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Journal of Asian Natural Products Research

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Synthesis and antitumor activity evaluation oflamiridosin A derivatives

Yan-Xia Yang, Jian-Wei Yan, Fu-Lin Yan, Yan-Yan Yin, Fang-Fang Zhuang & Zi-Yang Ji

To cite this article: Yan-Xia Yang, Jian-Wei Yan, Fu-Lin Yan, Yan-Yan Yin, Fang-Fang Zhuang & Zi-Yang Ji (2016) Synthesis and antitumor activity evaluation of lamiridosin A derivatives, Journalof Asian Natural Products Research, 18:1, 26-35, DOI: 10.1080/10286020.2015.1130037

To link to this article: http://dx.doi.org/10.1080/10286020.2015.1130037

Published online: 13 Jan 2016.

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Journal of asian natural Products research, 2016Vol. 18, no. 1, 26–35http://dx.doi.org/10.1080/10286020.2015.1130037

© 2016 taylor & francis

Synthesis and antitumor activity evaluation of lamiridosin A derivatives

Yan-Xia Yang, Jian-Wei Yan, Fu-Lin Yan, Yan-Yan Yin, Fang-Fang Zhuang and Zi-Yang Ji

Pharmacy college, Xinxiang Medical university, Xinxiang 453003, china

1. Introduction

Malignant tumor is one of the major threats to human health [1]. The less selective phar-macological approach commonly used was based on the principle of “killing” cancer cells utilizing all types of antineoplastic agents, such as antimetabolites, alkylating agents, anti-tumor antibiotics, blockers of mitosis, and even organ metallic compounds. Chemotherapy of human malignant tumor using conventional antineoplastic agents is accompanied by severe adverse side effects, including myelosuppression, hair loss, and transient damage of replicating tissues rapidly. Therefore, the treatment of malignant tumor is rapidly changing from conventional chemotherapy toward a more innovative individualized and targeted therapy. Novel exploitable targets for tumor therapy emerged, including natural products like camptothecin [2], paclitaxel [3], or reconstructing compounds for synthetic derivatives, such as irinotecan [4], docetaxel [5].

As the main active ingredients of traditional Chinese medicine Lamiophlomis rotata [6,7], lamiophlomiols A and B also exist in other plants, such as Phlomis umbrosa Turcz [8]. During the systemic isolation and identification of the chemical ingredients of Phlomis umbrosa Turcz, we obtained lots of epimers of lamiophlomiols A and B (3/1). The epimers cannot be separated by column chromatography or HPLC using a number of different solvent systems and conditions. Iridoids exhibit a variety of biological activities, such as anti-inflammatory [9], antitumor [10], antimicrobial [11], antioxidant [12], antihepatitis [13], and anti-HBV activity [14]. The reports on the biological activities of lamiophlomiols A and B, a class of important iridoids, are rare. The ring opening compounds of lamiophlo-miols A/B, named lamiridosins A/B, were found to significantly inhibit hepatitis C virus

ABSTRACTA series of lamiridosin A derivatives were synthesized through simple procedures. Their antitumor activities were evaluated against EC9706, MGC803, and B16 cell lines in vitro. Several compounds showed potent antitumor activity, especially compound 10, with IC50 value of 2.36 μmol/L against MGC803 cell lines, is more potent than marketed positive drug 5-fluorouridine (5-FU).

KEYWORDSlamiridosin a; iridoids; ether derivatives; triazole; antitumor

ARTICLE HISTORYreceived 31 august 2015 accepted 6 december 2015

CONTACT fu-lin Yan yanfulin03@xxmu.edu.cn; Yan Yan Yin yinyanyan7@hotmail.com

JoUrnAL oF ASIAn nATUrAL ProdUCTS rESEArCh 27

entry in vitro [15]. Using the mixtures of lamiophlomiols A/B (3/1) as the starting material, their modification and further derivation were explored. A series of derivatives, based on the basic skeleton structure of lamiridosin A, were synthesized. It is highly desirable to develop novel bioisosteres of lactol to improve the stability of lamiophlomiols A and B. C1-hydroxy ether derivation (compounds 1–3) plays an important role in drug design [16], without reducing the biological activity of lamiophlomiols A/B (3/1). The 1,2,3-triazole group and its derivatives also play vital function in the field of drug research and development because of their various biological activities [17], such as insecticide [18], antibacterial [19], antifungal [20], local anesthesia [21], antimalarial [22], antineoplastic [23] properties. The introduction of 1,2,3-triazole group may improve the biological activity and druggability. Based on these thoughts, we introduce the triazole group into the basic scaffold of lamiridosin A through click reaction [24] (compounds 4–17).

2. Results and discussion

Initially, the in vitro antitumor activity of the mixture of lamiophlomiols A/B (3/1) was examined against three human tumor cell lines including EC9706 (esophageal carcinoma), MGC803 (gastric cancer), and B16 (melanoma) by sulforhodamine B method. The slight activity against EC9706, MGC803, and B16 cells promoted us to further study the synthesis and antitumor activity of the derivatives of lamiophlomiols A/B (3/1). During the synthetic research of the derivatives of lamiophlomiols A/B (3/1), we found that treating the mixtures of lamiophlomiols A (1R-isomers) and B (1S-isomers) with 37% hydrochloric acid solution and different alcohol, interestingly generated the single enantiomer C1-R-ether derivatives by the oxygen-containing three-membered ring open loop and the attack of chloride ion (Scheme 1). Ethanol, isopropanol, and n-butyl alcohol reacted with raw material mixtures, resulting in compounds 1, 2, and 3, respectively, in 48–81% yields. In order to firmly estab-lish the structure, the HSQC, HMBC, and NOESY spectra of compound 3 (a selected exam-ple) were examined. According to the HSQC experiment, the 1H and 13C spectral signals of compound 3 were assigned. In the HMBC spectrum, the correlations between C-7 and H-7a, H-8, H-6, H-1, H-4a were observed. In the NOESY spectrum, the cross-peak between H-1 and H-8 of compound 3 confirmed the OR group of C-1 as β-orientation. Three compounds

OCl

HOO

O

O

HO

R1

H

HOO

HOO

OH

OH

H

a

1: R1=-CH2CH32: R1=-CH(CH3)23: R1=-CH2CH2CH2CH3

12

34

4a7a

56

7

a: ethanol/2-propanol/butyl alcohol, con. HCl, r.t., 48-81%.

Scheme 1. synthesis of compounds 1–3.

28 Y.-X. YAnG ET AL.

showed enhancing antitumor activity comparing with the mixtures of lamiophlomiols A/B (3/1), and especially, the isopropanol derivative 2 exhibited 12-fold enhancement against the cancer cells MGC803, closing to the activity of the positive drug 5-FU (as depicted in Table 1). Under the same conditions, the raw material mixture of lamiophlomiols A/B (3/1) in 3-butyn-1-ol gave the desired reactive intermediate alkynyl product 1A, then the interme-diate compound 1A reacted with azide producing the single enantiomer C1-R-1,2,3-triazole derivatives (compounds 4–17) in 77–97% yields (as depicted in Scheme 2). The introduction of 1,2,3-triazole into the lamiophlomiol skeleton, in general, exhibited enhancing effects in terms of antitumor activities, compared with the mixture of lamiophlomiols A/B (3/1) (as depicted in Table 1). Specifically, the substituent R2 of 1,2,3-triazole derivatives played an important role in enhancing anticancer activities. The electronic properties and steric hin-drance of the substituents on the benzene ring all affect the antitumor activity. Compound 4 with unsubstituted benzene did not show improved antitumor activity. Compounds with either electron-withdrawing substituents such as 4-nitrile, 4-fluoro, and 4-nitro groups (compounds 5, 6, and 7) or electron-donating substituents such as 4-methyl and 4-ethyl groups (compounds 8 and 9) in the benzene ring are all more effective than derivative 4. Particularly, the introduction of larger steric hindrance substituent to the benzene ring, such as tert-butyl group in compound 10, exhibited the significantly improved antitumor activity against the two cancer cell lines (EC9706, MGC803), with 10-fold (EC9706) and 38-fold (MGC803) more active than lamiophlomiols A/B (3/1), respectively. The IC50 of 10 was 2.36 μmol/L, less than the positive drug 5-FU. Changing the benzene ring to the pyridine ring (compounds 11–13) caused the loss of activity, compared with 10. Replacing

OCl

HOO

O

O

HO

H

HOO

HOO

OH

OH

H

b cO

Cl

HOO

O

O

HO

H

H

N NN

R21A

4: R2= 5: R2=CN

6: R2=

7: R2=NO2

8: R2= 9: R2=

10: R2= 11: R2=N

12: R2= N

13: R2=N

4-17

14: R2=O

15: R2=

16: R2= 17: R2=

F

Cl

O

b: 3-butyn-1-ol, con. HCl, 48%. c: R2CH

2-N

3, CuSO

4·5H

2O, sodium ascorbate, THF-H

2O or

t-BuOH-H2O, 77-97%.

Scheme 2. synthesis of compounds 4–17.

JoUrnAL oF ASIAn nATUrAL ProdUCTS rESEArCh 29

the pyridine ring with fatty chain groups gave mixed results (compounds 14–17). Among them, compound 17 showed remarkable apoptotic effect in three kinds of cell lines (EC9706, MGC803 and B16), that were fivefold (EC9706), sixfold (MGC803), and sevenfold (B16) more effective than the activity of lamiophlomiols A/B (3/1), respectively.

3. Experimental

3.1. General experimental procedures

All 1H NMR and 13C NMR spectra were measured in CDCl3 or DMSO-d6 using a Bruker ASCEND 400 spectrometer (Bruker Corporation, Faellanden, Switzerland). Chemical shifts are expressed in ppm and J values are given in Hz. High-resolution mass spectra were recorded on Bruker microTOF-QIII MS (ESI and APCI) (Bruker Corporation, German). Column chromatography was performed with 200–300 mesh silica gel (Qingdao Haiyang Chemical Company Limited, Qingdao, China) using flash column techniques. All the sol-vents and reagents were used directly as obtained commercially unless otherwise noted.

3.2. General procedure for the synthesis of compounds 1–3

To a solution of the mixed lamiophlomiols A/B (3/1, 48 mg, 0.2 mmol) in different alcohol (ethanol, isopropanol, or n-butyl alcohol) (1.0 ml), 37% hydrochloric acid (0.1 ml) was added. The reaction was stirred at room temperature for 12 h. The resulting solution was extracted with ethyl acetate. The organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography, eluting with petroleum ether (PE) and ethyl acetate (EA) in various ratios [PE:EA, 5:1, 3:1, 1:1, 0:1] to give the pure product.

Table 1. in vitro antitumor activity of lamiridosin a analogs.

Compound

IC50 (μmol/L) IC50 (μmol/L) IC50 (μmol/L)

EC9706 MGC803 B16lamiophlomiols >100 77.1 ± 1.9 68.6 ± 1.8a/B (3/1)1 >100 39.1 ± 1.5 >1002 90.4 ± 1.9 6.2 ± 0.7 >1003 52.8 ± 1.7 30.3 ± 1.4 81.6 ± 1.94 >100 94.3 ± 1.9 66.4 ± 1.85 65.2 ± 1.8 64.7 ± 1.8 84.8 ± 1.96 51.0 ± 1.7 >100 90.6 ± 1.97 35.5 ± 1.5 79.9 ± 1.9 60.9 ± 1.88 34.9 ± 1.5 31.6 ± 1.5 43.1 ± 1.69 33.6 ± 1.5 29.1 ± 1.4 20.1 ± 1.310 11.4 ± 1.0 2.3 ± 0.3 22.6 ± 1.311 44.4 ± 1.6 >100 >10012 87.9 ± 1.9 54.9 ± 1.7 >10013 >100 52.9 ± 1.7 85.8 ± 1.914 >100 23.2 ± 1.3 96.3 ± 1.915 >100 62.8 ± 1.8 >10016 >100 >100 77.3 ± 1.817 23.5 ± 1.3 12.1 ± 1.0 9.9 ± 0.95-fu 6.3 ± 0.1 3.24 ± 0.1 3.5 ± 2.6

30 Y.-X. YAnG ET AL.

3.2.1. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-ethoxy-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (1)Yield: 81%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.36 (s, 1H), 5.12 (s, 1H, H-1), 4.06 (t, J = 3.4 Hz, 1H), 3.99 (s, 1H, OH), 3.81–3.74 (m, 1H), 3.74 (s, 3H), 3.70 (dd, J = 9.3, 4.7 Hz, 1H), 3.59–3.50 (m, 1H), 2.84–2.79 (m, 1H), 2.69 (d, J = 11.9 Hz, 1H), 1.21 (s, 3H), 2.09 (s, 1H, OH), 1.17 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 168.9, 151.5, 110.0, 96.4, 81.6, 77.4, 72.5, 64.9, 51.9, 46.3, 35.7, 19.7, 15.2; HRESIMS: m/z 329.0770 [M + Na]+ (calcd for C13H19ClO6Na, 329.0762).

3.2.2. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-5,6-dihydroxy-1-isopropoxyl-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (2)Yield: 48%. Light yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.35 (s, 1H), 5.22 (s, 1H, H-1), 4.05 (d, J = 9.2 Hz, 1H), 3.97–3.90 (m, 1H), 3.73 (s, 3H), 3.69 (dd, J = 9.2, 4.7 Hz, 1H), 2.81 (dd, J = 11.8, 4.7 Hz, 1H), 2.63 (d, J = 11.8 Hz, 1H), 1.20 (s, 3H), 1.14 (dd, J = 8.7, 6.3 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 169.0, 151.7, 109.9, 94.7, 81.7, 77.4, 72.5, 71.2, 51.9, 46.6, 35.8, 23.5, 21.9, 19.6; HRESIMS: m/z 343.0917 [M + Na]+ (calcd for C14H21ClO6Na, 343.0919).

3.2.3. (1R,4aS,5S,6S,7R,7aR)-Methyl 1-butoxyl-7-chloro-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (3)Yield: 61%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.40 (s, 1H, H-3), 5.14 (s, 1H, H-1), 4.11 (d, J = 9.2 Hz, 1H, H-5), 3.84–3.67 (m, 5H, H-10, H-1′, H-6), 3.57–3.47 (m, H-1′), 2.84 (dd, J = 11.7, 4.3 Hz, 1H, H-4a), 2.72 (d, J = 11.7 Hz, 1H, H-7a), 2.20 (s, 1H, OH), 1.58–1.50 (m, 2H, H-2′), 1.39–1.29 (m, 2H, H-3′), 1.24 (s, 3H, H-8), 0.91 (t, J = 7.3 Hz, 3H, H-4′); 13C NMR (101 MHz, CDCl3): δ 168.9 (C-9), 151.5 (C-3), 109.9 (C-4), 96.5 (C-1), 81.5 (C-6), 77.3 (C-7), 72.4 (C-5), 69.0 (C-1′), 51.9 (C-10), 46.2 (C-7a), 35.7 (C-4a), 31.5 (C-2′), 19.5 (C-3′), 19.3 (C-8), 13.9 (C-4′); HRESIMS: m/z 357.1085 [M + Na]+ (calcd for C15H23ClO6Na, 357.1075).

3.3. General procedure for the synthesis of compounds 4–17

3.3.1. Step 1To a solution of mixed lamiophlomiols A/B (3/1, 48 mg, 0.2 mmol) in 3-butyn-1-ol (1.0 ml), 37% hydrochloric acid (0.1 ml) was added. The reaction was stirred at room temperature for 12 h. The resulting solution was extracted with ethyl acetate. Two layers were separated and the aqueous layer was extracted with additional ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography, eluting with petroleum ether and ethyl acetate in various ratios [PE:EA, 5:1, 3:1], to give the pure product 1A as a white solid, yield: 50%.

3.3.1.1. (1R,4aS,5S,6S,7R,7aR)-Methyl 1-(but-3-yn-1-yloxy)-7-chloro-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (1A). Yield: 50%. 1H NMR (400 MHz, CDCl3): δ 7.40 (d, J = 1.0 Hz, 1H), 5.07 (d, J = 1.9 Hz, 1H), 4.28 (dd, J = 6.1, 3.9 Hz, 1H), 4.01 (d, J = 3.8 Hz, 1H), 3.89–3.82 (m, 1H), 3.75 (s, 3H), 3.73–3.65 (m, 2H), 2.98 (dd, J = 11.8, 1.8 Hz, 1H), 2.83 (dd, J = 11.7, 5.3 Hz, 1H), 2.50–2.42 (m, 3H), 1.75 (s,

JoUrnAL oF ASIAn nATUrAL ProdUCTS rESEArCh 31

3H); 13C NMR (100 MHz, CDCl3): δ 169.8, 151.8, 110.3, 98.1, 81.0, 78.6, 77.4, 75.3, 69.9, 67.0, 52.0, 48.6, 38.2, 25.0, 20.0.

3.3.2. Step 2To a solution of compound 1A (62.4 mg, 0.2 mmol), azide (0.3 mmol), CuSO4·5H2O (5 mg), sodium ascorbate (5 mg) in 4 ml THF–water (v/v, 1/1) or 4 ml t-BuOH–water (v/v, 1/1), the mixture was stirred at 40 °C for 4 h. The reaction mixture was cooled to room temperature and treated with ethyl acetate. The layers were separated and the aqueous layer was extracted with additional ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chroma-tography, eluting with petroleum ether and ethyl acetate in various ratios [PE:EA, 10:1, 5:1, 3:1, 1:1, 0:1] to give the pure product.

3.3.2.1. (1R,4aS,5S,6S,7R,7aR)-Methyl 1-(2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethoxy)-7-chloro-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (4). Yield: 97%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.38–7.32 (m, 4H), 7.29 (s, 1H), 7.24 (s, 1H), 7.23 (s, 1H), 5.48 (d, J = 1.4 Hz, 2H), 4.99 (d, J = 1.7 Hz, 1H), 4.25 (dd, J = 5.9, 3.9 Hz, 1H), 4.05–3.99 (m, 1H), 3.98 (d, J = 3.8 Hz, 1H), 3.85–3.78 (m, 1H), 3.74 (s, 3H), 3.00 (t, J = 6.3 Hz, 2H), 2.83 (dd, J = 11.7, 1.4 Hz, 1H), 2.76 (dd, J = 11.7, 6.0 Hz, 1H), 1.64 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.8, 152.0, 144.7, 134.4, 129.4 × 2, 129.2, 128.3 × 2, 122.3, 110.0, 98.1, 81.0, 78.5, 75.1, 67.5, 54.7, 52.0, 48.6, 38.2, 26.2, 25.0; HRESIMS: m/z 486.1412 [M + Na]+ (calcd for C22H26ClN3O6Na, 486.1402).

3.3.2.2. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-(4-cyanobenzyl)-1H-1,2,3-triazol- 4-yl)ethoxy)-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (5). Yield: 89%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.64 (d, J = 7.7 Hz, 2H), 7.40–7.30 (m, 4H), 5.56 (s, 2H), 5.12 (s, 1H), 4.05 (d, J = 8.3 Hz, 1H), 4.00–3.94 (m, 1H), 3.81–3.75 (m, 1H), 3.73 (s, 3H), 3.68 (dd, J = 9.2, 4.3 Hz, 1H), 3.25–2.85 (m, 4H), 2.73 (dd, J = 11.6, 4.2 Hz, 1H), 2.66 (d, J = 11.9 Hz, 1H), 1.17 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.3, 145.4, 139.9, 133.1 × 2, 128.6 × 2, 122.6, 118.3, 113.0, 110.0, 96.3, 81.5, 77.4, 72.3, 67.5, 53.7, 52.0, 46.2, 35.7, 26.3, 19.5; HRESIMS: m/z 511.1354 [M + Na]+ (calcd for C23H25ClN4O6Na, 511.1355).

3.3.2.3. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-(4-fluorobenzyl)-1H-1,2,3-triazol- 4-yl)ethoxy)-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (6). Yield: 87%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.48 (s, 1H), 7.31 (s, 1H), 7.28 (dd, J = 8.5, 5.2 Hz, 2H), 7.05 (t, J = 8.5 Hz, 2H), 5.51 (s, 2H), 5.15 (s, 1H), 4.08 (d, J = 6.5 Hz, 1H), 3.40–3.93 (m, 1H), 3.82–3.75 (m, 1H), 3.73 (s, 3H), 3.67 (d, J = 9.3 Hz, 1H), 3.08–2.92 (m, 2H), 2.73 (d, J = 1.5 Hz, 2H), 1.16 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.6, 151.1, 144.2, 130.2 × 2, 129.9, 122.8, 116.3 × 2, 109.9, 96.1, 81.2, 77.2, 72.1, 67.0, 54.0, 51.8, 46.0, 35.4, 25.7, 19.2; HRESIMS: m/z 504.1296 [M + Na]+ (calcd for C22H25FClN3O6Na, 504.1308).

3.3.2.4. (1R,4aS,5S,6S,7R,7aR)-methyl 7-chloro-5,6-dihydroxy-7-methyl-1-(2-(1-(4-nitro benzyl)-1H-1,2,3-triazol-4-yl)ethoxy)-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (7). Yield: 81%. Colorless oil. 1H NMR (400  MHz, CDCl3): δ 8.21 (d,

32 Y.-X. YAnG ET AL.

J = 8.7 Hz, 2H), 7.55 (s, 1H), 7.43 (d, J = 8.6 Hz, 2H), 7.32 (s, 1H), 5.67 (s, 2H), 5.14 (s, 1H), 4.06 (d, J = 9.3 Hz, 1H), 3.99 (dt, J = 9.7, 5.9 Hz, 1H), 3.82–3.75 (m, 1H), 3.73 (s, 3H), 3.67 (dt, J = 9.3, 1.9 Hz, 1H), 3.10–2.95 (m, 2H), 2.72 (d, J = 1.7 Hz, 2H), 1.17 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.3, 148.4, 145.1, 141.4, 129.0 × 2, 129.0, 124.6 × 2, 110.1, 96.3, 81.4, 77.4, 72.3, 67.3, 53.7, 52.0, 46.2, 35.7, 31.2, 19.5; HRESIMS: m/z 531.1258 [M + Na]+ (calcd for C22H25ClN4O8Na, 531.1253).

3.3.2.5. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-5,6-dihydroxy-7-methyl-1-(2-(1-(4-methyl benzyl)-1H-1,2,3-triazol-4-yl)ethoxy)-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (8). Yield: 80%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.30 (s, 1H), 7.24 (s, 1H), 7.17–7.10 (m, 4H), 5.41 (s, 2H), 5.12 (s, 1H), 4.04 (d, J = 9.3 Hz, 1H), 3.97–3.90 (m, 1H), 3.79–3.74 (m, 1H), 3.72 (s, 3H), 3.67 (dd, J = 9.2, 3.6 Hz, 1H), 3.57–3.20 (m, 2H), 2.99–2.87 (m, 2H), 2.71 (dd, J = 11.8, 4.5 Hz, 1H), 2.62 (d, J = 11.9 Hz, 1H), 2.32 (s, 3H), 1.16 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.4, 144.8, 139.0, 131.6, 130.0 × 2, 128.3 × 2, 122.1, 110.0, 96.3, 81.5, 77.2, 72.3, 67.7, 54.3, 52.0, 46.3, 35.6, 26.2, 21.4, 19.4; HRESIMS: m/z 500.1545 [M + Na]+ (calcd for C23H28ClN3O6Na, 500.1559).

3.3.2.6. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-(4-ethylbenzyl)-1H-1,2,3-triazol- 4-yl)ethoxy)-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (9). Yield: 81%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.32 (d, J = 7.5 Hz, 2H), 7.21–7.14 (m, 4H), 5.45 (s, 2H), 5.13 (s, 1H), 4.05 (d, J = 9.3 Hz, 1H), 4.00–3.93 (m, 1H), 3.82–3.76 (m, 1H), 3.73 (s, 3H), 3.68 (dd, J = 9.3, 4.5 Hz, 1H), 3.62–3.38 (m, 2H), 3.06–2.87 (m, 2H), 2.73 (dd, J = 11.9, 4.5 Hz, 1H), 2.68–2.59 (m, 3H), 1.21 (t, J = 7.6 Hz, 3H), 1.17 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.4, 144.4, 135.5, 131.5, 128.9 × 2, 128.5 × 2, 122.6, 110.1, 96.3, 81.5, 77.4, 72.3, 67.4, 54.7, 52.0, 46.3, 35.7, 28.7, 26.0, 19.5, 15.6; HRESIMS: m/z 514.1713 [M + Na]+ (calcd for C24H30ClN3O6Na, 514.1715).

3.3.2.7. (1R,4aS,5S,6S,7R,7aR)-methyl 1-(2-(1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazol-4-yl)ethoxy)-7-chloro-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (10). Yield: 86%. Colorless oil. 1H NMR (400  MHz, CDCl3) δ 7.41 (d, J = 8.2 Hz, 2H), 7.38 (s, 1H), 7.35 (s, 1H), 7.19 (d, J = 8.2 Hz, 2H), 5.46 (d, J = 2.0 Hz, 2H), 5.14 (s, 1H), 4.07 (d, J = 9.3 Hz, 1H), 3.99–3.93 (m, 1H), 3.84–3.76 (m, 1H), 3.73 (s, 3H), 3.68 (dd, J = 9.3, 4.4 Hz, 1H), 3.44 (br s, 2H), 3.05–2.90 (m, 2H), 2.74 (dd, J = 11.9, 4.1 Hz, 1H), 2.68 (d, J = 12.0 Hz, 1H), 1.28 (s, 9H), 1.17 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.8, 152.4, 151.4, 144.4, 131.3, 128.2 × 2, 126.3 × 2, 122.6, 110.1, 96.3, 81.5, 77.4, 72.3, 67.5, 54.5, 52.0, 46.3, 35.7, 34.9, 31.5 × 3, 26.1, 19.5; HRESIMS: m/z 542.2015 [M + Na]+ (calcd for C26H34ClN3O6Na, 542.2028).

3.3.2.8. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-5,6-dihydroxy-7-methyl-1-(2-(1-(pyridin-4-ylmethyl)-1H-1,2,3-triazol-4-yl)ethoxy)-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (11). Yield: 91%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.74 (d, J = 4.9 Hz, 2H), 7.77 (s, 1H), 7.52 (J = 4.9 Hz, 2H), 7.34 (s, 1H), 5.83 (s, 2H), 5.13 (s, 1H), 4.16 (d, J = 9.1 Hz, 1H), 4.06–3.96 (m, 1H), 3.78–3.73 (m, 1H), 3.72 (s, 3H), 3.66 (dd, J = 9.2, 4.4 Hz, 1H), 3.08–2.89 (m, 2H), 2.81 (d, J = 12.0 Hz, 1H), 2.73 (dd, J = 11.8, 4.2 Hz, 1H), 1.11 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.4, 146.1 × 2, 145.2, 130.1, 124.6 × 2,

JoUrnAL oF ASIAn nATUrAL ProdUCTS rESEArCh 33

123.6, 110.0, 96.4, 81.4, 77.4, 72.5, 67.6, 52.5, 52.0, 46.5, 35.6, 26.4, 19.1; HRESIMS: m/z 487.1357 [M + Na]+ (calcd for C21H25ClN4O6Na, 487.1355).

3.3.2.9. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-((6-chloropyridin-3-yl)methyl)- 1 H - 1 , 2 , 3 - t r i a z o l - 4 - y l ) e t h ox y ) - 5 , 6 - d i hy d rox y - 7 - m e t hy l - 1 , 4 a , 5 , 6 , 7 , 7 a -hexahydrocyclopenta[c]pyran-4-carboxylate (12). Yield: 78%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 8.33 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.34 (s, 1H), 5.93–5.81 (m, 2H), 5.21 (s, 1H), 4.14 (d, J = 9.6 Hz, 1H), 4.05–4.00 (m, 1H), 3.85–3.77 (m, 1H), 3.73 (s, 3H), 3.65 (dd, J = 9.2, 4.7 Hz, 1H), 3.23 (s, 1H), 3.14–3.04 (m, 1H), 2.98 (d, J = 11.8 Hz, 1H), 2.74 (dd, J = 11.9, 4.4 Hz, 1H), 1.11 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 152.6, 151.1, 149.7, 143.3, 140.1, 128.5, 126.0, 125.4, 110.1, 96.2, 81.3, 77.4, 72.3, 66.2, 52.6, 52.0, 46.4, 35.5, 24.9, 18.9; HRESIMS: m/z 521.0955 [M + Na]+ (calcd for C21H24Cl2N4O6Na, 521.0965).

3.3.2.10. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-5,6-dihydroxy-7-methyl-1-(2-(1-(pyridin- 2-ylmethyl)-1H-1,2,3-triazol-4-yl)ethoxy)-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (13). Yield: 80%. Colorless oil. 1H NMR (400  MHz, CDCl3): δ 8.77 (d, J = 5.0 Hz, 1H), 8.18 (dt, J = 7.8, 3.9 Hz, 1H), 8.06 (s, 1H), 7.76–7.69 (m, 2H), 7.34 (s, 1H), 5.99 (d, J = 4.5 Hz, 2H), 5.11 (s, 1H), 4.19 (d, J = 9.3 Hz, 1H), 4.01 (dt, J = 9.8, 4.9 Hz, 1H), 3.73 (s, 3H), 3.67 (dd, J = 9.4, 4.8 Hz, 2H), 3.04–2.88 (m, 3H), 2.76 (dd, J = 11.9, 4.2 Hz, 1H), 1.11 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 168.9, 151.4, 151.3, 145.9, 144.8, 143.7, 126.5, 126.0, 123.8, 110.1, 96.7, 81.5, 77.3, 72.2, 67.8, 51.9, 51.5, 46.7, 35.6, 26.4, 18.9; HRESIMS: m/z 487.1351 [M + Na]+ (calcd for C21H25ClN4O6Na, 487.1355).

3.3.2.11. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-(4-ethoxy-4-oxobutyl)-1H-1,2,3-triazol-4-yl)ethoxy)-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (14). Yield: 81%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.34 (s, 1H), 7.33 (s, 1H), 5.11 (s, 1H), 4.36 (td, J = 6.8, 2.4 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 4.07 (d, J = 9.3 Hz, 1H), 3.99 (dt, J = 9.8, 6.1 Hz, 1H), 3.81–3.76 (m, 1H), 3.74 (s, 3H), 3.69 (dd, J = 9.3, 4.7 Hz, 1H), 3.00–2.94 (m, 2H), 2.78 (dd, J = 11.9, 4.4 Hz, 1H), 2.68 (d, J = 11.9 Hz, 1H), 2.31 (t, J = 7.0 Hz, 2H), 2.22–2.13 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.18 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 172.8, 168.8, 151.4, 144.7, 122.3, 110.1, 96.4, 81.5, 77.4, 72.3, 67.9, 61.1, 52.0, 49.5, 46.4, 35.7, 31.1, 26.4, 25.7, 19.5, 14.4; HRESIMS: m/z 510.1607 [M + Na]+ (calcd for C21H30ClN3O8Na, 510.1614).

3.3.2.12. (1R,4aS,5S,6S,7R,7aR)-Methyl 1-(2-(1-butyl-1H-1,2,3-triazol-4-yl)ethoxy)-7-chloro-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (15). Yield: 77%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.38 (s, 1H), 7.33 (s, 1H), 5.17 (s, 1H), 4.32 (t, J = 7.2 Hz, 2H), 4.08 (d, J = 9.1 Hz, 1H), 4.01–3.95 (m, 1H), 3.84–3.77 (m, 1H), 3.74 (s, 3H), 3.69 (dd, J = 9.3, 4.4 Hz, 1H), 3.00–2.95 (m, 2H), 2.77 (dd, J = 11.9, 4.3 Hz, 1H), 2.71 (d, J = 11.9 Hz, 1H), 1.90–1.79 (m, 2H), 1.37–1.28 (m, 2H), 1.19 (s, 3H), 0.93 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ: 168.8, 151.4, 144.2, 122.4, 110.1, 96.3, 81.5, 77.4, 72.3, 67.6, 52.0, 50.6, 46.3, 35.7, 32.3, 26.1, 19.9, 19.5, 13.6; HRESIMS: m/z 452.1552 [M + Na]+ (calcd for C19H28ClN3O6Na, 452.1559).

34 Y.-X. YAnG ET AL.

3.3.2.13. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-5,6-dihydroxy-1-(2-(1-isopentyl-1H- 1,2,3-triazol-4-yl)ethoxy)-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (16). Yield: 86%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.41 (s, 1H), 7.33 (s, 1H), 5.17 (s, 1H), 4.33 (t, J = 7.4 Hz, 2H), 4.08 (d, J = 9.2 Hz, 1H), 4.02–3.93 (m, 1H), 3.83–3.77 (m, 1H), 3.74 (s, 3H), 3.68 (dd, J = 9.3, 3.3 Hz, 1H), 3.64–3.40 (m, 2H), 3.09–2.88 (m, 2H), 2.77 (dd, J = 12.0, 3.9 Hz, 1H), 2.71 (d, J = 12.0 Hz, 1H), 1.78–1.72 (m, 2H), 1.59–1.51 (m, 1H), 1.19 (s, 3H), 0.93 (d, J = 6.5 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.4, 144.0, 122.4, 110.1, 96.3, 81.5, 77.4, 72.3, 67.6, 52.0, 49.3, 46.3, 39.1, 35.7, 26.1, 25.7, 22.4 × 2, 19.5; HRESIMS: m/z 466.1724 [M + Na]+ (calcd for C20H30ClN3O6Na, 466.1715).

3.3.2.14. (1R,4aS,5S,6S,7R,7aR)-Methyl 7-chloro-1-(2-(1-hexyl-1H-1,2,3-triazol-4-yl) ethoxy)-5,6-dihydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-4-carboxylate (17). Yield: 86%. Colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.37 (s, 1H), 7.32 (s, 1H), 5.17 (s, 1H), 4.29 (t, J = 7.2 Hz, 2H), 4.07 (d, J = 9.3 Hz, 1H), 4.01–3.93 (m, 1H), 3.84–3.76 (m, 1H), 3.73 (s, 3H), 3.68 (dd, J = 9.2, 4.2 Hz, 1H), 3.34 (br s, 2H), 3.06–2.92 (m, 2H), 2.77 (dd, J = 11.9, 4.3 Hz, 1H), 2.71 (d, J = 11.9 Hz, 1H), 1.90–1.79 (m, 2H), 1.32–1.24 (m, 6H), 1.18 (s, 3H), 0.85 (t, J = 5.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 168.8, 151.4, 144.3, 122.3, 110.1, 96.3, 81.5, 77.4, 72.3, 67.6, 52.0, 50.9, 46.3, 35.7, 31.3, 30.4, 26.3, 26.1, 22.6, 19.5, 14.1; HRESIMS: m/z 480.1883 [M + Na]+ (calcd for C21H32ClN3O6Na, 480.1872).

3.4. Cytotoxicity assay

The cytotoxicity of lamiophlomiols A/B (3/1) and its derivatives toward cells EC9706, MGC803 and B16 were determined in 96-well microtiter plates by the sulforhodamine B method described by Skehan et al. [25]. Briefly, exponentially growing EC9706, MGC803 and B16 cells were harvested and seeded in 96-well plates with the final volume 100 μl containing 4 × 103 cells per well. After 24 h incubation, cells were treated with various concentrations of lamiophlomiols A and B or its derivatives for 48 h. The cultures were fixed at 4 °C for 1 h by addition of ice-cold 50% trichloroacetic acid (TCA) to give a final concentration of 10%. Fixed cells were rinsed five times with deionized water and stained for 10 min with 0.4% sulforhodamine B dissolved in 0.1% acetic acid. The wells were washed five times with 0.1% acetic acid and left to dry overnight. The absorbed sulforhodamine B was dissolved in 150 μl unbuffered 1% Tris base [tris(hydroxymethyl) aminomethane] solution in water (pH 10.5). The absorbency of extracted sulforhodamine B at 515 nm was measured on a microplate reader (Bio-Rad, Shanghai, China). The experiments were carried out in triplicate. Each run entailed 5–6 concentrations of the compounds being tested. The percentage survival rates of cells exposed to the compounds were calculated by assuming the survival rate of untreated cells to be 100%.

Disclosure statement

No potential conflict of interest was reported by the authors.

JoUrnAL oF ASIAn nATUrAL ProdUCTS rESEArCh 35

Funding

This work was financially supported by the National Natural Science Foundation of China [grant number 81172953].

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