Optimization of the aromatase inhibitory activities of pyridylthiazole analogues of resveratrol

Article abstract of DOI:10.1016/j.bmc.2012.01.047

Aromatase is an established target not only for breast cancer chemotherapy, but also for breast cancer chemoprevention. The moderate and non-selective aromatase inhibitory activity of resveratrol (1) was improved about 100-fold by replacement of the ethyl

Full text of DOI:10.1016/j.bmc.2012.01.047

Bioorganic & Medicinal Chemistry 20 (2012) 2427–2434
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization of the aromatase inhibitory activities of pyridylthiazole
analogues of resveratrol
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, United States
b College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Aromatase is an established target not only for breast cancer chemotherapy, but also for breast cancer
chemoprevention. The moderate and non-selective aromatase inhibitory activity of resveratrol (1) was
improved about 100-fold by replacement of the ethylenic bridge with a thiadiazole and the phenyl rings
with pyridines (e.g., compound 3). The aromatase inhibitory activity was enhanced over 6000-fold by
using a 1,3-thiazole as the central ring and modifying the substituents on the ‘A’ ring to target the
Met374 residue of aromatase. On the other hand, targeting the hydroxyl group of Thr310 by a hydro-
gen-bond acceptor on the ‘B’ ring did not improve the aromatase inhibitory activity.
Received 2 October 2011
Revised 18 January 2012
Accepted 26 January 2012
Available online 11 February 2012
Keywords:
Aromatase
Inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Resveratrol
Chemotherapy
Chemoprevention
1. Introduction
anti-estrogen therapeutic modality involves the use of aromatase
inhibitors, including both steroidal6 and non-steroidal derivatives.7
Breast cancer is the most common cancer in the world. It is still
the most frequent cancer among females and it is the second lead-
ing cause of death from cancer in women.1 There were 192,370
cases of invasive breast cancer reported in the United States in
2009.2 The vast majority of both pre- and postmenopausal breast
cancers are classified as estrogen-dependent.3
Since the mid-1990s, a third generation of non-steroidal aromatase
inhibitors has became available that has shown therapeutic supe-
riority over steroidal derivatives.8 There are different classes of
non-steroidal aromatase inhibitors, including benzoflavanones,9
azoles and azines,10 and stilbene derivatives, which display both
anticancer11 and cancer chemopreventive effects.12
Normally estrogens control the development and maintenance
of the female sex organs, secondary sex characteristics, mammary
glands, and certain functions of the uterus and its accessory organs.
The pathological effect of the estrogens, as in the case of estrogen-
dependent breast cancer, occurs when the tumor cells express ex-
cess receptors for endogenous estrogens. The binding of estrogen
to its receptor activates transcription of its target genes, which
are responsible for cancer cell proliferation.4 Therefore, clinical
treatment focuses on decreasing the amount of estrogens either
by oophorectomy5 or blocking the pathological effect of endoge-
nous estrogens by using anti-estrogen chemotherapy. One
Resveratrol (1) is a natural stilbene derivative that occurs in
various edible plants such as grapes and nuts.13 It has several ther-
apeutic effects including improving postischemic ventricular per-
formance14 and cancer15 chemopereventive activities. However,
resveratrol exerts its chemopreventive effect via modulating many
biological pathways that are potentially capable of inhibiting carci-
nogenesis, and it has relatively low potency in each. In addition, it
is metabolized rapidly into inactive metabolites.16 For these rea-
sons, there is a need to investigate other resveratrol derivatives
that might show greater efficacy and selectivity.
This report describes recent efforts to develop more potent and
selective resveratrol cancer chemopreventive analogues. The res-
veratrol trans stilbene double bond was previously replaced with
a thiadiazole ring.17 This strategy afforded the lead compound 2
that had some chemopreventive activity against aromatase and
Abbreviations: IC50, sample concentration which causes 50% inhibition; iNOS,
inducible nitric oxide synthase; NF-jB, nuclear factor kappa-light-chain-enhancer
of activated B cells; QR1, NAD(P)H:quinone reductase.
NF-jB, and a good induction ratio with NAD(P)H:quinone reduc-
⇑
Corresponding author.
E-mail address: cushman@purdue.edu (M. Cushman).
tase (QR1).17 Further chemical optimization of the lead compound
2 furnished 3,5-dipyridyl-1,2,4-thiadiazoles (e.g., compound 3) as a
On leave from Al-Azhar University, College of Pharmacy, Cairo, Egypt.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.047

2428
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
new class of non-steroidal aromatase inhibitors.17 More attention
has recently been given to improve the aromatase inhibitory
activity of this newly discovered class of aromatase inhibitors since
aromatase is an established target in both breast cancer chemo-
therapy18 and chemoprevention.16
group or a halogen. The para position of the ‘B’ ring is calculated to
be 3.9 Å away from the hydroxyl group of Thr310 (Fig. 1), so instil-
lation of hydrogen-bond acceptors, such as a methoxy group or
halogens, might increase potency. In the same molecular model
(Fig. 1), the ortho position of the ‘A’ ring is close to a hydrophobic
Lead Compound
OH
A
N
S
S
N
N
N
N
HO
Resveratrol (1)
B
Aromatase, IC50 = 25 μM
NF-κB, IC50 = 0.98 μM
QR1, IR = 2.4
N
2
3
OH
Aromatase, IC50 > 50 μM
NF-κB, IC50 = 47.4 μM
QR1, IR = 8.6
Aromatase, IC50 = 0.24 μM
DPPH, IC50 = 251 μM
iNOS, IC50 = 23.2 μM
MCF-7, IC50 = 87.6 μM
promiscuous molecule
active against 3 targets
highly aromatase selective
The two peripheral rings are arbitrarily denoted “A” and “B” in structure 3.
All of the target compounds were tested for their aromatase
region (Leu477). Therefore, a small hydrophobic group is antici-
pated to improve the hydrophobic interactions. All of the sug-
gested modifications are summarized in Figure 2.
inhibitory activity. Resveratrol (1) and three clinically used non-
steroidal aromatase inhibitors were also tested as positive controls.
In addition, to test the chemopreventive selectivity of the com-
pounds, they were also evaluated against other enzymes that are
involved in chemopreventive pathways, such as NF-jB, inducible
nitric oxide synthase (iNOS), and QR1 (see Supplementary data).
Although there are abundant methods reported for synthesis of
3,5-disubstituted-1,2,4-thiadiazoles with identical substituents,21
there are limited ones for those with non-identical substituents.22
To prepare 3,5-disubstituted-1,2,4-thiadiazoles 6a and b, Howe’s
method22 was followed and oxathiazolone 5, prepared from nico-
tinamide (4) and (chlorocarbonyl)sulfenyl chloride, was allowed
to react with 3-bromobenzonitrile and its 3-methoxy analogue in
decalin at 200 °C (Scheme 1).
2. Results and discussion
2.1. Chemistry
Appropriate thioamides 8a–f were allowed to react with
bromoacetylpyridine hydrobromide 7 in dry DMF to afford the de-
sired thiazoles 9a–f (Scheme 2). The hydroxyl derivative 9g was
obtained from its corresponding methoxy analogue 9e by treat-
ment with hydrogen bromide in refluxing glacial acetic acid.
Compound 11 was prepared from thioamide 10 and bromoace-
tylpyridine 7 as shown in Scheme 3. The methoxy derivative 12
was obtained by allowing compound 11 to react with 10 equiv so-
dium methoxide and absolute methanol in a sealed tube at 120 °C.
Heating 3,4-dimethylpyridine (13) with selenium dioxide in
dioxane afforded the corresponding aldehyde 14 as outlined in
Scheme 4. The aldehyde was converted directly into its corre-
sponding amide using the Chill and Mebane method.23 The amide
To further enhance the aromatase inhibitory activity of com-
pound 3, a structure-based design strategy was adopted and com-
pound 3 was docked into the active site of aromatase (PDB ID
3eqm)19 using GOLD software.20 The hypothetical model of the
binding of compound 3 with aromatase is represented in Figure
1, which shows the possible interaction between the pyridine
nitrogen of the ‘A’ ring and the heme iron, while the other nitrogen
is calculated to hydrogen bond with the NH of Met374. The model
suggests several strategies to increase the aromatase inhibitory po-
tency. First, the nitrogen atom of the ‘A’ ring of compound 3 is near
to the NH group of Met374, suggesting replacement of the nitrogen
of the ligand by other hydrogen-bond acceptors, such as a methoxy
Figure 1. Hypothetical interaction between compound 3 and Met374 and heme in the human aromatase active site (PDB ID 3eqm). The stereoview is programmed for wall-
eyed viewing.

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
2429
N
S
Adding a H-bond
acceptor
Cl
S
N
NH2
a
N
S
O
Cl
N
NH
N
10
N
Met374
Replacement with
another H-bond
acceptor
11
HN
O
N
N
HO
MeO
Leu477
S
N
The310
Fe
Heme
O
b
N
Adding a small
hydrophobic group
Figure 2. Design of novel analogues to compound 3 based on its docking results.
12
Scheme 3. Reagents and conditions: (a) 2-bromoacetyl-3-pyridine hydrobromide
(7), DMF, Cs2CO3, 100 °C, 8 h, 67%; (b) NaOMe, MeOH, 120 °C, 24 h, 98%.
O
O
O
S
NH2
a
b
N
N
N
N
5
S
N
CHO
N
4
N
a
b
N
R
N
N
14
13
15
N
S
Scheme 4. Reagents and conditions: (a) SeO2, dioxane, 100 °C, 12 h, 80%; (b) (i)
NH2OHÁHCl, DMSO, 100 °C, 30 min; (ii) NaOH, H2O2, 100 °C, 7 min, 70%; (iii)
Lawesson’s reagent, THF, 50 °C, 3 h, 54%; (iv) 2-bromoacetyl-3-pyridine hydrobro-
mide (7), DMF, Cs2CO3, 100 °C, 8 h, 56%.
a; R = Br
b; R = OCH3
6a,b
Scheme 1. Reagents and conditions: (a) (chlorocarbonyl)sulfenyl chloride, toluene,
heat to reflux, 24 h, 31%; (b) 3-bromobenzonitrile or 3-methoxybenzonitrile,
decalin, 200 °C, 20 min, 8–13%.
O
O
a
O
OH
S
Br
Br
N
Br
N
NH2
a
+
17
16
Y
N
HBr
X
O
Br
7
8a-f
b
Br
N
HO
X
18
c
Y
S
S
b
N
N
N
9e
N
S
S
N
N
N
N
9a-f
N
9g
a; X = H, Y = C
b; X = F, Y = C
c; X = Cl, Y = C
d; X = Br, Y = C
N
R
R
19
20
e; X = OCH3, Y = C
f; XY = N
20; R = Br
19; R = Br
d
d
23; R = OMe
21; R = OMe
22; R = OEt
Scheme 2. Reagents and conditions: (a) DMF, Cs2CO3, heat to reflux, 3–6 h, 70–80%;
(b) (i) HBr, acetic acid, heat to reflux; (ii) K2CO3, 24 h, 44%.
d
Scheme 5. Reagents and conditions: (a) MeMgBr (2 equiv), dry THF, 23 °C, 10 min,
100%; (b) bromine, CCl4, heat to reflux, 3 h, 5%; (c) thionicotinamide or thioisonic-
otinamide, DMF, Cs2CO3, heat to 120 °C, 6 h; 55–57%; (d) NaOMe, MeOH or EtOH,
120 °C 46–58%.
was treated with Lawesson’s reagent in dry THF to yield a yellow
solid identified as 3-methylpyridine-4-carbothioamide, which
was treated with 2-bromoacetyl-3-pyridine hydrobromide (7) as
described in Scheme 2 to afford the desired thiazole 15 (Scheme 4).
3-Acetylpyridine 17 was obtained from the commercially avail-
able pyridine-3-carboxylate 16 using 2 equiv methyl magnesium
bromide (Scheme 5). Compound 17 was heated in CCl4 with bro-
mine to afford the corresponding monobromoacetyl derivative 18
in low yield. Changing the solvent from CCl4 to glacial acetic acid
or acetonitrile or the brominating agent from elemental bromine

2430
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
to NBS did not improve the yield. Compound 18 was allowed to re-
act with the suitable pyridine thioamides to afford 6-bromopyr-
idylthiazoles 19 and 20 (Scheme 5). The desired methoxypyridine
derivatives 21 and 23 were obtained by heating their correspond-
ing bromo analogues 19 and 20 with 5 equiv sodium methoxide
and absolute methanol in a sealed tube. Using absolute ethanol
as solvent under the same experimental conditions afforded the
ethoxy derivative 22 as the only isolable product (Scheme 5).
2.2. Biological results
First, thiadiazole derivatives 6a and 6b were prepared and both
showed weaker aromatase inhibitory activity than compound 3
(Table 1). This observed decrease in the anti-aromatase activity
might be due to the size congestion. The targeted Met374 NH
group appears to be closer to the ‘A’ ring meta position, but it is also
close enough to the para position for that to be considered. There-
fore, the attachment of hydrogen-bond acceptors to the ‘A’ ring
para position was considered. Originally, it was decided to perform
the aforementioned chemical modifications using a thiadiazole
central ring, but after the experience of preparation of compounds
6a and 6b, it was clear that synthesis of thiadiazoles with two non-
identical substituents in the required purity and enough quantities
would be difficult. For these reasons and since the thiadiazole
nitrogen at the 2-position apparently has no role in the interaction
with active site residues as shown in Figure 1, the thiadiazole
central ring was replaced with a thiazole ring and all subsequent
Figure 3. Dixon plot illustrating the inhibition of aromatase by compound 9f.
selectivity. Compound 9f had moderate NF-jB inhibitory activity
with an IC50 of 2.5 M.
l
Additional compounds were made and tested in an effort to fur-
ther probe the structure–activity-relationships. Hydrogen-bond
acceptor moieties were added to the ‘A’ ring meta position and
compounds 11 and 12 were prepared. Both compounds displayed
aromatase IC50 values (164 and 23 nM, respectively) that were bet-
ter than the lead compound in this study, that is, compound 2, as
shown in Table 1. Compound 12 had a higher aromatase IC50 value
(23 nM) than compound 9f, but it had complete aromatase target
modifications were performed using
scaffold.
a 2,4-diaryl-1,3-thiazole
Using this new framework provided higher accessibility and a
more feasible chemical pathway. Thiazole derivatives 9a–g with
different ‘A’ ring para substitutions were prepared and tested for
their aromatase inhibitory activity. Among this set of compounds,
the 4-pyridyl derivative 9f showed significantly greater inhibitory
activity with an IC50 value of 4 nM (Table 1) and a Ki value of 60 pM
(Table 1, Fig. 3). These values are comparable with those of the
clinically available non-steroidal aromatase inhibitors letrozole,
anastrozole and fadrozole (Table 1), suggesting this compound is
a good drug candidate. As mentioned earlier, all compounds were
subjected to a range of chemopreventive assays to test their target
selectivity, being inactive vs. quinone reductase 1, NF-jB, and ni-
tric oxide synthase (see Supplementary data). Figure 4 represents
the hypothetical binding mode of compound 12 within the aroma-
tase active site. The nitrogen atom of the ‘B’ ring is calculated to be
very close to the heme iron, similar to what is observed in case of
the lead compound 3 (Fig. 1). In contrast, the nitrogen atom of the
‘A’ ring was calculated to be displaced 2.7 Å away from the Met374
NH group. Instead, the methoxy group of the ‘A’ ring was hypothet-
ically docked 3.3 Å close to the Met374 NH group, which suggest-
ing a possibility of a hydrogen bond between the Met374 NH and
the oxygen atom of compound 12 methoxy group. This observation
might explain why compound 12 has weaker aromatase inhibitory
activity than 9f.
Table 1
Inhibition of human aromatase by thiadiazoles and their thiazole analoguesa
The last chemical modification on the ‘A’ ring resulted in com-
pound 15. In the molecular model (Fig. 1), the ortho position of
the ‘A’ ring is close to a hydrophobic region (Leu477). Therefore,
a small hydrophobic group was added to the structure of com-
pound 9f and that furnished its o-methyl analogue 15. Although
compound 15 had an aromatase IC50 value in the nanomolar range
(IC50 78 nM, Table 1), it was higher than that of 9f. The observed
decrease in the aromatase inhibitory potency might be because
introduction of an o-methyl in the ‘A’ ring pushes the molecule clo-
ser to Arg115 (Fig. 5); as a result, the nitrogen atom of the ‘B’ ring
shifted a little farther away from the heme iron.
Next, chemical modifications of the ‘B’ ring were considered. A
hydrogen-bond acceptor moiety was designed to be added to the
‘B’ ring in the para position in order to target the Thr310 hydroxy
group (Fig. 1). Since in the ‘A’ ring, nitrogen atoms at position-3
and position-4 have biological significance, both were taken into
consideration and the ‘B’ ring chemical modifications were per-
formed using both systems. Therefore, p-bromo and their alkoxy
derivatives 19–23 were prepared. This set of compounds showed
higher aromatase maximum percent inhibition than 9f when mea-
a
Compound
IC50
(lM)
Kib (nM)
3
6a
6b
9a
9b
9c
9d
9e
9f
9g
11
12
15
19
20
21
0.24 0.08
6.73 0.21
14.9 0.33
0.9 0.04
17.58 0.47
2.39 0.26
7.92 0.15
18 0.38
0.004 0.0005
11.21 0.27
0.164 0.06
0.023 0.004
0.078 0.007
2.68 0.36
7.41 0.42
1.04 0.19
1.36 0.24
0.59 0.11
25 0.52
11.43 0.91
1160 2.6
5321 22
63.38 5.3
12560 34
234.3 8.6
1722 5.4
15000 48
0.06 0.04
2548 15
8.20 0.73
0.65 0.07
3.17 0.22
285.1 4.5
1482 9.2
82.54 7.1
100.0 2.8
35.12 1.6
41670 27
0.02 0.01
0.13 0.04
0.05 0.008
22
23
Resveratrol (1)
Letrozole
Anastrozole
Fadrozole
0.002 0.0004
0.008 0.0007
0.003 0.001
a,b
sured at 20
lM, but the IC50 values ranged between 0.59 and
IC50
(lM) and Ki (nM) values are summarized for each of the tested compounds.
7.41 M (Table 1).
l
In each case, competitive inhibition was observed.

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
2431
Figure 4. Hypothetical interaction between compound 12 and Met374 and heme in the human aromatase active site (PDB ID 3eqm). The stereoview is programmed for wall-
eyed viewing.
Figure 5. Overlay of the top calculated binding poses of compounds 3 and 15 in the human aromatase active site (PDB ID 3eqm). C-Backbone of compound 3 was colored
pink, and compound 15 was colored gray. The stereoview is programmed for wall-eyed viewing.
HPLC analyses were performed on a Waters binary HPLC system
(Model 1525, 20 L injection loop) equipped with a Waters dual
wavelength absorbance UV detector (Model 2487) set for
3. Conclusion
l
Use of a 2,4-diaryl-1,3-thiazole scaffold provided more flexible
synthetic pathways to build novel aromatase inhibitors with IC50
values in the nanomolar range. Using the new scaffold and opti-
mizing the position of the hydrogen-bond acceptor on the ‘A’ ring
of the lead compound 2 afforded compound 9f, which had an aro-
matase IC50 value of 4 nM. In addition to the potent aromatase
254 nm, using a 5 lM C-18 reverse phase column. Compounds
9f,24 10,25 14,26 16,27 and 1728 are reported.
4.2. 5-(Pyridin-3-yl)-1,3,4-oxathiazol-2-one (5)
Nicotinamide (4, 490 mg, 4.0 mmol) was added to (chlorocar-
bonyl)sulfenyl chloride (780 mg, 6.39 mmol) in toluene (30 mL).
The reaction mixture was heated at reflux for 24 h. The solution
was allowed to cool and solvent was evaporated under reduced
pressure. The brown solid was collected and crystallized from ethyl
acetate to provide the required products as a yellowish solid
(223 mg, 30.9%): mp 117–118 °C. 1H NMR (CDCl3) d 9.09 (d,
J = 1.8 Hz, 1H), 8.71 (dd, J = 1.5, 4.8 Hz, 1H), 8.16 (dt, J = 1.8,
7.8 Hz, 1H), 7.39 (dd, J = 4.5, 7.8 Hz, 1H); 13C NMR (CDCl3) d
172.89, 155.29, 153.02, 148.40, 134.44, 123.65, 122.02; CIMS m/z
(rel intensity) 181 (MH+, 26), 106 (100); HRMS (EI), m/z
179.9991 M+, calcd for C7H4N2O2S 179.9994.
inhibitory activity of compound 9f, moderate NF-jB inhibitory
activity was also observed. The methoxy derivative 12 provided
complete aromatase inhibitory selectivity with potency in the
low nanomolar range (IC50 23 nM).
4. Experimental section
4.1. General
1H NMR spectra were recorded at 300 MHz and 13C NMR spec-
tra were acquired 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. High
resolution mass spectra for all ionization techniques were obtained
from a FinniganMAT XL95. Melting points were determined using
capillary tubes with a Mel-Temp apparatus and are uncorrected.
4.3. Preparation of thiadiazoles 6a,b
Oxathiazolone 5 (90 mg, 0.5 mmol) was added in portions, over
a 10 min time period, to a stirred solution of 3-bromobenzonitrile

2432
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
or 3-methoxybenzonitrile (5.0 mmol) in decalin (5 mL) at 200 °C.
The reaction mixture was stirred for an additional 10–15 min,
and then cooled to room temperature. The products were sepa-
rated and purified by silica gel flash chromatography, using hex-
ane–ethyl acetate (4:1).
129.12 (2C), 127.69 (2C), 123.58, 113.97; ESIMS m/z (rel intensity)
331 (MH+, 100); HRMS (ESI), m/z 273.0253 MH+, calcd for
C14H10ClN2S 273.0248; HPLC purity (C-18 reverse phase column):
98.19% (methanol–H2O, 95:5).
4.4.4. 2-(4-Bromophenyl)-4-(pyridin-3-yl)thiazole (9d)
Yellowish-brown solid (125 mg, 79.5%): mp 100 °C. 1H NMR
(CDCl3) d 9.13 (s, 1H), 8.55 (d, J = 4.6 Hz, 1H), 8.17 (d, J = 7.8 Hz,
1H), 7.89 (d, J = 8.7 Hz, 2H), 7.51 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H),
7.29 (m, 1H); 13C NMR (CDCl3) d 167.07, 153.17, 149.15, 147.67,
133.55, 132.13, 132.05, 129.94 (2C), 127.88 (2C), 124.51, 123.53,
113.99; ESIMS m/z (rel intensity) 319/317 (MH+, 96/100);
HRMS (ESI), m/z 316.9752 MH+, calcd for C14H10BrN2S 316.9748;
HPLC purity (C-18 reverse phase column): 95.27% (methanol–
H2O, 95:5).
4.3.1. 5-(3-Bromophenyl)-3-(pyridin-3-yl)-1,2,4-thiadiazole (6a)
White solid (20.5 mg, 12.8%): mp 120 °C. 1H NMR (CDCl3) d 9.59
(s, 1H), 8.72 (d, J = 3.3 Hz, 1H), 8.62 (dt, J = 1.8, 7.5 Hz, 1H), 9.22 (d,
J = 1.6 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.67 (dd, J = 1.0, 7.8 Hz, 1H),
7.41 (m, 2H); 13C NMR (CDCl3) d 186.99, 171.42, 151.18, 149.67,
135.43, 134.97, 132.05, 130.83, 130.17, 128.43, 126.10, 123.53,
123.41; CIMS m/z (rel intensity) 320/318 (MH+, 25/25), 105
(100); HRMS (EI), m/z 316.9625 M+, calcd for C13H8BrN3S
316.9622; HPLC purity (C-18 reverse phase column): 95.97%
(methanol–H2O, 95:5).
4.4.5. 2-(4-Methoxyphenyl)-4-(pyridin-3-yl)thiazole (9e)
White solid (95 mg, 71%): mp 104 °C. 1H NMR (CDCl3) d 9.19 (s,
1H), 8.57 (d, J = 3.9 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.95 (d,
J = 8.7 Hz, 2H), 7.48 (s, 1H), 7.35 (dd, J = 4.4, 7.5 Hz, 1H), 6.97 (d,
J = 8.7 Hz, 2H), 3.85 (s, 3H); 13C NMR (CDCl3) d 168.41, 161.31,
152.80, 148.97, 147.73, 133.61, 130.35, 128.07 (2C), 126.33 (2C),
123.52, 114.26, 112.87, 55.39; ESIMS m/z (rel intensity) 269
(MH+, 100); HRMS (ESI), m/z 269.0747 MH+, calcd for C15H13N2OS
269.0743; HPLC purity (C-18 reverse phase column): 95.05%
(methanol–H2O, 95:5).
4.3.2. 5-(3-Methoxyphenyl)-3-(pyridin-3-yl)-1,2,4-thiadiazole
(6b)
White solid (11 mg, 8.1%): mp 122–123 °C. 1H NMR (CDCl3) d
9.63 (s, 1H), 8.73 (s, 1H), 8.65 (dd, J = 2.0, 7.8 Hz, 1H), 8.16 (dd,
J = 3.5, 7.8 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.46–7.44 (m, 2H),
6.98 (dd, J = 3.0, 8.1 Hz, 1H), 3.93 (s, 3H); 13C NMR (CDCl3) d
185.32, 168.94, 159.02, 150.94, 149.62, 135.37, 134.72, 132.11,
128.41, 123.57, 119.40, 115.24, 114.08, 55.74; CIMS m/z (rel inten-
sity) 270 (MH+, 40), 136 (100); HRMS (CI), m/z 270.0698 MH+, calcd
for C14H12N3OS 270.0696; HPLC purity (C-18 reverse phase col-
umn): 95.02% (methanol–H2O, 95:5).
4.4.6. 4-[4-(Pyridin-3-yl)thiazol-2-yl]phenol (9g)
Aqueous HBr (48%, 3 mL) was added to compound 9e (50 mg,
0.2 mmol) in acetic acid (7 mL). The reaction mixture was heated
at reflux for 24 h. After the reaction mixture was cooled down to
room temperature, it was neutralized with K2CO3 powder until
pH 7. The solid was collected by filtration and purified by silica
gel flash chromatography, using dichloromethane–methanol
(95:5), to afford the product as a white solid (22 mg, 44%): mp
4.4. Preparation of thiazole derivatives 9a–e
Thioamides 8a–f (0.5 mmol), 3-(bromoacetyl)pyridine hydro-
bromide (7, 140 mg, 0.50 mmol), and cesium carbonate (165 mg,
0.51 mmol) were added to dry DMF (10 mL). The reaction mixture
was heated to reflux for 3–6 h and then allowed to cool, quenched
with water (20 mL), and filtered. The solid residues were purified
by silica gel flash chromatography, using hexane–ethyl acetate
(4:1).
271–273 °C. IR (KBr) 3434, 3045, 1608, 1586, 1472 cmÀ1 1H
;
NMR (DMSO-d6) d 9.23 (d, J = 1.5 Hz, 1H), 8.55 (d, J = 4.8 Hz, 1H),
8.35 (d, J = 7.8 Hz, 1H), 8.20 (s, 1H), 7.85 (d, J = 8.7 Hz, 2H), 7.48
(dd, J = 4.8, 7.8 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 3.8 (brs, 1H); 13C
NMR (DMSO-d6) d 168.95, 160.91, 152.74, 149.84, 148.19, 134.19,
130.78, 128.96 (2C), 124.80 (2C), 116.93, 115.46; ESIMS m/z (rel
intensity) 255 (MH+, 100); HRMS (ESI), m/z 255.0594 MH+, calcd
for C14H11N2OS 255.0592; HPLC purity (C-18 reverse phase col-
umn): 96.90% (methanol–H2O, 95:5).
4.4.1. 2-Phenyl-4-(pyridin-3-yl)thiazole (9a)
Off-white solid (83 mg, 70%): mp 82–83 °C. 1H NMR (CDCl3) d
9.18 (s, 1H), 8.56 (d, J = 3.6 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 8.01
(m, 2H), 7.51 (s, 1H), 7.44–7.26 (m, 3H), 7.33 (m, 1H); 13C NMR
(CDCl3) d 168.54, 152.99 (2C), 148.94, 147.62, 133.69, 133.29,
130.29, 128.96 (2C), 126.56 (2C), 123.60, 113.82; ESIMS m/z (rel
intensity) 239 (MH+, 100); HRMS (ESI), m/z 239.0641 MH+, calcd
for C14H11N2S 239.0637; HPLC purity (C-18 reverse phase column):
97.54% (methanol–H2O, 95:5).
4.5. 2-(2-Chloropyridin-4-yl)-4-(pyridin-3-yl)thiazole (11)
Thioamide 10 (60.0 mg, 0.35 mmol), 3-(bromoacetyl)pyridine
hydrobromide (7, 98 mg, 0.35 mmol), and cesium carbonate
(170 mg, 0.52 mmol) were added to dry DMF (10 mL). The reaction
mixture was heated at 100 °C for 8 h and then allowed to cool and
then quenched with distilled water (30 mL). The organic materials
were extracted with ethyl acetate (30 mL). The organic layer was
isolated and dried over anhydrous Na2SO4. Solvent was evaporated
under reduced pressure. The solid residue was purified by silica gel
flash chromatography, using ethyl acetate–hexane (1:1), and then
dichloromethane–methanol (95:5) to yield a yellowish-white solid
(64 mg, 67%): mp 182–183 °C. 1H NMR (DMSO-d6) d 9.28 (s, 1H),
8.56 (m, 3H), 8.42 (d, J = 7.2 Hz, 1H), 8.10 (s, 1H), 8.01 (d,
J = 7.0 Hz, 1H), 7.52 (dd, J = 3.9, 7.2 Hz, 1H); 13C NMR (DMSO-d6)
d 164.22, 154.03, 152.41, 152.06, 150.36, 148.35, 143.52, 134.50,
130.11, 124.88, 121.19, 120.72, 120.06; ESIMS (m/z, rel intensity)
274/276 (MH+, 100/24); HRMS (ESI), m/z MH+ 274.0201, calcd for
4.4.2. 2-(4-Fluorophenyl)-4-(pyridin-3-yl)thiazole (9b)
Yellowish-white solid (97 mg, 76%): mp 141–142 °C. 1H NMR
(CDCl3) d 9.18 (s, 1H), 8.56 (d, J = 4.5 Hz, 1H), 8.24 (d, J = 7.5 Hz,
1H), 7.99 (dd, JHH = 4.8 Hz, JHF = 7.8 Hz, 2H), 7.52 (s, 1H), 7.35 (dt,
J = 4.5, 7.8 Hz, 1H), 7.13 (dd, JHH = 4.5, JHF = 7.8 Hz, 2H); 13C NMR
(CDCl3) d 167.29, 165.61, 162.29, 153.10, 149.15, 147.72, 133.59,
130.09 (d, JCF = 32.2 Hz, 1C), 128.43 (d, JCF = 7.5 Hz, 2C), 123.53,
116.17 (d, JCF = 22.0 Hz, 2C), 113.67; ESIMS m/z (rel intensity) 257
(MH+, 100); HRMS (ESI), m/z 257.0545 MH+, calcd for C14H10FN2S
257.0543; HPLC purity (C-18 reverse phase column): 98.89%
(methanol–H2O, 95:5).
4.4.3. 2-(4-Chlorophenyl)-4-(pyridin-3-yl)thiazole (9c)
Yellowish-brown solid (102 mg, 75.0%): mp 141–142 °C. 1H
NMR (CDCl3) d 9.15 (s, 1H), 8.56 (s, 1H), 8.19 (d, J = 7.8 Hz, 1H),
7.87 (d, J = 8.7 Hz, 2H), 7.49 (s, 1H), 7.32 (m, 3H); 13C NMR (CDCl3)
d 167.06, 153.17, 149.11, 147.64, 136.16, 133.58, 131.73, 130.02,
C13H8ClN3S 274.0206; HPLC purity (C-18 reverse phase column):
98.93% (methanol–H2O, 95:5).

A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
2433
4.6. 2-(2-Methoxypyridin-4-yl)-4-(pyridin-3-yl)thiazole (12)
ethanone (18, 140 mg, 0.50 mmol), and cesium carbonate
(175 mg, 0.52 mmol) were added to dry DMF (10 mL). The reaction
mixture was heated at 120 °C for 6 h then allowed to cool and was
quenched with distilled water (30 mL). The organic materials were
extracted in a separatory funnel using ethyl acetate (30 mL). The
organic layer was isolated and dried over anhydrous Na2SO4. Sol-
vent was evaporated under reduced pressure. The solid residue
was purified by silica gel flash chromatography, using a gradient
of dichloromethane–methanol concentrations (95:5 then 9:1), to
yield the desired compounds.
Compound 11 (70.0 mg, 0.25 mmol) and sodium methoxide
(140 mg, 2.59 mmol) were added to absolute methanol (3 mL) in a
sealed tube. The reaction mixture was heated at 120 °C for 24 h
and then allowed to cool. The solvent was evaporated under reduced
pressure and the solid residue was partitioned between ethyl ace-
tate (10 mL) and water (10 mL). The organic layer was isolated and
dried over anhydrous Na2SO4. Solvent was evaporated under re-
duced pressure and the solid residue was purified by silica gel flash
chromatography, using dichloromethane–methanol (95:5), to yield
white solid (66 mg, 98%): mp 115–116 °C. 1H NMR (CDCl3) d 9.16 (s,
1H), 8.58 (s, 1H), 8.23 (d, J = 7.4 Hz, 2H), 7.63 (s, 1H), 7.44 (dd, J = 1.2,
4.8.1. 4-(6-Bromopyridin-3-yl)-2-(pyridin-4-yl)thiazole (19)
Yellowish-brown solid (89 mg, 55%): mp >300 °C. 1H NMR
(CDCl3) d 8.95 (d, J = 2.1 Hz, 1H), 8.75 (d, J = 8.0 Hz, 2H), 8.16 (dd,
J = 2.4, 7.4 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.71 (s, 1H), 7.59 (dd,
J = 0.6, 7.4 Hz, 1H); 13C NMR (CDCl3) d 163.44, 153.49, 149.06
(2C), 146.02, 145.63, 142.17, 137.79, 129.62, 129.30, 123.57,
123.18 (2C); ESIMS (m/z, rel intensity) 320/318 (MH+, 100/88);
HRMS (ESI), m/z MH+ 317.9708, calcd for C13H9BrN3S 317.9701;
HPLC purity (C-18 reverse phase column): 97.22% (methanol–
H2O, 95:5).
7.4 Hz, 1H), 7.34 (dd, J = 4.5, 7.5 Hz, 1H), 7.30 (s, 1H), 3.97 (s, 3H); 13
C
NMR (CDCl3) d 165.63, 164.93, 153.67, 149.37, 147.82, 147.70,
142.53, 133.67, 129.78, 123.60, 115.16, 113.87, 107.64, 53.72; ESIMS
(m/z, rel intensity) 270 (MH+, 100); HRMS (ESI), m/z MH+ 270.0704,
calcd for C14H12N3OS 270.0701; HPLC purity (C-18 reverse phase
column): 95.16% (methanol–H2O, 95:5).
4.7. 2-(3-Methylpyridin-4-yl)-4-(pyridin-3-yl)thiazole (15)
The aldehyde 14 (800 mg, 6.61 mmol) was added to a solution
of hydroxylamine hydrochloride (725 mg, 10.5 mmol) in DMSO
(15 mL), and the resulting reaction mixture was stirred and heated
for 30 min at 100 °C. The heat was then removed. Sodium hydrox-
ide (600 mg) was dissolved in distilled water (5 mL) and the result-
ing solution was slowly added to the reaction mixture over a 2-min
period with stirring, followed by the slow and careful addition of
H2O2 (50%, 4 mL) over a 5-min period. The reaction mixture was
further stirred for 5 min, quenched with distilled water (30 mL),
and then extracted with ethyl acetate (50 mL). The organic layer
was separated, dried over anhydrous Na2SO4, and evaporated un-
der reduced pressure to afford a white solid (495 mg, 70.4%). The
solid (135 mg, 1.00 mmol) and Lawesson’s reagent (490 mg,
1.20 mmol) were added to dry THF (15 mL). The reaction mixture
was stirred at 50 °C for 3 h. The solvent was evaporated under re-
duced pressure and the residue was partitioned between aqueous
NaHCO3 (25 mL) and ethyl acetate (25 mL). The organic solvent
was separated and dried over anhydrous Na2SO4. The crude prod-
uct was purified by silica gel flash chromatography, using dichloro-
methane–methanol (9:1), to yield the corresponding thioamide as
a yellow solid (82 mg, 54%). The solid (76 mg, 0.5 mmol), 3-
(bromoacetyl)pyridine hydrobromide (7, 140 mg, 0.50 mmol),
and cesium carbonate (165 mg, 0.50 mmol) were added to dry
DMF (10 mL). The reaction mixture was heated at 100 °C for 8 h
and then allowed to cool. The reaction mixture was quenched with
distilled water (30 mL). The organic materials were extracted using
ethyl acetate (30 mL). The organic layer was isolated and dried
over anhydrous Na2SO4. The solvent was evaporated under re-
duced pressure. The solid residue was purified by silica gel flash
chromatography, using dichloromethane–methanol (95:5), to yield
a brownish-white solid (70 mg, 56%): mp 104–105 °C. 1H NMR
(CDCl3) d 9.18 (d, J = 1.8 Hz, 1H), 8.57 (m, 2H), 8.52 (d, J = 5.1 Hz,
1H), 8.21 (dt, J = 1.8, 8.1 Hz, 1H), 7.71 (d, J = 4.8 Hz, 1H), 7.70 (s,
1H), 7.34 (dd, J = 5.1, 8.1 Hz, 1H), 2.66 (s, 3H); 13C NMR (CDCl3) d
165.38, 153.25, 152.81, 149.36, 147.83, 147.73, 138.80, 133.58,
130.55, 129.79, 123.62, 122.43, 115.44, 18.88; EIMS (m/z, rel inten-
sity) 253 (M+, 100); HRMS (EI), m/z M+ 253.0678, calcd for
C14H11N3S 253.0674; HPLC purity (C-18 reverse phase column):
96.10% (methanol–H2O, 95:5).
4.8.2. 4-(6-Bromopyridin-3-yl)-2-(pyridin-3-yl)thiazole (20)
Yellowish-brown solid (95 mg, 57%): mp >300 °C. 1H NMR
(CDCl3) d 9.23 (d, J = 1.8 Hz, 1H), 8.95 (d, J = 2.1 Hz, 1H), 8.70 (d,
J = 3.6 Hz, 1H), 8.32 (d, J = 8.1 Hz, 1H), 8.16 (dd, J = 2.4, 8.1 Hz,
1H), 7.65 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 4.8, 7.8 Hz,
1H); 13C NMR (CDCl3) d 165.41, 152.35, 151.20, 147.88, 147.74,
141.53, 136.21, 133.69, 129.24, 129.17, 128.12, 123.78, 114.87;
ESIMS (m/z, rel intensity) 320/318 (MH+, 97/100); HRMS (ESI), m/
z MH+ 317.9705, calcd for C13H9BrN3S 317.9701; HPLC purity (C-
18 reverse phase column): 96.50% (methanol–H2O, 95:5).
4.9. Preparation of 21–23
Bromopyridines 19 or 20 (30 mg, 0.1 mmol) and sodium meth-
oxide (26 mg, 0.5 mmol) were added to absolute methanol (2 mL)
in a sealed tube. In the case of 22, absolute ethanol was used.
The reaction mixture was heated at 120 °C for 8 h and then allowed
to cool. The solvent was evaporated under reduced pressure and
the solid residue was partitioned between ethyl acetate (10 mL)
and water (10 mL). The organic layer was isolated and dried over
anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the solid residue was purified by silica gel flash chro-
matography, using dichloromethane–methanol (95:5), to yield the
products as solids.
4.9.1. 4-(6-Methoxypyridin-3-yl)-2-(pyridin-4-yl)thiazole (21)
Off-white solid (15 mg, 58%): mp 180 °C. 1H NMR (CDCl3) d 8.95
(d, J = 2.4 Hz, 1H), 8.72 (d, J = 7.8 Hz, 2H), 8.15 (d, J = 7.4 Hz, 2H),
7.88 (s, 1H), 7.71 (s, 1H), 7.52 (dd, J = 0.6, 7.4 Hz, 1H), 3.99 (s,
3H); 13C NMR (CDCl3) d 163.77, 150.68 (2C), 147.91, 145.04,
136.87, 136.23, 128.16, 120.27 (2C), 115.85, 113.27, 110.90,
53.63; ESIMS (m/z, rel intensity) 270 (MH+, 100); HRMS (ESI), m/z
MH+ 270.0704, calcd for C14H12N3OS 270.0701; HPLC purity (C-
18 reverse phase column): 99.39% (methanol–H2O, 9:1).
4.9.2. 4-(6-Ethoxypyridin-3-yl)-2-(pyridin-4-yl)thiazole (22)
White solid (12 mg, 46%): mp 125–126 °C. 1H NMR (CDCl3) d
8.74 (m, 3H), 8.14 (d, J = 1.8 Hz, 1H), 8.15 (d, J = 7.8 Hz, 2H), 7.50
(s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.40 (q, J = 6.9 Hz, 2H), 1.41 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3) d 165.18, 163.88, 154.37, 150.67
(2C), 145.08, 140.12, 136.81, 123.44, 120.24 (2C), 113.13, 110.97,
61.97, 14.61; ESIMS (m/z, rel intensity) 284 (MH+, 100); HRMS
(ESI), m/z MH+ 284.0855, calcd for C15H14N3OS 284.0858; HPLC
purity (C-18 reverse phase column): 96.73% (methanol–H2O, 95:5).
4.8. General procedure for thiazoles 19 and 20
The appropriate thioamide (thionicotinamide or thioisonicoti-
namide, 60 mg, 0.5 mmol), 2-bromo-1-(6-bromopyridin-3-yl)

2434
A. S. Mayhoub et al. / Bioorg. Med. Chem. 20 (2012) 2427–2434
4.9.3. 4-(6-Methoxypyridin-3-yl)-2-(pyridin-3-yl)thiazole (23)
White solid (12 mg, 47%): mp 129–130 °C. 1H NMR (CDCl3) d
9.22 (d, J = 2.1 Hz, 1H), 8.77 (d, J = 2.4 Hz, 1H), 8.66 (dd, J = 1.8,
6.0 Hz, 1H), 8.29 (dt, J = 1.8, 6.0 Hz, 1H), 8.15 (dd, J = 2.4, 8.7 Hz,
1H), 7.44 (s, 1H), 7.28 (dd, J = 1.8, 8.7 Hz, 1H), 6.82 (d, J = 8.7 Hz,
1H), 3.98 (s, 3H); 13C NMR (CDCl3) d 164.69, 164.02 (2C), 153.87,
150.88, 147.70, 144.98, 136.83, 133.56, 129.49, 123.70, 112.21,
110.83, 53.61; ESI MS (m/z, rel intensity) 270 (MH+, 100); HRMS
(ESI), m/z MH+ 270.0699, calcd for C14H12N3OS 270.0701; HPLC
purity (C-18 reverse phase column): 98.01% (methanol–H2O, 9:1).
(excitation) and 530 nm (emission) every 10 s for at least 5 min.
Michaelis–Menten and Dixon plots were used to evaluate the
resulting data, and Ki values were calculated. Error limits represent
three independent experiments for each compound.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.047.
References and notes
4.10. Molecular modeling
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Kinetic analyses were performed essentially as described above.
Test compounds were preincubated with 30 lL of NADPH regener-
ating 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 phosphate buffer, pH 7.4)
for 10 min at 37 °C. A range of eight concentrations centered
around the IC50 was tested for each inhibitor. Substrate was added
at three concentrations: 800, 400, and 200 nM. Finally, CYP19
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(1 lM) was added, and fluorescence was measured at 485 nm