In vitro PAI-1 inhibitory activity of oxalamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2007.05.011

A number of oxalamide derivatives have been synthesized and evaluated for PAI-1 inhibitory activity. In vitro PAI-1 inhibitory activities of oxalamide derivatives are evaluated by chromogenic assay. Few compounds have shown significant PAI-1 inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2007.05.011

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 880e884
http://www.elsevier.com/locate/ejmech
Short communication
In vitro PAI-1 inhibitory activity of oxalamide derivatives*
*
Mukul R. Jain , Shankar Shetty, Ganes Chakrabarti, Vrajesh Pandya, Ajay Sharma,
Bhavesh Parmar, Soma Srivastava, Mehul Raviya, Hitesh Soni, Pankaj R. Patel
Zydus Research Centre, Sarkhej-Bavla NH No. 8A, Moraiya, Ahmedabad-382210, Gujarat, India
Received 1 March 2007; received in revised form 22 May 2007; accepted 29 May 2007
Available online 3 June 2007
Abstract
A number of oxalamide derivatives have been synthesized and evaluated for PAI-1 inhibitory activity. In vitro PAI-1 inhibitory activities of
oxalamide derivatives are evaluated by chromogenic assay. Few compounds have shown significant PAI-1 inhibitory activity.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: PAI-1; Oxalamide; Thrombosis; tPA; uPA
1. Introduction
[19], piperazine analogues 2 [20], and indole oxoacetic acid
3 [21] (Fig. 1).
The activity of tissue plasminogen activator (tPA) and uro-
kinase plasminogen activator (uPA) is negatively regulated by
a serine protease inhibitor (SERPIN) named plasminogen ac-
tivator inhibitor-1 (PAI-1) [1]. Platelets contain high amount
of PAI-1 [2,3] and release of PAI-1 from activated platelets
may lead to its high local concentration, which may be respon-
sible for higher incidence of tPA resistant thrombus formation
[4e6]. PAI levels are reported to be elevated in various throm-
botic disorders including deep vein thrombosis (DVT) [7,8]
and other diseases like diabetes [9,10], obesity [11,12], and
syndrome ‘X’ [11,13]. All of these disorders are associated
with an increased risk of systemic thrombosis; therefore, inhi-
bition of PAI-1 may represent a useful strategy for treating
thrombotic diseases. This hypothesis is supported by studies
suggesting that transgenic mice expressing high levels of
PAI-1 develops spontaneous thrombosis [14,15], whereas
PAI-1 knockout mice are resistant to venous or arterial
thrombosis [16,17].
These inhibitors are found to show good in vitro PAI-1 in-
hibitory activity and are under different stages of preclinical/
clinical development [20]. The process of finding a better
inhibitor by high throughput screening of various compound
libraries having carboxylic acid functionality culminated in the
identification of oxalamide derivative 4 (Fig. 2) that showed
an in vitro IC50 value of 96 mM in PAI-1 inhibitory assay.
Taking oxalamide derivative 4 as the suitable candidate for
modification, several compounds 9 and 15 having carboxylic
acid functionality were synthesized [22] and evaluated for
their PAI-1 inhibitory activity in chromogenic assay [23]
(Fig. 3). The PAI-1 protein and ligand interaction was also
studied by Native PAGE (Poly Acrylamide Gel Electrophore-
sis) experiment.
2. Chemistry
The oxalamide derivatives 4, 9 and 15 were prepared as
shown in Schemes 1 and 2. The aminobiphenyl intermediate 6
was prepared by Suzuki coupling of 4-trifluoromethoxyphenyl
boronic acid with 4-nitro-iodobenzene followed by reduction
of eNO2 group, which upon reductive amination with
substituted aldehyde produced 7. The reaction of 7 with methyl
chlorooxoacetate furnished compound 8. The conventional
Recently, several classes of small molecule PAI-1 inhibitors
have been reported [18], such as menthol based inhibitors 1
*
ZRC communication # 184.
* Corresponding author. Tel.: þ91 2717 250801; fax: þ91 2717 250606.
E-mail address: mukul.jain@zyduscadila.com (M.R. Jain).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.05.011

M.R. Jain et al. / European Journal of Medicinal Chemistry 43 (2008) 880e884
881
CF3
F3CO
NO2
H
OH
O
O
N
HOOC
O
NO2
O
N
O
N
N
O
OCH3
F3C
N
H
1
COOH
2
3
Fig. 1. PAI-1 inhibitors.
alkaline hydrolysis of compound 8 provided 9 (Scheme 1). Con-
trolled reaction of substituted sulfonyl chloride 10 with p-phe-
nylenediamine gave sulfonamide derivative 11. The reductive
amination of sulfonamide derivative 11 with 3,5-bistrifluoro-
methyl benzaldehyde gave 12. The reaction of 12 with methyl
chlorooxoacetate yielded oxalamide ester 13. The alkylation
of 13 with substituted alkyl halide provided compound 14 and
subsequent alkaline hydrolysis of compound 14 gave 15
(Scheme 2).
methoxy (15b) produced compounds with low PAI-1 inhibi-
tory activity. The compounds containing electron-withdrawing
groups such as trifluoromethyl and trifluoromethoxy groups on
phenyl ring demonstrated good PAI-1 inhibitory activity
[20,21]. Taking clue from the literature, first trifluoromethoxy
group was introduced on the phenyl ring, which resulted in
compound 15d and it inhibited PAI-1 activity with IC50
9.3 mM. However, both positional isomers 15e (meta) and
15f (ortho) displayed inferior inhibitory activity than 15d
( para). Introduction of a less bulky electron-withdrawing
fluoro substituent on phenyl ring produced compound 15c
with low PAI-1 inhibitory activity.
3. Results and discussion
In order to see the effect of substituents at free H of sulfon-
amide group (R2 of region 1), various compounds were syn-
thesized in which H atom was replaced by methyl in 15g,
propyl in 15h, pentyl in 15i and allyl in 15j. Results of PAI-
1 assay indicate that bulky alkyl groups help in improving ac-
tivity, as 15g is less active than 15h, which in turn is less active
than 15i. Compound 15j showed similar activity to that of 15i.
However, benzyl substituted compound 15k showed inferior
activity. Introduction of 4-OCF3 group on benzyl ring of 15k
produced compound 15l with very good activity (IC50
8.5 mM), which may be due to combined interaction of two
trifluoromethoxy groups.
Next, compounds containing trifluoromethyl group on phe-
nyl ring were synthesized. Introduction of 4-CF3 group on
phenyl ring (R3 of region 1) produced very less potent com-
pound 15m with IC50 of 86 mM. Substitution of methyl group
on sulfonamide linker of 15m gave compound 15n with mar-
ginally improved activity (IC50 61 mM). Further changing the
position of eCF3 group from para to meta, 15o showed dra-
matic improvement in potency (IC50 4.5 mM). Substitution
on sulfonamide linker of 15o to get 15p and 15q also gave
good compounds. The compound 15q was found to be as
The PAI-1 inhibitory activity of oxalamide derivatives 9ae
9d and 15ae15t is summarized in Tables 1 and 2.
The compound 4 inhibited PAI-1 activity with an IC50 of
96 mM in chromogenic assay [23], hence compound 4 was
selected for further modification in region 1 and region 2
(Fig. 2), which gave compounds 15ae15t and 9ae9d
respectively (Fig. 3).
The first synthesized compounds i.e. 3-methanesulfonate
phenyl derivative 9a and pyridyl derivative 9b did not show
PAI-1 inhibitory activity in the chromogenic assay. Introduc-
tion of an electron-withdrawing and bulky trifluoromethyl
group on phenyl ring 9c showed considerable improvement
in the activity with an IC50 of 23 mM. Additional increase in
bulk by introducing trifluoromethyl group on 9c gave 3,5-bis-
trifluoromethyl phenyl derivative 9d, which displayed even
better PAI-1 inhibitory activity with an IC50 14.4 mM (Table 1).
Based on these results, 3,5-bistrifluoromethyl-substituted phenyl
ring in region 2 was finalized.
Furthermore, the modification of region 1, we introduced e
SO2NR2e as a spacer group between two phenyl rings (Table 2).
Substitution of electron releasing groups at the para position
of the phenyl ring (R3 of region 1) such as chloro (15a) and
O
F3CO
F3CO
F3C
N
COOH
COOH
Region 1
COOH
O
N
O
CF3
N
Region 2
O
N
R1
S
R3
R2
O
4
IC50 = 96
M
9
15
Fig. 2. Lead compound from library.
Fig. 3. General structure derived from 4.

882
M.R. Jain et al. / European Journal of Medicinal Chemistry 43 (2008) 880e884
F3CO
F3CO
b
F3CO
F3CO
I
a
+
B(OH)2
NO2
NH2
R1 CHO
5
6
NO2
c
F3CO
F3CO
O
O
R1
N
COOCH3
N
H
e
N
COOH
7
8
9
R1
R1
d
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, K3PO4, (C4H9)4NþBrꢁ, DMF, 45e50 ꢀC, 16 h, 65%; (b) PdeC, H2 (60 psi), MeOH, 25e28 ꢀC, 3 h, 90%;
(c)NaBH4, EtOH, 40e45 ꢀC, 4e5 h, 80%; (d) ClCOCOOMe, pyridine, CH2Cl2, 5e10 ꢀC, 3 h, 85% and (e) KOH, THF, H2O, 25e28 ꢀC, 80%.
F3C
H2N
CF3
CF3
CHO
R3O2SHN
O
S
O
NH
R3O2SHN
NH2
+
R3
F3C
Cl
a
b
12
10
11
NH2
c
O
O
N
R2
R3O2SN
R2
O
N
COOH
CF3
COOCH3
CF3
COOCH3
CF3
R3O2SN
N
R3O2SHN
R2-X
d
e
14
13
15
CF3
CF3
CF3
Scheme 2. Reagents and conditions: (a) DIPEA, CH2Cl2, 25e28 ꢀC, 15e30 min, 75%; (b) NaBH4, EtOH, 40e45 ꢀC, 4e5 h, 80%; (c) ClCOCOOMe, pyridine,
CH2Cl2, 5e10 ꢀC, 3 h, 85%; (d) K2CO3, acetone, 25e28 ꢀC, 2e10 h, 80%; (e) KOH, THF, H2O, 25e28 ꢀC, 80%.
potent as 15o in PAI-1 inhibitory activity. Compound 15r with
3,5-bistrifluoromethyl-substituted ring (R2 of region 1) showed
a very promising PAI-1 inhibitory activity with an IC50 of
5 mM. However, changing methyl group from sulfonamide
linker (R2 of region 1) of 15r with bulky groups allyl (15t)
Table 1
PAI-1 inhibitory activity of oxalamide derivatives 9ae9d
F3CO
COOH
O
and propyl (15s) lead to compounds with mediocre in vitro ac-
tivity. This is in contrast to the observation in compounds
15ge15l, which may be due to crowding of alkyl groups
with meta-CF3 group. In Native PAGE (Poly Acrylamide
Gel Electrophoresis) experiment, we observed that PAI-1-li-
gand complex stays above the free PAI-1 (gel picture is not
given). The intensity of the PAI-1-ligand complex in Native
PAGE and unavailability of free PAI-1 after ligand interaction
clearly demonstrates that high concentration of the ligand does
not induce any PAI-1 inactivation.
N
R1
9
Compound
R1
IC50 (mM)a
9a
No inhibition
H3CO2SO
N
9b
9c
9d
No inhibition
4. Conclusion
23
In summary, we have explored the structureeactivity rela-
tionships around the PAI-1 inhibitor 4 by modification of the
regions 1 and 2. It was observed that regions 1 and 2 accom-
modate electron-withdrawing bulky groups on phenyl ring
with enhancement of potency, but electron releasing groups
in region 1 and polar groups in region 2 were not tolerated. In-
troduction of sulfonamide spacer retained its inhibitory
F3C
14.4
10
CF3
F3C
3 Tiplaxtinin
e
a
Values determined using in vitro chromogenic assay.

M.R. Jain et al. / European Journal of Medicinal Chemistry 43 (2008) 880e884
Table 2 (continued)
883
Table 2
PAI-1 inhibitory activity of oxalamide derivatives 15ae15t with sulfonamide
spacer
Compound
R2
R3
IC50 (mM)a
F3C
O
15p
F3C
11
N
COOH
F3C
F3C
15q
15r
F3C
CF3
5.4
5
O
N
CH3
S
R2
O
R3
15
F3C
F3C
R2
H
R3
IC50 (mM)a
15s
C3H7
Compound
8.4
15a
75
Cl
F3C
F3C
15b
15c
15d
15e
15f
H
H
H
H
H
>80
H3CO
F
15t
12.8
10
29.9
9.3
F
F3C
F3CO
F3CO
3 Tiplaxtinin
e
e
a
Values determined using in vitro chromogenic assay.
15.4
activity. Substitution at sulfonamide group with bulky alkyl
groups and benzyl group substituted with electron-withdraw-
ing groups resulted some good compounds 15l, 15o, 15q,
15r and 15s with good potency.
OCF3
114
25
15g
15h
15i
CH3
F3CO
F3CO
F3CO
F3CO
Acknowledgements
C3H7
C5H11
21
We are grateful to the management of Zydus Group for
encouragement and analytical department for their support.
We thank Dr. Brijesh Kumar Srivastava for his guidance in
preparing the manuscript.
14.9
13.3
15j
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determined using chromogenic assay that 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. Required quantity of phos-
phate buffer containing EDTA and tween 20 was added in each well and
incubated for 2 min at 27e28 ꢀC with gentle shaking in order to dissolve
tPA. Oxalamide derivatives were dissolved in DMSO and diluted to
a range of concentration between 1 and100 mM. Varying concentrations
oxalamide derivatives 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 27e28 ꢀC
with gentle shaking. The solution was aspirated from the plate, which
was then washed thrice with a buffer consisting of 0.05% tween 20
and 0.1% BSA in PBS. Aliquot of 100 mL of HRP substrate solution
was added and incubated for 5 min at 25 ꢀC. Reaction was terminated
with the addition of 50 mL of 1.6 M H2SO4 followed by the determina-
tion of absorbance at 490 nm. This assay detects only active inhibitory
PAI-1 (not latent or substrate) bound to the plate. The quantitation of re-
sidual active PAI-1 bound to t-PA at varying concentrations of oxalamide
derivative 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 de-
termined from a standard curve ranging from 0e100 ng/mL of human
PAI-1.
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[22] Characterization data: 9d. 96.5% purity by HPLC; 1H NMR (300 MHz,
DMSO-d6): d 8.04 (s, 1H), 7.9 (s, 2H), 7.78 (d, J ¼ 8.74 Hz, 2H), 7.73
(d, J ¼ 8.49 Hz, 2H), 7.44 (d, J ¼ 8.19 Hz, 2H), 7.33 (d, J ¼ 8.43 Hz,
2H), 5.19 (s, 2H); 15l. 98% purity by HPLC; 1H NMR (300 MHz,
DMSO-d6): d 7.91 (bs, 3H), 7.64 (d, J ¼ 8.82 Hz, 2H), 7.50 (d,
J ¼ 8.34 Hz, 2H), 7.31 (d, J ¼ 8.18 Hz, 2H), 7.1e7.18 (m, 4H), 6.99 (d,
J ¼ 8.72 Hz, 2H), 5.0 (s, 2H), 5.0 (s, 2H); 15r. 98.8% purity by HPLC;
1H NMR (300 MHz, DMSO-d6): d 8.49 (s, 1H), 7.92 (bs, 3H), 7.74 (bs,
2H), 7.24 (bs, 2H), 7.06 (d, 2H), 5.07 (s, 2H), 3.1 (s, 3H). The product for-
mation was supported by ESI-MS: 695.2 (M ꢁ H). 15o. 98% purity by