Discovery of pyridyl bis(oxy)dibenzimidamide derivatives as selective matriptase inhibitors

Article abstract of DOI:10.1021/ml400213v

Matriptase belongs to trypsin-like serine proteases involved in matrix remodeling/degradation, growth regulation, survival, motility, and cell morphogenesis. Herein, we report a structure-based approach, which led to the discovery of sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as potent and selective matriptase inhibitors. Co-crystal structures of selected compounds in complex with matriptase supported compound designing. Additionally, WaterMap analyses indicated the possibility of occupying a distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine proteases.

Full text of DOI:10.1021/ml400213v

Letter
pubs.acs.org/acsmedchemlett
Discovery of Pyridyl Bis(oxy)dibenzimidamide Derivatives as
Selective Matriptase Inhibitors
Rajeev Goswami,† Subhendu Mukherjee,† Gerd Wohlfahrt,‡ Chakshusmathi Ghadiyaram,† Jwala Nagaraj,†
Beeram Ravi Chandra,† Ramesh K. Sistla,† Leena K. Satyam,† Dodheri S. Samiulla,† Anu Moilanen,§
Hosahalli S. Subramanya,† and Murali Ramachandra*,†
†Aurigene Discovery Technologies Limited, 39-40 KIADB Industrial Area, Electronic City Phase II, Bangalore 560 100, India
‡Orion Corporation, Orionintie 1, FIN-02101 Espoo, Finland
§Orion Corporation, Tengstrominkatu 8, FIN-20101 Turku, Finland
̈
S
* Supporting Information
ABSTRACT: Matriptase belongs to trypsin-like serine proteases
involved in matrix remodeling/degradation, growth regulation,
survival, motility, and cell morphogenesis. Herein, we report
a structure-based approach, which led to the discovery of
sulfonamide and amide derivatives of pyridyl bis(oxy)-
benzamidine as potent and selective matriptase inhibitors.
Co-crystal structures of selected compounds in complex with
matriptase supported compound designing. Additionally,
WaterMap analyses indicated the possibility of occupying a
distinct pocket within the catalytic domain, exploration of which resulted in >100-fold improvement in potency. Co-crystal
structure of 10 with matriptase revealed critical interactions leading to potent target inhibition and selectivity against other serine
proteases.
KEYWORDS: Matriptase, pyridyl dibenzimidamide, SAR, crystal structure, cancer
etastasis is the principal event leading to death in individuals
Altogether, these findings imply that matriptase is a good target
1
Mwith cancer. Progression of metastasis embraces complex for the development of anticancer drugs.
Compounds for matriptase inhibition including bis-benzami-
processes that require multiple proteolytic steps for degradation
and remodeling of the extracellular matrix. Because proteases from
diverse classes are involved in these mechanisms,2−5 a possible
approach to prevent tumor growth and progression is by the use of
specific protease inhibitors.6−8
dines,19 amidinophenylalanines,8,20 and peptidomimetics21 have
been reported earlier. Here we describe the discovery of bis-
benzamidine based inhibitors that were conceptualized by
structure-guided design strategies. Initial design ideas were
drawn from a high-resolution crystal structure of matriptase
catalytic domain in complex with benzamidine22 (Figure 1).
From the binding mode of benzamidine as sulfate salt (PDB
code: 1EAX),22 it was apparent that benzamidine binds to S1 pocket
forming a salt-bridge interaction with the side-chain carboxylate
of Asp189 in addition to a number of hydrogen bonding contacts
directly or indirectly with different residues in the vicinity such as
Ser190, Gly219, and Lys224. Interestingly, this structure also
revealed the position of a sulfate ion close to the benzamidine
binding site, which has hydrogen bonding interactions with catalytic
residues His57, Ser195, and Gly193 either directly or mediated via
water molecules. This crystal structure revealed opportunity for
incorporating substituents to a benzamidine core to extend toward
the spacious and solvent exposed S3/S4 pocket close to Trp215.
Additionally, screening of a benzamidine focused compound
library of known protease inhibitors resulted in identification of 1
Matriptase or MT-SP1 is a type II transmembrane serine
protease (TTSP) that is mainly expressed in the epidermis,
thymic stroma, and other epithelia.9,10 The proteolytic activity of
matriptase is controlled by hepatocyte growth factor activator
inhibitor-1 (HAI-1).9,10 Knockout mouse studies have revealed
that matriptase plays a role in terminal differentiation of
epidermis, hair follicle development, and thymic homeostasis.9
Relative ratio of matriptase to HAI-1 is reported to be higher in a
range of epithelial tumors, in which active matriptase plays an
important role in cellular invasion and metastasis.11,12 Matriptase
is known to be overexpressed in inflammatory conditions such
as osteoarthritis,13 but downregulated in inflamed colonic
tissues from Crohn’s disease and ulcerative colitis patients.14
Recent findings suggest that matriptase activates hemagglutinin
of H9N2 and H1N1 influenza A viruses and promotes viral
replication.15,16 Matriptase is also known to activate proteases
such as receptor-bound urokinase-type plasminogen activator
(uPA), which plays a critical role in angiogenesis, tumor
invasion, and metastasis. Down-regulation of matriptase inhibits
tumor invasion through suppression of uPAR activation.17,18
Received: June 17, 2013
Accepted: October 7, 2013
Published: October 7, 2013
© 2013 American Chemical Society
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Figure 1. Crystallographic binding mode of benzamidine and a sulfate ion in matriptase catalytic domain (PDB code: 1EAX). Benzamidine showed a
Ki ≈ 400 μM for matriptase inhibition.
(Table 1) with a Ki value of 9.6 μM for matriptase. Compound 1
is one of the archetypical inhibitors of serine proteases previously
described as an inhibitor of factor Xa (Ki = 0.4 μM), thrombin
(Ki = 0.6 μM), and trypsin (Ki = 2.8 μM).23 Further screening
of pyridyl-substituted derivatives led to the identification of
aminopyridine 2 with a Ki value of 2.9 μM for matriptase but low
selectivity against other proteases.
also revealed conformational similarity of the pyridinyl bis-oxy-
dibenzimidamide moieties (docked model of 2 and binding
mode of 8) except for the P3/P4 benzamidine, which rotate
slightly inward resulting in a backbone contact with Phe97 in the
90-loop region (Figure 2b). The sulfone moiety of 8 occupies
the sulfate ion binding site making similar interactions, and the
fluoro-phenyl moiety was found to be packing against the Cys42-
Cys58 disulfide-bridge and Ile41 side-chain.
Structural modeling of 2 in the benzamidine binding pocket
(Figure 2a) suggested additional hydrogen bonding possibilities
with Ser195 and Gly216 apart from S1 pocket contacts similar to
benzamidine. These observations for 2 motivated us to explore
additional engagement of the sulfate ion binding pocket to
enhance the affinity. Thus, a 3-amino group focused library with
various aryl and heteroaryl sulfonamides was synthesized, and
chemistry of 2 and 8 is outlined in Scheme 1. Treatment of
2,6-dichloro-3-nitropyridine with 4-hydroxybenzonitrile yielded
3, followed by reduction of nitro to amine (4), which was
sulfonylated using 4-fluorobenzenesulfonylchloride with DIPEA
(N,N-diisopropylethylamine) in THF and this, resulted in
sulfonamide derivative 5. Refluxing nitrile intermediate 5 with
NH2OH·HCl in EtOH converted to N-hydroxyamidine (6),
followed by acetylation to form N-acetoxyamidine (7), which
was reduced to amidine analogue 8 using zinc. Zinc was used to
avoid dehalogenated side product resulting under the hydro-
genation conditions. Compounds 9−11 were prepared utilizing
the same synthetic route as 8 using different aryl sulfonamides.
In contrast, intermediate 3 was separately subjected for con-
version of its nitrile to amidine and nitro to amine in situ during
hydrogenation of N-acetoxyamidine to afford 2.
The crystallographic binding mode of 8 revealed opportunities
for binding affinity improvement through better van der Waals
(vdW) contacts with Cys42-Cys58 disulfide-bridge and Ile41.
Toward predicting the impact of such interactions on binding
affinity, we applied a new computational method, WaterMap
by Schrodinger LLC,26 which maps the arrangement and
̈
thermodynamic properties of water molecules that might solvate
binding sites. This method also estimates the entropic and
enthalpic forces involved in binding of small molecules and hence
help to elucidate the ability of inhibitors to differentially displace
specific water molecules from binding sites. WaterMap based
predictions have supported the rational design of enzyme
inhibitors in recent publications.27
Initially, we investigated whether the WaterMap model
generated for 8 crystal structure (Figure 2c) could explain binding
affinity differences between benzamidine, 2, and 8. From
Figure 2c, we can see that 4−5 unstable water sites (ΔG >
2 kcal/mol) are mapped collectively in the vicinity of 3 regions,
(i) ether linker to the P1 benzamidine, (ii) pyridyl moiety, and
(iii) P3/P4 benzamidine, suggesting that appending the pyridinyl
bis-oxy-benzamidine to P1 benzamidine is likely to displace
unstable water molecules resulting in free energy gain. Consistent
with this notion, Ki improved by >100-fold when changed from
benzamidine to 2. Figure 2c also shows unstable water sites in the
area occupied by the fluorophenyl sulfone substituent of 8 while
filling the hydrophobic S1′ region, thereby explaining the 13-fold
binding difference between compounds 2 and 8. Furthermore,
we could see an unstable water site close to the fluorine atom in 8
(≈2.4 Å) with more accessible space in the S1′ region suggesting
that the fluorine could be replaced by a bulkier group (Figure 2c).
Newly synthesized compounds were evaluated for potency
and selectivity (see Supporting Information) (Table 1).
Compound 8 with 4-fluorophenyl extending toward the
sulfate ion binding site showed improvement in matriptase
inhibition (Ki of 0.2 μM). A high resolution crystal structure of 8
(PDB code: 4JZ1) in complex with matriptase showed that
the compound binds in the known substrate binding pocket
similar to other known prototype matriptase inhibitors8,22
indicating that 8 is a competitive inhibitor. The crystal structure
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Table 1. SAR of Pyridyl Bis(oxy)benzamidines
a
b
Average of two values obtained from two independent dose response studies with standard deviation within 10%. Values from ref 23; NA
c
indicates that the compound was not screened against the mentioned enzyme. Indicates that Ki was not determined because of poor dose response
in biochemical assay. Reported with two decimal places and one significant digit because of the high binding affinity (Ki < 0.05 μM) of these
d
compounds
Hence, a compound was synthesized replacing the fluoro-phenyl
in 8 with a naphthyl to obtain 10, which exhibited a Ki of 0.04 μM.
Interestingly, 11 with a slightly different arrangement of the
bicyclic aryl moiety (R, Table 1) that would not have mapped onto
the unstable water in vicinity of 8 fluorine did not show any
improvement in binding. A high resolution crystal structure of the
most potent compound in this series, 10 (PDB code: 4JYT) in
matriptase catalytic domain was solved, which revealed a similar
binding mode as 2 (Figure 2d), thereby validating the working
hypothesis of hit generation. Additionally, orientation of the
P3/P4 benzamidine in the crystal structure of 10 was similar to
the docked model of 2 (Figure 2a) indicating that the Phe97
interaction observed in the binding mode of 8 may not be so
critical for binding. The napthyl was also observed to make vdW
contacts with Cys42-Cys58 disulfide-bridge and Ile41 as predicted.
These findings indicate that, apart from S1 region polar inter-
actions, adequate S1′ region occupancy and vdW contacts could
be vital for greater potency.
The crystallographic binding mode of 10 was further utilized
for understanding the structural basis for selectivity against
other serine proteases, factor Xa, and thrombin. This structure
shows that the napthyl moiety orients toward the 60-loop
region (Figure 2e) adjacent to Cys42-Cys58 disulfide-bridge.
These loop regions have different lengths and adopt diverse
conformations in different serine proteases. From structural
comparison of matriptase with factor Xa and thrombin, we
concluded that the observed selectivity could be due to the clash
of the napthyl group with Gln61 in factor Xa and Trp60D and
Lys60F in thrombin (Figure 2e) due to conformational dif-
ferences of the 60-loop regions. From the crystal structure of 10,
it appeared that the aryl sulfonamide group may be replaceable
with carboxamide and that extended saturated substituents
could be coupled in order to reach the 60-loop region while
occupying the region close to Cys42-Cys58 disulfide-bridge.
Thus, a focused library of compounds with various alkyl amines
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Figure 2. Overlays: (a) docked model of 2 (Ki = 2.9 μM) and benzamidine (Ki ≈ 400 μM) binding mode; (b) docked model of 2 against crystallographic
binding mode of 8 (Ki = 0.2 μM); (c) binding modes of 8 against that of benzamidine in surface mode depicting unstable WaterMap sites. Color codings
for spherical (water) representations: red, crystallographic water molecules and lemon green, unstable water sites (ΔG > 2 kcal/mol); (d) Overlay of 8
and 10 crystallographic binding modes in matriptase catalytic domain along with mapped water molecules; (e) crystallographic binding mode of 10 in
matriptase catalytic domain and structural basis for selectivity against thrombin (gray; PDB code: 1EB124) and factor Xa (orange; PDB code: 1EZQ25);
(f) crystallographic binding modes of 10 and 24 in matriptase catalytic domain.
was synthesized following the synthetic route described in
Scheme 2.
Scheme 1. Synthetic Scheme of 4,4′-(Pyridine-2,6-
a
diylbis(oxy))dibenzimidamide Derivatives
Treatment of ethyl 2,6-dichloronicotinate with 4-hydroxy-
benzonitrile resulted in ester 12, which was hydrolyzed using
LiOH to obtain acid 13. tert-Butyl ((1R,4R)-4-aminocyclohexyl)-
carbamate was coupled with 13 using benzotriazol-1-yl-
oxytripyrrolidinophosphonium-hexafluorophosphate (PyBop)
to obtain 14. Following the method described in Scheme 1
except using aq. NH2OH in place of NH2OH·HCl (Supporting
Information), nitriles of 14 were converted to bis-amidine 17.
The Boc group was removed using ethanolic HCl to obtain 18.
Compounds 19−24 were synthesized utilizing the similar
synthetic procedures as described for 18 by using nicotinic acid
or isonicotinic acid intermediate (13) and coupling with different
alkyl amines. The results of selected compounds are summarized
in Table 1.
Most of these R1 modifications (Table 1) lead to reduced
potency, while some of the R2 modifications as in 23 and 24
exhibited similar inhibition as 10. Among these two compounds,
24 exhibited a better selectivity profile. To understand this
structure−activity relationship (SAR) paradox, we determined the
crystallographic binding mode of 24 (PDB code: 4JZI; Figure 2f).
Its crystal structure revealed an unexpected inverse binding mode
in comparison to the pyridyl sulfonamides. In the case of pyridyl
sulfonamides (10), the 3-pyridyl sulfonamide substituents
(R1 in Table 1) occupy a region close to the Cys42-Cys58
a
Reagents and conditions: (a) K2CO3, DMF, 75 °C. (b) NH2OH·HCl,
DIPEA, EtOH, 6 h, 85 °C. (c) Ac2O, AcOH, RT. (d) H2 at balloon
pressure, 10% Pd/C, AcOH, 30 °C. (e) Zn powder, AcOH, 35 °C.
(f) 4-Fluorobenzene sulfonylchloride, DIPEA, THF, RT.
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a
Scheme 2. Synthetic Scheme of 2,6-Bis(4-carbamimidoylphenoxy)nicotinamide Derivatives
a
Reagents and conditions: (a) K2CO3, DMF, 75 °C. (b) LiOH, THF, MeOH, 2 h, 15−20 °C. (c) tert-Butyl ((1R,4R)-4-aminocyclohexyl)carbamate,
PyBop, DIPEA, DMF, RT. (d) Aq. NH2OH, EtOH, 4 h, 75 °C. (e) Ac2O in AcOH, RT. (f) Zn powder, AcOH, RT. (g) HCl(g)−EtOH at 5−10 °C.
Table 2. Analysis of Selected Compounds for Cellular Activity on PC-3 and DU-145, Aqueous Solubility, and Metabolic Stability
a
% inhibition at 10 μM
a
a
% cytotoxicity at 10 μM
PC-3
DU-145
aq sol (μM)
MLM stability (% parent remaining)
ID
PC-3
DU-145
migration
invasion
migration
invasion
pH 7.4
20 min
40 min
8
0
0
65 2.4
39 2.1
53 1.8
60 0.8
42 0.9
42 5.9
69 0.8
59 0.8
65 1.1
61 2.4
47 1.1
69 2.0
194
191
196
4
4
2
89.0 2.3
76.4 2.5
100 10.3
86.5 10.5
73.2 5.5
95.6 11.2
10
24
20
1
20
0
a
Values represent mean SD.
disulfide-bridge, but in this case (24), the R1 moiety trans-4-
aminocyclohexane orients away from that region, and instead,
the P3/P4 benzamidine moiety of 24 occupies the region
close to Cys42-Cys58 disulfide-bridge making two polar back-
bone contacts with His57 and Cys58 (Figure 2f). Molecular
modeling suggests that compounds including 18−21 with R1
substituents are likely to sterically interfere with either Phe99
or Trp215-Gly216 potentially leading to reduced matriptase
inhibition.
After establishing the SAR for matriptase inhibition and sel-
ectivity against other proteases, selected compounds were studied
for their effect on matriptase expressing prostate carcinoma cell
lines (PC-3 and DU-145), solubility, and metabolic stablility in
mouse liver microsomes (MLM) (Table 2).
Compounds 8 and 24 were not cytotoxic and all tested
compounds displayed good inhibition of invasion and migration
in PC-3 and DU-145 cells. All three compounds also showed
high aqueous solubility and good metabolic stability.
In conclusion, we have optimized a pyridyl bis(oxy)-
benzamidine series and achieved high potency toward matriptase
(Ki = 0.04 μM) and desirable selectivity over other proteases,
cellular activity, solubility, and metabolic stability. The optimized
compounds with sulfonamide and isonicotinamide derivatives
have the potential to be used as anti-invasive agents.
ASSOCIATED CONTENT
* Supporting Information
Experimental details for synthesis, protein expression, assay
methods, modeling, and crystallography. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(M.R.) Tel: +91 80 7102 5444. Fax: +91 80 2852 6285. E-mail:
murali_r@aurigene.com.
■
Funding
We gratefully acknowledge Orion Corporation for funding this
research.
Notes
The authors declare the following competing financial interest(s):
All authors are current or former employees of Aurigene Discovery
Technologies Limited or Orion Corporation as indicated.
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