English

• 1.   Introduction

  Rosins are the solid form of resins obtained from pines or similar types of plants belonging to the conifer family and they are important natural resources of China. Pine resin acids are the main components of rosin, and they have generated signcant interest from the medicinal, pharmacological and forestry communities for their wide variety of interesting biological activities.^[1] Dehydroabietic acid (DHAA) is a natural occurring diterpenic resin acid which can be easily obtained from Pinus rosin or from commercial disproportionated rosin.^[2] DHAA and its derivatives exhibited extensive pharmaceutical activities, such as antitumor, ^[3-7] antimicrobial, ^[8-10] antiprotozoal, ^[11] antiviral, ^[12] antifungal, ^[13-14] gastroprotective, ^[15] insecticidal^[16] anti-inflammatory^[17] antiulcer, ^[18] and anxiolytic^[19] activities. Furthermore, DHAA was reported to show properties of enhancing the inhibitory activity of an anticancer drug in cervical carcinoma cells, hepatocellular carcinoma cells, or breast cancer cells. These results suggest that DHAA is a promising starting material for the discovery of novel anticancer agents.

  In previous study, numerous reports demonstrate that nitric oxide (NO) is a multifunctional signaling and/or effector molecule with numerous functions in vasodilation, neurotransmission, the immune system, and cell apoptosis.^[20-21] It has been shown that high levels of NO can directly kill tumor cells, induce tumor cell apoptosis, impair tumor cell proliferation and invasion ability via nitric oxide-releasing, and prevent tumor cells from metastasizing and increase tumor cell response to radiochemotherapy. Particularly, it is reported that numerous NO-donating hybrids have the potential to provide novel anticancer candidates with low toxicity and high efficacy against drug-resistant cancers.^[22-27]

  Enlightened by these findings, together with our previous studies on the modification of natural products, ^[28-31] a modification via integrating DHAA and nitrate pharmacophore into one molecule to generate a novel dehydroabietic acid NO donor hybrids was conducted. The screening of the cytotoxicity of these hybrids against four human cancer cell lines and normal human cell were carried on. The NO released amounts of these hybrids by Griess assay to investigate the relationship between NO released amounts and cytotoxic activities were also detected.

  2.   Results and discussion

  2.1   Synthesis and characterization

  The synthetic route of these compounds is depicted in Scheme 1. Using dehydroabietic acid as starting material, the key intermediates 12-bromodehydroabietic acid (2) and 12, 14-dinitrodehydroabietic acid (3) were synthesized by reaction of DHAA with bromides and fuming nitric acid, respectively. Then they reacted with dibromohydrocarbon and silver nitrate to obtain the dehydroabietic acid-nitrate conjugates 5a~5r in a good yield. Synthetic compounds 5 need to be carried out protected from light.

  Scheme 1

  

  Scheme 1.  Synthetic route for dehydroabietic acid-based nitrate compounds 5a~5r

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  The FT-IR spectra of target compounds 5a~5r exhibited characteristic moderate absorption bands at about 1720 cm^-1 attributed to the stretching vibrations of C=O. The bands at about 1630 and 1280 cm^-1 were attributed to the stretching vibration of ONO₂. The bands at 1600~1450 cm^-1 were assigned to the vibration of the skeleton in benzene rings. The bands at 2950~2800 cm^-1 were assigned to the stretching vibrations of C—H in methyl or methylene. The ¹H NMR spectra of the target compounds 5a~5r showed characteristic signals at δ 7.56~6.89 assigned to the aromatic protons. The ¹³C NMR spectra of the target compounds 5a~5r showed peaks for C=O atδ 179~177, and for benzene rings at δ 152~121.

  2.2   Evaluation of cytotoxicity

  Given that nasopharyngeal carcinoma, liver cancer and cervical cancer are very common cancer types, CNE-2, HepG2, BEL-7402 and HeLa cells were chosen. (3-(4, 5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was applied to determine the effects of dehydroabietic acid nitrates on the growth inhibition against cancer cells. The results were listed in Table 1.

  Table 1

  

  Table 1.  Cytotoxic activity of dehydroabietic acid-based nitrate compounds

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  Compd.	IC50a/(μmol•L-1)
  	CNE-2	HepG2	BEL-7402	HeLa	HL-7702	NP69
  DHAA	88.64±0.73	80.36±0.84	46.70±0.55	37.40±0.64	> 100	> 100
  4i	75.84±1.42	61.55±0.37	53.18±1.83	88.28±3.12	> 100	> 100
  4j	60.33±2.54	45.96±0.77	69.24±1.63	73.04±1.08	> 100	> 100
  4k	42.29±0.86	57.58±1.26	48.35±0.48	50.28±0.57	> 100	> 100
  5a	43.88±0.15	50.44±0.08	38.25±0.44	44.32±0.28	> 100	> 100
  5b	35.64±0.48	66.93±0.20	42.11±0.64	38.12±0.15	> 100	> 100
  5c	33.09±0.23	57.34±0.15	24.02±0.62	40.10±0.23	> 100	> 100
  5d	36.32±0.57	34.62±0.17	32.22±0.37	33.44±0.72	> 100	> 100
  5e	20.63±0.66	25.84±0.22	50.78±0.32	14.14±0.21	> 100	> 100
  5f	25.44±0.74	21.94±0.54	14.88±0.30	27.34±0.64	> 100	> 100
  5g	27.26±0.55	30.17±0.32	18.40±0.24	67.33±0.46	> 100	> 100
  5h	18.20±0.16	20.46±0.18	17.52±0.64	55.45±0.40	74.39±0.19	89.35±0.56
  5i	34.16±0.43	30.64±0.24	44.72±0.46	65.15±0.74	> 100	> 100
  5j	8.36±0.14	13.00±0.61	55.33±0.42	23.36±0.44	> 100	> 100
  5k	16.84±0.34	8.83±0.65	35.14±0.13	12.06±0.52	87.66±0.43	> 100
  5l	22.33±0.56	15.29±0.42	38.78±0.36	7.88±0.58	> 100	> 100
  5m	9.24±0.40	34.88±0.10	32.47±0.42	8.42±0.70	> 100	> 100
  5n	66.89±0.67	10.40±0.27	11.23±0.21	15.16±0.81	88.18±0.23	> 100
  5o	25.00±0.11	30.53±0.82	14.66±0.40	42.51±0.18	> 100	> 100
  5p	15.66±0.42	22.08±0.45	44.05±0.30	14.06±0.16	> 100	> 100
  5q	9.63±0.33	13.90±0.16	33.88±0.08	4.57±0.41	65.28±0.35	92.86±0.75
  5r	20.87±0.08	28.66±0.43	18.93±0.37	34.78±0.13	> 100	> 100
  Cisplatin	8.75±0.24	6.42±0.18	12.68±0.33	1.94±0.20	20.76±0.83	10.74±0.52
  a Data represent the means±S.D. from three independent experiments.

  As tabulated in Table 1, most of dehydroabietic acid nitrates were more potent than the parent compounds dehydroabietic acid and displayed moderate cytotoxicity effects. It is found that compounds 5j and 5n showed significant inhibition enhancement against CNE-2 and BEL- 7402, respectively. Compared with DHAA, the inhibition activity of 5j against CNE-2 was increased by 10 times with IC₅₀ value of (8.36±0.14) μmol/L, which was the most active compound against CNE-2 cancer cell amongst all the tested derivatives; While 5n exhibited the best inhibition activity against BEL-7402 cancer cells with IC₅₀ value of (11.23±0.21) μmol/L, which was more active than cisplatin. Most impressively, the cytotoxicity of dehydroabietic acid nitrates is dependent on the length, kind of linkers and the substituent in benzene ring. For both CNE-2 and HepG2 cancer cells, the cytotoxicity of nitrate with unsaturated carbon chains is slightly better than that of saturated carbon chains, and the compounds with bromine substitution at position-12 exhibited greater potency than without substituents. The most of compounds with nitro substitution at position-12 and position-14 exhibited significant inhibition enhancement against the growth of all the tested cancer cells. Compound 5k showed the best inhibition activity against HepG2 with IC₅₀ value of (8.83±0.65) μmol/L, which was 9 times more active than the parent compound. Futhermore, it was found that the cytotoxicities of dehydroabietic acid-nitrate hybrid molecules against human normal liver cell line HL-7702 and nasopharyngeal epithelial cell NP69 are low, in which the IC₅₀ values are over (65.28±0.35) μmol/L.

  2.3   Evaluation of NO release

  To explore the association between NO release amounts of these nitrates and their cytotoxic activity, the NO release levels of these nitrates in vitro were detected using nitrate/nitrite assay. Thus, we decided to evaluate the cellular levels of NO of all nitrates at 4 mmol/L for 24 h. As shown in Figure 1, it was observed that the nitrates designed in this study could produce different levels of NO intracellularly which generally agreed with the cytotoxic activity.

  Figure 1

  

  Figure 1.  NO released amounts of the target compounds in CNE-2 cells for 24 h (mean±SD, n=3)

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  3.   Conclusions

  In conclusion, eighteen novel nitrates derivatives have been designed and synthesized using dehydroabietic acid as a starting material. All the compounds were confirmed with FT-IR, ESI-MS, ¹H NMR, ¹³C NMR and elemental analysis. Their cytotoxic activities against CNE-2, HepG2, BEL-7402 and HeLa cancer cell lines were determined. The preliminary bioassay showed that compound 5j was demonstrated as the most potent inhibitor against CNE-2 cell line with IC₅₀ value of (8.36±0.14) μmol/L; while 5n was confirmed as the most potent agent against BEL-7402 cancer cells with IC₅₀ value of (11.23±0.21) μmol/L. NO released amounts revealed that the potent compounds which exhibited high cytotoxic activities consistently produced high levels of NO in CNE-2. Therefore, the nitrate-hybrids could be a promising strategy for the synthesis of antitumor dehydroabietic acid derivatives.

  4.   Experimental section

  4.1   General

  All the chemicals and reagents were commercially available and used without further purification. Routine thin-layer chromatography (TLC) was performed on silica gel plates (silica gel GF254 from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), preparative flash column chromatography was performed on the 200~300 mesh silica gel (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) without calibration. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance Ⅲ HD 600 MHz spectrometer (Bruker Co., Ltd., Zurich, Switzerland) at room temperature with TMS as an internal standard and CDCl₃ as solvents. Mass spectra were recorded on a liquid chromatograph mass spectrometer (Shimazu Co. Ltd., Kyoto, Japan). Elemental analyses were carried out on a PE 2400II elemental analyzer (Perkin Elmer Instruments Co., Ltd., USA). The optical density was measured on a microplate reader (BioTek Instruments Co., Ltd., USA).

  4.2   Synthesis

  4.2.1   Synthetic route for intermediates 2

  According to literature method, ^[32] dehydroabietic acid (12.0 g, 40 mmol) was dissolved in glacial acetic acid (40 mL), then bromine (3.2 mL) dissolved in glacial acetic acid (20 mL) was added slowly to the above solution. The reaction solution continued stirring for 3 h at room temperature. Standing overnight, the reaction (pH=6) was adjusted with concentrated ammonia water, the sediments was filtered and recrystallized with 65% ethanol (50 mL) to obtain compound 2 (yield 63.8 %).

  3.2.2   Synthetic route for intermediates 3

  According to literature method, ^[33] to a mixture of concentrated sulfuric acid (60 mL) and fuming nitric acid (40 mL) cooled in ice bath, dehydroabietic acid (14 g) was added in batches. The mixture was stirred and allowed to come to room temperature. After completion of the reaction, the mixture slowly poured into 900 mL of ice water. Standing overnight, the reaction was drained and washed with distilled water until neutral. The crude products were recrystallized with ethanol three times to give compound 3 (yield 70.4%).

  3.2.3   Synthetic route for intermediates 4a~4r

  1, 12-Bromodehydroabietic acid (2) or 12, 14-dinitrode- hydroabietic acid (3) (21.12 mmol), dibromoalkane (40.00 mmol), and anhydrous K₂CO₃ (2.91 g) were added to 40 mL of N, N-dimethylformamide (DMF) and stired for 6 h at 40 ℃. After the reaction, the mixture was poured into cold water and extracted with ethyl acetate (40 mL×3), and then the combined organic layer was washed with saturated salt water and dried (Na₂SO₄) overnight. The solution was concentrated with a rotary evaporator, the intermediates 4a~4r were purified by silica gel chromatography with a mixture of petroleum ether and ethyl acetate (V:V=20:1) as eluent.

  Dehydroabietic acid-2'-bromoethyl ester (4a): White solid, yield 78.6%. m.p. 46.2~49.4 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.22 (d, J=8.4 Hz, 1H, H-11), 7.06 (d, J=8.4 Hz, 1H, H-12), 6.95 (s, 1H, H-14), 4.50~4.37 (m, 2H, H-21), 3.57~3.54 (t, J=6.0 Hz, 2H, H-22), 3.01~2.97 (m, 1H, H-15), 2.94~2.85 (m, 2H, H-7), 2.33~2.37 (m, 2H, He-1, H-5), 1.92~1.71 (m, 5H, H-2, H-3 and He-6), 1.47~1.57 (m, 2H, Ha-1, Ha-6), 1.35 (s, 3H, H-19), 1.28 (s, 3H, H-20), 1.27 (d, J=1.8 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 179.6, 148.2, 147.2, 136.1, 128.4, 125.6, 125.4, 65.5, 49.3, 46.1, 39.4, 38.4, 38.1, 34.9, 30.5, 25.5, 23.3, 20.1, 18.0; IR (KBr) ν: 2867, 1733 (C=O), 1496, 1498, 1387, 1243, 1172, 1006, 821, 682 cm^-1. Anal. calcd for C₂₂H₃₁BrO₂: C 64.86, H 7.67; found C 64.79, H 7.63.

  Dehydroabietic acid-3'-bromopropyl ester (4b): Color- less liquid, yield 75.4%. ¹H NMR (600 MHz, CDCl₃) δ: 7.20 (d, J=8.2 Hz, 1H, H-11), 7.04 (d, J=8.1 Hz, 1H, H-12), 6.92 (s, 1H, H-14), 4.24 (ddd, J=17.5, 11.0, 5.3 Hz, 2H, H-21), 3.47 (t, J=6.5 Hz, 2H, H-23), 2.91 (dd, J=10.6, 5.3 Hz, 2H, H-7), 2.86 (dt, J=13.8, 7.0 Hz, 1H, H-15), 2.34 (d, J=12.8 Hz, 1H, He-1), 2.26 (dd, J=12.5, 2.1 Hz, 1H, H-5), 2.22~2.17 (m, 2H, H-22), 1.94~1.69 (m, 5H, H-2, H-3, He-6), 1.53 (t, J=12.5 Hz, 1H, Ha-1), 1.43 (dtd, J=8.0, 6.0, 2.5 Hz, 1H, Ha-6), 1.31 (s, 3H, H-19), 1.26 (d, J=1.8 Hz, 6H, H-16, H-17), 1.24 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.4, 146.8, 145.8, 134.6, 127.0, 124.2, 124.0, 62.3, 47.8, 44.9, 37.9, 37.0, 36.7, 33.6, 33.5, 31.7, 31.6, 30.1, 29.5, 25.3, 24.5, 24.0, 21.8, 18.6, 16.5; IR (KBr) ν: 2916, 1724 (C=O), 1498, 1458, 1385, 1238, 1174, 1125, 1037, 883, 822 cm^-1. Anal. calcd for C₂₃H₃₃BrO₂: C 65.55, H 7.89; found C 65.58, H 7.84.

  Dehydroabietic acid-4'-bromobutyl ester (4c): Colorless liquid, yield 74.3%. ¹H NMR (600 MHz, CDCl₃) δ: 7.17 (d, J=8.4 Hz, 1H, H-11), 7.01 (d, J=8.4 Hz, 1H, H-12), 6.90 (s, 1H, H-14), 4.15~4.06 (m, 2H, H-21), 3.43 (t, J=6.0 Hz, 2H, H-24), 2.89~2.87 (m, 2H, H-7), 2.86~2.81 (m, 1H, H-15), 2.30 (d, J=12.0 Hz, 1H, He-1), 2.25(d, J=12.0 Hz, 1H, H-5), 1.95~1.93 (m, 2H, H-22), 1.86~1.63 (m, 7H, H-2, H-3, He-6, H-23), 1.52~1.48 (m, 1H, Ha-1), 1.42~1.39 (m, 1H, Ha-6), 1.28 (s, 3H, H-19), 1.23~1.24 (d, J=7.2 Hz, 6H, H-16, H-17), 1.22 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.5, 146.9, 145.8, 134.6, 127.0, 124.2, 124.0, 64.0, 63.6, 47.7, 44.8, 38.0, 37.0, 36.7, 33.5, 33.0, 30.1 29.3, 27.4, 25.5, 25.3, 24.0, 21.8, 18.6, 16.6; IR (KBr) ν: 2933, 1722 (C=O), 1496, 1463, 1384, 1246, 1174, 1126, 1035, 887, 823 cm^-1. Anal. calcd for C₂₄H₃₅BrO₂: C 66.20, H 8.10; found C 66.25, H 8.12.

  Dehydroabietic acid-5'-bromoamyl ester (4d): Colorless liquid, yield 80.4%. ¹H NMR (600 MHz, CDCl₃) δ: 7.20 (d, J=8.4 Hz, 1H, H-11), 7.03 (d, J=8.4 Hz, 1H, H-12), 6.92 (s, 1H, H-14), 4.15~4.06 (m, 2H, H-21), 4.44~3.42 (m, 2H, H-25), 2.91~2.83 (m, 3H, H-7, H-15), 2.32 (d, J=11.4 Hz, 1H, He-1), 2.26 (d, J=12.0 Hz, 1H, H-5), 1.94~1.54 (m, 11H, H-2, H-3, He-6, H-22, H-23 and H-24), 1.45~1.35 (m, 2H, Ha-1 and Ha-6), 1.30 (s, 3H, H-19), 1.24~1.26 (m, 9H, H-16, H-17 and H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.6, 146.9, 145.7, 134.7, 126.9, 124.2, 124.0, 64.2, 47.7, 44.8, 38.0, 37.0, 36.7, 33.5, 33.4, 30.1, 25.2, 24.7, 24.0, 21.8, 18.6 16.5; IR (KBr) ν: 2908, 1722 (C=O), 1496, 1463, 1388, 1246, 1174, 1126, 1035, 887, 823, 723 cm^-1. Anal. calcd for C₂₅H₃₇BrO₂: C 66.81, H 8.30; found C 66.77, H 8.26.

  Dehydroabietic acid-6'-bromohexyl ester (4e): Colorless liquid, yield 58.6%. ¹H NMR (600 MHz, CDCl₃) δ: 7.20 (d, 1H, J=8.1 Hz, H-11), 7.03 (d, 1H, J=8.1 Hz, H-12), 6.91 (s, 1H, H-14), 4.14~4.06 (m, 2H, H-21), 3.45~3.41 (m, 2H, H-26), 2.90 (dd, J=8.9, 4.3 Hz, 2H, H-7), 2.85 (dt, J=13.8, 6.9 Hz, 1H, H-15), 2.33 (d, 1H, J=12.2 Hz, He-1), 2.27 (dd, J=12.5, 2.0 Hz, 1H, H-5), 1.89~1.42 (m, 15H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24 and H-25), 1.29 (s, 3H, H-19), 1.25 (d, J=6.9 Hz, 6H, H-16 and H-17), 1.24 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.6, 147.0, 145.7, 134.7, 126.9, 124.2, 124.0, 64.5, 47.7, 44.8, 38.0, 37.0, 36.7, 33.7, 33.5, 32.6, 30.2, 28.5, 27.8, 25.3, 24.0, 21.8, 18.6, 16.5; IR (KBr) ν: 2933, 1722 (C=O), 1462, 1384, 1246, 1176, 1126, 1007, 975, 825, 726 cm^-1. Anal. calcd for C₂₆H₃₉BrO₂: C 67.38, H 8.48; found C 67.37, H 8.51.

  Dehydroabietic acid-8′-bromooctyl ester (4f): Colorless liquid, yield 72.8%. ¹H NMR (600 MHz, CDCl₃) δ: 7.11 (d, J=8.2 Hz, 1H, H-11), 6.93 (d, J=8.4 Hz, 1H, H-12), 6.82 (s, 1H, H-14), 4.05~3.91 (m, 2H, H-21), 3.33 (q, J=6.8, 6.8 Hz, 2H, H-28), 2.82~2.72 (m, 3H, H-7 and H-15), 2.23 (d, J=12.6 Hz, 1H, He-1), 2.18 (dd, J=12.5, 1.9 Hz, 1H, H-5), 1.82~1.25 (m, 19H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24, H-25, H-26 and H-27), 1.19 (s, 3H, H-19), 1.16 (s, 3H, H-20), 1.14 (d, J=3.1 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.7, 147.0, 145.7, 134.7, 126.9, 124.2, 124.0, 64.6, 47.7, 44.8, 38.0, 37.0, 36.6, 34.0, 33.5, 32.8, 30.2, 29.0, 28.6, 28.6, 28.6, 28.1, 25.9, 25.3, 24.0, 21.8, 18.7, 16.5; IR (KBr) ν: 2929, 2860, 1722 (C=O), 1496, 1462, 1382, 1244, 1176, 1124, 1035, 975, 823 cm^-1. Anal. calcd for C₂₈H₄₃BrO₂: C 68.42, H 8.82; found C 68.45, H 8.86.

  Dehydroabietic acid-12'-bromodecyl ester (4g): Color- less liquid, yield 62.8%. ¹H NMR (600 MHz, CDCl₃) δ: 7.20 (d, J=8.2 Hz, 1H, H-11), 7.03 (d, J=8.2 Hz, 1H, H-12), 6.91 (s, 1H, H-14), 4.08 (dq, J=17.4, 6.6 Hz, 2H, H-21), 3.43 (t, J=6.9 Hz, 2H, H-32), 2.90 (dd, J=10.8, 5.2 Hz, 2H, H-7), 2.85 (dt, J=13.8, 7.0 Hz, 1H, H-15), 2.33 (d, J=12.2 Hz, 1H, He-1), 2.28 (dd, J=12.5, 2.0 Hz, 1H, H-5), 1.91~1.29 (m, 30H, Ha-1, H-2, H-3, H-6, H-19, H-22, H-23, H-24, H-25, H-26, H-27, H-28, H-29, H-30 and H-31), 1.25 (d, J=6.9 Hz, 6H, H-16, H-17), 1.24 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.7, 147.0, 145.7, 134.7, 127.9, 124.2, 123.9, 64.7, 47.7, 44.8, 38.0, 37.0, 36.6, 34.1, 33.5, 32.9, 30.2, 29.5, 29.5, 29.4, 29.2, 28.8, 28.7, 28.2, 26.0, 25.3, 24.0, 21.8, 18.7, 16.5; IR (KBr) ν: 3074, 2954, 2872, 1724 (C=O), 1490, 1460, 1382, 1242, 1176, 1122, 1035, 981, 916, 823 cm^-1. Anal. calcd for C₃₂H₅₁BrO₂: C 70.18, H 9.39; found C 70.15, H 9.42.

  Dehydroabietic acid-4'-bromobutenyl ester (4h): Color- less liquid, yield 53.7%. ¹H NMR (600 MHz, CDCl₃) δ: 7.23 (s, 1H, H-11), 7.06 (d, J=8.0 Hz, 1H, H-12), 6.94 (s, 1H, H-14), 5.83 (dd, J=17.1, 10.3 Hz, 2H, H-22, H-23), 5.13 (dd, J=24.5, 13.7 Hz, 2H, H-21), 4.18 (td, J=17.4, 6.7 Hz, 2H, H-24), 2.95~2.86 (m, 3H, H-7 and H-15), 2.35 (d, J=12.3 Hz, 1H, He-1), 2.31 (d, J=11.2 Hz, 1H, H-5), 1.91~1.75 (m, 5H, H-2, H-3, He-6), 1.58~1.53 (m, 1H, Ha-1), 1.50~1.46 (m, 1H, Ha-6), 1.32 (s, 3H, H-19), 1.28 (d, J=6.9 Hz, 6H, H-16, H-17), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 179.9, 148.4, 147.1, 136.2, 135.6, 128.4, 125.6, 125.4, 118.6, 65.1, 49.1, 46.2, 39.4, 38.4, 38.1, 34.9, 34.6, 31.6, 26.7, 25.4, 23.2, 20.1, 17.9; IR (KBr) ν: 2954, 2872, 1726 (C=O), 1496, 1460, 1382, 1240, 1174, 1120, 1033, 979, 927, 823 cm^-1. Anal. calcd for C₂₄H₃₃BrO₂: C 66.51, H 7.67; found C 66.55, H 7.65.

  12-Bromodehydroabietic acid-2'-bromoethyl ester (4i): White solid, yield 80.0%. m.p. 102.5~103.8 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.38 (s, 1H, H-11), 6.93 (s, 1H, H-14), 4.47~4.32 (m, 2H, H-21), 3.51 (t, J=6.6 Hz, 2H, H-22), 3.28~3.25 (m, 1H, H-15), 2.87~2.82 (m, 2H, H-7), 2.27~2.20 (m, 2H, He-1, H-5), 1.83~1.67 (m, 5H, H-2, H-3, He-6), 1.51~1.46 (m, 2H, Ha-1, Ha-6), 1.30 (s, 3H, H-19), 1.20~1.23 (m, 9H, H-20, H-16, H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 179.4, 150.2, 145.5, 135.9, 129.9, 128.6, 123.0, 65.5, 49.1, 45.7, 39.2, 38.4, 38.0, 33.8, 31.0, 30.5, 25.6, 24.4, 24.2, 23.0, 19.9, 17.9; IR (KBr) ν: 2931, 2866, 1718 (C=O), 1462, 1386, 1386, 1238, 1182, 1105, 1033, 975, 883, 804 cm^-1. Anal. calcd for C₂₂H₃₀- Br₂O₂: C 54.34, H 6.22; found C 54.35, H 6.26.

  12-Bromodehydroabietic acid-3'-bromopropyl ester (4j): White solid, yield 77.0%. m.p. 137.7~139.7 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.94 (s, 1H, H-14), 4.27~4.20 (m, 2H, H-21), 3.46 (t, J=6.6 Hz, 2H, H-23), 3.29~3.26 (m, 1H, H-15), 2.89~2.84 (m, 2H, H-7), 2.26 (d, J=12.6 Hz, 1H, He-1), 2.17~2.20 (m, 3H, H-5, H-22), 1.88~1.67 (m, 5H, H-2, H-3, He-6), 1.51~1.53 (m, 1H, Ha-1), 1.43~1.46 (m, 1H, Ha-6), 1.29 (s, 3H, H-19), 1.22~1.25 (m, 9H, H-20, H-16, H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.2, 148.8, 144.1, 134.3, 128.54, 127.2, 121.5, 62.3, 47.6, 44.6, 37.8, 37.1, 36.6, 32.3, 31.6, 29.6, 29.5, 25.1, 22.8, 21.6, 18.5, 16.5; IR (KBr) ν: 2927, 1712 (C=O), 1463, 1240, 1107, 1039, 979 cm^-1. Anal. calcd for C₂₃H₃₂Br₂O₂: C 55.22, H 6.45; found C 55.24, H 6.41.

  12-Bromodehydroabietic acid-4'-bromobutyl ester (4k): Pale yellow solid, yield 66.8%. m.p. 95.5~97.9 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.94 (s, 1H, H-14), 4.16~4.09 (m, 2H, H-21), 3.44 (t, J=6.6 Hz, 2H, H-24), 3.31~3.26 (m, 1H, H-15), 2.86~2.83 (m, 2H, H-7), 2.26 (d, J=12.6 Hz, 1H, He-1), 2.19 (d, J=12.6 Hz, 1H, H-5), 1.95 (q, J=6.6 Hz, 2H, H-22), 1.81~1.67 (m, 7H, H-2, H-3, He-6 and H-23), 1.53~1.49 (m, 1H, Ha-1), 1.46~1.42 (m, 1H, Ha-6), 1.29 (s, 3H, H-19), 1.25~1.22 (m, 9H, H-20, H-16, H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.3, 148.9, 144.1, 134.4, 128.5, 127.2, 121.5, 63.6, 47.6, 44.6, 37.9, 37.0, 36.6, 33.0, 32.3, 29.6, 29.3, 27.4, 25.1, 23.0, 22.8, 21.5, 18.5, 16.5; IR (KBr) ν: 2956, 1708 (C=O), 1467, 1388, 1244, 1109, 883 cm^-1. Anal. calcd for C₂₄H₃₄Br₂O₂: C 56.04, H 6.66; found C 56.10, H 6.68.

  12-Bromodehydroabietic acid-5'-bromoamyl ester (4l): Pale yellow oil, yield 75.3%. ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.94 (s, 1H, H-14), 4.13~4.07 (m, 2H, H-21), 3.42 (t, J=6.6 Hz, 2H, H-25), 3.28~3.26 (m, 1H, H-15), 2.86~2.83 (m, 2H, H-7), 2.26 (d, J=12.6 Hz, 1H, He-1), 2.20 (d, J=12.6 Hz, 1H, H-5), 1.92~1.51 (m, 11H, H-2, H-3, He-6, H-22, H-23, H-24), 1.36~1.45 (m, 2H, Ha-1, Ha-6), 1.29 (s, 3H, H-19), 1.22~1.24 (m, 9H, H-20, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.4, 148.9, 144.0, 134.4, 128.5, 127.1, 121.5, 64.3, 47.6, 44.5, 37.9, 37.0, 36.6, 33.5, 32.3, 32.2, 29.6, 27.9, 25.1, 24.7, 23.0, 21.5, 18.5, 16.4; IR (KBr) ν: 2933, 1712 (C=O), 1465, 1388, 1246, 1107, 975 cm^-1. Anal. calcd for C₂₅H₃₆Br₂O₂: C 56.83, H 6.87; found C 56.89, H 6.88.

  12, 14-Dinitrodehydroabietic acid-2'-bromoethyl ester (4m): Yellow solid, yield 57.1%. m.p. 158.9~160.6 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.49~4.35 (m, 2H, H-21), 3.57~3.54 (m, 2H, H-22), 3.08~3.02 (m, 1H, H-15), 2.90~2.72 (m, 2H, H-7), 2.29 (d, J=12.7 Hz, 1H, He-1), 2.24 (d, J=12.7 Hz, 1H, H-5), 1.86~1.69 (m, 5H, H-2, H-3, He-6), 1.62~1.54 (m, 2H, Ha-1, Ha-6), 1.35~1.32 (m, 9H, H-16, H-17, H-19), 1.27 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.3, 152.1, 150.8, 149.5, 130.7, 128.5, 121.5, 64.3, 47.4, 43.4, 37.7, 37.6, 36.3, 29.1, 29.0, 25.0, 24.6, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2943, 1722 (C=O), 1533 (NO₂), 1463, 1361, 1290, 1249, 1178, 1116, 790, 734 cm^-1. Anal. calcd for C₂₂H₂₉BrN₂O₆: C 53.13, H 5.88, N 5.63; found C 53.10, H 5.86, N 5.67.

  12, 14-Dinitrodehydroabietic acid-3'-bromopropyl ester (4n): Yellow solid, yield 57.7%. m.p. 131.4~132.6 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.25~4.23 (t, J=6.0 Hz, 2H, H-21), 3.44~3.47 (t, J=6.0 Hz, 2H, H-23), 3.04~3.02 (m, 1H, H-15), 2.78~2.76 (m, 2H, H-7), 2.28 (d, J=12.6 Hz, 1H, He-1), 2.17~2.21 (m, 3H, H-5, H-22), 1.86~1.73 (m, 5H, H-2, H-3, He-6), 1.57~1.53 (m, 2H, Ha-1, Ha-6), 1.32 (d, J=7.2 Hz, 6H, H-16, H-17), 1.30 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.5, 150.8, 152.2, 149.5, 130.6, 128.5, 121.4, 62.6, 47.2, 43.6, 37.7, 37.6, 36.4, 31.5, 29.2, 29.0, 25.0, 24.6, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2937, 1718 (C=O), 1533(NO₂), 1363, 1242, 1178, 1132, 983, 902, 779, 732 cm^-1. Anal. calcd for C₂₃H₃₁BrN₂O₆: C 54.02, H 6.11, N 5.48; found C 53.96, H 6.15, N 5.47.

  12, 14-Dinitrodehydroabietic acid-4'-bromobutyl ester (4o): Yellow solid, yield 63.0%. m.p. 104.3~108.7 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.47 (s, 1H, H-11), 4.05 (t, J=6.3 Hz, 2H, H-21), 3.36 (t, J=6.5 Hz, 2H, H-24), 2.99~2.92 (m, 1H, H-15), 2.75~2.64 (m, 2H, H-7), 2.19 (d, J=12.6 Hz, 1H, He-1), 2.10 (dd, J=12.6, 2.0 Hz, 1H, H-5), 1.88~1.81 (m, 2H, H-22), 1.78~1.63 (m, 7H, H-2, H-3, He-6 and H-23), 1.48~1.41 (m, 2H, Ha-1 and Ha-6), 1.25 (d, J=7.2 Hz, 6H, H-17, H-16), 1.20 (s, 3H, H-19), 1.17 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.7, 152.1, 150.9, 149.5, 130.6, 128.5, 121.5, 64.0, 47.2, 43.5, 37.7, 37.6, 36.4, 32.9, 29.3, 29.0, 27.3, 25.0, 24.6, 20.7, 20.6, 20.2, 18.2, 16.5; IR (KBr) ν: 2935, 2875, 1716 (C=O), 1533 (NO₂), 1462, 1365, 1244, 1180, 1120, 1024, 905, 823, 734 cm^-1. Anal. calcd for C₂₄H₃₃BrN₂O₆: C 54.86, H 6.33, N 5.33; found C 54.91, H 6.36, N 5.37.

  12, 14-Dinitrodehydroabietic acid-5′-bromoamyl ester (4p): Yellow solid, yield 65.7%. m.p. 111.0~111.7 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H), 4.11 (t, J=6.4 Hz, 2H, H-21), 3.43 (t, J=6.6 Hz, 2H, H-25), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.77 (dd, J=9.7, 5.4 Hz, 2H, H-7), 2.28 (d, J=13.0 Hz, 1H, He-1), 2.20 (dd, J=12.6, 2.1 Hz, 1H, H-5), 1.95~1.49 (m, 13H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24), 1.34 (dd, J=7.1, 1.7 Hz, 6H, H-17, H-16), 1.29 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.7, 152.1, 150.9, 149.5, 130.6, 128.5, 121.5, 64.6, 47.2, 43.5, 37.7, 37.6, 36.4, 33.4, 32.2, 29.0, 27.8, 25.0, 24.7, 24.6, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2935, 1718 (C=O), 1533 (NO₂), 1460, 1365, 1247, 1180, 1130, 1047, 902, 736, 646, 561 cm^-1. Anal. calcd for C₂₅H₃₅BrN₂O₆: C 55.66, H 6.54, N 5.19; found C 55.70, H 6.56, N 5.15.

  12, 14-Dinitrodehydroabietic acid-6'-bromohexyl ester (4q): Yellow solid, yield 61.1%. m.p. 55.5~56.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.13~4.05 (m, 2H, H-21), 3.42 (t, J=6.7 Hz, 2H, H-26), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.76 (dd, J=8.9, 4.6 Hz, 2H, H-7), 2.28 (d, J=13.0 Hz, 1H, He-1), 2.19 (dd, J=12.6, 1.9 Hz, 1H, H-5), 1.79~1.37 (m, 15H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24, H-25), 1.34 (dd, J=7.1, 1.6 Hz, 6H, H-17, H-16), 1.29 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃CDCl₃) δ: 177.7, 152.1, 150.9, 149.5, 130.6, 128.5, 121.5, 64.8, 47.2, 43.5, 37.7, 37.6, 36.4, 33.7, 32.5, 29.0, 28.5, 27.7, 25.2, 25.0, 24.7, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2935, 1720 (C=O), 1533 (NO₂), 1462, 1365, 1246, 1180, 1130, 1047, 954, 902, 823, 731, 648, 563 cm^-1. Anal. calcd for C₂₆H₃₇Br- N₂O₆: C 56.42, H 6.74, N 5.06; found C 56.46, H 6.76, N 5.10.

  12, 14-Dinitrodehydroabietic acid-4'-bromobutenyl ester (4r). Yellow solid, yield 55.1%. m.p. 113.4~114.1 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 5.98 (dt, J=15.0, 7.4 Hz, 1H, H-22), 5.85 (t, J=13.5 Hz, 1H, H-23), 4.64~4.57 (m, 2H, H-21), 3.96 (d, J=7.4 Hz, 2H, H-24), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.77 (dd, J=10.0, 6.9 Hz, 2H, H-7), 2.28 (d, J=12.8 Hz, 1H, He-1), 2.20 (dd, J=12.6, 2.0 Hz, 1H, H-5), 1.78 (dd, J=27.9, 14.1 Hz, 5H, H-2, H-3, He-6), 1.57~1.51 (m, 2H, Ha-1, Ha-6), 1.34 (dd, J=7.1, 1.6 Hz, 6H, H-17 and H-16), 1.31 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.3, 152.1, 150.9, 149.5, 130.6, 130.4, 128.8, 128.6, 121.5, 63.9, 47.2, 43.5, 37.7, 37.6, 36.3, 31.2, 29.0, 25.0, 24.6, 20.7, 20.6, 20.1, 18.2, 16.4; IR (KBr) ν: 2929, 1724 (C=O), 1533 (NO₂), 1365, 1240, 1178, 1120, 960, 902, 732, 561 cm^-1. Anal. calcd for C₂₄H₃₁BrN₂O₆: C 55.07, H 5.97, N 5.35; found C 55.11, H 6.02, N 5.94.

  3.2.4   Synthetic route for target compounds 5a~5r

  Under anhydrous conditions, a mixture of the intermediates 4 (2.84 mmol) in CH₃CN (50 mL), coupled with AgNO₃ (1.0 g) in the presence of CH₃CN (50 mL) was heated with reflux for 5 h under dark condition. After completion of the reaction, the mixture was filtered and washed with CH₃CN, concentrated with a rotary evaporator and purified by silica gel chromatography with a mixture of petroleum ether and ethyl acetate (V:V=20:1) as eluent to give the target compounds 5a~5r.

  Dehydroabietic acid-2'-nitrooxyethyl ester (5a): White solid, yield 88.4%. 52.4~54.4 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.17 (d, J=8.4 Hz, 1H, H-11), 7.01 (d, J=8.4 Hz, 1H, H-12), 6.90 (s, 1H, H-14), 4.66~4.65 (t, J=4.2 Hz, 2H, H-22), 4.67~4.30 (m, 2H, H-21), 2.90~2.88 (m, 2H, H-7), 2.86~2.81 (m, 1H, H-15), 2.32 (d, J=12.6 Hz, 1H, He-1), 2.27~2.25 (d, J=12.6 Hz, 1H, H-5), 1.86~1.66 (m, 5H, H-2, H-3 and He-6), 1.53~1.39 (m, 2H, Ha-1 and Ha-6), 1.29 (s, 3H, H-19), 1.24 (s, 3H, H-20), 1.22 (d, J=3.6 Hz, 6H, H-16, H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.4, 146.8, 145.9, 134.7, 127.1, 124.3, 124.1, 70.7, 60.4, 47.9, 44.9, 38.0, 37.1, 36.6, 33.6, 30.1, 25.3, 24.1, 21.9, 18.6, 16.6; IR (KBr) ν: 2927, 1712 (C=O), 1643 (ONO₂), 1497, 1436, 1387, 1277 (ONO₂), 1246, 1174, 1125, 1052, 896, 843 cm^-1; ESI-MS m/z: 412.10 [M+ Na]⁺. Anal. calcd for C₂₂H₃₁NO₅: C 67.84, H 8.02, N 3.60; found C 67.78, H 8.06, N 3.61.

  Dehydroabietic acid-3'-nitrooxypropyl ester (5b): Color- less liquid, yield 85.2%. ¹H NMR (600 MHz, CDCl₃) δ: 7.20 (d, J=8.4 Hz, 1H, H-11), 7.04 (d, J=8.4 Hz, 1H, H-12), 6.93 (s, 1H, H-14), 4.55 (t, J=6.6 Hz, 2H, H-23), 4.24~4.17 (m, 2H, H-21), 2.92~2.89 (m, 2H, H-7), 2.88~2.84 (m, 1H, H-15), 2.35 (d, J=12.6 Hz, 1H, He-1), 2.26 (d, J=12.6 Hz, 1H, H-5), 2.10 (q, J=6.0 Hz, 2H, H-22), 1.90~1.68 (m, 5H, H-2, H-3, He-6), 1.44~1.36 (m, 2H, Ha-1, Ha-6), 1.31 (s, 3H, H-19), 1.27 (s, 3H, H-20), 1.25 (d, J=3.6 Hz, 6H, H-16, H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.4, 146.9, 145.9, 134.6, 127.1, 124.3, 124.1, 70.0, 60.6, 47.8, 44.9, 38.0, 37.1, 36.8, 33.6, 30.2, 25.3, 24.1, 21.9, 18.7 16.6; IR (KBr) ν: 2981, 1724 (C=O), 1637 (ONO₂), 1461, 1384, 1278 (ONO₂), 1102, 1076, 854, 756 cm^-1; ESI-MS m/z: 426.30 [M+Na]⁺. Anal. calcd for C₂₃H₃₃NO₅: C 68.46, H 8.24, N 3.47; found C 68.44, H 8.21, N 3.49.

  Dehydroabietic acid-4'-nitrooxybutyl ester (5c): Color- less liquid, yield 85.0%. ¹H NMR (600 MHz, CDCl₃) δ: 7.17 (d, J=8.4 Hz, 1H, H-11), 7.01 (d, J=8.4 Hz, 1H, H-12), 6.90 (s, 1H, H-14), 4.51~4.48 (m, 2H, H-24), 4.13~4.09 (m, 2H, H-21), 2.89~2.87 (m, 2H, H-7), 2.86~2.81 (m, 1H, H-15), 2.30 (d, J=12.0 Hz, 1H, He-1), 2.23 (d, J=12.0 Hz, 1H, H-5), 1.89~1.87 (m, 2H, H-22), 1.82~1.63 (m, 7H, H-2, H-3, He-6 and H-23), 1.52~1.48 (m, 1H, Ha-1), 1.42~1.39 (m, 1H, Ha-6), 1.28 (s, 3H, H-19), 1.24 (s, 3H, H-16), 1.23 (s, 3H, H-17), 1.22 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.6, 146.9, 145.9, 134.7, 127.0, 124.3, 124.1, 72.2, 63.7, 47.8, 44.9, 38.0, 37.1, 36.8, 33.6, 30.0, 25.3, 25.2, 23.5, 21.9, 18.7 16.6; IR (KBr) ν: cm^-1: 2983, 1720 (C=O), 1641 (ONO₂), 1446, 1384, 1278 (ONO₂), 1178, 1128, 1029, 984, 827 cm^-1; ESI-MS m/z: 417.10 [M]⁺. Anal. calcd for C₂₄H₃₅- NO₅: C 69.04, H 8.45, N 3.35; found C 69.09, H 8.44, N 3.32.

  Dehydroabietic acid-5'-nitoxypentyl ester (5d): Color- less liquid, yield 78.6%. ¹H NMR (600 MHz, CDCl₃) δ: 7.19 (d, J=8.4 Hz, 1H, H-11), 7.03 (d, J=8.4 Hz, 1H, H-12), 6.92 (s, 1H, H-14), 4.50~4.46 (m, 2H, H-25), 4.05~4.13 (m, 2H, H-21), 2.91~2.83 (m, 3H, H-7 and H-15), 2.32 (d, J=12.6 Hz, 1H, He-1), 2.26 (d, J=12.6 Hz, 1H, H-5), 1.84~1.48 (m, 11H, H-2, H-3, He-6, H-22, H-23 and H-24), 1.44~1.34 (m, 2H, Ha-1 and Ha-6), 1.29 (s, 3H, H-19), 1.24~1.26 (m, 9H, H-16, H-17 and H-20); ¹³C NMR (150 MHz, CDCl₃) δ:178.6, 146.9, 145.8, 134.61, 126.9, 124.2, 124.0, 72.7, 64.0, 47.7, 44.8, 38.0, 37.0, 36.7, 33.5, 30.1, 25.2, 25.0, 24.0, 21.3, 22.2, 21.7, 18.6 16.5; IR (KBr) ν: 2937, 1722 (C=O), 1629 (ONO₂), 1471, 1388, 1278 (ONO₂), 1244, 1176, 1009, 975, 882; ESI-MS m/z: 470.88 [M+K]⁺ cm^-1. Anal. calcd for C₂₅H₃₇NO₅: C 69.58, H 8.64, N 3.25; found C 69.55, H 8.61, N 3.27.

  Dehydroabietic acid-6'-nitrohexyl ester (5e): Colorless liquid, yield 84.8%. ¹H NMR (600 MHz, CDCl₃) δ: 7.14 (d, J=8.4 Hz, 1H, H-11), 7.01 (d, J=8.4 Hz, 1H, H-12), 6.90 (s, 1H, H-14), 4.50~4.47 (m, 2H, H-26), 4.13~4.07 (m, 2H, H-21), 2.89~2.86 (m, 2H, H-7), 2.86~2.82 (m, 1H, H-15), 2.31 (d, J=12.6 Hz, 1H, He-1), 2.23 (d, J=12.6 Hz, 1H, H-5), 1.89~1.87 (m, 4H, H-22 and H-25), 1.82~1.64 (m, 9H, H-2, H-3, He-6, H-23 and H-24), 1.38~1.52 (m, 2H, Ha-1 and Ha-6), 1.28 (s, 3H, H-19), 1.24 (s, 3H, H-20), 1.22 (d, J=4.8 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.6, 147.0, 145.8, 134.7, 127.0, 124.3, 124.0, 73.1, 64.4, 47.7, 44.9, 38.0, 37.0, 36.7, 33.5, 30.2, 25.7, 25.4, 25.3, 25.2, 24.0, 24.0, 21.8, 18.7 16.5; IR (KBr) ν: 2943, 1721 (C=O), 1625 (ONO₂), 1460, 1383, 1280 (ONO₂), 1176, 1128, 1009, 977, 806, 759 cm^-1; ESI-MS m/z: 445.88 [M]⁺. Anal. calcd for C₂₆H₃₉NO₅: C 70.08, H 8.82, N, 3.14; found C 70.10, H 8.85, N 3.11.

  Dehydroabietic acid-8'-nitoxyoctyl ester (5f): Colorless liquid, yield 80.7%. ¹H NMR (600 MHz, CDCl₃) δ: 7.17 (d, J=8.4 Hz, 1H, H-11), 7.00 (d, J=8.4 Hz, 1H, H-12), 6.89 (s, 1H, H-14), 4.46~4.41 (m, 2H, H-28), 4.10~4.00 (m, 2H, H-21), 2.88~2.85 (m, 2H, H-7), 2.84~2.80 (m, 1H, H-15), 2.25 (d, J=12.6 Hz, 1H, He-1), 2.25 (d, J=12.6 Hz, 1H, H-5), 1.82~1.32 (m, 17H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24, H-25, H-26 and H-27), 1.26 (s, 3H, H-19), 1.23 (s, 3H, H-20), 1.21 (d, J=4.8 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.7, 147.0, 145.8, 134.8, 127.0, 124.3, 124.0, 73.4, 64.7, 47.7, 44.9, 38.1, 37.0, 36.7, 34.1, 34.0, 32.8, 30.2, 29.1, 29.0, 28.7, 26.8, 25.9, 25.6, 25.3, 24.1, 24.0, 21.8, 18.7 16.6; IR (KBr) ν: 2933, 2852, 1720 (C=O), 1626 (ONO₂), 1463, 1382, 1280 (ONO₂), 1246, 1176, 1126, 974, 864, 758, 698 cm^-1; ESI-MS m/z: 474.20 [M+H]⁺. Anal. calcd for C₂₈H₄₃NO₅: C 71.00, H 9.15, N 2.96; found C 71.03, H 9.11, N 2.98.

  Dehydroabietic acid-12'-nitrooxydodecyl ester (5g): Colorless liquid, yield 83.5%. ¹H NMR (600 MHz, CDCl₃) δ: 7.21 (d, J=8.2 Hz, 1H, H-11), 7.04 (d, J=8.2 Hz, 1H, H-12), 6.92 (s, 1H, H-14), 4.47 (t, J=6.6 Hz, 2H, H-32), 4.15~4.02 (m, 2H, H-21), 2.91~2.82 (m, 3H, H-7 and H-15), 2.35~2.28 (m, 2H, He-1 and H-5), 1.83~1.61 (m, 10H, H-2, H-3, He-6, H-22, H-31 and Ha-1), 1.46~1.30 (m, 20H, Ha-6, H-19, H-23, H-24, H-25, H-26, H-27, H-28, H-29 and H-30), 1.27 (s, 3H, H-20), 1.25 (d, J=2.4 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.7, 147.0, 145.71, 134.7, 126.9, 124.3, 124.0, 73.5, 64.7, 47.7, 44.8, 38.0, 37.0, 36.6, 33.5, 30.2, 29.5, 29.4, 29.2, 29.1, 28.7, 26.8, 26.0, 25.7, 25.3, 24.0, 21.8, 18.7, 16.6; IR (KBr) ν: 2927, 2856, 1722 (C=O), 1631 (ONO₂), 1462, 1381, 1278 (ONO₂), 1244, 1176, 1126, 1035, 947, 862 cm^-1; ESI-MS m/z: 530.01 [M+H]⁺. Anal. calcd for C₃₂H₅₁NO₅: C 72.55, H 9.70, N 2.64; found C 72.53, H 9.72, N 2.61.

  Dehydroabietic acid-4'-nitrooxybutene ester (5h): Color- less liquid, yield 88.2%. ¹H NMR (600 MHz, CDCl₃) δ: 7.24 (d, J=8.4 Hz, 1H, H-11), 7.01 (d, J=8.4 Hz, 1H, H-12), 6.97 (s, 1H, H-14), 6.07~6.04 (m, 1H, H-23), 5.91~5.86 (m, 1H, H-22), 4.95 (d, J=6.6 Hz, 2H, H-24), 4.71~4.65 (m, 2H, H-21), 2.96~2.94 (m, 2H, H-7), 2.91~2.88 (m, 1H, H-15), 2.38 (d, J=12.0 Hz, 1H, He-1), 2.33 (d, J=12.0 Hz, 1H, H-5), 1.92~1.73 (m, 5H, H-2, H-3 and He-6), 1.59~1.47 (m, 2H, Ha-1 and Ha-6), 1.36 (s, 3H, H-19), 1.30 (s, 3H, H-20), 1.29 (d, J=3.0 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.1, 147.0, 145.9, 134.8, 133, 0, 127.1, 124.4, 124.2, 123.8, 72.5, 63.6, 47.9, 45.1, 38.2, 37.2, 36.8, 33.7, 30.2, 25.4, 24.2, 21.9, 18.8, 16.7; IR (KBr) ν: 2956, 2870, 1693 (C=O), 1639 (ONO₂), 1458, 1382, 1274 (ONO₂), 1188, 970, 852 cm^-1; ESI-MS m/z: 416.10 [M+H]⁺. Anal. calcd for C₂₄H₃₃NO₅: C 69.37, H 8.00, N 3.37; found C 69.32, H 8.02, N 3.33.

  12-Bromodehydroabietic acid-2'-nitrooxyethyl ester (5i): White solid, yield 64.4%. m.p. 134.1~136.7 ℃; IR (KBr) ν: 2927, 1710 (C=O), 1637 (ONO₂), 1496, 1382, 1244, 1128, 896; ¹H NMR (600 MHz, CDCl₃) δ: 7.37 (s, 1H, H-11), 6.93 (s, 1H, H-14), 4.53 (t, J=6 Hz, 2H, H-22), 4.19 (t, J=6 Hz, 2H, H-21), 3.29~3.25 (m, 1H, H-15), 2.85~2.80 (m, 2H, H-7), 2.26 (d, J=12.0 Hz, 1H, He-1), 2.17 (d, J=12.0 Hz, 1H, H-5), 2.10~1.81 (m, 5H, H-2, H-3 and He-6), 1.47~1.51 (m, 1H, Ha-1), 1.40~1.43 (m, 1H, Ha-6), 1.28 (s, 3H, H-19), 1.23 (s, 3H, H-20), 1.21 (d, J=6 Hz, 6H, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 179.6, 150.2, 145.5, 135.7, 129.9, 128.6, 123.0, 71.3, 62.0, 49.0, 46.0, 39.2, 38.4, 38.0, 33.8, 30.9, 26.5, 24.4, 24.2, 23.0, 19.8 cm^-1; ESI-MS m/z: 468.82 [M]⁺. Anal. calcd for C₂₂H₃₀BrNO₅: C 56.41, H 6.46, N 2.99; found C 56.44, H 6.42, N 3.03.

  12-Bromodehydroabietic acid-3'-nitrooxypropyl ester (5j): Pale yellow solid, yield 68.5%. m.p. 76.4~78.3 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.94 (s, 1H, H-14), 4.54 (t, J=6.6 Hz, 2H, H-23), 4.21~4.19 (m, 2H, H-21), 3.31~3.26 (m, 1H, H-15), 2.86~2.82 (m, 2H, H-7), 2.27 (d, J=12.6 Hz, 1H, He-1), 2.17 (d, J=12.6 Hz, 1H, H-5), 2.10~2.07 (m, 2H, H-22), 1.87~1.64 (m, 5H, H-2, H-3 and He-6), 1.53~1.49 (m, 1H, Ha-1), 1.44~1.41 (m, 1H, Ha-6), 1.29 (s, 3H, H-19), 1.25~1.22 (m, 9H, H-20, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.2, 148.8, 144.1, 134.3, 128.5, 127.2, 121.6, 69.9, 60.6, 47.6, 44.6, 37.8, 37.0, 36.6, 32.3, 29.5, 26.5, 25.1, 23.0, 22.8, 21.6, 18.4, 16.5; IR (KBr) ν: 2956, 1722 (C=O), 1641 (ONO₂), 1498, 1454, 1384, 1280 (ONO₂), 1174, 983 cm^-1; ESI-MS m/z: 504.70 [M+Na]⁺. Anal. calcd for C₂₃H₃₂BrNO₅: C 57.26, H 6.69, N 2.90; found C 57.22, H 6.70, N 2.92.

  12-Bromodehydroabietic acid-4'-nitrooxybutyl ester (5k): White solid, yield 63.6%. m.p. 101.5~102.8 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.94 (s, 1H, H-14), 4.50 (t, J=6.6 Hz, 2H, H-24), 4.15~4.11 (m, 2H, H-21), 3.31~3.26 (m, 1H, H-15), 2.86~2.79 (m, 2H, H-7), 2.26 (d, J=12.6 Hz, 1H, He-1), 2.18 (d, J=12.6 Hz, 1H, H-5), 1.88~1.67 (m, 9H, H-2, H-3, He-6, H-22 and H-23), 1.53~1.49 (m, 1H, Ha-1), 1.45~1.42 (m, 1H, Ha-6), 1.29 (s, 3H, H-19), 1.25~1.22 (m, 9H, H-20, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.3, 148.9, 144.1, 134.3, 128.5, 127.2, 121.6, 72.6, 63.7, 47.6, 44.6, 37.8, 37.0, 36.7, 32.3, 29.6, 29.3, 27.4, 25.1, 25.1, 23.7, 23.0, 22.8, 21.6, 18.5, 16.5 cm^-1; IR (KBr) ν: 2953, 1708 (C=O), 1471, 1631 (ONO₂), 1386, 1247 (ONO₂), 1024, 864; ESI-MS m/z: 518.85 [M+Na]⁺. Anal. calcd for C₂₄H₃₄BrNO₅: C 58.07, H 6.90, N 2.82; found C 58.11, H 6.87, N 2.84.

  12-Bromodehydroabietic acid-5'-nitoxypentyl ester (5l): White solid, yield 58.2%. m.p. 57.1~59.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.39 (s, 1H, H-11), 6.95 (s, 1H, H-14), 4.47 (t, J=6.6 Hz, 2H, H-25), 4.05~4.12 (m, 2H, H-21), 3.31~3.26 (m, 1H, H-15), 2.87~2.82 (m, 2H, H-7), 2.26 (d, J=12.6 Hz, 1H, He-1), 2.19 (d, J=12.6 Hz, 1H, H-5), 1.83~1.68 (m, 9H, H-2, H-3, He-6, H-22 and H-24), 1.51~1.42 (m, 4H, Ha-1, Ha-6 and H-23), 1.29 (s, 3H, H-19), 1.22~1.25 (m, 9H, H-20, H-16 and H-17); ¹³C NMR (150 MHz, CDCl₃) δ: 178.4, 148.9, 144.1, 134.4, 128.6, 127.2, 121.6, 72.9, 64.1, 47.6, 44.5, 37.9, 37.0, 36.6, 32.3, 29.6, 28.2, 25.1, 22.9, 22.8, 22.4, 21.6, 18.5, 16.5; IR (KBr) ν: 2935, 1722 (C=O), 1629 (ONO₂), 1471, 1388, 1278 (ONO₂), 1130, 1039, 975 cm^-1; ESI-MS m/z: 510.83 [M]⁺. Anal. calcd for C₂₅H₃₆BrNO₅: C 58.82, H 7.11, N 2.74; found C 58.81, H 7.13, N 2.74.

  12, 14-Dinitrodehydroabietic acid-2'-nitrooxyethyl ester (5m): Yellow solid, yield 73.2%. m.p. 110.5~111.5 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.47 (s, 1H, H-11), 4.24~4.08 (m, 2H, H-22), 3.75(t, J=4.8, 2H, H-21), 2.99~2.92(m, 1H, H-15), 2.71~2.67 (m, 2H, H-7), 2.20 (d, J=12.0 Hz, 1H, He-1), 2.13 (dd, J=12.0, 2.0 Hz, 1H, H-5), 1.80~1.66 (m, 5H, H-2, H-3 and He-6), 1.52~1.41 (m, 2H, Ha-1 and Ha-6), 1.26 (s, 3H, H-16), 1.24 (s, 3H, H-17), 1.22 (s, 3H, H-19), 1.18 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 178.3, 152.1, 150.9, 149.5, 130.7, 128.5, 121.5, 66.4, 61.4, 47.3, 43.6, 37.7, 37.6, 36.3, 29.0, 25.0, 24.7, 20.7, 20.6, 20.2, 18.2, 16.5; IR (KBr) ν: 2937, 1726 (C=O), 1641 (ONO₂), 1533 (NO₂), 1465, 1365, 1288 (ONO₂), 1242, 1180, 1136, 896, 848, 734, 665 cm^-1; ESI-MS m/z: 477.80 [M-H]^-. Anal. calcd for C₂₂H₂₉- N₃O₉: C 55.11, H 6.10, N 8.76; found C 55.08, H 6.13, N 8.73.

  12, 14-Dinitrodehydroabietic acid-3'-nitrooxypropyl ester (5n): Yellow solid, yield 76.0%. m.p. 123.6~125.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.55 (t, J=6.0 Hz, 2H, H-23), 4.25~4.23 (m, 2H, H-21), 3.07~3.02 (m, 1H, H-15), 2.78~2.76 (m, 2H, H-7), 2.28 (d, J=12.6 Hz, 1H, He-1), 2.19~2.17 (d, J=12.6 Hz, 1H, H-5), 2.08~2.12 (m, 2H, H-22), 1.85~1.74 (m, 5H, H-2, H-3 and He-6), 1.51~1.56 (m, 2H, Ha-1 and Ha-6), 1.34 (d, J=7.2 Hz, 6H, H-16 and H-17), 1.30 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.5, 152.1, 150.8, 149.5, 130.5, 128.6, 121.4, 69.8, 61.0, 47.2, 43.5, 37.7, 37.5, 36.4, 29.0, 26.5, 25.0, 24.6, 20.7, 20.6, 20.2, 18.1, 16.4; IR (KBr) ν: 2935, 1724 (C=O), 1627 (ONO₂), 1533 (NO₂), 1463, 1365, 1278 (ONO₂), 1244, 1180, 1122, 1045, 983, 864 cm^-1; ESI-MS m/z: 491.82 [M-H]^-. Anal. calcd for C₂₃H₃₁N₃O₉: C 55.98, H 6.33, N 8.51; found C 55.99, H 6.31, N 8.55.

  12, 14-Dinitrodehydroabietic acid-4′-nitrooxybutyl ester (5o): Yellow solid, yield 74.2%. m.p. 105.8~108.3 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.50 (t, J=6.2 Hz, 2H, H-24), 4.17~4.10 (m, 2H, H-21), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.77 (dd, J=9.0, 4.7 Hz, 2H, H-7), 2.28 (d, J=12.8 Hz, 1H, He-1), 2.18 (dd, J=12.6, 2.1 Hz, 1H, H-5), 1.87~1.71 (m, 9H, H-2, H-3, He-6, H-22 and H-23), 1.53 (t, J=14.9 Hz, 2H, Ha-1 and Ha-6), 1.34 (dd, J=7.1, 1.8 Hz, 6H, H-16 and H-17), 1.29 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.6, 152.1, 150.9, 149.5, 130.5, 128.6, 121.4, 72.5, 64.0, 47.2, 43.5, 37.7, 37.6, 36.4, 29.0, 25.1, 24.9, 24.6, 23.7, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2929, 2875, 1716 (C=O), 1618 (ONO₂), 1529 (NO₂), 1463, 1365, 1280 (ONO₂), 1244, 1180, 981, 948, 875 cm^-1; ESI-MS m/z: 506.87 [M-H]^-. Anal. calcd for C₂₄H₃₃N₃O₉: C 56.80, H 6.55, N 8.28; found C 56.83, H 6.56, N 8.29.

  12, 14-Dinitrodehydroabietic acid-5'-nitoxypentyl ester (5p): Yellow solid, yield 69.9%. m.p. 44.8~45.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H), 4.49 (d, J=6.6 Hz, 2H, H-25), 4.11 (t, J=5.7 Hz, 2H, H-21), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.76 (dd, J=9.2, 4.9 Hz, 2H, H-7), 2.28 (d, J=12.8 Hz, 1H, He-1), 2.19 (dd, J=12.6, 2.0 Hz, 1H, H-5), 1.90~1.45 (m, 13H, Ha-1, H-2, H-3, H-6, H-22, H-23 and H-24), 1.34 (dd, J=7.1, 2.0 Hz, 6H, H-16 and H-17), 1.29 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.7, 152.1, 150.9, 149.5, 130.6, 128.5, 121.5, 72.9, 64.4, 47.2 43.6, 37.7, 37.6, 36.4, 29.0, 28.2, 26.4, 24.9, 24.7, 22.3, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2943, 1718 (C=O), 1635 (ONO₂), 1533 (NO₂), 1463, 1365, 1280 (ONO₂), 1180, 1132, 1037, 979, 860, 758, 700, 659, 567 cm^-1; ESI-MS m/z: 522.74 [M+H]⁺. Anal. calcd for C₂₅H₃₅N₃O₉: C 57.57, H 6.76, N 8.06; found C 57.54, H 6.74, N 8.03.

  12, 14-Dinitrodehydroabietic acid-6'-nitrohexyl ester (5q): Yellow solid, yield 59%. m.p. 56.5~57.0 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 4.48 (d, J=6.5 Hz, 2H, H-26), 4.09 (td, J=10.9, 4.3 Hz, 2H, H-21), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.76 (dd, 2H, J=9.0, 4.7 Hz, H-7), 2.28 (d, J=12.8 Hz, 1H, He-1), 2.19 (dd, J=12.6, 2.0 Hz, 1H, H-5), 1.89~1.42 (m, 15H, Ha-1, H-2, H-3, H-6, H-22, H-23, H-24 and H-25), 1.36~1.32 (m, 6H, H-16 and H-17), 1.29 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.7, 152.1, 150.9, 149.5, 130.6, 128.5, 121.5, 73.0, 64.7, 47.2, 43.6, 37.7, 37.6, 36.4, 29.0, 28.4, 26.7, 26.6, 25.7, 25.4, 25.3, 25.0, 24.7, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2943, 1722 (C=O), 1625 (ONO₂), 1535 (NO₂), 1465, 1365, 1280 (ONO₂), 1182, 1134, 1045, 979, 867, 759, 698, 655, 569 cm^-1; ESI-MS m/z: 558.78 [M+Na]⁺. Anal. calcd for C₂₆H₃₇N₃O₉: C 58.31, H 6.96, N 7.85; found C 58.33, H 6.94, N 7.82.

  12, 14-Dinitrodehydroabietic acid-4'-nitrooxybutene ester (5r): Yellow solid, yield 81.6%. m.p. 114.9~115.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.56 (s, 1H, H-11), 6.08~5.95 (m, 1H, H-23), 5.90~5.80 (m, 1H, H-22), 4.94 (d, J=6.3 Hz, 2H, H-24), 4.64 (d, J=5.4 Hz, 2H, H-21), 3.04 (dt, J=14.3, 7.1 Hz, 1H, H-15), 2.77 (dd, J=8.9, 4.6 Hz, 2H, H-7), 2.29 (d, J=13.0 Hz, 1H, He-1), 2.20 (dd, J=12.6, 2.0 Hz, 1H, H-5), 1.86~1.73 (m, 5H, H-2, H-3 and He-6), 1.57~1.51 (m, 2H, Ha-1 and Ha-6), 1.34 (dd, J=7.1, 1.8 Hz, 6H, H-16 and H-17), 1.31 (s, 3H, H-19), 1.26 (s, 3H, H-20); ¹³C NMR (150 MHz, CDCl₃) δ: 177.2, 152.1, 150.8, 149.5, 132.3, 130.6, 128.6, 128.20, 124.0, 121.5, 72.2, 63.7, 47.3, 43.6, 37.7, 37.6, 36.4, 29.0, 25.0, 24.6, 20.7, 20.6, 20.2, 18.2, 16.4; IR (KBr) ν: 2935, 1726 (C=O), 1631 (ONO₂), 1533 (NO₂), 1465, 1365, 1276 (ONO₂), 1242, 1178, 1120, 923, 852, 732, 651, 567 cm^-1; ESI-MS m/z: 528.10 [M+Na]⁺. Anal. calcd for C₂₄H₃₁N₃O₉: C 57.02, H 6.18, N 8.31; found C 57.03, H 6.14, N 8.34.

  3.3   Cytotoxicity testing

  The cytotoxicity assay of the compounds was determined by MTT. Cells were grown in dulbecco's modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and grown at 37 ℃ in an atmosphere of 5% CO₂ and 95% air. Cells were routinely trypsinized (0.05% trypsin/EDTA). All experiments were repeated at least three times. The target compounds were dissolved in dimethyl sulfoxide (DMSO) and added into DMEM. The final DMSO concentration never exceeded 0.1%, and cisplatin was used as the control drug in each experiment.

  3.4   NO release evaluation

  Total NO release amounts in vitro were detected by nitrite/nitrate Assay Kit (Beyotime, China). Firstly, CNE-2 cells (3×100 μL, 5.0×10⁴ cells/mL) were incubated in 96 well-plates for 24 h at 37 ℃, and then treated with target compounds (4 mmol/L) for 24 h. Secondly, the amounts of NO in vitro were measured according to the manufacturer's instructions. The optical density (OD) value of each well was measured on a microplate reader (BioTek, USA) at 540 nm and calculated according to the standard curve. Data were mean of three independent experiments.

  Supporting Information  The ¹H NMR and ¹³C NMR spectra of compounds 4a~4rand 5a~5r. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

  
  
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  Scheme 1  Synthetic route for dehydroabietic acid-based nitrate compounds 5a~5r

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  Figure 1  NO released amounts of the target compounds in CNE-2 cells for 24 h (mean±SD, n=3)

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  Table 1.  Cytotoxic activity of dehydroabietic acid-based nitrate compounds

  Compd.	IC50a/(μmol•L-1)
  	CNE-2	HepG2	BEL-7402	HeLa	HL-7702	NP69
  DHAA	88.64±0.73	80.36±0.84	46.70±0.55	37.40±0.64	> 100	> 100
  4i	75.84±1.42	61.55±0.37	53.18±1.83	88.28±3.12	> 100	> 100
  4j	60.33±2.54	45.96±0.77	69.24±1.63	73.04±1.08	> 100	> 100
  4k	42.29±0.86	57.58±1.26	48.35±0.48	50.28±0.57	> 100	> 100
  5a	43.88±0.15	50.44±0.08	38.25±0.44	44.32±0.28	> 100	> 100
  5b	35.64±0.48	66.93±0.20	42.11±0.64	38.12±0.15	> 100	> 100
  5c	33.09±0.23	57.34±0.15	24.02±0.62	40.10±0.23	> 100	> 100
  5d	36.32±0.57	34.62±0.17	32.22±0.37	33.44±0.72	> 100	> 100
  5e	20.63±0.66	25.84±0.22	50.78±0.32	14.14±0.21	> 100	> 100
  5f	25.44±0.74	21.94±0.54	14.88±0.30	27.34±0.64	> 100	> 100
  5g	27.26±0.55	30.17±0.32	18.40±0.24	67.33±0.46	> 100	> 100
  5h	18.20±0.16	20.46±0.18	17.52±0.64	55.45±0.40	74.39±0.19	89.35±0.56
  5i	34.16±0.43	30.64±0.24	44.72±0.46	65.15±0.74	> 100	> 100
  5j	8.36±0.14	13.00±0.61	55.33±0.42	23.36±0.44	> 100	> 100
  5k	16.84±0.34	8.83±0.65	35.14±0.13	12.06±0.52	87.66±0.43	> 100
  5l	22.33±0.56	15.29±0.42	38.78±0.36	7.88±0.58	> 100	> 100
  5m	9.24±0.40	34.88±0.10	32.47±0.42	8.42±0.70	> 100	> 100
  5n	66.89±0.67	10.40±0.27	11.23±0.21	15.16±0.81	88.18±0.23	> 100
  5o	25.00±0.11	30.53±0.82	14.66±0.40	42.51±0.18	> 100	> 100
  5p	15.66±0.42	22.08±0.45	44.05±0.30	14.06±0.16	> 100	> 100
  5q	9.63±0.33	13.90±0.16	33.88±0.08	4.57±0.41	65.28±0.35	92.86±0.75
  5r	20.87±0.08	28.66±0.43	18.93±0.37	34.78±0.13	> 100	> 100
  Cisplatin	8.75±0.24	6.42±0.18	12.68±0.33	1.94±0.20	20.76±0.83	10.74±0.52
  a Data represent the means±S.D. from three independent experiments.

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