The synthesis of α,β-unsaturated carbonyl derivatives with the ability to inhibit both glutathione S-transferase P1-1 activity and the proliferation of leukemia cells

Article abstract of DOI:10.1016/j.bmc.2007.01.037

Ethacrynic acid (EA), an α,β-unsaturated carbonyl compound, is a glutathione S-transferase P1-1 (GSTP1-1) inhibitor. Twenty-one novel EA derivatives have been synthesized. The effects of these compounds on GSTP1-1 activity and on the proliferation of human leukemia HL-60 cells have been determined. Compounds with a halogen substitution at the 3′-position of the aromatic ring have greater inhibitory effects on GSTP1-1 activity than those of compounds with a methyl substitution there. Compounds with substitutions at both the 2′- and 3′-positions of the aromatic ring have more antiproliferative ability than those with one substitution at 3′-position. Esterification of the carboxyl group appears to increase the antiproliferative ability.

Full text of DOI:10.1016/j.bmc.2007.01.037

Bioorganic & Medicinal Chemistry 15 (2007) 2701–2707
The synthesis of a,b-unsaturated carbonyl derivatives with the
ability to inhibit both glutathione S-transferase P1-1 activity
and the proliferation of leukemia cells
Guisen Zhao,a Chuan Liu,a Rui Wang,b Dandan Song,b Xiaobing Wang,a
Hongxiang Loua,* and Yongkui Jingc,*
aCollege of Pharmacy, Shandong University, Ji’nan, Shandong Province 250012, PR China
bShenyang Pharmaceutical University, Shenyang 110016, PR China
cMount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
Received 10 December 2006; revised 18 January 2007; accepted 19 January 2007
Available online 24 January 2007
Abstract—Ethacrynic acid (EA), an a,b-unsaturated carbonyl compound, is a glutathione S-transferase P1-1 (GSTP1-1) inhibitor.
Twenty-one novel EA derivatives have been synthesized. The effects of these compounds on GSTP1-1 activity and on the prolifer-
ation of human leukemia HL-60 cells have been determined. Compounds with a halogen substitution at the 30-position of the
aromatic ring have greater inhibitory effects on GSTP1-1 activity than those of compounds with a methyl substitution there. Com-
pounds with substitutions at both the 20- and 30-positions of the aromatic ring have more antiproliferative ability than those with
one substitution at 30-position. Esterification of the carboxyl group appears to increase the antiproliferative ability.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
erative effects were determined in HL-60 cells. The struc-
ture–activity relationships between GSTP1-1 activity
inhibition and cell growth inhibition were analyzed.
Glutathione S-transferase P1-1 (GSTP1-1, GSTp), a
member of phase II detoxification enzymes, which cata-
lyzes the conjugation of glutathione with a broad range
of substrates, including chemotherapeutic agents,1 has
been reported to be overexpressed in a variety of cancer
cells and to be involved in multidrug resistance (MDR)
and the protection against cell death.2–12 GSTP1-1 inhib-
itors have the potential to overcome MDR and/or to in-
duce cell death in cancer cells with overexpressed
GSTP1-1. Ethacrynic acid has been found to be a
GSTP1-1 inhibitor and, at higher concentrations, has
antiproliferative effects in tumor cells.12,13 We previously
synthesized 15 EA derivatives and found that a substitu-
tion at the 30-position of EA is required for the inhibition
of GSTP1-1 activity.14 We have now designed and synthe-
sized 21 novel EA derivatives. The inhibitory effects of
these compounds on GSTP1-1 activity were determined
in human leukemia HL-60 cell lysates and the antiprolif-
2. Results and discussion
2.1. Chemistry
The synthesis of target compounds VI and VII was
accomplished by the steps shown in Figure 1. The target
compounds, as typified by the general structures of the
VI series and the intermediates III and IV, were pre-
pared by using methods we reported previously.14 VI
series compounds were esterified with methanol, with
para-toluenesulfonic acid as the catalyst to generate
compounds of the VII series. Twenty-one novel EA
derivatives of the VI and VII series with substitutions
termed R1, R2, R3, and R4 were synthesized (Table 1).
2.2. The inhibitory effects of EA derivatives on GSTP1-1
activity and on HL-60 cell proliferation
Keywords: Glutathione S-transferase; a,b-unsaturated carbonyl com-
pounds; Structure–activity relationship.
*
The inhibitory effects of these compounds on GSTP1-1
activity in HL-60 cell lysates were determined in vitro
Corresponding authors. Tel.: +86 531 88382019 (H.L.); fax: +1 212
996 5787 (Y.J.); e-mail: yongkui.jing@mssm.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.037

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G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
R2
R1
R2
R1
R2
R1
R3
O
ClCHCOOH
NaOH
HCl
R4H2C C Cl
OCHCOONa
R3
OH
R2
OCHCOOH
R3
III
AlCl3,CS2
I
II
R1
R2
R1
CH2
30%HCHO
K2CO3
HCl
R4H2C
C
OCHCOOH
R3
R4
C
C
O
OCHCOOK
O
R3
IV
V
R2
R1
R2
R1
CH2
CH2
ROH
PTS
R4
C
C
O
OCHCOOR
R3
R4
C
C
O
OCHCOOH
R3
VII
VI
Figure 1. The synthetic pathways of the VI and VII series.
Table 1. Substituted groups of compounds of the VI and VII series
as we reported previously.14 To compare the inhibitory
effects on GSTP1-1 activity of these derivatives with that
of EA, 40 lmol/L of each compound was incubated with
cell lysate for 30 min at 37 ꢁC. At this concentration, all
compounds had some inhibitory effects (Table 2). Com-
pounds of the VI-16–22 with a chloro or bromo at 30-
position, like EA, were potent GSTP1-1 activity inhibi-
tors. Replacement of the chloride or bromide by a meth-
yl group decreased inhibitory ability (VI-23–30). The
inhibitory ability was further decreased when the
carboxyl was esterified with methanol (VII series). The
length of the substitutions at 200 did not influence the
inhibitory effects (compare VI-27 with VI-28 or VII-1
with VII-2).
The antiproliferative ability of these compounds was
determined by counting total cells. Compounds with a
single substitution at the 30-position had less antiprolifer-
ative activity than those with two at the 20- and 30-posi-
tions (Table 2). Compounds with methyl substitutions
at the 30- and 20-positions (VI-27–30) had greater anti-
proliferative effects than EA. Esterification of the
carboxyl group (VII) with methanol significantly im-
proved the antiproliferative ability. The GI50 of the EA
methyl ester is 3.9 lM which is approximately 10-fold
lower than that of EA (44 lM). The length of the substi-
tutions at 200 did not influence the antiproliferative effects
(compare VI-27 with VI-28 and VII-1 with VII-2). How-
ever, some compounds (VII-1–4) with relatively low

G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
2703
Table 2. The inhibitory effects of EA derivatives on GSTP1-1 activity and on HL-60 cell growth
b
Code
Structure
Inhibition of GSTP1-1 activitya (%)
GI50 (lM)
Cl
Cl
CH2 O
EA
94.4
44.2
H3CH2C
C
C
OCH2COOH
Br
CH2 O
VI-16
92.9
>60
H3C
C
C
OCHCOOH
CH3
Br
Br
Cl
CH2 O
VI-17
VI-18
92.4
93.8
>60
>60
H3CH2C
C
C
OCHCOOH
CH3
CH2 O
H3CH2C
H3CH2C
C
C
OCH2COOH
CH2 O
VI-19
VI-20
91.7
95.1
>60
>60
C
C
OCH2COOH
Cl
CH2 O
H3C
H3C
C
C
OCH2COOH
Cl
CH2 O
VI-21
94.7
>60
C
C
OCHCOOH
CH3
Cl
CH2 O
VI-22
VI-23
94.8
84.4
>60
>60
H3CH2C
C
C
OCHCOOH
CH3
H3C
CH2 O
H3C
C
C
OCHCOOH
CH3
H3C
CH2 O
VI-24
VI-25
62.9
86.9
>60
>60
H3CH2C
C
C
OCHCOOH
CH3
H3C
CH2 O
H3C
C
C
OCH2COOH
H3C
CH2 O
VI-26
VI-27
46.1
54.8
>60
11.2
12.1
H3CH2C
C
C
OCH2COOH
CH3
H3C
CH2 O
H3CH2CH2C
C
C
OCHCOOH
CH3
CH3
H3C
CH2 O
VI-28
VI-29
51.2
62.5
H3CH2CH2CH2C
C
C
OCHCOOH
CH3
CH3
H3C
CH2 O
19.3
H3CH2CH2C
C
C
OCH2COOH
(continued on next page)

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G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
Table 2 (continued)
b
Code
Structure
Inhibition of GSTP1-1 activitya (%)
GI50 (lM)
CH3
OCH2COOH
H3C
CH2 O
VI-30
VII-1
61.1
17.8
14.4
5.6
H3CH2CH2CH2C
C
C
CH3
H3C
CH2 O
H3CH2CH2C
C
C
OCHCOOCH3
CH3
CH3
H3C
CH2 O
VII-2
VII-3
20.0
36.1
5.2
8.6
H3CH2CH2CH2C
C
C
OCHCOOCH3
CH3
CH3
H3C
CH2 O
H3CH2CH2C
C
C
OCH2COOCH3
CH3
H3C
CH2 O
VII-4
VII-5
34.8
74.9
8.6
4.6
H3CH2CH2CH2C
C
C
OCH2COOCH3
CH3
H3C
CH2 O
H3CH2C
C
C
OCHCOOCH3
CH3
CH3
H3C
CH2 O
VII-6
91.3
96.7
6.0
3.9
H3C
C
C
OCHCOOCH3
CH3
Cl
Cl
CH2 O
EA0
H3CH2C
C
C
OCH2COOCH3
a The inhibition rates of GSTP1-1 activity are calculated by the value of control group minus the value of treated group divided by the value of
control group and multiplying by 100. Data shown in each group are the means of triplicate samples.
b GI50 is the concentration that inhibits 50% of proliferation compared to untreated cells. The cells were treated with each derivative for 72 h and then
the cell number was determined using a haemocytometer.
GSTP1-1 inhibition ability have more potent antiprolif-
erative ability than compounds with potent GSTP1-1
inhibition ability (VI-16–26). These data suggest that
these compounds may act to inhibit cell proliferation
through a GSTP1-1 inhibition-independent pathway.
tion of GSTP1-1 activity and cell proliferation; (5)
esterification of the carboxyl group increases the anti-
proliferative effect; (6) some of these compounds may
inhibit cell proliferation independent of GSTP1-1
inhibition.
In summary, these data indicate that (1) compounds
with one substitution at the 30-position of the aromatic
ring, as we previously reported,14 are more potent
GSTP1-1 inhibitors than those without substitutions;
(2) compounds with one halogen substitution at the
30-position of the aromatic ring have greater GSTP1-
1 activity inhibition than those of compounds with
one methyl substitution at the same position; (3) com-
pounds with two substitutions (at 20- and 30-positions)
have greater antiproliferative effects than those com-
pounds with a single substitution at the 30-position;
(4) the length of the substitution at the 200-position does
not influence the effect of those compounds on inhibi-
3. Experimental
Melting points were determined on a Buchi capillary
¨
melting point apparatus. Infrared (IR) spectra were
measured on KBr pellets using a Nicolet Nexus
470FT-IR. Proton nuclear magnetic resonance (1H
NMR) spectra were recorded with a Bruker Avance
DRX600 spectrometer with DMSO-d6 as the solvent
and tetramethyl-silane (TMS) as the internal standard.
Mass spectra (MS) were measured with an API 4000.
Thin-layer chromatography (TLC) was performed on
silica gel GF254 plates (layer thickness, 0.2 mm).

G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
2705
3.1. Synthesis of the compound III, IV and VI series
v/v). 1H NMR (DMSO-d6) d: 7.33 (d, J = 8.52 Hz,
1H), 7.04 (d, J = 2.33 Hz, 1H), 6.92 (dd, J1 = 2.38 Hz,
J2 = 8.56 Hz, 1H), 6.10 (s, 1H), 5.50 (s, 1H), 4.99 (q,
J = 6.79 Hz, 1H), 1.94 (s, 3H), 1.51 (d, J = 6.77 Hz,
The intermediates III and IV and the target compounds
were synthesized as we previously described.14 The
parameters of the target compounds VI were as follows.
3H). IR (KBr, cmꢀ1): tCH: 2985.3, 2960.3, 2927.9; tC@O
:
1726.5, 1712.5; tC@C: 1660.9, 1599.6; tCAO: 1224.2;
VI-16: 2-[3-bromo-4-(2-methylene-1-oxopropyl)phenoxy]-
propionic acid, white power, yield 66.7%, mp 101.1–
104.0 ꢁC, TLC Rf = 0.45 (acetone/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 7.30 (d, J = 8.50 Hz,
1H), 7.18 (d, J = 2.44 Hz, 1H), 6.93 (dd, J1 = 2.45 Hz,
J2 = 8.52 Hz, 1H), 6.10 (s, 1H), 5.48 (s, 1H), 4.98 (q,
J = 6.81 Hz, 1H), 1.94 (s, 3H), 1.50 (d, J = 6.80 Hz,
c@CH : 1059.1, 867.8.
2
VI-22: 2-[3-chloro-4-(2-methylene-1-oxobutyl)phenoxy]-
propionic acid, yellow oil, yield 40.9%, TLC Rf = 0.41
(acetone/petroleum ether, 1:3, v/v). 1H NMR
(DMSO-d6) d: 7.29 (d, J = 8.59 Hz, 1H), 7.01 (d,
J = 2.52 Hz, 1H), 6.91 (dd, J1 = 2.44 Hz, J2 = 8.61 Hz,
1H), 5.97 (s, 1H), 5.51 (s, 1H), 4.95 (q, J = 6.87 Hz,
1H), 2.33 (q, J = 7.66 Hz, 2H), 1.52 (d, J = 7.13 Hz,
3H). IR (KBr, cmꢀ1): tCH: 2982.9, 2928.8; tC@O
:
1729.0, 1713.0; tC@C: 1659.9, 1595.0; tCAO: 1222.1;
c@CH : 1050.4, 954.1.
3H), 1.03 (t, J = 7.84 Hz, 3H). IR (KBr, cmꢀ1): t@CH
3089.3; tCH: 2975.7, 2936.3, 2875.1; tC@O: 1714.1;
C@C: 1660.4, 1600.8; tCAO: 1222.2; c@CH : 1097.4,
:
2
VI-17: 2-[3-bromo-4-(2-methylene-1-oxobutyl)phenoxy]-
propionic acid, yellow oil, yield 60.6%, TLC Rf = 0.42
(acetone/petroleum ether, 1:3, v/v). H NMR (DMSO-
t
2
862.0.
1
d6) d: 7.26 (d, J = 8.51 Hz, 1H), 7.16 (d, J = 2.47 Hz,
1H), 6.95 (dd, J1 = 2.35 Hz, J2 = 8.60 Hz, 1H), 5.98 (s,
1H), 5.51 (s, 1H), 4.94 (q, J = 6.82 Hz, 1 H), 2.33 (q,
J = 7.45 Hz, 2H), 1.51 (d, J = 7.03 Hz, 3H), 1.04 (t,
VI-23: 2-[3-methyl-4-(2-methylene-1-oxopropyl)phenoxy]-
propionic acid, yellow oil, yield 47.0%, TLC Rf = 0.26
(chloroform/glacial acetic acid, 80:1, v/v). 1H NMR
(DMSO-d6) d: 7.24 (d, J = 8.52 Hz, 1H), 6.79 (d,
J = 2.65 Hz, 1H), 6.68 (dd, J1 = 2.65 Hz, J2 = 8.54 Hz,
1H), 5.95 (s, 1H), 5.44 (s, 1H), 4.87 (q, J = 6.89 Hz,
1H), 2.22(s, 3H), 1.94 (s, 3H), 1.50 (d, J = 7.00 Hz,
3H). IR (KBr, cmꢀ1): t@CH: 3093.4; tCH: 2987.7,
J = 7.58 Hz, 3H). IR (KBr, cmꢀ1): t@CH: 3093.4; tCH
2969.5, 2937.4; tC@O: 1729.5; tC@C: 1595.5; tCAO
:
:
1224.6; c@CH : 1045.4, 954.7.
2
VI-18: [3-bromo-4-(2-methylene-1-oxobutyl)phenoxy]-
acetic acid, white power, yield 69.8%, mp 104.2–
106.0 ꢁC, TLC Rf = 0.45 (acetone/petroleum ether, 1:3,
2927.4; tC@O: 1728.4; tC@C: 1654.7, 1604.2; tCAO:
1244.5; c@CH : 1129.2, 802.4.
2
1
v/v). H NMR (DMSO-d6) d: 7.28 (d, J = 8.56 Hz, 1H),
VI-24: 2-[3-methyl-4-(2-methylene-1-oxobutyl)phenoxy]-
propionic acid, yellow oil, yield 57.3%, TLC Rf = 0.40
(acetone/petroleum ether, 1:3, v/v). H NMR (DMSO-
7.24 (d, J = 2.41 Hz, 1H), 7.00 (dd, J1 = 2.41 Hz,
J2 = 8.51 Hz, 1H), 6.03 (s, 1H), 5.49 (s, 1H), 4.81 (s,
2H), 2.34 (q, J = 7.44 Hz, 2H), 1.06 (t, J = 7.43 Hz,
3H). IR (KBr, cmꢀ1): t@CH: 3061.2; tCH: 2973.1,
1
d6) d: 7.76 (d, J = 8.50 Hz, 1H), 6.76 (m, 2H), 5.93 (s,
1H), 5.43 (s, 1H), 3.33 (q, J = 7.49 Hz, 1H), 2.41 (s,
3H), 1.56 (q, J = 7.12 Hz, 2H), 1.51 (d, J = 7.98 Hz,
2925.1; tC@O: 1738.2, 1709.7; tC@C: 1657.3, 1593.0; tCAO
:
1226.4; c@CH : 1081.8, 883.2.
3H), 0.86 (t, J = 7.34Hz, 3H). IR (KBr, cmꢀ1): tCH
:
2
2965.4, 2935.4, 2875.6; tC@O: 1735.0; tC@C: 1676.1,
VI-19: [3-chloro-4-(2-methylene-1-oxobutyl)phenoxy]-
acetic acid, white power, yield 78.7%, mp 102.5–
103.5 ꢁC, TLC Rf = 0.43 (acetone/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 7.32 (d, J = 8.51 Hz,
1H), 7.10 (d, J = 2.44 Hz, 1H), 6.97 (dd, J1 = 2.47 Hz,
J2 = 8.54 Hz, 1H), 6.02 (s, 1H), 5.52 (s, 1H), 4.80 (s,
2H), 2.34 (q, J = 7.5 Hz, 2H), 1.05 (t, J = 7.45 Hz,
3H). IR (KBr, cmꢀ1): t@CH: 3065.2; tCH: 2971.9,
2925.9, 2876.7; tC@O: 1737.3, 1712.0; tC@C: 1657.3,
1603.3; tCAO: 1246.2; c@CH : 1135.0.
2
VI-25: [3-methyl-4-(2-methylene-1-oxopropyl)phenoxy]-
acetic acid, white power, yield 27.3%, mp 112.0–
114.1 ꢁC, TLC Rf = 0.22 (acetone/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 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). IR (KBr, cmꢀ1): t@CH
3078.7, 3051.8; tCH: 2953.6, 2917.8, 2797.6; tC@O
:
:
:
1597.2; tCAO: 1228.1; c@CH : 1085.2, 885.3.
2
1733.1; tC@C: 1649.1, 1602.4; tCAO: 1242.4; c=CH
1121.3, 794.5.
2
VI-20: [3-chloro-4-(2-methylene-1-oxopropyl)phenoxy]-
acetic acid, white power, yield 56.1%, mp 107.4–
109.5 ꢁC, TLC Rf = 0.45 (acetone/petroleum ether, 1:3,
VI-26: [3-methyl-4-(2-methylene-1-oxobutyl)phenoxy]-
acetic acid, white power, yield 33.3%, mp 110.1–
113.1 ꢁC, TLC Rf = 0.25 (acetone/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 7.24 (d, J = 8.49 Hz,
1H), 6.83 (d, J = 2.48 Hz, 1H), 6.76 (dd, J1 =
2.62 Hz, J2 = 8.49 Hz, 1H), 5.86 (s, 1H), 5.42 (s,
1H), 4.72 (s, 2H), 2.22 (s, 3H), 1.55 (q, J = 7.31 Hz,
1
v/v). H NMR (DMSO-d6) d: 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). IR (KBr, cmꢀ1): t@CH: 3063.7; tCH
:
2983.2, 2920.1; tC@O: 1736.4, 1711.6; tC@C: 1654.6,
1596.4; tCAO: 1230.4; c@CH : 1085.5, 886.3.
2
2H), 1.02 (t, J = 7.40 Hz, 3H). IR (KBr, cmꢀ1): t@CH
3054.1; tCH: 2962.6, 2924.6, 2875.0; tC@O: 1732.6;
C@C: 1654.0, 1603.2; tCAO: 1244.3; c@CH : 1125.3,
:
VI-21: 2-[3-chloro-4-(2-methylene-1-oxopropyl)phenoxy]-
propionic acid, white power, yield 37.3%, mp 110.7–
112.8 ꢁC, TLC Rf = 0.51 (acetone/petroleum ether, 2:3,
t
2
756.5.

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G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
VI-27: 2-[2,3-dimethyl-4-(2-methylene-1-oxopentyl)phen-
oxy]propionic acid, yellow power, yield 86.2%, mp
83.6–85.2 ꢁC, TLC Rf = 0.75 (acetone/petroleum ether,
J = 8.50 Hz, 1H), 5.92 (s, 1H), 5.46 (s, 1H), 5.02 (q,
J = 6.72 Hz, 1H), 3.67 (s, 3H), 2.34 (t, J = 7.42 Hz,
2H), 2.15 (s, 3H), 2.10 (s, 3H), 1.54 (d, J = 6.84 Hz,
3H), 1.32 (m, 2H), 0.88 (t, J = 7.32 Hz, 3H). IR (KBr,
cmꢀ1): t@CH: 3087.0; tCH: 2988.4, 2955.4, 2930.8,
2871.9, 2860.9; tC@O: 1760.1, 1741.2; tC@C: 1656.6,
1
2:3, v/v). H NMR (DMSO-d6) d: 13.0 (s, 1H), 7.48 (d,
J = 8.66 Hz, 1H), 7.01 (d, J = 8.48 Hz, 1H), 5.93 (s,
1H), 5.48 (s, 1H), 4.84 (q, J = 6.80 Hz, 1H), 2.31 (t,
J = 7.43 Hz, 2H), 2.14 (s, 3H), 2.11 (s, 3H), 1.53 (d,
J = 6.74 Hz, 3H), 1.45 (m, 2H), 0.90 (t, J = 7.33 Hz,
1591.6, 1578.5; tCAO: 1262.0; c@CH : 1105.9, 799.3.
2
3H). IR (KBr, cmꢀ1): tCH: 2959.6, 2933.0, 2873.1; tC@O
1726.5, 1708.8; tC@C: 1653.4, 1591.7, 1577.1; tCAO
:
:
VII-2: methyl 2-[2,3-dimethyl-4-(2-methylene-1-oxohex-
yl) phenoxy]propionate, yellow oil, yield 61.0%, TLC
1
Rf = 0.55 (acetone/petroleum ether, 1:3, v/v). H NMR
1263.7; c@CH : 1105.0, 797.0.
2
(DMSO-d6) d: 7.46 (d, J = 8.65 Hz, 1H), 7.00 (d,
J = 8.48 Hz, 1H), 5.92 (s, 1H), 5.47 (s, 1H), 4.98 (q,
J = 6.76 Hz, 1H), 3.67 (s, 3H), 2.32 (t, J = 7.58 Hz,
2H), 2.15 (s, 3H), 2.11 (s, 3H), 1.55 (d, J = 7.95 Hz,
3H), 1.45 (m, 2H), 1.28 (m, 2H), 0.85 (t, J = 7.39 Hz,
3H). IR (KBr, cmꢀ1): t@CH: 3087.0; tCH: 2956.8,
2932.6, 2872.4; tC@O: 1759.8, 1741.0; tC@C: 1656.4,
VI-28: 2-[2,3-dimethyl-4-(2-methylene-1-oxohexyl)phen-
oxy]propionic acid, yellow power, yield 88.2%, mp 75.3–
76.7 ꢁC, TLC Rf = 0.51 (EtOAc/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 13.0 (s, 1H), 7.49 (d,
J = 8.65 Hz, 1H), 7.01 (d, J = 8.48 Hz, 1H), 5.93 (s,
1H), 5.48 (s, 1H), 4.88 (q, J = 6.74 Hz, 1H), 2.81 (t,
J = 7.27 Hz, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 1.53 (d,
J = 6.81 Hz, 3H), 1.44 (m, 4H), 0.85 (t, J = 7.39 Hz,
1591.6, 1578.8; tCAO: 1261.6; c@CH : 1105.2, 799.2.
2
3H). IR (KBr, cmꢀ1): tCH: 2959.6, 2932.8, 2873.0; tC@O
1726.5, 1708.8; tC@C: 1653.3, 1591.7, 1577.0; tCAO
:
:
VII-3: methyl [2,3-dimethyl-4-(2-methylene-1-oxopen-
tyl)phenoxy]acetate, yellow oil, yield 51.7%, TLC
1
Rf = 0.55 (acetone/petroleum ether, 1:3, v/v). H NMR
1263.7; c@CH : 1104.8, 797.0.
2
(DMSO-d6) d: 7.48 (d, J = 8.67 Hz, 1H), 7.02 (d,
J = 8.50 Hz, 1H), 5.92 (s, 1H), 5.49 (s, 1H), 4.84 (s,
2H), 3.70 (s, 3H), 2.32 (t, J = 7.42 Hz, 2H), 2.16 (s,
3H), 2.12 (s, 3H), 1.45 (m, 2H), 0.91 (t, J = 7.30 Hz,
3H). IR (KBr, cmꢀ1): t@CH: 3088.0; tCH: 2957.6,
2931.9, 2872.4; tC@O: 1764.1, 1743.3; tC@C: 1656.1,
VI-29: [2,3-dimethyl-4-(2-methylene-1-oxopentyl)phenoxy]-
acetic acid, yellow power, yield 74.3%, mp 75.1–78.3 ꢁC,
TLC Rf = 0.35 (EtOAc/petroleum ether, 1:3, v/v). 1H
NMR (DMSO-d6) d: 13.0 (s, 1H), 7.49 (d, J = 8.65 Hz,
1H), 7.02 (d, J = 8.48 Hz, 1H), 5.93 (s, 1H), 5.48 (s,
1H), 4.72 (s, 2H), 2.32 (t, J = 7.42 Hz, 2H), 2.15 (s,
3H), 2.11 (s, 3H), 1.44 (m, 2H), 0.91 (t, J = 7.33 Hz,
1591.8, 1579.5; tCAO: 1211.3; c@CH : 1124.2, 798.5.
2
3H). IR (KBr, cmꢀ1): tCH: 2959.7, 2931.5, 2874.8; tC@O
1747.6, 1714.0; tC@C: 1645.5, 1590.2, 1579.5; tCAO
:
:
VII-4: methyl [2,3-dimethyl-4-(2-methylene-1-oxohexyl)-
phenoxy]acetate, yellow oil, yield 32.9%, TLC
1
Rf = 0.57 (acetone/petroleum ether, 1:3, v/v). H NMR
1257.1; c@CH : 1123.3, 799.1.
2
(DMSO-d6) d: 7.48 (d, J = 8.67 Hz, 1H), 7.01 (d,
J = 8.50 Hz, 1H), 5.92 (s, 1H), 5.47 (s, 1H), 4.84 (s,
2H), 3.70 (s, 3H), 2.34 (t, J = 7.39 Hz, 2H), 2.16 (s,
3H), 2.12 (s, 3H), 1.40 (m, 2H), 1.31 (m, 2H), 0.88 (t,
VI-30: [2,3-dimethyl-4-(2-methylene-1-oxohexyl)phenoxy]-
acetic acid, yellow power, yield 72.3%, mp 79.8–
80.9 ꢁC, TLC Rf = 0.36 (EtOAc/petroleum ether, 1:3,
v/v). 1H NMR (DMSO-d6) d: 13.0 (s, 1H), 7.49 (d,
J = 8.63 Hz, 1H), 7.02 (d, J = 8.49 Hz, 1H), 5.93 (s,
1H), 5.47 (s, 1H), 4.72 (s, 2H), 2.34 (t, J = 7.46 Hz,
2H), 2.14 (s, 3H), 2.10 (s, 3H), 1.41 (m, 2H), 1.32 (m,
J = 7.28 Hz, 3H). IR (KBr, cmꢀ1): t@CH: 3087.6; tCH
2955.5, 2930.1, 2860.4; tC@O: 1764.3, 1743.4; tC@C
:
:
1656.3, 1591.6, 1579.1; tCAO: 1209.9; c@CH : 1124.0,
798.7.
2
2H), 0.89 (t, J = 7.30 Hz, 3H). IR (KBr, cmꢀ1): t@CH
3056.0; tCH: 2954.7, 2932.6, 2874.4; tC@O: 1743.1,
:
VII-5: methyl 2-[2,3-dimethyl-4-(2-methylene-1-oxobu-
tyl)phenoxy]propionate, yellow oil, yield 42.0%, TLC
1716.5; tC@C: 1673.2, 1644.4, 1591.3, 1580.2; tCAO
:
1
Rf = 0.55 (acetone/petroleum ether, 1:3, v/v). H NMR
1252.2; c@CH : 1124.5, 804.1.
2
(DMSO-d6) d: 7.01 (d, J = 8.48 Hz, 1H), 6.69 (d,
J = 8.47 Hz, 1H), 5.92 (s, 1H), 5.46 (s, 1H), 4.98 (q,
J = 6.74 Hz, 1H), 3.67 (s, 3H), 2.34 (q, J = 7.34 Hz,
2H), 2.15 (s, 3H), 2.11 (s, 3H), 1.54 (d, J = 6.82 Hz,
3.2. Synthesis of the target compounds of the VII series
A compound VI (2 mmol) and para-toluenesulfonic acid
(0.58 mmol) were dissolved in methanol (10 mL). The
mixture was heated up to 45 ꢁC for 40 min and diluted
by ether (15 mL) after the mixture was cooled down to
room temperature. Then the mixture was washed with
5% sodium bicarbonate solution (10 mL, 2·). The ether
layer was collected and dried with MgSO4 overnight.
After filtering, the ether was evaporated under reduced
pressure to obtain a yellow product VII.
3H), 1.04 (t, J = 7.42 Hz, 3H). IR (KBr, cmꢀ1): t@CH
:
3087.5; tCH: 2963.3, 2935.9, 2875.4; tC@O: 1759.7,
1740.9; tC@C: 1656.0, 1591.5, 1579.8; tCAO: 1261.2;
c@CH : 1136.2, 799.3.
2
VII-6: methyl 2-[2,3-dimethyl-4-(2-methylene-1-oxopro-
pyl)phenoxy]propionate, yellow oil, yield 41.0%, TLC
1
Rf = 0.52 (acetone/petroleum ether, 1:3, v/v). H NMR
(DMSO-d6) d: 7.02 (d, J = 8.48 Hz, 1H), 6.68 (d,
J = 8.56 Hz, 1H), 6.01 (s, 1H), 5.45 (s, 1H), 5.00 (q,
J = 6.77 Hz, 1H), 3.67 (s, 3H), 2.15 (s, 3H), 2.09 (s,
3H), 1.94 (s, 3H), 1.54 (d, J = 6.77 Hz, 3H). IR (KBr,
VII-1: methyl 2-[2,3-dimethyl-4-(2-methylene-1-oxopen-
tyl)phenoxy]propionate, yellow oil, yield 41.1%, TLC
1
Rf = 0.56 (acetone/petroleum ether, 1:3, v/v). H NMR
(DMSO-d6) d: 7.47 (d, J = 8.66 Hz, 1H), 7.00 (d,
cmꢀ1): t@CH
: 3089.2; tCH: 2987.0, 2953.6; tC@O:

G. Zhao et al. / Bioorg. Med. Chem. 15 (2007) 2701–2707
2707
1759.0, 1740.1; tC@C: 1657.2, 1591.7, 1580.2; tCAO
:
Acknowledgments
1261.4; c@CH : 1135.9, 799.3.
2
This work was partly supported by the Joint Research
Fund for Overseas Chinese Young Scholars of National
Natural Science Foundation of China (30328030) and
the National Natural Science Foundation of China
(20672069).
3.3. Biological activity methods
3.3.1. Cells. HL-60 cells were cultured in RPMI-1640
medium supplemented with 100 U/mL pencillin,
100 lg/mL streptomycin, 1 mmol/L L-glutamine, and
10% heat-inactivated fetal bovine serum.
References and notes
3.3.2. GSTP1-1 activity. GSTP1-1 activity was measured
using CDNB and GSH as substrates as we described
previously.14 Cells (3 · 106) in logarithmic growth were
collected, washed twice with PBS, resuspended in
300 lL of 100 mmol/L potassium phosphate buffer, pH
6.8, sonicated for 10 s at 4 ꢁC, centrifuged at
14,000 rpm for 30 min at 4 ꢁC, and the supernatant fluid
was used for enzyme activity assays. The protein content
in the cell lysates was determined by the Bradford Pro-
tein Assay, with bovine serum albumin as a standard.
The assay mixture contained 2.55 mL of 0.1 mol/L sodi-
um phosphate–1 mmol/L EDTA (pH 6.5), 150 lL of
20 mmol/L GSH, 150 lL of 20 mmol/L CDNB, and
150 lL of clear cell lysate. An assay mixture without
the cell lysate was used as a control. The absorbance
at 340 nm was continuously recorded for 2 min. The
nonenzymatic reaction was corrected by blanking the
spectrophotometer with the control cuvette before read-
ing a sample cuvette. The CDNB-GSH product has a
strong molar absorptivity at 340 nm (=9.6 mMꢀ1 cmꢀ1).
GSTP1-1 activity was defined as nanomoles of product
per min per milligram of protein.
1. Townsend, D. M.; Tew, K. D. Oncogene 2003, 22,
7369.
2. Kitahara, A.; Satoh, K.; Nishimura, K.; Ishikawa, T.;
Ruike, K.; Sato, K.; Tsuda, H.; Ito, N. Cancer Res. 1984,
44, 2698.
3. Jensson, H.; Eriksson, L. C.; Mannervik, B. FEBS Lett.
1985, 187, 115.
4. Di Ilio, C.; Del Boccio, G.; Aceto, A.; Casaccia,
R.; Mucilli, F.; Federici, G. Carcinogenesis 1988, 9,
335.
5. Howie, A. F.; Forrester, L. M.; Glancey, M. J.; Schlager,
J. J.; Powis, G.; Hayes, J. D.; Wolf, C. R. Carcinogenesis
1990, 11, 451.
6. Eimoto, H.; Tsutsumi, M.; Nakajima, A.; Yamamoto, K.;
Takashima, Y.; Maruyama, H.; Konishi, Y. Carcinogen-
esis 1988, 9, 2325.
7. Ranganathan, S.; Tew, K. D. Carcinogenesis 1991, 12,
2383.
8. Di Ilio, C.; Del Boccio, G.; Aceto, A.; Federici, G.
Carcinogenesis 1987, 8, 861.
9. Harrison, D. J.; Kharbanda, R.; Bishop, D.; McLellan, L.
I.; Hayes, J. D. Carcinogenesis 1989, 10, 1257.
10. Shiratori, Y.; Soma, Y.; Maruyama, H.; Sato, S.; Takano,
A.; Sato, K. Cancer Res. 1987, 47, 6806.
11. Moscow, J. A.; Fairchild, R. R.; Madden, M. J.; Ransom,
D. T.; Wieand, H. S.; O’Brien, E. E.; Poplack, D. G.;
Cossman, J.; Myers, C. E.; Cowan, K. H. Cancer Res.
1989, 49, 1422.
12. Zhao, G. S.; Wang, X. B. Curr. Med. Chem. 2006, 13,
1461.
13. Aizawa, S.; Ookawa, K.; Kudo, T.; Asano, J.; Hayakari,
M.; Tsuchida, S. Cancer Sci. 2003, 94, 886.
14. Zhao, G. S.; Yu, T.; Wang, R.; Wang, X. B.; Jing, Y. K.
Bioorg. Med. Chem. 2005, 13, 4056.
3.3.3. Cell proliferation inhibition. Cells were seeded at a
density of 1 · 105 cells/mL and incubated with various
concentrations of the tested compounds. Total cell num-
ber including trypan blue staining positive and negative
cells in each group was counted. The antiproliferative
ability was calculated and expressed as the ratio of the
cell number in the treated group to that of the untreated
group. The concentration (GI50) which inhibited half of
the cell proliferation was calculated.