Polyoxygenated Cyclohexenes and Other Constituents of

Article pubs.acs.org/jnp

Polyoxygenated Cyclohexenes and Other Constituents of Cleistochlamys kirkii Leaves Stephen S. Nyandoro,*,†,‡ Joan J. E. Munissi,† Amra Gruhonjic,‡,§ Sandra Duffy,⊥ Fangfang Pan,# Rakesh Puttreddy,# John P. Holleran,⊥ Paul A. Fitzpatrick,§ Jerry Pelletier,∥ Vicky M. Avery,⊥ Kari Rissanen,# and Máté Erdélyi*,‡,¶ †

Department of Chemistry, College of Natural and Applied Sciences, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania ‡ Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden § Sahlgrenska Cancer Centre, University of Gothenburg, Gothenburg, SE-405 30, Sweden ⊥ Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Q1d 4111, Australia ∥ Department of Biochemistry, McGill University, Montreal, QC, H3G 1Y6, Canada # University of Jyvaskyla, Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box. 35, Jyvaskyla FI-40014, Finland ¶ Swedish NMR Centre, University of Gothenburg, Gothenburg, SE-405 30, Sweden S Supporting Information *

ABSTRACT: Thirteen new metabolites, including the polyoxygenated cyclohexene derivatives cleistodiendiol (1), cleistodienol B (3), cleistenechlorohydrins A (4) and B (5), cleistenediols A−F (6−11), cleistenonal (12), and the butenolide cleistanolate (13), 2,5-dihydroxybenzyl benzoate (cleistophenolide, 14), and eight known compounds (2, 15− 21) were isolated from a MeOH extract of the leaves of Cleistochlamys kirkii. The purified metabolites were identified by NMR spectroscopic and mass spectrometric analyses, whereas the absolute configurations of compounds 1, 17, and 19 were established by single-crystal X-ray diffraction. The configuration of the exocyclic double bond of compound 2 was revised based on comparison of its NMR spectroscopic features and optical rotation to those of 1, for which the configuration was determined by X-ray diffraction. Observation of the cooccurrence of cyclohexenoids and heptenolides in C. kirkii is of biogenetic and chemotaxonomic significance. Some of the isolated compounds showed activity against Plasmodium falciparum (3D7, Dd2), with IC50 values of 0.2−40 μM, and against HEK293 mammalian cells (IC50 2.7−3.6 μM). While the crude extract was inactive at 100 μg/mL against the MDA-MB-231 triple-negative breast cancer cell line, some of its isolated constituents demonstrated cytotoxic activity with IC50 values ranging from 0.03−8.2 μM. Compound 1 showed the most potent antiplasmodial (IC50 0.2 μM) and cytotoxic (IC50 0.03 μM, MDAMB-231 cell line) activities. None of the compounds investigated exhibited translational inhibitory activity in vitro at 20 μM. Ellipeiopsis,32 Artabotrys,33 and Dasymaschalon34 genera of the Annonaceae. These metabolites possess antiproliferative,6,9,22,29 antimalarial,28 larval antifeedant,6 plant root growth inhibitory,21 and cytotoxic34 activities. Butenolides are unsaturated lactones possessing a five-membered, four-carbon heterocyclic core. From a few members of the family Annonaceae,4,33,35 analogues with an additional alicyclic three-carbon skeleton, and therefore termed heptenolides, have also been reported. They possess antimicrobial,4 cytotoxic,33,35 anti-inflammatory,36 and mosquito-larvae growth inhibitory37 activities. Due to their

Cleistochlamys kirkii (Benth.) Oliv. belongs to a monotypic genus of the family Annonaceae and is native to eastern and southern African countries including Malawi, Zambia, Mozambique, Zimbabwe, and Tanzania.1,2 In Mozambique, it is used in traditional medicine to treat wound infections, rheumatism, and tuberculosis.3 This plant was reported previously to contain butenolides and polyoxygenated cyclohexene derivatives that possess antimicrobial and cytotoxic activities.4 Polyoxygenated cyclohexenes are common metabolites of the family Annonaceae4,5 and were also found, albeit to a lesser extent, in the Zingiberaceae,6,7 Piperaceae,8−11 and Euphorbiaceae.12 Besides C. kirkii,4 polyoxygenated cyclohexenes have been reported from the Uvaria, 13−30 Monanthotaxis,31 © 2016 American Chemical Society and American Society of Pharmacognosy

Received: August 17, 2016 Published: December 21, 2016 114

DOI: 10.1021/acs.jnatprod.6b00759 J. Nat. Prod. 2017, 80, 114−125

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Spectroscopic Data for Cleistodienediol (1) and Cleistodienol B (3) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 1 δC, type

position 1′ OAc-1′

133.5, CH

OBz-1′ 1 2 OAc-2

163.3, 119.9, 64.1, 170.3, 21.3, 125.0, 134.4, 74.4,

3 4 5 OH-5 6 OH/OAc-6 1″ 2″/6″ 3″/5″ 4″

7.75

CO C CH CO CH3 CH CH CH

71.5, CH

128.7, 130.3, 128.8, 134.0,

3

δH

C CH CH CH

δC, type

(J in Hz) d (2.4)

88.5, 168.9, 21.1, 164.2, 129.1, 126.4,

6.49

d (4.0)

2.05 5.82 5.93 4.15 3.29 4.50 3.39

s ddd (2.4, 4.0, 10.4) d (10.4) d (8.0) s dd (2.4, 8.0) s

8.14 7.43 7.58

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

δH

CH CO CH3 CO C CH

123.7, CH 130.2, CH 69.5, CH 72.2, 171.2, 20.9, 129.0, 130.2, 128.7, 133.9,

CH CO CH3 C CH CH CH

(J in Hz)

7.58

s

2.10

s

6.47,

d (5.6)

6.12 6.07 4.42 2.21 5.83

dd (5.6, 9.6) dd, (4.0, 9.6) ddd, (4.0, 5.6, 6.4) d (6.4) d (5.6)

2.10

s

8.07 7.47 7.60

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

Table 2. 1H and 13C NMR Spectroscopic Data for Cleistenechlorohydrins A (4) and B (5) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 4 position 1′ OBz-1′ 1 OH-1 2 3 4 5 OAc-5 6 OH-6 1″ 2″/6″ 3″/5″ 4″

δC, type

δH

64.5, CH2

4.76 4.65

5 (J in Hz) d (12.1) d (12.1)

167.1, CO 76.0, C 62.1, 130.6, 126.7, 72.8, 171.3, 21.2, 75.8,

CHCl CH CH CH CO CH3 CH

129.5, 129.9, 128.7, 133.6,

C CH CH CH

δC, type

δH

64.7, CH2

4.78 4.66

d (12.2) d (12.2)

3.79 4.76 5.87 5.76 5.59

s ddd (2.4, 2.4, 2.4) ddd (2.4, 2.4, 10.3) dd (2.4, 2.4, 10.3) dddd (2.4, 2.4, 2.4, 8.4)

2.12 3.98 2.98

s dd (4.8, 8.4) d (4.8)

8.03 7.46 7.58

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

(J in Hz)

167.1, CO 76.0, C 3.86 4.76, 5.86 5.75 5.58

s ddd (2.4, 2.4, 2.4) ddd (2.4, 2.4, 10.3) ddd, (2.4, 2.4, 10.3) dddd, (2.4, 2.4, 2.4, 8.4)

2.11 3.98 3.12

s dd (4.8, 8.4) d (4.8)

8.02 7.45 7.57

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

■

62.1, 130.7, 126.8, 72.8, 171.2, 21.2, 75.9,

CHCl CH CH CH CO CH3 CH

129.5, 130.0, 128.7, 133.6,

C CH CH CH

RESULTS AND DISCUSSION By repeated silica gel column chromatographic separation of the MeOH extract of C. kirkii leaves, followed by purification on Sephadex LH-20 and on preparative HPLC, 21 secondary metabolites have been isolated and their structures were identified using NMR spectroscopic, mass spectrometric, and X-ray crystallographic analyses. Eleven new polyoxygenated cyclohexenes (1, 3−12), a new heptanolide (13), a new benzylbenzoate derivative (14), and eight previously known natural products (2, 15−21) were identified. The structures of the known compounds cleistodienol A (2),4 sootepenol B (15),34 ent-subglain C (16),31 (1S,4S,5S,6R)-5-[(benzyloxy)methyl]-5,6-dihydroxycyclohex-2-ene-1,4-diyl diacetate (17),31 Z-melodorinol (18),35 Z-acetylmelodorinol (19),35 tetramethylscutellarein (20),42 and 3-hydroxybenzaldehyde (21)43

bioactivity, heptenolides and polyoxygenated cyclohexene derivatives, also including those previously isolated from C. kirkii, have attracted the attention of synthetic chemists.14,38−41 On the basis of its expected richness in such bioactive secondary metabolites possibly useful in the development of novel antimalarial and anticancer agents, and as part of our ongoing search of new antimalarial and anticancer agents as well as our work on the phytochemical characterization of East African medicinal plants, C. kirkii has been reinvestigated. The constituents isolated from its leaves were evaluated for activity against Plasmodium falciparum (3D7, Dd2), human embryonic kidney cells (HEK-293), and human triple-negative breast cancer cells (MDA-MB-231) and luciferase mRNA translational inhibition. 115



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Table 3. 1H and 13C NMR Spectroscopic Data for Cleistenediol A (6) and Cleistenediol B (7) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 6 position 1′ OBz-1′ 1 OH-1 2 OMe-2 3 4 5 OAc/OH-5 6 OH/OAc-6 1″ 2″/6″ 3″/5″ 4″

δC, type

δH

65.0, CH2

4.74 4.58

7 (J in Hz) d (12.1) d (12.1)

167.2, CO 76.3, C 81.6, 59.4, 129.8, 125.8, 73.2, 171.2, 21.3, 75.1,

CH OCH3 CH CH CH CO CH3 CH

129.8, 129.9, 128.7, 133.4,

C CH CH CH

δC, type

δH

65.9, CH2

4.68 4.57

d (12.6) d (12.6)

3.89 4.02 3.59 5.82 5.85 4.47 2.30

s dd (2.4, 4.8) s ddd (2.4, 2.4, 10.5) ddd (2.4, 2.4, 10.5) m d (7.2)

5.05

d (8.0)

1.86

s

8.07 7.47 7.60

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

(J in Hz)

168.1, CO 76.7, C 3.56 3.98 3.55 5.93 5.76 5.51

s dd (2.4, 4.8) s ddd (2.4, 2.4, 10.5) ddd, (2.4, 2.4, 10.5) ddd, (2.4, 4.0, 6.4)

2.12 3.88 2.96

s dd (6.4, 6.4) d (6.4)

8.02 7.45 7.57

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

82.7, 60.0, 127.8, 129.2, 69.7,

CH OCH3 CH CH CH

78.5, 171.2, 20.9, 129.8, 129.9, 128.7, 133.6,


Table 4. 1H and 13C NMR Spectroscopic Data for Cleistenediol C (8) and Cleistenediol D (9) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 8 position 1′ OBz-1′ 1 OAc-1 2 OAc-2 3 4 OAc-4 5 OH-5 6 OH-6 1″ 2″/6″ 3″/5″ 4″ a

9

δC, type

δH

66.3, CH2

4.66 4.54

d (12.0) d (12.0)

5.49

dd (6.4)

2.00 5.59

s ddd, (2.2, 6.4)

2.12 5.79 5.96

s dd, (2.2, 10.1) ddd (1.6, 4.8, 10.1)

4.27 2.63

dd (4.8, 4.8) d (4.8)

167.4, 71.9, 170.6, 21.1, 70.8, 170.5, 21.0, 127.6, 129.1,

CO CH CO CH3 CH CO CH3 CH CH

69.3, CH

(J in Hz)

75.4, C 129.3, 129.9, 128.8, 133.8,

C CH CH CH

δC, type

δH

64.4, CH2

4.78 4.29

d (12.0) d (12.0)

5.37

d (8.0)

2.15 5.90a

s

166.8, 72.4, 170.9, 21.2, 126.3,

CO CH CO CH3 CH

130.3, 68.6, 171.2, 21.2, 74.3,

CH CH CO CH3 C

71.5, CH 2.82

s

8.06 7.47 7.60

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

129.4, 130.0, 128.7, 133.7,

C CH CH CH

(J in Hz)

5.90a 5.64

d (2.4)

2.15

s

2.96 4.18 2.83

s dd (3.2, 8.0) d (3.2)

8.09 7.47 7.59

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

Overlapping signals.

305.1069, calcd 305.1025). The 1H and 13C NMR spectroscopic data (Table 1) closely resembled those of (−)-cleistodienol (2),4 yet lacking the C-6 acetyl functionality. The atoms belonging to the spin system of the cyclohexene ring were assigned based on COSY and TOCSY correlations (Figures S3 and S4, Supporting Information) of H-1′ (δH 7.75) to H-6 (δH 4.50), H-6 to H-5 (δH 4.15), H-5 to H-4 (δH 5.93), H-4 to H-3 (δH 5.82), and H-3 to H-2 (δH 6.49). The hydroxy functionalities OH-6 (δH 3.39) and OH-5 (δH 3.29) were placed at the corresponding carbons based on their COSY

were confirmed by comparison of their observed and reported spectroscopic and physical data. Comparison of the NMR data of 1 and 3−12 and those of their known analogues 2, 15, 16, and 17 indicated their extensive structural similarities and the presence of oxybenzoyl, acetoxy, and hydroxy moieties in these compounds (Tables 1−7 and Figures S1−S159, Supporting Information). Compound 1 was obtained as white solid, when crystallized from MeOH−CH2Cl2 (1:1), and was assigned the molecular formula C16H16O6 based on HRESIMS analysis ([M + H]+ m/z 116



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Table 5. 1H and 13C NMR Spectroscopic Data for Cleistenediol E (10) and Cleistenediol F (11) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 10 δC, type

position 1′

62.8, CH2

OBz-1′ 1 OH/OAc-1

(J in Hz)

4.76 4.67

d (12.0) d (12.0)

166.6, CO 76.1, C−O

2 OAc-2

76.8, CH−O

3 4 5 OAc-5 6 OH-6 1″ 2″/6″ 3″/5″ 4″ 7″ 1‴ 2‴/6‴ 3″/5″ 4‴ a

11

δH

128.2, 128.0, 72.5, 171.2, 21.3, 75.3,

CH CH CH−O CO CH3 CH−O

129.6, 130.1, 128.6, 133.7, 167.3, 129.2, 129.8, 128.5, 133.2,

C CH CH CH CO C CH CH CH

4.42

br s

5.78

ddd (2.2, 2.4, 2.4)

5.81 5.87 5.58

ddd (2.4, 2.4, 10.3) ddd (2.4, 2.4, 10.3) dddd (2.2, 2.4, 2.4, 8.8)

2.15 4.12 3.25

s d (8.8) br s

7.97 7.33 7.50

dd (1.6, 8.8) dd (8.8, 8.8) tt (1.6, 8.8)

7.90 7.31 7.43

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

δC, type

δH

64.1, CH2

4.74 4.70

d (12.0) d (12.0)

5.51

d (8.4)

2.08 5.73

s ddd (1.6, 2.8, 8.4)

1.99 5.87a 5.89 5.88a

s m m

4.15

br s

8.99 7.37 7.53

m m m

7.97 7.37 7.53

m m m

166.8, 74.4, 170.5, 21.1, 70.5, 170.4, 20.8, 128.0, 128.1, 76.8,

CO CH−O CO CH3 CH−O CO CH3 CH CH CH−O

(J in Hz)

75.6, C−O 129.4, 130.1, 128.6, 133.9, 167.1, 129.2, 129.8, 128.5, 133.2,

C CH CH CH CO C CH CH CH

Overlapping signals.

Table 6. 1H and 13C NMR Spectroscopic Data for Cleistenonal (12) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] position 1′ OAc-1′ OBz-1′ 1 2 3 4 4a 5 6 7 CHO-7 8 8a 9 OH-9 10

1″ 2″/6″ 3″/5″ 4″

δC, type 88.4, 168.4, 21.0, 164.8, 53.2, 132.7, 133.9, 47.2, 148.5, 126.8, 129.6, 135.0, 192.1, 125.7, 137.6, 78.2,

CH CO CH3 CO C CH CH CH C CH CH C CO CH C CH

81.7, 172.9, 20.9, 128.7, 130.2, 128.8, 134.1,


δH

(J in Hz)

7.95

s

2.15

s

6.44, 6.65 4.09

d (8.0) dd (8.0, 8.0) dd (3.2, 8.0)

7.54 7.81

d (8.0) d (8.0)

10.1 8.44

s s

3.81 2.93 4.20

br s br s d (br s)

2.14

s

8.00 7.43 7.58

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

correlations to H-6 (δH 4.50) and H-5 (δH 4.15), respectively. The assignment of C-1 and C-1′ was supported by HSQC and 117



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Table 7. 1H and 13C NMR Spectroscopic Data for Cleistanolate (13) and Cleistophenolide (14) Acquired in CDCl3 at 25 °C [δH, Multiplicity (J in Hz)] 13 position 1′ OBz-1′ 1 1-OAc 2 2-OAc 3

δC, type

69.5, 169.3, 21.1, 70.1, 169.9, 20.9, 62.5,

CH CO CH3 CH CO CH3 CH2

OBz-3 4 5 6 2′ 3′ 3′-OMe 4′

166.4, CO

79.9, 76.3, 57.5, 34.7,

CH CH OCH3 CH2

5′ 1″ 2″/6″ 3″/5″ 4″

173.5, 129.6, 129.8, 128.7, 133.5,

CO C CH CH CH

δH

14 (J in Hz)

δC, type

δH 5.30

s

119.0, CH

6.85

d (8.8)

118.2, CH 149.4, C 118.3, CH

6.77

dd (3.2, 8.8)

6.86

d (3.2)

8.05 7.43 7.57

m m m

5.67

dd (5.6, 5.6)

63.7, CH2 168.8, CO 122.8, C

2.11 5.48

s ddd (3.4, 5.6, 7.2)

149.4, C

2.07 4.68 4.35

s dd (3.4, 12.2) dd (7.2, 12.2)

4.73 4.30 3.35 2.71 2.63

dd (5.6, 5.6) ddd (4.0, 5.6, 6.4) s dd (6.4, 17.6) dd (4.0, 17.6)

8.02 7.46 7.58

dd (1.6, 8.0) dd (8.0, 8.0) tt (1.6, 8.0)

129.3, 130.1, 128.6, 133.8,

C CH CH CH

(J in Hz)

The configuration of C-1′ of cleistodienol A (2) was proposed4 formerly to be opposite that derived for 1 based on X-ray diffraction analysis, based on the observation of the allylic 4JH1′,H6, but not of the 4JH1′,H2. Analogous observation of the allylic 4JH1′,H6 and the absence of 4JH1′,H2 (Table S6 and Figure S3, Supporting Information) for 1, however, suggested that 1 and 2 ought to have an identical relative configuration at C-1′, C-2, C-5, and C-6. Thus, the configuration of cleistodienol A (2) needs to be revisited. The observed 4 JH1′,H6 long-range coupling is likely developed by the fortuitous overlap of the C-1′C-1 π-orbital with the sp3-C−H σ-orbital at C-6, whereas the lack of some 4JH1′,H2 may be explained by the lack of orbital overlap of the C-1′C-1 π-system with the sp3-C−H σ-orbital at C-2 in the half-chair conformation preferred by the cyclohexene cores of 1 and 2. On the basis of the similar optical rotation values of 1 and 2, the absolute configuration of 2 is expected to be analogous to that of 1 shown in Figure 1. Compound 3 was obtained as a white solid and was assigned a molecular formula of C18H18O7 based on HRESIMS analysis ([M + H]+ m/z 347.1118, calcd 347.1131). NMR analysis supported the presence of an oxybenzoyl, a cyclohexadiene, and two acetate units (Table 1). Coupling of the olefinic protons H2 (δH 6.47), H-3 (δH 6.12), and H-4 (δH 6.07) was observed in the COSY spectrum (Figure S19, Supporting Information), whereas the corresponding carbons, C-2 (126.4), C-3 (δC 123.7), C-4 (δC 130.2), and C-1 (δC 129.1), were assigned using HSQC and HMBC correlations (Figures S22−23, Supporting Information). The cyclohexadiene core was established using the COSY correlations of H-4 (δH 6.07) to H-5 (δH 4.42) and of H-5 (δH 4.42) to H-6 (δH 5.83), while the position of OH-5 (δH 2.21) was proposed from its COSY correlation to H-5 (δH 4.42). The position of the acetoxy

HMBC correlations of H-1′ (δH 7.75) (Figure S7, Supporting Information), whereas the acetyl group was positioned at C-2 based on the HMBC correlation of H-2 (δH 6.49) to the acetyl CO (δC 170.3). The HMBC correlation of H-1′ (δH 7.75) to the benzoyloxy carbonyl COO-1′ (δC 163.3) allowed the assignment of the location of this functionality. A trans-diaxial configuration of H-6 (δH 4.50) and H-5 (δH 4.15) was indicated by the coupling constant value 3JH5,H6 = 8.0 Hz, similar to that observed for 2.4 From the lack of any diagnostic NOEs, the relative configurations at C-6 (δC 64.1) and C-1′ (δC 133.5) could not be elucidated based on NMR data alone, and therefore they were established by single-crystal X-ray diffraction analysis, which also demonstrated that the cyclohexene ring assumes a half-chair conformation (Figure 1). On the basis of the above spectroscopic data, the structure of the new compound cleistodienediol was characterized as 1.

Figure 1. X-ray structures of (a) cleistodienediol (1), (b) (1S,4S,5S,6R)-5-[(benzyloxy)methyl]-5,6-dihydroxycyclohex-2-ene1,4-diyl diacetate (17), and (c) Z-acetylmelodorinol (19). 118



Article

337.1287). The NMR data (Table 3) confirmed that 6 possesses a methoxy, an acetoxy, and two hydroxy substituents as well as a benzoyloxymethyl connected to a cyclohexene backbone. The relative configuration of H-6 (δH 3.88) and H-5 (δH 5.51) was established as being cis axial−equatorial based on the observed 3JH5,H6 coupling constant of 6.4 Hz. NOESY correlations (Figure S44, Supporting Information) between H1′ (δH 4.74) and H-6 (δH 3.88) and between H-1′ and H-5 (δH 5.51) indicated them to be cis, whereas the absence of any NOEs from H-6 and H-5 to H-2 (δH 3.98) revealed H-2 to be oriented toward the opposite side of the cyclohexene ring. The weak NOE observed for MeO-2 (δH 3.55) and H-5 (δH 5.51) provided further confirmation for the C-2 configuration. On the basis of the above spectroscopic data, the new compound cleistenediol A was characterized as 6. Compound 7 was obtained as a colorless gum and was assigned a molecular formula of C17H20O7 based on HRESIMS analysis ([M + H]+ m/z 337.1288, calcd 337.1287). Whereas its molecular formula was identical to that of 6, optical rotations of the two compounds were different, suggesting 7 to be an isomer of 6. The NMR data (Table 3) confirmed this hypothesis and indicated 7 to be acetylated at C-6, in contrast to 6, which is acetylated at C-5. Hence, H-6 (δH 5.05) gave an HMBC correlation (Figure S55, Supporting Information) to the CO of the acetyl functionality (δC 171.2). The transdiaxial configuration of H-5 and H-6 was indicated by the 3 JH2−H3 coupling constant of 8.0 Hz and by a more prominent NOE (Figure S52, Supporting Information) observed for H-6 (δH 5.05) and OH-5 (δH 2.30) as compared to that of H-6 (δH 5.05) and H-5 (δH 4.47). The NOE between H-6 (δH 5.05) and H-2 (δH 4.02) indicated that they are cis, revealing the relative configuration of C-2, which is the opposite of that of 6. On the basis of the above spectroscopic data, cleistenediol B was characterized as 7. Compound 8 was isolated as a colorless gum. The molecular formula was determined to be C18H20O8 on the basis of the HRESIMS (m/z 365.1235, calcd 365.1236) and NMR spectroscopic data (Table 4). The NMR spectra displayed spectroscopic features analogous to those of 6 and 7 and were thus typical of a polyoxygenated benzoyloxymethylcyclohexene skeleton bearing two hydroxy and two acetylated hydroxy groups. The HMBC correlations of H-1 (δH 5.49) to CO (δC 171.6) and of H-2 (δH 5.59) to CO (δC 170.5) revealed the positions of the acetyl groups. The relative stereochemical orientation of H-1 (δH 5.49) and H-2 (δH 5.59) was established to be cis axial−equatorial based on the coupling constant value 3 JH1,H2 of 6.4 Hz, whereas that of the substituent of C-6 was based on the strong NOE interactions observed between H-1 (δH 5.49) and H-1′ (δH 4.54 and 4.66) (Figure S60, Supporting Information). The weak NOE between H-1 (δH 5.49) and OH5 (δH 2.63) along with the NOE between H-5 (δH 4.27) and OH-6 (δH 2.82) provided information on the relative configuration of C-5. On the basis of the above spectroscopic evidence, this new compound, cleistenediol C, was characterized as 8. Compound 9 was obtained as a colorless gum and assigned the same molecular formula as 8 (C18H20O8), based on the HRESIMS analysis ([M + H]+ m/z 365.1223, calcd 365.1236). Optical rotation suggested 9 to be an isomer of 8. The positions of the acetyl functionalities were determined from the HMBC correlations of H-1 (δH 5.37) to CO (δC 170.9) and H-4 (δH 5.64) to CO (δC 171.2) (Figure S71, Supporting Information). The trans-diaxial orientation of H-6 and H-1 was

substituent at C-2 was determined from the observation of the HMBC correlation of H-6 (δH 5.83) to the acetyl carbonyl (δC 171.2) (Figure S23, Supporting Information). The H-1′ (δH 7.58) signal showed an HMBC correlation to the carbonyls of the C-1′ acetoxy (δC 168.9) and the C-1′ benzoyloxy (δC 164.2) carbonyls. The trans axial−equatorial relative configuration of H-5 and H-6 was established based on the 3JH5,H6 coupling value of 5.6 Hz. On the basis of the above-discussed spectroscopic features, the new compound cleistodienol B was therefore characterized as 3. It is structurally closely related to 1,6-deoxy-β-senepoxide, previously reported from U. ferruginea19,20 and from C. kirkii.4 Compounds 4 and 5 were obtained as pink and colorless gums, respectively, and were assigned the molecular formula C17H17O6Cl based on HRESIMS analysis ([M + H]+ m/z 341.0813 and 341.0774, respectively, calcd 341.0792). Their different optical rotation values suggested that they are diastereomers. Their chlorine content was revealed by the observation of the isotope mass peaks [M + H + 2]+ at m/z 343.0690 (4) and 343.0754 (5) in a 1:3 relative abundance as compared to the [M + H]+ peaks at m/z 341.0813 (4) and 341.0774 (5) (Figures S32 and S40, Supporting Information). Compounds 4 and 5 showed highly similar NMR spectroscopic features (Table 2), corroborating that they are stereoisomers. The benzoyloxymethylenecyclohexene skeleton of 4 was established based on the COSY and TOCSY correlations (Figure S27, Supporting Information) of H-6 (δH 3.98) to OH6 (3.12), H-6 to H-5 (δH 5.58), and H-5 (δH 5.58) to H-4 (δH 5.75), H-3 (δH 5.86), and H-2 (δH 4.76). These, along with the corresponding NOEs (Figure S28, Supporting Information) between OH-1 (δH 3.86) and H-6 (δH 3.98), H-6 and OH-6 (δH 3.13), OH-6 and H-5 (δH 5.59), H-5 and H-4 (δH 5.75), H4 and H-3 (δH 5.85), H-3 and H-2 (δH 4.75), H-2 and OH-1 (δH 3.86), and H-1′ (δH 4.65 and 4.76) and OH-1 (δH 3.86) as well as H-6 (δH 3.98), were used to confirm the assignment of the basic skeleton. The 3JH5,H6 value of 8.4 Hz suggested a transdiaxial configuration of H-5 (δH 5.59) and H-6 (δH 3.98), whereas the cis orientation of H-5 (δH 5.59), and the 1benzoyloxymethylene substituent (H-1′, δH 4.65) was revealed by their NOE correlations. Analogous COSY, TOCSY, NOESY, HSQC, and HMBC correlations of 5 (Figures S33− 39, Supporting Information) to those of 4 revealed their identical constitution. Compounds 4 and 5 showed slightly different chemical shifts (Table 2), with the largest shift difference observed for their OH-1 (Δδ 0.05 ppm), OH-6 (Δδ 0.14 ppm), and H-1′ (Δδ 0.02 ppm) resonances. Their different optical rotations, HPLC retention times, and 1H and 13 C NMR chemical shifts suggested they are diastereomers, with different chirality most likely at their quaternary C-1. Accordingly, the strong NOEs of H-6 (δH 3.98) and OH-1 (δH 3.79), H-6 (δH 3.98) and H-2 (δH 4.76), and OH-1 (δH 3.79) and H-2 (δH 4.76) of 5 suggested that OH-1, H-2, and H-6 are cis. Correspondingly, the weak NOE correlation of H-6 (δH 3.98) and OH-1 (δH 3.86) and of H-2 (δH 4.76) and OH-1 (δH 3.86) of 4 indicated that OH-1 of 4 is trans to H-2 and H-6. These new compounds, cleistenechlorohydrins A and B, were characterized therefore as 4 and 5, respectively. Structurally closely related polyoxygenated cyclohexenes were reported previously from Piper hookeri,8 P. nigrum,9 Uvaria calamistrata,15 U. grandif lora,22 and Dasymaschalon sootepense.34 Compound 6 was obtained as a colorless gum. Analysis by HRESIMS provided the exact mass of 337.1289 [M + H]+, compatible with the molecular formula C17H20O7 (calcd 119



Article

Scheme 1. Cleistenonal (12) Is Presumably Formed from Two Intermediates of the Shikimic Acid Pathway, Compound 3 and a Diene, via a Diels-Alder-Type Reaction, Followed by Dehydrative Aromatization of Ring B

established based on the observation of 3JH1,H6 = 8.0 Hz. The relative configuration of C-5 was established based on the NOEs between OH-6 (δH 2.83) and H-1′ (δH 4.78), revealing these functionalities to be cis, whereas that of C-4 was based on the NOE between H-4 (δH 5.64) and H-1′ (δH 4.29 and 4.78). The lack of any NOE between H-6 (δH 4.18) and H-4 (δH 5.64) provided further confirmation for the proposed relative configuration at C-6 and C-4. On the basis of the above spectroscopic evidence, this new compound, cleistenediol D, was characterized as 9. The new compounds 8 and 9 are closely related to (+)-senediol,24,30 uvaribonol,12 subglain C,26 and entsubglain C30 (16), (1S,4S,5S,6R)-5-[(benzoyloxy)methyl]-5,6dihydroxycyclohex-2-ene-1,4-diyl diacetate31 (17), with the latter two compounds having also been isolated in the present investigation. The structure and stereochemistry of 17 were confirmed by single-crystal X-ray diffraction analysis (Figure 1). Compound 10 was isolated as a colorless gum and was assigned the molecular formula C23H22O8 based on HRESIMS analysis ([M + H]+ m/z 427.1390, calcd 427.1393) and from its NMR data (Table 5). It showed NMR spectroscopic features consistent with a polyoxygenated benzoyloxymethylcyclohexene skeleton having two hydroxy groups, an acetoxy group, and an additional benzoyloxy substituent. The position of the substituents was determined based on the weak HMBC crosspeaks of H-5 (δH 5.58) to the acetyl carbonyl (δC 171.2), of H2 (δH 5.78) to the benzoyloxy carbonyl (δC 167.3), and of H-1′ (δH 4.67 and 4.76) to the other bezoyloxy carbonyl (δC 166.6) (Figure S79, Supporting Information). The relative configurations at C-6 and C-5 were established as trans-diaxial based on the 3JH5,H6 coupling constant value of 8.8 Hz and thus were determined as trans-diequatorial for the oxygenated substituents. The strong NOE correlations between H-6 (δH 4.12) and H-2 (δH 5.78) and between H-5 (δH 5.58) and H-1′ (δH 4.67 and 4.76) supported the assignment of the relative configurations at C-1, C-2, C-5, and C-6. On the basis of the above spectroscopic data, this new compound, cleistenediol E (10), was characterized as 5α-acetoxy-2β-(benzoyloxy)-1α,6β-dihydroxycyclohex-3-en-1-yl)methyl benzoate. Compound 11 was isolated as a colorless gum and was assigned the molecular formula C25H24O9 based on HRESIMS ([M + H]+ m/z 469.1502, calcd 469.1498) and NMR data analyses (Table 5). Its NMR spectra exhibited spectroscopic features similar to those of 10, yet with an additional set of signals corresponding to a second acetyl group for which the position was determined based on the HMBC correlation of H1 (δH 5.51) and the acetyl carbonyl (δC 170.5) (Figure S87, Supporting Information). The relative configurations at C-1 and C-2 were established as trans-diaxial based on the 3JH1,H2 8.4 Hz coupling constant value for the corresponding protons

and were thus trans-diequatorial for the acetoxy substituents. The relative configurations at C-1, C-2, C-5, and C-6 were established based on the NOEs between H-1 (δH 5.51) and H5 (δH 5.88) and between H-2 (δH 5.73) and H-1′ (δH 4.74) (Figure S84, Supporting Information). On the basis of the above spectroscopic evidence, this new compound, cleistenediol F, was characterized as 11, a diastereomer of a previously reported synthetic product.14 Compound 12 was obtained as a white solid and was assigned the molecular formula C25H22O8 based on HRESIMS ([M + H]+ m/z 451.1407, calcd 451.1393) and NMR data (Table 6) analyses. Its NMR spectra indicated it to possess an oxybenzoyl, a cyclohexene, two acetates, and a trisubstituted benzoyl unit having an aldehyde functionality (δH 10.1, δC 190.1). The HMBC correlations (Figure S95, Supporting Information) of H-1′ (δH 7.95) to C-2 (δC 132.7), C-8a (δC 137.6), C-1′-acetyl CO (δC 168.4), and C-1′-benzoyloxy CO (δC 164.8) indicated its position close to two of the bridging carbons of the tricyclic system. The HMBC correlations of H-10 (δH 4.20) to C-1 (δC 53.2), C-9 (δC 78.2), C-2 (δC 132.7), C-8a (δC 137.6), and acetyl CO (δC 172.9), along with those of H-9 (δH 3.81) to C-1 (δC 81.7), C10, and C-4a (δC 148.5), revealed the position of the deoxygenated C-9−C-10 unit in relation to the aromatic ring, and the oxymethine functionality at C-1′ (δ C 88.4). Analogously, the HMBC correlations of H-2 (δH 6.44) and H-3 (δH 4.20), given in Table S4 (Supporting Information), aided in locating the position of the cyclohexene ring of the tricyclic system. The trans-diequatorial configuration of H-9 and H-10 was indicated by the 100a 0.03 0.29 0.29 0.12 0.45 2.1 0.09 2.7 0.24

100%b

100%b

0.046 0.0075 0.045 0.000 44 0.000 15

4.2 60%b 1.8 0.36 75%c 50%c

8.2 1688 >4000b 494.5 15.9 >22.000b >22.000b

a μg/mL. bPercentage growth inhibition at 40 μM. cAt 20 μM. The inhibitory activities are given as the mean value of at least two independent measurements.

bond, as revealed by the HMBC correlations (Figure S111, Supporting Information) of H-1′ (δH 5.30) to C-1 (δC 122.8), C-2 (δC 149.4), and C-6 (δC 118.3) and of H-6 (δH 6.86) to C1′ (δC 63.7). The multiplicity pattern of the aromatic protons H-3 (δH 6.85, d), H-4 (δH 6.77, dd), and H-6 (δH 6.86, d) indicated it to be para-dihydroxylated. On the basis of the above spectroscopic evidence, this new compound, cleistophenolide, was characterized as 14. Compounds similar to 14 have been reported from Uvaria purpurea44 and U. kirkii,45 which also belong to the Annonaceae family. In addition to compounds 1−14, the known polyoxygenated cyclohexene derivatives sootepenol B (15),34 ent-subglain C (16),31 and 1S,4S,5S,6R-5-[(benzyloxy)methyl]-5,6-dihydroxycyclohex-2-ene-1,4-diyl diacetate (17),31 the heptenolides Zmelodorinol (18)4,35 and Z-acetylmelodorinol (19),4,35 the flavonoid tetramethylscutellarein (20),42 and 2-hydroxybenzaldehyde (21)43 were also isolated from the leaves of C. kirkii and were identified by comparison of their observed and reported spectroscopic and physical data. Whereas the connectivity of flexible, small molecules can be straightforwardly determined based on J- and NOE-connectivities, the determination of their configuration and conformation with NMR spectroscopy may be challenging. The availability of both NMR and X-ray data for compounds 1, 17, and 19 provides opportunity for verification of the NMR-based conclusions on molecular conformation and relative configuration. Thus, the half-chair conformation of cleistodienediol (1) determined by X-ray crystallography (Figure 1) is in excellent agreement with the trans diaxial 3JH5,H6 = 8.0 Hz and the gauche axial−equatorial 3JH2,H3 = 4.0 Hz (Table 1). The lack of NOE correlation between H-2 and H-6 is in agreement with the trans orientation of these protons. The X-ray-determined half-chair conformation of 17, shown in Figure 1, possessing two equatorial acetyl and two equatorial hydroxy groups, is in good agreement with the solution conformation and, hence, the trans diaxial orientation of H-6 and H-1 (3JH1,H6 = 8.7 Hz)

constituting a bicyclo[2.2.2]oct-2-enoid ring fused to benzaldehyde, with scarce previous examples having so far been reported from Kaempferia rotunda,6 Uvaria grandif lora,22 and U. zeylanica.29 The observation of the optically active 12 supports the presumption that these compounds may form via an enzymatic Diels−Alder reaction, as the nonenzymatic alternative would be expected to provide a racemic product.5,6 Compound 13 was obtained as a colorless gum and assigned the molecular formula C19H22O9 based on HRESIMS ([M + H]+ m/z 395.1327, calcd 395.1342) and NMR data (Table 7) analyses. Its NMR spectra indicated an oxybenzoyl group, two acetoxy groups, and a methoxy unit connected to a heptanolide moiety. The central butanolide core of 13 was established based on the COSY correlations (Figure S100, Supporting Information) of the diastereotopic methylene protons H-4′a (δH 2.71) and H-4′b (δH 2.63) to H-3′ (δH 4.30), with the latter correlating to H-2′ (δH 4.73). The position of the methoxy group (δH 3.35) was revealed by its HMBC correlation to C-3′ (δC 76.3) (Figure S103, Supporting Information), whereas the linear chain of the heptanolide skeleton was established based on COSY correlations. HMBC correlations of H-3a and H-3b to the carbonyl of the oxybenzoyl moiety (δC 166.4) revealed the position of the latter molecular unit. The relative configurations of C-3′, C-2′, C-1, and C-2 were proposed based on the magnitude of the vicinal coupling constants, 3JH2′,H3′ 5.6 Hz, 3JH1,H2′ 5.6 Hz, and 3 JH1,H2 5.6 Hz, revealing cis orientations of the corresponding protons, and on the NOE correlation of H-3′ (δH 4.30) with H2′ (δH 4.73). On the basis of the above spectroscopic evidence, this new compound, cleistanolate, was characterized as 13. Compound 14 was obtained as a colorless gum and was assigned a molecular formula of C14H12O4 based on HRESIMS analysis ([M + H]+ m/z 245.0788, calcd 245.0814) and the NMR data (Table 7, Figures S105−S112, Supporting Information). Its NMR spectra indicated an oxybenzoyl unit connected to a dihydroxybenzene moiety via the C-1−C-1′ 121



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Turbolon spray ion source and a Gemini 5 mm RP-C18 110 Å column using a H2O−MeCN gradient (80:20 to 20:80) in the presence of 0.2% HCO2H, with a separation time of 8 min. HRESIMS were obtained with a Q-TOF-LC/MS spectrometer with a lockmass-ESI source (Stenhagen Analysis Lab AB, Gothenburg, Sweden) using a 2.1 × 30 mm 1.7 μm RP-C18 column and a H2O−MeCN gradient (5:95 to 95:5, with 0.2% HCO2H). Analytical TLC was performed on silica gel 60 F254 (Merck) precoated aluminum plates, which after development with an appropriate solvent system were evaluated under UV light (254 and 366 nm) and then sprayed with 4anisaldehyde reagent (prepared by mixing 3.5 mL of 4-anisaldehyde with 2.5 mL of concentrated H2SO4, 4 mL of glacial HOAc, and 90 mL of MeOH) followed by heating for identification of UV-negative compounds and for detection of color change of the UV-positive spots. Column chromatography was carried out using silica gel 60 (230−400 mesh). Gel filtration was carried out over Sephadex LH-20 (Pharmacia) suspended in CH2Cl2−MeOH (1:1). Preparative HPLC was performed on a Waters 600E system using the Chromulan (Pikron Ltd.) software and an RP-C8 Kromasil column (250 mm × 25 mm) with a H2O−MeOH gradient (70:30 to 100:0) for 20−40 min with a flow rate of 7 mL/min. Plant Material. The leaves of C. kirkii were collected by S.S.N. and S. A. Yahaya in March 2013 from Nyamatumbili Hill at Mchakama village, Kilwa district, Lindi region, Tanzania. The plant species was identified by Mr. Yahaya in the field and confirmed at the Herbarium of the Botany Department at the University of Dar es Salaam, where a voucher specimen is deposited with the reference number YSA 3652. Extraction and Isolation. The air-dried and pulverized leaves (ca. 1 kg) were extracted with MeOH twice for 48 h at room temperature. The extracts were concentrated using a rotary evaporator at 40 °C, providing 83 g of a crude extract, which was subjected to gravitational column chromatography [silica gel, 30−100% (v/v)] with an EtOAc− isohexane gradient, collecting 20 fractions, ca. 250 mL each. These were concentrated and analyzed by TLC using a 4-anisaldehydecontaining spray reagent. Combined fractions 12 and 13, obtained by eluting with 75% EtOAc in isohexane, were subjected to chromatography on silica gel with a 50−75% EtOAc in isohexane gradient to yield 15 subfractions of ca. 50 mL each. Subfractions 6−10 were subjected to repeated column chromatography on Sephadex LH20 using MeOH−CH2Cl2 (1:1) as eluent, which, besides the removal of chlorophyll, afforded Z-acetylmelodorinol (19, 87.3 mg),4,35 cleistodienol A (2, 248.6 mg),4 cleistodienol F (11 42.4 mg), 2,5dihydroxybenzyl benzoate (14, 22.3 mg), 3-hydroxybenzaldehyde (21, 19.1 mg), and ent-subglain C (16, 132.2 mg).31 Z-Acetylmelodorinol (19) was further obtained by recrystallization from isohexane−-ethyl acetate (3:7). Fraction 14 from the first crude extract, upon removal of chlorophyll on Sephadex LH-20 with 1:1 MeOH−CH2Cl2, was subjected to column chromatography on silica gel with 75% EtOAc in isohexane, giving cleistenechlorohydrin A (4, 23.3 mg), cleistenechlorohydrin B (5, 16.8 mg), and cleistodienediol (1, 23.9 mg). Additional fractions obtained from the silica gel column were purified further by preparative HPLC on a C8 column using a H2O−MeOH gradient (70:30 to 100:0 for 20 min, with a flow rate of 7 mL/min) to give ent-subglain C (16, 12.0 mg),31 cleistodiendiol (1, 6.6 mg), Zmelodorinol (18, 12.4 mg),4,35 cleistodienol B (3, 14.7 mg), cleistenediol E (10, 8.8 mg), cleistanolate (13, 9.7 mg), cleistenediol C (8, 7.2 mg), cleistenediol A (6, 5.6 mg), and cleistenonal (12, 6.2 mg). Fraction 15 afforded (1S,4S,5S,6R)-5-[(benzyloxy)methyl]-5,6dihydroxycyclohex-2-ene-1,4-diyl diacetate (17, 14.1 mg),31 cleistenonal (12, 3.4 mg), tetramethylscutellarein (20, 6.4 mg),37 sootepenol (15, 8.6 mg), cleistenediol D (9, 10.9 mg), and cleistenediol B (7, 11.4 mg), following a similar chromatographic procedure to that described for fraction 14. Cleistodienediol, Z-[((2R,5S,6S)-2-acetoxy-5,6-dihydroxycyclohex3-en-1-ylidene)]methyl benzoate (1): white crystals (1:1 MeOH− CH2Cl2); mp 122−124 °C; [α]20D −128 (c 0.13, MeOH); HRESIMS [M + H]+ m/z 305.1069 (calcd for C16H16O6 305.1025); 1H and 13C NMR data, see Table 1. Cleistodienol A, Z-[2β,6α-diacetoxy-5β-hydroxycyclohex-3-en-1ylidene)]methyl benzoate (2): 4 colorless gum; [α]20D −73.6 (c 0.13,

(Table S6) and the cis axial−equatorial orientation of H-1 and H-2 (3JH1,H2 = 2.4 Hz) and of H-3 and H-4 (3JH3,H4 = 2.2 Hz). On the basis of the X-ray crystallographic structure of Zacetylmodorinol (19) (Figure 1), the absolute configuration of its stereogenic center and the geometry of its exocyclic double bond were confirmed. As expected, its NMR data differ from that reported for its transoid isomer, E-acetylmelodorinol,4 previously reported from C. kirkii. Owing to the bioactivity of previously reported polyoxygenated cyclohexene derivatives4,6,9,22,28,29,34 and heptenolides,4,33,35,36 the compounds isolated from C. kirkii were evaluated for activity against the parasite Plasmodium falciparum (3D7 and Dd2), the HEK-293 human embryonic kidney cell line, and the MDA-MB-231 triple-negative human breast cancer cell line. In addition, as part of an ongoing screening program for small-molecule inhibitors of eukaryotic protein synthesis, compounds were also tested in Krebs-2 in vitro translation extracts programmed with a bicistronic firefly-HCV IRESRenilla luciferase mRNA construct, to simultaneously monitor cap-dependent and cap-independent translation.46 In the initial screening for antiplasmodial activity, compounds 1−3 and 19 showed at least 90% activity at a 20 μM final concentration. Their effectiveness against the chloroquine-sensitive 3D7 and the chloroquine-resistant Dd2 clones of P. falciparum, as well as cytotoxicity against the HEK-293 mammalian cells, is given in Table 8. The compounds that possessed the best potencies against the malaria parasites were unfortunately nonselective (SI ratio 10% DMSO or 20 μM puromycin (100% inhibition) and 0.4% DMSO (no inhibition). All experiments were performed in triplicate, with n = 3. IC50 values were obtained by plotting the percent inhibition against log dose using the Prizm4 graphing package having nonlinear regression with variable slope plots. For compounds that showed appreciable antiplasmodial activity, their selectivity was computed with respect to an HEK/3D7 SI ratio in comparison to puromycin, a standard nonselective compound.47 Cytotoxic Activity against MDA-MB-231 Cells. The leaf methanol extract and its isolated constituents were evaluated against the MDA-MB-231, triple-negative, aggressive breast cancer cell line, as previously reported by Nyandoro et al.48 MDA-MB-231 human breast cancer cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM Lglutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in humidified 5% CO2. For cytotoxicity assays, cells were seeded in 96-well plates at optimal cell density (10 000 cells per well) to ensure exponential growth for the duration of the assay. After a 24 h preincubation growth, the medium was replaced with experimental medium containing the appropriate test compound concentrations (100−0.1 μg/mL) or vehicle controls (0.1% or 1.0% v/v DMSO). After 72 h of incubation, cell viability was measured using Alamar Blue reagent (Invitrogen Ab, Lidingö , Sweden) according to the manufacturer’s instructions. Absorbance was measured at 570 nm with 600 nm as a reference wavelength. Results were expressed as the mean ± standard error for six replicates as a percentage of vehicle control (taken as 100%). Experiments were performed independently at least six times. Statistical analyses were performed using a two-tailed Student’s t-test, with p < 0.05 considered to be statistically significant. Luciferase Inhibition Assay. Luciferase inhibition activity was performed following a previously established method.46 The compounds tested were first suspended at a concentration of 10 mM in DMSO and then diluted to 200 μM in water. Next, they were tested at a concentration of 20 μM in Krebs-II translation extracts

MeOH); ESIMS m/z 347.2 [M + H]+ (calcd C18H18O7, 347.11); 1H and 13C NMR data, see Tables S6 and S7, Supporting Information. Cleistodienol B, acetoxy(6α-acetoxy-5β-hydroxycyclohexa-1,3dien-1-yl)methyl benzoate (3): white solid; [α]20D +139.4 (c 0.06, MeOH); HRESIMS [M + H]+ m/z 347.1118 (calcd for C18H18O7, 347.1131); 1H and 13C NMR data, see Table 1. Cleistenechlorohydrin A, (5β-acetoxy-2α-chloro-1α,6αdihydroxycyclohex-3-en-1-yl)methyl benzoate (4): pinkish gum; [α]20D +286.7 (c 0.075 MeOH); HRESIMS [M + H]+ m/z 341.0813 (calcd for C17H17O6Cl, 341.0792); 1H and 13C NMR data, see Table 2. Cleistenechlorohydrin B, (5β-acetoxy-2α-chloro-1β,6αdihydroxycyclohex-3-en-1-yl)methyl benzoate (5): colorless gum; [α]20D +92.1 (c 0.33, MeOH); HRESIMS [M + H]+ m/z 341.0774 (calcd for C17H17O6Cl, 341.0792); 1H and 13C NMR data, see Table 2. Cleistenediol A, (5β-acetoxy-1,6β-dihydroxy-2α-methoxycyclohex-3-en-1-yl)methyl benzoate (6): colorless gum; [α]20D +190.0 (c 0.25, MeOH); HRESIMS [M + H]+ m/z 337.1289 (calcd for C17H20O7, 337.1287); 1H and 13C NMR data see Table 3. Cleistenediol B, (6β-acetoxy-1β-5α-dihydroxy-2β-methoxycyclohex-3-en-1-yl)methyl benzoate (7): colorless gum; [α]20D +120.0 (c 0.05, MeOH); HRESIMS [M + H]+ m/z 337.1288 [M + H]+ (calcd for C17H20O7, 337.1287); 1H and 13C NMR data, see Table 3. Cleistenediol C, 6α-[(benzoyloxy)methyl]-5β,6β-dihydroxycyclohex-3-ene-1,2-diyl diacetate (8): colorless gum; [α]20D +19.7 (c 0.06, MeOH); HRESIMS [M + H]+ m/z 365.1235 (calcd for C18H20O8, 365.1236); 1H and 13C NMR data, see Table 4. Cleistenediol D, 5α-[(benzoyloxy)methyl]-5β,6α-dihydroxycyclohex-2-ene-1,4-diyl diacetate (9): colorless gum; [α]20D +33.8 (c 0.26, MeOH); HRESIMS [M + H]+ m/z 365.1223 (calcd for C18H20O8, 365.1236); 1H and 13C NMR spectra data, see Table 4. Cleistenediol E, (5α-acetoxy-2β-(benzoyloxy)-1α,6β-dihydroxycyclohex-3-en-1-yl)methyl benzoate (10): colorless gum; [α]20D +228.2 (c 0.06, MeOH); HRESIMS [M + H]+ m/z 427.1390 (calcd for C23H22O8, 427.1393); 1H and 13C NMR data, see Table 5. Cleistenediol F, 5β-(benzoyloxy)-6β-[(benzoyloxy)methyl]-6αhydroxycyclohex-3-ene-1,2-diyl diacetate (11): colorless gum; [α]20D +4.2 (c 0.02, MeOH); HRESIMS [M + H]+ m/z 469.1502 (calcd for C25H24O9, 469.1498); 1H and 13C NMR data, see Table 5. Cleistenonal, acetoxy(10α-acetoxy-7-formyl-9β-hydroxy-1β,4βethanonaphthalen-1(4H)-yl)methyl benzoate (12): white solid; [α]20D −44 (c 0.025, MeOH); HRESIMS [M + H]+ m/z 451.1407 (calcd for C25H22O8, 451.1393); 1H and 13C NMR data, see Table 6. Cleistanolate, 3-(benzoyloxy)-1-(3β-methoxy-5-oxotetrahydrofuran-2β-yl)propane-1,2-diyl diacetate (13): colorless gum; [α]20D −9.7 (c 0.35, MeOH); HRESIMS [M + H]+ m/z 395.1327 (calcd for C19H22O9, 395.1342); 1H and 13C NMR data, see Table 7. Cleistophenolide, 2,5-dihydroxybenzyl benzoate (14): colorless gum; HRESIMS [M + H]+ m/z 245.0788 (calcd for C14H12O4, 245.0814); 1H and 13C NMR data, see Table 7. Sootepenol B (15): 34 colorless gum; [α]20D +14.2 (c 0.01, MeOH); ESIMS m/z 365.6 [M + H]+ (calcd C18H20O8, 365.3); 1H and 13C NMR data, see Table S5, Supporting Information. ent-Subglain C (16): 29 colorless gum; [α]20D +89.1 (c 0.22, MeOH); ESIMS m/z 365.6 [M + H]+ (calcd C18H20O8, 365.12); 1H and 13C NMR data, see Tables S6 and S7, Supporting Information. (1S,4S,5S,6R)-5-[(benzyloxy)methyl]-5,6-dihydroxycyclohex-2ene-1,4-diyl diacetate (17): 31 white solid; [α]20D +12.6 (c 0.02, MeOH); ESIMS m/z 365.6 [M + H]+ (calcd C18H20O8, 365.12); 1H and 13C NMR data, see Tables S6 and S7, Supporting Information. Z-Melodorinol (18):.4,34 white solid; [α]20D −5.0 (c 0.01, MeOH); ESIMS m/z 283.6 [M + Na]+ (calcd C14H12O5, 283.07); 1H and 13C NMR data, see Tables S8 and S9, Supporting Information. Z-Acetylmelodorinol (19):.4,35 colorless gum; [α]20D +2.5 (c 0.02, MeOH); ESIMS m/z 303.5 [M + H]+ (calcd C16H14O6, 303.08); 1H and 13C NMR data, see Tables S8 and S9, Supporting Information. Tetramethylscutellarein (20): 42 white solid; ESIMS m/z 343.2 [M + H]+ (calcd C19H18O6, 343.11); 1H and 13C NMR data, see Tables S8 and S9, Supporting Information. 123



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programed with a bicistronic Firefly-HCV IRES-Renilla mRNA construct that monitors cap-dependent translation (firefly levels, FF) as well as cap-independent translation through the HCV IRES (Renilla levels, Ren). The translation reaction was incubated at 30 °C for 60 min, and then the luciferase activities were measured. Compounds that inhibit only FF would be considered cap-dependent translation inhibitors, and compounds that inhibit expression of Ren would only be inhibitors of HCV IRES translation, while compounds that inhibit both FF and Ren would likely be translation elongation inhibitors. None of the compounds was observed to display significant inhibition of translation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00759. NMR and MS spectra (PDF) X-ray crystallography data (CIF) (CIF) (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +255-754-206560. E-mail: [email protected] (S. S. Nyandoro). *Tel: +46-31-786-9033. E-mail: [email protected] (M. Erdélyi). ORCID

Kari Rissanen: 0000-0002-7282-8419 Máté Erdélyi: 0000-0003-0359-5970 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Swedish Research Council (Swedish Research Links, 20126074), the Academy of Finland (KR, grant nos. 263256, 265328, 292746 and 298817), and the Australian Research Council (VMA, grant LP120200557) are gratefully acknowledged for financial support. S.S.N. is thankful to the Swedish Institute for a postdoctoral research award (00045/2014). We thank Mr. S. A. Yahaya in collaboration with Mr. F. M. Mbago, a curator at the Herbarium of the Department of Botany, University of Dar es Salaam, for locating and identifying the investigated plant species, and Dr. U. Brath (University of Gothenburg) for checking through the NMR assignments. We thank the Australian Red Cross Blood Service for the provision of human blood.

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REFERENCES

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