Design, synthesis, and biological evaluations of 2,5-diaryl-2,3-dihydro-1, 3,4-oxadiazoline analogs of combretastatin-A4

Article abstract of DOI:10.1021/jm901268n

A total of 24 novel 2,5-diaryl-1,3,4-oxadiazoline analogs of combretastatin A-4 (CA-4, 1) were designed, synthesized, and evaluated for biological activities. The compounds represent two structural classes; the Type I class has three methoxy groups on the A ring and the Type II class has a single methoxy group on the A ring. Biological evaluations demonstrate that multiple structural features control the biological potency. Four of the compounds, 2-(3′-bromophenyl)-5-(3″,4″,5″-trimethoxyphenyl) -2-acetyl-2,3-dihydro-1,3, 4-oxadiazoline (91), 2-(2′,5′- dimethoxyphenyl)-5-(3″-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3, 4-oxadiazoline (10h), 2-(3′,4′,5′-trimethoxyphenyl)-5- (3″-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (10i), and 2-(3′,5′-dimethoxyphenyl)-5-(3″-methoxyphenyl)-2-acetyl-2, 3-dihydro-1,3,4-oxadiazoline (10j), have potent antiproliferative activities against multiple cancer cell lines. Mechanistic studies indicate that they retain the microtubule disrupting effects of compound 1, including microtubule loss, the formation of aberrant mitotic spindles, and mitotic arrest. Compound 10i inhibits purified tubulin polymerization and circumvents drug resistance mediated by P-glycoprotein and βIII tubulin expression. The oxadiazoline analog 10i is a promising lead candidate worthy of further investigation.

Full text of DOI:10.1021/jm901268n

J. Med. Chem. 2010, 53, 325–334 325
DOI: 10.1021/jm901268n
Design, Synthesis, and Biological Evaluations of 2,5-Diaryl-2,3-dihydro-1,3,4-oxadiazoline Analogs of
Combretastatin-A4
Lauren Lee,† Lyda M. Robb,‡ Megan Lee,† Ryan Davis,† Hilary Mackay,† Sameer Chavda,† Balaji Babu,† Erin L. O’Brien,‡
April L. Risinger,‡ Susan L. Mooberry,*,‡ and Moses Lee*,†
†Department of Chemistry and the Division of Natural and Applied Sciences, Hope College, Michigan 49423 and ‡Department of Pharmacology,
University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229
Received August 24, 2009
A total of 24 novel 2,5-diaryl-1,3,4-oxadiazoline analogs of combretastatin A-4 (CA-4, 1) were designed,
synthesized, and evaluated for biological activities. The compounds represent two structural classes; the
Type I classhasthree methoxygroups on the A ring and the Type II class hasa single methoxygroup on the
A ring. Biological evaluations demonstrate that multiple structural features control the biological potency.
Four of the compounds, 2-(30-bromophenyl)-5-(300,400,500-trimethoxyphenyl)-2-acetyl-2,3-dihydro-1,3,
4-oxadiazoline (9l), 2-(20,50-dimethoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadia-
zoline (10h), 2-(30,40,50-trimethoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline
(10i), and 2-(30,50-dimethoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (10j),
have potent antiproliferative activities against multiple cancer cell lines. Mechanistic studies indicate that
they retain the microtubule disrupting effects of compound 1, including microtubule loss, the formation of
aberrant mitotic spindles, and mitotic arrest. Compound 10i inhibits purified tubulin polymerization and
circumvents drug resistance mediated by P-glycoprotein and βIII tubulin expression. The oxadiazoline
analog 10i is a promising lead candidate worthy of further investigation.
Introduction
has also been shown to disrupt VE-cadherin signaling in
HUVECs.12 Most recently, it was shown to rapidly upregulate
connective tissue growth factor (CTGF) in microvascular en-
dothelial cells in a cell density dependent manner.13 Upregula-
tion of CTGF, a pleotropic endothelial cell growth factor, was
observed only in subconfluent endothelial cell cultures or in
endothelial cells growing as capillary-like tubules. Prodrug 2-
mediated activation of the CTGF might explain why thin,
unstabilized tumor blood vessels are so susceptible to 1, while
other mature vessels are unaffected. It is not known which of
these compound 1-mediated signaling events are responsible for
the specific antivascular effects leading to rapid catastrophic
tumor vascular shutdown.
Significant efforts have been undertaken to develop analogs
of compound 1 that retain the biological actions of the parent
molecule but provide improved pharmacokinetic properties
and better water solubility.14 Inaddition to prodrug2, notable
analogsundergoingclinicalevaluationsinclude AVE 8062 (3),
the amino acid derivative of AC-7739 (4) and the CA-1 (5)
derivativeCA-1P (6;OXI-4503).10,15 Ourworkin thisareahas
yielded severalnew classes ofanalogsincluding pyrazoles16 (7)
and pyrazolines (8, Figure 1) of 1.17 These derivatives demon-
strate a wide range of potencies. A significant drop in activity
was observed in the pyrazole derivatives as compared to 1.
X-ray crystallography studies of a pyrazole analog of com-
pound 7 showed that it adopted a planar conformation, as
opposed to the nonplanar geometry of compound 1.16 The
structure of 1 contains two substituted benzene rings, referred
to as the A and B rings, which fit into the A and B pockets
within the colchicines binding site on tubulin. Modeling
studies of 1 within the colchicines binding site of tubulin
suggest that a geometrically planar analog of 1 would not
A worldwide effort led by the U.S. National Cancer Institute
to identify anticancer agents from plants led to the identification
of the combretastatins from the African bush willow Combre-
tum caffrum.1,2 Combretastatin A-4 (CA-4,a 1, Figure 1) is the
most potent of the natural combretastatins and early work
showed it inhibits tubulin polymerization and the proliferation
of murine and human cancer cells.3,4 Early reports of the
antitumor activity of the more water-soluble prodrug CA-4P
(2) against murine P388 and B16 tumors were not encouraging.5
However, detailed mechanistic studies showed that compound
1 has specific effects on tumor vasculature, causing rapid
vascular shutdown, leading to central tumor necrosis.6,7 The
effects of prodrug 2 on tumor vasculature have been verified in
human clinical trials.8,9 While compound 2 causes central
hemorrhagic necrosis in animal tumor models, a rim of viable
tumor cells remains, suggesting that the optimal use of 2 would
be in combination with agents that target the tumor periphery.
Compound 2 is currently being evaluated in phase II clinical
trials for activity in anaplastic thyroid carcinoma, nonsmall cell
lung cancer, and ovarian cancer in combination with conven-
tional cytotoxic drugs including paclitaxel, carboplatin, and the
antiangiogenic agent brevacizumab.10 Mechanistically, agent 1
rapidly alters HUVEC morphology, causing cell contraction,
membrane blebbing, and disruption of endothelial monolayer
permeability.11 These effects are potentially mediated by a Rho/
Rho kinase stimulated increase in actin stress fibers. Prodrug 2
*To whom correspondence should be addressed. Phone: 616-395-
8075 (M.L.); 210-567-4788 (S.L.M.). Fax: 616-395-7923 (M.L.); 210-
567-4300 (S.L.M.). E-mail: lee@hope.edu (M.L.); mooberry@uthscsa.
edu (S.L.M.).
r
2009 American Chemical Society
Published on Web 11/06/2009
pubs.acs.org/jmc

326 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Lee et al.
Figure 1. Structures of combretastatin-A4 (CA-4, 1), its water-soluble derivate (CA-4P, 2), its amino analogs (3 and 4), combretastatin-A1
(CA-1, 5), combretastatin-A1 diphosphate (CA-1P, 6), along with the pyrazole (7) and pyrazoline (8) analogs. Two general types of
oxadiazoline analogs were generated, including the Type I compounds 9a-l and the Type II compounds 10a-l.
Scheme 1. Representation of Hydrazone Intermediate and Oxadiazoline Synthesis
allow optimal interactions with this binding site.18,19 It is
reasonable to assume that the pyrazole’s loss of potency can
be attributed to its incompatible conformation within the
colchicines binding site of tubulin. Consistent with this rela-
tionship, pyrazolines have a less planar geometry than the
pyrazoles and are more potent inhibitors of cell proliferation
and antitubulin effects.
A new series of 2,5-diaryl-1,3,4-oxadiazoline analogs were
designed to retain the geometric features of 1. Compared to
the pyrazolines (8), the oxadiazoline moiety should provide an
optimal conformational geometry for interaction with the
colchicine binding site while increasing the number of het-
eroatoms in the core structure. The net effect is an increase in
the polarity of the molecule, which should enhance water
solubility. Accordingly, 24 specific oxadiazoline analogs were
synthesized and they are divided into two types (Figure 1).
Type I compounds (oxadiazoline 9a-l) contain a 3,4,5-tri-
methoxyphenyl group on the A ring and Type II compounds
(oxadiazoline 10a-l) contain a 4-methoxyphenyl moiety on
the A ring. These analogs were synthesized and evaluated for
biological activities. The most potent compounds were eval-
uated in mechanistic studies and attempts have been made to
understand the structure activity relationships (SAR) among
these compounds.
Chemistry
Oxadiazoline analogs were synthesized by adapting a pro-
cedure reported by Ali, Amer, and Abdel-Rahman.20 As an
example, the reaction of a hydrazide (11) with a benzaldehyde
(12) in reflux with water and glacial acetic acid in ethanol for
5 h produced the hydrazone intermediate 13 (Scheme 1). The
reaction mixture was poured into ice water and the precipitate
was collected and dried. When needed, the hydrazones were
purified by recrystallization in aqueous methanol. The yields
of the hydrazones ranged between 60 and 98%. The chemical
structures of the hydrazones were confirmed using 400 MHz
1H NMRand IR. Theoxadiazolineproductsweresynthesized
by refluxing the appropriate hydrazone intermediate in acetic
anhydride for 1 h under dry conditions (Scheme 1). The
reaction mixture was poured into ice-water, and the solid
product was collected and washed with water. Most products
1
were found to be homogeneous by TLC and 400 MHz H
NMR analyses, but when needed, heterogeneous products
were readily purified by silica gel column chromatography
using a methanol/chloroform eluent. Oxadiazoline product
yields ranged from 25-70%. The structures of compounds
9a-l and 10a-l were confirmed using NMR, IR, mass
spectrometry, and elemental analysis. The compounds that

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 327
Figure 2. Molecular ball and stick model of oxadiazoline 10i (A) and cis-4,30,40,50-tetramethoxystilbene (B). Black represents carbon, blue
nitrogen, gray hydrogen, and red oxygen.
Table 1. Sensitivity of L1210 Cells to Oxadiazoline Analogsa
showed a small amount (<5%) of rotational isomers (about
the amide bond) were analyzed by reversed phase HPLC
(Nova Pak C18 3.6 ꢀ 150 mm column using methanol as the
eluent). Each of the compounds showed a single peak with a
retention time of 2-3 min, confirming the purity of the
compounds.
To gain insight into the conformation of the oxadiazoline
analogs, molecular modeling studies on compound 10i were
conducted using MacSpartan, version ’04. The structure and
conformation of 10i was first optimized using molecular
mechanics (MMFF) and molecular dynamics (equilibrium
conformer search option and molecular mechanics). The
optimal structure was further optimized using Hartree-Fock
(equilibriumgeometry optimization usingthe 3-21 Gbasisset)
followed by density function theory (equilibrium geometry
optimization using the B3LYP and 6-31 G* basis set) calcula-
tions. The resulting conformation of analog 10i is shown in
Figure 2A. It is evident that the oxadiazoline-containing
compound adopts a nonplanar geometry similar to the parent
compound 1, unlike the previously reported pyrazole mole-
cules. This result is consistent with earlier results from single
X-ray diffraction studies indicating that the oxadiazoline
structure adopts a puckered conformation.21 In addition,
the conformation of cis-4,30,40,50-tetramethoxystilbene, a de-
hydroxy analog of 1, was also ascertained using the afore-
mentioned modeling method, and a model is given in
Figure 2B. It is worthy to note that both 10i and cis-
4,30,40,50-tetramethoxystilbene possess
a nonplanar or
“twisted” geometry similar to the pyrazolines (8),17 and they
show resemblance to the conformation of 1 itself.16
Biological Results and Discussion
Antiproliferative Effects of Oxadiazoline Compounds on
Murine Cancer Cells. The antiproliferative effects of the 24
compounds were evaluated in murine L1210 leukemia cells
and the IC50 values determined (Table 1). Additionally, the
compounds were evaluated in murine B16 melanoma cells
(data not shown) with general concordance with L1210 data.
The results reveal several interesting points including the
broad range of potencies in this series. The novel oxadiazo-
line compounds are substantially less potent than compound
1 itself, which has reported IC50 values ranging from 0.003 to
0.007 μM3-5 for L1210 and B16 cells. A correlation between
the potencies of the Type I and Type II compounds could be
expected, but this pattern was not observed. Not only is there
no trend in L1210 potency with A ring alterations but a single
adjustment to the B ring rarely affects a Type I compound in
the same way that it affects a Type II compound. For
example, a 30-acetyl, 40-methoxy B ring substitution in 9e
results in a compound with an IC50 of 3.9 μM while this same
substitution in the Type II compound (10e) results in a totally
inactive compound (IC50 > 100 μM). To explain these
a The standard deviation for the IC50 values is within 5-20% of the
reported values.
inconsistencies, one possibility is to consider that compound
1 can bind in two orientations within the colchicine binding
site of tubulin. For this reason the SAR of analogs of 1 gains
complexity, as the B ring could orient within the standard A
ring pocket of the binding site, if it results in a more
energetically favorable conformation. Consequently, the
effect of a single ring manipulation was less predictable
and the methodical alterations of the A and B ring substit-
uents were considered.
Compound 9i, with 300,400,500-trimethoxy A ring and
30,40,50-trimethoxy B ring substitutions, was found to be
entirely inactive (IC50 >100 μM), suggesting that this sym-
metric compound with two trimethoxy phenyl groups does

328 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Lee et al.
not interact well within the colchicine binding site. Com-
pound 1,with the 300,400,500-trimethoxy A ring is very potent,4
implying the unfavorable nature of the 30,40,50-trimethoxy B
ring. At the other extreme, 10d, a Type I compound with only
a 400-methoxy on the A ring and a 40-methoxy substitution on
the B ring is also inactive (IC50 > 100 μM). Interestingly,
shifting the 40-methoxy to the 30-position (10f) on the Type II
compound improves potency (IC50 41.0 μM). The methoxy
group in the 20-position gives the highest potency of these
three positions (10g, IC50 5.8 μM). Together, these data
suggest that a 4-methoxy substituted ring interacts poorly
with one pocket of the binding site. Because of the potency of
10i (IC50 0.5 μM), which contains a trisubstituted methoxy
ring that is predicted to only favorably interact with the A
site, the single methoxy-substituted ring must favorably
interact with the B site. This indicates that Type II com-
pounds might position the 400-methoxy A ring in the B ring
pocket of the colchicine binding site on tubulin.
Table 2. Antiproliferative and Microtubule Depolymerizing Effects of
Oxadiazoline Analogsa
IC50 (μM) ( SD EC50 (μM) microtubule
ratio
EC50/IC50
compound
MDA-MB-435
depolymerization
9e
9l
3.3 ( 0.2
10.2
1.5
3.1
10.0
6.5
3.6
9.1
2.3
0.15 ( 0.01
0.20 ( 0.02
0.14 ( 0.01
0.11 ( 0.01
0.003 ( 0.0002
10h
10i
10j
1
1.3
0.5
1.0
0.007
a The antiproliferative actions (IC50) of the oxadiazoline analogs and
compound 1 were measured using the SRB assay (n = 3-5) and EC50
values using indirect immunofluorescence techniques in at least three
independent experiments.
Compounds 10h and 10j have a 20,50-dimethoxy and a
30,50-dimethoxy substituted B ring, respectively. These com-
pounds are potent, with IC50 values of 500 nM. Compounds
9h and 9j have very different potencies (9h, IC50 55.0 μM; 9j,
IC50 5.9 μM). The 20,50-dimethoxy B ring on 9h would be
expected to interact poorly with the B ring pocket, whereas
the 30,50-dimethoxy B ring on 9j favorably interacts with the
binding site.
Figure 3. Effects of 10i on cellular microtubules. A-10 cells were
treated for 18 h with vehicle (A) or 1 μM 10i (B) and cellular
microtubule structures visualized with indirect immunofluores-
cence techniques. Normal interphase microtubules are visible in
panel A. The 10i-induced loss of interphase microtubules is shown
in panel B.
The pattern observed with 10d, 10f, and 10g is not
observed in the Type I compounds; 9d, 9f, and 9g and all
have low potencies with IC50s of 57, 45, and 48 μM,
respectively. The 40-methoxy B ring substitution is the least
potent in both Type I and Type II compounds. In an attempt
to improve 9d and 10d, the 40-methoxy substitution was
replaced with a 40-nitro group (9a, 10a). This resulted in a
modest improvement in potency of the Type II compound
(10a, IC50 68.0 μM). For the Type I compound, this change
resulted in a dramatic, 13-fold increase in potency (9a IC50
4.2 μM). The Type I compound (9a) demonstrates that the 40-
nitro group substitution is substantially more favorable than
the 40-methoxy in the B ring pocket. Shifting the nitro group
to the 30-position, 10a to 10b, results in a slight increase in
potency (10b, IC50 47.0 μM). In the Type I compound,
however, this change in the position of the nitro group
decreased potency 9-fold (9b, IC50 39.0 μM), suggesting that
in the B-ring pocket a p-nitro substitution is more favorable
than an m-nitro substitution. The 30-nitro substitution on
compound 10b may orient into the A-ring pocket, improving
interaction and biological potency. This is consistent with
the pattern observed with 10d, 10f, and 10g, which suggests a
preferable A-pocket interaction with an ortho or meta sub-
stitution, as compared to the para substitution.
Compounds 9c and 10c, with a 30-nitro, 40-methoxy B ring,
are less potent than a single 30-nitro substituted B ring.
Replacing the 30-nitro with a 30-bromo group produces a
more potent Type I compound (9b, IC50 39.0 μM; 9l, IC50
0.6 μM). In the Type II compounds, the 30-nitro a 30-bromo
group (10b, IC50 47.0 μM; 10l, IC50 37 μM) have similar
activities. Compounds 9k and 10k have no substitution on
the B ring, and they have dramatically different potencies
(9k, IC50 6.4 μM; 10k, IC50 >100 μM). Compound 9k could
favorably position the nonsubstituted ring in the B-ring
pocket, however, the low potency of 10k (>100 μM) is
consistent with the notion that the Type I and Type II
compounds might orient differently within the colchicine
binding site.
Antiproliferative Effects against MDA-MB-435. In the
development of chemical agents with potential application
in human anticancer therapeutics, it was important to de-
termine the activity of the most potent oxadiazolines in
human cancer cells. The five most potent inhibitors of
L1210 cell growth were evaluated in our reference human
cell line, MDA-MB-435, a cell line originally designated as
originating from breast but which has been shown defini-
tively to be derived from the M14 melanoma cell line.22 The
results, presented in Table 2, show that there is excellent
correlation between the L1210 and MDA-MB-435 poten-
cies, with 4 of the 5 compounds having submicromolar
potency against human cancer cells. These compounds were
evaluated further in mechanistic assays to determine if they
retain the microtubule disrupting and antimitotic effects of
the combretastatins.
Microtubule Depolymerizing Activity. With the goal of
ascertaining the mechanism of action of the oxadiazoline
compounds, the effects of 9e, 9l, 10h, 10i, and 10j on
interphase cellular microtubules were evaluated in A-10
smooth muscle cells. This cell line is valuable because the
cells are relatively large and flat, and they arrest in interphase
in response to tubulin binding antimitotics, consistent with
the responses of other smooth muscle cells to these drugs.23
All of the compounds caused a dose dependent loss of
cellular microtubules, similar to the effects of 1. Representa-
tive normal interphase microtubules and the microtu-
bule depolymerizing effects of compound 10i are shown in
Figure 3. A range of concentrations was evaluated for each
compound and the EC50, the concentration required to cause
50% loss of cellular microtubules, as visualized by micro-
scopy, was calculated (Table 2). The ratio of EC50 for
microtubule depolymerization and the IC50 for inhibition
of proliferation provides an indication of tight linkage of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 329
Figure 4. In vitro tubulin polymerization assays. The polymerization of bovine brain tubulin was monitored turbidimetrically by absorbance
at 340 nm at 37 °C in the presence of a range of 10i concentrations.
antiproliferative effects and the compound’s tubulin-depen-
dent mechanisms of action.24,25 Compound 10i had a ratio of
3.5, 9e a ratio of 3.1, 10h a ratio of 6.5, and 1 a ratio of 2.3
(Table 2). The ratios are within the ranges we have seen with
2-methoxyestradiol analogs24 and a large subset of polysub-
stituted pyrrole compounds.25 Interestingly, the other potent
antiproliferative compounds, and 10j and 9l did not demon-
strate correspondingly potent microtubule depolymerization
effects, and their ratios of EC50/IC50 ratios were 8.8 and 10,
respectively (Table 2). This suggests that these compounds
might exert cytotoxicity through mechanisms in addition to
tubulin inhibition as has been shown with similar molecules
that can bind directly to DNA.26
Interaction of Compound 10i with Purified Bovine Brain
Tubulin. The potent antiproliferative and microtubule depo-
lymerizing effects prompted further mechanistic studies on
10i. The effects of 10i on purified tubulin polymerization
were evaluated in a biochemical assay. In this assay, 10i
caused a concentration-dependent inhibition of tubulin po-
Figure 5. Effects of 10i on mitotic spindles and cell cycle distribu-
lymerization (Figure 4). The concentration required to cause
tion. HeLa cells were treated with vehicle (A) or 0.50 μM 10i (B) for
18 h, fixed and cellular structures visualized by indirect immuno-
fluorescence. Cellular microtubules were visualized with a β-tubulin
50% inhibition of tubulin polymerization was 9.6 μM. In
similar assays, 2-3 μM of 1 is needed to cause this effect.4
The tubulin data are consistent with the lower potency of 10i
for cellular microtubule and antiproliferative effects and
strongly implicate a direct interaction of 10i with tubulin.
Antimitotic Effects of Compound 10i. In addition to caus-
ing the loss of cellular microtubules, colchicine, compound 1
and their analogs initiate the formation of highly abnormal
mitotic spindles.27-29 The effects of 10i on mitotic spindles
were evaluated in HeLa cells. HeLa cells, unlike the smooth
muscle cells, arrest in the G2/M phase of the cell cycle in
response to tubulin binding drugs.28,29 Microtubules were
visualized 18 h after treatment with vehicle or 0.5 μM 10i.
Normal bipolar mitotic spindles emanating from γ-tubulin-
containing centrosomes were observed and they aligned
the chromosomes in the metaphase plate (Figure 5A). In
antibody (green), centrosomes using a γ-tubulin antibody (red), and
DNA using DAPI (blue). For the cell cycle distribution studies,
MDA-MB-435 cells were treated for 18 h with vehicle (A) or
0.50 μM 10i then stained with Kirshan’s reagent and evaluation
by flow cytometry.
contrast, compound 10i initiated the formation of highly
aberrant, multipolar mitotic spindles (Figure 5B). At least
seven mitotic spindles, three of which contained γ-tubulin,
were visible in a single representative focal plane (Figure 5B).
The DNA was not well organized into a metaphase plate,
suggesting these cells did not have the potential to success-
fully divide into two equivalent daughter cells with normal
complements of DNA.

330 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 3. Sensitivity of Cell Lines Expressing Pgp and βIII Tubulin
Lee et al.
Table 4. Relative Aqueous Solubility of Oxadiazoline Analogsa
cell line
IC50 (μM) ( SD 1
IC50 (μM) ( SD 10i
compound
aqueous solubility (μM)
SK-OV-3
SK-OV-3-MDR 1-6/6a
0.0033 ( 0.0002
0.0035 ( 0.0005
1.8
0.34 ( 0.01
0.45 ( 0.01
1.3
9e
9l
112
66
Rrc
10h
10i
10j
1
259
257
94
HeLa
WTβIIIb
Rrc
0.0029 ( 0.0003
0.0033 ( 0.0008
1.1
0.23 ( 0.03
0.27 ( 0.02
1.2
350
a SK-OV-3-MDR 1-6/6 cells were generated from SK-OV-3 by
adenoviral expression of MDR-1. b HeLa cells were transfected with
the βIII tubulin isotype to generate the WTβIII cells. c The Rr values
were calculated by dividing the IC50 of the genetically engineered line by
the IC50 of the parental cell line.
a The relative aqueous solubilities were measured using a protocol
based on Millipore’s MultiScreen aqueous solubility filter plate assay.
Maximal solubility in this assay is 500 μM.
synthesis and use of the more water-soluble prodrugs 2 and
6. Excellent water solubility would provide a significant
advantage for new analogs of 1. There was a good expecta-
tion that the increased number of heteroatoms in the core
structure of the oxadiazoline analogs would facilitate aqu-
eous solubility. An analytical assay derived from the shake-
flask solubility method was used to evaluate the relative
aqueous solubility of the most potent oxadiazoline analogs.
This assay is rapid and highly reproducible, requires only
small quantities of material, and provides good, but not
perfect, correlation with the shake-flask solubility method.
The results (Table 4) show that there was a wide range of
aqueous solubilities in this subset of compounds. Com-
pounds 10h and 10i have aqueous solubilities in the same
range as compound 1, while compounds 9e, 9l, and 10j have
lower aqueous solubility.
The cell cycle distribution of cells treated with 10i was
further evaluated by flow cytometry to determine whether
the formation of abnormal mitotic spindles caused cells to
accumulate in the G2/M phase of the cell cycle. MDA-MB-
435 cells were treated for 18 h with vehicle or 0.5 μM 10i. A
normal cell cycle distribution for MDA-MB-435 cells was
seen in the vehicle treated cells (Figure 5C). In contrast, in the
10i-treated cells complete G2/M accumulation occurred,
indicative of mitotic blockade. Paclitaxel caused an identical
effect (data not shown). These data, combined with the
cellular microtubule depolymerizing effects and the ability
of 10i to directly inhibit purified tubulin assembly, provide
strong evidence that this oxadiazoline retains the cellular
antitubulin mechanisms of action of 1.
Compound 10i can Circumvent Clinically Relevant Me-
chanisms of Drug Resistance. One major limitation of antic-
ancer drugs is intrinsic or acquired multidrug resistance.
Tumor expression of the ATP-dependent drug efflux pump,
P-glycoprotein (Pgp), is associated with anticancer treatment
failure.30 A second clinically relevant mechanism of drug
resistance to tubulin-targeted agents, including the taxanes
and vinorelbine, is the expression of the βIII isotype of
tubulin.31 Drugs that can overcome these mechanisms of
resistance might have advantages in the treatment of drug
resistant tumors. The ability of the oxadiazoline 10i to
overcome these mechanisms of resistance was tested in
isogenic cell line pairs. The sensitivity of the Pgp expressing
SK-OV-3-MDR-6-1-6/6 cell line was compared to the sensi-
tivity of the parental cell line, SK-OV-3.32 IC50s were mea-
sured for each cell line and relative resistance (Rr) values
were calculated by dividing the IC50 of the genetically
engineered cell line by the IC50 of the parental cell line. In
this cell line pair, the Pgp substrate paclitaxel had an Rr of
860 and the non-Pgp substrate 2-methoxyestradiol had an Rr
value of 2.6.32 As shown in Table 3, 1 has an Rr of 1.8 and 10i
has an Rr of 1.3, suggesting that 10i, like 1, is a poor substrate
for transport by Pgp. The effect of βIII tubulin expression
was evaluated using the WTβIII and HeLa cell line pair.32 In
this cell line pair, paclitaxel, which is susceptible to βIII-
mediated resistance, has an Rr of 4.7, while 2-methoxyestra-
diol, which is unaffected by βIII expression, has a Rr of 1.0.32
The Rr value for compound 10i was 1.2 and a Rr of 1.1 was
measured for compound 1 in these cell lines (Table 3). These
data suggest that the oxadiazoline 10i retains the ability of 1
to overcome drug resistance mediated by the expression of
Pgp and the βIII isotype of tubulin. Therefore, these drugs
would be expected to have advantages in the treatment of
multidrug resistant tumors.
In summary, the synthesis of novel 2,5-diaryl-1,3,4-oxa-
diazolines as antitubulin, antimitotic cytotoxins is described.
Compound 10i exhibits promising antiproliferative activities
and a mechanism of action identical to prodrug 2 in vitro.
The promising activities of this compound will be investi-
gated further with in vivo murine antitumor experiments,
which will be reported in due course.
Experimental Section
Solvents and organic reagents were purchased from Aldrich or
Fisher and were used without further purification. Melting points
(mp) were performed using a Mel-temp instrument, and the
results were uncorrected. Infrared (IR) spectra were recorded
with a Midac M1700 FT-IR instrument as films on KBr discs,
unless stated otherwise. Proton NMR spectra were recorded on a
Varian INOVA 400 MHz Fourier transform spectrometer using
an internal deuterium lock. Chemical shifts are quoted in parts
per million downfield from tetramethylsilane. High-resolution
mass spectra (HRMS) and low-resolution mass spectra (LRMS)
were provided by the Mass Spectrometry Laboratory, University
of South Carolina, Columbia, SC. Elemental analyses were
performed by Midwest Microlab, LLC, Indianapolis, IN. Reac-
tions were monitored using thin-layer chromatography (TLC)
using commercially available precoated plates (Merck Kieselgel
60 F254 silica). Visualization was achieved with UV light at
254 nm or I2 vapor staining. In addition to NMR and elemental
analysis, HPLC analysis was used to determine the purity (>95%)
of the compounds. Compounds were dissolved in methanol
(1.5 mL). A reversed phase Nova Pak C18 3.9 ꢀ 150 mm column
attached to a Perkin-Elmer Series 200 pump coupled to a Hitachi
UV-vis detector was used. Each sample was injected at a
volume of 5 μL, the eluent was methanol and the flow rate was
0.5 mL/min.
Representative Experimental Procedure for the Synthesis of
Oxadiazoline 9d via Hydrazone Intermediate 13. To a stirred
solution of hydrazide 11 (226 mg, 1 mmol) and p-anisaldehyde
(0.12 mL, 1 mmol) in ethanol (25 mL), water (5 mL) was added
followed by dropwise addition of glacial acetic acid (0.2 mL).
The resulting mixture was refluxed for 5 h, after which the
Analytical Measurement To Estimate Aqueous Solubility.
One major limitation of the combretastatins compounds 1
and 6 (CA-1) is low aqueous solubility; this led to the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 331
solution was poured into ice water. The mixture was stirred until
a precipitate formed, which was collected using suction filtration
and dried, followed by recrystallization in aqueous methanol,
giving hydrazone 13 as a pale yellow solid (0.19 g, 54%); Rf
[methanol/chloroform (2:98)] 0.47; mp 74-76 °C; δH (400 MHz,
CDCl3) 8.66 (1H, s br), 8.26 (1H, s), 7.53 (2H, d, J = 8.4), 7.24
(1H, s), 7.06 (1H, s), 6.94 (2H, d, J = 8.4), 3.93 (9H, s), and 3.86
(3H, s); υmax (CH2Cl2)/cm-1 3484, 1645, 1501, 1332, 1126, and
735; LRMS (EIþ) m/z (rel intensity) 344 (20%, Mþ), 211 (35%),
and 195 (100%); HRMS calcd for C18H20N2O5þ, 344.1372
(Mþ); found, 344.1373 (Mþ). 2-(40-Methoxyphenyl)-5-(300,400,
500-trimethoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline
9d was synthesized by addition of acetic acid (2 mL) to hydra-
zone 13 (100 mg, 0.29 mmol), and the resulting solution was
heated to reflux for 1 h. The reaction mixture was poured into ice
water, and the resulting solid product was filtered and washed
with copious amounts of water, followed by drying, to give 9d as
a white solid (74 mg, 66%); Rf [methanol/chloroform (2:98)]
0.31; mp 117-119 °C; δH (400 MHz, CDCl3) 7.41 (2H, dt, J =
8.8, 3.2), 7.11 (2H, s), 7.02 (1H, s), 6.92 (2H, dt, J = 8.8, 3.2),
3.91 (9H, s), 3.81 (3H, s), and 2.35 (3H, s); υmax (KBr disk)/cm-1
2939, 1667, 1583, 1462, 1416, 1233, 1127, 1001, 843, and 694;
LRMS (EIþ) m/z (rel intensity) 386 (30%, Mþ) and 195 (100%);
2-(20-Methoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-2-acet-
yl-2,3-dihydro-1,3,4-oxadiazoline (9g). Yellow powder (63 mg,
56%); Rf [methanol/chloroform (2:98)] 0.35; mp 151-153 °C;
δ
H (400 MHz, CDCl3) 7.38 (1H, t, J = 8.0), 7. 37 (1H, s), 7.32
(1H, d, 8.0), 7.10 (2H, s), 6.96 (1H, d, J = 8.0), 6.98 (1H, t, J =
8.8), 3.91 (6H, s), 3.90 (3H, s), 3.88 (3H, s), and 2.38 (3H, s); υmax
(KBr disk)/cm-1 2916, 2848, 1666, 1583, 1463, 1416, 1248, 1129,
911, 732, and 668; LRMS (EIþ) m/z (rel intensity) 386 (Mþ,
35%) and 195 (100%).
2-(20,50-Dimethoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-2-
acetyl-2,3-dihydro-1,3,4-oxadiazoline (9h). White powder
(40 mg, 36%); Rf [methanol/chloroform (2:98)] 0.37; mp
187-189 °C; δH (400 MHz, CDCl3) 7.33 (1H, s), 7.09 (2H, s),
6.90 (2H, s), 6.87 (1H, s), 3.94 (3H, s), 3.91 (6H, s), 3.89 (3H, s),
3.75 (3H, s), and 2.38 (3H, s); υmax (KBr disk)/cm-1 2917, 2848,
1665, 1613, 1583, 1515, 1462, 1416, 1130, 1033, and 732; LRMS
(EIþ) m/z (rel intensity) 416 (Mþ, 20%) and 195 (100%). Anal.
(C21H24N2O7) C, H.
2-(30,40,50-Trimethoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-
2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (9i). White solid (79 mg,
68%); Rf [methanol/chloroform (2:98)] 0.25; mp 162-163 °C;
δH (400 MHz, CDCl3) 7.12 (2H, s), 6.87 (1H, s), 6.70 (2H, s),
3.93 (3H, s), 3.91 (6H, s), 3.87 (6H, s), 3.85 (3H, s), and 2.39 (3H,
s); υmax (KBr disk)/cm-1 2916, 2848, 1666, 1582, 1503, 1331,
1234, 1127, 911, and 730; LRMS EI m/z (rel intensity) 466 (Mþ,
25%) and 195 (100%).
2-(30,50-Dimethoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-2-
acetyl-2,3-dihydro-1,3,4-oxadiazoline (9j). White powder (22
mg, 20%); Rf [methanol/chloroform (2:98)] 0.46; mp 80-
82 °C; δH (400 MHz, CDCl3) 7.11 (2H, s), 6.99 (1H, s), 6.64
(2H, d, J = 2.0), 6.48 (1H, t, J = 2.0), 3.93 (6H, s), 3.91 (3H, s),
3.79 (6H, s), and 2.37 (3H, s, Ac); υmax (KBr disk)/cm-1 2917,
2848, 2359, 2342, 1662, 1583, 1416, 1250, 1158,1173, 909, and
732; LRMS (ESþ) m/z (rel intensity) 416 (Mþ, 29%) and 195
(100%).
2-Phenyl-5-(300,400,500-trimethoxyphenyl)-2-acetyl-2,3-dihydro-
1,3,4-oxadiazoline (9k). White solid (77 mg, 77%); Rf [methanol/
chloroform (2:98)] 0.75; mp 175-177 °C; δH (400 MHz, CDCl3)
7.49 (2H, dd, J = 7.6, 2.4), 7.42 (3H, m), 7.13 (2H, s), 7.07 (1H,
s), 3.94 (6H, s), 3.91 (3H, s), and 2.37 (3H, s); υmax (KBr disk)/
cm-1 2916, 2848, 1667, 1582, 1462, 1416, 1250, 1127, and 762;
LRMS (EIþ) m/z 356 (rel intensity) (Mþ, 35%), 314 (15%), and
195 (100%).
2-(30-Bromophenyl)-5-(300,400,500-trimethoxyphenyl)-2-acetyl-2,
3-dihydro-1,3,4-oxadiazoline (9l). Off-white solid (60 mg, 54%);
Rf [methanol/chloroform (2:98)] 0.77; mp 134-136 °C; δH (400
MHz, CDCl3) 7.61 (1H, s), 7.54 (1H, d, J = 8.0), 7.44 (1H, d,
J = 8.0), 7.29 (1H, t, J = 8.0), 7.11 (2H, s), 7.01 (1H, s), 3.92
(6H, s), 3.91 (3H, s), and 2.38 (3H, s); υmax (KBr disk)/cm-1
2916, 2850, 1585, 1557, 1504, 1416, 1331, 1233, 1128, and 733;
LRMS (EIþ) m/z 434 (Mþ, 17%), 392 (10%), and 195 (100%);
HRMS calcd for C19H19BrN2O5þ, 434.0478 (Mþ); found,
434.0470 (Mþ). Anal. (C19H19Br N2O5) C, H.
2-(40-Nitrophenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-
1,3,4-oxadiazoline (10a). Yellow powder (50 mg, 44%); Rf
[methanol/chloroform (2:98)] 0.69; mp 99-101 °C; δH (400
MHz, CDCl3) 8.26 (2H, d, J = 8.9), 7.84 (2H, d, J = 8.9),
7.69 (2H, d, J = 8.8), 7.13 (1H, s), 6.97 (2H, d, J = 8.8), 3.89
(3H, s), and 2.36 (3H, s); υmax (KBr disk)/cm-1 2916, 2848, 1666,
1608. 1520, 1536, 1414, 1367, 1257, 1174, 1066, and 1027; LRMS
(EIþ) m/z 341 (Mþ, 12%), 299 (10%), and 135 (100%).
2-(30-Nitrophenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-
1,3,4-oxadiazoline (10b). Yellow solid (82 mg, 73%); Rf
[methanol/chloroform (2:98)] 0.51; mp 144-146 °C; δH (400
MHz, CDCl3) 8.34 (1H, s), 8.26 (1H, d, J = 8.0), 7.87 (1H, d,
J = 8.0), 7.85 (2H, d, J = 8.8), 7.61 (1H, t, J = 8.0), 7.13 (1H, s),
6.97 (2H, d, J = 8.8), 3.88 (3H, s, OCH3; ArB), and 2.37 (3 H, s,
CH3; Ac); υmax (KBr disk)/cm-1 2916, 2848, 2360, 1665, 1620,
1585, 1540, 1420, 1367, 1275, 1250, 1240, 1130, 1011, and 735;
LRMS m/z 341 (Mþ, 12%), 299 (10%), and 135 (100%).
þ
HRMS calcd for C20H22N2O5
,
386.1478 (Mþ); found,
386.1484 (Mþ).
2-(40-Nitrophenyl)-5-(300,400,500-trimethoxyphenyl)-2-acetyl-2,
3-dihydro-1,3,4-oxadiazoline (9a). Pale yellow solid (47 mg,
73%); Rf [methanol/chloroform (2:98)] 0.27; mp 141-142 °C;
δ
H (400 MHz, CDCl3) 8.28 (2H, d, J = 8.8), 7.69 (2H, d, J =
8.8), 7.14 (1H, s), 7.11 (2H, s), 3.95 (3H, s), 3.94 (6H, s), and 2.38
(3H, s); υmax (KBr disk)/cm-1 2940, 2847, 1666, 1584, 1525,
1505, 1416, 1175, 1233, 1129 1020, 910, and 731; LRMS (EIþ) m/
z (rel intensity) 401 (25%, Mþ), 195 (100%).
2-(30-Nitrophenyl)-5-(300,400,500-trimethoxyphenyl)-2-acetyl-2,
3-dihydro-1,3,4-oxadiazoline (9b). White powder (73 mg, 65%);
Rf [methanol/chloroform (2:98)] 0.44; mp 64-66 °C; δH (400
MHz, CDCl3) 8.35 (1H, s), 8.28 (1H, d, J = 8.4), 7.87 (1H, d,
J = 8.4), 7.65 (1H, t, J = 8.4), 7.14 (1H, s), 7.12 (2H, s), 3.92
(6H, s), 3.91 (3H, s), and 2.39 (3H, s); υmax (KBr disk)/cm-1
2916, 2848, 1662, 1584, 1536, 1504, 1462, 1416, 1351, 1130, 1001,
733, and 633; LRMS (EIþ) m/z 401 (25%, Mþ), 359 (20%), and
195 (100%).
2-(30-Nitro-4-methoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-
2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (9c). White solid (85 mg,
75%); Rf [methanol/chloroform (2:98)] 0.35; mp 176-178 °C;
δ
H (400 MHz, CDCl3) 7.95 (1H, d, J = 2.4), 7.71 (1H, dd, J =
8.4, 2.4), 7.14 (1H, d, J = 8.4), 7.10 (2H, s), 7.04 (1H, s), 3.98
(3H, s), 3.92 (6H, s), 3.91 (3H, s), and 2.37 (3H, s); υmax (KBr
disk)/cm-1 2917, 2846, 1666, 1622, 1578, 1540, 1500, 1468, 1420,
1359, 1250, 1233, 1135, 1001, and 733; LRMS (ESþ) m/z 441
(25%, Mþ), 389 (15%), and 195 (100%).
2-(30-Acetoxy-40-methoxyphenyl)-5-(300,400,500-trimethoxyphe-
nyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (9e). White powder
(73 mg, 65%); Rf [methanol/chloroform (2:98)] 0.35; mp
117-119 °C; δH (400 MHz, CDCl3) 7.38 (1H, d, J = 8.4),
7.14 (1H, s), 7.11 (2H, s), 7.01 (1H, s), 6.98 (1H, d, J = 8.4), 3.92
(6H, s), 3.91 (3H, s), 3.83 (3H, s), 2.35 (3H, s), and 2.30 (3H, s);
υ
max (KBr disk)/cm-1 3100, 2940, 2841, 1769, 1666, 1583, 1513,
1463, 1416; 1271, 1200, 1126, 1002, and 733; LRMS (ESþ) m/z
(rel intensity) 444 (30%, Mþ), 402 (15%), and 195 (100%). Anal.
(C22H24N2O8 H2O) C, H.
3
2-(30-Methoxyphenyl)-5-(300,400,500-trimethoxyphenyl)-2-acet-
yl-2,3-dihydro-1,3,4-oxadiazoline (9f). Yellow solid (16 mg,
14%); Rf [methanol/chloroform (2:98)] 0.45; mp 135-137 °C;
δ
H (400 MHz, CDCl3) 7.36 (1H, s), 7.33 (1H, t, J = 8.0), 7.11
(2H, s), 7.09 (1H, d, J = 8.0), 7.03 (1H, d, J = 8.0), 6.94 (1H, d,
J = 8.0), 3.91 (6H, s), 3.90 (3H, s), 3.82 (3H, s), and 2.39 (3H, s);
υ
max (KBr disk)/cm-1 2917, 2848, 1662, 1613, 1583, 1515, 1463,
1416, 1130, 1033, 732, and 696; LRMS (EIþ) m/z (rel intensity)
386 (25%, Mþ), 344 (15%), and 195 (100%).

332 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Lee et al.
2-(30-Nitro-40-methoxyphenyl)-5-(300-methoxyphenyl)-2-acet-
yl-2,3-dihydro-1,3,4-oxadiazoline (10c). White powder (27 mg,
24%); Rf [methanol/chloroform (2:98)] 0.47; mp 146-148 °C;
2917, 2849, 1731, 1609, 1456, 1257, 1158, 908, and 732; LRMS
(EIþ) m/z (rel intensity) 356 (30%, Mþ), 314 (6%), 179 (8%),
and 135 (100%); HRMS calcd for C19H20N2O5þ, 356.1372
(Mþ); found, 356.1376 (Mþ).
δ
H (400 MHz, CDCl3) 7.96 (1H, s), 7.83 (2H, d, J = 8.8), 7.72
(1H, d, J = 8.6), 7.13 (1H, d, J = 8.6), 6.98 (1H, s), 6.97 (2H, d,
J = 8.8), 3.98 (3H, s), 3.88 (3H, s), and 2.36 (3H, s, CH3); υmax
(KBr disk)/cm-1 2920, 2850, 1667, 1622, 1583, 1536, 1462, 1416,
1279, 1249, 1230, 1087, 1011, and 735; LRMS (ESþ) m/z (rel
intensity) 371 (Mþ, 12%), 329 (8%), and 135 (100).
2-(40-Methoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-di-
hydro-1,3,4-oxadiazoline (10d). Yellow oil (108 mg, 95%); Rf
[methanol/chloroform (2:98)] 0.37; δH (400 MHz, CDCl3) 7.83
(2H, d, J = 8.8), 7.41 (2H, d, J = 8.8), 7.02 (1H, s), 6.95 (2H, d,
J = 8.8), 6.91 (2H, d, J = 8.8), 3.89 (3H, s), 3.80 (3H, s), and 2.34
(3H, s); υmax (KBr disk)/cm-1 2919, 2849, 1630, 1461, 1255, 908,
and 733; LRMS (EIþ) m/z (rel intensity) 326 (Mþ, 18%), 282
(13%), and 135 (100%).
12-(30-Acetoxy-40-methoxyphenyl)-5-(300-methoxyphenyl)-2-
acetyl-2,3-dihydro-1,3,4-oxadiazoline (10e). Colorless oil (68
mg, 59%); Rf [methanol/chloroform (2:98)] 0.40; δH (400
MHz, CDCl3) 7.82 (2H, d, J = 8.9), 7.38 (1H, d, J = 8.4),
7.16 (1H, s), 7.02 (1H, s), 6.96 (2H, d, J = 8.9), 6.86 (1H, d, J =
8.4), 3.87 (3H, s), 3.83 (3H, s), 2.33 (3H), and 2.29 (3H, s); υmax
(KBr disk)/cm-1 3250, 2942, 2840, 1770, 1670, 1580, 1513, 1463,
1420, 1271, 1200, 1130, 1001, and 733; LRMS (EIþ) m/z (rel
intensity) 384 (Mþ, 15%), 342 (15%), 151 (10%), and 135
2-Phenyl-5-(300-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxa-
diazoline (10k). White powder (51 mg, 44%); RF [methanol:
chloroform (2:98)] 0.65; mp 149-151 °C; δH (400 MHz, CDCl3)
7.85 (2H, d, J = 8.8), 7.48 (2H, dd, J = 4.8, 8.0), 7.41-7.28 (3H,
m), 7.07 (1H, s), 6.95 (2H, d, J = 8.8), 3.87 (3H, s), and 2.36 (3H,
s); υmax (KBr disk)/cm-1 2917, 2849, 1630, 1606, 1510, 1461,
1312, 1257, 1174, 908, and 733; LRMS (EIþ; rel intensity) m/z
296 (Mþ, 10%), 254 (8%), 151 (18%), and 135 (100%). Anal.
(C17H16N2O3) C, H.
2-(30-Bromophenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-dihy-
dro-1,3,4-oxadiazoline (10l). White powder (56 mg, 63%); Rf
[methanol/chloroform (2:98)] 0.39; mp 115-117 °C; δH (400
MHz, CDCl3) 7.84 (2H, d, J = 8.8), 7.61 (1H, s), 7.53 (1H, d,
J = 8.0), 7.45 (1H, d, J = 8.0), 7.28 (1H, t, J = 8.0), 7.01 (1H, s),
6.96 (2H, d, J = 8.8), 3.88 (3H, s), and 2.36 (3H, s); υmax (KBr
disk)/cm-1 2917, 2849, 1644, 1557, 1509, 1462, 1425, 1364, 1316,
1256, 1174, 908, and 733; LRMS (EIþ) m/z (rel intensity) 296
(Mþ, 10%), 254 (8%), 151 (18%), and 135 (100%).
Inhibition of L1210 and B16 Cell Growth. Murine leukemia
L1210 cells were obtained from American Type Tissue Culture
Collection (ATCC). The murine melanoma B16-F0 cell line was
obtained from the Cancer Center of the Greenville Hospital
System. The cell lines were grown in Dulbecco’s Modified Eagle
Medium (DMEM, Atlanta Biochemicals) supplemented with
10% fetal bovine serum, HEPES buffer (2 mM, Mediatech
Cellgro), L-glutamine (2 mM, Mediatech Cellgro), and penicil-
lin/streptomycin (50000 units penicillin, 50000 mg streptomy-
cin, Atlanta Biologicals). Cells were maintained at 37 °C in a 5%
humidified CO2 atmosphere. Cultured cells (8000 cells/well)
were plated into 96-well flat-bottom tissue culture plates. The
drug solutions were diluted in DMEM and added to each well (5
μL/well). Quadruplicate wells were prepared for each drug
concentration. The plates were incubated for 72 h at 37 °C in
a 5% CO2 atmosphere. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) was dissolved in PBS (5 mg/mL).
After the indicated cell incubation period, 10 μL stock MTT
solution was added to each well, and the plates were further
incubated for 4 h at 37 °C in a 5% CO2 atmosphere. After this
final incubation, 100 μL acidic isopropanol solution (16 μL 12.1
N HCl in 5 mL isopropanol) was added to each well. The
contents of the wells were mixed and the plates developed at
room temperature for 15 min. The plates were then read on a
Dynatech Plate Reader, utilizing Dynex Revelation 3.2 soft-
ware, at a test wavelenth of 570 nm and a reference wavelength
of 630 nm. Dose response curves were generated and the IC50
values were extrapolated from curves.
Sulforhodamine B (SRB) Assay. The antiproliferative effects
of the oxadiazoline compounds against human cancer cells were
evaluated using the SRB assay33 as previously described.34 The
sources of the MDA-MB-435, SK-OV-3, SK-OV-3 MDR-1-6/6,
HeLa, and WTβIII cell lines and the procedures used to generate
the drug resistant clones have been described.32 Cells were
incubated with compounds for 48 h. Each IC50 value represents
the mean of 3-5 independent experiments, each performed in
triplicate.
Indirect Immunofluorescence and Calculation of EC50 Values.
Cellular structures were visualized in A-10 smooth muscle cells
using indirect immunofluorescence techniques, as previously
described.34 Cells were treated for 18-24 h with vehicle, com-
pound 1, or a wide range of concentrations of the oxadiazoline
analogs. Microtubules were visualized with a β-tubulin anti-
body, centrosomes with a γ-tubulin antibody, and DNA with
DAPI. Images were acquired with a digital camera and compiled
and colorized using NIS Elements AR 3.0 software. The EC50
values for cellular microtubule depolymerization were deter-
mined in A-10 cells by evaluating the microtubule depolymeriz-
(100%). Anal. (C20H20N2O6 2H2O) C, H.
3
2-(30-Methoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-di-
hydro-1,3,4-oxadiazoline (10f). Colorless oil (70 mg, 61%); Rf
[methanol/chloroform (2:98)] 0.44; δH (400 MHz, CDCl3) 7.84
(2H, d, J = 8.8), 7.32 (1H, t, J = 8.0), 7.07 (1H, d, J = 8.0), 7.02
(1H, s), 7.01 (1H, s), 6.94 (2H, d, J = 8.8), 6.92 (1H, d, J = 8.0),
3.86 (3H, s, OCH3; ArA/B), 3.83 (3H, s, OCH3; ArA/B), and 2.33
(3H, s, CH3; Ac); υmax (KBr disk)/cm-1 2916, 2848, 1666, 1463,
1416, 1248, 1129, 1027, 911, and 735; LRMS (EIþ) m/z (rel
intensity) 326 (Mþ, 20%), and 135 (100%).
2-(20-Methoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,3-di-
hydro-1,3,4-oxadiazoline (10g). White solid (290 mg, 93%); Rf
[methanol/chloroform (2:98)] 0.49; mp 187-189 °C; δH (400
MHz, CDCl3) 7.80 (2H, d, J = 8.8), 7.37-7.28 (2H, m), 6.97
(1H, s), 6.96-6.89 (2H, m), 6.91 (2H, d, J = 8.8), 3.86 (3H, s),
3.84 (3H, s), and 2.37 (3H, s); υmax (KBr disk)/cm-1 2916, 2848,
1666, 1583, 1463, 1416, 1249, 1129, 910, 732, and 669; LRMS
(EIþ) m/z (rel intensity) 326 (Mþ, 20%) and 135 (100%).
2-(20,50-Dimethoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,
3-dihydro-1,3,4-oxadiazoline (10h). Colorless oil (65 mg, 57%);
Rf [methanol/chloroform (2:98)] 0.56; δH (400 MHz, CDCl3)
7.80 (2H, d, J = 8.8), 7.31 (1H, s), 6.92 (2H, d, J = 8.8), 6.88
(2H, s), 6.85 (1H, s), 3.86 (3H, s), 3.83 (3H, s), 3.73 (3H, s), and
2.33 (3H, s); υmax (KBr disk)/cm-12920, 2850, 1660, 1614, 1580,
1515, 1461, 1250, 1235, 1173, 1129, 909, and 732; LRMS (EIþ)
m/z (rel intensity) 356 (Mþ, 20%) and 135 (100%); HRMS calcd
for C19H20N2O5þ, 356.1372 (Mþ); found, 356.1365 (Mþ). Anal.
(C19H20N2O5) C, H.
2-(30,40,50-Trimethoxyphenyl)-5-(300-methoxyphenyl)-2-acet-
yl-2,3-dihydro-1,3,4-oxadiazoline (10i). White powder (60 mg,
54%) Rf [methanol/chloroform (2:98)] 0.31; mp 148-150 °C; δH
(400 MHz, CDCl3) 7.84 (2H, d, J = 8.8), 6.97 (1H, s), 6.95 (1H,
s), 6.92 (2H, d, J = 8.8), 6.71 (1H, s), 3.91 (3H, s), 3.89 (3H, s),
3.87 (3H, s), 3.84 (3H, s), and 2.38 (3H, s); υmax (KBr disk)/cm-1
2918, 2849, 1610, 1503, 1463, 1327, 1257, 1174, 1128, 908, and
733; LRMS (EIþ) m/z (rel intensity) 386 (Mþ, 25%) and 135
(100%); HRMS calcd for C20H22N2O6þ, 386.1478 (Mþ); found,
386.1484 (Mþ). Anal. (C20H22N2O6) C, H.
2-(30,50-Dimethoxyphenyl)-5-(300-methoxyphenyl)-2-acetyl-2,
3-dihydro-1,3,4-oxadiazoline (10j). White solid (76 mg, 73%); Rf
[methanol/chloroform (2:98)] 0.48; mp 98-100 °C; δH (400
MHz, CDCl3) 7.82 (2H, d, J = 8.8), 6.98 (1H, s), 6.94 (2H, d,
J = 8.8), 6.63 (2H, d, J = 2.4), 6.47 (1H, t, J = 2.4, CH), 3.86
(3H, s), 3.78 (6H, s), and 2.36 (3H, s); υmax (KBr disk)/cm-1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 333
ing effects of a range of concentrations of each compound by
indirect immunofluorescence. The percent microtubule loss for
each concentration was estimated visually in at least three
independent experiments. A dose-response plot was generated
from the averages of the experiments and the EC50 values were
calculated by linear regression.
In Vitro Tubulin Polymerization. The kinetics of microtubule
assembly in vitro were measured using the tubulin polymeriza-
tion assay kit (Cytoskeleton Inc.) according to manufacturer’s
directions. Briefly, 300 μg of purified bovine brain tubulin was
added to tubulin assembly buffer (80 mM Na-Pipes, pH 6.9,
1 mM EGTA, 1 mM MgCl2, 1 mM GTP, and 5% glycerol) and
the indicated compound in a final volume of 100 μL. Micro-
tubule polymerization was followed by absorbance at 340 nm at
37 °C in a Spectromax Plus 96 well plate spectrophotometer
(Molecular Devices).
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M. D. MDA-MB-435 cells are derived from M14 melanoma cells;
A loss for breast cancer, but a boon for melanoma research. Breast
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Cell Cycle Analysis. MDA-MB-435 cells were incubated for
24 h with 0.05 μM 10i, vehicle, or 0.0125 μM paclitaxel as a
positive control. The cells were harvested, stained with
Krishan’s reagent, and analyzed using a FacsCalibur flow
cytometer. Data were plotted as propidium iodide intensity
versus the number of events.
Aqueous Solubility Assay. Our aqueous solubility assay is a
slightly modified version of the MultiScreen Solubility Filter
Plate protocol developed by Millipore. The assay is based on the
shake-flask solubility method that utilizes UV-vis spectroscopy
for quantification. Maximum aqueous solubility for this assay is
500 μM. The assay is conducted at room temperature at pH 7.4.
Tamoxifen, ketoconazole, and furosemide span the entire range
of aqueous solubilities in this assay (30-500 μM) and were used
as reference standards. Standard curves, covering the range of
3.13-500 μM, were generated for each compound using a 4:1
mixture of aqueous buffer (pH 7.4) to acetonitrile. Each com-
pound was dissolved in DMSO, and the final DMSO concen-
tration was 2.5% (v/v). Each sample was analyzed by UV-vis
spectroscopy at 10 nm increments, from 260-800 nm, to
determine the optimal wavelength and standard curves con-
structed. To measure the aqueous solubility of the reference
standards and the oxadiazoline compounds, a 500 μM solution
of each compound was prepared in aqueous buffer, mixed for 6
h, and briefly centrifuged to pellet any undissolved compound.
A (4:1) mixture of supernatant to acetonitrile was added to a 96-
well acrylic plate and analyzed by UV-vis spectroscopy. The
drug concentration in the supernatant was determined from the
standard curve.
Acknowledgment. The authors thank Conjura Pharma-
ceuticals, LLC, for support of this project (M.L.) and the
Cancer Therapy and ResearchCenter of the UTHSCSA (NCI
P30 CA054174; S.L.M.).
Supporting Information Available: 1H NMR (400 MHz)
spectra of the final compounds 9a-l and 10a-l and elemental
analysis data for 9e, 9h, 9l, 10e, 10h, and 10i are provided. HPLC
tracings of compounds 9b, 9c, 9f, 10c, 10d, and 10f are also
included. This material is available free of charge via the Internet
at http://pubs.acs.org.
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