A New Class of Cytotoxic Agents Targets Tubulin and Disrupts Microtubule Dynamics

Abstract

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC₅₀ values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI₅₀ values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Graphical Abstract

Introduction.

Every year ~500,000 people die from cancer in the United State.[1] Despite significant advances in prevention, detection and treatment over the last few decades, the five-year survival rate of some cancers is still very low.[2] Since many cancers are only detected after metastasis has occurred systemic treatments including small molecule chemotherapeutic agents are a main component of cancer treatment.[3] However, development of multiple drug resistance and undesirable side effects arising from off-target effects limit the clinical utility of the currently available cancer drugs. Cancer cells are characterized by high rates of proliferation; therefore, drugs that target various steps in the cell cycle are common in a therapeutic setting. Antimitotics, drugs that disrupt progression through mitosis, include a diversity of molecules currently in clinical use. Classical antimitotics are compounds that bind directly to tubulin to either stabilize polymerized microtubules or inhibit tubulin polymerization.[4,5] Defects in the mitotic spindle induced by these compounds cause a prolonged mitotic arrest that triggers apoptotic cell death.[6,7] This mitotic arrest is caused by the mitotic checkpoint, a multilayer signaling pathway that delays anaphase until all chromosomes are properly attached to the spindle.[8,9] The clinical success of antimitotics like paclitaxel suggests that targeting mitosis is a viable approach to develop new anti-cancer drugs.

In this report, we describe the fortuitous discovery of a novel class of antimitotic compounds with selective toxicity towards a wide range of cancer cell lines while continuing our efforts to develop potent and selective HDAC inhibitors,[10]. This approach was motivated by the fact that clinically used HDAC inhibitors such as vorinostat (SAHA), romidepsin (FK228),[11,12] belinostat (PXD101),[13] and panobinostat [14] suffer from undesirable side effects, which can be attributed to off-target effects of the promiscuous hydroxamic acid metal-binding group shared by most of them.[15] Replacing the hydroxamic acid group with alternative metal-binding groups such as benzamides has produced promising results leading to more selective inhibitors [16,17]. The 1-(1H-imidazol-2-yl)ethan-1-one moiety was introduced as a metal-binding group for potential HDAC inhibitory effect. The zinc-ion binding ability of imidazole has been reported [18]. The ketone moiety was introduced to provide an additional coordinating site for metal chelation. Structurally, these compounds are chalcone mimics. Chalcones are privileged structure scaffolds in medicinal chemistry and it should be noted that many chalcone mimics with antimitotic activity have been reported [19–22].

Consistent with our original aim, the compounds were capable of inhibiting purified HDACs with some analogues showing selective inhibition of purified HDAC8 with IC₅₀ values in low micromolar range. However, Western blot studies revealed that the acetylation of histone H3, tubulin and structural maintenance of chromosomes 3 (SMC3) protein was unchanged in HCT116 cells treated with the compounds. Despite this limited HDACi activity in cells, these new compounds showed cell growth inhibitory activity with single digit micromolar GI₅₀ values in the National Cancer Institute (NCI) 60 human tumor cell line screen. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B, our compounds display a striking ability to destabilize the mitotic spindle. In addition, these compounds reduce the rate of tubulin polymerization both in cells and of purified tubulin. Overall, these studies suggest that although the compounds possess limited in vitro HDAC inhibitory activity, their cytotoxicity is explained by disruption of microtubule dynamics leading to a lethal mitotic arrest.

Results and Discussion.

The compounds synthesized are shown Table 1.

Table 1.

Analogues synthesized and their effect on percentage mean growth of NCI sixty human tumor cell line panel at 10 μM.

Compound	R	Ar	Mean % growth at 10 μM
IPE-1			45.33
IPE-2			70.02
IPE-3			41.34
IPE-4			66.84
IPE-5			52.06
IPE-6			76.31
IPE-7			27.48
IPE-8			21.10
IPE-9			17.88
IPE-10			64.07
IPE-11			23.48
IPE-12			51.10
IPE-13			91.77
IPE-14			77.21
IPE-15			57.67
IPE-16			15.90
IPE-17			19.33
IPE-18			19.91
IPE-19			33.11
IPE-20			54.69
IPE-21			29.20
IPE-22			−18.79
IPE-23			12.97
IPE-24			22.66
IPE-25			61.43
IPE-26			68.98
IPE-27			53.49
IPE-28			92.81
IPE-29			98.67
TPE-30			84.29
PPE-31			99.14
HPE-32			85.87
HPE-33			91.98
HPE-34			89.56
HPE-35			85.87
HPE-36			96.15

Chemistry.

A general synthetic strategy as shown in the retrosynthetic analysis in Scheme 1 was used for the synthesis of all the analogues, except IPE-27. They can be synthesized by aldol reaction of N-tosyl imidazole ketone 1 with aldehydes 2. The aldehydes 2 can be obtained by Heck coupling of bromoester 7 with styrene 8, followed by ester hydrolysis, amide formation, and oxidative cleavage of olefinic double bond of the amide 4. The aldehyde 23 can also be obtained by direct palladium-catalyzed carbonylation of the bromoester 7, followed by ester hydrolysis and amide formation. However, this reaction gave low yields.

Scheme 1:

Retrosynthesis of IPE analogues

The imidazole ketone 1 was prepared as shown in Scheme 2A. 1-H-imidazole-2-caroxaldehyde 9 was tosylated to produce aldehyde 3, which was then treated with MeMgBr, followed by Dess-Martin periodinane oxidation to afford the N-tosyl imidazole ketone 1 (Scheme 2A).

Scheme 2.

General approach for IPE synthesis.

The synthesis of the aldehydes 2 began with palladium catalyzed Heck coupling between the bromoester 7 and styrene 8 to produce the ester 11, which was saponified and subjected to EDC coupling with different amines to produce the amides 4. They were subjected to oxidative cleavage using osmium tetroxide and sodium periodate to produce aldehydes 2 (Scheme 2B).

With both aldehydes 2 and ketone 1 in hand, aldol coupling and tosyl group removal were carried out in one step by heating with piperidine to produce the final products IPE-1 -IPE-14 in 50–70% yield. Palladium-catalyzed hydrogenation of IPE-1 gave IPE-28 (Scheme 2C). Cyclopropanation of the olefinic double bond of IPE-18 afforded the cyclopropyl derivative IPE-29. The N-alkyl imidazole derivatives were made by reacting the non-alkylated imidazole analogues with the corresponding alkyl halide in the presence of potassium carbonate in DMF to produce IPE-15-26 (Scheme 2C). Some of the N-methyl imidazole analogues were prepared using N-methylimidazole ketone instead of the N-tosyl imidazole ketone 1 in the aldol reaction. N-methyl imidazole ketone was prepared as reported earlier.[23]

An alternative approach was used in the synthesis of analogue IPE-27 (Scheme 3). Commercially available 4-hydroxymethylphenylacetic acid 12 was converted to the amide 13 using DEPBT as the coupling agent. It was oxidized to the aldehyde 14 with Dess-Martin periodinane and then subjected to aldol coupling as before to obtain IPE-27.

Scheme 3.

Synthesis of IPE-27.

To determine the importance of the imidazole moiety as the ZBG, we replaced it with other isosteric aromatic and heteroaromatic rings, thiazole, phenol and pyrimidine. The thiazole and pyrimidine analogues were synthesized by aldol coupling of the aldehyde 2 with the corresponding thiazole and pyrimidine ketones as in Scheme 1. The phenol analogues were synthesized by a slightly different approach as shown in Scheme 4. Palladium-catalyzed carbonylation of ethyl 2-(4-bromophenyl)-acetate 7 using paraformaldehyde produced the aldehyde 15. It was subjected to base-initiated aldol reaction with 2-hydroxy acetophenone 16, followed by acidification, to afford a mixture of the α,β-unsaturated carboxylic acid 17a and the β-hydroxyketone 17b (Scheme 4), which were separated. Amide coupling of these acids with the respective aromatic amines 19a–c in the presence of EDC produced the amides HPE-32, HPE-35, HPE-36 and HPE-34. Palladium-catalyzed hydrogenation of HPE-32 gave HPE-33.

Scheme 4.

Synthesis of HPE-32 – HPE-36.

Biology.

Preliminary screening for antiproliferative activity of several selected analogues on the human adenocarcinoma cell line HCT 116 was carried out by the MTS reduction assay using SAHA as the positive control. The cells were treated with different concentrations of the test compounds ranging from 0.01 μM to 100 μM for 96 hours. All the compounds inhibited the growth of HCT 116 cells with GI₅₀ values in single digit micromolar range (IPE-3: 6.85 μM, IPE-7: 6.95 μM, IPE-8: 5.14 μM, IPE-16: 5.91 μM, IPE-19: 6.56 μM) (Figure 1)

Figure 1.

Growth inhibition of HCT116 colorectal carcinoma cells treated with selected representative analogues. Cells were treated for 96 h with increasing concentrations of compounds from 10 nM to 100 μM. Data points represent the average of three biological replicates with error bars indicating the SEM. Curves were fit to the log(compound) vs. response and were calculated using a variable Hill slope model.

In order to assess the potential HDAC enzyme inhibition activity, selected analogues were tested at 100 μM against purified HDAC isoforms (Figure S1). This analysis included all of class I (HDAC1, 2, 3, and 8) and the well-studied class IIb protein, HDAC6. Of the compounds tested, IPE-25 was the most potent against all isoforms, with each isoform showing less than 20% activity remaining (Figure S1). IPE-7 and IPE-8 also showed similar pan inhibition, but with less potency, displaying 20–80% activity remaining (Figure S1). Interestingly, compounds IPE-24, IPE-26, IPE-29, IPE-27, and IPE-22 inhibited HDAC8 with 20–60% activity remaining, but with little effect on the other isoforms, suggesting possible HDAC8 selectivity when tested against the purified enzymes (Figure S1). Finally, compounds IPE-26 and IPE-18 showed only modest HDAC inhibition.

Next, the dose dependence (IC₅₀) of HDAC inhibition was determined for several analogues. IPE-25 and IPE-7 were assessed against all isoforms (Table S3/Figure S3 and Table S4/Figure S4, respectively), given the potency in the preliminary screen. In addition, IPE-22, IPE-24, IPE-26, IPE-27, and IPE-29 were tested only against HDAC8 (Table S2 and Figure S2), given the potency observed at 100 μM. The FDA-approved HDAC inhibitor SAHA (Vorinostat) was also tested for comparison. IPE-25 displayed low micromolar inhibition against all isoforms, consistent with pan inhibition (Table 2). The remaining inhibitors tested against HDAC8 also showed low micromolar potency. Given the poor >200 μM potency against the other HDAC isoforms, the compounds showed a range of 13–67-fold selectivity for HDAC8 compared to the other isoforms. The most potent and HDAC8-selective compound was IPE-22, which had at least a 67-fold selectivity towards HDAC8 with an IC₅₀ of 3.0±0.2 μM (Table 2). The dose dependence data showed that most compounds displayed HDAC8-selectivity, with IPE-25 and IPE-7 distinguishing themselves as pan HDAC inhibitors in in vitro studies using purified enzymes.

Table 2.

IC₅₀ values for SAHA and synthesized compounds

Compound ID	IC50 Value (μM)*
	HDAC1	HDAC2	HDAC3	HDAC6	HDAC8
SAHA	0.033±0.001	0.096±0.01	0.020±0.001	0.033±0.003	0.54±0.01
IPE-7	1.8±0.1	2.1±0.2	1.8±0.1	2.0±0.2	1.4±0.1
IPE-8	>100	>100	N.D.	>100	N.D.
IPE-18	>100	>100	>100	>100	>100
IPE-22	>200	>200	>200	>200	3.0±0.2
IPE-24	>200	>200	>200	>200	13±1
IPE-25	12±1	8.9±0.6	16±1.0	8.6±4.9	9.0±0.5
IPE-26	>200	>100	>200	>100	15±1
IPE-27	>100	>100	>100	>100	12±1
IPE-29	>200	>200	>200	>200	8.5±0.9

^*>100 or >200 indicates greater than 50% HDAC activity was observed at 100 or 200 μM concentrations. Recombinant HDAC isoforms were pre-incubated with serial dilutions of the inhibitors before deacetylase activity was monitored by chemiluminescence signal intensity. The activity after inhibition was then plotted and fit to a sigmoidal curve to calculate the IC₅₀ values. Higher concentrations were not tested due to solubility issues. N.D.= Not Determined

HDAC Inhibition in Cells.

Most HDACs exist in multimolecular complexes in cells; effectiveness of HDACi against purified enzymes cannot always be extrapolated to behavior in cells. Therefore, colorectal cancer cells HCT116 were treated with selected analogues IPE-7, IPE-16, and IPE-19 for 24 h, followed by Western blotting to assess histone H3 acetylation and tubulin acetylation as a measure of cellular HDAC activity. Surprisingly, no increase in global histone acetylation or tubulin acetylation was observed in treated cells exposed to any of the compounds at the highest concentration of 25 μM (Figure 2a). In contrast, global acetylation of histone H3 and tubulin was dramatically increased in cells exposed to the HDAC inhibitors SAHA or MS275. This result was particularly surprising with IPE-7 which, by inhibiting HDAC1, 2 and 3 (Table 2), would be expected to increase histone H3 acetylation in cells.

Figure 2.

Western blot analysis a) Levels of acetyl-histone H3 and acetyl-tubulin were analyzed in HCT116 cells treated with IPE-7, IPE-16, and IPE-19 at 1, 10, and 25 μM. Cells were treated for 24 hrs, after which total cellular proteins were isolated and analysis was completed. No increase in global histone H3 and tubulin acetylation was observed. b) Acetylation of SMC3 was analyzed in HeLaM cells after overnight treatment with IPE-8 or PCI34051.

Several of the compounds selectively inhibited purified HDAC8 (Table S2). Some studies suggest that HDAC8 does not deacetylate histone H3 or tubulin [24] and although this might explain the reason for not observing changes in these substrates, it is also possible that the compounds have no HDAC 8 inhibitory activity in cellular environment where HDAC enzymes exist in multimolecular complexes. Next, we applied Western blotting to measure acetylation of SMC3, a protein reported to be an HDAC8 substrate.[25] We observed an increase in SMC3 acetylation in cells exposed to PCI34051, an HDAC8 selective inhibitor, but only after treatment with high concentrations (25 μM). IPE-7 and IPE-8 had no effect on SMC3 acetylation (Figure 2b; our unpublished data) suggesting that HCAC8 is not a major target in cells. Next, we generated HDAC8-null HCT116 cells using CRISPR-Cas9-mediated genome editing (Figure 3a). In cytotoxicity assays, HDAC8-null cells were slightly more resistant to IPE-7 and PCI34051 compared to control HCT116 cells (Figure 3b). On the other hand, both parental and HDAC8-null cells were killed by 10 μM IPE-7 (Figure 3b). Taken together, these results suggest that IPE-7 and related compounds have only limited activity against HDACs in cells and that additional targets must contribute to cytotoxicity.

Figure 3.

Effect of HDAC8 knockout on IPE-7 sensitivity. a) HDC8 was knocked out in HCT116 using CRISPR-Cas9, b) and c) the cells were treated with IPE-7 or PCI34051. Knocking out HDAC8 rescued IPE-7 mediated cell death.

Cell biological analysis suggested that HDACs were likely not a major target of the compounds we designed. However, these compounds were capable of killing a variety of cancer cells. Furthermore, normal RPE cells were less sensitive to killing by these compounds than cancer cells such as HelaM (Figure 4). Cell survival was expressed as a percentage of the corresponding DMSO treated cells. In the case of both IPE-7 and IPE-22, HeLaM cells had 2–3 fold lower IC₅₀ compared to RPE cells, indicating the selectivity of these analogues for transformed cancer cells. Therefore, we expanded our toxicity studies and determined detailed SAR.

Figure 4.

Cytotoxicity of IPE-7 and IPE-22 was checked in malignant transformed HeLaM (a and c) and RPE (b and d) cells. Both compounds were at-least 2-fold more cytotoxic to HeLaM cells as compared to normal RPE cells. IC50s are shown in the inset.

All the synthesized compounds were screened for cancer cell growth inhibitory activity in the NCI sixty human tumor cell line panel. The compounds were first tested at a single dose of 10 μM.[26] The mean growth percent observed at 10 μM for each analogue is shown in Table 1 and the growth percent of each cell line is shown in Figure S5. In general, most of the analogues with aromatic cap groups were found to show significant growth inhibition activity. The nature of the cap group seemed to have a profound effect on activity. Analogue IPE-1 with an unsubstituted aniline moiety as cap group showed over 50% growth inhibition, whereas the corresponding cyclohexylamine analogue (IPE-2) was almost completely inactive. Analogue IPE-6 with a bulkier naphthylamine moiety too was less active than the aniline analogue. What was most noteworthy from the SAR study was that the presence of electron donating groups such as methyl (IPE-3), methoxy (IPE-4) and N,N-dimethyl (IPE-5) at the 4- position of the aniline moiety did not have a significant effect on activity, whereas electron withdrawing and hydrophobic groups such as, fluorine (IPE-7), chlorine (IPE-8), bromine (IPE-9) and trifluoromethyl (IPE-11) at the para position were found to increase activity significantly. However, fluorine substituents at 3,4,5 positions did not have a significant effect on activity in the unsubstituted imidazole analogue IPE-12 but were found to increase activity in the corresponding N-methylimidazole analogue IPE-24. Similarly, the pentafluoro analogue IPE-13 was less active than the unsubstituted aniline analogue IPE-1, but the corresponding N-methyl analogue IPE-22 was found to cause 100% growth inhibition at 10 μM. Trifluoromethyl substituents at 3 and 5-positions of the phenyl ring, unlike in the corresponding 4-substituted analogue, had a negative effect on activity in both unsubstituted imidazole and N-methylimidazole analogues. N-alkylation of the imidazole ring had no significant effect on activity. In general, the compounds seemed to have a higher growth inhibitory effect on leukemia cell lines, and most of colonic and CNS cancers. On the other hand, they showed weak growth inhibition activity on melanoma and renal cancer cell lines. The presence of an olefinic double bond in the linker was found to be crucial for activity as saturated analogue IPE-28 and the cyclopropyl derivative IPE-29 were virtually inactive. The presence of an alkynyl substituent at 4 position of the phenyl cap group as in IPE-27 made no contribution to activity. The presence of an imidazole ring was found to be essential as replacing it with thiazole (TPE-30), pyrimidine (PPE-32) and phenol (HPE-34 – HPE-38) groups gave inactive analogues.

Selected analogues (IPE-7 – IPE-9, IPE-11, IPE-12, IPE-16, IPE-17, IPE-19, and IPE-21 – IPE-24) were further tested in the NCI dose response assay (Figure 5). Most of the compounds inhibited the growth of multiple cell lines in a single digit micromolar range GI₅₀ values. Interestingly, most of the leukemia cell lines and some of the colon cancer cell lines in general appeared to be more sensitive to these compounds. Most strikingly, analogues IPE-22 and IPE-24 inhibited the growth of all leukemia cell lines with upper nanomolar GI₅₀ values, while analogue IPE-9 inhibited the growth of all but two of the cell lines each in the leukemia and colon cancer cell line panels with sub-micromolar GI₅₀ values. As the compounds have cell growth inhibitory activity, but limited HDAC inhibitory activity, it is likely that cells are killed by binding to additional higher affinity target(s).

Figure 5.

The half maximal cell growth inhibition concentration (GI₅₀) (μM) for representative IPEs in the NCI 60 cancer cell lines.

*ND= No Data

Antimitotic Effects of IPE Compounds.

We checked effects of IPEs on asynchronously growing HeLa cells by phase contrast time lapse microscopy. Images were taken at 12 min interval. Control/ DMSO treated cells start rounding up as they enter mitosis. They then divide into two daughter cells within an hour (pointed by white arrows in top panel of Figure 6a). Strikingly, HeLaM cells exposed to IPE analogues showed a strong mitotic arrest lasting for 10–12 hours (indicated by white arrows in bottom panel in Figure 6a and supplemental movies A and B).

Figure 6.

Compounds arrest HeLaM cells in mitosis. a): asynchronous HeLaM cells were treated with DMSO or IPE, images were taken every 12 minutes on a phase contrast microscope. The arrows indicate mitotic cells. b) Mitotic duration was calculated for every treatment and is displayed in the bar graph. c) Asynchronous HeLaM cells were treated with indicated inhibitors and cells were harvested at 24h, 48h and 72h post-treatment for cell cycle analysis using flow cytometry. Graphs for Cell count vs. PI intensity for each of the treatments are displayed. After treatment with IPE-7 and IPE-8, there is clear increase in G2/M i.e. 4N peak and post G2 population (i.e. >4N) confirming mitotic arrest and mitotic exit without division into two daughter cells. Also, cell death as seen in increased subG1 population.

Mitosis is a complex process controlled by a feedback mechanism called the spindle assembly checkpoint (SAC). The SAC arrests cells in mitosis until all chromosomes are bi-oriented on metaphase plate. The molecular mechanism of the SAC involves activity of mitotic checkpoint complex (MCC) composed of BubR1, cdc20, Bub3 and Mad2. This complex prevents the action of the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that must be active for cells to exit mitosis. MCC is generated when defects occur in the attachment of chromosomes to spindle microtubules. Therefore, drugs like taxol that disrupt microtubule dynamics lead to the accumulation of MCC, inhibition of APC/C and failure to exit mitosis. Furthermore, the ensuing prolonged mitotic block that occurs upon alterations in spindle microtubule dynamics can activate apoptotic cell death. Thus, as long as the SAC is active the cell will not proceed to anaphase.[8] We observed that after a prolonged mitotic arrest induced by IPEs, the cells either underwent apoptosis or exited without cytokinesis. Previous studies have linked aberrant mitosis to inhibition of HDACs..[27–30] We checked if other commercially available pan- or specific HDAC inhibitors (i.e. inhibitors of metal binding enzymes) would cause mitotic arrest. PCI34051 and SAHA showed lengthened mitoses of 1.5 h and 3 h, respectively, but this mitotic arrest was not to the same extent as caused by IPE analogues. Other commercially available pan- or specific HDAC inhibitors did not cause such arrest (Figure 6b). We did not observe any severe effect on interphase cells by phase contrast microscopy. Next, we observed the effects of the compounds on cell cycle distribution of HeLaM cells by measuring DNA content by flow cytometry. Asynchronous HeLaM cells were treated with DMSO, SAHA, Tubastatin A or IPE analogues and then harvested for flow-cytometry analysis at 24 h, 48 h or 72 h post treatment. SAHA caused an increase in the subG1 population compared to DMSO (72 hr: 37% vs 14%, respectively), indicating induction of cell death. subG1 cells were increased even more in cells exposed to either IPE-7 or IPE-8 (72 hr: 58% and 59% respectively) (Figure 6c). Tubastatin A had little effect on subG1 under these conditions. These experiments provide additional evidence for the toxic effects of these novel compounds. Next, we excluded subG1 cells and applied modeling software to estimate cell cycle distribution of live cells. IPE-7 and IPE-8 induced accumulation of cells with a G2/M content of DNA, an effect which was most evident at 48 hours after treatment (Figure 6c). Whereas 10% of DMSO, 8.2% of SAHA, and 12% of Tubastatin-treated cells were in G2/M, we observed 36% of IPE-7 and 33% of IPE-8-treated cells in G2/M. This redistribution into G2/M by flow cytometry corroborates our earlier observations showing mitotic arrest upon IPE treatment. Interestingly post G2(polyploid) population almost doubled after treatment with IPEs. Increase in post G2 population indicates that the cells that escaped mitosis without division reentered the cell-cycle.

Compounds that block cells in mitosis often do so by triggering the spindle assembly checkpoint (SAC), a feedback mechanism that ensures that mitosis is completed only after all chromosomes have aligned on the metaphase plate. For example, taxanes directly disrupt spindle function, triggering SAC-dependent mitotic arrest. Aurora B, a part of chromosome passenger complex (CPC), is required for chromosome bi-orientation and their correct segregation. Along with Aurora B, CPC consists of INCENP, Borealin and Survivin. The CPC accumulates at the centromeres where it destabilizes erroneous kinetochore-microtubule attachments allowing accurate bipolar attachment of chromosomes to spindle. By destabilizing incorrect attachments Aurora B keeps the SAC active until all chromosomes align at metaphase plate.[9] To test whether IPEs trigger the SAC to cause mitotic arrest, we used inhibitor of Aurora B (ZM447439) kinase. ZM447439 significantly reduced the mitotic arrest caused by exposure to IPE. To confirm these results we also used an inhibitor of Mps1 reversine. Mps1 is a kinase that activates Aurora B and thus, also control the SAC.[31] Reversine also overrode the SAC suggesting that IPEs do activate the SAC (Figure 7). A known spindle toxin, paclitaxel, was used as a positive control in this experiment.

Figure 7. Compounds activate the SAC in HeLaM cells.

a) HeLaM cells were synchronized in S phase by a single thymidine block. They were released from thymidine into IPE-7 (10 μM) or DMSO or paclitaxel (1 μM). 9 hours post thymidine release cells were treated with ZM447439 or reversine for 90 minutes and fixed. At-least 200 cells per treatment were counted from chromosome spreads in blinded manner. b) Treatment with IPE-7 arrested cells in mitosis. Mitotic arrest was relieved by ZM447438 (ZM) or reversine (Rev).

Since IPE compounds triggered the SAC, we tested whether they affected the overall morphology of the mitotic spindle. Single treatment with several of the analogues at 10 μM had no major effect on spindle morphology (our unpublished data). However, when combined with ZM447439, we observed dramatic reorganization of the spindle. For these experiments, cells were treated with the compounds for 30 min followed by treatment with MG132, a proteasome inhibitor that prevents mitotic exit. Next, ZM447439 was added and immunofluorescence with antibodies to tubulin used to assess the mitotic spindle. Upon initial examination, combining the compounds with ZM447439 led to a disorganized mass of microtubules that we call “spindle collapse” (examples shown in Figure 8). To investigate this phenomenon, mitotic cell phenotypes were quantified in a blinded manner and the spindles were categorized into 4 phenotypes: normal bipolar, monopolar, multipolar and collapsed spindles. At-least 100 mitotic cells were counted for each treatment. IPEs were examined in parallel to well characterized HDAC inhibitors Tubastatin A, SAHA and the HDAC8 inhibitor PCI34051.

Figure 8.

Spindle collapse in cells exposed to analogues in combination with Aurora B inhibition. a) HeLaM cells were treated with IPE-7 or IPE-8 or IPE-22 (10μM) or DMSO, followed by treatment with 20 μM MG132 to arrest their cell cycle progression. They were then treated with 2.5 μM ZM or Hesparadine (Hesp) for 90 minutes. b) Spindle phenotypes were quantified in blinded manner. In graph 3, the Aurora B inhibitor Hesperadin was used instead of ZM447439. For ease of presentation, normal spindles are omitted from the graphs c) Representative images of normal bipolar (IPE-7, MG, DMSO) and collapsed spindles (IPE, MG, ZM). Six z-planes from two confocal scans are shown.

Upon treatment with IPE analogues, HDACi, or ZM447439 alone did not induce monopolar or multipolar spindle or spindle collapse with the majority of cells having normal bipolar spindles as in DMSO treated cells. However, co-treatment of IPE and ZM447439 caused spindle collapse (3% in DMSO+MG132+ZM447439 vs 97% in IPE-7+MG132+ZM447439). Individual microtubules disappeared and only clumps of tubulin with scattered chromosomes could be seen in the cytoplasm, suggesting that co-treatment with compound and ZM abolished spindle structure and function. Representative images of these aberrant spindles are shown in Figure 8c and Supplemental Movies C and D. This phenotype was reproduced with other analogues, viz. IPE-16 and IPE-22. Data for IPE-7 is shown in all 3 graphs in Figure 8, and for IPE-8 and IPE-22 in graphs 1 and graph 3, respectively. Surprisingly, combining either Tubastatin A, SAHA or PCI34051 with ZM447439 did not produce the same aberrant spindle as that of IPE+ZM447439 (Figure 8). Combining the compounds with Hesperidin also induced spindle collapse suggesting that the Aurora B paralogue is more important in this response (Figure 8 Graph 3).

The spindle collapse morphology could be explained by depolymerization of microtubules in the presence of both IPE and Aurora inhibitors However, this could not be observed in static images from fixed cells. To address this idea, we observed tubulin dynamics in live cells by time-lapse fluorescence microscopy. For this purpose, we generated a HeLaM cell line expressing fluorescently tagged α-tubulin. As in the previous experiment, cells were treated with DMSO or analogues, followed by MG132 (MG) in 30 min. Thirty min after MG132, ZM447439 was added and images were taken every 5 min on SP8 Leica confocal microscope. As expected, control (DMSO+MG132) or analogue+MG132 treated cells showed normal bipolar spindles throughout the duration of imaging. However, cells treated with analogue+MG132+ZM447439 showed rapid disappearance of spindle microtubules within an hour of Aurora B inhibition (Figure 9) and Supplemental Movies E,F and G), explaining the “collapse” morphology observed earlier.

Figure 9.

Co-treatment with IPE-7+ZM447439 leads to collapsed mitotic spindle: HeLaM cells were treated with IPE-7 or DMSO (30 min) followed by MG132 for 30 min. Images were captured on Leica confocal microscope immediately after addition of ZM447439

Aurora B regulates microtubule dynamic via its effects on the kinesin MCAK. Aurora B phosphorylates MCAK thereby inhibiting its depolymerizing activity.[32] However, treatment with Aurora B inhibitors alone does not cause spindle collapse (Figure 8). It is possible that regulation of MCAK by Aurora B may contribute to our phenotype, yet it is clear that a second event is required (triggered by our compounds). Also, single treatment with our compounds did not cause spindle collapse suggesting that the effects may be more subtle or related to dynamic changes in microtubules. To investigate this idea, we analyzed tubulin dynamics using a cold treatment approach. Cold treatment induced microtubule depolymerization allowing the rate of repolymerization to be observed by rewarming cells to 37 °C.[33] HeLaM cells expressing fluorescently tagged α-tubulin were treated with DMSO or IPE-7 for 30 min at 37 °C. They were then incubated at 4 °C for 30 min before rewarming. After rewarming, cells were fixed at specific time points and observed by wide-field fluorescence microscopy. In the majority of the control cells, bipolar spindle was formed after 8 minutes of rewarming (Figure 10a). To quantify this phenotype, we repeated the experiment a total of 4 times, and collected images of between 10 and 30 cells per experiment per condition (total DMSO n = 72, IPE-7 n = 73). Images were assessed in a blinded manner and scored as either normal or abnormal spindle (examples are shown in Figure 10a). No significant differences were observed at either 0 minutes or 20 minutes post rewarming. However, at 8 minutes post-rewarming, we observed significantly more abnormal spindles in IPE-7-treated samples than control (Figure 10b). As a second method of quantitation, again using blinded images at the 8 minutes time-point, we used ImageJ to trace spindle outline and quantify spindle morphology (Figure 10c). IPE-7-treated cells had spindles that occupied significantly smaller area compared to control cells. These analyses suggest that IPE-7 delays spindle assembly during recovery after cold shock (Figure 10d).

Figure 10.

Treatment with IPE-7delays spindle reassembly after recovery from cold shock: HeLaM cells expressing mcherry-α-tubulin fusion were exposed to either DMSO or IPE-7 for 30 min at 37°C. Cells were then incubated at 4 °C for 30 min followed by rewarming at 37 °C. Cells were fixed and images collected at 0, 4, 8 or 20 post-rewarming. (a) Examples of fluorescent images of control and IPE-7-treated cells at the indicated time points. (b) Visual assessment of blinded images. Spindles were scored as either normal or abnormal and percent abnormal is shown. p-value using a paired t-test is indicated. (c) Examples of spindle morphology detection using the threshold feature and object identification features of ImageJ. Examples of normal and abnormal spindles in DMSO and IPE-7-treated cells respectively are shown. (d) Area of spindles identified using ImageJ. Average and standard errors along with p-value from a student’s t-test are shown.

Spindle toxins that interfere with microtubule dynamics usually cause mitotic arrest. Based on antimitotic effects, collapsed spindle phenotype with inhibition of Aurora B and delayed tubulin polymerization after cold shock, we predicted that tubulin may be directly targeted by IPEs. Hence, we measured the effect of IPE on polymerization of purified tubulin by spectrophotometry. Light scattering was measured every 30 seconds upon warming purified tubulin to 37 °C. The Vmax slope during the exponential phase was measured in mOD/min. 10 μM Taxol, which stabilizes microtubules, was used as a positive control and showed a higher V_max than DMSO.[34] (Figure 11a and b). In contrast, IPE-7 delayed tubulin polymerization. This effect was observed in dose-dependent manner as tubulin polymerization was slowed down significantly in both 10 and 50 μM IPE-7 as compared to DMSO (Vmax 10mOD/min in DMSO vs 5.5mOD/min in 10 μM IPE-7 and 4.5 mOD/min in 50 μM IPE-7). After warming samples to 37°C for an hour, the spectrometer was set to cool to ~15°C. Spectrophotometry was continued during this cooling phase to monitor microtubule depolymerization. Depolymerization was accelerated in 50 μM IPE-7 compared to the DMSO-treated sample (Figure 11 c and d). As a second way to visualize the extent of polymerization we graphed the rate of change in absorbance (change in absorbance for each time point versus time). IPE-7 reduced the amplitude of this change in absorbance compared to DMSO, again pointing to a reduction in the rate of polymerization (Figure 11e and f). These results suggest that IPE-7 destabilizes microtubules at 37°C and accelerates depolymerization upon cooling.

Figure 11:

IPE-7 affects in-vitro tubulin polymerization: Polymerization of tubulin heterodimers, purified from porcine brain, into microtubules was measured as increase in absorbance at 340nm at 37°C. Data points are mean absorbance from 3 experiments. a) IPE-7 destabilized microtubules and b) reduced Vmax. Vmax is expressed as mOD/min in exponential phase of polymerization. c) IPEs affected tubulin dynamics in dose dependent manner. d) p values for tubulin polymerization and depolymerization. e) and f) As IPE-7 destabilized microtubules, efficiency of tubulin polymerization was reduced as seen by lower amplitude for Δabsorbance vs time.

Evidence so far suggests that our compounds affect microtubule dynamics by targeting tubulin. In order to further validate this observation, we carried out molecular docking studies of IPE-7 on the surface of β-tubulin, specifically targeting the colchicine binding site. The occupation of this site by colchicine or various other molecules gives rise to a curved αβ-tubulin dimer that inhibits formation of microtubule assembly.[35]^,[36]

Experimentally, it has been established that colchicine binds to tubulin-β at the tubulin-α interacting interface. Most crystal structures used in the studies of colchicine binding site inhibition contained two units of αβ-tubulin heterodimers with Guanosine triphosphate (GTP) bound to tubulin-α and both Guanosine diphosphate (GDP) and 2-(N-Morpholino)-ethanesulfonic acid (MES) bound to tubulin-β in addition to the occupation of an inhibitor at the colchicine binding site. The recently evaluated D64131-bound αβ-tubulin dimer crystal structure[37] was chosen as the primary system on which various other potential docking sites were also tested. The colchicine binding site (occupied by D64131) containing β-tubulin was selected as the target protein for IPE-7 docking and in addition to the sites used by GDP, MES, and D64131, two other sites predicted by the OpenEye software were used for primary docking (See Figure 12a). The IPE-7 binding scores on these sites were given in Table S5a along with the score of the native ligand D64131 (or D3L as labeled in the pdb) for comparison. IPE-7 at the colchicine binding site exhibited the best binding score with a site simply above it (Site Y) also showing a similar score. This score was in par with the native inhibitor D64131 co-crystallized with β-tubulin. The other three sites showed relatively weaker docking scores. Also, the docking scores for IPE-7 remained relatively unchanged for most β-tubulin targets from various other crystal structures at the colchicine binding site (Table S5b). A slightly reduced binding score was observed when IPE-7 was docked at the taxane binding site. However, this score was relatively higher than that estimated for Taxol when the co-crystallized ligand was occupying the binding site. The best docking poses for IPE-7 at the colchicine and taxane binding sites as well as the contacting residues of β-tubulin are also shown in Figure 12.

Figure 12.

(a) Various ligand binding sites used in the present study for IPE-7 docking on the surface of β-tubulin (represented by the red ribbon). In the selected pdb structure (pdb ID: 6k9v), two units of α– and β-tubulin were present and the neighboring α-tubulin molecules are shown in white ribbons. (b) IPE-7 docked at the colchicine binding site. (c) the colchicine binding site residues of β-tubulin that are in contact with IPE-7. (d) IPE-7 docked at the taxane binding site. (e) the taxane binding site residues that are in contact with IPE-7.

Conclusion.

We designed chalcone mimetics containing 1-(1H-imidazol-2-yl)ethan-1-one moiety as the ZBG for potential HDAC inhibition. All compounds were prepared using a convergent synthetic approach. They showed cancer cell growth inhibition with a low micromolar potency. Imidazole moiety was found to be crucial for cytotoxic activity, as replacing it with thiazole, pyrimidine, or phenolic moieties caused total loss of cytotoxic activity. The olefin moiety in the linker is also essential for the cytotoxic activity, as hydrogenation of this double bond or conversion to a cyclopropyl moiety abolished the antiproliferative activity. The double bond, although is part of an α,β-unsaturated system, the Michel acceptor properties of this moiety is likely attenuated by electron donation from the imidazole ring. This is consistent with the fact that the compounds are not general toxins as some of the analogues, which differ only in the nature of the cap group, are not active. In fact, the nature of the cap group moiety was found to have a profound effect on cancer cell growth inhibition activity. Replacing the aniline moiety of the cap group with a cyclohexyl group or a bulky naphthyl moiety reduced antiproliferative activity; on the other hand, halogen atoms or trifluoromethyl group at para-position improved in vitro performance. Some of the analogues showed very distinctive cancer cell growth inhibition selectivity. Almost all leukemia cell lines, and most of the colon cancer cell lines were found to be particularly sensitive to these compounds. On the other hand, compounds exhibited low growth inhibitory activity on melanoma, renal cancer cell lines and normal RPE cells. Although the molecules contained a 1-(1H-imidazol-2-yl)ethan-1-one moiety as a metal-binding group for HDAC inhibition, Western blot analysis showed no increased global H3, tubulin or SMC3 acetylation in treated cells. Their most striking characteristic was mitotic arrest due to activation of the SAC. Also, spindle collapse when combined with an Aurora B inhibitor suggested a potential interaction with tubulin. Indeed, IPE-7 delayed spindle assembly after rewarming cells and reduced the rate of polymerization of purified tubulin. Molecular docking studies identified an interaction of IPE-7 with tubulin. Consistent with this observation, IPE-7 reduced the polymerization of purified tubulin indicating a potential direct interaction. Interestingly, 10 μM IPE on its own does not affect the overall appearance of the mitotic spindle as assessed by immunofluorescence microscopy even though mitotic arrest is robust at this concentration. We hypothesize that subtle changes in tubulin polymerization dynamics at this concentration may preclude effective kinetochore-microtubule attachments leading to activation of the spindle assembly checkpoint. These compounds represent a new class of antimitotic compounds that impact the function of the spindle. Other groups have designed dual inhibitors of HDACs and tubulin with potential as anti-cancer agents. [38–40] Given the inhibitory activity of our compounds against purified HDACs, it is possible that with further refinement this class of molecules may yield effective dual HDAC/tubulin inhibitors, however this has not yet been achieved in our case. It is important to note that while this ZBG was chosen to target HDACs, it is possible that these compounds might bind other zinc-containing proteins as do other zinc-binding groups, including hydroxamic acid;[41] this possibility was not directly tested. Chemical biology studies to directly identify intracellular targets using molecular probes are currently underway.

Experimental.

Material and Methods.

All chemicals and solvents were purchased from commercial suppliers and used without further purification, unless stated otherwise. Anhydrous tetrahydrofuran and diethyl ether were freshly distilled from sodium and benzophenone before use. ¹H and ¹³C NMR spectra were carried out on Brucker Avance 600MHz, INOVA 600 MHz and Varian VXRS 400 MHz NMR spectrometers in deuterated solvents using residual undeuterated solvents as internal standard. High-resolution mass spectra (HRMS) were recorded on a Waters Synapt high definition mass spectrometer (HDMS) equipped with nano-ESI source. Melting points were determined using a Fisher-Johns melting point apparatus. Crude products were purified by flash chromatography on silica gel (40–63 μ) from Sorbent Technologies and on a Teledyne ISCO CombiFlash Companion chromatography system on RediSep prepacked silica cartridges. Thin layer chromatography (TLC) plates (20 cm × 20 cm) were purchased from Sorbent Technologies (catalog #4115126) and were viewed under Model UVG-54 mineral light lamp UV-254 nm. Uniplates (1000 μm) purchased from Analtech Inc. were used for preparative thin layer chromatography. A Shimadzu Prominence HPLC with an LCT20AT solvent delivery system coupled to a Shimadzu Prominence SPD 20AV Dual wavelength UV/vis absorbance Detector, a Shimadzu C18 column (1.9 m, 2.1 mm × 50 m) and HPLC grade solvents (methanol, 1% formic acid and water) and a Waters 1525 binary pump HPLC system with Waters 2487 dual wavelength absorbance detector on a symmetry C18 column (5μ, 4.6 mm × 150 m)) were used to determine the purity of compounds by HPLC. A Biotage initiator was used for all microwave reactions in recommended vials of 10 mL and 20 mL capacity. Structural integrity and purity of the test compounds were determined by a combination of ¹H and ¹³C NMR, HRMS and HPLC and the purity of all test compounds were found to be >95%.

Synthesis of 1H-imidazole ketones.

1-Tosyl-1H-imidazole-2-carbaldehyde (3).

To a stirred solution of 1H-imidazole-2-carbaldehyde 9 (1.92 g, 20 mmol, 1 equiv) in dichloromethane (40 mL) were added tosyl chloride (4.2 g, 22 mmol, 1.1 equiv), triethylamine (4.05 g, 40 mmol, 2 equiv), and DMAP (244 mg, 2 mmol, 0.1 equiv). The reaction mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in 40% ethyl acetate in hexanes to give 3 as an off-white powder (3.8 g, 76%); mp 155 °C. ¹H NMR (600 MHz, CDCl₃) δ 9.81 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.86 (s, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 1.3 Hz, 1H), 2.47 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 178.71, 146.88, 143.55, 133.68, 130.54, 130.03, 128.93, 124.65, 21.84.

1-(1-Tosyl-1H-imidazol-2-yl)ethan-1-ol (10).

To a stirred solution of 1-tosyl-1H-imidazole-2-carbaldehyde 3 (1.1 g, 4.4 mmol, 1 equiv) in tetrahydrofuran (15 mL) at −78 °C was added methyl magnesium bromide (8.8 mL of 1.0 M solution in butyl ether, 8.8 mmol, 2 equiv). The reaction mixture was stirred for 1 h at −78 °C. After adding saturated ammonium chloride (10 mL), the reaction mixture was extracted with ethyl acetate (20 mL). The organic layer was separated, and the water layer was extracted with ethyl acetate (3 × 10 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–60%) to give 10 as a yellow oil (750 mg, 65%), recovered starting material (190 mg, 18%); mp 199–200 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (d, J = 8.3 Hz, 2H), 7.31 (dd, J = 18.8, 4.9 Hz, 3H), 6.89 (d, J = 1.5 Hz, 1H), 5.20 (q, J = 6.5 Hz, 1H), 2.36 (s, 3H), 1.51 (d, J = 6.5 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 151.90, 146.40, 134.98, 130.43, 128.19, 127.38, 119.89, 63.00, 22.25, 21.76. HRMS: (ESI) calcd for C₁₂H₁₄N₂O₃S [M + H]⁺ 267.0803; found, 267.0797.

1-(1-Tosyl-1H-imidazol-2-yl)ethan-1-one (1).

To a stirred solution of 1-(1-tosyl-1H-imidazol-2-yl)ethan-1-ol 10 (535 mg, 2 mmol, 1 equiv) in dichloromethane (25 mL) at 0 °C was added Dess-Martin Periodinane (1.02 g, 2.4 mmol, 1.2 equiv). The reaction mixture was stirred for 4 h at room temperature, filtered through a pad of celite, and concentrated in vacuo. The residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (5–20%) to give 1 as an off-white powder (528 mg, 100%); mp 103–104 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (t, J = 8.1 Hz, 6H), 7.87 (d, J = 1.3 Hz, 3H), 7.36 (d, J = 8.1 Hz, 7H), 7.18 (d, J = 1.3 Hz, 3H), 2.58 (s, 9H), 2.44 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ 187.71, 146.14, 143.96, 134.25, 129.98, 129.62, 129.10, 128.43, 127.41, 124.99, 44.64, 27.23, 21.82.

Ethyl (E)-2-(4-styrylphenyl)acetate (11).

To a solution of ethyl 2-(4-bromophenyl)acetate 7 (2.4 g, 10 mmol, 1 equiv) in dimethylformamide (10 mL) in a microwave vial was added styrene 8 (1.041 g, 30 mmol, 3 equiv), palladium acetate (80 mg, 3 mole%), triphenyl phosphine (160 mg, 6 mole%), and trimethylamine (2.024 mL, 20 mmol, 2 equiv). The reaction mixture was heated in a microwave synthesizer for 6 h at 120 °C. It was allowed to cool to room temperature. The resulting suspension was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (1–3%) to afford 11 as a white powder (1.73 g, 65%); mp 72 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.55 – 7.44 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.28 (dd, J = 6.3, 4.4 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 4.22 – 4.07 (m, 1H), 3.62 (s, 1H), 1.26 (td, J = 7.1, 3.2 Hz, 1H). ¹³C NMR (151 MHz, CDCl3) δ 171.55, 137.30, 136.21, 133.52, 129.61, 128.70, 128.64, 128.26, 127.64, 126.69, 126.51, 60.95, 41.21, 14.21. HRMS: (ESI) calcd for C₁₈H₁₈O₂ [M + H]⁺ 267.1385; found, 267.1374.

(E)-2-(4-Styrylphenyl)acetic acid (5).

To a stirred solution of ethyl (E)-2-(4-styrylphenyl)acetate 11 (1.5 g, 6.25 mmol, 1 equiv) in methanol (15 mL) at 0 °C was added sodium hydroxide (375 mg, 9.4 mmole, 1.5 equiv). The reaction mixture was stirred at room temperature for 6 h, and acidified with hydrochloric acid. The solid product was collected by filtration and dried to afford 5 as a white powder (1.35 g, 91%); mp 195 °C. ¹H NMR (400 MHz, CDCl3) δ 7.49 (q, J = 8.7 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H), 7.27 (dd, J = 12.0, 6.9 Hz, 2H), 7.10 (s, 1H), 3.67 (s, 1H). ¹³C NMR (151 MHz, CDCl3) δ 137.24, 136.55, 132.59, 129.73, 128.87, 128.71, 128.13, 127.70, 126.78, 126.53, 40.51.

General procedure for the synthesis of amides (4a-n).

To a stirred solution of (E)-2-(4-styrylphenyl)acetic acid 5 (1 equiv) in dichloromethane (20 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.5 equiv), DMAP (0.1 equiv), and the corresponding amines (1.5 equiv). The reaction mixture was stirred at room temperature overnight. Water (10 mL) was added, the organic layer was separated, and the water layer was extracted with dichloromethane (3 × 10 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (10–30 %) to give amides 4a-n.

(E)-N-Phenyl-2-(4-styrylphenyl)acetamide (4a).

White powder (70%); mp 70 °C. ¹H NMR (400 MHz, CDCl3) δ 7.53 (dd, J = 15.7, 7.4 Hz, 11H), 7.46 (t, J = 8.1 Hz, 3H), 7.42 (d, J = 7.9 Hz, 4H), 7.40 – 7.27 (m, 17H), 7.19 – 7.01 (m, 14H), 3.82 – 3.63 (m, 7H). ¹³C NMR (151 MHz, CDCl3) δ 168.93, 137.56, 137.10, 136.90, 133.57, 130.01, 129.95, 129.26, 128.98, 128.89, 128.75, 127.97, 127.86, 127.85, 127.55, 127.33, 127.08, 126.58, 124.53, 119.84, 119.80, 44.64. HRMS: (ESI) calcd for C₂₂H₁₉NO [M + H]⁺ 314.1545; found, 314.1552.

(E)-N-Cyclohexyl-2-(4-styrylphenyl)acetamide (4b).

Cream solid (50%); mp. 130 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.50 (d, J = 6.6 Hz, 1H), 7.36 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 5.7 Hz, 1H), 7.11 (s, 1H), 3.74 (s, 1H), 3.54 (s, 1H), 2.18 (s, 4H), 1.82 (s, 1H), 1.60 (s, 1H), 1.31 (s, 1H), 1.09 (d, J = 11.5 Hz, 1H), 1.01 (d, J = 9.7 Hz, 1H ppm. ¹³C NMR (151 MHz, CDCl₃) δ 169.87, 137.18, 136.41, 134.43, 129.77, 128.93, 128.74, 128.03, 127.76, 127.08, 126.53, 48.23, 43.79, 32.95, 25.46, 24.74 ppm. HRMS calcd for C₂₂H₂₅NO: 320.1936, found: 320.2024

(E)-2-(4-styrylphenyl)-N-(p-tolyl)acetamide (4c).

White solid (80%); mp. 220 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.55 (dd, J = 12.0, 7.7 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 7.32 – 7.28 (m, 1H), 7.14 (d, J = 3.6 Hz, 1H), 7.09 (d, J = 7.7 Hz, 1H), 6.99 (s, 1H), 3.75 (s, 1H), 2.30 (s, 1H), 1.57 (s, 1H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 168.81, 137.12, 136.84, 135.0, 134.18, 133.70, 129.96, 129.45, 129.22, 128.75, 127.86, 127.31, 126.57, 119.91, 44.59, 20.87 ppm.

(E)-N-(4-Methoxyphenyl)-2-(4-styrylphenyl)acetamide (4d).

White powder (77%); mp 202–203 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.54 (t, J = 8.3 Hz, 4H), 7.41 – 7.28 (m, 7H), 7.13 (d, J = 1.5 Hz, 2H), 6.94 (s, 1H), 6.85 – 6.79 (m, 2H), 3.77 (s, 3H), 3.74 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 168.80, 156.57, 137.11, 136.85, 133.74, 130.64, 129.96, 129.23, 128.75, 127.88, 127.84, 127.31, 126.57, 121.79, 114.09, 55.48, 44.45. HRMS: (ESI) calcd for C₂₃H₂₁NO₂ [M + H]⁺ 344.1651; found, 344.1682.

(E)-N-(4-(Dimethylamino)phenyl)-2-(4-styrylphenyl)acetamide (4e).

Off-white powder (73%); mp 229 °C. ¹H NMR (600 MHz, CDC_l3) δ 9.81 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.86 (s, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 1.3 Hz, 1H), 2.47 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 168.72, 137.15, 136.72, 134.00, 129.97, 129.13, 128.74, 127.95, 127.80, 127.25, 126.57, 121.84, 44.44. HRMS: (ESI) calcd for C₂₄H₂₄N₂O [M + H]⁺ 357.1967; found, 357.1967.

(E)-N-(Naphthalen-1-yl)-2-(4-styrylphenyl)acetamide (4f).

Yellow powder (73%); mp 206 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.94 (d, J = 7.5 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.43 (dd, J = 13.3, 5.8 Hz, 4H), 7.36 (ddd, J = 26.6, 15.4, 5.9 Hz, 5H), 7.26 (t, J = 6.7 Hz, 1H), 7.14 (d, J = 3.2 Hz, 2H), 3.89 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 169.44, 137.15, 137.05, 134.02, 133.80, 131.93, 130.16, 129.41, 128.80, 128.78, 127.91, 127.80, 127.53, 126.83, 126.60, 126.39, 125.93, 125.82, 125.76, 120.36, 119.99, 44.67. HRMS: (ESI) calcd for C₂₆H₂₁NO [M + H]⁺ 364.1701; found, 364.1712.

(E)-N-(4-Fluorophenyl)-2-(4-styrylphenyl)acetamide (4g).

White powder (72%); mp 223 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.60 – 7.48 (m, 4H), 7.42 – 7.35 (m, 4H), 7.35 – 7.31 (m, 2H), 7.29 (d, J = 7.4 Hz, 1H), 7.13 (d, J = 2.3 Hz, 2H), 7.02 (d, J = 7.6 Hz, 1H), 6.97 (dd, J = 12.0, 5.4 Hz, 2H), 3.75 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 168.94, 159.49 (d, J = 244.0 Hz), δ 137.02 (d, J = 12.4 Hz), 136.97, 133.47 (d, J = 14.4 Hz), 129.95, 129.34, 128.77, 127.84 (d, J = 14.6 Hz), 127.37, 126.58, 121.72 (d, J = 8.2 Hz), 115.63 (d, J = 22.8 Hz), 44.46. HRMS: (ESI) calcd for C₂₂H₁₈FNO [M + H]⁺ 332.1451; found, 332.1463.

General procedure for the synthesis of amides (25h-i).

A mixture of 2-(4-styrylphenyl)acetic acid 5 (2.0 equiv) and excess oxalyl chloride was refluxed at 65 °C for 2 h. After removing oxalyl chloride on rotary evaporator, corresponding amines (2.0 equiv) and anhydrous toluene (5.0 mL) were added via syringe and the reaction mixture was stirred overnight. The reaction was monitored by TLC (50% ethyl acetate/hexanes). The reaction was quenched with water (10 mL) and extracted with DCM (3 × 20 mL). The organic extract was dried over anhydrous sodium sulfate and DCM was removed on rotary evaporator to obtain a brown solid. It was chromatographed on silica gel in 20–50% ethyl acetate /hexanes to yield amides 25h-i.

(E)-N-(4-Chlorophenyl)-2-(4-styrylphenyl)acetamide (4h).

Light brown solid (0.5 g, 44%); mp. 210 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.55 (dd, J = 17.0, 7.8 Hz, 1H), 7.38 (dd, J = 8.1, 5.1 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 7.28 – 7.23 (m, 4H), 7.14 (d, J = 5.8 Hz, 1H), 7.04 (s, 1H), 3.76 (s, 1H), 1.59 (s, 3H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 169.04, 159.42, 140.88, 136.99, 136.29, 133.44, 129.93, 129.37, 128.85, 127.85, 127.32, 126.57, 125.15, 124.08, 121.04, 118.65, 110.84, 106.54, 55.92, 44.50, 43.17, 39.97, 36.50, 35.17, 25.42, 15.52 ppm. HRMS calcd for C₂₂H₁₈ClNO: 348.1076, found: 348.1152.

(E)-N-(4-Bromophenyl)-2-(4-styrylphenyl)acetamide (4i).

White powder (283 mg, 83%); mp 220–221 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.52 (dd, J = 16.4, 7.7 Hz, 4H), 7.39 – 7.33 (m, 4H), 7.30 (dd, J = 8.5, 3.6 Hz, 4H), 7.26 (t, J = 6.8 Hz, 1H), 7.10 (d, J = 5.7 Hz, 2H), 7.03 (s, 1H), 3.72 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 168.92, 137.04, 136.64, 133.24, 131.93, 129.94, 129.39, 128.77, 127.90, 127.77, 127.39, 126.59, 121.32, 117.09, 44.59. HRMS: (ESI) calcd for C₂₂H₁₈BrNO [M + H]⁺ 392.0650; found, 392.0652.

(E)-N-(4-Pentafluorosulfaneyl)-2-(4-styrylphenyl)acetamide (4j).

White powder (283 mg, 74%); mp 212–214 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, J = 9.1 Hz, 2H), 7.55 (dd, J = 11.8, 8.7 Hz, 6H), 7.34 (ddd, J = 20.8, 13.8, 7.3 Hz, 6H), 7.13 (d, J = 3.2 Hz, 2H), 3.78 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 169.22, 140.19, 137.18, 136.99, 132.87, 129.90, 129.51, 128.78, 127.95, 127.69, 127.45, 126.97 (d, J = 4.4 Hz), 126.60, 118.90, 44.60. HRMS: (ESI) calcd for C₂₂H₁₈F₅NOS [M + H]⁺ 440.1108; found, 440.1113.

(E)-2-(4-Styrylphenyl)-N-(4-(trifluoromethyl)phenyl)acetamide (4k).

Light brown solid; mp. 235 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.60 – 7.52 (m, 1H), 7.44 (s, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.19 – 7.13 (m, 1H), 7.10 (d, J = 11.6 Hz, 1H), 3.79 (s, 1H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 169.13, 140.58, 137.14, 137.02, 133.01, 129.93, 129.48, 128.78, 127.93, 127.73, 127.44, 126.60, 126.26, 126.24, 119.30, 44.66 ppm. HRMS calcd for C₂₃H₁₉F₃NO: 382.1418, found: 382.1089.

General procedure for the oxidative cleavage using OsO₄ for the synthesis of aldehydes (2a-n).

To a solution of 4a-n (1 equiv) in dioxane:water (2:1) were added 2,6-lutidine (2 equiv), OsO₄ (2.5 wt% in 2-methyl-2-propanol, 2 mole%), and NaIO4 (4 equiv). The reaction mixture was stirred at room temperature overnight. Water and dichloromethane were added. The organic layer was separated, and the water layer was extracted with dichloromethane. The combined organic extract was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuo. The residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–50%) to produce 2a-n.

2-(4-Formylphenyl)-N-phenylacetamide (2a).

White powder (93%); mp 183–185 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.03 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 7.9 Hz, 2H), 7.11 (t, J = 7.4 Hz, 2H), 3.82 (s, 2H). ¹³C NMR (151 MHz, CDCl3) δ 191.74, 167.72, 141.25, 137.37, 135.66, 130.44, 130.17, 129.07, 124.78, 119.87, 44.79. HRMS: (ESI) calcd for C₁₅H₁₃NO₂ [M + H]⁺ 240.1025; found, 240.1031.

N-Cyclohexyl-2-(4-formylphenyl)acetamide (2b).

White solid (40%); mp. 160 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.02 (s, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.1 Hz, 1H), 5.24 (s, 1H), 3.78 (s, 1H), 3.62 (s, 2H), 1.87 (d, J = 12.4 Hz, 3H), 1.71 – 1.62 (m, 7H), 1.60 (dd, J = 13.0, 3.7 Hz, 3H), 1.37 (d, J = 3.4 Hz, 1H), 1.34 (d, J = 13.4 Hz, 2H), 1.31 (s, 1H), 1.15 (s, 1H), 1.12 (d, J = 12.6 Hz, 1H), 1.08 (s, 1H), 1.05 (d, J = 11.1 Hz, 2H), 1.03 (s, 1H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 191.82, 142.15, 130.76, 130.28, 129.97, 129.49, 77.24, 77.03, 76.82, 48.49, 44.06, 32.99, 25.43, 24.74 ppm. HRMS calcd for C₁₅H₁₅NO₂: 246.1415, found: 246.1497.

2-(4-Formylphenyl)-N-(p-tolyl)acetamide (2c).

White crystals (55%); mp. 120 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.02 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 3.62 (s, 3H), 1.87 (d, J = 8.9 Hz, 3H), 1.65 (d, J = 13.6 Hz, 8H), 1.59 (d, J = 13.2 Hz, 3H), 1.34 (d, J = 13.4 Hz, 3H), 1.12 (d, J = 12.6 Hz, 1H), 1.05 (d, J = 11.1 Hz, 2H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 191.73, 167.57, 141.37, 135.64, 134.80, 134.48, 130.42, 130.17, 129.54, 119.97, 44.76, 20.88 ppm.

2-(4-Formylphenyl)-N-(4-methoxyphenyl)acetamide (2d).

White powder (99%); mp 193 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.05 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.39 – 7.33 (m, 2H), 7.03 (s, 1H), 6.88 – 6.79 (m, 2H), 3.82 (s, 2H), 3.80 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 191.74, 167.60, 156.74, 141.43, 135.63, 130.42, 130.17, 121.88, 114.17, 55.49, 44.61. HRMS: (ESI) calcd for C₁₆H₁₅NO₃ [M + H]⁺ 270.1130; found, 270.1141.

N-(4-(Dimethylamino)phenyl)-2-(4-formylphenyl)acetamide (2e).

The product was used in the next step without further purification.

2-(4-Formylphenyl)-N-(naphthalen-1-yl)acetamide (2f).

White powder (95%); mp 296 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.09 (s, 1H), 8.01 (d, J = 7.9 Hz, 2H), 7.94 (d, J = 7.5 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.53 – 7.40 (m, 5H), 4.01 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 191.66, 168.26, 141.45, 135.82, 134.06, 131.65, 130.64, 130.33, 128.90, 126.98, 126.52, 126.24, 126.07, 125.71, 120.93, 119.95, 44.83. HRMS: (ESI) calcd for C₁₉H₁₅NO₂ [M + H]⁺ 290.1181; found, 290.1186.

N-(4-Fluorophenyl)-2-(4-formylphenyl)acetamide (2g).

Yellow powder (99%); mp 183–184 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.05 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H), 7.53 (dd, J = 16.6, 6.4 Hz, 2H), 7.47 – 7.38 (m, 2H), 7.17 (s, 1H), 7.06 – 6.98 (m, 2H), 3.83 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 191.74, 167.77, 159.59 (d, J = 244.1 Hz), 141.14, 135.68, 133.35, 133.34, 130.31 (d, J = 44.0 Hz), 121.82 (d, J = 8.0 Hz), 115.74 (d, J = 22.4 Hz), 77.25, 77.04, 76.83, 44.59. HRMS: (ESI) calcd for C₁₅H₁₂FNO₂ [M + H]⁺ 258.0930; found, 258.0934.

N-(4-Chlorophenyl)-2-(4-formylphenyl)acetamide (2h).

Cream solid (76%); mp. 195 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.01 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.02 (s, 1H), 3.79 (s, 1H), 1.53 (s, 1H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 191.67, 167.71, 140.94, 135.93, 135.74, 130.47, 130.16, 129.78, 129.09, 121.10, 44.71 ppm.

N-(4-Bromophenyl)-2-(4-formylphenyl)acetamide (2i).

Yellow powder (96 mg, 89%); mp 193–196 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.06 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.9 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H), 7.09 (s, J = 8.4 Hz, 1H), 3.83 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 191.66, 167.70, 140.90, 136.45, 135.75, 132.04, 130.47, 130.16, 121.39, 117.39, 44.73. HRMS: (ESI) calcd for C₁₅H₁₂BrNO₂ [M + H]⁺ 318.0130; found, 318.0135.

N-(4-pentafluorosulfaneyl)-2-(4-formylphenyl)acetamide (2j).

Yellow powder (121 mg, 98%); mp 191–193 °C. ¹H NMR (600 MHz, CDCl₃) δ 10.05 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.71 (t, J = 6.0 Hz, 2H), 7.59 (d, J = 8.9 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.36 (s, 1H), 3.86 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 191.67, 168.03, 140.55, 140.03, 135.81, 130.50, 130.15, 127.13 – 127.05 (m), 119.00, 44.70. HRMS: (ESI) calcd for C₁₅H₁₂F₅NO₂S [M + H]⁺ 366.0587; found, 366.0591.

2-(4-Formylphenyl)-N-(4-(trifluoromethyl)phenyl)acetamide (2k).

Cream solid (46%); mp. 170 °C. ¹H NMR (400 MHz, CDCl₃) δ 10.05 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.56 (dd, J = 15.5, 5.2 Hz, 6H), 3.86 (s, 2H), 1.56 (s, 2H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 191.62, 140.64, 130.51, 130.17, 126.37, 126.34, 119.39, 44.79 ppm. HRMS calcd for C₁₆H₁₃F₃NO₂: 308.0898, found: 308.0907.

N-(4-Ethynylphenyl)-2-(4-formylphenyl)acetamide (2o).

To a stirred solution of 2-(4-(hydroxymethyl)phenyl)acetic acid (166 mg, 1 mmol, 1 equiv.) in THF (3 mL), was added DEPBT (600 mg, 2 mmol, 2 equiv.), followed by the addition of Et₃N (0.28 mL, 2 mmol, 2 equiv.). The resulting mixture was stirred at room temperature for 15 min. 4-ethynylaniline (117 mg, 1 mmol, 1 equiv.) was added and the reaction mixture was stirred overnight at room temperature. The reaction was quenched by the addition of brine and extracted with EtOAc. The combined organic extract was dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica in EtOAc/DCM to obtain N-(4-ethynylphenyl)-2-(4-hydroxymethyl)phenyl)acetamide as a pure product (151 mg, 57%) as a white solid. ¹H NMR (600 MHz, DMSO) δ 10.32 (s, 1H), 7.64 – 7.59 (m, 2H), 7.42 – 7.39 (m, 2H), 7.29 – 7.24 (m, 4H), 5.14 (br, 1H), 4.47 (s, 2H), 4.09 (s, 1H), 3.63 (s, 2H). ¹³C NMR (151 MHz, DMSO) δ 169.94, 141.33, 140.23, 134.49, 132.84, 129.28, 126.96, 119.35, 116.52, 84.02, 80.34, 63.14, 43.51.

To a stirred solution of N-(4-ethynylphenyl)-2-(4-(hydroxymethyl)phenyl)acetamide (60 mg, 0.22 mmol, 1 equiv.) in anhydrous DCM (3 mL) was added DMP (140 mg, 0.33mmol, 1.5 equiv). The resulting mixture was stirred at room temperature for 5 h. The crude product was purified by column chromatography on silica in EtOAc/ hexanes (0 – > 50 EtOAc) to obtain the pure product (52 mg, 90 %) as a white solid. ¹H NMR (600 MHz, DMSO) δ 10.44 (s, 1H), 9.99 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 4.10 (s, 1H), 3.81 (s, 2H). ¹³C NMR (151 MHz, DMSO) δ 193.24 (s), 169.03, 143.24, 140.06, 135.28, 132.88, 130.53, 130.05, 119.43, 116.71, 83.98, 80.40, 43.76.

General procedure for the aldol reaction using piperidine for the synthesis of final products (IPE-1–IPE-14).

To a mixture of aldehyde 2 (1 equiv) and methyl ketone 1 (1.2 equiv) in methanol in a microwave vial was added piperidine (4 equiv). The reaction mixture was heated in a microwave synthesizer for 4 h at 90 °C. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel in ethyl acetate/hexanes to obtain the final products (50–75%).

(E)-2-(4-(3-(1H-Imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-phenylacetamide (IPE-1).

Off-white powder (45%); mp 211–214 °C. ¹H NMR (600 MHz, acetone-d₆) δ 9.40 (s, 1H), 8.00 – 7.92 (m, 1H), 7.89 – 7.81 (m, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 7.9 Hz, 2H), 7.47 (d, J = 9.4 Hz, 3H), 7.26 (t, J = 7.8 Hz, 3H), 7.02 (t, J = 7.3 Hz, 1H), 3.75 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 178.60, 168.43, 168.34, 146.29, 142.79, 139.47, 139.38, 138.89, 133.52, 130.99, 130.92, 129.98, 129.10, 128.65, 128.62, 126.95, 123.34, 121.53, 121.30, 119.19, 119.10, 43.68, 43.64. HRMS: (ESI) calcd for C₂₀H₁₇N₃O₂ [M + H]⁺ 332.1399; found, 332.1401.

(E)-2-(4-(3-(1H-Imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-cyclohexylacetamide (IPE-2).

Cream solid (25%); mp. 208.5 °C. ¹H NMR (600 MHz, CDCl₃) δ 11.86 (t, J = 11.0 Hz, 1H), 11.66 (d, J = 8.2 Hz, 1H), 11.39 – 11.31 (m, 2H), 7.27 (dt, J = 3.3, 1.6 Hz, 3H), 5.81 (d, J = 9.6 Hz, 1H), 5.71 (dd, J = 10.3, 3.2 Hz, 1H), 5.59 (d, J = 12.8 Hz, 1H), 5.37 – 5.12 (m, 4H) ppm. ¹³C NMR (151 MHz, CDCl₃) δ 179.30, 169.31, 146.19, 144.25, 138.09, 133.76, 129.95, 129.42, 121.21, 77.25, 77.04, 76.82, 48.37, 43.91, 32.95, 29.72, 25.44, 24.72 ppm. HRMS calcd for C₂₀H₂₃N₃O₂: 338.1868, found: 338.1881.

(E)-2-(4-(3-(1H-Imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(p-tolyl)acetamide (IPE-3).

Cream solid (22%); mp. 218.5 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.86 (q, J = 15.9 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 3.26 (s, 9H), 2.25 (s, 2H) ppm. ¹³C NMR (151 MHz, CD₃OD) δ 178.62, 170.08, 145.98, 143.91, 143.81, 143.74, 138.77, 135.81, 133.70, 133.47, 129.67, 129.45, 128.94, 128.79, 128.70, 128.51, 120.70, 120.63, 120.53, 120.08, 120.03, 119.86, 48.02, 47.88, 47.73, 47.59, 47.45, 47.31, 47.17, 43.09, 19.51, 19.47 ppm. HRMS calcd for C₂₁H₁₉N₃O₂: 346.1555, found: 346.1573.

(E)-2-(4-(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-methoxyphenyl)acetamide (IPE-4).

White powder (30%); mp 224–226 °C. ¹H NMR (400 MHz, CD₃OD) δ 7.97 – 7.82 (m, 2H), 7.73 (d, J = 8.2 Hz, 2H), 7.44 (dt, J = 5.4, 4.7 Hz, 5H), 7.29 (s, 1H), 6.91 – 6.81 (m, 2H), 3.76 (s, 3H), 3.71 (s, 2H). ¹³C NMR (151 MHz, CD₃OD) δ 178.63, 169.99, 156.62, 143.85, 138.83, 133.46, 131.33, 129.55, 128.62, 121.69, 120.60, 113.54, 54.42, 42.99. HRMS: (ESI) calcd for C₂₁H₁₉N₃O₃ [M + H]⁺ 362.1505; found, 362.1515.

(E)-2-(4-(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-(dimethylamino)phenyl)acetamid (IPE-5).

Yellow powder (30%); mp 221 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.92 (q, J = 15.9 Hz, 1H), 7.74 (dd, J = 20.6, 5.3 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.43 – 7.34 (m, 2H), 6.80 (d, J = 9.0 Hz, 1H), 3.73 (s, 2H), 2.92 (s, 3H). ¹³C NMR (151 MHz, CD₃OD) δ 178.62, 169.86, 145.96, 143.87, 138.96, 133.43, 129.53, 128.61, 121.56, 120.59, 113.27, 42.99, 40.11. HRMS: (ESI) calcd for C₂₂H₂₂N₄O₂ [M + H]⁺ 375.1821; found, 375.1811.

(E)-2-(4-(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(naphthalen-1-yl)acetamide (IPE-6).

Yellow powder (55%); mp 229–230 °C. ¹H NMR (600 MHz, DMSO-d6) δ 10.22 (s, 2H), 8.06 (d, J = 8.0 Hz, 3H), 8.00 – 7.91 (m, 6H), 7.86 (dd, J = 13.8, 9.1 Hz, 3H), 7.81 (d, J = 8.0 Hz, 4H), 7.78 (d, J = 8.1 Hz, 2H), 7.67 (t, J = 10.9 Hz, 3H), 7.60 – 7.46 (m, 15H), 7.28 (s, 2H), 3.89 (s, 4H). ¹³C NMR (151 MHz, DMSO-d6) δ 178.94, 169.89, 146.29, 143.26, 139.69, 134.16, 133.82, 133.33, 130.42, 129.21, 128.65, 128.18, 126.53, 126.38, 126.03, 125.85, 123.02, 122.23, 121.92, 43.20. HRMS: (ESI) calcd for C₂₄H₁₉N₃O₂ [M + H]⁺ 382.1556; found, 382.1568.

(E)-2-(4-(3-(1H-Imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-fluorophenyl)acetamide (IPE-7).

White powder (30%); mp 224–226 °C. ¹H NMR (400 MHz, CD₃OD) δ 7.95 – 7.83 (m, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.58 – 7.53 (m, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 7.07 – 7.00 (m, 2H), 3.72 (s, 2H). ¹³C NMR (151 MHz, CD₃OD) δ 178.62, 170.16, 159.33 (d, J = 242.0 Hz), 145.96, 143.80, 138.59, 134.63, 133.51, 129.57, 128.62, 121.73 (d, J = 7.7 Hz), 120.64, 114.88 (d, J = 22.6 Hz), 43.00. HRMS: (ESI) calcd for C₂₀H₁₆FN₃O₂ [M + H]⁺ 350.1305; found, 350.1310.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-chlorophenyl)acetamide (IPE-8).

Cream solid (51%); mp. 244 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.92 – 7.84 (m, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.57 – 7.54 (m, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.35 (s, 1H), 7.29 – 7.26 (m, 1H), 3.71 (d, J = 10.5 Hz, 1H), 3.35 – 3.32 (m, 1H), 1.32 – 1.25 (m, 2H) ppm. ¹³C NMR (151 MHz, CD₃OD) δ 143.78, 138.49, 129.59, 128.72, 128.61, 128.40, 121.13, 120.67, 43.08 ppm. HRMS calcd for C₂₀H₁₆ClN₃O₂: 366.1009, found: 366.1037.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-bromophenyl)acetamide (IPE-9).

Yellow powder (41 mg, 41%); mp 209–212 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.91 (q, J = 15.9 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.56 – 7.52 (m, 2H), 7.48 – 7.42 (m, 5H), 7.35 (s, J = 7.5 Hz, 1H), 3.75 (s, 2H). ¹³C NMR (151 MHz, CD₃OD) δ 178.61, 170.23, 145.97, 143.77, 138.46, 137.79, 133.54, 131.42, 129.59, 128.61, 121.43, 120.68, 116.19, 43.10. HRMS: (ESI) calcd for C₂₀H₁₆BrN₃O₂ [M + H]⁺ 410.0504; found, 410.0508.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-pentafluorosulfaneyl)acetamide (IPE-10).

White powder (34 mg, 30%); mp 196–197 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.92 (q, J = 15.9 Hz, 2H), 7.82 – 7.72 (m, 7H), 7.48 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 16.4 Hz, 1H), 7.32 (s, 1H), 3.80 (s, 2H). ¹³C NMR (151 MHz, CD₃OD) δ 178.62, 170.54, 146.00, 143.75, 141.76, 138.18, 133.60, 129.65, 128.62, 126.54, 120.72, 118.88, 43.10. HRMS: (ESI) calcd for C₂₀H₁₆F₅N₃O₂S [M + H]⁺ 458.0962; found, 458.0974.

(E)-2-(4-(3-(1H-Imidazole-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-(trifluoromethyl)phenyl)acetamide (IPE-11).

Cream solid (30%); mp. 240.5 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.90 (q, J = 15.9 Hz, 2H), 7.78 (d, J = 8.6 Hz, 3H), 7.74 (d, J = 8.2 Hz, 3H), 7.60 (d, J = 8.7 Hz, 3H), 7.46 (d, J = 8.1 Hz, 3H), 7.42 (s, 1H), 7.29 (s, 1H), 5.50 (s, 1H), 3.78 (s, 3H), 2.01 (s, 1H) ppm. ¹³C NMR (151 MHz, CD₃OD) δ 178.62, 178.60, 170.52, 170.50, 145.97, 143.76, 142.06, 138.29, 133.58, 130.14, 129.63, 128.62, 125.66, 125.63, 125.16, 120.70, 119.37, 43.14 ppm. HRMS calcd for C₂₁H₁₇F₃N₃O₂: 400.1273, found: 413.2071.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(3,4,5-trifluorophenyl)acetamide (IPE-12).

Brown solid (22 mg, 21.7%). TLC R_f = 0.1 (50% ethyl acetate/hexanes), mp 255 °C. ¹H NMR (600 MHz, MeOD) δ 7.92 (q, J = 15.9 Hz, 3H), 7.75 (d, J = 8.2 Hz, 2H), 7.43 (ddd, J = 13.4, 8.5, 5.5 Hz, 5H), 7.31 (s, 1H), 3.75 (s, 2H). ¹³C NMR (151 MHz, MeOD) δ 178.60 (s), 170.35 (s), 151.70 – 151.55 (m), 150.07 – 149.92 (m), 145.97 (s), 143.71 (s), 138.06 (s), 133.63 (s), 130.14 (s), 129.61 (s), 128.62 (s), 121.40 (s), 120.74 (s), 103.64 (dd, J = 20.4, 5.6 Hz). HRMS calcd for C₂₀H₁₄F₃N₃O₂ [M + H]⁺ 386.1116, found: 386.1120.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(perfluorophenyl)acetamide (IPE-13).

Brown solid (16 mg, 13.07%). TLC R_f = 0.5 (50% ethyl acetate/hexanes), mp 235 °C. ¹H NMR (600 MHz, CD₃OD) δ 7.90 (q, J = 15.9 Hz, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H), 7.29 (s, 1H), 3.83 (s, 2H). ¹³C NMR (151 MHz, MeOD) δ 178.61 (s), 170.87 – 170.85 (m), 146.01 – 145.94 (m), 137.75 (s), 133.73 (s), 130.59 (s), 130.23 – 130.02 (m), 129.59 (s), 128.66 (s), 128.51 (s), 120.82 (s), 41.71 (s). HRMS calcd for C₂₀H₁₂F₅N₃O₂ [M + H]⁺ 422.0927 found: 422.0913.

(E)-2-(-4(3-(1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(bis(3,5-trifluoromethyl)phenyl)acetamide (IPE-14).

Brown solid (16 mg, 13.07%). TLC R_f = 0.5 (50% ethyl acetate/hexanes), mp 235 °C. ¹H NMR (600 MHz, CD₃OD) δ 8.19 (s,2H), 7.86 (q, J = 15.9 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.61 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.38 (s, 1H), 7.25 (s, 1H), 3.76 (s, 2H). ¹³C NMR (151 MHz, MeOD) δ 178.61 (s), 170.74 (s), 145.97 (s), 143.73 (s), 140.68 (s), 137.89 (s), 131.86 (q, J = 33.3 Hz), 130.12 (s), 129.67 (s), 128.65 (s), 124.20 (s), 122.39 (s), 121.40 (s), 120.77 (s), 119.02 (s), 116.32 (s), 43.10 (s). HRMS calcd for C₂₂H₁₅F₆N₃O₂ [M + H]⁺ 468.1146 found: 468.1164.

General procedure for the synthesis of the alkylated imidazole (IPE-15–IPE-26).

To a stirred solution of unalkylated imidazole based compounds (1 equiv) in dimethylformamide (0.5 mL) were added alkyl halide (1.2 equiv), and sodium carbonate (18 mg, 0.12 mmol, 2 equiv). The reaction mixture was stirred overnight at room temperature. Water (1 mL) and dichloromethane (2 mL) were added. The organic layer was separated, and the water layer was extracted with dichloromethane (3 × 2 mL). The combined organic extract was washed with brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuo. The residue was purified by column chromatography on silica gel in methanol/dichloromethane (0–2%), followed by reversed phase chromatography on C18 in methanol/water (20–60%) to produce the alkylated final compounds.

(E)-N-Cyclohexyl-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-15).

White crystals (6.0 mg, 38%), mp. 182.5 °C, ¹H NMR (600 MHz, CD₃OD) δ 8.13 (d, J = 6.7 Hz, 1H), 7.98 (d, J = 15.9 Hz, 1H), 7.79 (d, J = 15.9 Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.49 (s, 1H), 7.42 – 7.35 (m, 3H), 7.21 (s, 1H), 7.09 (s, 1H), 4.09 (s, 3H), 3.69 – 3.58 (m, 1H), 3.52 (s, 2H), 1.91 – 1.59 (m, 7H), 1.42 – 1.28 (m, 3H), 1.27 – 1.14 (m, 4H) ppm. ¹³C NMR (151 MHz, CD₃OD) δ 179.85, 171.01, 143.20, 138.98, 133.43, 129.33, 128.47, 127.91, 121.87, 48.54, 42.34, 35.33, 32.32, 25.22, 24.71 ppm. HRMS calcd for C₂₁H₂₅N₃O₂: 352.2025, found: 352.2038

(E)-2-(4-(3-(1-Methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-phenylacetamide (IPE-16).

The starting material were IPE-1 and methyl iodide. A white powder was produced (6 mg, 60%); mp 184–186 °C. ¹H NMR (400 MHz, acetone-d₆) δ 9.40 (s, 1H), 8.11 (d, J = 16.1 Hz, 1H), 7.81 – 7.68 (m, 3H), 7.66 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 7.9 Hz, 2H), 7.16 (s, 1H), 7.04 (t, J = 7.4 Hz, 1H), 4.08 (s, 3H), 3.77 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 179.74, 168.44, 143.95, 141.85, 139.47, 138.68, 133.63, 129.95, 128.97, 128.64, 128.52, 127.90, 123.34, 122.84, 119.19, 43.68, 35.45. HRMS: (ESI) calcd for C₂₁H₁₉N₃O₂ [M + H]⁺ 346.1556; found, 346.1554.

(E)-N-(4-chlorophenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-17).

Cream solid (10 mg, 12%); mp. 250 °C. ¹H NMR (600 MHz, CD₃OD) δ 8.01 – 7.78 (m, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.59 – 7.55 (m, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.40 (s, 1H), 7.31 – 7.28 (m, 1H), 7.20 (s, 1H), 4.08 (s, 1H), 3.72 (d, J = 9.6 Hz, 1H) ppm. ¹³C NMR (151 MHz, CD₃OD) δ 179.83, 170.25, 143.13, 143.05, 138.29, 137.31, 133.62, 129.55, 128.71, 128.55, 128.40, 128.35, 127.93, 121.99, 121.12, 43.07, 35.33 ppm. HRMS calcd for C₂₁H₁₈ClN₃O₂ [M + H]⁺ 380.1165, found: 380.1170.

(E)-N-(4-Bromophenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-18).

The starting material were IPE-9 and methyl iodide. A white powder was produced (7 mg, 60%); mp 221–223 °C. ¹H NMR (600 MHz, acetone-d₆) δ 9.53 (s, 1H), 8.14 (d, J = 16.2 Hz, 1H), 7.79 (dd, J = 16.3, 12.1 Hz, 3H), 7.67 (d, J = 16.1 Hz, 2H), 7.50 – 7.43 (dd, J = 12.1, 12.5 Hz, 4H), 7.39 (s, 1H), 7.18 (s, 1H), 4.11 (s, 3H), 3.78 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 179.72, 168.65, 143.95, 141.79, 138.77, 138.36, 133.70, 131.58, 129.97, 128.98, 128.54, 127.91, 122.90, 121.05, 115.21, 43.61, 35.44. ¹³C NMR (151 MHz, acetone-d₆) δ 179.72, 168.65, 143.95, 141.79, 138.77, 138.36, 133.70, 131.58, 129.97, 128.98, 128.54, 127.91, 122.90, 121.05, 115.21, 43.61, 35.44. HRMS: (ESI) calcd for C₂₁H₁₈BrN₃O₂ [M + H]⁺ 424.0661; found, 424.0668.

(E)-N-(4-Fluorophenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-19).

The starting material were IPE-7 and methyl iodide. A white powder was produced (7.5 mg, 33%); mp 213 °C. ¹H NMR (600 MHz, acetone-d₆) δ 9.45 (s, 1H), 8.12 (d, J = 16.1 Hz, 1H), 7.77 (dd, J = 16.4, 12.1 Hz, 3H), 7.72 – 7.65 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.42 (s, 1H), 7.18 (d, J = 0.8 Hz, 1H), 7.11 – 7.04 (m, 2H), 4.10 (s, 3H), 3.77 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 179.73, 168.37, 158.70 (d, J = 240.5 Hz),143.96, 141.80, 138.55, 135.76, 133.66, 129.95, 128.98, 128.52, 127.90, 122.87, 120.96 (d, J = 7.7 Hz), 115.06 (d, J = 22.8 Hz), 43.55, 35.44. HRMS: (ESI) calcd for C21H18FN3O2 [M + H]⁺ 364.1461; found, 364.1468.

(E)-2-(4-(3-(1-Benzyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-fluorophenyl)acetamide (IPE-20).

The starting material were IPE-7 and benzyl bromide. A white powder was produced (5.5 mg, 67%); mp 200–202 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.14 (d, J = 16.0 Hz, 1H), 7.83 (d, J = 15.9 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.44 – 7.35 (m, 6H), 7.33 (t, J = 7.2 Hz, 1H), 7.26 (d, J = 7.4 Hz, 2H), 7.17 (s, 1H), 7.08 (s, 1H), 7.01 (t, J = 8.6 Hz, 2H), 5.78 (s, 2H), 3.78 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 180.29, 168.38, 160.33, 158.72, 143.55, 142.62, 136.62, 136.47, 134.48, 133.46, 130.05, 129.85, 129.53, 128.93, 128.13, 127.60, 126.45, 123.34, 121.79, 121.73, 115.73, 115.59, 51.98, 44.55. HRMS: (ESI) calcd for C₂₇H₂₂FN₃O₂ [M + H]⁺ 440.1774; found, 440.1771.

(E)-2-(4-(3-(1-Ethyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-fluorophenyl)acetamide (IPE-21).

The starting material were IPE-7 and the alkyl halide is ethyl iodide. A white powder was produced (6.5 mg, 63%); mp 219–220 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 16.0 Hz, 1H), 7.80 (d, J = 16.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.43 – 7.34 (m, 4H), 7.24 (s, 1H), 7.17 (s, 1H), 7.07 (s, 1H), 6.98 (t, J = 8.6 Hz, 2H), 4.55 (q, J = 7.2 Hz, 2H), 3.76 (s, 2H), 1.48 (t, J = 7.2 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 180.11, 168.39, 159.53 (d, J = 244.2 Hz), 143.40, 142.29, 136.53, 134.56, 133.45, 130.04, 129.64, 129.52, 125.73, 123.50, 121.76 (d, J = 8.1 Hz), 115.66 (d, J = 22.6 Hz), 44.56, 44.06, 29.72, 16.55. HRMS: (ESI) calcd for C₂₂H₂₀FN₃O₂ [M + H]⁺ 379.1618; found, 379.1618.

(E)-2-(4-(3-(1-Methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-(pentafluoro- λ⁶-sulfanyl)phenyl)acetamide (IPE-22).

The starting material were IPE-10 and methyl iodide. A white powder was produced (5 mg, 56%); mp 215–216 °C. ¹H NMR (600 MHz, acetone-d₆) δ 9.81 (s, 1H), 8.09 (d, J = 16.1 Hz, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.79 (d, J = 9.2 Hz, 2H), 7.73 (dd, J = 12.2, 8.3 Hz, 3H), 7.46 (d, J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.14 (s, 1H), 4.06 (s, 3H), 3.81 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 179.71, 169.22, 143.95, 142.51, 141.74, 138.00, 133.79, 130.02, 128.99, 128.56, 127.92, 126.81, 122.96, 118.70, 43.58, 35.44. HRMS: (ESI) calcd for C₂₁H₁₈F₅N₃O₂S [M + H]⁺ 472.1118; found, 472.1119.

(E)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(4-(trifluoromethyl)phenyl)acetamide (IPE-23).

Brown solid (27 mg, 27%). TLC R_f = 0.2 (50% ethyl acetate/hexanes), mp 218 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.08 (d, J = 16.0 Hz, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.57 (dd, J = 24.3, 8.3 Hz, 5H), 7.41 (br, J = 12.3 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.24 (s, 1H), 7.12 (s, 1H), 4.12 (s, 3H), 3.79 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 180.30 (s), 168.60 (s), 143.97 (s), 142.33 (s), 140.51 (s), 136.14 (s), 134.64 (s), 130.02 (s), 129.57 (s), 129.43 (s), 127.45 (s), 126.27 (q, J = 3.8 Hz), 123.36 (s), 119.34 (s), 44.73 (s), 36.44 (s).). HRMS calcd for C₂₂H₁₈F₃N₃O₂ [M + H]⁺ 414.1429, found: 414.1441.

(E)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(3,4,5-trifluorophenyl)acetamide (IPE-24).

Brown solid (49 mg, 23.4%). TLC R_f = 0.1 (50% ethyl acetate/hexanes), mp 222 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.09 (d, J = 16.0 Hz, 1H), 7.82 (d, J = 16.0 Hz, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.25 (s, 1H), 7.18 (dt, J = 10.5, 5.3 Hz, 2H), 7.12 (s, 2H), 4.12 (s, 3H), 3.77 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 180.19 (s), 168.56 (s), 151.98 – 151.79 (m), 150.32 – 150.15 (m), 142.39 (s), 137.58 – 137.19 (m), 135.94 (s), 134.69 (s), 133.20 – 132.93 (m), 129.99 (s), 129.59 (s), 127.43 (s), 123.38 (s), 104.19 (dd, J = 20.1, 6.0 Hz), 44.55 (s), 36.48 (s). HRMS (ESI) calcd for C₂₁H₁₆F₃N₃O₂ [M + H]⁺ 400,1272, found: 400.1273.

(E)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-(perfluorophenyl)acetamide (IPE-25).

Brown solid (30 mg, 13.1%). TLC R_f = 0.7 (50% ethyl acetate/hexanes), mp 248 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.07 (d, 1H), 7.79 (d, J = 13.4 Hz, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 13.5 Hz, 2H), 7.22 (s, 1H), 7.11 (s, 1H), 6.87 (br, 1H), 4.11 (s, 3H), 3.86 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 180.27 (s), 168.84 (s), 143.94 (s), 142.33 (s), 141.28 – 141.05 (m), 139.62 – 139.26 (m), 138.76 – 138.40 (m), 137.09 – 136.77 (m), 129.95 (s), 129.58 (s), 129.40 (s), 127.47 (s), 123.32 (s), 43.28 (s), 36.45 (s). HRMS (ESI) calcd for C₂₁H₁₄F₅N₃O₂ [M + H]⁺ 436.1084, found: 436.1093.

(E)-N-(3,5-bis(trifluoromethyl)phenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-26).

Brown solid (30 mg, 30%). TLC R_f = 0.7 (50% ethyl acetate/hexanes), mp 215 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.11 (d, J = 16.0 Hz, 1H), 7.99 (s, 2H), 7.82 (d, J = 16.0 Hz, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.59 (s, 1H), 7.56 (br, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 7.13 (s, 1H), 4.13 (s, 3H), 3.82 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 168.89 (s), 138.99 (s), 135.85 (s), 134.69 (s), 132.35 (q, J = 33.5 Hz), 130.02 (s), 129.67 (s), 127.44 (s), 127.44 (s), 123.91 (s), 123.33 (s), 122.10 (s), 119.49 (s), 117.79 (s), 44.55 (s), 36.52 (s). HRMS (ESI) calcd for C₂₃H₁₇F₆N₃O₂ [M + H]⁺ 482,1303, found: 482.1321.

(E)-N-(4-Ethynylphenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (IPE-27).

White solid (59 mg, 23%), m.p 242 °C ¹H NMR (600 MHz, DMSO) δ 10.39 (s, 1H), 8.02 (d, J = 16.1 Hz, 1H), 7.74 (m, , 3H), 7.63 (d, J = 8.7 Hz, 2H), 7.58 (s, 1H), 7.44 – 7.40 (m, 4H), 7.21 (d, J = 0.7 Hz, 1H), 4.10 (s, 1H), 4.02 (s, 3H), 3.73 (s, 2H), ¹³C NMR (151 MHz, DMSO) δ 179.85, 169.41, 143.75, 142.51, 140.14, 139.00, 133.42, 132.87, 130.40, 129.50, 129.13, 129.09, 123.02, 119.39, 116.63, 83.99, 80.39, 43.65, 36.32. HRMS (ESI) calcd for C₂₃H₁₉N₃O₂ [M + H]⁺ 370.1556, found: 370.1563.

2-(4-(3-(1H-imidazol-2-yl)-3-oxopropyl)phenyl)-N-phenylacetamide (IPE-28).

A mixture of (E)-2-(4-(3-(1H-Imidazol-2-yl)-3-oxoprop-1-en-1-yl)phenyl)-N-phenylacetamide IPE-1 (7 mg, 0.021 mmol, 1 equiv) and 5% Pd/C (10 wt. %) in MeOH (1 mL) was stirred under hydrogen at 3 atm for 5 h. After disappearance of starting material (TLC), the reaction mixture was filtered through a pad of celite, concentrated in vacuo, and the residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–50%) to give IPE-28 as a white powder (5.5 mg, 79%); mp 193–195 °C. ¹H NMR (400 MHz, acetone-_d6) δ 9.28 (s, 1H), 7.64 (d, J = 7.6 Hz, 2H), 7.40 (s, 1H), 7.26 (dt, J = 17.9, 6.5 Hz, 6H), 7.18 (s, 1H), 7.03 (t, J = 7.4 Hz, 1H), 3.64 (s, 2H), 3.36 (dd, J = 9.4, 5.9 Hz, 2H), 3.00 (t, J = 7.6 Hz, 2H). ¹³C NMR (151 MHz, acetone-_d6) δ 190.04, 168.99, 145.34, 139.86, 139.56, 133.59, 130.60, 129.27, 129.16, 128.60, 128.39, 126.76, 123.21, 120.89, 120.74, 119.11, 43.51, 39.02. HRMS: (ESI) calcd for C₂₀H₁₉N₃O₂ [M + H]⁺ 334.1555; found, 334.1559.

N-(4-bromophenyl)-2-(4-(2-(1-methyl-1H-imidazole-2-carbonyl)cyclopropyl)phenyl)acetamide (IPE-29).

A mixture of (Z)-3-(4-(2-(4-bromophenyl)-2-oxoethyl)phenyl)-1-(1-methyl-1H-imidazol-2-yl)prop-2-en-1-one (43 mg , 0.0912 mmol) and palladium acetate (2 mg, 0.045 mmol, 10 mol%) were dissolved in anhydrous diethyl ether (1.5 mL) at −78 °C. A saturated solution of diazomethane in anhydrous ether (5 mL) was added dropwise. The reaction mixture was allowed to slowly warm up to room temperature and stirred at this temperature for 12 h. Water (1 mL) was added and the mixture was extracted with EtOAc (3 × 2 mL). The combined organic extract was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–60%) to obtain IPE-29 (7 mg, 16%) as a dark brown semi solid: TLC Rf = 0.6 (80% EtOAc-hexanes); ¹H NMR (600 MHz, acetone-_d6) δ 9.46 (s, 1H), 7.66 (m, 2H), 7.47 (m, 2H), 7.37 (s, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.22 (m, 2H), 7.09 (d, J = 0.8 Hz, 1H), 4.02 (s, 3H), 3.68 (s, 2H), 3.61 (ddd, J = 8.3, 5.4, 4.1 Hz, 1H), 2.54 (ddd, J = 9.1, 6.5, 4.1 Hz, 1H), 2.10 (s, 1H), 1.71 (ddd, J = 9.2, 5.4, 3.9 Hz, 1H), 1.52 (ddd, J = 8.3, 6.5, 3.9 Hz, 1H). ¹³C NMR (151 MHz, acetone-d₆) δ 189.28, 169.12, 143.44, 139.41, 138.84, 133.81, 131.53, 129.27, 129.00, 127.64, 126.14, 121.02, 115.09, 76.33, 55.04, 43.43, 35.27, 18.29; HRMS: (ESI) calcd for C₂₂H₂₀ Br N₃O₂ [M + H]⁺ 437.0701; found, 438.0817.

(E)-2-(4-(3-Oxo-3-(thiazol-2-yl)prop-1-en-1-yl)phenyl)-N-phenylacetamide (TPE-30).

To a mixture of 2-(4-formylphenyl)-N-phenylacetamide 2a (30 mg, 0.125 mmol, 1 equiv) and 1-(thiazol-2-yl)ethan-1-one 15a (19 mg, 0.15 mmol, 1.2 equiv) in methanol (4 mL) in a microwave vial was added piperidine (33 mg, 0.375 mmol, 3 equiv). The reaction mixture was heated in a microwave synthesizer for 6 h at 90 °C. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–60%) to give TPE-30 as a yellow powder (23 mg, 55%); mp 185–187 °C. ¹H NMR (400 MHz, CD₃OD) δ 8.12 (d, J = 3.0 Hz, 6H), 8.01 – 7.97 (m, 19H), 7.74 (d, J = 8.2 Hz, 14H), 7.58 – 7.53 (m, 15H), 7.47 (d, J = 8.2 Hz, 14H), 7.30 (t, J = 8.0 Hz, 17H), 7.09 (t, J = 7.4 Hz, 8H), 3.74 (s, 13H). ¹³C NMR (151 MHz, CD₃OD) δ 181.50, 170.17, 168.30, 145.20, 144.65, 139.03, 138.41, 133.37, 129.64, 128.79, 128.43, 126.93, 123.96, 120.00, 119.87, 43.12. HRMS: (ESI) calcd for C₂₀H₁₆N₂O₂S [M + H]⁺ 349.1011; found, 349.1027.

(E)-2-(4-(3-Oxo-3-(pyrimidin-2-yl)prop-1-en-1-yl)phenyl)-N-phenylacetamide (TPE-31).

To a mixture of 2-(4-formylphenyl)-N-phenylacetamide 2a (30 mg, 0.125 mmol, 1 equiv) and 1-(pyrimidin-2-yl)ethan-1-one 15b (18 mg, 0.15 mmol, 1.2 equiv) in methanol (4 mL) in a microwave vial was added piperidine (33 mg, 0.375 mmol, 3 equiv). The reaction mixture was heated in a microwave synthesizer for 3 h at 85 °C. The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel in ethyl acetate/hexanes (30–80%) to give TPE-31 as a yellow oily (24 mg, 45%). ¹H NMR (600 MHz, acetone-d₆) δ 9.40 (s, 1H), 9.04 (d, J = 4.8 Hz, 2H), 8.06 (d, J = 16.1 Hz, 1H), 7.86 (d, J = 16.1 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.71 (t, J = 4.8 Hz, 1H), 7.67 (d, J = 7.7 Hz, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.30 (t, J = 8.0 Hz, 2H), 7.06 (t, J = 7.4 Hz, 1H), 3.79 (s, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 187.31, 168.36, 161.54, 157.76, 144.70, 139.47, 139.19, 133.44, 130.02, 128.74, 128.64, 123.34, 123.05, 121.94, 119.18, 43.68. HRMS: (ESI) calcd for C₂₁H₁₇N₃O₂ [M + H]⁺ 344.1399; found, 344.1404.

(E)-2-(4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)-N-phenylacetamide (HPE-32), and 2-(4-(1-Hydroxy-3-(2-hydroxyphenyl)-3-oxopropyl)phenyl)-N-phenylacetamide (HPE-34).

To a stirred solution of a mixture of (E)-2-(4-(3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)acetic acid 17a and 2-(4-(1-hydroxy-3-(2-hydroxyphenyl)-3-oxopropyl)phenyl)acetic acid 17b (4:1) (65 mg, 0.17 mmol, 1 equiv) in dichloromethane (2 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (51 mg, 0.34 mmole, 2 equiv), DMAP (4 mg, 0.034 mmol, 0.2 equiv), and aniline 19a (34 mg, 0.34 mmole, 2 equiv). The reaction mixture was stirred at room temperature overnight. Water (3 mL) was added, the organic layer was separated, and the water layer was extracted with dichloromethane (3 × 5 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/dichloromethane (0.5–4%) to give HPE-32 as a yellow powder (32 mg, 70%); mp 233–235 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.91 (dd, J = 8.5, 7.1 Hz, 2H), 7.67 (t, J = 12.3 Hz, 3H), 7.53 – 7.47 (m, 1H), 7.42 (dd, J = 7.4, 6.3 Hz, 4H), 7.28 (dd, J = 16.5, 8.3 Hz, 2H), 7.09 (dd, J = 14.4, 6.9 Hz, 2H), 7.03 (dd, J = 8.4, 0.8 Hz, 1H), 6.97 – 6.91 (m, 1H), 3.77 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 193.63, 168.21, 163.64, 144.64, 137.46, 137.34, 136.55, 134.04, 130.22, 129.66, 129.39, 129.04, 124.68, 120.46, 119.98, 119.83, 118.93, 118.71, 44.68. HRMS: (ESI) calcd for C₂₃H₁₉NO₃ [M + H]⁺ 358.1143; found, 358.1441. And HPE-34 as a white powder (10 mg, 77%); mp 211–212 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.99 – 7.94 (m, 1H), 7.58 – 7.52 (m, 3H), 7.46 (t, J = 7.6 Hz, 4H), 7.32 (t, J = 7.9 Hz, 2H), 7.12 (ddd, J = 17.3, 16.2, 9.9 Hz, 4H), 5.54 (dd, J = 13.3, 2.7 Hz, 1H), 3.80 (d, J = 8.2 Hz, 2H), 3.11 (dd, J = 16.8, 13.3 Hz, 1H), 2.94 (dd, J = 16.8, 2.9 Hz, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 191.83, 168.64, 161.45, 138.21, 137.52, 136.33, 135.06, 130.01, 129.02, 127.11, 127.00, 124.61, 121.78, 120.93, 119.89, 118.14, 79.22, 44.63, 44.51. HRMS: (ESI) calcd for C₂₃H₂₁NO₄ [M + H]⁺ 376.1549; found, 376.1553.

4-(3-(2-Hydroxyphenyl)-3-oxopropyl)phenyl)-N-(naphthalen-1-yl)acetamide (HPE-33).

A mixture of HPE-32 (5.4 mg, 0.015 mmol, 1 equiv) was dissolved in MeOH (1 mL) and Pd/C (10 wt. %) was stirred under hydrogen at 3 atm for 4 h. After disappearance of starting material (TLC), the reaction mixture was filtered through a pad of celite, concentrated in vacuo, and the residue was purified by column chromatography on silica gel in dichloromethane to give HPE-33 as a white powder (4.5 mg, 85%); mp 186–187 °C. ¹H NMR (600 MHz, CDCl₃) δ 12.30 (s, 1H), 7.78 (dd, J = 8.0, 1.5 Hz, 1H), 7.49 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.43 (d, J = 7.7 Hz, 2H), 7.34 – 7.29 (m, 6H), 7.11 (t, J = 7.4 Hz, 1H), 7.05 (s, 1H), 7.01 (dd, J = 8.4, 0.9 Hz, 1H), 6.91 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 3.74 (s, 2H), 3.38 (t, J = 7.6 Hz, 2H), 3.12 (t, J = 7.6 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 205.18, 169.06, 162.47, 140.29, 137.57, 136.46, 132.36, 129.80, 129.35, 128.97, 124.50, 119.81, 119.26, 118.99, 118.63, 44.49, 39.82, 29.58. HRMS: (ESI) calcd for C₂₃H₂₁NO₃ [M + H]⁺ 382.1419; found, 382.1422.

(E)-N-(4-(Dimethylamino)phenyl)-2-(4-(3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)acetamide (HPE-35).

To a stirred solution of (E)-2-(4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)acetic acid 17a (36 mg, 0.13 mmol, 1 equiv) in dichloromethane (2 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (51 mg, 0.26 mmole, 2 equiv), DMAP (3 mg, 0.026 mmol, 0.2 equiv), and N₁,N₁-dimethylbenzene-1,4-diamine 19b (35 mg, 0.26 mmole, 2 equiv). The reaction mixture was stirred at room temperature overnight. Water (3 mL) was added, the organic layer was separated, and the water layer was extracted with dichloromethane (3 × 5 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/ dichloromethane (0.5–10%) to give HPE-35 as a yellow powder (38 mg, 74%); mp 217–219 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.94 – 7.87 (m, 2H), 7.70 – 7.61 (m, 3H), 7.49 (t, J = 7.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 4.7 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 13.3, 5.3 Hz, 2H), 6.65 (t, J = 10.7 Hz, 2H), 3.73 (s, 2H), 2.88 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 193.65, 168.05, 163.62, 148.25, 144.77, 137.85, 136.51, 133.84, 130.22, 129.67, 129.32, 127.14, 121.91, 120.29, 120.00, 118.92, 118.68, 112.89, 44.46, 40.86. HRMS: (ESI) calcd for C₂₅H₂₄N₂O₃ [M + H]⁺ 401.1865; found, 401.1861.

(E)-2-(4-(3-(2-Hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)-N-(naphthalen-1-yl)acetamide (HPE-36).

To a stirred solution of (E)-2-(4-(3-(2-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl)acetic acid 17a (36 mg, 0.13 mmol, 1 equiv) in dichloromethane (2 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (51 mg, 0.26 mmole, 2 equiv), DMAP (3 mg, 0.026 mmol, 0.2 equiv), and 1-naphthylamine 19c (38 mg, 0.26 mmole, 2 equiv). The reaction mixture was stirred at room temperature overnight. Water (3 mL) was added, the organic layer was separated, and the water layer was extracted with dichloromethane (3 × 5 mL). The combined organic extract was washed with brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel in ethyl acetate/ dichloromethane (0.5–2%) to give HPE-36 as a yellow powder (37 mg, 76%); mp 253–254 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 10.16 (s, 3H), 8.23 (d, J = 8.0 Hz, 3H), 8.02 (t, J = 12.0 Hz, 6H), 7.94 – 7.85 (m, 8H), 7.83 (d, J = 15.5 Hz, 3H), 7.73 (d, J = 8.1 Hz, 3H), 7.65 (d, J = 7.3 Hz, 3H), 7.58 – 7.48 (m, 13H), 7.45 (t, J = 7.8 Hz, 3H), 6.98 (t, J = 8.9 Hz, 6H), 3.87 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 194.10, 169.82, 162.35, 145.20, 140.03, 136.83, 134.16, 133.85, 133.33, 131.35, 130.31, 129.77, 128.66, 128.16, 126.52, 126.35, 126.04, 125.82, 123.03, 122.19, 121.75, 121.21, 119.66, 118.22, 43.25. HRMS: (ESI) calcd for C₂₇H₂₁NO₃ [M + H]⁺ 408.1560; found, 408.1579.

Microtubule Polymerization Assay

Turbidity-based tubulin polymerization assay kit (Cytoskeleton Inc. catalog #BK004P) was used. Tubulin was resuspended at 4mg/ml concentration in ice-cold G-PEM buffer [80mM PIPES, 2mM MgCl₂, 0.5mM EGTA, 1mM GTP, 5% glycerol]. In triplicate 100 μl of 4 mg/ml tubulin was used for each treatment. IPE stock concentrations were adjusted to have exactly the same amount of DMSO in the final assay. Half area 96 well plate was warmed to 37°C along with the compounds or DMSO. Immediately after addition of tubulin, light scattering was measured in temperature-controlled spectrophotometer at 340 nm every 30 seconds for 3.5 hours. 1 hour into the assay, the spectrophotometer was cooled down to measure depolymerization. Graph-pad Prism was used for data analysis.

Cell Culture and Cell Viability Assays.

HCT116 colorectal carcinoma cells (ATCC # CCL-247) were maintained in McCoy’s 5A medium (Thermo # 16600082) supplemented with 10% fetal bovine serum (Gemini Bio-Products # 100–106) at 37 °C with 5 % CO₂. For viability assays, cells were seeded in triplicate per experimental condition (3 × 10³ cells/well) in 96-well plates and allowed to adhere overnight. The medium was then replaced with 100 μL of fresh medium containing the appropriate concentration of each compound or DMSO. Following incubation for 96 hrs, 20 μL of CellTiter 96 Aqueous One Solution (Promega # G3582) was added per well and plates were mixed then incubated for 2 hrs at 37 °C. Resulting absorbance was measured at 490 nm using a SpectraMax M5 (Molecular Devices). In some experiments cell viability was assessed using methylene blue staining: cells were plated at 25000/well or 5000/well in 24 or 96 well plates respectively and treated the next day. Three days post treatment cells were fixed and stained in methylene blue saturated in 50% ethanol for 30 min at RT. Plates were rinsed to wash off excessive dye. Retained dye was dissolved in 0.1N HCl and absorbance was measured at 668nm. Cell viability was expressed as a percentage of absorbance of control cells.

For Figures. 6–11, cells were cultured in a humidified 37°C atmosphere containing 10% CO₂ in Dulbecco’s Modified Eagle’s medium (Mediatech, Inc.) supplemented with 10% calf serum (Atlanta Biologicals) and 1000 U/ml of both Penicillin and Streptomycin (Mediatech, Inc.). HeLaM cells were obtained from Ganes Sen (Cleveland Clinic) and HCT116 used for HDAC8 knockout were obtained from Bert Vogelstein (Johns Hopkins). HDAC8 Knock-out HCT-116 cell were generated by transferring plasmid coding for cas9 and dual gRNA against HDAC8. Plasmid expressing non-coding Safe Harbor (SH) gRNA was used as control. The plasmids were a generous gift from E. Chen’s lab at University of Washington.[42] After antibiotic selection isolated clones were used for further experiments. For experiments to study tubulin dynamics, a plasmid coding for mCherry-α-tubulin courtesy of Dr. Song-Tao Liu, University of Toledo was transferred in HeLaM cells, and an appropriate antibiotic resistant clone showing enough fluorescence without any cytotoxic effect was selected for further experiments.

Western Blot Analysis of Histone Acetylation Levels.

HCT116 cells were seeded into T-25 flasks (2 × 10⁶ cells) and allowed to adhere overnight. Medium was then replaced with 5 mL of fresh medium containing either experimental compounds or DMSO. After incubation for 24 hrs, cell lysates were extracted using 1X RIPA buffer (Thermo # 89900) and resulting protein concentrations were determined using a BCA Protein Assay (Thermo # 23225). Reduced protein samples (25 μg/sample) were separated on a Bolt 4–12% Bis-Tris Gel (Thermo # NW04120BOX), transferred to a PVDF membrane (BioRad # 1620177), and blocked using standard western blotting techniques. Primary antibodies used were acetyl-Histone H3 (Sigma # 06–599), acetyl-Tubulin Lys40 (Sigma # ABT241), and β-actin (Santa-Cruz # sc-8432). Fluorescent-conjugated secondary antibodies from Li-Cor Biosciences were used for detection and visualization was conducted using a fluorescent Odyssey scanning system (LiCor Biosciences). To detect acetylated SMC3, antibodies were obtained from EMD Millipore (MABE1073), and detection was carried out using horse radish peroxidase secondary antibodies (Biorad) and enhanced chemiluminescence reagents (Biorad). Images were obtained by exposure to film.

Inhibitor Testing with HDAC Isoforms.

In a half-area 96-well white opaque plate (Corning), recombinant HDAC1 (1 μL; 3 ng/μL, BPS Bioscience), HDAC2 (1 μL; 1 ng/μL, BPS Bioscience), HDAC3 (1 μL; 30 ng/μL, BPS Bioscience), HDAC6 (1 μL; 35 ng/μL, BPS Bioscience), HDAC8 (1 μL; 70 ng/μL, BPS Bioscience), or buffer alone (1 μL) was added to HDAC-Glo^™ buffer (43 μL, Promega). Serial dilutions or single concentrations of inhibitors (1 μL in DMSO, concentrations described in supplement) or DMSO alone (1 μL) were added to the enzyme solution, followed by 3 hr. incubation at room temperature. The HDAC-Glo^™ reagent (5 μL), prepared per manufacturer recommendations, was added to each reaction. Luminescent signal was measured every 3 minutes over the course of 30 minutes using an M-Plex Infinite 200 Pro (Tecan). To determine IC₅₀, the luminescent signal at peak reading was first background corrected with the signal from a background control reaction where HDAC was excluded. The background corrected luminescence signal of each inhibitor-containing reaction was divided by the signal of the reaction without inhibitor for each HDAC enzyme to generate a percent deacetylase activity remaining value. IC₅₀ values were calculated by fitting the percent deacetylase activity remaining as a function of inhibitor concentration to a sigmoidal dose-response curve (y = 100/(1+(x/IC₅₀)^z), where y = percent deacetylase activity and x = inhibitor concentration) using non-linear regression with KaleidaGraph 4.1.3 software.

Chromosome Drops to Determine Mitotic Index.

HeLaM cells were synchronized in S-phase by a single thymidine block (2mM thymidine treatment overnight). Then they were washed with PBS and released into IPEs or Paclitaxel for about 9 hrs. Then they were treated with ZM for 90 min. For harvesting both attached and floating cells were collected without discarding the culture medium or PBS after wash. Cells were trypsinized and harvested in DMEM +10%CS and P/S, then spun down. To swell the cells, cell pellets were resuspended in 0.075 M KCl for 10min at 37°C and fixed with pre-chilled methanol: acetic acid (3:1 v/v). The cells were dropped onto slides and stained with DAPI (1:2000) and then rinsed three times in PBS. Following this a drop of mounting medium [1% w/v p-phenylenediamine, in 90% glycerol plus 10mM TRIS (pH 9)] was placed on the slides and covered with coverslip. Chromosome morphology and mitotic index was determined for each sample in a blinded manner and include data from at least three experiments.

Microscopy.

For phase contrast time lapse imaging, cells were plated at ~70% density and were treated the next day. Cells were maintained in a sealed flask containing growth medium+ drug pre-equilibrated to 10% CO2. Flasks were placed on a 37°C heated stage on an inverted microscope. Images were captured using a ×40 microscope objective and an Olympus C740 digital camera controlled by AmScope software. Image analysis was done using ImageJ. Mitotic duration of at-least 50 cells was counted.[7] For immunofluorescence, cells were plated on sterilized coverslips. After treatments they were fixed with 2% formaldehyde in phosphate buffered saline (PBS) for 10 min, followed by permeabilization [150 mM NaCl, 10 mM Tris (pH 7.7), 0.1% Triton X-100, and 0.1% BSA] for 9 min. Fixed cells were blocked with PBS containing 0.1% BSA for 1 hr at room temperature. Cells were then stained with antibodies to α-tubulin (Sigma-Aldrich), and human anti-centromere antibodies (ACA). Antibodies were visualized by incubating samples with Alexafluor-conjugated secondary antibodies (Invitrogen). DNA was visualized by staining with Hoechst 33342. Phenotypes were counted in blinded manner. Representative images were captured on SP8 Leica Confocal microscope. For mCherry-α-tubulin time lapse imaging, cells were plated on 35mm culture dish with glass bottom at about 70% confluence. They were treated the next day. Images were captured SP8 Leica confocal microscope right after addition of ZM447439 at the interval of 5 min. During imaging, cells were maintained in 37°C humidified chamber and in Ham’s F-12 medium with additional 20mM HEPES (pH 7.4) and 10% calf serum.

Flow Cytometry.

Five hundred thousand HeLaM cells were plated on 7cm dishes and treated with the compounds the next day. After 24h, 48h, or 72h of treatment, all cells (floating and attached) were collected by trypsinization and centrifugation. Cells were then fixed by pre-chilled 70% ethanol (final concentration). Fixed cells were collected by centrifugation and resuspended in PBS. Then they were treated with RNase H (10μg/ml) for 30 min at 37°C. Cells were again collected by centrifugation and resuspended in PBS+2% FBS +propidium iodide. Cells were analyzed using BD LSR Fortessa FACScanner and FloJo software. Twenty thousand cells were analyzed for each treatment.

Molecular Docking.

In order to further validate this observation, we carried out molecular docking studies using the crystal structure of tubulin in complex with inhibitor D64131 (pdb ID: 6k9v) as receptor configuration. This structure contained two α-tubulin and two β-tubulin molecules with Guanosine triphosphate (GTP) bound to α-tubulin and Guanosine diphosphate (GDP) and 2-(N-morpholino)-ethenesulfonic acid (MES) bound to β-tubulin in addition to D64131 bound at the colchicine binding site of β-tubulin. All ligand binding sites of β-tubulin were tested for IPE-7 binding and the sites were prepared for docking using the Make_Receptor module (Release 3.5.0.4) of the OpenEye software package (OpenEye Scientific Software, Inc., Santa Fe, NM, U.S.A.) with the help of bound ligand in defining the binding cavity while docking studies were carried out using either the Hybrid or Fred module (Release 3.5.0.4). Additional sites (Site X and Site Y in Figure 12) were defined by searching potential ligand binding sites on β-tubulin with the help of the Make_Receptor module. All possible conformations of IPE-7 were constructed with Omega2 module (Release 4.0.0.4) and the scorings were done using the default ChemGauss4 scoring function used by the Hybrid/Fred modules. This scoring function is based on the shape of the ligand, hydrogen bonding between ligand and receptor, hydrogen bonding interactions with implicit solvent, and metal-chelator interactions.

In addition, multiple other X-ray crystal structures with the colchicine binding site of β-tubulin occupied by various inhibitors were subjected to the docking study for further evaluation of the binding of IPE-7 to this side and for the comparison of IPE-7 binding scores with those co-crystallized ligands of β-tubulin. Additionally, the taxol-bound β-tubulin (pdb ID:6wvr) was used to evaluate the possibility of IPE-7 binding at the taxol binding site

Highlights.

• Synthesis of chalcone mimics containing an imidazole-based metal-binding group intended for HDAC inhibition

• Compounds showed cytotoxicity with IC50 values in the upper nanomolar to low micromolar range

• Compounds caused mitotic arrest of HeLa cells

• Representative analogues induced selective inhibition of purified HDAC8 with IC50 values in low micromolar range.

• Western blot analysis suggests that HDAC inhibition is not a major mechanism responsible for cytotoxicity.

• In combination with an aurora B inhibitor, compounds led to dramatic disassembly of the mitotic spindle

• The compounds reduced the rate of tubulin polymerization both in cells and of purified tubulin

• Compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity