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Prenylated Acylphloroglucinols with Leishmanicidal Activity from the Fern Elaphoglossum lindbergii Cecilia Socolsky,*,† Efrain Salamanca,‡ Alberto Giménez,‡ Susana A. Borkosky,§ and Alicia Bardón† †

INQUINOA−CONICET, Ayacucho 471, Tucumán 4000, Argentina Instituto de Investigaciones Fármaco Bioquímicas (IIFB), Universidad Mayor de San Andrés, Avenida B. Saavedra 2224, La Paz 3239, Bolivia § Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471, Tucumán 4000, Argentina ‡

S Supporting Information *

ABSTRACT: Purification of a diethyl ether extract of the Argentinian fern Elaphoglossum lindbergii afforded five new prenylated acylphloroglucinols, lindbergins E−I (1−5), of which two showed significant in vitro leishmanicidal activity against promastigotes of Leishmania braziliensis and L. amazonensis. The structures of compounds 1−5 were elucidated based on analysis of their spectroscopic data and comparison with values previously reported for other phloroglucinol derivatives isolated from plant species of the genera Hypericum, Dryopteris, and Elaphoglossum. Fragmentation and rearrangement patterns of prenylated acylphloroglucinols were analyzed, and some mechanisms were proposed to rationalize the peaks observed in the mass spectra of these natural products produced by EI and FAB. Compounds isolated from E. lindbergii show the opposite absolute configuration when compared to those reported from E. crassipes. Empirical evidence indicates that acylphloroglucinols carrying a prenylated acylfilicinic acid residue possess a high-amplitude configuration-dependent Cotton effect centered at 350−360 nm in their CD curves, from which the absolute configuration of the sole chiral center of the prenylated acylfilicinic acid moiety can be deduced.

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species and discuss the absolute configuration assignment of acylphloroglucinols carrying a prenylated acylfilicinic acid residue. Leishmaniasis, a neglected disease according to the WHO, is caused by protozoa of over 20 species of the genus Leishmania and is transmitted to humans through the bites of infected female sandflies.8 The illness affects 98 countries around the globe, with 12 million people infected and 350 million at risk.9 The standard treatments for this disease include the use of intralesional injections of pentavalent antimony, topical amphotericin B, or oral treatment with drugs such as miltefosine.10 However, the emergence of drug-resistant parasites and the serious side effects of the available drugs has encouraged the search for new effective and safe drugs for the treatment of leishmaniasis.10,11 Plants have been used for centuries for the treatment of many diseases, and they are

erns belonging to the genus Elaphoglossum are a rich source of prenylated acylphloroglucinols.1 More than 25 compounds of this class were isolated from five Argentinian species in this genus studied in our laboratory in the past few years.2−6 These compounds seem to be the main constituents of the exudates produced by secreting glands located on the scales, which usually cover the rhizomes of these plants.1 They show interesting biological effects, such as molluscicidal activity against the potential schistosomiasis vector snail Biomphalaria peregrina.2,3,7 Some of these compounds also display antibacterial activity against pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa and even antidepressant-like effects, as determined by a forced-swimming test in mice.4,6 In an earlier article, we reported on the isolation, characterization, and antibacterial activity of four acylphloroglucinols from Elaphoglossum lindbergii (Mett. ex Kuhn) Rosenst (Dryopteridaceae).5 Herein, we present the structures of another five new phloroglucinol derivatives from this fern © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 27, 2015

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DOI: 10.1021/acs.jnatprod.5b00767 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

important sources of drug candidates.12 A great number of plant species have shown activity against different forms of the parasite.13 Some pure compounds such as certain alkaloids, terpenoids, and flavonoids display leishmanicidal activity against protozoa of the genus Leishmania.13 To the best of our knowledge, no records on the leishmanicidal activity of acylphloroglucinols are present in the literature. Accordingly, it was decided to evaluate the effects of the new acylphloroglucinols isolated from E. lindbergii against promastigotes of Leishmania amazonensis and L. braziliensis.

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isoprenyl moiety could be constructed through 1H−1H COSY correlations of H-10 to H-9, H-12, and H-13 and HMBC correlations of H-12 and H-13 to C-10 and C-11. The location of this residue at C-4 was deduced from HMBC correlations of H-9 to C-3, C-4, C-5, and C-8. The high degree of unsaturation and the several quaternary sp2 carbons detected in the 13C NMR spectrum of 1 suggested the presence of an aromatic ring. 1H−13C HMBC correlations of OH-5′ to C-5′, C-6′, and C-10′, OH-7′ to C-6′, C-7′, and C-8′, CH-4′ to C-5′, C-9′, and C-10′, and CH3-11′ and CH3-12′ to C-2′ and C-3′, allowed construction of a 2,2-dimethylchromene ring (Figure 1).

RESULTS AND DISCUSSION

The crude diethyl ether extract of the rhizomes and roots of E. lindbergii was purified by column chromatography over silica gel, and a fraction of interest was further processed by means of normal-phase HPLC to afford five new prenylated acylphloroglucinols, lindbergins E−I (1−5). Compound 1 was obtained as a yellow gum. The HREIMS of this compound showed a molecular ion peak [M]+ centered at m/z 552.2723, consistent with the molecular formula C32H40O8 (calcd 552.2724) and accounting for 13 degrees of unsaturation. A broad IR absorption band at 3171 cm−1 was assigned to O−H stretching, and broad bands at 2708, 2644, and 2602 cm−1 pointed to the presence of enol hydroxy groups.14 This evidence, together with duplicated signals observed in the 1H NMR spectrum of 1, suggested the coexistence of tautomers in solution. The 1H NMR spectrum of lindbergin E (1) showed resonances of four phenol/enol hydroxy groups (δH 18.66, 16.26, 11.38, and 9.88, 1H each, s), five singlet methyl groups (δH 1.53, 1.52, 1.51, 1.36, and 1.31), two triplet methyl groups (δH 1.01 and 0.99), an isolated methylene (AB system, δH 3.55 and 3.52, 1H each, d, J = 15.5 Hz), a trisubstituted double bond (δH 4.63, 1H, m), and a cis-disubstituted double bond (δH 6.65 and 5.59, 1H each, d, J = 10.0 Hz). The 13C NMR spectrum showed 32 carbon resonances and, together with HSQC data, revealed the presence of six methylenes, three olefinic methines (δC 127.2, 119.3, and 118.6), and 16 quaternary carbons, including three oxygenated olefinic carbons (δC 163.3, 160.9, and 157.5), six sp2 carbons (δC 137.5, 115.1, 111.8, 111.1, 106.0, and 105.4), an sp3 oxygen-bearing carbon (δC 80.2), and two ketone carbonyl carbons (δC 208.5 and 207.6). An

Figure 1. Selected 2D correlations for lindbergin E (1).

Likewise, the acylfilicinic acid residue was built based on HMBC correlations of OH-3, OH-5, CH2-7, CH3-8, and CH2-9 to the ring carbons (Figure 1, Table 1). The isolated methylene showed HMBC correlations with C-1, C-2, C-3, C-5′, C-6′, and C-7′, indicating it connected both rings through C-2 and C-6′. It is worth mentioning that the proton of the OH group attached to C-5 is strongly linked to the oxygen atom of the carbonyl C-14 by hydrogen bonding, giving rise to a sixmembered chelate. The above-mentioned proton is “shared” by the oxygen atoms on C-5 and C-14 due to tautomerism, as shown in Figure 2.3 This is evidenced by the J2 correlation of OH-5 to C-14 present in the HMBC spectrum of 1. At the same time, this correlation was used to locate the acyl group linked to carbonyl C-14 to the filicinic acid-type ring. The chemical shift of OH-7′ (δH 16.26) indicated the proton of this hydroxy group is engaged in hydrogen bonding. Then, the remaining acyl group was located at C-8′ so that carbonyl C-1″ and OH-7′ could give a six-membered arrangement by B



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Table 1. NMR Spectroscopic Data of Compounds 1 and 2 (Acetone-d6, 500 MHz)a 1 δH (J in Hz)

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 1″ 2″ 3″ 4″ OH-3 OH-5 OH-5′ OH-7′ a

3.55 3.52 1.52 2.58 2.72 4.63

d (15.5) d (15.5) s dd (13.5, 6.8) dd (13.5, 9.0) m

1.31 brs 1.36 brs 3.21 3.10 1.69 0.99

ddd (16.0, 8.0, 7.0) ddd (16.0, 10.0, 7.5) m t (7.5)

5.59 d (10.0) 6.65 d (10.0)

1.51 s 1.53 s 3.12 m 1.72 m 1.01 t (7.5) 9.88 s 18.66 s 11.38 s 16.26 s

2

δC, type 189.7, 115.1, 171.8, 50.7, 199.8, 111.8, 17.8,

C C C C C C CH2

24.1, CH3 40.1, CH2 119.3, 137.5, 26.6, 18.6, 207.6, 44.3,

CH C CH3 CH3 C CH2

19.8, CH2 15.1, CH3

80.2, 127.2, 118.6, 160.9, 111.1, 163.3, 106.0, 157.5, 105.4, 28.9,

C CH CH C C C C C C CH3

29.0, 208.5, 47.5, 20.1, 15.2,

CH3 C CH2 CH2 CH3

δH (J in Hz)

HMBC

1, 1, 3, 3, 3,

2, 2, 4, 4, 4,

3, 3, 5, 5, 5,

5′, 6′, 7′ 5′, 6′, 7′ 9 10, 11 8, 10, 11

3.55 3.52 1.52 2.72 2.58 4.63

d (16.0) d (16.0) s dd (13.5, 9.0) dd (13.5, 7.0) m

10, 11, 13 10, 11, 12

1.32 brs 1.36 brs

14 14 14, 15 15

3.23 ddd (15.5, 8.5, 7.5) 3.17−3.08b 1.65 mc 1.40−1.36b 1.40−1.36b 0.92 t (7.0)

2′, 10′, 11′/12′ 2′, 5′, 9′, 10′

5.59 d (10.0) 6.65 d (10.0)

2′, 3′

1.51 s

2′, 3′

1.53 s

1″ 1″, 2″ 2″ 4 4, 5, 6, 14 5′, 6′, 10′ 6′, 7′, 8′

3.16−3.10b 1.72 sextet (7.0) 1.01 t (7.0) 9.88 s 18.67 s 11.38 s 16.25 s

δC, type 189.6, 115.2, 171.8, 50.7, 200.0, 111.7, 17.8,

HMBC

C C C C C C CH2

24.2, CH3 40.1, CH2

1, 1, 3, 3, 3,

2, 2, 4, 4, 4,

3, 3, 5, 5, 5,

5′, 6′, 7′ 5′, 6′, 7′ 9 8, 10, 11 8, 10, 11

119.3, 137.5, 26.6, 18.6, 207.8, 42.3,

CH C CH3 CH3 C CH2

26.3, 33.2, 24.1, 15.2, 80.2, 127.2, 118.6, 160.9, 108.1, 163.3, 106.0, 157.5, 105.4, 28.9,

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

14, 15, 17, 18

29.0, 208.5, 47.5, 20.1, 15.2,

CH3 C CH2 CH2 CH3

2′, 3′, 11′

10, 11, 13 10, 11, 12 14, 16, 17

17, 18 2′, 10′, 11′/12′ 2′, 5′, 9′, 10′

2′, 3′, 12′

1″, 3″, 4″ 1″, 2″, 4″ 2″, 3″ 4 4, 5, 6, 14 5′, 6′, 10′ 6′, 7′, 8′

Only signals belonging to the most stable tautomer are listed. bOverlapping signals. cObscured due to tautomerism.

one of the most intense peaks in their mass spectra is produced by a fragmentation α to the methylene bridge and β to the aromatic ring, leading to the loss of the acylfilicinic acid moiety as a radical (Figures S47 and S48, Supporting Information).15,16 The mass loss may be used to assess the length of the acyl residue attached to this ring and the mass-charge ratio of the peak to deduce the length of the acyl residue attached to the aromatic ring. The EIMS of lindbergin E (1) was found to display a base peak at m/z 275, [M − 277]+, consistent with the presence of butanoyl residues attached to both rings. Another typical loss for this type of natural products comes from a McLafferty rearrangement involving the carbonyl C-5 (this carbon is a carbonyl in at least one tautomer) and the prenyl residue attached to C-4 (Figure 3). In acylphloroglucinols carrying an isoprenyl moiety at C-4, this rearrangement gives a

Figure 2. Two of the tautomers of lindbergin E (1).

intramolecular hydrogen bonding. 1H−1H COSY correlations of CH2-16 with CH3-17 and CH2-15 and of CH2-3″ with CH22″ and CH3-4″, along with HMBC correlations of H-15 to C14 and of H-2″ to C-1″, revealed the presence of two butanoyl residues (Figure 1). Analysis of the EIMS of prenylated acylphloroglucinols from ferns indicates that the base peak or C



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The HREIMS of lindbergin F (2) showed a molecular ion peak at m/z 580.3041, indicating the molecular formula to be C34H44O8 (calcd 580.3037). The 1D NMR spectra of 2 were very similar to those of compound 1, with the differences concerning solely the acyl moieties. 1H−1H COSY correlations of H-3″ to H-2″ and of H-4″ and H-15 to H-16 and HMBC correlations of H-2″ to C-1″, H-15 to C-14, C-16, and C-17, and H-19 to C-17 and C-18 allowed the identification of a butanoyl residue and a hexanoyl residue. A long-range correlation of OH-5 to C-14 was observed in the HMBC spectrum of 2, indicating that the longer acyl chain is attached to C-6. Further evidence was obtained from the EIMS of lindbergin F (2), which showed a base peak at m/z 275, [M − 305]+, consistent with a hexanoyl residue attached to the prenylated filicinic acid ring and a butanoyl residue attached to the chromene ring. Thus, the structure of compound 2 was established as depicted. The absolute configuration of 2 at C-4 was assigned as being identical to that of 1 based on the similarities between their ECD spectra (Figure 5).

Figure 3. McLafferty rearrangement in acylphloroglucinols carrying a prenylated acylfilicinic acid moiety.

peak at [M − 68]+, and those having a geranyl residue attached to C-4, like yungensin A, show a peak at [M − 136]+ (Figures S47 and S48, Supporting Information).3−5,15 It was observed previously for other acylphloroglucinols that the chemical shift of the methyl group of a butanoyl residue is typically located at 0.96−1.02 ppm, whereas the methyl group of a hexanoyl residue is shifted upfield (0.90−0.93 ppm).2−6,17−21 The chemical shifts of the terminal methyl groups of the two butanoyl moieties present in lindbergin E (1) (δH 1.01 and 0.99) were used to confirm this observation. Moreover, while the shape of the triplet arising from the terminal methyl group of a butanoyl residue is well defined, that of a hexanoyl moiety is distorted (second-order signal) (Figure S49, Supporting Information). This information can be used as evidence to assess the length of acyl groups in tri- and polycyclic acylphloroglucinols, where the signals usually overlap. The ECD (electron circular dichroism) spectrum of lindbergin E in EtOH showed positive Cotton effects at Δε358 +7.1 and Δε236 +4.5 and negative Cotton effects at Δε314 −4.9, Δε255 −0.8, and Δε208 −4.0. The structure of lindbergin E (1) was almost identical to that of crassipin A, a phloroglucinol derivative isolated from E. crassipes, except that an isoprenyl side chain instead of a geranyl residue is attached to C-4.6 The absolute configuration of crassipin A was assigned as R by VCD spectroscopy in combination with an in silico method.6 Comparison of the CD spectrum of 1 with that previously recorded for crassipin A (Figure 4) showed that they display opposite-sign bands all through their spectra. This pattern suggests these two natural products have an opposite configuration at C-4, the only chiral center present in these molecules. Thus, the absolute configuration of lindbergin E (1) at C-4 was assigned as S. On the basis of the foregoing evidence, the structure of 1 was established as depicted.

Figure 5. Comparison of the ECD spectra of lindbergins E−I (1−5).

Lindbergin G (3) was obtained as a yellow oil, for which the molecular formula, C37H52O8, was deduced from its HREIMS (found m/z 624.3664, calcd 624.3664). An additional isoprenyl residue was detected when analyzing the proton NMR spectrum of 3, in comparison with those of 1 and 2 (δH 5.23, 1H, tsept, J = 6.5, 1.5 Hz; 3.32, 2H, d, J = 6.5 Hz; 1.65, 3H, brs; 1.77, 3H, brs). The location of this moiety at C-5′ was established through long-range H−C correlations between H-7′ and C-4′, C-5′, and C-6′. The presence of a methoxy group was clear from the 1H NMR signal at δH 3.79 (3H, s). A J3 correlation between the protons of the −OCH3 group to C-4′ indicated its location at the carbon atom mentioned. The EIMS of lindbergin G (3) showed a base peak at m/z 319, [M − 305]+, due to the fragmentation α to the methylene bridge and β to the aromatic ring and consistent with the presence of a hexanoyl residue attached to each ring. There were some discrepancies when comparing the ECD spectra of 1 and 3. The negative Cotton effect observed at 315 nm for 1 was overlapped with a band centered at 288 nm, and the bands below 250 nm showed the opposite sign for these two compounds (Figure 5). Nevertheless, comparison of the specific rotation of compounds 1 and 3 (−50.1 and −44.6, respectively) indicate they display the same sign. Due to the fact that these two compounds possess only one chiral carbon, this would mean they show the same absolute configuration at

Figure 4. Comparison of the ECD spectra of crassipin A and lindbergin E (1). The spectrum of the former compound was multiplied by a scale factor (0.4) for comparison purposes. D



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fragmentations occurred α to the methylene bridges, giving sequential fragments that allowed identification of the acyl substituents linked to the different rings. The CD spectrum of 4 showed a positive Cotton effect at 356 nm (Δε356 +6.1), indicating the absolute configuration at C-4 to be S (Figure 5). Moreover, both chiral centers should have the same configuration (S); otherwise the structure would correspond to that of a meso-compound and would show in consequence no CD absorption bands. On the basis of the presented evidence, the structure of lindbergin H (4) was elucidated as shown. For compound 4, the location of the respective acyl moieties was not evident from the HMBC correlations. However, it has been observed for other Elaphoglossum acylphloroglucinols bearing a butanoyl or hexanoyl side chain that the chemical shift of the carbonyl group attached to the filicinic acid-type ring is 207.6−207.8 ppm in all cases (taken from 15 examples).3,5,6 Then, by analogy, the butanoyl residues (δC 207.7) should be attached to these rings, while the only hexanoyl moiety (δC 209.8) would be linked to the phloroglucinol-type ring. As explained in the previous paragraph, this was exactly the case, as confirmed by FABMS. This means that the chemical shift of the carbonyl carbon could be used as evidence to locate an acyl residue to one of the rings. The molecular formula of lindbergin I (5) was deduced as C48H64O12 from a molecular ion peak [M]+ at m/z 832.4420 in its HRFABMS (calcd 832.4399). The 1D NMR spectra of 5 resembled those of compound 4. The LRFABMS of lindbergin I (5) displayed a base peak at m/z 251, indicating that a hexanoyl residue is attached to a filicinic acid-type ring. The fragmentation β to the aromatic ring with the loss of an acylfilicinic acid residue as a radical was located at m/z 527, providing evidence of a butanoyl residue attached to the phloroglucinol-type ring. The absolute configuration of this natural product at C-4 was assigned as S by analysis of its CD spectrum (Figure 4). As a result, the structure of compound 5 was established as depicted. The absolute configuration of lindbergins A−D, described in a previous article,5 was also determined to be S by analysis of their ECD spectra, as they all showed a positive Cotton effect at 350−360 nm (Figure S50, Supporting Information). It is important to point out that acylphloroglucinols from E. lindbergii show opposite absolute configurations at C-4 when compared to those isolated from E. crassipes. The leishmanicidal activity of the new compounds (1−5) was evaluated against promastigotes of Leishmania braziliensis and L. amazonensis (Table 4). Lindbergins E−H (1−4) displayed higher potency against L. braziliensis than against L. amazonensis. Compounds 1 and 2 showed significant leishmanicidal activity (IC50 13.6 and 29.9 μM against L. braziliensis, respectively), with their potency being much weaker than that of amphotericin B, used as a positive control. Acylphloroglucinols 3 and 4 showed low activity (IC50 66.5 and 87.7 μM against L. braziliensis, respectively), while lindbergin I (5) resulted in inactivity against both Leishmania strains at the concentrations tested (IC50 > 100 μg/mL). Apparently, an increase in hydrophobicity of the tested substance leads to decreased activity. Compound 2 differs from 1 only in the length of one of the acyl substituents, and as a consequence of the presence of a hexanoyl residue instead of a butanoyl moiety, the leishmanicidal activity was found to be greatly reduced. Further studies with amastigote forms, the clinically relevant

the chiral center (S). Therefore, the structure of lindbergin G (3) was assigned as shown. It seems that both the ECD band located at 350−360 nm and that centered at 300−320 nm can be used as diagnostic observations for the absolute configuration assignment of the prenylated acylfilicinic acid chromophore. On the basis of the empirical evidence presented, it can be concluded that (a) bands below 250 nm are substitution-dependent and are, therefore, unsuitable for configurational assignment and (b) for a compound possessing the S-configuration at C-4, the ECD band centered at 350−360 nm is positive and that at 300−320 nm is negative, whereas these bands show opposite signs for compounds showing an R-configuration at the asymmetric carbon. The positive HRFABMS of lindbergin H (4) suggested the molecular formula C46H60O12 (found m/z 804.4088, calcd 804.4086). Some of the proton NMR signals of this compound were broad, as was previously observed for acylphloroglucinols carrying three or more rings (Figure S11, Supporting Information).5,19 The presence of two equivalent methylene bridges at δH 3.60 and 3.56 (2H each, d, J = 16.0 Hz) in the proton NMR spectrum of 4 suggested the presence of three rings and a certain symmetry in the molecule. Moreover, despite the molecule containing 46 carbon atoms, only 27 carbon resonances were observed in the 13C NMR spectrum of lindbergin H (4). Three triplet methyl groups presumably belonging to three acyl groups were detected in the 1H NMR spectrum of 4 at δH 1.02 (6H, J = 7.2 Hz) and 0.93 (3H, J = 7.0 Hz). A proton resonance at δH 18.71 revealed the presence of at least one acylfilicinic acid moiety. The area under this signal indicated two such protons were present, suggesting the trimer to display two filicinic acid-type rings. Acylfilicinic acid-type rings show only one free position to accommodate a methylene bridge and are thus always at the side of the molecule. Signals assigned to two sp2 oxygen-bearing carbons (δC 162.1 and 160.8) suggested the presence of a phloroglucinol-type ring in the structure of lindbergin H (4). The 1H and 13C NMR spectra of 4 resembled those of the previously reported compound lindbergin C (6), a tricyclic phloroglucinol derivative composed of two prenylated acylfilicinic acid moieties on the sides and a phloroglucinol-type ring in the middle, with these rings connected together through methylene bridges.5 The only differences in the NMR spectra of these two compounds (4 and 6) were related to the acyl groups. The FABMS of compound 4 showed a low-intensity peak at [M − 68]+, assigned to a McLafferty rearrangement involving the isoprenyl residue attached to C-4 and the carbonyl carbon C-5 (Figure 3), as was previously presented for compound 1. The base peak was located at m/z 223, and it apparently comes from a fragmentation α to the methylene bridge and β to the acylfilicinic acid residue, followed or preceded by a McLafferty rearrangement (loss of 68 mass units). These data are consistent with the presence of a butanoyl residue attached to the filicinic acid-type ring. Another fragmentation was observed β to the aromatic ring with the loss of an acylfilicinic acid residue as a radical (m/z 527), as was previously observed in the EIMS of this type of compound. The mass of this peak confirmed the presence of a hexanoyl moiety attached to the phloroglucinol-type ring. Fragmentations observed in the FABMS of bi- and polycyclic fern acylphloroglucinols have been studied in the past.22 Although the natural products investigated did not possess a prenyl group attached to the only sp3 carbon of the acylfilicinic acid ring, it was observed that all E



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Table 2. NMR Spectroscopic Data of Compound 3 (Acetone-d6, 500 MHz) δH (J in Hz)

position 1 2 3 4 5 6 7

3.60 3.57 1.52 2.71 2.56 4.61

8 9 10 11 12 13 14 15

1.29 brs 1.31 brs

16 17 18 19 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 1″ 2″ 3″ 4″ 5″ 6″ CH3OOH-3 OH-5 OH-2′ OH-6′ a

d (15.8) d (15.8) s dd (13.5, 9.0) dd (13.5, 7.0) m

3.25 ddd (15.2, 8.0, 6.5) 3.08 ddd (15.2, 8.0, 7.0) 1.73−1.65a 1.41−1.32a 1.41−1.32a 0.90 t (7.5)b

3.32 d (6.5) 5.23 tsept (6.5, 1.5) 1.65 brs 1.77 brs 3.16 m 1.73−1.65a 1.41−1.32a 1.41−1.32a 0.92 t (7.2)b 3.79 s 9.94 s 18.66 s 15.74 s 11.35 s

δC, type 189.6, 114.9, 171.8, 50.8, 200.0, 111.6, 18.2,

C C C C C C CH2

23.9, CH3 40.2, CH2 119.2, 137.6, 26.6, 18.5, 207.8, 42.3,

CH C CH3 CH3 C CH2

26.5, 33.2, 24.2, 15.2, 111.5, 161.5, 111.7, 162.2, 118.4, 164.4, 24.6, 125.2, 132.5, 26.8, 19.0, 209.0, 43.6, 26.3, 33.3, 24.2, 15.2, 64.4,

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

Table 3. 1D NMR Spectroscopic Data of Compounds 4 and 5 (Acetone-d6)

HMBC

4 position

1, 1, 3, 3, 3,

2, 2, 4, 4, 4,

3, 3, 5, 5, 5,

1 2 3 4 5 6 7

5′, 6′, 7′ 5′, 6′, 7′ 9 10, 11 10, 11

3.60 d (16.0) 3.56 d (16.0) 1.54 s 2.74 dd (13.5, 8.0) 2.70−2.63a 4.70 brsa

8 9

10, 11 10, 11

10 11 12 13 14 15

14, 16 14, 16

1.39 s 1.43 s 3.29−3.20b 3.15−3.07b 1.71 sext (7.2) 1.02 t (7.2)

16 17 18 19 1′,5′ 2′,4′ 3′ 6′ 7′ 8′

18

4′, 5′, 6′, 8′, 9′ 7′, 10′, 11′ 8′, 9′, 11′ 8′, 9′, 10′

9′ 10′ 11′ 12′ OH-3 OH-5 OH-2′, OH-4′

1″

a

4′ 4, 5, 6, 14 10′

δH (J in Hz)

3.29−3.21b 3.19−3.10b 1.76−1.65b 1.47−1.33b 1.47−1.33b 0.93 t (7.0) 10.08 s 18.71 s 13.19 s

5 δC, type 190.0, 115.2, 172.2, 50.9, 200.4, 111.5, 18.8,

C C C C C C CH2

24.6, CH3 39.8, CH2

119.3, 137.5, 26.7, 18.7, 207.7, 44.3,

CH C CH3 CH3 C CH2

20.0, CH2 15.1, CH3

108.0, 160.8, 107.1, 162.1, 209.8, 45.5,

C C C C C CH2

26.3, 33.4, 24.2, 15.3,

CH2 CH2 CH2 CH3

3.58 d (15.8) 3.54 d (15.8) 1.53 s 2.73 dd (13.5, 8.5) 2.69−2.61a 4.68 brsa 1.39 s 1.42 s 3.23 m 3.14−3.06a 1.67 ma 1.46−1.30b 1.46−1.30b 0.93 t (6.6)

3.18 m 1.72 sext (7.5) 0.99 t (7.5)

δC, type 189.9, 115.2, 172.6, 50.9, 200.4, 111.5, 18.8,

C C C C C C CH2

24.5, CH3 39.8, CH2

119.3, 137.5, 26.7, 18.7, 207.8, 42.3,

CH C CH3 CH3 C CH2

26.5, 33.3, 24.1, 15.3, 108.0, 160.8, 107.2, 162.1, 209.6, 47.5,

CH2 CH2 CH2 CH3 C C C C C CH2

19.8, CH2 15.2, CH3

10.06 s 18.70 s 13.17 s

Obscured due to tautomerism. bOverlapping signals.

Table 4. Effects of Acylphloroglucinols 1−5 from Elaphoglossum lindbergii against Promastigotes of Leishmania braziliensis and L. amazonensis

Overlapping signals. bSignals may be exchangeable.

IC50 (μM)a

stage of Leishmania pathogens, will show whether these compounds have potential as new leads against leishmaniasis.

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δH (J in Hz)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1030 polarimeter. UV spectra were obtained on a JASCO V-550 spectrophotometer. ECD spectra were recorded on a JASCO J-815 spectropolarimeter using EtOH as solvent. IR spectra were recorded on a Bruker Vektor 22 FT-IR spectrophotometer, in the attenuated total reflectance mode. NMR experiments were performed with standard pulse sequences and parameters on a Varian Unity 500 using acetone-d6 as solvent and internal reference. Low- and high-resolution mass spectra were registered on a Finnigan MAT 95 spectrometer. Column chromatography (CC) was carried

compound

L. braziliensis

L. amazonensis

1 2 3 4 5 amphotericin B

13.6 ± 1.3 29.9 ± 0.7 66.5 ± 11.0 87.7 ± 8.4 inactiveb 0.12 ± 0.01

16.3 ± 1.3 52.4 ± 1.7 151.3 ± 19.7 inactiveb inactiveb 0.11 ± 0.01

a Results are expressed as mean ± standard deviation. bCompounds were considered to be inactive when showing an IC50 higher than 100 μg/mL.

out over silica gel (70−230 mesh) with an n-hexane−EtOAc gradient as eluent. Preparative HPLC was performed on a Gilson instrument equipped with a Chemcopak silica gel column (Develosil 60-5, 5 μm, F



Article

250 × 10 mm i.d.) and a refractive index detector. TLC was carried out on aluminum plates precoated with silica gel 60 F254 (Merck). Detection of spots was accomplished by observing the TLC under UV light (254 nm) and further spraying with Godin reagent23 followed by heating. Plant Material. E. lindbergii was collected near Tiraxi, Jujuy, Argentina, in March 2009 (GPS data can be found in ref 5). The plant material was identified by Lic. Marcela A. Hernández, and a reference voucher specimen (LIL 609963) was deposited at the Herbarium of Fundación Miguel Lillo, Tucumán, Argentina. Extraction and Isolation. The procedure used for the extraction of the plant material was described elsewhere.5 Dried rhizomes and roots of E. lindbergii (56 g) were ground and extracted twice with diethyl ether at room temperature for 1 week. The plant material was filtered out, and the combined diethyl ether solutions were then concentrated at reduced pressure. The resulting reddish gum (2.3 g, 3.9% yield) was fractionated by column chromatography, using SiO2 as adsorbent and hexane−EtOAc (95:5) as eluent, to give a phloroglucinol-containing fraction (956.2 mg, 42% of the extract), fraction II. A portion of this sample (220 mg) was further purified by normal-phase HPLC (n-hexane−EtOAc, 98:2, 3.5 mL/min) to yield four fractions. Processing of fraction 1 (10 mg) by HPLC (n−hexane− EtOAc, 99:1, 0.4% HOAc, 4.0 mL/min) afforded 2 (5.5 mg) and 3 (1.6 mg). Fraction 2 (64.2 mg) gave 1 (3.4 mg) upon HPLC purification (n-hexane−EtOAc, 99.5:0.5, 0.2% HOAc, 4.0 mL/min). Further purification of fractions 3 (13.2 mg) and 4 (23.2 mg) by normal-phase HPLC (n-hexane−EtOAc, 99:1, 0.4% HOAc, 4.0 mL/ min) furnished 5 (10.2 mg) and 4 (7.0 mg), respectively. Lindbergin E (1): yellow gum; [α]22D −50.1 (c 1.0, CHCl3); UV (EtOH) λmax (log ε) 360 (4.16), 290 (4.25), 225 (4.38) nm; ECD (EtOH) λmax nm (Δε) 358 (+7.1), 314 (−4.9), 255 (−0.8), 236 (+4.5), 208 (−4.0), see Figure 3; IR νmax (neat) 3171, 3059, 2708, 2644, 2602, 1643, 1601, 1367, 1198, 1132 cm−1; 1H NMR data (500 MHz, acetone-d6), see Table 1; 13C NMR data (125 MHz, acetoned6), see Table 1; LREIMS m/z 554 (6), 553 (28), 552 (84), 484 (12), 465 (43), 275 (100), 247 (97); HREIMS, m/z 552.2723 (calcd for C32H40O8, 552.2724). Lindbergin F (2): yellow gum; [α]22D −42.9 (c 1.0, CHCl3); ECD (EtOH) λmax nm (Δε) 358 (+9.8), 312 (−6.8), 253 (−1.2), 236 (+6.0), 210 (−5.5), see Figure 4; IR νmax (neat) 3165, 3059, 2706, 2644, 2596, 1643, 1601, 1366, 1196, 1132 cm−1; 1H NMR data (500 MHz, acetone-d6), see Table 1; 13C NMR data (125 MHz, acetoned6), see Table 1; LREIMS m/z 582 (6), 581 (26), 580 (75), 565 (10), 512 (12), 493 (43), 275 (100), 247 (95); HREIMS, m/z 580.3041 (calcd for C34H44O8, 580.3037). Lindbergin G (3): yellow oil; [α]21D −44.6 (c 1.0, CHCl3); ECD (EtOH) λmax nm (Δε) 363 (+3.7), 288 (−2.6), 238 (−3.9), see Figure 4; IR νmax (neat) 3167, 3050, 2719, 2650, 1641, 1603, 1373, 1193 cm−1; 1H NMR data (500 MHz, acetone-d6), see Table 2; 13C NMR data (125 MHz, acetone-d6), see Table 2; LREIMS m/z 626 (8), 625 (32), 624 (75), 555 (48), 537 (93), 481 (16), 319 (100), 318 (56), 263 (40), 235 (40); HREIMS, m/z 624.3664 (calcd for C37H52O8, 624.3664). Lindbergin H (4): yellow gum; [α]23D −0.3 (c 1.0, CHCl3); ECD (EtOH) λmax nm (Δε) 356 (+6.1), 303 (−5.5), 270 (+0.8), 242 (−11.4), 212 (+3.2), see Figure 4; IR νmax (neat) 3157, 2702, 2638, 2604, 1639, 1612, 1554, 1377, 1236, 1196 cm−1; 1H NMR data (500 MHz, acetone-d6), see Table 3; 13C NMR data (125 MHz, acetoned6), see Table 3; HRFABMS, m/z 804.4088 (calcd for C46H60O12, 804.4086). Lindbergin I (5): yellow gum; [α]22D +1.0 (c 1.0, CHCl3); ECD (EtOH) λmax nm (Δε) 356 (+6.8), 302 (−5.9), 270 (+1.5), 242 (−12.7), 215 (+3.1), see Figure 4; IR νmax (neat) 3155, 3036, 2723, 2644, 2611 1637, 1612, 1458, 1441, 1373, 1238, 1196 cm−1; 1H NMR data (500 MHz, acetone-d6), see Table 3; 13C NMR data (125 MHz, acetone-d6), see Table 3; HRFABMS, m/z 832.4420 (calcd for C48H64O12, 832.4399). Leishmanicidal Activity Assay.24,25 The activity was evaluated in vitro against the promastigote form of complex L. amazonensis (clone 1, AML, MHOM/BR/76/LTB-012) and complex L. braziliensis

(strand M2904 C192 RJA). Parasites obtained from in vitro cultures of IIFB (Instituto de Investigaciones Fármaco Bioquı ́micas, Universidad Mayor de San Andrés) were grown at 26 °C in Schneider’s insect medium (pH 6.8) supplemented with 10% fetal bovine serum inactivated at 56 °C for 30 min. Promastigotes in logarithmic phase of growth, at a concentration of 1 × 106 parasites/mL (100 μL), were distributed on a 96-well flat bottom microtiter plate, and different concentrations of the test substances (0.75−100 μg/mL) dissolved in DMSO were added. The microplates were incubated for 72 h at 26 °C. After incubation, a solution of XTT (1 mg/mL) in PBS (pH 7.0 at 37 °C) with PMS (Sigma-Aldrich, 0.06 mg/mL) was added (50 μL/well), and the microtiter plate was then incubated again for 4 h at 26 °C. DMSO (1%) and amphotericin B (Sigma-A2411) (0.5 μg/mL) were used as negative and positive controls, respectively. All reagents were used without further purification. Optical density of each well was recorded on a Synergy HT microplate reader (Biotec) at λ 450 nm. The IC50 values, concentrations of a compound that causes a 50% decrease in parasite viability, were calculated using Microsoft Excel 2007 and are expressed as mean values ± standard error (Table 4). All assays were carried out in triplicate.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00767. 1D and 2D NMR, MS, and IR spectra of compounds 1− 5, LREIMS data of yungensins A, B, and F and elaphopilosins D and E, and ECD spectra of lindbergins A−D (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +54-381-4247752. Fax: +54-381-4248169. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Lic. M. A. Hernández for the identification of the plant material, the Analytical Chemistry Department of the University of Stuttgart for the measurement of mass and NMR spectra, and financial support from ANPCyT and CONICET, Argentina. C.S. thanks the Alexander von Humboldt Foundation for a fellowship and an equipment subsidy. Used equipment donations made by the companies Knauer, Dr. Kebelmann, and Heidolph, Germany, are greatly acknowledged.

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REFERENCES

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