A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics

Bioorganic Chemistry 116 (2021) 105297

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

A new class of cytotoxic agents targets tubulin and disrupts

microtubule dynamics

,

Ayad A. Al-Hamashi^{a 1}, Radhika Koranne^{b 1}, Samkeliso Dlamini^a, Abdulateef Alqahtani^a,

Endri Karaj^a, Maisha S. Rashid^b, Joseph R. Knoff^c, Matthew Dunworth^d, Mary Kay H. Pﬂum^c,

Robert A. Casero Jr^d, Lalith Perera^e, William R. Taylor^{b *}, L.M. Viranga Tillekeratne^a

,

,*

^aDepartment of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, 2801, W. Bancroft Street, Toledo, OH-

43606, USA

^bDepartment of Biological Sciences, College of Natural Sciences and Mathematics, The University of Toledo, 2801, W. Bancroft Street, Toledo, OH-43606, USA

^cDepartment of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA

^dThe Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Bunting/Blaustein Cancer Research Building 1 1650 Orleans

Street - Room 551, Baltimore, MD 21231, USA

^eLaboratory of Genome Integrity and Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and

Human Services, Research Triangle Park, NC 27709, USA

A R T I C L E I N F O

A B S T R A C T

Keywords:

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 mecha-

nisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of anti-

mitotic 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 IC₅₀values 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 poly-

merization 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.

Antimitotic agents

Anticancer agents

Mitotic spindle

Chalcones

Mitotic arrest

1. Introduction

development of multiple drug resistance and undesirable side effects

arising from off-target effects limit the clinical utility of the currently

Every year ~500,000 people die from cancer in the United States. [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 chemothera-

peutic agents are a main component of cancer treatment. [3] However,

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

* Corresponding authors.

E-mail addresses: william.tayor2@utoledo.edu (W.R. Taylor), ltillek@utnet.utoledo.edu (L.M.V. Tillekeratne).

1

A.A.A (Design and synthesis) and RK (biological assay) contributed equally to this work.

https://doi.org/10.1016/j.bioorg.2021.105297

Received 16 July 2021; Received in revised form 3 August 2021; Accepted 17 August 2021

Available online 30 August 2021

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

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 chromo-

somes 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.

Table 1

Analogues synthesized and their effect on percentage mean growth of NCI sixty

human tumor cell line panel at 10

μ

M.

.

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 se-

lective HDAC inhibitors, [10]. This approach was motivated by the fact

that clinically used HDAC inhibitors such as vorinostat (SAHA), romi-

depsin (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 bind-

ing ability of imidazole has been reported [18]. The ketone moiety was

introduced to provide an additional coordinating site for metal chela-

tion. 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].

Compound

R

Ar

Mean %

growth

at 10

μ

M

IPE-1

IPE-2

IPE-3

IPE-4

45.33

70.02

41.34

66.84

IPE-5

IPE-6

52.06

76.31

Consistent with our original aim, the compounds were capable of

inhibiting puriﬁed HDACs with some analogues showing selective in-

hibition of puriﬁed HDAC8 with IC₅₀values 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.

IPE-7

IPE-8

IPE-9

IPE-10

27.48

21.10

17.88

64.07

IPE-11

IPE-12

23.48

51.10

2. Results and Discussion

The compounds synthesized are shown Table 1.

2.1. Chemistry

IPE-13

IPE-14

91.77

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 ke-

tone 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 hydro-

lysis and amide formation. However, this reaction gave low yields.

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 peri-

odinane oxidation to afford the N-tosyl imidazole ketone 1 (Scheme 2A).

The synthesis of the aldehydes 2 began with palladium catalyzed

Heck coupling between the bromoester 7 and styrene 8 to produce the

77.21

57.67

IPE-15

IPE-16

15.90

(continued on next page)

2

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Table 1 (continued)

Compound

R

Ar

Mean %

growth

at 10

Compound

R

Ar

Mean %

growth

at 10

μM

IPE-17

IPE-18

IPE-19

IPE-20

19.33

19.91

33.11

54.69

HPE-33

HPE-34

91.98

89.56

HPE-35

HPE-36

85.87

96.15

IPE-21

IPE-22

29.20

ꢀ 18.79

IPE-23

IPE-24

12.97

22.66

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). Cyclo-

propanation of the oleﬁnic double bond of IPE-18 afforded the cyclo-

propyl derivative IPE-29. The N-alkyl imidazole derivatives were made

by reacting the non-alkylated imidazole analogues with the corre-

sponding alkyl halide in the presence of potassium carbonate in DMF to

produce IPE-15–26 (Scheme 2C). Some of the N-methyl imidazole an-

alogues were prepared using N-methylimidazole ketone instead of the N-

tosyl imidazole ketone 1 in the aldol reaction. N-methyl imidazole ke-

tone was prepared as reported earlier.[23]

IPE-25

IPE-26

61.43

68.98

IPE-27

IPE-28

53.49

92.81

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.

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 corre-

sponding 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-bromo-

phenyl)-acetate 7 using paraformaldehyde produced the aldehyde 15. It

was subjected to base-initiated aldol reaction with 2-hydroxy aceto-

phenone 16, followed by acidiﬁcation, to afford a mixture of the

IPE-29

98.67

TPE-30

PPE-31

84.29

99.14

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.

HPE-32

85.87

3

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Scheme 1. Retrosynthesis of IPE analogues.

2.2. Biology

IPE-7 distinguishing themselves as pan HDAC inhibitors in in vitro

studies using puriﬁed enzymes.

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

2.3. 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 acetyla-

tion 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

compounds ranging from 0.01 μM to 100 μM for 96 h. All the compounds

inhibited the growth of HCT 116 cells with GI₅₀values in single digit

micromolar range (IPE-3: 6.85 M, IPE-7: 6.95 M, IPE-8: 5.14 M,

IPE-16: 5.91 M, IPE-19: 6.56 M) (Fig. 1)

In order to assess the potential HDAC enzyme inhibition activity,

selected analogues were tested at 100 M against puriﬁed HDAC iso-

μ

forms (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 iso-

form showing less than 20% activity remaining (Figure S1). IPE-7 and

IPE-8 also showed similar pan inhibition, but with less potency, dis-

playing 20–80% activity remaining (Figure S1). Interestingly, com-

pounds 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 iso-

forms, suggesting possible HDAC8 selectivity when tested against the

puriﬁed enzymes (Figure S1). Finally, compounds IPE-26 and IPE-18

showed only modest HDAC inhibition.

μ

M (Fig. 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.

Several of the compounds selectively inhibited puriﬁed HDAC8

(Table S2). Some studies suggest that HDAC8 does not deacetylate his-

tone 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 re-

ported to be an HDAC8 substrate.[25] We observed an increase in SMC3

acetylation in cells exposed to PCI34051, an HDAC8 selective inhibitor,

Next, the dose dependence (IC₅₀) of HDAC inhibition was deter-

mined 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 com-

parison. IPE-25 displayed low micromolar inhibition against all iso-

forms, 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 com-

pounds 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

but only after treatment with high concentrations (25 μM). IPE-7 and

IPE-8 had no effect on SMC3 acetylation (Fig. 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

(Fig. 3a). In cytotoxicity assays, HDAC8-null cells were slightly more

resistant to IPE-7 and PCI34051 compared to control HCT116 cells

(Fig. 3b). On the other hand, both parental and HDAC8-null cells were

killed by 10 μM IPE-7 (Fig. 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.

an IC₅₀of 3.0 ± 0.2

μM (Table 2). The dose dependence data showed

that most compounds displayed HDAC8-selectivity, with IPE-25 and

Cell biological analysis suggested that HDACs were likely not a major

4

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

10

Scheme 2. General approach for IPE synthesis.

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 (Fig. 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 IC₅₀compared to RPE cells, indicating

the selectivity of these analogues for transformed cancer cells. There-

fore, we expanded our toxicity studies and determined detailed SAR.

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

5

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Scheme 3. Synthesis of IPE-27.

Scheme 4. Synthesis of HPE-32 – HPE-36.

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-

in the corresponding 4-substituted analogue, had a negative effect on

activity in both unsubstituted imidazole and N-methylimidazole ana-

logues. 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 ac-

tivity 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 (Fig. 5). Most of the compounds inhibited the growth of

multiple cell lines in a single digit micromolar range GI₅₀values.

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 GI₅₀values,

22 was found to cause 100% growth inhibition at 10

μM. Tri-

ﬂuoromethyl substituents at 3 and 5-positions of the phenyl ring, unlike

6

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 1. Growth inhibition of HCT116 colorectal carcinoma cells treated with selected representative analogues. Cells were treated for 96 h with increasing con-

centrations 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.

exposed to IPE analogues showed a strong mitotic arrest lasting for

Table 2

10–12 h (indicated by white arrows in bottom panel in Fig. 6a and

IC₅₀values for SAHA and synthesized compounds.

supplemental movies A and B).

Compound

ID

IC₅₀Value (

HDAC1

μ

M)*

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, inhi-

bition of APC/C and failure to exit mitosis. Furthermore, the ensuing

prolonged mitotic block that occurs upon alterations in spindle micro-

tubule 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 un-

derwent 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. in-

hibitors 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 (Fig. 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. Asynchro-

nous 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 popula-

tion 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)

HDAC2

HDAC3

HDAC6

HDAC8

SAHA

0.033 ±

0.001

1.8 ± 0.1

>100

0.096 ±

0.01

0.020 ±

0.001

0.033 ±

0.003

0.54 ±

0.01

IPE-7

2.1 ± 0.2

>100

1.8 ± 0.1

N.D.

2.0 ± 0.2

>100

1.4 ± 0.1

N.D.

IPE-8

IPE-18

IPE-22

IPE-24

IPE-25

IPE-26

IPE-27

IPE-29

>100

>200

3.0 ± 0.2

13 ± 1

9.0 ± 0.5

15 ± 1

12 ± 1

8.5 ± 0.9

>200

12 ± 1

>200

8.9 ± 0.6

>100

16 ± 1.0

>200

8.6 ± 4.9

>100

>200

* >100 or > 200 indicates>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 chem-

iluminescence signal intensity. The activity after inhibition was then plotted and

ﬁt to a sigmoidal curve to calculate the IC₅₀values. Higher concentrations were

not tested due to solubility issues. N.D. = Not Determined

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 GI₅₀values. As the compounds have cell growth inhibi-

tory activity, but limited HDAC inhibitory activity, it is likely that cells

are killed by binding to additional higher afﬁnity target(s).

2.4. 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 Fig. 6a). Strikingly, HeLaM cells

7

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 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.

Fig. 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.

(Fig. 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 h after treatment (Fig. 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

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 chromo-

somes align at metaphase plate. [9] To test whether IPEs trigger the SAC

to cause mitotic arrest, we used an 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

8

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

a) Cytotoxicity of IPE-7 in HeLa

c) Cytotoxicity of IPE-22 in HeLa

100

50

0

100

50

3.6+0.3 x 10^-6M

4.1+0.06 x 10^-6M

0

-7

-6

-5

-4

-7

-6

-5

-4

Concentration(log[M])

b)

d) Cytotoxicity of IPE-22in RPE

Cytotoxicity of IPE-7 in RPE

100

50

0

10.1+0.2 x 10^-6M

8.5+0.5 x 10^-6M

0

-7

-6

-5

-4

-7

-6

-5

-4

Concentration(log[M])

Fig. 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.

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 (Fig. 7). A known spindle toxin, paclitaxel, was

used as a positive control in this experiment.

clumps of tubulin with scattered chromosomes could be seen in the

cytoplasm, suggesting that co-treatment with compound and ZM abol-

ished spindle structure and function. Representative images of these

aberrant spindles are shown in Fig. 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 Fig. 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

(Fig. 8). Combining the compounds with Hesperidin also induced spin-

dle collapse suggesting that the Aurora B paralogue is more important in

this response (Fig. 8 Graph 3).

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 (20

μ

M), a proteasome inhibitor that

prevents mitotic exit. Next, ZM447439 (2.5

μ

M) was added and immu-

noﬂuorescence 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 Fig. 8). To investigate this phe-

nomenon, 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 in-

hibitor PCI34051.

The spindle collapse morphology could be explained by depoly-

merization of microtubules in the presence of both IPE and Aurora in-

hibitors 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, fol-

lowed by MG132 in 30 min. Thirty min after MG132, ZM447439 was

added and images were taken every 5 min on SP8 Leica confocal mi-

croscope. 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 (Fig. 9) and Supplemental Movies E,F and G),

explaining the “collapse” morphology observed earlier.

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

Aurora B regulates microtubule dynamic via its effects on the kinesin

9

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 5. The half maximal cell growth inhibition concentration (GI₅₀) (µM) for representative IPEs in the NCI 60 cancer cell lines. *ND = No Data.

MCAK. Aurora B phosphorylates MCAK thereby inhibiting its depoly-

merizing activity. [32] However, treatment with Aurora B inhibitors

alone does not cause spindle collapse (Fig. 8). It is possible that regu-

lation 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 min of rewarming

(Fig. 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-7n = 73). Images

were assessed in a blinded manner and scored as either normal or

abnormal spindle (examples are shown in Fig. 10a). No signiﬁcant dif-

ferences were observed at either 0 min or 20 min post rewarming.

10

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 6. Compounds arrest HeLaM cells in mitosis. a): asynchronous HeLaM cells were treated with DMSO or IPE, images were taken every 12 min 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 24 h, 48 h and 72 h 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. 4 N peak and post G2

population (i.e. > 4 N) conﬁrming mitotic arrest and mitotic exit without division into two daughter cells. Also, cell death as seen in increased subG1 population.

However, at 8 min post-rewarming, we observed signiﬁcantly more

abnormal spindles in IPE-7-treated samples than control (Fig. 10b). As a

second method of quantitation, again using blinded images at the 8 min

time-point, we used ImageJ to trace spindle outline and quantify spindle

morphology (Fig. 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 (Fig. 10d).

(Fig. 11e and f). These results suggest that IPE-7 destabilizes microtu-

bules at 37 ^◦C and accelerates depolymerization upon cooling.

Evidence so far suggests that our compounds affect microtubule

dynamics by targeting tubulin. In order to further validate this obser-

vation, 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

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 polymeri-

zation after cold shock, we predicted that tubulin may be directly tar-

geted by IPEs. Hence, we measured the effect of IPE on polymerization

of puriﬁed tubulin by spectrophotometry. Light scattering was measured

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 β-tubulin heterodimers with Guanosine triphosphate (GTP)

bound to tubulin-

α

and both Guanosine diphosphate (GDP) and 2-(N-

◦

every 30 s upon warming puriﬁed tubulin to 37 C. The Vmax slope

Morpholino)-ethanesulfonic acid (MES) bound to tubulin-β in addition

during the exponential phase was measured in mOD/min. 10

μ

M Taxol,

to the occupation of an inhibitor at the colchicine binding site. The

which stabilizes microtubules, was used as a positive control and

showed a higher V_maxthan DMSO. [34] (Fig. 11a and b). In contrast,

IPE-7 delayed tubulin polymerization. This effect was observed in dose-

dependent manner as tubulin polymerization was slowed down signiﬁ-

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 Fig. 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

cantly in both 10 and 50

μ

M IPE-7 as compared to DMSO (Vmax

10mOD/min in DMSO vs 5.5mOD/min in 10

μ

M IPE-7 and 4.5 mOD/

min in 50 μ

M 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 depoly-

merization. Depolymerization was accelerated in 50 μM IPE-7 compared

to the DMSO-treated sample (Fig. 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

11

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 6. (continued).

(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 Fig. 12.

2.5. Conclusion

We designed chalcone mimetics containing 1-(1H-imidazol-2-yl)

12

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

docking studies identiﬁed an interaction of IPE-7 with tubulin. Consis-

tent 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 inhibi-

tory 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.

3. Experimental.

3.1. Material and Methods.

All chemicals and solvents were purchased from commercial sup-

pliers and used without further puriﬁcation, unless stated otherwise.

Anhydrous tetrahydrofuran and diethyl ether were freshly distilled from

Fig. 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

1

sodium and benzophenone before use. H and ¹³C NMR spectra were

thymidine into IPE-7 (10 μM) or DMSO or paclitaxel (1 μM). 9 h post thymidine

carried out on Brucker Avance 600 MHz, 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 Com-

panion chromatography system on RediSep prepacked silica cartridges.

Thin layer chromatography (TLC) plates (20 cm × 20 cm) were pur-

chased from Sorbent Technologies (catalog #4115126) and were viewed

under Model UVG-54 mineral light lamp UV-254 nm. Uniplates (1000

release cells were treated with ZM447439 or reversine for 90 min 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).

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 ac-

μ

m) 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 com-

bination of ¹H and ¹³C NMR, HRMS and HPLC and the purity of all test

compounds were found to be > 95%.

tivity. 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 in-

hibition selectivity. Almost all leukemia cell lines, and most of the colon

cancer cell lines were found to be particularly sensitive to these com-

pounds. 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

3.2. Synthesis of 1H-imidazole ketones.

3.2.1. 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 chroma-

tography on silica gel in 40% ethyl acetate in hexanes to give 3 as an off-

13

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 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 (10

μ

M) or

DMSO, followed by treatment with 20 μM MG132 (MG) to arrest their cell cycle progression. They were then treated with 2.5 μM ZM447439 (ZM) or Hesparadine

(Hesp) for 90 min. 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.

14

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 9. Co-treatment with IPE-7 þ ZM447439 leads to collapsed mitotic spindle: HeLaM cells were treated with IPE-7 or DMSO (30 min) followed by MG132

(MG) for 30 min. Images were captured on Leica confocal microscope immediately after addition of ZM447439 (ZM).

Fig. 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.

1

◦

white powder (3.8 g, 76%); mp 155 C. H 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.

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

3.2.2. 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

15

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

Fig. 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 340 nm 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.

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.

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.

3.2.4. 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 mol

%), triphenyl phosphine (160 mg, 6 mol%), 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 chroma-

tography on silica gel in ethyl acetate/hexanes (1–3%) to afford 11 as a

3.2.3. 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 re-

action 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

1

◦

white powder (1.73 g, 65%); mp 72 C. H 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,

16

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

b)

c)

a)

Tubulin α

GDP

binding

site

Tubulin β

Site X

Taxol

binding

site

d)

e)

Site Y

MES

binding

site

colchicine

binding

site

Tubulin α

Fig. 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.

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.

3.3.1. (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). ¹³

C

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

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.

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.

3.3.2. (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. ¹³

C

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

3.3. 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-dimethylamino-

propyl)carbodiimide (1.5 equiv), DMAP (0.1 equiv), and the corre-

sponding 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 chroma-

tography on silica gel in ethyl acetate/hexanes (10–30 %) to give amides

4a-n.

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

1

◦

White solid (80%); mp. 220 C. H 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.

3.3.4. (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),

17

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

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.

127.39, 126.59, 121.32, 117.09, 44.59. HRMS: (ESI) calcd for

C₂₂H₁₈BrNO [M + H]⁺392.0650; found, 392.0652.

3.4.3. (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.

3.3.5. (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

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

(4k).

C

₂₄H₂₄N₂O [M + H]⁺357.1967; found, 357.1967.

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.

3.3.6. (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

3.5. General procedure for the oxidative cleavage using OsO₄for the

synthesis of aldehydes (2a-n).

C

₂₆H₂₁NO [M + H]⁺364.1701; found, 364.1712.

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 mol%),

and NaIO4 (4 equiv). The reaction mixture was stirred at room tem-

perature 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 evapo-

rated under vacuo. The residue was puriﬁed by column chromatography

on silica gel in ethyl acetate/hexanes (30–50%) to produce 2a-n.

3.3.7. (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.

3.5.1. 2-(4-Formylphenyl)-N-phenylacetamide (2a).

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

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.

A mixture of 2-(4-styrylphenyl)acetic acid 5 (2.0 equiv) and excess

oxalyl chloride was reﬂuxed at 65 ⁰C 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.

3.5.2. 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.

3.4.1. (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. ¹³

C

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.

3.5.3. 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.

3.4.2. (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,

3.5.4. 2-(4-Formylphenyl)-N-(4-methoxyphenyl)acetamide (2d).

White powder (99%); mp 193 ^◦C. ¹H NMR (600 MHz, CDCl₃) δ 10.05

18

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

(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

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.

C

₁₆H₁₅NO₃[M + H]⁺270.1130; found, 270.1141.

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

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

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, 0.33 mmol, 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.

3.5.6. 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.

3.5.7. 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),

3.6. General procedure for the aldol reaction using piperidine for the

7.47 – 7.38 (m, 2H), 7.17 (s, 1H), 7.06 – 6.98 (m, 2H), 3.83 (s, 2H). ¹³

C

synthesis of ﬁnal products (IPE-1–IPE-14).

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.

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/hex-

anes to obtain the ﬁnal products (50–75%).

3.5.8. 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.

3.6.1. (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, ace-

tone‑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.

3.5.9. 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.

3.5.10. 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.

3.6.2. (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 ⁰C. 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.

3.5.11. 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.

3.6.3. (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 ⁰C. 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.

3.5.12. 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 reac-

tion was quenched by the addition of brine and extracted with EtOAc.

The combined organic extract was dried over anhydrous Na₂SO₄,

19

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

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

methoxyphenyl)acetamide (IPE-4).

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.

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). ¹³

C

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.

3.6.11. (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 ⁰C. 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.

3.6.5. (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.

3.6.12. (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/hex-

anes), 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.

3.6.6. (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.

3.6.13. (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.

3.6.7. (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). ¹³

C

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

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

(bis(3,5-triﬂuoromethyl)phenyl)acetamide (IPE-14).

C

₂₀H₁₆FN₃O₂[M + H]⁺350.1305; found, 350.1310.

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.

3.6.8. (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 ⁰C. 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.

3.7. General procedure for the synthesis of the alkylated imidazole (IPE-

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

bromophenyl)acetamide (IPE-9).

15–IPE-26).

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

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 res-

idue 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.

(m, 2H), 7.48 – 7.42 (m, 5H), 7.35 (s, J = 7.5 Hz, 1H), 3.75 (s, 2H). ¹³

C

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.

3.6.10. (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

20

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

3.7.1. (E)-N-Cyclohexyl-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-

1-en-1-yl)phenyl)acetamide (IPE-15).

(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

White crystals (6.0 mg, 38%), mp. 182.5 ⁰C, 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

C

₂₇H₂₂FN₃O₂[M + H]⁺440.1774; found, 440.1771.

3.7.7. (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.

3.7.2. (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

1

◦

was produced (6 mg, 60%); mp 184–186 C. H NMR (400 MHz, ace-

tone‑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.

3.7.8. (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

3.7.3. (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.

= 8.0 Hz, 2H), 7.39 (s, 1H), 7.14 (s, 1H), 4.06 (s, 3H), 3.81 (s, 2H). ¹³

C

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.

3.7.9. (E)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-oxoprop-1-en-1-yl)

phenyl)-N-(4-(triﬂuoromethyl)phenyl)acetamide (IPE-23).

3.7.4. (E)-N-(4-Bromophenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-

oxoprop-1-en-1-yl)phenyl)acetamide (IPE-18).

Brown solid (27 mg, 27%). TLC R_f= 0.2 (50% ethyl acetate/hex-

anes), 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

The starting material were IPE-9 and methyl iodide. A white powder

1

◦

was produced (7 mg, 60%); mp 221–223 C. H NMR (600 MHz, ace-

tone‑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.

C

₂₂H₁₈F₃N₃O₂[M + H]⁺414.1429, found: 414.1441.

3.7.10. (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/hex-

anes), 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.

3.7.5. (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, ace-

tone‑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.

3.7.11. (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/hex-

anes), 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

3.7.6. (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

1

◦

powder was produced (5.5 mg, 67%); mp 200–202 C. H NMR (600

MHz, CDCl₃) δ 8.14 (d, J = 16.0 Hz, 1H), 7.83 (d, J = 15.9 Hz, 1H), 7.73

21

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

(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.

121.02, 115.09, 76.33, 55.04, 43.43, 35.27, 18.29; HRMS: (ESI) calcd

for C₂₂H₂₀Br N₃O₂[M + H]⁺437.0701; found, 438.0817.

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

phenylacetamide (TPE-30).

3.7.12. (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).

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

Brown solid (30 mg, 30%). TLC R_f= 0.7 (50% ethyl acetate/hex-

anes), 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

◦

heated in a microwave synthesizer for 6 h at 90 C. The solvent was

removed under reduced pressure, and the residue was puriﬁed by col-

umn 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),

C

₂₃H₁₇F₆N₃O₂[M + H]⁺482,1303, found: 482.1321.

7.30 (t, J = 8.0 Hz, 17H), 7.09 (t, J = 7.4 Hz, 8H), 3.74 (s, 13H). ¹³

C

3.7.13. (E)-N-(4-Ethynylphenyl)-2-(4-(3-(1-methyl-1H-imidazol-2-yl)-3-

oxoprop-1-en-1-yl)phenyl)acetamide (IPE-27).

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.

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.

3.7.17. (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 col-

umn chromatography on silica gel in ethyl acetate/hexanes (30–80%) to

3.7.14. 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.

1

give TPE-31 as a yellow oily (24 mg, 45%). H NMR (600 MHz, ace-

tone‑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.

3.7.18. (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 anhy-

drous 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 –

3.7.15. 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 re-

action 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 sol-

vent 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,

22

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

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.

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.

3.7.19. 4-(3-(2-Hydroxyphenyl)-3-oxopropyl)phenyl)-N-(naphthalen-1-

yl)acetamide (HPE-33).

3.8. Microtubule polymerization assay

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.

Turbidity-based tubulin polymerization assay kit (Cytoskeleton Inc.

catalog #BK004P) was used. Tubulin was resuspended at 4 mg/ml

concentration in ice-cold G-PEM buffer [80 mM PIPES, 2 mM MgCl₂, 0.5

mM EGTA, 1 mM 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 s for 3.5 h. 1 h into the assay, the spectrophotometer was cooled

down to measure depolymerization. Graph-pad Prism was used for data

analysis.

3.9. Cell culture and cell viability Assays.

HCT116 colorectal carcinoma cells (ATCC # CCL-247) were main-

tained 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 × 10³cells/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

3.7.20. (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-dimethylamino-

propyl)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-

◦

plates were mixed then incubated for 2 hrs at 37 C. Resulting absor-

bance was measured at 490 nm using a SpectraMax M5 (Molecular

Devices). In some experiments cell viability was assessed using methy-

lene 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 0.1 N HCl and absorbance was

measured at 668 nm. Cell viability was expressed as a percentage of

absorbance of control cells.

1

◦

35 as a yellow powder (38 mg, 74%); mp 217–219 C. H 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.

For Figs. 6-11, cells were cultured in a humidiﬁed 37 ^◦C atmosphere

containing 10% CO₂in Dulbecco’s Modiﬁed Eagle’s medium (Medi-

atech, 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 Uni-

versity of Washington.[42] After antibiotic selection isolated clones

were used for further experiments. For experiments to study tubulin

3.7.21. (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-dimethylamino-

propyl)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 over-

night. Water (3 mL) was added, the organic layer was separated, and the

water layer was extracted with dichloromethane (3 × 5 mL). The com-

bined 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 –

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.

4. Western blot analysis of histone acetylation Levels.

HCT116 cells were seeded into T-25 ﬂasks (2 × 10⁶cells) and

allowed to adhere overnight. Medium was then replaced with 5 mL of

fresh medium containing either experimental compounds or DMSO.

23

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

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 (Bio-

Rad # 1620177), and blocked using standard western blotting tech-

niques. 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 peroxi-

dase secondary antibodies (Biorad) and enhanced chemiluminescence

reagents (Biorad). Images were obtained by exposure to ﬁlm.

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 permeabiliza-

tion [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 35 mm 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 in-

terval of 5 min. During imaging, cells were maintained in 37 ^◦C hu-

midiﬁed chamber and in Ham’s F-12 medium with additional 20 mM

HEPES (pH 7.4) and 10% calf serum.

4.1. Inhibitor testing with HDAC Isoforms.

In a half-area 96-well white opaque plate (Corning), recombinant

HDAC1 (1

μ

L; 3 ng/

μ

L, BPS Bioscience), HDAC2 (1

μ

L; 1 ng/

μ

L, BPS

L; 35

ng/μL, BPS Bioscience), HDAC8 (1 μL; 70 ng/μL, BPS Bioscience), or

Bioscience), HDAC3 (1 μL; 30 ng/μL, BPS Bioscience), HDAC6 (1 μ

4.4. Flow Cytometry.

buffer alone (1 µL) was added to HDAC-Glo™ buffer (43

μ

L, Promega).

L in DMSO,

L) were

Serial dilutions or single concentrations of inhibitors (1

μ

Five hundred thousand HeLaM cells were plated on 7 cm dishes and

treated with the compounds the next day. After 24 h, 48 h, or 72 h of

treatment, all cells (ﬂoating and attached) were collected by trypsini-

zation and centrifugation. Cells were then ﬁxed by pre-chilled 70%

ethanol (ﬁnal concentration). Fixed cells were collected by centrifuga-

tion and resuspended in PBS. Then they were treated with RNase H

concentrations described in supplement) or DMSO alone (1

μ

added to the enzyme solution, followed by 3 hr. incubation at room

temperature. The HDAC-Glo™ reagent (5 L), prepared per manufac-

μ

turer recommendations, was added to each reaction. Luminescent signal

was measured every 3 min over the course of 30 min using an Mꢀ 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 back-

ground corrected luminescence signal of each inhibitor-containing re-

action was divided by the signal of the reaction without inhibitor for

each HDAC enzyme to generate a percent deacetylase activity remaining

value. IC₅₀values 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 × = inhibitor concentration) using non-linear

regression with KaleidaGraph 4.1.3 software.

(10μ

g/ml) for 30 min at 37 ^◦C. Cells were again collected by centrifu-

gation 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.

4.5. Molecular Docking.

In order to further validate this observation, we carried out molec-

ular 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

-tubulin and Guanosine

4.2. Chromosome drops to determine mitotic Index.

Guanosine triphosphate (GTP) bound to

α

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 Fig. 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 con-

structed 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.

HeLaM cells were synchronized in S-phase by a single thymidine

block (2 mM thymidine treatment overnight). Then they were washed

with PBS and released into IPEs or Paclitaxel for about 9 hrs. Then they

were treated with ZM447439 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 10 min 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-phenyl-

enediamine, in 90% glycerol plus 10 mM 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.

4.3. Microscopy.

In addition, multiple other X-ray crystal structures with the colchi-

cine binding site of β-tubulin occupied by various inhibitors were sub-

jected 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

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

24

A.A. Al-Hamashi et al.

Bioorganic Chemistry 116 (2021) 105297

immune cell-mediated tumor cell cytotoxicity, Cancer Chemother Pharmacol. 69

(4) (2012) 901–909, https://doi.org/10.1007/s00280-011-1766-x.

[18] A. Vasudevan, Z. Ji, R.R. Frey, C.K. Wada, D. Steinman, H.R. Heyman, Y. Guo, M.

L. Curtin, J. Guo, J. Li, L. Pease, K.B. Glaser, P.A. Marcotte, J.J. Bouska, S.

K. Davidsen, M.R. Michaelides, Heterocyclic ketones as inhibitors of histone

deacetylase, Bioorganic Med. Chem. Lett. 13 (22) (2003) 3909–3913, https://doi.

org/10.1016/j.bmcl.2003.09.007.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

[19] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chalcone: A Privileged

Structure in Medicinal Chemistry, Chem. Rev. 117 (12) (2017) 7762–7810,

https://doi.org/10.1021/acs.chemrev.7b00020.

Acknowledgements

[20] M.L. Edwards, D.M. Stemerick, P.S. Sunkara, Chalcones: A New Class of

Antimitotic Agents, J. Med. Chem. 33 (7) (1990) 1948–1954, https://doi.org/

10.1021/jm00169a021.

This research is supported by NIH grants R15CA213185 to L.M.V.T,

grant R15GM120712 to W.R.T, grants GM131821 and GM121061 to

MKHP and grant Z01 ES043010 of the intramural research program of

the National Institute of Environmental Health Sciences to L.P. The

authors thank the Developmental Therapeutics Program of the National

Cancer Institute, Bethesda, MD, USA for performing cytotoxicity studies

in the human tumor cell lines screes and Gloria Basil for help with time-

lapse imaging.

[21] S. Ducki, R. Forrest, J.A. Hadﬁeld, A. Kendall, N.J. Lawrence, A.T. McGown,

D. Rennison, Potent antimitotic and cell growth inhibitory properties of substituted

chalcones, Bioorganic Med. Chem. Lett. 8 (9) (1998) 1051–1056, https://doi.org/

10.1016/S0960-894X(98)00162-0.

[22] N.J. Lawrence, R.P. Patterson, L.-L. Ooi, D. Cook, S. Ducki, Effects of

α

-substitutions on structure and biological activity of anticancer chalcones,

Bioorganic Med. Chem. Lett. 16 (22) (2006) 5844–5848, https://doi.org/10.1016/

j.bmcl.2006.08.065.

[23] N. Duchemin, E. Benedetti, L. Bethge, S. Vonhoff, S. Klussmann, J.-J. Vasseur,

J. Cossy, M. Smietana, S. Arseniyadis, Expanding Biohybrid--Mediated Asymmetric

Catalysis into the Realm of RNA, Chem. Commun. 52 (55) (2016) 8604 –8607 .

[24] A. Chakrabarti, I. Oehme, O. Witt, G. Oliveira, W. Sippl, C. Romier, R.J. Pierce,

M. Jung, HDAC8: A multifaceted target for therapeutic interventions, Trends

Pharmacol. Sci. 36 (7) (2015) 481–492, https://doi.org/10.1016/j.

tips.2015.04.013.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.105297.

[25] M.A. Deardorff, M. Bando, R. Nakato, E. Watrin, T. Itoh, M. Minamino, K. Saitoh,

M. Komata, Y. Katou, D. Clark, K.E. Cole, E. De Baere, C. Decroos, N. Di Donato,

S. Ernst, L.J. Francey, Y. Gyftodimou, K. Hirashima, M. Hullings, Y. Ishikawa,

C. Jaulin, M. Kaur, T. Kiyono, P.M. Lombardi, L. Magnaghi-Jaulin, G.R. Mortier,

N. Nozaki, M.B. Petersen, H. Seimiya, V.M. Siu, Y. Suzuki, K. Takagaki, J.J. Wilde,

P.J. Willems, C. Prigent, G. Gillessen-Kaesbach, D.W. Christianson, F.J. Kaiser, L.

G. Jackson, T. Hirota, I.D. Krantz, K. Shirahige, HDAC8 Mutations in Cornelia de

Lange Syndrome Affect The Cohesin Acetylation Cycle, Nature. 489 (7415) (2012)

313–317, https://doi.org/10.1038/nature11316.

References.

[1] (Accessed Feb 16 (2021) (n.d.). https://www.cancer.gov.

[2] D. Mahalingam, A. Mita, M.M. Mita, S.T. Nawrocki, F.J. Giles, Targeted therapy for

advanced non-small cell lung cancers: historical perspective, current practices, and

future development, Curr Probl Cancer. 33 (2) (2009) 73–111, https://doi.org/

10.1016/j.currproblcancer.2009.03.001.

¨

[3] M. Rocken, Early Tumor Dissemination, But Late Metastasis: Insights Into Tumor

[26] R.H. Shoemaker, The NCI60 human tumour cell line anticancer drug screen, Nat

Rev Cancer. 6 (10) (2006) 813–823, https://doi.org/10.1038/nrc1951.

[27] F.E. Stevens, H. Beamish, R. Warrener, B. Gabrielli, Histone deacetylase inhibitors

induce mitotic slippage, Oncogene. 27 (10) (2008) 1345–1354, https://doi.org/

10.1038/sj.onc.1210779.

Dormancy, J. Clin. Invest. 120 (6) (2010) 1800–1803, https://doi.org/10.1172/

JCI43424.

[4] I. Arnal, R.H. Wade, How Does Taxol Stabilize Microtubules? Curr. Biol. 5 (8)

(1995) 900–908, https://doi.org/10.1016/S0960-9822(95)00180-1.

[5] D.R. Matson, P.T. Stukenberg, Cdt1 Throws Kinetochore-Microtubule Attachments

for a Loop, Nat. Cell Biol. 14 (6) (2012) 561–563, https://doi.org/10.1038/

ncb2513.

[28] H.-J. Shin, K.-H. Baek, A.-H. Jeon, S.-J. Kim, K.-L. Jang, Y.-C. Sung, C.-M. Kim, C.-

W. Lee, Inhibition of histone deacetylase activity increases chromosomal instability

by the aberrant regulation of mitotic checkpoint activation, Oncogene. 22 (25)

(2003) 3853–3858, https://doi.org/10.1038/sj.onc.1206502.

[6] C.H. Topham, S.S. Taylor, Mitosis and Apoptosis: How is The Balance Set? Curr.

Opin. Cell Biol. 25 (6) (2013) 780–785, https://doi.org/10.1016/j.

ceb.2013.07.003.

[29] L. Magnaghi-Jaulin, G. Eot-Houllier, G. Fulcrand, C. Jaulin, Histone Deacetylase

Inhibitors Induce Premature Sister Chromatid Separation and Override the Mitotic

Spindle Assembly Checkpoint, Cancer Res. 67 (13) (2007) 6360–6367, https://doi.

org/10.1158/0008-5472.CAN-06-3012.

[7] M.E. Bekier, R. Fischbach, J. Lee, W.R. Taylor, Length of Mitotic Arrest Induced by

Microtubule-Stabilizing Drugs Determines Cell Death After Mitotic Exit, Mol.

Cancer Ther. 8 (6) (2009) 1646–1654, https://doi.org/10.1158/1535-7163.MCT-

08-1084.

[30] M. Cornago, C. Garcia-Alberich, N. Blasco-Angulo, N. Vall-llaura, M. Nager,

J. Herreros, J.X. Comella, D. Sanchis, M. Llovera, Histone deacetylase inhibitors

promote glioma cell death by G2 checkpoint abrogation leading to mitotic

catastrophe, Cell Death Dis. 5 (10) (2014) e1435, https://doi.org/10.1038/

cddis.2014.412.

[8] A.P. Joglekar, A cell biological perspective on past, present and future

investigations of the spindle assembly checkpoint, Biology (Basel). 5 (2016) 1–19,

https://doi.org/10.3390/biology5040044.

[9] E.A. Foley, T.M. Kapoor, Microtubule Attachment and Spindle Assembly

Checkpoint Signalling at The Kinetochore, Nat. Rev. Mol. Cell Biol. 14 (1) (2013)

25–37, https://doi.org/10.1038/nrm3494.

[31] A.T. Saurin, M.S. van der Waal, R.H. Medema, S.M.A. Lens, G.J.P.L. Kops, Aurora B

potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset

of mitosis, Nat. Commun. 2 (1) (2011), https://doi.org/10.1038/ncomms1319.

[32] P.D. Andrews, Y. Ovechkina, N. Morrice, M. Wagenbach, K. Duncan, L. Wordeman,

J.R. Swedlow, Aurora B Regulates MCAK at the Mitotic Centromere, Dev. Cell. 6

(2) (2004) 253 –268 .

[10] J. Almaliti, A.A. Al-Hamashi, A.T. Negmeldin, C.L. Hanigan, L. Perera, M. Kay, H.

Pﬂum, R.A. ⊥ Casero, L.M. Viranga Tillekeratne, Largazole Analogues Embodying

Radical Changes in the Depsipeptide Ring: Development of a More Selective and

Highly Potent Analogue, (n.d.).

[33] L.U. Cassimeris, P. Wadsworth, E.D. Salmon, Dynamics of microtubule

depolymerization in monocytes, J. Cell Biol. 102 (1986) 2023–2032, https://doi.

org/10.1083/jcb.102.6.2023.

[11] W.K. Kelly, O.A. O’Connor, L.M. Krug, J.H. Chiao, M. Heaney, T. Curley,

B. MacGregore-Cortelli, W. Tong, J.P. Secrist, L. Schwartz, S. Richardson, E. Chu,

S. Olgac, P.A. Marks, H. Scher, V.M. Richon, Phase I Study of an Oral Histone

Deacetylase Inhibitor, Suberoylanilide Hydroxamic Acid, in Patients With

Advanced Cancer, J. Clin. Oncol. 23 (17) (2005) 3923–3931, https://doi.org/

10.1200/JCO.2005.14.167.

[34] P.B. Schiff, S.B. Horwitz, Taxol Stabilizes Microtubules in Mouse Fibroblast Cells,

Proc. Natl. Acad. Sci. U. S. A. 77 (3) (1980) 1561 –1565 .

[35] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,

M. Knossow, Insight Into Tubulin Regulation From A Complex With Colchicine and

A Stathmin-Like Domain, Nature. 428 (6979) (2004) 198–202, https://doi.org/

10.1038/nature02393.

[12] K.M. VanderMolen, W. McCulloch, C.J. Pearce, N.H. Oberlies, Romidepsin

(Istodax, NSC 630176, FR901228, FK228, depsipeptide): a natural product recently

approved for cutaneous T-cell lymphoma, J. Antibiot. (Tokyo) 64 (8) (2011)

525–531, https://doi.org/10.1038/ja.2011.35.

[36] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An Overview of Tubulin Inhibitors That

Interact With The Colchicine Binding Site, Pharm. Res. 29 (11) (2012) 2943–2971,

https://doi.org/10.1007/s11095-012-0828-z.

[13] P. Campbell, C.M. Thomas, Belinostat for the treatment of relapsed or refractory

peripheral T-cell lymphoma, J. Oncol. Pharm. Pract. 23 (2) (2017) 143–147,

https://doi.org/10.1177/1078155216634178.

[37] A. Fedoriw, S.R. Rajapurkar, S. O’Brien, S.V. Gerhart, L.H. Mitchell, N.D. Adams,

N. Rioux, T. Lingaraj, S.A. Ribich, M.B. Pappalardi, N. Shah, J. Laraio, Y. Liu,

M. Butticello, C.L. Carpenter, C. Creasy, S. Korenchuk, M.T. McCabe, C.F. McHugh,

R. Nagarajan, C. Wagner, F. Zappacosta, R. Annan, N.O. Concha, R.A. Thomas, T.

K. Hart, J.J. Smith, R.A. Copeland, M.P. Moyer, J. Campbell, K. Stickland, J. Mills,

S. Jacques-O’Hagan, C. Allain, D. Johnston, A. Raimondi, M. Porter Scott,

N. Waters, K. Swinger, A. Boriack-Sjodin, T. Riera, G. Shapiro, R. Chesworth, R.

K. Prinjha, R.G. Kruger, O. Barbash, H.P. Mohammad, Anti-tumor Activity of the

Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through

MTAP Loss, Cancer Cell. 36 (1) (2019) 100–114.e25, https://doi.org/10.1016/j.

ccell.2019.05.014.

[14] K.P. Garnock-Jones, Panobinostat: First Global Approval, Drugs. 75 (6) (2015)

695–704, https://doi.org/10.1007/s40265-015-0388-8.

[15] B.E. Gryder, Q.H. Sodji, A.K. Oyelere, Targeted cancer therapy: giving histone

deacetylase inhibitors all they need to succeed, Futur. Med Chem. 4 (4) (2012)

505–524, https://doi.org/10.4155/fmc.12.3.

[16] A. Saito, T. Yamashita, Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando, T. Suzuki,

T. Tsuruo, O. Nakanishi, A synthetic inhibitor of histone deacetylase, MS-27-275,

with marked in vivo antitumor activity against human tumors, Proc. Natl. Acad.

Sci. U. S. A. 96 (8) (1999) 4592–4597, https://doi.org/10.1073/pnas.96.8.4592.

[17] Z.-Q. Ning, Z.-B. Li, M.J. Newman, S. Shan, X.-H. Wang, D.-S. Pan, J. Zhang,

M. Dong, X. Du, X.-P. Lu, Chidamide (CS055/HBI-8000): a new histone deacetylase

inhibitor of the benzamide class with antitumor activity and the ability to enhance

[38] M.J. Lai, R. Ojha, M.H. Lin, Y.M. Liu, H.Y. Lee, T.E. Lin, K.C. Hsu, C.Y. Chang, M.

C. Chen, K. Nepali, J.Y. Chang, J.P. Liou, 1-Arylsulfonyl indoline-benzamides as a

25

A.A. Al-Hamashi et al.

new antitubulin agents, with inhibition of histone deacetylase, Eur. J. Med. Chem.

Bioorganic Chemistry 116 (2021) 105297

inhibitor, MPT0B451, displays anti-tumor ability in human cancer cells in vitro and

in vivo, Front. Pharmacol. 9 (2018) 1–16, https://doi.org/10.3389/

fphar.2018.00205.

162 (2019) 612–630, https://doi.org/10.1016/j.ejmech.2018.10.066.

[39] X. Peng, J. Chen, L. Li, Z. Sun, J. Liu, Y. Ren, J. Huang, J. Chen, Efﬁcient Synthesis

and Bioevaluation of Novel Dual Tubulin/Histone Deacetylase 3 Inhibitors as

Potential Anticancer Agents, J. Med. Chem. 64 (12) (2021) 8447–8473, https://

doi.org/10.1021/acs.jmedchem.1c0041310.1021/acs.jmedchem.1c00413.

s00110.1021/acs.jmedchem.1c00413.s00210.1021/acs.jmedchem.1c00413.

s00310.1021/acs.jmedchem.1c00413.s004 .

[41] L. Zhang, J. Zhang, Q. Jiang, L.i. Zhang, W. Song, Zinc binding groups for histone

deacetylase inhibitors, J. Enzyme Inhib. Med. Chem. 33 (1) (2018) 714–721,

https://doi.org/10.1080/14756366.2017.1417274.

[42] M.P. Phelps, J.N. Bailey, T. Vleeshouwer-Neumann, E.Y. Chen, CRISPR screen

identiﬁes the NCOR / HDAC3 complex as a major suppressor of differentiation in

rhabdomyosarcoma, PNAS. 113 (52) (2016) 15090–15095, https://doi.org/

10.1073/pnas.1610270114.

[40] Y.W. Wu, K.C. Hsu, H.Y. Lee, T.C. Huang, T.E. Lin, Y.L. Chen, T.Y. Sung, J.P. Liou,

W.W. Hwang-Verslues, S.L. Pan, W.C. HuangFu, A novel dual HDAC6 and tubulin

26

Article Doi

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics

DOI: 10.1016/j.bioorg.2021.105297

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2021.105297

Full text of DOI:10.1016/j.bioorg.2021.105297

Products guided by the article

R&D Labs maybe for 875553-56-3

Relevant to this article

Hot Product