Benzimidazolone as potent chymase inhibitor: Modulation of reactive metabolite formation in the hydrophobic (P1) region

Article abstract of DOI:10.1016/j.bmcl.2011.05.126

A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P₁ hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation of a GSH conjugate on the benzothiophene P₁ moiety. Replacement of the benzothiophene with different heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P₁ replacements, benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials agreed with the experimental outcome.

Full text of DOI:10.1016/j.bmcl.2011.05.126

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4533–4539
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzimidazolone as potent chymase inhibitor: Modulation of reactive
metabolite formation in the hydrophobic (P1) region
Ho Yin Lo a, , Peter A. Nemoto a, Jin Mi Kim a, Ming-Hong Hao a, Kevin C. Qian a, Neil A. Farrow a,
⇑
Daniel R. Albaugh d, Danielle M. Fowler b, Richard D. Schneiderman b, E. Michael August c, Leslie Martin c,
Melissa Hill-Drzewi c, Steven S. Pullen b, Hidenori Takahashi a, Stéphane De Lombaert a,
a Boehringer Ingelheim Pharmaceuticals Inc., Medicinal Chemistry, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
b Boehringer Ingelheim Pharmaceuticals Inc., Cardiometabolic Diseases, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
c Boehringer Ingelheim Pharmaceuticals Inc., High Throughput Biology, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
d Boehringer Ingelheim Pharmaceuticals Inc., Drug Discovery Support, 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P1
hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation
of a GSH conjugate on the benzothiophene P1 moiety. Replacement of the benzothiophene with different
heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P1 replacements,
benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials
agreed with the experimental outcome.
Received 15 April 2011
Revised 27 May 2011
Accepted 31 May 2011
Available online 17 June 2011
Keywords:
Chymase
Ó 2011 Elsevier Ltd. All rights reserved.
Cathepsin
Serine protease
GSH
Indole
Benzoisothiazole
Chymase is a chymotrypsin-like serine protease that is stored in
a latent form in secretory granules of mast cells.1 Upon stimulation,
mast cells degranulate and release an active form of chymase into
the local tissue.2 In addition to angiotensin converting enzyme, chy-
mase has been demonstrated to catalyze the formation of angioten-
sin II (Ang II) from angiotensin I,3 although the physiological
significance of this remains unclear since the role of chymase on
the regulation of blood pressure is minimal.4 Besides generating
Ang II, chymase is also believed to be involved in the activation of
transforming growth factor-b (TGF-b)5 and the production of colla-
gen type I.6 During pathological states, the release of chymase in lo-
cal tissues such as the heart may result in vascular injury, fibrosis,
and cardiac remodeling upon chronic exposure.
Heart failure is one of the leading causes of death in the United
States and continues to represent an unmet medical need. A link
between heart failure and chymase has been documented,7 and
there is an interest to develop a specific chymase inhibitor as a
new therapeutic regimen for the disease. A number of small
molecules inhibitors for chymase have been reported.8 TPC-806
(Fig. 1) is the first reported non-covalent chymase inhibitor9 and
has been in phase II clinical trials for heart failure since 2007. In sil-
ico analysis of the binding mode of TPC-806 with human chymase
suggested a possible replacement of the benzimidazole core of
TPC-806 with a benzimidazolone. As shown in Figure 1, lead com-
pounds 1 and 2 were prepared and tested against TPC-806 in a
human chymase (h-Chy) enzyme assay,10 compound 1 is only
threefold weaker than TPC-806 (IC50 of 70 nM vs 22 nM). We be-
lieve that benzimidazolones represent an interesting new class of
inhibitor with perhaps better physical chemical properties than
TPC-806 (c log P for TPC-806: 5.5; c log P for 1: 3.7).
An X-ray co-crystal structure of lead compound 2 bound to
human chymase was obtained which allowed determination of
the binding mode of the inhibitor (Fig. 2). The benzothiophene,
namely the P1 moiety of the inhibitor, occupies a well defined
hydrophobic pocket, namely the S1 pocket with a size of approxi-
mately 10 Å deep, 7 Å wide and 7 Å high. The benzimidazolone
core orientates perpendicular to the P1 moiety and anchors the
carboxylic acid side chain to interact with two basic amino acid
residues, LYS40 and LYS192. This acid–base interaction is critical to
the affinity of the inhibitor.11
Assessment of the target independent properties of this new
class of inhibitor revealed reactive metabolite formation as a liabil-
ity of compound 1. Upon incubation of compound 1 with glutathi-
one (GSH) in the presence of human liver microsomes, a GSH
⇑
Corresponding author.
E-mail address: ho-yin.lo@boehringer-ingelheim.com (H.Y. Lo).
Present address: Karos Pharmaceuticals.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.126

4534
H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
O
OH
OH
O
O
OH
n=1, 2
N
N
De Novo Design
N
N
N
O
N
O
S
S
S
S
+2 H
HO
365
S
O
O
TPC-806
h-Chymase IC50: 22 nM
1: n=1: h-Chymase IC50: 70 nM
2: n=2: h-Chymase IC50: 180 nM
H
N
H2N
HO
N
H
OH
Figure 1. De novo design for benzimidazolone core.
O
O
561
337
458
337
615
458
Figure 3. GSH conjugate of 1 (with LC/MS/MS fragments).
O
O
a, b
N
N
O
+
O
O
N
H
N
H
O
3
I
N
c
S
4
O
HO
O
O
d
N
N
N
N
O
O
Figure 2. X-ray co-crystal structure of 2 in human chymase.
S
S
conjugate was formed.12 Identification of the pathways leading to
potential reactive metabolites is considered as one of the key stud-
ies in understanding the mechanism of toxicity associated with a
compound of interest because there are numerous reports linking
the formation of glutathione adducts with xenobiotics to either
liver toxicity or idiosyncratic reaction.13 Glutathione has been
frequently used to trap potential reactive metabolites generated
through bioactivation in systems such as liver microsomes. Gluta-
thione adducts are able to be detected by a mass spectrometer, and
the results are used not only to confirm the presence of reactive
metabolites, but also to provide information on their potential
structures. To further elucidate the structure of the glutathione
adduct, an enhanced product ion scan was performed. Based on
the enhanced product ion spectra, the site of conjugation was
tentatively assigned to the P1 benzothiophene group (Fig. 3).
With this information in hand, it is possible to alleviate this
liability by replacing the benzothiophene P1 with other systems
that are resistant to glutathione conjugation. In this Letter, an ef-
fort to identify an alternative P1 moiety is reported.
1
5
Scheme 1. Synthesis of benzimidazolone 1; key: Reagents and conditions: (a)
benzyltrimethylammonium hydroxide, MeOH, rt, 76%; (b) HCl, MeOH/H2O, 60 °C,
92%; (c) 4, K2CO3, DMF, 90 °C, 50%; (d) LiOH, 1,4-dioxane/H2O, rt, 100%.
HCl gave benzimidazolone 3. SN2 reaction of 3 with benzothio-
phene iodide 4 provided benzimidazolone ester 5. The acid moiety
in lead compound 1 was finally unmasked with LiOH in quantita-
tive yield.
Based on molecular modeling, the first set of alternative P1s was
designed to focus on ligand similarity with respect to benzothio-
phene. Different bicyclic systems, which have appropriate steric
requirements, were prepared. The syntheses of those analogs are
similar to 1 with replacement of intermediate 4 with other bicyclic
systems (Scheme 1). The synthetic routes of some representative
bicyclic P1 intermediates and analogs are described in Schemes
2–6.
As shown in Scheme 2, the synthesis of indole P1 analog 31
started with methylation of 3-methyl indole to give dimethyl
indole 6. Friedel–Crafts formylation of 6 with pyrophosphoryl
The general synthetic route of compound 1 is depicted in
Scheme 1. Starting with 1-isopropenyl-2-benzimidazolidinone,
Michael addition to ethyl acrylate followed by deprotection with

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
4535
dine P1 analog 12, the synthesis began with a SN2 displacement of
bromo-malonaldehyde with 2-amino-6-picoline followed by
in situ cyclization to form imidazopyridine aldehyde 9 (Scheme
3). Reduction of aldehyde in 9 with LiAlH4 gave alcohol 10. The
imidazopyridine P1 could then be incorporated into the benzimi-
dazolone core by Mitsunobu reaction, followed by hydrolysis to
give analog 12 in fair yield. The preparation of pyrazolopyridine
16 is described in Scheme 4. Amino-pyridinium salt 13 was first
prepared from 3-picoline and was then subjected to ethyl propio-
late to give cyclization products 14a and 14b in about 2 to 1 ratio.
The desirable regioisomer 14a was then reduced to alcohol 15 by
DIBAL-H. Following similar procedures to the preparation of 12,
pyrazole–pyridine 16 was obtained in moderate yield. The first
step of the preparation of indolizine P1 was the formation of pyrid-
imium salt 17 from 2-picoline and chloroacetic acid. It was reacted
with acrylonitrile under oxidative conditions to give cycloadduct
18 in moderate yield. Following similar procedures to the indole
P1 intermediate (8), indolizine P1 intermediate 20 was obtained
accordingly (Scheme 5). Finally, the preparation of benzoisothiaz-
ole P1 intermediate was depicted in Scheme 6. Starting from
3,5-dimethylthiophenol, Friedel–Crafts acylation with oxalyl chlo-
ride gave thioisatin 22 in fair yield. The rearrangement reaction of
22 to benzoisothiazole 23 was accomplished under NH3 in MeOH
and H2O2 conditions. Hydrolysis of the amide group in 23 followed
by esterification provided benzoisothiazole ester 24 in good yield.
Reduction of ester 24 followed by SN2 displacement with a bro-
mide ion source gave bromo-benzoisothiazole intermediate 26.
The synthesis of the final benzoisothiazole analogs were completed
with coupling method described in Scheme 1.
I
O
N
H
c, d
a
b
N
N
N
N
H
6
7
8
Scheme 2. Synthesis of indole P1 intermediate; key: Reagents and conditions: (a)
NaH, MeI, DMF, 0 °C to rt; 100%; (b) pyrophosphoryl chloride, DMF, 5 °C, 76%; (c)
Me2NH, NaBH(OAc)3, CH2Cl2, 0 °C to rt, 57%; (d) MeI, CH3CN, rt, 82%.
O
O
HO
N
O
H
H2N
Br
a
b
N
N
N
N
9
10
c
O
HO
O
O
d
N
N
N
N
O
O
N
N
N
N
The analogs were tested in the human chymase enzyme assay
and a human cathepsin G enzyme assay14 to evaluate the selectiv-
ity profile. The results are shown in Table 1. Firstly, steric bulk in
the 50 position of the benzothiophene in 1 is important for chymase
activity because of hydrophobic interactions. Removal of the
methyl group as in unsubstituted benzothiophene 27, resulted in
a significant loss of activity. Next, the benzothiophene P1 in 27
can either be replaced by different heterocycles (such as benzoiso-
thiazole 28; indole 29) or a carbocycle (such as naphthalene 30)
without significant lost of activity to chymase. Combining the
above findings, 50-methyl indole 31 was prepared and showed
comparable activity profile to 1 (only about twofolds difference
in both chymase and cathepsin G activities). Due to the fact that
12
11
Scheme 3. Synthesis of imidazopyridine P1; key: Reagents and conditions: (a)
EtOH, 90 °C, 68%; (b) LiAlH, THF, ꢀ10 °C, 74%; (c) 3, DIAD, PPh3, THF, rt, 30%; (d)
LiOH, 1,4-dioxane/H2O, rt, 100%.
chloride provided aldehyde 7. Indole P1 intermediate 8 was
prepared by sequential reactions of reductive amination and iodide
salt formation of 7. The synthesis of analog 31 was then completed
according to the method depicted in Scheme 1. As for imidazopyri-
O
O
O
O
a
b
+
N
N
N
N
N
N
I
NH2
14a
14b
13
HO
c
O
OH
d, e
N
N
O
N
N
15
N
N
16
Scheme 4. Synthesis of indazolopyridine P1 key: Reagents and conditions: (a) hydroxylamine-O-sulfonic acid, HI, H2O, 87 °C to rt, 30%; (b) ethyl propiolate, K2CO3, DMF, rt,
14a: 31%, 14b: 17%; (c) DIBAL-H, THF, 0 °C, 51%; (d) 3, DIAD, PPh3, THF, rt, 42%; (e) LiOH, 1,4-dioxane/H2O, 100%.

4536
H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
O
NH2
O
O
N
a
a
b
O
N
+
HO
O
N
N
N
b
OH
S
S
Cl
SH
Cl
22
23
N
18
17
c, d
O
c
O
OH
Br
OH
O
O
I
H
N
f
e
N
N
f, g
N
N
d, e
N
N
N
O
S
S
S
26
25
24
N
N
Scheme 6. Synthesis of benzoisothiazole P1 intermediate; key: Reagents and
conditions: (a) AlCl3, oxalyl chloride, CH2Cl2, 120 °C microwave, 35%; (b) NH3/
MeOH, H2O2, rt, 33%; (c) KOH, EtOH/H2O, 85 °C, 75%; (d) H2SO4, MeOH, 60 °C, 94%;
(e) LiBH4, THF, 0 °C; (f) CBr4, PPh3, CH2Cl2, rt, 68% for two steps.
N
20
N
19
21
Different analogs in the ‘6–5’ indole family were tested in the
chymase and cathepsin G enzyme assays as shown in Table 2.
Methylindole 37 showed about fourfold loss in chymase inhibitory
activity compared with 31. In contrast to our prediction from
molecular modeling, systematically building up bulk with methyl
groups around the indole 10, 20 and 30 positions did not improve po-
tency dramatically, with the best case of 140 nM for 41, in which
three methyl groups were included. Surprisingly, installation of
an additional methyl group in the 50 position as in 42 provided a
small improvement of chymase activity compared with 41. Electron
donating and withdrawing groups in the 50 position were then
investigated (43 and 36). The chloro-substituted analog 36 showed
the most promising result with an IC50 of 25 nM against chymase,
which is equivalent to TPC-806. The selectivity profile of chymase
over cathepsin G in 36 is also encouraging (>800-fold). Interest-
ingly, relocating the chlorine group from the 50 to 60 position as in
44 completely eliminated the chymase activity. This could be ratio-
nalized, based on molecular modeling, the steric effects of the 60-
chlorine which forces the P1 group into an unfavorable orientation
for binding with respect to the core as illustrated in Figure 2.
Up to this point, we have demonstrated that benzothiophene P1
can be replaced by various heterocyclic P1s in terms of chymase
activity. ‘5–6’ Indole, indolzine and benzoisothiazole P1 moieties
showed comparable chymase activity and selectivity over cathep-
sin G, while ‘6–5’ indoles moiety (36) showed superior chymase
activity than the benzothiophene P1 moiety.
We next addressed if the new P1 systems prevent the formation
of GSH conjugates, which was the primary goal for this research on
the P1 moiety. Selected analogs, based on the chymase activity,
were tested in the GSH adduct assay, initially to monitor for GSH
conjugation, and then to identify the site for conjugation with
LC/MS/MS technique. The results are summarized in Table 3. To
our disappointment, all the indole P1 analogs (31, 41 and 36)
formed GSH conjugates readily. However, the site of conjugation
for the indoles shifted from the P1 aromatic region to the benzylic
‘linker’ position. A review of the literature revealed that this conju-
gation pattern has been documented.16 Indolizine 21 showed con-
jugation in both the P1 and benzylic region. Only the
benzoisothiazole P1 (32) was demonstrated to not form any GSH
conjugate. Although compound 32 is about 10-fold less potent
than TPC-806, subsequent research on the acid side chain area
suggested that the potency for this benzoisothiazole P1 family
could be rescued by appropriate acid side chain modification.17
Therefore, the benzothiazole P1 is still a very promising candidate
on this novel series of chymase inhibitors.
Scheme 5. Synthesis of indolizine P1 key: Reagents and conditions: (a) EtOAc, rt,
90%; (b) acrylonitrile, MnO2, Et3N, toluene, 90 °C, 52%; (c) pyrophosphoryl chloride,
DMF, rt, 89%; (d) dimethylamine, NaBH(OAc)3, CH2Cl2, 0 °C to rt, 65%; (e) MeI,
CH3CN, rt, 60%; (f) 3, K2CO3, DMF, 90 °C, 60%; (g) LiOH, 1,4-dioxane/H2O, rt, 100%.
the S1 pocket is mainly hydrophobic in nature, more polar P1
replacements such as imidazopyridine (12) and pyrazolopyridine
(16) are not tolerated even though they have similar shape as the
50-methylbenzothiophene (1) in molecular modeling. This observa-
tion is supported by the substantial drop in c log P values for 12
(c log P = 1.49) and 16 (c log P = 1.79) compared with
1
(c log P = 3.71). Indolizine is often regarded as a bioisoster for
indole. An attempt to prepare an unsubstituted indolizine failed
because of intrinsic instability of electron rich indolizine ring. An
electron withdrawing group, such as nitrile in 21, was introduced
to improve the overall chemical stability. Not surprisingly, indoli-
zine 21 has very similar profile to indole 31 with only slight drop
in chymase inhibitory activity (ꢁ4-fold). Lastly, studying the
X-ray co-crystal structure of 2 led to the realization that there is
an unoccupied space within the S1 pocket. Extension from the 30-
position of benzothiophene P1 was predicted to occupy that space
and possibly improve the binding affinity for chymase. Therefore,
dimethyl benzoisothiazole P1 (32) was designed to address this
hypothesis. The chymase IC50 for 32 was 280 nM, which is about
15-fold better than the unsubstituted benzoisothiazole (28).
Up to this point, we have described several potential replace-
ments for the benzothiophene P1 such as an indole and benzoiso-
thiazole. However, the new analogs are generally less potent
than the clinical compound TPC-806. Based on the structural infor-
mation obtained either from the X-ray crystallography or molecu-
lar modeling, we believed that the potency of this new series of
compounds could be improved by further optimization of the P1
group.
Besides the ‘5–6’ aromatic system, as the benzothiophene, we
decided to explore another aromatic system such as the ‘6–5’ sys-
tem. Encouraged by the good results from the ‘5–6’ indole analog
(31), the ‘reverse indole’ (i.e., ‘6–5’ indole system) was evaluated.
The general synthesis of the 6–5 indole P1 analog is shown in
Scheme 7. Starting from 3-chloro-5-nitrobenzaldehyde, acetal
formation with n-butanol under acidic condition gave 33. The
indole system in 34 was formed using the Bartoli’s protocol15 with
1-methyl-1-propenylmagnesium bromide. Methylation of the
indole followed by reduction of the aldehyde provided trimethylin-
dole 35, which in turn was coupled to the benzimidazolone core
using the Mitsunobu protocol. Hydrolysis of the ester with LiOH
produced chloro-trimethylindole 36.
To further understand the tendency of GSH conjugation, we
hypothesized that the propensity of the P1 systems towards

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
4537
Table 1
Bicyclic P1 systems
O
OH
N
O
N
P1
c log Pb
a
a
Compounds
P1=
Chymase IC50 (nM)
Cathepsin G IC50 (lM)
*
5
4
1
1
*
70
2.1
3.71
3
S
2
27
28
1900
4200
>10
>10
3.22
3.26
S
S
N
*
N
*
29
30
31
2600
600
>10
>10
4.8
2.22
3.24
2.51
*
*
170
N
*
*
*
12
16
N
>10000
>10000
>10
>10
1.49
1.79
N
N
N
N
21
640
280
>10
9.5
2.12
4.23
NC
*
32
N
S
a
Values are means of three experiments.
c log P calculation based on Pipeline Pilot Webport.
b
oxidation is a key parameter for the conjugation to occur. We cal-
culated the HOMO, LUMO energy states of the various P1 systems
and the oxygenation energy of the P1 group (DE) as shown in Table
4. The oxygenation energy of P1 group is defined as the energy
difference between the neutral molecule and the same molecule
that losses one electron.18 The higher the oxygenation energy
means the lower tendency to be oxidized and hence attract GSH
conjugation. The result of this exercise suggested that benzoiso-
thiazole 32 had indeed, the highest oxygenation energy
(215.4 kcal/mol), and therefore the least likelihood to form a GSH
conjugate, which is in line with the experimental outcome.
Although this in silico analysis provided some interesting insight
related to the reactive metabolite formation in this case, a larger
set of compound pool analysis is needed before we can draw any

4538
H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
Table 3
GSH conjugates formation
MgBr
b
H
O
O
O
O
H
Compounds
GSH conjugationa
GSH attachment positionb
H
a
NO2
N
NO2
1
Detected
Detected
Detected
Detected
Detected
Not detected
P1 aromatic
Benzylic
Benzylic
Benzylic
P1 aromatic and benzylic
–
31
41
36
21
32
Cl
Cl
O
Cl
33
34
HO
c, d
a
Experimental procedure refer to Ref. 12.
Determine by LC/MS/MS experiments.
b
N
N
HO
O
e, f
N
Cl
conclusion on the predictive value of this method. We hope that
we will be able to report new research results related to this topic
in the near future.
Cl
N
35
36
In summary, we have identified a novel class of benzimidazo-
lone chymase inhibitors. After a thorough study of the P1 region
of the inhibitors, replacements for the benzothiophene moiety
which form GSH conjugates were discovered. Among the newly
identified heteroaromatic P1s, ‘6–5’ chloro-trimethylindole is the
Scheme 7. Synthesis of ‘6–5’ indole; key: Reagents and conditions: (a) nBuOH,
p-TsOH, toluene, reflux, 22%; (b) 1-methyl-1-propenylmagnesium bromide, THF,
ꢀ40 °C to rt, 21%; (c) NaH, MeI, DMF, 0 °C to rt, 99%; (d) NaBH4, MeOH, 0 °C, 58%; (e)
3, DIAD, PPh3, THF, rt, 60%; (f) LiOH, 1,4-dioxane/H2O, rt, 90%.
Table 2
6–5 Indole systems (general structure refer to Table 1)
c log Pb
a
a
Compounds
P1=
Chymase IC50 (nM)
Cathepsin G IC50 (lM)
*
1
N
2
6
37
620
>10
2.02
3
5
*
4
N
38
39
40
41
570
640
>10
8.8
2.35
2.51
2.51
2.84
*
*
*
*
N
2800
140
>10
>10
N
N
N
42
43
65
7.0
3.33
2.72
*
N
220
>10
O
*
N
36
44
25
22
3.40
3.40
Cl
Cl
*
N
>10,000
>10
a
Values are means of three experiments.
c log P calculation based on Pipeline Pilot Webport.
b

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4533–4539
4539
Table 4
Miyazaki, M. Life Sci. 2002, 71, 437; (c) Jin, D.; Takai, S.; Yamada, M.; Sakaguchi,
M.; Kamoshita, K.; Ishida, K.; Sukenaga, Y.; Miyazaki, M. Cardiovasc. Res. 2003,
60, 413; (d) Palaniyandi, S. S.; Nagai, Y.; Watanbe, K.; Ma, M.; Veeraveedu, P. T.;
Prakash, P.; Kamal, F. A.; Abe, Y.; Yamaguchi, K.; Tachikawa, H.; Kodama, M.;
Aizawa, Y. Exp. Biol. Med. 2007, 232, 1213.
Molecular calculation of oxidative energy of P1
Compounds
HOMOa (kcal/mol)
LUMOa (kcal/mol)
D
Ea (kcal/mol)
1
31
41
36
21
32
ꢀ133.6
ꢀ121.7
ꢀ118.8
ꢀ125.7
ꢀ129.8
ꢀ141.9
ꢀ20.08
ꢀ10.04
ꢀ9.933
ꢀ12.65
ꢀ34.34
ꢀ23.38
173.0
159.6
155.0
160.9
167.4
215.4
8. Aoyama, Y. Expert Opin. Ther. Patents 2001, 11, 1423.
9. Refer to homepage of Teijin Pharma Limited: www.teijin-pharma.co.jp./
English/.
10. Chymase was assayed in a 15
human chymase and 100 nM rhodamine 110, bis-(succinoyl-
-prolyl- -phenylalanylamide) (American Peptide) in buffer containing
20 mM Tris HCl pH 8.0, 50 mM NaCl, 100 M TCEP, 0.01% CHAPS, and 1%
l
L volume containing 500 pM recombinant
L
-alanyl- -alanyl-
L
L
L
a
l
a
Molecular calculation using quantum chemistry DFT/B3LYP method.
DMSO for 1 h at 28 °C/80% humidity, and fluorescence was read at 485 nm
excitation and 530 nm emission.
11. The coordinates of co-crystal structure of 2 with human chymase are deposited
in the RCSB protein data bank (RCSD ID: RCSB065611; PDB ID: 3S0N).
12. To assess reactive metabolite formation, compound 1 (50 lM) was added to
most potent. However, only the benzoisothiazole P1 prevents the
formation of a GSH conjugate. The calculated oxygenation energies
of the new P1 analogs agree with the experimental results. The
progress of this new class of chymase inhibitor toward a new drug
candidate will be reported in the near future.
human liver microsomes (1 mg/mL) in phosphate buffer (100 mM, pH 7.4).
Pooled human liver microsomes were purchased from BD Biosciences (San
Jose, CA). After preincubation at 37 °C for 3 min, the reaction was initiated by
the addition of NADPH (2.5 mM). The final incubation volume was 1 mL.
Samples without compound or NADPH were used as negative controls. After
incubation for 2 h, samples were quenched with 2 mL of acetonitrile. Samples
were centrifuged at 2095ꢂg for 5 min and the supernatant was transferred to a
Acknowledgment
glass tube and the solvent was evaporated using
(Hopkinton, MA). Samples were redissolved in 500
a
Caliper TurboVap LV
l
L water/acetonitrile (1:1,
The authors would like to thank Dr. Anil Padyana for the depo-
sition of X-ray crystal coordinates.
v:v) and analyzed by mass spectrometry for the detection of glutathione
adducts. A glutathione adduct of compound 1 was detected after analysis of the
microsomal incubate.
13. (a) Uetrecht, J. P. Curr. Drug Metab. 2000, 1, 133; (b) Zhou, S.; Chan, E.; Duan,
W.; Huang, M.; Chen, Y.-Z. Drug Metab. Rev. 2005, 1, 41; (c) Walgren, J. L.;
Mitchell, M. D.; Thompson, D. C. Crit. Rev. Toxicol. 2005, 35, 325.
Reference and notes
1. Bacani, C.; Frishman, W. H. Cardiol. Rev. 2006, 14, 187.
14. Cathepsin G was assayed as described for chymase in 15 lL containing 4 nM
2. (a) McEuen, A. R.; Sharama, B.; Walls, A. F. Biochim. Biophys. Acta 1995, 1267,
115; (b) Galli, S. J.; Wershil, B. K. Exp. Dermatol. 1995, 4, 240; (c) Reiling, K. K.;
Krucinski, J.; Miercke, L. J., et al Biochemistry 2003, 42, 2616.
3. (a) Nishimoto, M.; Takai, S.; Kim, S.; Jin, D.; Yuda, A.; Sakaguchi, M.; Yamada,
M.; Sawada, Y.; Kondo, K.; Asada, K.; Iwao, H.; Sasaki, S.; Miyazaki, M.
Circulation 2001, 104, 1274; (b) Nishimoto, M.; Takai, S.; Sawada, Y.; Yuda, A.;
Kondo, K.; Yamada, M.; Jin, D.; Sakaguchi, M.; Asada, K.; Sasaki, S.; Miyazaki, M.
J. Thorac. Cardiovasc. Surg. 2001, 121, 729.
4. (a) Takai, S.; Miyazaki, M. Curr. Vasc. Pharmacol. 2003, 1, 217; (b) Doggrell, S. A.;
Wanstall, J. C. Cardiovasc. Res. 2004, 61, 653.
5. Taipale, J.; Lohi, J.; Saarinen, J.; Kovanen, P. T.; Keski-Oja, J. J. Biol. Chem. 1995,
270, 4689.
cathepsin G (Athens Research and Technology) and 300 nM chymase substrate
in a buffer containing 1ꢂ phosphate-buffered saline, 10 mM HEPES pH 7.4,
100 lM TCEP, 0.01% CHAPS, and 1% DMSO.
15. Dalpozzo, R.; Bartoli, G. Curr. Org. Chem. 2005, 9, 163.
16. (a) Kubow, S.; Janzen, E. G.; Bray, T. M. J. Biol. Chem. 1984, 259, 4447; (b)
Nocerini, M. R.; Yost, G. S.; Carlson, J. R.; Liberato, D. J.; Breeze, R. G. Drug Metab.
Dispos. 1985, 13, 690; (c) Thornton-Manning, J.; Appleton, M. L.; Gonzalez, F. J.;
Yost, G. S. J. Pharmacol. Exp. Ther. 1996, 276, 21; (d) Zhang, K. E.; Kari, P. H.;
Davis, M. R.; Doss, G.; Baillie, T. A.; Vyas, K. P. Drug Metab. Dispos. 2000, 28, 633.
17. Unpublished results for the acid side chain modification.
18. Energy calculations were carried out using quantum chemistry DFT/B3LYP
method. The basis-set function used in the calculation was 6-31⁄⁄G++. The
geometry of each group was optimized by the DFT method and the energy
value is for the fully optimized molecule.
6. Kofford, M. W.; Schwartz, L. B.; Schechter, N. M., et al J. Biol. Chem. 1997, 272,
7127.
7. (a) Jin, D.; Takai, S.; Yamada, M.; Sakaguchi, M.; Yao, Y.; Miyazaki, M. Jpn. J.
Pharmacol. 2001, 86, 203; (b) Jin, D.; Takai, S.; Yamada, M.; Sakaguchi, M.;