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A library of 1,2,3-triazole-substituted oleanolic acid derivatives as anticancer agents: design, synthesis, and biological evaluation 5
Gaofei Wei, Shuai Wang, Weijing Luan, Shanshan Cui, Fengran Li, Yongxiang Liu, Yang Liu* and Maosheng Cheng* Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x
A series of novel oleanolic acid coupled 1,2,3-triazole derivatives have been designed and synthesized by employing a Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction. 10 The anti-proliferative evaluation indicated that some compounds exhibited excellent anticancer activity against the examined cancer cell lines. In all derivatives, compound 3t, possesses the best inhibitory activity against HT1080 cells. A series of pharmacology experiments show that compound 3t significantly induced HT1080 cell apoptosis. Therefore, this compound can serve as a promising lead candidate for further study.
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Introduction Cancer, a multi-factorial disease characterized by uncontrolled growth of cells in the body, is a leading cause of deaths worldwide after cardiovascular diseases.1 Although, several anticancer drugs are used clinically, they fail to achieve the desired therapeutic effect because of multi-drug resistance, toxicity or poor bioavailability. Therefore, it is clinically necessary to identify and develop more effective and safe anticancer drugs. Naturally occurring compounds are an important source of potential drug substances with multifaceted effects and targets for cancer therapy. In this study we focused our attention towards oleanolic acid (OA, 1). OA is a natural pentacyclic triterpenoid compound that is synthesized in many plants by the cyclization of squalene.2 OA also exhibits highly potent anti-inflammatory, antitumor, anti-HIV, cardiovascular, and glycogen phosphorylase inhibitory activities.3 Because of its favorable properties, OA was used as a base molecule for further synthetic modifications to its three “active” portions: the C3 hydroxy, the C12-C13 double bond, and the C-28 carboxylic acid.4-7 Through the efforts of many medicinal chemists, over 300 new derivatives of OA have been synthesized.8 Compared to the parent OA, most synthesized analogues demonstrate increased anticancer activity. A synthetically modified OA derivative CDDO-Me (Fig. 1), has successfully completed a Phase I clinical trial for cancer treatment.9 Mechanistic studies have revealed that OA can inhibit proliferation, induce apoptosis and suppress inflammation to
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prevent and treat cancer. (1) OA can inhibit the proliferation of malignant cells involved in several cell-cycle proteins, including cyclin D1, p21, p27, PCNA, caveolin 1, and MYC.10-14 (2) OA can selectively induce the apoptosis of human cancer cell lines through both the extrinsic death receptor-mediated and the intrinsic mitochondrial dependent pathways.15-17 (3) OA can suppress the induction of both iNOS and COX2 in primary macrophages stimulated with various pro-inflammatory molecules.18, 19
Figure 1. Structures of oleanolic acid and CDDO-Me
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According to previous reports, polar substituents at the C-28 position of OA can enhance its anticancer potential. Herein, we describe the preparation of a library of novel OA derivatives with a coupled 1,2,3-triazole moiety that is appended to the C28 carboxylic of OA. Using Huisgen [3+2] cycloaddition between a terminal alkyne and an azide, a series of novel regioselective 1,2,3-triazole OA derivatives have been synthesized and screened for anticancer activity against a panel of human cancer cell lines. The IC50 values indicated that the compounds retained their anticancer activity with improved cytotoxicity compared to the parent compound OA. The synthesis and anticancer activity of [journal], [year], [vol], 00–00 | 1
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the synthesized compounds are presented in this paper.
Results and discussion
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Chemistry
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As illustrated in Scheme 1, naturally abundant oleanolic acid was treated with propargyl bromide, potassium carbonate solution, and TBAB in dry CH2Cl2 to fabricate a good yield of 2. Aromatic azides were prepared from their corresponding anilines by diazotization with sodium nitrite in acidic conditions followed by displacement with sodium azide. Huisgen [3+2] cycloaddition of 2 with aromatic azides in the presence of copper ion and sodium ascorbate in a ter-butyl alcohol aqueous solution resulted in the formation of 1,4 substituted-triazolyl derivatives.
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Compd
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In vitro anticancer activity The oleanolic acid-coupled 1,2,3-triazole moiety derivatives were screened for their in vitro anticancer activities against five human cancer cell lines: cervical carcinoma HeLa cells, hepatocellular carcinoma HepG2 cells, colon carcinoma HCT116 cells, melanoma A375-S2 cells, and fibrosarcoma HT1080 cells. The MTT analysis is summarized in Table 1. The IC50 values revealed that most of the conjugates exhibited more potent inhibitory activities against the five cancer cell lines than oleanolic acid. Compounds 3t, 3w and 3i, with p-NO2, p-CN and p-F substitutions at an aromatic ring, were the most promising. From the IC50 values, we concluded the following: (a) Most of the conjugates exhibited more potent inhibitory activities against the five cancer cell lines than oleanolic acid. 3t possesses strong inhibitory activity against A375-S2 and HT1080 cells with IC50 values of 4.97 and 3.51 µM; (b) Compounds with p-substitutions at an aromatic ring (3i, 3k, 3n, 3q, 3t, and 3w) are more active than corresponding compounds without substitutions or substitutions at an ortho- or meta- position. However, compounds with p-Ac substitutions (3q) are less active than compounds with o-Ac substitutions (3o); and (c) Compounds with electron withdrawing groups at an aromatic ring are generally more active than compounds without substitutions or substitutions with an electron donating group at the identical position. Selective killing of cancer cells without affecting normal cell growth is an important characteristic in cancer chemotherapy. Therefore, the most promising compound 3t was evaluated for 2 | Journal Name, [year], [vol], 00–00
Table 1. IC50 Values (µM) of Synthetic OA Derivatives (3a-3y) against Human Cancer Cell Lines
Scheme 1. Synthesis of OA derivatives 3a-3y 15
possible cytotoxicity towards normal cells, human brain microvascular endothelial cells HBMEC, human liver cells L-O2, human colonic epithelial cells 841, human immortalized keratinocyte cells Hacat, and human bronchial epithelial cells BEAS-2B. The growth of all five cell lines were not significantly affected by 3t, suggesting that 3t selectively inhibit the growth of cancer cells (Table 2).
R
HeLa
HepG2
HCT116
A375S2
HT1080
3a
o-OMe
>200
>200
144.32
>200
>200
3b
m-OMe
>200
>200
87.92
>200
>200
3c
p-OMe
>200
>200
>200
>200
>200
3d
o-Me
131.26
111.49
178.04
>200
>200
3e
m-Me
>200
>200
>200
>200
>200
3f
p-OEt
>200
>200
>200
>200
>200
3g
o-F
109.13
117.38
102.35
>200
>200
3h
m-F
53.87
131.36
77.04
157.37
65.61 17.02
3i
p-F
23.27
32.02
36.11
13.88
3j
m-Cl
122.01
112.39
80.62
>200
>200
3k
p-Cl
75.04
87.64
72.26
65.05
59.57
3l
o-Br
148.36
>200
98.1
148.36
163.27
3m
m-Br
124.36
132.54
>200
138.45
134.73
3n
p-Br
86.42
81.6
76.68
81.34
86.89
3o
o-Ac
54.63
36.54
134.05
122.45
74.16
3p
m-Ac
124.12
97.44
42.31
133.76
135.26
3q
p-Ac
74.86
37.71
76.48
53.79
64.8
3r
o-NO2
81.3
88.74
95.77
89.3
79.61
3s
m-NO2
75.04
97.64
62.26
55.05
59.57
3t
p-NO2
10.85
24.15
12.28
4.97
3.51
3u
o-CN
96.47
123.54
117.45
107.53
93.93
3v
m-CN
109.02
117.73
109.35
83.87
91.36
3w
p-CN
34.31
44.17
38.65
22.45
17.14
166,43
146.95
89.69
151.93
73.52
>200
>200
>200
>200
>200
3y
oCOOMe H
OA
---
>200
>200
>200
>200
>200
5-Fu
---
26.18
67.64
35.16
90.74
25.46
3x
Table 2. IC50 Values (µM) of 3t Against Human Normal Cell Lines Compd
HBMEC
L-O2
841
Hacat
BEAS-2B
3t
>100
>100
>100
>100
>100
Cell morphology
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The effect of 3t on HT1080 cell morphology was determined. As shown in Fig. 2, cell morphology remained consistent when the cells were treated with vehicle. However, concentrations of 3t up to 6.2 µM resulted in spherical cells and decreased cell size. Only a small number of living cells can be observed at concentrations exceeding 20 µM. Analysis of apoptosis by Annexin V-FITC/PI An annexin V-FITC / PI binding assay was performed to
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determine whether the 3t induced anti-proliferative activity of the cancer cells was the result of apoptosis. HT1080 cells were treated with vehicle alone or with various concentrations (1.5, 3.0, and 6.0 µM) of compound 3t for 72 h and then stained with FITC-annexin V and propidium iodide (PI). The percentages of apoptotic HT1080 cells were determined by flow cytometry. As shown in Figure 3, conjugate 3t resulted in significant apoptosis in a concentration-dependent manner. When treated with 1.5, 3.0, and 6.0 µM of compound 3t for 72 h, the percentages of apoptotic cells were 12.60%, 35.67%, and 60.88% (Q2 + Q4), respectively. The vehicle control contained 7.29% apoptotic cells. This result demonstrated that the anti-proliferative activity observed when HT1080 cells were treated with 3t could be the result of apoptosis.
Figure 3. Effects of 3t on HT1080 Cells apoptosis
Fluorescence microscopy 20
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Apoptosis is characterized by chromatin condensation that results in compact small nuclei and/or the formation of apoptotic bodies. HT1080 cells were cultured with 3t, and the chromatin condensation and the formation of apoptotic bodies was verified using DAPI staining. Compact nuclei were evident, indicating chromatin condensation (Fig. 4).
Figure 2. Effects of 3t on HT1080 Cells morphology
Figure 4. Effects of 3t on HT1080 Cells nuclei
Changes in mitochondrial membrane potential
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In general, mitochondria are necessary mediators that play a vital role in the execution of both intrinsic and extrinsic apoptotic pathways. Disruption of the mitochondrial membrane potential (MMP) decrease is the symbolic event in the process of apoptosis. To estimate the role of mitochondria in 3t-induced HT1080 cell apoptosis, we explored mitochondrial membrane potential changes by flow cytometric analysis after JC-1 staining (Fig. 5). After treatment with 0 to 6.0 µM of compound 3t, flow cytometry studies revealed a concentration-dependent depression in MMP. This decrease was first detected after treatment with 1.5 µM and Journal Name, [year], [vol], 00–00 | 3
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was most notable with 6.0 µM. The loss of MMP indicated that 3t induces HT1080 apoptosis via a mitochondrial apoptotic deathsignal pathway.
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2489. Melting points were obtained on a Büchi melting point B540 apparatus. 1H and 13C NMR spectra were recorded on a Bruker ARX 600 MHz spectrometer. ESI-MS were obtained on Agilent ESI-QTOF instrument. HRMS were obtained on a Agilent 1260-G6530B Q_TOF. Synthesis of compound 2
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Figure 5. Effects of 3t on HT1080 Cells MMP 60
Conclusions
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A series of oleanolic acid coupled 1,2,3-triazole derivatives have been designed and synthesized by employing a Cu(I)catalyzed Huisgen 1,3-dipolar cycloaddition reaction of terminal alkyne derivatives of 2 with various aromatic azides. The synthesized compounds were screened for anticancer activity against a panel of five human cancer cell lines using an MTT assay. Some compounds exhibited better anti-cancer activity against the tested cancer cell lines compared to positive controls 5-fluorouracil and oleanolic acid. Compound 3t was the most promising derivative. Our biological evaluations suggest that compound 3t is a potent apoptosis inducer in HT1080 cells. Therefore, this compound can serve as a promising lead candidate for further study.
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Experimental
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Reagents were used without further purification unless otherwise specified. Solvents were dried and redistilled prior to use in the usual way. Analytical TLC was performed using silica gel HF254. Preparative column chromatography was performed with silica gel H. HPLC spectra were obtained on a Waters 15254 | Journal Name, [year], [vol], 00–00
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To a solution of 1 (2.0 g, 4.4 mmol) in DCM, potassium carbonate solution (1.8 g, 13.1 mmol/15ml), TBAB (290.0 mg, 0.9mmol) and propargyl bromide (1.0 g, 8.8 mmol) were added and the reaction mixture was stirred at room temperature for 5 hours. Reaction was monitored by TLC and the crude product was subjected to column chromatography to give pure 2 (1.93 g, 89.1% yield). General procedure for the synthesis of compounds 3a-3y To a solution of compound 2 (100.0 mg, 0.202 mmol) in tBuOH:H2O (2:1, 10ml), sodium ascorbate (32.0 mg, 0.162 mmol) and CuSO4.5H2O (20.2 mg, 0.08 mmol) were added at room temperature. To this mixture, aryl azide (1.0 mmol) was added and the reaction mixture was sonicated at 60 ºC still its completion. The crude mixture was extracted with DCM (3×20 ml) and the combined organic layer was dried over Na2SO4 and purified through column chromatography to offer pure 3a-3y. All analogues synthesized by us gave single sharp peaks on HPLC and they were judged at least 95% pure. HPLC Methods: Diamonsil C18(2) column 5u 250×4.6mm, wavelength 254nm, injection volume 20 µL, flow 1.0 mL/min; mobile phase MeOHH2O/95-5, injection time 20min. 3a White solid, 86.9 % yield; HPLC: Purity 95.79%, Rt 11.139 min; Mp 87.0 – 88.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.15 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.13 – 7.07 (m, 2H), 5.33 – 5.22 (m, 3H), 3.89 (s, 3H), 3.21 – 3.16 (m, 1H), 2.87 (d, J = 10.5 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.80 (s, 3H), 0.76 (s, 3H), 0.51 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.69, 150.98, 143.67, 142.57, 130.09, 126.28, 126.23, 125.42, 122.44, 121.19, 112.23, 78.96, 57.53, 55.96, 55.18, 47.56, 46.73, 45.87, 41.68, 41.35, 39.27, 38.74, 38.43, 37.00, 33.85, 33.07, 32.67, 32.39, 30.68, 28.10, 27.64, 27.18, 25.81, 23.63, 23.36, 22.96, 18.28, 16.76, 15.57, 15.23; ESI-MS(m/z): 1309.48 [2M+Na]+; HRMS (ESI): Calcd. for [M+ H]+ C40H58N3O4: 644.4422, found 644.4425. 3b Yellow solid, 80.7 % yield; HPLC: Purity 96.67%, Rt 12.336 min; Mp 86.5 – 88.2 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.40 (t, J = 8.2 Hz, 1H), 7.32 (t, J = 2.1 Hz, 1H), 7.24 (dd, J = 7.9, 1.7 Hz, 1H), 6.97 (dd, J = 8.3, 2.3 Hz, 1H), 5.31 – 5.21 (m, 3H), 3.88 (s, 3H), 3.18 (dd, J = 11.6, 3.8 Hz, 1H), 2.86 (dd, J = 13.8, 4.1 Hz, 1H), 1.09 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.81, 160.65, 143.76, 143.62, 137.97, 130.51, 122.67, 122.49, 114.64, 112.36, 106.40, 78.97, 57.34, 55.64, 55.17, 47.54, 46.75, 45.87, 41.68, 41.38, 39.27, 38.73, 38.41, 36.99, 33.83, 33.06, 32.64, 32.42, 30.67, 28.09, 27.63, 27.18, 25.76, 23.61, 23.35, 22.97, 18.25, 16.78, 15.53, 15.16; ESI-MS(m/z): 1309.49 [2M+Na]+; HRMS(ESI): Calcd. for [M+ H]+ C40H58N3O4: 644.4422, found 644.4425. 3c White solid, 81.9 % yield; HPLC: Purity 96.10%, Rt 11.723 min; Mp 173.4 – 174.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.94 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 5.31 – This journal is © The Royal Society of Chemistry [year]
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5.21 (m, 3H), 3.87 (s, 3H), 3.19 (s, 1H), 2.87 (d, J = 10.7 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.79 (s, 3H), 0.75 (s, 3H), 0.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.53, 160.04, 143.61, 130.92, 128.84, 122.65, 122.46, 122.13, 114.99, 114.75, 110.71, 78.96, 61.61, 57.41, 55.65, 55.16, 47.53, 46.74, 45.86, 41.67, 41.37, 39.27, 38.73, 38.41, 36.99, 33.82, 33.06, 32.64, 32.41, 30.67, 28.09, 27.63, 25.77, 23.61, 22.97, 18.26, 16.79, 15.55, 15.20, 14.11; ESI-MS(m/z): 1308.76 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O4: 644.4422, found 644.4423. 3d White solid, 74.1% yield; HPLC: Purity 95.41%, Rt 11.214 min; Mp 102.4 – 105.2 °C; 1H NMR (600 MHz, CDCl3) δ 7.78 (s, 1H), 7.45 – 7.40 (m, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 4.0 Hz, 2H), 5.29 (q, J = 12.8 Hz, 3H), 3.20 (dd, J = 11.4, 3.9 Hz, 1H), 2.87 (dd, J = 13.6, 3.9 Hz, 1H), 2.22 (s, 3H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H), 0.77 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.87, 155.79, 143.65, 143.05, 136.34, 133.49, 131.57, 129.93, 129.62, 126.85, 125.92, 125.61, 122.50, 120.57, 115.34, 79.08, 77.24, 77.03, 76.82, 57.48, 55.19, 47.57, 46.82, 45.88, 41.72, 41.33, 39.33, 38.75, 38.44, 37.02, 33.84, 33.06, 32.69, 32.43, 30.67, 28.11, 27.68, 27.16, 25.81, 23.60, 23.38, 22.99, 18.29, 17.90, 17.00, 15.58, 15.27; ESI-MS(m/z): 1277.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O3: 628.4473, found 628.4491. 3e White solid, 79.2 % yield; HPLC: Purity 96.55%, Rt 13.418 min; Mp 106.1 – 108.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.54 (s, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.25 (d, J = 10.5 Hz, 1H), 5.26 (q, J = 12.2 Hz, 3H), 3.19 (d, J = 9.7 Hz, 1H), 2.86 (d, J = 11.3 Hz, 1H), 2.44 (s, 3H), 1.09 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.83, 143.63, 143.59, 139.96, 136.87, 129.57, 129.50, 124.19, 122.75, 122.46, 121.22, 117.61, 78.94, 57.33, 55.14, 47.51, 46.74, 45.85, 41.66, 41.36, 39.25, 38.71, 38.40, 36.97, 33.82, 33.05, 32.62, 32.42, 32.39, 30.66, 28.08, 27.60, 27.13, 25.75, 23.60, 23.33, 22.95, 21.38, 18.23, 16.75, 15.54, 15.16; ESI-MS(m/z): 1277.52 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O3: 628.4473, found 628.4478. 3f Yellow solid, 81.2 % yield; HPLC: Purity 96.21%, Rt 13.527 min; Mp 98.4 – 100.3 °C; 1H NMR (600 MHz, CDCl3) δ 7.93 (s, 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 5.32 – 5.19 (m, 3H), 4.08 (q, J = 7.0 Hz, 2H), 3.22 – 3.15 (m, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.79 (s, 3H), 0.75 (s, 3H), 0.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.81, 159.28, 143.64, 143.59, 130.27, 122.66, 122.47, 122.13, 115.27, 78.98, 63.96, 57.43, 55.17, 47.55, 46.74, 45.88, 41.68, 41.38, 39.28, 38.73, 38.42, 37.00, 33.83, 33.07, 32.64, 32.42, 30.67, 28.09, 27.63, 27.18, 25.77, 23.62, 23.36, 22.98, 22.66, 18.26, 16.80, 15.55, 15.21, 14.72, 14.12; ESI-MS(m/z): 1337.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H60N3O4: 658.4578, found 658.4585. 3g White solid, 83.9 % yield; HPLC: Purity 95.96%, Rt 13.505 min; Mp 66.4 – 68.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.12 (d, J = 2.7 Hz, 1H), 7.96 (t, J = 7.1 Hz, 1H), 7.44 (dd, J = 12.2, 7.0 Hz, 1H), 7.37 – 7.28 (m, 2H), 5.41 – 5.18 (m, 3H), 3.19 (dd, J = 11.5, 4.1 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.80 (s, 3H), 0.75 (s, 3H), 0.50 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.67, 153.30 (d, J = 249.0 This journal is © The Royal Society of Chemistry [year]
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Hz), 143.63, 143.60, 130.24 (d, J = 7.5 Hz), 128.86, 125.28 (d, J = 19.5 Hz), 125.27 (d, J = 7.5 Hz), 124.84, 122.53, 117.07 (d, J = 21.0 Hz), 78.99, 57.32, 55.18, 47.56, 46.77, 45.86, 41.68, 41.36, 39.28, 38.74, 38.43, 37.00, 33.83, 33.07, 32.65, 32.40, 30.67, 28.10, 27.63, 27.18, 25.80, 23.61, 23.34, 22.98, 18.27, 16.70, 15.56, 15.18, 14.11; ESI-MS(m/z): 1285.27 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55FN3O3: 632.4222, found 632.4224. 3h White solid, 84.1 % yield; HPLC: Purity 96.58%, Rt 11.972 min; Mp 101.3 – 103.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.61 – 7.44 (m, 3H), 7.21 – 7.09 (m, 1H), 5.33 – 5.10 (m, 3H), 3.19 (dd, J = 11.5, 4.1 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.45 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 177.84, 163.12 (d, J = 247.5 Hz), 144.07, 143.57, 138.10 (d, J = 10.5 Hz), 131.20 (d, J = 9.0 Hz), 122.59, 122.52, 115.77, 115.73 (d, J = 18.0 Hz), 108.34 (d, J = 25.5 Hz), 78.97, 57.24, 55.16, 47.52, 46.77, 45.84, 41.68, 41.39, 39.28, 38.72, 38.41, 36.98, 33.82, 33.06, 32.63, 32.43, 30.67, 28.08, 27.62, 27.17, 25.76, 23.61, 23.34, 22.97, 18.23, 16.78, 15.51, 15.13; ESI-MS(m/z): 1285.44 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55FN3 O3: 632.4222, found 632.4228. 3i White solid, 81.90 % yield; HPLC: Purity 98.36%, Rt 11.120 min; Mp 173.4 – 174.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.98 (s, 1H), 7.74 – 7.66 (m, 2H), 7.25 – 7.19 (m, 2H), 5.31 – 5.22 (m, 3H), 3.22 – 3.15 (m, 1H), 2.86 (dd, J = 13.8, 4.2 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H), 0.46 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 177.85, 162.49 (d, J = 246.0 Hz), 143.96, 143.59, 133.23, 128.86, 122.72, 122.48 (d, J = 7.5 Hz), 116.72 (d, J = 24.0 Hz), 78.96, 57.31, 55.16, 47.52, 46.77, 45.84, 41.69, 41.39, 39.29, 38.73, 38.42, 36.99, 33.82, 33.06, 32.64, 32.43, 30.67, 28.09, 27.63, 27.18, 26.92, 25.77, 23.61, 23.35, 22.97, 18.25, 16.77, 15.54, 15.18; ESI-MS(m/z): 1284.65 [2M+Na]+; HRMS(ESI): Calcd. for [M+ H]+ C39H55FN3O3: 632.4222, found 632.4226. 3j Yellow solid, 84.3 % yield; HPLC: Purity 96.05%, Rt 14.310 min; Mp 88.0 – 90.6 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.77 (t, J = 1.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.49 – 7.39 (m, 2H), 5.31 – 5.19 (m, 3H), 3.18 (dd, J = 11.5, 4.0 Hz, 1H), 2.86 (dd, J = 13.8, 4.2 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.42 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.08, 143.58, 137.79, 135.66, 130.83, 128.90, 122.69, 122.52, 120.87, 118.47, 78.98, 57.22, 55.17, 47.51, 46.77, 45.85, 41.68, 41.39, 39.28, 38.73, 38.41, 36.99, 33.83, 33.06, 32.64, 32.44, 30.67, 28.09, 27.62, 27.18, 25.74, 23.60, 23.34, 22.97, 18.23, 16.79, 15.52, 15.18; ESI-MS(m/z): 1317.57 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55ClN3O3: 648.3926, found 648.3925. 3k White solid, 82.1 % yield; HPLC: Purity 95.07%, Rt 13.892 min; Mp 203.3 – 205.5 °C; 1H NMR (600 MHz, CDCl3) δ 8.00 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 5.29 – 5.21 (m, 3H), 3.21 – 3.16 (m, 1H), 2.86 (d, J = 10.6 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.75 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.06, 143.57, 135.44, 134.67, 130.93, 129.93, 122.51, 121.64, 78.95, 61.62, 57.27, 55.16, 47.51, 46.76, 45.84, 41.68, 41.38, 39.28, 38.73, 38.41, 36.98, 33.81, 33.06, 32.63, 32.42, 30.67, 28.09, 27.62, 27.17, 25.76, 23.61, 23.34, 22.97, 18.25, 16.77,
Journal Name, [year], [vol], 00–00 | 5
Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/C4OB01605J
Organic & Biomolecular Chemistry
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15.55, 15.17, 14.11; ESI-MS(m/z): 1318.48 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55ClN3 O3: 648.3926, found 648.3929. 3l White solid, 89.45 % yield; HPLC: Purity 98.44%, Rt 12.259 min; Mp 59.6 – 61.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.00 (s, 1H), 7.76 (dd, J = 8.0, 0.9 Hz, 1H), 7.56 (dd, J = 7.9, 1.4 Hz, 1H), 7.49 (dd, J = 11.0, 4.3 Hz, 1H), 7.40 (td, J = 7.9, 1.5 Hz, 1H), 5.35 – 5.24 (m, 1H), 3.20 (dd, J = 11.4, 4.3 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.70, 143.66, 142.97, 136.45, 134.01, 131.19, 128.46, 128.16, 126.13, 122.49, 118.37, 78.98, 57.42, 55.20, 47.58, 46.79, 45.88, 41.71, 41.32, 39.34, 38.75, 38.44, 37.03, 33.85, 33.06, 32.70, 32.42, 30.67, 28.11, 27.68, 27.19, 25.82, 23.61, 23.39, 23.00, 18.31, 16.98, 15.59, 15.30; ESIMS(m/z): 1406.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3O3: 692.3421, found 692.3419. 3m White solid, 75.74 % yield; HPLC: Purity 95.62%, Rt 15.092 min; Mp 119.6 – 201.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.02 (s, 1H), 7.92 (t, J = 1.8 Hz, 1H), 7.67 (dd, J = 8.1, 1.2 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 5.31 – 5.20 (m, 3H), 3.18 (dd, J = 11.5, 4.1 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H), 0.42 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.86, 144.07, 143.57, 137.86, 131.85, 131.06, 123.69, 123.35, 122.71, 122.51, 118.97, 78.98, 57.21, 55.17, 47.51, 46.77, 45.86, 41.68, 41.39, 39.28, 38.73, 38.42, 36.99, 33.83, 33.05, 32.64, 32.44, 30.67, 28.09, 27.61, 27.18, 26.93, 25.74, 23.60, 23.34, 22.97, 18.24, 16.78, 15.55, 15.23, 14.12; ESI-MS(m/z): 1406.54 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3 O3: 692.3421, found 692.3420. 3n White solid, 86.44 % yield; HPLC: Purity 97.14%, Rt 14.889 min; Mp 194.9 – 196.5 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 5.32 – 5.20 (m, 1H), 3.19 (d, J = 10.9 Hz, 1H), 2.86 (dd, J = 13.8, 4.0 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.75 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.10, 143.58, 135.93, 132.91, 122.51, 122.45, 121.87, 78.97, 57.27, 55.16, 47.51, 46.77, 45.84, 41.68, 41.38, 39.28, 38.73, 38.41, 36.99, 33.81, 33.05, 32.63, 32.42, 30.67, 28.09, 27.62, 27.18, 25.76, 23.60, 23.35, 22.97, 18.25, 16.78, 15.55, 15.17; ESI-MS(m/z): 1406.49 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3O3: 692.3421, found 692.3419. 3o White solid, 71.94 % yield; HPLC: Purity 98.18%, Rt 8.032 min; Mp 66.1 – 68.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.90 (s, 1H), 7.71 (dd, J = 7.5, 1.6 Hz, 1H), 7.64 – 7.58 (m, 2H), 7.45 (dd, J = 7.7, 1.3 Hz, 1H), 5.29 – 5.25 (m, 3H), 3.19 (dd, J = 11.4, 4.4 Hz, 1H), 2.85 (dd, J = 13.6, 4.5 Hz, 1H), 2.17 (s, 3H), 1.11 (s, 3H), 0.96 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.75 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 199.19, 177.64, 143.74, 143.55, 136.15, 134.22, 131.79, 129.90, 129.00, 125.52, 125.47, 122.40, 78.88, 60.32, 57.25, 55.08, 47.47, 46.68, 45.75, 41.61, 41.23, 39.22, 38.65, 38.33, 36.92, 33.72, 32.96, 32.55, 30.57, 29.08, 28.00, 27.57, 27.07, 25.72, 23.51, 23.28, 22.88, 18.19, 16.86, 15.49, 15.18; ESI-MS(m/z): 1332.56 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O4: 656.4422, found 656.4449. 3p White solid, 79.63 % yield; HPLC: Purity 96.86%, Rt
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10.065 min; Mp 137.6 – 139.4 °C; 1H NMR (600 MHz, CDCl3) δ 8.28 (s, 1H), 8.11 (s, 1H), 8.04 – 7.97 (m, 2H), 7.65 (t, J = 7.9 Hz, 1H), 5.32 – 5.21 (m, 3H), 3.18 (dd, J = 11.4, 3.8 Hz, 1H), 2.87 (dd, J = 13.7, 4.0 Hz, 1H), 2.67 (s, 3H), 1.10 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.72 (s, 3H), 0.45 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 196.43, 177.79, 144.14, 143.52, 138.56, 137.34, 130.20, 128.45, 124.65, 122.59, 122.52, 119.71, 78.92, 57.25, 55.14, 47.50, 46.76, 45.83, 41.67, 41.38, 39.27, 38.71, 38.39, 36.97, 33.81, 33.05, 32.63, 32.42, 30.66, 28.08, 27.62, 27.16, 26.72, 25.75, 23.61, 23.33, 22.97, 18.23, 16.75, 15.53, 15.15; ESI-MS(m/z): 1332.75 [2M+Na]+; HRMS(ESI): － Calcd. for [M+COOH] C42H58N3O6: 700.4331, found 700.4449. 3q White solid, 71.72 % yield; HPLC: Purity 97.60%, Rt 10.573 min; Mp 142.4 – 145.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.15 – 8.08 (m, 3H), 7.86 (d, J = 8.6 Hz, 2H), 5.30 – 5.23 (m, 3H), 3.18 (dd, J = 11.6, 4.0 Hz, 1H), 2.86 (dd, J = 13.8, 4.1 Hz, 1H), 2.65 (s, 3H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.73 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 196.44, 177.85, 144.26, 143.56, 139.96, 136.94, 130.06, 122.51, 120.04, 78.94, 57.25, 57.22, 55.13, 47.50, 46.78, 45.83, 41.68, 41.39, 39.27, 38.72, 38.39, 36.97, 33.80, 33.05, 32.62, 32.42, 30.67, 28.07, 27.63, 27.16, 26.67, 25.76, 23.60, 23.34, 22.97, 18.23, 16.76, 15.54, 15.16; ESI-MS(m/z): 1332.76 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O4: 656.4422, found 656.4421. 3r Yellow solid, 83.02 % yield; HPLC: Purity 95.95%, Rt 8.016 min; Mp 57.1 – 59.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 5.32 – 5.23 (m, 3H), 3.20 (dd, J = 11.3, 4.2 Hz, 1H), 2.86 (dd, J = 13.7, 3.9 Hz, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.76 (s, 3H), 0.58 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.77, 144.41, 143.81, 143.65, 133.72, 130.83, 130.16, 127.87, 125.70, 125.64, 122.49, 78.97, 57.23, 55.18, 47.57, 46.80, 45.88, 41.72, 41.33, 39.34, 38.75, 38.43, 37.03, 33.82, 33.06, 32.65, 32.35, 30.67, 28.10, 27.67, 27.18, 25.81, 23.61, 23.37, 23.00, 18.30, 16.93, 15.59, 15.28; ESI-MS(m/z): 1338.69 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4074. 3s Yellow solid, 78.70 % yield; HPLC: Purity 97.20%, Rt 10.763 min; Mp 75.4 – 78.1 °C; 1H NMR (600 MHz, CDCl3) δ 8.58 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 9.5 Hz, 2H), 7.76 (t, J = 8.1 Hz, 1H), 5.33 – 5.22 (m, 3H), 3.18 (dd, J = 11.1, 3.8 Hz, 1H), 2.87 (d, J = 10.4 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.72 (s, 3H), 0.45 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 177.87, 148.96, 143.50, 137.64, 130.98, 128.84, 125.89, 123.27, 122.67, 122.55, 115.29, 78.92, 61.61, 57.15, 55.13, 47.48, 46.80, 45.81, 41.69, 41.39, 39.29, 38.71, 38.39, 36.96, 33.80, 33.04, 32.63, 32.43, 30.66, 28.07, 27.62, 27.15, 25.75, 23.59, 23.33, 22.97, 18.22, 16.76, 15.51, 15.11, 14.11; ESI-MS(m/z): 1338.67 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4073. 3t Yellow solid, 75.42 % yield; HPLC: Purity 97.24%, Rt 11.263 min; Mp 218.4 – 200.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.42 (d, J = 9.0 Hz, 2H), 8.14 (s, 1H), 7.96 (d, J = 9.0 Hz, 2H), 5.34 – 5.23 (m, 3H), 3.18 (d, J = 10.9 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.76 (s, 3H), 0.73 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz,
This journal is © The Royal Society of Chemistry [year]
Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/C4OB01605J
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CDCl3) δ 177.91, 147.33, 144.72, 143.53, 141.07, 125.53, 122.55, 120.48, 78.94, 57.11, 55.14, 47.49, 46.82, 45.80, 41.70, 41.41, 39.29, 38.72, 38.40, 36.98, 33.79, 33.04, 32.63, 32.43, 30.67, 28.07, 27.63, 27.16, 25.77, 23.60, 23.34, 22.98, 18.25, 16.75, 15.54, 15.17; ESI-MS(m/z): 1338.48 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4054. 3u White solid, 83.47 % yield; HPLC: Purity 97.43%, Rt 8.221 min; Mp 52.4 – 55.6 °C; 1H NMR (600 MHz, CDCl3) δ 8.26 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.80 (t, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 5.34 – 5.26 (m, 3H), 3.20 (d, J = 11.2 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.81 (s, 3H), 0.76 (s, 3H), 0.53 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.66, 144.15, 143.59, 138.44, 134.40, 134.23, 129.59, 125.38, 124.56, 122.53, 115.51, 106.68, 78.97, 57.22, 55.17, 47.56, 46.80, 45.86, 41.69, 41.35, 39.32, 38.74, 38.42, 37.01, 33.81, 33.07, 32.65, 32.39, 30.67, 28.10, 27.66, 27.18, 25.81, 23.62, 23.36, 22.98, 18.30, 16.82, 15.58, 15.25, 13.70; ESI-MS(m/z): 1298.71 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O3: 639.4269, found 639.4267. 3v White solid, 79.26 % yield; HPLC: Purity 96.55%, Rt 9.590 min; Mp 58.0 – 60.1 °C; 1H NMR (600 MHz, CDCl3) δ 8.08 (s, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 5.30 – 5.22 (m, 3H), 3.18 (dd, J = 11.3, 3.6 Hz, 1H), 2.86 (dd, J = 13.7, 4.0 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.43 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.86, 144.50, 143.52, 137.47, 132.11, 130.90, 124.36, 123.62, 122.54, 122.50, 117.25, 114.27, 78.93, 57.14, 55.15, 47.48, 46.79, 45.81, 41.68, 41.39, 39.29, 38.72, 38.40, 36.98, 33.80, 33.04, 32.63, 32.43, 30.66, 28.08, 27.61, 27.16, 25.75, 23.60, 23.34, 22.97, 18.24, 16.79, 15.55, 15.20; ESI-MS(m/z): 1298.79 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C41H55N4O5: 683.4178, found 683.4170. 3w White solid, 76.05 % yield; HPLC: Purity 98.84%, Rt 9.530 min; Mp 196.5 – 198.8 °C; 1H NMR (600 MHz, CDCl3) δ 8.10 (s, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 5.31 – 5.23 (m, 3H), 3.22 – 3.15 (m, 1H), 2.89 – 2.82 (m, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.76 (s, 3H), 0.75 (s, 3H), 0.43 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.91, 144.57, 143.52, 139.72, 133.92, 122.54, 122.40, 120.55, 117.61, 112.57, 78.94, 57.12, 55.14, 47.49, 46.80, 45.80, 41.69, 41.40, 39.28, 38.72, 38.41, 36.98, 33.79, 33.04, 32.62, 32.43, 30.66, 28.08, 27.62, 27.16, 25.76, 23.59, 23.34, 22.97, 18.24, 16.74, 15.56, 15.17; ESI-MS(m/z): 1298.57 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H55N4O5: 683.4178, found 683.4182. 3x White solid, 67.1 % yield; HPLC: Purity 97.01%, Rt 8.172 min; Mp 65.1 – 68.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 5.32 – 5.24 (m, 3H), 3.72 (s, 3H), 3.20 (dd, J = 11.4, 4.3 Hz, 1H), 2.89 – 2.85 (m, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.60 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.71, 165.48, 143.67, 143.01, 136.10, 132.64, 131.31, 129.91, 127.39, 126.68, 125.90, 122.49, 78.98, 61.62, 57.48, 55.19, 52.56, 47.58, 46.76, 45.88, 41.72, 41.35, 39.34, 38.75, 38.44, 37.03, 33.85, 33.07, 32.68, 32.37, 30.67, 28.10, 27.69, 27.19, 25.82, 23.62, 23.01, 18.30, 16.97, 15.58, 15.28, 14.11; ESI-MS(m/z): 1364.75 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O5: 672.4371, found 672.4370.
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Cell viability assay
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Cell viability was evaluated using an MTT assay. HT1080 cells were seeded into 96-well plates at a density of 5 × 104 cells per well and stabilized at 37 °C for 24 h. Compounds 3a-3y were added to each well at various concentrations, and then the cells were incubated for 72 h. The MTT solution (20 µL 5 mg/mL) was added to each well, and the cells were incubated for another 4 h. Formazan crystals were dissolved in 150 µL of DMSO. Cell viability was assessed by measuring the absorbance at 540 nm wavelength using a microplate reader (BioTek ELx800 USA). Annexin V/PI staining assay
85
HT1080 cells were treated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. Then, cells were collected, washed with annexin-binding buffer, and stained with annexin V fluorescein isothiocyanate (FITC) and PI for 15 min at RT (25 °C). After that, the samples were analyzed by flow cytometry (BD FACSCalibur, USA). DAPI staining assay
90
95
HT1080 cells were treated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. The cells were collected, resuspended, and fixed in 4% paraformaldehyde in PBS. After PBS washing, cells were stained with DAPI for 30 min followed by another PBS wash. The stained cells were examined under a fluorescent microscope (IX51, OLYMPUS, Japan). Mitochondrial membrane potential assay
100
The mitochondrial membrane potential was assessed using JC1 dye. HT1080 cells were plated at 1 × 106 cells per well in 24well plates and incubated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. The cells were subsequently incubated with JC-1 dye and finally, analyzed by flow cytometry (BD FACSCalibur, USA).
Acknowledgments 105
The authors wish to express their thanks for the financial support received from the National Natural Science Foundation of China (No. 81273358), the Program for Innovative Research Team of the Ministry of Education, and the Program for Liaoning Innovative Research Team in University.
Notes 110
This journal is © The Royal Society of Chemistry [year]
3y White solid, 83.03 % yield; HPLC: Purity 97.02%, Rt 11.248 min; Mp 79.8 – 82.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.53 (t, J = 7.7 Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H), 5.38 – 5.09 (m, 3H), 3.19 (d, J = 11.2 Hz, 1H), 2.94 – 2.77 (m, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.46 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.68, 143.71, 143.27, 133.83, 133.13, 131.80, 128.90, 126.11, 122.49, 79.01, 57.38, 55.23, 47.63, 46.81, 45.92, 41.75, 41.27, 39.36, 38.76, 38.46, 37.04, 33.86, 33.04, 32.70, 32.30, 30.66, 28.11, 27.71, 27.21, 26.93, 25.83, 23.58, 23.41, 23.05, 18.31, 17.06, 15.58, 15.32; ESI-MS(m/z): 1249.40 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H56N3O3: 614.4316, found 614.4314.
a
Key Laboratory of Structure-Based Drugs Design & Discovery of
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Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China. E-mail: [email protected]; Tel: +86 24 23986413 (Yang Liu) E-mail: [email protected]; Tel: +86 24 23986419 (Maosheng Cheng) † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/
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A library of 1,2,3-triazole-substituted oleanolic acid derivatives as anticancer agents: design, synthesis, and biological evaluation 5
Gaofei Wei, Shuai Wang, Weijing Luan, Shanshan Cui, Fengran Li, Yongxiang Liu, Yang Liu* and Maosheng Cheng* Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x
A series of novel oleanolic acid coupled 1,2,3-triazole derivatives have been designed and synthesized by employing a Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction. 10 The anti-proliferative evaluation indicated that some compounds exhibited excellent anticancer activity against the examined cancer cell lines. In all derivatives, compound 3t, possesses the best inhibitory activity against HT1080 cells. A series of pharmacology experiments show that compound 3t significantly induced HT1080 cell apoptosis. Therefore, this compound can serve as a promising lead candidate for further study.
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Introduction Cancer, a multi-factorial disease characterized by uncontrolled growth of cells in the body, is a leading cause of deaths worldwide after cardiovascular diseases.1 Although, several anticancer drugs are used clinically, they fail to achieve the desired therapeutic effect because of multi-drug resistance, toxicity or poor bioavailability. Therefore, it is clinically necessary to identify and develop more effective and safe anticancer drugs. Naturally occurring compounds are an important source of potential drug substances with multifaceted effects and targets for cancer therapy. In this study we focused our attention towards oleanolic acid (OA, 1). OA is a natural pentacyclic triterpenoid compound that is synthesized in many plants by the cyclization of squalene.2 OA also exhibits highly potent anti-inflammatory, antitumor, anti-HIV, cardiovascular, and glycogen phosphorylase inhibitory activities.3 Because of its favorable properties, OA was used as a base molecule for further synthetic modifications to its three “active” portions: the C3 hydroxy, the C12-C13 double bond, and the C-28 carboxylic acid.4-7 Through the efforts of many medicinal chemists, over 300 new derivatives of OA have been synthesized.8 Compared to the parent OA, most synthesized analogues demonstrate increased anticancer activity. A synthetically modified OA derivative CDDO-Me (Fig. 1), has successfully completed a Phase I clinical trial for cancer treatment.9 Mechanistic studies have revealed that OA can inhibit proliferation, induce apoptosis and suppress inflammation to
This journal is © The Royal Society of Chemistry [year]
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prevent and treat cancer. (1) OA can inhibit the proliferation of malignant cells involved in several cell-cycle proteins, including cyclin D1, p21, p27, PCNA, caveolin 1, and MYC.10-14 (2) OA can selectively induce the apoptosis of human cancer cell lines through both the extrinsic death receptor-mediated and the intrinsic mitochondrial dependent pathways.15-17 (3) OA can suppress the induction of both iNOS and COX2 in primary macrophages stimulated with various pro-inflammatory molecules.18, 19
Figure 1. Structures of oleanolic acid and CDDO-Me
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According to previous reports, polar substituents at the C-28 position of OA can enhance its anticancer potential. Herein, we describe the preparation of a library of novel OA derivatives with a coupled 1,2,3-triazole moiety that is appended to the C28 carboxylic of OA. Using Huisgen [3+2] cycloaddition between a terminal alkyne and an azide, a series of novel regioselective 1,2,3-triazole OA derivatives have been synthesized and screened for anticancer activity against a panel of human cancer cell lines. The IC50 values indicated that the compounds retained their anticancer activity with improved cytotoxicity compared to the parent compound OA. The synthesis and anticancer activity of [journal], [year], [vol], 00–00 | 1
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the synthesized compounds are presented in this paper.
Results and discussion
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Chemistry
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As illustrated in Scheme 1, naturally abundant oleanolic acid was treated with propargyl bromide, potassium carbonate solution, and TBAB in dry CH2Cl2 to fabricate a good yield of 2. Aromatic azides were prepared from their corresponding anilines by diazotization with sodium nitrite in acidic conditions followed by displacement with sodium azide. Huisgen [3+2] cycloaddition of 2 with aromatic azides in the presence of copper ion and sodium ascorbate in a ter-butyl alcohol aqueous solution resulted in the formation of 1,4 substituted-triazolyl derivatives.
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Compd
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In vitro anticancer activity The oleanolic acid-coupled 1,2,3-triazole moiety derivatives were screened for their in vitro anticancer activities against five human cancer cell lines: cervical carcinoma HeLa cells, hepatocellular carcinoma HepG2 cells, colon carcinoma HCT116 cells, melanoma A375-S2 cells, and fibrosarcoma HT1080 cells. The MTT analysis is summarized in Table 1. The IC50 values revealed that most of the conjugates exhibited more potent inhibitory activities against the five cancer cell lines than oleanolic acid. Compounds 3t, 3w and 3i, with p-NO2, p-CN and p-F substitutions at an aromatic ring, were the most promising. From the IC50 values, we concluded the following: (a) Most of the conjugates exhibited more potent inhibitory activities against the five cancer cell lines than oleanolic acid. 3t possesses strong inhibitory activity against A375-S2 and HT1080 cells with IC50 values of 4.97 and 3.51 µM; (b) Compounds with p-substitutions at an aromatic ring (3i, 3k, 3n, 3q, 3t, and 3w) are more active than corresponding compounds without substitutions or substitutions at an ortho- or meta- position. However, compounds with p-Ac substitutions (3q) are less active than compounds with o-Ac substitutions (3o); and (c) Compounds with electron withdrawing groups at an aromatic ring are generally more active than compounds without substitutions or substitutions with an electron donating group at the identical position. Selective killing of cancer cells without affecting normal cell growth is an important characteristic in cancer chemotherapy. Therefore, the most promising compound 3t was evaluated for 2 | Journal Name, [year], [vol], 00–00
Table 1. IC50 Values (µM) of Synthetic OA Derivatives (3a-3y) against Human Cancer Cell Lines
Scheme 1. Synthesis of OA derivatives 3a-3y 15
possible cytotoxicity towards normal cells, human brain microvascular endothelial cells HBMEC, human liver cells L-O2, human colonic epithelial cells 841, human immortalized keratinocyte cells Hacat, and human bronchial epithelial cells BEAS-2B. The growth of all five cell lines were not significantly affected by 3t, suggesting that 3t selectively inhibit the growth of cancer cells (Table 2).
R
HeLa
HepG2
HCT116
A375S2
HT1080
3a
o-OMe
>200
>200
144.32
>200
>200
3b
m-OMe
>200
>200
87.92
>200
>200
3c
p-OMe
>200
>200
>200
>200
>200
3d
o-Me
131.26
111.49
178.04
>200
>200
3e
m-Me
>200
>200
>200
>200
>200
3f
p-OEt
>200
>200
>200
>200
>200
3g
o-F
109.13
117.38
102.35
>200
>200
3h
m-F
53.87
131.36
77.04
157.37
65.61 17.02
3i
p-F
23.27
32.02
36.11
13.88
3j
m-Cl
122.01
112.39
80.62
>200
>200
3k
p-Cl
75.04
87.64
72.26
65.05
59.57
3l
o-Br
148.36
>200
98.1
148.36
163.27
3m
m-Br
124.36
132.54
>200
138.45
134.73
3n
p-Br
86.42
81.6
76.68
81.34
86.89
3o
o-Ac
54.63
36.54
134.05
122.45
74.16
3p
m-Ac
124.12
97.44
42.31
133.76
135.26
3q
p-Ac
74.86
37.71
76.48
53.79
64.8
3r
o-NO2
81.3
88.74
95.77
89.3
79.61
3s
m-NO2
75.04
97.64
62.26
55.05
59.57
3t
p-NO2
10.85
24.15
12.28
4.97
3.51
3u
o-CN
96.47
123.54
117.45
107.53
93.93
3v
m-CN
109.02
117.73
109.35
83.87
91.36
3w
p-CN
34.31
44.17
38.65
22.45
17.14
166,43
146.95
89.69
151.93
73.52
>200
>200
>200
>200
>200
3y
oCOOMe H
OA
---
>200
>200
>200
>200
>200
5-Fu
---
26.18
67.64
35.16
90.74
25.46
3x
Table 2. IC50 Values (µM) of 3t Against Human Normal Cell Lines Compd
HBMEC
L-O2
841
Hacat
BEAS-2B
3t
>100
>100
>100
>100
>100
Cell morphology
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The effect of 3t on HT1080 cell morphology was determined. As shown in Fig. 2, cell morphology remained consistent when the cells were treated with vehicle. However, concentrations of 3t up to 6.2 µM resulted in spherical cells and decreased cell size. Only a small number of living cells can be observed at concentrations exceeding 20 µM. Analysis of apoptosis by Annexin V-FITC/PI An annexin V-FITC / PI binding assay was performed to
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determine whether the 3t induced anti-proliferative activity of the cancer cells was the result of apoptosis. HT1080 cells were treated with vehicle alone or with various concentrations (1.5, 3.0, and 6.0 µM) of compound 3t for 72 h and then stained with FITC-annexin V and propidium iodide (PI). The percentages of apoptotic HT1080 cells were determined by flow cytometry. As shown in Figure 3, conjugate 3t resulted in significant apoptosis in a concentration-dependent manner. When treated with 1.5, 3.0, and 6.0 µM of compound 3t for 72 h, the percentages of apoptotic cells were 12.60%, 35.67%, and 60.88% (Q2 + Q4), respectively. The vehicle control contained 7.29% apoptotic cells. This result demonstrated that the anti-proliferative activity observed when HT1080 cells were treated with 3t could be the result of apoptosis.
Figure 3. Effects of 3t on HT1080 Cells apoptosis
Fluorescence microscopy 20
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Apoptosis is characterized by chromatin condensation that results in compact small nuclei and/or the formation of apoptotic bodies. HT1080 cells were cultured with 3t, and the chromatin condensation and the formation of apoptotic bodies was verified using DAPI staining. Compact nuclei were evident, indicating chromatin condensation (Fig. 4).
Figure 2. Effects of 3t on HT1080 Cells morphology
Figure 4. Effects of 3t on HT1080 Cells nuclei
Changes in mitochondrial membrane potential
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In general, mitochondria are necessary mediators that play a vital role in the execution of both intrinsic and extrinsic apoptotic pathways. Disruption of the mitochondrial membrane potential (MMP) decrease is the symbolic event in the process of apoptosis. To estimate the role of mitochondria in 3t-induced HT1080 cell apoptosis, we explored mitochondrial membrane potential changes by flow cytometric analysis after JC-1 staining (Fig. 5). After treatment with 0 to 6.0 µM of compound 3t, flow cytometry studies revealed a concentration-dependent depression in MMP. This decrease was first detected after treatment with 1.5 µM and Journal Name, [year], [vol], 00–00 | 3
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was most notable with 6.0 µM. The loss of MMP indicated that 3t induces HT1080 apoptosis via a mitochondrial apoptotic deathsignal pathway.
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2489. Melting points were obtained on a Büchi melting point B540 apparatus. 1H and 13C NMR spectra were recorded on a Bruker ARX 600 MHz spectrometer. ESI-MS were obtained on Agilent ESI-QTOF instrument. HRMS were obtained on a Agilent 1260-G6530B Q_TOF. Synthesis of compound 2
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Figure 5. Effects of 3t on HT1080 Cells MMP 60
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A series of oleanolic acid coupled 1,2,3-triazole derivatives have been designed and synthesized by employing a Cu(I)catalyzed Huisgen 1,3-dipolar cycloaddition reaction of terminal alkyne derivatives of 2 with various aromatic azides. The synthesized compounds were screened for anticancer activity against a panel of five human cancer cell lines using an MTT assay. Some compounds exhibited better anti-cancer activity against the tested cancer cell lines compared to positive controls 5-fluorouracil and oleanolic acid. Compound 3t was the most promising derivative. Our biological evaluations suggest that compound 3t is a potent apoptosis inducer in HT1080 cells. Therefore, this compound can serve as a promising lead candidate for further study.
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Reagents were used without further purification unless otherwise specified. Solvents were dried and redistilled prior to use in the usual way. Analytical TLC was performed using silica gel HF254. Preparative column chromatography was performed with silica gel H. HPLC spectra were obtained on a Waters 15254 | Journal Name, [year], [vol], 00–00
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To a solution of 1 (2.0 g, 4.4 mmol) in DCM, potassium carbonate solution (1.8 g, 13.1 mmol/15ml), TBAB (290.0 mg, 0.9mmol) and propargyl bromide (1.0 g, 8.8 mmol) were added and the reaction mixture was stirred at room temperature for 5 hours. Reaction was monitored by TLC and the crude product was subjected to column chromatography to give pure 2 (1.93 g, 89.1% yield). General procedure for the synthesis of compounds 3a-3y To a solution of compound 2 (100.0 mg, 0.202 mmol) in tBuOH:H2O (2:1, 10ml), sodium ascorbate (32.0 mg, 0.162 mmol) and CuSO4.5H2O (20.2 mg, 0.08 mmol) were added at room temperature. To this mixture, aryl azide (1.0 mmol) was added and the reaction mixture was sonicated at 60 ºC still its completion. The crude mixture was extracted with DCM (3×20 ml) and the combined organic layer was dried over Na2SO4 and purified through column chromatography to offer pure 3a-3y. All analogues synthesized by us gave single sharp peaks on HPLC and they were judged at least 95% pure. HPLC Methods: Diamonsil C18(2) column 5u 250×4.6mm, wavelength 254nm, injection volume 20 µL, flow 1.0 mL/min; mobile phase MeOHH2O/95-5, injection time 20min. 3a White solid, 86.9 % yield; HPLC: Purity 95.79%, Rt 11.139 min; Mp 87.0 – 88.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.15 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.13 – 7.07 (m, 2H), 5.33 – 5.22 (m, 3H), 3.89 (s, 3H), 3.21 – 3.16 (m, 1H), 2.87 (d, J = 10.5 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.80 (s, 3H), 0.76 (s, 3H), 0.51 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.69, 150.98, 143.67, 142.57, 130.09, 126.28, 126.23, 125.42, 122.44, 121.19, 112.23, 78.96, 57.53, 55.96, 55.18, 47.56, 46.73, 45.87, 41.68, 41.35, 39.27, 38.74, 38.43, 37.00, 33.85, 33.07, 32.67, 32.39, 30.68, 28.10, 27.64, 27.18, 25.81, 23.63, 23.36, 22.96, 18.28, 16.76, 15.57, 15.23; ESI-MS(m/z): 1309.48 [2M+Na]+; HRMS (ESI): Calcd. for [M+ H]+ C40H58N3O4: 644.4422, found 644.4425. 3b Yellow solid, 80.7 % yield; HPLC: Purity 96.67%, Rt 12.336 min; Mp 86.5 – 88.2 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.40 (t, J = 8.2 Hz, 1H), 7.32 (t, J = 2.1 Hz, 1H), 7.24 (dd, J = 7.9, 1.7 Hz, 1H), 6.97 (dd, J = 8.3, 2.3 Hz, 1H), 5.31 – 5.21 (m, 3H), 3.88 (s, 3H), 3.18 (dd, J = 11.6, 3.8 Hz, 1H), 2.86 (dd, J = 13.8, 4.1 Hz, 1H), 1.09 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.81, 160.65, 143.76, 143.62, 137.97, 130.51, 122.67, 122.49, 114.64, 112.36, 106.40, 78.97, 57.34, 55.64, 55.17, 47.54, 46.75, 45.87, 41.68, 41.38, 39.27, 38.73, 38.41, 36.99, 33.83, 33.06, 32.64, 32.42, 30.67, 28.09, 27.63, 27.18, 25.76, 23.61, 23.35, 22.97, 18.25, 16.78, 15.53, 15.16; ESI-MS(m/z): 1309.49 [2M+Na]+; HRMS(ESI): Calcd. for [M+ H]+ C40H58N3O4: 644.4422, found 644.4425. 3c White solid, 81.9 % yield; HPLC: Purity 96.10%, Rt 11.723 min; Mp 173.4 – 174.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.94 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 5.31 – This journal is © The Royal Society of Chemistry [year]
Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/C4OB01605J
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5.21 (m, 3H), 3.87 (s, 3H), 3.19 (s, 1H), 2.87 (d, J = 10.7 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.79 (s, 3H), 0.75 (s, 3H), 0.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.53, 160.04, 143.61, 130.92, 128.84, 122.65, 122.46, 122.13, 114.99, 114.75, 110.71, 78.96, 61.61, 57.41, 55.65, 55.16, 47.53, 46.74, 45.86, 41.67, 41.37, 39.27, 38.73, 38.41, 36.99, 33.82, 33.06, 32.64, 32.41, 30.67, 28.09, 27.63, 25.77, 23.61, 22.97, 18.26, 16.79, 15.55, 15.20, 14.11; ESI-MS(m/z): 1308.76 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O4: 644.4422, found 644.4423. 3d White solid, 74.1% yield; HPLC: Purity 95.41%, Rt 11.214 min; Mp 102.4 – 105.2 °C; 1H NMR (600 MHz, CDCl3) δ 7.78 (s, 1H), 7.45 – 7.40 (m, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.33 (d, J = 4.0 Hz, 2H), 5.29 (q, J = 12.8 Hz, 3H), 3.20 (dd, J = 11.4, 3.9 Hz, 1H), 2.87 (dd, J = 13.6, 3.9 Hz, 1H), 2.22 (s, 3H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H), 0.77 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.87, 155.79, 143.65, 143.05, 136.34, 133.49, 131.57, 129.93, 129.62, 126.85, 125.92, 125.61, 122.50, 120.57, 115.34, 79.08, 77.24, 77.03, 76.82, 57.48, 55.19, 47.57, 46.82, 45.88, 41.72, 41.33, 39.33, 38.75, 38.44, 37.02, 33.84, 33.06, 32.69, 32.43, 30.67, 28.11, 27.68, 27.16, 25.81, 23.60, 23.38, 22.99, 18.29, 17.90, 17.00, 15.58, 15.27; ESI-MS(m/z): 1277.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O3: 628.4473, found 628.4491. 3e White solid, 79.2 % yield; HPLC: Purity 96.55%, Rt 13.418 min; Mp 106.1 – 108.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.54 (s, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.25 (d, J = 10.5 Hz, 1H), 5.26 (q, J = 12.2 Hz, 3H), 3.19 (d, J = 9.7 Hz, 1H), 2.86 (d, J = 11.3 Hz, 1H), 2.44 (s, 3H), 1.09 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.83, 143.63, 143.59, 139.96, 136.87, 129.57, 129.50, 124.19, 122.75, 122.46, 121.22, 117.61, 78.94, 57.33, 55.14, 47.51, 46.74, 45.85, 41.66, 41.36, 39.25, 38.71, 38.40, 36.97, 33.82, 33.05, 32.62, 32.42, 32.39, 30.66, 28.08, 27.60, 27.13, 25.75, 23.60, 23.33, 22.95, 21.38, 18.23, 16.75, 15.54, 15.16; ESI-MS(m/z): 1277.52 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H58N3O3: 628.4473, found 628.4478. 3f Yellow solid, 81.2 % yield; HPLC: Purity 96.21%, Rt 13.527 min; Mp 98.4 – 100.3 °C; 1H NMR (600 MHz, CDCl3) δ 7.93 (s, 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 5.32 – 5.19 (m, 3H), 4.08 (q, J = 7.0 Hz, 2H), 3.22 – 3.15 (m, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.79 (s, 3H), 0.75 (s, 3H), 0.47 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.81, 159.28, 143.64, 143.59, 130.27, 122.66, 122.47, 122.13, 115.27, 78.98, 63.96, 57.43, 55.17, 47.55, 46.74, 45.88, 41.68, 41.38, 39.28, 38.73, 38.42, 37.00, 33.83, 33.07, 32.64, 32.42, 30.67, 28.09, 27.63, 27.18, 25.77, 23.62, 23.36, 22.98, 22.66, 18.26, 16.80, 15.55, 15.21, 14.72, 14.12; ESI-MS(m/z): 1337.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H60N3O4: 658.4578, found 658.4585. 3g White solid, 83.9 % yield; HPLC: Purity 95.96%, Rt 13.505 min; Mp 66.4 – 68.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.12 (d, J = 2.7 Hz, 1H), 7.96 (t, J = 7.1 Hz, 1H), 7.44 (dd, J = 12.2, 7.0 Hz, 1H), 7.37 – 7.28 (m, 2H), 5.41 – 5.18 (m, 3H), 3.19 (dd, J = 11.5, 4.1 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.80 (s, 3H), 0.75 (s, 3H), 0.50 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.67, 153.30 (d, J = 249.0 This journal is © The Royal Society of Chemistry [year]
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Hz), 143.63, 143.60, 130.24 (d, J = 7.5 Hz), 128.86, 125.28 (d, J = 19.5 Hz), 125.27 (d, J = 7.5 Hz), 124.84, 122.53, 117.07 (d, J = 21.0 Hz), 78.99, 57.32, 55.18, 47.56, 46.77, 45.86, 41.68, 41.36, 39.28, 38.74, 38.43, 37.00, 33.83, 33.07, 32.65, 32.40, 30.67, 28.10, 27.63, 27.18, 25.80, 23.61, 23.34, 22.98, 18.27, 16.70, 15.56, 15.18, 14.11; ESI-MS(m/z): 1285.27 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55FN3O3: 632.4222, found 632.4224. 3h White solid, 84.1 % yield; HPLC: Purity 96.58%, Rt 11.972 min; Mp 101.3 – 103.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.61 – 7.44 (m, 3H), 7.21 – 7.09 (m, 1H), 5.33 – 5.10 (m, 3H), 3.19 (dd, J = 11.5, 4.1 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.45 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 177.84, 163.12 (d, J = 247.5 Hz), 144.07, 143.57, 138.10 (d, J = 10.5 Hz), 131.20 (d, J = 9.0 Hz), 122.59, 122.52, 115.77, 115.73 (d, J = 18.0 Hz), 108.34 (d, J = 25.5 Hz), 78.97, 57.24, 55.16, 47.52, 46.77, 45.84, 41.68, 41.39, 39.28, 38.72, 38.41, 36.98, 33.82, 33.06, 32.63, 32.43, 30.67, 28.08, 27.62, 27.17, 25.76, 23.61, 23.34, 22.97, 18.23, 16.78, 15.51, 15.13; ESI-MS(m/z): 1285.44 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55FN3 O3: 632.4222, found 632.4228. 3i White solid, 81.90 % yield; HPLC: Purity 98.36%, Rt 11.120 min; Mp 173.4 – 174.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.98 (s, 1H), 7.74 – 7.66 (m, 2H), 7.25 – 7.19 (m, 2H), 5.31 – 5.22 (m, 3H), 3.22 – 3.15 (m, 1H), 2.86 (dd, J = 13.8, 4.2 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H), 0.46 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 177.85, 162.49 (d, J = 246.0 Hz), 143.96, 143.59, 133.23, 128.86, 122.72, 122.48 (d, J = 7.5 Hz), 116.72 (d, J = 24.0 Hz), 78.96, 57.31, 55.16, 47.52, 46.77, 45.84, 41.69, 41.39, 39.29, 38.73, 38.42, 36.99, 33.82, 33.06, 32.64, 32.43, 30.67, 28.09, 27.63, 27.18, 26.92, 25.77, 23.61, 23.35, 22.97, 18.25, 16.77, 15.54, 15.18; ESI-MS(m/z): 1284.65 [2M+Na]+; HRMS(ESI): Calcd. for [M+ H]+ C39H55FN3O3: 632.4222, found 632.4226. 3j Yellow solid, 84.3 % yield; HPLC: Purity 96.05%, Rt 14.310 min; Mp 88.0 – 90.6 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.77 (t, J = 1.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.49 – 7.39 (m, 2H), 5.31 – 5.19 (m, 3H), 3.18 (dd, J = 11.5, 4.0 Hz, 1H), 2.86 (dd, J = 13.8, 4.2 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.42 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.08, 143.58, 137.79, 135.66, 130.83, 128.90, 122.69, 122.52, 120.87, 118.47, 78.98, 57.22, 55.17, 47.51, 46.77, 45.85, 41.68, 41.39, 39.28, 38.73, 38.41, 36.99, 33.83, 33.06, 32.64, 32.44, 30.67, 28.09, 27.62, 27.18, 25.74, 23.60, 23.34, 22.97, 18.23, 16.79, 15.52, 15.18; ESI-MS(m/z): 1317.57 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55ClN3O3: 648.3926, found 648.3925. 3k White solid, 82.1 % yield; HPLC: Purity 95.07%, Rt 13.892 min; Mp 203.3 – 205.5 °C; 1H NMR (600 MHz, CDCl3) δ 8.00 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 5.29 – 5.21 (m, 3H), 3.21 – 3.16 (m, 1H), 2.86 (d, J = 10.6 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.75 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.06, 143.57, 135.44, 134.67, 130.93, 129.93, 122.51, 121.64, 78.95, 61.62, 57.27, 55.16, 47.51, 46.76, 45.84, 41.68, 41.38, 39.28, 38.73, 38.41, 36.98, 33.81, 33.06, 32.63, 32.42, 30.67, 28.09, 27.62, 27.17, 25.76, 23.61, 23.34, 22.97, 18.25, 16.77,
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Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/C4OB01605J
Organic & Biomolecular Chemistry
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15.55, 15.17, 14.11; ESI-MS(m/z): 1318.48 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C39H55ClN3 O3: 648.3926, found 648.3929. 3l White solid, 89.45 % yield; HPLC: Purity 98.44%, Rt 12.259 min; Mp 59.6 – 61.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.00 (s, 1H), 7.76 (dd, J = 8.0, 0.9 Hz, 1H), 7.56 (dd, J = 7.9, 1.4 Hz, 1H), 7.49 (dd, J = 11.0, 4.3 Hz, 1H), 7.40 (td, J = 7.9, 1.5 Hz, 1H), 5.35 – 5.24 (m, 1H), 3.20 (dd, J = 11.4, 4.3 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.70, 143.66, 142.97, 136.45, 134.01, 131.19, 128.46, 128.16, 126.13, 122.49, 118.37, 78.98, 57.42, 55.20, 47.58, 46.79, 45.88, 41.71, 41.32, 39.34, 38.75, 38.44, 37.03, 33.85, 33.06, 32.70, 32.42, 30.67, 28.11, 27.68, 27.19, 25.82, 23.61, 23.39, 23.00, 18.31, 16.98, 15.59, 15.30; ESIMS(m/z): 1406.53 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3O3: 692.3421, found 692.3419. 3m White solid, 75.74 % yield; HPLC: Purity 95.62%, Rt 15.092 min; Mp 119.6 – 201.9 °C; 1H NMR (600 MHz, CDCl3) δ 8.02 (s, 1H), 7.92 (t, J = 1.8 Hz, 1H), 7.67 (dd, J = 8.1, 1.2 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 5.31 – 5.20 (m, 3H), 3.18 (dd, J = 11.5, 4.1 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H), 0.42 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.86, 144.07, 143.57, 137.86, 131.85, 131.06, 123.69, 123.35, 122.71, 122.51, 118.97, 78.98, 57.21, 55.17, 47.51, 46.77, 45.86, 41.68, 41.39, 39.28, 38.73, 38.42, 36.99, 33.83, 33.05, 32.64, 32.44, 30.67, 28.09, 27.61, 27.18, 26.93, 25.74, 23.60, 23.34, 22.97, 18.24, 16.78, 15.55, 15.23, 14.12; ESI-MS(m/z): 1406.54 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3 O3: 692.3421, found 692.3420. 3n White solid, 86.44 % yield; HPLC: Purity 97.14%, Rt 14.889 min; Mp 194.9 – 196.5 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 5.32 – 5.20 (m, 1H), 3.19 (d, J = 10.9 Hz, 1H), 2.86 (dd, J = 13.8, 4.0 Hz, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.75 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.85, 144.10, 143.58, 135.93, 132.91, 122.51, 122.45, 121.87, 78.97, 57.27, 55.16, 47.51, 46.77, 45.84, 41.68, 41.38, 39.28, 38.73, 38.41, 36.99, 33.81, 33.05, 32.63, 32.42, 30.67, 28.09, 27.62, 27.18, 25.76, 23.60, 23.35, 22.97, 18.25, 16.78, 15.55, 15.17; ESI-MS(m/z): 1406.49 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H55BrN3O3: 692.3421, found 692.3419. 3o White solid, 71.94 % yield; HPLC: Purity 98.18%, Rt 8.032 min; Mp 66.1 – 68.9 °C; 1H NMR (600 MHz, CDCl3) δ 7.90 (s, 1H), 7.71 (dd, J = 7.5, 1.6 Hz, 1H), 7.64 – 7.58 (m, 2H), 7.45 (dd, J = 7.7, 1.3 Hz, 1H), 5.29 – 5.25 (m, 3H), 3.19 (dd, J = 11.4, 4.4 Hz, 1H), 2.85 (dd, J = 13.6, 4.5 Hz, 1H), 2.17 (s, 3H), 1.11 (s, 3H), 0.96 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.75 (s, 3H), 0.59 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 199.19, 177.64, 143.74, 143.55, 136.15, 134.22, 131.79, 129.90, 129.00, 125.52, 125.47, 122.40, 78.88, 60.32, 57.25, 55.08, 47.47, 46.68, 45.75, 41.61, 41.23, 39.22, 38.65, 38.33, 36.92, 33.72, 32.96, 32.55, 30.57, 29.08, 28.00, 27.57, 27.07, 25.72, 23.51, 23.28, 22.88, 18.19, 16.86, 15.49, 15.18; ESI-MS(m/z): 1332.56 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O4: 656.4422, found 656.4449. 3p White solid, 79.63 % yield; HPLC: Purity 96.86%, Rt
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10.065 min; Mp 137.6 – 139.4 °C; 1H NMR (600 MHz, CDCl3) δ 8.28 (s, 1H), 8.11 (s, 1H), 8.04 – 7.97 (m, 2H), 7.65 (t, J = 7.9 Hz, 1H), 5.32 – 5.21 (m, 3H), 3.18 (dd, J = 11.4, 3.8 Hz, 1H), 2.87 (dd, J = 13.7, 4.0 Hz, 1H), 2.67 (s, 3H), 1.10 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.72 (s, 3H), 0.45 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 196.43, 177.79, 144.14, 143.52, 138.56, 137.34, 130.20, 128.45, 124.65, 122.59, 122.52, 119.71, 78.92, 57.25, 55.14, 47.50, 46.76, 45.83, 41.67, 41.38, 39.27, 38.71, 38.39, 36.97, 33.81, 33.05, 32.63, 32.42, 30.66, 28.08, 27.62, 27.16, 26.72, 25.75, 23.61, 23.33, 22.97, 18.23, 16.75, 15.53, 15.15; ESI-MS(m/z): 1332.75 [2M+Na]+; HRMS(ESI): － Calcd. for [M+COOH] C42H58N3O6: 700.4331, found 700.4449. 3q White solid, 71.72 % yield; HPLC: Purity 97.60%, Rt 10.573 min; Mp 142.4 – 145.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.15 – 8.08 (m, 3H), 7.86 (d, J = 8.6 Hz, 2H), 5.30 – 5.23 (m, 3H), 3.18 (dd, J = 11.6, 4.0 Hz, 1H), 2.86 (dd, J = 13.8, 4.1 Hz, 1H), 2.65 (s, 3H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.73 (s, 3H), 0.44 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 196.44, 177.85, 144.26, 143.56, 139.96, 136.94, 130.06, 122.51, 120.04, 78.94, 57.25, 57.22, 55.13, 47.50, 46.78, 45.83, 41.68, 41.39, 39.27, 38.72, 38.39, 36.97, 33.80, 33.05, 32.62, 32.42, 30.67, 28.07, 27.63, 27.16, 26.67, 25.76, 23.60, 23.34, 22.97, 18.23, 16.76, 15.54, 15.16; ESI-MS(m/z): 1332.76 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O4: 656.4422, found 656.4421. 3r Yellow solid, 83.02 % yield; HPLC: Purity 95.95%, Rt 8.016 min; Mp 57.1 – 59.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 5.32 – 5.23 (m, 3H), 3.20 (dd, J = 11.3, 4.2 Hz, 1H), 2.86 (dd, J = 13.7, 3.9 Hz, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.76 (s, 3H), 0.58 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.77, 144.41, 143.81, 143.65, 133.72, 130.83, 130.16, 127.87, 125.70, 125.64, 122.49, 78.97, 57.23, 55.18, 47.57, 46.80, 45.88, 41.72, 41.33, 39.34, 38.75, 38.43, 37.03, 33.82, 33.06, 32.65, 32.35, 30.67, 28.10, 27.67, 27.18, 25.81, 23.61, 23.37, 23.00, 18.30, 16.93, 15.59, 15.28; ESI-MS(m/z): 1338.69 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4074. 3s Yellow solid, 78.70 % yield; HPLC: Purity 97.20%, Rt 10.763 min; Mp 75.4 – 78.1 °C; 1H NMR (600 MHz, CDCl3) δ 8.58 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 9.5 Hz, 2H), 7.76 (t, J = 8.1 Hz, 1H), 5.33 – 5.22 (m, 3H), 3.18 (dd, J = 11.1, 3.8 Hz, 1H), 2.87 (d, J = 10.4 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.74 (s, 3H), 0.72 (s, 3H), 0.45 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 177.87, 148.96, 143.50, 137.64, 130.98, 128.84, 125.89, 123.27, 122.67, 122.55, 115.29, 78.92, 61.61, 57.15, 55.13, 47.48, 46.80, 45.81, 41.69, 41.39, 39.29, 38.71, 38.39, 36.96, 33.80, 33.04, 32.63, 32.43, 30.66, 28.07, 27.62, 27.15, 25.75, 23.59, 23.33, 22.97, 18.22, 16.76, 15.51, 15.11, 14.11; ESI-MS(m/z): 1338.67 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4073. 3t Yellow solid, 75.42 % yield; HPLC: Purity 97.24%, Rt 11.263 min; Mp 218.4 – 200.7 °C; 1H NMR (600 MHz, CDCl3) δ 8.42 (d, J = 9.0 Hz, 2H), 8.14 (s, 1H), 7.96 (d, J = 9.0 Hz, 2H), 5.34 – 5.23 (m, 3H), 3.18 (d, J = 10.9 Hz, 1H), 2.86 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.76 (s, 3H), 0.73 (s, 3H), 0.45 (s, 3H); 13C NMR (150 MHz,
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CDCl3) δ 177.91, 147.33, 144.72, 143.53, 141.07, 125.53, 122.55, 120.48, 78.94, 57.11, 55.14, 47.49, 46.82, 45.80, 41.70, 41.41, 39.29, 38.72, 38.40, 36.98, 33.79, 33.04, 32.63, 32.43, 30.67, 28.07, 27.63, 27.16, 25.77, 23.60, 23.34, 22.98, 18.25, 16.75, 15.54, 15.17; ESI-MS(m/z): 1338.48 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O7: 703.4076, found 703.4054. 3u White solid, 83.47 % yield; HPLC: Purity 97.43%, Rt 8.221 min; Mp 52.4 – 55.6 °C; 1H NMR (600 MHz, CDCl3) δ 8.26 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.80 (t, J = 7.8 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 5.34 – 5.26 (m, 3H), 3.20 (d, J = 11.2 Hz, 1H), 2.87 (dd, J = 13.7, 4.1 Hz, 1H), 1.11 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.81 (s, 3H), 0.76 (s, 3H), 0.53 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.66, 144.15, 143.59, 138.44, 134.40, 134.23, 129.59, 125.38, 124.56, 122.53, 115.51, 106.68, 78.97, 57.22, 55.17, 47.56, 46.80, 45.86, 41.69, 41.35, 39.32, 38.74, 38.42, 37.01, 33.81, 33.07, 32.65, 32.39, 30.67, 28.10, 27.66, 27.18, 25.81, 23.62, 23.36, 22.98, 18.30, 16.82, 15.58, 15.25, 13.70; ESI-MS(m/z): 1298.71 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C40H55N4O3: 639.4269, found 639.4267. 3v White solid, 79.26 % yield; HPLC: Purity 96.55%, Rt 9.590 min; Mp 58.0 – 60.1 °C; 1H NMR (600 MHz, CDCl3) δ 8.08 (s, 1H), 8.05 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 5.30 – 5.22 (m, 3H), 3.18 (dd, J = 11.3, 3.6 Hz, 1H), 2.86 (dd, J = 13.7, 4.0 Hz, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.43 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.86, 144.50, 143.52, 137.47, 132.11, 130.90, 124.36, 123.62, 122.54, 122.50, 117.25, 114.27, 78.93, 57.14, 55.15, 47.48, 46.79, 45.81, 41.68, 41.39, 39.29, 38.72, 38.40, 36.98, 33.80, 33.04, 32.63, 32.43, 30.66, 28.08, 27.61, 27.16, 25.75, 23.60, 23.34, 22.97, 18.24, 16.79, 15.55, 15.20; ESI-MS(m/z): 1298.79 [2M+Na]+; HRMS (ESI): Calcd. for [M+H]+ C41H55N4O5: 683.4178, found 683.4170. 3w White solid, 76.05 % yield; HPLC: Purity 98.84%, Rt 9.530 min; Mp 196.5 – 198.8 °C; 1H NMR (600 MHz, CDCl3) δ 8.10 (s, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 5.31 – 5.23 (m, 3H), 3.22 – 3.15 (m, 1H), 2.89 – 2.82 (m, 1H), 1.10 (s, 3H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.76 (s, 3H), 0.75 (s, 3H), 0.43 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.91, 144.57, 143.52, 139.72, 133.92, 122.54, 122.40, 120.55, 117.61, 112.57, 78.94, 57.12, 55.14, 47.49, 46.80, 45.80, 41.69, 41.40, 39.28, 38.72, 38.41, 36.98, 33.79, 33.04, 32.62, 32.43, 30.66, 28.08, 27.62, 27.16, 25.76, 23.59, 23.34, 22.97, 18.24, 16.74, 15.56, 15.17; ESI-MS(m/z): 1298.57 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H55N4O5: 683.4178, found 683.4182. 3x White solid, 67.1 % yield; HPLC: Purity 97.01%, Rt 8.172 min; Mp 65.1 – 68.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 7.7 Hz, 1H), 7.86 (s, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 5.32 – 5.24 (m, 3H), 3.72 (s, 3H), 3.20 (dd, J = 11.4, 4.3 Hz, 1H), 2.89 – 2.85 (m, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.60 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.71, 165.48, 143.67, 143.01, 136.10, 132.64, 131.31, 129.91, 127.39, 126.68, 125.90, 122.49, 78.98, 61.62, 57.48, 55.19, 52.56, 47.58, 46.76, 45.88, 41.72, 41.35, 39.34, 38.75, 38.44, 37.03, 33.85, 33.07, 32.68, 32.37, 30.67, 28.10, 27.69, 27.19, 25.82, 23.62, 23.01, 18.30, 16.97, 15.58, 15.28, 14.11; ESI-MS(m/z): 1364.75 [2M+ Na]+; HRMS(ESI): Calcd. for [M+H]+ C41H58N3O5: 672.4371, found 672.4370.
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Cell viability assay
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Cell viability was evaluated using an MTT assay. HT1080 cells were seeded into 96-well plates at a density of 5 × 104 cells per well and stabilized at 37 °C for 24 h. Compounds 3a-3y were added to each well at various concentrations, and then the cells were incubated for 72 h. The MTT solution (20 µL 5 mg/mL) was added to each well, and the cells were incubated for another 4 h. Formazan crystals were dissolved in 150 µL of DMSO. Cell viability was assessed by measuring the absorbance at 540 nm wavelength using a microplate reader (BioTek ELx800 USA). Annexin V/PI staining assay
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HT1080 cells were treated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. Then, cells were collected, washed with annexin-binding buffer, and stained with annexin V fluorescein isothiocyanate (FITC) and PI for 15 min at RT (25 °C). After that, the samples were analyzed by flow cytometry (BD FACSCalibur, USA). DAPI staining assay
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HT1080 cells were treated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. The cells were collected, resuspended, and fixed in 4% paraformaldehyde in PBS. After PBS washing, cells were stained with DAPI for 30 min followed by another PBS wash. The stained cells were examined under a fluorescent microscope (IX51, OLYMPUS, Japan). Mitochondrial membrane potential assay
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The mitochondrial membrane potential was assessed using JC1 dye. HT1080 cells were plated at 1 × 106 cells per well in 24well plates and incubated with 3t at various concentrations (0, 1.5, 3.0 and 6.0 µM) for 72 h. The cells were subsequently incubated with JC-1 dye and finally, analyzed by flow cytometry (BD FACSCalibur, USA).
Acknowledgments 105
The authors wish to express their thanks for the financial support received from the National Natural Science Foundation of China (No. 81273358), the Program for Innovative Research Team of the Ministry of Education, and the Program for Liaoning Innovative Research Team in University.
Notes 110
This journal is © The Royal Society of Chemistry [year]
3y White solid, 83.03 % yield; HPLC: Purity 97.02%, Rt 11.248 min; Mp 79.8 – 82.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.72 (d, J = 8.0 Hz, 2H), 7.53 (t, J = 7.7 Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H), 5.38 – 5.09 (m, 3H), 3.19 (d, J = 11.2 Hz, 1H), 2.94 – 2.77 (m, 1H), 1.10 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.77 (s, 3H), 0.74 (s, 3H), 0.46 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.68, 143.71, 143.27, 133.83, 133.13, 131.80, 128.90, 126.11, 122.49, 79.01, 57.38, 55.23, 47.63, 46.81, 45.92, 41.75, 41.27, 39.36, 38.76, 38.46, 37.04, 33.86, 33.04, 32.70, 32.30, 30.66, 28.11, 27.71, 27.21, 26.93, 25.83, 23.58, 23.41, 23.05, 18.31, 17.06, 15.58, 15.32; ESI-MS(m/z): 1249.40 [2M+Na]+; HRMS(ESI): Calcd. for [M+H]+ C39H56N3O3: 614.4316, found 614.4314.
a
Key Laboratory of Structure-Based Drugs Design & Discovery of
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Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China. E-mail: [email protected]; Tel: +86 24 23986413 (Yang Liu) E-mail: [email protected]; Tel: +86 24 23986419 (Maosheng Cheng) † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/
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