RGD mimetics containing phthalimidine fragment as novel ligands of fibrinogen receptor

Article abstract of DOI:10.1016/j.bmcl.2011.07.063

The novel RGD mimetics with phthalimidine central fragment were synthesized with the use of 4-piperidine-4-yl-butyric, 4-piperidine-4-yl-benzoic, 4-piperazine-4-yl-benzoic and 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acids as surrogates of Arg motif. T

Full text of DOI:10.1016/j.bmcl.2011.07.063

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5971–5974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
RGD mimetics containing phthalimidine fragment as novel ligands of
fibrinogen receptor
Andrei A. Krysko a, , Georgiy V. Samoylenko a, Pavel G. Polishchuk a,b, Sergei A. Andronati a,
⇑
Tatyana A. Kabanova a, Tetiana M. Khristova a,b, Victor E. Kuz’min a, Vladimir M. Kabanov a,
Olga L. Krysko a, Alexandre A. Varnek b, Ruslan Ya. Grygorash a
a A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Lustdorfskaya doroga 86, Odessa 65080, Ukraine
b Laboratory of Chemoinformatics, University of Strasbourg, 1, rue B. Pascal, Strasbourg 67000, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel RGD mimetics with phthalimidine central fragment were synthesized with the use of 4-
piperidine-4-yl-butyric, 4-piperidine-4-yl-benzoic, 4-piperazine-4-yl-benzoic and 1,2,3,4-tetrahydroiso-
quinoline-7-carboxylic acids as surrogates of Arg motif. The synthesized compounds potently inhibited
Received 19 May 2011
Revised 15 July 2011
Accepted 17 July 2011
Available online 26 July 2011
platelet aggregation in vitro and blocked FITC-Fg binding to
aIIbb3 integrin in a suspension of washed
human platelets. The key
a
IIbb3 protein–ligand interactions were determined in docking experiments.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Fibrinogen receptor antagonists
aIIbb3
RGD mimetics
Phthalimidine
Platelet aggregation
Over the last twenty years, the fibrinogen receptor GP IIb/IIIa (or
integrin IIbb3), has attracted a considerable attention as a promising
therapeutic target and its antagonists were applied for treatment of
thrombotic disorders such as unstable angina, myocardial infarc-
tion, ischemic disease, atherosclerosis, and stroke.1 In the majority
serotonin 5-HT12a–c and dopamine D12c receptors, inhibition of
TNF-a12d and thromboxane A212e as well as documented anti-
fungal and antibacterial activity.12f Indobufen, a phthalimidine
derivative, is a platelet aggregation inhibitor.12g The antagonist of
a
a
IIbb3 L-709,780 containing phthalimidine fragment inhibits ADP-
induced platelet aggregation of human gel-filtered platelets with
an IC50 of 0.025
M.12h
of cases, the design of aIIbb3 antagonists has been based on the mod-
eling of Arg-Gly-Asp (RGD) sequence.1b,2 The main binding sites of
RGD sequence are d-guanidine of arginine and b-carboxylic group
of aspartyl. Previously, to create non-peptide fibrinogen receptor
antagonists mimicking RGD sequence, p-benzamidine, piperidine,
isoindoline, tetrahydroisoquinoline1b,2,3 and b-alanine4 containing
fragments were successfully utilised as bioisosteres of arginine
and Asp moieties correspondingly. This approach led the develop-
ment of Tirofiban (AggrastatÒ) approved for treatment of patients
with unstable angina.5 Numerous bicyclic scaffolds have been also
used as RGD peptidomimetic blocks including indole,6 3,4-dihy-
dro-2H-benzopyran,7 tetrahydronaphthalene,7b 1,2,3,4-tetrahydro-
isoquinoline,7c 3-oxo-1,4-benzodiazepine,8 3,4-dihydro-2H-1,
4-benzoxazine,9 benzimidazole, benzoxazole,10 1,2,4-triazolo[3,4-
a]pyridine,11 etc.1b
l
This Letter describes the synthesis of new RGD mimetics
containing a phthalimidine fragment and the study of their
anti-aggregative properties. Furthermore, we demonstrate the
utility of 4-piperidine-4-yl-butyric, 4-piperidine-4-yl-benzoic,
4-piperazine-4-yl-benzoic and 1,2,3,4-tetrahydroisoquinoline-7-
carboxylic acid residues as Arg surrogates for RGD mimetic design.
Initial Boc derivatives of 4-piperidine-4-yl-butyric (6a),13
4-piperidine-4-yl-benzoic (6b),14 4-piperazine-4-yl-benzoic (6c)15
and 1,2,3,4-tetrahydroisoquinoline-7-carboxylic (6d)16 acids have
been synthesized using previously published methods.
The synthesis of aminophthalimidine building blocks 5a–d is
shown in Scheme 1 and the synthesis of target RGD mimetics
8a–i is presented in Scheme 2. The central step of formation of
phthalimidines 5a–d is the reduction of phthalimides 1a–d using
zinc amalgam. The nitro derivatives 3a–d obtained by nitration
of the compounds 2a–d were further reduced by H2/Pd(C).
Condensation of acids 6a–c with amines 5a–d was carried out
using HATU. Subsequent saponification of ester groups of com-
pounds 7a–n and elimination of Boc-protective groups yielded
Phthalimidines (2,3-dihydroisoindol-1-one) exhibit
a wide
spectrum of biological activities, for example, antagonism of
⇑
Corresponding author.
E-mail addresses: peptides@paco.net, peptides@physchem.od.ua (A.A. Krysko).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.063

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A. A. Krysko et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5971–5974
O
R
R
R
b
a
N
O
N
O
N
O
( )n
( )n
O2N
( )n
OH
O
OH
O
OH
1a-d
O
3a-d
2a-d
c
R
R
N
O
O
d
N
O
O
( )n
O2N
( )n
H2N
O
O
4a-d
5a-d
R = H, CH3; n = 0, 1, 2
Scheme 1. Synthesis of aminophthalimidines. Reagents and conditions: (a) Zn (Hg), HCl, reflux, 4 h, 65–70%; (b) HNO3, H2SO4, À5 °C, 6 h, 54–65%; (c) MeOH (H2SO4), reflux,
3 h, 74–87%; (d) H2/Pd (C), MeOH, room temperature, 7 h, 82–92%.
R
( )n
a
N
O
O
Вoc
OH
Aa
O
Aa
N
H
O
O
b
R
N
O
O
Aa
( )n
N
H
c
OH
O
O
R
N
O
HX*H
Aa
N
( )n
N
H
OH
O
O
N
O
N
=
Aa
O
N
N
O
R = H, CH3; n = 0, 1, 2; HX = HCl, CF COOH
3
Scheme 2. Synthesis of RGD mimetics. Reagents and conditions: (a) NEt3, HATU, 5a–d, room temperature, overnight, 46–58%; (b) (i) 1 M NaOH, H2O, MeOH, room
temperature, overnight; (ii)1 M HCl, 73–81%; (c) CH2Cl2, HCl gas, 0 °C, 1 h or CH2Cl2, TFA, 0 °C, 2 h, 92–96%.
the target mimetics 9a–n. Mimetics 9h–k were obtained only
as racemic mixtures in order to reveal potent compounds and
to determinate general characteristics of structure–activity
relationships.
fragments of 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid and
b-alanine, demonstrated that these two antagonists inhibit platelet
aggregation with similar potencies. The affinity of the mimetic 9g
for aIIbb3 on activated platelets was slightly higher than the affinity
Biological activity was assessed in vitro by measuring the ability
of synthesized compounds to inhibit the binding of fluoresceiniso-
of the mimetic 10. The antiaggregative activity of 9f was found
to be twice higher than that of 9g. Replacing b-alanine
with b-methyl-b-alanine leads to compounds 9h–k. Their
antiaggregative properties were generally similar to those of
unsubstituted ones, with the only exception of the mimetic 9k.
This compound possessed the highest antiaggregative activity
among all compounds synthesized in this study. Furthermore, anti-
aggregative activity of 9k is comparable to those of compounds 11
and 12 containing bicyclic cores reported in the literature, but in
the case of L-709,780 the comparison is not valid because of the
difference in biomaterials used in the assays.
thiocyanate-labeled fibrinogen (FITC-Fg)17 to
aIIbb3 (in a suspen-
sion of human washed platelets).18 Functional activity was
determined by measuring the inhibition of ADP induced platelet
aggregation in human platelet-rich plasma (PRP) by Born’s
method.19
Experimental data (Table 1) demonstrate that mimetics 9 are
potent inhibitors of FITC-Fg binding to aIIbb3 in activated platelets.
We found that peptidomimetics 9d–k with the b-alanine fragment
exhibited higher antiaggregative activities compared to com-
pounds 9a–c and 9l–n. This suggests that b-amino acids might
Ligand to protein docking study for all synthesized compounds
be the optimal blocks for the
a
IIbb3 antagonists design. The com-
has been performed with the MOE20 program. The structure of
aIIbb3
pounds 9f and 9g displayed the highest antiaggregative activity
among the synthesized mimetics with unsubstituted b-alanine as
the Asp bioisostere (Table 1), whereas the antiaggregative activity
of 9g exceeds that of 9d nearly fivefold, and that of 9e nearly
ninefold.
receptor-Tirofiban complex (2VDM) was extracted from the Protein
Data Bank.21 For docking, all water molecules were removed from
the binding pocket of 2VDM with the exception of two molecules
coordinated with the metal ion-dependent adhesive site (MIDAS)
of a
IIbb3 receptor — Mg2+, which is involved in the interaction with
Comparison of antiaggregative properties of the mimetic 9f
with the previously described compound 10 (Fig. 1) containing
carboxyl group of Tirofiban. Then, structure of the complex was opti-
mized using the MMFF94x force field. For initial ligand Tirofiban,

A. A. Krysko et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5971–5974
5973
Table 1
Biological properties of RGD mimetics 9 and RGDS peptide
Compound
HX
Aa
n
0
0
R
IC50
,
l
M (PRP)a
IC50
, lM (FITC-Fg/a
IIbb3)b
HN
O
O
9a
9b
TFA
TFA
H
H
66.0 9.0
24.0 3.0
—
O
0.27 0.06
1.2 0.1
HN
HN
HN
9c
HCl
0
H
120.0 20.0
O
9d
9e
9f
TFA
TFA
TFA
1
1
1
H
H
H
5.9 0.6
9.6 1.9
0.0055 0.009
0.0068 0.0012
—
O
O
HN
HN
0.54 0.06
N
HN
HN
9g
HCl
1
H
1.1 0.1
0.0065 0.0005
O
O
9h
9i
HCl
HCl
HCl
1
1
1
CH3
CH3
CH3
5.4 1.0
6.2 1.2
0.35 0.03
—
O
O
HN
HN
9j
3.74 0.51
0.037 0.08
N
HN
HN
9k
HCl
1
CH3
0.086 0.007
0.0065 0.0012
O
O
O
9l
TFA
TFA
2
2
H
H
51.0 30.0
—
—
O
9m
410.0 60.0
HN
HN
9n
HCl
2
H
330.0 50.0
31.0 2.0
—
RGDS
13.0 1.6
a
Concentration required to reduce ADP-induced human platelet aggregation response by 50%.
b
Concentration required to reduce binding of FITC-Fg to
aIIbb3 in suspensions of washed human platelets by 50%. The IC50 values are expressed as averages of at least two
determinations.
NHTs
H
H
H
N
OH
HCl
HN
N
N
OH
O
O
O
O
O
N
10
IC50, μM (PRP)a: 1.1
IC50, μM (FITC-Fg/αIIbβ3)a: 0.022
NH
11
IC50, μMb: 4.5
HCl HN
O
O
O
TFA
N
H
OH
H2N
N
H
N
HN
O
N
O
OH
L-709,780
IC50, μMc: 0.025
12
IC50, μMa: 0.09
Figure 1. Structure and in vitro activities of a
IIbb3 antagonists 10,16 11,6 127c and L-709,780.12h aSee Table 1; binhibition of guinea-pig PRP aggregation induced by collagen;
cinhibition of ADP-induced platelet aggregation of human gel-filtered platelets.
which was a part of 2VDM, re-docking had been carried out with
and 1,2,3,4-tetrahydroisoquinoline-7-carboxylic acids) interacts
with two amino acid residues of chain of fibrinogen receptor,
good result (RMSD = 0.6 Å). Thus
aIIbb3 cavity was prepared, and
a
all further studies of compounds 9 docking were carried out for this
binding site of the receptor.
namely with carboxyl group of D224 side chain and S225 amide
bond. Carboxyl group of mimetics 9 is involved in coordination
sphere of Mg2+, and also interacts with Y122 amide bond and amide
The docking studies have revealed some general patterns for
binding of mimetics 9 to their receptor
aIIbb3. It has been shown that
group of N215 side chain incorporated in the aIIbb3 b-chain. These
the nitrogen atom of Arg-isosteres (the fragments of 4-piperidine-4-
interactions are illustrated in Figure 2 using the complex of 9g as
yl-butyric, 4-piperidine-4-yl-benzoic, 4-piperazine-4-yl-benzoic
an example.

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A. A. Krysko et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5971–5974
Figure 2. Binding of mimetic 9g to the aIIbb3 receptor observed in docking experiments.
Duggan, M. E.; Hartman, G. D.; Lo, M.; Murphy, G. M.; Deckelbaum, L. I.; Sax, F.
L.; Barr, E. Cardiovasc. Drug Rev. 1999, 17, 199.
6. Grumel, V.; Mérour, J.-Y.; Lesur, B.; Giboulot, T.; Frydman, A.; Guillaumet, G.
Eur. J. Med. Chem. 2002, 37, 45.
7. (a) Anderluh, M.; Marc, G.; Dolenc, M. S. Synth. Commun. 2005, 35, 1461; (b)
Okumura, K.; Shimazaki, T.; Aoki, Y.; Yamashita, H.; Tanaka, E.; Banba, S.;
Yazawa, K.; Kibayashi, K.; Banno, H. J. Med. Chem. 1998, 41, 4036; (c) Fisher, M.
J.; Arfstan, A. E.; Giese, U.; Gunn, B. P.; Harms, C.; Khau, S. V.; Kinnick, M. D.;
Lindstrom, T. D.; Martinelli, M. J.; Mest, H.-J.; Mohr, M. J.; Morin, J. M., Jr.;
Mullaney, J. T.; Nunes, A.; Paal, M.; Rapp, A.; Rühter, G.; Ruterbories, K. J.; Sall,
D. J.; Scarborough, R. M.; Schotten, T.; Sommer, B.; Stenzel, W.; Towner, R. D.;
Um, S. L.; Utterback, B. G.; Vasileff, R. T.; Vöelkers, S.; Wyss, V. L.; Jakubowski, J.
A. J. Med. Chem. 1999, 42, 4875.
Docking studies revealed that S-enantiomers of compounds 9i
and 9j bind to IIbb3 stronger than corresponding R-enantiomers.
In the case of compounds 9h and 9k, no significant differences in
values of scoring function for S and R-enantiomers was observed.
Detailed investigation of the relationship between the stereochem-
a
istry of the mimetics 9 and their affinity for
aIIbb3 will be the
purpose of future studies.
To summarize, for a series of 4-piperidine-4-yl-butyric, 4-piper-
idine-4-yl-benzoic and 1,2,3,4-tetrahydroisoquinoline-7-carbox-
ylic acids derivatives, no significant influence of Arg isostere
8. (a) Ku, T. W.; Ali, F. E.; Bondinell, W. E.; Erhard, K. F.; Huffman, W. F.;
Venslavsky, J. W.; Yuan, C. C.-K. Tetrahedron Lett. 1997, 38, 3131; (b) Atkins, R.
J.; Banks, A.; Bellingham, R. K.; Breen, G. F.; Carey, J. S.; Etridge, S. K.; Hayes, J. F.;
Hussain, N.; Morgan, D. O.; Oxley, P.; Passey, S. C.; Walsgrove, T. C.; Wells, A. S.
Org. Process Res. Dev. 2003, 7, 663.
structure on affinity for
the compounds 9d–k have demonstrated a high affinity for
and acceptable antiaggregative activity. A combination of tetrahy-
droisoquinoline and b-methyl-b-alanine as Arg and Asp bioisoster-
es, respectively, within the given phthalimidine series, leads to the
best prospective inhibitor of the platelet aggregation. It has been
a
IIbb3 was observed. At the same time,
aIIbb3
ˇ
9. (a) Anderluh, M.; Cesar, J.; Štefanic, P.; Kikelj, D.; Janeš, D.; Murn, J.; Nadrah, K.;
Tominc, M.; Addicks, E.; Giannis, A.; Stegnar, M.; Dolenc, M. S. Eur. J. Med. Chem.
2005, 40, 25; (b) Anderluh, P. Š.; Anderluh, M.; Ila, J.; Mravljak, J.; Dolenc, M. S.;
Stegnar, M.; Kikelj, D. J. Med. Chem. 2005, 48, 3110.
shown by docking studies that
a:D224, a:S225 and b:Y22,
10. Xue, C.-B.; Rafalski, M.; Roderick, J.; Eyermann, C. J.; Mousa, S.; Olson, R. E.;
DeGrado, W. F. Bioorg. Med. Chem. Lett. 1996, 6, 339.
11. Lawson, E. C.; Hoekstra, W. J.; Addo, M. F.; Andrade-Gordon, P.; Damiano, B. P.;
Kauffman, J. A.; Mitchell, J. A.; Maryanoff, B. E. Bioorg. Med. Chem. Lett. 2001, 11,
2619.
b:N215 residues of IIbb3 integrin play the key role in binding of
a
the mimetics 9 to the receptor.
Acknowledgments
12. (a) Zhuang, Z.-P.; Kung, M.-P.; Mu, M.; Kung, H. F. J. Med. Chem. 1998, 41, 157;
(b) Hamprecht, D.; Fabrizio, M.; Tedesco, G.; Checchia, A.; Donati, D.; Petrone,
M.; Terreni, S.; Wood, M. Bioorg. Med. Chem. Lett. 2007, 17, 428; (c) Norman, M.
H.; Rigdon, G. C.; Navas, F.; Cooper, B. R. J. Med. Chem. 1994, 37, 2552; (d) Luo,
W.; Yu, Q.-S.; Salcedo, I.; Holloway, H. W.; Lahiri, D. K.; Brossi, A.; Tweedie, D.;
Greig, N. H. Bioorg. Med. Chem. 2011, 19, 3965; (e) Kato, Y.; Ebiike, H.; Achiwa,
K.; Ashizawa, N.; Kurihara, T.; Kobayashi, F. Chem. Pharm. Bull. 1990, 38, 2060;
(f) Cai, S.-L.; Chen, Y.; Sun, W.-X.; Li, H.; Chen, Y.; Yuan, S.-S. Bioorg. Med. Chem.
Lett. 2010, 20, 5649; (g) Cimminiello, C.; Milani, M.; Uberti, T.; Arpaia, G.;
Bonfardeci, G. J. Int. Med. Res. 1989, 17, 514; (h) Brashear, K. M.; Cook, J. J.;
Bednar, B.; Bednar, R. A.; Gould, R. J.; Halczenko, W.; Holahan, M. A.; Lynch, R.
J.; Hartman, G. D.; Hutchinson, J. H. Bioorg. Med. Chem. Lett. 1997, 7, 2793.
13. Klein, S. I.; Molino, B. F.; Czekaj, M.; Gardner, C. J.; Chu, V.; Brown, K.; Sabatino,
R. D.; Bostwick, J. S.; Kasiewski, C.; Bentley, R.; Windisch, V.; Perrone, M.;
Dunwiddie, C. T.; Leadley, R. J. J. Med. Chem. 1998, 41, 2492.
14. Frank, H.; Guenter, L.; Volkhard, A.; Helmut, P.; Thomas, M.; Johannes, W.;
Brian, G. Eur. Patent 0604800, 1993.
15. Duggan, M. E.; Egbertson, M. S.; Hartman, G. D.; Young, S. D.; Ihle, N. C. US
Patent 5854245, 1998.
16. Malovichko, O. L.; Krysko, A. A.; Kabanova, T. A.; Andronati, S. A.; Grishkovets,
V. I.; Kachala, V. V.; Panov, D. A. Med. Chem. 2009, 5, 158.
17. Hantgan, R. Biochim. Biophys. Acta 1987, 927, 55.
The author thanks Dr. M. Kopanitsa (Cambridge, United
Kingdom) for helpful discussions and the proof reading of the
manuscript.
References and notes
1. (a) Tsikaris, V. J. Pept. Sci. 2004, 10, 589; (b) Andronati, S. A.; Karaseva, T. L.;
Krysko, A. A. Curr. Med. Chem. 2004, 11, 1183.
2. Perdih, A.; Sollner Dolenc, M. Curr. Med. Chem. 2010, 17, 2371.
3. (a) Masic, L. P.; Kikelj, D. Tetrahedron 2001, 57, 7073–7105; (b) Masic, L. P. Curr.
Med. Chem. 2006, 13, 3627; (c) Krysko, A. A.; Chugunov, B. M.; Malovichko, O.
L.; Andronati, S. A.; Kabanova, T. A.; Karaseva, T. L.; Kiriyak, A. V. Bioorg. Med.
Chem. Lett. 2004, 14, 5533; (d) Andronati, S. A.; Krysko, A. A.; Chugunov, B. M.;
Kabanova, T. A.; Artemenko, A. G. Russ. J. Org. Chem. 2006, 42, 1174; (e)
Malovichko, O. L.; Petrus, A. S.; Krysko, A. A.; Kabanova, T. A.; Andronati, S. A.;
Karaseva, T. L.; Kiriyak, A. V. Bioorg. Med. Chem. Lett. 2006, 16, 5294; (f) Krysko,
A. A.; Krysko, O. L.; Kabanova, T. A.; Andronati, S. A.; Kabanov, V. M. Bioorg. Med.
Chem. Lett. 2010, 20, 4444.
4. Stefanic, P.; Dolenc, M. S. Curr. Med. Chem. 2004, 11, 945.
18. Xia, Z.; Wong, T.; Liu, Q.; Kasirer-Friede, A.; Brown, E.; Frojmnvic, M. M. Br. J.
Haematol. 1996, 93, 204.
19. Born, G. V. R. Nature 1962, 194, 927.
20. Molecular Operating Environment (MOE), 2010.10; Chemical Computing Group
Inc.: 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2010.
21. http://dx.doi.org/10.2210/pdb2vdm/pdb.; (b) Xiao, T.; Takagi, J.; Coller, B. S.;
Wang, J.-H.; Springer, T. A. Nature 2004, 432, 59.
5. (a) Hartman, G. D.; Egbertson, M. S.; Halczenko, W.; Laswell, W. L.; Duggan, M.
E.; Smith, R. L.; Naylor, A. M.; Manno, P. D.; Lynch, R. J.; Zhang, G.; Chang, C. T.-
C.; Gould, R. J. J. Med. Chem. 1992, 35, 4640; (b) Egbertson, M. S.; Chang, C. T.-C.;
Duggan, M. E.; Gould, R. J.; Halczenko, W.; Hartman, G. D.; Laswell, W. L.;
Lynch, J. J., Jr.; Lynch, R. J.; Manno, P. D.; Naylor, A. M.; Prugh, J. D.; Ramjit, D. R.;
Sitko, G. R.; Smith, R. S.; Turchi, L. M.; Zhang, G. J. Med. Chem. 1994, 37, 2537; (c)
Cook, J. J.; Bednar, B.; Lynch, J. J.; Gould, R. J.; Egbertson, M. S.; Halczenko, W.;