Asymmetric 1,5-Diarylpenta-1,4-dien-3-ones: Antiproliferative Activity in Prostate Epithelial Cell Models and Pharmacokinetic Studies

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.

Graphical Abstract

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.

Fig. 1.

Structures, antiproliferative potency, and pharmacokinetic profiles of curcumin and symmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (2–7) [7]

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.

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.

Scheme 1.

Synthesis of asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25–58)

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.

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, 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.

Table 3.

Anti-proliferative activity of the asymmetric 1,5-diheteroarylpenta-1,4-dien-3-ones (25–58) toward three prostate cancer cell lines

Compd	IC50 (μM)a			IC50 (curcumin)/IC50(dienone)
	PC-3b	DU145c	LNCaPd	PC-3b	DU145c	LNCaPd
Curcumin	25.43 ± 2.15	26.23 ± 0.65	13.61 ± 2.69	1	1	1
25	0.39 ± 0.08	0.42 ± 0.09	0.17 ± 0.02	64	62	80
26	0.33 ± 0.02	0.28 ± 0.04	0.31 ± 0.01	76	94	44
27	0.31 ± 0.03	0.53 ± 0.04	0.24 ± 0.04	82	50	57
28	0.23 ± 0.02	0.35 ± 0.04	0.22 ± 0.05	110	75	62
29	0.37 ± 0.02	0.75 ± 0.03	0.24 ± 0.04	69	35	57
30	0.72 ± 0.01	0.67 ± 0.02	0.66 ± 0.04	35	39	21
31	1.33 ± 0.20	0.85 ± 0.04	0.91 ± 0.08	19	31	15
32	0.26 ± 0.02	0.29 ± 0.03	0.26 ± 0.02	98	90	52
33	0.55 ± 0.05	1.13 ± 0.12	0.45 ± 0.07	46	23	30
34	0.30 ± 0.04	0.34 ± 0.05	0.24 ± 0.02	85	77	57
35	0.31 ± 0.03	0.50 ± 0.03	0.22 ± 0.01	82	53	62
36	0.45 ± 0.04	0.70 ± 0.10	0.31 ± 0.11	57	38	44
37	6.86 ± 0.37	3.68 ± 0.08	4.05 ± 0.76	4	7	3
38	3.40 ± 0.21	2.55 ± 0.23	2.17 ± 0.19	8	10	6
39	2.27 ± 0.12	1.32 ± 0.27	1.41 ± 0.13	11	20	10
40	0.80 ± 0.04	0.66 ± 0.16	0.54 ± 0.05	32	40	25
41	0.18 ± 0.01	0.13 ± 0.10	0.26 ± 0.07	141	138	52
42	0.24 ± 0.02	0.35 ± 0.05	0.33 ± 0.05	106	75	41
43	0.20 ± 0.05	0.37 ± 0.12	0.56 ± 0.05	127	71	24
44	0.21 ± 0.04	0.77 ± 0.20	0.93 ± 0.33	121	34	41
45	0.14 ± 0.04	0.28 ± 0.04	0.37 ± 0.14	182	94	37
46	0.10 ± 0.01	0.22 ± 0.03	0.22 ± 0.11	254	119	62
47	0.19 ± 0.04	0.31 ± 0.08	0.40 ± 0.03	134	85	34
48	0.48 ± 0.02	0.78 ± 0.06	0.34 ± 0.09	53	34	40
49	0.23 ± 0.00	0.50 ± 0.02	0.25 ± 0.03	110	53	54
50	0.55 ± 0.03	0.84 ± 0.14	0.46 ± 0.07	46	31	30
51	0.34 ± 0.05	0.57 ± 0.04	0.30 ± 0.19	75	46	45
52	0.84 ± 0.03	0.83 ± 0.09	0.40 ± 0.02	30	32	34
53	0.54 ± 0.04	0.63 ±0.06	0.30 ± 0.07	47	42	45
54	1.70 ± 0.20	2.05 ± 0.15	1.76 ± 0.15	15	13	8
55	0.56 ± 0.05	0.68 ± 0.27	0.18 ± 0.04	45	39	76
56	0.23 ± 0.02	0.46 ± 0.13	0.12 ± 0.05	111	57	113
57	0.25 ± 0.02	0.32 ± 0.05	0.17 ± 0.08	102	82	80
58	0.04 ± 0.01	0.12 ± 0.05	0.03 ± 0.01	636	219	454

^aIC₅₀ 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.

^bHuman androgen-insensitive prostate cancer cell line

^cHuman androgen-insensitive prostate cancer cell line

^dHuman androgen-sensitive prostate cancer cell line

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.

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 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	1
25	0.14 ± 0.03	63
26	0.14 ± 0.07	63
27	0.11 ± 0.03	81
28	0.11 ± 0.04	81
32	0.13 ± 0.01	68
34	0.17 ± 0.01	52
35	0.10 ± 0.02	89
41	0.03 ± 0.01	293
45	0.04 ± 0.00	221
46	0.02 ± 0.01	443
49	0.08 ± 0.03	111
51	0.05 ± 0.01	177
56	0.04 ± 0.03	221
57	0.02 ± 0.03	443
58	0.01 ± 0.01	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.

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)
	LNCaP95	VCaP	22Rv1
28	0.37	0.35	0.85
46	0.35	1.12	0.22
49	0.38	1.41	0.22
58	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 rings of the dienones resulted in markedly improved anti-proliferative potency, but little enhancement in bioavailability.

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	0.04
1 hr	0.33	360.7 ± 3.8	113.9 ± 2.2	112.1 ± 5.9	822.6 ± 62.1
2 hr	0.57
3 hr		672.4 ± 23.4	196.5 ± 16.7	982.3 ± 44.7	4264.1 ± 185.3
4 hr	0.13
6 hr		1943.8 ± 47.3	280.9 ± 23.7	1225.1 ± 43.0	456.3 ± 15.2
1 day	0.03	230.3 ± 5.6	86.0 ± 5.9	314.1 ± 1.5	102.7 ± 4.4
tmax (hrs)	2	6	6	6	3
Cmax (ng/mL)	0.57	1943.8 ± 47.3	280.9 ± 23.7	1225.1 ± 1.5	4264.1 ± 185.3
Area under curve (AUC) (ng/mL*h)	2.85	24704.65	4385.55	18314.35	17609.60

^aThe data for curcumin have been reported in our previous paper [7]

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.

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.

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 or metabolite	Retention time (min)	MH+: Theoretical	Parent ion (observed in positive ion mode)	Mass error (ppm)	Major Fragment ions
28	4.35min	370.1201	370.1195	1.6	121, 246, 314 (M-CHCH3CH2CH3)+2H),
28-MO1	3.32min	386.1150	386.1145	1.3	314, 121, 294, 332, 136, 179
28-MO2	3.7min	386.1150	386.1145	1.3	151, 167, 332, 220
28-MO3	3.97min	386.1150	386.1145	1.3	330, 310, 151, 121, 167
49	5.05min	392.1044	392.1039	1.3	199 (M-CHCH-C5H3F3NS); 171 (M+H-CHCH-C5H3F3NS-CH2CH3), 372, 325, 352
49-MO1	4.54min	408.0994	408.0988	1.5	215(M-CHCH-C5H3F3NS); 187 (M+H-CHCH-C5H3F3NS-CH2CH3), 136, 341, 388
49-MO2	4.66min	408.0994	408.0988	1.5	199 (M-CHCH-C5H3F3NS-O); 171 (M+H-CHCH-C5H3F3NS-CH2CH3-O), 388, 215, 171

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.

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 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.

Fig. 3.

Evolution of viable, apoptotic, and necrotic PC-3 cells populations in response to 46.

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 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 0°C for 30 min. The corresponding aromatic carbaldehyde (0.4 mmol, 1 equiv.) was added dropwise to the soltion. 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 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)

Yellow oil, 82 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.95 (1H, d, J = 3.3 Hz, thiazole H-4), 7.86 (1H, d, J = 15.9 Hz, vinyl H), 7.65 (1H, d, J = 15.0 Hz, vinyl H), 7.59 (1H, d, J = 15.0 Hz, vinyl H), 7.47 (1H, d, J = 3.3 Hz, thiazole H-5), 7.24 (1H, s, imidazole H-4), 7.23 (1H, d, J = 15.9 Hz, vinyl H), 7.11 (1H, s, imidazole H-5), 4.40 (1H, sextet, J = 7.2 Hz, sec-butyl CH), 1.88-1.72 (2H, m, sec-butyl CH₂CH₃), 1.47 (3H, d, J = 6.6 Hz, sec-butyl CH₃CH), 0.84 (3H, t, J = 7.2 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.1, 164.1, 145.2, 143.1, 134.4, 131.3, 130.6, 127.5, 125.8, 121.9, 119.2, 53.6, 31.0, 22.0, 10.7. IR (film) ν_max: 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 Hz, sec-butyl CH), 3.95 (3H, s, N-CH₃), 1.89-1.73 (2H, m, sec-butyl CH₂CH₃), 1.49 (3H, d, J = 6.9 Hz, sec-butyl CH₃CH), 0.86 (3H, t, J = 7.2 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.8, 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) ν_max: 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) δ: 7.69 (1H, d, J = 16.5 Hz, vinyl H), 7.65 (1H, d, J = 16.2 Hz, vinyl H), 7.60 (1H, d, J = 16.2 Hz, vinyl H), 7.25 (1H, s, imidazole H-4), 7.12 (1H, s, imidazole H-5), 6.93 (1H, d, J = 16.5 Hz, vinyl H), 6.23 (1H, s, isoxazole H-4), 4.41 (1H, sextet, J = 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, J = 6.6Hz, sec-butyl CH₃CH), 0.85 (3H, t, J = 7.2 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 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) ν_max: 3128, 2970, 2931, 1657, 1630, 1599, 1458, 1268 cm⁻¹. HR-MS (ESI) m/z: 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 (1E,4E)-1-(1-(sec-Butyl)-1H-imidazol-2-yl)-5-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)penta-1,4-dien-3-one (28)

Yellow solid, mp 79–80 °C, 79 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.96 (1 H, d, J = 15.6 Hz, vinyl H), 7.63 (1H, d, J = 17.4 Hz, vinyl H), 7.58 (1H, d, J = 17.4 Hz, vinyl H), 7.27 (1H, s, imidazole H-4), 7.12 (1H, d, J = 0.6 Hz, imidazole H-5), 6.72 (1H, d, J =15.6 Hz, vinyl H), 4.40 (1H, sextet, J = 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, 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 (J_CF = 35 Hz), 142.7, 136.5, 132.3, 130.4, 129.7, 126.8, 126.7, 120.8 (J_CF = 270.8 Hz), 119.3, 54.0, 31.0, 22.0, 19.8, 10.7. IR (film) ν_max: 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) δ: 7.71 (1H, d, J = 15.0 Hz, vinyl H), 7.59 (1H, d, J = 15.0 Hz, vinyl H), 7.57 (1H, d, J = 16.2 Hz, vinyl H), 7.28 (1H, s, imidazole H-4), 7.12 (1H, s, imidazole H-5), 7.07 (1H, d, J =16.2 Hz, vinyl H), 6.38 (1H, s, isoxazole H-5), 4.41 (1H, sextet, J = 7.2 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) ν_max: 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) δ: 8.19 (1H, d, J = 15.3 Hz, vinyl H), 7.94 (1H, d, J = 16.2 Hz, vinyl H), 7.55 (1H, d, J = 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, J = 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 (3H, d, J = 6.6Hz, sec-butyl CH₃CH), 0.87 (3H, t, J = 7.5 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 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, 54.5, 30.8, 21.8, 10.7. IR (film) ν_max: 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) δ: 8.14 (1H, d, J = 15.0 Hz, vinyl H), 7.95 (1H, d, J = 16.2 Hz, vinyl H), 7.56 (1H, d, J = 15.0 Hz, vinyl H), 7.34 (1H, s, imidazole H-4), 7.26 (1H, dd, J =7.2, 1.8 Hz, phenyl H-6), 7.18 (1H, d, J = 1.8 Hz, phenyl H-2), 7.14 (1H, s, imidazole H-5), 6.91 (1H, d, J = 8.1 Hz, phenyl H-5), 6.85 (1H, d, J = 16.2 Hz, vinyl H), 4.46 (1H, sextet, J = 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, 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) ν_max: 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) δ: 7.87 (1H, d, J = 15.3 Hz, vinyl H), 7.80 (1H, d, J = 15.9 Hz, vinyl H), 7.60 (1H, t, J = 7.8 Hz, pyridine 4-H), 7.59 (1H, d, J = 15.0 Hz, vinyl H), 7.44 (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), 7.13 (1H, d, 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, sec-butyl CH₃CH), 0.84 (3H, t, J = 7.5 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 189.1, 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) ν_max: 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)

Yellow solid, mp 78–79 °C, 84 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.70 (1H, d, J = 3.9 Hz, pyridine H-6), 7.98 (1H, d, J = 3.0 Hz, thiazole H-4), 7.89 (1H, d, J = 15.9 Hz, vinyl H), 7.80-7.73 (2H, overlapped, vinyl H; pyridine H-4), 7.61 (1H, d, J = 15.6 Hz, vinyl H), 7.51 (1H, d, J = 7.2 Hz, pyridine H-3), 7.49 (1H, d, J = 2.7 Hz, thiazole H-5), 7.39 (1H, d, J = 15.9 Hz, vinyl H), 7.32 (1H, dd, J = 8.4, 5.7 Hz, pyridine H-5). ¹³C NMR (75 MHz, CDCl₃) δ: 188.8, 164.2, 153.1, 150.4, 145.2, 142.5, 137.2, 134.7, 129.4, 129.5, 125.3, 124.8, 121.8. IR (film) ν_max: 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) δ: 8.69 (1H, d, J = 4.5 Hz, pyridine H-6), 7.80 (1H, d, J = 15.6 Hz, vinyl H), 7.75–7.72 (2H, overlapped, vinyl H; pyridine H-4), 7.63 (1H, d, J = 15.0 Hz, vinyl H), 7.50 (1H, d, J = 7.8 Hz, pyridine H-3), 7.47 (1H, d, J = 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, J = 0.9 Hz, imidazole H-5), 4.43 (1H, sextet, J = 6.9 Hz, sec-butyl CH), 1.90–1.74 (2H, m, sec-butyl CH₂CH₃), 1.49 (3H, d, J = 6.6 Hz, sec-butyl CH₃CH), 0.86 (3H, t, J = 7.2 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 189.1, 153.3, 150.4, 143.2, 142.3, 137.0, 131.0, 130.2, 127.0, 126.3, 125.1, 124.5, 118.9, 53.6, 31.0, 22.0, 10.7. IR (film) ν_max: 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)

Yellow solid, mp 81–82 °C, 71 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.67 (1H, d, J = 4.2 Hz, pyridine H-6), 7.76 – 7.74 (1H, dt, J = 8.1, 1.8 Hz, pyridine H-4), 7.68 (1H, s, pyrazole H-5), 7.67 (d, J = 16.2 Hz, vinyl H), 7.56 (1H, d, J = 15.9 Hz, vinyl H), 7.50-7.42 (2H, overlapped, vinyl H; pyridine H-3), 7.45 (1H, d, J = 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) δ: 189.4, 153.4, 150.2, 145.3, 141.7, 137.1, 132.5, 132.1, 129.1, 126.6, 125.0, 124.5, 96.1, 40.2. IR (film) ν_max: 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)

Yellow solid, mp 77–78 °C, 89 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.68 (1H, d, J = 4.0 Hz, pyridine H-6), 7.78–7.74 (1H, m, pyridine H-4), 7.72 (1H, d, J = 16.2 Hz, vinyl H), 7.69 (1H, d, J = 16.2 Hz, vinyl H), 7.62 (1H, d, J = 15.6 Hz, vinyl H), 7.49 (1H, d, J = 7.8 Hz, pyridine H-3), 7.33–7.29 (1H, m, pyridine H-5), 7.04 (1H, d, J = 16.2 Hz, vinyl H), 6.24 (1H, s, isoxazole H-3), 2.47 (3H, s, isoxazole 5-CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.9, 170.6, 160.3, 153.0, 150.4, 142.8, 137.1, 132.1, 130.9, 127.6, 125.3, 124.8, 99.7, 12.5. IR (neat) ν_max: 3071, 1640, 1603, 1575, 1493, 1387 cm⁻¹. IR (film) ν_max: 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)

Brown oil, 28% yield. ¹H 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, J = 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) δ: 187.8, 169.4, 149.0, 143.1, 135.5, 130.7, 126.8, 125.5, 124.2, 122.9, 118.3, 50.0, 47.7, 25.8, 24.0. IR (neat) ν_max: 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)

Brown-red oil, 63% yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.83 (1H, d, J = 15.3 Hz, vinyl H), 7.57 (1H, d, J = 15.3 Hz, vinyl H), 7.52 (1H, d, J = 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, J = 15.3 Hz, vinyl H), 4.68 (1H, hept, J = 6.6 Hz, isopropyl CH), 3.58 (4H, br.s, piperidine 2 × CH₂), 1.70 (6H, br.s, piperidine 3 × CH₂), 1.48 (6H, d, J = 6.6 Hz, isopropyl 2 × CH₃). ¹³C NMR (75 Hz, CDCl₃) δ: 187.8, 172.8, 148.6, 143.0, 135.3, 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)

Brown-red oil, 77% yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.82 (1H, d, J = 15.3 Hz, vinyl H), 7.59 (1H, d, J = 15.0 Hz, vinyl H), 7.53 (1H, d, J = 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, J = 15.3 Hz, vinyl H), 4.69 (1H, hept, J = 6.6 Hz, isopropyl CH), 3.83 (4H, t, J = 4.8 Hz, morpholine 2 × OCH₂), 3.59 (4H, t, J = 4.8 Hz, morpholine 2 × NCH₂), 1.48 (6H, d, J = 6.6 Hz, isopropyl 2 × CH₃). ¹³C NMR (75 Hz, CDCl₃) 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) ν_max: 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) δ: 8.73 (2H, d, J = 6.0 Hz, pyridine 2-H & 6-H), 7.92 (1H, d, J = 15.3 Hz, vinyl H), 7.81 (2H, d, J = 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, J = 15.6 Hz, vinyl H), 4.77-4.66 (1H, m, isopropyl CH), 2.65 (3H, s, thiazole 4-CH₃), 1.51 (6H, d, J = 6.6 Hz, isopropyl 2 × CH₃). ¹³C NMR (75MHz, CDCl₃) δ: 187.6, 164.7, 158.3, 150.9, 142.6, 139.8, 132.3, 131.4, 131.1, 129.2, 127.5, 127.0, 120.5, 118.9, 47.8, 24.0, 16.1. IR (film) ν_max: 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) δ: 7.84 (1H, d, J = 15.2 Hz, vinyl H), 7.82–7.75 (overlapped, 1H, benzoimidazole H), 7.78 (1H, d, J = 15.2 Hz, vinyl H), 7.68 (1H, d, J = 15.2 Hz, vinyl H), 7.56–7.51 (1H, overlapped, benzoimidazole H), 7.54 (1H, d, J = 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, J = 7.2 Hz, isopropyl CH), 4.70 (1H, hept, J = 6.9 Hz, 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) ν_max: 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, J = 15.3 Hz, vinyl H), 7.60–7.50 (2H, 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, J = 7.2 Hz, sec-butyl CH₃CH), 1.49 (3H, d, J = 6.6 Hz, sec-butyl CH₃CH), 0.86 (3H, t, J = 7.4 Hz, sec-butyl CH₂CH₃), 0.80 (3H, t, J = 7.4 Hz, sec-butyl CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.1, 148.5, 143.9, 143.0, 134.6, 132.2, 131.4, 127.9, 127.7, 127.2, 123.7, 123.2, 120.7, 119.2, 112.2, 54.4, 53.6, 31.0, 28.7, 22.1, 20.4, 11.3, 10.7. IR (film) ν_max: 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) δ: 7.81 (1H, d, J = 15.2 Hz, vinyl H), 7.83–7.76 (1H, m, benzoimidazole H), 7.73 (1H, d, J = 15.3 Hz, vinyl H), 7.61 (1H, d, J = 15.3 Hz, vinyl H), 7.49 (1H, d, J = 15.3 Hz, vinyl H), 7.39–7.30 (3H, m, 3 × benzoimidazole H), 7.23 (1H, d, J = 0.9 Hz, imidazole H-4), 7.04 (1H, d, J = 0.9 Hz, imidazole H-5), 4.12 (2H, d, J = 7.5 Hz, isobutyl CH₂), 3.89 (2H, d, J = 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, J = 6.6 Hz, isobutyl 2 × CH₃), 0.95 (6H, d, J = 6.6 Hz, isobutyl 2 × CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 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) ν_max: 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) δ: 7.81 (1H, d, J = 15.3 Hz, vinyl H), 7.81–7.78 (1H, m, benzoimidazole H), 7.75 (1H, d, J = 15.3 Hz, vinyl H), 7.64 (1H, d, J = 15.0 Hz, vinyl H), 7.50 (1H, d, J = 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, J = 7.4 Hz, isopentyl N-CH₂), 4.10 (2H, t, J = 7.5 Hz, isopentyl N-CH₂), 1.71–1.60 (6H, m, 2 × isopentyl N-CH₂CH₂CH), 1.01 (6H, d, J = 5.9 Hz, isopentyl CH(CH₃)₂), 0.97 (6H, d, J = 6.2 Hz, isopentyl CH(CH₃)₂). ¹³C NMR (75 MHz, CDCl₃) δ: 187.9, 148.1, 143.5, 143.0, 135.9, 131.8, 131.1, 127.7, 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) ν_max: 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) δ: 7.84–7.78 (3H, m, 2 × vinyl H; 1 × benzoimidazole), 7.69 (1H, d, J = 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, J = 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, J = 7.6 Hz, 1-(pentan-2-yl) CHCH₂CH₂CH₃), 1.70 (3H, d, J = 6.9 Hz, 1-(pentan-2-yl) CH₃CH), 1.49 (3H, d, J = 6.7 Hz, 1-(pentan-2-yl) CH₃CH), 1.28 – 1.15 (3H, m, 1-(pentan-2-yl) CHCH₂CH₂CH₃), 0.91 (3H, t, J = 7.3 Hz, 1-(pentan-2-yl) CHCH₂CH₂CH₃), 0.87 (3H, t, J = 7.2 Hz, 1-(pentan-2-yl) CHCH₂CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.1, 148.4, 144.0, 143.0, 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) ν_max: 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).

4.3.22 (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)

This compound was prepared in 38% yield as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ: 7.83 (1H, d, J = 15.2 Hz, vinyl H), 7.84–7.78 (1H, m, benzoimidazole H), 7.74 (1H, d, J = 15.2 Hz, vinyl H), 7.63 (1H, d, J = 15.2 Hz, vinyl H), 7.51 (1H, d, J = 15.2 Hz, vinyl H), 7.42–7.30 (3H, m, 3 × benzoimidazole H), 7.23 (1H, d, J = 0.7 Hz, imidazole H-4), 7.08 (1H, d, J = 1.0 Hz, imidazole H-5), 4.30 (2H, t, J = 7.2 Hz, propyl NCH₂), 4.08 (2H, t, J = 7.2 Hz, propyl NCH₂), 1.95–1.79 (4H, m, 2 × CH₂CH₂CH₃), 0.982 (3H, t, J = 7.5 Hz, propyl CH₃), 0.977 (3H, t, J = 7.5 Hz, propyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 187.9, 148.3, 143.4, 143.1, 136.0, 131.7, 131.1, 127.7, 127.2, 127.1, 124.1, 123.5, 123.3, 120.5, 110.2, 48.0, 45.4, 24.9, 24.0, 11.5, 11.3. IR (film) ν_max: 2966, 2932, 1652, 1624, 1597, 1446, 1408, 1096 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₁H₂₅N₄O [M+H]⁺: 349.2028; found 349.2023. HPLC purity 95.9% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.23 (1E,4E)-1-(1-Butyl-1H-benzo[d]imidazole-2-yl)-5-(1-butyl-1H-imidazole-2-yl)penta-1,4-dien-3-one (47)

This compound was prepared in 50% yield as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ: 7.83 (1H, d, J = 15.3 Hz, vinyl H), 7.83–7.80 (1H, m, benzoimidazole H), 7.75 (1H, d, J = 15.3 Hz, vinyl H), 7.64 (1H, d, J = 15.3 Hz, vinyl H), 7.51 (1H, d, J = 15.3 Hz, vinyl H), 7.40–7.30 (3H, m, 3 × benzoimidazole H), 7.24 (1H, d, J = 0.7 Hz, imidazole H-4), 7.07 (1H, d, J = 1.0 Hz, imidazole H-5), 4.33 (2H, t, J = 7.2 Hz, butyl NCH₂), 4.10 (2H, t, J = 7.2 Hz, butyl NCH₂), 1.87–1.74 (4H, m, 2 × butyl NCH₂CH₂), 1.39–1.32 (4H, m, 2 × butyl NCH₂CH₂CH₂), 0.97 (3H, t, J = 7.2 Hz, butyl CH₃), 0.96 (3H, t, J = 7.2 Hz, butyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.0, 148.3, 143.6, 143.1, 136.1, 131.9, 131.2, 127.8, 127.32, 127.25, 124.2, 123.6, 123.3, 120.6, 110.2, 46.4, 43.8, 33.7, 32.9, 20.4, 20.1, 13.9, 13.8. IR (film) ν_max: 2960, 2931, 2874, 1653, 1624, 1446, 1408, 1264 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₃H₂₉N₄O [M+H]⁺: 377.2341; found 377.2336. HPLC purity 97.3% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.24 (1E,4E)-1-(1-Methyl-1H-benzo[d]imidazol-2-yl)-5-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)penta-1,4-dien-3-one (48)

Yellow solid, mp 134–135 °C, 34 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.98 (1H, d, J = 15.6 Hz, vinyl H), 7.77 (1H, d, J = 8.7 Hz, benzoimidazole H), 7.76 (2H, s, 2 × vinyl H), 7.35-7.31 (3H, overlapped, 3 × benzoimidazole H), 6.73 (1H, d, J = 15.6 Hz, vinyl H), 3.90 (3H, s, benzoimidazole CH₃), 2.75 (3H, s, thiazole CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 186.7, 168.1, 148.2, 143.7 (J_CF = 35.2 Hz), 143.2, 136.5, 136.3, 131.8, 130.3, 130.1, 127.8, 124.5, 123.8, 120.8 (J_CF = 270.8 Hz), 120.4, 110.0, 30.2, 19.8. IR (film) ν_max: 2920, 1669, 1623, 1588, 1458, 1367, 1334, 1169, 1111 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₈H₁₅N₃OSF₃ [M+H]⁺: 378.0888; found 378.0878. HPLC purity 97.0% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.25 (1E,4E)-1-(1-Ethyl-1H-benzo[d]imidazol-2-yl)-5-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)penta-1,4-dien-3-one (49)

Yellow solid, mp 133–134°C, 89 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.02 (1H, d, J = 15.9 Hz, vinyl H), 7.93-7.82 (2H, m, 1 × vinyl H; 1 × benzoimidazole H), 7.76 (1H, d, J = 15.0 Hz, vinyl H), 7.41-7.34 (3H, overlapped, 3 × benzoimidazole H), 6.78 (1H, d, J = 15.6 Hz, vinyl H), 4.40 (2H, q, J = 7.5 Hz, ethyl CH₂), 2.77 (3H, s, thiazole CH₃), 1.49 (3H, t, J = 7.5 Hz, ethyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 186.7, 168.1, 147.5, 143.7 (q, J_CF = 35.3 Hz), 143.4, 136.3, 135.5, 131.9, 130.3, 130.2, 127.7, 124.4, 123.8, 120.8 (q, J_CF = 270.8 Hz), 120.5, 110.0, 38.8, 19.8, 16.0. IR (film) ν_max: 2980, 1653, 1621, 1540, 1409, 1363 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₉H₁₇N₃OSF₃ [M+H]⁺: 392.1044; found 392.1037. HPLC purity 97.1% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.26 (1E,4E)-1-(2-Methyl-4-(trifluoromethyl)thiazol-5-yl)-5-(1-propyl-1H-benzo[d]imidazol-2-yl)penta-1,4-dien-3-one (50)

Yellow solid, mp 145–146 °C, 59 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 7.99 (1H, d, J = 15.6 Hz, vinyl H), 7.81 (1H, d, J = 15.0 Hz, vinyl H), 7.79 (1H, t, J = 4.2 Hz, benzoimidazole H), 7.73 (1H, d, J = 15.0 Hz, vinyl H), 7.40-7.28 (3H, overlapped, 3 × benzoimidazole H), 6.76 (1H, d, J = 15.6 Hz, vinyl H), 4.27 (2H, t, J = 7.2 Hz, propyl N-CH₂), 2.72 (3H, s, thiazole CH₃), 1.93-1.83 (2H, m, propyl N-CH₂CH₂), 0.95 (3H, t, J = 7.2 Hz, propyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 186.7, 168.1, 147.9, 143.7 (q, J_CF = 36 Hz), 143.3, 136.3, 136.0, 131.9, 130.3, 130.1, 127.9, 124.4, 123.7, 120.8 (q, J_CF = 270.8 Hz), 120.5, 110.3, 45.5, 24.1, 19.8, 11.5. IR (film) ν_max: 2968, 1699, 1622, 1507, 1472, 1363 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₀H₁₉N₃OSF₃ [M+H]⁺: 406.1201; found 406.1194. HPLC purity 96.2% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.27 (1E,4E)-1-(1-Isopropyl-1H-benzo[d]imidazol-2-yl)-5-(2-methyl-4-(trifluoromethyl)thiazol-5-yl)penta-1,4-dien-3-one (51)

Yellow syrup, 54 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.00 (1H, d, J = 15.9 Hz, vinyl H), 7.87-7.7.75 (3H, overlapped, 2 × vinyl H; 1 × benzoimidazole H), 7.57-7.54 (1H, m, benzoimidazole H), 7.31-7.26 (2H, overlapped, 2 × benzoimidazole H), 6.76 (1H, d, J = 15.6 Hz, vinyl H), 4.99-4.92 (1H, m, isopropyl CH), 2.74 (3H, s, thiazole CH₃), 1.69 (6H, d, J = 7.2 Hz, isopropyl 2 × CH₃). IR (film) ν_max: 2980, 1653, 1618, 1488, 1386, 1165 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₀H₁₉N₃OSF₃ [M+H]⁺: 406.1201; found 406.1197. HPLC purity 95.0% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.28 (1E,4E)-1-(Pyridin-2-yl)-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (52)

Yellow oil, 42 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.70 (1H, d, J = 4.8 Hz, pyridine H-6), 7.80-7.71 (4H, overlapped, 3 × vinyl H; pyridine H-4), 7.51 (1H, d, J = 7.7 Hz, pyridine H-3), 7.32 (1H, dd, J = 7.1, 5.2 Hz, pyridine H-5), 6.99 (1H, d, J = 16.0 Hz, vinyl H), 6.86, (2H, s, phenyl H-2, H-6), 3.93 (6H, s, phenyl 3-OCH₃, 5-OCH₃), 3.91 (3H, s, phenyl 4-OCH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 189.2, 153.7, 153.3, 150.1, 144.3, 141.2, 140.6, 137.4, 130.3, 128.5, 125.6, 125.4, 124.6, 105.8, 61.2, 56.3. IR (film) ν_max: 3071, 2939, 2839, 1653, 1623, 1579, 1503, 1464, 1431, 1419, 1320 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₉H₂₀NO₄ [M+H]⁺: 326.1392; found 326.1387. HPLC purity 98.4% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.29 (1E,4E)-1-(6-Methylpyridin-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (53)

Brown solid, mp 43–44 °C, 80 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.68 (1H, d, J = 4.5 Hz, pyridine H-6), 7.79-7.72 (3H, overlapped, 2 × vinyl H, pyridine H-4), 7.67-7.58 (3H, overlapped, 2 × vinyl H, methylpyridine H-4), 7.51 (1H, d, J = 7.8 Hz, pyridine H-3), 7.34-7.27 (2H, overlapped, pyridine H-5, methylpyridine H-3), 7.16 (1H, d, J = 7.7 Hz, methylpyridine H-5), 2.62 (3H, s, methylpyridine CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 189.8, 159.2, 153.4, 152.6, 150.3, 142.5, 142.2, 137.2, 137.0, 129.0, 128.8, 125.0, 124.5, 124.5, 122.2, 24.5. IR (film) ν_max: 3054, 1655, 1628, 1603, 1582, 1451, 1331 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₆H₁₅N₂O [M+H]⁺: 251.1184; found 251.1179. HPLC purity 95.2% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.30 (1E,4E)-1-(3,4-Dimethoxyphenyl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (54)

Yellow oil, 21 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.71 (1H, d, J = 4.7 Hz, pyridine H-6), 7.82–7.72 (4H, overlapped, 3 × vinyl H, pyridine H-4), 7.52 (1H, d, J = 7.8 Hz, pyridine H-3), 7.34 (1H, dd, J = 7.2, 5.1 Hz, pyridine H-5), 7.23 (1H, dd, J = 8.3, 1.8 Hz, phenyl H-6), 7.16 (1H, d, J = 1.8 Hz, phenyl H-2), 6.97 (1H, d, J = 16.2 Hz, vinyl H), 6.91 (1H, d, J = 8.4 Hz, phenyl H-5), 3.95 (3H, s, OCH₃), 3.94 (3H, s, OCH₃). IR (film) ν_max: 2925, 1651, 1623, 1588, 1265 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₈H₁₈NO₃ [M+H]⁺: 296.1287; found 296.1281. HPLC purity 96.3% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.31 (1E,4E)-1-(1-Methyl-1H-benzo[d]imidazol-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (55)

Yellow solid, mp 65–66°C, 52 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.70 (1H, d, J = 4.2 Hz, pyridine H-6), 8.02 (1H, d, J = 15.0 Hz, vinyl H), 7.85 (1H, d, J = 15.9 Hz, vinyl H), 7.81 (1H, d, J = 15.3 Hz, vinyl H), 7.85-7.75 (2H, overlapped, pyridine H-4; 1 × benzoimidazole H), 7.53 (1H d, J = 15.0 Hz, vinyl H), 7.51 (1H, d, J = 8.1 Hz, pyridine H-3), 7.39-7.29 (4H, overlapped, pyridine H-5; 3 × benzoimidazole H), 3.95 (3H, s, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.7, 153.2, 150.5, 148.6, 143.2, 137.1, 136.5, 130.7, 130.0, 127.0, 125.3, 124.8, 124.4, 123.8, 120.4, 110.0, 30.3. IR (film) ν_max: 3053, 2928, 1655, 1615, 1471, 1330 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₈H₁₆N₃O [M+H]⁺: 290.1293; found 290.1287. HPLC purity 98.5% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.32 (1E,4E)-1-(1-Ethyl-1H-benzo[d]imidazol-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (56)

Yellow oil, 34 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.69 (1H, d, J = 3.9 Hz, pyridine H-6), 7.99 (1H, d, J = 15.3 Hz, vinyl H), 7.85-7.77 (4H, overlapped, 2 × vinyl H; pyridine H-4; 1 × benzoimidazole H), 7.51 (1H, d, J = 15.9 Hz, vinyl H), 7.50 (1H, d, J = 8.1 H, pyridine H-3), 7.42-7.26 (4H, overlapped, pyridine H-5; 3 × benzoimidazole H), 4.38 (2H, q, J = 7.5 Hz, ethyl CH₂), 1.48 (3H, t, J = 7.2 Hz, ethyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.6, 153.1, 150.4, 147.8, 143.4, 143.0, 136.9, 135.5, 130.4, 129.9, 127.1, 125.1, 124.6, 124.1, 123.5, 120.4, 109.9, 38.7, 16.0. IR (film) ν_max: 3058, 2976, 1655, 1627, 1601, 1564, 1470, 1326 cm⁻¹. HR-MS (ESI) m/z: calcd for C₁₉H₁₈N₃O [M+H]⁺: 304.1450; found 304.1444. HPLC purity 99.0% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.33 (1E,4E)-1-(1-Propyl-1H-benzo[d]imidazol-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (57)

Brown syrup, 39 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.69 (1H, d, J = 4.5 Hz, pyridine H-6), 8.02 (1H, d, J = 15.3 Hz, vinyl H), 7.86-7.73 (4H, overlapped, 2 × vinyl H; pyridine H-4; 1 × benzoimidazole H), 7.51 (1H, d, J = 9.9 Hz, pyridine H-3), 7.50 (1H, d, J = 15.6 Hz, vinyl H), 7.42-7.28 (4H, overlapped, pyridine H-5; 3 × benzoimidazole H), 4.30 (2H, t, J = 7.2 Hz, propyl N-CH₂), 1.89 (2H, sextet, J = 7.2 Hz, propyl N-CH₂CH₂), 0.98 (3H, t, J = 7.2 Hz, propyl CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.7, 153.2, 150.5, 148.3, 143.4, 143.0, 137.0, 136.0, 130.4, 130.1, 127.3, 125.1, 124.7, 124.1, 123.5, 120.5, 110.2, 45.4, 24.1, 11.5. IR (film) ν_max: 3069, 2964, 2930, 1655, 1627, 1599, 1465, 1407, 1326 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₀H₂₀N₃O [M+H]⁺: 318.1606; found 318.1601. HPLC purity 96.3% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.3.34 (1E,4E)-1-(1-Isopropyl-1H-benzo[d]imidazol-2-yl)-5-(pyridin-2-yl)penta-1,4-dien-3-one (58)

Brown oil, 79 % yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.67 (1H, d, J = 4.5 Hz, pyridine H-6), 7.98 (1H, d, J = 15 Hz, vinyl H), 7.85 (1H, d, J = 15.0 Hz, vinyl H), 7.823 (1H, t, J = 5.7 Hz, benzoimidazole H), 7.817 (1H, d, J = 15.3 Hz, vinyl H), 7.71 (1H, t, J = 7.5 Hz, pyridine H-4), 7.58-7.47 (3H, overlapped, 1 × vinyl H; pyridine H-3; 1 × benzoimidazole H ), 7.30-7.26 (3H, overlapped, 2 × benzoimidazole H; pyridine H-5), 4.99 (1H, septet, J = 6.9 Hz, isopropyl CH), 1.69 (6H, d, J = 6.9 Hz, isopropyl 2 × CH₃). ¹³C NMR (75 MHz, CDCl₃) δ: 188.7, 153.1, 150.4, 147.7, 143.8, 142.9, 136.9, 134.4, 130.7, 129.9, 127.9, 125.1, 124.6, 123.7, 123.1, 120.6, 112.1, 48.2, 22.0. IR (film) ν_max: 3052, 2977, 2933, 1654, 1624, 1596, 1564, 1462, 1383, 1321 cm⁻¹. HR-MS (ESI) m/z: calcd for C₂₀H₂₀N₃O [M+H]⁺: 318.1606; found 318.1603. HPLC purity 95.0% (30 min run of 45–80% CH₃CN in H₂O, with 15 min gradient, 1.0 mL/min).

4.4 Cell culture

All cell lines were initially purchased from American Type Culture Collection (ATCC). The PC-3 and LNCaP prostate cancer cell lines were routinely cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a high humidity environment supplemented with 5% carbon dioxide at a temperature of 37°C. The DU-145 prostate cancer cells were routinely cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. LNCaP95, VCaP, and 22Rv1 cells were maintained in Dr. Yan Dong’s laboratory at Tulane School of Medicine and have been previously characterized [18,19].

4.5 WST-1 cell proliferation assay

PC-3, DU-145, or LNCaP were plated in 96-well plates at a density of 3,200 each well in 200 μL of culture medium. PWR-1E was plated in 96-well plates at a density of 5,000 each well in 200 μL of culture medium. The cells were then treated with curcumin, or synthesized analogues separately at different doses for 3 days, while equal treatment volumes of DMSO were used as vehicle control. The cells were cultured in a CO₂ incubator at 37 °C for three days. 10 μL of the premixed WST-1 cell proliferation reagent (Clontech) was added to each well. After mixing gently for one minute on an orbital shaker, the cells were incubated for additional 3 hours at 37 °C. To ensure homogeneous distribution of color, it is important to mix gently on an orbital shaker for one minute. The absorbance of each well was measured using a microplate-reader (Synergy HT, BioTek) at a wavelength of 430 nm. The IC₅₀ value is the concentration of each compound that inhibits cell proliferation by 50% under the experimental conditions and is the average from triplicate determinations that were reproducible and statistically significant. For calculating the IC₅₀ values, a linear proliferative inhibition was made based on at least five dosages for each compound.

4.6 Cell cycle analysis

PC-3 cells were plated in 24-well plates at a density of 200,000 each well in 400 μL of culture medium. After 3 h of cell attachment, the cells were then treated with dienone 28 at 5 μM and dienone 46 at 4 μM, while equal treatment volumes of DMSO were used as vehicle control. The cells were cultured in CO₂ incubator at 37 °C for 16 h. Both attached and floating cells were collected in a centrifuge tube by centrifugation at rcf 450 g for 5 min. After discarding the supernatant, the collected cells were re-suspended with 500 μL 80% cold ethanol to fix for 30 min in 4 °C. The fixed cells could be stored at −20 °C for one week. After fixation, the ethanol was removed after centrifuging and the cells were washed with PBS. The cells were then re-suspended with 100 μL of 100 mg/mL ribonuclease and were cultured at 37 °C for 30 min to degrade all RNA. The cells were stained with 200 μL of 50 μg/mL propidium iodide (PI) stock solution for 30 min at −20 °C, and then the fluorescence intensity of PI was detected in individual PC-3 cells using an Attune flow cytometer (Life Technologies) within 0.5–1 h after staining.

4.7 F2N12S and CYTOX AADvanced double staining assay

PC-3 cells were plated in 24-well plates at a density of 200,000 each well in 400 μL of culture medium. After 3 h of cell attachment, the cells were then treated with the test compound at different concentrations and cultured in CO₂ incubator at 37 °C for 16 h, while equal treatment volumes of DMSO were used as vehicle control. Both attached and floating cells were collected in a centrifuge tube by centrifugation at rcf value of 450 g for 5 min. The collected cells were re-suspended with 500 μL HBSS to remove proteins which may affect flow signal and centrifuged again. After discarding the supernatant, the collected cells were re-suspended with 300 μL HBSS and stained with 0.3 μL of F2N12S for 3–5 min followed by 0.3 μL SYTOX AADvanced for an additional 5 min. The fluorescence intensity of the two probes was further measured in individual PC-3 cells using an Attune flow cytometer (Life Technologies) within 0.5–1 h after staining.

4.8 Pharmacokinetic Study (Sampling and Analysis) [23]

Male Sprague-Dawley rats, weighing between 250 and 300 g (Charles River Laboratories, Portage, MI) were used for the pharmacokinetic study of compounds 28, 46, 49, and 58. Rats (n = 4) were given oral gavage containing 5% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 400, 55% saline-dissolved 28, 46, 49, and 58 at a single dose of 10 mg/kg. After oral administration, blood samples were collected from the lateral tail vein of the rats at 1, 3, 6, and 24 h. Rat blood was collected with a capillary into 1.5 mL microcentrifuge tubes containing 0.01 mL of 10% EDTA anticoagulant. Plasma was then separated from red cells by centrifugation in a refrigerated centrifuge at 4 °C and transferred to a separate tube. The plasma samples were frozen at −80 °C until analysis. All procedures involving these animals were conducted in compliance with state and federal laws, standards of the U.S. Department of Health and Human Services, and guidelines established by Xavier University Animal Care and Use Committee. The facilities and laboratory animals program of Xavier University are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.

4.9 High Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS/MS) for Drug Analysis in Plasma Samples

Plasma samples were extracted with chloroform/methanol (2:1) using traditional Folch method for lipid extraction. Methanol (1mL) and chloroform (2 mL) were added to each plasma sample followed by addition of 5 ng of trans-tamoxifen-¹³C2,¹⁵N to each sample as the internal standard. The mixtures were stored at −20 °C overnight. Next the samples were sonicated for 5 min and centrifuged with a Thermo Scientific Heraeus Megafuge16 centrifuge. The top layer was transferred to another test tube. The bottom layer was washed with 1 mL of chloroform/methanol (2:1), centrifuged, and the top layer was transferred and combined with the previous top layer. Eight-tenth of a milliliter of HPLC grade water was added to the extracts. After vortexing, the mixture was centrifuged. The bottom layer was dried out with nitrogen and re-suspended in 100 μL of HPLC grade acetonitrile. An aliquot of 10 μL of sample was injected onto a Hypersil Gold column (50 mm × 2.1 mm; particle size 1.9 μm, Thermo Scientific) on a Dionex Ultimate 3000 UPLC system equipped with a TSQ Vantage triple quadrupole mass spectrometer for analysis. A binary mobile phase (A, water with 0.05% formic acid; B, acetonitrile with 0.05% formic acid) was used to achieve the gradient of initial 30% B for 1 min and then to 80% B at 8 min, to 100% B at 9 min, and returned to 30% B for 4 min. The flow rate was controlled at 0.6 mL/min. The settings of HESI source were as follows: spray voltage (3200 V); vaporizer temperature (365 °C); sheath gas pressure (45 psi); auxiliary gas pressure (10 psi); capillary temperature (330 °C). Nitrogen was used as the sheath gas and auxiliary gas. Argon was used as the collision gas.

4.10. Incubation of compound 28 (or 49) with rat liver microsomes (Total 300 μL)

The pre-incubation solution was prepared by adding 30 μL of potassium phosphate buffer (pH 7.4; 10X), 241.5 μL water, 15 μL of NADPH solution A, 3μL of NADPH solution B, 7.5 μL pooled human liver microsomes from Corning Gentest into a 1.5 mL microcentrifuge. The mixtures were incubated at 37 °C for 5 min in an incubator. Then 3 μL of 10 mM 28 (or 49) was added, mixed, and incubated at 37 °C for 60 min in the incubator. After incubation, 300 μL MeOH was added to terminate the reaction. The final mixture was then centrifuged at 10,000 × g for 4 min at 4 °C. The supernatant was analyzed on a high resolution mass spectrometer Q-Exactive from Thermo Fisher Scientific connected with a UHPLC ultimate 3000 from Dionex.

4.11 Statistical analysis

All data are represented as the mean ± standard deviation (S.D.) for the number of experiments indicated. Other differences between treated and control groups were analyzed using the Student’s t-test. A p-value < 0.05 was considered statistically significant.

Table 1.

Structures for (E)-diethyl(2-oxo-4-heteroaryl-but-3-en-1-yl)phosphonates 10–24

Table 2.

Structures for asymmetric 1,5-diarylpenta-1,4-dien-3-ones (25–58)

Highlights.

• 34 New asymmetric dienones were evaluated in prostate epithelial cell models.

• The asymmetric dienones are more potent than the corresponding symmetric ones.

• The optimal trienone is 219- to 636-fold more potent than curcumin.

• Two promising compounds exhibit improved potency and bioavailability.

• Four dienones show cytotoxicity in enzalutamide-resistant prostate cancer cells.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://.