HPLC-Based Activity Profiling for Antiprotozoal Compounds in the

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

HPLC-Based Activity Profiling for Antiprotozoal Compounds in the Endemic Iranian Medicinal Plant Helichrysum oocephalum Maryam Akaberi,†,‡ Ombeline Danton,† Zahra Tayarani-Najaran,§ Javad Asili,‡ Mehrdad Iranshahi,‡ S. Ahmad Emami,*,‡ and Matthias Hamburger*,† †

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Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland ‡ Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran § Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran S Supporting Information *

ABSTRACT: In a screening of Iranian plants for antiprotozoal activity a dichlomethane extract from the aerial parts of Helichrysum oocephalum showed in vitro antiprotozoal activity against Plasmodium falciparum and Leishmania donovani, with IC50 values of 4.01 ± 0.50 and 5.08 ± 0.07 μg/mL, respectively. The activity in the extract was tracked by HPLC-based activity profiling, and subsequent targeted preparative isolation afforded 24 compounds, including pyrones 22−24, phloroglucinol derivatives 12−19, and compounds containing both structural motifs (1−11, 20, and 21). Of these, 15 compounds were new natural products. The in vitro antiprotozoal activity of isolates was determined. Compound 3 showed good potency and selectivity in vitro against L. donovani (IC50 1.79 ± 0.17 μM; SI 53). compounds, including α-pyrones, phloroglucinols, benzofurans, and flavonoids, has all been reported.8−10 Reported herein are the activity-guided isolation and identification of new heterodimeric α-pyrones and phloroglucinol derivatives from H. oocephalum and their in vitro antiprotozoal activity.

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ccording to the WHO, tropical parasitic diseases such as leishmaniasis and malaria affect more than one billion people worldwide in tropical and subtropical regions. For malaria alone, more than 216 million new cases and about 445 000 deaths were reported in 2016.1 Artemisinin combination therapies (ATCs) are currently the primary treatment for malaria, but artemisinin-resistant Plasmodium falciparum strains have already emerged in Asia. As for the treatment of cutaneous and visceral leishmaniasis, only a few drugs are available, and they are all associated with severe side effects and increasing drug resistance. There is thus a need for new drugs with novel modes of action to treat these infectious diseases. Natural products have in many instances provided interesting leads to combat tropical parasitic diseases.2 In an ongoing search for antiprotozoal compounds from endemic plants of Iran,3−7 the dichloromethane extract of Helichrysum oocephalum L. (Asteraceae) showed promising activity against P. falciparum and Leishmania donovani (IC50 4.01 ± 0.50 and 5.08 ± 0.07 μg/mL, respectively). The genus Helichrysum comprises over 600 species that are distributed mainly in Africa (244 species in South Africa), Madagascar, Australasia, and Eurasia. Helichrysum species have been used traditionally for the treatment of infections, digestive and hepatic disorders, and colds and coughs and for wound healing. As to the phytochemistry of the genus, the presence of an essential oil, sesquiterpenes, diterpenes, and various classes phenolic © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The active compounds in the dichloromethane extract of H. oocephalum were tracked with the aid of HPLC-based activity profiling.11,12 An activity profile of 34 1 min fractions and the corresponding LC−ELSD traces are shown in Figure 1. Major activity in both assays was observed for the time window between 7 and 20 min. For the isolation of the compounds of interest, the extract was separated by open column chromatography on silica gel into 28 fractions (F1−F28). On the basis of the in vitro activity, fractions F9−F15 were selected for further purification. Due to the low amount of the active fractions (2.3 g in total) and similar LC-UV-ELSD patterns, F9 and F10, and F11−F14 were combined (fractions A and B, respectively). Further separation by normal- and reversedphase chromatography afforded compounds 1−24: 1−6, 8, and 12−24 from fraction A and compounds 7 and 9−11 from fraction B. Received: December 7, 2018

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DOI: 10.1021/acs.jnatprod.8b01031 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. HPLC-based activity profiling of the dichloromethane extract of H. oocephalum. The ELSD chromatogram of a separation of 300 μg of extract on an analytical RP-HPLC column is shown. Activity of 1 min microfractions is indicated with colored columns for antileishmanial (A) and antiplasmodial (B) activity, expressed as % inhibition.

H-16), 2.66 ppm (dd, J = 9.5 Hz, 7.6 Hz, H-17), 1.79 ppm (overlapped, H-19b), 2.03 ppm (m, H-19a), 1.65 ppm (m, H20b), 1.74 ppm (overlapped, H-20a), 2.44 ppm (t, J = 7.3 Hz, H-21), 1.39 ppm (s, H3-23), and 0.78 ppm (s, H3-24). HMBC correlations from H-16 to C-3 (δC 104.1) and to the oxygenbearing tertiary carbon C-4 (δC 154.4), from H3-23 and H3-24 to C-16 (δC 35.9) and C-21 (δC 46.2), from H-21 to C-16 (δC 35.9), C-23 (δC 33.5), and to the oxygen-bearing tertiary carbon C-18 (δC 87.3), and from H2-20 to C-17 (δC 37.9) and C-18 (δC 87.3) indicated the presence of a 3,4-dihydro-2Hpyran unit fused to a five-membered ring at C-17 and C-18. In addition, a four-membered ring fused at C-16 and C-21 was present, as indicated by a quaternary carbon (δC 39.1 ppm, C22), bearing a gem-dimethyl group [δH 1.39 (s, H3-23); δC 33.5, C-23; 0.78 (s, H3-24); δC 17.7, C-24]. Such cyclol rearrangements have been reported in other natural products, such as clusiacyclols and cannabicyclol.20,21 A resonance at 3.65 ppm (s, H2-7) was indicative of a methylene bridge between an aromatic (phloroglucinol) and a pyrone ring.22 The nature and location of three substituents on both rings were deduced from the 13 C NMR data and HMBC correlations from H2-7 to carbons at 154.4 ppm (C-4), 105.1 ppm (C-5), and 157.4 ppm (C-6) for the aromatic ring and at 102.5 ppm (C-8), 167.0 ppm (C-9), and 168.8 ppm (C-15) for the pyrone ring. Substituents of the pyrone ring at C-10 (δC 107.7) and C-11 (δC 161.3) were deduced from HMBC

Compounds 3, 5, 8−19, and 22−24 were located in the active time windows, while 1, 2, 4, 6, 7, 20, and 21 were located in the nonactive regions (Figure 1). By means of 1D and 2D NMR data and HRMS measurements, their structures were identified as α-pyrone and phloroglucinol derivatives. For NMR and MS data of the compounds, see Tables 1−6, Tables S1−S5 (Supporting Information), and Figures S1−S69 (Supporting Information). Known acyl phloroglucinol pyrone derivatives were identified as achyroclinopyrone A (4),13 achyroclinopyrone B (5),13 23-methyl-6-O-desmethylauricepyrone(8),14 norauricepyrone (desmethylauricepyrone) (9),15 italidipyrone (20), 23methylitalidipyrone (21),16 helipyrone A (22), helipyrone B (23), and helipyrone C (24).17−19 Compound 1 exhibited a molecular formula of C29H36O7, as determined by the HRESIMS data (molecular ion at m/z 519.2357 [M + Na]+; calcd for C29H36NaO7+ 519.2353). The molecular formula accounted for 12 indices of hydrogen deficiency. Analysis of the 1H and 2D NMR spectra (Table 1) indicated an acyl α-pyrone phloroglucinol scaffold similar to that of achyroclinopyrone A13 (4), but with additional rings derived from the geranyl moiety. As the scaffold already accounted for nine indices of hydrogen deficiency, the remaining three were in the rearranged portion of the compound. This part of the molecule was characterized by signals in the 1H NMR spectrum at 3.14 ppm (d, J = 9.5 Hz, B



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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−3 (CDCl3 for 1 and 3, CD3OD for 2; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 1 position

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19a 19b 20a 20b 21 22 23 24 25 26 27 28 29 OH-2 OH-4 OH-6 OH-9

106.0, C 163.3, C 104.1, C 154.4, C 105.1, C 157.4, C 18.0, CH2 102.5, C 167.0, C 107.7, C 161.3, C 24.3, CH2 11.6, CH3 9.4, CH3 168.8, C 35.9, CH 37.9, CH 87.3, C 38.5, CH2

a

25.6, CH2 46.2, C 39.1, C 33.5, CH3 17.7, CH3 27.1, CH3 212.1, C 39.4, CH 19.4, CH3 19.3, CH3

2 δH (J in Hz)

δC, type

3 δH (J in Hz)

a

b

3.65, s

2.57, q (7.4) 1.22, t (7.3) 1.96, s 3.14, d (9.5) 2.66, dd (9.5, 7.6) 2.03, m 1.79c 1.74c 1.65, m 2.44, t (7.3) 1.39, s 0.78, s 1.57, s 4.11, m 1.20, d (6.4) 1.17, d (6.7) 13.84, s

155.0, C 104.9, C 158.4, C 108.9, C 160.0, C 18.9, CH2 102.0, C 174.8, C 111.4, C 161.9, C 25.2, CH2 12.0, CH3 9.9, CH3 170.0, C 119.2, CH 124.6, CH 81.4, C 42.5, CH

3.58, s

2.54, q (7.6) 1.17c 1.91, s 6.68 (d, 10.1) 5.40 (d, 10.1) 1.74, m 1.66, m 2.08, m

24.1, CH2 125.2, CH 132.8, C 25.8, CH3 17.7, CH3 26.6, CH3 212.0, C 41.2, CH 19.2, CH3 19.4, CH3

5.08, tqq (7.3, 1.5, 1.3)

δC, type

δH (J in Hz)

104.0, C 156.0, C 103.7, C 159.1, C 103.6, C 161.0, C 17.3, CH2 99.0, C 167.4, C 108.9, C 158.7, C 17.2, CH3 9.8, CH3 169.5, C 117.2, CH 124.9, CH 78.1, C 27.7, CH3 27.7, CH3

3.64, s

2.24, s 1.95, s 6.70, d (9.8) 5.44, d (10.1) 1.49, sc 1.49, sc

203.8, C 32.6, CH3

2.68, s

1.62, br s 1.54, br s 1.36, s 3.68, qq (6.9, 6.9) 1.15, d (6.9) 1.14, d (6.6) 10.54, s 15.80, s 9.82, s

10.46, s 9.34, s

a

Extracted from H−C 2D inverse-detected experiments due to a low amount of sample. bSignal not visible. cOverlapping signals.

(Table 1) were very similar to those of 1, with some differences in the portion of C-16−C-25 (Table 1). In the HMBC spectrum, two olefinic protons [6.68 ppm (d, J = 10.1 Hz, H-16) and 5.40 ppm (d, J = 10.1 Hz, H-17)] showed cross-peaks with C-3 (δC 104.9) and with the oxygen-bearing tertiary carbon C-18 (δC 81.4). The molecular formula indicated 12 indices of hydrogen deficiency, of which nine were already accounted for with the known part of the structure. Therefore, a 2H-pyran ring was deduced. In the COSY spectrum, the third olefinic proton at 5.08 ppm (tqq, J = 7.3, 1.5, 1.3 Hz, H-21) coupled with two methyl groups [δH 1.62 (br s, H3-23), 1.54 (br s, H3-24)] and a methylene group [δH 2.08 (m, H2-20)]. COSY correlations between H2-20 and H2-19 (δH 1.74; 1.66) and HMBC correlations from H3-25 (δH 1.36) and H2-19 to C-18 (Figure 2) were indicative of a methyl group and an isopent-3-enyl residue both attached at C-18. The chemical shifts for C-2 (δC 155.0) and C-4 (δC 158.4) indicated a ring closure at C-2 rather than C-4, as they were in good agreement with those reported for (S)-3-[{5,7dihydroxy-2,2-dimethyl-8-(2-methylbutanoyl)-2H-chromen-6yl}methyl]-6-ethyl-4-hydroxy-5-methyl-2H-pyran-2-one.23 Due

correlations from H3-14 (δH 1.96) to C-9, C-10, and C-11, from H2-12 (δH 2.57) to C-11 and C-10, and from H3-13 (δH 1.22) to C-11 (Figure 2). H-27 (δH 4.11), H3-28 (δH 1.20), and H3-29 (δH 1.17) showed COSY correlations characteristic of an isopropyl group (Figure 2). An HMBC correlation of H27 with a carbonyl carbon (δC 212.1, C-26) was indicative of a 2-methyl oxopropyl group attached to C-1 (106.0 ppm). The 1 H NMR data showed three hydroxy groups (δH 13.84, 10.46, and 9.34), which, according to HMBC correlations, corresponded to OH-2, OH-6 and OH-9, respectively. Based on NOESY correlations (Figures 2 and S5, Supporting Information), the relative configurations of carbons C-16, C-17, C-18, and C-21 were established. A specific rotation close to 0 and the absence of any Cotton effect in the experimental electronic circular dichroism (ECD) spectrum (Figure S70, Supporting Information) suggested that the compound was isolated as a racemic mixture. Compound 1 is reported here for the first time and named helicyclol. Compound 2 gave the same molecular formula as 1, as deduced from its HRMS data ([M + Na]+ at m/z 519.2358, calcd for C29H36NaO7+ 519.2353). The NMR spectra of 2 C



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Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 6 and 7 (CD3OD; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 6 δC, type position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27a 27b 28 29

E 105.98; 106.01, C 161.3, C 109.8; 109.9, C 160.8, C 106.9; 107.0, C 159.3, C 18.80; 18.83, CH2 102.93; 102.94, C 170.1; 170.2, C 110.61; 110.63, C 158.3, C 17.2, CH3 10.05; 10.06, CH3 170.7, C 22.4; 22.6, CH2 123.8, CH 136.1, C 40.8, CH2 27.57, CH2 125.4, CH 132.0, C 17.7, CH3 25.8, CH3 16.3, CH3 212.9, C 40.3, 40.4, CH 19.8, CH3 19.8, CH3

7 δH (J in Hz)

a

δC, type

a

Z

E

δH (J in Hz)a

a ,c

Z

E

Z

E

Z

d

3.51, s

2.16, s 1.87, s

124.2, CH 136.6, C 32.9, CH2 27.61, CH2 125.7, CH 132.2, C 17.8, CH3 25.9, CH3 23.7, CH3

3.31, m 5.135b

5.16

1.92, m 2.00, m 4.98, t (6.2)

2.21, m 2.08, m 5.126b

1.49, br s 1.55, br s 1.74, br s

1.60, br s 1.65, br s 1.63, br s

b

4.03, qq (6.5, 6.4) 1.14, d (6.6) 1.14, d (6.6)

161.0, C 109.93; 109.95, C 161.3, C 106.73; 106.79, C 159.5, C 19.2, CH2 102.82; 102.86, C 171.9, C 111.1, C 158.2, C 17.4, CH3 10.3, CH3 170.9, C 22.6; 22.8, CH2 124.2, CH 124.6, CH 135.9, C 136.4, C 41.0, CH2 33.1, CH2 27.80, CH 27.75, CH 125.6, CH 125.9, CH 132.0, C 132.2, C 17.8, CH 17.9, CH 25.9, CH3 26.1, CH3 16.5, CH3 23.8, CH3 212.8, C 47.2, CH 28.4, CH2 12.5, CH3 17.4, CH3

3.57, s

2.20, s 1.91, s 3.30b; 3.29b 5.130b

5.17b

1.93a 2.01, m 4.99, t (7.0)

2.19a 2.07, m 5.122b

1.49, br s 1.54, br s 1.73, br s

1.60, br s 1.64, br s 1.63, br s

3.93, 1.79, 1.36, 0.88, 1.12,

qt (6.7, 6.7) m m t (7.3) d (6.7)

a

Signals could not be assigned to E or Z unless specified. bOverlapping signals. cExtracted from H−C 2D inverse-detected experiments due to low amount of sample. dSignal not visible.

compound 6. NMR data (Table 2) indicated the presence of an acyl α-pyrone phloroglucinol skeleton as in 4 (achyroclinopyrone A), but with a methyl group (δH 2.16 (s, H3-12); δC 17.2, C-12) at C-11 (Table 2) and a geranyl chain attached at C-3. The NMR data indicated that 6 is an inseparable mixture of E and Z isomers. The diagnostic chemical shifts of carbons 18 and 24 [δC 40.8, C-18E/δC 32.9, C-18Z; δC 16.3, C-24E/δC 23.7; C-24Z] enabled an unambiguous assignment of the two isomers that were named 16E- and 16Zachyroclinopyrone C, respectively. A molecular formula of C29H38O7 (HRESIMS m/z 521.2507 [M + H]+, calcd for C29H38NaO7+ 521.2510) was determined for 7. The NMR data (Table 2) indicated an acyl α-pyrone phloroglucinol skeleton bearing a methyl group at C-11. The only difference from 6 was the presence of a 2-methyl oxobutyl moiety attached to C-1. Again, 7 was obtained as an inseparable mixture of E and Z isomers, named 16E- and 16Z-achyroclinopyrone D, respectively. Compounds 10 and 11 were assigned as possessing both an acyl α-pyrone phloroglucinol skeleton with a prenyl chain at C3 and an acyl group at C-1, similar to compounds 8 (23methyl-6-O-desmethylauricepyrone)14 and 9 (6-O-desmethylauricepyrone)15 (Tables 3 and S2, Supporting Information). The 1H NMR data and HMBC correlations of compound 10

to a specific rotation close to 0, compound 2 was considered to be a racemic mixture and named helicepyrone. Compound 3 showed a molecular formula of C21H22O7 (HRESIMS m/z 409.1266 [M + Na]+, calcd for C21H22NaO7+ 409.1258). The NMR data (Table 1) indicated the same scaffold as for compound 2, but with different substituents at the three rings. Compound 3 was found to bear a methyl group at C-11 [δH 2.24 (s, H3-12)] instead of an ethyl group in 2, and an oxoethyl (δH 2.68 (s, H3-21); δC 32.6, C-21; δC 203.8, C20) showed an HMBC correlation with the quaternary carbon at 104.0 ppm (C-1). A gem-dimethyl group [δH 1.49 (overlapped, H3-18 and H3-19)] showed HMBC correlations with C-17 (δC 78.1) and C-16 (δC 124.9). The chemical shifts of C-2 (δC 156.0) and C-4 (δC 159.1) indicated that the ring closure was at C-2 as in compound 2.23 This was also supported by resonances in the 1H NMR spectrum at 15.80 and 10.54 ppm attributed to OH-6 and OH-4, respectively, and the absence of a resonance at δH 13.8 as observed for OH2 in 1. The shifts were also in agreement with data reported for cycloarzanol B,24 whereby 3 differed from cycloarzanol B in having a Δ15(16) double bond and a methyl group instead of an ethyl moiety at C-11. Compound 3 was named cycloarzanol C. A molecular formula of C28H36O7 (HRESIMS m/z 507.2357 [M + Na]+, calcd for C28H36NaO7+ 507.2353) was assigned to D



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C-6 of the phloroglucinol. A sharp signal at δH 13.41 (OH-2) and a broad one at δH 6.88 (OH-4) indicated that the spiroketal closed at C-6. Compound 12 was named helispiroketal A. Compound 13, with a molecular formula of C21H24O6 (HRMS m/z 395.1473 [M + Na]+; calcd for C21H24NaO6+ 395.1465), differed from 12 by the presence of an oxopropyl group [C-19 (δC 205.9), CH2-20 (δH 2.87; δC 37.0) and CH321 (δH 1.09; δC 8.6)] at C-1 (Table 4). Compound 13 was named helispiroketal B. Compound 14 exhibited a molecular formula of C23H28O6, as deduced from its 13C NMR and HRMS data (molecular ion at m/z 423.1794 [M + Na]+; calcd C23H28NaO6+ 423.1778). Comparison of the NMR data with those of 13 revealed the presence of a 2-methyl oxopropyl moiety [C-20 (δC 209.7), CH-21 (δH 3.54; δC 39.9), CH3-22 (δH 1.09; δC 19.8), and CH3-23 (δH 1.08; δC 19.1)] at C-1 and an ethyl group [CH2− 12 (δH 2.69; δC 23.3) and CH3-13 (δH 1.23; δC 10.6)] at C-11 (Table 4). This new natural product was named helispiroketal C. Compounds 15 (C23H28O6; HRESIMS at m/z 423.1799 [M + Na]+, calcd for C23H28NaO6+ 423.1778), 16 (C22H26O6; HRESIMS m/z 409.1634 [M + Na]+, calcd for C22H26NaO6+ 409.1621), and 17 (C21H24O6; HRESIMS m/z 395.1473 [M + Na]+, calcd for C21H24NaO6+ 395.1471) were all observed to possess very similar NMR data (Table 5) to helispiroketal A (12). The main differences were in the substituents at C-1, namely, a 2-methyl-oxobutyl residue [C-19 (δC 208.2), CH-20 (δH 3.44; δC 45.2), CH2-21 (δH 1.78, 1.35; δC 26.9, 27.3), CH3-22 (δH 0.85, 0.87; δC 11.6, 11.7), and CH3-23 (δH 1.10, 1.11; δC 15.6, 16.0)] for 15 and a 2-methyl-oxopropyl group [C-19 (δC 208.3), CH-20 (δH 3.58; δC 38.5), CH3-21 (δH 1.14; δC 18.74), and CH3-22 (δH 1.13; δC 18.67)] for 16. In the case of 17, the methyl group at C-11 was replaced by an ethyl group [CH2-12 (δH 2.58; δC 21.6) and CH3-13 (δH 1.23; δC 9.7)]. Compound 15 was found to be an inseparable mixture of two diastereoisomers due to the stereogenic C-20. This was also evidenced by the presence of two signals for OH-2 (δH 13.81, 13.82). These three compounds were named helispiroketals D (15), E (16), and F (17). Compounds 18 (C27H34O6; HRESIMS m/z 477.2256 [M + Na]+; calcd for C27H34O6Na+, 477.2247) and 19 (C25H30O6; HRESIMS m/z 449.1938 [M + Na]+; calcd for C25H30O6Na+, 449.1934) were found to contain an acyl spiroketal phloroglucinol scaffold as in compounds 12−17. The difference was in the presence of a geranyl moiety at C-3 instead of a prenyl chain (Table 6). Analysis of the 1D and 2D NMR data showed that the two compounds again were inseparable mixtures of E and Z isomers. The moiety attached to C-1 consisted of a 2-methyl-oxopropyl (18) or an acetyl group (19). The compounds were named 15E- and 15Z-helispiroketal G and 15E- and 15Z-helispiroketal H, respectively. In their 1H NMR spectra, the integrals of the methyl (15, 16, 18) and methylene group (17) attached to C-11 were lower than expected. A fast exchange between the two forms of the keto−enol tautomers could explain this observation. ECD spectra were measured for compounds 12 and 16, as enough sample was available. Despite an α,β-unsaturated carbonyl group near the stereogenic C-8, no Cotton effect was observed (Figures S72 and S73, Supporting Information). Therefore, the spiroketals had to be racemic mixtures of both enantiomers. A possible biosynthesis of these compounds is outlined in Scheme 1. Acyl α-pyrone phloroglucinols would be

Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 10 and 11 (DMSO-d6; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 10 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22a 22b 23 24 OH-2

δC, type

a

δH (J in Hz)

11 δC, type

a

b

b

161.6, C 106.1, C 161.5, C 109.4, C 158.6, C 19.4, CH2 98.3, C 177.2, C 112.3, C 153.9, C 16.9, CH3 9.9, CH3 168.1, C 21.5, CH2 123.9, CH 129.2, C 17.6, CH3 25.4, CH3 210.4, C 37.9, CH 19.5, CH3

161.5, C 106.4, C 160.4, C 108.1, C 158.1, C 19.5, CH2 98.2, C 175.8, C 111.9, C 153.7, C 17.0, CH3 10.0, CH3 168.1, C 21.5, CH2 124.0, CH 129.1, C 17.7, CH3 25.5, CH3 209.5, C 44.5, CH 26.5, CH2

19.5, CH3

3.35, s

2.07, s 1.76, s 3.11, d (6.7) 5.11, br t (5.8) 1.68, br s 1.58, br s 4.08, qq (6.8, 6.7) 1.07, d (6.7) 1.07, d (6.7) 13.87, br s

11.9, CH3 16.9, CH3

δH (J in Hz)

3.37, s

2.09, s 1.78, s 3.12, d (7.0) 5.11, br t (7.0) 1.69, br s 1.59, br s 3.97, qt (6.7, 6.6) 1.73c 1.33, m 0.84, t (7.3) 1.06, d (6.7) 13.77, br s

a

Extracted from H−C 2D inverse-detected experiments due to a low amount of sample. bSignal not visible. cOverlapping signals.

revealed the presence of two protons at 3.11 ppm (d, J = 6.7 Hz, H2-15), a vinylic hydrogen at 5.11 ppm (t, J = 5.8 Hz, H16), and two methyl groups at 1.68 (s, H3-18) and 1.58 (s, H319), indicative of a prenyl chain. Compared to 8 and 9, compounds 10 and 11 were shown to bear a methyl group [10 (δH 2.07 (s, H3-12); δC 16.9, C-12); 11 (δH 2.09 (s, H3-12); δC 17.0, C-12)] at C-11 instead of an ethyl residue. The only difference between 10 and 11 was in the substituent at C-1. For 10, a molecular formula of C23H28O7 (HRMS m/z 439.1746 [M + Na]+; calcd for C23H28NaO7+ 439.1727) was determined and the NMR data indicated the occurrence of a 2methyl oxopropyl moiety, while compound 11 [C24H30O7 (HRMS m/z 453.19037 [M + Na]+; calcd for C24H30NaO7+ 453.183)] showed a 2-methyl oxobutyl residue at C-1. The structures of 10 and 11 were similar to that of arenol from H. arenarium,23 differing only in the nature of the side chain at C1. Compounds 10 and 11 were therefore named as arenol B and arenol C, respectively. Compound 12 had a molecular formula of C20H22O6, as deduced from its 13C NMR and HRMS data (molecular ion at m/z 381.1316 [M + Na]+; calcd for C20H22NaO6+ 381.1308). Analysis of the 1H and 13C NMR spectra (Table 4) indicated an acyl phloroglucinol scaffold as in compound 10, but with an oxoethyl group at C-1 [C-19 (δC 201.7) and CH3-20 (δH 2.53; δC 31.3)]. Furthermore, an α-pyrone moiety was lacking. The remaining four indices of hydrogen deficiency and HMBC correlations (Figure 2) were indicative of an α,β-unsaturated spiroketal unit with five-membered rings attached at C-5 and E



Article

Table 4. 1H and 13C NMR Spectroscopic Data for Compounds 12, 13, and 14 (CDCl3 for 12, CD3OD for 13 and 14; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 12 position 1 2 3 4 5 6 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 OH-2 OH-4

δC, type 102.0, 162.4, 107.7, 157.7, 101.2, 158.6, 34.29,

C C C C C C CH2

109.1, C 198.3, C 109.4, C 184.7, C 15.1, CH3 5.7, CH3 21.7, CH2 121.8, CH 135.5, C 17.9, CH3 25.8, CH3 201.7, C 31.3, CH3

13 δH (J in Hz)

3.37a 3.17, d (16.2)

2.23, s 1.75, s 3.34a 5.23, t (6.9) 1.79, s 1.73, s 2.53, s

δC, type

14 δH (J in Hz)

b

δC, type

b

c

c

163.2, C 111.4, C 158.5, C 102.0, C 159.7, C 35.4, CH2

164.0, C 111.6, C 159.1, C 102.2, C 156.9, C 35.5, CH2

3.31, d (16.1) 3.22, d (16.1)

110.5, C 199.9, C 110.3, C 187.8, C 15.0, CH3 5.5, CH3 22.4, CH2 123.9, CH 131.9, C 26.0, CH3 17.9, CH3 205.9, C 37.0, CH2 8.6, CH3

2.29, 1.74, 3.27, 5.18,

s s d (6.9) t (6.6)

1.65, s 1.75, s 2.87, m 1.09, t (7.3)

110.8, C 201.1, C 109.3, C 191.9, C 23.3, CH2 10.6, CH3 5.5, CH3 22.6, CH2 124.0, CH 132.3, C 26.1, CH3 18.1, CH3 209.7, C 39.9, CH 19.8, CH3 19.1, CH3

13.61, sd 6.43, br sd

13.41, s 6.88, br s

δH (J in Hz)

3.32, d (16.1) 3.21, d (16.1)

2.69, 1.23, 1.75, 3.27, 5.18,

q (7.6) t (7.6) s d (6.9) br t (6.9)

1.65, br s 1.76, br s 3.54, qq (6.7, 6.6) 1.09, d (6.9) 1.08, d (6.6) 13.79, sd 6.43, br sd

a Overlapping signals. bExtracted from H−C 2D inverse-detected experiments due to a low amount of sample. cSignal not visible. dMeasured in CDCl3.

■

obtained by formation of a methylene bridge between a 2methyl α-pyrone unit and a prenylated phloroglucinol.25−27 The spiroketals are likely derived from such acyl α-pyrone phloroglucinols, whereby a cleavage of the α-pyrone would lead to a benzofuran intermediary. In a nonenzymatic reaction, the attack of electrons of the hydroxy group on either face of the ring would result in a racemic mixture. Compounds 1−20 and 22−24 were tested for in vitro antiprotozoal activity against L. donovani and P. falciparum. In parallel, cytotoxicity of the compounds in L6 cells was determined in order to obtain an initial assessment of their selectivity (Table 7). Of the two parasites tested, L. donovani was generally found to be more sensitive. Compounds 1, 2, 3, 15, 16, 18, 19, and 20 were active at low micromolar concentrations (IC50 1.7−2.8 μM) and possessed moderate to good selectivity (SI 12−57). Compound 3 (IC50 1.8 μg/mL, SI 53) had the best profile against L. donovani. Spiroketals 15, 16, and 18 were also active against P. falciparum, with IC50s of 2.5, 2.6, and 1.2 μg/mL, respectively. It is interesting to note that a number of phloroglucinol derivatives reportedly possess antiparasitic activity. (±)-Rhodomyrtosone F from Syncarpia glomulifera (Myrtaceae),28 mallatojaponin C from Mallotus oppositifolius (Euphorbiaceae),29 and tomentosone A from Rhodomyrtus tomentosa (Myrtaceae)30 are examples of antiplasmodial phloroglucinol derivatives, while antileishmanial activity has been described for lindbergins E and F from Elaphoglossum lindbergii (Dryopteridaceae),31 and spiranthenones A and B from Spiranthera odoratissima (Rutaceae).32

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 (100−400 μg/mL) on a Chirascan CD spectrometer (Applied Photophysics, Leatherhead, UK) using 110 QS 1 mm path precision cells (Hellma Analytics, Müllheim, Germany). NMR spectra were recorded on a Bruker Avance III NMR spectrometer (Billerica, CA, 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. Spectra were analyzed by Bruker TopSpin 3.5 software. HRESIMS data were measured in the positive mode on a Thermo Scientific Orbitrap LQT XL mass spectrometer (Waltham, MA, USA) via direct injection. HPLC-grade solvents were obtained from Macron Fine Chemicals (Avantor Performance Materials, Phillipsburg, NJ, USA), SigmaAldrich (St. Louis, MO, USA), and Merck Millipore (Darmstadt, Germany). For extraction and preparative separation, technical grade solvents Scharlau (Scharlab S. L., Barcelona, Spain) were used after distillation. Analytical-grade formic acid, trifluoroacetic acid, and sulfuric acid were sourced from Merck Millipore (Darmstadt, Germany). CDCl3 and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water and ultrapure water were prepared using Elix and Milli-Q water purification systems (Merck Millipore, Darmstadt, Germany). Methanol-d4, CDCl3, or DMSO-d6 were purchased from Armar Chemicals (Döttingen, Switzerland). Silica gel 60 F254 coated aluminum TLC plates and silica gel (230− F



Article

Table 5. 1H and 13C NMR Spectroscopic Data for Compounds 15, 16, and 17 (CDCl3; 500.13 MHz for 1H and 125.77 MHz for 13C NMR; δ in ppm) 15 position 1 2 3 4 5 6 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 OH-2 OH-4

δC, type

a

101.6, C 163.3, C 107.9, C 157.4, C 101.4, C 158.1, C 34.0, CH2 109.2, C 197.9, C 109.4, C 184.4, C 14.5, CH3 5.6, CH3 21.7, CH2 121.8, CH 135.8, C 17.9, CH3 25.7, CH3 208.2, C 45.2, CH 26.9; 27.3, CH2c 11.6; 11.7, CH3c 15.6; 16.0, CH3c

16 δH (J in Hz)

3.38b 3.16, d (16.2)

2.22, m 1.76b 3.39b 5.28, br t (7.2) 1.82, br s 1.76b 3.44, mc 1.78b; 1.35, mc 0.85, t (7.3); 0.87, t (7.3)c 1.10, d (6.4); 1.11, d (6.4)c 13.81, s; 13.82, sc 6.62, br s

δC, type

a

101.0, C 163.1, C 107.6, C 157.3, C 101.3, C 157.8, C 34.1, CH2 109.1, C 197.8, C 109.3, C 184.2, C 14.9, CH3 5.5, CH3 21.8, CH2 121.7, CH 135.9, C 17.8, CH3 25.7, CH3 208.3, C 38.5, CH 18.74, CH3 18.67, CH3

17 δH (J in Hz)

3.38, d (16.2) 3.16, d (16.2)

2.24, s 1.78b 3.40, d (7.0) 5.29, tqq (7.3, 1.4, 1.2) 1.83, br s 1.78b 3.58, qq (6.7, 6.7) 1.14, d (6.7) 1.13, d (6.7) 13.78, s

δC, type

a

102.0, C 162.3, C 107.2, C 157.6, C 101.2, C 158.6, C 34.0, CH2 109.0, C 198.3, C 108.2, C 188.5, C 21.6, CH2 9.7, CH3 5.3, CH3 21.6, CH2 121.5, CH 136.0, C 17.7, CH3 25.5, CH3 201.4, C 31.0, CH3

δH (J in Hz)

3.38b 3.16, d (16.2)

2.58c 1.23, s 1.77, s 3.41b 5.29, br t (7.3) 1.84, br s 1.79, br s 2.56, s

13.49, s 6.49, br s

a Extracted from H−C 2D inverse-detected experiments due to a low amount of sample. bOverlapping signals. cSignals belonging to both diastereoisomers.

400 μm) for open-column chromatography were obtained from Merck (Darmstadt, Germany). HPLC-PDA-ELSD-ESIMS were obtained in the positive- and negative-ion modes (scan range of m/z 200−1500) on a Shimadzu LC-MS/MS 8030 triple quadrupole MS system, connected via Tsplitter (1:10) to a Shimadzu HPLC system (Kyoto, Japan) consisting of degasser, binary mixing pump, autosampler, column oven, and a diode array detector and to an Alltech (Büchi, Flawil, Switzerland) 3300 ELSD detector using a SunFire C18 (3.5 μm, 150 × 3.0 mm i.d.) column equipped with a guard column (10 mm × 3.0 mm i.d.) (Waters, Milford, MA, USA). Data acquisition and processing were performed with LabSolution software (Kyoto, Japan). Microfractions were evaporated with a Genevac EZ-2 plus vacuum centrifuge (Ipswich, UK). Semipreparative HPLC-DAD separations were carried out with an Agilent HP 1100 Series system (Santa Clara, CA, USA) consisting of a quaternary pump, autosampler, column oven, and a diode array detector. SunFire C18 (5 μm, 10 × 150 mm i.d.) columns (Waters) were used for reversed-phase semipreparative separations. Data acquisition and processing were performed with Chemstation software. Preparative HPLC was carried out on a Puriflash 4100 system (Interchim, Montluçon, France) or a Reveleris PREP purification system (Büchi, Flawil, Switzerland). Plant Material. H. oocephalum aerial parts were collected from Mashhad, Razavi Khorasan, Iran, in May 2015. A voucher specimen (voucher number 13249) has been deposited at the Herbarium of Mashhad University of Medical Sciences. After harvesting, the plants were dried at room temperature in the shade and stored in paper bags until extraction. Extraction and Isolation. The aerial parts (4 kg) were milled and exhaustively percolated with methanol (20 L, 24 h). The extract was filtered and dried under reduced pressure to afford 55 g of solid residue. The methanol extract was suspended in water and partitioned

successively with petroleum ether, dichloromethane, ethyl acetate, and n-butanol, to afford 10, 12, 13, and 20 g fractions, respectively. Microfractionation for Activity Profiling. HPLC-based activity profiling of the dichloromethane extract was performed according to a previously established protocol.11,12 The extract (10 mg/mL in DMSO) was submitted to HPLC-PDA in three portions of 300 μg (30 μL). The mobile phase was H2O (A) and CH3CN (B), both containing 0.1% trifluoroacetic acid (TFA), and the following gradient profile was used: 55% B isocratic (0−1 min), 55% → 90% B (1−11 min), 90% → 93% B (11−21 min), 93% → 100% B (21−23 min), and 100% B (23−35 min). The flow rate was 0.4 mL/min, and 1 min fractions (between min 1 and 35) were collected into a 96 deep well plate with the aid of an FC 204 fraction collector (Gilson). Corresponding microfractions from the three separations were combined. After drying the plate in a vacuum centrifuge (Genevac), microfractions were tested for antiprotozoal activity. Preparative Isolation. The dried CH2Cl2 extract (12 g) was fractionated by column chromatography (CC) on silica gel (40 × 5 cm, 230−400 mesh), using a gradient of n-hexane−chloroform (100:0 → 0:100). Fractions 1−28 (F1−F28) were combined on the basis of TLC patterns (silica gel; n-hexane−CHCl3, 4:1, 1:1, and 1:9; detection with vanillin sulfuric acid reagent and heating to 105 °C). Fractions were analyzed by HPLC-PDA-ELSD-MS to track peaks previously seen in the active time windows of the activity profile. The mobile phase was H2O (solvent A) and CH3CN (solvent B), both containing 0.1% TFA, and the following gradient was used: 55% B isocratic (0−1 min), 55% → 90% B (1−11 min), 90% → 93% B (11− 21 min), 93% → 100% B (21−23 min), 100% B (23−35 min). The flow rate was 0.4 mL/min. Fractions F9 and F10 (A) were combined (764.58 mg) and submitted to column chromatography on silica gel (460 × 26 mm, 230−400 mesh) using a gradient of n-hexane and EtOAc (100:0 → G



Article

Table 6. 1H and 13C NMR Spectroscopic Data for Compounds 18 and 19 (MeOD; 500.13 MHz for 1H and 125.77 MHz for 13 C NMR; δ in ppm) 18 δC, typea position 1 2 3 4 5 6 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 OH-2 OH-4

E

19 δH (J in Hz)a

Z

101.3, C 164.29; 164.33, C 111.7, C 159.2, C 102.1, C 158.8, C 35.4, CH2 110.5, C 200.1, C 110.30; 110.33, C 187.8, C 15.0, CH3 5.5, CH3 22.2; 22.4, CH2 124.0, CH 124.5, CH 135.3, C 135.8, C 40.9, CH2 33.1, CH2 27.7, CH2 27.8, CH2 125.5, CH 125.8, CH 132.0, C 17.70, CH3 17.8, CH3 25.9, CH3 26.0, CH3 16.4, CH3 23.7, CH3 209.5, C 39.5, CH 19.3, CH3 19.3, CH3

δC, typea,d

E

Z

E

δH (J in Hz)a Z

E

Z

e e

111.2, C 159.7, C e e

3.30b 3.21, d (16.1)

3.30b 3.22, d (16.1)

35.5, CH2 e

2.28, s 1.74, s 3.27b 5.20b

5.22b

1.94, m 2.04, m 5.04, tqq (7.1, 1.4, 1.2)

2.22, m 2.10, m 5.16b

1.54, br s 1.59, br s 1.75, br s

1.63, br s 1.67, br s 1.65, br s

3.54, qq (6.7, 6.7) 1.07, d (6.9) 1.09, d (6.9) 13.77; 13.79, sc

199.8, C 110.3, C 187.8, C 15.0, CH3 5.5, CH3 22.1; 22.3, CH2 123.9, CH 135.9, C 40.9, CH2 27.7, CH2 125.5, CH 131.4, C 17.69, CH3 25.8, CH3 16.2, CH3 202.7, C 31.3, CH3

124.4, CH

2.30, s 1.75, s 3.27b 5.19b

5.21b

32.9, CH2 27.8, CH2 125.8, CH

1.95, m 2.05, m 5.05, tqq (6.9, 1.4, 1.2)

2.23, m 2.11, m 5.17b

17.74, CH3 25.9, CH3 23.7, CH3

1.55, br s 1.61, br s 1.76, br s

1.64, br s 1.68, br s 1.66, br s

2.48, s

13.49; 13.51, sc 6.56, br sc

a

Signals could not be assigned to E or Z unless specified. bOverlapping signals. cMeasured in CDCl3. dExtracted from H−C 2D inverse-detected experiments due to a low amount of sample. eSignal not visible.

0:100) and a final wash with methanol (500 mL). Subfractions A1− A23 were combined based on TLC analysis and analyzed by HPLCPDA-ELSD-MS to track the active peaks. Fraction A4 (12 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3OH (B); 80% B (0−1 min), 80 → 100% B (1−30 min), 100% B (30−40 min); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 100 μL] to afford compounds 1 (0.5 mg, tR 26.0 min) and 2 (1.0 mg, tR 30.0 min). Fraction A7 (22 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3OH (B); 75% B (0−1 min), 75 → 90% B (1−30 min), 90 → 93% B (30−35 min), 93 → 100% B (35−36 min), 100% B (36−40); flow rate 4 mL/min; sample concentration 50 mg/mL in DMSO; injection volume 100 μL] to yield compound 3 (0.5 mg, tR 18.6 min). Final purification was achieved on a Sephadex LH-20 column (80 × 1 cm) using methanol as eluent. Fraction A11 (24 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3OH (B); 80% B (0−1 min), 80 → 95% B (1−25 min), 95 → 100% B (25−30 min), 100% B (30−35 min); flow rate 4 mL/min; sample concentration 30 mg/mL in DMSO; injection volume 100 μL] to afford compounds 14 (0.7 mg, tR 14.5 min) and 18 (2.2 mg, tR 17.9 min). Final purification was on a Sephadex LH-20 column (80 × 1 cm) with methanol as mobile phase. Fraction A12 (26 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3OH (B); 75% B (0−1 min), 55 → 90% B (1−30 min), 90 → 93% B (30−35 min), 93 → 100% B (35−36 min), 100%

B (36−40 min); flow rate 4 mL/min; sample concentration 30 mg/ mL in DMSO; injection volume 100 μL] to afford compounds 17 (1.2 mg, tR 12.8 min), 16 (1.0 mg, tR 15.4 min), and 15 (1.0 mg, tR 17.8 min). Fraction A13 (29 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3OH (B); 75% B (0−1 min), 55 → 90% B (1−30 min), 90 → 93% B (30−35 min), 93 → 100% B (35−36 min), 100% B (36−40 min); flow rate 4 mL/min; sample concentration 30 mg/ mL in DMSO; injection volume 100 μL] to afford compounds 12 (1.9 mg, tR 10.3 min), 13 (0.9 mg, tR 13.8 min), and 19 (1.1 mg, tR 22.2 min), which were purified on a Sephadex LH-20 column (80 × 1 cm) using methanol as eluent. Fraction A16,17 (23 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3CN (B); 55% B (0−1 min), 55 → 90% B (1−11 min), 90 → 93% B (11−21 min), 93 → 100% B (21−23 min), 100% B (23−40 min); flow rate 4 mL/min; sample concentration 20 mg/ mL in DMSO; injection volume 100 μL] to afford compounds 22 (2.7 mg, tR 10.8 min), 20 (0.8 mg, tR 23.7 min), and 21 (1.5 mg, tR 26.3 min). Fraction A21 (41 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3CN (B); 55% B (0−1 min), 55 → 90% B (1−11 min), 90 → 93% B (11−21 min), 93 → 100% B (21−23 min), 100% B (23−40 min); flow rate 4 mL/min; sample concentration 50 mg/ mL in DMSO; injection volume 100 μL] to afford compounds 24 (1.9 mg, tR 7.5 min), 23 (0.4 mg, tR 9.1 min), 8 (0.3 mg, tR 17.7 min), 6 (1.1 mg, tR 21.5 min), 4 (1.5 mg, tR 24.2 min), and 5 (1.2 mg, tR H



Article

Chart 1

26.9 min). Final purification was on a Sephadex LH-20 column (80 × 1 cm) with methanol as eluent. F11, F12, F13, and F14 (B) were combined (550 mg) and submitted to column chromatography on silica gel (460 × 26 mm, 230−400 mesh) using a gradient of n-hexane and EtOAc (100:0 → 0:100) as mobile phase and a final wash with methanol (500 mL). Subfractions B1−B19 were combined based on TLC analysis and analyzed by HPLC-PDA-ELSD-MS to track the active peaks. Fraction B15 (13.38 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3CN (B); 55% B (0−1 min), 55 → 88% B (1−11 min), 88% B (11−16 min), 88 → 90% B (16−26 min), 90 → 91% B (26-31 min), 91 → 100% B (31−36 min), 100% B (36−45 min); flow rate 4 mL/min; sample concentration 20 mg/mL in DMSO; injection volume 100 μL] to afford compounds 10 (0.4 mg, tR 14.9 min), 11 (0.8 mg, tR 16.4 min), and 9 (0.5 mg, tR 16.9 min). Final purification was on a Sephadex LH-20 column (80 × 1 cm) with methanol as mobile phase. Fraction B17 (20.18 mg) was purified by semipreparative RP-HPLC [H2O (A), CH3CN (B); 55% B (0−1 min), 55 → 88% B (1−11

min), 88% B (11−16 min), 88 → 90% B (16−26 min), 90 → 91% B (26−31 min), 91 → 100% B (31−36 min), 100% B (36−45 min); flow rate 4 mL/min; sample concentration 20 mg/mL in DMSO; injection volume 100 μL] to afford compound 7 (2.1 mg, tR 26.5 min). Helicyclol (1): white solid; [α]25D +1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 219 (4.18), 323 (3.95) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 519.2357 [M + Na]+ (calcd for C29H36O7Na+, 519.2353). Helicepyrone (2): white solid; [α]25D +1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 223 (4.22), 313 (3.99) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 519.2358 [M + Na]+ (calcd for C29H36O7Na+, 519.2353). Cycloarzanol (3): white solid; UV λmax (MeOH) (log ε) 225 (4.16), 310 (4.10) nm; 1H and 13C NMR, see Table 1; HRESIMS m/ z 409.1266 [M + Na]+ (calcd for C21H22O7Na+, 409.1258). Achyroclinopyrone A (4): white solid; 1H and 13C NMR see (Table S1 Supporting Information); ESIMS m/z 499 [M + H]+. I



Article

Figure 2. Key COSY (blue bonds) HMBC (red arrows) correlations for compounds 1, 2, 6, 12, and 16 and key NOESY (green arrows) correlations for compound 1.

Scheme 1. Proposed Biosynthesis of Spiroketal Derivativesa

For compounds with a methyl group at C-11, an acetyl CoA instead of a propionyl-CoA unit would serve as starter for the α-pyrone ring.

a

Achyroclinopyrone B (5): white solid; 1H and 13C NMR see (Table S1 Supporting Information); ESIMS m/z 513 [M + H]+. 16Z/E-Achyroclinopyrone C (6): white solid; UV λmax (MeOH) (log ε) 223 (4.47), 323 (4.14) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 507.2357 [M + Na]+ (calcd for C28H36O7Na+, 507.2353). 16Z/E-Achyroclinopyrone D (7): white solid; [α]25D −1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 222 (4.35), 319 (4.06) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 521.2507 [M + Na]+ (calcd for C29H38O7Na+, 521.2510). 23-Methyl-6-O-desmethylauricepyrone (8): white solid; 1H and 13 C NMR, see (Table S2 Supporting Information); ESIMS m/z 485 [M + H]+.

Norauricepyrone (9): white solid; 1H and 13C NMR, see (Table S2 Supporting Information); ESIMS m/z 431 [M + H]+. Arenol B (10): white solid; UV λmax (MeOH) (log ε) 223 (4.15), 318 (3.88) nm; 1H and 13C NMR, see Table 3; HRESIMS m/z 439.1746 [M + Na]+ (calcd for C23H28O7Na+, 439.1727). Arenol C (11): white solid; [α]25D +1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 223 (4.31), 322 (4.05) nm; 1H and 13C NMR, see Table 3; HRESIMS m/z 453.1904 [M + Na]+ (calcd for C24H30O7Na+, 453.1884). Helispiroketal A (12): white solid; [α]25D +1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 218 (4.13), 240 (sh, 4.02), 311 (4.15) nm; 1H and 13C NMR, see Table 4; HRESIMS m/z 381.1316 [M + Na]+ (calcd for C20H22O6Na+, 381.1308). J



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Helipyrone A (22): white solid; 1H and 13C NMR, see (Table S4 Supporting Information); ESIMS m/z 321 [M + H]+. Helipyrone B (23): white solid; 1H and 13C NMR, see (Table S4 Supporting Information); ESIMS m/z 307 [M + H]+. Helipyrone C (24): white solid; 1H and 13C NMR, see (Table S4 Supporting Information); ESIMS m/z 293 [M + H]+. In Vitro Biological Testing. Assays for in vitro activities against the protozoan parasites P. falciparum (NF54) erythrocytic stage, L. donovani (MHOM-ET-67/L82) axenically grown amastigotes, and cytotoxicity in L6 cells (rat skeletal myoblasts) were carried out according to the procedures described by Witschel et al.33 For this purpose, serial drug dilutions of 11 3-fold dilution steps covering a range from 100 to 0.001 μg/mL were prepared. The IC50 values were calculated by linear regression.34 Assays were run singly and repeated at least two times. Using 1H NMR spectroscopy, the purity of the compounds was determined as >95%. Miltefosine and chloroquine as positive controls, with a purity of >95%, were purchased from Sigma. The selectivity index was calculated as IC50 for L6-cells/IC50 for parasites.

Table 7. In Vitro Activity of Compounds 1−20 and 22−24 against L. donovani (MHOM-ET-67/L82), P. falciparum (NF54), and Cytotoxicity in L6 Cells compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 23 24 positive controls

L. donovani IC50 (μM)a

P. falciparum IC50 (μM)a

L6 cells IC50 (μM)a

2.0 ± 0.1 (6.0)b 1.9 ± 0.1 (11.2)b 1.8 ± 0.2 (29.7)b 2.7 ± 0.4 (15.0)b 5.6 ± 0.4 (6.4)b 3.1 ± 0.1 (12.3)b 4.4 ± 1.8 (11.1)b 16.8 ± 2.1 (2.25)b 13.7 ± 3.9 (3.8)b 5.8 ± 0.5 (10.4)b 4.8 ± 0.5 (11.5)b 5.1 ± 0.7 (11.1)b 7.1 ± 0.6 (6.2)b 6.0 ± 0.3 (7.2)b 1.7 ± 0.4 (7.6)b 1.9 ± 0.5 (10.5)b 5.8 ± 0.6 (9.5)b 2.2 ± 0.1 (8.5)b 2.8 ± 0.3 (15.6)b 2.5 ± 0.7 (22.9)b 14.9 ± 3.1 (3.6)b 15.5 ± 2.0 (2.8)b 15.7 ± 1.9 (1.7)b 0.285 ± 0.065c

7.12 ± 1.2 (1.7)b 9.3 ± 1.2 (2.3)b 11.3 ± 0.3 (4.7)b 3.8 ± 0.6 (11.3)b 1.2 ± 0.01 (15.7)b 5.7 ± 1.8 (9.6)b 2.6 ± 0.1 (7.7)b 2.5 ± 0.09 (5.36)b 7.4 ± 0.6 (7.6)b 8.0 ± 1.4 (5.5)b 5.5 ± 0.3 (7.9)b 14.6 ± 0.07 (3.9)b 31.7 ± 1.7 (0.8)b 18.0 ± 6.0 (2.1)b 7.6 ± 0.1 (4.8)b 22.7 ± 3.2 (1.9)b 23.1 ± 3.2 (2.3)b 7.2 ± 0.4 (5.5)b 23.9 ± 1.4 (2.2)b 8.6 ± 0.5 (4.4)b 8.9 ± 1.2 (5.4)b 18.1 ± 0.3 (3.4)b 22.3 ± 1.7 (2.5)b 0.004 ± 0.000d

12.0 ± 4.2 21.4 ± 1.5 53.1 ± 8.5 39.7 ± 22.7 36.3 ± 22.3 38.4 ± 0.6 48.3 ± 10.5 37.8 ± 17.3 52.2 ± 0.0 61.1 ± 8.3 55.5 ± 1.1 56.3 ± 2.0 44.1 ± 21.3 43.0 ± 17.0 13.2 ± 7.6 20.1 ± 1.0 55.1 ± 0.7 18.8 ± 0.7 43.4 ± 21.6 57.6 ± 3.7 54.3 ± 9.5 43.9 ± 10.7 26.5 ± 7.5 0.005 ± 0.003e

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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.8b01031.

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a

Average of two independent assays. bSelectivity index (SI): IC50 in L6 cells divided by IC50 in the titled parasitic strain. cMiltefosine. d Chloroquine. ePodophyllotoxin.

1D and 2D NMR spectra of new compounds 1−3, 6, 7, and 10−19; ECD spectra of compounds 1, 12, and 16; computational methods; HPLC chromatogram of the dichloromethane extract of H. oocephalum aerial parts; NMR tables of known compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +98 513 605 97 05. E-mail: [email protected]. *Tel: +41 61 207 14 25. Fax: +41 61 207 14 74. E-mail: [email protected].

[α]25D

Helispiroketal B (13): white solid; +2 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 223 (3.88), 243 (sh, 3.82), 310 (3.87) nm; 1H and 13C NMR, see Table 4; HRESIMS m/z 395.1473 [M + Na]+ (calcd for C21H24O6Na+, 395.1465). Helispiroketal C (14): white solid; [α]25D +2 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 221 (3.90), 244 (sh, 3.80), 313 (3.92) nm; 1H and 13C NMR, see Table 4; HRESIMS m/z 423.1794 [M + Na]+ (calcd for C23H28O6Na+, 423.1778). Helispiroketal D (15): white solid; [α]25D +8 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 219 (4.16), 243 (sh, 4.05), 313 (4.21) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 423.1799 [M + Na]+ (calcd for C23H28O6Na+, 423.1778). Helispiroketal E (16): white solid; [α]25D −1 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 219 (4.08), 243 (sh, 3.97), 313 (4.11) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 409.1634 [M + Na]+ (calcd for C22H26O6Na+, 409.1621). Helispiroketal F (17): white solid; [α]25D −2 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 220 (4.03), 240 (sh, 3.93), 312 (4.06) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 395.1473 [M + Na]+ (calcd for C21H24O6Na+, 395.1465). Helispiroketal G (18): white solid; [α]25D 0 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 219 (4.29), 243 (sh, 4.09), 313 (4.23) nm; 1H and 13C NMR, see Table 6; HRESIMS m/z 477.2256 [M + Na]+ (calcd for C27H34O6Na+, 477.2247). Helispiroketal H (19): white solid; [α]25D +2 (c 0.1, MeOH); UV λmax (MeOH) (log ε) 220 (4.10), 243 (sh, 3.94), 312 (4.02) nm; 1H and 13C NMR, see Table 6; HRESIMS m/z 449.1938 [M + Na]+ (calcd for C25H30O6Na+, 449.1934). Italidipyrone (20): white solid; 1H and 13C NMR, see (Table S3 Supporting Information); ESIMS m/z 529 [M + H]+. 23-Methylitalidipyrone (21): white solid; 1H and 13C NMR, see (Table S3 Supporting Information); ESIMS m/z 543 [M + H]+.

ORCID

Maryam Akaberi: 0000-0002-3971-2377 Ombeline Danton: 0000-0002-9323-7079 Zahra Tayarani-Najaran: 0000-0001-8899-0886 Javad Asili: 0000-0002-4635-474X Mehrdad Iranshahi: 0000-0002-3018-5750 S. Ahmad Emami: 0000-0003-4298-3132 Matthias Hamburger: 0000-0001-9331-273X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by a grant from Mashhad University of Medical Sciences (Grant No. 930705). Thanks are due to M. Cal, S. Keller-Marki, and R. Rocchetti for the in vitro assay results.

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