5/15.4
24.3/26.9
0.9/1.4
28a
28b
8.9/10.0
9.4/10.8
10.9/12.1
0.8/1.0
28c
28d
28e
Apixabanc
a EC2x values represent the average of at least three independent experiments.
b The concentration of the compound required to double the clotting time in the PT assay using human/rabbit
plasma.
c Used as a positive control.
As illustrated in Table 2, all target compounds showed moderate to significant anticoagulant
activity with EC2X values ranging from 40 to 0.5 µM. The most promising compounds 15c and
22b showed strongly potent anticoagulant effects with EC2X values of PT = 0.5, APTT = 0.7 and

PT = 0.7, APTT = 1.0, respectively, which were superior to apixaban (PT = 0.8, APTT = 0.9).
The introduction of different triazole groups on the C-3 pyrazole showed little impact on the
anticoagulant activity. First, the introduction of 1,3,4-triazole groups was important for the
anticoagulant activity. Particularly, compounds with methyl and morpholinylmethyl at the 2-C
position of 1,3,4-triazole (15b and 15c) showed marked potency (PT = 1.2, APTT = 2.8 and PT =
0.5, APTT = 0.7, respectively), while the introduction of other substituents on 1,3,4-triazole (15a
and 15d-15f) resulted in a negative potency, suggesting the steric effect could influence the
anticoagulant activity. Based on the excellent activity of 15c, a closer look at the model of 15c in
the active site of FXa clearly showed morpholinylmethyl was very suitable to P2 pocket (Fig. 5).
As expected, when one carbon atom was inserted between 1,2,4-triazole and the nucleus, the
activity of target compounds 20a-20f was comparable to 1,2,4-triazole compound 14a (PT = 0.5,
APTT = 0.8).16 The introduction of substituents on the phenyl group of 1,2,4-triazole showed
similar anticoagulant activity. In particular, compound 20b (PT = 0.8, APTT = 1.1) showed
activity equal to that of apixaban (PT = 0.8, APTT = 0.9).
To our delight, 1,2,3-triazolylmethyl derivatives 22a-22c showed excellent anticoagulant
activity. It is worth mentioning that the introduction of the cyano and amide groups to the 4-C
position of 1,2,3-triazolylmethyl (22b and 22c) showed higher anticoagulant activity (PT = 0.7,
APTT = 1.0 and PT = 1.4, APTT = 3.1, respectively) than compound 22a did (PT = 2.4, APTT =
3.7), which contained unsubstituted 1,2,3-triazolylmethyl. However, compound 23, with the
benzene ring into triazole, showed a dramatic loss in activity (PT = 25.2, APTT = 31.6). A
reasonable explanation for this was that the electric effect may influence the anticoagulant activity.
To increase hydrogen bond action, triazole ketones 26a and 26b were synthesized.
Unfortunately, both compounds showed lost potency (PT > 11, APTT > 19). Meanwhile, a series
of partially saturated five-, six-, and seven-membered nitrogenous heterocyclics were introduced
into the C-3 pyrazole (28a-28e) that resulted in decreased anticoagulation activity. The results also
supported the influence of electric effect on compound activity.
3.2. FXa enzymatic assays in vitro
Based on the anticoagulant potency, as shown in Table 2, the five tested compounds 15b, 15c,
20b, 20c, and 22b exhibited moderate to excellent anticoagulant activity. Therefore, these
compounds were tested at two concentrations of 5 µM and 0.5 µM against human FXa. The
percentage inhibition values were summarized in Table 3, as mean values of experiments
performed in triplicate.
Table 3
The percentage inhibition of six compounds against human FXa.
Inhibition(%) SDa
Compd.
5 µM
0.5 µM
15b
15c
65.25 1.08
98.96 0.60
76.38 0.24
70.31 0.31
94.01 0.21
98.36 0.31
55.12 0.46
94.53 0.50
66.12 0.25
60.62 1.06
86.68 0.31
91.36 0.66
20b
20c
22b
Apixabanb
a SD: standard deviation.

b Used as a positive control.
Subsequently, compounds 15c and 22b were tested the IC50 values against human FXa in
vitro, as listed in Table 4. The IC50 curves along with IC50 values of compounds 15c and 22b were
summarized in Fig. 4 below. Compound 15c displayed the highest potency and was superior to
that of the positive control.
Table 4
In vitro FXa enzymatic assays.
IC50 on FXa (µM)a SDb
Compd.
0.14 0.05
0.36 0.08
0.21 0.05
15c
22b
Apixabanc
a Inhibitory activity against human FXa.
b SD: standard deviation.
c Positive control.
Fig.4. IC50 determination of apixaban, 15c, and 22b against human FXa.
3.3. Antithrombotic effect in vivo
In order to evaluate the antithrombotic effect of compound 15c and 22b in vivo, we
conducted the rat venous thrombosis test. The antithrombotic effect and inhibition rate of
compounds 15c and 22b were listed in Table 5. Compound 15c showed higher inhibition rate than
apixaban.
Table 5
In vivo antithrombotic effect and inhibition rate.
Thrombus weight (mg)
Compd.a
Inhibition rate (%)
SDb
0.1 0.2
0.5 0.3
0.2 0.2
4.4 3.9
98
89
95
15c
22b
Apixabanc
model
a Orally administered to rats at a dose of 10 mg/kg.
b SD: standard deviation.
c Positive control.

3.4. Binding model analysis
To further elucidate the binding mode of compounds, a docking analysis was performed. In
our study, the co-crystal structure of apixaban with human FXa was selected as the docking model
(PDB ID: 2P16). The docking simulation was conducted using an Accelrys DS Visualizer 3.0
system. The properties and shape of the receptor were represented on a grid by using several
different sets of fields that provided progressively more accurate scoring of the ligand poses. The
image files were generated using an Accelrys DS Visualizer 4.0 system. The binding model was
exemplified by the interaction of compound 15c with human FXa. As shown in Fig. 5,
1,3,4-triazole linked to the C-3 pyrazole formed two H-bonds with Ser195 and Asp194, and
morpholinylmethyl was suitable to P2 pocket. Additionally, the tetrahydropyrazolopyridone
scaffold (P3 moiety) interacted with Gln192. The pendant P4 phenyllactam showed interaction
with Try99 and Trp215.
Fig. 5. The human FXa active site in the complex of compound 15c.
4. Conclusions
In summary, we designed and prepared a series of novel apixaban derivatives and evaluated
their anticoagulant potency. Our preliminary investigation showed that several compounds (15b,
15c, 20b, 20c, and 22b) showed excellent anticoagulant potency in human and rabbit plasma in
vitro. The SARs analysis indicated that the electric and steric effects in P2 could influence
anticoagulant activity. Meanwhile, compounds 15b, 15c, 20b, 20c, and 22b were further tested for
their activity against human FXa, and compounds 15c and 22b were examined for venous
thrombosis in rat. The most promising compound 15c showed excellent activity with an IC50 value
of 0.14 µM against human FXa in vitro and an inhibition rate of 98% in vivo. Further SARs and
mechanistic studies of these compounds are in progress, and the results will be reported in due
course.
5. Experimental
5.1. Chemistry

Unless otherwise specified, all materials were obtained from commercial suppliers and were
used without further purification. Reactions’ time and purity of the products were monitored by
TLC on FLUKA silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm.
Column chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals
(Qingdao, Shandong, China). All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS)
1
were taken in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). H NMR and
13C NMR spectra were recorded on Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience,
Billerica, MA, USA) with TMS as an internal standard.
5.2. Preparation of ethyl 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-t
etrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate (11)
The preparation of the key intermediate 11 has been illustrated in detail in our previous
study.15 So the synthesis method would not be listed here.
5.3. Synthesis of target compounds 15a-15f and 24
5.3.1. Preparation of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetra
hydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (12)
A mixture of CH3ONa (4.1 g, 0.08 mol), formamide (15.8 g, 0.35 mol) and intermediate 11
(24.4 g, 0.05 mol) in DMF (100 mL) was stirred at 50 °C for 4 h. Upon cooling to room
temperature (rt.), the reaction mixture was poured into ice-water (50 mL). The resulting precipitate
was filtered, washed with H2O (20 mL) and dried under reduced pressure to afford product 12 as a
white solid (17.5 g, 76.2%). Mp: 177.4-178.3 °C; MS (ESI) m/z (%): 482.2 [M+Na]+.
5.3.2. Preparation of 3-amino-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-te
trahydro-7H-pyrazolo[3,4-c]pyridin-7-one (13)
Iodobenzene diacetate (4.2 g, 15.0 mmol) and KOH (0.9 g, 16.0 mmol) were added to a
solution of intermediate 12 (3.0 g, 6.0 mmol) in MeOH (60 mL). The mixture was stirred at rt. for
5 h. After evaporating in vacuo, the residue was dissolved in a solution of H2O (30 mL), dioxane
(60 mL) and NaOH (0.8 g, 0.02 mol). The mixture was heated to 100 °C for 6 h. Upon cooling to
rt., the reaction mixture was poured into ice-water (20 mL). The aqueous layer was extracted with
CH2Cl2 (3 × 10 mL). The organic phase was washed with brine (2 × 20 mL). Then the organic
layer was dried with Na2SO4, filtered and evaporated in vacuo to afford 13 as a brown solid (2.3 g,
62.4%). Mp: 178.4-179.9 °C; MS (ESI) m/z (%): 432.3 [M+H]+.
5.3.3. Preparation of (E)-N'-(1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,
6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridin-3-yl)-N,N-dimethylformimidamide (14)
DMF-DMA (20 mL, 0.15 mol) was added to a stirring solution of 13 (2.0 g, 4.6 mmol) in
CH2Cl2 (30 mL) and heated to reflux for 3 h. After being cooled to rt., the mixture was
concentrated under reduced pressure to afford 14 as a light yellow solid (2.1 g, 90.0%). Mp:
178-182 °C; MS (ESI) m/z (%): 487.5 [M+H]+.
5.3.4. General procedure for preparation of triazole compounds (15a–15f)
A mixture of different substituted hydrazides (0.36 mmol), HOAc (1 mL) and intermediate
14 (0.15 g, 0.30 mmol) was stirred at 80 °C for 2 h. After being cooled to rt., the reaction mixture
was poured into ice-water (10 mL). The precipitate was filtered and purified by silica gel column
chromatography (eluent, CH2Cl2/MeOH = 30:1 to 20:1) to afford target products 15a–15f.

5.3.4.1. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(4H-1,2,4-triazol-4-yl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15a)
1
White solid; Yield: 70.1%; Mp: 188-195 °C; MS (ESI) m/z (%): 506.1 [M+Na]+. H NMR
(400 MHz, DMSO-d6) δ 9.11 (s, 2H), 7.53 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.30 (d, J
= 8.8 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 4.12 (t, J = 6.6 Hz, 2H), 3.81 (s, 3H), 3.60 (t, J = 5.5 Hz,
2H), 3.15 (t, J = 6.6 Hz, 2H), 2.39 (t, J = 6.0 Hz, 2H), 1.85-1.75 (m, 4H).
5.3.4.2. 1-(4-methoxyphenyl)-3-(3-methyl-4H-1,2,4-triazol-4-yl)-6-(4-(2-oxopiperidin-1-yl)phenyl)
-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15b)
1
White solid; Yield: 67.0%; Mp: 157-162 °C; MS (ESI) m/z (%): 498.1 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 8.88 (s, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.30 (d, J
= 8.6 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 3.80 (s, 3H), 3.60 (t, J = 5.5 Hz,
2H), 3.02 (t, J = 6.4 Hz, 2H), 2.47 (m, 3H), 2.39 (t, J = 6.3 Hz, 2H), 1.85-1.75 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) δ 169.33, 159.65, 156.76, 150.70, 143.82, 142.03, 140.05, 139.20, 133.33,
132.57, 127.08, 126.88, 126.54, 118.23, 114.01, 55.96, 51.22, 33.05, 23.46, 21.36, 19.86, 11.02.
5.3.4.3. 1-(4-methoxyphenyl)-3-(3-(morpholinomethyl)-4H-1,2,4-triazol-4-yl)-6-(4-(2-oxopiperidi
n-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15c)
1
White solid; Yield: 65.2%; Mp: 194-198 °C; MS (ESI) m/z (%): 583.3 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 8.91 (s, 1H), 7.50 (dd, J = 6.7, 4.7 Hz, 2H), 7.36 (dd, J = 8.6, 4.6 Hz, 2H),
7.28 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.9 Hz, 2H), 4.08 (t, J = 6.7 Hz, 2H), 3.78 (s, 3H), 3.58 (t, J
= 5.5 Hz, 4H), 3.50-3.27 (m, 6H), 3.17-2.89 (m, 2H), 2.35 (dt, J = 8.5, 5.3 Hz, 4H), 1.83 (dd, J =
14.6, 10.3 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 169.35, 159.57, 156.72, 151.14, 144.85,
142.01, 141.92, 139.99, 139.24, 133.10, 132.61, 127.15, 127.09, 127.02, 126.86, 126.54, 119.24,
114.05, 114.01, 66.60, 55.96, 53.16, 51.61, 51.29, 33.06, 23.46, 21.36, 19.82.
5.3.4.4. 3-(3-amino-4H-1,2,4-triazol-4-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)
-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15d)
1
Yellow solid; Yield: 72.0%; Mp: 188-195 °C; MS (ESI) m/z (%): 499.1 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 8.88 (s, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.30 (d, J
= 8.8 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 3.81 (s, 3H), 3.60 (t, J = 5.5 Hz,
2H), 3.03 (t, J = 6.5 Hz, 2H), 2.39 (t, J = 6.3 Hz, 2H), 1.83-1.75 (m, 4H).
5.3.4.5. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(3-(pyrrolidin-1-ylmethyl)-4H-
1,2,4-triazol-4-yl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15e)
1
Yellow solid; Yield: 71.3%; Mp: 182-190 °C; MS (ESI) m/z (%): 567.2 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 9.00 (s, 1H), 7.54 (d, J = 8.9 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.30 (d, J
= 8.8 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 4.10 (t, J = 4.9 Hz, 2H), 3.81 (s, 3H), 3.60 (t, J = 5.5 Hz,
2H), 3.01 (t, J = 4.9 Hz, 2H), 2.39 (t, J = 6.3 Hz, 2H), 2.09 (t, J = 6.9 Hz, 4H), 1.91(s, 2H),
1.85-1.75 (m, 4H), 1.74 (t, J = 6.9 Hz, 4H).
5.3.4.6. 3-(3-((diethylamino)methyl)-4H-1,2,4-triazol-4-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiper
idin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (15f)
1
Yellow solid; Yield: 71.0%; Mp: 184-190 °C; MS (ESI) m/z (%): 569.2 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 8.90 (s, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.29 (d, J
= 8.8 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 4.09 (t, J = 6.5 Hz, 2H),3.80 (s, 3H), 3.60 (t, J = 5.7 Hz,
2H), 2.92 (t, J = 6.5 Hz, 2H), 2.39 (t, J = 6.3 Hz, 2H), 2.38 (q, J = 7.1Hz,4H), 2.09 (s, 2H),
1.85-1.75 (m, 4H), 0.79 (t, J=7.1 Hz, 6H).
5.3.5. Preparation of 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-tetrazol-1-yl)

-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (24)
Compound 13 (0.20 g, 0.46 mmol) was added to a solution of triethyl orthoformate (0.12 mL,
0.7 mmol) in HOAc (20 mL). The mixture was stirred at 45 °C for 2 h. Then sodium azide (0.05 g,
0.7 mmol) was added to the reaction mixture and was slowly heated to 100 °C. Progress of the
reaction was monitored by TLC and after 5 h the solvent was evaporated in vacuo. The mixture
was diluted with CH2Cl2 (20 mL) and washed with H2O (2 × 10 mL). The organic phase was dried
with Na2SO4 and evaporated in vacuo to afford 24 as a yellow solid (0.14 g, 65.0%). Mp: 187-192
°C; MS (ESI) m/z (%): 483.5 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.45 (d, J =
8.8 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 4.07 (t, J
= 6.3 Hz, 2H), 3.79 (s, 3H), 3.59 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.3 Hz, 2H), 2.38 (t, J = 6.1 Hz,
2H), 1.85-1.75 (m, 4H).
5.4. Synthesis of target compounds 20a-20f and 26a-26b
5.4.1. Preparation of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetra
hydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylic acid (16)
Intermediate 11 (30.0 g, 0.06 mol) and NaOH (9.4 g, 0.18 mol) were added to a solution of
MeOH and H2O (300 mL, MeOH:H2O = 5:1). The mixture was stirred at 60 °C for 1 h. Upon
cooling to rt., the solvent was removed under vacuum, and then H2O (100 mL) was added to the
mixture. The solution was adjusted to pH 2-3 with 3N HCl, and then the resulting precipitate was
filtered and dried under reduced pressure to afford a white solid (25.4 g, 90.0%). Mp: 129-132 °C;
MS (ESI) m/z (%): 459.1 [M-H]-.
5.4.2. Preparation of 3-(hydroxymethyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-
1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (17)
Triethylamine (2.3 mL, 0.02 mol) was added to a solution of 16 (5.0 g, 0.01 mol) in THF (50
mL) at rt.. After 30 min, ethyl chloroformate (2 mL, 0.02 mol) was added drop-wise to the
solution and stirred for 4 h at 30 °C. After being cooled to rt., filtrate was obtained and added
drop-wise to a solution of sodium borohydride (0.6 g, 0.02 mol) in H2O (5 mL) at 0 °C for 30 min.
The solvent was removed in vacuo and the mixture was diluted with CH2Cl2 (30 mL) and washed
with brine (2 × 10 mL). The organic phase was dried with Na2SO4 and evaporated in vacuo to
yield a light yellow solid (3.6 g, 75.0%). Mp: 115-120 °C; MS (ESI) m/z (%): 447.2 [M+H]+.
5.4.3. Preparation of 3-(chloromethyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,
4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18)
A solution of intermediate 17 (1.0 g, 2.3 mmol) in CH2Cl2 (10 mL) was stirred to dissolve,
SOCl2 (0.32 mL, 4.6 mmol) was added at rt. for 1 h. The precipitate was filtered and dried under
reduced pressure to afford a light yellow solid (1.2 g, 95.0%). Mp: 104-108 °C; MS (ESI) m/z (%):
465.2 [M+H]+.
5.4.4. General procedure for preparation of triazole intermediates (19a–19f)
A mixture of different amides A1-A6 (8.1 mmol) and DMF-DMA (10 mL, 0.08 mol) was
stirred at 120 °C for 2 h.The mixture was concentrated under reduced pressure to afford white
solid, which was dissolved in HOAc (10 mL), and then 80% hydrazine hydrate (0.61 g, 9.8 mmol)
was drop-wise to the solution. The mixture was stirred at 90 °C for 2 h. The solvent was removed
in vacuo and the mixture was added in ether at 0 °C for 30 min. The precipitate was filtered and
dried under reduced pressure to afford 19a-19f as white solid.
5.4.4.1. 3-phenyl-1H-1,2,4-triazole(19a)
White solid; Yield: 80.0%; Mp: 176-178 °C; MS (ESI) m/z (%): 146.1 [M+H]+.

5.4.4.2. 3-(4-methoxyphenyl)-1H-1,2,4-triazole (19b)
White solid; Yield: 76.5%; Mp: 185-187 °C; MS (ESI) m/z (%): 176.2 [M+H]+.
5.4.4.3. 3-(4-fluorophenyl)-1H-1,2,4-triazole(19c)
White solid; Yield: 78.8%; Mp: 182-185 °C; MS (ESI) m/z (%): 164.1 [M+H]+.
5.4.4.4. 3-(4-chlorophenyl)-1H-1,2,4-triazole(19d)
White solid; Yield: 76.5%; Mp: 179-185 °C; MS (ESI) m/z (%): 180.1 [M+H]+.
5.4.4.5. 4-(1H-1,2,4-triazol-3-yl)pyridine(19e)
White solid; Yield: 78.6%; Mp: 284-285 °C; MS (ESI) m/z (%): 147.1 [M+H]+.
5.4.4.6. 3-(1H-1,2,4-triazol-3-yl)pyridine(19f)
White solid; Yield: 72.1%; Mp: 172-175 °C; MS (ESI) m/z (%): 147.1 [M+H]+.
5.4.5. General procedure for preparation of target compounds (20a–20f)
A mixture of DMF (10 mL), NaH (0.37 g, 1.7mmol), lithium bromide (0.14 g, 1.6 mmol),
and 19a-19f (1.6 mmol) was stirred at rt. for 30 min. Compound 18 was added to the solution and
then was stirred at 90 °C for 6 h. After being cooled to rt., H2O (10 mL) was added to the mixture.
The precipitate was filtered and purified by silica gel column chromatography (eluent,
CH2Cl2/MeOH = 50:1 to 30:1) to obtain products 20a-20f.
5.4.5.1. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-((3-phenyl-1H-1,2,4-triazol-1-y
l)methyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20a)
1
White solid; Yield: 65.0%; Mp: 157-160 °C; MS (ESI) m/z (%): 574.3 [M+H]+. H NMR
(400 MHz, CDCl3) δ 8.20 (s, 1H), 8.05 (d, J = 6.9 Hz, 2H), 7.41-7.35 (m, 5H), 7.24 (d, J = 8.5 Hz,
2H), 7.16 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 6.9 Hz, 2H), 5.43 (s, 2H), 4.01 (t, J = 6.3 Hz, 2H), 3.74
(s, 3H), 3.51 (t, J = 6.4 Hz, 2H), 2.98 (t, J = 6.4 Hz, 2H), 2.48 (t, J = 5.4 Hz, 2H), 1.88-1.85 (m,
4H).
5.4.5.2. 1-(4-methoxyphenyl)-3-((3-(4-methoxyphenyl)-1H-1,2,4-triazol-1-yl)methyl)-6-(4-(2-oxop
iperidin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20b)
1
White solid; Yield: 63.0%; Mp: 161-163 °C; MS (ESI) m/z (%): 604.3 [M+H]+. H NMR
(400 MHz, CDCl3) δ 8.19 (s, 1H), 8.04 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.31 (d, J =
7.8 Hz, 2H), 7.23 (d, J = 7.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 5.47 (s,
2H), 4.10 (m, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.58 (t, J = 5.5 Hz, 2H), 3.03 (t, J = 5.5 Hz, 2H),
2.54 (t, J = 5.2 Hz, 2H), 1.94-1.92 (m, 4H).
5.4.5.3. 3-((3-(4-fluorophenyl)-1H-1,2,4-triazol-1-yl)methyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopip
eridin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20c)
1
Light yellow solid; Yield: 55.2%; Mp: 155-159 °C; MS (ESI) m/z (%): 614.2 [M+Na]+. H
NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 8.16 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.32 (d,
J = 6.3 Hz, 2H), 7.26-7.22 (d, J = 6.3 Hz, 2H), 7.16 (t, J = 7.8 Hz, 2H), 6.92 (d, J =8.0 Hz, 2H),
5.53 (s, 2H), 4.11 (m, 2H), 3.81 (s, 3H), 3.58 (t, J = 6.7 Hz, 2H), 3.06 (t, J = 6.7Hz, 2H), 2.55 (t, J
= 5.2 Hz, 2H), 1.94-1.92 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 169.30, 159.80, 159.25,
157.21, 150.60, 147.30, 146.42, 143.32, 141.81, 140.35, 133.61, 133.12, 132.80, 127.11, 126.90,
126.76, 126.55, 124.44, 123.63, 113.83, 55.88, 51.29, 45.80, 33.05, 23.46, 21.36, 20.27.
5.4.5.4. 3-((3-(4-chlorophenyl)-1H-1,2,4-triazol-1-yl)methyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopip
eridin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20d)
1
Light yellow solid; Yield: 59.0%; Mp: 157-160 °C; MS (ESI) m/z (%): 608.2 [M+H]+. H
NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 9.1 Hz, 2H), 7.42 (d,
J = 9.1 Hz, 2H), 7.32 (d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.6 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 5.49

(s, 2H), 4.10 (t, J = 4.0Hz, 2H), 3.81 (s, 3H), 3.60 (t, J = 6.5 Hz, 2H), 3.04 (t, J = 6.5 Hz, 2H),
2.56 (t, J = 5.2 Hz, 2H), 1.94-1.92 (m, 4H).
5.4.5.5. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-((3-(pyridin-4-yl)-1H-1,2,4-tria
zol-1-yl)methyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20e)
1
Light yellow solid; Yield: 77.0%; Mp: 143-147 °C; MS (ESI) m/z (%): 575.2 [M+H]+. H
NMR (400 MHz, CDCl3) δ 8.75 (d, J = 6.4 Hz, 2H), 8.36 (s, 1H), 8.28 (d, J = 6.4 Hz, 2H),7.44 (d,
J = 8.8 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 5.55
(s, 2H), 4.12 (m, 2H), 3.81 (s, 3H), 3.59 (t, J = 6.4 Hz, 2H), 3.06 (t, J = 6.4 Hz, 2H), 2.55 (s, J=
5.2 Hz, 2H), 1.94-1.92 (m, 4H).
5.4.5.6. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-((3-(pyridin-3-yl)-1H-1,2,4-tria
zol-1-yl)methyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (20f)
1
Light yellow solid; Yield: 73.5%; Mp: 139-142 °C; MS (ESI) m/z (%): 575.2 [M+H]+. H
NMR (400 MHz, CDCl3) δ 9.47 (s, 1H), 8.98 (d, J = 6.6 Hz, 1H), 8.74 (d, J = 6.2 Hz, 1H), 8.37 (s,
1H), 7.91 (dd, J = 6.6 Hz, 6.2 Hz, 1H),7.44 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.24 (d, J
= 8.1 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 5.53 (s, 2H), 4.14 (m, 2H), 3.81 (s, 3H), 3.59 (t, J = 6.4
Hz, 2H), 3.08 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 5.1 Hz, 2H), 1.94-1.92 (m, 4H).
5.4.6. General procedure for preparation of triazole ketone intermediates (25a–25b)
A mixture of 4-substituted benzaldehydes B1-B2 (9.4 mmol) dissolved in MeOH (10 mL) was
added to a well-stirred solution of semicarbazide (0.75 g, 0.01 mol) and sodium acetate (0.82 g,
0.01 mol) in H2O (10 mL) at rt. for 2 h. The precipitate was filtered and dried under reduced
pressure to yield compounds C1-C2 as white solid. A solution of bromine (0.38 mL, 0.01 mol)
diluted with acetic acid (2 mL) was added to a mixture of C1-C2 (8.0 mmol) and sodium acetate
(0.82 g, 0.01 mol) in acetic acid (10 mL) at rt. for 2 h. The reaction mixture was poured into
ice-water (10 mL). The resulting precipitate was filtered and dried under reduced pressure to
afford D1-D2 as light yellow solid. A solution of D1-D2 (6.0 mmol), KOH (1.0 g, 0.02 mol) in
EtOH was heated at reflux for 5 h. Upon cooling to rt., the solvent was removed under vacuum,
and H2O (5 mL) was added to the mixture. The solution was adjusted to pH 4-5 with HOAc, and
then the resulting precipitate was filtered and dried under reduced pressure to obtain white solid
E1-E2. Compounds E1-E2 (5.0 mmol) were added to 2 N HCl and then refluxed for 3 h. After
being cooled to rt., the precipitate was filtered and dried under reduced pressure to afford 25a-25b.
5.4.6.1. 5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one (25a)
White solid; Yield: 45.8%; Mp: 314-316 °C; MS (ESI) m/z (%): 162.1 [M+H]+.
5.4.6.2. 5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (25b)
White solid; Yield: 43.4%; Mp: 333-335 °C; MS (ESI) m/z (%): 192.1 [M+H]+.
5.4.7. General procedure for preparation of target compounds (26a–26b)
The preparation of target compounds 26a-26b was similar to 20a-20f, so the synthesis
methods would not be listed here.
5.4.7.1. 1-(4-methoxyphenyl)-3-((5-oxo-3-phenyl-4,5-dihydro-1H-1,2,4-triazol-1-yl)methyl)-6-(4-
(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (26a)
1
White solid; Yield: 58.2%; Mp: 159-162 °C; MS (ESI) m/z (%): 590.3 [M+H]+. H NMR
(400 MHz, CDCl3) δ 7.78 (d, J = 7.4 Hz, 2H), 7.50 (m, 3H), 7.44 (s, 1H), 7.35 (d, J = 8.8 Hz, 2H),
7.31 (d, J = 5.1 Hz, 2H), 7.24 (d, J = 5.2 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 5.06 (s, 2H), 4.05 (t, J
= 6.4 Hz, 2H), 3.80 (s, 3H), 3.60 (t, J = 5.6 Hz, 2H), 3.04-2.97 (t, J = 5.6 Hz, 2H),2.57 (t, J = 5.2
Hz, 2H), 1.94-1.92 (m, 4H).

5.4.7.2. 1-(4-methoxyphenyl)-3-((3-(4-methoxyphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)m
ethyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (26
b)
1
White solid; Yield: 50.5%; Mp: 163-166 °C; MS (ESI) m/z (%): 620.1 [M+H]+. H NMR
(400 MHz, CDCl3) δ 7.74 (d, J = 8.2 Hz, 2H), 7.54-7.47 (m, 1H), 7.37 (d, J = 8.4Hz, 2H), 7.32 (d,
J = 7.6 Hz, 2H), 7.23 (d, J = 7.6 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.2Hz, 2H), 5.04 (s,
2H), 4.08 (m, 2H), 3.86 (s, 3H), 3.81 (s, 3H), 3.61 (t, J = 4.9 Hz, 1H), 3.03 (t, J = 4.9 Hz, 2H),
2.59 (t, J = 4.3 Hz, 2H), 1.95-1.93 (m, 4H).
5.5. Synthesis of target compounds of 22a-22c and 23
5.5.1. Preparation of 3-(azidomethyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,
4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (21)
Sodium azide (0.4 g, 6.4 mmol) was added to a solution of 18 (2.0 g, 4.3 mmol) in DMF (10
mL), and then stirred at rt. for 6 h. The reaction mixture was poured into ice-water (20 mL) and
the resulting precipitate was filtered and dried under reduced pressure to abtain 21 as a white solid
(1.4 g, 70.0%). Mp: 135-142 °C; MS (ESI) m/z (%): 488.2 [M+H]+.
5.5.2. Preparation of 3-((1H-1,2,3-triazol-1-yl)methyl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidi
n-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (22a)
A mixture of DMF (10 mL), 21 (1.0 g, 2.1 mmol), propiolic acid (0.17 g, 2.5 mmol), cuprous
iodide (0.24 g, 1.2 mmol) and cesium carbonate (0.68 g, 2.1 mmol) was stirred at 120 °C for 5 h.
After being cooled to rt., H2O (10 mL) was added to the solution. The precipitate was filtered and
dried under reduced pressure to yield compound 22a as a light yellow solid (0.69 g, 65.7%). Mp:
180-185 °C; MS (ESI) m/z (%): 498.0 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 4.4 Hz,
2H), 7.44 (d, J =8.7 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 8.6 Hz,
2H), 5.73 (s, 2H), 4.05 (t, J = 4.3 Hz, 2H), 3.81 (s, 3H), 3.59 (t, J = 5.1 Hz, 2H), 2.93 (t, J = 5.1
Hz, 2H), 2.59 (t,J = 5.4 Hz, 2H), 1.94-1.92 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 169.32,
159.51, 156.83, 153.11, 141.93, 140.18, 138.88, 136.87, 133.24, 132.74, 127.07, 126.83, 126.53,
117.79, 113.94, 55.94, 51.25, 33.05, 23.46, 21.36, 21.02.
5.5.3. Preparation of 1-((1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-t
etrahydro-1H-pyrazolo[3,4-c]pyridin-3-yl)methyl)-1H-1,2,3-triazole-4-carbonitrile (22b)
A mixture of 21 (1.5 g, 3.2 mmol), cuprous iodide (0.31 g, 1.6 mmol) and
2-chloroacrylonitril (15 mL) was stirred at 80 °C for 4 h. After being cooled to rt., the solvent was
removed under vacuum and ethyl ether (5 mL) was added to the mixture. The resulting precipitate
was filtered and dried under reduced pressure to obtain 22b as agray solid (1.2 g, 68.4%) Mp:
1
188-193 °C; MS (ESI) m/z (%): 523.2 [M+H]+. H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 1H),
7.44 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H),7.27 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H),
5.88 (s, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.79 (s, 3H),3.58 (t, J = 6.3 Hz, 2H), 2.92 (t, J = 6.4 Hz, 2H),
2.38 (t, J = 6.1 Hz, 2H), 1.87-1.83 (m, 4H).
5.5.4. Preparation of 1-((1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-t
etrahydro-1H-pyrazolo[3,4-c]pyridin-3-yl)methyl)-1H-1,2,3-triazole-4-carboxamide (22c)
A solution of 22b (1.0 g, 1.6mmol) in MeOH (10 mL) was stirred to dissolve, 30% hydrogen
peroxide (1 mL) and 6 N aqueous solution of NaOH (1 mL) were added and stirred for 2 h at 50
°C. After cooling to rt., the precipitate was filtered and dried under reduced pressure to provide a
1
light yellow solid (0.86 g, 80.0%). Mp: 190-198 °C;MS (ESI) m/z (%): 563.0 [M+H]+. H NMR

(400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.88 (s, 1H), 7.50 (s, 2H), 7.44 (d, J = 8.9 Hz, 1H), 7.33 (d,
J = 8.7 Hz, 2H), 7.26 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 5.79 (s, 2H), 4.03 (t, J = 6.5 Hz,
2H), 3.79 (s, 3H), 3.58 (t, J = 6.4 Hz, 2H), 2.89 (t, J = 6.5 Hz, 2H), 2.38 (t, J = 6.2 Hz, 2H),
1.87-1.83 (m,4H).
5.5.5. Preparation of3-((1H-benzo[d][1,2,3]triazol-1-yl)methyl)-1-(4-methoxyphenyl)-6-(4-(2-ox
opiperidin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (23)
A mixture of DMF (10 mL), K2CO3 (0.23 g, 1.7 mmol), benzotriazole (0.14 g, 1.2 mmol) and
silica (1.0 g) was stirred at rt. for 30 min. Then 18 (0.5 g, 0.8 mmol) was added to the solution,
which was stirred at 90 °C for 6 h. After being cooled to rt., H2O (10 mL) was added to the
solution. The resulting precipitate was filtered and purified by silica gel column chromatography
(eluent, CH2Cl2/MeOH = 30:1) to afford product 23 as a white solid (0.37 g, 63.0%). Mp: 186-189
1
°C;MS (ESI) m/z (%): 547.9 [M+H]+. H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 8.4 Hz, 1H),
7.73 (d, J = 8.3 Hz, 1H), 7.51 (m, 1H), 7.46-7.40 (m, 3H), 7.26-7.19 (m, 4H), 6.93 (d, J = 9.0 Hz,
2H), 5.99 (s, 2H), 3.97 (t, J = 6.6 Hz, 2H), 3.82 (s, 3H), 3.58 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.6
Hz, 2H), 2.58 (t, J = 5.5 Hz, 2H),1.94-1.92 (m, 4H).
5.6. Synthesis of target compounds 28a-28e
5.6.1. Preparation of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetra
hydro-1H-pyrazolo[3,4-c]pyridine-3-carbonitrile (27)
Triethylamine (18 mL, 0.13 mol) was added to a solution of 12 (15.0 g, 33.0 mmol) in
CH2Cl2 (225 mL) at 30 °C. After 30 min, trifluoroacetic anhydride (18 mL, 0.13 mol) was added
drop-wise to the solution and stirred for 2 h. The solution was adjusted to pH 7 with H2O and the
organic phase was evaporated in vacuo. The residue was crystallized with MeOH to afford the title
compound 27 as a white solid (10.5 g, 72.9%). Mp: 171.3-173.6 °C; MS (ESI) m/z (%): 435.3
[M+H]+.
5.6.2. General procedure for preparation of nitrogenous heterocycliccompounds (28a–28e)
A mixture of compound 27 (0.2 g, 0.37 mmol), different diamines (0.14 mol), and sublimed
sulfur (3.6 mg, 1.0 nmol) was stirred at 116 °C for 2 h. After being cooled to rt., the precipitate
was filtered and dried under reduced pressure to afford target compounds 28a–28e.
5.6.2.1. 3-(4,5-dihydro-1H-imidazol-2-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)
-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (28a)
White solid; Yield: 77.6%; Mp: 150-152 °C; MS (ESI) m/z (%): 484.2 [M+H]+, 485.2
[M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 7.49 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.27
(d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.9 Hz, 2H), 4.04 (t, J = 6.5 Hz, 2H), 3.80 (s, 3H), 3.74 (t, J = 9.8
Hz, 2H), 3.59 (t, J = 5.5 Hz, 2H), 3.36 (t, J = 9.8 Hz, 2H), 3.16 (t, J = 6.5 Hz, 2H), 2.38 (t, J = 6.2
Hz, 2H), 1.85-1.75 (m, 4H).
5.6.2.2. 1-(4-methoxyphenyl)-3-(1-methyl-4,5-dihydro-1H-imidazol-2-yl)-6-(4-(2-oxopiperidin-1-
yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (28b)
1
White solid; Yield: 82.8%; Mp: 157-162 °C; MS (ESI) m/z (%): 499.1 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 7.50 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.7 Hz,
2H), 7.00 (d, J = 8.9 Hz, 2H), 4.04 (t, J = 6.5 Hz, 2H), 3.80 (s, 3H), 3.75 (t, J = 9.8 Hz, 2H), 3.59
(t, J = 5.5 Hz, 2H), 3.37 (t, J = 9.8 Hz, 2H), 3.16 (t, J = 6.5 Hz, 2H), 3.12 (s, 3H), 2.39 (t, J = 6.2
Hz, 2H), 1.85-1.75 (m, 4H).
5.6.2.3. 1-(4-methoxyphenyl)-3-(4-methyl-4,5-dihydro-1H-imidazol-2-yl)-6-(4-(2-oxopiperidin-1-

yl)phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (28c)
1
White solid; Yield: 79.3%; Mp: 165-169 °C; MS (ESI) m/z (%): 499.1 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 7.48 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz,
2H), 6.99 (d, J = 8.9 Hz, 2H), 4.04 (t, J = 6.7 Hz, 2H),3.80 (s, 3H), 3.59 (t, J = 5.4 Hz, 2H), 3.37
(m, 2H), 3.19 (t, J = 6.6 Hz, 2H), 2.89(m, 1H), 2.80 (m, 1H), 2.38 (t, J = 6.2 Hz, 2H), 1.84-1.75
(m, 4H), 1.12 (d, J = 5.9 Hz, 3H).
5.6.2.4. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1,4,5,6-tetrahydropyrimidin-2-
yl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (28d)
1
White solid; Yield: 76.5%; Mp: 153-157 °C; MS (ESI) m/z (%): 499.2 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 7.46 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.7 Hz,
2H), 6.98 (d, J = 8.9 Hz, 2H), 4.00 (t, J = 6.5 Hz, 2H) , 3.80 (s, 3H), 3.74 (t, J = 7.3 Hz, 2H), 3.59
(t, J = 5.4 Hz, 2H), 3.36 (t, J = 7.3 Hz, 2H), 3.18 (t, J = 6.5 Hz, 2H), 2.38 (t, J = 6.1 Hz, 2H),
1.85-1.75 (m, 4H), 1.71 (m, 2H).
5.6.2.5. 1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(4,5,6,7-tetrahydro-1H-1,3-dia
zepin-2-yl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (28e)
1
White solid; Yield: 78.9%; Mp: 147-153 °C; MS (ESI) m/z (%): 412.2 [M+H]+. H NMR
(400 MHz, DMSO-d6) δ 7.47 (d, J = 8.9 Hz, 2H), 7.33 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz,
2H), 6.97 (d, J = 8.9 Hz, 2H), 4.01 (t, J = 6.6 Hz, 2H), 3.80 (s, 3H), 3.76 (t, J = 7.6 Hz, 2H), 3.59
(t, J = 5.3 Hz, 2H), 3.35 (t, J = 7.3 Hz, 2H), 3.18 (t, J = 6.6 Hz, 2H), 2.38 (t, J = 6.0 Hz, 2H),
1.85-1.75 (m, 4H), 1.70 (m, 4H).
5.7. Pharmacology
5.7.1. In vitro coagulation assays
PT and APTT were measured using commercially available kits. Blood was obtained from
healthy human volunteers or rabbits and anticoagulated with 3.8% sodium citrate. Plasma was
obtained after centrifugation at 2000 g for 10 min. An initial stock solution of the inhibitor was
prepared in DMSO, and serial dilutions were performed. Clotting time was determined using the
control plasma and plasma containing five to seven different concentrations of the inhibitor. PT
measurement was performed in a temperature-controlled automated coagulation device
(SysmexCA50, Dade-Behring) using a Thromborel-S (Dade-Behring) kit according to the
supplied instructions. For each plasma concentration, measurements were performed in
duplicate.15,27
5.7.2. In vitro human FXa inhibition assays
The potency of the inhibition of six test compounds against human FXa was evaluated using
a chromogenic substrate in a 96-well microtiter plate. The enzymatic action of human FXa was
measured using the conversion of an FXa-specific chromogenic substrate. FXa cleaved
p-nitroaniline from the chromogenic substrate (Table 6).
Table 6
Reaction setup for human FXa inhibition assay.
Mix step(10 µL)
Compounds
Sample
5 µL
Negative Control
5 µL 10%DMSO/buffer
0 µL
Positive Control
5 µL 10%DMSO/buffer
5 µL
Human FXa
5 µL
Seal plate and incubate at 25 ºC 10 min
Enzyme step(40 µL)

Tris-buffer
35 µL
5 µL
40 µL
5 µL
35 µL
5 µL
Pefachrome FXa
Seal plate and incubate for 20 min at 25 ºC
5.7.3. Data Analysis
The inhibition rate of the compounds was determined as follows:
Inhibition (%) = (ODpositive-ODsample)/(ODpositive-ODnegative) × 100%
ODpositive: absorbance of positive wells (FXa + Substrate);
ODsample: absorbance of compound wells (FXa + Substrate +Compound);
ODnegative: absorbance of negative wells (Buffer + Substrate).
The IC50 curves were generated along with the IC50 values by using GraphPad Prism 5.0
software, and the IC50 value was obtained from the plot of anti-FXa activity against inhibitor
concentration.
5.7.4. In vivo experimental venous thrombosis
The male SD rats (n=6 per dose group) weighing 280-320 g were used in the study. In rats,
the test drug was administered to animals orally at 10 mg/kg. Vein thrombosis was induced using a
modification of the method described by Reyerset al.28 Briefly, the animals were anesthetized with
pentobarbital (40 mg/kg, ip) and the abdomen was opened. The vena cava inferior was carefully
separated from the surrounding tissues and ligated tightly with a cotton thread just below the left
renal vein. The abdomen was then closed with a double layer of sutures. After 2 h, the abdomen
was reopened, and the vena cava was dissected longitudinally, and the formed thrombus was
removed. The obtained thrombi were retained at 37 °C for 24 h, after which their dry weight was
measured.
Acknowledgements
This work was supported by the Program for Innovative Research Teams by the Ministry of
Education of the People’s Republic of China and the Program for Liaoning Innovative Research
Team in University (IRT1073), Natural Science Foundation of Liaoning province (No.
201602697), Grants from Long-term Training of Young Teachers of Shenyang Pharmaceutical
University (No. ZCJJ2014403), and the Student’s Platform for Innovation and Entrepreneurship
Training Program of Shenyang Pharmaceutical University.
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A series of compounds was synthesized and evaluated for anticoagulant activity.
Enzymatic assay and venous thrombosis test in vivo analysis of potent compounds.
Most promising 15c showed an IC50 value of 0.14 µM and 98% inhibition rate in vivo.