Design, synthesis and biological evaluation of anthranilamide derivatives as potential factor Xa (fXa) inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.09.012

Factor Xa (fXa) is a crucial player in various thromboembolic disorders. Inhibition of fXa can provide safe and effective antithrombotic effects. In this study, a series of anthranilamide compounds were designed by utilizing structure-based design strategies. Optimization at P1 and P4 groups led to the discovery of compound 16g: a highly potent, selective fXa inhibitor with pronounced in vitro anticoagulant activity. Moreover, 16g also displayed excellent in vivo antithrombotic activity in the rat venous thrombosis (VT) and arteriovenous shunt (AV-SHUNT) models. The bleeding risk evaluation showed that 16g had a safer profile than that of betrixaban at 1 mg/kg and 5 mg/kg dose. Additionally, 16g also exhibited satisfactory PK profiles. Eventually, 16g was selected to investigate its effect on hypoxia-reoxygenation- induced H9C2 cell viability. MTT results showed that H9C2 cell viability can be remarkably alleviated by 16g.

Full text of DOI:10.1016/j.bmc.2018.09.012

Accepted Manuscript
Design, synthesis and biological evaluation of anthranilamide derivatives as
potential factor Xa (fXa) inhibitors
Junhao Xing, Lingyun Yang, Jinpei Zhou, Huibin Zhang
PII:
S0968-0896(18)30840-X
https://doi.org/10.1016/j.bmc.2018.09.012
BMC 14537
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
1 May 2018
27 August 2018
10 September 2018
Please cite this article as: Xing, J., Yang, L., Zhou, J., Zhang, H., Design, synthesis and biological evaluation of
anthranilamide derivatives as potential factor Xa (fXa) inhibitors, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.09.012
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Design, synthesis and biological evaluation of anthranilamide derivatives as
potential factor Xa (fXa) inhibitors
Junhao Xinga, Lingyun Yangb, Jinpei Zhouc, Huibin, Zhangb, d,
∗
aDepartment of Organic Chemistry, School of Science, China Pharmaceutical University, 639 Longmian Avenue,
Nanjing 211198, PR China.
bCenter of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China.
cDepartment of Medicinal Chemistry, China Pharmaceutical University, TongjiaXiang 24, 210009 Nanjing, PR
China
dJiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, Nanjing
210009, PR China
Abstract
Factor Xa (fXa) is a crucial player in various thromboembolic disorders. Inhibition of fXa can
provide safe and effective antithrombotic effects. In this study, a series of anthranilamide
compounds were designed by utilizing structure-based design strategies. Optimization at P1
and P4 groups led to the discovery of compound 16g: a highly potent, selective fXa inhibitor
with pronounced in vitro anticoagulant activity. Moreover, 16g also displayed excellent in vivo
antithrombotic activity in the rat venous thrombosis (VT) and arteriovenous shunt
(AV-SHUNT) models. The bleeding risk evaluation showed that 16g had a safer profile than
that of betrixaban at 1 mg/kg and 5 mg/kg dose. Additionally, 16g also exhibited satisfactory
PK profiles. Eventually, 16g was selected to investigate its effect on hypoxia-reoxygenation-
induced H9C2 cell viability. MTT results showed that H9C2 cell viability can be remarkably
alleviated by 16g.
Keywords factor Xa; venous thrombosis; arteriovenous shunt; bleeding risk; H9C2 cell.
1. Introduction
Factor Xa (fXa), a trypsin-like serine protease, plays a key role in the blood coagulation
cascade1. It can form pro-thrombinase complex with fVa, phospholipids, and Ca2+ to produce
thrombin by activating prothrombin during blood clotting in intrinsic and extrinsic coagulation
pathways2, 3. fXa is upstream from thrombin in the coagulation cascade. Inhibition of fXa
instead of thrombin can be more effective in diminishing the coagulation cascade. Moreover,
Inhibition of fXa has lower risk of bleeding due to its ability to reduce the further generation of
thrombin without affecting the existing level of thrombin which should be sufficient to ensure
∗
Corresponding authors. Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Tel: +86-25-83271302; Fax:
+86-25-83271480.
E-mail addresses: hb_zhang@hotmail.com (H. Zhang) or zhanghb80@163.com, xingjhcpu@163.com (J. Xing)
1

primary hemostasis4. Therefore, fXa is a particularly promising target for the development of
novel anticoagulants and has attracted great interest in the past decades.
Currently, four oral, direct and selective fXa inhibitors have been approved by the U.S.
Food and Drug Administration (FDA): rivaroxaban5, 6, apixaban7, edoxaban8 and betrixaban9.
Compared with traditional anticoagulants, these novel fXa inhibitors exhibited good oral
bioavailability, high specificity and minor drug and food interactions10, 11. However, they still
faced with challenging scenarios due to lack of required monitoring and specific reversal
agents for significant bleeding12. In addition, they have narrow clinical indications and should
not be used in patients with severe renal and hepatic impairment13, 14, patients with mechanical
heart valves15. Therefore, it is also necessary to further develop novel and safer fXa inhibitors
to promote their use in clinic.
Fig. 1. The structures of rivaroxaban, apixaban, edoxaban, betrixaban and eribaxaban.
In our previous studies, we have reported a series of anthranilamide and 2,
3-dihydroquinazolin- 4(1H)-one derivatives as fXa inhibitors16, 17. As a part of our ongoing
research program to develop safer and more effective fXa inhibitors, guided by X-ray
crystallography and structure-based design strategies, a series of novel anthranilamide fXa
inhibitors were designed and synthesized. The following optimization resulted in the discovery
of compound 16g, which was identified as a highly potent and selective FXa inhibitor and
warrants further evaluation as a potential candidate for the prevention and treatment of
thromboembolic diseases. Additionally, myocardial ischemia (MI) is known to induce the
formation of thrombosis, and thrombus is also a cause of MI18-20. Thus, compounds possessing
both anticoagulant and cardioprotective effects in MI are desirable. The most excellent
compound 16g was selected to investigate its effect on hypoxia–reoxygenation -induced H9C2
cell viability. MTT results showed that 16g could inhibite hypoxia– reoxygenation-induced
H9C2 cell viability loss.
2. Drug design
Over the past decades, the development of synthetic, direct fXa inhibitors has undergone
four phases 21. Although these fXa inhibitors possess various scaffolds, most of them bind to
2

the active site in a characteristic L-shaped conformation21. In other word, they have a
three-component system including a central scaffold and two hydrophobic fragments (P1 and
P4) which provides a similar non-linear geometry considered to play a crucial role in fXa
recognition. Many novel fXa inhibitors such as apixaban and rivaroxaban adopt very similar
conformations including the length and orientation of P1 and P4. As shown in Fig.2A,
apixaban (pink, PDB code 2P16) and rivaroxaban (green, PDB code 2W26) bind with fXa in
a quite similar conformational space. Although their scaffolds could not be overlaid and
formed significantly different interactions with fXa, their aromatic rings in P4 fragments
superimposed very well, and length and orientation of P1 and P4 are also very similar. In
addition, the binding conformation of betrixaban with fXa was predicted by glide in
Schrodinger 2009 which has been demonstrated to successfully predict the binding modes of
many reported inhibitors to fXa. It was also found that the P1 and P4 fragments of betrixaban
and rivaroxaban, especially P1 group, adopted very similar conformations (Fig. 2B).
Fig.2. The binding mode of apixaban, rivaroxaban and betrixaban with the active sites of fXa. (A):
Superimposition of structures of rivaroxaban (green, PDB code 2W26) and apixaban (pink, PDB code
2P16); (B). Overlay of the binding conformations of rivaroxaban and betrixaban. PDB entry 2W26 was
used for molecular docking simulation of betrixaban. Hydrogen atoms have been hidden for clarity. The
figures were prepared using PyMol (www.pymol.org).
Based on these modeling and findings, it was postulated that the highly basic amidine
group of betrixaban can be replaced by less basic P4 moieties of rivaroxaban, apixaban and
edoxaban. Meanwhile, the P1 fragment of betrixaban also can be changed to other substituted
aromatic or heteroaromatic rings. As a result, a novel series of anthranilamide derivatives
were designed (Fig. 3), synthesized and evaluated for their fXa inhibitory activity.
Fig. 3. Design of the target compounds
3

3. Results and discussion
3.1 Chemistry
The synthetic procedure for 7a-7l is shown in Scheme 1. To synthesize intermediates
3a-3f, commercial available carboxylic acid 1 was used as the starting material. This material
was treated with aromatic amines and EDCI in the presence of DIPEA to form intermediates
2a-2f, which were reduced by using hydrogen in the presence of Pd/C catalyst (10%) in
anhydrous EtOH to be converted to 3a-3f. The intermediates 5a-5d was formed by the
copper-catalysed coupling reaction of the corresponding β-Lactam with commercially
available 4 in the presence of N, N’-dimethyl ethylenediamine as a ligand22. 5a-5d were
hydrolyzed in aqueous sodium hydroxide to yield 6a-6d, respectively, which were treated
with 3a-3f and EDCI in the presence of DIPEA to obtain products 7a-7l.
Scheme 1. Synthesis routes and structures of intermediates and target compounds 7a-7l. (a) Arylamine,
EDCI, DIPEA, CH2Cl2, 0-r.t., 12 h; (b) H2, 10% Pd/C, ethonal, r.t., 15 h; (c) Morpholin-3-one or
Piperidin-2-one, N,N’-dimethyl ethylenediamine, CuI, K3PO4, N2, PhMe, reflux, 16 h; (d) NaOH,
MeOH/H2O, 0 oC, 1 h; (e) EDCI, DIPEA, DMF, 0-r.t., 24 h.
The synthetic procedure for 16a-16j is presented in Scheme 2. In this route, intermediates
15a-15h were synthesized in the same method as that described for 3a-3f. The synthesis of
carboxylic acid 13 started with compound 8, which was transformed to intermediate 9 by
bromination23. Treatment of 9 with ethyl thioxamate and then deprotection with trifluoroacetic
acid afforded amine 11. Subsequent reaction with methyl iodide afforded intermediate 12.
After hydrolysis of 12 with aqueous sodium hydroxide, the key intermediate 13 was obtained
in good yield. Then, compounds 16a-16j were synthesized from 13 and 3a, 3b and 15a-15h
following the approach used for compounds 7a-7l.
4

Scheme 2. Synthesis routes and structures of intermediates and target compounds 16a-16j. (a) Bromine, CH2Cl2,
0-r.t., 0.5 h; (b) Ethyl thiooxamate, NaHCO3, IPA, reflux, 12 h; (c) F3CCO2H, CH2Cl2, 0 oC-r.t., 3 h; (d)
iodomethane, CH2Cl2, 30 oC, 18 h; (e) 1 M NaOH, MeOH, r.t., 1.5 h; (f) Arylamine, EDCI, DIPEA, CH2Cl2, 0-r.t.,
12 h; (g) H2, 10% Pd/C, ethonal, r.t., 15 h; (h) EDCI, DIPEA, DMF, 0-r.t., 48 h.
3.2 Biological activities and discussion
3.2.1 In vitro fXa inhibition activities studies
All the designed compounds were evaluated for their human fXa enzyme inhibitory
activity. Shown in Table1 are IC50 values of the targeted compounds 7a-7j. The assay data
displayed compounds with morpholin-3-one as P4 fragment showed slightly increase of
activity compared with molecules possessing piperidin-2-one, pyridin-2(1H)-one or
pyrrolidin-2-one as P4 surrogate (7a-7f vs 7g-7j, 7g vs 7e, 7k, 7l). In general, compounds (7e
and 7g) with 4-chlorophenyl as the P1 surrogate showed strong inhibitory activity. Derivative
7d with 3-chlorophenyl as the P1 fragment showed a sharply decreased fXa inhibitory activity
compared with that of 7e and 7g. Additionally, compared with 4-chlorophenyl derivatives, a
loss of potency to varying extents was observed for the methoxy, fluoro and tert-butyl
derivatives. The results were consistent with our precious study of SAR which the Cl−π
interaction formed by the chlorine atom and the phenyl ring of Tyr228 plays a crucial role in
potency16.
5

Table 1. fXa inhibitory effect of compounds 7a-7l.
Compd.
R1
R2
IC50 (nM)a
246 47
Compd.
R1
R2
IC50 (nM)a
7a
7g
18
5
7b
513 113
7h
77 15
7c
1079 208
272 65
7i
2087 676
1543 592
7d
7j
7e
7f
45 11
7k
7l
179 42
3189 790
653 136
Betrixaban
-
-
4.5 1.8
a The data represent the mean of at least three independent determinations.
Further modifications were made to P1 surrogates with 5-methyl-4,5,6,7-
tetrahydrothiazolo [5,4-c]pyridin as P4 substituent. Overall, these compounds had a moderate
6

to high potency against fXa. Among them, the derivatives (16e-16g) with substituted pyridine
as P1 fragment were more potent than their corresponding phenyl and chlorothiophene
derivatives. Compound 16g with 5-chloropyridin as the P1 surrogate displayed the strongest
potency against fXa with the IC50 value of 3.5 nM. Moreover, 5-fluoropyridin (16e) and
5-methylpyridin (16f) derivatives also exhibited excellent potency. When the substituted
phenyls were used as P1 fragment, 4-chlorophenyl (16i) was found to be the best surrogate.
Compounds with 4- methyl phenyl (16a) or 4-ethynylphenyl (16c) as the P1 substituent also
showed a potent fXa inhibitory activity. However, changing 4-chlorophenyl to 3-chlorophenyl
(16h) resulted in a 66-fold loss of potency, whereas 4-methoxy and 3-methoxy derivatives
(16c and 16d) lost potency by about 49 and 200-fold, respectively. Additionally, Compound
16j with 5-chlorothiophen as the P1 surrogate displayed low potency.
Table 2. fXa inhibitory effect of compounds 16a-16j.
Compd.
R1
IC50 (nM)a
30 7.0
Compd.
R1
IC50 (nM)a
3.5 1.0
16a
16g
16b
16c
234 63
20 5.1
16h
16i
315 63
4.8 1.4
16d
16e
16f
981 185
24 6.3
10 3.2
16j
138 7 362
4.5 1.8
Betrixaban
a The data represent the mean of at least three independent determinations.
3.2.2 Selectivity and in vitro clotting activity assay
7

Based on the results of fXa inhibition activities, compounds 16f, 16g and 16i were
selected to evaluate their selectivity against related serine proteases. All tested compounds
were found to be more than 1,000 fold selective versus fIIa, fVIIa, fIXa, and trypsin (Table 3).
In addition, the in vitro clotting activities of these compounds were measured by the
prothrombin time (PT) assay and activated partial thromboplastin time (aPTT). Compounds
16f displayed moderate clotting activity judged by 2 × PT value of 5.6 µM and 2 × aPTT
value of 2.2 µM. For its counterpart, 16g and 16i demonstrated potent activity in both assays.
Especially, compound 16g, which exhibited the best clotting activity with 2 × PT value of 0.8
µM and 2 × aPTT value of 0.5 µM, was better than betrixaban.
Table 3 Selectivity and anticoagulant activity of compounds selected.
Compd.
Thrombin
fVIIa
2 × PT
2 × aPTT
fIXa
trypsin
IC50(µM)a
IC50(µM)a
IC50(µM)a
IC50(µM)a
(µM)b
(µM)c
16g
16i
4.2
7.1
32
12
4.7
11
36
26
42
9.1
18
54
0.8
1.3
5.6
2.4
0.5
0.8
2.2
1.1
16f
Betrixaban
18
aThe data represent the mean of at least three independent determinations.
bPT values are defined as the inhibitor concentration required to double fibrin formation the time and
using rabbit plasma.
caPPT values are defined as the inhibitor concentration required to double activated partial
thromboplastin time using rabbit plasma.
3.2.3 In vivo antithrombotic activity in rats and bleeding risk of 16g in rats.
As a result of the excellent in vitro potency and selectivity, compound 16g was selected
to further evaluate its in vivo antithrombotic effect in FeCl3-induced venous thrombosis (VT),
arteriovenous shunt (AV-SHUNT) thrombosis model in rats and bleeding risk in a rat
tail-bleeding time study. As shown in Fig. 4, 16g displayed significant in vivo antithrombotic
activity in both thrombosis models in a dose-dependent manner. In VT model (Fig. 4A), 16g
reduced thrombus weight by 23%, 36% and 54% at 1 mg/kg, 5 mg/kg and 10 mg/kg
compared with 27%, 38% and 59% for betrixaban, respectively. In AV-SHUNT thrombosis
model (Fig. 4B), compound 16g was found to be more potent than betrixaban. Reduction in
thrombus weight was 30%, 50% and 67% at 1 mg/kg, 5 mg/kg and 10 mg/kg respectively.
Betrixaban reduced thrombus weight by 24%, 43% and 61% at 1 mg/kg, 5 mg/kg and 10
8

mg/kg, respectively. Additionally, a rat tail-bleeding time assay was implemented to evaluate
the bleeding risk of 16g. The test results appearing in Fig. 5 showed that 16g and betrixaban
prolonged bleeding time in a dose-dependent manner, and 16g had safer profile than that of
betrixaban at the doses of 1 mg/kg and 5 mg/kg. When administrated with 10 mg/kg, 16g and
betrixaban prolonged the bleeding time to the same extent.
Fig. 4. In vivo antithrombotic activity of 16g and betrixaban in rats (A): Antithrombotic profile of 16g
and betrixaban in FeCl3-induced venous thrombosis model; (B): Antithrombotic profile of 16g and
betrixaban in the rat arteriovenous shunt model. Values are mean SEM (n = 8). *P < 0.05, **P < 0.01,
and ***P < 0.001 compared to vehicle group by Student’s t-test.
Fig. 5. Effect of 16g and betrixaban on rat tail-bleeding time. Values are mean SEM (n = 8). *P < 0.05,
**P < 0.01, and ***P < 0.001 compared to vehicle group by Student’s t-test.
3.2.4 PK profiles of compound 16g.
Given the excellent in vitro potency, the PK profiles of compound 16g were studied in rats
with the data for betrixaban included for comparison. As summarized in Table 4, compound
16g displayed favorable PK profiles, with high plasma exposure (AUC0−∞ = 1160 ng·h/mL),
high maximal plasma concentration (Cmax = 221 ng/mL), lower clearance (CLp = 0.92 L/h/kg)
and good oral bioavailability (F = 51.3%) after oral administration. These values were
comparable or better than that of betrixaban.
9

Table 4. PK profiles of 16g and betrixaban in male ratsa.
Compd.
Route
Dose
Cmax
Tmax
(h)
T1/2
(h)
CLp
Vss
F
AUC0-
∞
(mg/kg) (ng/mL)
(L/h/kg) (L/kg) (%)
(ng⋅h/mL)
1160
p.o.
i.v.
3
1
3
1
221
146
3.2
9.6
8.8
8.6
7.8
51.3
16g
2258
0.92
1.36
0.201
0.191
p.o.
2.6
571
30.2
Betrixaban
i.v.
1832
aAbbreviations: p.o., per oral; i.v., intravenous; Cmax, peak plasma concentration of the compound after
administration; Tmax, time to reach Cmax; T1/2, elimination half-life; AUC, area under the
concentration-time curve; CLp, plasma clearance; Vss, volume of distribution at steady state; F,
bioavailability.
3.2.5 MTT assay
Compound 16g was selected to investigate its effect on hypoxia–reoxygenation-induced
H9C2 cell viability. MTT results showed that hypoxia–reoxygenation significantly inhibited H9C2
cell viability, which was remarkably alleviated by 16g. By contrast, the assay demonstrated H9C2
cell viability could not be improved by betrixaban.
or better than rivaroxaban
Fig. 6. MTT results. All values given are the mean SDs. ##P < 0.01 vs. control group. **P < 0.01 vs.
hypoxia–reoxygenation vehicle group.
3.3 Docking study for compound 16g
To elucidate the binding mode compound 16g to fXa, molecular docking was conducted
using glide in Schrodinger 2009 with the crystal structure of fXa (PDB code: 2w26). As shown
in Fig.7A, The 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine as P4 fragment occupy S4
pocket which is formed by Tyr99, Trp215, and Phe174 and did not interact with any residues
10

in the S4 pocket. For the central scaffold, two hydrogen bonds are formed with the residues in
this region of the enzyme. The carbonyl oxygen forms a hydrogen bond with Gly216. The NH
of the scaffold carboxamide forms a hydrogen bond with the carbonyl oxygen of Gly219. The
5-chloropyridin is orientated to the S1 pocket and interacts with the aromatic ring of Tyr228
located at the bottom of the S1 pocket to form the key Cl-π interaction, which is considered to
be important for the potent fXa inhibitory activity24. In addition, when the binding
conformations of 16g and rivaroxaban were overlaid within the same coordinate system
(Fig.7B), it can be found that they adopted very similar conformations. The two P4 fragments
have similar length and orientation and their P1groups superimposed very well. All the results
above confirmed the reliability of our drug design strategy.
Fig.7. The docking mode of 16g with the active sites of fXa (PDB code 2w26). (A) 3D interactions of
16g with the active sites of fXa; (B) Superimposition of the binding conformations of 16g and
rivaroxaban.
4. Conclusion
In summary, a series of novel anthranilamide derivatives were designed and synthesized
as potent and selective fXa inhibitors by utilizing structure-based design strategies. Most of
the target compounds displayed some degree of fXa inhibitory activity. Of these molecules,
the compound 16g was found to possess the best fXa inhibition with an IC50 value of 3.5 nM,
significant selectivity against other serine protease, and pronounced in vitro and in vivo
anticoagulant efficacy. The bleeding risk evaluation exhibited that 16g had a safer profile than
that of betrixaban at 1 mg/kg and 5 mg/kg dose. The pharmacokinetic profiles of 16g were
studied and presented favorable results. In addition, in MTT assay, 16g displayed significant
ability to improve hypoxia–reoxygenation-induced H9C2 cell viability. The effects of 16g on
myocardial injury are being evaluated by our lab and will be reported in due course.
5. Experimental section
5.1 Docking study
Ligand molecules for docking studies were drawn using ChemBioDraw Ultra 16.0
software. The compounds were energy minimized and refined with CHARMm force field
using Prepare Ligands tool of DS3.0. Docking simulation was performed with Glide in
Schrodinger 200925. The crystal complex was downloaded from the protein data bank (PDB
code: 2w26) and processed with water removed and hydrogen added at pH=7.0. Then a 10-Å
11

box was defined by the geometrical center of the ligand binding site. Protein structure was
prepared using the “protein preparation wizard” to generate the grid file in the binding site.
Other parameters were in default.
5.2 Chemistry
5.2.1 General chemistry
Solvents, reagents and starting materials were obtained from commercial sources in the
appropriate grade and used without further purification. NMR spectra were recorded on Bruker
AVANCE AV-300 spectrometer (300 MHz for 1H, 75 MHz for 13C) using DMSO-d6 or CDCl3.
Chemical shifts were reported in δ (ppm). Multiplicity was indicated as follows: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), br (broad). Coupling constants were reported in
Hertz (Hz). Mass spectrometry (MS): HewlettePackard 1100 LC/MSD spectrometer, in m/z;
Elemental analyses were carried out on CHN-O-Rapid instrument. For thin layer
chromatography (TLC), Yantai pre-coated TLC plates (HSGF 254) were used, and compounds
were visualized with a UV light at 254 nm. Column chromatography separations were
performed on silica gel (300–400 mesh) eluting with n-hexane/ethyl acetate (8/1 to 1/1) or
dichloromethane/methanol (100/1, 60/1 or 30/1).
5.2.2 N-(4-chlorophenyl)-5-methoxy-2-nitrobenzamide(2a)
To a mixture of compound 1 (2 g, 10.1 mmol) and EDCI (2.3 mg, 12.2 mmol) in CH2Cl2
(25 mL) cooled to 0 oC was added dropwise a solution of 4-chloroaniline (1.3 g, 10.1 mmol)
and DIPEA (2.5 mL) in CH2Cl2 (10 mL). The reaction mixture was stirred at 0 oC for 15 min
and then kept at room temperature for 12 h, and quenched by the addition of water (20 mL).
The organic layer was washed with 15 mL brine, dried over anhydrous Na2SO4 and filtered, and
then the filtrate was concentrated in vacuo. The resulted residue was chromatographed on silica
gel eluting with n-hexane/ethyl acetate (5/1-2/1) to give the desired compound 2a as faint
yellow solid (2.3 g, 74 %). 1H NMR (300 MHz, DMSO-d6): δ 10.73 (s, 1H), 8.20 (d, J = 8.9 Hz,
1H), 7.70 (d, J = 8.9 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.29 – 7.20 (m, 2H), 3.94 (s, 3H).
5.2.3 5-methoxy-N-(4-methoxyphenyl)-2-nitrobenzamide (2b)
Compound 2b was synthesized from 1 (1.7 g, 8.6 mmol) and 4-methoxyaniline (1.1 g,
8.6 mmol) by the similar manner to that described for 2a as yellow solid (1.5 g, 75 %).1H
NMR (300 MHz, DMSO-d6): δ 9.83 (s, 1H), 8.16 (d, J = 9.1 Hz, 1H), 8.01 (d, J = 7.7 Hz, 1H),
7.26 – 7.12 (m, 3H), 7.07 (d, J = 7.5 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 3.93 (s, 3H), 3.80 (s,
3H).
5.2.4 N-(4-fluorophenyl)-5-methoxy-2-nitrobenzamide (2c)
Compound 2c was synthesized from 1 (1.8 g, 9.1 mmol) and 4-fluoroaniline (1.0 g, 9.1
mmol) by the similar manner to that described for 2a as yellow solid (2.0 g, 75 %). 1H NMR
(300 MHz, DMSO-d6): δ 10.68 (s, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.48
(d, J = 8.8 Hz, 2H), 7.15 (m, 2H), 3.93 (s, 3H).
5.2.5 N-(4-(tert-butyl)phenyl)-5-methoxy-2-nitrobenzamide (2d)
12

Compound 2d was synthesized from 1 (1.8 g, 9.1 mmol) and 4-(tert-butyl)aniline (1.4 g,
9.1 mmol) bythe similar manner to that described for 2a as yellow solid (2.7 g, 90 %). 1H NMR
(300 MHz, DMSO-d6): δ 10.61 (s, 1H), 8.32 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 8.6 Hz, 2H),
7.52-7.47 (m, 3H), 7.22 (dd, J = 9.1, 2.8 Hz, 1H), 3.87 (s, 3H), 1.31 (s, 9H).
5.2.6 N-(3-fluorophenyl)-5-methoxy-2-nitrobenzamide (2e)
Compound 2e was synthesized from 1 (2.0 g, 10.1 mmol) and 3-fluoroaniline (1.1 g, 10.1
mmol) by the similar manner to that described for 2a as yellow solid (1.6 g, 65 %).1H NMR
(300 MHz, DMSO-d6): δ 10.72 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.78 (s, 1H), 7.54 (d, J = 8.2
Hz, 1H), 7.48– 7.7.35 (m, 4H), 3.94 (s, 3H).
5.2.7 5-methoxy-N-(2-methoxyphenyl)-2-nitrobenzamide (2f)
Compound 2f was synthesized from 1 (2.0 g, 10.1 mmol) and 2-methoxyaniline (1.3 g,
10.1 mmol) by the similar manner to that described for 2a as yellow solid (2.7 g, 86 %).1H
NMR (300 MHz, DMSO-d6): δ 10.58 (s, 1H), 8.32 (d, J = 9.6 Hz, 1H), 7.51-7.46 (m, 4H),
7.17 (d, J = 8.2 Hz, 1H), 6.91(s 1H), 3.91(s, 3H), 3.76 (s, 3H).
5.2.8 2-amino-N-(4-chlorophenyl)-5-methoxybenzamide (3a)
A mixture of compound 2a (1.0 g, 3.3 mmol) and Pd/C (10%, 0.1 g) in ethanol (15 mL)
was stirred under 1 atm of H2 at room temperature for 15 h. The reaction was filtered through a
celite pad, and the filtrate was concentrated to give crude product, which was chromatographed
on silica gel eluting with CH2Cl2/MeOH (100/1-60/1) to give the desired compound 3a as white
solid (0.7 g, 80 %). 1H NMR (300 MHz, DMSO-d6) δ 10.11 (s, 1H), 7.74 (d, J = 8.6 Hz, 2H),
7.39 (d, J = 8.6 Hz, 2H), 7.17 (s, 1H), 6.94 (s, 1H), 6.74 (s, 1H), 5.93 (s, 2H), 3.73 (s, 3H).
5.2.9 2-amino-5-methoxy-N-(4-methoxyphenyl)benzamide (3b)
Compound 3b was prepared from 2b (1.1 g, 3.6 mmol) by the similar method to that
1
described for 3a as white solid (0.8 g, 81 %). H NMR (300 MHz, DMSO-d6): δ 9.87 (s, 1H),
7.58 (d, J = 8.9 Hz, 2H), 7.16 (d, J = 2.7 Hz, 1H), 6.96 – 6.86 (m, 3H), 6.71 (d, J = 8.9 Hz, 1H),
5.84 (s, 2H), 3.74 (s, 3H), 3.72 (s, 3H).
5.2.10 2-amino-N-(4-fluorophenyl)-5-methoxybenzamide (3c)
Compound 3c was prepared from 2c (1.0 g, 3.5 mmol) by the similar method to that
described for 3a as white solid (0.6 g, 67 %). 1H NMR (300 MHz, DMSO-d6): δ 10.64 (s, 1H),
7.67 (d, J = 9.0 Hz, 1H), 7.59 – 7.35 (m, 3H), 7.22 (d, J = 9.0, 2H), 6.96 (s, 1H), 3.86 (s, 3H).
5.2.11 2-amino-N-(4-(tert-butyl)phenyl)-5-methoxybenzamide (3d)
Compound 3d was prepared from 2d (0.7 g, 2.1 mmol) by the similar method to that
described for 3a as white solid (0.4 g, 63%). 1H NMR (300 MHz, DMSO-d6): δ 10.46 (s, 1H),
7.93 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 2.8 Hz, 1H), 7.38 (d, J = 8.7 Hz,
1H), 7.22 (s, 1H), 6.01 (s, 2H), 3.87 (s, 3H).
5.2.12 2-amino-N-(3-fluorophenyl)-5-methoxybenzamide (3e)
13

Compound 3e was prepared from 2e (0.8 g, 2.8 mmol) by the similar method to that
1
described for 3a as white solid (0.5 g, 70%). H NMR (300 MHz, DMSO-d6): δ 10.62 (s, 1H),
7.88 (d, J = 9.0 Hz, 2H), 7.66 (m, 1H), 7.51 – 7.38 (m, 5H), 6.96(m, 1H), 5.94(s, 2H), 3.87 (s,
3H).
5.2.13 2-amino-5-methoxy-N-(2-methoxyphenyl)benzamide (3f)
Compound 3f was prepared from 2f (1.0 g, 3.3 mmol) by the similar method to that
described for 3a as white solid (0.4 g, 44%). 1H NMR (300 MHz, DMSO-d6): δ 10.75 (s, 1H),
7.73 (m, 1H), 7.28-7.16 (m, 3H), 7.07 (s, 1H), 7.00 (s, 1H), 6.87 (s, 1H), 3.84 (s, 3H), 3.76 (s,
3H).
5.2.14 ethyl 4-(2-oxopiperidin-1-yl)benzoate (5a)
Compound 4 (5.0 g, 18 mmol) was dissolved in toluene (100 mL), followed by the
addition of piperidin-2-one (3.6 g, 36 mmol), anhydrous potassium phosphate (7.8 g, 36 mmol),
CuI (0.34 g, 1.8 mmol) and N,N’-dimethylethanediamine (0.39 mL, 3.6 mmol). The mixture
o
was degassed and refilled with N2 three times and was heated to 115 C under N2 for 16 h,
cooled, and quenched with water (100 mL). The organics were extracted with ethyl acetate (2 ×
75 mL) and dried with Na2SO4. Purification by silica gel column chromatography
(n-hexane/ethyl acetate, 6/1 to 2/1, as eluent) afforded compound 5a as off-white solid (3.5 g,
1
65 %). H NMR (300 MHz, CDCl3): δ 8.07-8.02 (m, 2H), 7.38-7.33 (m, 2H), 4.36 (q, J = 7.1
Hz, 2H), 3.70-3.65 (m, 2H), 2.60-2.55 (m, 2H), 2.03-1.90 (m, 4H), 1.39 (t, J = 7.1 Hz, 3H).
5.2.15 ethyl 4-(3-oxomorpholino)benzoate (5b)
Compound 5b was prepared by the similar method to that described for 5a as faint yellow
solid (4.0 g, 69%). 1H NMR (300 MHz, CDCl3): δ 8.11(d, J = 9.0 Hz, 2H), 7.48(d, J = 9.0 Hz,
2H), 4.43-4.36 (m, 4H), 4.07(t, J = 4.7 Hz, 2H), 3.83 (t, J = 5.2 Hz, 2H), 1.41(t, J = 7.1 Hz, 3H).
5.2.16 ethyl 4-(2-oxopyridin-1(2H)-yl)benzoate(5c)
Compound 5c was prepared by the similar method to that described for 5a as yellow
solid (2.1 g, 52%).1H NMR (300 MHz, CDCl3): δ 8.38 (m, 1H), 8.13 (m, 1H), 7.95 – 7.77 (m, 2H),
7.44 (m, 1H), 7.39 – 7.11 (m, 2H), 6.06 (m, 1H), 4.31 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H).
5.2.17 ethyl 4-(2-oxopyrrolidin-1-yl)benzoate(5d)
Compound 5d was prepared by the similar method to that described for 5a as white solid
(3.6 g, 71%).1H NMR (300 MHz, CDCl3): δ 8.90 – 8.69 (m, 2H), 8.04 – 7.68 (m, 2H), 4.30 (q, J =
7.2Hz, 2H), 3.79 (m, 2H), 2.45 (m, 2H), 2.18 – 1.87 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H).
5.2.18 4-(2-oxopiperidin-1-yl)benzoic acid (6a)
Compound 5a (3.0 g, 12.1 mmol) was dissolved in methanol (36 mL). To the solution was
added 1 M NaOH (5 mL), and the reaction mixture was stirred at room temperature for 1 h. The
14

mixture was adjusted to pH 5 with 1 M hydrochloric acid. The resulting precipitate was filtered,
washed with H2O/EtOH (6 mL, v/v, 2:1), and dried to afford the desired product which was
pure enough for the next step (2.4 g, 90%). 1H NMR (300 MHz, CDCl3): δ 8.07-8.02 (m, 2H),
7.38-7.33 (m, 2H), 4.36 (q, J = 7.1 Hz, 2H), 3.70-3.65 (m, 2H), 2.60-2.55 (m, 2H), 2.03-1.90 (m,
4H), 1.39 (t, J = 7.1 Hz, 3H).
5.2.19 4-(3-oxomorpholino)benzoic acid (6b)
Compound 6b was prepared by the similar method to that described for 6a as colorless
solid (2.1 g, 81%). 1H NMR (300 MHz, DMSO-d6): δ 12.96 (s, 1H), 7.95 (d, J = 9.0 Hz, 2H),
7.57 (d, J = 9.0 Hz, 2H), 4.23 (s, 2H), 3.98 (t, J = 4.6 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H).
5.2.20 N-(4-chlorophenyl)-5-methoxy-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide (7a)
To a solution of 6a (95 mg, 0.43 mmol) and EDCI (91 mg, 0.48 mmol) in DMF (2 mL) at
0 °C was added 3a (120 mg, 0.43 mmol) and DIPEA (91 µL, 0.52 mmol) in DMF (1 mL).
The mixture was stirred for 24 h at room temperature. The reaction was quenched by addition
of cold water (3 mL). The resulting precipitate was filtered, washed with water (5 mL), and
dried to afford the crude product which was purified by chromatography on silica gel with
1
CH2Cl2/MeOH (30:1) afforded 7a as colorless solid (170 mg, 82%). H NMR (300MHz,
DMSO-d6): δ 11.23 (s, 1H), 10.89 (s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 8.5 Hz, 2H),
7.81 (d, J = 8.8 Hz, 2H), 7.43 (dd, J = 16.5, 8.8 Hz, 5H), 7.18 (dd, J = 9.0, 2.8 Hz, 1H), 3.87
(s, 3H), 3.66 (t, J = 5.2 Hz, 2H), 2.42 (t, J = 6.0 Hz, 2H), 1.97 – 1.75 (m, 4H); 13C NMR (75
MHz, DMSO -d6): δ 169.03, 166.80, 164.04, 155.03, 146.30, 137.72, 131.81, 131.21, 128.41,
127.56, 127.46, 125.81, 123.98, 122.52, 117.53, 115.63, 113.92, 55.60, 50.31, 32.66, 22.87,
20.75; MS (ESI) m/z: 500.2 [M+H]+; Anal. calcd. for C26H24ClN3O4: C, 65.34; H, 5.06; N,
8.79. Found: C, 65.31; H, 5.08; N, 8.76.
5.2.21 5-methoxy-N-(4-methoxyphenyl)-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide
(7b)
Compound 7b was prepared by the similar method to that described for 7a as colorless
solid (112 mg, 90%). mp: 236-239 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.47 (s, 1H), 10.40
(s, 1H), 8.35 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2H), 7.46 (dd, J
= 8.1, 5.7 Hz, 3H), 7.21 (dd, J = 9.1, 2.8 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 3.86 (s, 3H), 3.75
(s, 3H), 3.66 (t, J = 5.3 Hz, 2H), 2.43 (t, J = 6.1 Hz, 2H), 1.94 – 1.76 (m, 4H); 13C NMR (75
MHz, DMSO-d6): δ 169.02, 166.57, 163.77, 156.02, 154.80, 146.52, 131.74, 131.66, 131.30,
127.31, 125.89, 124.46, 123.11, 122.83, 117.36, 113.77, 55.55, 55.17, 50.28, 32.66, 22.87,
20.76; MS (ESI) m/z: 496.2 [M+Na]+; Anal. calcd. for C27H27N3O5: C, 68.49; H, 5.75; N, 8.87.
Found: C, 68.41; H, 5.78; N, 8.82.
5.2.22 N-(4-fluorophenyl)-5-methoxy-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide (7c)
Compound 7c was prepared by the similar method to that described for 7a as colorless
1
solid (114 mg, 83%). mp: 212-215 °C; H NMR (300 MHz, DMSO-d6): δ 11.00 (s, 1H),
10.64 (s, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.67 (dd, J = 11.6 Hz, 2.1 Hz,
1H), 7.57 – 7.32 (m, 5H), 7.22 (dd, J = 9.0, 2.9 Hz, 1H), 6.96 (td, J = 8.4, 2.2 Hz, 1H), 3.86 (s,
3H), 3.66 (t, J = 5.3 Hz, 2H), 2.43 (t, J = 6.1 Hz, 2H), 1.97 – 1.78 (m, 4H); 13C NMR (75
15

MHz, DMSO-d6): δ 169.03, 166.74, 163.92, 154.94, 146.50, 134.84, 131.74, 131.39, 127.38,
125.86, 125.05, 123.55, 122.98, 122.87, 117.35, 115.36, 115.07, 113.93, 55.56, 50.29, 32.66,
22.87, 20.76; MS (ESI) m/z: 484.2 [M+Na]+; Anal. calcd. for C26H24FN3O4: C, 67.67; H, 5.24;
N, 9.11. Found: C, 67.63; H, 5.28; N, 9.14.
5.2.23 N-(4-(tert-butyl)phenyl)-5-methoxy-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide
(7d)
Compound 7d was prepared by the similar method to that described for 7a as colorless
solid (88 mg, 90%). mp: 238-242 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.39 (s, 1H), 10.46
(s, 1H), 8.32 (d, J = 9.0 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 7.46 (dd, J
= 11.7 Hz, 5.6 Hz, 3H), 7.38 (d, J = 8.6 Hz, 2H), 7.22 (dd, J = 9.1, 2.8 Hz, 1H), 3.86 (s, 3H),
3.66 (s, 2H), 2.42 (t, J = 6.0 Hz, 2H), 1.86 (d, J = 2.6 Hz, 4H), 1.28 (s, 9H); 13C NMR (75
MHz, DMSO-d6): δ 169.00, 166.77, 163.80, 154.82, 146.65, 146.52, 135.79, 131.70, 131.54,
127.32, 125.88, 125.23, 124.74, 123.23, 120.90, 117.30, 113.96, 55.55, 50.27, 33.96 32.66,
31.13, 22.87, 20.77; MS (ESI) m/z: 522.3 [M+Na]+; Anal. calcd. for C30H33N3O4: C, 72.12; H,
6.66; N, 8.41. Found: C, 72.17; H, 6.58; N, 8.46.
5.2.24 N-(3-fluorophenyl)-5-methoxy-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide (7e)
Compound 7e was prepared by the similar method to that described for 7a as colorless
solid (91 mg, 69%). mp: 202-205 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.98 (s, 1H), 10.62
(s, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.88 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2.1 Hz, 1H),
7.52 – 7.37 (m, 5H), 7.20 (m, 1H), 6.94 (m, 1H), 3.86 (s, 3H), 3.65 (t, J = 5.3 Hz, 2H), 2.42
(t, J = 6.1 Hz, 2H), 1.85 (m, 4H); 13C NMR (75 MHz, DMSO-d6): δ 169.04, 166.87, 164.05,
155.22, 146.41, 140.29, 131.60, 130.84, 130.20, 127.47, 125.84, 124.10, 117.15, 116.26,
113.99, 110.66, 107.08, 55.57, 50.30, 32.66, 22.87, 20.77; MS (ESI) m/z: 484.2 [M+H]+; Anal.
calcd. for C26H24FN3O4: C, 67.67; H, 5.24; N, 9.11. Found: C, 67.72; H, 5.28; N, 9.13.
5.2.25 5-methoxy-N-(2-methoxyphenyl)-2-(4-(2-oxopiperidin-1-yl)benzamido)benzamide
(7f)
Compound 7f was prepared by the similar method to that described for 7a as colorless
solid (96 mg, 79%). mp: 221-224 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.49 (s, 1H), 9.77 (s,
1H), 8.26 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 9.0 Hz, 1H), 7.47 (dd, J =
8.1, 5.7 Hz, 3H), 7.21 (m, 2H), 6.95 (m, 2H), 3.85 (s, 3H), 3.70 (s, 3H), 3.64 (s, 2H), 2.42 (s,
2H), 1.86 (s, 4H); 13C NMR (75 MHz, DMSO-d6): δ 169.03, 166.78, 163,45, 156.13, 150.80,
146.89, 134.71, 132.56, 128.31, 127.67, 125.93, 125.87, 125.45, 123.83, 123.45, 121.78,
120.22, 115.36, 112.87, 55.55, 55.27, 50.29, 32.66, 22.85, 21.76; MS (ESI) m/z: 496.2
[M+Na]+; Anal. calcd. for C27H27N3O5: C, 68.49; H, 5.75; N, 8.87. Found: C, 68.43; H, 5.72;
N, 8.84.
5.2.26 N-(4-chlorophenyl)-5-methoxy-2-(4-(3-oxomorpholino)benzamido)benzamide (7g)
Compound 7g was prepared by the similar method to that described for 7a as colorless
solid (54 mg, 50%). mp: 202- 204 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.28 (s, 1H), 10.50
(s, 1H), 8.23 (dd, J = 22.8, 9.0 Hz, 1H), 8.01 – 7.86 (m, 2H), 7.74 (dd, J = 11.9, 8.4 Hz, 2H),
7.62 (dd, J = 8.6, 1.9 Hz, 2H), 7.41 (dd, J = 6.8, 4.9 Hz, 2H), 7.27 – 7.07 (m, 2H), 4.25 (s,
2H), 4.08 – 3.94 (m, 2H), 3.86 (d, J = 1.4 Hz, 3H), 3.83 – 3.71 (m, 2H); 13C NMR (75 MHz,
DMSO-d6): δ 166.81, 166.12, 163.74, 154.97, 144.43, 138.48, 132.04, 131.29, 128.61, 128.52,
127.60, 127.55, 124.91, 122.37, 121.02, 117.32, 113.97, 67.73, 63.38, 55.56, 48.44; MS (ESI)
16

m/z: 478.1 [M-H]+; Anal. calcd. for C25H22ClN3O5: C, 62.57; H, 4.62; N, 8.76. Found: C,
62.51; H, 4.68; N, 8.81.
5.2.27 5-methoxy-N-(4-methoxyphenyl)-2-(4-(3-oxomorpholino)benzamido)benzamide (7h)
Compound 7h was prepared by the similar method to that described for 7a as colorless
solid (89 mg, 42%). mp: 191- 193 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.54 (s, 1H), 10.53
(s, 1H), 8.33 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.63 (dd, J = 8.6, 6.3 Hz, 4H), 7.49
(d, J = 2.6 Hz, 1H), 7.20 (dd, J = 9.1, 2.8 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 4.24 (s, 2H), 3.99
(s, 2H), 3.87 (s, 3H), 3.84 – 3.77 (m, 2H); 13C NMR (75 MHz, DMSO-d6): δ 166.56, 166.13,
163.67, 155.98, 154.87, 144.43, 132.08, 131.59, 131.37, 127.50, 124.91, 124.63, 123.23,
122.85, 117.44, 113.80, 113.73, 67.73, 63.38, 55.57, 55.17, 48.43; MS (ESI) m/z: 498.2
[M+Na]+; Anal. calcd. for C26H25N3O6: C, 65.68; H, 5.30; N, 8.84. Found: C, 65.65; H, 5.81;
N, 8.87.
5.2.28 N-(4-(tert-butyl)phenyl)-5-methoxy-2-(4-(3-oxomorpholino)benzamido)benzamide
(7i)
Compound 7i was prepared by the similar method to that described for 7a as colorless
1
solid (87 mg, 63%). mp: 208-211 °C; H NMR (300MHz, DMSO-d6): δ 11.42 (s, 1H), 10.46
(s, 1H), 8.33 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 8.6 Hz, 4H), 7.45 (d, J
= 2.8 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.22 (dd, J = 9.1, 2.9 Hz, 1H), 4.25 (s, 2H), 3.99 (dd,
J = 5.9, 4.0 Hz, 2H), 3.87 (s, 3H), 3.83 – 3.76 (m, 2H), 1.28 (s, 9H); 13C NMR (75 MHz,
DMSO-d6): δ 166.77, 166.11, 163.68, 154.86, 146.66, 144.44, 135.79, 132.02, 131.50, 127.49,
125.23, 124.90, 124.79, 123.26, 120.90, 117.28, 113.98, 67.73, 63.37, 55.54, 48.42, 34.06,
31.13; MS (ESI) m/z: 524.3 [M+Na]+; Anal. calcd. for C29H31N3O5: C, 69.44; H, 6.23; N, 8.38.
Found: C, 69.49; H, 6.19; N, 8.32.
5.2.29 5-methoxy-N-(2-methoxyphenyl)-2-(4-(3-oxomorpholino)benzamido)benzamide (7j)
Compound 7j was prepared by the similar method to that described for 7a as colorless
solid (101 mg, 61%). mp: 203-206 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.57 (s, 1H), 9.81 (s,
1H), 8.28 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 6.8 Hz, 1H), 7.61 (d, J = 8.6
Hz, 2H), 7.54 (d, J = 2.4 Hz, 1H), 7.31 – 7.16 (m, 2H), 7.07 (d, J = 7.5 Hz, 1H), 6.99 (t, J = 7.6
Hz, 1H), 4.24 (s, 2H), 4.06 – 3.93 (m, 2H), 3.86 (s, 3H), 3.83 – 3.75 (m, 2H), 3.71 (s, 3H); 13C
NMR (75 MHz, DMSO-d) δ 166.51, 166.11, 163.97, 155.24, 151.84, 144.49, 131.83, 131.32,
127.56, 126.38, 126.04, 124.97, 124.89, 123.93, 120.22, 117.89, 113.60, 111.43, 67.73, 63.36,
55.54, 55.50, 48.42; MS (ESI) m/z: 498.2 [M+H]+; Anal. calcd. for C26H25N3O6: C, 65.68; H,
5.30; N, 8.84. Found: C, 65.60; H, 5.35; N, 8.87.
5.2.30 N-(4-chlorophenyl)-5-methoxy-2-(4-(2-oxopyridin-1(2H)-yl)benzamido)benzamide(7k
)
Compound 7k was prepared by the similar method to that described for 7a as faint yellow
solid (76 mg, 46%). mp: 215-218 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.54 (s, 1H), 10.50 (s,
1H), 8.30 (m, 1H), 8.13 (m, 1H), 7.81 – 7.65 (m, 5H), 7.56 (m 1H), 7.31 (m, 5H), 6.08 (m, 1H),
3.85 (s, 3H); 13C NMR (75 MHz, DMSO-d) δ 166.58, 166.70, 165.82, 160.16, 144.92, 139.17,
138.48, 136.72, 136.46, 134.70, 130.37, 128.67, 128.23, 125.66, 123.46, 123.25, 122.88,
121.71, 121.23, 115.24, 110.25, 56.18; MS (ESI) m/z: 474.1 [M+H]+; Anal. calcd. for :
C26H20ClN3O4: C, 65.90; H, 4.25; N, 8.87; Found: C, 65.81; H, 4.30; N, 8.81.
17

5.2.31 N-(4-chlorophenyl)-5-methoxy-2-(4-(2-oxopyrrolidin-1-yl)benzamido)benzamide(7l)
Compound 7l was prepared by the similar method to that described for 7a as white yellow
solid (91 mg, 74%). mp: 187-191 °C; 1H NMR (300 MHz, DMSO-d6): δ 10.92 (s, 1H), 10.60 (s,
1H), 8.10 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2.1 Hz, 1H), 7.50 –
7.33(m, 5H), 7.18 (m, 1H), 6.92 (m, 1H), 3.89 – 3.65 (m, 5H), 2.45 (m, 2H), 2.30 (m, 2H); 13C
NMR (75 MHz, DMSO-d6): δ 170.16, 166.80, 164.82, 155.62, 146.20, 140.20, 131.51, 130.62,
130.41, 127.32, 125.75, 124.32, 117.20, 116.31, 113.63, 110.76, 108.14, 55.68, 45.14, 31.46,
18.87; MS (ESI) m/z: 464.1 [M+H]+; Anal. calcd. for : C26H20ClN3O4: C, 64.73; H, 4.78; N,
9.06; Found: C, 64.79; H, 4.70; N, 8.76.
5.2.32 tert-butyl 3-bromo-4-oxopiperidine-1-carboxylate (9)
To a solution of N-(tert-Butoxycarbonyl)-4-piperidone (8 g, 48.2 mmol) in CH2Cl2 (80
mL) was added dropwise a solution of bromine (7.06 g, 44.17 mmol) in CH2Cl2 (80 mL) over
a period of 1 h. After addition, the reaction mixture was stirred for 0.5 h at room temperature.
The reaction mixture was quenched by the addition of 10% aqueous sodium bisulfite. The
layers were separated, and the organic layer was washed with water and brine, dried over
anhydrous Na2SO4, and filtered, and then the filtrate was concentrated in vacuo. The residue
was recrystallized from ethyl acetate/petroleum (16 mL, v/v, 1:1) to afford 9 as colorless solid
(4.5 g, 41%). 1H NMR (300 MHz, CDCl3): δ 4.35 (br, 1H), 4.03 (br, 2H), 3.50 (br, 2H), 3.03 (br,
1H), 2.43 (m, 1H), 1.52 (s, 9H).
5.2.33 5-(tert-butyl) 2-ethyl 6,7-dihydrothiazolo[5,4-c]pyridine-2,5(4H)-dicarboxylate (10)
Compound 9 (4.0 g, 14.4 mmol) was dissolved in IPA (25 mL). Ethyl thioxamate (1.60 g,
12.0 mmol) and NaHCO3 (2.01 g, 24.0 mmol) were added. The mixture was refluxed for 12 h,
followed by concentration. The residue was taken in water and extracted with
dichloromethane. The organic layer was washed with water, brine and dried over anhydrous
Na2SO4 and concentrated to the desired compound (2.2 g, 60%), which is pure enough for the
1
next step. mp: 138-140 °C; H NMR (300 MHz, CDCl3): δ 4.71(s, 2H), 4.48(q, J = 12Hz, 2H,),
3.72(m, 2H), 2.96(t, J = 5.4Hz, 2H), 2.41(t, J = 6.3Hz, 2H), 1,51(s, 9H), 1,44(t, J = 1.2Hz, 3H,); MS
(ESI) m/z: 335.1 [M+Na]+.
5.2.34 Ethyl 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylate (11)
To a solution of 10 (2.23 g, 7.1 mmol) in AcOEt (15 mL) was added trifluoroacetic acid
(1.6 mL, 21.3 mmol). The mixture was stirred at room temperature for 3 h. Then, the solution
was evaporated, and the residue was triturated with Et2O, filtrated and dried in vacuo to give
compound 11 as a paint yellow solid (1.62 g, 91%), which is pure enough for the next step. mp:
1
148-151 °C; H NMR (300 MHz, CDCl3): δ 4.32 (s, 2H), 4.21 (m, 5H), 3.32 (s, 2H), 2.76 (s, 2H),
1.41 (s, 2H), 1.32 (s, 3H); MS (ESI) m/z: 213.2 [M+H]+.
5.2.35 Ethyl 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylate (12)
To a solution of 11 (1.6 g, 7.1 mmol) and triethyl amine (1.5 mL, 10.6 mmol eq) in CH2Cl2
(15 mL) was added iodomethane (0.48 mL, 7.8 mmol) and the reaction mixture was stirred for
o
l8 h at 30 C. Reaction mixture was then diluted with CH2Cl2 (10 mL) and washed with water.
Organic layer was then separated, dried and concentrated under reduced pressure to afford the
18

desired compound (1.4 g, 88%). 1H NMR (300 MHz, CDCl3,): δ 4.91-4.78 (m, 2H), 4.49 (d, J
= 7.1 Hz, 2H) 3.99-3.80 (m, 2H), 3.09-3.00 (m, 2H), 2.46 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H).
5.2.36 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylic acid (13)
Compound 12 (1.3 g, 5.8 mmol) was dissolved in methanol (10 mL). To this solution was
added 1 M NaOH (5 mL), and the reaction mixture was stirred at 0 oC for 1.5 h. The mixture
was adjusted to pH 4 with 0.5 M hydrochloric acid. The methanol was removed in vacuo, and
the resulting precipitate was filtered, washed with H2O/EtOH (9 mL, v/v, 2:1), and dried to
afford the desired product. 1H-NMR (DMSO-d6): δ2.36 (3H, s), 2.63-2.76 (4H, m), 3.54 (2H,
s).
5.2.37 N-(4-ethynylphenyl)-5-methoxy-2-nitrobenzamide (14a)
Compound 14a was prepared by the similar method to that described for 2a as yellow solid
(0.56 g, 75%). 1H NMR (300 MHz, DMSO-d6): δ 10.75 (s, 1H), 8.20 (d, J = 9.1 Hz, 1H), 7.77 –
7.67 (m, 3H), 7.31 – 7.17 (m, 1H), 7.16 (d, J = 8.1 Hz, 2H), 3.94 (s, 3H), 2.55 (s, 1H).
5.2.38 5-methoxy-N-(3-methoxyphenyl)-2-nitrobenzamide (14b)
Compound 14b was prepared bythe similar method to that described for 2a as faint yellow
solid (0.51 g, 67%). 1H NMR (300 MHz, DMSO-d6): δ 10.54 (s, 1H), 8.19 (d, J = 9.6 Hz, 1H),
7.35 (s, 1H), 7.31 – 7.16 (m, 4H), 6.70 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.75 (s, 3H).
5.2.39 5-methoxy-2-nitro-N-(p-tolyl)benzamide (14c)
Compound 14c was prepared by the similar method to that described for 2a as faint yellow
solid (0.61 g, 83%). 1H NMR (300 MHz, DMSO-d6): δ 10.46 (s, 1H), 8.18 (d, J = 10.0 Hz, 1H),
7.54 (d, J = 8.4 Hz, 2H), 7.27 – 7.20 (m, 2H), 7.16 (d, J = 7.8 Hz, 2H), 3.94 (s, 3H), 2.28 (s, 3H).
5.2.40 N-(5-fluoropyridin-2-yl)-5-methoxy-2-nitrobenzamide (14d)
Compound 14d was prepared by the similar method to that described for 2a as tan solid
(0.43 g, 32 %). 1H NMR (300 MHz, DMSO-d6): δ 11.22 (s, 1H), 8.36 (d, J = 2.2 Hz, 1H), 8.28
– 8.14 (m, 2H), 7.82 (td, J = 8.9, 3.1 Hz, 1H), 7.26 – 7.17 (m, 2H), 3.93 (s, 4H).
5.2.41 5-methoxy-N-(5-methylpyridin-2-yl)-2-nitrobenzamide (14e)
Compound 14e was prepared by the similar method to that described for 2a as yellow solid
(0.48 g, 38 %). 1H NMR (300 MHz, DMSO-d6): δ 11.00 (s, 1H), 8.22 – 8.11 (m, 2H), 8.08 (d, J
= 8.5 Hz, 1H), 7.72 – 7.56 (m, 1H), 7.29 – 7.13 (m, 2H), 3.93 (s, 3H), 2.28 (s, 3H).
5.2.42 N-(5-chloropyridin-2-yl)-5-methoxy-2-nitrobenzamide (14f)
Compound 14f was prepared by the similar method to that described for 2a as yellow solid
(0.37 g, 28 %). 1H NMR (300 MHz, DMSO-d6): δ 11.30 (s, 1H), 8.40 (s, 1H), 8.19 (t, J = 8.2 Hz,
2H), 8.06 – 7.87 (m, 1H), 7.24 (s, 1H), 7.20 (d, J = 2.5 Hz, 1H), 3.93 (s, 3H).
5.2.43 N-(3-chlorophenyl)-5-methoxy-2-nitrobenzamide (14g)
Compound 14g was prepared by the similar method to that described for 2a as faint yellow
solid (0.63 g, 81 %). 1H NMR (300 MHz, DMSO-d6): δ 10.78 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H),
19

7.56 – 7.15 (m, 5H), 3.94 (s, 3H).
5.2.44 N-(5-chlorothiophen-2-yl)-5-methoxy-2-nitrobenzamide (14h)
Compound 14h was prepared by the similar method to that described for 2a as tan solid
(0.25 g, 52 %). 1H NMR (300 MHz, DMSO-d6): δ 10.65 (s, 1H), 8.05 (d, J = 9.1 Hz, 1H), 8.01
(s, 1H), 7.64 (s, 1H), 7.14 (dd, J = 9.0, 2.8 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 3.90 (s, 3H).
5.2.45 2-amino-N-(4-ethynylphenyl)-5-methoxybenzamide (15a)
Compound 15a was prepared by the similar method to that described for 3a as faint yellow
solid (0.32 g, 65 %). 1H NMR (300 MHz, DMSO-d6): δ 9.92 (s, 1H), 7.86 – 7.69 (m, 1H), 7.69
– 7.49 (m, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.16 (s, 2H), 6.90 (d, J = 7.5 Hz, 1H), 6.82 – 6.51 (m,
1H), 5.85 (s, 2H), 3.73 (s, 3H), 2.58 (s, 1H).
5.2.46 2-amino-5-methoxy-N-(3-methoxyphenyl)benzamide (15b)
Compound 15b was prepared by the similar method to that described for 3a as faint yellow
solid (0.51 g, 89 %). 1H NMR (300 MHz, DMSO-d6): δ 9.96 (s, 1H), 7.38 (s, 1H), 7.35 – 7.20
(m, 2H), 7.15 (s, 1H), 6.95 – 6.85 (m, 1H), 6.76 – 6.61 (m, 2H), 5.83 (s, 2H), 3.74 (s, 3H), 3.73
(s, 3H).
5.2.47 2-amino-5-methoxy-N-(p-tolyl)benzamide (15c)
Compound 15c was prepared by the similar method to that described for 3a as faint yellow
solid (0.45 g, 85 %). 1H NMR (300 MHz, DMSO-d6): δ 9.91 (s, 1H), 7.57 (d, J = 8.1 Hz, 2H),
7.24 – 7.04 (m, 3H), 6.90 (dd, J = 8.9, 2.6 Hz, 1H), 6.71 (d, J = 9.2 Hz, 1H), 5.84 (s, 2H), 3.72
(s, 3H), 2.28 (s, 4H).
5.2.48 2-amino-N-(5-fluoropyridin-2-yl)-5-methoxybenzamide (15d)
Compound 15d was prepared by the similar method to that described for 3a as faint yellow
solid (0.32 g, 72 %). 1H NMR (300 MHz, DMSO-d6): δ 10.67 (s, 1H), 8.38 (d, J = 9.0 Hz, 1H),
8.12 (m, 1H), 7.77 (m, 1H), 7.28 (d, J = 8.9 Hz, 1H), 6.95 – 6.84 (m, 1H), 6.73 (d, J = 8.5 Hz,
1H), 6.00 (s, 2H), 3.73 (s, 3H).
5.2.49 2-amino-5-methoxy-N-(5-methylpyridin-2-yl)benzamide (15e)
Compound 15e was prepared by the similar method to that described for 3a as faint yellow
solid (0.21g, 76 %). 1H NMR (300 MHz, DMSO-d6): δ 10.47 (s, 1H), 8.20 (s, 1H), 7.99 (d, J =
8.3 Hz, 1H), 7.63 (dd, J = 8.3, 2.0 Hz, 1H), 7.28 (d, J = 2.7 Hz, 1H), 6.88 (dd, J = 8.8, 2.7 Hz,
1H), 6.72 (d, J = 8.8 Hz, 1H), 5.98 (s, 2H), 3.73 (s, 3H), 2.28 (s, 3H).
5.2.50 2-amino-N-(5-chloropyridin-2-yl)-5-methoxybenzamide (15f)
Compound 15f was prepared by the similar method to that described for 3a as faint yellow
solid (0.19 g, 63%). 1H NMR (300 MHz, DMSO-d6): δ 10.74 (s, 1H), 8.42 (d, J = 2.5 Hz, 1H),
8.15 (d, J = 9.0 Hz, 1H), 7.93 (dd, J = 9.0, 2.7 Hz, 1H), 7.28 (d, J = 2.8 Hz, 1H), 6.91 (dd, J = 8.9,
2.8 Hz, 1H), 6.78 – 6.70 (m, 1H), 6.03 (s, 2H), 3.74 (s, 3H).
5.2.51 2-amino-N-(3-chlorophenyl)-5-methoxybenzamide (15g)
Compound 15g was prepared by the similar method to that described for 3a as faint yellow
20

solid (0.32 g, 86%). 1H NMR (300 MHz, DMSO-d6): δ 10.20 (s, 1H), 7.95 (s, 1H), 7.69 (s, 1H),
7.42 (s, 1H), 7.32 – 7.11 (m, 2H), 7.00 (s, 1H), 6.79 (s, 1H), 5.93 (s, 2H), 3.78 (s, 3H).
5.2.52 2-amino-N-(5-chlorothiophen-2-yl)-5-methoxybenzamide (15h)
Compound 15h was prepared by the similar method to that described for 3a as faint yellow
solid (0.15 g, 61%). 1H NMR (300 MHz, DMSO-d6): δ 10.13 (s, 1H), 7.83 (d, J = 8.8 Hz, 1H),
7.62 (s, 1H), 7.33 (s, 1H), 7.02 (m, 1H), 6.89 (d, J = 2.5 Hz, 1H), 5.91 (s, 2H), 3.75 (s, 3H).
5.2.53 N-(4-methoxy-2-(p-tolylcarbamoyl)phenyl)-5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c
]pyridine-2-carboxamide (16a)
To a solution of 15c (100 mg, 0.39 mmol) and EDCI (82 mg, 0.43 mmol) in DMF (2 mL)
at 0 °C was added 13 (77 mg, 0.39 mmol ) and DIPEA (82 µL, 0.47 mmol) in DMF (1 mL).
The mixture was stirred for 48 h at room temperature. The reaction was quenched by addition
of cold water (3 mL). The resulting precipitate was filtered, washed with water (5 mL), and
dried to afford the crude product which was purified by chromatography on silica gel with
CH2Cl2/MeOH (60/1-30/1) afforded 16a as colorless solid (91 mg, 53%). mp: 183-186 °C; 1H
NMR (300 MHz, DMSO-d6): δ11.53 (s, 1H), 10.52 (s, 1H), 8.37 (d, J = 9.1 Hz, 1H), 7.52 –
7.07 (m, 5H), 6.73 (s, 1H), 3.85 (s, 3H), 3.76 (s, 3H), 3.68 (s, 2H), 2.94 – 2.82 (m, 2H), 2.74
(t, J = 5.5 Hz, 2H), 2.38 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 166.28, 160.38, 157.19,
154.94, 150.20, 135.78, 133.56, 133.37, 130.34, 129.03, 124.61, 122.74, 121.17, 117.31,
114.04, 55.54, 51.87, 51.43, 44.45, 26.45, 20.49; MS (ESI) m/z: 459.2 [M + Na]+; Anal. calcd.
for C23H24N4O3S: C, 63.28; H, 5.54; N, 12.83. Found: C, 63.32; H, 5.57; N, 12.88.
5.2.54 N-(4-methoxy-2-((4-methoxyphenyl)carbamoyl)phenyl)-5-methyl-4,5,6,7-tetrahydroth
iazolo[5,4-c]pyridine-2-carboxamide (16b)
Compound 16b was prepared by the similar method to that described for 16a as faint
1
yellow solid (105 mg, 60%). mp: 206-210 °C; H NMR (300 MHz, DMSO-d6): δ 11.63 (s,
1H), 10.48 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 7.56 (d, J = 8.3 Hz, 2H), 7.41 (s, 1H), 7.19 (d, J
= 7.5 Hz, 3H), 3.85 (s, 3H), 3.67 (s, 2H), 2.85 (s, 2H), 2.73 (s, 2H), 2.37 (s, 3H), 2.29 (s, 3H);
13C NMR (75 MHz, DMSO-d6): δ 166.09, 160.39, 157.18, 156.03, 154.92, 150.20, 133.55,
131.25, 130.39, 124.45, 122.81, 122.65, 117.29, 113.96, 113.79, 55.55, 55.18, 51.88, 51.44,
44.46, 26.45; MS (ESI) m/z: 475.1 [M+Na]+; Anal. calcd. for C23H24N4O4S: C, 61.05; H, 5.35;
N, 12.38. Found: C, 61.12; H, 5.41; N, 12.32.
5.2.55 N-(2-((4-ethynylphenyl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahydrothi
azolo[5,4-c]pyridine-2-carboxamide (16c)
Compound 16c was prepared by the similar method to that described for 16a as faint
1
yellow solid (65 mg, 70%). mp: 221-226 °C; H NMR (300 MHz, DMSO-d6): δ 11.64 (s, 1H),
10.49 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 7.72 (s, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.51 (s, 1H), 7.41
(s, 1H), 7.21 (d, J = 7.7 Hz, 2H), 3.85 (s, 3H), 3.67 (s, 2H), 2.85 (s, 2H), 2.73 (s, 2H), 2.58 (d, J
= 7.2 Hz, 1H), 2.37 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ166.28, 158.75, 157.19, 154.93,
150.20, 133.55, 132.24, 130.34, 127.85, 124.51, 122.72, 121.23, 120.70, 117.30, 114.09, 81.24,
75.44, 55.55, 51.87, 51.44, 44.45, 26.44; MS (ESI) m/z: 445.1 [M-H]+; Anal. calcd. for
C24H22N4O3S: C, 64.56; H, 4.97; N, 12.55. Found: C, 64.58; H, 4.92; N, 12.51.
21

5.2.56 N-(4-methoxy-2-((3-methoxyphenyl)carbamoyl)phenyl)-5-methyl-4,5,6,7-tetrahydroth
iazolo[5,4-c]pyridine-2-carboxamide (16d)
Compound 16d was prepared by the similar method to that described for 16a as faint
1
yellow solid (89 mg, 74%). mp: 194-198 °C; H NMR (300 MHz, DMSO-d6): δ 11.56 (s, 1H),
10.49 (s, 1H), 8.37 (d, J = 7.7 Hz, 1H), 7.32 (dd, J = 34.7, 17.4 Hz, 5H), 6.74 (s, 1H), 4.81 (d, J
= 13.8 Hz, 2H), 3.81 (m, 8H), 2.90 (m, 2H), 2.11 (s 3H); 13C-NMR (75 MHz, DMSO-d6): δ
166.25, 160.15, 157.08, 155.03, 151.99, 150.23, 139.59, 139.24, 133.63, 130.12, 129.44,
124.75, 122.88, 117.16, 113.11, 109.74, 106.84, 55.57, 55.04, 51.88, 51.46, 44.42, 26.18; MS
(ESI) m/z: 475.1 [M+Na]+; Anal. calcd. for C23H24N4O4S: C, 61.05; H, 5.35; N, 12.38. Found: C,
61.12; H, 5.37; N, 12.35.
5.2.57 N-(2-((5-fluoropyridin-2-yl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahydr
othiazolo[5,4-c]pyridine-2-carboxamide (16e)
Compound 16e was prepared by the similar method to that described for 16a as faint
1
yellow solid (49 mg, 41%). mp: 178-182 °C; H NMR (300 MHz, DMSO-d6): δ 11.56 (s, 1H),
11.23 (s, 1H), 8.43 (s, 1H), 8.32 (d, J = 8.9 Hz, 1H), 8.12 (d, J = 5.3 Hz, 1H), 7.84 (s, 1H), 7.47
(s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 3.85 (s, 3H), 3.66 (s, 2H), 2.85 (s, 2H), 2.73 (s, 2H), 2.37 (s,
3H); 13C NMR (75 MHz, DMSO-d6): δ 166.98, 160.30, 157.29, 154.98, 150.17, 135.62, 135.29,
133.60, 130.42, 125.38, 125.12, 123.98, 123.06, 118.47, 116.80, 113.90, 55.50, 51.87, 51.43,
44.44, 26.44; MS (ESI) m/z: 464.1 [M+Na]+; Anal. calcd. for C21H20FN5O3S: C, 57.13; H, 4.57;
N, 15.86. Found: C, 57.11; H, 4.56; N, 15.84.
5.2.58 N-(4-methoxy-2-((5-methylpyridin-2-yl)carbamoyl)phenyl)-5-methyl-4,5,6,7-tetrahydr
othiazolo[5,4-c]pyridine-2-carboxamide (16f)
Compound 16f was prepared by the similar method to that described for 16a as faint
1
yellow solid (32 mg, 33%). mp: 186-189 °C; H NMR (300 MHz, DMSO-d6): δ 11.63 (s, 1H),
11.03 (s, 1H), 8.33 (d, J = 9.2 Hz, 1H), 8.25 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 7.9 Hz,
1H), 7.47 (s, 1H), 7.18 (d, J = 8.8 Hz, 1H), 3.86 (s, 3H), 3.68 (s, 2H), 2.87 (s, 2H), 2.75 (s, 2H),
2.38 (s, 3H), 2.30 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 166.98, 160.26, 157.27, 154.95,
150.16, 149.41, 147.74, 138.48, 133.54, 130.46, 129.27, 124.07, 122.93, 118.42, 115.10,
113.76, 55.48, 51.87, 51.43, 44.43, 26.43, 17.31. MS (ESI) m/z: 438.2 [M+H]+; Anal. calcd. for
C22H23N5O3S: C, 60.40; H, 5.30; N, 16.01. Found: C, 60.45; H, 5.32; N, 16.07.
5.2.59 N-(2-((5-chloropyridin-2-yl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahydr
othiazolo[5,4-c]pyridine-2-carboxamide (16g)
Compound 16g was prepared by the similar method to that described for 16a as faint
1
yellow solid (51 mg, 37%). mp: 178-182 °C; H NMR (300 MHz, DMSO-d6): δ 11.50 (s, 1H),
11.29 (s, 1H), 8.47 (s, 1H), 8.29 (d, J = 9.2 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 8.8 Hz,
1H), 7.46 (s, 1H), 7.19 (d, J = 8.3 Hz, 1H), 3.85 (s, 3H), 3.67 (s, 2H), 2.86 (s, 2H), 2.74 (s, 2H),
2.38 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 167.17, 160.27, 157.17, 154.99, 150.37, 150.17,
146.38, 137.86, 133.62, 130.36, 126.02, 124.09, 123.09, 118.55, 116.43, 113.94, 55.50, 51.88,
51.43, 44.45, 26.45; MS (ESI) m/z: 458.1 [M+H]+; Anal. calcd. for C21H20ClN5O3S: C, 55.08; H,
4.40; N, 15.29. Found: C, 55.05; H, 4.46; N, 15.34.
22

5.2.60 N-(2-((3-chlorophenyl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahydrothia
zolo[5,4-c]pyridine-2-carboxamide (16h)
Compound 16h was prepared by the similar method to that described for 16a as faint
1
yellow solid (62 mg, 68%). mp: 201-204 °C; H NMR (300 MHz, DMSO-d6): δ 11.42 (s, 1H),
10.67 (s, 1H), 8.32 (s, 1H), 7.84 (s, 1H), 7.64 (s, 1H), 7.40 (s, 2H), 7.22 (s, 2H), 3.85 (s, 3H),
3.67 (s, 2H), 2.86 (s, 2H), 2.74 (s, 2H), 2.37 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 167.17,
160.28, 157.29, 155.08, 150.35, 150.15, 139.98, 137.87, 133.66, 130.35, 126.02, 124.85,
123.17, 120.39, 117.50, 116.43, 114.20, 55.58, 51.86, 51.42, 44.45, 26.43; MS (ESI) m/z: 479.1
[M+Na]+; Anal. calcd. for C22H21ClN4O3S: C, 57.83; H, 4.63; N, 12.26. Found: C, 57.89; H,
4.68; N, 12.21.
5.2.61 N-(2-((4-chlorophenyl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahydrothia
zolo[5,4-c]pyridine-2-carboxamide (16i)
Compound 16i was prepared by the similar method to that described for 16a as colorless
solid (87 mg, 74%). mp: 194-197 °C; 1H NMR (300 MHz, DMSO-d6): δ 11.49 (s, 1H), 10.64 (s,
1H), 8.35 (d, J = 9.1 Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 2.8
Hz, 1H), 7.22 (dd, J = 9.2, 2.8 Hz, 1H), 3.85 (s, 3H), 3.68 (s, 2H), 2.86 (t, J = 5.1 Hz, 2H), 2.74
(t, J = 5.6 Hz, 2H), 2.38 (s, 3H); 13C NMR (75 MHz, DMSO-d6): δ 166.47, 160.31, 157.22,
155.00, 150.18, 137.40, 133.56, 130.18, 128.58, 127.91, 124.71, 123.00, 122.53, 117.44,
114.18, 55.57, 51.86, 51.42, 44.44, 26.42; MS (ESI) m/z: 479.1 [M+H]+; Anal. calcd. for
C22H21ClN4O3S: C, 57.83; H, 4.63; N, 12.26. Found: C, 57.81; H, 4.66; N, 12.29.
5.2.62 N-(2-((5-chlorothiophen-2-yl)carbamoyl)-4-methoxyphenyl)-5-methyl-4,5,6,7-tetrahy
drothiazolo[5,4-c]pyridine-2-carboxamide (16j)
Compound 16j was prepared by the similar method to that described for 16a as colorless
1
solid (32 mg, 22%). mp: 166-170 °C; H NMR (300MHz, DMSO-d6): δ 1H NMR (300 MHz,
DMSO) δ 11.98 (s, 1H), 11.60 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 7.44 (s, 1H), 7.23 (s, 1H), 6.96
(s, 1H), 6.75 (s, 1H), 3.85 (s, 3H), 3.72 (s, 2H), 2.91 (s, 2H), 2.80 (s, 2H), 2.41 (s, 3H); 13C
NMR (75 MHz, DMSO-d6): δ 164.27, 161.38, 157.29, 155.01, 151.88, 150.08, 137.25, 133.28,
130.65, 123.52, 123.23, 120.10, 117.80, 113.92, 111.64, 55.68, 51.80, 51.32, 44.29, 40.33,
40.05, 39.77, 39.50, 39.22, 38.94, 26.36; MS (ESI) m/z: 461.1 [M-H]+; Anal. calcd. for
C20H19ClN4O3S2: C, 51.89; H, 4.14; N, 12.10. Found: C, 51.82; H, 4.20; N, 12.17.
5.3 Biology
5.3.1 In vitro enzyme assays.
In vitro enzymatic activity against human fIIa, fVIIa, fIXa, fXa and trypsin was
measured using chromogenic or fluorogenic substrates 17. The final enzyme concentrations
were 0.003 U/mL (human fXa from Enzyme Research Laboratories) and 0.125 U/mL (human
thrombin fVIIa, fIXa, and trypsin from Sigma Chemical Co.). The reagents containing 25µL
of buffer (0.02 M Bis-Tris；0.7M NaCl; pH 6.0),10 µL compound dilutions and enzyme were
mixed, centrifuged, and incubated for 30 min at 37 °C in 96-well microtiter plates. The
appropriate substrate was added to initiate enzyme reaction. The time course of the reaction
was monitored continuously for 20 min at 405 nm in Synergy H1 MultMode Reader (BioTek,
USA). The IC50 value was determined by nonlinear regression using GraphPad Prism 5.
23

5.3.2 In Vitro Anticoagulant Activity.
Commercially available kits were used for the measurement of PT and aPTT26. All tested
Compounds or DMSO (3 µL) were added to citrated rabbit plasma containing 3.8% sodium
citrate (100 µL, v/v, 1:9) and incubated for 10 min at 37 °C. Clotting time was recorded using
a coagulometer (A52 Semi-auto Coagulometer 2-channel analyzer) according the
manufacturer’s instructions and each experiment was repeated three times. Anticoagulant
activity was defined as the concentration of inhibitor needed to double PT and aPTT.
5.3.3 In vivo venous thrombosis model assay.
The protocol for this assay was approved bythe Animal Care and Use Committee of China
Pharmaceutical University (Nanjing, China) and similar to the method reported previously
with minor modifications27, 28. The vena cava was isolated by using a midline abdominal
incision, and the surface was cleared by blunt dissection between the renal and iliolumbar veins.
Male rats (n = 10 per dose group) were treated orally with 16g or betrixaban suspended in
0.5% carboxymethylcellulose sodium (CMC-Na) solution at 1, 5 and 10 mg/kg or vehicle w
60 min before thrombus formation. A 1.5×2.5 mm strip of filter paper saturated with 25%
FeCl3 in water was kept on the vena cava for 1.5 min. One hour after application of the filter
paper, the vena cava was dissected free. The thrombus was excised, cleaned in saline, blotted of
excess blood, and weighed.
5.3.4 In vivo AV-shunt model assays
The protocol for this test was approved by the Animal Care and Use Committee of China
Pharmaceutical University (Nanjing, China) and similar to the method reported previously
with slightly modifications16, 27, 28. The right common carotid artery and left jugular vein were
cannulated with 80 mm long, saline-filled polyethylene tube. Then the tube was connected
with a 50 mm long polyethylene tube containing rough nylon thread (50 × 0.15 mm) which
was folded into a double string. Male rats (n = 10 per dose group) were treated orally with
16g or betrixaban dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution or
vehicle was treated orally 90 min before the shunt was opened for 15 min. The nylon thread
was withdrawn rapidly, cleaned in saline, dried and weighed.
5.3.5 Tail-Bleeding Model
Betrixaban, 16g or vehicle was treated orally 90 min before the tails of anesthetized rats
according the reported protocols with minor modifications26. Compound 16g, betrixaban or
vehicle was given orally 60 min before the tails of anesthetized rats were transected 4 mm from
the tip and immersed vertically immediately into saline at 37 °C. The time until continuous
blood flow ceased for >30 s was recorded with a maximum observation time of 30 min. The use
of rats in this study was approved by the Animal Care and Use Committee of China
Pharmaceutical University (Nanjing, China).
5.3.6 Pharmacokinetic Studies
Compounds 16g (5% DMSO + 5% Tween-80 in 90% saline) and betrixaban (10% DMSO
+ 10% Tween-80 + 80% PEG400 (40% solution in saline)) were selected to PK studies on male
SD rats (200−225 g) with five animals in each group28. Test compounds were administered via
24

the oral route at 3 mg/kg or administered via the intravenous route at 1 mg/kg. Blood samples
were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48 h after the dose via posterior orbital
venous plexus and were into microcentrifuge tubes containing heparin sodium. Then blood
samples were centrifuged at 3,000 rpm for 10 min to separate plasma. Serial specimens (0.4 mL)
−mass
were collected via the retrobulbar vein and quantified by liquid chromatography
spectrometry (LC-MS). PK parameters were calculated from the mean plasma concentration by
noncompartmental analyses of DAS 3.2.8 statistical software. The protocol for this study was
reviewed and approved by the Animal Care and Use Committee of China Pharmaceutical
University (Nanjing, China).
5.3.7 MTT assay (Cell Viability Assay)
H9C2 cell viability was measured by cell proliferation Kit (Sigma, St. Louis, MO)
according the manufacturer’s instructions. The MTT cell viability OD value was detected.
Acknowledgment
This research was supported by the Natural Science Foundation of Jiangsu Province (No.
BK 20141349) and the China National Key Hi-tech Innovation Project for the R&D of Novel
Drugs (No.2013ZX09301303-002).
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Highlights
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A series of anthranilamide derivatives were designed and synthesized.
Most of the compounds had significant fXa inhibitoy activity.
16g displayed great in vitro and in vivo antithrombotic activity and low bleeding risk.
16g exhibited satisfactory PK profile.
16g was found to improve hypoxia–reoxygenation-induced H9C2 cell viability.
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27