Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with two different terminal rings in prostate epithelial cell models

Article abstract of DOI:10.1016/j.ejmech.2017.03.067

To systematically investigate the structure-activity relationships of 1,7-diheteroarylhepta-1,4,6-trien-3-ones in three human prostate cancer cell models and one human prostate non-neoplastic epithelial cell model, thirty five 1,7-diarylhepta-1,4,6-trien-

Full text of DOI:10.1016/j.ejmech.2017.03.067

Accepted Manuscript
Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with
two different terminal rings in prostate epithelial cell models
Rubing Wang, Xiaojie Zhang, Chengsheng Chen, Guanglin Chen, Cristian Sarabia,
Qiang Zhang, Shilong Zheng, Guangdi Wang, Qiao-Hong Chen
PII:
S0223-5234(17)30235-0
10.1016/j.ejmech.2017.03.067
EJMECH 9327
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2017
Revised Date: 20 February 2017
Accepted Date: 26 March 2017
Please cite this article as: R. Wang, X. Zhang, C. Chen, G. Chen, C. Sarabia, Q. Zhang, S. Zheng, G.
Wang, Q.-H. Chen, Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with
two different terminal rings in prostate epithelial cell models, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.03.067.
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Graphical Abstract

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Structure-Activity Relationship Studies of 1,7-Diheteroarylhepta-1,4,6-trien-3-ones
with Two Different Terminal Rings in Prostate Epithelial Cell Models
Rubing Wanga, Xiaojie Zhanga, Chengsheng Chena, Guanglin Chena, Cristian Sarabiaa, 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 and cRCMI Cancer Research Center, Xavier University of Louisiana, 1
Drexel Drive, New Orleans, LA 70125, USA
ABSTRACT:
To systematically investigate the structure-activity relationships of 1,7-
diheteroarylhepta-1,4,6-trien-3-ones in three human prostate cancer cell models and one human
prostate non-neoplastic epithelial cell model, thirty five 1,7-diarylhepta-1,4,6-trien-3-ones with
different terminal heteroaromatic rings have been designed for evaluation of their anti-proliferative
potency in vitro. These target compounds have been successfully synthesized through two sequential
Horner-Wadsworth-Emmons reactions starting from the appropriate aldehydes and tetraethyl (2-
oxopropane-1,3-diyl)bis(phosphonate). Their anti-proliferative potency against PC-3, DU-145 and
LNCaP human prostate cancer cell lines can be significantly enhanced by the manipulation of the
terminal heteroaromatic rings, further demonstrating the utility of 1,7-diarylhepta-1,4,6-trien-3-one as a
potential scaffold for the development of anti-prostate cancer agents. The optimal analog 40 is 82-, 67-,
and 39-fold more potent than curcumin toward the three prostate cancer cell lines, respectively. The
experimental data also reveal that the trienones with two different terminal aromatic rings possess
greater potency toward three prostate cancer cell lines, but also have greater capability of suppressing
the proliferation of PWR-1E benign human prostate epithelial cells, as compared to the corresponding
counterparts with two identical terminal rings and curcumin. The terminal aromatic rings also affect the
cell apoptosis perturbation.
Key words: 1,7-diheteroarylhepta-1,4,6-trien-3-one, antiproliferative activity, prostate cancer,
structure-activity relationship, cell apoptosis
____________
* Corresponding author.
E-mail: qchen@csufresno.edu.
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1. Introduction
Curcumin (1) is the major chemical component of well-known turmeric (Curcuma longa) that has
long been used as a yellow spicy curry ingredient and as a traditional Ayurvedic, Chinese, and Hindu
medicine [1]. Curcumin is a well-established lead compound for the development of new anti-prostate
cancer agents due to its safety profiles in human and its demonstrated potential in treating prostate
cancer [1-4]. Several strategies have been applied to optimize curcumin in the hope of improving its
potency and/or bioavailability. Chemical manipulations on curcumin have been established as a
meaningful approach to the development of curcumin-based anti-prostate cancer agents with more
drug-like properties [5]. 1,5-Diheteroarylpenta-1,4-dienones [6,7], 1,7-diheteroarylhepta-1,4,6-
trienones [8], and 1,9-diheteroarylnona-1,4,6,8-tetraenones [9] have been identified by us as potential
scaffolds for developing curcumin-based anti-prostate cancer agents because of their apparently
improved potency as compared with curcumin. Among them, the 1,4,6-trienone motif was envisioned
and illustrated as a good bioisostere of the keto-enol central spacer in curcumin due to the
indistinguishable shape and size [8]. Our previous study demonstrates that 1-alkyl-1H-imidazol-2-yl in
compounds 2-6 and 1-alkyl-1H-benzo[d]imidazole-2-yl in compounds 7-11 are significantly beneficial
to the in vitro antiproliferative potency in three prostate cancer cell models [8]. All 1,7-diheteroaryl-
1,4,6-trienones investigated in our previous study have two identical nitrogen-containing terminal
aromatic rings.
To systematically investigate the structure-activity relationships of 1,7-diheteroaryl-1,4,6-heptatrien-
3-ones in prostate epithelial cell models, this study aims to further explore the effect of two different
terminal aromatic rings of the trienones on anti-proliferative potency in androgen-sensitive and
androgen-insensitive prostate cancer cell models, as well as on PWR-1E non-neoplastic prostate
epithelial cells. Consequently, thirty five new (1E,4E,6E)-heptatrien-3-ones (12-46, see Schemes 1 and
2, Table 1) that retain the 7-carbon central trienone linker but with two different nitrogen-containing
heteroaromatic rings were designed and synthesized for the in vitro evaluation of their inhibitory ability
towards cell proliferation of cancerous and noncancerous prostate epithelial cells.
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O
OH
1
O
7
1
3
6
5
5
7
4
2
4
3
6
BHR
BHR
2
HO
OH
OCH3
OCH3
curcumin (1):
enol-ketone tautomer
1,7-diaryl-1,4,6-heptatrien-3-ones
(2-11)
N
N
N
N
N
N
N
N
N
N
N
N
BHR for 2 BHR for 3
BHR for 4
BHR for 5
BHR for 7
BHR for 6
N
N
N
N
N
N
N
N
BHR for 11
BHR for 9
BHR for 10
BHR for 8
Fig. 1. Structures of curcumin and its analogues
2. Results and Discussion
2.1 Chemistry
Initially, the protocol developed in our previous study was used to synthesize fourteen 1,7-diaryl-1,4,6-
heptatrien-3-ones (12-16, 32-35, 42-46) with two different terminal heteroaromatic rings. Specifically,
the syntheses were completed via the Horner-Wadsworth-Emmons reaction of (E)-diethyl(2-oxo-4-(1-
BHR)-but-3-en-1-yl)phosphonates (47) with (2E)-3-(7-BHR)-2-propenals (48) using potassium
carbonate as base (Scheme 1). Both phosphonates (47) and propenals (48) can be readily obtained
using the procedure previously described by us [8]. Due to the unsatisfactory yields (7-39%) for the
Horner-Wadsworth-Emmons reaction as illustrated in Scheme 1, the new reaction sequence with high
efficiency was developed as shown in Scheme 2 and applied to the synthesis of the remaining twenty-
one 1,7-diaryl-1,4,6-heptatrien-3-ones (17-31, 36-41). This synthetic sequence started with the
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preparation of four common ((3E,5E)-2-oxo-6-(aryl)hexa-3,5-dien-1-yl)phosphonates (50-53) by
treating the appropriate (2E)-3-(7-BHR)-2-propenal (48) with
1
equivalent of 1,3-
bis(diethylphosphonato)acetone (49). The 1,7-diaryl-1,4,6-heptatrien-3-ones (17-31, 36-41) were
obtained in 35-86% yields by the Horner-Wadsworth-Emmons reaction of ((3E,5E)-2-oxo-6-
(aryl)hexa-3,5-dien-1-yl)phosphonates (50-53) with the appropriate heteroarylformaldehyde (54). It
should be noted that we had already synthesized enough amount of compounds 12-16, 32-35, and 42-
46 via Scheme 1 (Method A) for our structure-activity relationship studies when we figured out the
more efficient synthetic method (Scheme 2, Method B). That is the reason why these compounds were
not synthesized using Method B. According to the structural similarity, the method B described in
Scheme 2 could be an efficient method for the synthesis of compounds 12-16, 32-35, and 42-46 as
well.
Scheme 1. Synthesis of 1,7-diheteroaryl-1,4,6-heptatrien-3-ones (12-16, 32-35, and 42-46) (Method
A).
Scheme 2. Synthesis of 1,7-diheteroaryl-1,4,6-heptatrien-3-ones (17-31 and 36-41) (Method B).
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Table 1. Structures of 1,7-diarylhepta-1,4,6-trien-3-ones with different nitrogen-containing
heteroaromatic rings (12-46)
Compd 1-BHR
12
7-BHR
Compd 1-BHR
13
7-BHR
14
16
18
20
22
24
15
N
17
19
21
23
25
N
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26
27
CF3
CF3
N
28
30
32
29
S
S
CF3
N
N
31
N
F
S
33
35
34
36
37
39
N
38
F
40
42
41
43
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44
46
45
2.2 Anti-proliferative activity towards prostate cancer cell lines and structure-activity
relationships.
The in vitro anti-proliferative activity of these thirty-five new trienones was first evaluated on human
prostate cancer cell models, employing both androgen-sensitive and androgen-insensitive prostate
cancer cell lines (PC-3, DU145, and LNCaP). WST-1 cell proliferation assay was employed for the
potency assessment, in which a water-soluble tetrazolium salt can be converted to formazan catalyzed
by cell mitochondrial dehydrogenases. Consequently, the amount of produced formazan dye directly
associates with live cell populations in the culture. Curcumin was used as a positive control for
comparison in the parallel experiments and the anti-proliferative potency of each test trienone was
represented as IC50 values. As illustrated in Table 2, all these trienones with two different aromatic
rings have greater anti-proliferative potency than curcumin as indicated in their IC50 values. Their IC50
values against PC-3, DU-145 and LNCaP human prostate cancer cell lines are in the ranges of 0.29-
14.02 µM, 0.33-8.68 µM, and 0.27-6.32 µM, respectively. They are up to 88-, 80-, 50-fold more potent
than curcumin in the three prostate cancer cell models.
The structure-antiproliferative activity relationships of trienones on the basis of this study in
combination with our previous study [8] can be summarized as below:
− It can be clearly concluded that 1,7-diaryl-1,4,6-heptatrien-3-ones with two different
heteroaromatic rings are more potent towards both androgen-sensitive and androgen-insensitive
prostate cancer cell lines than the corresponding ones with two identical aromatic rings. This
conclusion is supported by the following data: i) the optimal trienone (7) with two identical
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terminal heteroaromatic rings is 36-, 29-, and 11-fold more potent than curcumin toward PC-3,
DU145, and LNCaP prostate cancer cell lines [8]; while the optimal trienones with two
different terminal aromatic rings (40 and 41) are 82-88, 44-67, and 39-51 times more potent
than curcumin; and ii) trienones (42 and 43) having two different terminal heteroaromatic rings
are apparently more potent than their corresponding counterparts (7 & 2, and 9 & 4) with two
identical heteroaromatic rings (Table 3).
− 1-(α-Branched alkyl)-1H-imidazol-2-yl in trienones 22-26, 1-(α-branched alkyl)-1H-
benzo[d]imidazole-2-yl in compounds 40-45, and pyridin-2-yl in trienones 32-35 serve as the
optimal heteroaromatic rings at the C-1 terminal (1-BHR) for enhanced in vitro potency of this
scaffold.
− 2-Methyl-4-trifluoromethyl-thiazole-5-yl in trienones 16, 22, 36, and 40 and 1-(α-branched
alkyl)-1H-imidazol-2-yl in trienones 14, 15, 32, 33, 34, and 35 are the optimal heteroaromatic
rings at the C-7 terminal for great potency.
− If both 1-BHR and 7-BHR are five-membered aromatic rings, swapping the location of two
terminal rings has no effect on their potency (Table 4). For example, 12 and 17, 14 and 20, 13
and 18, 15 and 23 exhibited approximately similar potency based on the ratios of
IC50(curcumin)/IC50(trienone). The only exception is compound 16 with the 2-methyl-4-
trifluoromethyl-thiazole-5-yl moiety on C-7, which is two-fold more potent than its counterpart
29 possessing thiazol-2-yl unit on C-7.
− If one side terminal is a five-membered aromatic ring and the other side is a six-membered ring,
location of the five-membered ring to C-7 (as 7-BHR) significantly contributes to greater anti-
proliferative potency toward three prostate cancer cell lines as described in Table 4. For
example, compounds 32, 33, 34, 35, and 36 having a five-membered ring on C-7 exhibited
greater potency than their respective counterparts (19, 21, 24, 26, or 30) with a six-membered
ring on C-7.
− A generalized pharmarcophore for the 1,7-diheteroarylhepta-1,4,6-trien-3-ones was proposed in
Fig. 2.
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Fig. 2. Proposed pharmarcophore for the 1,7-diheteroarylhepta-1,4,6-trien-3-ones
Table 2. Anti-proliferative activity of 1,7-diheteroarylhepta-1,4,6-trien-3-ones
IC50 (µM)a
IC50 (curcumin)/IC50(trienone)
Compd
PC-3b
DU145c
LNCaPd
PC-3b DU145c
LNCaPd
25.43 ± 2.15
26.23 ± 0.65
1.59 ± 0.42
13.61 ± 2.69
1
1
1
Curcumin
12
1.52 ± 0.47
1.13 ± 0.10
1.37 ± 0.18
1.23 ± 0.04
1.28 ± 0.20
1.09 ± 0.17
1.65 ± 0.22
1.91 ± 0.07
1.21 ± 0.14
1.18 ± 0.25
0.75 ± 0.07
0.58 ± 0.08
0.73 ± 0.03
0.54 ± 0.09
1.10 ± 0.24
0.42 ± 0.04
0.67 ± 0.06
17
16
18
24
19
25
12
32
20
13
12
21
13
14
15
16
17
18
19
20
21
1.53 ± 0.29
1.14 ± 0.28
0.81 ± 0.11
1.79 ± 0.27
1.06 ± 0.37
1.40 ± 0.25
23
19
21
20
23
15
17
23
32
15
25
19
1.82 ± 0.14
1.27 ± 0.04
1.07 ± 0.21
1.15 ± 0.06
13
21
14
21
1.12 ± 0.24
0.66 ± 0.16
22
23
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22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
0.63 ± 0.04
1.16 ± 0.03
1.47 ± 0.32
0.99 ± 0.09
1.03 ± 0.25
3.19 ± 1.09
1.93 ± 0.07
3.63 ± 0.98
4.73 ± 0.31
2.69 ± 0.02
1.02 ± 0.03
0.86 ± 0.17
0.70 ± 0.07
0.64 ± 0.06
1.81 ± 0.08
14.02 ± 3.98
1.79 ± 0.07
1.67 ± 0.27
0.31 ± 0.01
0.29 ± 0.06
0.53 ± 0.06
0.42 ± 0.13
0.54 ± 0.07
0.51 ± 0.04
0.40 ± 0.09
1.17 ± 0.21
1.12 ± 0.01
0.74 ± 0.08
1.00 ± 0.22
5.74 ± 0.12
1.75 ± 0.24
3.61 ± 0.87
6.14 ± 0.42
3.16 ± 0.32
0.71 ± 0.07
0.75 ± 0.15
0.89 ± 0.27
0.66 ± 0.05
1.72 ± 0.29
8.68 ± 0.67
2.07 ± 0.10
2.00 ± 0.26
0.39 ± 0.08
0.60 ± 0.04
0.33 ± 0.08
0.64 ± 0.37
0.79 ± 0.15
0.61 ± 0.06
0.41 ± 0.07
0.74 ± 0.06
1.29 ± 0.14
0.69 ± 0.05
0.55 ± 0.18
4.33 ± 0.89
1.42 ± 0.35
2.10 ± 0.31
4.99 ± 0.67
1.28 ± 0.34
0.37 ± 0.06
0.38 ± 0.01
0.28 ± 0.02
0.38 ± 0.03
1.16 ± 0.23
6.32 ± 0.27
1.58 ± 0.50
2.09 ± 0.34
0.35 ± 0.03
0.27 ± 0.01
0.60 ± 0.16
0.30 ± 0.06
0.55 ± 0.05
0.72 ± 0.10
41
22
17
26
25
8
66
22
23
35
26
5
33
18
11
20
25
3
13
7
15
7
10
6
5
4
3
9
8
11
37
36
49
36
12
2
25
30
36
39
14
2
37
35
30
40
15
3
14
15
82
88
48
60
47
50
13
13
67
44
80
41
33
43
9
7
39
50
23
45
25
19
10

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46
IC50 is the drug concentration effective in inhibiting 50% of the cell viability measured by WST-1 cell proliferation assay after 3 days
2.74 ± 0.28
2.40 ± 0.36
1.88 ± 0.34
9
11
7
a
exposure. The data were presented as the mean ± standard deviation 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
Table 3. Comparison of potency for trienones with identical and different aromatic rings
Structure
IC50(curcumin)/IC50(trienone)
Compd.
PC-3
DU145 LNCaP PWR-1E
48
79
29
16
23
11
9
7
42
7a
36
10
10
O
N
N
N
2a
0.9-1.3
N
O
N
N
43
60
25
87
26
45
11
28
24
N
N
9a
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4a
14
25
11
2.5-5.6
aThe IC50(curcumin)/IC50(trienone) ratios for compounds 7, 2, 9, and 4 have been reported in our previous paper [8]
Table 4. Effects of the location of terminal aromatic rings on the potency
IC50(curcumin)/IC50(trienone)
Compd
1-BHR
7-BHR
PC-3
DU145
LNCaP
N
17
16
18
12
N
N
N
23
19
21
23
15
25
23
21
17
19
32
19
12
24
20
17
14
20
13
18
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21
22
32
22
25
18
15
23
20
7
15
7
12
7
16
29
CF3
N
S
13
25
22
30
17
14
37
23
35
23
13
37
21
36
11
19
32
21
33
24
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36
25
30
26
49
25
34
26
39
40
36
35
CF3
N
5
4
3
30
36
S
14
15
12
2.3 Anti-proliferative activity towards PWR-1E non-neoplastic human prostate epithelial cell
line.
PWR-1E human prostatic epithelial cells were initially originated from a non-malignant prostate with
mild hyperplasia and immortalized by a hybrid virus of adenovirus 12 and Simian 40. This cell line
expresses markers of normal prostatic epithelial cells, including prostate specific antigen and androgen
receptor, and mimics normal growth and differentiation responses to androgen [10]. Curcumin was
used as a positive control, which has generally been considered to possess human safety profile as
validated by human clinical trials [3,11] and animal studies [12]. No anti-proliferative potency of
curcumin against non-cancerous PWR-1E cells has so far been reported. However, it has been revealed
that the apoptotic cell death pathway could be triggered by androgen deprivation in both normal human
primary prostate epithelial cells and androgen-sensitive human prostate cancer cells [13]. In this study,
we observed the IC50 values for curcumin fall into the range of 8.14-9.54 µM, suggesting no
differential responses of curcumin to LNCaP prostate cancer epithelial cell lines and PWR-1E benign
human prostate epithelial cells. This is reasonable given that curcumin can downregulate the expression
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and activity of androgen receptor and suppress the gene expression of prostate-specific antigen in
LNCaP prostate cancer cells [14] and that both LNCaP prostate cancer cells and PWR-1E non-
neoplastic prostate epithelial cells express androgen receptor and androgen specific antigen. As shown
in Table 5, the 1,7-diaryl-1,4,6-heptatrien-3-ones (compounds 2-6) with two identical 1-alkyl-1H-
imidazol-2-yl as terminal aromatic rings have equivalent or slightly greater potency (within 3 folds) in
inhibiting PWR-1E cell proliferation, as compared with curcumin. In contrast, those 1,7-diaryl-1,4,6-
heptatrien-3-ones with two identical 1-alkyl-1H-benzo[d]imidazole-2-yl as terminal aromatic rings
(compounds 7-11) and those with two different aromatic rings ( compounds 13, 15, 17, 22, 23, 25, 26,
32-35, and 40-43) possess significantly stronger ability in inhibiting PWR-1E cell proliferation.
Considering both potency towards prostate cancer cells and selectivity between prostate cancer cells
and non-neoplastic cells, compounds 2-6 and 42 are the optimal 1,7-diaryl-1,4,6-heptatrien-3-ones for
further investigation as anti-prostate cancer agents.
Table 5. Anti-proliferative activity of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with two identical
terminal aromatic rings (2-11) and those with two different aromatic rings (13, 15, 17, 22, 23, 25, 26,
32-35, and 40-43) toward PWR-1E non-neoplastic prostate epithelial cells
IC50 (curcumin) /
IC50 (trienone)
Compd
IC50 (curcumin) /
IC50 (trienone)
IC50 (µM)a
8.14-9.54
6.39-10.16
4.85-5.80
1.45-3.54
2.72-4.75
4.99
Compd
13
IC50 (µM)a
0.49
Curcumin
1
18
20
14
0.9-1.3
1.7
0.44
2
15
0.62
3
17
2.5-5.6
2.0-3.0
0.39
4
22
23
16
0.57
5
23
0.50
6
25
1.9
10
18
29
0.80
0.31
7
26
0.23
36
24
>8
34
0.54
8
32
16
0.33
0.40
9
33
22
10
11
<1
34
0.41
21
0.24
0.038
0.048
0.195
35
239
186
46
40
41
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1.34
0.32
7
42
43
28
a IC50 is the drug concentration effective in inhibiting 50% of the normal cell viability measured by WST-1 cell proliferation assay after 3
days exposure.
b Human prostatic epithelial cells derived from prostate with mild hyperplasia.
2.3 Effects of trienone 40 on PC-3 cell cycle progression and apoptosis
Curcumin has been demonstrated to block PC-3 prostate cancer cell cycle progression by arresting the
G1/S phase [15]. Our previous study revealed that treatment of PC-3 cells with compound 7, the most
potent 1,7-diheteroarylhepta-1,4,6-trien-3-one with two identical aromatic rings, led to appreciable cell
cycle arrest at the G1/G0 phase [8]. To evaluate the effect of terminal aromatic rings of the trienone
scaffold on the PC-3 prostate cancer cell cycle regulation, compound 40, as one of the most potent
trienones with two different aromatic rings, was selected for further assessment using flow cytometry
analysis with propidium iodide DNA staining. The similar cell cycle regulation has been identified in
this study when exposing PC-3 cells to trienone 40 at 5 µM concentration (Fig. 3). Specifically,
trienone 40 was found to cause accumulation of PC-3 cells in the G0/G1 phase from 49% and 61% for
control cells at 16 hours and 24 hours, respectively, to 65% and 64% for the compound-treated cells.
The cell population in the G2 phase decreased from 33% in control cells to 23% at 16 hours, and from
26% in control cells to 23% at 24 hours.
The growth suppression of PC-3 prostate cancer cells by curcumin and trienone 7 has been
demonstrated to be, at least in part, associated with its capability of inducing PC-3 prostate cancer cell
apoptosis [2, 16]. Consequently, F2N12S and CYTOX AADvanced double staining flow cytometry-
based assay was chosen to discriminate PC-3 cells dying from apoptosis from those dying from
necrosis in response to increasing concentrations of trienone 40. Straurosporine was used as specific
apoptotic inducer and positive apoptotic control in all these experiments (data were shown in Fig. 4).
As illustrated in Fig. 5, trienone 40 induced appreciably higher levels of late apoptotic/necrotic cell
death rather than apoptotic cell death in the androgen-insensitive PC-3 prostate cancer cell line after a
16-hour treatment. Specifically, 3-5 µM of trienone 40 could induce 43% late apoptotic/necrotic cells
but merely 13-15% early apoptotic cells as compared with control cells; treatment with 10 µM of
trienone 40 led to 55% late apoptotic/necrotic cells but only 27% early apoptotic cells; 20 µM of
trienone 40 activated necrosis as well, with 63% late apoptotic/necrotic cells and 34% early apoptotic
cells. Both apoptotic and necrotic cell population increased in response to increasing concentration of
16

ACCEPTED MANUSCRIPT
trienone 40 (0-30 µM final concentration range). The apoptotic cell population reached maximum
when treating PC-3 cancer cells with trienone 40 at 20 µM concentration. Trienone 40 is more potent
than trienone 7 in inhibiting PC-3 cell proliferation but has less inductive effect on PC-3 cell apoptosis,
indicating the cell death path way induced by the 1,7-diheteroarylhepta-1,4,6-trien-3-ones varies with
different terminal heteroaromatic rings.
Cell Cycle Regulation of 40 toward PC-3
G0/G1:
G0/G1:
49.1%
65.2%
16 Hours Control
16 Hours 5µM Treatment
G0/G1:
63.9 %
G0/G1:
61.0%
24 Hours Control
24 Hours 5µM Treatment
Fig. 3. Cell cycle analysis of PC-3 cells. PC-3 cancer cells were untreated or treated with trienone 40.
Cells were harvested after 16 and 24 hours then they were fixed, stained, and analyzed for DNA
content. The distribution and percentage of cells in G1/G0, and G2 phase of the cell cycle are indicated.
17

ACCEPTED MANUSCRIPT
Apoptotic Effect of strauroporine against PC-3 Cell Line
95
100
90
80
70
60
50
40
30
20
10
0
87
84
76
68
60
47
46
Control
5 µM
31
24
15
10
10
9
9
8
8
6
4
3
2
Control
DMSO
5µM
10µM
20µM
30µM
40µM
Viable Cell
Apoptotic Cell
Necrotic Cell
20 µM
40 µM
Fig. 4. Evolution of viable, apoptotic, and necrotic PC-3 cells populations in response to increasing
dosages of straurosporine.
Apoptotic Effect of 40 against PC-3 Cell Line
95
100
90
80
70
60
50
40
30
20
10
0
92
89
70
63
55
Control
1 µM
46
43
43 43
34
29
27
18
15
11
8
5
3
3
3
3
2
1
Control DMSO
Viable Cell
1µM
3µM
5µM
10µM
20µM
30µM
5 µM
20 µM
Apoptotic Cell
Necrotic Cell
Fig. 5. Evolution of viable, apoptotic, and necrotic PC-3 cells populations in response to increasing
dosages of compound 40.
18

ACCEPTED MANUSCRIPT
3. Conclusion
In summary, thirty five 1,7-diarylhepta-1,4,6-trien-3-ones with different terminal heteroaromatic rings
have been designed and assessed for their anti-proliferative potency against three prostate cancer cell
lines and one benign prostate epithelial cell line. Our findings provide further evidence that this group
of curcumin-based analogs possesses promising antiproliferative activities toward both androgen-
sensitive and androgen-insensitive prostate cancer cell lines. The optimal analog 40 is 82-, 67-, 39-fold
more potent than curcumin against PC-3, DU-145 and LNCaP human prostate cancer cell lines.
Structure-activity relationships demonstrate that the trienones with two different terminal aromatic
rings possess greater potency toward three prostate cancer cell lines, but also have higher capability in
suppressing the proliferation of PWR-1E benign human prostate epithelial cells, as compared to those
with two identical terminal rings. The terminal aromatic rings are a determining factor of this scaffold
in the ability to suppress proliferation and to induce apoptosis in prostate epithelial cells. Considering
both potency towards prostate cancer cells and selectivity between prostate cancer cells and non-
neoplastic cells, compounds 2-6 and 42 are identified as the optimal 1,7-diaryl-1,4,6-heptatrien-3-ones
for further investigation.
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). Curcumin was synthesized by Claisen-Schmidt condensation of
aromatic aldehyde with acetylacetone according to the procedure described in the literature [17]. 1,3-
Bis(diethylphosphonato)acetone (49) was synthesized using the procedure illustrated in the literature
[18]. (E)-3-(aryl)-acryladehydes (48) and (E)-diethyl(2-oxo-4-aryl-but-3-en-1-yl)phosphonates (47)
were synthesized according to the procedure described in the literature [8]. To compare the anti-
19

ACCEPTED MANUSCRIPT
proliferative activity toward PWR-1E prostate non-neoplastic epithelial cells, seven known 1,7-
diheteroarylhepta-1,4,6-trien-2-ones (2-11) with two identical terminal aromatic rings have also been
synthesized according to the procedure illustrated in the literature [8].
4.2 General procedure for the synthesis of diethyl ((3E,5E)-2-oxo-6-(aryl)hexa-3,5-dien-1-
yl)phosphonates (50-53).
To the solution of tetraethyl (2-oxopropane-1,3-diyl)bis(phosphonate) (49) (0.5 mmol) in ethanol (0.4
o
mL), potassium carbonate (1 mmol) in water (1.0 mL) was added dropwise at 4 C. After 30 min at
room temperature, (E)-(aryl)acrylaldehyde (48, 0.8 mmol) in ethanol (0.4 mL) was added dropwise to
the solution. The inorganic solids were removed by filtration after 2 h, and the filtrate was diluted with
water (10 mL) and extracted with dichloromethane (10 mL × 3). The combined organic extracts were
dried over anhydrous magnesium sulfate, and the volatile components were evaporated under vacuum
to give the crude product, which was subjected to the PTLC purification using
dichloromethane/methanol (100/3, v/v) as eluent to give the respective product. The yields and NMR
data for each product were listed below:
4.2.1 Diethyl ((3
1H NMR (300 MHz, CDCl3)
7.22–7.08 (m, 2H), 6.51 (d,
6.9 Hz, 6H). 13C NMR (75 MHz, CDCl3)
130.3, 119.4, 61.8 (d, = 6.4 Hz), 40.3 (d, JCP = 126.8 Hz), 15.4 (d,
E
,5
E
)-2-oxo-6-(thiazol-2-yl)hexa-3,5-dien-1-yl)phosphonate (50): Yellow oil, 40%.
7.84 (d, = 3.2 Hz, 1H), 7.41–7.31 (m, 1H), 7.35 (d, = 3.2 Hz, 1H),
= 15.2 Hz, 1H), 4.20–4.04 (m, 4H), 3.24 (d, = 22.7 Hz, 2H), 1.31 (q,
189.9 (d, = 6.0 Hz), 164.1, 143.6, 141.6, 132.3, 130.6,
= 6.2 Hz, 1H).
δ
J
J
J
J
J =
δ
J
J
J
4.2.2
yl)phosphonate (51): Yellow oil, 51%. H NMR (300 MHz, CDCl3)
1H), 7.22 (d, = 15.3 Hz, 1H), 6.62 (dd, = 15.3, 11.0 Hz, 1H), 6.45 (d,
(m, 4H), 3.23 (d, = 22.7 Hz, 2H), 2.70 (s, 3H), 1.31 (t, δ
= 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3)
Diethyl
((3E,5E)-6-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)-2-oxohexa-3,5-dien-1-
1
δ
7.31 (dd,
J = 15.3, 11.0 Hz,
J
J
J
= 15.3 Hz, 1H), 4.17–4.07
J
J
190.8, 166.4, 142.5, 141.8 (q, JCF = 35.1 Hz), 137.6, 132.5, 131.3, 128.2, 121.0 (q, JCF = 270.3 Hz),
62.8, 41.2 (d, JCP = 126.8 Hz), 19.6, 16.4.
4.2.3 Diethyl ((3
1H NMR (300 MHz, CDCl3)
E
,5
E
)-2-oxo-6-(pyridin-2-yl)hexa-3,5-dien-1-yl)phosphonate (52): Yellow oil, 58%.
8.61 (d, = 4.0 Hz, 1H), 7.68 (td, = 7.7, 1.8 Hz, 1H), 7.50–7.31
δ
J
J
20

ACCEPTED MANUSCRIPT
(overlapped, 3H), 7.20 (ddd,
J
= 7.5, 4.8, 1.0 Hz, 1H), 7.02 (d,
= 22.7 Hz, 2H), 1.32 (t,
= 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3)
191.0 (d, JCP = 6.5 Hz), 153.9, 149.9, 143.6, 140.9, 136.6, 130.9, 130.1, 123.4, 123.3, 62.5 (d, = 6.3
J = 14.0 Hz, 1H), 6.50 (d, J = 14.0 Hz,
1H), 4.23–4.07 (m, 4H), 3.26 (d,
J
J
δ
J
Hz), 40.8 (d, J = 127.0 Hz), 16.2 (d, J = 6.1 Hz).
4.2.4 Diethyl ((3
E
1
,5
E
)-6-(3-fluoropyridin-4-yl)-2-oxohexa-3,5-dien-1-yl)phosphonate (53): Light
8.43 (s, 1H), 8.35 (d, = 4.0 Hz, 1H), 7.40–7.25
yellow oil, 51%. H NMR (300 MHz, CDCl3)
δ
J
(overlapped, 2H), 7.09 (dd,
4.15–4.06 (m, 4H), 3.22 (d,
191.0 (d, = 6.0 Hz), 146.0 (d,
= 2.2 Hz), 130.7 (d, = 9.7 Hz), 121.1, 62.7 (d,
Hz).
J
= 15.7, 10.2 Hz, 1H), 6.99 (d,
= 22.7 Hz, 2H), 1.28 (t,
= 5.1 Hz), 143.0, 139.1 (d,
= 6.5 Hz), 40.2 (d,
J
= 15.7 Hz, 1H), 6.49 (d, J = 15.3 Hz, 1H),
13
J
J
= 7.1 Hz, 6H). C NMR (75 MHz, CDCl3)
δ
J
J
J
= 24.2 Hz), 133.2, 133.1, 131.9, 131.3 (d,
= 126.7 Hz), 16.3 (d, = 6.2
J
J
J
J
J
4.3 General procedure for the synthesis of the (1E,4E,6E)-1,7-diarylhepta-1,4,6-trien-3-ones (12-
46):
Method A: A 5 mL flask was charged with the appropriate (
E)-diethyl (2-oxo-4-aryl-but-3-en-1-
yl)phosphonate (47, 0.25 mmoL) and the appropriate ( )-3-aryl-acrylaldehyde (48, 0.25 mmoL). To
E
the flask was added a solution of potassium carbonate (0.035 g, 0.25 mmoL) in water (0.2 mL) and
ethanol (0.15 mL). The biphasic mixture was stirred rapidly at cold room (4 oC) overnight before being
extracted with dicloromethane (10 mL × 3). The combined extracts were dried over anhydrous
magnesium sulfate and concentrated. The residue was purified over repetitive PTLC eluting with
dicloromethane/methanol (100:5, v/v), hexane/EtOAc (1:1, v/v), and EtOAc/methanol (100:5, v/v) to
furnish the respective final product.
Method B: A 5 mL flask was charged with the appropriate (3E,5E)-2-oxo-6-arylhexa-3,5-dien-1-
yl)phosphonate (50-53, 0.6 mmol) and the appropriate heteroaromatic carboxaldehyde (54, 0.5 mmol).
To the flask was added a solution of potassium carbonate (1.5 mmol) in water (5 mL) and ethanol (5.0
o
mL). The subsequent biphasic mixture was stirred rapidly at cold room (4 C) overnight. The reaction
was quenched by adding water (10 mL), and the mixture was extracted with dichloromethane (10 mL ×
3). The combined extracts were dried over anhydrous magnesium sulfate and concentrated under
reduced pressure. The residue was subjected to PTLC purification eluting with
dichloromethane/methanol (5% methanol in dichloromethane, v/v) to give the respective product.
21

ACCEPTED MANUSCRIPT
4.3.1 (1
E
,4E
,6
E
)-7-(1-Ethyl-1
H-imidazol-2-yl)-1-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (12). This
compound was obtained in 7% yield as a yellow oil from (
yl)phosphonate and ( )-3-(1-ethyl-1
MHz, CDCl3) 7.95 (d, = 3.2 Hz, 1H), 7.76 (d,
7.47 (d, = 3.2 Hz, 1H), 7.40 (dd, = 14.7, 11.6 Hz, 1H), 7.32 (d,
= 1.0 Hz, 1H), 6.81 (d, = 14.6 Hz, 1H), 6.65 (d,
= 7.3 Hz, 3H). C NMR (75 MHz, CDCl3) 188.2, 164.3, 145.1, 143.9, 143.5, 133.7,
E
)-diethyl (2-oxo-4-(thiazol-2-yl)but-3-en-1-
1
E
H
-imidazol-2-yl)acrylaldehyde using Method A. H NMR (300
δ
J
J
= 15.7 Hz, 1H), 7.54 (dd,
= 15.7 Hz, 1H), 7.16 (d,
= 14.8 Hz, 1H), 4.06 (q, J
J
= 14.7, 11.6 Hz, 1H),
= 0.7 Hz,
= 7.3 Hz,
J
J
J
J
1H), 6.99 (d,
2H), 1.45 (t,
J
J
J
J
13
δ
130.5, 130.1, 129.8, 129.2, 125.5, 121.8, 121.1, 41.1, 16.7. IR (film) νmax: 1647, 1609, 1577, 1478,
1077 cm-1; HRMS (ESI): m/z calculated for C15H16N3OS [M+H]+: 286.1014. Found: 286.1008.
4.3.2 (1
compound was obtained in 15% yield as a yellow oil from (
1-yl)phosphonate and ( )-3-(1-propyl-1
-imidazol-2-yl)acrylaldehyde using Method A. 1H NMR (300
MHz, CDCl3) 7.95 (d, = 3.2 Hz, 1H), 7.76 (d, = 15.7 Hz, 1H), 7.54 (dd, = 14.9, 11.6 Hz, 1H),
7.47 (d, = 3.2 Hz, 1H), 7.40 (dd, = 14.6, 11.6 Hz, 1H), 7.32 (d, = 15.7 Hz, 1H), 7.15 (s, 1H), 6.97
(d, = 0.9 Hz, 1H), 6.80 (d, = 14.6 Hz, 1H), 6.64 (d, = 14.8 Hz, 1H), 3.96 (t, = 7.2 Hz, 2H), 1.85–
1.74 (m, 2H), 0.96 (t, 188.3, 164.3, 145.1, 144.1, 143.6,
= 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3)
E
,4
E
,6
E
)-7-(1-Propyl-1
H-imidazol-2-yl)-1-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (13). This
E)-diethyl (2-oxo-4-(thiazol-2-yl)but-3-en-
E
H
δ
J
J
J
J
J
J
J
J
J
J
J
δ
133.7, 130.4, 130.0, 129.7, 129.1, 125.6, 121.9, 121.8, 47.9, 24.8, 11.32. IR (film) νmax: 1646, 1609,
1577, 1263, 1077 cm-1; HRMS (ESI): m/z calculated for C16H18N3OS [M+H]+: 300.1171. Found:
300.1168.
4.3.3 (1
This compound was obtained in 17% yield as a yellow oil from (
3-en-1-yl)phosphonate and ( )-3-(1-isopropyl-1
NMR (300 MHz, CDCl3) 7.96 (d, = 3.2 Hz, 1H), 7.77 (d,
Hz, 1H), 7.48 (d, = 3.2 Hz, 1H), 7.44 (dd, = 14.5, 11.6 Hz, 1H), 7.33 (d,
1H), 7.07 (d, = 0.9 Hz, 1H), 6.87 (d, = 14.4 Hz, 1H), 6.66 (d, = 14.7 Hz, 1H), 4.59–4.49 (m, 1H),
1.50 (d, 188.2, 164.3, 145.0, 143.6, 133.75, 130.6, 130.0,
= 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3)
E,4E,6E)-7-(1-Isopropyl-1H-imidazol-2-yl)-1-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (14).
E)-diethyl (2-oxo-4-(thiazol-2-yl)but-
1
E
H
-imidazol-2-yl)acrylaldehyde using Method A. H
δ
J
J
= 15.7 Hz, 1H), 7.56 (dd,
J = 14.5, 11.6
J
J
J
= 15.7 Hz, 1H), 7.19 (s,
J
J
J
J
δ
129.8, 129.2, 125.7, 121.8, 117.6, 47.57, 23.78. IR (film) νmax: 1647, 1609, 1577, 1463, 1078 cm-1;
HRMS (ESI): m/z calculated for C16H18N3OS [M+H]+: 300.1171. Found: 300.1163.
4.3.4 (1
E,4E,6E)-7-(1-(sec-Butyl)-1H-imidazol-2-yl)-1-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (15).
This compound was synthesized in 19% yield as a yellow oil from (E)-diethyl (2-oxo-4-(thiazol-2-
22

ACCEPTED MANUSCRIPT
yl)but-3-en-1-yl)phosphonate and (
A. 1H NMR (300 MHz, CDCl3)
7.96 (d,
11.6 Hz, 1H), 7.46 (d, = 14.5 Hz, 1H), 7.44 (dd,
7.20 (s, 1H), 7.03 (d, = 1.0 Hz, 1H), 6.86 (d, = 14.5 Hz, 1H), 6.65 (d,
(m, 1H), 1.85–1.72 (m, 2H), 1.48 (d, = 6.7 Hz, 3H), 0.86 (t,
= 7.4 Hz, 3H). 13C NMR (75 MHz,
CDCl3) 188.2, 164.3, 145.0, 144.1, 143.7, 133.6, 130.8, 129.9, 129.8, 129.2, 125.8, 121.8, 117.8, 53.5,
E
)-3-(1-(sec-butyl)-1
= 3.2 Hz, 1H), 7.76 (d,
= 14.5, 11.6 Hz, 1H), 7.33 (d,
= 14.5 Hz, 1H), 4.33–4.18
H
-imidazol-2-yl)acrylaldehyde using Method
= 15.7 Hz, 1H), 7.56 (dd, = 14.5,
= 15.7 Hz, 1H),
δ
J
J
J
J
J
J
J
J
J
J
J
δ
31.0, 21.8, 10.8. IR (film) νmax: 1647, 1610, 1577, 1459, 1264, 1078, 904 cm-1; HRMS (ESI): m/z
calculated for C17H20N3OS [M+H]+: 314.1327. Found: 314.1325.
4.3.5
one (16). This compound was synthesized in 39% yield as a yellow oil from (
(thiazol-2-yl)but-3-en-1-yl)phosphonate and )-3-(2-methyl-4-(trifluoromethyl)thiazol-5-
yl)acrylaldehyde using Method A. 1H NMR (300 MHz, CDCl3)
7.96 (d, = 3.1 Hz, 1H), 7.76 (d,
15.7 Hz, 1H), 7.49 (d, = 3.5 Hz, 1H), 7.45 (dd, = 15.3, 11.0 Hz, 1H), 7.35–7.24 (overlapped, 2H),
6.69 (dd, = 15.2, 11.0 Hz, 1H), 6.61 (d,
= 15.3 Hz, 1H), 2.72 (s, 3H). 13C NMR (75 MHz, CDCl3)
188.0, 166.4, 164.0, 145.1, 142.3, 140.9 (q, = 35.1 Hz), 137.7, 134.1, 132.9, 131.2, 128.4, 128.0,
(1
E
,4
E
,6
E
)-7-(2-Methyl-4-trifluoromethyl-thiazol-5-yl)-1-thiazol-2-yl-hepta-1,4,6-trien-3-
E)-diethyl (2-oxo-4-
(E
δ
J
J =
J
J
J
J
δ
J
122.1, 120.0 (q, JCF = 270.1 Hz). IR (film) νmax: 1650, 1612, 1481, 1363, 1125, 1001 cm-1; HRMS
(ESI): m/z calculated for C15H12F3N2OS2 [M+H]+: 357.0343. Found: 357.0333.
4.3.6 (1
compound was synthesized in 40% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-ethyl-1
-imidazole-2-carbaldehyde using Method B. 1H
NMR (300 MHz, CDCl3) 7.87 (d, = 3.2 Hz, 1H), 7.55–7.53 (overlapped, 2H), 7.51 (dd, = 15.3,
10.2 Hz, 1H), 7.36 (d, = 3.2 Hz, 1H), 7.28–7.18 (overlapped, 2H), 7.14 (d,
= 1.0 Hz, 1H), 6.60 (d, = 15.3 Hz, 1H), 4.13 (q, = 7.3 Hz, 2H), 1.46 (t,
E
,4E
,6
E
)-1-(1-Ethyl-1
H-imidazol-2-yl)-7-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (17): This
E
,5E)-2-oxo-6-(thiazol-2-
H
δ
J
J
J
J
= 15.4 Hz, 1H), 7.06 (d,
J
J
J
J
= 7.3 Hz, 3H). 13C NMR
(75 MHz, CDCl3)
δ 188.4, 165.4, 144.5, 143.0, 141.8, 133.3, 132.6, 132.1, 130.9, 126.7, 125.8, 122.2,
120.2, 41.2, 16.9. IR (film) νmax: 1646, 1613, 1579, 1244, 1082 cm-1; HRMS (ESI): m/z calculated for
C15H16N3OS [M+H]+: 286.1014. Found: 286.1009.
4.3.7 (1
compound was synthesized in 71% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-propyl-1 -imidazole-2-carbaldehyde using Method B.
1H NMR (300 MHz, CDCl3)
7.84 (d, = 3.2 Hz, 1H), 7.60–7.43 (overlapped, 3H), 7.34 (d,
E
,4
E
,6
E
)-1-(1-Propyl-1
H-imidazol-2-yl)-7-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (18). This
E
,5E)-2-oxo-6-(thiazol-2-
H
δ
J
J
= 3.2
23

ACCEPTED MANUSCRIPT
Hz, 1H), 7.26–7.16 (overlapped, 2H), 7.12 (d,
15.3 Hz, 1H), 4.01 (t, = 7.2 Hz, 2H), 1.86–1.72 (m, 2H), 0.92 (t,
CDCl3) 188.3, 165.4, 144.5, 143.2, 141.8, 133.3, 132.5, 132.0, 130.6, 126.6, 125.8, 123.0, 120.2, 48.0,
J
= 15.3 Hz, 1H), 7.03 (d,
J = 1.0 Hz, 1H), 6.57 (d, J =
J
J
= 7.4 Hz, 3H). 13C NMR (75 MHz,
δ
24.9, 11.2. IR (film) νmax: 1644, 1611, 1578, 1442, 1277, 1077, 993 cm-1; HRMS (ESI): m/z calculated
for C16H18N3OS [M+H]+: 300.1171. Found: 300.1166.
4.3.8 (1
compound was synthesized in 63% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (52) and 1-propyl-1
-imidazol-2-carbaldehyde using Method B. 1H
NMR (300 MHz, CDCl3) 8.62 (d, = 4.7 Hz, 1H), 7.68 (td, = 7.7, 1.8 Hz, 1H), 7.63–7.41
(overlapped, 4H), 7.36 (d,
E
,4
E
,6
E
)-1-(1-Propyl-1
H-imidazol-2-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-one (19). This
E
,5 )-2-oxo-6-(pyridin-2-
E
H
δ
J
J
J
= 7.8 Hz, 1H), 7.20 (d,
J
= 5.8 Hz, 1H), 7.19 (d,
J
= 6.5 Hz, 1H), 7.01 (d,
J
= 14.5 Hz, 2H), 6.59 (d,
J
= 14.9 Hz, 1H), 4.03 (t,
J
= 7.2 Hz, 2H), 1.87–1.74 (m, 2H), 0.94 (t,
J
= 7.4
13
Hz, 3H). C NMR (75 MHz, CDCl3)
δ 188.7, 154.4, 150.1, 143.3, 142.9, 140.3, 136.8, 132.8, 131.0,
130.5, 126.3, 126.0, 123.6, 123.4, 122.9, 48.0, 24.9, 11.2. IR (film) νmax: 1646, 1605, 1467, 1080 cm-1;
HRMS (ESI): m/z calculated for C18H20N3O [M+H]+: 294.1606. Found: 294.1602.
4.3.9 (1
This compound was synthesized in 35% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-isopropyl-1 -imidazole-2-carbaldehyde using Method B.
1H NMR (300 MHz, CDCl3)
7.87 (d, = 3.2 Hz, 1H), 7.74 (d, = 15.0 Hz, 1H), 7.59 (dd, = 15.1,
10.8 Hz, 1H), 7.57 (d, = 14.6 Hz, 1H), 7.37 (d,
= 15.4 Hz, 1H), 4.74–4.64 (m, 1H), 1.50 (d,
E
,4
E
,6
E
)-1-(1-Isopropyl-1
H
-imidazol-2-yl)-7-(thiazol-2-yl)-hepta-1,4,6-trien-3-one (20).
E
,5E)-2-oxo-6-(thiazol-2-
H
δ
J
J
J
J
J
= 3.2 Hz, 1H), 7.28–7.15 (overlapped, 4H), 6.59 (d,
13
J
J
= 6.7 Hz, 6H). C NMR (75 MHz, CDCl3)
δ
188.4,
165.4, 144.5, 142.7, 141.7, 133.3, 132.5, 132.1, 131.0, 126.8, 125.89, 120.2, 118.7, 47.8, 24.0. IR
(film) νmax: 1646, 1612, 1461, 1265, 1081, 905, 725 cm-1; HRMS (ESI): m/z calculated for C16H18N3OS
[M+H]+: 300.1171. Found: 300.1170.
4.3.10 (1
This compound was synthesized in 64% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (52) and 1-isopropyl-1 -imidazol-2-carbaldehyde using Method B.
1H NMR (300 MHz, CDCl3)
8.60 (d, = 4.2 Hz, 1H), 7.66 (t, = 7.7 Hz, 1H), 7.62–7.58 (overlapped,
2H), 7.56 (d, = 15.8 Hz, 1H), 7.44 (dd, = 14.8, 11.4 Hz, 1H), 7.35 (d, = 7.8 Hz, 1H), 7.24–7.14
(overlapped, 2H), 7.11 (s, 1H), 7.00 (d, = 14.9 Hz, 1H), 6.58 (d, = 15.1 Hz, 1H), 4.70–4.61 (m, 1H),
1.46 (d,
= 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3)
E
,4E
,6
E
)-1-(1-Isopropyl-1
H-imidazol-2-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-one (21).
E
,5 )-2-oxo-6-(pyridin-2-
E
H
δ
J
J
J
J
J
J
J
J
δ
188.7, 154.3, 150.1, 142.9, 142.7, 140.2, 136.8,
24

ACCEPTED MANUSCRIPT
132.7, 131.0, 130.7, 126.3, 126.0, 123.6, 123.4, 118.6, 47.7, 23.9. IR (film) νmax: 1646, 1605, 1462,
1269, 1081 cm-1; HRMS (ESI): m/z calculated for C18H20N3O [M+H]+: 294.1606. Found: 294.1605.
4.3.11
(1E,4E,6E)-1-(1-Isopropyl-1H-imidazol-2-yl)-7-(2-methyl-4-(trifluoromethyl)thiazol-5-
yl)hepta-1,4,6-trien-3-one (22). This compound was synthesized in 45% yield as a yellow oil from
diethyl
((3E
,5
E)-6-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)-2-oxohexa-3,5-dien-1-yl)phosphonate
1
(51) and 1-isopropyl-1
7.62 (d, = 15.0 Hz, 1H), 7.58 (s, 1H), 7.49 (dd,
6.70 (dd, = 15.2, 11.0 Hz, 1H), 6.52 (d, = 15.5 Hz, 1H), 4.73–4.64 (m, 1H), 2.73 (s, 3H), 1.50 (d,
= 6.6 Hz, 6H). 13C NMR (75 MHz, CDCl3)
188.3, 166.3, 142.7, 141.8, 140.8 (q, JCF = 35.1 Hz),
H
-imidazole-2-carbaldehyde using Method B. H NMR (300 MHz, CDCl3)
δ
J
J = 15.0, 10.6 Hz, 1H), 7.28–7.14 (overlapped, 3H),
J
J
J
δ
137.8, 133.1, 132.9, 131.1, 127.6, 126.9, 125.5, 121.1 (q, JCF = 270.4 Hz), 118.8, 47.8, 23.9, 19.7. IR
(film) νmax: 1647, 1613, 1489, 1364, 1126, 1083 cm-1; HRMS (ESI): m/z calculated for C18H19F3N3OS
[M+H]+: 382.1201. Found: 382.1200.
4.3.12 (1
E
,4
E
,6
E
)-1-(1-(sec-Butyl)-1
H
-imidazol-2-yl)-7-(thiazol-2-yl)hepta-1,4,6-trien-3-one (23).
,5 )-2-oxo-6-(thiazol-2-
-imidazole-2-carbaldehyde using Method
This compound was synthesized in 35% yield as a yellow oil from diethyl ((3
E
E
yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-(sec-butyl)-1
H
1
B. H NMR (300 MHz, CDCl3)
δ
7.87 (d,
J = 3.2 Hz, 1H), 7.64–7.56 (overlapped, 2H), 7.52 (dd, J =
15.4, 10.1 Hz, 1H), 7.36 (d,
7.14 (d, = 15.4 Hz, 1H), 7.09 (d,
1.76 (m, 2H), 1.47 (d,
J
= 3.2 Hz, 1H), 7.24 (d,
J
= 0.8 Hz, 1H), 7.23 (dd,
J
= 15.4, 10.2 Hz, 1H),
J
J
= 1.1 Hz, 1H), 6.59 (d,
J
= 15.3 Hz, 1H), 4.45–4.34 (m, 1H), 1.83–
13
J
= 6.7 Hz, 3H), 0.84 (t,
J
= 7.4 Hz, 3H); C NMR (75 MHz, CDCl3)
δ
188.4,
165.5, 144.5, 143.3, 141.7, 133.4, 132.5, 132.1, 131.2, 126.9, 125.8, 120.2, 119.0, 53.6, 31.0, 22.0,
10.7. IR (film) νmax: 1646, 1610, 1459, 1245, 1085, 967 cm-1; HRMS (ESI): m/z calculated for
C17H20N3OS [M+H]+: 314.1327. Found: 314.1320.
4.3.13 (1
E
,4
E
,6
E
)-1-(1-(sec-Butyl)-1
H
-imidazol-2-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-one (24).
,5 )-2-oxo-6-(pyridin-2-
-imidazol-2-carbaldehyde using Method
This compound was synthesized in 62% yield as a yellow oil from diethyl ((3
E
E
yl)hexa-3,5-dien-1-yl)phosphonate (52) and 1-(sec-butyl)-1
H
1
B. H NMR (300 MHz, CDCl3)
δ
8.60 (d,
= 7.8 Hz, 1H), 7.23–7.14 (m, 2H), 7.07 (s, 1H), 7.00 (d,
= 14.9 Hz, 1H), 4.44–4.29 (m, 1H), 1.86–1.69 (m, 2H), 1.44 (d, = 6.7 Hz, 3H), 0.82 (t, J =
J
= 4.4 Hz, 1H), 7.70–7.51 (overlapped, 4H), 7.44 (dd,
J =
14.7, 11.3 Hz, 1H), 7.35 (d,
6.58 (d,
J
J
= 14.9 Hz, 1H),
J
J
25

ACCEPTED MANUSCRIPT
7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ
188.7, 154.3, 150.1, 143.2, 142.9, 140.2, 136.8, 132.8,
131.0, 130.9, 126.5, 126.0, 123.6, 123.4, 118.8, 53.5, 31.0, 22.0, 10.7. IR (film) νmax: 1646, 1605, 1459,
1263, 1081, 906 cm-1; HRMS (ESI): m/z calculated for C19H22N3O [M+H]+: 308.1763. Found:
308.1758.
4.3.14
(1
E
,4
E
,6
E
)-1-(1-(Pentan-2-yl)-1
H
-imidazol-2-yl)-7-(thiazol-2-yl)hepta-1,4,6-trien-3-one
,5 )-2-oxo-6-
-imidazole-2-carbaldehyde
(25). This compound was synthesized in 75% yield as a yellow oil from diethyl ((3
E
E
(thiazol-2-yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-(pentan-2-yl)-1
H
1
using Method B. H NMR (300 MHz, CDCl3)
δ
7.85 (d,
= 3.2 Hz, 1H), 7.24–7.03 (overlapped, 4H), 6.58 (d,
= 7.7 Hz, 2H), 1.44 (d, = 6.7 Hz, 3H), 1.28–1.07 (m, 2H), 0.87 (t,
188.4, 165.4, 144.5, 143.1, 141.8, 133.3, 132.5, 132.0,
J
= 3.2 Hz, 1H), 7.57 (s, 2H), 7.51 (dd,
J =
15.4, 10.1 Hz, 1H), 7.34 (d,
4.53–4.38 (m, 1H), 1.72 (q,
7.3 Hz, 3H). 13C NMR (75 MHz, CDCl3)
J
J
= 15.3 Hz, 1H),
J
J
J =
δ
131.0, 126.7, 125.9, 120.2, 118.9, 52.0, 40.0, 22.4, 19.4, 13.8. IR (film) νmax: 1645, 1610, 1456, 1264,
1082, 996 cm-1; HRMS (ESI): m/z calculated for C18H22N3OS [M+H]+: 328.1483. Found: 328.1480.
4.3.15
(26). This compound was synthesized in 52% yield as a yellow oil from diethyl ((3
(pyridin-2-yl)hexa-3,5-dien-1-yl)phosphonate (52) and 1-(pentan-2-yl)-1 -imidazol-2-carbaldehyde
using Method B. 1H NMR (300 MHz, CDCl3)
8.60 (d, = 4.7 Hz, 1H), 7.66 (td, = 7.7, 1.7 Hz, 1H),
7.62–7.49 (m, 3H), 7.44 (dd, = 14.7, 11.3 Hz, 1H), 7.34 (d, = 7.9 Hz, 1H), 7.22–7.12 (m, 2H), 7.07
(d, = 0.9 Hz, 1H), 6.99 (d, = 14.9 Hz, 1H), 6.58 (d, = 14.8 Hz, 1H), 4.53–4.37 (m, 1H), 1.76–1.68
(m, 2H), 1.43 (d, = 6.7 Hz, 3H), 1.30–1.06 (m, 2H), 0.86 (t,
= 7.3 Hz, 3H). 13C NMR (75 MHz,
CDCl3) 188.7, 154.3, 150.1, 143.2, 142.8, 140.2, 136.8, 132.8, 131.1, 131.0, 126.5, 125.9, 123.5,
(1
E
,4
E
,6E
)-1-(1-(Pentan-2-yl)-1
H-imidazol-2-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-one
E
,5 )-2-oxo-6-
E
H
δ
J
J
J
J
J
J
J
J
J
δ
123.3, 118.8, 51.9, 40.0, 22.4, 19.4, 13.8. IR (film) νmax: 1646, 1604, 1458, 1258, 1080 cm-1; HRMS
(ESI): m/z calculated for C20H24N3O [M+H]+: 322.1919. Found: 322.1912.
4.3.16 (1
This compound was synthesized in 60% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (50) and 1-(isopentyl)-1 -imidazole-2-carbaldehyde using Method
B. 1H NMR (300 MHz, CDCl3)
7.87 (d, = 3.2 Hz, 1H), 7.56–7.53 (overlapped, 2H), 7.52 (dd,
15.4, 10.1 Hz, 1H), 7.36 (d, = 3.2 Hz, 1H), 7.28–7.19 (overlapped, 2H), 7.15 (d, = 15.3 Hz, 1H),
7.04 (d, = 1.0 Hz, 1H), 6.60 (d, = 15.3 Hz, 1H), 4.07 (t, = 7.2 Hz, 2H), 1.70–1.58 (m, 3H), 0.96 (d,
E
,4E
,6
E
)-1-(1-Isopentyl-1
H-imidazol-2-yl)-7-(thiazol-2-yl)hepta-1,4,6-trien-3-one (27).
E
,5E)-2-oxo-6-(thiazol-2-
H
δ
J
J =
J
J
J
J
J
26

ACCEPTED MANUSCRIPT
J
= 6.4 Hz, 6H); 13C NMR (75 MHz, CDCl3)
δ 188.4, 165.5, 144.5, 143.2, 141.8, 133.3, 132.6, 132.1,
130.7, 126.6, 125.7, 122.8, 120.3, 44.8, 40.4, 25.8, 22.4. IR (film) νmax: 1649, 1614, 1264, 730 cm-1;
HRMS (ESI): m/z calculated for C18H22N3OS [M+H]+: 328.1484. Found: 328.1475.
4.3.17 (1
This compound was synthesized in 57% yield as a yellow oil from diethyl ((3
yl)hexa-3,5-dien-1-yl)phosphonate (52) and 1-isopentyl-1 -imidazol-2-carbaldehyde using Method B.
1H NMR (300 MHz, CDCl3)
8.62 (dd, = 4.8, 0.9 Hz, 1H), 7.68 (td,
15.0 Hz, 1H), 7.58 (dd, = 15.0, 11.3 Hz, 1H), 7.51 (d, = 14.9 Hz, 1H), 7.45 (dd,
1H), 7.36 (d, = 7.9 Hz, 1H), 7.23–7.16 (m, 2H), 7.03 (s, 1H), 7.01 (d,
14.8 Hz, 1H), 4.07 (t, = 7.2 Hz, 2H), 1.71–1.55 (m, 3H), 0.96 (d,
CDCl3) 188.7, 154.4, 150.1, 143.2, 142.9, 140.3, 136.8, 132.7, 131.0, 130.6, 126.3, 126.0, 123.6,
E
,4
E
,6E
)-1-(1-isopentyl-1
H-imidazol-2-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-one (28).
E
,5 )-2-oxo-6-(pyridin-2-
E
H
δ
J
J
= 7.7, 1.8 Hz, 1H), 7.58 (d,
J
=
=
J
J
J
= 14.8, 11.3 Hz,
J
J
= 14.9 Hz, 1H), 6.59 (d, J
J
J
= 6.4 Hz, 6H). 13C NMR (75 MHz,
δ
123.4, 122.7, 44.8, 40.4, 25.7, 22.4. IR (film) νmax: 1674, 1605, 1468, 1274, 1081 cm-1; HRMS (ESI):
m/z calculated for C20H23N3O [M+H]+: 322.1919. Found: 322.1918.
4.3.18 (1
E
,4
E
,6
E
)-1-(2-Methyl-4-trifluoromethyl-thiazol-5-yl)-7-thiazol-2-yl-hepta-1,4,6-trien-3-
,5 )-2-oxo-6-
one (29). This compound was synthesized in 84% yield as a yellow oil from diethyl ((3
E
E
(thiazol-2-yl)hexa-3,5-dien-1-yl)phosphonate (50) and 2-methyl-4-(trifluoromethyl)-thiazol-2-
1
carbaldehyde using Method B. H NMR (300 MHz, CDCl3)
δ
7.90 (d,
J
= 16.2 Hz, 1H), 7.88 (d,
= 15.0, 9.6 Hz, 1H),
= 15.5 Hz, 1H), 2.76 (s, 3H). 13C NMR
J =
6.3 Hz, 1H), 7.47 (dd,
7.18 (d, = 15.1 Hz, 1H), 6.73 (d,
(75 MHz, CDCl3)
J
= 15.0, 9.6 Hz, 1H), 7.38 (d,
J
= 3.2 Hz, 1H), 7.25 (dd, J
J
J
= 16.2 Hz, 1H), 6.67 (d,
J
δ
187.2, 167.7, 165.2, 144.6, 143.4 (q, JCF = 35.6 Hz), 142.5, 136.3, 133.2, 131.5,
130.9, 130.5, 129.6, 120.8 (q, JCF = 270.8 Hz), 120.5, 19.8. IR (film) νmax: 1664, 1608, 1476, 1324,
1076, 1001 cm-1; HRMS (ESI): m/z calculated for C15H12F3N2OS2 [M+H]+: 357.0343. Found:
357.0341.
4.3.19 (1
one (30). This compound was synthesized in 86% yield as a yellow solid from diethyl ((3
6-(pyridin-2-yl)hexa-3,5-dien-1-yl)phosphonate (52) and 2-methyl-4-trifluoromethyl-thiazol-5-
carbaldehyde using Method B. Yellow solid, 86% yield. 1H NMR (300 MHz, CDCl3)
8.61 (d, = 4.7
Hz, 1H), 7.87 (dd, = 15.6, 0.9 Hz, 1H), 7.67 (td, = 7.7, 1.8 Hz, 1H), 7.57–7.38 (m, 2H), 7.34 (d,
E
,4
E
,6
E
)-1-(2-Methyl-4-trifluoromethyl-thiazol-5-yl)-7-pyridin-2-yl-hepta-1,4,6-trien-3-
E
,5 )-2-oxo-
E
δ
J
J
J
J =
27

ACCEPTED MANUSCRIPT
7.8 Hz, 1H), 7.23–7.16 (m, 1H), 7.03 (d,
J
= 14.4 Hz, 1H), 6.72 (d,
J = 15.6 Hz, 1H), 6.63 (d, J = 14.3
13
Hz, 1H), 2.73 (s, 3H). C NMR (75 MHz, CDCl3)
δ
187.5, 167.6, 154.2, 150.2, 143.5, 143.3 (q, JCF =
35.2 Hz), 141.0, 136.8, 136.4, 131.0, 130.5, 130.0, 129.3, 126.2, 123.5, 120.8 (q, JCF = 270.7 Hz), 19.7.
IR (film) νmax: 1650, 1604, 1585, 1345, 1165 cm-1; HRMS (ESI): m/z calculated for C17H14F3N2OS
[M+H]+: 351.0779. Found: 351.0777.
4.3.20
1,4,6-trien-3-one (31). This compound was synthesized in 76% yield as a yellow solid from diethyl
((3 ,5 )-6-(3-fluoropyridin-4-yl)-2-oxohexa-3,5-dien-1-yl)phosphonate (53) and 2-methyl-4-
trifluoromethyl-thiazol-5-carbaldehyde using Method B. 1H NMR (300 MHz, CDCl3)
8.49 (d, = 2.1
Hz, 1H), 8.41 (d, = 5.0 Hz, 1H), 7.89 (d, = 15.6 Hz, 1H), 7.47 (dd, = 15.1, 10.6 Hz, 1H), 7.39 (t,
= 5.7 Hz, 1H), 7.20 (dd, = 15.7, 10.7 Hz, 1H), 7.06 (d, = 15.7 Hz, 1H), 6.72 (d,
6.67 (d,
= 15.9 Hz, 1H), 2.75 (s, 3H). 13C NMR (75 MHz, CDCl3)
(1E,4E,6E)-7-(3-Fluoro-pyridin-4-yl)-1-(2-methyl-4-trifluoromethyl-thiazol-5-yl)-hepta-
E
E
δ
J
J
J
J
J
J
J
J
= 15.9 Hz, 1H),
J
δ
187.3, 167.8, 156.9 (d, JCF
=
=
259.6 Hz), 146.1 (d, JCF = 5.2 Hz), 143.5 (q, JCF = 35.3 Hz), 142.9, 139.4, 139.1, 136.3, 133.4 (d, JCF
6.0 Hz), 131.5 (d, JCF = 2.1 Hz), 130.9, 130.8, 129.7, 121.2, 120.7 (q, JCF = 270.7 Hz), 19.8. IR (film)
νmax: 1650, 1607, 1583, 1489, 1417, 1122, 1055 cm-1; HRMS (ESI): m/z calculated for C17H13F4N2OS
[M+H]+: 369.0684. Found: 369.0675.
4.3.21 (1
compound was synthesized in 33% yield as a yellow oil from (
en-1-yl)phosphonate and ( )-3-(1-propyl-1
(300 MHz, CDCl3) 8.66 (d, = 4.7 Hz, 1H), 7.72 (td,
7.55 (d, = 15.5 Hz, 1H), 7.54 (dd, = 14.9, 11.5 Hz, 1H), 7.46 (d,
11.5 Hz, 1H), 7.28 (dd, = 4.8, 1.1 Hz, 1H), 7.15 (s, 1H), 6.95 (d,
1H), 6.67 (d, = 14.8 Hz, 1H), 3.95 (t, = 7.2 Hz, 2H), 1.85–1.73 (m, 2H), 0.95 (t,
NMR (75 MHz, CDCl3) 189.2, 153.4, 150.3, 144.2, 143.1, 141.4, 137.0, 130.8, 130.1, 130.1, 128.5,
E,4E,6E)-7-(1-Propyl-1H-imidazol-2-yl)-1-pyridin-2-yl-hepta-1,4,6-trien-3-one (32). This
E)-diethyl (2-oxo-4-(pyridin-2-yl)but-3-
1
E
H
-imidazol-2-yl)acrylaldehyde using Method A. H NMR
δ
J
J
= 7.7, 1.8 Hz, 1H), 7.65 (d,
= 5.6 Hz, 1H), 7.40 (dd,
= 1.0 Hz, 1H), 6.78 (d, = 14.6 Hz,
= 7.4 Hz, 3H). 13C
J
= 15.6 Hz, 1H),
J
J
J
J
= 14.7,
J
J
J
J
J
J
δ
125.3, 125.1, 124.5, 121.8, 47.9, 24.8, 11.3. IR (film) νmax: 1649, 1617, 1578, 1430, 1080, 995 cm-1;
HRMS (ESI): m/z calculated for C18H20N3O [M+H]+: 294.1606. Found: 294.1601.
4.3.21 (1E,4E,6E)-7-(1-Isopropyl-1H-imidazol-2-yl)-1-pyridin-2-yl-hepta-1,4,6-trien-3-one (33).
This compound was synthesized in 29% yield as a yellow oil from (
E
)-diethyl (2-oxo-4-(pyridin-2-
28