Synthesis and Bioactivity of Natural Occurring Petasin-Like Derivatives as Antitumor Agents

Petasin is a potential antitumor against human neuroblastoma cell SK-N-SH by inhibiting the ERK1/2 phosphorylation. In view of its great activity and new antiproliferative mechanisms, a series of petasin derivatives were designed and synthesized, which showed great antiproliferative activity. Among them compounds 1h and 1f were more effective against SK-N-SH cells than petasin with the IC ₅₀ values of 0.87 and 2.63 μM, respectively.

• Petasin
• SK-N-SH
• Mechanism
• Derivative
• Antiproliferative Activity

1. Introduction

Neuroblastoma (neuroblastoma, NB) [1] is the most common extracranial tumor in infants and young children. Recent clinical statistics show that the neuroblastoma has become the third most common cause of death after renal tumor and leukemia during the childhood [2] [3] . Due to the diversity of clinical manifestations, its treatment varies accordingly. In general, neuroblastoma needs surgery and radioactive therapy, chemotherapy and other treatments are also used for the residual tumors.

Petasin (Figure 1) is the active component of Petasites hybridus L. (Petasites Tricholobus, Compositae), which has been reported to possess many bioactivities including analgesic, anti-inflammation, and inhibition of ileum

Figure 1

The structures of petasin and petasin derivatives (R 1 , R 2 , R 3 = alkyl group)

contraction. In our group, the great antitumor activity against Sk-N-SH was reported for the first time [4] . Petasin successfully inhibited the proliferation but not induced the apoptosis. The inhibition of ERK1/2 phosphorylation might be involved in the antiproliferation.

Based on the above results, a series of petasin derivatives were designed and synthesized (Figure 1) for investigating the structure-activity relationship and discovering new antiproliferative agents.

2. Chemistry

Petasin derivatives were efficiently synthesized as described in Scheme 1. Compounds 1a -h and 3 were obtained from the isopetasinol by acylation reactions with carboxylic acids or maleic anhydride [5] [6] . Isopetasinol was prepared by the hydrolysis [7] of petasin in the presence of NaOH. Compounds 2 were prepared by an epoxidation [8] -[10] of compounds 1. Compounds 4a-b with an unsaturated side chain were synthesized by the reaction of compound 3 and different alkyl halides.

To further investigate SARs, compounds 8a-c with an amide functional group were designed and synthesized [11] -[17] . Tosylation of isopetasinol resulted in compound 5, followed by nucleophilic substitution with NaN₃ giving the corresponding azide 6. Zn-mediated reduction of compound 6 produced compound 7, which was then subjected to prepare compounds 8a-c by acylation reactions.

3. Results and Discussion

The IC₅₀ values for all obtained petasin analogues against three human cancer cell lines including SK-N-SH, MGC-803 and HepG-2 were determined using MTT assay [18] -[22] . Natural occurring petasin, isopetasinol and cisplatin were used as positive controls. The results were demonstrated in Table 1. The cell-based investigation demonstrated that some synthetic compounds showed much more potent or comparable antiproliferative activity compared to the petasin. Especially they showed selective antiproliferative activities toward SK-N-SH cells with weak inhibition against other tumor cells.

Compared to petasin, the antiproliferative activities of isopetasinol against the tested cancer cell lines was decreased significantly (IC₅₀ > 100 µM), which indicated that an additional unsaturated ester group is beneficial for the activity. Therefore, substituted benzoic ester group was introduced as represented in compounds 1a -h. Interestingly, some of these compounds showed great cytotoxicity against SK-N-SH and were much more potent than petasin and cisplatin. Compounds with electron-donating groups attached to the phenyl ring were much

Scheme 1. Reagents and conditions: 1) NaOH, CH₃OH, 62˚C, 4 - 6 h; 2) DMAP, DCC, CH₂Cl₂, 0˚C, 6 h; 3) m- ClC₆H₅COOOH, CH₂Cl₂, rt, 3 h; 4) (C₂H₅)₃N, CH₂Cl₂, 0˚C, 2 h; 5) Na₂CO₃, DMF, 65˚C, 2 h; 6) m-CH₃C₆H₅SO₂Cl, (C₂H₅)₃N, CH₂Cl₂, 0˚C, 6 h; 7) NaN₃, DMF, 95˚C, 8 h; 8) Zn, NH₄Cl, THF/H₂O, 2 h, rt; 9) (C₂H₅)₃N, CH₂Cl₂, 0˚C, 2 h.

Table 1

In vitro inhibitory activity of petasin derivatives against SK-N-SH, MGC-803 and HepG-2 cell lines

Compd	IC50 (µmol/L)
	SK-N-SH	MGC-803	HepG-2
1a	12.01 ± 1.27	>100	38.02 ± 1.87
1b	5.98 ± 1.06	12.99 ±2.62	64.17 ± 2.06
1c	14.91 ± 1.85	12.84 ± 1.87	34.55 ± 3.51
1d	>100	13.73 ± 2.46	30.30 ± 2.43
1e 1f 1g 1h	31.36 ± 2.01 2.63 ± 0.76 61.25 ± 3.21 0.87 ± 0.28	33.84 ± 2.85 24.83 ± 1.97 40.40 ± 3.41 >100	33.82 ± 2.01 57.33 ± 2,53 32.02 ± 1.98 27.46 ± 1.63
2a	34.78 ± 3.56	43.75 ± 3.78	>100
2b 2c	76.23 ± 2.18 26.87 ± 1.87	>100 >100	>100 49.34 ± 1.76
2d	48.95 ± 2.14	>100	>100
2e	>100	>100	>100
4a	14.54 ± 1.43	43.19 ± 2.86	58.17 ± 3.01
4b	12.76 ± 1.52	>100	>100
8a	>100	>100	>100
8b	31.80 ± 2.07	36.86 ± 3.41	27.63 ± 2.43
8c	64.31 ± 2.65	78.37 ± 4.01	43.98 ± 3.01
Isopetasinol	>100	>100	>100
Petasin Cisplatin	5.76 ± 0.98 12.32 ± 1.06	34.41 ± 2.75 10.88 ± 1.86	26.84 ± 1.65 8.14 ± 0.89

more potent than those with electron-withdrawing groups. Among them, compound 1h and 1f showed the best cytotoxicity with the IC₅₀ values of 0.87 and 2.63 µM, respectively, which were about 14 or 15 fold more potent than cisplatin (IC₅₀ = 12.32 µM).

Compounds 2a -e with epoxy ring showed bad antiproliferative activity against SK-N-SH with the IC₅₀ values more than 26 µM and were less potent than cisplatin and petasin (IC₅₀ = 5.76 µM), which indicated that exo- double bond were important for the antiproliferative activity. Besides, the antiproliferative activity of compounds 4a-b and compounds 8a-c also become to be worse than that of compounds 1a-1h and the positive control.

In conclusion, SAR of new petasin derivatives against SK-N-SH showed that an additional ester group and the exo-double bond were important for the antiproliferative activity. Furthermore the isomerization of the double bond and unsaturated ester unaffected the antiproliferative activity. But if the ester group was substituted with amide group, the antiproliferative activity against Sk-N-SH showed a certain extent effects. And compounds 1h and 1f were discovered, which showed selective and great antiproliferative activity with IC₅₀ values of 0.87 and 2.63 µM against SK-N-SH cells. The result in this study is valuable for the design and optimization of petasin derivative and the discovery of new antiproliferative agent.

4. Experimental Section

4.1. Chemistry

Petasin, extracted from Ligularia fischeri with traditional ethanol reflux. Reagents and solvents were purchased from commercial sources and were used without further purification. Thin-layer chromatography (TLC) was performed on silica gel GF₂₅₄ plates. Silicagel GF₂₅₄ and H (200 - 300 mesh) from Qingdao Haiyang Chemical Company was used for TLC, preparative TLC, and column chromatography respectively. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker 400 MHz and 100 MHz spectrometer respectively. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray ionization (ESI).

4.1.1. General Procedure for the Synthesis of Compounds 1a-h

A mixture of isopetasinol (1 equiv) and Carboxylic acid (1.5 equiv) in CH₂Cl₂ in the presence of DMAP (1 equiv) and DCC (1 equiv) was stirred at 0˚C for 6 h. The disappearance of compound isopetasinol was monitored by TLC. Upon completion, the solvent was removed and water was added, the reaction mixture was extracted with CH₂Cl₂. The combined organic layer was washed with brine, dried over anhydrous MgSO₄ and concentrated under vacuum to afford the crude product. The crude product was purified by a flash column to yield the pure product 1a-h.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-4-methylbenzoate (1a). Yellow solid, yield 75%, mp: 71.2˚C - 72.1˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.94 - 7.25 (m, 4H, ArH), 5.81 (s, 1H, HC = C), 5.14 - 5.01 (m, 1H, CH), 2.96 (d, J = 13.7 Hz, 1H, CH₂), 2.52 (d, J = 13.6, 1H, CH₂), 2.42 (s, 3H, CH₃ ), 2.39 - 2.26 (m, 2H, CH₂), 2.23 (d, J = 11.0 Hz, 1H, CH₂), 2.11 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.81 (d, J = 11.0, 1H, CH₂), 1.63 - 1.54 (m, 1H, CH), 1.08 (s, 3H, CH₃), 1.04 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.94, 191.66, 166.23, 165.15, 143.73, 143.43, 129.60, 129.11, 127.59, 127.14, 126.76, 74.01, 46.34, 42.27, 41.17, 31.65, 30.91, 30.13, 22.61, 22.13, 21.65, 17.19, 10.83. HR-MS (ESI): Calcd. C₂₃H₂₈O₃, [M+H]⁺ m/z: 352.2126, found: 352.2128.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-4-methoxybenzoate (1b). Yellow solid, yield 70%, mp: 107.7˚C - 110.4˚C, ¹H NMR (400 MHz, CDCl₃) δ 8.03 - 6.95 (m, 4H, ArH), 5.83 (s, 1H, HC=C), 5.13 - 5.04 (m, 1H, CH), 3.89 (s, 3H, CH₃), 2.98 (d, J = 13.7 Hz, 1H, CH₂), 2.56 (d, J = 13.7, 1H, CH₂), 2.46 - 2.29 (m, 2H, CH₂), 2.25 (d, J = 11.0 Hz, 1H, CH₂), 2.13 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 1.82 (d, J = 11.0, 1H, CH₂), 1.63 - 1.57 (m, 1H, CH), 1.10 (s, 3H, CH₃), 1.06 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.99, 191.71, 165.93, 165.26, 163.45, 143.46, 131.59, 127.14, 126.72, 122.71, 113.65, 73.85, 55.46, 46.38, 42.28, 41.17, 31.70, 30.91, 30.15, 22.62, 22.14, 17.19, 10.83. HR-MS (ESI): Calcd. C₂₃H₂₈O₄, [M+H]⁺ m/z: 368.2047, found: 368.2049.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-3-nitrobenzoate (1c). White solid, yield 74%, mp: 128.9˚C - 132.8˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.95 - 7.64 (m, 4H, ArH), 5.82 (s, 1H HC = C), 5.12 - 5.01 (m, 1H, CH), 2.95 (d, J = 13.7 Hz, 1H, CH₂), 2.56 (d, J = 13.7 Hz, 1H, CH₂), 2.47 - 2.39 (m, 2H, CH₂), 2.21 (d, J = 11.1 Hz, 1H, CH₂), 2.12 (s, 3H, CH₃), 1.88 (s, 3H, CH₃), 1.78 - 1.70 (m, 1H, CH), 1.59 (d, J = 11.0 Hz, 1H, CH₂), 1.09 (s, 3H, CH₃), 1.06 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 207.02, 191.53, 165.14, 164.52, 147.95, 143.63, 132.98, 131.74, 129.65, 127.88, 126.99, 123.96, 76.40, 45.85, 42.28, 41.04, 30.95, 29.94, 22.63, 22.16, 17.17, 10.79. HR-MS (ESI): Calcd. C₂₂H₂₅NO₅, [M+H]⁺ m/z: 383.1807, found: 383.1809.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-4-nitrobenzoate (1d). White solid yield 75%, mp: 88.1˚C - 91.4˚C, ¹H NMR (400 MHz, CDCl₃) δ 8.36 - 8.21 (m, 4H, ArH), 5.84 (s, 1H, HC = C), 5.17 - 5.09 (m, 1H, CH), 2.99 (d, J = 13.7 Hz, 1H, CH₂), 2.58 (d, J = 13.7 Hz, 1H, CH₂), 2.48 - 2.31 (m, 2H), 2.26 (d, J = 11.0 Hz, 1H, CH₂), 2.14 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 1.86 (d, J = 11.2, 1H, CH₂), 1.72 - 1.64 (m, 1H, CH), 1.11 (s, 3H, CH₃), 1.08 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.86, 191.48, 164.32, 164.29, 150.61, 143.79, 135.67, 130.71, 126.99, 126.92, 123.60, 75.66, 46.16, 42.25, 41.13, 31.52, 30.93, 29.99, 22.65, 22.17, 17.17, 10.93. HR-MS (ESI): Calcd. C₂₂H₂₅NO₅, [M+H]⁺ m/z: 383.1807, found: 383.1813.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-3-fluorobenzoate (1e). White solid, yield 73%, mp: 115.1˚C - 116.7˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.85 - 7.27 (m, 4H, ArH), 5.82 (s, 1H, HC = C), 5.11 - 5.03 (m, 1H, CH), 2.98 (d, J = 13.7 Hz, 1H, CH₂), 2.57 (d, J = 13.7 Hz, 1H, CH₂), 2.46 - 2.29 (m, 2H, CH₂), 2.26 (d, J = 11.2 Hz, 1H, CH₂), 2.12 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.82 (d, J = 11.4, 1H, CH₂), 1.61 - 1.53 (m, 1H, CH), 1.09 (s, 3H, CH₃), 1.05 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.99, 191.60, 164.90, 143.60, 127.04, 126.87, 125.31, 120.23, 120.01, 116.58, 116.35, 74.83, 46.21, 42.25, 41.13, 31.55, 30.94, 30.05, 22.64, 22.15, 17.17, 10.87. HR-MS (ESI): Calcd. C₂₂H₂₅FO₃, [M+H]⁺ m/z: 356.1863, found: 356.1865.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-2-hydroxybenzoate (1f). White solid, yield 73%, mp: 128.8˚C - 134.6˚C, ¹H NMR (400 MHz, CDCl₃) δ 10.81 (s, 1H, OH), 7.84 - 6.89 (m, 4H, ArH), 5.82 (s, 1H, HC = C), 5.14 - 5.06 (m, 1H, CH), 2.99 (d, J = 13.7 Hz, 1H, CH₂), 2.56 (d, J = 13.7 Hz, 1H, CH₂), 2.48 - 2.30 (m, 2H, CH₂), 2.26 (d, J = 11.2 Hz, 1H, CH₂), 2.12 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.83 (d, J = 11.2, 5.5 Hz, 1H, CH₂), 1.72 ? 1.62 (m, 1H, CH), 1.09 (s, 3H, CH₃), 1.06 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.91, 191.53, 164.42, 161.86, 143.72, 135.89, 129.75, 126.96, 119.23, 117.68, 112.47, 75.08, 46.18, 42.24, 41.15, 31.58, 30.96, 30.08, 22.66, 22.41, 17.61, 10.97. HR-MS (ESI): Calcd. C₂₂H₂₆O₄, [M+H]⁺ m/z: 354.1906, found: 354.1907.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-4-chlorobenzoate (1g). Yellow solid, yield 63%, mp: 69.2˚C - 72.8˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.98 - 7.43 (m, 4H, ArH), 5.81 (s, 1H, HC = C), 5.10 - 5.02 (m, 1H, CH), 2.98 (d, J = 13.7 Hz, 1H, CH₂), 2.56 (d, J = 13.7, 1H, CH₂), 2.44 - 2.30 (m, 2H, CH₂), 2.25 (d, J = 11.1 Hz, 1H, CH₂), 2.11 (s, 3H, CH₃), 1.86 (s, 3H, CH₃), 1.84 - 1.76 (m, 1H, CH), 1.58 (d, J = 11.2 Hz, 1H, CH₂), 1.08 (s, 3H, CH₃), 1.04 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.99, 191.61, 165.32, 164.83, 143.62, 139.52, 130.97, 128.77, 128.74, 127.04, 126.84, 74.65, 46.26, 42.26, 41.14, 31.59, 30.92, 30.07, 22.63, 22.15, 17.17, 10.86. HR-MS (ESI): Calcd. C₂₂H₂₅ClO₃, [M+H]⁺ m/z: 372.1570, found: 372.1571.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-2-methoxybenzoate (1h). White solid, yield 73%, mp: 102.1˚C - 103.7˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.77 - 6.96 (m, 4H, ArH), 5.80 (s, 1H HC = C), 5.11 - 5.01 (m, 1H, CH), 3.91 (s, 3H, CH₃), 2.97 (d, J = 13.7 Hz, 1H, CH₂), 2.56 (d, J = 13.7, 1H, CH₂), 2.45 - 2.31 (m, 2H, CH₂), 2.24 (d, J = 11.0 Hz, 1H, CH₂), 2.11 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.78 (d, J = 11.0 Hz, 1H, CH₂), 1.62 - 1.51 (m, 1H, CH), 1.10 (s, 3H, CH₃), 1.08 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 206.89, 191.67, 165.95, 165.27, 159.13, 143.36, 133.47, 131.31, 127.18, 126.70, 120.13, 112.09, 74.07, 55.90, 46.27, 42.29, 41.17, 31.61, 30.16, 22.59, 22.12, 17.19, 10.74. HR-MS (ESI): Calcd. C₂₃H₂₈O₄, [M+H]⁺ m/z: 368.2071, found: 368.2072.

4.1.2. General Procedure for the Synthesis of Compounds 2a-e

A mixture of petasin derivatives (1 equiv) which were synthesized as the route II and m-ClC₆H₅COOOH (1.5 equiv) in CH₂Cl₂ was stirred at room temperature for 3 h. The disappearance of petasin derivatives was monitored by TLC. Upon completion, the solvent was removed and water was added, the reaction mixture was extracted with CH₂Cl₂. The combined organic layer was washed with brine, dried over anhydrous MgSO₄ and concentrated under vacuum to afford the crude product. The crude product was purified by a flash column to yield the pure product 2a-e.

3',3',8,8a-tetramethyl-3-oxo-3,5,6,7,8,8a-hexahydro-1H-spiro[naphthalene-2,2'-oxiran]-7-ylbenzoate (2a). White solid, 90%, mp: 136.1˚C - 137.6˚C, ¹H NMR (400 MHz, CDCl₃) δ 8.07 - 7.48 (m, 5H, ArH), 5.97 (s, 1H, HC = C), 5.14 (m, 1H, CH), 2.66 (d, J = 14.5 Hz, 1H, CH₂), 2.54 - 2.45 (d, J = 14.5 Hz, 1H, CH₂), 2.38 (d, J = 11.2 Hz, 1H, CH₂), 2.28 - 2.15 (m, 2H, CH₂), 1.89 (d, J = 11.2, 1H, CH₂), 1.72 - 1.64 (m, 1H, CH), 1.48 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.05 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.74, 167.29, 166.14, 133.17, 130.13, 129.59, 129.59, 128.46, 128.46, 125.42, 73.47, 65.79, 65.15, 47.00, 42.12, 40.48, 31.47, 30.48, 21.20, 19.31, 18.93, 10.68. HR-MS (ESI): Calcd. C₂₂H₂₆O₄, [M+H]⁺ m/z: 354.1925, found: 354.1924.

3',3',8,8a-tetramethyl-3-oxo-3,5,6,7,8,8a-hexahydro-1H-spiro[naphthalene-2,2'-oxiran]-7-yl-4-methoxybenzoate (2b). White solid, yield 92%, mp: 206.4˚C - 208.8˚C, ¹H NMR (400 MHz, CDCl₃) δ 8.02 - 6.95 (m, 4H, ArH), 5.96 (s, 1H, HC = C), 5.10 (m, 1H, CH), 3.89 (s, 3H, CH₃), 2.66 (d, J = 14.5 Hz, 1H, CH₂), 2.49 (d, J = 14.5 Hz, 1H, CH₂), 2.40 - 2.33 (d, J = 11.2 Hz, 1H, CH₂), 2.27 - 2.08 (m, 2H, CH₂), 1.87 (d, J = 11.2 Hz, 1H, CH₂), 1.76 - 1.68 (m, 1H, CH), 1.48 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.29 (s, 3H, CH₃), 1.03 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.76, 167.47, 165.89, 163.55, 131.63, 131.63, 125.36, 122.50, 113.70, 113.70, 73.04, 65.81, 65.14, 55.48, 47.07, 42.13, 40.49, 31.55, 30.51, 21.19, 19.31, 18.93, 10.66. HR-MS (ESI): Calcd. C₂₃H₂₈O₅, [M+H]⁺ m/z: 384.1995, found: 384.1996.

3',3',8,8a-tetramethyl-3-oxo-3,5,6,7,8,8a-hexahydro-1H-spiro[naphthalene-2,2'-oxiran]-7-yl-2-nitrobenzoate (2c). White solid, yield 91%, mp: 169.8˚C - 171.7˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.97 - 7.68 (m, 4H, ArH), 5.96 (s, 1H, HC=C), 5.15 (m, 1H, CH), 2.63 (d, J = 14.8 Hz, 1H, CH₂), 2.52 (d, J = 14.7 Hz, 1H, CH₂), 2.47 (d, J = 11.2 Hz, 1H, CH₂), 2.13 (m, 2H, CH₂), 1.78 (d, J = 11.2 Hz, 1H, CH₂), 1.70 - 1.63 (m, 1H, CH), 1.48 (s, 3H, CH₃), 1.32 (s, 3H, CH₃), 1.29 (s, 3H, CH₃), 1.04 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.62, 166.79, 165.14, 147.90, 133.03, 131.82, 129.58, 127.77, 125.49, 124.01, 75.51, 65.72, 65.13, 46.56, 42.13, 40.36, 30.65, 30.30, 21.19, 19.27, 18.92, 10.59. HR-MS (ESI): Calcd. C₂₂H₂₅NO₆ [M+H]⁺ m/z: 399.2785, found: 399.2783.

3',3',8,8a-tetramethyl-3-oxo-3,5,6,7,8,8a-hexahydro-1H-spiro[naphthalene-2,2'-oxiran]-7-yl-2-fluorobenzoate (2d). White solid, yield 87%, mp: 136.4˚C - 137.8˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.86 - 7.32 (m, 4H, ArH), 5.97 (s, 1H HC = C), 5.16 - 5.05 (m, 1H, CH), 2.66 (d, J = 14.4 Hz, 1H, CH₂), 2.46 (d, J = 14.4 Hz, 1H, CH₂), 2.41 - 2.33 (d, J = 11.1 Hz, 1H, CH₂), 2.26 - 2.11 (m, 2H, CH₂), 1.89 (d, J = 11.2 Hz, 1H, CH₂), 1.72 - 1.63 (m, 1H, CH), 1.48 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.04 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.68, 167.0, 164.99, 163.73, 161.27, 132.31, 130.17, 125.34, 120.14, 116.61, 74.01, 65.76, 65.16, 46.91, 42.11, 40.46, 31.39, 30.41, 21.19, 19.29, 18.93, 10.69. HR-MS (ESI): Calcd. C₂₂H₂₅FO₄, [M+H]⁺ m/z: 372.1805, found: 372.1806.

3',3',8,8a-tetramethyl-3-oxo-3,5,6,7,8,8a-hexahydro-1H-spiro[naphthalene-2,2'-oxiran]-7-yl-2-fluorobenzoate (2e). White solid, yield 87%, mp: 123.8˚C - 124.1˚C, ¹H NMR (400 MHz, CDCl₃) δ 7.23 - 6.87 (m, 4H, ArH), 5.92 (s, 1H HC=C), 4.86 (m, 1H, CH), 3.82 (s, 3H, CH₃), 3.58 (s, 2H, CH₂), 2.53 (d, J = 14.4 Hz, 1H, CH₂), 2.46 (d, J = 14.4 Hz, 1H, CH₂), 2.23 - 2.11 (d, J = 11.2 Hz, 1H, CH₂), 2.04 (m, 2H, CH₂), 1.68 (d, J = 11.2 Hz, 1H, CH₂), 1.54 - 1.47 (m, 1H, CH), 1.45 (s, 3H, CH₃), 1.30 (s, 3H, CH₃), 1.21 (s, 3H, CH₃), 0.86 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 194.68, 171.53, 167.21, 158.76, 130.19, 130.19, 125.95, 125.35, 114.03, 114.03, 73.20, 65.74, 65.08, 55.26, 46.73, 42.02, 40.82, 40.39, 31.28, 30.37, 21.16, 19.21, 18.91, 10.37. HR-MS (ESI): Calcd. C₂₄H₃₀O₅, [M+H]⁺ m/z: 398.2160, found: 398.2161.

4.1.3. General Procedure for the Synthesis of Compounds 4a-b

A mixture of petsasinol (1 equiv) and Maleic anhydride (0.9 equiv) in CH₂Cl₂ in the presence of (C₂H₅)₃N (1 equiv) was stirred at 0˚C for 2 h. The disappearance of compound petsasinol was monitored by TLC. Upon completion, the solvent was removed and water containing HCl was added, the reaction mixture was extracted with CH₂Cl₂. The combined organic layer was dried over anhydrous MgSO₄ and concentrated under vacuum to afford the crude product. The crude product was added to a stirred solution of Excessive halids in the presence of NaCO₃ in DMF at 65˚C. The disappearance of the crude product was monitored by TLC. Upon completion, the solvent was removed and water containing NaCl was added, the reaction mixture was extracted with EA. The combined organic layer was dried over anhydrous MgSO₄ and concentrated under vacuum to afford the crude product. The crude product was purified by a flash column to yield the pure product 4a-b.

allyl (1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)-fumarate (4a). Yellow oil, yield 68%, ¹H NMR (400 MHz, CDCl₃) δ 6.31 (d, J = 12.2 Hz, 1H, HC = C), 6.28 (d, J = 12.1 Hz, 1H, HC = C), 5.97 (m, 1H, HC = C), 5.80 (s, 1H, HC = C), 5.38 (d, J = 17.2 Hz, 1H, HC = C), 5.30 (d, J = 10.4 Hz, 1H, HC = C), 4.96 (m, 1H, CH), 4.71 (d, J = 5.8 Hz, 2H, CH₂), 2.94 (d, J = 13.7 Hz, 1H, CH₂), 2.49 (d, J = 13.6, 4.4 Hz, 1H, CH₂), 2.34 (m, 2H, CH₂), 2.21 (d, J = 11.2 Hz, 1H, CH₂), 2.11 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.74 - 1.67 (d, J = 11.2 Hz, 1H, CH₂), 1.51 (m, 1H, CH), 1.05 (s, 3H, CH₃), 1.02 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 191.64, 164.89, 164.84, 163.42, 143.65, 131.53, 130.02, 129.58, 127.01, 126.76, 118.97, 75.03, 65.92, 45.99, 42.23, 41.10, 31.23, 29.99, 22.63, 22.14, 17.17, 10.70. HR-MS (ESI): Calcd. C₂₂H₂₈O₅, [M+H]⁺ m/z: 372.2015, found: 372.2013.

1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl-propylfumarate (4b). Yellow oil, yield 69%, ¹H NMR (400 MHz, CDCl₃) δ 6.29 (d, J = 12.3 Hz, 1H, HC = C), 6.25 (d, J = 12.3 Hz, 1H, HC = C), 5.80 (s, 1H, HC = C), 4.95 (m, 1H, CH), 4.16 (t, J = 6.8 Hz, 2H, CH₂), 2.94 (d, J = 13.7 Hz, 1H, CH₂), 2.50 (d, J = 13.7 Hz, 1H, CH₂), 2.41 - 2.27 (m, 2H, CH₂), 2.21 (d, J = 11.1 Hz, 1H, CH₂), 2.11 (s, 3H, CH₃), 1.87 (s, 3H, CH₃), 1.74 (dd, J = 14.3, 7.1 Hz, 2H, CH₂), 1.69 - 1.64 (d, J = 11.1 Hz, 1H, CH₂), 1.56 - 1.48 (m, 1H, CH), 1.05 (s, 3H, CH₃), 1.02 (d, J = 6.7 Hz, 3H, CH₃), 0.98 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 191.66, 171.60, 165.32, 164.88, 143.55, 130.10, 129.54, 127.04, 126.76, 74.91, 66.92, 51.64, 45.99, 42.22, 41.09, 31.23, 30.00, 22.62, 21.81, 20.72, 17.15, 10.70. HR-MS (ESI): Calcd. C₂₂H₃₀O₅, [M+H]⁺ m/z: 374.2174, found: 374.2172.

4.1.4. General Procedure for the Synthesis of Compounds 8a-c

A mixture of petasinol (1 equiv) and TsOCl (1.5 equiv) in CH₂Cl₂ in the presence of (C₂H₅)₃N (1 equiv) was stirred at 0˚C for 6 h. Then the mixture was filtered and concentrated in vacuum. The residue was purified by flash column to yield the pure product 5. The NaN₃ was added to a stirred solution of product 5 in DMF at 95˚C for 8 h. The disappearance of product 5 was monitored by TLC. After purification, the product 6 was afforded and was reduced with Zn power (1 equiv) in the presence of NH₄Cl (2 equiv) to produce 7. Condensation of product 7 with different kinds of acyl chloride (2 equiv) in CH₂Cl₂ in the presence of (C₂H₅)₃N (1 equiv) resulted in acyl derivatives. Upon completion, the solvent was removed and water was added, the reaction mixture was extracted with CH₂Cl₂. The combined organic layer was dried over anhydrous MgSO₄ and concentrated under vacuum to afford the crude product. The crude product was purified by a flash column to yield the pure product 8a-c.

N-(1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)acetamide (8a). Yellow oil, yield 66%, ¹H NMR (400 MHz, CDCl₃) δ 5.96 (s, 1H, NH), 5.75 (s, 1H, HC = C), 4.21 (m, 1H, CH), 2.87 (d, J = 14.4 Hz, 1H, CH₂), 2.44 (d, J = 14.6, Hz, 1H, CH₂), 2.24 - 2.18 (d, J = 11.1 Hz, 1H, CH₂), 2.15 - 2.04 (m, 2H, CH₂), 2.09 (s, 3H, CH₃), 2.02 (s, 3H, CH₃), 1.82 (s, 3H, CH₃), 1.75 - 1.67 (d, J = 11.1 Hz, 1H, CH₂), 1.24 (m, 1H, CH), 1.03 (s, 3H, CH₃), 1.01 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 191.44, 169.98, 165.75, 144.53, 127.17, 127.11, 49.39, 43.82, 41.67, 41.03, 30.95, 27.18, 23.77, 22.80, 22.31, 18.18, 12.02. HR-MS (ESI): Calcd. C₁₇H₂₅NO₂, [M+H]⁺ m/z: 275.1972, found: 275.1975.

2-chloro-N-(1,8a-dimethyl-6-oxo-7-(propan-2-ylidene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)acetamide (8b). Yellow oil, yield 66%, ¹H NMR (400 MHz, CDCl₃) δ 6.98 (s, 1H, NH), 5.83 (s, 1H, HC = C), 4.20 (m, 1H, CH), 4.13 (s, 2H, CH₂), 2.93 (d, J = 14.2 Hz, 1H, CH₂), 2.47 (d, J = 14.2 Hz, 1H, CH₂), 2.32 - 2.23 (d, J = 11.1 Hz, 1H, CH₂), 2.18 - 2.03 (m, 2H, CH₂),2.13 (s, 3H, CH₃), 1.85 (s, 3H, CH₃), 1.75 (d, J = 11.1 Hz, 1H, CH₂), 1.26 (m, 1H, CH), 1.11 (s, 3H, CH₃), 1.07 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 191.40, 165.75, 165.17, 144.61, 127.17, 127.06, 50.17, 43.76, 43.01, 41.58, 40.89, 30.75, 27.03, 22.78, 22.26, 17.88, 12.09. HR-MS (ESI): Calcd. C₁₇H₂₄ClNO₂, [M+H]⁺ m/z: 309.1579, found: 309.1580.

4-chloro-N-(1,8a-dimethyl-6-oxo-7-(propan-2-yli-dene)-1,2,3,4,6,7,8,8a-octahydronaphthalen-2-yl)benzamide (8c). Yellow oil, yield 66%, ¹H NMR (400 MHz, CDCl₃) δ 7.71 - 7.45 (m, 4H, ArH), 6.33 (s, 1H, NH), 5.86 (s, 1H, HC = C), 4.43 (m, 1H, CH), 2.95 (d, J = 14.2 Hz, 1H, CH₂), 2.49 (d, J = 14.2 Hz, 1H, CH₂), 2.33 - 2.28 (d, J = 11.1 Hz,), 2.22 - 2.19 (m, 2H, CH₂), 2.15 (s, 3H, CH₃), 1.92 (d, J = 11.3 Hz,), 1.87 (s, 3H, CH₃), 1.31 - 1.25 (m, 1H, CH), 1.17 (s, 3H, CH₃), 1.13 (d, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 191.37, 166.38, 165.29, 144.75, 137.92, 133.09, 131.46, 129.05, 128.77, 128.13, 127.24, 127.06, 50.12, 44.03, 41.59, 41.05, 30.89, 27.20, 22.83, 22.32, 18.29, 12.18. HR-MS (ESI): Calcd. C₂₂H₂₆ClNO₂, [M+H]⁺ m/z: 371.1710, found: 371.1708.

4.2. Bioassays

4.2.1. Grow Inhibition Assay

The petasin derivatives were assayed for in vitro anticancer activity by using MTT method. Cells were seeded in 96-well plates at a density of 4.6 × 10³ cells/well for 24 h. The cancer cell lines included SK-N-SH, MGC-803 and HepG-2. The seeded cells were treated with various concentrations of the test derivatives for 48 h. After 20 ml of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) solution (5 mg/mL) was added to each well and incubated for 4 h at 37˚C. The medium containing MTT was discarded; then 150 mL of dimethyl sulfoxide (DMSO) was added to each well. And the plates were agitated until the dark blue crystals (formazan) had completely dissolved. The absorbance was measured using a microplate reader at a wavelength of 490 nm. The average 50% inhibitory concentration (IC₅₀) was determined from the concentration response curves according to the inhibition ratio for each concentration.

4.2.2. Statistical Analysis

Cell activity data were analysed by the SPSS 17.0 software and were expressed as means ± standard error (SE).

Acknowledgements

The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 81273393).

References

1. Berbegall, A.P., Villamon, E., Tadeo, I., Martinsson, T., Canete, A., Castel, V., Navarro, S. and Noguera, R. (2014) Neuroblastoma after Childhood: Prognostic Relevance of Segmental Chromosome Aberrations, ATRX Protein Status, and Immune Cell Infiltration. Neoplasia, 16, 471-480. http://dx.doi.org/10.1016/j.neo.2014.05.012

2. Agarwala, S., Mandelia, A., Bakhshi, S., Srinivas, M., Bajpai, M., Gupta, A.K., Gupta, D.K. and Bhatnagar, V. (2014) Neuroblastoma: Outcome over a 14 Year Period from a Tertiary Care Referral Centre in India. Journal of Pediatric Surgery, 49, 1280-1285. http://dx.doi.org/10.1016/j.jpedsurg.2014.03.017

3. Spix, C., Pastore, G., Sankila, R., Stiller, C.A. and Steliarova-Foucher, E. (2006) Neuroblastoma Incidence and Survival in European Children (1978-1997): Report from the Automated Childhood Cancer Information System Project. European Journal of Cancer, 42, 2081-2091. http://dx.doi.org/10.1016/j.ejca.2006.05.008

4. Cheng, Y.F., Wang, C.F., Li, W.J., Lin, Q. and Zhang, Q. (2012) Antiproliferation Effects of Petasin on Human Neuroblastoma SK-N-SH Cells. Acta Neuropharmacologica, 1, 1-6.

5. Yi, W.-B., Zhu, Y.-W. and Cai, C. (2013) Fluorous 4-N,N-Dimethylaminopyridium Iodide: Recyclable Organocatalysts by Precipitation for Acylation Reaction at Room Temperature. Catalysis Communications, 41, 26-28. http://dx.doi.org/10.1016/j.catcom.2013.06.031

6. Liu, Z., Ma, Q., Liu, Y. and Wang, Q. (2014) 4-(N,N-Dimethylamino)Pyridine Hydrochloride as a Recyclable Catalyst for Acylation of Inert Alcohols: Substrate Scope and Reaction Mechanism. Organic Letters, 16, 236-239. http://dx.doi.org/10.1021/ol4030875

7. Ozaki, A., Sumita, K., Goto, K. and Matsumoto, A. (2013) Synthesis of Poly(Decahydro-2-Naphthyl Methacrylate)s with Different Geometric Structures and Effects of Side-Group Dynamics on Polymer Properties Investigated by Thermal and Dynamic Mechanical Analyses and DFT Calculations. Macromolecules, 46, 2941-2950. http://dx.doi.org/10.1021/ma400254d

8. Vyas, D.J., Larionov, E., Besnard, C., Guenee, L. and Mazet, C. (2013) Isomerization of Terminal Epoxides by a [Pd-H] Catalyst: A Combined Experimental and Theoretical Mechanistic Study. Journal of the American Chemical Society, 135, 6177-6183. http://dx.doi.org/10.1021/ja400325w

9. Hyun, M.Y., Kim, S.H., Song, Y.J., Lee, H.G., Jo, Y.D., Kim, J.H., Hwang, I.H., Noh, J.Y., Kang, J. and Kim, C. (2012) Terminal and Internal Olefin Epoxidation with Cobalt(II) as the Catalyst: Evidence for an Active Oxidant Co(II)-Acylperoxo Species. The Journal of Organic Chemistry, 77, 7307-7312. http://dx.doi.org/10.1021/jo3009963

10. Elbert, B.L., Lim, D.S., Gudmundsson, H.G., O’Hanlon, J.A. and Anderson, E.A. (2014) Synthesis of Cyclic Alkenylsiloxanes by Semihydrogenation: A Stereospecific Route to (Z)-Alkenyl Polyenes. Chemistry, 20, 8594-8598. http://dx.doi.org/10.1002/chem.201403255

11. Maury, J., Feray, L., Bertrand, M.P., Kapat, A. and Renaud, P. (2012) Unexpected Conversion of Alkyl Azides to Alkyl Iodides and of Aryl Azides to N-Tert-Butyl Anilines. Tetrahedron, 68, 9606-9611. http://dx.doi.org/10.1016/j.tet.2012.09.066

12. Jurícek, M., Felici, M., Contreras-Carballada, P., Lauko, J., Bou, S.R., Kouwer, P.H.J., Brouwer, A.M. and Rowan, A.E. (2011) Triazole-Pyridine Ligands: A Novel Approach to Chromophoric Iridium Arrays. Journal of Materials Chemistry, 21, 2104-2111. http://dx.doi.org/10.1039/C0JM03117H

13. Ogawa, S., Asada, M., Ooki, Y., Mori, M., Itoh, M. and Korenaga, T. (2005) Design and Synthesis of Glycosidase Inhibitor 5-Amino-1,2,3,4-cyclohexanetetrol Derivatives from (−)-Vibo-Quercitol. Bioorganic & Medicinal Chemistry, 13, 4306-4314. http://dx.doi.org/10.1016/j.bmc.2005.04.003

14. Reed, C.S., Huigens III, R.W., Rogers, S.A. and Melander, C. (2010) Modulating the Development of E. coli Biofilms with 2-Aminoimidazoles. Bioorganic & Medicinal Chemistry Letters, 20, 6310-6312. http://dx.doi.org/10.1016/j.bmcl.2010.08.075

15. Ma, L., Li, S., Zheng, H., Chen, J., Lin, L., Ye, X., Chen, Z., Xu, Q., Chen, T., Yang, J., Qiu, N., Wang, G., Peng, A., Ding, Y., Wei, Y. and Chen, L. (2011) Synthesis and Biological Activity of Novel Barbituric and Thiobarbituric Acid Derivatives against Non-Alcoholic Fatty Liver Disease. European Journal of Medicinal Chemistry, 46, 2003-2010. http://dx.doi.org/10.1016/j.ejmech.2011.02.033

16. Kumar, B.R.P. and Nanjan, M.J. (2010) Novel Glitazones: Design, Synthesis, Glucose Uptake and Structure-Activity Relationships. Bioorganic & Medicinal Chemistry Letters, 20, 1953-1956. http://dx.doi.org/10.1016/j.bmcl.2010.01.125

17. Zhou, L. and Lu, W. (2014) Palladium-Catalyzed Beta-Acyloxylation of Simple Amide via sp3 C-H Activation. Organic Letters, 16, 508-511. http://dx.doi.org/10.1021/ol403393w

18. Jin, Y., Chen, S.W. and Tian, X. (2006) Synthesis and Biological Evaluation of New Spin-Labeled Derivatives of Podophyllotoxin. Bioorganic & Medicinal Chemistry, 14, 3062-3068. http://dx.doi.org/10.1016/j.bmc.2005.12.025

19. Perez-Areales, F.J., Di Pietro, O., Espargaro, A., Vallverdu-Queralt, A., Galdeano, C., Ragusa, I.M., Viayna, E., Guillou, C., Clos, M.V., Perez, B., Sabate, R., Lamuela-Raventos, R.M., Luque, F.J. and Munoz-Torrero, D. (2014) Shogaol-Huprine Hybrids: Dual Antioxidant and Anticholinesterase Agents with β-Amyloid and Tau Anti-Aggregating Properties. Bioorganic & Medicinal Chemistry, 22, 5298-5307. http://dx.doi.org/10.1016/j.bmc.2014.07.053

20. Kawano, H., Shakushiro, K., Nakata, M., Kita, A., Maeda, A., Watanabe, S., Sako, K. and Oku, N. (2014) Antitumor Efficacy and Biodistribution of Liposomal Sepantronium Bromide (YM155), a Novel Small-Molecule Survivin Suppressant. European Journal of Pharmaceutics and Biopharmaceutics, 88, 283-289. http://dx.doi.org/10.1016/j.ejpb.2014.06.015

21. Rodriguez-Berna, G., Mangas-Sanjuan, V., Gonzalez-Alvarez, M., Gonzalez-Alvarez, I., Garcia-Gimenez, J.L., Diaz Cabanas, M.J., Bermejo, M. and Corma, A. (2014) A Promising Camptothecin Derivative: Semisynthesis, Antitumor Activity and Intestinal Permeability. European Journal of Medicinal Chemistry, 83, 366-373. http://dx.doi.org/10.1016/j.ejmech.2014.06.050

22. Zhang, D., Debnath, B., Yu, S., Sanchez, T.W., Christ, F., Liu, Y., Debyser, Z., Neamati, N. and Zhao, G. (2014) Design and Discovery of 5-Hydroxy-6-Oxo-1,6-Dihydropyrimidine-4-Carboxamide Inhibitors of HIV-1 Integrase. Bioorganic & Medicinal Chemistry, 22, 5446-5453. http://dx.doi.org/10.1016/j.bmc.2014.07.036