Asymmetric 1,5-diarylpenta-1,4-dien-3-ones: Antiproliferative activity in prostate epithelial cell models and pharmacokinetic studies

Article abstract of DOI:10.1016/j.ejmech.2017.05.062

To further engineer dienones with optimal combinations of potency and bioavailability, thirty-four asymmetric 1,5-diarylpenta-1,4-dien-3-ones (25–58) have been designed and synthesized for the evaluation of their in vitro anti-proliferative activity in three human prostate cancer cell lines and one non-neoplastic prostate epithelial cell line. All these asymmetric dienones are sufficiently more potent than curcumin and their corresponding symmetric counterparts. The optimal dienone 58, with IC₅₀ values in the range of 0.03–0.12 μM, is 636-, 219-, and 454-fold more potent than curcumin in three prostate cancer cell models. Dienones 28 and 49 emerged as the most promising asymmetric dienones that warrant further preclinical studies. The two lead compounds demonstrated substantially improved potency in cell models and superior bioavailability in rats, while exhibiting no acute toxicity in the animals at the dose of 10 mg/kg. Dienones 28 and 46 can induce PC-3 cell cycle regulation at the G₀/G₁ phase. However, dienone 28 induces PC-3 cell death in a different way from 46 even though they share the same scaffold, indicating that terminal heteroaromatic rings are critical to the action of mechanism for each specific dienone.

Full text of DOI:10.1016/j.ejmech.2017.05.062

Accepted Manuscript
Asymmetric 1,5-diarylpenta-1,4-dien-3-ones: Antiproliferative activity in prostate
epithelial cell models and pharmacokinetic studies
Xiaojie Zhang, Shanchun Guo, Chengsheng Chen, German Ruiz Perez, Changde
Zhang, Manee Patanapongpibul, Nithya Subrahmanyam, Rubing Wang, Joshua
Keith, Guanglin Chen, Yan Dong, Qiang Zhang, Qiu Zhong, Shilong Zheng, Guangdi
Wang, Qiao-Hong Chen
PII:
S0223-5234(17)30433-6
10.1016/j.ejmech.2017.05.062
EJMECH 9492
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2017
Revised Date: 18 May 2017
Accepted Date: 31 May 2017
Please cite this article as: X. Zhang, S. Guo, C. Chen, G.R. Perez, C. Zhang, M. Patanapongpibul, N.
Subrahmanyam, R. Wang, J. Keith, G. Chen, Y. Dong, Q. Zhang, Q. Zhong, S. Zheng, G. Wang, Q.-
H. Chen, Asymmetric 1,5-diarylpenta-1,4-dien-3-ones: Antiproliferative activity in prostate epithelial cell
models and pharmacokinetic studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.062.
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ACCEPTED MANUSCRIPT
Table of Contents Graphic
Asymmetric 1,5-Diarylpenta-1,4-dien-3-ones: Antiproliferative Activity in Prostate Epithelial
Cell Models and Pharmacokinetic Studies, Xiaojie Zhang, Shanchun Guo, Chengsheng Chen,
German Ruiz Perez, Changde Zhang, Manee Patanapongpibul, Nithya Subrahmanyam, Rubing Wang,
Joshua Keith, Guanglin Chen, Yan Dong, Qiang Zhang, Qiu Zhong, Shilong Zheng, Guangdi Wang,
and Qiao-Hong Chen
Poor Bioavailability
Good Bioavailability
Good Bioavailability
1

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Asymmetric 1,5-Diarylpenta-1,4-dien-3-ones: Antiproliferative Activity in Prostate
Epithelial Cell Models and Pharmacokinetic Studies
Xiaojie Zhanga, Shanchun Guob,c, Chengsheng Chena, German Ruiz Pereza, Changde Zhangb,c, Manee
Patanapongpibula, Nithya Subrahmanyama, Rubing Wanga, Joshua Keitha, Guanglin Chena, Yan Dongd,
Qiang Zhangb,c, Qiu Zhongb,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 and cRCMI Cancer Research Center, Xavier University of Louisiana, 1
Drexel Drive, New Orleans, LA 70125, USA
dDepartment of Structural and Cellular Biology, Tulane University School of Medicine, New Orleans,
LA 70112, USA
ABSTRACT: To further engineer dienones with optimal combinations of potency and bioavailability,
thirty-four asymmetric 1,5-diarylpenta-1,4-dien-3-ones (25-58) have been designed and synthesized for
the evaluation of their in vitro anti-proliferative activity in three human prostate cancer cell lines and
one non-neoplastic prostate epithelial cell line. All these asymmetric dienones are sufficiently more
potent than curcumin and their corresponding symmetric counterparts. The optimal dienone 58, with
IC50 values in the range of 0.03-0.12 µM, is 636-, 219-, and 454-fold more potent than curcumin in
three prostate cancer cell models. Dienones 28 and 49 emerged as the most promising asymmetric
dienones that warrant further preclinical studies. The two lead compounds demonstrated substantially
improved potency in cell models and superior bioavailability in rats, while exhibiting no acute toxicity
in the animals at the dose of 10 mg/kg. Dienones 28 and 46 can induce PC-3 cell cycle regulation at the
G0/G1 phase. However, dienone 28 induces PC-3 cell death in a different way from 46 even though
they share the same scaffold, indicating that terminal heteroaromatic rings are critical to the action of
mechanism for each specific dienone.
Key words: 1,5-diheteroarylpenta-1,4-dien-3-one, prostate cancer, cell proliferation, pharmacokinetic
study, cell apoptosis
____________
Corresponding author.
*E-mail: qchen@csufresno.edu. Phone: (+1)5592782394. Fax: (+1)5592784402.
1

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1. Introduction
Curcumin (1), a pleiotropic diarylheptanoid, is the major chemical component contributing to the
diverse bioactivities of turmeric (the rhizomes of Curcuma longa L.) [1]. The potential of curcumin in
treating prostate cancer has been intensively investigated since 2000 when its capability in suppressing
prostate cancer cell proliferation was first revealed by Dorai and co-workers [2-4]. To address its key
weaknesses as a drug candidate, a plethora of research efforts have been devoted to the development of
its analogues with improved potency and/or bioavailability [3,5]. Monoketone curcumin mimics, in
which the metabolically unstable diketone moiety in curcumin is substituted with a monoketone, have
been demonstrated as a group of promising anti-cancer agents with 10-20 times improved in vitro
potency relative to curcumin [3,5]. Our laboratory has systematically investigated the effect of central
monoketone-containing linker and terminal rings on the in vitro potency of the monoketone curcumin
mimics in prostate cancer cell models [6-9]. Our previous findings have revealed that terminal basic
nitrogen-containing heteroaromatic rings are obviously beneficial to the enhanced cytotoxic and anti-
proliferative potency and that the 1,5-diheteroarylpenta-1,4-dien-3-one is the most promising class of
curcumin-based anti-prostate cancer agents, with the most potent compounds being over 100 folds
more potent than curcumin against prostate cancer cell lines [6,7]. Most monoketone curcumin mimics
are symmetric with two identical terminal aromatic rings, but a few of recent reports suggest that
asymmetric monoketone curcumin mimics might exhibit more desirable biological profile as compared
to the corresponding symmetric counterparts [10,11]. All 1,5-diheteroarylpenta-1,4-dien-3-ones
previously reported by us are symmetric with two identical terminal nitrogen-containing
heteroaromatic rings [7], but we have noticed from our previous data that different terminal
heteroaromatic rings can bring in varied benefits to the scaffold of 1,5-diheteroarylpenta-1,4-dien-3-
one. For example, 1-alkyl-1H-imidazol-2-yl moiety in analogues 2-4 and 1-alkyl-1H-
benzo[d]imidazole-2-yl moiety in analogues 5-6 (Figure 1) are beneficial to the optimal potency of
these compounds, whereas 1-alkyl-1H-imidazol-2-yl moiety in analogue 3 affords little enhancement in
its pharmacokinetic profile. On the other hand, 2-methyl-4-(trifluoromethyl)thiazol-5-yl bestows
analogue 7 with an attractive in vivo pharmacokinetic profile but only with a moderate increase in
potency [7]. These data prompted us to explore a new group of asymmetric 1,5-diheteroarylpenta-1,4-
dien-3-ones (25-58) with the hope of integrating optimal potency and pharmacokinetic profile by
incorporating two different heteroaromatic rings into a single curcumin dienone mimic.
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The standard of care for prostate cancer has been androgen deprivation therapy (ADT) to block
androgen-dependent prostate cancer growth. However, after varying duration of progression free
period, most late stage prostate cancers eventually progress to castration-resistant tumors that are no
longer responsive to ADT. Further treatment with CYP17A1 inhibitors such as abiraterone or AR
antagonists such as enzalutamide has clinically proven to prolong patient survival but the disease
remains incurable beyond this stage. Expression of truncated AR variant proteins via AR alternative
splicing emerged as an important mechanism of abiraterone and enzalutamide resistance in prostate
cancer. Therefore, new anticancer agents that can overcome resistance to current CRPC regimens are
highly desirable. To this end, we also evaluated the activities of selected asymmetric curcumin mimics
in three prostate cancer cell lines that harbor AR splicing variants and are resistant to enzalutamide
treatment.
O
O
O
7
1
5
1
N
N
2
6
2
4
5
3
3
4
N
R
N
HO
OH
OCH3
R
metabolic
unstable
OCH3
2, R = sec-Butyl, 34-55 times more potent
3, R = isopentyl, 20-37 times more potent
AUC = 5.04 ng/mL.h
curcumin (1)
AUC = 2.43 ng/mL.h
4, R = pentan-2-yl, 57-90 times more potent
O
CF3
N
CF3
S
O
N
N
N
N
N
R
S
R
7 23-36 times more potent
5, R = Methyl
AUC = 892.77 ng/mL.h
6, R = isopropyl
Fig. 1. Structures, antiproliferative potency, and pharmacokinetic profiles of curcumin and symmetric
1,5-diheteroarylpenta-1,4-dien-3-ones (2-7) [7]
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2. Results and Discussion
2.1 Chemistry
The desired thirty-four asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25-58) have been
synthesized through two sequential Horner-Wadsworth-Emmons reactions of 1,3-
bis(diethylphosphonato)acetone (9) with the appropriate aromatic carbadehyde (8) (Scheme 1). 1-
Alkyl-1H-imidazole-2-carbaldehydes and 1-alkyl-1H-benzo[d]imidazole-2-carbaldehydes were
synthesized according to the procedure illustrated in the literature [7,12]. All other aromatic
carbaldehydes were obtained from commercial sources. In the case of preparing fifteen (E)-diethyl(2-
oxo-4-heteroaryl-but-3-en-1-yl)phosphonates (10-24) as the intermediates through the Horner-
Wadsworth-Emmons reaction, 1 equivalent of the appropriate carbaldehye was added slowly to the
reaction mixture of 1 equivalent of 1,3-bis(diethylphosphonato)acetone (9) and potassium carbonate in
ethanol and water (0.1 M). This strategy was implemented to reduce the formation of symmetric 1,5-
diheteroarylpenta-1,4-dien-3-ones.
Note: BHR: Basic nitrogen containing heteroaromatic ring.
For the structures of phosphonates 10-24, refer to Table 1.
For the structures of dienones 25-58, refer to Table 2.
Scheme 1. Synthesis of asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25-58)
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Table 1
Structures for (E)-diethyl(2-oxo-4-heteroaryl-but-3-en-1-yl)phosphonates 10-24
O
O
P
OEt
OEt
BHR
BHR
compd
10
11
12
13
14
BHR
compd
15
16
17
18
19
BHR
compd
20
21
22
23
24
Table 2
Structures for asymmetric 1,5-diarylpenta-1,4-dien-3-ones (25-58)
Compd
1-BHR
5-BHR or 5-AR
Compd
1-BHR
5-BHR or 5-AR
N
N
25
26
Br
5

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27
29
31
33
35
28
30
32
34
36
N
N
Br
37
39
41
38
40
42
43
45
44
N
N
46
6

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47
48
49
51
50
52
53
55
57
54
56
58
2.2 Antiproliferative Activity towards Prostate Cancer Cell Lines
The in vitro anti-proliferative activity of the thirty-four asymmetric 1,5-diheteroarylpenta-1,4-dien-3-
ones (25-58) against both androgen-sensitive and androgen-insensitive prostate cancer cell lines
(LNCaP, DU145, and PC-3) were assessed by WST-1 cell proliferation assay according to the
procedure as described in the Experimental Section. Curcumin was used as a positive control for
comparison and the anti-proliferative potency of each test dienone was represented as IC50 values. As
shown in Table 3, all the asymmetric dienones exhibit much greater potency than curcumin in
suppressing prostate cancer cell proliferation. Their IC50 values towards PC-3, DU145, and LNCaP
human prostate cancer cell line are in the ranges of 0.04-6.86 µM, 0.12-3.68 µM, and 0.03-4.05 µM,
7

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respectively. The optimal dienone 58 with IC50 values in the range of 0.03-0.12 µM is 636-, 219-, and
454-fold more potent than curcumin in three prostate cancer cell models.
The structure-antiproliferative activity relationships of the asymmetric 1,5-diheteroarylpenta-1,4-dien-
3-ones can be summarized as below:
− All thirty-four asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones exhibit far greater potency
towards three human prostate cancer cell lines (Table 3) than curcumin, implying that
asymmetric (1E, 4E)-1,5-diheteroarylpenta-1,4-dien-3-one is an optimal scaffold for curcumin
mimics with substantially improved potency in inhibiting prostate cancer cell proliferation.
− Asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25-58) are significantly more potent than
their symmetric counterparts reported previously by us [7]. For example, (1E,4E)-1,5-bis(1-
isopropyl-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-one (6, symmetric) is 30-111 times more
potent [7] and (1E,4E)-1,5-di(pyridin-2-yl)penta-1,4-dien-3-one (symmetric) is 15-60 folds
more potent [6] than curcumin towards three prostate cancer cell lines; in contrast, the
asymmetric version (dienone 58) with pyridine-2-yl and 1-isopropyl-1H-benzo[d]imidazole-2-
yl moieties is 219-636 folds more potent than curcumin.
− The following four pairs of heteroaromatic rings serve as the optimal combinations of the
terminal rings for the promising potency: i) 1-alkyl-1H-imidazol-2-yl and 2-methyl-4-
(trifluoromethyl)thiazol-5-yl moieties in dienone 28, ii) 1-alkyl-1H-imidazol-2-yl and 1-alkyl-
1H-benzo[d]imidazole-2-yl moieties in dienones 41-47, iii) 1-alkyl-1H-benzo[d]imidazole-2-yl
and 2-methyl-4-(trifluoromethyl)thiazol-5-yl moieties in dienone 49, and iv) 1-alkyl-1H-
benzo[d]imidazole-2-yl and pyridine-2-yl moieties in dienones 56-58.
Table 3
Anti-proliferative activity of the asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25-58) toward
three prostate cancer cell lines
IC50 (µM)a
DU145c
26.23 ± 0.65 13.61 ± 2.69
IC50 (curcumin)/IC50(dienone)
Compd
PC-3b
LNCaPd PC-3b DU145c
LNCaPd
Curcumin
25.43 ± 2.15
1
1
1
25
0.39 ± 0.08
0.42 ± 0.09
0.17 ± 0.02
64
62
80
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26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
0.33 ± 0.02
0.31 ± 0.03
0.23 ± 0.02
0.37 ± 0.02
0.72 ± 0.01
1.33 ± 0.20
0.26 ± 0.02
0.55 ± 0.05
0.30 ± 0.04
0.31 ± 0.03
0.45 ± 0.04
6.86 ± 0.37
3.40 ± 0.21
2.27 ± 0.12
0.80 ± 0.04
0.18 ± 0.01
0.24 ± 0.02
0.20 ± 0.05
0.21 ± 0.04
0.14 ± 0.04
0.10 ± 0.01
0.19 ± 0.04
0.28 ± 0.04
0.53 ± 0.04
0.35 ± 0.04
0.75 ± 0.03
0.31 ± 0.01
0.24 ± 0.04
0.22 ± 0.05
0.24 ± 0.04
76
82
94
50
75
35
44
57
62
57
21
15
52
30
57
62
44
3
110
69
0.67 ± 0.02
0.85 ± 0.04
0.66 ± 0.04
0.91 ± 0.08
35
19
39
31
0.29 ± 0.03
1.13 ± 0.12
0.34 ± 0.05
0.50 ± 0.03
0.70 ± 0.10
3.68 ± 0.08
2.55 ± 0.23
1.32 ± 0.27
0.66 ± 0.16
0.13 ± 0.10
0.35 ± 0.05
0.37 ± 0.12
0.77 ± 0.20
0.28 ± 0.04
0.22 ± 0.03
0.31 ± 0.08
0.26 ± 0.02
0.45 ± 0.07
0.24 ± 0.02
0.22 ± 0.01
0.31 ± 0.11
4.05 ± 0.76
2.17 ± 0.19
1.41 ± 0.13
0.54 ± 0.05
0.26 ± 0.07
0.33 ± 0.05
0.56 ± 0.05
0.93 ± 0.33
0.37 ± 0.14
0.22 ± 0.11
0.40 ± 0.03
98
46
90
23
77
53
38
7
85
82
57
4
8
10
20
40
138
75
71
34
94
119
85
6
11
10
25
52
41
24
41
37
62
34
32
141
106
127
121
182
254
134
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48
49
50
51
52
53
54
55
56
57
58
0.48 ± 0.02
0.23 ± 0.00
0.55 ± 0.03
0.34 ± 0.05
0.84 ± 0.03
0.54 ± 0.04
1.70 ± 0.20
0.56 ± 0.05
0.23 ± 0.02
0.25 ± 0.02
0.04 ± 0.01
0.78 ± 0.06
0.50 ± 0.02
0.84 ± 0.14
0.57 ± 0.04
0.83 ± 0.09
0.63 ±0.06
2.05 ± 0.15
0.68 ± 0.27
0.46 ± 0.13
0.32 ± 0.05
0.12 ± 0.05
0.34 ± 0.09
0.25 ± 0.03
0.46 ± 0.07
0.30 ± 0.19
0.40 ± 0.02
0.30 ± 0.07
1.76 ± 0.15
0.18 ± 0.04
0.12 ± 0.05
0.17 ± 0.08
0.03 ± 0.01
53
110
46
34
53
31
46
32
42
13
39
57
82
219
40
54
30
45
34
45
8
75
30
47
15
45
76
113
80
454
111
102
636
a IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay (WST-1) after
3 days exposure. The data were presented as the mean ± standard derivation of the mean.
b Human androgen-insensitive prostate cancer cell line
c Human androgen-insensitive prostate cancer cell line
d Human androgen-sensitive prostate cancer cell line
2.3 Antiproliferative Activity towards PWR-1E Non-neoplastic Human Prostate Epithelial Cell Line
Fifteen dienones (25-28, 32, 34-35, 41, 45-46, 49, 51, and 56-58) were selected as the representatives
of different subgroups for further evaluation of their ability in inhibiting PWR-1E benign human
prostatic epithelial cell proliferation. 25-28 are the optimal dienones with two different five-membered
heteroaromatic rings; 32, 34, and 35 are the representatives of the dienones with one five-membered
and one six-membered heteroaromatic rings; 41, 45-46, 49, and 51 are those good examples with
bicyclic 1-alkyl-1H-benzo[d]imidazole-2-yl as one terminal ring and a five-membered heteromatic as
another terminal ring; and 56-58 represent the subgroup having one bicyclic 1-alkyl-1H-
benzo[d]imidazole-2-yl as one terminal ring and one six-membered heteromatic as the other terminal
ring. PWR-1E human prostatic epithelial cell line expresses prostate specific antigen (PSA) and
androgen receptor (AR) and mimics normal growth and differentiation responses to androgen [13]. The
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PWR-1E cell line was originally isolated from a non-malignant prostate with mild hyperplasia and
immortalized by adenovirul 12/Simian 40. Curcumin was used as a positive control as curcumin's
general human safety profile has been validated by clinical trials [4,14] and animal studies [15]. The
apoptotic cell death pathway in both normal human primary prostate epithelial cells and androgen-
sensitive human prostate cancer cells could be triggered by androgen deprivation [16]. In this study, we
observed the IC50 values for curcumin are 8.55 µM for PWR-1E cells and 13.61 µM for LNCaP cells,
suggesting no differential responses to LNCaP and PWR-1E cells. This is reasonable considering that
curcumin can downregulate the expression and activity of AR and PSA in LNCaP prostate cancer cells
[17] and that both LNCaP prostate cancer cells and PWR-1E non-neoplastic prostate epithelial cell
lines express androgen receptor and androgen specific antigen. As shown in Table 4, the asymmetric
dienones have 63- to 885-fold greater potency in suppressing PWR-1E benign prostate cell
proliferation, as compared with curcumin. The asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25,
26-28, 32, 34-35) having five-membered and six-membered heteroaromatic rings as their terminal rings
display approximately equivalent potency towards the PWR-1E non-neoplastic prostate epithelial cell
line and the three human prostate cancer cell lines; while the asymmetric 1,5-diheteroarylpenta-1,4-
dien-3-ones (41, 45-46, 49, 51, and 56-58) possessing a bicyclic 1-alkyl-1H-benzo[d]imidazole-2-yl as
one terminal ring exhibit substantially higher anti-proliferative potency against the PWR-1E non-
neoplastic prostate epithelial cell line than three human prostate cancer cell lines.
Table 4
Anti-proliferative activity of selected asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones toward PWR-
1E prostate epithelial cells that express AR and PSA.
Compd
IC50 (µM)
IC50 (curcumin)/IC50(dienone)
Curcumin
8.85 ± 0.70
0.14 ± 0.03
1
25
63
26
27
28
32
0.14 ± 0.07
0.11 ± 0.03
0.11 ± 0.04
0.13 ± 0.01
63
81
81
68
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34
35
41
45
46
49
51
56
57
58
0.17 ± 0.01
0.10 ± 0.02
0.03 ± 0.01
0.04 ± 0.00
0.02 ± 0.01
0.08 ± 0.03
0.05 ± 0.01
0.04 ± 0.03
0.02 ± 0.03
0.01 ± 0.01
52
89
293
221
443
111
177
221
443
885
2.4 In vitro cytotoxicity in enzalutimide-resistant prostate cancer cell models expressing AR splice
variants:
To test if the asymmetric curcumin mimics are effective against ligand independent prostate cancer,
we treated three cell lines, LNCaP95, VCaP, and 22Rv1 [18,19] with four potent compounds, 28, 46,
49, and 58. The IC50 values, the concentrations for test compounds effective in suppressing 50% of the
cell viability, were measured by the trypan blue exclusion assay after 5 days exposure. As shown in
Table 5, these four mimics also exhibited impressive cytotoxicity against LNCaP95, VCaP, and 22Rv1
prostate cancer cell lines, with IC50 values ranging from 0.22 µM to 1.41 µM.
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Table 5
Cytotoxicity of selected asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones against three ligand
independent prostate cancer cell lines expressing AR splice variants.
Compd
IC50 (µM)
VCaP
0.35
LNCaP95
0.37
22Rv1
0.85
28
46
49
58
0.35
1.12
0.22
0.38
1.41
0.22
0.26
0.64
0.45
2.5. In vivo pharmacokinetic studies and acute toxicity in rat:
The overarching goal of this research is to engineer curcumin mimics with improved potency and
bioavailability. To evaluate if the asymmetric curcumin mimics with markedly improved anticancer
activities could also possess greater bioavailability, we chose four most promising mimics, 28, 46, 49,
and 58, for pharmacokinetic studies. Among them, dienones 28 and 49 were highly expected to have
good bioavailability because they both contain 2-methyl-4-(trifluoromethyl)thizaol-5-yl moiety that has
been demonstrated by us to confer analogue 7 with an attractive in vivo pharmacokinetic profile in mice
[7]. In this study we sought to evaluate the pharmacokinetic profiles for these lead compounds in
Sprague Dawley rats, a species that are more suited for bioavailability study. The animals administered
with 28, 46, 49, or 58, via oral gavage at a single dose of 10 mg/kg, and blood samples were collected
at 1, 3, 6, and 24 hours after oral administration. Plasma was prepared from the blood samples and was
analyzed by HPLC-MS/MS for determination of drug concentrations as described in the Experimental
Section. Summarized in Table 6 are the plasma concentrations of 28, 46, 49, and 58 at different
sampling time points, which lead to the conclusion that, among the four dienones, compound 46 gains
the least improvement in its bioavailability with the peak concentration at 280.9 ng/mL. The relatively
poor pharmacokinetic results of (1E,4E)-1-(1-propyl-1H-benzo[d]imidazole-2-yl)-5-(1-propyl-1H-
imidazole-2-yl)penta-1,4-dien-3-one (46) are consistent with our previous reports where (1E,4E)-1,5-
bis(1-isopentyl-1H-imidazol-2-yl)penta-1,4-dien-3-one (3) only showed very little improvement in its
peak concentration and AUC value as compared with curcumin.7 These data collectively suggest that
location of 1-alkyl-1H-imidazole-2-yl and/ or 1-alkyl-1H-benzo[d]imidazole-2-yl to both terminal
13

ACCEPTED MANUSCRIPT
rings of the dienones resulted in markedly improved anti-proliferative potency, but little enhancement
in bioavailability.
Conversely, assignment of a 1-alkyl-1H-imidazole-2-yl or 1-alkyl-1H-benzo[d]imidazole-2-yl
moiety as one terminal ring and incorporation of a 2-methyl-4-(trifluoromethyl)thiazol-5-yl or pyridin-
2-yl moiety as the other terminal ring lead to the very promising dienones 28, 49, and 58 with
substantially improved bioavailability, in addition to the great potency. The peak concentration for each
of these three dienones (28, 49, and 58) is 1943.8 ng/mL (5.27 µM), 1225.1 ng/mL (3.25 µM), and
4264.1 ng/mL (13.45 µM), respectively, far exceeding their IC50 values ranging from 0.03-0.50 µM in
three human prostate cancer cell lines. It is thus reasonable to conclude that the excellent bioavailability
of 28, 49, and 58, as demonstrated by their high peak plasma concentration and AUC values, will
provide the therapeutic efficacy necessary to block tumor growth.
The acute in vivo toxicity in rats indicates that the animals were able to tolerate the dose of 10 mg/kg
of dienones 28 and 49 without observed toxicity. However, we noticed that the rats died in about 48
hours and 72 hours, respectively, after orally given 46 or 58 at 10 mg/kg. Therefore, 28 and 49 are
more promising asymmetric dienones worthy for further development.
Table 6
24-Hour mouse-plasma concentrations of curcumin, 28, 46, 49, and 58
Times
Concentration in Plasma (ng/mL)
Curcumina
28
46
49
58
30 min
1 hr
0.04
0.33
0.57
360.7 ± 3.8
113.9 ± 2.2
196.5 ± 16.7
112.1 ± 5.9
982.3 ± 44.7
822.6 ± 62.1
4264.1 ± 185.3
2 hr
3 hr
672.4 ± 23.4
4 hr
0.13
6 hr
1943.8 ± 47.3 280.9 ± 23.7
1225.1 ± 43.0 456.3 ± 15.2
1 day
tmax (hrs)
Cmax (ng/mL)
0.03
2
230.3 ± 5.6
6
86.0 ± 5.9
6
314.1 ± 1.5
6
102.7 ± 4.4
3
0.57
1943.8 ± 47.3 280.9 ± 23.7
24704.65 4385.55
1225.1 ± 1.5
18314.35
4264.1 ± 185.3
17609.60
Area under curve 2.85
(AUC)
(ng/mL*h)
a.
The data for curcumin have been reported in our previous paper [7]
14

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Note: Single oral dose for 28, 46, 49, and 58 is 10 mg/kg in rats.
Single oral dose for curcumin is 1 mg/kg in mice.
2.6.Metabolic profiling of dienones 28 and 49
A preliminary metabolic transformation study was conducted for dienones 28 and 49 by in vitro
microsomal incubation experiments to identify major metabolites of these compounds. As shown in
Table 7, a total of three major metabolic products of dienone 28 were detected and identified based on
chromatographic and mass spectral data collected. All three metabolites, assigned as 28-MO1, 28-
MO2, and 28-MO3 (Scheme 2), were monohydroxylation products. The polarity of the metabolites
with various oxidation sites is consistent with the retention time indicating 28-MO1 is the most polar
metabolite followed by 28-MO2 and 28-MO3. For dienone 49, only two major metabolic products
were observed, 49-MO1 and 49-MO2 (Scheme 3). While the assignment of hydroxylation sites may
not be definitive with available analytical information, the mono-oxidation of both parent compounds
has been confirmed by their respective high resolution mass spectra (Table 7).
Table 7.
Analytical results of dienones 28, 49, and their respective metabolic products.
Compound Retention MH+:
Parent ion
Mass
error
(ppm)
Major Fragment ions
or
time
Theoretical (observed in
positive ion
metabolite (min)
mode)
4.35min 370.1201
370.1195
1.6
121, 246, 314 (M-
28
CHCH3CH2CH3)+2H),
3.32min 386.1150
3.7min 386.1150
3.97min 386.1150
5.05min 392.1044
386.1145
386.1145
386.1145
392.1039
1.3
1.3
1.3
1.3
314, 121, 294, 332, 136, 179
151, 167, 332, 220
330, 310, 151, 121, 167
28-MO1
28-MO2
28-MO3
49
199 (M-CHCH-C5H3F3NS);
171 (M+H-CHCH-C5H3F3NS-
CH2CH3), 372, 325, 352
215(M-CHCH-C5H3F3NS); 187
(M+H-CHCH-C5H3F3NS-
CH2CH3), 136, 341, 388
199 (M-CHCH-C5H3F3NS-O);
171 (M+H-CHCH-C5H3F3NS-
CH2CH3-O), 388, 215, 171
4.54min 408.0994
4.66min 408.0994
408.0988
408.0988
1.5
1.5
49-MO1
49-MO2
15

ACCEPTED MANUSCRIPT
Scheme 2. The metabolites of 28 from incubation with liver microsome
Scheme 3. The metabolites of 49 from incubation with liver microsome
2.7.Cell cycle regulation and cell apoptosis.
Curcumin has been reported to arrest PC-3 cell cycle at the G1/S phase [20]. The PC-3 cell cycle
regulations of dienones 28 and 46 were assessed using flow cytometry analysis with propidium iodide
DNA staining. The data illustrated in Figure 2 suggest that both of them induce cell cycle arrest at the
G0/G1 phase by accumulating PC-3 cell population in the G0/G1 phase, while fewer cells were observed
in the G2 phase. Specifically, dienone 46 (4 µM) increases the population of PC-3 cells in the G0/G1
phase from 51% and 59% (control cells) at 16 h and 24 h, respectively, to 78% and 76%. The
population of cells in the G2 phase decreases from 25% to 13% at 16 h, and from 24% in control cells
to 14% at 24 h. Similarly, treatment of PC-3 cells with dienone 28 (5 µM) led to 14% (at 16 h) and 4%
(at 24 h) higher cell population at the G0/G1 phase and 10% (at 16 h) and 3% (at 24 h) lower cell
population at the G2 phase, as compared with their control cells.
16

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G0/G1
78
76
68
66
80
60
40
20
0
62
59
52
51
G0/G1
G0/G1
G2
28
25
24
24
30
21
18
14
20
10
0
13
G2
G2
Fig. 2. Cell cycle analysis of PC-3 prostate cancer cells. PC-3 cancer cells were untreated or treated
with 28 at 5 µM and 46 at 4 µM, respectively. Cells were harvested after 16 h or 24 h, fixed, stained,
and analyzed for DNA content.
The growth suppression of PC-3 prostate cancer cells by curcumin has been demonstrated to be, at
least in part, associated with its cell apoptosis activation [2]. The violet excitable dye F2N12S can
detect membrane asymmetry changes during apoptosis and SYTOX AADVanced dead cell stain can
distinguish the cells with compromised membrane (late apoptotic and necrotic cells) from the viable
cells. The F2N12S and SYTOX AADVanced double staining assay in a flow cytometer was employed
for the discrimination between early apoptotic PC-3 cells and late apoptotic/necrotic PC-3 cells when
17

ACCEPTED MANUSCRIPT
treated with dienones 28 and 46 at the concentrations specified in Figures 3 and 4 for 16 h.
Staurosporine, a known apoptotic inducer, was used as positive apoptotic control in all experiments
(data not shown). As summarized in Figure 3, dienone 46 can simultaneously activate apoptotic and
necrotic cell death in the androgen-insensitive PC-3 prostate cancer cell line after a 16-hour treatment.
Specifically, exposure of PC-3 cells to 46 at 4 µM can lead to 22% of PC-3 cells in early phase of
apoptosis, as well as 23% late apoptotic/necrotic cells, as compared with control cells; treatment with
46 at 6-15 µM induces 43-52% early apoptotic cells together with 35-47% late apoptotic/necrotic cells.
It is worth noting that 28 induces PC-3 cell death in a different way from 46 even though they possess
same 1,5-diheteroarylpenta-1,4-dien-3-one scaffold, indicating that terminal heteroaromatic rings might
be very important to the action of mechanism for each specific dienone. As shown in Figure 4,
incubation of the PC-3 cells with dienone 28 for 16 h induced considerable levels of late
apoptotic/necrotic cells rather than early apoptotic cells. For example, 5 µM of dienone 28 can induce
39% late apoptotic/necrotic cells but only 11% early apoptotic cells.
Apoptotic Effect of 46 toward PC-3 Cells
120
96
95
93
100
80
60
40
20
0
55
52
47
Control
2 µM
49
43
45
42
43
35
23
22
22
13
8
4
3
3
2
2
2
1
Control DMSO
Viable Cell
2 µM
4 µM
6 µM
8 µM
10 µM 15 µM
Apoptotic Cell
Necrotic Cell
4 µM
8 µM
Fig. 3. Evolution of viable, apoptotic, and necrotic PC-3 cells populations in response to 46.
18

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Apoptotic Effect of 28 toward PC-3 Cells
120
95
95
100
80
60
40
20
0
54
53
51
50
49
46
Control
5 µM
39
37
34
20
33
13
26
23
25
22
14
11
3
3
2
2
Control DMSO
Viable Cell
5μM
10μM
15μM
20μM
30μM
40μM
Apoptotic Cell
Necrotic Cell
15 µM
30 µM
Fig. 4. Evolution of viable, apoptotic, and necrotic PC-3 cells populations in response to 28.
3. Conclusion
To optimize monoketone curcumin mimics as anti-prostate cancer agents, thirty-four asymmetric 1,5-
diheteroarylpenta-1,4-dien-3-ones (25-58) have been designed and synthesized for the evaluation of
their in vitro antiproliferative activity in three human prostate cancer cell lines and one human non-
neoplastic prostate epithelial cell line. All these asymmetric dienones are sufficiently more potent than
curcumin and their corresponding symmetric counterparts. The optimal dienone 58 with IC50 values in
the range of 0.03-0.12 µM is 636-, 219-, and 454-fold more potent than curcumin in three prostate
cancer cell models. However, its in vivo acute toxicity in mice may limit the further development of
dienone 58. Dienones 28 and 49 have been identified as more-promising asymmetric dienones based on
their substantially improved potency in cell models and excellent bioavailability in mice, as well as the
lack of apparent acute toxicity in the animals at the dose of 10 mg/kg. Importantly, these asymmetric
curcumin mimics also demonstrate potent antiproliferative activities against ligand independent, AR-V
harboring prostate cancer cells that are resistant to all forms of hormonal therapy. Dienones 28 and 46
can induce PC-3 cell cycle regulation at the G0/G1 phase, but dienone 28 induces PC-3 cell death in a
different way from 46 even though they possess the same 1,5-diheteroarylpenta-1,4-dien-3-one scaffold,
suggesting that the terminal heteroaromatic rings may play a critical role in the underlying mechanism
19

ACCEPTED MANUSCRIPT
of action for each specific dienone. The further study on anticancer mechanism of dienone 28 is
ongoing.
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. The chemical shifts are given in
ppm referenced to the respective solvent peak, and coupling constants are reported in Hz. Anhydrous
THF and dichloromethane were 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). Curcumin was synthesized by Claisen-Schmidt condensation of aromatic
aldehyde with acetylacetone according to the procedure described in the literature [21]. 1,3-
Bis(diethylphosphonato)acetone was synthesized using the procedure illustrated in the literature [22].
The purities of thirty-two out of thirty-four biologically tested compounds are
≥ 95% as determined by
HPLC. Specifically, the major peak accounted for 95% of the combined total peak area when
≥
monitored by a Diode Array Detector (DAD) 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. The purity of two compounds, 37 and 38, cannot be
measured using the above-mentioned conditions. We did not further pursuit their purity because they
exhibited the poorest anti-proliferative potency.
4.2 General procedure for the synthesis of (E)-diethyl(2-oxo-4-aryl-but-3-en-1-yl)phosphonates 10-24
[8]
A solution of tetraethyl(2-oxopropane-1,3-diyl)bis(phosphonate) (0.4 mmol, 1 equiv.) and potassium
carbonate (55 mg, 0.4 mmol, 1 equiv.) in ethanol (1.4 mL) and water (2.1 mL) was stirred at 0oC for 30
min. The corresponding aromatic carbaldehyde (0.4 mmol, 1 equiv.) was added dropwise to the soltion.
o
The subsequent mixture was stirred at 0 C for 4-10 hours as determined by TLC prior to being
quenched with aqueous ammonium chloride solution (15 mL). The mixture was extracted with
dichloromethane (10 mL × 3), and the combined dichloromethane layers were dried over anhydrous
20

ACCEPTED MANUSCRIPT
magnesium sulfate and concentrated under reduced pressure. The crude product was subjected to PTLC
purification using DCM:MeOH (100:10, v/v) as eluent to give the respective phosphonate. The NMR
data of phosphonates 11 and 16-20 are in consistent with those reported in the literature [8]. The NMR
data for other phosphonates (10, 12-15, and 21-24) are listed in Supplementary Data.
4.3 General procedure for the synthesis of asymmetric (1E,4E)-1,5-diheteroarylpenta-1,4-dien-3-ones
(25-58) [8]
To a solution of the corresponding (E)-diethyl(2-oxo-4-aryl-but-3-en-1-yl)phosphonate (0.122
mmoL, 1 equiv.) in ethanol (0.6 mL) and water (0.9 mL) was added potassium carbonate (16 mg, 0.122
mmol, 1 equiv.) and the corresponding aromatic carbaldehyde(0.122 mmol, 1 equiv.), and the mixture
was stirred at room temperature for 2-48 h as determined by TLC. The reaction was quenched with
brine (15 mL), and the subsequent mixture was extracted with dichloromethane (10 mL × 3). The
combined extracts were dried over anhydrous magnesium sulfate and concentrated in vacuum. The
residue was purified over preparative thin layer chromatography, eluting with
dichloromethane/methanol (100:5-10, v/v) and/or ethyl acetate/methanol (100:5, v/v), to give the
respective product.
4.3.1 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(thiazol-2-yl)penta-1,4-dien-3-one (25)
1
Yellow oil, 82 % yield. H NMR (300 MHz, CDCl3)
δ
: 7.95 (1H, d,
= 15.0 Hz, vinyl H), 7.59 (1H, d,
= 3.3 Hz, thiazole H-5), 7.24 (1H, s, imidazole H-4), 7.23 (1H, d,
7.11 (1H, s, imidazole H-5), 4.40 (1H, sextet,
CH2CH3), 1.47 (3H, d, = 6.6 Hz, sec-butyl CH3CH), 0.84 (3H, t,
NMR (75 MHz, CDCl3) : 188.1, 164.1, 145.2, 143.1, 134.4, 131.3, 130.6, 127.5, 125.8, 121.9, 119.2,
J = 3.3 Hz, thiazole H-4), 7.86
(1H, d,
J
= 15.9 Hz, vinyl H), 7.65 (1H, d,
J
J
= 15.0 Hz, vinyl H),
7.47 (1H, d,
J
J = 15.9 Hz, vinyl H),
J
= 7.2 Hz, sec-butyl CH), 1.88-1.72 (2H, m, sec-butyl
= 7.2 Hz, sec-butyl CH2CH3). 13C
J
J
δ
53.6, 31.0, 22.0, 10.7. IR (film) νmax: 3106, 2969, 2930, 1651, 1615, 1589, 1505, 1457, 1419 cm-1. HR-
MS (ESI) m/z: calcd for C15H18N3OS [M+H]+: 288.1170; found 288.1166. HPLC purity 95.3% (30 min
run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.2
(1E,4E)-1-(4-Bromo-1-methyl-1H-pyrazol-3-yl)-5-(1-(sec-butyl)-1H-imidazol-2-yl)penta-1,4-
dien-3-one (26)
Yellow oil, 69 % yield. 1H NMR (300 MHz, CDCl3)
δ: 7.69 (1H, d, J = 16.2 Hz, vinyl H), 7.68 (1H,
d,
J = 15.6 Hz, vinyl H), 7.62 (1H, d, J = 15.0 Hz, vinyl H), 7.44 (1H, s, pyrazole H), 7.34 (1H, d, J =
16.2 Hz, vinyl H), 7.28 (1H, s, imidazole H-4), 7.11 (1H, s, imidazole H-5), 4.43 (1H, sextet,
J
= 7.2
21

ACCEPTED MANUSCRIPT
Hz, sec-butyl CH), 3.95 (3H, s,
sec-butyl CH3CH), 0.86 (3H, t,
N
J
-
CH3), 1.89-1.73 (2H, m, sec-butyl CH2CH3), 1.49 (3H, d,
J
= 6.9 Hz,
: 188.8,
13
= 7.2 Hz, sec-butyl CH2CH3). C NMR (75 MHz, CDCl3)
δ
145.3, 143.3, 132.3, 132.1, 131.1, 128.1, 126.8, 126.3, 118.8, 96.2, 53.5, 40.2, 31.0, 22.0, 10.7. IR
(film) νmax: 3116, 2971, 2933, 1669, 1623, 1507, 1489, 1457, 1418 cm-1. HR-MS (ESI) m/z: calcd for
C16H20BrN4O [M+H]+: 363.0820, 365.0800; found 363.0817, 365.0796. HPLC purity 95.8% (30 min
run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.3 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(5-methylisoxazol-3-yl)penta-1,4-dien-3-one (27)
Yellow oil, 49 % yield. 1H NMR (300 MHz, CDCl3)
= 16.2 Hz, vinyl H), 7.60 (1H, d, = 16.2 Hz, vinyl H), 7.25 (1H, s, imidazole H-4), 7.12 (1H, s,
imidazole H-5), 6.93 (1H, d, = 16.5 Hz, vinyl H), 6.23 (1H, s, isoxazole H-4), 4.41 (1H, sextet,
6.9 Hz, sec-butyl CH), 2.47 (3H, s, isoxazole 5-CH3), 1.87-1.83 (2H, m, sec-butyl CH2CH3), 1.48 (3H,
d, = 6.6Hz, sec-butyl CH3CH), 0.85 (3H, t,
= 7.2 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz,
CDCl3) : 188.2, 170.5, 160.4, 143.1, 133.1, 131.4, 130.7, 127.8, 125.1, 119.2, 99.7, 53.7, 31.1, 22.0,
12.5, 10.7. IR (film) νmax: 3128, 2970, 2931, 1657, 1630, 1599, 1458, 1268 cm-1. HR-MS (ESI) m/z
δ: 7.69 (1H, d, J = 16.5 Hz, vinyl H), 7.65 (1H,
d,
J
J
J
J =
J
J
δ
:
calcd for C16H20N3O2 [M+H]+: 286.1556; found 286.1550. HPLC purity 98.7% (30 min run of 45-80%
CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.4
1,4-dien-3-one (28)
Yellow solid, mp 79-80 C, 79 % yield. H NMR (300 MHz, CDCl3)
vinyl H), 7.63 (1H, d, = 17.4 Hz, vinyl H), 7.58 (1H, d, = 17.4 Hz, vinyl H), 7.27 (1H, s, imidazole
H-4), 7.12 (1H, d, = 0.6 Hz, imidazole H-5), 6.72 (1H, d, =15.6 Hz, vinyl H), 4.40 (1H, sextet,
6.9 Hz, sec-butyl CH), 2.75 (3H, s, thiazole 2-CH3), 1.87-1.75 (2H, m, sec-butyl CH2CH3), 1.48 (3H, d,
(1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)penta-
o
1
δ: 7.96 (1 H, d, J = 15.6 Hz,
J
J
J
J
J =
J
= 6.6 Hz, sec-butyl CH3CH), 0.84 (3H, t,
J
= 7.2 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz, CDCl3)
δ: 187.1, 167.7, 143.5 (JCF = 35 Hz), 142.7, 136.5, 132.3, 130.4, 129.7, 126.8, 126.7, 120.8 (JCF
=
270.8 Hz), 119.3, 54.0, 31.0, 22.0, 19.8, 10.7. IR (film) νmax: 3071, 1640, 1603, 1575, 1493, 1387 cm-1.
HR-MS (ESI) m/z: calcd for C17H19N3OF3S [M+H]+: 370.1201; found 370.1198. HPLC purity 96.0%
(30 min run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.5 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(3-methylisoxazol-4-yl)penta-1,4-dien-3-one (29)
Yellow oil, 51 % yield. 1H NMR (300 MHz, CDCl3)
= 15.0 Hz, vinyl H), 7.57 (1H, d, = 16.2 Hz, vinyl H), 7.28 (1H, s, imidazole H-4), 7.12 (1H, s,
imidazole H-5), 7.07 (1H, d, =16.2 Hz, vinyl H), 6.38 (1H, s, isoxazole H-5), 4.41 (1H, sextet,
δ: 7.71 (1H, d, J = 15.0 Hz, vinyl H), 7.59 (1H,
d,
J
J
J
J
= 7.2
22

ACCEPTED MANUSCRIPT
Hz, sec-butyl CH), 2.33 (3H, s, isoxazole 3-CH3), 1.87-1.74 (2H, m, sec-butyl CH2CH3), 1.48 (3H, d,
J
= 6.6 Hz, sec-butyl CH3CH), 0.84 (3H, t,
J
= 7.5 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz, CDCl3)
δ: 187.8, 165.9, 160.6, 142.8, 130.7, 130.5, 126.8, 126.6, 126.4, 119.2, 107.9, 53.9, 31.0, 22.0, 11.5,
10.7. IR (film) νmax: 2925, 1647, 1623, 1616, 1577, 1558, 1521, 1507 cm-1. HR-MS (ESI) m/z: calcd
for C16H20N3O2 [M+H]+: 286.1555; found 286.1552. HPLC purity 95.8% (30 min run of 45-80%
CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.6 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (30)
Yellow oil, 37 % yield. 1H NMR (300 MHz, CDCl3)
= 16.2 Hz, vinyl H), 7.55 (1H, d, = 15.0 Hz, vinyl H), 7.35 (1H, s, imidazole H-4), 7.15 (1H, s,
imidazole H-5), 6.90 (2H, s, phenyl H-2, H-6), 6.86 (1H, d, = 16.2 Hz, vinyl H), 4.52-4.41 (1H, m,
sec-butyl CH), 3.92 (6H, s, 2 × OCH3), 3.90 (3H, s, OCH3), 1.93-1.76 (2H, m, sec-butyl CH2CH3), 1.52
δ: 8.19 (1H, d, J = 15.3 Hz, vinyl H), 7.94 (1H,
d,
J
J
J
(3H, d,
CDCl3)
J
= 6.6Hz, sec-butyl CH3CH), 0.87 (3H, t,
: 188.5, 153.6, 145.7, 142.7, 140.8, 130.3, 128.9, 128.1, 126.7, 123.5, 118.8, 106.0, 61.2, 56.4,
J
= 7.5 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz,
δ
54.5, 30.8, 21.8, 10.7. IR (film) νmax: 3071, 2927, 1647, 1616, 1581, 1504, 1456, 1419 cm-1. HR-MS
(ESI) m/z: calcd for C21H26N2O4 [M+H]+: 371.1971; found 371.1973. HPLC purity 95.3% (30 min run
of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.7 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (31)
Yellow oil, 19 % yield. 1H NMR (300 MHz, CDCl3)
= 16.2 Hz, vinyl H), 7.56 (1H, d, = 15.0 Hz, vinyl H), 7.34 (1H, s, imidazole H-4), 7.26 (1H, dd,
=7.2, 1.8 Hz, phenyl H-6), 7.18 (1H, d, = 1.8 Hz, phenyl H-2), 7.14 (1H, s, imidazole H-5), 6.91
= 8.1 Hz, phenyl H-5), 6.85 (1H, d, = 16.2 Hz, vinyl H), 4.46 (1H, sextet, = 6.9 Hz, sec
butyl CH), 3.95 (3H, s, OCH3), 3.94 (3H, s, OCH3), 1.90-1.78 (2H, m, sec-butyl CH2CH3), 1.51 (3H, d,
δ: 8.14 (1H, d, J = 15.0 Hz, vinyl H), 7.95 (1H,
d,
J
J
J
J
(1H, d,
J
J
J
-
J
= 6.6 Hz, sec-butyl CH3CH), 0.87 (3H, t,
J
= 7.2 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz, CDCl3)
δ: 188.5, 151.9, 149.5, 145.6, 142.9, 129.2, 128.8, 127.8, 125.5, 124.0, 123.9, 118.6, 111.3, 110.2, 56.2,
56.1, 54.4, 30.9, 21.9, 10.7. IR (film) νmax: 2966, 2934, 1645, 1615, 1582, 1508, 1458, 1421 cm-1. HR-
MS (ESI) m/z: calcd for C20H25N2O3 [M+H]+: 341.1865; found 341.1860. HPLC purity 97.6% (30 min
run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.8 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(6-methylpyridin-2-yl)penta-1,4-dien-3-one (32)
Yellow oil, 72 % yield. 1H NMR (300 MHz, CDCl3)
= 15.9 Hz, vinyl H), 7.60 (1H, t, = 7.8 Hz, pyridine 4-H), 7.59 (1H, d, J
δ
: 7.87 (1H, d,
J
= 15.3 Hz, vinyl H), 7.80 (1H,
= 15.0 Hz, vinyl H),
d,
J
J
23

ACCEPTED MANUSCRIPT
7.44 (1H, d,
7.13 (1H, d,
J
= 15.9 Hz, vinyl H), 7.30 (1H, d,
J
= 7.8 Hz, pyridine 3-H), 7.27 (1H, s, imidazole H),
J
= 7.8 Hz, pyridine 5-H), 7.10 (1H, s, imidazole H), 4.41 (1H, sextet, J = 7.2 Hz, sec-
butyl CH), 2.58 (3H, s, pyridine CH3), 1.86-1.72 (2H, m, sec-butyl CH2CH3), 1.47 (3H, d,
J
= 6.9 Hz,
: 189.1,
sec-butyl CH3CH), 0.84 (3H, t,
J
= 7.5 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz, CDCl3)
δ
159.3, 152.6, 143.2, 143.0, 137.1, 130.2, 130.0, 127.3, 125.8, 124.4, 122.4, 118.8, 53.9, 30.9, 24.8,
21.9, 10.7. IR (film) νmax: 2970, 2932, 1653, 1623, 1616, 1590, 1457 cm-1. HR-MS (ESI) m/z: calcd for
C18H22N3O [M+H]+: 296.1763; found 296.1758. HPLC purity 97.7% (30 min run of 45-80% CH3CN in
H2O, with 15 min gradient, 1.0 mL/min).
4.3.9 (1E,4E)-1-(Pyridin-2-yl)-5-(thiazol-2-yl)penta-1,4-dien-3-one (33)
o
1
Yellow solid, mp 78-79 C, 84 % yield. H NMR (300 MHz, CDCl3)
pyridine H-6), 7.98 (1H, d, = 3.0 Hz, thiazole H-4), 7.89 (1H, d, = 15.9 Hz, vinyl H), 7.80-7.73 (2H,
overlapped, vinyl H; pyridine H-4), 7.61 (1H, d, = 15.6 Hz, vinyl H), 7.51 (1H, d, = 7.2 Hz,
pyridine H-3), 7.49 (1H, d, = 2.7 Hz, thiazole H-5), 7.39 (1H, d, = 15.9 Hz, vinyl H), 7.32 (1H, dd,
= 8.4, 5.7 Hz, pyridine H-5). 13C NMR (75 MHz, CDCl3)
: 188.8, 164.2, 153.1, 150.4, 145.2, 142.5,
δ: 8.70 (1H, d, J = 3.9 Hz,
J
J
J
J
J
J
J
δ
137.2, 134.7, 129.4, 129.5, 125.3, 124.8, 121.8. IR (film) νmax: 3078, 1655, 1622, 1597, 1582, 1467,
1431, 1329 cm-1. HR-MS (ESI) m/z: calcd for C13H11N2OS [M+H]+: 243.0592; found 243.0587. HPLC
purity 98.5% (30 min run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.10 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (34)
Yellow oil, 77 % yield. 1H NMR (300 MHz, CDCl3)
(1H, d, = 15.6 Hz, vinyl H), 7.75–7.72 (2H, overlapped, vinyl H; pyridine H-4), 7.63 (1H, d,
Hz, vinyl H), 7.50 (1H, d, = 7.8 Hz, pyridine H-3), 7.47 (1H, d, = 15.6 Hz, vinyl H), 7.31–7.27 (1H,
m, pyridine H-5), 7.27 (1H, s, imidazole H-4), 7.11 (1H, d, = 0.9 Hz, imidazole H-5), 4.43 (1H, sextet,
= 6.9 Hz, sec-butyl CH), 1.90–1.74 (2H, m, sec-butyl CH2CH3), 1.49 (3H, d, = 6.6 Hz, sec-butyl
= 7.2 Hz, sec-butyl CH2CH3). 13C NMR (75 MHz, CDCl3)
: 189.1, 153.3,
31.0, 22.0, 10.7. IR
δ: 8.69 (1H, d,
J
= 4.5 Hz, pyridine H-6), 7.80
= 15.0
J
J
J
J
J
J
J
CH3CH), 0.86 (3H, t,
J
δ
150.4, 143.2, 142.3, 137.0, 131.0, 130.2, 127.0, 126.3, 125.1, 124.5, 118.9, 53.6
,
(film) νmax: 3105, 2970, 2932, 1651, 1622, 1585, 1503, 1458, 1431 cm-1. HR-MS (ESI) m/z: calcd for
C17H20N3O [M+H]+: 282.1606; found 282.1601. HPLC purity 96.0% (30 min run of 45-80% CH3CN in
H2O, with 15 min gradient, 1.0 mL/min).
4.3.11 (1E,4E)-1-(4-Bromo-1-methyl-1H-pyrazol-3-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (35)
o
1
Yellow solid, mp 81-82 C, 71 % yield. H NMR (300 MHz, CDCl3)
δ: 8.67 (1H, d, J = 4.2 Hz,
pyridine H-6), 7.76 – 7.74 (1H, dt, = 8.1, 1.8 Hz, pyridine H-4), 7.68 (1H, s, pyrazole H-5), 7.67 (d,
J
J
24

ACCEPTED MANUSCRIPT
= 16.2 Hz, vinyl H), 7.56 (1H, d,
H-3), 7.45 (1H, d, = 16.2 Hz, vinyl H), 7.30–7.26 (1H, m, pyridine H-5), 3.93 (3H, s, pyrazole 1-
CH3). 13C NMR (75 MHz, CDCl3)
: 189.4, 153.4, 150.2, 145.3, 141.7, 137.1, 132.5, 132.1, 129.1,
J = 15.9 Hz, vinyl H), 7.50-7.42 (2H, overlapped, vinyl H; pyridine
J
δ
126.6, 125.0, 124.5, 96.1, 40.2. IR (film) νmax: 3123, 1654, 1628, 1599, 1507, 1466, 1323, 1309 cm-1.
HR-MS (ESI) m/z: calcd for C14H13N3OBr [M+H]+: 318.0242, 320.0222; found 318.0239, 320.0218.
HPLC purity 96.4% (30 min run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.12 (1E,4E)-1-(5-Methylisoxazol-3-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (36)
o
1
Yellow solid, mp 77-78 C, 89 % yield. H NMR (300 MHz, CDCl3)
pyridine H-6), 7.78– 7.74 (1H, m, pyridine H-4), 7.72 (1H, d, = 16.2 Hz, vinyl H), 7.69 (1H, d,
16.2 Hz, vinyl H), 7.62 (1H, d, = 15.6 Hz, vinyl H), 7.49 (1H, d, = 7.8 Hz, pyridine H-3), 7.33–7.29
(1H, m, pyridine H-5), 7.04 (1H, d, = 16.2 Hz, vinyl H), 6.24 (1H, s, isoxazole H-3), 2.47 (3H, s,
isoxazole 5-CH3). 13C NMR (75 MHz, CDCl3)
: 188.9, 170.6, 160.3, 153.0, 150.4, 142.8, 137.1,
δ: 8.68 (1H, d, J = 4.0 Hz,
J
J =
J
J
J
δ
132.1, 130.9, 127.6, 125.3, 124.8, 99.7, 12.5. IR (neat) νmax: 3071, 1640, 1603, 1575, 1493, 1387 cm-1.
IR (film) νmax: 3051, 2927, 1680, 1660, 1634, 1603, 1463, 1451, 1434 cm-1. HR-MS (ESI) m/z: calcd
for C14H13N2O2 [M+H]+: 241.0977; found 241.0972. HPLC purity 97.2% (30 min run of 45-80%
CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.13 (1E,4E)-1-(1-isopropyl-1H-imidazol-2-yl)-5-(2-(pyrrolidin-1-yl)thiazol-5-yl)penta-1,4-dien-3-
one (37)
1
Brown oil, 28% yield. H NMR (300 MHz, CDCl3)
δ: 7.83 (1H, d,
J
= 15.0 Hz, vinyl H), 7.55 (2H,
s, 2 × vinyl H), 7.49 (1H, s, thiazole H-4), 7.19 (1H, s, imidazole H-4), 7.10 (1H, s, imidazole H-5),
6.28 (1H, d, = 15.0 Hz, vinyl H), 4.75-4.60 (1H, m, isopropyl CH), 3.59-3.49 (4H, m, pyrrolidine 2 ×
CH2), 2.13-2.04 (4H, m, pyrrolidine 2 × CH2), 1.53-1.41 (6H, broad peak, isopropyl 2 × CH3). 13C
NMR (75 Hz, CDCl3) : 187.8, 169.4, 149.0, 143.1, 135.5, 130.7, 126.8, 125.5, 124.2, 122.9, 118.3,
J
δ
50.0, 47.7, 25.8, 24.0. IR (neat) νmax: 2923, 2853, 1641, 1607, 1537, 1502, 1458, 1264 cm-1. HR-MS
(ESI) m/z: calcd for C18H23N4OS [M+H]+: 343.1593; found 343.1594.
4.3.14 (1E,4E)-1-(1-isopropyl-1H-imidazol-2-yl)-5-(2-(piperidin-1-yl)thiazol-5-yl)penta-1,4-dien-3-one
(38)
1
Brown-red oil, 63% yield. H NMR (300 MHz, CDCl3)
δ
: 7.83 (1H, d,
= 15.3 Hz, vinyl H), 7.45 (1H, s, thiazole H-4), 7.19 (1H,
s, imidazole H-4), 7.11 (1H, s, imidazole H-5), 6.27 (1H, d, = 15.3 Hz, vinyl H), 4.68 (1H, hept,
6.6 Hz, isopropyl CH), 3.58 (4H, br.s, piperidine 2 × CH2), 1.70 (6H, br.s, piperidine 3 × CH2), 1.48
J = 15.3 Hz, vinyl H), 7.57
(1H, d,
J = 15.3 Hz, vinyl H), 7.52 (1H, d, J
J
J =
25

ACCEPTED MANUSCRIPT
(6H, d, J δ: 187.8, 172.8, 148.6, 143.0, 135.3,
= 6.6 Hz, isopropyl 2 × CH3). 13C NMR (75 Hz, CDCl3)
130.7, 126.7, 125.5, 124.2, 123.1, 118.3, 49.9, 47.7, 25.3, 24.2, 24.0. HR-MS (ESI) m/z: calcd for
C19H25N4OS [M+H]+: 357.1749; found 357.1747.
4.3.15
(1E,4E)-1-(1-Isopropyl-1H-imidazol-2-yl)-5-(2-morpholinothiazol-5-yl)penta-1,4-dien-3-one
(39)
1
Brown-red oil, 77% yield. H NMR (300 MHz, CDCl3)
δ
: 7.82 (1H, d,
= 15.0 Hz, vinyl H), 7.47 (1H, s, thiazole H-4), 7.20 (1H,
s, imidazole H-4), 7.12 (1H, s, imidazole H-5), 6.32 (1H, d, = 15.3 Hz, vinyl H), 4.69 (1H, hept,
6.6 Hz, isopropyl CH), 3.83 (4H, t, = 4.8 Hz, morpholine 2 × CH2), 3.59 (4H, t, = 4.8 Hz,
morpholine 2 × CH2), 1.48 (6H, d,
= 6.6 Hz, isopropyl 2 × CH3). 13C NMR (75 Hz, CDCl3)
J = 15.3 Hz, vinyl H), 7.59
(1H, d,
J = 15.0 Hz, vinyl H), 7.53 (1H, d, J
J
J =
J
O
J
N
J
δ:
187.8, 172.8, 147.8, 142.9, 134.8, 130.8, 126.4, 125.9, 125.2, 124.1, 118.5, 66.2, 48.5, 47.7, 24.0. IR
(film) νmax: 2937, 2855, 1640, 1608, 1529, 1504, 1461, 1308, 1267 cm-1. HR-MS (ESI) m/z: calcd for
C18H23N4O2S [M+H]+: 359.1542; found 359.1538. HPLC purity 98.2% (30 min run of 45-80% CH3CN
in H2O, with 15 min gradient, 1.0 mL/min).
4.3.16 (1E,4E)-1-(1-Isopropyl-1H-imidazol-2-yl)-5-(4-methyl-2-(pyridine-4-yl)thiazol-5-yl)penta-1,4-
dien-3-one (40)
Yellow oil, 55% yield. 1H NMR (300 MHz, CDCl3)
7.92 (1H, d, = 15.3 Hz, vinyl H), 7.81 (2H, d, = 6.0 Hz, pyridine H-3 & H-5), 7.64 (2H, s, 2 × vinyl
H), 7.25 (1H, s, imidazole H-4), 7.16 (1H, s, imidazole H-5), 6.71 (1H, d, = 15.6 Hz, vinyl H), 4.77-
4.66 (1H, m, isopropyl CH), 2.65 (3H, s, thiazole 4-CH3), 1.51 (6H, d, = 6.6 Hz, isopropyl 2 × CH3).
13C NMR (75MHz, CDCl3)
: 187.6, 164.7, 158.3, 150.9, 142.6, 139.8, 132.3, 131.4, 131.1, 129.2,
δ: 8.73 (2H, d, J = 6.0 Hz, pyridine 2-H & 6-H),
J
J
J
J
δ
127.5, 127.0, 120.5, 118.9, 47.8, 24.0, 16.1. IR (film) νmax: 2979, 2923, 1647, 1611, 1595, 1578, 1461,
1270 cm-1. HR-MS (ESI) m/z: calcd for C20H21N4OS [M+H]+: 365.1436; found 365.1431. HPLC
purity 96.0% (30 min run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.17 (1E,4E)-1-(1-Isopropyl-1H-benzo[d]imidazole-2-yl)-5-(1-isopropyl-1H-imidazole-2-yl)penta-
1,4-dien-3-one (41)
This compound was prepared in 43% yield as a yellow oil. 1H NMR (300 MHz, CDCl3)
= 15.2 Hz, vinyl H), 7.82–7.75 (overlapped, 1H, benzoimidazole H), 7.78 (1H, d, = 15.2 Hz
vinyl H), 7.68 (1H, d, = 15.2 Hz, vinyl H), 7.56–7.51 (1H, overlapped, benzoimidazole H), 7.54
(1H, d, = 15.2 Hz, vinyl H), 7.31–7.26 (2H, m, 2 × benzoimidazole H), 7.24 (1H, s, imidazole H-4),
7.15 (1H, s, imidazole H-5), 4.99 (1H, hept, = 7.2 Hz, isopropyl CH), 4.70 (1H, hept, = 6.9 Hz
δ: 7.84 (1H,
d,
J
J
,
J
J
J
J
,
26

ACCEPTED MANUSCRIPT
isopropyl CH), 1.69 (6H, d,
J
= 6.9 Hz, isopropyl 2 × CH3), 1.49 (6H, d, J = 6.6 Hz, isopropyl 2 ×
CH3). 13C NMR (75 MHz, CDCl3)
δ: 188.0, 147.8, 144.0, 142.4, 134.5, 132.2, 131.3, 127.8, 127.6,
127.1, 123.7, 123.2, 120.7, 119.0, 112.2, 48.2, 47.8, 24.0, 22.0. IR (film) νmax: 2978, 2935, 2878, 1651,
1619, 1589, 1460, 1383, 1266, 1100 cm-1. HR-MS (ESI) m/z: calcd for C21H25N4O [M+H]+:
349.2028; found 349.2027. HPLC purity 99.4% (30 min run of 45-80% CH3CN in H2O, with
15 min gradient, 1.0 mL/min).
4.3.18 (1E,4E)-1-(1-(sec-Butyl)-1H-benzo[d]imidazole-2-yl)-5-(1-(sec-butyl)-1H-imidazole-2-yl)penta-
1,4-dien-3-one (42)
This compound was prepared in 49% yield as a yellow oil. 1H NMR (300 MHz, CDCl3)
δ: 7.83–7.81
(3H, m, 2 × vinyl H; 1 × benzoimidazole H), 7.69 (1H, d, = 15.3 Hz, vinyl H), 7.60–7.50 (2H,
J
overlapped, 1× vinyl H; 1 × benzoimidazole H), 7.33–7.25 (3H, m, 2 × benzoimidazole H; imidazole
H-4), 7.13 (1H, s, imidazole H-5), 4.75–4.62 (1H, m, sec-butyl CH), 4.48–4.38 (1H, m, sec-butyl CH),
2.28–2.12 (1H, m, sec-butyl CH2CH3 ), 2.05–1.95 (1H, m, sec-butyl CH2CH3), 1.88–1.75 (2H, m, sec
butyl CH2CH3), 1.70 (3H, d, = 7.2 Hz, sec-butyl CH3CH), 1.49 (3H, d, = 6.6 Hz, sec-butyl CH3CH),
0.86 (3H, t, = 7.4 Hz, sec-butyl CH2CH3), 0.80 (3H, t,
= 7.4 Hz, sec-butyl CH2CH3). 13C NMR (75
MHz, CDCl3) : 188.1, 148.5, 143.9, 143.0, 134.6, 132.2, 131.4, 127.9, 127.7, 127.2, 123.7, 123.2,
-
J
J
J
J
δ
120.7, 119.2, 112.2, 54.4, 53.6, 31.0, 28.7, 22.1, 20.4, 11.3, 10.7. IR (film) νmax: 2969, 2934, 2877,
1651, 1620, 1457, 1384, 1290, 1178 cm-1. HR-MS (ESI) m/z: calcd for C23H29N4O [M+H]+:
377.2341; found 377.2337. HPLC purity 95.0% (30 min run of 45-80% CH3CN in H2O, with
15 min gradient, 1.0 mL/min).
4.3.19 (1E,4E)-1-(1-Isobutyl-1H-benzo[d]imidazole-2-yl)-5-(1-isobutyl-1H-imidazole-2-yl)penta-1,4-
dien-3-one (43)
This compound was prepared in 35% yield as a yellow oil. 1H NMR (300 MHz, CDCl3)
= 15.2 Hz, vinyl H), 7.83–7.76 (1H, m, benzoimidazole H), 7.73 (1H, d, = 15.3 Hz, vinyl H),
7.61 (1H, d, = 15.3 Hz, vinyl H), 7.49 (1H, d, = 15.3 Hz, vinyl H), 7.39–7.30 (3H, m, 3 ×
benzoimidazole H), 7.23 (1H, d, = 0.9 Hz, imidazole H-4), 7.04 (1H, d, = 0.9 Hz, imidazole H-5),
4.12 (2H, d, = 7.5 Hz, isobutyl CH2), 3.89 (2H, d, = 7.5 Hz, isobutyl CH2), 2.30–2.18 (1H, m,
isobutyl CH), 2.09– 2.00 (1H, m, isobutyl CH2, 0.97 (6H, d, = 6.6 Hz, isobutyl 2 × CH3), 0.95 (6H, d,
= 6.6 Hz, isobutyl 2 × CH3). 13C NMR (75 MHz, CDCl3)
: 187.9, 148.6, 143.4, 136.3, 131.8, 131.0,
127.8, 127.5, 127.2, 124.1, 123.7, 123.5, 120.5, 110.5, 53.8, 51.2, 30.8, 30.1, 20.4, 20.1. IR (film) νmax
δ: 7.81 (1H,
d,
J
J
J
J
J
J
J
J
J
J
δ
:
27

ACCEPTED MANUSCRIPT
3043, 2962, 2873, 1652, 1622, 1592, 1404, 1091 cm-1. HR-MS (ESI) m/z: calcd for C23H29N4O
[M+H]+: 377.2341; found 377.2333. HPLC purity 95.0% (30 min run of 45-80% CH3CN in
H2O, with 15 min gradient, 1.0 mL/min).
4.3.20 (1E,4E)-1-(1-Isopentyl-1H-benzo[d]imidazole-2-yl)-5-(1-isopentyl-1H-imidazole-2-yl)penta-
1,4-dien-3-one (44)
This compound was prepared in 35% yield as a yellow oil. 1H NMR (300 MHz, CDCl3)
= 15.3 Hz, vinyl H), 7.81–7.78 (1H, m, benzoimidazole H), 7.75 (1H, d, = 15.3 Hz, vinyl H),
7.64 (1H, d, = 15.0 Hz, vinyl H), 7.50 (1H, d, = 15.0 Hz, vinyl H), 7.38–7.27 (3H, m, 3 ×
benzoimidazole H), 7.23 (1H, s, imidazole H-4), 7.07 (1H, s, imidazole H-5), 4.31(2H, t, = 7.4 Hz,
isopentyl -CH2), 4.10 (2H, t, = 7.5 Hz, isopentyl -CH2), 1.71–1.60 (6H, m, 2 × isopentyl
CH2CH2CH), 1.01 (6H, d, = 5.9 Hz, isopentyl CH(CH3)2), 0.97 (6H, d, = 6.2 Hz, isopentyl
CH(CH3)2). 13C NMR (75 MHz, CDCl3)
: 187.9, 148.1, 143.5, 143.0, 135.9, 131.8, 131.1, 127.7,
δ: 7.81 (1H,
d,
J
J
J
J
J
N
J
N
N-
J
J
δ
127.24, 127.19, 124.2, 123.5, 123.1, 120.6, 110.0, 44.8, 42.4, 40.5, 39.6, 26.1, 25.8, 22.6, 22.5. IR
(film) νmax: 2956, 2928, 2870, 1652, 1622, 1447, 1406, 1094 cm-1. HR-MS (ESI) m/z: calcd for
C25H33N4O [M+H]+: 405.2654; found 405.2648. HPLC purity 96.1% (30 min run of 45-80%
CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
4.3.21 (1E,4E)-1-(1-(Pentan-2-yl)-1H-benzo[d]imidazole-2-yl)-5-(1-(pentan-2-yl)-1H-imidazole-2-
yl)penta-1,4-dien-3-one (45)
This compound was prepared in 81% yield as a yellow oil. 1H NMR (300 MHz, CDCl3)
(3H, m, 2 × vinyl H; 1 × benzoimidazole), 7.69 (1H, d, = 15.0 Hz, vinyl H), 7.58–7.52 (2H, m, 1×
vinyl H; 1× benzoimidazole H), 7.34– 7.28 (3H, m, 2 × benzoimidazole H; imidazole H-4), 7.13 (1H,
d, = 0.9 Hz, imidazole H-5), 4.81–4.74 (1H, m, 1-(pentan-2-yl) CH), 4.56–4.49 (1H, m, 1-(pentan-2-
yl) CH), 2.23–2.12 (2H, m, 1-(pentan-2-yl) CHCH2CH2CH3), 1.97–1.89 (1H, m, 1-(pentan-2-yl)
CHCH2CH2CH3), 1.77 (2H, q, = 7.6 Hz, 1-(pentan-2-yl) CHCH2CH2CH3), 1.70 (3H, d, = 6.9 Hz, 1-
(pentan-2-yl) CH3CH), 1.49 (3H, d, = 6.7 Hz, 1-(pentan-2-yl) CH3CH), 1.28 – 1.15 (3H, m, 1-
(pentan-2-yl) CHCH2CH2CH3), 0.91 (3H, t, = 7.3 Hz, 1-(pentan-2-yl) CHCH2CH2CH3), 0.87 (3H, t,
= 7.2 Hz, 1-(pentan-2-yl) CHCH2CH2CH3). 13C NMR (75 MHz, CDCl3)
: 188.1, 148.4, 144.0, 143.0,
δ: 7.84–7.78
J
J
J
J
J
J
J
δ
134.6, 132.2, 131.5, 127.9, 127.8, 127.2, 123.7, 123.2, 120.8, 119.2, 112.2, 52.7, 52.0, 40.1, 37.8, 22.5,
20.7, 20.1, 19.5, 13.9, 13.8. IR (film) νmax: 2959, 2932, 2873, 1651, 1619, 1455, 1382, 1175 cm-1. HR-
MS (ESI) m/z: calcd for C25H33N4O [M+H]+: 405.2654; found 405.2650. HPLC purity 97.9%
(30 min run of 45-80% CH3CN in H2O, with 15 min gradient, 1.0 mL/min).
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