Synthesis, biological evaluation and molecular docking studies of 2-piperazin-1-yl-quinazolines as platelet aggregation inhibitors and ligands of integrin αIIbβ3

Article abstract of DOI:10.1016/j.bmcl.2016.02.011

A series of 2-piperazin-1-yl-quinazolines were synthesized and evaluated for their antiaggregative activity. The synthesized small molecule compounds have potently inhibited platelet aggregation in vitro and blocked FITC-Fg binding to α_IIbβ₃integrin in a suspension of washed human platelets. The key α_IIbβ₃protein–ligand interactions were determined in docking experiments and some correlations have been observed between values of the affinity and docking scores.

Full text of DOI:10.1016/j.bmcl.2016.02.011

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and molecular docking studies
of 2-piperazin-1-yl-quinazolines as platelet aggregation
inhibitors and ligands of integrin
aIIbb3
a
b
a
Andrei A. Krysko a, , Alexander Yu. Kornylov , Pavel G. Polishchuk , Georgiy V. Samoylenko ,
Olga L. Krysko a, Tatyana A. Kabanova a, Victor Ch. Kravtsov c, Vladimir M. Kabanov a, Barbara Wicher d,
Sergei A. Andronati a
⇑
a A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Lustdorfskaya doroga 86, Odessa 65080, Ukraine
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky´ University and University Hospital in Olomouc, Hnevotínská 1333/5, Olomouc
b
ˇ
77900, Czech Republic
c Institute of Applied Physics, Academy of Sciences of Moldova, Academiei 5, Chisinau 2028, Republic of Moldova
d Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-piperazin-1-yl-quinazolines were synthesized and evaluated for their antiaggregative activ-
ity. The synthesized small molecule compounds have potently inhibited platelet aggregation in vitro and
blocked FITC-Fg binding to aIIbb3 integrin in a suspension of washed human platelets. The key aIIbb3 pro-
tein–ligand interactions were determined in docking experiments and some correlations have been
observed between values of the affinity and docking scores.
Received 10 December 2015
Revised 2 February 2016
Accepted 4 February 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Fibrinogen receptor antagonists
aIIbb3
2-Piperazin-1-yl-quinazoline
Platelet aggregation
Nowadays, it is known the leading role of platelet aggregation
in the formation of thrombus, as a consequence emerge of danger-
ous cardiovascular diseases such as unstable angina, myocardial
infarction, ischaemic disease, atherosclerosis, and stroke.1 One of
the rational approaches for treatment and prevention of cardiovas-
cular diseases associated with thrombosis is the creation of drugs
which inhibit platelet aggregation—antiplatelet agents. The final
obligatory step in the platelet aggregation is fibrinogen binding
to its receptor on the activated platelets. Over the past twenty
years, the most interesting objects among antiplatelet agents are
fibrinogen receptor antagonists.2 Key domains of fibrinogen are
batide and tirofiban (Fig. 1) which were approved for intravenous
administration during percutaneous coronary intervention and in
acute coronary syndromes.4 Crystallographic analysis of ‘RGD
mimetics–aIIbb3’ complexes showed that during the binding of a
ligand (RGD mimetic) a ‘swing-out’ motion of the b3 subunit occurs
resulting in a substantial change in conformation, namely adopting
a high-affinity ligand-binding conformation.5 It was supposed that
such conformational changes are associated with thrombocytope-
nia that occurs in some patients after treatment with classical
antagonists.6,7 The rational approach can be used to avoid the
problem of thrombocytopenia—design of antagonists that prevent
receptor activation upon binding to the intracellular domain of
RGD-sequences found on the
575) and the dodecapeptide sequence on the
QAGDV 400–411), by means of which the fibrinogen interacts to
IIbb3 integrin on the surface of activated platelets.3 In the majority
of cases, the design of IIbb3 antagonists is based on the mimicking
a-chain (RGDF 95–98, RGDS 572–
c
-chain (HHLGGAK-
a
IIbb3.8 Recently, a number of compounds was identified which
belong to the novel class of ‘ion displacement ligands’. These com-
pounds are antagonists (RUC-1, RUC-2 and RUC-4) of IIbb3 and
a
a
a
they limit conformational reorganization of the receptor and keep
it in a low-affinity state (closed form) (Fig. 1).9–11 The second part
of our previous article was devoted to the design of potential
of RGD motif, and alternative approach represents the use of dode-
capeptide sequence. These two are classic approaches. Currently
three
aIIbb3 inhibitors exist including the small molecules eptifi-
ligands for closed form of aIIbb3, we proposed three derivatives of
2-piperazin-1-yl-quinazoline based on 3D topological pharma-
cophore containing two positively charged centres separated by,
at least, the distance of 15.8 Å.12 In order to test this hypothesis,
⇑
Corresponding author.
E-mail address: peptides@physchem.od.ua (A.A. Krysko).
http://dx.doi.org/10.1016/j.bmcl.2016.02.011
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Krysko, A. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.011

2
A. A. Krysko et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
H2N
piperazin-1-yl)-6-amino-3H-quinazolin-4-one (1) was given in
the previous report.12 Condensation of acids with amine 1 has been
conducted using the HBTU or HATU. The elimination of Boc-protec-
tive groups yielded the compounds 3.
O
O
HN
O
S
O
S
H
S
O
N
HN
OH
O
N
HN
O
O
H
N
O
H
N
Tirofiban
N
H
HN
NH
Single crystals of di-Boc-derivative 2b were grown from metha-
nol, and their structure was confirmed by single crystal X-ray
diffraction analysis (Fig. 2).13 The amide fragment is about coplanar
with quinazoline moiety (dihedral angle 1.63°) but almost perpen-
dicular to the flat carbamate fragment (dihedral angle 84.27°)
resulting in angular conformation of molecule. Torsion angle
N6AC20AC19AC18 equals 60.8(3)°. In the crystal the classical
NAHÁ Á ÁO and NAHÁ Á ÁN intermolecular hydrogen bonds unite
molecules 2b in well defined layer with hydrophobic tert-butyl
groups on the surfaces of layer. The layers are stacked in parallel
fashion.
O
N
N
O
S
N
O
OH
N
HN
RUC-1
NH
O
Eptifibatide
H2N
NH2
H
N
NH2
H
HN
HN
N
N
N
O
O
S
N
N
S
O
N
N
N
N
N
RUC-2
RUC-4
O
Figure 1. Structures of small molecules—antagonists of aIIbb3.
The synthetic route to the target 4-phenyl-2-piperazin-1-yl-
quinazolines (9) is summarized in Scheme 2. 6-Nitro-4-phenyl-
1H-quinazolin-2-one (4) was obtained by heating 2-amino-5-
nitrobenzophenone and urea. Chlorination of compound 4 using
POCl3/PCl5 was carried out in order to obtain 6-nitro-2-chloro-4-
phenyl-quinazoline (5). Then the piperazinyl ring was conve-
niently introduced to the 2-position of the intermediate 5 by the
reaction of the latter with the 1-Boc-piperazine, which gave the
compound 6. The reduction of the nitro group of compound 6 using
H2–Pd(C) gave the amine 7 as a crucial substrate for the assembly
of the target molecules. Acylation of the amine and subsequent
deprotection resulted in the compounds 9.
compounds with varying distance between the positively charged
groups have been synthesized. Also, in order to find out impor-
tance of the presence of the carbonyl group in 4-position of the
quinazoline core, 4-phenyl-2-piperazin-1-yl-quinazoline deriva-
tives have been studies. Here, we report about antiaggregative
properties of the expanded set of compounds (2-piperazin-1-yl-
quinazoline derivatives) and observed structure–activity
relationships.
For the synthesis of a set of 2-(piperazin-1-yl)-3H-quinazolin-4-
one derivatives (3), the following synthetic route was applied as
outlined in Scheme 1. Description of the synthesis of 2-(4-Boc-
O
O
NH+
N
O
2 Cl
N
O
i
ii
N
O
N
N
O
N
N
O
N
O
O
NH
NH
NH
3a
H2N
N
H
2a
N
H
O
1
iii
NH+
N
O
2 Cl
O
N
O
N
N
O
N
ii
O
Aa
NH
Aa
HCl*H
NH
3b-m
N
N
H
H
2b-m
O
O
H
H
N
H
O
H
N
N
,
N
,
,
Aa
,
N
,
=
e
b
c
d
f
O
O
O
O
O
N
N
O
N
,
,
,
N
g
i
h
H
O
O
H
O
O
O
N
HN
O
HN
O
HN
O
H
N
O
H
H
,
,
N
,
N
O
m
O
l
j
k
O
Scheme 1. Synthesis of compounds 3. Reagents and conditions: (i) NEt3, propionic acid anhydride, ACN, 50 °C, overnight, 40%; (ii) CH2Cl2, HCl gas, room temperature, 1 h, 90–
96%; (iii) Boc-acids, NEt3, HBTU or HATU, ACN, 50 °C, overnight, 30–58%.
Figure 2. The X-ray crystal structure of compound 2b.
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A. A. Krysko et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
H
NH2
O
N
O
N
Cl
N
O
ii
iii
i
N
N
N
N
O2N
O2N
O
O2N
N
4
5
6
O2N
O
iv
N
O
N
N
NH+
N
O
2 Cl
N
N
N
N
N
H2N
O
O
7
N
N
vi
N
H
N
H
9a
8a
v
O
N
O
NH+
2 Cl
O
N
N
N
N
vi
Aa
Aa
Aa
O
N
HCl*H
N
N
H
N
H
9b-e
8b-e
O
O
H
N
H
H
N
H
N
,
N
,
,
=
e
d
b
O
c
O
Scheme 2. Synthesis of compounds 9. Reagents and conditions: (i) (H2N)2CO, 150 °C, 3 h, 7%; (ii) POCl3, PCl5, 100 °C, 4 h; (iii) 1-Boc-piperazine, NEt3, ACN, 50 °C, 2 h, 87%, (iv)
H2/Pd(C), MeOH, room temperature, 3 h, 90%; (v) Boc-acids, NEt3, HBTU, ACN, 50 °C, overnight, 43–50%; (vi) CH2Cl2, HCl gas, room temperature, 1 h, 93–97%.
Functional inhibitory activity of synthesized compounds 3 and
9 were evaluated by measuring the inhibition of ADP induced pla-
telet aggregation in human platelet-rich plasma (PRP) by Born’s
method.14 All results of ADP induced platelet aggregation assay
are listed in Table 1.
First, we have focused on the optimization of 3 by structural
modification of the amide group in 6-positions of 2-(piperazin-1-
yl)-3H-quinazolin-4-one scaffold. The compound 3a, that we have
accepted as an analogue of RUC-1 showed IC50 = 0.17 lM. For bind-
ing to b3-subunit ligands have to bear a second amino group. On
Table 1
Biological properties of compounds 3, 9 and tirofiban
H
H
H2N+
N
N
N
N
N
Cl H2N+
Cl
N
N
R
R
N
H
O
3
9
Compound
R
IC50
,
l
M (PRP)a IC50
,
l
M (FITC-Fg/
a
IIbb3)b Compound
R
IC50
,
l
M (PRP)a
IC50
, lM (FITC-Fg/a
IIbb3)b
O
O
O
HN
O
Cl H3N+
3a
0.17 0.02
0.055 0.004
0.0050 0.000812
0.0022 0.000312
—
3j
0.06 0.003
0.040 0.006
0.019 0.002
—
H3N+
O
O
Cl
Cl
Cl
HN
3b
3c
3d
0.15 0.02512
0.011 0.00112
1.40 0.17
3k
3l
0.0018 0.0002
O
H3N+
O
O
O
O
H3N+
Cl
HN
H3N+
0.00153 0.00006
O
Cl H3N+
O
O
H3N+
Cl
O
3m
0.0100 0.0008 0.00157 0.00011
HN
O
O
O
H3N+
Cl
3e
3f
1.30 0.17
—
9a
9b
0.72 0.07
—
—
H2N+
H3N+
Cl
Cl
Cl
0.100 0.01512
0.0038 0.000312
0.57 0.071
O
O
O
O
H3N+
H3N+
Cl
3g
3h
1.9 0.3
—
—
9c
0.049 0.006
—
Cl H3N+
O
O
H2N+
Cl
0.57 0.08
9d
9e
48.0 6.0
58.0 6.0
—
—
O
O
H2N+
Cl
H3N+
Cl
3i
1.3 0.1
0.020 0.005
N
Tirofiban
0.030 0.003
0.0019 0.0005
a
Concentration required to reduce ADP-induced human platelet aggregation response by 50%. The IC50 values are expressed as the average of at least three determinations.
Concentration required to reduce binding of FITC-Fg to aIIbb3 on the suspension of washed human platelets by 50%. The IC50 values are expressed as the average of at least
b
three determinations.
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4
A. A. Krysko et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3c
3b
3l
3h
9b
Figure 3. Binding of 3b, 3c, 3h, 3l and 9b to aIIbb3 integrin estimated by molecular docking (aIIb chain is marked by pink surface, b3 by green surface).
fragment with a residue of isonipecotic acid has led to the com-
pound 3f. However, antiaggregation activity of the compound 3f
is comparable to 3b and is an order of magnitude less than that
for the compound 3c. Use of 4-aminomethylbenzoyl group pro-
vides a negative effect on antiaggregative activity, so IC50 value
for 3g is the smallest one in a series of 2-(piperazin-1-yl)-3H-
quinazolin-4-ones. Two compounds 3h and 3i, containing residues
of 4-(piperidin-4-yl)benzoic and 4-(piperazine-4-yl)benzoic acids,
respectively, showed low in vitro antiaggregative activity. It should
be noted that we observed a significant increase in in vitro activity
upon introduction of a substituent in
fragment.
a-position of b-alanine
Addition of ethoxycarbonylamino group to the b-alanine resi-
due led to the 2.5 times increase in the activity of analogue 3j in
comparison with the compound 3b. Changing from ethyl to iso-
butyl in 3j produced the analogue 3k with equivalent activity,
and replacement with benzyl group (analogue 3l) gave an increase
in the activity of about 2 times. Homologation of compound 3l
resulted in increased antiaggregative activity for analogue 3m con-
taining a residue of 4-amino-2-benzyloxycarbonylaminobutyric
acid. In addition, the value for antiaggregation activity of com-
Figure 4. Plot of docking score vs. measured values of affinity for
compounds 3a–c,f,i,k,l.
aIIbb3 integrin of
pound 3m (IC50 = 0.01 lM) was slightly higher than the value for
compound 3c. Finally, the replacement of an oxo group with phe-
nyl in the heterocyclic scaffold has been done.
the base of this concept, compounds 3a–m have been synthesized
and studied. The compound 3b, containing a residue of b-alanine,
showed an insignificant increase of antiaggregative activity com-
pared to the compound 3a. Change from b-alanine residue to the
All 2-(piperazin-1-yl)-4-phenylquinazoline derivatives (9)
showed lower antiaggregative activity as compared to the sub-
group of compounds 3 containing a 2-(piperazin-1-yl)-3H-quina-
zolin-4-one scaffold. However it is important to note, that
influence of the structure of substituents in the 6-position on the
activity remained the same. Although activity of the compound
9c is in the desired range, but the value is less than that of its direct
analogue 3c.
c
-aminobutyric acid residue leads to the more active compound
3c. Interestingly, the compounds 3d and 3e, containing residues
of d-aminovaleric or -aminocaproic acid, respectively, exhibit sig-
nificantly lower antiaggregative activity. Replacement of b-alanine
e
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A. A. Krysko et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Mode of action for randomly selected representatives of com-
pounds 3 was subsequently determined in vitro by assessment of
their ability to inhibit the binding of fluorescein isothiocyanate-
For compounds with measured affinity for
a
IIbb3 integrin the
reasonable correlation between those values and docking scores
is observed with values of Pearson correlation coefficient À0.81
and Kendall correlation coefficient À0.79 (Fig. 4).
labelled fibrinogen (FITC-Fg) to
aIIbb3 (in a suspension of human
washed platelets). Tirofiban was used as a positive control.
In summary, analysis of the structure–activity relationship of
Molecular docking of synthesized compounds in the pocket of
IIbb3 integrin (PDB code: 3T3M) was performed with PLANTS
the series of studied
tance of the optimal distance between the two positively charged
centres of ligands. The -aminobutyric derivative 3c is more potent
than b-aminopropionic derivative 3b and much more potent than
d-aminovaleric or -aminocaproic derivatives 3d and 3e, respec-
tively. Introduction of substituent in -position of b-aminopropi-
onic or -aminobutyric fragment has a positive impact on the
antiaggregative activity and affinity for IIbb3. Docking studies
aIIbb3 antagonists demonstrated the impor-
a
docking software.15,16 The SPORES utility was used for protonation
of the protein and all ligands. In our previous study it was shown
that water molecules near Asp232 can be important for proper ori-
entation of ligands inside the binding pocket.12 Therefore these
two water molecules were remained. In the docking setup these
water molecules kept only rotational degrees of freedom and were
replaceable. Self-docking of X-ray ligand RUC-2 showed good
reproducibility of its pose (RMSD = 1.01 Å).
c
e
a
c
a
revealed that ligand–protein interactions of studied compounds
mainly correspond to those observed in the crystal structure of
Docking of the investigated compounds 3a–m and 9a–e
revealed their most probable binding poses and corresponding
ligand–protein interactions. The main anchor residues in the
the complex RUC-2–aIIbb3 and water molecules may participate
in these interactions.
pocket of
aIIbb3 integrin in its closed from are
aIIbAsp224 and
Acknowledgments
b3Glu220 to which ligands bind with positively charged groups.
All investigated compounds except 9d bind to the former residue
and just seven of them bind to the latter one mainly due to the
too short distance between two positively charged groups in
ligands or conformational constraints. However, almost all com-
pounds bind near the b3Glu220 residue and thus probably may
compete with Mg2+ ion for binding with b3 chain of the integrin.
Only two compounds cannot adopt the binding pocket due to their
size and conformational rigidity, 3h and 3i. The docking pose of the
compound 3h is shown in Figure 3. Other residues mainly involved
in ligand–protein interactions are Phe160, Ser161, Tyr190, Leu192,
Phe231 of the aIIb chain and Asn215, Arg216, Asp217 of the b3
chain.
Selected water molecules participate in binding of almost all
ligands 3 and coordinate mainly carbonyl group of quinazoline
ring. However, the compounds 9 are not coordinated with the
receptor with the participation of two water molecules. This may
explain low antiaggregative values for the subgroup 9. As it was
found in our previous study12 ligands can adopt either orientation
relative to the binding pocket, cf. poses of 3b and 3c on Figure 3
(piperazine group can bind either aIIb or b3 chain of the integrin).
It should be noted that one of the most active compounds, 3l, is ori-
ented in the ‘opposite’ direction and its benzyl group is outside the
binding pocket (Fig. 3).
This work was financially supported by the Bilateral Moldova–
Ukraine Project 14.820.18.04.05/U and No M/161-2014. Authors
also thank reviewers whose comments and remarks helped us to
significantly improve the manuscript quality.
References and notes
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Cambridge Crystallographic Data Centre (CCDC1437058) and can be obtained
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Compounds bearing a phenyl substituent in quinazoline ring
(9a–e) have similar docking poses relatively to compounds with
carbonyl group at the same position (3a–e) (Fig. 3). The size of
the binding pocket is quite large and contains hydrophobic resi-
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dues
aIIbPhe160 and aIIbPhe231 involved in the ligand–protein
interactions that probably could explain the similarity of ligands
of these two groups in binding modes.
Please cite this article in press as: Krysko, A. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.02.011