Discovery of inhibitors of plasminogen activator inhibitor-1: Structure-activity study of 5-nitro-2-phenoxybenzoic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.08.031

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.

Full text of DOI:10.1016/j.bmcl.2011.08.031

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5701–5706
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of inhibitors of plasminogen activator inhibitor-1: Structure–activity
study of 5-nitro-2-phenoxybenzoic acid derivatives q
Vrajesh Pandya a,b, , Mukul Jain a, Ganes Chakrabarti a, Hitesh Soni a, Bhavesh Parmar a, Balaji Chaugule a,
⇑
Jigar Patel a, Jignesh Joshi a, Nirav Joshi a, Akshyaya Rath a, Mehul Raviya a, Mubeen Shaikh a,
Kalapatapu V.V.M. Sairam a, Harilal Patel a, Pankaj Patel a
a Zydus Research Centre, Sarkhej-Bavla N.H. 8A, Moraiya, Ahmedabad 382 210, India
b Department of Chemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors
using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo
types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral
bioavailability in the rodents.
Received 8 April 2011
Revised 22 July 2011
Accepted 5 August 2011
Available online 11 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PAI-1
Hybridization
Thrombosis
Tissue plasminogen activator
Plasminogen activator inhibitor-1 (PAI-1), a member of serpin
(serine protease inhibitor) superfamily, prevents the formation of
plasmin by inhibiting the activity of tissue plasminogen activator
(tPA) and urokinase plasminogen activator (uPA), the key enzymes
F3CO
CF3
OH
O
O
HOOC
O
NO2
N
N
N
OCH3
F3C
N
H
1(Tiplaxtinin)
CF3
2
O
S
NH
O
O
O
CF3
N
HO
HO
S
S
OH
OH
O O
HOOC
N
CF3
O
4
3
Figure 1. Structures of known PAI-1 inhibitors.
q
ZRC communication: 329.
⇑
Corresponding author. Tel.: +91 2717 250801; fax: +91 2717 250606.
E-mail address: vrajeshpandya@zyduscadila.com (V. Pandya).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.031

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V. Pandya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5701–5706
whose proteolytic action converts plasminogen to plasmin.1 Plas-
min dissolves fibrin clots by degrading insoluble fibrin molecules
to small soluble fragments. Deficiency of PAI-1 in humans results
in a hyperfibrinolytic state suggesting its important role in the fibro-
sis.2–4 PAI-1 has been reported to have its implications in various
patho-physiological processes, such as cancer,5 diabetic nephropa-
thy,6 obesity,7 metabolic syndrome,8 venous thrombosis,9 and ath-
erosclerosis.10,11 The therapeutic potential of PAI-1 inhibitors has
been reviewed recently.12 Several small molecules have been iden-
tified using the concept of the high throughput screening or virtual
screening.13 The most studied PAI-1 inhibitor tiplaxtinin 1 could
reach up to Phase I.14 Consistent research efforts resulted in the
development of inhibitors with improved potency over tiplaxtinin
exemplified by piperazine derivative 2,15 and aryl sulfonamide
derivative 416 (Fig. 1). There are few PAI-1 inhibitors which, demon
strated good antithrombotic efficacy in the various preclinical mod-
els however, none of them could advance to clinic.17–26
As a part of our research endeavor to develop viable therapies
for the treatment of thrombotic diseases, we have previously
reported the structure-activity relationship of a series of oxalamide
derivatives 327 (Fig. 1) identified by highthroughput screening of
our compound library. However, none of those oxalamide deriva-
tives could be further evaluated due to their poor oral bioavailibil-
ity. In continuation of our efforts to identify potent and orally
bioavailable PAI-1 inhibitors, we further opted hybridization and
conformational restriction strategies using known chemotypes. In
order to achieve this objective, we selected tiplaxtinin
1(IC50 = 2.7
potent PAI-1 inhibitory activity (IC50 = 0.5
l
M, Lit. value),14 and piperazine derivative 2, with
l
M, Lit. value).15 The
known PAI-1 inhibitors reported mostly possess a carboxylic acid
Strategy 1
Acid part
F3CO
CF3
Acid part
OH
O
HOOC
O
NO2
O
N
lipophilic part
lipophilic part
Central core
N
N
OCH3
F3C
N
H
Central core
1
2
R1
NO2
HOOC
1
2
5a
5b
5c
: R = OCF3, R = OCH3
1
: R = OCF3, R2 = H
O
N
R2
1
2
: R = H, R = OCH3
5a-5c
Strategy 2
cyclised to make constrained analogue
H
F3C
N
N
R2
OCH3
O
O
N
N
R3
N
N
HOOC
NO2
HOOC
NO2
Constrained analogs
CF3
6a-6g
2
6a
6b
6c
6d
6e
6f
6g
Central
ring
N
N
N
N
N
N
N
N
R2
R3
H
H
H
H
OCH3
OCH3
p-CF3
H
H
m-CF3
m-CF3
m-CF3
m-CF3
m-CF3
Figure 2. Strategies for the synthesis of PAI-1 inhibitors.

V. Pandya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5701–5706
5703
or an acid equivalent group attached to a lipophilic aromatic ring
as a key structural feature. Tiplaxtinin 1 contains indole oxoacetic
acid scaffold and published SAR14 suggests importance of 4-
trifloromethoxyphenyl group (lipophilic part) and also its position
on indole. Piperazine derivative 2 contain 5-nitro-2-phenoxyben-
zoic acid part as an acid group, which was found to be optimum
after employing various acid groups.15 We thus proceeded and
designed the compounds by incorporating the acid part of 2 in
the 1 as a probable replacement of oxoacetic acid group of 1, to
get the hybridized molecules 5a–5c (Fig. 2, strategy 1). Further, ra-
tional has been derived from docking study28 of 5a and tiplaxtinin,
which reveled that both compounds possess similar orientation in
the ligand binding pocket of PAI-1. The H bond interaction of
carboxylic group of 5a with Arg118 was found to be the key inter-
action (Fig. 3). As an alternative strategy, we intended to make the
constrained analogues of 2 and subsequently few analogue 6a–6g
(Fig. 2, strategy 2) were synthesized. The compounds 5a–5c and
6a–6g were evaluated for their PAI-1 inhibitory activity (Tables 1
and 2). The pharmacokinetics parameters29 of the potent com-
pounds 5a, 6c, and 6e were studied in the male wistar rats (Table
3).
The compounds 5a–5c were prepared as shown in the Scheme
1. The salicylaldehyde derivative 8 was reacted with methyl
2-chloro-5-nitrobenzoate 7 using NaH as a base to give diphenyl
ether derivative 9, which upon reduction with sodium borohydride
followed by bromination with PBr3 afforded the bromo derivative
10. The BOC protected 5-bromoindole 11 was reacted with the
appropriate boronic acids 12 under Suzuki coupling30 reaction con-
ditions to afford the indole derivative 13 after the BOC deprotec-
tion using the TFA. The coupling of indole derivative 13 with the
10 in presence of t-BuOK provided the ester derivative 14, which
upon basic hydrolysis with KOH afforded the target compounds
5a–5c.
The compounds 6a–6g were prepared as depicted in the
Scheme 2. The piperazine derivative 15 was coupled with the
BOC protected heterocycle 16 using Pd(OAc)2 as a catalyst and BIN-
AP as a ligand to give compound 17,31 which was subsequently
deprotected using either TFA or concd sulfuric acid to furnish 18.
The coupling of 18 with the halogen derivative 10 in presence of
t-BuOK or K2CO3 as a base provided the ester derivative 19. The ba-
sic hydrolysis of derivative 19 with KOH afforded the compounds
6a–6g.
All the compounds 5a–5c and 6a–6g synthesized32 were evalu-
ated for their in vitro PAI-1 inhibitory activities33 (Tables 1 and 2).
The hybridized derivative 5a synthesized as a part of strategy 1,
Table 2
PAI-1 inhibitory activity of compounds 6a–6g
R3
N
N
N
O
R2
HOOC
NO2
R2
H
R3
Central ring
IC50 (lM)
a
Figure 3. Overlay of docking images of tiplaxtinin (orange) and 5a (yellow) into
active site of PAI-1: H bond interaction of 5a with Arg118 is shown in dashed line.
Compound
6a
m-CF3
29
N
Table 1
PAI-1 inhibitory activity of compounds 5a–5c
6b
H
H
H
m-CF3
63.8
N
N
R1
NO2
HOOC
N
6c
6d
6e
6f
m-CF3
m-CF3
m-CF3
p-CF3
3.2
14.6
2.4
22
O
R2
N
N
OCH3
OCH3
N
N
a
Compound
R1
R2
IC50 (lM)
5a
5b
5c
OCF3
OCF3
H
OCH3
H
OCH3
3.4
4.9
98
6g
H
—
H
—
No inhibition
14.8
N
Tiplaxtinin (1)
—
Tiplaxtinin (1)
14.8
a
a
Values determined using in vitro chromogenic assay.
Values determined using in vitro chromogenic assay.

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V. Pandya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5701–5706
NO2
NO2
H3COOC
BrH2C
Cl
H3COOC
OHC
OH
R2
b,c
a
COOCH3
OHC
R2
O
O
+
R2
NO2
9
7
10
8
R1
R1
NO2
R1
Br
H3COOC
N
f
d,e
+
N
BOC
R2
B(OH)2
O
N
H
11
13
14
12
R1
NO2
HOOC
N
: R1 = OCF3, R2 = OCH3
g
5a
R2
O
5b: R1 = OCF3, R2 = H
5c: R1 = H, R2 = OCH3
5a-5c
Scheme 1. Reagents and conditions: (a) NaH, DMSO, 0–25 °C, 60–70%; (b) NaBH4, MeOH, 10–25 °C, 90–95%; (c) PBr3, CH2Cl2, 10–25 °C, 75–80%; (d) Pd(OAc)2, MeOH, reflux,
70–80%; (e) TFA, CH2Cl2, 25 °C, 80–90%; (f) 10, t-BuOK, DMF, 10–25 °C, 60–75%; (g) KOH, MeOH, H2O, 25 °C, 85–95%.
R3
R3
R3
Br
N
N
a
N
N
b
+
N
N
BOC
NH
N
BOC
NH
15
17
16
18
N
N
N
c
N
N
N
d
R3
R3
R2
O
R2
O
H3COOC
HOOC
19
6a-6g
NO2
NO2
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, BINAP, K3PO4, DME, reflux, 20–50%; (b)TFA, CH2Cl2 or neat H2SO4, 0–25 °C, 80–90%; (c) 10, t-BuOK, DMF or K2CO3, CH3CN,
10–25 °C, 70–80%; (g) KOH, MeOH, H2O, 25 °C, 85–95%.
compound 5c (IC50 = 98 lM). Further more the compounds 6a–6g
were synthesized (Table 2) as part of strategy 2 (Fig. 2). The confor-
mational restriction of 2 with two carbon atoms produced indoline
Table 3
Pharmacokinetic parameters for compoundsa 5a, 6c, 6e
derivative 6a, which showed less potency (IC50 = 29
to the tiplaxtinin (IC50 = 14.8 M). The ring expansion in the 6a to
get tetrahydroquinoline derivative 6b found to be detrimental to
PAI-1 inhibitory activity (IC50 = 63.8 M), probably due to confor-
lM) compared
Compound
Cmax
(l
g/mL)
Tmax(h)
T1/2 (h)
AUC(0–24) (h lg/mL)
l
5a
6c
6e
2.4
6.8
1.78
1
6
4
3.27
9.86
6.88
10.38
80.18
5.39
l
mational misfit of the molecule (six-membered ring of 6b vs five-
membered ring of 6a). The introduction of the double bond in the
indoline derivative 6a to get indole derivative 6c inhibited the PAI-
a
Compounds were dosed in fasted male wistar rats at 30 mpk po formulated
with a Tween-80: PEG: CMC(5:5: 90% v/v).
1 activity with impressive IC50 of 3.2
tion of an extra N atom in the ring gave indazole derivative 6d, which
showed inferior potency with an IC50 of 14.6 M. Further, a methoxy
group was introduced in the most potent compound 6c to get the
compound 6e, interestingly the compound 6e exhibited slightly
lM, (Table 2). The incorpora-
inhibited PAI-1 with an IC50 of 3.4
removal of methoxy group from 5a gave 5b, which exhibited slightly
lower potency (IC50 = 4.9 M) than 5a. The removal of triflorometh-
oxy group is found to be detrimental for activity as evident from the
lM in the chromogenic assay. The
l
l

V. Pandya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5701–5706
5705
higher potency (IC50 = 2.4
translocation of m-CF3 group of 6e (IC50 = 2.4
was found to be detrimental in terms of potency as witnessed from
IC50 value of 6f, (IC50 = 22 M). The removal of CF3 group from 6c to
l
M) compared to 6c (IC50 = 3.2
lM). The
Acknowledgments
l
M) at para position
We are grateful to the management of Zydus Group for encour-
agement and analytical department for their support.
l
get the compound 6g resulted in the deterioration of the PAI-1 inhi-
bition (Table 2), which further supported the importance of the of
the CF3 group. The compounds with potent PAI-1 inhibitory activity,
5a, 6c, and 6e were evaluated for their pharmacokinetic parameters
in rats (Table 3).
References and notes
1. Yepes, M.; Loskutoff, D. F.; Lawrence, D. A. Hemostasis Thrombosis: Basic
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The compound 5a showed good plasma levels (Cmax = 2.4 lg/
2. Schleef, R. R.; Higgins, D. L.; Pillemer, E.; Levitt, L. J. J. Clin. Invest. 1989, 83, 1747.
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mL) and a half life (T1/2 = 3.27 h) when dosed orally at 30 mg/kg
in wistar rats (Table 3). The compound 6c showed impressive plas-
ma levels (Cmax = 6.8 lg/mL) and a long (T1/2 = 9.86 h), which is
favorable for this class of compounds. However, plasma concentra-
tion of the methoxy derivative 6e was found to be modest when
compared to 6c. The significant plasma concentration and long half
life of the compounds 5a, 6c, and 6e prompted us to study the com-
pounds for their in vivo efficacy in rats using FeCl3 induced arterial
thrombosis model using Clopidogrel, a well known antiplatelet
agent as a positive control.34 However, compound 6c exhibited
moderate antithrombotic efficacy while compounds 5a and 6e
failed to show any in vivo efficacy, inspite of their impressive
in vitro PAI-1 inhibitory activity and favorable pharmacokinetic
parameters (Fig. 4). The further optimization efforts for this class
of compounds to get the appropriate pharamacodynamics and
pharmacokinetics correlation are in progress.
In summary, the novel 5-nitro-2-phenoxybenzoic acid deriva-
tives derived using hybridization and conformational restriction
strategies display potent PAI-1 inhibitory activity and favorable
pharmacokinetic parameters. Oxoacetic acid part of Tiplaxtinin 1
has been effectively replaced with 5-nitro-2-phenoxybenzoic acid
part of 2 producing potent PAI inhibitor 5a. The docking study con-
firmed the similar orientation of 5a and tiplaxtinin in PAI-1 ligand
binding site. Conformational restriction of 2 with indole as a cen-
tral core (6c) showed potent PAI-1 inhibitory activity and excellent
pharmacokinetic profile with moderate efficacy in rats using FeCl3
induced arterial thrombosis model. These findings provided the
impetus for further studies on the refinement of these templates
which will be reported in due course.
12. Brown, N. J. Ther Adv Cardiovasc Dis. 2010, 4(5), 315.
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Charles van Ypersele de Strihou, C.; Hirayama, N.; Miyata, T. Arterioscler.
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Med. Chem. Lett. 2001, 11, 2589.
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Thromb. Haemost. 2002, 88, 137.
19. Einholm, A. P.; Pedersen, K. E.; Wind, T.; Kulig, P.; Overgaard, M. T.; Jensen, J. K.;
Bodker, J. S.; Christensen, A.; Charlton, P.; Andreasen, P. A. Biochem. J. 2003, 373,
723.
20. Crandall, D. L.; Elokdah, H.; Di, L.; Hennan, J. K.; Gorlatova, N. V.; Lawrence, D.
A. J. Thromb. Haemost. 2004, 2, 1422.
21. Liang, A.; Wu, F.; Tran, K.; Jones, S. W.; Deng, G.; Ye, B.; Zhao, Z.; Snider, R. M.;
Dole, W. P.; Morser, J.; Wu, Q. Thromb. Res. 2005, 115, 341.
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D. L.; Lawrence, D. A. J. Biol. Chem. 2007, 282, 9288.
23. Gardell, S. J.; Krueger, J. A.; Antrilli, T. A.; Elokdah, H.; Mayer, S.; Orcutt, S. J.;
Crandall, D. L.; Vlasuk, G. P. Mol. Pharmacol. 2007, 72, 897.
24. Rupin, A.; Gaertner, R.; Mennecier, P.; Richard, I.; Benoist, A.; De Nanteuil, G.;
Verbeuren, T. J. Thromb. Res. 2008, 122, 265.
25. Izuhara, Y.; Takahashi, S.; Nangaku, M.; Takizawa, S.; Ishida, H.; Kurokawa, K.;
de Strihou, C.; Hirayama, N.; Miyata, T. Arterioscler. Thromb. Vasc. Biol. 2008, 28,
672.
26. Lucking, A. J.; Visvanathan, A.; Philippou, H.; Fraser, S.; Grant, P. J.; Connolly, T.
M.; Gardell, S. J.; Feuerstein, G. Z.; Fox, K. A.; Booth, N. A.; Newby, D. E. J.
Thromb. Haemost. 2010, 8, 1333.
60
*
Cut off time
50
40
30
20
10
0
27. Jain, M. R.; Shetty, S.; Chakrabarti, G.; Pandya, V.; Sharma, A.; Parmar, B.;
Srivastava, S.; Raviya, M.; Soni, H.; Patel, P. R. Eur. J. Med. Chem. 2008, 43, 880.
28. Protocol for docking study: (a) X-ray crystal structure of PAI-1 (PDB code: 3Q03)
was obtained from PDB database. Protein crystal structure of PAI-1 was
prepared using the Schrödinger’s protein preparation wizard module. The
binding site of the protein structure was identified using the sitemap module
of the Schrödinger software. The probable site according to the literature was
used as the centroid to generate the grid files for the docking. Docking study
was carried out by using the induced fit docking; (b) Schrödinger Suite 2010
Induced Fit Docking protocol; Glide version 5.6, Schrödinger, LLC, New York,
NY, 2010; Prime version 2.2, Schrödinger, LLC, New York, NY, 2010; (c) Docking
study of Tiplaxtinin using the Glide software in PAI-1 protein crystal structure
(PDB code: 1B3K) obtained from PDB did not gave a reported binding mode,22
hence PAI-1 crystal structure recently reported by Jensen et al. was used for
docking study of compounds along with tiplaxtinin; (d) Jensen, J. K.;
Thompson, L. C.; Bucci, J. C.; Nissen, P.; Gettins, P. G. W.; Peterson, C. B.;
Andreason, P. A.; Morth, J. P. J. Biol. Chem. 2011, in press. doi:10.1074/
jbc.M111.236554.
*
29. Pharmacokinetic study: Compounds were formulated with
a
Tween-
80:PEG:CMC (5:5:90% v/v), A graduated dose volume (5 ml/kg) of suspension
was administered to fasted male Wistar rats at 30 mg/kg po. The animals were
anesthetized for blood sample collection from retro-orbital plexus. Serial blood
samples were collected into heparinised containers at various time points and
blood centrifuged to yield plasma. Plasma concentration was determined by
using LC–MS/MS method.
Figure 4. Effects of the compound 5a and 6c and 6e on time to thrombus formation
in FeCl3-induced arterial thrombosis in rats at 30 mpk. Each value represents
mean SEM (n = 6). ⁄ indicates p < 0.05 versus vehicle control. Clopidogrel was used
as positive control and administered orally 2 h before application of FeCl3 paper on
the carotid artery.
30. Kotha, S.; Lahiri, K.; Kashinath, D. Tetrahedron 2002, 58, 9633.

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V. Pandya et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5701–5706
31. Wolfe, J. P.; Tomori, H.; Sadighi, J. P.; Yin, J.; Buchwald, S. L. J. Org. Chem. 2000,
65, 1158.
20 and 0.1% BSA in PBS. This assay detects only active inhibitory PAI-1 (not
latent or substrate) bound to the plate. 100 l aliquot of HRP substrate solution
was added and incubated for 5 min at 25 °C. Reaction was terminated with the
addition of 50 l of 1.6 (M) H2SO4 followed by the determination of absorbance
at 490 nm. The quantization of residual active PAI-1 bound to t-PA at varying
concentrations of phenoxybenzoic acid was used to determine the IC50 by
fitting the results to a logistic dose–response program (Graphpad Prism, CA,
USA). IC50 was defined as the concentration of compound required to achieve
50% inhibition of PAI-1 activity. The assay sensitivity was 5 ng/ml of human
PAI-1 as determined from a standard curve ranging from 0-100 ng/ml of
human PAI-1.
l
32. Spectroscopic characterization of selected compounds: 5a: 97.8% by HPLC; mp
225–228 °C; 1H NMR (400 MHz, DMSO-d6): 8.5 (d, J = 2.8 Hz, 1H), 8.08 (dd, J =
2.8 and 9.4 Hz, 1H), 7.7 (m, 3H), 7.48 (d, J = 8.8 Hz, 1H), 7.44 (d, J = 3.2 Hz, 1H),
7.40 (d, J = 8 Hz, 2H), 7.33 (dd, J = 1.6 and 8.8 Hz, 1H), 7.26 (m, 1H), 7.16 (dd,
J = 1.2 and 8.4 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 6.49 (d, J = 9.2 Hz, 1H), 6.41 (d,
J = 2.8 Hz, 1H), 5.34 (s, 2H), 3.68 (s, 3H); ESI-MS: 578.9 (M+H)+, 6c: 97% by
HPLC; mp 180–185 °C; 1H NMR (300 MHz, DMSO-d6): 8.56 (d, J = 3.2 Hz, 1H),
8.23 (dd, J = 2.9 and 9.1 Hz, 1H), 7.42 (m, 2H), 7.33 (m, 3H), 7.21 (m, 2H), 7.21
(m, 2H), 7.12 (m, 3H), 7.0 (m, 2H), 6.78 (d, J = 9.1 Hz, 1H), 6.3 (d, J = 3 Hz, 1H),
5.34 (s, 2H), 3.37 (t, J = 4.2 Hz, 4H), 3.16 (t, J = 4.4 Hz, 4H); ESI-MS: 617.2
(M+H)+; 6e: 97.5% by HPLC; mp 120–125 °C; 1H NMR (400 MHz, DMSO-d6):
8.51 (d, J = 2.8 Hz, 1H), 8.07 (dd, J = 2.8 and 9.2 Hz, 1H), 7.44 (m, 1H), 7.28 (m,
3H), 7.24 (m, 2H), 7.18 (dd, J = 0.8 and 8 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.95 (d,
J = 2 Hz, 1H), 6.87 (dd, J = 2 and 8.8 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.45 (d,
J = 9.2 Hz, 1H), 6.24 (d, J = 3.2 Hz, 1H), 5.26 (s, 2H), 3.69 (s, 3H), 3.38 (t, J = 4 Hz,
4H), 3.14 (t, J = 4.8 Hz, 4H); ESI-MS: 647 (M+H)+.
l
34. Protocol for in vivo study: In this study FeCl3 induced chemical injury was used
as a model of arterial thrombosis in rat model. Rats (n = 6) were anaesthetized
with urethane (1.25 g/kg, ip) and secured in supine position. A midline cervical
incision was made on the ventral side of the neck, and left carotid artery was
isolated by blunt dissection. Compound 5a, 6c and 6e (each 30 mg/kg) were
formulated in polyethylene glycol (PEG) and 0.5% sodium carboxymethyl
cellulose (1:10) and administered by oral gavage. Exactly after 2 h of
administration, a 2 Â 3 mm strip of Whatman # 1 filter paper saturated with
35% (w/v) FeCl3 was placed on the carotid artery for 5 min. A temperature
probe (Thermalert-TH8, Physitemp Instruments Inc., Clifton, N.J., USA) was
placed distal to filter paper to monitor the temperature of carotid artery. A
sudden fall in temperature (about 1–2 °C) was taken as an indication of
cessation of blood flow as a result of thrombus formation. Time to occlusion
(TTO) was defined as the time from FeCl3 application to time of thrombus
formation (indicated by sudden fall in carotid temperature). In case if no
thrombus formation was seen in drug-treated animals, a cutoff time was fixed
at 1 h.
33. Chromogenic assay: The chromogenic assay was based upon the interaction
between tPA and active PAI-1. tPA coated assay plates obtained from Trinity
Biotech., NY, USA were kept at 4 °C overnight. phenoxybenzoic acid derivatives
were dissolved in DMSO and diluted to a range of concentration between 1
and100 lM. Varying concentrations of phenoxybenzoic acid were then
incubated with human PAI-1 (50 nM, Molecular Innovations, MI, USA) for
30 min at 25 °C. An aliquot of this solution along with a monoclonal antibody
against human PAI-1 conjugated with HRP (Trinity Biotech., NY, USA) was
added to the t-PA-coated plate. The Plate was then incubated for 30 min at
room temperature with gentle shaking. The solution was aspirated from the
plate, which was then washed thrice with a buffer consisting of 0.05% Tween