Quinazoline based α1-adrenoreceptor antagonists with potent antiproliferative activity in human prostate cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2017.05.003

New α₁-adrenoreceptor (α₁-AR) antagonists related to prazosin and doxazosin were synthesized by replacing piperazine ring with (S)- or (R)-3-aminopiperidine. Binding studies indicated that the S configuration at the 3-C position of the piperidine ring is crucial for an optimal interaction of the compounds at all three α₁-AR subtypes. Quinazolines 9 and 10, bearing a quinone ring on the lateral chain, exhibited also potent antiproliferative activity in LNCaP androgen-sensitive prostate cancer cell lines, higher than that of doxazosin. Compound 10 increased apoptosis, in terms of DNA fragmentation, without triggering cell necrosis. The prooxidant activity found in compound 10 may underlie its ability to inhibit cell proliferation in synergy with the effect mediated by α₁-AR antagonism. Due to its better biological profile compared to doxazosin for LNCaP cell line, compound 10 might be a valuable lead compound for the design of new prostate antitumor agents.

Full text of DOI:10.1016/j.ejmech.2017.05.003

European Journal of Medicinal Chemistry 136 (2017) 259e269
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Quinazoline based a1-adrenoreceptor antagonists with potent
antiproliferative activity in human prostate cancer cell lines
Valentina Maestri a, Andrea Tarozzi b, Elena Simoni a, Antonio Cilia c, Elena Poggesi c,
,
Marina Naldi a d, Benedetta Nicolini b, Letizia Pruccoli b, Michela Rosini a,
,
*
Anna Minarini a
a Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso D'Augusto 237, 47921 Rimini, Italy
c Recordati S.p.A., Drug Discovery, Pharmacology Department, Via Civitali 1, 20148, Milano, Italy
d Center for Applied Biomedical Research (C.R.B.A.), S. Orsola-Malpighi Hospital, Via Massarenti 9, 40138 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
New a1-adrenoreceptor (a1-AR) antagonists related to prazosin and doxazosin were synthesized by
replacing piperazine ring with (S)- or (R)-3-aminopiperidine. Binding studies indicated that the S
configuration at the 3-C position of the piperidine ring is crucial for an optimal interaction of the
compounds at all three a1-AR subtypes. Quinazolines 9 and 10, bearing a quinone ring on the lateral
chain, exhibited also potent antiproliferative activity in LNCaP androgen-sensitive prostate cancer cell
lines, higher than that of doxazosin. Compound 10 increased apoptosis, in terms of DNA fragmentation,
without triggering cell necrosis. The prooxidant activity found in compound 10 may underlie its ability to
inhibit cell proliferation in synergy with the effect mediated by a1-AR antagonism. Due to its better
biological profile compared to doxazosin for LNCaP cell line, compound 10 might be a valuable lead
compound for the design of new prostate antitumor agents.
Received 26 January 2017
Received in revised form
27 March 2017
Accepted 1 May 2017
Available online 4 May 2017
Keywords:
Quinazoline
Quinone
a1-Adrenoreceptor antagonist
DNA fragmentation
Antiproliferative activity
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
AR subtype, reducing prostatic smooth muscle tone, thereby
relieving bladder outlet obstruction and increasing urinary flow.
a1-adrenoreceptor (a1-AR) antagonists (
a
-blockers) are the first
The different localization of a-AR subtypes suggests the possibility
line treatment for lower urinary tract symptoms (LUTS) and benign
prostatic hyperplasia (BPH). In fact, a1-ARs play an important role
in the sympathetic control of prostatic muscle contraction [1].
of designing drugs able to interact with a particular subtype in an
effort to limit the adverse effect profile and improve efficacy. A
great deal of interest now lies in the development of a-blockers
There are three known subtypes of a1-ARs, namely a1A
,
a1B and a1D
.
which target only one receptor subtype while not affecting the
others.
a1A-ARs are mainly expressed in the prostate (70%) and mediate its
contractile response. The a1B-AR subtype is found in vascular tissue
regulating blood pressure. The a1D-AR subtype is associated with
bladder muscle contraction, suggesting that it also may have a role
It is well known that quinazoline-based a1-AR antagonists exert
antitumor activity on prostate cancer cell lines [3,4]. In particular,
doxazosin and terazosin, used to treat BPH, were shown to induce
apoptosis in human prostate cancer cells DU-145, that lack a1-AR,
providing evidence that the apoptotic activity on malignant pros-
tate cells is independent by their capacity to antagonize a1-ARs. The
androgen-sensitive LNCaP cells were slightly more sensitive to the
cytotoxic effects of prazosin and doxazosin than the castration-
resistant PC-3 cells [5]. It was also found that the apoptotic effect
of quinazolines on the androgen-sensitive prostate cancer cells
LNCaP is not affected by androgens and does not interfere with cell
cycle progression [6e8]. Interestingly, human normal prostate
epithelial cells displayed a very low sensitivity to the apoptotic
in the control of BPH symptoms [2]. All a-blockers bind to the a1A-
Abbreviations used: a1-AR, a1-adrenoreceptor; BPH, benign prostatic hyperpla-
sia; CHO, Chinese hamster ovary; Dox, doxazosin; GSH, glutathione; LUTS, lower
urinary tract symptoms; MCB, monochlorobimane; MTT, 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NCI, National Cancer Institute;
PBS, phosphate buffered saline; ROS, reactive oxygen species; TGI, total growth
inhibition.
* Corresponding author.
E-mail address: anna.minarini@unibo.it (A. Minarini).
http://dx.doi.org/10.1016/j.ejmech.2017.05.003
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

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V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
effects of doxazosin in comparison with that elicited in the malig-
nant prostate cells. Among the cellular pathways affected by pra-
zosin and doxazosin, also autophagy contributes to cytotoxic effects
in PC-3 cells but not in LNCaP cells [9]. All these findings may have
potential therapeutic implications on the use of quinazoline-based
a1-AR antagonists for the treatment of androgen dependent and
independent human prostate cancer, with a possible role in the
prevention of tumor development.
Our research group has long been involved in designing new
quinazoline-based a1-AR antagonists with the aim to discover more
subtype selective a1-blockers [9e13]. In particular, since the
insertion of chiral features in the lateral chain of quinazoline has
already represented a useful approach when searching for clues
about binding site topography [14], we modified quinazoline-based
drugs by replacing the piperazine ring of prazosin and doxazosin
with the 3-aminopiperidine chiral unit, obtaining compounds 3
and 4 (Fig. 1) and 5e8 (Fig. 2), respectively.
naphthoquinone moiety (compounds 9 and 10, Fig. 2), as
quinone-based scaffolds are endowed with antiproliferative activ-
ity [9].
Receptor subtype selectivity of compounds 1e10 was deter-
mined at a1-ARs by radioreceptor binding assays. In addition, their
antiproliferative activities were assessed on different prostate
cancer cells (LNCaP, DU-145 and PC-3) and the most potent com-
pound 10 was also investigated for its ability to induce cell death in
terms of necrosis, apoptosis, and to impair the redox status.
2. Chemistry
The syntheses of the new quinazoline derivatives 3e10 are
described in Scheme 1. Intermediates 11 and 12 were obtained
through aromatic nucleophilic substitution of (S)-3-(Boc-amino)
piperidine and (R)-3-(Boc-amino)piperidine, both commercially
available,
on
4-amino-2-chloro-6,7-dimethoxyquinazoline,
Compounds 1 (R enantiomer) and 2 (S enantiomer) (Fig. 1),
which represent intermediates in the synthetic pathway, were
included in the assays as reference compounds. In compounds 5e8,
the presence of a second chiral center due to the 1,4-benzodioxane-
2-carbonyl group of doxazosin could result in additional structural
respectively. To shortening the reaction time the syntheses were
performed under microwave irradiation by following an adapted
procedure described by Althuis et al. [15] Boc-deprotection of 11
and 12 with 4 M HCl in dioxane led to the primary amines 1 and 2
that by amidation with 2-furoyl chloride afforded compounds 3 and
4, respectively, in good yields. Compounds 5e8 were obtained by
coupling amines 1 and 2 and (R)-2,3-dihydro-benzo[1,4]dioxine-2-
carbonyl chloride 13 or (S)-2,3-dihydro-benzo[1,4]dioxine-2-
carbonyl chloride 14, achieved by reacting the respective carbox-
ylic acids with thionyl chloride. Moreover, the reaction between 1
and 2 with 1,4-naphthoquinone led to the quinazoline-quinones 9
and 10, respectively. Finally, doxazosin was also synthesized by
following an adapted procedure described by Althuis et al. [15], in
which the aromatic nucleophilic substitution of piperazine on 4-
elements that might affect affinity for a-ARs and might confer also
antiapoptotic and antiproliferative properties, as shown by dox-
azosin and cyclazocin. In this regard, doxazosin was included in the
study to compare its activity with that of the new molecules.
The antitumor activity of quinazoline-based
a-blockers has
generated interest in the development of more potent novel com-
pounds that maintain the quinazoline moiety conjugated with
different scaffolds [14]. On this basis, we sought to strengthen the
antiproliferative effect by conjugating
1 and 2 with a 1,4-
Fig. 1. Design strategy for compounds 3 and 4.

V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
261
Fig. 2. Design strategy for compounds 5e10.
amino-2-chloro-6,7-dimethoxyquinazoline was performed under
microwave irradiation.
even though the differences in binding affinity were not so marked
as between 3 and 4. Moreover, compound 10 seemed to be 13efold
and 4efold more selective for the a1B-ARs than for a1A- and a1D
-
ARs, respectively. Compounds 1 and 2 lacking the furoyl moiety and
thus bearing a primary amine function on the lateral chain, have
lower affinity for a1-AR subtypes compared to their respective
amide derivatives 3e10. The present finding was in good agree-
3. Results and discussion
The affinity profile of compounds 1e10 was evaluated in radi-
oreceptor binding assays on membranes of Chinese hamster ovary
(CHO) cells expressing human a1-AR subtypes. Binding affinities
were expressed as pKi values derived using the Cheng-Prusoff
equation. The results are reported in Table 1. Doxazosin and pra-
zosin were also included in the study as reference compounds. An
analysis of the results reveals that most of the new compounds
show a valuable affinity toward all a1-AR subtypes, albeit to a
different extent, with 4 and 10 being as active as doxazosin and
prazosin. However, the stereochemistry at the 3-C position of the
piperidine ring plays an important role for the binding at a1-ARs.
Thus, we can observe that 4 possesses an affinity for all a1-AR
subtypes that is more than 100-fold higher compared to its enan-
tiomer 3. The same observation applies to 10 vs its enantiomer 9
ment with
quinazoline-related compounds. They found that the prazosin de-
rivative lacking the furoyl moiety displayed a low affinity for a1
a study conducted by Cambpell et al. [16] on
-
ARs. It derives that the substituent on the amine group is important
for the interaction with a1-ARs. However, compound 2 displayed a
higher affinity for all three a1-AR subtypes than its enantiomer 1.
Derivative 2 is slightly more selective for the a1D-relative to the a1B
-
ARs. This finding is interesting because also a1D-ARs are thought to
have a relevant role in the control of the symptoms associated with
BPH, while a1B subtype is responsible for the regulation of blood
pressure. In conclusion, the S configuration at the 3-C position of
the piperidine ring is an important feature that drives the

262
V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
Scheme 1. Synthesis of compounds 1e10.a
aReagents and conditions: a) Isoamyl alcohol, 50 min, 130 ꢀC, mw, quantitative yield; b) HCl 4 M/dioxane, MeOH, rt, 18 h, NaOH 4 N, quantitative yield; c) CH2Cl2, Et3N, 2 h, rt,
55e100% yield; d) EtOH, 8 h, rt, 85% yield; Boc ¼ (CH3)3COCO-.
interaction with a1-ARs.
cancer cells both 9 and 10 caused 54% of cell death. In PC-3 cells, 9
and 10 inhibited cellular growth by 45.51% and 77.05%, respectively.
Due to the promising a1-AR affinity of most compounds, we
focused on their antiproliferative effect in different human prostate
cancer cells.
Therefore, the antiproliferative activity of compounds 2e4 and
7e10 in comparison to doxazosin was more deeply investigated in
prostate cancer cell lines, DU-145, PC-3 and LNCaP, the results being
expressed as IC50 (concentration of compound resulting in 50%
inhibition of cell proliferation). LNCaP cells, but not DU-145 and PC-
3 cells, express mainly a1A-ARs and are androgen-sensitive prostate
cells [17,18].
As reported in Fig. 3A, treatment of DU-145, PC-3 and LNCaP
cells with compounds 2e4, 7 and 8 did not show modified cell
proliferation, confirming NCI data. By contrast, the treatment of
various prostate cancer cells with compounds 9, 10 and doxazosin
produced a decrease of cell proliferation. In particular, both 9
From the analysis of the binding assay results for compounds
5e8 bearing a benzodioxane ring, we can remark that the stereo-
chemistry at the benzodioxane ring does not markedly affect the
affinity and selectivity for the a1-AR subtypes. A prominent dif-
ference in affinity was observed between diasteroisomers 5 and 6
with respect to their corresponding enantiomers 7 and 8. In fact,
also in these derivatives, inverting the R configuration at the 3-C
position of the piperidine ring of 5 and 6 into the S configuration,
as in 7 and 8, increased the affinity towards all a1-AR subtypes.
Compounds 7 and 8 showed a similar biological profile, but 7 dis-
played a 10efold increase in affinity for a1B-ARs compared to
compound 8.
Antiproliferative activities of compounds 1e10 were firstly
in vitro evaluated at National Cancer Institute (NCI) according to the
Developmental Therapeutics Program (DTP), in order to select the
most promising compounds. Compounds were tested at a con-
centration of 10
m
M on 60 different human tumor cell lines, rep-
(IC50 ¼ 5.78
proliferative activity than doxazosin (IC50 ¼ 10.48
cells (Fig. 3B). Similar differences in antiproliferative activity be-
tween these compounds (9, IC50 ¼ 17.82 M; 10, IC50 ¼ 18.54 M;
doxazosin, IC50 ¼ 30.56 M) were also recorded on DU-145
(Fig. 4b), while doxazosin (IC50 ¼ 12.10 M) showed slightly
higher antiproliferative activity than 9 (IC50 ¼ 18.58 M) and 10
(IC50 ¼ 18.23 M) on PC-3.
Taken together, these results show the highest antiproliferative
m
M) and 10 (IC50 ¼ 6.32
m
M) exerted higher anti-
resenting leukemia, melanoma and cancers of the lung, colon,
brain, ovary, breast, prostate, and kidney. Among all the com-
pounds, 9 and 10 were found to be the most promising in the one-
dose assays. An analysis of the results (data not shown) revealed
that 9 and 10 possessed an antiproliferative activity that is more
marked for tissue specific cell lines, that is colon, brain and prostate.
It emerged that there was not a noteworthy change in activity be-
tween the two enantiomers. In particular, in DU-145 prostate
mM) on LNCaP
m
m
m
m
m
m

V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
263
Table 1
(SI) in terms of ratio of IC50 and IC10 obtained from the experiments
on human fibroblasts (HGFs), a control of normal cells, versus
Binding affinity constants (pKi)a of compounds 1e10, prazosin and doxazosin at
human cloned a1-AR subtypes expressed in CHO cells.
LNCaP cells. In particular, the SI were 4.7 and 9.1 at IC50 and IC10
,
respectively, suggesting a high selective action of compound 10 on
cancer cells at low concentrations.
These results are in agreement with recent studies that report
the ability of novel anticancer compounds with the quinone moiety
to induce cytotoxicity, apoptosis, mitochondrial damage as well as
inhibition of cell proliferation in various prostate cancer cell lines,
including DU-145, PC-3 and LNCaP cells [18e23].
Nowadays cytotoxic quinones represent an important group of
anticancer drugs. Among various anticancer mechanisms, they can
mainly induce the formation of the semiquinone radical which can
transfer an electron to oxygen to produce superoxide. Both radical
species can then generate the hydroxyl radical which is known to
cause glutathione (GSH) depletion, DNA strand breaks and cell
death [24].
Compound
pKia
R
H
H
a1a
<6
6.72
6.96
a1b
a1d
1
2
3
6.75
7.26
7.37
6.99
7.55
7.43
On the basis of these considerations, we determined the ability
of 10 and 2 (analogue without the quinone moiety) to induce cell
death in terms of apoptosis and necrosis in LNCaP cells. We also
included in the study doxazosin, which is known to induce
apoptosis in prostate cancer cell lines through a1-AR independent
mechanisms [25].
4
5
9.20
9.55
7.33
9.48
6.53
<6
As shown in Fig. 4, treatment of LNCaP cells with 1e10
but not 2, increased apoptosis, in terms of DNA fragmentation into
oligosomes, in concentration-dependent manner with
mM 10,
a
a
maximum of 9.9 fold, indicating that the quinone moiety has
conferred significant proapoptotic properties to 10. By contrast,
doxazosin increased the DNA fragmentation of 2.2 fold only at
6
7
8
9
<6
6.50
8.13
7.20
7.60
<6
10 mM. In the same experimental conditions, compounds 2, 10 and
doxazosin didn't induce necrosis (data not shown) suggesting a
selective apoptotic death elicited by 10 and doxazosin. Further, the
lack of necrosis associated to 10 can be an added value to the profile
of bioactivity of this compound. The necrosis processes can
contribute to trigger an inflammatory response that is also involved
in regulation of cellular events in prostate cancer progression
through control of the tumor micro-environment [26,27].
7.48
7.74
6.92
7.88
7.31
7.47
Subsequently, in the same prostate cancer cells we also evalu-
ated the redox influence of the quinone moiety present in the
compound 10. First, the antioxidant activity of 10 and 2 against
formation of ROS in LNCaP cells after concomitantly short and
prolonged oxidant treatment with t-BuOOH was assessed. The
short treatment with compound 10, but not 2, showed an antiox-
idant activity, in a concentration-dependent manner, with a
maximum activity of 34% (Fig. S1 in Supplementary information).
In contrast to short treatment, the prolonged treatment of LNCaP
cells with compound 10 didn't record antioxidant effects against t-
BuOOH (data not shown). Interestingly, the prolonged treatment of
LNCaP cells with same concentrations of 10 in the absence of
oxidant treatment showed prooxidant effects, in a concentration-
dependent manner, with a maximum intracellular ROS formation
of 56% (Fig. 5A). To confirm these intracellular prooxidant effects,
we also evaluated the GSH levels, an indicator of intracellular redox
state, in similar experimental conditions of treatment. Remarkably,
10, but not 2, induced a strong depletion of intracellular basal GSH
level (Fig. 5B). These results suggest that the transient antioxidant
activity of 10 is prodromal to its prooxidant activity [28]. In
particular, the prooxidant activity of 10 may underlie its ability to
induce DNA fragmentation and inhibition of cell proliferation in
synergy with the effect mediated by a1-AR antagonism.
10
8.15
9.28
8.67
Doxazosin
Prazosinb
9.11
9.23
9.20
9.39
9.49
9.65
a
Equilibrium dissociation constants (Ki) were derived from IC50 values using the
Cheng-Prusoff equation. Ki values were from two to three experiments, which
agreed within 20%.
b
Data from Ref. [12].
activity of the compounds 9 and 10, bearing the quinone moiety, in
LNCaP cells, which express a1A-ARs. In particular, the anti-
proliferative effects of the compounds 9 and 10 are similar although
the compound 10 shows a higher affinity for a1A-ARs than 9. This
evidence therefore suggests that the quinone moiety present in 9
and 10 could contribute to a1-AR independent antiproliferative
mechanisms. Compound 10 also showed a good selectivity index
4. Conclusions
New quinazoline analogues (1e10) related to prazosin and
doxazosin obtained by replacing piperazine ring with (S)- or (R)-3-

264
V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
Fig. 3. Antiproliferative effects of the compounds 2e4, 7e10 and doxazosin (Dox) on prostate cancer cell lines. The DU-145, PC-3 and LNCaP cells were incubated with 0.1e50 mM
compound concentrations for 120 h and then evaluated for the cell number as reported in method section. (A) The values are expressed as percentage of untreated cells. Results are
reported as mean of three independent experiments. (B) The values are expressed as concentration of compound resulting in 50% inhibition of prostate cancer cell proliferation
(IC50). Results are reported as mean SD of three independent experiments.
aminopiperidine were investigated to assess their a1-AR antago-
nism and antiproliferative effects. Binding studies indicated that
the S configuration at the 3-C position of the piperidine ring is
fundamental for an optimal interaction of the compounds at all
three a1-AR subtypes. However, treatment of DU-145, PC-3 and
LNCaP prostate cancer cells with compounds 2e4, 7 and 8 did not
show modified cell proliferation. In contrast, compounds 9 and 10
bearing a quinone ring on the lateral chain exhibited potent anti-
proliferative activity in LNCaP androgen-sensitive prostate cancer
cell lines, higher than that of doxazosin, suggesting that the
quinone moiety present in 9 and 10 contributes to a1-AR inde-
pendent antiproliferative mechanisms.
In particular, quinone moiety confers both antioxidant and
prooxidant activity to compound 10. The prooxidant activity of 10
may underlie its ability to induce DNA fragmentation and inhibition
of cell proliferation and synergize with a1-AR antagonist activity in
prostate tissue. Further, early and transient antioxidant activity of
10 against ROS can be an added value to bioactivity of this com-
pound. It should be noted that ROS play an increasingly important
role not only in neoplastic transformation, but also in progression
and aggressive phenotype of prostate cancer [29]. Due to its better
Fig. 4. Apoptotic effect of compounds 2, 10 and doxazosin (Dox) in LNCaP cells. Cells
were treated with various concentrations of compounds for 24 h, after which
biological profile compared to doxazosin in LNCaP cancer cell line,
compound 10 might be of interest as a lead compound for the
design of new prostate antitumor agents.
apoptosis, in terms of DNA fragmentation into oligosomes, was determined by the Cell
Death Detection ELISAplus as described in the method section. The values in the treated
samples are expressed as fold increases with respect to corresponding untreated
controls. Results are reported as mean
SD of three independent experiments
(*p < 0.05, **p < 0.01 and ***p < 0.001 vs untreated cells at t-test).

V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
265
recorded at 200e400 and 50e100 MHz, respectively, on Varian
instruments (S2 in Supplementary information). Chemical shifts
were measured in ppm and are quoted as d, relative to TMS. Mul-
tiplicities are quoted as s (singlet), d (doublet), t (triplet), q (quar-
tet), quintet and m (multiplet), br (broad) with coupling constants
defined as J given in Hz. High mass resolution (HRMS) analyses
were performed using a Q-ToF Micro quadrupole time of-flight (Q-
TOF) hybrid analyser (Micromass, Manchester, UK) with a Z-spray
electrospray ion source (ESI) coupled to an Agilent 1200 HPLC
system (Walbronn, Germany), consisting of a binary pump with
degasser and autosampler. Data acquisition was performed with
MassLynx 4.1 software (Waters, Milford, MA, USA). Final com-
pounds were >97% pure, as determined by high performance liquid
chromatography (HPLC) analyses (S3 in Supplementary informa-
tion). Liquid chromatography was performed by using a Jasco PU-
1585HPLC system (Jasco Corporation, Tokyo, Japan) equipped
with a Rheodyne 7281 injection valve (50
mL sample loop). The
detection was performed with a Jasco UV-1575 detector set at
247 nm. The flow rate was 0.2 ml/min. The analyses were per-
formed under reversed-phase conditions by using a Zorbax Eclipse
XDB-C18 (2.1 ꢁ 150 mm; 3.5
mm) column. The composition of the
mobile phase was H2O/CH3CN/TFA, 91/9/0.1 (v/v/v) for compound
2, 80/20/0.1 (v/v/v) for compounds 3 and 4, 70/30/0.1 (v/v/v) for
compounds 5e8 and 65/35/0.1 (v/v/v) for compound 9.
5.1.1. General procedure for the synthesis of 1 and 2
A solution of 4 M HCl in dioxane (25 mL) was added dropwise at
0
ꢀC to a solution of 11 and 12 (3 mmol) in MeOH (3 mL). The
mixture was stirred at r.t. overnight. The solvent was then evapo-
rated under reduced pressure and the residue was basified with 4 N
NaOH. The aqueous layer was saturated with NaCl and extracted
with EtOAc (10 ꢁ 30 mL). The combined organic phases were dried
over Na2SO4 and evaporated to dryness affording 1 and 2, respec-
tively, as a white solid in quantitative yield. 1 and 2 were then
converted into the dihydrochloride salt.
5.1.1.1. (R)-2-(3-Aminopiperidin-1-yl)-6,7-dimethoxyquinazolin-4-
Fig. 5. Redox influence of compounds 2 and 10 in LNCaP cells. The cells were treated
with various concentrations of compounds for 24 h. At the end of incubation, the redox
influence in terms of intracellular reactive oxygen species (ROS) formation and
decreased glutathione (GSH) level was determined as described in the method section.
(A) The results are expressed as fold increase of intracellular ROS evoked by exposure
to compounds. Values are reported as mean SD of three independent experiments
(**p < 0.01 vs untreated cells at t-test). (B) The results are expressed as arbitrary unit of
fluorescence (AUF). Values are reported as mean SD of three independent experi-
ments (*p < 0.05, **p < 0.01 and ***p < 0.001 vs untreated cells at t-test).
20
amine (1). mp (dihydrochloride salt) ¼ 240 ꢀC; [
a
]
¼ ꢂ49.99; 1H
D
NMR (free base, 400 MHz, CD3OD)
d 1.23e1.33 (1H, m, CHH),
1.43e1.54 (1H, m, CHH), 1.69 (1H, dt, J ¼ 13.3, 3.9 Hz, CHH),
1.90e1.94 (1H, m, CHH), 2.67e2.75 (2H, m, CHH-N, CH-NH2), 2.92
(1H, ddd, J ¼ 12.9, 11.3, 3.1 Hz, CHH-N), 3.81 (3H, s, OCH3), 3.86 (3H,
s, OCH3), 4.38e4.32 (1H, m, CHH-N), 4.54 (1H, dd, J ¼ 17.6, 9.0 Hz,
CHH-N), 6.82 (1H, s, Ar), 7.24 (1H, s, Ar); 13C NMR (free base,
100 MHz, CD3OD)
d 23.65, 33.26, 44.04, 47.37, 51.74, 54.80, 55.24,
102.86, 102.90, 104.29, 145.50, 148.96, 154.81, 159.07, 161.70; Anal.
Calcd for C15H29Cl2N5O5: C 41.87, H 6.79, N 16.27, found C 42.12, H
6.65, N 15.94. HRMS-ESI (m/z): calcd for C15H22N5O2 [MþH]þ
304.1773, found 304.1775.
5. Experimental section
5.1. Chemistry
All reactions were carried out at atmospheric pressure with
stirring unless otherwise indicated. All solvents and chemicals were
purchased from suppliers and used without further purification.
For microwave-assisted organic synthesis a CEM Discover Bench-
Mate reactor was used. Reaction progress was monitored by TLC
plates pre-coated with silica gel 60 F254 (Sigma Aldrich) visualized
by UV (254 nm) or chemical stain (KMnO4, bromocresol green and
cerium-molybdate). Flash and gravity column chromatography
5.1.1.2. (S)-2-(3-Aminopiperidin-1-yl)-6,7-dimethoxyquinazolin-4-
20
amine (2). mp (dihydrochloride salt) ¼ 240 ꢀC; [
a
]
¼ þ49.03; 1H
D
NMR (free base, 400 MHz, CD3OD)
d 1.29e1.38 (1H, m, CHH),
1.46e1.57 (1H, m, CHH), 1.69e1.76 (1H, m, CHH), 1.94e1.98 (1H, m,
CHH), 2.72e2.81 (2H, m, CHH-N, CH-NH2), 2.97 (1H, ddd, J ¼ 12.9,
11.0, 3.1 Hz, CHH-N), 3.84 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.39 (1H,
dd, J ¼ 9.4, 3.9 Hz, CHH-N), 4.49e4.56 (1H, m, CHH-N), 6.83 (1H, s,
Ar), 7.27 (1H, s, Ar); 13C NMR (free base, 100 MHz, CD3OD)
d 23.53,
were performed on silica gel (particle size 40e63
mM, Merck).
32.98, 44.04, 47.31, 51.49, 54.78, 55.24, 102.84, 102.93, 104.20,
Melting points were determined using Büchi SMP-20 apparatus.
Compounds were named relying on the naming algorithm devel-
oped by CambridgeSoft Corporation and used in Chem-BioDraw
Ultra 14.0. Optical activities of the hydrochloride salts of the com-
pounds 1e10 dissolved in methanol were determined using a
Perkin Elmer instrument. 1H NMR and 13C NMR spectra were
145.56, 148.84, 154.86, 159.03, 161.73. HRMS-ESI (m/z): calcd for
C
15H22N5O5 [MþH]þ 304.1773, found 304.1770.
5.1.2. General procedure for the synthesis of 3e8
A solution of the appropriate acyl chloride (0.5 mmol) in CH2Cl2
(2 mL) was added dropwise at 0 ꢀC to a solution of 1 or 2 (0.5 mmol)

266
V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
and Et3N (0.5 mmol) in CH2Cl2 (4 mL). The mixture was stirred at r.t.
for 2 h. The solvent was evaporated under reduced pressure and the
crude was purified by flash chromatography column affording
compound 3e8 as white solids that were converted into the cor-
responding hydrochloride salts.
6 as a white solid (95% yield). mp (hydrochloride salt) ¼ 223 ꢀC;
20
[a
]
¼ ꢂ89.68; 1H NMR (free base, 400 MHz, CDCl3)
d 1.60e1.65
D
(1H, m, piperidine ring), 1.73e1.81 (1H, m, piperidine ring),
1.87e1.92 (2H, m, piperidine ring), 3.62e3.68 (1H, m, piperidine
ring), 3.77 (1H, d, J ¼ 12.9 Hz, piperidine ring), 3.83 (3H, s, OCH3),
3.91 (3H, s, OCH3), 3.95 (1H, dd, J ¼ 12.9, 4.7 Hz, piperidine ring),
4.01e4.06 (1H, m, piperidine ring), 4.07e4.10 (1H, m, piperidine
ring), 4.10 (1H, dd, J ¼ 11.3, 7.8 Hz, CHH-O), 4.54 (1H, dd, J ¼ 2.7,
11.3 Hz, CHH-O), 4.59 (1H, dd, J ¼ 7.8, 2.7 Hz, CH-C¼O), 5.53 (2H, br
s, NH2), 6.51 (1H, d, J ¼ 7.8 Hz, Ar), 6.68 (1H, td, J ¼ 7.8, 1.6 Hz, Ar),
6.77 (1H, t, J ¼ 7.8 Hz, Ar), 6.83 (1H, dd, J ¼ 7.8, 1.6 Hz, Ar), 6.85 (1H,
br s, Ar), 6.93 (1H, s, Ar), 7.18 (1H, d, J ¼ 7.0 Hz, NH-C¼O); 13C NMR
5.1.2.1. ((R)-N-(1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperidin-
3-yl)furan-2-carboxamide (3). Compound 3 was synthesized by
reacting 2-furoyl chloride with 1. The crude was purified by flash
chromatography eluting with CHCl3/MeOH, (9:1) affording 3 (85%
20
yield). mp (hydrochloride salt) ¼ 226 ꢀC; [
a
]
¼ ꢂ96.87; 1H NMR
D
(free base, 400 MHz, CDCl3)
d 1.58e1.66 (1H, m, piperidine ring),
1.73e1.83 (1H, m, piperidine ring), 1.86e1.95 (2H, m, piperidine
ring), 3.68e3.75 (1H, m, piperidine ring), 3.91e3.99 (3H, m,
piperidine ring), 3.91 (3H, s, OCH3), 3.94 (3H, s, OCH3), 4.17e4.24
(1H, m, piperidine ring), 5.76 (2H, br s, NH2), 6.42 (1H, dd, J ¼ 3.5,
2.0 Hz, Ar), 6.94 (1H, s, Ar), 7.02 (1H, d, J ¼ 3.5 Hz, Ar), 7.06 (1H, s,
Ar), 7.14 (1H, d, J ¼ 7.0 Hz, NH-C¼O), 7.29e7.30 (1H, m, Ar); 13C NMR
(free base, 100 MHz, CDCl3) d 22.20, 29.68, 44.72, 46.12, 48.28,
55.91, 56.13, 65.61, 73.08, 101.56, 102.81, 105.62, 117.11, 117.28,
121.62, 122.20, 141.42, 142.98, 145.85, 155.04, 160.78, 166.71. HRMS-
ESI (m/z): calcd for C24H28N5O5 [MþH]þ 466.2090, found 466.2093.
5.1.2.5. (S)-N-((S)-1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piper-
(free base, 100 MHz, CD3OD)
d
23.39, 29.41, 44.95, 45.72, 48.58,
idin-3-yl)-2,3-dihydrobenzo
[b][1,4]dioxine-2-carboxamide
(7).
55.62, 55.68, 98.22, 101.27, 103.75, 111.61, 114.26, 135.69, 145.13,
Compound 7 was synthesized by reacting 2 with 14. The crude was
purified by flash chromatography eluting with CHCl3/MeOH
147.28, 151.06, 155.78, 158.97, 161.57. HRMS-ESI (m/z): calcd for
C
20H24N5O4 [MþH]þ 398.1828, found 398.1826.
(9.5:0.5) affording 7 (65% yield). mp (hydrochloride salt) ¼ 221 ꢀC;
¼ þ20.46. 1H NMR (free base, 400 MHz, CDCl3)
d 1.42e1.47
20
[a
]
D
5.1.2.2. ((S)-N-(1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piper-
idin-3-yl)furan-2-carboxamide (4). Compound 4 was synthesized
by reacting 2-furoyl chloride with 2. The crude was purified by flash
(2H, m, piperidine ring), 1.73e1.77 (2H, m, piperidine ring),
3.52e3.58 (1H, m, piperidine ring), 3.78 (1H, dd, J ¼ 12.9, 2.7 Hz,
piperidine ring), 3.93 (3H, s, OCH3), 3.97 (3H, s, OCH3), 3.97e4.07
(3H, m, piperidine ring), 4.20 (1H, dd, J ¼ 11.3, 6.7 Hz, CHH-O), 4.39
(1H, dd, J ¼ 11.3, 2.7 Hz, CHH-O), 4.61 (1H, dd J ¼ 6.7, 2.7 Hz, CH-
C¼O), 5.53 (2H, br s, NH2), 6.44 (1H, d, J ¼ 8.2, 1.2 Hz, Ar), 6.63 (1H,
ddd, J ¼ 8.2, 7.0, 2.0 Hz), 6.79 (1H, td, J ¼ 7.0,1.2 Hz, Ar), 6.84 (1H, dd,
J ¼ 8.2, 2.0 Hz), 6.94 (1H, s, Ar), 6.96 (1H, s, Ar), 7.0 (1H, d, J ¼ 7.0 Hz,
chromatography eluting with CHCl3/MeOH (9:1) affording 4 (85%
20
yield). mp (hydrochloride salt) ¼ 226 ꢀC; [
a
]
¼ þ96.54; 1H NMR
D
(free base, 400 MHz, CDCl3)
d 1.59e1.62 (1H, m, piperidine ring),
1.74e1.78 (1H, m, piperidine ring), 1.86e1.91 (2H, m, piperidine
ring), 3.68e3.74 (1H, m, piperidine ring), 3.90e3.96 (3H, m,
piperidine ring), 3.90 (3H, s, OCH3), 3.93 (3H, s, OCH3), 4.15e4.22
(1H, m, piperidine ring), 5.81 (2H, br s, NH2), 6.40 (1H, dd, J ¼ 3.6,
1.7 Hz, Ar), 6.93 (1H, s, Ar), 7.01 (1H, d, J ¼ 3.6 Hz, Ar), 7.06 (1H, s,
Ar), 7.13 (1H, d, J ¼ 7.1 Hz, NH-C¼O), 7.26e7.28 (1H, m, Ar); 13C NMR
NH-C¼O); 13C NMR (free base, 100 MHz, CDCl3)
d 21.90, 29.72,
44.63, 46.13, 48.12, 56.07, 56.18, 65.22, 73.19, 101.60, 102.88, 105.62,
116.90, 117.38, 121.67, 122.18, 141.39, 143.17, 145.87, 155.13, 160.78,
166.89. HRMS-ESI (m/z): calcd for C24H28N5O5 [MþH]þ 466.2090,
found 466.2092.
(100 MHz, CD3OD)
d 23.50, 29.50, 45.03, 45.76, 48.60, 55.80, 55.86,
98.24, 101.14, 103.70, 111.70, 114.37, 135.55, 145.31, 147.11, 147.18,
150.87,155.58,158.97,161.37. HRMS-ESI (m/z): calcd for C20H24N5O4
[MþH]þ 398.1828, found 398.1826.
5.1.2.6. (R)-N-((S)-1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piper-
idin-3-yl)-2,3-dihydrobenzo
[b][1,4]dioxine-2-carboxamide
(8).
Compound 8 was synthesized by reacting 2 with 13. The crude was
purified by flash chromatography eluting with CHCl3/MeOH
5.1.2.3. (R)-N-((R)-1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piper-
idin-3-yl)-2,3-dihydrobenzo
[b][1,4]dioxine-2-carboxamide
(5).
(9.5:0.5) affording 8 in quantitative yield. mp (hydrochloride
20
Compound 5 was synthesized by reacting 1 with 13. The crude was
purified by flash chromatography (CHCl3-MeOH, 9.5:0.5) affording
salt) ¼ 223 ꢀC; [
a
]
D
¼ þ89.71. 1H NMR (free base, 400 MHz, CDCl3)
d
1.56e1.61 (1H, m, piperidine ring), 1.73e1.81 (1H, m, piperidine
5 as a white solid (83% yield).; mp (hydrochloride salt) ¼ 221 ꢀC;
ring), 1.83e1.88 (2H, m, piperidine ring), 3.63e3.70 (1H, m, piper-
idine ring), 3.81 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.91e3.99 (3H, m,
piperidine ring), 4.04e4.09 (1H, m, piperidine ring), 4.08 (1H, dd,
J ¼ 11.3, 7.8 Hz, CHH-O), 4.50 (1H, dd, J ¼ 11.3, 2.7 Hz, CHH-O), 4.55
(1H, dd, J ¼ 7.8, 2.7 Hz, CH-C¼O), 5.63 (2H, br s, NH2), 6.52 (1H, d,
J ¼ 8.2 Hz, Ar), 6.67 (1H, td, J ¼ 8.2, 1.6 Hz, Ar), 6.76 (1H, td, J ¼ 8.2,
1.6 Hz, Ar), 6.81 (1H, dd, J ¼ 8.2, 1.6 Hz, Ar), 6.84 (1H, s, Ar), 6.94 (1H,
s, Ar), 7.14 (1H, d, J ¼ 7.0 Hz, NH-C¼O). 13C NMR (free base, 100 MHz,
20
[
a]
¼ ꢂ20.66; 1H NMR (free base, 400 MHz, CDCl3)
d 1.43e1.49
D
(2H, m, piperidine ring), 1.75e1.79 (2H, m, piperidine ring),
3.54e3.60 (1H, m, piperidine ring), 3.80 (1H, dd, J ¼ 12.9, 2.7 Hz,
piperidine ring), 3.91 (3H, s, OCH3), 3.91e3.95 (1H, m, piperidine
ring) 3.95 (3H, s, OCH3), 4.01 (1H, dd, J ¼ 13.3, 5.9, piperidine ring),
4.05e4.10 (1H, m, piperidine ring), 4.22 (1H, dd, J ¼ 11.3, 6.4 Hz,
CHH-O), 4.42 (1H, dd, J ¼ 11.3, 2.7 Hz, CHH-O), 4.63 (1H, dd, J ¼ 6.4,
2.7 Hz, CH-C¼O), 5.60 (2H, br s, NH2), 6.44 (1H, dd, J ¼ 8.2, 1.2 Hz,
Ar), 6.63 (1H, td, J ¼ 8.2, 2.0 Hz, Ar), 6.79 (1H, td, J ¼ 7.0, 1.2 Hz, Ar),
6.83 (1H, dd, J ¼ 8.2, 2.0 Hz, Ar), 6.96 (2H, s, Ar), 7.03 (1H, d,
CDCl3) d 22.26, 29.71, 44.66, 46,14, 48.30, 55.88, 56.11, 65.58, 73.09,
101.69, 102.87, 105.61, 117.10, 117.27, 121.63, 122.21, 141.40, 142.96,
145.80, 154.97, 160.86, 166.75. HRMS-ESI (m/z): calcd for
J ¼ 7.0 Hz, NH-C¼O); 13C NMR (free base, 100 MHz, CDCl3)
d
21.90,
C
24H28N5O5 [MþH]þ 466.2090, found 466.2092.
29.72, 44.62, 46.14, 48.12, 56.07, 56.17, 65.21, 73.19, 101.65, 102.90,
105.59, 116.90, 117.38, 121.68, 122.18, 141.38, 143.16, 145.86, 155.13,
160. HRMS-ESI (m/z): calcd for C24H28N5O5 [MþH]þ 466.2090,
found 466.2095.
5.1.3. General procedure for the synthesis of 9 and 10
A solution of 1,4-naphthoquinone (0.5 mmol) in EtOH (3 mL)
was added dropwise to a solution of 1 or 2 (0.5 mmol) in EtOH
(1 mL). The mixture was stirred at r.t. for 8 h. The solvent was
evaporated under reduced pressure and the crude was purified by
flash chromatography eluting with a mixture of CHCl3/MeOH
(9.5:0.5) affording 9 and 10, respectively, as a red solid. 9 and 10
were then converted into the corresponding hydrochloride salts.
5.1.2.4. (S)-N-((R)-1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piper-
idin-3-yl)-2,3-dihydrobenzo
Compound 6 was synthesized by reacting 1 with 14. The crude was
purified by flash chromatography (CHCl3-MeOH, 9.5:0.5) affording
[b][1,4]dioxine-2-carboxamide
(6).

V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
267
5.1.3.1. (R)-2-((1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperidin-
5.1.5. General procedure for the synthesis of 13 and 14
3-yl)amino)naphthalene-1,4-dione
(9). 85%
yield;
mp
Thionyl chloride (4 eq) was added dropwise at 0 ꢀC to a solution
of (R)- or (S)-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic acid (1
eq) in toluene (5 mL). The mixture was stirred at reflux for 4 h. The
solvent was then evaporated to dryness affording 13 and 14,
respectively, as a yellow oil in quantitative yield.
20
(salt) > 250 ꢀC; [
CDCl3)
a]
¼ ꢂ94.46; 1H NMR: (free base, 400 MHz,
D
d
1.59e1.69 (1H, m, CHH), 1.75e1.84 (2H, m, CH2), 2.02e2.06
(1H, m, CHH), 3.13 (1H, dd, J ¼ 12.9, 8.6 Hz, CHH-N), 3.35e3.48 (2H,
m, CH, CHH-N), 3.89 (3H, s, OCH3), 4.00 (3H, s, OCH3), 4.32e4.36
(1H, m, CHH-N), 4.61 (1H, d, J ¼ 12.9 Hz, CHH-N), 5.73 (2H, s, NH2),
6.14 (1H, d, J ¼ 7.0 Hz, NH-Ar), 6.58 (1H, s, Ar), 6.88 (1H, s, Ar), 7.04
(1H, s, Ar), 7.58 (1H, td, J ¼ 7.7, 1.2 Hz, Ar), 7.69 (1H, td J ¼ 7.7, 1.2 Hz,
Ar), 8.00 (1H, dd, J ¼ 7.7 Hz, 1.0, Ar), 8.07 (1H, dd, J ¼ 7.7, 1.0 Hz, Ar);
5.1.5.1. (R)-2,3-Dihydrobenzo[b][1,4]dioxine-2-carbonyl
(13). 1H NMR: (400 MHz, CDCl3)
chloride
d
4.34 (1H, dd, J ¼ 12.0, 2.8 Hz,
CHH-O), 4.76 (1H, dd, J ¼ 12.0, 2.8 Hz, CHH-O), 5.10 (1H, t, J ¼ 2.8 Hz,
13C NMR: (free base, 100 MHz, CDCl3)
d 23.49, 30.00, 45.50, 48.33,
CH-C¼O), 6.93e7.09 (4H, m, Ar); 13C NMR: (100 MHz, CDCl3)
48.91, 56.10, 56.17, 101.34, 102.49, 102.92, 105.87, 125.93, 126.23,
130.69, 131.90, 133.55, 134.59, 145.93, 147.24, 155,10, 160.97, 181.89,
183.48. HRMS-ESI (m/z): calcd for C25H26N5O4 [MþH]þ 460.1985,
found 460.1995.
d 63.87, 78.70, 117.40, 117.72, 122.61, 122.89, 142.54, 142.99, 170.44.
5.1.5.2. (S)-2,3-Dihydrobenzo[b][1,4]dioxine-2-carbonyl
(14). 1H NMR: (400 MHz, CDCl3)
chloride
d
4.35 (1H, dd, J ¼ 11.8, 2.7 Hz,
CHH-O), 4.75 (1H, dd, J ¼ 11.8, 2.7 Hz, CHH-O), 5.10 (1H, t, J ¼ 2.7 Hz,
5.1.3.2. (S)-2-((1-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperidin-
CH-C¼O), 6.91e6.98 (3H, m, Ar), 7.02e7.04 (1H, m, Ar); 13C NMR:
3-yl)amino)naphthalene-1,4-dione (10). 86% yield; mp (hydrochlo-
(100 MHz, CDCl3)
d 63.77, 78.58, 117.27, 117.58, 122.47, 122.74,
20
ride salt) > 250 ꢀC; ES [M þ Hþ]: 460; [
a
]
D
¼ þ93.51; 1H NMR: (free
141.39, 142.82, 170.26.
base, 400 MHz, CDCl3)
d 1.64e1.65 (1H, m, CHH), 1.74e1.81 (2H, m,
CH2), 2.02e2.06 (1H, m, CHH), 3.15 (1H, dd, J ¼ 12.9, 8.2 Hz, CHH-N),
3.39e3.44 (2H, m, CHH-N, CH), 3.88 (3H, s, OCH3), 3.99 (3H, s,
OCH3), 4.32e4.35 (1H, m, CHH-N), 4.61 (1H, d, J ¼ 12.9 Hz, CHH-N),
5.68 (2H, br s, NH2), 6.13 (1H, d, J ¼ 7.0 Hz, NH-Ar), 6.58 (1H, s, Ar),
6.87 (1H, s, Ar), 7.02 (1H, s, Ar), 7.58 (1H, td, J ¼ 7.6, 1.2 Hz, Ar), 7.68
(1H, td, J ¼ 7.6, 1.2 Hz, Ar), 7.99 (1H, dd, J ¼ 7.6, 0.8 Hz, Ar), 8.06 (1H,
5.2. Biological evaluation methods
5.2.1. Radioligand binding assays
Binding to cloned human a1-AR subtypes was performed in
membranes from CHO cells transfected by electroporation with
DNA expressing the gene encoding each a1-AR subtype. Cloning
and stable expression of the human a1-AR gene was performed as
dd, J ¼ 7.6, 0.8 Hz, Ar); 13C NMR: (free base,100 MHz, CDCl3)
d 23.63,
30.14, 45.61, 48.45, 49.05, 56.24, 56.31, 101.42, 102.64, 103.06,
106.08,126.06,126.35,129.60,130.82,132.02,133.69,134.71,146.03,
147.37, 155.20, 161.07, 182.05, 183.58; Anal. Calcd for C25H28ClN5O5:
C 58.42, H 5.49, N 13.63, found C 58.46, H 5.29, N 13.21. HRMS-ESI
(m/z): calcd for C25H26N5O4 [MþH]þ 460.1985, found 460.1995.
previously described [30]. CHO cell membranes (30 mg proteins)
were incubated in 50 mM Tris-HCl (pH 7.4) with 0.2 nM [3H]pra-
zosin, in a final volume of 1.02 mL for 30 min at 25 ꢀC, in absence or
presence of competing drugs (1 pMe10
was determined in the presence of 10
m
M). Non-specific binding
mM phentolamine. The in-
cubation was stopped by addition of ice-cold Tris-HCl buffer and
rapid filtration through 0.2% polyethylenimine pretreated What-
man GF/B or Schleicher & Schuell GF52 filters. Binding data were
analyzed using the non-linear curve-fitting program Allfit. Equi-
librium inhibition constants (Ki) were derived from the Chenge-
Prusoff equation, Ki ¼ IC50/(1 þ L/Kd), where L and Kd are the
concentration and the equilibrium dissociation constant of the
radioligand. pKi values are the mean SE of 2e3 separate experi-
ments performed in triplicate.
5.1.4. General procedure for the synthesis of 11 and 12
4-Amino-2-chloro-6,7-dimethoxyquinazoline (1 eq) and (R) or
(S)-3-(Boc-amino)piperidine (2 eq) were dissolved in isoamyl
alcohol (20 mL). The mixture was stirred under microwave irradi-
ation at 130 ꢀC for 50 min. After evaporating the solvent under
reduced pressure, the crude was purified by flash chromatography
eluting with a mixture of petrol/EtOAc/CHCl3/MeOH (3:2:4:1)
affording, respectively, 11 and 12 as a white solid in quantitative
yield.
5.2.2. Cell cultures
5.1.4.1. tert-Butyl
(R)-(1-(4-amino-6,7-dimethoxyquinazolin-2-yl)
1.41
DU-145, PC-3 and LNCaP human prostate cell lines were
routinely grown at 37 ꢀC in a humidified incubator with 5% CO2 in
RPMI medium supplemented with 10% fetal calf serum, 2 mM
piperidin-3-yl)carbamate (11). 1H NMR: (400 MHz, CDCl3)
d
(9H, s, OtBu), 1.56e1.63 (2H, m, piperidine ring), 1.70e1.77 (1H, m,
piperidine ring), 1.84e1.89 (1H, m, piperidine ring), 3.53 (1H, dd,
J ¼ 12.7, 6.8 Hz, piperidine ring), 3.63e3.73 (2H, m, piperidine ring),
3.83 (1H, dd, J ¼ 7.0, 2.7 Hz, piperidine ring), 3.87 (3H, s, OCH3), 3.92
(3H, s, OCH3), 4.02 (1H dd, J ¼ 12.8, 2.7 Hz, piperidine ring), 4.91
(1H, d, J ¼ 6.7 Hz, NH-C¼O), 5.69 (2H, s, NH2), 6.91 (1H, s, Ar), 6.95
glutamine, 50 U/ml penicillin and 50
mg/mL streptomycin. To
determine the cell proliferation and apoptosis the human prostate
cells were seeded in 96-well plates at 5 ꢁ 103 cells/well and
1 ꢁ 103 cells/well, respectively. To evaluate the necrosis the cells
were seeded in 6-well plates at 1 ꢁ 106 cells/well. To determine the
redox influence in terms of intracellular ROS formation and
decreased GSH level, the cells were seeded in 96-well plates at
2 ꢁ 104 cells/well. All experiments were performed after 24 h of
incubation at 37 ꢀC in 5% CO2.
(1H, s, Ar); 13C NMR: (100 MHz, CDCl3)
d 22.67, 28.41, 30.61, 44.40,
46.70, 49.21, 55.96, 56.10, 79.21, 101.83, 102.91, 105.47, 145.66,
154.93, 155.38, 158.88, 160.80.
5.1.4.2. tert-Butyl
(S)-(1-(4-amino-6,7-dimethoxyquinazolin-2-yl)
1.26
piperidin-3-yl)carbamate (12). 1H NMR: (400 MHz, CDCl3)
d
5.2.3. Determination of cell proliferation
(9H, s, OtBu), 1.36e1.38 (2H, m, piperidine ring), 1.52e1.58 (1H, m,
piperidine ring), 1.67e1.72 (1H, m, piperidine ring), 3.23e3.32 (2H,
m, piperidine ring), 3.54e3.57 (1H, br m, piperidine ring), 3.61 (3H,
s, OCH3), 3.69 (3H, s, OCH3), 3.80e3.84 (1H, m, piperidine ring),
4.00e4.03 (1H, br m, piperidine ring), 5.09 (1H, br s, NH-C¼O), 6.15
(2H, s, NH2), 6.76 (1H, s, Ar), 7.00 (1H, s, Ar); 13C NMR: (100 MHz,
5.2.3.1. NCI cancer cell line screening. The NCI screening is a two-
stage process beginning with the evaluation of all compounds
against the 60 cell lines at a single concentration of 10ꢂ5 M. Com-
pounds exhibiting significant growth inhibition will progress to the
full five-dose assay, according to standard procedures [31]. In both
cases the exposure time is 48 h. The one-dose data will be reported
as a mean graph of the percent growth of treated cells. The number
reported for the one-dose assay is growth relative to the no-drug
CDCl3)
d 22.74, 28.20, 30.47, 44.15, 46.62, 49.08, 55.54, 55.70, 78.90,
102.34,102.90, 105.03, 145.27, 148.93,154.50,155.33,158.69,160.99.

268
V. Maestri et al. / European Journal of Medicinal Chemistry 136 (2017) 259e269
control, and relative to the time zero number of cells. This allows
detection of both growth inhibition (values between 0 and 100) and
lethality (values less than 0). A value of 100 means no growth in-
hibition [32].
monochlorobimane (MCB), as previously described [35]. Briefly,
LNCaP cells were treated with compounds (1e10 M) for 24 h at
37 ꢀC in 5% CO2. After treatment, the cells were washed with PBS
and then incubated with 50
M of MCB in PBS at 37 ꢀC in 5% CO2 for
m
m
30 min. At the end of incubation, the fluorescence of the cells from
each well was measured (lexcitation ¼ 360 nm, lemission ¼ 465 nm)
with a spectrofluorometer (Wallac Victor® Multilabel Counter,
Perkin Elmer Inc.). The results were expressed as arbitrary unit of
fluorescence (AUF).
5.2.3.2. Prostate cancer cell line screening. The DU-145, PC-3 and
LNCaP cell proliferation was evaluated with the MTT [3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
assay as previously described [33]. Briefly, the cells were treated
with compounds (0.1e50
M) for 120 h at 37 ꢀC in 5% CO2. After
bromide]
m
treatment, the cells were washed with phosphate buffered saline
(PBS) and then incubated with MTT (5 mg/mL) in PBS for 4 h. After
removal of MTT and further washing, the formazan crystals were
dissolved with isopropanol. The amount of formazan was measured
(570 nm) with a spectrophotometer (TECAN®, Spectra model
Classic, Salizburg, Austria). The cell viability was expressed as a
Acknowledgement
This work was supported by the University of Bologna (RFO).
Professor Carlo Melchiorre is gratefully acknowledged for fruitful
discussions. We thank the National Cancer Institute (Bethesda, MD,
USA) for the anticancer assays.
percentage of control cells and calculated by the formula Ft/Fnt
x
100, where Ft ¼ absorbance of treated cells and Fnt ¼ absorbance of
untreated cells. At least three independent dose-response curves
were plotted and the concentration of compound resulting in 50%
inhibition of cell proliferation (IC50) was calculated.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.003.
5.2.4. Determination of apoptosis and necrosis
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