Antimycobacterial Activity of Milemaronol,a New Squalene-Type Triterpene,and Other Isolate?

Abstract

A new triterpene, named milemaronol (1), was isolated from Homalolepis suffruticosa Engl., Simaroubaceae, along with 10 known metabolites, chaparrinone (2), scopoletin (3), 5-methoxycanthin-6-one (4), eurylene (5), hispidol A (6), hispidol B (7), nilocitine (8), α-dihydronylocytine (9), β-dihydronylocytine (10), and teurilene (11). These compounds were characterized based on their spectral data, mainly 1D (¹H, ¹³C-APT) and 2D (¹H-¹H-COSY, NOESY, HSQC, HMBC) NMR and their mass spectra (HR-ESI-MS), in comparison with data from the literature. Compounds 1 to 6, 8, and 9 were evaluated for their antimycobacterial activity against 2 strains (H37Rv and M299).

Keywords

Simaroubaceae triterpenes quassinoid alkaloids

The family Simaroubaceae contains 117 reported species of trees, whose characteristic is the bitter taste of their cortex.^1,2 This can be attributed to the presence of quassinoids, which is also considered a taxonomic marker of this family.³ The genus Homalolepis Turcz., recently segregated from Simaba Aub., comprises 28 species, spread mainly in tropical South America.²

Besides the quassinoids, other types of triterpenes, alkaloids, and other classes of compounds were also observed.⁴ These compounds showed many biological activities like antibacterial,^5,6 anticancer,^7,8 antileishmanial^9
-11; antiviral,^12
-14 and others.^11,15

-19

Thus, in view of the high biological potential of the compounds of the Simaroubaceae, a phytochemical study of the roots of Homalolepis suffruticosa was carried out, during which a new unusual squalene triterpene, named milemaronol (1), was characterized and 10 known compounds were identified; their antimycobacterial and cytotoxicity activities were evaluated.

Results and Discussion

Elaboration of the MeOH and n-hexane extracts of the roots of H. suffruticosa through classical chromatographic methods resulted in the isolation of 11 compounds, 1 to 11, whose structures are shown in Figure 1. A new triterpenoid, named milemaronol,¹ along with 10 known compounds, chaparrinone (2),²⁰ scopoletin (3),²¹ 5-methoxycanthin-6-one (4),²² eurylene (5),²³ hispidol A (6),²⁴ hispidol B (7),²⁴ nilocitine (8),²⁵ α-dihydronylocitine (9),²⁶ β-dihydronilocitine (10),²⁷ and meso-teurilene (11),²³ was characterized based on their ¹H- and ¹³C-NMR spectral data, especially 2D-NMR, and by comparison of the mass spectral data with the literature values.

Figure 1

Compounds isolated from roots of Homalolepis suffruticosa (1-11) and teurilene (12).

Milemaronol (1) was isolated as an yellow oil. Its HR-ESI-MS showed peaks due to the presence of a sodium ion at m/z 515.3606 ([M+Na]⁺, calc. m/z 515.3712) and a potassium ion at m/z 531.3481 ([M+K]⁺, calc. m/z 531.3452) (Supplemental Scheme 1S). These data, combined with the information obtained by 1D and 2D NMR spectral analysis, were used to deduce the molecular formula C₃₀H₅₂O₅, indicating 5 degrees of unsaturation (2 C=C bonds and 3 rings).

The ¹³C-APT-NMR spectrum of 1 revealed signals corresponding to 30 carbon atoms, including 6 quaternary [C₆: 2 sp² (double bond) and 4 sp³ linked to oxygen atoms], 6 methines [(CH)₆: 4 sp³carbinolics and 2 sp² olefinics at δ _C 124.8 and 124.5], 10 methylenes [(CH₂)₁₀], and 8 methyls [(CH₃)₈ including 4 linked to sp² carbon atoms (2 at δ _C 25.7 and 2 at δ _C 17.6)]. ¹H NMR spectral analysis of 1 showed 2 triplet signals in the region of the olefinic hydrogens at δ _H 5.11 (H-3, t, 6.7) and 5.14 (H-22, t,7.1); 4 double doublet signals in the region of carbinolic hydrogens δ _H 3.90 (H-7, dd, 7.8, 5.0), 3.69 (H-18, dd, 11.5, 3.9), 3.71 (H-11, dd, 12.1, 1.9), and 3.55 (H-14, dd, 7.6, 5.0); and 4 singlet signals corresponding to methyl groups linked to sp² carbon atoms at δ _H 1.71 (3H-24), δ _H 1.69 (3H-1), δ _H 1.64 (3H-25), and δ _H 1.62 (3H-30). All ¹H and ¹³C chemical shifts are assigned in Table 1.

Table 1

¹H (500 MHz) and ¹³C (125 MHz) NMR Data of Compound 1, in CDCl₃, Including HSQC and HMBC (² J _HC and ³ J _HC). δ in ppm and J (in Parentheses) in Hz.

	1
C	δ _c	δ _H	HMBC
1	25.7	1.69, s	3, 25
2	131.3	-	1, 4, 25
3	124.8	5.11, t (6.7)	1, 4, 5, 25
4	22.3	2.08 (m)	3
5	38.6	1.58 (m), 1.48 (m)	26
6	73.8	-	7, 26
7	84.4	3.90, dd (7.8, 5.0)	9, 26
8	26.0	2.08 (m), 1.90 (m)	9
9	31.2	2.25 (m), 1.48 (m)	11, 27
10	85.2	-	7, 9, 11, 27
11	75.1	3.71, dd (12.1, 1.9)	27
12	20.3	2.03 (m), 1.80 (m)	14
13	27.2	1.82 (m), 1.60 (m)	12
14	78.0	3.55, dd (7.6, 5.0)	13, 28
15	75.9	-	28
16	38.1	1.55 (m), 1.40 (m)	28
17	24.7	1.90 (m), 1.75 (m)	18
18	72.9	3.69, dd (11.5, 3.9)	14, 29
19	73.2	-	18, 29
20	35.1	1.90 (m), 1.75 (m)	21, 29
21	22.5	2.03 (m), 1.78 (m)	22
22	124.5	5.14, t (7.1)	20, 21, 24, 30
23	131.9	-	21, 24, 30
24	25.7	1.71, s	22, 30
25	17.6	1.64, s	1, 3
26	24.6	1.24, s
27	24.9	1.14, s
28	23.2	1.20, s	-
29	17.8	1.31, s	18, 20
30	17.6	1.62, s	22, 24

The location of these 2 C=CH bonds was established through analysis of the HMBC spectrum, which revealed cross-peaks of δ _C 131.3 (C-2) with 3H-1 (δ _H 1.69, ² J _CH), 3H-25 (δ _H 1.64, ² J _CH), and 2H-4 (δ _H 2.08, ³ J _CH) and δ _C 131.9 (C-23) with 3H-24 (δ _H 1.71, ² J _CH), 3H-30 (δ _H 1.71, ² J _CH), and 2H-21 (δ _H 2.03, 1.78, ³ J _CH). Additional HMBC couplings via ³ J _CH of CH-3 (δ _C 124.8) with 2H-5 (δ _H 2.08) and CH-22 (δ _C 124.5) with 2H-20 (δ _H 1.90, 1.75) confirmed the terminal units (Me₂C=CH-CH₂-CH₂-5 and Me₂C=CH-CH₂-CH₂-20). The tetrahydrofuran ring was observed based on the carbon shift of C-10 (δ _C 85.2) with cross-peak with H-7 (δ _H 3.90), 2H-9 (δ _H 2.25 and 1.48), H-11 (δ _H 3.71), and 3H-27 (δ _H 1.14). This allowed to recognize the identical unit from C-1 to C-10 of 11, confirmed by comparative analysis of spectral data.²³

In the region of the carbinolic carbons of 1, signals were observed at δ _C 73.8 (C-6) and δ _C 73.2 (C-19), which displayed correlations in the HMBC spectrum with methyl groups 3H-26 (δ _H 1.24) and 3H-29 (δ _H 1.31), and 2 carbinolic hydrogens H-7 (δ _H 3.90, dd, 7.8, 5.0) and H-18 (3.69, dd, 11.5, 3.9), which showed a system with a methyl linked to a carbinolic carbon previously observed in the equivalent spectra of eurylene and teurilene.²³ Moreover, in the region of the carbinolic carbons, signals were observed at δ _C 84.4 (CH-7), 85.2 (C-10), 75.1 (CH-11), 78.0 (CH-14), 75.9 (C-15), and 72.9 (C-18), corresponding to an ether function.

The location of this unit linked to C-11 (δ _C 75.1) was deduced by heteronuclear interaction of this nonprotonated carbon with 3H-27 (δ _H 1.14, ³ J _CH). The other terminal unit involving carbon atom C-19 was linked to CH-18 [δ _C 72.9/δ _H 3.69 (dd, J = 11.5, 3,9)] by correlation (³ J _CH) of this methinic carbon with 3H-29 (δ _H 1.31) and H-14 (δ _H 3.55). The HMBC correlation (³ J _CH) of CH-14 (δ _C 78.0) with 3H-28 (δ _H 1.20) and the 3H-28 with CH₂-16 (δ _C 38.1) allowed us to postulate the presence of 2 pyran rings, 2-alkyl-octahydro-4a-methyl-6-alkylpyrano[3,2-b]pyran (1). This deduction is in agreement and does not deny the necessary biogenetic arguments involving squalene as a precursor.

Finally, 2 tetrahydropyran rings formed by the ether bond CH-11/C-15 and CH-14/CH-18 attached to the tetrahydrofuran ring between C-10/CH-11 were established from the HMBC spectrum which exposed cross-peaks of δ _H 3.55 (H-14, dd, 7.6, 5.0) with CH-18 (δ_C 72.9) and CH₂-12 (δ_C 20.3); δ _H 1.20 (3H-28, s) with CH-14 (δ_C 78.0), C-15 (δ_C 75.9), and CH₂-16 (δ_C 38.1); δ _H 2.03 (1Ha-12) and 1.80 (1Hb-12) with CH₂-13 (δ_C 27.2); and δ _H 3.71 (H-11, dd, 12.1, 1.9) with CH-9 (δ _C 31.2) and C-10 (δ _C 85.2). The complete assignment of the ¹H and ¹³C signals of 1 was successfully performed with the use of the HSQC and ¹H-¹H COSY spectra (Figure 1; Table 1).

The relative stereochemistry of 1 was determined from the coupling constants of the relevant hydrogens and from the observed ¹H-¹H-NOESY spectra. The values corresponding to vicinal interaction (³ J _H,H) between hydrogens H-11ax (J = 12.1 Hz, axial-axial interaction with H-12ax), H-14 (J = 7.6 Hz, axial-axial interaction with H-13ax), and H-18 (J = 11.5 Hz, axial-axial interaction with H-17ax) are consistent with the relative configuration shown in 1 and 11 (Table 1). In accordance with these observations, the NOESY spectrum of 1 showed cross-peaks assigned to dipolar interaction (spatial proximity, Figure 2) of 3H-26 (δ _H 1.24) with H-7 (δ _H 3.90); 3H-27 (δ _H 1.14) with H-7 (δ _H 3.90), H-11 (δ _H 3.71), and H-14 (δ _H 3.55); and 3H-28 (δ _H 1.20) with H-14 (δ _H 3.55).

Figure 2

Important ¹H-¹H-NOESY correlations for milemaronol (1).

The antimycobacterial activity against Mycobacterium tuberculosis strains H37Rv and M299, and cytotoxic activities of compounds 1 to 6, 8, and 9 were evaluated; the results are shown in Table 2. Compounds 2 and 5 showed excellent growth inhibition of 2 Mycobacterial strains, but revealed toxicity in viable cells. Compounds 4, 6, and 9 yielded a good response in the inhibition of both strains, but high cytotoxicity. On the other hand, compound 1 showed the best result, being sensitive to both mycobacteria and presenting low cell toxicity (Table 2).

Table 2

Antimycobacterial Activities of Compounds 1-6, 8, and 9.

	MIC₅₀		IC₅₀
	H37Rv	M299	MTT
1	42.1 ± 0.7	64.4 ± 0.8	307.7 ± 0.1
2	3.0 ± 1.2	7.6 ± 1.4	3.95 ± 1.0
3	≥500	≥500	≥500
4	30.4 ± 0.5	34.9 ± 0.3	46.9 ± 0.1
5	1.4 ± 0.9	2.0 ± 0.6	40.1 ± 0.7
6	14.6 ± 1.9	7.2 ± 1.9	3.4 ± 0.3
8	≥500	253.8 ± 1,0	79.5 ± 0.1
9	25.5 ± 0.7	24.4 ± 0.7	51.6 ± 0,2
Rifampicin	0.2 ± 0.1	1.1 ± 0.1	-

Material and Methods

General Experimental Procedures

Column chromatography (CC) was performed on silica gel 60 (0.063-0.200 mm, Merck), and n-hexane (98.5%), methanol (99.8%), ethyl acetate (99.5%), and dichloromethane (99.5%) were used as mobile phase solvents, purchased from Synth (São Paulo, Brazil). 1D and 2D NMR analysis was performed on a 500 MHz Bruker Ascend 500 NMR spectrometer operating at 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR. Deuterated chloroform (CDCl₃), tetradeuterated methanol (CD₃OD), and pyridine (pyridine-d ₅), containing TMS (tetramethylsilane) as an internal standard, were used. HR-ESI-MS were obtained on a micrOTOF-Q II Bruker Daltonics mass spectrometer, with the use of the positive ion mode of analysis.

Plant Material

Roots of H. suffruticosa were collected in September 2017, in Araguari City, Minas Gerais. The species was identified by the taxonomist José Rubens Pirani from the Universidade de São Paulo (USP). A voucher specimen (HUENF-10840) was deposited in the herbarium of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF).

Extraction and Isolation

Homalolepis suffruticosa roots were dried and powdered. The extraction was performed first with n-hexane (CH, 20.5 g) and then with methanol (CM, 23.4 g). The methanolic extract (23.4 g) was fractionated by silica gel CC, with a polar gradient of CH₂Cl₂:MeOH, obtaining 9 fractions (CM1-CM9). CM4 was similarly rechromatographed, generating 10 fractions (CM4.1-CM4.10). Chaparrinone (2) and scopoletin (3) were identified in fractions CM4.7 and CM4.4, respectively. Fraction CM4.8 was also chromatographed with hexane:acetone, generating 17 fractions (CM4.8.1-CM4.8.17); fractions CM4.8.3 (30.3 mg) and CM4.8.11 were identified as milemaronol (1) and 5-methoxicantin-6-one (4), respectively.

The hexane extract (CH, 20.5 g) was fractionated by silica gel CC with a gradient of hexane:acetone to yield 14 fractions (CH1-CH14). The CH5 fraction was similarly rechromatographed, yielding 9 fractions (CH5.1-CH5.9). Nilocitine (8) was identified in fraction CH5.3 (4.5 mg). The CH8 fraction (1.9 g) was subjected to CC with CH₂Cl₂:MeOH as eluent, yielding 10 fractions (CH8.1-CH8.10). In the CH8.5 fraction, a precipitate was observed, which was recrystallized and identified as eurylene (5). Fraction CH8.5 was chromatographed with hexane:EtOAc yielding α-dihydronylocytin (9) and β-dihydronylocytin (10) as a mixture. CH10 (1.46 g) was chromatographed with hexane:EtOAc, obtaining 8 fractions (CH 10.1-CH10.8). Hispidol A (6) and hispidol B (7) were identified as a mixture in CH10.3 (24.9 mg), and compound 6 was isolated from CH10.4 (14.5 mg). Meso-teurilene (11) was isolated from CH12 (29.1 mg).

Culture of Mycobacteria and Evaluation of Bacterial Growth

Two strains of Mycobacterium tuberculosis were used in this study (a virulent laboratory strain H37Rv, ATCC 27294 and a highly virulent Mtb strain Beijing M299, isolated from a TB patient in Mozambique), which were evaluated for virulence in a previous study.²⁸ Middlebrook 7H9 broth, containing 10% dextrose albumin complex (ADC), 0.5% glycerol, and 0.05% Tween-80, was used for the growth of the mycobacterial strains at 37°C, under conditions of containment of Biosafety 3. The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay was performed to quantify bacterial growth,²⁹ using the procedures described by Ventura et al.³⁰ The samples were analyzed by optical density at 570 nm. For negative control, untreated bacterial suspensions were used, while for positive control rifampicin was applied.

Evaluation of Cytotoxicity by MTT Assay

RAW 264.7 macrophages were treated with the compounds at concentrations of 4, 20, 100, and 500 µg/mL. After 24 hours, the levels of cytotoxicity of the samples were assessed using mitochondrial functionality using the MTT method and compared with negative (macrophages stimulated by lipopolysaccharide - LPS) and positive (macrophages stimulated by LPS and treated with 1% Triton X-100) controls. Values are reported as mean ± standard deviation, and different groups were considered significant according to P < .001 (***), P < .01 (**), and P < .05 (*).

Supplemental Material

Supplementary Material 1 - Supplemental material for Antimycobacterial Activity of Milemarinol, a New Squalene-Type Triterpene, and Other Isolate?

Supplemental material, Supplementary Material 1, for Antimycobacterial Activity of Milemarinol, a New Squalene-Type Triterpene, and Other Isolate? by Samyra I. S. Boeno, Michel de S. Passos, Mariana Félix, Sanderson D. Calixto, Almir R. C. Júnior, Lara F. Barbosa Siqueira, Michelle F. Muzitano, Raimundo Braz-Filho and Ivo J. Curcino Vieira in Natural Product Communications

Footnotes

Acknowledgment

The authors are grateful to Fundação de Amparo à Pesquisado Estado do Rio de Janeiro, to CNPq, and to CAPES (Finance Code 001).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Supplementary Material

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