Accepted Manuscript Antiproliferative glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus Nahyun Kim, Kyu-Won Cho, Seong Su Hong, Bang Yeon Hwang, Taehoon Chun, Dongho Lee PII: DOI: Reference:

S0960-894X(14)01304-3 http://dx.doi.org/10.1016/j.bmcl.2014.12.007 BMCL 22252

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

1 September 2014 3 December 2014 4 December 2014

Please cite this article as: Kim, N., Cho, K-W., Hong, S.S., Hwang, B.Y., Chun, T., Lee, D., Antiproliferative glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.007

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Antiproliferative glabretal-type triterpenoids from the root bark of Dictamnus dasycarpus

Nahyun Kima, Kyu-Won Choa,† Seong Su Honga,b, Bang Yeon Hwangc, Taehoon Chuna,*, and Dongho Leea,*

a

College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea

b

Natural Products Research Institute, Gyeonggi Institute of Science and Technology

Promotion, Suwon 443-270, Korea c

College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea

* Corresponding authors. Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail address: [email protected]. (D. Lee) Tel: +82-2-3290-3069. Fax; +82-2-3290-3499. E-mail address: [email protected] (T. Chun)

1

ABSTRACT

Four new glabretal-type triterpenoids, dictabretols A−D (1−4), were isolated by activityguided fractionation from the root bark of Dictamnus dasycarpus T. (Rutaceae) using an in vitro antiproliferative assay on T cells using splenocytes. The structures of these compounds were determined by spectroscopic methods, including 2D NMR experiments. Compounds were evaluated for their immunosuppressive activity on T cells and demonstrated inhibition of proliferation of activated T cells, up to IC50 of 1.5 µM.

Keywords: Dictamnus dasycarpus; Rutaceae; glabretal-type triterpenoid; antiproliferative effect

2

Dictamnus dasycarpus T. (Rutaceae) is a perennial herb that is widespread across Asia and Europe; it is known as the ‘gas plant’ or ‘burning-bush’ because of the volatile oils it produces.1,2 D. dasycarpus has been used as a traditional medicine for amenorrhea, antifertilization, cough, jaundice, rheumatism, and skin disorders. Phytochemical studies of the Dictamnus genus have shown it to contain alkaloids, limonoids, flavonoids, coumarins, sesquiterpene glycosides, and essential oils.3-10 Immunosuppressive agents have been used to treat immunologically mediated diseases and prevent activities related to the immune system, such as organ transplantation rejection. Among these agents, cyclosporin A and FK506, initially isolated from the fungus Cylindrocarbon lucidum and Streptomyces tsukubaensis is the most widely used and effective immunosuppressive drug in clinical use today.11-14 Given its side effects on vascular tension and plasma lipoprotein, there is a need for new immunosuppressive agents.15 In our search for immunosuppressive agents from natural origin, methanolic extract of the root bark of D. dasycarpus was found to inhibit the proliferation of T cells. The bioassayguided fractionation and purification of CHCl3-soluble fraction led to the isolation of four new glabretal-type triterpenoids, dictabretols A−D (1−4).16 The effects of the compounds on activated T cells were examined by measuring the proliferation of T cells and the secretion level of cytokines, IL-2 and IFN-γ.17 This report describes the isolation and structure elucidation of 1−4 along with their biological evaluation. Dictabretol A (1)18 was isolated as a white amorphous powder, mp 234.4 °C. The molecular formula was determined as C35H56O7 by HRESIMS at m/z 587.3951 [M - H](calcd for C35H55O7, 587.3948), with eight degrees of unsaturation in the molecule. The IR spectrum showed the presence of hydroxy (3402 cm-1) and ester (1723 cm-1) functionalities. The 1H NMR spectrum of 1 (in CDCl3, Table 1) displayed the signals for seven methyl 3

groups at δH 0.88 (H-19), 1.31 (H-27), 0.84 (H-28), 0.87 (H-29), 1.04 (H-30), 0.96 (H-4′), and 0.95 (H-5′). Proton signals for six oxygenated methines at δH 4.65 (H-3), 3.75 (H-7), 5.43 (H-21), 3.94 (H-23), 3.18 (H-24), and 3.65 (H-26) as well as overlapping proton signals for aliphatic methines and methylenes were exhibited. In addition, characteristic signals were observed for a cyclopropyl methylene group in a relatively high-field region at δH 0.71 (2H, doublet, J = 4.5 Hz, H-18a) and 0.46 (2H, doublet, J = 5.0 Hz, H-18b). Detailed analysis of 1

H and

13

C NMR spectra (Tables 1 and 2) as well as the HMBC spectrum revealed that

compound 1 has a glabretal triterpene skeleton.19,20 The 1H and

13

C NMR spectra of 1

displayed the signals of a trisubstituted epoxy group at C-24 [δH 3.18 (1H, doublet, J = 7.5 Hz) and δC 63.4] and C-25 (δC 60.7). In addition, the chemical shifts at δC 98.3 (C-21) and δH 5.43 (1H, overlap, H-21) suggested the presence of a hemi-acetal group. The 1H NMR signals at δH 2.21 (2H, broad doublet, J = 7.0 Hz, H-2′), 2.13 (1H, multiplet, H-3′), 0.96 (3H, doublet, J = 2.0 Hz, H-4′), and 0.95 (3H, doublet, J = 2.0 Hz, H-5′) demonstrated the presence of an isovaleric group. The position of the isovaleric acid was determined as C-3 from the longrange HMBC correlation of δH 0.84 (H-28) and δH 0.87 (H-29) with δC 77.8 (C-3), 36.2 (C-4), and 41.3 (C-5), as well as that of δH 4.65 (H-3) with δC 33.9 (C-1), 22.9 (C-2), 36.2 (C-4), 41.3 (C-5), 27.8 (C-28), 21.9 (C-29) and 172.9 (C-1′).19 The signal of the broad singlet at δH 4.65 (1H, broad singlet, H-3) and 3.75 (1H, broad singlet, H-7) indicated an α-orientation of the isovaleric acid and hydroxy moieties, respectively.21,22 Further evidence for the relative configuration of compound 1 was proven by the NOE correlations between H-3/H-19 and H-7/H-30 and the comparison of chemical shifts with literature values of glabretal-type triterpenoids.19,21,23 In addition, the HSQC spectra exhibited correlation of H-21 (δH 5.43 overlap) with C-21 (δC 98.3 and 102.2), and the 13

C NMR spectrum exhibited pairs of closely spaced signals, accounting for an inseparable

epimeric mixture at C-21.19-21,23,24 Furthermore, with the effort to address the absolute 4

configuration, a modified Mosher’s method was applied; however, it was unsuccessful to decide the structure with the product decomposition after the reaction. Therefore, the structure of this new glabretal-type triterpenoid, dictabretol A (1), was elucidated as shown. Dictabretol B (2),25 obtained as a white amorphous powder, mp 229.9 °C showed a molecular formula of C35H58O7 by HRESIMS at m/z 589.4103 [M - H]- (calcd for C35H57O7, 589.4104), with seven degrees of unsaturation in the molecule. The 1H and 13C NMR spectra of compound 2 (Tables 1 and 2) were very similar to those of compound 1, and suggested that it is also a glabretal triterpene. The additional singlet methyl group of C-26 [δH 1.28 (3H, singlet) and δC 26.9] replaced the methylene group of 1 [δH 3.65 (2H, doublet, J = 12.5 Hz) and δC 64.9], accounting for the loss of a hydroxy group at C-26. The downfield shifts of C24 (δC 75.3) and C-25 (δC 73.7) signals of 2 compared to those of 1 (δC 63.4 and 60.7) suggested an epoxide ring-opening,24 which was substantiated by the decreased degrees of unsaturation. Accordingly, the structure of compound 2 was unambiguously determined to have a singlet methyl group at C-26 and a 24,25-dihydroxy functionality, which replaced the methylene group after losing the hydroxy group and the 24,25-epoxy group in 1, respectively. In addition, compound 2 was also isolated as an inseparable epimeric mixture; however, only the 1H and 13C NMR data of the major epimer are shown in Tables 1 and 2 since the 1D and 2D NMR signals for the minor isomer were ambiguous to complete the assignments. Dictabretol C (3)26 was isolated as a colorless oil. The molecular formula was determined as C40H66O7 by HRESIMS at m/z 657.4731 [M - H]- (calcd for C40H65O7, 657.4730), with eight degrees of unsaturation in the molecule. The 1H and

13

C NMR spectra of compound 3

(Tables 1 and 2) also showed very similar profiles to those of 1 indicating that 3 has the same glabretal triterpene skeleton, except for the absence of the signals for an isovaleric acid moiety. The 1 H NMR spectrum for a long aliphatic chain of methylene groups at δH 1.24– 1.61 and a methyl group at δH 0.87 and the 13C NMR spectrum around δC 29, indicating C-4′– 5

C-7′ resonances, suggested the presence of a decanoic acid moiety, which was confirmed by the mass spectrum. Additionally, the HMBC correlation of δH 4.63 (H-3) with δC 33.9 (C-1), 36.2 (C-4), 41.3 (C-5), 27.7 (C-28), 21.8 (C-29), and 173.7 (C-1′) as well as that of δH 2.32 (H-2′) with δC 173.7 (C-1′), 25.2 (C-3′), and 29.2 (C-4′) supported the presence of a decanoic acid at the C-3 position. Thus, the results obtained from the 1D and 2D NMR spectra indicated that compound 3 is a glabretal triterpene with a decanoic acid substitution at C-3. Dictabretol D (4)27 was isolated as a yellow oil. The molecular formula was determined as C41H68O7 by HRESIMS at m/z 671.4887 [M - H]- (calcd for C41H67O7, 671.4887), with eight degrees of unsaturation in the molecule. The 1H and

13

C NMR spectra of compound 4

(Tables 1 and 2) could nearly be superimposed on those of compound 3, except for the characteristic signals for an anteiso-type methyl branched chain at C-8′. The presence of an 8-methyl decanoate moiety was confirmed by observation of the HMBC correlation of δH 0.86 (H-10′) with δC 34.4 (C-8′) and δC 29.7 (C-9′), as well as that of δH 0.84 (H-11′) with δC 36.6 (C-7′), δC 34.4 (C-8′), and δC 29.7 (C-9′). Furthermore, due to the γ-effect of methyl substitution at C-8′, the upfield shift of the terminal methyl group at C-10′ (δC 11.4) was examined, which indicated the presence of an anteiso-type methyl branched chain rather than a n-alkyl side chain as seen in 3.28,29 Accordingly, the structure of compound 4 was unambiguously determined to have a methyl substitution for the decanoic acid moiety at C-8′ in 3. The immunosuppressive activity of all isolates (1–4) were evaluated in terms of proliferation and cytokine secretion in T cells, using splenocytes from 8–10-week-old OT-II T cell receptor transgenic mice.30 Cell viability, as measured by propidium iodide staining, showed that none of the compounds had any significant cytotoxicity to T cells at their effective concentrations for cell proliferation and cytokine secretion (data not shown). In the 6

proliferation assay, the relative rate of cell growth was decreased, with IC50 values of 1.5, 1.8, and 1.5 µM for compounds 1, 3, and 4, respectively, using cyclosporin A (IC50 0.1 µM) as a positive control (Table 3); compound 2 was inactive (IC50 > 20 µM). In addition, the secretion levels of cytokines IL-2 and IFN-γ by activated T cells were examined; however, none of the compounds showed any significant effect on cytokine secretion. These results demonstrated that compounds 1, 3, and 4 inhibited T cell proliferation without affecting cytokine secretion levels. Further study is required to confirm the mechanism of action of dictabretols A, C, and D and their in vivo activity.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of

Korea

(NRF)

funded

by the

Ministry of

Education

(2013R1A6A3A01064888) and a Korea University Grant.

Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/.

References and notes

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Storer, R.; Young, D. W. Tetrahedron 1973, 29, 1217.

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Chang, J.; Xuan, L. J.; Xu, Y. M.; Zhang, J. S. J. Nat. Prod. 2001, 64, 935. 7

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Chang, J.; Xuan, L. J.; Xu, Y. M.; Zhang, J. S. Planta Med. 2002, 68, 425.

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Jeong, S. H.; Han, X. H.; Hong, S. S.; Hwang, J. S.; Hwang, J. H.; Lee, D.; Lee, M. K.; Ro, J. S.; Hwang, B. Y. Arch. Pharmacal Res. 2006, 29, 1119.

6.

Lei, J.; Yu, J.; Yu, H.; Liao, Z. Food Chem. 2008, 107.

7.

Miyazawa, M.; Shimamura, H.; Nakamura, S.-i.; Kameoka, H. J. Agric. Food Chem. 1995, 43, 1428.

8.

Nam, K. W.; Je, K. H.; Shin, Y. J.; Kang, S. S.; Mar, W. Arch. Pharm. Res. 2005, 28, 675.

9.

Yoon, J. S.; Jeong, E. J.; Yang, H.; Kim, S. H.; Sung, S. H.; Kim, Y. C. J. Enzyme Inhib. Med. Chem. 2012, 27, 490.

10. Zhao, W.; Wolfender, J.-L.; Hostettmann, K.; Xu, R.; Qin, G. Phytochemistry 1998, 47, 7. 11. Borel, J. F.; Feurer, C.; Gubler, H.; Stähelin, H. Inflammation Res. 1994, 43, 179. 12. Herold, K.; Lancki, D.; Moldwin, R.; Fitch, F. J. Immunol. 1986, 136, 1315. 13. Krönke, M.; Leonard, W. J.; Depper, J. M.; Arya, S. K.; Wong-Staal, F.; Gallo, R. C.; Waldmann, T. A.; Greene, W. C. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 5214. 14. Mann, J. Nat. Prod. Rep. 2001, 18, 417. 15. Huang, H. C.; Chang, J. H.; Tung, S. F.; Wu, R. T.; Foegh, M. L.; Chu, S. H. Eur. J. Pharmacol. 1992, 211, 359. 16. The root bark of D. dasycarpus was purchased from Kyung Dong market, Seoul, Korea, in January 2008, and was identified by Emeritus Professor Kyong Soon Lee, College of Pharmacy, Chungbuk National University, Cheongju, Korea. A voucher specimen (accession number SS45-01142008) has been deposited at the College of Life Sciences and Biotechnology, Korea University, Seoul, Korea. The dried and ground root bark (5 kg) of D. dasycarpus was extracted with MeOH (3 × 18 L) at room temperature. After filtration and evaporation of the solvent in vacuo, the MeOH extract was suspended in distilled water and then partitioned, in turn, with n-hexane, CHCl3, and EtOAc. Given the 8

immunosuppressive assay results, the CHCl3 extract (95 g) was subjected to column chromatography over silica gel (CHCl3–MeOH, 100:0 to 50:50), yielding five fractions (SS45-47-1–SS45-47-5). Subfraction SS45-47-4 (12.8 g) was further purified on Sephadex LH-20, eluted with CHCl3–MeOH (1:1), yielding nine fractions (SS45-51-1– SS45-51-9). Fraction SS45-51-3 (4.4 g) was separated by silica gel column chromatography, eluted with hexane–acetone (10:1 to 1:1, then pure acetone), yielding 11 fractions (SS45-56-1–SS45-56-11). Fraction SS45-56-5 (900 mg) was subjected to vacuum liquid chromatography on RP-18, eluted with acetonitrile–water (40–100% acetonitrile, then acetonitrile–acetone 1:1), and was finally purified by silica gel column chromatography, eluted with CH2Cl2–MeOH (1:0, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, and 30:1), to give compound 1 (dictabretol A, 65 mg). Fraction SS45-56-4 (203 mg) was subjected to flash column chromatography on RP-18, eluted with acetonitrile–water (3:7, 5:5, 7:3, 8:2, 9:1, and then pure acetonitrile), yielding nine fractions (SS45-57-1–SS4557-9). Fraction SS45-57-2 (44 mg) was purified by silica gel column chromatography, eluted with hexane–acetone (10:1 to 1:1), to give compound 2 (dictabretol B, 33 mg). Fraction SS45-57-6 (36 mg) was further separated over silica gel column chromatography, eluted with CH2Cl2–MeOH (1:0, 80:1, 50:1, 30:1, 20:1, and 10:1), to give compound 3 (dictabretol C, 23 mg). Fraction SS45-57-8 (9 mg) was purified by silica gel column chromatography using CHCl3–MeOH (1:0, 80:1, 50:1, 30:1, 20:1, and 10:1) to afford compound 4 (dictabretol D, 5 mg). All the isolates were monitored by two-dimensional TLC on silica gel plates using CHCl3: MeOH (15:1) and n-Hexane: Acetone (2:1) as eluent and analyzed by HPLC-ELSD using Acetonitrile− 0.05% TFA in H2O (from 50:50 to 100:0 for 20 min, flow rate 1.0 mL/min) to confirm their purity (Supplementary data). 17. Lee, H. H.; Lee, S. J.; Kim, S.; Jeong, S.; Na, M.; Lee, D. M.; Cheon, Y. P.; Lee, K. H.; 9

Choi, I.; Chun, T. Biotechnol. Lett. 2012, 34, 1225. 18. Dictabretol A (1): White amorphous powder; mp 234.4 °C; [α]27D −22.6 (c 0.3, CHCl3); IR (ATR) νmax 3402, 2967, 1723, 1216, 1054 cm-1; 1H and 13C NMR data (500 MHz, CDCl3), see Tables 1 and 2; ESIMS (negative) m/z 587 [M - H] -, 633 [M + HCOO]-; ESIMS (positive) m/z 611 [M + Na]+; HRESIMS m/z 587.3951 [M - H]- (calcd for C35H55O7, 587.3948) 19. Kamperdick, C.; Lien, T. P.; Adam, G.; Sung, T. V. J. Nat. Prod. 2003, 66, 675. 20. Mulholland, D. A.; Monkhe, T. V. Phytochemistry 1993, 34, 579. 21. Ntalli, N. G.; Cottiglia, F.; Bueno, C. A.; Alché, L. E.; Leonti, M.; Vargiu, S.; Bifulco, E.; Menkissoglu-Spiroudi, U.; Caboni, P. Molecules 2010, 15, 5866. 22. Phuwapraisirisan, P.; Sombund, S.; Tip-pyang, S.; Siripong, P. Nat. Prod. Res. 2013, 27, 753. 23. Ochi, M.; Tatsukawa, A.; Seki, N.; Kotsuki, H.; Shibata, K. Bull. Chem. Soc. Jpn. 1988, 61, 3225. 24. Su, B. N.; Chai, H.; Mi, Q.; Riswan, S.; Kardono, L.; Afriastini, J. J.; Santarsiero, B. D.; Mesecar, A. D.; Farnsworth, N. R.; Cordell, G. A.; Swanson, S. M.; Kinghorn, A. D. Bioorg. Med. Chem. 2006, 14, 960. 25. Dictabretol B (2): White amorphous powder; mp 229.9 °C; [α]27D −20.2 (c 0.3, CHCl3); IR (ATR) νmax 3444, 2930, 1727, 1387, 1095 cm-1; 1H and 13C NMR data (500 MHz, CDCl3), see Tables 1 and 2; ESIMS (negative) m/z 589 [M - H] -, 635 [M + HCOO]-; ESIMS (positive) m/z 613 [M + Na]+; HRESIMS m/z 589.4103 [M - H]- (calcd for C35H57O7, 589.4104) 26. Dictabretol C (3): Colorless oil; [α]27D −33.2 (c 0.3, CHCl3); IR (ATR) νmax 3421, 2931, 1721, 1215, 1025 cm-1; 1H and 13C NMR data (500 MHz, CDCl3), see Tables 1 and 2; ESIMS (negative) m/z 657 [M - H] -, 703 [M + HCOO]-; ESIMS (positive) m/z 681 [M + 10

Na]+; HRESIMS m/z 657.4731 [M - H]- (calcd for C40H65O7, 657.4730) 27. Dictabretol D (4): Yellow oil; [α]27D −17.9 (c 0.3, CHCl3); IR (ATR) νmax 3392, 2931, 1718, 1216, 1023 cm-1; 1H and 13C NMR data (500 MHz, CDCl3), see Tables 1 and 2; ESIMS (negative) m/z 671 [M - H] -, 717 [M + HCOO]-; ESIMS (positive) m/z 695 [M + Na]+; HRESIMS m/z 671.4887 [M - H]- (calcd for C41H67O7, 671.4887) 28. Kim, N.; Shin, J. C.; Kim, W.; Hwang, B. Y.; Kim, B. S.; Hong, Y. S.; Lee, D. J. Antibiot. 2006, 59, 797. 29. Wang, C. Y.; Wang, B. G.; Wiryowidagdo, S.; Wray, V.; van Soest, R.; Steube, K. G.; Guan, H. S.; Proksch, P.; Ebel, R. J. Nat. Prod. 2003, 66, 51. 30. Splenocytes (1 × 106 cells/well) from 8–10-week-old OT-II T cell receptor transgenic mice (H-2b haplotype; The Jackson Laboratory, Bar Harbor, ME, USA) were seeded in 96-well plates. After seeding, splenocytes were stimulated with the OVA323–339 peptide (323ISQAVHAAHAEINEAGR339; AnaSpec, Fremont, CA, USA) and cultured at 37 °C and 5% CO2 in RPMI-10 medium in the presence of each compound. DMSO (0.1%) was added as a control. The following day, propidium iodide staining was performed to assess cell viability, and the results were analyzed by flow cytometry using a FACSCaliburTM system with CellQuest softwareTM (BD Biosciences, San Jose, CA). Three days after treatment with each compound, the relative rate of cell growth was assessed using a MTT assay kit according to the manufacturer’s instruction (Promega, Madison, WI, USA). The supernatants from cell cultures were harvested, and the levels of secreted IL-2 and IFN-γ were measured using sandwich ELISA kits according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). For a statistical analysis, a Student’s t-test was adopted to compare mean values for independent variables. In addition, all the data are representative of triplicate experiments and represent mean values.

11

Figure legend

Figure 1. Compounds 1–4.

12

Figure 1.

2

1

4

3

13

Table 1. 1H NMR Data for Compounds 1–4a 1

No.

2

3

4

1

1.36 m, 1.13 m

1.37 m, 1.15 m

1.35 m, 1.12 m

1.36 m, 1.13 m

2

1.89 m, 1.58 m

1.89 m, 1.58 m

1.88 m, 1.56 m

1.89 m, 1.58 m

3

4.65 brs

4.65 brs

4.63 brs

4.65 brs

5

1.96 m [1.93m]

1.95 m

1.96 m [1.93 m]

1.96 m [1.95 m]

6

1.61 m

1.61 m

1.56 m

1.63 m

7

3.75 brs

3.75 brs

3.75brs

3.76 brs

1.30 overlap

1.32 m

1.30 overlap

1.31 m

11

1.30 overlap

1.29 overlap

1.30 overlap

1.33 m

12

2.12 m; 1.79 m

2.10 m; 1.77 m

15

1.93 m; 1.55 m

1.93 m

16

1.64 m

1.66 m

1.63 m

1.64 m

17

2.18 m [2.02 m]

2.17 m

2.20 m

2.21 m [2.04 m] 0.72 d (4.5); 0.48 d (5.0) [0.81 d (4.5); 0.50 d (4.5)] 0.90 s 1.88 m [2.15 m]

4

8 9 10

2.09 m; 1.77 m

13 14 1.93 m; 1.55 m

1.93 m; 1.55 m

19

0.71 d (4.5); 0.46 d (5.0) [0.79 d (5.0); 0.49 (5.0)] 0.88 s

0.89 s

0.70 d (4.5); 0.46 d (4.5) [0.78 d (5.0); 0.48 (5.0)] 0.88 s

20

1.86 m [2.13 m]

1.86 m

1.86 m [2.14 m]

21

5.43

5.35 brs

5.43 overlap [5.42 overlap]

5.44 overlap [5.45 overlap]

22

1.96 m; 1.68 m 3.94 dt (9.5, 7.0) [4.03 ddd (10.5, 7.5, 5.0)] 3.18 d (7.5) [3.06 d (7.5)]

1.85 m, 1.98 m 4.48 t (7.5) 3.15 s

1.98 m; 1.69 m 3.93 dt (9.5, 7,5)[4.03 ddd (10.5, 7.5, 5.5)] 3.17 d (7.5) [3.05 d (7.5)]

1.98 m; 1.70 m 3.97 dt (9.5, 7.5) [4.05 ddd (10.5, 7.5, 5.0)] 3.17 d (7.5) [3.07 d (7.5)]

26

3.65 d (12.5) [3.57 d (12.5)]

1.28 s

3.64 d (12.5) [3.56 d (12.5)]

3.67 d (12.0) [3.59 d (12.0)]

27

1.31 s

1.26 s

1.30 s

1.33 s

28

0.84 s

0.85 s

0.83 s

0.85 s

29

0.87 s

0.88 s

0.87 s

0.89 s

30

1.04 s [1.03 s]

1.04 s

1.03 s [1.02 s]

1.06 s [1.05 s]

2′

2.21 brd (7.0)

2.23 brd (6.0)

2.32 m

2.34 t (7.5) [2.34 t (7.5)]

3′

2.13 m

2.13 m

1.61 m

1.62 m

4′

0.96 d (6.5)

0.97 d (7.0)

1.26 m

1.31 m

5′

0.95 d (6.5)

0.97 d (6.5)

1.26 m

1.26 m

6′

1.26 m

1.29 m

7′

1.26 m

1.28 m; 1.07 m

8′

1.24 m

1.29 m

9′

1.27 m

1.31 m

10′

0.87 m

18

23 24

overlap [5.43 overlap]

0.72 brd (4.5), 0.47 d (4.5)

25

1′

11′

0.86 m 0.84 m

a Measured at 500 MHz; obtained in CDCl 3 with TMS as an internal standard; chemical shifts are shown in the δ scale with J values (Hz) in parentheses; shifts of the minor components in brackets when different from the main components. The assignments were based on DEPT, 1 H–1H COSY, HSQC, and HMBC experiments.

14

Table 2. 13C NMR Data for Compounds 1–4a 1

2

3

4

No. Major

Minor

Major

Major

Minor

Major

1

33.9

33.7

34.1

33.9

33.7

33.9

Minor

2

22.9

22.8

23.1

22.8

22.8

22.8

22.8

3

77.8

78.0

77.8

77.7

77.8

77.7

4

36.2

36.1

36.4

36.2

36.2

36.2

36.2

5

41.3

41.3

41.5

41.3

41.3

41.3

41.3

6

24.2

24.5

24.2

7

74.3

74.2

74.5

74.3

74.2

74.3

74.2

8

39.0

39.1

39.2

39.0

39.1

39.0

39.1

9

44.0

43.9

44.3

44.1

43.9

44.1

43.9

10

37.2

37.3

37.5

37.2

37.3

37.3

37.3

11

16.2

16.0

16.4

16.2

16.0

16.2

16.0

b

25.6

25.7b

29.0

28.6

24.2

12

25.6

25.6

26.0

25.6

25.6

13

29.0

28.6

29.2

29.0

28.6

14

37.0

37.3

37.0

15

26.3

26.5

26.3

25.9b

26.3

25.9b

b

27.5

26.2b

25.9

37.0

16

27.5

26.2

27.7

27.5

26.1

17

44.7

48.3

45.2

44.7

48.2

44.7

48.3

18

13.8

13.6

14.0

13.7

13.6

13.7

13.6

19

15.7

15.6

15.9

15.7

15.6

15.7

15.6

20

49.4

50.8

49.0

49.4

50.8

49.4

50.8

21

98.3

102.2

97.7

98.3

102.2

98.3

102.2

22

30.7

32.9

29.5

30.7

32.8

30.7

32.9

23

78.1

76.9

78.9

78.1

76.9

78.1

76.9

24

63.4

61.0

75.3

63.5

61.1

63.3

60.9

25

60.7

60.0

73.7

60.7

60.0

60.6

59.9

26

64.9

64.7

26.9

65.0

64.7

65.0

64.6

27

14.6

14.8

27.0

14.6

14.8

14.6

14.8

28.0

27.7

27.7

22.1

21.8

21.9

28

27.8

29

21.9

21.9

30

19.5

19.4

19.7

19.5

1′

172.9

173.1

173.7

173.6

2′

43.8

44.1

34.8

34.8

3′

25.8

26.0

25.2

25.2

4′

22.5

22.7

29.2b

29.3b

5′

22.5

22.7

29.3b

29.3

29.5b

b

29.3

27.0

25.8

6′

29.4

7′

29.5b

19.4

19.6

36.6

8′

31.9

34.4

9′

22.7b

29.7b

10′

14.1

11.4

11′

19.4

19.2

Measured at 125 MHz; obtained in CDCl 3 with TMS as an internal standard. The assignments were based on DEPT, 1H–1H COSY, HSQC, and HMBC experiments. b Assignments are interchangeable in the same column. a

15

Table 3. Inhibitory effects of the isolated compounds 1–4 on proliferation of T cells using mouse splenocytes Compound

IC50 (µM)

1

1.5

2

> 20

3

1.8

4

1.5

Cyclosporin A

0.1

16

Graphical abstract

HO O

OH

HO H H O

H O

Dictabretol A (1)

OH

Dictabretol B (2)

Dictabretol C (3)

Dictabretol D (4)

17