Structure-guided discovery of 1,3,5 tri-substituted benzenes as potent and selective matriptase inhibitors exhibiting in vivo antitumor efficacy

Article abstract of DOI:10.1016/j.bmc.2014.04.013

Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is frequently dysregulated in human cancers and matriptase has been reported to activate latent growth factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer. Here we report a structure-based approach which led to the discovery of selective and potent matriptase inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge interactions in the S1 pocket, as well as strong CH-π contacts between the P2/P4 cyclohexyl and Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain results in substantial improvement in matriptase inhibition and selectivity against other related serine proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate cancer mouse model. These compounds could be useful as tools to further explore the biology of matriptase as a drug target.

Full text of DOI:10.1016/j.bmc.2014.04.013

Bioorganic & Medicinal Chemistry 22 (2014) 3187–3203
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-guided discovery of 1,3,5 tri-substituted benzenes
as potent and selective matriptase inhibitors exhibiting in vivo
antitumor efficacy
Rajeev Goswami a, Subhendu Mukherjee a, Chakshusmathi Ghadiyaram a, Gerd Wohlfahrt b,
Ramesh K. Sistla a, Jwala Nagaraj a, Leena K. Satyam a, Krishnaprasad Subbarao a, Rajendra K. Palakurthy a,
Sreevalsam Gopinath a, Narasimha R. Krishnamurthy a, Tarja Ikonen b, Anu Moilanen c,
Hosahalli S. Subramanya a, Pekka Kallio c, Murali Ramachandra a,
⇑
a Aurigene Discovery Technologies Limited, 39-40 KIADB Industrial Area, Electronic City Phase II, Bangalore 560 100, India
b Orion Corporation, Orionintie 1, FIN-02101 Espoo, Finland
c Orion Corporation, Tengströminkatu 8, FIN-20101 Turku, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Matriptase is a serine protease implicated in cancer invasion and metastasis. Expression of matriptase is
frequently dysregulated in human cancers and matriptase has been reported to activate latent growth
factors such as hepatocyte growth factor/scatter factor, and proteases such as urokinase plasminogen
activator suggesting that matriptase inhibitors could have therapeutic potential in treatment of cancer.
Here we report a structure-based approach which led to the discovery of selective and potent matriptase
inhibitors with benzene as central core having 1,3,5 tri-substitution pattern. X-ray crystallography of one
of the potent analogs in complex with matriptase revealed strong hydrogen bonding and salt-bridge
Received 3 March 2014
Revised 4 April 2014
Accepted 4 April 2014
Available online 18 April 2014
Keywords:
Matriptase
Amidine
interactions in the S1 pocket, as well as strong CH–
p contacts between the P2/P4 cyclohexyl and
Trp215 side-chain. An additional interaction of the pendant amine at cyclohexyl with Gln175 side-chain
results in substantial improvement in matriptase inhibition and selectivity against other related serine
proteases. Compounds 15 and 26 showed tumor growth inhibition in a subcutaneous DU-145 prostate
cancer mouse model. These compounds could be useful as tools to further explore the biology of matrip-
tase as a drug target.
Molecular docking
Crystallography
Cancer
In vivo activity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
during their life spans.1 Number of cancer deaths worldwide is pro-
jected to increase, with an anticipated 13 million deaths in 2030.2
According to the American Cancer Society, cancer is the second
principal cause of death in the U.S. with nearly half of all male and
one-third of all female population developing some form of cancer
Patients with cancer die largely because of metastasis, rapid
creation of abnormal cells that grow beyond their usual boundaries
and spread from one organ to another. Proteases are involved in
cancer spread by mediating the degradation of basement
membrane and extracellular matrix constituents thus facilitating
migration of cells into the extracellular environment. Matriptase
is a type II transmembrane serine protease broadly expressed in
epithelial tissues and commonly overexpressed in many tumor
types,3,4 such as in prostate adenocarcinomas.5,6 The oncogenic
and metastatic activities of matriptase derive from its ability to
degrade extracellular matrix and to cleave and activate various
pro-oncogenic and pro-metastatic factors including inactive pre-
cursor of hepatocyte growth factor/scatter factor (pro-HGF/SF).7
The potent oncogenic activity of matriptase was demonstrated
using a transgenic mice model in which mild overexpression of
Abbreviations: Ac, acetyl; Boc, tert-butoxycarbonyl; DCM, dichloromethane;
DIPEA, di-iso-propylethylamine; DMF, N,N-dimethylformamide; DMAP, 4-dimeth-
ylaminopyridine; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; EtOAc, ethyl acetate; EtOH, ethanol; Et2O, diethyl ether; HOBt,
1-N-hydroxybenzotriazole; Py, pyridine; SAR, structure–activity relationship; HPLC,
high pressure liquid chromatography; LCMS, liquid chromatography–mass spec-
trometry; MeOH, methanol; mp, melting point; rt, room temperature; TEA,
triethylamine; TLC, thin layer chromatography; THF, tetrahydrofuran; ADME,
absorption distribution metabolism and excretion; PPB, plasma protein binding;
LLE, ligand-lipophilicity efficiency; CYP, cytochrome P450 enzymes; MLM, mouse
liver microsomes; RLM, rat liver microsomes; HLM, human liver microsomes.
⇑
Corresponding author. Tel.: +91 80 7102 5444; fax: +91 80 2852 6285.
E-mail address: murali_r@aurigene.com (M. Ramachandra).
http://dx.doi.org/10.1016/j.bmc.2014.04.013
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

3188
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
matriptase in the skin resulted in spontaneous squamous cell car-
cinoma and a very high susceptibility to carcinogen-induced tumor
formation.8 Interestingly, the oncogenic activity of matriptase was
completely negated when HAI-1, the endogenous inhibitor of
matriptase, was simultaneously overexpressed in the transgenic
mice.8 Matriptase also activates other serine proteases such as uro-
kinase-type plasminogen activator (uPA) that plays a vital role in
angiogenesis, tumor invasion and metastasis. Downregulation of
matriptase has also been shown to inhibit tumor invasion through
suppression of uPAR activation.9,10 Taken together, the available
data demonstrate that matriptase is functionally engaged in tumor
progression and thus represents a potential target for anti-cancer
therapy.
In the past decade, potent and selective matriptase inhibitors
from diverse chemical classes (Fig. 1) including bis-benzami-
dines,11 amidinophenylalanines12–14 and peptidomimetics15 have
been reported. A simple bis-benzamidine compound such as 1
(Fig. 1) is known to inhibit matriptase with a Ki of ꢀ200 nM and
>10 fold selectivity over uPA and thrombin.11 A very potent tribasic
matriptase inhibitor 2 (Fig. 1) with a Ki of 80 pM and >100 fold
selectivity over related serine proteases including plasmin, uPA,
thrombin and factor Xa was reported by Curacyte.13 Recently, a
peptidomimetic15 has also been demonstrated to inhibit matrip-
tase with a Ki of 11 nM with several fold selectivity over other
tested serine proteases including thrombin. However, only limited
information is available on the activity of selective small molecule
matriptase inhibitors (3,12 412 and 516) in the cellular and in vivo
tumor models.
around 4th and 5th positions of the pyridyl core to attempt substi-
tutions (Fig. 2). In addition, since the pyridyl nitrogen was not
engaged in any polar contacts, we decided to replace this nitrogen
with CH as a benzene core (Fig. 2).
This article describes the synthesis of compounds from a chem-
ical series with benzene as central core having 1,3,5 tri-substitu-
tion pattern (prepared as shown in Schemes 1–3) as potent and
selective inhibitors. SAR was explored for achieving greater
selectivity for matriptase over other trypsin-like serine proteases.
Two of the early leads exhibited potent inhibition of matriptase
with selectivity over related serine proteases. Furthermore, the
lead compounds displayed inhibition of invasion, migration and
matrix-independent growth in cellular assays and a desirable
DMPK profile, which in turn contributed to significant tumor
growth inhibition in a xenograft model of prostate cancer.
2. Chemistry
The synthetic scheme for the preparation of 3,5-bis(4-carbam-
imidoylphenoxy)phenyl derivatives is outlined in Scheme 1.
The commercially available ethyl 3,5-dihydroxybenzoate (44)
was treated with 4-fluorobenzonitrile (4 equiv) to afford ethyl
3,5-bis(4-cyanophenoxy)benzoate (45), followed by its hydrolysis
with LiOH to get 3,5-bis(4-cyanophenoxy)benzoic acid (46), which
was subjected to Curtius Rearrangement using diphenylphosphory-
lazide18 to afford 4,40-((5-amino-1,3-phenylene)bis(oxy))dibenzo-
nitrile (47). Acid 46 was coupled with commercially available
amines using EDCI to give an amide 48. Treatment of nitrile groups
of 48 with aq NH2OH17 in ethanol converted it to N-hydroxyami-
dine (49), followed by acetylation and reduction with zinc afforded
amidine (51). The amino group (if present as Boc-protected) or
hydroxyl group (if present as THP-protected) of 51 was deprotec-
ted using ethanolic-HCl to obtain 9–22. The inverted-amide ana-
logs (23–27) were synthesized by the coupling of 47 with a
carboxylic acid, followed by its nitrile group conversion to the
amidine and then deprotection of amino/hydroxyl group to give
the desired compounds.
The sulfonamide analogs (28–29) were synthesized (Scheme 1)
by treating 47 with an arylsulfonyl chloride and then conversion of
their nitrile group to amidine provide the 4,40-((5-(sulfonamido)-
1,3-phenylene)bis(oxy))dibenzimidamide derivatives. However,
(1r,4r)-4-aminocyclohexyl 3,5-bis(4-carbamimidoylphenoxy)ben-
zoate (30) was prepared by the coupling of 46 with an alcohol,
followed by its nitrile groups conversion to the amidine and then
Boc-protected amino group was deprotected to give the desired
compound. The binding affinity data for selected compounds are
summarized in Table 1.
We have previously identified selective matriptase inhibitors17
through structure-guided design strategies based on the pyridyl
bis(oxy) dibenzimidamide scaffold. Notably, these compounds
exhibited selective target inhibition, favorable aqueous solubility
and microsomal stability along with good potencies on prostate
carcinoma cell lines.17 However, only limited SAR was discussed
in that report.17 In this article, we report extensive SAR of structur-
ally related matriptase inhibitors identified through structure-
guided design approach. In addition, we have also discussed about
the in vivo efficacies of the two early lead compounds from this
series.
During optimization of the chemical series described
previously,17 we were able to solve a high resolution co-crystal
structure of one of the preliminary hits (compound 6, matriptase
Ki = 218 nM) in complex with matriptase catalytic domain (PDB
ID: 4JZ1).17 Apart from providing information on the different tar-
get recognition moieties, this crystal structure also revealed inter-
esting opportunities for further SAR exploration. The binding mode
of compound 6 showed that there is sufficient space available
NH2
NH2
H2N
N
O
N
HN
HN
HN
H2N
O
S2
O
O
N
H
O
HN
OH
O
NH2
1
2
NH
O
O
H
NH2
N
NH2
NH
NH2
O
HN
H
O
N
H2N
O
O
N
O
O
O
S2
HN
S2
N
N
H
H
NH
HN
HN
HN
NH2
3
4
NH2
5
NH2
Figure 1. Structures of known matriptase inhibitors.

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3189
Figure 2. Key learnings from crystallographic binding mode of compound 6 (PDB ID: 4JZ1). Regions highlighted in green indicate moieties engaging polar contacts with
different residues.
Unsymmetrical compounds were synthesized (Scheme 2) by
treating 44 with 4-fluorobenzonitrile (1.5 equiv) to obtain ethyl
3-(4-cyanophenoxy)-5-hydroxybenzoate (63), which was hydro-
lyzed to give an acid 64, followed by its coupling with tert-butyl
((1r,4r)-4-aminocyclohexyl)carbamate resulted in an amide 65.
Using the hydroxyl group of 65 to couple with an alkyl or aryl
halide afforded 66. Using the method described in Scheme 1,
nitrile/s of 66 was/were converted to the amidine and then Boc-
protected amino group was deprotected using ethanolic-HCl to
give 31–39. The nitrile group in 65 was reduced to a Boc-protected
aminomethyl19 group to afford 67. The free hydroxyl group of 67
was treated with an aryl halide followed by the nitrile group (if
present) conversion to the amidine and then deprotection of Boc-
protected amino group yielded 40–41.
Compounds with methylene linker were synthesized as out-
lined in Scheme 3. Compound 44 was treated with 3/4-(bromo-
methyl)benzonitrile to get an ester (75), followed by the ester
group hydrolysis to form the acid (76), and then coupling with
tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate afforded an
amide (77). Using the hydroxylamine method described in
Scheme 1, the nitriles were converted to the amidine, and then
deprotection of Boc-protected amino group afforded 42–43.
7–11 and 23 (Table 1) to understand the tolerability of different
linkers such as amide, inverted-amide, ester and sulphonamide
with various cyclic/acyclic groups at the 5-position for matriptase
inhibition. Interestingly, all these compounds with such diverse R
groups (Table 1) demonstrated moderate inhibition of matriptase
with Ki in the range of 410–1600 nM, which indicated that the
5-position is not only amenable to substitution but can also accom-
modate fairly bulky and conformationally puckered groups.
Additionally, the fact that most of these initial modifications (7–
11 and 23) with diverse R groups (Table 1) exhibited moderate
matriptase binding, indicates that the R groups possibly project
towards a solvent exposed S2/S4 (spacious) region of the protein.
Further molecules in this series were primarily designed through
de novo approach based on predicted binding modes of these pre-
liminary hits. Figure 3 illustrates the docked model of one such
compound, 11 (Table 1). This model indicated possibilities of
salt-bridge interaction in the S1 pocket involving one of the ami-
dines as observed earlier17,20 and the other amidine orienting
towards the S10 region making hydrogen bonding interaction with
the backbone carbonyl of Ile60. The 4-methyl cyclohexyl amide
appendage was anticipated to project towards S2/S4 region and
the 4-methyl group occupying a region close to the polar side-
chain of Gln175. We therefore targeted the region close to the
polar side-chain of Gln175 with small hydrophilic groups on the
cyclohexyl amide. Additionally, to develop an understanding on
potential selectivity hot-spots, we compared the catalytic sites of
factor Xa, thrombin, plasmin, trypsin and matriptase (Fig. 4).
3. Results and discussion
Initially, simple but diverse 5-substituted phenylene1,3-
bis(oxy) dibenzimidamide analogs were synthesized, for example,

3190
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
HO
O
O
S2
NH2
46
NHBoc
R
O
NH
CN
CN
EtO
O
CN
d
OH
CN
a, b
c
O
O
O
O
O
O
j
O
HO
OH
BocHN
56
47
46
44
O
O
CN
O
CN
CN
i
59
d
e
R
O
R
O
NH
CN
57 (N-hydroxyamidine)
e
CN
CN
f
60 (N-hydroxyamidine)
O
O
O
58 (N-acetoxyamidine)
f
48
52
g
61
(N-acetoxyamidine)
CN
CN
e
e
g, h
O
S2
R
O
NH
NH2
NH
49 (N-hydroxyamidine)
53 (N-hydroxyamidine)
O
O
NH2
f
f
H2N
NH
O
50 (N-acetoxyamidine)
54 (N-acetoxyamidine)
O
O
g, h
g, h
O
28-29
R
O
30
NH2
R
O
NH
NH2
NH
HN
NH2
HN
NH2
NH
O
O
O
9-22
23-27
HN
NH2
HN
NH2
Scheme 1. Synthesis of 3,5-bis(4-carbamimidoylphenoxy)phenyl derivatives. Reagents and conditions: (a) 4-fluorobenzonitrile, K2CO3, DMF, 80 °C, 6 h; (b) LiOH, THF, water,
5–10 °C, 2 h; (c) diphenylphosphorylazide, DIPEA, t-BuOH, toluene, 80 °C, 12 h; (d) cycloalkyl or aryl amine/alcohol (with or without pendant amine masked with Boc), EDCI,
HOBt, DIPEA, DMF, rt, 6 h; (e) aq NH2OH, EtOH, 75 °C, 6 h; (f) Ac2O,AcOH, rt, 2 h; (g) Zn, AcOH, rt, 6 h; (h) HCl(g)–EtOH, 5–10 °C, 2 h; (i) cycloalkylcarboxylic acid(with or
without pendant amine masked with Boc), PyBop, DIPEA, DMF, rt, 6 h; (j) arylsulfonyl chloride, Py, DMAP, DCM, rt, 12 h.
H
H
HO
O
N
O
N
O
EtO
O
BocHN
BocHN
a, b
OH
c
d
R
O
OH
O
OH
O
O
HO
66
64
65
44
CN
CN
CN
i
e-h
H
H
H
N
N
O
N
O
O
H2N
BocHN
H2N
d, e-h
R
R
O
O
O
OH
O
O
31 - 39
40 - 41
67
H2N
BocHN
HN
NH2
Scheme 2. Synthesis of N-(4-aminocyclohexyl)benzamide derivatives. Reagents and conditions: (a) 4-fluorobenzonitrile (1 equiv), K2CO3, DMF, 45 °C, 6 h; (b) LiOH, MeOH,
water, 10 °C, 2 h; (c) tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate, EDCI, HOBt, DIPEA, DMF, rt, 6–8 h; (d) R-X, K2CO3,DMF, 65 °C, 4 h; (e) aq NH2OH, EtOH, 75 °C, 6 h; (f)
Ac2O, AcOH, RT, 2 h; (g) Zn, AcOH, rt, 3 h; (h) amino group deprotection: HCl(g)–EtOH at 5–10 °C, 2 h; (i) NiCl2ꢁ6H2O, NaBH4, methanol, 0–10 °C, 4 h.
Gln175 in matriptase was observed to be non-conserved and the
loop harboring Gln175 (corresponding to Ile in factor Xa, Arg in
thrombin and plasmin, Lys in trypsin) as well as the adjoining
90-loop was also found to have different lengths in these proteases,
which further indicated that the S4 site might also be different
than in all these trypsin-like serine proteases. Therefore, com-
pounds with terminal polar groups which might project towards
S4 site and interact with the Gln175 side-chain were synthesized

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3191
H
N
O
H
N
O
HO
O
EtO
O
H2N
BocHN
NC
d-g
a, b
c
O
O
O
O
O
O
HO
OH
HN
NH2
CN
NH2
HN
NC
CN
76
77
42 - 43
44
Scheme 3. Synthesis of N-(4-aminocyclohexyl)-3,5-bis((3/4-carbamimidoylbenzyl)oxy)benzamide derivatives. Reagents and conditions: (a) 3/4-(bromomethyl)benzonitrile,
K2CO3, DMF, 65 °C, 6–7 h; (b) LiOH, THF, MeOH, 10 °C, 2 h; (c) tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate, EDCI, HOBt, DIPEA, DMF, rt, 6–8 h; (d) aq NH2OH, EtOH, 75 °C,
6 h; (e) Ac2O, AcOH, rt, 2 h; (f) Zn, AcOH, rt, 3 h; (g) amino group deprotection: HCl(g)–EtOH at 5–10 °C, 2 h.
Table 1
SAR of 5-substituted phenylene 1,3-bis(oxy) dibenzimidamide derivatives 7–30
O
O
H2N
NH2
NH
NH
R
ID
R
Kia (nM)/% inhibitionb at 5
lM
Matriptase
uPA (%)
f Xa
Thrombin
Plasmin
Trypsin
7
8
–CONH2
–NH2
O
1617
1060
13
22
785
1056
2816
5%
6%
640
534
851
190
9
592
411
770
9
1636
2406
744
2395
2160
3610
179
571
253
HN
NH
O
10
11
13
19
73
O
282
HN
O
CH3
OH
12
13
14
15
301
204
141
10
15
9
538
640
415
516
1240
1785
1285
2105
15%
14%
3%
ndc
HN
O
ndc
OH
HN
O
N
NH2
9
1035
1625
HN
O
N
14
12%
HN
NH2
NH2
O
N
16
17
94
4
1289
920
4061
2852
17%
18%
545
NH
O
1182
16
1550
OH
O
O
O
18
1850
6
1026
23%
2630
471
HN
N
O
N
OH
19
20
20%
12
13
29%
5%
0%
718
232
O
O
N
305
336
501
1530
O
N
21
730
12
273
1562
3855
225
O
22
23
591
545
20
10
1877
743
2105
2343
2665
1205
1636
516
HN
NH
F
CH3
O
(continued on next page)

3192
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
Table 1 (continued)
ID
R
Kia (nM)/% inhibitionb at 5
lM
Matriptase
61
uPA (%)
f Xa
Thrombin
Plasmin
2590
Trypsin
541
NH2
NH2
NH
24
9
95
2680
N
O
NH
O
N
25
26
18
3
12
23
62
2530
2216
2410
1338
894
O
NH
NH2
503
28%
O
NH
OH
N
27
28
18
17
10
1160
1164
4202
4850
18%
7%
1468
1388
O
NH
O2S
1982
F
NH
O2S
29
30
1790
1
13
25
876
44
25%
0%
3000
264
O
O
2467
28%
NH2
a
b
c
Ki: Mean of at least two independent experiments with standard deviation within 10%.
% Inhibition: Ki was not determined for compounds which show 630% inhibition at 5
lM concentration.
nd: not determined against the mentioned enzyme.
Figure 3. Overlay of the docked model of 11 (pink) on the crystallographic binding mode of 15 (green) in matriptase catalytic domain (PDB ID: 4O9V). Grey spheres indicate
the positions of stable water sites (
D
G <ꢂ1 Kcal/mol) from WaterMap analyses on co-crystal structure of a structurally related matriptase inhibitor (PDB ID: 4JZ1).17
to improve matriptase inhibition as well as selectivity over other
serine proteases. An improvement in matriptase inhibition was
observed by replacing the terminal methyl group with a hydroxyl
group (11 vs 12). This motivated us to synthesize further analogs
including 13–16 having different linkers and other terminal polar
groups. Interestingly, some of these analogs with 4-amin-
ocyclohexyl appendage, for example, 15, 26 and 30 exhibited
significantly improved matriptase inhibition with the Ki of 10, 3
and 1 nM respectively and also P40 fold selectivity over other
serine proteases (Table 1). Improvement in target inhibition and

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3193
Figure 4. Sequence comparison of matriptase (m), factor Xa (f), thrombin (th), plasmin (p) and trypsin (tr) catalytic domains.
selectivity over other related proteases indicated that different
linkers including amide, inverted-amide and ester are well toler-
ated as these did not exhibit significant difference in matriptase
inhibition. Compound 30 with an ester linker showed most potent
inhibition of matriptase (Ki = 1 nM). However, because of low met-
abolic stability, further optimization of compound 30 was discon-
tinued. Compounds 15, 26 and 30 did not show significant
improvement in their Ligand Efficiency (LE) in comparison to
compound 7 (LE ~ 0.3 for all 4 compounds) but interestingly these
compounds 15, 26 and 30 exhibited markedly improved
Ligand-Lipophilicity Efficiency (LLE) by ꢀ2 units in comparison to
7 (LLE = 3.9), thereby demonstrating that incorporation of the
amide/inverted-amide/ester linked cyclohexyl amine not only
improved matriptase affinity and selectivity but also ‘drug-
likeness’.
Since compound 15 showed potent matriptase inhibition
(Ki = 10 nM) and selectivity (>50 fold) over other related proteases,
a high resolution crystal structure of matriptase catalytic domain
in complex with 15 was solved (PDB ID: 4O9V). This crystal struc-
ture revealed a similar binding mode to the docking model of 11
(Fig. 3), thereby validating the working hypothesis followed during
lead identification. Most significantly (and as expected), the 4-
aminocyclohexyl was observed to engage in polar contact with
the Gln175 side-chain carbonyl.
CH–p contacts between P2/P4 cyclohexyl and Trp215 side-chain.
Thus, bicyclic analogs 20 and 21 at R (Table 1) were synthesized
to test whether incorporation of bicyclic systems can improve
matriptase inhibition in comparison to 9. However, except for 20,
which also showed only moderate matriptase inhibition, none of
the other compounds exhibited any improvement in binding.
Therefore, 4-aminocyclohexyl with amide linker (15) was retained
as R for further optimization with an option of introducing the
inverted-amide analog (as in 26). While one of the amidines of
15 was observed to form a strong bidentate salt-bridge in S1
pocket, the other amidine was found to be involved in a relatively
weak hydrogen bond with Ile60 backbone in the S10 region (Fig. 3).
Therefore, we studied the effect of P10 alterations on matriptase
inhibition keeping other features unchanged as in 15 (Table 2). A
couple of modifications were tested by synthesizing compounds
31 and 32 with R1 = 4-aminophenoxy and 6-aminopyridin-3-oxy
respectively. These substitutions were not expected to engage in
a hydrogen bond with the Ile60 backbone having the P10 oriented
like that in 15, but the basic objective was to assess whether the
P10 end can adopt a slightly different orientation so that the amine
in 31 or 32 might be involved in the same Ile60 backbone contact
as it is observed in 15. However, none of these demonstrated good
matriptase binding and in fact showed >1000 fold reduced inhibi-
tion. Compound 33 was synthesized by replacing the P10 4-car-
Owing to the high binding affinities observed with 15 and 26,
further analogs were synthesized replacing the P2/P4 amino cyclo-
hexyl amine of 15 with appendages having terminal carboxylates
as in 18 and 19 assuming that the Gln175 might flip and the
side-chain NH could make a polar contact with carboxylate oxy-
gen. However, all these analogs surprisingly showed reduced
matriptase inhibition. Further analysis of the crystallographic
binding mode of 15 with matriptase indicated that the Gln175
side-chain NH makes a long intra-molecular polar interaction with
the backbone carbonyl of neighboring Pro173 (Fig. 3) and because
of this the Gln175 side-chain likely remains stabilized in only one
rotameric state. Such phenomenon could contribute to the lower
inhibition observed for compounds with terminal P2/P4 hydrogen
bond acceptors, while the presence of a terminal hydrogen bond
donor such as primary amine might be critical. Apart from the dif-
ferent hydrogen bonding or salt-bridge interactions observed in
the binding mode of 15, the crystal structure also revealed strong
bamimidoylphenoxy of 15 with a 4-carbamoylphenoxy but
unexpectedly this also lead to >800 fold reduced potency. We
hypothesized that it could be due to orientation of the amide car-
bonyl towards the Asp96 side-chain carboxylate (Fig. 3), which
would create a repulsive interaction. Reduced potency could also
be due to the fact that the non Ile60 interacting amidine NH (ami-
dine NH not engaged in polar contact with Ile60 back-bone) in 15
is involved in water-mediated contacts that were not resolved in
the crystal structure and replacing this NH with a carbonyl as in
33 likely displace a stable water molecule due to water-amide car-
bonyl lone pair repulsions. The latter possibility seemed plausible
because WaterMap analysis carried out on a co-crystal structure
of our earlier compounds17 predicted stable water molecules (
DG
<ꢂ1 Kcal/mol) in the S10 site (Fig. 3). This rationale was supported
by the improved inhibition observed with R1 (Table 2) as 4-(amino-
methyl)phenoxy (40), which exhibited improved inhibition possi-
bly because the hypothesized stable water molecule does not get

3194
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
Table 2
SAR of 5-substituted N-((1r,4r)-4-aminocyclohexyl)-3,5-(bis-alkyl/aryloxy) benzamide derivatives
O
O
R1
R2
O
H2N
NH
ID
R1
R2
Kia (nM)/% inhibitionb at 5
lM
Matriptase
uPA (%)
f Xa
Thrombin
Plasmin (%)
Trypsin
>3000d
31
32
NH2
NH2
4-Benzimidamide
4-Benzimidamide
1131
2032
4
28%
18%
0
5
1
14%
26%
1%
0
8
>3000d
>3000d
N
NH2
O
33
34
4-Benzimidamide
4-Benzimidamide
824
10
3540
NH
7
25%
2808
2677
22
13
1868
1541
NH2
NH2
35
4-Benzimidamide
6
11
1140
HN
O
36
37
4-Benzimidamide
4-Benzimidamide
116
793
2
8
3580
3685
3250
3985
8
5
>3000d
>3000d
NH
Br
NH2
N
38
39
Cl
4-Benzimidamide
4-Benzimidamide
470
260
15
23
ndc
ndc
ndc
ndc
Br
2089
3973
13
2064
NH2
40
41
4-Benzimidamide
40
5
4
21%
21%
3125
8
0
1541
3850
1698
9%
NH2
NH
NH2
NH
42
43
23
3
15
11
1558
157
1642
1289
16
10
1022
901
NH2
NH2
NH
NH
H2N
H2N
a
b
c
Ki: Mean of at least two independent experiments with standard deviation within 10%.
% Inhibition: Ki was not determined for compounds which show 630% inhibition at 5
nd: not determined against the mentioned enzyme.
lM concentration.
d
Indicates that Ki was not determined because of the poor dose response in the biochemical assay.
displaced from the S10 site. At the same time, the binding was
slightly different that of 15, likely due to the missing water-mediated
interactions. Further modeling studies suggest the possibility of
water-mediated interactions of the non Ile60 interacting P10
amidine NH of 15 with Asp96 in the 90-loop region. The Ile60
interacting P10 amidine NH2 of 15 was also anticipated to engage
in an additional water-mediated contact with Asp60B in the
60-loop region. Subsequently, additional analogs were designed
to interact with Asp96 or Asp60B apart from making the Ile60
backbone contact, like 34–35. Interestingly, most of these inhibi-
tors exhibited similar potency as 15, which further validated the
hypothesis on water-mediated interactions with a 60-loop residue.
Figure 5 depict the likely binding modes of 34 and 35.
target inhibition of 37–39 suggested that vdW interactions alone
in this region might not have significant contribution in binding.
Furthermore, a few symmetric compounds were synthesized to
understand the effect of P1 alterations as shown in Table 2. The
non-amidine compound 41 resulted in a 40-fold reduced matrip-
tase inhibition in comparison with 40 (R1 = 4-(aminomethyl)phen-
oxy, Table 2), thereby signifying the importance of P1 amidine in
binding. However, compounds 42 and 43 with P1-benzamidine
but with additional methylene spacer showed similar inhibition
to 34 and 35 respectively, suggesting that spacers may be tolerated
between P1 benzamidine and the ether linker. Among these, 43
showed the highest inhibition of matriptase with the Ki of 3 nM
and >50 fold selectivity over other trypsin-like serine proteases.
A crystal structure of matriptase catalytic domain in complex
with 43 was solved (PDB ID: 4O97). The binding mode of 43 was
different in comparison to 15 because the 4-aminocyclohexyl
amide appendage (P2/P4 appendage of 15) pointed away from
To understand the impact of van der Waals (vdW) contacts in
the S10 region, which is slightly hydrophobic in nature due to the
presence of a disulfide bridge and a tyrosine residue (Fig. 2), com-
pounds 37–39 (Table 2) were synthesized. However, the reduced

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3195
Figure 5. Predicted binding modes of 34 (a, marine blue) & 35 (b, light pink) compared to the crystallographic binding mode of 15 (green) in matriptase catalytic domain (PDB
ID: 4O9V). Grey spheres indicate projected distribution of stable water molecules (
D
G ~ ꢂ1 Kcal/mol) from WaterMap analyses on co-crystal structure of a structurally related
matriptase inhibitor (PDB ID: 4JZ1).17
the S2/S4 site (Fig. 6). This indicated that the SAR trend observed
for the 3,5-bis(4-carbamimidoylphenoxy) analogs may not trans-
late to the 3,5-bis((4-carbamimidoylbenzyl)oxy analogs and hence
its further advancement will be discussed separately in due course.
4. Cellular activity and biopharmaceutical properties
After establishing SAR for matriptase inhibition and selectivity
against other proteases, we evaluated the effect of selected com-
pounds on migration and invasion of matriptase expressing pros-
tate cancer cell lines5,21,22 DU-145, PC-3 and LNCaP C42B4
(Table 3). An androgen-sensitive prostate cancer cell line, LNCaP
C42B4 was evaluated after confirmation of matriptase protein
expression (data not shown). This LNCaP C42B4 is a subline of
LNCaP that is more invasive than the parental cells. Compounds
that were potent in these assays without exhibiting cytotoxicity
were further evaluated for inhibition of soft agar colony formation
in matriptase expressing prostate cancer cell lines (Table 4). Impor-
tantly, most of the compounds which exhibited good biochemical
potency for matriptase also displayed good cellular potency with-
out any signs of cytotoxicity.
The cellular potency for this series of compounds varied
depending on the pendant amine at amide appendage and also
on P1 amidine/non-amidine motif. At 10 lM concentration, com-
pound 15 (with amide linker) and 30 (with ester linker) exhibited
Figure 6. Overlay of 15 (green) and 43 (white) crystallographic binding modes.
no cytotoxicity and caused >80% inhibition of invasion and
Table 3
Effect of selected compounds on DU145, LNCaP C42B4 and PC-3 cells
ID
% Cell survival at 10
LNCaP C42B4
lM
% Inhibitiona at 10
LNCaPC42B4
lM
DU-145
PC-3
DU-145
PC-3
Migration
Invasion
Migration
Invasion
Migration
Invasion
15
16
20
24
26
30
39
40
100
100
78
98
100
92
95
100
79
99
90
95
65
100
96
93
87
80
94
82
93
100
88
63
18
98
96
81
50
53
4
2
2
2
1
2
7
2
81
62
45
94
98
89
61
49
1
3
3
2
2
1
4
3
91
73
19
69
61
98
63
60
2
1
1
2
9
3
2
4
84
61
15
60
63
90
76
59
1
3
1
3
2
1
1
4
98
48
60
54
84
91
66
73
2
6
5
2
4
4
3
5
97
54
47
37
86
95 10
51
54
8
3
1
2
1
78
90
7
2
a
Values represent mean SD.

3196
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
Table 4
due to the presence of an ester group (ester cleavage was indicated
by metabolite identification studies). In plasma protein binding
studies, surprisingly compound 30 showed >3 fold more free
fraction. Therefore 15, 26 and 30 were selected for in vivo mouse
pharmacokinetics studies. Though 30 showed poor in vitro
microsomal stability, it was also selected to further validate the
in vitro results.
In the subcutaneous PK study (Fig. 7) at 1 mg/kg dose, com-
pounds 15 and 26 showed good exposure and therefore were taken
up further for in vivo efficacy studies. As expected 30 exhibited
very poor exposure and was discontinued from further profiling.
EC50a (nM) values for inhibition of soft agar colony formation of DU145, LNCaP C42B4
and PC-3 cells
ID
DU-145
LNCaP C42B4
PC-3
15
24
26
30
40
200
130
41
9
3
1
5
1364 20
1580 18
1536 13
48
580
105
81
6
8
3
5
152
135
9
324 16
1698 19
485 14
a
Values represent mean SD.
migration in both DU-145, LNCaP C42B4 and PC-3 cell lines while
compound 26 (inverted-amide) showed good inhibition of invasion
and migration in DU-145 and PC-3 cells but moderate inhibition in
LNCaP C42B4 cells. The compounds with lower matriptase inhibi-
tory activity in biochemical assays (16, 20 and 39) tend to display
reduced activity in migration and invasion assays as compared to
the compounds with high activity towards matriptase (1, 3, 10).
The soft agar colony formation assay measures the long-term
survival and anchorage-independent growth of tumor cells. Com-
pounds that were potent inhibitors of invasion and migration were
evaluated for inhibition of soft agar colony formation by DU-145,
LNCaP C42B4 and PC-3 cells (Table 4). Compound 15 displayed
the EC50 of 200 nM, 1364 nM and 48 nM in the soft agar colony for-
mation with DU-145, LNCaP C42B4 and PC-3 cells respectively.
Compound 30 showed relatively similar potency in these three cell
lines. Compound 26 exhibited the EC50 of 41 nM and 105 nM with
DU-145 and PC-3 cells. Ester analog 30 showed the EC50 of 135 nM
on LNCaP C42B4 cells in soft agar colony formation, however other
analogs (amides and inverted-amides) exhibited only moderate
activity.
5. In vivo evaluation of compounds 15 and 26 in a DU-145
prostate cancer model
Since selective matriptase inhibitors 15 and 26 showed signifi-
cant inhibition of migration, invasion and soft agar colony forma-
tion, we investigated the efficacy of these compounds on tumor
growth inhibition in SCID mice bearing subcutaneous DU-145
prostate cancer xenograft tumors.
The compounds 15 and 26 were dosed subcutaneously at 1 and
0.5 mg/kg respectively twice daily for 31 days. Both compounds 15
and 26 exhibited significant inhibition of the median primary
tumor growth of 48% and 52% respectively (Fig. 8). Furthermore,
none of the mice treated with compound 15 and 26 experienced
any significant weight loss (<5%) or showed other clinical signs
of toxicity during or after the treatment.
6. Conclusion
In this work, we have identified highly potent phenyl
bis(oxy)benzamidine compounds as matriptase inhibitors, which
are 20–1000 fold selective against other trypsin-like serine prote-
ases. Replacement of the terminal methyl group (11) with an
amino group at the 4-position of cyclohexyl (15) significantly
improved matriptase inhibition along with selectivity against
In vitro ADME properties for compounds 15, 26 and 30 are
shown in Table 5. All three compounds 15, 26 and 30 showed high
aqueous solubility along with a clean CYP inhibition profile. 15 and
26 exhibited high microsomal stability in MLM, RLM and HLM but
30 showed poor microsomal stability in all three species probably
Table 5
ADME profile of selected compounds (in vitro)
Solubilitya
(
lM)
Metabolic stabilitya (% remaining after 20 min)
CYP Inhibition—IC50
(
lM)
PPB (% free)a
b
ID
MLM
RLM
HLM
3A4
2D6
2C9
2C19
15
26
30
194
200
182
3
2
3
81
83
24
5
1
4
100
91
34
1
4
5
85
97
54
1
3
6
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
8.1
7.2
26.7
1
1
2
a
Values represent mean SD.
SD values for CYP inhibition assays will be 0 since all triplicates showed >30
b
lM IC50
.
1000
(a)
15
24
26
40
(b)
100
10
1
Tmax
(h)
Cmax
(ng/ml)
Beta
t1/2 (h)
AUC (0-inf)
(ng.h/ml)
ID
15
26
0.27
0.75
487
1.6
1000
1852
484
3.3
0
1
2
3
4
5
6
7
8
Time (hours)
Figure 7. Pharmacokinetic profile of 15 and 26 in mice. Male NMRI mice were dosed subcutaneous at 1 mg/kg with compound formulated with 100% PBS and time-points
were pre-dose, 5, 15, 30 min, 1, 2, 4, 6 and 8 h; (a) graphical presentation and (b) tabular representation of the PK data.

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3197
were visualized using UV (254 nm) and/or by staining with potas-
sium permanganate followed by heating. NMR spectra were
recorded on a Varian (1H, 300 or 400 MHz; 13C, 75 or 100 MHz)
spectrometer, using tetramethylsilane as an internal reference,
and chemical shifts were expressed in d (ppm) units (in 1H NMR
description, s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet, and br s = broad singlet). Liquid chromatography mass spec-
tra were obtained from Agilent 1100-LC/MSD VL using mobile
phase: 0.1% TFA-acetonitrile or water–methanol (either by positive
or negative ionization mode). The melting points were determined
on a DBK programmed melting point apparatus from Servewell
Instruments and were uncorrected. The purity values for all biolog-
ically evaluated compounds were >95% (or mentioned otherwise)
as determined by the analysis on high performance liquid
chromatography (HPLC) performed on Agilent 1100 series HPLC
instrument with
a Agilent XDB C18 reverse phase column
(21.2 ꢃ 150 mm, 5micron). The mobile phases were 30% acetoni-
trile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA). The
nomenclature used in this section is based on ChemDraw. Bio-
chemical assays were performed at least in duplicates while func-
tional and ADME assays were performed in triplicates. Matriptase
protein expression, purification, crystallization & data collection
of crystal structures, aqueous solubility and metabolic stability
were determined using the methods as described previously.17
7.2. Chemistry
7.2.1. General procedure for synthesis of 3,5-bis(4-carbamimi
doylphenoxy)benzamides (9–22)
Figure 8. Antitumor activity of compounds 15 and 26 in the DU-145 prostate
cancer xenograft model in SCID mice. Tumors were grown to an average of 70 mm3
and compounds 15 and 26 were administered subcutaneously twice daily for
31 days at 1.0 and 0.5 mg/kg, respectively. (a) Tumor growth. (b) Animal body
weight. Values shown are mean SEM. Statistical analysis was performed by one-
way ANOVA followed by Dunnett’s test,⁄p <0.05.
7.2.1.1. Synthesis of ethyl 3,5-bis(4-cyanophenoxy)benzoate
(45).
A solution of ethyl 3,5-dihydroxybenzoate (44) (1.5 g,
8.23 mmol) in DMF (150 ml) at rt was treated with K2CO3
(4.55 g, 32.92 mmol) and stirred for 10 min. A solution of 4-fluoro-
benzonitrile (4.0 g, 32.92 mmol) in DMF (100 ml) was added drop
wise and the resultant solution was stirred for 4 h at 80 °C. The
reaction mixture temperature was brought down to rt and
quenched with ice-cold water and diluted with EtOAc. The aqueous
layer was extracted with EtOAc (3 ꢃ 150 ml), and the combined
organics were washed with 1 M Na2CO3 solution followed by brine.
Organic layer was dried over anhydrous sodium sulfate and evap-
orated to dryness to get crude oily residue, which was purified by
column chromatography and eluting with 10–20% EtOAc/hexane
to afford ethyl 3,5-bis(4-cyanophenoxy)benzoate as an off-white
solid (2.1 g; yield 66%). 1H NMR (300 MHz, DMSO-d6): d 1.30 (t,
3H), 4.32 (q, 2H), 6.92 (s, 1H), 7.18 (d, 4H), 7.45 (s, 2H), 7.84 (d, 4H).
related proteases by projecting towards S4 site and engaging in an
additional Gln175 side-chain interaction. Potent inhibition of
matriptase by compound 15 could also be due to additional hydro-
gen bonding or salt-bridge interactions. Potent inhibition and
selectivity for matriptase observed for 15 were also supported by
strong CH–p contacts between the P2/P4 cyclohexyl and Trp215
side-chain as observed by X-ray crystallography. Among several
potent and selective matriptase inhibitors, compound 26 also
exhibited excellent activities in several prostate cancer cell lines.
Selected compounds exhibited acceptable DMPK properties that
allowed for their use as tool compounds after subcutaneous
administration. Compound 15 and 26 exhibited primary tumor
growth inhibition of 48% (at 1 mg/kg) and 52% (0.5 mg/kg) respec-
tively in a DU-145 prostate cancer xenograft model. In summary,
high potency, selectivity and in vivo efficacy of the compounds
15 and 26 support their use as pharmacological tools for under-
standing the role of matriptase in cancer as well as in inflammatory
conditions such as osteoarthritis23 where matriptase is known to
play a key role.
7.2.1.2. Synthesis of 3,5-bis(4-cyanophenoxy)benzoic acid
(46).
A solution of ester 45 (2.1 g, 5.46 mmol) in THF & water
mixture (30 ml, 1:1) at rt was treated with LiOH (0.4 g,
16.38 mmol) and stirred for 2 h. Reaction mixture was washed
with ether and the aqueous layer was cooled to 5 °C followed by
pH adjusted to 2–3 using 6 N HCl resulted in white precipitate,
which was filtered off and dried under vacuum to afford the title
compound 46 (1.6 g; yield 82%). 1H NMR (300 MHz, DMSO-d6) d
7.21 (d, 4H), 7.31 (s, 1H), 7.38 (d, 2H), 7.86 (d, 4H), 13.45 (br s, 1H).
7. Materials and methods
7.1. Experimental section
7.2.1.3. General procedure for the synthesis of 3,5-bis(4-cyano-
phenoxy)benzamide derivatives (48).
A solution of 46
All commercially available starting materials and solvents were
used as received. All anhydrous reactions were performed under a
nitrogen atmosphere in oven-dried glassware with magnetic stir-
ring. Column chromatography was performed using silica gel of
60–120 mesh and solvent (hexane/DCM/EtOAc) mixture/gradient.
Thin layer chromatography was carried out using silica gel 60
F254 plates (Merck, Darmstadt) and developed chromatograms
(0.84 mmol) in DMF (15 ml) at 5 °C was treated with EDCI
(0.84 mmol), HOBt (0.84 mmol) and DIPEA (0.84 mmol). The mix-
ture was stirred for 10 min and a solution of an amine or an alcohol
(0.84 mmol), dissolved in DMF (3 ml), was added drop wise and
stirred at rt for 8 h. Reaction mixture was quenched with ice-cold
water and aqueous layer was extracted with ether. Combined
organics were washed with 1 M solution of Na2CO3 followed with

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R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
brine, dried over anhydrous Na2SO4 and concentrated to give a
semi solid residue, which was purified by CC, eluting with 20%
EtOAc/hexane gave pure compound (48).
3,5-Bis(4-carbamimidoylphenoxy)-N-(1-(2-hydroxyethyl)piperi-
din-4-yl)benzamide 13: Yield 62%; mp 134–136 °C; HPLC: 95.44%;
LCMS (ES, m/z): 517.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.92 (m, 3H), 3.11 (m, 4H), 3.50–3.78 (m, 6H), 7.14 (s, 1H), 7.28
(d, 4H), 7.51 (s, 2H), 7.86 (d, 4H), 8.62 (d, 1H), 9.16 (br s, 4H),
9.28 (br s, 4H), 9.42 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d
25.30, 44.78, 51.21, 54.98, 57.73, 113.91, 114.67, 117.93, 122.85,
130.37, 137.76, 156.18, 158.13, 158.55, 150.58, 163.79, 164.76.
N-(1-(3-Aminopropyl)piperidin-4-yl)-3,5-bis(4-carbamimidoyl-
phenoxy)benzamide 14: Yield 64%; mp 162–164 °C; HPLC: 95.27%;
LCMS (ES, m/z): 530.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.78 (m, 2H), 1.98 (m, 4H), 2.86 (m, 2H), 3.12 (m, 4H), 3.50 (d,
2H), 3.98 (m, 1H), 7.14 (s, 1H), 7.26 (d, 4H), 7.51 (s, 2H), 7.85 (d,
4H), 7.95 (br s, 3H), 8.67 (d, 1H), 9.22 (br s, 4H), 9.29 (br s, 4H);
13C NMR (100 MHz, DMSO-d6): d 21.54, 28.35, 36.07, 50.68,
52.68, 114.39, 114.75, 117.89, 122.83, 130.45, 137.51, 156.15,
158.30, 158.73, 160.63, 163.62, 164.62.
N-(4-Aminocyclohexyl)-3,5-bis(4-carbamimidoylphenoxy)benz-
amide 15: Yield 72%; mp 181–183 °C; HPLC: 99.04%; LCMS (ES, m/
z): 487.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.35 (m, 4H),
1.80 (m, 2H), 1.96 (m, 2H), 2.84 (m, 1H), 3.62 (m, 1H), 7.11 (s,
1H), 7.24 (d, 4H), 7.50 (s, 2H), 7.78 (d, 4H), 8.12 (br s, 3H), 8.48
(d, 1H), 9.14 (br s, 4H), 9.36 (br s, 4H). 13C NMR (75 MHz, DMSO-
d6): d 29.59, 30.14, 48.01, 49.01, 115.31, 118.54, 123.49, 131.07,
138.51, 156.81, 159.41, 159.84, 161.32, 163.87, 165.43.
7.2.1.4. General procedure for the conversion of nitrile to
N-hydroxybenzimidamide derivatives (49).
Nitrile 48
(0.81 mmol), dissolved in EtOH (10 ml), at rt was added aq NH2OH
(3.24 mmol). The reaction mixture was stirred at 75 °C for 6 h and
then concentrated to afford the N-hydroxy compound as a white
solid, which was used as such into the next step without further
purification.
7.2.1.5. General procedure for the synthesis of N-acetoxybenzi-
midamide derivatives (50).
A solution of N-hydroxyamidine
49 (0.65 mmol), dissolved in AcOH (3 ml), was treated with Ac2O
(2.6 mmol) and stirred at rt for 2 h. Reaction mixture was concen-
trated to give acetoxy compound as a white solid (50), which was
used into the next step as such without further purification.
7.2.1.6. General procedure for the synthesis of dibenzimidamide
derivatives (51, 9–11, 18–22).
To the solution of N-acetoxy
benzimidamide 50 (0.57 mmol), in AcOH (5 ml), zinc (1.71 mmol)
was added and stirred at rt for 6–8 h. Reaction mixture was filtered
through celite and concentrated to give crude solid, which was
purified by the reversed-phase preparative HPLC to give the target
compounds.
4,40-((5-(4-(2-Aminoethyl)piperidine-1-carbonyl)-1,3-pheny-
lene)bis(oxy))dibenzimidamide 16: Yield 55%; mp 145–147 °C;
HPLC: 97.85%; LCMS (ES, m/z): 501.2 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.08 (m, 2H), 1.52 (m, 6H), 2.76 (m, 3H), 2.98 (m,
1H), 3.34 (m, 1H), 4.30 (m, 1H), 6.91 (m, 3H), 7.28 (d, 4H), 7.84
(m, 6H), 9.22 (br s, 4H), 9.31 (br s, 4H). 13C NMR (75 MHz,
DMSO-d6): d 32.56, 33.26, 36.43, 41.82, 45.72, 111.76, 113.67,
115.73, 118.39, 123.10, 130.68, 140.11, 156.59, 158.47, 160.60,
164.77, 166.78.
7.2.1.7. General procedure for the deprotection of a Boc-
protected amino group or a THP-protected hydroxyl group
(12–17).
Boc-protected amino group or THP-protected hydro-
xyl group containing compound 51 (0.34 mmol) was dissolved in
5 ml of ethanol (saturated with HCl gas) and stirred at 5–10 °C
for 2 h. Solvent was removed to give the crude solid, which was
purified by reversed-phase preparative HPLC to give the target
compounds 12–17.
3,5-Bis(4-carbamimidoylphenoxy)-N-((4-hydroxycyclohexyl)
3,5-Bis(4-carbamimidoylphenoxy)-N-cyclohexylbenzamide
9:
methyl)benzamide 17: Yield 65%; mp 128–130 °C; HPLC: 92.74%;
LCMS (ES, m/z): 502.0 (M+H)+; 1H NMR (300 MHz, DMSO-d6):
d 1.38 (m, 6H), 1.56 (m, 3H), 3.12 (t, 2H), 3.62 (m, 1H), 4.32 (br s,
1H), 7.16 (s, 1H), 7.32 (d, 4H), 7.49 (s, 2H), 7.90 (d, 4H), 8.64
(t, 1H), 9.26 (br s, 4H), 9.31 (br s, 4H). 13C NMR (75 MHz, DMSO-d6):
d 24.65, 31.68, 35.94, 44.58, 64.81, 114.17, 114.47, 115.18, 118.29,
123.08, 130.68, 138.12, 156.50, 158.45, 160.80, 164.02, 164.79.
Ethyl 4-(3,5-bis(4-carbamimidoylphenoxy)benzamido)piperidine-
1-carboxylate 18: Yield 64%; mp 252 °C; HPLC: 94.24%; LCMS (ES,
m/z): 545.5 (M+H)+; 1H NMR (400 MHz, DMSO-d6): d 1.15 (t, 3H),
1.36 (m, 2H), 1.74 (m, 2H), 2.87 (m, 2H), 3.93 (m, 2H), 4.01 (q,
2H), 7.15 (s, 1H), 7.29 (d, 4H), 7.48 (s, 2H), 7.86 (d, 4H), 8.41 (m,
1H), 9.18 (br s, 4H), 9.26 (br s, 4H). 13C NMR (75 MHz, DMSO-d6):
Yield 74%; mp 148–150 °C; HPLC: 95.03%; LCMS (ES, m/z): 472.1
(M+H)+; 1H NMR (400 MHz, DMSO-d6): d 1.23 (m, 4H), 1.68 (m,
6H), 3.71 (m, 1H), 7.15 (s, 1H), 7.30 (d, 4H), 7.49 (s, 2H), 7.88 (d,
4H), 8.37 (d, 1H), 9.18 (br s, 4H), 9.28 (br s, 4H); 13C NMR
(75 MHz, DMSO-d6): d 24.82, 25.14, 32.20, 48.60, 114.18, 114.71,
118.11, 122.94, 130.61, 138.31, 156.31, 158.24, 158.57, 160.85,
163.09, 164.76.
3,5-Bis(4-carbamimidoylphenoxy)-N-(cyclohexylmethyl)benzam-
ide 10: Yield 75%; mp 138–140 °C; HPLC: 98.98%; LCMS (ES, m/z):
485.9 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 0.84 (m, 2H), 1.12
(m, 3H), 1.58 (m, 6H), 3.04 (t, 2H), 7.20 (s, 1H), 7.28 (d, 4H), 7.46
(s, 2H), 7.88 (d, 4H), 8.58 (t, 1H), 9.28 (br s, 8H). 13C NMR (75 MHz,
DMSO-d6): d 25.12, 25.78, 30.29, 37.09, 45.38, 113.61, 114.27,
118.08, 122.86, 130.39, 138.13, 156.29, 160.59, 163.97, 164.74.
3,5-Bis(4-carbamimidoylphenoxy)-N-(4-methylcyclohexyl)benz-
amide 11: Yield 70%; mp 140–142 °C; HPLC: 97.74%; LCMS (ES, m/
z): 486.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 0.91 (d, 2H),
1.20-1.81 (m, 9H), 3.78 (m, 1H), 7.12 (s, 1H), 7.26 (d, 4H), 7.48 (s,
2H), 7.84 (d, 4H), 8.34 (d, 1H), 9.13 (br s, 4H), 9.25 (br s, 4H). 13C
NMR (75 MHz, DMSO-d6): d 21.84, 27.47, 29.62, 33.48, 47.00,
48.53, 113.60, 114.49, 114.60, 117.98, 122.81, 130.39, 138.45,
156.15, 160.66, 164.76.
d
14.35, 30.82, 42.37, 46.59, 60.39, 114.24, 117.99, 122.85,
130.42, 138.03, 154.50, 156.24, 160.66, 163.35, 164.75.
1-(3,5-Bis(4-carbamimidoylphenoxy)benzoyl)piperidine-4-carbox-
ylic acid 19: Yield 42%, mp 95–97 °C; HPLC: 95.33%; LCMS (ES, m/z):
501.7 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.34 (m, 2H), 1.70
(m, 2H), 2.83 (m, 1H), 3.05 (m, 1H), 3.51 (m, 1H), 4.16 (m, 2H),
6.92 (s, 2H), 7.00 (s, 1H), 7.29 (d, 4H), 7.85 (d, 4H), 9.08 (br s,
4H), 9.24 (br s, 4H).13C NMR (100 MHz, DMSO-d6): d 27.36,
27.94, 46.05, 111.74, 113.43, 118.43, 123.13, 130.61, 139.87,
156.64, 160.52, 164.72, 166.87, 175.32.
3,5-Bis(4-carbamimidoylphenoxy)-N-(4-hydroxycyclohexyl)benz-
amide 12: Yield 58%; mp 120–122 °C; HPLC: 97.19%; LCMS (ES, m/
z): 488.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.18 (m, 4H),
1.64 (m, 4H), 3.32 (m, 1H), 3.65 (m, 1H), 7.14 (s, 1H), 7.26 (d,
4H), 7.48 (s, 2H), 7.68 (d, 4H), 8.34 (d, 1H), 9.18 (br s, 4H), 9.26
(br s, 4H). 13C NMR (100 MHz, DMSO-d6): d 30.14, 34.17, 48.19,
68.27, 114.35, 114.80, 115.32, 118.13, 123.02, 130.65, 138.23,
156.36, 158.45, 158.77, 159.09, 160.91, 163.31, 164.89.
4,40-((5-(Decahydroquinoline-1-carbonyl)-1,3-phenylene)bis(oxy))
dibenzimidamide 20: Yield 68%; mp 238–240 °C; HPLC: 93.43%;
LCMS (ES, m/z): 512.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
1.32 (m, 11H), 1.78 (m, 1H), 3.54 (m, 4H), 6.80 (s, 1H), 6.88 (s,
1H), 6.98 (s, 1H), 7.29 (d, 4H), 7.85 (d, 4H), 8.96 (br s, 4H), 9.24
(br s, 4H); 13C NMR (100 MHz, DMSO-d6): d 21.47, 24.38, 25.49,
29.03, 33.66, 35.79, 40.94, 45.65, 46.21, 51.35, 111.45, 113.33,
118.54, 123.17, 130.60, 140.14, 156.68, 160.47, 164.70

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3199
4,40-((5-(1,2,3,4-Tetrahydroisoquinoline-2-carbonyl)-1,3-pheny-
lene)bis(oxy))dibenzimidamide 21: Yield 65%; mp 112–114 °C;
HPLC: 93.50%; LCMS (ES, m/z): 506.1 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 2.75 (m, 2H), 3.54 (m, 1H), 3.74 (m, 1H), 4.67 (s,
2H), 6.94 (m, 2H), 7.03 (m, 1H), 7.14 (m, 4H), 7.32 (d, 4H), 7.84
(d, 4H), 9.06 (br s, 4H), 9.22 (br s, 4H); 13C NMR (75 MHz, DMSO-
d6): d 28.15, 44.50, 49.87, 111.65, 113.28, 118.39, 123.04, 126.26,
128.31, 130.39, 132.67, 134.08, 139.56, 156.64, 157.96, 158.38,
160.30, 164.72, 167.25
3,5-Bis(4-carbamimidoylphenoxy)-N-(4-fluorophenyl)benzamide
22: Yield 70%; mp 286–288 °C; HPLC: 96.58%; LCMS (ES, m/z):
484.0 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 7.16 (m, 3H), 7.33
(d, 4H), 7.57 (s, 2H), 7.71 (m, 2H), 7.89 (d, 4H), 9.29 (br s, 4H),
9.36 (br s, 4H), 10.41 (br s, 1H). 13C NMR (75 MHz, DMSO-d6): d
114.11, 114.60, 114.83, 115.13, 118.17, 122.30, 122.40, 123.02,
130.45, 134.77, 138.02, 156.42, 160.48, 163.14, 164.76
4H), 9.09 (br s, 4H), 9.23 (br s, 4H). 13C NMR (75 MHz, DMSO-d6):
d 98.18, 101.06, 117.86, 122.19, 130.38, 151.92, 156.98, 161.28,
164.71.
7.2.5. General procedure for the synthesis of N-(3,5-bis(4-
cyanophenoxy)phenyl)carboxamide derivatives (52)
A solution of a carboxylic acid (1.07 mmol) in DMF (15 ml) at
5 °C was treated with PyBop (1.07 mmol) and DIPEA (1.07 mmol).
The reaction mixture was stirred for 10 min and a solution of
amino compound 47 (1.07 mmol) dissolved in DMF (5 ml) was
added drop wise. Resulting mixture was stirred at RT for 6 h,
quenched with ice-cold water and extracted with EtOAc. Combined
organics were washed with 1 M solution of Na2CO3 followed with
brine, dried over Na2SO4 and concentrated to give oily residue,
which was purified by CC using hexane–EtOAc (10:2) to give the
desired compound 52.
4-Aminocyclohexyl 3,5-bis(4-carbamimidoylphenoxy)benzoate 30:
Yield 68%; mp 113–115 °C; HPLC: 99.26%; LCMS (ES, m/z): 488.1
(M+H)+; 1H NMR (400 MHz, DMSO-d6): d 1.29 (m, 2H), 1.44 (m,
2H), 1.86 (m, 2H), 1.96 (m, 2H), 2.81 (m, 1H), 4.77 (m, 1H), 7.25
(s, 1H), 7.29 (d, 4H), 7.40 (s, 2H), 7.86 (d, 4H), 9.05 (br s, 6H). 13C
NMR (75 MHz, DMSO-d6): d 24.21, 28.75, 29.26, 48.05, 73.02,
115.85, 116.14, 118.40, 124.03, 130.45, 133.59, 156.90, 158.54,
158.75, 160.28, 163.67, 165.04, 175.90
Further, following the procedure of nitrile group conversion to
amidine (48?51), the nitrile groups of 52 were converted to ami-
dine to give N-(3,5-bis(4-carbamimidoylphenoxy)phenyl)carbox-
amide derivatives (55 and 23). Boc-protected amino group or
THP-protected hydroxyl group of compound (55) was deprotected,
following the procedure of Example 12, to give the free amino or
the hydroxyl group derivatives (24–27).
N-(3,5-bis(4-carbamimidoylphenoxy)phenyl)-4-methylcyclohex-
anecarboxamide 23: Yield 74%; mp 146–148 °C; HPLC: 98.43%;
LCMS (ES, m/z): 486.2 (M+H)+; 1H NMR (400 MHz, DMSO-d6): d
0.84 (m, 5H), 1.31 (m, 3H), 1.67 (m, 4H), 2.16 (m, 1H), 6.64 (s,
1H), 7.24 (m, 6H), 7.86 (d, 4H), 9.08 (br s, 4H), 9.24 (br s, 4H),
10.18 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d 22.87, 29.37,
31.97, 34.31, 45.15, 106.02, 106.28, 118.77, 123.27, 131.01,
142.83, 157.09, 161.31, 165.22, 175.28.
1-(3-Aminopropanoyl)-N-(3,5-bis(4-carbamimidoylphenoxy)
phenyl)piperidine-4-carboxamide 24: Yield 52%; mp 142–144 °C;
HPLC: 97.97%; LCMS (ES, m/z): 544.2 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.32 (m, 3H), 1.74 (m, 3H), 2.11 (m, 3H), 2.95 (m,
3H), 4.36 (m, 1H), 6.60 (s, 1H), 7.24 (s, 2H), 7.31 (d, 4H), 7.64 (br
s, 3H), 7.86 (d, 4H), 9.22 (br s, 4H), 9.32 (br s, 4H), 10.44 (br s,
1H). 13C NMR (75 MHz, DMSO-d6): d 27.62, 29.47, 35.18, 42.25,
43.86, 105.46, 105.98, 118.07, 122.63, 130.29, 141.99, 156.45,
157.94, 158.36, 160.65, 164.71, 167.79, 173.10.
7.2.2. Synthesis of 3,5-bis(4-carbamimidoylphenoxy)benzamide
(7)
Acid 46 (0.075 g, 0.21 mmol) was treated with oxalyl chloride
(0.32 g, 0.25 mmol), followed with aq NH3 to yield its amide analog
and then by converting its nitrile groups to amidine afforded crude
solid, which was purified by reverse-phase preparative HPLC and
afforded 0.04 g of 7. Yield 62%; mp 240–242 °C; HPLC: 98.09%;
LCMS (ES, m/z): 390.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
7.15 (s, 1H), 7.32 (d, 4H), 7.46 (s, 2H), 7.61 (br s, 1H), 7.89 (d,
4H), 8.12 (br s, 1H) 9.18 (br s, 3H), 9.26 (br s, 3H); 13C NMR
(75 MHz, DMSO-d6): d 113.70, 114.36, 118.24, 122.96, 130.42,
137.85, 156.41, 160.53, 164.78, 165.72.
7.2.3. Synthesis of 4,40-((5-amino-1,3-
phenylene)bis(oxy))dibenzonitrile (47)
A solution of acid 46 (1.0 g, 2.8 mmol), diphenylphosphorylaz-
ide (0.6 ml, 2.8 mmol) and DIPEA (0.5 ml, 2.8 mmol) in DMF
(50 ml) at rt t-BuOH (15 ml) was added and stirred at 80 °C for
12 h. Reaction mixture was cooled to rt, diluted with 50 ml of sat-
urated solution of NaHCO3 and extracted with EtOAc. The com-
bined organics were washed with brine, dried over Na2SO4 and
concentrated to give a yellowish residue, which was purified by
CC and eluted with 10–20% EtOAc/hexane to afford a Boc-protected
amine. Thus obtained product was treated with 6 N HCl (1 ml) in
EtOH (5 ml) at rt and stirred for 2 h. Solvent was evaporated and
residue was dissolved in ice-cold water, which was basified with
Na2CO3 solution to pH 7–8 and then extracted with EtOAc. Com-
bined organics were washed with brine, dried over Na2SO4 and
concentrated to afford the title compound 47 as a light-yellow
solid (0.51 g, yield 55%). LCMS (ES, m/z) 328.10 (M+H)+; 1H NMR
(300 MHz, DMSO-d6) d 5.52 (br s, 2H), 5.94 (t, 1H), 6.18 (d, 2H),
7.11 (d, 4H), 7.28 (d, 4H).
1-(2-Aminoethyl)-N-(3,5-bis(4-carbamimidoylphenoxy)phenyl)
piperidine-4-carboxamide 25: Yield 56%; mp 122–124 °C; HPLC:
98.91%; LCMS (ES, m/z): 516.0 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.74 (m, 2H), 1.85 (m, 2H), 2.41 (m, 2H), 2.56 (m,
2H), 3.18 (m, 5H), 6.61 (s, 1H), 7.25 (m, 6H), 7.86 (d, 4H), 8.08
(br s, 3H), 9.13 (br s, 4H), 9.22 (br s, 4H), 10.40 (br s, 1H). 13C
NMR (75 MHz, DMSO-d6): d 25.75, 33.90, 51.39, 53.21, 58.74,
105.74, 106.13, 118.09, 122.69, 130.38, 141.88, 156.49, 158.62,
160.70, 164.71, 172.36.
(1r,4r)-4-Amino-N-(3,5-bis(4-carbamimidoylphenoxy)phenyl)
cyclohexanecarboxamide 26: Yield 68%; mp 118–120 °C; HPLC:
99.18%; LCMS (ES, m/z) 487.2(M+H)+; 1H NMR (300 MHz, DMSO-
d6): d 1.28 (m, 4H), 1.80 (m, 2H), 1.92 (m, 2H), 2.21 (m, 1H), 2.93
(m, 1H), 6.60 (s, 1H), 7.24 (m, 6H), 7.85 (d, 7H), 9.16 (m, 8H),
10.26 (br s, 1H). 13C NMR (100 MHz, DMSO-d6): d 26.90, 29.29,
43.46, 48.56, 105.71, 105.88, 118.08, 118.30, 122.85, 130.57,
142.22, 156.66, 158.26, 158.59, 160.83, 164.76, 173.89
N-(3,5-Bis(4-carbamimidoylphenoxy)phenyl)-1-(2-hydroxyethyl)
piperidine-4-carboxamide 27: Yield 60%; mp 128–130 °C; HPLC:
97.18%; LCMS (ES, m/z): 517.1 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.84 (m, 4H), 2.78-3.21 (m, 5H), 3.52-3.78 (m, 5H),
6.60 (s, 1H), 7.25 (m, 6H), 7.85 (d, 4H), 9.18 (br s, 4H), 9.26 (br s,
4H), 10.41 (br s, 1H). 13C NMR (75 MHz, DMSO-d6): d 25.31,
42.22, 51.25, 54.91, 57.91, 105.67, 106.06, 118.06, 122.66, 130.31,
141.78, 156.46, 158.05, 158.48, 160.63, 164.70, 172.01.
7.2.4. Synthesis of 4,40-((5-amino-1,3-
phenylene)bis(oxy))dibenzimidamide 8
Following the procedure of nitrile group conversion to amidine
(48?51), nitrile groups of 47 were converted to amidine to give
the title compound 8. Yield 45%; mp 256–258 °C; HPLC 96.69%;
LCMS (ES, m/z): 362.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
4.68 (br s, 3H), 5.95 (s, 1H), 6.12 (s, 2H), 7.22 (d, 4H), 7.84 (d,

3200
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
7.2.6. General procedure for the synthesis of N-(3,5-bis(4-
cyanophenoxy)phenyl)sulfonamide derivatives (56)
Further, following the general procedure of nitrile group con-
version to amidine (48?51), nitrile group/s of 66 were converted
to amidine and then amino group deprotection afforded 31–39.
N-((1r,4r)-4-Aminocyclohexyl)-3-(4-aminophenoxy)-5-(4-car-
bamimidoylphenoxy)benzamide 31: Yield 38%; mp 95–97 °C; HPLC:
97.05%; LCMS (ES, m/z): 460.0 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.36 (m, 4H), 1.80 (m, 2H), 1.94 (m, 2H), 3.00 (m,
1H), 3.68 (m, 1H), 6.85 (s, 1H), 7.01 (s, 4H), 7.25 (d, 2H), 7.63 (s,
2H), 7.84 (m, 5H), 8.38 (d, 1H), 9.08 (br s, 2H), 9.25 (br s, 2H).
To the solution of 47 (1.68 mmol), pyridine (3.36 mmol), DMAP
(0.35 mmol) in 50 ml of DCM, arylsulfonyl chloride (1.68 mmol,
dissolved in 5 ml of DCM) was added under N2 atmosphere and
resulting mixture was stirred at rt for 12 h. After the reaction com-
pletion, mixture was concentrated and thus obtained residue was
dissolved in 100 ml of EtOAc. Organic layer was washed with 1 N
HCl (50 ml) followed by brine (2 ꢃ 50 ml) and dried over anhy-
drous Na2SO4. Solvent was evaporated to give crude solid, which
was purified by CC (silica gel, 10% ethyl acetate in hexane) to give
the title compound 56.
13C NMR (75 MHz, DMSO-d6):
d 29.00, 29.49, 47.40, 48.55,
111.62, 112.72, 112.84, 114.28, 117.82, 118.19, 119.88, 120.10,
122.68, 130.37, 137.86, 150.27, 155.90, 158.50, 158.75, 160.78,
163.75, 164.84.
N-((1r,4r)-4-Aminocyclohexyl)-3-((6-aminopyridin-3-yl)oxy)-5-
(4-carbamimidoylphenoxy)benzamide 32: Yield 48%; mp 113–
115 °C; HPLC: 95.48%; LCMS (ES, m/z): 461.2 (M+H)+; 1H NMR
(400 MHz, DMSO-d6): d 1.38 (m, 4H), 1.85 (m, 2H), 1.96 (m, 2H),
2.95 (m, 1H), 3.67 (m, 1H), 6.94 (d, 1H), 7.02 (s, 1H), 7.20 (d, 2H),
7.33 (d, 2H), 7.77 (dd, 1H), 7.85 (d, 2H), 7.93 (br s, 3H), 7.98 (s,
1H), 8.40 (d, 1H), 9.27 (br s, 2H), 9.34 (br s, 2H); 13C NMR
Further, following the procedure of nitrile group conversion to
amidine (48?51), nitrile groups of 56 were converted to amidine
and afforded the 4,40-((5-(arylsulfonamido)-1,3-phenylene)bis
(oxy))dibenzimidamide derivatives 28–29.
4,40-((5-(4-Fluorophenylsulfonamido)-1,3-phenylene)bis(oxy))dib-
enzimidamide 28: Yield 47%; mp 210–212 °C; HPLC: 95.72%; LCMS
(ES, m/z): 520.0 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 6.58 (s,
1H), 6.63 (s, 2H), 7.15 (d, 4H), 7.45 (t, 2H), 7.76 (m, 2H), 7.82 (d,
4H), 9.12 (br s, 3H), 9.25 (br s, 3H), 10.68 (br s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 106.31, 116.57, 116.80, 118.28, 123.12,
129.74, 129.84, 130.55, 135.18, 140.64, 156.94, 160.29, 164.67
4,40-((5-(Naphthalene-2-sulfonamido)-1,3-pheny-
(100 MHz, DMSO-d6):
d 29.14, 29.66, 47.53, 48.56, 111.71,
112.35, 113.72, 113.86, 117.87, 122.82, 129.90, 130.57, 136.76,
137.89, 142.42, 153.70, 156.04, 158.54, 158.66, 159.19, 160.94,
163.64, 164.84.
lene)bis(oxy))dibenzimidamide 29: Yield 52%; mp 232–232 °C;
HPLC: 96.10%; LCMS (ES, m/z): 551.8 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 6.48 (s, 1H), 6.65 (s, 2H), 7.06 (d, 4H), 7.68 (m, 7H),
8.02 (d, 1H), 8.12 (t, 2H), 8.39 (s, 1H), 8.82 (br s, 4H), 9.18 (br s,
4H), 10.80 (br s, 1H). 13C NMR (75 MHz, DMSO-d6): d 105.92,
106.19, 118.08, 121.68, 122.94, 127.70, 127.88, 129.05, 129.50,
130.33, 131.40, 134.24, 140.72, 156.77, 160.17, 164.66.
N-((1r,4r)-4-Aminocyclohexyl)-3-(4-carbamimidoylphenoxy)-5-
(4-carbamoylphenoxy)benzamide 33: Yield 42%, mp 114–116 °C;
HPLC: 97.22%; LCMS (ES, m/z): 488.2 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.32 (m, 4H), 1.84 (m, 4H), 2.86 (m, 1H), 3.61 (m,
1H), 7.01 (s, 1H), 7.08 (d, 2H), 7.25 (m, 3H), 7.40 (d, 2H), 7.74 (br
s, 2H), 7.82 (d, 2H), 7.91 (d, 2H), 8.37 (d, 1H), 8.88 (br s, 2H),
9.21 (br s, 2H). 13C NMR (100 MHz, DMSO-d6): d 29.03, 29.53,
47.45, 48.54, 113.31, 113.98, 114.17, 117.61, 118.00, 122.83,
129.63, 130.44, 137.97, 156.15, 157.03, 158.51, 160.72, 163.52,
164.80, 166.92.
7.2.7. Synthesis of 3-(4-cyanophenoxy)-5-hydroxybenzoic acid
(64)
A solution of 44 (2.25 g, 12.4 mmol) in DMF (50 ml) at rt was
treated with K2CO3 (2.57 g, 18.6 mmol) and stirred for 10 min. A
solution of 4-fluorobenzonitrile (2.25 g, 18.6 mmol) in DMF
(100 ml) was added drop wise and the resultant solution was stir-
red at 45 °C for 6 h. The reaction mixture was quenched with ice-
cold water and extracted with EtOAc. The organic layer was
washed with 1 M Na2CO3 solution, followed with brine and then
dried over anhydrous sodium sulfate. Solvent was evaporated to
dryness to get crude oily residue, which was purified by CC and
eluted with 30% EtOAc/hexane to afford the white solid ester
(1.57 g; yield 45%). The resulted ester was hydrolyzed, following
the procedure of Example 46, to give the title compound 64
(1.1 g; yield 70%). 1H NMR (300 MHz, DMSO-d6): d 6.95 (s, 1H),
7.21 (d, 2H), 7.56 (s, 2H), 7.88 (d, 2H), 9.28 (br s, 1H), 13.6 (br s,
1H).
N-((1r,4r)-4-Aminocyclohexyl)-3-((4-carbamimidoylbenzyl)oxy)-
5-(4-carbamimidoylphenoxy)benzamide 34: Yield 72%, mp 263–
265 °C; HPLC: 91.03%; LCMS (ES, m/z): 501.1 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 1.40 (m, 4H), 1.82 (m, 4H), 2.86 (m, 1H),
3.60 (m, 1H), 5.32 (s, 2H), 7.04 (s, 1H), 7.21 (m, 3H), 7.44 (s, 1H),
7.68 (d, 2H), 7.88 (m, 7H), 8.37 (d, 1H), 9.20 (br s, 7H); 13C NMR
(75 MHz, DMSO-d6): d 29.68, 30.21, 48.02, 49.22, 69.53, 109.88,
111.02, 111.69, 115.54, 118.39, 123.14, 128.16, 128.73, 131.02,
138.09, 143.11, 156.43, 158.56, 158.98, 159.97, 161.62, 164.66,
165.38, 166.02.
N-((1r,4r)-4-Aminocyclohexyl)-3-((3-carbamimidoylbenzyl)oxy)-
5-(4-carbamimidoylphenoxy)benzamide 35: Yield 65%; mp 96–
98 °C; HPLC: 96.67%; LCMS (ES, m/z): 501.1 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 1.39 (m, 4H), 1.86 (m, 2H), 1.95 (m, 2H),
2.99 (m, 1H), 3.70 (m, 1H), 5.27 (s, 2H), 7.06 (s, 1H), 7.22 (m,
3H), 7.47 (s, 1H), 7.66 (d, 1H), 7.80 (t, 2H), 7.81 (m, 5H), 8.38 (d,
1H), 9.23 (br s, 2H), 9.29 (br s, 2H), 9.35 (br s, 2H), 9.40 (br s,
2H); 13C NMR (75 MHz, DMSO-d6): d 29.00, 29.53, 47.36, 48.57,
69.06, 109.21, 110.40, 111.06, 117.72, 118.57, 122.47, 126.84,
127.42, 128.39, 128.99, 130.31, 132.48, 137.44, 155.77, 158.15,
159.38, 160.97, 164.03, 164.79, 165.65.
N-((1r,4r)-4-Aminocyclohexyl)-3-(4-(3-aminopropanamido)phen-
oxy)-5-(4-carbamimidoylphenoxy)benzamide 36: Yield 52%; mp
110–112 °C; HPLC: 96.79%; LCMS (ES, m/z): 531.2 (M+H)+; 1H
NMR (300 MHz, DMSO-d6): d 1.32 (m, 4H), 1.82 (m, 4H), 2.71 (t,
2H), 2.95 (m, 2H), 3.12 (q, 2H), 3.65 (m, 1H), 6.89 (s, 1H), 7.08 (d,
2H), 7.22 (d, 2H), 7.31 (d, 2H), 7.62 (d, 2H), 7.82 (m, 8H), 8.38 (d,
1H), 9.16 (br s, 2H), 9.25 (br s, 2H), 10.25 (br s, 1H); 13C NMR
(100 MHz, DMSO-d6): d 29.11, 29.63, 33.09, 34.90, 47.44, 48.48,
112.06, 112.85, 117.91, 119.84, 120.85, 122.79, 130.61, 135.39,
137.72, 150.89, 155.97, 157.95, 158.27, 158.80, 160.91, 163.69,
164.67, 168.11.
7.2.8. Synthesis of tert-butyl ((1r,4r)-4-(3-(4-cyanophenoxy)-5-
hydroxybenzamido)cyclohexyl)carbamate (65)
Following the procedure of Example 48, the product of Example
64 (0.34 g, 1.35 mmol) was coupled with tert-butyl ((1r,4r)-4-
aminocyclohexyl)carbamate (0.29 g, 1.35 mmol) to afford 0.41 g
(yield 55%) of 65. 1H NMR (300 MHz, DMSO-d6): d 1.23 (m, 4H),
1.37 (s, 9H), 1.78 (m, 4H), 3.12 (m, 1H), 3.61 (m, 1H), 6.68 (br s,
1H), 7.01 (s, 1H), 7.18 (d, 2H), 7.31 (s, 2H), 7.85 (d, 2H), 8.28 (br
s, 1H).
7.2.9. General procedure for the coupling of alkyl/aryl halide
with tert-butyl ((1r,4r)-4-(3-(4-cyanophenoxy)-5-
hydroxybenzamido)cyclohexyl)carbamate (66)
Following the general procedure for the synthesis of 46 except
heating the reaction mixture at 65 °C, the alkoxy analogs 66 were
synthesized (yield 45-65%).

R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
3201
N-((1r,4r)-4-Aminocyclohexyl)-3-((6-bromopyridin-3-yl)methoxy)
-5-(4-carbamimidoylphenoxy)benzamide 37: Yield 64%; mp 192–
194 °C; HPLC: 95.49%; LCMS (ES, m/z): 538.1 & 540.1 (Bromo pat-
tern, M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.38 (m, 4H), 1.88
(m, 2H), 1.95 (m, 2H), 2.99 (m, 1H), 3.69 (m, 1H), 5.20 (s, 2H),
7.02 (s, 1H), 7.17 (s, 1H), 7.19 (d, 2H), 7.42 (s, 1H), 7.70 (d, 1H),
7.85 (m, 6H), 8.35 (d, 1H), 8.50 (s, 1H), 9.16 (br s, 2H), 9.28 (br s,
2H). 13C NMR (100 MHz, DMSO-d6): d 29.16, 29.70, 47.44, 48.59,
66.69, 109.35, 110.42, 111.24, 117.89, 122.68, 127.95, 130.54,
132.07, 137.42, 139.06, 140.91, 149.71, 155.92, 159.30, 161.07,
164.02, 164.83.
N-((1r,4r)-4-Aminocyclohexyl)-3,5-bis(4-(aminomethyl)phenoxy)
benzamide 41: Yield 48%; mp 93–95 °C; HPLC: 98.87%; LCMS (ES,
m/z): 461.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.31 (m,
4H), 1.85 (m, 2H), 1.94 (m, 2H), 2.99 (m, 1H), 3.65 (m, 1H), 4.05
(s, 4H), 6.79 (s, 1H), 7.14 (d, 4H), 7.26 (s, 2H), 7.48 (d, 4H), 7.82
(br s, 3H), 8.18 (br s, 6H), 8.38 (d, 1H). 13C NMR (100 MHz,
DMSO-d6): d 28.95, 29.45, 41.57, 47.34, 48.49, 111.32, 112.28,
118.75, 129.41, 130.71, 137.69, 156.00, 157.51, 163.80.
7.2.11. General procedure for the coupling of an alkyl halide
with ethyl 3,5-dihydroxybenzoate (75)
N-((1r,4r)-4-Aminocyclohexyl)-3-(4-carbamimidoylphenoxy)-5-
((4-chlorobenzyl)oxy)benzamide 38: Yield 75%; mp 252–254 °C;
HPLC: 95.85%; LCMS (ES, m/z): 493.2 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 1.35 (m, 4H), 1.87 (m, 2H), 1.94 (m, 2H), 2.97 (m,
1H), 3.67 (m, 1H), 6.97 (s, 1H), 7.14 (s, 1H), 7.17 (d, 2H), 7.40 (s,
1H), 7.45 (s, 4H), 7.84 (m, 4H), 8.34 (d, 1H), 9.17 (br s, 2H), 9.25
(br s, 2H). 13C NMR (100 MHz, DMSO-d6): d 29.13, 29.69, 47.39,
48.53, 68.91, 109.28, 110.39, 110.96, 115.40, 117.86, 122.67,
128.44, 129.58, 130.53, 132.55, 135.46, 137.29, 155.86, 158.44,
159.49, 161.06, 164.03, 164.85.
Following
the
procedure
of
Example
66,
ethyl
3,5-dihydroxybenzoate (2.4 mmol) was coupled with 3 or 4-
(bromomethyl)benzonitrile (2.4 mmol) to give ethyl 3,5-bis((3 or
4-cyanobenzyl)oxy)benzoate analogs (yield 75%).
7.2.12. General procedure for the synthesis of 3,5-bis((3 or
4-cyanobenzyl)oxy)benzoic acid (76)
Following the procedure of Example 46, ethyl 3,5-bis((3 or
4-cyanobenzyl)oxy)benzoate (1.8 mmol) was hydrolyzed to give
3,5-bis((3 or 4-cyanobenzyl)oxy)benzoic acid derivatives (yield
85%).
N-((1r,4r)-4-Aminocyclohexyl)-3-((4-bromobenzyl)oxy)-5-(4-car-
bamimidoylphenoxy)benzamide 39: Yield 68%; mp 211–213 °C;
HPLC: 96.42%; LCMS (ES, m/z): 537.1
&
539.1 (Bromo
7.2.13. General procedure for the synthesis of tert-butyl ((1r,4r)-
4-(3,5-bis((3 or 4-cyanobenzyl)oxy)benzamido)cyclohexyl)
carbamate (77)
Following the procedure of Example 48, acid 76 (1.5 mmol) was
coupled with tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate
(1.5 mmol) to give amide 77 (yield 65–70%).
pattern,M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.32 (m, 4H),
1.81 (m, 2H), 1.95 (m, 2H), 2.92 (m, 1H), 3.64 (m, 1H), 5.15 (s,
2H), 6.99 (s, 1H), 7.16 (s, 1H), 7.21 (d, 2H), 7.44 (m, 3H), 7.61 (d,
2H), 7.78 (d, 2H), 8.04 (br s, 3H), 8.38 (d, 1H), 9.05 (br s, 2H),
9.30 (br s, 2H); 13C NMR (100 MHz, DMSO-d6): d 29.00, 29.53,
47.34, 48.56, 68.99, 109.08, 110.41, 110.90, 117.76, 120.88,
122.49, 129.56, 130.31, 131.17, 135.80, 137.38, 155.78, 159.37,
160.93, 164.02, 164.80.
Further, following the procedure of nitrile group conversion to
amidine (48?51), nitrile groups of 77 were converted to amidine
and then Boc-protected amino group deprotection afforded N-
3,5-bis((4-cyanobenzyl)oxy)benzamide derivatives (42–43).
N-((1r,4r)-4-Aminocyclohexyl)-3,5-bis((4-carbamimidoylbenzyl)
oxy)benzamide 42: Yield 72%; mp 171–173 °C; HPLC: 98.01%; LCMS
(ES, m/z): 515.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.41 (m,
4H), 1.88 (m, 2H), 1.98 (m, 2H), 3.01 (m, 1H), 3.71 (m, 1H), 5.28 (s,
4H), 6.88 (s, 1H), 7.14 (s, 2H), 7.67 (d, 4H), 7.85 (d, 4H), 7.92 (br s,
3H), 8.28 (d, 1H), 9.29 (br s, 4H), 9.38 (br s, 4H); 13C NMR (75 MHz,
DMSO-d6): d 29.01, 29.57, 47.25, 48.60, 68.63, 104.60, 106.69,
127.38, 127.99, 136.82, 142.75, 158.58, 158.90, 164.69, 165.46.
N-((1r,4r)-4-Aminocyclohexyl)-3,5-bis((3-carbamimidoylbenzyl)
oxy)benzamide 43: Yield 68%; mp 210–212 °C; HPLC: 98.63%; LCMS
(ES, m/z): 515.2 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 1.41 (m,
4H), 1.89 (m, 4H), 2.92 (m, 1H), 3.61 (m, 1H), 5.22 (s, 4H), 6.91 (s,
1H), 7.18 (s, 2H), 7.67 (d, 2H), 7.80 (m, 4H), 7.92 (m, 5H), 8.29 (d,
1H), 9.26 (br s, 4H), 9.38 (br s, 4H); 13C NMR (100 MHz, DMSO-
d6): d 29.21, 29.79, 47.39, 48.66, 68.85, 104.54, 106.70, 115.59,
118.56, 127.04, 127.61, 128.61, 129.21, 132.68, 136.84, 137.82,
158.67, 158.99, 159.14, 164.84, 165.80.
7.2.10. General procedure for the conversion of nitrile group to
Boc-protected aminomethyl group (67)
A solution of 65 (1.6 g, 3.53 mmol) in MeOH (100 ml) at 0 °C
was added nickel(II) chloride hexahydrate (0.046 g, 0.02 mmol),
di-tert-butyl dicarbonate (1.15 g, 5.29 mmol), followed by sodium
borohydride (0.94 g, 24.72 mmol) in portions during 15 min and
then resulting mixture was stirred at rt for 1 h. Reaction mixture
was cooled to 0 °C and added diethylenetriamine (3.64 g,
35.32 mmol) drop wise and then stirred at rt for 2 h. Solvent was
evaporated and residue was dissolved in water and then extracted
with EtOAc. Combined organics were washed with water, followed
with brine and dried over anhydrous sodium sulfate. Solvent was
evaporated to yield crude solid, which was purified by CC and
eluted with 20–30% EtOAc/hexane to give the off-white compound
67 (0.8 g; yield 45%). 1H NMR (300 MHz, DMSO-d6): d 1.32 (m, 4H),
1.36 (s, 18H), 1.74 (m, 4H), 3.14 (m, 1H), 3.58 (m, 1H), 4.46 (d, 2H),
6.75 (br s, 1H), 6.94 (s, 1H), 7.26 (d, 2H), 7.34 (m, 4H), 8.05 (br s,
2H), 8.28 (br s, 1H).
Further, following the general procedure of alkylation of Exam-
ple 66, the hydroxyl group of 67 was alkylated with an aryl halide,
followed by the nitrile group (if present) conversion to amidine
(like 48?51) and then Boc-protected amino group deprotection
to give compounds 40–41.
N-((1r,4r)-4-Aminocyclohexyl)-3-(4-(aminomethyl)phenoxy)-5-
(4-carbamimidoylphenoxy)benzamide 40: Yield 56%; mp 192–
194 °C; HPLC: 96.64%; LCMS (ES, m/z): 474.2 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 1.32 (m, 4H), 1.79 (m, 2H), 1.81 (m, 2H),
2.84 (m, 1H), 3.62 (m, 1H), 4.01 (d, 2H), 6.95 (t, 1H), 7.15 (d, 2H),
7.22 (d, 2H), 7.34 (s, 1H), 7.38 (s, 1H), 7.48 (d, 2H), 7.84 (d, 4H),
8.16 (br s, 2H), 8.38 (d, 1H), 9.14 (br s, 2H), 9.26 (br s, 2H); 13C
NMR (100 MHz, DMSO-d6): d 29.19, 29.73, 41.69, 47.56, 48.58,
113.59, 115.06, 118.00, 119.25, 122.95, 129.96, 130.68, 137.89,
156.05, 156.13, 158.09, 158.50, 161.00, 163.68, 164.91.
7.3. Molecular modeling
Publicly accessible matriptase co-crystal structures (viz., PDB
IDs: 2GV6 & 1EAX) were employed for setting-up protocols for
molecular docking. Prior to this, the protein coordinates were opti-
mized through assignment of suitable bond-orders, water mole-
cules elimination, addition of hydrogen atoms, internal hydrogen
bonds refinement and restrained minimization through OPLS
2005 force-field. Hydrogen bonding constraints were applied to
Asp189 and Ser195 side-chains during receptor grid generation
so that they can be used in docking (if required). The molecules
were energy minimized through LigPrep and then docked in to
generated grid (matriptase active-site) employing standard precision
mode using Glide software. The matriptase crystal structure in com-
plex with compound 6 was employed for WaterMap estimations,

3202
R. Goswami et al. / Bioorg. Med. Chem. 22 (2014) 3187–3203
as described previously.17 Briefly, the calculation was done in
default mode which included a simulation run of 2.0 ns at 300 K
for computing the hydration site statistics. All software names
mentioned in this section are products of Schrödinger Inc and all
images were prepared using PyMOL.24
Tumor volume, body weight and clinical symptoms were
monitored. Tumor volumes were calculated assuming tumors to
be ellipsoid using the formula V = (D ꢃ d2)/2 where V (mm3) is
tumor volume, D is longest diameter in mm, and d is shortest
diameter in mm.
7.4. Cytochrome P450 inhibition
Accession codes
Cytochrome p450 inhibition assay was done using the probe-
substrate method.25–28 A mixture of five substrates was prepared
by pooling caffeine (CyP1A2), tolbutamide (CyP2C9), dextrometh-
orphan (CyP2D6), midazolam (CyP3A4) and testosterone
(CyP3A4).25,26 Serial dilutions of the test compounds ranging from
Protein crystal structure coordinates and structure factors are
deposited in the RCSB Protein Data Bank with the accession
numbers 4O9V and 4O97.
Acknowledgment
100 to 0.005 lM were incubated with human liver microsomes in
the presence of a pool of probe substrates specific for five major
isoforms. The reaction was initiated by the addition of NADPH
and terminated by adding acetonitrile. The supernatant samples
were analyzed for the metabolites of the probe substrates by
LCMS:MS and the IC50 for selected compounds screened was deter-
mined using the software Graphpad Prism.
We gratefully acknowledge Orion Corporation for funding this
research.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.013.
7.5. Plasma protein binding
References and notes
Plasma protein binding assay was carried out by the ultra-filtra-
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lM solutions
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