Structure-based design, synthesis, and binding mode analysis of novel and potent chymase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.11.031

Based on insight from the X-ray crystal structure of human chymase in complex with compound 1, a lactam carbonyl of the diazepane core was exchanged with O-substituted oxyimino group, leading to amidoxime derivatives. This modification resulted in highly potent chymase inhibitors, such as O-phenylamidoxime 5f. X-ray crystal structure analysis indicated that compound 5f induced movement of the Leu99 and Tyr94 side chains at the S2 site, and the increase in inhibitory activity of O-phenyl amidoxime derivatives suggested that the O-phenyl moiety interacted with the Tyr94 residue. Surface plasmon resonance experiments showed that compound 5f had slower association and dissociation kinetics and the calculated residence time of compound 5f to human chymase was extended compared to that of amide compound 1.

Full text of DOI:10.1016/j.bmcl.2017.11.031

Accepted Manuscript
Structure-based design, synthesis, and binding mode analysis of novel and po-
tent chymase inhibitors
Junko Futamura-Takahashi, Taisaku Tanaka, Hajime Sugawara, Shinzo
Iwashita, Seiichi Imajo, Yoshiaki Oyama, Tsuyoshi Muto
PII:
S0960-894X(17)31118-6
https://doi.org/10.1016/j.bmcl.2017.11.031
BMCL 25437
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 October 2017
14 November 2017
15 November 2017
Please cite this article as: Futamura-Takahashi, J., Tanaka, T., Sugawara, H., Iwashita, S., Imajo, S., Oyama, Y.,
Muto, T., Structure-based design, synthesis, and binding mode analysis of novel and potent chymase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.031
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Bioorganic & Medicinal Chemistry Letters
Structure-based design, synthesis, and binding mode analysis of novel and potent
chymase inhibitors
Junko Futamura-Takahashi, Taisaku Tanaka†, Hajime Sugawara, Shinzo Iwashita, Seiichi Imajo, Yoshiaki
Oyama, Tsuyoshi Muto*
Asubio Pharma Co., Ltd, 6-4-3 Minatojima-Minamimachi, chuo-ku, kobe 650-0047, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Based on insight from the X-ray crystal structure of human chymase in complex with compound
1, a lactam carbonyl of the diazepane core was exchanged with O-substituted oxyimino group,
leading to amidoxime derivatives. This modification resulted in highly potent chymase
inhibitors, such as O-phenylamidoxime 5f. X-ray crystal structure analysis indicated that
compound 5f induced movement of the Leu99 and Tyr94 side chains at the S2 site, and the
increase in inhibitory activity of O-phenyl amidoxime derivatives suggested that the O-phenyl
moiety interacted with the Tyr94 residue. Surface plasmon resonance experiments showed that
compound 5f had slower association and dissociation kinetics and the calculated residence time
of compound 5f to human chymase was extended compared to that of amide compound 1.
Keywords:
chymase
inhibitor
structure-based design
surface plasmon resonance
2009 Elsevier Ltd. All rights reserved.
Chymase is a chymotrypsin-like serine protease stored in TC type mast cells that are localized in connective tissue such as the lung,
small intestine, and skin.1,2 Chymase is released from mast cell granules by various stimuli and is involved in inflammatory diseases,
including dermatitis,3,4 allergic conjunctivitis,5,6 and inflammatory bowel disease.7 In previous studies of chymase inhibitors as
therapeutic agents for atopic dermatitis, we identified 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as selective
chymase inhibitors.8,9 Oral dosing of the representative compound SUN13834 improved spontaneous dermatitis in NC/Nga mice.10
Furthermore, in a mouse dermatitis model induced by repeated painting of 2,4-dinitrofluorobenzene, SUN13834 inhibited not only skin
swelling, but also accumulation of inflammatory cells in the skin and significantly decreased scratching behavior.7 Based on these
results, we carried out further structural modifications to improve the inhibitory activity and/or pharmacokinetic profile of this series of
compounds and identified novel amidoxime derivatives as potent novel chymase inhibitors. In addition, we found that a substituent on
the amidoxime moiety affected the binding mode of the inhibitor to chymase. In this study, we designed and synthesized amidoxime
derivatives based on the crystal structure of human chymase in complex with compound 1 (Fig. 1) and analyzed the binding structure
and binding kinetics of amidoxime derivatives.
We previously reported the X-ray crystal structure of human chymase in complex with compound 1 (Fig. 1).9 The key structural
features are 1) a benzyl unit located in the hydrophobic S1 pocket, 2) a lactam NH that forms a hydrogen bond with the main chain
carbonyl of Ser214, 3) an ethyl moiety orientated in the S1’ site, and 4) amino group and carboxyl group of an anthranic acid, which
cooperatively interact with the main chain carbonyl of Phe41 and side chain of Arg143, respectively. Further investigation of the crystal
structures of human chymase bound to 1 or related inhibitors revealed the presence of a water molecule in the hydrophobic S2 site
comprised of Leu99, Asp102, Ser214, and Tyr215. This water molecule may interact with the carbonyl oxygen at the 2-position of 1
and phenolic hydroxyl group of the Tyr94 side chain. The interaction distances were 3.21 and 2.96 Å, respectively. Examination of
other crystal structures of human chymase in complex with our compounds showed that this water molecule was not always observed.
In other known X-ray crystal structures of chymase in complex with its inhibitor (e.g. 3S0N, 4K69, 4KP0), this water molecule was
also not observed. These findings and the longer distance of these hydrogen bonds suggest that the contribution of the water molecule to
binding affinity of 1 is relatively weak. To confirm this prediction, we evaluated the thermodynamic properties of each water molecule11
in the active site of chymase (details are described in the supplementary content). Analysis of the calculated interaction energies and
excess entropy terms of each hydration site indicated that the water molecule in the hydration site of the S2 region was
———
 Corresponding authors.
E-mail addresses: futamura.junko.b6@asubio.co.jp (J. Futamura-Takahashi), muto.tsuyoshi.ba@asubio.co.jp (T. Muto)
†Present address: Shinagawa R&D Center, Daiichi-Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan

thermodynamically unfavorable, showing a relatively poor interaction energy despite the large entropy reduction. Based on this result,
we expected that displacement of the water molecule with a suitable substituent favorably contributed to the binding free energy,
resulting in improved potency. To introduce a substituent that can interact with the S2 site, we designed the O-substituted amidoxime
derivatives shown in Table 1, maintaining the key hydrogen bond between NH of the seven-membered ring of the inhibitor and
backbone carbonyl of Ser214.
Figure 1. Schematic view of compound 1 in the active site of human chymase and design of amidoxime derivatives.
The amidoxime compounds in Table 1 were prepared from the known lactams 2a and 2c (Scheme 1).8 To synthesize benzoic acid
derivatives 5b and 5g, the lactam carbonyl of 2a was selectively thionated with Belleau’s reagent to generate thioamide 3a, which was
converted to amidoxime compounds 4b and 4g in a reaction with mercury (II) acetate and the corresponding hydroxylamines. Next, a
2,4,6-trimethoxybenzyl (TMB) group and tert-butyl group were deprotected simultaneously under acidic conditions to afford 5b and 5g.
The benzoic acid derivatives 5c–e were synthesized in the same manner as above using 2b, which was prepared from 2a by
deprotection of the TMB group with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). This method, including initial deprotection of
the TMB group, has the advantage that final product is easily isolated and purified, although this results in an additional synthetic step.
In the case of anthranic acid compounds 5a and 5f, the TMB group of compound 2c was initially deprotected by DDQ, and then the
nitro group was reduced with zinc powder in acetic acid to give compound 2d, which was converted to compounds 5a and 5f in the
same manner as above. The reference compound 6 was prepared from 2a by deprotection of the TMB group and tert-butylester under
acidic condition.
Scheme 1. Reagents and conditions: (a) DDQ, CH2Cl2-H2O, rt; (b) Zn, AcOH, rt; (c) Belleau's reagent, THF, 0℃; (d) R2ONH2･HCl,
Hg(OAc)2, Et3N, THF-MeOH, 0℃; (e) HCl/AcOEt, rt; (f) TFA, anisole, rt.

The compounds shown in Table 1 were evaluated for their inhibitory activity against recombinant human chymase (detailed assay
conditions are described in the supplementary content). O-Ethylamidoximes 5a and 5b showed more potent inhibitory activities than
the corresponding amide compounds 1 and 6, as expected. In the both cases of the amide (1 and 6) and amidoxime (5a and 5b)
derivatives, introduction of amino group to benzoic acid moiety slightly increase the inhibitory activity. Although the hydrogen bond
between the amino group and Phe41 main chain would contribute to improve the potency, we initially examined the substituent effect
of amidoxime moiety by using readily prepared benzoic acid derivatives (R1 = H). As a result, the inhibitory activity of bulkier O-alkyl
amidoximes such as isopropyl 5c, isobutyl 5d, and cyclopentyl 5e decreased as the size of the alkyl substituent increased. However, in
contrast to the alkyl substituents, the bulky O-phenylamidoxime 5g showed more potent inhibitory activities. Furthermore, 5f, the
anthranic acid derivative of O-phenylamidoxime, exhibited the most potent, a one digit nanomolar activity. This result shows the
importance of amino group of anthranic acid moiety again, and also the preservation of hydrogen bond with Phe41 in amidoxime
derivatives.
Table 1. Inhibitory activity of amidoxime derivatives against human chymasea.
Compound
R1
A
IC50 (M)
NH2
H
O
0.30
0.57
0.078
0.24
1
O
6
NH2
H
N-O-Et
N-O-Et
5a
5b
H
H
H
N-O-iso-Pr
0.58
1.2
5c
5d
5e
N-O-iso-Bu
N-O-cyclo-Pen
2.4
NH2
H
N-O-Ph
N-O-Ph
0.0089
0.050
5f
5g
a: Detailed assay conditions are described in the supplementary content.
To understand these substituent effects, we examined the X-ray crystal structures of O-ethylamidoxime 5b and O-phenylamidoxime
5f bound to human chymase (Fig. 2 and 3). The X-ray crystal structure of O-ethylamidoxime 5b in the active site of human chymase
(Figure 2a, PDB ID: 5YJP) revealed that nearly all key interactions were maintained compared to that of amide compound 1: 1) the
benzyl unit occupies the S1 pocket, 2) the lactam NH forms a hydrogen bond with Ser214, 3) the ethyl moiety is located in the S1’ site,
and 4) the benzoic acid interacts with Arg143. At the S2 site, the Leu99 side chain slightly moves and accepts the ethyl moiety of
compound 5b (Fig. 3a). Therefore, the water molecule is released into bulk solution, as expected. The increase in inhibitory activity of
5b may be attributable to the free energy gain from the release of water molecules. Since the inhibitory activity decreased as the size of
alkyl substituent of amidoxime moiety was increased, it is speculated that the movement of the Leu99 side chain is restricted. In the
case of the complex structure with O-phenylamidoxime 5f (Fig. 2b, PDB ID: 5YJM), it is observed that in addition to the movement of
Leu99, the side chain structure of Tyr94 is also changed at S2 site (Fig. 3b). The conformational change of Tyr94 was thought to be the
result of an interaction with the phenyl moiety, and, as a result, the inhibitory activity of O-phenylamidoximes was improved.

Figure 2. X-ray crystal structures of human chymase in complex with O-ethylamidoxime 5b (a; PDB ID: 5YJP) and O-
phenylamidoxime 5f (b; PDB ID: 5YJM). The molecular surfaces of chymase are represented by gray mesh and the key amino acids are
shown in stick form. The inhibitors 5b and 5f are shown as a ball-and-stick representation (C: Green, O: Red, N: Blue, Cl: Dark
Green).
Figure 3. Enlarged view at S2 site of superposition of X-ray co-crystal structures of chymase and its inhibitors. The molecular surface
of the inhibitors are represented by a colored mesh: (a) 1 (cyan) and 5b (gray), and (b) 1 (cyan) and 5f (gray). The inhibitors are shown
as ball-stick representations and the key amino acid residues, Leu99 and Tyr94, are highlighted by colored stick representations.

To evaluate the influence of the conformational change at the S2 site on inhibitor binding kinetics, surface plasmon resonance (SPR)
experiments for compounds 1 and 5f were performed. Representative sensorgrams are shown in Figure 4. Comparison of each
sensorgram pattern revealed apparently different binding profiles. The calculated kinetic parameters indicate that 5f has a 10-fold
slower association rate and 40-fold slower dissociation rate than those of 1, respectively. The slower binding kinetics of 5f can be
attributed to an induced conformational change at the S2 site by 5f and/or to the resulting strong hydrophobic interaction. The slower
off-rate of 5f, indicating a longer residence time to the active site of chymase, is attractive from a pharmacologic perspective, as it is
expected that the inhibitor inhibits chymase for longer times in vivo.12,13
Figure 4. SPR sensorgrams and binding kinetics of componds 1 and 5f. Residence time τ was calculated as the reciprocal of the mean
koff
value
(τ
=
1/koff)
In conclusion, we identified a novel series of potent human chymase inhibitors by structure-based design based on the X-ray crystal
structure of compound 1 and human chymase. We exchanged the amide carbonyl of the seven-membered ring to the O-substituted
oxyimino group, generating amidoxime derivatives. The X-ray crystal structures of chymase with amidoxime derivatives such as 5b
and 5f showed that the water molecule observed in the S2 site of the complex structure of chymase bound to compound 1 is released
into bulk water and that the side chain conformations of the S2 site amino acids are changed to accept the substituents of inhibitors.
Furthermore, SPR analysis showed that 5f had slower association and dissociation profiles than compound 1.The conformational
change at the S2 site and/or resulting strong hydrophobic interaction is thought to explain the slower binding kinetics. Further structure-
activity relationship studies of this series of amidoxime derivatives will be conducted.
4. Watanabe N, Tomimori Y, Saito K, Miura K, Wada A, Tsudzuki M,
Acknowledgments
Fukuda Y. Chymase inhibitor improves dermatitis in NC/Nga mice. Int
Arch Allergy Immunol. 2002;128:229-234.
5. Nabe T, Kijitani Y, Kitagawa Y, Sakano E, Ueno, T, Fujii M, Nakao S,
Sakai M,Takai S. Involvement of chymase in allergic conjunctivitis of
guinea pigs. Exp. Eye Res. 2013;113:74-79.
6. Ebihara N, Funaki T, Miyazaki M, Fujiki K, Murakami A. Tear chymase
in vernal keratoconjunctivitis. Curr Eye Res. 2004;28:417-420.
7. Liu WX, Wang Y, Sang LX, Zhang S, Wang T, Zhou F, Gu SZ.
Chymase inhibitor TY-51469 in therapy of inflammatory bowel disease.
World J Gastroenterol. 2016;22:1826-1833.
We are grateful to Dr. T. Nakatsuka for his helpful advice and
valuable discussion during this study. We also thank Dr. Y.
Tomimori for his assistance in preparing the manuscript.
Supplementary Material
8. Maruoka H, Muto T, Tanaka T, Imajo S, Tomimori Y, Fukuda Y,
Nakatsuka T. Development of 6-benzyl substituted 4-aminocarbonyl-1,4-
diazepane-2,5-diones as orally active human chymase inhibitors. Bioorg
Med Chem Lett. 2007;17:3435-3439.
Supplementary content associated with this article can be found,
in the online version, at
9. Tanaka T, Sugawara H, Maruoka H, Imajo S, Muto T. Discovery of
novel series of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-
diones as human chymase inhibitors using structure-based drug design.
Bioorg Med Chem. 2013;21:4233-4249.
10. Watanabe N, Tomimori Y, Terakawa M, Ishiwata K, Wada A, Muto T,
Tanaka T, Maruoka H, Nagahira K, Nakatsuka T, Fukuda Y. Oral
administration of chymase inhibitor improves dermatitis in NC/Nga mice.
J Invest Dermatol. 2007;127:971-973.
References
1. Schwartz BL, Austen FK. Enzymes of the mast cell granule. J Invest
Dermatol. 1980;74:349-353.
2. Irani AM, Bradford TR, Kepley CL, Schechter NM, Schwartz LB.
Detection of MCT and MCTC types of human mast cells by
immunohistochemistry using new monoclonal anti-tryptase and anti-
chymase antibodies. J Histochem Cytochem. 1989;37:1509-1515.
3. Tomimori Y, Muto T, Fukami H, Saito K, Horikawa C, Tsuruoka N,
Saito M, Sugiura, N, Yamashiro K, Sumida M, Kakutani S, Fukuda Y.
Chymase participates in chronic dermatitis by inducing eosinophil
infiltration. Lab Invest. 2002; 82:789-794.
11. Abel R, Young T, Farid R, Berne BJ, Friesner RA. Role of the active-site
solvent in the thermodynamics of factor Xa ligand binding. J Am Chem
Soc. 2008;130:2817-2831.

12. Copeland RA, Pompliano DL, Meek TD. Drug-target residence time and
its implications for lead optimization. Nat Rev Drug Discov. 2006;5:730-
739.
13. Copeland RA. The drug-target residence time model: a 10-year
retrospective. Nat Rev Drug Discov. 2016;15:87-95.
Graphical Abstract
Structure-based design, synthesis,
and binding mode analysis of novel
and potent chymase inhibitors
Leave this area blank for abstract info.
Junko Futamura-Takahashi, Taisaku Tanaka, Hajime Sugawara, Shinzo Iwashita, Seiichi Imajo,
Yoshiaki Oyama, Tsuyoshi Muto