Antiprotozoal Isoprenoids from Salvia hydrangea - Journal of Natural

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Antiprotozoal Isoprenoids from Salvia hydrangea Marzieh Tabefam,†,‡ Mahdi Moridi Farimani,*,† Ombeline Danton,‡ Justine Ramseyer,‡ Samad Nejad Ebrahimi,† Markus Neuburger,§ Marcel Kaiser,⊥,∥ Peyman Salehi,† Olivier Potterat,‡ and Matthias Hamburger*,‡

J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 12/19/18. For personal use only.

†

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran ‡ Department of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland § Inorganic Chemistry, Department of Chemistry, University of Basel, Mattenstrasse 24a, 4058 Basel, Switzerland ⊥ Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland ∥ University of Basel, 4001 Basel, Switzerland S Supporting Information *

ABSTRACT: Fractionation of the n-hexane extract of Salvia hydrangea afforded seven isoprenoids including six new compounds (1−6) and salvadione A (7). Their structures were established by comprehensive spectroscopic and spectrometric data analysis (1D and 2D NMR, HRMS). The absolute configuration of salvadione A (7) was established by single-crystal X-ray diffraction analysis with Cu/Kα radiation. In addition, the absolute configuration of all compounds was determined by electronic circular dichroism spectroscopy. A biosynthetic pathway for the formation of the scaffold of 1 is proposed. The antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum was determined, and cytotoxicity was assessed in rat myoblast L6 cells. Perovskone C (2) exhibited good activity against P. falciparum (IC50 0.6 μM) and a selectivity index of 62.2.

P

was done, and herein the isolation and structure elucidation of six new isoprenoids (1−6) along with known salvadione A (7) and their in vitro antiprotozoal activity are reported.

arasitic diseases are among the most significant human health burdens, especially in developing countries. Protozoal diseases such as malaria, trypanosomiasis, and leishmaniasis have disability adjusted life years (DALYs) in the millions in tropical and subtropical regions of the world.1,2 Owing to the emergence of parasite resistance to nearly all available drugs, there is a need for the identification of new lead compounds and the development of drugs with novel modes of action. With more than 1000 species, the genus Salvia is the largest genus of the family Lamiaceae. Of the 61 Salvia species growing in Iran, 17 are endemic.3 Several Iranian Salvia, such as S. urmiensis, S. reuterana, S. lachnocalyx, and S. sahendica, have been investigated from a phytochemical and pharmacological perspective, and a broad range of isoprenoids with antitrypanosomal, antiplasmodial, antimicrobial, apoptosisinducing, and cytotoxic activities were reported.4−9 S. hydrangea DC. ex Benth. (Persian name “Gol-e Arooneh”) is a well-known medicinal plant that grows widely in Iran, Anatolia, and Transcaucasia. The aerial parts of the plant are used as an anthelmintic and antileishmanial remedy. In the traditional medicine of Pars Province infusions of the flowers are prepared to treat colds. We previously reported on several new antiplasmodial isoprenoids with unique scaffolds that were isolated from the aerial parts.10,11 For the characterization of minor isoprenoids, a large-scale re-collection of the aerial parts © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Isolation and Structure Elucidation. The aerial parts of S. hydrangea were extracted with n-hexane. The resulting extract showed an IC50 of 3.2 μg/mL against Plasmodium falciparum. Fractionation by a combination of open-column chromatography on silica gel and preparative and semipreparative RP-HPLC afforded six new isoprenoids (1−6) and salvadione A (7).12 Their structures were established on the basis of extensive spectroscopic analysis, including 1D and 2D NMR, X-ray crystallographic analysis, and electronic circular dichroism (ECD), and by comparison with literature data. Salvadione A (7) had been previously reported from Salvia bucharica.12 Its scaffold and relative configuration were at that time proposed on the basis of NMR data and single-crystal Xray diffraction using Mo Kα radiation. Comparing the specific rotation and NMR spectroscopic data of 7 with those of the original compound indicated that they had to be identical. XReceived: June 29, 2018

A

DOI: 10.1021/acs.jnatprod.8b00498 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

ECD spectra. In the experimental ECD, three positive Cotton effects (CEs) were observed at 339, 241, and 202 nm along with a negative CEs at 300 nm, corresponding to π → π* and n → π* transitions of double bonds and carbonyl groups, respectively. The calculated ECD spectrum showed three positive CEs at 339, 250, and 200 nm and a negative CE at 302 nm, respectively (Figure 2). Thus, the absolute configuration of salvadione A was defined for the first time.

Figure 2. Comparison of experimental and calculated ECD spectra of compound 7. TDDFT at the level of cam-B3LYP/6-31G** theory in MeOH was used.

Compound 1 possessed a molecular formula of C30H42O5 (HRAPCIMS m/z 483.3108 [M + H]+; calcd for C30H43O5+, 483.3105), accounting for 10 indices of hydrogen deficiency. The 13C NMR spectrum showed 30 carbon resonances, which were assigned to seven methyls, eight methylenes, four methines, five quaternary carbons, four oxygenated tertiary carbons, and two carbonyl carbons (Table 1). Thus, 41 hydrogen atoms could be accounted for, while the remaining one was assigned to a hydroxy group. The 13C NMR spectrum displayed resonances for an α,β-unsaturated carbonyl moiety (δC 201.8, 171.5, 121.6) bearing a β-oxygen substituent, a ketocarbonyl group (δC 210.1), and four oxygen-bearing sp3 carbons (δC 93.9, 89.8, 89.7, 82.9). The absence of other sp or sp2 carbon signals and the 10 indices of hydrogen deficiency indicated that 1 contained seven rings. Taking into account the number of oxygenated carbons and given that the compound contained only five oxygens, two rings had to be cyclic ethers. The 1H NMR spectrum showed resonances for five methyl singlets at δH 0.77, 0.80, 1.39, 1.61, and 2.31, respectively. Two additional methyl doublets at δH 1.13 (d, J = 7.1 Hz) and 1.16 (d, J = 7.1 Hz), along with a signal at δH 3.16 (sept, J = 7.1 Hz), indicated the presence of an isopropyl moiety. These structural features were reminiscent of hydrangenone, an isoprenoid with a unique arrangement of 6/7/6/5/5membered carbon rings fused with two tetrahydrofuran rings that had been recently reported from S. hydrangea.11 However, notable differences in the 1H and 13C NMR spectra of 1 and hydrangenone were observed, such as the resonance of an oxygenated methine [δH 3.80 (1H, dd, J = 12.2, 5.7 Hz), δC 82.9] replacing the methine group at C-22 in hydrangenone. The chemical shift of C-23 in 1 was observed at δC 69.7 and thus appeared upfield shifted by ca. 18 ppm. A COSY

ray analysis of the newly isolated material utilizing Cu Kα radiation established the absolute configuration of 7 as (5S,8R,9S,10S,11R,13R,24R,25R) (Figure 1), which is enantiomeric to the reported structure. 12 This finding was corroborated by comparison of the experimental and timedependent density functional theory (TDDFT)-calculated

Figure 1. Single-crystal X-ray structure of salvadione A (7). B



Article

Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−3 (CDCl3; 500 MHz for 1H and 125 MHz for 13C NMR; δ in ppm) 1

2

3

δH (J in Hz)

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

1

1.56a

42.7, CH2

42.3, CH2

1.42a 1.71a 1.14a 1.34a

19.5, CH2

1.70a 2.05, br d (11.6) 1.50a 1.76a 1.20, ddd (13.1, 13.1, 3.1) 1.39a

42.1, CH2

2

1.67a 1.96a 1.48a 1.75a 1.19, ddd (13.1, 13.1, 3.1) 1.39a

position

3 4 5 6

0.94, dd (12.2, 4.3) 1.11a 1.65a 1.54a 2.32a

7 8 9 10 11 12 13 14 15 16 17 18 19 20 20 21 21 22 23 24 24 25 25 26 27 28 29 30

α β α β

α β α β

3.16, sept (7.1) 1.16, d (7.1) 1.13, d (7.1) 0.80, s 0.77, s 2.61, d (13.5) 1.52a 2.34a 2.55, dd (12.2, 5.8) 3.80, dd (12.2, 5.8) 2.84, dd (14.3, 7.3) 1.52a 2.48, dd (12.2, 7.3)

1.61, s 1.39, s 2.31, s

42.2, CH2 34.1, C 52.6, CH 22.3, CH2 34.5, CH2 53.5, C 65.1, C 89.7, C 93.9, C 171.5, C 121.6, C 201.8, C 24.2, CH 20.3, CH3 19.8, CH3 31.8, CH3 21.1, CH3 48.8, CH2 50.5, CH2

89.8, C 26.7, CH3 24.2, CH3 210.1, C 31.8, CH3

41.7, CH2 33.8, C 54.7, CH 21.4, CH2

1.01, br d (10.7) 1.43a 1.54a 1.38a 2.17a

42.2, CH2 50.5, C 57.3, C 90.0, C 96.9, C 167.3, C 123.4, C 199.0, C 24.5, CH 20.4, CH3 19.9, CH3 32.0, CH3 22.1, CH3 46.4, CH2

3.04, sept (7.0) 1.08, d (7.0) 0.96, d (7.0) 0.87, s 0.84, s 2.46, d (13.7) 1.96a 2.14a 3.15, d (17.1)

46.4, CH2

82.9, CH 69.7, C 38.5, CH2 57.5, CH

19.4, CH2

1.69, s 1.40, s 1.63, s

41.7, CH2 33.8, C 54.7, CH 21.4, CH2

1.03a 1.48a 1.57a 1.42a 2.16a

41.9, CH2

3.02, sept (7.0) 1.07, d (7.0) 0.95, d (7.0) 0.89, s 0.85, s 2.54, d (13.7) 2.39, d (13.7) 2.19, d (16.8) 3.14, d (16.8)

195.8, C 132.4, C 160.1, C 2.82, dd (19.5, 11.9) 2.46a 2.68, d (11.9, 6.7)

19.3, CH2

50.9, C 56.5, C 94.2, C 94.4, C 167.0, C 123.3, C 198.8, C 24.4, CH 20.4, CH3 19.9, CH3 32.0, CH3 22.0, CH3 46.8, CH2 46.4, CH2 196.8, C 135.1, C 158.1, C

29.2, CH2

4.70, d (4.3)

72.0, CH

52.6, CH 90.4, C 29.1, CH3 24.6, CH3 10.7, CH3

2.67, d (4.3)

63.4, CH 89.8, C 29.2, CH3 24.3, CH3 10.2, CH3

1.74, s 1.53, s 1.77, s

a

Overlapping signals.

configuration showed a good fit with the experimental data (Figure 5). Compound 1 was named hydrangenone B. The biosynthesis of hydrangenone B (1) can be rationalized as shown in Scheme 1. The skeleton of the compound is presumably formed via a [3+2] cycloaddition-type reaction of a putative icetexone moiety (B)13−15 and a branched C10 unit (A)16 to form the five-membered ring D, followed by an intramolecular ene-type reaction and successive formation of the tetrahydrofuran rings, similar to hydrangenone.11 A molecular formula of C30H40O4 was assigned to compound 2 on the basis of a molecular ion peak (m/z 465.3000 [M + H]+, calcd for C30H41O4+, 465.2999) in the HRAPCIMS, indicating 11 indices of hydrogen deficiency. The 13 C NMR spectrum showed 30 carbon resonances, which were assigned to seven methyls, eight methylenes, three methines, six quaternary carbons, four oxygenated tertiary carbons, and two carbonyl carbons. Thus, 40 hydrogens were accounted for,

correlation between H2-21 (δH 2.34, 2.55) and H-22 (δH 3.80) and HMBC correlations from H-30 (δH 2.31), H2-24 (δH 1.52, 2.84), and Hβ-21 (δH 2.55) to C-23 (δC 69.7) and from H-22 and H2-24 to C-29 (δC 210.1) (Figure 3) confirmed that the positions of the hydroxy and acetyl groups were inverted compared to hydrangenone. The relative configuration was deduced from a NOESY spectrum. NOESY correlations of H318/H-5 and H-5/Hα-20 corroborated the linkage of rings A and B. Diagnostic cross-peaks between Hβ-20/H-25 and H220/H3-30 confirmed their cofacial orientation. In addition, cross-peaks between Hβ-24/H-22, H-22/Hβ-21, and H3-17/H22 indicated the α-orientation of the hydroxy group at C-22 (Figure 3). The experimental ECD spectrum of 1 showed two sequential negative CEs at 272 and 240 nm, along with positive CEs at 315 and 211 nm. The overall pattern of the calculated ECD spectrum for the (5S,8R,9R,10S,11R,22R,23S,25S) C



Article

sp2 carbon signals and the 11 indices of hydrogen deficiency implied that the compound contained seven rings. Three oxygenated tertiary carbons at δC 96.9, 90.4, and 90.0, respectively, suggested the presence of two cyclic ethers. The 1 H NMR data (Table 1) exhibited three signals [δH 3.04 (1H, sept, J = 7.0 Hz, H-15), 1.08 (3H, d, J = 7.0 Hz, H-16), 0.96 (3H, d, J = 7.0 Hz, H-17)] of an isopropyl group. 1D and 2D NMR data suggested that compound 2 had the same scaffold as perovskone and perovskone B which had been reported from Perovskia abrotanoides and S. hydrangea.10,14 Compared to perovskone B, NMR resonances of the C-24 aliphatic and C-22 olefinic methine signals were absent in 2, but the spectra indicated the presence of an additional methylene (δH 3.15, 2.14, δC 46.4) and an olefinic quaternary carbon (δC 160.1) instead. HMBC correlations from H3-30 (δH 1.63) to C-23 (δC 132.4), C-24 (δC 160.1), C-22 (δC 195.8), and C-21 (δC 46.4), from Hβ-21 (δH 3.15) to C-22, and from H2-25 (δH 2.46, 2.82) and H-26 (δH 2.68) to C-24 (Figure 3) showed that the only difference between perovskone B and compound 2 was in the position of the α,β-unsaturated carbonyl group in ring D. The relative configuration of 2 was determined from the NOESY spectrum. Diagnostic cross-peaks between H3-18/H-5, H-5/ Hα-20, Hβ-20/H-26, and H-26/Hα-25 confirmed their cofacial orientation (Figure 3). The absolute configuration of 2 was determined by ECD (Figure 5). The experimental spectrum showed two positive CEs at 320 and 248 nm and a negative CE at 276 nm. The calculated ECD spectrum for the (5S,8R,9S,10S,11R,26R) stereoisomer (positive CEs at 310 and 240 nm and a negative CE at 276 nm) was in good agreement. Compound 2 was named perovkone C. A molecular formula of C30H40O5 (HRAPCIMS m/z 481.2950 [M + H]+; calcd for C30H41O5+, 481.2949) was assigned to compound 3. The NMR data of 3 (Table 1) were similar to those of 2. The notable difference was in the presence of an oxygenated methine (δH 4.70, δC 72.0) instead of a methylene group, suggesting that compound 3 was a oxygenated analogue of 2. Key HMBC correlation from H-26 (δH 2.67) to the signal at δC 72.0 and a COSY correlation of H-26 with a proton signal at δH 4.70 indicated that the oxygenated methine was at C-25 (Figure 3). A small coupling constant between H-25 and H-26 (J = 2.7 Hz) corresponded to a dihedral angle of ca. 60° and indicated a β-orientation of the hydroxy group. This was also supported by NOESY correlations between H-26/H-25, H-25/H3-29, and H-25/H330 (Figure 3). In the experimental ECD spectrum of 3, two positive CEs at 321 and 244 nm along with a negative CE at 275 resulted from π → π* transitions of α,β-unsaturated carbonyl moieties (Figure 5). The data were in good agreement with the calculated ECD spectrum for the (5S,8R,9S,10S,11R,25R,26R) stereoisomer (positive CEs at 315 and 250 and a negative CE at 275 nm). Compound 3 was named perovskone D. Compound 4 had a molecular formula of C30H40 O5 (HRAPCIMS m/z 481.2947 [M + H]+; calcd for C30H41O5+, 481.2949). Analysis of the NMR data (Table 2) confirmed the same carbon skeleton as for 2. Diagnostic differences included the presence of two oxygen-bearing tertiary carbons resonating at δC 94.2 and 64.6 and the absence of a Δ12(13) double bond. In comparison to compound 2, the molecular formula of 4 contained an extra oxygen atom. Given that 11 indices of hydrogen deficiency were needed, compound 4 had to contain an epoxy moiety. Key HMBC correlations from H-15 (δH 2.21), H-16 (δH 1.10), and H-17 (δH 0.80) to C-13 (δC 64.6)

Figure 3. (A) Key HMBC (red arrows), COSY (green bonds), and (B) NOESY correlations for compounds 1−3 and 5.

Figure 4. Key HMBC correlations for compounds 4 and 6.

and the molecule did not contain free hydroxy groups. Two series of signals [δC 199.0 (C), 167.3 (C), 123.4 (C) and 195.8 (C), 160.1 (C), 132.4 (C)] indicated the presence of two α,βunsaturated carbonyl groups. The absence of additional sp and D



Article

Figure 5. Comparison of experimental and calculated ECD spectra of compounds 1−6. TDDFT at the level of cam-B3LYP/6-31G** theory in MeOH was used.

(12S,13R and 12R,13S) were calculated and compared with the experimental spectrum of 4. The experimental ECD showed positive CEs at 326 and 260 and two negative CEs at 221 and 203 nm, which resulted from the π → π* and n → π* transitions of the α,β-unsaturated carbonyl and carbonyl group, respectively (Figure 5) and showed an excellent match with the calculated spectrum for the (12R,13S) stereoisomer

and from H-26 (δH 2.75) to C-12 (δC 94.2, C) located the epoxy ring at C-12 and C-13 (Figure 4). Key NOESY correlations of H3-18/H-5, H-5/Hα-20, Hβ-20/H-26, H-26/ Hα-25, and Hα-25/H3-30 indicated that the configuration of 4 was the same as for 2 (Figure 4). However, the configuration of the epoxy ring at C-12 and C-13 could not be determined at this stage. ECD spectra of the two possible stereoisomers E



Article

Scheme 1. Proposed Biosynthetic Pathway of Compound 1

These observations suggested an α-orientation of the hydroxy group at C-22 and of the epoxy ring at C-23 and C-24. Moreover, NOESY correlations of H-26 with Hβ-20 and H3-28 indicated their location on the same face of the molecule. NOESY cross-peaks of H-5/H3-18 and H-5/Hα-20 confirmed the linkage of rings A and B (Figure 3). The absolute configuration of perovskone F (5) was established as (5S,8R,9R,10S,11R,22R,23R,24S,26R) on the basis of a good match of the experimental and calculated ECD spectra (Figure 5). Compound 6 had a molecular formula of C30H38 O4 (HRAPCIMS m/z 463.2845 [M + H]+; calcd for C30H39O4+, 463.2843), which differed by 18 amu from that of salvadione C, previously reported from this plant.10 Notable differences in the 1H and 13C NMR spectra of compound 6 (Table 2) and salvadione C were observed, such as the absence of the C-28 methylene group in 6. The presence of an olefinic proton (δH 6.11, δC 140.6) suggested that 6 was a dehydration product of salvadione C. HMBC correlations from H-26 (δH 2.36) and H3-29 (δH 1.68) to both olefinic carbons C-27 (δC 120.7) and C-28 (δC 140.6), and between H-25 (δH 2.92) and C-27 confirmed the position of the extra double bond (Figure 4). The relative configuration of 6 was deduced from the NOESY spectrum and was in agreement with that of salvadione C,10 and the absolute configuration of salvadione D (6) was established via ECD data (Figure 5) as (5S,8R,9S,10S,11R,13R,25R,26S). Antiprotozoal Activity. Compounds 1−7 were tested for their in vitro inhibitory activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, and Leishmania donovani. Cytotoxicity was determined in rat skeletal myoblast L6 cells (Table 3). Among the parasites tested, P. falciparum was generally found to be the most sensitive to the test compounds, with IC50 values ranging from 0.6 (2) to 7.9 μM and selectivity indices (SI) between 1.8 (3) and 62.2 (2).

(positive CEs at 325 and 255 nm and negative CEs at 235 and 202 nm). Thus, the absolute configuration of perovskone E (4) was established as (5S,8R,9S,10S,11R,12R,13S,26R). The HRAPCIMS of compound 5 showed an [M + H]+ ion at m/z 483.3108 (calcd for C30H43O5+, 483.3105). In combination with the 13C NMR data, the molecular formula was established as C30 H42 O 5. The molecular formula accounted for 10 indices of hydrogen deficiency, and seven methyls, eight methylenes, four methines, four quaternary carbons, six oxygenated tertiary carbons, and one carbonyl carbon were identified from the NMR data (Table 2). Thus, 41 hydrogen atoms could be accounted for, with the remaining one belonging to a hydroxy group. Resonances at δC 200.4 (C),168.6 (C), and 122.9 (C) indicated the presence of an α, β-unsaturated carbonyl with an oxygen substituent at the βposition. Five oxygen-bearing tertiary carbons appeared at δC 95.4, 90.4, 89.3, 73.1, and 64.7, together with an oxygenated methine (δH 3.43 and δC 68.1). The absence of other sp or sp2 carbon signals and the 10 indices of hydrogen deficiency implied that 5 contained eight rings including three cyclic ethers moieties. Comparison of the NMR data of 5 with those of 2 revealed structural similarities, but also some notable differences. Compound 5 was found to lack the α,βunsaturated carbonyl group in ring D, but to contain an oxygenated methine and an epoxy unit instead. Key HMBC correlations from H2-25 (δH 1.98, 2.00) to C-24 (δC 73.1) and C-23 (δC 64.7), from H3-30 (δH1.25) to C-23, and from Hβ-20 (δH 2.1) to C-24 located the epoxy ring at C-23 and C-24. A COSY correlation of Hα-21 (δH 2.14) and the signal at δH 3.43, and HMBC correlations from Hα-21 (δH 2.14) and H3-30 to the signal at δC 68.1 confirmed the position of the hydroxy group at C-22 (Figure 3). The relative configuration was determined from the NOESY spectrum. Diagnostic cross-peaks of H3-29/Hβ-25, Hβ-25/H3-30, H3-30/H-22, and H-22 with H-15, H3-16, and H3-17 confirmed their cofacial orientation. F



Article

Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 4−6 (CDCl3; 500 MHz for 1H and 125 MHz for 13C NMR; δ in ppm) 4 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20α 20β 21 22 23 24 25α 25β 26 27 28 29 30

5

δH (J in Hz)

δC

1.63a 1.91a 1.54a 1.94a 1.22a 1.44, m

42.2, CH2

b

19.7, CH2 41.7, CH2 34.3, C 54.3, CH 20.5, CH2

1.14, m 1.58a 2.06, m 1.21a 2.41


2.21, sept (7.0) 1.10, d (7.0) 0.80, d (7.0) 0.89, s 1.07, s 2.38, d (13.7) 1.89, d (13.7) 2.34a 2.91, d (16.8)

43.6, CH2 195.7, C 132.4, C 159.1, C 30.0, CH2

2.86, dd (19.3, 11.0) 2.62, dd (19.3, 7.6) 2.75, dd (11.0, 7.6)

54.2, CH 83.1, C 29.0, CH3 25.1, CH3 10.6, CH3

1.68, s 1.35, s 1.63, s

6

δH (J in Hz)

δC

δH (J in Hz)

δC

1.67a 1.95, m 1.47a 1.73a 1.18a 1.37a

42.2, CH2

1.46a 2.06a 1.49a 2.05a 1.18a 1.43a

41.7, CH2

19.4, CH2 41.8, CH2

0.93, dd (11.5, 3.5) 1.34a 1.51a 1.27a 2.08a

3.13, sept (7.0) 1.15, d (7.0) 1.08, d (7.0) 0.85, s 0.80, s 2.30, d (14.0) 2.10, d (14.0) 1.25a 2.14, dd (12.5, 5.2) 3.43, dd (11.5, 5.2)

1.98, m 2.00, m 2.66, dd (10.5, 9.5) 1.67, s 1.40, s 1.25, s

33.7, C 53.8, CH 21.5, CH2

39.5, CH2 68.1, CH 64.7, C 73.1, C 27.9, CH2 52.0, CH 90.4 28.5, CH3 24.5, CH3 16.1, CH3

41.2, CH2 35.9, C 50.0, CH 20.6, CH2

1.31a 1.89a 1.94a 1.28a 1.90a


19.6, CH2


2.16, sept (6.8) 1.21, d (6.8) 1.08, d (6.8) 0.89, s 1.01, s 2.03a 5.07, d (11.0) 5.25, d (17.7) 6.30, dd (17.6, 10.8)

113.2, CH2 137.8, CH 135.7, C 128.6, CH 44.7, CH

5.68, d (6.4) 2.92, d (6.1) 2.36, s

47.4, CH 120.7, C 140.6, CH 18.9, CH3 27.3, CH2

6.11, m 1.68, d (1.3) 2.50, d (16.8) 2.60, d (16.8)

a

Overlapping signals. bExtracted from H−C 2D inverse-detected experiments due to a low amount of sample.

Table 3. In Vitro Activity of Compounds 1−7 against T. b. rhodesiense (STIB900), T. cruzi (Tulahuen C4 LacZ), L. donovani (MHOM-ET-67/L82), and P. falciparum (NF54) and Cytotoxicity in L6 Cells compound 1 2 3 4 5 6 7 positive controls

T.b. rhodesiense IC50 (μM)a 85.8 56.0 45.3 30.5 74.6 24.6 66.9

(92.9, 78.8); (65.7, 46.3); (47.5, 43.1); (33.9, 27.1); (84.6, 64.7); (28.8, 20.4); (86.4, 47.4); 0.04c

b

0.5 0.7b 0.3b 2.4b 0.2b 1.7b 0.7b

T. cruzi IC50 (μM)a

L. donovani IC50 (μM)a

b

b

33.9 (33.5, 34.3);1.2 3.5 (1.5, 1.2, 7.7); 10.7b 3.8 (2.0, 2.1, 7.3); 3.6b 11.5 (16.4, 6.6); 6.3b 19.8 (25.7, 14.0); 2.4b 47.9 (52.5, 43.3); 0.9b 35.8 (40.9, 30.7); 1.3b 5.1d

12.3 (11.3, 13.3); 3.3 15.5 (14.5, 16.4); 2.4b 7.5 (4.9, 10.0);1.8b 12.3 (12.7, 11.8); 5.9b 44.1 (35.7, 52.5); 1.1b 15.6 (13.0, 18.2); 2.7b 21.3 (16.6, 26.0); 2.2b 0.9e

P. falciparum IC50 (μM)a b

7.9 (8.8, 6.9); 5.2 0.6 (0.6, 0.6); 62.2b 7.5 (7.6, 7.4); 1.8b 1.3 (1.0, 1.5); 56.9b 3.6 (2.8, 4.5); 12.9b 1.9 (1.9, 1.9); 22b 1.1 (1.1,1.1); 44.2b 0.01f 0.008h

L6 cells IC50 (μM)a 41.1 37.2 13.6 72.2 46.9 41.6 47.2

(42.2, 40.1) (38.1, 36.3) (14.1, 13.1) (77.6, 66.8) (49.4, 44.4) (42.3, 41.0) (49.1, 45.3) 0.009g

a Values are expressed in μM. Each value corresponds to the mean of at least two independent assays, with the individual values indicated in brackets. bSelectivity index (SI): IC50 in L6 cells divided by IC50 in the title parasitic strain. cMelarsoprol. dBenznidazole. eMiltefosine. f Chloroquine. gPhodophyllotoxin. hArtemisinin.

G



Article

acetone (100:0 to 10:90) as mobile phase. On the basis of TLC analysis, fractions with similar composition were combined to yield 25 main fractions (F1 to F25). A portion (12.0 g) of fraction F2 (40.0 g) was separated on a silica gel column (60 × 4 cm, 70−230 mesh) with CHCl3−n-hexane (25:75) to afford seven fractions (F2a−F2g). Fraction F2b (4.0 g) was further purified by silica gel column chromatography (60 × 3.5 cm, 70−230 mesh) with CHCl3−n-hexane (55:45) to afford seven subfractions (F2b‑1 to F2b‑7). F2b‑1 (760 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 80% → 97% B (0−15 min), 97% B (15−35 min); flow rate 20 mL/min; sample concentration 100 mg/mL in DMSO; injection volume 1 mL] to yield compounds 1 (42 mg, tR 14.8 min) and 2 (6 mg, tR 17.6 min). F2b‑3 (235 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 80% → 95% B (0−30 min); flow rate 20 mL/min; sample concentration 150 mg/mL in DMSO; injection volume 1 mL] to afford compounds 6 (2 mg, tR 16.3 min) and 7 (28 mg, tR 20.6 min). F2b‑7 (358 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 80% → 90% B (0−10 min), 97% B (10−20 min); flow rate 20 mL/min; sample concentration 100 mg/mL in THF; injection volume 300 μL] to afford compound 4 (0.3 mg, tR 13.1 min). Fraction F9 (3.2 g) was separated on a silica gel column with CHCl3−EtOAc (99:1) to afford 11 subfractions (F9a−F9k). F9k (242 mg) was purified by preparative RP-HPLC [H2O (A), MeCN (B); 60% → 80% B (0−15 min), 80% B (15−35 min), 100% B (35−45 min); flow rate 20 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 1 mL] to yield subfractions F9k‑1 and F9k‑2. F9k‑1 (8 mg) was further purified by semipreparative RP-HPLC [H2O (A), MeOH (B); 72% → 81% B (0−30 min), 100% B (30−40 min); flow rate 4 mL/min; sample concentration 40 mg/mL in DMSO; injection volume 90 μL] to afford compound 3 (2 mg, tR 30.6 min). F9k‑2 (15 mg) was further purified by semipreparative RP-HPLC [H2O (A), MeOH (B); 50% → 70% B (0−20 min), 70% → 72% B (20−50 min); flow rate 4 mL/min; sample concentration 75 mg/mL in DMSO; injection volume 50 μL] to afford compounds 3 (1 mg, tR 32.7 min) and 5 (2 mg, tR 36.1 min). Fraction 12 (50 mg) was recrystallized from CHCl3−MeOH to yield salvigenin (5 mg). Hydrangenone B (1): white, amorphous powder; [α]25D +74 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 272 (4.05) nm; ECD (MeOH, c 4.1 × 10−4 M) [θ]211 + 7109, [θ]240 −37 295, [θ]257 −27 777, [θ]272 −42 160, [θ]315 +38 944; 1H and 13C NMR, see Table 1; HRAPCIMS m/z 483.3108 [M + H]+ (calcd for C30H43O5+, 483.3105). Perovskone C (2): colorless oil; [α]25D +69 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 252 (3.76), 271 (3.79) nm; ECD (MeOH, c 4.3 × 10−4 M) [θ]248 +10 543, [θ]276 −35 154, [θ]320 +23 838; 1H and 13 C NMR, see Table 1; HRAPCIMS m/z 465.3 [M + H]+ (calcd for C30H41O4+, 465.2999). Perovskone D (3): white, amorphous solid; [α]25D +83 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 246 (4.07) nm; ECD (MeOH, c 4.2 × 10−4 M) [θ]244 +68 074, [θ]275 −72 216, [θ]321 +27 385 nm; 1H and 13C NMR, see Table 1; HRAPCIMS m/z 481.2950 [M + H]+ (calcd for C30H41O5+, 481.2949). Perovskone E (4): white, amorphous solid; [α]25D +22 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 247 (4.11) nm; ECD (MeOH, c 8.3 × 10−4 M) [θ]203 −41 110, [θ]221 −21 846, [θ]232 −23 034, [θ]260 +23 856, [θ]326 +6758; 1H and 13C NMR, see Table 2; HRAPCIMS m/z 481.2947 [M + H]+ (calcd for C30H41O5+, 481.2949). Perovskone F (5): white, amorphous solid; [α]25D +26 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 273 (3.95) nm; ECD (MeOH, c 4.1 × 10−4 M) [θ]205 −2184, [θ]213 +1116, [θ]258 −24 646, [θ]275 −37 350, [θ]320 +26 726; 1H and 13C NMR, see Table 2; HRESIMS m/z 483.3108 [M + H]+ (calcd for C30H43O5+, 483.3105). Salvadione D (6): colorless oil; [α]25D +68 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 196 (4.02), 229 (3.96) nm; ECD (MeOH, c 4.3 × 10−4 M) [θ]202 +35 380, [θ]221 −10 093, [θ]241 +21 734, [θ]277 −9275, [θ]314 +6963; 1H and 13C NMR, see Table 2; HRAPCIMS m/ z 463.2845 [M + H]+ (calcd for C30H39O4+, 463.2843). Salvadione A (7): white crystals; [α]25D +42 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 197 (4.08), 243 (3.03) nm; ECD (MeOH, c 4.3 × 10−4 M) [θ]202 +97 903, [θ]223 +2647, [θ]241 +19 586, [θ]300 −7490, [θ]339 +5171; 1H and 13C NMR, see Table S1, Supporting

Perovskone C (2) and perovskone D (3) were the most active compounds against T. cruzi (IC50 values of 3.5 and 3.8 μM and SIs of 10.7 and 3.6, respectively). None of the compounds exhibited selective toxicity against T. b. rhodesiense and L. donovani (SI ≤ 2.4 and SI ≤ 5.9, respectively). The limited amount of perovskone C (2) precluded in vivo studies. However, a preliminary in silico evaluation was performed using Percepta software (ACD Laboratories).17 Favorable Hdonor (0) and H-acceptor (4) values but high lipophilicity (log P 5.65) were predicted. High Caco-2 permeability (Pe 173 × 10−6 cm/s) and human intestinal absorption (100%) were also anticipated. Considering the sub-micromolar potency and good selectivity of perovskone C (2) against P. falciparum, together with a predicted good intestinal absorption, this compound may represent a starting point for further optimization by medicinal chemistry.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH on a Jasco P-2000 digital polarimeter (Tokyo, Japan) equipped with a sodium lamp (589 nm) and a 10 cm length temperature-controlled microcell. UV and ECD spectra were recorded in MeOH on a Chirascan spectrometer (Applied Photophysics, Leatherhead, UK), with 110 QS precision cells (1 mm path, Hellma Analytics, Müllheim, Germany), and data were analyzed with Pro-Data V2.4 software. NMR spectra were recorded in CDCl3 (Armar Chemicals, Döttingen, Switzerland) on a Bruker AVANCE III 500 MHz spectrometer (Billerica, MA, USA) operating at 500.13 MHz for 1H and 125.77 MHz for 13C. 1H NMR, COSY, HSQC, HMBC, and NOESY spectra were measured at 18 °C in a 1 mm TXI probe with a z-gradient, using standard Bruker pulse sequences. 13C NMR/DEPTQ spectra were recorded at 23 °C in a 5 mm BBO probe with a z-gradient. Data were processed with Bruker TopSpin 3.5 software. HRAPCIMS data were recorded in positive ion mode with an Agilent 6540 UHD Accurate-Mass Quadrupole time-of-flight detector connected to an Agilent 1290 Infinity system (Santa Clara, CA, USA). Formic acid and solvents were obtained from Scharlau (Scharlab S. L., Barcelona, Spain) or from Macron Fine Chemicals (Avantor Performance Materials, Phillipsburg, NJ, USA). HPLC-grade solvents and ultrapure water from a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany) were used for HPLC. For extraction and preparative separation, technical grade solvents were used after redistillation. Silica gel (70−230 mesh) for open-column chromatography and silica gel 60 F254 coated aluminum TLC plates were obtained from Merck (Darmstadt, Germany). TLC plates were visualized under UV light or by spraying with 5% phosphomolybdic acid in EtOH and subsequent heating. Preparative HPLC was carried out with a Puriflash 4100 system (Interchim, Montluçon, France) utilizing a SunFire C18 (5 μm, 150 × 30 mm i.d.) column with guard column (10 mm × 20 mm i.d.) (Waters, Milford, MA, USA). Semipreparative HPLC was performed on an Agilent 1100 Series instrument with a PDA detector. A SunFire C18 (5 μm, 150 × 10 mm i.d.) column with a guard column (10 mm × 10 mm i.d.) (Waters) was used. Data acquisition and processing was performed using ChemStation software. Plant Material. The aerial parts of S. hydrangea DC. ex Benth. were collected from the Koohin region in Qazvin Province, Iran, in May 2012, and identified by Dr. G. R. Amin, Tehran University of Medical Sciences, Tehran, Iran. A voucher specimen (6719-TEH) has been deposited at the herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Extraction and Isolation. The air-dried aerial parts of S. hydrangea (15 kg) were milled and successively macerated at room temperature with n-hexane (7 × 100 L, 24 h). The dried n-hexane extract (315 g) was fractionated by column chromatography on silica gel (90 × 5.5 cm, 70−230 mesh) with a step gradient of CHCl3− H



Article

Information; HRAPCIMS m/z 467.3157[M + H]+ (calcd for C30H43O4+, 467.3156). X-ray Crystallographic Analysis of Salvadione A (7). Formula C30H42O4, M = 466.66, F(000) = 1016, colorless plate, size 0.05 × 0.12 × 0.21 mm3, orthorhombic, space group P212121, Z = 4, a = 9.20900(10) Å, b = 14.6102(2) Å, c = 18.3937(3) Å, α = β = γ = 90°, V = 2474.79(6) Å3, Dcalc = 1.252 Mg·m−3. The crystal was measured on a Stoe Stadi Vari diffractometer at 123 K; the source was a Metaljet D2 generator equipped with graded multilayer mirrors and Ga Kα radiation with λ = 1.341 43 Å, θmax = 57.292°. Minimal/ maximal transmission 0.97/0.98, μ = 0.406 mm−1. The STOE XAREA suite18 has been used for data collection and integration. From a total of 50 769 reflections, 5079 were independent (merging r = 0.025). From these, 4945 were considered as observed (I > 2.0σ(I)) and were used to refine 312 parameters. The structure was solved by the charge flipping method using the program Superflip.19 Leastsquares refinement against F was carried out on all non-hydrogen atoms using the program CRYSTALS.20 R = 0.0250 (observed data), wR = 0.0297 (all data), GOF = 1.0761. Minimal/maximal residual electron density = −0.14/0.25 e Å−3. Chebychev polynomial weights were used to complete the refinement.21 Plots were produced using Mercury.22 The Flack parameter refined to a value of −0.05(12). With a target value of 0 indicating the correct handedness of the crystallographic model our refined value is with great probability confirming that the handedness of the crystal structure has been determined correctly. Crystallographic data for the structure in this paper have been deposited with the Cambridge Crystallographic Data Center with the deposition number of 1843863. Copies of the data can be obtained, free of charge, on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-1223-336033 or email: [email protected]]. Computational Methods. Conformational analysis of compounds 1−7 was performed with MacroModel 9.8 software (Schrödinger LLC) with the Gaussian 09 program package,23 and ECD curves were obtained using SpecDis v1.64.24 More details are provided in the Supporting Information. In Vitro Antiprotozoal Assays. In vitro inhibitory activity against the protozoan parasites T. b. rhodesiense (STIB900) trypomastigotes, T. cruzi (Tulahuen C4) amastigotes, L. donovani (MHOM-ET-67/ L82) axenically grown amastigotes, and P. falciparum (NF54) IEF stage and cytotoxicity in L6 cells (rat skeletal myoblasts) were determined as reported previously.25,26 The selectivity index was calculated as IC50 for L6-cells/IC50 for the parasites.

■

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS M. Tabefam is grateful for a visiting research fellowship from the Ministry of Science, Research and Technology of Iran (MSRT). Financial support by the Shahid Beheshti University Research Council and Iran National Science Foundation (INSF; Grant No. 95835801) is gratefully acknowledged. ECD spectra were measured at the Biophysics Facility, Biozentrum, University of Basel. Thanks are due to M. Cal, S. KellerMaerki, and R. Rocchetti (Swiss Tropical and Public Health Institute) and to T. Hettich (Group of Prof. Dr. Götz Schlotterbeck, School of Life Sciences, University of Applied Sciences, Institute for Chemistry and Bioanalytics, Muttenz) for in vitro assays and the HRAPCIMS data, respectively. O. Fertig (Division of Pharmaceutical Biology, University of Basel) is gratefully acknowledged for technical assistance.

■

(1) World Health Organization. Working to overcome the global impact of neglected tropical diseases; First WHO report on neglected tropical diseases, 2010. (2) Andrews, K. T.; Fisher, G.; Skinner-Adams, T. S. Int. J. Parasitol.: Drugs Drug Resist. 2014, 4, 95−111. (3) Jamzad, Z. Lamiaceae. In Flora of Iran; Assadi, M, Maassoumi, A, Mozaffarian, V, Eds.; Research Institute of Forest and Rangelands: Tehran, Iran. 2012; Vol. 76. (4) Moridi Farimani, M.; Abbas-Mohammadi, M. Nat. Prod. Res. 2016, 30, 2648−2654. (5) Farimani, M. M.; Abbas-Mohammadi, M.; Esmaeili, M.-A.; Salehi, P.; Nejad-Ebrahimi, S.; Sonboli, A.; Hamburger, M. Planta Med. 2015, 81, 1290−1295. (6) Farimani, M. M.; Taleghani, A.; Aliabadi, A.; Aliahmadi, A.; Esmaeili, M. A.; Sarvestani, N. N.; Khavasi, H. R.; Smieško, M.; Hamburger, M.; Ebrahimi, S. N. Planta Med. 2016, 82, 1279−1285. (7) Farimani, M. M.; Mazarei, Z. Fitoterapia 2014, 98, 234−240. (8) Moridi Farimani, M.; Nejad Ebrahimi, S.; Salehi, P.; Bahadori, M. B.; Sonboli, A.; Khavasi, H. R.; Zimmermann, S.; Kaiser, M.; Hamburger, M. J. Nat. Prod. 2013, 76, 1806−1809. (9) Ebrahimi, S. N.; Zimmermann, S.; Zaugg, J.; Smiesko, M.; Brun, R.; Hamburger, M. Planta Med. 2013, 29, 150−156. (10) Moridi Farimani, M.; Bahadori, M. B.; Taheri, S.; Ebrahimi, S. N.; Zimmermann, S.; Brun, R.; Amin, G.; Hamburger, M. J. Nat. Prod. 2011, 74, 2200−2205. (11) Farimani, M. M.; Taheri, S.; Ebrahimi, S. N.; Bahadori, M. B.; Khavasi, H. R.; Zimmermann, S.; Brun, R.; Hamburger, M. Org. Lett. 2012, 14, 166−169. (12) Ahmad, V. U.; Zahid, M.; Ali, M. S.; Ali, Z.; Jassbi, A.; Abbas, M.; Clardy, J.; Lobkovsky, E.; Tareen, R.; Iqbal, M. Z. J. Org. Chem. 1999, 64, 8465−8467. (13) Jiang, Z.-Y.; Yu, Y.-J.; Huang, C.-G.; Huang, X.-Z.; Hu, Q.-F.; Yang, G.-Y.; Wang, H.-B.; Zhang, X.-Y.; Li, G.-P. Planta Med. 2015, 81, 241−246. (14) Parvez, A.; Choudhary, M. I.; Akhter, F.; Noorwala, M.; Mohammad, F. V.; Hasan, N. M.; Zamir, T.; Ahmad, V. U. J. Org. Chem. 1992, 57, 4339−4340. (15) Ahmad, V. U.; Parvez, A.; Hassan, N. Tetrahedron Lett. 1993, 34, 5337−5340. (16) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach; John Wiley & Sons, 2002. (17) Structure Elucidator; Advanced Chemistry Development, Inc.: Toronto, ON, Canada, www.acdlabscom, 2015. (18) Stoe & Cie. X-area Software, V1.08; S.C.X., 2011. (19) Palatinus, L.; Chapuis, G. J. Appl. Crystallogr. 2007, 40, 786− 790.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00498. 1D and 2D NMR spectra of compounds 1−3, 5, and 6, 1 H and 2D NMR spectra of compound 4, 1H NMR data and 13C NMR data of compound 7, and details of computational methods (PDF) X-ray crystallographic data (CIF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel: +982129904043. Fax: +982129902679. E-mail: m_ [email protected]. *Tel: +41 61 207 14 25. Fax: +41 61 207 14 74. E-mail: [email protected]. ORCID

Mahdi Moridi Farimani: 0000-0001-9273-1019 Samad Nejad Ebrahimi: 0000-0003-2167-8032 Olivier Potterat: 0000-0001-5962-6516 Matthias Hamburger: 0000-0001-9331-273X I



Article

(20) Betteridge, P. W.; Carruthers, J. R.; Cooper, R. I.; Prout, K.; Watkin, D. J. Appl. Crystallogr. 2003, 36, 1487−1487. (21) Carruthers, J. R.; Watkin, D. J. Acta Crystallogr., Sect. A: Cryst. Phys., Diffr., Theor. Gen. Crystallogr. 1979, A35, 698−699. (22) Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.; Taylor, R.; Streek, J. v.; Wood, P. A. J. Appl. Crystallogr. 2008, 41, 466−470. (23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.Gaussian 09; Gaussian, Inc.: Wallingford, CT, USA, 2009. (24) Bruhn, T. H., A, S. Y.; Bringmann, G. SpecDis, 1.64; University of Wuerzburg: Germany, 2015. (25) Orhan, I.; Ş ener, B.; Kaiser, M.; Brun, R.; Tasdemir, D. Mar. Drugs 2010, 8, 47−58. (26) Huber, W.; Koella, J. C. Acta Trop. 1993, 55, 257−261.

J


Antiprotozoal Isoprenoids from Salvia hydrangea - Journal of Natural

Recommend Documents