N -((1-benzyl-1 H -1,2,3-triazol-4-yl)methyl)arylamide as a new scaffold that provides rapid access to antimicrotubule agents: Synthesis and evaluation of antiproliferative activity against select cancer cell lines

Article abstract of DOI:10.1021/jm1000979

A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example, compound 13e had an IC ₅₀ of 46 nM against MCF-7 human breast tumor cells. Structure-activity relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of tubulin polymerization, (ii) G₂/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii) perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.

Full text of DOI:10.1021/jm1000979

J. Med. Chem. 2010, 53, 3389–3395 3389
DOI: 10.1021/jm1000979
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a New Scaffold that Provides
Rapid Access to Antimicrotubule Agents: Synthesis and Evaluation of Antiproliferative
Activity Against Select Cancer Cell Lines
Jonathan A. Stefely,† Rahul Palchaudhuri,‡ Patricia A. Miller,† Rebecca J. Peterson,†
Garrett C. Moraski,† Paul J. Hergenrother,‡ and Marvin J. Miller*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, and
‡Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, Illinois 61801
Received January 24, 2010
A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed
azide-alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example,
compound 13e had an IC50 of 46 nM against MCF-7 human breast tumor cells. Structure-activity
relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is
important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group
of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the
NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents
such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of
tubulin polymerization, (ii) G2/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii)
perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The
results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible
small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.
Introduction
Herein we report the synthesis, in vitro antiproliferative
activity against select cancer cell lines, and structure-activity
relationships of compounds containing the N-((1-benzyl-1H-
1,2,3-triazol-4-yl)methyl)arylamide scaffold. We also report
mode of action studies based on in silico, in vitro, and cell
culture experiments, which reveal the potent antimicrotubule
activity of this scaffold. The discovery of this scaffold
stemmed from our work on Mycobactin S (1),10 a natural
product produced by Mycobacterium smegmatis that exhibits
antituberculosis activity11 (Chart 1). All synthetic intermedi-
ates encountered during our group’s total synthesis of Myco-
bactin S12 were screened for biological activity. Surprisingly,
benzyl ester 2, a small fragment of the natural product,
exhibited antituberculosis activity similar to that of Myco-
bactin S. Furthermore, in contrast to Mycobactin S,
compound 2 provided a scaffold that was amenable to rapid
structure-activity relationship studies, which led to the discov-
ery of more potent antituberculosis agents.13 While exploring
derivatives of 2, we found that 2-phenyl-oxazole-4-carboxamide
derivative 3 was also active against M. tuberculosis. 2-Phenyl-
oxazole-4-carboxamides are known inhibitors of histone
deacetylase,14 Stat3,15 phosphodiesterase,16 phosphatase,17
thromboxane synthase,18 kinase proteins,19 and known
activators of cellular caspase activity.20 We synthesized
derivatives of 3 to identify a more potent antituberculosis
agent.13 One of the derivatives, compound 4e, had weak
antituberculosis activity, but broader biological screening
serendipitously revealed that 4e and related derivatives
have potent antimicrotubule activity in cancer cells, as
disclosed in this report.
Microtubules, dynamic protein polymers composed of
R-tubulin and β-tubulin heterodimers, are a well-established
cellular target for anticancer drugs.1 Dynamic polymerization
of tubulin is a necessary and tightly controlled process during
mitosis.2 Perturbing microtubule dynamics with small mole-
cules blocks the cell cycle in the metaphase/anaphase transi-
tion and leads to apoptosis.3 Thus, molecules4 that target
tubulin halt rapid cell division, a characteristic of cancer cells.5
This therapeutic strategy has been validated by the clinical
success of antimicrotubule drugs such as paclitaxel, docetaxel,
vincristine, and vinblastine. Nonetheless, neurotoxicityand P-
glycoprotein-mediated drug resistance limit the clinical utility
of these drugs.6 New-generation taxoids, vinca alkaloids, and
other novel chemotypes that modulate microtubule dynamics
have been synthesized in efforts to overcome these limita-
tions.7 For example, small molecule modulators of tubulin
polymerization that do not elicit neurotoxicity in mice have
been identified,8 suggesting that neurotoxicity is not intrinsi-
cally linked to antimicrotubule agents. Nonetheless, few of
these new antimicrotubule agents have produced useful clin-
ical results. The key limitation to the development of new
antimicrotubule drugs is a narrow therapeutic window.9 A
new antimicrotubule scaffold amenable to rapid derivatiza-
tion and combinatorial library synthesis would provide an
exceptional opportunity for the discovery of an efficacious
antimicrotubule agent with an improved therapeutic window.
*To whom correspondence should be addressed. Phone: þ1 574-631-
7571. Fax: þ1 574-631-6652. E-mail: mmiller1@nd.edu.
r
2010 American Chemical Society
Published on Web 03/24/2010
pubs.acs.org/jmc

3390 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Stefely et al.
Chart 1. Structures of Antituberculosis Compounds 2 and 3 and Antimicrotubule Compound 4e, All Derived from a Fragment of
Mycobactin S (1)a
a MIC values indicate in vitro antituberculosis activity against M. tuberculosis H37Rv in GAST medium,21 and IC50 values indicate in vitro
antiproliferative activity against human breast cancer cell line MCF-7.
Scheme 1. Synthesis of 2-Phenyloxazole-4-carboxylic Acid
Scheme 2. Synthesis of N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-phenyl-oxazole-4-carboxamide Scaffold
Results and Discussion
(derived from the corresponding carboxylic acid 7) pro-
vided alkyne 8. With the terminal alkyne precursor in hand,
we turned our attention to the syntheses of azides 10a-e.
Benzyl bromides 9a-e were treated with NaN3 to afford
benzyl azides 10a-e.26 Exposing terminal alkyne 8 to
benzyl azides 10a-e in the presence of catalytic Cu(I)
produced 1,4-disubstituted triazoles 4a-e in high regios-
electivity. Aqueous CuAAC conditions (H2O/t-BuOH, 2:1)
facilitated precipitation of the products, which were
isolated with high purity.24
Chemistry. 2-Phenyl-oxazole-4-carboxylic acid 7 was synt-
hesized according to the protocols shown in Scheme 1.11,12
Coupling benzoyl chloride to serine benzyl ester hydrochlor-
ide afforded β-hydroxy amide 5. Dehydrative cyclization and
oxidation of β-hydroxy amide 5 with diethylaminosulfurtri-
fluoride (DAST) and DBU/BrCCl3 yielded oxazole 6.22
Catalytic hydrogenolysis of the benzyl ester provided
2-phenyl-oxazole-4-carboxylic acid 7.
Our strategy for exploring the chemical space around the
2-phenyl-oxazole-4-carboxamide fragment employed “click
chemistry.”23 More specifically, we selected the Cu(I)-cata-
lyzed azide-alkyne cycloaddition (CuAACa) reaction24 because
of its wide scope, high efficiency, and recognized utility for drug
discovery.25 Following this strategy, N-((1-benzyl-1H-1,2,
3-triazol-4-yl)methyl)-2-phenyl-oxazole-4-carboxamides 4a-e
were synthesized as shown in Scheme 2. Coupling propargyla-
mine to freshly prepared 2-phenyloxazole-4-carboxyl chloride
To explore the structure-activity relationships of the aryl
amide group, a more general N-((1-benzyl-1H-1,2,3-triazol-
4-yl)methyl)arylamide scaffold was synthesized according to
the protocols shown in Scheme 3. The simplicity of reaction
Schemes 2 and 3 is anticipated to allow the synthesis of a
large library of 1,2,3-triazole-based structures. Moreover,
the CuAAC reaction is the convergent synthetic step. Both
the arylamide and benzyl groups, attached to opposite sides
of the central triazole, are important components of the
pharmacophore, as shown by the structure-activity studies
described below. The convergence of the synthesis will allow
both sides of the scaffold to be systematically varied in future
SAR studies.
a Abbreviations: CuAAC, copper-catalyzed azide-alkyne cycloaddi-
tion; MIC, minimum inhibition concentration to kill 90% of the
bacterium; GI50, 50% growth inhibition; TGI, total growth inhibition;
LC50, 50% lethal concentration, IC50, half-maximal inhibitory concen-
tration; TB, Mycobacterium tuberculosis; GAST, medium of glycerol-
alanine-salts-Tween 80 without added iron; DBU, 1,8-Diazabicyclo-
[5.4.0]undec-7-ene; SEM, standard error of the mean.
In Vitro Antiproliferative Activity. The antiproliferative
activity of compounds 4a-e against cancer cells was

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3391
Scheme 3. Synthesis of N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide Scaffold
Table 1. In Vitro Antiproliferative Activity of Compounds 4a-e,
Colchicine, and 2-Methoxyestradiol against Human Breast Cancer Cell
Line MCF-7
Table 2. Antiproliferative Activities (IC50) of Compounds 4e, 13a-f,
14, Colchicine, and 2-Methoxyestradiol against Human Breast Cancer
Cell Line MCF-7 and Human Lymphoma Cell Line U937
IC50 (μM)
MCF-7
compd
R1
4a
4b
4c
4d
4e
H
p-CH3
15.9
7.59
7.33
8.35
0.56
0.013
0.84
p-CF3
m-OCH3
m-OPh
colchicine
2-methoxyestradiol
discovered during broad biological screening of a series of
compounds originally anticipated to have antituberculosis
activity. Although compounds 4a-e had negligible anti-
tuberculosis activity, they inhibited the proliferation of
cancer cells in vitro. Therefore, we determined the antipro-
liferative activities (IC50 values) of hit compounds 4a-e
against the breast tumor-derived cell line MCF-7 (Table 1).
Because the IC50 of 4e (0.56 μM) was significantly lower
than those of 4a-d (IC50 = 7.3-16 μM), we conserved
meta-phenoxy benzyl substitution at triazole N-1 in the
second series of analogues (Scheme 3).
The structure-activity relationships of the triazole C-4
substituent were investigated by changing the carboxamide
group (Table 2). In this SAR study, antiproliferative activity
was investigated with the MCF-7 cell line and human
lymphoma cell line U937. Addition of electron withdrawing
or electron donating groups to the para-position of the
2-phenyloxazole group (13a-c) had a significant effect on
antiproliferative activity, revealing an avenue for future
optimization. Before performing an extensive SAR study
via substitution of the 2-phenyloxazole group, we wanted to
know if the 2-phenyloxazole was necessary for antiprolifera-
tive activity. If simpler aryl groups could replace the
2-phenyloxazole, the three-step synthesis of the 2-phenyl-
oxazole-4-carboxylic acids could be bypassed by using
commercial aryl acids. Thus, we conducted a systematic
truncation of the 2-phenyloxazole-4-carboxamide group
a IC50 values represent the concentration at which the cell count was
inhibited to 50% of that measured in the vehicle control. Error is SEM,
n g 3.
found in 4e. Removing the 2-phenyl group (13d) did not
significantly change the activity. Following this lead, which
suggested that we could use simpler aryl groups, we synthe-
sized 2-pyridyl derivative 13e. Against the MCF-7 cell line,
the IC50 of 13e (46 nM) was significantly lower than that of 4e
(560 nM). Likewise, a significant decrease in IC50 was
observed with the U937 cell line. Therefore, replacing the
2-phenyloxazole group with simpler aryl groups represents a
significant opportunity to improve antiproliferative activity.
Phenyl derivative 13f had improved antiproliferative activity
compared to 4e but was less active than 2-pyridyl derivative 13e.

3392 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Stefely et al.
Table 4. Matrix COMPARE Analysis of 4b, 4c, 4d, and 4ea
compd
4b
4c
4d
4e
4b
4c
4d
4e
1.000
0.600
0.583
0.489
0.600
1.000
0.968
0.475
0.583
0.968
1.000
0.491
0.489
0.475
0.491
1.000
a Matrix values (r values) are Pearson’s correlation coefficients.30
Table 5. Standard COMPARE Analysis of 4ea
rank
compd
r
Based on GI50 Mean Graph
paclitaxel
maytansine
1
2
3
4
5
0.541
0.534
0.500
0.480
0.460
Figure 1. Time and concentration dependence of the antiprolifera-
tive activity of 13e against MCF-7 tumor cells. Time is in terms of
time elapsed after addition of the compound. Cell counts are shown
relative to the cell count observed in the vehicle control 96 h after
addition of the 0.5% DMSO solution.
vincristine sulfate
trimetrexate
soluble Baker’s Antifol
Based on TGI Mean Graph
paclitaxel (hiConc = 10-5 M)
paclitaxel (hiConc = 10-6 M)
paclitaxel (hiConc = 10-4.6 M)
maytansine
1
2
3
4
5
0.654
0.646
0.642
0.621
0.606
Table 3. Antiproliferative Activity of Compounds 4b, 4c, 4d, and 4e
against Selected Cell Lines in the NCI-60 Screen
GI50 (μM)
vinblastine sulfate
compd
mean
HL-60
MCF-7
MDA-MB-435
Based on LG50 Mean Graph
4b
4c
4d
4e
21.9
16.6
17.8
0.87
1.53
2.34
2.37
0.39
8.93
3.83
4.16
0.36
3.71
1.94
2.12
0.18
1
2
3
4
5
rhizoxin (hiConc = 10-9 M)
rhizoxin (hiConc = 10-4 M)
vinblastine sulfate (hiConc = 10-7.6 M)
R-20-deoxythioguanosine
0.681
0.650
0.558
0.545
0.540
vinblastine sulfate (hiConc = 10-4 M)
Replacing the aryl group with a methyl group (14) gave a
meaningful loss of antiproliferative activity and demon-
strated the importance of the aryl carboxamide group.
Time Dependence of In Vitro Cellular Antiproliferative
Activity. To distinguish cytotoxic activity from cytostatic
activity, the time dependence of the effect of 13e on MCF-7
cells was determined (Figure 1). At a concentration of 39 nM,
13e slowed the cell proliferation rate. At higher concentra-
tions (78 nM and 156 nM), cellular proliferation was halted,
but 13e did not decrease the number of cells. Thus, at
concentrations moderately higher than the IC50, 13e is
cytostatic rather than cytotoxic against MCF-7 cells.
Broad-Spectrum In Vitro Antiproliferative Activity. The
NCI-60 anticancer drug screen is an in vitro assay consisting
of 60 different human tumor cell lines.27 Organized by
disease type, the NCI-60 panel includes various leukemia
cell lines and cell lines derived from solid tumor sources. Cell
line selectivity guides further biological evaluation. In the
NCI-60 panel, compounds 4b, 4c, 4d, and 4e induced broad-
spectrum antiproliferative activity against tumor cell lines
derived from leukemia, nonsmall cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal can-
cer, prostate cancer, and breast cancer (see Supporting
Information). Of these four compounds, 4e (NCI-60 mean
GI50 = 870 nM) was more than 20 times more potent than
the other three compounds (Table 3). This result is consistent
with the results from our MCF-7 and U937 assays and
further emphasizes the importance of meta-phenoxy benzyl
substitution of triazole N-1 for optimal activity within this
scaffold. With respect to selectivity among the 60 cell lines in
the NCI-60 panel, all four compounds tested in the panel
showed greater than average potency against human leukemia
cell line HL-60, breast cancer cell line MCF-7, and melano-
ma28 cell line MDA-MB-435 (Table 3).
a The target set was the standard agent database and the target set end
points were set equal to the seed end points (GI50, TGI, and LC50).
Correlation values (r) are Pearson’s correlation coefficients. Some hits
appear multiple times because they were tested by the NCI for the
Standard Agent Database at multiple concentration ranges.
activity measured in the NCI-60 differs by cell line. Further-
more, antitumor agents with similar mechanisms of action can
produce similar patterns of differential antiproliferative data.
We used the matrix COMPARE algorithm29 to measure the
correlations between compounds 4b, 4c, 4d, and 4e with respect
to differential antiproliferative activity. The matrix produced
by the analysis showed that 4c and 4d have highly correlated
activities (r = 0.968) (Table 4). On the contrary, 4e (the most
potent compound tested in the NCI-60) had low correlations (r
< 0.5) with the other three compounds. These matrix COM-
PARE results suggest that 4c and 4d have the same mechanism
of action, but the mechanism of 4e is distinct. Future studies will
further investigate the importance of the meta-phenoxy sub-
stituent found in 4e for its mechanism action. In the studies
reported here, we focused on 4e and related analogues, which
also have the meta-phenoxy substituent, because of their
superior potency.
The COMPARE algorithm can also compare the differ-
ential antiproliferative activity of a new compound to those
of compounds with known mechanisms of action in the NCI
Standard Agent Database.31 Standard COMPARE analysis
has been used previously to identify the cellular targets of
antitumor agents.32 Thus, the pattern of differential anti-
proliferative activity of 4e was used to probe the NCI
Standard Agent Database for correlations. We used all three
measures of activity provided by the NCI-60 screen (GI50,
50% growth inhibition; TGI, total growth inhibition; LC50,
50% lethal concentration). A standard COMPARE analysis
of 4e (Table 5) showed correlations to paclitaxel, maytansine,
vincristine, vinblastine, and rhizoxin, all of which affect
COMPARE Analysis Revealed a Correlation to Anti-
microtubule Drugs. For a given compound, the antiproliferative

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3393
microtubule polymerization. Given this in silico result, we
hypothesized that 4e targets microtubules and directly tested
this hypothesis in vitro. In contrast, the first-ranked COM-
PARE hits for 4b-d did not include antimicrotubule agents
(see Supporting Information).
Inhibition of Tubulin Polymerization In Vitro. The poly-
merization of microtubules from purified tubulin can be
monitored in vitro by measuring an increase in light scatter-
ing. This in vitro experiment removes complicating factors,
such as microtubule-associated proteins (MAPs), which
might be part of a putative target that leads to disruption
of microtubules as observed with microscopy. To test our
hypothesis that the target of the N-((1-benzyl-1H-1,2,
3-triazol-4-yl)methyl)arylamide scaffold is tubulin, and not
a MAP, we monitored the polymerization of tubulin
after treatment with 4e, 13a, and 13e (Figure 2). In this
experiment, paclitaxel, a microtubule stabilizer, enhanced
the rate of tubulin polymerization, while nocodazole, a
microtubule destabilizer, prevented the polymerization of
tubulin. Similar to nocodazole, 4e, 13a, and 13e completely
inhibited tubulin polymerization at 10 μM. Thus, the tria-
zole-based compounds prevent the formation of microtu-
bules in vitro. We followed this in vitro assay with cell culture
experiments to see if microtubules are the primary cellular
target.
Figure 2. Inhibition of tubulin assembly by 4e, 13a, and 13e in
vitro. All compounds were tested at a concentration of 10 μM.
Effects of compounds on tubulin polymerization were assessed by
monitoring the increase in light scattering, measured as optical
density (OD), at 340 nm. Standards of 10 μM nocodazole (a tubulin
assembly inhibitor) and 10 μM paclitaxel (a tubulin assembly
promoter) were used for direct comparison.
Cell Cycle Analysis Demonstrated G2/M-Phase Arrest.
Antimicrotubule agents induce M-phase arrest. Flow cyto-
metry can quantitatively determine the population of cells in
each phase of the cell cycle by measuring the DNA content of
individual cells. Cells in G2-phase or M-phase have twice as
much DNA as cells in G1-phase. Thus, we conducted flow
cytometric cell cycle analysis of HeLa cells treated with 4e, 13a,
13d, 13e, and 14. Consistent with the hypothesized mechanism
of action, compounds 4e, 13a, 13d, and 13e significantly
increased the population of cells in G2/M-phase (Figure 3).
Upon treatment with these four compounds, the population of
G2/M-phase cells increased from 13% in the control to over
90%. Compound 14, however, did not induce significant G2/
M-phase arrest, which suggested that the arylamide moiety is
important for the antimitotic activity of the N-((1-benzyl-1H-
1,2,3-triazol-4-yl)methyl)arylamide scaffold.
Figure 3. Effects of 4e, 13a, 13d, 13e, and 14 on the cell cycle
distribution of HeLa cells as measured by propidium iodide staining
and flow cytometry. HeLa cells were treated with 5 μM compound
for 18 h in triplicate.
Confocal Microscopy Showed M-phase Arrest and Disrup-
tion of Microtubules. Visual evidence for M-phase arrest can
Figure 4. Confocal microscopy images of HeLa cells after 18 h incubation in the presence of 5 μM compound. Nocodazole is a known tubulin
polymerization inhibitor. Nuclear DNA was stained with propidium iodide (red channel), and tubulin was stained with FITC-conjugated anti-
R-tubulin antibody (green channel). Compounds 4e and 13e disrupted normal microtubule structures, caused fragmentation of mitotic
spindles, and induced M-phase arrest.

3394 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Stefely et al.
be obtained with confocal microscopy due to DNA conden-
sation, resulting in enhanced staining by propidium iodide.
This outcome is in contrast to the diffuse staining of DNA in
interphase cells. Visual evidence for the disruption of micro-
tubules can be obtained concurrently using a fluorescein
isothiocyanate-conjugated antitubulin antibody. We there-
fore used confocal microscopy to examine HeLa cancer cells
treated with compounds 4e and 13e. Both DNA condensa-
tion and disruption of microtubules were observed at 5 μM
of 4e or 13e (Figure 4). Together, these images show that 4e
and 13e induce M-phase arrest and interfere with microtu-
bule formation in whole cells.
EtOAc/CH2Cl2 stepwise elution, 1:1 to 10:1) to give 2-(4-
methoxyphenyl)-N-(prop-2-ynyl)oxazole-4-carboxamide (12c)
as an off-white solid (0.788 g, 77%); mp 151-152 °C. 1H
NMR (300 MHz, CDCl3) δ 8.06 (s, 1H), 7.82 (d, J = 8.5 Hz,
2H), 7.13 (bs, NH, 1H), 6.84 (d, J = 8.5 Hz, 2H), 4.15-4.07 (m,
2H), 3.72 (s, 3H), 2.15 (s, 1H). 13C NMR (126 MHz, CDCl3) δ
161.82, 161.57, 160.40, 140.49, 136.44, 128.83, 128.29, 119.13,
114.26, 113.69, 79.17, 71.69, 55.37, 28.66. HRMS-FAB (m/z)
[M þ H]þ calcd for C14H12N2O3, 257.0921; found, 257.0930.
2-(4-Methoxyphenyl)-N-(prop-2-ynyl)oxazole-4-carboxa-
mide (12c, 300 mg, 1.17 mmol) and 1-(azidomethyl)-3-phenoxy-
benzene (290 mg, 1.29 mmol) were suspended in a 2:1 mixture of
water and tert-butyl alcohol (4.7 mL total volume). Sodium
ascorbate (0.12 mmol, 0.12 mL, 1 M) and copper(II) sulfate
(0.012 mmol, 0.12 mL, 0.1 M) were added sequentially. After
stirring for 4 days at room temperature, TLC analysis indicated
complete consumption of the reactants. The reaction mixture
was diluted with water (5 mL) and cooled on ice. The white
precipitate was isolated by vacuum filtration and washed with
cold water (3 ꢀ 5 mL) and cold diethyl ether (3 ꢀ 3 mL) to afford
506 mg (90%) of pure product (13c) as a white powder. TLC
Rf = 0.42 (EtOAc); HPLC tr = 6.82 min (9:1 hexanes/2-
Conclusion
In summary, we identified N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)arylamide as a novel and proprietary33 small mole-
cule scaffold for potential antitumor agents. Elucidating
structure-activity relationships by subtraction from initial
hit compound4e(MCF-7IC50 = 560 nM) led tothe discovery
of 13e (MCF-7 IC50 = 46 nM), a foundational compound for
further study. Compound 13e (and related compounds) in-
duced M-phase arrest in HeLa cells at 5 μM and inhibited
tubulin polymerization in vitro at 10 μM, providing strong
support for antimicrotubule activity as the primary mecha-
nism of action. The NCI-60 screen demonstrated broad-
spectrum antitumor activity and prompted further biological
evaluation. Compound 13c was recently evaluated by the
National Cancer Institute Developmental Therapeutics Pro-
gram (NCI DTP) for acute toxicity in vivo, and 100, 200, and
400 mg/kg intraperitoneal (IP) doses were well tolerated in
nontumor-bearing mice. Ongoing studies in collaboration
with the NCI DTP will evaluate in vivo efficacy in hollow
fiber assays.34 Extensive SAR studies and the development of
a combinatorial library are accessible because compounds
based on the N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)aryl-
amide scaffold are readily synthesized with the CuAAC re-
action. Our findings will facilitate the design and optimization
of potent, cell-permeable antimicrotubule agents.
1
propanol); mp 119.1-119.4 °C. H NMR (300 MHz, CDCl3)
δ 8.18 (s, 1 H), 7.99-7.93 (m, 2 H), 7.60 (bs, NH, 1 H), 7.55 (s, 1
H), 7.39-7.28 (m, 3 H), 7.16-7.09 (m, 1 H), 7.03-6.90 (m, 7 H),
5.47 (s, 2 H), 4.72 (d, J = 6.1 Hz, 2 H), 3.88 (s, 3 H). 13C NMR
(151 MHz, CDCl3) δ 161.83, 161.56, 160.84, 158.04, 156.36,
145.05, 140.26, 136.70, 136.29, 130.46, 129.85, 128.31, 123.79,
122.46, 122.27, 119.26, 119.18, 118.56, 118.04, 114.28, 55.40,
53.84, 34.46. HRMS-FAB (m/z) [Mþ H]þ calcd for C27H23N5O4,
482.1823; found, 482.1803.
Acknowledgment. Initial support by the NIH (R01 AI
05419) of the antituberculosis research program that gener-
ated the lead compounds is gratefully acknowledged. We
thank the University of Notre Dame, especially the Mass
Spectrometry & Proteomics Facility (Bill Boggess, Nonka
Sevova, Michelle Joyce), which is supported by the by grant
CHE-0741793 from the National Science Foundation, and
Dr. Jaroslav Zajicek for assistance in obtaining mass spectral
and NMR data, respectively. We thank Dr. Kamlesh Gupta
and Prof. Holly Goodson for advice regarding the tubulin
experiments and Dr. Timothy Mitchison for the gift of
purified tubulin. We also thank Dr. Jed Fisher for helpful
discussions. We acknowledge financial support from the
University of Notre Dame (J.A.S. was supported by a Notre
Dame College of Science Summer Undergraduate Research
Fellowship). Wewould alsolike toacknowledge theassistance
of the Cell Flow Cytometry facility of the Biotechnology
Center at the University of Illinois at Urbana-Champaign.
Experimental Section
Purity of allsamples areg95% as determined by HPLC or LC/
HRMS.
2-(4-Methoxyphenyl)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-tria-
zol-4-yl)methyl)oxazole-4-carboxamide (C27H23N5O4, 13c). 2-(4-
Methoxyphenyl)oxazole-4-carboxylic acid (11c, 0.951 g, 4.3 mmol)
was suspended in anhydrous CH2Cl2 (12 mL) under argon. Oxalyl
chloride (0.45 mL, 5.2 mmol) and N,N-dimethylformamide
(20 μL) were added carefully to the mixture because of gas
evolution. The reaction slowly turned to a light-yellow homoge-
neous solution over 3 h. The solution was concentrated in vacuo to
give 2-(4-methoxyphenyl)oxazole-4-carbonyl chloride (1.0 g, 97%)
as an yellow solid, which was used immediately in the next reaction
without characterization.
Supporting Information Available: Materials and methods,
characterization data for all compounds, cell proliferation
curves, cell cycle arrest profiles, and COMPARE analysis
results. This material is available free of charge via the Internet
at http://pubs.acs.org.
2-(4-Methoxyphenyl)oxazole-4-carbonyl chloride (1.0 g,
4.0 mmol) was dissolved in anhydrous CH2Cl2 (15 mL) under
argon and cooled to 0 °C (ice bath). Propargyl amine hydro-
chloride (0.443 g, 4.8 mmol) and N,N-diisopropylethylamine
(2.1 mL, 12.0 mmol) were added with stirring. The reaction was
allowed to warm to room temperature. After stirring for 20 h,
TLC analysis indicated completion of the reaction. The mixture
was poured into a solution of 10% aqueous NaHCO3 and
extracted with CH2Cl2 (2ꢀ). The organic layer was separated,
washed with 10% aqueous NaHCO3 and brine, dried with
Na2SO4, filtered, and concentrated in vacuo. The resultant
crude material was purified by column chromatography (SiO2,
References
(1) Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer
Drugs. Nat. Rev. Cancer 2004, 4, 253–265.
(2) (a) Mitchison, T. J. Microtubule Dynamics and Kinetochore
Function in Mitosis. Annu. Rev. Cell Biol. 1988, 4, 527–549.
(b) Rusan, N. M.; Fagerstrom, C. J.; Yvon, A. C.; Wadsworth, P. Cell
Cycle-Dependent Changes in Microtubule Dynamics in Living Cells
Expressing Green Fluorescent Protein-R Tubulin. Mol. Biol. Cell 2001,
12, 971–980.
(3) Jordan, M. A. Mechanism of Action of Antitumor Drugs that
Interact with Microtubules and Tubulin. Curr. Med. Chem.: Anti-
Cancer Agents 2002, 2, 1–17.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3395
(4) (a) Islam, M. N.; Iskander, M. N. Microtubulin Binding Sites as
Target for Developing Anticancer Agents. Mini-Rev. Med. Chem.
2004, 4, 1077–1104. (b) Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown,
A. T. Tubulin and Microtubules as Targets for Anticancer Drugs. Prog.
Cell Cycle Res. 2003, 5, 309–325. (c) Hamel, E. Antimitotic Natural
Products and Their Interactions with Tubulin. Med. Res. Rev. 1996, 16,
207–231.
(21) GAST medium is glycerol-alanine-salts-Tween 80 medium without
added iron. See, (a) Voss, J. J. D.; Rutter, K.; Schroeder, B. G.; Su,
H.; Zhu, Y.; Barry, C. E. The salicylate-derived mycobactin side-
rophores of Mycobacterium tuberculosis are essential for growth in
macrophages. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1252–1257.
(b) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.; Franzblau,
S. G. Low-Oxygen-Recovery Assay for High-Throughput Screening of
Compounds against Nonreplicating Mycobacterium tuberculosis. Anti-
microb. Agents Chemother. 2007, 51, 1380–1385.
(5) Hanahan, D.; Weinberg, R. A. The Hallmarks of Cancer. Cell
2000, 100, 57–70.
(6) Kuppens, I. E. L. M. Current State of the Art of New Tubulin
Inhibitors in the Clinic. Curr. Clin. Pharmacol. 2006, 1, 57–70.
(7) (a) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T.
Tubulin as a Target for Anticancer Drugs: Agents Which Interact
with the Mitotic Spindle. Med. Res. Rev. 1998, 18, 259–296.
(b) Kiselyov, A.; Balakin, K. V.; Tkachenko, S. E.; Savchuk, N.;
Ivachtchenko, A. V. Recent Progress in Discovery and Development
of Antimitotic Agents. Anti-Cancer Agents Med. Chem. 2007, 7, 189–
208.
(8) Backer, G.; Beckers, T.; Emig, P.; Klenner, T.; Kutscher, B.;
Nickel, B. New small-molecule tubulin inhibitors. Pure Appl.
Chem. 2001, 73, 1459–1464.
(9) Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Past and future of
the mitotic spindle as an oncology target. Curr. Opin. Pharmacol.
2001, 1, 370–377.
(10) (a) Snow, G. A. Mycobactins: Iron-Chelating Growth Factors
from Mycobacteria. Bacteriol. Rev. 1970, 34, 99–125. (b) White, A.
J.; Snow, G. A. Isolation of Mycobactins form Various Mycobacteria:
The Properties of Mycobactins S and H. Biochem. J. 1969, 111, 785–
792.
(11) (a) Maurer, P. J.; Miller, M. J. Total Synthesis of a Mycobactin:
Mycobactin S2. J. Am. Chem. Soc. 1983, 105, 240–245. (b) Hu, J.;
Miller, M. J. Total Synthesis of a Mycobactin S, a Siderophore and
Growth Promoter of Mycobacterium smegmatis, and Determination of
its Growth Inhibitory Activity against Mycobacterium tuberculosis.
J. Am. Chem. Soc. 1997, 119, 3462–3468.
(22) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R.
Synthesis of Functionalized Oxazolines and Oxazoles with DAST
and Deoxo-Fluor. Org. Lett. 2000, 2, 1165–1168.
(23) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry:
Diverse Chemical Function from a Few Good Reactions. Angew.
Chem., Int. Ed. 2001, 40, 2004–2021. (b) Moses, J. E.; Moorhouse,
A. D. The Growing Applications of Click Chemistry. Chem. Soc. Rev.
2007, 36, 1249–1262.
(24) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed
Regioselective “Ligation” of Azides and Terminal Alkynes. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599. (b) Tornøe, C. W.; Christensen,
C.; Meldal, M. Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by
Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of
Terminal Alkynes to Azides. J. Org. Chem. 2002, 67, 3057–3064.
(25) Kolb, H. C.; Sharpless, K. B. The Growing Impact of Click
Chemistry on Drug Discovery. Drug Discovery Today 2003, 8,
1128–1137.
(26) Alvarez, S. G.; Alvarez, M. T. A Practical Procedure for the
Synthesis of Alkyl Azides at Ambient Temperature in Dimethyl
Sulfoxide in High Purity and Yield. Synthesis 1997, 4, 413–414.
(27) (a) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer
Drug Discovery Screen. Drug Dev. Res. 1995, 34, 91–109.
(b) Shoemaker, R. H. The NCI60 Human Tumor Cell Line Anticancer
Drug Screen. Nature Rev. Cancer 2006, 6, 813–823.
(12) Vergne, A. F.; Walz, A. J.; Miller, M. J. Iron chelators from
mycobacteria (1954-1999) and potential therapeutic applications.
Nat. Prod. Rep. 2000, 17, 99–116.
(28) (a) Ross, D. T.; Scherf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.;
Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van de Rijn, M.; Waltham, M.;
Pergamenschikov, A.; Lee, J. C. F.; Lashkari, D.; Shalon, D.;
Myers, T. G.; Weinstein, J. N.; Botstein, D.; Brown, P. O.
Systematic variation of gene expression patterns in human cancer
cell lines. Nat. Genet. 2000, 24, 227–235. (b) Ellison, G.; Klinowska,
T.; Westwood, R. F. R.; Docter, E.; French, T.; Fox, J. C. Further
evidence to support the melanocytic origin of MDA-MB-435. J. Clin.
Pathol.: Mol. Pathol. 2002, 55, 294–299.
(29) (a) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display
and Analysis of Patterns of Differential Activity of Drugs Against
Human Tumor Cell Lines: Development of Mean Graph and
COMPARE Algorithm. J. Natl. Cancer Inst. 1989, 81, 1088–
1092. (b) Zaharevitz, D. W.; Holbeck, S. L.; Bowerman, C.; Svetlik,
P. A. COMPARE: a web accessible tool for investigating mechanisms of
cell growth inhibition. J. Mol. Graphics Modell. 2002, 20, 297–303.
(30) (a) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display
and Analysis of Patterns of Differential Activity of Drugs Against
Human Tumor Cell Lines: Development of Mean Graph and
COMPARE Algorithm. J. Natl. Cancer Inst. 1989, 81, 1088–
1092. (b) Zaharevitz, D. W.; Holbeck, S. L.; Bowerman, C.; Svetlik,
P. A. COMPARE: a web accessible tool for investigating mechanisms
of cell growth inhibition. J. Mol. Graphics Modell. 2002, 20, 297–
303.
(13) (a) Moraski, G. C.; Chang, M.; Villegas-Estrada, A.; Franzblau, S.;
€
Mollmann, U.; Miller, M. J. Structure-Activity Relationship of
New Antituberculosis Agents Derived from Oxazoline and Oxa-
zole Benzyl Esters. Eur. J. Med. Chem. 2010, 45(5), 1703-1716.
(b) Moraski, G. C.; Franzblau, S. G.; Miller, M. Utilization of the Suzuki
Coupling to Enhance the Antituberculosis Activity of Aryl Oxazoles.
Heterocyles 2010, 80, 977–988.
(14) Fennell, K. A.; Miller, M. J. Synthesis of Amamistatin Fragments
and Determination of Their HDAC and Antitumor Activity. Org.
Lett. 2007, 9, 1683–1685.
(15) Siddiquee, K. A. Z.; Gunning, P. T.; Glenn, M.; Katt, W. P.;
Zhang, S.; Schroeck, C.; Sebti, S. M.; Jove, R.; Hamilton, A. D.;
Turkson, J. An Oxazole-Based Small-Molecule Stat3 Inhibitor
Modulates Stat3 Stability and Processing and Induces Antitumor
Cell Effects. ACS Chem. Biol. 2007, 2, 787–798.
(16) Kuang, R.; Shue, H. J.; Blythin, D. J.; Shih, N. Y.; Gu, D.; Chen,
X.; Schwerdt, J.; Lin, L.; Ting, P. C.; Zhu, X.; et al. Discovery of a
highly potent series of oxazole-based phosphodiesterase 4 inhibi-
tors. Bioorg. Med. Chem. Lett. 2007, 17, 5150–5154.
(17) (a) Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.;
Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. A Targeted
Library of Small-Molecule, Tyrosine, and Dual-Specificity Phos-
phatase Inhibitors Derived from a Rational Core Design and
Random Side Chain Variation. Biochemistry 1997, 36, 15965–
15974. (b) Ducruet, A. P.; Rice, R. L.; Tamura, K.; Yokokawa, F.;
Yokokawa, S.; Wipf, P.; Lazo, J. S. Identification of New Cdc25 Dual
Specificity Phosphatase Inhibitors in a Targeted Small Molecule Array.
Bioorg. Med. Chem. 2000, 8, 1451–1466.
(18) Takeuchi, K.; Kohn, T. J.; True, T. A.; Mais, D. E.; Wikel, J. H.;
Utterback, B. G.; Wyss, V. L.; Jakubowski, J. A. Development of
Dual-Acting Agents for Thromboxane Receptor Antagonism and
Thromboxane Synthase Inhibition. 3. Synthesis and Biological
Activities of Oxazolecarboxamide-Substituted ω-Phenyl-ω-(3-pyr-
idyl)alkenoic Acid Derivatives and Related Compounds. J. Med.
Chem. 1998, 41, 5362–5374.
(19) Morwick, T.; Berry, A.; Brickwood, J.; Cardozo, M.; Catron, K.;
DeTuri, M.; Emeigh, J.; Homon, C.; Hrapchak, M.; Jacober, S.
Evolution of the Thienopyridine Class of Inhibitors of IκB Kinase-
β, Part I: Hit-to-Lead Strategies. J. Med. Chem. 2006, 49, 2898–2908.
(20) Tai, V. W. F.; Sperandio, D.; Shelton, E. J.; Litvak, J.; Pararaja-
singham, K.; Cebon, B.; Lohman, J.; Eksterowicz, J.; Kantak, S.;
Sabbatini, P.; et al. Discovery and structure-activity relationship
of 2-phenyl-oxazole-4-carboxamide derivatives as potent apotosis
inducers. Bioorg. Med. Chem. Lett. 2006, 16, 4554–4558.
(31) DTP Human Tumor Cell Line Screen, Standard Agent Database;
Online: http://dtp.nci.nih.gov/docs/cancer/searches/standard_agent.
html.
(32) (a) Bai, R.; Paull, K. D.; Herald, C. L.; Malspeis, L.; Pettit, G. R.;
Hamel, E.; Halichondrin, B; Homohalichondrin, B Marine Nat-
ural Products Binding in the Vinca Domain of Tubulin. J. Biol.
Chem. 1991, 266, 15882–15889. (b) Paull, K. D.; Lin, C. M.; Malspeis,
L.; Hamel, E. Identification of Novel Antimitotic Agents Acting at the
Tubulin Level by Computer-Assisted Evaluation of Differential Cyto-
toxicity Data. Cancer Res. 1992, 52, 3892–3900. (c) Kuo, S. C.; Lee, H.
Z.; Juang, J. P.; Lin, Y. T.; Wu, T. S.; Chang, J. J.; Lednicer, D.; Paull, K.
D.; Lin, C. M.; Hamel, E.; Lee, K. H. Synthesis and Cytotoxicity of
1,6,7,8-Substituted 2-(40-Substituted phenyl)-4-quinolones and Related
Compounds: Identification as Antimitotic Agents Interacting with
Tubulin. J. Med. Chem. 1993, 36, 1146–1156.
(33) Stefely, J. A.; Moraski, G. A.; Miller, M. J. Anti-cancer Com-
pounds, Synthesis Thereof, And Methods Of Using Same. Patent
WO/2009/111502, 2009.
(34) Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott, B. J.;
Mayo, J. G.; Malspeis, L.; Grever, M. R. In vivo cultivation of
tumor cells in hollow fibers. Life Sci. 1995, 57, 131–141.