The synthesis of l-carvone and limonene derivatives with increased antiproliferative effect and activation of ERK pathway in prostate cancer cells

Article abstract of DOI:10.1016/j.bmc.2006.06.013

Thirty-one novel derivatives of carvone, carveol, and limonene were designed and synthesized using l-carvone as a starting material via chlorination, nucleophilic substitution, and reduction. The structures of these derivatives were characterized by MS an

Full text of DOI:10.1016/j.bmc.2006.06.013

Bioorganic & Medicinal Chemistry 14 (2006) 6539–6547
The synthesis of L-carvone and limonene derivatives with increased
antiproliferative effect and activation of ERK pathway
in prostate cancer cells
Jiaojiao Chen,a Min Lu,b Yongkui Jingb,* and Jinhua Donga,*
aSchool of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
bDepartment of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
Received 16 May 2006; revised 5 June 2006; accepted 6 June 2006
Available online 27 June 2006
Abstract—Thirty-one novel derivatives of carvone, carveol, and limonene were designed and synthesized using L-carvone as a start-
ing material via chlorination, nucleophilic substitution, and reduction. The structures of these derivatives were characterized by MS
1
and H NMR. The antiproliferative effect was evaluated in human prostate cancer LNCaP cells. L-Carvone, L-carveol, and L-lim-
onene were weak cell growth inhibitors and introduction of 4-(2-methoxyphenyl)piperazine to carvone, carveol or limonene signif-
icantly increased their antiproliferative effect. The antiproliferative effect was correlated with ERK activation and p21waf1 induction.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
OH
O
Natural isoprenoids have been found to have effects of
inhibiting tumor cell growth in vivo and in vitro.1–3
Among these isoprenoids, monoterpene D-limonene
and its in vivo metabolite, perillyl alcohol (Fig. 1), have
been observed with preventive and therapeutic effects
against a variety of tumors in animal models.4–6
Although the mechanism of D-limonene and perillyl
alcohol of inhibiting tumor cell growth is unclear, it
has been proposed that inhibition of farnesyl transferase
might be one of their action targets.7–9 Both agents have
been put into Phase I and phase II clinical trials and the
preliminary results indicated that both agents were well
tolerated in cancer patients.10–13 Based on the in vitro
results, higher concentrations of D-limonene are needed
to reach a therapeutic effect. The low polarity of limo-
nene structure might limit its ability to transverse cellu-
lar membranes and resulted in the less activity.9
Structural modifications with increased polarity of limo-
nene might lower its effective concentrations. Some hy-
droxy-containing derivatives of limonene have been
D-limonene
D-perillyl alcohol
L-carvone
Figure 1. Structures of D-limonene, D-perillyl alcohol, and L-carvone.
synthesized and their inhibitory effects on tumor cell
growth have been compared. It was found that those
derivatives with improved polarity without breaking
unsaturated double bond of limonene have increased
antiproliferative effect.9 Based on this observation, we
designed and synthesized a new group of limonene ana-
logues containing polar groups in the structure.
Since direct structural modification from limonene
might result in multiple by-products, L-carvone was cho-
sen as the starting reagent and derivatives of carvone,
carveol, and limonene were generated. The structural
modification of L-carvone was focused on the 10-carbon
and the carbonyl moiety. Lipophilic benzoates and
hydrophilic amines were linked to the terpenoid moiety
in order to enhance polarity and to alter hydrophilicity.
In this study, thirty-one novel derivatives of limonene,
carvone, and carveol were synthesized. Among these
Keywords: L-Limonene; L-Carvone; Synthesis; Antiproliferative effect;
ERK; Prostate cancer.
*
Corresponding authors. Tel.: +862423986402; fax: +862423904249
(J.D.); e-mail addresses: yongkui.jing@mssm.edu; dongjh66@
hotmail.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.013

6540
J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
derivatives, 21 compounds contain carvone skeleton,
five compounds contain carveol skeleton, and five com-
pounds contain limonene skeleton. The cell growth
inhibitory effect of these compounds was measured in
prostate cancer cells in vitro and the potential mecha-
nism was explored.
carvone, carveol, and limonene were in L-
configuration.
2.2. Antiproliferative effect
The antiproliferative effects of these compounds were
measured in human prostate LNCaP cancer cells using
MTT assay. The IG50 (the concentration which inhibits
50% of the cell growth) was calculated. As shown in Ta-
ble 1, the IG50s of L-carvone, L-carveol, and L-limonene
were more than 100 lM (the highest concentration used
in this experiment). The addition of a group of 4-meth-
ylbenzoic acid (II2), 4-methoxybenzoic acid (II6) or 4-
aminobenzoic acid (II7) significantly increased the anti-
proliferative activity of L-carvone. Addition of substi-
2. Results and discussion
2.1. Chemistry
The synthetic route of these derivatives starting with L-
carvone is listed in Figure 2. The intermediate com-
pound, 5-(1-chloromethyl)vinyl-2-methylcyclohex-2-en-
one (I), was prepared by chlorination of L-carvone
with tert-butyl hypochlorite in n-hexane at room tem-
perature.14 Compound I reacted with a substituted
potassium benzoate in presence of potassium carbonate
in N,N-dimethylformamide and a corresponding ester
derivative (II1–II7) was obtained. Reaction of com-
pound I with N-alkylpiperazines or N-arylpiperazines
in presence of potassium carbonate in boiling ethanol
afforded the derivatives containing a piperazine moiety
(III1–III8). Compounds IV1–IV6 were prepared by
reacting compound I with an aliphatic amine or hetero-
cyclic amine. Sodium borohydride reduction of ketones
III1, III2, or III5–III7 in methanol at room temperature
yielded V1–V5, respectively. Compounds VI1–VI5 were
prepared from related ketone derivatives using 85%
hydrazine hydrate and potassium hydroxide based on
the method of Wolff–Kishner reduction.15 Since the
conditions of both reduction methods could not change
the configuration of L-carvone, all the derivatives of
tuted
groups
of
N-alkylpiperazine
or
N-
benzylpiperazine in carvone (III1–III5) did not increase
the antiproliferative effect of L-carvone evidently. How-
ever, introduction of N-(4-methoxyphenyl)piperazine
(III6), N-(2-methoxyphenyl)piperazine (III7), or N-(2-
chlorophenyl)piperazine (III8) significantly increased
the antiproliferative effect of L-carvone. Based on these
data, it seems that the compounds with increased activ-
ity have increased the polarity of L-carvone. The addi-
tion of a substituted amine into L-carvone did not
increase the antiproliferative effect (IV) except for IV4
which had a 2-thiopheneethylamine substitute. Since
some N-arylpiperazine-substituted carvone derivatives
(III) have an increased antiproliferative effect, we have
synthesized related carveol and limonene derivatives
by reduction of III. As shown in Table 1, although
N-methylpiperazine or N-ethylpiperazine-substituted
carvone (III1 and III2) and carveol (V1 and V2) did
not increase the antiproliferative effect of L-carvone
O
b
R1
O
HO
O
e
II 1-7
R2
N
N
O
O
O
V 1-5
a
c
R2
N
N
Cl
III1-8
L-carvone
I
f
R2
N
N
O
VI 1-5
d
NR3R4
IV1-6
Figure 2. The synthetic route of L-carvone, L-carveol, and L-limonene derivatives. Reagents: (a) tert-butyl hypochlorite; (b) a substituted potassium
benzoate (II1–II7: R1 = H, CH3, F, Cl, Br, OCH3, or NH2, respectively) and K2CO3; (c) a N-alkylpiperazines or N-arylpiperazine (III1–III8:
R2 = CH3, C2H5, isopropyl, isobutyl, benzyl, 4-methoxyphenyl, 2-methoxyphenyl, or 2-chlorophenyl, respectively) and K2CO3; (d) an aliphatic
amine or heterocyclic amine (IV1–IV6: NR3R4 = pyrrolidinyl, piperidinyl, cyclohexylamino, 2-thiopheneethylamino, dimethylamino, or 1-
adamantanamino, respectively) and K2CO3; (e) NaBH4; (f) NH2NH2ÆH2O (85%) and KOH.

J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
6541
Table 1. The structures and IG50 values of carvone, carveol, and
limonene derivatives in LNCaP cells
Table 1 (continued)
(continued on next page)

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J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
Table 1 (continued)
evidently, the addition of N-methylpiperazine or N-eth-
ylpiperazine into limonene (VI1 and VI2) significantly
increased the antiproliferative effect of limonene. Based
on compounds III, it was found that introduction of
4-arylpiperazine moiety significantly increased the
antiproliferative effect of carvone than addition of other
groups. By focusing on compounds III6, III7, V4, V5,
VI4, and VI5, it seems that derivatives of limonene are
more potent than the derivatives of carvone and carveol
with same substitute.
2.3. The effects of some potent compounds on farnesyl
transferase and MAP kinases
It has been suggested that limonene-like monoterpenes
are inhibitors of farnesyl transferase and their tumor
growth inhibitory effect might result from the inhibi-
tion of farnesyl transferase.8,16 To investigate this pos-
sibility, the farnesyl transferase inhibition was
investigated by measuring the HDJ2 unfarnesyl form
using Western blot analyses as reported.17 Compounds
II7, III6, IV4, V4, VI4, L-limonene, and L-carvone were
selected for this purpose. LNCaP cells were treated
with these compounds at a concentration of 50 lM
for 1–3 days. Surprisingly, no one of these compounds
at this concentration increased the unfarnesyl form of
HDJ2 (data not shown). Since compounds II7, III6,
IV4, V4, and VI4 significantly inhibited LNCaP cell
growth at this concentration (Table 1), it suggests that
the cell growth inhibitory effects of these compounds
might not be through inhibition of farnesyl transfer-
ase. Since these compounds showed cytostatic but
not cytotoxic effect, we suspect that the cell growth
survival pathways might be regulated by these com-
pounds. It has been found that ERK and p38 MAP
kinases are regulators of cell growth and some natural
compounds inhibited cell growth through activation or
inhibition of these kinases.18–20 The protein and phos-
phorylated forms of both p38 and ERK were investi-
gated in LNCaP cells. As shown in Figure 3,
phosphorylated ERK was significantly increased in
LNCaP cells after treatment with compounds II7,
III6, IV4, V4, and VI4, but not by L-limonene or L-
carvone. The increased levels of phosphorylated
ERK sustained for three days. Unlike phosphorylated
ERK, phosphorylated p38 was increased transiently.
Since compounds II7, III6, IV4, V4, and VI4, but not
by L-limonene or L-carvone, significantly inhibited
growth of LNCaP cells, these data suggest that acti-
vated ERK would participate in antiproliferative effect
of these compounds. Recently, it has been found that
activated ERK leads to an increase of cell cycle regu-
21,22
latory protein p21waf1
,
the protein level of p21waf1
was compared. As shown in Figure 3, p21waf1 protein
level was increased in LNCaP cells after treatment
with II7, III6, IV4, V4, and VI4, but not by L-limonene
or L-carvone. The p21waf1 induction was correlated
with the increased levels of phosphorylated ERK. This
result indicates that these derivatives might inhibit cell
growth through activation of ERK which leads to
upregulation of p21waf1 protein and cell cycle arrest.
The mechanism of these compounds which activate
ERK is worthy of further study.

J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
6543
(2H, s, –CH2Cl), 5.03 (1H, s, @CH2), 5.23 (1H, s,
@CH2), 6.73 (1H, m, @CH–).
3.1.2. General procedure for the preparation of com-
pounds II1–II7
3.1.2.1. Benzoic acid 2-(4-methyl-5-oxocyclohex-3-
enyl)allyl ester (II1). Compound I (1.5 g, 8.1 mmo1),
potassium carbonate (1.2 g, 17.8 mmol), and potassium
benzoate (1.4 g, 8.1 mmol) were dissolved in 15 mL
dry N,N-dimethylformamide and heated to 80–85 ꢁC
for 6–8 h. Then water (100 mL) was added and the reac-
tion mixture was extracted with methylene chloride (5·
30 mL). The combined organic extracts were washed
with brine, dried over anhydrous sodium sulfate, and fil-
tered. The filtrate was concentrated in vacuo. The resi-
due was purified on a silica gel column with petroleum
ether–ethylacetate as eluent to give the target product
1
II1 (0.48 g, yield 22.0%), mp 65–67 ꢁC. H NMR 1.79
(3H, s, –CH3), 2.31–2.51 (2H, m, –CH2–), 2.59–2.70
(2H, m, –CH2–), 2.90 (1H, m, >CH–), 4.81 (1H, d,
–CH2–O–), 4.87 (1H, d, –CH2–O–), 5.11 (1H, s,
@CH2), 5.29 (1H, s, @CH2), 6.70 (1H, m, @CH–),
7.44 (2H, dd, Ar-H), 7.56 (1H, t, Ar-H), 8.04 (2H, d,
Ar-H). MS (EI) m/z: 270 (M+, 11), 105 (100).
3.1.2.2. 4-Methylbenzoic acid 2-(4-methyl-5-oxocyclo-
hex-3-enyl)allyl ester (II2). Compound II2 was obtained
in 24.5% yield, mp 63–64 ꢁC. 1H NMR 1.79 (3H, s,
–CH3), 2.30–2.50 (5H, m, –CH2–, -CH3), 2.58–2.65
(2H, m, –CH2–), 2.88 (1H, m, >CH–), 4.77 (1H, d,
–CH2–O–), 4.88 (1H, d, –CH2–O–), 5.10 (1H, s,
@CH2), 5.27 (1H, s, @CH2), 6.75 (1H, m, @CH–),
7.25 (2H, d, Ar-H), 7.93 (2H, d, Ar-H). MS (EI) m/z:
284 (M+, 20), 119 (100).
Figure 3. The effect of some synthetic derivatives on the phosphory-
lation of ERK and p38 as well as p21waf1 protein levels. LNCaP cells
were treated with indicated compounds at 50 lM for indicated times.
Whole cellular protein lysates were prepared with RIPA buffer and
Western blot analysis was used to analyze each protein level using
indicated antibodies. Lim, L-limonene; Cav, L-carvone.
3.1.2.3. 4-Fluorobenzoic acid 2-(4-methyl-5-oxocyclo-
hex-3-enyl)allyl ester (II3). Compound II3 was obtained
in 29.1% yield, mp 73–75 ꢁC. 1H NMR 1.79 (3H, s,
–CH3), 2.31–2.50 (2H, m, –CH2–), 2.58–2.69 (2H, m,
–CH2–), 2.88 (1H, m, >CH–), 4.82 (1H, d, –CH2–O–),
4.84 (1H, d, –CH2–O–), 5.12 (1H, s, @CH2), 5.27 (1H,
s, @CH2), 6.76 (1H, m, @CH–), 7.13 (2H, t, Ar-H),
8.04 (1H, d, Ar-H), 8.07 (1H, d, Ar-H). MS (EI) m/z:
288 (M+, 6), 123 (100).
3. Experimental
3.1. Chemistry
Melting points were determined on a Yanaco micro
melting point apparatus and are uncorrected. Mass
spectra were determined on an Agilent-5973A spectrom-
1
eter. H NMR spectra were recorded on Varian mercu-
ry-300 NMR instrument using tetramethylsilane (TMS)
as the internal standard (chemical shifts in d, ppm) in
deuteriochloroform. All the reagents and solvents used
in the experiments were obtained from commercial
sources and used without further purification.
3.1.2.4. 4-Chlorobenzoic acid 2-(4-methyl-5-oxocyclo-
hex-3-enyl)allyl ester (II4). Compound II4 was obtained
in 18.6% yield, mp 56–58 ꢁC. 1H NMR 1.79 (3H, s,
–CH3), 2.30–2.50 (2H, m, –CH2–), 2.57–2.69 (2H, m,
–CH2–), 2.87 (1H, m, >CH–), 4.83 (1H, d, –CH2–O–),
4.84 (1H, d, –CH2–O–), 5.12 (1H, s, @CH2), 5.27 (1H,
s, @CH2), 6.75 (1H, m, @CH–), 7.44 (2H, d, Ar-H),
7.97 (2H, d, Ar-H). MS (EI) m/z: 304 (M+, 6), 148 (100).
3.1.1. 5-(1-Chloromethyl)vinyl-2-methylcyclohex-2-enone
(I). tert-Butyl hypochlorite (13.3 g, 0.11 mol) was added
dropwise into a solution of L-carvone (15.0 g, 0.1 mol)
in n-hexane (300 mL) at 0 ꢁC. The mixture was stirred
for 3 h at room temperature, washed with aqueous sodi-
um sulfite and brine, dried over anhydrous sodium sul-
fate, and filtered. The filtrate was concentrated in
vacuo to give brown oil, which was purified on silica
gel column with petroleum ether as eluent to give the ti-
tle compound (10.7 g, 76.0% yield) as yellow oil. 1H
NMR 1.76 (3H, s, –CH3), 2.33–2.40 (2H, m, –CH2–),
2.49–2.66 (2H, m, –CH2–), 2.94 (1H, m, >CH–), 4.06
3.1.2.5. 4-Bromobenzoic acid 2-(4-methyl-5-oxocyclo-
hex-3-enyl)allyl ester (II5). Compound II5 was obtained
in 23.4% yield, mp 56–58 ꢁC. 1H NMR 1.79 (3H, s,
–CH3), 2.30–2.50 (2H, m, –CH2–), 2.57–2.69 (2H, m,
–CH2–), 2.83 (1H, m, >CH–), 4.80 (1H, d, –CH2–O–),
4.86 (1H, d, –CH2–O–), 5.11 (1H, s, @CH2), 5.27 (1H,
s, @CH2), 6.75 (1H, m, @CH–), 7.60 (2H, d, Ar-H),
7.90 (2H, d, Ar-H). MS (EI) m/z: 349 (M+, 3), 148 (100).

6544
J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
3.1.2.6. 4-Methoxybenzoic acid 2-(4-methyl-5-oxocy-
d, –CH(CH3)2), 1.64 (3H, s, –CH3), 1.88 (1H, m,
–CH(CH3)2), 2.21–2.43 (6H, m, –CH2–, –N–CH2–),
2.49 (1H, m, >CH–), 2.57 (4H, m, –N–CH2–), 2.72
(4H, m, –N–CH2–), 2.90 (2H, s, –N–CH2–), 4.84 (1H,
s, @CH2), 4.91 (1H, s, @CH2), 6.64 (1H, m, @CH–).
MS (EI) m/z: 290 (M+, 38), 247 (100).
clohex-3-enyl)allyl ester (II6). Compound II6 was ob-
tained in 22.7% yield, oily material. 1H NMR 1.79
(3H, s, –CH3), 2.30–2.50 (2H, m, –CH2–), 2.58–2.67
(2H, m, –CH2–), 2.89 (1H, m, >CH–), 3.87 (3H, s,
–OCH3), 4.80 (1H, d, –CH2–O–), 4.84 (1H, d, –CH2–
O–), 5.09 (1H, s, @CH2), 5.27 (1H, s, @CH2), 6.76
(1H, m, @CH–), 6.92 (2H, d, Ar-H), 7.99 (2H, d, Ar-
H). MS (EI) m/z: 300 (M+, 20), 135 (100).
3.1.3.5. 5-[1-(4-Benzylpiperazin-1-yl)methyl]vinyl-2-
methylcyclohex-2-enone (III5). Compound III5 was ob-
1
tained in 77.3% yield. H NMR (DMSO-d6), 1.76 (3H,
3.1.2.7. 4-Aminobenzoic acid 2-(4-methyl-5-oxocyclo-
hex-3-enyl)allyl ester (II7). Compound II7 was obtained
in 24.3% yield, mp 64–65 ꢁC. 1H NMR 1.79 (3H, s,
–CH3), 2.30–2.50 (4H, m, –CH2–, –NH2), 2.56–2.73 (2H,
m, –CH2–), 2.88 (1H, m, >CH–), 4.81 (1H, d, –CH2–O–),
4.85 (1H, d, –CH2–O–), 5.10 (1H, s, @CH2), 5.27 (1H, s,
@CH2), 6.75 (1H, m, @CH–), 7.25 (2H, d, Ar-H), 7.94
(2H, d, Ar-H). MS (EI) m/z: 284 (M+À1, 14), 119 (100).
s, –CH3), 2.36 (2H, m, –CH2–), 2.70 (2H, m, –CH2–),
3.33 (1H, m, >CH–), 3.45–3.63 (4H, m, –N–CH2–),
3.76 (2H, s, –CH2Ph), 4.03 (4H, m, –N–CH2–), 4.27
(2H, s, –N–CH2–), 5.44 (1H, s, @CH2), 5.58 (1H, s,
@CH2), 6.74 (1H, m, @CH–), 7.47 (3H, m, Ar-H),
7.67 (2H, m, Ar-H). MS (EI) m/z: 324 (M+, 22), 91 (100).
3.1.3.6. 5-{1-[4-(4-Methoxyphenyl)piperazin-1-yl]-
methyl}vinyl-2-methylcyclohex-2-enone (III6). Com-
3.1.3. General procedure for the preparation of com-
pounds III1–III8
1
pound III6 was obtained in 65.9% yield. H NMR 1.79
(3H, s, –CH3), 2.37 (2H, m, –CH2–), 2.77 (2H, m,
–CH2–), 3.36 (1H, m, >CH–), 3.79 (9H, m, –N–CH2–,
–OCH3), 4.27 (2H, m, –N–CH2–), 4.89 (2H, m, –N–
CH2–), 5.50 (1H, s, @CH2), 5.67 (1H, s, @CH2), 6.76
(1H, m, @CH–), 7.00 (2H, d, Ar-H), 7.86 (2H, d, Ar-
H). MS (EI) m/z: 340 (M+, 100).
3.1.3.1. 2-Methyl-5-{[1-(4-methylpiperazin-1-yl)meth-
yl]vinyl}cyclohex-2-enone (III1). Compound I (1.5 g,
8.1 mmo1), potassium carbonate (1.2 g, 8.9 mmol), and
N-methylpiperazine (0.8 g, 8.1 mmol) were dissolved in
ethanol (15 mL) and refluxed for 6–10 h. Then the sol-
vent was removed by evaporation. The residue was dis-
solved in 30 mL of methylene chloride, washed with 3 N
hydrochloric acid (3· 30 mL). The combined aqueous
solution was neutralized with aqueous sodium carbon-
ate to pH 9.0–10.0, extracted with methylene chloride
(5· 30 mL). The combined organic extracts were washed
with brine, dried over anhydrous sodium sulfate, and fil-
tered. The filtrate was concentrated in vacuo. The resi-
due was purified on a silica gel column with petroleum
ether–ethylacetate as eluent to afford the target product
3.1.3.7. 5-{1-[4-(2-Methoxyphenyl)piperazin-1-yl]-
methyl}vinyl-2-methylcyclohex-2-enone (III7). Com-
1
pound III7 was obtained in 70.5% yield. H NMR 1.79
(3H, s, –CH3), 2.35–2.51 (2H, m, –CH2–), 2.56–2.66
(6H, m, –CH2–, –N–CH2–), 2.89 (1H, m, >CH–), 3.00
(2H, m, –N–CH2–), 3.05 (4H, m, –N–CH2–), 3.84 (3H,
s, –OCH3), 4.93 (1H, s, @CH2), 5.05 (1H, s, @CH2),
6.75 (1H, m, @CH–), 6.83–7.00 (4H, m, Ar-H). MS
(EI) m/z: 340 (M+, 84), 150 (100).
1
III1 (1.79 g, yield 67.3%). H NMR (DMSO-d6), 1.75
(3H, s, –CH3), 2.30–2.55 (15H, m, –CH2–, –N–CH2–,
–N–CH3), 2.88 (1H, m, >CH–), 2.90 (2H, m, –CH2–
N–), 4.88 (1H, s, @CH2), 4.98 (1H, s, @CH2), 6.75
(1H, m, @CH–). MS (EI) m/z: 248 (M+, 86), 58 (100).
3.1.3.8. 5-{1-[4-(2-Chlorophenyl)piperazin-1-yl]meth-
yl}vinyl-2-methylcyclohex-2-enone (III8). Compound
1
III8 was obtained in 66.1% yield. H NMR 1.79 (3H,
s, –CH3), 2.20–2.42 (2H, m, –CH2–), 2.66–2.86 (2H,
m, –CH2–), 3.40 (1H, m, >CH–), 3.44–3.81 (8H, m,
–N–CH2–), 4.40 (2H, m, –N–CH2–), 5.48 (1H, s,
@CH2), 5.63 (1H, s, @CH2), 6.77 (1H, m, @CH–),
7.24 (1H, m, Ar-H), 7.35 (2H, m, Ar-H), 7.47 (1H, m,
Ar-H). MS (EI) m/z: 340 (M+, 84), 150 (100).
3.1.3.2. 5-[1-(4-Ethylpiperazin-1-yl)methyl]vinyl-2-
methylcyclohex-2-enone (III2). Compound III2 was ob-
1
tained in 61.5% yield. H NMR (DMSO-d6), 1.51 (3H,
t, –CH2–CH3), 1.79 (3H, s, –CH3), 2.23–2.45 (2H, m,
–CH2–), 2.68–2.74 (2H, m, –CH2–), 3.22 (2H, q,
–CH2–CH3), 3.31 (1H, m, >CH–), 3.58 (4H, m, –N–
CH2–), 3.76 (2H, m, –CH2–N–), 3.95–4.20 (4H, m,
–N–CH2–), 5.47 (1H, s, @CH2), 5.61 (1H, s, @CH2),
6.75 (1H, m, @CH–). MS (EI) m/z: 262 (M+, 100).
3.1.4. General procedure for the preparation of com-
pounds IV1–IV6. The title compounds were synthesized
using the method described for the synthesis of com-
pound III1, substituting an aliphatic amine or a hetero-
cyclic amine for N-methylpiperazine.
3.1.3.3. 5-[1-(4-Isopropylpiperazin-1-yl)methyl]vinyl-2-
methylcyclohex-2-enone (III3). Compound III3 was ob-
tained in 70.4% yield. 1H NMR 1.50 (6H, d, –CH(CH3)2),
1.79 (3H, s, –CH3), 2.30 (2H, m, –CH2–), 2.71 (2H, m,
–CH2–), 3.25 (1H, m, >CH–), 3.40–4.40 (11H, m, –N–
CH2–, –CH(CH3)2), 5.46 (1H, s, @CH2), 5.63 (1H, s,
@CH2), 6.76 (1H, m, @CH–). MS (EI) m/z: 276 (M+, 100).
3.1.4.1. 2-Methyl-5-[1-(pyrrolidin-1-ylmethyl)vinyl]-
cyclohex-2-enone (IV1). Compound IV1 was obtained
1
in 69.3% yield. H NMR 1.80 (3H, s, –CH3), 2.08 (2H,
m, –CH2–), 2.29 (3H, m, –CH2–), 2.42 (1H, m, –CH2–),
2.67 (1H, m, –CH2–), 2.81 (3H, m, –CH2–,
–N–CH2–), 3.30 (1H, m, >CH–), 3.57–3.80 (4H, m,
–N–CH2–), 5.35 (1H, s, @CH2), 5.56 (1H, s, @CH2),
6.77 (1H, m, @CH–). MS (EI) m/z: 218 (M+, 25), 84
(100).
3.1.3.4. 5-[1-(4-Isobutylpiperazin-1-yl)methyl]vinyl-2-
methylcyclohex-2-enone (III4). Compound III4 was ob-
1
tained in 75.4% yield. H NMR (DMSO-d6), 0.89 (6H,

J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
6545
3.1.4.2. 2-Methyl-5-[1-(piperidin-1-ylmethyl)vinyl]-
cyclohex-2-enone (IV2). Compound IV2 was obtained
umn with petroleum ether–ethylacetate–methanol as
eluent to afford the target product V1 (0.42 g, yield
1
in 74.8% yield. H NMR 1.43 (1H, m, –CH2–), 1.78
1
16.4%). H NMR 1.57 (1H, m, –CH2–), 1.77 (3H, s,
(3H, s, –CH3), 1.86 (3H, m, –CH2–), 2.18–2.27 (1H,
m, –CH2–), 2.35–2.69 (6H, m, –CH2–, –N–CH2–), 2.84
(1H, m, –N–CH2–), 3.34 (1H, m, >CH–), 3.43 (2H, m,
–N–CH2–), 3.56 (1H, m, –N–CH2–), 3.70 (1H, m,
–N–CH2–), 5.38 (1H, s, @CH2), 5.54 (1H, s, @CH2),
6.76 (1H, m, @CH–). MS (EI) m/z: 232 (M+, 43), 98
(100).
–CH3), 1.96 (2H, m, –CH2–), 2.14 (2H, m, –CH2–), 2.21
(1H, m, >CH–), 2.29 (3H, s, –N–CH3), 2.42–2.44 (8H,
m, –N–CH2–), 2.91–2.95 (2H, d, –N–CH2–), 4.17 (1H,
m, >CHOH), 4.93 (1H, s, @CH2), 4.95 (1H, s, @CH2),
5.51 (1H, m, @CH–). MS (EI) m/z: 251 (M+, 100).
3.1.5.2. 5-{[1-(4-Ethylpiperazin-1-yl)methyl]vinyl}-2-
methylcyclohex-2-enol (V2). Compound V2 was obtained
in 19.2% yield. 1H NMR 1.09 (3H, m, –CH3), 1.57 (1H, m,
–CH2–), 1.76 (3H, s, –CH3), 1.88 (1H, m, –CH2–), 2.10
(1H, m, –CH2–), 2.44 (1H, m, –CH2–), 2.65 (1H, m,
–CH2–), 2.87 (1H, m, >CH–), 2.35–2.44 (10H, m, –N–
CH2–), 2.90–2.92 (2H, d, –N–CH2–), 4.16 (1H, m,
>CHOH), 4.92 (1H, s, @CH2), 4.96 (1H, s, @CH2), 5.50
(1H, m, @CH–). MS (EI) m/z: 265 (M+, 80), 72 (100).
3.1.4.3. 5-(1-Cyclohexylaminomethyl)vinyl-2-methyl-
cyclohex-2-enone (IV3). Compound IV3 was obtained
1
in 69.2% yield. H NMR 1.24 (3H, m, –CH2–), 1.65
(3H, m, –CH2–), 1.79 (3H, s, –CH3), 1.86 (2H, m,
–CH2–), 2.18–2.27 (3H, m, –CH2–), 2.39 (1H, m,
–CH2–), 2.65 (1H, m, –CH2–), 2.72 (1H, m, –CH2–),
2.92 (1H, m, >CH–), 3.07 (1H, m, >CH–), 3.55 (1H,
d, –N–CH2–), 3.64 (1H, d, –N–CH2–), 5.28 (1H, s,
@CH2), 5.52 (1H, s, @CH2), 6.75 (1H, m, @CH–). MS
(EI) m/z: 246 (M+, 32), 56 (100).
3.1.5.3. 5-{[1-(4-Benzylpiperazin-1-yl)methyl]vinyl}-2-
methylcyclohex-2-enol (V3). Compound V3 was obtained
1
in 12.8% yield, mp 179–180 ꢁC. H NMR (DMSO-d6),
3.1.4.4. 2-Methyl-5-{1-[(2-thiophen-2-ylethylamino)-
methyl]vinyl}cyclohex-2-enone (IV4). Compound IV4
was obtained in 66.3% yield. 1H NMR 1.77 (3H, s,
–CH3), 2.30 (2H, m, –CH2–), 2.64 (2H, m, –CH2–),
2.97 (1H, m, >CH–), 3.20 (2H, m, –N–CH2–), 3.52
(2H, m, –N–CH2–), 3.62 (2H, m, Ar-CH2–), 5.30 (1H,
s, @CH2), 5.50 (1H, s, @CH2), 6.71 (1H, m, @CH–),
6.94 (2H, s, Ar-H), 7.18 (1H, s, Ar-H). MS (EI) m/z:
275 (M+, 1), 178 (100).
1.56 (1H, m, –CH2–), 1.76 (3H, s, –CH3), 1.95 (1H, m,
–CH2–), 2.15 (2H, m, –CH2–), 2.44 (10H, br, –CH2–,
–N–CH2–, >CH2–), 2.90–2.94 (2H, d, –N–CH2–), 3.50
(2H, s, –CH2Ph), 4.15 (1H, m, >CHOH), 4.86 (1H, s,
@CH2), 4.92 (1H, s, @CH2), 5.49 (1H, m, @CH–),
7.30 (3H, m, Ar-H), 7.31 (2H, m, Ar-H). MS (EI) m/z:
326 (M+, 19), 91 (100).
3.1.5.4. 5-{1-[4-(4-Methoxyphenyl)piperazin-1-yl]-
methyl}vinyl-2-methylcyclohex-2-enol (V4). Compound
1
V4 was obtained in 15.8% yield. H NMR 1.59 (1H,
3.1.4.5. 5-(1-Dimethylaminomethyl)vinyl-2-methylcy-
clohex-2-enone (IV5). Compound IV5 was obtained in
m, –CH2–), 1.77 (3H, s, –CH3), 1.95 (2H, m, –CH2–),
2.20 (2H, m, –CH2–), 2.45 (1H, m, >CH2–), 2.55 (4H,
m, –N–CH2–), 2.95 (2H, m, –N–CH2–), 3.25 (4H, m,
–N–CH2–), 3.75 (3H, s, –OCH3), 4.19 (1H, m,
>CHOH), 4.95 (1H, s, @CH2), 5.02 (1H, s, @CH2),
5.50 (1H, m, @CH–), 6.82 (2H, d, Ar-H), 6.90 (2H, d,
Ar-H). MS (EI) m/z: 342 (M+, 38), 150 (100).
1
65.9% yield. H NMR 1.78 (3H, s, –CH3), 2.15 (6H, s,
–N(CH3)2), 2.27–2.43 (2H, m, –CH2–), 2.48–2.65 (2H,
m, –CH2–), 2.80–2.91 (3H, m, –N–CH2–, >CH–), 4.90
(1H, s, @CH2), 5.01 (1H, s, @CH2), 6.76 (1H, m,
@CH–). MS (EI) m/z: 192 (M+, 33), 58 (100).
3.1.4.6.
5-[1-(Adamantan-1-ylamino)methyl]vinyl-2-
methylcyclohex-2-enone (IV6). Compound IV6 was ob-
tained in 64.8% yield. 1H NMR 1.60–1.62 (3H, m,
–CH2–), 1.69 (2H, m, –CH2–), 1.78 (7H, m, –CH2–),
1.79 (3H, s, –CH3), 2.08 (3H, m, >CH–), 2.34 (1H, m,
–CH2–), 2.47 (1H, m, –CH2–), 2.52 (1H, m, –CH2–),
2.64 (1H, m, –CH2–), 2.88 (1H, m, >CH–), 3.25 (2H,
m, –N–CH2–), 4.91 (1H, s, @CH2), 5.10 (1H, s,
@CH2), 6.75 (1H, m, @CH–). MS (EI) m/z: 299 (M+,
25), 135 (100).
3.1.5.5. 5-{1-[4-(2-Methoxyphenyl)piperazin-1-yl]-
methyl }vinyl-2-methylcyclohex-2-enol (V5). Compound
1
V5 was obtained in 17.3% yield. H NMR 1.62 (1H,
m, –CH2–), 1.77 (3H, s, –CH3), 2.02 (2H, m, –CH2–),
2.19 (2H, m, –CH2–), 2.46 (1H, m, >CH2–), 2.59 (4H,
br, –N–CH2–), 2.99 (2H, m, –N–CH2–), 3.07 (4H, br,
–N–CH2–), 3.85 (3H, s, –OCH3), 4.18 (1H, m,
>CHOH), 4.93 (1H, s, @CH2), 4.99 (1H, s, @CH2),
5.51 (1H, m, @CH–), 6.84–6.99 (4H, m, Ar-H). MS
(EI) m/z: 342 (M+, 3), 150 (100).
3.1.5. General procedure for the preparation of com-
pounds V1–V5
3.1.6. General procedure for the preparation of com-
pounds VI1–VI5
3.1.5.1. 2-Methyl-5-{[1-(4-methylpiperazin-1-yl)meth-
yl]vinyl}cyclohex-2-enol (V1). Compound III1 (2.0 g,
8.0 mmo1) was dissolved in 15 mL methanol, then sodi-
um borohydride (20 mmol) was added in three portions
within 1 h. The mixture was stirred at room temperature
for 2 h, then dissolved in 30 mL brine and extracted with
methylene chloride (3· 30 mL). The combined organic
extracts were washed with brine, dried over anhydrous
sodium sulfate, and filtered. The filtrate was concentrat-
ed in vacuo. The residue was purified on a silica gel col-
3.1.6.1.
1-Methyl-4-[2-(4-methylcyclohex-3-enyl)al-
lyl]piperazine (VI1). A solution of compound III1
(2.0 g, 8.0 mmo1), 80% hydrazine hydrate (3.9 mL,
40 mmo1) in 15 mL ethylene glycol was heated at
120 ꢁC for 2 h. The mixture was cooled to 70 ꢁC and
treated with potassium hydroxide (3.7 g, 56 mmo1),
then heated at 180–185 ꢁC for 4–6 h. Then, the mixture
was cooled to the room temperature, diluted with brine
(100 mL), and extracted with methylene chloride

6546
J. Chen et al. / Bioorg. Med. Chem. 14 (2006) 6539–6547
(3· 30 mL). The combined organic extracts were washed
with brine, dried over anhydrous sodium sulfate, and fil-
tered. The filtrate was concentrated in vacuo. The resi-
due was purified on a silica gel column with petroleum
ether–acetate as eluent to afford the target product
VI1, (12.5% yield). 1H NMR 1.51 (2H, m, –CH2–),
1.65 (3H, s, –CH3), 1.79 (1H, m, –CH2–), 1.90 (1H, m,
–CH2–), 2.12 (1H, m, –CH2–), 2.15 (1H, m, –CH2–),
2.21 (1H, m, >CH–), 2.27 (3H, s, –N–CH3), 2.46
(8H, m, –N–CH2–), 2.91–2.93 (2H, d, –N–CH2–), 4.87
(1H, s, @CH2), 4.94 (1H, s, @CH2), 5.39 (1H, m,
@CH–). MS (EI) m/z: 234 (M+, 28), 113 (100).
spread for 24 h. The various concentrations of the com-
pounds to be tested were then added and cultured for 4
days at 37 ꢁC. Fifty microliters of 2 mg/mL 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) solution was added per well and the cells were
continued to be cultured for an additional 4 h. The
medium was removed by aspiration. The cells were dis-
solved in 200 ll DMSO, and absorbance at 570 nm was
measured in the 96-well plate reader. Growth inhibition
was determined as compared to untreated cells and cal-
culated as % of untreated cells.23
3.2.3. Western blot analysis. Protein extracts (50 lg) pre-
pared with RIPA lysis buffer (50 mM Tris-HCl, 150 mM
NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycho-
late, 1 mM PMSF, 100 lM leupeptin, and 2 lg/mL
aprotinin, pH 8.0) were separated on an 8 or 12%
SDS–polyacrylamide gel, and then transferred to nitro-
cellulose membranes. The membranes were stained with
0.2% Ponceau S red to assure equal protein loading and
transfer. After blocking with 5% non-fat milk, the mem-
branes were incubated with antibodies to phospho-
ERK1/2, ERK, HDJ-2, phospho-p38, p38, and
p21waf1 (Signal Transduction Lab). Immunocomplexes
were visualized by chemiluminescence (ECL kit,
Amersham).
3.1.6.2. 1-Ethyl-4-[2-(4-methylcyclohex-3-enyl)allyl]pi-
perazine (VI2). Compound VI2 was obtained in 10.8%
yield. 1H NMR 1.09 (3H, m, –CH3), 1.51 (2H, m,
–CH2–), 1.65 (3H, s, –CH3), 1.80 (1H, m, –CH2–),
1.93 (1H, m, –CH2–), 2.04 (1H, m, –CH2–), 2.13 (1H,
m, –CH2–), 2.21 (1H, m, >CH–), 2.43 (10H, m, –N–
CH2–), 2.91–2.94 (2H, d, –N–CH2–), 4.87 (1H, s,
@CH2), 4.94 (1H, s, @CH2), 5.40 (1H, m, @CH–). MS
(EI) m/z: 248 (M+, 100).
3.1.6.3. 1-Benzyl-4-[2-(4-methylcyclohex-3-enyl)al-
lyl]piperazine (VI3). Compound VI3 was obtained in
1
14.9% yield. H NMR 1.49 (2H, m, –CH2–), 1.65 (3H,
s, –CH3), 1.77–2.21 (5H, m, –CH2–, >CH–), 2.44 (8H,
br, –N–CH2–), 2.91 (2H, m, –N–CH2–), 3.51 (2H, s,
–CH2Ph), 4.85 (1H, s, @CH2), 4.93 (1H, s, @CH2),
5.39 (1H, m, @CH–), 7.30 (3H, m, Ar-H), 7.31 (2H,
m, Ar-H). MS (EI) m/z: 310 (M+, 40), 91 (100).
Acknowledgment
This work was partly supported by National Natural
Science Foundation of China (30328030).
3.1.6.4. 1-(4-Methoxyphenyl)-4-[2-(4-methylcyclohex-
3-enyl)allyl]piperazine (VI4). Compound VI4 was ob-
1
tained in 11.7% yield. H NMR (DMSO-d6), 1.58 (2H,
References and notes
m, –CH2–), 1.70 (3H, s, –CH3), 1.86–2.25 (5H, m,
–CH2–, >CH–), 2.59 (4H, br, –N–CH2–), 3.03 (2H, m,
–N–CH2–), 3.12 (4H, br, –N–CH2–), 3.80 (3H, s,
–OCH3), 4.95 (1H, s, @CH2), 5.03 (1H, s, @CH2), 5.45
(1H, m, @CH–), 6.85 (2H, d, Ar-H), 6.94 (2H, d, Ar-
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