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Cytotoxic phloroglucinol meroterpenoid from Eugenia umbelliflora fruits
Phytochemistry Letters 27 (2018) 187–192
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Cytotoxic phloroglucinol meroterpenoid from Eugenia umbelliflora fruits a
a
a
T
b
Ingrid V. Farias , Larissa G. Faqueti , Vania Floriani Noldin , Gilberto Franchi Junior , Alexandre E. Nowilb, Ivania T.A. Schuquelc, Franco Delle Monachea, Pablo A. Garcíad, ⁎ José L. López-Pérezd, Arturo San Felicianod, Valdir Cechinel-Filhoa, Christiane Meyre-Silvaa,e, a
Universidade do Vale do Itajaí (UNIVALI), Centro de Ciências da Saúde, Rua Uruguai, 458, Itajaí, Santa Catarina State, 88302-202, Brazil Centro Integrado de Pesquisas Oncohematológicas na Infância (CIPOI), UNICAMP, Campinas, São Paulo, Brazil c Universidade Estadual de Maringá (UEM), Departamento de Química, Maringá, Paraná State, Brazil d Departamento de Ciencias Farmacéuticas. Área de Química Farmacéutica, Facultad de Farmacia, CIETUS, IBSAL, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain e Universidade Federal de Santa Catarina (UFSC), Curso de Farmácia – Bloco JK, Campus Reitor João David Ferreira Lima, s/n - Trindade, Florianópolis, Santa Catarina State, 88040-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eugenia umbelliflora Myrtaceae Phloroglucinol Eugenial E NMR
Eugenia umbelliflora is a member of Myrtaceae family, with antimicrobial and anti-leishmanial properties related to the presence of phloroglucinol derivatives in its extracts. This study describe the isolation and structure elucidation of a sesquiterpenyl phloroglucinol (eugenial E), that is described here for the first time. Eugenial E (5) was isolated from the n-hexane extract of fruits, and its structural elucidation was carried out through the combined analysis of MS, IR and 1D-(¹H, ¹³C BB and DEPT, and NOEDIFF) and 2D-(COSY, HSQC, HMBC and ROESY) NMR spectra, with conformational analysis support. Cytotoxicity was assessed using MTT analysis against human leukemic cells (k562 and Nalm-6 cells) and murine melanoma cells (B16F10 cell). The extracts showed high cytotoxicity, while eugenial C with 0.38 μM, eugenial D 1.9 μM and eugenial E 4.97 μM of IC50 for K562 and for B16F10 cells 6.0; 3.2 and 8.8 μM, respectively. The eugenials were evaluated in silico for prediction of bioactivity, toxicity and molecular properties using the ChemDoodle, Molinspiration and PreADMET services. The results of in silico analysis have shown that eugenial C and eugenial D may be mutagenic. In rats eugenial C, eugenial D and eugenial E may be carcinogenic, and eugenial D may have low-to-medium risk of environmental / developmental toxicity.
1. Introduction The importance of medicinal plants and natural products as strategic sources for the discovery of new and effective anticancer agents is well known, as is their use as models to design compounds with improved properties. In fact, almost 80% of all clinical anticancer agents are structurally related to natural products (Newman and Cragg, 2012). Eugenia constitutes one of the most representative genus of Myrtaceae family, distributed mainly in tropical areas, such as the north of Brazil, where the existence of 350 species is estimated (Landrum and Kawasaki, 1997). In terms of tree species, the genus is one of the richest on the Atlantic coast of Brazil and many species are appreciated in the diet due to their edible fruits, including E. uniflora (“pitanga”), E. edulis (“jaboticaba”) and E. brasiliensis (“grumixama”) (Mazine et al., 2014). Eugenia umbelliflora O. Berg, known as “baguaçu”, is a tree with
edible fruits that grows in southeastern Brazil. Pharmacological in-vivo and in-vitro studies have exhibited promising gastroprotective effects and antibacterial, antifungal and antileishmanial activity of its fruit extracts (Meyre-Silva et al., 2009; Machado et al., 2005, 2009; Cechinel-Filho et al., 2013), prompting phytochemical studies with E. umbelliflora fruits, which identified the presence of anthocyanin in the methanol ripe fruit extract (Kuskoski et al., 2003) and four previously unkown terpenyl-phloroglucinolaldehydes: eugenials A–D, in the CH2Cl2 unripe fruit extract (Faqueti et al., 2015). It was also demonstrated that eugenials C and D possess excellent antibacterial activity against different methicillin-resistant strains of Staphylococcus aureus, which corroborates the antibacterial activity shown by the extract (Machado et al., 2005; Faqueti et al., 2015). The literature reports that phloroglucinols from species of Myrtaceae family are promising compounds in drug discovery, mainly
⁎ Corresponding author.at: Universidade Federal de Santa Catarina (UFSC), Curso de Farmacia - Bloco JK, Campus Reitor Joao David Ferreira Lima, s/n - Trindade, Florianopolis, Santa Catarina State, 88040-900, Brazil. E-mail address: [email protected] (C. Meyre-Silva).
https://doi.org/10.1016/j.phytol.2018.07.004 Received 6 May 2018; Received in revised form 20 June 2018; Accepted 10 July 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of eugenials A–E (1–5) isolated from Eugenia umbelliflora fruits. (The numbering of compound 5 has been changed from the used for 4 in our preceding paper, to facilitate the follow-up of discussions on the terpene stereochemical assignment).
as antineoplastic agents (Wang et al., 2012; Soliman et al., 2014; Nisa et al., 2016). Considering this fact and the biological potential of E. umbelliflora fruits, this study aims to evaluate the cytotoxicity of extracts and components of E. umbelliflora fruits against several tumor cells.
Table 1 1 H and 13C NMR spectral data for Eugenial E (5).a
2. Results and discussion Five phologlucinol derivates were isolated. Four of which have already been reported by our research group and named eugenials A (1), B (2), C (3) and D (4), as mentioned previously. Structure elucidation of a sequiterpenyl phloroglucinol (eugenial E) (5) is described here for the first time (Fig. 1). It was isolated from the hexane extract of E. umbelliflora fruits in the form of a white amorphous solid (mp: 132–137 °C, not recrystallized), whose standard mass spectrum (EI-MS) showed peak at m/z 440, corresponding to the formula C27H36O5; whereas negative and positive electrospray techniques, NESI-MS and POSI-MS, yielded values of m/z 457 [M−H]− and m/z 459 [M+H]+ respectively, incremented in 18 mass units of a H2O molecule, and supporting the molecular formula C27H38O6 for 5. The IR spectrum exhibited a strong carbonyl band at 1625 cm-1 and the NMR data are summarized in Table 1. All 1H and 13C chemical shifts were assigned by combined use of one- and two-dimensional NMR methods. According to previous results for other eugenials, the spectral data suggested that compound 5 consisted of two parts: a sesquiterpene fragment, numbered as the main unit (Fig. 1, Table 1) to facilitate discussion of stereochemical assignment, and a fully substituted phloroglucinol moiety, similar to those previously reported for other eugenials and numbered as the prime (‘) unit, including the connecting methylene group as C-1′. The 13C NMR spectrum of 5 (Table 1) showed 27 signals which were associated with 5 methyls, 7 methylenes, 5 methines and 10 non-protonated carbons, with the help of DEPT spectra. Seven signals [δC: 193.4 (d, C-12′), 171.6 (s, C-3′), 168.9 (s, C-5′), 165.2 (s, C-7′), 108.0 (s, C-2′), 104.6 (s, C-4′) and 105.2 (s, C-6′)] arose from a typical formylphloroglucinol unit, while signals for a linear butiryl substituent [δC: 207.5 (s, C-8′), 46.7 (t, C-9′), 18.4 (t, C-10′) and 14.2 (q, C-11′)] were also observed. The remaining 13C NMR signals were attributed to the sesquiterpene part, with two methyls (δC 22.7 × 2), four methylenes (δC 21.1, 23.1, 34.0 and 36.8), three methines (δC 40.5, 46.6 and 48.3) and one quaternary
Position
δC, ppma multiplicityb
δH, ppm multiplicity, (J in Hz)
1 2
48.3 d 23.1 t
3 4 5 6 7 8 9
34.0 t 40.5 d 150.3 s 123.7 d 46.6 d 21.1 t 36.8 t
10 11 12 13 14 15 1’
38.7 73.0 28.0 27.9 22.7 22.7 23.1
2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 11’ 12’
108.0 s 171.6 s 104.6 s 168.9 s 105.2 s 165.2 s 207.5 s 46.7 t 18.4 t 14.2 q 193.4 d
1.50 m 1.10 m 1.27 dd (13.2, 3.3) 1.48-1.32 m 2.45 m – 5.55 d (3.3) 2.05 mc 1.75 m 1.65 m 1.80 m – – 1.15 s 1.15 s 1.13 s 1.13 d (7.6) 2.45 dd (13.8, 11.4) 2.79 dd (13.8, 2.7) – – – – – – – 3.10 t (7.2) 1.70 dt (7.2) 0.98 t (7.2) 10.15 s
a b c
s s q q q q t
in Acetone-d6 at 300 MHz (1H) and 75.5 MHz (13C). assigned by DEPT method, s = C, d = CH, t = CH2 and q = CH3. Overlapped with the residual signal of the solvent.
carbon (δC 38.7), along with signals for a trisubstituted double bond (δC150.3 and 123.7) and a not totally free rotating isopropanol residue (δC 27.9, 28.0 and 73.0). Comparison of these 13C NMR spectral data with those of eugenial D (4) (Faqueti et al., 2015), revealed a close similarity except for the presence of the 2-hydroxy-2-propyl group at C-7 in compound 5, 188
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et al., 2015). The NOE correlations of H-14 with H-1′ and H-8β indicated that these protons were co-facially oriented, thus establishing the β-orientation of Me-14. In addition, NOEs were found between the olefinic proton H-6 and the methine protons H-4 and H-7, and between the methyl protons H-12/13 and H-9α, while a narrow multiplicity pattern was observed for the H-7 signal, a multiplet with J-coupling values under 5 Hz. All these data supported the proposal of an almost coplanar pseudo-equatorial arrangement for H-4, H-6 and H-7, and the pseudo-axial orientation of both the methyl group at C-4β and the 2hydroxy-2-propyl group attached to C-7α. Some significant interprotonic distances calculated for the main conformer of 5, close to those found for the other three main conformers and in fairly close agreement with the experimentally observed NOEs, are indicated in Fig. 3. Therefore, the structure 5 was assigned to eugenial E. Aiming to obtain additional proofs of the structure of 5, some molecular modelling studies were performed (see Experimental part for details). A conformational study, followed by an optimization at the DFT level of stable conformers, provided four main low energy conformers for this compound (Fig. 4). These conformers mainly differed in the pattern of intramolecular hydrogen bonding of phenols to carbonyls in the phloroglucinol moiety, while all of them displayed very similar conformational energies and spatial arrangements of the bicyclic terpene system. Small differences in conformational energy (≤ 0.5 kcal mol−1) found within this group of conformers ensure the practical free rotation of the C-7 – C-11 bond and the hydroxypropyl fragment, thus justifying the experimental NOE observed between the H-12/H-13 methyl protons and H-9α. H-bonds between phenols of the phloroglucinol fragment and the neighboring carbonyl groups are also depicted in Fig. 4. The permanent involvement of the 5′−OH group in Hbonding and its preference towards the formyl group at C-6′ were noted, while the butiryl carbonyl binds to the 3′−OH group in such cases, according the numbering system used in this article. As depicted in Fig. 1, the phloroglucinols isolated from E. umbelliflora have global structural similarity, but differ in the associated terpenoid, and reveal the existence of congener series that might be a consequence of the plant’s need to generate its own chemical diversity, which assists in the discovery of new bioactive compounds. In order to establish the antineoplastic potential of this plant and relate it to the constituents, the extracts and the isolated eugenials were evaluated for their ability to inhibit the growth of three types of cancer cells: human chronic myelogenous leukemia (K562) and acute lymphoblastic leukemia (Nalm-6) cells, and murine melanoma (B16F10) cells. The extracts analyzed showed good cytotoxicity, while eugenial C, eugenial D and eugenial E displayed IC50 values in the low range for K562 and Nalm-6 cells as shown in Tables 2 and 3. When the cytotoxicity of isomers 1 and 2 was compared, it was observed that 1 was slightly more potent than 2 on K562 cells (IC50 30.5 μM versus 42.8 μM), but was inactive against B16F10 cells. The main difference in the structure is the exchange of positions between the formyl and butiryl groups, slightly affecting the value for lipophilicity (log P), as shown in Table 4. The inhibition results found for K562 cells underscore the differences in cytotoxicity shown by the fused phloroglucinol-monoterpene eugenials A and B (1, 2), compared with
Fig. 2. Key HMBC correlations for the new compound 5.
replacing the 2-propenyl group present in 4. Based on connectivities observed in the HSQC and HMBC spectra (Fig. 2), the signals were collectively assembled in a sesquiterpene with the eudesmane skeleton, as depicted in the structure of 5, based on the following findings. Were observed the correlations of methyl protons H-15 with C-5, of H-4 with C-5 and C-6, and of H-6 with C-4, C-7 and C-8, as well as of H-7 with the olefinic C-5, that linked the trisubstituted double bond Δ5 to C-7. The correlations of methyl protons H-14 with C-1, C-5, C-9 and C-10 allowed the connections of C-1, C-5 and C-9 to the quaternary carbon C10. The correlations of methyl protons H-13 with C-7, C-11 and C-12, and of methyl protons H-12 with C-7, C-11 and C-13, located the hydroxypropyl group at C-7. In addition, the HMBC and COSY correlations revealed that the two parts of eugenial E were linked by a methylene bridge (C-1′). One or both signals of the connecting gem-methylene protons at position C-1′ (δH 2.45 dd and 2.79 dd, coupled to H-1) showed correlations with those of carbons C-2′, C-3′, and C-7′ of the aromatic phloroglucinol fragment, and with those of C-1, C-2 and C-10 of the eudesmane skeleton. Furthermore, HMBC correlations of the aldehyde proton H-12′ (δH 10.15) allowed the differential assignment of signals for the neighboring C-5′, C-6′ and C-7′ atoms with respect to those of C-2′, C-3′ and C-4′, close to the butiryl carbonyl. Other HMBC correlations were also observed for the methyl protons H-12 and H-13 with C-7; the methylene protons H-8 with C11 and C10; the olefinic H-6 with C-4 and C-10, and the methyl protons H-14 and H-15 with the non-protonated olefinic carbon C-5 and, respectively, with C10 and C-4. Stereochemical assignment of compound 5 (Fig. 3) was based on combined analysis of vicinal interproton couplings of protons H-6, H-7 and H-8, and the NOE/ROE effects observed through 1D-NOEDIFF and 2D-ROESY experiments, carried out in different solvent conditions (acetone-d6, methanol-d4, pyridine-d5 and mixtures) to resolve signal overlapping in different parts of the 1H spectrum. The result was in agreement with that previously reported for eugenial D (4) (Faqueti
Fig. 3. NOE correlations observed for the terpenic part of compound 5 and relevant distances (Å) calculated for the main conformer (in chloroform), which displayed very small conformational energy differences (ΔCE) from its C-7/C-11 rotamers.
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Fig. 4. Selected stereochemical and energy calculation results from the conformational analysis performed for compound 5 in three solvents. Two pairs of low-energy rotamers, A/B (lowest) and C/D around the C1′-C2′ bond, corresponding to more stable conformers of compound 5 are represented. in Conformational energy differences (ΔCE, kcal·mol−1) with respect to the most stable conformers (A in chloroform and acetone and B in pyridine, to which the 0.00 value was arbitrarily assigned) are shown in the color of the corresponding solvent. Preferential phenol-carbonyl H-bonds in each case, are also depicted.
three sesquiterpenyl-phloroglucinols, whereas relative to structure-activity aspects, the eudesmadiene 4 was the most potent cytotoxic for either Nalm-6 or B16F10 cells. It could be of interest to note the beneficial influence for the activity associated with the presence of a larger terpenoidal (higher lipohilic) fragment attached to the phoroglucinol moiety. This observation appears to be confirmed by the activity reduction in the eudesmenol 5, in comparison with the more lipophilic eudesmadiene 4. This is probably due to the presence of the hydroxyl group at C-11 in the former. Tomoeones A–H, phloroglucinol derivatives isolated from Hypericum ascyron, showed cytotoxicity against K562 cells with IC50 values ranging from 40 to 69 μM and doxorubicin used as positive control showed an IC50 value of 0.83 μM. In comparison with this reference drug, it should be noted that the eugenial C (3, IC50 0.38 μM) shows higher cytotoxicity against such K562 cells. Consequently, it is necessary to establish the mechanism of action and to evaluate the toxicity of compound 3, in order to perform a better comparison, as doxorubicin is highly myelotoxic (Hashida et al., 2008). To obtain some information on the pharmaco-therapeutic profile of eugenials, some virtual studies were carried out for online calculation/ prediction of their main physico-chemical, pharmacological and toxicological properties. The most representative results are summarized in Table 4. Interestingly, according to the predicted molinspiration bioactivity (Molinspiration, 2017), all five eugenials qualified as potential nuclear-receptor-ligands, with homogeneus positive bioactivity scores in the range of 0.43 to 0.63. This finding could serve to rule out such a possible mechanism of cytotoxicity for eugenials, since the most potently cytotoxic of them, compound 3, achieved the lowest score. On the other hand, the fact that the cytotoxic potency of eugenials occurs in parallel with lipophilicity (Table 3), with compound 3 also displaying the highest log P value, may suggest the membrane, or another lipidic region of the cell, as the probable target zone for eugenials. In relation to the online prediction of toxicity via PreADMET (PreADMET, 2017), eugenials A and B were practically devoid of mutagenic (Ames test) carcinogenic (mouse & rat), environmental (algae, insect, minnow), or developmental (medaka embryos) effects, with a low risk of cardiac toxicity related to human Ether-à-go-go-Related Gene (hERG) inhibition. Eugenials C, D and E proved similarly non-toxic in most of the assays, though in agreement with their higher cytotoxicity, were
Table 2 Cytotoxicity of extracts from fruits of E. umbelliflora. Extract
IC50 values (MD ± SD. μg/mL) K562
EuH EuD EuA EuM EuMb
2.50 2.50 0.45 79.3 34.1
± ± ± ± ±
0.05 0.10 0.0 0.8 0.8
Nalm – 6
B16F10
12.0 4.74 8.75 83.5 78.1
35.7 ± 1.0 > 1000 Inactive 36.7 ± 1.4 > 1000 Inactive 24.1 ± 0.8
± ± ± ± ±
0.1 0.09 0.05 0.1 0.8
Note: EuH: n-hexane extract; EuD: dichloromethane extract; EuA: ethyl acetate extract; EuM: methanol extract; EuMb: methanol crude extract. Table 3 Cytotoxicity of isolated compound from fruits of E. umbelliflora. Compound
IC50 values (MD ± SD. μM) K562
1 2 3 4 5 Vincristine Doxorubicin*
30.5 42.8 0.38 1.90 4.97 0.01 0.83
± ± ± ± ± ± ±
6.9 0.9 0.3 0.1 0.3 0.07 0.02
Nalm – 6
B16F10
> 125 70.5 ± 0.3 10.5 ± 0.1 7.75 ± 0.4 29.1 ± 0.2 0.25 ± 0.12 Not rated
> 125 12.0 ± 2.1 6.00 ± 0.3 3.20 ± 0.1 8.80 ± 1.9 Not rated Not rated
Note: 1: eugenial A; 2: eugenial B; 3: eugenial C; 4: eugenial D; 5: eugenial E. * Value from Hashida et al., 2008.
the more potent eugenials C, D and E (3-5), which did not have fused, but simply attached phloroglucinol-sesquiterpene arrangements. Such differences in activity could be associated with the presence of the additional free phenol group in compounds 3 - 5. Another interesting observation relates to the comparison within the group of sesquiterpenyl derivatives. Eugenial C (3, IC50 = 0.38 μM), bears a tricyclic aromadendrene fragment, revealed as 5-fold and 13-fold respectively, more potent than those with the bicyclic eudesmadiene (eugenial D, 4, IC50 = 1.9 μM) and the eudesmenol (eugenial E, 5 IC50 = 4.97 μM) fragments. Relative to the other cell lines, the results found were parallel, with the Nalm-6 leukemia cells being the least sensitive to the 190
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Table 4 Selected calculated and predicted molecular, pharmaco-toxicological and ADME data for eugenials.A–E(1–5) Comp.
Formula
MW
Log Sa
clog Pa
NRLsb
Mutc
MCarc
EDTc
HIAc
SkPc
Caco2c
1 2 3 4 5
C22H28O5 C22H28O5 C27H36O5 C27H36O5 C27H38O6
372.459 372.459 440.578 440.578 458.593
−4.84 −4.84 −5.76 −5.62 −5.26
5.54 5.11 6.27 5.58 5.37
0.55* 0.55* 0.43 0.61* 0.63*
non non mut mut non
neg neg neg# neg# neg#
low low low l-m low
93.6 93.6 91.3 91.7 82.3
−2.47 −2.47 −1.87 −1.73 −1.52
11.91 11.91 19.28 19.17 18.44
MW: Molecular Weight; Log S: log Molar aqueous solubility at pH = 7; clog P: calculated log of n-octanol/water partition; NRLs: Nuclear-Receptor-Ligand score (* potential enzyme inhibitor); Mut: mutagenic (Ames test); MCar: Mouse-Carcinogenicity (neg = negative, # = positive in rat); EDT: Environmental/Developmental Toxicity (l–m = low-to-medium risk); HIA: Human Intestinal Absorption (%); SkP: skin permeability (log Kp in cm/h); Caco2: colon adenocarcinoma cell permeability (nm/sec). a ChemDoodle. b Molinspiration. c PreADMET.
4.3. Extraction and isolation
predicted as potentially mutagenic and carcinogenic (to rats only). Additionally, a medium risk of cardiac incidence associated with hERG inhibition was predicted for eugenial D. In relation to the bioavailability and ADME properties of eugenials A – E, high levels (from 82.2% for 5 to 93.6% for 1 and 2) of human intestinal absorption (HIA) and middle Caco2 adenocarcinoma cell permeability (from 11.9 nm/sec for 1 and 2 to 19.3 for 3) were predicted through PreADMET algorithms. The physico-chemical parameters also demonstrated that there is no violation of Egan (Egan et al., 2000) or Veber (Veber et al., 2002) rules, and one violation (log P > 5) of the Lipinski rule of five (Lipinski, 2004), which along with the bioavailability score (BS) of 0.17 for all the compounds (ChemDoodle, 2016), likely indicates their good oral bioavailability.
The unripe fruits were dried and powdered using a knife grinder. The material (100 g) were extracted successively by maceration at room temperature with n-hexane, dichloromethane, ethyl acetate and methanol for five days. The solvent was removed under reduced pressure with a rotatory evaporator, resulting in the n-hexane (EuH) (21.29 g), dichloromethane (EuD) (14.14 g), ethyl acetate (EuA) (4.68 g) and methanol extracts (EuM) (28.93 g). Another crude methanol extract (EuMb) (81.62 g) was obtained by maintaining the ground, dried fruits with methanol at room temperature for seven days. Another extract was obtained by direct maceration with methanol at room temperature for seven days (81.62 g), identified as crude methanolic extract (EuMb). The EuH (21.29 g) was subjected to silica gel 60 (Merck) open column chromatography (4 x 50 cm) and eluted with 400 mL each of gradients n-hexane:CH2Cl2 in ratios 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50, respectively. Based on similar Rf values on TLC developed with the eluent n-hexane:CH2Cl2 (3 :7) and sprayed with 10% sulfuric acid three fractions (C1 − C3) were obtained. Fractions C1 (1.5 g) and C2 (6 g) were resolved as described by Faqueti et al., 2015, furnishing the compounds eugenial A (1), eugenial B (2), eugenial C (3) and eugenial D (4). Fraction C3 (10 g) was submitted to purification by silica gel 60 open column chromatography (2.5 x 30 cm) and eluted with 400 mL each of mixtures of n-hexane:CH2Cl2 at ratios of 80:20, 70:30, 60:40, respectively, yielding 90 mg of a powder eluted in the 70:30 fraction, which was identified as a new compound and denominated eugenial E (5): amorphous white solid; mp 132–137 °C; [α]D −36° (c 0.20%, MeOH); IR (KBr) 3500, 3000, 2950, 1625, 1500, 1450, 1250, 800 cm−1; NMR data, see Table 1; HRESIMS, found 459.2730 (calcd. for C27H38O6+H: 459.2741).
3. Conclusion In conclusion, the study resulted in the isolation and characterization of a new sesquiterpenyl phloroglucinol eugenial E, and determination of promising cytotoxicities of the phloroglucinols meroterpenoids isolated from the extracts of Eugenia umbelliflora that merits further study, in search of a candidate for pre-clinical evaluation of efficacy and toxicity. 4. Experimental 4.1. General experimental procedures Melting points were determined in a MQ APF-301 (Microquímica) and are uncorrected. Optical rotations were measured at room temperature in CHCl3 solution using a 341 Perkin-Elmer digital polarimeter equipped with a sodium lamp. IR spectra were recorded on an MB-100 Bomen spectrometer. HRESIMS analyses were performed on an Agilent 1100 HPLC-ESI-Q-TOF system. NMR spectra were recorded on DRX-400 (1H at 400 MHz) (Bruker) and Mercury plus 300 (1H at 300 MHz) (Varian) spectrometers. Acetone-d6 was mainly used as solvent, while other deuterated solvents were used to avoid signal overlapping. Column chromatography was performed with silica gel (70–230 mesh) from Merck. TLC analysis was carried out using pre-coated plates (Merck, silica gel GF-254, 0.2 mm): the spots were visualized either using a UV lamp λ254 nm or by spraying with 10% sulfuric acid in ethanol.
4.4. Cytotoxicity assay K562 Homo sapiens bone marrow chronic myelogenous leukemia (ATCC® CCL243™), Nalm-6 Homo sapiens peripheral blood acute lymphoblastic leukemia (DSMZ® ACC 128) B16F10 and murine melanoma (ATCC® CRL6475™) cells were obtained from the cell bank of Rio de Janeiro, Brazil. B16F10 and K562 cells were cultured in Dulbecco's modified Eagle's medium (DMEM); NALM-6 cells were cultured in RPMI 1640 medium. The culture media were supplemented with 10% fetal bovine serum, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin. Cell cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. In all experiments, viable cells were checked in the beginning of the experiment by trypan blue. Cells were seeded in 96-well plate with density 30,000 cell/well and pre-incubated for 24 h. The extracts and compounds were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 0.5%). After removal of pre-incubated culture medium, 100 μL of medium containing various concentrations of samples were
4.2. Plant material The unripe fruits of E. umbelliflora were collected in June 2010 in the town of Balneário Camboriú, Santa Catarina, Brazil, and identified by Prof. Msc. Renê Artur Ferreira (Universidade do Vale do Itajaí, ItajaíSC). A voucher specimen (No. VC Filho 50) was deposited at Barbosa Rodrigues Herbarium, Itajaí, Santa Catarina, Brazil. 191
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added and further incubated for 24 h for B16F10 cells and 48 h for Nalm-6 and K562 cells. Cell viability was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Mosmann, 1983). The optical density (OD) of each well was detected using a Microplate reader at 540 nm. The 50% inhibition concentration (IC50 value) was determined by curve fitting with the Graphpad Prism.
J. Comput. Chem. 17, 1571–1586. 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., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., 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., 2009. Gaussian 09, revision d.01 ed. Gaussian, Inc., Wallingford, CT. Hashida, W., Tanaka, N., Kashiwada, Y., Sekiya, M., Ikeshiro, Y., Takaishi, Y., 2008. Tomoeones A–H, cytotoxic phloroglucinol derivatives from Hypericum ascyron. Phytochemistry 69, 2225–2230. Koch, W., Holthausen, M.C., 2000. A Chemist’s Guide to Density Functional Theory, 2nd edition. Wiley-VCH, Weinheim, Germany. Kuskoski, E.M., Vega, J.M., Rios, J.J., Fett, R., Troncoso, A.M., Asuero, A.G., 2003. Characterization of anthocyanins from the fruits of baguaçu (Eugenia umbelliflora Berg.). J. Agric. Food Chem. 51, 5450–5454. Landrum, L.R., Kawasaki, M.L., 1997. The genera of Myrtaceae in Brazil: an illustrated synoptic treatment and identification keys. Brittonia 49, 508–536. Lee, C., Yang, W., Parr, R.G., 1988. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785–789. Lipinski, C.A., 2004. Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov. Today Technol. 1, 337–464. Lynch, B.J., Zhao, Y., Truhlar, D.G., 2003. Effectiveness of diffuse basis functions for calculating relative energies by density functional theory. J. Phys. Chem. A 107, 1384–1388. Machado, K.E., Cechinel-Filho, V., Tessarolo, M.L., Mallmann, R., Meyre-Silva, C., Bella Cruz, A., 2005. Potent antibacterial activity of Eugenia umbelliflora. Pharm. Biol. 43, 636–639. Machado, K.E., Cechinel-Filho, V., Cruz, R.C., Meyre-Silva, C., Bella Cruz, A., 2009. Antifungal activity of Eugenia umbelliflora against dermatophytes. Nat. Prod. Commun. 4, 1181–1184. Marenich, A.V., Cramer, C.J., Truhlar, D.G., 2009. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113, 6378–6396. Mazine, F.F., Souza, V.C., Sobral, M.F., Forest, E., Lucas, A., 2014. Preliminary phylogenetic analysis of Eugenia (Myrtaceae: Myrteae), with a focus on Neotropical species. Kew Bull. 69, 94–97. Meyre-Silva, C., Petry, C.M., Berté, T.E., Becker, R.G., Zanatta, F., Delle-Monache, F., Cechinel-Filho, V., Andrade, S.F., 2009. Phytochemical analyses and gastroprotective effects of Eugenia umbelliflora (Myrtaceae) on experimental gastric ulcers. Nat. Prod. Commun. 4, 911–916. Molinspiration Cheminformatics. URL http://www.molinspiration.com/, 2017 (Accessed March 2017). Mosmann, D.J., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Newman, D.J., Cragg, G.M.J., 2012. Natural products as sources of new drugs over the 30 years from 1981 to 2010. Nat. Prod. 75, 311–335. Nisa, K., Ito, T., Kodama, T., Tanaka, M., Asakawa, Y., Imagawa, H., Morita, H., 2016. New cytotoxic phloroglucinols, baeckenones D-F, from the leaves of Indonesian Baeckea frutescens. Fitoterapia 109, 236–240. Parr, R.G., Yang, W., 1989. Density Functional Theory of Atoms and Molecules. Clarendon Press, Oxford, UK. PreADMET. URL. https://preadmet.bmdrc.kr/, 2017 (Accessed March, 2017). Schuchardt, K.L., Didler, B.T., Elsethagen, T., Sun, L., Gurumoorthi, V., Chase, J., Li, J., Windus, T.L., 2007. Basis set Exchange: a Community database for computational sciences. J. Chem. Inf. Model. 47, 1045–1052. Soliman, F.M., Fathy, M.M., Salama, M.M., Al-Abd, A.M., Saber, F.R., El-Halawany, A.M., 2014. Cytotoxic activity of acyl phloroglucinols isolated from the leaves of Eucalyptus cinerea F. Muell. ex Benth. cultivated in Egypt. Sci. Rep. 4, 5410. Stephens, P.J., Devlin, F.J., Chabalowski, C.F., Frisch, M.J., 1994. Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields. J. Phys. Chem. 98, 11623–11627. Veber, D.F., Johnson, S.R., Cheng, H.Y., Smith, B.R., Ward, K.W., Kopple, K.D., 2002. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 45, 2615–2623. Wang, J., Zhai, W.Z., Zou, Y., Zhu, J.J., Xiong, J., Zhao, Y., Yang, G.X., Fan, H., Hamann, M.T., Xia, G., Hu, J.F., 2012. Eucalyptals D and E, new cytotoxic phloroglucinols from the fruits of Eucalyptus globulus and assignment of absolute configuration. Tetrahedron Lett. 53, 2654–2658.
4.5. Molecular modelling studies The structure of compound 5 was built using Spartan'08 (Spartan’08. Wavefunction, Inc., Irvine, CA). Conformational analysis was performed by a Monte Carlo random search with the MMFF Force Field implemented in that Package. Geometry optimizations and energy calculations were performed with GAUSSIAN 09 (Frisch et al.,2009) using DFT (Lynch et al., 2003; Koch and Holthausen, 2000; Parr and Yang, 1989) at the B3LYP/6-31+g (d,p) (Stephens et al., 1994; Becke, 1993; Lee et al., 1988; Schuchardt et al., 2007; Feller, 1996) level of theory. To simulate the NMR experimental conditions, the solvent effect (acetone, dichloromethane or pyridine) was considered during the calculation using the SMD continuum model (Marenich et al., 2009). 4.6. Prediction of molecular properties The five structures of eugenials were evaluated in silico for prediction of bioactivity, toxicity and molecular properties using the ChemDoodle (ChemDoodle, 2016), Molinspiration (Molinspiration, 2017) and PreADMET (PreADMET, 2017) services. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgments The authors are grateful to CNPq-Brazil, ProPPEC/UNIVALI, MINECO-Spain (Project: AGL2016-79813-C2-2-R) and JCyL-Spain (Project: SA221U13) for providing the financial support, Prof. Rene A. Ferreira (UNIVALI) for the classification and collection of plant material. Free access to online services of ChemDoodle Free Tral, Molinspiration and PreADMET predictive systems are also acknowledged. This manuscript is dedicated to Prof. Dr. Mahabir P. Gupta on the occasion of his 75th anniversary References Becke, A.D., 1993. Density functional thermochesmistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. Cechinel-Filho, V., Meyre-Silva, C., Niero, R., Mariano, L.N.B., Nascimento, F.G., Farias, I.V., Gazoni, V.F., Silva, B.S., Giménez, A., Gutierrez-Yapu, D., Salamanca, E., Malheiros, A., 2013. Evaluation of antileishmanial activity of selected brazilian plants and identification of the active principles. Evid. Complement. Alternat. Med. https:// doi.org/10.1155/2013/265025. advance online publication 9 June 2013. ChemDoodle Free Trial. URL. https://www.chemdoodle.com/free-trial/, 2016 (Accessed November 2016). Egan, W.J., Merz, K.M., Baldwin, J.J., 2000. Prediction of drug absorption using multivariate statistics. J. Med. Chem. 43, 3867–3877. Faqueti, L.G., Farias, I.V., Sabedot, E.C., Delle-Monache, F., San-Feliciano, A., Schuquel, I.T., Cechinel-Filho, V., Cruz, A.B., Meyre-Silva, C., 2015. Macrocarpal-like compounds from Eugenia umbelliflora fruits and their antibacterial activity. J. Agric. Food Chem. 63, 8151–8155. Feller, D., 1996. The role of databases in support of computational chemistry calculations.
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Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Cytotoxic phloroglucinol meroterpenoid from Eugenia umbelliflora fruits a
a
a
T
b
Ingrid V. Farias , Larissa G. Faqueti , Vania Floriani Noldin , Gilberto Franchi Junior , Alexandre E. Nowilb, Ivania T.A. Schuquelc, Franco Delle Monachea, Pablo A. Garcíad, ⁎ José L. López-Pérezd, Arturo San Felicianod, Valdir Cechinel-Filhoa, Christiane Meyre-Silvaa,e, a
Universidade do Vale do Itajaí (UNIVALI), Centro de Ciências da Saúde, Rua Uruguai, 458, Itajaí, Santa Catarina State, 88302-202, Brazil Centro Integrado de Pesquisas Oncohematológicas na Infância (CIPOI), UNICAMP, Campinas, São Paulo, Brazil c Universidade Estadual de Maringá (UEM), Departamento de Química, Maringá, Paraná State, Brazil d Departamento de Ciencias Farmacéuticas. Área de Química Farmacéutica, Facultad de Farmacia, CIETUS, IBSAL, Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain e Universidade Federal de Santa Catarina (UFSC), Curso de Farmácia – Bloco JK, Campus Reitor João David Ferreira Lima, s/n - Trindade, Florianópolis, Santa Catarina State, 88040-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eugenia umbelliflora Myrtaceae Phloroglucinol Eugenial E NMR
Eugenia umbelliflora is a member of Myrtaceae family, with antimicrobial and anti-leishmanial properties related to the presence of phloroglucinol derivatives in its extracts. This study describe the isolation and structure elucidation of a sesquiterpenyl phloroglucinol (eugenial E), that is described here for the first time. Eugenial E (5) was isolated from the n-hexane extract of fruits, and its structural elucidation was carried out through the combined analysis of MS, IR and 1D-(¹H, ¹³C BB and DEPT, and NOEDIFF) and 2D-(COSY, HSQC, HMBC and ROESY) NMR spectra, with conformational analysis support. Cytotoxicity was assessed using MTT analysis against human leukemic cells (k562 and Nalm-6 cells) and murine melanoma cells (B16F10 cell). The extracts showed high cytotoxicity, while eugenial C with 0.38 μM, eugenial D 1.9 μM and eugenial E 4.97 μM of IC50 for K562 and for B16F10 cells 6.0; 3.2 and 8.8 μM, respectively. The eugenials were evaluated in silico for prediction of bioactivity, toxicity and molecular properties using the ChemDoodle, Molinspiration and PreADMET services. The results of in silico analysis have shown that eugenial C and eugenial D may be mutagenic. In rats eugenial C, eugenial D and eugenial E may be carcinogenic, and eugenial D may have low-to-medium risk of environmental / developmental toxicity.
1. Introduction The importance of medicinal plants and natural products as strategic sources for the discovery of new and effective anticancer agents is well known, as is their use as models to design compounds with improved properties. In fact, almost 80% of all clinical anticancer agents are structurally related to natural products (Newman and Cragg, 2012). Eugenia constitutes one of the most representative genus of Myrtaceae family, distributed mainly in tropical areas, such as the north of Brazil, where the existence of 350 species is estimated (Landrum and Kawasaki, 1997). In terms of tree species, the genus is one of the richest on the Atlantic coast of Brazil and many species are appreciated in the diet due to their edible fruits, including E. uniflora (“pitanga”), E. edulis (“jaboticaba”) and E. brasiliensis (“grumixama”) (Mazine et al., 2014). Eugenia umbelliflora O. Berg, known as “baguaçu”, is a tree with
edible fruits that grows in southeastern Brazil. Pharmacological in-vivo and in-vitro studies have exhibited promising gastroprotective effects and antibacterial, antifungal and antileishmanial activity of its fruit extracts (Meyre-Silva et al., 2009; Machado et al., 2005, 2009; Cechinel-Filho et al., 2013), prompting phytochemical studies with E. umbelliflora fruits, which identified the presence of anthocyanin in the methanol ripe fruit extract (Kuskoski et al., 2003) and four previously unkown terpenyl-phloroglucinolaldehydes: eugenials A–D, in the CH2Cl2 unripe fruit extract (Faqueti et al., 2015). It was also demonstrated that eugenials C and D possess excellent antibacterial activity against different methicillin-resistant strains of Staphylococcus aureus, which corroborates the antibacterial activity shown by the extract (Machado et al., 2005; Faqueti et al., 2015). The literature reports that phloroglucinols from species of Myrtaceae family are promising compounds in drug discovery, mainly
⁎ Corresponding author.at: Universidade Federal de Santa Catarina (UFSC), Curso de Farmacia - Bloco JK, Campus Reitor Joao David Ferreira Lima, s/n - Trindade, Florianopolis, Santa Catarina State, 88040-900, Brazil. E-mail address: [email protected] (C. Meyre-Silva).
https://doi.org/10.1016/j.phytol.2018.07.004 Received 6 May 2018; Received in revised form 20 June 2018; Accepted 10 July 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of eugenials A–E (1–5) isolated from Eugenia umbelliflora fruits. (The numbering of compound 5 has been changed from the used for 4 in our preceding paper, to facilitate the follow-up of discussions on the terpene stereochemical assignment).
as antineoplastic agents (Wang et al., 2012; Soliman et al., 2014; Nisa et al., 2016). Considering this fact and the biological potential of E. umbelliflora fruits, this study aims to evaluate the cytotoxicity of extracts and components of E. umbelliflora fruits against several tumor cells.
Table 1 1 H and 13C NMR spectral data for Eugenial E (5).a
2. Results and discussion Five phologlucinol derivates were isolated. Four of which have already been reported by our research group and named eugenials A (1), B (2), C (3) and D (4), as mentioned previously. Structure elucidation of a sequiterpenyl phloroglucinol (eugenial E) (5) is described here for the first time (Fig. 1). It was isolated from the hexane extract of E. umbelliflora fruits in the form of a white amorphous solid (mp: 132–137 °C, not recrystallized), whose standard mass spectrum (EI-MS) showed peak at m/z 440, corresponding to the formula C27H36O5; whereas negative and positive electrospray techniques, NESI-MS and POSI-MS, yielded values of m/z 457 [M−H]− and m/z 459 [M+H]+ respectively, incremented in 18 mass units of a H2O molecule, and supporting the molecular formula C27H38O6 for 5. The IR spectrum exhibited a strong carbonyl band at 1625 cm-1 and the NMR data are summarized in Table 1. All 1H and 13C chemical shifts were assigned by combined use of one- and two-dimensional NMR methods. According to previous results for other eugenials, the spectral data suggested that compound 5 consisted of two parts: a sesquiterpene fragment, numbered as the main unit (Fig. 1, Table 1) to facilitate discussion of stereochemical assignment, and a fully substituted phloroglucinol moiety, similar to those previously reported for other eugenials and numbered as the prime (‘) unit, including the connecting methylene group as C-1′. The 13C NMR spectrum of 5 (Table 1) showed 27 signals which were associated with 5 methyls, 7 methylenes, 5 methines and 10 non-protonated carbons, with the help of DEPT spectra. Seven signals [δC: 193.4 (d, C-12′), 171.6 (s, C-3′), 168.9 (s, C-5′), 165.2 (s, C-7′), 108.0 (s, C-2′), 104.6 (s, C-4′) and 105.2 (s, C-6′)] arose from a typical formylphloroglucinol unit, while signals for a linear butiryl substituent [δC: 207.5 (s, C-8′), 46.7 (t, C-9′), 18.4 (t, C-10′) and 14.2 (q, C-11′)] were also observed. The remaining 13C NMR signals were attributed to the sesquiterpene part, with two methyls (δC 22.7 × 2), four methylenes (δC 21.1, 23.1, 34.0 and 36.8), three methines (δC 40.5, 46.6 and 48.3) and one quaternary
Position
δC, ppma multiplicityb
δH, ppm multiplicity, (J in Hz)
1 2
48.3 d 23.1 t
3 4 5 6 7 8 9
34.0 t 40.5 d 150.3 s 123.7 d 46.6 d 21.1 t 36.8 t
10 11 12 13 14 15 1’
38.7 73.0 28.0 27.9 22.7 22.7 23.1
2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 11’ 12’
108.0 s 171.6 s 104.6 s 168.9 s 105.2 s 165.2 s 207.5 s 46.7 t 18.4 t 14.2 q 193.4 d
1.50 m 1.10 m 1.27 dd (13.2, 3.3) 1.48-1.32 m 2.45 m – 5.55 d (3.3) 2.05 mc 1.75 m 1.65 m 1.80 m – – 1.15 s 1.15 s 1.13 s 1.13 d (7.6) 2.45 dd (13.8, 11.4) 2.79 dd (13.8, 2.7) – – – – – – – 3.10 t (7.2) 1.70 dt (7.2) 0.98 t (7.2) 10.15 s
a b c
s s q q q q t
in Acetone-d6 at 300 MHz (1H) and 75.5 MHz (13C). assigned by DEPT method, s = C, d = CH, t = CH2 and q = CH3. Overlapped with the residual signal of the solvent.
carbon (δC 38.7), along with signals for a trisubstituted double bond (δC150.3 and 123.7) and a not totally free rotating isopropanol residue (δC 27.9, 28.0 and 73.0). Comparison of these 13C NMR spectral data with those of eugenial D (4) (Faqueti et al., 2015), revealed a close similarity except for the presence of the 2-hydroxy-2-propyl group at C-7 in compound 5, 188
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et al., 2015). The NOE correlations of H-14 with H-1′ and H-8β indicated that these protons were co-facially oriented, thus establishing the β-orientation of Me-14. In addition, NOEs were found between the olefinic proton H-6 and the methine protons H-4 and H-7, and between the methyl protons H-12/13 and H-9α, while a narrow multiplicity pattern was observed for the H-7 signal, a multiplet with J-coupling values under 5 Hz. All these data supported the proposal of an almost coplanar pseudo-equatorial arrangement for H-4, H-6 and H-7, and the pseudo-axial orientation of both the methyl group at C-4β and the 2hydroxy-2-propyl group attached to C-7α. Some significant interprotonic distances calculated for the main conformer of 5, close to those found for the other three main conformers and in fairly close agreement with the experimentally observed NOEs, are indicated in Fig. 3. Therefore, the structure 5 was assigned to eugenial E. Aiming to obtain additional proofs of the structure of 5, some molecular modelling studies were performed (see Experimental part for details). A conformational study, followed by an optimization at the DFT level of stable conformers, provided four main low energy conformers for this compound (Fig. 4). These conformers mainly differed in the pattern of intramolecular hydrogen bonding of phenols to carbonyls in the phloroglucinol moiety, while all of them displayed very similar conformational energies and spatial arrangements of the bicyclic terpene system. Small differences in conformational energy (≤ 0.5 kcal mol−1) found within this group of conformers ensure the practical free rotation of the C-7 – C-11 bond and the hydroxypropyl fragment, thus justifying the experimental NOE observed between the H-12/H-13 methyl protons and H-9α. H-bonds between phenols of the phloroglucinol fragment and the neighboring carbonyl groups are also depicted in Fig. 4. The permanent involvement of the 5′−OH group in Hbonding and its preference towards the formyl group at C-6′ were noted, while the butiryl carbonyl binds to the 3′−OH group in such cases, according the numbering system used in this article. As depicted in Fig. 1, the phloroglucinols isolated from E. umbelliflora have global structural similarity, but differ in the associated terpenoid, and reveal the existence of congener series that might be a consequence of the plant’s need to generate its own chemical diversity, which assists in the discovery of new bioactive compounds. In order to establish the antineoplastic potential of this plant and relate it to the constituents, the extracts and the isolated eugenials were evaluated for their ability to inhibit the growth of three types of cancer cells: human chronic myelogenous leukemia (K562) and acute lymphoblastic leukemia (Nalm-6) cells, and murine melanoma (B16F10) cells. The extracts analyzed showed good cytotoxicity, while eugenial C, eugenial D and eugenial E displayed IC50 values in the low range for K562 and Nalm-6 cells as shown in Tables 2 and 3. When the cytotoxicity of isomers 1 and 2 was compared, it was observed that 1 was slightly more potent than 2 on K562 cells (IC50 30.5 μM versus 42.8 μM), but was inactive against B16F10 cells. The main difference in the structure is the exchange of positions between the formyl and butiryl groups, slightly affecting the value for lipophilicity (log P), as shown in Table 4. The inhibition results found for K562 cells underscore the differences in cytotoxicity shown by the fused phloroglucinol-monoterpene eugenials A and B (1, 2), compared with
Fig. 2. Key HMBC correlations for the new compound 5.
replacing the 2-propenyl group present in 4. Based on connectivities observed in the HSQC and HMBC spectra (Fig. 2), the signals were collectively assembled in a sesquiterpene with the eudesmane skeleton, as depicted in the structure of 5, based on the following findings. Were observed the correlations of methyl protons H-15 with C-5, of H-4 with C-5 and C-6, and of H-6 with C-4, C-7 and C-8, as well as of H-7 with the olefinic C-5, that linked the trisubstituted double bond Δ5 to C-7. The correlations of methyl protons H-14 with C-1, C-5, C-9 and C-10 allowed the connections of C-1, C-5 and C-9 to the quaternary carbon C10. The correlations of methyl protons H-13 with C-7, C-11 and C-12, and of methyl protons H-12 with C-7, C-11 and C-13, located the hydroxypropyl group at C-7. In addition, the HMBC and COSY correlations revealed that the two parts of eugenial E were linked by a methylene bridge (C-1′). One or both signals of the connecting gem-methylene protons at position C-1′ (δH 2.45 dd and 2.79 dd, coupled to H-1) showed correlations with those of carbons C-2′, C-3′, and C-7′ of the aromatic phloroglucinol fragment, and with those of C-1, C-2 and C-10 of the eudesmane skeleton. Furthermore, HMBC correlations of the aldehyde proton H-12′ (δH 10.15) allowed the differential assignment of signals for the neighboring C-5′, C-6′ and C-7′ atoms with respect to those of C-2′, C-3′ and C-4′, close to the butiryl carbonyl. Other HMBC correlations were also observed for the methyl protons H-12 and H-13 with C-7; the methylene protons H-8 with C11 and C10; the olefinic H-6 with C-4 and C-10, and the methyl protons H-14 and H-15 with the non-protonated olefinic carbon C-5 and, respectively, with C10 and C-4. Stereochemical assignment of compound 5 (Fig. 3) was based on combined analysis of vicinal interproton couplings of protons H-6, H-7 and H-8, and the NOE/ROE effects observed through 1D-NOEDIFF and 2D-ROESY experiments, carried out in different solvent conditions (acetone-d6, methanol-d4, pyridine-d5 and mixtures) to resolve signal overlapping in different parts of the 1H spectrum. The result was in agreement with that previously reported for eugenial D (4) (Faqueti
Fig. 3. NOE correlations observed for the terpenic part of compound 5 and relevant distances (Å) calculated for the main conformer (in chloroform), which displayed very small conformational energy differences (ΔCE) from its C-7/C-11 rotamers.
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Fig. 4. Selected stereochemical and energy calculation results from the conformational analysis performed for compound 5 in three solvents. Two pairs of low-energy rotamers, A/B (lowest) and C/D around the C1′-C2′ bond, corresponding to more stable conformers of compound 5 are represented. in Conformational energy differences (ΔCE, kcal·mol−1) with respect to the most stable conformers (A in chloroform and acetone and B in pyridine, to which the 0.00 value was arbitrarily assigned) are shown in the color of the corresponding solvent. Preferential phenol-carbonyl H-bonds in each case, are also depicted.
three sesquiterpenyl-phloroglucinols, whereas relative to structure-activity aspects, the eudesmadiene 4 was the most potent cytotoxic for either Nalm-6 or B16F10 cells. It could be of interest to note the beneficial influence for the activity associated with the presence of a larger terpenoidal (higher lipohilic) fragment attached to the phoroglucinol moiety. This observation appears to be confirmed by the activity reduction in the eudesmenol 5, in comparison with the more lipophilic eudesmadiene 4. This is probably due to the presence of the hydroxyl group at C-11 in the former. Tomoeones A–H, phloroglucinol derivatives isolated from Hypericum ascyron, showed cytotoxicity against K562 cells with IC50 values ranging from 40 to 69 μM and doxorubicin used as positive control showed an IC50 value of 0.83 μM. In comparison with this reference drug, it should be noted that the eugenial C (3, IC50 0.38 μM) shows higher cytotoxicity against such K562 cells. Consequently, it is necessary to establish the mechanism of action and to evaluate the toxicity of compound 3, in order to perform a better comparison, as doxorubicin is highly myelotoxic (Hashida et al., 2008). To obtain some information on the pharmaco-therapeutic profile of eugenials, some virtual studies were carried out for online calculation/ prediction of their main physico-chemical, pharmacological and toxicological properties. The most representative results are summarized in Table 4. Interestingly, according to the predicted molinspiration bioactivity (Molinspiration, 2017), all five eugenials qualified as potential nuclear-receptor-ligands, with homogeneus positive bioactivity scores in the range of 0.43 to 0.63. This finding could serve to rule out such a possible mechanism of cytotoxicity for eugenials, since the most potently cytotoxic of them, compound 3, achieved the lowest score. On the other hand, the fact that the cytotoxic potency of eugenials occurs in parallel with lipophilicity (Table 3), with compound 3 also displaying the highest log P value, may suggest the membrane, or another lipidic region of the cell, as the probable target zone for eugenials. In relation to the online prediction of toxicity via PreADMET (PreADMET, 2017), eugenials A and B were practically devoid of mutagenic (Ames test) carcinogenic (mouse & rat), environmental (algae, insect, minnow), or developmental (medaka embryos) effects, with a low risk of cardiac toxicity related to human Ether-à-go-go-Related Gene (hERG) inhibition. Eugenials C, D and E proved similarly non-toxic in most of the assays, though in agreement with their higher cytotoxicity, were
Table 2 Cytotoxicity of extracts from fruits of E. umbelliflora. Extract
IC50 values (MD ± SD. μg/mL) K562
EuH EuD EuA EuM EuMb
2.50 2.50 0.45 79.3 34.1
± ± ± ± ±
0.05 0.10 0.0 0.8 0.8
Nalm – 6
B16F10
12.0 4.74 8.75 83.5 78.1
35.7 ± 1.0 > 1000 Inactive 36.7 ± 1.4 > 1000 Inactive 24.1 ± 0.8
± ± ± ± ±
0.1 0.09 0.05 0.1 0.8
Note: EuH: n-hexane extract; EuD: dichloromethane extract; EuA: ethyl acetate extract; EuM: methanol extract; EuMb: methanol crude extract. Table 3 Cytotoxicity of isolated compound from fruits of E. umbelliflora. Compound
IC50 values (MD ± SD. μM) K562
1 2 3 4 5 Vincristine Doxorubicin*
30.5 42.8 0.38 1.90 4.97 0.01 0.83
± ± ± ± ± ± ±
6.9 0.9 0.3 0.1 0.3 0.07 0.02
Nalm – 6
B16F10
> 125 70.5 ± 0.3 10.5 ± 0.1 7.75 ± 0.4 29.1 ± 0.2 0.25 ± 0.12 Not rated
> 125 12.0 ± 2.1 6.00 ± 0.3 3.20 ± 0.1 8.80 ± 1.9 Not rated Not rated
Note: 1: eugenial A; 2: eugenial B; 3: eugenial C; 4: eugenial D; 5: eugenial E. * Value from Hashida et al., 2008.
the more potent eugenials C, D and E (3-5), which did not have fused, but simply attached phloroglucinol-sesquiterpene arrangements. Such differences in activity could be associated with the presence of the additional free phenol group in compounds 3 - 5. Another interesting observation relates to the comparison within the group of sesquiterpenyl derivatives. Eugenial C (3, IC50 = 0.38 μM), bears a tricyclic aromadendrene fragment, revealed as 5-fold and 13-fold respectively, more potent than those with the bicyclic eudesmadiene (eugenial D, 4, IC50 = 1.9 μM) and the eudesmenol (eugenial E, 5 IC50 = 4.97 μM) fragments. Relative to the other cell lines, the results found were parallel, with the Nalm-6 leukemia cells being the least sensitive to the 190
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Table 4 Selected calculated and predicted molecular, pharmaco-toxicological and ADME data for eugenials.A–E(1–5) Comp.
Formula
MW
Log Sa
clog Pa
NRLsb
Mutc
MCarc
EDTc
HIAc
SkPc
Caco2c
1 2 3 4 5
C22H28O5 C22H28O5 C27H36O5 C27H36O5 C27H38O6
372.459 372.459 440.578 440.578 458.593
−4.84 −4.84 −5.76 −5.62 −5.26
5.54 5.11 6.27 5.58 5.37
0.55* 0.55* 0.43 0.61* 0.63*
non non mut mut non
neg neg neg# neg# neg#
low low low l-m low
93.6 93.6 91.3 91.7 82.3
−2.47 −2.47 −1.87 −1.73 −1.52
11.91 11.91 19.28 19.17 18.44
MW: Molecular Weight; Log S: log Molar aqueous solubility at pH = 7; clog P: calculated log of n-octanol/water partition; NRLs: Nuclear-Receptor-Ligand score (* potential enzyme inhibitor); Mut: mutagenic (Ames test); MCar: Mouse-Carcinogenicity (neg = negative, # = positive in rat); EDT: Environmental/Developmental Toxicity (l–m = low-to-medium risk); HIA: Human Intestinal Absorption (%); SkP: skin permeability (log Kp in cm/h); Caco2: colon adenocarcinoma cell permeability (nm/sec). a ChemDoodle. b Molinspiration. c PreADMET.
4.3. Extraction and isolation
predicted as potentially mutagenic and carcinogenic (to rats only). Additionally, a medium risk of cardiac incidence associated with hERG inhibition was predicted for eugenial D. In relation to the bioavailability and ADME properties of eugenials A – E, high levels (from 82.2% for 5 to 93.6% for 1 and 2) of human intestinal absorption (HIA) and middle Caco2 adenocarcinoma cell permeability (from 11.9 nm/sec for 1 and 2 to 19.3 for 3) were predicted through PreADMET algorithms. The physico-chemical parameters also demonstrated that there is no violation of Egan (Egan et al., 2000) or Veber (Veber et al., 2002) rules, and one violation (log P > 5) of the Lipinski rule of five (Lipinski, 2004), which along with the bioavailability score (BS) of 0.17 for all the compounds (ChemDoodle, 2016), likely indicates their good oral bioavailability.
The unripe fruits were dried and powdered using a knife grinder. The material (100 g) were extracted successively by maceration at room temperature with n-hexane, dichloromethane, ethyl acetate and methanol for five days. The solvent was removed under reduced pressure with a rotatory evaporator, resulting in the n-hexane (EuH) (21.29 g), dichloromethane (EuD) (14.14 g), ethyl acetate (EuA) (4.68 g) and methanol extracts (EuM) (28.93 g). Another crude methanol extract (EuMb) (81.62 g) was obtained by maintaining the ground, dried fruits with methanol at room temperature for seven days. Another extract was obtained by direct maceration with methanol at room temperature for seven days (81.62 g), identified as crude methanolic extract (EuMb). The EuH (21.29 g) was subjected to silica gel 60 (Merck) open column chromatography (4 x 50 cm) and eluted with 400 mL each of gradients n-hexane:CH2Cl2 in ratios 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50, respectively. Based on similar Rf values on TLC developed with the eluent n-hexane:CH2Cl2 (3 :7) and sprayed with 10% sulfuric acid three fractions (C1 − C3) were obtained. Fractions C1 (1.5 g) and C2 (6 g) were resolved as described by Faqueti et al., 2015, furnishing the compounds eugenial A (1), eugenial B (2), eugenial C (3) and eugenial D (4). Fraction C3 (10 g) was submitted to purification by silica gel 60 open column chromatography (2.5 x 30 cm) and eluted with 400 mL each of mixtures of n-hexane:CH2Cl2 at ratios of 80:20, 70:30, 60:40, respectively, yielding 90 mg of a powder eluted in the 70:30 fraction, which was identified as a new compound and denominated eugenial E (5): amorphous white solid; mp 132–137 °C; [α]D −36° (c 0.20%, MeOH); IR (KBr) 3500, 3000, 2950, 1625, 1500, 1450, 1250, 800 cm−1; NMR data, see Table 1; HRESIMS, found 459.2730 (calcd. for C27H38O6+H: 459.2741).
3. Conclusion In conclusion, the study resulted in the isolation and characterization of a new sesquiterpenyl phloroglucinol eugenial E, and determination of promising cytotoxicities of the phloroglucinols meroterpenoids isolated from the extracts of Eugenia umbelliflora that merits further study, in search of a candidate for pre-clinical evaluation of efficacy and toxicity. 4. Experimental 4.1. General experimental procedures Melting points were determined in a MQ APF-301 (Microquímica) and are uncorrected. Optical rotations were measured at room temperature in CHCl3 solution using a 341 Perkin-Elmer digital polarimeter equipped with a sodium lamp. IR spectra were recorded on an MB-100 Bomen spectrometer. HRESIMS analyses were performed on an Agilent 1100 HPLC-ESI-Q-TOF system. NMR spectra were recorded on DRX-400 (1H at 400 MHz) (Bruker) and Mercury plus 300 (1H at 300 MHz) (Varian) spectrometers. Acetone-d6 was mainly used as solvent, while other deuterated solvents were used to avoid signal overlapping. Column chromatography was performed with silica gel (70–230 mesh) from Merck. TLC analysis was carried out using pre-coated plates (Merck, silica gel GF-254, 0.2 mm): the spots were visualized either using a UV lamp λ254 nm or by spraying with 10% sulfuric acid in ethanol.
4.4. Cytotoxicity assay K562 Homo sapiens bone marrow chronic myelogenous leukemia (ATCC® CCL243™), Nalm-6 Homo sapiens peripheral blood acute lymphoblastic leukemia (DSMZ® ACC 128) B16F10 and murine melanoma (ATCC® CRL6475™) cells were obtained from the cell bank of Rio de Janeiro, Brazil. B16F10 and K562 cells were cultured in Dulbecco's modified Eagle's medium (DMEM); NALM-6 cells were cultured in RPMI 1640 medium. The culture media were supplemented with 10% fetal bovine serum, 100 U/mL benzylpenicillin and 100 μg/mL streptomycin. Cell cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. In all experiments, viable cells were checked in the beginning of the experiment by trypan blue. Cells were seeded in 96-well plate with density 30,000 cell/well and pre-incubated for 24 h. The extracts and compounds were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 0.5%). After removal of pre-incubated culture medium, 100 μL of medium containing various concentrations of samples were
4.2. Plant material The unripe fruits of E. umbelliflora were collected in June 2010 in the town of Balneário Camboriú, Santa Catarina, Brazil, and identified by Prof. Msc. Renê Artur Ferreira (Universidade do Vale do Itajaí, ItajaíSC). A voucher specimen (No. VC Filho 50) was deposited at Barbosa Rodrigues Herbarium, Itajaí, Santa Catarina, Brazil. 191
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added and further incubated for 24 h for B16F10 cells and 48 h for Nalm-6 and K562 cells. Cell viability was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Mosmann, 1983). The optical density (OD) of each well was detected using a Microplate reader at 540 nm. The 50% inhibition concentration (IC50 value) was determined by curve fitting with the Graphpad Prism.
J. Comput. Chem. 17, 1571–1586. 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4.5. Molecular modelling studies The structure of compound 5 was built using Spartan'08 (Spartan’08. Wavefunction, Inc., Irvine, CA). Conformational analysis was performed by a Monte Carlo random search with the MMFF Force Field implemented in that Package. Geometry optimizations and energy calculations were performed with GAUSSIAN 09 (Frisch et al.,2009) using DFT (Lynch et al., 2003; Koch and Holthausen, 2000; Parr and Yang, 1989) at the B3LYP/6-31+g (d,p) (Stephens et al., 1994; Becke, 1993; Lee et al., 1988; Schuchardt et al., 2007; Feller, 1996) level of theory. To simulate the NMR experimental conditions, the solvent effect (acetone, dichloromethane or pyridine) was considered during the calculation using the SMD continuum model (Marenich et al., 2009). 4.6. Prediction of molecular properties The five structures of eugenials were evaluated in silico for prediction of bioactivity, toxicity and molecular properties using the ChemDoodle (ChemDoodle, 2016), Molinspiration (Molinspiration, 2017) and PreADMET (PreADMET, 2017) services. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgments The authors are grateful to CNPq-Brazil, ProPPEC/UNIVALI, MINECO-Spain (Project: AGL2016-79813-C2-2-R) and JCyL-Spain (Project: SA221U13) for providing the financial support, Prof. Rene A. Ferreira (UNIVALI) for the classification and collection of plant material. Free access to online services of ChemDoodle Free Tral, Molinspiration and PreADMET predictive systems are also acknowledged. This manuscript is dedicated to Prof. Dr. Mahabir P. Gupta on the occasion of his 75th anniversary References Becke, A.D., 1993. Density functional thermochesmistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. Cechinel-Filho, V., Meyre-Silva, C., Niero, R., Mariano, L.N.B., Nascimento, F.G., Farias, I.V., Gazoni, V.F., Silva, B.S., Giménez, A., Gutierrez-Yapu, D., Salamanca, E., Malheiros, A., 2013. Evaluation of antileishmanial activity of selected brazilian plants and identification of the active principles. Evid. Complement. Alternat. Med. https:// doi.org/10.1155/2013/265025. advance online publication 9 June 2013. ChemDoodle Free Trial. URL. https://www.chemdoodle.com/free-trial/, 2016 (Accessed November 2016). Egan, W.J., Merz, K.M., Baldwin, J.J., 2000. Prediction of drug absorption using multivariate statistics. J. Med. Chem. 43, 3867–3877. Faqueti, L.G., Farias, I.V., Sabedot, E.C., Delle-Monache, F., San-Feliciano, A., Schuquel, I.T., Cechinel-Filho, V., Cruz, A.B., Meyre-Silva, C., 2015. Macrocarpal-like compounds from Eugenia umbelliflora fruits and their antibacterial activity. J. Agric. Food Chem. 63, 8151–8155. Feller, D., 1996. The role of databases in support of computational chemistry calculations.
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