Design and synthesis of cyclopropylamide analogues of combretastatin-A4 as novel microtubule-stabilizing agents

Article abstract of DOI:10.1021/jm301864s

A series of novel cyclopropylamide analogues of combretastatin-A4 (CA-4) were designed and synthesized. Most of them had significant in vitro antiproliferative activities, particularly for compounds 7i4, 7c4, 8a4, and 8c4. Moreover, compound 8c4 was also equally potent against paclitaxel resistant cancer cells. Interestingly, the novel cyclopropylamide analogues had different binding mechanisms from CA-4. Instead of inhibiting tubulin polymerization, these CA-4 derivatives were able to stimulate tubulin polymerization. Flow cytometry revealed that compound 8c4 arrested A549 cancer cells in the G2/M phase and resulted in cellular apoptosis. Further immunofluorescence assays revealed that compound 8c4 induced mitotic arrest in A549 cells through disrupting microtubule dynamics. In addition, compound 8c4 also effectively inhibited the tumor growth in the A549 xenograft model without causing significant loss of body weight. Compound 8c4 represents a novel class of microtubule-stabilizing agent and can be used as a promising lead for the development of new antitumor agents.

Full text of DOI:10.1021/jm301864s

Article
pubs.acs.org/jmc
Design and Synthesis of Cyclopropylamide Analogues of
Combretastatin-A4 as Novel Microtubule-Stabilizing Agents
Huan Chen,† Yongmei Li,‡ Chunquan Sheng,*,† Zhiliang Lv,† Guoqiang Dong,† Tiantian Wang,† Jia Liu,†
Mingfeng Zhang,† Lingzhen Li,† Tao Zhang,† Dongping Geng,† Chunjuan Niu,† and Ke Li*,†
†Department of Medicinal Chemistry, College of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China
‡Department of Oncology, Changhai Hospital, Second Military Medical University, Shanghai 200433, People’s Republic of China
S
* Supporting Information
ABSTRACT: A series of novel cyclopropylamide analogues of
combretastatin-A4 (CA-4) were designed and synthesized. Most of
them had significant in vitro antiproliferative activities, particularly for
compounds 7i4, 7c4, 8a4, and 8c4. Moreover, compound 8c4 was also
equally potent against paclitaxel resistant cancer cells. Interestingly, the
novel cyclopropylamide analogues had different binding mechanisms
from CA-4. Instead of inhibiting tubulin polymerization, these CA-4
derivatives were able to stimulate tubulin polymerization. Flow cytometry
revealed that compound 8c4 arrested A549 cancer cells in the G2/M
phase and resulted in cellular apoptosis. Further immunofluorescence
assays revealed that compound 8c4 induced mitotic arrest in A549 cells
through disrupting microtubule dynamics. In addition, compound 8c4
also effectively inhibited the tumor growth in the A549 xenograft model
without causing significant loss of body weight. Compound 8c4 represents a novel class of microtubule-stabilizing agent and can
be used as a promising lead for the development of new antitumor agents.
mitotic arrest that could lead to cell apoptosis.10−12
INTRODUCTION
■
Combretastatin-A4 (CA-4, Figure 1), isolated from the bark
Microtubules are cytoskeletal filaments consisting of α,β-
tubulin heterodimers and are involved in a wide range of
cellular processes such as cell shape organization, transportation
of vesicles, mitochondria, and other cellular organs, cell
of African willow tree Combretum caffrum in 1982,13 is a natural
cis-stilbene product that strongly inhibits tubulin polymer-
ization by binding to the colchicine binding site. The work of
Hsieh and Nam14,15 showed that 3,4,5-trimethoxy substitution
signaling, cell division, and mitosis, which are required for
on the A-ring and the cis-orientation between the two aryl rings
cell life cycle.1,2 Because of the multifunction of microtubules in
were essential for efficient binding to tubulin. Up to now, a
the cell cycle, tubulin has become an attractive target in
anticancer drug discovery. Natural-products-based drugs
great number of CA-4 analogues have been reported.16−21
The cyclopropyl group is generally considered as an alkene
targeting tubulin and the microtubule system are referred as
bioisostere.22,23 The cyclopropyl group can maintain the cis-
antimitotics, and they are also important components in
orientation of the pharmacophoric substructures in lead
combination chemotherapy for many pediatric and adult
compounds. On the other hand, many TBAs containing the
malignancies. There are many chemically diverse compounds
amide moieties are now first-line clinical drugs or currently in
targeting the tubulin−microtubule system. Tubulin binding
clinical trials for the treatment of solid tumors.24,25 Alterations
agents (TBAs) are potent mitotic poisons that are broadly
classified as microtubule-stabilizing drugs (such as taxanes and
epothilones) and microtubule-destabilizing drugs (such as
in microtubule dynamics that occur in the development of
resistance to paclitaxel and other microtubule-stabilizing drugs
have become an emerging issue in drug resistance,26−28 and
vincristine, vinblastine, and vinorelbine).3 There are three
some microtubule-destabilizing drugs also show drug resist-
ligand binding sites in tubulin α/β-heterodimer: paclitaxel
ance.29−31 In order to search for novel compounds that possess
excellent antitumor activities and less drug resistance, a series of
novel cyclopropylamide analogues of CA-4 were designed and
synthesized (Figure 1) by introducing the 3,4,5-trimethylphenyl
group as A-ring (which was the same as CA-4), a cyclopropyl
binding site,4 vinblastine binding site,4,5 and colchicine binding
site.5,6 The binding mechanisms of the clinically important
TBAs with α,β-tubulin heterodimer have been clarified after
determining the crystal structure of the TBA−tubulin
heterodimer.7,8 TBAs suppress spindle microtubule dynamics,
causing a delay or block at the metaphase−anaphase transition
during mitosis.9 Disruption of the mitotic spindle does not
satisfy the spindle assembly checkpoint, causing an extended
Received: June 1, 2012
Published: January 28, 2013
© 2013 American Chemical Society
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Figure 1. Structures of selected antimitotic compounds and the design of the target compound 8c4.
group as the linking group, and a substituted phenyl, benzyl, or
phenethyl group to connect the cyclopropyl moiety through an
amide functional group. As shown in Figure 2, the crystal
compared to CA-4. Several compounds also showed potent in
vitro and in vivo antitumor activities and represent promising
leads for the development of novel antitumor agents.
RESULTS AND DISCUSSION
■
Chemistry. The syntheses of the key intermediates trans-5
and cis-5 are outlined in Scheme 1. Diethyl 2-(3,4,5-
trimethoxybenzylidene)malonate (compound 2) was prepared
by reacting commercially available 3,4,5-trimethoxybenzalde-
hyde (compound 1) with diethyl malonate through Knoeve-
nagel condensation.32,33 Then compound 2 was reacted with
trimethylsulfoxonium iodide (TMSOI) to give intermediate
3.34,35 Compound 3 was hydrolyzed by KOH to generate the
racemes of compound 4. Optical compounds trans-5 and cis-5
were obtained by the chiral separation of compound 4 using the
preparative HPLC equipped with a chiral column. Acyl
chlorides were generated in situ from compound 5 and reacted
with various substituted amines to give the target compounds
6a−6n (Scheme 2). Compounds 6a, 6c, and 6i were selected to
prepare the chiral isomers 7a1−4, 7c1−4, and 7i1−4 from
preparative HPLC (Scheme 3). The ethyl ester groups of
Figure 2. Three-dimensional structures of 8c4 (left) and CA-4 (right).
structure of optical isomer 8c4 possesses a spatial configuration
similar to that of CA-4. These novel CA-4 derivatives were
expected to inhibit tubulin polymerization and bind to the
colchicine binding site. Interestingly, the results of the tubulin
polymerization assay show that they could effectively stimulate
tubulin polymerization, which was a totally different mechanism
a
Scheme 1
a
Reagents and conditions: (a) piperidine/HOAc, diethyl malonate, toluene, reflux, 12 h; (b) 1.1 equiv of (CH3)3SOI, 1.1 equiv of NaH, microwave,
10 min; (c) KOH/EtOH, room temperature, 30 min; (d) separation with HPLC equipped with chiral column.
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a
Scheme 2
a
Reagents and conditions: (a) (COCl)2/pyridine/CH2Cl2, amine/CH2Cl2.
a
Scheme 3
a
Reagents and conditions: (a) separation with HPLC equipped with chiral column; (b) LiAlH4, THF, 5 min.
compounds 7X (optical isomers 7a1−7i4) were reduced to the
hydroxymethyl group to afford compounds 8X (optical isomers
8a1−8i4) which maintained the same spatial configuration as
compounds 7X. The methods of determination of absolute
configuration for the compounds can be found in Supporting
Information, and the X-ray crystal structure of compound 8c4
(Figure 3) demonstrated that the corroboration methods were
reliable and correct.
In Vitro Antiproliferative Activity and Structure−
Activity Relationships (SARs). The antiproliferative activity
of the target compounds was evaluated in A549 (non-small-cell
lung cancer line), HeLa (human epithelial cervical cancer),
QGY (human hepatoma cancer), SW480 (human colon
cancer), and K562 (leukemia) cell lines using MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
5-Fluorouracil (5-Fu), CA-4, and paclitaxel were used as
reference drugs. The results are summarized in Table 1. The
antiproliferative activity of each compound was expressed as the
Figure 3. ORTEP drawing of compound 8c4.
concentration of compound that achieved 50% inhibition
(IC50) of cancer cell growth. As shown in Table 1, most of the
synthesized compounds possessed better antiproliferative
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Journal of Medicinal Chemistry
Article
a
Table 1. Antiproliferative Activity of Target Compounds against Five Human Cancer Cell Lines
b
IC50 (μM)
compd
R1
R2
A549
HeLa
>100
SW480
>100
QGY
>100
K562
>100
trans-6a
cis-6a
trans-6b
cis-6b
trans-6c
cis-6c
trans-6d
cis-6d
trans-6e
cis-6e
trans-6f
cis-6f
trans-6g
cis-6g
trans-6h
cis-6h
trans-6i
cis-6i
trans-6j
cis-6j
trans-6k
cis-6k
trans-6l
cis-6l
trans-6m
cis-6m
trans-6n
cis-6n
7a1
phenyl
phenyl
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CH2OH
48.6 6.7
31.5 2.1
30.9 2.0
24.5 2.7
18.5 2.9
18.0 1.2
45.2 2.6
25.6 3.4
80.3 4.6
76.4 10.3
>100
>100
49.0 3.5
31.8 3.1
34.1 1.6
66.7 4.3
27.2 1.0
28.7 3.1
37.4 2.6
49.5 6.8
>100
59.3 4.6
>100
>100
>100
2-NO2-phenyl
2-NO2-phenyl
2-Cl-phenyl
2-Cl-phenyl
3-Cl-phenyl
3-Cl-phenyl
3-NO2-phenyl
3-NO2-phenyl
4-OCH3-phenyl
4-OCH3-phenyl
4-F-phenyl
4-F-phenyl
benzyl
35.1 2.7
40.2 2.4
76.4 2.4
22.1 1.8
>100
54.1 2.9
48.8 3.1
36.1 1.4
>100
56.3 3.4
14.2 2.1
41.4 2.4
>100
35.1 2.1
>100
46.3 3.1
>100
52.1 1.2
>100
59.4 2.6
>100
>100
>100
>100
>100
>100
61.4 3.6
28.0 3.4
22.4 4.3
>100
81.6 6.4
36.8 6.0
62.4 3.1
76.3 5.6
46.5 2.4
25.6 2.6
33.2 3.5
27.8 4.6
32.3 4.6
>100
66.3 5.2
42.0 5.4
46.7 1.2
>100
86.1 7.3
36.1 5.7
79.1 10.3
>100
>100
>100
63.7 4.1
>100
benzyl
79.5 2.4
21.4 3.4
18.7 2.4
34.1 0.3
29.4 7.2
73.8 7.0
>100
89.6 5.2
>100
>100
>100
4-CH3-benzyl
4-CH3-benzyl
4-F-benzyl
37.9 3.7
>100
>100
29.0 6.4
28.4 7.6
34.1 5.2
>100
>100
>100
43.1 5.3
45.1 5.6
>100
4-F-benzyl
59.4 4.1
>100
4-OCH3-benzyl
4-OCH3-benzyl
2-F-phenethyl
2-F-phenethyl
3-F-phenethyl
3-F-phenethyl
4-F-phenethyl
4-F-phenethyl
phenyl
>100
>100
>100
>100
31.1 2.4
27.3 4.3
64.3 1.2
52.3 0.5
>100
>100
16.9 2.5
86.3 2.0
79.3 2.0
35.5 7.2
40.3 5.1
75.1 4.5
31.7 2.6
>100
46.8 3.1
34.1 1.6
>100
79.1 5.2
>100
58.2 5.1
58.2 4.2
28.1 0.8
35.0 4.2
58.1 3.3
>100
>100
39.9 4.6
>100
76.1 5.1
>100
49.3 4.2
21.3 1.3
65.9 2.4
>100
>100
>100
38.1 4.2
48.7 2.6
>100
>100
7a2
phenyl
>100
>100
7a3
phenyl
>100
>100
>100
7a4
phenyl
37.3 3.4
50.9 5.6
17.6 0.4
29.0 2.4
13.4 1.3
34.9 2.6
17.1 4.3
>100
>100
>100
>100
>100
7c1
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
4-CH3-benzyl
4-CH3-benzyl
4-CH3-benzyl
4-CH3-benzyl
phenyl
>100
>100
34.7 4.7
34.3 3.1
>100
19.5 3.5
>100
7c2
14.4 4.3
>100
30.1 2.4
46.7 5.3
25.6 2.4
>100
7c3
49.4 4.7
20.7 2.1
20.4 5.4
>100
7c4
42.4 3.1
>100
28.0 3.0
>100
7i1
7i2
19.7 5.1
>100
>100
26.4 4.3
>100
7i3
>100
>100
7i4
13.3 2.3
>100
28.5 4.1
>100
36.4 2.3
>100
32.1 4.6
>100
>100
8a1
>100
8a2
phenyl
CH2OH
43.2 1.6
>100
86.4 11.4
>100
76.8 2.5
>100
36.7 3.7
>100
>100
8a3
phenyl
CH2OH
>100
8a4
phenyl
CH2OH
24.1 2.1
36.7 2.4
11.4 4.3
>100
32.0 1.8
45.5 3.5
13.3 2.2
>100
33.3 1.5
40.2 2.1
10.7 1.2
>100
51.3 3.4
40.7 0.9
24.5 1.6
>100
>100
8c1
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
4-CH3-benzyl
4-CH3-benzyl
4-CH3-benzyl
4-CH3-benzyl
CH2OH
>100
8c2
CH2OH
18.4 2.3
>100
8c3
CH2OH
8c4
CH2OH
4.4 2.1
60.7 5.1
46.3 5.4
63.8 5.3
10.5 4.4
56.0 1.2
8.1 1.3
57.8 6.2
41.2 6.2
>100
13.2 2.4
94.0 1.5
76.3 4.8
>100
15.7 2.2
56.1 5.2
58.7 5.3
>100
16.9 2.4
>100
8i1
CH2OH
8i2
CH2OH
>100
8i3
CH2OH
>100
8i4
CH2OH
17.4 2.9
142.6 3.6
26.8 4.3
158.2 2.1
25.1 1.2
126.2 3.1
>100
5-Fu
114.5 2.1
c
c
c
c
c
CA-4
5.6 1.4
2.1 0.6
18.2 2.8
26.5 6.9
4.8 4.5
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Table 1. continued
b
c
IC50 (μM)
SW480
compd
R1
R2
A549
HeLa
QGY
K562
c
c
c
c
paclitaxel
8.2 3.4
6.3 4.6
12.1 4.3
11.3 5.3
15.7 5.7
a
Cell lines were treated with different concentrations of the compounds for 48 h as described in the Experimental Section. Cell viability was
b
c
measured by MTT assay. IC50 values are indicated as the mean SD (standard error) of three independent experiments. The unit of IC50 for CA-
4 and paclitaxel is nM.
Table 2. In Vitro Growth Inhibitory Effect of Compound 8c4 on Paclitaxel-Resistant Cell Lines A549 and HeLa
IC50 (μM)
A549 (resistant)
IC50 (μM)
HeLa (resistant)
a
a
compd
8c4
paclitaxel
A549
resistance index
HeLa
resistance index
4.4 2.1
8.2 3.4
5.3 1.4
1.2
8.1 1.3
6.3 4.6
10.7 2.5
1.3
b
b
b
b
1037.8 33.4
126.6
1375.0 49.6
218.3
a
b
Resistance index = IC50(resistant)/IC50(sensitive) . IC50 unit for paclitaxel is nM.
activities than the positive control 5-Fu. The importance of the
substitutions on the phenyl, benzyl, or phenethyl group for the
antiproliferative activity was also analyzed. Compounds cis-6a−
6n possessed the best antiproliferative activity against the five
human cancer lines. When the chlorine atom of compound cis-
6c was replaced by a nitro group (compound cis-6f) or moved
to position 3 (compound cis-6d), the analogues maintained
good antiproliferative activity. When the chlorine atom was
removed (compound cis-6a), the antiproliferative activity was
greatly decreased. For the benzyl derivatives, they were
generally less active than the phenyl analogues. The
introduction of a methoxyl group or a fluorine atom on the
benzyl group (e.g., compounds cis-6i and cis-6j) had positive
effects on the antiproliferative activity. However, the addition of
a 4-methoxyl group on both phenyl and benzyl groups led to a
loss of the activity. The overall antiproliferative activity of
compounds trans-6a−6n was lower than that of the cis-type
compounds, but the SAR was similar. Moreover, the
antiproliferative activity was also maintained for the phenethyl
derivatives (compounds cis-6l−6n).
resistant A549 cell line and 1.3 for resistant HeLa cell line) than
paclitaxel (126.6 for resistant A549 cell line and 218.3 for
resistant HeLa cell line).
Stimulation of Tubulin Polymerization in Vitro. The
CA-4 derivatives showed potent antiproliferative effects in vitro
(Table 1), and thus, their cytotoxic mechanisms were further
investigated. Ten compounds, including cis-6a, cis-6f, trans-6k,
7i4, and 8c4 (Table 3), were selected to evaluate their ability to
Table 3. In Vitro Effects of the Selected Compounds on
a
Tubulin Polymerization
compd
(10 μM)
tubilin polymerization
(% stimulation)
compd
(10 μM)
tubilin polymerization
(% stimulation)
control
cis-6a
0
trans-6k
7c4
50.0
28.6
79.3
31.5
24.3
245.3
23.2
50.0
25.0
40.8
7.9
cis-6d
cis-6f
7i4
8a4
cis-6n
trans-6g
8c4
paclitaxel
a
The final concentration of the compounds was 10 μM. The
For the optical isomers 7a1−8i4, the antiproliferative activity
of compounds 7X2 was higher than that of compounds 7X1,
and compounds 7X4 were more potent than 7X3. The same
trend was observed for the comparison of compounds 8X1/
8X2 and 8X3/8X4, which highlighted that the 2S configuration
was preferred for the antiproliferative activity. In addition, the
antiproliferative activity of compounds 8X4 was higher than
that of compounds 8X2, suggesting that the best spatial
configuration of these derivatives was (1S,2S)-8x4 and (1R,2S)-
7x4. Among the target compounds, compounds 7i4 and 8c4
possessed the best antiproliferative activity and were selected
for further study.
Compound 8c4 Is Effective against Paclitaxel Resist-
ant A549 and HeLa Cells. Paclitaxel is used as first-line
chemotherapy for non-small-cell lung cancer and human
epithelial cervical cancer but acquired resistance becomes a
critical problem. We compared the activity of compound 8c4
for paclitaxel resistant and nonresistant cancer cells (A549 and
HeLa). As shown in Table 2, the IC50 value of paclitaxel for
A549 and HeLa resistant cell lines was increased to 1037.8 and
1375.0 nM, respectively. Interestingly, the activity of compound
8c4 was not significantly changed for these two paclitaxel
resistant cancer cell lines compared with the nonresistant ones.
The IC50 value of compound 8c4 against paclitaxel resistant
A549 and HeLa cell-lines was 5.3 and 10.7 μM, respectively.
Compound 8c4 had a much lower resistance index (1.2 for
compounds were preincubated with tubulin at a final concentration
of 3.0 mg/mL.
stimulate tubulin polymerization activity. All the compounds
were employed at 10 μM in the assays. Paclitaxel was used as
the positive control (10 μM). Among them, compound 7i4 was
found to stimulate tubulin polymerization the most (79.3%),
while the percent of stimulation for the positive control
paclitaxel was 245.3%. It is well-known that the mechanism of
tubulin-binding agents can be divided into two types:
stimulating agents (e.g., paclitaxel) and inhibiting agents (e.g.,
CA-4). In order to confirm which type the cyclopropylamide
derivatives belonged to, the microtubule dynamics was
investigated for compounds 7i4 and 8c4. Unexpectedly, the
results demonstrated that the two tested compounds could
concentration-dependently stimulate tubulin polymerization
rather than inhibit tubulin polymerization (Figure 4). The
activity of compound 7i4 was higher than that of compound
8c4, which correlated well with the tubulin polymerization
assay. Next, we determined the EC50 value of compounds 7i4,
7c4, 8a4, and 8c4 for their ability to stimulate tubulin
polymerization assembly. Compound 7i4 revealed the max-
imum stimulation of tubulin assembly with the lowest EC50
value (5.83 μM) (Table 4), while the EC50 value of the positive
control paclitaxel was 1.23 μM. Moreover, molecular docking
studies also showed that compound 8c4 was more preferable to
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microtubules dynamic processes. The chromosomes and
microtubules were stained with diamidinophenylindole
(DAPI) and anti-α-tubulin conjugated with fluorescein
isothiocyanate (FITC), respectively, and images were acquired
using laser confocal microscope. A549 cells were allowed to
adhere to acid-washed glass coverslips and then treated with
compound 8c4 or DMSO for 22 h. Next, the cells were washed,
fixed in formaldehyde, and subjected to a direct immuno-
fluorescence staining. Sufficient images of compound 8c4 and
DMSO treated cells were obtained for manually scoring the
mitotic index based on DNA staining. More than 2500 cells
treated with compound 8c4 and DMSO were analyzed. A
paired t test was applied to analyze whether treatment of
unsynchronized A549 cells with compound 8c4 would cause a
significant increase in the number of mitotic cells compared
with the cells treated with DMSO. Statistical data indicated that
the mitotic index was significantly increased in the cells treated
with compound 8c4 compared with the cells treated with
DMSO. The results showed that the percentage of cell mitotic
arrest was 1.1% at 0 μM 8c4 (SD = 1.3%, N = 3), and the
percentage was increased to 37.4% (SD = 2.5%, N = 3), 53.1%
(SD = 3.2%, N = 3), and 78.8% (SD = 4.1%, N = 3) at 1, 5, and
10 μM, respectively. All these data along with the results of cell
cycle analysis described in Table 5 revealed that compound 8c4
can convincingly induce G2/M phase arrest in A549 cells.
Compound 8c4 Induces Mitotic Arrest in A549 Cells
through Disrupting Microtubule Dynamics. As the
tubulin−microtubule system has a major role in maintaining
the cellular morphology,1,2 it is interesting to know whether
compound 8c4 could affect microtubule dynamics in living
cells. As shown in Figure 5, confocal images showed that the
cells treated with compound 8c4 were largely arrested at the
G2/M phase compared with the DMSO control. DMSO-
treated cells were observed to be predominantly in the
interphase of the cell division cycle which was characterized
by uncondensed chromosomes (Figure 5G,H). In contrast,
cells treated with compound 8c4 (Figure 5A−F) were enriched
in the G2/M phase. In the interphase, the microtubules from
cells treated with compound 8c4 were disorderly dispersed in
the cytoplasm, whereas the microtubules from cells treated with
DMSO showed regular assembly. When the concentration of
compound 8c4 was increased, the percentage of metaphase
arrest cells was increased accordingly, indicating that compound
8c4 had significant influence on cell mitosis (Figure 5A,B).
More interestingly, at a higher concentration of 5 μM (Figure
5C,D), a significant reduction of microtubule density occurred
for compound 8c4. The reduction in microtubule density at the
periphery of the cells was apparent with disorganized central
networks. Compound 8c4 strongly disrupted interphase
microtubule in A549 cells at 10 μM (Figure 5E,F), while the
irregularity of cytoplasm and chromosomes was more obvious
than cells treated with the other two concentrations. These
results indicated that compound 8c4 might also perturb
microtubule dynamics.
Figure 4. Effects of compounds 7i4 and 8c4 on microtubule dynamics.
Polymerization of tubulin at 37 °C in the presence of paclitaxel (15
μM), CA-4 (7.5 μM), 7i4 (7.5 μM, 15 and 30 μM), and 8c4 (7.5, 15,
and 30 μM) were monitored continuously by recording the
absorbance at 340 nm over 60 min. The reaction was initiated by
the addition of tubulin to a final concentration of 3.0 mg/mL. All the
concentrations of compounds 7i4 and 8c4 as well as paclitaxel could
stimulate tubulin polymerization.
Table 4. Stimulating Activity of Tubulin Polymerization for
Selected Compounds and Paclitaxel
compd
EC50 (μM)
compd
8c4
paclitaxel
EC50 (μM)
7i4
7c4
8a4
5.83
16.91
14.30
19.68
1.23
bind with the paclitaxel binding site and formed hydrophobic
and hydrogen-bonding interactions with tubulin (see Support-
ing Information for details).
Compound 8c4 Induces Cell Cycle Arrest on G2/M
Phase. To determine whether the cytotoxicity induced by the
cyclopropylamide CA-4 derivatives was due to cell cycle arrest,
flow cytometry analysis was performed. A549 cells were treated
with compound 8c4 at 0, 0.1, 1, 5, or 10 μM for 24 h,
respectively. The cells were harvested, and the cell cycle phases
were analyzed by flow cytometry. The results revealed that
compound 8c4 significantly arrested cell cycle at the G2/M
phase in a dose-dependent manner (Table 5). At a
Table 5. Distribution of A549 Cells at G1 and G2/M Phases
after the Treatment of Compound 8c4
% of cells in G1
phase
% of cells in S
phase
% of cells in G2/M
phase
concentration
control
87.56
78.7
9.12
9.54
2.78
12.53
51.68
60.85
79.92
0.1
1
38.41
28.62
9.85
9.96
5
10.81
10.29
10
concentration of 1 μM, 51.68% of G2/M phase was arrested.
When the concentration was increased to 5 and 10 μM, 60.85%
and 79.92% G2/M phase arrest was observed, respectively.
These data confirmed the growth inhibitory effect of compound
8c4 on lung cancer cells.
Compound 8c4 Induces Mitotic Arrest in A549 Cell.
To assess the effect of G2/M phase arrest for compound 8c4,
cultured A549 cells were treated with compound 8c4 using
dimethyl sulfoxide (DMSO) as control. Immunofluorescence
assays were employed to study the chromosomes and
In vitro tubulin assembly experiments showed that
compound 8c4 and paclitaxel could promote tubulin polymer-
ization despite the significant difference of the structures
between them. The above experiments showed that compound
8c4 can strongly induce mitotic arrest in A549 cells through
disruption of microtubule dynamics. Next, we want to know
whether the structures of spindle fibers and nucleus are similar
or not when the A549 cells were treated with compound 8c4 or
paclitaxel. The confocal images shown in Figure 6 revealed that
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Figure 5. Effects of compound 8c4 on microtubule dynamics in vitro. A549 cells were treated with 1 μM 8c4 (A, B), 5 μM 8c4 (C, D), 10 μM 8c4
(E,F), and DMSO (G, H) for 22 h, respectively. Nucleuses were stained with DAPI (blue), and microtubules were stained with mouse anti-α-tubulin
conjugated with FITC (green). Images were taken using LSM 570 laser confocal microscope (Carl Zeiss, Germany).
Figure 6. Effects of compound 8c4 and paclitaxel on spindle microtubules assembly and chromosomes condensation. A549 cells were treated with
compound 8c4 at 10 μM and paclitaxel at 10 nM for 22 h, respectively. Following the termination of the experiment, cells were fixed and stained for
tubulin. DAPI was used as counterstain.
the two compounds can alter spindle microtubule dynamics.
Although similar multipolar spindles phenotypes were
generated in the cells treated with compound 8c4 and
paclitaxel, the configurations of the astral structures and
concentrated chromosomes of the two regiment treated cells
were significantly different. Chromosomes were located in the
cells and microtubule spindles were concentrated as multipolar
when A549 cells were treated with compound 8c4. In contrast,
the chromosomes around the cells and microtubule spindles
were concentrated at the center of the cells when treated with
paclitaxel.
Compound 8c4 Induces Cell Apoptosis on A549 Cells.
The results of cell cycle and mitotic arrest showed that
compound 8c4 can arrest the A549 cell in the G2/M phase and
can cause spindle microtubule disorder. As described above,
disruption of the mitotic spindle does not satisfy the spindle
assembly checkpoint and cause an extended mitotic arrest that
could lead to cell apoptosis.10−12 Cell apoptosis was assayed by
flow cytometry to determine whether the target derivatives
could arrest cell cycle progression at mitosis and lead to cell
death. A549 cells were treated with compound 8c4 at 1, 5, or
10 μM, respectivel, and DMSO served as a control. The results
showed that the percentage of cell apoptosis was 0.73% at 0 μM
for compound 8c4, and cell apoptosis was increased to 15.57%,
37.40%, and 55.23% at 1, 5, and 10 μM, respectively (Figure 7).
Like other tubulin-binding agents, compound 8c4 could induce
mitotic arrest at the G2/M phase and cause cell apoptosis.
Compound 8c4 Effectively Inhibits Tumor Growth in
Vivo. The in vivo antitumor efficacy of compound 8c4 was
further investigated in a BALB/c male nude mouse model
established by subcutaneous inoculation of the A549 solid
tumor (3 mm3) in the right armpit of the mice. Compound 8c4
was intragastrically administered daily at a dose of 100 and 500
mg/kg, respectively. Paclitaxel at a dose of 30 mg/kg and the
vehicle were administered when the tumor reached a volume of
100−200 mm3 on day 13 after model establishment. As shown
in Figure 8A, treatment with compound 8c4 or paclitaxel
resulted in a significant reduction in the tumor volume
compared with that observed in the vehicle group (∗∗, P <
0.05; ∗, P < 0.01). Compound 8c4 inhibited tumor growth in a
dose-dependent manner, and the tumor inhibitory rates on day
16 after treatment were 44.6% and 55.9% at 100 and 500 mg/
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totally different from CA-4. Compound 8c4 induced cell cycle
arrest at the G2/M phase and led to cell apoptosis. Further
immunofluorescence assays revealed that compound 8c4
induced mitotic arrest in A549 cells through disruption of
microtubule dynamics. More importantly, compound 8c4
showed similar activity against A549 and HeLa cell lines that
were resistant to paclitaxel. The resistance index of compound
8c4 was much lower than paclitaxel. In particular, compound
8c4 was proved to have a potent inhibitory effect on A549
tumor growth in vivo without causing significant body weight
loss. In summary, this is the first report that CA-4 analogues
containing a 3,4,5-trimethoxybenzyl moiety could stimulate
tubulin polymerization as microtubule-stabilizing agents. These
findings make this scaffold an attractive target for further study
on its binding mode to tubulin.
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All the chemicals and reagents
were commercially available without pretreatment. Silica gel thin-layer
chromatography was performed on precoated plates GF-254 (Qingdao
Haiyang Chemical, China). All solvents and reagents were analytically
pure, and no further purification was needed. IR spectra were recorded
1
on a PE Spectrum One FI-IR spectrometer as KBr pellets. H NMR
and 13C NMR spectra were recorded on a Bruker Avance 300
spectrometer (Bruker Company, Germany), using TMS as an internal
standard and DMSO-d6 as solvents. Chemical shifts were given in ppm
(parts per million). For data reporting, the following was used: s =
singlet, d = doublet, t = triplet, m = multiplet. High-resolution mass
spectra were recorded on a QSTAR XL hybrid MS/MS mass
spectrometer (Agilent Technolohies). Crystal structures were
determined on a Bruker SMART CCD (Bruker Company, Germany).
The optical rotation was measured on a polarmeter, and the circular
dichroism spectra were recorded on a B027760750 instrument. The
purity of each compound (>95%) was determined on an Agilent 1100
series LC system (column, ZORBAX Eclipse XDB C8, 4.6 mm ×150
mm, 5 μm; mobile phase, methanol (70%)/H2O (30%); low rate, 1.0
mL/min; UV wavelength, maximal absorbance at 254 nm; temper-
ature, ambient; injection volume, 20 μL).
Figure 7. Effects of compound 8c4 on cells apoptosis. A549 cells were
harvested after the treatment with compound 8c4 at 0, 1, 5, and 10
μM for 24 h, respectively. The percentage of cells in each part of cell
apoptosis was quantitated by flow cytometry: (A−D) (upper left
quadrant) detection error cells; (upper right quadrant) thanatosis and
proapoptosis cells; (bottom left quadrant) live cells; (bottom right
quadrant) apoptosis cells.
kg, respectively. The inhibitory effect of paclitaxel was 81.3% at
the dose of 30 mg/kg on A549 solid tumor cells. Compared
with the control vehicle-treated mice, compound 8c4 at
different doses did not cause obvious body weight loss, whereas
significant weight loss was observed in the paclitaxel group
(Figure 8B).
Synthesis of Diethyl 2-(3,4,5-Trimethoxybenzylidene)-
malonate (2). A 1000 mL three-neck round-bottom flask was
charged with 3,4,5-trimethoxylbenzaldehyde compound 1 (98.1g, 0.5
mol) and toluene (500 mL). The flask was fixed in an oil bath and
equipped with a condenser pipe. Diethyl malonate (80 mL, 0.505 mol)
was added. Then piperidine (9 mL, 15 mmol) and HOAc (15 mL, 25
mmol) were added dropwise into the flask. The reaction mixture was
heated to reflux for 12 h. Then toluene was evaporated, and the
mixture was extracted three times with CH2Cl2. The combined organic
CONCLUSION
■
In this investigation, a series of novel cyclopropylamide CA-4
analogues were designed and synthesized. Compounds 7i4,
7c4, 8a4, and 8c4 showed excellent antiproliferative activities
against five human cancer cell lines. The results of tubulin
polymerization assay showed that the novel cyclopropylamide
analogues could stimulate tubulin polymerization, which was
Figure 8. Effects of compound 8c4 include inhibition of tumor growth in vivo and changes in body weight. Dots represent the mean of six mice, and
bars represent the SD: (∗∗) P < 0.01; (∗) P < 0.05. (A) Compound 8c4 and paclitaxel inhibited tumor growth in the A549 xenograft model. (B)
Body weight changes after xenograft model treated with compound 8c4, paclitaxel, and the vehicle.
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layers were washed with saturated Na2CO3 aqueous solution, brine
and dried over MgSO4. The solvent was removed under reduced
pressure to obtain a crude solid, which was recrystallized from ethyl
acetate/hexane to give compound 2 (155.64 g, yield 92.0%). White
crystal, mp 81.6−82.1 °C. 1H NMR (300 MHz, DMSO-d6): δ 7.86 (s,
1H), 6.46 (s, 2H), 3.98−3.74 (m, 4H), 3.73 (s, 6H), 3.60 (s, 3H),
0.83−0.74 (m, 6H).
60.71, 59.95, 55.87, 37.90, 32.95, 18.59, 13.44. HRMS (EI+) calcd
C22H25NO6 (M+) 399.1682, found 399.1692.
Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5-trimethoxyphenyl)-
cyclopropanecarboxylate (cis-6a). White solid, mp 158.3−159.0
°C. 1H NMR (DMSO-d6, 300 MHz): δ 9.93 (s, 1H), 7.33−7.30 (d, J =
8.4 Hz, 2H), 7.21−7.16 (t, J = 7.8 Hz, 2H), 6.98−6.93 (t, J = 5.4 Hz,
1H), 6.56 (s, 2H), 4.19−4.16 (m, 2H), 3.64 (s, 6H), 3.50 (s, 3H),
3.07−3.01 (t, J = 7.8 Hz, 1H), 2.09−2.05 (dd, J1 = 4.5 Hz, J2 = 5.4 Hz,
1H), 1.61−1.56 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.22−1.17 (t, J =
8.7 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ 170.50, 163.25,
152.38, 139.09, 136.51, 131.19, 128.19, 128.19, 123.39, 120.58, 119.27,
105.95, 61.29, 55.83, 32.25, 19.26, 14.10. HRMS (EI+) calcd
C22H25NO6 (M+) 399.1682, found 399.1687.
Synthesis of 2-(3,4,5-Trimethoxyphenyl)cyclopropane-1,1-
dicarboxylic Acid Diethyl Ester (3). A 500 mL round-bottom flask
was charged with NaH (1.584 g, 0.066 mol) and anhydrous DMSO
(100 mL). Then TMSOI in anhydrous DMSO (50 mL) was added
dropwise for 5 min. Compound 2 (20.30 g, 0.06 mol) was dissolved in
anhydrous DMSO (50 mL) and added dropwise over 10 min. Then
the flask was placed in a microwave reaction equipment at 60 °C for
10 min, and the power was 400 W. When the reaction was completed,
the whole mixture was added dropwise into water (500 mL) and
extracted three times with CH2Cl2. The combined organic layers were
dried with MgSO4. Removal of solvent under reduced pressure
afforded a crude solid, which was recrystallized from ethyl acctate/
hexane to give compound 3 (19.66 g, yield 93.0%). White solid, mp
78.6−79.3 °C. 1H NMR (500 MHz, DMSO-d6): δ 6.47 (s, 1H), 4.19−
4.13 (m, 2H), 3.87−3.82 (m, 2H), 3.73 (s, 6H), 3.60 (s, 3H), 3.09−
3.05 (t, J = 8.5 Hz, 1H), 2.13−2.10 (dd, J1 = 5.0 Hz, J2 = 5.0 Hz, 1H),
1.64−1.61 (dd, J1 = 5.0 Hz, J2 = 5.5 Hz, 1H), 1.22−1.21 (t, J = 3.5 Hz,
3H), 0.84−0.81 (t, J = 6.0 Hz, 3H).
S y n t h e s i s o f 1 - ( E t h o x y c a r b o n y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylic Acid (4). A 500 mL
round-bottom flask was charged with compound 3 (17.63 g, 0.05 mol)
and ethanol (200 mL). KOH (2.94 g, 0.0505 mol) was dissolved in
water (50 mL) and added dropwise, and the reaction temperature was
increased to 30 °C for 30 min. 3% HCl was added dropwise into the
reaction mixture, and then the whole mixture was added dropwise into
water (500 mL). The solvent was extracted three times with CH2Cl2.
The combined organic layers were dried with MgSO4. The solvent was
removed under reduced pressure to give a crude solid, which was then
recrystallized with ethyl acctate/hexane to give compound 4 (14.59 g,
yield 90%). White solid, mp 98.3−99.5 °C. 1H NMR (300 MHz,
DMSO-d6): δ 13.04 (s, 1H), 6.44 (s, 2H), 3.88−3.83 (m, 2H), 3.72 (s,
6H), 3.58 (s, 3H), 3.02−2.96 (t, J = 8.1 Hz, 1H), 2.08−2.04 (dd, J1 =
5.7 Hz, J2 = 5.1 Hz, 1H), 1.60−1.56 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H),
0.85−0.84 (t, J = 8.1 Hz, 3H).
E t h y l 1 - ( 2 - N i t r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6b). Yellow
1
solid, mp 132.2−132.4 °C. H NMR (DMSO-d6, 300 MHz): δ 11.46
(s, 1H), 8.25−8.22 (t, J = 7.8 Hz, 1H), 8.12−8.08 (t, J = 5.4 Hz, 1H),
7.77−7.72 (m, 1H), 7.37−7.32 (m, 1H), 6.58 (s, 2H), 3.91−3.77 (m,
2H), 3.74 (s, 6H), 3.60 (s, 3H), 3.18−3.12 (t, J = 7.8 Hz, 1H), 2.35−
2.31 (dd, J1 = 4.8 Hz, J2 = 4.8 Hz, 1H), 1.94−1.90 (dd, J1 = 5.4 Hz, J2 =
4.5 Hz, 1H), 0.79−0.74 (t, J = 8.1 Hz, 3H). 13C NMR (DMSO-d6, 300
MHz): δ 169.95, 163.70, 152.33, 140.97, 136.68, 133.84, 131.60,
130.23, 124.82, 124.13, 106.19, 61.50, 59.90, 55.72, 33.55, 18.63,
13.85. HRMS (EI+) calcd C22H24N2O8 (M+) 444.1533, found
444.1543.
E t h y l 1 - ( 2 - N i t r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6b). Yellow
1
solid, mp 95.9−96.3 °C. H NMR (DMSO-d6, 300 MHz): δ 10.48
(s, 1H), 7.91−7.87 (m, 1H), 7.57−7.51 (t, J = 7.8 Hz, 1H), 7.28−7.22
(t, J = 8.1 Hz, 2H), 6.56 (s, 2H), 4.25−4.18(m, 2H), 3.63 (s, 6H), 3.53
(s, 3H), 3.13−3.08 (t, J = 7.8 Hz, 1H), 2.20−2.16 (dd, J1 = 5.1 Hz, J2 =
5.1 Hz, 1H), 1.71−1.66 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.28−1.23
(t, J = 8.7 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ 169.95,
163.70, 152.33, 140.97, 136.68, 133.84, 131.60, 130.23, 124.82, 124.13,
106.19, 61.50, 59.90, 55.72, 33.55, 18.63, 13.85. HRMS (EI+) calcd
C22H24N2O8 (M+) 444.1533, found. 444.1538.
E t h y l 1 - ( 2 - C h l o r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6c). White
crystal, mp 110.2−110.4 °C. 1H NMR (DMSO-d6, 300 MHz): δ 10.82
(s, 1H), 8.24−8.21 (d, J = 8.7 Hz, 1H), 7.54−7.51 (m, 1H), 7.37−7.32
(t, J = 7.8 Hz, 1H), 7.18−7.13 (m, 1H), 6.60 (s, 2H), 3.83−3.78 (m,
2H), 3.74 (s, 6H), 3.60 (s, 3H), 3.20−3.14 (t, J = 8.7 Hz, 1H), 2.38−
2.34 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 2.04−1.99 (dd, J1 = 4.8 Hz, J2 =
4.8 Hz, 1H), 0.76−0.74 (t, J = 7.8 Hz, 3H). HRMS (EI+) calcd
C22H24ClNO8 (M+) 433.1292, found 433.1298.
General Procedures for the Synthesis of trans-(6a−6n) and
cis-(6a−6n). A solution of compound 4 (6.70g, 25 mmol) and oxalyl
chloride (2.40 mL, 25.25 mmol) in dry CH2Cl2 was stirred at room
temperature. Then the solvent was removed under reduced pressure.
The residue was washed with dry CH2Cl2, and the solvent was
removed under reduced pressure. The above procedure was repeated
three times to fully remove the residual oxalyl chloride. The crude acyl
chloride could be used in the next step without further purification.
The obtained acyl chloride was added dropwise to a solution of dry
pyridine (3 equiv, 5.25 mL, 5 mmol) and various amines (1.2 equiv, 30
mmol) in dry CH2Cl2 (4.0 equiv, 100 mL). After 30 min, the reaction
mixture was stirred at room temperature overnight. Then water (10
mL) was added to the solution, and the pH was adjusted to 7.0 with
3% HCl. The mixture was extracted with CH2Cl2 and water (v:v = 1:1,
100 mL) three times. The organic layer was separated, washed with
saturated NaHCO3 aqueous solution, and dried over anhydrous
MgSO4. The solvent was removed under reduced pressure and the
residue was purified by flash column chromatography (gradient
elution, EtOAc/hexane = 0−3%, 3−6%, 6−10%, v/v) to give
compounds trans-(6a−6n) and cis-(6a−6n).
E t h y l 1 - ( 2 - C h l o r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6c). White
1
solid, mp 168.5−169.4 °C. H NMR (DMSO-d6, 300 MHz): δ 9.76
(s, 1H), 7.41−7.37 (t, J = 5.1 Hz, 1H), 7.16−7.13 (t, J = 7.8 Hz, 1H),
7.10−7.06 (m, 2H), 6.60 (s, 2H), 4.24−4.17 (dd, J1 = 6.9 Hz, J2 = 7.2
Hz, 2H), 3.65 (s, 6H), 3.57 (s, 3H), 3.11−3.06 (t, J = 8.7 Hz, 1H),
2.19−2.15 (dd, J1 = 4.5 Hz, J2 = 5.4 Hz, 1H), 1.67−1.63 (dd, J1 = 4.8
Hz, J2 = 4.8 Hz, 1H), 1.28−1.23 (t, J = 6.6 Hz, 3H). HRMS (EI+)
calcd C22H24ClNO8 (M+) 433.1292, found 433.1291.
E t h y l 1 - ( 3 - C h l o r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6d). White
crystal, mp 110.9−111.1 °C. IR (KBr, cm−1): 3305, 3264, 3219, 3116,
3066, 2998, 2966, 2934, 2874, 2843, 2824, 1701, 1661, 1585, 1542,
1507, 1458, 1425, 1371, 1333, 1308, 1273, 1197, 1146, 1127, 1044,
1
1011, 912, 884, 842, 792, 741, 716, 688, 656, 591, 558, 507, 421. H
NMR (DMSO-d6, 300 MHz): δ 10.33 (s, 1H), 7.85 (s, 1H), 7.53−
7.51 (d, J = 8.1 Hz, 1H), 7.36−7.31 (t, J = 7.8 Hz, 1H), 7.14−7.11 (d,
J = 7.8 Hz, 1H), 6.53 (s, 2H), 3.87−3.78 (m, 2H), 3.74 (s, 6H), 3.60
(s, 3H), 3.25−3.19 (t, J = 7.8 Hz, 1H), 2.22−2.17 (dd, J1 = 5.1 Hz, J2 =
5.1 Hz, 1H), 1.74−1.69 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 0.80−0.76
(t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C22H24ClNO6 (M+) 433.1292,
found 433.1296.
E t h y l 1 - ( 3 - C h l o r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6d). White
solid, mp 144.1−144.5 °C. 1H NMR (DMSO-d6, 300 MHz): δ
Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5-trimethoxyphenyl)-
cyclopropanecarboxylate (trans-6a). White crystal, mp 120.0−
1
120.2 °C. H NMR (DMSO-d6, 300 MHz): δ 10.18 (s, 1H), 7.65−
7.62 (d, J = 7.8 Hz, 2H), 7.34−7.29 (t, J = 7.8 Hz, 2H), 7.09−7.04 (d,
J = 7.2 Hz, 1H), 6.54 (s, 2H), 3.85−3.82 (m, 2H), 3.74 (s, 6H), 3.60
(s, 3H), 3.24−3.19 (t, J = 7.8 Hz, 1H), 2.22−2.17 (dd, J1 = 4.8 Hz, J2 =
5.1 Hz, 1H), 1.75−1.70 (dd, J1 = 5.1 Hz, J2 = 4.8 Hz, 1H), 0.81−0.76
(t, J = 7.8 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ 168.13,
165.88, 152.45, 138.82, 136.76, 130.89, 128.66, 123.56, 119.72, 106.54,
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10.12 (s, 1H), 7.51−7.50 (t, J = 7.8 Hz, 1H), 7.26−7.18 (m, 2H),
7.04−7.00 (m, 1H), 6.55 (s, 2H), 4.22−4.14 (m, 2H), 3.65 (s, 6H),
3.50 (s, 3H), 3.09−3.03 (t, J = 7.2 Hz, 1H), 2.12−2.08 (dd, J1 = 5.1
Hz, J2 = 5.1 Hz, 1H), 1.63−1.58 (dd, J1 = 5.1 Hz, J2 = 4.8 Hz, 1H),
1.22−1.17 (t, J = 7.8 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ
170.08, 163.49, 152.27, 140.37, 136.49, 132.82, 130.78, 122.86, 118.43,
117.32, 105.84, 61.18, 59.81, 55.69, 32.16, 19.01, 13.94. HRMS (EI+)
calcd C22H24ClNO6 (M+) 433.1292, found 433.1298.
8.4 Hz, 1H), 2.10−2.06 (dd, J1 = 5.4 Hz, J2 = 4.8 Hz, 1H), 1.61−1.57
(dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.22−1.18 (t, J = 6.9 Hz, 3H). 13
C
NMR (DMSO-d6, 300 MHz): δ 170.32, 163.03, 152.45, 152.30,
136.47, 135.45, 131.02, 121.58, 120.87, 120.76, 115.25, 114.96, 106.57,
105.86, 61.16, 59.88, 56.03, 55.92, 55.75, 32.11, 19.11, 18.55, 14.01,
13.48. HRMS (EI+) calcd C22H24FNO6 (M+) 417.1588, found
417.1596.
Ethyl 1-(Benzylcarbamoyl)-2-(3,4,5-trimethoxyphenyl)-
cyclopropanecarboxylate (trans-6h). White solid, mp 98.1−98.5
°C. IR (KBr, cm−1): 3366, 3066, 2987, 2933, 2826, 1714, 1643, 1585,
1519, 1503, 1457, 1428, 1374, 1333, 1313, 1276, 1239, 1156, 1123,
E t h y l 1 - ( 3 - N i t r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6e). Yellow
solid, mp 144.9−145.3 °C. IR (KBr, cm−1): 3308, 3274, 3230, 3137,
3087, 3004, 2965, 2937, 2834, 1704, 1668, 1591, 1543, 1520, 1457,
1427, 1379, 1341, 1312, 1276, 1238, 1180, 1133, 1098, 1071, 1015,
1
1021, 1001, 924, 865, 826, 773, 740, 701, 577, 523, 494, 448. H
NMR(DMSO-d6, 300 MHz): δ 8.71−6.72 (t, J = 6.0 Hz, 1H), 7.36−
7.23 (m, 5H), 6.49 (s, 2H), 4.38−4.32 (m, 2H), 3.84−3.75 (m, 2H),
3.73 (s, 6H), 3.59 (s, 3H), 3.10−3.04 (t, J = 8.7 Hz, 1H), 2.11−2.07
(dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.63−1.58 (dd, J1 = 4.8 Hz, J2 = 4.8
Hz, 1H), 0.78−0.76 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6, 300
MHz): δ 168.17, 167.29, 152.42, 139.37, 136.68, 131.00, 128.24,
127.07, 126.72, 106.72, 60.60, 59.94, 55.85, 42.75, 37.09, 32.58, 18.01,
13.37. HRMS (EI+) calcd C23H27NO6 (M+) 413.1838, found
413.1845.
Ethyl 1-(Benzylcarbamoyl)-2-(3,4,5-trimethoxyphenyl)-
cyclopropanecarboxylate (cis-6h). White solid, mp 144.8−145.1
°C. 1H NMR(DMSO-d6, 300 MHz): δ 8.45−8.41 (t, J = 6.0 Hz, 1H),
7.17−7.13 (m, 3H), 6.90−6.86 (m, 2H), 6.52 (s, 2H), 4.18−4.08 (m,
4H), 3.67 (s, 6H), 3.63 (s, 3H), 3.00−2.95 (t, J = 8.7 Hz, 1H), 2.06−
2.02 (dd, J1 = 5.4 Hz, J2 = 4.8 Hz, 1H), 1.54−1.49 (dd, J1 = 5.1 Hz, J2 =
5.1 Hz, 1H), 1.23−1.21 (t, J = 8.1 Hz, 3H). 13C NMR (DMSO-d6, 300
MHz): δ 170.64, 164.36, 152.44, 139.02, 136.64, 131.13, 127.89,
126.70, 126.70, 126.45, 106.01, 61.06, 59.97, 56.06, 55.78, 42.54,
38.23, 31.90, 18.75, 18.56, 13.96. HRMS (EI+) calcd C23H27NO6 (M+)
413.1838, found 413.1838.
1
1003, 888, 841, 796, 734, 673, 504, 417. H NMR (DMSO-d6, 300
MHz): δ 10.61 (s, 1H), 8.70−8.69 (t, J = 2.1 Hz, 1H), 8.01−7.92 (m,
2H), 7.64−7.59 (t, J = 7.8 Hz, 1H), 6.54 (s, 2H), 3.87−3.83 (m, 2H),
3.74 (s, 6H), 3.60 (s, 3H), 3.29−2.23 (t, J = 7.8 Hz, 1H), 2.24−2.22
(dd, J1 = 5.1 Hz, J2 = 4.8 Hz, 1H), 1.75−1.73 (dd, J1 = 4.8 Hz, J2 = 5.1
Hz, 1H), 0.81−0.76 (t, J = 7.2 Hz, 3H). HRMS (EI+) calcd
C22H24N2O8 (M+) 444.1533, found 444.1539.
E t h y l 1 - ( 3 - N i t r o p h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6e). Yellow
solid, mp 150.6−151.4 °C. 1H NMR (DMSO-d6, 300 MHz): δ
10.44 (s, 1H), 8.36−8.35 (t, J = 8.1 Hz, 1H), 7.84−7.81 (m, 1H),
7.69−7.66 (m, 1H), 7.53−7.48 (t, J = 8.1 Hz, 1H), 6.56 (s, 2H), 4.23−
4.16 (m, 2H), 3.65 (s, 6H), 3.46 (s, 3H), 3.31−3.07 (t, J = 8.7 Hz,
1H), 2.17−2.13 (dd, J1 = 4.8 Hz, J2 = 5.1 Hz, 1H), 1.66−1.62 (dd, J1 =
5.1 Hz, J2 = 4.8 Hz, 1H), 1.23−1.22 (t, J = 7.8 Hz, 3H). HRMS (EI+)
calcd C22H24N2O8 (M+) 444.1533, found 444.1538.
Ethyl 1-(4-Methoxyphenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (trans-6f). White
solid, mp 110.2−110.4 °C. IR (KBr, cm−1): 3308, 3281, 3230, 3071,
3004, 2972, 2937, 2842, 2826, 1700, 1645, 1592, 1550, 1508, 1461,
1376, 1340, 1315, 1244, 1183, 1147, 1124, 1012, 908, 860, 830, 698,
660, 554, 525, 463. 1H NMR (DMSO-d6, 300 MHz): δ 10.00 (s, 1H),
7.55−7.52 (d, J = 9.3 Hz, 2H), 6.90−6.87 (d, J = 9.0 Hz, 2H), 6.53 (s,
2H), 3.86−3.78 (m, 2H), 3.74 (s, 6H), 3.72 (s, 3H), 3.60 (s, 3H),
3.22−3.16 (t, J = 8.7 Hz, 1H), 2.19−2.15 (dd, J1 = 5.1 Hz, J2 = 4.5 Hz,
1H), 1.71−1.67 (dd, J1 = 4.5 Hz, J2 = 5.1 Hz, 1H), 0.85−0.77 (t, J =
7.2 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ 168.19, 165.46,
155.46, 152.45, 136.73, 131.96, 130.97, 121.36, 113.77, 106.51, 60.69,
59.96, 55.87, 55.14, 37.81, 32.79, 18.54, 13.45. HRMS (EI+) calcd
C23H27NO7 (M+) 429.1788, found 429.1793.
E t h y l 1 - ( 4 - M e t h y l b e n z y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6i). White
solid, mp 95.9−96.3 °C. IR (KBr, cm−1): 3370, 3050, 3016, 2959,
2932, 2825, 1703, 1660, 1589, 1537, 1511, 1470, 1415, 1381, 1316,
1281, 1248, 1209, 1183, 1151, 1125, 1091, 1056, 1010, 914, 884, 846,
1
807, 753, 646, 574, 509, 472. H NMR (DMSO-d6, 300 MHz): δ
8.66−8.62 (t, J = 5.7 Hz, 1H), 7.21−7.19 (m, 4H), 6.50 (s, 2H), 4.33−
4.31 (d, J = 5.7 Hz, 2H), 3.83−3.80 (m, 2H), 3.74 (s, 6H), 3.60 (s,
3H), 3.09−3.04 (t, J = 8.7 Hz, 1H), 2.26 (s, 3H), 2.11−2.07 (dd, J1 =
4.8 Hz, J2 = 4.8 Hz, 1H), 1.63−1.58 (dd, J1 = 4.5 Hz, J2 = 5.1 Hz, 1H),
0.79−0.76 (t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+)
427.1995, found 427.2002.
E t h y l 1 - ( 4 - M e t h y l b e n z y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6i). White
solid, mp 138.7−139.3 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.40−8.38 (d, J = 5.7 Hz, 1H), 6.98−6.95 (d, J = 7.8 Hz, 2H), 6.81−
6.78 (d, J = 8.1 Hz, 2H), 6.50 (s, 2H), 4.16−4.04 (m, 2H), 4.06−4.04
(t, J = 5.7 Hz, 2H), 3.67 (s, 6H), 3.62 (s, 3H), 2.98−2.93 (t, J = 7.8
Hz, 1H), 2.20 (s, 3H), 2.04−2.00 (dd, J1 = 5.1 Hz, J2 = 4.8 Hz, 1H),
1.53−1.50 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.24−1.21 (t, J = 8.1 Hz,
3H). 13C NMR (DMSO-d6, 300 MHz): δ 170.61, 164.22, 152.35,
136.55, 135.91, 135.38, 131.10, 128.41, 126.69, 106.33, 105.92, 61.00,
59.91, 55.85, 55.71, 42.28, 38.14, 31.84, 20.60, 18.76, 13.91. HRMS
(EI+) calcd C24H29NO6 (M+) 427.1995, found 427.1994.
E t h y l 1 - ( 4 - F l u o r o b e n z y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6j). White
solid, mp 100.2−100.8 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.96−8.97 (t, J = 5.4 Hz, 1H), 7.34−7.26 (m, 1H), 7.12−6.94 (m,
3H), 6.47 (s, 2H), 4.67−4.49 (m, 2H), 4.02−3.96 (m, 2H), 3.84 (s,
6H), 3.80 (s, 3H), 3.26−3.21 (t, J = 9.0 Hz, 1H), 2.27−2.22 (dd, J1 =
5.1 Hz, J2 = 4,8 Hz, 1H), 2.21−2.06 (dd, J1 = 4.8 Hz, J2 = 5.1 Hz, 1H),
0.77−0.72 (t, J = 7.2 Hz, 3H). HRMS (EI+) calcd C23H26FNO6 (M+)
431.1744, found 431.1751.
Ethyl 1-(4-Methoxyphenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (cis-6f). White
1
solid, mp 168.5−169.4 °C. H NMR (DMSO-d6, 300 MHz): δ 9.78
(s, 1H), 7.23−7.20 (d, J = 8.7 Hz, 2H), 6.79−6.76 (d, J = 9.0 Hz, 2H),
6.56 (s, 2H), 4.25−4.13 (m, 2H), 3.67 (s, 6H), 3.66 (s, 3H), 3.53 (m,
3H), 3.10−2.96 (t, J = 7.8 Hz, 1H), 2.10−2.06 (dd, J1 = 5.4 Hz, J2 =
5.1 Hz, 1H), 1.65−1.57 (dd, J1 = 5.1 Hz, J2 = 5.4 Hz, 1H), 1.24−1.19
(t, J = 7.8 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ 170.59,
162.78, 155.39, 152.37, 136.50, 132.22, 131.28, 120.97, 113.76, 105.92,
61.23, 60.01, 55.83, 55.22, 32.15, 19.24, 14.09. HRMS (EI+) calcd
C23H27NO7 (M+) 429.1788, found 429.1791.
E t h y l 1 - ( 4 - F l u o r o p h e n yl c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (trans-6g). White
solid, mp 137.9−138.3 °C. IR (KBr, cm−1): 3330, 3161, 3091, 3018,
2962, 2938, 2837, 1716, 1649, 1619, 1591, 1556, 1506, 1421, 1382,
1339, 1316, 1268, 1242, 1207, 1127, 1019, 840, 779, 652, 581, 523,
501, 418. 1H NMR (DMSO-d6, 300 MHz): δ 10.19 (s, 1H), 7.67−7.63
(m, 2H), 7.18−7.12 (t, J = 9.3 Hz, Hz, 2H), 6.52 (s, 2H), 3.86−3.79
(m, 2H), 3.74 (s, 6H), 3.59 (s, 3H), 3.23−3.18 (t, J = 8.7 Hz, 1H),
2.20−2.16 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.72−1.67 (dd, J1 = 5.1
Hz, J2 = 5.1 Hz, 1H), 0.80−0.76 (t, J = 7.2 Hz, 3H). HRMS (EI+)
calcd C22H24FNO6 (M+) 417.1588, found 417.1593.
E t h y l 1 - ( 4 - F l u o r o p h e n yl c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
E t h y l 1 - ( 4 - F l u o r o b e n z y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (cis-6j). White
solid, mp 154.9−155.6 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.05−8.01 (t, J = 5.4 Hz, 1H), 7.19−7.14 (m, 1H), 6.92−6.86 (m,
1H), 6.75−6.59 (m, 2H), 6.44 (s, 2H), 4.49−4.42 (m, 2H), 4.29−4.15
trimethoxyphenyl)cyclopropanecarboxylate (cis-6g). White
1
solid, mp 126.0−127.4 °C. H NMR (DMSO-d6, 300 MHz): δ 9.97
(s, 1H), 7.34−7.30 (m, 2H), 7.07−7.01 (t, J = 9.0 Hz, 2H), 6.55 (s,
2H), 4.20−4.13 (m, 2H), 3.64 (s, 6H), 3.51 (s, 3H), 3.07−3.01 (t, J =
694
dx.doi.org/10.1021/jm301864s | J. Med. Chem. 2013, 56, 685−699

Journal of Medicinal Chemistry
Article
(m, 2H), 3.84 (s, 6H), 3.80 (s, 3H), 3.02−2.97 (t, J = 9.0 Hz, 1H),
2.51−2.46 (dd, J1 = 5.1 Hz, J2 = 4.5 Hz, 1H), 2.08−2.03 (dd, J1 = 4.5
Hz, J2 = 5.1 Hz, 1H), 1.35−1.31 (t, J = 7.2 Hz, 3H). HRMS (EI+)
calcd C23H26FNO6 (M+) 431.1744, found 431.1752.
8.08−8.04 (t, J = 5.7 Hz, 1H), 7.28−7.20 (m, 1H), 7.00−6.97 (dd, J1 =
2.1 Hz, J2 = 2.4 Hz, 1H), 6.82−6.78 (t, J = 5.7 Hz, 2H), 6.50 (s, 2H),
4.34−4.27 (m, 2H), 3.72 (s, 6H), 3.53 (s, 3H), 3.17−3.13 (t, J = 6.9
Hz, 1H), 3.01−2.90 (m, 2H), 2.32−2.26 (m, 2H), 2.04−1.99 (dd, J1 =
4.8 Hz, J2 = 5.4 Hz, 1H), 1.53−1.48 (dd, J1 = 5.4 Hz, J2 = 4.8 Hz, 1H),
1.19−1.15 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6, 300 MHz): δ
170.53, 164.21, 163.75, 160.53, 152.32, 142.43, 142.34, 136.47, 131.22,
130.07, 129.96, 124.51, 115.16, 114.88, 112.89, 112.62, 105.80, 60.95,
59.83, 56.04, 55.76, 34.57, 31.81, 18.77, 18.53, 13.93. HRMS (EI+)
calcd C24H28FNO6 (M+) 445.1901, found 445.1907.
Ethyl 1-(4-Methoxybenzylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (trans-6k). White
1
crystal, mp 73.5−73.8 °C. H NMR (DMSO-d6, 300 MHz): δ 9.86−
9.85 (t, J = 7.8 Hz, 1H), 7.88−7.85 (d, J = 8.7 Hz, 2H), 7.14−7.11 (d,
J = 8.7 Hz, 2H), 6.43 (s, 2H), 4.28−4.24 (m, 2H), 3.94−3.92 (t, J =
7.2 Hz, 2H), 3.71 (s, 6H), 3.64 (s, 3H), 3.59 (s, 3H), 3.09−3.08 (t, J =
8.7 Hz, 1H), 1.98−1.96 (dd, J1 = 4.5 Hz, J2 = 4.5 Hz, 1H), 1.76−1.73
(dd, J1 = 4.8 Hz, J2 = 4.8 Hz, 1H), 1.48−1.46 (t, J = 8.7 Hz, 3H).
HRMS (EI+) calcd C24H29NO7 (M+) 443.1845, found 443.1836.
Ethyl 1-(4-Methoxybenzylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (cis-6k). White
solid, mp 125.1−125.4 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.38−8.34 (t, J = 8.1 Hz, 1H), 6.81−6.78 (d, J = 8.7 Hz, 2H), 6.73−
6.69 (m, 2H), 6.50 (s, 2H), 4.15−4.08 (m, 2H), 4.05−4.00 (t, J = 6.3
Hz, 2H), 3.68 (s, 3H), 3.66 (s, 6H), 3.62 (s, 3H), 2.98−2.92 (t, J = 8.7
Hz, 1H), 2.04−2.00 (dd, J1 = 4.5 Hz, J2 = 4.5 Hz, 1H), 1.52−1.48 (dd,
J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.48−1.17 (t, J = 8.7 Hz, 3H). 13C NMR
(DMSO-d6, 300 MHz): δ 170.63, 164.16, 157.95, 152.37, 136.56,
131.09, 130.90, 127.96, 113.27, 105.95, 61.00, 59.93, 55.71, 54.92,
41.95, 38.15, 31.86, 18.59, 13.93. HRMS (EI+) calcd C24H29NO7 (M+)
443.1845, found 443.1838.
Ethyl 1-(4-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (trans-6n). White
solid, mp 132.9−133.3 °C. IR (KBr, cm−1): 3376, 3068, 3032, 2998,
2941, 2833, 1698, 1651, 1584, 1506, 1456, 1428, 1410, 1369, 1330,
1311, 1267, 1240, 1156, 1129, 1011, 834, 776, 653, 605, 572, 529, 503,
1
420. H NMR (DMSO-d6, 300 MHz): δ 8.21−8.20 (t, J = 8.1 Hz,
1H), 7.27−7.22 (m, 2H), 7.13−7.07 (m, 2H), 6.44 (s, 2H), 3.82−3.76
(m, 2H), 3.75 (s, 6H), 3.58 (s, 3H), 3.40−3.33 (m, 2H), 3.00−2.94 (t,
J = 8.7 Hz, 1H), 2.78−2.73 (t, J = 7.5 Hz, 2H), 2.07−2.03 (dd, J1 = 4.5
Hz, J2 = 4.5 Hz, 1H), 1.59−1.55 (dd, J1 = 4.8 Hz, J2 = 4.8 Hz, 1H),
0.73−0.69 (t, J = 7.2 Hz, 3H). HRMS (EI+) calcd C24H28FNO6 (M+)
445.1901, found 445.1907.
Ethyl 1-(4-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (cis-6n). White
solid, mp 144.5−145.1 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.03−8.02 (t, J = 8.1 Hz, 1H), 7.23−7.05 (m, 1H), 7.01−6.97 (m,
3H), 6.45 (s, 2H), 4.17−4.10 (m, 2H), 3.61 (s, 6H), 3.60 (s, 3H),
2.98−2.92 (m, 2H), 2.76−2.74 (t, J = 7.8 Hz, 1H), 2.28−2.32 (t, J =
7.5 Hz, 2H), 2.04−2.00 (dd, J1 = 4.8 Hz, J2 = 5.1 Hz, 1H), 1.53−1.48
(dd, J1 = 5.1 Hz, J2 = 4.8 Hz, 1H), 1.20−1.15 (m, 3H). 13C NMR
(DMSO/TMS, 300 MHz): δ 170.49, 164.11, 152.28, 136.47, 135.49,
131.18, 130.10, 130.00, 114.96, 114.69, 105.84, 60.91, 59.83, 55.77,
33.98, 31.76, 18.65, 13.92. HRMS (EI+) calcd C24H28FNO6 (M+)
445.1901, found 445.1909.
Ethyl 1-(2-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (trans-6l). White
solid, mp 99.2−99.7 °C. IR (KBr, cm−1): 3386, 3302, 2997, 2940,
2901, 2832, 1700, 1655, 1584, 1535, 1457, 1426, 1408, 1369, 1328,
1309, 1266, 1235, 1155, 1128, 1022, 1003, 876, 841, 818, 782, 757,
653, 569, 481, 421. 1H NMR (DMSO-d6, 300 MHz): δ 8.25−8.21 (t, J
= 5.7 Hz, 1H), 7.32−7.23 (m, 2H), 7.16−7.10 (m, 2H), 6.46 (s, 2H),
3.82−3.75 (m, 2H), 3.73 (s, 6H), 3.45 (s, 3H), 3.44−3.35 (m, 2H),
2.98−2.93 (t, J = 8.7 Hz, 1H), 2.83−2.78 (t, J = 6.9 Hz, 2H), 2.07−
2.03 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.60−1.56 (dd, J1 = 5.1 Hz, J2 =
5.1 Hz, 1H), 0.76−0.74 (t, J = 8.1 Hz, 3H). 13C NMR (DMSO-d6, 300
MHz): δ 168.35, 167.15, 162.41, 159.19, 152.45, 136.76, 131.32,
131.26, 131.00, 128.31, 128.21, 126.08, 125.87, 124.25, 115.17, 114.88,
106.30, 60.52, 59.91, 55.81, 36.69, 33.14, 28.33, 17.78, 13.30. HRMS
(EI+) calcd C24H28FNO6 (M+) 445.1901, found 445.1907.
Ethyl 1-(2-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (cis-6l). White
solid, mp 145.8−146.2 °C. 1H NMR (DMSO-d6, 300 MHz): δ
8.10−8.06 (t, J = 5.7 Hz, 1H), 7.22−7.17 (m, 1H), 7.08−6.96 (m,
3H), 6.51 (s, 2H), 4.17−4.07 (m, 2H), 3.73 (s, 6H), 3.52 (s, 3H),
3.17−3.10 (t, J = 8.7 Hz, 1H), 2.99−2.91 (m, 2H), 2.31−2.05 (m,
2H), 2.05−2.01 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.53−1.48 (dd, J1 =
5.1 Hz, J2 = 5.1 Hz, 1H), 1.54−1.35 (t, J = 9.0 Hz, 3H). 13C NMR
(DMSO-d6, 300 MHz): δ 170.53, 164.20, 152.34, 136.52, 131.17,
130.78, 128.28, 128.17, 125.87, 125.67, 124.27, 115.18, 114.89, 105.90,
60.98, 59.82, 55.80, 38.22, 31.82, 28.15, 18.61, 13.96. HRMS (EI+)
calcd C24H28FNO6 (M+) 445.1901, found 445.1909.
( 1 R , 2 R ) - E t h y l 1 - ( P h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (7a1). 99.8% ee.
1H NMR (DMSO-d6, 300 MHz): δ 10.179 (s, 1H), 7.654−7.629 (d,
J = 7.5 Hz, 2H), 7.343−7.290 (t, J = 7.8 Hz, 2H), 7.092−7.043 (t, J =
7.2 Hz, 1H), 6.543 (s, 2H), 3.856−3.824 (m, 2H), 3.748 (s, 6H),
3.608 (s, 3H), 3.248−3.191 (t, J = 8.4 Hz, 1H), 2.224−2.181 (dd, J1 =
5.4 Hz, J2 = 5.4 Hz, 1H), 1.752−1.706 (dd, J1 = 5.7 Hz, J2 = 5.1 Hz,
1H), 0.816−0.769 (t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C22H25NO6
(M+) 399.1682, found 399. 1689. [α]22 +80.1 (c 0.1, CHCl3).
D
( 1 S , 2 S ) - E t h y l 1 - ( P h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (7a2). 99.9% ee.
1H NMR (DMSO-d6, 300 MHz): δ 10.181 (s, 1H), 7.654−7.628 (d,
J = 7.8 Hz, 2H), 7.341−7.290 (t, J = 7.5 Hz, 2H), 7.093−7.066 (t, J =
7.2 Hz, 1H), 6.542 (s, 2H), 3.855−3.823 (m, 2H), 3.746 (s, 6H),
3.607 (s, 3H), 3.248−3.191 (t, J = 8.4 Hz, 1H), 2.223−2.180 (dd, J1 =
5.4 Hz, J2 = 5.4 Hz, 1H), 1.750−1.704 (dd, J1 = 5.7 Hz, J2 = 5.1 Hz,
1H), 0.814−0.767(t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C22H25NO6
(M+) 399.1682, found 399.1693. [α]22 −80.77 (c 0.1, CHCl3).
( 1 S , 2 R ) - E t h y l 1 - ( P h e n y lDc a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (7a3). 99.1% ee.
1H NMR (DMSO-d6, 300 MHz): δ 9.940 (s, 1H), 7.345−7.316 (t, J
Ethyl 1-(3-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (trans-6m). White
solid, mp 111.3−111.9 °C. IR (KBr, cm−1): 3378, 3063, 2997, 2976,
2940, 2831, 1699, 1651, 1585, 1525, 1457, 1409, 1379, 1369, 1311,
1238, 1212, 1158, 1127, 1013, 937, 844, 818, 781, 755, 696, 653, 607,
= 7.5 Hz, 2H) 7.219−7.167 (t, J = 7.5 Hz, 2H), 6.985−6.936 (t, J = 7.2
Hz, 1H), 6.571 (s, 2H), 4.219−4.138 (m, 2H), 3.643 (s, 6H), 3.507 (s,
3H), 3.075−3.020 (t, J = 8.1 Hz, 1H), 2.098−2.056 (dd, J1 = 4.8 Hz, J2
= 4.8 Hz, 1H), 1.612−1.565 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.226−
1.179 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO/TMS, 300 MHz): δ
171.03, 163.69, 152.91, 139.70, 137.02, 131.72, 129.14, 123.83, 119.73,
106.47, 61.77, 60.49, 56.34, 32.77, 19.84, 14.65. HRMS (EI+) calcd
1
568, 522, 417. H NMR (DMSO-d6, 300 MHz): δ 8.22−8.18 (t, J =
5.7 Hz, 1H), 7.33−7.89 (m, 1H), 7.07−7.01 (m, 3H), 6.45 (s, 2H),
4.09−4.01 (m, 2H), 3.72 (s, 6H), 3.64 (s, 3H), 3.41−3.38 (m, 2H),
3.17−3.13 (t, J = 6.9 Hz, 1H), 2.82−2.77 (t, J = 8.1 Hz, 2H), 2.08−
2.03 (dd, J1 = 4.8 Hz, J2 = 5.4 Hz, 1H), 1.60−1.56 (dd, J1 = 5.4 Hz, J2 =
4.8 Hz, 1H), 0.74−0.69 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6, 300
MHz): δ 168.28, 167.05, 163.77, 160.55, 152.40, 142.42, 142.32,
136.71, 130.93, 130.08, 129.97, 124.85, 115.55, 115.27, 112.95, 112.67,
106.29, 60.50, 59.92, 55.82, 36.71, 34.46, 32.99, 17.76, 13.29. HRMS
(EI+) calcd C24H28FNO6 (M+) 445.1901, found 445.1906.
C22H25NO6 (M+) 399.1682, found 399.1686. [α]22 +24.8 (c 0.1,
D
CHCl3).
( 1 R , 2 S ) - E t h y l 1 - ( P h e n y l c a r b a m o y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)cyclopropanecarboxylate (7a4). 99.8% ee.
1H NMR (DMSO-d6, 300 MHz): δ 9.939 (s, 1H), 7.344−7.316 (t, J
= 7.5 Hz, 2H), 7.219−7.166 (t, J = 7.5 Hz, 2H), 6.988−6.960 (t, J =
4.8 Hz, 1H), 6.570 (s, 2H), 4.208−4.138 (m, 2H), 3.643 (s, 6H),
3.510 (s, 3H), 3.076−3.019 (t, J = 8.4 Hz, 1H), 2.099−2.057 (dd, J1 =
Ethyl 1-(3-Fluorophenethylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (cis-6m). White
solid, mp 146.9−147.2 °C. 1H NMR (DMSO-d6, 300 MHz): δ
695
dx.doi.org/10.1021/jm301864s | J. Med. Chem. 2013, 56, 685−699

Journal of Medicinal Chemistry
Article
4.8 Hz, J2 = 4.8 Hz, 1H), 1.613−1.566 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz,
1H), 1.226−1.179(t, J = 7.2 Hz, 3H). HRMS (EI+) calcd C22H25NO6
= 5.1 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+) 427.1995, found
427.2007. [α]22 +46.6 (c 0.1, CHCl3).
D
(M+) 399.1682, found 399.1680. [α]22 −25.4 (c 0.1, CHCl3).
(1R,2S)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7i4). 99.7% ee.
1H NMR (DMSO-d6, 300 MHz): δ 8.413−8.374 (t, J = 5.7 Hz,
D
(1R,2R)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7c1). 99.0% ee.
1H NMR (DMSO-d6, 300 MHz): δ 10.838 (s, 1H), 8.246−8.218 (d,
1H), 6.983−6.783 (m, 4H), 6.505 (s, 2H), 4.163−4.093 (m, 2H),
4.054−4.043 (m, 2H), 3.668 (s, 6H), 3.624 (s, 3H), 2.987−2.930 (t, J
= 8.4 Hz, 1H), 2.218 (s, 3H), 2.044−2.001 (dd, J1 = 5.1 Hz, J2 = 5.1
Hz, 1H), 1.531−1.484 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.234−1.186
(t, J = 4.8 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+) 427.1995,
J = 0.84 Hz, 1H), 7.546−7.508 (m, 1H), 7.379−7.322 (m, 1H),
7.184−7.127 (m, 1H), 6.605 (s, 2H), 3.873−3.806 (m, 2H), 3.740 (s,
6H), 3.600 (s, 3H), 3.206−3.147 (t, J = 9.0 Hz, 1H), 2.391−2.348 (dd,
J1 = 4.8 Hz, J2 = 5.1 Hz, 1H), 2.045−1.998 (dd, J1 = 5.1 Hz, J2 = 5.1
Hz, 1H), 0.761−0.715 (t, J = 6.9 Hz, 3H). 13C NMR (DMSO/TMS,
300 MHz): δ 170.27, 166.81, 153.14, 137.63, 135.56, 131.13, 129.99,
128.38, 125.95, 124.10, 123.21, 107.37, 61.75, 60.62, 56.58, 37.86,
36.67, 19.99, 13.83. HRMS (EI+) calcd C22H24ClNO6 (M+) 433.1306,
found 433.1300. [α]22D+46.1 (c 0.1, CHCl3).
found 427.2001. [α]22 −38.6 (c 0.1, CHCl3).
D
(1S, 2R)-1-(Hydroxymethyl)-N-phenyl-2-(3, 4, 5-
trimethoxyphenyl)cyclopropanecarboxamide (8a1). White
solid, mp 147.2−147.7 °C. 1H NMR (DMSO-d6, 300 MHz): δ
9.687 (s, 1H), 7.635−7.606 (t, J = 8.7 Hz, 2H), 7.337−7.284 (m, 2H),
7.078−7.025 (m, 1H), 6.578 (s, 2H), 5.324 (s, 1H), 3.765 (s, 6H),
3.634 (s, 3H), 3.403−3.382 (d, J = 6.3 Hz, 2H), 2.777−2.736 (t, J =
7.2 Hz, 1H), 1.520−1.476 (dd, J1 = 4.8 Hz, J2 = 5.1 Hz, 1H), 1.413−
1.375 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300
MHz): δ 171.91, 153.18, 139.53, 136.89, 133.02, 129.35, 123.92,
120.18, 107.14, 60.82, 56.48, 32.93, 30.52, 17.15. HRMS (EI+) calcd
(1S,2S)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7c2). 99.9% ee.
1H NMR (DMSO-d6, 300 MHz): δ 10.841 (s, 1H), 8.249−8.222 (d,
J = 0.51 Hz, 1H), 7.544−7.513 (m, 1H), 7.374−7.322 (t, J = 7.8 Hz,
1H), 7.182−7.126 (m, 1H), 6.603 (s, 2H), 3.871−3.804 (m, 2H),
3.746 (s, 6H), 3.606 (s, 3H), 3.205−3.147 (t, J = 8.7 Hz, 1H), 2.390−
2.346 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 2.044−1.997 (dd, J1 = 4.5 Hz,
J2 = 5.7 Hz, 1H), 0.759−0.712 (t, J = 6.9 Hz, 3H). HRMS (EI+) calcd
C20H23NO5 (M+) 357.1576, found 357.1579. [α]22 +57.8 (c 0.1,
D
CHCl3).
C22H24ClNO6 (M+) 433.1306, found 433.1315. [α]22 −57.0 (c 0.1,
(1R, 2S)-1-(Hydroxymethyl)-N-phenyl-2-(3, 4, 5-
trimethoxyphenyl)cyclopropanecarboxamide (8a2). White
solid, mp 147.2−148.2 °C. 1H NMR (DMSO-d6, 300 MHz): δ
9.689 (s, 1H), 7.637−7.608 (t, J = 3.3 Hz, 2H), 7.337−7.284 (m, 2H),
7.078−7.025 (m, 1H), 6.580 (s, 2H), 5.323 (s, 1H), 3.766 (s, 6H),
3.636 (s, 3H), 3.408−3.378 (t, J = 4.5 Hz, 2H), 2.781−2.728 (t, J = 7.2
Hz, 1H), 1.523−1.479 (dd, J1 = 4.8 Hz, J2 = 4.8 Hz, 1H), 1.418−1.376
(d, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ
171.91, 153.18, 139.53, 136.89, 133.03, 129.36, 123.92, 120.18, 107.14,
60.82, 56.48, 32.94, 30.53, 17.15. HRMS (EI+) calcd C20H23NO5 (M+)
357.1576, found 357.1582. [α]22 −75.1 (c 0.1, CHCl3).
D
CHCl3).
(1S,2R)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7c3). 99.7% ee.
1H NMR (DMSO-d6, 300 MHz): δ 9.797 (s, 1H), 7.439−7.382 (t, J
= 8.4 Hz, 1H), 7.218−7.056 (m, 3H), 6.606 (s, 2H), 4.244−4.173 (m,
2H), 3.659 (s, 6H), 3.574 (s, 3H), 3.120−3.064 (t, J = 8.4 Hz, 1H),
2.201−2.159 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.674−1.627 (dd, J1 =
5.1 Hz, J2 = 5.1 Hz, 1H), 1.283−1.236 (t, J = 7.8 Hz, 3H). 13C NMR
(DMSO-d6, 300 MHz): δ 171.10, 164.14, 152.94, 137.28, 135.54,
131.23, 129.98, 127.72, 126.72, 125.97, 106.98, 61.60, 60.54, 56.39,
38.96, 33.94, 19.34, 14.62. HRMS (EI+) calcd C22H24ClNO6 (M+)
(1R, 2R)-1-(HydroxymDethyl)-N-phenyl-2-(3, 4, 5-
trimethoxyphenyl)cyclopropanecarboxamide (8a3). White
solid, mp 129.2−130.4 °C. 1H NM R(DMSO-d6, 300 MHz): δ
9.638 (s, 1H), 7.423−7.396 (t, J = 8.4 Hz, 2H), 7.235−7.183 (t, J = 7.5
Hz, 2H), 6.995−6.946 (t, J = 7.5 Hz, 1H), 6.506 (s, 2H), 5.394 (s,
1H), 3.986−3.933 (m, 1H), 3.506 (s, 6H), 3.489 (s, 3H), 3.387 (s,
1H), 2.348−2.297 (t, J = 8.4 Hz, 1H), 1.822−1.783 (dd, J1 = 5.7 Hz, J2
= 5.1 Hz, 1H), 1.197−1.152 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C
NMR (DMSO/TMS, 300 MHz): δ 168.73, 152.84, 139.60, 136.45,
134.10, 129.12, 123.77, 120.14, 106.26, 66.22, 60.50, 56.23, 38.26,
28.94, 15.32. HRMS (EI+) calcd C20H23NO5 (M+) 357.1576, found
357.1581. [α]22 +25.5 (c 0.1, CHCl3).
433.1306, found 433.1298. [α]22 +44.0 (c 0.1, CHCl3).
D
(1R,2S)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7c4). 100% ee.
1H NMR(DMSO-d6, 300 MHz): δ 9.771 (s, 1H), 7.403−7.378 (t, J
= 5.4 Hz, 1H), 7.168−7.050 (m, 3H), 6.603 (s, 2H), 4.241−4.170 (m,
2H), 3.656 (s, 6H), 3.571 (s, 3H), 3.117−3.060 (t, J = 8.4 Hz, 1H),
2.197−2.155 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.670−1.623 (dd, J1 =
5.1 Hz, J2 = 5.1 Hz, 1H), 1.280−1.233 (t, J = 7.8 Hz, 3H). HRMS
(EI+) calcd C22H24ClNO6 (M+) 433.1306, found 433.1305. [α]22
D
−39.5 (c 0.1, CHCl3).
(1R,2R)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5-
(1S, 2S)-1D-(H ydroxymethyl )-N -phenyl-2-(3, 4, 5-
trimethoxyphenyl)cyclopropanecarboxamide (8a4). White
solid, mp 129.2−130.0 °C. 1H NMR (DMSO-d6, 300 MHz): δ
9.618 (s, 1H), 7.416−7.384 (dd, J1 = 2.1 Hz, J2 = 2.4 Hz, 2H), 7.231−
7.179 (t, J = 7.5 Hz, 2H), 6.995−6.942 (m, 1H), 6.494 (s, 2H), 5.385−
5.348 (t, J = 5.7 Hz, 1H), 3.982−3.925 (dd, J1 = 3.9 Hz, J2 = 4.2 Hz,
1H), 3.622 (s, 6H), 3.535 (s, 3H), 3.351 (s, 1H), 2.336−2.285 (t, J =
6.9 Hz, 1H), 1.808−1.769 (dd, J1 = 5.7 Hz, J2 = 5.1 Hz, 1H), 1.189−
1.114 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300
MHz): δ 168.71, 152.83, 139.60, 136.44, 134.10, 129.12, 123.76,
120.13, 106.25, 66.20, 60.50, 56.23, 38.28, 28.90, 15.32. HRMS (EI+)
1
trimethoxyphenyl)cyclopropanecarboxylate (7i1). 100% ee. H
NMR (DMSO-d6, 300 MHz): δ 8.653−8.615 (t, J = 5.7 Hz, 1H),
7.196−7.112 (m, 4H), 6.493 (s, 2H), 4.329−4.310 (d, J = 5.7 Hz, 2H),
3.833−3.789 (m, 2H), 3.735 (s, 6H), 3.599 (s, 3H), 3.099−3.043 (t, J
= 8.7 Hz, 1H), 2.269 (s, 3H), 2.113−2.071 (dd, J1 = 4.8 Hz, J2 = 4.8
Hz, 1H), 1.632−1.586 (dd, J1 = 4.5 Hz, J2 = 5.1 Hz, 1H), 0.796−0.769
(t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+) 427.1995,
found 427.2011. [α]22 +16.8 (c 0.1, CHCl3).
D
(1S,1S)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7i2). 99.7% ee.
1H NMR (DMSO-d6, 300 MHz): δ 8.656−8.616 (t, J = 6.0 Hz,
calcd C20H23NO5 (M+) 357.1576, found 357.1580. [α]22 −28.5 (c
1H), 7.199−7.112 (m, 4H), 6.495 (s, 2H), 4.332−4.313 (d, J = 5.7 Hz,
2H), 3.835−3.787 (m, 2H), 3.735 (s, 6H), 3.599 (s, 3H), 3.102−3.045
(t, J = 8.7 Hz, 1H), 2.269 (s, 3H), 2.115−2.073 (dd, J1 = 4.8 Hz, J2 =
4.8 Hz, 1H), 1.635−1.589 (dd, J1 = 4.5 Hz, J2 = 5.1 Hz, 1H), 0.793−
0.766 (t, J = 7.8 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+)
D
0.1, CHCl3).
(1S,2R)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropyl)benzamide (8c1). White solid,
1
mp 161.2−161.6 °C. H NMR (DMSO-d6, 300 MHz): δ 10.323 (s,
427.1995, found 427.1998. [α]22 −21.3 (c 0.1, CHCl3).
1H), 8.375−8.343 (m, 1H), 7.507−7.481 (m, 1H), 7.353−7.301 (m,
1H), 7.120−7.068 (m, 1H), 6.582 (s, 2H), 5.687 (s, 1H), 3.677 (s,
6H), 3.628 (s, 3H), 3.584−3.531 (m, 1H), 3.427−3.380 (m, 1H),
2.830−2.776 (t, J = 8.1 Hz, 1H), 1.497−1.421 (m, 2H). 13C NMR
(DMSO-d6, 300 MHz): δ 172.48, 153.18, 136.93, 136.14, 132.72,
129.88, 128.34, 125.03, 122.20, 107.18, 60.57, 56.49, 32.20, 30.78,
17.74. HRMS (EI+) calcd C20H22ClNO5 (M+) 391.1187, found
D
(1S,2R)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxylate (7i3). 99.9% ee.
1H NMR(DMSO-d6, 300 MHz): δ 8.414−8.375 (t, J = 5.7 Hz, 1H),
6.983−6.783 (m, 4H), 6.506 (s, 2H), 4.164−4.093 (m, 2H), 4.055−
4.044 (m, 2H), 3.667 (s, 6H), 3.624 (s, 3H), 2.988−2.931 (t, J = 8.4
Hz, 1H), 2.218 (s, 3H), 2.044−2.002 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz,
1H), 1.531−1.484 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.234−1.186 (t, J
391.1189. [α]22 +85.5 (c 0.1, CHCl3).
D
696
dx.doi.org/10.1021/jm301864s | J. Med. Chem. 2013, 56, 685−699

Journal of Medicinal Chemistry
Article
(1R,2S)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropyl)benzamide (8c2). White solid,
mp 161.2−161.9 °C. H NMR (DMSO-d6, 300 MHz): δ 10.331 (s,
1.771−1.719 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H), 1.082−1.036 (dd, J1 =
5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ 169.72,
152.83, 137.02, 136.53, 135.95, 133.93, 129.11, 127.31, 106.49, 66.33,
60.54, 56.26, 37.23, 28.86, 21.28, 14.64. HRMS (EI+) calcd
1
1H), 8.377−8.350 (d, J = 8.1 Hz, 1H), 7.503−7.477 (d, J = 9.0 Hz,
1H), 7.352−7.301 (t, J = 7.2 Hz, 1H), 7.116−7.066 (t, J = 7.5 Hz,
1H), 6.586 (s, 2H), 5.700 (s, 1H), 3.783 (s, 6H), 3.631 (s, 3H),
3.581−3.544 (m, 1H), 3.436−3.397 (m, 1H), 2.840−2.788 (t, J = 7.8
Hz, 1H), 1.504−1.440 (m, 2H). 13C NMR (DMSO-d6, 300 MHz): δ
172.49, 153.19, 136.95, 132.72, 129.86, 128.32, 125.01, 122.75, 122.21,
107.19, 60.95, 56.49, 32.21, 30.80, 17.74. HRMS (EI+) calcd
C22H27NO5 (M+) 385.1889, found 385.1907. [α]22 +119.5 (c 0.1,
D
CHCl3).
(1S,2S)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxamide (8i4). White solid,
mp 97.4−98.2 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.044−8.003 (t,
J = 6.0 Hz, 1H), 6.953−6.928 (d, J = 7.8 Hz, 2H), 6.751−6.722 (d, J =
7.8 Hz, 2H), 6.460 (s, 2H), 5.227−5.192 (t, J = 5.4 Hz, 1H), 4.263−
4.197 (m, 1H), 4.021−3.958 (m, 1H), 3.653 (s, 6H), 3.623 (s, 3H),
2.987−2.930 (t, J = 8.4 Hz, 1H), 2.489−2.476 (m, 2H), 2.204 (s, 3H),
1.771−1.716 (dd, J1 = 5.7 Hz, J2 = 5.1 Hz, 1H), 1.085−1.036 (dd, J1 =
5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ 169.70,
152.83, 137.02, 136.59, 135.90, 133.91, 129.11, 127.34, 106.49, 66.33,
60.53, 56.24, 37.23, 28.87, 21.28, 14.62. HRMS (EI+) calcd
C20H22ClNO5 (M+) 391.1187, found 391.1185. [α]22 −101.5 (c
D
0.1, CHCl3).
(1R,2R)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropyl)benzamide (8c3). White solid,
1
mp 116.4−117.0 °C. H NMR (DMSO-d6, 300 MHz): δ 10.133 (s,
1H), 7.888−7.856 (m, 1H), 7.437−7.406 (m, 1H), 7.197−7.165 (m,
1H), 7.048−6.991 (m, 1H), 6.528 (s, 2H), 6.132 (s, 1H), 4.149−4.110
(d, J = 11.7 Hz, 1H), 3.644 (s, 6H), 3.552 (s, 3H), 3.384−3.360 (t, J =
11.1 Hz, 1H), 2.501−2.482 (m, 1H), 1.863−1.823 (dd, J1 = 4.5 Hz, J2
= 4.8 Hz, 1H), 1.163−1.118 (dd, J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C
NMR (DMSO-d6, 300 MHz): δ 169.05, 152.80, 136.63, 135.97,
133.40, 129.75, 128.14, 125.11, 123.12, 122.63, 106.82, 66.08, 60.51,
56.21, 36.41, 31.28, 14.80. HRMS (EI+) calcd C20H22ClNO5 (M+)
C22H27NO5 (M+) 385.1889, found 385.1890. [α]22 −129.5 (c 0.1,
D
CHCl3).
Cell Culture and Maintenance. Five normal cancer cell lines in
this study were purchased from China Life Science Collage (Shanghai,
PRC). Two paclitaxel-resistant sublines (A549/paclitaxel and HeLa/
paclitaxel) were derived from the parental-sensitive cell lines A549 or
HeLa by paclitaxel-based stepwise selection. A549 (non-small-cell lung
cancer line), HeLa (human epithelial cervical cancer), QGY (human
hepatoma cancer), SW480 (human colon cancer), K562 (leukemia),
A549/paclitaxel, and HeLa/paclitaxel cell lines were grown in the
Dulbecco’s modified Eagle medium (DMEM, containing 10% fetal calf
serum) in culture dish in a humidified atmosphere of 5% CO2 at 37
°C.
Antiproliferative Activity Assays in Vitro. The antiproliferative
activity of the target compounds were examined in five human cancer
cell lines (A549, HeLa, QGY, SW480, and K562) and two paclitaxel-
resistance cell lines (A549/paclitaxel, HeLa/paclitaxel). About 40000−
50000 cells/mL cells, which were in the logarithmic phase, were plated
into each well of 96-well plates and exposed to five different
concentrations of a test compound for 48 h in three to five replicates.
Cell numbers at the end of the drug treatment were measured using
MTT assay. Briefly, the cells were fixed with 10% trichloroacetic acid
and stained with 20 μL of MTT (5 mg/mL), and the absorbance at
570 nm was measured using a plate reader (BD Biosciences, San Jose,
CA). Percentages of cell survival versus drug concentrations were
plotted.
Tubulin Polymerization Assay in Vitro. Tubulin polymerization
assay was monitored by the change in optical density at 340 nm using
a modification of methods described by Jordan et al.4 Purified brain
tubulin polymerization kit was purchased from Cytoskeleton
(BK006P, Denver, CO). The final buffer concentrations for tubulin
polymerization contained 80.0 mM piperazine-N,N′-bis(2-ethanesul-
fonic acid)sequisodium salt (pH 6.9), 2.0 mM MgCl2, 0.5 mM
ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
(EGTA), 1 mM GTP, and 10.2% glycerol. Test compounds were
added in different concentrations, and then all components except the
purified tubulin were warmed to 37 °C. The reaction was initiated by
the addition of tubulin to a final concentration of 3.0 mg/mL.
Paclitaxel and CA-4 were used as positive controls under similar
experimental conditions. To determine the EC50 values of the
compounds to stimulate tubulin polymerization, the compounds
were preincubated with tubulin at various concentrations (3.75, 7.5,
15, 30, and 60 μM). The optical density was measured for 1 h at 1 min
intervals in BioTek’s Synergy 4 multifunction microplate spectropho-
tometer with a temperature controlled cuvette holder. Assays were
performed according to the manufacturer’s instructions and under
conditions similar to those employed for the tubulin polymerization
assays described above.36−38
391.1187, found 391.1192. [α]22 +73.2 (c 0.1, CHCl3).
D
(1S,2S)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropyl)benzamide (8c4). White solid,
1
mp 116.3−117.2 °C. H NMR (DMSO-d6, 300 MHz): δ 10.134 (s,
1H), 7.892−7.860 (m, 1H), 7.436−7.409 (t, J = 8.4 Hz, 1H), 7.221−
7.169 (m, 1H), 7.052−6.995 (m, 1H), 6.531 (s, 2H), 6.110−6.100 (d,
J = 0.15 Hz, 1H), 4.164−4.103 (m, 1H), 3.648 (s, 6H), 3.557 (s, 3H),
3.389−3.361 (d, J = 8.4 Hz, 1H), 2.505−2.153 (m, 1H), 1.868−1.828
(dd, J1 = 4.5 Hz, J2 = 4.8 Hz, 1H), 1.016−1.121 (dd, J1 = 5.1 Hz, J2 =
5.1 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ 169.05, 152.81,
136.65, 135.98, 133.40, 129.74, 128.13, 125.11, 123.13, 122.64, 106.84,
66.09, 60.51, 56.22, 36.31, 31.28, 14.81. HRMS (EI+) calcd
C20H22ClNO5 (M+) 391.1187, found 391.1196. [α]22 −85.1 (c 0.1,
D
CHCl3).
(1S,2R)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxamide (8i1). White solid,
1
mp 114.5−115.3 °C. H NMR (DMSO-d6, 300 MHz: δ 8.107−8.068
(t, J = 6.0 Hz, 1H), 7.200−7.109 (dd, J1 = 8.1 Hz, J2 = 7.8 Hz, 4H),
6.507 (s, 2H), 4.925−4.896 (t, J = 3.9 Hz, 1H), 4.435−4.365 (m, 1H),
4.284−4.216 (m, 1H), 3.751 (s, 6H), 3.617 (s, 3H), 3.543−3.487 (m,
1H), 3.241−3.189 (m, 1H), 2.664−2.612 (t, J = 7.5 Hz, 1H), 2.270 (s,
3H), 1.385−1.341 (dd, J1 = 4.8 Hz, J2 = 4.8 Hz, 1H), 1.307−1.263 (dd,
J1 = 4.5 Hz, J2 = 4.5 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ
168.86, 167.84, 153.06, 137.32, 136.95, 136.44, 131.65, 129.44, 127.76,
106.95, 61.25, 60.59, 56.49, 37.69, 33.27, 21.18,18.65, 14.01. HRMS
(EI+) calcd C22H27NO5 (M+) 385.1889, found 385.1901. [α]22D +54.5
(c 0.1, CHCl3).
(1R,2S)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxamide (8i2). White solid,
mp 114.5−115.0 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.107−8.068
(t, J = 5.7 Hz, 1H), 7.200−7.110 (dd, J1 = 8.1 Hz, J2 = 7.8 Hz, 4H),
6.507 (s, 2H), 4.925−4.896 (t, J = 1.2 Hz, 1H), 4.435−4.216 (m, 1H),
4.285−4.214 (m, 1H), 3.751 (s, 6H), 3.617 (s, 3H), 3.543−3.487 (m,
1H), 3.241−3.189 (m, 1H), 2.665−2.612 (t, J = 5.7 Hz, 1H), 2.267 (s,
3H), 1.385−1.341 (dd, J1 = 4.8 Hz, J2 = 5.1 Hz, 1H), 1.301−1.263 (dd,
J1 = 5.1 Hz, J2 = 5.1 Hz, 1H). 13C NMR (DMSO-d6, 300 MHz): δ
173.05, 153.13, 137.21, 136.76, 136.42, 133.35, 129.50, 127.82, 107.00,
60.96, 60.57, 56.46, 42.98, 32.28, 29.97, 21.29, 16.74. HRMS (EI+)
calcd C22H27NO5 (M+) 385.1889, found 385.1900. [α]22 −69.5 (c
D
0.1, CHCl3).
(1R,2R)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5-
trimethoxyphenyl)cyclopropanecarboxamide (8i3). White solid,
mp 97.5−98.0 °C. 1H NMR (DMSO-d6, 300 MHz): δ 8.045−8.006 (t,
J = 6.0 Hz, 1H), 6.951−6.925 (d, J = 7.8 Hz, 2H), 6.748−6.722 (d, J =
7.8 Hz, 2H), 6.466 (s, 2H), 5.227−5.190 (t, J = 5.4 Hz, 1H), 4.267−
4.196 (m, 1H), 4.020−3.952 (m, 1H), 3.652 (s, 6H), 3.623 (s, 3H),
2.987−2.930 (t, J = 8.4 Hz, 1H), 2.497−2.479 (m, 2H), 2.207 (s, 3H),
Analysis of Cell Cycle. A549 cells in 96-well plates were incubated
for 24 h in the presence or absence of compound 8c4. Cells were
harvested with trypsin−ethylenediamintetraacetic acid (trypsin−
EDTA) and fixed with ice-cold 70% ethanol at 4 °C for 2 h. Ethanol
was removed by centrifugation and washed with cold 10% phosphate
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dx.doi.org/10.1021/jm301864s | J. Med. Chem. 2013, 56, 685−699

Journal of Medicinal Chemistry
Article
buffer solution (PBS). Then cells were stained with 0.5 mL of DNA
staining solution (propidium iodide, PI) at 37 °C for 30 min and at 4
°C for 24 h. The DNA contents of 50 000 events were measured by
BD Canto III flow cytometer (BD Biosciences, San Jose, CA) at 488
nm, which was also used to determine the percentage of cells in
different phases of the cell cycle.
AUTHOR INFORMATION
Corresponding Author
*For C.S.: phone/fax, 86-21-81871233; e-mail, shengcq@
hotmail.com. For K.L.: phone/fax, 86-21-81871237; e-mail,
proflike@sina.com.
■
Notes
Laser Fluorescence Confocal Microscopy. For laser fluores-
cence confocal microscopy, the method of this assay according to the
work of Leslie et al.39 The 0.5 × 106 A549 cells were grown on a nitric
acid washed culture plate overnight in a 5% CO2 incubator at 37 °C.
Compound in DMSO or DMSO vehicle alone (0.2% DMSO final)
was added to the cells at 70% confluency and further incubated for 22
h. Cells were washed briefly with PBS. After being washed three times
in PBS (pH 7.4), cells were stained with 4% paraformaldehyde (PME).
Cells were given three rinses with a solution of 0.5% Triton X-100
(sigma) in PBS and blocked for 20 min in 10% goat serum (sigma).
FITC conjugated anti-α-tubulin (Sigma) was added at a 1:100 dilution
in 10% goat serum, and the cells were incubated 2 h at 37 °C and then
washed 3 × 10 min in PBS. Goat anti-mouse IgG/Alexa-Fluor 488
(Invitrogen, U.S.) conjugate was diluted 1:200 in 10% goat serum and
incubated with cells for 25 min. DAPI (Roche, U.S.) was diluted 1:100,
joined with the cells, and then washed four times with PBS. Samples
were visualized immediately on a Zeiss LSM 570 laser scanning
confocal microscope. Wide-field images were acquired by moving the
stage to 10−15 random locations on each slide, thereafter only
adjusting the stage in the z-direction to bring a maximal number of
cells into focus.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of China (Grant No. 21172259). We also thank
the Key laboratory of drug research for special environments,
PLA. We are grateful to Dr. Youheng Wei (State Key
Laboratory of Genetic Engineering, Fudan University, Shang-
hai, China) for the assistance with laser fluorescence confocal
microscopy mitotic arrest experiments.
ABBREVIATIONS USED
■
CA-4, combretastatin-A4; TBA, tubulin-binding agent; TMSOI,
trimethylsulfoxonium iodide; SAR, structure−activity relation-
ship; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; 5-Fu, 5-fluorouracil; SD, standard error;
DMSO, dimethyl sulfoxide; DAPI, diamidinophenylindole;
FITC, fluorescein isothiocyanate; PI, propidium iodide;
EDTA, ethylenediamintetraacetic acid; PME, paraformalde-
hyde; PBS, phosphate buffered solution; EGTA, ethylene glycol
bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
Flow Cytometry with Apoptotic Cells. A549 cells at 5 × 106
cell/mL were incubated with various concentrations of compound 8c4
for 24 h at 37 °C to induce cell apoptosis. The percentage of apoptotic
cells was estimated by staining with annexin V-FITC and PI. A549
cells without compound 8c4 treatment were used as a vehicle control.
After cells were induced with compound 8c4 for 24 h, both compound
8c4 treated and untreated cells were harvested and washed once with
PBS. The cells were incubated with 5 μL of annexin V-FITC in
binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at
pH 7.4) for 15 min at room temperature. PI was then dropwise added
to the medium at cold temperature, and the mixture was incubated for
10 min. The cells were analyzed using a BD Canto III flow cytometer.
The data were acquired with Cell Quest acquisition software, version
3.3 (BD Biosciences, San Jose, CA).
Antitumor Activities in Vivo. The animals were purchased from
Animal Centre of Chinese Academy of Science (Shanghai, PRC). Five-
week-old male BALB/c nude mice (18−20 g) were housed at constant
room temperature and fed a standard rodent diet and water. A549
tumor animal model was established by subcutaneously inoculating the
A549 solid tumor (3 mm3) in the right armpit of 5-week-old male
BALB/c nude mice. When the tumors reached a volume of 100−200
mm3 in all mice on day 13, treated mice were intragastrically injected
with compound 8c4 and paclitaxel, which were first dissolved in
Tween-80 and then diluted to the needed concentration with
physiological saline at a dosage of 30 mg/kg (paclitaxel), 100 mg/
kg, or 500 mg/kg (compound 8c4), whereas the vehicle control mice
were intraperitoneally (ip) injected with Tween-80 and physiological
saline. The tumor volumes were measured with electronic digital
calipers and determined by measuring length (l) and width (w) to
calculate volume (V = lw2/2).
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