Cytotoxic Clerodane Diterpenes from - American Chemical Society

Article pubs.acs.org/jnp

Cytotoxic Clerodane Diterpenes from Zuelania guidonia Carlos Calderón,† Christian De Ford,‡,§ Victor Castro,† Irmgard Merfort,*,‡,§ and Renato Murillo*,† †

Escuela de Quimica and CIPRONA, Universidad de Costa Rica, 2060 San José, Costa Rica Department of Pharmaceutical Biology and Biotechnology, Albert Ludwigs University Freiburg, Stefan-Meier-Strasse 19, D-79104 Freiburg, Germany § Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Albertstrasse 19a, D-79104 Freiburg, Germany ‡

S Supporting Information *

ABSTRACT: The leaves of Zuelania guidonia yielded eight new clerodane diterpenes, namely, zuelaguidins A−H (1−8), and the known clerodane diterpene esculentin A (9). Some of these structures contained a 3,6dihydro-1,2-dioxin moiety. The new compounds were isolated and identified using 1D- and 2D-NMR experiments. All compounds were evaluated for cytotoxicity against the CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), and MIA-PaCa-2 (human pancreatic carcinoma) cell lines as well as for their selectivity against peripheral blood mononuclear cells from healthy human subjects. Zuelaguidins B, C, and E were the most potent compounds against the CCRF-CEM cell line, with IC50 values ranging from 1.6 to 2.5 μM.

D

spectrometric data of compound 9 agreed with published values.18 The HRESIMS of zuelaguidin A (1) revealed a molecular formula of C33H40O8 (Δ 0.7 ppm), based on the sodiated ion peak at m/z 587.2617 [M + Na]+ (calcd 587.2621). An initial interpretation of the NMR data suggested the presence of two acetyl groups [δC 170.2 and 21.4, δH 2.04 (3H, s) δC 170.1 and 21.8, δH 1.98 (3H, s)] and one cinnamoyl group (see Tables 1 and 2). The 20 remaining carbons in the 13C NMR spectrum suggested that the compound is a diterpenoid. Two methyl groups [δH 1.00 (3H, s, H-20), δC 25.6 (C-20) and δH 0.93 (3H, d, 6.6 Hz, H-17), δC 15.7 (C-17)], two acetalacyloxy methines [δH 6.55 (1H, dd, J = 1.8, 1.8 Hz, H-18), δC 95.5 (C18) and δH 6.65 (1H, s), δC 98.5 (C-19)] and one 2-substituted butadiene [(δC 145.3 (C-13), δH 6.43 (1H, dd, J = 18.0, 10.8 Hz, H-14), δC 140.4 (C-14), δH 5.22 (1H, d, J = 18 Hz, H-15a) and δH 5.03 (1H, d, J = 10.8 Hz, H-15b), δC 112.7 (C-15), δH 4.95 (1H, brs, H-16a) and δH 5.05 (1H, brs, H-16b), δC 115.5 (C-16)] were assigned and are consistent with a clerodane diterpenoid carbon skeleton; this skeleton is prevalent in the Salicaceae (formerly Flacourtiaceae) family.8,9,14,19,20 The longrange correlations of the signal at δH 6.55 (H-18) with those at δC 98.5 (C-19), δC 170.2 (CH3COOH at C-18), δC 126.6 (C3), and δC 142.4 (C-4) as well as at δH 6.01 (H-3) with that at δC 95.5 (C-18) confirmed the presence of a double bond between C-3 and C-4, while the long-range correlations for the signal at δH 6.65 (H-19) with resonances at δC 170.1

uring a continuing search to discover bioactive compounds from plants in the Costa Rican rainforest, Zuelania guidonia (SW.) Britton et Millsp. has been reinvestigated, a species formerly grouped into the Flacourtiaceae family but recently grouped into the Salicaceae sensu lato family.1 This plant is known as “cagajón”, “arbol caspa”, or “caraño”2 and is a shrub or small tree found in Guanacaste Province, Costa Rica. The stem bark contains large quantities of clerodane diterpenes, including zuelanin and isozuelanin derivatives.3,4 This type of diterpenoid commonly appears in the genus Casearia5−8 and is known for a broad range of biological activities,9 such as antitumor6 and cytotoxic activities.8−16 Recently, apoptotic activity15 and synergistic effects with TRAIL leading to cell death have been reported.17 Phytochemical investigations of leaf extracts yielded eight new clerodane diterpenes (1−8) and the known clerodane diterpene esculentin A (9). The cytotoxic activity of the isolated compounds was tested against three tumor cell lines (CCRF-CEM, CEM-ADR5000, and MIA-PaCa-2) and peripheral blood mononuclear cells (PBMCs) from healthy human subjects to evaluate their selectivity against tumor cells.

■

RESULTS AND DISCUSSION

The MeOH phase of the methyl tert-butyl ether (MTBE)− MeOH extract of the leaves of Z. guidonia was investigated, leading to the isolation of eight new clerodane diterpenoids [zuelaguidins A−H (1−8)] and the previously known esculentin A (9). The structures of 1−8 were elucidated based on 1D- and 2D-NMR (1H, 13C, COSY, HSQC, HMBC) and HRMS (ESI, APCI) data analysis. The spectroscopic and © 2014 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Otto Sticher Received: August 19, 2013 Published: January 31, 2014 455

dx.doi.org/10.1021/np400672g | J. Nat. Prod. 2014, 77, 455−463

Journal of Natural Products

Article

Chart 1

(CH3COOH at C-19), δC 142.4 (C-4), δC 52.3 (C-5), δC 74.4 (C-6), and δC 36.3 (C-10) suggested that an ester group occurs at C-6. Moreover, the long-range correlations of the signal at δH 5.05 (H-6) with δC 166.2 (C-9′ of the cinnamate group) confirmed that the latter group is attached to C-6, and the coupling constants of H-6 with H-7α (J = 13.0 Hz) and H-7β (J = 4.2 Hz) indicated it is in the α-position.20 The C-2 alcohol was located via a correlation in the COSY spectrum between the oxymethine proton at δH 4.44 and the methine at δH 6.01 (H-3). The relative configuration proposed was determined using NOESY data (see Figure 1) as well as coupling patterns and constants observed for the key protons. The J values (3.6 and 13.8 Hz) for the coupling between H-10 and H-1 suggested H-10 (rel-α) is in an axial position. A small coupling constant (J1,2 = 4.2 Hz) suggested H-2 has an equatorial and therefore a rel-β orientation, agreeing with the broad triplet at δH 4.44 and the chemical shift of C-2 at δC 63.9, as indicated by literature data.9 Moreover, the chemical shift at δC 63.9 matched the literature data for an α-hydroxy group at C-2 occurring in the range δC 63.6−64.2,3−6,8,9,14,17,21,22 whereas the chemical shifts for a β-hydroxy group at C-2 would be in the range δC 67.9−68.3.3,4,21 The dipolar interactions between H-19 and H-7α (δH 1.73); H-19 and H-17; H-19 and H-6; and H-18 and H-6 revealed that the acetyl groups at C-19 and C-18 have an α-configuration. The syn orientation of the acetyl groups was also confirmed by the dipolar interactions between H-18 and H-19. The cinnamoyl group at C-6 and the methyl group at C-8 are α-oriented, according to the dipolar interactions between H-6 and H-8 and between H-2 and H6. Overall, the configuration proposed for zuelaguidin A (1) agrees with the structure published for casearborin E, which has a similarly substituted diterpenoid skeleton8 and for which an X-ray structure was published. Moreover, NOESY correlations between H3-20 (δH 1.00) and H-8 (δH 1.93) as well as between H3-17 (δH 0.93) and H-11a (δH 1.31) corroborated the relative configuration at C-9. Therefore, the new diterpenoid zuelaguidin A (1) was assigned as rel-(2R,5S,6S,8R, 9R,10S,18R,19S)-18,19-diacetoxy-18,19-epoxy-6-cinnamoyloxy2-hydroxyclero-3,13(16),14-triene. The molecular formula for zuelaguidin B (2) was determined as C33H40O8 (Δ 0.3 ppm), according to the sodiated ion peak at m/z 587.2619 [M + Na]+ (calcd 587.2621) in the HRESIMS, indicating that 2 is an isomer of zuelaguidin A. The 1H and 13C NMR spectra were found to be very similar to

those of 1, except for the chemical shifts for C-1, -2, -3, -7, and -10, as well as for H-2. The long-range correlations of the signal at δH 5.75 (H-2) and δC 134.4 (C-1′) confirmed that the cinnamoyl moiety is appended to C-2-OH. The 13C NMR chemical shifts and 1H NMR coupling constants allowed the inference to be made that the configuration of 2 is similar to that of 1, apart from C-2. The α-ester group at C-2 correlated with a 13 C NMR chemical shift at δ C 66.1− 67.53−5,7−9,11,13,14,17,19,23,24 and a β-ester group at δC 70.3− 72.1;3−5,11,12,16,20,21,24,25 consequently, the cinnamoyl moiety was assigned as being attached to the C-2-OH in a βorientation [δC 71.0 (C-2)]. Therefore, zuelaguidin B (2) was assigned as rel-(2S,5S,6S,8R,9R,10S,18R,19S)-18,19-diacetoxy18,19-epoxy-2-cinnamoyloxy-6-hydroxyclero-3,13(16),14-triene. Zuelaguidin C (3) gave the molecular formula C36H56O9 (Δ −0.3 ppm), as determined by HRAPCIMS (observed m/z 650.4270 [M + NH4]+, calcd 650.4268). The 1H and 13C NMR data for compound 3 were very similar to those of 1, except that the cinnamoyl was replaced by a 3-hydroxydodecanoyl (=3-hydroxylaureate) group (see Tables 1 and 2). The 1H and 13 C NMR assignments for compound 3 were validated by 2DNMR correlations. Its relative configuration was established by dipolar interactions between H-18 and H-19; H-19 and H-7α; and H-6 and H-2. The relative stereochemistry at C-3′ could not be elucidated. Zuelaguidin C was assigned, therefore, as rel(2R,5S,6S,8R,9R,10S,18R,19S)-18,19-diacetoxy-18,19-epoxy-2hydroxy-6-(3-hydroxylaureate)clero-3,13(16),14-triene. Zuelaguidin D (4) gave an elemental formula of C36H56O10 (Δ −0.9 ppm), based on the sodiated ion at m/z 671.3777 ([M + Na]+ (calcd 671.3771) in the HRESIMS. The 1H and 13C NMR data of compound 4 were comparable to those of compound 3. However, the chemical shifts for C-12 (δC 83) and H-12 [δH 4.77 (1H, dd, J = 7.2, 2.4)] were strongly shifted downfield relative to those signals for compound 3; these differences may be explained by a hydroxy group being attached to C-12. The long-range correlations of H-12 with resonances at δC 38.6 (C-11), δC 146.1 (C-13), δC 135.2 (C-14), and δC 116.3 (C-15) confirmed its position at C-12. The chemical shifts δH 4.77 (H-12) and δC 83 (C-12) suggested that C-12 has an R configuration. An S configuration at C-12 would result in chemical shifts of about δH 4.46 and δC 67.7, as described by Whitson et al. in the Supporting Information for deoxoargutin F, which has a similar skeleton.17 The absolute configuration at 456



Article

Table 1. 13C NMR Spectroscopic Data for Compounds 1−8a position

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 18-OAc

29.6 63.9 126.6 142.4 52.3 74.4 33.1 36.9 37.5 36.3 28.1 23.9 145.3 140.4 112.7 115.5 15.7 95.5 98.5 25.6 170.2 21.4 170.1 21.8

CH2 CH CH C C CH CH2 CH C CH CH2 CH2 CH CH CH2 CH2 CH3 CH CH CH3 C CH3 C CH3

26.6 71.0 124.4 144.6 53.8 74.4 37.6 37.7 38.3 41.2 27.8 23.9 145.1 140.4 112.8 115.5 15.8 95.3 97.6 25.6 170.1 21.3 169.9 21.7


134.3 128.5 128.9 130.6 128.9 128.5 146.2 117.8 166.2

C CH CH CH CH CH CH CH C

134.4 128.3 129.1 130.6 129.1 128.3 145.6 117.9 166.8

C CH CH CH CH CH CH CH C

19-OAc Cin 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ (3-OH)-laur 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ Dec 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ Xyl 1″ 2″

2

3 29.9 63.9 126.6 142.4 52.4 74.5 33.2 36.9 37.5 36.4 28.1 24.0 145.3 140.4 112.8 115.6 15.7 95.4 98.3 25.6 170.3 21.4 170.0 21.8


4 29.6 63.8 126.5 142.0 52.4 74.5 33.3 36.1 38.8 41.9 38.6 83.0 146.1 135.2 116.3 117.0 15.7 95.4 98.0 24.0 170.3 21.4 169.6 22.0

CH2 CH CH C C CH CH2 CH C CH CH2 CH CH CH CH2 CH2 CH3 CH CH CH3 C CH3 C CH3

5 29.6 63.9 126.6 142.5 52.3 74.2 33.1 36.9 37.4 36.2 27.4 25.5 134.9 116.9 70.3 73.2 15.7 95.5 98.6 25.6 170.2 21.4 170.0 21.8 134.4 128.6 129.0 130.7 129 CH 128.6 146.4 117.8 166.2

172.3 C 42.5 CH2 68.5 CH 37.1 CH2 25.6 CH2 29.7b CH2 29.7b CH2 29.6b CH2 29.5b CH2 32.0 CH2 22.8 CH2 14.3 CH3

6 CH2 CH CH C C CH CH2 CH C CH CH2 CH2 CH CH CH2 CH2 CH3 CH CH CH3 C CH3 C CH3

27.0 66.0 122.8 144.9 53.1 81.4 36.5 36.2 38.6 41.7 41.6 68.1 150.3 136.5 114.6 115.3 15.9 96.0 98.5 24.6 170.8 21.4 170.0 22.0


7 27.0 65.9 122.9 144.5 53.0 81.3 36.4 36.3 38.4 41.2 40.6 68.5 139.4 118.5 70.0 70.4 15.7 96.1 98.5 24.9 170.1 21.4 170.0 21.8


8 26.9 66.0 122.6 144.6 52.9 81.6 36.0 36.2 38.2 42.0 36.0 81.9 136.5 121.6 69.9 70.0 15.9 96.3 98.4 24.1 171.1 21.4 170.0 21.7


C CH CH CH CH CH CH C

172.3 C 42.5 CH2 68.5 CH 37.1 CH2 25.6 CH2 29.7b CH2 29.7b CH2 29.6b CH2 29.5b CH2 32.1 CH2 22.8 CH2 14.3 CH3

457

173.4 C 34.7 CH2 25.2 CH2 29.6a CH2 29.5a CH2 29.4a CH2 29.2a CH2 32.0 CH2 22.8 CH2 14.2 CH3



104.7 CH 73.3 CH

105.0 CH 73.3 CH

104.9 CH 73.2 CH



Article

Table 1. continued position

1

2

3

4

3″ 4″ 5″ a

5

6

7

8

76.0 CH 69.6 CH 65.2 CH2

76.5 CH 69.5 CH 65.4 CH2

76.4 CH 69.6 CH 65.4 CH2

Measured in CDCl3 at 125 MHz, δ. bInterchangeable signal.

range correlation of the signal at δH 4.14 (H-1″) with δC 81.4 (C-6), as well as between δH 3.66 (H-6) and δC 104.7 (C-1″). Compound 6 was a ssigned as rel-( 2R, 5S,6S ,8R , 9R,10S,12S,18R,19S)-18,19-diacetoxy-2-decanoate-18,19epoxy-6-β-xylosylclero-3,13(16),14-triene. Until now, clerodanes glycosylated with xylose have not been reported. Moreover, glycosylated diterpenes in the genera Casearia and Zuelania are rare, with one example in Casearia sylvestris.31 Interestingly, reports of glycosylated clerodanes in other genera lacked the clerodane-18,19-acetal nucleus.32−42 The 1H and 13C NMR spectra of zuelaguidin G (7) were very similar to those of 6, including the xylosyl and decanoyl units; however, the 2-substituted butadiene was replaced with a 3,6-dihydro-1,2-dioxin structural element, which was confirmed by its 1H,1H-COSY, HSQC, and HMBC spectra, as described for compound 5, agreeing with the molecular formula (C39H60O15Na) (Δ −0.9 ppm) deduced from the sodiated ion at m/z 791.3823 [M + Na]+ (calcd 791.3830). According to the chemical shifts, C-12 has an S orientation, as proposed for compound 6. Therefore, zuelaguidin G was assigned as rel(2R,5S,6S,8R,9R,10S,12S,18R,19S)-18,19-diacetoxy-2-decanoate-18,19-epoxy-12-hydroxy-6-xylosylclero-3,13-diene15,16-endoperoxide. The 1H and 13C NMR data of zuelaguidin (8) were almost identical to those of compound 7, including the molecular formula (C39H60O15Na), which was established using the sodiated ion m/z 791.3828 [M + Na]+ (calcd 791.3830). The NMR spectra differed only in the chemical shift of C-12 (δC 81.9); this resonance was similar to that determined for compound 4, indicating that C-12 has an R configuration.17,24 Therefore, compound 8 was assigned as rel-(2R,5S,6S, 8R,9R,10S,12R,18R,19S)-18,19-diacetoxy-2-decanoate-18,19epoxy-12-hydroxy-6-β-xylosylclero-3,13-diene-15,16-endoperoxide. Zuelaguidins A−H (1−8) are new compounds that share common structural features with previously reported diterpenoids from Salicaceae sensu lato (formerly Flacourtiaceae) with novel structural components including in some cases a xylose unit and a 3,6-dihydro-1,2-dioxin moiety. Since clerodane diterpenoids are known for their cytotoxic activity, zuelaguidins A−H (1−8) and esculentin A (9) were evaluated in vitro against three tumor cell lines using an MTT assay (see Table 3): human acute lymphocytic leukemia (CCRF-CEM and CEM-ADR5000) and human pancreatic carcinoma (MIA PaCa-2). The most potent cytotoxic activity was obtained for the CCRF-CEM cells; their IC50 values ranged from 1.6 to 9.1 μM. The observed cytotoxicity decreased for the doxorubicin-resistant CEM-ADR5000 cells; in many cases, cytotoxicity was not apparent (Table 3). The MIA PaCa-2 cells were less responsive toward the diterpenes tested, with IC50 values ranging from 4.6 to >10 μM. Clerodane diterpenoids seem to have some selectivity for leukemia cells. The same trend was observed with other clerodane diterpenoids, including the casearins, caseargrewiins, and casearupestrins.9,10,15 Overall, compounds 2, 3, and 5 were the most

C-12 for deoxoargutin F was determined by using the Mosher ester method. It should be noted that the structural formula of deoxoargutin F is in error and has to be corrected.17 Moreover, the dipolar interactions between H-18 and H-6; H-10 and H11; H-19 and H-7α; and H-11 and H-18 agreed with the proposed relative configuration. Therefore, compound 4 was assigned as rel-(2R,5S,6S,8R,9R,10S,12R,18R,19S)-18,19-diacetoxy-18,19-epoxy-2,12-dihydroxy-6-(3-hydroxylaureate)clero3,13(16),14-triene. Zuelaguidin E (5) gave a molecular formula of C33H40O10 (Δ 1.2 ppm), which was established using the sodiated ion at m/z 619.2512 [M + Na]+ (calcd 619.2519). The 1H and 13C NMR data for the clerodane skeleton were very similar to those of 1, but the signals for the 2-substituted butadiene unit were missing and replaced by signals indicating a 3,6-dihydro-1,2dioxin unit.26,27 This assignment was performed using the longrange correlations of the signals at δH 4.55 (H-15α), δH 4.61 (H-15β), δH 4.39 (H-16α), δH 4.43 (H-16β), δH 1.94 (H-12a), and δH 1.85 (H-12b) with C-13 (δC 134.9) and C-14 (δC 116.9). The 13C NMR data agreed with the relevant literature data.26−28 Relative stereochemistry was established by NOESY correlations between H-19 and H-11; H-19 and H-7α; H-18 and H-19; and H-6 and H-2. Therefore, compound 5 was assigned as rel-(2R,5S,6S,8R,9R,10S,18R,19S)-18,19-diacetoxy18,19-epoxy-6-cinnamoyloxy-2-hydroxyclero-3,13-diene-15,16endoperoxide. This is the first time that a 3,6-dihydro-1,2dioxin structural element has been described within a diterpenoid. Previously, this endoperoxide structural element was reported as an intermediate during the enzymatic synthesis of furanomonoterpenoids starting from β-myrcene and its analogues.26 This functionality may arise from a Diels−Alder reaction between compound 1 and molecular oxygen, as suggested by Yong et al. during plakortolide biosynthesis in the marine sponge Plakinastrella clathrata (see also Figure S1, Supporting Information).29 The molecular formula of zuelaguidin F (6) was established as C39H60O13 (Δ −0.2 ppm) from the sodiated ion at m/z 759.3933 ([M + Na]+, calcd 759.3932) in the HRESIMS. The 1 H and 13C NMR data, as well as the H,H-COSY, HSQC, and HMBC spectra, were very similar to those of compound 4 (see Tables 1 and 2) except for three differences. One main difference was observed at C-12 (δC 68.1) and H-12 (δH 4.45), suggesting an S orientation at C-12.17,24 Moreover, the position of the long-chain fatty acid was altered, and the hydroxy group at C-3′ was missing. Long-range correlations of the signal at δH 5.46 (H-2) with δC 173.4 (C-1′) revealed an esterification on the C-2-OH group. Additionally, signals for a xylose unit appeared. The sequence H-1″ → H-2″ → H-3″ → H-4″ and H5″(α, β) was elucidated using the 1H,1H-COSY spectrum; the corresponding coupling constants (Table 2) and the couplings derived from the HSQC suggest that this sugar unit is βxylose. 30 Although its absolute configuration was not determined, it is most likely that D-xylose is present. Up to now only this configuration of xylose has been found in plants. The pentose was attached at C-6, as confirmed by the long458


459

5.03 5.22 4.95 5.05 0.93 6.55

6.65 1.00 2.04 1.91

15a 15b 16a 16b 17 18

19 20 18-OAc 19-OAc Cin 2′ 3′ 4′ 5′ 6′ 7′ 8′ (3-OH)-laur 2′

3′ 4′ 5′ 6′

7.54 7.36 7.36 7.36 7.54 7.76 6.39

1.31 1.54 2.11 2.11 6.43

11a 11b 12a 12b 14

8 10

m m m m m d (16.0) d (16)

s s s s

d (10.8) d (18.0) brs brs d (6.6) dd (1.8, 1.8)

m m m m dd (18.0,10.8)

1.73 ddd (13.0, 13.0, 13.0), 1.83 ddd (13.0, 4.2, 4.2) 1.93 m 2.38 dd (13.8, 3.6)

7α

7β

4.44 brt (4.2) 6.01 brd (4.2) 5.05 dd (13.0, 4.2)

2 3 6

1

1.96−2.08 m

1

position

7.54 7.40 7.40 7.40 7.54 7.72 6.43

m m m m m d (16.2) d (16.2)

1.88 m 2.40 dd (14.4, 2.4) 1.19 m 1.50 m 2.08 m 2.08 m 6.44 dd (17.4, 10.8) 5.06 d (10.8) 5.25 d (17.4) 4.92 s 5.05 s 0.93 d (6.6) 6.73 dd (1.2, 1.2) 6.44 s 0.97 s 2.07 s 1.91 s

1.77 m

5.75 m 5.98 brs 3.96 dd (13.2, 3.6) 1.67 m

1.80 m, 2.26 m

2

m m brt (7.2) brt (7.2) dd (17.4, 10.8)

s s s s

2.41 dd (16.2, 9.6), 2.52 dd (16.2, 3.0) 3.99 m 1.42 m, 1.50 m 1.42 m 1.26 m

6.54 0.99 2.07 1.90

5.04 d (10.8) 5.22 d (17.4) 4.93 brs 5.05 brs 0.93 d (6.6) 6.6 dd (1.8, 1.8)

1.53 1.53 2.11 2.11 6.43

1.90 m 2.35 dd (12.6, 4.8)

1.72 m

1.69 m

4.44 brs 6.02 brd (3.6) 5.02 dd (12.0, 4.8)

1.98 m, 2.01 m

3

Table 2. 1H NMR Spectroscopic Data for Compounds 1−8a 4

2.41 dd (16.2, 3.99 m 1.43 m, 1.42 m, 1.26 m

1.50 m 1.32 m

(16.0, 9.6), 2.51 dd 2.4)

d (10.8) d (17.4) s s d (7.2) dd (1.8, 1.8)

6.49 s 1.11 s 2.08 1.99

5.17 5.49 5.19 5.29 1.09 6.58

6.28 dd (17.4, 10.8)

1.28 m 1.81 m 4.77 dd (7.2, 2.4)

1.83 m 2.03 m

1.73 m

1.73 m

2.07 m, 1.93 ddd (13.8, 4.8, 4.8) 4.41 brs 6.00 brd (3.6) 4.99 m

5

7.56 7.38 7.38 7.38 7.56 7.78 6.41

m m m m m d (16.2) d (16.2)

4.55 brd (15.6) 4.61 brd (15.6) 4.39 brd (16.2) 4.43 brd (16.2) 0.94 d (6.6) 6.56 dd (1.8, 1.8) 6.68 s 0.97 s 2.07 s 2.01 s

1.97 m 2.34 dd (6.0, 2.4) 1.33 m 1.49 m 1.85 m 1.94 m 5.55 brs

1.85 brt (4.2)

4.45 brt (3.6) 6.03 brd (3.6) 5.07 dd (12.0, 4.2) 1.70 m

1.97 m, 2.01 m

6.50 1.05 2.06 1.93

s s s s

d (11.4) d (18.0) s s d (7.2) brs

6.50 1.03 2.06 1.98

4.53 4.67 4.52 4.60 1.03 6.88

s s s s

d (15.6) d (15.6) d (15.6) d (15.6) brs s

4.2 brd (7.2) 5.8 s

1.73 m 2.01 m

1.91 m

1.80 m

4.45 brd (7.2) 6.28 dd (11.4, 18.0) 5.15 5.43 5.13 5.15 1.08 6.87

7

5.46 brs 6.00 brd (3.6) 3.66 dd (10.8, 3.6)

1.83−2.04 m

1.39 d (14.4) 1.59 m

6

1.36 m 1.71 m

1.74 m 2.03 m

1.93−1.80 m

1.93−1.80 m

5.46 m 5.98 brd (3.6) 3.66 m

1.88−2.04 m

8

6.47 1.00 2.04 2.00

s s s s

4.45−4.65 4.45−4.65 4.45−4.65 4.45−4.65 1.01 brs 6.92 s

5.87 s

1.26 m 1.82 m 4.47 m

1.72 m 1.93 m

1.91 m

m m m m

5.43 brs 5.96 brs 3.66 dd (10.8, 3.6) 1.62 m

1.89−1.98 m

Journal of Natural Products Article


a

460

1

2

Measured in CDCl3 at 600 MHz, J in Hz.

7′ 8′ 9′ 10′ 11′ 12′ Dec 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ Xyl 1″ 2″ 3″ 4″ 5″

position

Table 2. continued 1.26 1.26 1.26 1.25 1.29 0.88

m m m m m t (7.2)

3 1.26 1.26 1.26 1.26 1.29 0.88

m m m m m t (7.2)

4

5

2.35 t (7.2) 1.63 m 1.22−1.34 m 1.22−1.34 m 1.22−1.34 m 1.22−1.34 m 1.22−1.34 m 1.22−1.34 m 0.87 t (7.2) 4.12 d (7.2) 3.45 m 3.50 m 3.70 m 3.18 dd (10.8, 10.8), 3.94 brd (10.8)

4.14 d (6.6) 3.41 dd (9.6, 6.6) 3.49 dd (9.6, 9.6) 3.73 m 3.22 dd (9.6, 11.4), 3.98 dd (4.8, 11.4)

7

2.35 t (7.2) 1.64 m 1.24−1.36 m 1.24−1.36 m 1.24−1.36 m 1.24−1.36 m 1.24−1.36 m 1.28 m 0.88 t (7.2)

6

4.14 3.38 3.43 3.65 3.18

m m m m m, 3.92 m

2.34 t (7.2) 1.62 m 1.20−1.35 m 1.20−1.35 m 1.20−1.35 m 1.20−1.35 m 1.24 m 1.27 m 0.89 t (7.2)

8

Journal of Natural Products Article



Article

decanoate group at C-2 in compounds 6−8 greatly decreased the resultant cytotoxic activity (see Table 3). It was already reported that bulkiness at C-6 is detrimental for the cytotoxicity of clerodane diterpenes.43 Additionally, sugar moieties are less able to penetrate biomembranes due to their polar hydroxy groups. Accordingly, the mono- and diglycosidic clerodanes from Casearia sylvestris were not cytotoxic against four carcinoma cell lines.31 However, a bulky residue at C-2 is more favorable than at C-6; compound 2 was more cytotoxic than 1. Interestingly, the rarely reported 3,6-dihydro-1,2-dioxin residue increases the resultant cytotoxicity of 5 compared to 1. Although several studies describing the cytotoxic potential of clerodane diterpenes have been published,8−16 a comprehensive quantitative structure−activity study must be developed to finally evaluate the potential of clerodane diterpenes as leads for anticancer drugs. Moreover, studies of the molecular mechanism are also important for obtaining insights into the targeting of tumor cells by the clerodane diterpenes.

■

Figure 1. Key NOESY correlations supporting the configuration of 1.

Table 3. Cytotoxic Activity (IC50 μM) of Clerodane Diterpenoids 1−9 Isolated from Zuelania guidonia against Three Human Tumor Cell Lines and PBMCs after a 24 h Incubation Using an MTT Assay compound 1 2 3 4 5 6 7 8 9 CPTb Doxob

CCRF-CEMa 5.3 1.6 2.5 5.5 1.9 9.1 8.0 8.4 9.5 0.08 0.5

± ± ± ± ± ± ± ± ± ± ±

0.2 0.3 0.1 1.2 0.1 0.1 0.8 0.5 1.2 0.02 0.03

CEM-ADR5000a 8.6 4.6 5.7 >10 7.5 >10 >10 >10 >10 0.3 >10

± 0.6 ± 0.1 ± 0.1 ± 1.1

± 0.1

MIA-PaCa2a >10 4.6 7.4 >10 6.9 >10 >10 >10 >10 >10 4.8

± 0.1 ± 0.4 ± 0.1

± 0.6

EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded in CDCl3 on a Bruker Ascend 600 MHz instrument at 600 MHz (1H) and 125 MHz (13C). HRESIMS were measured with a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Fisher), and APCIMS were collected with a Thermo Exactive with an Orbitrap analyzer (Thermo Fisher). Open-column liquid chromatography was carried out on silica gel 60 (Merck) using an MPLC instrument with an FMI pump (model QD410-2). Aluminum sheets with silica gel 60 F254 (Merck) were used for preparative TLC. Preparative-scale HPLC was performed using a Knauer 6400 apparatus with a differential refractive index detector using an Alltech Econosil C18 (250 mm × 10 mm) column at 2 mL/min with a 500 μL sample. Plant Material. Leaves from Zuelania guidonia were collected in La Cruz, Guanacaste, Costa Rica, during March 2010. The plant was identified by Dr. L. Poveda. A voucher specimen was deposited at the Juvenal Valerio Rodriguez herbarium in Heredia, Costa Rica, code JVR 13919. Extraction and Isolation. Ground Z. guidonia leaves (2 kg) were extracted with MTBE−MeOH (90:10, 4.5 L:0.5 L) for 24 h. On removal of solvent, the crude extract was filtered and concentrated under vacuum at 40 °C, yielding 50 g of extract; the extract was treated with MeOH at −20 °C, generating 18 g of soluble material after the solvent was removed. This dried extract was fractionated by open column chromatography using silica gel and elution with different mixtures of hexane−MTBE (100:0, 75:25, 50:50, 25:75, 0:100) and MTBE−MeOH (80:20) to collect seven fractions (F1−F7); the fractions were examined using analytical TLC (hexane−MTBE, 50:50, UV detection at 254 nm). Fraction F5 (5.1 g) was separated by MPLC (hexane−MTBE−MeOH, using the elution gradient mentioned above) to obtain nine subfractions (F5/1−F5/9). Subfraction F5/3 (250 mg) was separated by preparative TLC using benzene− CH2Cl2−MTBE (80:10:10) as the mobile phase to afford 9 (Rf 0.65, 6 mg). Subfraction F5/5 (200 mg) was further separated by preparative TLC using CH2Cl2−MTBE (90:10) to yield 2 (Rf 0.56, 1 mg). Subfraction F5/6 (1.2 g) was separated by MPLC using a hexane− MTBE−MeOH elution gradient, as mentioned above, to produce five subfractions (F 5/6/1−F5/6/5). Subfraction F5/6/2 (466 mg) was separated by preparative TLC and eluted with hexane−MTBE (25:75) to give 1 (Rf 0.55, 1 mg). Subfraction F5/6/4 (83 mg) was separated by HPLC on an RP-18 column using MeOH−H2O (75:25) as the eluent, followed by preparative TLC eluted with CH2Cl2− benzene−MTBE (60:20:20) to yield 3 (Rf 0.55, 4 mg) and 5 (Rf 0.65, 2 mg). Fraction F6 (4.2 g) was separated by MPLC using an elution gradient of hexane−MTBE−MeOH (condition as mentioned above) to obtain seven subfractions (F6/1−F6/7). Subfraction F6/4 (1.3 g) was separated using preparative TLC (CHCl3−EtOAc, 60:40) to produce

PBMCsa >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

The data represent the IC50 values (μM) ± standard deviation obtained from the nonlinear regression of three independent experiments. bCamptothecin (CPT) and doxorubicin (Doxo) were used as positive controls. a

active against CCRF-CEM and MIA PaCa-2 cells, whereas the doxorubicin-resistant CEM-ADR5000 cell line was far less sensitive. Every compound was significantly less cytotoxic against human PBMCs (p < 0.001) relative to the human cancer cells studied (Table 3). Therefore, the most active diterpenes (2, 3, and 5) have high selectivity indexes (the ratio IC50 for PBMCs/ IC50 for tumor cells) toward CCRF-CEM cells, respectively, revealing their lower toxicity toward normal human blood cells. A hydroxy group moiety was present in the butadiene moiety (at R3), eliminating the hydrophobicity of this side chain and decreasing the cytotoxic activity, as demonstrated by compounds 3 and 4 (a 2-fold reduction in the IC50 for CCRF-CEM and MIA-PaCa-2 cells and a complete loss of activity for the CEM-ADR5000 cells). The hydroxy group at C2 was not essential for the cytotoxic activity, but substitution by a carbonyl group caused a decrease in cytotoxicity, as shown with compound 9. Substitution at C-2 and C-6 is important for the cytotoxic activity. The xylose unit moiety at C-6 and the 461



Article

Prism 5 program (Intuitive Software for Science, San Diego, CA, USA). The data are expressed as means ± SD.

two subfractions (F6/4/1 and F6/4/2). Subfraction F6/4/2 was separated by HPLC (2.5 mL/min flow rate with MeOH−H2O, 80:20) to obtain 4 (tR 12.1 min, 2 mg). Fraction F7 (6.3 g) was separated by MPLC (hexane−MTBE− MeOH, elution gradient mentioned above) to obtain three subfractions, F7/1−F7/3. Subfraction F7/1 (1.5 g) was separated by preparative TLC and CHCl3−EtOAc (60:40) as eluant to produce two subfractions (F7/1/1 and F7/1/2). Subfraction F7/1/2 (550 mg) was separated by HPLC (flow rate 1.5 mL/min, MeOH−H2O, 80:20) to obtain 6 (tR 36.2 min, 2 mg), 7 (tR 18.9 min, 4 mg), and 8 (tR 22.1 min, 9 mg). Proof for purity of 1−9 was done by TLC analysis with anisaldehyde/sulfuric acid as detection reagent and was at least 95%. Zuelaguidin A (1): [α]20D −1.2 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 223 (1.87), 280 (1.26) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 587.2617 [M + Na]+ (calcd for C33H40O8Na, 587.2621). Zuelaguidin B (2): [α]20D −38 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 223 (2.81), 280 (2.48) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 587.2619 [M + Na]+ (calcd for C33H40O8Na, 587.2621). Zuelaguidin C (3): [α]20D −7 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 223 (2.31) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRAPCIMS m/z 650.4270 [M + NH4]+ (calcd for C36H60NO9 650.4268). Zuelaguidin D (4): [α]20D +10 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 223 (2.71) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 671.3777 [M + Na]+ (calcd C36H56O10Na, 671.3771). Zuelaguidin E (5): [α]20D +4 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 223 (2.19), 280 (2.14) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 619.2512 [M + Na]+ (calcd C33H40O9Na, 619.2519). Zuelaguidin F (6): [α]20D +8 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 223 (2.45) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 759.3933 [M + Na]+ (calcd C39H60O13Na, 759.3932). Zuelaguidin G (7): [α]20D +28 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 202 (2.66) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 791.3823 [M + Na]+ (calcd C39H60O15Na, 791.3830). Zuelaguidin H (8): [α]20D +15 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 202 (2.84) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS (positive mode) m/z 791.3828 [M + Na]+ (calcd C39H60O15Na, 791.3830). Cytotoxicity Assay. The cytotoxicity of the clerodane diterpenes (1−9) was tested against three tumor cell lines and against healthy PBMCs to calculate selectivity indexes: CCRF-CEM (human acute lymphocytic leukemia), CEM-ADR5000 (human acute lymphocytic leukemia resistant to doxorubicin), MIA-PaCa-2 (human pancreatic carcinoma). The MIA-PaCa-2 cells were kindly provided by Dr. Ralph Graeser (Tumour Biology Center, Freiburg, Germany), the CCRFCEM and CEM-ADR5000 cells were a gift from Prof. T. Efferth, Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany, and the PBMCs were isolated from human buffy coats obtained from the Freiburg University Clinic, Freiburg, Germany (ethical permission number from the ethics commission, University of Freiburg: 356/13; 2013). The cells were maintained at 37 °C under 5% CO2 in RPMI 1640 medium that was supplemented with 10% heat-inactivated fetal bovine serum, 100 mg/mL streptomycin, and 100 U/mL penicillin. The cells were seeded in 96-well plates (1.2 × 104 cells/well for MIA-PaCa-2 cells, 4 × 104 cells/well for leukemic cells, and 2 × 105 PBMCs/well in 150 μL complete medium). Compounds 1−9 were dissolved in DMSO, and the cells were incubated for 24 h with various concentrations of a clerodane diterpene or the positive control, respectively. Camptothecin and doxorubicin were used as the positive controls, and DMSO 0.1% was the solvent control. The viability of the tumor cells was quantified using an MTT assay, as previously described.44 The IC50 values were obtained by nonlinear regression using the GraphPad

■

ASSOCIATED CONTENT

S Supporting Information *

The NMR spectra for compounds 1−8 are available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +49-761-203-8373. E-mail: irmgard.merfort@pharmazie. uni-freiburg.de. *Tel: +506-2511-4477. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to Dr. J. Wörth and C. Warth at the Institute of Organic Chemistry, University of Freiburg, for the HRMS data, M. Wagner at the Department of Pharmaceutical and Medicinal Chemistry, University of Freiburg, for measuring the optical rotation, botanist L. Poveda at the Universidad Nacional, Heredia, Costa Rica, for support while collecting and identifying the plant material, Dr. R. Graeser at the Tumour Biology Center, Freiburg, Germany, for providing the MIA PaCa-2 cells, and Prof. T. Efferth at the Department of Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany, for the CCRF-CEM and CEM-ADR5000 cells. C.D.F. is grateful for a DAAD scholarship.

■

DEDICATION Dedicated to Prof. Dr. Otto Sticher of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

■

REFERENCES

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