Novel oxadiazole analogues derived from ethacrynic acid: Design, synthesis, and structure - Activity relationships in inhibiting the activity of glutathione S-transferase P1-1 and cancer cell proliferation

Article abstract of DOI:10.1021/jm9011565

Ethacrynic acid (EA) is a glutathione S-transferase P1-1 (GST P1-1) inhibitor with weak antiproliferative ability in tumor cells. By use of the principle of bioisosterism, a series of novel EA oxadiazole analogues were designed and synthesized. The struct

Full text of DOI:10.1021/jm9011565

J. Med. Chem. 2010, 53, 1015–1022 1015
DOI: 10.1021/jm9011565
Novel Oxadiazole Analogues Derived from Ethacrynic Acid: Design, Synthesis, and Structure-Activity
Relationships in Inhibiting the Activity of Glutathione S-Transferase P1-1 and Cancer Cell
Proliferation
Xinmei Yang,† Guyue Liu,‡ Hongcai Li,† Yun Zhang,† Dandan Song,‡ Chunmin Li,† Rui Wang,§ Bo Liu,† Wen Liang,†
Yongkui Jing,§ and Guisen Zhao*,†
†School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, P. R. China, ‡Shenyang Pharmaceutical University,
Shenyang 110016, P. R. China, and §Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029
Received August 4, 2009
Ethacrynic acid (EA) is a glutathione S-transferase P1-1 (GST P1-1) inhibitor with weak antiprolifera-
tive ability in tumor cells. By use of the principle of bioisosterism, a series of novel EA oxadiazole
analogues were designed and synthesized. The structure-activity relationships of inhibiting GST P1-1
activity and cell proliferation of those EA analogues were investigated in human leukemia HL-60 cells.
Our data revealed that those EA oxadiazole analogues had improved antiproliferative activity and most
of them had similar or better inhibitory effects on GST P1-1 activity than EA. Compound 6u was one of
the potent antiproliferative agents without inhibition of GST P1-1 activity. Compounds 6r and 6s were
two potent cell growth inhibitors in several solid tumor cell lines with the concentrations inhibiting half
of cell growth of less than 5 μM. Our data suggest that these EA oxadiazole analogues are promising
antitumor agents that may act through GST P1-1 inhibition-dependent and/or -independent pathways.
Introduction
mitogen activated stress kinase (MAPK) family, which med-
iates many important pathways in the transduction of cell
survival and apoptotic signals initiated by stress or toxic
stimuli.8 Considering the multiple functions of GST P1-1 in
regulating multidrug resistance (MDR), cell proliferation,
and apoptosis, it has been considered as a target for develop-
ing therapeutic agents for cancer treatment.9,10
Glutathione S-transferases (GSTs), a family of phase II
detoxification enzymes, play an important role in the protec-
tion of cells from injury by toxic chemical including some
chemotherapeutic agents through catalyzingconjugationwith
glutathione.1 Among these enzymes, glutathione S-transfer-
ase P1-1 (GST P1-1a) is the predominant isozyme of this
family and has been found to overexpress in a number types of
human cancers including stomach carcinoma, colon cancer,
pancreatic cancer, and lung cancer.2,3 The increased levels of
GST P1-1 have been found to be relevant to the development
of resistance toward chemotherapeutic agents by catalyzing
their conjugation with glutathione (GSH) to form either
inactivated conjugates or conjugates that can be easily efflu-
xed.4 In addition, GST P1-1 has been shown to decrease the
levels of hydrogen peroxide by promoting its catabolism and
to protect against hydrogen peroxide-induced cell death.5,6
Hydrogen peroxide is one member of reactive oxygen species
(ROS) that play important roles in regulating cell prolifera-
tion, apoptosis, and senescence.7 Recently it was found that
GST P1-1 acted as an endogenous inhibitor that suppresses
the activation of c-Jun N-terminal kinase (JNK) through a
noncatalytic protein-protein interaction and protects cells
against stress-induced apoptosis.7,8 JNK is a member of the
Ethacrynic acid (EA) is a known GST P1-1 inhibitor with
an R,β-unsaturated carbonyl functional group.11,12 EA by
itself inhibits tumor cell growth at high concentrations, and it
enhances the therapeutic efficiencies of several anticancer
agents.13 The low efficacy and side effects of EA limit its
clinical use.14,15 It has been found that the ability of EA to
inhibit GST P1-1 activity results from its R,β-unsaturated
carbonyl group and that the main diuretic side effect of EA
results from its carboxyl group.16 We have done structure
modifications of EA and found that EA derivatives with
double substitutions on the phenyl cycle together with the
R,β-unsaturated carbonyl group were required to maintain
GST P1-1 activity inhibition and cell growth inhibition
ability.17,18 Moreover, we found that EA esters, such as EA
butyl ester, have increased cell growth inhibitory ability
significantly.18,19 These esters are unstable in vivo and can
be metabolized rapidly to EA.19 The similarities of electrolyte
and conformational properties as well as the physicochemical
properties such as pKa and logP of EA denote that an
authentic bioisosteric relationship exists between the ester
(CO2R)- and heterocyclic oxadiazole-containing analo-
gues.20 On the basis of the bioisosterism principle, wedesigned
and synthesized 28 novel EA derivatives containing a hetero-
cyclic oxadiazole. The inhibitory effects of these compounds
on GST P1-1 activity in HL-60 cell lysates and on HL-60 cell
proliferation were determined. The relationship between GST
*To whom correspondence should be addressed. Phone: þ86 531
88382009. Fax: þ86 531 88382835. E-mail: guisenzhao@sdu.edu.cn.
a Abbreviations: CDNB, 1-cholro-2,4-dinitrobenzene; EA, ethacry-
nic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; GSH,
glutathione; GST P1-1, glutathione S-transferase P1-1; HPLC, high
performance liquid chromatography; JNK, c-jun N-terminal kinase;
MAPK, mitogen-activated protein kinase; MDR, multidrug resistance;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
ROS, reactive oxygen species; SAR, structure-activity relationship.
r
2010 American Chemical Society
Published on Web 01/07/2010
pubs.acs.org/jmc

1016 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Scheme 1. Synthetic Routes of Target Compoundsa
Yang et al.
previously described.18 Cell growth inhibition ability was
determined by counting cells with the aid of a hemocytometer,
and the concentrations that inhibit growth of HL-60 cells by
50% (GI50) were determined.
Substituents of those lead compounds are shown in Table 1.
From the results obtained, it was noticed that all compounds
in this series, except compound 6u, inhibited GST P1-1
activity with effects comparable to or better than EA. The
most potent compounds in inhibiting the activity of GST P1-1
were compounds 6o, 6p, and 6q. The influences of the
substituents chlorine, bromide, or methyl on the middle
phenyl group on the GST P1-1 activity inhibition were
analyzed. Compounds with one chlorine substitution (6n-q)
atthe R2-position ofthe phenyl ring inhibited GST P1-1better
than compounds containing one methyl substitution (6a-f)
or one bromide substitution (6y, 6z, 6aa, and 6ab) at the same
position. Compounds with double chlorine substitutions
(6r-s) at the R2- and R1- positions of the phenyl ring inhibited
GST P1-1 poorly than compounds containing one chlorine
substitution (6n-q). The influences of substituents at the
oxadiazole ring on abilities of inhibiting GST P1-1 activity
were also analyzed. Compounds substituted with a phenyl
group at the R4-position on the oxadiazole ring were more
potent GST P1-1 activity inhibitors than those with one
methyl substitution. Among compounds 6h-m, those con-
taining either electron-withdrawing or electron-donating
para-substituent groups (e.g., NO2, MeO, CF3, and F) at
the phenyl ring (6i-m) had less ability in inhibiting GST P1-1
activity than the compound (6h) containing nonsubstituted
benzene at the R4-position. It has been shown that EA inhibits
the GST P1-1 activity by binding to GST P1-1 through
the R,β-unsaturated carbonyl group.12,24 All our synthesized
compounds kept the R,β-unsaturated carbonyl group and
exhibited the ability to inhibit GST P1-1 activity except
compound 6u. Our results suggest that additional reactions
may be needed for GST P1-1 activity inhibition and those
compounds with or without the ability to inhibit GST P1-1
activity could be used as lead molecules to dissect the relation-
ship between the GST P1-1 binding and activity inhibition.
Although those lead compounds had slight improvement
onthe GSTP1-1activityinhibition, theyexhibited much more
improved antiproliferative activities over EA in HL-60 cells
(Table 1). Our principle is to use a heterocyclic oxadiazol to
replace the ester (CO2R)- for improving the antiproliferative
ability of EA based on an authentic bioisosteric relationship
existing between the ester (CO2R)- and the heterocyclic
oxadiazole-containing analogues. The antiproliferative data
shown here support this principle. EA analogues containing a
heterocyclic oxadiazole have similar cell growth inhibition
abilities with that of the EA esters.18 Structure-activityanaly-
sis of those oxadiazole-containing analogues on cell growth
inhibition indicated that substituents at the end phenyl group
of the oxadiazole ring did not influence cell growth inhibition.
Trifluoromethyl substituents showed a modest improvement
in HL-60 cell growth inhibition (e.g., 6k, 6q, 6u, and 6ab) with
GI50 < 2.0 μM that was 20-fold more active than the lead
compound, EA. It is unclear whether the antiproliferative
effects of EA and its derivatives are mediated through GST
P1-1 activity inhibition. The glutathione conjugates of EA
and its esters have increased ability in inhibiting GST P1-1
activity.24,25 However, they were not effective, or less effective,
in inhibiting tumor cell growth.19 EA and its derivatives form
conjugates with GSH both spontaneously and by GST-driven
catalysis, and the R,β-unsaturated carbonyl moiety of EA is
a Reagents and conditions: (i) paraformaldehyde, dimethylamine
hydrochloride, HAc, 100 °C; (ii) 10% NaHCO3 (aq), reflux, HCl; (iii)
NH2OH HCl, K2CO3, alcohol, reflux; (iv) SOCl2, toluene, 90-100 °C;
3
(v) pyridine, 100 °C.
P1-1 activity inhibition and the antiproliferative abilities was
analyzed. Compounds6r and 6s were two of the most effective
inhibitors on HL-60 cellgrowthwiththe abilitytoinhibitGST
P1-1 activity. The antiproliferative effects of both compounds
6r and 6s were determined in additional cancer cell lines
including two prostate cancer cell lines, PC-3 and DU145,
three breast cancer cell lines, T47D, MCF-7, and MDA-MB-
231, and one nontumorigenic epithelial cell line, MCF 10A.
Chemistry
The schematic pathways of synthesizing R,β-unsaturated
carbonyl EA analogues containing a heterocyclic oxadiazole
are shown in Scheme 1. The starting material compounds 1
were prepared from the appropriate phenol in two steps as we
previously described.17 Compounds 1 with paraformaldehyde
and dimethylamine hydrochloride in the presence of a cata-
lytic amount of acetic acid gave Mannich compounds which
were then converted to compounds 2 under alkaline condition
via an elimination reaction.21 EA was obtained from the
commercial source (TCI, Tokyo Kasei Kogyo Co., Ltd.).
Acyl chlorides 5 were produced from compounds 2 using
thionyl chloride as a chlorinating agent and toluene as the
solvent. The reaction mixture was refluxed for 4 h and then
cooled to room temperature. After evaporation under redu-
ced pressure to remove toluene, the obtained acyl chlorides 5
were used in the next step without further purification.
The key intermediates, amidoximes 4, were synthesized
from the corresponding nitrile. Suitable nitriles 3 in an alco-
holic solution containing hydroxylamine hydrochloride and
anhydrous potassium carbonate were heated to reflux for
4-16 h. Filtration and purification on silica gel chromato-
graphy afforded good yields of compounds 4.22
The target compounds, 1,2,4-oxadiazoles 6, were obtained
from compounds 4 and compounds 5 by cyclization of O-
acyamidoximes according to a reported method.23 Flash
column chromatography, eluting with different proportions
of petroleum ether-acetone, gave pure types of compounds 6.
The R4-group of intermediates of compounds 4 was used as
a handle to introduce structural diversity. A substitutive
benzene ring was introduced at this position in most of these
compounds.
Results and Discussion
The structures of R,β-unsaturated carbonyl EA analogues
containing a heterocyclic oxadiazoleprepared in this study are
shown in Table 1. The inhibition of these compounds on GST
P1-1 activity in HL-60 cell lysate was investigated in vitro as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1017
Table 1. Substituents of Synthesized EA Analogues Containing an Oxadiazole Group and Their Inhibitory Effects on GST P1-1 Activity and
Proliferation of HL-60 Cells
IC50 (μM) for
inhibiting GST
P1-1 activitya
GI50 (μM) for
inhibiting HL-60
cell growthb
compd
R1
Cl
R2
Cl
R3
R4
EA
6a
6b
6c
6d
6e
6f
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CH3
CH3
CH3
CH3
-
CH3
3.4 ( 0.7
5.0 ( 2.0
1.97 ( 0.3
2.1 ( 1.1
3.5 ( 1.3
3.3 ( 0.6
2.9 ( 0.1
4.0 ( 1.0
1.8 ( 0.9
3.4 ( 1.1
2.4 ( 0.6
3.9 ( 0.4
2.0 ( 0.5
2.3 ( 0.5
2.1 ( 0.6
0.6 ( 0.1
0.7 ( 0.5
0.7 ( 0.1
4.0 ( 0.3
3.6 ( 0.7
3.9 ( 0.8
>40
45.0 ( 1.1
3.82
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
H
phenyl
6.7 ( 3.0
3.5 ( 0.8
4.6 ( 0.9
3.2 ( 0.8
6.1 ( 3.6
3.2 ( 0.4
3.5 ( 0.6
3.5 ( 0.5
2.1 ( 0.6
1.4 ( 0.5
3.1 ( 0.7
2.4 ( 0.7
9.7 ( 1.6
5.4 ( 1.5
2.8 ( 0.8
1.9 ( 0.5
2.3 ( 0.2
1.7 ( 0.1
1.7 ( 1.0
1.1 ( 0.2
1.5 ( 0.3
1.6 ( 0.3
1.6 ( 0.2
3.2 ( 0.7
7.9 ( 1.7
3.2 ( 0.8
1.7 ( 0.7
H
4-OH phenyl
4-F phenyl
4-CF3 phenyl
4-NO2 benzyl
CH3
H
H
H
6g
6h
6i
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
phenyl
4-OH phenyl
4-F phenyl
4-CF3 phenyl
4-Cl benzyl
4-NO2 benzyl
CH3
6j
6k
6l
6m
6n
6o
6p
6q
6r
H
Cl
Cl
phenyl
H
4-F phenyl
4-CF3 phenyl
CH3
H
Cl
Cl
Cl
6s
Cl
Cl
Cl
Cl
phenyl
6t
4-F phenyl
4-CF3 phenyl
4-OCH3 phenyl
4-Cl benzyl
4-NO2 benzyl
CH3
6u
6v
6w
6x
6y
6z
6aa
6ab
Cl
Cl
Cl
Cl
3.8 ( 1.2
5.4 ( 2.0
3.3 ( 0.6
3.2 ( 1.3
2.0 ( 0.5
1.4 ( 0.5
1.3 ( 0.3
Cl
Cl
Cl
Cl
H
Br
Br
H
phenyl
4-F phenyl
4-CF3 phenyl
H
Br
Br
H
a IC50 indicates the concentration of the tested compound that inhibits 50% of GST P1-1 activity. b GI50 indicates the concentration of the tested
compound that inhibits 50% of HL-60 cell growth.
the target for conjugation.26 GSH is a major regulator of the
redox equilibrium and protects against mitochondrial-
mediated apoptosis.27 The depletion and reduction of GSH
increases the sensitivity of cells to toxic insults by regulating
the apoptosis-associated redox potential and by inducing
changes in mitochondrial function.28 EA and EA derivatives
depleted intracellular GSH which was correlated with their
antitumor activity.19 Recently it has been shown that EA and
its derivatives inhibited the signaling pathways of NF-kB and
Wnt/β-catenin signaling through covalently modifying sulf-
hydryl groups.29,30 Reduction of the R,β-unsaturated carbo-
nyl group abrogated the inhibition on the Wnt/β-catenin
signaling.30 These data suggest that the antiproliferative
effects of EA and its derivatives including the compounds
described here may inhibit cell growth through a GST P1-1
inhibition-independent pathway due to their binding to GSH
or the thiol groups of functional proteins. Compound 6u con-
taining the R,β-unsaturated carbonyl group without the abi-
lity of inhibiting GST P1-1 ability supports this expectation.
Compounds 6r and 6s have the same substituents as EA
esters in R1, R2, and R3 positions and are two of the most
effective EA oxadiazole-containing analogues in inhibiting
HL-60 cell growth (Table 1). The growth inhibitory effects of
Table 2. Growth Inhibitory Effects of 6r and 6s in Several Solid Tumor
Cell Lines
GI50 (μM)a
cell lines
PC-3
DU145
6r
6s
1.4 ( 0.2
2.1 ( 0.2
2.0 ( 0.2
1.6 ( 0.1
4.0 ( 0.2
5.0 ( 0.4
1.5 ( 0.1
3.7 ( 0.3
2.6 ( 0.2
2.0 ( 0.2
4.5 ( 0.3
5.9 ( 0.7
T47D
MCF-7
MDA-MB-231
MCF-10A
a Cell growth inhibition was determined using the MTT assay. Cells
were treated with various concentrations of 6r and 6s for 4 days, and
GI50 was calculated. The data shown are the mean ( SD of three
independent experiments.
both compounds in additional cancer cell lines were investi-
gated (Table 2). Our data showed that PC-3, DU-145, T47D,
and MCF-7 cells were more sensitive than nontumorigenic
MCF 10A cells to the treatment of both agents.
Conclusion
By use of the principle of bioisosterism, R,β-unsaturated
carbonyl EA analogues containing a heterocyclic oxadiazole

1018 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Yang et al.
were synthesized. On the basis of their inhibitory effects on
GST P1-1 activity and HL-60 cell growth, it was revealed that
(1) the replacement of the carboxyl group by an oxadiazole
group in the structure of EA improved its antiproliferative
ability more efficiently than previously published carboxylic
acid and ester derivatives18 and (2) most of those compounds
had similar or better inhibitory effects on GST P1-1 activity as
EA. Some of those compounds may inhibit cell proliferation
independent of GST P1-1 activity inhibition. The connection
between GST P1-1 activity inhibition and cell growth inhi-
bition of these compounds needs to be further investigated.
We anticipate that these analogues represent a new group of
antitumor candidates.
J=9.00 Hz, 1H), 6.02 (s, 1H), 5.46 (s, 1H), 4.73 (s, 2H), 2.15 (s,
3H), 2.10 (s, 3H), 1.95 (s, 3H). HRMS (ESI) m/z calcd for
C14H17O4 [M þ H]þ: 249.1121, found 249.1108.
[3-Chloro-4-(2-methylene-1-oxopropyl)phenoxy]acetic Acid (2c).
White powder, yield 34.3%, mp 107.4-109.5 °C, TLC Rf=0.45
(acetone/petroleum ether, 1:3, v/v). 1H NMR (DMSO-d6) δ
(ppm): 7.33 (d, J=8.53 Hz, 1H), 7.10 (d, J=2.44 Hz, 1H), 6.97
(dd, J1=2.45 Hz, J2=8.50 Hz, 1H), 6.10 (s, 1H), 5.51 (s, 1H),
4.80 (s, 2H), 1.94 (s, 3H). HRMS (ESI) m/z calcd for
C12H12O4Cl [M þ H]þ: 255.0419, found 255.0406.
[3-Bromo-4-(2-methylene-1-oxopropyl)phenoxy]acetic Acid (2d).
White powder, yield 28.3%, mp 123.5-124.4 °C, TLC Rf=0.44
(acetone/petroleum ether, 1:2, v/v). 1H NMR (DMSO-d6) δ
(ppm): 13.15 (s, 1H), 7.32 (d, J=9.00 Hz, 1H), 7.10 (d, J=1.80
Hz, 1H), 7.02 (dd, J1=2.40 Hz, J2=8.40 Hz, 1H), 6.11 (s, 1H),
5.50 (s, 1H), 4.80 (s, 2H), 1.94 (s, 3H). HRMS (ESI) m/z calcd for
C12H12O4Br [M þ H]þ: 298.9913, found 298.9901.
Experimental Section
Synthesis of the Compound 4 Series. Hydroxylamine hydro-
chloride (50 mmol) and anhydrous potassium carbonate
(50 mmol) were dissolved in 40 mL of dry alcohol and stirred
for 0.5 h at room temperature. The corresponding nitriles
(10 mmol) 3 were added, and the reaction mixture was heated
to reflux for 4-16 h. After filtration of inorganic salts, the
solvent was evaporated under reduced pressure. The products 4
were purified by column chromatography (petroleum ether/
acetone, 2:1, v/v).
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. All
€
melting points were determined in a Buchi capillary melting point
apparatus and are uncorrected. The infrared (IR) spectra were
measured on KBr pellets using a Nicolet Nexus 470FT-IR
spectrometer and were expressed in cm-1. The proton nuclear
magnetic resonance (1H NMR) spectra were recorded with a
Bruker Avance DRX600 instrument with tetramethylsilane
(TMS) as the internal standard. The chemical shifts (δ) were
reportedinparts per million (ppm) andwererelativetothe central
peak of the solvent, which was DMSO-d6 or CDCl3. Coupling
constants (J) were given in Hz. Data were reported as follows:
chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,
q=quartet, br=broad, m=multiplet), coupling constants, and
number of protons. Mass spectra (MS) were measured with an
API 4000, and the high resolution mass spectra data were
obtained using a Accela UPLC-LTQ Orbitrap mass spectro-
meter. Column chromatography was carried out with silica gel
in the solvents indicated. Thin-layer chromatography (TLC) was
performed on silica gel GF254 plates (layer thickness, 0.2 mm),
and compounds were visualized using UV light. Petroleum ether
usedfor TLC andcolumn chromatography had a boiling range of
60-90 °C. All tested compounds were more than 95% pure on
the basis of HPLC analysis. The specific method for HPLC
analysis and a table of the data for all tested compounds are in
Supporting Information.
Synthesis of the Compound 2 Series. The starting materials 1
were synthesized via methods reported previously.17 In a 50 mL
round-bottomed flask equipped with an outlet tube suitable
for application of intermittent suction, an intimate mixture of
1 (9.6 mmol), paraformaldehyde (0.38 g, 11.5 mmol), dimethyl-
amine hydrochloride (0.91 g, 10.4 mmol), and acetic acid
(0.05 mL) was heated to 100 °C for about 1.5 h, during which
period suction was applied. When the mixture was cooled to
room temperature, the Mannich compounds obtained were
dissolved in 10% sodium bicarbonate and modulated to slight
alkalinity. The resulting solution was heated to 100 °C for 0.5 h,
cooled, and acidified using concentrated hydrochloric acid. The
products obtained were filtered and dried. The crude products
were purified by column chromatography by eluting with
acetone/petroleum ether (1:3). The parameters of compounds
2 were as follows.
N-Hydroxyacetimidamide (4a). White crystalline solid, yield
56.5%, mp 136.3-137.7 °C. 1H NMR (CDCl3) δ (ppm): 8.66 (s,
1H), 5.34 (s, 2H), 1.61 (s, 3H).
N-Hydroxybenzimidamide (4b). White crystalline solid, yield
55.8%, mp 72.1-73.2 °C, TLC Rf =0.35 (acetone/petroleum
ether, 1:2, v/v). 1H NMR (CDCl3) δ (ppm): 9.62 (s, 1H), 7.67(m,
2H), 7.36 (m, 3H), 5.80 (s, 2H).
N-Hydroxy-4-hydroxybenzimidamide (4c). Light-yellow crys-
talline solid, yield 55.0%, TLC Rf = 0.24 (acetone/petroleum
ether, 1:1, v/v). 1H NMR (DMSO-d6) δ (ppm): 9.59 (s, 1H), 9.35
(s, 1H), 7.47 (d, J=8.40 Hz, 2H), 6.72 (d, J=9.00 Hz, 2H), 5.63
(s, 2H). MS (ESI) m/z: 158.2 [M þ H]þ.
4-Fluoro-N-hydroxybenzimidamide (4d). White crystalline
solid, yield 56.5%, mp 79.4-80.1 °C, TLC Rf=0.47(acetone/
petroleum ether, 1:1, v/v). 1H NMR (DMSO-d6) δ (ppm): 9.66
(s, 1H), 7.71 (m, 2H), 7.20 (m, 2H), 5.84 (s, 2H).
N-Hydroxy-4-(trifluoromethyl)benzimidamide (4e). White
crystalline solid, yield 86.0%, mp 94.7-95.3 °C, TLC Rf =
0.57(acetone/petroleum ether, 1:1, v/v). H NMR (DMSO-d6)
1
δ (ppm): 9.92 (s, 1H), 7.89 (d, J=7.80 Hz, 2H), 7.74 (d, J=8.40
Hz, 2H), 5.98 (s, 2H).
2-(4-Chlorophenyl)-N-hydroxyacetimidamide (4f). White crys-
talline solid, yield 54.8%, mp 92.3-93.0 °C, TLC Rf = 0.51-
(acetone/petroleum ether, 1:1, v/v). 1H NMR (DMSO-d6) δ
(ppm): 8.91 (s, 1H), 7.33 (d, J=8.40 Hz, 2H), 7.28 (d, J=8.40
Hz, 2H), 5.43 (s, 2H), 3.24 (s, 2H). MS (ESI) m/z: 184.6[M þ H]þ.
N-Hydroxy-2-(4-nitrophenyl)acetimidamide (4g). Light-yel-
low crystalline solid, yield 90.0%, mp 166.2-166.9 °C, TLC Rf
1
=0.34 (acetone/petroleum ether, 1:1, v/v). H NMR (DMSO-
d6) δ (ppm): 9.01 (s, 1H), 8.16 (d, J=9.00 Hz, 2H), 7.28 (d,
J = 9.00 Hz, 2H), 5.43 (s, 2H), 3.42 (s, 2H). MS (ESI) m/z:
195.9[M þ H]þ.
N-Hydroxy-4-methoxybenzimidamide (4h). White crystalline
solid, yield 57.5%, mp 123.6-124.2 °C, TLC Rf=0.48 (acetone/
petroleum ether, 1:1, v/v). 1H NMR (DMSO-d6) δ (ppm): 9.46
(s, 1H), 7.61 (d, J=9.00 Hz, 2H), 6.92 (d, J=9.00 Hz, 2H), 5.74
(s, 2H), 3.76 (s, 3H).
[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxy]acetic Acid (2a).
White powder, yield 83.5%, mp 112.1-114.1 °C, TLC Rf=0.22
(acetone/petroleum ether, 1:3, v/v). 1H NMR (DMSO-d6) δ
(ppm): 7.25 (d, J=8.47 Hz, 1H), 6.79 (d, J=2.36 Hz, 1H), 6.68
(dd, J1=2.54 Hz, J2=8.48 Hz, 1H), 5.99 (s, 1H), 5.44 (s, 1H),
4.72 (s, 2H), 2.23 (s, 3H), 1.94 (s, 3H). HRMS (ESI) m/z calcd for
C13H15O4 [M þ H]þ: 235.0965, found 235.0953.
Synthesis of the Compound 5 Series. To a suspension of 2 (2
mmol) in anhydrous toluene (10 mL) was added thionyl chloride
(0.15 mL, 2 mmol). The reaction mixture was refluxed for 4 h
and then cooled to room temperature. The resulting solution of
5 in toluene was suitable for use without further purification.
Synthesis of the Target Compounds of the 6 Series. A solution
of 5 (2 mmol) in toluene (10 mL) was added dropwise to a
[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxy]acetic
Acid (2b). White powder, yield 74.5%, mp 119.5-121.3 °C, TLC
Rf=0.38 (acetone/petroleum ether, 1:2, v/v). 1H NMR (DMSO-
d6) δ (ppm): 13.0 (s, 1H), 7.05 (d, J = 8.40 Hz, 1H), 6.74 (d,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1019
solution of amidoximes 4 (2 mmol) in pyridine (5 mL). The
mixture was stirred at 100 °C for about 1 h. The supernatant
fluid was decanted, and the residue was washed with CH2Cl2.
The combined organic solvent phase was evaporated at reduced
pressure to yield the crude product which was purified by flash
chromatography with acetone/petroleum ether (1:3) and ace-
tone/petroleum ether (1:6) to give 6.
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-nitrobenzyl)-1,2,4-oxadiazol (6f). White powder, yield
47.9%, mp 55.8-56.7 °C, TLC Rf =0.33 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.20 (m, 2H), 7.51
(m, 2H), 7.26 (m, 1H), 6.83 (m, 1H), 6.77 (m, 1H), 5.93 (s, 1H),
5.53 (s, 1H), 5.28 (s, 2H), 4.23 (s, 2H), 2.31 (s, 3H), 2.04 (s, 3H).
13C NMR (CDCl3) δ (ppm): 199.59, 175.05, 168.54, 158.31,
147.30, 145.35, 142.34, 139.50, 133.00, 130.51, 130.00, 129.01,
123.99, 117.28, 110.73, 60.83, 32.04, 20.25, 17.58. IR (KBr,
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-methyl-1,2,4-oxadiazole (6a). Yellow powder, yield 57.0%, mp
60.3-61.5 °C, TLC Rf=0.61 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 7.28 (d, J=8.30 Hz, 1H), 6.86 (s, 1H),
6.79 (d, J=8.42 Hz, 1H), 5.92 (s, 1H), 5.55 (s, 1H), 5.28 (s, 2H),
cm-1): υdCH, 3064.17; υCH, 2925.85; υCdO, 1652.01; υCdC
1627.66, 1607.43, 1571.97; υNO2, 1522.50, 1350.14; δCH
1494.70, 1447.68, 1371.74; υC-O, 1238.84, 1125.47; γdCH
,
,
,
2.45 (s, 3H), 2.33 (s, 3H), 2.04 (s, 3H). IR (KBr, cm-1): υdCH
,
979.84, 841.49, 797.17. HRMS (ESI) m/z calcd for C21H20-
3060.52; υCH, 2978.06, 2954.75, 2924.05; υCdO, 1644.31; υCdC
,
N3O5 [M þ H]þ: 394.1397, found 394.1401.
1605.92; υC-O, 1249.34; γdCH2, 1131.81, 803.12. HRMS (ESI) m/z
calcd for C15H17N2O3 [M þ H]þ: 273.2866, found 273.2871.
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-phenyl-1,2,4-oxadiazole (6b). White powder, yield 51.3%, mp
56.7-57.9 °C, TLC Rf=0.42 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 8.09 (d, J=7.80 Hz, 2H), 7.51 (m,
3H), 7.28 (d, J=8.40 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J=8.40 Hz,
1H), 5.92 (s, 1H), 5.55 (s, 1H), 5.37 (s, 2H), 2.33 (s, 3H), 2.03 (s,
3H). IR (KBr, cm-1): υdCH, 3088.19, 3076.41; υCH, 2955.08,
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-methyl-1,2,4-oxadiazole (6g). Yellow powder, yield 40.4%, mp
68.7-69.9 °C, TLC Rf=0.58 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 7.08 (d, J=8.42 Hz, 1H), 6.74 (d,
J=8.44 Hz, 1H), 5.95 (s, 1H), 5.58 (s, 1H), 5.29 (s, 2H), 2.46 (s,
3H), 2.24 (s, 3H), 2.20 (s, 3H), 2.05 (s, 3H). IR (KBr, cm-1):
υ
dCH, 3097.90; υCH, 2984.24, 2954.83, 2925.30, 2861.81; υCdO,
1656.36; υCdC, 1592.41; υC-O, 1295.77; γdCH2, 1086.35, 801.36.
HRMS (ESI) m/z calcd for C16H19N2O3 [M þ H]þ: 287.1390,
found 287.1394.
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-phenyl-1,2,4-oxadiazole (6h). White powder, yield 64.6%, mp
77.2-78.3 °C, TLC Rf=0.47 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 8.09 (d, J = 6.60 Hz, 2H), 7.52
(m, 3H), 7.07 (d, J=8.40 Hz, 1H), 6.78 (d, J=8.40 Hz, 1H), 5.93
(s, 1H), 5.56 (s, 1H), 5.37 (s, 2H), 2.25 (s, 3H), 2.19 (s, 3H), 2.03
(s, 3H). IR (KBr, cm-1): υCH, 2990.63, 2961.44, 2927.94,
2922.63; υCdO, 1646.10; υCdC, 1605.92; υC-O, 1242.07; γdCH2
1126.13, 717.16, 693.14. HRMS (ESI) m/z calcd for
,
C20H19N2O3 [M þ H]þ: 335.1390, found 335.1394.
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-hydroxyphenyl)-1,2,4-oxadiazol (6c). White powder, yield
45.3%, mp 123.3-123.9 °C, TLC Rf=0.30 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 7.96 (d, J=8.40 Hz,
2H), 7.28 (d, J=8.40 Hz, 1H), 6.93 (d, J=8.40 Hz, 2H), 6.88 (s,
1H), 6.82 (d, J=8.40 Hz, 1H), 6.28 (s, 1H), 5.94 (s, 1H), 5.57 (s,
1H), 5.34 (s, 2H), 2.33 (s, 3H), 2.04 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 200.26, 174.20, 168.28, 158.78, 158.62, 145.28, 139.58,
132.67, 130.68, 129.49, 129.43, 118.49, 117.38, 115.95, 110.82,
60.92, 20.30, 17.62. IR (KBr, cm-1): υOH, 3293.53; υCH, 2957.00,
2924.12; υCdO, 1641.16; υCdC, 1602.60, 1584.13; δCH, 1481.33,
1447.80, 1347.23; υC-O, 1225.23, 1078.11; γdCH, 844.93, 752.98.
HRMS (ESI) m/z calcd for C20H19N2O4 [M þ H]þ: 351.1339,
found 351.1345.
2862.21; υCdO, 1651.83; υCdC, 1598.43; υC-O, 1256.25; γdCH2
,
1131.24, 721.53. HRMS (ESI) m/z calcd for C21H21N2O3 [M þ
H]þ: 349.1547, found 349.1552.
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-hydroxyphenyl)-1,2,4-oxadiazol (6i). White powder, yield
46.6%, mp 149.3-150.2 °C, TLC Rf=0.27 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 7.96 (d, J=9.00 Hz,
2H), 7.08 (d, J=8.40 Hz, 1H), 6.91 (d, J=8.40 Hz, 2H), 6.78 (d,
J=8.40 Hz, 1H), 6.00 (br, 1H), 5.96 (s, 1H), 5.59 (s, 1H), 5.34 (s,
2H), 2.24 (s, 3H), 2.19 (s, 3H), 2.05 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 201.29, 174.47, 168.23, 158.63, 156.51, 145.49, 136.93,
133.79, 130.22, 129.38, 127.09, 126.42, 118.68, 115.92, 108.12,
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-fluorophenyl)-1,2,4-oxadiazol (6d). White powder, yield
64.5%, mp 73.2-74.0 °C, TLC Rf =0.41 (acetone/petroleum
ether, 1:4, v/v). 1H NMR (CDCl3) δ: 8.10 (m, 2H), 7.28 (m, 1H),
7.18 (m, 2H), 6.89 (s, 1H), 6.83 (m, 1H), 5.92 (s, 1H), 5.55 (s, 1H),
5.37 (s, 2H), 2.34 (s, 3H), 2.04 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 199.62, 174.57, 167.81, 164.72 (d, JC-F = 250.65 Hz),
158.47, 145.32, 139.55, 132.88, 130.59, 129.74 (d, JC-F =9.15
61.52, 17.38, 17.23, 11.83. IR (KBr, cm-1): υOH, 3269.34; υCH
2928.45, 2861.80; υCdO, 1629.63; υCdC, 1616.22, 1599.31; δCH
1484.13, 1434.40, 1355.00; υC-O, 1260.27, 1083.12; γdCH
,
,
,
846.99, 753.37. HRMS (ESI) m/z calcd for C21H21N2O4 [M þ
H]þ: 365.1496, found 365.1501.
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-fluorophenyl)-1,2,4-oxadiazol (6j). White powder, yield
53.6%, mp 92.7-93.5 °C, TLC Rf =0.57 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.10 (m, 2H), 7.18
(m, 2H), 7.08 (d, J=8.40 Hz, 1H), 6.79 (d, J=8.40 Hz, 1H), 5.94
(s, 1H), 5.57 (s, 1H), 5.37 (s, 2H), 2.25 (s, 3H), 2.20 (s, 3H), 2.04
(s, 3H). 13C NMR (CDCl3) δ (ppm): 200.70, 174.88, 167.78,
164.72 (d, JC-F =251.25 Hz), 156.40, 145.56, 136.94, 134.04,
129.73 (d, JC-F =9.15 Hz), 129.69, 127.03, 126.36, 122.58 (d,
Hz), 129.01, 122.48 (d, JC-F=3.3 Hz), 117.33,116.16 (d, JC-F
=
,
,
21.75 Hz), 110.75, 60.88, 20.29, 17.61. IR (KBr, cm-1): υdCH
3057.09; υCH, 2968.33, 2930.51, 2858.33; υCdO, 1642.69; υCdC
1622.13, 1607.41, 1586.18; δCH, 1485.45, 1420.06, 1355.35;
υ
C-O, 1241.51, 1016.44; γdCH, 964.64, 856.83, 803.66, 754.22.
HRMS (ESI) m/z calcd for C20H18FN2O3 [M þ H]þ: 353.1296,
found 353.1300.
5-[3-Methyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol (6e). White power,
yield 66.4%, mp 87.9-88.8 °C, TLC Rf=0.48 (acetone/petro-
leum ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.23 (d, J=8.40
Hz, 2H), 7.76 (d, J=7.80 Hz, 2H), 7.29 (d, J=8.40 Hz, 1H), 6.90
(d, J=7.80 Hz, 1H), 6.84 (m, 1H), 5.93 (s, 1H), 5.55 (s, 1H), 5.39
(s, 2H), 2.34 (s, 3H), 2.04 (s, 3H). 13C NMR (CDCl3) δ (ppm):
199.61, 175.02, 167.66, 158.43, 145.35, 139.58, 133.32, 130.59,
129.64, 129.02, 127.94, 125.95 (q, JC-F = 3.80 Hz), 124.60,
122.80, 117.35, 110.77, 60.89, 20.29, 17.60. IR (KBr, cm-1):
J
C-F =2.85 Hz), 116.16 (d, JC-F =21.75 Hz), 108.13, 61.50,
17.37, 17.20, 11.83. IR (KBr, cm-1): υdCH, 3066.30; υCH
2985.69, 2956.57, 2923.40, 2870.25; υCdO, 1645.28; υCdC
,
,
1607.26, 1594.56, 1583.07; δCH, 1483.45, 1455.92, 1357.80;
υ
C-O, 1261.62, 1080.80; γdCH, 849.60, 792.73. HRMS (ESI)
m/z calcd for C21H20FN2O3 [M þ H]þ: 367.1456, found
367.1452.
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol (6k). White powder,
yield 55.9%, mp 123.6-124.2 °C, TLC Rf = 0.60 (acetone/
petroleum ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.24 (d,
J=8.40 Hz, 2H), 7.77 (d, J=8.40 Hz, 2H), 7.08 (d, J=8.40 Hz,
1H), 6.79 (d, J=9.00 Hz, 1H), 5.94 (s, 1H), 5.57 (s, 1H), 5.40
υ
dCH, 3069.12; υCH, 2966.74, 2926.34; υCdO, 1640.08; υCdC
,
1624.44, 1610.78, 1571.70; δCH, 1451.65, 1421.08, 1328.32;
υ
C-O, 1247.66, 1067.29; γdCH, 980.34, 853.44. HRMS (ESI)
m/z calcd for C21H18F3N2O3 [M þ H]þ: 403.1264, found
403.1270.

1020 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Yang et al.
(s, 2H), 2.26 (s, 3H), 2.20 (s, 3H), 2.04 (s, 3H). 13C NMR
(CDCl3) δ (ppm): 200.70, 175.30, 167.62, 156.33, 145.55, 136.98,
134.12, 133.29, 133.07, 129.77, 129.72, 127.92, 127.05, 126.34,
125.95 (q, JC-F=3.70 Hz), 108.13, 61.49, 17.36, 17.21, 11.84. IR
(KBr, cm-1): υCH, 2925.80; υCdO, 1643.63; υCdC, 1625.44,
1592.07, 1578.76; δCH, 1483.35, 1420.57, 1330.58; υC-O, 1267.83,
1068.02; γdCH, 850.64, 820.64, 758.75. HRMS (ESI) m/z calcd for
C22H20F3N2O3 [M þ H]þ: 417.1421, found 417.1422.
5-[3-Chloro-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol (6q). White powder,
yield 46.6%, mp 86.7-87.2 °C, TLC Rf=0.32 (acetone/petro-
leum ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.23 (d, J=7.80
Hz, 2H), 7.77 (d, J=8.40 Hz, 2H), 7.28 (m, 1H), 7.10 (m, 1H),
6.97 (m, 1H), 5.99 (s, 1H), 5.59 (s, 1H), 5.40 (s, 2H), 2.04 (s, 3H).
13C NMR (CDCl3) δ (ppm): 196.63, 174.40, 167.72, 158.65,
144.64, 133.39, 133.17, 132.89, 132.80, 130.40, 130.16, 129.51,
127.95, 125.99 (q, JC-F=3.75 Hz), 116.54, 112.87, 61.12, 17.06.
IR (KBr, cm-1): υdCH, 3082.30; υCH, 2929.58; υCdO, 1646.25;
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-chlorobenzyl)-1,2,4-oxadiazol (6l). White powder, yield
51.3%, mp 54.5-55.4 °C, TLC Rf =0.52 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 7.30 (d, J=8.40 Hz,
2H), 7.26 (d, J=8.40 Hz, 2H), 7.04 (d, J=9.00 Hz, 1H), 6.70 (d,
J=8.40 Hz, 1H), 5.94 (s, 1H), 5.55 (s, 1H), 5.26 (s, 2H), 4.08 (s,
2H), 2.21 (s, 3H), 2.18 (s, 3H), 2.02 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 200.70, 174.97, 169.32, 156.27, 145.52, 136.89, 133.99,
133.52, 133.24, 130.37, 129.75, 128.92, 126.99, 126.28, 108.09,
61.46, 31.62, 17.36, 17.18, 11.80. IR (KBr, cm-1): υCH, 2982.16,
υ
CdC, 1624.55, 1597.35, 1561.72; δCH, 1490.25, 1444.04,
1324.81; υC-O, 1228.19, 1065.21; γdCH, 853.57, 717.77. HRMS
(ESI) m/z calcd for C20H15ClF3N2O3 [M þ H]þ: 423.0718,
found 423.0706.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-methyl-1,2,4-oxadiazole (6r). White powder, yield 52.9%, mp
78.5-79.4 °C, TLC Rf=0.37 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 7.15 (d, J=8.40 Hz, 1H), 6.96 (d, J=
8.40 Hz, 1H), 5.96 (s, 1H), 5.60 (s, 1H), 5.38 (s, 2H), 2.47 (q, J=
7.80 Hz, 2H), 2.45 (s, 3H), 1.15 (t, J=7.20 Hz, 3H). 13C NMR
(CDCl3) δ (ppm): 195.89, 173.49, 167.84, 155.16, 150.30, 129.09,
127.03, 111.55, 62.28, 23.58, 12.59, 11.80. IR (KBr, cm-1):
2923.62; υCdO, 1644.29; υCdC, 1624.15, 1591.60, 1579.26; δCH
1491.43, 1448.66, 1366.13; υC-O, 1264.92, 1096.68; γdCH
927.43, 798.32, 787.16. HRMS (ESI) m/z calcd for C22H22-
,
,
ClN2O3 [M þ H]þ: 397.1313, found 397.1317.
5-[2,3-Dimethyl-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-nitrobenzyl)-1,2,4-oxadiazol (6m). White powder, yield
38.0%, mp 114.8-115.6 °C, TLC Rf=0.39 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.20 (d, J=9.00 Hz,
2H), 7.51 (d, J=8.40 Hz, 2H), 7.05 (d, J=8.40 Hz, 1H), 6.71 (d,
J=8.40 Hz, 1H), 5.94 (s, 1H), 5.55 (s, 1H), 5.28 (s, 2H), 4.22
(s, 2H), 2.21 (s, 3H), 2.18 (s, 3H), 2.04 (s, 3H). 13C NMR
(CDCl3) δ (ppm): 200.66, 175.31, 168.46, 156.18, 145.52,
142.36, 136.92, 134.08, 129.99, 129.76, 126.98, 126.23, 123.97,
υ
υ
dCH, 3104.30; υCH, 2970.72, 2945.48, 2883.28; υCdO, 1667.98;
CdC, 1599.02; υC-O, 1278.91; γdCH2, 1063.05, 802.53. HRMS
(ESI) m/z calcd for C15H15Cl2N2O3 [M þ H]þ: 341.0454, found
341.0461.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-phenyl-1,2,4-oxadiazole (6s). White powder, yield 67.2%, mp
93.2-93.8 °C, TLC Rf=0.45 (acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 8.09 (m, 2H), 7.52 (m, 3H), 7.16 (d,
J=8.40 Hz, 1H), 7.03 (d, J=8.40 Hz, 1H), 5.95 (s, 1H), 5.60 (s,
1H), 5.47 (s, 2H), 2.45 (q, J=7.20 Hz, 2H), 1.14 (t, J=7.20 Hz,
3H). 13C NMR (CDCl3) δ (ppm): 195.68, 173.52, 168.68, 155.02,
150.07, 134.63, 131.67, 131.61, 128.97, 128.90, 127.54, 126.84,
108.04, 61.40, 32.04, 17.35, 17.17, 11.78. IR (KBr, cm-1): υdCH
,
3101.26; υCH, 2982.54, 2922.70; υCdO, 1644.24; υCdC, 1624.18,
1579.31; υNO2, 1591.73, 1366.14; δCH, 1491.34, 1448.69,
126.05, 111.48, 62.21, 23.36, 12.37. IR (KBr, cm-1): υdCH
3084.65; υCH, 2969.39, 2938.28; υCdO, 1670.22; υCdC, 1586.23;
,
1375.08; υC-O
, 1265.04, 1096.70; γdCH, 927.36, 798.31,
787.21. HRMS (ESI) m/z calcd for C22H22N3O5 [M þ H]þ:
408.1554, found 408.1558.
υ
C-O, 1281.15; γdCH2, 1070.47, 722.22. HRMS (ESI) m/z calcd
5-[3-Chloro-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-methyl-1,2,4-oxadiazole (6n). Yellowish oil, yield 20.5%, TLC
Rf=0.40 (acetone/petroleum ether, 1:3, v/v). 1H NMR (CDCl3)
δ (ppm): 7.26 (d, J=8.40 Hz, 1H), 7.04 (s, 1H), 6.91 (d, J=8.40
Hz, 1H), 5.98 (s, 1H), 5.58 (s, 1H), 5.29 (s, 2H), 2.45 (s, 3H), 2.04
(s, 3H). IR (KBr, cm-1): υdCH, 3088.06; υCH, 2980.30, 2926.79;
for C20H17Cl2N2O3 [M þ H]þ: 403.0611, found 403.0617.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-(4-fluorophenyl)-1,2,4-oxadiazol (6t). White powder, yield
44.5%, mp 94.6-95.4 °C, TLC Rf =0.48 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.10 (m, 2H), 7.16
(m, 3H), 7.04 (d, J=9.00 Hz, 1H), 5.96 (s, 1H), 5.60 (s, 1H), 5.47
υ
CdO, 1664.71; υCdC, 1599.58; υC-O, 1228.90; γdCH2, 1060.84.
(s, 2H), 2.47 (q, J=7.40 Hz, 2H), 1.15 (t, J=7.50 Hz, 3H). 13
NMR (CDCl3) δ (ppm): 195.65, 173.65, 167.90, 164.79 (d, JC-F
=251.10 Hz), 155.01, 150.12, 134.74, 131.72, 129.77 (d, JC-F
C
HRMS (ESI) m/z calcd for C14H14ClN2O3 [M þ H]þ: 293.0474,
found 293.0479.
=
5-[3-Chloro-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-phenyl-1,2,4-oxadiazole (6o). White powder, yield 55.3%, mp
56.2-57.2 °C, TLC Rf=0.55(acetone/petroleum ether, 1:3, v/v).
1H NMR (CDCl3) δ (ppm): 8.10 (m, 2H), 7.51 (m, 3H), 7.27 (d,
J=8.40 Hz, 1H), 7.10 (d, J=2.40 Hz, 1H), 6.96 (dd, J1=8.40 Hz,
J2=2.40 Hz, 1H), 5.98 (s, 1H), 5.59 (s, 1H), 5.38 (s, 2H), 2.04 (s,
9.15 Hz), 128.85, 126.84, 123.88, 122.31, 116.22 (d, JC-F=21.75
Hz), 111.52, 62.21, 23.38, 12.38. IR (KBr, cm-1): υdCH, 3079.94;
υ
CH, 2971.08, 2942.01; υCdO, 1667.37; υCdC, 1606.97, 1596.92,
1584.78; δCH, 1484.99, 1467.65, 1385.17; υC-O, 1278.69,
1048.64; γdCH, 912.48, 849.06, 755.82. HRMS (ESI) m/z calcd
for C20H16Cl2FN2O3 [M þ H]þ: 421.0517, found 421.0523.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol (6u). White power,
yield 51.8%, mp 121.2-121.9 °C, TLC Rf = 0.46 (acetone/
petroleum ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.23 (d,
J=8.40 Hz, 2H), 7.77 (d, J=7.80 Hz, 2H), 7.18 (d, J=9.00 Hz,
1H), 7.04 (d, J=8.40 Hz, 1H), 5.96 (s, 1H), 5.60 (s, 1H), 5.50 (s,
2H), 2.47 (q, J=7.40 Hz, 2H), 1.15 (t, J=7.50 Hz, 3H). 13C
NMR (CDCl3) δ (ppm): 195.63, 174.08, 167.73, 154.95, 150.11,
3H). IR (KBr, cm-1): υdCH, 3071.14; υCH, 2924.87; υCdO
,
1664.10; υCdC, 1599.61; υC-O, 1229.05; γdCH2, 1060.09,
721.03. HRMS (ESI) m/z calcd for C19H16ClN2O3 [M þ H]þ:
355.0844, found 355.0850.
5-[3-Chloro-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-fluorophenyl)-1,2,4-oxadiazol (6p). White powder, yield
56.8%, mp 78.9-79.5 °C, TLC Rf =0.34 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.10 (m, 2H), 7.28
(m, 1H), 7.19 (m, 2H), 7.10 (m, 1H), 6.97 (m, 1H), 5.99 (s, 1H),
5.59 (s, 1H), 5.37 (s, 2H), 2.04 (s, 3H). 13C NMR (CDCl3) δ
(ppm): 196.64, 173.92, 167.88, 164.78 (d, JC-F = 250.80 Hz),
134.82, 131.76, 129.48, 128.91, 127.95, 126.86, 125.99 (q, JC-F=
3.80 Hz), 124.58, 123.90, 122.77, 111.54, 62.20, 23.38, 12.38. IR
(KBr, cm-1): υdCH, 3084.58; υCH, 2974.95, 2936.48, 2879.35;
158.72, 144.63, 132.80, 132.77, 130.37, 130.12, 129.76 (d, JC-F
9.44 Hz), 122.37 (d, JC-F=3.15 Hz), 116.54, 116.21 (d, JC-F
=
=
υCdO, 1661.10; υCdC, 1623.11, 1603.46, 1585.57; δCH, 1471.07,
1421.92, 1328.83; υC-O, 1169.28, 1065.62; γdCH, 856.46, 802.50,
758.93. HRMS (ESI) m/z calcd for C21H16Cl2F3N2O3 [M þ H]þ:
471.0485, found 471.0487.
21.75 Hz), 112.86, 61.13, 17.06. IR (KBr, cm-1): υdCH, 3084.41;
υ
CH, 2963.53, 2917.41; υCdO, 1651.03; υCdC, 1609.08, 1597.38,
1587.12; δCH, 1482.76, 1419.08, 1359.67; υC-O, 1234.81,
1055.99; γdCH, 917.50, 843.18, 749.66. HRMS (ESI) m/z calcd
for C19H15ClFN2O3 [M þ H]þ: 373.0750, found 373.0734.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-(4-methoxyphenyl)-1,2,4-oxadiazol (6v). White podwer, yield
48.6%, mp 87.6-88.1 °C, TLC Rf =0.40 (acetone/petroleum

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1021
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.03 (d, J=8.40 Hz,
2H), 7.17 (d, J=9.00 Hz, 1H), 7.04 (d, J=8.40 Hz, 1H), 7.00 (d,
J=9.00 Hz, 2H), 5.95 (s, 1H), 5.60 (s, 1H), 5.45 (s, 2H), 3.87 (s,
3H), 2.47 (q, J=7.40 Hz, 2H), 1.15 (t, J=7.50 Hz, 3H). 13C
NMR (CDCl3) δ (ppm): 195.64, 173.19, 168.36, 162.19, 155.04,
150.07, 134.58, 131.63, 129.17, 128.82, 126.82, 123.79, 118.45,
116.27, 116.20 (d, JC-F =21.75 Hz), 113.35, 61.13, 17.07. IR
,
(KBr, cm-1): υdCH, 3084.06; υCH, 2961.47, 2918.03; υCdO
1652.06; υCdC, 1626.31, 1607.83, 1596.43; δCH, 1483.47,
1417.55, 1360.58; υC-O, 1233.89, 1049.56; γdCH, 909.40,
843.84, 749.64. HRMS (ESI) m/z calcd for C19H15BrFN2O3
[M þ H]þ: 417.0245, found 417.0250.
114.33, 111.47, 62.21, 55.39, 23.35, 12.36. IR (KBr, cm-1): υCH
2967.20, 2937.32, 2873.93, 2839.76; υCdO, 1662.69; υCdC
,
,
5-[3-Bromo-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-trifluoromethylphenyl)-1,2,4-oxadiazol (6ab). White pow-
der, yield 55.2%, mp 104.7-105.5 °C, TLC Rf=0.32 (acetone/
petroleum ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.24 (d,
J=8.40 Hz, 2H), 7.77 (d, J=8.40 Hz, 2H), 7.29 (d, J=8.40 Hz,
1H), 7.25 (s, 1H), 7.02 (d, J=9.00 Hz, 1H), 6.00 (s, 1H), 5.58 (s,
1H), 5.40 (s, 2H), 2.05 (s, 3H). 13C NMR (CDCl3) δ (ppm):
197.35, 174.40, 167.73, 158.46, 144.39, 134.96, 133.18, 130.46,
130.43, 130.15, 129.52, 127.95, 125.98 (q, JC-F = 3.75 Hz),
1610.68, 1597.01, 1585.31; δCH, 1481.95, 1472.74, 1360.51;
υ
C-O, 1260.62, 1065.35; γdCH, 910.38, 846.54, 755.24. HRMS
(ESI) m/z calcd for C21H19Cl2N2O4 [M þ H]þ: 433.0716, found
433.0721.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-(4-chlorobenzyl)-1,2,4-oxadiazol (6w). White powder, yield
54.7%, mp 57.7-58.3 °C, TLC Rf =0.40 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 7.30 (d, J=8.40 Hz,
2H), 7.25 (d, J=8.40 Hz, 2H), 7.13 (d, J=8.40 Hz, 1H), 6.94 (d,
J=9.00 Hz, 1H), 5.96 (s, 1H), 5.58 (s, 1H), 5.36 (s, 2H), 4.08 (s,
2H), 2.47 (q, J=7.40 Hz, 2H), 1.15 (t, J=7.60 Hz, 3H). 13C
NMR (CDCl3) δ (ppm): 195.62, 173.74, 169.47, 154.90, 150.08,
134.69, 133.36, 133.30, 131.65, 130.37, 128.94, 128.88, 126.77,
123.85, 111.53, 62.18, 31.58, 23.36, 12.38. IR (KBr, cm-1):
120.74, 119.64, 113.39, 61.14, 17.07. IR (KBr, cm-1): υdCH
3078.22; υCH, 2929.49; υCdO, 1643.72; υCdC, 1624.33, 1594.17;
CH, 1487.87, 1443.20, 1325.04; υC-O, 1226.80, 1136.00; γdCH
,
δ
,
853.19, 717.72. HRMS (ESI) m/z calcd for C20H15BrF3N2O3 [M
þ H]þ: 467.0217, found 467.0213.
Biological Activity Methods. Cell Lines. HL-60, PC-3, and
DU-145 cells were cultured in RPMI-1640 medium supple-
mented with 100 U/mL pencillin, 100 μg/mL streptomycin,
1 mmol/L L-glutamine, and 10% (v/v) heat-inactivated fetal
bovine serum (FBS). T47D and MDA-MB-231 cells were
cultured in RPMI 1640 medium supplemented with 10%
FBS, 5 μg/mL insulin, 100 U/mL penicillin, and 100 μg/mL
streptomycin. MCF-7 cells were cultured in DMEM medium
supplemented with 10% FBS, 5 μg/mL insulin, 100 U/mL
penicillin, and 100 μg/mL streptomycin. MCF-10A cells were
maintained in DMEM/F12 supplemented with 5% donor
horse serum, 20 ng/mL epidermal growth factor (EGF),
10 μg/mL insulin, 1 ng/mL cholera toxin, 100 μg/mL
hydrocortisone, 50 U/mL penicillin, and 50 μg/mL strepto-
mycin.
υ
υ
dCH, 3086.99; υCH, 2970.79, 2933.93, 2876.00; υCdO, 1661.23;
CdC, 1619.26, 1585.62; δCH, 1492.16, 1468.85, 1382.35; υC-O
,
1287.26, 1037.36; γdCH, 945.48, 802.96, 751.57. HRMS (ESI)
m/z calcd for C21H18Cl3N2O3 [M þ H]þ: 451.0378, found
451.0382.
5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl)phenoxymethyl]-
3-(4-nitrobenzyl)-1,2,4-oxadiazol (6x). White powder, yield
60.4%, mp 67.9-68.8 °C, TLC Rf =0.40 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.20 (d, J=7.80 Hz,
2H), 7.51 (d, J=7.80 Hz, 2H), 7.14 (d, J=7.80 Hz, 1H), 6.96 (d,
J=8.40 Hz, 1H), 5.96 (s, 1H), 5.78 (s, 1H), 5.38 (s, 2H), 4.23 (s,
2H), 2.47 (q, J=6.60 Hz, 2H), 1.15 (t, J=6.90 Hz, 3H). 13C
NMR (CDCl3) δ (ppm): 195.58, 174.10, 168.65, 154.86, 150.12,
147.32, 142.22, 134.83, 131.70, 130.02, 128.87, 126.78, 123.99,
123.91, 111.58, 62.18, 32.02, 23.36, 12.38. IR (KBr, cm-1):
GST P1-1 Activity Assay. GST P1-1 activity was measured
spectrophotometrically at 25 °C using 1-cholro-2,4-dinitroben-
zene (CDNB) and GSH as substrates as described before.17 The
CDNB-GSH product has a strong molar absorptivity at 340 nm,
and the extinction coefficient of 9.6 mM-1 cm-1 was used to
calculate GST P1-1 activity. GST P1-1 activity was expressed as
nanomoles of product per minute per milligram of protein. The
percent inhibition rates were calculated as [(Vc - Vt)/Vc] ꢀ 100.
Vc represents GST P1-1 activity of the control group, and Vt
represents GST P1-1 activity of the treated group. The concen-
tration of the tested compound that inhibits 50% of GST P1-1
activity (IC50) was calculated.
Cell Proliferation Inhibition of HL-60 Cells. Cell proliferation
was determined as reported previously.18 Cells were plated in 12-
well microplates at a density of 1 ꢀ 105 cells/mL and incubated
with various concentrations of the test compounds. Cell number
in each group was counted with the aid of a hemocytometer. The
percent cell growth inhibition rate was calculated as [(Nc - Nt)/
Nc] ꢀ 100. Nc represents the cell number of the control group,
and Nt represents the cell number of the treated group. The
concentration inhibiting 50% of the cell proliferation (GI50) was
calculated.
υ
υ
dCH, 3083.36; υCH, 2974.62, 2937.10, 2876.43; υCdO, 1658.64;
NO2, 1528.05, 1348.95; υCdC, 1608.29, 1594.37, 1585.54; δCH
,
1469.26, 1427.81, 1378.71; υC-O, 1266.53, 1040.13; γdCH
803.73, 721.79. HRMS (ESI) m/z calcd for C21H18Cl2N3O5 [M
,
þ H]þ: 462.0618, found 462.0624.
5-[3-Bromo-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-methyl-1,2,4-oxadiazole (6y). White powder, yield 38.6%, mp
63.5-64.5 °C, TLC Rf = 0.45 (acetone/ petroleum ether, 1:3,
v/v). 1H NMR (CDCl3) δ (ppm): 7.23 (m, 2H), 6.96 (dd, J1=8.40
Hz, J2=2.40 Hz, 1H), 5.99 (s, 1H), 5.57 (s, 1H), 5.28 (s, 2H), 2.45
(s, 3H), 2.04 (s, 3H). IR (KBr, cm-1): υdCH, 3087.83; υCH
,
2979.57, 2925.71; υCdO, 1664.55; υCdC, 1596.68; υC-O, 1225.30;
γ
dCH2, 1050.30. HRMS (ESI) m/z calcd for C14H14BrN2O3
[M þ H]þ: 337.0182, found 337.0187.
5-[3-Bromo-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-phenyl-1,2,4-oxadiazole (6z). White powder, yield 45.1%, mp
52.3-53.5 °C, TLC Rf = 0.51 (acetone/ petroleum ether, 1:3,
v/v). 1H NMR (CDCl3) δ (ppm): 8.09 (d, J=6.60 Hz, 2H), 7.51
(m, 3H), 7.29 (s, 1H), 7.24 (s, 1H), 7.01 (d, J=8.40 Hz, 1H), 5.99
(s, 1H), 5.58 (s, 1H), 5.38 (s, 2H), 2.05 (s, 3H). IR (KBr, cm-1):
υ
dCH, 3070.23; υCH, 2979.31, 2924.44; υCdO, 1664.13; υCdC
,
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide
(MTT) Assay. PC-3, DU-145, T47D, MCF-7, MDA-MB-231,
and MCF 10A cells (2 ꢀ 103) were plated in each well of a 96-well
plate and allowed to adhere and spread for 24 h. Then various
concentrations (20, 10, 5, 2.5, 1.25, 0.625, and 0 μM) of 6r and 6s
were added, and then the cells were cultured for 4 days at 37 °C.
MTT solution (50 μL of 2 mg/mL) was added per well, and the
cultures were continued for an additional 4 h. The medium was
aspirated, the cells were dissolved in 200 μL of DMSO, and the
optical density (OD) at 570 nm was determined in each well with
a 96-well plate reader. The percent cell growth inhibition rate
was calculated as [(ODc - ODt)/ODc] ꢀ 100. ODc represents the
OD of the control group, and ODt represents the OD of the
1596.64; υC-O, 1225.52; γdCH2, 1048.82, 720.90. HRMS (ESI)
m/z calcd for C19H16BrFN2O3 [M þ H]þ: 399.0339, found
399.0345.
5-[3-Bromo-4-(2-methylene-1-oxopropyl)phenoxymethyl]-
3-(4-fluorophenyl)-1,2,4-oxadiazol (6aa). White powder, yield
42.3%, mp 83.5-84.5 °C, TLC Rf =0.32 (acetone/petroleum
ether, 1:3, v/v). 1H NMR (CDCl3) δ (ppm): 8.10 (m, 2H), 7.28 (s,
1H), 7.25 (d, J=8.40 Hz, 1H), 7.18 (m, 2H), 7.01 (d, J=8.40 Hz,
1H), 5.99 (s, 1H), 5.58 (s, 1H), 5.37 (s, 2H), 2.05 (s, 3H). 13C
NMR (CDCl3) δ (ppm): 197.37, 173.97, 167.85, 164.75 (d,
JC-F = 250.50 Hz), 158.50, 144.35, 134.81, 130.46, 130.12,
129.75 (d, JC-F=9.15 Hz), 122.36 (d, JC-F=2.85 Hz), 119.62,

1022 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Yang et al.
treated group. The concentration inhibiting 50% of the cell
proliferation (GI50) was calculated.
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Acknowledgment. This work was supported by grants
from the National Natural Science Foundation of China
(Grant No. 20672069) and Major Projects of Science and
Technology for Drug Innovation (Grant No. 2009ZX09103-
116).
(17) Zhao, G.; Yu, T.; Wang, R.; Wang, X.; Jing, Y. Synthesis and
structure-activity relationship of ethacrynic acid analogues on
glutathione-S-transferase P1-1 activity inhibition. Bioorg. Med.
Chem. 2005, 13, 4056–4062.
Supporting Information Available: Purity data for all tested
compounds and the methods for HPLC analysis. This material
is available free of charge via the Internet at http://pubs.acs.org.
(18) Zhao, G.; Liu, C.; Wang, R.; Song, D.; Wang, X.; Lou, H.; Jing, Y.
The synthesis of alpha,beta-unsaturated carbonyl derivatives with
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