Phenylcinnamides as novel antimitotic agents

Article abstract of DOI:10.1021/jm901805m

Compound 8H is a phenylcinnamide that induces G2/M-phase cell cycle arrest and cell death in cancer cell lines. Here we show that 8H exerts its cytotoxic activity through disruption of microtubule dynamics in vitro and in cell culture. A series of cinnamide derivatives were synthesized and evaluated, and several new compounds were identified that improve on the activity of the parent compound, with IC₅₀ values for induction of cell death ranging from 1 to 10 μM. Notably, these compounds retain potency in the HL-60/VCR leukemia cell line, which is resistant to antimitotic cancer drugs vincrisitine and paclitaxel through up-regulation of P-glycoprotein drug efflux pumps. As P-glycoprotein expression is often responsible for drug resistance in cancer and the exclusion of compounds from the central nervous system, 8H and its derivatives merit further examination as potential antimitotic therapeutics, specifically for brain cancers and cancers that are resistant to standard antimitotic agents.

Full text of DOI:10.1021/jm901805m

3964 J. Med. Chem. 2010, 53, 3964–3972
DOI: 10.1021/jm901805m
Phenylcinnamides as Novel Antimitotic Agents
Benjamin J. Leslie, Clinton R. Holaday, Tran Nguyen, and Paul J. Hergenrother*
Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana—Champaign, 600 S. Mathews Avenue,
Urbana, Illinois 61801
Received December 5, 2009
Compound 8H is a phenylcinnamide that induces G2/M-phase cell cycle arrest and cell death in cancer
cell lines. Here we show that 8H exerts its cytotoxic activity through disruption of microtubule dynamics
in vitro and in cell culture. A series of cinnamide derivatives were synthesized and evaluated, and several
new compounds were identified that improve on the activity of the parent compound, with IC50 values
for induction of cell death ranging from 1 to 10 μM. Notably, these compounds retain potency in the
HL-60/VCR leukemia cell line, which is resistant to antimitotic cancer drugs vincrisitine and paclitaxel
through up-regulation of P-glycoprotein drug efflux pumps. As P-glycoprotein expression is often
responsible for drug resistance in cancer and the exclusion of compounds from the central nervous
system, 8H and its derivatives merit further examination as potential antimitotic therapeutics,
specifically for brain cancers and cancers that are resistant to standard antimitotic agents.
Introduction
been argued that microtubules represent the single most im-
portant protein target for anticancer therapy.3-5
Microtubules are large, dynamic copolymers comprised of
alternating R- and β-tubulin subunits that form long, tubular,
rodlike structures inside eukaryotic cells. Their highly ordered
structures, rigidity, and their ability to grow and shrink via
polymerization/depolymerization mechanisms are critical to
their function in several cellular processes, including the
maintenance of cell shape, motility, and cell signaling.1 Per-
haps the most important role of microtubules is during mitosis,
where they serve to organize and segregate chromosomes.
Several classes of small molecules are known that perturb
microtubule dynamics (and thus mitosis) by binding tubulin
or microtubules (Figure 1). Compounds that inhibit tubulin
polymerization and destabilize microtubules include colchi-
cine (1), the combretastatins (2), the cryptophycins, estramus-
tine, the halichondrins, nocodazole and the Vinca alkaloids
(3, 4). Compounds that stabilize microtubules include disco-
dermolide, eleutherobins, the epothilones, laulimalide, the
sarcodictyins, and the taxanes (5).1-3 Both microtubule stabi-
lizers and destabilizers alter the tubulin-microtuble equilibri-
um causing mitotic arrest and ultimately apoptotic cell death.
Because of the clinical success of microtubule-affecting com-
pounds such as paclitaxel, the Vinca alkaloids, and epothilone
derivatives in the treatment of a wide variety of cancers, it has
These antimitotic drugs, however, are not without limita-
tions. Many, including paclitaxel (5) and the Vinca alkaloids
(3 and 4), are large (MW>700 Da) natural products that dis-
play ADME-Toxa shortcomings (including poor water solu-
bility, bioavailability, and significant dose-limiting toxicity).
In addition, these large natural products are substrates for
the P-glycoprotein (P-gp) drug efflux pump, and multidrug
resistance (MDR) after drug exposure is a common problem
observed with this class of compounds.2,6-9 Furthermore,
drugs such as taxanes are typically poor chemotherapeutics
for the treatment of many brain cancers, as high levels of P-gp
in the blood-brain barrier (BBB) and the chemical properties
of the molecules themselves prevent significant accumulation
ofdrug inthe brain. Because ofthese factors, therehasbeen an
intense search for more effective antimitotics.1,2,5,8,10-16
We report herein the characterization of a novel anticancer
compound, 8H, and its derivatives, which cause cell death
through destabilization of microtubules. These low molecular
weight compounds are readily synthesized, show efficacy in
cell lines resistant to conventional antimitotics, and are pre-
dicted to have increased blood-brain barrier (BBB) perme-
ability relative to many clinically used antimitotics.
Results
*To whom correspondence should be addressed. Phone: 1-217-333-
0363. Fax: 1-217-244-8024. E-mail: hergenro@illinois.edu.
We have previously reported the discovery of compound
8H (Figure 1), a small molecule that induces cell death in
cancer cell lines at low micromolar concentrations.17 Struc-
tural features of 8H, including the trimethoxyphenyl moiety
(found in colchicine, combretastatin A-4, and several syn-
thetic derivatives), suggested that 8H exerted cytotoxic action
through microtubule binding and mitotic arrest. Given the
documented need for antimitotics that are not substrates for
P-gp drug efflux, our goal was to determine the mechanism
of 8H-induced cell death, to elucidate structure-activity
a Abbreviations: ADME-Tox, absorption, distribution, metabolism,
excretion, and toxicity; BBB, blood-brain barrier; CNS, central ner-
vous system; DIPEA, diisopropylethylamine; DMAP, 4-(dimethyl-
amino)pyridine; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol
tetraacetic acid; FBS, fetal bovine serum; HATU, 2-(1H-7-azabenzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; IC50
,
half maximal inhibitory concentration; IgG, immunoglobulin G; M-
phase, metaphase; MDR, multidrug resistant; MeOH, methanol; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H-tetrazolium, inner salt; P-gp, P-glycoprotein; PBS, phosphate-
buffered saline; PI, propidium iodide; PIPES, piperazine-1,4-bis(2-ethane-
sulfonic acid); SAR, structure-activity relationship.
r
pubs.acs.org/jmc
Published on Web 04/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3965
Figure 2. Cell cycle analysis of synchronized HeLa cells treated for
9 h with 8H, colchicine, or vehicle. Double thymidine-blocked cells
were released by washing two times in fresh media and plated in
fresh media with compound or vehicle. After 9 h, cells were fixed in
3% formaldehyde-PBS, permeabilized in 90% MeOH, treated
with a primary antibody raised against phospho(Ser10) histone
H3, a mitosis-specific cellular marker, and a green fluorescent
2° antibody, and propidium iodide (PI). (A-D), Alexa-Fluor 488
2° antibody-conjugate fluorescence intensity vs propidium iodide
fluorescence intensity. (A) Vehicle control shows primarily G1-
arrested cells (bottom left quadrant), with no M-phase cells (top
right quadrant). (B) Colchicine (100 nM) produces strong G2/M
arrest (right quadrants), with 59% of cells in M-phase. (C) Com-
pound 8H (25 μM) produces strong G2/M arrest (right quadrants),
with 49% of cells in M-phase. (D) M-Phase fluorescence staining is
specific for cells containing Ser10 phosphorylation on histone H3,
as cells treated with 25 μM 8H but stained with PI and 2° Ab only
(no primary Ab) show strong G2/M arrest (bottom right quadrant)
and low 2° Ab background (top quadrants).
DNA, washed with PBS, and analyzed in a dual-laser flow
cytometer. In this experiment the G2/M population was
positive by PI staining (upper and lower right quadrants),
and M-phase arrested cells were further distinguished from
G2-arrested cells through staining with an antibody to
phospho(Ser10) histone H3. Cells arrested in mitosis exhi-
bited intense fluorescence staining in both the PI channel
(G2/M population) and the AF-488 channel (M-phase cells),
the upper right quadrant of the graphs in Figure 2.
After 9 h of treatment with DMSO vehicle, 57% of the cell
population had returned to the G1 phase (bottom left
quadrant), and less than 1% of the cell population was in
M-phase (top right quadrant, Figure 2A). Treatment with
colchicine caused 59% of the cell population to arrest in
metaphase (top right quadrant), with all but 4% of the
remaining population halted in G2 (Figure 2B). Similarly,
a 9 h treatment with 8H caused significant G2/M arrest
as assessed by propidium iodide staining, with 49% of the
cell population staining positive for histone H3 Ser10 phos-
phorylation, meaning that 8H, like colchicine (1), arrests
cells in mitosis (Figure 2C). Similar results for 8H and
control compounds were obtained in U-937 (human
lymphoma) cells, indicating that mitotic arrest is not limited
to a specific cell line (data not shown). M-Phase fluorescence
staining is specific for cells containing Ser10 phosphoryla-
tion on histone H3, as cells treated with 25 μM 8H but
stained with PI and 2° antibody only (no primary Ab) show
strong G2/M arrest in the PI channel (bottom right quad-
rant) but no staining in the AF-488 channel (top quadrants)
(Figure 2D).
Figure 1. Structures of selected antimitotic compounds.
relationships, and to create more potent versions that would
be suitable for in vivo evaluation.
8H-Induced Cell Death Is Preceded by M-Phase Arrest.
Flow cytometric analysis of DNA content in 8H-treated cells
shows that this compound arrests cells in either the G2- or
M-phase of the cell cycle in a dose-dependent manner.17
To determine if 8H induces arrest in the M-phase of the
cell cycle, we used an antibody specific for Ser10 phos-
phorylation of histone H3, a common mitotic marker.18,19
Double thymidine-blocked G1/S-arrested HeLa cells were
released by washing two times in fresh media and grown in
fresh media in the presence of 25 μM 8H, 100 nM 1, or an
equal volume of DMSO vehicle (Figure 2). After 9 h, cells
were fixed in 3% formaldehyde-PBS, permeabilized with
90% MeOH, treated with a mouse antibody raised against
phospho(Ser10) histone H3, followed by an Alexa-Fluor
488-conjugated goat antimouse 2° antibody. Cells were
then briefly treated with propidium iodide (PI) to stain

3966 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Leslie et al.
Figure 3. Effects of 8H on microtubule dynamics in vivo. (A-E) Representative microscopic images of HeLa cells treated with (A) DMSO,
(B) 10 nM paclitaxel, (C) 100 nM colchicine, and (D-F) 25 μM 8H for 6 h. Note the stray chromosomes that have not progressed to the
metaphase plate in the 8H-treated cells. Chromosomes are stained with propidium iodide (red), and microtubules were visualized with mouse
anti-R-tubulin, FITC conjugate (green). All cells are roughly 15 μm in diameter.
8H Induces Mitotic Arrest in HeLa Cells As Assessed by
Microscopy. As another means of assessing the degree of
M-phase arrest induced by 8H, cells were treated with
compound or vehicle control, the DNA and microtubules
were stained, and cells were imaged using fluorescence con-
focal microscopy. For this experiment HeLa cells were
allowed to adhere to acid-washed glass coverslips and then
treated for 6 h with compound or DMSO vehicle. Next, the
cells were washed, fixed in formaldehyde, and treated with
an indirect immunofluorescent stain for microtubules in
conjunction with PI staining to visualize DNA. Widefield
images were obtained for DMSO- and 8H-treated slides and
manually scored for mitotic index based on DNA staining.
Roughly 2000 cells were analyzed each for DMSO and 8H
treatment over three identical experiments performed on
different days with different cell preparations. A paired
t test was performed to determine if treatment of unsynchro-
nized HeLa cells with 8H caused a statistically significant
increase in the number of mitotic cells observed compared
to DMSO treatment. Mitotic index increase from 2.2% in
DMSO-treated cells (SD = 1.1%, N = 3 with ∼2000 cells
counted each time) to 18.2% in cells treated with 8H (SD =
4.0%, N = 3 with ∼2000 cells counted each time) was highly
statistically significant (t(4)=6.80, two-tail p=0.002). These
data, when combined with the results described in Figure 2,
convincingly demonstrate that 8H treatment induces M-
phase arrest in cancer cells.
8H Induces Mitotic Arrest in HeLa Cells through Disrup-
tion of Microtubule Dynamics. We next tested whether 8H
could directly affect microtubule dynamics in living cells. High-
resolution images of cells treated with DMSO (Figure 3A),
10 nM paclitaxel (Figure 3B), 100 nM colchicine (Figure 3C),
and 25 μM 8H (Figure 3D-F) were obtained on a fluores-
cence confocal microscope. Vehicle-treated cells were ob-
served to be predominantly in interphase, characterized by
diffuse cytoplasmic tubulin staining and diffuse staining of
uncondensed DNA (Figure 3A). However, treatment with 8H
produced a large increase in the amount of mitotic cells,
Figure 4. Effects of 8H on microtubule dynamics. Polymerization
of tubulin at 37 °C in the presence of paclitaxel (10 μM), nocodazole
(10 μM), or 8H (25 and 100 μM) was monitored continuously by
recording the absorbance at 340 nm over 13 min. Data shown are
representative of three independent experiments.
evidenced by nuclear tubulin staining and chromosomal
condensation (Figure 3D-F). The irregularity of the mitotic
cells observed, asevidenced by straychromosomes notaligned
atthe metaphase plate, suggests that8H treatmentarrests cells
in mitosis by affecting microtubule dynamics.1
8H Destabilizes Tubulin Polymerization in Vitro. Although
several new targets have emerged, most M-phase arresting
compounds target tubulin and/or microtubules.1,20-23 We
monitored the spontaneous polymerization of bovine brain
tubulin in the presence of 8H in vitro in order to determine
if the antimitotic properties of 8H were due to direct
interference with microtubule dynamics and whether this
effect was due to the stabilization or destabilization of
microtubule polymerization (Figure 4). In vitro, microtubule
polymerization can be quantitated spectrophotometrically

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3967
Scheme 1a
mechanistic role of 8H as an electrophile, as has been seen
for other antimitotics.26,27 Removal of the methoxy group
from R2 results in compounds (8, 9) with greatly diminished
activity. Likewise, compounds that have alterations to the
3,4,5-trimethoxy motif (23-30) also have reduced activity.
Compounds lacking substitution at R7 showed a complete
lack of activity (23-25, 27, 29, 30), whereas substitution
on R7 alone provided compounds of intermediate potency
(26, 28). The entire phenylcinnamide core structure of 8H
was required for activity, as neither compound 34 nor 35
showed anticancer activity.
a Reagents and reaction conditions: (a) (COCl)2, DMF, anhydrous
€
CH2Cl2, room temp, 12 h; (b) (R5-R9)PhNH2, Hunig’s base, anhydrous
MeCN, DMAP, microwave, 150 °C, 1.5 h ; (c) (R5-R9)PhNH2, Hunig’s
base, HATU, microwave, 150 °C, 1.5 h.
€
In contrast, structural and chemical modifications to the
part of the molecule derived from the carboxylic acid (8-22)
produced a few derivatives that retained or surpassed the
potency of 8H. In particular, compounds with substitution at
R1 (16) and larger groups at R2 (20-22) still induce cell
death. Overall, the most active compounds (8H, 6, 16,
20-22) contained the 3,4,5-trimethoxy substitution derived
from the aniline building block, with ortho- or meta-sub-
stitution on the cinnamic acid side of the molecule, with
increasing bulk in the meta-position leading to the most
potent compounds (20-22).
as an increase in turbidity at 340 nm.24 DMSO vehicle is
known to slightly stabilize microtubules and produces an
intermediate polymerization phenotype.24 As expected, the
microtubule stabilizer paclitaxel induces a strong increase in
turbidity, whereas nocodazole destabilizes growing micro-
tubules such that depolymerization at the (-)-end is faster
than polymerization at the (þ)-end, giving rise to a net
“depolymerization” phenotype and no rise in turbidity
(Figure 4).1 Attempted polymerization of tubulin in the
presence of 8H produced a depolymerization phenotype,
suggesting that 8H and related phenylcinnamides may exert
cytoxicity through destabilization of microtubule polymer-
ization. This effect was maximal at 100 μM, while at 25 μM
an intermediate effect was observed. The 100 μM concentra-
tion of 8H used in this experiment is approximately 1 order of
magnitude higher than its IC50 in cell culture (Tables 2
and 3); however, paclitaxel and nocodazole are routinely
used at concentrations 103 times their IC50 in cell culture for
optimal results in these in vitro tubulin polymerization
experiments.12,13,15
Structure-Activity Relationship and Cytotoxicity of Deri-
vatives of 8H. In an effort to define a structure-activity
relationship (SAR) for 8H and to identify more potent
versions, several derivatives of this compound were synthe-
sized and evaluated. Specifically, compounds that had dif-
ferential substitutions on the two phenyl rings were created,
as was one in which the backbone unsaturation was re-
moved. The general synthetic route to the new derivatives
of 8H is depicted in Scheme 1, and the structures of new
derivatives are shown in Table 1. Amidation was accom-
plished either by treatment of the corresponding acid with
oxalyl chloride and catalytic N,N-dimethylformamide in
CH2Cl2 followed by DMAP-promoted amidation via micro-
wave irradiation at elevated temperature and pressure or via
direct amidation of the acid in the presence of HATU and
DIPEA to provide phenylcinnamides 6-33 in modest yields
after automated purification on a normal-phase medium
pressure liquid chromatography system. Increasing reaction
temperature led to increased percentages of conjugate-addi-
tion side products, whereas decreasing reaction temperature
resulted in decreased product yield.
Predicted Blood-Brain Barrier Penetration. The general
hydrophilicity and large polar surface areas of several classes
of antimitotics contributes to their poor penetration of the
blood-brain barrier (BBB).28 As the number of compounds
for which experimental data on BBB penetration are pub-
lically available is extremely small, computational methods
have been developed to predict a molecule’s ability to enter
the central nervous system (CNS). Many compounds that
penetrate the blood-brain barrier (BBB) share common
features: their calculated partition coefficient octanol/water
(ClogP) values are typically between 1 and 3, and their pre-
dicted topological polar surface area (TPSA) is usually less
2 29
˚
than 90 A . Values for ClogP and TPSA were calcu-
lated using ClogP29 and TPSA30 algorithms. These two values
were substituted into eq 1 to calculate logBB, the predicted
partition coefficient between the brain and the blood.
logBB ¼ ð- 0:0148TPSAÞ þ ð0:152ClogPÞ þ 0:139 ð1Þ
Compounds with predicted values of log BB less than -0.3
are not considered capable of crossing the BBB, while values
greater than zero are predicative of concentration in the
brain higher than in the blood.29,31 As shown in Table 3, the
antimitotic colchicine is not predicted to cross the BBB. In
contrast, the most potent 8H derivative (compound 22) is
predicted to be brain-permeant, with a log BB value of -0.12
(Table 3).6 These values are comparable to those of 2-methoxy-
estradiol, an antimitotic compound currently being investi-
gated for treatment of CNS cancers (Table 3).32-36
8H and Its Derivatives Are Not Substrates of P-Glycopro-
tein (P-gp). In addition to the physical barriers to entry into
the CNS, drug efflux pumps such as P-glycoprotein actively
exclude drugs from the brain. In addition to complicating
treatments of cancers of the CNS, up-regulation of these
same proteins has also been observed in drug-resistant
cancers from several other cell types.37 We tested the cyto-
toxicity of 8H derivatives in the HL-60 (human leukemia)
cell line, as well as in HL-60/VCR, a daughter cell line that
is resistant to vincrisitine and colchicine by virtue of P-gp
up-regulation.38,39 Colchicine (1) is a potent cytotoxin in
HL-60 cells, with an IC50 value of 53 nM (Table 3). As 1 is a
known substrate of P-gp, we found the HL-60/VCR cell
line to be extremely resistant to 1, with IC50 values greater
All compounds were assessed for their ability to induce
death in two human cancer cell lines: U-937 (human lym-
phoma) and HeLa (human cervical cancer) using either the
MTS bioreduction assay (Promega) or sulforhodamine B
biomass assay25 (Table 2). Toxicity of each compound was
typically assessed three times on different days, and IC50 data
are reported as the average ( SD. Compound 8H induces
death in the U-937 and HeLa cell lines with IC50 values of
3.0 ( 1.2 and 11.3 ( 2.3 μM, respectively. The derivatives
lacking the olefin (compounds 31 and 32) are largely devoid
of cell death inducing properties, possibly suggesting a

3968 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Table 1. Structures and Isolated Yields of Cinnamides 6-35
Leslie et al.
than 100 μM. The extreme difference in the sensitivity of
these two cell lines gives resistant:sensitive (R:S) IC50 ratios
greater than 1000:1; i.e., 1 is significantly less cytotoxic in the
HL-60/VCR cell line presumably because P-gp efflux pre-
vents it from reaching threshold concentrations inside the
cell needed to produce microtubule disregulation. In con-
trast, compounds 8H, 6, and 22 retain almost full potency in
the HL-60/VCR cells compared to the parent HL-60 cell line
(R:S ratios of 0.98 to 3.74), indicating that these compounds
are not substrates of P-glycoprotein drug transporters
(Table 3).
Compound 22 Induces M-Phase Arrest. Compound 22, the
most potent compound in its class in all cell lines tested,
retains almost full activity in the HL-60/VCR cell line, with
an IC50 of 5.5 μM, indicating that it is not a substrate of P-gp
(Table 3). This value is comparable to that of 2-methoxy-
estradiol, a compound that binds to the colchicine domain of
tubulin; this compound is currently in phase I and phase II
clinical trials for the treatment of multiple myeloma, glio-
blastoma, multiforme carcinoid, prostate, and breast can-
cers.40 Like the parent 8H, compound 22 causes microtubule
disruption in cell culture (Figure 5). Given that 22 is readily

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3969
Table 2. Inhibition of Growth of U-937 (Lymphoma) and HeLa
(Cervical) Human Cancer Cell Lines
U-937a,c
HeLab,c
compd
IC50 ( SD (μM)
IC50 ( SD (μM)
8H
6
3.0 ( 1.2
13.2 ( 7.5
40.4 ( 9.1
56.7 ( 1.3
45.0 ( 7.1
15.3 ( 2.5
9.7 ( 4.8
>50
11.3 ( 2.3
4.4 ( 1.2
9.8 ( 7.5
18.7 ( 8.0
>50
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
>50
38.9 ( 21.9
ND
>50
Figure 5. Effects of 22 on microtubule dynamics in vivo. (A, B)
Representative microscopic images of HeLa cells treated with
25 μM 22 for 6 h, showing metaphase cells with misaligned
chromosomes (A) and mitotic cells lacking microtubule structures
(B). Chromosomes are stained with propidium iodide (red), and
microtubules were visualized with mouse anti-R-tubulin, FITC
conjugate (green). All cells are roughly 15 μm in diameter.
>50
>50
>50
>50
22.4 ( 22.4
5.1 ( 0.4
>50
6.1 ( 1.34
36.1 ( 6.2
>50
32.0 ( 13.3
>50
>50
6.0 ( 1.4
13.3 ( 0.1
1.8 ( 0.8
>50
2.0 ( 1.7
5.4 ( 1.2
2.1 ( 1.4
ND
synthesized and that possess improved properties (solubility,
stability, toxicity, brain permeability) over those of natural
products. We have therefore synthesized a small library of
derivatives of lead molecule 8H via convenient one- or two-
step protocols to provide useful quantities of compounds
(10-100 mg) in modest yields. We have shown that 8H and
its phenylcinnamide derivatives induce M-phase cell cycle
arrest leading to cell death and cause disruption of micro-
tubule dynamics in vitro and in cell culture. On the basis of its
structural similarity to colchicine and combretastatin A-4, we
hypothesize that tubulin is the primary molecular target of 8H
and its derivatives, causing cell cycle arrest through destabi-
lizationof microtubule assembly, similartoa“depolymerizer”
compound such as colchicine or vincristine.
>50
>50
ND
>50
18.1 ( 6.8
>50
ND
>50
15.9 ( 3.5
>50
>50
20.1 ( 7.0
ND
>50
ND
>50
>50
>50
>50
>50
>50
>50
>50
>50
a Cytotoxicity in human U-937 (lymphoma) cells as assessed by MTS
assay. b Cytotoxicity in human HeLa (cervical cancer) cells as assessed by
SRB assay. c Error equals standard deviation of the mean from at least
three separate experiments. Values without SD represent the average of
two IC50 values generated on separate days. ND = not determined.
One major limitation of antimitotic drugs such as vincris-
tine and paclitaxel is that they are substrates of P-gp efflux
pumps and thus are actively excluded from cell lines that
express P-gp, where it acts to excrete xenobiotics.41 Therefore,
cell types that natively express P-gp, such as epithelial cells of
the gastrointestinal tract, liver, kidneys, and the capillaries of
the brain and gonads, are innately resistant to some degree.
Furthermore the presence of mdr-1 gene transcripts (which
code for P-gp) have been observed in 39 of the 60 cell lines that
comprise the NCI-60 panel of cancer cell lines, and a strong
correlation exists between its expression level and cellular
resistance to several chemical therapeutics.42 Thus, our find-
ing that several compounds containing the phenylcinnamide
core retain potency in the HL-60/VCR cell line, a model cell
line for multidrugresistant cancers, is significant. The fact that
8H and its derivatives do not appear to be substrates of the P-
gp efflux pump, coupled with the fact that calculated logBB
values predict increased brain permeability over several nat-
ural product antimitotics, makes this an exciting new class of
anticancer compounds.
Table 3. Inhibition of Growth HL-60 and HL-60/VCR (Leukemia)
Human Cancer Cell Lines
a Predicted ClogP and log BB values for all compounds calculated
using ClogP and TPSA algorithms (see text). b Cytotoxicity in human
HL-60 and HL-60/VCR (leukemia) cells as assessed by MTS assay.
Error equals standard deviation of the mean from at least three separate
experiments. Values without SD represent the average of two IC50 values
generated on separate days. c Ratio of toxicity in resistant HL-60/VCR
to sensitive HL-60 cell line.
Materials and Methods
Chemistry. Unless otherwise stated chemical reagents were
purchased from Sigma-Aldrich and used without further purifica-
tion. Solvents (HPLC grade, no inhibitors) were passed through
activated alumina in an Integrated Technologies solvent drying
system under UHP N2. All NMR experiments were recorded on
Varian Unity 400 and 500 MHz spectrometers with residual
undeuterated solvent as an internal reference. The following data
are reported: chemical shift (ppm), coupling constant J (Hz),
multiplicity (s=singlet, d=doublet, t=triplet, q=quintet, m=
multiplet), and integration. High-resolution mass spectral data
synthesized via O-alkylation of compound 8, we envision this
as a lead pharmacophore for future molecule development.
Discussion
Antimitotics are an especially important class of anticancer
agents, and there is a great need for compounds that are easily

3970 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Leslie et al.
were recorded on Micromass Q-TOF Ultima hybrid quadrupole/
time-of-flight (ESIþ) and Micromass 70-VSE (EI) mass spectro-
meters at the University of Illinois Mass Spectrometry Labo-
ratory. Thin layer chromatography was visualized by UV or
stained with cerium ammonium molybdate (CAM), ninhydrin,
or DPIP (0.5 mg/mL 2,6-dichloroindophenolate hydrate in
EtOH). Flash chromatography was performed on EMD Bio-
sciences silica gel 60 (230-400 mesh) using reagent-grade solvents.
All new chemical entities were analyzed for purity on Rainin
Dynamax SD-200 or Finnigan LCQ Deca XP HPLCs detecting
eluted solute at 254 nm equipped with an Alltech Alltima analy-
tical C18 column (20 mm ꢀ 2.1 mm ꢀ 3 μm) with an acet-
onitrile-0.1% trifluoroacetic acid in Millipore Milli-Q-filtered
water solvent gradient. All compounds were purified to at least
95% purity before characterization and use in biological assays. In
the event that a compound was not of sufficient purity after flash
chromatography, the compound was repurified via preparatory
HPLC on a Rainin Dynamax SD-200 fitted with a Vydac
218TP1022 (250 mm ꢀ 22 mm ꢀ 5 μm) C18 column.
by aspiration, and cells were resuspended in 100 μL of blocking
solution containing mouse anti-phospho(Ser10) histone H3
antibody at 1:25 dilution and incubated for 30 min at 25 °C.
A portion of the 8H-treated cells (0.5 ꢀ 106) were separated
before treatment and were incubated in blocking solution alone
as a control for nonspecific 2° antibody binding. After incuba-
tion, cells were washed 3ꢀ1 mL in blocking solution and were
resuspended in 200 μL of blocking solution containing goat
antimouse IgG/Alexa-Fluor 488 conjugate (1:1000) and incu-
bated for 30 min at 25 °C. After washing 3 ꢀ 1 mL in blocking
solution, cells were resuspended in 200 μL of PBS containing
100 μg/mL RNase A and incubated for 30 min at 25 °C, at
which point 100 μL of propidium iodide (1 mg/mL in PBS) was
added and incubated for an additional 30 min. Cells were
washed 3 ꢀ 1 mL of PBS, resuspended in 400 μL of PBS, and
immediately analyzed on a BD Biosciences LSR II flow cyto-
meter using a 488 nm excitation laser, monitoring green and red
channels with 530 ( 15 and 695 ( 20 nm bandpass filters,
respectively.
Assessment of Cell Viability via Sulforhodamine B Assay.
Cytotoxicity of compounds against the HeLa cell line was
assessed using the sulforhodamine B (SRB) assay in 96-well
plate format using the optimized protocol of Vichai and
Kirtikara.25 Briefly, 2 μL of a dilution series of each compound
dissolved in DMSO were added to 98 μL of appropriate growth
medium at five replicates per concentration. Then cells sus-
pended in 100 μL of growth medium at a concentration of
5 ꢀ 104 cells/mL were added, and the plate was incubated in a
5% CO2 incubator for 72 h at 37 °C. After 72 h, media were
removed from the plates and cells were fixed by addition of
100 μL of ice-cold 10% (w/v) trichloroacetic acid (TCA) in water
and placed in a refrigerator. After overnight incubation, TCA
was removed by washing plates four times with distilled water,
and plates were allowed to dry at room temperature overnight.
Sulforhodamine B, sodium salt (100 μL of 0.06% w/v solution
dissolved in 1% acetic acid) was added to each well, and the
plates were incubated at room temperature for 30 min, after
which time unbound sulforhodamine B was removed by wash-
ing 4 times with 1% acetic acid. SRB bound to proteins was
released by addition of 200 μL of 10 mM Tris, pH 10.5, and the
absorbance of each well was measured at 510 nm on a Molecular
Devices SpectraMax 384 Plus plate reader after 30 min of
incubation at room temperature. Five replicates of each com-
pound were added at 10 different concentrations ranging from
100 μM to 5 nM in a 96-well plate. Each plate contained internal
positive (known cytotoxic compound at 100 μM) and negative
(DMSO vehicle) controls for calibration of the percentage of
cell death observed in each well, which were used to construct
a dose-response curve and calculate an IC50 by fitting the data
to a four-parameter logistic curve of the equation y = (A - D)/
[1 þ (x/C)B] þ D.
Biological Materials. Purified bovine brain tubulin was a
gift of Professor Tim Mitchison (Harvard Medical School).
Before use, polymerization-competent tubulin was repurified
following Mitchison’s polymerization/depolymerization cycl-
ing protocol and quantitated spectrophotometrically using
ε
280nm = 115 000 M-1 cm-1 43
. MTS/PMS CellTiter 96 cell pro-
liferation assay reagent was purchased from Promega (Madison,
WI). Fetal bovine serum was purchased from Biomeda (Foster
City, CA). FITC-conjugated mouse anti-R-tubulin antibody,
sulforhodamine B sodium salt, formaldehyde (37% solution in
water), glutaraldehyde (50% aqueous solution, photographic
grade) were purchased from Sigma-Aldrich (St. Louis, MO).
Goat antimouse IgG/Alexa-Fluor 488 conjugate and propidium
iodide were from Molecular Probes (Eugene, OR). Goat serum
(10% solution) was from Invitrogen (Carlsbad, CA). Vectashield
mounting medium was from Vector Laboratories (Burlingame,
CA). RPMI-1640 cell culture medium was obtained from the
UIUC School of Chemical Sciences Cell Media Facility. Micro-
titer plates (96-well, tissue culture-treated), microscope slides, no.
1 microscope coverslips, Eppendorf tubes, and all other reagents
were purchased from Fisher (Chicago, IL).
Cell Culture. HeLa and U-937 cell lines were purchased from
American Type Culture Collection (Manassas, VA). HL-60 and
HL-60/VCR cell lines were a generous gift from Professor
Russell J. Mumper (University of North Carolina). For all
experiments, cell lines were cultured in RPMI-1640 supplemen-
ted with 10% FBS and 1% penicillin/streptomycin in tissue-
culture treated flasks and Petri dishes and maintained at 37 °C in
a humidified 5% CO2 incubator.
Flow Cytometric Analysis. HeLa cells were synchronized in
G1/S following a standard double thymidine-block protocol.44
Arrested cells were released by washing three times with thymi-
dine-free medium and immediately harvested by trypsinization,
counted using a hemocytometer, and plated (1.5 ꢀ 106 per plate)
in 10 cm cell-culture-treated Petri dishes containing 12 mL of cell
growth medium. Compound 8H (25 μM), colchicine (100 nM),
cycloheximide (2.5 μM), or an equal volume of DMSO vehicle
was added, and cells were incubated for 9 h at 37 °C in a
humidified 5% CO2 incubator. Cells were rapidly harvested
by scraping, washed with PBS, pH 7.4, and fixed for 10 min in
500 μL of PBS, pH 7.4, containing 3% formaldehyde in sealed
1.7 mL Eppendorf tubes in a 37 °C water bath. After fixation,
cells were incubated on ice for 1 min and centrifuged for 5 min at
200g, and PBS was removed via aspiration. Cells were gently
resuspended in 300 μL of MeOH by dropwise addition of ice-
cold 100% MeOH to a gently vortexed tube. Cell suspensions
were incubated on ice for 30 min and stored overnight at -20 °C
to permeabilize cells. Permeabilized cells were then washed twice
with blocking solution (0.5% BSA in PBS, pH 7.4) to remove
MeOH and resuspended in 500 μL of blocking solution and
incubated at 25 °C for 10 min. Blocking solution was removed
Assessment of Cell Viability via MTS Assay. Cytotoxicity of
compounds against the U-937, HL-60, and HL-60/VCR cell
lines was assessed using the MTS assay according to the
manufacturer’s specifications (Promega). Briefly, an amount
of 2 μL of a dilution series of each compound dissolved in
DMSO was added to 98 μL of appropriate growth medium at
five replicates per concentration. Then cells suspended in 100 μL
of growth medium at a concentration of 1 ꢀ 105 (U-937 and
HL-60) or 1.5 ꢀ 105 (HL-60/VCR) cells/mL were added, and the
plate was incubated in a 5% CO2 incubator at 37 °C. After 72 h,
plates were removed and processed as per the MTS protocol,
wherein a 20 μL solution of MTS ((3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt) and PMS (phenazine methosulfate) in PBS was added
to each well, and plates were returned to a 5% CO2 incubator
until the signal from vehicle-treated cells reached the upper end of
the linear range of the assay (OD ≈ 1.5, roughly 30 min for U-937
and HL-60 cell lines, up to 2 h for HL-60/VCR cell line).
Absorbance of each well was measured at 490 nm on a Molecular

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3971
Devices SpectraMax 384 Plus plate reader. Five replicates of each
compound were added at 10 different concentrations ranging
from 100 μM to 5 nM in a 96-well plate. Each plate contained
internal positive (known cytotoxic compound at 100 μM) and
negative (DMSO vehicle) controls for calibration of the percen-
tage of cell death observed in each well, which were used to
construct a dose-response curve and to calculate an IC50 by
fitting the data to a four-parameter logistic curve of the equation
y = (A - D)/[1 þ (x/C)B] þ D.
Acknowledgment. We thank Professor Russell J. Mumper
(University of North Carolina) for the generous gift of HL-60
and HL-60/VCR cell lines, and Professor Timothy Mitchison
(Harvard Medical School) for the generous gift of soluble
bovine brain tubulin. This work was supported by the Uni-
versity of Illinois. B.J.L. acknowledges The American Che-
mical Society, Division of Medicinal Chemistry, for an ACS
MEDI graduate fellowship.
Tubulin Polymerization Assay. Polymerization of tubulin was
monitored by measuring absorbance at 340 nm in a 384-well
plate Molecular Devices Spectramax 384 Plus spectrophoto-
meter (Sunnydale, CA) preheated to 37 °C. To a 0.5 mL
polypropylene tube on ice was added 40 μL of ice-cold
1.25ꢀ BRB-80 buffer (100 mM PIPES, pH 6.8, 1.25 mM MgCl2,
1.25 mM EGTA, 1.25 μM GTP (added from 100 mM stock
immediately before use), 6.25% v/v glycerol). To individual
aliquots of buffer was also added DMSO or compound in
DMSO (DMSO 0.6% final in buffer, paclitaxel, and noco-
dazole were used at final concentrations of 10 μM, 8H at 25
and 100 μM). To this solution was rapidly added 10 μL of
15 mg/mL polymerization-competent tubulin in ice-cold
500 mM K-PIPES, pH 6.8, 0.5 mM MgCl2, buffer, which had
been thawed on ice immediately before use. Solutions were mixed
rapidly on ice and then immediately transferred to a 384-well
plate. Final concentrations of reagentsin polymerization reaction
were as follows: 80 mM PIPES, pH 6.8, 1.0 mM MgCl2, 1.0 mM
EGTA, 1.0 μM GTP, 5% glycerol, 0.5% DMSO, 3 mg/mL
tubulin. Turbidity at 340 nm corresponding to polymerization
was assessed every minute for 60 min.
Laser Fluorescence Confocal Microscopy. For laser fluores-
cence confocal microscopy, HeLa cells were grown on nitric
acid-washed no. 1 coverslips overnight in a 5% CO2 incubator h
at 37 °C. Compound in DMSO or DMSO vehicle alone (0.2%
DMSO final) was added to the cells at 40% or 70% confluency
and further incubated for 6 h (70% confluent cells) or 16 h (40%
confluent cells). Cells were washed briefly with BRB-80 and
fixed for 10 min with 0.5% glutaraldehyde in BRB-80, then
permeabilized for 15 min in 1% Triton X-100 in PBS. After
washing three times in PBS, pH 8.0, unreacted aldehydes were
reduced with three 7 min incubations of 1 mg/mL NaBH4
dissolved in PBS, pH 8, immediately before use. Cells were given
three rinses with a solution of 0.1% Triton X-100 in PBS, pH 8.0
(PBST), and blocked 20 min in 10% goat serum. FITC-
conjugated anti-R-tubulin was added at a 1:100 dilution in
10% goat serum, and the cells were incubated for 1 h, then
washed 3 ꢀ 10 min in PBST. Goat antimouse IgG/Alexa-Fluor
488 conjugate was diluted 1:200 in 10% goat serum and
incubated with cells for 1 h, then washed three times with PBST.
Cells were incubated in PBS containing 10 μg/mL propidium
iodide and 1 μg/mL RNase A for 15 min, washed twice with
PBS, once with H2O, and mounted onto microscope slides using
8 μL of Vectashield mounting medium and sealed with colorless
nail polish.45 Samples were visualized immediately on a Zeiss
LSM 510 laser scanning confocal microscope, 63ꢀ oil DIC
objective, 1.4 NA. Wide-field images were acquired by moving
the stage to 10-15 random locations on each slide, thereafter
only adjusting the stage in the z-direction to bring a maximal
number of cells into focus.
Supporting Information Available: Additional information on
materials and methods, including protocols for compound
synthesis, NMR spectra of all new compounds, and supporting
figures. This material is available free of charge via the Internet
at http://pubs.acs.org.
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