Synthesis of a new class of pyrrolo[3,4-h]quinazolines with antimitotic activity

Article abstract of DOI:10.1016/j.ejmech.2013.10.014

A new series of pyrrolo[3,4-h]quinazolines was conveniently prepared with a broad substitution pattern. A large number of derivatives was obtained and the cellular cytotoxicity was evaluated in vitro against 5 different human tumor cell lines with GI₅₀ values reaching the low micromolar level (1.3-19.8 μM). These compounds were able to induce cell death mainly by apoptosis through a mitochondrial dependent pathway. Selected compounds showed antimitotic activity and a reduction of tubulin polymerization in a concentration-dependent manner. Moreover, they showed anti-angiogenic properties since reduced in vitro endothelial cell migration and disrupted HUVEC capillary-like tube network in Matrigel.

Full text of DOI:10.1016/j.ejmech.2013.10.014

European Journal of Medicinal Chemistry 74 (2014) 340e357
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of a new class of pyrrolo[3,4-h]quinazolines with antimitotic
activity
Virginia Spanò a, Alessandra Montalbano a, Anna Carbone a, Barbara Parrino a,
Patrizia Diana a, Girolamo Cirrincione a, Ignazio Castagliuolo b, Paola Brun b,
Olaf-Georg Issinger c, Silvia Tisi d, Irina Primac d, Daniela Vedaldi d, Alessia Salvador d,
,
Paola Barraja a
*
a Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
b Dipartimento di Medicina Molecolare, Università degli Studi di Padova, Via Gabelli 63, 35121 Padova, Italy
c Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova, Via Marzolo 5, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrrolo[3,4-h]quinazolines was conveniently prepared with a broad substitution pattern.
A large number of derivatives was obtained and the cellular cytotoxicity was evaluated in vitro against 5
different human tumor cell lines with GI50 values reaching the low micromolar level (1.3e19.8 mM).
These compounds were able to induce cell death mainly by apoptosis through a mitochondrial depen-
dent pathway. Selected compounds showed antimitotic activity and a reduction of tubulin polymeri-
zation in a concentration-dependent manner. Moreover, they showed anti-angiogenic properties since
reduced in vitro endothelial cell migration and disrupted HUVEC capillary-like tube network in Matrigel.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 8 July 2013
Received in revised form
4 October 2013
Accepted 5 October 2013
Available online 16 October 2013
Keywords:
Pyrrolo[3,4-h]quinazolines
Antiproliferative activity
Antimitotic activity
Tubulin polymerization
Vascular disrupting activity
1. Introduction
the quinazoline moiety, such as the pyrazolo[4,3-h]quinazoline-
3-carboxamides of type 2, have been largely investigated as ki-
Quinazolines are considered an attractive target for medicinal
chemists, because they are the scaffold of several potent anti-
tumor drugs. Leading examples are the well known Erlotinib
(trade name Tarceva) [1] and Gefitinib (trade name Iressa) [2],
but many other examples are currently under clinical investiga-
tion. Among these, the quinazoline derivative LZ-11 (1) has
recently emerged as effective and selective telomeric G-quad-
ruplex ligand [3]. Moreover, tricyclic compounds incorporating
nases inhibitors [4]. Considering our interest in pyrrole and
indole systems with antitumor properties [5e14], we have
recently reported the synthesis of pyrrolo[3,2-h]quinazolines 3
with the aim of developing a new series of quinazolines con-
taining the pyrrole moiety. These compounds expressed their
activity upon light activation of a proper wavelength, showing
potent growth inhibitory effects in cell proliferation in several
human tumor cell lines reaching submicromolar level (GI50 0.2e
15.2 mM) [15]. Herein, we report the synthesis of a new series of
pyrrolo[3,4-h]quinazolines of type 4 and the evaluation of their
antitumor properties (Chart 1).
At first, considering our previous results on the pyrroloquina-
zolines of type 3, the new derivatives of the title ring system were
screened to evaluate the growth inhibitory effect in cell prolifera-
tion upon light irradiation. However, pyrrolo[3,4-h]quinazolines 4
did not undergo photoactivation, and the antitumor effect was very
poor. In contrast, they gave interesting results “in the dark”
showing antimitotic activity.
Abbreviations: AO, acridine orange; EGFR, Epidermal Growth Factor Receptor;
FITC, fluorescein isothiocyanate; H2DCFDA, 20,70-dichlorodihydrofluorescein diac-
etate; HE, hydroethidine; HUVEC, human umbilical vein endothelial cell; JC1,
5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine; MTT test, (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test; PI, propidium
iodide; PS, phosphatidylserine; RI, Resistance Index; ROS, reactive oxygen species.
* Corresponding author. Tel.: þ39 091 6161606; fax: þ39 091 23860854.
E-mail address: paola.barraja@unipa.it (P. Barraja).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.014

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
341
We further studied the title annelation and those compounds
were re-prepared, together with several new derivatives of the ring
system properly decorating the pyrimidine moiety at the 2 position.
Thus, the annelation of the pyrimidin-2-one ring was achieved
by refluxing in anhydrous ethanol the hydroxymethylene de-
rivatives 6ees with urea leading to the desired compounds 9ees in
good yields (55e75%) (Table 1, Supp. info). In agreement with our
previous results on the pyrrolo[3,2-h]quinazoline series, among the
three possible tautomeric structures, compounds 9 exist in the A
form (Chart 2) as demonstrated by the presence of two doublets in
the 1H NMR spectra attributable to the exchangeable amidic proton
at position 3 and to H-4 adjacent to it. This contrasts with the
behavior of quinazolines, which generally exist in C form [26].
Moreover, the enaminoketones 7b,e,f,hem,pes were used as
starting materials for the synthesis of 2-amino-pyrrolo[3,4-h]qui-
nazolines of type 10 (Table 1, Supp. info, 50e75%), through reaction
with guanidine nitrate in the presence of sodium methoxide as
base to promote nucleophilic attack of the amino groups to the
electrophilic centers. Similarly, upon heating under reflux enami-
noketones 7b,eem,pes with phenylguanidine in DMF, the 2-
anilino-pyrrolo[3,4-h]quinazolines 11 were obtained in good
yields (Table 1, Supp. info, 60e84%).
N
N
O
HN
Cl
N
N
O
RHN
N
N
R1
N
N
HN
(CH2)3 N
2
1
2
X
N1
X
3 N
N
N
R
N
9
7
4
N R
8
5
6
4
3
X=CH, C=O, C-NH2, C-NHPh
Chart 1. Structures of LZ11 1, pyrazolo[4,3-h]quinazolines 2, pyrrolo[3,2-h]quinazo-
lines 3 and pyrrolo[3,4-h]quinazolines 4.
The 57 synthesized derivatives of the title ring system were
screened to evaluate the growth inhibitory effect in cell prolifera-
tion against a panel of human tumor cell lines and the mechanism
of action was investigated.
2. Chemistry
Tetrahydroindol-7-ones 5aes, suitable substrates for our pur-
pose, were prepared by different synthetic routes with a good
substitution pattern on the pyrrole moiety by methods previously
3. Results and discussion
reported by us (Scheme 1) [16e18]. Functionalization in
a
position
3.1. Antiproliferative assays
to the carbonyl group, transformated ketones 5aes either into the
corresponding hydroxymethylketones or into enaminoketones
which have demonstrated versatile key intermediates for further
annelation upon reaction with several dinucleophiles. In fact, the
Cellular cytotoxicity was carried out in vitro in 5 different human
tumor cell lines: K562, Jurkat, LoVo, MCF-7 and A-431. Cytotoxicity
was evaluated by the MTT test after 72 h of cellular incubation in
the presence of different concentrations of compounds as described
in experimental section (5.2). For each compound, GI50 value was
calculated and is shown in Table 1. The compounds whose GI50 was
annular carbonyl group and the exocyclic carbon in
a position
represent the two electrophilic centers, readily available for further
cyclization [19e25].
Thus, hydroxymethylketones 6ees were conveniently prepared
in good yields (52e86%) by formylation of the corresponding ke-
tones 5ees under nitrogen atmosphere at 0 ꢀC, with ethyl formate
as formylating agent and potassium tert-butoxide as the base in
toluene. A selection of enaminoketones 7, in terms of best avail-
ability of the precursors, were prepared by different routes. In
particular, compounds 7eei,pes (R3 ¼ Et, 50e96%) were obtained
by reaction of the corresponding hydroxymethylketones with
diethylamine at room temperature in toluene. Alternatively, com-
pounds 7b,jem (R3 ¼ Me, 57e87%) were achieved by direct
introduction of the enamino functionality from the corresponding
ketones 5b,jem, with the Bredereck reagent, tert-butoxy-bis
(dimethylamino) methane (TBDMAM) in refluxing toluene. Some of
these intermediates were previously reported by us [16,17].
In the light of the previously reported attracting results on
pyrrolo[3,2-h]quinazolines 3, we planned the annelation of the
pyrimidine ring on the isoindole moiety in order to obtain de-
rivatives of the pyrrolo[3,4-h]quinazolines ring system of type 4
(X ¼ CH, C]O, CeNH2, CeNHPh), properly functionalized at posi-
tion 2 of the pyrimidine moiety.
higher than 20 mM in all cell lines were not included in Table 1.
Most compounds reduced cell survival in at least one or more
cell lines. The most cytotoxic compounds were 8l, 8m, 8r, 8s and
11j. Owing to the large number of molecules, some considerations
about structureeactivity relationship can be made. For the de-
rivatives with no substitution on position 2, the most active ones
present lipophilic substituents with high steric hindrance in posi-
tions 7 and 8: an ethyl ester (8l and 8m) or a phenyl (8r and 8s) in 7
and a p-methylbenzyl group (8l and 8r) or a p-methoxybenzyl
group (8m and 8s) in 8. It is interesting to note how the presence of
only one of the two substitutions makes the molecule inactive (see
for example 8b, 8h or 8i) or significantly reduces the cytotoxicity
(8d or 8p).
With the introduction of a substitution in position 2 in the
quinazoline structure, we did obtain some active derivatives even if
their GI50 were usually higher than the 8l and 8m. In particular,
derivatives bearing a carbonyl in 2 resulted cytotoxic in Jurkat cells.
The introduction of an amine group led to some compounds with
GI50 lower than 5 mM in Jurkat cell line (10p and 10r). However, no
significant increase in cytotoxicity was observed in comparison
with compounds 8.
In analogy with quinazoline molecules which demonstrated
inhibitory activity towards tyrosine kinases [27], we analysed some
derivatives with an aniline group in position 2 (11be11s); this latter
substitution does not seem to lead to more cytotoxic compounds
but interestingly, most of them were active in the Epidermal
Growth Factor Receptor (EGFR) over-expressing A-431 cell line (see
for example 11b).
Actually, the synthesis of the tricyclic compounds of type 8
bearing the unsubstituted position 2 of the pyrimidine moiety was
already reported by us, as a new ring system, and the growth
inhibitory properties of these compounds were investigated [18].
However, only derivative 8s showed activity against all the 59
tested cell lines at micromolar concentration (1.46e18.4 mM) in the
in vitro growth inhibition assays performed by the National Cancer
Institute of Bethesda.

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V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
R1
O
R1
O
R1
O
HO
ii
(R3)2N
i
NR
R2
NR
R2
NR
R2
7e-i,p-s (R3= Et)
7b,j-m (R3=Me)
5a-s
6e-s
iii
iv
v
vi
NHR4
O
2
R1
N
N
R1
NR
R2
HN
N
3
R1
N
N1
9
7
4
8
NR
R2
8a,b,d-f,h-m,p-s
NR
5
6
R2
9e-s
10b,e,f,h-m,p-s (R4=H)
11b,e-m,p-s (R4=Ph)
(Table 1, Supp. Info)
(Table 1, Supp. Info)
(Table 1, Supp. Info)
a R=R1=R2=H; b R=H, R1=Me, R2 =CO2Et; c R=R2=H, R1=Me; d R=R1 =H, R2 =Ph; e R=Me, R1 =R2=H; f R=Bn,
R1=R2=H; g R=Ph, R1=R2=H; h R=BnpMe, R1=R2=H; i R=BnpOMe, R1=R2=H; j R=R1=Me, R2 =CO2Et; k R=Bn,
R1=Me, R2=CO2Et; l R=BnpMe, R1=Me, R2=CO2Et; m R= BnpOMe, R1=Me, R2=CO2Et; n R=R1=Me, R2=H; o R=Bn,
R1=Me, R2 =H; p R=Me, R1 =H, R2 =Ph; q R=Bn, R1 =H, R2 =Ph; r R=Bn pMe, R1 =H, R2 =Ph; s R=Bn pOMe, R1 =H,
R2=Ph.
Scheme 1. Reagents and conditions: i) t-BuOK, HCOOEt, toluene, rt, 24 h, 70e86%; ii) from 6eei,pes: HNEt2, toluene, rt, 24 h, 50e96%; iii) from 5b,jem: TBDMAM, toluene, reflux,
24 h, 57e87%; iv) CH(NHCHO)3, TsOH, formamide, N2, reflux, 24 h, 45e80%; v) urea, EtOH, reflux, 24 h, 55e75%; vi) (NH2)2eC]NH$HNO3, EtOH, NaOMe, reflux, 2e5 h, 50e75%
(10b,e,f,hem,pes) or (NH2)2eC]NHPh, DMF, 100 ꢀC, 1.5e2 h, 55e84% (11b,eem,pes).
3.1.1. Cytotoxicity in resistant cell line
treated with a known EGFR inhibitor (AG1478). This test was per-
formed with the cytotoxic compounds 8l and 8m but also with
some aniline-substituted structures, such as 11b,11e and 11f, which
were quite cytotoxic towards A431 cells (Fig. 1). However, these
compounds were not able to inhibit the phosphorylation and the
activity of EGFR, as can be clearly observed in the western blot
pictures. Moreover, the phosphorylation of the downstreaming
proteins ERK1/2 and AKT was also not inhibited in the pyrrolo-
quinazoline treated cells; as a consequence, we hypothesized that
EGFR is not the target for these compounds.
Although many anticancer drugs in clinical use are effective in
the treatment of different kinds of tumors, their potential is limited
by the development of drug resistance. Resistance can be intrinsic
or acquired, but in either case, tumors become refractory to a va-
riety of structurally different drugs [28]. Thus, the antiproliferative
effects of the most active pyrroloquinazolines were evaluated in a
human cancer cell line expressing high level of 170-kDa P-glyco-
protein (P-gp) drug efflux such as LoVodoxo subclone. Then, RI
(Resistance Index: GI50LoVodoxo/GI50LoVowt) was also calculated. As
reported in Table 2, pyrroloquinazolines 8l, 8m, 8r and 8s were
almost equivalently potent in LoVodoxo subclone.
3.3. Evaluation of cell cycle
3.2. Evaluation of EGFR inhibition
To better understand the mechanism of action and the mode of
cellular death, a cell cycle analysis was performed. After fixation in
ethanol, cells were stained with PI which made possible to do rapid
determinations of DNA per cell. These tests were performed after
24 h of treatment of Jurkat cells with 8l, 8m, 8r, 8s and 11j. These
compounds were chosen since they are the most cytotoxic and 11j
as representative of the aniline substituted pyrroloquinazoline.
Deep changes in cell cycle profile can be detected (Table 3): in
The human skin cancer cell line A-431, which overexpresses the
EGFR, was used to investigate whether these compounds were able
to block EGF receptor signaling. After treating these cells with
compounds for 3 h, the blockage in EGF receptor signaling was
detected by immunoblotting using phospho-specific antibodies
against the EGFR receptor and its downstream signaling molecules
ERK1/2 and AKT. As a positive control, some cell samples were
fact, for all compounds, there is
a concentration-dependent
O
OH
O
R1
R1
R1
HN
N
N
N
N
NH
NR
R2
NR
R2
NR
R2
A
B
C
Chart 2. Tautomeric forms of compounds 9.

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
343
Table 1
Cytotoxicity of pyrrolo[3,4-h]quinazolines 8e11 in vitro.
3.3.1. Mitotic index
Since pyrroloquinazoline compounds caused a massive cell
accumulation in the G2/M phase, the appearance of mitotic cells
was quantified. Exponentially growing Jurkat cells were incubated
GI50
CPD
(
m
M)a
K562b
Jurkat
LoVo
MCF-7
A-431
with 20 mM 8l, 8m, 8r, 8s and 11j for 24 h. After fixation, samples
were stained with Giemsa and analyzed by optical microscopy for
the presence of dispersed chromosomes in the cytoplasm and
disappearance of the nuclear membrane. It was found that the
percentage of mitotic cells (the mitotic index) increased for all
compounds in comparison to control (Fig. 2, lower panel), in
agreement with the cell cycle analysis data. Pyrroloquinazolines
could act as antimitotic drugs so further investigations have been
performed to confirm their mechanism of action.
8d
8k
8l
8m
8p
8r
>20
>20
3.9 ꢁ 0.6
3.8 ꢁ 0.4
>20
8.4 ꢁ 0.5
>20
6.9 ꢁ 1.9
11.3 ꢁ 1.8
5.0 ꢁ 0.4
5.4 ꢁ 0.3
9.1 ꢁ 1.4
9.9 ꢁ 1.8
7.1 ꢁ 0.7
>20
>20
7.5 ꢁ 0.3
14.1 ꢁ 0.9
>20
>20
>20
11.3 ꢁ 1.8
17.3 ꢁ 2.3
>20
3.7 ꢁ 0.5
3.1 ꢁ 0.1
10.7 ꢁ 1.3
3.2 ꢁ 0.5
1.3 ꢁ 0.2
13.1 ꢁ 2.5
11.7 ꢁ 0.4
>20
>20
8s
10.9 ꢁ 2.3
7.0 ꢁ 0.4
3.4. Perturbation of microtubule network
We next evaluated the effect of test compounds on cellular
microtubule network by using an anti-b-tubulin antibody FITC
conjugated. As shown in Fig. 3, pyrroloquinazolines disrupted the
organization and arrangement of tubulin network. Moreover, with
the exception of 11j, all compounds induced a clear modification in
cell morphology which “rounded-up”, a typical feature of cells in
which microtubule disaggregation occurs. Similar phenotype can
be observed in vinblastine treated cells.
9g
9j
9l
9p
9q
9r
>20
>20
>20
>20
>20
>20
9.4 ꢁ 0.8
12.5 ꢁ 1.7
8.5 ꢁ 2.1
6.2 ꢁ 0.6
7.4 ꢁ 0.4
9.2 ꢁ 1.3
>20
>20
>20
>20
>20
>20
>20
>20
17.9 ꢁ 1.6
>20
>20
>20
>20
>20
8.5 ꢁ 1.2
14.6 ꢁ 1.6
>20
>20
3.5. Effects on microtubule polymerization in vitro
As the immunofluorescence studies suggested that pyrroloqui-
nazolines impaired the formation or the stability of microtubules in
the cells, their effects on tubulin polymerization in vitro were stud-
ied. Polymerization was followed by fluorescent enhancement
because of the incorporation of a fluorescent reporter into the mi-
crotubules as polymerization occurred. Results are shown in Fig. 4.
While in the control sample, the fluorescence increased over time,
when the reaction was carried out in the presence of vinblastine or
Taxol, an inhibition or enhancement of tubulin polymerization
occurred, respectively. Pyrroloquinazolines, with the exception of
11j, reduced tubulin polymerization in concentration-dependent
manner. Hence, these compounds showed an antimitotic action.
10b
10h
10i
10k
10l
10p
10q
10r
10s
>20
>20
>20
10.7 ꢁ 1.1
6.8 ꢁ 0.4
>20
12.8 ꢁ 0.2
9.4 ꢁ 0.2
>20
10.5 ꢁ 1.2
9.0 ꢁ 1.7
8.0 ꢁ 0.9
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
19.1 ꢁ 2.1
>20
8.1 ꢁ 1.9
11.7 ꢁ 1.1
6.3 ꢁ 0.4
5.0 ꢁ 1.0
5.9 ꢁ 0.5
3.9 ꢁ 0.8
8.1 ꢁ 1.2
>20
>20
>20
>20
11.8 ꢁ 1.5
>20
>20
>20
>20
>20
15.3 ꢁ 3.0
15.3 ꢁ 0.8
>20
>20
3.6. Evaluation of antivascular activity in vitro
11b
11e
11f
11g
11h
11i
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
3.0 ꢁ 0.5
7.7 ꢁ 0.5
7.6 ꢁ 1.3
>20
9.2 ꢁ 1.6
11.7 ꢁ 0.8
9.7 ꢁ 0.4
>20
8.6 ꢁ 1.9
7.4 ꢁ 1.2
Since many antimitotic drugs such as colchicine and com-
bretastatin can also have an effect on tumor immature and neo-
formed vessel network, an antivascular effect was assessed per-
forming preliminary tests in vitro. In fact, due to their action on
microtubules, many antimitotic compounds can disrupt cytoskel-
eton of the rapidly dividing tumor vessel endothelial cells. This can
>20
>20
11.0 ꢁ 1.0
>20
>20
>20
7.3 ꢁ 1.5
13.0 ꢁ 1.6
>20
8.8 ꢁ 1.0
>20
>20
9.9 ꢁ 1.0
9.2 ꢁ 0.6
>20
>20
>20
11j
8.8 ꢁ 1.4
11k
11m
11p
11q
11r
>20
>20
>20
16.7 ꢁ 2.4
15.5 ꢁ 0.9
>20
>20
Table 2
18.6 ꢁ 1.6
>20
6.9 ꢁ 0.4
>20
19.8 ꢁ 3.2
Effect of pyrrolo[3,4-h]quinazolines 8l, 8m, 8r and 8s on multidrug-resistant cell
line.
>20
10.9 ꢁ 0.7
>20
a
Cytotoxicity as GI50 for each cell line is the concentration of compound which
reduced by 50% the optical density of treated cells with respect to untreated cells
using the MTT assay.
GI50
CPD
(m
M)a
RIb
LoVoc
LoVodoxo
b
Cell lines include human chronic myelogenous leukemia (K562), T-cell leukemia
8l
5.0 ꢁ 0.4
5.4 ꢁ 0.3
9.9 ꢁ 1.8
7.1 ꢁ 0.7
0.12 ꢁ 0.03
15.2 ꢁ 1.0
8.6 ꢁ 0.1
9.5 ꢁ 2.5
12.5 ꢁ 1.9
13.5 ꢁ 0.1
3.0
1.6
1.0
1.8
112.5
(Jurkat), colon carcinoma (LoVo), breast adenocarcinoma (MCF-7) and squamous
carcinoma (A431) cells.
8m
8r
8s
Doxod
a
increase of cells in G2/M phase together with a concomitant
decrease of G1 phase cells. This profile is consistent with the one of
the antimitotic drug vinblastine, as a reference. Moreover, the onset
of a subG1 peak can be observed (Fig. 2, upper panel): subG1 phase
represents apoptotic cells and is a consequence of the ordered
degradation of DNA during cell death.
Cytotoxicity as GI50 for each cell line, is the concentration of compound which
reduced by 50% the optical density of treated cells with respect to untreated cells
using the MTT assay.
b
RI ¼ Resistance Index.
c
Cell lines include colon carcinoma (LoVo) and its doxorubicin resistant subclone
(LoVodoxo) cells.
d
Doxo ¼ doxorubicin.

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V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
Fig. 1. Pyrroloquinazoline effect on EGFR signaling. Cells were serum-starved and either treated for 10 min with 100 ng/mL EGF after 3 h of compound incubation. Cell lysates
(100 g) were separated by SDS-PAGE and the membrane probed with phospho-specific antibodies against the EGF receptor (P-Tyr1173) or AKT (Thr308) or ERK1/2 (P-Thr202/P-
Tyr204) (B). In addition, the membranes were probed with antibodies against total EGFR, AKT-1 and ERK1/2 (A). a) 1 (minus EGF), 2 (plus EGF), 3 (AG1478, 5
M þ EGF), 4 (8l,
20 M þ EGF) c) 1e3 same as a), 4 (11b, 20 M þ EGF), 5 (11e, 20 M þ EGF), 6 (11f, 20 M þ EGF).
M þ EGF) b) 1e3 same as a), 4 (8m, 20
m
m
m
m
m
m
m
affect their morphology and lead to interferences of cell-to-cell
junctional proteins, causing an increase in vascular permeability
and a final blood collapse [29]. These preliminary assays were
carried out using human umbilical vein endothelial cells, HUVECs.
First of all, a study of the effect of these molecules on cellular
motility was performed by a scratch test. HUVEC monolayers were
scratched with a tip and after different times of incubation in the
absence or presence of pyrroloquinazolines, the width of the
wound was measured. Quantitative results are shown in the upper
panel of Fig. 5, while in the lower panel representative micropho-
tographs of scratch with 8l and scratch control are shown.
the same figure as representative. Quantitative image analysis
showed that while 8l and 8s significantly decreased both dimen-
sional (percent of area covered by HUVECs, total length per field
and percent of area covered by meshes) and topological parameters
(number of mesh per field, number of branching points) of the
capillary-like network, 8m and 8r were mainly responsible for the
decrease of the number of meshes and the percent of their area. 11j
was not able to induce significant changes in HUVEC morphology.
3.7. Evaluation of the mechanism of cellular death
With the exception of 11j, all compounds were active at 20
m
M:
We performed a flow cytometry test to investigate the kind of
cellular death (necrosis or apoptosis) induced by pyrroloquina-
zolines. Changes to the plasma membrane are among the earliest
events of morphological changes in apoptosis. In apoptotic cells,
the membrane phospholipid phosphatidylserine (PS) is trans-
located from the inner to the outer leaflet of the plasma mem-
brane, thereby exposing PS to the external cellular environment
[31]. We performed bi-parametric cytofluorimetric analysis us-
ing propidium iodide (PI) and Annexin V-FITC conjugates, which
stain DNA and PS residues, respectively. Experiments were per-
formed in Jurkat cells after 6 and 24 h of incubation with the
most active compounds.
in fact, they inhibited the closure of scratch in comparison to
control (after 24 h, the less active compound 8r induced a closure
only of the 40%, while control was completely closed after 20 h). 8l
was still very active at 10
mM whereas the other compounds
reduced cell motility in a weaker way (after 24 h, they induced a
closure of 70e80% of the scratch).
The effect of compound in vessel disruption was evaluated
in vitro after growing HUVECs on Matrigel, which stimulates the
attachment, migration and differentiation of endothelial cells into
tubule like structures [30]. After the formation of these capillary-
like tubules, HUVECs were incubated with compounds at 20 mM.
Pictures of capillary network were taken after 3 h and analysed by
AngioJ plug-in of ImageJ software. Five parameters which are
associated with morphology of capillary network were analyzed
and results were presented in Fig. 6, upper panel. The pictures of
HUVECs control and treated with 8l are shown in the lower panel of
After 24 h, high percentage of treated cells presented both red
and green fluorescence (late apoptosis or necrosis), with the
exception of those treated with 10 mM 11j which were still in early
apoptosis (only green fluorescence) (Fig. 7, upper panel). As 11j is
the less cytotoxic, cell death could be a less rapid event. This

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
345
Table 3
3.7.1. Mitochondrial involvement in cellular death
Cell cycle analysis of Jurkat cells treated with compounds 8l, 8m, 8r, 8s, 11j and the
Mitochondria play an important role in apoptosis [32] and thus
some tests such as the assessment of membrane mitochondrial
potential and the production of reactive oxygen species were per-
formed. Impairment in mitochondrial function is an early event in
the executive phase of programmed cell death in various cell types,
and appears as the result of a preliminary decrease in the mito-
chondrial transmembrane potential (DjM). The determination of
DjM was carried out through a flow cytometric analysis using
5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine
(JC-1), as a probe. In normal cells (high DjM), JC-1 accumulates
selectively in mitochondria and, as a consequence of the electro-
chemical gradient, forms orange fluorescence aggregates, whereas
when the mitochondrial membrane is depolarized (low DjM), JC-1
forms monomers which emit at 530 nm [33].
reference compound vinblastine.
% G1
59.6
% S
% G2/M
22.4
Control
18.0
8l
10
20
10
20
mM
mM
mM
mM
25.5
12.8
32.6
18.9
57.4
50.7
55.9
42.3
36.7
27.4
15.2
24.9
8.4
49.6
78.8
47.4
64.0
26.1
31.6
28.9
44.1
51.1
64.7
74.8
8m
20.0
17.1
16.5
17.7
15.2
13.6
12.2
7.9
8r
5
mM
10
mM
8s
5
mM
10
10
20
m
m
m
M
M
M
11j
Vinblastine
20 nM
10.0
This assay was performed after 6 and 24 h of treatment with 8l,
8m, 8r, 8s and 11j and results are shown in Fig. 8, upper panel. After
the treatment with pyrroloquinazolines, a clear concentration- and
time-dependent increase of cells with collapsed mitochondrial
membrane potential was detected. The mitochondrial membrane
depolarization has been associated with mitochondrial production
of reactive oxygen species (ROS) [34]. The determination of ROS
generation during apoptosis was performed using the fluorescence
indicators hydroethidine (HE) and 20,70-dichlorodihydrofluorescein
diacetate (H2DCFDA), whose fluorescence appeared if reactive ox-
ygen species were produced [35]. This test was performed after
24 h of treatment of Jurkat cells with the selected compounds
(Fig. 8, lower panel). The production of ROS was clearly increased in
treated cells in comparison with control. As these data are consis-
tent with the ones of the previous experiments, we hypothesize a
mitochondrial involvement in cell death.
analysis was also performed after 6 h of treatment to verify
whether apoptosis was the main mechanism of cellular death. The
reduction of treatment time resulted in a higher occurrence of early
apoptotic cells (Fig. 7, lower panel).
3.7.2. Lysosomal involvement in cell death
To investigate the integrity of lysosomes after the treatment
with pyrroloquinazoline derivatives, flow cytometric analysis was
performed using the fluorescent dye acridine orange (AO). AO is a
lysosomotropic base and a metachromatic fluorochrome exhibiting
red fluorescence when highly concentrated, as in the case of intact
lysosomes. When lysosomes are damaged, AO relocates to the
cytosol and it shows green fluorescence [36]. The percentage of
cells with intact lysosomes was evaluated by assessing red fluo-
rescence after AO staining. Lysosomial integrity was assessed after
6 or 24 h of treatment of Jurkat cells with 8l, 8m, 8r, 8s and 11j
(Fig. 9). From these data, it is clear that lysosomes are also impli-
cated in pyrroloquinazoline apoptosis even if these organelles seem
to be involved in a later stage than mitochondria.
4. Conclusion
In conclusion, a new series of pyrrolo[3,4-h]quinazolines has
been successfully prepared by annelation of the pyrimidine ring on
the isoindole moiety. The synthetic pathway presented herein was
never described before in the isoindole series and proved to be
versatile allowing the preparation of a large number of derivatives.
Some of them showed an interesting cytotoxicity in many tumor
cell lines and were active even in a multi-drug resistant cell line.
The most cytotoxic derivatives 8l, 8m, 8r, 8s and 11j induced cell
death mainly by apoptosis, as demonstrated by the onset of the
apoptotic peak in cell cycle analysis and by the classical Annexin-V/
PI test. Moreover, the involvement of mitochondria and in a later
stage of lysosomes was demonstrated.
Fig. 2. Quantitative assessment of apoptotic cells and mitotic arrest by pyrroloquina-
zolines. Upper panel: Jurkat cells were treated with different concentrations of pyr-
roloquinazoline for 24 h. Then cell cycle analysis was performed and the percentage of
cells with DNA content lower than G1 phase was quantified. Lower panel: Jurkat cells
were treated with the compounds (20 mM) for 24 h. The mitotic index was calculated
as the proportion of cells with mitotic figures (400 cells/treatment). Data are expressed
as mean ꢁ SEM of three independent experiments.
Even if quinazoline moiety is often associated with the inhibi-
tion of EGFR or of other tyrosine kinases, we found out that the
most cytotoxic ones acted as antimitotics and inhibited tubulin
polymerization in vitro: in fact, other compounds with quinazoline

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V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
Fig. 3. Effects of pyrroloquinazolines on microtubule network in cells. Immunofluorescence images of LoVo cells treated with anti-
microscopy. Cells were either untreated or exposed to 20 M pyrroloquinazoline or 2 M vinblastine for 18 h.
b-tubulin-FITC and then observed by confocal
m
m
structure showed a similar mechanism as reported in Refs. [37,38].
Like other antimitotic drugs, these derivatives also presented some
antiangiogenic effects which could be useful for disrupting tumor
vascular network and thus inducing an extensive cell death in its
core.
5.1.1.2. 2-Benzyl-1-phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one
(5q). This compound was obtained from 5d in anhydrous DMF
and benzyl bromide, after 1 h. Yield: 90%; m.p. 128e129 ꢀC; IR
cmꢂ1: 1655 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 1.88e
1.99 (m, 2H, CH2), 2.37 (t, J ¼ 6.1 Hz, 2H, CH2), 2.56 (t, J ¼ 6.1 Hz,
2H, CH2), 5.19 (s, 2H, CH2), 6.88e6.93 (m, 2H, Ar), 6.95e7.45 (m,
8H, Ar), 7.54 (s, 1H, H-3); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
5. Experimental
d
21.4 (t), 24.7 (t), 39.9 (t), 50.5 (t), 120.8 (s), 123.3 (d), 124.6 (s),
5.1. Synthesis and characterization
126.8 (2ꢃ d), 127.4 (d), 127.5 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ d), 128.9
(s), 129.6 (2ꢃ d), 130.9 (s), 137.7 (s), 193.8 (s, CO). Anal calcd for
All melting points were taken on a Buchi-Tottoli capillary
apparatus and were uncorrected; IR spectra were determined, in
CHBr3, with a Jasco FT/IR 5300 spectrophotometer; 1H and 13C NMR
spectra were measured in DMSO-d6 or CDCl3 solutions, at 200 and
50.3 MHz respectively, using a Bruker Avance II series 200 MHz
spectrometer. Column chromatography was performed with Merck
silica gel 230e400 mesh ASTM or with a SEPACORE chromatog-
raphy apparatus BÜCHI. Elemental analyses (C, H, N) were within
ꢁ0.4% of the theoretical values.
C21H19NO: C, 83.69; H, 6.35; N, 4.65. Found: C, 83.45; H, 6.01; N,
4.88.
5.1.1.3. 2-(4-Methylbenzyl)-1-phenyl-2,5,6,7-tetrahydro-4H-iso-
indol-4-one (5r). This compound was obtained from 5d in anhy-
drous DMF and 4-methylbenzyl chloride, after 1 h. Yield: 85%; m.p.
122e123 ꢀC; IR cmꢂ1: 1653 (CO); 1H NMR (DMSO-d6, 200 MHz,
ppm):
d
1.86e1.98 (m, 2H, CH2), 2.22 (s, 3H, CH3), 2.36 (t, J ¼ 6.0 Hz,
2H, CH2), 2.55 (t, J ¼ 6.0 Hz, 2H, CH2), 5.13 (s, 2H, CH2), 6.81 (d,
J ¼ 7.7 Hz, 2H, H-30 and H-50), 7.05 (d, J ¼ 7.7 Hz, 2H, H-20 and H-60),
7.28e7.46 (m, 5H, Ar), 7.51 (s, 1H, H-3); 13C NMR (DMSO-d6,
5.1.1. General procedure for the preparation of 1-phenyl-2,5,6,7-
tetrahydro-4H-isoindol-4-one (5pes)
50.3 MHz, ppm):
d 20.6 (q), 21.4 (t), 24.7 (t), 38.9 (t), 50.3 (t), 120.7
To a solution of 5d (18 mmol) in anhydrous THF or DMF (22 mL),
sodium hydride (0.9 g, 39 mmol) was added at 0 ꢀC. After 1 h
stirring at room temperature the iodomethane or the proper aryl
halide (20 mmol) was added at 0 ꢀC. The reaction mixture was
stirred at room temperature for 1e2 h then it was poured onto
crushed ice. The solid was filtered off, dried and purified by chro-
matography using dichloromethane:ethyl acetate (98:2) as eluent.
(s),123.2 (d),124.6 (s), 126.8 (2ꢃ d),127.5 (d),128.5 (2ꢃ d), 128.8 (s),
129.0 (2ꢃ d), 129.5 (2ꢃ d), 131.0 (s), 134.6 (s), 136.6 (s), 193.8 (s, CO).
Anal calcd for C22H21NO: C, 83.78; H, 6.71; N, 4.44. Found: C, 83.63;
H, 6.91; N, 4.67.
5.1.1.4. 2-(4-Methoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-4H-iso-
indol-4-one (5s). This compound was obtained from 5d in anhy-
drous DMF and 4-methoxybenzyl chloride, after 2 h. Yield: 85%;
m.p. 135e136 ꢀC; IR cmꢂ1: 1655 (CO); 1H NMR (DMSO-d6, 200 MHz,
5.1.1.1. 2-Methyl-1-phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one
(5p). This compound was obtained from 5d in anhydrous THF and
iodomethane, after 1 h. Yield: 90%; m.p. 139e140 ꢀC; IR cmꢂ1: 1653
ppm):
d
1.85e1.97 (m, 2H, CH2), 2.36 (t, J ¼ 6.1 Hz, 2H, CH2), 2.55 (t,
J ¼ 6.1 Hz, 2H, CH2), 3.68 (s, 3H, CH3), 5.10 (s, 2H, CH2), 6.79 (d,
J ¼ 8.9 Hz, 2H, H-30 and H-50), 6.87 (d, J ¼ 8.9 Hz, 2H, H-20 and H-60),
7.28e7.47 (m, 5H, Ar), 7.49 (s, 1H, H-3); 13C NMR (DMSO-d6,
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.90e1.94 (m, 2H, CH2),
2.35e2.58 (m, 4H, 2ꢃ CH2), 3.61 (s, 3H, CH3), 7.41e7.46 (m, 6H, Ar
and H-3); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
21.7 (t), 24.8 (t),
d
50.3 MHz, ppm): d 21.4 (t), 24.7 (t), 38.9 (t), 50.0 (t), 55.0 (q), 113.8
35.2 (q), 38.9 (t),120.3 (s),124.0 (d),124.3 (s), 127.2 (d),128.5 (2ꢃ d),
(2ꢃ d), 120.7 (s), 123.0 (d), 124.6 (s), 127.5 (d), 128.4 (2ꢃ d), 128.5
(2ꢃ d), 128.7 (s), 129.4 (s), 129.6 (2ꢃ d), 131.0 (s), 158.6 (s), 193.7 (s,
CO). Anal calcd for C22H21NO2: C, 79.73; H, 6.39; N, 4.23. Found: C,
80.01; H, 6.12; N, 4.45.
128.8 (s), 129.4 (2ꢃ d), 130.9 (s), 193.6 (s, CO). Anal calcd for
C
15H15NO: C, 79.97; H, 6.71; N, 6.22. Found: C, 79.84; H, 6.89; N,
6.05.

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
347
Fig. 4. Effects of pyrroloquinazolines on microtubule assembly. Purified tubulin was incubated in the absence or in the presence of the compound at the indicated concentrations.
Fluorescence measurement (
IF ¼ intensity of fluorescence.
l
ex ¼ 355 nm and
l
em ¼ 450 nm) was determined every minute for 60 min at 37 ꢀC. Polymerization was initiated by the addition of tubulin.
5.1.2. General procedure for the preparation of 5-
(hydroxymethylidene)-2,5,6,7-tetrahydro-4H-isoindol-4-one (6je
m,pes)
evaporated under reduced pressure. The residue was purified by
chromatography using dichloromethane:ethyl acetate (95:5) as
eluent.
To a suspension of t-BuOK (36 mmol) in anhydrous toluene
(30 mL), a solution of 5pes (12 mmol) in anhydrous toluene
(40 mL) was added dropwise under N2 at 0 ꢀC. After 3 h stirring at
room temperature the reaction was cooled at 0 ꢀC and a solution of
ethyl formate (36 mmol) in toluene (20 mL) was added and the
mixture was kept stirring at room temperature for 24 h, then the
solvent was removed under vacuum. The residue was dissolved in
water and the solution was washed with diethyl ether. The aqueous
solution was then acidified with 6 M HCl and extracted with
dichloromethane. The organic extracts were dried (Na2SO4) and
5.1.2.1. Ethyl 5-(hydroxymethylidene)-2,3-dimethyl-4-oxo-4,5,6,7-
tetrahydro-2H-isoindole-1-carboxylate (6j). This compound was
obtained from 5j. Yield: 72%; m.p. 63e65 ꢀC; IR cmꢂ1: 2976 (OH),
1689 (CO), 1635 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
d 1.37 (t,
J ¼ 7.3 Hz, 3H, CH3), 2.45 (t, J ¼ 6.3 Hz, 2H, CH2), 2.59 (s, 3H, CH3),
2.94 (t, J ¼ 6.3 Hz, 2H, CH2), 3.81 (s, 3H, CH3), 4.30 (q, J ¼ 7.3 Hz, 2H,
CH2), 7.42 (s, 1H, CH), 14.34 (bs, 1H, OH); (CDCl3, 50.3 MHz, ppm):
d
11.3 (q), 14.3 (q), 22.6 (t), 25.0 (t), 32.5 (q), 59.8 (t), 109.1 (s), 117.3
(s), 134.4 (s), 140.0 (s), 161.5 (s), 164.2 (d), 187.6 (s, CO), 200.1 (s, CO).

348
V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
Fig. 5. Pyrroloquinazolines inhibit HUVEC migration. Confluent HUVEC monolayers were scratch wounded and then they were treated with 10 and 20
different times cells were photographed (10ꢃ magnification) and the scratch width was quantified (upper panel). Data are presented as the mean ꢁ SEM of three independent
experiments. Lower panel shows representative pictures for 20 M 8l and control after 0 and 20 h from the scratch.
mM of compounds. After
m
Anal calcd for C14H17NO4: C, 63.87; H, 6.51; N, 5.32. Found: C, 63.60;
H, 6.78; N, 5.05.
117.7 (s), 125.7 (2ꢃ d), 127.2 (d), 128.7 (2ꢃ d), 134.9 (s), 137.1 (s),
140.4 (s), 161.3 (s), 164.8 (d), 187.7 (s, CO), 201.2 (s, CO). Anal calcd
for C20H21NO4: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.65; H, 6.52; N,
4.35.
5.1.2.2. Ethyl
2-benzyl-5-(hydroxymethylidene)-3-methyl-4-oxo-
4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate (6k). This compound
was obtained from 5k. Yield: 80%; m.p. 78e79 ꢀC; IR cmꢂ1: 2981
5.1.2.3. Ethyl 5-(hydroxymethylidene)-3-methyl-2-(4-methylbenzyl)-
(OH), 1691 (CO), 1635 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
1.58
4-oxo-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate
compound was obtained from 5l. Yield: 60%; m.p. 90e91 ꢀC; IR
cmꢂ1 2978 (OH), 1691 (CO), 1635 (CO); 1H NMR (CDCl3,
200 MHz, ppm):
(6l). This
(t, J ¼ 7.2 Hz, 3H, CH3), 2.77 (t, J ¼ 6.9 Hz, 2H, CH2), 2.85 (s, 3H, CH3),
3.31 (t, J ¼ 6.9 Hz, 2H, CH2), 4.52 (q, J ¼ 7.2 Hz, 2H, CH2), 5.92 (s, 2H,
CH2), 7.24 (d, J ¼ 7.2 Hz, 2H, H-20 and H-60), 7.51e7.62 (m, 3H, H-30,
H-40 and H-50), 7.76 (s,1H, CH),14.66 (bs,1H, OH); (CDCl3, 50.3 MHz,
:
d
1.30 (t, J ¼ 7.1 Hz, 3H, CH3), 2.30 (s, 3H, CH3),
2.51 (t, J ¼ 6.9 Hz, 2H, CH2), 2.56 (s, 3H, CH3), 3.01 (t, J ¼ 6.9 Hz,
2H, CH2), 4.23 (q, J ¼ 7.1 Hz, 2H, CH2), 5.58 (s, 2H, CH2), 6.85 (d,
ppm): d 11.5 (q), 14.2 (q), 22.8 (t), 25.0 (t), 48.2 (t), 60.0 (t), 109.3 (s),

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
349
Fig. 6. Disruption of capillary-like structures by pyrroloquinazolines. HUVEC cells were incubated on Matrigel until the formation of capillary-like tubes. The disrupting effects of
20 M pyrroloquinazolines was quantified in microscope pictures after 3 h of incubation. Upper panel shows the quantitative effect of compounds on dimensional and topological
m
parameters of HUVEC morphology in comparison to time 0 h. Data are presented as the mean ꢁ SEM of three independent experiments. Lower panel shows representative pictures
(10ꢃ magnification) of 8l treated cells and control.
J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.09 (d, J ¼ 8.0 Hz, 2H, H-20 and H-
5.1.2.6. 2-Benzyl-5-(hydroxymethylidene)-1-phenyl-2,5,6,7-
tetrahydro-4H-isoindol-4-one (6q). This compound was obtained
from 5q. Yield: 85%; oil; IR cmꢂ1: 2931 (OH), 1637 (CO); 1H NMR
60), 7.46 (s, 1H, CH), 14.37 (bs, 1H, OH); (CDCl3, 50.3 MHz, ppm):
d
11.6 (q), 14.3 (q), 21.0 (q), 22.9 (t), 25.1 (t), 48.0 (t), 60.0 (t), 109.3
(s), 117.7 (s), 117.9 (s), 125.8 (2ꢃ d), 129.4 (2ꢃ d), 134.1 (s), 134.9
(s), 136.9 (s), 140.5 (s), 161.4 (s, CO), 164.7 (d), 187.8 (s, CO). Anal
calcd for C21H23NO4: C, 71.37; H, 6.56; N, 3.96. Found: C, 71.39; H,
6.37; N, 4.19.
(CDCl3, 200 MHz, ppm):
d
2.49 (t, J ¼ 6.0 Hz, 2H, CH2), 2.62 (t,
J ¼ 6.0 Hz, 2H, CH2), 5.05 (s, 2H, CH2), 6.96e7.01 (m, 2H, Ar), 7.19e
7.39 (m, 9H, Ar and H-3), 7.53 (s, 1H, CH), 14.38 (bs, 1H, OH); 13C
NMR (CDCl3, 50.3 MHz, ppm):
d 21.1 (t), 25.8 (t), 51.4 (t), 109.5 (s),
120.5 (s), 123.1 (d), 123.3 (s), 127.0 (2ꢃ d), 127.9 (2ꢃ d), 128.6 (2ꢃ d),
128.8 (2ꢃ d), 130.0 (2ꢃ d), 130.1 (s), 130.9 (s), 136.9 (s), 166.0 (d),
186.7 (s, CO). Anal calcd for C22H19NO2: C, 80.22; H, 5.81; N, 4.25.
Found: C, 80.37; H, 6.00; N, 4.09.
5.1.2.4. Ethyl 5-(hydroxymethylidene)-2-(4-methoxybenzyl)-3-
methyl-4-oxo-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylate (6m).
This compound was obtained from 5m. Yield: 65%; m.p. 128e
129 ꢀC; IR cmꢂ1: 2973 (OH), 1678 (CO), 1628 (CO); 1H NMR (CDCl3,
200 MHz, ppm):
d
1.41 (t, J ¼ 7.3 Hz, 3H, CH3), 2.80 (s, 3H, CH3),
5.1.2.7. 5-(Hydroxymethylidene)-2-(4-methylbenzyl)-1-phenyl-
2,5,6,7-tetrahydro-4H-isoindol-4-one (6r). This compound was ob-
tained from 5r. Yield: 77%; oil; IR cmꢂ1: 2929 (OH), 1633 (CO); 1H
3.70e3.92 (m, 7H, 2ꢃ CH2 and CH3), 4.36 (q, J ¼ 7.3 Hz, 2H, CH2),
5.87 (s, 2H, CH2), 6.79 (d, J ¼ 7.2 Hz, 2H, H-30 and H-50), 6.92 (d,
J ¼ 7.2 Hz, 2H, H-20 and H-60), 7.61 (s, 1H, CH), 13.37 (bs, 1H, OH);
NMR (CDCl3, 200 MHz, ppm):
d
2.30 (s, 3H, CH3), 2.49 (t, J ¼ 6.3 Hz,
(CDCl3, 50.3 MHz, ppm):
d
12.5 (q), 14.5 (q), 29.7 (t), 48.8 (t), 55.3
2H, CH2), 2.61 (t, J ¼ 6.3 Hz, 2H, CH2), 5.00 (s, 2H, CH2), 6.89 (d,
J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.08 (d, J ¼ 8.0 Hz, 2H, H-20 and H-60),
7.19e7.44 (m, 6H, Ar and H-3), 7.52 (s, 1H, CH), 14.39 (bs, 1H, OH);
(q), 59.9 (t), 60.0 (t), 111.7 (s), 112.8 (s), 114.2 (2ꢃ d), 126.6 (s), 127.4
(2ꢃ d), 128.5 (s), 132.4 (s), 136.2 (s), 158.9 (s), 161.6 (s, CO), 163.7 (d),
194.9 (s, CO). Anal calcd for C21H23NO5: C, 68.28; H, 6.28; N, 3.79.
Found: C, 67.88; H, 6.54; N, 3.57.
13C NMR (CDCl3, 50.3 MHz, ppm):
d 21.0 (q), 21.1 (t), 25.8 (t), 51.2 (t),
109.5 (s), 120.4 (s), 123.0 (d), 123.2 (s), 127.1 (2ꢃ d), 127.8 (d), 128.5
(2ꢃ d), 128.6 (s), 129.5 (2ꢃ d), 130.1 (2ꢃ d), 131.0 (s), 133.8 (s), 137.6
(s), 166.0 (d), 186.6 (s, CO). Anal calcd for C23H21NO2: C, 80.44; H,
6.16; N, 4.08. Found: C, 80.12; H, 6.41; N, 4.26.
5.1.2.5. 5-(Hydroxymethylidene)-2-methyl-1-phenyl-2,5,6,7-
tetrahydro-4H-isoindol-4-one (6p). This compound was obtained
from 5p. Yield: 86%; m.p. 114e115 ꢀC; IR cmꢂ1: 2931 (OH), 1635
(CO); 1H NMR (CDCl3, 200 MHz, ppm):
d
2.49 (t, J ¼ 6.5 Hz, 2H, CH2),
5.1.2.8. 5-(Hydroxymethylidene)-2-(4-methoxybenzyl)-1-phenyl-
2,5,6,7-tetrahydro-4H-isoindol-4-one (6s). This compound was ob-
tained from 5s. Yield: 70%; m.p. 106e107 ꢀC; IR cmꢂ1: 2927 (OH),
2.63 (t, J ¼ 6.5 Hz, 2H, CH2), 3.61 (s, 3H, CH3), 7.26e7.50 (7H, m, Ar,
H-3 and CH), 14.39 (bs, 1H, OH); 13C NMR (CDCl3, 50.3 MHz, ppm):
d
21.2 (t), 25.9 (t), 35.6 (q), 109.5 (s), 120.0 (s), 123.3 (s), 123.8 (d),
1645 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
CH2), 2.61 (t, J ¼ 6.2 Hz, 2H, CH2), 3.77 (s, 3H, CH3), 4.97 (s, 2H, CH2),
6.78 (d, J ¼ 8.7 Hz, 2H, H-30 and H-50), 6.93 (d, J ¼ 8.7 Hz, 2H, H-20
and H-60), 7.2e7.45 (m, 6H, Ar and H-3), 7.52 (s, 1H, CH), 14.39 (bs,
d
2.48 (t, J ¼ 6.2 Hz, 2H,
127.7 (d), 128.6 (2ꢃ d), 128.8 (2ꢃ d), 129.9 (s), 131.0 (s), 165.5 (d),
186.8 (s, CO). Anal calcd for C16H15NO2: C, 75.87; H, 5.97; N, 5.53.
Found: C, 76.05; H, 6.21; N, 5.35.

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V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
1H, OH); 13C NMR (CDCl3, 50.3 MHz, ppm):
d 21.1 (t), 25.8 (t), 51.0
(t), 55.3 (q), 109.5 (s), 114.2 (2ꢃ d), 120.3 (s), 122.9 (d), 123.3 (s),
127.8 (d), 128.5 (2ꢃ d),128.6 (2ꢃ d), 128.8 (s), 129.9 (s), 130.1 (2ꢃ d),
131.1 (s), 159.3 (s), 165.9 (d), 186.7 (s, CO). Anal calcd for C23H21NO3:
C, 76.86; H, 5.89; N, 3.90. Found: C, 76.53; H, 6.06; N, 3.77.
5.1.3. General procedure for the preparation of 5-[(diethylamino)
methylidene]-1-phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (7pe
s)
To a solution of diethylamine (2.5 mL, 25 mmol) in anhydrous
toluene (30 mL), a solution of 6pes (25 mmol) in anhydrous
toluene (30 mL) was added dropwise under N2 at 0 ꢀC. After 24 h
stirring at room temperature the solvent was removed and the
residue was washed with diethyl ether and filtered off.
5.1.3.1. 5-[(Diethylamino)methylidene]-2-methyl-1-phenyl-2,5,6,7-
tetrahydro-4H-isoindol-4-one (7p). This compound was obtained
from 6p. Yield: 90%; oil; IR cmꢂ1: 1637 (CO); 1H NMR (DMSO-d6,
200 MHz, ppm):
d
1.15 (t, J ¼ 7.1 Hz, 6H, 2ꢃ CH3), 2.55 (t, J ¼ 6.1 Hz,
2H, CH2), 2.71 (t, J ¼ 6.1 Hz, 2H, CH2), 3.30 (q, J ¼ 7.1 Hz, 4H, 2ꢃ CH2),
3.60 (s, 3H, CH3), 7.27e7.48 (m, 7H, Ar, H-3 and CH); 13C NMR
(DMSO-d6, 50.3 MHz, ppm):
d
14.6 (2ꢃ q), 21.6 (t), 24.9 (t), 35.0 (q),
47.2 (2ꢃ t),102.6 (s),122.0 (s),122.5 (s),123.5 (d),126.8 (d),127.8 (s),
128.4 (2ꢃ d), 129.2 (2ꢃ d), 131.2 (s), 144.9 (d), 182.7 (s, CO). Anal
calcd for C20H24N2O: C, 77.89; H, 7.84; N, 9.08. Found: C, 77.66; H,
7.74; N, 9.29.
5.1.3.2. 2-Benzyl-5-[(diethylamino)methylidene]-1-phenyl-2,5,6,7-
tetrahydro-4H-isoindol-4-one (7q). This compound was obtained
from 6q. Yield: 92%; oil; IR cmꢂ1: 1635 (CO); 1H NMR (DMSO-d6,
200 MHz, ppm):
d
1.14 (t, J ¼ 7.0 Hz, 6H, 2ꢃ CH3), 2.55 (t, J ¼ 6.0 Hz,
2H, CH2), 2.73 (t, J ¼ 6.0 Hz, 2H, CH2), 3.33 (q, J ¼ 7.0 Hz, 4H, 2ꢃ
CH2), 5.18 (s, 2H, CH2), 6.91e6.95 (m, 2H, Ar), 7.22e7.43 (m, 10H, Ar,
H-3 and CH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
14.6 (2ꢃ q),
21.4 (t), 24.8 (t), 47.3 (2ꢃ t), 50.3 (t), 102.7 (s), 122.6 (s), 122.8 (d),
126.7 (2ꢃ d), 126.8 (s), 127.1 (d), 127.3 (d), 127.9 (s), 128.4 (2ꢃ d),
128.5 (2ꢃ d), 129.4 (2ꢃ d), 131.3 (s), 138.2 (s), 145.1 (d), 182.8 (s, CO).
Anal calcd for C26H28N2O: C, 81.21; H, 7.34; N, 7.29. Found: C, 81.42;
H, 7.57; N, 7.02.
Fig. 7. Percentage of Annexin-V positive and Annexin-V and PI positive cells after 24 h
(upper panel) and 6 h (lower panel) of incubation at the indicated concentrations of
compounds. Data are expressed as mean ꢁ SEM of three independent experiments.
5.1.3.3. 5-[(Diethylamino)methylidene]-2-(4-methylbenzyl)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (7r). This compound
was obtained from 6r. Yield: 95%; oil; IR cmꢂ1: 1633 (CO); 1H
NMR (DMSO-d6, 200 MHz, ppm):
d
1.14 (t, J ¼ 7.1 Hz, 6H, 2ꢃ CH3),
(2ꢃ d), 129.9 (s), 130.9 (s), 131.4 (s), 145.1 (d), 158.5 (s), 182.8 (s, CO).
Anal calcd for C27H30N2O2: C, 78.23; H, 7.29; N, 6.76. Found: C,
78.51; H, 7.10; N, 6.99.
2.22 (s, 3H, CH3), 2.54 (t, J ¼ 6.1 Hz, 2H, CH2), 2.73 (t, J ¼ 6.1 Hz,
2H, CH2), 3.30 (q, J ¼ 7.1 Hz, 4H, 2ꢃ CH2), 5.11 (s, 2H, CH2), 6.82
(d, J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.05 (d, J ¼ 8.0 Hz, 2H, H-20 and
H-60), 7.25e7.45 (m, 7H, Ar, H-3 and CH); 13C NMR (DMSO-d6,
5.1.4. General procedure for the preparation of pyrrolo[3,4-h]
quinazolin-2-one (9ees)
50.3 MHz, ppm):
d
14.6 (2ꢃ q), 21.6 (q), 22.4 (t), 25.9 (t), 48.3 (2ꢃ
t), 55.9 (t), 103.7 (s), 123.5 (s), 123.7 (s), 127.8 (2ꢃ d), 128.1 (d),
128.9 (s), 129.3 (2ꢃ d), 129.5 (d), 130.0 (2ꢃ d), 130.4 (2ꢃ d), 132.3
(s), 136.2 (s), 137.5 (s), 146.1 (d), 183.8 (s, CO). Anal calcd for
To a suspension of the suitable hydroxymethylketones 6ees
(3 mmol) in anhydrous ethanol urea (6 mmol) was added under N2.
The reaction mixture was heated under reflux for 24 h. Then the
reaction mixture was cooled at room temperature and the solid
formed was filtered off and purified by flash chromatography using
dichloromethane:methanol (98:2) as eluent.
C
27H30N2O: C, 81.37; H, 7.59; N, 7.03. Found: C, 81.18; H, 7.71; N,
6.83.
5.1.3.4. 5-[(Diethylamino)methylidene]-2-(4-methoxybenzyl)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (7s). This compound
was obtained from 6s. Yield: 96%; oil; IR cmꢂ1: 1635 (CO); 1H NMR
5.1.4.1. 8-Methyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinazolin-2-
one (9e). This compound was obtained from 6e. Yield: 62%; m.p.
250e251 ꢀC; IR cmꢂ1: 3160 (NH), 1703 (CO); 1H NMR (DMSO-d6,
(DMSO-d6, 200 MHz, ppm):
d
1.13 (t, J ¼ 7.0 Hz, 6H, 2ꢃ CH3), 2.49e
2.55 (m, 4H, 2ꢃ CH2), 3.29 (q, J ¼ 7.0 Hz, 4H, 2ꢃ CH2), 3.69 (s, 3H,
CH3), 5.10 (s, 2H, CH2), 6.80 (d, J ¼ 8.7 Hz, 2H, H-30 and H-50), 6.89 (d,
J ¼ 8.7 Hz, 2H, H-20 and H-60), 7.29e7.51 (m, 6H, Ar and H-3), 7.71 (s,
200 MHz, ppm):
H-7), 7.18e7.26 (m, 2H, H-4 and H-9), 10.63 (d, J ¼ 11.0 Hz, 1H, NH);
13C NMR (DMSO-d6, 50.3 MHz, ppm):
21.4 (t), 30.2 (t), 36.1 (q),
d
2.54 (s, 4H, 2ꢃ CH2), 3.63 (s, 3H, CH3), 6.55 (s, 1H,
d
1H, CH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
14.6 (2ꢃ q), 20.5
109.0 (s), 118.1 (d), 122.7 (s), 123.2 (d), 124.9 (s), 134.7 (d), 154.6 (s),
185.7 (s, CO). Anal calcd for C11H11N3O: C, 65.66; H, 5.51; N, 20.88.
Found: C, 65.88; H, 5.35; N, 21.02.
(t), 24.9 (t), 47.3 (2ꢃ t), 50.0 (t), 55.0 (q),102.7 (s), 113.9 (2ꢃ d),122.8
(s), 123.3 (s), 128.3 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ d), 129.4 (d), 129.6

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
351
Fig. 9. Assessment of lysosomal dysfunction after the treatment with pyrroloquina-
zolines. Jurkat cells were treated for 6 or 24 h with pyrroloquinazolines at the indicated
concentrations, were stained then with AO (acridine orange) and analysed by flow
cytometry. Data are expressed as mean ꢁ SEM of three independent experiments.
(d), 119.8 (d), 120.0 (2ꢃ d), 124.8 (s), 126.3 (s), 126.4 (d), 129.7 (2ꢃ
d), 135.6 (d), 139.2 (s), 154.5 (s), 185.8 (s, CO). Anal calcd for
C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C, 73.31; H, 4.63; N,
15.86.
5.1.4.4. 8-(4-Methylbenzyl)-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinazolin-2-one (9h). This compound was obtained from 6h.
Yield: 60%; m.p. 259e260 ꢀC; IR cmꢂ1: 3153 (NH), 1709 (CO); 1H
NMR (DMSO-d6, 200 MHz, ppm):
d
2.27 (s, 3H, CH3), 2.53 (s, 4H, 2ꢃ
CH2), 5.06 (s, 2H, CH2), 6.63 (s, 1H, H-7), 7.12e7.24 (m, 5H, Ar and H-
4), 7.40 (s, 1H, H-9), 10.61 (d, J ¼ 11.0 Hz, 1H, NH); 13C NMR (DMSO-
d6, 50.3 MHz, ppm):
d 21.2 (q), 22.2 (t), 30.6 (t), 52.9 (t), 109.4 (s),
112.2 (s), 117.5 (d), 122.8 (d), 123.3 (s), 125.5 (s), 128.2 (2ꢃ d), 129.6
Fig. 8. Assessment of mitochondrial dysfunction after treatment with pyrroloquina-
zolines. Upper panel: induction of loss of mitochondrial membrane potential after 6
and 24 h of incubation of Jurkat cells by pyrroloquinazolines at the indicated con-
centrations. Cells were stained with the fluorescent probe JC-1(5,50,6,60-tetrachloro-
1,10,3,30-tetraethylbenzimidazol-carbocyanine) and analyzed by flow cytometry. Lower
panel: mitochondrial production of ROS in Jurkat cells. After 24 h of incubation, cells
were stained with HE (Hydroethidine) and with H2DCFDA (20,70-dichlorodihydro-
fluorescein diacetate) and analyzed by flow cytometry. Data are expressed as
mean ꢁ SEM of three independent experiments.
(2ꢃ d), 135.4 (d), 137.4 (s), 155.1 (s), 186.3 (s, CO). Anal calcd for
C18H17N3O: C, 74.20; H, 5.88; N, 14.42. Found: C, 74.03; H, 6.10; N,
14.25.
5.1.4.5. 8-(4-Methoxybenzyl)-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinazolin-2-one (9i). This compound was obtained from 6i. Yield:
55%; m.p. 233e234 ꢀC; IR cmꢂ1: 3166 (NH), 1711 (CO); 1H NMR
(DMSO-d6, 200 MHz, ppm):
d
2.52 (s, 4H, 2ꢃ CH2), 3.73 (s, 3H, CH3),
5.03 (s, 2H, CH2), 6.64 (s, 1H, H-7), 6.89e7.70 (m, 6H, Ar, H-4 and H-
9), 10.60 (d, J ¼ 10.9 Hz, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz,
5.1.4.2. 8-Benzyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinazolin-2-
one (9f). This compound was obtained from 6f. Yield: 64%; m.p.
240e241 ꢀC; IR cmꢂ1: 3167 (NH), 1716 (CO); 1H NMR (DMSO-d6,
ppm):
d
21.4 (t), 30.2 (t), 52.1 (t), 55.1 (q), 108.9 (s), 114.0 (2ꢃ d),
116.9 (d), 122.2 (d), 122.7 (s), 125.0 (s), 129.2 (2ꢃ d), 134.7 (d), 154.6
(s), 158.8 (s), 166.9 (s), 185.8 (s, CO). Anal calcd for C18H17N3O2: C,
70.34; H, 5.58; N, 13.67. Found: C, 70.19; H, 5.79; N, 13.35.
200 MHz, ppm):
H-7), 7.20e7.43 (m, 7H, Ar, H-4 and H-9), 10.63 (d, J ¼ 11.0 Hz, 1H,
NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
21.5 (t), 30.2 (t), 52.6
d
2.54 (s, 4H, 2ꢃ CH2), 5.12 (s, 2H, CH2), 6.66 (s, 1H,
d
(t), 109.0 (s), 117.2 (d), 122.5 (d), 122.9 (s), 125.1 (s), 127.6 (2ꢃ d),
127.7 (d), 128.6 (2ꢃ d), 134.8 (d), 137.9 (s), 154.6 (s), 185.8 (s, CO).
Anal calcd for C17H15N3O: C, 73.63; H, 5.45; N, 15.15. Found: C,
73.85; H, 5.33; N, 14.98.
5.1.4.6. Ethyl 8,9-dimethyl-2-oxo-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-
h]quinazoline-7-carboxylate (9j). This compound was obtained
from 6j. Yield: 60%; m.p. 268e269 ꢀC; IR cmꢂ1: 3397 (NH), 1691
(CO), 1649 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 1.29 (t,
J ¼ 6.9 Hz, 3H, CH3), 2.51e2.57 (m, 5H, CH3 and CH2), 2.88 (t,
J ¼ 6.2 Hz, 2H, CH2), 3.76 (s, 3H, CH3), 4.22 (q, J ¼ 6.9 Hz, 2H, CH2),
7.25 (d, J ¼ 10.9 Hz, 1H, H-4), 10.64 (d, J ¼ 10.9 Hz, 1H, NH); 13C NMR
5.1.4.3. 8-Phenyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinazolin-2-
one (9g). This compound was obtained from 6g. Yield: 66%; m.p.
223e224 ꢀC; IR cmꢂ1: 3405 (NH), 1675 (CO); 1H NMR (DMSO-d6,
(DMSO-d6, 50.3 MHz, ppm): d 11.0 (q), 14.2 (q), 23.1 (t), 29.1 (t), 32.3
200 MHz, ppm):
d
2.60e2.65 (m, 4H, 2ꢃ CH2), 7.25e7.70 (m, 7H, Ar,
(q), 59.6 (t),108.4 (s),116.7 (s), 118.9 (s), 134.0 (s),135.2 (d),140.0 (s),
154.6 (s), 160.9 (s, CO), 186.2 (s, CO). Anal calcd for C15H17N3O3: C,
62.71; H, 5.96; N, 14.63. Found: C, 62.95; H, 5.79; N, 14.40.
H-4 and H-7), 7.85 (s, 1H, H-9), 10.68 (d, J ¼ 11.1 Hz, 1H, NH); 13C
NMR (DMSO-d6, 50.3 MHz, ppm): d 21.4 (t), 29.9 (t), 108.8 (s), 115.6

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V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
5.1.4.7. Ethyl 8-benzyl-9-methyl-2-oxo-3,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinazoline-7-carboxylate (9k). This compound was ob-
tained from 6k. Yield: 60%; m.p. 272e273 ꢀC; IR cmꢂ1: 3398 (NH),
5.1.4.12. 8-Methyl-7-phenyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinazolin-2-one (9p). This compound was obtained from 6p.
Yield: 60%; m.p. 263e264 ꢀC; IR cmꢂ1: 3305 (NH), 1660 (CO); 1H
1712 (CO), 1682 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.22 (t,
NMR (DMSO-d6, 200 MHz, ppm):
d
2.55 (s, 4H, 2ꢃ CH2), 3.63 (s, 3H,
J ¼ 6.8 Hz, 3H, CH3), 2.51 (s, 3H, CH3), 2.59 (t, J ¼ 6.0 Hz, 2H, CH2),
2.95 (t, J ¼ 6.0 Hz, 2H, CH2), 4.16 (q, J ¼ 6.8 Hz, 2H, CH2), 5.64 (s, 2H,
CH2), 6.94e7.32 (m, 6H, Ar and H-4), 10.65 (d, J ¼ 11.3 Hz, 1H, NH);
CH3), 7.24 (d, J ¼ 11.0 Hz, 1H, H-4), 7.33e7.51 (m, 6H, Ar and H-9),
10.66 (d, J ¼ 11.0 Hz, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
22.3 (t), 30.6 (t), 35.7 (q), 109.4 (s), 122.4 (s), 123.4 (s), 124.8 (d),
13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
11.0 (q), 14.1 (q), 23.2 (t),
127.7 (d), 129.0 (2ꢃ d), 129.2 (2ꢃ d), 129.9 (s), 131.3 (s), 135.3 (d),
155.1 (s), 186.2 (s, CO). Anal calcd for C17H15N3O: C, 73.63; H, 5.45;
N, 15.15. Found: C, 73.95; H, 5.67; N, 14.94.
29.0 (t), 47.4 (t), 59.7 (t), 108.4 (s), 116.4 (s), 119.4 (s), 125.8 (2ꢃ d),
127.0 (d), 128.6 (2ꢃ d), 134.6 (s), 135.5 (d), 137.6 (s), 140.1 (s), 154.5
(s), 160.7 (s, CO), 186.3 (s, CO). Anal calcd for C21H21N3O3: C, 69.41;
H, 5.82; N, 11.56. Found: C, 69.64; H, 5.65; N, 11.62.
5.1.4.13. 8-Benzyl-7-phenyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinazolin-2-one (9q). This compound was obtained from 6q.
Yield: 62%; m.p. 253e254 ꢀC; IR cmꢂ1: 3322 (NH), 1702 (CO); 1H
5.1.4.8. Ethyl 9-methyl-8-(4-methylbenzyl)-2-oxo-3,5,6,8-
tetrahydro-2H-pyrrolo[3,4-h]quinazoline-7-carboxylate
(9l).
NMR (DMSO-d6, 200 MHz, ppm):
d
2.56 (s, 4H, 2ꢃ CH2), 5.21 (s, 2H,
This compound was obtained from 6l. Yield: 70%; m.p. 261e262 ꢀC;
CH2), 6.90e7.44 (m, 11H, Ar and H-4), 7.54 (s, 1H, H-9), 10.64 (d,
IR cmꢂ1: 3401 (NH), 1712 (CO), 1678 (CO); 1H NMR (DMSO-d6,
J ¼ 11.1 Hz, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 21.6 (t),
200 MHz, ppm):
d
1.23 (t, J ¼ 7.1 Hz, 3H, CH3), 2.25 (s, 3H, CH3), 2.51
30.0 (t), 50.5 (t), 108.9 (s), 122.4 (s), 123.2 (s), 123.7 (d), 126.6 (2ꢃ d),
127.3 (d),127.5 (d),128.4 (2ꢃ d),128.5 (2ꢃ d),128.7 (s),129.5 (2ꢃ d),
131.0 (s), 135.1 (d), 138.0 (s), 154.6 (s), 185.8 (s, CO). Anal calcd for
(s, 3H, CH3), 2.58 (t, J ¼ 6.3 Hz, 2H, CH2), 2.94 (t, J ¼ 6.3 Hz, 2H, CH2),
4.17 (q, J ¼ 7.1 Hz, 2H, CH2), 5.59 (s, 2H, CH2), 6.85 (d, J ¼ 8.0 Hz, 2H,
H-30 and H-50), 7.12 (d, J ¼ 8.0 Hz, 2H, H-20 and H-60), 7.29 (d,
J ¼ 11.1 Hz, 1H, H-4), 10.66 (d, J ¼ 11.1 Hz, 1H, NH); 13C NMR (DMSO-
C23H19N3O: C, 78.16; H, 5.42; N, 11.89. Found: C, 78.34; H, 5.53; N,
11.51.
d6, 50.3 MHz, ppm): d 11.0 (q),14.1 (q), 20.6 (q), 23.2 (t), 29.0 (t), 47.2
(t), 59.6 (t), 108.3 (s), 116.4 (s), 119.4 (s), 125.8 (2ꢃ d), 129.1 (2ꢃ d),
134.5 (s), 134.6 (s). 135.4 (d), 136.1 (s), 140.0 (s), 154.5 (s), 160.7 (s,
CO), 186.3 (s, CO). Anal calcd for C22H23N3O3: C, 70.01; H, 6.14; N,
11.13. Found: C, 70.24; H, 5.95; N, 10.99.
5.1.4.14. 8-(4-Methylbenzyl)-7-phenyl-3,5,6,8-tetrahydro-2H-pyrrolo
[3,4-h]quinazolin-2-one (9r). This compound was obtained from 6r.
Yield: 75%; m.p. 261e262 ꢀC; IR cmꢂ1: 3315 (NH), 1701 (CO); 1H
NMR (DMSO-d6, 200 MHz, ppm):
d
2.22 (s, 3H, CH3), 2.55 (s, 4H, 2ꢃ
CH2), 5.15 (s, 2H, CH2), 6.81 (d, J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.06 (d,
J ¼ 8.0 Hz, 2H, H-20 and H-60), 7.21e7.46 (m, 6H, Ar and H-4), 7.51 (s,
1H, H-9), 10.63 (d, J ¼ 11.1 Hz, 1H, NH); 13C NMR (DMSO-d6,
5.1.4.9. Ethyl 8-(4-methoxybenzyl)-9-methyl-2-oxo-3,5,6,8-
tetrahydro-2H-pyrrolo[3,4-h]quinazoline-7-carboxylate
(9m).
This compound was obtained from 6m. Yield: 60%; m.p. 250e
50.3 MHz, ppm): d 20.6 (q), 21.6 (t), 30.3 (t), 50.3 (t), 108.9 (s), 122.4
251 ꢀC; IR cmꢂ1: 3388 (NH), 1708 (CO), 1684 (CO); 1H NMR
(s),123.2 (s), 123.6 (d), 126.7 (2ꢃ d),127.5 (d), 128.5 (2ꢃ d),128.7 (s),
129.0 (2ꢃ d), 129.5 (2ꢃ d), 130.9 (s), 134.8 (s), 135.0 (d), 136.5 (s),
154.6 (s), 185.8 (s, CO). Anal calcd for C24H21N3O: C, 78.45; H, 5.76;
N, 11.44. Found: C, 78.77; H, 5.65; N, 11.13.
(DMSO-d6, 200 MHz, ppm):
d
1.24 (t, J ¼ 7.1 Hz, 3H, CH3), 2.53 (s,
3H, CH3), 2.58 (t, J ¼ 6.1 Hz, 2H, CH2), 2.94 (t, J ¼ 6.1 Hz, 2H, CH2),
3.71 (s, 3H, CH3), 4.18 (q, J ¼ 7.1 Hz, 2H, CH2), 5.56 (s, 2H, CH2),
6.87 (d, J ¼ 9.1 Hz, 2H, H-30 and H-50), 6.93 (d, J ¼ 9.1 Hz, 2H, H-20
and H-60), 7.28 (d, J ¼ 11.1 Hz, 1H, H-4), 10.65 (d, J ¼ 11.1 Hz, 1H,
5.1.4.15. 8-(4-Methoxybenzyl)-7-phenyl-3,5,6,8-tetrahydro-2H-pyr-
rolo[3,4-h]quinazolin-2-one (9s). This compound was obtained
from 6s. Yield: 60%; m.p. 252e253 ꢀC; IR cmꢂ1: 3320 (NH), 1703
NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 11.1 (q), 14.1 (q),
23.2 (t), 29.0 (t), 46.8 (t), 55.0 (q), 59.7 (t), 108.3 (s), 114.0 (2ꢃ d),
116.3 (s), 119.4 (s), 127.3 (2ꢃ d), 129.4 (s), 134.5 (s), 135.4 (d),
140.0 (s), 154.5 (s), 158.3 (s), 160.8 (s, CO), 186.3 (s, CO). Anal calcd
for C22H23N3O4: C, 67.16; H, 5.89; N, 10.68. Found: C, 67.02; H,
6.14; N, 10.49.
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.54 (s, 4H, 2ꢃ CH2),
3.69 (s, 3H, CH3), 5.12 (s, 2H, CH2), 6.78 (d, J ¼ 8.1 Hz, 2H, H-30 and
H-50), 6.88 (d, J ¼ 8.1 Hz, 2H, H-20 and H-60), 7.21e7.48 (m, 6H, Ar
and H-4), 7.50 (s, 1H, H-9), 10.62 (d, J ¼ 11.2 Hz, 1H, NH); 13C NMR
(DMSO-d6, 50.3 MHz, ppm):
d 21.6 (t), 30.0 (t), 50.0 (t), 55.0 (q),
5.1.4.10. 8,9-Dimethyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]quinazo-
lin-2-one (9n). This compound was obtained from 6n. Yield: 60%;
m.p. 260e261 ꢀC; IR cmꢂ1: 3394 (NH), 1704 (CO); 1H NMR
108.9 (s), 113.9 (2ꢃ d), 122.3 (s), 123.2 (s), 123.4 (d), 127.5 (d), 128.2
(2ꢃ d), 128.5 (2ꢃ d), 128.7 (s), 129.6 (2ꢃ d), 129.7 (s), 130.9 (s), 135.0
(d), 154.6 (s), 158.5 (s), 185.8 (s, CO). Anal calcd for C24H21N3O2: C,
75.18; H, 5.52; N, 10.96. Found: C, 75.37; H, 5.29; N, 10.82.
(DMSO-d6, 200 MHz, ppm):
d
2.47 (s, 3H, CH3), 2.50 (s, 4H, 2ꢃ
CH2), 3.48 (s, 3H, CH3), 6.45 (s, 1H, H-7), 7.17 (d, J ¼ 10.9 Hz, 1H,
H-4), 10.71 (d, J ¼ 10.9 Hz, 1H, NH); 13C NMR (DMSO-d6,
5.1.5. General procedure for the preparation of pyrrolo[3,4-h]
quinazolin-2-amine (10b,e,f,hem,pes)
50.3 MHz, ppm):
d 11.0 (q), 22.1 (t), 30.9 (t), 33.3 (q), 110.1 (s),
116.9 (d), 119.0 (s), 123.4 (s), 133.7 (s), 134.6 (d), 155.2 (s), 187.2 (s,
CO). Anal calcd for C12H13N3O: C, 66.96; H, 6.09; N, 19.52. Found:
C, 66.78; H, 6.32; N, 19.69.
To a suspension of NaOMe (30 mmol) in anhydrous ethanol
(12 mL) guanidine nitrate (15 mmol) was added. A solution of the
suitable dialkylamino derivative 7 (3 mmol) in anhydrous ethanol
(12 mL) was added dropwise to the suspension and the reaction
mixture was heated under reflux for the suitable time. The reaction
mixture was cooled and dried under reduced pressure, then it was
added with water and ice. The solid formed was filtered off and
purified by flash chromatography using dichloromethane:ethyl
acetate (9:1) as eluent.
5.1.4.11. 8-Benzyl-9-methyl-3,5,6,8-tetrahydro-2H-pyrrolo[3,4-h]
quinazolin-2-one (9o). This compound was obtained from 6o.
Yield: 60%; m.p. 255e256 ꢀC; IR cmꢂ1: 3388 (NH), 1676 (CO); 1H
NMR (DMSO-d6, 200 MHz, ppm):
d
2.44 (s, 3H, CH3), 2.53 (s, 4H, 2ꢃ
CH2), 5.10 (s, 2H, CH2), 6.60 (s, 1H, H-7), 7.10e7.39 (m, 6H, Ar and H-
4), 10.71 (d, J ¼ 10.9 Hz, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz,
ppm):
d
10.9 (q), 21.7 (t), 30.4 (t), 49.2 (t), 109.6 (s), 116.2 (d), 123.4
5.1.5.1. Ethyl 2-amino-9-methyl-6,8-dihydro-5H-pyrrolo[3,4-h]qui-
nazoline-7-carboxylate (10b). This compound was obtained from
7b after 2 h. Yield: 50%; m.p. 253e254 ꢀC; IR cmꢂ1: 3292 (NH),
3280e3161 (NH2), 1666 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
(s), 127.0 (2ꢃ d), 127.5 (d), 128.7 (2ꢃ d), 133.0 (s), 134.5 (d), 137.7 (s),
154.8 (s), 159.8 (s), 187.0 (s, CO). Anal calcd for C18H17N3O: C, 74.20;
H, 5.88; N, 14.42. Found: C, 74.33; H, 5.76; N, 14.09.

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
353
d
1.29 (t, J ¼ 7.1 Hz, 3H, CH3), 2.59 (s, 3H, CH3), 2.62 (t, J ¼ 7.5 Hz, 2H,
5.1.5.7. Ethyl
[3,4-h]quinazoline-7-carboxylate (10k). This compound was ob-
tained from 7k after 4 h. Yield: 60%; m.p. 143e144 ꢀC; IR cmꢂ1
3511e3409 (NH2), 1687 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
2-amino-8-benzyl-9-methyl-6,8-dihydro-5H-pyrrolo
CH2), 2.88 (t, J ¼ 7.5 Hz, 2H, CH2), 4.24 (q, J ¼ 7.1 Hz, 2H, CH2), 6.11 (s,
2H, NH2), 7.95 (s, 1H, H-4), 11.74 (s, 1H, NH); 13C NMR (DMSO-d6,
:
50.3 MHz, ppm):
d 12.8 (q), 14.4 (q), 20.6 (t), 24.4 (t), 59.3 (t), 114.9
(s), 115.3 (s),116.5 (s), 130.4 (s), 134.0 (s), 155.1 (d),159.0 (s), 160.6 (s,
CO), 162.4 (s). Anal calcd for C14H16N4O2: C, 61.75; H, 5.92; N, 20.58.
Found: C, 61.95; H, 5.79; N, 20.33.
d
1.22 (t, J ¼ 7.1 Hz, 3H, CH3), 2.63e2.70 (m, 5H, CH2 and CH3), 2.98
(t, J ¼ 7.7 Hz, 2H, CH2), 4.16 (q, J ¼ 7.1 Hz, 2H, CH2), 5.65 (s, 2H, CH2),
6.21 (s, 2H, NH2), 6.94 (d, J ¼ 6.7 Hz, 2H, H-20 and H-60), 6.97e7.36
(m, 3H, H-30, H-40 and H-50), 8.00 (s, 1H, H-4); 13C NMR (DMSO-d6,
5.1.5.2. 8-Methyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazolin-2-amine
(10e). This compound was obtained from 7e after 5 h. Yield: 60%;
m.p. 206e207 ꢀC; IR cmꢂ1: 3515e3409 (NH2); 1H NMR (DMSO-d6,
50.3 MHz, ppm): d 11.2 (q), 14.1 (q), 21.5 (t), 24.1 (t), 47.3 (t), 59.5 (t),
115.1 (s), 116.1 (s), 116.7 (s), 125.6 (2ꢃ d), 126.8 (d), 128.6 (2ꢃ d),
132.0 (s), 136.6 (s), 138.1 (s), 155.5 (d), 158.4 (s), 160.8 (s, CO), 170.3
(s). Anal calcd for C21H22N4O2: C, 69.59; H, 6.12; N, 15.46. Found: C,
69.37; H, 5.85; N, 15.68.
200 MHz, ppm):
NH2), 6.56 (s, 1H, H-7), 7.13 (s, 1H, H-9), 7.93 (s, 1H, H-4); 13C NMR
(DMSO-d6, 50.3 MHz, ppm): 20.4 (t), 25.6 (t), 36.5 (q), 115.4 (s),
d
2.59 (s, 4H, 2ꢃ CH2), 3.63 (s, 3H, CH3), 6.19 (s, 2H,
d
118.7 (d), 119.6 (s), 120.1 (d), 122.4 (s), 155.7 (d), 158.8 (s), 163.1 (s).
Anal calcd for C11H12N4: C, 65.98; H, 6.04; N, 27.98. Found: C, 65.79;
H, 5.86; N, 28.35.
5.1.5.8. Ethyl
2-amino-9-methyl-8-(4-methylbenzyl)-6,8-dihydro-
5H-pyrrolo[3,4-h]quinazoline-7-carboxylate (10l). This compound
was obtained from 7l after 5 h. Yield: 62%; m.p. 122e123 ꢀC; IR
cmꢂ1
:
3513e3409 (NH2), 1687 (CO); 1H NMR (DMSO-d6,
5.1.5.3. 8-Benzyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazolin-2-amine
(10f). This compound was obtained from 7f after 3 h. Yield: 52%;
m.p. 148e149 ꢀC; IR cmꢂ1: 3365e3299 (NH2); 1H NMR (DMSO-d6,
200 MHz, ppm):
d
1.23 (t, J ¼ 7.1 Hz, 3H, CH3), 2.25 (s, 3H, CH3),
2.62e2.69 (m, 5H, CH2 and CH3), 2.97 (t, J ¼ 7.2 Hz, 2H, CH2), 4.16
(q, J ¼ 7.1 Hz, 2H, CH2), 5.59 (s, 2H, CH2), 6.17 (s, 2H, NH2), 6.84 (d,
J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.11 (d, J ¼ 8.0 Hz, 2H, H-20 and H-
200 MHz, ppm):
NH2), 6.68 (s, 1H, H-7), 7.26e7.35 (m, 6H, Ar and H-9), 7.94 (s, 1H, H-
4); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
20.4 (t), 25.5 (t), 53.0 (t),
d
2.59 (s, 4H, 2ꢃ CH2), 5.11 (s, 2H, CH2), 6.20 (s, 2H,
60), 8.00 (s, 1H, H-4); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 11.2
d
(q), 14.1 (q), 20.6 (q), 21.5 (t), 24.2 (t), 47.1 (t), 59.5 (t), 115.2 (s),
116.1 (s), 116.7 (s), 125.6 (2ꢃ d), 129.1 (2ꢃ d), 131.9 (s), 135.1 (s),
135.9 (s), 136.6 (s), 155.4 (d), 158.4 (s), 160.8 (s, CO), 162.3 (s).
Anal calcd for C22H24N4O2: C, 70.19; H, 6.43; N, 14.88. Found: C,
69.97; H, 6.59; N, 14.72.
115.4 (s), 117.9 (d), 119.3 (s), 119.8 (d), 122.6 (s), 128.1 (d), 128.2 (2ꢃ
d), 129.1 (2ꢃ d), 138.8 (s), 155.8 (d), 158.7 (s), 163.1 (s). Anal calcd for
C
17H16N4: C, 73.89; H, 5.84; N, 20.27. Found: C, 73.63; H, 5.99; N,
20.38.
5.1.5.4. 8-(4-Methylbenzyl)-6,8-dihydro-5H-pyrrolo[3,4-h]quinazo-
lin-2-amine (10h). This compound was obtained from 7h after 4 h.
Yield: 56%; m.p. 154e155 ꢀC; IR cmꢂ1: 3313e3168 (NH2); 1H NMR
5.1.5.9. Ethyl 2-amino-8-(4-methoxybenzyl)-9-methyl-6,8-dihydro-
5H-pyrrolo[3,4-h]quinazoline-7-carboxylate (10m). This compound
was obtained from 7m after 2 h. Yield: 55%; m.p. 236e237 ꢀC; IR
cmꢂ1: 3375e3315 (NH2), 1639 (CO); 1H NMR (DMSO-d6, 200 MHz,
(DMSO-d6, 200 MHz, ppm):
d
2.27 (s, 3H, CH3), 2.58 (s, 4H, 2ꢃ CH2),
5.04 (s, 2H, CH2), 6.12 (s, 2H, NH2), 6.65 (s, 1H, H-7), 7.13e7.22 (m,
ppm):
d
1.24 (t, J ¼ 7.1 Hz, 3H, CH3), 2.61e2.68 (m, 5H, CH2 and CH3),
5H, H-20, H-30, H-50, H-60 and H-9), 7.92 (s, 1H, H-4); 13C NMR
2.96 (t, J ¼ 7.3 Hz, 2H, CH2), 3.70 (s, 3H, CH3), 4.17 (q, J ¼ 7.1 Hz, 2H,
CH2), 5.56 (s, 2H, CH2), 6.17 (s, 2H, NH2), 6.84e6.96 (m, 4H, H-20, H-
30, H-50 and H-60), 7.99 (s, 1H, H-4); 13C NMR (DMSO-d6, 50.3 MHz,
(DMSO-d6, 50.3 MHz, ppm):
d 20.4 (t), 21.1 (q), 25.5 (t), 52.8 (t),
115.9 (s), 118.0 (d), 119.1 (d), 119.8 (s), 122.5 (s), 128.2 (2ꢃ d), 129.6
(2ꢃ d),135.7 (s),140.1 (s),155.7 (d),159.1 (s),163.5 (s). Anal calcd for
ppm): d 11.3 (q), 14.2 (q), 21.5 (t), 24.2 (t), 46.8 (t), 55.0 (q), 59.5 (t),
C
18H18N4: C, 74.46; H, 6.25; N, 19.30. Found: C, 74.32; H, 6.02; N,
114.0 (2ꢃ d),114.6 (s),115.2 (s),116.1 (s),116.7 (s), 127.1 (2ꢃ d),129.9
(s), 131.9 (s), 136.5 (s), 155.5 (d), 158.2 (s), 160.8 (s, CO), 162.3 (s).
Anal calcd for C22H24N4O3: C, 67.33; H, 6.16; N, 14.28. Found: C,
67.49; H, 6.28; N, 14.06.
19.66.
5.1.5.5. 8-(4-Methoxybenzyl)-6,8-dihydro-5H-pyrrolo[3,4-h]quina-
zolin-2-amine (10i). This compound was obtained from 7i after
3 h. Yield: 65%; m.p. 163e164 ꢀC; IR cmꢂ1: 3294e3151 (NH2); 1H
5.1.5.10. 8-Methyl-7-phenyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazo-
lin-2-amine (10p). This compound was obtained from 7p after 5 h.
Yield: 75%; m.p. 234e235 ꢀC; IR cmꢂ1: 3384e3294 (NH2); 1H NMR
NMR (DMSO-d6, 200 MHz, ppm):
d
2.58 (s, 4H, 2ꢃ CH2), 3.73 (s,
3H, CH3), 5.01 (s, 2H, CH2), 6.12 (s, 2H, NH2), 6.66 (s, 1H, H-7),
6.91 (d, J ¼ 8.7 Hz, 2H, H-30 and H-50), 7.21 (s, 1H, H-9), 7.26 (d,
J ¼ 8.7 Hz, 2H, H-20 and H-60), 7.92 (s, 1H, H-4); 13C NMR (DMSO-
(DMSO-d6, 200 MHz, ppm):
6.15 (s, 2H, NH2), 7.32 (s, 1H, H-9), 7.35e7.51 (m, 5H, Ar), 7.96 (s, 1H,
H-4); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
20.2 (t), 25.1 (t), 35.1
d
2.61 (s, 4H, 2ꢃ CH2), 3.63 (s, 3H, CH3),
d6, 50.3 MHz, ppm):
d
20.4 (t), 25.5 (t), 52.5 (t), 55.6 (q), 114.4 (2ꢃ
d
d), 115.4 (s), 117.6 (d), 119.0 (d), 119.7 (s), 122.4 (s), 129.8 (2ꢃ d),
(q), 115.1 (s), 118.5 (s), 120.2 (s), 120.8 (d), 127.0 (d), 128.4 (2ꢃ d),
129.0 (s), 129.3 (2ꢃ d), 131.2 (s), 155.4 (d), 158.2 (s), 162.7 (s). Anal
calcd for C17H16N4: C, 73.89; H, 5.84; N, 20.27. Found: C, 73.69; H,
5.99; N, 20.06.
130.7 (s), 155.8 (d), 158.7 (s), 159.3 (s), 163.1 (s). Anal calcd for
C
18H18N4O: C, 70.57; H, 5.92; N, 18.29. Found: C, 70.67; H, 6.11; N,
18.08.
5.1.5.6. Ethyl 2-amino-8,9-dimethyl-6,8-dihydro-5H-pyrrolo[3,4-h]
quinazoline-7-carboxylate (10j). This compound was obtained from
7j after 2 h. Yield: 55%; m.p. 227e228 ꢀC; IR cmꢂ1: 3300e3184
5.1.5.11. 8-Benzyl-7-phenyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazo-
lin-2-amine (10q). This compound was obtained from 7q after
2 h. Yield: 65%; m.p. 210e211 ꢀC; IR cmꢂ1: 3325e3174 (NH2); 1H
(NH2), 1685 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.30 (t,
NMR (DMSO-d6, 200 MHz, ppm):
5.19 (s, 2H, CH2), 6.18 (s, 2H, NH2), 6.96e7.47 (m, 11H, Ar and H-
9), 7.97 (s, 1H, H-4); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
20.0
d
2.59e2.67 (m, 4H, 2ꢃ CH2),
J ¼ 7.1 Hz, 3H, CH3), 2.60 (t, J ¼ 6.8 Hz, 2H, CH2), 2.72 (s, 3H, CH3),
2.91 (t, J ¼ 6.8 Hz, 2H, CH2), 3.35 (s, 3H, CH3), 4.23 (q, J ¼ 7.1 Hz, 2H,
CH2), 6.18 (s, 2H, NH2), 7.96 (s, 1H, H-4); 13C NMR (DMSO-d6,
d
(t), 24.9 (t), 50.3 (t), 115.2 (s), 119.1 (s), 120.0 (d), 120.5 (s), 126.9
(2ꢃ d), 127.2 (d), 127.3 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ d), 129.1 (s),
129.5 (2ꢃ d), 131.2 (s), 138.2 (s), 155.6 (d), 158.0 (s), 162.7 (s). Anal
calcd for C23H20N4: C, 78.38; H, 5.72; N, 15.90. Found: C, 78.16; H,
5.87; N, 16.09.
50.3 MHz, ppm):
d 11.3 (q), 14.3 (q), 21.4 (t), 24.3 (t), 32.2 (q), 59.4
(t),115.0 (s),115.5 (s),116.9 (s),131.2 (s),136.6 (s),155.4 (d),158.5 (s),
161.0 (s, CO), 162.3 (s). Anal calcd for C15H18N4O2: C, 62.92; H, 6.34;
N, 19.57. Found: C, 63.15; H, 6.17; N, 19.33.

354
V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
5.1.5.12. 8-(4-Methylbenzyl)-7-phenyl-6,8-dihydro-5H-pyrrolo[3,4-
h]quinazolin-2-amine (10r). This compound was obtained from 7r
after 5 h. Yield: 55%; m.p. 205e207 ꢀC; IR cmꢂ1: 3276e3151 (NH2);
d6, 200 MHz, ppm):
d
2.65e2.70 (m, 4H, 2ꢃ CH2), 5.16 (s, 2H, CH2),
6.73 (s, 1H, H-7), 6.88 (t, J ¼ 7.3 Hz, 1H, Ar), 7.21e7.39 (m, 8H, H-9
and Ar), 7.82 (d, J ¼ 7.8 Hz, 2H, Ar), 8.16 (s, 1H, H-4), 9.25 (s, 1H, NH);
1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.23 (s, 3H, CH3), 2.55e2.60
13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 19.7 (t), 25.0 (t), 52.5 (t),
(m, 4H, 2ꢃ CH2), 5.13 (s, 2H, CH2), 6.19 (s, 2H, NH2), 6.89 (d,
J ¼ 8.0 Hz, 2H, H-30 and H-50), 7.08 (d, J ¼ 8.0 Hz, 2H, H-20 and H-60),
7.30e7.48 (m, 6H, Ar and H-9), 7.97 (s,1H, H-4); 13C NMR (DMSO-d6,
117.2 (s), 117.7 (d), 118.1 (2ꢃ d), 119.1 (d), 120.3 (d), 122.1 (s), 127.5
(2ꢃ d), 127.6 (d), 128.3 (2ꢃ d), 128.6 (2ꢃ d), 136.1 (s), 138.3 (s), 141.3
(s), 155.0 (d), 158.2 (s), 159.0 (s). Anal calcd for C23H20N4: C, 78.38;
H, 5.72; N, 15.90. Found: C, 78.57; H, 5.88; N, 15.74.
50.3 MHz, ppm): d 20.0 (t), 20.6 (q), 24.9 (t), 50.1 (t), 108.6 (s), 115.1
(s), 119.0 (s), 119.8 (d), 120.5 (s), 127.0 (2ꢃ d), 127.2 (d), 128.5 (2ꢃ d),
129.1 (2ꢃ d), 129.5 (2ꢃ d), 131.2 (s), 135.1 (s), 136.5 (s), 155.5 (d),
158.0 (s), 162.7 (s). Anal calcd for C24H22N4: C, 78.66; H, 6.05; N,
15.29. Found: C, 78.50; H, 6.19; N, 15.48.
5.1.6.4. N,8-Diphenyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazolin-2-
amine (11g). This compound was obtained from 7g after 1.5 h.
Yield: 60%; m.p. 229e230 ꢀC; IR cmꢂ1: 3253 (NH); 1H NMR
(DMSO-d6, 200 MHz, ppm):
d
2.65e2.80 (m, 4H, 2ꢃ CH2), 6.90 (t,
5.1.5.13. 8-(4-Methoxybenzyl)-7-phenyl-6,8-dihydro-5H-pyrrolo
[3,4-h]quinazolin-2-amine (10s). This compound was obtained
from 7s after 2 h. Yield: 75%; m.p. 215e216 ꢀC; IR cmꢂ1: 3319e3168
J ¼ 7.3 Hz, 1H, Ar), 7.24e7.35 (m, 4H, Ar), 7.51 (t, J ¼ 7.8 Hz, 2H,
Ar), 7.67 (d, J ¼ 7.7 Hz, 2H, Ar), 7.81e7.88 (m, 1H, Ar, H-7 and H-
9), 8.26 (s, 1H, H-4), 9.34 (s, 1H, NH); 13C NMR (DMSO-d6,
(NH2); 1H NMR (DMSO-d6, 200 MHz, ppm):
3.70 (s, 3H, CH3), 5.09 (s, 2H, CH2), 6.17 (s, 2H, NH2), 6.83 (d,
J ¼ 8.6 Hz, 2H, H-30 and H-50), 6.94 (d, J ¼ 8.6 Hz, 2H, H-20 and H-60),
7.28e7.48 (m, 6H, Ar and H-9), 7.96 (s, 1H, H-4); 13C NMR (DMSO-
d
2.60 (s, 4H, 2ꢃ CH2),
50.3 MHz, ppm): d 19.7 (t), 24.9 (t), 116.1 (d), 116.4 (d), 117.7 (s),
118.2 (2ꢃ d), 119.8 (2ꢃ d), 120.5 (d), 121.5 (s), 124.0 (s), 126.0 (d),
128.4 (2ꢃ d), 129.8 (2ꢃ d), 139.5 (s), 141.2 (s), 155.6 (d), 157.7 (s),
159.1 (s). Anal calcd for C22H18N4: C, 78.08; H, 5.36; N, 16.56.
Found: C, 78.29; H, 5.08; N, 16.45.
d6, 50.3 MHz, ppm):
d
20.0 (t), 25.0 (t), 49.8 (t), 55.5 (q),113.9 (2ꢃ d),
115.1 (s), 119.0 (s), 119.6 (d), 120.5 (s), 127.2 (d), 128.5 (2ꢃ d), 128.6
(2ꢃ d), 129.0 (s), 129.5 (2ꢃ d), 129.9 (s), 131.3 (s), 155.5 (d), 158.0 (s),
158.5 (s), 162.6 (s). Anal calcd for C24H22N4O: C, 75.37; H, 5.80; N,
14.65. Found: C, 75.51; H, 6.00; N, 14.82.
5.1.6.5. 8-(4-Methylbenzyl)-N-phenyl-6,8-dihydro-5H-pyrrolo[3,4-h]
quinazolin-2-amine (11h). This compound was obtained from 7h
after 1.5 h. Yield: 65%; m.p. 216e217 ꢀC; IR cmꢂ1: 3280 (NH); 1H
NMR (DMSO-d6, 200 MHz, ppm): d 2.29 (s, 3H, CH3), 2.61e2.74 (m,
5.1.6. General procedure for the preparation of 2-(phenylamino)-
pyrrolo[3,4-h]quinazoline (11b,eem,pes)
4H, 2ꢃ CH2), 5.10 (s, 2H, CH2), 6.71 (s,1H, H-7), 6.88 (t, J ¼ 7.3 Hz,1H,
Ar), 7.15e7.29 (m, 6H, Ar), 7.37 (s, 1H, H-9), 7.82 (d, J ¼ 7.4 Hz, 2H,
Ar), 8.16 (s, 1H, H-4), 9.26 (s, 1H, NH); 13C NMR (DMSO-d6,
To a suspension of the suitable dialkylamino derivative 7
(1.5 mmol) in anhydrous DMF (7 mL) phenylguanidine (4.5 mmol)
was added. The reaction mixture was heated at 100 ꢀC for the
suitable time. The reaction mixture was poured onto crushed ice
and the formed solid was filtered off and recrystallized from
ethanol.
50.3 MHz, ppm):
d 19.7 (t), 20.7 (q), 25.1 (t), 52.3 (t), 117.1 (s), 117.6
(d), 118.1 (2ꢃ d), 119.0 (s), 119.1 (d), 120.3 (d), 122.1 (s), 127.7 (2ꢃ d),
128.3 (2ꢃ d), 129.1 (2ꢃ d), 135.2 (s), 136.8 (s), 141.3 (s), 154.9 (d),
158.3 (s), 159.0 (s). Anal calcd for C24H22N4: C, 78.66; H, 6.05; N,
15.29. Found: C, 78.89; H, 5.96; N, 15.45.
5.1.6.1. Ethyl
[3,4-h]quinazoline-7-carboxylate (11b). This compound was ob-
tained from 7b after 1.5 h. Yield: 65%; m.p. 253e254 ꢀC; IR cmꢂ1
3298 (NH), 3280 (NH), 1674 (CO); 1H NMR (DMSO-d6, 200 MHz,
ppm):
J ¼ 7.1 Hz, 2H, CH2), 2.95 (t, J ¼ 7.1 Hz, 2H, CH2), 4.26 (q, J ¼ 7.1 Hz,
2H, CH2), 6.90 (t, J ¼ 7.3 Hz, 1H, H-400), 7.26 (t, J ¼ 7.3 Hz, 2H, H-300
and H-500), 7.79 (d, J ¼ 7.3 Hz, 2H, H-200 and H-600), 8.18 (s, 1H, H-4),
9.21 (s, 1H, NH), 11.87 (s, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz,
9-methyl-2-(phenylamino)-6,8-dihydro-5H-pyrrolo
5.1.6.6. 8-(4-Methoxybenzyl)-N-phenyl-6,8-dihydro-5H-pyrrolo[3,4-
h]quinazolin-2-amine (11i). This compound was obtained from 7i
after 1.5 h. Yield: 77%; m.p. 200e201 ꢀC; IR cmꢂ1: 3283 (NH); 1H
:
NMR (DMSO-d6, 200 MHz, ppm):
d
2.59e2.73 (m, 4H, 2ꢃ CH2), 3.74
d
1.31 (t, J ¼ 7.1 Hz, 3H, CH3), 2.67 (s, 3H, CH3), 2.73 (t,
(s, 3H, CH3), 5.08 (s, 2H, CH2), 6.72 (s, 1H, H-7), 6.85e6.97 (m, 3H,
Ar), 7.21e7.32 (m, 4H, Ar), 7.36 (s, 1H, H-9), 7.82 (d, J ¼ 7.6 Hz, 2H,
Ar), 8.16 (s, 1H, H-4), 9.26 (s, 1H, NH); 13C NMR (DMSO-d6,
50.3 MHz, ppm):
d
19.7 (t), 25.1 (t), 52.0 (t), 55.1 (q), 114.0 (2ꢃ d),
117.1 (s), 117.5 (d), 118.1 (2ꢃ d), 118.9 (d), 119.0 (s), 120.3 (d), 122.1
(s), 128.3 (2ꢃ d), 129.2 (2ꢃ d), 130.1 (s), 141.3 (s), 154.9 (d), 158.3 (s),
158.8 (s), 159.0 (s). Anal calcd for C24H22N4O: C, 75.37; H, 5.80; N,
14.65. Found: C, 75.16; H, 5.67; N, 14.80.
ppm): d 13.0 (q),14.4 (q), 20.4 (t), 24.5 (t), 59.4 (t),115.5 (s),116.6 (s),
117.1 (s), 118.6 (2ꢃ d), 120.6 (d), 128.2 (2ꢃ d), 130.5 (s), 134.2 (s),
141.0 (s), 155.0 (d), 158.7 (s), 158.9 (s), 160.6 (s, CO). Anal calcd for
C
20H20N4O2: C, 68.95; H, 5.79; N, 16.08. Found: C, 68.88; H, 5.66; N,
16.23.
5.1.6.7. Ethyl 8,9-dimethyl-2-(phenylamino)-6,8-dihydro-5H-pyrrolo
[3,4-h]quinazoline-7-carboxylate (11j). This compound was ob-
tained from 7j after 2 h. Yield: 75%; m.p. 172e173 ꢀC; IR cmꢂ1: 3260
5.1.6.2. 8-Methyl-N-phenyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazo-
lin-2-amine (11e). This compound was obtained from 7e after 2 h.
Yield: 60%; m.p. 203e204 ꢀC; IR cmꢂ1: 3287 (NH); 1H NMR (DMSO-
(NH), 1679 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 1.30 (t,
J ¼ 7.0 Hz, 3H, CH3), 2.69 (t, J ¼ 6.8 Hz, 2H, CH2), 2.74 (s, 3H, CH3),
2.94 (t, J ¼ 6.8 Hz, 2H, CH2), 3.78 (s, 3H, CH3), 4.23 (q, J ¼ 7.0 Hz, 2H,
CH2), 6.91 (t, J ¼ 7.7 Hz,1H, H-400), 7.26 (t, J ¼ 7.7 Hz, 2H, H-300 and H-
500), 7.75 (d, J ¼ 7.9 Hz, 2H, H-200 and H-600), 8.18 (s, 1H, H-4), 9.23 (s,
d6, 200 MHz, ppm):
d
2.61e2.74 (m, 4H, 2ꢃ CH2), 3.67 (s, 3H, CH3),
6.61 (s,1H, H-7), 6.88 (t, J ¼ 7.3 Hz,1H, H-400), 7.22e7.30 (m, 3H, H-9,
H-300 and H-500), 7.83 (d, J ¼ 7.6 Hz, 2H, H-200 and H-600), 8.15 (s, 1H,
H-4), 9.24 (s, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
19.7
1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 11.4 (q), 14.2 (q),
(t), 25.2 (t), 36.1 (q), 117.1 (s), 118.1 (2ꢃ d), 118.5 (d), 118.9 (s), 120.0
(d), 120.3 (d), 121.9 (s), 128.3 (2ꢃ d), 141.4 (s), 154.9 (d), 158.3 (s),
159.0 (s). Anal calcd for C17H16N4: C, 73.89; H, 5.84; N, 20.27. Found:
C, 74.03; H, 5.69; N, 20.38.
21.1 (t), 24.3 (t), 32.3 (q), 59.4 (t), 115.5 (s), 117.1 (s), 117.3 (s), 118.6
(2ꢃ d), 120.7 (d), 128.3 (2ꢃ d), 131.3 (s), 136.7 (s), 141.0 (s), 155.0 (d),
158.3 (s), 158.7 (s), 160.9 (s, CO). Anal calcd for C21H22N4O2: C,
69.59; H, 6.12; N, 15.46. Found: C, 69.71; H, 5.96; N, 15.59.
5.1.6.3. 8-Benzyl-N-phenyl-6,8-dihydro-5H-pyrrolo[3,4-h]quinazo-
lin-2-amine (11f). This compound was obtained from 7f after 1.5 h.
Yield: 62%; m.p. 227e228 ꢀC; IR cmꢂ1: 3280 (NH); 1H NMR (DMSO-
5.1.6.8. Ethyl 8-benzyl-9-methyl-2-(phenylamino)-6,8-dihydro-5H-
pyrrolo[3,4-h]quinazoline-7-carboxylate (11k). This compound was
obtained from 7k after 1.5 h. Yield: 60%; m.p. 195e196 ꢀC; IR cmꢂ1
:

V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
355
3266 (NH), 1685 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.23 (t,
d),128.6 (2ꢃ d), 129.4 (s), 129.5 (2ꢃ d), 131.1 (s), 151.2 (s), 155.1 (d),
158.1 (s), 159.0 (s). Anal calcd for C29H24N4: C, 81.28; H, 5.65; N,
13.07. Found: C, 81.41; H, 5.76; N, 12.93.
J ¼ 7.1 Hz, 3H, CH3), 2.69 (s, 3H, CH3), 2.75 (t, J ¼ 7.2 Hz, 2H, CH2),
3.03 (t, J ¼ 7.2 Hz, 2H, CH2), 4.17 (q, J ¼ 7.1 Hz, 2H, CH2), 5.67 (s, 2H,
CH2), 6.85e6.99 (m, 3H, Ar), 7.20e7.37 (m, 5H, Ar), 7.73 (d,
J ¼ 7.7 Hz, 2H, Ar), 8.23 (s, 1H, H-4), 9.24 (s, 1H, NH); 13C NMR
5.1.6.13. 8-(4-Methylbenzyl)-N,7-diphenyl-6,8-dihydro-5H-pyrrolo
[3,4-h]quinazolin-2-amine (11r). This compound was obtained
from 7r after 1.5 h. Yield: 67%; m.p. 195e196 ꢀC; IR cmꢂ1: 3274
(DMSO-d6, 50.3 MHz, ppm): d 11.4 (q), 14.1 (q), 21.3 (t), 24.3 (t), 47.4
(t), 59.6 (t), 116.1 (s), 116.9 (s), 117.5 (s), 118.7 (2ꢃ d), 120.8 (d), 125.7
(2ꢃ d),126.9 (d),128.3 (2ꢃ d), 128.6 (2ꢃ d),132.0 (s),136.8 (s),138.0
(s), 140.9 (s), 155.3 (d), 158.2 (s), 158.8 (s), 160.7 (s, CO). Anal calcd
for C27H26N4O2: C, 73.95; H, 5.98; N, 12.78. Found: C, 74.12; H, 6.07;
N, 12.65.
(NH); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 2.25 (s, 3H, CH3), 2.68e
2.72 (m, 4H, 2ꢃ CH2), 5.19 (s, 2H, CH2), 6.89e7.12 (m, 5H, Ar), 7.22e
7.45 (m, 8H, Ar), 7.82 (d, J ¼ 7.3 Hz, 2H, Ar), 8.21 (s, 1H, H-4), 9.31 (s,
1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 19.8 (t), 20.6 (q),
25.0 (t), 50.1 (t), 117.4 (s), 118.1 (2ꢃ d), 118.7 (s), 120.4 (d), 120.5 (s),
126.9 (2ꢃ d), 127.3 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ d), 128.8 (d), 129.1
(2ꢃ d), 129.4 (s), 129.5 (2ꢃ d), 131.1 (s), 135.1 (s), 136.6 (s), 141.3 (s),
155.1 (d), 158.1 (s), 159.0 (s). Anal calcd for C30H26N4: C, 81.42; H,
5.92; N, 12.66. Found: C, 81.55; H, 6.09; N, 12.36.
5.1.6.9. Ethyl 9-methyl-8-(4-methylbenzyl)-2-(phenylamino)-6,8-
dihydro-5H-pyrrolo[3,4-h]quinazoline-7-carboxylate
(11l). This
compound was obtained from 7l after 2 h. Yield: 84%; m.p. 211e
212 ꢀC; IR cmꢂ1: 3260 (NH), 1692 (CO); 1H NMR (DMSO-d6,
200 MHz, ppm):
d
1.25 (t, J ¼ 7.1 Hz, 3H, CH3), 2.26 (s, 3H, CH3), 2.69
(s, 3H, CH3), 2.76 (t, J ¼ 7.1 Hz, 2H, CH2), 3.02 (t, J ¼ 7.1 Hz, 2H, CH2),
4.18 (q, J ¼ 7.1 Hz, 2H, CH2), 5.63 (s, 2H, CH2), 6.87 (d, J ¼ 8.0 Hz, 2H,
H-30 and H-50), 7.13 (d, J ¼ 8.0 Hz, 2H, H-20 and H-60), 7.21e7.32 (m,
3H, H-300, H-400 and H-500), 7.72 (d, J ¼ 7.7 Hz, 2H, H-200 and H-600),
8.23 (s, 1H, H-4), 9.25 (s, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz,
5.1.6.14. 8-(4-Methoxybenzyl)-N,7-diphenyl-6,8-dihydro-5H-pyrrolo
[3,4-h]quinazolin-2-amine (11s). This compound was obtained
from 7s after 1.5 h. Yield: 65%; m.p. 151e152 ꢀC; IR cmꢂ1: 3280
(NH); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.67e2.73 (m, 4H, 2ꢃ
CH2), 3.71 (s, 3H, CH3), 5.15 (s, 2H, CH2), 6.78e6.99 (m, 5H, Ar),
7.21e7.43 (m, 8H, Ar), 7.82 (d, J ¼ 7.7 Hz, 2H, Ar), 8.21 (s, 1H, H-4),
ppm):
d 11.4 (q), 14.1 (q), 20.6 (q), 21.3 (t), 24.3 (t), 47.2 (t), 59.6 (t),
116.0 (s), 116.9 (s), 117.5 (s), 118.7 (2ꢃ d), 125.7 (2ꢃ d), 128.3 (2ꢃ d),
128.8 (d), 129.1 (2ꢃ d), 132.0 (s), 135.0 (s), 136.0 (s), 136.8 (s), 140.9
(s), 155.2 (d), 158.2 (s), 158.7 (s), 160.7 (s, CO). Anal calcd for
9.31 (s, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 19.8 (t),
25.0 (t), 49.9 (t), 55.0 (q), 113.9 (2ꢃ d), 117.4 (s), 118.1 (2ꢃ d), 118.7
(s), 120.1 (s), 120.4 (d), 120.6 (s), 127.3 (d), 128.3 (2ꢃ d), 128.4 (2ꢃ d),
128.5 (2ꢃ d), 128.8 (d), 129.3 (s), 129.6 (2ꢃ d), 129.9 (s), 131.1 (s),
141.3 (s), 155.4 (d), 158.1 (s), 159.0 (s). Anal calcd for C30H26N4O: C,
78.58; H, 5.72; N, 12.22. Found: C, 78.36; H, 5.85; N, 12.07.
C
28H28N4O2: C, 74.31; H, 6.24; N, 12.38. Found: C, 74.55; H, 6.08; N,
12.26.
5.1.6.10. Ethyl 8-(4-methoxybenzyl)-9-methyl-2-(phenylamino)-6,8-
dihydro-5H-pyrrolo[3,4-h]quinazoline-7-carboxylate
(11m).
5.2. Antiproliferative assays
This compound was obtained from 7m after 2 h. Yield: 55%; m.p.
190e191 ꢀC; IR cmꢂ1: 3253 (NH), 1692 (CO); 1H NMR (DMSO-d6,
Human chronic myelogenous leukemia (K562) and human T-cell
leukemia (Jurkat) cells were grown in RPMI-1640 medium. Human
colon carcinoma (LoVo) cells were grown in Ham’s F12 medium.
Human breast adenocarcinoma (MCF-7) and human squamous
carcinoma (A-431) cells were grown in DMEM medium. All media
200 MHz, ppm):
d
1.26 (t, J ¼ 7.1 Hz, 3H, CH3), 2.71 (s, 3H, CH3), 2.75
(t, J ¼ 7.2 Hz, 2H, CH2), 3.02 (t, J ¼ 7.2 Hz, 2H, CH2), 3.71 (s, 3H, CH3),
4.20 (q, J ¼ 7.1 Hz, 2H, CH2), 5.60 (s, 2H, CH2), 6.86e6.97 (m, 5H, Ar),
7.21e7.32 (m, 2H, Ar), 7.72 (d, J ¼ 7.6 Hz, 2H, Ar), 8.23 (s, 1H, H-4),
9.24 (s, 1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d
11.4 (q),
were supplemented with 115 units/mL of penicillin, 115 mg/mL of
14.2 (q), 21.3 (t), 24.3 (t), 46.9 (t), 55.0 (q), 59.6 (t),114.0 (2ꢃ d),116.1
(s), 116.8 (s), 117.5 (s), 118.7 (2ꢃ d), 120.8 (d), 127.1 (2ꢃ d), 128.3 (2ꢃ
d), 128.8 (s), 129.8 (s), 132.0 (s), 136.7 (s), 140.9 (s), 155.3 (d), 158.2
(s), 158.8 (s), 160.8 (s, CO). Anal calcd for C28H28N4O3: C, 71.78; H,
6.02; N, 11.96. Found: C, 71.91; H, 5.89; N, 12.08.
streptomycin and 10% fetal bovine serum (Invitrogen, Milano, Italy).
A doxorubicin resistant subclone of LoVo cells (LoVodoxo) was
grown in complete Ham’s F12 medium supplemented with doxo-
rubicin (0.1
mg/mL). Individual wells of a 96-well tissue culture
microtiter plate were inoculated with 100 mL of complete medium
containing 5 ꢃ 103 cells. The plates were incubated at 37 ꢀC in a
5.1.6.11. 8-Methyl-N,7-diphenyl-6,8-dihydro-5H-pyrrolo[3,4-h]qui-
nazolin-2-amine (11p). This compound was obtained from 7p after
1.5 h. Yield: 60%; m.p. 204e205 ꢀC; IR cmꢂ1: 3269 (NH); 1H NMR
humidified 5% CO2 incubator for 18 h prior to the experiments.
After medium removal,100 mL of fresh medium containing different
concentrations of compounds were added to each well and incu-
bated at 37 ꢀC for 72 h. The percentage of DMSO in the medium
never exceeded 0.5%. Cell viability was assayed by the (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test as
previously described [39]. The GI50 was defined as the compound
concentration required to inhibit cell proliferation by 50% in com-
parison to control.
(DMSO-d6, 200 MHz, ppm):
d
2.61e2.76 (m, 4H, 2ꢃ CH2), 3.68 (s,
3H, CH3), 6.90 (t, J ¼ 7.3 Hz, 1H, Ar), 7.28 (t, J ¼ 7.9 Hz, 2H, Ar), 7.33e
7.53 (m, 6H, Ar), 7.86 (d, J ¼ 7.7 Hz, 2H, Ar), 8.20 (s, 1H, H-4), 9.31 (s,
1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 20.0 (t), 25.1 (t),
35.2 (q), 117.3 (s), 118.1 (2ꢃ d),118.3 (s), 120.3 (s),120.4 (d),121.2 (d),
127.1 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ d), 129.2 (s), 129.4 (2ꢃ d), 131.1 (s),
141.3 (s), 155.0 (d), 158.2 (s), 159.0 (s). Anal calcd for C23H20N4: C,
78.38; H, 5.72; N, 15.90. Found: C, 78.44; H, 5.89; N, 15.67.
5.3. Protein extraction and western blotting
5.1.6.12. 8-Benzyl-N,7-diphenyl-6,8-dihydro-5H-pyrrolo[3,4-h]qui-
nazolin-2-amine (11q). This compound was obtained from 7q after
1.5 h. Yield: 75%; m.p. 212e213 ꢀC; IR cmꢂ1: 3264 (NH); 1H NMR
106 A-431 cells were seeded in Petri dishes; after 24 h, they were
washed with PBS and incubated in medium without serum over-
night. Then, cells were incubated with or without 20
pounds for 3 h and stimulated with EGF 100 ng/mL for 10 min. Cell
extracts were prepared as previously described [7] and 100 g of
mM of com-
(DMSO-d6, 200 MHz, ppm):
d
2.68 (t, J ¼ 5.0 Hz, 2H, CH2), 2.72 (t,
J ¼ 5.0 Hz, 2H, CH2), 5.24 (s, 2H, CH2), 6.84e7.02 (m, 3H, Ar), 7.20e
7.48 (m,11H, Ar), 7.82 (d, J ¼ 7.9 Hz, 2H, Ar), 8.21 (s, 1H, H-4), 9.29 (s,
m
proteins were loaded in 12.5% SDS-PAGE and then blotted to a PVDF
membrane (Bio-Rad). Membranes were incubated with polyclonal
anti-Phospho-EGFR (Tyr1173) (Santa Cruz), anti-Phospho-AKT
(Thr308) (Cell Signaling), anti-Phospho-ERK1/2 (P-p42/p44)
1H, NH); 13C NMR (DMSO-d6, 50.3 MHz, ppm):
d 19.8 (t), 25.0 (t),
50.4 (t), 112.5 (s), 117.4 (s), 118.1 (2ꢃ d), 118.8 (s), 120.3 (d), 120.4 (d),
120.6 (s), 126.8 (2ꢃ d), 127.3 (d), 127.4 (d), 128.4 (2ꢃ d), 128.5 (2ꢃ

356
V. Spanò et al. / European Journal of Medicinal Chemistry 74 (2014) 340e357
(Thr202/Tyr204) (Cell Signaling) overnight or with anti-EGFR
(Santa Cruz), anti-AKT (BD Biosciences) and anti-ERK1/2 (Cell
Signaling) for 2 h. The membranes were incubated with the proper
secondary antibody coupled to alkaline phosphatase (Jackson
immunoresearch laboratories) for 1 h. Visualization was done with
CDP-star (Tropix) according to the manufacturer’s instructions.
5.9. Endothelial cell vessel formation on a Matrigel matrix
A Matrigel matrix was kept at 4 ꢀC overnight. Two hundred
microliters of Matrigel (BD Biosciences) was added to each well of a
24-well plate. After gelling at 37 ꢀC for 30 min, gels were overlaid
with 500
m
L of medium containing 6 ꢃ 104 HUVEC cells. Cells were
incubated over Matrigel until the formation of the capillary-like
tubes. Then, compounds were added and continuously incubated
for additional times, and the possible antivascular effect was
monitored by optical microscope (at least five fields for each well;
10ꢃ magnification). Pictures were taken using a digital camera.
Image analysis was carried out using the ImageJ image analysis
software and evaluating both dimensional parameters (percent
area covered by cells, total length of cell network per field and
percent area inside meshes) and topological parameters (number of
meshes and branching points per fields) [40].
5.4. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 5 ꢃ 105 Jurkat cells
in exponential growth were treated with different concentrations
of the test compounds for 24 h. After the incubation period, cells
were collected, centrifuged and fixed with ice-cold ethanol (70%).
Cells were then treated with lysis buffer containing RNAse A and
0.1% Triton X-100, and then stained with PI. Samples were analysed
with BD FACS Calibur flow cytometer. Results of cell-cycle analysis
were examined using WinMDI 2.9 (Windows Multiple Document
Interface for Flow Cytometry).
5.10. Externalization of phosphatidylserine
5.5. Mitotic index determinations
The mode of cellular death was checked after 6 and 24 h of Jurkat
treatment with different concentrations of tested compounds. Sur-
face exposure of phosphatidylserine (PS) by apoptotic cells was
measured by flow cytometry with BD FACS Calibur flow cytometer
by adding Annexin-V-FITC to cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostic, Monza, Italy).
Concurrently, cells were stained with propidium iodide (PI).
Exponentially growing Jurkat cells were incubated with test
compound for 24 h. Then, cells were fixed in coverslips as described
previously [11] and were stained with Giemsa solution. A total of
400 cells/treatment was scored for the presence of mitotic figures
by optical microscopy, and the mitotic index was calculated as the
proportion of cells with mitotic figures.
5.11. Assessment of mitochondrial changes
5.6. Immunofluorescence detection of microtubule perturbation
Mitochondrial changes were assessed after 6 and 24 h of incu-
bation of Jurkat cells in the presence of the most active compounds.
The mitochondrial membrane potential was measured with the
lipophilic cation 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-
dazol-carbocyanine (JC-1, Molecular Probes, Eugene, OR, USA),
while the production of reactive oxygen species (ROS) was followed
by flow cytometry using the fluorescent dyes hydroethidine (HE,
Molecular Probes) and 20,70-dichlorodihydro-fluorescein diacetate
(H2DCFDA), as previously described [23].
LoVo cells were seeded on sterile microscope coverslips. After
24 h, the test compounds (20
mM) and vinblastine (1 mM), as
reference compound, were added to the culture medium and cells
were then incubated for 18 h. Afterward, cells were washed twice
with PBS, and treated with cold methanol for 10 min at ꢂ20 ꢀC.
Then cell layer was rinsed twice for 10 s with cold acetone and after
two PBS washes, cells were rehydrated in PBS for 30 min. Cells were
then incubated with an anti-b-tubulin-FITC labeled mouse mono-
clonal antibody diluted in PBS containing 1% bovine serum albumin
(BSA) for 1 h at room temperature. Slides were then washed
repeatedly with PBS, mounted with mounting medium, and
analyzed by confocal microscopy (SP1 Leica).
5.12. Lysosome dysfunction
After 6 and 24 h of treatment with different concentrations
of test compounds, Jurkat cells were stained with AO at a concen-
5.7. Tubulin polymerization
tration of 1 m
M in RPMI-1640 at 37 ꢀC for 15 min. The fluorescence
was directly recorded with a flow cytometer (BD FACS Calibur)
using 488 nm wavelength as excitation and emission in the FL3
channel [36].
The effects of test compounds on the polymerization of
microtubule protein isolated from porcine brain were analyzed by
using
a microtubule polymerization assay kit (Cytoskeleton)
following the recommended protocol. Polymerization was fol-
5.13. Statistic analysis
lowed by fluorescence enhancement as a consequence of the
incorporation of
polymerization occurred [39]. Vinblastine and Taxol were used as
reference compounds.
a
fluorescent reporter into microtubules as
Unless indicated otherwise, results are presented as
mean ꢁ SEM. The differences between different treatments were
analyzed using the two-sided Student’s t test. p values lower than
0.05 were considered significant (*p < 0.05).
5.8. Motility assay
Acknowledgments
HUVEC cells were purchased from Invitrogen and were cultured
in 0.3% collagen I-coated flasks in M200 medium supplemented
with Low Serum Growth Supplement (Invitrogen). HUVEC cells
(1 ꢃ 105) were seeded into twelve-well plates until a confluent
monolayer was formed. Cell monolayer was scratch wounded using
a pipette tip. Then, cells were treated with the test compounds, and
at different times after the scratch, they were photographed under
a light microscope (10ꢃ magnification). Wound closure was eval-
uated by Optika pro-vision software.
This work was financially supported by Ministero dell’Istruzione
dell’Università e della Ricerca. We would like to thank Tine D.
Rasmussen for excellent technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.014.

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