Synthesis and in vitro evaluation of a series of diketopiperazine inhibitors of plasminogen activator inhibitor-1

Article abstract of DOI:10.1016/S0960-894X(01)00508-X

We have synthesized and evaluated a series of diketopiperazine-based inhibitors of PAI-1. These studies resulted in the identification of 34 which inhibited PAI-1 in vitro with an IC₅₀ = 0.2 μM. The synthesis and SAR of these compounds are desc

Full text of DOI:10.1016/S0960-894X(01)00508-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2589–2592
Synthesis and In Vitro Evaluation of a Series of Diketopiperazine
Inhibitors of Plasminogen Activator Inhibitor-1
Adrian Folkes,* Michael B. Roe, Sukhjit Sohal, Julian Golec, Richard Faint,
Teresa Brooks and Peter Charlton
Xenova Limited, 957 Buckingham Avenue, Slough, Berks SL1 4NL, UK
Received 15May 2001; revised 9 July 2001; accepted 18 July 2001
Abstract—We have synthesized and evaluated a series of diketopiperazine-based inhibitors of PAI-1. These studies resulted in the
identification of 34 which inhibited PAI-1 in vitro with an IC50=0.2 mM. The synthesis and SAR of these compounds are described.
# 2001 Elsevier Science Ltd. All rights reserved.
A key event in the regulation of thrombus formation
and clearance is tissue plasminogen activator (tPA)
generation of plasmin.1 Plasminogen activator inhib-
itor-1 (PAI-1), a member of the serpin superfamily of
proteinase inhibitors,2,3 is the primary physiological
regulator of tPA and is an essential regulatory protein
of the fibrinolytic system. Several studies have linked
increased PAI-1 activity with thromboembolic disease4,5
and elevated levels of PAI-1 in transgenic mice are
associated with severe venous thrombosis.6 Further-
more, anti-PAI-1 antibodies have been shown to
enhance fibrinolysis in vivo.7 PAI-1 is also associated
with a poor prognosis in a variety of cancers,8 and is
believed to play a role in angiogenesis,9 metastasis10 and
invasion.9
of tPA is mediated through a bait peptide bond
(Arg346-Met347), present within the RCL, which
mimics the natural substrate for tPA, plasminogen.13
We have previously reported XR5118 (1) which was
identified from a medicinal chemistry program based on
a natural product lead.14 XR5118 has demonstrated
PAI-1 inhibitory activity in vitro15 and in vivo16 and has
been shown to attenuate the binding of a PAI-1 inhi-
biting monoclonal antibody CLB-2C8,16 suggesting that
binding of XR5118 to PAI-1 may occur at the epitope
for CLB-2C8 which is the domain spanning amino acids
110–145. We describe here the synthesis and biological
activities of a series of bis-arylidenediketopiperazines
based on XR5118.
PAI-1 is unique amongst serpins in that the protein can
spontaneously convert from its active conformation into
a latent, inactive form that is unable to irreversibly bind
to tPA.11 The reactive center loop (RCL), which is
responsible for the inhibitory activity of PAI-1 is
exposed in the active form, but is inserted into the major
b-sheet A in the latent confirmation.12 PAI-1 inhibition
Scheme 1.
*Corresponding author. Fax:+44-1753-706607; e-mail: adrian_folkes@xenova.co.uk
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00508-X

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A. Folkes et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2589–2592
The general synthetic route used to prepare analogues
of XR5118 is outlined in Scheme 1.17ꢀ20 Glycine anhy-
dride was bis-acetylated in acetic anhydride to yield the
intermediate 2 (79% yield). Condensation of 2 with a
variety of aromatic aldehydes using potassium tert-but-
oxide in THF yielded mono-arylidenes of general for-
mula 15 in 60–80% yield. Performing this reaction in
THF rather than DMF prevented formation of the bis-
arylidene. Reaction of 15 with a second aldehyde using
Cs2CO3 in DMF yielded the desired bis-arylidenediketo-
piperazines in reasonable yield (30–60%). NMR studies
and comparison with known compounds confirmed that
this route resulted in formation of only the 3Z,6Z iso-
mer. The aldehydes used for both the first and second
condensation were either available commercially or
were prepared as shown in Scheme 2. Aldehydes 3–6
were conveniently prepared by reaction of 5-bromo-2-
thiophene carboxaldehyde with the appropriate
nucleophile. Direct reaction between the nucleophile
and 5-bromo-2-thiophenecarboxaldehyde yielded 3, 4
and 6, whilst the preparation of 5 required protection
of the aldehyde as the acetal, followed by Cu(I) cata-
lyzed reaction with the sodium salt of N,N-
dimethylethanolamine and then acid-catalyzed depro-
tection. To prepare aldehydes 7–10, 4-nitrobenzalde-
hyde was protected as the acetal, which was then
reduced using H2/PtO2 in THF to yield the corre-
sponding amine. Reaction with the appropriate acid
chloride followed by acid-catalyzed deprotection yielded
the desired aldehydes. Reaction of the sodium salt of 4-
hydroxybenzaldehyde with the appropriate alkylhalide
ester afforded aldehydes 11–14. Compound 27 was pre-
pared by mCPBA oxidation of XR5118 in DCM/
MeOH. Bis-arylidenediketopiperazines containing an
ester functionality (e.g., 35) were hydrolysed to their
corresponding carboxylic acids (e.g., 34) using NaOH in
H2O/THF/MeOH.
Compounds were tested in a plasmin generation chro-
mogenic assay as reported previously,21 utilizing the
S2251 tripeptide as the chromogenic substrate.
Our initial investigations focused on the introduction of
a variety of substituents onto the left-hand ring of
XR5118 (Table 1).
In general, it was found that substitution on the left-
hand ring with a variety of electron withdrawing and
donating substituents was not well tolerated, with most
analogues showing at least a 3-fold reduction in
potency. However, increasing the size of the substituent,
as with amides 24 and 25, yielded compounds of similar
activity to XR5118.
Modifications to the dimethylamino ethylsulfanyl side
chain were also not tolerated (Table 2). Small changes
to the linker and manipulation of the amine group
resulted in a loss of activity indicating the sensitive nat-
ure of the side chain.
Bjorquist et al. have previously reported a series of site
directed mutagenesis experiments which suggested that
the binding site on PAI-1 for an anthranilic acid
derivative, AR-H029953XX, was located in the region
of the epitope for the antibody CLB-2C8.22 It was also
reported that the acidic moiety of AR-H029953XX was
essential for activity, and it was postulated that there
was an interaction between the carboxylic acid group
and one or more of three arginines (Arg76, Arg115and
Arg118) present within the epitope region. As our own
studies have suggested that this region on PAI-1 may be
important for the binding of XR5118, we introduced an
acidic group onto the diketopiperazine template to
explore whether potency could be enhanced through a
possible interaction with a putative arginine residue.
Scheme 2.

A. Folkes et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2589–2592
Table 1. PAI-1 inhibitory activity for compounds 1 and 16–25 Table 3. PAI-1 inhibitory activity for compounds 29–38
2591
Compound no.
R00
X
n
R000 IC50 (mM)
Compound no.
R
IC50 (mM)
29
30
31
32
33
34
35
36
37
38
H
H
H
H
—
O
O
O
O
O
O
—
1
3
4
4
7
7
6
6
8
H
H
H
H
H
H
>25
>25
20.0ꢁ0.27
15( n=1)
1.75ꢁ0.02
0.20ꢁ0.015
1, XR5118
H
4-Me
4-OMe
4-Br
3-Cl
3.5ꢁ0.19
12.4ꢁ 0.28
10.9ꢁ0.49
13.1ꢁ 0.71
>25
16
17
18
19
20
21
22
23
24
25
NHCO(2-thiophene)
NHCO(2-thiophene)
NHCO(2-thiophene)
NHCO(2-thiophene) NHCO
NHCO(2-thiophene) NHCO
NHCO(2-thiophene) NHCO
Me 1.93ꢁ0.25
4-CN
2-Br
>25
>25
H
0.52ꢁ0.035
Me 4.77ꢁ0.40
4-CO2Me
4-NMe2
4-NHCOPh
4-NHCO(2-thiophene)
17.7ꢁ3.25
H
0.39ꢁ0.012
>25
6.1ꢁ1.48
3.1ꢁ0.31
Table 4. Activity of 34 in various assay systems
Compound no.
Chromogenic
assay
IC50 (mM)
Fibrin
plate assay
IC50 (mM)
Complex
assay
IC50 (mM)
Table 2. PAI-1 inhibitory activity for compounds 1 and 26–28
34
0.20ꢁ0.0150.26
ꢁ01.050.5
ꢁ0.08
The excellent activity of 34 was confirmed in the fibrin
plate assay and in a tPA/PAI-1 complex assay (Table 4).
In the fibrin plate assay the PAI-1 inhibitory activity of
the compounds is quantified by measuring the rate of
fibrinolysis,23 and in the solid-phase tPA/PAI-1 complex
assay,24 pre-incubation of PAI-1 with an inhibitor pre-
vents tPA/PAI-1 complex formation in a concentration
dependant manner.
Compound no.
X
R0
IC50 (mM)
1, XR5118
S
S
S(O)
O
Me
Et
Me
Me
3.5ꢁ0.19
>25
>25
26
27
28
>25
Consequently carboxylic acid groups were introduced at
varying distances from the diketopiperazine template.
In summary, we have developed several diketopiper-
azine-based inhibitors of PAI-1 showing a marked
improvement in potency over XR5118. Compound 34
demonstrated excellent potency (0.2 mM) in a mechan-
istic plasmin generation assay. This level of activity was
retained in a functional fibrinolysis assay, and in addi-
tion 34 prevented complex formation between tPA and
PAI-1. We believe that 34 is the most potent inhibitor of
PAI-1 reported to date, and this compound may prove
useful in further evaluating the therapeutic potential of
inhibiting PAI-1 activity.
Parallel studies had shown that the thiophenyl-thioether
linkage of XR5118 could be successfully replaced by a
phenyl-ether moiety, and for reasons of synthetic acces-
sibility this grouping was used in these studies. Our
initial efforts, in which the left-hand ring was left
unsubstituted, produced compounds of very modest
activity, with a marginal improvement in potency as the
carboxylic acid functionality was extended away from
the diketopiperazine ring (Table 3). However introduc-
tion of the thiophene 2-carbonylamino group, shown
earlier to be tolerated, to the 4-position of the left-hand
ring resulted in a 9-fold improvement in activity (cf. 32
and 33). Extending the chain length from four to seven
carbons yielded a further 9-fold improvement in
potency (34). Exploration of the linker showed that
replacement of the ether with an amide was well toler-
ated, and led to potent compounds such as 36 and 38.
The importance of the acidic functionality is clearly
demonstrated by comparison of 34 and 36 with their
corresponding methyl esters 35 and 37.
Acknowledgements
We are grateful for the excellent support we received
from the following colleagues: Justin Bryans, Chris
Latham, Julie Brumwell, Cristina Visintin and John
Spencer.
References and Notes
On the basis of marginal superiority in potency and
improved physicochemical properties, the ether 34 was
selected for further assay studies.
1. Declerk, P. J.; Juhan-Vague, I.; Felez, J.; Wiman, B. J.
Intern. Med. 1994, 236, 425.
2. Van Meijer, M.; Pannekoek, H. Fibrinolysis 1995, 9, 263.

2592
A. Folkes et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2589–2592
3. Potempa, J.; Korzus, E.; Travis, J. J. Biol. Chem. 1994, 269,
15957.
14. Bryans, J.; Charlton, P. A.; Chicarelli-Robinson, I.; Col-
lins, M.; Faint, R.; Latham, C.; Shaw, I.; Trew, S. J. Antibiot.
1996, 49, 1014.
15. Charlton, P.; Faint, R.; Barnes, C.; Bent, F.; Folkes, A.;
Templeton, D.; Mackie, I.; Machin, S.; Bevan, P. Fibrinol.
Proteol. 1997, 11, 5 1.
16. Friederich, P. W.; Levi, M.; Biemond, B. J.; Charlton, P.;
Templeton, D.; van Zonneveld, A. J.; Bevan, P.; Pannekoek,
H.; ten Cate, J. W. Circulation 1997, 96, 916.
17. Gallina, C.; Liberatori, A. Tetrahedron 1974, 30, 667.
18. Gallina, C.; Liberatori, A. Tetrahedron Lett 1973, 1135.
19. Yokoi, T.; Yang, L. M.; Wu, R. Y.; Lee, K. H. J. Antibiot.
1988, 41, 494.
4. Dawson, S.; Henney, A. Atherosclerosis 1992, 95, 105.
5. Aznar, J.; Estelles, A. Haemostasis 1994, 24, 243.
6. Erikson, L. A.; Fici, G. J.; Lund, J. E.; Boyle, T. P.; Polites,
H. G.; Marotti, K. R. Nature 1990, 346, 74.
7. Biemond, B. J.; Levi, M.; Coronel, R.; Janse, M. J.; ten
Cate, J. W.; Pannekoek, H. Circulation 1995, 91, 1175.
8. Pappot, H.; Gardsvoll, H.; Rømer, J.; Pedersen, A.; Grøn-
dahl-Hansen, J.; Pyke, C.; Brunner, N. Biol. Chem. Hoppe-
Seyler 1995, 376, 259.
9. Bajou, K.; Noel, A.; Gerard, R. D.; Masson, V.; Brun-
ner, N.; Holst-Hansen, C.; Skobe, M.; Fusenig, N. E.;
Carmeliet, P.; Collen, D.; Foidart, J. M. Nat. Med. 1998, 4,
923.
20. Katritsky, A. R.; Fan, W. Q.; Szajda, M.; Li, Q.-L.;
Caster, K. C. J. Heterocycl. Chem. 1988, 25, 591.
21. Charlton, P. A.; Faint, R. F.; Bent, F.; Bryans, J.;
Chicarelli-Robinson, I.; Mackie, I.; Machin, S.; Bevan, P.
Thromb. Haemost. 1996, 75, 808.
10. Tsuchiya, H.; Katsuo, S.; Matsuda, E.; Sunamaya, C.;
Tomita, K.; Veda, Y.; Binder, B. Gen. Diagn. Pathol. 1995,
141, 41.
11. Sancho, E.; Declerck, P. J.; Price, N. C.; Kelly, S. M.;
Booth, N. A. Biochemistry 1995, 34, 1064.
12. Eitzman, D. T.; Fay, W. P.; Lawrence, D. A.; Francis-
Chmura, A. M.; Shore, J. D.; Olson, S. T.; Ginsburg, D. J.
Clin. Invest. 1995, 95, 2416.
22. Bjorquist, P.; Ehnebom, J.; Inghardt, T.; Hansson, L.;
Lindberg, M.; Linschoten, M.; Stromqvist, M.; Deinum, J.
Biochemistry 1998, 37, 1227.
23. Ehnebom, J.; Kristianssen, C.; Bjorquist, P.; Deinum, J.;
Bostrom, S. Thromb. Haemost. 1993, 69, 1330a.
24. Nieuwenhuizen, W.; Laterveer, R.; Hoegee, B. Blood
Coagul. Fibrinolysis 1995, 6, 520.
13. Lindahl, T. L.; Ohlsson, P. I.; Wiman, B. Biochem. J.
1990, 265, 109.