Identification of novel plasminogen activator inhibitor-1 inhibitors with improved oral bioavailability: Structure optimization of N-acylanthranilic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.11.016

Novel plasminogen activator inhibitor-1 (PAI-1) inhibitors with highly improved oral bioavailability were discovered by structure-activity relationship studies on N-acyl-5-chloroanthranilic acid derivatives. Because lipophilic N-acyl groups seemed to be important for the anthranilic acid derivatives to strongly inhibit PAI-1, synthesis of compounds in which 5-chloroanthranilic acid was bound to a variety of highly lipophilic moieties with appropriate linkers was investigated. As the result it appeared that some of the derivatives possessing aryl- or heteroaryl-substituted phenyl groups in the acyl chain had potent in vitro PAI-1 inhibitory activity. Oral absorbability of typical compounds was also evaluated in rats, and compounds 40, 55, 60 and 76 which have diverse chemical structure with each other were selected for further pharmacological evaluation.

Full text of DOI:10.1016/j.bmcl.2017.11.016

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel plasminogen activator inhibitor-1 inhibitors
with improved oral bioavailability: Structure optimization of
N-acylanthranilic acid derivatives
a
a
a
b
Nagahisa Yamaoka a, , Kenji Murano , Hidehiko Kodama , Akihisa Maeda , Takashi Dan ,
⇑
Tetsuo Nakabayashi b, Toshio Miyata b, Kanji Meguro a
a CT Laboratory, Hamari Chemicals, Ltd., 1-4-29 Kunijima, Higashiyodogawa-ku, Osaka 533-0024, Japan
b United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2-1 Seiryo-Machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel plasminogen activator inhibitor-1 (PAI-1) inhibitors with highly improved oral bioavailability were
discovered by structure-activity relationship studies on N-acyl-5-chloroanthranilic acid derivatives.
Because lipophilic N-acyl groups seemed to be important for the anthranilic acid derivatives to strongly
inhibit PAI-1, synthesis of compounds in which 5-chloroanthranilic acid was bound to a variety of highly
lipophilic moieties with appropriate linkers was investigated. As the result it appeared that some of the
derivatives possessing aryl- or heteroaryl-substituted phenyl groups in the acyl chain had potent in vitro
PAI-1 inhibitory activity. Oral absorbability of typical compounds was also evaluated in rats, and com-
pounds 40, 55, 60 and 76 which have diverse chemical structure with each other were selected for further
pharmacological evaluation.
Received 14 September 2017
Revised 3 November 2017
Accepted 8 November 2017
Available online xxxx
Keywords:
Plasminogen activator inhibitor-1 (PAI-1)
PAI-1 inhibitor
N-Acylanthranilic acid derivatives
Oral bioavailability
Ó 2018 Elsevier Ltd. All rights reserved.
Structure-activity relationship
Plasminogen activator inhibitor-1 (PAI-1), also known as SER-
PINE1, is a serine protease inhibitor belonging to the serpin super-
family. PAI-1 deactivates tissue-type plasminogen activator (tPA)
and urokinase-type plasminogen activator (uPA) by covalently
forming complexes with these serine protease enzymes which play
central roles in the vascular thrombotic and thrombolytic sys-
tem.1–3 Inhibition of PAI-1 could therefore increase the activity of
tPA and uPA to elevate the level of a fibrinolytic enzyme plasmin
which is derived from its proenzyme plasminogen. Consequently,
PAI-1 inhibitors are expected to be useful for treating various car-
diovascular diseases4,5 such as myocardial infarction, brain infarc-
tion, arteriosclerosis, deep vein thrombosis, and restenosis after
coronary angioplasty. Many recent studies6–9 have also revealed
that PAI-1 is involved in various biological disorders including dia-
betes, tissue fibrosis (lung, kidney and liver), inflammation, cancer,
Alzheimer’s disease and so on. A number of small molecule PAI-1
inhibitors have been widely investigated as promising targets for
new drug discovery,5,10,11 while none of them is yet applied in a
clinical setting. We have extensively studied small molecule PAI-
1 inhibitors12–15 and demonstrated a variety of intriguing biologi-
cal properties suggestive of possible clinical applications not only
as an anti-thrombotic agent devoid of risk of bleeding,13 but also
as drugs capable of preventing or treating senescence,16 inflama-
tion,17 multiple sclerosis18 and tumor.19 Furthermore, inhibition
of PAI-1 enhanced rapid and sustainable hematopoietic regenera-
tion20 suggesting a possible application of PAI-1 inhibitors in the
field of regenerative medicine.
In the previous papers,14,15 we have reported syntheses of N-
acylanthranilic acid derivatives as new PAI-1 inhibitors which
were designed based on the structure of original hits, TM500112
and TM500712 (Fig. 1), discovered as the outcome of a structure-
based drug design followed by in silico screening focused on the
binding affinity to the b-sheet A21 of PAI-1 protein, a cleft which
is thought to play an essential role in determining PAI-1’s enzy-
matic activity.22 Introduction of the 4-diphenylmethyl-1-piper-
azinyl moiety into the acyl side chain of 5-chloroanthranilic acid
as in TM527515 (Fig. 1) was found to be an effective way to amelio-
rate oral bioavailability and pharmacokinetic (PK) profile of this
series of PAI-1 inhibitors, since introduction of, for example,
diphenylmethylamino, diphenylamino or p-chloroanilino group
such as in compounds 1, 2 or 3 (Fig. 1) in place of the 4-diphenyl-
methylpiperazin-1-yl group resulted in decrease of activity.15 In
spite of these imperfections observed, we continued to explore
on anilide type compounds with more lipophilic substituents on
the phenyl ring, expecting to identify new lead compounds with
⇑
Corresponding author.
E-mail address: nagahisa-yamaoka@hamari.co.jp (N. Yamaoka).
https://doi.org/10.1016/j.bmcl.2017.11.016
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamaoka N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2017.11.016

2
N. Yamaoka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
COOH
HOOC
HN
Ar
Ar
NH
O
TM5001: Ar=Ph
TM5007: Ar=2-Thienyl
S
S
O
CH(Ph)2
COOH
NH
COOH
R1
COOH
NH
N
Cl
NH
O
N
R2
Cl
O
N
O
Cl
R
L
O
O
O
O
1: R1=H; R2=-CH(Ph)2
TM5275
IC50 µM
A
1
2
2
: R =R =Ph
=
7.94
3: R1=H; R2=p-Cl-Ph
Fig. 1. Chemical modification of PAI-1 inhibitors TM5001 and TM5007.
more potent PAI-1 inhibitory activity as well as higher oral
bioavailability than those of TM5275.
Thus, novel N-acyl-5-chloroanthranilic acid derivatives
depicted as the general structure A (Fig. 1) were synthesized and
evaluated as new PAI-1 inhibitors. Some of these compounds were
found to exhibit potent PAI-1 inhibitory activity and excellent oral
bioavailability in rats. This paper describes structure-optimization
study on anthranilic acid derivatives (A).
Table 1
Profiles of new 5-chloroanthranilic acid derivatives.
COOH
2
3
5
R1
N
R3
4
Cl
NH
O
O
R2
O
Compd.
R1
R2
R3
CLog Pa
PAI-1 activity (%)b
Rat PK (50 mg/kg, p.o.)c
50 mM
20 mM
Cmax (mM)d
Tmax (h)
T1/2 (h)
24
25
26
27e
28e
29
30
31
32
33
34
35g
36
37
38e
39e
40
H
H
H
H
H
H
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-F
3-CF3
2-(4F-Ph)
3-(4F-Ph)
4-(4F-Ph)
3-(4F-Ph)
3-(4F-Ph)
3-(2-MeO-Ph)
3-(3-MeO-Ph)
3-(4-MeO-Ph)
3-(4-AcNH-Ph)
2-(4-Pyridyl)
3-(4-Pyridyl)
4-(4-Pyridyl)
3-(3-Pyridyl)
3-(2-Pyridyl)
3-(3-Furyl)
3.71
4.65
4.37
5.40
5.40
5.25
6.21
4.47
5.01
5.01
4.08
2.85
3.88
3.88
3.88
4.09
4.38
79.4 12.3
39.8 6.3
39.6 3.3
12.4 3.9
15.6 3.3
8.1 3.6
8.0 2.5
11.3 4.9
6.9 0.7
11.2 2.5
6.7 1.7
24.3 3.7
12.8 4.2
0.6 1.0
22.0 2.7
41.3 8.5
6.4 2.6
99.7 0.3
93.2 7.3
82.4 7.3
95.6 5.8
92.2 6.3
48.6 9.0
58.5 5.8
73.7 10.1
64.3 7.5
61.5 14.5
41.4 14.4
65.1 10.5
14.6 6.2
28.7 4.7
74.2 7.0
76.7 4.6
16.2 8.1
ND
ND
1
1
1
ND
1
ND
0.8
0.8
0.9
ND
1.8
0.9f
2.5f
1.5f
1.0f
1.4
5.3
>18
>18
>22
2.4
2.1
2.3f
4.2f
4.0f
1.9
3.1f
9.9
ND
6.0f
ND
1.9f
ND
2.2f
2.5
58.6 10.9
41.8 7.0
62.8 35.1
ND
H
Me
Me
Me
Me
Me
Me
H
H
H
H
H
92.7 24.0
0.82 0.43f
2.08 0.83f
9.4 3.8f
2.2 0.5f
0.32 0.1
3.5 1.24
18.0 5.2
7.8 1.7
15.1 3.73
136.5 23.0
149.3 34.8
17.9 6.4f
17.59 7.3f
11.5 1.9f
86.5 20.4
1.70 1.75f
20.5 6.0
ND
1f
2f
2f
2f
2
1
6
6
2
1
1
H
1f
2f
2f
2
41
42
43
44
45
46
47
48
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-(3-Furyl)
3-(2-Furyl)
3-(3-Thienyl)
4.38
4.59
4.89
3.46
3.61
3.26
3.56
3.56
3.29
3.53
4.75
3.37
19.2 4.2
13.7 2.0
6.0 1.4
9.4 3.5
19.6 1.2
63.3 6.6
36.6 2.7
78.0 h
35.8 2.1
59.6 5.3
14.5 3.3
6.0 1.4
71.6 2.2
69.9 2.3
79.0 8.4
20.7 10.4
96.1 4.4
94.2 10.3
77.9 5.9
100.4i
74.6 12.9
92.6 4.9
64.0 3.2
79.0 8.4
3-(4-Pyrazolyl)
3-(1-Me-4-Pyrazolyl)
3-(5-Oxazolyl)
3-(5-Isoxazolyl)
3-(3-Isoxazolyl)
3-(3,5-Me2-1-Isoxazolyl)
3-(1-Imidazolyl)
3-(1-Pyrrolyl)
1f
2
ND
1f
ND
2f
ND
2f
2.0
14.0 5.0f
ND
49
50
51
2.0 0.6f
ND
7.4 1.9f
34.2 2.6
TM5275e,j
a
b
c
d
e
f
CLogP was obtained from ChemDraw Ultra 10.0 for free carboxylic acid/amine.
Remaining PAI-1 activity after incubation with test compound in Method A (see Ref. 23) is shown. Data are expressed as mean S.D.
ND = not determined.
Data are expressed as mean S.D.
Na salt.
Obtained at 5 mg/kg, p.o.
HCl salt.
Obtained from single experiment (n = 1) at 10 mM.
Obtained from single experiment (n = 1) at 2.5 mM.
See Ref. 15.
g
h
i
j
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N. Yamaoka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Chemistry23
large variety of compounds (13) were synthesized directly and
more conveniently by Route B (6 ? 12 ? 13). Using similar reac-
tion procedures, compounds expressed by the general formula 18
were also synthesized easily via Route C (4 ? 15?17 ? 18) or D
5-Chloroanthranilic acid derivatives (Tables 1–3) were in gen-
eral prepared by the methods shown in Scheme 1 starting with
5-chloroanthranilic acid esters (4). Compounds 6 which we pre-
pared15 previously by acylation of 4 with cyclic anhydrides (5)
were coupled with various substituted-amines 7 and 8 to afford
9 and 12, respectively. While 9 was then converted to 11 applying
the Suzuki-Miyaura coupling followed by hydrolysis (Route A), a
(4 ? 17 ? 18). Compound 57 (Table 2) was prepared in
a
different way (Route E); 4 was acylated with chloroacetyl chloride
followed by reaction with 3-bromophenol (20) afforded 21
which was then subjected to the Suzuki-Miyaura coupling with
3-furyl-boronic acid (22) to yield 23. Hydrolysis of 23 furnished
Table 2
N-Acyl-5-chloroanthranilic acid derivatives possessing m-(3-furyl)phenyl group combined by different linkers (L).
O
COOH
NH
O
Cl
L
Compd.
L
CLogPa
PAI-1 activity (%)b,c
IC50
Rat PK (5 mg/kg, p.o.)c
(mM)
50 mM
20 mM
Cmax (mM)b
Tmax (h)
T1/2 (h)
52
53
54
55
56
57
58
59
ACH2OCH2CONHCH2A
ACH2SCH2CONHA
ACH2CONHA
ACONHA
ACONHCH2A
ACH2OA
4.41
4.75
4.63
3.09
3.24
5.24
5.26
5.21
17.7 2.1
47.3 8.3
25.7 9.8
8.4 2.7
8.6 3.6
8.2 4.7
19.6 2.7
8.7 1.8
59.2 9.3
83.9 13.9
72.3 9.8
47.8 2.1
46.9 4.4
39.9 12.4
63.7 13.4
49.0 4.0
7.45
ND
ND
3.56
3.55
4.74
ND
14.0 4.5
5.4 7.0
ND
22.5 4.3
41.6 25.5
56.3 4.1
43.1 8.2
29.4 5.9
2
1
ND
2
2
2
2
2
2.7
2.6
ND
1.7
>22
1.9
1.9
2.0
ACH2A
d
–
7.39
a
b
c
CLogP was obtained from ChemDraw Ultra 10.0.
Remaining PAI-1 activity after incubation with test compound in Method A (see Ref. 23) is shown.
Data are expressed as mean S.D. ND = not determined.
Direct bond.
d
Table 3
5-Chloro-N-(substituted)benzoylanthranilic acids.
COOH
NH
R1
Cl
R2
O
Compd.
R1
R2
CLogPa
IC50 (mM)b
Rat PK (5mg/kg, p.o.)
Cmax (mM)c
Tmax (h)
T1/2 (h)
60
61
62
63
64
65
66
67
Ph
H
H
H
H
6.04
6.04
5.08
6.25
5.98
6.77
6.47
6.77
4.37
5.39
5.39
4.62
5.91
8.44
8.44
4.58
5.92
5.96
5.71
5.71
7.21
2.82
2.98
>10
17.0 4.9
73.5 13.0
22.3 3.1
64.2 1.4
64.9 22.9
3.3 2.8
5.20 0.9
15.2 1.9
45.5 0.8
35.0 17.7
34.4 25.8
26.7 5.1
38.4 6.5
13.8 0.6
4.4 1.2
2
2
1
6
1
1
1
1
1
1
2
2
2
2
2
2
1
2
2
6
2
1.6
10.4
5.0
13.3
3.4
1.4
0.7
0.7
1.8
0.9
11.3
8.0
1.9
4.3
2.3
1.6
2.5
3.5
10.5
9.2
1.7
Ph
iPrOA
PhOA
5.52
5.20
2.42
2.31
2.40
21.6
4.11
6.41
7.80
>20
1.17
1.86
>40
3.63
1.67
1.84
3.19
0.90
H
tBu
Cyclohexyl
1-Cyclohexenyl
H
H
H
H
H
H
H
H
Cyclohexyl
4-Morpholinyl
PhCOA
1-Pyrrolyl
1-Pyrrolidinyl
2-Thienyl
1-Adamantyl
H
H
H
H
H
H
H
68
69
70d
71d
72
73
74
H
1-Adamantyl
4-Pyridyl
8-Quinolinyl
3-Quinolinyl
5-Isoquinolinyl
4-Isoquinolinyl
1-Naphthyl
75e
76e
77e
78e
79e
80
5.2 0.5
22.2 7.8
1.1 0.3
1.2 2.2
1.0 1.3
6.3 6.8
a
b
c
CLogP was obtained from ChemDraw Ultra 10.0 for free carboxylic acid/amine.
Calculated based on PAI-1 activities observed in Method B at various concentrations (see Ref. 23).
Data are expressed as mean S.D. ND = not determined.
HCl salt.
Na salt.
d
e
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4
N. Yamaoka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Me
O
Me
R3
B
Me
Me
O
or
COOMe
NH
R2
COOMe
NH
R2
COOH
NH
R2
OH- or H+ and OH-
R3B(OH)2
Pd(0)
H
N
H
N
H
N
Route A
Cl
O
Cl
O
Cl
O
O
O
Br
O
O
R3
O
O
R3
9a, b
R2
10a-1 − 10a-3
11A (31 − 33, 38, 43 − 45)
(R=Me,
X=O)
H2N
Br
R2
7a, b
R3
R1HN
COOR
NH
COOR
NH
R2
COOH
NH
R2
( )n
R3
R3
R1
OH-
R1
(n=0, 1)
Cl
X
Cl
X
N
Cl
X
N
Route B
( )n
( )n
COOH
8a-w
O
O
O
O
O
6a, b
12a
w
−
13B (24 30, 34 37, 39 42, 46 53)
− − − −
Br or I
X
(X=O, S)
HOOC
L
COOR
NH
X=O, S
R=Me, PhCH2 -
O
O
O
14-Br, 14-I
Br or I
5
Cl
n=0 ,1
(R=Me)
L
R1=H, Et
R2=H, Me,
O
COOR
NH2
15-Br, 15-I
R3=F, CF3, cyclohexyl, aryl, heteroaryl, et c.
Cl
L=direct bond, -CH2-, -CONH-, -CH2CONH-
Pd(0)
R3B(OH)2
Route C
Route D
4a, b
R3
HOOC
L
COOR
NH
COOH
OH- or H2/Pd-C
16a-t
R3
R3
Cl
Cl
O
NH
O
O
(R=Me, PhCH2-)
L
L
(R=Me)
Cl
O
Cl
18C and 18D (54 − 56, 58 − 80)
17a
z
−
Br
O
O
COOMe
COOMe
Br
COOMe
COOH
HO
(HO)2B
Pd(0)
OH-
22
20
Route E
Cl
NH Cl
O
Cl
NH
O
O
Cl
NH
O
O
Cl
NH
O
O
19
21
23
Scheme 1. Synthesis of 5-chloroanthranilic acid derivatives.
57
57. Biological properties of these compounds are summarized in
Tables 1–3.
derivative (26) failed to recover the activity of TM5275, almost
complete recovery of the activity was observed in m- or p-bipheny-
lamide derivatives (27–34) (Table 1). The results suggest that the
cavity of PAI-1 protein can accommodate these types of com-
pounds (27–34) better than 24–26. Since compounds 27–34 have
rather high CLogP values (Table 1), we synthesized compounds
35–51 in order to lower Clog P values by introducing heteroaryl
groups instead of phenyl groups. However, CLogP itself does not
seem to be is a significant determinant of in vitro potency as can
be seen in Table 1. Among these compounds, 36, 37, and 40 were
worthy of attention due to their favorable in vitro potencies.
Brief pharmacokinetic (PK) profiles23 of some of the compounds
were determined in rats by oral gavage (Table 1). Compound 40
which has m-(3-furyl)anilide structure was the most attractive in
terms of both potent in vitro activity and high oral absorbability
superior than TM5275.
In further studies, the linker section of 40 was replaced by var-
ious linkages (L) as shown in Table 2. While in vitro activities at 20
mM of compounds 52–59 thus prepared were significantly
decreased relative to 40, oral absorbability of these compounds
was comparable or rather improved (Table 2). The length or the
kinds of L were not major determinants both in vitro PAI-1 inhibi-
tory activity and oral PK profile. Of particular interest, even the
simplest compound 59, which is the smallest in molecular size
without any linker, showed considerably potent in vitro activity
as well as high oral absorbability. Therefore, we next synthesized
N-(substituted)benzoyl-5-chloroanthranilic acid derivatives
(60–80, Table 3) possessing a different type of substituent at the
Results and discussion
As we have previously reported,15 the strategy in our drug
design was to combine substituted anthranilic acid and lipophilic
moieties with proper length of acyl-type linkers, which led to the
discovery of TM5275. The results from our series of the previous
work14,15 suggested that the carboxyl group in the anthranilic acid
was essential to bind to the PAI-1 protein at the target cleft prob-
ably by ionic binding while the lipophilic group played a secondary
effect to interact with a section of the cleft by van der Waals inter-
actions or
p-p stacking. Although co-crystallizing our inhibitors
with PAI-1 for X-ray crystallography is still in progress to analyze
their binding modes, computer-assisted docking simulation which
we have reported on TM500112 and TM527524 denotes our specu-
lation as being reasonable. Our subsequent study is directed
towards finding smaller and simpler compounds than TM5275,
expecting to induce high oral bioavailability sufficient to clearly
exhibit in vivo biological action after oral administration.
Since the in vitro PAI-1 inhibitory activity23 of simple anilides
such as 24 and 25 (Table 1) was lower than that of TM5275 as
was the case with compound 315 (Fig 1), we next synthesized phe-
nyl-substituted anilides, namely biphenylamide-type compounds,
mimicking in part the structure of TM5275 which possesses two
phenyl rings in the N-acyl side chain. While an o-biphenylamide
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N. Yamaoka et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
meta- or para-position on the benzoyl-benzene ring. The PAI-1
inhibitory activity (IC50) and PK profiles in rats orally administered
with 5 mg/kg of these test compounds are shown in Table 3.
Generally speaking, benzoyl derivatives (Table 3) with CLogP
values of more than 6.0, such as compounds 60, 61, 63, 65, 66,
67, 73, 74 and 80, tended to exhibit high in vitro PAI-1 inhibitory
activity irrespective of the position of substituents (R1 or R2). Con-
versely oral absorbability decreased in compounds 65, 66, 74 and
80 which have lipophilic substituents at the meta-position (R1).
Interestingly, highly lipophilic compounds 67 and 73 with cyclo-
hexyl and 1-adamantyl group as a para-substituent, respectively,
exhibited moderate oral absorbability. Although the CLogP values
of compounds 76–79 possessing a quinoline or an isoquinoline
moiety are lower than 6.0, these compounds, in contrast to the
above-observed trends, exhibited potent in vitro activities and
somewhat decreased oral absorbability with the exception of 76
which has the 8-quinolinyl moiety as a substituent at R1. On the
other hand, compound 75 with a 4-pyridyl moiety at R1 surpris-
ingly lost in vitro activity. Although structure-activity relationship
studies on derivatives having nitrogen heterocycles as the benzoyl-
phenyl substituents have not been studied in details, the current
results suggest that the position of nitrogen atom is an important
determinant of biological activities in this series of compounds.
While many of the compounds which had high potential with
respect to both in vitro activity and PK profile as mentioned above
violate Lipinski’s Rule of Five25 on drug-likeness in terms of CLogP,
some fulfilled our criteria for further pharmacological evaluation,
and are expected to exhibit high oral bioavailability in various ani-
mal models.
of the cavity of PAI-1, as we have observed a difference between
TM5007 and TM527513. Although the exact interaction modes for
the newly identified inhibitors to associate with PAI-1 protein
are uncertain at present, further structure-based studies on this
series of compounds targeting not only the above-mentioned cleft
but also another potential binding site such as the one proposed by
Gorlatova et al.26 for tiplaxtinin are going underway to unveil this
issue with the aim of designing novel PAI-1 inhibitors.
In summary, structure-optimization starting with original hits
(TM5001, TM5007)12 to discover more drug-like compounds was
achieved by identifying orally bioavailable 5-chloroanthranilic acid
derivatives by our intense structure-activity-relationship studies,
in particular, not only by converting the thiophene ring in the orig-
inal hits to its isosteric benzene ring15 but also by substantially
diminishing molecular size.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.11.016.
References
1. Dellas C, Loskutoff DJ. Thromb Haemost. 2005;93:631–640.
2. Crandall DL, Quinet EM, El Ayachi S, et al. Arterioscler Thromb Vasc Biol.
2006;26:2209–2215.
3. Westrick RJ, Eitzman DT. Curr Drug Targets. 2007;8:966–1002.
4. Ha H, Oh EY, Lee HB. Nat Rev Nephrol. 2009;5:203–211.
5. Suzuki J, Ogawa M, Muto S, et al. Expert Opin Invest Drugs. 2011;20:255–261.
6. East E, Baker D, Pryce G, Lijnen HR, Cuzner ML, Djordje D. Am
2005;167:545–554.
J Pathol.
7. Aso Y. Front Biosci. 2007;12:2957–2966.
8. Ma LJ, Fogo AB. Front Biosci. 2009;14:2028–2041.
9. Jacobsen JS, Comery TA, Martone RL, et al. Proc Natl Acad Sci USA.
2008;105:8754–8759.
Conclusion
10. Fjellström O, Deinum J, Stögren T, et al. J Biol Chem. 2013;288:873–885.
Novel 5-chloroanthranilic acid derivatives with lipophilic N-
acyl groups were synthesized and evaluated as PAI-1 inhibitors
in vitro. In addition, oral PK profiles of some of the compounds
which exhibited potent in vitro activities were also evaluated in
rats to select candidate compounds for further evaluation as poten-
tial new drugs. Taking diversity of chemical structure and relative
ease of synthesis in consideration, compounds 40, 55, 60 and 76
were chosen as valuable tools to evaluate pharmacological profiles
PAI-1 inhibitors in detail in order to develop useful drugs, in partic-
ular, with expectation of identifying unprecedentedly novel clinical
applications.16–21 Extensive pharmacological and toxicological
studies on these compounds are in progress.
11. Rouch A, Vanucci-Bacque C, Bedos-Belval F, Baltas M. Eur
J Med Chem.
2015;92:619–636.
12. Izuhara Y, Takahashi S, Nangaku M, et al. Arterioscler Thromb Vasc Biol.
2008;28:672–677.
13. Izuhara Y, Yamaoka N, Kodama H, et al.
2010;30:904–912.
J Cereb Blood Flow Metab.
14. Yamaoka N, Kawano Y, Izuhara Y, Miyata T, Meguro K. Chem Pharm Bull.
2010;58:615–619.
15. Yamaoka N, Kodama H, Izuhara Y, Miyata T, Meguro K. Chem Pharm Bull.
2011;59:215–224.
16. Eren M, Boe AE, Murphy SB, et al. PNAS. 2014;111:7090–7095.
17. Ichimura A, Matsumoto S, Suzuki S, et al. Arterioscler Thromb Vasc Biol.
2013;33:935–942.
18. Pelisch N, Dan T, Ichimura A, et al. PLoS One. 2015;10 e0124510.
19. Placencio VR, Ichimura A, Miyata T, DeClerck YA. PLoS One. 2015;10 e0133786.
20. Ibrahim AA, Yahata T, Onizuka M, et al. Stem Cells. 2014;32:946–958.
21. Mottonen J, Strand A, Symersky J, et al. Nature. 1992;355:270–273.
22. Eitzman DT, Fay WP, Lawewnce DA, et al. J Clin Invest. 1995;95:2416–2420.
23. For experimental details see Supplementary material of this article.
24. Miyata T, Kikuchi K, Kiyomoto H, van Ypersele de Strihou C. Nat Rev Nephrol.
2011;7:469–477.
Our previous14,15 and present investigations on N-acylan-
thranilic acid derivatives suggest that the cleft of the PAI-1 protein
has considerably variable capacity in accommodating a variety of
compounds which has a carboxylate group together with a proper
lipophilic functionality in their molecular structures, because the
relative differences in distance or position between the carboxylate
and lipophilic moieties do not seem to affect PAI-1 inhibitory activ-
ity in a direct fashion. This fact may suggest that these anthranilic
acid derivatives do not necessarily accommodate at the same site
25. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Adv Drug Deliv Rev.
1997;23:3–25.
26. Gorlatova MV, Cale JM, Elokdah H, et al. J Biol Chem. 2007;282:9288–9296.
Please cite this article in press as: Yamaoka N., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2017.11.016