Optimizing thiadiazole analogues of resveratrol versus three chemopreventive targets

Article abstract of DOI:10.1016/j.bmc.2011.09.031

Chemoprevention is an approach to decrease cancer morbidity and mortality through inhibition of carcinogenesis and prevention of disease progression. Although the trans stilbene derivative resveratrol has chemopreventive properties, its action is compromised by weak non-specific effects on many biological targets. Replacement of the stilbene ethylenic bridge of resveratrol with a 1,2,4-thiadiazole heterocycle and modification of the substituents on the two aromatic rings afforded potential chemopreventive agents with enhanced potencies and selectivities when evaluated as inhibitors of aromatase and NF-κB and inducers of quinone reductase 1 (QR1).

Full text of DOI:10.1016/j.bmc.2011.09.031

Bioorganic & Medicinal Chemistry 20 (2012) 510–520
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimizing thiadiazole analogues of resveratrol versus three
chemopreventive targets
Abdelrahman S. Mayhoub a, , Laura Marler b, Tamara P. Kondratyuk b, Eun-Jung Park b, John M. Pezzuto b,
Mark Cushman a,
⇑
a Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
b College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2011
Revised 13 September 2011
Accepted 19 September 2011
Available online 25 September 2011
Chemoprevention is an approach to decrease cancer morbidity and mortality through inhibition of car-
cinogenesis and prevention of disease progression. Although the trans stilbene derivative resveratrol
has chemopreventive properties, its action is compromised by weak non-specific effects on many biolog-
ical targets. Replacement of the stilbene ethylenic bridge of resveratrol with a 1,2,4-thiadiazole hetero-
cycle and modification of the substituents on the two aromatic rings afforded potential
chemopreventive agents with enhanced potencies and selectivities when evaluated as inhibitors of aro-
Keywords:
matase and NF-jB and inducers of quinone reductase 1 (QR1).
Thiadiazole
Cancer chemoprevention
Aromatase
Ó 2011 Elsevier Ltd. All rights reserved.
QR1
NF-jB
1. Introduction
by a known cancer-promoting kinase IkB kinase b (IKKb), its inhib-
itors are also considered to be chemopreventive agents.8 iNOS is
involved in the immune response by catalyzing a high level of
NO production.9 However, excessive and persistent expression of
iNOS and production of NO can contribute to pathological condi-
tions including inflammatory diseases as well as cancer. In fact,
the overexpression of iNOS has been reported in many cancer
types.10 On the other hand, chemoprevention could also be
achieved by activating cytoprotective enzymes such as quinone
reductase 1 (QR1).11 An additional target for development of can-
Drugs that prevent disease from developing offer attractive
alternatives to those used in treatment. The use of aspirin to pre-
vent coronary heart diseases is a familiar example.1 Tamoxifen2
and fenasteride3 are examples of drugs that have been employed
as breast and prostate cancer chemopreventive agents,
respectively.
The concept of cancer chemoprevention refers to delaying and/
or preventing cancer development.4 Cancer chemoprevention can
theoretically be achieved by reducing the effects of carcinogens
by inhibiting or down-regulating phase I enzymes such as aroma-
tase and inducible nitric oxide synthase (iNOS) that are capable of
generating carcinogenic species.5,6 Aromatase, the key enzyme in
endogenous estrogen production, is an established target in breast
cancer chemotherapy.7 Since aromatase transcription is mediated
cer chemopreventive agents is NF-
sion of anti-apoptotic genes and is up-regulated in cancer cells due
to gene mutation or secretion of NF-
B activating factors.12,13
Therefore, down-regulation of NF- B hinders cancer progression
and cell differentiation via apoptosis activation. In addition, NF-
B plays a pivotal role in immune system regulation.14 It can be
activated by variable stimuli including interleukin 1b,15 oxidants,16
jB, which regulates the expres-
j
j
j
and some small molecules such as cocaine.17 The NF-
jB pathway is
Abbreviations: ARE, antioxidant response element; BBO, multinuclear broad-
band observe; CD, concentration to double QR1 activity; DMEM, Dulbecco’s
Modified Eagle Medium; DRE, dioxin response element; IC50, sample concentration
which causes 50% inhibition; DPPH, 2,2-diphenyl-1-picrylhydrazyl; iNOS, inducible
also involved in HIV-1 replication and pathogenesis.18 Based on our
understanding of these various biological pathways, cancer chemo-
prevention could be achieved by selectively targeting any of the
proteins involved.
nitric oxide synthase; IR, QR1 induction ratio; LPS, lipopolysaccharide; NF-jB,
nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor
erythroid 2-related factor 2; QNP, quattro nucleus probe; QR1, quinone reductase 1;
TSO, trans-stilbene oxide; SRB, sulforhodamine B.
Grapes have been reported to reduce the risk of certain types of
cancer.19 Grapes contain resveratrol,20 a naturally occurring trans
stilbene with anticancer21 and cancer chemopreventive poten-
tial.22,23 However, resveratrol lacks potency and specificity, and is
⇑
Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790.
E-mail address: cushman@purdue.edu (M. Cushman).
On leave from Al-Azhar University, College of Pharmacy, Cairo, Egypt.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.031

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
511
converted rapidly into inactive metabolites.8,24 Resveratrol’s main
disadvantage in chemoprevention is a poor pharmacokinetic pro-
file due to the presence of free hydroxyl groups that are conjugated
in vivo. Methylated analogues of resveratrol such as DMU-21225
and trimethoxystilbene26 have been used as chemopreventative
agents that show enhanced potency due to tubulin binding and
favorable pharmacokinetics. There is therefore interest in design
and synthesis of resveratrol analogues with enhanced potencies,
selectivities, and metabolic stabilities.
breast cancer cell line MCF-7 and their antioxidant capacities were
evaluated using a DPPH assay.
trans-Resveratrol was used as a reference compound in this
study. In addition, its cis and double bond-reduced analogues were
also tested to investigate whether the constrained trans geometry
of the two aromatic rings on the alkene are necessary for biological
activity in each case.
OH
Resveratrol target specificity versus aromatase and quinone
reductase 2 (QR2) has been improved by varying the substituents
on the two phenyl rings.27 Replacement of the alkene linker with
heterocyclic systems is a complementary strategy that has been
used to enhance the antitumor efficacy of the cis stilbene derivative
combretastatin A-4.28 In this report, both strategies have been
combined. First, the trans stilbene ethylenic bridge of the resvera-
trol scaffold was replaced with five-membered heterocycles.
Replacement of the trans stilbene ethylenic bridge with a 1,2-di-
phenyl heterocyclic system would change the geometric configura-
tion of the two peripheral rings to resemble a cis stilbene (Fig. 1).
To keep the geometry of these two phenyl rings relatively un-
changed and close to that of the trans stilbene template, the phenyl
rings were attached on the 1 and 3 positions of a five-membered
heterocycle (Fig. 1). Based on this design, it was hypothesized that
1,3-diaryl five-membered heterocycles would retain the chemo-
preventive properties of the trans stilbene nucleus. This hypothesis
has been tested, a lead compound was obtained, and then a series
of analogues with different substituents were prepared. In order to
address the chemopreventive target specificity, all of the com-
pounds have been evaluated against a number of enzymes that
are involved in chemopreventive pathways such as aromatase,
HO
OH
OH
OH
HO
trans-resveratrol
cis-resveratrol
OH
HO
OH
dihydroresveratrol
2. Results and discussion
2.1. Chemistry
NF-jB, iNOS, and QR1. Moreover, the direct antiproliferative effects
of the compounds were evaluated in the estrogen receptor-positive
Methyl bromocyanoacetate (1), prepared by bromination of
commercially available methyl cyanoacetate with N-bromosuccin-
imide (NBS), was allowed to react with a set of thioamides 2a-ii in
absolute methanol at room temperature (Scheme 1). Non-commer-
cially available thioamides 2ff, 2gg, 2hh, and 2ii were prepared
from the reactions of their corresponding amides with Lawesson’s
reagent in dry THF.
The formation of the thiadiazole ring was confirmed by NMR
and mass spectral data. For example, the 13C NMR spectra of all
of the 1,2,4-thiadiazoles 3a–ii showed two distinct downfield qua-
ternary carbon signals at about d 182 and 175 ppm corresponding
to the thiadiazole-C3 and -C5 carbons, respectively. The non-equiv-
alence of these two carbons eliminates the corresponding 1,3,4-
thiadiazole structure.
Stilbene nucleus
Het.
Het.
The diphenols 3jj and 3kk were prepared from their methoxy
analogues 3g and 3ii by treatment with hydrogen bromide in gla-
cial acetic acid at reflux temperature for 8 h (Scheme 2).
Catalytic hydrogenation of the dinitro derivative 3ff afforded
the corresponding diamino derivative 3ll (Scheme 3).
trans-like configuration
cis-like configuration
2.2. Biological results
A preliminary study was conducted to test the hypothesis that
1,3-diaryl five-membered heterocycles can retain the chemopre-
ventive properties of the stilbene nucleus. The stilbene analogue
3a with a thiadiazole system at the position of the stilbene double
bond was synthesized and tested for its potential chemopreventive
activity. Interestingly, compound 3a showed moderate to weak
Het.
Het.
activity against aromatase and NF-
with QR1. Based on these initial results, it could be concluded that
jB, and a good induction ratio
Figure 1. Design of 1,3-diaryl five-membered heterocycles from the stilbene
template.

512
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
Next, to increase the selectivity towards aromatase, a structure-
O
S
S
N
based drug design approach was conducted using the available
enzyme crystal structure.29 The existing SAR data of the stilbene
series on aromatase could not be directly applied here because
the two peripheral rings of the stilbene and 3,5-disubstituted-
1,2,4-thiadiazole systems do not overlap precisely, and the bridge
provided by the heterocyclic system is slightly longer than an eth-
ylenic bridge.
N
NC
R
O
NH2
R
+
R
Br
2a-ii
3a-ii
1
a, R = C6H5
v, R = 3,4-Cl2C6H3
w, R = 3-Cl-o-toluyl
x, R = 4-CH3C6H4
y, R = 2-naphthyl
z, R = 4-n-BuC6H4
aa, R = 5-Cl-o-toluyl
b, R = 3-pyridyl
c, R = 4-pyridyl
d, R = 4-ClC6H5
e, R = 4-FC6H5
f, R = 4-BrC6H4
g, R = 4-CH3OC6H4
h, R = 2-BrC6H4
i, R = 2-ClC6H4
j, R = 4-CF3C6H4
k, R = 4-tBuC6H4
l, R = 4-IC6H4
m, R = 3-IC6H4
n, R = 3-FC6H4
Compound 3a was docked into the active pocket of aromatase
(PDB ID 3eqm) using GOLD software.30 The initial expectation
was that the mild activity of the lead compound 3a could be due
to the interaction between the thiazole and the heme iron. How-
ever, none of the top 10 poses showed such an interaction. Instead,
the hypothetical binding mode of the lead compound 3a with aro-
matase (Fig. 2) indicated that one of the phenyl rings lies over the
heme iron and the other one is surrounded by the Met374 and
Val373 residues without any interaction with the heme iron.
The model provided a rationale for synthesis of pyridyl ana-
logues in which the nitrogen of one pyridine ring would coordinate
with the heme iron of aromatase and the other pyridine nitrogen
would hydrogen bond with the NH of Met374 (Fig. 3). The aroma-
tase active site is known to be large and quiet flexible,31 so it was
very difficult to predict the best nitrogen positions from our simple
computational model. Therefore, all of the possible 2-, 3- and 4-
pyridyl derivatives were taken into consideration. Both 3-pyridyl
and 4-pyridyl derivatives 3b and 3c were synthesized successfully;
however, the 2-pyridyl analogue could not be synthesized and it
has never been reported. The 3-pyridyl analogue 3b was found to
be greater than 250 times more potent than the lead compound
bb, R = 4-Br-o-toluyl
cc, R = 2-IC6H4
dd, R = 2-Br-p-toluyl
ee, R = 2,4-Cl2C6H3
ff, R = 4-NO2C6H4
gg, R = 4-NCC6H4
hh, R = 3-NCC6H4
ii, R = 3-CH3OC6H4
o, R = 2-FC6H4
p, R = 3-BrC6H4
q, R = 2,3-Cl2C6H3
r, R = 4-Cl-o-toluyl
s, R = 3-CH3C6H4
t, R = 2-CF3C6H4
u, R = 2,5-Cl2C6H3
Scheme 1. Reagent and conditions: (a) Methanol, 23 °C, 1 min, 10–100%.
3a as an aromatase inhibitor and it displayed an IC50 of 0.2 lM
(Table 1). Moving the nitrogen atom to position-4 (compound 3c)
partially suppressed the aromatase inhibitory activity (IC50
R1
R2
R3
R4
0.8
of structural specificity, being the only compounds in the series
that were aromatase inhibitors with IC50 values less than 50
except of the diamino compound 3ll, which had an IC50 of
26.5 M. Both 3a and 3b had a high degree of selectivity for aroma-
lM, Table 1). Compounds 3b and 3c displayed a high degree
S
S
l
M
N
N
a
N
N
l
tase versus the other targets investigated.
R2
R4
Based on these results, it seems that 3,5-dipyridyl-1,2,4-thi-
adiazoles could serve as a new class of non-steroidal aromatase
inhibitors. Non-steroidal aromatase inhibitors in general have
superior therapeutic benefits compared with their steroidal
counterparts.32
R1
R3
3g; R1= OCH3, R2= H
3ii; R1= H, R2= OCH3
3jj; R3= OH, R4= H
3kk; R3= H, R4= OH
a
(a) HBr, glacial acetic acid, heat to
Reagents and conditions:
reflux for 8 h, 33-40%.
As discussed earlier, the unsubstituted thiadiazole 3a exhibited
some NF-
jB inhibitory activity (IC50 47
lM) plus a high QR1 induc-
tion ratio (IR 8.6). Although the crystal structure of NF-
jB is avail-
Scheme 2. Reagents and conditions: (a) HBr, glacial acetic acid, heat to reflux for
8 h, 33–40%.
able,33 a structure-based design approach to optimize 3a is not
possible because the mechanism of inhibition is not known. Simi-
larly, the mechanism of QR1 induction by 3a is yet to be deter-
mined. Therefore,
thiadiazoles was prepared and both QR1 and NF-
a
large set of mono- and disubstituted
B assays were
H2N
j
conducted in order to eventually elucidate more extensive struc-
ture–activity relationships (SAR). The results are summarized in
Table 3. In addition, the same set of compounds was tested in a
number of other indicative chemoprevention assays including
DPPH free radical quenching, inhibition of NO production, and
antiproliferative activity in the MCF-7 breast cancer cell line to test
for activity and selectivity. The results are listed in Table 3.
QR1 plays an important role in carcinogen deactivation.34 The
thiadiazoles reported here have proven to be very effective QR1
inducers, with 23 of the tested compounds increasing the QR1
activity by a factor of two or more (Table 2). The top thiadiazole
QR1 inducers include the unsubstituted thiadiazole 3a and the
2-halogen-substituted thiadiazoles such as 3h and 3i. The most
potent QR1 inducer is the 2-fluoro derivative 3o (Table 2), which
S
N
N
a
3ff
NH2
3ll
Scheme 3. Reagents and conditions: (a) H2/Pd, methanol, 23 °C, 24 h, 40%.
the 3,5-diaryl-1,2,4-thiadiazole scaffold retains some of the che-
mopreventive activities of resveratrol.
displayed
a QR1 induction ratio of 10.5. The more bulky

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
513
Figure 2. Hypothetical interaction between lead compound 3a, and Met374, Arg115, Val370 and heme in the human aromatase active site. The heme backbone is colored
orange. Many amino acid residues are deleted for the sake of clarity (PDB ID 3eqm). The stereoview is programmed for wall-eyed viewing.
Figure 3. Hypothetical interaction between lead compound 3b and Met374, Arg115 and heme in the human aromatase active site. Hydrogen bonds are represented by yellow
dashed line. Many amino acid residues are deleted for the sake of clarity (PDB ID 3eqm). The stereoview is programmed for wall-eyed viewing.
Table 1
Inhibition of the human aromatase by thiadiazole 3 derivatives
Compound
Aromatase assay
% Max inhiba
Compound
Aromatase assay
% Max inhiba
b
b
IC50
(l
M)
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
60.2 2.8
97.2 1.4
98.0 2.2
9.7 0.9
>50
3v
3w
3x
3y
3z
3aa
3bb
3cc
3dd
3ee
3ff
3gg
3hh
3ii
3jj
3kk
23.0 0.8
19.8 1.2
21.3 0.4
15.8 0.9
35.5 0.7
22.0 1.1
47.8 1.6
39.4 1.0
48.3 0.9
40.3 0.4
29.4 0.8
38.5 1.4
22.1 0.6
26.6 0.7
50.4 1.6
24.4 0.8
70.4 1.9
67.8 2.0
44.6 1.7
37.8 0.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.2 0.04
0.8 0.02
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
45.2 3.7
37.4 2.3
11.1 1.1
44.8 0.8
49.7 1.9
41.0 1.2
43.5 2.1
36.7 0.6
30.6 1.4
41.8 0.7
53.2 1.8
48.4 1.3
45.9 0.5
35.3 0.6
32.9 1.1
21.6 0.7
16.6 0.3
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3ll
26.5 1.4
25.0 0.8
>50
trans-Resveratrol
cis-Resveratrol
Dihydroresveratrol
>50
>50
>50
>50
3u
a
The inhibition percentage was tested at a concentration of 50
l
M of each compound.
b
The IC50 values (lM) are the concentrations that cause 50% inhibition of aromatase.
ortho-substituted derivatives such as the trifluoromethyl and iodo
derivatives 3t and 3cc had lower activity. The size and the elec-
tronic properties of the meta substituents also have a large impact
on the QR1 induction ratio. The smaller and the more electronega-
tive the substituent, the higher the IR, as could be observed for the
3-fluoro derivative 3n (IR = 8.4). As the size of the meta substitu-

514
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
Table 2
Induction of QR1 by thiadiazole 3 derivatives
Compound
Hepa 1c1c7 cells
CDc,d
M)
Tao c1 cells
BPrc1 cells
IRa,b
(
l
% Survivalb
IRa,b
CDc,d
(lM)
% Survivalb
IRa,b
CDc,d
(lM)
% Survivalb
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
3z
3aa
3bb
3cc
3dd
3ee
3ff
8.63 0.21
2.70 0.09
1.00 0.13
3.60 0.18
3.00 0.07
2.20 0.05
1.20 0.20
7.00 0.14
7.40 0.03
2.00 0.12
3.00 0.22
1.30 0.08
4.40 0.04
8.40 0.16
10.50 0.25
7.30 0.23
5.10 0.18
4.90 0.07
4.80 0.24
0.70 0.06
4.20 0.10
1.50 0.13
2.10 0.09
0.80 0.06
1.00 0.12
0.80 0.14
3.80 0.16
2.30 0.24
2.30 0.17
1.50 0.15
4.50 0.21
1.30 0.11
1.80 0.19
1.60 0.07
6.30 0.36
0.70 0.13
0.1
2.1 0.24
53.1 1.3
>50
4.0 0.36
2.07 0.17
16.0 0.89
50.28
73.5
2.2
0.6
NTe
1
18.6
85.9
71.6
2.0
0.5
NTe
0.7
0.6
0.9
NTe
0.5
2.8
0.8
0.7
NTe
1
0.9
1.7
3.4
1.7
5.6
0.6
0.7
2.4
NTe
2.2
NTe
0.9
NTe
5.7
1.2
3.9
NTe
1.4
NTe
NTe
NTe
0.8
49.7
77.8
87.8
86.60
78.26
96.26
86.95
94.24
72.55
75.23
97.91
47.5
104.7
102.9
77.62
86.2
77.8
83
80.1
79.3
86.4
60.3
1.1
1.1
NTe
1.6
2.0
1.2
1.1
NTe
1.2
1.4
1
5.4
1.4
6.3
0.8
0.8
2.2
NTe
2.0
NTe
0.9
NTe
7.1
1.7
4.0
NTe
1.2
NTe
NTe
NTe
0.6
NTe
1.2
NTe
0.7
0.8
NTe
>50
1.73 0.24
1.72 0.09
53.3 1.4
27.7 1.8
99.1
100.0
91.0
100.0
83.8
71.0
85.1
44.3
34.6
74.1
>50
3.10 0.42
4.90 0.22
1.80 0.36
0.059 0.002
2.00 0.14
0.44 0.08
4.00 0.16
2.10 0.11
18.20 0.74
>50
4.00 0.25
>50
16.00 0.41
>50
0.470 0.04
0.570 0.06
0.540 0.03
>50
5.23 0.22
>50
>50
>50
4.03 0.31
>50
0.98 0.07
>50
21.00 0.84
>50
>50
73.6
77.1
82.5
77.7
74.5
71.0
94.9
89.8
82.8
77.3
73.5
88.4
92.3
56.3
94.7
100.4
81.0
94.7
79.27
47.05
77.3
2.5
3.8
6.5
2.8
33.71
88.78
88.13
80.03
96.02
96.81
88.78
99.97
37.93
71.35
48.77
49.63
30.65
78.93
46.20
57.9
41
44.5
38.2
48.7
68.0
96.9
100.2
98.1
2
80.2
68.6
69.8
4.4
8.1
91.5
95.9
95.1
6.3
82.4
89.8
3gg
3hh
3ii
3jj
3kk
3ll
63.7
36.45
4.0
66.13
88.7
89.3
76.3
63.7
97.1
94.3
1.4
0.70 0.04
2.40 0.12
1.90 0.14
1.80 0.21
trans-Resveratrol
cis-Resveratrol
Dihydro-resveratrol
86.3
100.0
1.1
2.0
NTe
86.3
77.8
49.7
82.6
a
b
c
IR, induction ratio.
Testing concentration, 50 lM.
CD is the concentration that doubles the activity.
CD values were determined for compounds with induction ratios > 2.
NT, not tested.
d
e
ents became larger and the electronegativity decreased, the IR de-
creased too, as seen in the case of the 3-bromo 3p and 3-iodo 3m
analogues with IR values of 7.3 and 4.4, respectively. Thiadiazoles
with more polar and larger meta substituents such as the 3-nitrile
analogue 3hh had much lower induction ratios. Replacement of the
fluoride in the meta position with methyl or methoxy led to lower
IR values as seen in the 3-tolyl and 3-methoxy analogues 3s and
3ii. The para-substituted diphenyl thiadiazoles had weak or mod-
erate IR values regardless the nature of the substituents as seen
in the cases of 3d–g, 3j–l, 3z, 3ff and 3gg. Furthermore, adding a
para substituent to the ortho-substituted active thiadiazoles such
as 3h and 3i afforded 3dd and 3ee and the IR values dropped from
7.0 and 7.4 to 1.5 and 4.5, respectively. In terms of selectivity, the
most potent QR1 inducer 3o has high QR1 selectivity since it has
very low activities when tested versus the other chemopreventive
targets. In terms of the concentrations of the compounds required
to double the QR1 activity (CD values), many compounds showed
QR1 induction activities in the sub-micromolar range, such as 3r
of the antioxidant responsive element (ARE) without accompany-
ing induction of phase I enzymes. Inducers which act through the
XRE are said to be bifunctional; those activating ARE are mono-
functional. A QR1 assay in mutant Hepa cells was used (Table 2)
to determine whether the observed induction is monofunctional
or bifunctional. TAOc1 and BPrc1 mutant cells are defective in a
functional Ah receptor or unable to translocate the receptor–ligand
complex to the nucleus, respectively. Induction of QR1 seen in
these two cell lines indicates that the sample does not work
through the XRE pathway.35
Based on a QR1 assay in two mutant cell lines TAOc1 and BPrc1
(Table 2), eight compounds were found to be monofunctional induc-
ers of phase II enzymes. Inducers that are monofunctional are typi-
cally preferred, since they do not induce potentially toxic phase I
enzymes. However, it should be noted that some quite potent
bifunctional inducers, such as 40-bromoflavone, have been found
to significantly induce QR1, glutathione S-transferase, glutathione
peroxidase, and other phase II enzymes without cytotoxic effects.36
Among the substituted 3a derivatives, the hydrophobic thi-
adiazoles with meta-substituted phenyl rings exhibited potent
(CD 0.44
lM), 3aa (CD 0.47 lM), 3bb (CD 0.57 lM) and 3cc (CD
0.54 M) as shown in Table 2.
l
Phase II enzymes may be induced by the xenobiotic responsive
element (XRE) along with phase I enzymes, or through activation
NF-
j
B inhibitory activity in the sub-micromolar range (Table 3).
M) was
The 3-methylphenyl thiadiazole derivative 3s (IC50 0.8
l

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
515
Table 3
Evaluation of NF-
j
B inhibition, antioxidant, antiproliferative, and anti-inflammatory potentials of thiadiazole 3 derivatives
Compounds
NF-
j
B-luciferase
DPPH assay
% Inhibitiond
IC50
SRB assay (MCF-7)
IC50 (lM)
Nitrite assay
c
c
c
c
Lucif. act. % inhiba,b
IC50
47.4
(l
M)
(l
M)
% Inhibitione
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
82.4 5.8
30.9 8.6
7.2 7.0
0.00
0.00
0.00
21.2 4.3
0.00
25.3 9.6
0.00
0.00
0.00
8.26 3.48
ꢀ16.1 1.1
ꢀ2.9 3.2
2.7 6.3
6.9 4.4
0.8 4.9
3.1 5.5
1.8 7.3
2.8 5.3
2.5 5.5
2.9 4.6
16.2 1.4
14.3 0.4
1.3 1.4
3.8 3.0
1.0 0.5
11.1 3.1
3.7 1.1
1.9 1.1
2.9 2.9
1.0 1.7
9.6 1.4
2.3 0.5
7.2 2.6
1.1 0.7
2.1 0.2
1.3 2.3
2.2 0.1
3.4 1.0
3.4 0.4
2.9 0.2
7.3 3.3
6.0 2.3
1.4 1.3
ꢀ2.1 3.1
3.4 5.9
NTf
24.4 3.3
55.7 6.5
1.3 2.6
0.9 3.3
5.1 1.3
18.8 1.7
22.2 2.5
17.1 1.7
1.7 4.4
17.4 2.1
35.0 2.5
35.8 4.3
9.1 2.2
ꢀ5.9 5.0
8.5 0.7
0.5 4.4
20.3 3.3
15.6 4.7
3.2 2.3
24.6 1.9
22.8 2.3
21.4 4.5
10.1 3.0
0.9 2.2
0.00
37.1 3.6
34.5 2.4
31.4 3.0
31.0 6.0
10.2 2.5
39.5 0.07
22.1 7.1
15.4 12.0
28.3 4.1
0.00
0.00
0.00
84.4 2.9
0.00
0.8
6.7 2.4
40.3 2.8
38.3 5.3
20.3 12.1
35.4 6.1
48.5 5.9
39.4 11.2
29.7 5.5
6.7 8.3
10.9 8.3
0.00
45.8 6.6
15.9 2.8
18.2 6.9
46.5 15.3
93.9 1.8
78.9 2.8
60.4 9.3
90.6 8.0
79.0 6.3
38.0 8.3
16.7 2.4
17.8 5.6
23.5 3.2
30.3 5.7
17.9 4.2
21.5 5.0
24.5 0.8
9.6 2.9
3y
3z
0.5 3.7
ꢀ3.5 5.6
25.9 5.6
23.3 6.8
29.0 2.7
25.0 3.3
25.0 2.5
1.6 2.9
3aa
3bb
3cc
3dd
3ee
3ff
3gg
3hh
3ii
3jj
3kk
3ll
29.0 2.8
39.8 0.2
30.0 0.1
31.3 4.1
ꢀ0.4 7.2
26.4 3.5
17.6 3.1
93.7 0.5
47.4 13.2
69.6 3.4
87.6 0.7
31.5 3.1
36.2 4.4
0.4 0.2
19.3 4.4
38.1 6.7
7.6 2.1
0.98 0.2
24.2 1.6
4.7 0.7
13.50 2.22
33.1 2.4
65.3 2.3
33.9 1.8
14.7 1.3
30.8 3.3
30.3 3.5
23.27 2.42
30.71 1.96
trans-Resveratrol
cis-Resveratrol
Dihydro-resveratrol
251.1 7.6
a
b
c
d
e
f
Testing concentration, 50 lM.
NF-
The IC50 values are the concentrations corresponding to 50% inhibition.
The inhibition percentage was tested at a concentration of 400 M of each compound.
M of each compound.
jB IC50 calculated when inhibition > 60%.
l
l
The inhibition percentage was tested at a concentration of 40
Not tested.
found to be slightly more potent than trans-resveratrol, while its
methoxy analogues 3ii (IC50 0.4 M) is a more potent NF- B inhib-
itor. Both 3s and 3ii retained some QR1 induction activity (IRs 4.8
and 6.3, respectively). The more polar 3-hydroxy derivative 3kk
(IC50 38 lM) is less potent than its methoxy analogue 3ii; however,
it is still more active than the unsubstituted analogue 3a. Unlike
the meta-methyl or -methoxy derivatives, their para analogues
3g and 3x are weakly active. Finally, the para amino and hydroxyl
In normal physiology, a large amount of NO is produced by iNOS
as an inflammatory mediator in host the immune system. How-
ever, NO, as one of reactive nitrogen species, can damage DNA
and form nitrative (8-nitroguanine) or oxidative DNA (8-oxodG)
species that eventually result in carcinogenesis.37 In view of this,
the inhibitory activities of the compounds on NO production were
evaluated using the RAW 264.7 cell line-based assay to determine
anti-inflammatory and cancer chemopreventive capacity. As a re-
sult, 3jj and 3ll, which have hydroxyl or amino groups in the
para-position, showed better inhibition than the reference com-
l
j
derivatives showed moderate NF-
19 M) and 3ll (IC50 7.6 M).
Free radicals can cause oxidative damage to DNA, proteins, car-
jB IC50 values, that is, 3jj (IC50
l
l
pound (trans-resveratrol) with IC50 values of 13.5 and 23.3 lM,
bohydrates and lipids, with high chemical reactivity, and conse-
quently lead to various diseases including cancer. To evaluate the
antioxidant capacity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-
radical scavenging was performed according to the method of
Lee et al. as described in the experimental section. In general, all
of the compounds had weak or no antioxidant properties. The only
compound that showed some antioxidant activity was the 4-amino
derivative 3ll (Table 3).
respectively. It is noteworthy that these are the only compounds
that have hydrogen-bond donor moieties in the para-position, sug-
gesting that a hydrogen bond in this position might play an impor-
tant role in inhibition of NO production.
Lastly, the antiproliferative potentials of the newly synthesized
thiadiazoles and the reference compounds were tested with
cultured MCF-7 breast cancer cells and the results are summarized
in Table 3. Except for the moderately cytotoxic 4-hydroxy

516
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
Me: active (IC50 = 0.8 µM).
OH: inactive.
Moving OMe to p-position:
abolish the activity
Replacement with C l& Br:
retains the activity, while I
decreases activity
F
S
S
S
H3CO
N
N
N
Moving F to m or p
positions: decrease activity
N
N
N
F
More bulky (CF3): abolish
the activity
H3CO
3a
3o
Aromatase, IC50 > 20 µM
NF- B, IC50 = 47.4 µM
QR1, IR = 8.6
QR1, IR = 10.5
3ii
Aromatase: inactive
NF- B: inactive
NF- B, IC50 = 0.4 µM
Aromatase: inactive
QR1, IR = weakly active
N
S
N
N
Aromatase, IC50 = 0.2 µM
NF B, IC50: inactive
QR1, IR: weakly active
N
3b
Figure 4. Summary of the chemopreventive activities of 3,5-diaryl-1,2,4-thiadiazoles.
derivative 3jj (IC50 4.7
lM), all of the compounds had weak anti-
performed using a 5 lM C-18 reverse phase column; the default
proliferative activities.
setting was used (k 254).
4.2. Preparation of thioamides 2. General procedure
3. Conclusion
Amides (1 mmol) and Lawesson’s reagent (490 mg, 1.2 mmol)
were added to dry THF (15 mL). The reaction mixture was stirred
at room temperature for 1 h, or heated under reflux for 5 h in
the case of the 4-nitro derivative. The solvent was evaporated
under reduced pressure and the residue was partitioned be-
tween aq NaHCO3 (25 mL) and ethyl acetate (25 mL). The organ-
ic solvent was separated and dried over anhydrous MgSO4. The
crude product was further purified by silica gel flash chromatog-
raphy, using hexane–ethyl acetate (4:1), to yield the correspond-
ing thioamides as yellow solids (42–60%). 4-Nitrothioamide
(2ff),38 4-cyanothiobenzamide (2gg)39 and 3-cyanothiobenza-
mide (2hh),40 3-methoxythiobenzamide (2ii)41 have been previ-
ously reported.
The attempted optimization of the relatively weak activities of
resveratrol against its many biological targets led to the 3,5-dia-
ryl-1,2,4-thiadiazole scaffold, which provided a means to increase
both potency and selectivity in aromatase and NF-jB inhibition as-
says, as well as in a QR1 induction assay as summarized in Figure
4. The pyridyl derivatives 3b and 3c had 30–125 times higher aro-
matase inhibitory activity (IC50 values of 0.2 and 0.8 lM, respec-
tively) than trans-resveratrol, in addition to the advantage of the
higher degree of target selectivity. ortho-Halo substituted deriva-
tives 3h, 3i, and 3o had very high QR1 induction ratios (IR values
7.0, 7.4, and 10.5, respectively) with a very high degree of selectiv-
ity. It is noteworthy that these IR values ranked among or even
above those of the best known QR1 inducers. Finally, the meta-
methyl or methoxy substituted derivatives 3s and 3ii showed
4.3. Preparation of 1,2,4-thiadiazole derivatives 3a–ii
equivalent or onefold more NF-
jB inhibitory activity (IC50 values
0.8 and 0.4 M, respectively) than trans-resveratrol. However, both
3s and 3ii are more selective than trans-resveratrol towards NF-jB,
but they still have QR1 induction properties.
l
Thioamide (1 mmol) was added to a solution of methyl bro-
mocyanoacetate (1, 215 mg, 1.2 mmol) in absolute methanol
(5 mL) at room temperature. The reaction mixture was stirred for
15 s to 1 min. The precipitate was collected by filtration, washed
with methanol–water (5 mL), and dried. In the case of oily com-
pound 3z, it was purified by silica gel chromatography using hex-
ane–ethyl acetate (9:1).42 Compounds 3a,43,44 3b,45,46 3c,47 3d,48
3e,49 3f, 3g,50 3h,51 3s,52 3ff,53 3jj,54 3kk55 have previously been
reported.
4. Experimental section
4.1. General
The integrated intensity of the major peak was P95% of the
combined intensities of all peaks in the HPLC traces of all biologi-
cally tested compounds. 1H NMR spectra were run at 300 MHz and
13C NMR spectra were run at 75.46 MHz in deuterated chloroform
(CDCl3) or dimethyl sulfoxide (DMSO-d6). Chemical shifts are given
in parts per million (ppm) on the delta (d) scale. Chemical shifts are
related to that of the solvent. Mass spectra were recorded at 70 eV.
Melting points were determined using capillary tubes with a Mel-
Temp apparatus and are uncorrected. HPLC analyses were
4.3.1. 3,5-Bis(2-bromophenyl)-1,2,4-thiadiazole (3h)
White solid (99%): mp 98 °C. 1H NMR (CDCl3) d 8.51 (d,
J = 7.8 Hz, 1H), 7.96 (t, J = 5.1 Hz, 2H), 7.85 (d, J = 7.5 Hz, 1H), 7.56
(m, 4H); 13C NMR (CDCl3) d 187.01, 172.78, 138.14, 136.55,
131.12, 129.60, 128.95, 128.66; ESIMS (m/z, rel intensity) 399/
397/395 (MH+, 12/60/100); HRMS (ESI), m/z MH+ 394.8858, calcd
for C14H8Br2N2S 394.8853; HPLC purity 99.61% (MeOH–H2O, 95:5).

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
517
4.3.2. 3,5-Bis(4-(trifluoromethyl)phenyl)-1,2,4-thiadiazole (3j)
White solid (99%): mp 81 °C. 1H NMR (CDCl3) d 8.49 (d,
J = 5.1 Hz, 2H), 8.16 (d, J = 5.1 Hz, 2H), 7.80 (d, J = 5.1 Hz, 2H),
4.3.9. 3,5-Bis(2,3-dichlorophenyl)-1,2,4-thiadiazole (3q)
White solid (100%): mp 165–166 °C. 1H NMR (CDCl3) d 8.53 (dd,
J = 8.1, 1.2 Hz, 1H), 7.86 (dd, J = 7.6, 1.3 Hz, 1H), 7.66 (dd, J = 8.1,
1.3 Hz, 1H), 7.60 (dd, J = 7.8, 1.3 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H),
7.34 (t, J = 7.5 Hz, 1H); 13C NMR (CDCl3) d 182.92, 169.34, 134.42,
134.29, 134.09, 132.80, 132.05, 131.70, 131.42, 130.32, 128.90,
127.99, 127.20; CIMS m/z (rel intensity) 379/377/375 (MH+, 43/
100/78); HRMS (EI), m/z 373.9009 M+, calcd for C14H6Cl4N2S
373.9006; HPLC purity 100% (MeOH–H2O, 95:5).
7.76 (d, J = 5.1 Hz, 2H); 13C NMR (CDCl3)
d 186.85, 172.54,
135.42, 133.30, 132.80, 128.55, 127.72, 126.30, 125.65, 125.63;
CIMS (m/z, rel intensity) 374 (MH+), 355 (MH+–HF, 100); HRMS
(ESI), m/z MH+ 374.0316, calcd for C10H8F6N2S 374.0312; HPLC pur-
ity 98.30% (MeOH–H2O, 95:5).
4.3.3. 3,5-Bis(4-(tert-butyl)phenyl)-1,2,4-thiadiazole (3k)
White solid (99%): mp 91–92 °C. 1H NMR (CDCl3) d 8.35 (d,
J = 5.1 Hz, 2H), 7.98 (d, J = 5.1 Hz, 2H), 7.53 (m, 4H), 1.38 (s, 9H);
4.3.10. 3,5-Bis(4-chloro-2-methylphenyl)-1,2,4-thiadiazole (3r)
White solid (100%): mp 73–74 °C. 1H NMR (CDCl3) d 8.12 (d,
J = 8.4 Hz, 1H), 8.00 (d, J = 8.4, 1.3 Hz, 1H), 7.36–7.26 (m, 4H),
2.71 (s, 3H), 2.65 (s, 3H); 13C NMR (CDCl3) d 185.28, 172.58,
139.81, 138.72, 136.97, 135.44, 132.25, 131.59, 131.27, 130.92,
130.42, 128.43, 126.69, 126.00, 22.11, 21.84; CIMS m/z (rel inten-
sity) 337/ 335 (MH+, 16/51); HRMS (EI), m/z 334.0092 M+, calcd
for C16H12Cl2N2S 334.0098; HPLC purity 100% (MeOH–H2O, 95:5).
13C NMR (CDCl3)
d 187.73, 174.15, 155.37, 153.57, 130.68,
128.00, 127.20, 126.10, 125.40, 36.01, 31.15; ESIMS (m/z, rel inten-
sity) 351 (MH+, 100); HRMS (ESI), m/z MH+ 351.1892, calcd for
C22H26N2S 351.1895; HPLC purity 96.26% (MeOH–H2O, 95:5).
4.3.4. 3,5-Bis(4-iodophenyl)-1,2,4-thiadiazole (3l)
Pale yellow solid (100%): mp 224–226 °C. 1H NMR (CDCl3) d
8.10 (d, J = 5.4 Hz, 2H), 7.88 (d, J = 5.2 Hz, 2H), 7.86 (d, J = 5.4 Hz,
2H), 7.76 (d, J = 5.2 Hz, 2H); 13C NMR (CDCl3) d 187.30, 173.04,
138.50, 137.89, 136.75, 132.08, 129.89, 128.76, 98.77, 97.21; CIMS
m/z (rel intensity) 491 (MH+, 52); HRMS (ESI), m/z 489.8489 MH+,
calcd for C14H8I2N2S 489.8498; HPLC purity 96.8% (MeOH–H2O,
95:5).
4.3.11. 3,5-Bis[2-(trifluoromethyl)phenyl]-1,2,4-thiadiazole (3t)
White solid (10%): mp >260 °C. 1H NMR (CDCl3) d 7.94–7.79 (m,
4H), 7.73–7.59 (m, 4H); 13C NMR (CDCl3) d 184.88, 171.18, 132.07,
131.94, 131.65, 130.92, 129.85, 129.39, 129.21, 128.96, 128.44,
126.91, 126.85, 126.77, 125.49, 121.87; CIMS m/z (rel intensity)
375 (MH+, 13); HRMS (EI), m/z 374.0307 M+, calcd for C16H8F6N2S
374.0312; HPLC purity 100% (MeOH–H2O, 95:5).
4.3.5. 3,5-Bis(3-iodophenyl)-1,2,4-thiadiazole (3m)
White solid (100%): mp 102–103 °C. 1H NMR (CDCl3) d 8.74 (dd,
J = 1.6, 1.8 Hz, 1H), 8.41 (dd, J = 1.6, 1.6 Hz, 1H), 8.36 (dd, J = 7.8,
1.6 Hz, 1H), 7.98 (dd, J = 7.8, 2.1 Hz, 1H), 7.90 (dd, J = 7.8, 1.2 Hz,
1H), 7.83 (dd, J = 7.8, 1.2 Hz, 1H), 7.26 (m, 2H); 13C NMR (CDCl3)
d 186.59, 172.14, 140.81, 139.33, 137.16, 135.94, 134.38, 132.18,
130.84, 130.38, 127.43, 126.71, 94.81, 94.37; CIMS m/z (rel inten-
sity) 491 (MH+, 52); HRMS (ESI), m/z 489.8501 MH+, calcd for
4.3.12. 3,5-Bis(2,5-dichlorophenyl)-1,2,4-thiadiazole (3u)
Off-white solid (100%): mp 116–117 °C. 1H NMR (CDCl3) d 8.61
(d, J = 2.7 Hz, 1H), 8.08 (d, J = 2.7 Hz, 1H), 7.54–7.37 (m, 4H); 13C
NMR (CDCl3) d 182.04, 168.53, 133.80, 132.74, 132.11, 132.03,
131.94, 131.53, 130.84, 130.54, 130.06; APCIMS m/z (rel intensity)
379/377/375 (MH+, 52/100/64); HRMS (ESI), m/z 374.9082 MH+,
calcd for C14H7Cl2N2S 374.9079; HPLC purity 96.8% (MeOH–H2O,
95:5).
C14H8I2N2S 489.8498; HPLC purity 97.20% (MeOH–H2O, 95:5).
4.3.6. 3,5-Bis(3-fluorophenyl)-1,2,4-thiadiazole (3n)
4.3.13. 3,5-Bis(3,4-dichlorophenyl)-1,2,4-thiadiazole (3v)
Off-white solid (100%): mp 127–128 °C. 1H NMR (CDCl3) d 8.44
(d, J = 1.8 Hz, 1H), 8.18 (dd, J = 1.8, 8.1 Hz, 1H), 8.14 (d, J = 2 Hz, 1H),
7.82 (dd, J = 2, 8.1 Hz, 1H), 7.56 (dd, J = 1.7, 8.1 Hz, 1H); 13C NMR
(CDCl3) d 186.01, 171.68, 136.44, 134.82, 133.90, 133.07, 132.19,
131.36, 130.78, 130.19, 129.97, 128.97, 127.34, 126.47; CIMS m/z
(rel intensity) 379/377/375 (MH+, 58/100/75); HRMS (CI), m/z
373.8999 M+, calcd for C14H6 Cl4N2S 373.9006; HPLC purity 100%
(MeOH–H2O, 95:5).
White solid (100%): mp 112–113 °C. 1H NMR (CDCl3) d 8.15 (d,
J = 7.8 Hz, 1H), 8.08 (dd, J = 9, 0.9 Hz, 1H), 7.77 (s, 1H), 7.76 (d,
J = 1.8 Hz, 1H), 7.50–7.45 (m, 2H), 7.26–7.17 (m, 2H); 13C NMR
(CDCl3) d 186.87, 172.52, 163.92, 161.95, 134.55, 132.26, 130.97,
130.26, 123.90, 123.27, 118.95, 117.42, 115.29, 114.22; CIMS m/z
(rel intensity) 275 (MH+, 49); HRMS (ESI), m/z 274.0374 M+, calcd
for C14H8F2N2S 274.0376; HPLC purity 100% (MeOH–H2O, 95:5).
4.3.7. 3,5-Bis(2-fluorophenyl)-1,2,4-thiadiazole (3o)
White solid (100%): mp 96–97 °C. 1H NMR (CDCl3) d 8.47 (dt,
J = 0.5, 7.5, 1 Hz, 1H), 8.33 (dt, J = 0.5, 7.5, 1 Hz, 1H), 7.56 (dt,
J = 1.5, 8, 5.5 Hz, 1H), 7.49 (dt, J = 1.5, 8, 5.5 Hz, 1H), 7.37 (t,
J = 8 Hz, 1H), 7.31 (m, 3H); 13C NMR (CDCl3) d 182.03, 172.89,
163.92, 133.21, 133.14, 131.82, 131.74, 128.73, 124.97, 124.14,
116.87, 116.70, 116.02, 115.85; CIMS m/z (rel intensity) 275
(MH+, 45); HRMS (ESI), m/z 274.0378 M+, calcd for C14H8F2N2S
274.0376; HPLC purity 100% (MeOH–H2O, 95:5).
4.3.14. 3,5-Bis(3-chloro-2-methylphenyl)-1,2,4-thiadiazole (3w)
Off-white solid (100%): mp 82–83 °C. 1H NMR (CDCl3) d 7.90 (d,
J = 6 Hz, 1H), 7.88 (d, J = 6 Hz, 1H), 7.54 (d, J = 6 Hz, 1H), 7.48 (d,
J = 6 Hz, 1H), 7.28 (q, J = 6, 8.7 Hz, 2H), 2.697 (s, 3H), 2.691(s,
3H); 13C NMR (CDCl3) d 186.53, 171.91, 136.41, 135.77, 134.36,
131.88, 130.74, 129.50, 128.65, 127.07, 126.55, 18.01, 17.87; CIMS
m/z (rel intensity) 337/335 (MH+, 71/100); HRMS (EI), m/z
334.0092 M+, calcd for C16H12Cl2N2S 334.0098; HPLC purity 100%
(MeOH–H2O, 95:5).
4.3.8. 3,5-Bis(3-bromophenyl)-1,2,4-thiadiazole (3p)
White solid (100%): mp 114–115 °C. 1H NMR (CDCl3) d 8.53 (t,
J = 1.5 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.21(t, J = 1.8 Hz, 1H), 7.93
(dt, J = 8.7, 1.6 Hz, 1H), 7.68 (dt, J = 7.8, 1.8, 1 Hz, 1H), 7.60 (dt,
J = 8.7, 1.8, 1 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.35 (d, J = 7.8 Hz,
1H); 13C NMR (CDCl3) d 186.26, 172.28, 134.88, 134.34, 133.40,
132.13, 131.33, 130.78, 130.26, 130.14, 126.81, 126.10, 123.38,
122.81; CIMS m/z (rel intensity) 399/397/395 (MH+, 45/100/49);
HRMS (ESI), m/z 393.8775 MH+, calcd for C14H8Br2N2S 393.8775;
HPLC purity 100% (MeOH–H2O, 95:5).
4.3.15. 3,5-Di-p-tolyl-1,2,4-thiadiazole (3x)
White solid (100%): mp 124–125 °C. 1H NMR (CDCl3) d 8.30 (d,
J = 8.4 Hz, 2H), 7.92 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.29
(d, J = 8.1 Hz, 2H), 2.43 (s, 3H), 2.41 (s, 3H); 13C NMR (CDCl3) d
187.89, 173.73, 142.39, 140.41, 130.34, 129.85, 129.36, 128.27,
128.06, 127.36, 21.62, 21.52; ESIMS m/z (rel intensity) 267 (MH+,
100); HRMS (ESI), m/z 267.0956 MH+, calcd for C16H15N2S
267.2950; HPLC purity 99.62% (MeOH–H2O, 95:5).

518
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
4.3.16. 3,5-Di(naphthalen-2-yl)-1,2,4-thiadiazole (3y)
White solid (100%): mp 120–121 °C. 1H NMR (CDCl3) d 8.98 (s,
1H), 8.61 (s, 1H), 8.50 (dd, J = 1.5, 7.5 Hz, 1H), 8.12 (dd, J = 1.5,
7.5 Hz, 1H), 8.02–7.89 (m, 6H), 7.61–7.54 (m, 4H); 13C NMR (CDCl3)
d 188.15, 173.90, 134.96, 134.30, 133.24, 133.06, 130.23, 129.18,
128.97, 128.61, 128.43, 127.94, 127.78, 127.65, 127.10, 126.44,
125.18, 124.19; CIMS m/z (rel intensity) 339 (MH+, 100); HRMS
(CI), m/z 338.0876 M+, calcd for C22H14N2S 338.0878; HPLC purity
100% (MeOH–H2O, 95:5).
4.3.22. 3,5-Bis(2,4-dichlorophenyl)-1,2,4-thiadiazole (3ee)
White solid (100%): mp 80–81 °C. 1H NMR (CDCl3) d 8.56 (d,
J = 5.7 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 2.1 Hz, 1H),
7.58 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 2.1, 8.4 Hz, 1H), 7.39 (dd,
J = 2.1, 8.4 Hz, 1H); 13C NMR (CDCl3) d 185.47, 168.82, 137.91,
136.27, 134.33, 134.05, 133.07, 131.36, 130.77, 130.20, 128.06,
127.17; APCIMS m/z (rel intensity) 381/379/377/375 (MH+, 12/
41/100/66); HRMS (CI), m/z 374.9082 MH+, calcd for C14H7Cl4N2S
374.9084; HPLC purity 98.28% (MeOH–H2O, 95:5).
4.3.23. 4,40-(1,2,4-Thiadiazole-3,5-diyl)dibenzonitrile (3gg)
4.3.17. 3,5-Bis(4-butylphenyl)-1,2,4-thiadiazole (3z)
Colorless viscous oil (96%). 1H NMR (CDCl3) d 8.29 (d, J = 8.0 Hz,
2H), 7.95 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.31 (d,
J = 8.0 Hz, 2H), 2.69 (t, J = 7.5 Hz, 4H), 1.66 (q, J = 7.5 Hz, 4H), 1.63
(q, J = 7.5 Hz, 4H), 0.96 (t, J = 7.5 Hz, 6H); 13C NMR (CDCl3) d
187.91, 173.78, 147.38, 145.40, 130.49, 129.23, 128.70, 128.24,
127.40, 35.65, 35.58, 33.40, 33.29, 29.66, 22.28, 13.90; ESIMS m/z
(rel intensity) 373 (MNa+, 100), 351 (MH+, 98); HRMS (ESI), m/z
351.1901 MH+, calcd for C12H27N2S 351.1889; HPLC purity
96.59% (MeOH–H2O, 95:5).
White solid (100%): mp >260 °C. IR (KBr) 3096, 2923, 2230,
1607, 1466 cmꢀ1 1H NMR (CDCl3) d 8.50 (d, J = 8.7 Hz, 2H), 8.17
;
(d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.7 Hz, 2H);
13C NMR (CDCl3)
d
186.61, 172.27, 136.01, 133.82, 133.15,
132.61, 128.84, 127.97, 118.41, 117.78, 115.56, 114.05; CIMS m/z
(rel intensity) 289 (MH+, 100); HRMS (CI), m/z 288.0474 M+, calcd
for C16H8N4S 288.0470; HPLC purity 98.44% (MeOH–H2O, 95:5).
4.3.24. 3,30-(1,2,4-Thiadiazole-3,5-diyl)dibenzonitrile (3hh)
White solid (100%): mp 176–177 °C. 1H NMR (CDCl3) d 8.70 (s,
1H), 8.62 (d, J = 8.4 Hz, 1H), 8.37 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H),
7.82 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.69–7.64 (m, 2H);
4.3.18. 3,5-Bis(5-chloro-2-methylphenyl)-1,2,4-thiadiazole (3aa)
Off-white solid (100%): mp 80–81 °C. 1H NMR (CDCl3) d 8.18 (d,
J = 0.9 Hz, 1H), 8.10 (d, J = 0.9 Hz, 1H), 7.38 (dd, J = 2.1, 7.8 Hz, 1H),
7.29 (dd, J = 0.9, 7.8 Hz, 1H), 7.26 (dd, J = 0.9, 7.5 Hz, 1H), 2.69 (s,
3H), 2.64 (s, 3H); 13C NMR (CDCl3) d 185.08, 172.31, 136.42,
135.38, 133.27, 133.06, 132.83, 132.30, 131.53, 131.33, 130.94,
130.69, 129.70, 129.23, 21.68, 21.52; CIMS m/z (rel intensity)
337/335 (MH+, 66/100); HRMS (CI), m/z 334.0096 M+, calcd for
13C NMR (CDCl3)
d 186.32, 171.78, 135.11, 133.76, 133.38,
132.30, 132.01, 131.44, 130.77, 130.37, 129.71, 118.27, 117.54,
114.00, 113.15; CIMS m/z (rel intensity) 289 (MH+, 100); HRMS
(CI), m/z 288.0476 M+, calcd for C16H8N4S 288.0470; HPLC purity
95.01% (MeOH–H2O, 95:5).
4.3.25. 3,5-Bis(3-methoxyphenyl)-1,2,4-thiadiazole (3ii)
C16H12Cl2N2S 334.0098; HPLC purity100% (MeOH–H2O, 95:5).
Pale yellow solid (148 mg, 100%): mp 71 °C. 1H NMR (CDCl3) d
7.99 (d, J = 7.8 Hz, 1H), 7.92 (t, J = 2.1, 3.3 Hz, 1H), 7.55 (m, 2H),
7.38 (m, 2H), 7.02 (m, 2H); 13C NMR (CDCl3) d 187.86, 173.44,
160.04, 159.79, 134.04, 131.69, 130.26, 129.69, 120.90, 120.00,
117.84, 116.65, 112.96, 112.05, 55.42, 55.34; CIMS m/z (rel inten-
sity) 299 (MH+, 100); HRMS (ESI), m/z 298.0781 M+, calcd for
4.3.19. 3,5-Bis(4-bromo-2-methylphenyl)-1,2,4-thiadiazole
(3bb)
Off-white solid (100%): mp 74 °C. 1H NMR (CDCl3) d 8.17 (d,
J = 2.1 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 2.1, 7.5 Hz,
1H), 7.33–7.25 (m, 4H), 2.69 (s, 3H), 2.65 (s, 3H); 13C NMR (CDCl3)
d 185.07, 172.29, 136.36, 135.34, 133.24, 133.01, 132.78, 132.25,
131.48, 131.29, 130.90, 130.64, 129.66, 129.20, 21.60, 21.46; CIMS
m/z (rel intensity) 427/425/423 (MH+, 18/100/53); HRMS (CI), m/z
421.9090 M+, calcd for C16H12Br2N2S 421.9088; HPLC purity 100%
(MeOH–H2O, 95:5).
C16H14N2O2S 298.0776; HPLC purity 97.91% (MeOH–H2O, 95:5).
4.3.26. 3,5-Bis(4-aminophenyl)-1,2,4-thiadiazole (3ll)
Orange-yellow solid (40%): mp 208–209 °C. 1H NMR
(CD3COCD3) d 8.07 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 6.78
(d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 5.52 (br s, 2H), 5.15 (br
s, 2H); 13C NMR (CD3COCD3) d 188.17, 174.30, 153.12, 151.28,
130.15, 129.57, 122.75, 119.72, 114.55, 114.33; CIMS m/z (rel
intensity) 269 (MH+, 100); HRMS (CI), m/z 268.0789 M+, calcd for
4.3.20. 3,5-Bis(2-iodophenyl)-1,2,4-thiadiazole (3cc)
Yellowish-white solid (100%): mp 112 °C. 1H NMR (CDCl3) d
8.34 (d, J = 6.9 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 7.8 Hz,
1H), 7.84 (d, J = 6.9 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.56 (d,
J = 7.2 Hz, 1H), 7.34 (dt, J = 2.4, 7.5 Hz, 1H), 7.28 (dt, J = 2.4,
7.8 Hz, 1H); 13C NMR (CDCl3) d 186.69, 172.17, 141.17, 140.74,
137.57, 135.78, 132.07, 131.79, 131.51, 130.92, 128.55, 128.10,
97.19, 95.59; CIMS m/z (rel intensity) 491 (MH+, 100); HRMS (CI),
m/z 489.8501 M+, calcd for C14H8I2N2S 489.8498; HPLC purity
100% (MeOH–H2O, 95:5).
C14H12N4S 268.0783; HPLC purity 98.99% (MeOH–H2O, 80:20).
4.4. Molecular modeling
Compounds of interest were built with Sybyl 7.1 software and
minimized to 0.01 kcal/mol by the Powell method, using
Gasteiger–Hückel charges and the Tripos force field. The energy-
optimized compounds were docked into the androgen binding
pocket of aromatase after removal of the structure of the natural
ligand. The parameters were set as the default values for GOLD.
The maximum distance between hydrogen bond donors and accep-
tors for hydrogen bonding was set to 3.5 Å. After docking, the first
pose conformations of compounds of interest were merged into the
ligand–free protein. The new ligand–protein complex was subse-
quently subjected to energy minimization using the Amber force
field with Amber charges. During the energy minimization, the
structure of the compounds of interest and a surrounding 10 Å
sphere of the protein were allowed to move. The structure of
remaining protein was kept frozen. The energy minimization was
performed using the Powell method with a 0.05 kcal/(mol Å)
4.3.21. 3,5-Bis(2-bromo-4-methylphenyl)-1,2,4-thiadiazole
(3dd)
Off-white solid (100%): mp 73 °C. 1H NMR (CDCl3) d 8.49 (d,
J = 8.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.58 (s, 2H), 7.29 (d,
J = 8.4 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 2.41 (s, 3H), 2.40 (s, 3H);
13C NMR (CDCl3)
d 184.33, 170.50, 143.14, 141.35, 134.55,
134.27, 131.97, 131.14, 128.99, 128.84, 128.15, 123.25, 121.77,
21.05, 20.96; CIMS m/z (rel intensity) 427/425/423 (MH+, 18/100/
51); HRMS (CI), m/z 421.9090 M+, calcd for C16H12Br2N2S
421.9087; HPLC purity 95.01% (MeOH–H2O, 95:5).

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
519
energy gradient convergence criterion and a distance dependent
dielectric function.
emitting light, which is detected with a luminometer. Data were
expressed as % inhibition at 50 M or IC50 values (i.e., concentra-
tion of test sample required to inhibit TNF- activated NF- B activ-
l
a
j
4.5. Aromatase assay
ity by 50%). After incubating treated cells, they were lysed in
Reporter Lysis buffer. The luciferase assay was performed using
the Luc assay system from Promega, following the manufacturer’s
Aromatase activity was assayed as previously reported, with the
necessary modifications to assay in a 384-well plate.56 Briefly, the
instructions. In this assay, N-a-tosyl-L-phenylalanine chloromethyl
test compound (3.5
lL) was preincubated with 30
lL of NADPH-
ketone (TPCK) was used as a positive control; IC50 = 5.09 lM.
regenerating system (2.6 mM NADP+, 7.6 mM glucose-6-phos-
phate, 0.8 U/mL glucose-6-phosphate dehydrogenase, 13.9 mM
MgCl2, and 1 mg/mL albumin in 50 mM potassium phosphate buf-
fer, pH 7.4) for 10 min at 37 °C. As a source of aromatase we use
BD-Supersomes™ a well-established source of cDNA-expressed
enzymes. BD recommends using NADPH generating system for
cytochrome P450 activity. An NADPH solution can also be used
but depletion of this cofactor might happen during longer incuba-
4.8. Nitrite assay
RAW 264.7 mouse macrophage cells were incubated in a 96-
well culture plate for 24 h. The cells were pretreated with various
concentrations of compounds dissolved in phenol red-free DMEM
for 30 min followed by 1 lg/mL of LPS treatment for 24 h. The level
of nitrite, a stable end product of NO, in the cultured media was
measured using a colorimetric reaction with Griess reagent. The
optical density (OD) was measured at 540 nm and the level of
tions. Before addition of the enzyme and substrate mixture (33
lL
of 1 M CYP19 enzyme, BD Biosciences, 0.4 M dibenzylfluoresce-
l
l
in, 4 mg/mL albumin in 50 mM potassium phosphate, pH 7.4) was
added, and the plate was incubated for 30 min at 37 °C before
nitrite was estimated using
concentrations of sodium nitrite. The positive control in this assay
was NG-
-monomethyl arginine ( -NMMA); IC50 = 19.7 M. In par-
a standard curve with known
quenching with 25
lL of 2 N NaOH. After termination of the reac-
L
L
l
tion and shaking for 5 min, the plate was further incubated for 2 h
at 37 °C. This enhances the ratio of signal to background. Fluores-
cence was measured at 485 nm (excitation) and 530 nm (emis-
sion). IC50 values were based on three independent experiments
performed in duplicate using five concentrations of test substance.
allel, the cytotoxic effects of compounds were evaluated by SRB
assay
4.9. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging assay
Naringenin (IC50 = 0.23
lM) was used as a positive control.
Kinetic aromatase assays were carried out in order to determine
To evaluate antioxidant capacity, 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) free-radical scavenging was performed accord-
ing to the method described by Lee et al. In this assay,57
antioxidants convert the stable free radical DPPH to an inactive
hydrazine form, accompanied by color fading from purple to pale
inhibitor mechanism. Test compound was preincubated with 30 lL
of NADPH-regenerating system (2.6 mM NADP+, 7.6 mM glucose 6-
phosphate, 0.8 U/mL glucose 6-phosphate dehydrogenase,
13.9 mM MgCl2, and 1 mg/mL albumin in 50 mM potassium phos-
phate buffer, pH 7.4) for 10 min at 37 °C. The IC50 was used as the
final concentration for each inhibitor. Substrate was added at five
yellow. Briefly, 95
l
L of DPPH radical solution in ethanol (316
lM)
was added in a 96-well plate containing 5
lL of each compound
concentrations: 16, 8, 4, 2, and 1
l
M. CYP19 (1
l
M) was added
dissolved in 100% DMSO, and the mixture incubated for 30 min
at 37 °C. The OD of each well was measured at 515 nm using a
microplate reader. The DPPH radical scavenging activity of each
sample was evaluated by calculating % inhibition as follows: %
inhibition = (1 ꢀ ODsample/ODcontrol) ꢁ 100.
and fluorescence was measured at 485 nm (excitation) and
530 nm (emission) every 10 s for at least 5 min. Error values repre-
sent three independent experiments for each compound.
4.6. Quinone reductase 1 (QR1) assay
4.10. SRB assay58
Hepa 1c1c7 (mouse hepatoma) cells, TAOc1 mutant Hepa cells,
or BPrC1 mutant Hepa cells were used in the assay. Cells were incu-
bated in a 96-well plate with test compounds at a maximum con-
Serially diluted test compounds in DMSO were transferred to
96-well plates and incubated for 72 h at 37 °C in a CO2 incubator.
The incubation was stopped by adding trichloroacetic acid (10%),
which fixes cells. The cells were washed, air-dried, stained with
0.4% SRB solution, and optical densities were determined at
515 nm using a microplate reader. In each case, a zero-day control
was performed by adding an equivalent number of cells (1 ꢁ 104
cells per well), incubating at 37 °C for 30 min, and processing as de-
scribed above. Percent of cell survival was calculated using the for-
mula: (ODtested compound ꢀ ODzero-day)/ODcontrol ꢀ ODzero-day) ꢁ 100.
centration of 50 lM, digitonin was used to permeabilize cell
membranes, and enzyme activity was measured by the reduction
of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to a blue formazan. Production was measured by absorption
at 595 nm. A total protein assay using crystal violet staining was
run in parallel. 40-Bromoflavone (CD = 0.01
lM) was used as a po-
sitive control. Mutant cell lines TAOc1 and BPrc1 were supplied by
Dr. J.P. Whitlock, Jr. (Department of Molecular Pharmacology, Stan-
ford University School of Medicine, Stanford, California 94305,
USA). All cells were maintained and passaged according to ATCC
Acknowledgment
instructions in MEM-a containing 5% antibiotic–antimycotic and
10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. The assay
in these cells follows the procedure of the QR1 assay described in
wild type Hepa1c1c7 cells.
This work was supported by Program Project Grant P01
CA48112 awarded by the National Cancer Institute.
References and notes
4.7. NF-
jB luciferase assay
1. Catella-Lawson, F.; Fitzgerald, G. A. Drug Saf. 1995, 13, 69–75.
2. (a) Fisher, B.; Costantino, J. P.; Wickerham, D. L.; Cecchini, R. S.; Cronin, W. M.;
Robidoux, A.; Bevers, T. B.; Kavanah, M. T.; Atkins, J. N.; Margolese, R. G.;
Runowicz, C. D.; James, J. M.; Ford, L. G. J. Natl. Cancer Inst. 1998, 90, 1371–
1388; (b) Kramer, R.; Brown, P. Drug Saf. 2004, 27, 979–989.
Studies were performed with NF-
human embryonic kidney cells 293 from Panomics (Fremont, CA).
This cell line contains chromosomal integration of a luciferase re-
j
B reporter stably-transfected
3. Parnes, H. L.; Thompson, I. M.; Ford, L. G. J. Clin. Oncol. 2005, 23, 368–377.
4. Hong, W. K.; Sporn, M. B. Science 1997, 278, 1073–1077.
porter construct regulated by NF-
jB response element. The gene
product, luciferase enzyme, reacts with luciferase substrate,

520
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 510–520
5. Conda-Sheridan, M.; Marler, L.; Park, E.; Kondratyuk, T. P.; Jermihov, K.;
29. Ghosh, D.; Griswold, J.; Erman, M.; Pangborn, W. Nature 2009, 457, 219–223.
30. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Protein:
Struct., Funct., Genet. 2003, 52, 609–623.
Mesecar, A. D.; Pezzuto, J. M.; Asolkar, R. N.; Fenical, W.; Cushman, M. J. Med.
Chem. 2010, 53, 8688–8699.
6. Aggarwal, B. B.; Shishodia, S. Biochem. Pharmacol. 2006, 71, 1397–1421.
7. Strasser-Weippl, K.; Goss, P. E. J. Clin. Oncol. 2005, 23, 1751–1759.
8. (a) Hoshino, J.; Park, E.; Kondratyuk, T. P.; Marler, L.; Pezzuto, J. M.; van
Breemen, R. B.; Mo, S.; Li, Y.; Cushman, M. J. Med. Chem. 2010, 53, 5033–5043;
(b) Ghosh, S.; Choudary, A.; Musi, N.; Hu, Y. F.; Li, R. Mol. Endocrinol. 2009, 23,
662–670.
31. Ekroos, M.; Sjogren, T. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13682–13687.
32. (a) Buzdar, A.; Douma, J.; Davidson, N.; Elledge, R.; Morgan, M.; Smith, R.;
Porter, L.; Nabholtz, J.; Xiang, X.; Brady, C. J. Clin. Oncol. 2001, 19, 3357–3366;
(b) Eisen, A.; Trudeau, M.; Shelley, W.; Messersmith, H.; Pritchard, K. I. Cancer
Treat. Rev. 2008, 34, 157–174.
33. Ghosh, G.; Van Duyne, G.; Ghosh, S.; Sigler, P. B. Nature 1995, 373, 303–310.
34. Kennelly, E. J.; Gerhauser, C.; Song, L. L.; Graham, J. G.; Beecher, C. W. W.;
Pezzuto, J. M.; Kinghorn, A. D. J. Agric. Food Chem. 1997, 45, 3771–3777.
35. Gerhäuser, C.; You, M.; Liu, J.; Moriarty, R. M.; Hawthorne, M.; Mehta, R. G.;
Moon, R. C.; Pezzuto, J. M. Cancer Res. 1997, 57, 272–278.
36. Marler, L.; Conda-Sheridan, M.; Cinelli, M. A.; Morrell, A. E.; Cushman, M.;
Chen, L.; Huang, K.; Van Breemen, R.; Pezzuto, J. M. Anticancer Res. 2010, 30,
4873–4882.
9. Aktan, F. Life Sci. 2004, 75, 639–653.
10. Aggarwal, B. B.; Gehlot, P. Curr. Opin. Pharmacol. 2009, 9, 351–369.
11. Ross, D.; Kepa, J. K.; Winski, S. L.; Beall, H. D.; Anwar, A. H. D.; Siegel, D. Chem.
Biol. Interact. 2000, 129, 77–97.
12. Sheikh, M. S.; Huang, Y. Cell Cycle 2003, 2, 550–552.
13. Escárcega, R. O.; Fuentes-Alexandro, S.; García-Carrasco, M.; Gatica, A.; Zamora,
A. Clin. Oncol. (R. Coll. Radiol.) 2007, 19, 154–161.
14. Hayden, M. S.; West, A. P.; Ghosh, S. Oncogene 2006, 25, 6758–6780.
15. Fitzgerald, D. C.; Meade, K. G.; McEvoy, A. N.; Lillis, L.; Murphy, E. P.; MacHugh,
D. E.; Baird, A. W. Vet. Immunol. Immunopathol. 2007, 116, 59–68.
16. Chandel, N. S.; Trzyna, W. C.; McClintock, D. S.; Schumacker, P. T. J. Immunol.
2000, 165, 1013–1021.
37. Hiraku, Y.; Kawanishi, S.; Ichinose, T.; Murata, M. Ann. N.Y. Acad. Sci. 2010, 1203,
15–22.
38. Ciureanu, M.; Hillebrand, M.; Volanschi, E.; Ghetu, D. Rev. Roum. Chim. 1987, 32,
467–475.
39. Zhu, B.; Bauer, S. M.; Jia, Z. J.; Probst, G. D.; Zhang, Y.; Scarborough, R. M. 2006,
PCT Int. Appl. WO 2006002099.
40. Oda, K.; Sakai, M.; Machida, M. Chem. Pharm. Bull. 1997, 45, 584–589.
41. Michael, N.; Claudia, P.; Andrea, B.; Walther, S. Synthesis 2008, 24, 4012–4018.
42. Mayhoub, A. S.; Kiselev, E.; Cushman, M. Tetrahedron Lett. 2011, 52, 4941–4943.
43. Toland, W. G. J. Org. Chem. 1962, 27, 869–871.
17. Hargrave, B. Y.; Tiangco, D. A.; Lattanzio, F. A.; Beebe, S. J. Cardiovasc. Toxicol.
2003, 3, 141–151.
18. Hiscott, J.; Kwon, H.; Génin, P. J. Clin. Invest. 2001, 107, 143–151.
19. Pezzuto, J. M. J. Agric. Food Chem. 2008, 56, 6777–6784. Ref. 37 cited therein.
20. Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W. W.;
Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.;
Mezzuto, J. M. Science 1997, 275, 218–220.
44. Kresze, G.; Horn, A.; Philippson, R.; Trede, A. Chem. Ber. 1965, 98, 3401–3409.
45. Boeini, H. Z. J. Iran. Chem. Soc. 2009, 6, 547–551.
21. (a) Pizarro, J. G.; Verdaguer, E.; Ancrenaz, V.; Junyent, F.; Sureda, F.; Pallàs, M.;
Folch, J.; Camins, A. Neurochem. Res. 2011, 36, 187–194; (b) Oi, N.; Jeong, C. H.;
Nadas, J.; Cho, Y. Y.; Pugliese, A.; Bode, A. M.; Dong, Z. Cancer Res. 2010, 70,
9755–9764.
46. Yan, M.; Chen, Z.; Zheng, Q. J. Chem. Res., Synop. 2003, 10, 618–619.
47. Meltzer, R. I.; Lewis, A. D.; King, J. A. J. Am. Chem. Soc. 1955, 77, 4062–4066.
48. Kresze, G.; Mashke, A.; Albrecht, R.; Bederke, K.; Patzchke, H. P.; Smalla, H.;
Trede, A. Angew. Chem. 1962, 74, 135–144.
22. (a) Carbó, N.; Costelli, P.; Baccino, F. M.; López-Soriano, F. J.; Argilés, J. M.
Biochem Biophys. Res. Commun. 1999, 254, 739–743; (b) Chen, Y.; Tseng, S. H.;
Lai, H. S.; Chen, W. J. Surgery 2004, 136, 57–66; (c) Roy, P.; Kalra, N.; Prasad, S.;
George, J.; Shukla, Y. Pharm. Res. 2009, 26, 211–217; (d) Aziz, M. H.; Kumar, R.;
Ahmad, N. Int. J. Oncol. 2003, 23, 17–28.
49. Khosropour, A. R.; Noei, J. Monatsh. Chem. 2010, 141, 649–651.
50. Rafiqul-Islam, M.; Shimada, K.; Aoyagi, S.; Takikawa, Y.; Kabuto, C. Heteroat.
Chem. 2004, 15, 175–186.
51. Komatsu, M.; Shibata, J.; Ohshiro, Y.; Agawa, T. Bull. Chem. Soc. Jpn. 1983, 56,
180–183.
23. Pezzuto, J. M. Pharm. Biol. 2008, 46, 443–573.
24. Pezzuto, J. M. Biochem. Pharmacol. 1997, 53, 121–133.
25. Ma, Z.; Molavi, O.; Haddadi, A.; Lai, R.; Gossage, R. A.; Lavasanifar, A. Cancer
Chemother. Pharmacol. 2008, 63, 27–35.
26. Deng, Y. H.; Alex, D.; Huang, H. Q.; Wang, N.; Yu, N.; Wang, Y. T.; Leung, G. P.;
Lee, S. M. Phytother. Res. 2011, 25, 451–457.
52. Cheng, D.; Chen, Z. Synth. Commun. 2002, 32, 2155–2159.
53. Patil, P. C.; Bhalerao, D. S.; Dangate, P. S.; Akamanchi, K. G. Tetrahedron Lett.
2009, 50, 5820–5822.
54. Hartmann, R.; Frotscher, M.; Oberwinkler, S.; Bey, E. PCT Int. Appl. 2009, WO
2009027346 A2 20090305.
55. Bey, E.; Marchais-Oberwinkler, S.; Werth, R.; Negri, M.; Al-Soud, Y. A.;
Kruchten, P.; Oster, A.; Frotscher, M.; Birk, B.; Hartmann, R. W. J. Med. Chem.
2008, 51, 6725–6739.
27. Sun, B.; Hoshino, J.; Jermihov, K.; Marler, L.; Pezzuto, J. M.; Mesecar, A. D.;
Cushman, M. Bioorg. Med. Chem. 2010, 18, 5352–5366.
28. (a) Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.; Rondanin, R.; Piccagli, L.;
Baruchello, R.; Rossi, M.; Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.; Jung, M.
K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Di Cristina, A.; Dusonchet,
L.; Meli, M.; Tolomeo, M. J. Med. Chem. 2005, 48, 723–736; (b) Romagnoli, R.;
Baraldi, P. G.; Cruz-Lopez, O.; Cara, C. L.; Carrion, M. D.; Brancale, A.; Hamel, E.;
Chen, L.; Bortolozzi, R.; Basso, G.; Viola, G. J. Med. Chem. 2010, 53, 4248–4258;
(c) Schobert, R.; Biersack, B.; Dietrich, A.; Effenberger, K.; Knauer, S.; Mueller, T.
J. Med. Chem. 2010, 53, 6595–6602.
56. Maiti, A.; Cuendet, M.; Croy, V. L.; Endringer, D. C.; Pezzuto, J. M.; Cushman, M.
J. Med. Chem. 2007, 50, 2799–2806.
57. Lee, S. K.; Mbwambo, Z. H.; Chung, H.; Luyengi, L.; Gamez, E. J.; Mehta, R. G.;
Kinghorn, A. D.; Pezzuto, J. M. Comb. Chem. High Throughput Screen 1998, 1, 35–
46.
58. You, M.; Wickramaratne, D. B.; Silva, G. L.; Chai, H.; Chagwedera, T. E.;
Farnsworth, N. R.; Cordell, G. A.; Kinghorn, A. D.; Pezzuto, J. M. J. Nat. Prod.
1995, 58, 598–604.