Synthesis and Identification of New 4-Arylidene Curcumin Analogs as Potential Anticancer Agents Targeting Nuclear Factor-κB Signaling Pathway

Abstract

A series of curcumin analogues including new 4-arylidene curcumin analogs (4-arylidene-1,7-bisaryl-hepta-1,6-diene-3,5-diones) were synthesized. Cell growth inhibition assays revealed that most 4-arylidene curcumin analogs can effectively decrease the growth of a panel of lung cancer cells in sub- and low micromolar concentration ranges. High content analysis technology coupled with biochemical studies showed that this new class of 4-arylidene curcumin analogs exhibits significantly improved NF-κB inhibition activity over the parent compound curcumin, at least in part by inhibiting IκB phosphorylation and degradation via IKK blockage; selected 4-arylidene curcumin analogs also reduced the tumorigenic potential of cancer cells in a clonogenic assay.

Introduction

Nuclear factor-κB (NF-κB) proteins are a family of structurally related eukaryotic transcription factors with a conserved reticuloendotheliosis (Rel) domain.¹ They influence gene expression events that impact a large number of physiological processes including immune response, cell survival, differentiation, and proliferation.² Before cell stimulation, NF-κB dimers are inactive in the cytoplasm and mainly regulated by two distinct signaling pathways.³ The canonical NF-κB activation pathway has been extensively studied. Prior to activation, NF-κB dimers composed of RelA (also known as p65), c-REL and p50 are retained in the cytoplasm by inhibitory κB (IκB) proteins. Various extracellular signals, such as those from microbial infections, viral infections and proinflammatory cytokines, activate the IκB kinase (IKK) complex. The IKK complex targets NF-κB-bound IκBs by phosphorylating two conserved serine residues, which drives ubiquitin-mediated degradation of IκB and leads to the liberation and subsequent nuclear translocation of released NF-κB dimers. Phosphorylation of IκB mainly depends on the IKKβ catalytic subunit of the IKK complex in the canonical pathway. In the second pathway of NF-κB activation, NF-κB, typically exists as an RelB/p100 dimer and remains inactivated by p100 instead of IκB. After stimulation by certain signals, such as the tumor-necrosis factor (TNF) cytokine family member lymphotoxin B (LTβ), an IKKα homodimer, along with NF-κB inducing kinase (NIK), induces the phosphorylation and proteasome-mediated processing of p100 to generate p52, resulting in the production of an active p52/RelB dimer and its translocation to the nucleus.

Recent advances have revealed key roles of NF-κB signaling in pathological conditions such as oncogenesis and inflammation. NF-κB is commonly overexpressed and constitutively activated in different types of hematologic cancers and solid tumors.^4-7 Constitutive activation of NF-κB promotes tumor proliferation, invasion and metastasis;^8-9 moreover, NF-κB activation allows malignant cells to escape apoptosis and, therefore, contributes to radiation and chemotherapy resistance in cancer cells.^10-12 Noticeably, many chemotherapeutic agents have been found to activate NF-κB via various approaches, leading to chemoresistance and subsequent failure of chemotherapy.^13-14 Accumulating evidence suggests that inhibition of NF-κB activation can prevent tumor resistance to chemotherapeutic agents, shift the death–survival balance towards apoptosis and improve the efficacy of current chemotherapeutic regimens.^15-16

A large number of compounds have been reported to inhibit NF-κB by interacting with key macromolecules in the signaling pathway. Many natural dietary agents, such as Soya isoflavone¹⁷, Resveratrol¹⁸ and Curcumin¹⁹, have been found to inhibit NF-κB and induce apoptosis in tumor cells. Curcumin (diferuloylmethane), a yellow spice and pigment isolated from the rhizome of Curcuma longa, has been used as a spice in south Asia for centuries. It has attracted much attention due to its broad range of bioactivities²⁰, including anti-oxidative, anti-inflammatory and anti-cancer activities, as well as its pharmacological safety and potency as chemopreventive agent.²¹ One of the predominant targets of curcumin is the NF-κB cell signaling pathway.²² Curcumin directly inhibits IKK and the 26S proteasome^23-24 to block NF-κB activation. Unfortunately, the clinical potential of curcumin remains limited due to its relatively low potency and poor bioavailability.²⁵ Attempts have been made by others to chemically modify curcumin in order to increase its activity against cancer and NF-κB.^26-31 However, most of these investigations have focused on design, synthesis and activity of 1,3-diketones and monoketone analogs of curcumin. However, curcuminoids, such as Knoevenagel condensates of curcumin analogues, contain a novel skeleton and remain quite less investigated.^{28, 32-33} In 2006, Ajit P. Zambre et al reported that Copper (II) conjugates of Knoevenagel condensates of curcumin showed potential in inhibiting TNFα induced NF-κB activation. ²⁸ Nevertheless, highly active and clinically promising curcuminoids remain to be developed. A systematic investigation of current curcumin analogues would greatly facilitate the development of new curcumin analogs for therapeutic interventions. In the present study, we describe the synthesis and identification of new 4-arylidene curcumin analogs (Knoevenagel condensates of 1,3-diketones curcumin analogs with aromatic aldehydes) as a new class of potential anticancer agents. Screening of the synthesized compounds with high content analysis technology, coupled with biochemical studies, revealed 4-arylidene curcumin analogs have significantly improved IKK/NF-κB inhibition activity and elevated cytotoxicity over the parent compound curcumin. These compounds effectively decreased growth and reduced the tumorigenic potential of cancer cells, as seen in a clonogenic assay.

Results and Discussion

Chemistry

To search for potent analogues with favorable medicinal properties, we extended the molecular diversity of curcuminiods by designing three types of curcuminoids: 1,3-diketones curcumin analogs, monoketone curcumin analogs and 4-arylidene curcumin analogs (4-arylidene-1,7-bisaryl-hepta-1,6-diene-3,5-diones) (Fig. 1).

Figure 1. The structures of 1,3-diketones curcumin analogs (1-6), monoketone curcumin analogs (7-10), 4-arylidene crucumin analogs (13-37) and 4-hydroxymethylene curcumin analogs (38-40).

Classical 1,3-diketones (1-6) including curcumin (1) and a representative 1,3-diketones derivative with known activity (3)³⁴,, were synthesized using a previously reported procedure³⁵ with slight modification (Scheme 1). Briefly, to protect the C-3 of acetylacetone from an unwanted Knoevenagel reaction, a boric acetylacetone anhydride complex was prepared first by refluxing acetylacetone with boric anhydride in EtOAc. The final products 1-6 were synthesized by aldol condensation of protected acetylacetone aromatic aldehydes as described.

Scheme 1. Synthesis of compounds 1-6 and 13-37.

Monoketone curcumin analogs have been extensively investigated due to their potential anti-cancer activities. 7 has been reported to have high activity in antitumor assays³⁶ and was therefore considered; 9 and 10 are asymmetric analogs of 7. These three compounds were included in our work to represent monoketone curcumin analogs. 7 and the intermediate 11 were synthesized by treating 3,4,5-trimethoxybenzaldehyde with excess acetone in the presence of KOH. 9-10 were synthesized by condensing 11 with veratraldehyde and 3-methylthiophene-2-carbaldehyde, respectively. Furthermore, another new compound, 8, was prepared by condensing 12 and 3,4,5-trimethoxybenzaldehyde. The intermediate 12 was obtained using the same conditions as in the snythesis 1-6, except the excess protected acetylacetone (Scheme 2).

Scheme 2. Synthesis of compounds 7-10.

Compared to the large number of traditional 1,3-diketones and monoketone curcumin analogs that have been designed and evaluated, investigation of Knoevenagel condensates of curcuminoids remains scarce. In our current work, 23 new 4-arylidene curcumin analogs (Knoevenagel condensates 13-17, 19-28, 30-37) were designed and synthesized by coupling 1-4 with different aromatic aldehydes in toluene with AcOH and piperidine as a catalyst. The known 4-arylidene curcumin analogs 18 and 29 were synthesized under the same conditions, except with acetylacetone and aldehydes as reactants (Scheme 1).

The 4-arylidene modification can induce the change of a partial enolic diketone, which is universal in 1,3-diketones curcumin analogs, to an absolute diketones moiety, possibly affecting bioactivity. To explore the effect of this transition, three new 4-hydroxymethylene curcumin analogs (38-40, Fig. 1), which possess absolute diketone moieties, were designed as references. 38-40 were synthesized by refluxing triethoxymethane and Ac₂O with 1, 2 and 3, respectively (Scheme 3).

Scheme 3. Synthesis of compound 38-40.

New 4-arylidene crucumin analogs exhibit potent inhibitory activity against caner cells

In order to evaluate the anti-cancer activities of new curcumin derivatives, we tested the effects of synthesized compounds on the growth of A549 lung adenocarcinoma cells. For comparison, dose-response experiments were carried out to obtain GI₅₀ values for each compound as previously described³⁷, with curcumin (1) as a control. As summarized in Table 1, all the traditional 1,3-diketones cucumin analogs (1-6) showed moderate to poor activity against A549 growth, while improved anti-proliferation activities of monoketone analogues 7 and 9 were observed (GI₅₀: 0.53, 1.21μM respectively).

Table 1. Effect of curcumin and its analogs on growth of A549 cells^*.

Growth inhibition of A549 cells
Compd.	GI50 (μM)	Compd.	GI50 (μM)	Compd.	GI50 (μM)
Curcumin	15.23	16	0.23	29	2.00
2	>25	17	0.53	30	0.43
3	5.80	18	0.31	31	0.69
4	19.26	19	2.62	32	0.36
5	>25	20	0.58	33	0.78
6	>25	21	0.55	34	0.55
7	0.55	22	0.42	35	0.42
8	0.53	23	0.67	36	0.64
9	1.21	24	0.63	37	1.31
10	9.31	25	0.67	38	>25
13	1.64	26	0.37	39	>25
14	0.93	27	0.53	40	>25
15	1.12	28	0.65

^*Human adenocarcinoma A549 cells were seeded for 12 hr, subsequently incubated with varying concentrations of curcumin and its analogs for 48 h and then examined for cell viability by the SRB assay. GI₅₀ is the concentration of compounds which causes a 50% inhibition as compared to the vehicle control (0.25% DMSO). Samples were run in quintuplicate and the results are means of at least two independent tests.

At the same time, the thiophene ring moiety bearing 10 (GI₅₀, 9.31μM) showed decreased activity compared to 7 and 9, indicating S replacement of the O atom in the heterocycle of 9 made a negative contribution to its activity. As a regio-isomer of 4, the Knoevenagel condensate 8 exhibited much higher activity (GI₅₀: 0.53μM) than 4. 8 can also be regarded as a monoketone curumin analog with an acetyl substitution. When the activities of 7-9 and 1-6 were compared, it was revealed that the spacing of the two aromatic rings most likely influences the activity of curcumin analogs against A549 growth. The close activities of 8 and 7 indicated that the acetyl group has no apparent effect on activity against A549 growth.

Remarkably, most of the 4-arylidene curcumin analogs (4-arylidene-1,7-bisaryl-hepta-1,6-diene-3, 5-diones, 13-37) showed potent anticancer activities with a GI₅₀ in the submicromolar range (0.23∼0.93μM), except 15, 19, 29 and 37 which exhibited slightly lower activities (GI₅₀: 1.12∼2.62μM). The potency of 4-arylidene analogs was improved by 10∼60 fold over the parent compound curcumin in this assay. 4-hydroxymethylene curcumin analogs (38-40), however, showed poor activity, suggesting the importance of the third arylvinyl moiety. Importantly, the cytotoxic activity of these 4-arylidene curcumin analogs was not limited to A549 cells. In additional to A549 cells, other adenocarcinoma cells H1944, squamous cell carcinoma cells H157, and large cell carcinoma cells H460, were tested with selected 4-arylidene curcumin analogs. These compounds effectively decreased viability of all three major types of lung cancer cells tested. Most selected 4-arylidene analogs potently inhibited the growth of lung cancer cells with GI₅₀ values at submicromolar concentrations (low to 0.07 μM) and induced apoptotic cell death. Data of representative compounds 17, 21, 35 are shown in Fig. 2 (see Supporting information for additional data from viability and apoptosis assays). These studies identified a new class of curcumin analogs with significantly improved activity against cancer cell growth.

Figure 2.

Inhibition of lung cancer cell viability by curcumin and its analogs. Cells were grown in 384 well plates and were treated with curcumin, 17, 21 or 35 as indicated for 72 hr. Cell viability was assessed by the Alamar Blue method and expressed as % control (DMSO). Results with a panel of lung cancer cells are shown (lung adenocarcinoma: A549 H1944, H1792; squamous cell carcinoma: H157; and squamous cell carcinoma: H157). Results from one representative experiment are shown.

Inhibition of TNFα induced nuclear translocation of NF-κB by new curcuminoids

One of the major curcumin targets critical for cancer survival is IκB kinase, IKK.³⁸ IKK is a key regulator of NF-κB activation. Activated NF-κB localizes to the nucleus to promote transcription, which can be triggered by TNFα.³⁹ Thus, we utilized nuclear translocation of NF-κB in response to TNFα as a primary readout to examine the mechanism of action of new curcuminoids in comparison to curcumin. An automated imaging system for high content analysis was employed to monitor the cytoplasmic/nuclear localization of NF-κB in response to TNFα.³⁷ A549 cells in a 384-well plate format were treated with various test compounds before TNFα was added to trigger the nuclear translocation of the NF-κB p65 subunit. As shown in Fig. 3 and Table 2, curcumin (1) attenuated TNFα-induced nuclear redistribution of NF-κB with an IC₅₀ of 11.6 μM, consistent with previous reports.³⁷

Figure 3.

Inhibition of the TNFα induced NF-κB activation by new curcuminoids, 17, 18, 20, and 21. Results from one representative experiment are shown. (A) Example images of NF-κB subcellular localization. A549 cells were treated with compounds, or vehicle (DMSO) for 30 min, followed by stimulation with TNFα (10 ng/ml) for 30 min. In the vehicle (DMSO) treatment, NF-κB is located at cytoplasm. Upon TNFα treatment, NF-κB is activated and translocated to nucleus. Pre-incubation of the cells with increasing concentrations of compounds, with compound 21 as an example, dose-dependently inhibited the TNFα-induced NF-κB translocation to the nucleus. (B) Dose-response curves of the inhibitory effect of test compounds on TNFα-induced NF-κB activation. The inhibitory effect of the compound on TNFα induced NF-κB translocation (activation) was expressed as: % of Control = ΔFI _(compound)/ ΔFI _{(TNFα only control)}. ΔFI is the measured NF-κB fluorescence (green) intensity difference between nucleus and cytoplasm. Data showed are average values from triplicate samples with SD.

Table 2.

The inhibitory effect of compounds on NFκB activation/translocation^*.

Compd.	IC50 (μM)		Compd.	IC50 (μM)		Compd	IC50 (μM)
	mean	SD		mean	SD		mean	SD
Curcumin	9.5	3.00	16	1.8	1.12	29	1.0	1.05
2	> 40	\	17	1.0	0.55	30	> 40	\
3	> 40	\	18	1.5	0.95	31	1.0	1.05
4	> 40	\	19	> 40	\	32	1.4	1.56
5	8.5	2.19	20	1.3	1.01	33	2.7	3.32
6	> 40	\	21	1.1	0.45	34	1.8	2.14
7	7.9	4.26	22	1.4	0.26	35	1.7	1.0
8	4.8	1.85	23	1.4	1.48	36	1.3	1.35
9	9.0	3.44	24	4.9	1.96	37	2.8	1.02
10	24.3	7.01	25	1.5	0.04	38	39.3
13	3.0	2.01	26	1.0	0.05	39	> 40	\
14	3.3	2.37	27	1.5	0.04	40	> 40	\
15	3.8	2.13	28	1.7	0.17

^*A549 cells were seeded in 384-well plates for 15 h, subsequently incubated with varying concentrations of curcumin and its analogs for 30min and then stimulated with TNFα. The inhibitory effect of test compounds on TNFα induced NF-κB translocation was expressed as percentage of fluorescence intensity difference (in nucleus and in cytoplasm) in the control wells (TNFα only) after subtracting background (no TNFα treatment). The IC₅₀ of NF-κB translocation stands for the concentration of a compound required to induce 50% inhibitory effect. Data points from each experiment were obtained as average values from triplicate samples. Data shown are average from three independent experiments with standard deviation (SD).

The synthesized compounds were tested in the same assay and showed different degrees of activity against TNFα induced NF-κB activation. IC₅₀ values of the tested curcuminoids are summarized in Table 2. As shown in Table 2, most of the traditional 1, 3-diketones curcumin analogs (2-4, 6) were less active than curcumin. Three monoketone curcumin analogs (7-9) showed improved activities (IC₅₀ 4.8-9.0μM). Consistent with its cytotoxity profile, 10 was much less active (IC₅₀ 24.3μM) than 7-9 in NF-κB inhibition, evidencing the disadvantages of thiophene replacement with respect to NF-κB inhibition.

Interestingly, except 19 and 30, the newly designed 4-arylidene crucumin analogs (13-37) exhibited significant inhibitory activities against NF-κB translocation with IC₅₀ values in the low micromolar range (1.0∼4.9 μM). 17 (IC₅₀ 1.0 μM) and 21 (IC₅₀ 1.1 μM) showed more than 10 fold greater potency in blocking the nuclear localization of NF-κB than that of curcumin. The low activity of 19 may have resulted from its dimethyl amine moiety, which might be protonized under the test conditions used, leading to unfavorable target interactions. 30, however, remained unclear. Nevertheless, these synthesized 4-arylidene curcumin analogs generally showed superior activity to the classical 1,3-diketones analogs, (1-6) while the monoketone analogs (7-9) displayed moderate activity, suggesting a strong correlation between a compound's molecular skeleton and its activity. Comparing the chemical structures of all three types of curcuminoids, the presence of absolute diketone moieties and 4-arylidene substitutions in 13-37 are unique over others (1-7, 9-10). Both factors may contribute to the increased activities of the tested 4-arylidene curcumin analogs. To explore the nature of these phenomena, we further designed and evaluated the NF-κB inhibitory activity of 4-hydroxymethylene curcumin analogs 38-40, 38-40 showed only poor inhibitory activity against NF-κB activation (Table 2), indicating the importance of the 4-arylidene substitution for activity. On the other hand, although the inhibitory activities varied, most of tri-arylvinyl condensates with different substituted groups (except 19 and 30) were active at low micromolar concentrations. It might also be concluded that the tri-arylvinyl scaffold of 4-arylidene curcumin analogs play a key role in their NF-κB inhibition activities as a pharmacophore, though further investigation is still needed to confirm that hypothesis.

Noticeably, data in Tables 1 and 2 suggest an important correlation between the antiproliferative activity of tested compounds and their NF-κB inhibitory activity. Just like 1-6 and 10 showed low activities in both A549 cell growth and NF-κB activation inhibition, 7-9, 38-40 and most 4-arylidene curcumin analogs behaved consistently in both assays. These results strongly support the notion that the NF-κB pathway is one of the major molecular targets for curcumin and its analogs.

Enhanced inhibition of IκB kinase activity by 4-arylidene curcumin analogs

NF-κB is mainly activated by IKKβ in the well defined canonical signaling pathway.⁴⁰ Because of this, we selected three potent curcumin analogs 17, 21 and 35 to directly test their effects on IKK enzymatic activity, with the parent compound curcumin as a control. Two assays were utilized: (i) a cell based IKK activation assay, which detects phosphorylated IκB and subsequent IκB degradation, and (ii) a reconstituted in vitro kinase assay which directly measures the transfer of γ-³²P from ATP to recombinant IκB. Upon stimulation of A549 cells with TNFα, activated IKKβ can catalyze the phosphorylation of IκB at Ser32 and Ser36, followed by degradation of phosphorylated IκB, leading the release and nuclear translocation of NF-κB. Antibodies against both IκB and Ser32 phosphorylated IκB were used to detect the status of IκB as a readout of IKK activity.

Figure 4 shows the Western blotting results of IκB phosphorylation (A) and subsequent degradation (B). Without the stimuli of TNFα, IκB showed basal level of phosphorylation and no degradation (the first column). TNFα induced significant IκB phosphorylation and subsequent degradation of the protein (the second column). After treatment of cells with different compounds for 4 hr, curcumin could block IκB phosphorylation and degradation when high concentrations were used, whereas 17, 21 and 35 potently block the IκB phosphorylation and degradation in a lower concentration. Dose-response curves for IκB phosphorylation suggested IC50 values of 2.8 (17), 2.2 (21) and 5.0 μM (35) respectively, with a similar trend for IkB degradation. Those results suggest that 17, 21 and 35 block TNF-α induced NF-κB activation at least in part by inhibiting IκB phosphorylation and degradation. Again, the 4-arylidene curcumin analogs show much higher activities than curcumin.

Figure 4.

Effect of curcumin analogs on in vivo activity of IKK. (A) In vivo IKK activity as revealed by phosphorylation of IκB. A549 cells were pretreated with test compounds for 30 min prior to adding TNFα (10 ng/ml). Whole cell lysates were prepared after 7 min of TNFα treatment and analyzed for the phosphorylation state of IκB with anti-pS³² antibody via Western blotting. Then, antibodies on the membrane were stripped and the membrane re-probed for total IκB with antiserum against IκB as indicated. (B) IκB stability assay. A549 cells were pretreated with test compounds at indicated dosage prior to the addition of TNFα. Cells were cultured in the presence of TNFα (10 ng/ml) for an additional 20 minutes, lysed and analyzed for total IκB levels by Western blot.

The phosphorylation of IκB mainly depends on the IKKβ catalytic subunit of the IKK complex in the canonical pathway. It is possible that the inhibitory activities of curcumin and derivatives may be due to direct inhibition to the IKKβ kinase. To test this model, an in vitro reconstituted IKK inhibition assay was performed with recombinant IKKβ and its substrate GST-IκB.³⁷ The addition of curcumin in the range of tested concentrations had no obvious effect on IKKβ catalyzed incorporation of ³²P into its substrate GST-IκB (Fig. 5). On the other hand, incubation of compounds 17, 21, and 35 induced a dose-dependent inhibition of IKKβ. Thus, the structural modifications of these novel curcumin analogs led to drastically enhanced inhibitory activity over curcumin in this defined in vitro IKK kinase assay.

Figure 5.

Effect of curcumin analogs on the catalytic activity of recombinant IKK in vitro. . Recombinant IKKβ was incubated with increasing concentrations of test compounds as indicated. Addition of Mg/[γ-³²P] cocktail with purified GST-I-κB started the reactions, which were continued for 30 min at 30°C. Proteins were separated by SDS-PAGE and processed for radiolabeled GST-I-κB (³²P- I-κB) and stained total GST-I-κB (GST- I-κB). Controls include reactions without IKKβ (lane 1 from left) or without compound (lane 2).

Inhibition of lung cancer clonogenic activity by 4-arylidene curcumin analogs

To compare the anticancer efficacy of curcumin and its new analogs, we investigated the effect of these compounds on colony formation properties of lung cancer cells, a readout for tumorigenesis.⁴¹ A549 adenocarcinoma cells readily formed colonies in a solid matrix. The addition of the new tri-arylvinyl curcumin analogs 17, 21 and 35 drastically inhibited the number of colonies formed by A549 cells (Fig. 6). At concentrations less than 0.2 μM, 17 and 21 reached ∼50% inhibition, while 35 required approximately 0.4 μM to reach the same level of inhibition. For comparison, curcumin inhibited colony forming activity at much higher concentrations, with detectable inhibition at 4 μM. These studies demonstrate that these 4-arylidene curcumin analogs have significantly improved anticancer activity over the parental curcumin.

Figure 6.

Compound 17, 21, and 35 inhibit the colony formation of A549 cells. A549 cells were seeded in 12-well plate at density of 200 cells/well and incubated overnight. The cells were then treated with compounds, or vehicle (DMSO) for 3 days. The medium and compound were replaced for every 3 days. After a total of 9 day incubation, cells were fixed and stained with SRB. The image of the plate was scanned and the colonies were counted. (B) The colonies were counted and normalized to DMSO control. The data shown are average of triplicate samples with SD.

Molecular modeling of the IKKβ/17complex

To gain insights into the potential binding pose of the 4-arylidene curcumin analogs in IKKβ, a molecular docking analysis was carried out. Since three dimensional structural data are unavailable for IKKβ, a homology model of the IKKβ catalytic domain was built based on four templates (PDB code: 2JC6, 1A06, 2QNJ and 2A2A) from the Protein Data Bank (PDB) (see Experimental section for details). Discovery Studio 2.1 (Accelrys Inc.) and Sybyl 7.3.5 (Tripos Inc.) were used for the homology model building and energy minimization of the IKKβ catalytic domain, respectively. The docking of the selected compound, 17, to the IKKβ homology model suggests that 17 could potentially bind to the ATP pocket of IKKβ (Fig. 7). Compound 17 adopts a propeller-shaped conformation in the pocket. The 4-arylidene moiety of 17 occupied the main hydrophobic adenine binding pockets (Fig. 7, region A), the 4′-OH and 3′-OMe in 4-Arylidene moiety of 17 have hydrogen bonds respectively with hinge region residues Glu97 and Cys99, which are the same residues targeted by the adenine moiety during the interaction of ATP with IKKβ catalytic domain. Furthermore, one of the 1,7-diaryl moieties of compound 17 lies along with the phosphate binding pockets (Fig. 7, region P) with a hydrogen bond between a 3′-OMe and residue Thr180. The other side of 1,7-diaryl moieties extends from the ribose binding pockets (Fig.7, region R) to the hydrophilic solvent-exposed entrance (Fig. 7, region E), keeps orthogonal with the 4-arylidene moiety of 17, and has a hydrogen bond between a 4′-OMe and residue Asp103 in the entrance region of the catalytic domain. It should be cautioned that this computer model provides a starting point and details of the Structure-Activity Relationship (SAR) have to be experimentally tested in future work.

Figure 7.

Binding of compound 17 and ATP with the IKKβ catalytic domain homology model. Left, overlay of compound 17 (colored by atom type) and ATP (purple) in the ATP binding pocket, the molecules were represented in stick; Right, Surface representation of the IKKβ catalytic domain homology model with compound 17 (colored by atom type) and ATP (purple) docking into the ATP binding pocket, the kinase surface was mapped by lipophilic potential (from orange to blue, lipophilic potential decrease), and Z-Clipping to remove the N terminal region for visualization facility. Compound 17 adopts a propeller-shaped conformation in the pocket. Typical hydrogen bonds of compound 17 (green dotted line) and ATP (brown dotted line) with the homology model were shown.

In summary, our design and synthesis have led to the generation of a novel series of curcumin analogs. Biochemical and cell biology based studies coupled with high content analysis have led to the identification of a novel class of 4-arylidene curcumin analogs as a new generation of highly potent curcuminoids. These compounds exhibited significantly improved potency in blocking IKK activity and the NF-κB pathway as revealed by both in vivo and in vitro kinase assays and pathway analysis.

Consistent with their enhanced activity against a major molecular target, the representative 4-arylidene curcumin analogs effectively inhibited the viability of three types of lung cancer cells and attenuated the clonogenic activity of A549 cells. Based on our finding, it can be concluded that the tri-arylvinyl skeleton of 4-arylidene curcumin analogs acts as a phamacophore, and the 4-arylmethylene modification is an effective strategy to improve the NF-κB inhibitory activity of curcumin analogs.

Although presented data support an important role of IKKβ in the NF-κB pathway in mediating the effect of reported compounds, some observed difference in compound potency between cell viability assay and the IKKβ/NF-κB assays could be due to a number of factors. For example, (i) the status of compounds in cell culture and in vitro conditions could be different. The employed compounds could be further metabolized in cells to generate multiple active species while in vitro kinase assays only measure the activity of the test compounds. (ii) Although IKKβ could be an important target of the tested curcumin analogs as proposed, the efficacy of the test compounds on cell survival are also determined by cell's genetic background. Cells harboring different oncogene mutations, deletions, or amplifications could show different sensitivities to test compounds that target IKKβ. Our data in Fig. 2 strongly support this model. (iii) The suppression of cell growth by these curcumin analogs could be due to their inhibition of other targets in addition to IKKβ. These potential targets include the cytoprotective antioxidant system. Whether and how these 4-Arylidene curcumin analogs inhibit other targets in addition to IKKβ in cancer cells requires further investigation. Regardless, due to their potent IKKβ inhibitory activity, these novel tri-arylvinyl curcuminoids represent new opportunities for therapeutic development against cancer and other NF-κB dependent disorders, including various inflammatory diseases. Detailed SAR analysis is still required and is being actively pursued by our laboratory.

Experiments

General

All reagents used were commercially available. Solvents were treated using standard techniques. Reactions were monitored by TLC on glass plate coated with silica gel with fluorescent indicator (GF₂₅₄). Column chromatography was performed on silica gel (100-200 mesh). ¹H NMR, ¹³C NMR spectra were recorded with Varian INOVA 500NB or Bruker Ultrashield 400 MHz spectrometers using TMS as an internal standard. Purity of target compounds was determined by reversed-phase HPLC analyses (Hypersil BDS-C18, 250×4.6μm) with a flow rate of 0.5 mL/min. The samples were eluted with a gradient of constant 70% A (2-4 and 40), 90% A (38-39) or 50% A (others), where Solvent A was CH₃CN and solvent B was 0.1% TFA in water; using UV monitor at 254 nm for detection. Retention times (t_R) were calculated in minutes and purity was calculated as % total area. All compounds were >95% purity by HPLC. The HR-MS analysis was performed on Thermo Finnigan MAT95XP-Trap specifications.

Chemistry

3.2.1 Synthesis of d1-d10

5-hydroxy-1, 7-bis(4-hydroxy-3-methoxyphenyl)hepta-1, 4, 6-trien-3-one (1), 5-hydroxy-1, 7-bis(4-methoxyphenyl) hepta-1, 4, 6-trien-3-one (2), 1, 7-bis(3, 4-dimethoxyphenyl)-5-hydroxyhepta-1, 4, 6-trien-3-one (3), 5-hydroxy-1, 7-bis(3, 4, 5-trimethoxyphenyl)hepta-1, 4, 6-trien-3-one (4), 5-hydroxy-1, 7-bis(5-methylfuran-2-yl)hepta-1,4,6-trien-3-one (5) and 1, 7-di(furan-2-yl)-5-hydroxyhepta-1, 4, 6-trien-3-one (6) were synthesized according to our previous report.³⁵

(E)-4-(3,4,5-trimethoxyphenyl)but-3-en-2-one (11) and (1E,4E)-1,5-bis(3,4,5-trimethoxyphenyl) penta-1, 4-dien-3-one (7)

To a solution of 3mL acetone in 10mL EtOH, 0.84g KOH (15mmol, dissolved in 5mL H₂O) was added in 0°C and the mixture was stirred for 20min. After that, 2.94g 3,4,5-trimethoxybenzaldehyde (15mmol, dissolved in 25mL EtOH) was added dropwise, then the reaction was brought to room temperature for 2h and monitored by TLC. Once the 3,4,5-trimethoxybenzaldehyde was consumed, the reaction solution was poured into water and the resulted solution was neutralized by 2N HCl. Then the raw product was extracted from water by acetic ether and purified with column chromatography (petroleum ether/acetic ether 5:1) to give 1.23g intermediate 11 (35%) and 1.53g yellow powder 7 (49%) respectively. HPLC t_R = 17.8 min; R_f 0.38 (petroleum ether/acetic ether 2:1). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J=15.8Hz, 2H), 6.97 (d, J=15.8Hz, 2H), 6.84 (s, 4H), 3.91 (s, 12H), 3.89 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 188.48, 153.51, 143.34, 140.52, 130.29, 124.82, 105.72, 61.00, 56.25.

(5E)-3-(3,4,5-trimethoxybenzylidene)-6-(3,4,5-trimethoxyphenyl)hex-5-ene-2,4-dione (8)

Compound 8 was synthesized in two steps. First the intermediate 12 was synthesized with the following procedures. Acetyl acetone (5.01g, 50mmol), boric anhydride (2.1g, 30 mmol) and tri-n-butyl borate (23 g, 100 mmol) were mixed in ethyl acetate (100 mL) and stirred in 0°C for 1hour. Then a solution of 3,4,5-trimethoxybenzaldehyde (1.96g, 10mmol) and n-butylamine (0.5 mL) in ethyl acetate was added dropwise in 1h. Next the reaction mixture was stirred in room temperature for 2 days. Followed the resulted solution was neutralized by 0.4 N HCl and stirred for an additional 30 min in 60°C. Then the raw product was extracted with ethyl acetate, and the organic layer was washed with water (30 mL, 2 times), dried (Na₂SO₄) and evaporated under vacuum. Finally the product was isolated by column chromatography(petroleum ether/acetic ether 9:1) to give 0.93g intermediate 12 (33%). ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 15.8 Hz, 1H), 6.77 (s, 2H), 6.39 (d, J = 15.8 Hz, 1H), 5.67 (s, 1H), 3.91 (s, 6H), 3.90 (s, 3H), 2.18 (s, 3H). After this, 557mg 12 (2mmol) and 3,4,5-trimethoxybenzaldehyde (1.18g, 6mmol) were dissolved in 10mL DMF and stirred in 105°C overnight, then the DMF was evaporated under vacuum. Finally the crude mixture were purified with column chromatography (petroleum ether/acetic ether 5:1) to give 280mg 8 as pale yellow powder, yield 31%. HPLC t_R = 11.1 min; R_f 0.31 (petroleum ether/acetic ether 2:1). HRMS calcd for C₂₅H₂₈O₈: 456.1779, found 456.1780. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 1H), 7.40 (d, J=16.1Hz, 1H), 6.75 (d, J=16.1Hz, 1H), 6.71 (s, 2H), 6.68 (s, 2H), 3.87 (s, 3H), 3.85 (s, 6H), 3.84 (s, 3H), 3.79 (s, 6H), 2.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.82, 195.75, 153.48, 153.19, 146.41, 141.04, 140.97, 140.36, 139.37, 129.35, 128.23, 126.27, 107.92, 105.91, 60.96, 60.90, 56.20, 56.14, 27.30.

General procedure for the synthesis of 9 and 10

11 (236mg, 1mmol) and 1mmol corresponding benzaldehyde were dissolved in 5mL EtOH, and KOH (56mg, 1mmol, dissolved in 0.1mL H₂O) was added. The reaction mixture was stirred for 1h in room temperature. Then the solution was poured into water and the resulted solution was neutralized by 2N HCl. The raw product was extracted with ethyl acetate, and the organic layer was washed with water (10 mL, 2 times), dried (Na₂SO₄) and evaporated under vacuum. Finally the raw product was purified with column chromatography to give final product respectively.

(1E,4E)-1-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (9)

3,4-dimethoxybenzaldehyde (166mg, 1mmol) was used as benzaldehyde and the column chromatography (petroleum ether/acetic ether 2:1) gave 324mg d9 as yellow powder, yield 84%. HPLC t_R = 13.5 min; R_f 0.14 (petroleum ether/acetic ether 2:1). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J=15.8Hz, 1H), 7.64 (d, J=15.8Hz, 1H), 7.20 (dd, J=1.9, 8.3Hz, 1H), 7.13 (d, J=1.9Hz, 1H), 6.96 (d, J=15.8Hz, 1H), 6.95 (d, J=15.8Hz, 1H), 6.88 (d, J=8.3Hz, 1H), 6.84 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 188.60, 153.51, 151.49, 149.33, 143.40, 143.03, 140.41, 130.41, 127.82, 125.00, 123.51, 123.21, 111.20, 110.04, 105.66, 61.02, 56.25, 56.03, 55.99.

(1E,4E)-1-(3-methylthiophen-2-yl)-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (10)

3-methylthiophene-2-carbaldehyde (126mg, 1mmol) was used as benzaldehyde and the column chromatography (petroleum ether/acetic ether 9:1) gave 266mg d10 as yellow powder, yield 77%. HPLC t_R = 22.9 min; R_f 0.39 (petroleum ether/acetic ether 3:1). HR-MS calcd for C₁₉H₂₀O₄S₁: 344.1077, found 344.1074. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 15.3 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.30 (d, J = 5.1 Hz, 1H), 6.91 (d, J = 5.1 Hz, 1H), 6.88 (d, J = 15.9 Hz, 1H), 6.86 (d, J = 15.3 Hz, 1H), 6.85 (s, 2H), 3.92 (s, 6H), 3.90 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 188.07, 153.48, 142.94, 142.68, 134.48, 134.16, 131.48, 130.34, 127.29, 125.87, 122.85, 105.57, 61.00, 56.20, 14.32.

General procedure for the Synthesis of 13-17, 19-28 and 30-37

1.0 mmol 1, 2, 3 or 4 and 2mmol corresponding benzaldehyde as well as 25mL toluene were added to a three neck rounded flask equipped a water dispenser. Pyridine (4.0mg, 0.05mmol, in 0.1mL toluene) and acetic acid (4.8mg, 0.08mmol, in 0.1mL toluene) were added as catalysts. The reaction mixture was stirred in 140°C overnight and the generated water was removed by water dispenser during the whole reaction. Then the reaction mixture was washed with water (10mL, twice) to remove pyridine and acetic acid. Next the organic layer was evaporated under vacuum to get raw product. Finally the product was purified by using silica gel column chromatography.

(1E,6E)-4-(4-hydroxy-3-methoxybenzylidene)-1,7-bis(4-methoxyphenyl)hepta-1,6-diene-3,5-dione (13)

4-hydroxy-3-methoxybenzaldehyde (304mg, 2mmol) and 2 (336mg, 1mmol) were used as reactants and the raw product was purified by column chromatograph (petroleum ether/acetic ether 2:1) to give 249mg 13 as yellow powder, yield 53%. HPLC t_R = 19.5 min; R_f 0.20 (petroleum ether/acetic ether 2:1). HR-MS calcd for C₂₉H₂₆O₆: 470.1724, found 470.1727. ¹H NMR (500 MHz, CDCl₃) δ 7.81 (s, 1H), 7.77 (d, J=15.4Hz, 1H), 7.53 (d, J=16.1Hz, 1H), 7.52 (d, J=8.9Hz, 2H), 7.42 (d, J=8.8Hz, 2H), 7.08 (dd, J=2.0, 8.3Hz, 1H), 7.03 (d, J=2.0Hz, 1H), 6.99 (d, J=15.4Hz, 1H), 6.89 (d, J=8.8Hz, 2H), 6.86 (d, J=8.3Hz, 1H), 6.86 (d, J=8.9Hz, 2H), 6.81 (d, J=16.1Hz, 1H), 3.83 (s, 3H), 3.81 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 198.62, 186.81, 162.15, 161.79, 148.14, 146.64, 146.54, 144.48, 140.79, 138.69, 130.49, 130.42, 127.62, 126.90, 125.97, 125.75, 125.42, 120.11, 114.82, 114.50, 114.39, 112.45, 55.94, 55.41.

(1E,6E)-4-(3,4-dimethoxybenzylidene)-1,7-bis(4-methoxyphenyl)hepta-1,6-diene-3,5-dione (14)

3,4-dimethoxybenzaldehyde (332mg, 2mmol) and 2 (336mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 4:1) to give 312mg 14 as yellow powder, yield 64%. HPLC t_R = 27.3 min; R_f 0.33 (petroleum ether/acetic ether 2:1). HR-MS calcd for C₃₀H₂₈O₆: 484.1880, found 484.1886. ¹H NMR (500 MHz, CDCl₃) δ 7.82 (s, 1H), 7.78 (d, J = 15.4 Hz, 1H), 7.53 (d, J = 8.9 Hz, 2H), 7.53 (d, J = 15.4 Hz, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.13 (dd, J = 8.4, 1.8 Hz, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.99 (d, J = 15.4 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.9 Hz, 2H), 6.83 (d, J = 16.1 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 – 3.81 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 198.73, 186.73, 162.13, 161.77, 151.09, 148.81, 146.81, 144.57, 140.65, 138.80, 130.53, 130.47, 127.52, 126.79, 126.31, 125.35, 125.05, 119.95, 114.47, 114.35, 112.82, 111.02, 55.90, 55.82, 55.43, 55.40.

(1E,6E)-1,7-bis(4-methoxyphenyl)-4-(2,3,4-trimethoxybenzylidene)hepta-1,6-diene-3,5-dione (15)

3,4,5-trimethoxybenzaldehyde (392mg, 2mmol) and 2 (336mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 4:1) to give 283mg 15 as yellow powder, yield 55%. HPLC t_R = 32.0 min; R_f 0.39 (petroleum ether/acetic ether 2:1). HR-MS calcd for C₃₁H₃₀O₇: 514.1986, found 514.1987. ¹H NMR (500 MHz, CDCl₃) δ 7.78 (d, J=15.4Hz, 1H), 7.76 (s, 1H), 7.53 (d, J=8.7Hz, 2H), 7.53 (d, J=16.1Hz, 1H), 7.43 (d, J=8.7Hz, 2H), 6.98 (d, J=15.4Hz, 1H), 6.90 (d, J=8.8Hz, 2H), 6.87 (d, J=8.8Hz, 2H), 6.81 (d, J=16.1Hz, 1H), 6.76 (s, 2H), 3.84 (m, 6H), 3.82 (s, 3H), 3.80 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 198.24, 186.77, 162.23, 161.89, 153.13, 146.82, 144.94, 140.46, 140.28, 140.10, 130.54, 128.87, 127.47, 126.76, 125.26, 119.88, 114.54, 114.41, 107.92, 60.93, 56.14, 55.45, 55.43.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxybenzylidene)hepta-1,6-diene-3,5-dione (16)

3,4,5-trimethoxybenzaldehyde (392mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 2:1) to give 486mg 16 as yellow powder, yield 85%. HPLC t_R = 17.0 min; R_f 0.39 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₃H₃₄O₉: 574.2197, found 574.2201. ¹H NMR (500 MHz, CDCl₃) δ 7.75 (s, 1H), 7.76 (d, J=15.4Hz, 1H), 7.51 (d, J=16.1Hz, 1H), 7.18 (dd, J=8.4, 2.0Hz, 1H), 7.07 (d, J=2.0Hz, 1H), 7.05 (dd, J=8.4, 2.0Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.98 (d, J=15.4Hz, 1H), 6.86 (d, J=8.4Hz, 1H), 6.82 (d, J=8.4Hz, 1H), 6.80 (d, J=16.1Hz, 1H), 6.76 (s, 2H) 3.90 (s, 3H), 3.89(s, 3H), 3.88(s, 3H), 3.86 (s, 3H), 3.84(s, 3H), 3.78(s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 197.96, 186.86, 153.18, 152.10, 151.78, 149.42, 149.33, 146.96, 145.23, 140.46, 140.40, 140.27, 128.84, 127.77, 127.07, 125.48, 123.56, 123.37, 120.21, 111.24, 110.76, 110.33, 108.02, 60.89, 56.17, 56.02, 55.97.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-hydroxy-3-methoxybenzylidene)hepta-1,6-diene-3,5-dione (17)

4-hydroxy-3-methoxybenzaldehyde (304mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 2:1) to give 320mg 17 as yellow powder, yield 60%. HPLC t_R = 11.6 min; R_f 0.27 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₁H₃₀O₈: 530.1935, found 530.1941. ¹H NMR (500 MHz, CDCl₃) δ 7.80 (s, 1H), 7.75 (d, J=15.4Hz, 1H), 7.50 (d, J=16.1Hz, 1H), 7.18 (dd, J=8.5, 2.0Hz, 1H), 7.09 (dd, J=8.5, 2.0Hz, 1H), 7.06 (dd, J=8.0, 2.0Hz, 1H), 7.06 (d, J=2.0Hz, 1H), 7.04 (d, J=2.0Hz, 1H), 6.98 (d, J=2.0Hz, 1H), 6.97 (d, J=15.4Hz, 1H), 6.87 (d, J=8.5Hz, 1H), 6.86 (d, J=8.0Hz, 1H), 6.82 (d, J=8.5Hz, 1H), 6.81 (d, J=16.1Hz, 1H), 3.91 (m, 6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.61, 186.89, 151.96, 151.61, 149.31, 149.24, 148.17, 147.02, 146.54, 144.92, 140.96, 138.58, 127.81, 127.09, 125.90, 125.71, 125.57, 123.60, 123.31, 120.20, 114.84, 112.42, 111.15, 111.12, 110.61, 110.19, 56.04, 56.03, 56.01, 55.94.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-(dimethylamino)benzylidene)hepta-1,6-diene-3,5-dione (19)

4-(dimethylamino)benzaldehyde (298mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 2:1) to give 258mg 19 as red powder, yield 49%. HPLC t_R = 18.1 min; R_f 0.39 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₂H₃₃O₆N₁: 527.2302, found 527.2301. ¹H NMR (500 MHz, CDCl₃) δ 7.73 (d, J=15.4Hz, 1H), 7.52 (d, J=16.0Hz, 1H), 7.42 (d, J=8.9Hz, 2H), 7.17 (dd, J=8.4, 1.8Hz, 1H), 7.07 (dd, J=8.5, 1.9Hz, 1H), 7.06 (d, J=1.8Hz, 1H), 7.00 (d, J=1.9Hz, 1H), 6.99 (d, J=15.4Hz, 1H), 6.84 (d, J=8.5Hz, 1H), 6.85 (d, J=16.0Hz, 1H), 6.81 (d, J=8.4Hz, 1H), 6.61 (d, J=8.9Hz, 2H), 3.90 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 2.99 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 199.52, 186.63, 151.80, 151.69, 151.33, 149.23, 149.20, 146.50, 143.88, 142.14, 135.43, 133.08, 128.14, 127.43, 126.16, 123.47, 123.05, 120.87, 120.72, 111.66, 111.15, 111.06, 110.62, 110.28, 56.06, 56.02, 56.01, 55.93, 39.97.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(3-fluorobenzylidene)hepta-1,6-diene-3,5-dione (20)

3-fluorobenzaldehyde (248mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:1) to give 413mg 20 as yellow powder, yield 82%. HPLC t_R = 22.5 min; R_f 0.36 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₀H₂₇O₆F₁: 502.1786, found 502.1785. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (d, J = 15.4 Hz, 1H), 7.78 (s, 1H), 7.46 (d, J = 16.2 Hz, 1H), 7.30 (dd, J =8.0, 7.5 Hz, 1H), 7.29 (d, J = 8.0Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.10-7.04 (m, 2H), 7.03 (dd, J = 8.3 Hz, J =1.8 Hz, 1H ), 6.99 (s, 1H), 6.95 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 3.92 (s, 6H), 3.90 (s, 3H), 3.88 (s, 3H). ¹³C NMR (101MHz, CDCl₃) δ 197.47, 186.74, 162.68 (d, J = 247.2 Hz), 152.09, 151.83, 149.29, 149.24, 147.83, 145.92, 141.94, 138.75, 135.71 (d, J = 7.9 Hz), 130.39 (d, J = 8.3 Hz), 127.54, 126.89, 126.06 (d, J = 2.9 Hz), 125.40, 123.85, 123.62, 119.84, 117.16 (d, J = 21.4 Hz), 116.60 (d, J = 22.3 Hz), 111.10, 111.04, 110.54, 110.04, 56.05, 56.04, 55.93.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-fluorobenzylidene)hepta-1,6-diene-3,5-dione (21)

4-fluorobenzaldehyde (248mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:1) to give 390mg 21 as yellow powder, yield 78%. HPLC t_R = 21.9 min; R_f 0.33 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₀H₂₇O₆F₁: 502.1786, found 502.1789. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (s, 1H), 7.78 (d, J = 15.4 Hz, 1H), 7.48-7.51 (m, 2H), 7.47 (d, J = 16.2 Hz, 1H), 7.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.05-7.06 (m, 2H), 7.03 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.96 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.88 (s, 3H). ¹³C NMR (101MHz, CDCl₃) δ 197.92, 186.76, 163.73 (d, J = 252.5 Hz), 152.09, 151.77, 149.32, 149.25, 147.63, 145.59, 140.57, 139.18, 132.38 (d, J = 8.6 Hz), 129.79 (d, J = 3.3 Hz), 127.63, 126.93, 125.43, 123.79, 123.52, 119.97, 116.07 (d, J = 21.9 Hz), 111.12, 111.07, 110.58, 110.09, 56.06, 56.04, 55.94.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-hydroxybenzylidene)hepta-1,6-diene-3,5-dione (22)

4-hydroxybenzaldehyde (244mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 2:1) to give 187mg 22 as orange powder, yield 37%. HPLC t_R = 11.6 min; R_f 0.30 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₇: 500.1830, found 500.1826. ¹H NMR (400 MHz, DMSO) δ 7.76 (d, J = 15.4 Hz, 1H), 7.50 (d, J = 16.1 Hz, 1H), 7.39 (d, J = 8. 7 Hz, 2H), 7.19 (dd, J = 8.4, 1.9 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 7.07 (dd, J = 8.2, 2.1 Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.97 (d, J = 15.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 16.1 Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 198.22, 187.83, 159.85, 151.39, 151.14, 148.95, 148.93, 145.93, 143.26, 140.22, 137.93, 132.55, 127.48, 126.78, 125.42, 124.23, 123.51, 123.29, 119.29, 115.81, 111.62, 111.49, 111.16, 110.68, 55.71, 55.54.

(1E,6E)-4-(2,5-dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-di one (23)

2,5-dimethoxybenzaldehyde (332mg, 2mmol) and 1 (368mg, 1mmol)were used as reactants and the column chromatography (petroleum ether/acetic ether 2:1) gave 177mg 23 as yellow powder, yield 34%. HPLC t_R = 10.7 min; R_f 0.20 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₈[M-H]⁻: 515.1706, found 515.1701. ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.74 (d, J = 15.5 Hz, 1H), 7.44 (d, J = 16.1 Hz, 1H), 7.17 (dd, J = 8.2, 1.8 Hz, 1H), 7.05 (d, J = 1.8 Hz, 1H), 7.01 (d, J = 15.5 Hz, 1H), 6.99 (dd, J = 8.1, 1.9 Hz, 1H), 6.94-6.95 (m, 2H), 6.92 (d, J = 8.2 Hz, 1H), 6.84-6.87 (m, 2H), 6.80 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 16.1 Hz, 1H), 5.92 (s, 1H), 5.92 (s, 1H), 3.93 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ197.61, 187.76, 153.21, 152.49, 148.63, 148.41, 146.76, 145.17, 140.88, 136.27, 127.41, 126.87, 125.31, 123.96, 123.58, 123.43, 120.15, 117.70, 114.91, 114.83, 114.76, 111.92, 110.31, 109.72, 56.05, 56.01, 55.95, 55.74.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-ethylbenzylidene)hepta-1,6-diene-3,5-dione (24)

4-ethylbenzaldehyde (268mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 4:1) to give 316mg 24 as yellow powder, yield 62%. HPLC t_R = 27.9 min; R_f 0.37 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₂H₃₂O₆: 512.2193, found 512.2196. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 7.78 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.19 (dd, J = 8.5, 1.9 Hz, 1H), 7.16 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 1.9 Hz, 1H), 7.06 (dd, J = 8.3, 1.8 Hz, 1H), 6.99 (d, J = 1.8 Hz, 1H), 7.00 (d, J = 15.4 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.2 Hz, 1H), 3.91 (s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 2.62 (q, J = 7.6 Hz, 2H), 1.19 (t, J =7.6 Hz, 3H). ¹³C NMR (101MHz, CDCl₃) δ 198.33, 187.04, 151.85, 151.60, 149.20, 149.18, 147.21, 147.17, 145.18, 140.76, 139.81, 130.87, 130.61, 130.23, 128.40, 127.92, 127.71, 127.10, 125.67, 123.64, 123.39, 120.08, 111.08, 110.99, 110.51, 110.09, 55.99, 55.88, 28.74, 15.09.

(1E,6E)-4-(2,3-dimethoxybenzylidene)-1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (25)

2,3-dimethoxybenzaldehyde (332mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:1) to give 225mg 25 as yellow powder, yield 41%. HPLC t_R = 21.0 min; R_f 0.26 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₂H₃₂O₈: 544.2092, found 544.2090. ¹H NMR (400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.43 (d, J = 16.1 Hz, 1H), 7.20 (dd, J = 8.3, 1.9 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H), 7.02 (dd, J = 8.4, 1.9 Hz, 1H), 7.01 (d, J = 15.4 Hz, 1H), 6.98 (dd, J = 7.8, 1.8Hz, 1H), 6.96 (d, J = 1.9 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.90 (dd, J = 7.8, 1.8 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.72 (d, J = 16.1 Hz, 1H), 3.93 – 3.90 (m, 9H), 3.89 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.35, 187.65, 152.70, 151.77, 151.65, 149.25, 148.34, 146.66, 145.20, 141.65, 136.09, 128.30, 127.79, 127.27, 125.78, 124.10, 123.51, 123.38, 121.89, 120.42, 114.42, 111.16, 111.06, 110.54, 110.06, 61.35, 56.01, 55.90, 55.86.

(1E,6E)-4-(3,4-dimethoxybenzylidene)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,6-diene-3,5-dione (26)

3,4-dimethoxybenzaldehyde (332mg, 2mmol) and 4 (456mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:2) to get 323mg 26 as yellow powder, yield 53%. HPLC t_R = 18.6 min; R_f 0.16 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₄H₃₆O₁₀: 604.2303, found 604.2313. ¹H NMR (500 MHz, CDCl₃) δ 7.82 (s, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.47 (d, J = 16.1 Hz, 1H), 7.13 (dd, J = 8.5, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 15.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 16.1 Hz, 1H), 6.80 (s, 2H), 6.70 (s, 2H), 3.89 (s, 6H), 3.88 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.25, 186.95, 153.53, 151.43, 149.05, 146.86, 145.03, 141.21, 140.95, 138.79, 130.24, 129.51, 126.87, 126.27, 125.07, 121.54, 113.04, 111.24, 106.24, 106.13, 60.99, 56.38, 56.27, 55.97, 55.93.

(1E,6E)-4-(2,5-dimethoxybenzylidene)-1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (27)

2,5-dimethoxybenzaldehyde (332mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:1) to give 211mg 27 as yellow powder, yield 39%. HPLC t_R = 20.4 min; R_f 0.24 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₃₂H₃₂O₈: 544.2092, found 544.2096. ¹H NMR (400 MHz, CDCl₃) δ 8.15 (s, 1H), 7.76 (d, J = 15.5 Hz, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.20 (dd, J = 8.3, 1.5 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 8.0, 1.5 Hz, 1H), 7.04 (d, J = 15.5 Hz, 1H), 6.95-6.96 (m, 2H), 6.87 (d, J = 8.3 Hz, 1H), 6.85-6.87 (m, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 3.92 (s, 6H), 3.89 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.67 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.49, 187.78, 153.20, 152.46, 151.70, 151.54, 149.18, 146.47, 144.96, 140.90, 136.31, 127.78, 127.25, 125.53, 123.48, 123.39, 123.30, 120.38, 117.67, 114.94, 111.91, 111.11, 111.02, 110.47, 109.97, 55.98, 55.88, 55.70.

(1E,6E)-4-(2,4-dimethoxybenzylidene)-1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (28)

2,4-dimethoxybenzaldehyde (332mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography(petroleum ether/acetic ether 3:1) to give 337mg 28 as yellow powder, yield 62%. HPLC t_R = 19.7 min; R_f 0.40 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₂H₃₂O₈: 544.2092, found 544.2094. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.74 (d, J = 15.5 Hz, 1H), 7.48 (d, J = 16.1 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.3, 1.8 Hz, 1H), 7.08 (d, J = 1.8 Hz, 1H), 7.05 (dd, J = 8.3, 1.8 Hz, 1H), 7.03 (d, J = 15.5 Hz, 1H), 6.98 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 16.1 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 6.40 (dd, J = 8.4, 2.4 Hz, 1H), 3.91 (s, 6H), 3.89 (s, 3H), 3.87 (s, 6H), 3.79 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.32, 187.60, 163.17, 159.83, 151.62, 151.37, 149.17, 149.15, 146.10, 144.34, 138.40, 136.26, 131.78, 127.96, 127.37, 125.73, 123.41, 123.10, 120.62, 115.80, 111.09, 111.00, 110.52, 110.04, 105.16, 98.27, 55.99, 55.89, 55.53, 55.43.

(1E,6E)-4-(3,4-dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-di one (30)

3,4-dimethoxybenzaldehyde (332mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography(petroleum ether/acetic ether 3:2) gave 347mg 30 as yellow powder, yield 67%. HPLC t_R = 9.3 min; R_f 0.20 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₈: 516.1779, found 516.1783. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (s, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.1 Hz, 1H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H), 7.14 (dd, J = 8.2, 2.0 Hz, 1H), 7.03-7.06 (m, 2H), 7.03 (dd, J = 8.2, 1.9 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.96 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 16.1 Hz, 1H), 5.96 (s, 1H), 5.94 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.79, 186.90, 151.13, 148.98, 148.83, 148.55, 147.49, 146.91, 146.83, 145.25, 140.74, 138.74, 127.31, 126.57, 126.28, 125.20, 125.02, 124.04, 123.59, 119.76, 114.94, 114.91, 112.84, 111.05, 110.44, 109.99, 56.04, 55.95, 55.91, 55.84.

(1E,6E)-4-(2,4-dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-di one (31)

2,4-dimethoxybenzaldehyde (332mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 2:1) gave 268mg 31 as yellow powder, yield 52%. HPLC t_R = 11.1 min; R_f 0.29 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₈: 516.1779, found 516.1782. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 7.72 (d, J = 15.4 Hz, 1H), 7.45 (d, J = 16.1 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.05 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 15.4 Hz, 1H), 6.96 (s, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.73 (d, J = 16.1 Hz, 1H), 6.41 (s, 1H), 6.39 (d, J = 8.4 Hz, 1H), 5.89 (m, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H), 3.79 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.45, 187.61, 163.15, 159.85, 148.55, 148.24, 146.76, 146.72, 146.41, 144.56, 138.37, 136.21, 131.77, 127.56, 126.97, 125.46, 123.87, 123.37, 120.34, 115.82, 114.79, 114.75, 110.36, 109.81, 105.15, 98.28, 56.05, 55.95, 55.53, 55.43.

(1E,6E)-4-(3-fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (32)

3-fluorobenzaldehyde (248mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 3:1) gave 329mg 32 as yellow powder, yield 69%. HPLC t_R = 12.2 min; R_f 0.32 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₂₈H₂₃O₆F₁: 474.1473, found 474.1476. ¹H NMR (400 MHz, acetone) δ 7.90 (s, 1H), 7.69 (d, J = 15.5 Hz, 1H), 7.50 (d, J = 16.2 Hz, 1H), 7.39 (m, 2H), 7.39 (d, J = 15.5 Hz, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.2, 1.8Hz, 1H), 7.28 (dd, J = 8.4, 1.8Hz, 1H), 7.12-7.17 (m, 1H), 7.11 (dd, J = 8.2, 1.8 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 16.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H). ¹³C NMR (101 MHz, acetone) δ 197.59, 188.25, 163.48 (d, J = 244.6 Hz), 150.86, 150.62, 148.83, 148.77, 148.08, 145.78, 143.93, 138.30, 137.46 (d, J = 7.9 Hz), 131.54 (d, J = 8.4 Hz), 127.86, 127.33, 127.00 (d, J = 2.8 Hz), 125.68, 124.95, 124.35, 119.85, 117.47 (d, J = 21.4 Hz), 117.00 (d, J = 22.6 Hz), 116.31, 116.18, 112.43, 111.66, 56.42, 56.35.

(1E,6E)-4-(4-fluorobenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (33)

4-fluorobenzaldehyde (248mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 3:1) gave 311mg 33 as yellow powder, yield 66%. HPLC t_R = 12.3 min; R_f 0.31 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₂₈H₂₃O₆F₁: 474.1473, found 474.1474. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (s, 1H), 7.77 (d, J = 15.4 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 16.2 Hz, 1H), 7.16 (dd, J = 8.2, 1.8 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.03 (dd, J = 8.2, 2.0 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 15.4 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 16.2 Hz, 1H), 5.94 (s, 2H), 3.93 (s, 3H), 3.90 (s, 3H) ¹³C NMR (101 MHz, acetone) δ 197.89, 188.22, 164.33 (d, J = 249.7 Hz), 150.76, 150.49, 148.78, 148.73, 147.77, 145.41, 142.61, 138.69, 133.26 (d, J = 8.7 Hz), 131.51 (d, J = 3.1Hz ), 127.89, 127.35, 125.74, 124.81, 124.23, 119.89, 116.58 (d, J = 21.9 Hz), 116.27, 116.15, 112.36, 111.63, 56.38, 56.31

(1E,6E)-4-(4-ethylbenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (34)

4-ethylbenzaldehyde (268mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 3:1) gave 288mg 34 as yellow powder, yeild 59%. HPLC t_R = 16.9 min; R_f 0.36 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₆: 484.1880, found 484.1881. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (s, 1H), 7.75 (d, J = 15.4 Hz, 1H), 7.47 (d, J = 16.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.13-7.16 (m, 3H), 7.03 (d, J = 1.5 Hz, 1H), 7.01 (dd, J = 8.2, 1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz,1H), 6.96 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 2.61 (q, J = 7.56 Hz, 2H), 1.19 (t, J = 7.59 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 198.43, 187.03, 148.84, 148.54, 147.51, 147.16, 146.82, 146.79, 145.39, 140.69, 139.77, 130.86, 130.61, 128.40, 127.28, 126.66, 125.38, 124.03, 123.67, 119.77, 114.86, 114.83, 110.36, 109.97, 56.06, 55.95, 28.75, 15.10.

(1E,6E)-4-(2,3-dimethoxybenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-di one (35)

2,3-dimethoxybenzaldehyde (332mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 2:1) gave 180mg 35 as yellow powder, yield 35%. HPLC t_R = 18.8 min; R_f 0.24 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₃₀H₂₈O₈ [M-H]⁻: 515.1706, found 515.1696. ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.75 (d, J = 15.5 Hz, 1H), 7.41 (d, J = 16.1 Hz, 1H), 7.16 (dd, J = 8.3, 1.5 Hz, 1H), 7.06 (d, J = 1.5 Hz, 1H), 6.98 (dd, J = 8.2, 1.5 Hz, 1H), 6.98 (d, J = 15.5 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 6.88-6.96 (m, 4H), 6.86 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 16.1 Hz, 1H), 5.92 (s, 1H), 5.90 (s, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.56, 187.64, 152.67, 148.72, 148.54, 148.29, 147.05, 146.81, 145.46, 141.56, 136.00, 128.25, 127.30, 126.76, 125.45, 124.11, 123.95, 123.73, 121.82, 120.01, 114.86, 114.80, 114.35, 110.26, 109.81, 61.37, 56.04, 55.93, 55.83..

(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-4-(3-methoxybenzylidene)hepta-1,6-diene-3,5-dione (36)

3-methoxybenzaldehyde (272mg, 2mmol) and 1 (368mg, 1mmol) were used as reactants and the column chromatography (petroleum ether/acetic ether 2:1) gave 285mg 36 as yellow powder, 59%. HPLC t_R = 11.6 min; R_f 0.24 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₂₉H₂₆O₇: 486.1679, found 486.1674. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (s, 1H), 7.76 (d, J = 15.4 Hz, 1H), 7.45 (d, J = 16.1 Hz, 1H), 7.24 (dd, J =8.0, 8.0 Hz, 1H), 7.16 (dd, J = 8.2, 1.9 Hz, 1H), 7.0-7.09 (m, 1H), 7.04 (d, J = 1.9 Hz, 1H), 7.02-7.03 (m, 1H), 7.02 (dd, J = 8.2, 1.9 Hz, 1H), 6.96 (d, J = 15.4 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.87-6.89 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 16.1 Hz, 1H), 5.97 (s, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.76 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.92, 187.01, 159.64, 148.86, 148.59, 147.51, 146.80, 146.77, 145.67, 141.09, 140.31, 134.86, 129.81, 127.25, 126.64, 125.28, 124.07, 123.77, 122.80, 119.74, 116.34, 115.15, 114.85, 114.83, 110.33, 109.90, 56.07, 55.96, 55.27.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-((5-methylfuran-2-yl)methylene)hepta-1,6-diene-3,5-dione (37)

5-methylfuran-2-carbaldehyde (220mg, 2mmol) and 3 (396mg, 1mmol) were used as reactants and the raw product was purified by column chromatography (petroleum ether/acetic ether 3:1) to give 276mg 37 as yellow powder, yield 57%. HPLC t_R = 16.3 min; R_f 0.32 (petroleum ether/acetic ether 1:1). HR-MS calcd for C₂₉H₂₈O₇: 488.1830, found 488.1828. ¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J = 15.4 Hz, 1H), 7.60 (s, 1H), 7.46 (d, J = 16.1 Hz, 1H), 7.17 (dd, J = 8.4, 1.9 Hz, 1H), 7.11 (dd, J = 8.4, 1.9 Hz, 1H), 7.05 (d, J = 1.9 Hz, 2H), 6.93 (d, J = 15.4 Hz, 1H), 6.90 (d, J = 16.1 Hz, 1H), 6.84 (d, J =8.4 Hz, 2H), 6.72 (d, J = 3.4 Hz, 1H), 6.08 (dq, J = 3.4, 0.9 Hz, 1H), 3.89-3.90 (m, 12H), 2.26(d, J =0.9Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 197.53, 185.99, 157.42, 151.68, 151.55, 149.31, 149.22, 148.45, 146.25, 144.64, 134.83, 127.86, 127.39, 126.67, 126.25, 123.30, 123.25, 120.24, 120.16, 111.13, 110.60, 110.21, 109.74, 56.05, 56.03, 56.00, 55.95, 13.99.

General procedures for synthesis of 18 and 29

3,4-dimethoxybenzaldehyde (831 mg, 5 mmol) or 4-hydroxy-3-methoxybenzaldehyde (760mg, 5mmol) was mixed with pentane-2,4-dione (42 mg, 1 mmol) and 25mL toluene in a three neck rounded flask equipped a water dispenser. Then pyridine (7.9 mg, 0.1 mmol, in 0.2 mL toluene) and acetic acid (9.6 mg, 0.16 mmol, in 0.2 mL toluene) were added in as catalysts. The reaction mixture was stirred in 140°C overnight and the generated water was removed by water dispenser during the whole reaction. After washed with water to remove pyridine and acetic acid, the solvent were evaporated to get raw product. Finally the raw product was purified with column chromatograph to give 18 and 29 respectively.

(1E,6E)-4-(3,4-dimethoxybenzylidene)-1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (18)

The column chromatography (petroleum ether/acetic ether 3:2) give 381mg 18 as yellow powder, yield 70%. HPLC t_R = 15.7 min; R_f 0.28 (petroleum ether/acetic ether 1:1). ¹H NMR (500 MHz, CDCl₃) δ 7.80 (s, 1H), 7.75 (d, J=15.4Hz, 1H), 7.51 (d, J=16.1Hz, 1H), 7.18 (dd, J=8.4, 2.0Hz, 1H), 7.13 (dd, J=8.4, 2.0Hz, 1H), 7.05-7.06 (m, 3H), 6.98 (d, J=2.0Hz, 1H), 6.99 (d, J=15.4Hz, 1H), 6.85 (d, J=8.4Hz, 1H), 6.81 (d, J=8.4Hz, 2H), 6.81 (d, J=16.1Hz, 1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.86 (s, 6H), 3.80 (s, 3H). ¹³C NMR (101MHz, CDCl3) δ 198.39, 186.84, 152.01, 151.66, 151.21, 149.37, 149.30, 148.95, 146.90, 144.88, 140.65, 139.00, 127.86, 127.14, 126.39, 125.61, 124.98, 123.54, 123.27, 120.29, 112.99, 111.24, 111.19, 111.16, 110.75, 110.33, 56.05, 56.01, 55.94, 55.90, 55.87.

(1E,6E)-4-(4-hydroxy-3-methoxybenzylidene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene -3,5-dione (29)

The column chromatography (petroleum ether/acetic ether 2:3) give 223mg t17 as yellow powder, yield 44%. HPLC t_R = 7.9 min; R_f 0.39 (petroleum ether/acetic ether 1:2). HR-MS calcd for C₂₉H₂₆O₈: 502.1628, found 502.1623. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (s, 1H), 7.75 (d, J = 15.4 Hz, 1H), 7.49 (d, J = 16.1 Hz, 1H), 7.16 (dd, J = 8.2, 1.7 Hz, 1H), 7.09 (dd, J = 8.3, 1.8 Hz, 1H), 7.04 (m, 2H), 7.03 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.7 Hz, 1H), 6.95 (d, J = 15.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 16.1 Hz, 1H), 5.95 (s, 1H), 5.93 (s, 1H), 5.89 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H). ¹³C NMR (101 MHz, acetone) δ 198.90, 188.17, 150.69, 150.37, 150.05, 148.84, 148.77, 148.37, 147.20, 144.77, 140.74, 140.11, 128.05, 127.48, 126.61, 126.13, 126.01, 124.64, 124.05, 120.15, 116.31, 116.25, 116.23, 114.39, 112.38, 111.75, 56.43, 56.35, 56.25.

General procedures for the synthesis of 38-40

triethoxymethane (1.48g, 10mmol) and 1 mmol corresponding symmetrical curcuminoids were dissolved in 5mL acetic anhydride. The reaction mixture was stirred in 140°C for 5h then transferred to room temperature and 10mL H₂O was added for further 10min. the raw product was extracted by ethyl acetate then purified by column chromatography or recrystallation.

4,4′-((1E,6E)-4-(hydroxymethylene)-3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-phenylen e) diacetate (38)

1 (368mg, 1mmol) was used as reactant and the raw product was purified by column chromatography (petroleum ether/acetic ether 4:1) to get 140mg 38 as yellow powder, yield 29%. HPLC t_R = 10.4 min; R_f 0.44 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₂₆H₂₄O₉: 480.1415, found 480.1409. ¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.93 (d, J = 15.5 Hz, 2H), 7.76 (d, J = 15.5 Hz, 2H), 7.27 (dd, J = 8.2, 1.6 Hz, 2H), 7.21 (d, J = 1.6 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 3.91 (s, 6H), 2.34 (s, 6H). ¹³C NMR (400 MHz, CDCl₃) δ 189.36, 186.95, 168.67, 151.55, 145.09, 142.17, 133.63, 123.44, 122.18, 120.47, 112.77, 111.94, 56.03, 20.67.

(1E,6E)-4-(hydroxymethylene)-1,7-bis(4-methoxyphenyl)hepta-1,6-diene-3,5-dione (39)

2 (336mg, 1mmol) was used as reactant and the raw product was purified by column chromatography (petroleum ether/acetic ether 9:1) to get 255mg 39 as yellow powder, yield 70%. HPLC t_R = 9.9 min; R_f 0.37 (petroleum ether/acetic ether 3:1). HR-MS calcd for C₂₂H₂₀O₅: 364.1305, found 364.1292. ¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.94 (d, J = 15.4 Hz, 2H), 7.72 (d, J = 15.4 Hz, 2H), 7.61 (d, J = 8.2 Hz, 4H), 6.94 (d, J = 8.2 Hz, 4H), 3.87 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 189.48, 187.14, 162.15, 145.44, 130.79, 127.60, 117.94, 114.54, 55.46.

(1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(hydroxymethylene)hepta-1,6-diene-3,5-dione (40)

3 (396mg, 1mmol) was used as reactant and the raw product was purified by recrystallization in acetic anhydride to get 273mg 40 as orange powder, yield 64%. HPLC t_R = 14.6 min; R_f 0.40 (petroleum ether/acetic ether 3:2). HR-MS calcd for C₂₄H₂₄O₇, 424.1517; found, 424.1518; ¹H NMR (400 MHz, CDCl₃) δ 10.37 (s, 1H), 7.93 (d, J = 15.4 Hz, 2H), 7.71 (d, J = 15.4 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 7.17 (s, 2H), 6.91 (d, J = 8.1 Hz, 2H), 3.97 (s, 6H), 3.95 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 189.39, 187.23, 151.98, 149.35, 145.80, 127.83, 124.03, 118.04, 111.12, 110.26, 56.04, 56.01.

Biology

Cell viability assay Sulforhodamine B (SRB) method: Cells were maintained in RPMI with 5% fetal bovine serum (FBS) and penicillin/streptomycin in a 37°C incubator with 5% CO₂. Cells were plated in 96-well plates and were treated with test agents in the following day and further incubated for 48 before viability assay was carried out. The SRB assay was performed to evaluate cell viability with Thermo Scientific's Multiskan MK3 plate reader to obtain the GI50 values. Alamar Blue Method: Cells were maintained in RPMI with 10% fetal bovine serum (FBS) and penicillin/streptomycin in a 37°C incubator with 5% CO₂. Cells were plated in 384-well plates and were treated with test agents in the following day and further incubated for 72 hr before viability assay was carried out. The Alamar Blue assay was performed to evaluate cell viability with PerkinElmer's EnVision multi-mode plate reader to obtain the GI₅₀ values ^42-43. The GI₅₀ is defined as the concentration of agents that decrease viability by 50% in a total cell population as compared to control cells with solvent vehicle at the end of the incubation period.

NF-κB translocation assay In brief, A549 cell were seeded in 384-well plates, incubated for 15 h, then incubated with various concentrations of compounds for 30 min. TNF-α (10ng/ml, final) was added to cells to stimulate the NF-κB cell signaling pathway for 30 min. Cells were then fixed with paraformaldehyde (2%, 100μl) and permeabilized with Triton X-100 (0.1%, 100μl). Finally, rabbit anti-p65 NF-κB antibody and goat anti-rabbit IgG with conjugated Alexa Fluor 488 were used to stain NF-κB, and Hoechst 33342 was used to stain nucleus. Fluorescence intensity of NF-κB and nuclear staining were recorded with an automated imaging system, ImageXpress5000A (Molecular Devices). The levels of NF-κB translocation were calculated and expressed as the difference between average fluorescence intensity in nucleus and in cytoplasm. After stimulating with TNF-α, the inhibitory effect of test compounds on TNFα induced NF-κB translocation was expressed as percentage of fluorescence intensity difference (in nucleus and in cytoplasm) in the control wells (TNFα only) after subtracting background (no TNFα treatment). The IC₅₀ of test compounds in this NF-κB translocation assay stands for the concentration of a compound required to induce 50% inhibition. All data are obtained as average values from triplicate samples and the experiments were repeated at least three times.

Clonogenic assay A549 cells were plated in low density in a 12-well plate and treated with test compounds the following day and every 3 days thereafter. On day 9 after colonies were formed, cells were fixed with trichloroacetic acid (TCA) (10%) for 30 min at 4°C, washed with water, stained with SRB, and then washed with acetic acid (1%). The images of the plate were scanned and colonies were counted using Image Processing and Analysis in Java (Image J, Research Service Branch, NIH).

IKK assays

Cell based IKK assay Cells were treated with various test agents for defined period of time as described in each figure and were lysed for detection of phosphorylation status of IκB, a readout for IKK activity, and total IκB protein. The NP-40 lysates buffer was used (1.0% NP-40, 10 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM NaF, 2 mM Na₃VO₄, 5 mM Na₄P₂O₇, 10 μg/mlaprotinin, 10 μg/ml leupeptin, 1 mM PMSF). For Western blotting, equal volumes of cell lysate were subject to electrophoresis on SDS-PAGE (12.5%). Proteins were then electrotransfered to a nitrocellulose membrane (GE water and Process Technologies, Trevose, PA ) as described previously ⁴⁴. Membranes were blocked in a solution of 5% non-fat dry milk in TBS-T buffer (20 mM Tris pH 7.6, 500 mM NaCl, 0.5% Tween-20) for 30 min followed by incubation with anti-pS³²-I-κB or I-κB antibodies from Cell Signaling (Beverly, MA) for at least 2 hr. The membrane was then washed and treated with the corresponding horseradish peroxidase-conjugated anti-mouse immunoglobulin [Ig] or anti-rabbit Ig as indicated. Immunodetection was performed using West Pico (Pierce, Rockford, IL) or West Dura (Pierce) followed by imaging on Kodak's Image Station 2000R (New Haven, CT).

In vitro IKK assay Activated recombinant IKKβ in MOPS buffer (8 mM MOPS-NaOH, pH 7.0, 200 μ,M EDTA, 15 mM MgCl₂) from Upstate Cell Signaling Solutions (Lake Placid, NY) was used for the in vitro kinase assay. The test compounds were incubated with IKKβ (40 ng) for 30 minutes at room temperature. The addition of Mg-ATP mix (15 mM MgCl₂, 100 μM ATP, 8 mM MOPS-NaOH, pH 7.0, 5 mM β-glycero-phosphate, 1 mM EGTA, 200 nM sodium orthovanadate, 200 nM DTT), purified GST-I-κBα, (5μg), and [γ-³²P]ATP (0.5 μCi) in a final volume of 25 μl started the reaction, which was allowed to proceed at 30°C for 30 min. Reactions were terminated by boiling the kinase reaction solution in a 6 × SDS sample buffer for 5 min. The samples were resolved on SDS-PAGE (12.5%). The gel was stained with Coomassie Blue dye (0.05%). ³²P incorporation into GST-I-κBα, was assessed by autoradiography. In addition, the radiolabeled GST-I-κBα, protein bands were excised for quantification with a scintillation counter (Beckman LS 6500, Beckman Coulter, Fullerton, CA).

Molecular modeling

Homology model building IKKβ catalytic domain sequence (code O14920) was collected from UniProt (http://www.uniprot.org/), and C terminal amino acid residues after G218, which are difficult to be modeled, were deleted since it's far away from ATP binding pocket. The sequence similarity search of IKKβ catalytic domain was performed using the BLAST protocol which is built in Discovery Studio 2.1 (Accelrys Inc.). Four templates 2JC6, 1A06, 2QNJ and 2A2A were chosen from Protein Data Bank (PDB). The C terminal amino acids after a conserved glycine was deleted from all the templates. The automated sequence alignment and analysis of template and target was carried out by Discovery Studio 2.1.

The homology modeling of IKKβ catalytic domain was carried out by using built-in Modeler package of Discovery Studio 2.1. The four different kinase templates sequences were aligned together with the IKKβ sequence and this alignment was supplied along with the 3D coordinates of templates as an input to the program. The best model given by the program was chosen, and energy minimization by using the Powell method for about 1000 iterations, with tripos force field in Sybyl 7.3.5. The structural analysis was carried out with the Ramanchandran Plot in Sybyl 7.3.5.

Small molecular preparation The 3D structure of ATP was extracted from the crystal structures of the phosphorylase kinase (PDB code 1PHK, 28.6% sequence identity with IKKβ catalytic domain). The geometries of compound 17 were energy minimized using density functional theory. These calculations were performed with the B3LYP hybrid functional and the 6-31G basis set implemented in Gaussian03 E01 software package.⁴⁵ After energy minimization, it adopts a propeller-shaped conformation.

Docking Molecular docking studies of compounds with the ATP binding pocket were performed with Surflex-Dock in Sybyl 7.3.5. Automatic was chosen as the mode of construction for the protomol, threshold is 0.5 and bloat is 0.

Abbreviations

NF-κB

nuclear factor-κB

Rel

reticuloendotheliosis

IκB

inhibitor of κB

IKK

inhibitor of κB kinase

NIK

NF-κB inducing kinase

TNF

tumor-necrosis factor

LTβ

lymphotoxin B

GST

glutathione S-transferase

TCA

trichloroacetic acid

SRB

sulforhodamine B