Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and Human Lung Epithelial Cells

Vineet Kumar; Sarvesh Kumar; Mohammad Hassan; Hailong Wu; Rajesh K Thimmulappa; Amit Kumar; Sunil K Sharma; Virinder S Parmar; Shyam Biswal; Sanjay V Malhotra

doi:10.1021/jm2002348

. Author manuscript; available in PMC: 2012 Jun 26.

Published in final edited form as: J Med Chem. 2011 May 19;54(12):4147–4159. doi: 10.1021/jm2002348

Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and Human Lung Epithelial Cells

Vineet Kumar ^a,^ψ, Sarvesh Kumar ^b,^ψ, Mohammad Hassan ^a, Hailong Wu ^b, Rajesh K Thimmulappa ^b, Amit Kumar ^c, Sunil K Sharma ^c, Virinder S Parmar ^c, Shyam Biswal ^b,^*, Sanjay V Malhotra ^a,^*

PMCID: PMC3212436 NIHMSID: NIHMS298141 PMID: 21539383

Abstract

Nrf2-mediated activation of antioxidant response element is a central part of molecular mechanisms governing the protective function of phase II detoxification and antioxidant enzymes against carcinogenesis, oxidative stress and inflammation. Nrf2 is sequestered in the cytoplasm by its repressor, Keap1. We have designed and synthesized novel chalcone derivatives as Nrf2 activators. The potency of these compounds was measured by the expression of Nrf2 dependent antioxidant genes, GCLM, NQO1 and HO1, in human lung epithelial cells; while the cytotoxicity was analyzed using MTT assay. In vivo potency of identified lead compounds to activate Nrf2 was evaluated using mouse model. Our studies showed 2-trifluoromethyl-2’-methoxychalone (2b) to be a potent activator of Nrf2, both, in vitro and in mice. Additional experiments showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed a quantitative structure-activity relationship and proposed a possible mechanism of Nrf2 activation.

INTRODUCTION

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a basic-leucine zipper (b-ZIP) transcription factor present in the cytoplasm of normal cells. Upon activation in response to inflammatory stimulus, environmental toxicant, oxidative and electrophilic stress, Nrf2 detaches from its cytosolic inhibitor, Kelch-like ECH-associated protein 1 (Keap1) and translocates to the nucleus and binds to the antioxidant response element (ARE) of target genes along with other binding partners leading to their transcriptional induction.^1–4 The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation.^5–7 In addition to Keap1, the activation of diferent protein kinases has been shown to activate Nrf2.^8–12 The Nrf2-regulated genes include almost all of the relevant antioxidants and cytoprotective genes such as heme oxygenase-1 (HO-1), NAD (P)H:quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), γ-glutamyl cysteine synthase, glutathione peroxidase (GPx), and several members of the glutathione S-transferase family ^{6, 13–18} that express an ARE in their promoter.¹⁹ Small molecules that activate Nrf2 signaling are being investigated as potential anti-cancer or anti-inflammatory agents. A wide variety of dietary and synthetic compounds that function as potent inducers of ARE-regulated gene expression have been shown to exert chemopreventive activities, e.g., sulforaphane^{4, 20–22}, dithiolethione^23–25, curcumin²⁶, and caffeic acid phenethyl ester (CAPE)²⁶. It is notable that both curcumin and CAPE bear an α, β-unsaturated ketone moiety and can therefore act as Michael acceptors that are able to modify cysteine thiols present in Keap1.

Chalcones or 1,2-diphenyl-2-propen-1-ones are Michael acceptors and constitute an important group of natural products belonging to the flavonoid family.^{27, 28} They have been reported to possess many biological properties including anti-cancer^{29, 30}, anti-malarial^{31, 32}, anti-inflammatory^33–35, antileishmanial^33–35, anti-tuberclulosis³⁶, nitric oxide inhibition^{37, 38}, anti-mitotic³⁹, analgesic, antipyretic, antioxidant^40–43, antibacterial, anti-HIV⁴⁴, antifungal⁴⁵ and antiprotozoal activities.^46–48 They are also reported to be gastric protectant⁴⁹, anti-mutagenic, and anti-tumorogenic.^50–52 Natural and synthetic chalcones have been reported to possess strong antiproliferative effects in primary and established ovarian cancer cells⁵³ and in gastric cancer cells.⁵² Chalcones contain two aromatic rings separated by α, β -unsaturated ketone and this unique structure is responsible for various activities of these molecules.²⁷ It is well known that α, β unsaturated carbonyl entity in chalcones is a soft electrophile and would attract soft nucleophiles like thiols, rather than hard nucleophiles like amino and hydroxyl groups. Chalcones are unlikely to react with the amino and hydroxyl groups on nucleic acids and thus would unlikely induce mutagenicity and carcinogenicity commonly associated with alkylating agents used in cancer chemotherapy.²⁸

The remarkable biological potential of chalcones is due to their possible interactions with various proteins related to cell apoptosis and proliferation.^{54, 55} A number of recent studies have indicated that the anti-inflammatory effect of chalcones is due to the inhibition of the NF-κB pathway, which is mediated by IκB degradation and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun.^56–58 It has been reported that electrophilic α, β-unsaturated carbonyl moiety on chalcone resulted in the activation of Nrf2/ARE pathway and the induction of phase II detoxifying enzyme expression.^{59, 60} This moiety acts as an electrophile and reacts with free sulfhydryl groups of thioredoxin and cysteine residues in proteins.^{58, 59, 61} It is also reported that electrophilic phytochemicals could give rise to thiyl radicals, which could also interact with sulfhydryl residues of intracellular targets, including Nrf2.⁶² These studies demonstrate that the endogenous electrophilic activity, through its α, β-unsaturated carbonyl moiety, is involved in the anti-oxidant and anti-inflammatory properties of chalcone. In the present study, we synthesized novel chalcone derivatives and tested their Nrf2 activating activity in human bronchial epithelial cells. Furthermore, to draw a structure-activity relationship, a possible mechanism for Nrf2 activation is proposed.

RESULTS AND DISCUSSION

Chemistry

Chaclones can be readily synthesized by the base-catalyzed Claisen-Schmidt condensation of an aldehyde and ketone in a polar solvent like ethanol or methanol. The traditional synthesis of chalcones involves the use of strong bases such as NaOH^{31, 35, 39, 63}, KOH^{16, 64}, Ba(OH)₂^{65, 66}, hydrotalcites⁶⁷, and LiHMDS⁶⁸, calcined NaNO₃/natural phosphate⁶⁹. They can also be synthesized by acid-catalyzed aldol condensations, e.g., AlCl₃⁷⁰, BF₃⁷¹, dry HCl⁷², Zn(bpy)(OAc)₂⁷³, Cp₂ZrH₂/NiCl₂⁷⁴, and RuCl₃ (for cyclic and acyclic ketones).⁷⁵ Suzuki coupling has also been employed for the synthesis of chalcone derivatives.⁷⁶ Several disadvantages of these procedures include long reaction time, high reaction temperature, complex reaction conditions and the use of expensive and non-commercial reagents. Recently, an efficient and facile synthesis of chalcones by condensation of aldehydes and ketones has been reported using LiOH.H₂O as a dual activation catalyst under mild conditions.⁷⁷ We employed similar conditions for the synthesis of all chalcone derivatives used in the present study. In short, the appropriately substituted acetophenone was dissolved in ethanol followed by addition of catalytic amount of LiOH.H₂O. The reaction mixture was stirred at room temperature for 15 minutes and the desired substituted benzaldehyde was added. The reaction was carried out at room temperature until completion and the corresponding chalcone derivative (1a–5l) was isolated by crystallization or by silica gel flash chromatography (Table 1). All chalcone derivatives were characterized by ¹H, ¹³C NMR, and HRMS. To the best of our knowledge, compounds 2b, 2f, 2i, 2k, 2l, 3b, 3c, 3f, 3i, 3k, 5f, 5g, and 5i have never been reported before, whereas compounds 2e, 2g, 2h, 3g, and 3h are commercially available as per the SciFinder search; however, there is no report on these compounds in mice and human lung epithelial cells.

Table 1.

Synthesis of chalcone derivatives by Claisen-Schmidt condensation and their Nrf2 induction activity


Entry	Chalcones	Substituents on Ring A					Substituents on Ring B			Relative fold Change^a

		R¹	R²	R³	R⁴	R⁵	R⁶	R⁷	R⁸	GCLM	NQO1
1	1a	H	H	H	H	H	H	H	H	0.7	1.8
2	1b	OMe	H	H	H	H	H	H	H	3.6	3.2
3	1c	H	OMe	H	H	H	H	H	H	1.5	2.4
4	1d	H	H	OMe	H	H	H	H	H	1.9	2.8
5	1e	OMe	H	OMe	H	H	H	H	H	2.7	2.8
6	1f	OMe	H	H	H	OMe	H	H	H	2.5	1.9
7	1g	OMe	H	H	OMe	H	H	H	H	3.0	2.6
8	1h	H	OMe	OMe	H	H	H	H	H	2.3	2.5
9	1i	H	OMe	H	OMe	H	H	H	H	3.0	2.4
10	1j	H	OMe	OMe	OMe	H	H	H	H	0.7	0.7
11	1k	OMe	OMe	OMe	H	H	H	H	H	2.4	2.4
12	1l	OMe	H	OMe	H	OMe	H	H	H	3.1	1.8
13	2a	H	H	H	H	H	CF₃	H	H	5.0	5.3
14	2b	OMe	H	H	H	H	CF₃	H	H	4.5	4.6
15	2c	H	OMe	H	H	H	CF₃	H	H	5.6	4.3
16	2d	H	H	OMe	H	H	CF₃	H	H	5.4	4.6
17	2e	OMe	H	OMe	H	H	CF₃	H	H	5.4	4.5
18	2f	OMe	H	H	H	OMe	CF₃	H	H	5.7	5.3
19	2g	OMe	H	H	OMe	H	CF₃	H	H	4.4	2.7
20	2h	H	OMe	OMe	H	H	CF₃	H	H	4.3	2.7

21	2i	H	OMe	H	OMe	H	CF₃	H	H	4.0	3.7
22	2j	H	OMe	OMe	OMe	H	CF₃	H	H	3.0	3.1
23	2k	OMe	OMe	OMe	H	H	CF₃	H	H	5.4	4.1
24	2l	OMe	H	OMe	H	OMe	CF₃	H	H	4.4	4.3
25	3a	H	H	H	H	H	H	CF₃	H	3.1	2.8
26	3b	OMe	H	H	H	H	H	CF₃	H	3.9	2.9
27	3c	H	OMe	H	H	H	H	CF₃	H	4.6	3.5
28	3d	H	H	OMe	H	H	H	CF₃	H	2.7	2.8
29	3e	OMe	H	OMe	H	H	H	CF₃	H	4.6	4.0
30	3f	OMe	H	H	H	OMe	H	CF₃	H	1.8	0.6
31	3g	OMe	H	H	OMe	H	H	CF₃	H	1.8	0.8
32	3h	H	OMe	OMe	H	H	H	CF₃	H	4.1	2.9
33	3i	H	OMe	H	OMe	H	H	CF₃	H	3.8	3.9
34	3j	H	OMe	OMe	OMe	H	H	CF₃	H	1.7	0.8
35	3k	OMe	OMe	OMe	H	H	H	CF₃	H	3.6	3.2
36	3l	OMe	H	OMe	H	OMe	H	CF₃	H	1.4	0.7
37	4a	H	H	H	H	H	H	H	CF₃	3.1	2.9
38	4b	OMe	H	H	H	H	H	H	CF₃	4.3	5.4
39	4c	H	OMe	H	H	H	H	H	CF₃	2.9	3.1
40	4d	H	H	OMe	H	H	H	H	CF₃	3.5	4.6
41	4e	OMe	H	OMe	H	H	H	H	CF₃	5.0	4.8
42	4f	OMe	H	H	H	OMe	H	H	CF₃	1.4	0.6
43	4g	OMe	H	H	OMe	H	H	H	CF₃	4.1	4.0
44	4h	H	OMe	OMe	H	H	H	H	CF₃	3.5	2.7
45	4i	H	OMe	OMe	OMe	H	H	H	CF₃	4.1	3.7
46	4j	OMe	OMe	OMe	H	H	H	H	CF₃	4.1	3.9
47	4k	OMe	H	OMe	H	OMe	H	H	CF₃	2.4	2.4
48	5a	H	H	H	H	H	NO₂	H	H	2.1	1.6

49	5b	OMe	H	H	H	H	NO₂	H	H	1.9	1.4
50	5c	H	OMe		H	H	NO₂	H	H	2.9	2.7
51	5d	H	H	OMe	H	H	NO₂	H	H	4.6	4.3
52	5e	OMe	H	OMe	H	H	NO₂	H	H	2.7	3.3
53	5f	OMe	H	H	H	OMe	NO₂	H	H	4.1	3.7
54	5g	OMe	H	H	OMe	H	NO₂	H	H	1.7	0.7
55	5h	H	OMe	OMe	H	H	NO₂	H	H	2.3	2.7
56	5i	H	OMe	H	OMe	H	NO₂	H	H	3.4	4.1
57	5j	H	OMe	OMe	OMe	H	NO₂	H	H	1.2	0.7
58	5k	OMe	OMe	OMe	H	H	NO₂	H	H	2.9	3.3
59	5l	OMe	H	OMe	H	OMe	NO₂	H	H	2.6	2.0
	DMSO									1.0	1.0
	Sulforaphane									2.7	3.6

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Data presented are representative of 3 independent experiments.

Values shown are mean ± SD of quadruplicate wells.

Biology

Potency of chalcone derivatives to activate the expression of Nrf2-regulated cytoprotective genes in human lung epithelial cells

To investigate the potency of chalcone derivatives to activate Nrf2, we measured the expression of antioxidant genes, GCLM and NADPH-NQO1, two well characterized transcriptional targets of Nrf2, as surrogate markers. Previously, we and others have shown that oxidants or small molecule activators of Nrf2 increase GCLM and NQO1 in cells or tissues of wild-type but not in Nrf2-deficient mice.⁷⁸ In this study, to screen novel Nrf2 activators, we treated normal human bronchial epithelial cells (Beas-2B) with chalcone derivatives (10 µM) for 16 h and analyzed the expression of GCLM and NQO1 by quantitative RT-PCR (qRT-PCR). We included sulforaphane, a well known potent activator of Nrf2, as a positive control. We identified 59 chalcone derivatives that induce the expression of GCLM and NQO1 (Table 1). Concurrent with the gene expression analysis, we analyzed the cytotoxicity of the chalcone derivatives using the MTT assay. A total of 20 chalcones showed a higher induction of Nrf2-regulated transcriptional targets than the positive control i.e., sulforaphane (Table 2).

Table 2.

List of positive leads from primary screening and their effect on cell viability

Entry	Compound	Relative Fold Change^a		% Cell Viability
Entry	Compound	GCLM	NQO1	% Cell Viability
1	DMSO	1	1	100
2	sulforaphane	2.7	3.6	97.3
3	2a	5	5.3	144.8
4	2b	4.5	4.6	100.9
5	2c	5.6	4.3	96.6
6	2d	5.4	4.6	96
7	2e	5.4	4.5	105
8	2f	5.7	5.3	101.1
9	2i	4	3.7	87.1
10	2k	5.4	4.1	101.6
11	2l	4.4	4.3	145.3
12	3c	4.6	3.5	95.3
13	3i	3.8	3.9	108.2
14	4b	4.3	5.4	91
15	4d	3.5	4.6	104.1
16	4e	5	4.8	92.6
17	4g	4.1	4	91.6
18	4j	4.1	3.7	100.1
19	5d	4.6	4.3	93.2
20	5f	4.1	3.7	75.8
21	5i	3.4	4.1	90.9

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Data presented are representative of 3 independent experiments.

Values shown are mean ± SD of quadruplicate wells.

Preliminary structure-activity relationship

The structure-activity relationship analysis showed that the chalcone derivatives 1a–l without any substitution on ring B were not active. The activity of similar derivatives with trifluoromethyl (CF₃) substitution on ring B enhanced the activity dramatically. The position of CF₃ substitution was also crucial for the activity and cytotoxicity of these compounds. In general, the chalcone derivatives with CF₃ substitution at ortho position on ring B were the most active compounds (entries 13–24, Table 1), followed by para (entries 37–47, Table 1), and meta (entries 25–36, Table 1) substitution. Also, the cytotoxicity data show that the ortho CF₃-substituted chalcones were non cytotoxic. This shows that 4-bond separation between carbonyl and CF₃ is crucial for the induction activity. Interestingly, with nitro (NO₂) substitution at ortho position on ring B, the activity decreased and the toxicity increased significantly (entries 48–59, Table 1 and entries 19–21, Table 2). Thus, based on these data, we selected only those chalcone derivatives that showed > 4-fold induction of GCLM and NQO1 genes and > 95% cell viability. Based on these stringent criteria, of the 20 compounds that showed potency to increase Nrf2 activity, we selected compounds 2a–f, 2k, and 2l for further analysis.

In vivo potency of identified lead compounds to activate Nrf2 using mouse models

Next, we evaluated the potency of the 8 lead chalcones identified in the in vitro screening to activate Nrf2 pathway in mouse models. First, we tested various formulations to dissolve the compounds, and the DCP (10% DMSO+ 10% Cremophor EL+ 80% phosphate buffered saline) formulation offered the maximum solubility for delivery of these compounds by oral route. Mice (C57BL/6) were administrated with a single dose of vehicle or test compound(s) or sulforaphane as the positive control at a dose of 50 mg/kg body weight by gavage and small intestines were harvested 24 h later. The expression of Nrf2-regulated genes GCLM and NQO1 was analyzed in the tissue by qRT-PCR. All the 8 lead compounds increased the expression of GCLM and NQO1 in small intestine. However, 2b was the most potent inducer of Nrf2 activity (Figure 2). The expression of GCLM and NQO1 in the small intestine of mice treated with 2b was 6-fold and 10-fold higher compared to vehicle, respectively. Similarly, the expression of GCLM and NQO1 in the small intestine treated with 2b was 3-fold and 5-fold higher compared to sulforaphane, respectively. Taken together, we selected 2b as the most potent activator of Nrf2 for further studies.

Mice (n=4) were fed with vehicle (DCP-10% DMSO + 10% Cremophor + 80% phosphate buffered saline) or chalcone derivatives or sulforaphane (50 mg/kg body weight) by gavage, and the small intestines were harvested 24 h later. The expression of Nrf2-regulated genes GCLM and NQO1 was analyzed in the tissues by qRT-PCR as a surrogate marker of Nrf2 activity. β-actin was used for normalization. Data are representative of 3 independent experiments (P ≤ 0.05).

Nrf2 is essential for induction of antioxidant genes by compound 2b

We further characterized Nrf2 induction by 2b using cell-based assays. Nrf2 increases the expression of NQO1 and GCLM by binding to the ARE present in the promoter region of these genes.⁷⁹ We determined whether the ARE mediates the transcriptional regulation of NQO1 by 2b. We measured the expression of the luciferase gene under the control of NQO1-ARE sequence using stably transfected Beas-2B cells treated with 2b. The exposure to compound 2b resulted in a significant concentration-dependent increase in luciferase activity as measured by the chemiluminescence-based assay (Figure 3). These results implicate the ARE element in the induction of NQO1 gene by compound 2b. The transcriptional activation of antioxidant genes through an ARE is largely dependent upon Nrf2, suggesting that 2b upregulates antioxidant genes via Nrf2 activation.

NQO1-ARE luciferase activity was measured by using stably transfected Beas-2B cells after treatment with compound 2b or sulforaphane (SFN) or dimethyl sulfoxide (DMSO). The exposure to compound 2b resulted in a significant concentration-dependent increase in luciferase activity as relative luminescence intensity (RLI). Data are representative of 3 independent experiments. Values shown are mean ± SD of quadruplicate wells (P ≤ 0.05).

Concentration and time-course studies

Next, we examined the concentration-dependent effect of 2b on the mRNA levels of Nrf2-driven antioxidant genes, GCLM, NQO1, and HO1. We measured the expression of these genes at 24 h after treatment with various concetrations (2.5, 5, 10, 20 µM) of 2b in Beas-2B cells. As shown in Figure 5, compound 2b significantly increased the Nrf2-regulated gene expression in a concentration-dependent manner. There was ~5- and 10-fold increase in the expression of GCLM and NQO1, respectively, at the highest concentration (20 µM) with no cytotoxicity. Interestingly, we found a dramatic concentration-dependent activation of Nrf2 genes. At 10 µM concentration of 2b, the expression of HO-1 was 6-fold higher compared to sulforaphane (Figure 4).

Human bronchial epithelial cells (Beas-2B) were treated with compound 2b (10 µM) at various time points. The expression of Nrf2-regulated genes GCLM, HO1, and NQO1 was analyzed in the tissues by qRT-PCR. β-actin was used for normalization. Data are representative of 3 independent experiments. Values shown are mean ± SD of triplicate wells (P ≤ 0.05).

Human bronchial epithelial cells (Beas-2B) were treated with compound 2b at the indicated concentrations for 16–20 h. The expression of Nrf2-regulated genes GCLM, HO1, and NQO1 was analyzed in the tissues by qRT-PCR as a surrogate marker of Nrf2 activity. β-actin was used for normalization. Data are representative of 3 independent experiments. Values shown are mean ± SD of triplicate wells (P ≤ 0.05).

For the time-course studies, we measured the expression of antioxidant genes (GCLM, NQO1, and HO-1) at 6, 12, 24 and 48 h after treatment with 2b (10 µM) in Beas-2B cells. The time-course studies showed the highest induction of GCLM (~7-fold) and HO-1 (~150-fold) at 6 h after treatment with 2b (Figure 5). The expression of GCLM and HO-1 decreased at 6 h after treatment with 2b and was comparable to vehicle by 48 h. The expression of NQO1 was highest at 24 h and remained significantly elevated even at 48 h after treatment with compound 2b compared to vehicle. Taken together, these results suggest that 2b is a potent activator of Nrf2-regulated antioxidant defenses.

Activation of Nrf2 by compound 2b is independent of ROS generation

The activation of Nrf2 by various electrophiles and compounds that are Michael acceptor is attributed to changes in ROS production and or redox environment and or direct cysteine modification in Keap1.^{80, 81} We examined whether 2b activates Nrf2 by generating ROS or redox changes. Beas-2B cells were co-incubated with compound 2b and with or without N-acetyl-cysteine (NAC, 10 mM), and the expression of GCLM, NQO1, and HO1 was measured 24 h later. We found that 2b potentially increases the expression of Nr2-regulated antioxidant genes in the presence of NAC (Figure 6). NAC alone showed no induction of Nrf2-regulated genes. Taken together, these results suggest that the activation of Nrf2 by 2b is independent of ROS or redox changes. Further studies are required to determine whether compound 2b activates Nrf2 by direct thiol modification of Keap1.

Human bronchial epithelial cells (Beas-2B) were treated with compound 2b (10 µM) in the presence of an antioxidant, N-acetyl cystiene (10 mM, NAC). Cells were harvested 24 h after the treatment, and the Nrf2-driven expression of NQO1, HO-1, and GCLM was quantified. β-actin was used for normalization. Data are representative of 3 independent experiments. Values shown are mean ± SD of triplicate wells (P ≤ 0.05).

CONCLUSIONS

In conclusion, we have identified a novel chalcone 2b as a potent activator of Nrf2 signaling pathway after screening a series of chalcone derivatives using cell-based and mouse models. Further studies are needed to evaluate and develop chalcone 2b as a potential drug for the treatment of inflammatory disorders.

EXPERIMENTAL SECTION

Chemistry

General methods

TLCs were run on pre-coated Merck silica gel 60F254 plates and observed under UV light. The products were isolated and purified by crystallization or using a Teledyne ISCO Rf Flash chromatography system with hexanes and ethyl acetate as eluents. The ¹H (400 MHz), ¹³C (101 MHz), gCOSY, and gHSQC NMR spectra were taken on a Varian 400-MR spectrophotometer using TMS as an internal standard. Chemical shifts (δ) are expressed in ppm, coupling constants (J) are expressed in Hz, and splitting patterns are described as follows: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; dd = doublet of doublets; dt = doublet of triplets; td = triplet of doublets; ddd = doublet of doublet of doublets. For the verification of the product and purity analysis, the LC-MS was taken on an Agilent 1200 series system with an Agilent 6210 Time-Of-Flight (TOF) mass detector using Agilent Eclipse XDB-C-18 column (5 mm, 4.6 × 150 mm) using a flow rate of 0.9 mL/min and solvent system water (with 0.1% formic acid)/acetonitrile (ACN) (Gradient: 50% ACN @ 0 min, 80% ACN @ 7 min, 80% ACN @ 10 min and 50% ACN @ 15 min). All synthesized compounds were >95% pure, (exact purity is given in the subsequent sections with the characterization data). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used without further purification.

General procedure for synthesis of chalcones

In a 14-ml vial, the substituted acetophenone (1.25 mmol) and lithium hydroxide monohydrate (0.25 mmol) were dissolved in ethanol (5 ml) and the mixture was stirred at RT for 10 min followed by addition of substituted benzaldehyde (1.27 mmol). The reaction mixture was then stirred at RT and monitored by TLC using 25% ethyl acetate/hexanes as the solvent system. The reaction was quenched after 2 hrs by pouring into 50 ml of stirring ice cold water. If the product precipitated out after quenching with cold water, it was filtered off and crystallized with hot ethanol. In some examples, a sticky mass was observed in the aqueous solution after quenching. In those cases, the product was extracted by ethyl acetate (3 × 50 ml), dried over sodium sulfate, and concentrated under vacuum. The crude product was purified by flash chromatography using ethyl acetate/hexanes as the solvent system in increasing order of polarity.

(E)-2,3-diphenylprop-2-en-1-one (1a)

It was obtained as light yellow solid in 80% yield. ¹H NMR (400 MHz, DMSO) δ 8.16 – 8.14 (m, 1H), 8.13 (t, J = 1.71, 1.71 Hz, 1H), 7.92 (d, J = 15.67 Hz, 1H), 7.89 – 7.85 (m, 2H), 7.74 (d, J = 15.68 Hz, 1H), 7.69 – 7.63 (m, 1H), 7.59 – 7.53 (m, 2H), 7.47 – 7.42 (m, 3H). ¹³C NMR (101 MHz, DMSO) δ 189.67, 144.48, 137.99, 135.08, 133.60, 131.09, 129.37, 129.34, 129.24, 128.96, 122.51. LC-MS (ESI-TOF): m/z 209.0963 ([C₁₅H₁₂O + H]⁺ calcd. 209.0961). Purity 100.00% (rt 7.39 min).

(E)-1-(2-methoxyphenyl)-3-phenylprop-2-en-1-one (1b)

It was obtained as yellow oil in 71% yield. ¹H NMR (400 MHz, DMSO) δ 7.76 – 7.67 (m, 2H), 7.56 – 7.46 (m, 3H), 7.44 – 7.37 (m, 4H), 7.18 (d, J = 7.9 Hz, 1H), 7.05 (td, J = 7.5, 0.9 Hz, 1H), 3.85 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 192.60, 158.17, 142.97, 135.01, 133.53, 130.94, 129.98, 129.45, 129.23, 128.97, 127.41, 121.01, 112.79, 56.27. LC-MS (ESI-TOF): m/z 239.1072 ([C₁₆H₁₄O₂ + H]⁺ calcd. 239.1067). Purity 98.02% (rt 7.21 min).

(E)-1-(3-methoxyphenyl)-3-phenylprop-2-en-1-one (1c)

It was obtained as yellow oil in 54% yield. ¹H NMR (400 MHz, DMSO) δ 7.95 – 7.85 (m, 3H), 7.78 – 7.70 (m, 2H), 7.62 – 7.58 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.47 – 7.42 (m, 3H), 7.23 (ddd, J = 8.2, 2.6, 0.8 Hz, 1H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 189.36, 159.99, 144.57, 139.42, 135.07, 131.12, 130.40, 129.42, 129.36, 122.51, 121.52, 119.69, 113.41, 55.83. LC-MS (ESI-TOF): m/z 239.1071 ([C₁₆H₁₄O₂ + H]⁺ calcd. 239.1067). Purity 98.52% (rt 7.84 min).

(E)-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one (1d)

It was obtained as white solid in 76% yield. ¹H NMR (400 MHz, DMSO) δ 8.16 (d, J = 9.0 Hz, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.90 – 7.83 (m, 2H), 7.69 (d, J = 15.6 Hz, 1H), 7.44 (dd, J = 5.1, 1.9 Hz, 3H), 7.07 (d, J = 9.0 Hz, 2H), 3.85 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 187.79, 163.68, 143.60, 135.24, 131.39, 130.88, 130.85, 129.33, 129.24, 122.43, 114.48, 56.03. LC-MS (ESI-TOF): m/z 239.1068 ([C₁₆H₁₄O₂ + H]⁺ calcd. 239.1067). Purity 100.00% (rt 7.36 min).

(E)-3-phenyl-1-(2,4-dimethoxyphenyl)prop-2-en-1-one (1e)

It was obtained as yellow oil in 40% yield. ¹H NMR (400 MHz, CDCl₃) δ = 7.76 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 15.8 Hz, 1H), 7.60 (dd, J = 7.3, 1.8 Hz, 2H), 7.52 (d, J = 15.8 Hz, 1H), 7.43 – 7.34 (m, 3H), 6.57 (dd, J = 8.6, 2.2 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 189.73, 164.44, 160.71, 141.62, 135.32, 132.51, 130.65, 129.43, 128.78, 127.52, 121.79, 106.46, 99.07, 56.42, 56.06. LC-MS (ESI-TOF): m/z 269.1171 ([C₁₇H₁₆O₃ + H]⁺ calcd. 269.1172). Purity 96.00% (rt 7.25 min).

(E)-3-phenyl-1-(2,6-dimethoxyphenyl)prop-2-en-1-one (1f)

It was obtained as white solid in 60% yield. ¹H NMR (400 MHz, DMSO) δ = 7.72 – 7.58 (m, 2H), 7.45 – 7.31 (m, 4H), 7.17 (d, J = 16.2 Hz, 1H), 6.97 (d, J = 16.2 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 194.53, 157.29, 144.91, 134.61, 131.35, 131.11, 129.43, 129.04, 129.01, 118.25, 104.86, 56.24. LC-MS (ESI-TOF): m/z 269.1175 ([C₁₇H₁₆O₃ + H]⁺ calcd. 269.1172). Purity 100.00% (rt 6.19 min).

(E)-3-phenyl-1-(2,5-dimethoxyphenyl)prop-2-en-1-one (1g)

It was obtained as yellow oil in 62% yield. ¹H NMR (400 MHz, DMSO) δ = 7.76 – 7.67 (m, 2H), 7.50 (d, J = 16.0 Hz, 1H), 7.43 (d, J = 2.7 Hz, 3H), 7.40 (d, J = 12.2 Hz, 1H), 7.14 – 7.09 (m, 2H), 7.03 (dd, J = 2.6, 0.8 Hz, 1H), 3.80 (s, 3H), 3.73 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 192.17, 153.47, 152.36, 143.16, 135.00, 130.98, 129.67, 129.46, 128.99, 127.25, 119.02, 114.36, 114.33, 56.80, 56.00. LC-MS (ESI-TOF): m/z 269.1170 ([C₁₇H₁₆O₃ + H]⁺ calcd. 269.1172). Purity 98.59% (rt 7.31 min).

(E)-3-phenyl-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (1h)

It was obtained as yellow oil in 62% yield. ¹H NMR (400 MHz, DMSO) δ 7.95 (d, J = 15.6 Hz, 1H), 7.93 – 7.85 (m, 3H), 7.70 (d, J = 15.6 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 1.9, 5.1 Hz, 3H), 7.09 (d, J = 8.5 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 187.77, 153.67, 149.23, 143.53, 135.25, 130.89, 130.87, 129.32, 129.27, 123.87, 122.36, 111.31, 111.11, 56.23, 56.02. LC-MS (ESI-TOF): m/z 269.1173 ([C₁₇H₁₆O₃ + H]⁺ calcd. 269.1172). Purity 97.87% (rt 6.33 min).

(E)-3-phenyl-1-(3,5-dimethoxyphenyl)prop-2-en-1-one (1i)

It was obtained as yellow oil in 66% yield. ¹H NMR (400 MHz, DMSO) δ 7.94 – 7.86 (m, 3H), 7.73 (d, J = 15.6 Hz, 1H), 7.44 (dd, J = 2.6, 3.8 Hz, 3H), 7.25 (d, J = 2.3 Hz, 2H), 6.78 (t, J = 2.3 Hz, 1H), 3.82 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 189.16, 161.14, 144.70, 140.05, 135.06, 131.12, 129.48, 129.33, 122.46, 106.71, 105.53, 56.01. LC-MS (ESI-TOF): m/z 269.1176 ([C₁₇H₁₆O₃ + H]⁺ calcd. 269.1172). Purity 100.00% (rt 8.09 min).

(E)-3-phenyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (1j)

It was obtained as white solid in 68% yield. ¹H NMR (400 MHz, DMSO) δ 7.99 – 7.85 (m, 3H), 7.73 (d, J = 15.5 Hz, 1H), 7.52 – 7.36 (m, 5H), 3.89 (s, 6H), 3.75 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 188.34, 153.37, 144.31, 142.44, 135.15, 133.39, 131.03, 129.42, 129.32, 122.35, 106.62, 60.64, 56.67. LC-MS (ESI-TOF): m/z 299.1284 ([C₁₈H₁₈O₄ + H]⁺ calcd. 299.1278). Purity 100.00% (rt 6.97 min).

(E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (1k)

It was obtained as white solid in 66% yield. ¹H NMR (400 MHz, DMSO) δ = 7.73 (dd, J = 6.8, 2.8 Hz, 2H), 7.54 (d, J = 15.9 Hz, 1H), 7.48 – 7.39 (m, 4H), 7.37 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 190.50, 157.16, 153.34, 142.86, 142.07, 135.08, 130.88, 129.47, 128.89, 126.98, 126.58, 125.60, 108.34, 62.16, 60.96, 56.54. LC-MS (ESI-TOF): m/z 299.1275 ([C₁₈H₁₈O₄ + H]⁺ calcd. 299.1278). Purity 100.00% (rt 7.19 min).

(E)-3-phenyl-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (1l)

It was obtained as yellow oil in 71% yield. ¹H NMR (400 MHz, DMSO) δ = 7.64 (dd, J = 6.6, 3.1 Hz, 2H), 7.39 (dd, J = 5.1, 1.8 Hz, 3H), 7.19 (d, J = 16.1 Hz, 1H), 6.94 (d, J = 16.1 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 193.70, 162.41, 158.53, 143.97, 134.81, 130.90, 129.46, 129.40, 128.91, 111.45, 91.53, 56.26, 55.91. LC-MS (ESI-TOF): m/z 299.1276 ([C₁₈H₁₈O₄ + H]⁺ calcd. 299.1278). Purity 100.00% (rt 6.23 min).

(E)-1-phenyl-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2a)

It was obtained as yellow solid in 64% yield. ¹H NMR (400 MHz, DMSO) δ = 8.35 (d, J = 7.8 Hz, 1H), 8.25 – 8.15 (m, 2H), 8.05 (d, J = 15.3 Hz, 1H), 7.98 (dd, J = 15.5 Hz, 2.0 Hz, 1H), 7.89 – 7.76 (m, 2H), 7.76 – 7.65 (m, 2H), 7.61 (t, J = 7.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ = 189.33, 138.24 (d, J = 1.8 Hz), 137.47, 134.01, 133.42, 133.22 (d, J = 1.5 Hz), 130.99, 129.32, 129.26, 129.16, 127.96 (q, J = 29.2 Hz), 126.64, 126.62 (q, J = 6.0 Hz), 124.61 (q, J = 274.5 Hz). LC-MS (ESI-TOF): m/z 277.0833 ([C₁₆H₁₁F₃O + H]⁺ calcd. 277.0835). Purity 100.00% (rt 6.97 min).

(E)-1-(2-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2b)

It was obtained as yellow oil in 72% yield. ¹H NMR (400 MHz, CDCl₃) δ = 7.90 – 7.82 (m, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.55 (dd, J = 7.6 Hz, 1.8, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.22 (d, J = 15.7 Hz, 1H), 6.97 (td, J = 7.5 Hz, 0.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 3.82 (s, 4H). ¹³C NMR (101 MHz, DMSO) δ = 192.21, 158.40, 136.83 (d, J = 2.1 Hz), 134.05, 133.58, 133.29 (d, J = 1.6 Hz), 131.30, 130.82, 130.15, 128.79, 128.58, 127.81 (q, J = 29.2 Hz), 126.68 (q, J = 5.2 Hz), 124.55 (q, J = 274.5 Hz), 121.07, 112.76, 56.31. LC-MS (ESI-TOF): m/z 304.0940 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 96.40% (rt 8.69 min).

(E)-1-(3-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2c)

It was obtained as yellow solid in 26% yield. ¹H NMR (400 MHz, DMSO) δ = 8.35 (d, J = 7.9 Hz, 1H), 8.06 – 7.94 (m, 2H), 7.88 – 7.76 (m, 3H), 7.72 – 7.63 (m, 2H), 7.52 (t, J = 7.9 Hz, 1H), 7.28 (ddd, J = 8.2 Hz, 2.7 Hz, 0.8 Hz, 1H), 3.87 (s, 3H). ¹H NMR (400 MHz, CDCl₃) δ = 8.13 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.60 (dd, J = 13.7 Hz, 7.1 Hz, 2H), 7.55 – 7.47 (m, 2H), 7.41 (dd, J = 15.9 Hz, 8.5 Hz, 2H), 7.15 (dd, J= 8.2 Hz, 1.9 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 189.12, 160.04, 138.92, 138.33 (d, J = 2.2 Hz), 133.41, 133.21 (d, J = 1.7 Hz), 131.00, 130.47, 129.32, 127.96 (d, J = 29.2 Hz), 126.71, 126.62 (d, J = 6. 0 Hz), 124.62 (d, J = 273.5 Hz), 121.69, 120.04, 113.64, 55.86. LC-MS (ESI-TOF): m/z 304.0945 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 9.13 min).

(E)-1-(4-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2d)

It was obtained as yellow solid in 37% yield. ¹H NMR (400 MHz, DMSO) δ = 8.34 (d, J = 7.9 Hz, 1H), 8.24 – 8.17 (m, 2H), 8.04 (d, J = 15.3 Hz, 1H), 7.95 (dd, J = 15.4 Hz, 2.2, 1H), 7.87 – 7.76 (m, 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.16 – 7.07 (m, 2H), 3.89 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 187.42, 164.00, 137.46 (d, J = 2.2 Hz), 133.45 (d, J = 1.6 Hz), 133.38, 131.63, 130.78, 130.40, 129.21, 127.87 (d, J = 29.2 Hz), 126.67, 126.58 (d, J = 6.0 Hz), 124.64 (d, J = 274.5 Hz), 114.58, 56.08. LC-MS (ESI-TOF): m/z 304.0944 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 8.75 min).

(E)-1-(2,4-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2e)

It was obtained as yellow solid in 50% yield. ¹H NMR (400 MHz, DMSO) δ = 8.07 (d, J = 7.6 Hz, 1H), 7.79 (dt, J = 14.8 Hz, 7.9 Hz, 3H), 7.69 – 7.60 (m, 3H), 6.71 (d, J = 2.3 Hz, 1H), 6.67 (dd, J = 8.6 Hz, 2.3 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 189.18, 164.88, 161.01, 135.47 (d, J = 2.0 Hz), 133.64 (d, J = 1.3 Hz), 133.59, 132.74, 131.55, 130.55, 128.69, 127.74 (d, J = 29.2 Hz), 126.64 (d, J = 6.0 Hz), 124.62 (d, J = 274.5 Hz). 121.23, 106.69, 99.04, 56.50, 56.13. LC-MS (ESI-TOF): m/z 337.1050 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 8.71 min).

(E)-1-(2,6-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2f)

It was obtained as white solid in 67% yield. ¹H NMR (400 MHz, DMSO) δ 8.07 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.30 (d, J = 16.3 Hz, 1H), 7.14 (d, J = 16.2 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 194.45, 157.36, 142.84, 135.92, 132.43, 131.45, 130.69, 130.41, 130.26 (q, J = 31.1 Hz), 127.17 (q, J = 3.6 Hz), 125.92 (q, J = 3.8 Hz), 124.38 (q, J = 272.5 Hz), 118.22, 104.90, 56.27. LC-MS (ESI-TOF): m/z 337.1045 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 7.68 min).

(E)-1-(2,5-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2g)

It was obtained as yellow solid in 80% yield. ¹H NMR (400 MHz, DMSO) δ = 8.09 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.80 – 7.72 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 1.8 Hz, 2H), 7.09 (t, J = 1.8 Hz, 1H), 3.83 (s, 3H), 3.76 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 191.72, 153.50, 152.64, 136.95 (d, J = 2.1 Hz), 133.59, 133.28 (d, J = 1.7 Hz), 131.14, 130.86, 128.96, 128.79, 127.82 (q, J = 29.2 Hz), 126.69 (q, J = 16.6 Hz), 124.56 (d, J = 273.5 Hz), 119.72, 114.41, 114.33, 56.80, 56.04. LC-MS (ESI-TOF): m/z 337.1049 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 8.76 min).

(E)-1-(3,4-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2h)

It was obtained as yellow solid in 72% yield. ¹H NMR (400 MHz, DMSO) δ = 8.33 (d, J = 7.8 Hz, 1H), 8.04 (d, J =15.3 Hz, 1H), 7.96 (dd, J = 6.2 Hz, 2.1 Hz, 1H), 7.94 (d, J= 2.0 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 187.44, 154.02, 149.31, 137.42 (d, J = 2.0 Hz), 133.49 (d, J = 1.0 Hz), 133.37, 130.76, 130.44, 129.26, 127.85 (q, J = 29.2 Hz), 126.66, 126.58 (q, J = 5.2 Hz), 124.65 (q, J = 273.5 Hz), 124.22, 111.40, 111.28, 56.28, 56.07. LC-MS (ESI-TOF): m/z 337.1048 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 7.77 min).

(E)-1-(3,5-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2i)

It was obtained as yellow oil in 72% yield. ¹H NMR (400 MHz, DMSO) δ = 8.36 (d, J = 7.9 Hz, 1H), 7.98 (s, 2H), 7.85 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 2.2, Hz 2H), 6.83 (t, J = 2.2 Hz, 1H), 3.85 (s, 6H). ¹H NMR (400 MHz, CDCl₃) δ = 8.12 (d, J = 13.9 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 15.6 Hz, 1H), 7.14 (d, J = 2.2 Hz, 2H), 6.69 (t, J = 2.1 Hz, 1H), 3.86 (s, 7H). ¹³C NMR (101 MHz, DMSO) δ = 188.97, 161.20, 139.53, 138.45 (d, J = 2.2 Hz), 133.39, 133.18 (d, J = 1.6 Hz), 131.00, 129.40, 127.96 (q, J = 29.2 Hz), 126.67, 129.60 (q, J = 5.2 Hz), 124.62 (q, J = 273.5 Hz), 106.93, 105.88, 56.05. LC-MS (ESI-TOF): m/z 337.1047 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 9.37 min).

(E)-1-(3,4,5-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2j)

It was obtained as yellow solid in 80% yield. ¹H NMR (400 MHz, DMSO) δ = 8.31 (d, J = 7.8 Hz, 1H), 8.02 (d, J = 15.2 Hz, 1H), 7.95 (dd, J = 15.4 Hz, 2.1 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.44 (s, 2H), 3.88 (s, 6H), 3.76 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 188.07, 153.41, 142.77, 138.14 (d, J=2.0), 133.37, 132.86, 130.91, 129.40, 127.77 (q, J = 29.2 Hz), 126.61, 126.61 (q, J = 5.0 Hz), 124.63 (q, J = 274.5 Hz), 106.86, 60.66, 56.68. LC-MS (ESI-TOF): m/z 367.1160 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 100.00% (rt 8.38 min).

(E)-1-(2,3,4-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2k)

It was obtained as yellow solid in 58% yield. ¹H NMR (400 MHz, DMSO) δ = 8.10 (d, J = 7.7 Hz, 1H), 7.80 (dt, J = 22.4, 7.7 Hz, 3H), 7.66 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 8.9 Hz, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.80 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 189.74, 157.66, 153.66, 142.08, 136.44 (d, J = 2.1 Hz), 133.60, 133.46 (d, J = 2.0 Hz), 131.02, 130.73, 128.71, 127.78 (d, J = 29.2 Hz), 126.66 (d, J = 5.0 Hz), 125.95 (d, J = 7.0 Hz), 124.59 (d, J = 274.5 Hz), 108.48, 62.21, 60.98, 56.60. LC-MS (ESI-TOF): m/z 367.1152 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 96.17% (rt 8.70 min).

(E)-1-(2,4,6-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2l)

It was obtained as yellow solid in 72% yield. ¹H NMR (400 MHz, DMSO) δ = 8.04 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.48 (dd, J = 15.9, 2.2 Hz, 1H), 7.01 (d, J = 15.8 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 193.37, 162.78, 162.78, 158.71, 138.33 (d, J = 2.2 Hz), 133.51, 133.07, 130.80, 128.83, 127.58 (q, J = 29.2 Hz), 126.61 (q, J = 6.0 Hz), 124.46 (q, J = 274.5 Hz), 110.73, 91.34, 56.23, 55.96. LC-MS (ESI-TOF): m/z 367.1157 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 96.17% (rt 7.58 min).

(E)-1-phenyl-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3a)

It was obtained as white solid in 68% yield. ¹H NMR (400 MHz, DMSO) δ 8.36 (s, 1H), 8.26 – 8.18 (m, 3H), 8.15 (d, J = 15.7 Hz, 1H), 7.89 – 7.78 (m, 2H), 7.75 – 7.67 (m, 2H), 7.64 – 7.57 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 189.51, 142.61, 137.72, 136.28, 133.83, 133.36, 130.38, 130.26 (q, J = 28.1 Hz), 127.14 (q, J = 3.8 Hz), 125.62 (q, J = 3.7 Hz), 124.15 (q, J = 272.5 Hz), 124.38. LC-MS (ESI-TOF): m/z 277.0840 ([C₁₆H₁₁F₃O + H]⁺ calcd. 277.0835). Purity 100.00% (rt 9.20 min).

(E)-1-(2-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3b)

It was obtained as yellow oil in 45% yield. ¹H NMR (400 MHz, DMSO) δ 8.57 (t, J = 1.9 Hz, 1H), 8.29 – 8.19 (m, 2H), 7.73 (t, J = 8.0 Hz, 1H), 7.69 – 7.61 (m, 2H), 7.61 – 7.57 (m, 1H), 7.57 – 7.52 (m, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.89 (s, 3H). LC-MS (ESI-TOF): m/z 304.0941 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 96.00% (rt 8.88 min).

(E)-1-(3-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3c)

It was obtained as white solid in 21% yield. ¹H NMR (400 MHz, DMSO) δ 8.35 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 8.11 (d, J = 15.7 Hz, 1H), 7.87 – 7.77 (m, 3H), 7.70 (t, J = 7.78 Hz, 1H), 7.66 (dd, J = 1.6, 2.5 Hz, 1H), 7.52 (t, J = 7.94 Hz, 1H), 7.27 (ddd, J = 0.8, 2.6, 8.2 Hz, 1H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 189.28, 160.03, 142.68, 139.18, 136.27, 133.32, 130.41, 130.36, 130.25 (q, J = 31.2 Hz), 127.15 (q, J = 3.7 Hz), 125.72 (q, J = 3.7 Hz), 124.50 (q, J = 272.5 Hz), 124.46, 121.70, 119.76, 113.68, 55.85. LC-MS (ESI-TOF): m/z 304.0945 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 9.35 min).

(E)-1-(4-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3d)

It was obtained as white solid in 59% yield. ¹H NMR (400 MHz, DMSO) δ 8.33 (s, 1H), 8.24 – 8.20 (m, 2H), 8.18 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 15.7 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.14 – 7.07 (m, 2H), 3.88 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 187.69, 163.85, 141.74, 136.45, 133.23, 131.57, 130.67, 130.34, 130.24 (q, J = 32.7 Hz), 126.93 (q, J = 3.8 Hz), 125.49 (q, J = 3.7 Hz), 124.52 (q, J = 272.5 Hz), 124.45, 114.49, 56.05. LC-MS (ESI-TOF): m/z 304.0951 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 8.99 min).

(E)-1-(2,4-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3e)

It was obtained as white solid in 52% yield. ¹H NMR (400 MHz, DMSO) δ 8.11 – 8.03 (m, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.72 – 7.58 (m, 4H), 6.70 (d, J = 2.3 Hz, 1H), 6.66 (dd, J = 2.3, 8.6 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H). ¹³C NMR (101 MHz, d₂o) δ 189.57, 164.52, 160.77, 139.58, 136.52, 132.49, 131.97, 130.39, 130.18 (q, J = 31.2 Hz), 129.38, 126.69 (q, J = 3.7 Hz), 125.52 (q, J = 3.8 Hz, H), 124.37 (q, J = 272.5 Hz), 121.55, 106.44, 99.03, 56.38, 56.02. LC-MS (ESI-TOF): m/z 337.1044 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 8.93 min).

(E)-1-(2,6-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3f)

It was obtained as light yellow solid in 72% yield. ¹H NMR (400 MHz, DMSO) δ = 8.07 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8, 1H), 7.71 (dd, J = 11.4 Hz, 4.0 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 11.1 Hz, 5.7 Hz, 1H), 7.02 (d, J = 15.9 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 194.52, 157.33, 139.62 (d, J = 2.2 Hz), 133.52, 132.81 (d, J = 1.6 Hz), 132.57, 131.77, 131.02, 128.90, 127.59 (q, J = 30.2 Hz), 126.64 (q, J = 5.7 Hz), 124.38 (q, J = 274.5 Hz), 117.44, 104.69, 56.22. LC-MS (ESI-TOF): m/z 337.1050 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 98.65% (rt 7.83 min).

(E)-1-(2,5-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3g)

It was obtained as yellow oil in 71% yield. ¹H NMR (400 MHz, DMSO) δ 8.12 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 16.1 Hz, 1H), 7.55 (dd, J = 0.9, 16.0 Hz, 1H), 7.18 – 7.12 (m, 2H), 7.09 – 7.03 (m, 1H), 3.82 (d, J = 0.8 Hz, 3H), 3.76 (d, J = 0.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 192.15, 153.49, 152.47, 141.17, 136.27, 132.29, 130.48, 130.27 (q, J = 31.2 Hz), 129.50, 129.14, 127.06 (q, J = 3.7 Hz), 125.83 (q, J = 3.3 Hz), 124.42 (d, J = 272.5 Hz), 119.20, 114.42, 114.37, 56.84, 56.04. LC-MS (ESI-TOF): m/z 337.1048 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 96.60% (rt 8.96 min).

(E)-1-(3,4-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3h)

It was obtained as white solid in 64% yield. ¹H NMR (400 MHz, d₂o) δ 8.11 (dd, J = 11.7, 8.7 Hz, 3H), 7.98 – 7.91 (m, 1H), 7.79 (dd, J = 19.6, 12.0 Hz, 3H), 7.63 (d, J = 1.9 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, d₂o) δ 187.59, 153.86, 149.24, 141.43, 139.26 (d, J = 1.3 Hz), 130.59, 130.23 (d, J = = 31.2 Hz), 129.74, 126.02 (q, J = 3.7 Hz), 125.06, 124.45 (d, J = 272.5 Hz), 124.08, 111.29, 111.13, 56.21, 56.00. LC-MS (ESI-TOF): m/z 337.1041 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 98.47% (rt 8.03 min).

(E)-1-(3,5-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3i)

It was obtained as light yellow solid in 72% yield. ¹H NMR (400 MHz, DMSO) δ 8.34 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 15.7 Hz, 1H), 7.88 – 7.77 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 2.3 Hz, 2H), 6.83 (t, J = 2.2 Hz, 1H), 3.85 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 189.08, 161.18, 142.83, 139.77, 136.24, 133.33, 130.33, 130.24 (q, J = 32.2 Hz), 127.17 (q, J = 3.6 Hz), 125.85 (q, J = 3.7 Hz), 124.50 (q, J = 273.5 Hz), 124.36, 106.98, 105.52, 56.03. LC-MS (ESI-TOF): m/z 337.1049 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 9.55 min).

(E)-1-(3,4,5-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3j)

It was obtained as white solid in 60% yield. ¹H NMR (400 MHz, DMSO) δ 8.28 – 8.19 (m, 2H), 8.06 (d, J = 15.7 Hz, 1H), 7.86 – 7.75 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.44 (s, 2H), 3.89 (s, 6H), 3.76 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 188.34, 153.39, 142.69, 142.48, 136.33, 133.16, 132.95, 130.33, 130.24 (q, J = 32.2 Hz), 127.13 (q, J = 3.7 Hz), 126.02 (q, J = 3.7 Hz), 124.49 (q, J = 272.5 Hz), 124.34, 106.90, 60.66, 56.75. LC-MS (ESI-TOF): m/z 367.1155 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 96.17% (rt 8.61 min).

(E)-1-(2,3,4-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3k)

It was obtained as yellow oil in 46% yield. ¹H NMR (400 MHz, DMSO) δ 8.13 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.71 – 7.56 (m, 3H), 7.42 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.9 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 190.46, 157.30, 153.41, 142.11, 140.87, 136.36, 132.23, 130.49, 130.27 (q, J = 31.2 Hz), 128.92, 126.96 (q, J = 3.7 Hz), 126.42, 125.69, 125.66 (q, J = 5.0 Hz), 124.43 (q, J = 272.5 Hz), 108.33, 62.14, 60.97, 56.57. LC-MS (ESI-TOF): m/z 367.1157 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 97.99% (rt 8.83 min).

(E)-1-(2,4,6-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3l)

It was obtained as light yellow solid in 69% yield. ¹H NMR (400 MHz, DMSO) δ 8.05 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 16.2 Hz, 1H), 7.11 (d, J = 16.2 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.70 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 193.58, 162.54, 158.63, 141.94, 136.11, 132.29, 130.39, 130.24 (q, J = 30.2 Hz), 127.00 (d, J = 4.0 Hz), 125.81 (d, J = 4.0 Hz), 124.40 (q, J = 272.5 Hz), 111.35, 91.55, 56.27, 55.93. LC-MS (ESI-TOF): m/z 367.1154 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 100.00% (rt 7.86 min).

(E)-1-phenyl-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4a)

It was obtained as white solid in 59% yield. ¹H NMR (400 MHz, DMSO) δ 8.20 – 8.11 (m, 3H), 8.08 (d, J = 15.7 Hz, 2H), 7.83 – 7.75 (m, 3H), 7.72 – 7.65 (m, 1H), 7.62 – 7.54 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 189.53, 142.43, 139.13 (d, J = 1.3 Hz), 137.68, 133.87, 130.49 (q, J = 32.2 Hz), 129.91, 129.30, 129.11, 126.14 (q, J = 3.8 Hz), 125.13, 124.44 (q, J = 272.5 Hz). LC-MS (ESI-TOF): m/z 277.0834 ([C₁₆H₁₁F₃O + H]⁺ calcd. 277.0835). Purity 100.00% (rt 9.35 min).

(E)-1-(2-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4b)

It was obtained as yellow oil in 79% yield. ¹H NMR (400 MHz, DMSO) δ 7.97 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.2 Hz, 1H), 7.61 – 7.48 (m, 6H), 7.22 (d, J = 8.0 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 2H). LC-MS (ESI-TOF): m/z 304.0948 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 94.00% (rt 9.09 min).

(E)-1-(3-methoxyphenyl)-4-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4c)

It was obtained as white solid in 61% yield. ¹H NMR (400 MHz, DMSO) δ 8.14 (d, J = 8.1 Hz, 2H), 8.07 (d, J = 15.7 Hz, 1H), 7.85 – 7.80 (m, 3H), 7.79 (d, J = 4.3 Hz, 1H), 7.67 – 7.62 (m, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.27 (dd, J = 8.2, 2.6 Hz, 1H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 189.29, 160.04, 142.49, 139.13, 13.50 (d, J = 31.2 Hz), 130.45, 129.95, 126.12 (q, J = 3.7 Hz), 124.5 (d, J = 272.46 Hz), 125.20, 121.65, 119.90, 113.58, 55.86. LC-MS (ESI-TOF): m/z 304.0943 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 9.52 min).

(E)-1-(4-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4d)

It was obtained as white solid in 56% yield. ¹H NMR (400 MHz, DMSO) δ 8.23 – 8.01 (m, 5H), 7.76 (dd, J = 21.4, 11.6 Hz, 3H), 7.08 (d, J = 8.2 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 187.70, 163.89, 141.58, 139.30, 131.55, 130.60, 130.32 (d, J = 32.2 Hz), 129.78, 126.11 (dd, J = 3.8 Hz), 125.18, 124.5 (d, J = 272.5 Hz), 114.55, 56.06. LC-MS (ESI-TOF): m/z 304.0940 ([C₁₇H₁₃ F₃O₂ + H]⁺ calcd. 307.0940). Purity 100.00% (rt 9.13 min).

(E)-1-(2,4-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4e)

It was obtained as off white solid in 40% yield. ¹H NMR (400 MHz, DMSO) δ 7.92 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.70 – 7.50 (m, 3H), 6.74 – 6.57 (m, 2H), 3.89 (s, 3H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 189.45, 164.74, 160.94, 139.41, 132.65, 130.12) q, J = 32.2 Hz), 130.10, 129.33, 126.21 (dd, J = 3.5 Hz), 124.50 (d, J = 272.5 Hz), 121.48, 106.62, 99.07, 56.48, 56.10. LC-MS (ESI-TOF): m/z 337.1046 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 96.35% (rt 9.13 min).

(E)-1-(2,6-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4f)

It was obtained as light yellow solid in 64% yield. ¹H NMR (400 MHz, DMSO) δ 7.92 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.41 (t, J = 8.4 Hz, 1H), 7.29 (d, J = 16.2 Hz, 1H), 7.13 (d, J = 16.2 Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 3.73 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 194.25, 157.40, 142.53, 138.76 (d, J = 1.4 Hz), 131.59, 131.29, 130.55 (q, J = 31.2 Hz), 126.15 (q, J = 3.8 Hz), 124.42 (q, J = 272.5 Hz), 118.12, 104.93, 56.29. LC-MS (ESI-TOF): m/z 337.1047 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 100.00% (rt 7.97 min).

(E)-1-(2,5-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4g)

It was obtained as yellow oil in 45% yield. ¹H NMR (400 MHz, DMSO) δ 7.95 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 16.0 Hz, 1H), 7.54 (d, J = 16.0 Hz, 1H), 7.17 – 7.12 (m, 2H), 7.08 – 7.04 (m, 1H), 3.82 (s, 3H), 3.74 (s, 3H). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (dd, J = 17.3, 8.7 Hz, 5H), 7.51 (d, J = 15.9 Hz, 1H), 7.23 (d, J = 3.1 Hz, 1H), 7.06 (dd, J = 9.0, 3.2 Hz, 1H), 6.96 (d, J = 9.0 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 191.85, 153.50, 152.62, 140.78, 139.13 (d, J = 1.4 Hz), 130.37 (d, J = 32.2 Hz), 129.72, 129.54, 129.28, 126.22 (q, J = 3.7 Hz), 124.46 (d, J = 272.5 Hz), 119.51, 114.44, 114.37, 56.83, 56.01. LC-MS (ESI-TOF): m/z 337.1048 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 96.42% (rt 9.16 min).

(E)-1-(3,4-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4h)

It was obtained as white solid in 64% yield. ¹H NMR (400 MHz, DMSO) δ 8.31 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 15.6 Hz, 1H), 7.99 (dd, J = 2.0, 8.5 Hz, 1H), 7.83 – 7.75 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 187.70, 153.90, 149.31, 141.68, 136.46, 133.10, 130.72, 130.33, 130.24 (d, J = 32.2 Hz), 126.93 (q, J = 3.6 Hz), 125.64 (q, J = 3.6 Hz), 124.52 (d, J = 272.4 Hz), 124.39, 124.24, 111.30, 111.21, 56.27, 56.09. LC-MS (ESI-TOF): m/z 337.1042 ([C₁₈H₁₅ F₃O₃ + H]⁺ calcd. 337.1046). Purity 95.94% (rt 8.19 min).

(E)-1-(3,4,5-trimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4i)

It was obtained as white solid in 77% yield. ¹H NMR (400 MHz, DMSO) δ 8.11 (d, J = 8.2 Hz, 2H), 8.06 (d, J = 15.6 Hz, 1H), 7.83 – 7.74 (m, 3H), 7.43 (s, 2H), 3.89 (s, 6H), 3.76 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 188.24, 153.40, 142.69, 142.27, 139.19 (d, J = 1.3 Hz), 133.08, 130.44 (q, J = 31.2 Hz), 129.95, 126.08 (q, J = 3.7 Hz), 125.04, 124.50 (q, J = 271.4 Hz), 106.80, 60.65, 56.70. LC-MS (ESI-TOF): m/z 367.1158 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 96.17% (rt 8.78 min).

(E)-1-(2,3,4-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4j)

It was obtained as white solid in 63% yield. ¹H NMR (400 MHz, DMSO) δ 7.95 (d, J = 6.8 Hz, 2H), 7.77 (d, J = 7.0 Hz, 2H), 7.58 (s, 2H), 7.40 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.77 (s, 3H). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (dd, J = 23.7, 8.2 Hz, 5H), 7.59 (d, J = 15.9 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 3.94 (s, 4H), 3.92 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 190.22, 157.48, 153.53, 142.08, 140.54, 139.21, 130.30 (q, J = 32.2 Hz), 126.26, 126.23, 125.79, 124.48 (q, J = 271.5 Hz), 108.41, 62.17, 60.97, 56.58. LC-MS (ESI-TOF): m/z 367.1158 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 100.00% (rt 9.05 min).

(E)-1-(2,4,6-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4k)

It was obtained as yellow oil in 86% yield. ¹H NMR (400 MHz, DMSO) δ 7.91 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 16.2 Hz, 1H), 7.11 (d, J = 16.2 Hz, 1H), 6.32 (s, 2H), 3.84 (s, 3H), 3.73 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 193.27, 162.66, 158.73, 141.53, 138.97 (d, J = 1.4 Hz), 130.33 (q, J = 32.2 Hz), 126.14 (q, J = 3.8 Hz), 124.45 (q, J = 271.4 Hz), 111.28, 91.57, 91.40, 56.29, 55.94. LC-MS (ESI-TOF): m/z 367.1152 ([C₁₉H₁₇ F₃O₄ + H]⁺ calcd. 367.1152). Purity 96.54% (rt 8.03 min).

(E)-1-phenyl-3-(2-nitrophenyl)prop-2-en-1-one (5a)

It was obtained as off white solid in 46% yield. ¹H NMR (400 MHz, DMSO) δ = 8.24 – 8.20 (m, 1H), 8.20 – 8.15 (m, 2H), 8.11 (dd, J = 8.1, 1.2 Hz, 1H), 8.00 (d, J = 15.5 Hz, 1H), 7.91 (d, J = 15.5 Hz, 1H), 7.88 – 7.81 (m, 1H), 7.76 – 7.68 (m, 2H), 7.60 (t, J = 7.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ = 189.52, 149.24, 139.03, 137.48, 134.22, 133.98, 131.53, 130.19, 129.98, 129.33, 129.18, 126.83, 125.16. LC-MS (ESI-TOF): m/z 254.0819 ([C₁₅H₁₁NO₃ + H]⁺ calcd. 254.0812). Purity 100.00% (rt 6.68 min).

(E)-1-(2-methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5b)

It was obtained as light yellow solid in 49% yield. ¹H NMR (400 MHz, DMSO) δ = 8.09 (dd, J = 8.2, 1.2 Hz, 1H), 7.99 (dd, J = 7.9, 1.2 Hz, 1H), 7.84 – 7.76 (m, 2H), 7.73 – 7.66 (m, 1H), 7.61 – 7.57 (m, 1H), 7.55 (dd, J = 7.7, 1.7 Hz, 1H), 7.42 (d, J = 15.7 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 192.08, 158.46, 149.07, 137.47, 134.36, 134.07, 131.33, 130.24, 130.22, 129.62, 128.56, 125.20, 121.06, 112.82, 56.32. LC-MS (ESI-TOF): m/z 284.0916 ([C₁₆H₁₃NO₄ + H]⁺ calcd. 284.0917). Purity 97.28% (rt 6.52 min).

(E)-1-(3-methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5c)

It was obtained as off white solid in 21% yield. ¹H NMR (400 MHz, DMSO) δ = 8.18 (d, J = 7.7 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 15.5 Hz, 1H), 7.83 (dd, J = 17.6, 11.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.61 (s, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.25 (d, J = 6.4 Hz, 1H), 3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 189.36, 160.02, 149.23, 139.13, 138.91, 134.21, 131.53, 130.50, 130.16, 130.02, 126.91, 125.14, 121.70, 120.06, 113.60, 55.87. LC-MS (ESI-TOF): m/z 284.0918 ([C₁₆H₁₃NO₄ + H]⁺ calcd. 284.0917). Purity 100.00% (rt 6.91 min).

(E)-1-(4-methoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5d)

It was obtained as off white solid in 32% yield. ¹H NMR (400 MHz, DMSO) δ = 8.17 (t, J = 7.9 Hz, 3H), 8.07 (d, J = 8.1 Hz, 1H), 7.97 – 7.85 (m, 2H), 7.81 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H). ¹H NMR (400 MHz, CDCl₃) δ = 8.15 – 8.00 (m, 3H), 7.74 (d, J = 7.6 Hz, 1H), 7.68 (t, J= 7.6 Hz, 1H), 7.56 (t, J = 7.1 Hz, 1H), 7.32 (d, J = 15.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 2H), 3.90 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 187.58, 163.98, 149.23, 138.00, 134.15, 131.63, 131.36, 130.38, 130.26, 129.91, 126.89, 125.09, 114.59, 56.08. LC-MS (ESI-TOF): m/z 284.0919 ([C₁₆H₁₃NO₄ + H]⁺ calcd. 284.0917). Purity 100.00% (rt 6.55 min).

(E)-1-(2,4-dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5e)

It was obtained as yellow solid in 57% yield. ¹H NMR (400 MHz, DMSO) δ = 8.06 (dd, J = 8.2 Hz, 1.2 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.82 – 7.73 (m, 2H), 7.69 – 7.61 (m, 2H), 7.51 (d, J = 15.6 Hz, 1H), 6.68 (d, J = 2.2 Hz, 1H), 6.64 (dd, J = 8.6, 2.3 Hz, 1H), 3.90 (s, 4H), 3.84 (s, 4H). ¹³C NMR (101 MHz, DMSO) δ = 189.17, 164.87, 161.02, 149.09, 136.03, 134.33, 132.76, 131.63, 131.12, 130.48, 129.55, 125.14, 121.20, 106.68, 99.03, 56.48, 56.13. LC-MS (ESI-TOF): m/z 314.1027 ([C₁₇H₁₅NO₅ + H]⁺ calcd. 314.1023). Purity 97.57% (rt 6.62 min).

(E)-1-(2,6-dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5f)

It was obtained as white solid in 82% yield. ¹H NMR (400 MHz, DMSO) δ = 8.06 (dd, J = 8.1, 1.2 Hz, 1H), 7.98 (dd, J = 7.8, 1.3 Hz, 1H), 7.78 (ddd, J = 7.8, 1.2, 0.6 Hz, 1H), 7.70 – 7.64 (m, 1H), 7.52 (d, J = 16.0 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.96 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 3.75 (s, 7H). ¹³C NMR (101 MHz, DMSO) δ = 194.37, 157.44, 148.75, 140.06, 134.33, 132.66, 131.72, 131.52, 129.86, 129.66, 125.21, 117.59, 104.80, 56.26. LC-MS (ESI-TOF): m/z 314.1024 ([C₁₇H₁₅NO₅ + H]⁺ calcd. 314.1023). Purity 100.00% (rt 5.74 min).

(E)-1-(2,5-dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5g)

It was obtained as yellow solid in 49% yield. ¹H NMR (400 MHz, d₂o) δ = 8.09 (dd, J = 8.1, 1.1 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.85 – 7.76 (m, 2H), 7.73 – 7.65 (m, 1H), 7.41 (d, J = 15.8 Hz, 1H), 7.16 (d, J = 1.8 Hz, 2H), 7.09 (t, J = 1.8 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H). ¹³C NMR (101 MHz, d₂o) δ = 191.59, 153.41, 152.61, 137.66, 134.31, 131.31, 131.11, 130.19, 129.56, 128.86, 125.14, 119.67, 114.33, 114.32, 56.74, 55.96. LC-MS (ESI-TOF): m/z 314.1029 ([C₁₇H₁₅NO₅ + H]⁺ calcd. 314.1023). Purity 100.00% (rt 6.63 min).

(E)-1-(3,4-dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5h)

It was obtained as yellow solid in 17% yield. ¹H NMR (400 MHz, DMSO) δ = 8.20 (d, J = 7.8 Hz, 1H), 8.10 (dd, J = 8.1, 1.0 Hz, 1H), 7.99 – 7.88 (m, 3H), 7.84 (t, J = 7.6 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H). ¹H NMR (400 MHz, CDCl₃) δ = 8.06– 8.12 (m, 2H), 7.74 (d, J = 7.7 Hz, 1H), 7.72 – 7.65 (m, 2H), 7.62 (d, J = 1.7 Hz, 1H), 7.56 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 15.7 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 3.98 (s, 3H). ¹³C NMR (101 MHz, d₂o) δ = 187.54, 153.92, 149.23, 149.15, 137.91, 134.06, 131.25, 130.35, 130.26, 129.89, 126.80, 125.02, 124.14, 111.33, 111.21, 56.22, 56.00. LC-MS (ESI-TOF): m/z 314.1027 ([C₁₇H₁₅NO₅ + H]⁺ calcd. 314.1023). Purity 100.00% (rt 5.63 min).

(E)-1-(3,5-dimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5i)

It was obtained as off white solid in 57% yield. ¹H NMR (400 MHz, DMSO) δ = 8.21 (dd, J = 7.8, 1.3 Hz, 1H), 8.10 (dd, J = 8.1, 1.2 Hz, 1H), 7.98 (d, J = 15.5 Hz, 1H), 7.89 – 7.80 (m, 2H), 7.74 – 7.68 (m, 1H), 7.28 (d, J = 2.3 Hz, 2H), 6.82 (t, J = 2.3 Hz, 1H), 3.85 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 189.21, 161.19, 149.24, 139.53, 139.25, 134.18, 131.52, 130.15, 130.09, 126.89, 125.13, 106.91, 105.89, 56.05. LC-MS (ESI-TOF): m/z 314.1024 ([C₁₇H₁₅NO₅ + H]⁺ calcd. 314.1023). Purity 100.00% (rt 7.21 min).

(E)-1-(3,4,5-trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5j)

It was obtained as off white solid in 31% yield. ¹H NMR (400 MHz, DMSO) δ = 8.13 (dd, J = 27.6, 8.0 Hz, 2H), 7.97 (d, J = 15.4 Hz, 1H), 7.92 – 7.79 (m, 2H), 7.70 (t, J = 7.7 Hz, 1H), 7.42 (s, 2H), 3.88 (s, 6H), 3.76 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ = 188.38, 153.39, 149.18, 142.64, 139.01, 134.20, 132.84, 131.45, 130.35, 130.12, 126.80, 125.16, 106.80, 60.66, 56.65. LC-MS (ESI-TOF): m/z 344.1130 ([C₁₈H₁₇NO₆ + H]⁺ calcd. 344.1129). Purity 99.00% (rt 6.25 min).

(E)-1-(2,3,4-trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5k)

It was obtained as light white solid in 32% yield. ¹H NMR (400 MHz, DMSO) δ = 8.07 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.85 – 7.75 (m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.43 (dd, J = 12.2 Hz, 3.2 Hz, 2H), 6.95 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.77 (s, 3H). LC-MS (ESI-TOF): m/z 344.1127 ([C₁₈H₁₇NO₆ + H]⁺ calcd. 344.1129). Purity 100.00% (rt 6.52 min).

(E)-1-(2,4,6-trimethoxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (5l)

It was obtained as yellow solid in 61% yield. ¹H NMR (400 MHz, DMSO) δ = 8.03 (dd, J = 8.1 Hz, 1.0, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 15.9 Hz, 1H), 6.92 (d, J = 15.9 Hz, 1H), 6.30 (s, 2H), 3.82 (s, 3H), 3.72 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ = 193.25, 162.76, 158.80, 148.78, 138.79, 134.30, 133.17, 131.33, 130.06, 129.61, 125.18, 110.82, 91.42, 56.26, 55.94. LC-MS (ESI-TOF): m/z 344.1141 ([C₁₈H₁₇NO₆ + H]⁺ calcd. 344.1129). Purity 100.00% (rt 5.77 min).

Biology

Cell culture and treatment

Human bronchial epithelial (Beas-2B) cells were cultured in DMEM:F12 (pH 7.4) supplemented with 10% (v/v) FBS, 100 mg/l gentamicin and genetisin. Beas-2B cells were grown in 48-well plates for 24 h and then treated with a series of chalcone derivatives dissolved in DMSO for various time points. The concentration of DMSO did not exceed 0.1%. RNA was isolated and gene expression was measured after 16 h.

Cell viability assay

The cytotoxicity of chalcone derivatives was analyzed by using trypan blue exclusion test and was further confirmed by colorimetric methylthiazolydiphenyl-tetrazolium bromide (MTT) assay as described⁸². Briefly, Beas-2B cells were treated with chalcone analogs or DMSO alone (0.1%, as vehicle) for 24 h. Four hours before the end of incubation, the medium was removed and 100 µl of MTT (5 mg/ml in serum free medium) was added to each well. The MTT was removed after 4 h, cells were washed with PBS, and 100 µl DMSO was added to each well to dissolve the water-insoluble MTT-formazan crystals. The absorbance was recorded at 570 nm in a plate reader (Molecular Devices, Sunnyvale, CA).

Generation of stable transfectants

Beas-2B cells overexpressing ARE luciferase reporter plasmid were obtained by transfecting Beas-2B cells with 3 µg of NQO1-ARE reporter plasmid and 0.3 µg of pUB6 empty vector (Invitrogen, Carlsbad, CA). Stable transfectants were selected using blasticidin at a concentration of 6 µg/ml. Stable clones were expanded and screened for the expression of ARE luciferase.⁸³

ARE reporter assay

Beas-2B cells stably expressing NQO1-ARE luciferase were seeded onto a 96-well plate at a density of 10,000 cells/well for 16 h before incubation with test compounds. Next day, cells were treated with the indicated concentrations of compound 2b. Cells were also treated with DMSO, which was used as the solvent. The reporter activity was measured after 16 h exposure using the luciferase assay kit (Promega, Madison, WI). The level of increase in luciferase activity reflects the degree of Nrf2 activity.⁸³

Mice in vivo study

All experiments in mice were performed in accordance with the standards established by the US Animal Welfare Acts, set forth in NIH guidelines and the Policy and Procedures Manual of the Johns Hopkins University Animal Care and Use Committee. C57BL/6 mice (male, 7 weeks) were maintained on AIN 76A diet (Harlan Tekland, Madison, WI) and water ad libitum and housed at a temperature (range, 20–23°C) under 12 h light/dark cycles. The mice were treated with chalcone analogs (50 mg/kg body weight) or vehicle or sulforaphane as a positive control by gavage. After 24 h treatment, the small intestines were harvested and stored at −80°C until analysis.

RNA extraction and gene expression analysis

Total RNA was extracted from cells/tissue using Qiagen RNeasy kit (Qiagen Corporation, Valencia, CA) and reverse transcription was performed by using random hexamers and MultiScribe reverse transcriptase according to the manufacturer’s recommendations (Applied Biosystems, Foster City, CA, USA). Quantitative real-time RT-PCR analyses of Nrf2, NQO1, HO1, and GCLM were performed by using Assay-on-Demand primers and probe sets from Applied Biosystems. Assays were performed using the ABI 7000 Taqman system (Applied Biosystems, Foster City, CA). β-actin was used for normalization.

Statistics

The values are represented as mean ± SE and analyzed by student’s t-test. Differences were considered significant at P ≤ 0.05.

ACKNOWLEDGEMENTS

VK, MH, and SVM thank the National Cancer Institute (NCI) Developmental Therapeutics Program. This project has been funded in whole or in part with funds from the NCI, National Institutes of Health, Grant HSN261200800001E. This work was also supported by National Institutes of Health Grant HL081205 (SB), National Heart, Lung, and Blood Institute Specialized Centers of Clinically Oriented Research Grant P50HL084945, the Flight Attendant Medical Research Institute (SB and RKT), and National Institute on Environmental Health Sciences Grants P50ES015903, P01 ES018176, and ES03819 (SB). VSP and SKS would like to thank the University of Delhi and the Department of Scientific and Industrial Research (DSIR, New Delhi) for financial support.

ABBREVIATION USED

Nrf2: Nuclear factor-erythroid 2 p45-related factor 2
b-ZIP: basic-leucine zipper
Keap 1: Kelch-like ECH-associated protein 1
ARE: antioxidant response element
HO-1: heme oxygenase-1
NQO1: NAD (P)H:quinone oxidoreductase 1
GCLM: glutamate-cysteine ligase modifier subunit

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PERMALINK

Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and Human Lung Epithelial Cells

Vineet Kumar

Sarvesh Kumar

Mohammad Hassan

Hailong Wu

Rajesh K Thimmulappa

Amit Kumar

Sunil K Sharma

Virinder S Parmar

Shyam Biswal

Sanjay V Malhotra

Abstract

INTRODUCTION

RESULTS AND DISCUSSION

Chemistry

Table 1.

Biology

Potency of chalcone derivatives to activate the expression of Nrf2-regulated cytoprotective genes in human lung epithelial cells

Table 2.

Preliminary structure-activity relationship

In vivo potency of identified lead compounds to activate Nrf2 using mouse models

Figure 2. Expression of Nrf2-regulated genes in small intestine after treatment with chalcone derivatives.

Nrf2 is essential for induction of antioxidant genes by compound 2b

Figure 3. Levels of NQO1-ARE luciferase activity after treatment with compound 2b.

Concentration and time-course studies

Figure 5. Time-dependent increase in Nrf2-regulated genes after treatment with compound 2b.

Figure 4. Expression of Nrf2-regulated genes after treatment with compound 2b.

Activation of Nrf2 by compound 2b is independent of ROS generation

Figure 6. Activation of Nrf2 genes by compound 2b is independent of ROS generation.

CONCLUSIONS

EXPERIMENTAL SECTION

Chemistry

General methods

General procedure for synthesis of chalcones

(E)-2,3-diphenylprop-2-en-1-one (1a)

(E)-1-(2-methoxyphenyl)-3-phenylprop-2-en-1-one (1b)

(E)-1-(3-methoxyphenyl)-3-phenylprop-2-en-1-one (1c)

(E)-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one (1d)

(E)-3-phenyl-1-(2,4-dimethoxyphenyl)prop-2-en-1-one (1e)

(E)-3-phenyl-1-(2,6-dimethoxyphenyl)prop-2-en-1-one (1f)

(E)-3-phenyl-1-(2,5-dimethoxyphenyl)prop-2-en-1-one (1g)

(E)-3-phenyl-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (1h)

(E)-3-phenyl-1-(3,5-dimethoxyphenyl)prop-2-en-1-one (1i)

(E)-3-phenyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (1j)

(E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (1k)

(E)-3-phenyl-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (1l)

(E)-1-phenyl-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2a)

(E)-1-(2-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2b)

(E)-1-(3-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2c)

(E)-1-(4-methoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2d)

(E)-1-(2,4-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2e)

(E)-1-(2,6-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2f)

(E)-1-(2,5-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2g)

(E)-1-(3,4-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2h)

(E)-1-(3,5-dimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2i)

(E)-1-(3,4,5-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2j)

(E)-1-(2,3,4-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2k)

(E)-1-(2,4,6-trimethoxyphenyl)-3-(2-(trifluoromethyl)phenyl)prop-2-en-1-one (2l)

(E)-1-phenyl-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3a)

(E)-1-(2-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3b)

(E)-1-(3-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3c)

(E)-1-(4-methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3d)

(E)-1-(2,4-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3e)

(E)-1-(2,6-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3f)

(E)-1-(2,5-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3g)

(E)-1-(3,4-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3h)

(E)-1-(3,5-dimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3i)

(E)-1-(3,4,5-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3j)

(E)-1-(2,3,4-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3k)

(E)-1-(2,4,6-trimethoxyphenyl)-3-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (3l)

(E)-1-phenyl-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4a)

(E)-1-(2-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4b)

(E)-1-(3-methoxyphenyl)-4-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4c)

(E)-1-(4-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4d)

(E)-1-(2,4-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4e)

(E)-1-(2,6-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4f)

(E)-1-(2,5-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4g)

(E)-1-(3,4-dimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4h)

(E)-1-(3,4,5-trimethoxyphenyl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4i)