Synthesis and evaluation of platelet aggregation inhibitory activity of some 3-phenyl-pyrroloquinazolinones

Article abstract of DOI:10.1016/j.ejmech.2011.12.026

A series of 3-phenyl-2,7-dihydro-1H-pyrrolo[3,2-f]quinazolin-1-one derivatives (3-PPyQZ) was synthesized starting from 5-amino-indoles, via condensation with N-ethoxycarbonylthiobenzamides followed by thermal cyclization. On the basis of their structural analogy with reported anti-thrombin pyrroloquinazolines, the derivatives were first tested for their capacity to inhibit platelet aggregation. Some of them had in vitro inhibitory effects on collagen and thrombin-induced aggregation in the micromolar range, and much higher inhibition than that shown by some phenyl-pyrroloquinolinones. Experiments to determine the mechanism of action of the most potent inhibitor (compound 18) indicated that it acts in at least two sites: one preceding the agonist-induced increase of cytosolic [Ca²⁺], and one following this step of the platelet activation cascade. The compound also inhibited thrombin-evoked protein-Tyr-phosphorylation. Although it is premature to draw definitive conclusions, the present results indicate that 3-PPyQZ structure, with the quite potent inhibitor of platelet aggregation compound 18, might constitute a starting point for the synthesis of potential anti-thrombosis agents.

Full text of DOI:10.1016/j.ejmech.2011.12.026

European Journal of Medicinal Chemistry 48 (2012) 275e283
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of platelet aggregation inhibitory activity of some
3-phenyl-pyrroloquinazolinones
,
b
b
Maria Grazia Ferlin a , Christian Borgo , Renzo Deana
*
a Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2011
Received in revised form
14 December 2011
Accepted 16 December 2011
Available online 23 December 2011
A series of 3-phenyl-2,7-dihydro-1H-pyrrolo[3,2-f]quinazolin-1-one derivatives (3-PPyQZ) was synthe-
sized starting from 5-amino-indoles, via condensation with N-ethoxycarbonylthiobenzamides followed
by thermal cyclization. On the basis of their structural analogy with reported anti-thrombin pyrrolo-
quinazolines, the derivatives were first tested for their capacity to inhibit platelet aggregation. Some of
them had in vitro inhibitory effects on collagen and thrombin-induced aggregation in the micromolar
range, and much higher inhibition than that shown by some phenyl-pyrroloquinolinones. Experiments to
determine the mechanism of action of the most potent inhibitor (compound 18) indicated that it acts in
at least two sites: one preceding the agonist-induced increase of cytosolic [Ca2þ], and one following this
step of the platelet activation cascade. The compound also inhibited thrombin-evoked proteineTyr
ephosphorylation. Although it is premature to draw definitive conclusions, the present results indi-
cate that 3-PPyQZ structure, with the quite potent inhibitor of platelet aggregation compound 18, might
constitute a starting point for the synthesis of potential anti-thrombosis agents.
Keywords:
Phenyl-pyrroloquinazolinones
Synthetic methods
Platelet aggregation
Cytosolic Ca2þ concentration
Tyrosine phosphorylation
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
quinazolinone moiety, comprise different structures, (depending
on the fused sides), both natural (e.g., vasicine and its keto-analog
Heterocycle quinazoline (Fig. 1), a bicyclic fused benzene and
pyrimidine structure, is known to be a very interesting and useful
scaffold, as it can be suitably modified with a variety of chemicals in
order to obtain pharmacologically active compounds [1]. Quina-
zolinones are products deriving from oxidation of quinazolines, and
like the latter, have attracted much attention due to their wide
range of pharmacological properties [2]. Among the quinazolinone
structures (Fig. 1), the most frequently encountered in medicinal
chemistry is that of 4(3H)-quinazolinone, which has turned out to
be particularly important, both as a synthetic intermediate [3] and
as a natural product [4]. Its derivatives have been reported to
possess various biological applications including antimicrobial [5],
analgesic and anti-inflammatory [6,7], anti-tubercular [8], anti-
malaria [9] and anti-cancer [10] activities.
vasicinone [11], and the luotonine family [12]) and synthetic ones
[13,14].
Recently, continuing a research line aiming at synthesizing and
studying anticancer compounds [15], some 3-phenyl-2,7-dihydro-
1H-pyrrolo[3,2-f]quinazolin-1-ones
(3-PPyQZs)
have
been
prepared in our laboratory as analogs of antimitotic 7-phenyl-
pyrrolo[3,2-f]quinolin-9-ones (7-PPyQs) modified at position 8 by
replacing CH with N (Fig. 1) [16]. The 3-PPyQZs assayed against
a panel of human cancer cell lines did not show significant cyto-
toxicity (data not shown). As 3-PPyQZs share the tricyclic nucleus
with some amino-PyQZs having anti-thrombin activity [17e19], we
thought it would be interesting to evaluate their potential inhibi-
tory activity on platelet aggregation. Inhibition of platelet aggre-
gation in humans is predicted to be beneficial in thrombotic
disorders, and safer and resistance-free new drugs are needed in
this field. Four 3-PPyQZs were tested, one unsubstituted at pyrrolic
N (18) and three bearing alkyl groups (19, 21 and 22) at this position
(Fig. 2). In this screening, we also included four previously
described non-cytotoxic PPyQs 24e27 [15,20] belonging to the two
isomeric [3,2-f] and [2,3-h] structures (Fig. 2). These derivatives
were selected because they share the 3-phenyl-quinolin-4-one (3-
PQ) moiety with some 3-PQs reported to have anti-platelet activity
Pyrroloquinazolines and pyrroloquinazolinones (PyQZs),
consisting of
a pyrrole ring fused to the quinazoline or
Abbreviations: PPyQ, phenyl-pyrroloquinolinones; 3-PPyQZ, 3-phenyl-2,7-
dihydro-1H-pyrrolo[3,2-f]quinazolin-1-ones; ACD, citric-anticoagulant; PMA,
phorbol myristate acetate; PKC, protein kinase C; LDH, lactate dehydrogenase.
* Corresponding author. Tel.: þ390498271603; fax: þ390498275366.
E-mail address: mariagrazia.ferlin@unipd.it1 (M.G. Ferlin).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.026

276
M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
O
2.1.1. Synthesis
O
The new 3-PPyQZs were obtained from 5-amino-indole but
N
NH
N
NH
applying another previously reported method for 3-PQZs [23,24].
First, N-substituted nitroindoles 2e4 (Scheme 2) were prepared by
reaction with the adequate bromoalkane followed by catalytic
hydrogenation to give the corresponding aminoindoles 5e8,
according to previously described procedures [16]. Aminoindole
derivatives were then condensed with N-ethoxycarbonylth-
iobenzamides 11 and 12. These were prepared in parallel by means
of FriedeleCrafts thioacylation of 9 and 10 with ethoxycarbonyl
isothiocyanate in the presence of anhydrous AlCl3 [24]. The ob-
tained N-indole-N0-ethoxycarbonylamidine compounds 13e17
were then cyclized in boiling diphenyl ether to final pyrroloqui-
nazolinones 18e22 (Scheme 2A).
1
1
1
N
1
N
N
O
N
O
H
H
quinazoline
quinazolin-4(3H)-one
quinazolin-2(1H)-one
quinazoline-2,4(1H,3H)-dione
O
O
HN
NH
NH
N
H
N
PyQZ
PyQ
Fig. 1. Structures of heterocycles mentioned in the text.
The general structure of the amidine intermediates 13e17 is
supported by their IR spectra, which contain an absorption band at
1660e1680 cmꢀ1 consistent with a conjugated carbonyl group
[21]. We describe here the synthesis of these new 3-PPyQZs and
preliminary results of a study assessing their anti-platelet activity
and mechanism(s) of action.
(unsaturated
a
,
b
ꢀamide), as well as 1H NMR signals corresponding
to the protons of the three aromatic rings and their substituents.
The exact structure of the newly synthesized PPyQZs was
confirmed by 1-D and 2-D experiments. The 1H NMR (CDCl3)
spectrum of compound 19, taken as a sample, showed a singlet at
2. Results and discussion
d
12.31 which, by analogy with literature data, was assigned to the
2.1. Chemistry
proton in the 2 position (2-HN). Confirmation of this inference and
complete structure assignments were achieved by 13C NMR, HMQC
and HMBC experiments. HMBC experiments revealed correlations
within a distance of 2e3 CeH bonds (Fig. 3A, blue arrows). Diag-
nostic HMBC correlations were observed between the proton
Two synthetic pathways leading to the pyrroloquinazoline
molecular structure have been described: one, more complicated,
exploits the well-established anthranilic acid pathway to quina-
zoline, and PyQZs are then obtained by various methods for con-
structing the indole nucleus [14]; the second starts from
aminoeindole and, via a condensation reaction with sodium
dicyanamide, yields pyrrolequinazolines (Scheme 1) [22].
signals at
d
4.36 (CH3eCH2e) and carbon resonances at
8.03 (6-H) and carbons
d 123.7 (9a-C), 144.8 (4a-C). Further correlations were observed
d 129.8 (8-
C) and 133.1 (6aeC) and from the signal at
d
at
R2
R2
O
1
O
O
1
N
N
R1
R1
NH
f
f
h
1
NH
8-PPyQs
R1
R1
N
NH
N
3-PPyQZs
3-PPyQs
R2
R1
H
19 C2H5
R2
H
H
H
R2
H
R1
H
27 C2H5
R2
H
H
18
24
H
26
25 C2H5
H
21
-CH2
22 C2H5
OCH3
Fig. 2. Structures of tested compounds.
O
O
O
C
NO2
NH2
NH
H2N
HN
HN
R1
1
B
reagents
B
A
N
B
A
R1
H2N
N
quinazolinone derivative
PyQZs
R2
3
C
NR
H2N
N
A
C
NaN(CN)2
B
B
1
N
R1
N
H
aminoindole
pyrroloquinazolines
Scheme 1. Previously reported two pathways leading to pyrroloquinazoline derivatives.

M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
277
H2N
O2N
O2N
b
a
R2
N
COOC2H5
N
N
N
C
R1
H
R1
HN
1
5-8
2-4
d
N
R1
S
13-17
C
NHCOOC2H5
c
e
SCNCOOC2H5
R2
R2
O
11, 12
9, 10
N
R1
HN
N
R2
18-22
compounds
5, 13, 18
2, 6, 14, 19
3, 7, 15, 20
4, 8, 16, 21
9, 11
R1
H
R2
Reagents and conditions:
H
H
a) R-Br, NaH, toluene, reflux, 2-24 h;
b) H2, Pd/C 10%, 30-40°C, 4-14 h;
c) AlCl3, 0°C, 4 h;
C2H5
C3H7
H
CH2-cyclopropyl
H
d) absolute ethanol, reflux, 6-8 h;
e) diphenyl ether, 250°C, 15 min;
---
-
H
---
10, 12
-
OCH3
OCH3
17, 22
C2H5
Scheme 2. Synthetic route to phenyl-pyrroloquinazolinone derivatives 18e22.
from the signal at
correlations from 20- and 60-H of the side phenyl ring (
observed with carbon at 149.6 (3-C). In the NOESY spectrum
(Fig. 3A, red arrows) correlations were observed between signal at
4.36 (CH3eCH2e) and proton signals at 7.64 and 8.03, confirming
the aliphatic side-chain position and allowing the signal (t,
J ¼ 3.1 Hz) at 7.64 to be assigned to the proton in the 8 position
d
7.32 (9-H) and carbon at
d
117.5 (6-C). HMBC
and the doublet of doublets (J ¼ 8.8 and 0.6 Hz) at
d
8.03 to the
d
8.22) were
proton in the 6 position. In addition, the NOESY spectrum showed
only one correlation between the signal at 12.31 (2-HN) and the
multiplet at
8.22 (integral two protons), due to the 20- and 60-H of
d
d
d
d
d
the side phenyl ring in the 3 position (Fig. 3B). In the case of the
tautomer in which the proton is on the 4-N, two NOESY correlations
should be observed, one between 4-H and the 2- and 6-H of the
side phenyl ring, and the other between 4-H and 5-H.
d
2.2. Biology
The effect of 3-PPyQZs 18e22 and PPyQs 24e27 on platelet
aggregation induced by thrombin is shown in Fig. 4. Compound 20
was not included in the analysed list because of its poor solubility in
the test solvent. The concentration of the PPyQZs (8
mM) was
chosen after some preliminary experiments with a range of various
PPyQZ concentrations. Fig. 4 shows that, in the experimental
conditions adopted, compound 18 caused complete inhibition of
thrombin-induced platelet aggregation, whereas compounds 19, 21
and 22 displayed lower inhibitory action and PPyQs very little
inhibition on this process. Similar results were also obtained when
the platelets were stimulated with collagen (data not shown).
Thrombin and collagen were selected as agonists, since they are
considered two of the most important platelet-stimulating agents
[26e28]. As 18 turned out to be the strongest inhibitor of platelet
aggregation, it was chosen for the following experiments, to
elucidate its mechanism of action. Fig. 5 shows that 18, in the
experimental conditions adopted, inhibited thrombin-induced
platelet aggregation in
causing complete inhibition at a concentration above 4
M. Inhibition was only slightly less in aspirin-
treated platelets, indicating that 3-PPyQZ had only a slight effect
on the cyclo-oxygenase level.
a
concentration-dependent manner,
mM with an
Fig. 3. (A) HMBC (blue arrows) and NOESY (red arrows) main correlations for
compound 19; (B) NOESY correlations at aromatic zone. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
IC50 of 1.79 ꢁ 0.28
m

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M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
Fig 5. Inhibition of thrombin-induced platelet aggregation caused by increasing
concentrations of compound 18. Aggregation extent (magnitude) in untreated
(Control) or aspirin-treated platelets, was measured 10 min after thrombin-addition
(40 mU/mL), which was added at the reported concentrations 3 min after 3-PPyQZ
18. Acetylsalicylic acid treatment was performed as described in the methods
section. Data are means of 4 experiments with S.E.M. indicated by the vertical bars.
as well as by collagen, another physiological agonist. The results
reported in Fig. 6 show that the compound 18 inhibited the platelet
aggregation promoted by collagen, on the one hand, slightly more
efficiently than that promoted by thrombin (Fig. 5), and on the
other hand, more strongly compared to that elicited by the artificial
activators ionomycin or phorboleester. The latter results suggest
that the compound acts at least at two sites: one preceding the
agonist-induced cytosolic Ca2þ increase and protein-kinase acti-
vation and one following these events.
The action of 18 was then tested on the thrombin-induced
[Ca2þ
] increase, which is considered as the pivotal step of the
c
platelet activation cascade [26,30]. Compound 18 alone caused
a slight increase in [Ca2þ]c and a large increment in the [Ca2þ]c rise
produced by thrombin added 1 min after 3-PPyQZ (Fig. 7A). Instead,
18 caused a lowering of the [Ca2þ
] increase caused by thrombin
c
added at least 3 min after the pyrroloquinazolinone (Fig. 7B).
In order to determine whether 3-PPyQZ exerted its action
upstream of the thrombin-elicited intracellular Ca2þ movement,
i.e., endoplasmic reticulum Ca2þ release, or on the subsequent entry
of Ca2þ from the extracellular compartment [31], we carried out the
above experiments with platelets suspended in a medium virtually
devoid of Ca2þ, i.e., in the presence of the extracellular Ca2þchelator
EGTA. As expected under these conditions, the amplitude of the
thrombin-induced [Ca2þ]c increase was less than that observed in
the presence of external calcium (Fig. 8A and B), although the
effects of 18 were substantially similar to those obtained with
platelets suspended in a calcium-containing medium (Fig. 7A and
B). These results indicate that 3-PPyQZ exerts its action mainly
upstream of the intracellular Ca2þ movement from the endo-
plasmic reticulum to the cytosolic compartment.
Fig. 4. Inhibitory effect of the new 3-PPyQZs 18e22 and the PPyQs 24e27 on
thrombin-induced platelet aggregation. (A): representative traces of the experiments
of platelet aggregation induced by thrombin (Th) (40 mU/mL), added at the arrow,
3 min after the 3-PPyQZs or PPyQ (8
aggregation rates (velocities) of aggregation induced by thrombin (40 mU/mL) added
3 min after the indicated compounds (8 M). Results are means of four separate
mM) or vehicle (DMSO, Control) (B): initial
m
determinations with the S.E.M. indicated by vertical bars.
Platelet activation may also be caused by non-physiological
agonists such as calcium ionophores, which give rise to an artifi-
cial increase in cytosolic Ca2þ concentration ([Ca2þ]c), or protein-
kinase C (PKC) activators, such as phorboleesters [29]. The
concentration-dependent inhibitory effect of 18 was also studied
As on one hand protein phosphorylation is an outstanding event
associated with platelet activation [32e34] and, on the other, it has
been reported that the anti-thrombotic properties of certain
on platelet aggregation elicited by the non-physiological Ca2þ
-
ionophore ionomycin and the PKC activator phorboleester (PMA),

M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
279
Fig. 6. Effect of 18 on platelet aggregation induced by collagen, or the cal-
ciumeionophore ionomycin, or the PKC activator phorboleester (PMA). The indicated
concentrations of 18 (or equivalent volume of the vehicle DMSO) were incubated for
3 min and then 40 mg/mL collagen, or 20 nM PMA or 0.5 mM ionomycin were added.
The maximal aggregation rate measured after the agonist addition was considered
100% in the absence of 3-PPyQZ. Data are means of 4 experiments with S.E.M. indicated
by the vertical bars.
pyrroloquinazolines are probably due to their inhibitory effect on
proteinetyrosineekinases [14,35], we investigated the effect of 3-
PPyQZs on the levels of proteinetyrosine-phosphorylation in
resting and thrombin-stimulated platelets. Fig. 9 shows that 18
added to resting platelets did not significantly alter the degree of
tyrosine phosphorylation, but did produce a marked inhibition in
the phosphorylation pattern obtained after thrombin stimulation.
The inhibitory effect was particularly evident on proteins of about
58 and 80 kDa, the chemical nature of which is still undetermined.
Lastly, we analysed the possible damaging effects of 3-PPyQZs on
platelet membrane integrity by monitoring the release of lactate
dehydrogenase (LDH) [36] from platelets incubated with the
compound. No appreciable release of LDH was found after platelet
incubation for 1 h with 4 mM 18 (data not shown). We also moni-
tored the effect of this compound on platelet viability determined
by the Alamar Blue method [37]. At the concentration used, 18
caused only a slight decrease in cellular viability after 4e6 h of
incubation (not shown).
Fig. 7. Cytosolic [Ca2þ] changes produced by thrombin in platelets preincubated with
vehicle or the 3-PPyQZ 18. (A): traces of [Ca2þ]c increase induced by addition (at the
arrow) of 40 mU/mL thrombin 1 min after the 3-PPyQZ 18 (2 mM) or an equivalent
volume of DMSO (control). (B): changes of [Ca2þ]c produced by thrombin added 3 min
3. Conclusions
after 18. Traces are representative of three separate experiments.
Two small series of tricyclic compounds, the newly synthesized
PPyQz 18e22 and the previously reported PPyQ 24e27 derivatives
were evaluated as anti-aggregation agents.
None of the tested PPyQ derivatives at the concentration
adopted (8 mM), showed significant inhibitory effects on thrombin-
induced platelet aggregation. This means that enlargement of the
3-phenylequinolinone model structure by fusing a pyrrole ring is
detrimental for anti-platelet activity. At the same concentration
substitutions on the 3-PPyQZ scaffold are unfavorable for anti-
platelet activity, as unsubstituted compound 18 is the most
active. Regarding the alkyl substitutions at pyrrolic N, both ethyl
and cycloprolylmethyl groups, cause a decrease of the inhibitory
action of the compounds 19, 22 and 21 in comparison with
compound 18, the decrease of inhibitory effect being greater for 21
with the bulky cyclopropylmethyl group (Fig. 4B). Moreover, the
methoxylic group on the side phenyl ring causes a further decrease
(8
mM), 3-PPyQZs had interesting inhibitory effects, although to
different extents. In general, it seems that the here made

280
M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
Fig. 9. Effect of 18 on protein tyrosine phosphorylation of resting and thrombin-
stimulated platelets. Anti-P-Tyr immunostaining of proteins from platelets pre-
treated with vehicle, (DMSO, control) or 2 M 18 for 3 min and then stimulated
with thrombin (40 mU/mL). Mass molecular markers were used for the determination
of actin apparent molecular masses of the proteic bands. Anti immunoblotting was
m
b
performed in parallel to control the amounts of proteins loaded in the SDS/PAGE lanes.
Blots are representative of four experiments performed with different platelet samples.
Thefinding that compound18 inhibitedthe [Ca2þ]c increase induced
by physiological agonists, even in the absence of extracellular
calcium, indicates that it acts at a level preceding the agonist-
induced increase in cytosolic [Ca2þ] deriving from the endo-
plasmic reticulum. In addition, the inhibition displayed on aggre-
gation elicited by compounds which caused direct increases in
[Ca2þ]c or protein-kinase activity indicates that the compound also
acts downstream of the agonist-induced increase in cytosolic [Ca2þ].
It is still difficult to establish whether the inhibitory effect caused by
compound 18 on thrombin-elicited proteineTyr phosphorylation is
due to direct action on kinase/phosphatase enzymes or is a conse-
quence of the reduced rise in [Ca2þ]c. In conclusion, the present
results indicate that 3-PPyQZ structure, with the quite potent
inhibitor of platelet aggregation compound 18, might constitute
astarting point for thesynthesis ofpotentialanti-thrombosisagents.
Fig. 8. Thrombin-induced [Ca2þ]c increase in platelets pre-incubated with vehicle or
the 3-PPyQZ 18 in the absence of extra-cellular calcium. (A) and (B): traces of [Ca2þ]c
increase induced by thrombin-addition (40 mU/mL at the arrow) 1 min or 3 min
4. Experimental
4.1. Materials and methods
respectively after 18 (2 mM) which, in turn, was added 1 min after 0.5 mM EGTA. Traces
are representative of three separate experiments.
Melting points were determined on a Gallenkamp MFB 595
010M/B capillary melting point apparatus, and are uncorrected.
Infrared spectra were recorded on a PerkineElmer 1760 FTIR
spectrometer with potassium bromide pressed disks; all values are
expressed in cmꢀ1. 1H NMR spectra were determined on Bruker 300
and 400 MHz spectrometers, with the solvents indicated; chemical
in the inhibitory activity (22 vs 19). As 21 is the least active of all
compounds, it is clear that aliphatic bulky groups at pyrrolic N
should be avoided anyway (Fig. 4B).
However, in contrast with what has previously been described
for some pyrroloquinazolines [18,38], compound 18 does not
specifically interfere with the thrombin receptor PAR-1. In fact, the
inhibitory effect of 3-PPyQZ is similar in platelet aggregation
induced by PAR-interacting thrombin or PAR-independent collagen.
shifts are reported in
d (ppm) downfield from tetramethylsilane as
internal reference. Coupling constants are given in Hertz. In the
case of multiplets, chemical shifts were measured starting from the
approximate center. Integrals were satisfactorily in line with those

M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
281
expected on the basis of compound structure. Elemental analyses
were performed in the Microanalytical Laboratory, Department of
Pharmaceutical Sciences, University of Padova, on a PerkineElmer
C, H, N elemental analyzer model 240B, and analyses indicated by
the symbols of the elements were within ꢁ0.4% of the theoretical
values. Mass spectra were obtained on a Mat 112 Varian Mat Bre-
men (70 ev) mass spectrometer and Applied Biosystems Mariner
System 5220 LC/Ms (nozzle potential 250.00). Column flash chro-
matography was performed on Merck silica gels (250e400 mesh
ASTM); chemical reactions were monitored by analytical thin-layer
chromatography (TLC) on Merck silica gel 60 F-254 glass plates.
Solutions were concentrated on a rotary evaporator under
reduced pressure. Starting materials were purchased from Aldrich
Chimica and Acros, and solvents from Carlo Erba, Fluka and Lab-
Scan.
gel Flash Chromatography (eluant ethyl acetate/n-hexane 8:2).
Yield 40%; mp 151e155 ꢂC; rf 0.76 (ethyl acetate/n-hexane 8:2); IR
nmax (KBr): 1662 (C]O) cmꢀ1 1H NMR (DMSO-d6):
; d 0.95 (t, 3H,
J ¼ 7.1 Hz; C(O)OCH2CH3), 3.82 (q, 2H, J ¼ 7.1 Hz, C(O)OCH2CH3),
6.41e7.91 (m, 10H, Ar), 9.77 (s, 1H, N]CeNH), 11.06 (bs, 1H, NH).
4.1.2.1. Sinthesis of (E,Z)-ethyl(1-ethyl-1H-indol-5-ylamino)(phenyl)
methylenecarbamate (14)
The synthesis of compound 14 was carried out by the same
procedure as described for 13 by reaction of aminoindole derivative
6 (1.6022 g, 10 mmol) and 11 (2.0926 g, 5 mmol). Yield 50%; mp
148e150 ꢂC; rf 0.80 (ethyl acetate/n-hexane 8:2); IR nmax (KBr):
1679 (C]O) cmꢀ1
C(O)OCH2CH3,), 1.35 (t, 3H, J ¼ 7.2 Hz, NCH2CH3), 3.82 (q, 2H,
J ¼ 7.1 Hz, C(O)OCH2CH3), 4.18 (q, 2H, J ¼ 7.2 Hz; NCH2CH3),
6.41e7.92 (m, 10 H, Ar), 9.77 (bs, 1H, N]CeNH).
;
1H NMR (DMSO-d6):
d
0.94 (t, 3H, J ¼ 7.1 Hz;
Apyrase, prostacyclin, a-thrombin and sodium p-nitrophenyl-
phosphate (pNPP) were purchased from Sigma Chemicals (St. Louis,
MO) and fura 2/AM, ionomycin and sodium orthovanadate from
Calbiochem (Darmstadt, Germany). Anti-phosphotyrosine mono-
clonal antibody PY20 was from ICN Biotechnology (Irvine, CA). All
other reagents were of analytical grade.
4.1.2.2. (E,Z)-Ethyl(1-propyl-1H-indol-5-ylamino)(phenyl)
methylenecarbamate (15)
The synthesis of compound 15 was carried oput by the same
procedure as described for 13 by reaction of aminoindole derivative
7 (1.4652 g, 8.4 mmol) and 11 (0.878 g, 4.2 mmol). Yield 46%; mp
149e150 ꢂC; rf 0.85 (ethyl acetate/n-hexane 8:2); IR nmax (KBr):
4.1.1. Synthesis of N-ethoxycarbonylthiobenzamides (11)
To 15 mL of a stirred cold (ice bath) solution of CH2Cl2 containing
0.3901 g (d 0.88, 0.44 mL, 5 mmol) of benzene (9) and 0.6557 g
(5 mmol) of ethoxycarbonyl isothiocyanate, was added 10 mmol of
anhydrous AlCl3 (1.3334 g) in small portions (10e15 min) at 0e3 ꢂC.
The reaction mixture was stirred at this temperature for 4 h (TLC
ethyl acetate/n-hexane 8:2) and was then hydrolysed by careful
addition of ice and 2 M hydrochloric acid. Volumes of CH2Cl2
sufficient to dissolve any solid organic material were added, and the
resulting solution was extracted with four portions of 10% aqueous
NaOH. This extract was washed with ethyl ether and acidified with
concentrated hydrochloric acid (ice bath) to yield an oil, which
solidified upon cooling. The solid material was then washed with
1 M hydrochloric acid and water, dried and washed again with cold
aqueous ethanol (ethanol/water 6:4). The resulting dried solid
product was used as such in the following reactions. Yield 69%
[literature [25] 52%]; orange solid; mp 63e64 ꢂC [literature [25]
61e61.5 ꢂC], rf 0.69 (ethyl acetate/n-hexane 8:2); 1H NMR
1672 (C]O) cmꢀ1
;
1H NMR (DMSO-d6):
d
0.82 (t, 3H, J ¼ 7.2 Hz,
NCH2CH2CH3), 0.94 (t, 3H, J ¼ 7.1 Hz, C(O)OCH2CH3), 1.71e1.83 (m,
2H, NCH2CH2CH3), 3.82 (q, 2H, J ¼ 7.1 Hz, C(O)OCH2CH3), 4.11 (t, 2H,
J ¼ 7.2 Hz, NCH2CH2CH3), 6.41e7.91 (m, 10H, Ar), 9.76 (bs, 1H, N]
CeNH).
4.1.2.3. Synthesis of (E,Z)-ethyl(1-(cyclopropylmethyl)-1H-indol-5-
ylamino)(phenyl)methylenecarbamate (16)
The synthesis of compound 16 was carried out by the same
procedure as for 13 by reaction of aminoindole derivative 8
(1.2953 g, 6.95 mmol) and 11 (0.7272 g, 3.47 mmol). Yield 45%; mp
155e158 ꢂC; rf 0.84 (ethyl acetate/n-hexane 8:2); IR nmax (KBr):
1675 (C]O) cmꢀ1 1H NMR (DMSO-d6):
; d 0.37e0.52 (m, 4H,
CHCH2CH2), 0.95 (t, 3H, J ¼ 7.1 Hz, C(O)OCH2CH3), 1.17e1.28 (m, 1H,
CHCH2CH2), 3.82 (q, 2H, J ¼ 7.1 Hz, C(O)OCH2CH3), 4.02 (d, 2H,
J ¼ 7.2 Hz, N-CH2,), 6.41e7.94 (m, 10H, Ar), 9.70 (bs, 1H, N]CeNH).
(DMSO-d6)
d
1.34 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 4.18 (q, 2H, J ¼ 7.1 Hz,
4.1.2.4. Synthesis of (E,Z)-Ethyl(1-ethyl-1H-indolo-5-ylamino-4-
methoxy-phenyl)methylenecarbamate (17)
NCH2CH3), 7.36e7.64 (m, 5H, Ar), 12.18 (bs, 1H, S]CeNH); HRMS
(ESI) [M þ H]þ calculated for C10H12NO2S m/z 210.0544; found
210.0559.
The synthesis of compound 17 was carried out by the same
procedure as for 13 by reaction of aminoindole derivative 6
(1.9410 g, 12.11 mmol) and compound 12 1.5554 g, 6.2 mmol. Yield
43%; mp 137e140 ꢂC; rf 0.78 (ethyl acetate/n-hexane 8:2); IR nmax
4.1.1.1. Synthesis of ethyl p-methoxy-phenyl-carbothionylcarbamate
(12)
(KBr): 1667 (C]O) cmꢀ1 1H NMR (DMSO-d6):
; d 1.00 (t, 3H,
Compound 12 was prepared by the procedure of compound 11
by reaction of 0.6705 g (d 0.995, 0.674 mL, 6.2 mmol) and 1.643 g
(12.4 mmol). Yield 88% [literature 90%]; orange yellowish solid; mp
90e91 ꢂC [literature [26] 88e90 ꢂC]; rf 0.77 (ethyl acetate/n-hexane
J ¼ 7.2 Hz, NCH2CH3), 1.35 (t, 3H, J ¼ 7.1 Hz, C(O)OCH2CH3),
3.81e3.85 (m, 5H, OCH3 and C(O)OCH2CH3), 4.18 (q, 2H, J ¼ 7.2 Hz,
NCH2CH3), 6.40e7.86 (m, 9H, Ar), 9.70 (bs, 1H, N]CeNH).
8:2); 1H NMR (DMSO-d6)
d
1.25 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 3.82 (s,
4.1.3. Synthesis of 3-phenyl-2,7-dihydro-1H-pyrrolo[3,2-f]
quinazolin-1-one (18)
3H, OCH3), 4.18 (q, 2H, J ¼ 7.1 Hz, NCH2CH3), 6.93 (d, 2H, J ¼ 8.8 Hz,
Ar), 7.70 (d, 2H, J ¼ 8.8 Hz, Ar.), 11.90 (bs, 1H, S]CeNH); HRMS (ESI)
[M þ H]þ calculated for C11H13NO3S m/z 240.0650; found 240.0665.
In a two-necked, 50-mL, round-bottomed flask, 10e15 mL of
diphenyl ether were heated to boiling temperature; 0.3326 g
(1.08 mmol) of amidine 13 were then added portion-wise and the
mixture was refluxed for 15e20 min. After cooling to 50 ꢂC, the
separated precipitate was collected by filtration and washed many
times with diethyl ether. The collected product was purified by
flash chromatography (ethyl acetate/n-hexane 8:2). Yield 54%,
brown solid; mp >300 ꢂC; rf 0.79 (ethyl acetate/n-hexane 8:2); IR
4.1.2. Synthesis of N-indole-N0-ethoxycarbonylamidine derivative
(13)
In a two-necked round-bottomed flask, a solution of amino-
indole derivatives 5 [16] (2.6433 g, 20 mmol) and N-ethox-
ycarbonylthioamides 11 (2.0926 g, 10 mmol) in 30 mL of absolute
ethanol was refluxed until hydrogen sulfite evolution had stopped
(6e8 h), as indicated by lead acetate paper and TLC (ethyl acetate/n-
hexane 8:2). At the end of the reaction, the solvent was evaporated
under reduced pressure and the raw material was purified by silica
nmax (KBr): 3227 (NH), 3217 (NH), 1690 (C]O) cmꢀ1 1H NMR
;
(DMSO-d6)
d
7.33 (td, 1H, J ¼ 2.7 and 0.8 Hz, 9H), 7.49 (d, 1H,
J ¼ 8.6 Hz, 5eH), 7.54 (m, 3H, J ¼ 7.9 and J ¼ 1.9 Hz, 30-H-, 40-H-,
50-H), 7.59 (t, 1H, J ¼ 2.8 Hz, 8-H), 7.89 (dd, 1H, J ¼ 0.76 and

282
M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
J ¼ 8.6 Hz, 6-H), 8.20 (m, 2H, 20-H and 6’-H), 11.69 (s, 1H, NH
NHC(O)); 13C NMR (DMSO-d6)
d 15.8, 40.6, 54.8, 102.9, 112.7, 115.1,
pyrrole), 12.39 (bs, 1H, NHC(O)); 13C NMR (DMSO-d6)
d
102.9, 112.7,
117.5, 120.6, 123.7, 127.5, 129.8, 130.8, 133.0, 133.1, 144.9, 149.6,
117.5, 120.6, 123.7, 127.5, 128.6, 129.8, 130.8, 133.0, 133.1, 144.9,
149.6, 162.4; HRMS (ESI) [M þ H]þ calculated for C16H12N3O m/z
262.0936; found 262.0948; anal. calcd. for C16H11N3O: C, H, N.
162.4; HRMS (ESI) [M þ H]þ calculated for C19H17N3O2 m/z
C19H18N3O2
C19H17N3O2: C, H, N.
¼
320,1354; found 320.1366; anal. calcd. for
4.1.3.1. Synthesis 7-ethyl-3-phenyl-2,7-dihydro-1H-pyrrolo[3,2-f]
quinazolin-1-one (19)
4.2. Biological assays
The synthesis of compound 19 was carried out by the same
procedure as for 18 by thermal cyclization of amidine derivative 14
(0.4450 g, 1.33 mmol). Yield 47%; light yellow solid; mp>300 ꢂC; rf
0.72 (ethyl acetate/n-hexane 8:2); IR nmax (KBr): 3207 (NH), 1683
4.2.1. Preparation of platelet suspensions
Blood samples were collected from healthy volunteers who had
taken no medications during the preceding 2 weeks, with their
informed consent and in accordance with the Helsinki Declaration.
Platelet isolation was performed by differential centrifugation, as
previously described [39]. Briefly: blood was immediately mixed
with citric-anticoagulant ACD (75 mM sodium citrate, 40 mM citric
acid, 130 mM dextrose, pH 6.0), supplemented with 0.8 mU/mL
apyrase and 20 mg/mL prostacyclin, and centrifuged for 20 min at
200 g. The supernatant platelet-rich plasma (PRP) was further
centrifuged for 20 min at 750 g. Spun-down platelets were re-
suspended, unless otherwise indicated, in basal buffer consisting
of (mM) 145 NaCl, 5 KCl, 1 MgCl2, 10 glucose, and 10 Tris/Hepes, (pH
7.4), at a concentration of 2 e 4 ꢃ 108 cells/mL. Where indicated,
acetylsalicylic acid treatment was performed by addition (0.1 mM
final concentration) of a freshly prepared solution in 0.1 M NaHCO3
to platelet-rich plasma, followed by 20 min incubation at 36 ꢂC.
(C]O) cmꢀ1
;
1H NMR (DMSO-d6)
d
1.41 (t, 3H, J ¼ 7.2 Hz,
NCH2CH3,), 4.36 (q, 2H, J ¼ 7.2 Hz, NCH2CH3), 7.32 (dd, 1H, J ¼ 3.1
and J ¼ 0.6 Hz, 9-H), 7.51 (d, 1H, J ¼ 8.8 Hz, 5-H), 7.54 (m, 3H, 30-H,
40-H, 50-H), 7.64 (d, 1H, J ¼ 3.6 Hz, 8-H), 8.03 (dd, 1H, J ¼ 8.8 and
J ¼ 0.6 Hz, 6-H), 8.22 (m, 2H, 20-H and 6’-H), 12.31 (bs, 1H, NHC(O));
13C NMR (DMSO-d6)
d 15.8, 40.6, 102.9, 112.7, 117.5, 120.6, 123.7,
127.5, 128.6, 129.8, 130.8, 133.0, 133.1, 144.8, 149.6, 162.4; HRMS
(ESI) [M þ H]þ calculated for C18H16N3O m/z 290.1249; found
290.1262; anal. calcd. for C18H15N3O: C, H, N.
4.1.3.2. Synthesis of 7-propyl-3-phenyl-2,7-dihydro-1H-pyrrolo
[3,2-f]quinazolin-1-one (20)
The synthesis of compound 20 was carried out by the same
procedure as for 18 by thermal cyclization of amidine derivative 15
(0.3493 g, 1.00 mmol). Yield 62%, white solid; mp 295-96 ꢂC; rf 0.81
(ethyl acetate/n-hexane 8:2); IR nmax (KBr): 3212 (NH), 1671 (C]O)
4.2.2. Determination of platelet aggregation
Platelet aggregation was followed turbidometrically on an Elvi-
Logos aggregometer as previously reported [40].
cmꢀ1
;
1H NMR (DMSO-d6)
d
0.85 (t, 3H, J ¼ 7.2 Hz, NCH2CH2CH3),
1.76e1.88 (m, 2H, NCH2CH2CH3), 4.28 (t, 2H, 7.0 Hz,
J
¼
NCH2CH2CH3), 7.32 (dd, 1H, J ¼ 3.1 and J ¼ 0.6 Hz, 9-H), 7.50 (d, 1H,
J ¼ 8.8 Hz, 5-H), 7.54 (m, 3H, 30-H, 40-H, 50-H), 7.61 (d, 1H, J ¼ 3.1 Hz,
8-H), 8.03 (dd, 1H, J ¼ 8.8 and J ¼ 0.6 Hz, 6-H), 8.22 (m, 2H, 20-H and
4.2.3. Analysis of cytosolic Ca2þ concentration
Cytosolic [Ca2þ] was determined by means of the fluorescent
probe fura 2, as previously described [41]. Fluorescence changes
were measured in a thermostated, magnetically stirred quartz
cuvette, at dual excitation wavelengths of 340 and 385 nm and an
emission wavelength of 505 nm. Calibration was performed with
the calciumeionophore ionomycin and calciumechelator EGTA.
6’-H), 12.42 (bs, 1H, NHC(O)); 13C NMR (DMSO-d6)
d 11.5, 33.4, 42.7,
102.9, 112.7, 117.5, 120.6, 123.7, 127.5, 128.6, 129.8, 130.8, 133.0, 133.1,
144.9, 149.6, 162.4; HRMS (ESI) [M þ H]þ calculated for C19H18N3O
m/z 304.1405; found 304.1412; anal. calcd. for C19H17N3O:C, H, N.
4.1.3.3. Synthesis of 7-cyclopropylmethyl-3-phenyl-2,7-dihydro-
1H-pyrrolo[3,2-f]quinazolin-1-one (21)
4.2.4. Detection of tyrosine phosphorylation of platelet proteins
Platelets suspended in the basal medium, at a count of
300 ꢃ 106 cells/mL, were incubated according to the experimental
The synthesis of compound 21 was carried out by the same
procedure as for 18 by thermal cyclization of amidine derivative 16
(0.4802 g, 1.33 mmol). Yield 61%, brown solid; mp>300 ꢂC; rf 0.74
(ethyl acetate/n-hexane 8:2); IR nmax (KBr): 3237 (NH), 1665 (C]O)
protocol. Aliquots of 30
ml (protein concentrations determined
according to Bradford [42]) were withdrawn from the differently
treated platelet suspensions and transferred into Eppendorf
test tubes containing 15 mL of Laemmli solution supplemented
cmꢀ1
;
1H NMR (DMSO-d6)
d
0.40e0.56 (m, 4H, CHCH2CH2),
1.23e1.31 (m, 1H, CHCH2CH2), 4.19 (d, 2H, J ¼ 7.0 Hz, N-CH2), 7.33
(dd, 1H, J ¼ 3.1 and J ¼ 0.6 Hz, 9-H), 7.50 (d, 1H, J ¼ 8.8 Hz, 5-H), 7.54
(m, 3H, 30-H, 40-H, 50-H), 7.68 (d, 1H, J ¼ 3.1 Hz, 8-H), 8.10 (dd, 1H,
J ¼ 8.8 and J ¼ 0.6 Hz, 6-H), 8.21 (m, 2H, 20-H and 60-H), 12.44 (bs,
with 2-mercaptoethanol (5%), anti-phosphatase sodium orthova-
nadate (1 mM), anti-protease EDTA (2 mM) and electrophoretic
tracer pyronin. Samples were subjected to SDS/PAGE (10%
gels) and separated proteins were immediately electrophoretically
transferred to nitrocellulose membranes, treated with anti-
phosphotyrosine PY20 (ICN Biotechnology), followed by
secondary peroxidase-conJugate antibody and detected by the
enhanced chemi-luminescence technique (ECL, Amersham Phar-
1H, NHC(O)); 13C NMR (DMSO-d6)
d 4.10, 12.22, 50.22, 102.9, 112.7,
117.5, 120.6, 123.7, 127.5, 128.6, 129.8, 130.8, 133.0, 133.1, 144.9,
149.6, 162.4; HRMS (ESI) [M þ H]þ calculated for C20H18N3O m/z
316.1405; found. 316.1408; anal. calcd. for C20H17N3O: C, H, N.
macia Biotech, San Francisco, CA) [39]. Anti-bactin immunoblotting
4.1.3.4. Synthesis of 7-ethyl-3-(4-methoxy-phenyl)-2,7-dihydro-
1H-pyrrolo[3,2-f]quinazolin-1-one (22)
was performed in parallel to check the amounts of proteins in SDS/
PAGE lanes.
The synthesis of compound 22 was carried out by the same
procedure as for 18 by thermal cyclization of amidine derivative 17
(0.5006 g, 1.36 mmol). Yield 65%, brown solid; mp>300 ꢂC; rf 0.70
(ethyl acetate/n-hexane 8:2); IR nmax (KBr): 3217 (NH), 1680 (C]O)
4.2.5. Platelet damage
Possible damage caused by 3-PPyQZ on platelet membranes was
analysed by monitoring the release of lactate dehydrogenase
(LDH)] from platelets incubated with the compound [36]. LDH
cmꢀ1; 1H NMR (DMSO-d6)
d
1.41 (t, 3H, J ¼ 7.2 Hz, NCH2CH3), 3.83 (s,
3H, OCH3), 4.34 (q, 2H, J ¼ 7.2 Hz, NCH2CH3,), 7.08 (d, 2H, J ¼ 9.0 Hz,
30-H and 50-H), 7.31 (dd, 1H, J ¼ 3.05 and J ¼ 0.6 Hz, 9-H), 7.51 (d, 1H,
J ¼ 8.8 Hz, 5-H), 7.62 (d, 1H, J ¼ 3.1 Hz, 8-H), 8.02 (dd, 1H, J ¼ 8.8 and
J ¼ 0.6 Hz, 6-H), 8.21 (d, 2H, J ¼ 9.0 Hz, 20-H and 60-H), 12.31 (bs, 1H,
release was measured spectrophotometrically by recording b-
NADH oxidation following pyruvate addition. Platelet suspensions
(1 mL, 2 ꢃ 108 cells/mL) were incubated at 37 ꢂC for different times,
with various concentrations of tested compounds, and then

M.G. Ferlin et al. / European Journal of Medicinal Chemistry 48 (2012) 275e283
283
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Analysis of statistical significance of differences was performed
with the non-parametric ManneWhitney U-test.
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