Structure and Absolute Configuration of Abietane ... - ACS Publications

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

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Structure and Absolute Configuration of Abietane Diterpenoids from Salvia clinopodioides: Antioxidant, Antiprotozoal, and Antipropulsive Activities Celia Bustos-Brito,† Pedro Joseph-Nathan,‡ Eleuterio Burgueño-Tapia,§ Diego Martínez-Otero,⊥ Antonio Nieto-Camacho,† Fernando Calzada,∥ Lilian Yépez-Mulia,# Baldomero Esquivel,*,† and Leovigildo Quijano*,†

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Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Mexico City, 04510 Mexico ‡ Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, Mexico City, 07000 Mexico § Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala, Col. Santo Tomás, Mexico City, 11340 Mexico ⊥ Centro Conjunto de Investigación en Química Sustentable UAEM-UNAM, Carretera Toluca-Atlacomulco, km. 14.5, Toluca, 50200 Mexico ∥ Unidad de Investigación Médica en Farmacología del Hospital de Especialidades and #Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias del Hospital de Pediatría, UMAE, Centro Médico Nacional Siglo XXI, IMSS, Avenue Cuauhtémoc 330, Mexico City, 06725 Mexico S Supporting Information *

ABSTRACT: The aerial parts of Salvia clinopodioides afforded abietanes 1a, 2a, and 3 (clinopodiolides A−C), two of which possess an unusual lactol moiety at C-19−C-20, together with an icetexane named clinopodiolide D (4a). Their structures were established by spectroscopic means, mainly 1H and 13C NMR, including 1D and 2D homo- and heteronuclear experiments. The antioxidant, antiprotozoal, and antidiarrheal effects of the isolates were evaluated. Compounds 2a and 3 showed better effects than α-tocopherol in the inhibition of lipid peroxidation with IC50 (μM) = 5.9 ± 0.1 and 2.7 ± 0.2, respectively, and moderate activity in the DPPH assay. All tested compounds showed moderate antiamoebic and antigiardial activity, as well as a good antipropulsive effect.

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been described in the leaves and roots of plants in this genus, the most distinguishing feature of Salvia is the significant number or rearranged diterpenoid skeletons.6 Among the produced metabolites, the diterpenoids could be part of the active principles responsible for the ethnobotanic uses of sages in the folk medicine systems of several parts of the world. Cytotoxic, antimicrobial, antioxidant, antiprotozoal, allelopathic, and antifeedant activities, among others, have been documented for some diterpenoids isolated from sages.7 Recently we described the diterpenoid content and biological activity of Salvia ballotif lora Benth.8 In continuation of a systematic phytochemical study of Mexican Salvia spp. in search for biologically active diterpenoids, in this paper we

he Lamiaceae family is highly diverse in Mexico, with 32 genera and over 590 species totaling 65.8% endemism.1 The genus Salvia L. (tribe Mentheae) is the largest of the family, with nearly 1000 species widespread worldwide, although the major area of diversity is the New World,2 with over 317 species described in Mexico. The family was organized into four subgenera by Bentham (Salvia, Leonia, Sclarea, and Calosphace),3 although the diterpenoid content4 and taxonomic and molecular phylogenetic analysis suggest a reconsideration of this classification and also the possibility to split this large group of species into six genera.5 Phytochemical studies of several Salvia species, commonly known as sages, established that the most diversified and characteristic secondary metabolites of the genus are diterpenoids. Although several labdane, pimarane, kaurane, totarane, clerodane, abietane, and icetexane constituents have © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 14, 2018

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DOI: 10.1021/acs.jnatprod.8b00952 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

RESULTS AND DISCUSSION The CH2Cl2 extract of S. clinopodiodes afforded the three new abietanes 1a, 2a, and 3, as well as the new icetexane 4a. The compounds were fully characterized by physical and spectroscopic properties, including 1H and 13C NMR data, whose signals were assigned based on COSY, HSQC, NOESY, and HMBC correlations. Compound 1a was isolated as white powder that revealed absorption maxima due to hydroxy (3592 and 3219 cm−1) and carbonyl groups (1686 cm−1) in the IR spectrum. Its mass spectrum indicated the molecular formula C21H28O5 and a high degree of unsaturation. The 13C NMR data (Table 1) showed the presence of 21 carbons grouped, according to the HSQC and DEPT experiments, into four methyls, five methylenes, four methines including an aromatic one, and eight nonprotonated carbons comprising two sp3, a carbonyl, and five aromatic ones. In the 1H NMR data (Table 1) the four methyl signals were observed as a methoxy group at δ 3.86 (δ 61.1) attached to C-12 (δ 146.6), Me-18 (δH1.14, δC 24.3) located at C-4, and two characteristic signals of an isopropyl group connected to an aromatic ring at δ 1.19 and 1.21 (3H each, d, J = 6.9 Hz) attached to CH-15 at δ 3.28 (1H, sept, J = 6.9 Hz) of an abietane skeleton. This was confirmed by the HMBC correlation of C-15 with the aromatic hydrogen atom at δ 6.55, which was assigned as H-14 in agreement with a pentasubstituted aromatic ring. Two singlets at δ 9.27 and 4.36, disappearing upon addition of D2O, were assigned to a

report the chemical composition and biological activity of extractives from the leaves of Salvia clinopodioides Kunth. (section Cucullatae).9 This perennial herb is endemic to Mexico, where it is mainly found in the states of Chihuahua, Durango, Jalisco, Mexico, and Michoacan. From the aerial parts of this species an icetexane and three new abietane diterpenoids were isolated and assayed for their antioxidant, antiprotozoal, and antipropulsive effects. Chart 1

Table 1. NMR Data (1H 700 MHz and 13C 176 MHz, CDCl3) of 1a and 2a 1a position

δC

type

1a 1b 2a 2b 3a 3b 4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe 11-OH 12-OH 19-OH

35.2

CH2

21.2

CH2

32.8

CH2

37.0 50.4 21.6

C CH CH2

31.1

CH2

133.1 123.7 50.4 150.0 146.6 141.9 118.3 26.9 23.4 23.6 24.3 102.8 179.7 61.1

C C C C C C CH CH CH3 CH3 CH3 CH C CH3

2a

δH (J in Hz)

HMBC

3.45, 1.45, 1.74, 1.74, 2.29, 1.21,

brd (14.3) m m m brd (13.9) m (obs)

2, 2, 1, 1, 1, 4,

3, 3, 4, 4, 2, 5,

5, 9 5, 20 5a 5a 5 18

1.58, 2.11, 1.32, 2.83, 2.77,

dd (11.8, 1.0) m qd (12.5, 5.1) ddd (16.7, 4.8, 1.8) ddd (16.7, 12.3, 4,8)

1, 5, 5, 5, 5,

7, 7, 7 6, 6,

19, 20 8

6.55, 3.28, 1.19, 1.21, 1.14, 5.60,

brs hept (6.9) d (6.9) d (6.9) s brs

3.86, s 9.27, s

8, 9, 14 8, 9, 14

7, 9, 12, 15 12, 13, 14, 16, 17 13, 15 13, 15 3, 4, 5, 19 3, 18, 20 12 9, 12

δC

type

35.5

CH2

21.3

CH2

32.7

CH2

37.1 50.4 21.8

C CH CH2

30.8

CH2

128.2 122.4 50.0 142.3 143.5 134.1 119.0 27.3 22.4 22.5 24.2 102.9 180.4

C C C C C C CH CH CH3 CH3 CH3 CH C

δH (J in Hz) 1.48, 3.35, 1.72, 1.77, 1.23, 2.30,

td (13.4, 5.1) ddt (13.6, 4.2, 1.9) qt (14.4, 3.7) m td (12.6, 6.3) ddt (13.9, 4.1, 1.9)

HMBC 2, 2, 3, 3, 1, 1,

3, 3, 4, 4, 2, 2,

5, 9 5, 9 10 10 5, 18, 19 5, 19

1.61, dd (13.0, 1.6) 1.29, qd (12.7, 4.8) 2.10 ddt (13.0, 4.5, 2.0) 2.75, ddd (16.6, 12.3, 5.0) 2.80, ddd (16.5, 5.0, 2.0)

4, 5, 5, 5, 5,

6, 7, 7, 6, 6,

7, 19 8 8 8, 9, 14 8, 9, 13,a 11,a 14

6.55, 3.24, 1.23, 1.23, 1.14, 5.60,

7, 9, 10,a 12, 15

s hept (7.0) d (7.0) d (7.0) s brs

9.31, s 6.10, s 4.53, brs

4.36, s

a

Through four-bond interaction. B



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Table 2. NMR Data (1H 700 MHz and 13C 176 MHz, CDCl3) of 3 and 4a 3 position

δC

type

1a 1b 2a 2b 3a 3b 4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19a 19b 20a 20b OMe 11-OH 12-OH

35.2

CH2

21.8

CH2

40.5

CH2

33.1 49.3 22.2

C CH CH2

30.5

CH2

128.2 122.8 50.1 142.3 143.6 134.0 119.0 27.3 22.4 22.5 23.8 77.3

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

179.9

C

4a

δH (J in Hz)

HMBC

δC

type

35.4

CH2

19.8

CH2

35.7

CH2

48.8 53.8 23.8

C CH CH2

31.3

CH2

138.0 118.4 84.4 147.1 142.7 139.6 117.2 26.6 24.0 23.9 17.2 180.7

C C C C C C CH CH CH3 CH3 CH3 C

33.3

CH2

62.0

CH3

3.34, 1.46, 1.82, 1.72, 1.84, 1.56,

brd (14.4) td (13.7, 4.1) m qt (14.1, 4.4) m m

2, 2, 1, 1, 1, 2,

3, 5, 9 3, 5, 9, 20 4, 10 4, 10 5,19 4, 19

1.58, 2.10, 1.31, 2.81,

d (12.4) brd (12.6) qd (12.5, 5.3) m

1, 7, 5, 5, 5,

4, 8, 7, 6, 6,

6.55, 3.25, 1.23, 1.23, 1.05, 4.37, 4.23,

brs sept (7.0) d (7.0) d (7.0 s d (11.6) d (11.6)

9.35, s 6.10, s

7, 10, 19, 20 10 8 8, 9, 14 8, 9, 14

7, 9, 10,a 12, 15 12, 13, 14, 16, 17 13, 15 13, 15 3, 4, 5, 19 3, 4, 5, 18, 20

8,a 9, 11 12, 13

δH (J in Hz) 1.99, 1.75, 1.76, 1.64, 1.65, 1.50,

brd (14.3) m m m m m

HMBC 2, 3, 5, 10, 20 3, 10 1, 4, 10 1, 4, 5, 10, 19, 2, 4, 5, 10, 19

1.81, dd (12.7, 4.2) 1.92, dq (14.7 6.5) 1.4, tt (13.8, 7.2) 2.90, dt (15.3, 6.0) 2.77, dt (14.8, 7.2)

1, 4, 4, 5, 5,

6.49, 3.21, 1.22, 1.22, 1.07,

7, 9, 11,a 12, 15, 16, 17 13, 14, 16, 17 13, 15 13, 15 3, 4, 5, 19

s sept (6.9) d (6.9) d (6.9) s

3.21, d (14.1) 3.14, d (14.1) 3.76, s 5.68

3, 5, 5, 6, 6,

4, 7, 7, 8, 8,

6, 19 8, 10 8, 10 14 14

8, 9, 11, 10, 5 8, 9, 11, 10, 5 12 9, 11, 12, 20a

a

Through four-bond interaction.

showed the presence of the acetyl methyl group (δ 2.20) and the downfield shift of H-19 to δ 6.44, while the IR spectrum displayed absorption bands for two carbonyls due to the acetate and the δ-lactone (νmax 1769, 1700 cm−1, respectively) functionalities. Diacetate 1c showed two acetate methyl signals (s, δ 2.17 and 2.25) in the 1H NMR data and a broad carbonyl absorption band at 1765 cm−1 in the IR spectrum. The lactol moiety is an uncommon chemical functionality in natural products. Only a few compounds with this functional group have been isolated from Isodon species (Lamiaceae),10,11 this being the first time such a hydroxy-δ-lactone is isolated from a Salvia species. Treatment of 1a with diazomethane in Et2O gave 5a, whose high-resolution direct analysis in real-time mass spectrometry (HR-DARTMS) data showed an [M + H]+ ion at m/z 375.216 46 (calculated for C22H31O5, 375.21715), which supported the molecular formula C22H30O5. The 13C NMR spectrum showed 22 carbon signals, 20 of them due to the diterpenoid skeleton and two to the methyl ether and methyl ester groups. The 1H and 13C NMR spectra of 5a displayed features similar to those of 1a, except for the presence of a formyl hydrogen signal at δH 9.60 (δC 203.8) instead of the lactol group and an additional sharp singlet at δ 3.76 (δC 61.6) due to the methyl ester group. Compound 2a displayed an [M + H]+ ion at m/z 347.184 38, as determined by HR-DARTMS, which supports the molecular formula C20H26O5. The IR spectrum revealed

phenolic group attached to C-11 (δ 150.0) and a hemiacetalic hydroxy group at C-19 (δC 102.8, δH5.60), respectively. The signal at δ 179.7 was ascribed to the δ-lactone carbonyl (C-20) in accordance with the IR absorption. The above data suggested the presence of the C-19 lactol functionality. The signal assigned to C-18 (δC 24.3) showed HMBC correlations with the methylene protons at δ 1.21 and 2.29 and the methine signal at δ 1.58 (1H, dd, J = 11.8, 1.0 Hz), thus assigning these signals to CH2-3 and CH-5, respectively, the latter being α-axially oriented. In addition, the hemiacetalic proton at δ 5.60 (H-19) showed HMBC correlations with C-3 (δC 32.8) and C-5 (δC 50.4), and therefore, the lactol group must be located at C-19. Furthermore, the carbonyl group at δ 179.7 (C-20) showed three-bond HMBC correlations with H5 and four-bond correlations with the C-2 hydrogens of the methylene multiplet at δ 1.74, and accordingly the lactone group must be attached to C-10. In the COSY spectrum H-5 showed correlation peaks with signals at δ 2.11 (1H, m) and 1.32 (1H, dq, J = 12.5, 5.1 Hz), which in turn correlated with the methylene protons at δ 2.83 (1H, ddd, J = 16.7, 4.8, 1.8 Hz) and 2.77 (1H, ddd, J = 16.7, 12.3, 4.8 Hz), assigning these signals to CH2-6 and CH2-7, respectively. Therefore, it follows that clionopodiolide A is a new abietane derivative identified as 11,19-dihydroxy-12-methoxyabietatrien-19,20-olide. Acetylation of 1a with pyridine and Ac2O gave monoacetate 1b, while diacetate 1c was obtained by acetylation catalyzed with 4-dimethylaminopyridine. The 1H NMR data of 1b C



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absorption bands for −CO and −OH groups at 1685, and 3593, 3517, and 3214 cm−1. The 1H NMR data (Table 1) displayed features similar to those of 1a, except for the absence of the signal at δ 3.86, corresponding to the methoxy function, and an additional singlet exchangeable with D2O at δ 6.10 assigned to the hydroxy group at C-12, instead of the methoxy group. Consequently, the structure of clinopodiolide B (2a) was defined as 12-O-demethylclinopodiolide A. As in the case of 1a, removal of the labile hydrogen atom from the lactol functionality of 2a under mild reaction conditions with diazomethane caused ring opening to generate a formyl and a carboxylic acid functionality, the latter in the presence of diazomethane transforming into the corresponding methyl ester 5b. The HR-DARTMS showed an [M + H]+ ion at m/z 361.200 31, which together with the 13C NMR data indicates the molecular formula C21H28O5. The 1H and 13C NMR spectra of 5b were similar to those of 5a, the main differences being the lack of methoxy group signals. Compound 3 was isolated as a yellow oil, whose molecular composition was determined as C20H26O4 based on its HRDARTMS protonated molecular ion at m/z 331.18964 [M + H]+ (calculated for C20H27O4 331.190 93), indicating eight indices of hydrogen deficiency. The 1H NMR data of 3 (Table 2) were similar to those of 2a, except for the lack of the lactol proton and the presence of an AB spin system with doublets at δ 4.37 and 4.23 (J = 11.6 Hz), which was attributed to the C19 methylene protons, carrying a δ-lactone functionality as indicated by the HMBC correlations of the C-19 methylene protons with C-3 (δC 40.5), C-5 (49.3), C-18 (23.8), and C-20 (179.9). Accordingly, clinopodiolide C (3) is a novel abietane. Compound 4a was identified as an icetexane derivative with the molecular formula C21H28O4, as determined by HRDARTMS data. Its IR spectrum exhibited characteristic absorptions due to γ-lactone and hydroxy groups at 1761 and 3423 cm−1, respectively. In the 1H NMR data (Table 2), the characteristic proton signals with a large coupling constant assignable to H-20 appeared as an AB system at δH 3.21, 3.14 (1H each d, J = 14.1 Hz), suggesting compound 4a possesses an icetexane-type skeleton with the typical seven-membered B ring. Additionally, the signals for an isopropyl group at δ 1.22 (6H, d, J = 6.9 Hz) and 3.21 (1H, sept, J = 6.9 Hz) were observed. The 13C NMR spectrum showed four methyls, including a methoxy, six methylenes, three methines, including an aromatic one, four quaternary, three oxygenated tertiary carbons, and a carbonyl carbon. The signals (Table 2) at δC 180.7 and 17.2 were attributed to the γ-lactone carbonyl carbon of an icetexone-type derivative and the methyl group at C-4, respectively. The location of the γ-lactone ring closure, i.e., C-10, is observed at δC 84.4, which is characteristic of icetexane-type diterpenoids. Compound 4a possesses the same C-ring substitution as 1a with a hydroxy and a methoxy group located at C-11 and C-12, respectively. Thus, the structure of clinopodiolide D (4a) is defined as 6,7,11,14-tetrahydro-14deoxy-12-O-methylicetexone. A single-crystal X-ray diffraction study of 1a permitted confirmation of its structure and absolute configuration (AC). Suitable crystals were obtained by slow evaporation of a CH2Cl2 solution. The selected colorless prism was orthorhombic, space group P212121, with four molecules in the unit cell. The absolute structure parameters calculated (PLATON)12 for the analyzed crystal were Flack13 = 0.01(3), Parsons-Flack14 = 0.01(3), and Hooft15 = 0.01(3), thus confirming the sample is enantiopure. The molecular structure

shows the (4S,5S,10R,19R) configuration (chiral SPGR) (Figure 1). Concerning the conformation of 1a, the A ring

Figure 1. Molecular structure of compound 1a with 50% thermal ellipsoids.

has a chair conformation with C-19 and C-20 axially oriented being part of the hydroxy-δ-lactone moiety and the 19-hydroxy group. The six-membered C-19−C-4−C-5−C-10−C-20−O-4 lactone ring adopts a flattened chair conformation with a dihedral angle C-19−O-4−C-20−C-10 of −5.2(2)°, close to planarity, while for the eight-membered ring C-1−C-2−C-3− C-4−C-19−O-4−C-20−C-10 a boat conformation is observed with C-4 and C-10 in the highest positions and C-2 and O-4 in the lowest positions. An intramolecular hydrogen bond between the 11-phenolic group and the lactone carbonyl with a 1.76(2) Å distance forms a seven-membered ring that adopts a boat conformation described as an S117 in terms of graph set.16 This explains the strong absorption band for the δlactone at 1686 cm−1 in the IR spectrum, the uncommon chemical shift of the 11-phenolic hydrogen atom at δ 9.27 in the 1H NMR spectrum, and its resistance to acetylation under mild reaction conditions. Considering that naturally occurring 2a, 3, and 4a share a common biosynthetic origin with 1a, it could be assumed that the four molecules also have the same AC. The AC of 1a was independently verified by vibrational circular dichroism (VCD), while that of 2a was determined in the same way. The VCD methodology has been used successfully for the AC determination of several types of natural products,17,18 and it was recently used for the AC determination of abietanes and icetexanes from S. ballotiflora.8,19 Since it is well known that the presence of a hydroxy group complicates VCD studies due to intermolecular associations, acetates 1b and 2b were selected. Their molecular models were constructed in the Spartan’08 suite and subjected to initial Monte Carlo conformational searches using molecular mechanics force field MMFF94. This afforded 10 conformers in a 9.06 kcal/mol gap for 1b and eight conformers in a 9.59 kcal/mol gap for 2b. Single-point calculations of all these conformers were done using density functional theory (DFT) at the B3LYP/6-31G(d) level of theory as implemented in the Spartan’08 software package, which maintained in both cases only three conformers. In the case of 1b they appear in the initial 2.18 kcal/mol window and contribute 99.8% of the total conformational population, the next conformer being 3.77 kcal/mol above the global energy minimum. In the case of 2b they appear in the initial 2.47 kcal/mol window and contribute almost 100% of the conformational itinerary, since the next D



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conformer is 4.94 kcal/mol over the global minimum. The three conformers of 1b and 2b were geometry optimized using the Gaussian03 software, which revealed that two conformers of 1b and 2b (Figure 2) contribute 99.2% and 99.4%,

Figure 2. Most stable conformers of (4S,5S,10R,19R)-1b and (4S,5S,10R,19R)-2b.

respectively, of the total conformational population. An inspection of the four conformers shown in Figure 2 reveals that the scaffold of the diterpenoids is quite rigid since the conformers differ only in the isopropyl group orientation, as evidenced by the C-12−C-13−C-15−H-15 dihedral angles for conformers 1b−A, 1b−B, 2b−A, and 2b−B, which are −4.8°, 179.6°, 8.0°, and 177.9°, respectively. The thermochemical parameters for both molecules are given in Table 3. Finally, geometry-optimized conformers of (4S,5S,10R,19R)-1b and 2b were the subject of calculation of the IR and VCD spectra with the Gaussian03 software using DFT at the same B3LYP/ DGDZVP level of theory. The ΔG values (Table 3) for each conformer were used as the criterion for obtaining the Boltzmann distribution weighted IR and VCD spectra of 1b (Figure 3) and 2b (Figure 4). A comparison between experimental and calculated IR and VCD spectra was done using the CompareVOA software,20 providing the statistical data given in Table 4. The confidence level (C) for the (4S,5S,10R,19R) AC was 100%. The lipid peroxidation in rat brain homogenate was evaluated using the thiobarbituric acid-reactive substances (TBARS) assay. The antioxidant effect of compounds 1b, 1c, 2b, 3, 4a, and 5a was assessed as the inhibition of TBARS produced (Table 5), and their radical scavenging activity was

Figure 3. Comparison of the experimental (B) IR and (D) VCD spectra of (−)-1b and DFT B3LYP/DGDZVP-calculated (A) IR and (C) VCD spectra for (4S,5S,10R,19R)-1b.

tested using the DPPH test.21 Quercetin, butylhydroxytoluene (BHT), and α-tocopherol were used as positive controls. Compounds 2a and 3 exhibit in the TBARS assay the best inhibition effects, with an IC50 value of 5.9 ± 0.4 and 2.7 ± 0.2 μM, respectively. Both compounds showed a better effect than α-tocopherol but lower than quercetin and BHT. Compound

Table 3. Thermochemical Parameters of (4S,5S,10R,18R)-1b and (4S,5S,10R,18R)-2b 1b−A 1b−B 2b−A 2b−B

ΔEMMFFa

%b

ΔE6‑31G(d)c

%b

ΔEDGDZVPd

%b

ΔGDGDZVPe

%f

0.00 1.04 0.00 1.04

82.6 1.3 84.7 14.5

0.00 1.49 0.00 1.32

90.3 7.3 88.9 9.7

0.00 0.91 0.00 1.37

81.7 17.5 90.4 9.0

0.00 2.09 0.00 2.00

97.2 2.8 96.7 3.3

Relative to 1b−A (83.09 kcal/mol), 2b−A (77.59 kcal/mol). bCalculated according to ΔE ≅ −RT ln K. cRelative to 1b−A (−940 332.97 kcal/ mol), 2b−B (−1 011 468.33 kcal/mol). dRelative to 1b−A (−940 456.26 kcal/mol), 2b−A (−1 011 599.96 kcal/mol). eRelative to 1b−A (−940 158.31 kcal/mol), 2b−A (−1 011 298.48 kcal/mol). fCalculated according to ΔG = −RT ln K. a

E



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Table 5. Antioxidant Activity of Isolates from Salvia clinopodioides and Derivativesa

a

compound

DPPH (IC50), μM

TBARS (IC50), μM

1a 1b 1c 2a 2b 3 4a 5a BHT (n = 5) quercetin (n = 3) α-tocopherol (n = 4)

NA NA NA 24.8 ± 1.2 NA 29.9 ± 0.6 NA NA

NA NA NA 5.9 ± 0.4 NA 2.7 ± 0.2 40.9 ± 2.7 19.2 ± 1.8 1.2 ± 0.4 1.5 ± 0.0 6.8 ± 2.2

10.9 ± 0.5 31.7 ± 1.0

Values represent the mean ± SD, n = 3. NA, not active.

important for the antioxidant activity.22 The only difference between 1a and 2a is the presence of a methoxy group at C-12 in 1a instead of the hydroxy group in 2a, and therefore 1a was not active in the TBARS model. The antioxidant effect of 11,12-dihydroxyabietane-type diterpenoids has been documented in carnosol derivatives and other compounds isolated from S. ballotif lora.22,8 Compounds 4a and 5a showed moderate antioxidant activity with IC50 (μM) = 19.2 ± 1.8 and 40.9 ± 2.7, respectively, while 1a, 1b, 1c, and 2b showed no antioxidant activity in the TBARS assay. In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay 2a and 3 were the only active compounds, with IC50 (μM) values of 24.8 ± 1.2 and 29.9 ± 0.6, respectively (Table 5). Compounds 1a, 1b, 2a, 2b, 3, and 5a were also tested for their antiprotozoal activity (Table 6) against Entamoeba histolytica and Giardia lamblia and on the charcoal-gum acacia-induced hyperperistalsis model in rats. The compounds showed moderate inhibitory activity on hyperpropulsive movement of the small intestine in rats, while 1a and 2b displayed activities comparable to quercetin (Table 6).

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Figure 4. Comparison of the experimental (B) IR and (D) VCD spectra of (−)-2b and DFT B3LYP/DGDZVP-calculated (A) IR and (C) VCD spectra for (4S,5S,10R,19R)-2b.

Table 4. Confidence Level Data for the IR and VCD Spectra Comparison of (4S,5S,10R,18R)-1b and (4S,5S,10R,18R)2b compound

anHa

SIRb

SEc

S−Ed

ESIe

Cf

(4S,5S,10R,18R)-1a (4S,5S,10R,18R)-2a

0.988 0.991

94.2 94.6

86.4 92.6

7.4 4.2

79.0 88.7

100 100

EXPERIMENTAL SECTION

General Experimental Procedures. The melting points (uncorrected) were determined on a Fisher-Johns apparatus. Optical rotations were measured on a PerkinElmer 341 polarimeter. The UV spectra were recorded on a Shimadzu UV 160U spectrophotometer. The IR spectra were obtained on a Bruker Tensor 27 spectrometer; 1D and 2D NMR experiments were performed on a Bruker Advance III HD spectrometer at 700 MHz for 1H and 175 MHz for 13C. Chemical shifts were referred to CHCl3 (δH = 7.26, δC = 77.16). The DARTMS data were obtained on a Jeol AccuTOF JMS-T100LC mass spectrometer; silica gel 230−400 mesh (Macherey-Nagel; Düren, Germany), Sephadex LH-20 (Pharmacia Biotech; Uppsala, Sweden), and octadecyl-functionalized silica gel (Sigma-Aldrich; Steinheim, Germany) were used for column chromatography. The X-ray data were collected on a Bruker APEX II DUO diffractometer. The VCD spectra were measured using a BioTools dualPEM ChiralIR spectrophotometer. Plant Material. Salvia clinopodioides was collected from Valle de Bravo, State of Mexico, Mexico, 19°6′36″ N, 100°3′32″ W, and 2227 m altitude in September 2017. The plant material was identified by ́ Dr. Martha Martinez-Gordillo, and a voucher specimen (FCME 161792) was deposited at the Herbarium (FCME) of Facultad de Ciencias, UNAM. Extraction and Isolation. The dried and powdered aerial parts of S. clinopodioides (600 g) were extracted exhaustively by percolation with petroleum ether followed by CH2Cl2. The CH2Cl2 extract was

a

Anharmonicity factor. bIR spectral similarity in percentage. cVCD spectral similarity for the correct enantiomer in percentage. dVCD spectral similarity for the opposite enantiomer in percentage. e Enantiomer similarity index, calculated as the SE − S−E difference. f Confidence level for the absolute configuration determination in percentage.

2a displayed IC50 values 5 and 4 times higher than BHT and quercetin, respectively, and 3 showed an IC50 twice as high as the antioxidant drugs used as positive controls. It is well documented that the presence of ortho-dihydroxy groups is F



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Table 6. In Vitro Antiprotozoal Activity and Inhibition of Hyperperistalsis of Isolates from Salvia clinopodioides and Derivatives ID50 (μmol/kg ± SD)b

IC50 (μM) (CI)a compound

Entamoeba histolytica

Giardia lamblia

1a 1b 2a 2b 3 5a emetinec metronidazolec quercetinc loperamide hydrochloridec

43.0 (44.5−42.5) 34.9 (36.2−33.6) 37.8 (41.0−34.5) 39.5 (40.5−38.5) 31.3 (32.6−29.0) 48.5 (51.1−46.0) 2.2 (2.2−2.1) 0.2 (0.58−0.2)

67.1 (68.1−66.2) 46.4 (47.8−45.1) 63.0 (64.1−61.9) 47.7 (50.0−45.4) 49.0 (49.9−48.1) 61.8 (64.5−59.2) 0.8 (0.9−0.8) 1.2 (1.6−0.8)

inhibition of hyperperistalsis 2.2 ± NT 4.1 ± 2.7 ± 5.5 ± NT

0.0 0.0 0.0 0.0

1.1 ± 0.0 0.2 ± 0.0

Results are expressed as mean (n = 6), CI = 95% confidence intervals. bResults are expressed as mean (n = 6) ± SEM, p < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). cPositive controls. NT, not tested.

a

concentrated at reduced pressure to yield 30 g of residue, a portion (20 g) of which was subjected to column chromatography (CC) on silica gel using CH2Cl2 as the mobile phase to obtain 50 fractions, 300 mL each, which were combined into nine major fractions (A−I) after thin-layer chromatography (TLC) evaluation. Fraction D (717 mg) was subjected to CC on silica gel using gradient elution with petroleum ether/EtOAc (100:0−0:100) to obtain 29 subfractions, 50 mL each, which were combined into six major subfractions (DA−DF) based on TLC evaluation. Subfraction DE (48 mg) was purified by TLC on silica gel, eluting with petroleum ether/acetone (9:1) as the mobile phase, to give 3 (16.3 mg). Fraction E (530 mg) was subjected to CC on silica gel using petroleum ether/acetone (9:1) as the mobile phase to give 4a (125 mg). Compound 2a (250 mg) was obtained from fraction F (530 mg). Compound 1a (345 mg) crystallized from fraction G (967 mg). Clinopodiolide A (1a): colorless crystals; mp 215−218 °C; [α]25D −136 (c 0.2, CHCl3); UV (MeOH) λmax nm (log ε) 210 (4.61), 283 (3.41); IR (CHCl3) νmax 3592, 3219, 2963, 2937, 2878, 1686, 1176, 1110 cm−1; 1H and 13C NMR, see Table 1; HR-DARTMS m/z 361.20022 (calcd for C21H29O5, 361.20150). 19-O-Acetylclinopodiolide A (1b). Acetylation of 1a (20 mg) with pyridine/Ac2O gave 16 mg of 1b as a white powder: mp 90−93 °C; [α]25D −131 (c 0.2, CHCl3); UV (MeOH) λmax nm (log ε) 212 (4.19), 213 (3.14); IR (CHCl3) νmax 3229, 2960, 2874, 1769, 1700, 1168, 1017 cm−1; 1H NMR (CDCl3,700 MHz) δ 9.08 (1H, s, 11OH), 6.55 (1H, s, H-14), 6.44 (1H, s, H-19), 3.86 (3H, s, OCH3), 3.49 (1H, ddt, J = 13.8, 4.2, 1.9 Hz, H-1a), 3.28 (1H, hept, J = 6.9 Hz, H-15), 2.87 (1H, ddd, J = 16.8, 4.8, 1.9 Hz, H-7a), 2.79 (1H, ddd, J = 17.0, 12.6, 5.2 Hz, H-7b), 2.23 (1H, d, J = 14.0 Hz, H-3a), 2.20 (3H, s, OCOCH3), 2.17 (1H, d, J = 12.6 Hz, H-6a), 1.79 (1H, d, J = 15.9 Hz, H-2a), 1.73 (1H, qt, J = 14.3, 4.6 Hz, H-2b), 1.64 (1H, dd, J = 13.1, 1.7 Hz, H-5), 1.47 (1H, td, J = 13.7, 4.6 Hz, H-1b), 1.43 (1H, dd, J = 12.8, 4.8 Hz, H-6b), 1.31 (1H, td, J = 13.7, 5.7 Hz, H-3b), 1.19, 1.21 (3H, d, J = 6.9 Hz, CH3-16, 17); 13C NMR (CDCl3, 176 MHz) δ 178.2 (C, C-20), 169.2 (C, OCOCH3), 149.9 (C, C-11), 146.7 (C, C-12), 142.1 (C, C-13), 132.9 (C, C-8), 123.3 (C, C-9), 118.5 (C, C-14), 97.3 (CH, C-19), 61.1 (CH3, OCH3), 50.4 (C, C10), 50.3 (CH, C-5), 36.1 (C, C-4), 35.2 (CH2, C-1), 34.1 (CH2, C3), 30.9 (CH2, C-7), 26.9 (CH, C-15), 23.8 (CH3, C-18), 23.6 (CH3, C-16), 23.4 (CH3, C-17), 21.6 (CH2, C-6), 21.2 (CH2, C-2), 21.0 (CH3, OCOCH3); HR-DARTMS m/z 403.21211 (calcd for C23H31O6, 403.21206). Diacetylclinopodiolide A (1c). Acetylation of 1a (20 mg) with pyridine/Ac2O and 4-dimethylaminopyridine gave 1c (18 mg) as a white powder: mp 205−208 °C; [α]25D −113 (c 0.3, CHCl3); UV (MeOH) λmax nm (log ε) 208 (4.38), 279 (3.09); IR (film) νmax 2965, 2939, 2879, 1765, 1017 cm−1; 1H NMR (CDCl3,700 MHz) δ 6.84 (1H, s, H-14), 6.38 (1H, s, H-19), 3.72 (3H, s, OCH3), 3.27 (1H, hept, J = 7.2 Hz, H-15), 2.90 (1H, ddd, J = 17.1, 5.0, 2.1 Hz, H-7a), 2.82 (1H, ddd, J = 17.0, 12.3, 5.5 Hz, H-7a), 2.25 (3H, s, OCOCH3-

11), 2.19 (1H, m, H-6a), 2.18 (1H, m, H-3a), 2.17 (3H, s, OCOCH319), 1.90 (1H, qt, J = 14.2, 4.5 Hz, H-2a), 1.75 (1H, brs, H-2b), 1.62 (1H, m, H-5), 1.60 (1H, m, 3a), 1.48 (1H, qd, J = 12.7, 5.0 Hz, H6b), 1.29 (1H, td, J = 13.9, 5.3), 1.211, 1.206 (each CH3, d, J = 6.9 Hz, CH3-16, 17), 1.07 (3H, s, CH3-18); 13C NMR (CDCl3, 176 MHz) δ 170.9 (C, C-20), 169.2 (C, OCOCH3-19), 167.2 (C, OCOCH3-11), 149.5 (C, C-12), 144.3 (C, C-11), 142.1 (C, C-13), 133.5 (C, C-8), 128.0 (C, C-9), 124.5 (CH, C-14), 97.1 (CH, C-19), 61.5 (CH3, OCH3), 50.0 (C, C-10), 49.4 (CH, 5), 35.8 (CH2, C-1), 34.5 (CH2, C-3), 30.9 (CH2, C-7), 26.7 (CH, C-15), 23.7 (2CH3, C16, 17), 23.6 (CH3, C-18), 21.6 (CH2, C-6), 21.1 (2CH3-OCOCH311,19), 20.9 (CH2, C-2); HR-DARTMS m/z 445.22137 (calcd for C25H33O7, 445.22263). Aldehyde 5a. Treatment of 1a (20 mg) with diazomethane gave a mixture, which was subjected to TLC using petroleum ether/EtOAc (2:1) as the mobile phase to provide 5a (8.2 mg) as a yellow oil: [α]25D +90 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 212 (4.2), 283 (3.2) nm; IR (CHCl3) νmax 3513, 2965, 2874, 1718, 1028 cm−1; 1H NMR (CDCl3,700 MHz) δ 9.60 (1H, s, H-19), 7.02 (1H, s, OH-11), 6.53 (1H, s, H-14), 3.76 (3H, s, OCH3), 3.61 (3H, s, COOCH3), 3.50 (1H, dt, J = 14.2, 3.8 Hz, H-1a), 3.21 (1H, hept, J = 7.0 Hz, H15), 2.91 (1H, dd, J = 16.5, 4.8 Hz, H-7a), 2.84 (1H, ddd, J = 16.9, 12.1, 6.2 Hz, H-7b), 2.37 (1H, qd, J = 12.6, 5.4 Hz, H-6a), 2.25 (1H, dt, J = 13.7, 3.8 Hz, H-3a), 2.18 (1H, d, J = 13.0, 5.9 Hz, H-6b), 1.95 (1H, qt, J = 13.7, 4.1 Hz, H-2a), 1.75 (1H, d, J = 12.7 Hz, H-5), 1.63 (1H, dt, J = 14.1, 3.5 Hz, H-2b), 1.30 (1H, td, J = 13.5, 4.1 Hz, H-1b), 1.21, 1.20 (3H each, d, J = 7.0 Hz, CH3-16,17), 1.15 (3H, s, CH3-18); 13 C NMR (CDCl3, 176 MHz) δ 203.8 (CH, C-19), 176.3 (C, C-20), 148.7 (C, C-11), 143.9 (C, C-12), 140.6 (C, C-13), 133.6 (C, C-8), 124.0 (C, C-9), 118.2 (CH, C-14), 61.6 (CH3-OCH3), 53.3 (CH, C5), 52.0 (CH3-COOCH3), 49.7 (C, C-10), 48.8 (C, C-4), 34.5 (CH2, C-1), 34.0 (CH2, C-3), 32.4 (CH2, C-7), 26.7 (CH, C-15), 24.7 (CH3, C-18), 23.8, 23.5 (each CH3, C-16, C-17), 20.5 (CH2, C-2), 19.6 (CH2, C-6); HR-DARTMS m/z 375.21646 (calcd for C22H31O5, 375.21715). Clinopodiolide B (2a): white powder; mp 118−121 °C [α]25D −168 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 210 (41.87), 289 (0.87), 332 (3.77) nm; IR (CHCl3) νmax 3593, 3517, 3214, 2962, 2878, 1685, 1179, 1119 cm−1; 1H and 13C NMR, see Table 1; HRDARTMS m/z 347.18438 (calcd for C20H27O5, 347.18585). Triacetylclinopodiolide B (2b). Acetylation of 2a (20 mg) with pyridine/Ac2O gave 2b (18 mg) as a white powder; mp 200−202 °C; [α]25D −111 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 205 (4.89), 279 (3.67), 280 (5.05) nm; IR (film) νmax 3021, 2937, 2875, 1764, 1208, 1173, 1017 cm−1; 1H NMR (CDCl3,700 MHz) δ 6.93 (1H, s, H-14), 6.37 (1H, s, H-19), 3.47 (1H, brs, H-1a), 2.94 (1H, hept, J = 7.9 Hz, H-15), 2.93 (1H, ddd, J = 16.7, 12.3, 4,8 Hz, H- 7a), 2.86 (1H, ddd, J = 17.2, 12.7, 5.5 Hz, H-7b), 2.28 (3H, s, CH3-2‴), 2.21 (3H, s, CH3-2′), 2.17 (3H, s, CH3-2″), 1.86 (1H, qt, J = 14.2, 4.4 Hz, H-2a), 1.75 (1H, brd, J = 14.3 Hz, H-2b), 1.66 (1H, d, J = 13.0 Hz, G



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H-5), 1.60 (1H, td, J = 13.8, 4.4 Hz, H-1b), 1.48 (1H, qd, J = 12.8, 5.1 Hz, H-6a), 1.29 (1H, td, J = 13.9, 5.2 Hz, H-3b), 1.20. 1.16 (each 3H, d, J = 6.9 Hz, CH3-16,17), 1.07 (3H, s, CH3-18); 13C NMR (CDCl3, 176 MHz) 171.2 (C, C-20), 169.1 (C, C1″), 169.0 (C, C1‴), 167.2 (C, C1′), 143.0 (C, C-11), 141.0 (C, C-13), 140.3 (C, C-12), 136.1 (C, C-8), 128.0 (C, C-9), 124.6 (CH, C-14), 97.1 (CH, C-19), 49.9 (CH, C-5), 49.7 (C, C-10), 36.0 (C, C-4), 35.4 (CH2, C-1), 34.4 (CH2, C-3), 31.0 (CH2, C-7), 27.6 (CH, C-15), 23.6 (CH3, C-18), 23.1, 22.8 (CH3 each, C-16,17), 21.4 (CH2, C-6), 21.3 (CH3, C2‴), 21.0 (CH3, C-2′), 20.9 (CH2, C-2), 20.7 (CH3, 2″); HR-DARTMS m/z 473.21748 (calcd for C26H33O8, 473.21754). Aldehyde 5b. Treament of 2a (40 mg) with diazomethane gave a complex mixture, which was subjected to TLC using petroleum ether/ EtOAc (2:1) as the mobile phase to afford 5b (1.5 mg) as a yellow oil together with an unresolved complex mixture: [α]25D +13 (c 0.2, CHCl3); IR (film) νmax 3454, 3260, 2959, 2871, 1718, 1682, 1249 cm−1; 1H NMR (CDCl3, 700 MHz) δ 9.59 (1H, d, J = 1.2, H-19), 8.75 (1H, s, OH-11), 6.56 (1H, s, H-14), 5.97 (1H, s, OH-12), 3.67 (3H, s, COOCH3), 3.38 (1H, dt, J = 13.8, 3.5 Hz, H-1a), 3.23 (1H, hept, J = 6.9 Hz, H-15), 2.90 (1H, dd, J = 12.8, 3.9 Hz, H-7a), 2.85 (1H, m, H-7b), 2.29 (2H, m, CH2-6), 2.25 (1H, dt, J = 13.8, 3.4 Hz, H-3a), 1.78 (1H, m, H-5), 1.68 (1H, dq, J = 14.8, 3.6 Hz, H-2a), 1.75 (1H, d, J = 12.7 Hz, H-5), 1.63 (1H, dt, J = 14.4, 3.7 Hz, H-2b), 1.30 (1H, td, J = 13.6, 3.3 Hz, H-1b), 1.22, 1.21 (3H each, d, J = 6.9 Hz, CH3-16,17), 1.19 (3H, s, CH3-18); 13C NMR (CDCl3, 176 MHz) δ 202.7 (CH, C-19), 178.1 (C, C-20), 142.7 (C, C-12), 142.4 (C, C11), 134.1 (C, C-13), 128.0 (C, C-8), 121.3 (C, C-9), 119.3 (CH, C14), 53.4 (CH, C-5), 52.3 (CH3-COOCH3), 50.8 (C, C-10), 48.7 (C, C-4), 35.4 (CH2, C-1), 33.1 (CH2, C-3), 32.3 (CH2, C-7), 27.3 (CH, C-15), 24.76 (CH3, C-18), 22.5, 22.3 (CH3 each, C-16,17), 21.0 (CH2, C-2), 20.1 (CH2, C-6); HR-DARTMS m/z 361.20031 (calcd for C21H29O5, 361.20150). Clinopodiolide C (3): yellow oil; [α]25D −101 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 208 (4.5), 280 (3.5) nm; IR (film) νmax 3510, 3160, 2958, 2917, 1680, 1428, 1177 cm−1; 1H and 13C NMR, see Table 2; HR-DARTMS m/z 331.18964 (calcd for C20H27O4, 331.19093). Clinopodiolide D (4a): white powder; mp 78−81 °C; [α]25D +31 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 208 (4.1), 280 (3.1) nm; IR (film) νmax 3422, 2959, 2872, 2761, 1761, 1452, 1424, 1133, 1035, 756 cm−1; 1H and 13C NMR, see Table 2; HR-DARTMS m/z 345.20558 (calcd for C21H29O4, 345.20658). Acetylclinopodiolide D (4b). Acetylation of 4a (20 mg) with pyridine/Ac2O gave 4b (18.3 mg) as white powder: mp 70−73 °C; [α]25D +8 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 209 (3.8), 283 (3.1) nm; IR (film) νmax 2960, 2938, 2872, 1771, 1450, 1421, 1198 cm−1; 1H NMR (CDCl3,700 MHz) δ 6.85 (1H, s, H-14), 3.73 (3H, s, OCH3), 3.27 (1H, hept, J = 6.9 Hz, H-15), 3.01 (1H, d, J = 14.1 Hz, H-20a), 2.98 (1H, m, H-7b), 2.97 (1H, d, J = 14.1 Hz, H-20a), 2.82 (1H, dt, J = 15.2, 7.5 Hz, H-7b), 2.35 (3H, s, OCOCH3), 1.93 (1H, d, J = 12.6 Hz, H-6a), 3.49 (1H, ddt, J = 13.8, 4.2, 1.9 Hz, H-1a), 1.80 (1H, dd, J = 13.3, 5.0, Hz, H-1a), 1.78 (1H, m, H-5), 1.75 (2H, m, 2H-2), 1.66 (1H, m, H-1b), 1.62 (1H, m, H-3a), 1.49 (1H, td, J = 12.7, 6.8 Hz, H-3b), 1.48 (1H, m, H-6b), 1.22, 1.21 (3H, d, J = 6.9 Hz, CH3-16, 17); 13C NMR (CDCl3, 176 MHz) δ 180.4 (C, C-19), 169.0 (C, OCOCH3), 147.5 (C, C-12), 142.9 (C, C-11), 141.4 (C, C13), 136.9 (C, C-8), 125.2 (C, C-9), 124.3 (C, C-14), 84.2 (C, C-10), 61.6 (CH3, OCH3), 53.2 (CH, C-5), 48.7 (C, C-4), 35.5 (CH2, C-3), 35.3 (CH2, C-1), 34.4 (CH2, C-20), 31.1 (CH2, C-7), 26.7 (CH, C15), 23.8, 23.7 (2CH3, C-16,17), 23.1 (CH2, C-6), 20.8 (CH3, OCOCH3), 19.7 (CH2, C-2), 17.2 (CH3, C-18); HR-DARTMS m/z 387.21653 (calcd for C23H31O5, 387.21715). Single-Crystal X-ray Diffraction Analysis. Crystallographic data for 1a were collected on a Bruker SMART APEX DUO three-circle diffractometer equipped with an Apex II CCD detector using Cu Kα radiation (λ = 1.541 78 Å, Incoatec IμS microsource and Helios optic monochromator), for a correct estimation of the anomalous dispersion and an adequate determination of the absolute structure parameter due to the nature of the sample (only carbon, oxygen, and hydrogen atoms), at −173 °C.23 A suitable crystal of 1a was coated

with Paratone hydrocarbon oil, picked up with a nylon cryoloop, and mounted on the diffractometer. The structure was solved by a dualspace algorithm (SHELXT)24 and refined by full-matrix least-squares on F225 using the shelXle GUI software.26 The hydrogen atoms of the C−H bonds were placed in idealized positions, whereas the hydrogen atoms from the O−H moieties were localized from the difference electron density map, and their positions were refined with Uiso tied to the oxygen atom with distance restraints. The molecular graphics were prepared using Ortep3, POV-RAY, GIMP, and Mercury.27 The crystal data and structure refinement for 1a are shown in Tables S1 and S2 (Supporting Information) and deposited at the CCDC under deposition number 1874959. Vibrational Circular Dichroism. For the measurement of VCD and their associated IR spectra, using a BioTools dualPEM ChiralIR spectrophotometer, samples of 7.6 mg of 1b and 5.0 mg of 2b were dissolved in 150 μL of 100% atom-D CDCl3, placed in a cell having BaF2 windows and a path length of 0.1 mm, and the data acquisition was performed at a resolution of 4 cm−1 for 6 h. Baseline corrections were performed by subtracting the spectrum of the solvent acquired under identical conditions. The sample stability was monitored by 1H NMR analysis at 300 MHz immediately before and after the VCD measurements. For spectra calculations the initial conformational searches for 1b and 2b were carried out using the Monte Carlo protocol and molecular mechanics force field (MMFF94) calculations considering an energy cutoff of 10 kcal/mol. Single-point energy calculations were done using DFT, the B3LYP functional, and the 6-31G(d) basis set as implemented in the Spartan’08 software (Wavefunction, Inc., Irvine, CA, USA). The geometry of the conformers was optimized using the Gaussian03 (Gaussian, Inc., Wallingford, CT, USA) software with DFT at the B3LYP/DGDZVP level of theory. For calculating final vibrational normal modes and rotational strengths the same functional and basis set were used. Molecular visualization was carried out with the GaussView 5.0 software. The estimated ΔG value for each conformer was used as a criterion for weighting the IR and VCD spectra for 1b and 2b. The band-shapes were generated with Lorentzian functions and a bandwidth of 6 cm−1. Calculated and experimental spectra were compared using the CompareVOA (BioTools, Jupiter, FL, USA) software. Lipid Peroxidation Inhibition. Animals. Adult male Wistar rats (200−250 g) were provided by Instituto de Fisiologiá Celular, Universidad Nacional Autónoma de México. Procedures and care of animals were conducted in agreement with Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999). They were maintained at 23 ± 2 °C on a 12/12 h light−dark cycle with free access to food and water. Rat Brain Homogenate Preparation. Animal sacrifice was carried out avoiding unnecessary pain. Rats were sacrificed with CO2. The whole brain cerebral tissue was rapidly dissected and homogenized in phosphate-buffered saline (PBS) solution (0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl, and 2.16 g of NaHPO4·7H2O, pH adjusted to 7.4), as reported elsewhere28,29 to produce a 1/10 (w/v) homogenate. The homogenate was centrifuged for 10 min at 800 rcf (relative centrifugal field) to yield a pellet that was discarded. The supernatant protein content was measured using Folin and Ciocalteu’s phenol reagent30 and adjusted with PBS at 2.666 mg of protein/mL. Induction of Lipid Peroxidation and Thiobarbituric Acid Reactive Substances Quantification. As an index of lipid peroxidation, TBARS levels were measured using rat brain homogenates according to the method described31 with some modifications. Supernatant (375 μL) was added with 50 μL of 20 μM EDTA and 25 μL of each sample concentration dissolved in DMSO (25 μL of DMSO for control group) and incubated at 37 °C for 30 min. Lipid peroxidation was started by adding 50 μL of freshly prepared 100 μM FeSO4 solution (final concentration 10 μM) and incubated at 37 °C for 1 h. The TBARS content was determined as described32 with some modifications. TBA reagent (500 μL) (0.5% 2thiobarbituric acid in 0.05 N NaOH and 30% trichloroacetic acid, in 1:1 proportion) was added to each tube, and the final suspension was cooled on ice for 10 min, centrifuged at 13 400 rcf for 5 min, and H



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heated at 80 °C in a water bath for 30 min. After cooling at room temperature, the absorbance of 200 μL of supernatant was measured at 540 nm in a Synergy HT Bio-Tek microplate reader. The TBARS concentration was calculated by interpolation in a standard curve of tetramethoxypropane (TMP) as a precursor of malondialdehyde (MDA).33 Results were expressed as nanomoles of TBARS per mg of protein. The inhibition ratio (RI [%]) was calculated using the equation RI = (C − E) × 100/C, where C is the absorbance of the control and E is the absorbance of the test sample. BHT and αtocopherol were used as positive standards. All data were represented as mean ± standard error (SEM). They were analyzed by one-way ANOVA followed by Dunnett’s test for comparison against control. Values of p ≤ 0.05 (*) and p ≤ 0.01 (**) were considered statistically significant. The IC50 was estimated by linear regression. Scavenging Activity on Free Radical 2,2-Diphenyl-1picrylhydrazyl. The scavenging activity was measured using an adapted method of Mellors and Tappel.21 The test was carried out on 96-well microplates. A 50 μL aliquot of the solution of the test compound was mixed with 150 μL of an EtOH solution of DPPH (final concentration 100 μM). This mixture was incubated at 37 °C for 30 min, and the absorbance was measured at 515 nm using a Synergy HT BioTek microplate reader. The percent inhibition of each compound was determined by comparison with a 100 μM DPPH EtOH blank solution. Antiprotozoal Assays. E. histolytica strain HM1-IMSS used in all experiments was grown axenically at 37 °C in TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum. In the case of G. lamblia, strain IMSS: 8909:1 was grown in TYI-S-33-modified medium supplemented with 10% calf serum and bovine bile. The trophozoites were axenically maintained and were employed in the log phase of growth for assays. In vitro susceptibility tests were performed using a subculture method.34 Briefly, E. histolytica (6 × 103) or G. lamblia (5 × 104) trophozoites were incubated for 48 h at 37 °C in the presence of different concentrations (2.5−200 μg/mL) of pure compounds in DMSO. Each test included metronidazole, kaempferol, and emetine (Sigma) as standard amoebicidal and giardicidal drugs, a control (culture medium plus trophozoites and DMSO), and a blank (culture medium). After incubation, the trophozoites were detached by chilling, and 50 μL samples of each tube were subcultured in fresh medium for another 48 h, without antiprotozoal samples. The final number of parasites was determined with a hemocytometer, and the percentages of trophozoite growth inhibition were calculated by comparison with the control culture. The results were confirmed by a colorimetric method: The trophozoites were washed and incubated for 45 min at 37 °C in PBS with 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide and phenazine methosulfate. The formazan dye was extracted, and the absorbance was determined at 570 nm. The experiments were performed in duplicate for each protozoan and repeated at least three times. The in vitro results were classified as follows: If the samples displayed a 50% inhibitory concentration (IC50) < 20.0 μg/mL, the antiprotozoal activity was considered good; from 21.0 to 60 μg/mL, the antiprotozoal activity was considered moderate; from 61 to 200 μg/mL, the antiprotozoal activity was considered weak; and over 200 μg/mL, the samples were considered inactive. Data were analyzed using probit analysis. The percentage of trophozoites surviving was calculated by comparison with the growth in the control group. The plot of probit against log concentration was made, the best straight line was determined by regression analysis, and the IC50 values were calculated. The regression coefficient, its level of significance (p < 0.05 indicates significant difference between group), and correlation coefficient were calculated and 95% confidence interval (CI) values determined. Animals. Male Sprague−Dawley rats (200−250 g) were obtained from the animal house of the IMSS. The experimental protocols were approved by Animal Care and Use Committee of Hospital de Pediatria del Centro Medico Nacional Siglo XXI, IMSS. Investigation using experimental animals was conducted in accordance with the official Mexican norm NOM 0062-ZOO-1999 entitled Technical specifications for the production, care and use of laboratory animals.35

They were fasted overnight, but tap water was available ad libitum until the start of the experiments. Effect on Charcoal−Gum Acacia-Induced Hyperperistalsis. The method described by Calzada et al. (2010) was adopted to study the effect of compounds on hyperperistalsis in rats.36 The test animals were divided into control group and test groups containing six rats in each group. Rats were treated orally with each compound (0.01, 0.1, 1.0, 10, 20, 40 mg/kg in 1 mL of a 2% DMSO solution in water), vehicle (1 mL of a 2% DMSO solution in water), loperamide hydrochloride (Sigma), or quercetin (Sigma) (0.1, 1.0, 10, 20, 40 mg/ kg in 1 mL of a 2% DMSO solution in water). After 20 min, each of these animals was given 1 mL of charcoal meal (10% charcoal suspension in 5% aqueous arabic gum) by the oral route. All animals were sacrificed after 30 min, the stomach and small intestine were removed and extended on a clean glass surface. The distance moved by the charcoal meal from the pylorus was measured and expressed as a percentage of the distance from the pylorus to the cecum. After the plot of percentage of inhibition against concentration was made, the best straight line was determined by regression analysis and the IC50 values were calculated. The regression coefficient, its level of significance (p), and correlation coefficient were calculated. The experiments were performed six times for each concentration. IC50 values are mean ± SEM. p < 0.05 (one-way ANOVA followed by Dunnett’s post hoc test). GraphPad Prism version 5.03 (GraphPad Software Inc., La Jolla, CA, USA) was used.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00952. Copies of 1D and 2D NMR spectra of 1a, 2a, 3, 4a, and 5a (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +52-55-5622-4442. Fax: +52-55-5616-2217. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Pedro Joseph-Nathan: 0000-0003-3347-3990 Leovigildo Quijano: 0000-0002-4804-3013 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge H. Rios, B. Quiroz, E. Huerta, A. Peña, R. Patiño, L. Velasco, C. Garcia,́ and J. Pérez for collecting NMR, UV, IR, and MS data. The authors are ́ indebted to Dr. M. Martinez-Gordillo (Herbarium of the Faculty of Sciences of UNAM) for plant identification. The authors thank C. Rivera Cerecedo and H. Malagón (Bioterium of the Institute of Cellular Physiology, UNAM) for the donation of the biological material for the TBARS assay. This study made use of UNAM’s NMR lab: LURMN at IQ-UNAM, which is funded by CONACYT-Mexico (Project 0224747), and UNAM. Partial financial support from CONACYT (Mexico) grant 284194 is also acknowledged.

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REFERENCES

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