Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent

Article abstract of DOI:10.1016/j.bmc.2008.02.031

The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI₅₀ values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI₅₀ value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI₅₀ = 39.8 μM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI₅₀ = 850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.

Full text of DOI:10.1016/j.bmc.2008.02.031

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3519–3529
Potent anti-prostate cancer agents derived from a novel
androgen receptor down-regulating agent
Puranik Purushottamachar,a Aakanksha Khandelwal,a Tadas S. Vasaitis,a
Robert D. Bruno,a Lalji K. Gediyaa and Vincent C. O. Njara,b,
*
aDepartment of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine,
685 West Baltimore Street, Baltimore, MD 21201-1559, USA
bUniversity of Maryland Marlene and Stewart Greenebaum Cancer Center, School of Medicine, Baltimore, MD 21201-1559, USA
Received 16 October 2007; revised 5 February 2008; accepted 8 February 2008
Available online 14 February 2008
Abstract—The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to
the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not
correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimiza-
tion of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer
agents. Some compounds (1–6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI50 values in the nanomolar
range (1.45–83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI50 value of 1.45 nM is
27,000 times more potent than our lead compound BTB01434 (GI50 = 39.8 lM). In addition, some of the compounds exhibited modest
anti-androgenic activities and one was also a potent inhibitor (GI50 = 850 nM) of PC-3 (AR-null) cell growth. A clear structure–activity
relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted
aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treat-
ment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
mastia, loss of libido, and erectile dysfunction.3–5 The
nonsteroidal antiandrogens show fewer side effects and
have improved oral bioavailability; therefore, they are
favored over the steroidal antiandrogens. However, anti-
androgen withdrawal syndrome has been discovered in
patients receiving nonsteroidal antiandrogens for several
months.6,7 Long-term drug usage probably leads to
mutation of the AR, and the nonsteroidal antiandrogens
now exhibit agonistic activity to the mutant AR.8 In
addition, the clinically available antiandrogens are un-
able to kill prostate cancer cells, and within one to three
years of drug administration, the cancer usually pro-
gresses into an androgen-refractory phenotype which is
not curable. The poor clinical outcome of advanced met-
astatic PC highlights the urgent need to develop effective
novel agents for prevention and treatment of this disease.
Prostate cancer (PC) is the most common cancer among
males of Western countries1 and is a complex heteroge-
neous disease that acts differently in different men. The
underlying cause of prostate cancer is still unknown.
However, androgen and the androgen receptor (AR)
are postulated to play crucial roles in the development
of prostate cancer.2 The current treatment for prostate
cancer is a combination of surgery, radiation, and che-
motherapy. The therapeutic agents used clinically in-
clude steroidal antiandrogens, such as cyproterone
acetate, and nonsteroidal antiandrogens, such as flutam-
ide and bicalutamide. The steroidal antiandrogens pos-
sess partial agonistic activity and overlapping effects
with other hormonal systems, leading to many complica-
tions including severe cardiovascular problems, gyneco-
The addition of novel and potent drug candidates to the
existing therapy would certainly improve the outcome of
PC therapy. Androgen receptor down-regulating agents
(ARDAs) have emerged as an attractive target for the
development anti-prostate cancer drugs.9–13 Until re-
cently most of the agents known as ARDAs were natu-
Keywords: Anti-prostate cancer agents; Sulfonamides; Antiprolifera-
tive; Structure–activity relationship (SAR).
*
Corresponding author. Tel.: +1 410 706 5885; fax: +1 410 706
0032; e-mail: vnjar001@umaryland.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.031

3520
P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
ral chemicals.14–18 Recently, we discovered six synthetic
small molecules, including BTB01434 (see Fig. 1a and b)
as ARDAs (EC50 values 17.5–212 lM range) through
the development of a qualitative 3D pharmacophore
modeling strategy.19 These compounds were also shown
to be modest inhibitors (GI50 values 4.5–39.8 lM) of the
growth of LNCaP cells.19 However, the anti-prolifera-
tive effects on LNCaP cells by these molecules did not
correlate to their ARDA activities.
Since it is well known that sulfonamides have a variety
of biological activities, there appeared to be the possibil-
ity that new anti-cancer sulfonamides could be discov-
ered from our new lead compounds. Because of the
relatively simple chemical structure of BTB01434 and
also potential ease of its chemical modifications, this
compound was selected for lead optimization and subse-
quent evaluations of the new analogs for anti-prostate
cancer activities. We have now prepared three series of
a
OH
OH
OH
OH
HO
O
HO
O
HO
O
O
NH
CF
3
OH
OH
OH
OH
1. (-) Epicatechin
EC50 = 13 M
2. Quercetin
3. Flufenamic acid
EC50 = 100-200 M
EC50 = 25 M
μ
μ
μ
CH
CH
O
3
3
O
O
O
CH
CH
HO
CH
3
O
3
O
3
H C
3
CH
3
O
H C
CH
O
3
3
HO
OH
4. Curcumin
EC50 = 35 M
5. Vitamin E succinate
EC50 = 38 M
μ
μ
b
HipHop Pharmacophore
Training set
Cl
H C
3
CH
3
H C
3
O
O
O
O
HN
HO
N
O
O
O
HO
O
O
O
OH
Cl
NCI0002815
NCI0001009
NCI0002091
HN
S
O
S
CH
N
CH
3
3
O
NO
H
N
O
O
2
N
B
S
O
S
A
O
HO
H C
3
OH
BTB01434
KM06622
NCI0004355
Novel ARDAs retrieved from NCI and MayBridge databases
Figure 1. (a) Chemical structures of five known androgen receptor down-regulating agents (ARDAs) used to generate the pharmacophore model.19
(b) Synthetic ARDAs discovered through generation of HipHop 3D pharmacophore modeling.19

P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
3521
new BTB01434 analogs, including diaryl sulfonamide
analogs bearing various substituents on the phenyl rings
(series A), aryl amide analogs (series B), and amine ana-
logs (series C). These new compounds were evaluated
for cell growth inhibition in two human prostate cancer
cell lines, (LNCaP and PC-3) by the MTT assay. Based
on the structures and anti-prostate cancer activities of
the new compounds, a structure–activity relationship
(SAR) was formulated. This SAR clearly reveals the
structural features of the new compounds responsible
for the growth inhibitory activities in human PC cells.
This paper describes the discovery of novel anti- PC sul-
fonamide agents, including design, synthesis, and struc-
ture–activity relationship.20
zene sulfonyl chloride at 80 ꢁC (Scheme 1b).22
Treatment of the cyano compound 8 with sodium azide
and ammonium chloride in DMF at 120 ꢁC afforded the
corresponding tetrazole 9 (Scheme 1c).23 Compounds
10–12 were synthesized by simply triturating the appro-
priate substituted anilines with the corresponding aro-
matic sulfonyl chlorides (Scheme 1d).24 The tertiary
sulfonamide compound 13 was obtained by treatment
of 5 with methyl iodide in the presence of 1-butyl-3-
methyl imidazolium hexafluro phosphate at room tem-
perature (Scheme 1e).25 Reaction of the appropriate aryl
amine with aryl aldehyde in the presence of triethyl
amine afforded benzamide 14 (Scheme 2).26 Finally,
the aryl amines 16 and 18 were obtained by reducing
intermediate imines 15 and 17, respectively, with sodium
borohydride. The intermediate imines (15 and 17) were
prepared by condensation of appropriate aryl amines
and benzaldehydes (Scheme 3).27 All compounds were
properly characterized by physical and spectroscopic
analyses. However, with regard to compound 6, we ob-
served a significant difference in the melting point re-
ported for this compound in the literature (130 ꢁC)
from that obtained in this study (197–198 ꢁC). The spec-
tral data (1H NMR, 13C NMR, and HRMS) confirm
with certainty the integrity of this compound. Except
compounds 4, 7, 8, and 9, all other compounds de-
scribed in the study have previously been re-
ported.21,25,28–35
2. Chemistry
A total of 16 compounds described in this study were
prepared following straightforward chemistry as out-
lined in Scheme 1a–e (series A), Scheme 2 (series B),
and Scheme 3 (series C). Compounds 1–5 and 8 were
synthesized by refluxing the appropriate substituted aryl
amines with the corresponding aryl sulfonyl chlorides in
pyridine (Scheme 1a).21 The carboxylic acid containing
compounds 6 and 7 were synthesized by treatment of
2-amino-5-methylbenzoic acid in aqueous Na2CO3 solu-
tion at 60 ꢁC followed by reaction with appropriate ben-
a
w
x
y
z
CH3
H
H
F
CH3
z
w
w
y
NO2
NO2
NO2
NO2 CH3
H
1
2
3
4
5
8
H
H
H
H
H
H
SO Cl
NH
NH
O
2
i
2
S
CH3
a
b
c
+
O
H
z
x
x
y
CH3
1-5, 8
CN
H
CH3
F
z
COOH
x
z
b
COOH
NH
O
SO Cl
2
ii
NH
2
S
6
7
CH3
CH3
H
F
+
O
x
z
x
6-7
N
N
HN
N
F
NH
S
iii
O
8
O
CH
3
9
x
z
d
z
10 CH3
H
CH3
H
NH
2
SO Cl
2
NH
S
iv
11
12
H
H
d
+
O
O
x
z
x
10-12
CH
3
CH
N
3
v
S
e
5
O
O
H C
3
13
Scheme 1. Synthesis of sulfonamide compounds (series A) (1–13). Reagents and conditions: (i) pyridine, reflux, 125 ꢁC, 6 h; (ii) Na2CO3, reflux,
80 ꢁC, 6 h; (iii) NH4Cl, NaN3, DMF, 120 ꢁC, 20 h; (iv) NaHCO3, rt, 5–10 min; (v) CH3I, 1-butyl-3-methyl imidazolium hexafluoro phosphate, rt, 2 h.

3522
P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
CH
3
O
H
N
NH
2
Cl
i
+
O
H C
3
H C
3
H C
3
14
Scheme 2. Synthesis of arylamide derivative (series B) (14). Reagents and conditions: (i) TEA, EtOAc, rt, 12 h.
z
z
O
H
N
NH
N
2
H
+
z
x
x
x
16: x, z = CH3
18: x = CH3, z = H
15: x, z = CH3
17: x = CH3, z = H
Scheme 3. Synthesis of aryl imines and amines (series C) (15–18). Reagents and conditions: (i) EtOH, reflux, 12 h; (ii) MeOH, NaBH4, rt, 10 min.
3. Results and discussion
39,800 nM of BTB01434 ! 2.95 nM of 2), while
replacement of the methyl group of ring A with H also
yielded excellent enhancement of activity (GI50 from
39,800 nM of BTB01434 ! 83.0 nM of 1). Interest-
ingly, removal of both CH3 groups from rings A and
B resulted in compound 3, with GI50 of 14.8 nM, a va-
lue 5-fold less potent than 2 and 5.6-fold more potent
than 1. Introduction of the electron withdrawing fluo-
3.1. Inhibition of prostate cancer cell proliferation and
SAR
All 16 compounds were tested for their ability to inhi-
bit cell viability of both the LNCaP (mutant AR) and
PC-3 (AR null) human prostate cancer cell lines using
the MTT assay as we had previously described19 and
the GI50 values are presented in Table 1. Clearly,
the LNCaP cell line was exquisitely more sensitive
than the PC-3 cell line to the growth inhibitory effects
of our compounds. The GI50 values for the LNCaP
cell line ranged from 1.45 to >100,000 nM, while those
for PC-3 cell line ranged from 850 to >100,000 nM.
Figure 2 shows the dose–response curves for the ef-
fects of compounds 4 and 5 on inhibition of growth
of both LNCaP and PC-3 human prostate cancer cell
lines. The reason(s) underlying this difference in sensi-
tivity between the two cell lines is unknown at this
time. One possibility is that the mutant androgen
receptor present in the LNCaP cell may be responsible
for its sensitivity to our compounds. Therefore, the
structure–activity relationship (SAR) discussed below
is with respect to their activities against the LNCaP
cell line.
rine in ring
B
of
2
yielded compound
4
(GI50 = 2.34 nM) with no significant change in activity.
The strong electron withdrawing NO2 group appears to
cause a decrease in activity, because its replacement
with H led to exquisite enhancement of activity (GI50
from 39,800 nM of BTB01434 ! 1.45 nM for 5). How-
ever, its replacement with the moderate electron with-
drawing CO2H in the absence of the CH3 group of
ring B resulted in a 6-fold decrease in activity (GI50
from 2.95 of 2 ! 18.2 nM for 6). Introduction of a
p-fluoro group in ring B of compound 6 resulted in 7
with a drastic decrease in activity. In addition, as
shown for compounds 8 and 9, replacement of the ni-
tro group with either CN or 1H-tetrazole also afforded
deleterious effects on inhibitory activity.
We further investigated the effects of the methyl substit-
uents on rings A and B of our most potent compound 5
(GI50 = 1.45 nM). As shown in compounds 10, 11, and
12, removal of either or both methyl groups resulted
in a drastic loss of activity. Clearly, the effects of the
methyl groups in 5 are in contrast to their effects on
the potency of compound 1 described above. To probe
the contribution of the –NH-group, we synthesized com-
pound 13, an N-methyl derivative of 5. Introduction of
the N-methyl group resulted in a drastic decrease in
activity (GI50 from 1.45 nM of 5 to 89, 150 nM of 13).
Furthermore, replacement of the sulfonamide linker
(–SO2NH–) with two other types of linkers, that is,
amide (14), and amines (16 and 18) resulted in a
significant loss of activity.
The strategy for the design of new molecules was devised
using
lead
compound
BTB01434
(Fig.
1b,
GI50 = 39,800 nM) on the basis of its simple structure
and also envisioned ease of optimization. Thus, we
planned to prepare a series of analogs by substituting
and/or removing the functional groups on the two aro-
matic rings (A and B; see Fig. 1b) and also by introduc-
ing different linkers between the two aromatic rings.
Firstly, examination of the growth inhibitory data re-
veals that substituting any of the three aryl substituents
(i.e., CH3 of ring B and CH3 or NO2 of ring A) with
either H, F, or CO2H led to interesting SAR informa-
tion. Clearly, these substitutions led to excellent
enhancement (480- to 27,000-fold) in growth inhibitory
activities. Substitution of CH3 of ring B with H re-
sulted in the largest enhancement of activity (GI50 from
3.2. Other biological studies
3.2.1. Effects of 1 and 5 on AR protein expression. As sta-
ted above, some compounds, such as 4 and 6 also

P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
3523
Table 1. In vitro growth inhibitory activities of LNCaP and PC-3 human prostate cancer cells by BTB01434 analogs
z
w
H
N
B
S
A
O
O
x
y
Compound
w
x
y
z
Cytotoxicity GI50 (nM)
ARDA
LNCaP
PC-3
EC50 (lM)
A
BTB01434
1
NO2
NO2
NO2
NO2
NO2
H
CH3
H
H
H
H
H
H
H
H
H
CH3
CH3
H
39,800
83.00
2.95
43,670
50,110
60,250
22,900
850.0
76.0
>150
NT
NT
NT
150
NT
NT
NT
NT
NT
NT
NT
2
CH3
H
3
H
14.8
2.34
4
CH3
CH3
CH3
CH3
H
F
5
CH3
H
1.45
30,200
1350
6
COOH
COOH
CN
18.20
10,700
30,200
>100,000
21,300
24,000
53,500
7
F
>100,000
ꢀ100,000
>100,000
13,800
38,900
>100,000
8
CH3
CH3
H
F
9
1H-tetrazole
H
H
F
10
11
12
CH3
H
H
H
H
CH3
H
H
H
H
Structure
Cytotoxicity GI50 (nM)
ADRA (lM)
LNCaPa
PC-3a
CH
CH
3
CH
N
3
13
89,150
44,660
NT
S
O
O
H C
3
B
3
NH
14
54,950
26,920
NT
O
H C
3
X
x
z
Compound
x
X
z
Cytotoxicity GI50 (nM)
LNCaP PC-3
ADRA (lM)
C
15
16
17
18
CH3
CH3
CH3
CH3
N@CH
HN–CH2
CH3
CH3
H
NT
NT
NT
NT
NT
NT
19,050
NT
>100,000
29,510
NT
52,400
N@CH
HN–CH2
H
a Values are means of three independent experiments, SEM = 10%, NT, not tested. Compounds 15 and 17 were unstable under the assay conditions.
showed potent inhibition of PC-3 cell growth which
suggests that they might have mechanisms of action
other than interrupting the AR signaling pathway in
human prostate cancer cells. Because these compounds
LNCaP cells, 5 (GI50 = 1.45 nM), and 1 (GI50 =
83.0 nM), respectively, were assessed for their AR
down-regulating activity as we have previously de-
scribed.19 Both compounds were not effective at
decreasing AR protein expression since they each
caused less than 50% AR protein expression at rela-
tively high concentrations (150 lM). Although the
other four compounds were not tested, it is probable
that they may also behave in a similar fashion. These
results demonstrates that the growth inhibitory effects
of these compounds are due to properties not related
to AR down-regulation.
are analogs of BTB01434,
a
modest ARDA
(EC50 = 76 lM),19 it was of interest to evaluate the ef-
fects of selected compounds on AR protein expression
and also on their abilities to bind to and cause transac-
tivation of the AR.
For comparison purposes, the most and least effective
of the first series of compounds (i.e., 1–6) in the

3524
P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
100
75
50
25
0
a
LNCaP
PC3
-6 -5 -4 -3 -2 -1
0
1
2
3
log [4] (μM)
LNCaP
PC3
100
75
50
25
0
b
-6 -5 -4 -3 -2 -1
0
1
2
3
log [5] (μM)
Figure 3. Dose–response for the competition of 4 and 5 for AR
binding to [3H]-R1881 in (a) LNCaP cells and in (b) PC-3-AR
transfected cells. Data plotted are mean values SEM for three
independent experiments.
Figure 2. The effect of 4 (a) and 5 (b) on LNCaP and PC3 cell viability.
Data plotted are mean values
experiments.
SEM for three independent
3.2.2. LNCaP and PC-3AR androgen receptor binding
and AR-mediated transcription studies. Competitive
androgen binding studies were carried out utilizing the
labeled synthetic androgen [3H]-R1881 in androgen-
dependent LNCaP cells that express a mutated AR,
and androgen independent PC-3 cells stably transfected
with the wild-type AR (PC-3-AR) as previously de-
scribed by our group.36 In this assay, we selected the
crease at 0.1 lM. In the presence of DHT, compounds
4 and 5 reduced luciferase expression by 50% and 80%
at 10 lM, respectively. Taken together, these results sug-
gest that both compounds do not possess AR agonistic
activity and may be considered as modest, pure andro-
gen receptor antagonists. It is relevant to point out that
the potency of compound 5 is similar to potencies of our
steroidal CYP17/antiandrogens and casodex, a clinically
used anti-androgen that were previously tested by us in a
similar assay36 as that used in this study. Although we
did not test the compounds with PC-3AR/LU cells,
most active compound
5
in LNCaP cells
(GI50 = 1.45 nM) and 4 that was active in both LNCaP
(GI50 = 2.34 nM) and PC-3 (GI50 = 850 nM) cell lines.
In both cell lines, compounds 4 and 5 were able to effec-
tively compete with labeled R1881 in a dose-dependent
fashion, albeit at high concentrations (Fig. 3). In PC-
3-AR cells 5 reduced [3H]-R1881 binding by 65% and
80% at 10 and 20 lM, respectively. However, 5 favored
binding to the wild-type AR over the mutated receptor,
with an approximately 20% greater inhibition of [3H]-
R1881 binding to the wild-type AR at 10 lM and above.
Compound 4 reduced [3H]-R1881 binding by 50% and
65% at 10 and 20 lM, respectively, and was equipotent
in both cell lines.
110
100
4
5
90
80
70
60
50
40
30
20
10
0
To determine if the compounds act as AR agonists or
antagonists, androgen regulated transcriptional activa-
tion studies were performed in LNCaP cells transiently
transfected with the probasin luciferase reported
construct ARR2-Luc.36 The results are presented in
Figure 4. 5 nM DHT stimulated luciferase activity
approximately 200-fold. Compound 5 alone had no ef-
fect on luciferase activity at 0.1 and 10 lM concentra-
+ 5nM DHT
Figure 4. The effects of 4 and 5 on transcriptional activity of luciferase
mediated through LNCaP-AR in LNCaP-ARR2-lu prostate cancer
cells. Cells in steroid-free medium were treated with vehicle, or
increasing concentrations of either 4 or 5 with and without 5 nM DHT
for 18 h. Cells were then assayed for luciferase activity as described
previously.36 Data plotted are mean values SEM for three indepen-
dent experiments.
tions. Compound
4
did not increase luciferase
expression at 10 lM, however, there was a slight in-

P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
3525
which express wild-type AR, it is likely that they may
also behave in a similar fashion. In general, these agents
interacted modestly with both AR types, an indication
that the compounds may be useful for the treatment of
patients with tumors expressing either wild-type or mu-
tated AR.
treated with various concentrations of compounds and
whole cell lysates were prepared using lysis buffer con-
taining 0.1 M Tris, 0.5% Triton X-100, and protease
inhibitor. Protein content was determined using the
Bradford Assay (Bio-Rad, Hercules, CA, USA). Protein
was subjected to SDS–PAGE (10% acrylamide) and
transferred onto nitrocellulose membrane. The blots
were blocked overnight in 5% nonfat milk in PBS-T buf-
fer at 4 ꢁC. Monoclonal antibody was used against the
AR (AR441; sc-7305; Santa Cruz Biotechnology, Santa
Cruz, CA; 1:500 dilution) at room temperature for 1 h.
Membranes were then incubated with a goat anti-mouse
IgG secondary antibody conjugated to horseradish per-
oxidase (Bio-Rad cat # 170-6516; 1:2000 dilution) at
room temperature for 1 h. Blots were rinsed with PBS-
T between each step and specific bands were visualized
by enhanced chemiluminescence (ECL; Amersham Bio-
sciences, Arlington Heights, IL, USA). Equivalent load-
ing of samples was determined by reprobing membranes
with b-actin (Calbiochem, USA). Protein expression was
normalized to b-actin.
The biological studies with these lead-modified com-
pounds (1–14, 16, and 18) suggest that their PC growth
inhibitory activities are not due to effects on AR expres-
sion, but in some cases may involve AR antagonistic
properties. However, either with or without anti-andro-
genic activities, these compounds have the potential to
treat both androgen-dependent and androgen-refractory
PC. The anti-proliferative properties of some of these
diaryl sulfonamides in both LNCaP and PC-3 cell lines
suggest that the compounds are capable of interacting
with multiple cellular targets. There are extensive litera-
ture precedents that support the potential of sulfon-
amide compounds as a useful class of drugs, with a
variety of biological activities.37–40
5.3. Cell growth inhibition (MTT colorimetric assay)
4. Conclusions
LNCaP cells were seeded in 24-well plates (Corning
Costar) at a density of 2 · 104 cells per well per 1 mL
of medium. Cell were allowed to adhere to the plate
for 24 h and then treated with different concentrations
of compounds dissolved in DMSO. Cells were treated
for five days with renewal of compounds and media
on day 3. On the fifth day, the medium was renewed
and 100 lL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-2H-tetrazolium bromide from Sigma] solution
(0.5 mg MTT/mL of media) was added to the medium
such that the ratio of MTT:medium was 1:10. The cells
were incubated with MTT for 2 h. The medium was then
aspirated and 500 lL of DMSO was added to solubilize
the violet MTT-formazan product. The absorbance at
560 nm was measured by spectrophotometer (Victor
1420 multilabel counted, Wallac). For each concentra-
tion of compounds there were triplicate wells in each
independent experiment. GI50 values (concentrations
that cause 50% cell growth inhibition) were calculated
by nonlinear regression analysis using GraphPad Prism
software.
Lead optimization of BTB01434 previously identified
from a qualitative 3D pharmacophore modeling study
afforded sulfonamide compounds that are extremely po-
tent inhibitors of LNCaP cell growth and some are strong
inhibitors of PC-3 cell growth as well. Interestingly, the
compounds generated in this study possess new scaffolds
that are different from those of molecules used for gener-
ation of pharmacophore model (see Ref. 19). Because of
their exceptional prostate cancer growth inhibitory
potencies and high synthetic feasibility, these compounds
are strong candidates for development for treatment of
prostate cancer. On the basis of these impressive results,
our next focus will be towards large-scale syntheses of
compounds 4 and 5 and their molecular and pharmaco-
logical characterization and anti-prostate tumor (LNCaP
and PC-3) evaluations in suitable model systems.
5. Experimental
5.1. Cell culture
5.4. Competitive androgen receptor (AR) binding and
luciferase transactivation assays
Androgen-dependent LNCaP cells were obtained from
American Type Culture Collection (Rockville, MD,
USA). Cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum (Atlanta
Biologicals, Lawrenceville, GA, USA) and 1% penicil-
lin/streptomycin. Cells were grown as a monolayer in
T75 or T150 tissue culture flasks in a humidified incuba-
tor (5% CO2, 95% air) at 37 ꢁC.
These assays were performed as previously described by
our group.36
5.4.1. Chemistry. General procedures and techniques
were identical with those previously reported.21–27 1H
NMR spectra were recorded in CDCl3 (unless men-
tioned) with Me4Si as an internal standard and 13C
NMR were recorded in DMSO-d6 using Varian Inova
500 MHz spectrometer operating at 150 MHz. High-res-
olution mass spectra (HRMS) were determined on a 12T
Bruker FTICR–MS with an Apollo II ion source, by po-
sitive ion electrospray. Low-resolution mass spectra
(LRMS) were determined on a Finnegan LCR–MS.
Infrared spectra were recorded on a Perkin-Elmer 1600
The lead compound (BTB01434) was from Maybridge
(Ryan Scientific, Inc., Isle of Palms, SC, USA) databases.
5.2. Western blot analysis
For immunoblot detection of the AR, LNCaP cells were
cultured as described above in T25 flasks. Cells were

3526
P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
FTIR spectrometer using solutions in CHCl3. Melting
points (mp) were determined with fisher Johns melting
point apparatus and are uncorrected. As a criterion of
purity for key target compounds, we provided high-res-
olution mass spectral data with HPLC chromatographic
data indicating compound homogeneity. All the precur-
sors required for synthesis were purchased from Al-
drich–Sigma.
1533, 1351, 1173, 1156, 661 cmꢁ1
;
1H NMR
(500 MHz, CDCl3) d 2.35 (s, 3H, CH3), 7.10–7.89 (m,
7H, aromatic) and 9.59 (s, 1H, NH). 13C NMR
(500 MHz, DMSO-d6)
d 165.51, 163.50, 144.072,
137.23, 135.66, 134.58, 129.85, 127.01, 126.76, 125.36,
116.58, 20.01. HRMS calcd 333.0315(C13H11FN2O4SÆ
Na+), found 333.0314.
5.4.6. (4-Methylphenyl)[(4-methylphenyl)sulfonyl]amine
(5). The method followed that described for compound
1 but using p-toluedine (0.5 g, 4.66 mmol) and 4-
methyl-benzenesulfonyl chloride (0.89 g, 4.66 mmol)in
dry pyridine (2 mL), to give 5 (1.0 g, 82%). Crystalliza-
tion with EtOH gave (0.73 g) white crystals, mp: 114–
115 ꢁC (lit30 116 ꢁC); IR (CHCl3) 3256, 1510, 1332,
5.4.2. [(4-Methylphenyl)sulfonyl](2-nitrophenyl)amine
(1).21 To a solution of 2-nitroaniline (1 g, 7.24 mmol) in
dry pyridine (4 mL) was added 4-methyl-benzenesulfo-
nyl chloride (1.38 g, 7.24 mmol) over a period of
3 min, and the reaction mixture was stirred for 6 h at
125 ꢁC. The reaction mixture was concentrated under re-
duced pressure and the concentrate was poured into ice
cold water (300 mL). The resulting reddish brown solid
precipitate was filtered, washed with water and dried to
give 1 (1.12 g, 52.9%). Crystallization from EtOH gave
(0.82 g) yellow needle shaped crystals, mp: 112–113 ꢁC
(lit21 111–112 ꢁC); IR (CHCl3) 3289, 1530, 1349, 1170,
1160, 766ꢁ1 1H NMR (500 MHz, DMSO-d6) d 2.18
;
(s, 3H, CH3), 2.33 (s, 3H, CH3), 6.94–7.60 (m, 8H, aro-
matic) and 10.01 (s, 1H, NH). 13C NMR (500 MHz,
DMSO-d6) d 143.03, 136.74, 135.16, 133.22, 129.52,
126.71, 120.47, 20.91, 20.26. HRMS calcd 284.0715
(C14H15NO2SÆNa+), found 284.0716.
1
1147, 757 cmꢁ1; H NMR (500 MHz, CDCl3) d 2.38
(s, 3H, CH3), 7.14-8.11 (m, 8H, aromatic), and 9.84 (s,
1H, NH). 13C NMR (500 MHz, DMSO-d6) d 143.79,
142.99, 136.33, 134.20, 130.39, 129.78, 126.84, 126.14,
125.48, 125.23, 21.00. HRMS calcd 315.0409
(C13H12N2O4SÆNa+), found 315.0407.
5.4.7. 5-Methyl-2-[(phenylsulfonyl)amino]benzoic acid
(6).22 To a solution of 2-amino-5-methyl-benzoic acid
(0.2 g, 1.32 mmol), sodium carbonate (0.34 g,
3.17 mmol) in water (2 mL) at 60 ꢁC was added benzene-
sulfonyl chloride (0.28 g, 1.58 mmol) over a period of
2 min, and the reaction mixture was stirred for 6 h at
80 ꢁC. The reaction mixture was cooled to room temper-
ature and acidified with 6 N HCl and the resulting cream
colored solid precipitate was filtered, washed with water
and dried to give 6 (0.39 g, 98.7%). Crystallization from
EtOH gave (0.22 g) white needle shaped crystals, mp:
197–198 ꢁC (lit28 130 ꢁC); IR (nujol) 3202, 1669, 1341,
5.4.3. (4-Methyl-2nitrophenyl)(phenylsulfonyl)amine (2).
The method followed that described for compound 1
but using 2-nitro-4-methylaniline (1 g, 6.57 mmol) and
benzenesulfonyl chloride (1.17 g, 6.57 mmol) in dry pyr-
idine (4 mL), to give 2 (1.22 g, 63.48%). Crystallization
with EtOH gave (1.1 g) yellow needle shaped crystals,
mp: 95–96 ꢁC (lit29 101–102 ꢁC); IR (CHCl3) 3292,
1
1167, 1149, cmꢁ1; H NMR (500 MHz, DMSO-d6) d
1532, 1349, 1171, 1147, 686 cmꢁ1
;
1H NMR
2.50 (s, 3H, CH3), 7.35–7.77 (m, 8H, aromatic) and
1093 (br, 1H, COOH). 13C NMR (500 MHz, DMSO-
d6) d 169.74, 138.55, 137.23, 135.03, 133.45, 132.84,
131.50, 129.43, 126.79, 118.98, 116.98, 19.99. HRMS
calcd 314.0457 (C14H13NO4SÆNa+), found 314.0460.
(500 MHz, CDCl3) d 2.34 (s, 3H, CH3), 7.26–7.87 (m,
8H, aromatic) and 9.62 (s, 1H, NH). 13C NMR
(500 MHz, DMSO-d6) d 143.76, 139.29, 136.94, 134.55,
133.19, 129.31, 127.33, 126.71, 126.34, 125.31, 19.95.
HRMS calcd 315.0409 (C13H12N2O4SÆNa+), found
315.0412.
5.4.8. {[(4-Fluorophenyl)sulfonyl]amino}-5-methylbenzo-
icacid (7). The title compound was synthesized by react-
ing 2-amino-5-methylbenzoic acid (0.25 g, 1.65 mmol),
4-fluoro-benzenesulfonyl chloride (0.39 g, 1.98 mmol),
and sodium carbonate (0.36 g, 3.39 mmol) in 3 mL of
water, following the reaction procedure of 6. The white
solid obtained after acidification of reaction mixture was
filtered, washed with water and dried to give 7 (0.44 g,
85.42%). Crystallization with EtOH gave (0.3 g) white
amorphous solid, mp: 159–160 ꢁC; IR (CHCl3) 3177,
5.4.4. (2-Nitrophenyl)(phenylsulfonyl)amine (3). The
method followed that described for compound 1 but
using 2-nitroaniline (1 g, 7.24 mmol) and benzenesulfo-
nyl chloride (1.28 g, 7.24 mmol) in dry pyridine (4 mL)
to give 3 (1.54 g, 66.51%). Crystallization with EtOH
gave (1.34 g) yellow crystals, mp: 102 ꢁC (lit21 101–
102 ꢁC); IR (CHCl3) 3286, 1530, 1350, 1172, 1147,
686 cmꢁ1 1H NMR (500 MHz, CDCl3) d 7.16–8.11
;
(m, 9H, aromatic), and 9.85 (s, 1H, NH). 13C NMR
(500 MHz, DMSO-d6) d 143.30, 139.20, 134.16, 133.32,
130.14, 129.36, 126.76, 126.38, 125.64, 125.47. HRMS
calcd 301.0253 (C12H10N2O4SÆNa+), found 301.0257.
1662, 1342, 1148, 667 cmꢁ1 1H NMR (500 MHz,
;
CDCl3) d 2.31 (s, 3H, CH3), 7.08–7.86 (m, 7H, aro-
matic), 9.32 (br, 1H, NH), and 10.18 (s, 1H, COOH).
13C NMR (500 MHz, DMSO-d6)d 169.61, 165.59,
163.59, 136.91, 135.00, 133.10, 131.54, 130.02, 129.95,
119.34, 117.42, 116.75, 116.58, 20.00. HRMS calcd
332.0363 (C14H12FNO4SÆNa+), found 332.0372.
5.4.5. [(4-Flurophenyl)sulfonyl](4-methyl-2-nitrophenyl)
amine (4). The method followed that described for com-
pound
1
but using 2-nitro-4-methylaniline (1 g,
6.57 mmol) and 4-fluoro-benzenesulfonyl chloride
(1.4 g, 6.57 mmol)in dry pyridine (4 mL), to give 4
(1.7 g, 83.33%). Crystallization with EtOH gave (1.4 g)
white crystals, mp: 109–112 ꢁC; IR (CHCl3) 3289,
5.4.9. 2-{[(4-Fluorophenyl)sulfonyl]amino}-4-methylben-
zenecarbonitrile (8). The title compound was synthesized
by reacting 2-amino-4-methylbenzonitrile (0.25 g,
1.89 mmol) and 4-fluoro-benzenesulfonyl chloride

P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
3527
(0.37 g, 1.89 mmol) in 2 mL of dry pyridine, following
the reaction procedure of 1.21 The concentrated reaction
mixture was poured into ice cold water (300 mL), and
resulting pale white solid was filtered, washed with
water, and dried to gave 8 (0.24 g, 42.8%). Crystalliza-
tion with EtOH gave (0.15 g) white amorphous solid,
mp: 130–132 ꢁC; IR (CHCl3) 3260, 2226, 1376, 1340,
water, and dried to give 11 (1.13 g, 84.9%). The product
was purified by FCC by using eluent as in 10 to give 11
as a white powder (0.9 g), mp: 95–97 ꢁC (lit32 96 ꢁC); IR
(CHCl3) 3255, 1599, 1342, 1160 cmꢁ1
;
1H NMR
(500 MHz, CDCl3) d 2.37 (s, 3H, CH3), 6.53 (br, 1H,
NH), and 7.04–7.65 (m, 9H, aromatic). 13C NMR
(500 MHz, DMSO-d6) d 143.18, 137.83, 136.72, 129.63,
129.10, 126.70, 123.92, 119.90, 20.90. HRMS calcd
270.05592 (C13H13NO2SÆNa+), found 270.05589.
1
1171, 1156 cmꢁ1; H NMR (500 MHz, CDCl3) d 2.42
(s, 3H, CH3) and 6.90–7.83 (m, 7H, aromatic). 13C
NMR (500 MHz, DMSO-d6) d 165.51, 163.51, 144.83,
138.27, 135.88, 133.69, 129.93, 129.86, 128.04, 127.31,
116.62, 116.58, 116.44, 106.96, 21.20. HRMS calcd
313.0417 (C14H11FN2O2SÆNa+), found 313.0418.
5.4.13. Phenyl(phenylsulfonyl)amine (12). The title com-
pound was synthesized by triturating aniline (0.5 g,
5.36 mmol), powdered sodium bicarbonate (0.9 g,
1.07 mmol), and benzene sulfonyl chloride (0.95 g,
5.36 mmol) for 10 min at rt. Then the reaction mixture
was stirred with water at 60 ꢁC for 30 min, filtered,
washed with water, and dried to gave 12 (0.85 g, 68%).
The product was purified by FCC by using eluent as
in 10 to give 12 as white flakes (0.79 g), mp: 107–
109 ꢁC (lit33 110 ꢁC); IR (CHCl3) 3256, 1599, 1331,
5.4.10. (2-(1H-1,2,3,4-Tetrazole-5-yl)-5-methylphenyl)
[(4-fluorophenyl)sulfonyl]amine (9). The title compound
was synthesized by reacting 2-{[(4-fluorophenyl)sulfo-
nyl]amino}-4-methylbenzenecarbonitrile (8) (0.1 g,
0.34 mmol), sodium azide (0.09 g, 1.37 mmol), and
ammonium chloride (0.74 g, 1.37 mmol) in 3 mL of
DMF at 120 ꢁC for 20 h.23 The reaction mixture was
poured into ice cold water (30 mL) and acidified with di-
lute HCl solution. The white precipitate obtained on
acidification was extracted with ethyl acetate. The or-
ganic layer was washed (H2O), dried (Na2SO4), and con-
centrated to give crude product (0.1 g), which was
purified by FCC (silica gel, CH2Cl2, MeOH,
CH3COOH, 10:2:0.1, v/v/v) to give 9 as a white solid
(0.086 g, 74.9%), mp: 252–254 ꢁC; IR (CHCl3) 3136,
1162 cmꢁ1 1H NMR (500 MHz, CDCl3) d 6.42 (br,
;
1H, NH), and 7.04–7.77 (m, 10H, aromatic). 13C
NMR (500 MHz, DMSO-d6) d 139.63, 137.75, 132.75,
129.13, 126.69, 124.09, 120.13. HRMS calcd 256.04027
(C12H11NO2SÆNa+), found 256.04016.
5.4.14. Methyl(4-methylphenyl)[(4-methylphenyl)sulfo-
nyl]amine (13). The title compound was synthesized by
adding methyl iodide (0.09 g, 0.63 mmol) to a stirring
mixture of approximately 0.5 mL of 1-butyl-3-methyl-
imidazolium-hexa-fluorophosphate, powdered KOH
(0.04 g, 0.72 mmol), and 5 (0.15 g, 0.57 mmol) at rt.25
The reaction mixture was stirred for 2 h and then diluted
with water, extracted with ethyl acetate. The organic
layer was treated with brine solution, dried with
Na2SO4, and evaporated to get a thick liquid. The prod-
uct was purified by FCC (silica gel, petroleum ether,
ethyl acetate, 9:1, v/v) to give 13 as a colorless viscous
liquid, which solidified after several days (0.14 g,
88.5%), mp: 52–54 ꢁC (lit25 59–60 ꢁC); IR (Neat) 1598,
1457, 1376, 1348, 1165, 1153 cmꢁ1
;
1H NMR
(500 MHz, DMSO-d6) d 2.33 (s, 3H, CH3), 6.60–7.77
(m, 7H, aromatic) and 10.54 (br, 1H, NH). 13C NMR
(500 MHz, DMSO-d6) d 154.00, 152.79, 142.40, 136.53,
128.56, 128.50, 124.08, 122.88, 120.11, 110.70, 109.92,
21.31. HRMS calcd 356.0587 (C14H12FN5O2SÆNa+),
found 356.0587.
5.4.11. (4-Methylphenyl)(phenylsulfonyl)amine (10). The
title compound was synthesized by triturating powdered
mixture of p-toluedine (0.5 g, 4.66 mmol) and sodium
bicarbonate (0.78 g, 9.33 mmol) with benzene sulfonyl
chloride (0.82 g, 4.66 mmol) for 10 min at rt.24 Then
the reaction mixture was stirred with water, filtered,
washed with water to remove sodium bicarbonate, and
dried to give 10 (1.1 g, 95.14%). The product was puri-
fied by FCC using solvent gradient (silica gel, petroleum
ether, ethyl acetate, triethyl amine, 9:1:0.2, v/v/v, and
petroleum ether and ethyl acetate 8:2, v/v) to give 10
as white needles (0.89 g), mp: 118–119 ꢁC (lit31 120–
121 ꢁC); IR (CHCl3) 3254, 1510, 1389, 1330,
1510, 1348, 1171, 1154, 679 cmꢁ1
;
1H NMR
(500 MHz, CDCl3) d 2.32 (s, 3H, CH3), 2.41 (s, 3H,
CH3), 3.13 (s, 3H, CH3), and 6.95–7.44 (m, 8H, aro-
matic). 13C NMR (500 MHz, DMSO-d6) d 143.51,
138.65, 136.56, 133.20, 129.58, 129.32, 127.47, 126.03,
37.89, 21.00, 20.51. HRMS calcd 298.08722
(C15H17NO2SÆNa+), found 298.08699.
5.4.15. (4-Methylphenyl)-N-(4-methylphenyl)carboxam-
ide (14). The title compound was synthesized by adding
(drop wise) a solution of p-toluoyl chloride (0.86 g,
5.59 mmol) in 11 mL ethyl acetate (2 mL/mmol) to a
solution of TEA (0.66 g, 6.5 mmol), and p-toluidine
(0.5 g, 4.6 mmol) in 25 mL ethyl acetate (5 mL/mmol)
at rt.26 The white precipitate obtained was stirred
over-night. The next day, most of the solvent was re-
moved in vacuo. Petroleum ether was added, and the
solids were collected on a filter, washed twice with a
mixture of petroleum ether/ethyl acetate (5/1), and three
times with water. The residue was dried to obtain white
crystalline needles (0.92 g, 85%). Crystallization from
EtOH gave (0.75 g) white needle shaped shining crystals,
mp: 163 ꢁC (lit34 163–164 ꢁC); IR (CHCl3) 3437, 3323,
1
1163 cmꢁ1; H NMR (500 MHz, CDCl3) d 2.27 (s, 3H,
CH3), 6.42 (br, 1H, NH), and 6.92–7.74 (m, 9H, aro-
matic). 13C NMR (500 MHz, DMSO-d6) d 139.60,
135.03, 133.41, 132.68, 129.55, 129.10, 126.67, 120.66,
20.24. HRMS calcd 270.05592 (C13H13NO2SÆNa+),
found 270.05568.
5.4.12. [(4-Methylphenyl)sulfonyl]phenylamine (11). The
title compound was synthesized by triturating aniline
(0.5 g, 5.36 mmol), powdered sodium bicarbonate
(0.9 g, 1.07 mmol), and powdered p-toluene sulfonyl
chloride (1.02 g, 5.36 mmol) for 5 min at rt. The reaction
mixture was stirred with water, filtered, washed with

3528
P. Purushottamachar et al. / Bioorg. Med. Chem. 16 (2008) 3519–3529
1665, 1516, 1217, 1212 cmꢁ1
;
1H NMR (500 MHz,
methanol with sodium borohydride (0.6 g, 15.8 mmol)
at rt for 10–20 min.27 Methanol evaporated under vacuo
and residue treated with saturated sodium bicarbonate
solution and extracted with MDC. Organic layer dried
with sodium sulfate and evaporated under vacuo to
get crude 18. Crude product was purified by FCC (silica
gel, petroleum ether, ethyl acetate, 9:1, v/v) to give 18 as
colorless low melting solid (0.32 g, 86%), mp: 20 ꢁC; IR
CDCl3) d 2.33 (s, 3H, CH3), 2.42 (s, 3H, CH3), 7.16–
7.77 (m, 8H, aromatic) and 7.72 (s, 1H, NH). 13C
NMR (500 MHz, DMSO-d6) d 165.14, 141.39, 136.75,
132.45, 132.19, 128.99, 128.85, 127.64, 120.39, 20.97,
20.47. HRMS calcd 248.10458 (C15H15NOÆNa+), found
248.10446.
5.4.16. (1E)-1-Aza-1,2-bis(4-methylphenyl)ethane (15).
The title compound was synthesized by reacting p-tolu-
idine (0.05 g, 0.46 mmol) and p-tolualdehyde (0.056 g,
0.46 mmol) for 5 min, then to this reaction mixture
3 mL of ethanol was added and refluxed over-night.27
The next day the solvent was evaporated and the crude
product was purified by FCC (silica gel, petroleum
ether, ethyl acetate, 9:1, v/v) to give 15 as a yellow fluffy
solid (0.045 g, 46%), mp: 83–85 ꢁC (lit35 107 ꢁC); IR
(CHCl3) 3018, 2923, 2399, 1626, 1512, 1503, 1214,
(CHCl3) 3414, 3025, 2357, 1616, 1520, 806 cmꢁ1 1H
;
NMR (500 MHz, CDCl3) d 2.22 (s, 3H, CH3), 3.88 (s,
1H, NH), 4.29 (s, 2H), and 6.54–7.36 (m, 9H, aromatic).
13C NMR (500 MHz, DMSO-d6) d 146.49, 140.52,
129.30, 128.25, 127.19, 126.53, 124.15, 112.50, 46.88,
20.12. HRMS calcd 220.10967 (C14H15NÆNa+), found
220.10936.
Acknowledgments
1
751 cmꢁ1; H NMR (500 MHz, CDCl3) d 2.36 (s, 3H,
This research was supported in part by a grant from US
National Institutes of Health and National Cancer Insti-
tute (R21 CA117991-01). ROB was supported by a
training grant from the National Institute of Environ-
mental Health Sciences (2T32ES007263-16A1). We
thank the agencies for their generous support. We also
thank Dr. Kellie Hom, Manager NMR facility, School
of Pharmacy, University of Maryland, Baltimore for
her help with acquisition of 13C NMR spectra.
CH3), 2.41 (s, 3H, CH3), 7.11–7.79 (m, 8H, aromatic)
and 8.42 (s, 1H). 13C NMR (500 MHz, DMSO-d6) d
159.47, 148.93, 141.27, 135.14, 133.63, 129.67, 129.39,
128.57, 120.91, 21.14, 20.58. HRMS calcd 232.10967
(C15H15NÆNa+), found 232.12772.
5.4.17. (4-Methylphenyl)[(4-methylphenyl)methyl]amine
(16). The title compound was synthesized by reducing
the crude (1E)-1-aza-1,2-bis(4-methyl phenyl)ethane
(15) in 4 mL methanol with sodium borohydride (0.15 g,
3.96 mmol) at rt for 10–20 min.27 Methanol was evapo-
rated under vacuo and the residue treated with saturated
sodium bicarbonate solution and extracted with MDC.
The organic layer was dried with sodium sulfate and evap-
orated under vacuo to get crude 16. The crude product
was purified by FCC (silica gel, petroleum ether, ethyl ace-
tate, 9:1, v/v) to give 16 as a pale yellow solid (0.079 g,
80%), mp: 50–51 ꢁC; IR (CHCl31) 3416, 3015, 2921,
2864, 1615, 1518, 1215, 810 cmꢁ1; H NMR (500 MHz,
CDCl3) d 2.23 (s, 3H, CH3), 2.33 (s, 3H, CH3), 3.84 (br,
1H, NH), 4.25 (s, 2H), and 6.55–7.25 (m, 8H, aromatic).
13C NMR (500 MHz, DMSO-d6) d 146.45, 137.36,
135.46, 129.21, 128.77, 127.12, 123.89, 112.44, 46.51,
20.64, 20.06. HRMS calcd 234.12532 (C15H17NÆNa+),
found 234.12517.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.031.
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(CHCl3) 3023, 1626, 1512, 1504, 1190, 814, 691 cmꢁ1
;
1H NMR (500 MHz, CDCl3) d 2.36 (s, 3H, CH3), 7.13–
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