Design, synthesis, and structure–activity relationship of novel and effective apixaban derivatives as FXa inhibitors containing 1,2,4-triazole/pyrrole derivatives as P2 binding element

Article abstract of DOI:10.1016/j.bmc.2016.09.024

Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2 binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising compound 14a, with an IC₅₀(FXa) value of 0.15?μM and 99% inhibition rate, was identified for further evaluation as an FXa inhibitor.

Full text of DOI:10.1016/j.bmc.2016.09.024

Accepted Manuscript
Design, synthesis, and structure-activity relationship of novel and effective
apixaban derivatives as FXa inhibitors containing 1, 2, 4-triazole/pyrrole deriv-
atives as P2 binding element
Yong Wang, Xiaoqing Sun, Di Yang, Zhuang Guo, Xuxu Fan, Minhua Nie,
Feng Zhang, Yue Liu, Yue Li, Yulin Wang, Ping Gong, Yajing Liu
PII:
S0968-0896(16)30713-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.024
BMC 13277
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 August 2016
9 September 2016
10 September 2016
Please cite this article as: Wang, Y., Sun, X., Yang, D., Guo, Z., Fan, X., Nie, M., Zhang, F., Liu, Y., Li, Y., Wang,
Y., Gong, P., Liu, Y., Design, synthesis, and structure-activity relationship of novel and effective apixaban
derivatives as FXa inhibitors containing 1, 2, 4-triazole/pyrrole derivatives as P2 binding element, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.024
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Design, synthesis, and structure-activity relationship of novel and effective apixaban
derivatives as FXa inhibitors containing 1, 2, 4-triazole/pyrrole derivatives as P2 binding
element
Yong Wanga, Xiaoqing Suna, Di Yanga, Zhuang Guoa, Xuxu Fana, Minhua Niea, Feng Zhanga, Yue Liua, Yue Lia,
Yulin Wangb, Ping Gonga, Yajing Liua,*
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry
of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
aDepartment of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, PR China
bDepartment of Parasitology, College of Basic Medical Sciences, Dalian Medical University, 9 South Lvshun Roa
d Western Section, Dalian 116044, Liaoning, China.
Abstract
Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2
binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed,
synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds
showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for
their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising
compound 14a, with an IC50 (FXa) value of 0.15 μM and 99% inhibition rate, was identified for further evaluation
as an FXa inhibitor.
Keywords: FXa; Anticoagulant activity; Synthesis; Structure-activity relationships.
1. Introduction
Thromboembolic events are a leading cause of mortality worldwide, and play a pivotal role in the
pathogenesis of numerous cardiovascular disorders, including unstable angina, venous thromboembolism (VTE)
ischemic stroke, acute coronary syndrome (ACS), deep venous thrombosis (DVT), and pulmonary embolism
(PE).1-5 One of the most feasible methods for treating these fatal diseases is to prevent the generation of
thrombus.6 Warfarin and heparin are traditional pharmacotherapies and have been used extensively, however,
numerous limitations exist such as monitoring clotting time, bleeding, and interactions with other drugs and
foods.7
Coagulation enzyme factor Xa (FXa), as the promising target of anticoagulation, is located at the
convergence point of the extrinsic and intrinsic coagulation cascade. Over the past decades, several oral, direct
and selective FXa inhibitors have been approved or are in clinical research, such as rivaroxaban, apixaban,
edoxaban, darexaban, and betrixaban (Fig. 1). All of these possess lower risks of bleeding and more specific
mechanisms of preventing thrombin generation, instead of affecting platelet function and the level of thrombin.8-10
On the basis of data obtained for apixaban, we endeavored to create novel scaffolds for further exploration such as
decreasing dosages and reducing side effects of bleeding. Thus, a series of derivatives were synthesized as novel
FXa inhibitors.
* Corresponding author. Tel./fax: +86 24 23986429 (Y.L.).
E-mail address:
1

Fig. 1. Structures of oral FXa inhibitors.
The X-ray structure of FXa bound to apixaban (Fig. 2) 11 shows that the 4-methoxy group in the S1 pocket is
oriented in a planar manner relative to the phenyl P1 moiety. The pendant P4 phenyllactam in the S4 pocket is
appropriately positioned in a hydrophobic pocket. The hydrogen bonds between the protein and ligand
contributed to its high affinity, involving the pyrazole N-2 atom with Gln192, carbonyl oxygen (scaffold
carboxamide) with Gly216, and the pyrazole C-3 carboxamido with Glu146.
Fig. 2. X-ray structure of FXa bound to apixaban.
According to the structure-activity relationships (SARs), the 1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-
one scaffold (P3 moiety) was retained. To occupy the P2 cavity on a larger scale and enhance the hydrogen
bonding to Glu146, a series of nitrogenous heterocyclic were introduced into pyrazole C-3 to replace the
carboxamido moiety. In the P1 moiety, fluoromethoxy and halogens were introduced into the methoxy group to
accommodate the narrow cavity and investigate the influence of the electric effect of the substituents. Furthermore,
to increase the hydrophilicity of the lactam moiety (P4), imidazoline and tetrahydropyrimidine groups were
incorporated into the phenyl group.
2

Fig. 3. Structure of the series I~IV target compounds
2. Chemistry
The majority of the target compounds listed in Fig. 3 were synthetized at variable yields, as shown in
Schemes 1-5. In the process of synthesis, the most critical steps were the synthesis of key intermediates 11a-11f
and 32a.
2.1. Synthesis of intermediates 11a-11f
The synthesis of intermediates 11a-11f was achieved using a convenient eleven-step procedure starting from
4-nitroaniline, outlined in Scheme 1. The commercially available 4-nitroaniline was reacted with 4-
chlorobutanoyl chloride to provide compound 1, which was treated with potassium carbonate to obtain
intermediate 2. Then, compound 2 was chlorinated with PCl5 and substituted with morpholine to obtain compound
4. Different substituted anilines (5a-5f) were treated with NaNO2/HCl, and the corresponding diazoniums were
condensed with ethyl 2-chloro-3-oxobutanoate in the presence of sodium acetate to provide chlorohydrazones 6a-
6f. Reaction of 6a-6f with compound 4 using excess triethylamine afforded the cycloadducts 7a-7f. Compounds
7a-7f were treated with TFA in dichloromethane to obtain compounds 8a-8f, respectively. Finally, the cardinal
intermediates 11a-11f were prepared from compounds 8a-8f through reduction, amidation, and cyclization
reactions.
3

Scheme 1. Reagents and conditions: (i) 4-chlorobutanoyl chloride, THF, 0 ºC, 30 min, r.t., 6 h; (ii) K2CO3, DMSO, 80 ºC, 6 h; (iii)
POCl5, CH2Cl2, 85 ºC, 5 h; (iv) morpholine, 130 ºC, 1.5 h; (v) NaNO2, HCl, H2O, 0 ºC, 30 min; (vi) ethyl 2-chloro-3-oxobutanoate,
sodium acetate, r.t., 6 h; (vii) triethylamine, ethyl acetate, 80 ºC, 6 h; (viii) TFA, CH2Cl2, r.t., 1 h; (ix) Fe powder, HCl, NH4Cl,
EtOH/H2O, reflux, 2.5 h; (x) 4-chlorobutanoyl chloride, THF, 0 ºC, 30 min, r.t., 6 h; (xi) NaH, DMF, 0 ºC, 1 h.
2.2. Synthesis of apixaban derivatives of series I: substituted 1,2,4-triazole
The target compounds of series I were synthesized according to the procedures outlined in Scheme 2.
Compounds 14a-14f, 15, and 16 were synthesized as follows: firstly, the key intermediates 11a-11f were
aminolysized with formamide to provide 12a-12f. 12 Compounds 13a-13f were obtained from 12a-12f in CH2Cl2.
Next, the target compounds 14a-14f were available via cyclization of 13a-13f and 80% hydrazine monohydrate.
Compounds 14a-14f were methylated with iodomethane to obtain the target compounds 15 and 16. 13-15
For obtaining the derivatives 18a-18e and 21a-21d with substituents at different positions of triazole, two
synthetic routes were used; as depicted in Scheme 2. The compounds 18a-18e were obtained by converting
compound 17 using different hydrazides followed by dehydration of 12a.16-17 The target compounds 21a-21d were
created by a three-step procedure and treatment of compound 11a with hydrazine hydrate afforded intermediate 19,
which was treated with DMF-DMA to afford compound 20. The desired compounds 21a-21d were obtained from
20 and different amines via intramolecular cyclization. 18
4

Scheme 2. Reagents and conditions: (i) CH3ONa, HCONH2, DMF, 50 C, 5 h; (ii) DMF-DMA, CH2Cl2, reflux, 3 h; (iii)
NH2NH2·H2O, acetic acid, reflux, 3 h; (iv) CH3I, NaH, DMF, 40 C, 5 h; (v) triethylamine, trifluoroacetic anhydride, CH2Cl2, reflux,
3 h; (vi) K2CO3, hydrazides, n-butyl alcohol, reflux, 20 h; (vii) NH2NH2·H2O, reflux, 4 h; (viii) DMF-DMA, CH2Cl2, reflux, 4 h; (ix)
R2NH2, acetic acid, reflux, 10 h.
2.3. Synthesis of apixaban of series II:N-4-substituted-1,2,4-triazolin-3-one derivatives
The desired compounds 23a-23c were synthesized as outlined in Scheme 3. Treatment of compound 19 with
substituted phenyl isocyanates in anhydrous tetrahydrofuran afforded intermediates 22a–22c, which were treated
with anhydrous potassium carbonate in dimethyl sulfoxide (DMSO) to afford target compounds 23a–23c via
intramolecular cyclization .19
Scheme 3. Reagents and conditions: (i) substituted phenyl isocyanates, tetrahydrofuran, 50 C, 3 h; (ii) potassium carbonate,
dimethyl sulfoxide, 80 C, 6 h.
2.4. Synthesis of apixaban derivatives of series III: pyrrole derivatives
The preparation of compounds 26a-26c is illustrated in Scheme 4. Firstly, amino derivative 24 was obtained
by the Hofmann rearrangement of 12a under basic conditions in the presence of iodobenzene diacetate.20 Then,
treatment of 24 with 2,5-dimethoxytetrahydrofuran and p-toluenesulfonic acid in tetrahydrofuran afforded
compound 25.21 Finally, the target compounds 26a-26c were obtained via the Mannich reaction of 25,
formaldehyde, and appropriate secondary amines.
5

Scheme 4. Reagents and conditions: (i) KOH, MeOH, r.t., 10 min, PhI(OAc)2, dioxane, reflux, 6-10 h; (ii) 2,5-
dimethoxytetrahydrofuran, p-toluenesulfonic, tetrahydrofuran, 60 C, 1 h; (iii) HCHO, HR5, HOAc, r.t. 30 min.
2.5. Synthesis of apixaban derivatives of series IV: dihydroimidazole/tetrahydropyrimidine groups in P4
The synthesis of the intermediates 27-30, 31a, and 32a is shown in Scheme 1. As described in Scheme 5, the
target compounds 35a-35b were prepared from 32a through hydrolysis, ammoniation, and cyclization with
corresponding diamines. 22
Scheme 5. Reagents and conditions: (i) 4-chlorobutanoyl chloride, THF, 0 C, 30 min, r.t., 6 h; (ii) K2CO3, DMSO, 80 C, 6 h; (iii)
POCl5, CH2Cl2, 85 C, 5 h; (iv) morpholine, 130 C, 1.5 h; (v) NaNO2, HCl, H2O, 0 C, 30 min, 2-chloro-3-oxobutanoate, sodium
acetate, r.t., 6 h; (vi) triethylamine, ethyl acetate, 80 C, 6 h; (vii) TFA, CH2Cl2, r.t., 1 h; (viii) Sodium hydroxide, methanol/water
(5:1 v/v), reflux, 3h; (ix) triethylamine, ethyl chlorocarbonate, ammonia solution rt, 3 h; (x) ethylenediamine or trimethylenediamine,
sulfur, 130 C, 0.5 h.
3. Results and discussion
3.1. In vitro anticoagulant activity
All target compounds were evaluated for their anticoagulant activity by using human and rabbit plasma in
vitro. The results were expressed as prothrombin time (PT) and activated partial thromboplastin time (APTT). PT
measures the effect of a compound on the extrinsic pathway of coagulation, whereas APTT represents the effect
on the intrinsic pathway. The results were expressed as EC2x values and are summarized in Table 1. The EC2x
values in Table 1 are the average of at least three independent experiments.
Table 1 Anticoagulant activity of series I-IV.
EC2x( μM )
Compd.
P2
P1
P4
PTa(human/rabbit)
APTTa(human/rabbit)
14a
0.5/0.7
0.8/1.2
6

5.5/6.4
3.6/4.0
1.8/2.8
4.3/4.2
6.4/7.9
10.5/12.0
6.6/7.8
14b
14c
14d
14e
14f
4.5/5.1
8.4/9.9
11.3/10.2
3.3/4.1
0.8/0.9
1.6/1.9
2.4/3.0
10.0/15.1
1.9/3.0
3.7/3.2
4.1/6.0
15
16
18a
18b
18c
18d
1.0/1.4
2.3/4.1
3.5/4.9
5.1/6.0
3.9/5.0
6.3/7.9
18e
3.9/4.4
4.5/5.7
7.0/8.0
21a
21b
9.0/10.1
5.0/6.1
10.0/11.1
21c
5.5/7.5
10.5/15.0
20.3/22.1
21d
23a
12.9/13.3
7

14.8/9.2
21.3/19.2
23b
16.8/17.9
1.5/1.9
30.1/32.1
3.5/3.2
23c
26a
26b
1.6/2.1
3.7/3.9
26c
35a
1.1/1.7
0.9/0.7
2.4/3.9
0.9/1.0
35b
0.9/1.1
0.8/0.6
1.1/2.0
0.9/1.4
Apixabanb
a Concentration of the compound required to double the clotting time in the PT assay using human/rabbit plasma.
b Used as a positive control.
As shown in Table 1, most of the target compounds exhibited moderate to excellent activity, with the EC2x
value from 30 to 0.5 μM, wherein the activity of compounds 14a (PT = 0.5, APTT = 0.8), 16 (PT= 0.8, APTT =
1.9), 18c (PT = 1.0, APTT = 2.3), 26c (PT = 1.1, APTT = 2.4), 35a (PT = 0.9, APTT = 0.9), and 35b (PT = 0.9,
APTT = 1.1) was comparable to that of apixaban (PT = 0.8, APTT = 0.9). Particularly, compound 14a was the
most potent candidate for further research in these series. Furthermore, the anticoagulant activity in rabbit and
human plasma was correlative.
During the investigation of the C-3 pyrazole position, as illustrated in Table 1, 14a (PT = 0.5, APTT = 0.8)
with unsubstituted 1,2,4-triazole showed superior anticoagulant activity. Unfortunately, different electron-
withdrawing groups (EWGs) in the methoxyl position decreased the activity obviously. The order of anticoagulant
activity was -OCH3 (14a) > -F (14d) > -OCHF2 (14c) > -Cl (14e) > -OCF3 (14b) > -Br (14f), suggesting the
importance of methoxyl as an electron-donating group (EDG) in the P1 ligand.
Upon comparing the isomers 15 and 16, significant differences were observed between 2-N-methyl and 1-N-
methyl in anticoagulant activity. By switching from the N-methyl to C-methyl position, 18a was produced by
introducing 5-C-methyl into 1,2,4-triazole. The activity of 18a was between that of compounds 15 and 16 (16 >
18a > 15). Subsequently, a series of derivatives 18b-18e were synthesized with different substituents at 5-C. They
showed a decrease in anticoagulant potency except for compound 18c with t-butyl (PT = 1.0, APTT = 2.3),
indicating that the 5-C-substituted EDG and steric effects were factors that could not be neglected.
Compounds 21a-21d were also synthetized to increase the hydrophilicity or solubility by adding an alkaline
8

hydrophilic group. All of them displayed decreased potency. For increasing hydrogen bond action, 5-oxo-
substituted 1,2,4-triazole with 4-N-phenyl was introduced into the C-3 pyrazole to obtain compounds 23a, 23b,
and 23c. The pharmacological data indicated that the activity was lost completely (PT > 12, APTT > 20). A
plausible explanation for this is that the steric hindrance produced by the introduction of large groups influenced
the activity.
Further studies were performed to examine the effect of different nitrogenous heterocyclics on the C-3
pyrazole. Compounds 26a, 26b, and 26c were synthetized with alkaline-substituted pyrrole moieties. The
biological data showed that compounds 26c (PT = 1.1, APTT = 2.4), 26a (PT = 1.5, APTT = 3.5), and 26b (PT =
1.6, APTT = 3.7) possessed high anticoagulant activity.
As illustrated in Table 1, the introduction of dihydroimidazol and tetrahydropyrimidine groups into the
hydrophobic pocket (P4 region) could improve the anticoagulant activity, such as compounds 35a (PT = 0.9,
APTT = 0.9) and 35b (PT = 0.9, APTT = 1.1). More studies on the P4 moiety are under way.
The SARs based on the EC2x values in Table 1 showed that the hydrogen bond action and size of the
nitrogenous heterocyclic in P2 and the lipophilic region in P4 were responsible for the anticoagulant activity.
3.2. In vitro FXa enzymatic assays and in vivo antithrombotic effect
Based on the anticoagulant potency, as shown in Table 1, the six tested compounds 14a, 16, 18c, 26c, 35a,
and 35b showed excellent anticoagulant activity. Therefore, these compounds, at their IC50 values, were tested
against human FXa in vitro and rat venous thrombosis in vivo, as listed in Table 2. The fitting curve, indicating
the percent of inhibition, and the curve detailing the IC50 values of 14a and 35b are summarized in Fig. 4 and Fig.
5, respectively. Wherein compound 14a displays the most potent activity against human FXa with an IC50 value of
0.15 μM and 99% inhibition rate in the rat venous thrombosis test, which is superior to that of apixaban.
Table 2 In vivo FXa inhibition and antithrombotic effect.
Compd.
IC50 on FXa (μM)a
Thrombus weight (mg)
0.07±0.3
1.6±0.3
Inhibition rate (%)
99
0.15
2.40
0.85
2.40
1.99
0.35
0.23
14a
65
70
57
66
82
96
16
1.4±1.2
18c
1.9±1.0
26c
1.5±0.2
35a
0.8±0.3
35b
Apixabanb
0.2±0.2
model
4.4±3.9
a Inhibitory activity against human FXa. IC50values shown were the mean of duplicate measurements.
b Used as positive control.
9

Fig. 4. Preliminary screening of seven compounds against human FXa
Fig. 5. Determination of the IC50 of apixaban, 14a, and 35b against human FXa
3.4. Molecular modeling study
To clarify the binding mode of the compounds, a detailed docking analysis was performed. The co-crystal
structure of apixaban with human FXa was selected as the docking model (PDB ID code: 2P16).11 The docking
simulation was conducted using Accelrys DS visualizer 3.0 systems. The image files were generated using
Accelrys DS visualizer 4.0 systems. The binding model was exemplified by the interaction of compound 14a with
human FXa. As shown in Fig. 6, the model further suggested that the 1-NH group of the 1,2,4-triazole linked to
C-3 pyrazole formed a H-bond with Glu146. Additionally, the pyrazole N-2 nitrogen atom interacted with the
Gln192. The pendant phenyllactam positioned between the Tyr215 and Phe174 occupies the P4 pocket in the FXa
hydrophobic cavity.
10

Fig. 6. The human FXa active site in the complex of compound 14a.
Furthermore, some physicochemical properties of the target compounds and apixaban were predicted using
free online software (http://www.molinspiration.com/) for their adaptability with Lipinski's rule of five. As shown
in Table 3, compounds 14a and 35b conformed well to the Lipinski's rule of five.23 Both of them were predicted
to possess strong drug resistance.
Table 3 Prediction of physicochemical propertiesa of the target compounds
Compound
miLogP
TPSA
natoms
MW
nON
nOHNH nviolations nrotb
volume
Accepted range
﹤5
﹤140
﹤500
﹤10
﹤5
≤10
14a
16
2.17
2.24
3.98
3.42
0.99
1.26
1.78
109.25
98.40
36
37
40
43
32
33
34
483.53
497.56
539.64
580.69
430.47
444.50
459.51
10
10
10
10
9
1
0
1
0
3
3
2
0
0
1
1
0
0
0
5
5
6
7
5
5
5
424.36
441.30
490.55
527.99
376.84
393.64
406.55
18c
109.25
85.08
26c
35a
114.85
114.85
110.77
35b
Apixabanb
9
9
a miLogP: molinspiration predicted Log P; TPSA: topological polar surface area; natoms: no. of atoms; MW: molecular weight;
nON: no. of hydrogen bond acceptors; nOHNH: no. of hydrogen bond donors; nviolations: no. of violations; nrotb: no. of rotatable
bonds; volume: molar volume.
b Used as positive control.
4. Conclusions
In this study, four series of novel potent FXa inhibitors based on apixaban were designed and synthesized.
The pharmacological data indicated that several compounds (14a, 16, 18c, 26c, 35a, and 35b) exhibited moderate
to excellent anticoagulant potency in human and rabbit plasma in vitro. The SARs, based on the EC2x values
shown in Table 1, showed that the hydrogen bond action and size of the nitrogenous heterocyclic in P2 and the
lipophilic region in P4 were responsible for the anticoagulant activity. Eventually, compound 14a possessing a
novel scaffold was selected for further exploration owing to its pronounced enzymatic anticoagulant activity with
an IC50 value of 0.15 μM in vitro and 99% inhibition rate in rat venous thrombosis test in vivo. The docking model
indicated that compound 14a formed a hydrogen bond with Glu146, and 1,2,4-triazole provided a much more
suitable size (5-membered triazole ring) accommodating the position of P2 compared with apixaban.
11

5. Experimental
5.1. Chemistry
Unless otherwise noted, all materials were used without further purification and were obtained from
commercial suppliers. 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. All melting points were obtained on a
Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland). Column chromatography was
run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). Mass spectra (MS)
were taken in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). Elemental analysis was
1
determined on a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, Milan, Italy). H NMR spectra were
recorded on Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an
internal standard.
5.2. The preparation of the key intermediates 11a-11f
5.2.1. Preparation of 5-Chloro-N-(4-nitrophenyl)pentanamide (1)
P-nitroaniline (50.0 g, 0.36 mol) and trimethylamine (100 mL, 0.72 mol) were solubilized in tetrahydrofuran
(200 mL), then a solution of 5-chlorovaleryl chloride (70 mL, 0.54 mol) was added drop-wise at 0-5C. After
stirring for 6 h at r.t., the mixture was poured into ice-water (200 mL). The resulting precipitate was filtered,
washed with diethyl ether and dried under reduced pressure to yield the title compound as a yellow solid (72.3 g,
77.8%), M.p.: 121.4-123.3C; MS (ESI) m/z (%): 257.06 [M+H]+.
5.2.2. Preparation of 1- (4-Nitrophenyl) piperidin-2-one (2)
A mixture of compound 1 (72.3 g, 0.28 mol), K2CO3 and DMSO (360 mL) was stirred at 80C for 6 h. Upon
cooling to r.t., the mixture was poured into ice-water (200 mL). The resulting precipitate was filtered, washed with
water and dried under reduced pressure to yield the title compound as a yellow powder (55.7 g, 89.6%)，M.p.:
96.4-98.3C; MS (ESI) m/z (%): 221.08 [M+H]+.
5.2.3. Preparation of 3,3-Dichloro-1-(4-nitrophenyl)piperidin-2-one (3)
A solution of intermediate 2 (55.7 g, 0.25 mol) in dichloromethane (330 mL) was stirred to dissolve, PCl5
(158.0 g, 0.76 mol) was added and refluxed for 5 h. After cooling to r.t., the reaction mixture was poured into ice-
water (300 mL). The aqueous layer was extracted with CH2Cl2 (3 × 100 mL). The organic phase was washed with
water (3 × 100 mL), brine (200 mL). The organic layer was dried with Na2SO4, filtered and evaporated in vacuo.
The residue was triturated with methanol to give a white powder (50.3 g, 69.0%), M.p.: 170.0-172.1C; MS (ESI)
m/z (%): 289.01 [M+H]+.
5.2.4. Preparation of 3-Morpholino-1-(4-nitrophenyl)-5,6-dihydropyridin-2(1H)-one (4)
A mixture of compound 3 (50.3 g, 0.17mol) and morpholine (200 mL) was stirred at 130C for 1.5 h , and then
cooled to room temperature. The solvent was removed under vacuum, and the precipitate was collected by
filtration and washed with water to afford the desired product 4 as a yellow solid (46.3g，87.6%), M.p.: 157.3-
1
160.3C; MS (ESI) m/z (%): 304.0 [M+H]+, 326.0 [M+Na]+. H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 3.0
Hz, 1H, Ar-H), 8.18 (d, J = 3.0 Hz, 1H, Ar-H), 7.62 (d, J = 3.0 Hz, 1H, Ar-H), 7.59 (d, J = 3.0 Hz, 1H, Ar-H), 5.77
(t, J = 4.5 Hz, 1H, C = CH-C), 3.79 (t, J = 7.5 Hz, 2H, N-CH2-C), 3.62-3.59 (m, 4H, O-CH2-C), 2.73-2.77 (m, 4H,
N-CH2-C), 2.47-2.41(m, 2 H, =C-CH2-C).
5.2.5. General procedure for preparation of 2-Chloro-2-(2-(4-substituted phenyl)hydrazono ) acetate 6a–6f
HCl (0.6 mol, 50 mL) was added to a well-stirred solution of 4-substituted phenyl amines 5a-5f (0.20 mol) in
water (100 mL) at r.t.. After the completion of addition, NaNO2 (15.2 g, 0.22 mol) in H2O (30 mL) was added
12

dropwise at -5C. Then the mixture was stirred at 0C for 30 min. Ethyl 2-chloroacetoacetate (34.5 g, 0.21 mol)
and sodium acetate (49.2 g, 0.60 mol) were added to the solution and stirred for 30 min. The reaction mixture was
then heated to 25C for 2 h. The resulting precipitate was filtered, washed with methano and dried under reduced
pressure to yield the title compound 6a-6f.
5.2.5.1. Ethyl (Z)-2-Chloro-2-(2-(4-methoxyphenyl)hydrazono)acetate (6a)
Light yellow solid; Yield: 85.1%; M.p.: 92.3-94.3C; MS (ESI) m/z (%): 279.3 [M+Na]+.
5.2.5.2. Ethyl (Z)-2-Chloro-2-(2-(4-trifluoromethoxyphenyl)hydrazono)acetate (6b)
Light yellow solid; Yield: 80.7%; M.p.: 91.3-93.3C; MS (ESI) m/z (%): 333.6 [M+Na]+.
5.2.5.3. Ethyl (Z)-2-Chloro-2-(2-(4-difluoromethoxyphenyl)hydrazono)acetate (6c)
Light yellow solid; Yield: 83.3%; M.p.: 94.1-96.3C; MS (ESI) m/z (%): 293.2 [M+H]+, 315.2 [M+Na]+.
5.2.5.4. Ethyl (Z)-2-Chloro-2-(2-(4-fluorophenyl)hydrazono)acetate (6d)
Light yellow solid; Yield: 83.3%; M.p.: 90.3-92.0C; MS (ESI) m/z (%): 245.1 [M+H]+, 268.0 [M+Na]+.
5.2.5.5. Ethyl (Z)-2-Chloro-2-(2-(4-chlorophenyl)hydrazono)acetate (6e)
Light yellow solid; Yield: 84.5%; M.p.: 91.5-93.0C; MS (ESI) m/z (%): 261.2 [M+H]+, 283.1 [M+Na]+.
5.2.5.6. Ethyl (Z)-2-Chloro-2-(2-(4-bromophenyl)hydrazono)acetate (6f)
Light yellow solid; Yield: 82.5%; M.p.: 91.7-93.9C; MS (ESI) m/z (%): 305.2[M+H]+.
5.2.6. General procedure for the preparation of intermediates 7a-7f
Trimethylamine (32 mL, 0.23 mol) and 6a-6f (0.18 mol) were added to a solution of intermediate 4 (45.5 g,
0.15 mol) in ethyl acetate (500 mL). The mixture was stirred at 80C for 6 h. After cooling to r.t., yellow solid was
precipitated. The resultant precipitate was filtered and washed with ethyl acetate to afford corresponding
compounds 7a-7f.
5.2.6.1. Ethyl1-(4-methoxyphenyl)-7a-morpholino-6-(4-nitrophenyl)-7-oxo-3a, 4, 5, 6, 7, 7a-hexahydro-1H-pyrazo
lo[3,4-c]pyridine-3-carboxylate (7a)
Yellow solid; Yield: 97.5%; M.p.: 175.3-177.3C; MS (ESI) m/z (%): 524.21 [M+H]+.
5.2.6.2. Ethyl7a-morpholino-6-(4-nitrophenyl)-7-oxo-1-(4-(trifluoromethoxy) phenyl)-3a,4,5,6,7,7a-hexahydro-1H
-pyrazolo[3,4-c]pyridine-3-carboxylate (7b)
Yellow solid; Yield: 94.6%; M.p.: 174.4-175.7C; MS (ESI) m/z (%): 578.18 [M+H]+.
5.2.6.3. Ethyl1-(4-(difluoromethoxy) phenyl)-7a-morpholino-6-(4-nitrophenyl)-7-oxo-3a,4,5,6,7,7a-hexahydro-1H
-pyrazolo[3,4-c]pyridine-3-carboxylate (7c)
Yellow solid; Yield: 96.3%; M.p.: 175.8-176.9C; MS (ESI) m/z (%): 560.19 [M+H]+.
5.2.6.4. Ethyl1-(4-fluorophenyl)-7a-morpholino-6-(4-nitrophenyl)-7-oxo-3a,4,5,6,7,7a-hexahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxylate (7d)
Yellow solid; Yield: 94.1%; M.p.: 173.3-174.3C; MS (ESI) m/z (%): 512.19 [M+H]+.
13

5.2.6.5. Ethyl1-(4-chlorophenyl)-7a-morpholino-6-(4-nitrophenyl)-7-oxo-3a,4,5,6,7,7a-hexahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxylate (7e)
Yellow solid; Yield: 93.0%; M.p.: 175.6-176.3C; MS (ESI) m/z (%): 528.16 [M+H]+.
5.2.6.6 Ethyl1-(4-bromophenyl)-7a-morpholino-6-(4-nitrophenyl)-7-oxo-3a,4,5,6,7,7a-hexahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxylate (7f)
Yellow solid; Yield: 94.6%; M.p.: 176.3-177.9C; MS (ESI) m/z (%): 572.11 [M+H]+.
5.2.7. General procedure for the preparation of intermediates 8a-8f
Trifluoroacetic acid (TFA) was added drop-wise in ice-bath to a solution of intermediates 7a-7f (0.12 mol) in
CH2Cl2 (650 mL). The resulting solution was stirred at r.t. for 1 h . The reaction mixture was poured into ice-water
(300 mL). The organic phase was washed with water (3 × 100 mL), brine (200 mL) and dried with Na2SO4,
filtered and evaporated in vacuo to get red-brown solid. Then the solid was triturated in ether to afford
intermediates 8a-8f.
5.2.7.1. Ethyl1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carb
oxylate (8a)
Light yellow powder; Yield: 62.1%; M.p.: 159.6-161.3C; MS (ESI) m/z (%): 459.0 [M+Na]+.
5.2.7.2. Ethyl6-(4-nitrophenyl)-7-oxo-1-(4-(trifluoromethoxy)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo [3,4-c]
pyridine-3-carboxylate (8b)
Light yellow powder; Yield: 59.8%; M.p.: 158.5-159.9C; MS (ESI) m/z (%): 513.0 [M+Na]+.
5.2.7.3. Ethyl1-(4-(difluoromethoxy)phenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carboxylate (8c)
Light yellow powder; Yield: 57.7%; M.p.: 160.6-161.3C; MS (ESI) m/z (%): 495.0 [M+Na]+.
5.2.7.4. Ethyl1-(4-fluorophenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxylate (8d)
Light yellow powder; Yield: 64.4%; M.p.: 160.5-161.2C; MS (ESI) m/z (%): 446.9 [M+Na]+.
5.2.7.5. Ethyl1-(4-chlorophenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxylate (8e)
Light yellow powder; Yield: 63.8%; M.p.: 160.2-162.3C; MS (ESI) m/z (%): 463.0 [M+Na]+.
5.2.7.6. Ethyl1-(4-bromophenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxylate (8f)
Light yellow powder; Yield: 63.0%; M.p.: 161.6-162.3C; MS (ESI) m/z (%): 506.8 [M+Na]+.
5.2.8. General procedure for the preparation of intermediates 9a-9f
A mixture of Fe powder (22.4 g, 0.40 mol), HCl (1.7 mL, 0.02 mol) and ammonium chloride (2.7 g, 0.05 mol)
in ethanol (90%, 350 mL) was heated to 80C with vigorous agitation for 30 min. Then corresponding
intermediates 8a-8f (0.1 mol) were added portion-wise until TLC showed the completion of the reaction. The hot
solution was filtered and washed with hot methanol. The filtrate was evaporated to afford 9a-9f.
14

5.2.8.1. Ehyl6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-Pyrazolo[3,4-c]
pyridine-3-carboxylate (
)
9a
White solid; Yield: 80.5%; M.p.: 150.6-152.3C; MS (ESI) m/z (%): 407.0 [M+H]+, 429.1 [M+Na]+.
5.2.8.2.Ehyl6-(4-aminophenyl)-7-oxo-1-(4-(trifluoromethoxy)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carboxylate (9b)
White solid; Yield: 75.6%; M.p.: 151.4-153.2C; MS (ESI) m/z (%): 461.0 [M+H]+, 483.1 [M+Na]+.
5.2.8.3. Ehyl6-(4-aminophenyl)-1-(4-(difluoromethoxy)phenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carboxylate (9c)
White solid; Yield: 76.4%; M.p.: 151.6-153.3C; MS (ESI) m/z (%): 443.1 [M+H]+, 465.2 [M+Na]+.
5.2.8.4. Ehyl6-(4-aminophenyl)-1-(4-fluorophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carbo
xylate (9d)
White solid; Yield: 82.7%; M.p.: 149.6-151.3C; MS (ESI) m/z (%): 395.0 [M+H]+, 417.1 [M+Na]+.
5.2.8.5. Ehyl6-(4-aminophenyl)-1-(4-chlorophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carbo
xylate (9e)
White solid; Yield: 77.8%; M.p.: 148.8-150.9C; MS (ESI) m/z (%): 411.0 [M+H]+, 433.0 [M+Na]+.
5.2.8.6. Ehyl6-(4-aminophenyl)-1-(4-bromophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carbo
xylate (9f)
White solid; Yield: 82.1%; M.p.: 149.6-151.3C; MS (ESI) m/z (%): 454.9 [M+H]+, 477.0 [M+Na]+.
5.2.9. General procedure for the preparation of compounds 10a-10f
Trimethylamine (19.5 mL, 0.14 mol) and a solution of 5-chlorovalerylchloride (13.5 mL, 0.11 mol) in
anhydrous tetrahydrofuran (60 mL) were added to a well-stirred mixture of 9a-9f (0.07 mol) in anhydrous
tetrahydrofuran (120 mL) at 0C. After the completion of addition, the mixture was warmed to 25C for 6 h, and
poured into ice-water (200 mL). The resulting precipitate was filtered, washed with water and dried under reduced
pressure to yield yellow solid. Then the solid was triturated in ether to afford corresponding compounds 10a-10f.
5.2.9.1. Ethyl6-(4-(5-chloropentanamido)phenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10a)
Gray solid; Yield: 86.7%; M.p.: 139.6-141.3C; MS (ESI) m/z (%): 547.1 [M+Na]+.
5.2.9.2. Ethyl6-(4-(5-chloropentanamido)phenyl)-7-oxo-1-(4-(trifluoromethoxy)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10b)
Gray solid; Yield: 84.0%; M.p.: 138.6-139.6C; MS (ESI) m/z (%): 600.9 [M+Na]+.
5.2.9.3. Ethyl6-(4-(5-chloropentanamido)phenyl)-1-(4-(difluoromethoxy)phenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10c)
Gray solid; Yield: 83.1%; M.p.: 140.5-142.3C; MS (ESI) m/z (%): 583.1 [M+Na]+.
5.2.9.4 Ethyl6-(4-(5-chloropentanamido)phenyl)-1-(4-fluorophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10d)
Gray solid; Yield: 84.5%; M.p.: 138.1-140.3C; MS (ESI) m/z (%): 535.2 [M+Na]+.
15

5.2.9.5 Ethyl6-(4-(5-chloropentanamido)phenyl)-1-(4-chlorophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10e)
Gray solid; Yield: 87.1%; M.p.: 138.4-139.7C; MS (ESI) m/z (%):550.8[M+Na]+.
5.2.9.6 Ethyl1-(4-bromophenyl)-6-(4-(5-chloropentanamido)phenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (10f)
Gray solid; Yield: 86.7%; M.p.: 137.6-138.8C; MS (ESI) m/z (%): 595.0 [M+Na]+.
5.2.10. General procedure for the preparation of compounds 11a-11f
Compounds 10a-10f (0.05 mol) and NaH (3.6 g, 0.15 mol) were added in DMF (270 mL) at 0C, and then
stirred for 1 h. The reaction mixture was poured into ice-water (200 mL).The aqueous layer was extracted with
CH2Cl2 (3 × 100 mL). The organic phase was washed with water (3 × 100 mL), brine (200 mL) and dried with
Na2SO4, filtered and evaporated in vacuo. The residue was triturated with methanol to give the title compounds
11a-11f as white solid.
5.2.10.1. Ethyl1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (11a)
White solid; Yield: 80.3%; M.p.: 126.4-128.3C; MS (ESI) m/z (%): 489.21 [M+H]+. H NMR (400 MHz,
1
CDCl3) δ 7.46 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.24 (d, J =8.4 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 4.46
(q, J = 7.1 Hz, 2H), 4.13 (t, J = 6.7 Hz, 2H), 3.81 (s, 3H),3.62 (t, J = 6.6 Hz 2H), 3.32 (t, J = 6.6 Hz, 2H), 2.66 (t,
J = 6.2 Hz, 2H), 1.95-1.92 (m, 4H), 1.43(t, J = 7.1 Hz, 2H); Anal. calcd. for C27H28N4O5 (%): C, 66.38; H, 5.78; N,
11.47. Found (%): C, 66.36; H, 5.79; N, 11.43.
5.2.10.2. Ethyl 7-oxo-6-(4-(2-oxopiperidin-1-yl) phenyl)-1-(4-(trifluoromethoxy)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (11b)
+
White solid; Yield: 82.3%; M.p.: 125.6-127.3C; MS (ESI) m/z (%): 543.18 [M+H] ; Anal. calcd. for
C27H25F3N4O5 (%): C, 59.78; H, 4.65; N, 10.33. Found (%): C, 59.79; H, 4.68; N, 10.35.
5.2.10.3. Ethyl 1-(4-(difluoromethoxy) phenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (11c)
White solid; Yield: 79.7%; M.p.: 127.6-129.4C; MS (ESI) m/z (%): 523.19 [M+H]+; Anal. calcd. for
C27H26F2N4O5 (%): C, 61.83; H, 5.00; N, 10.68. Found (%): C, 61.86; H, 5.02; N, 10.69.
5.2.10.4 Ethyl1-(4-fluorophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl) phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (11d)
White solid; Yield: 76.0%; M.p.: 128.2-129.7C; MS (ESI) m/z (%): 477.19 [M+H]+; Anal. calcd. for
C26H25FN4O4 (%): C, 65.54; H, 5.29; N, 11.76. Found (%): C, 65.56; H, 5.27; N, 11.78.
5.2.10.5 Ethyl1-(4-chlorophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carboxylate (11e)
White solid; Yield: 81.7%; M.p.: 127.5-129.5C; MS (ESI) m/z (%): 493.16 [M+H]+; Anal. calcd. for
C26H25ClN4O4 (%): C, 63.35; H, 5.11; N, 11.37. Found (%): C, 63.37; H, 5.13; N, 11.39.
5.2.10.6 Ethyl1-(4-bromophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
16

pyridine-3-carboxylate (11f)
White solid; Yield: 81.7%; M.p.: 125.6-127.3C; MS (ESI) m/z (%): 537.11 [M+H]+; Anal. calcd. for
C26H25BrN4O4 (%): C, 58.11; H, 4.69; N, 10.43. Found (%): C, 58.14; H, 4.71; N, 10.44.
5.3. Synthesis of Apixaban derivatives of series I
5.3.1. General procedure for the preparation of intermediates 12a-12f
A mixture of CH3ONa (4.10 g, 0.075 mol), methanamide (15.8 g, 0.35 mol), intermediates 11a-11f (0.05 mol)
and DMF (100 mL) was stirred at 50C for 4 h. After being cooled to r.t., the reaction mixture was poured into
ice-water. The precipitate was filtered and dried to afford products 12a-12f.
5.3.1.1. 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide (12a)
White solid; yield: 76.2%; M.p.: 177.4-178.3C; MS (ESI) m/z (%): 482.2 [M+Na]+.
5.3.1.2. 7-Oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-1-(4-(trifluoromethoxy)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide (12b)
White solid; yield: 76.4%; M.p.: 176.5-178.6C; MS (ESI) m/z (%): 436.2 [M+Na]+.
5.3.1.3. 1-(4-(Difluoromethoxy)phenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide (12c)
White solid; yield: 78.8%; M.p.: 177.3-178.3C; MS (ESI) m/z (%): 518.2 [M+Na]+.
5.3.1.4. 1-(4-Fluorophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide (12d)
White solid; yield: 80.5%; M.p.: 176.8-178.9C; MS (ESI) m/z (%): 470.2 [M+Na]+.
5.3.1.5. 1-(4-Chlorophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide(12e)
White solid; yield: 79.9%; M.p.: 177.8-179.3C; MS (ESI) m/z (%): 486.2 [M+Na]+.
5.3.1.6. 1-(4-Bromophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo
[3,4-c]pyridine-3-carboxamide (12f)
White solid; yield: 77.3%; M.p.: 178.4-179.2C; MS (ESI) m/z (%): 530.0 [M+Na]+.
5.3.2. General procedure for the preparation of intermediates 13a–13f
DMF-DMA (8.0 mL, 0.06 mol) was added to a solution of 12a-12f (4.59 g, 0.01 mol) in CH2Cl2 (80.0 mL).
The reaction was heated to 40°C for 3 h. After cooling to r.t., the resultant solid 13a-13f were collected by
concentrating under vacuum.
5.3.2.1. (E)-N-((Dimethylamino)methylene)-1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7
-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13a)
Light yellow solid; yield: 96.1%; M.p.: 178.6-179.9C; MS (ESI) m/z (%): 515.2 [M+H]+.
5.3.2.2. (E)-N-((Dimethylamino)methylene)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-1-(4-(trifluoromethoxy)
phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13b)
Light yellow solid; yield: 98.7%; M.p.: 178.3-180.3C; MS (ESI) m/z (%): 569.2 [M+H]+.
17

5.3.2.3. (E)-1-(4-(Difluoromethoxy)phenyl)-N-((dimethylamino)methylene)-7-oxo-6-(4-(2-oxopiperidin-1-yl)
phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13c)
Light yellow solid; yield: 99.8%; M.p.: 179.4-180.6C; MS (ESI) m/z (%): 551.2 [M+H]+.
5.3.2.4. (E)-N-((Dimethylamino)methylene)-1-(4-fluorophenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13d)
Light yellow solid; yield: 97.0%; M.p.: 179.1-181.8C; MS (ESI) m/z (%): 503.2 [M+H]+.
5.3.2.5. (E)-1-(4-Dhlorophenyl)-N-((dimethylamino)methylene)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13e)
Light yellow solid; yield: 95.9%; M.p.: 179.9-181.7C; MS (ESI) m/z (%): 519.2 [M+H]+.
5.3.2.6. (E)-1-(4-Bromophenyl)-N-((dimethylamino)methylene)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-
tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (13f)
Light yellow solid; yield: 93.0%; M.p.: 180.4-181.9C; MS (ESI) m/z (%): 562.1 [M+H]+.
5.3.3. General procedure for the preparation of compounds 14a–14f
A solution of intermediates 13a-13f (1.54 g, 3.0 mmol) and hydrazine hydrate (1.5 g, 30.0 mmol) in glacial
acetic acid (20.0 mL) was refluxed on an oil-bath for approximate 3 h. After cooling to ambient temperature, the
contents were concentrated under reduced pressure, and the residue solution was adjusted to pH 9 with saturated
sodium carbonate solution. The resulting precipitate was filtered, washed with ether, and dried under vacuum to
afford the title compounds 14a-14f .
5.3.3.1. 1-(4-Methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1,4,5,6-tetrahydro-7H-
pyrazolo[3,4-c]pyridin-7-one (14a)
White solid; yield: 82.8%; M.p.: 176.1-178.2C; MS (ESI) m/z (%): 505.7 [M+Na]+; 1H NMR (400 MHz, DMSO)
δ 8.46 (s, 1H), 7.52 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H),
4.11 (t, J = 6.5 Hz, 2H), 3.81 (s, 3H), 3.59 (t, J = 5.4 Hz, 2H), 3.29 (t, J = 6.5 Hz, 2H), 2.39 (t, J = 6.2 Hz, 2H),
1.99-1.75 (m, 4H); 13C NMR (101 MHz, DMSO) δ 169.34, 159.43, 157.21, 141.83, 140.36, 133.22, 133.03,
127.16, 126.79, 126.50, 123.68, 113.86, 55.92, 51.46, 51.30, 33.06, 23.46, 21.68, 21.36. Anal. calcd. for
C26H25N7O3 (%): C, 64.58; H, 5.21; N, 20.28. Found (%): C, 64.61; H, 5.24; N, 20.30.
5.3.3.2. 6-(4-(2-Oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1-(4-(trifluoromethoxy)phenyl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (14b)
White solid; yield: 74.5%; M.p.: 174.4-176.5C; MS (ESI) m/z (%): 560.0 [M+Na]+; H NMR (400 MHz,
1
CDCl3) δ 8.10 (s, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6
Hz, 2H), 4.10 (t, J = 6.5 Hz, 2H), 3.63-3.60 (m, 2H), 3.34 (t, J = 6.4 Hz, 2H), 2.60-2.57 (m, 2H), 1.95-1.93 (m,
4H); Anal. calcd. for C26H22F3N7O3 (%): C, 58.10; H, 4.13; N, 18.24. Found (%): C, 58.13; H, 4.16; N, 18.30.
5.3.3.3. 1-(4-(Difluoromethoxy)phenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (14c)
White solid; yield: 70.6%; M.p.: 174.3-176.7C; MS (ESI) m/z (%): 542.0 [M+Na]+; H NMR (400 MHz,
1
CDCl3) δ 8.11 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.6
Hz, 2H), 6.69-6.32 (s, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.62-3.60 (m, 2H), 3.34 (t, J = 6.4 Hz, 2H), 2.60-2.57 (m, 2H),
18

1.96-1.94 (m, 4H); Anal. calcd. for C26H23F2N7O3 (%): C, 60.11; H, 4.46; N, 18.87. Found (%): C, 60.14; H, 4.48;
N, 18.89.
5.3.3.4. 1-(4-Fluorophenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1,4,5,6-tetrahydro-7H-
pyrazolo[3,4-c]pyridin-7-one (14d)
White solid; yield: 84.9%; M.p.: 186.5-188.7C; MS (ESI) m/z (%): 493.9 [M+Na]+; H NMR (400 MHz,
1
CDCl3) δ 8.14 (s, 1H), 7.59 (dd, J = 8.8, 4.7 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.6 Hz, 3H), 7.09 (t, J
= 8.6 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 3.63-3.60 (m, 2H), 3.37 (t, J = 6.5 Hz, 2H), 2.60-2.57 (m, 2H), 1.96-1.94
(m, 4H); Anal. calcd. for C25H22FN7O2 (%): C, 63.69; H, 4.70; N, 20.80. Found (%): C, 63.71; H, 4.73; N, 20.82.
5.3.3.5. 1-(4-Chlorophenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1,4,5,6-tetrahydro-7H-
pyrazolo[3,4-c]pyridin-7-one (14e)
White solid; yield: 82.1%; M.p.: 188.4-190.3C; MS (ESI) m/z (%): 512.3 [M+Na]+; H NMR (400 MHz,
1
CDCl3) δ 8.14 (s, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.38–7.32 (m, 4H), 7.26（d, J = 8.7 Hz, 2H), 4.13 (t, J = 6.6 Hz,
2H), 3.63-3.60 (m, 2H), 3.36 (t, J = 6.6 Hz, 2H), 2.60-2.57 (m, 2H), 1.96-1.94 (m, 4H); Anal. calcd. for
C25H22ClN7O2 (%): C, 61.54; H, 4.54; N, 20.09. Found (%): C, 61.57; H, 4.57; N, 20.11.
5.3.3.6. 1-(4-Bromophenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-1,2,4-triazol-3-yl)-1,4,5,6-tetrahydro-7H-
pyrazolo[3,4-c]pyridin-7-one (14f)
White solid; yield: 91.6%; M.p.: 185.5-187.2C; MS (ESI) m/z (%): 554.1 [M+Na]+; H NMR (400 MHz,
1
CDCl3) δ 8.13 (s, 1H), 7.72 (d, J = 8.4 Hz, 0.7H), 7.54-7.49 (m, 2.6H), 7.38 (d, J = 8.6 Hz, 0.7H), 7.34 (d, J = 8.6
Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 4.14 (t, J = 6.6 Hz, 2H), 3.63-3.61 (m, 2H), 3.37 (t, J = 6.6 Hz, 2H), 2.60–2.57
(m, 2H), 1.96-1.94 (m, 4H); Anal. calcd. for C25H22BrN7O2 (%): C, 56.40; H, 4.17; N, 18.42. Found (%): C, 56.44;
H, 4.19; N, 18.45.
5.3.4. General procedure for the preparation of intermediates 15–16
NaH (0.03 g, 1.2 mmol) was added dropwise to the mixture of 14a (0.2 g, 0.4 mmol) in dry DMF (9 mL) at
0°C. Then the mixture was warmed to 40°C for 5 h. The solution was adjusted to pH 7 with acetic acid. The
reaction mixture was poured into ice-water (10 mL). The aqueous layer was extracted with ethyl acetate (3 ×10
mL). The organic phase was washed with water (3 × 10 mL), brine (20 mL). The organic layer was dried with
Na2SO4, filtered and evaporated in vacuo to afford white solid. The solid was purified by silica gel column
chromatography (eluent, ethyl acetate/MeOH = 80:1 to 50:1) to afford corresponding 15 and 16.
5.3.4.1. 1-(4-Methoxyphenyl)-3-(1-methyl-1H-1,2,4-triazol-5-yl)-5-(4-(2-oxopiperidin-1-yl)phenyl)-1,5,6,7-
tetrahydro-4H-pyrazolo[4,3-c]pyridin-4-one (15)
White solid; Yield: 93.2%; M.p.: 183.3-185.1C; MS (ESI) m/z (%): 498.5 [M+H]+; H NMR (400 MHz,
1
CDCl3) δ 7.89 (s, 1H), 7.46 (d, J = 8.9 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 3.6 Hz, 2H), 6.88 (d, J = 8.9
Hz, 2H), 4.23 (s, 3H), 4.09 (t, J = 7.0 Hz, 2H), 3.76 (s, 3H), 3.56 (t, J = 17.4 Hz, 2H), 3.36 (t, J = 6.6 Hz, 2H),
2.49 (t, J = 5.8 Hz, 2H), 1.85 (m, J = 13.0 Hz, 4H); Anal. calcd. for C27H27N7O3 (%): C, 65.18; H, 5.47; N, 19.71.
Found (%): C, 65.20; H, 5.49; N, 19.74.
5.3.4.2. 1-(4-Methoxyphenyl)-3-(1-methyl-1H-1,2,4-triazol-3-yl)-5-(4-(2-oxopiperidin-1-yl)phenyl)-1,5,6,7-
tetrahydro-4H-pyrrolo[3,4-c]pyridin-4-one (16)
White solid; Yield: 93.2%; M.p.: 184.2-186.1°C; MS (ESI) m/z (%): 498.5 [M+H]+; 1H NMR (400 MHz, DMSO-
d6) δ 8.59 (s, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 9.0 Hz,
19

2H), 4.10 (t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 3H), 3.60 (t, J = 5.5 Hz, 2H), 3.27 (t, J = 6.5 Hz, 2H), 2.39 (t, J
= 6.2 Hz, 2H), 1.92-1.78 (m, 4H); 13C NMR (101 MHz, DMSO) δ 169.31, 159.31, 157.27, 156.72, 145.84, 141.81,
140.41, 140.17, 133.32, 132.75, 126.99, 126.78, 126.48, 123.68, 113.83, 55.90, 51.44, 51.30, 36.48, 33.06, 23.47,
21.83, 21.37. Anal. calcd. for C27H27N7O3 (%): C, 65.18; H, 5.47; N, 19.71. Found (%): C, 65.21; H, 5.46; N,
19.73.
5.3.5. Preparation of 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carbonitrile 17
Triethylamine (0.13 mol, 18.1 mL) was added to a solution of 12a (15.0 g, 33.0 mmol) in DCM (225.0 mL)
at 30°C. After 30 min, trifluoroacetic anhydride (0.13 mol, 18.3 mL) was added dropwise to the reaction and
stirred for 2 h. The organic layer was washed with water and filtered to give a solid. The residue was crystallized
with methanol to afford the title compound 17 (10.5 g, 72.9%), M.p.: 171.3-173.6 C；MS (ESI) m/z (%): 435.3
[M+H]+.
5.3.6. General procedure for the preparation of compounds 18a-18e
A mixture of 17 (1.0 g, 2.3 mmol), K2CO3 (0.3 g, 2.3 mmol) and hydrazide (4.5 mmol) in n-butanol (10 mL)
was refluxed for 20-30 h. The reaction mixture was poured into ice-water (10 mL). The aqueous layer was
extracted with CH2Cl2 (3 ×10 mL). The organic phase was washed with water (3 × 10 mL), brine (20 mL). The
organic layer was dried with Na2SO4, filtered and evaporated in vacuo to give the title compounds 18a-18e.
5.3.6.1. 1-(4-Methoxyphenyl)-3-(5-methyl-1H-1,2,4-triazol-3-yl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18a)
1
Light yellow solid; Yield: 43.7%; M.p.: 172.2-174.5C; MS (ESI) m/z (%): 520.2 [M+Na]+; H NMR(400
MHz, CDCl3) δ 7.51 (d, J = 8.9 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.9 Hz,
2H), 4.09 (t, J = 6.5 Hz, 2H), 3.80 (s, 3H), 3.62-3.59 (m, 2H), 3.31 (t, J = 6.5 Hz, 2H), 2.59-2.56 (m, 2H), 2.41 (s,
3H), 1.95-1.93 (m, 4H); Anal. calcd. for C27H27N7O3 (%): C, 65.18; H, 5.47; N, 19.71. Found (%): C, 65.22; H,
5.44; N, 19.75.
5.3.6.2. 3-(5-(Methoxymethyl)-1H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-
1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18b)
Light yellow solid; Yield: 41.2%; M.p.: 164.4-166.3C; MS (ESI) m/z (%): 549.5 [M+Na] +, 526.1 [M-H]-,
562.1 [M+Cl]-; 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.25 (d, J = 8.7
Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 4.65 (s, 2H), 4.12 (t, J = 6.6 Hz, 2H), 3.80 (s, 3H), 3.61-3.58 (m, 2H), 3.48 (s,
3H), 3.38 (t, J = 6.6 Hz, 2H), 2.58-2.55 (m, 2H), 1.94-1.92 (m, 4H); Anal. calcd. for C28H29N7O4 (%): C, 63.74; H,
5.54; N, 18.58. Found (%): C, 63.76; H, 5.56; N, 18.57.
5.3.6.3. 3-(5-(Tert-butyl)-1H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18c)
Light yellow solid; Yield: 40.3%; M.p.: 172.6-174.4C; MS (ESI) m/z (%): 562.2 [M+Na] +; H NMR (400
1
MHz, CDCl3) δ 7.49 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4，2H), 6.90 (d, J = 8.7 Hz, 2H),
4.13 (t, J = 6.5 Hz, 2H), 3.80 (s, 3H), 3.60 (s, 2H), 3.39 (t, J = 6.4 Hz, 2H), 2.56 (s, 2H), 1.93 (s, 4H), 1.44 (s, 9H);
Anal. calcd. for C30H33N7O3 (%): C, 66.77; H, 6.16; N, 18.17. Found (%): C, 66.79; H, 6.19; N, 18.19.
5.3.6.4. 3-(5-Cyclopropyl-1H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-
20

tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18d)
Light yellow solid; Yield: 41.6%; M.p.: 170.4-172.1C; MS (ESI) m/z (%): 546.1 [M+Na] +, 562.0 [M+K] +;
1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 6.89 (d,
J = 8.7 Hz, 2H), 4.10 (t, J = 5.8 Hz, 2H), 3.79 (s, 3H), 3.60 (s, 2H), 3.38-3.31 (m, 2H), 2.56 (s, 2H), 1.94 (s, 4H),
1.25 (s, 1H), 1.09-1.08 (m, 2H), 0.98-0.96 (m, 2H); Anal. calcd. for C29H29N7O3 (%): C, 66.52; H, 5.58; N, 18.73.
Found (%): C, 66.54; H, 5.60; N, 18.74.
5.3.6.5. 1-(4-Methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(5-(tetrahydrofuran-2-yl)-1H-1, 2, 4-triazol-3-
yl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (18e)
Light yellow solid; Yield: 39.3%; M.p.: 169.4-171.7C; MS (ESI) m/z (%): 553.6 [M+H] +, 575.6 [M+Na] +;
1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 6.90 (d,
J = 8.9 Hz, 2H), 5.18 (dd, J = 7.4, 6.0 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 4.08 (dd, J = 14.7, 6.8 Hz, 1H), 3.96 (dd,
J = 15.0, 7.1 Hz, 1H), 3.80 (s, 3H), 3.61-3.58 (m, 2H), 3.38 (dd, J = 7.0, 5.4 Hz, 2H), 2.57-2.54 (m, 2H), 2.45-
2.38 (m, 1H), 2.35-2.27 (m, 1H), 2.04 (dd, J = 14.0, 7.0 Hz, 2H), 1.94-1.91 (m, 4H); Anal. calcd. for C30H31N7O4
(%): C, 65.09; H, 5.64; N, 17.71. Found (%): C, 65.10; H, 5.66; N, 17.74.
5.3.7. Preparation of 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carbohydrazide (19)
A solution of 11a (10.0 g, 20.0 mmol) in hydrazine hydrate (60.0 mL) was stirred at 80°C for 3.5 h. The
hydrazine hydrate was distilled. Solid was filtered and washed with water to afford 19 (7.8 g, 80.3%), M.p.:
168.3-170.6 C; MS (ESI) m/z (%): 497.2 [M+Na]+.
5.3.8. Preparation of (Z)-N'-(1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H
-pyrazolo[3,4-c]pyridine-3-carbonyl)-N,N-dimethylformohydrazonamide (20)
DMF-DMA (0.1 mol, 14.0 mL) was added to a stirring solution of 19 (8.3 g, 18.0 mmol) in DCM (160 mL)
and heated to reflux for 4 h. The reaction mixture was poured into ice-water (150 mL). The aqueous layer was
extracted with CH2Cl2 (3 × 50 mL). The organic phase was washed with water (3 × 50 mL), brine (50 mL) and
dried with Na2SO4, filtered and evaporated in vacuo.The yellow residue was purified by silica gel column
chromatography to give a white solid (7.5 g, 81.0%), M.p.: 169.3-171.4C; MS (ESI) m/z (%): 530.24 [M+H]+.
5.3.9. General procedure for the preparation of compounds 21a-21d
Compound 20 (0.3 g, 0.57 mmol) was dissolved with acetic acid (6 mL). Then appropriate amine (3 mL) was
added dropwise to the solution and heated to 90 °C for 10 h. The solution was poured into ice-water (5 mL), and
then adjusted to pH 9 with saturated sodium carbonate. The aqueous layer was extracted with CH2Cl2 (3 × 10 mL).
The organic phase was washed with water (3 × 10 mL), brine (20 mL) and dried with Na2SO4, filtered and
evaporated in vacuo to give the title compounds 21a-21d.
5.3.9.1. 3-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)
phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (21a)
Light yellow solid; Yield: 60.1%; M.p.: 164.3-166.4C; MS (ESI) m/z (%): 555.1 [M+H]+, 577.2, 578.1
[M+Na] +; 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.26 (d,
J = 8.6 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 4.60 (t, J = 6.5 Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.62-3.59
(m, 2H), 3.50 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.57-2.54 (m, 2H), 2.28 (s, 6H), 1.94-1.93 (m, 4H);
Anal. calcd. for C30H34N8O3 (%): C, 64.96; H, 6.18; N, 20.20. Found (%): C, 64.99; H, 6.17; N, 20.23.
21

5.3.9.2. 3-(4-(3-(Dimethylamino)propyl)-4H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)
phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (21b)
Light yellow solid; Yield: 55.6%; M.p.: 160.5-162.8C; MS (ESI) m/z (%): 568.0 [M+H]+; H NMR (400
1
MHz, CDCl3) δ 8.26 (s, 1H), 7.51 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 8.7 Hz, 2H), 6.94 (d, J
= 8.9 Hz, 2H), 4.54 (t, J = 6.8 Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.62-3.59 (m, 2H), 3.50 (t, J = 6.7
Hz, 2H), 2.56-2.55 (m, 2H), 2.27 (t, J = 6.7 Hz, 2H), 2.19 (s, 6H), 2.05-2.01 (m, 2H), 1.94-1.93 (m, 4H); Anal.
calcd. for C31H36N8O3 (%): C, 65.47; H, 6.38; N, 19.70. Found (%): C, 65.49; H, 6.40; N, 19.73.
5.3.9.3. 3-(4-(3-(Diethylamino)propyl)-4H-1,2,4-triazol-3-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)
phenyl)-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (21c)
Light yellow solid; Yield: 58.9%; M.p.: 161.4-163.7C; MS (ESI) m/z (%): 596.3 [M+H]+; H NMR(400
1
MHz, CDCl3) δ 8.27(s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.7 Hz, 2H), 6.94 (d, J
= 8.9 Hz, 2H), 4.53 (t, J = 7.0 Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.62-3.59 (m, 2H), 3.50 (t, J = 6.6
Hz, 2H), 2.61-2.46 (m, 8H), 2.11-2.04 (m, 2H), 1.95-1.93 (m, 4H), 0.98 (t, J = 7.1 Hz, 6H); Anal. calcd. for
C33H40N8O3 (%): C, 66.42; H, 6.76; N, 18.78. Found (%): C, 66.44; H, 6.78; N, 18.80.
5.3.9.4. 1-(4-Methoxyphenyl)-3-(4-(3-morpholinopropyl)-4H-1,2,4-triazol-3-yl)-6-(4-(2-oxopiperidin-1-yl)phenyl)
-1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (21d)
Light yellow solid; Yield: 57.5%; M.p.: 156.3-158.6C; MS (ESI) m/z (%): 610.9 [M+H]+, 632.8 [M+Na]+;
1H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 7.49 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.7 Hz,
2H), 6.94 (d, J = 8.9 Hz, 2H), 4.54 (t, J = 6.8 Hz, 2H), 4.17 (t, J = 6.6 Hz, 2H), 3.83 (s, 3H), 3.66-3.57 (m, 6H),
3.49 (t, J = 6.6 Hz, 2H), 2.57-2.54 (m, 2H), 2.40-2.31 (m, 6H), 2.10-2.03 (m, 2H), 1.94-1.93 (m, 4H); Anal. calcd.
for C33H38N8O4 (%): C, 64.90; H, 6.27; N, 18.35. Found (%): C, 64.93; H, 6.29; N, 18.37.
5.4. The preparation of Apixaban derivatives of series II: 3-substituted-1,2,4-triazolin-5-one derivatives 23a-23c
5.4.1 General procedure for preparation of 22a–22c
A solution of substituted phenyl isocyanates (0.25 g, 0.0021 mol) was added to a solution of 19 (1.0 g,
0.0019 mol) in THF (10.0 mL). The resulting reaction mixture was heated to 50C for 3 h. After cooling to r.t, the
precipitate was collected by filtration, washed with diethyl ether and dried to afford 22a-22c.
5.4.1.1. 2-(1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carbonyl)-N-phenylhydrazine-1-carboxamide (22a)
White solid; Yield: 90%; M.p.: 176.3-178.2C; MS (ESI) m/z (%): 594.2 [M+H]+
5.4.1.2. N-(4-Methoxyphenyl)-2-(1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro
-1H-pyrazolo[3,4-c]pyridine-3-carbonyl)hydrazine-1-carboxamide (22b)
White solid; Yield: 90%; M.p.: 177.3-179.5C; MS (ESI) m/z (%): 624.2 [M+H]+
5.4.1.3. 2-(1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]
pyridine-3-carbonyl)-N-(2-(trifluoromethyl)phenyl)hydrazine-1-carboxamide (22c)
White solid; Yield: 90%; M.p.: 175.6-177.7C; MS (ESI) m/z (%): 662.2 [M+Na]+
5.4.2 General procedure for preparation of 23a–23c
K2CO3 (0.3 g, 2.4 mmol) was added to a solution of 22a-22c (1.7 mmol) in DMSO (6 mL). The reaction
mixture was stirred at 80C for 6 h. After cooling to r.t., the mixture was poured into water with vigorously
22

stirring. The resulting precipitate was filtered, washed with water and dried under vacuum to afford 23a–23c.
5.4.2.1. 1-(4-Methoxy-phenyl)-3-(5-oxo-4-phenyl-4,5-dihydro-1H-[1,2,4]triazol-3-yl)-6-[4-(2-oxo-piperidin-1-yl)-
phenyl]-1,4,5,6-tetrahydro-pyrazolo[3,4-c]pyridin-7-one (23a)
White solid; Yield: 90%; M.p.: 184.2-190.6C; MS (ESI) m/z (%): 598.1 [M+Na]+. H NMR (400 MHz,
1
DMSO-d6) δ 10.27 (s, 1H, triazol-H), 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.35-7.27 (m,
5H, Ar-H), 7.09 (d, J = 8.8 Hz, 2H, Ar-H), 7.03 (d, J = 8.8 Hz, 2H, Ar-H), 4.09 (t, J = 6.6 Hz, 2H, pyrazolo
pyridine N-CH2), 3.82 (s, 3H, OCH3), 3.60 (t, J = 5.5 Hz, 2H, piperidone N-CH2), 3.27 (t, J = 6.5 Hz, 2H,
pyrazolo pyridine=C-CH2), 2.39 (t, J = 6.2 Hz, 2H, OC-CH2), 1.85–1.75 (m, 4H, CH2CH2); Anal. calcd. for
C32H29N7O4 (%): C, 66.77; H, 5.08; N, 17.03. Found (%): C, 66.79; H, 5.10; N, 17.04.
5.4.2.2. 1-(4-Methoxy-phenyl)-3-[4-(4-methoxy-phenyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl]-6-[4-(2-oxo-
piperidin-1-yl)-phenyl]-1,4,5,6-tetrahydro-pyrazolo[3,4-c]pyridin-7-one(23b)
White solid; Yield: 92%; M.p.: 186.2-194.5C; MS (ESI) m/z (%): 605.6 [M+H]+. H NMR (400 MHz,
1
DMSO-d6) δ 10.23 (s, 1H, triazol-H), 7.57 (d, J = 8.9 Hz, 2H, Ar-H), 7.45 (d, J = 8.2 Hz, 2H, Ar-H), 7.36 (d, J =
8.6 Hz, 2H, Ar-H), 7.28 (d, J = 8.7 Hz, 2H, Ar-H), 7.22 (d, J = 8.1 Hz, 2H, Ar-H), 7.02 (d, J = 8.9 Hz, 2H, Ar-H),
4.09 (t, J = 6.5 Hz, 2H, pyrazolo pyridine N-CH2), 3.81 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.59 (t, J = 5.3 Hz,
2H, piperidone N-CH2), 3.26 (t, J = 6.5 Hz, 2H, pyrazolo pyridine=C-CH2), 2.39 (t, J= 6.1 Hz, 2H, OC-CH2),
1.84-1.75(m, 4H,CH2CH2); Anal. calcd. for C33H31N7O5 (%): C, 65.44; H, 5.16; N, 16.19. Found (%): C, 65.46; H,
5.15; N, 16.18.
5.4.2.3. 1-(4-Methoxy-phenyl)-6-[4-(2-oxo-piperidin-1-yl)-phenyl]-3-[5-oxo-4-(2-trifluoromethyl-phenyl)-4,5-
dihydro-1H-[1,2,4]triazol-3-yl]-1,4,5,6-tetrahydro-pyrazolo[3,4-c]pyridin-7-one(23c)
White solid; Yield: 89%; M.p.: 186.4-194.1C; MS (ESI) m/z (%): 644.1 [M+H]+. H NMR (400 MHz,
1
DMSO-d6) δ 9.85 (s, 1H, triazol-H), 7.88-7.70 (m, 3H, Ar-H), 7.56 (d, J = 8.9 Hz, 2H, Ar-H), 7.49 (s, 1H, Ar-H),
7.36 (d, J = 8.7 Hz, 2H, Ar-H), 7.29 (d, J = 8.7 Hz, 2H, Ar-H), 7.04 (d, J = 9.0 Hz, 2H, Ar-H), 4.09 (t, J = 6.5 Hz,
2H, pyrazolo pyridine N-CH2), 3.81 (s, 3H, OCH3), 3.59 (t, J = 5.5 Hz, 2H, piperidone N-CH2), 3.24 (t, J = 6.5 Hz,
2H, pyrazolo pyridine=C-CH2), 2.39 (t, J = 6.3 Hz, 2H, OC-CH2), 1.85-1.75(m, 4H, CH2CH2); Anal. calcd. for
C33H28F3N7O4 (%): C, 61.58; H, 4.39; N, 15.23. Found (%): C, 61.59; H, 4.36; N, 15.25.
5.5. The preparation of Apixaban derivatives of series III: pyrazole derivatives 26a-26c
5.5.1. Preparation of 3-Amino-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-tetrahydro-7H-
pyrazolo[3,4-c]pyridin-7-one (24)
A mixture of 12a (3.0 g, 6 mmol), Potassium hydroxide (0.9 g, 16.0 mmol), iodobenzene diacetate (4.2 g,
15.0 mmol) and methanol (60 mL) was stirred at r.t. for 5 h. After evaporating in vacuum, the residue was
dissolved in a solution of H2O (30.0 mL), dioxane (60.0 mL) and NaOH (0.8 g, 0.02 mol). The mixture was
heated to 100C for 6 h. The residue was treated with water 10 mL, extracted with CH2Cl2 (3 × 10 mL), washed
with brine (2 × 10 mL), dried over anhydrous MgSO4 and concentrated under reduced pressure to afford 24 (2.3 g,
62.4%), M.p.: 178.4-179.9C; MS (ESI) m/z (%): 432.3 [M+H]+.
5.5.2. Preparation of 1-(4-Methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-3-(1H-pyrrol-1-yl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (25)
24 (1.0 g, 2.8 mmol) and catalytic amount of p-toluenesulfonic acid (0.01g, 0.06mmol) were added to a
solution of 2,5-dimethoxytetrahydrofuran (0.34 mL, 2.3 mmol) in THF. The mixture was heated to 60C for 1 h.
After cooling to r.t., the solution was exacted by CH2Cl2. The organic layer was washed by water and then dried
23

under vacuum to yield 25 (0.73 g, 70.0%), M.p.: 178.6-180.3C; MS (ESI) m/z (%): 504.1 [M+Na]+.
5.5.3. General procedure for preparation of 26a–26c.
A mixture of corresponding secondary amines (5.2 mmol), 37% formaldehyde solution (0.2 g, 2.2 mmol) and
25 (1 g, 2.0 mmol) was added in a sequence to a well-stirred solution of acetic acid (20.0 mL) at r.t.. The mixture
was stirred for 1 h. The residue was poured into water and adjusted pH to 8 by 10% NaOH in ice bath. The
separated solid was collected by filtration and the residue was dried to afford 26a–26c.
5.5.3.1. 3-(2-((Dimethylamino)methyl)-1H-pyrrol-1-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,
4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (26a)
White solid; Yield: 67%; M.p.: 178.4-179.6C; MS (ESI) m/z (%): 539.2 [M+H]+. H NMR (400 MHz,
1
CDCl3) δ 7.51 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.25 (d, J =8.7 Hz 2H), 6.92 (d, J =8.9 Hz, 2H), 6.88
(dd, J = 2.5, 1.8 Hz, 1H), 6.32 (d, 1.8 Hz, 1H), 6.29 (d, J = 2.5 Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.81 (s, 3H), 3.76
(s, 2H), 3.59 (t, J = 5.3 Hz, 2H), 3.00 (t, J =5.3 Hz, 2H), 2.55 (t, J = 5.5 Hz, 2H), 2.28 (s, 6H), 1.95-1.92 (m, 4H);
Anal. calcd. for C31H34N6O3 (%): C, 69.12; H, 6.36; N, 15.60. Found (%): C, 69.14; H, 6.38; N, 15.61.
5.5.3.2. 3-(2-((Diethylamino)methyl)-1H-pyrrol-1-yl)-1-(4-methoxyphenyl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-
1,4,5,6-tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (26b)
White solid; Yield: 65%; M.p.: 179.1-180.6C; MS (ESI) m/z (%): 567.2 [M+H]+. H NMR (400 MHz,
1
CDCl3) δ 7.49 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.25 (d, J =8.7 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 6.86
(dd, J = 2.9, 1.6 Hz 1H), 6.34 (d, J =1.6 Hz 1H), 6.28 (d, J = 2.9 Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 3.91 (s, 2H),
3.81 (s, 3H), 3.60 (t, J = 6.5 Hz ,2H), 2.97 (t, J = 6.5 Hz, 2H), 2.58 (d, J = 6.4 Hz, 4H), 2.55 (t, J = 3.1 Hz, 2H),
1.94-1.92 (m, 4H), 0.98 (t, J =6.4 Hz, 6H); Anal. calcd. for C33H38N6O3 (%): C, 69.94; H, 6.76; N, 14.83. Found
(%): C, 69.96; H, 6.75; N, 14.84.
5.5.3.3. 1-(4-Methoxyphenyl)-3-(2-(morpholinomethyl)-1H-pyrrol-1-yl)-6-(4-(2-oxopiperidin-1-yl)phenyl)-1,4,5,6-
tetrahydro-7H-pyrazolo[3,4-c]pyridin-7-one (26c)
Light yellow solid; Yield: 65%; M.p.: 178.7-181.9C; MS (ESI) m/z (%): 581.5 [M+H]+. H NMR (400 MHz,
1
CDCl3) δ 7. 49 (d, J = 9.0 Hz, 2H), 7.35(d, J = 8.6 Hz ,2H), 7.28 (d, J =8.5 Hz 2H), 7.26 (dd, J = 2.4,1.8 Hz, 1H),
6.92 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 1.8 Hz, 1H), 6.28 (d, J = 2.4 Hz ,1H), 4.12 (t, J = 6.5 Hz, 2H), 3.81 (s, 3H),
3.71 (m,6H), 3.61 (t, J = 6.5 Hz ,2H), 3.00 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 5.2 Hz, 4H), 2.46 (t, J = 5.5 Hz, 2H),
1.95-1.92 (m, 4H); Anal. calcd. for C33H36N6O4 (%): C, 68.26; H, 6.25; N, 14.47. Found (%): C, 68.27; H, 6.28; N,
14.45.
5.6. Synthesis of Apixaban derivatives of series IV (compounds 35a-35b)
5.6.1. Procedure for the preparation of compound 32a
The preparation of the intermediate 32a was similar to 8a, so their synthetic methods was not be listed here.
5.6.1.1. 5-Chloro-N-(4-cyanophenyl)pentanamide (27)
White solid. M.p.: 121.7-123.4C; MS (ESI) m/z (%): 237.07 [M+H]+.
5.6.1.2. 4-(2-Oxopiperidin-1-yl)benzonitrile (28)
White solid. M.p.: 101.4-103.3C; MS (ESI) m/z (%): 201.09 [M+H]+.
5.6.1.3. 4-(3,3-Dichloro-2-oxopiperidin-1-yl)benzonitrile (29)
24

White solid. M.p.: 171.4-173.3C; MS (ESI) m/z (%):269.02 [M+H]+.
5.6.1.4. 4-(5-Morpholino-6-oxo-3,6-dihydropyridin-1(2H)-yl)benzonitrile (30)
White solid. M.p.: 157.1-159.3C; MS (ESI) m/z (%): 284.13 [M+H]+.
5.6.1.5. Ethyl6-(4-Cyanophenyl)-1-(4-methoxyphenyl)-7a-morpholino-7-oxo-3a,4,5,6,7,7a-hexahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxylate (31a)
Yellow solid. M.p.: 176.4-178.3C; MS (ESI) m/z (%): 504.22 [M+H]+.
5.6.1.6. Ethyl6-(4-Cyanophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxylate (32a)
Yellow solid. M.p.: 165.5-167.3C; MS (ESI) m/z (%): 417.15 [M+H]+.
5.6.2. Procedure for the preparation of compound 33a
A mixture of 32a (1.0 g, 2.4 mmol) , NaOH (0.29 g, 7.3 mmol) in the solution of methanol and water
(methanol : water = 5 : 1) was stirred at 60C for 3 h. The solvent was removed under vacuum, and water (20 mL)
was added. The solution was adjusted to pH 4-5 with 3N hydrochloric acid, filtered and dried to afford a white
solid.
5.6.2.1. 6-(4-Cyanophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxylic acid (33a)
White solid; M.p.: 168.3-170.3C; MS (ESI) m/z (%): 387.12 [M-H]-.
5.6.3. Procedure for the preparation of compound 34a
TEA (1.4 mL) and ethyl chloroformate (1 mL) were added to the solution of compound 33a (2.7 g, 7.7 mmol)
in THF (5 mL) at 15C. The mixture was warmed to r.t. for 3 h. Then NH4OH (15 mL) was added to the reaction
and stirred for another 2 h. The mixture was then poured into water (30 mL) and stirred for 30 min. The
precipitate was filtered, washed with water and dried to give the title compound 34a.
5.6.3.1. 6-(4-Cyanophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-
carboxamide (34a)
White solid; M.p.: 170.3-172.3C; MS (ESI) m/z (%): 388.13 [M+H]+.
5.6.4. General procedure for the preparation of compounds 35a-35b
A stirring mixture of an appropriate 34a (0.2 g, 0.5 mmol), ethylenediamine or propanediamine (1 mL) and S
(0.13 mmol) was refluxed for 0.5 h. After cool to r.t., water was added to the solution and stirred for 30 min. The
precipitate was filtered to give the corresponding 35a-35b as a white solid.
5.6.4.1. 6-(4-(4,5-Dihydro-1H-imidazol-2-yl)phenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxamide (35a)
White solid; M.p.: 170.3-172.5C; MS (ESI) m/z (%): 431.18 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s,
2H), 8.07 (d, J = 8.8 Hz, 2H), 7.76 (d, 2H), 7.63 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 5.7 Hz, 2H), 6.99 (d, J = 9.0 Hz,
2H), 4.14 (t, J = 6.5 Hz, 2H), 3.96 (d, 2H), 3.97-3.93 (d, 2H), 3.78 (s, 3H), 3.20 (t, J = 6.4 Hz, 2H); 13C NMR (101
MHz, DMSO) δ 163.63, 163.50, 159.62, 156.96, 144.13, 141.97, 133.40, 133.03, 128.47, 127.73, 127.34, 125.75,
125.56, 113.88, 55.73, 50.96, 21.30, 14.10. Anal. calcd. for C23H22N6O3 (%): C, 64.18; H, 5.15; N, 19.52. Found
25

(%): C, 64.19; H, 5.17; N, 19.54.
5.6.4.2. 1-(4-Methoxyphenyl)-7-oxo-6-(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl)-4,5,6,7-tetrahydro-1H-
pyrazolo[3,4-c]pyridine-3-carboxamide (35b)
White solid; M.p.: 171.2-173.1C; MS (ESI) m/z (%): 445.19 [M+H]+. H NMR (400 MHz, DMSO-d6) δ
1
10.12 (s, 2H), 7.81 (d, J = 8.6 Hz, 2H), 7.76 (s, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 7.4 Hz, 2H), 7.01 (d, J =
9.0 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 3.81 (s, 3H), 3.50 (t, 2H), 3.45 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 6.5 Hz, 2H),
1.97 (m, J = 5.2 Hz, 2H); Anal. calcd. for C24H24N6O3 (%): C, 64.85; H, 5.44; N, 18.91. Found (%): C, 64.86; H,
5.47; N, 18.90.
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. Subsequent dilutions were
done. 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
24
reagent instructions. Determinations at each plasma concentration were done in duplicate.
5.7.2. In vitro human FXa inhibition assays
The potent inhibitory activity of six test compounds against human FXa was evaluated by using chromogenic
substrate in 96-well microtiter plate. The enzymatic action of human factor Xa was measured using the conversion
of a chromogenic substrate specific for FXa. FXa cleaved p-nitroaniline from the chromogenic substrate.
Table 4 Setting up reactions for human FXa inhibiton 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 25ºC 10 min
Enzyme step(40 μL)
40 μL
Tris-buffer
35 μL
35 μL
Pefachrome FXa
5 μL
5 μL
5 μL
Seal plate and incubate 20 min at 25ºC
5.7.3 Data Analysis
Determination of the inhibition rate of compounds was as follows:
Inhibition (%) = (ODpositivea -ODsampleb)/(ODpositive-ODnegativec) × 100%
a ODpositive: absorbance of positive wells (FXa + Substrate); b ODsample: absorbance of compound wells (FXa + Substrate
+Compound); c ODnegative: absorbance of negative wells (Buffer + Substrate)
The IC50 curves were generated along with the IC50 values by using GraphPad Prism 5.0 software.
The IC50 value was obtained by plotting the anti-FXa activity against the inhibitor concentration.
5.7.4. In vivo experimental vein thrombosis
Vein thrombosis was induced according to Reyers et al.25 with modifications. Briefly, the animals were
anesthetized with pentobarbital (40 mg/kg, i.p.) and the abdomen was opened. The venacava inferior was carefully
separated from surrounding tissues and ligated tightly with a cotton thread just below the left renal vein. The
26

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 kept at 37 °C for
24 h and after this time, their dry weight was measured.
Acknowledge
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),
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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Graphical abstract
Four series of Apixaban derivatives containing 1, 2, 4-triazole/ pyrrole moietys as P2 binding element and
dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized and evaluated
for their biological activity.
29