Design and Synthesis of Cyclopropylamide Analogues of

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.

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mitotic arrest that could lead to cell apoptosis. 10−12 Combretastatin-A4 (CA-4, Figure 1), isolated from the bark of African willow tree Combretum caf frum in 1982,13 is a natural cis-stilbene product that strongly inhibits tubulin polymerization by binding to the colchicine binding site. The work of Hsieh and Nam14,15 showed that 3,4,5-trimethoxy substitution on the A-ring and the cis-orientation between the two aryl rings were essential for efficient binding to tubulin. Up to now, a great number of CA-4 analogues have been reported.16−21 The cyclopropyl group is generally considered as an alkene bioisostere.22,23 The cyclopropyl group can maintain the cisorientation of the pharmacophoric substructures in lead compounds. On the other hand, many TBAs containing the amide moieties are now first-line clinical drugs or currently in clinical trials for the treatment of solid tumors.24,25 Alterations 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 some microtubule-destabilizing drugs also show drug resistance.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

INTRODUCTION Microtubules are cytoskeletal filaments consisting of α,βtubulin 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 signaling, cell division, and mitosis, which are required for cell life cycle.1,2 Because of the multifunction of microtubules in the cell cycle, tubulin has become an attractive target in anticancer drug discovery. Natural-products-based drugs targeting tubulin and the microtubule system are referred as antimitotics, and they are also important components in combination chemotherapy for many pediatric and adult malignancies. There are many chemically diverse compounds targeting the tubulin−microtubule system. Tubulin binding 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 vincristine, vinblastine, and vinorelbine).3 There are three ligand binding sites in tubulin α/β-heterodimer: paclitaxel 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 © 2013 American Chemical Society

Received: June 1, 2012 Published: January 28, 2013 685

dx.doi.org/10.1021/jm301864s | J. Med. Chem. 2013, 56, 685−699

Journal of Medicinal Chemistry

Article

Figure 1. Structures of selected antimitotic compounds and the design of the target compound 8c4.

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.

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

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RESULTS AND DISCUSSION Chemistry. The syntheses of the key intermediates trans-5 and cis-5 are outlined in Scheme 1. Diethyl 2-(3,4,5trimethoxybenzylidene)malonate (compound 2) was prepared by reacting commercially available 3,4,5-trimethoxybenzaldehyde (compound 1) with diethyl malonate through Knoevenagel 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 Scheme 1a

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. 686



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Scheme 2a

a

Reagents and conditions: (a) (COCl)2/pyridine/CH2Cl2, amine/CH2Cl2.

Scheme 3a

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-(4,5dimethylthiazol-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 687



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Table 1. Antiproliferative Activity of Target Compounds against Five Human Cancer Cell Linesa

IC50 (μM)b compd

R1

R2

A549

HeLa

SW480

QGY

K562

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 7a2 7a3 7a4 7c1 7c2 7c3 7c4 7i1 7i2 7i3 7i4 8a1 8a2 8a3 8a4 8c1 8c2 8c3 8c4 8i1 8i2 8i3 8i4 5-Fu CA-4

phenyl phenyl 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 benzyl 4-CH3-benzyl 4-CH3-benzyl 4-F-benzyl 4-F-benzyl 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 phenyl phenyl phenyl 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 phenyl phenyl phenyl 2-Cl-phenyl 2-Cl-phenyl 2-Cl-phenyl 2-Cl-phenyl 4-CH3-benzyl 4-CH3-benzyl 4-CH3-benzyl 4-CH3-benzyl

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 CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH 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 61.4 ± 3.6 28.0 ± 3.4 22.4 ± 4.3 >100 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 31.1 ± 2.4 27.3 ± 4.3 64.3 ± 1.2 52.3 ± 0.5 >100 49.3 ± 4.2 21.3 ± 1.3 65.9 ± 2.4 >100 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 13.3 ± 2.3 >100 43.2 ± 1.6 >100 24.1 ± 2.1 36.7 ± 2.4 11.4 ± 4.3 >100 4.4 ± 2.1 60.7 ± 5.1 46.3 ± 5.4 63.8 ± 5.3 10.5 ± 4.4 56.0 ± 1.2 5.6 ± 1.4c

>100 >100 35.1 ± 2.7 40.2 ± 2.4 76.4 ± 2.4 22.1 ± 1.8 >100 35.1 ± 2.1 >100 59.4 ± 2.6 >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 >100 >100 58.2 ± 5.1 58.2 ± 4.2 28.1 ± 0.8 35.0 ± 4.2 58.1 ± 3.3 >100 >100 >100 >100 >100 14.4 ± 4.3 >100 42.4 ± 3.1 >100 19.7 ± 5.1 >100 28.5 ± 4.1 >100 86.4 ± 11.4 >100 32.0 ± 1.8 45.5 ± 3.5 13.3 ± 2.2 >100 8.1 ± 1.3 57.8 ± 6.2 41.2 ± 6.2 >100 17.4 ± 2.9 142.6 ± 3.6 2.1 ± 0.6c

>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 >100 66.3 ± 5.2 42.0 ± 5.4 46.7 ± 1.2 >100 89.6 ± 5.2 >100 29.0 ± 6.4 28.4 ± 7.6 34.1 ± 5.2 >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 >100 >100 >100 30.1 ± 2.4 46.7 ± 5.3 25.6 ± 2.4 >100 >100 >100 36.4 ± 2.3 >100 76.8 ± 2.5 >100 33.3 ± 1.5 40.2 ± 2.1 10.7 ± 1.2 >100 13.2 ± 2.4 94.0 ± 1.5 76.3 ± 4.8 >100 26.8 ± 4.3 158.2 ± 2.1 18.2 ± 2.8c

>100 59.3 ± 4.6 >100 54.1 ± 2.9 48.8 ± 3.1 36.1 ± 1.4 >100 46.3 ± 3.1 >100 >100 >100 86.1 ± 7.3 36.1 ± 5.7 79.1 ± 10.3 >100 >100 37.9 ± 3.7 >100 >100 59.4 ± 4.1 >100 >100 46.8 ± 3.1 34.1 ± 1.6 >100 39.9 ± 4.6 >100 >100 38.1 ± 4.2 48.7 ± 2.6 >100 >100 34.7 ± 4.7 34.3 ± 3.1 >100 28.0 ± 3.0 >100 26.4 ± 4.3 >100 32.1 ± 4.6 >100 36.7 ± 3.7 >100 51.3 ± 3.4 40.7 ± 0.9 24.5 ± 1.6 >100 15.7 ± 2.2 56.1 ± 5.2 58.7 ± 5.3 >100 25.1 ± 1.2 126.2 ± 3.1 26.5 ± 6.9c

>100 >100 >100 56.3 ± 3.4 14.2 ± 2.1 41.4 ± 2.4 >100 52.1 ± 1.2 >100 >100 >100 >100 >100 63.7 ± 4.1 >100 >100 >100 >100 43.1 ± 5.3 45.1 ± 5.6 >100 >100 79.1 ± 5.2 >100 >100 76.1 ± 5.1 >100 >100 >100 >100 >100 >100 19.5 ± 3.5 >100 49.4 ± 4.7 20.7 ± 2.1 20.4 ± 5.4 >100 >100 >100 >100 >100 >100 >100 >100 18.4 ± 2.3 >100 16.9 ± 2.4 >100 >100 >100 >100 114.5 ± 2.1 4.8 ± 4.5c

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Table 1. continued IC50 (μM)b compd

R1

R2

paclitaxel

A549

HeLa

SW480

QGY

K562

8.2 ± 3.4c

6.3 ± 4.6c

12.1 ± 4.3c

11.3 ± 5.3c

15.7 ± 5.7c

a

Cell lines were treated with different concentrations of the compounds for 48 h as described in the Experimental Section. Cell viability was measured by MTT assay. bIC50 values are indicated as the mean ± SD (standard error) of three independent experiments. cThe unit of IC50 for CA4 and paclitaxel is nM.

Table 2. In Vitro Growth Inhibitory Effect of Compound 8c4 on Paclitaxel-Resistant Cell Lines A549 and HeLa IC50 (μM)

a

IC50 (μM)

compd

A549

A549 (resistant)

resistance indexa

HeLa

HeLa (resistant)

resistance indexa

8c4 paclitaxel

4.4 ± 2.1 8.2 ± 3.4b

5.3 ± 1.4 1037.8 ± 33.4b

1.2 126.6

8.1 ± 1.3 6.3 ± 4.6b

10.7 ± 2.5 1375.0 ± 49.6b

1.3 218.3

Resistance index = IC50(resistant)/IC50(sensitive) . bIC50 unit for paclitaxel is nM.

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

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 cis6c 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). 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 Resistant 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

Table 3. In Vitro Effects of the Selected Compounds on Tubulin Polymerizationa compd (10 μM)

tubilin polymerization (% stimulation)

compd (10 μM)

tubilin polymerization (% stimulation)

control cis-6a cis-6d cis-6f cis-6n trans-6g

0 23.2 50.0 25.0 40.8 7.9

trans-6k 7c4 7i4 8a4 8c4 paclitaxel

50.0 28.6 79.3 31.5 24.3 245.3

a The final concentration of the compounds was 10 μM. The 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 maximum 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 689



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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 immunofluorescence 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. DMSOtreated 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. In vitro tubulin assembly experiments showed that compound 8c4 and paclitaxel could promote tubulin polymerization 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

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

EC50 (μM)

7i4 7c4 8a4

5.83 16.91 14.30

8c4 paclitaxel

19.68 1.23

bind with the paclitaxel binding site and formed hydrophobic and hydrogen-bonding interactions with tubulin (see Supporting 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 concentration

% of cells in G1 phase

% of cells in S phase

% of cells in G2/M phase

control 0.1 1 5 10

87.56 78.7 38.41 28.62 9.85

9.12 9.54 9.96 10.81 10.29

2.78 12.53 51.68 60.85 79.92

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 690



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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.

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/

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 691



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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.

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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 on a PE Spectrum One FI-IR spectrometer as KBr pellets. 1H 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; temperature, ambient; injection volume, 20 μL). 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

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).

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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. 692



Article

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. Ethyl 1-(2-Nitrophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (trans-6b). Yellow solid, mp 132.2−132.4 °C. 1H 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. Ethyl 1-(2-Nitrophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6b). Yellow solid, mp 95.9−96.3 °C. 1H 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. Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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. Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6c). White solid, mp 168.5−169.4 °C. 1H 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. Ethyl 1-(3-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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, 1011, 912, 884, 842, 792, 741, 716, 688, 656, 591, 558, 507, 421. 1H 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. Ethyl 1-(3-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6d). White solid, mp 144.1−144.5 °C. 1H NMR (DMSO-d6, 300 MHz): δ

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). Synthesis of 2-(3,4,5-Trimethoxyphenyl)cyclopropane-1,1dicarboxylic 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). Synthesis of 1-(Ethoxycarbonyl)-2-(3,4,5trimethoxyphenyl)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). 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). Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5-trimethoxyphenyl)cyclopropanecarboxylate (trans-6a). White crystal, mp 120.0− 120.2 °C. 1H 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, 693



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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. Ethyl 1-(3-Nitrophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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, 1003, 888, 841, 796, 734, 673, 504, 417. 1H 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. Ethyl 1-(3-Nitrophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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-(3,4,5trimethoxyphenyl)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. Ethyl 1-(4-Methoxyphenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6f). White solid, mp 168.5−169.4 °C. 1H 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. Ethyl 1-(4-Fluorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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. Ethyl 1-(4-Fluorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6g). White solid, mp 126.0−127.4 °C. 1H 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 =

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). 13C 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, 1021, 1001, 924, 865, 826, 773, 740, 701, 577, 523, 494, 448. 1H 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. Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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, 807, 753, 646, 574, 509, 472. 1H 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. Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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. Ethyl 1-(4-Fluorobenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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-Fluorobenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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), 4.29−4.15 694



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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-Fluorophenethylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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, 420. 1H 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-(3,4,5trimethoxyphenyl)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. (1R,2R)-Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7a1). 99.8% ee. 1 H 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]22D +80.1 (c 0.1, CHCl3). (1S,2S)-Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7a2). 99.9% ee. 1 H 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]22D −80.77 (c 0.1, CHCl3). (1S,2R)-Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7a3). 99.1% ee. 1 H NMR (DMSO-d6, 300 MHz): δ 9.940 (s, 1H), 7.345−7.316 (t, J = 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 C22H25NO6 (M+) 399.1682, found 399.1686. [α]22D +24.8 (c 0.1, CHCl3). (1R,2S)-Ethyl 1-(Phenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7a4). 99.8% ee. 1 H 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 =

(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. Ethyl 1-(4-Methoxybenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (trans-6k). White crystal, mp 73.5−73.8 °C. 1H 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-(3,4,5trimethoxyphenyl)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-(2-Fluorophenethylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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-(3,4,5trimethoxyphenyl)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. Ethyl 1-(3-Fluorophenethylcarbamoyl)-2-(3,4,5trimethoxyphenyl)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, 568, 522, 417. 1H 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. Ethyl 1-(3-Fluorophenethylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (cis-6m). White solid, mp 146.9−147.2 °C. 1H NMR (DMSO-d6, 300 MHz): δ 695



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= 5.1 Hz, 3H). HRMS (EI+) calcd C24H29NO6 (M+) 427.1995, found 427.2007. [α]22D +46.6 (c 0.1, CHCl3). (1R,2S)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7i4). 99.7% ee. 1 H NMR (DMSO-d6, 300 MHz): δ 8.413−8.374 (t, J = 5.7 Hz, 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, found 427.2001. [α]22D −38.6 (c 0.1, CHCl3). (1S,2R)-1-(Hydroxymethyl)-N-phenyl-2-(3,4,5trimethoxyphenyl)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 C20H23NO5 (M+) 357.1576, found 357.1579. [α]22D +57.8 (c 0.1, CHCl3). (1R,2S)-1-(Hydroxymethyl)-N-phenyl-2-(3,4,5trimethoxyphenyl)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]22D −75.1 (c 0.1, CHCl3). (1R,2R)-1-(Hydroxymethyl)-N-phenyl-2-(3,4,5trimethoxyphenyl)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]22D +25.5 (c 0.1, CHCl3). (1S,2S)-1-(Hydroxymethyl)-N-phenyl-2-(3,4,5trimethoxyphenyl)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+) calcd C20H23NO5 (M+) 357.1576, found 357.1580. [α]22D −28.5 (c 0.1, CHCl3). (1S,2R)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5trimethoxyphenyl)cyclopropyl)benzamide (8c1). White solid, mp 161.2−161.6 °C. 1H NMR (DMSO-d6, 300 MHz): δ 10.323 (s, 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 391.1189. [α]22D +85.5 (c 0.1, CHCl3).

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 (M+) 399.1682, found 399.1680. [α]22D −25.4 (c 0.1, CHCl3). (1R,2R)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7c1). 99.0% ee. 1 H NMR (DMSO-d6, 300 MHz): δ 10.838 (s, 1H), 8.246−8.218 (d, 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). (1S,2S)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7c2). 99.9% ee. 1 H 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 C22H24ClNO6 (M+) 433.1306, found 433.1315. [α]22D −57.0 (c 0.1, CHCl3). (1S,2R)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7c3). 99.7% ee. 1 H 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+) 433.1306, found 433.1298. [α]22D +44.0 (c 0.1, CHCl3). (1R,2S)-Ethyl 1-(2-Chlorophenylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7c4). 100% ee. 1 H 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]22D −39.5 (c 0.1, CHCl3). (1R,2R)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7i1). 100% ee. 1H 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]22D +16.8 (c 0.1, CHCl3). (1S,1S)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7i2). 99.7% ee. 1 H NMR (DMSO-d6, 300 MHz): δ 8.656−8.616 (t, J = 6.0 Hz, 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+) 427.1995, found 427.1998. [α]22D −21.3 (c 0.1, CHCl3). (1S,2R)-Ethyl 1-(4-Methylbenzylcarbamoyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxylate (7i3). 99.9% ee. 1 H 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 696



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(1R,2S)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5trimethoxyphenyl)cyclopropyl)benzamide (8c2). White solid, mp 161.2−161.9 °C. 1H NMR (DMSO-d6, 300 MHz): δ 10.331 (s, 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 C20H22ClNO5 (M+) 391.1187, found 391.1185. [α]22D −101.5 (c 0.1, CHCl3). (1R,2R)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5trimethoxyphenyl)cyclopropyl)benzamide (8c3). White solid, mp 116.4−117.0 °C. 1H 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+) 391.1187, found 391.1192. [α]22D +73.2 (c 0.1, CHCl3). (1S,2S)-2-Chloro-N-(1-(hydroxymethyl)-2-(3,4,5trimethoxyphenyl)cyclopropyl)benzamide (8c4). White solid, mp 116.3−117.2 °C. 1H 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]22D −85.1 (c 0.1, CHCl3). (1S,2R)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5trimethoxyphenyl)cyclopropanecarboxamide (8i1). White solid, mp 114.5−115.3 °C. 1H 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-(3,4,5trimethoxyphenyl)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]22D −69.5 (c 0.1, CHCl3). (1R,2R)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5trimethoxyphenyl)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),

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 C22H27NO5 (M+) 385.1889, found 385.1907. [α]22D +119.5 (c 0.1, CHCl3). (1S,2S)-1-(Hydroxymethyl)-N-(4-methylbenzyl)-2-(3,4,5trimethoxyphenyl)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 C22H27NO5 (M+) 385.1889, found 385.1890. [α]22D −129.5 (c 0.1, 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 paclitaxelresistance 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-ethanesulfonic 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 spectrophotometer 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 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 697



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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. Laser Fluorescence Confocal Microscopy. For laser fluorescence 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. 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). Fiveweek-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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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, profl[email protected]. Notes

The authors declare no competing financial interest.

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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, Shanghai, China) for the assistance with laser fluorescence confocal microscopy mitotic arrest experiments.

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ABBREVIATIONS USED CA-4, combretastatin-A4; TBA, tubulin-binding agent; TMSOI, trimethylsulfoxonium iodide; SAR, structure−activity relationship; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5-Fu, 5-fluorouracil; SD, standard error; DMSO, dimethyl sulfoxide; DAPI, diamidinophenylindole; FITC, fluorescein isothiocyanate; PI, propidium iodide; EDTA, ethylenediamintetraacetic acid; PME, paraformaldehyde; PBS, phosphate buffered solution; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

General chiral resolution assay, determination of configuration, and he binding mode of compound 8c4 with tubulin. This material is available free of charge via the Internet at http:// pubs.acs.org. 698



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