Synthesis and structure-activity relationship of potent, selective and orally active anthranilamide-based factor Xa inhibitors: Application of weakly basic sulfoximine group as novel S4 binding element

Article abstract of DOI:10.1016/j.ejmech.2012.10.005

A novel series of potent and efficacious factor Xa inhibitors which possesses sulfoximine moiety as novel S4 binding element in anthranilamide chemotype has been identified. Lead optimization at this novel P4 group led to many potent factor Xa inhibitors with excellent anticoagulant activity in human plasma. Selected compounds were dosed orally in rats and checked for their ex vivo prothrombin time prolonging activity, which resulted in identification of compound 5-chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(diethylamino)acetyl)-S- methylsulfonimidoyl)benzamido)benzamide (18f). The detailed pharmacokinetic evaluation and subsequent metabolism study of 18f suggested the presence of an active metabolite. The compound 18f and its active metabolite 18b demonstrated excellent in vivo efficacy in both arterial and venous thrombosis model in rats and were found to be highly selective against related serine proteases. Based on this promising profile, compound 18f was selected for further evaluation.

Full text of DOI:10.1016/j.ejmech.2012.10.005

European Journal of Medicinal Chemistry 58 (2012) 136e152
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationship of potent, selective and orally active
anthranilamide-based factor Xa inhibitors: Application of weakly basic
sulfoximine group as novel S4 binding elementq
,
b
,
a
a
a
a
a
Vrajesh Pandya a , Mukul Jain , Ganes Chakrabarti , Hitesh Soni , Bhavesh Parmar , Balaji Chaugule ,
Jigar Patel a, Tushar Jarag a, Jignesh Joshi a, Nirav Joshi a, Akshyaya Rath a, Vishal Unadkat a,
Bhavesh Sharma a, Haresh Ajani a, Jeevan Kumar a, Kalapatapu V.V.M. Sairam a, Harilal Patel a,
Pankaj Patel a
*
a Zydus Research Centre, Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382210, India
b Department of Chemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of potent and efficacious factor Xa inhibitors which possesses sulfoximine moiety as novel
S4 binding element in anthranilamide chemotype has been identified. Lead optimization at this novel P4
group led to many potent factor Xa inhibitors with excellent anticoagulant activity in human plasma.
Selected compounds were dosed orally in rats and checked for their ex vivo prothrombin time prolonging
activity, which resulted in identification of compound 5-chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-
(diethylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide (18f). The detailed pharmacoki-
netic evaluation and subsequent metabolism study of 18f suggested the presence of an active metabolite.
The compound 18f and its active metabolite 18b demonstrated excellent in vivo efficacy in both arterial
and venous thrombosis model in rats and were found to be highly selective against related serine
proteases. Based on this promising profile, compound 18f was selected for further evaluation.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 24 May 2012
Received in revised form
4 October 2012
Accepted 4 October 2012
Available online 16 October 2012
Keywords:
Factor Xa
Anticoagulant
Anthranilamide
Sulfoximine
1. Introduction
VTE (Venous Thromboembolism). It affects more than one million
individuals in the European union each year and is responsible for at
Thrombosis is a pathological process and it occurs when the
functioning of hemostatic pathway exceeds the normal regulatory
counterbalance by fibrinolytic pathway or other anticoagulant
factors, which are supposed to restrict and localize thrombus
formation to the area of injury. Occlusions of coronary and cerebral
arteries are associated with platelet activation and coagulation
triggered by the rupture of atherosclerotic plaque, resulting in
myocardial infarction and ischemic stroke [1]. There is a significant
association between myocardial ischemia commonly known as ACS
(Acute Coronary Syndrome) and AF (Atrial Fibrillation), one form of
cardiac arrhythmia and patients with AF are at higher risk of
cerebral stroke [2].
least 500,000 deaths [3]. Platelets play a major role in arterial
thrombosis which is a leading cause of MI (Myocardial Infarction)
and stroke, and antiplatelet therapy improves survival of patients
with this arterial thrombotic disorders [4]. Anticoagulants hold
promise in both arterial and venous thrombotic disorders as they
prevent fibrin formation required for stable clot formation. The
drawbacks of warfarin, an oral anticoagulant has provoked extensive
research for identification of safe and orally bioavailable anticoagu-
lant which can fulfill the high unmet need of oral anticoagulants.
The discovery process for oral anticoagulant has been domi-
nated by directly targeting two specific protease enzymes, coagu-
lation factor Xa (FXa) and thrombin (FIIa). Due to its unique
position in coagulation cascade and limited role outside, FXa has
always been considered to have an edge over thrombin in terms of
safety in bleeding [5,6]. However, race to be the first selective oral
anticoagulant in the market won by Dabigatran etexilate [7],
a thrombin inhibitor. Enormous research activity in developing
novel orally bioavailable Factor Xa inhibitor resulted in several
clinical candidates, which are listed in Fig. 1.
Venous thrombotic occlusions are associated with DVT (Deep
Vein Thrombosis) and PE (Pulmonary Embolism), which comprises
q
ZRC communication: 387.
* Corresponding author. Zydus Research Centre, Sarkhej-Bavla N.H. 8A, Moraiya,
Ahmedabad 382210, India. Tel.: þ91 2717 250801; fax: þ91 2717 250606.
E-mail address: vrajeshpandya@zyduscadila.com (V. Pandya).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.005

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
137
to prepare sulfoximine fragment. Controlled oxidation of
compounds 6aed using H2O2 gave sulfoxides 7aed, which upon
treatment with sodium azide and sulfuric acid [26] were converted
into sulfoximine derivatives 8aed. Sulfoximine group was then
protected by reacting it with benzyloxycarbonyl chloride using
pyridine as base to get intermediates 9aed. Alkaline hydrolysis of
9aed gave key acid intermediates 10aed. Commercially available
2-nitrobenzoic acid derivatives 11aec were condensed with 5-
chloro-2-aminopyridine (12) to get intermediates 13aec, which
upon reduction using SnCl2 furnished anilines 14aec. Synthesis of
14d was accomplished by chlorination of 14c using N-chloro suc-
cinimide (NCS). The compounds 15aeg were obtained by con-
verting 10aed into acid chlorides using oxalyl chloride followed by
condensation with appropriate anilines (14aed) and subsequent
deprotection of Cbz group using sulfuric acid or BBr3.
Fig. 1. Oral factor Xa inhibitors.
The compounds 16aee were synthesized as shown in Scheme 2.
Compound 15a was reacted with methanesulfonyl chloride, acetyl
chloride and methoxyacetyl chloride to obtained 16a, 16b and 16c
respectively. Reaction of 15a with cyanogen bromide (BrCN) gave
16d, which after treatment with sulfuric acid produced amide
derivative 16e.
The aminoacyl derivatives (18aes) and aminoalkyl derivatives
(22aec) were synthesized as shown in Scheme 3. Anthranilamide
derivatives 15a, 15e, and 15f were reacted with the chloroacetyl
chloride to get key intermediates 17a, 17e, and 17f respectively. The
displacement of chloro group from 17a, 17e, and 17f with appro-
priately substituted amines gave desired compounds 18aes.
Alternatively, 15a was reacted with methyl bromoacetate using
sodium hydride to get 19. Ester group of 19 was reduced using
NaBH4 to get alcohol derivative 20. The hydroxyl group of 20 was
then converted into leaving group using triphenylphosphine (TPP)
and CBr4 as brominating agent to get the bromo derivative 21. The
compounds 22aec were obtained by displacement of bromo group
from 21 with appropriate amines.
Rivaroxaban (1) is the first oral FXa inhibitor to be approved in
Europe and US for the prevention of VTE in patients who have had
elective total hip or knee replacement surgery [8]. Recently, it has
also been approved by US for stroke prophylaxis in patients with
non-valvular AF. Clinical trial for secondary prevention of ACS is in
progress. Apixaban (2) is approved in Europe for the prevention of
VTE in patients who have had elective total hip or knee replacement
surgery [9]. Edoxaban (3) is approved in Japan for the prevention of
VTE after major orthopedic surgery [10]. Recently, Darexaban (4),
which was in Phase III for the prevention of VTE after major ortho-
pedic surgery is discontinued by Astellas due to commercial reason
[11]. Betrixaban (5) is in Phase II for the treatment of DVTand PE, and
for the prevention of stroke related to chronic AF [12]. Letaxaban
which was in Phase II for VTE prevention in patients underwent knee
replacement surgery is discontinued by Takeda [13].
These newly discovered FXa inhibitors are devoid of a highly
basic amidine group (except Betrixaban), a known culprit for poor
oral bioavailibility and was earlier used successfully as both S1 and
S4 ligand [14]. Vicinal diamide based FXa inhibitors (Anthranilamide
and cis-diamine based inhibitors) have been explored by many
research groups as they provide U or V shape of the molecule, which
is ideal for FXa binding [15e20]. Three molecules based on vicinal
diamide scaffold are already in clinical trials. Edoxaban (3), which is
recently approved in Japan has the 5-chloro-2-aminopyridine group
as S1 ligand and the methyltetrahydrothiazolo[5,4-c]pyridine group
as S4 ligand [21]. Betrixaban (5) has the 5-chloro-2-aminopyridine
group as S1 ligand, but possesses classical amidine group as S4
ligand [22]. Darexaban (4) contains the 4-methoxyphenyl group as
S1 ligand and the 4-methylhomopiperazine group as S4 ligand [11].
Several vicinal diamide-based FXa inhibitors with diversified S4
ligands are reported by many research groups [23e25].
3. Results and discussion
The compounds synthesized were evaluated for their in vitro
inhibitory activity of human FXa, expressed as IC50 or Ki values or %
inhibition at 0.1 mM and their anticoagulant activity in human and
rat plasma was measured as prolongation of prothrombin time (PT),
expressed as the concentration of the compound required to double
the clotting time (PTCT2) in the PT assay. Prediction of oral
bioavailability and simultaneous efficacy of the compounds has
been achieved by measuring ex vivo PT prolonging activity in rats.
PT prolongation was measured at 2 h after oral administration to rat
at a dose of 30 mg/kg and expressed as fold prolongation with
respect to control group.
As a part of our research endeavor to develop viable therapies
for the treatment of thrombotic diseases through inhibition of
coagulation FXa, we envisioned less basic sulfoximine group in
anthranilamide chemotype as S4 binding element, and that works
as novel replacement of highly basic amidine group of Betrixaban.
The optimized 5-chloro-2-aminopyridine group as S1 ligand has
been retained in our designed compounds. In this paper, we
describe the synthesis and structureeactivity relationships for I
(Fig. 2), and identified compound 18f and its active metabolite 18b
as potent and selective FXa inhibitor with excellent in vivo efficacy
and favorable oral bioavailability, thus making it a promising drug
candidate for further evaluation.
3.1. In vitro and ex vivo structureeactivity relationships
Table 1 shows the SAR of initial compounds 15aeg, where the
effect of introduction of the sulfoximine group, effect of alkyl
substituent at S atom and effect of hydrophilicity in the central
phenyl ring was studied.
The simplest sulfoximine compound 15a with methyl substituent
at S atom and chloro substituent at central phenyl ring displayed 76%
inhibition of FXa activity at 0.1 mM. Insertion of methylene group
between phenyl ring and sulfoximine group in 15a was not tolerated
and led to the inactive compound 15b. Increasing alkyl chain at S
atom was also not tolerated as reflected from the data of 15c (S-ethyl)
and 15d (S-hydroxyethyl). We then evaluated SAR at central phenyl
ring of compound 15a (Table 1). Replacement of neutral chloro
substituent in 15a with hydrophilic methoxy substituent produced
2. Chemistry
The synthesis of first set of compounds 15aeg is outlined in
Scheme 1. Ester derivatives 6aed were chosen as starting materials
compound 15e with diminished potency (42% inhibition at 0.1 mM).

138
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
Fig. 2. Anthranilamide derivatives possessing sulfoximine group as novel S4 binding element.
Scheme 1. Reagents and conditions: (a) H2O2, cat. V2O5, acetonitrile, 0e25 ꢀC; (b) NaN3, H2SO4, CHCl3, ꢁ20 ꢀC to 45 ꢀC; (c) ClCOOBn, CH2Cl2, pyridine, 0e25 ꢀC; (d) NaOH, THF, H2O,
25 ꢀC; (e) POCl3, pyridine, acetonitrile, 25 ꢀC; (f) SnCl2, ethyl acetate, 25 ꢀC; (g) NCS, benzene, 60e65 ꢀC; (h) Oxalyl chloride, DCM, 25e30 ꢀC, then 14aed, THF, 25e30 ꢀC, 2 h; (i)
sulfuric acid, 0 ꢀCe25 ꢀC, or BBr3, DCM, ꢁ30 ꢀC to 25 ꢀC, (for 15d).

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
139
Table 1
In vitro FXa inhibitory activity data of compounds 15aeg.
Compound
n
R2
R3
R4
% Inhibitiona at 0.1
mM
15a
15b
15c
15d
15e
15f
0
1
0
0
0
0
0
CH3
CH3
C2H5
CH2CH2OH
CH3
Cl
Cl
Cl
Cl
OCH3
Cl
H
H
H
H
H
H
OCH3
OCH3
76
No inhibition
No inhibition
No inhibition
42
Scheme 2. Reagents and conditions: (a) MeSO2Cl, pyridine, DCM, 0e25 ꢀC (for 16a);
CH3COCl, pyridine, DCM, 0e25 ꢀC (for 16b); CH3OCH2COCl, pyridine, DCM, 0 ꢀCe25 ꢀ
(for 16c); (b) BrCN, DCM, 25e30 ꢀC; (c) Sulfuric acid, 0e30 ꢀC.
C
CH3
CH3
69
15g
No inhibition
a
Inhibitory activity against human FXa. Values shown are the mean of duplicate
Further addition of methoxy substituent in central ring of 15a had no
measurements.
advantage in improving FXa inhibitory activity, which can be seen
from the data of compound 15f (69% inhibition at 0.1
m
M). Removal
lipophilicity and plasma protein binding. Compound 16b with
acetyl substituent showed 61% inhibition of FXa activity at 0.1 M,
but displayed poor anticoagulant activity (PTCT2 M).
Methoxyacetyl derivative 16c with increased polarity showed
improvement in anticoagulant activity (PTCT2 ¼ 3 M). Compound
16d with cyano group showed improved inhibition of FXa (76%
M).
of chloro substituent from 15f produced inactive compound (15g)
suggesting the importance of chloro substituent and its position on
the central phenyl ring.
Next, SAR at N atom of sulfoximine group was evaluated.
Initially, simple substituents were introduced at N atom to get
compounds 16aee (Table 2). Compound 16a with methanesulfonyl
substituent inhibited FXa with slightly lower potency compare to
unsubstituted sulfoximine derivative 15a. However, anticoagulant
activity of 16a in human plasma was found to be much better than
m
7 m
¼
m
inhibition) and appreciable anticoagulant activity (PTCT2 ¼ 2.3
m
Conversion of cyano group of 16d to amide group (16e) was found
to be detrimental for both FXa inhibitory activity (41% inhibition)
and anticoagulant activity (PTCT2 ¼ 4.1
mM).
15a (PTCT2 ¼ 7.2
m
M for 15a vs 2.6
m
M for 16a), which was in line
Taking clue from the difference observed in anticoagulant
activity for compound 16b and 16c, we decided to replace methoxy
group of 16c with several alkylamino substituents to see its effect
with the fact that anticoagulant activity of FXa inhibitors has been
observed to be a function not only of potency, but also of
Scheme 3. Reagents and conditions: (a) chloroacetyl chloride, TEA, THF, 25e30 ꢀC; (b) NHR5R6, DMF, 25e30 ꢀC; (c) NaH, Methyl bromoacetate, DMF, 40 ꢀC; (d) NaBH4, DMSO, 60 ꢀC;
(e) TPP, CBr4, DCM, 25e30 ꢀC.

140
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
Table 2
Table 3
In vitro FXa inhibitory and anticoagulant activity data of compounds 15a, 16aee.
In vitro FXa inhibitory and anticoagulant activity data of compounds 18aes.
R1
% Inhibitiona
PTCT2 using human
b
Compound
at 0.1
m
M
plasma (mM)
15a
16a
16b
16c
16d
16e
H
76
55
61
48
76
41
7.2
2.6
7
3
2.3
4.1
Compound NR5R6
R3
R4
% Inhibitiona PTCT2b using human
SO2Me
COCH3
COCH2OMe
CN
at 0.1
mM
plasma (mM)
CONH2
18a
18b
Cl
Cl
H
H
93
0.77
0.87
a
Inhibitory activity against human FXa. Values shown are the mean of duplicate
measurements.
100
b
Concentration of the compound required to double the clotting time in the PT
assay using human plasma. PTCT2 values shown are the mean of duplicated
measurements.
18c
Cl
H
97
0.95
2.26c
1.2
on anticoagulant potential and subsequently several aminoacyl
derivatives 18aes were synthesized. In vitro FXa inhibitory activity
and anticoagulant activity in human plasma of 18aes are listed in
Table 3. Compound 18a with methylamino substituent showed 93%
18d
18e
Cl
Cl
H
H
100
85
inhibition at 0.1
plasma (PTCT2 ¼ 0.77
PTCT2 ¼ 0.77 M) to ethyl (18b, PTCT2 ¼ 0.87
(18c, PTCT2 ¼ 0.95 M) was well tolerated for both FXa inhibitory
m
M with improved anticoagulant activity in human
M). Increasing alkyl chain from methyl (18a,
M) and to isopropyl
18f
Cl
Cl
H
H
100
79
0.68
1.45
m
m
m
18g
m
and anticoagulant activity as shown in Table 3. Further increase in
bulk with cyclopentylamino substituent (18d) was found to be
detrimental for anticoagulant activity even though FXa inhibitory
18h
18i
18j
18k
18l
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
95
97
45
88
75
0.46
1.73c
NDd
potency was retained (PTCT2 ¼ 2.26
were also well tolerated with comparable anticoagulant activity as
evident from data of 18e (dimethylamino, PTCT2 ¼ 1.2 M) and 18f
(diethylamino, PTCT2 ¼ 0.68 M). However, compound 18g with
diisopropylamino substituent showed decreased anticoagulant
activity (PTCT2 ¼ 1.45 M). Compound 18h with pyrrolidinyl
substituent showed potent FXa inhibitory activity and excellent
M). Ring expansion in 18h (5
mM). Dialkyl amino groups
m
m
2.03c
1.82c
m
anticoagulant activity (PTCT2 ¼ 0.46
m
membered pyrrolidine) to 18i (6 membered piperidine) retained
FXa inhibitory activity, but showed significant reduction in anti-
18m
18n
18o
18p
Cl
H
H
H
69
49
56
2.16c
2.54c
1.44
0.58
coagulant activity (PTCT2 ¼ 0.46
Substitution with morpholine group (18j) resulted in a diminished
activity with only 45% FXa inhibition at 0.1 M. Compound 18k with
mM of 18h vs 1.73 mM of 18i).
Cl
m
OCH3
Cl
4-methylpiperazine substituent showed 88% inhibition of FXa
activity, but failed to show improvement in anticoagulant activity
OCH3 92
OCH3 100
OCH3 97
(PTCT2 ¼ 2.03
(4-methylhomopiperazine derivative) with similar anticoagulant
M). Introduction of additional hydrophilic
mM). Ring expansion in 18k produced compound 18l
18q
18r
Cl
Cl
0.74
0.92
activity (PTCT2 ¼ 1.82
m
substituent in 18i afforded compounds 18m (4-hydroxypiperidine)
and 18n (piperidine-4-carboxamide), with inferior FXa inhibitory
and anticoagulant activity (18m: PTCT2
PTCT2 ¼ 2.54 M).
¼
2.16 mM, 18n:
m
18s
Cl
OCH3 90
0.53
Further refining of FXa inhibition and anticoagulant effect have
been achieved by studying effect of chloro and methoxy substitu-
ents on central phenyl ring, which represent neutral and hydro-
philic substituents respectively and are also frequently used in
anthranilamide compounds.
a
Inhibitory activity against human FXa. Values shown are the mean of duplicate
measurements.
b
Concentration of the compound required to double the clotting time in the PT
assay using human plasma. PTCT2 values shown are the mean of duplicated
measurements unless otherwise indicated.
c
Replacement of chloro substituent in 18f with methoxy
substituent produced compound 18o with reduced potency
Single determination.
Not determined.
d

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
141
Table 5
(56% inhibition at 0.1
m
M, PTCT2 ¼ 1.44
mM), which matches with
In vitro and ex vivo anticoagulant activities of potent factor Xa inhibitors.
our earlier observation for compound 15e. Incorporating additional
methoxy substituent along with chloro substituent has produced
compounds with almost similar FXa inhibitory and anticoagulant
activity, when compared with only chloro substituted compounds
as evident from compounds 18pes (18p vs 18a, 18q vs 18b, 18r vs
18f, and 18s vs 18h).
We then synthesized compounds 22a, 22b and 22c by
replacing aminoacyl group with aminoalkyl group in compounds
18e, 18f and 18h respectively and their biological data is
summarized in Table 4. Compound 22a with dimethyl amino
IC50 (nM)a
Human
PTCT2
Rat PTCT2
Ex vivo rat
b
Compound
PT ratioc
b
18a
18b
18c
18e
18f
18h
18p
18q
18r
18s
22a
1
5.4
2.7
4
0.77
0.81
0.95
1.2
0.68
0.46
0.58
0.74
0.92
0.53
1.14
0.39
1.7
2.1
1.9
1.4
0.8
1.57
0.9
1.15
1.3
1.1
1.1
1.3
1.5
1.6
2.2
1.4
1.1
1.1
1.1
1.1
1.0
NDd
9
2.1
3.2
5.6
2.4
3.4
6.4
11.6
1.6
substituent showed 82% inhibition of FXa activity at 0.1
mM and
moderate anticoagulant activity (PTCT2 ¼ 1.14
m
M). Compounds
2.01
1.5
with diethylamino substituent (22b) and pyrrolidinyl substituent
(22c) showed similar FXa inhibition but their anticoagulant
a
Inhibitory activity against human FXa. IC50 values shown are the mean of
duplicate measurements.
potency decreased significantly (PTCT2 ¼ 1.78
mM for 22b and
b
Concentration of the compound required to double the clotting time in the PT
2.16 M for 22c).
m
assay using human and rat plasma. PTCT2 values shown are the mean of duplicated
measurements.
The compounds selected on the basis of FXa inhibition and
anticoagulant activity from Tables 3 and 4 were evaluated for their
ex vivo PT prolonging activity in rats, in vitro anticoagulant activity
in rat plasma, and IC50 value determination. Results are summa-
rized in Table 5. All tested compounds have their IC50 value for
human FXa inhibition less than 12 nM. They all displayed relatively
inferior anticoagulant activity in rat plasma compare to human
plasma as reflected in their PTCT2 values. However, compound 18f
(IC50 ¼ 2.1 nM) showed balanced anticoagulant activity in both rat
c
The ex vivo PT prolonging activity was determined 2 h after oral administration
to rat at a dose of 30 mg/kg (n ¼ 4).
d
Not determined.
3.2. Profiling studies including in vivo antithrombotic efficacy
Based on ex vivo anticoagulant activity of 18f, it was selected for
further evaluation which includes pharmacokinetics evaluation in
rats, selectivity against related proteases and CYP3A4 inhibition.
However, to the contrary, hydrochloride salt of 18f showed low
plasma levels (less than 100 ng/mL) when dosed orally at 30 mg/kg
in Wistar rats. The lack of correlation between plasma concentra-
tion and ex vivo anticoagulant activity prompted us to search the
possibility for an active metabolite. From thorough metabolism
study, it was found that de-ethylated compound is generated from
18f, which is same to compound 18b (Table 3), further confirmed by
matching UPLC retention time. In a separate study, plasma levels of
compounds 18f and its metabolite 18b were estimated by dosing
hydrochloride salt of 18f orally in rats at 30 mg/kg (Table 6). The
and human plasma (PTCT2 ¼ 0.8
Compound 18e (IC50 ¼ 9 nM) is another compound with similar
anticoagulant activity in both species (PTCT2 ¼ 1.4 M for rat vs
1.2 M for human).
The ex vivo PT prolonging activity in rats was determined at 2 h
mM for rat vs 0.68 mM for human).
m
m
after oral administration to rat at a dose of 30 mg/kg and the data
are summarized in Table 5. Compounds 18f, 18e, and 18c have
shown PT prolongation 2.2, 1.6 and 1.5 fold respectively, while
remaining compounds failed to show significant PT prolongation
which could be due to their poor oral bioavailability.
plasma levels of 18b were found to be excellent (Cmax ¼ 3.28
mg/mL)
in comparison with parent compound 18f. The absorption and
elimination pattern of 18f with simultaneous formation of metab-
olite 18b are shown in Fig. 3. The half life of 18b (4.79 h) justifies
once a day dosage regime and the low peak vs trough ratio in PK
profile may mitigate the bleeding liability associated with this
therapeutic class.
Table 4
In vitro FXa inhibitory and anticoagulant activity data of compounds 22ae22c.
Selectivity against related serine proteases is a decisive criteria
for developing FXa inhibitors and then we examined the effect of
compounds 18f and its metabolite 18b against related serine
proteases. Both the compounds 18f and 18b were found to be more
than 104 fold selective against thrombin, plasmin, trypsin, t-PA and
aPC (Table 7). Additionally, compounds 18f and 18b were checked
for their effect on human cytochrome P450 enzyme (CYP3A4),
which is known to be a primary factor responsible of metabolism of
most drugs. Both the compounds showed low inhibition of CYP3A4
NR5R6
% Inhibitiona
PTCT2 using human
b
Compound
at 0.1
mM
plasma (mM)
at 10 mM (18% for 18f and 27% for 18b).
22a
22b
82
77
1.14
1.78
Table 6
Pharmacokinetic parametersa for compound 18fb and its metabolite 18b.
Compound
Cmax
(
m
g/mL)
Tmax (h)
T1/2 (h)
AUC (0e24)
(h g/mL)
43.51 ꢂ 2.81
m
22c
74
2.16
18f (18b)c
3.28 ꢂ 0.26
2.75 ꢂ 0.47
4.79 ꢂ 0.2
a
a
Inhibitory activity against human FXa. Values shown are the mean of duplicate
Data are expressed as the mean ꢂ SEM.
b
c
measurements.
Monohydrochloride salt was used.
b
Concentration of the compound required to double the clotting time in the PT
Compound 18f was dosed in fasted male Wistar rats at 30 mg/kg po formulated
assay using human plasma. PTCT2 values shown are the mean of duplicated
measurements.
with a Tween-80:PEG:CMC (5:5:90% v/v) and plasma levels of metabolite 18b was
determined (n ¼ 4).

142
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
H
N
O
S
N
O
N
O
S
N
O
O
NH
80
70
60
50
40
30
20
10
0
4500
4000
3500
3000
2500
2000
1500
1000
500
O
O
N
NH
HN
Cl
O
Cl
N
18b (from 18f)
HN
Cl
Cl
18f
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hr)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (hr)
Fig. 3. Absorption and elimination pattern of 18f and its metabolite 18b in fasted male Wistar rats at 30 mg/kg po.
Further, the in vivo antithrombotic efficacy of compound 18f
4. Conclusion
was evaluated in rats using FeCl3-induced arterial and venous
thrombosis model. Dose dependent thrombus weight reduction
was found in both arterial and venous thrombosis model, when
18f was dosed orally at 10 mg/kg and 30 mg/kg in rats (Fig. 4 and
Fig. 5). In an arterial thrombosis model, 18f reduced thrombus
weight by 32% and 71% at 10 mg/kg and 30 mg/kg respectively
(Fig. 4). In venous thrombosis model, reduction in thrombus
weight was 45% and 81% at 10 mg/kg and 30 mg/kg respectively
(Fig. 5). Rivaroxaban (1) was used as positive standard at single
dose of 10 mg/kg. In an arterial thrombosis model, 1 reduced
thrombus weight by 50% and in venous thrombosis model,
reduction in thrombus weight was 63%.
Application of sulfoximine group as novel S4 ligand in anthra-
nilamide chemotype resulted in identification of lead molecule 15a.
Further optimization was achieved by synthesizing several deriv-
atives with substituents at both S and N atom of sulfoximine group.
Biological evaluation of synthesized compounds led to identifica-
tion of compound 18f, which has shown strong human factor Xa
inhibitory activity and anticoagulant activities in both rat and
human plasma. Oral dosing of this compound in rats produced
active metabolite 18b, which was responsible for potent ex vivo
anticoagulant activity of compound 18f. In vivo antithrombotic
efficacy was determined using FeCl3-induced arterial and venous
thrombosis model in rats. Dose dependent reduction in thrombus
weight was observed with the compound 18f. Both the compounds
(18f and its metabolite 18b) were found to be more than 104 fold
selective against related serine proteases and have low effect on
CYP3A4. The PK profile of 18f along with its metabolite showed
fairly long half life and low peak vs trough ratio. Based on these
encouraging data compound 18f was selected for further
evaluation.
3.3. Molecular modeling study
To predict the binding mode of compound 18f and its active
metabolite 18b, a docking study was carried out. Fig. 6 shows
inhibitors 18f (A) and 18b (B) docked in the active site of FXa. The
proposed binding model indicated that the 5-chloro-2-
aminopyridyl moiety deeply occupied the S1 pocket with chlo-
rine atom pointing toward the center of the Tyr228 aromatic ring,
while the substituted sulfoximine group fits in the S4 pocket
formed by the residues Phe174, Trp215, and Tyr99.
5. Experimental section
We have also observed that there are several
interactions in 18f and 18b with Trp215 and Tyr99. These two
molecules form a CHe interaction with Tyr99 with a distance of
3.6 A between aromatic CH and centroid of Tyr99, at the same time
an inclined interaction between Trp215 and phenyl ring
attached to sulfoximine group with a distance of 5.4 A. The eNHe
of ethylamino group attached to sulfoximine group of 18b showed
H-bond interaction with Glu97, which is at the periphery of the S4
site. The model further suggested that the NH group of the amide
bond linked to the 5-chloro-2-aminopyridyl moiety forms H-bond
with Gly218 and likewise, the carbonyl group of the amide bond
connected to the sulfoximine group substituted phenyl ring forms
H-bond with Gly216. Additionally, carbonyl group of amide bond
linked to the sulfoximine group substituted phenyl ring forms a H-
bond with Gln192.
p
e
p
and CHe
p
5.1. Chemistry
p
5.1.1. General methods
ꢀ
Reagents and solvents were obtained from commercial suppliers
and used without further purification. Flash chromatography was
performed using commercial silica gel (100e200 or 230e400 mesh).
Melting points were determined on a capillary melting point appa-
ratus and are uncorrected. IR spectra were recorded on a Shimadzu
FT IR 8300 spectrophotometer (Vmax in cmꢁ1, using KBr pellets, CCl4
or CHCl3). The 1H NMR spectra were recorded on a Brucker Avance-
300 spectrometer (300 MHz) and Brucker Avance-400 spectrometer
pep
ꢀ
(400 MHz). The chemical shifts (d) are reported in parts per million
(ppm) relative to TMS, in either CDCl3 or DMSO-d6 solution. Signal
multiplicities are represented by s (singlet), d (doublet), dd (doublet
of doublet), t (triplet), q (quartet), bs (broad singlet), bd (broad
Table 7
Selectivity profile for compounds 18f and 18b against related serine proteasesa and effect on CYP3A4.
Compound Ki (nM)
FXa
Thrombin
Plasmin
Trypsin
t-PA
aPC
CYP3A4
18f
18b
1.1
1.5
>20000b
>20000b
>20000b
>20000b
>20000b
18% Inhibition at 10
27% Inhibition at 10
m
m
M
M
>20000b
>20000b
>20000b
>20000b
>20000b
a
Inhibitory constant for human enzymes. Ki values shown are the mean of duplicate measurements.
Single determination.
b

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
143
Vehicle treated (5 ml/kg, p.o.)
5.1.1.3. Methyl 4-(ethylsulfinyl)benzoate (7c). This compound was
prepared from 6c by means of the procedure similar to that
reported for 7a as off white solid; yield: 80%; mp 72e74 ꢀC; purity
by HPLC: 91.35%; IR (KBr) 2952, 2933, 1718, 1595, 1488, 1436, 1398,
3.0
2.5
2.0
1.5
1.0
0.5
0.0
18f (10 mg/kg, p.o.)
18f (30 mg/kg, p.o.)
1 (10 mg/kg p.o.)
1278, 1045, 860, 758 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d: 1.00
(t, 3H), 2.74e2.83 (m, 1H), 3.04e3.13 (m, 1H), 3.87 (s, 3H), 7.78
(d, J ¼ 8.4 Hz, 2H), 8.12 (d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 212.9
(M þ H)þ.
*
*
*
5.1.1.4. Methyl4-((2-methoxyethyl)sulfinyl)benzoate (7d). This compound
was prepared from 6d by means of the procedure similar to that
reported for 7a as off white solid; yield: 72%; mp 60e63 ꢀC; purity by
UPLC: 94.50%; IR (KBr) 2842, 1724, 1595, 1571, 1442, 1396, 1280, 1195,
Fig. 4. Effect of 18f (monohydrochloride) and
1 on FeCl3-induced carotid artery
1
1172, 1047, 860, 758 cmꢁ1; H NMR (DMSO-d6, 400 MHz)
d: 2.94e3.0
thrombus weight after 2 h of oral administration in male Wistar rats (n ¼ 10). *p < 0.01
vs vehicle control, ANOVA followed by Dunnett’s test.
(m, 1H), 3.19e3.22 (m, 1H), 3.24 (s, 3H), 3.53e3.58 (m, 1H), 3.68e3.74
(m, 1H), 3.87 (s, 3H), 7.82 (d, J ¼ 8.4 Hz, 2H), 8.12 (d, J ¼ 8.4 Hz, 2H);
ESI/MS m/z 242.9 (M þ H)þ.
doublet), and m (multiplet). 13C NMR spectra were recorded on
Brucker Avance-400 at 100 MHz in DMSO-d6 solution. Mass spectra
(ESI-MS) were obtained on Shimadzu LC-MS 2010-A spectrometer.
Purity of compounds were determined by Ultra Performance Liquid
Chromatography (UPLC) (Column, BEH C-18, 2.1 ꢃ 100 mm; UV
detection, 220 nm; eluent, 0.05% TFA buffer:ACN (gradient); flow
rate, 0.4 mL/min) or by HPLC analysis (column ODS C-18,
5.1.1.5. Methyl 4-(S-methylsulfonimidoyl)benzoate (8a). To a stirring
solution of 7a (6.8 g, 0.034 mol) in chloroform (10 v/w) at 25 ꢀC was
added sodium azide (6.63 g, 0.102 mol). To this was added sulfuric
acid (20 g, 0.204 mol) at ꢁ20 to ꢁ25 ꢀC under nitrogen atmosphere
in 20e30 min. The reaction mixture was stirred at 25e30 ꢀC for
12 h and then at 45e50 ꢀC for 3 h. Chloroform was removed from
reaction mixture and remaining residue was made alkaline by
using aqueous potassium carbonate solution. Ethyl acetate was
added to it and organic layer was separated out. The organic phase
was dried over sodium sulfate and evaporated to afford the title
compound 8a (6.75 g, 92%) as white solid: mp 113e115 ꢀC; purity by
HPLC: 97.48%; IR (KBr) 3280, 2999, 2923, 1703, 1595, 1573, 1438,
150 nm ꢃ 4.6 nm ꢃ 4
m on AGILENT 1100 series; UV detection,
220 nm; eluent, 0.05% TFA buffer:ACN (gradient); flow rate,
1 mL/min) purity of all tested compounds was found to be >95%.
5.1.1.1. Methyl 4-(methylsulfinyl)benzoate (7a). To a stirring solution
of methyl 4-(methylthio)benzoate (6a) (10 g, 0.0548 mol) and V2O5
(100 mg, 0.0004 mol) in acetonitrile (10 v/w) at ꢁ20 to ꢁ25 ꢀC under
nitrogen atmosphere, 50% hydrogen peroxide (2.05 g, 0.0604 mol)
was added drop wise to the above solution in 20e30 min. The
reaction mixture was stirred at 25e30 ꢀC for 5 h. Reaction mixture
was diluted with water (30 v/w) and extracted with ethyl acetate
(30 v/w). The organic phase was dried over sodium sulfate and
evaporated to afford the title compound 7a (9.5 g, 88%) as white
solid: mp 120e122 ꢀC; purity by HPLC: 94.19%; IR (KBr) 2991, 2952,
1398, 1298, 1220, 1097, 1008, 856, 756 cmꢁ1 1H NMR (DMSO-d6,
;
400 MHz)
d
: 3.10 (s, 3H), 3.89 (s, 3H), 4.42 (s, 1H), 8.06 (d, J ¼ 8.4 Hz,
2H), 8.14 (d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 213.9 (M þ H)þ.
5.1.1.6. Methyl 4-((S-methylsulfonimidoyl)methyl)benzoate (8b).
This compound was prepared from 7b by means of the proce-
dure similar to that reported for 8a as off white solid; yield: 65%;
mp 131e133 ꢀC; purity by HPLC: 91.68%; IR (KBr) 3255, 2956,
1718, 1595, 1429, 1400, 1373, 1274, 1045, 860, 758 cmꢁ1 1H NMR
;
2914, 1718, 1612, 1573, 1510, 1436, 1417, 1280, 864, 705 cmꢁ1
;
1H
(DMSO-d6, 400 MHz)
d
: 2.78 (s, 3H), 3.87 (s, 3H), 7.83 (d, J ¼ 8.4 Hz,
2H), 8.12 (d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 198.9 (M þ H)þ.
NMR (DMSO-d6, 400 MHz)
d: 2.77 (s, 3H), 3.72 (s, 1H), 3.84
(s, 3H), 4.42e4.50 (q, J ¼ 12.8 Hz, 2H), 7.57 (d, J ¼ 8 Hz, 2H), 7.94e
7.96 (dd, J ¼ 2.0 and 6.8 Hz, 2H); ESI/MS m/z 227.7 (M þ H)þ.
5.1.1.2. Methyl 4-((methylsulfinyl)methyl)benzoate (7b). This compound
was prepared from 6b by means of the procedure similar to that re-
ported for 7a as off white solid; yield: 52%; mp 80e83 ꢀC; purity by
HPLC: 95.35%; IR (KBr) 2999, 2949, 1718, 1610, 1575, 1508, 1434, 1280,
5.1.1.7. Methyl 4-(ethylsulfonimidoyl)benzoate (8c). This compound
was prepared from 7c by means of the procedure similar to that
reported for 8a as off white solid; yield: 80%; mp 77e80 ꢀC; purity
by HPLC: 93.33%; IR (KBr) 3267, 2956, 2916, 1720, 1593, 1571, 1434,
1396, 1296, 1217, 972, 866, 731 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
1182, 1031, 862, 709 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 2.48 (s, 3H),
3.84 (s, 3H), 4.03 (d, J ¼ 12.8 Hz, 1H), 4.24 (d, J ¼ 12.4 Hz, 1H), 7.45
(d, J ¼ 8.4 Hz, 2H), 7.94e7.96 (dd, J ¼ 1.6 and 6.4 Hz, 2H); ESI/MS m/z
212.8 (M þ H)þ.
d
: 1.04 (t, J ¼ 7.2 Hz, 3H), 3.13e3.18 (q, J ¼ 7.2 Hz, 2H), 3.88 (s, 3H),
4.40 (s, 1H), 8.01 (d, J ¼ 8.4 Hz, 2H), 8.14 (d, J ¼ 8.4 Hz, 2H); ESI/MS
m/z 228 (M þ H)þ.
Vehicle treated (5 ml/kg, p.o.)
15
18f (10 mg/kg, p.o.)
18f (30 mg/kg, p.o.)
1 (10 mg/kg p.o.)
5.1.1.8. Methyl 4-(2-methoxyethylsulfonimidoyl)benzoate (HCl salt)
(8d). This compound was prepared from 7d by means of the
procedure similar to that reported for 8a. Crude product was stirred
with HCl:diethyl ether to obtained hydrochloride salt of title
compound as off white solid; yield: 67%; mp 143e145 ꢀC; purity by
UPLC: 95.16%; IR (KBr) 3441, 1732, 1573, 1550, 1438, 1282, 1114, 864,
10
*
*
5
*
0
734 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 2.99 (s, 3H), 3.65e3.69
(m, 2H), 3.90 (s, 3H), 4.06e4.13 (m, 2H), 8.13e8.26 (m, 4H); ESI/MS
m/z 257.9 (M þ H)þ.
5.1.1.9. Methyl 4-(N-((benzyloxy)carbonyl)-S-methylsulfonimidoyl)
benzoate (9a). To a stirring solution of 8a (2 g, 0.0093 mol) in
dichloromethane (5 v/w) was added pyridine (1.1 g, 0.0139 mol).
Fig. 5. Effect of 18f (monohydrochloride) and 1 on partial stasis combined with FeCl3-
induced venous thrombus weight after 2 h of oral administration in male Wistar rats
(n ¼ 10). *p < 0.01 vs vehicle control, ANOVA followed by Dunnett’s test.

144
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
Fig. 6. Docking study of 18f (A) and 18b (B) in the active site of FXa depicting the surface of FXa.
To this was added benzyloxycarbonyl chloride (50% in toluene)
(1.9 g, 0.0116 mol) at 15e20 ꢀC under nitrogen atmosphere. The
reaction mixture was stirred at 25e30 ꢀC for 3 h and then diluted
with dichloromethane (10 v/w). The organic phase was separated,
dried over sodium sulfate and evaporated to afford the title
compound 9a (3.1 g, 95%) as off white solid: mp 141e143 ꢀC; purity
by UPLC: 98.13%; IR (KBr) 1718, 1668, 1573, 1496, 1286, 1255, 1219,
means of the procedure similar to that reported for 9a; yield: 85%; mp
116e118 ꢀC; purity by HPLC: 96.13%; IR (KBr) 1720, 1666, 1610, 1573,
1500, 1288, 1209, 1114, 869, 785 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d
: 3.19 (s, 3H), 3.85 (s, 3H), 4.97e5.06 (m, 4H), 7.30e7.38 (m, 5H), 7.54
(d, J ¼ 8 Hz, 2H), 7.95 (d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 384 (M þ Na)þ.
5.1.1.11. Methyl 4-(N-((benzyloxy)carbonyl)ethylsulfonimidoyl)benzoate
(9c). This compound was prepared from 8c by means of the proce-
dure similar to that reported for 9a; yield: 82%; purity by HPLC:
94.98%; IR (KBr) 2976, 2943, 1720, 1658, 1575, 1498, 1382, 1271, 1087,
1085, 896, 752 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 3.52 (s, 3H),
3.91 (s, 3H), 4.88e4.97 (q, J ¼ 12 Hz, 2H), 7.19e7.21 (m, 2H), 7.27e
7.33 (m, 3H), 8.06e8.09 (dd, J ¼ 2 and 6.8 Hz, 2H), 8.16e8.18
(dd, J ¼ 2 and 6.8 Hz, 2H); ESI/MS m/z 369.9 (M þ Na)þ.
860, 752 cmꢁ1
;
1H NMR (DMSO-d6, 400 MHz)
d
: 1.09 (t, J ¼ 7.2 Hz,
3H), 3.58e3.69 (m, 2H), 3.91 (s, 3H), 4.87e4.97 (q, J ¼ 12.4 Hz, 2H),
7.18e7.20 (m, 2H), 7.28e7.37 (m, 3H), 8.03 (d, J ¼ 8.4 Hz, 2H), 8.18
(d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 362 (M þ H)þ.
5.1.1.10. Methyl
4-((N-((benzyloxy)carbonyl)-S-methylsulfonimidoyl)
methyl)benzoate (9b). This compound was prepared from 8b by

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
145
5 . 1. 1. 1 2 . M e t h y l 4 - ( N - ( ( b e n z y l o x y ) c a r b o n y l ) - 2 -
methoxyethylsulfonimidoyl)benzoate (9d). This compound was
prepared from 8d by means of the procedure similar to that re-
ported for 9a; yield: 88%; mp 95e97 ꢀC; purity by UPLC: 92.66%; IR
(DMSO-d6, 400 MHz)
d
: 7.80e7.83 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.92
(s, 1H), 7.96e7.99 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.16e8.19 (dd, J ¼ 4.0
and 8.8 Hz, 2H), 8.41 (s,1H), 11.43 (s,1H); ESI/MS m/z 309.8 (M ꢁ H).
(KBr) 3020, 1728, 1678, 1517, 1400, 1215, 1116, 759, 669 cmꢁ1
;
1H
5.1.1.18. N-(5-Chloropyridin-2-yl)-5-methoxy-2-nitrobenzamide
(13b). This compound was prepared from 11b by means of the
procedure similar to that reported for 13a as off white solid; yield:
94%; mp 155e158 ꢀC; purity by UPLC: 97.65%; IR (KBr) 1691, 1579,
1517, 1461, 1377, 1234, 1110, 1070, 835, 750 cmꢁ1; 1H NMR (DMSO-
NMR (DMSO-d6, 400 MHz)
d: 2.98 (s, 3H), 3.60e3.67 (m, 2H), 3.91
(s, 3H), 3.94e3.99 (m, 2H), 4.89e4.98 (q, J ¼ 12.4 Hz, 2H), 7.19e7.21
(m, 2H), 7.27e7.30 (m, 3H), 8.02e8.04 (dd, J ¼ 2.0 and 6.8 Hz, 2H),
8.13e8.15 (dd, J ¼ 2.0 and 6.8 Hz, 2H); ESI/MS m/z 391.8 (M þ H)þ.
d6, 400 MHz) d: 3.91 (s, 3H), 7.19e7.24 (m, 2H), 7.96e7.99 (dd,
5.1.1.13. 4-(N-((Benzyloxy)carbonyl)-S-methylsulfonimidoyl)benzoic
acid (10a). To a stirring solution of sodium hydroxide (0.518 g,
0.01296 mol) in the solvent mixture of 15 mL water and 15 mL THF
was added 9a (3 g, 0.00864 mol). Reaction mixture was stirred at
25e30 ꢀC for 3 h and then diluted with water (10 v/w). Aqueous
layer was washed with methyl tert-butyl ether (10 v/w). Aqueous
layer was then cooled to 0 ꢀC and acidified with diluted HCl.
Aqueous layer was extracted with ethyl acetate, which on drying
over sodium sulfate and evaporation afforded title compound
(2.6 g, 90%) as off white solid: mp 172e174 ꢀC; purity by HPLC:
95.20%; IR (KBr) 3435, 1693, 1664, 1602, 1575, 1375, 1272, 1228,
J ¼ 2.4 and 8.8 Hz, 1H), 8.16e8.22 (m, 2H), 8.40 (d, J ¼ 1.6 Hz, 1H),
11.30 (s, 1H); ESI/MS m/z 329.8 (M þ Na)þ.
5.1.1.19. N-(5-Chloropyridin-2-yl)-3-methoxy-2-nitrobenzamide
(13c). This compound was prepared from 11c by means of the
procedure similar to that reported for 13a as off white solid; yield:
97%; mp 198e200 ꢀC; purity by UPLC: 99.22%; IR (KBr) 1691, 1577,
1537, 1473, 1375, 1307, 1276, 1058, 852, 792 cmꢁ1; 1H NMR (DMSO-
d6, 400 MHz)
d
: 3.92 (s, 3H), 7.44 (d, J ¼ 7.6 Hz, 1H), 7.53
(d, J ¼ 8.4 Hz, 1H), 7.66 (t, J ¼ 8 Hz, 1H), 7.94e7.96 (dd, J ¼ 2.4 and
8.8 Hz, 1H), 8.07 (d, J ¼ 8.8 Hz, 1H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.47
(s, 1H); ESI/MS m/z 306.7 (M ꢁ H).
1087, 746, 698 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 3.51 (s, 3H),
4.89e4.97 (q, J ¼ 12.4 Hz, 2H), 7.19e7.21 (m, 2H), 7.25e7.33 (m, 3H),
8.06 (d, J ¼ 8.8 Hz, 2H), 8.16 (d, J ¼ 8.4 Hz, 2H),13.56 (bs,1H); ESI/MS
m/z 333.8 (M þ H)þ.
5.1.1.20. 2-Amino-5-chloro-N-(5-chloropyridin-2-yl)benzamide
(14a). To a stirring solution of 13a (5 g, 0.016 mol) in 50 mL ethyl
acetate at 25e30 ꢀC was added stannous chloride dihydrate (18 g,
0.08 mol). After stirring at same temperature for 2 h, reaction
mixture was diluted with ethyl acetate (10 v/w) and made alkaline
with aqueous ammonia solution. Reaction mixture was then
filtered through hyflow bed and organic layer was dried over
sodium sulfate. Evaporation of solvent afforded 14a (3.2 g, 71%) as
pale yellow solid; mp 182e184 ꢀC; purity by UPLC: 99.22%; IR (KBr)
5.1.1.14. 4-((N-((Benzyloxy)carbonyl)-S-methylsulfonimidoyl)methyl)
benzoic acid (10b). This compound was prepared from 9b by means
of the procedure similar to that reported for 10a; yield: 82%; mp
180e183 ꢀC; purity by HPLC: 95.55%; IR (KBr) 3444, 1676, 1641,
1427, 1382, 1259, 1103, 790, 696 cmꢁ1
400 MHz)
;
1H NMR (DMSO-d6,
: 3.19 (s, 3H), 4.96 (s, 2H), 4.98e5.06 (q, J ¼ 12.4 Hz, 2H),
d
7.30e7.38 (m, 5H), 7.50 (d, J ¼ 8 Hz, 2H), 7.93 (d, J ¼ 8 Hz, 2H);
3489, 3377, 1658, 1614, 1571, 1373, 1294, 1087, 850, 746 cmꢁ1
;
1H
: 6.54 (s, 2H), 6.78 (d, J ¼ 8.8 Hz, 1H),
ESI/MS m/z 370 (M þ Na)þ.
NMR (DMSO-d6, 400 MHz)
d
7.20e7.23 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.76 (d, J ¼ 2.4 Hz, 1H), 7.90e
7.93 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.08e8.10 (dd, J ¼ 0.4 and 8.8 Hz,
1H), 8.412e8.419 (dd, J ¼ 0.4 and 2.8 Hz, 1H), 10.78 (s, 1H); ESI/MS
m/z 303.7 (M þ Na)þ.
5.1.1.15. 4-(N-((Benzyloxy)carbonyl)ethylsulfonimidoyl)benzoic acid
(10c). This compound was prepared from 9c by means of the
procedure similar to that reported for 10a; yield: 84%; purity by
HPLC: 97.91%; IR (KBr) 3435, 1720, 1633, 1500, 1456, 1392, 1294,
1228, 1085, 902, 734 cmꢁ1
;
1H NMR (DMSO-d6, 400 MHz)
d: 1.07
5.1.1.21. 2-Amino-N-(5-chloropyridin-2-yl)-5-methoxybenzamide
(14b). This compound was prepared from 13b by means of the
procedure similar to that reported for 14a as off white solid; yield:
75%; mp 69e71 ꢀC; purity by UPLC: 99.16%; IR (KBr) 3462, 3367,
(t, J ¼ 7.6 Hz, 3H), 3.58e3.67 (m, 2H), 4.88e4.98 (q, J ¼ 12.4 Hz, 2H),
7.18e7.20 (dd, J ¼ 2.0 and 7.6 Hz, 2H), 7.25e7.32 (m, 3H), 7.98 (d,
J ¼ 8.4 Hz, 2H), 8.15 (d, J ¼ 8.8 Hz, 2H); ESI/MS m/z 347.9 (M þ H)þ.
1658, 1593, 1571, 1514, 1375, 1298, 1159, 1091, 856, 748 cmꢁ1
;
1H
5.1.1.16. 4-(N-((Benzyloxy)carbonyl)-2-methoxyethylsulfonimidoyl)
benzoic acid (10d). This compound was prepared from 9d by means
of the procedure similar to that reported for 10a as oily compound;
yield: 73%; purity by UPLC: 97.37%; IR (KBr) 3433, 1718, 1637, 1500,
1388, 1286, 1230, 1105, 1082, 898, 796, 731 cmꢁ1; 1H NMR (DMSO-
NMR (DMSO-d6, 400 MHz) d: 3.72 (s, 3H), 6.01 (s, 2H), 6.73
(d, J ¼ 8.8 Hz, 1H), 6.87e6.90 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 7.20
(d, J ¼ 2.8 Hz, 1H), 7.90e7.93 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.11e8.14
(dd, J ¼ 0.4 and 8.8 Hz, 1H), 8.40e8.41 (dd, J ¼ 0.8 and 2.8 Hz, 1H),
10.73 (s, 1H); ESI/MS m/z 277.8 (M þ H)þ.
d6, 400 MHz) d: 3.00 (s, 3H), 3.57e3.68 (m, 2H), 3.90e4.01 (m, 2H),
4.90e4.98 (q, J ¼ 12.4 Hz, 2H), 7.20e7.32 (m, 5H), 8.02 (d, J ¼ 8.4 Hz,
5.1.1.22. 2-Amino-N-(5-chloropyridin-2-yl)-3-methoxybenzamide
(14c). This compound was prepared from 13c by means of the
procedure similar to that reported for 14a as off white solid; yield:
60%; mp 137e139 ꢀC; purity by UPLC: 98.34%; IR (KBr) 3493, 3367,
2H), 8.14 (d, J ¼ 8.4 Hz, 2H); ESI/MS m/z 377.8 (M þ H)þ.
5.1.1.17. 5-Chloro-N-(5-chloropyridin-2-yl)-2-nitrobenzamide (13a).
To a stirring solution of 11a (5 g, 0.02487 mol) and 2-amino-5-
chloro pyridine (12) (3.2 g, 0.02487 mol) in 20 mL acetonitrile at
25e30 ꢀC was added pyridine (5.9 g, 0.0746 mol). The reaction
mixture was then cooled to 0e10 ꢀC under nitrogen atmosphere. To
this was added POCl3 (4.57 g, 0.0298 mol) drop wise by maintaining
exothermicity. After stirring at 25e30 ꢀC for 1 h, reaction mixture
was poured in cooled water and filtered. Solid obtained was stirred
in saturated solution of sodium bicarbonate for 10 min. Filtration
and drying afforded title compound 13a (7 g, 90%) as pale yellow
solid: mp 182e183 ꢀC; purity by HPLC: 96.28%; IR (KBr) 1691, 1575,
1525, 1461, 1375, 1299, 1114, 1016, 914, 842, 761,678 cmꢁ1; 1H NMR
1658, 1612, 1587, 1552, 1500, 1373, 1226, 1080, 846, 740 cmꢁ1
;
1H
NMR (DMSO-d6, 400 MHz)
d: 3.80 (s, 3H), 6.14 (s, 2H), 6.55
(t, J ¼ 8 Hz, 1H), 6.96 (t, J ¼ 6.8 Hz, 1H), 7.37e7.40 (dd, J ¼ 1.2 and
8.4 Hz, 1H), 7.90e7.93 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.10e8.12
(dd, J ¼ 0.4 and 9.2 Hz, 1H), 8.40e8.41 (dd, J ¼ 0.8 and 2.8 Hz,
1H), 10.54 (s, 1H); ESI/MS m/z 277.9 (M þ H)þ.
5.1.1. 23. 2-Amino-5-chloro-N-(5-chloropyridin-2-yl)-3-
methoxybenzamide(14d). To a stirring solution of 14c (15.5 g, 0.0558 mol)
in 155 mL benzene at 25e30 ꢀC was added NCS (8.17 g, 0.0614 mol).
Reaction mixture was then heated at 60e65 ꢀC for 24 h. Excess solvent

146
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
was removed under vacuum. Water was added to it and product was
extracted with ethyl acetate. Ethyl acetate was dried over sodium sulfate
and evaporated to afford the title compound14d (12.3 g, 71%) as off white
solid: mp 116e118 ꢀC; purity by UPLC: 98.15%; IR (KBr) 3485, 3367, 1712,
1666, 1593, 1573, 1519, 1375, 1303, 1238, 1051, 833, 740 cmꢁ1; 1H NMR
2-yl)benzamide (300 mg, 0.00046 mol) in 3 mL DCM cooled at ꢁ30 ꢀC
was added BBr3 (0.577 g. 0.0023 mol) under nitrogen atmosphere.
Reaction mixture was stirred at 25e30 ꢀC for 3 h. Reaction mixture was
diluted with water and basified with aq. sodium carbonate solution.
Organic layer was separated, dried and distilled out to get crude product,
which was column purified using 100e200 silica gel and 2% methanol in
chloroform as mobile phase producing title compound as off white solid;
yield: 28%; mp 207e210 ꢀC; purity by UPLC: 96.94%; IR (KBr) 3019, 1731,
(DMSO-d6, 400 MHz)
d
: 3.83 (s, 3H), 6.24 (s, 2H), 6.99 (d, J ¼ 2 Hz, 1H),
7.47 (d, J ¼ 2 Hz, 1H), 7.91e7.94 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.07e8.09
(dd, J ¼ 0.8 and 9.2 Hz, 1H), 8.41e8.42 (dd, J ¼ 0.8 and 2.8 Hz, 1H),
10.73 (s, 1H); ESI/MS m/z 312 (M þ H)þ.
1
1672, 1601, 1584, 1513, 1403, 1375, 1215, 1024, 757, 699 cmꢁ1; H NMR
(DMSO-d6, 400 MHz)
d
: 3.33 (t, J ¼ 6.4 Hz, 2H), 3.62e3.67 (m, 2H), 4.47 (s,
5.1.1.24. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-
sulfonimidoyl)benzamido)benzamide (15a). To a stirring solution of
10a (2 g, 0.0060 mol) and DMF (one drop) in DCM (10 v/w) cooled
at 10e15 ꢀC was added oxalyl chloride (0.91 g, 0.0072 mol) under
nitrogen atmosphere. Reaction mixture was stirred at 25e30 ꢀC for
3 h and then evaporated to dryness. Acid chloride obtained was
dissolved in dry THF (8 mL) and then added to the solution con-
taining 14a (1.52 g, 0.0054 mol) in 6 mL THF cooled at 10e15 ꢀC.
After stirring at 25e30 ꢀC for 2 h, reaction mixture was quenched
with water (10 v/w). Product was extracted with chloroform which
on drying over sodium sulfate, evaporation gave solid product.
The crude product obtained above was added to sulfuric acid
(5 v/w) cooled at 0e5 ꢀC. The reaction mixture was stirred at same
temperature for 15e20 min. Above reaction mixture was slowly
poured in chilled water and basified using aqueous potassium
carbonate solution. Precipitated product was filtered and washed
with water. Drying afforded 15a (1 g, 64%) as off white solid: mp
239e242 ꢀC; purity by UPLC: 98.19%; IR (KBr) 3315, 1685, 1651,
1602, 1573, 1519, 1460, 1371, 1292, 1217, 1030, 1006, 833, 746,
1H), 4.79 (t, 1H), 7.65e7.68 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz,
1H), 7.93e7.95 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.01e8.04 (m, 4H), 8.10e8.12
(dd, J ¼ 2.8 and 8.8 Hz, 2H), 8.43 (d, J ¼ 2.4 Hz, 1H), 11.18 (s, 1H), 11.26
(s, 1H); ESI/MS m/z 492.6 (M þ H)þ.
5.1.1.28. N-(5-Chloropyridin-2-yl)-5-methoxy-2-(4-(S-methyl-
sulfonimidoyl)benzamido)benzamide (15e). This compound was
prepared from 10a and 14b by means of the procedure similar to
that reported for 15a as off white solid; yield: 61%; mp 247e250 ꢀC;
purity by UPLC: 95.78%; IR (KBr) 3435, 3288, 1676, 1651, 1608, 1523,
1458, 1373, 1298, 1218, 1072, 835, 744 cmꢁ1
400 MHz)
9.2 Hz, 1H), 7.40 (d, J ¼ 2.8 Hz, 1H), 7.92e7.97 (m, 2H), 8.04 (s, 4H),
8.14 (d, J ¼ 8.8 Hz, 1H), 8.42 (d, J ¼ 2.4 Hz, 1H), 10.97 (s, 1H), 11.11
(s, 1H); ESI/MS m/z 458.9 (M þ H)þ.
;
1H NMR (DMSO-d6,
: 3.10 (s, 3H), 3.84 (s, 3H), 7.16e7.19 (dd, J ¼ 2.8 and
d
5.1.1.29. 5-Chloro-N-(5-chloropyridin-2-yl)-3-methoxy-2-(4-(S-
methylsulfonimidoyl)benzamido)-benzamide (15f). This compound
was prepared from 10a and 14d by means of the procedure similar
to that reported for 15a as off white solid; yield: 75%; mp 188e
190 ꢀC; purity by UPLC: 97.66%; IR (KBr) 3269, 1664, 1577, 1458,
677 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 3.11 (s, 3H), 4.40 (s, 1H),
7.66e7.69 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.92 (d, J ¼ 2.4 Hz, 1H), 7.94e
7.97 (dd, J ¼ 2.4 and 8.8 Hz,1H), 8.07 (bs, 4H), 8.12 (d, J ¼ 4.4 Hz,1H),
8.14 (d, J ¼ 4.4 Hz, 1H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.19 (s, 1H), 11.27
(s, 1H); ESI/MS m/z: 462.9 (M þ H)þ.
;
1375, 1307, 1228, 1064, 839, 748 cmꢁ1 1H NMR (DMSO-d6,
400 MHz)
d
: 3.08 (s, 3H), 3.84 (s, 3H), 4.36 (s, 1H), 7.28 (d, J ¼ 2.0 Hz,
1H), 7.37 (d, J ¼ 2.0 Hz, 1H), 7.85e7.88 (dd, J ¼ 2.4 and 8.8 Hz, 1H),
7.98e8.06 (m, 5H), 8.36 (d, J ¼ 2.4 Hz, 1H), 9.98 (s, 1H), 10.87 (s, 1H);
ESI/MS m/z 492.6 (M þ H)þ.
5.1.1.25. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-((S-methyl-
sulfonimidoyl)methyl)benzamido)benzamide (15b). This compound
was prepared from 10b and 14a by means of the procedure similar
to that reported for 15a as off white solid; yield: 63%; mp 217e
219 ꢀC; purity by HPLC: 96.07%; IR (KBr) 3178, 1674, 1652, 1602,
5.1.1.30. N-(5-Chloropyridin-2-yl)-3-methoxy-2-(4-(S-methyl-
sulfonimidoyl)benzamido) benzamide (15g). This compound was
prepared from 10a and 14c by means of the procedure similar to
that reported for 15a as off white solid; yield: 65%; mp 185e187 ꢀC;
purity by UPLC: 98.82%; IR (KBr) 3277, 1662, 1583, 1518, 1469, 1375,
1575, 1521, 1460, 1375, 1296, 1209, 1035, 831, 750 cmꢁ1
;
1H NMR
: 2.78 (s, 3H), 3.69 (s, 1H), 4.41e4.50
(DMSO-d6, 400 MHz)
d
(q, J ¼ 13.6 Hz, 2H), 7.59 (d, J ¼ 8.8 Hz, 2H), 7.64e7.67 (dd, J ¼ 2.4
and 8.8 Hz, 1H), 7.89 (d, J ¼ 8 Hz, 2H), 7.91e7.95 (m, 2H), 8.11
(d, J ¼ 8 Hz, 1H), 8.19 (d, J ¼ 8.8 Hz, 1H), 8.43 (d, J ¼ 2.4 Hz, 1H), 11.18
(s, 1H), 11.27 (s, 1H); ESI/MS m/z 499 (M þ Na)þ.
1266, 1062, 1004, 833, 746 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d:
3.08 (s, 3H), 3.82 (s, 3H), 4.36 (s, 1H), 7.26 (d, J ¼ 8 Hz, 1H), 7.29 (d,
J ¼ 7.6 Hz, 1H), 7.40 (d, J ¼ 8 Hz, 1H), 7.85e7.88 (dd, J ¼ 2.8 and
9.2 Hz, 1H), 7.99e8.10 (m, 5H), 8.35 (d, J ¼ 2.8 Hz, 1H), 9.92 (s, 1H),
10.62 (s, 1H); ESI/MS m/z 458.8 (M þ H)þ.
5.1.1.26. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(ethyl-
sulfonimidoyl)benzamido)benzamide (15c). This compound was
prepared from 10c and 14a by means of the procedure similar to
that reported for 15a as off white solid; yield: 67%; mp 213e215 ꢀC;
purity by HPLC: 97.47%; IR (KBr) 3259, 1735, 1666, 1602, 1583, 1571,
5.1.1.31. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(meth-
ylsulfonyl)sulfonimidoyl)benzamido)benzamide (16a). To a stirring
solution of 15a (0.5 g, 0.0010 mol) and pyridine (0.12 g, 0.0015 mol)
in 3 mL DCM cooled at 10e15 ꢀC was added methanesulfonyl chlo-
ride (0.137 g, 0.0012 mol) under nitrogen atmosphere. Reaction
mixture was stirred at 25e30 ꢀC for 3 h and then quenched with
water (10 v/w). Product was extracted with DCM which on drying
over sodium sulfate and evaporation gave solid product. Product
obtained was column purified using ethyl acetate:hexane mobile
phase (0e60%) and 100e200 silica gel to get title compound as off
white solid; yield: 51%; mp: 220e222 ꢀC; purity by UPLC: 98.96; IR
(KBr) 3342, 1676, 1653, 1599, 1502, 1460, 1371, 1307, 1072, 848,
1517, 1438, 1373, 1218, 1002, 829, 756 cmꢁ1
400 MHz)
(s, 1H), 7.65e7.68 (dd, J ¼ 2.4 and 8.8 Hz,1H), 7.90 (d, J ¼ 2.4 Hz, 1H),
7.92e7.95 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.01 (d, J ¼ 8.8 Hz, 2H), 8.07
(d, J ¼ 8.4 Hz, 2H), 8.09e8.12 (dd, J ¼ 2.4 and 8.8 Hz, 2H), 8.43 (d,
J ¼ 2.8 Hz, 1H), 11.17 (s, 1H), 11.25 (s, 1H); ESI/MS m/z 477 (M þ H)þ.
;
1H NMR (DMSO-d6,
: 1.05 (t, J ¼ 7.2 Hz, 3H), 3.14e3.19 (q, J ¼ 7.2 Hz, 2H), 4.37
d
5 .1.1. 2 7 . 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(2-
hydroxyethylsulfonimidoyl)benzamido)benzamide (15d). This compound
was prepared from 10d and 14a by means of the procedure similar to that
reported for 15a. Deprotection of CBZ group and methoxy group was
achieved as follows: To a stirring solution of 2-(4-(N-benzyloxycarbonyl-
2-methoxyethylsulfonimidoyl)benzamido)-5-chloro-N-(5-chloropyridin-
752 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 3.04 (s, 3H), 3.66 (s, 3H),
7.67e7.70 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.93e
7.96 (dd, J ¼ 2.8 and 8.8 Hz,1H), 8.08e8.19 (m, 6H), 8.44 (d, J ¼ 2.4 Hz,
1H), 11.21 (s, 1H), 11.27 (s, 1H); ESI/MS m/z: 540.9 (M þ H)þ.

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
147
5.1.1.32. 2-(4-(N-Acetyl-S-methylsulfonimidoyl)benzamido)-5-
chloro-N-(5-chloropyridin-2-yl) benzamide (16b). This compound
was prepared from 15a and acetyl chloride by means of the
procedure similar to that reported for 16a as off white solid; yield:
61%; mp 215e217 ꢀC; purity by UPLC: 96.00%; IR (KBr) 3431, 1637,
8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.94e7.96 (dd, J ¼ 2.4 and 8.8 Hz,
1H), 8.08e8.13 (m, 6H), 8.44 (d, J ¼ 2.8 Hz, 1H), 11.19 (s, 1H), 11.27
(s, 1H); ESI/MS m/z 538.9 (M þ H)þ.
5.1.1.37. 2-(4-(N-(2-Chloroacetyl)-S-methylsulfonimidoyl)benzamido)-
N-(5-chloropyridin-2-yl)-5-methoxybenzamide (17e). This compound
was prepared from 15e by means of the procedure similar to that
reported for 17a as off white solid; yield: 80%; mp 178e180 ꢀC; purity
by UPLC: 98.74%; IR (KBr) 1681, 1652, 1610, 1573, 1515, 1461, 1373,
1602, 1575, 1512, 1460, 1373, 1296, 1217, 1033, 831, 746 cmꢁ1
;
1H
NMR (DMSO-d6, 400 MHz)
d: 1.97 (s, 3H), 3.46 (s, 3H), 7.67e7.69
(dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.92 (d, J ¼ 2.4 Hz, 1H), 7.93e7.96
(dd, J ¼ 2.4 and 8 Hz, 1H), 8.09e8.13 (m, 6H), 8.44 (d, J ¼ 2.4 Hz,
1H), 11.19 (s, 1H), 11.26 (s, 1H); ESI/MS m/z 503.1 (M ꢁ H).
1207, 1028, 839, 744 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 3.56 (s,
3H), 3.84 (s, 3H), 4.26 (s, 2H), 7.16e7.19 (dd, J ¼ 2.8 and 9.2 Hz, 1H),
7.39 (d, J ¼ 2.8 Hz, 1H), 7.92 (d, J ¼ 2.4 Hz, 1H), 7.93e7.95 (dd, J ¼ 2.8
and 5.6 Hz, 1H), 8.10e8.14 (m, 5H), 8.42 (d, J ¼ 2.4 Hz, 1H), 10.97
(s, 1H), 11.11 (s, 1H); ESI/MS m/z 557.1 (M þ Na)þ.
5.1.1.33. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-
methoxyacetyl)-S-methylsulfonimidoyl) benzamido)benzamide (16c).
This compound was prepared from 15a and methoxyacetyl chloride
by means of the procedure similar to that reported for 16a as off
white solid; yield: 69%; mp: 206e208 ꢀC; purity by UPLC: 96.98%;
IR (KBr) 2926, 2823, 1666, 1604, 1573, 1516, 1462, 1373, 1296, 1217,
5.1.1.38. 5-Chloro-2-(4-(N-(2-chloroacetyl)-S-methylsulfonimidoyl)
benzamido)-N-(5-chloropyridin-2-yl)-3-methoxybenzamide (17f).
This compound was prepared from 15f by means of the procedure
similar to that reported for 17a as off white solid; yield: 82%; mp
143e145 ꢀC; purity by UPLC: 90%; IR (KBr) 1656, 1573, 1521,
14,601, 1375, 1315, 1226, 1064, 842, 750 cmꢁ1; 1H NMR (DMSO-d6,
1116, 922, 837, 746 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d: 3.26
(s, 3H), 3.52 (s, 3H), 3.94 (d, J ¼ 2.8 Hz, 2H), 7.66e7.69 (dd, J ¼ 2.4
and 8.8 Hz, 1H), 7.92 (d, J ¼ 2.0 Hz, 1H), 7.93e7.96 (dd, J ¼ 2.4 and
8.8 Hz, 1H), 8.09e8.13 (m, 6H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.20 (s, 1H),
11.25 (s, 1H); ESI/MS m/z 532.9 (M ꢁ H).
400 MHz)
d
: 3.54 (s, 3H), 3.85 (s, 3H), 4.26 (s, 2H), 7.29 (d, J ¼ 2 Hz,
1H), 7.38 (d, J ¼ 2 Hz, 1H), 7.85e7.88 (dd, J ¼ 2.8 and 8.8 Hz, 1H),
8.05e8.10 (m, 5H), 8.36 (d, J ¼ 2.8 Hz, 1H), 10.09 (s, 1H), 10.89 (s,
1H); ESI/MS m/z 592.6 (M þ Na)þ.
5.1.1.34. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-cyano-S-meth-
ylsulfonimidoyl)benzamido)benzamide (16d). To a stirring solution
of 15a (200 mg, 0.000432 mol) in 3 mL DCM was added catalytic
amount of DMAP followed by cyanogen bromide (58 mg,
0.000476 mol) under nitrogen atmosphere. Reaction mixture was
stirred at 25e30 ꢀC for 6 h and then quenched with water (10 v/w).
Product was extracted with DCM which on drying over sodium
sulfate and evaporation gave solid product. Product obtained was
column purified using ethyl acetate:hexane mobile phase (0e40%)
and 100e200 silica gel to get off white solid; yield: 57%; mp: 166e
168 ꢀC; purity by UPLC: 96.44%; IR (KBr) 2195, 1710, 1676, 1654,
1602, 1516, 1460, 1375, 1246, 1114, 827, 746 cmꢁ1; 1H NMR (DMSO-
5.1.1.39. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-
(methylamino)acetyl)sulfonimidoyl)benzamido)benzamide (18a).
To a stirring solution of 17a (1 g, 0.0018 mol) in 5 mL DMF was
added 40% aq. CH3NH2 solution (0.576 g, 0.0185 mol) followed by
catalytic amount of KI. The reaction mixture was stirred at 25e
30 ꢀC for 12 h and then diluted with water. Filtration and
drying afforded title compound, which was column purified using
230e400 silica gel and methanol:DCM mobile phase (0e3%);
yield: 42%; mp: 174e176 ꢀC; purity by UPLC: 98.72%; IR (KBr)
3392, 1683, 1649, 1602, 1573, 1525, 1460, 1373, 1296, 1217, 1114,
d6, 400 MHz)
d
: 3.80 (s, 3H), 7.67e7.70 (dd, J ¼ 2.4 and 8.4 Hz, 1H),
7.90 (d, J ¼ 2.4 Hz, 1H), 7.93e7.96 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.08
(d, J ¼ 8.8 Hz, 1H), 8.14 (d, J ¼ 8.8 Hz,1H), 8.19e8.24 (m, 4H), 8.44 (d,
J ¼ 2.4 Hz 1H),11.21 (s,1H),11.27 (s,1H); ESI/MS m/z 487.8 (M þ H)þ.
833, 746 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 2.38 (s, 3H), 3.47
(bs, 2H), 3.57 (s, 3H), 7.65 (d, J ¼ 6.8 Hz, 1H), 7.93e7.96 (m, 2H),
8.12e8.20 (m, 6H), 8.45 (d, J ¼ 2.8 Hz, 1H); 13C NMR (DMSO-d6,
100 MHz) d: 33.64, 42.87, 53.56, 116.17, 124.65, 125.68, 126.41,
5.1.1.35. 2-(4-(N-Carbamoyl-S-methylsulfonimidoyl)benzamido)-5-
chloro-N-(5-chloropyridin-2-yl)benzamide (16e). 16d (100 mg,
0.000205 mol) was added to 2 mL sulfuric acid. Reaction mixture
was stirred at 25e30 ꢀC for 6 h and then quenched with water (10
v/w). Solid obtained was filtered, washed with water and dried to
get off white solid; yield: 45%; mp 205e208 ꢀC; purity by HPLC:
96.78%; IR (KBr) 3423, 1654, 1600, 1577, 1510, 1460, 1375, 1298,
126.87, 127.58, 128.51, 129.15, 131.52, 137.72, 140.42, 140.75,
146.41, 151.01, 164.01, 166.01, 175.67; ESI/MS m/z 533.9 (M þ H)þ.
5.1.1.40. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(ethyl-
amino)acetyl)-S-methylsulfonimidoyl) benzamido)benzamide (18b).
10 mol eq. 70% ethylamine was used and this compound was
prepared from 17a by means of the procedure similar to that re-
ported for 18a as off white solid; yield: 48%; mp: 158e160 ꢀC;
purity by UPLC: 99.38%; IR (KBr) 3433, 1681, 1639, 1602, 1523,
1458, 1373, 1296, 1224, 1116, 922, 835, 744 cmꢁ1; 1H NMR (DMSO-
1226, 1118, 835, 748 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d: 3.37
(s, 3H), 6.05 (bs, 1H), 6.45 (bs, 1H), 7.65e7.68 (dd, J ¼ 2.4 and 8.4 Hz,
1H), 7.91 (d, J ¼ 2.8 Hz, 1H), 7.92e7.95 (dd, J ¼ 2.4 and 8.8 Hz, 1H),
8.03e8.11 (m, 5H), 8.13 (d, J ¼ 4.4 Hz, 1H), 8.43 (d, J ¼ 0.4 Hz, 1H),
11.19 (s, 1H), 11.26 (s, 1H); ESI/MS m/z 527.8 (M þ Na)þ.
d6, 400 MHz)
d
: 1.0 (t, J ¼ 7.2 Hz, 3H), 2.57e2.66 (m, 2H), 3.42
(d, J ¼ 9.6 Hz, 2H), 3.54 (s, 3H), 7.60 (bd, J ¼ 9.2 Hz, 1H), 7.91 (d,
J ¼ 2.4 Hz, 1H), 7.93e7.95 (dd, J ¼ 2.8 and 8.0 Hz, 1H), 8.10
(d, J ¼ 8.8 Hz, 2H), 8.18 (d, J ¼ 8.8 Hz, 1H), 8.25 (bs, 3H), 8.45 (d,
5.1.1.36. 5-Chloro-2-(4-(N-(2-chloroacetyl)-S-methylsulfonimidoyl)
benzamido)-N-(5-chloropyridin-2-yl)benzamide (17a). To a stirring
solution of 15a (2 g, 0.0042 mol) and TEA (1.06 g, 0.0105 mol) in
20 mL THF cooled at 0 ꢀC was added chloroacetyl chloride (0.71 g,
0.0063 mol). Reaction mixture was stirred at 25e30 ꢀC for 2 h.
Reaction mixture was diluted with water (10 v/w). Precipitated
product was filtered and purified by refluxing in solvent mixture of
6 v/w hexane and 3 v/w ethyl acetate. Filtration at 25e30 ꢀC
afforded 17a (1.86 g, 80%) as off white solid; mp 190e193 ꢀC;
purity by UPLC: 95.83%; IR (KBr) 1687, 1660, 1602, 1573, 1516,
1460, 1404, 1375, 1298, 1209, 1008, 839, 748 cmꢁ1; 1H NMR (DMSO-
J ¼ 2.8 Hz, 1H); 13C NMR (DMSO-d6, 100 MHz)
d: 13.92, 42.67,
42.95, 53.30, 116.17, 124.60, 125.35, 126.40, 126.78, 127.37, 128.65,
129.11, 131.39, 137.65, 140.40, 140.77, 146.44, 151.25, 164.37, 165.97,
178.05; ESI/MS m/z 547.8 (M þ H)þ.
5.1.1.41. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(iso-
propylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide
(18c). 2 mol eq. isopropylamine was used and this compound was
prepared from 17a by means of the procedure similar to that re-
ported for 18a as off white solid; yield: 57%; mp: 176e178 ꢀC; purity
by UPLC: 99.24%; IR (KBr) 3313, 1681, 1637, 1600, 1523, 1458, 1373,
d6, 400 MHz)
d
: 3.57 (s, 3H), 4.27 (s, 2H), 7.67e7.69 (dd, J ¼ 2.4 and

148
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
1296, 1226, 1093, 833, 744 cmꢁ1
;
1H NMR (DMSO-d6, 400 MHz)
d
:
5.1.1.45. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(diisopropylamino)
acetyl)-S-methylsulfonimidoyl)benzamido)benzamide (18g). 2 mol eq. dii-
sopropylamine was used and this compound was prepared from 17a by
means of the procedure similar to that reported for 18a as off white solid;
yield: 86%; mp: 151e153 ꢀC; purity by UPLC: 97.94%; IR (KBr) 1680, 1656,
0.97e1.00 (q, 6H), 2.78e2.84 (m, 1H), 3.42 (s, 2H), 3.54 (s, 3H), 7.62
(bd, 1H), 7.91e7.95 (m, 2H), 8.11 (d, J ¼ 8.4 Hz, 2H), 8.18 (d,
J ¼ 8.8 Hz, 1H), 8.31 (bs, 3H), 8.45 (d, J ¼ 2.8 Hz, 1H); 13C NMR
(DMSO-d6, 100 MHz) d: 21.71, 42.91, 48.09, 51.35, 116.16, 124.66,
125.43, 126.32, 126.61, 127.40, 128.62, 129.13, 131.42, 137.69, 140.40,
140.72, 146.44, 151.10, 164.33, 165.96, 178.23; ESI/MS m/z 561.9
(M þ H)þ.
1602, 1518, 1460, 1375, 1296, 1213, 1116, 920, 831, 746 cmꢁ1
;
1H NMR
(DMSO-d6, 400 MHz)
d
: 0.92 (d, J ¼ 4.8 Hz, 12H), 2.95e2.98 (m, 2H), 3.17
(s, 2H), 3.46 (s, 3H), 7.67 (d, J ¼ 8 Hz, 1H), 7.92e7.95 (dd, J ¼ 2.4 and
8.8 Hz, 2H), 8.06e8.14 (m, 6H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.25 (bs, 2H);
ESI/MS m/z 604 (M þ H)þ.
5.1.1.42. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(cyclo-
pentylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide
(18d). 2 mol eq. cyclopentylamine was used and this compound
was prepared from 17a by means of the procedure similar to that
reported for 18a as off white solid; yield: 40%; mp: 141e143 ꢀC;
purity by UPLC: 97.52%; IR (KBr) 3416, 1681, 1651, 1602, 1510,
5.1.1.46. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-
(pyrrolidin-1-yl)acetyl) sulfonimidoyl)benzamido)benzamide (18h).
2 mol eq. pyrrolidine was used and this compound was prepared
from 17a by means of the procedure similar to that reported for 18a
as off white solid; yield: 69%; mp: 133e135 ꢀC; purity by UPLC:
99.47%; IR (KBr) 1685,1656,1602,1573,1518,1458,1373,1296,1203,
1458, 1373, 1296, 1219, 1114, 920, 833, 746 cmꢁ1
;
1H NMR (DMSO-
d6, 400 MHz) : 1.31e1.35 (m, 2H), 1.43e1.46 (m, 2H), 1.54e1.59 (m,
d
2H), 1.67e1.71 (m, 2H), 3.05e3.08 (m, 1H), 3.41 (s, 2H), 3.54 (s, 3H),
7.64 (bd, 1H), 7.92e7.94 (m, 2H), 8.09e8.19 (m, 6H), 8.45
(d, J ¼ 2.8 Hz, 1H); ESI/MS m/z 587.8 and 589.9 (M þ H)þ.
1116, 922, 839, 748 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 1.67 (bs,
4H), 2.60e2.61 (bd, 4H), 3.51 (s, 3H), 7.65e7.68 (dd, J ¼ 2.0 and
8.4 Hz, 1H), 7.92 (d, J ¼ 2.8 Hz, 1H), 7.93e7.96 (dd, J ¼ 2.4 and 8.8 Hz,
1H), 8.08e8.14 (m, 6H), 8.44 (d, J ¼ 2.8 Hz, 1H), 11.25 (bs, 2H); 13C
5.1.1.43. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(dimethy-
lamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide
(18e). 10 mol eq. 50%. dimethylamine solution was used and this
compound was prepared from 17a by means of the procedure
similar to that reported for 18a as off white solid; yield: 66%; mp:
134e136 ꢀC; purity by UPLC: 99.19%; IR (KBr) 1683, 1647, 1602,
NMR (DMSO-d6, 100 MHz) d: 23.26, 42.94, 53.25, 60.20, 116.20,
124.60, 125.80, 126.95, 127.51, 127.93, 128.43, 129.20, 131.63, 137.79,
139.06,141.49,146.39,150.61,164.01,166.06,177.39; ESI/MS m/z 574
and 575.7 (M þ H)þ.
5.1.1.47. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-
(piperidin-1-yl)acetyl) sulfonimidoyl)benzamido)benzamide (18i).
2 mol eq. piperidine was used and this compound was prepared
from 17a by means of the procedure similar to that reported for 18a
as off white solid; yield: 91%; mp: 125e127 ꢀC; purity by UPLC:
98.90%; IR (KBr) 1681, 1654, 1600, 1510, 1458, 1374, 1296, 1217, 1112,
1518, 1458, 1373, 1294, 1217, 1116, 922, 839, 746 cmꢁ1
;
1H NMR
(DMSO-d6, 400 MHz) : 2.20 (s, 6H), 3.09 (s, 2H), 3.48 (s, 3H),
d
7.64e7.66 (dd, J ¼ 2 and 8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.92e
7.94 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.06e8.13 (m, 6H), 8.43 (d,
J ¼ 2.8 Hz, 1H), 11.23 (bs, 2H); 13C NMR (DMSO-d6, 100 MHz)
d:
42.94, 44.70, 63.86, 116.18, 124.47, 125.80, 126.70, 127.47, 127.87,
128.39, 129.17, 131.63, 137.76, 139.01, 141.58, 146.35, 150.58, 163.97,
166.06, 177.91; ESI/MS m/z 548 (M þ H)þ.
920, 833, 746 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 1.29 (bs, 2H),
1.34e1.46 (bs, 4H), 2.42e2.50 (bs, 4H), 3.15 (bs, 2H), 3.50 (s, 3H),
7.68 (d, J ¼ 7.2 Hz, 1H), 7.91e7.96 (m, 2H), 8.07e8.13 (m, 6H), 8.44
(d, J ¼ 2.8 Hz, 1H), 11.27 (bd, 2H); ESI/MS m/z 587.9 and 589.9
(M þ H)þ.
5.1.1.44. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(dieth-
ylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide (18f).
To a stirring solution of 17a (1 g, 0.0018 mol) in 5 mL DMF was
added diethylamine (0.263 g, 0.0036 mol) followed by catalytic
amount of KI. The reaction mixture was stirred at 25e30 ꢀC for 16 h
and then diluted with water. Filtration and drying afforded title
compound, which was column purified using 230e400 silica gel
and 0e3% methanol in chloroform as mobile phase to get 18f
(0.710 g, 66%) as off white solid; mp: 170e172 ꢀC; purity by UPLC:
98.91%; IR (KBr) 1687,1666,1600,1573,1510,1464, 1398,1375,1294,
5.1.1.48. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-
morpholinoacetyl) sulfonimidoyl)benzamido)benzamide (18j). 2 mol eq.
morpholine was used and this compound was prepared from 17a by
means of the procedure similar to that reported for 18a as off white
solid; yield: 60%; mp: 135e137 ꢀC; purity by UPLC: 98.71%; IR (KBr)
1681, 1656, 1600, 1575, 1510, 1458, 1373, 1296, 1215, 1112, 918, 833,
746 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d
: 2.45 (t, J ¼ 4.4 Hz, 4H), 3.12
(s, 2H), 3.50 (s, 3H), 3.53 (t, J ¼ 4.8 Hz, 4H), 7.66e7.69 (dd, J ¼ 2.4 and
8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.93e7.96 (dd, J ¼ 2.4 and 8.8 Hz, 1H),
8.07e8.13 (m, 6H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.19 (s, 1H), 11.27 (s, 1H);
ESI/MS m/z 590.1 (M þ H)þ.
1217,1114, 920, 831, 748 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d: 0.94
(t, J ¼ 7.2 Hz, 6H), 2.54e2.59 (q, J ¼ 7.2 Hz, 4H), 3.26 (s, 2H), 3.49
(s, 3H), 7.65e7.68 (dd, J ¼ 2.0 and 8.4 Hz,1H), 7.92 (d, J ¼ 2.4 Hz,1H),
7.92e7.95 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.07e8.14 (m, 6H), 8.44
(d, J ¼ 2.8 Hz, 1H), 11.24 (bs, 2H); 13C NMR (DMSO-d6, 100 MHz)
d:
5.1.1.49. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-(4-
12.16, 42.95, 46.87, 57.70, 116.19, 124.52, 125.80, 126.79, 127.48,
127.85, 128.40, 129.19, 131.63, 137.77, 139.07, 141.66, 146.37, 150.64,
164.02, 166.07, 178.89; ESI/MS m/z 576 and 577.6 (M þ H)þ.
To a stirring solution of 18f (0.5 g, 0.000869 mol) in 5 mL DCM
cooled at 10e15 ꢀC was added HCl:diethyl ether (12% w/w, 0.2 mL).
The reaction mixture was stirred at 25e30 ꢀC for 1 h and then diluted
with diethyl ether. Precipitated solid was filtered and dried to get
monohydrochloride salt of 18f (0.490 g, 92%) as off white solid;
methylpiperazin-1-yl)acetyl)
sulfonimidoyl)benzamido)benzamide
(18k). 2 mol eq. N-methylpiperazine was used and this compound
was prepared from 17a by means of the procedure similar to that
reported for 18a as off white solid; yield: 60%; mp: 178e180 ꢀC;
purity by UPLC: 98.83%; IR (KBr) 1689, 1653, 1602, 1516, 1460,
1375, 1296, 1224, 1010, 920, 823, 742 cmꢁ1
400 MHz)
;
1H NMR (DMSO-d6,
: 2.14 (s, 3H), 2.33 (bs, 4H), 2.45 (bs, 4H), 3.10 (s, 2H),
d
3.49 (s, 3H), 7.67 (d, J ¼ 8.0 Hz,1H), 7.92 (d, J ¼ 2.4 Hz,1H), 7.92e7.95
(dd, J ¼ 2.8 and 9.2 Hz, 1H), 8.07e8.15 (m, 6H), 8.44 (d, J ¼ 2.4 Hz,
1H), 11.25 (bs, 2H); ESI/MS m/z 603.2 (M þ H)þ.
purity by UPLC: 98.14%; 1H NMR (DMSO-d6, 400 MHz)
d: 1.15e1.23
(m, 6H), 3.06e3.17 (m, 4H), 3.66 (s, 3H), 4.08 (d, J ¼ 5.2 Hz, 2H),
7.67e7.70 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.92 (d, J ¼ 2.4 Hz, 1H), 7.94e
7.97 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.09 (d, J ¼ 4.8 Hz, 1H), 8.11
(d, J ¼ 4.8 Hz, 1H), 8.15e8.21 (m, 4H), 8.45 (d, J ¼ 2.4 Hz, 1H), 9.43
(bs, 1H), 11.28 (s, 2H); ESI/MS m/z 575.9 (M þ H)þ.
5.1.1.50. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-(4-
methyl-1,4-diazepan-1-yl)acetyl)sulfonimidoyl)benzamido)benza-
mide (18l). 2 mol eq. N-methylhomopiperazine was used and this

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
149
compound was prepared from 17a by means of the procedure
similar to that reported for 18a as off white solid; yield: 69%; mp:
177e180 ꢀC; purity by UPLC: 98.57%; IR (KBr) 1654, 1600, 1575,
was prepared from 17f by means of the procedure similar to that
reported for 18a as off white solid; yield: 53%; mp: 178e180 ꢀC;
purity by UPLC: 96.77%; IR (KBr) 3433, 1664, 1573, 1529, 1460,
1510, 1460, 1373, 1298, 1215, 1112, 920, 833, 746 cmꢁ1
;
1H NMR
1375, 1307, 1224, 1114, 1064, 840, 748 cmꢁ1 1H NMR (DMSO-d6,
;
(DMSO-d6, 400 MHz)
d
: 1.86 (bs, 2H), 2.68 (s, 3H), 2.78 (bs, 2H), 2.91
400 MHz)
d
: 1.10 (t, J ¼ 7.2 Hz, 3H), 2.79 (t, J ¼ 7.2 Hz, 2H), 3.58 (s,
(bs, 2H), 2.99e3.15 (m, 4H), 3.50 (s, 2H), 3.65 (s, 3H), 7.69 (d,
J ¼ 7.6 Hz, 1H), 7.92e7.96 (bd, 2H), 8.10e8.12 (bd, 6H), 8.45 (s, 1H),
11.27 (bs, 2H); ESI/MS m/z 616.8 and 618.8 (M þ H)þ.
3H), 3.86 (s, 3H), 7.31 (s, 1H), 7.39 (s, 1H), 7.90 (d, J ¼ 8.4 Hz, 1H),
8.06 (d, J ¼ 8.8 Hz, 1H), 8.12 (s, 4H), 8.38 (s, 1H), 10.10 (bs, 1H), 10.81
(bs, 1H); 13C NMR (DMSO-d6, 100 MHz)
d: 11.83, 42.05, 42.85, 51.04,
56.66, 114.35, 115.28, 120.19, 123.07, 125.51, 127.44, 128.70, 131.47,
135.22, 137.83, 138.74, 140.59, 146.38, 150.62, 155.37, 164.82, 177.63;
ESI/MS m/z 578.0 (M þ H)þ.
5.1.1.51. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(4-
hydroxypiperidin-1-yl)acetyl)-S-methylsulfonimidoyl)benzamido)
benzamide (18m). 2 mol eq. 4-hydroxyopiperidine was used and
this compound was prepared from 17a by means of the procedure
similar to that reported for 18a as off white solid; yield: 75%; mp:
204e206 ꢀC; purity by UPLC: 96.75%; IR (KBr) 3417, 1681, 1653,
5.1.1.56. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(dieth-
ylamino)acetyl)-S-methylsulfonimidoyl)benzamido)-3-
methoxybenzamide (18r). 2 mol eq. diethylamine was used and this
compound was prepared from 17f by means of the procedure
similar to that reported for 18a as off white solid; yield: 64%; mp:
168e170 ꢀC; purity by UPLC: 98.31%; IR (KBr) 1666, 1573, 1521,
1458, 1375, 1307, 1220, 1114, 1066, 839, 750 cmꢁ1; 1H NMR (DMSO-
1604, 1573, 1521, 1460, 1373, 1294, 1215, 1114, 920, 837, 746 cmꢁ1
1H NMR (DMSO-d6, 400 MHz)
: 1.37 (bs, 2H), 1.66 (bs, 2H), 2.20 (bs,
2H), 2.72 (bs, 2H), 3.12 (bs, 2H), 3.35e3.40 (m, 1H), 3.50 (s, 3H), 4.55
(s, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.92 (d, J ¼ 2.4 Hz, 1H), 7.93e7.96 (dd,
J ¼ 2.4 and 8.8 Hz, 1H), 8.08e8.14 (m, 6H), 8.44 (d, J ¼ 2.4 Hz, 1H),
11.20 (s, 1H), 11.26 (s, 1H); ESI/MS m/z 604.1 and 605.8 (M þ H)þ.
;
d
d6, 400 MHz)
d
: 0.95 (t, J ¼ 3.2 Hz, 6H), 2.49e2.52 (m, 4H), 3.47 (s,
2H), 3.86 (s, 3H), 7.30 (d, J ¼ 2.0 Hz, 1H), 7.39 (d, J ¼ 2.4 Hz, 1H),
7.86e7.89 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.01e8.10 (m, 5H), 8.37 (d,
J ¼ 2.4 Hz, 1H), 10.07 (s, 1H), 10.87 (s, 1H); 13C NMR (DMSO-d6,
5.1.1.52. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(4-
aminocarbonyl-1-piperidinyl)acetyl)-S-methylsulfonimidoyl)benza-
mido)benzamide (18n). 2 mol eq. piperidine-4-carboxamide was
used and this compound was prepared from 17a by means of the
procedure similar to that reported for 18a as off white solid; yield:
42%; mp: 246e248 ꢀC; purity by UPLC: 98.55%; IR (KBr) 3408, 3155,
1689, 1662, 1602, 1521, 1460, 1373, 1294, 1215, 1120, 922, 839,
100 MHz) d: 12.14, 42.98, 46.88, 56.63, 57.64, 114.29, 115.33, 120.45,
122.95, 125.46, 127.20, 128.62, 131.44, 135.24, 137.85, 138.43, 141.46,
146.31, 150.59, 155.36, 164.75, 177.83; ESI/MS m/z 605.9 (M þ H)þ.
5.1.1.57. 5-Chloro-N-(5-chloropyridin-2-yl)-3-methoxy-2-(4-(S-
methyl-N-(2-(pyrrolidin-1-yl)acetyl)sulfonimidoyl)benzamido)ben-
zamide (18s). 2 mol eq. pyrrolidine was used and this compound
was prepared from 17f by means of the procedure similar to that
reported for 18a as off white solid; yield: 74%; mp: 154e156 ꢀC;
purity by UPLC: 97.66%; IR (KBr) 1687, 1656, 1575, 1521, 1460,
746 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d: 1.49e1.55 (m, 2H),
1.59e1.62 (m, 2H), 1.96e2.08 (m, 3H), 2.82 (bs, 2H), 3.11 (bs, 2H),
3.49 (s, 3H), 6.7 (s, 1H), 7.19 (s, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.91
(d, J ¼ 2.4 Hz, 1H), 7.93e7.96 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.07e8.14
(m, 6H), 8.44 (d, J ¼ 2.8 Hz, 1H), 11.19 (s, 1H), 11.25 (s, 2H); ESI/MS
m/z 630.8 (M þ H)þ.
1377, 1309, 1215, 1066, 842, 750 cmꢁ1
400 MHz) : 1.67 (bs, 4H), 2.58 (bs, 4H), 3.48 (s, 3H), 3.86 (s, 3H),
;
1H NMR (DMSO-d6,
d
7.30 (s,1H), 7.38 (s,1H), 7.86e7.89 (dd, J ¼ 2.4 and 8.8 Hz,1H), 8.02e
5.1.1.53. N-(5-Chloropyridin-2-yl)-2-(4-(N-(2-(diethylamino)acetyl)-
S-methylsulfonimidoyl) benzamido)-5-methoxybenzamide (18o).
2 mol eq. diethylamine was used and this compound was prepared
from 17e by means of the procedure similar to that reported for 18a
as off white solid; yield: 41%; mp: 113e115 ꢀC; purity by UPLC:
99.58%; IR (KBr) 1656,1610,1573,1529,1460,1375,1299,1220,1074,
8.10 (m, 5H), 8.37 (d, J ¼ 1.6 Hz, 1H), 10.07 (s, 1H), 10.87 (s, 1H); 13
C
NMR (DMSO-d6, 100 MHz) d: 23.28, 42.97, 53.19, 56.62, 60.29,
114.29, 115.23, 120.12, 123.10, 125.45, 127.20, 128.62, 131.41, 135.20,
137.84, 138.45, 140.97, 146.31, 150.58, 155.35, 164.74, 177.43; ESI/MS
m/z 604.0 (M þ H)þ.
837, 744 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d
: 0.92 (t, J ¼ 7.2 Hz,
5.1.1.58. 2-(4-(N-Methoxycarbonylmethyl-S-methylsulfonimidoyl)ben-
zamido)-5-chloro-N-(5-chloropyridin-2-yl)benzamide (19). To a stir-
ring solution of 15a (5 g, 0.0108 mol) in 15 mL DMF cooled at 10e
15 ꢀC was added sodium hydride (1.76 g, 0.0367 mol) under
nitrogen atmosphere. To this was added methyl bromoacetate
(2.48 g, 0.0162 mol) in one lot. Reaction mixture was stirred at 40 ꢀC
for 3 h and cooled to 20 ꢀC. Product was diluted with water and
extracted with DCM. Crude product was column purified using 100e
200 silica gel and 0e50% ethyl acetate in hexane as mobile phase to
get 19 (1.1 g, 20%) as off white solid; mp: 211e213 ꢀC; purity by UPLC:
94.47%; IR (KBr) 1747, 1678, 1656, 1600, 1572, 1512, 1460, 1375, 1296,
6H), 2.49e2.54 (m, 4H), 3.21 (s, 2H), 3.47 (s, 3H), 3.84 (s, 3H), 7.18 (d,
J ¼ 8 Hz, 1H), 7.39 (s, 1H), 7.94 (d, J ¼ 8.4 Hz, 2H), 8.05e8.14 (m, 5H),
8.41 (s, 1H), 10.96 (s, 1H), 11.09 (s, 1H); ESI/MS m/z 572 (M þ H)þ.
5.1.1.54. 5-Chloro-N-(5-chloropyridin-2-yl)-3-methoxy-2-
(4-(S-methyl-N-(2-(methylamino)acetyl)sulfonimidoyl)benzamido)
benzamide (18p). 10 mol eq. 40% aq. CH3NH2 solution was used and
this compound was prepared from 17f by means of the procedure
similar to that reported for 18a as off white solid; yield: 64%; mp:
157e160 ꢀC; purity by UPLC: 97.68%; IR (KBr) 3263, 1662, 1573,
1529, 1458, 1375, 1307, 1222, 1064, 840, 750 cmꢁ1; 1H NMR (DMSO-
1153, 837, 746 cmꢁ1 1H NMR (DMSO-d6, 400 MHz)
; d: 3.26 (s, 3H),
d6, 400 MHz)
d
: 2.22 (s, 3H), 3.20 (d, 2H), 3.47 (s, 3H), 3.84 (s, 3H),
3.49e3.66 (q, J ¼ 16.8 Hz, 2H), 3.55 (s, 3H), 7.66e7.69 (dd, J ¼ 2.8 and
8.8 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.94e7.97 (dd, J ¼ 2.8 and 8.8 Hz,
1H), 8.01e8.13 (m, 6H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.17 (s, 1H), 11.26 (s,
1H); ESI/MS m/z 532.9 (M ꢁ H).
7.30 (d, J ¼ 2.4 Hz,1H), 7.36 (s,1H), 7.85e7.88 (dd, J ¼ 2.4 and 8.8 Hz,
1H), 8.02e8.09 (m, 5H), 8.36 (d, J ¼ 2.4 Hz, 1H), 10.10 (bs, 1H), 10.90
(bs,1H); 13C NMR (DMSO-d6, 100 MHz)
d: 34.63, 42.92, 54.96, 56.63,
114.31, 115.24, 120.14, 123.03, 125.47, 127.32, 128.65, 131.40, 135.17,
137.83, 138.61, 141.03, 146.34, 150.60, 155.35, 164.77, 177.61; ESI/MS
m/z 563.7 (M þ H)þ.
5.1.1.59. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-
hydroxyethyl)-S-methylsulfonimidoyl)benzamido)benzamide (20).
To a stirring solution of 19 (2 g, 0.00374 mol) in 10 mL DMSO at
20e25 ꢀC was added sodium borohydride (0.71 g, 0.0149 mol).
Reaction mixture was stirred at 60 ꢀC for 3 h and cooled to 25 ꢀC.
Product was diluted with water and extracted with DCM.
5.1.1.55. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(ethylamino)
acetyl)-S-methylsulfonimidoyl) benzamido)-3-methoxybenzamide
(18q). 10 mol eq. 70% ethylamine was used and this compound

150
V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
The organic phase was dried over sodium sulfate and evaporated
to afford crude product. Product obtained was column purified
using 100e200 silica gel and 0e2% methanol in DCM to get 20
(1.02 g, 54%) as off white solid; mp: 210e212 ꢀC; purity by UPLC:
96.52%; IR (KBr) 3416, 1680, 1656, 1602, 1514, 1460, 1373, 1294,
(d, J ¼ 2.4 Hz, 1H), 7.77e7.80 (dd, J ¼ 2.8 and 9.2 Hz, 1H), 8.08
(d, J ¼ 8.4 Hz, 2H), 8.17 (d, J ¼ 8.4 Hz, 2H), 8.19e8.32 (m, 2H), 8.87
(d, J ¼ 8.8 Hz, 1H), 11.95 (bs, 1H); ESI/MS m/z 559.6 and 561.4
(M þ H)þ.
1220, 1139, 1070, 920, 831, 748 cmꢁ1
;
1H NMR (DMSO-d6,
: 2.70e2.75 (m, 1H), 2.83e2.89 (m, 1H), 3.18 (s, 3H),
5.2. Biology
400 MHz)
d
3.37e3.42 (m, 2H), 4.42 (t, J ¼ 6 Hz, 1H), 7.66e7.69 (dd, J ¼ 2.8 and
8.8 Hz, 1H), 7.92 (d, J ¼ 2.8 Hz, 1H), 7.94e7.97 (dd, J ¼ 2.4 and
8.8 Hz, 1H), 8.02 (d, J ¼ 8.4 Hz, 2H), 8.10 (d, J ¼ 8.4 Hz, 2H), 8.13 (d,
J ¼ 8.8 Hz, 2H), 8.44 (d, J ¼ 2.0 Hz, 1H), 11.18 (s, 1H), 11.26 (s, 1H);
ESI/MS m/z 506.9 and 508.6 (M þ H)þ.
5.2.1. In vitro factor Xa inhibition assay
The inhibitory activity of different compounds against purified
serine proteases was measured using chromogenic substrates in
96-well microtiter plates at RT. The enzymes were incubated with
test compound or its solvent, dimethyl sulfoxide (DMSO). The plate
was incubated at 37 ꢀC for 45 min. At the end of incubation the plate
was read at 405 nm using a Spectra Max. The following buffer (final
concentrations) was used in the assay: human FXa (10 ng), 50 mM
5.1.1.60. 2-(4-(N-(2-Bromoethyl)-S-methylsulfonimidoyl)benza-
mido)-5-chloro-N-(5-chloropyridin-2-yl) benzamide (21). To a stir-
ring solution of 20 (1.2 g, 0.0023 mol) in 15 mL DCM at 25e30 ꢀC
was added triphenylphosphine (0.92 g, 0.0035 mol) and carbon-
tetrabromide (1.1 g, 0.0035 mol). Reaction mixture was stirred at
30 ꢀC for 3 h and cooled to 20 ꢀC. Product was diluted with water
and extracted with DCM. The organic phase was dried over sodium
sulfate and evaporated to afford crude product. Product obtained
was column purified using 100e200 silica gel and 0e40% ethyl
acetate in hexane to get 21 (0.270 g, 20%) as off white solid; mp:
198e200 ꢀC; purity by HPLC: 96.15%; IR (KBr) 1676, 1600, 1573,
1514, 1458, 1373, 1294, 1130, 920, 833, 748 cmꢁ1; 1H NMR (DMSO-
TriseHCl buffer pH 7.5, 150
mM NaCl, and 1 mM calcium chloride
and 500 M substrate (S-2765) (Hyphen Biomed), 2.5% DMSO with
m
varying concentrations of test compound. The % inhibition was
calculated using wells without any inhibitor [only substrate,
enzyme and buffer (Test Well)], wells without any enzyme [only
substrate and buffer (served as substrate blank wells)]. The IC50 was
the amount of inhibitor required to inhibit 50% enzymatic activity
compared to control.
5.2.2. In vitro prothrombin time (PT) prolongation in human and
rat plasma
d6, 400 MHz) d: 3.02e3.09 (m, 1H), 3.16e3.22 (m, 1H), 3.24 (s, 3H),
3.48 (t, J ¼ 6.8 Hz, 2H), 7.66e7.69 (dd, J ¼ 2.4 and 8.8 Hz,1H), 7.91 (d,
J ¼ 2.4 Hz,1H), 7.93e7.96 (dd, J ¼ 2.8 and 9.2 Hz,1H), 8.02e8.04 (bd,
2H), 8.09e8.13 (m, 4H), 8.44 (d, J ¼ 2.4 Hz, 1H), 11.18 (s, 1H), 11.26
(s, 1H); ESI/MS m/z 570.6 (M þ H)þ.
Blood was obtained from healthy volunteers or rat and anti-
coagulated with 3.8% sodium citrate. Plasma was obtained after
centrifugation at 2000g for 10 min. An initial stock solution of the
inhibitor was prepared in DMSO. Subsequent dilutions were done
in plasma. Clotting time was determined on control plasma and
plasma containing five to seven different concentrations of inhib-
itor. Prothrombin time (PT) measurement was performed in
a temperature-controlled automated coagulation device (Sysmex
CA50, Dade-Behring) using Thromborel-S (Dade Behring) kit
according to the reagent instructions. Determinations at each
plasma concentration were done in duplicate. Anticoagulant
activity was defined as the concentration required to double the
prothrombin time [PTCT2].
5.1.1.61. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(dimethyla-
mino)ethyl)-S-methylsulfonimidoyl)benzamido)benzamide (22a).
10 mol eq. 50% dimethylamine was used and this compound was
prepared from 21 by means of the procedure similar to that re-
ported for 18a as off white solid; yield: 26%; mp: 131e133 ꢀC;
purity by UPLC: 99.06%; IR (KBr) 1684, 1635, 1575, 1507, 1459,
1374, 1296, 1229, 835, 744 cmꢁ1; 1H NMR (DMSO-d6, 400 MHz)
d:
2.11 (s, 6H), 2.31e2.38 (bd, 2H), 2.72e2.78 (m, 1H), 2.87e2.93
(m, 1H), 3.17 (s, 3H), 7.64 (d, J ¼ 8.8 Hz, 1H), 7.91 (bs, 2H), 7.99
(d, J ¼ 8.4 Hz, 2H), 8.12e8.14 (bd, 4H), 8.42 (d, J ¼ 2.0 Hz, 1H), 11.93
(bs, 1H); ESI/MS m/z 533.9 (M þ H)þ.
5.2.3. Ex vivo PT prolongation in rats
Male rats weighing 230e280 g were used in these studies. In
rats, the test drug was formulated in polyethyleneglycol (PEG)-
400:0.5% sodium carboxymethylcellulose (1:9) and administered to
animals orally at 30 mg/kg and 5 mL/kg dosing volume using
a gastric tube. Citrated blood was collected from retro orbital plexus
2 h after oral administration. Platelet-poor plasma was prepared by
centrifugation for measurement of PT. All data were expressed as
relative fold values, compared with the baseline value of the vehicle
group in rat.
5.1.1.62. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(dieth-
ylamino)ethyl)-S-methylsulfonimidoyl)benzamido)benzamide (22b).
2 mol eq. diethylamine was used and this compound was prepared
from 21 by means of the procedure similar to that reported for 18a
as off white solid; yield: 60%; mp: 63e65 ꢀC; purity by UPLC:
98.34%; IR (KBr) 1654, 1577, 1506, 1458, 1373, 1294, 1072, 831,
744 cmꢁ1
;
1H NMR (CDCl3, 400 MHz)
d
: 0.98 (t, J ¼ 7.2 Hz, 6H),
2.46e2.53 (m, 4H), 2.64 (t, J ¼ 4.0 Hz, 2H), 2.87e2.95 (m, 1H), 3.04e
3.09 (m, 1H), 3.12 (s, 3H), 7.58e7.61 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 7.73
(d, J ¼ 2.0 Hz, 1H), 7.76e7.79 (dd, J ¼ 2.8 and 8.8 Hz, 1H), 8.08 (d,
J ¼ 8.4 Hz, 2H), 8.18 (d, J ¼ 8.4 Hz, 2H), 8.28e8.38 (m, 2H), 8.87 (d,
J ¼ 8.8 Hz, 1H), 11.93 (bs, 2H); ESI/MS m/z 561.6 (M þ H)þ.
5.2.4. Pharmacokinetic study in rats
The hydrochloride salt of 18f was formulated with Tween-
80:PEG:CMC (5:5:90% v/v), A graduated dose volume (5 mL/kg) of
suspension was administered to fasted male Wistar rats at 30 mg/
kg po. The animals were anesthetized for blood sample collection
from retro-orbital plexus. Serial blood samples were collected into
heparinised containers at various time points and blood centri-
fuged to yield plasma. Plasma concentration was determined by
using LCeMS/MS method.
5.1.1.63. 5-Chloro-N-(5-chloropyridin-2-yl)-2-(4-(S-methyl-N-(2-
(pyrrolidin-1-yl)ethyl)sulfonimidoyl)benzamido)benzamide (22c).
2 mol eq. pyrrolidine was used and this compound was prepared
from 21 by means of the procedure similar to that reported for
18a as off white solid; yield: 64%; mp: 88e90 ꢀC; purity by UPLC:
99.03%; IR (KBr) 1656, 1573, 1521, 1460, 1375, 1315, 1226, 1064,
5.2.5. Enzyme selectivity of 18f and 18b
842, 750 cmꢁ1
;
1H NMR (CDCl3, 400 MHz)
d
: 1.72 (bs, 4H), 2.47
Reaction mixtures were prepared in 96-well plates containing
the chromogenic substrate and test compound. The reaction was
initiated by the addition of enzyme, and the color was continuously
(bs, 4H), 2.59e2.74 (m, 2H), 2.93e3.00 (m, 1H), 3.06e3.18 (m, 1H),
3.13 (s, 3H), 7.58e7.61 (dd, J ¼ 2.4 and 9.2 Hz, 1H), 7.74

V. Pandya et al. / European Journal of Medicinal Chemistry 58 (2012) 136e152
151
monitored at 405 nm using a microplate reader Spectra Max 340PC
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We are grateful to the management of Zydus Group for
encouragement and analytical department for their support.

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