Synthesis and biological evaluation of N-mercaptoacylproline and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives as leukotriene A4 hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.043

We studied the synthetic modification of structurally similar N-mercaptoacyl-l-proline and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A₄ (LTA₄) hydrolase inhibitors. An N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Additional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C₄ position of proline yielded much more potent LTA₄ hydrolase inhibitors (IC₅₀; 52, 31, and 34 nM, respectively) than captopril (IC₅₀; 630,000 nM).

Full text of DOI:10.1016/j.bmcl.2008.07.043

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4529–4532
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of N-mercaptoacylproline
and N-mercaptoacylthiazolidine-4-carboxylic acid derivatives
as leukotriene A4 hydrolase inhibitors
*
Hiroshi Enomoto , Yuko Morikawa, Yurika Miyake, Fumio Tsuji, Maki Mizuchi, Hiroshi Suhara,
Ken-ichi Fujimura, Masato Horiuchi, Masakazu Ban
Nara Research & Development Center, Santen Pharmaceutical Co., Ltd, 8916-16, Takayama-cho, Ikoma-shi, Nara 630-0101, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We studied the synthetic modification of structurally similar N-mercaptoacyl-L-proline and (4R)-N-mer-
Received 2 May 2008
Revised 9 July 2008
Accepted 11 July 2008
Available online 15 July 2008
captoacylthiazolidine-4-carboxylic acid to obtain potent leukotriene A4 (LTA4) hydrolase inhibitors. An
N-mercaptoacyl group, (2S)-3-mercapto-2-methylpropionyl group, was effective for both scaffolds. Addi-
tional introduction of a large substituent such as 4-isopropylbenzylthio (3f), 4-tert-butylbenzylthio (3l)
or 4-cyclohexylbenzylthio group (3m) with (S)-configuration at the C4 position of proline yielded much
more potent LTA4 hydrolase inhibitors (IC50; 52, 31, and 34 nM, respectively) than captopril (IC50
630,000 nM).
;
Keywords:
LTA4 hydrolase
LTA4 hydrolase inhibitor
Epoxide hydrolase
Ó 2008 Elsevier Ltd. All rights reserved.
N-mercaptoacyl-L-proline
N-mercaptoacylthiazolidine-4-carboxylic
acid
Labile epoxide leukotriene A4 (LTA4) is transformed into leuko-
triene B4 (LTB4) by LTA4 hydrolase in the 5-lipooxygenase (5-LO)
pathway of arachidonic acid metabolism. LTB4 is a lipid mediator
that induces chemokinetic, chemotactic and aggregation responses
in polymorphonuclear leukocytes,1,2 and it promotes adhesion of
these cells to endothelial cell monolayers.3 LTB4 is produced in var-
ious cells, such as neutrophils, monocytes, macrophages, keratino-
cytes, lymphocytes and mast cells.4 It plays important pathological
roles in inflammatory diseases, such as rheumatoid arthritis,5 pso-
riasis6 and bowel disease.7 Recent studies suggest that LTB4 is in-
volved in vascular inflammation and arteriosclerosis.8 Prevention
of either LTB4 biosynthesis by inhibiting the 5-LO pathway, or
LTB4 binding to receptors, would be a suitable approach for design-
ing anti-inflammatory drugs.
The other function is intrinsic arginyl aminopeptidase activity,
which acts on various substrates.14
Several LTA4 hydrolase inhibitors have been reported.15 The
general aminopeptidase inhibitor, bestatin, inhibits the epoxy
hydrolase activity (IC50; 4
detracts the activity (IC50; 14
l
M).16 The ACE inhibitor captopril also
l
M).17
Since captopril retained a weak inhibitory activity against LTA4
hydrolase, we examined other thiol compounds including SA446,
which is a potent ACE inhibitor containing (4R)-thiazolidine-4-car-
boxylic acid (Fig. 1).18–23 However, these compounds did not show
any inhibitory activity.24
We examined the effects of the N-mercaptoacyl part of N-mer-
captoacyl-L
-proline 125 and (4R)-N-mercaptoacylthiazolidine-4-
carboxylic acid 223 (Table 1). Among the derivatives, only
(2S)-3-mercapto-2-methylpropionyl group (1e [captopril] and 2e)
LTA4 hydrolase (EC 3.3.2.6) locates in the cytosol9 and nu-
cleus.10 Its membrane-bound form11 has also been reported. This
enzyme is a bifunctional zinc metalloenzyme,12 which is a member
of the MA clan of metallopeptidases such as angiotensin-convert-
ing enzyme (ACE) and thermolysin.13 One of the functions is highly
substrate-specific epoxide hydrolase activity. This catalytic reac-
tion is rate-determining, and is the final step in LTB4 biosynthesis.
OH
S
O
H
N
O
HS
H
N
N
HS
OH
HS
OH
O
O
OH
O
SH
O
tiopronin
bucillamine
(Rimatil®)
SA446
(Thiola®)
* Corresponding author. Tel.: +81 743 79 4527; fax: +81 743 79 4608.
E-mail address: hiroshi.enomoto@santen.co.jp (H. Enomoto).
Figure 1. Structures of tiopronin, bucillamine, and SA446.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.043

4530
H. Enomoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4529–4532
Table 1
pionyl group. Only compound 2g that has a large substituent
(R2 = 1-naphtyl) showed an equipotent inhibitory activity with
compound 2e. Because the modification of R1 or R2 gave no more
potent compound, we introduced R3 to captopril (1e) (Table 3).
LTA4 hydrolase inhibitory activities of N-mercaptoacyl-L-proline and (4R)-N-mercap-
toacylthiazolidine-4-carboxylic acid derivatives
S
N
O
N
O
N-Mercaptoacyl-4-substituted-L-proline derivatives have been re-
R1
R1
ported to be ACE inhibitors;26 the inhibition of LTA4 hydrolase
has not been observed.
1
2
OH
OH
Compound 3a (R3 = benzylthio) increased the inhibitory activity
against LTA4 hydrolase (175-fold) over captopril (1e, R3 = H), as
R1–
Compound
LTA4 hydrolase
% inhibition at
1 mM
Compound
LTA4 hydrolase
% inhibition at
1 mM
1a
2b
Table 3
HS
LTA4 hydrolase and ACE inhibitory activities of N-[(2S)-3-mercapto-2-methylpropi-
1a
1b
1c
1d
NI
ND
NI
5
2a
2b
2c
2d
2e
NI
NI
NI
18
58
onyl]-L-proline derivatives
O
R3
*
HS
HS
N
O
O
O
O
OH
HS
HS
Compound –R3
Configuration
IC50 (nM)
a
LTA4 hydrolase ACE
1e
3a
–H
—
630,000
3600
23b
(S)
2.4
O
O
S
S
HS
1e
59c
3b
3c
3d
(S)
(S)
(S)
5100
1100
9200
0.18
0.029
3.2
(captopril)
a
b
c
Ref. 25.
Ref. 23.
IC50: 630
S
l
M, NI, no inhibition, ND, no data.
S
S
exhibited similar inhibitory potency against LTA4 hydrolase, sug-
gesting a strict steric requirement around the mercaptoacyl group.
This result also suggests equivalency of the scaffolds of compounds
1 and 2 for the inhibition of LTA4 hydrolase.
3e
3f
3g
3h
3i
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(R)
1100
52
4.2
8.4
Table 2 shows the effect of R2 on the (4R)-thiazolidine-4-car-
boxylic acid derivatives having the (2S)-3-mercapto-2-methylpro-
S
S
Table 2
LTA4 hydrolase inhibitory activities of (4R)-N-[(2S)-3-mercapto-2-methylpropio-
nyl]thiazolidine-4-carboxylic acid derivativesa
790
2700
>10,000
840
120
31
19
R2
S
HS
N
O
15
S
S
O
OH
Compound
R2–
H-
LTA4 hydrolase % inhibition at 1 mM
58
0.9
2e
2f
NI
3j
2.3
4.1
S
S
3k
3l
S
2g
58
410
S
S
S
2h
2i
25
36
14
N
3m
4
34
280
O
S
290
3.3
2j
a
Configuration of C4 position of proline.
Ref. 25.
b
Ref. 23, NI, no inhibition. Absolute configuration of R2 is not determined.
a

H. Enomoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4529–4532
4531
well as with ACE27 (10-fold) (Table 3). ACE inhibitory activities of
thesis of trans-4-arylalkylthio-L-proline 4 was completed by a
compounds 3b and 3c increased remarkably with increasing bulk-
iness of R3, yet LTA4 hydrolase inhibition decreased (3b) or in-
creased threefold (3c). Compound 3d (R3 = 1-naphtylmethylthio)
was three times less potent than compound 3a; compound 3e
(R3 = 2-naphtylmethylthio) was three times more potent than
compound 3a against LTA4 hydrolase, though these did not show
significant change with ACE inhibitory activity.
The introduction of an isopropyl group (3f) to compound 3a
notably improved the inhibitory activity of LTA4 hydrolase
(12,000-fold) over captopril (1e). Conversions of the methylene
moiety of the 4-isopropylbenzylthio group (3f) to ethylene (3g)
or trimethylene (3h) reduced the inhibitory activities against
LTA4 hydrolase, but did not affect ACE. The 3-methylbenzylthio
group (3i) lost the inhibitory activity completely. 2-Methylbenzyl-
thio (3j) and 4-methythiobenzylthio groups (3k) exhibited moder-
ate inhibitory activities against LTA4 hydrolase. The replacement of
4-isopropyl of compound 3f by tert-butyl (3l) or cyclohexyl group
(3m) resulted in a potent LTA4 hydrolase inhibitory activity similar
to that of compound 3f. The LTA4 hydrolase inhibitory activities of
these compounds were 20,000-fold more potent than that of cap-
topril (1e). These substitutions were allowed to diminish the inhib-
itory ability against ACE.
similar procedure using trans-4-mercapto-
material, which was prepared from cis-4-hydroxy-
L
-proline 6 as a starting
-proline.
L
X-ray crystallography of LTA4 hydrolase with a bestatin mole-
cule in the active site was reported, in which carbonyl O and the
adjacent hydroxyl O of the ligand coordinated with the catalytic
Zn ion.30 Other small molecules, such as captopril, thioamine and
amino hydroxamic acid derivatives, were also known to bind
LTA4 hydrolase by a similar ability to coordinate with Zn.31 Several
potent compounds (3f, 3l, 3m, and 4) and captopril were subjected
to a docking study to examine their binding mode in LTA4 hydro-
lase (Fig. 2). The docking was performed within GOLD32 with a dis-
tance constraint (1.5–3.5 Å) between the terminal S of the ligand
and the catalytic Zn (tetrahedral) of LTA4 hydrolase (1H6S.pdb).
The obtained pose of captopril was similar to that of X-ray crystal-
lography; coordination of the sulfhydryl S to the Zn ion, binding of
the carboxyl O to Arg563, and the carbonyl O to Gly268 and
Gly269.31 The pyrrolidinyl and the mercaptopropionyl parts of
the docked four compounds located over the captopril structure,
and the substituted benzylthio group extended in a wide groove
toward Phe340 (pose a) in the enzyme. Another pose (pose b)
whose benzylthio group extended toward Arg568 was also found
for the three compounds (3f, 3l, and 4). Torsional angle (C3–C4–
S-C) classifying pose a and b of compound 3l was 161° and 108°,
respectively. The docking score ordinarily prioritizing binding
poses was not effective between the two poses. Both the poses
located deep inside the groove occupied different positions from
the suggested binding position of LTA4 in the enzyme.30 The acyl
part of compound 3l was closely surrounded by several amino acid
residues (Tyr267, Gly269, His295, Glu296, and Tyr383), suggesting
limited tolerance of R1 (Table 1). Substituent R2 of compounds 2f–j
would be too close to the side chain of Tyr383 and be inappropriate
to bind for them having particularly a twisted aromatic ring to the
thiazolidine (Table 2). The wide cleft appeared to accept even lar-
ger R3 than tert-butylbenzylthio group and be tolerable to steri-
cally inversed compound 4 (Table 3). Analysis of ACE-captopril
cocrystal revealed that the amide carbonyl oxygen of the
The inversion of the stereochemistry of compound 3f made
compound 4 six times less potent against LTA4 hydrolase.
N-Mercaptoacyl-4-arylalkylthio-
and 4 were prepared as shown in Scheme 1. cis-4-Mercapto-
L
-proline derivatives 3a–m
-pro-
L
line 528 was treated with the corresponding arylalkyl chlorides or
bromides in the presence of aqueous sodium hydroxide to give
compounds 7a–m. The coupling reaction of compounds 7a–m
with 4-nitrophenyl (2S)-3-benzoylthio-2-methylpropionate29
yielded cis-4-arylalkylthio-N-[(2S)-3-benzoylthio-2-methylpropio-
nyl]-L-proline derivatives 9a–m. A reaction of compounds
9a–m with aqueous ammonia gave cis-4-arylalkylthio-N-[(2S)-3-
mercapto-2-methylpropionyl]-
L-proline derivatives 3a–m. Syn-
R4
R5
R4
R5
e
ligand was hydrogen-bonded by two N 2Hs of the imidazole parts
of His353 and His513.33 More flexible and efficient coordinating
ability of these hydrogen-bonds than that of the two NHs
(Gly268 and Gly269) of LTA4 hydrolase main chain would have
captopril more potent against ACE than LTA4 hydrolase. Phen-
ylalkylthio groups of compounds 3a–c poses obtained by docking
study with ACE (1UZF.pdb) located near hydrophobic area
(Val379, Val380, Phe457, and Phe527), which appeared deficient
in LTA4 hydrolase. This suggests the preference for hydrophobicity
a
HCl .
HN
O
HN
O
OH
OH
7a - 7m R4 = S(CH2)nAr, R5 = H
5
6
R4 = SH, R5 = H
R4 = H, R5 = SH
(n = 1 - 3)
8
R4 = H, R5 = 4-isopropylbenzylthio
R4
R5
b
S
N
O
O
O
OH
9a - 9m R4 = S(CH2)nAr, R5 = H
(n = 1 - 3)
10 R4 = H, R5 = 4-isopropylbenzylthio
R4
R5
c
HS
N
O
O
OH
3a - 3m R4 = S(CH2)nAr, R5 = H
(n = 1 - 3)
4
R4 = H, R5 = 4-isopropylbenzylthio
Scheme 1. Reagents and conditions: (a) corresponding arylalkyl chlorides or
corresponding arylalkyl bromides, 2 M NaOH aq, EtOH, rt, overnight; (b) 4-
nitrophenyl (2S)-3-benzoylthio-2-methylpropionate, triethylamine, DMF, rt, over-
night; (c) 28% NH3 aq, rt, 1h.
Figure 2. Possible poses of compound 3l docked into LTA4 hydrolase (pose a;
purple, pose b; green). Captopril; blue

4532
H. Enomoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4529–4532
of R3 of these compounds to ACE inhibitory activity and the differ-
ence between the two enzymes. Instead of these hydrophobic res-
idues, the aminobutyl chain of Lys565 lying close to the
phenylalkylthio groups may contribute to activity increase against
LTA4 hydrolase.
14. Orning, L.; Gierse, J. K.; Fitzpatrick, F. A. J. Biol. Chem. 1994, 269, 11269.
15. Penning, T. D. Curr. Pharm. Des. 2001, 7, 163.
16. Orning, L.; Krivi, G.; Fitzpatrik, F. A. J. Biol. Chem. 1991, 266, 1375.
17. Orning, L.; Krivi, G.; Bild, G.; Gierse, J.; Aykent, S.; Fitzpatrick, F. A. J. Biol. Chem.
1991, 266, 16507.
18. Tsuji, F.; Matsuoka, H.; Aono, H.; Takai, M.; Horiuchi, M.; Nishimura, K.; Mita, S.
Biol. Pharm. Bull. 2000, 23, 663.
19. Oya, M.; Matsumoto, J.; Takashina, H.; Iwao, J.; Funae, Y. Chem. Pharm. Bull.
1981, 29, 63.
20. Oya, M.; Matsumoto, J.; Takashina, H.; Watanabe, T.; Iwao, J. Chem. Pharm. Bull.
1981, 29, 940.
21. Oya, M.; Kato, E.; Matsumoto, J.; Kawashima, Y.; Iwao, J. Chem. Pharm. Bull.
1981, 29, 1203.
22. Iso, T.; Yamauchi, H.; Suda, H.; Nakata, K.; Nishimura, K.; Iwao, J. Jpn. J.
Pharmacol. 1981, 31, 875.
23. Oya, M.; Baba, T.; Kato, E.; Kawashima, Y.; Watanabe, T. Chem. Pharm. Bull.
1982, 30, 440.
In conclusion, the inhibitory activities of N-mercaptoacyl-L-pro-
line and (4R)-N-mercaptoacylthiazolidine-4-carboxylic acid deriv-
atives against LTA4 hydrolase were studied. The N-(2S)-3-
mercapto-2-methylpropionyl group was necessary to bear the
inhibitory activity for the L-proline and (4R)-thiazolidine-4-carbox-
ylic acid derivatives. In addition to this chiral acyl group, the intro-
duction of (S)-benzylthio group at the C4 position of proline gave a
potent LTA4 hydrolase inhibitor, compound 3a (IC50; 3600 nM).
Larger benzylthio groups such as 4-isopropylbenzylthio (3f), 4-
tert-butylbenzylthio (3l) or 4-cyclohexylbenzylthio group (3m)
yielded much more potent LTA4 hydrolase inhibitors (IC50; 52,
31, and 34 nM, respectively) than captopril (1e). In particular, com-
pounds 3l and 3m decreased inhibitory ability against ACE.
24. Ohishi, N.; Izumi, T.; Minami, M.; Kitamura, S.; Seyama, Y.; Ohkawa, S.; Terao,
S.; Yotsumoto, H.; Takaku, F.; Shimizu, T. J. Biol. Chem. 1987, 262, 10200. LTA4
hydrolase was purified from guinea pig lung and subjected to the enzyme
assay according to Ref. 24. LTA4 hydrolase activity was determined as follows.
The reaction mixture (150 lL) consisting of 60 mM HEPES buffer (pH 7.8),
3 mM dithiothreitol and LTA4 hydrolase with or without LTA4 hydrolase
inhibitor was preincubated at 26 °C for 1 min. Ethanol solution of LTA4
containing less than 50 mM lithium hydroxide was added to final
concentration of 60
terminated by addition of acidic acetonitrile (acetonitrile/ethanol/acetic acid,
150:50:3, 100 L) and then was added PGB2 as an internal standard for HPLC
analysis. The mixture was kept at -20 °C for 30 min and centrifuged at 10,000g
for 5 min at 4 °C. An aliquot (25 L) of the supernatant was analyzed by HPLC
(column; TSK ODS-80TS 4.6 ꢀ 75 mm, mobile phase; acetonitrile/methanol/
water/acetic acid, 900:300: 800:1.8, containing 0.05% EDTA, pH 5.6, flow rate;
1.0 mL/min, detection; UV at 270 nm). IC50 values were calculated by linear
regression analysis of at least three independent dose–response titration of
each compound in duplicate.
lM. After 1 min incubation at 26 °C, the reaction was
Supplementary data
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.043.
l
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