Pharmacological exploitation of the alpha1-adrenoreceptor antagonist doxazosin to develop a novel class of antitumor agents that block intracellular protein kinase B/Akt activation.

Article abstract of DOI:10.1021/jm049752k

The alpha1-adrenoreceptor antagonist doxazosin induces apoptosis in malignant cells with moderate potency via an alpha1-adrenoreceptor-independent mechanism. Here, we demonstrate that the ability of doxazosin to induce apoptosis in PC-3 prostate cancer cells was, in part, attributable to the inhibition of protein kinase B (PKB)/Akt activation. The separation of the effect of doxazosin on apoptosis from its original pharmacological activity provides molecular underpinnings to develop novel antitumor agents. Replacement of the (2,3-dihydro-benzo[1,4]dioxane)-carbonyl moiety of doxazosin with aryl-sulfonyl functions dramatically improves the potency in facilitating Akt deactivation and inducing apoptosis. The optimal compounds, 33 and 44, were effective in apoptosis induction at low micromolar concentrations irrespective of androgen dependency and p53 functional status. Both agents were active in suppressing the growth of a panel of 60 cancer-cell lines with IC50 values of 2.2 and 1.5 microM, respectively. Together, these in vitro efficacy data suggest the translational potential of these agents in prostate cancer treatment.

Full text of DOI:10.1021/jm049752k

J . Med. Chem. 2004, 47, 4453-4462
4453
P h a r m a cologica l Exp loita tion of th e r1-Ad r en or ecep tor An ta gon ist Doxa zosin
to Develop a Novel Cla ss of An titu m or Agen ts Th a t Block In tr a cellu la r P r otein
Kin a se B/Ak t Activa tion
Yeng-J eng Shaw,† Ya-Ting Yang,† J ason B. Garrison,‡ Natasha Kyprianou,‡ and Ching-Shih Chen*,†
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
and Division of Urology, Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky 40536
Received March 30, 2004
The R1-adrenoreceptor antagonist doxazosin induces apoptosis in malignant cells with moderate
potency via an R1-adrenoreceptor-independent mechanism. Here, we demonstrate that the
ability of doxazosin to induce apoptosis in PC-3 prostate cancer cells was, in part, attributable
to the inhibition of protein kinase B (PKB)/Akt activation. The separation of the effect of
doxazosin on apoptosis from its original pharmacological activity provides molecular underpin-
nings to develop novel antitumor agents. Replacement of the (2,3-dihydro-benzo[1,4]dioxane)-
carbonyl moiety of doxazosin with aryl-sulfonyl functions dramatically improves the potency
in facilitating Akt deactivation and inducing apoptosis. The optimal compounds, 33 and 44,
were effective in apoptosis induction at low micromolar concentrations irrespective of androgen
dependency and p53 functional status. Both agents were active in suppressing the growth of
a panel of 60 cancer-cell lines with IC50 values of 2.2 and 1.5 µM, respectively. Together, these
in vitro efficacy data suggest the translational potential of these agents in prostate cancer
treatment.
In tr od u ction
kinase B (PKB)/Akt (designated as Akt from this point)
activation. Our data indicate that the apoptosis-induc-
ing potency of doxazosin was correlated with its efficacy
in facilitating Akt dephosphorylation and that the
overexpression of constitutively active Akt could par-
tially protect cells from drug-induced apoptosis. Con-
sequently, we carried out structural modifications of
doxazosin to generate a novel class of apoptosis-inducing
agents with improved efficacy in blocking intracellular
Akt activation.
The R1-adrenoreceptor antagonist doxazosin (Car-
dura) has been safely used for the treatment of benign
prostatic hyperplasia (BPH).1 It relaxes prostate smooth
muscle through the blockade of R1-adrenergic innerva-
tion to the prostate. In addition, this alpha-blocker also
exhibits moderate potency in inducing apoptosis in
prostate cancer cells2-5 and shows synergistic antitumor
effects in conjunction with radiation6 or certain chemo-
therapeutics such as adriamycin and etoposide7 against
prostate cancer cells. In light of its potential use in the
prevention/treatment of prostate cancer, the mechanism
by which doxazosin mediates apoptosis has been the
focus of many recent publications.8 It is noteworthy that
the in vitro antitumor activity of doxazosin is mediated
via an R1-adrenoreceptor-independent pathway.9 Puta-
tive mechanisms underlying doxazosin-mediated apo-
ptosis include the upregulation of transforming growth
factor â (TGF-â) signaling and increased gene expres-
sion of p21 and IκBR (inhibitor of NF-κB R).10,11 From a
drug discovery perspective, the separation of the effect
of doxazosin on apoptosis in prostate cancer cells from
its original pharmacological activity in normal cells
provides a molecular basis to develop a novel class of
apoptosis-inducing agents through lead optimization.
Ch em istr y
To optimize the apoptosis-inducing activity of dox-
azosin, we carried out the structural modifications in a
systematic manner (Figure 1).
In strategy A, we replaced the 2,3-dihydrobenzo[1,4]-
dioxane moiety with different aromatic acyl side chains
to produce compounds 1-10 (Figure 2, Scheme 1; Table
1). In strategy B, we substituted the aryl carboxamide
function with aryl sulfonamides to generate compounds
11-40 (Figure 2, Scheme 2; Tables 2 and 3). In strategy
C, the piperazine moiety of the optimal compounds (23
and 33) was replaced by an ethylenediamine linker,
generating compounds 41 and 42, respectively (Figure
2, Scheme 3; Chart 1). In strategy D, we modified the
methoxy side chains on the quinazoline ring of com-
pound 33 to prepare compounds 43-46 (Figure 2,
Scheme 4; Table 4).
All compounds were evaluated for their ability to
induce apoptotic death in human androgen-independent
PC-3 prostate cancer in RPMI 1640 medium containing
10% fetal bovine serum (FBS). For representative
compounds tested in DU-145 and LNCaP prostate
cancer cells, the IC50 values for inhibiting cell prolifera-
tion were similar in these three cell lines irrespective
In this study, we obtained evidence that the ability
of doxazosin to induce apoptotic death in PC-3 androgen-
independent prostate cancer cells was, at least in part,
attributable to the inhibition of intracellular protein
* To whom correspondence should be addressed. Address: Parks
Hall, Rm 336, 500 West 12th Avenue, The Ohio State University,
Columbus, Ohio 43210-1291. Tel: (614) 688-4008. Fax: (614) 688-8556.
E-mail: chen.844@osu.edu.
† The Ohio State University.
‡ University of Kentucky Medical Center.
10.1021/jm049752k CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/06/2004

4454 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Shaw et al.
F igu r e 1. Overall strategy for the structural modification of doxazosin. A, B, C, and D denote four modification strategies that
target the 2,3-dihydro-benzo[1,4]dioxane moiety, the terminal acyl function, the piperazine linker, and the methoxy side chain of
the quinazoline base, respectively. The numbers indicate the designation of doxazosin derivatives.
Ta ble 1. Structures and IC50 Values of Compounds 1-10
a
a
IC50
IC50
entry
Ar
(PC-3)
entry
Ar
(PC-3)
doxazosin
2,3-dihydrobenzo[1,4]dioxane
4-chlorophenyl
4-cyanophenyl
benzyloxy
3-cyanophenyl
45 ( 5
60 ( 8
68 ( 6
63 ( 6
82 ( 5
75 ( 11
6
7
8
9
3,4-dimethoxyphenyl
1-naphthyl
4-aminophenyl
4-tert-butylphenyl
4-(trifluoromethyl)phenyl
71 ( 8
59 ( 4
>100
47 ( 6
>100
1
2
3
4
5
10
4-nitrophenyl
a
Values represent means + sd (n ) 6).
of differences in androgen sensitivity, PTEN mutation,
the functional status of p53 and Rb, and other biomar-
kers.
specificity of the drug action on intracellular signaling
pathways. It is also noteworthy that doxazosin exhibited
no inhibitory effects on the kinase activity of immuno-
precipitated Akt.
Resu lts
To examine the causal relationship between Akt
deactivation and doxazosin-mediated apoptosis, we as-
sessed the protective effect of the transient transfection
of constitutively active Akt, AktT308D/S473D (ref 14), on
drug-induced PC-3 cell death. Western blot analysis
using antibodies against Akt and the HA tag confirmed
that transient transfection of AktT308D/S473D led to a
severalfold increase in Akt expression (Figure 4A).
These transient transfectants were exposed to 25 µM
doxazosin in a 1% FBS-supplemented medium to ex-
amine their susceptibility to drug-induced cell death vis-
a`-vis transfectants with an empty pcDNA vector (panel
B). As shown, AktT308D/S473D gave partial, yet significant,
protection against doxazosin-induced apoptotic death.
Together, these data suggest that doxazosin-induced
apoptosis in PC-3 cells was mediated, in part, through
the inhibition of intracellular Akt activation. This
premise was in line with the finding that the apoptosis-
inducing potency of doxazosin was attenuated in 10
versus 1% FBS-supplemented medium, with the IC50
value increasing from 20 to 45 µM. This precipitous drop
Doxa zosin -In d u ced Ap op tosis, in P a r t, by F a -
cilita tin g Ak t Dep h osp h or yla tion . Exposure of PC-3
cells to doxazosin in 1% FBS-supplemented RPMI 1640
medium resulted in time- and dose-dependent apoptotic
death, as evidenced by the disappearance of the native
form of PARP (Figure 3A). The potency of doxazosin in
inducing apoptosis, however, was moderate. Although
PC-3 cells were susceptible to the drug-induced apop-
tosis at 25 µM and up, no appreciable apoptotic death
was noted at 10 µM.
To shed light on the mechanism of doxazosin-medi-
ated apoptosis, we investigated the effect of doxazosin
on the phosphorylation state of Akt and ERKs, two
signaling kinases that play a pivotal role in cell prolif-
eration and survival12,13 in PC-3 cells. The exposure of
PC-3 cells to doxazosin caused Akt dephosphorylation
in a dose- and time-dependent manner (Figure 3B,
upper and lower panels, respectively). In contrast,
doxazosin, even at 50 µM, did not affect the phospho-
rylation status of ERKs (Figure 3B), suggesting the

Doxazosin-Derived Apoptosis-Inducing Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4455
F igu r e 2. Synthetic schemes employed for the structural modifications of doxazosin.
Ta ble 2. Structures and IC50 Values of Compounds 11-35
a
a
IC50
IC50
entry
Ar
(PC-3)
entry
Ar
3-carboxyphenyl
4-carboxyphenyl
2,5-dichlorophenyl
(PC-3)
11
12
13
14
15
16
17
18
19
20
21
22
23
4-chlorophenyl
4-bromophenyl
4-iodophenyl
5-chlorothienyl
2-nitrophenyl
3-nitrophenyl
4-nitrophenyl
4-methylphenyl
4-(trifluoromethyl)phenyl
4-methoxyphenyl
4-(trifluoromethoxy)phenyl
4-(methylsulfonyl)phenyl
4-tert-butylphenyl
23 ( 3
20 ( 2
15 ( 2
25 ( 3
35 ( 4
32 ( 5
36 ( 3
30 ( 2
27 ( 3
39 ( 4
25 ( 2
17 ( 3
4.1 ( 0.7
24
25
26
27
28
29
30
31
32
33
34
35
53 ( 4
53 ( 5
60 ( 7
67 ( 5
56 ( 4
52 ( 6
14 ( 2
15 ( 2
29 ( 4
4.2 ( 0.8
24 ( 3
5.2 ( 0.9
2,4-diaminophenyl
3-carboxy-4-chloro-5-fluorophenyl
3-carboxy-4,6-dichlorophenyl
1-naphthyl
2-naphthyl
1-(5-dimethylamino)naphthyl
biphenyl-
2,4,6-tri-isopropylphenyl
4-(phenanthren-9-yl)phenyl
a
Values represent means + sd (n ) 6).
in potency was reminiscent for that noted for cyclooxy-
genase 2 inhibitor celecoxib,15 which might be attribut-
able to several factors. First, like celecoxib, doxazosin
displays a high binding affinity with serum proteins,16
resulting in lower intracellular drug concentrations.
Second, continuous stimulation of phosphoinositide

4456 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Ch a r t 1. Structures and IC50 Values of Compounds 41 and 42
Shaw et al.
Ta ble 3. Structures and IC50 Values of Compounds 36-40
a
a
IC50
(PC-3) entry
IC50
entry
R
R
(PC-3)
36 4-methyl
3.4 ( 0.4 39 4-n-butyl
3.4 ( 0.2
37 4-trifluoromethyl 3.3 ( 0.3 40 4-tert-butyl 10 ( 2
38 4-methylsulfonyl 7.2 ( 0.5
a
Values represent means + sd (n ) 6).
Ta ble 4. Structures and IC50 Values of Compounds 43-46
a
a
IC50
IC50
entry
R
(PC-3)
entry
R
(PC-3)
43
44
allyl
n-propyl
3.3 ( 0.4
2.5 ( 0.3
45
46
isopropyl
n-butyl
24 ( 5
F igu r e 3. Induction of apoptosis in PC-3 cells by doxazosin.
(A, left panel) Time- and dose-dependent effect of doxazosin
on the cell viability of PC-3 cells in 1% FBS-supplemented
RPMI 1640 medium. Values obtained from six replicates were
plotted at each time point at the indicated concentrations of
doxazosin. (A, right panel) Induction of poly(ADP-ribose)
polymerase (PARP) cleavage by doxazosin at the indicated
concentrations after 48 h of treatment. PARP proteolysis to
the apoptosis-specific 85-kd fragment was monitored by West-
ern blotting. Although there was no substantial accumulation
of the 85-kd fragment, a significant decrease in the level of
native protein was noted. (B) Dose- and time-dependent (upper
and lower panels, respectively) effects of doxazosin on Akt
phosphorylation. (C) dose-dependent effect of doxazosin on
ERK phosphorylation. PC-3 cells were treated with doxazosin
at the indicated concentrations for 24 h or at 25 µM for the
indicated times and lysed, and proteins in the resulting
supernatants were resolved on SDS-PAGE and subjected to
Western blot analysis. The phosphorylation status of Akt and
ERKs was determined by immunoblotting with the respective
phosphospecific antibodies. Unphosphorylated Akt and ERKs,
as immunostained by anti-Akt and anti-ERK antibodies, were
used as internal standards for the comparison of phospho-Akt
and phospho-ERK levels among samples of different prepara-
tions. The blots are representative of three independent
experiments.
3.5 ( 0.4
a
Values represent means + sd (n ) 6).
3-kinase (PI3K)/Akt signaling through various growth
factor receptors counters the inhibitory effect of dox-
azosin on Akt. Third, serum could upregulate Bcl-xL,
which enhances the threshold to apoptotic signals
emanating from PI3K/Akt inhibition.17
Role of th e Ar om a tic Acyl Sid e Ch a in s in Ap o-
p tosis In d u ction (Str a tegy A). Substitution of the 2,3-
dihydro-benzo[1,4]dioxane moiety of doxazosin with
different aromatic acyl side chains gave derivatives with
varying potency in apoptosis induction (Table 1). In
general, analogues with hydrophilic side chains exhib-
ited lower apoptosis-inducing activity, whereas that of
a hydrophobic aromatic system, for example, tert-
butylphenyl, retained the in vitro efficacy. These find-
ings, however, provided a proof of principle that dox-
azosin was amenable to structural optimization to
develop a new class of apoptosis-inducing agents.
Ar yl Su lfon a m id e Der iva tives Exh ibited High
P oten cy in Tr igger in g Ap op tosis (Str a tegy B). To
explore the functional role of the acyl function in
apoptosis induction further, we replaced the carboxa-
mide moiety of compounds 1, 5, 7, and 9 with sulfona-
mide, yielding compounds 11, 17, 30, and 23, respec-
tively. As shown in Table 2, this substitution resulted
in a substantial increase in apoptosis-inducing potency.
As shown, the core structural component, that is, the
quinazoline base and the adjacent piperazine ring,
conferred a high degree of structural rigidity to the
molecule. The boat conformation of the piperazine ring
oriented the N1 appendage, that is, carbonyl or sulfonyl,
perpendicular to the quinazoline planar structure. We
rationalized that the discrepancy in potency was at-
tributable to the transition from a trigonal-planar
structure of a carboxamide moiety (upper panel) to a
tetrahedronlike structure of sulfonamide (lower panel).
As a result, the spatial arrangement of the aromatic
Among these four pairs of compounds, 23 exhibited 1
order of magnitude higher potency than its carboxamide
counterpart 9. To understand the structural basis for
this improvement in potency, we compared the energy-
minimized structures of compounds 9 and 23 (Figure
5).

Doxazosin-Derived Apoptosis-Inducing Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4457
F igu r e 6. (A) Time- and dose-dependent effects of compound
33 on the cell viability of PC-3 cells in 1% FBS-supplemented
RPMI 1640 medium. (B) Western blot analysis of RAPR
proteolysis and Akt dephosphorylation in PC-3 cells treated
with the indicated concentrations of 33 for 48 h. (C) Effects of
compound 44 on Akt phosphorylation in PC-3 cells treated
with the indicated concentrations for 48 h.
F igu r e 4. Protective effect of constitutive active Akt on
doxazosin-induced apoptotic death in PC-3 cells. (A) Expression
of AktT308D/S473D in PC-3 transient transfection. Western blot
analysis used antibodies against Akt and the HA tag. (B)
Viability of PC-3 cells overexpressing AktT308D/S473D vis-a`-vis
cells transfected with empty pcDNA vector (mock) in the
presence of 25 µM doxazosin in 1% FBS-supplemented medium
for 24 h. Values are means ( sd (n ) 3).
compounds 23, 33, and 35, with the side chains of tert-
butylphenyl, biphenyl, and phenanthren-9-yl-phenyl,
respectively, represented the optimal compounds, with
IC50 values in the range of 4-5 µM in 10% serum-
containing medium at 48 h.
Figure 6A indicates a dose-dependent effect of com-
pound 33 on apoptosis in a 1% FBS-supplemented
medium, as evidenced by PARP proteolysis (Figure 6B),
with an IC50 value of approximately 2.5 µM at 48 h.
Western blot analysis confirmed that this apoptotic
effect was attributable, in part, to the inhibition of Akt
activation (Figure 6B).
Further modifications of the biphenyl ring of com-
pound 33 by adding alkyl chains such as CH3, CF3, or
n-C4H9 at the 4′ position did not further improve the
apoptosis-inducing potency (Table 3). However, a sig-
nificant drop in potency was noted with the bulky tert-
butyl substitution.
Im p or ta n ce of th e P ip er a zin e Rin g to th e Ap o-
p tosis-In d u cin g P oten cy (Str a tegy C). The 4-(4-
amino-6,7-dimethoxy-quinaolin-2-yl)-piperazine moiety
provided structural rigidity to the molecule, which
might play a role in the ligand-protein interactions. To
examine this premise, we replaced the piperazine ring
of compounds 23 and 33 with an ethylenediamine
linker, generating 41 and 42 (Chart 1).
This replacement resulted in a 2-fold decrease in
apoptosis-inducing potency, suggesting the importance
of this unique structural feature in maintaining the
efficacy.
Role of th e Alk oxy Su bstitu en t on th e Qu in a zo-
lin e Rin g in th e In d u ction of Ap op tosis (Str a tegy
D). To optimize the activity of compound 33 in inducing
apoptosis further, we replaced the methoxy side chains
with alkoxy functions with different stereochemical
properties (Table 4).
Among the four derivatives, compound 44 represented
the optimal compound with a slight improvement in
potency (IC50 ) 2.5 µM in 10% FBS-supplemented
medium), whereas its isopropyl counterpart 45 dis-
played a precipitous drop in potency (IC50 ) 24 µM).
F igu r e 5. Comparison of chemical and 3D structures of
compounds 9 and 23. The 3D structures of small molecules
were generated using the software SYBYL 6.9 (Tripos Associ-
ate; St. Louis, MO) on Silicon Graphics O2 (Silicon Graphics
Inc.; Mountain View, CA). Energy minimization was carried
out with default parameters (minimum rms gradient, 0.005
kcal/mol; maximum iterations, 1000; minimum energy change,
0.05 kcal/mol).
sidearm relative to the neighboring plane of the quinazo-
line system differed.
Further examinations of the impact of the aryl sul-
fonamide function on apoptosis-inducing potency con-
firmed the preference for bulky, hydrophobic aromatic
systems (Table 2). Among the 25 derivatives examined,

4458 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Shaw et al.
These data suggest a very subtle impact of the quinazo-
line side-chain structure on target binding. Again, the
induction of apoptosis by compound 44 is characterized
by the dephosphorylation of phospho-Akt in a dose-
dependent manner, which was evident at concentrations
as low as 1 µM (Figure 6C).
clearly demonstrate the in vitro efficacy of these agents
and their potential application in cancer prevention and/
or treatment. Testing of the in vivo efficacy of these
agents in xenograft models constitutes the focus of the
present investigation.
Con clu sion s
Discu ssion
The present study demonstrates that doxazosin could
be pharmacological exploited to develop a novel class
of antitumor agents that mediate apoptosis in prostate
cancer cells via the inhibition of intracellular Akt
activation. In light of the importance of Akt signaling
in prostate cancer carcinogenesis and progression, these
novel agents may have translation potential in prostate
cancer prevention and/or therapy.
The effect of doxazosin on apoptosis in benign and
malignant prostate cancer cells suggests a plausible
extension of its clinical use from BPH management to
prostate cancer prevention.8 Thus, elucidating the un-
derlying mechanism will provide a molecular basis for
developing more efficacious antitumor agents. In this
paper, we obtained several lines of evidence that dox-
azosin mediated apoptosis, in part, through the down-
regulation of Akt signaling in PC-3 cells. Because Akt
plays a pivotal role in regulating cell growth and
survival in cancer cells, this finding underpinned the
pharmacological exploitation of doxazosin to develop a
novel class of apoptosis-inducing agents that block
intracellular Akt activation. However, the target whereby
doxazosin and its derivatives mediate Akt downregu-
lation is still under investigation, but kinase assay data
indicate that these agents displayed no direct inhibition
in vitro on protein kinase C isozymes or any of the
upstream kinases of Akt, including phosphoinositide-
dependent kinase-1 and phosphoinositide 3-kinase (data
not shown). It is plausible that these quinazoline-based
derivatives, through competing with ATP binding,
interfere with a yet unidentified tyrosine kinase that
uses Akt, but not ERKs, as a downstream effector. It is
noteworthy that these doxazosin-derived agents are
structurally distinct from existing quinazoline-based
inhibitors of epidermal growth receptor tyrosine ki-
nases18 such as Iressa (ZD1839) and CP-358,744.
We found that the replacement of the (2,3-dihydro-
benzo[1,4]dioxane)-carbonyl moiety of doxazosin with
aryl sulfonyl substituents dramatically improved the
potency in facilitating Akt dephosphorylation and in-
ducing apoptosis. Structurally optimized agent 33 ex-
hibited an order of magnitude higher potency than
parent compound doxazosin in triggering apoptotic
death in PC-3 cells. It is noteworthy that the structural
rigidity incurred by the piperazine linker was integral
in maintaining the high potency of these derivatives.
Consequently, the use of a flexible linker such as
ethylenediamine substantially reduced the apoptosis-
inducing activity of 33. Further structural optimization
was accomplished by replacing the methoxy side chains
on the quinazoline ring with propoxy functions. Both
33 and 44 were effective in suppressing the proliferation
of different prostate cancer cell lines at low micromolar
concentration levels. In addition, both agents were
submitted to the Developmental Therapeutic Program
(DTP) at the National Cancer Institute (NCI) for
screening against 60 human tumor cells lines, repre-
senting leukemia, melanoma, and cancers of lung, colon,
brain, ovary, breast, prostate, and kidney (http://dtp.n-
ci.nih.gov/index.html). All of the tested cell lines showed
high degree of sensitivity to the growth inhibitory effects
of 33 and 44. The mean GI50 values (concentration
resulting in 50% growth inhibition) among these 60 cell
lines were 2.2 and 1.5 µM, respectively. These data
Exp er im en ta l Section
Chemical reagents and organic solvents were purchased
from Aldrich unless otherwise mentioned. Nuclear magnetic
resonance spectra (1H NMR) were measured on a Bruker 250-
or 400-MHz spectrometer. Chemical shifts (δ) are reported in
ppm relative to the TMS peak. Electrospray ionization (ESI)
mass spectrometry analyses were performed with
a 3-T
Finnigan FTMS-2000 Fourier transform mass spectrometer.
Elemental analyses were within (0.4% of the calculated
values. Flash column chromatography was performed with
silica gel (230-400 mesh). Rabbit polyclonal antibodies against
Akt, phospho-Ser473-Akt, ERKs, and phospho-ERKs were
purchased from New England Biolabs (Beverly, MA). Rabbit
antipoly(ADP-ribose) polymerase (PARP) antibodies were from
BD PharMingen (San Diego, CA). Mouse antiactin monoclonal
antibody was from ICN Pharmaceuticals (Costa Mesa, CA).
Goat antirabbit immunoglobulin G (IgG)-horseradish peroxi-
dase conjugates were from J ackson ImmunoResearch Labo-
ratories.
Gen er a l P r oced u r es for th e Syn th esis of Am id es 1-10
(Sch em e 1). 4-(4-Am in o-6,7-d im eth oxy-qu in a zolin -2-yl)-
p ip er a zin e-1-ca r boxylic Acid Ben zyl Ester (iii). A mixture
of 4-amino-2-chloro-6,7-dimethoxyquinazoline (2.51 g, 10 mmol)
and benzyl 1-piperazine-carboxylate (2.24 g, 10 mmol) in
1-butanol (15 mL) was stirred under reflux overnight and
cooled to 80 °C. The crude solid product was collected, washed
with cold 1-butanol (2 × 10 mL), added to methanol (30 mL),
and heated under reflux for 1 h. The white solid was filtered
and washed with methanol (2 × 10 mL) to yield compound
1
iii. H NMR (DMSO-d6) δ 3.59-3.61 (m, 4 H), 3.83-3.89 (m,
4 H), 3.85 (s, 3 H), 3.91 (s, 3 H), 5.14 (s, 2 H), 7.14 (s, 1 H),
7.34-7.88 (m, 5 H), 7.89 (s, 1 H). HRMS (M + H)+ calcd for
C
22H26N5O4 424.1979; found 424.1989. Anal. (C22H25N5O4‚HCl)
C, H, N.
[4-(4-Am in o-6,7-d im eth oxy-qu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-ch lor op h en yl)-m eth a n on e (1). Compound iii (2.12
g, 5.0 mmol) was dissolved in methanol (15 mL), and 10%
palladium on charcoal (20 mg, 10% w/w) and triethylamine
(0.2 mL) were added. The mixture was treated with hydrogen
under atmospheric pressure for 6 h and filtered. The solvent
was evaporated to obtain the intermediate 6,7-dimethoxy-2-
piperazin-1-yl-quinazolin-4-ylamine (iv) without purification.1H
NMR (DMSO-d6) δ 3.22 (br s, 4 H), 3.83 (s, 3 H), 3.87 (s, 3 H),
3.98 (br s, 4 H), 7.54 (s, 1 H), 7.69 (s, 1 H). The intermediate
amine (0.578 g, 2.0 mmol) was dissolved in dry DMF (10 mL),
and triethylamine (0.202 g, 2.0 mmol) was added. The result-
ing mixture was treated dropwise with 4-chlorobenzoyl chlo-
ride (0.35 g, 2.0 mmol) over 15 min, stirred at room temper-
ature for 4 h, and then concentrated. The crude solid product
was washed with methanol, filtered, and recrystallized from
ethanol to give compound 1. 1H NMR (DMSO-d6) δ 3.32-3.34
(m, 4 H), 3.38 (s, 3 H), 3.84 (s, 3 H), 3.77-3.88 (m, 4 H), 7.48-
7.57 (m, 5 H), 7.73 (s, 1 H), 8.66 (br s, 1H), 8.88 (br s, 1 H).
HRMS (M + H)+ calcd for C21H23ClN5O3 428.1484; found
428.1492. Anal. (C21H22ClN5O3‚HCl) C, H, N.

Doxazosin-Derived Apoptosis-Inducing Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4459
Gen er a l P r oced u r e for th e Syn th esis of Su lfon a m id es
(Sch em e 2). Meth od A. 2-[4-(4-Ch lor oben zen esu lfon yl)-
piper azin -1-yl]-6,7-dim eth oxy-qu in azolin -4-yl-am in e (11).
To a solution of intermediate amine iv (0.578 g, 2.0 mmol) and
triethylamine (0.276 g, 2.0 mmol) in methanol (10 mL)
4-chlorobenzenesulfonyl chloride (0.443 g, 2.1 mmol) was added
to the solution. The mixture was stirred at room temperature
for 1 h. The resulting solid was filtered and then washed with
ethyl acetate (2 × 10 mL) to obtain the crude solid product.
The crude product was stirred in methanol (10 mL) under
reflux for 1 h, filtered and dried to obtain compound 11. 1H
NMR (DMSO-d6) δ 3.27 (s, 4 H), 3.35 (s, 3 H), 3.85 (s, 3 H),
3.99 (s, 4 H), 7.55 (s, 1 H), 7.60-7.80 (m, 5 H), 8.63 (s, 1 H),
8.80 (s, 1 H). HRMS (M + H)+ calcd for C20H23ClN5O4S
464.1154, found 464.1158. Anal. (C20H22ClN5O4S‚HCl) C, H,
N.
C22H30N5O4S 460.2013; found 460.2010. Anal. (C22H29N5O4S‚
HCl) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Su lfon a m id es
(Sch em e 4). 6,7-Bis-a llyloxy-2-[4-(bip h en yl-4-su lfon yl)-
p ip er a zin -1-yl]-qu in a zolin -4-yla m in e (43). A solution of
4-amino-2-chloro-6,7-dimethoxyquinazoline (2.51 g, 10.0 mmol)
in CH2Cl2 (30 mL) was cooled to -70 °C under argon, and
boron tribromide (6.01 g, 12.0 mmol) was added. The mixture
was allowed to warm to room temperature over a period of 4
h and was then cooled to -70 °C; methanol (30 mL) was added,
and the solution was concentrated. The solid residue was
washed with ethyl acetate to obtain 4-amino-2-chloro-6,7-
dihydroxyquinazoline [1H NMR (DMSO-d6) δ 7.04 (s, 1 H), 7.51
(s, 1 H)]. A mixture of the first intermediate (0.21 g, 1.0 mmol),
allyl bromide (0.432 g, 3.6 mmol), and K2CO3 (0.331 g, 2.4
mmol) in methanol (10 mL) was stirred under reflux for 12 h,
concentrated, and purified by silica gel chromatography to
afford 4-amino-2-chloro-6,7-diallyloxyquinazoline [1H NMR
(DMSO-d6) δ 4.63 (d, J ) 5.4 Hz, 2 H), 4.70 (d, J ) 1.5 Hz, 2
H), 5.28 (d, J ) 1.3 Hz, 2 H), 5.42 (d, J ) 1.3 Hz, 1 H), 5.49 (d,
J ) 1.3 Hz, 1 H), 6.07-6.11 (m, 2 H), 7.06 (s, 1 H), 7.62 (s, 1
H)]. A solution of the second intermediate (0.291 g, 1.0 mmol)
and 1-(biphenyl-4-sulfonyl)-piperazine (0.302 g, 1.0 mmol) in
1-butanol (5 mL) was stirred under reflux for 8 h and
concentrated. The solid residue was stirred with methanol
under reflux for 30 min, filtered, and washed with methanol
Meth od B (14, 20, 23, 32). 2-[4-(5-Ch lor oth iop h en e-2-
su lfon yl)-p ip er a zin -1-yl]-6,7-d im et h oxy-q u in a zolin -4-
yla m in e (14). A solution of piperazine (0.517 g, 6.0 mmol) and
5-chloro-thiophene-2-sulfonyl chloride (0.436 g, 2.0 mmol) in
methanol (10 mL) was stirred at room temperature for 1 h.
The solvent was evaporated, and the residue was purified with
silica gel chromatography to obtain 1-(5-chloro-thiophene-2-
sulfonyl)-piperazine. The intermediate (0.266 g, 1.0 mmol) and
4-amino-2-chloro-6,7-dimethoxy-quinazoline (0.251 g, 1.0 mmol)
in 1-butanol (5 mL) were stirred under reflux overnight and
cooled to 80 °C. The collected solid product was washed with
ethyl acetate (2 × 10 mL), stirred in methanol (30 mL) under
reflux for 1 h, filtered, and washed with methanol (2 × 10 mL)
1
to yield compound 44. H NMR (DMSO-d6) δ 3.36 (br s, 4 H),
4.01 (br s, 4 H), 4.60-4.64 (m, 4 H), 5.26-5.35 (m, 4 H), 6.02-
6.13 (m, 2 H), 7.30-7.52 (m, 4 H), 7.72-7.75 (m, 3 H), 7.83-
7.86 (m, 4 H), 8.63 (s, 1 H), 8.83 (s, 1 H). HRMS (M + H)+
calcd for C30H32N5O4S 558.2169; found 558.2169. Anal.
(C30H31N5O4S‚HCl) C, H, N.
1
to yield compound 14. H NMR (DMSO-d6) δ 3.06-3.08 (m, 4
H), 3.80 (s, 3 H), 3.84 (s, 3 H), 3.94 (s, 4 H),7.36 (s, 1 H), 7.37
(s, 1 H), 7.59 (s,1 H), 7.60 (s, 1 H). HRMS (M + H)+ calcd for
4-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-ca r bon yl]-ben zon itr ile (2). Compound 2 was synthesized
from the procedure described for compound 1. 1H NMR
(DMSO-d6) δ 3.46-3.47 (m, 4 H), 3.84 (s, 3 H), 3.88 (s, 3 H),
4.03-4.17 (m, 4 H), 7.51 (s, 1 H), 7.66 (d, J ) 8.0 Hz, 2 H),
7.97 (d, J ) 8.0 Hz, 2 H). HRMS (M + H)+ calcd for C22H23N6O3
419.1826; found 419.1812. Anal. (C22H22N6O3‚HCl) C, H, N.
3-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-ca r bon yl]-ben zon itr ile (4). Compound 4 was synthesized
from the procedure described for compound 1. 1H NMR
(DMSO-d6) δ 3.44-3.46 (m, 4 H), 3.77 (s, 3 H), 3.82 (s, 3 H),
3.77-3.99 (m, 4 H), 6.97 (s, 1 H), 7.45 (s, 1 H), 7.60-7.64 (m,
1 H), 7.70-7.72 (m, 1 H), 7.82-7.93 (m, 2 H). HRMS (M +
H)+ calcd for C22H23N6O3 419.1826; found 419.1823.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-n itr op h en yl)-m eth a n on e (5). Compound 5 was
synthesized from the procedure described for compound 1. 1H
NMR (DMSO-d6) δ 3.21-3.35 (m, 4 H), 3.85(s, 3 H), 3.89 (s, 3
H), 3.93-3.96 (m, 4 H), 7.41 (s, 1 H), 7.73-7.76 (m, 3 H), 7.93-
8.39 (m, 2 H), 8.83 (br s, 1 H), 8.90 (br s, 1 H). HRMS (M +
H)+ calcd for C21H23N6O5 439.1724; found 439.1718. Anal.
(C21H22N6O5‚HCl) C, H, N.
C18H21ClN5O4S2 470.0718; found 470.0740. Anal. (C18H20
-
ClN5O4S2‚HCl) C, H, N.
Met h od C. 6,7-Dim et h oxy-2-[4-(4-p h en a n t h r en -9-yl-
b en zen esu lfon yl)-p ip er a zin -1-yl]-q u in a zolin -4-yla m in e
(35). To a solution of cbz-protected N-piperazine (2.24 g, 10.0
mmol) and 4-bromobenzenesulfonyl chloride (2.55 g, 10.0
mmol) in methanol (20 mL) triethylamine (1.38 g, 10.0 mmol)
was added to the solution. The mixture was stirred at room
temperature for 2 h, concentrated, and purified by silica gel
chromatography to afford 4-(4-bromo-benzenesulfonyl)-pipera-
zine-1-carboxylic acid benzyl ester (v). Under argon, compound
v (0.439 g, 1.0 mmol), K2CO3 (0.345 g, 2.5 mmol), Bu4NBr
(0.322 g, 1.0 mmol), and Pd(OAc)2 (11 mg, 5 mol %) were added
to a stirred solution of 4-phenanthrenylboronic acid (0.243 g,
1.1 mmol) in H2O (5 mL). The reaction mixture was vigorously
stirred at 70 °C for 1 h and cooled to room temperature, and
ethyl acetate was added (10 mL). The organic layer was dried
and concentrated to obtain compound vi. To a solution of
compound vi (0.389 g, 0.5 mmol) in methanol (5 mL) 10%
palladium on charcoal (5 mg, 10% w/w) was added. The
mixture was treated with hydrogen under atmospheric pres-
sure for 6 h and filtered. The solvent was evaporated to yield
product vii. Following the procedure for the synthesis of
compound 14, compound 35 was synthesized.1H NMR (DMSO-
d6) δ 3.20 (s, 4 H), 3.81 (s, 3 H), 3.86 (s, 3 H), 3.99 (s, 4 H),
7.28 (s, 1 H), 7.60-7.78 (m, 7 H), 7.81 (d, J ) 8.4 Hz, 2 H),
8.02 (d, J ) 8.1 Hz), 8.4 (s, 1 H), 8.87 (d, J ) 8.1 Hz, 1 H),
8.94 (d, J ) 8.4 Hz, 1 H). HRMS (M + H)+ calcd for
C34H32N5O4S 606.2169; found 606.2164. Anal. (C34H31N5O4S‚
HCl) C, H, N.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(3,4-d im eth oxyp h en yl)-m eth a n on e (6). Compound 6
was synthesized from the procedure described for compound
1
1. H NMR (DMSO-d6) δ 3.68 (s, 4 H), 3.79(s, 3 H), 3.81 (s, 3
H), 3.84(s, 3 H), 3.88 (s, 3 H), 4.26 (s, 4 H), 7.41 (s, 1 H), 7.00-
7.05 (m, 3 H), 7.74 (s, 1 H), 8.54 (br s, 1 H), 8.90 (br s, 1 H).
HRMS (M + H)+ calcd for C23H28N5O5 454.2085; found
454.2071.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-n a p h th a len -1-yl-m eth a n on e (7). Compound 7 was
synthesized from the procedure described for compound 1. 1H
NMR (DMSO-d6) δ 3.18-3.30 (m, 4 H), 3.67 (s, 3 H), 3.78 (s,
3 H), 3.97 (br s, 2 H), 4.08 (br s, 2 H), 7.48 (s, 1 H), 7.53-7.59
(m, 1 H), 7.60-7.61 (m, 3 H), 7.65 (s, 1 H), 7.96 (s, 1 H). HRMS
(M + H)+ calcd for C25H26N5O3 444.2030; found 444.2030.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-a m in op h en yl)-m eth a n on e (8). Compound 8 was
synthesized from the procedure described for compound 1. 1H
NMR (DMSO-d6) δ 3.71(s, 4 H), 3.86(s, 3 H), 3.87 (s, 3 H),
3.92-3.97(m, 4 H), 6.65-6.68 (m, 2 H), 7.13 (d, J ) 3.2 Hz, 1
Gen er a l P r oced u r e for th e Syn th esis of Su lfon a m id es
(Sch em e 3). N-[2-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-
yla m in o)-eth yl]-4-ter t-bu tylben zen e Su lfon a m id e (41). A
mixture of ethylenediamine (0.36 g, 6.0 mmol) and tert-
butylbenzenesulfonyl chloride (0.464 g, 2.0 mmol) in methanol
(15 mL) was stirred for 3 h, concentrated, and purified by silica
gel chromatography to yield N-(2-amino-ethyl)-4-tert-butyl-
benzenesulfonamide. Following the procedure for the synthesis
of compound 14, compound 42 was obtained. 1H NMR (DMSO-
d6) δ 1.17 (s, 9 H), 3.11 (s, 2 H), 3.48 (s, 2 H), 3.86 (s, 3 H),
3.93 (s, 3 H), 6.90 (s, 1 H), 7.50 (d, J ) 8.4 Hz, 2 H), 7.55 (s, 1
H), 7.70 (d, J ) 8.4 Hz, 2 H). HRMS (M + H)+ calcd for

4460 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Shaw et al.
1
H), 7.25 (d, J ) 3.4 Hz, 1 H), 7.27 (d, J ) 3.3 Hz, 1 H), 7.62 (d,
J ) 3.2 Hz, 1 H). HRMS (M + H)+ calcd for C21H25N6O3
409.1983; found 409.1984.
11. H NMR (DMSO-d6) δ 3.08 (s, 4 H), 3.79 (s, 3 H), 3.84 (s,
3 H), 3.90 (s, 4 H), 7.56 (s, 1 H), 7.80-8.03 (m, 5 H). HRMS
(M + H)+ calcd for C21H23F3N5O3S 498.1417; found 498.1420.
Anal. (C21H22F3N5O3S‚HCl) C, H, N.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-ter t-bu tylp h en yl)-m eth a n on e (9). Compound 9 was
synthesized from the procedure described for compound 1. 1H
NMR (DMSO-d6) δ 1.32 (s, 9 H), 3.66-3.74 (m, 4 H), 3.85 (s,
3 H), 3.88 (s, 3 H), 3.93 (s, 4 H), 7.31-7.51 (m, 5 H), 7.73 (s, 1
H), 8.51 (s, 1 H), 8.97 (s, 1 H). HRMS (M + H)+ calcd for
C25H32N5O3 450.2500; found 450.2485. Anal. (C25H31N5O3‚HCl)
C, H, N.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-t r iflu or om et h ylp h en yl)-m et h a n on e (10). Com-
pound 10 was synthesized from the procedure described for
compound 1. 1H NMR (DMSO-d6) δ 3.50 (s, 4 H), 3.85 (s, 3 H),
3.88 (s, 3 H), 4.0 (s, 4 H), 7.56 (s, 1 H), 7.70 (d, J ) 7.7 Hz, 1
H), 7.87 (d, J ) 7.7 Hz, 2 H), 8.59 (s, 1 H), 8.94 (s, 1 H). HRMS
(M + H)+ calcd for C22H23 F3N5O3 462.1748; found 462.1708.
2-[4-(4-Br om ob e n ze n e su lfon yl)-p ip e r a zin -1-yl]-6,7-
d im eth oxyqu in a zolin -4-yl-a m in e (12). Compound 12 was
synthesized from the procedure described for compound 11.
1H NMR (DMSO-d6) δ 3.13-3.14 (m, 4 H), 3.48-3.49 (m, 4
H), 3.83 (s, 3 H), 3.90 (s, 3 H), 6.99 (s, 1 H), 7.49 (s, 1 H), 7.74
(d, J ) 8.4 Hz, 2 H), 7.83 (d, J ) 8.4 Hz, 2 H). HRMS (M +
H)+ calcd for C20H23BrN5O4S 508.0649; found 508.0646. Anal.
(C20H22BrN5O4S‚HCl) C, H, N.
6,7-Dim et h oxy-2-[4-(4-m et h oxyb en zen esu lfon yl)-p ip -
er a zin -1-yl]-qu in a zolin -4-yl-a m in e (20). Compound 20 was
synthesized from the procedure described for compound 14.
1H NMR (DMSO-d6) δ 2.95-2.97 (m, 4 H), 3.73 (s, 3 H), 3.75
(s, 3 H), 3.82 (s, 3 H), 3.73-3.82 (m, 4 H), 6.98 (s, 1 H), 7.06
(d, J ) 21.1 Hz, 2 H), 7.57 (d, J ) 19.1 Hz, 2 H). HRMS (M +
H)+ calcd for C21H26N5O5S 460.1649; found 460.1652. Anal.
(C21H25N5O5S‚HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(4-tr iflu or om eth oxyben zen esu lfo-
n yl)-p ip er a zin -1-yl]-q u in a zolin -4-yl-a m in e (21). Com-
pound 21 was synthesized from the procedure described for
1
compound 11. H NMR (DMSO-d6) δ 3.40 (s, 4 H), 3.66 (s, 3
H), 3.69 (s, 3 H), 3.71 (s, 4 H), 6.68 (s, 1 H), 7.00 (br s, 2 H),
7.23 (s, 1 H), 7.54-755 (m, 2 H), 7.86-7.88 (m, 2 H). HRMS
(M + H)+ calcd for C21H23F3N5O5S 514.1367; found 514.1363.
Anal. (C21H22F3N5O5S‚HCl) C, H, N.
2-[4-(4-Meth a n esu lfon yl-ben zen esu lfon yl)-p ip er a zin -
1-yl]-6,7-d im eth oxy-qu in a zolin -4-yla m in e (22). Compound
22 was synthesized from the procedure described for compound
1
11. H NMR (DMSO-d6) δ 3.00 (s, 4 H), 3.21 (s, 3 H), 3.76 (s,
3 H), 3.80 (s, 3 H), 3.82 (s, 4 H), 6.69 (s, 1 H), 7.07 (br s, 2 H),
7.26 (s, 1 H), 8.01 (d, J ) 8.3 Hz, 2 H), 8.16 (d, J ) 8.4 Hz, 2
H). HRMS (M + H)+ calcd for C21H26N5O6S2 508.1319; found
508.1317. Anal. (C21H25N5O6S2‚HCl) C, H, N.
2-[4-(4-I o d o b e n ze n e s u lfo n y l)-p ip e r a zin -1-y l]-6,7-
d im eth oxyqu in a zolin -4-yl-a m in e (13). Compound 13 was
synthesized from the procedure described for compound 11.
1H NMR (DMSO-d6) δ 2.95 (s, 4 H), 3.77 (s, 3 H), 3.78-3.82
(m, 4 H), 3.82 (s, 3 H), 7.26-39 (m, 2 H), 7.38 (d, J ) 8.3 Hz,
2 H), 8.01 (d, J ) 8.5 Hz, 2 H). HRMS (M + H)+ calcd for
2-[4-(4-ter t-Bu tylben zen esu lfon yl)-p ip er a zin -1-yl]-6,7-
d im eth oxyqu in a zolin -4-yla m in e (23). Compound 23 was
synthesized from the procedure described for compound 14.
1H NMR (DMSO-d6) δ 1.28 (s, 9 H), 2.90 (s, 4 H), 3.76 (s, 3 H),
3.80 (s, 3 H), 3.90-3.98 (m, 4 H), 6.70 (s, 3 H), 7.17 (br s, 1 H),
7.26 (s, 1 H), 7.45-7.68 (m, 3 H). HRMS (M + H)+ calcd for
C
20H23IN5O4S 556.0510; found 556.0496. Anal. (C20H22IN5O4S‚
HCl) C, H, N.
C
24H32N5O4S 486.2170; found 486.2173. Anal. (C24H31N5O4S‚
HCl) C, H, N.
2-[4-(5-Ch lor ot h iop h en e-2-su lfon yl)-p ip er a zin -1-yl]-
6,7-d im eth oxyqu in a zolin -4-yla m in e (14). Compound 14
was synthesized from the procedure described for compound
11. 1H NMR (DMSO-d6) δ 3.06-3.08 (m, 4 H), 3.80 (s, 3 H),
3.84 (s, 3 H), 3.94 (s, 4 H),7.36 (s, 1 H), 7.37 (s, 1 H), 7.59 (s,1
H), 7.60 (s, 1 H). HRMS (M + H)+ calcd for C18H21ClN5O4S2
470.0718; found 470.0740.
3-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-su lfon yl]-ben zoic a cid (24). Compound 24 was synthesized
from the procedure described for compound 11. 1H NMR
(DMSO-d6) δ 2.99 (s, 4 H), 3.77 (s, 3 H), 3.82 (s, 7 H), 6.78 (s,
1 H), 7.38 (br s, 1 H), 7.44 (s, 1 H), 7.78 (t, J ) 7.6 Hz, 1 H),
8.00 (d, J ) 7.4 Hz, 1 H), 8.21-8.23 (m, 2 H). HRMS (M +
H)+ calcd for C21H24N5O6S 474.1442; found 474.1426.
[4-(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)-p ip er a zin -
1-yl]-(4-t r iflu or om et h ylp h en yl)-m et h a n on e (25). Com-
pound 25 was synthesized from the procedure described for
compound 11.1H NMR (DMSO-d6) δ 3.34 (br s, 4 H), 3.77 (s, 3
H), 3.82 (s, 3 H), 3.95 (s, 4 H), 6.83 (s, 1 H), 7.41-7.51 (br s,
2 H), 7.66-7.71(m, 2 H), 7.85-7.94 (m, 2 H), 10.20 (br s, 1 H).
HRMS (M + H)+ calcd for C21H24N5O6S 474.1442; found
474.1479.
2-[4-(2,5-Dich lor oben zen esu lfon yl)-p ip er a zin -1-yl]-6,7-
d im eth oxyqu in a zolin -4-yla m in e (26). Compound 26 was
synthesized from the procedure described for compound 11.
1H NMR (DMSO-d6) δ 3.33 (br s, 4 H), 3.64 (s, 3 H), 3.78-
3.82 (m, 4 H), 3.82 (s, 3 H), 6.74 (s, 1 H), 7.21 (br s, 2 H), 7.43-
(s, 1 H), 7.69-7.96 (m, 2 H), 7.97 (s, 1 H). HRMS (M + H)+
calcd for C20H22Cl2N5O4S 498.0764; found 498.0768.
6,7-Dim eth oxy-2-[4-(2-n itr oben zen esu lfon yl)-piper azin -
1-yl]-qu in a zolin -4-yl-a m in e (15). Compound 15 was syn-
thesized from the procedure described for compound 11. 1H
NMR (DMSO-d6) δ 3.16 (s, 4 H), 3.34 (s, 3 H), 3.43 (s, 3 H),
3.74 (s, 4 H), 7.64 (s, 1 H), 7.95 (t, J ) 8.1 Hz, 1 H), 8.21 (d, J
) 7.8 Hz, 1 H), 8.41 (s, 1 H), 8.53 (s, 1 H), 8.56 (s, 1 H). HRMS
(M + H)+ calcd for C20H23N6O6S 475.1394; found 475.1394.
6,7-Dim eth oxy-2-[4-(3-n itr oben zen esu lfon yl)-piper azin -
1-yl]-qu in a zolin -4-yl-a m in e (16). Compound 16 was syn-
thesized from the procedure described for compound 11. 1H
NMR (DMSO-d6) δ 3.22 (s, 4 H), 3.68 (s, 3 H), 3.77 (s, 3 H),
3.82 (s, 4 H), 6.72 (s, 1 H), 7.19 (br s, 2 H), 7.42 (s, 1 H), 7.82-
7.88 (m, 2 H), 7.98-8.07 (m, 2 H). HRMS (M + H)+ calcd for
C
20H23N6O6S 475.1394; found 475.1392. Anal. (C20H22N6O6S‚
HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(4-n itr oben zen esu lfon yl)-piper azin -
1-yl]-qu in a zolin -4-yl-a m in e (17). Compound 17 was syn-
thesized from the procedure described for compound 11. 1H
NMR (DMSO-d6) δ 3.00 (s, 4 H), 3.66 (s, 3 H), 3.76 (s, 3 H),
3.83 (s, 4 H), 6.75 (s, 1 H), 7.16 (br s, 2 H), 7.38 (s, 1 H), 8.01
(d, J ) 8.6 Hz, 2 H), 8.20 (d, J ) 8.5, 2H). HRMS (M + H)+
calcd for C20H23N6O6S 475.1394; found 475.1379.
6,7-Dim et h oxy-2-[4-(t olu en e-4-su lfon yl)-p ip er a zin -1-
yl]-qu in a zolin -4-yla m in e (18). Compound 18 was synthe-
sized from the procedure described for compound 11. 1H NMR
(DMSO-d6) δ 29 (s, 3 H), 3.03-3.07 (m, 4 H), 3.82 (s, 3 H),
3.85(s, 3 H), 3.98-4.05 (m, 4 H), 7.45 (d, J ) 7.6 Hz, 2 H),
7.52 (s, 1 H), 7.65 (d, J ) 7.3 Hz, 2 H), 7.73 (s, 1 H), 8.55 (s, 1
H), 8.92 (s, 1 H). HRMS (M + H)+ calcd for C21H26N5O4S
444.1700; found 444.1706. Anal. (C21H25N5O4S‚HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(4-tr iflu or om eth ylben zen esu lfon yl)-
p ip er a zin -1-yl]-qu in a zolin -4-yl-a m in e (19). Compound 19
was synthesized from the procedure described for compound
4-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-su lfon yl]-ben zen e-1,3-d ia m in e (27). Compound 27 was
synthesized from the procedure described for compound 11
followed by hydrogenation to get the diamine product. 1H NMR
(DMSO-d6) δ 3.09 (s, 4 H), 3.34 (s, 3 H), 3.67 (s, 3 H), 3.86-
3.89 (m, 4 H), 6.28 (d, J ) 8.5 Hz, 1 H), 6.74 (s, 1 H), 7.31 (d,
J ) 9.0 Hz, 1 H), 7.66 (s, 1 H), 8.23 (s, 1 H), 8.64 (s, 1 H), 8.85
(s, 1 H), 8.99 (s, 1 H). HRMS (M + H)+ calcd for C20H26N7O6S
460.1761; found 460.1758. Anal. (C20H25N7O6S‚HCl) C, H, N.
5-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-su lfon yl]-2-ch lor o-4-flu or oben zoic Acid (28). Compound
28 was synthesized from the procedure described for compound
1
11. H NMR (DMSO-d6) δ 3.15 (s, 4 H), 3.70 (s, 3 H), 3.76 (s,
3 H), 3.80 (s, 4 H), 6.76 (s, 1 H), 7.37 (br s, 1 H), 7.42 (s, 1 H),
7.82 (s, 1 H), 8.12 (s, 1 H). HRMS (M + H)+ calcd for C21H22
-
ClFN5O6S 526.0958; found 526.0943.

Doxazosin-Derived Apoptosis-Inducing Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4461
5-[4-(4-Am in o-6,7-dim eth oxyqu in azolin -2-yl)-piper azin e-
1-su lfon yl]-2,4-d ich lor oben zoic Acid (29). Compound 29
was synthesized from the procedure described for compound
for C27H30N5O6S2 584.1632; found 584.1658. Anal. (C27H29-
N5O6S2‚HCl) C, H, N.
2-[4-(4′-Bu t ylbip h en yl-4-su lfon yl)-p ip er a zin -1-yl]-6,7-
d im eth oxyqu in a zolin -4-yl-a m in e (39). Compound 39 was
synthesized from the procedure described for compound 35.
1H NMR (DMSO-d6) δ 0.89 (t, J ) 7.3 Hz, 3 H), 1.29-1.34 (m,
2 H), 1.55-1.59 (m, 2 H), 2.60-2.64 (m, 2 H), 3.13 (s, 4 H),
3.81 (s, 3 H), 3.86 (s, 3 H), 3.97 (s, 4 H), 7.31-7.33 (m, 3 H),
7.63-7.69 (m, 3 H), 7.82 (d, J ) 8.3 Hz, 2 H), 7.91 (d, J ) 8.4
Hz, 2 H), 8.66 (br s, 1 H), 8.79 (br s, 1 H). HRMS (M + H)+
calcd for C30H36N5O4S 562.2483; found 562.2458. Anal.
(C30H35N5O4S‚HCl) C, H, N.
1
11. H NMR (DMSO-d6) δ 3.37 (br s, 4 H), 3.81 (s, 3 H), 3.85
(s, 3 H), 3.95 (s, 4 H), 7.47 (s, 1 H), 7.71 (s, 2 H), 7.43(s, 1 H),
8.03 (s, 1 H), 8.33 (s, 1 H), 8.62 (br s, 1 H), 8.86 (br s, 1 H).
HRMS (M + H)+ calcd for C21H22Cl2N5O6S 542.0662; found
542.0657.
6,7-Dim eth oxy-2-[4-(n aph th alen e-1-su lfon yl)-piper azin -
1-yl]-qu in a zolin -4-yl-a m in e (30). Compound 30 was syn-
thesized from the procedure described for compound 11. 1H
NMR (DMSO-d6) δ 3.08 (s, 4 H), 3.81 (s, 3 H), 3.83 (s, 3 H),
3.95 (s, 4 H), 7.66 (s, 1 H), 7.68-7.78 (m, 4 H), 8.11 (d, J ) 8.0
Hz, 1 H), 8.18 (d, J ) 7.4 Hz, 1 H), 8.31 (d, J ) 8.2 Hz, 1 H),
8.71 (d, J ) 8.6 Hz, 1 H), 10.29 (br s, 2 H). HRMS (M + H)+
calcd for C24H26N5O4S 480.1700; found 480.1696. Anal.
(C24H25N5O4S‚HCl) C, H, N.
2-[4-(4′-ter t-Bu tylbip h en yl-4-su lfon yl)-p ip er a zin -1-yl]-
6,7-d im et h oxyq u in a zolin -4-yl-a m in e (40). Compound 40
was synthesized from the procedure described for compound
1
35. H NMR (DMSO-d6) δ 1.31 (s, 9 H), 3.14 (s, 4 H), 3.81 (s,
3 H), 3.86 (s, 3 H), 3.95 (s, 4 H), 7.19 (s, 1 H), 7.45-7.53 (m, 2
H), 7.60-7.66 (m, 3 H), 7.82-7.84 (m, 2 H), 7.92 (d, J ) 8.3
Hz, 2 H), 8.66 (br s, 1 H), 8.81 (br s, 1 H). HRMS (M + H)+
calcd for C30H36N5O4S 562.2483; found 562.2471. Anal.
(C30H35N5O4S‚HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(n aph th alen e-2-su lfon yl)-piper azin -
1-yl]-qu in a zolin -4-yl-a m in e (31). Compound 31 was syn-
thesized from the procedure described for compound 11. 1H
NMR (DMSO-d6) δ 3.00-3.08 (m, 4 H), 3.39 (s, 3 H), 3.43 (s,
3 H), 3.74-3.81 (m, 4 H), 6.67 (s, 1 H), 7.12 (br s, 2 H), 7.36 (s,
1 H), 7.66-7.73 (m, 2 H), 7.77 (d, J ) 8.7 Hz, 1 H), 8.05 (d, J
) 7.7 Hz, 1 H), 8.14 (d, J ) 8.7 Hz, 1 H), 8.20 (d, J ) 7.7 Hz,
1 H), 8.45 (s, 1 H). HRMS (M + H)+ calcd for C24H26N5O4S
480.1700; found 480.1708. Anal. (C24H25N5O4S‚HCl) C, H, N.
2-[4-(5-Dim eth yla m in on a p h th a len e-1-su lfon yl)-p ip er -
a zin -1-yl]-6,7-d im eth oxyqu in a zolin -4-yla m in e (32). Com-
pound 32 was synthesized from the procedure described for
N-[2-(4-Am in o-6,7-d im et h oxyq u in a zolin -2-yla m in o)-
eth yl]-4-bip h en ylsu lfon a m id e (42). Compound 42 was syn-
thesized from the procedure described for compound 41. 1H
NMR (DMSO-d6) δ 3.05 (s, 2 H), 3.46 (s, 2 H), 3.80 (s, 3 H),
3.83 (s, 3 H), 6.91(br s, NH), 7.40-7.47 (m, 3 H), 7.60-7.64
(m, 3 H), 7.78-7.83 (m, 2 H), 7.86-7.88 (m, 2 H), 8.01 (s, 1
H). HRMS (M + H)+ calcd for C24H26N5O4S 480.1700; found
480.1687. Anal. (C24H25N5O4S‚HCl) C, H, N.
2-[4-(Bip h en yl-4-su lfon yl)-p ip er a zin -1-yl]-6,7-d ip r o-
p oxyqu in a zolin -4-yl-a m in e (44). Compound 44 was syn-
thesized from the procedure described for compound 43. 1H
NMR (DMSO-d6) δ 0.99 (t, J ) 7.3 Hz, 6 H), 1.71-1.83 (m, 4
H), 3.12 (br s, 4 H), 3.93-4.14 (m, 8 H), 7.33 (s, 1 H), 7.44-
7.54 (m, 3 H), 7.65 (s, 1 H), 7.75-7.79 (m, 2 H), 7.86 (d, J )
8.3 Hz, 2 H), 7.94 (d, J ) 8.3 Hz, 2 H). HRMS (M + H)+ calcd
for C30H36N5O4S 562.2483; found 562.2466. Anal. (C30H35N5O4S‚
HCl) C, H, N.
2-[4-(Bip h en yl-4-su lfon yl)-p ip er a zin -1-yl]-6,7-d iisop r o-
p oxyqu in a zolin -4-yl-a m in e (45). Compound 45 was syn-
thesized from the procedure described for compound 43. 1H
NMR (DMSO-d6) δ 1.25 (s, 3 H), 1.27 (s, 3 H), 1.33 (s, 3 H),
1.35 (s, 3 H), 3.12 (br s, 4 H), 3.96 (br s, 4 H), 4.41-4.66 (m, 2
H), 7.36 (s, 1 H), 7.41-7.53 (m, 3 H), 7.68-7.84 (m, 3 H), 7.83-
7.86 (m, 4 H), 8.59 (br s, NH), 8.76 (br s, NH). HRMS (M +
H)+ calcd for C30H36N5O4S 562.2483; found 562.2478. Anal.
(C30H35N5O4S‚HCl) C, H, N.
1
compound 14. H NMR (DMSO-d6) δ 2.82 (s, 6 H), 3.27-3.29
(m, 4 H), 3.44 (s, 3 H), 3.81 (s, 3 H), 3.85-3.91 (m, 4 H), 7.27
(d, J ) 7.6 Hz, 1 H), 7.61 (s, 1 H), 7.61-7.70 (m, 3 H), 8.17 (d,
J ) 7.4 Hz, 1 H), 8.35 (d, J ) 8.64 Hz, 1 H), 8.53 (d, J ) 8.5
Hz, 1 H), 8.63 (br s, 1 H), 8.85 (br s, 1 H). HRMS (M + H)+
calcd for C26H31N6O4S 523.2122; found 523.2153.
2-[4-(Biph en yl-4-su lfon yl)-piper azin -1-yl]-6,7-dim eth oxy-
qu in a zolin -4-yla m in e (33). Compound 33 was synthesized
from the procedure described for compound 11. 1H NMR
(DMSO-d6) δ 3.02-3.03 (m, 4 H), 3.81 (s, 3 H), 3.86 (s, 3 H),
3.98-3.46 (m, 4 H), 7.38-7.52 (m, 4 H), 7.68-7.74 (m, 3 H),
7.85 (d, J ) 8.2 Hz, 2 H), 7.94 (d, J ) 8.2 Hz, 2 H). HRMS (M
+ H)+ calcd for C26H28N5O4S 506.1857; found 506.1840. Anal.
(C26H27N5O4S‚HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(2,4,6-tr iisopr opylben zen esu lfon yl)-
p ip er a zin -1-yl]-qu in a zolin -4-yl-a m in e (34). Compound 34
was synthesized from the procedure described for compound
11. 1H NMR (DMSO-d6) δ 1.22 (s, 9 H), 1.24 (s, 9 H), 2.92-
2.98 (m, 1 H), 3.23 (s, 4 H), 3.84 (s, 3 H), 3.87 (s, 3 H), 3.94 (s,
4 H), 4.01-4.15 (m, 2 H), 7.32 (s, 2 H), 7.44 (s, 1 H), 7.76 (s, 1
H), 8.69 (br s, 1 H), 8.96 (br s, 1 H). HRMS (M + H)+ calcd for
2-[4-(Biph en yl-4-su lfon yl)-piper azin -1-yl]-6,7-dibu toxy-
qu in a zolin -4-yla m in e (46). Compound 46 was synthesized
from the procedure described for compound 43. 1H NMR
(DMSO-d6) δ 0.934 (t, J ) 7.5 Hz, 6 H), 1.44 (q, J ) 7.5 Hz, 4
H), 1.66-1.812 (m, 4 H), 3.13 (br s, 4 H), 3.91 (br s, 4 H), 3.91-
4.08 (m, 4 H), 7.21 (s, 1 H), 7.41-7.53 (m, 3 H), 7.64 (s, 1 H),
7.73 (d, J ) 8.0 Hz, 1 H), 7.74 (s, 1 H), 7.829-7.864 (m, 3 H),
7.94 (d, J ) 8.0 Hz, 2 H). HRMS (M + H)+ calcd for
C
29H42N5O4S 556.2952; found 556.2944.
6,7-Dim eth oxy-2-[4-(4′-m eth ylbip h en yl-4-su lfon yl)-p ip -
er a zin -1-yl]-qu in a zolin -4-yl-a m in e (36). Compound 36 was
synthesized from the procedure described for compound 35.
1H NMR (CD3OD-d4) δ 2.28 (s, 3 H), 3.12 (s, 4 H), 3.78 (s, 3
H), 3.84 (s, 3 H), 3.86 (s, 4 H), 6.88-6.90 (m, 3 H), 7.18 (d, J
) 8.1 Hz, 1 H), 7.36 (s, 1 H), 7.38-7.40 (m, 2 H), 7.44 (d, J )
8.1 Hz, 1 H), 7.46-7.78 (m, 2 H). HRMS (M + H)+ calcd for
C
32H40N5O4S 590.2795; found 590.2770. Anal. (C32H39N5O4S‚
HCl) C, H, N.
Cell Cu ltu r e. PC-3 (p53-/-) human androgen-nonrespon-
sive prostate cancer cells were purchased from the American
Type Tissue Collection (Manassas, VA). Cells were cultured
in an RPMI 1640 medium (Gibco, Grand Island, NY) supple-
mented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in
a humidified incubator containing 5% CO2.
C
27H30N5O4S 520.2013; found 520.2040. Anal. (C27H29N5O4S‚
HCl) C, H, N.
6,7-Dim eth oxy-2-[4-(4′-tr iflu or om eth ylbip h en yl-4-su l-
fon yl)-p ip er a zin -1-yl]-qu in a zolin -4-yl-a m in e (37). Com-
pound 37 was synthesized from the procedure described for
1
compound 35. H NMR (DMSO-d6) δ 3.05 (s, 4 H), 3.78 (s, 3
Cell Via bility Assa y. Effects of the test agent on cell
viability were assessed by the MTT ([3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide]) assay in 96-well,
flat-bottomed plates in which 8000 PC-3 or DU-145 cells/well
were seeded. Cells were exposed to the test agent at the
indicated concentrations, in six replicates, in 10% FBS-
supplemented RPMI-1640 medium at 37 °C in 5% CO2 for 48
h. The medium was removed and was replaced by 150 µL of
0.5 mg/mL MTT in RPMI 1640 medium, and the cells were
incubated in the CO2 incubator at 37 °C for 2 h. Supernatants
were removed from the wells, and the reduced MTT dye was
H), 3.82 (s, 2 H), 3.91 (s, 3 H),7.53 (s, 1 H), 7.71-7.89 (m, 4
H), 7.94-8.07 (m, 4 H), 10.16 (s, 1 H). HRMS (M + H)+ calcd
for C27H26 F3N5O4S 574.1730; found 574.1728. Anal. (C27H25
F3N5O4S‚HCl) C, H, N.
2-[4-(4′-Meth an esu lfon yl-biph en yl-4-su lfon yl)-piper azin -
1-yl]-6,7-d im eth oxy-qu in a zolin -4-yla m in e (38). Compound
38 was synthesized from the procedure described for compound
1
35. H NMR (DMSO-d6) δ 2.91 (s, 4 H), 3.26 (s, 3 H), 3.78 (s,
2 H), 3.83 (s, 3 H), 3.91 (s, 4 H), 6.97 (s, 1 H), 7.55 (s, 1 H),
7.88 (d, J ) 8.2 Hz, 2 H), 8.08 (m, 6 H). HRMS (M + H)+ calcd

4462 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
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solubilized with 200 µL/well DMSO. The absorbance was
determined on a plate reader at 570 nm.
Wester n Blot An a lysis. PC-3 cells (1.5 × 106) treated with
the test agent at the indicated concentrations in the RPMI
1640 medium for 24 h were collected and sonicated. Protein
concentrations of the lysates were determined by using a
Bradford protein assay kit (Bio-Rad, Hercules, CA); equivalent
amounts of proteins from each lysate were resolved in 10%
SDS-polyacrylamide gel and then transferred onto Immobilon-
nitrocellulose membranes (Millipore, Bellerica, MA) in
a
semidry transfer cell. The transblotted membrane was washed
twice with tris-buffered saline (TBS) containing 0.1% Tween
20 (TBST). After blocking with TBST containing 5% nonfat
milk for 40 min, the membrane was incubated with the
primary antibody (1:1000 dilution) in TBST-1% nonfat milk
at 4 °C overnight. After treatment with the primary antibody,
the membrane was washed three times with TBST for a total
of 15 min, followed by goat antirabbit or antimouse IgG-
horseradish peroxidase conjugates (diluted 1:3000) for 1 h at
room temperature and was washed three times with TBST
for a total of 1 h. The immunoblots were visualized by
enhanced chemiluminescence.
Tr a n sien t Tr a n sfection . The constitutively active Akt
construct HA-PKB-T308D/S473D was kindly provided by Dr.
Brain Hemmings (Friedrich Miescher Institute, Basel, Swit-
zerland). PC-3 cells were seeded into T-75 flasks (1.5 × 106/
flask). Aliquots containing 3 µg of each plasmid or a control
pcDNA3.1(+) vector in 750 µL of Opti-MEM medium (Invit-
rogen-Life Technologies, Inc.,) were incubated with 9 µL of
FuGene 6 reagent (Roche Diagnostics Corp., Indianapolis, IN)
for 15 min. Each flask was washed with Opti-MEM medium
and then received the plasmid-FuGene 6 mixture and 4 mL
of Opti-MEM medium. The flask was placed in a CO2 incubator
for 4 h, and the transfection medium was replaced with 10%
FBS-supplemented RPMI 1640. After 24 h, Mock- and Akt-
transfected PC-3 cells were seeded into 96-well plates at 5000
cells/well in 10% FBS-supplemented RPMI 1640. On the next
day, cells were treated in four replicates with the indicated
concentrations of OSU-03012 in 1% FBS-containing medium
for 24 h. An MTT assay was used to determine the cell
viability.
Ack n ow led gm en t . This investigation was sup-
ported by National Institutes of Health grant CA94829
and Army Grant DAMD17-02-1-0117.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data for compounds 1-46. This material is available free of
charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Kirby, R. S.; Pool, J . L. Alpha Adrenoceptor Blockade in the
Treatment of Benign Prostatic Hyperplasia: Past, Present and
Future. Br. J . Urol. 1997, 80, 521-532.
J M049752K