Optimization of diarylpentadienones as chemotherapeutics for prostate cancer

Article abstract of DOI:10.1016/j.bmc.2018.08.018

Our earlier studies indicate that (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d]imidazol-2-yl)penta-1,4-diene-3-ones exhibit up to 121-fold greater antiproliferative potency than curcumin in human p

Full text of DOI:10.1016/j.bmc.2018.08.018

Accepted Manuscript
Optimization of diarylpentadienones as chemotherapeutics for prostate cancer
Manee Patanapongpibul, Changde Zhang, Guanglin Chen, Shanchun Guo,
Qiang Zhang, Shilong Zheng, Guangdi Wang, Qiao-Hong Chen
PII:
S0968-0896(18)31130-1
https://doi.org/10.1016/j.bmc.2018.08.018
BMC 14505
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 June 2018
4 August 2018
12 August 2018
Please cite this article as: Patanapongpibul, M., Zhang, C., Chen, G., Guo, S., Zhang, Q., Zheng, S., Wang, G., Chen,
Q-H., Optimization of diarylpentadienones as chemotherapeutics for prostate cancer, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Optimization of diarylpentadienones as chemotherapeutics for prostate cancer
Manee Patanapongpibula, Changde Zhangb,c, Guanglin Chena, Shanchun Guo b,c, Qiang Zhangb,c,
Shilong Zhengb,c, Guangdi Wangb,c, and Qiao-Hong Chena,*
aDepartment of Chemistry, California State University, Fresno, 2555 E. San Ramon Avenue, M/S
SB70, Fresno, CA 93740, USA
bDepartment of Chemistry, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA 70125,
USA
cRCMI Cancer Research Center, Xavier University of Louisiana, 1 Drexel Drive, New Orleans, LA
70125, USA
ABSTRACT: Our earlier studies indicate that (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-
diene-3-ones and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d] imidazol-2-yl)penta-1,4-diene-3-ones exhibit up
to 121-fold greater antiproliferative potency than curcumin in human prostate cancer cell models, but
only 2-10 fold increase in mouse plasma concentrations. The present study aims to further optimize
them as anti-prostate cancer agents with both good potency and bioavailability. (1E,4E)-1,5-Bis(1H-
imidazol-2-yl)penta-1,4-diene-3-one, the potential metabolic product of (1E,4E)-1,5-bis(1-alkyl-1H-
imidazol-2-yl)penta-1,4-diene-3-ones, was synthesized and evaluated for its anti-proliferative activity.
The promising potency of 1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones was completely
abolished by removing the 1-alkyl group, suggesting the critical role of an appropriate group on the N1
position. We then envisioned that N-aryl substitution to exclude the C-H bond on the carbon adjacent to
the N1 position (α-H) may increase the metabolic stability. Consequently, seven (1E,4E)-1,5-bis(1-aryl-
1H-imidazol-2-yl)penta-1,4-dien-3-ones and three (1E,4E)-1,5-bis(1-aryl-1H-benzo[d]imidazol-2-
yl)penta-1,4-dien-3-ones, as well as three (1E,4E)-1,5-bis(1-aryl-1H-pyrrolo[3,2-b]pyridine-2-yl)penta-
1,4-dien-3-ones, were synthesized through a three-step transformation, including N-arylation via
Ullmann condensation, formylation, and Horner-Wadsworth-Emmons reaction. Six optimal (1E,4E)-
1

1,5-bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones exhibit 24- to 375-fold improved potency as
compared with curcumin. Replacement of the imidazole with bulkier benzoimidazole and 4-azaindole
results in a substantial decrease in the potency. (1E,4E)-1,5-Bis(1-(2-methoxyphenyl)-1H-imidazol-2-
yl)penta-1,4-dien-3-one (17d) was established as an optimal compound with both superior potency and
good bioavailability that is sufficient to provide the therapeutic efficacy necessary to suppress in vivo
tumor growth.
Key words: diarylpentadienone, prostate cancer, antiproliferative activity, pharmacokinetic study
____________
* Corresponding author.
E-mail: qchen@csufresno.edu.
2

1. Introduction
Diferuloylmethane (1, Figure 1), commonly known as curcumin, is a naturally occurring
diarylheptanoid from the rhizomes of Curcuma longa L (turmeric) of the Zingiberaceae family.
Turmeric, with curcumin as its major and active chemical component, has long been used as curry
ingredient and as traditional medicines in China and India.1 Curcumin was first revealed by Dorai and
co-workers to have the capability of suppressing human prostate cancer proliferation and inducing
prostate cancer apoptosis.2-4 Additionally, the safety profile of curcumin in humans has been validated
by the Food and Drug Administration (FDA) in the USA.4-5 However, the low bioavailability of
curcumin together with its moderate potency has hindered its clinical advancement.6-7 The reported IC50
values of curcumin in suppressing cell proliferation against three prostate cancer cell lines (LNCaP,
DU145, and PC-3) range from 2.0 µM to 39.6 µM.2 A phase I trial indicates that curcumin
concentration in plasma falls below the detection limit after oral administration of 450-3600 mg of
curcumin daily.7 One way to address these weaknesses is to develop analogs with improved potency
and/or bioavailability.2,8 For example, replacement of the unstable diketone moiety with monokeone
leads to a 10-20 fold increase in in vitro potency.2,8 Our earlier studies have identified (1E,4E)-1,5-
diheteroarylpenta-1,4-diene-3-ones (2-6, Fig. 1) as the most impressive and promising class of
curcumin-based anti-prostate cancer agents due to their superior in vitro potency in prostate cancer cell
models.9-10 The 1-alkyl-1H-imidazol-2-yl group in compounds 2-4 (Fig. 1) and the 1-alkyl-1H-
benzo[d]imidazole-2-yl group in compounds 5-6 (Fig. 1) have been demonstrated to be significantly
beneficial to the in vitro antiproliferative potency in three prostate cancer cell models.10 The most
potent compounds showed up to 121-fold improved anti-proliferative potency toward both androgen-
dependent and androgen-independent prostate cancer cells. However, their bioavailability is only
slightly increased, as evidenced by the 2-10 fold increase in mouse plasma concentration.9-10
The present study aims to further optimize (1E,4E)-1,5-bis(1-alkyl-1H)-imidzaole-2-yl)penta-1,4-
diene-3-ones (e.g. 2-4) and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d]imidazole-2-yl)penta-1,4-diene-3-ones
3

(e.g. 5-6). New groups of 1,5-diheretoarylpenta-1,4-dien-3-ones (7) with the hope of integrating
optimal potency and pharmacokinetic profile were thus designed by incorporating a metabolically
stable group to the N1 position of imidazole and benzoimidazole moiety.
Fig. 1. Structures of curcumin and its mimics
2. Results and Discussion
2.1 Design, Synthesis, and Antiproliferative Activity of (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-
1,4-diene-3-one (14):
To optimize the potency and bioavailability of 1,5-bis(1H-imidazol-2-yl)penta-1,4-dien-3-ones (2-4),
we first evaluated the in vitro anti-proliferative potency of (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-1,4-
diene-3-one (14). Compound 14 was first studied because it may be the metabolic product of 1,5-
bis(1H-imidazol-2-yl)penta-1,4-dien-3-ones through N-dealkylation catalyzed by cytochrome P45011
and it can be used to test the necessity of an 1-alkyl group for the in vitro potency. Consequently, we
4

started our optimization with the synthesis and anti-proliferative evaluation of (1E, 4E)-1,5-bis(1H-
imidazol-2-yl)penta-1,4-diene-3-one (14). As shown in Scheme 1, potential metabolic product (14) of
(1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones (2-4) was synthesized through the
Horner-Wadsworth-Emmons reaction of 1-trityl-1H-imidazole-2-carbaldehyde (9) with 1,3-
bis(diethylphosphonato)acetone (12) followed by removal of the trityl protecting group of 13 in the
presence of hydrochloric acid. 1-Trityl-1H-imidazole-2-carbaldehyde (9) was initially synthesized by
tritylation of 2-imidazolecarboxaldehyde (8) with trityl chloride using triethylamine as base in 20%
yield. Replacement of triethylamine with potassium carbonate did not improve the yield. Alternately,
aldehyde 9 was achieved in 60% yield by formylation of 1-trityl-1H-imidazole (11) that was derived
from tritylation of imidazole (10).
The anti-proliferative activity of (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-1,4-diene-3-one (14) against
androgen-insensitive prostate cancer cell lines (PC-3 and DU145) and androgen-sensitive prostate
cancer cell line (LNCaP) has been evaluated by WST-1 cell proliferation assay following the procedure
as described in the experimental section. The IC50 values for (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-
1,4-diene-3-one (14) towards three prostate cancer cell lines range from 73 µM to 305 µM, which
indicates that (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-1,4-diene-3-one (14) is even less potent than
curcumin (IC50, 14-26 µM) and that removal of the 1-alkyl group diminishes the antiproliferative
activity of (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones (2-4). This suggests that
introduction of an appropriate metabolically stable group to N1 position of imidazole moiety is
indispensable for both potency and bioavailability.
2.2 Design and Synthesis of (1E,4E)-1,5-bis(1-aryl-1H)-heteroaryl-2-yl)penta-1,4-diene-3-ones:
In order to further optimize (1E,4E)-1,5-bis(1-alkyl-1H)-imidzaole-2-yl)penta-1,4-diene-3-ones (2-4)
and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d]imidazole-2-yl)penta-1,4-diene-3-ones (5-6), we envisioned
that N-aryl substitution to exclude the C-H bond on the carbon adjacent to the N1 position (α-H) may
5

increase the metabolic stability. Seven 5- and 6-membered aryl rings (a – g, Scheme 2) were thus
chosen to substitute the alkyl groups at the N1 position of the imidazole or benzo[d]imidazole moiety.
Consequently, seven (1E,4E)-1,5-bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones (17a-17g, Scheme
2) and three (1E,4E)-1,5-bis(1-aryl-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-ones (21a, 21b, and
21f, Scheme 3), as well as three (1E,4E)-1,5-bis(1-aryl-1H-pyrrolo[3,2-b]pyridine-2-yl)penta-1,4-dien-
3-ones (25a, 25c, and 25f, Scheme 4), were synthesized for the evaluation of their antiproliferative
activity against three prostate cancer cell lines. Compounds 25a, 25c, and 25f were also selected due to
the similar shape and size between 4-azaindole and benzimidazole. Each of the thirteen pentadienones
was synthesized through a three-step transformation as illustrated in Schemes 2-4. Specifically, the 1-
aryl-heteroaromatics (15a-15g, 19a, 19b, 19f, 23a, 23c, and 23f) were prepared by arylation of
imidazole (10), benzimidazole (18), and 4-azaindole (22), respectively, with an appropriate aryl
bromide through Ullmann condensation catalyzed by copper iodide and cesium carbonate.12 The aryl
substituted heteroaromatics (15a-15g, 19a, 19b, 19f, 23a, 23c, and 23f) have their most acidic
hydrogen at C-2, which can be deprotonated by tert-butyllithium under argon. The generated
carbanions at C-2 are allowed to react with electrophile dimethylformamide (DMF)13 to give 1-aryl-
heteroaromatic carbaldehydes (16a-16g, 20a, 20b, 20f, 24a, 24c, and 24f), which were then subjected
to the Horner-Wadsworth-Emmons (HWE) reaction with 1,3-bis(diethylphosphonato)acetone (12)14 to
give the desired 1,5-diaryl-1,4-pentadienones (17a-17g, 21a, 21b, 21f, 25a, 25c, and 25f) according to
the reported procedure.10
6

Scheme 1. Synthesis of (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-1,4-diene-3-one (14).
Scheme 2. Synthesis of (1E,4E)-1,5-bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones (17a-17g).
7

Scheme 3. Synthesis of (1E,4E)-1,5-bis(1-aryl-1H-benzo[d]imidazole-2-yl)penta-1,4-dien-3-ones (21a,
21b, & 21f)
Scheme 4. Synthesis of (1E,4E)-1,5-bis(1-aryl-1H-pyrrolo[3,2-b]pyridine-2-yl)penta-1,4-dien-3-ones
(25a, 25c, 25f).
2.3 Anti-proliferative activities of (1E,4E)-1,5-bis(1-aryl-1H)-heteroaryl-2-yl)penta-1,4-diene-3-
ones (17a-17g, 21a, 21b, 21f, 25a, 25c, and 25f) towards prostate cancer cell lines.
8

The anti-proliferative activities of these 1,5-diaryl-1,4-dien-3-ones toward prostate cancer cells were
evaluated. The WST-1 cell proliferation assay was used to assess the potency change resulting from
replacing the 1-alkyl group in (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones (2-4)
and (1E,4E)-1,5-bis(1-alkyl-1H-benzo[d]imidazole-2-yl)penta-1,4-diene-3-ones (5-6) with a 1-aryl
moiety. The IC50 values for these newly made compounds are summarized in Table 1. (1E,4E)-1,5-
Bis(1-aryl-1H-imidazol-2-yl)penta-1,4-dien-3-ones (17a-17f) are six optimal compounds, exhibiting
24- to 375-fold improved potency as compared with curcumin. These compounds (e.g. 17a and 17d)
possess even greater potency than (1E,4E)-1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones (2-
4). Replacement of the imidazole (17a, 17b, 17c, and 17f) with a bulkier benzoimidazole (21a, 21b,
and 21f) and 4-azaindole (25a, 25c, and 25f) resulted in substantial decreases in the potency.
To further study the potency toward castration resistant prostate cancer models, compound 17a, one of
the optimal compounds, was evaluated towards enzalutamide-resistant LNCaP95, VCaP and 22Rv1
cell lines15-16 by the trypan blue exclusion assay after 5 days exposure. Compound 17a is also very
promising in suppressing cell proliferation towards LNCaP95, VCaP and 22Rv1 cell lines, with IC50
values of 0.15 µM, 0.18 µM, and 0.30 µM, respectively.
Table 1. Anti-proliferative activity of 1,5-diheteroarylpenta-1,4-dien-3-ones
IC50 (µM)a
IC50 (curcumin)/IC50(dienone)
Compd
PC-3b
DU145c
LNCaPd
PC-3b
DU145c
LNCaPd
curcumin
14
25.43 ± 2.15
26.23 ± 0.65
13.61 ± 2.69
_
_
_
304.89 ± 34.95
0.12 ± 0.03
0.23 ± 0.09
0.22 ± 0.09
236.30 ± 19.33 72.82 ± 1.85
0.08
0.11
0.19
62
0.07 ± 0.02
0.21 ± 0.04
0.18 ± 0.06
0.22 ± 0.11
0.58 ± 0.03
0.23 ± 0.07
212
111
375
125
17a
24
17b
116
146
59
17c
9

0.11 ± 0.04
0.26 ± 0.07
0.26 ± 0.08
7.76 ± 0.69
2.73 ± 0.53
23.14 ± 0.54
1.14 ± 0.19
1.02 ± 0.25
> 50
0.14 ± 0.01
0.22 ± 0.09
0.10 ± 0.02
7.62 ± 0.49
2.82 ± 0.82
19.25 ± 6.74
2.01 ± 0.75
0.80 ± 0.11
47.28 ± 11.10
> 10
0.29 ± 0.06
0.32 ± 0.07
0.32 ± 0.03
6.61 ± 1.57
5.36 ± 1.79
30.47 ± 3.53
7.50 ± 0.66
1.71 ± 0.36
38.21 ± 1.35
> 10
231
98
187
119
262
3.4
9
47
43
17d
17e
17f
17g
21a
21b
21f
25a
25c
25f
98
43
3.3
9
4.1
2.5
0.5
2
1.1
22
1.4
13
25
33
8
< 0.5
< 2.5
0.6
0.4
> 10
< 3
< 1.4
a IC50 is the compound concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3
days exposure. The data were presented as the mean ± standard deviation of the mean.
b Human androgen-insensitive prostate cancer cell line derived from bone metastasis of prostate tumor
c Human androgen-insensitive prostate cancer cell line derived from brain metastasis of prostate tumor
d Human androgen-sensitive prostate cancer cell line
2.4 Pharmacokinetic Studies: (1E,4E)-1,5-Bis(1-phenyl-1H-imidazol-2-yl)penta-1,4-dien-3-one (17a)
was selected for pharmacokinetic studies in Sprague Dawley rats due to its promising in vitro anti-
proliferative potency in three prostate cancer cell models. The blood samples of rats, administered with
17a via oral gavage at a single dose of 10 mg/kg, were collected at 1, 3, 6, and 24 h after oral
administration. Plasma was prepared from the blood samples and was analyzed by HPLC-MS/MS for
determination of drug concentration as described in the Experimental Section. The PK parameters for
17a could not be calculated because detectable plasma concentrations could barely be achieved at the
1h time point, suggesting the poor pharmacokinetic profile of 17a. This may be due to its poor water
solubility caused by its structural coplanarity. At this point, (1E,4E)-1,5-bis(1-(2-methoxyphenyl)-1H-
imidazol-2-yl)penta-1,4-dien-3-one (17d) with similar in vitro antiproliferative potency was chosen for
10

further pharmacokinetic studies because the methoxyl group at the 2-position of the phenyl ring can
break the coplanarity of the phenyl and the imidazole and lead to enhanced water solubility. The
pharmacokinetic parameters for 17d, as summarized in Table 2, imply that 17d does afford greater
bioavailability than 17a. Its peak plasma concentration at 149.6 ng/mL (0.35 µM) exceeds its IC50
values of 0.11-0.29 µM in three human prostate cancer cell lines. Compound 17d affords an AUC
value of 1800.45 ng/mL*h versus 5.04 ng/mL*h for (1E,4E)-1,5-bis(1-isopentyl-1H-imidazol-2-
yl)penta-1,4-dien-3-one (3)10 and 2.43 ng/mL*h for curcumin10, suggesting its appreciably improved
therapeutic potential as compared with curcumin and parental compound 3.” The bioavailability of 17d,
as demonstrated by its peak plasma concentration and AUC value, is thus sufficient to provide the
therapeutic efficacy necessary to suppress in vivo tumor growth at doses that are ≥ 10 mg/kg.
Table 2. Pharmacokinetic parameters for 17d
Parameter
Lambda_z
t1/2
Unit
1/h
17d
0.14 ± 0.02
4.82 ± 0.06
2.67 ± 0.58
h
Tmax
h
Cmax
ng/ml
h
149.62 ± 5.70
0
Tlag
Clast_obs/Cmax
AUC 0-t
0.050 ± 0.006
1748.44 ± 164.51
1800.45 ± 170.09
0.97 ± 0.02
ng/ml*h
ng/ml*h
AUC 0-inf_obs
AUC 0-t/0-inf_obs
AUMC 0-inf_obs
MRT 0-inf_obs
Vz/F_obs
Cl/F_obs
ng/ml*h^2
h
11086.59 ± 1195.10
6.16 ± 0.14
(mg/kg)/(ng/ml)
(mg/kg)/(ng/ml)/h
0.039 ± 0.003
0.0056 ± 0.001
11

2.5 Metabolic stability studies
To compare the metabolic stability, under both phase I oxidation and phase II glucuronidation
conditions, of our newly synthetic compounds (17a and 17d) with that of curcumin and (1E,4E)-1,5-
bis(1-isopentyl-1H-imidazol-2-yl)penta-1,4-dien-3-one (3), each of curcumin, 3, 17a, and 17d were
incubated with rat liver microsomes, NADPH, and UGT reaction mixtures. The incubation
concentrations of each test compound in liver microsomes were measured by a UHPLC-TSQ mass
spectrometer. The incubation concentrations of curcumin, 3, 17a, and 17d in rat liver microsomes at
different time points were listed in Table 3. We can conclude from these data that the intrinsic
clearance is 161.2 mL/min/kg for curcumin, 39.2 mL/min/kg for 3, 60.4 mL/min/kg for 17a, 7.96
mL/min/kg for 17d, indicating all these three curcumin analogues (3, 17a, 17d) have higher metabolic
stability than curcumin. Among them, 17d has highest metabolic stability.
Table 3. The incubation concentrations of curcumin, 3, 17a, and 17d in rat liver microsomes
Concentration of test compound (nM)
Time points (min)
Curcumin
3
17a
17d
0
1135.2±20.0 1017.6±24.8
1039.7±6.9
624.9±24.4
311.5±11.3
84.5±6.4
34.8±2.0
19.6±1.7
1000.6±35.5
922.1±29.8
914.7±22.8
879.8±58.5
827.8±16.1
757.1±32.8
5
589.6±20.4
200.0±7.0
54.6±3.4
17.6±1.0
9.5±0.4
850.0±26.0
706.2±6.7
644.6±19.0
387.9±7.2
296.0±10.2
15
30
45
60
3 Conclusion
The poor antiproliferative potency of (1E,4E)-1,5-bis(1H-imidazol-2-yl)penta-1,4-diene-3-one (14) in
three human prostate cancer cell lines suggests the critical role of an appropriate group on the N1
position for the potency of 1,5-bis(1-alkyl-1H-imidazol-2-yl)penta-1,4-diene-3-ones (2-4). Thirteen
new diarylpentadienones (17a-17g, 21a, 21b, 21f, 25a, 25c, and 25f) were then designed and
12

synthesized through a three-step transformation. The six optimal (1E,4E)-1,5-bis(1-aryl-1H-imidazol-
2-yl)penta-1,4-dien-3-ones (17a-17f) exhibit 24- to 375-fold improved potency in three prostate cancer
cell models, as compared with curcumin. Replacement of the imidazole (17a, 17c, and 17f) with
bulkier benzoimidazole (21a, 21b, and 21f) and 4-azaindole (25a, 25c, and 25f) resulted in substantial
decrease in the potency. (1E,4E)-1,5-Bis(1-phenyl-1H-imidazol-2-yl)penta-1,4-dien-3-one (17a)
possesses a poor pharmacokinetic profile probably due to its poor water solubility caused by its
structural coplanarity. Instead, (1E,4E)-1,5-bis(1-(2-methoxyphenyl)-1H-imidazol-2-yl)penta-1,4-dien-
3-one (17d) was demonstrated to have both good potency and bioavailability sufficient to provide the
therapeutic efficacy necessary to suppress in vivo tumor growth.
4 Experimental
4.1 General Procedures. HRMS were obtained on an Orbitrap mass spectrometer with electrospray
ionization (ESI). NMR spectra were obtained on a Bruker Fourier 300 spectrometer in CDCl3, CD3OD,
or DMSO-d6. The chemical shifts are given in ppm referenced to the respective solvent peak, and
coupling constants are reported in Hz. Anhydrous THF was purified by PureSolv MD 7 solvent
purification system from Innovative Technologies (MB-SPS-800). All other reagents and solvents were
purchased from commercial sources and were used without further purification. Silica gel column
chromatography was performed using silica gel (32-63 µM). Preparative thin-layer chromatography
(PTLC) separations were carried out on thin layer chromatography plates loaded with silica gel 60
GF254 (EMD Millipore Corporation, MA, USA). 1,3-Bis(diethylphosphonato)acetone was synthesized
using the procedure illustrated in the literature.14 The purities of compounds 14, 17a-17g, 21a, 21b, 21f,
and 25f were determined by HPLC. The peaks were monitored by a diode array detector (ADA) at 325
± 100 nm. The HPLC analyses were performed on an Agilent Hewlett-Packard 1100 series HPLC
DAD system using a 5 µM C18 reversed phase column (4.6 mm × 250 mm) and a diode array detector.
13

4.2 Synthesis of 1-trityl-1H-imidazole-2-carbaldehyde (9): A solution of triphenylmethylchloride
(640 mg, 2.3 mmol) in dichloromethane (1 mL) was added to a mixture of 2-imidazolecarboxadehyde
(200 mg, 2.1 mmol) and potassium carbonate (1.44 g, 10.4 mmol) in dimethylformamide (1.5 mL). The
mixture was stirred at 60 oC overnight and then cooled down to room temperature. The reaction mixture
was extracted with ethyl acetate, the combined extracts were dried over anhydrous sodium sulfate and
concentrated in vacuo. The residue was purified by preparative thin layer chromatography eluting with
1
40% ethyl acetate in hexane to provide 9 as a white to light-yellow solid in 32% yield. H NMR (300
MHz, CDCl3) δ 9.23 (s, 1H), 7.38-7.29 (m, 10 H), 7.13-7.10 (m, 6 H), 7.02 (s, 1H). The 1H NMR data
are consistent with those reported in the literature.17
4.3 Synthesis of (1E,4E)-1,5-bis(1-trityl-1H-imidazol-2-yl)penta-1,4-dien-3-one (13): A solution of
1-trityl-1H-imidazole-2-carbaldehyde (9, 0.61 mmol) and 1,3-bis(diethylphophonato)acetone (12, 0.10
g, 0.29 mmol) in ethanol (1.2 mL) was added a solution of potassium carbonate (0.20 g, 1.4 mmol) in
water (1.7 mL). The reaction was stirred at room temperature and monitored by TLC. When the
reaction was complete, the reaction mixture was then extracted with ethyl acetate. The organic extracts
were dried over anhydrous sodium sulfate and concentrated. The crude product was subjected to PTLC
purification eluting with 5% methanol in DCM to generate the desired product in 51% yield. 1H NMR
(300 MHz, CDCl3) δ 7.32-7.25 (m, 18H), 7.09-7.06 (m, 14H), 6.85 (d, J = 1.1 Hz, 2H), 6.72 (d, J =
13
15.2 Hz, 2H), 6.50 (d, J = 15.2 Hz, 2H). C NMR (75 MHz, CDCl3) δ 187.1, 145.5, 142.5, 130.6,
129.9, 128.32, 128.27, 128.1, 126.7, 124.7, 75.9. IR (film) νmax 1650, 1615, 1596, 1492, 1444, 1403,
1336, 1262, 1220, 1168, 1121, 1097, 1032, 1001, 975, 744, 700, 675, 650, 616 cm-1; HRMS (ESI): m/z
calculated for C49H39N4O [M+H]+: 699.3124. Found: 699.3107.
14

4.4 Synthesis of (1E,4E)-1,5-di(1H-imidazol-2-yl)penta-1,4-dien-3-one (14): A solution of (1E,4E)-
1,5-bis(1-trityl-1H-imidazol-2-yl)penta-1,4-dien-3-one (13, 1 mmol) in 1M hydrochloric acid (10 mmol)
was refluxed for 2 hours. Then the reaction mixture was cooled down and extracted with ethyl acetate.
The combined organic layer was washed with saturated potassium bicarbonate. Light yellow solid was
obtained in 100% yield. M.p. 200 °C (decomposed). 1H NMR (300 MHz, CD3OD) δ 7.77 (s, 4H), 7.70
13
(s, 2H), 7.68 (s, 2H). C NMR (75 MHz, CD3OD) δ 187.1, 142.1, 133.6, 124.1, 123.0. IR (film) νmax
1653, 1618, 1440, 1265, 1156, 1100, 976, 730, 699, 668, 644, 617 cm-1. HRMS (ESI): m/z calculated
for C11H11N4O [M+H]+: 215.0933. Found: 215.0925. HPLC purity 97.6% (30 min run of 1-20%
CH3CN in H2O with 30 min gradient, 1.0 mL/min).
4.5. General Procedure for N-Arylation: To a solution of heteroaromatic compound (8.0 mmol),
copper iodide (20 mol%), and cesium carbonate (11 mmol) in DMF (11 mL) was added aryl bromide
(5.7 mmol) under argon atmosphere, and the reaction mixture was stirred at room temperature for 30
min prior to being refluxed for 48 hours. The reaction mixture was cooled down to room temperature
and diluted with ethyl acetate. The subsequent mixture was filtered through a pad of silica gel. The
filtrate was concentrated to give a residue, which was subjected to preparative thin layer
chromatography eluting with 50% ethyl acetate in hexane.
4.5.1 1-Pheny-1H-imidazole (15a): The residue was purified by column chromatography eluting with
1
5% methanol in dichloromethane12 to provide the title compound as a clear oil in 40% yield. H NMR
(300 MHz, CDCl3) δ 7.89 (s, 1H), 7.52 – 7.45 (m, 2H), 7.42 – 7.34 (m, 3H), 7.29 (s, 1H), 7.22 (s, 1H).
13C NMR (75 MHz, CDCl3) δ 137.4, 135.7, 130.4, 129.9, 127.6, 121.5, 118.3.
15

1
4.5.2 1-(3-Chlorophenyl)-1H-imidazole (15b): Yellow oil was obtained in 98% yield. H NMR (300
MHz, CDCl3) δ 7.93 (s, 1H), 7.47 – 7.39 (m, 2H), 7.38 – 7.31 (m, 1H), 7.30 – 7.27 (m, 2H), 7.24 (s,
1H). 13C NMR (75 MHz, CDCl3) δ 138.4, 135.8, 135.6, 131.2, 130.5, 127.9, 121.9, 119.7, 118.3.
4.5.3 1-(3-Methoxyphenyl)-1H-imidazole (15c): Yellow oil was obtained in 88% yield. 1H NMR (300
MHz, CDCl3) δ 7.91 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.21 (s, 1H), 6.98 (d, J = 7.8 Hz,
13
1H), 6.92 (s, 1H), 6.91 (d, J = 7.8 Hz, 1H), 3.86 (s, 3H). C NMR (75 MHz, CDCl3) δ 160.9, 138.5,
135.7, 130.9, 130.2, 118.6, 113.9, 113.0, 108.0, 55.7.
1
4.5.4 1-(2-Methoxyphenyl)-1H-imidazole (15d): Yellow oil was obtained in 55% yield. H NMR
(300 MHz, CDCl3) δ 7.82 (s, 1H), 7.36 (ddd, J = 9.3, 7.5, 1.8 Hz, 1H), 7.28 (dd, J = 7.5, 1.5 Hz, 1H),
7.21 (t, J = 1.2 Hz, 1H), 7.18 (s, 1H), 7.05 (d, J = 8.7 Hz, 1H), 7.04 (dt, J = 7.2, 1.2 Hz, 1H), 3.85 (s,
3H).
4.5.5 1-(Thiazole-2-yl)-1H-imidazole (15e): Yellow solid was obtained in 85% yield. 1H NMR (300
MHz, CDCl3) δ 8.23 (s, 1H), 7.59 (d, J = 3.6 Hz, 1H), 7.52 (s, 1H), 7.20 (s, 1H), 7.17 (d, J = 3.6 Hz,
1H). 13C NMR (75 MHz, CDCl3) δ 157.7, 140.9, 135.7, 130.8, 118.0, 115.9.
1
4.5.6 1-(Thiophen-2-yl)-1H-imidazole (15f): Yellow oil was obtained in 66% yield. H NMR (300
MHz, CDCl3) δ 7.81 (s, 1H), 7.21 (s, 1H), 7.19 (s, 1H), 7.16 (dd, J = 5.1, 1.5 Hz, 1H), 7.03– 6.97
(overlapped, 2H). 13C NMR (75 MHz, CDCl3) δ 138.9, 137.1, 129.8, 126.6, 122.2, 120.6, 119.5.
1
4.5.7 (1-(Thiophen-3-yl)-1H-benzo[d]imidazole (15g): Yellow solid was obtained in 35% yield. H
NMR (300 MHz, CDCl3) δ 7.96 (s, 1H), 7.46 (dd, J = 5.2, 3.2 Hz, 1H), 7.28-7.26 (overlapped, 1H),
7.27 (s, 1H), 7.21 (s, 1H), 7.21-7.19 (overlapped, 1H).
16

4.5.8 1-Phenyl-1H-benzo[d]imidazole (19a): Iodobenzene instead of bromobenzene was used in a
same equivalent. The residue was purified by preparative thin layer chromatography eluting with 5%
1
methanol in dichloromethane to provide the title compound as a yellow oil in 57.5% yield. H NMR
13
(300 MHz, CDCl3) δ 8.15 (s, 1H), 7.91 – 7.88 (m, 1H), 7.61 – 7.43 (m, 6H), 7.37 – 7.32 (m, 2H). C
NMR (75 MHz, CDCl3) δ 143.8, 142.3, 136.4, 133.8, 130.2, 128.3, 124.3, 123.9, 123.1, 120.6, 110.7.
1
4.5.9 1-(3-Chlorophenyl)-1H-benzo[d]imidazole (19b): Beige solid was obtained in 25% yield. H
NMR (300 MHz, CDCl3) δ 8.37 (s, 1H), 7.94 (d, J = 5.2 Hz, 1H), 7.58 (t, J = 1.8 Hz, 1H), 7.54 (d, J =
7.5 Hz, 1H), 7.48 (dt, J = 6.3, 1.8 Hz, 2H), 7.43 – 7.39 (overlapped, 3H). 13C NMR (75 MHz, CDCl3) δ
141.7, 137.1, 136.1, 131.4, 128.9, 124.8, 124.5, 124.0, 122.4, 120.3, 110.8.
1
4.5.10 1-(Thiophen-2-yl)-1H-benzo[d]imidazole (19f): Yellow oil was obtained in 60% yield. H
NMR (300 MHz, CDCl3) δ 8.16 (s, 1H), 7.90-7.87 (m, 1H), 7.59-7.55 (m, 1H), 7.40-7.36 (m, 2H), 7.33
(d, J = 5.1 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.12 (dd, J = 5.3, 3.7 Hz, 1H). 13C NMR (75 MHz, CDCl3)
δ 143.1, 142.9, 136.9, 134.7, 126.7, 124.6, 123.9, 123.7, 122.4, 120.5, 110.8.
4.5.11 1-Phenyl-1H-pyrrolo[3,2-b]pyridine (23a): Iodobenzene instead of bromobenzene was used
in a same equivalent. Yellow oil was obtained in 62% yield. 1H NMR (300 MHz, CDCl3) δ 8.54 (d, J =
4.6 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 3.4 Hz, 1H), 7.61-7.55 (m, 2H), 7.49-7.41 (m, 3H),
7.25 - 7.20 (m, 1H), 7.01 (d, J = 3.2 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 147.1, 143.5, 139.0, 131.5,
130.2, 130.1, 127.3, 124.3, 118.5, 117.3, 104.4.
4.5.12 1-(3-Methoxyphenyl)-1H-pyrrolo[3,2-b]pyridine (23c): Yellow oil was obtained in 52% yield.
1H NMR (300 MHz, CDCl3) δ 8.52 (d, J = 4.4 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 3.3 Hz,
17

1H), 7.45 (t, J = 8.1 Hz, 1H), 7.19 (dd, J = 8.3, 4.8 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H), 6.95
13
(d, J = 8.6 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H). C NMR (75 MHz, CDCl3) δ 160.9, 146.7,
143.1, 140.0, 131.8, 130.9, 129.4, 118.9, 117.2, 116.4, 112.7, 110.4, 104.2, 55.7.
4.5.13 1-(Thiophen-2-yl)-1H-pyrrolo[3,2-b]pyridine (23f): Yellow oil was obtained in 70% yield. 1H
NMR (300 MHz, CDCl3) δ 8.53 (d, J = 4.4 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 3.3 Hz, 1H),
7.26 – 7.22 (m, 2H), 7.09 – 7.06 (overlapped, 2H), 6.90 (t, J = 3.0 Hz, 1H). 13C NMR (75 MHz, CDCl3)
δ 147.2, 144.3, 141.6, 140.3, 132.5, 128.9, 124.2, 120.6, 118.3, 117.8, 105.2.
4.6 General Procedure for formylation. t-BuLi (1.9 M, 0.87 mmol) was added to a solution of
arylatedheteroaromatic (0.78 mmol) in tetrahydrofuran (7.6 mL) dropwise under argon atmosphere at -
78 C, and the reaction mixture was stirred for 1 hour at the same temperature before adding DMF
(0.98 mmol). The reaction mixture was then warmed up to room temperature and the completion of
reaction was monitored by TLC. The reaction was quenched with saturated ammonium chloride
solution and diluted with brine, and the subsequent mixture was extracted by dichloromethane. The
organic layer was dried with sodium sulfate followed by evaporation of the solvent. The residue was
purified by preparative thin layer chromatography eluting with 50% ethyl acetate in hexane.
4.6.1 1-Phenyl-1H-imidazole-2-carbaldehyde (16a): n-BuLi (2.5 M) and diethyl ether were used
instead of t-BuLi (1.9 M) and tetrahydrofuran in the same equivalent. White solid was obtained in 66%
1
yield. H NMR (300 MHz, CDCl3) δ 9.78 (s, 1H), 7.47 – 7.41 (m, 3H), 7.37 (s, 1H), 7.33 – 7.28 (m,
13
2H), 7.25 (s, 1H). C NMR (75 MHz, CDCl3) δ 180.2, 143.3, 136.7, 131.9, 129.20, 129.18, 127.2,
125.7.
18

4.6.2 1-(3-Chlorophenyl)-1H-imidazole-2-carbaldehyde (16b): Diethyl ether was used instead of
tetrahydrofuran. Light yellow solid was obtained in 18% yield. 1H NMR (300 MHz, CDCl3) δ 9.85 (s,
1H), 7.49 (t, J = 8.1, 1.5 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 1.8 Hz,
13
1H), 7.27 – 7.23 (overlapped, 2H). C NMR (75 MHz, CDCl3) δ 180.4, 143.2, 137.8, 135.0, 131.9,
130.4, 129.7, 127.1, 126.3, 124.4.
4.6.3 1-(3-Methoxyphenyl)-1H-imidazole-2-carbaldehyde (16c): Yellow oil was obtained in 62%
yield. 1H NMR (300 MHz, CDCl3) δ 9.84 (s, 1H), 7.42 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.28 (s, 1H),
7.02 (dd, J = 7.8, 1.8 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.87 (t, J = 2.1 Hz, 1H), 3.84 (s, 3H). 13C NMR
(75 MHz, CDCl3) δ 180.3, 160.2, 143.5, 137.8, 131.9, 130.1, 127.2, 118.1, 115.0, 112.0, 55.7.
4.6.4 1-(2-Methoxyphenyl)-1H-imidazole-2-carbaldehyde (16d): Yellow oil was obtained in 54%
1
yield. H NMR (300 MHz, CDCl3) δ 9.81 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.25 (d, J =
13
8.4 Hz, 1H), 7.19 (s, 1H), 7.07 (t, J = 7.8 Hz, 2H), 3.76 (s, 3H). C NMR (75 MHz, CDCl3) δ 180.2,
154.1, 144.0, 131.3, 130.8, 127.3, 127.1, 126.1, 120.8, 112.1, 55.9.
4.6.5 1-(Thiazol-2-yl)-1H-imidazole-2-carbaldehyde (16e): Yellow solid was obtained in 26% yield.
1H NMR (300 MHz, CDCl3) δ 9.67 (s, 1H), 7.78 (s, 1H), 7.71 (d, J = 3.0 Hz, 1H), 7.43 (s, 1H), 7.41 (d,
J = 3.3 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 180.7, 156.1, 143.4, 140.4, 132.1, 126.2, 120.4.
4.6.6 1-(Thiophen-2-yl)-1H-imidazole-2-carbaldehyde (16f): Brown oil was obtained in 39% yield.
1H NMR (300 MHz, CDCl3) δ 9.84 (s, 1H), 7.40 (s, 1H), 7.32 (dd, J = 5.7, 1.5 Hz, 1H), 7.30 (s, 1H),
13
7.12 (dd, J = 3.6, 1.2 Hz, 1H), 7.03 (dd, J = 5.7, 3.9 Hz, 1H). C NMR (75 MHz, CDCl3) δ 180.1,
144.2, 136.9, 131.9, 128.4, 126.0, 125.03, 125.01.
19

4.6.7 1-(Thiophen-3-yl)-1H-imidazole-2-carbaldehyde (16g): Brown oil was obtained in 12% yield.
1H NMR (300 MHz, CDCl3) δ 9.88 (s, 1H), 7.94 (s, 1H), 7.85 (d, J = 5.1 Hz, 1H), 7.30 (s, 2H), 7.22 (d,
J = 5.2 Hz, 1H).
4.6.8 1-Phenyl-1H-benzo[d]imidazole-2-carbaldehyde (20a): n-BuLi (2.5 M) and diethyl ether were
used instead of t-BuLi (1.9 M) and tetrahydrofuran in the same equivalent. Yellow solid was obtained
1
in 44% yield. H NMR (300 MHz, CDCl3) δ 10.10 (s, 1H), 8.02 – 7.96 (m, 1H), 7.60-7.57 (m, 3H),
13
7.46 – 7.42 (m, 2H), 7.40 – 7.37 (m, 2H), 7.29 – 7.25 (m, 1H). C NMR (75 MHz, CDCl3) δ 182.9,
145.9, 142.6, 137.5, 135.2, 130.1, 129.6, 127.4, 127.1, 124.6, 122.3, 111.7.
4.6.9 1-(3-Chlorophenyl)-1H-benzo[d]imidazole-2-carbaldehyde (20b): Beige solid was obtained in
1
53% yield. H NMR (300 MHz, CDCl3) δ 10.09 (s, 1H), 7.99 (t, J = 6.0 Hz, 1H), 7.57-7.52 (m, 2H),
13
7.49 – 7.44 (m, 2H), 7.40 (s, 1H), 7.31 – 7.24 (overlapped, 2H). C NMR (75 MHz, CDCl3) δ 183.0,
147.6, 145.9, 142.7, 136.5, 135.3, 130.7, 129.8, 127.8, 127.53, 125.6, 125.0, 122.5, 111.6.
4.6.10 1-(Thiophen-2-yl)-1H-benzo[d]imidazole-2-carbaldehyde (20f): Yellow oil was obtained in
1
45% yield. H NMR (300 MHz, CDCl3) δ 10.03 (s, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.43-7.37
(overlapped, 3H), 7.30-7.27 (m, 1H), 7.08-7.05 (overlapped, 2H). 13C NMR (75 MHz, CDCl3) δ 182.5,
146.7, 142.4, 138.5, 135.2, 127.9, 126.3, 126.0, 125.0, 122.4, 111.9.
4.6.11 1-Phenyl-1H-pyrrolo[3,2-b]pyridine-2-carbaldehyde (24a): Diethyl ether was used instead of
tetrahydrofuran. Yellow solid was obtained in 57% yield. 1H NMR (300 MHz, CDCl3) δ 10.55 (s, 1H),
8.73 (dd, J = 4.5, 0.9 Hz, 1H), 8.26 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.3 (t, J = 6.9 Hz, 2H), 7.56 – 7.48
(m, 3H), 7.34 (dd, J = 8.4, 4.8 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 185.0, 145.6, 137.5, 136.7, 130.6,
130.5, 129.0, 127.7, 124.7, 120.0, 119.0, 118.2.
20

4.6.12 1-(3-Methoxyphenyl)-1H-pyrrolo[3,2-b]pyridine-2-carbaldehyde (24c): Beige solid was
1
obtained in 12% yield. H NMR (300 MHz, CDCl3) δ 10.46 (s, 1H), 8.71 (d, J = 4.6 Hz, 1H), 8.22 (s,
1H), 7.88 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.29 (overlapped, 1H), 7.09 (d, J = 8.4 Hz, 1H),
7.04 (d, J = 8.7 Hz, 1H), 7.03 (s, 1H), 3.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.1, 161.1, 146.4,
144.7, 138.7, 136.3, 131.2, 130.3, 119.4, 119.0, 118.5, 116.6, 114.2, 110.6, 55.8.
4.6.13 1-(Thiophen-2-yl)-1H-pyrrolo[3,2-b]pyridine-2-carbaldehyde (24f): Yellow solid was
obtained in 57% yield. 1H NMR (300 MHz, CDCl3) δ 9.90 (s, 1H), 8.59 (d, J = 4.2 Hz, 1H), 8.12 (d, J
= 8.4 Hz, 1H), 7.78 (d, J = 4.1 Hz, 1H), 7.66 (d, J = 3.5 Hz, 1H), 7.31 (dd, J = 8.4, 4.8 Hz, 1H), 7.19 (d,
J = 4.1 Hz, 1H), 7.02 (dd, J = 3.5, 0.7 Hz, 1H).
4.7. General Procedure for the Synthesis of (1E,4E)-1,5-bis(1-aryl-1H-heteroaromatic-2-yl)penta-
1,4-dien-3-ones:
A
solution
of
appropriate
aldehyde
(0.61
mmol)
and
1,3-
bis(diethylphophonato)acetone (0.10 g, 0.29 mmol) in ethanol (1.2 mL) was added a solution of
potassium carbonate (0.20 g, 1.4 mmol) in water (1.7 mL). The reaction was stirred at room
temperature and monitored by TLC until the completion. The reaction mixture was then extracted with
ethyl acetate, the combined extracts were dried over anhydrous sodium sulfate and concentrated in
vacuo. The residue was subjected to PTLC purification eluting with 7% methanol in dichloromethane.
4.7.1 (1E,4E)-1,5-Bis(1-phenyl-1H-imidazol-2-yl)penta-1,4-dien-3-one (17a): The yellow solid
was obtained in 26% yield. M.p. 200 – 204 °C. 1H NMR (300 MHz, CDCl3) δ 7.55-7.48 (m, 6H), 7.40
13
(d, J = 15.3 Hz, 2H), 7.34 (d, J = 15.3 Hz, 2H), 7.31-7.28 (m, 6H), 7.22 (s, 2H). C NMR (75 MHz,
CDCl3) δ 188.0, 143.6, 136.7, 131.0, 130.0, 129.4, 128.3, 128.0, 126.2, 124.2. IR (film) νmax 1651,
1618, 1596, 1498, 1450, 1418, 1344, 1311, 1247, 1175, 1094, 978, 765, 732, 696, 646 cm-1; HRMS
21

(ESI): m/z calculated for C23H19N4O [M+H]+: 367.1559. Found: 367.1558. HPLC purity 93.7% (30 min
run of 10-85% CH3CN in H2O with 30 min gradient, 1.0 mL/min).
4.7.2 (1E,4E)-1,5-Bis(1-(3-chlorophenyl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17b): Yellow
solid was obtained in 62% yield. M.p. 214 °C (decomposed). 1H NMR (300 MHz, CDCl3) δ 7.51–7.47
(m, 4H), 7.41 (d, J = 15.3 Hz, 2H), 7.34 (s, 2H), 7.34-7.31 (overlapped, 2H), 7.32 (d, J = 15.3 Hz, 2H),
7.23-7.20 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 187.6, 143.5, 137.6, 135.6, 131.3, 130.9, 129.6, 128.4,
127.5, 126.4, 124.5, 123.9. IR (film) νmax 1625, 1592, 1480, 1388, 1338, 1097, 765, 744, 693, 669 cm-1.
HRMS (ESI): m/z calculated for C23H17Cl2N4O [M+H]+: 435.0779; 437.0750; 439.0720. Found:
435.0777; 437.0745; 439.0716. HPLC purity 95.1% (30 min run of 10-85% CH3CN in H2O with 30
min gradient, 1.0 mL/min).
4.7.3 (1E,4E)-1,5-Bis(1-(3-methoxyphenyl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17c): Yellow
1
solid was obtained in 30% yield. M.p. 134-135 °C. H NMR (300 MHz, CDCl3) δ 7.46 (t, J = 7.8 Hz,
2H), 7.33 – 7.19 (overlapped, 6H), 7.19 (s, 2H), 7.12 (s, 2H), 7.06 (d, J = 7.3 Hz, 4H), 3.77 (s, 6H). 13C
NMR (75 MHz, CDCl3) δ 188.4, 154.4, 144.4, 131.1, 130.6, 128.5, 128.4, 127.6, 125.3, 124.7, 121.2,
112.6, 56.0. IR (film) νmax 1651, 1621, 1596, 1507, 1466, 1448, 1420, 1346, 1313, 1283, 1247, 1180,
1134, 1093, 1047, 1022, 1001, 977, 755, 734, 669, 644 cm-1. HRMS (ESI): m/z calculated for
C25H23N4O3 [M+H]+: 427.1770. Found: 427.1771. HPLC purity 98.4% (30 min run of 30-85% CH3CN
in H2O with 30 min gradient, 1.0 mL/min).
4.7.4 (1E,4E)-1,5-Bis(1-(2-methoxyphenyl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17d): Yellow
solid was obtained in 30% yield. M.p. 98-99 °C. 1H NMR (300 MHz, CDCl3) δ 7.47 (t, J = 8.1 Hz, 2H),
7.39 (d, J = 15.3 Hz, 2H), 7.38 (s, 2H), 7.25 (d, J = 1.2 Hz, 2H), 7.09 – 6.97 (m, 4H), 6.92 (d, J = 7.8
Hz, 2H), 6.83 (t, J = 2.1 Hz, 2H), 3.86 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 187.9, 160.8, 143.4, 137.4,
22

130.9, 129.2, 127.7, 127.1, 124.1, 118.5, 115.2, 112.2, 55.8. IR (film) νmax 1649, 1619, 1605, 1590,
1492, 1469, 1446, 1418, 1344.2, 1289, 1236, 1223, 1174, 1126, 1097, 1045, 1015, 978, 866, 817, 781,
732, 695, 681, 650 cm-1. HRMS (ESI): m/z calculated for C25H23N4O3 [M+H]+: 427.1770. Found:
427.1768. HPLC purity 96.9% (30 min run of 15-70% CH3CN in H2O with 30 min gradient, 1.0
mL/min).
4.7.5 (1E,4E)-1,5-Bis(1-(thiazol-2-yl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17e): Yellow solid
was obtained in 95% yield. M.p. 135-137 °C. 1H NMR (300 MHz, CDCl3) δ 8.09 (d, J = 15.6 Hz, 2H),
7.79 (d, J = 3.3 Hz, 2H), 7.72 (d, J = 15.9 Hz, 2H), 7.47 (s, 2H), 7.39 (d, J = 3.3 Hz, 2H), 7.35 (s, 2H).
13C NMR (75 MHz, CDCl3) δ 188.2, 156.9, 144.1, 141.6, 131.6, 129.9, 128.7, 122.8, 118.9. IR (film)
νmax 1649, 1618, 1591, 1525, 1509, 1462, 1400, 1328, 1290, 1254, 1178, 1154, 1095, 973, 754, 725 cm-
1; HRMS (ESI): m/z calculated for C17H13N6OS2 [M+H]+: 381.0592. Found: 381.0587. HPLC purity
95.0% (30 min run of 15-85% CH3CN in aqueous solution of ammonium formate (1.26 g ammonium
formate in 1000 mL water), with 30 min gradient, 1.0 mL/min).
4.7.6 (1E,4E)-1,5-Bis(1-(thiophen-2-yl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17f): Yellow
solid was obtained in 59% yield. M.p. 190 °C (decomposed). 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J =
13.8 Hz, 2H), 7.46 (d, J = 15.6 Hz, 2H), 7.37 (d, J = 4.8 Hz, 2H), 7.34 (s, 2H), 7.28-7.21 (overlapped,
2H), 7.11-7.08 (overlapped, 4H). 13C NMR (75 MHz, CDCl3) δ 188.0, 145.0, 136.9, 131.2, 128.7, 127.5,
126.6, 125.5, 125.4, 125.2. IR (film) νmax 1651, 1621, 1590, 1550, 1456, 1439, 1409, 1335, 1291, 1256,
1220, 1173, 1122, 1094, 975, 874, 729, 702, 669, 639 cm-1. HRMS (ESI): m/z calculated for
C19H15N4OS2 [M+H]+: 379.0687. Found: 379.0682. HPLC purity 92.4% (30 min run of 10-85% CH3CN
in H2O with 30 min gradient, 1.0 mL/min).
23

4.7.7 (1E,4E)-1,5-Bis(1-(thiophen-3-yl)-1H-imidazol-2-yl)penta-1,4-dien-3-one (17g): Orange solid
1
was obtained in 10% yield. M.p. 200.0 °C (decomposed). H NMR (300 MHz, DMSO-d6) δ 8.00 (s,
2H), 7.93 (dd, J = 5.4, 0.6 Hz, 2H), 7.54 (s, 2H), 7.48 (dd, J = 15.6, 0.6 Hz, 2H), 7.34 (d, J = 5.4 Hz,
13
2H), 7.18 (s, 2H), 7.07 (d, J = 15.6 Hz, 2H). C NMR (75 MHz, DMSO-d6) δ 186.3, 138.0, 137.7,
131.0, 130.1, 129.8, 129.4, 126.6, 126.2, 121.2. IR (film) νmax 1600, 1541, 1487, 1315, 1246, 1101,
1069, 1022, 734, 658 cm-1 HRMS (ESI): m/z calculated for C19H15N4OS2 [M+H]+: 379.0687. Found:
379.0676. HPLC purity 95.2% (30 min run of 10-85% CH3CN in H2O with 30 min gradient, 1.0
mL/min).
4.7.8 (1E,4E)-1,5-Bis(1-phenyl-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-one (21a): The yellow
solid was obtained in 27% yield. M.p. 184-191 °C. 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 7.8 Hz,
2H), 7.78 (d, J = 16.2 Hz, 2H), 7.61 (t, J = 7.5 Hz, 6H), 7.49 (d, J = 15.6 Hz, 2H), 7.42-7.30
13
(overlapped, 8H), 7.23 (d, J = 7.8 Hz, 2H). C NMR (75 MHz, CDCl3) δ 187.4, 148.1, 137.0, 134.8,
130.5, 130.4, 129.8, 127.6, 125.1, 124.5, 123.8, 120.9, 120.3, 111.1. IR (film) νmax 1683, 1614, 1596,
1529, 1498, 1454, 1414, 1396, 1340, 1267, 1230, 1017, 760, 744, 696 cm-1. HRMS (ESI): m/z
calculated for C31H23N4O [M+H]+: 467.1872. Found: 467.1869. HPLC purity 94.8% (30 min run of 30-
85% CH3CN in H2O with 30 min gradient, 1.0 mL/min).
4.7.9 (1E,4E)-1,5-Bis(1-(3-chlorophenyl)-1H-benzo[d]imidazole-2-yl)penta-1,4-dien-3-one (21b):
Yellow solid was obtained in 20% yield. M.p. 201-203 °C. 1H NMR (300 MHz, CDCl3) δ 7.88 (dd, J =
6.9, 0.6 Hz, 2H), 7.81 (d, J = 15.6 Hz, 2H), 7.59-7.57 (overlapped, 4H), 7.45 (d, J = 15.3 Hz, 2H),
7.45-7.42 (overlapped, 2H), 7.39 (dt, J = 7.2, 1.2 Hz, 2H), 7.36-7.29 (overlapped, 4H), 7.22 (dd, J = 7.5,
13
1.5 Hz, 2H). C NMR (75 MHz, CDCl3) δ 187.2, 147.9, 142.5, 136.7, 136.1, 135.9, 132.4, 131.5,
130.2, 128.1, 127.8, 126.0, 125.4, 124.7, 120.4, 110.8. IR (film) νmax 1654, 1591, 1389, 1338, 1268.
HRMS (ESI): m/z calculated for C31H21Cl2N4O [M+H]+: 535.1092; 537.1063; 539.1033. Found:
24

535.1090; 537.1055; 539.1028. HPLC purity 98.3% (30 min run of 15-85% CH3CN in aqueous
solution of ammonium formate (1.26 g ammonium formate in 1000 mL water), with 30 min gradient,
1.0 mL/min).
4.7.10 (1E,4E)-1,5-Bis(1-(thiophen-2-yl)-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-one
(21f):
1
Yellow solid was obtained in 55% yield. M.p. 175 °C (decomposed). H NMR (300 MHz, CDCl3) δ
7.85 (dd, J = 6.3, 1.2 Hz, 2H), 7.69 (d, J = 15.6 Hz, 2H), 7.58 (d, J = 15.6 Hz, 2H), 7.47 (dd, J = 5.1,
1.8 Hz, 2H), 7.38–7.30 (overlapped, 6H), 7.19–7.15 (overlapped, 4H). 13C NMR (75 MHz, CDCl3) δ
187.4, 149.4, 143.1, 138.1, 135.0, 131.9, 128.4, 126.9, 126.8, 126.7, 125.4, 124.5, 120.6, 111.1. IR
(film) νmax 1616, 1548, 1453, 1386, 1356, 1324, 1228, 1176, 764, 746, 700, 669 cm-1; HRMS (ESI):
m/z calculated for C27H19N4OS2 [M+H]+: 479.1000. Found: 479.0996. HPLC purity 93.6% (30 min run
of 10-85% CH3CN in H2O with 30 min gradient, 1.0 mL/min).
4.7.11 (1E,4E)-1,5-Bis(1-phenyl-1H-pyrrolo[3,2-b]pyridin-2-yl)penta-1,4-dien-3-one (25a): The
yellow solid was obtained in 23% yield. M.p. 195-197 °C. 1H NMR (300 MHz, CDCl3) δ 8.52 (dd, J =
4.6, 1.1 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 3.3 Hz, 2H), 7.55 (t, J = 7.8 Hz, 6H), 7.49 – 7.45
13
(m, 4H), 7.40 (dt, J = 7.2, 1.2 Hz, 2H), 7.16 (dd, J = 8.4, 4.5 Hz, 2H), 6.92 (d, J = 3.3 Hz, 2H). C
NMR (75 MHz, CDCl3) δ 186.8, 144.0, 135.6, 134.0, 131.2, 130.4, 129.5, 128.5, 128.2, 128.1, 127.4,
119.6, 118.8, 104.6. IR (film) νmax 1654, 1617, 1595, 1507, 1489, 1456, 1412, 1332, 1297.1, 1240.6,
1187.0, 1147.0, 1101.0, 1057.1, 1033.1, 980.8, 950.7, 772.5, 728.1, 700.9, 668.7, 579.2, 564.3 cm-1.
HRMS (ESI): m/z calculated for C31H23N4O [M+H]+: 467.1872. Found: 467.1867.
4.7.12.
(1E,4E)-1,5-Bis(1-(3-methoxyphenyl)-pyrrolo[3,2-b]pyridine-2-yl)penta-1,4-dien-3-one
(25c): Orange solid was obtained in 53% yield. M.p.109 – 112 °C. 1H NMR (300 MHz, CDCl3) δ 8.53
25

(d, J = 3.9 Hz, 2H), 7.53 (d, J = 15.9 Hz, 2H), 7.45 (t, J = 8.4 Hz, 4H), 7.32 (s, 2H), 7.11 (dd, J = 8.4,
4.5 Hz, 2H), 7.06 (ddd, J = 8.4, 2.4, 0.6 Hz, 2H), 6.95 (d, J = 15.9 Hz, 2H), 6.94 – 6.90 (overlapped,
13
2H), 6.86 (t, J = 2.1 Hz, 2H), 3.84 (s, 6H). C NMR (75 MHz, CDCl3) δ 187.1, 160.9, 145.6, 145.5,
138.6, 137.0, 133.5, 131.5, 130.8, 127.7, 120.4, 118.9, 118.4, 114.7, 114.1, 105.3, 55.8. IR (film) νmax
1669, 1589, 1559, 1527, 1492, 1457, 1427, 1404, 1368, 1310, 1283, 1260, 1196, 1175, 1127, 1085,
1037, 996, 850, 773, 732, 715, 689, 629, 562, 535 cm-1. HRMS (ESI): m/z calculated for C33H27N4O3
[M+H]+: 527.2083. Found: 527.2081.
4.7.13 (1E,4E)-1,5-Bis(1-(thiophen-2-yl)-1H-pyrrolo[3,2-b]pyridin-2-yl)penta-1,4-dien-3-one (25f):
1
Orange solid was obtained in 52% yield. M.p. 156 °C (decomposed). H NMR (300 MHz, CDCl3) δ
8.52 (s, 2H), 7.97 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 15.3 Hz, 2H), 7.56 (d, J = 2.7 Hz, 2H), 7.29 (d, J =
3.6 Hz, 2H), 7.20 (dd, J = 8.1, 4.5 Hz, 2H), 7.03 (d, J = 3.6 Hz, 2H), 6.89 (s, 2H), 6.75 (d, J = 15.3 Hz,
2H). 13C NMR (75 MHz, CDCl3) δ 187.0, 147.7, 144.8, 143.3, 135.7, 135.5, 131.8, 131.6, 124.1, 119.9,
118.3, 118.0, 116.5, 106.4. IR (film) νmax 1661, 1599, 1559, 1541, 1514, 1187, 1028, 777, 668 cm-1.
HRMS (ESI): m/z calculated for C27H19N4OS2 [M+H]+: 479.1000. Found: 479.0998. HPLC purity 98.1%
(30 min run of 10-85% CH3CN in H2O with 30 min gradient, 1.0 mL/min).
4.8 Cell culture.
All cell lines were initially purchased from American Type Culture Collection (ATCC). The PC-3 and
LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640 medium supplemented with
10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a high humidity environment
supplemented with 5% carbon dioxide at a temperature of 37oC. The DU-145 prostate cancer cells were
routinely cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and
1% penicillin/ streptomycin.
26

4.9 WST-1 cell proliferation assay.
PC-3, DU-145, or LNCaP cells were plated in 96-well plates at a density of 3,200 each well in 200 µL
of culture medium. The cells were then treated with curcumin, or synthesized analogs separately at
different doses for 3 days, while equal treatment volumes of DMSO were used as vehicle control. The
o
cells were cultured in a CO2 incubator at 37 C for three days. 10 µL of the premixed WST-1 cell
proliferation reagent (Clontech) was added to each well. After mixing gently for one minute on an
o
orbital shaker, the cells were incubated for additional 3 hours at 37 C. To ensure homogeneous
distribution of color, it is important to mix gently on an orbital shaker for one minute. The absorbance
of each well was measured using a microplate-reader (Synergy HT, BioTek) at a wavelength of 430
nm. The IC50 value is the concentration of each compound that inhibits cell proliferation by 50% under
the experimental conditions and is the average from triplicate determinations that were reproducible
and statistically significant. For calculating the IC50 values, a linear proliferative inhibition was made
based on at least five dosages for each compound.
4.10. Pharmacokinetic study (sampling and analysis).18
Male Sprague-Dawley rats, weighing between 250 and 300 g (Charles River Laboratories, Portage, MI)
were used for the pharmacokinetic studies of 17a and 17d. Rats (n = 4) were given oral gavage
containing 5% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400, 55% saline-dissolved
compound at a single dose of 10 mg/kg. After oral administration, blood samples were collected from
the lateral tail vein of the rats at 1, 3, 6, and 24 h. Rat blood was collected with a capillary into 1.5 mL
microcentrifuge tubes containing 0.01 mL of 10% EDTA anticoagulant. Plasma was then separated
o
from red cells by centrigugation in a refrigerated centrifuge at 4 C and transferred to a separate tube.
o
The plasma samples were frozen at -80 C until analysis. All procedures involving these animals were
conducted in compliance with state and federal laws, standards of the U.S. Department of Health and
27

Human Services, and guidelines established by Xavier University are accredited by the Association for
the Assessment and Accreditation of Laboratory Animal Care.
4.11. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for
drug analysis in plasma Samples.
Plasma samples were extracted with chloroform/methanol (2:1) using traditional Folch method for lipid
extraction. Methanol (1 mL) and chloroform (2 mL) were added to each plasma sample followed by
15
addition of 5 ng of trans-tamoxifen-13C2, N to each sample as the internal standard. The mixtures
o
were stored at -20 C overnight. Next the samples were sonicated for 5 min and centrifuged with a
Thermo Scientific Heraeus Megafuge 16 centriguge. The top layer was transferred to another test tube.
The bottom layer was washed with 1 mL of chloroform/methanol (2:1), centrifuged, and the top layer
was transferred and combined with the previous top layer. Eight-tenth of a milliliter of HPLC grade
water was added to the extracts. After vortexing, the mixture was centrifuged. The bottom layer was
dried out with nitrogen and re-suspended in 100 µL of HPLC grade acetonitrile. An aliquot of 10 µL of
sample was injected onto a Hypersil Gold column (50 mm × 2.1 mm; particle size 1.9 µM, Thermo
Scientific) on a Dionex Ultimate 3000 UPLC system equipped with a TSQ Vantage triple quadrupole
mass spectrometer for analysis. A binary mobile phase (A, water with 0.05% formic acid; B,
acetonitrile with 0.05% formic acid) was used to achieve the gradient of initial 30% B for 1 min and
then to 80% B at 8 min. The settings of HESI source were as follows: spray voltage (3200 V);
o
vaporizer temperature (365 C); sheath gas pressure (45 psi); auxiliary gas pressure (10 psi); capillary
o
temperature (330 C). Nitrogen was used as the sheath gas and auxiliary gas. Argon was used as the
collision gas.
4.12. Incubation of test compounds with liver microsomes (Total volume 500 µL).
28

Each test compound (curcumin, 3, 17a, and 17b) was incubated with rat liver microsomes, NADPH,
and UGT reaction mixtures to evaluate their metabolic stability under both phase I oxidation and phase
II glucuronidation conditions. The incubation solution was prepared by adding 50 µL of potassium
phosphate buffer (pH 7.4; 10×), 25 µL of NADPH solution A, 5 µL of NADPH solution B, 25 µL of
pooled rat liver microsomes (Female; 20 mg/mL), 250 µL water, 100 µL of UGT Reaction Mix
solution B, and 40 µL of UGT Reaction Mix solution A. The mixture was incubated at 37 oC for 5 min
in incubator. Then, 5 µL of a 0.1 mM test compound dissolved in ethanol was added to the above
mixture and incubated at 37 oC for 60 min in a temperature-controlled incubator. At the following time
points an aliquot of 50 µL incubation mixture was sampled: 0, 5, 15, 30, 45, 60 mins. To the aliquot of
incubation mixture was added 150 µL of MeOH to terminate the reaction and 5 ng of E-tamoxifen-
2C13, N15 as internal standard for quantitative analysis. The MeOH-incubation mixture was then
centrifuged at 10,000 × g for 4 min at 4 oC. The clear top layer supernatant was injected on a UHPLC-
TSQ mass spectrometer to measure the concentration of the test compounds present in the incubation
solution at different time points.
4.13. Statistical analysis
All data are represented as the mean ± standard deviation (S.D.) for the number of experiments
indicated. Other differences between treated and control groups were analyzed using the Student’s t-
test. A p-value < 0.05 was considered statistically significant.
ACKNOWLEDGMENT
This work was financially supported by California State University (CSU)-Fresno and CSU Program
for Education and Research in Biotechnology (CSUPERB) Research Development Grant (2015). This
study was also supported in part by NIH RCMI program at Xavier University of Louisiana through
Grant 2G12MD007595 (G. Wang). We are also grateful to the Graduate Net Initiative at CSU-Fresno
29