New Isoflavonoids from the extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity

Author’s Accepted Manuscript New Isoflavonoids from the Extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity Enrique Wenceslao Coronado-Aceves, Giulia Gigliarelli, Adriana Garibay-Escobar, Ramón Enrique Robles Zepeda, Massimo Curini, Jaime López Cervantes, Clara Inés Espitia-Pinzón, Stefano Superchi, Stefania Vergura, Maria Carla Marcotullio

PII: DOI: Reference:

www.elsevier.com/locate/jep

S0378-8741(16)30870-4 http://dx.doi.org/10.1016/j.jep.2017.05.019 JEP10860

To appear in: Journal of Ethnopharmacology Received date: 16 September 2016 Revised date: 5 May 2017 Accepted date: 11 May 2017 Cite this article as: Enrique Wenceslao Coronado-Aceves, Giulia Gigliarelli, Adriana Garibay-Escobar, Ramón Enrique Robles Zepeda, Massimo Curini, Jaime López Cervantes, Clara Inés Espitia-Pinzón, Stefano Superchi, Stefania Vergura and Maria Carla Marcotullio, New Isoflavonoids from the Extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.05.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New Isoflavonoids from the Extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity. Enrique Wenceslao Coronado-Acevesa,b, Giulia Gigliarellic, Adriana Garibay-Escobara, Ramón Enrique Robles Zepedaa, Massimo Curinic, Jaime López Cervantesb, Clara Inés Espitia-Pinzónd, Stefano Superchie, Stefania Vergurae, Maria Carla Marcotullioc.*

a

Department of Chemistry-Biology, University of Sonora, Blvd. Luis Encinas y Rosales s/n,

Hermosillo, Sonora 83000, Mexico b

Department of Biotechnology and Alimentary Sciences, Technological Institute of Sonora, 5 de

Febrero 818 Sur, 85000 Ciudad Obregon, Sonora, Mexico c

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo, 1-06123 Perugia,

Italy d

Immunology Department, Biomedical Research Institute, National Autonomous University of

Mexico, 04510, DF, Mexico e

Department of Sciences, University of Basilicata, Via dell’Ateneo Lucano 10, 85100, Potenza,

Italy

*Corresponding author: Maria Carla Marcotullio, Department of Pharmaceutical Sciences, University of Perugia-Via del Liceo, 1-06123 Perugia, Italy. Tel: +39-075-5855100; Fax: +39-0755855116. Email: [email protected]

Abstract. Ethnopharmacology relevance 1

The evaluation of the antimycobacterial activity of extracts of medicinal plants used by Mayos against tuberculosis and respiratory problems, allowed the identification of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC (Fabaceae) as the best candidate to find new antimycobacterial compounds. Aim of the study To isolate and characterize the compounds of R. precatoria responsible for the inhibitory and bactericidal activity against Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis ATCC 700084. To determine antimycobacterial synergistic effect of pure compounds and their selectivity index towards Vero cells. Materials and Methods A total of six flavonoids were purified by silica gel column chromatography. Structural elucidation of the isolated compounds was achieved by using 1D and 2D NMR spectroscopy techniques. The configuration at the C-3 chiral center was established by quantum mechanical calculation of the electronic circular dichroism (ECD) spectrum. In vitro inhibitory and bactericidal activity against M. tuberculosis and M. smegmatis were determined with the redox indicator Alamar Blue (resazurin). Synergy was determined by X/Y quotient. Cytotoxicity was measured by MTT assay. Results The isolated compounds were identified as precatorin A (1), precatorin B (2), precatorin C (3), lupinifolin (4), cajanone (5) and lupinifolinol (6). Compounds 1-3 are new. Compounds 1 to 5 inhibited the growth of M. tuberculosis (MIC ≥31.25 µg/mL); compounds 1, 2, 4 and 5 killed the bacteria (MBC ≥31.25 µg/mL) and also inhibited M. smegmatis (MIC ≥125 µg/mL), while 1 and 4 also resulted bactericidal (MBC ≥125 µg/mL). Compounds 4 and 5 presented synergistic effect (X/Y quotient value <0.5) at a concentration of 1/2 MIC of each compound in the combination. Cytotoxicity in murine macrophages (RAW 264.7 cells) gave IC50 values of 13.3 to 46.98 µM, for compounds 1-5. Conclusions 2

In this work we isolated two new isoflavanones (1 and 2), and one new isoflavone (3) with a weak antimycobacterial activity. The (3R) absolute configuration was assigned to 1 by computational analysis of its ECD spectrum and to 2 and 5 by similarity of their ECD spectra with that of 1. We are also reporting by first time, activity against virulent strain of M. tuberculosis for compounds 4 and 5 and their antimycobacterial synergistic effect.

Keywords:

Rhynchosia

precatoria;

Fabaceae;

Mycobacterium

tuberculosis;

Precatorins;

Isoflavonoids; Absolute configuration;.

3

1. Introduction Tuberculosis (TB) is an ancient disease as there are evidences of infection that date back to around 8000 BC (Herzog, 1998). Despite the success of antimycobacterial drugs over the past decades, TB is still responsible for millions of deaths especially in developing countries (WHO, 2014). After an initial decline of TB in industrialized countries due to an improvement in socio-economic conditions, and to the development of antimycobacterial drugs, today there is a recrudescence of the disease due to multi-drug resistant strains of Mycobacterium tuberculosis and to an increase of HIV-infected patients (Jones, 2005). For these reasons, it is important to find new compounds to treat TB. Plants have been proved as an important source of bioactive compounds and almost 75% of all antibacterial molecules are of natural origin (Newman and Cragg, 2016). Natural products are multitarget compounds which may reduce drug-resistance and, up to now, several natural metabolites have been tested for their antimycobacterial activity (Farah et al., 2015; Newton et al., 2000). It has been proved that a relationship exists between pulmonary TB and respiratory diseases such as bronchial asthma, and 1% of the population with predominant pulmonary TB also has allergic bronchial asthma and vice versa (Committee on Allergy, 1960). Recently, in a large study aimed to evaluate the antimycobacterial activity of extracts of medicinal plants used by the Mayo population against TB and respiratory diseases, Rhynchosia precatoria dichloromethane root extract was found to be an excellent candidate for the isolation of antimycobacterial compounds (Coronado-Aceves et al., 2016), confirming the traditional use of this root made by Mayo Indians from Sonora, Mexico. As a matter of fact, they brew R. precatoria root into a tea and drink it for asthma and bronchitis (Yetman and Van Devender, 2002), symptoms that are closely related to pulmonary tuberculosis (Campos et al., 2008; Unger et al., 1960). Rhynchosia (Fabaceae) is a pantropical legume genus widely distributed across Africa, Madagascar, the Americas, Asia, and Australia (Schrire, 2005). The genus Rhynchosia comprises 4

230 species, grouped into two sections and six series: Copisma, comprising four series, and Arcyphyllum, with two (Grear, 1978). Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) is known by several common names such as frijolillo (Veracruz), ojos de cangrejo (Guerrero, Morelos), ojo de zanate (Sinaloa), chanate pusi (Mayo language) and ojo de chanate (Sonora) (Yetman and Van Devender, 2002). English common names are crab’s-eyes (CONABIO, 2011), blackbird’s eye and rosary bean. Its seeds are used as food and as a traditional medicine to cure some aches; some people also use the seeds to make necklaces and bracelets (CONABIO, 2011). Analysis of the literature showed that R. volubilis [syn. R. precatoria, (The Plant List, 2017)] was studied by several researchers that isolated isoflavones such as daidzein, calycosin, biochanin A, isoflavanones, such as cajanone, and several known flavonoids (quercetin, tricin, apigenin) (Guo et al., 2011; Li and Xiang, 2011). The aim of the present study was to determine the composition of the dichloromethane extract of R. precatoria roots and to evaluate the antimycobacterial activity of the isolated compounds against Mycobacterium tuberculosis (Mtb) H37Rv ATCC 27294 and M. smegmatis (Msm) ATCC 700084. Two new isoflavanones, precatorin A (1) and precatorin B (2), and a new isoflavone, precatorin C (3), were isolated along with the known lupinifolin (4) (Smalberger et al., 1974), cajanone (5) (Preston, 1977) and lupinifolinol (6) (Smalberger et al., 1974) (Figure 1).

5

HO 2' OH O 5

O 2''' HO OH O

10 4

O

9 O

2"

1'

O

3''' 4'''

2

O

O

4" 3"

1

2 OH O O

HO OH O

O O

O

O

OH

3

4

HO OH O

O

OH

O

OH O OH O

O OH

5

6

Fig. 1: Structures of compounds isolated from R. precatoria

2. Material and Methods 2.1 General Experimental Procedures. The melting points were measured on a Koffler micro hot-stage apparatus and are uncorrected. Optical rotations ([α]D) were measured on a JASCO DIP-1000 digital polarimeter. UV spectra were obtained using a UV/Vis T70+ (PG Instruments Ltd) spectrophotometer. Electronic Circular Dichroism (ECD) spectra were recorded at room temperature on a JASCO J815 spectropolarimeter. IR spectra were recorded on a JASCO 410 spectrophotometer equipped with a diffuse reflectance 6

accessory. NMR spectra were recorded using a Bruker Avance DRX-400 spectrometer operating at frequencies of 400 MHz (1H) and 100 MHz (13C). The spectra were measured in CDCl3. The 1H and 13

C NMR chemical shifts (δ) were expressed in ppm with reference to the solvent signals [CDCl3:

δH 7.26 and δC 77.0]. Coupling constants are given in Hz. Column chromatography was performed using Merck silica gel 60 (70-230 mesh ASTM). Fractions were monitored by TLC (Silica gel 60 F254, Merck) and spots on TLC were visualized under UV light and after staining with panisaldehyde-H2SO4-EtOH (1:1:98) followed by heating at 110 °C. Combustion analyses were carried out on a Fisons EA1108 elemental analyser.

2.2 Chemicals Chemical compounds studied in this article: lupinifolin (Pubchem CID: 13846826), cajanone (Pubchem CID: 325518), n-hexane (PubChem CID: 8058), methanol (PubChem CID: 887), dichloromethane (PubChem CID: 6344), and ethyl acetate (EtOAc) (PubChem CID: 8857). Rifampicin (PubChem CID: 5381226), gentamicin sulfate (PubChem CID: 3467; 72396; 588785), resazurin sodium salt (PubChem CID: 112939), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alamar blue (PubChem CID: 11077) was purchased from AbD Serotec (Oxford, UK).

2.3. Computational methods Preliminary conformational analysis was performed by Spartan02 package (SPARTAN 02) employing MMFF94s molecular mechanics (MM) force field with Monte Carlo searching and assuming the (S) absolute configuration for 1. All possible conformers were searched, considering the degrees of freedom of the system within a range of 30 kcal/mol and retaining only the structures in an energy range of 10 kcal/mol with respect to the most stable one. The minimum energy conformers found by MM were further fully optimized by Gaussian09 package (Frisch et al., 2009) using the Density Functional Theory (DFT) at the DFT/B3LYP/TZVP level in solvent employing the IEFPCM solvation model (Tomasi et al. 2005). All conformers are real minima, no imaginary 7

vibrational frequencies have been found and the free energy values have been calculated and used to get the Boltzmann population of conformers at 298.15 K. The DFT geometries were then employed as input for Time Dependent DFT (TDDFT) calculations of ECD spectrum in gas phase. The theoretical ECD spectrum was obtained as average over the conformers Boltzmann populations. The ECD spectrum was obtained from calculated excitation energies and rotational strengths, as a sum of Gaussian functions centered at the wavelength of each transition, with a parameter σ (width of the band at ½ height) of 0.3 eV using SpecDis v1.60 program (Bruhn et al., 2013).

2.4 Plant Material. The roots of R. precatoria were collected around the municipality of Etchojoa, Sonora, Mexico (26° 55' 0.5 N, 109° 40' 0.2 W) in July 2015. Plant name verified on February 1, 2017 (The Plant List, 2017). A botanical specimen was identified by Professor Jesús Sánchez-Escalante (Herbarium of the University of Sonora) (voucher specimen No 22022). All the roots were washed with distilled water, finely cut, air dried in the shade at room temperature and powdered in an electric blender (JR model LP-12; Torrey; Monterrey, Mexico).

2.5 Extraction and Isolation. The plant roots (305 g) were extracted by maceration in CH2Cl2 (DCM) (1 × 3 L) at room temperature for 5 days. After filtration, the organic solutions were concentrated under vacuum at 40 °C to give a crude DCM extract (10.22 g). An aliquot of this extract (2.00 g) was purified over a SiO2 gel chromatography column (C1) using a gradient of DCM-EtOAc (0-20%), followed by MeOH, collecting 5 mL fractions that were evaluated by TLC and combined as a result of their similar appearance, yielding 10 pooled fractions (F1C1-F10C1) (Scheme S1, Supplementary Material). Fraction F6C1 (248 mg) was constituted by pure precatorin A (1). Fraction F7C1 (353.4 mg) was purified by SiO2 column chromatography (C2) and elution with DCM led to five fractions 8

(F1C2-F5C2). Fraction F4C2 (161.2 mg), by purification over SiO2 column chromatography (C3) with n-hexane-EtOAc (95:5-90:10) eluent, gave six fractions and fraction F5C3 was lupinifolin (4) (9.7 mg) (Scheme S2, Supplementary Material). Fraction F9C1 (91.5 mg) was purified by column chromatography (C4) and elution with DCM-EtOAc (95:5) gave five fractions (F1C4-F5C4). Fraction F3C4 after column chromatography purification (C5) gave 6.2 mg of cajanone (5) and 2.1 mg of lupinifolinol (6) (Scheme S3, Supplementary Material). Fraction F4C1 (31.8 mg) by purification by SiO2 column (C6) with n-hexane-EtOAc (95:5) eluent gave eight fractions (F1C6F8C6). Fraction F4C6 contained pure precatorin C (3) (10.1 mg) (Scheme S4, Supplementary Material). Fraction F5C1 (192.8 mg) after purification by SiO2 column (C7) with n-hexane: EtOAc 95:5 eluent, gave eleven fractions (F1C7-F11C7). Fraction F7C7 contained pure precatorin B (21.7 mg) (Scheme S5, Supplementary Material). Precatorin A (1): pale yellow solid; m.p.187-188 °C; [α]25D +0.91 (c 0.005, CHCl3); UV (EtOH) λmax nm (log ε): 295 (4.18), 271 (4.60), 222 (4.50); IR (KBr) νmax 3225, 2970, 2927, 1647, 1627, 1500, 1369, 1114 cm-1; 1H and

13

C NMR, see Table 1; anal. C 71.15%, H 5.64%, calcd for

C25H24O6, C 71.42%, H 5.30%. Precatorin B (2): pale yellow solid; m.p.133-134 °C; [α]25D -4.76 (c 0.004, CHCl3); UV (EtOH) λmax nm (log ε): 295 (4.23), 273 (4.13), 222 (4.43); IR (KBr) νmax 3349, 2982, 2928, 1646, 1631, 1491, 1388, 1159 cm-1; 1H and

13

C NMR, see Table 1; anal. C 71.34%, H 5.47%, calcd for

C25H24O6, C 71.42%, H 5.30%. Precatorin C (3): yellow solid; m.p. 96-98 °C; UV (EtOH) λmax nm (log ε): 268.5 (4.60), 246 (4.39), 225 (4.49); IR (KBr) νmax 3082, 2977, 2922, 1653, 1576, 1482,1145 cm-1; 1H and 13C NMR, see Table 2; anal. C 71.76%, H 5.30%, calcd for C25H22O6, C 71.55%, H 5.48%.

2.6 Antimycobacterial Assay. 2.6.1. Mycobacterial strains growth conditions

9

M. tuberculosis H37Rv and M. smegmatis reference strains were cultivated in Middlebrook agar 7H10 supplemented 2% (v/v) glycerol and 10% (v/v) OADC (oleic acid, albumin, dextrose and catalase; Becton Dickinson) at 37 °C (Thermo Scientific; Waltham, MA, USA) until reaching the logarithmic growth phase, 3-4 and 12-14 days, respectively; in a biosafety level (BSL) 3 and 2 laboratories, respectively.

2.6.2 Mycobacterium tuberculosis H37Rv. Evaluation of the activity against M. tuberculosis H37Rv ATCC 27294 was performed by the visual Microplate Alamar Blue Assay (MABA) according to the protocols previously described (Coronado-Aceves et al., 2016; Franzblau et al., 1998; Molina-Salinas et al., 2006).

2.6.3 Mycobacterium smegmatis. The activity against Mycobacterium smegmatis ATCC 700084 (Msm) was performed by the fluorometric Resazurin Microplate Assay (fREMA) as previously described (Coronado-Aceves et al., 2016; Palomino et al., 2002) with some modifications. Briefly, the assay was set up as for MABA, wells containing drug only were used to detect autofluorescence of compounds and were used to subtract out the background. After 48 h incubation all wells were revealed with 30 µL of resazurin 0.01% (weight/volume) (Sigma Aldrich, St. Louis, MO) and the microplate was reincubated for additional 48 h. Fluorescence was measured in a plate fluorometer (Fluoroskan Ascent, Thermo, Finland) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm, and relative fluorescence units (RFU) were recorded. MIC was defined as the lowest drug concentration that presented RFU values lower than those presented by the 10% growth control (Luna-Herrera et al., 2007).

2.6.3.1. Determination of synergistic antimycobacterial activity by fREMA X/Y quotient analysis.

10

The MICs found for the individual compounds against Mtb were similar to the activity of the crude extract (except for lupinifolin); as well as the MICs against Msm (except for precatorin A and cajanone). Therefore, synergistic antimycobacterial activity of compounds in combinations was tested against M. smegmatis by fREMA, using compounds 1, 2, 4 and 5 alone and in combinations at one-half, one-fourth and one-eighth of the MIC previously determined in the antimycobacterial assay described earlier. RFU were measured in the plate fluorometer, and the X/Y quotient analysis was performed as described by Luna-Herrera et al. (2007). X represents the RFU value obtained with the combination of both compounds, and Y is the RFU value of the compound that presented the lowest RFU value, when alone. Synergy was considered when the X/Y value was <0.5, additive activity when X/Y was >0.5 and <1.0, no activity when X/Y was 1–2, and antagonism when X/Y was >2 (Luna-Herrera et al., 2007).

2.7. Cell viability assay and selectivity index. Cell viability was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] reduction assay with some modifications (Mosmann, 1983; Rascón-Valenzuela et al., 2015). RAW 264.7 (murine macrophages transformed by virus Abelson leukemia) (ATCC number: TIB-71) was provided by Dr. Emil A. Unanue (Department of Pathology and Immunology, Washington University in St. Louis, MO, USA). Vero cell line (African green monkey kidney cells) was kindly provided by Dr. Jesús Hernández (Centro de Investigación en Alimentación y Desarrollo, A.C., CIAD, Hermosillo, Sonora, México). Maximum cytotoxicity (100%) was determined by lysing the cells with sodium dodecyl sulfate (Sigma Aldrich, St. Louis, MO) (Cantrell et al., 1996). The selectivity index (SI) was calculated by dividing the 50% inhibitory concentration (IC50) by the MIC; if the SI was >10, the compound was considered selective (Orme et al., 2001).

3. Results and Discussion 3.1 Structure elucidation of new isolated compounds 11

Compound 1 was obtained as a pale yellow solid with [α]25D +0.91° (c 0.005, CHCl3). Its molecular formula, C25H24O6, was deduced by elemental analysis indicating 14 degrees of unsaturation. The IR spectrum showed an absorption band for a carbonyl group (1647 cm-1) and a broad band for OH stretching at 3325 cm-1. The proton NMR spectrum (Figure S1, Supplementary Material) showed four methyls (δH 1.38, 1.41, 1.43 and 1.45), two sets of coupled olefinic protons at δH 5.53 and 6.57 (J = 10.1 Hz) and at δH 5.47 and 6.25 (J = 9.8 Hz) and three aromatic singlets (δH 5.97, 6.39 and 7.01). Two phenolic hydroxyl groups were present at δH 7.62 and 11.8, the latter diagnostic for a hydrogen-bonded proton. Finally, the presence of a methylene group at δH 4.70 (dd, J = 4.7, 11.7 Hz) and 4.86 (dd, J =. 4.7, 11.7 Hz) coupled with a methine at δH 3.97 (t, J = 4.7 Hz) was diagnostic for the isoflavane skeleton. From

13

C NMR and JMODXH (J-modulated spin echo) spectra analysis it was evident that

compound 1 had 12 quaternary carbons, one of which was connected with a carbonyl (δC 196.7) and two with tetrasubstituted oxygenated carbons (δC 78.5 and 76.5). In the

13

C NMR spectrum there

were eight methines (one of which was at δC 97.8), one methylene carbon at δC 69.7 and four methyls (δH 27.9, 28.2, 28.4 and 28.5) (Table 1). Furthermore, there were five signals diagnostic for aromatic carbons bonded to oxygens (δC 154.1, 155.9, 156.7, 163.2 and 164.6) (Figure S2, Supplementary Material). Analysis of the 1D and 2D NMR (HMQC, H-H COSY, HMBC) data allowed the structure of compound 1 to be determined (Table 1). COSY data were used to establish the spin system sequence from H-2 to H-3, from H-3’’ to H-4’’, and from H-3’’’ to H-4’’’ (Figures S3 and S4, Supplementary Material). Further support for these assignments was provided by HMBC correlations observed between H-2 and C-4 and C-1’, H-3 and C-4 and C-2, H-6 and C-7 and C-8, 5-OH and C-6, H-3’ and C-2’ and C-5’, H-6’ and C-2’ and C-4’’’, H-4’’ and C-8, C-7, C-3’’ and C8, H-4’’’ and C-6’ and C-5’. Furthermore, 2’’-Mes were coupled with C-2’’ and C-3’’ and 2’’’-Mes with C-2’’’ and C-3’’’ (Figures S6 and S7, Supplementary Material). The presence of a 7-hydroxy2,2-dimethylchromen-6-yl group bound at C-3 of the isoflavane ring was confirmed by analysis of 12

the 13C NMR data of kraussianone 1 (Drewes et al., 2002; Selepe et al., 2010) and licoisoflavanone (McKee et al., 1997). From the above data it was possible to establish structure 1 for precatorin A. The relative configuration of compound 1 was established through analysis of its NOESY spectrum (Figure S8, Supplementary Material). NOESY interactions were observed between H-6’ and H-2, H-4’’’ and H-3’’’ and between this last and 2’’’-Mes, 2’-OH and H-3, H-4’’’ and H-6’, H-3’’ and H-4’’. From all these observations we established that precatorin A (1) was 5-hydroxy-3-(7hydroxy-2,2-dimethyl-2H-chromen-6-yl)-8,8-dimethyl-2,3-dihydro-4H,8H-pyrano[2,3-f]chromen4-one

5,2’-dihydroxy-2’’,2’’-dimethylpyrano[7,8:6’,5’]-2’’’,2’’’-

or

dimethylpyrano[4’,5’:6’’’,5’’’]isoflavanone. Compound 2 was obtained as a pale yellow solid with [α]25D -4.76° (c 0.004, CHCl3). Its molecular formula, C25H24O6, was deduced by elemental analysis, indicating 14 degrees of unsaturation. The IR spectrum showed absorption bands for a carbonyl group (1646 cm-1) and OH stretching as a broad band at 3349 cm1. Careful examination of the NMR spectra (Table 1) showed that compound 2 is closely related to 1. The location of the pyrane ring fused to an A ring (Buckingham, 1993) was confirmed by HMBC correlations between δH 5.94 (H-8) and δC 162.3 (C9), 5-OH and δC 101.6 (C-6), δH 6.57 (H-4’’) and C-5 (δ 159.0) and C-6 (δ 101.6). NOESY interactions (Figure S16, Supplementary Material) were observed between 5-OH and H-6, H-3 and H-2 and H-6’, H-2 and H-6’, H-3’’ and H-4’’, H-3’’’ and H-4’’’. According to these data, compound 2 (precatorin B) was identified as the previously unreported 5-hydroxy-7-(7-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-2,2-dimethyl-7,8-dihydro-2H,6Hpyrano[3,2-g]chromen-6-one

or

5,2’-dihydroxy-2’’,2’’-dimethylpyrano[5,6:8,7]-2’’’,2’’’-

dimethylpyrano[5,6:5,4]isoflavanone.

Table 1. NMR Data for Compounds 1 and 2.a

position 2a 2b

Precatorin A (1) δC, type δH, (J in Hz) 4.70, dd (4.7, 11.7) 69.7, CH2 4.86, dd (4.7, 11.7)

Precatorin B (2) δC, type δH, (J in Hz) 4.65, dd (4.8, 11.6) 69.5, CH2 4.78, dd (4.8, 11.6) 13

3 44.7, CH 3.97, t (4.7) 44.9, CH 3.96, t (4.6) 4 196.7, C 196.8, C 5 156.7, C 159.0, C 6 97.8, CH 5.97, s 101.6, C 7 163.2, C 162.9, C 8 101.8, C 96.1, CH 5.943, s 9 164.6, C 162.3, C 10 101.6, C 103.1, C 1’ 114.5, C 114.5, C 2’ 155.9, C 155.8, C 3’ 105.7, CH 6.39, s 105.6, CH 6.37, s 4’ 154.1, s 154.1, C 5’ 115.1, C 115.1, C 6’ 125.1, CH 7.01, s 125.2, CH 6.99, s 2’’ 78.5, C 1.61, s 78.6, C 3’’ 126.6, CH 5.53, d (10.1) 126.2, CH 5.46 d (10.1) 4’’ 115.2, CH 6.57, d (10.1) 115.1, CH 6.57, d (10.1) 2’’’ 76.5, C 1.63, s 76.5, C 3’’’ 128.3, CH 5.47, d (9.8) 128.3, CH 5.45, d (9.8) 4’’’ 121.6, CH 6.25, d (9.8 121.7, CH 6.23, d (9.8) 2’’-Me 27.9, CH3 1.45, s 27.9, CH3 1.44, s 2’’-Me 28.2, CH3 1.43, s 28.1, CH3 1.42, s 2’’’-Me 28.4, CH3 1.37, s 28.4, CH3 1.37, s 2’’’-Me 28.5, CH3 1.41, s 28.5, CH3 1.40, s 5-OH 11.8, s 12.3, s 2’-OH 7.62, s 7.66, s a Chemical shifts: δ values are given in ppm with reference to the signal of CHCl3 (δ 7.26 ppm) for 1 H and to the center peak of the signal of CDCl3 (δ 77.0 ppm) for 13C.

Precatorin C (3) was isolated as a pale yellow solid. Its molecular formula, C25H22O6, was deduced by elemental analysis, indicating 15 degrees of unsaturation The IR spectrum established the presence of a carbonyl (1653 cm-1) and OH groups (3082 cm-1). Analysis of the 1H (Figure S17, Supplementary Material) and

13

C NMR (Figure S18, Supplementary Material) spectra were very

similar to those of precatorin A (1) (Table 2). The main differences were the disappearance in the 1

H NMR spectrum of the oxymethylene protons and the aliphatic methine and the presence of an

olefinic proton at δH 8.00 (s). Table 2. NMR Data for Compound 3.a position 2 3 4 5 6 7 8 9 10 1’

δC, type 154.7, CH 23.2, C 182.1, C 161.8, C 101.0, CH 160.4, C 101.19, C 152.0, C 105.4, C 111.9, C

δH, (J in Hz) 8.00, s

6.34, s

14

2’ 157.1, C 3’ 107.3, CH 6.53, s 4’ 155.6, C 5’ 115.2, C 6’ 127.1, CH 6.75, s 2’’ 78.5, C 3’’ 127.8, CH 5.62, d (10.1) 4’’ 114.2, CH 6.69, d (10.1) 2’’’ 76.7, C 3’’’ 128.8, CH 5.52d (9.8) 4’’’ 121.3, CH 6.27, d (9.8) 2’’-Me 28.1, CH3, 1.48, s 2’’-Me 28.3, CH3 1.43, s 2’’’-Me 28.1, CH3, 1.48, s 2’’’-Me 28.3, CH3 1.43, s 5-OH 12.3, s 2’-OH3 8.30, s a Chemical shifts: δ values are given in ppm with reference to the signal of CHCl3 (δ 7.26 ppm) for 1H NMR and to the center peak of the signal of CDCl3 (δ 77.0 ppm).

COSY correlations (Figures S19 and S20, Supplementary Material) showed only couplings between H-3’’ and H-4’’, and between H-3’’’ and H-4’’’. HMBC correlations (Figures S22 and S23, Supplementary Material) were shown for H-2 with C-3, C-4 and C-1’, H-6 and C-7, C-8 and C-10, 5-OH and C-6 and C-10, H-3’ and C-2’ and C-5’, H-6’ and C-3, C-4’’’ and C-4’, H-4’’ and C-7, C-2’’ and C-8, and H-3’’ and C-2’’ and C -8, H-3’’’ and C-2’’’ and H-4’’’ and C-2’’’. NOESY correlations (Figure S24, Supplementary Material) enabled the relative configuration of compound 3 to be determined. NOESY correlations were observed between H-2 and H-6’ and between the latter and H-4’’’. H-4’’’ was coupled with H-3’’’ and H-4’’ with H-3’’. Finally, 2’’Mes were coupled with H-3’’ and 2’’’-Mes with H-3’’’ From these observations compound 3 was established as

5-hydroxy-3-(7-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-8,8-dimethyl-4H,8H-pyrano[2,3-

f]chromen-4-one

or

5,2’-dihydroxy-2’’,2’’-dimethylpyrano[7,8:6’,5’]-2’’’,2’’’-

dimethylpyrano[4’,5’:6’’’,5’’’]isoflavone. The absolute configuration at C-3 for isoflavanones 1, 2, and 5 was assigned by analysis of their ECD spectra. In the literature, ECD has been widely employed for absolute configuration assignment to isoflavanones (Galeffi et al., 1997; Yenesew et al., 2000) by applying an empirical rule derived from the Snatzke’s one for cyclic aryl ketones (Snatzke, 1965) and relying on the sign 15

of the n-π* Cotton effect in ECD spectrum (Slade et al., 2005; Kurtan et al. 2012). According to this rule, if the heterocyclic ring, adopting the lowest energy envelope conformation, displays a P helicity, then a positive n-π* Cotton effect is observed in the ECD spectrum, vice versa for a M helicity of the ring (Snatzke and Snatzke 1973; Slade et al., 2005). The ring helicity depends on both the lowest energy conformation and the absolute configuration at C-3. For the same absolute configuration at this stereocenter opposite helicity, and then n-* band sign, is expected if the C-3 substituent is either in equatorial or axial conformation, respectively. It follows that a correct application of the method requires the knowledge of the most stable conformer and the correct identification of the n-* band. This is often difficult, because the n-* transition wavelength can be shifted by intramolecular hydrogen bonding involving the carbonyl and by aryl substitution. Moreover, the presence of multiple conformations prevents a reliable identification of the most stable one. More recent studies have been in fact demonstrated that application of similar empirical rules for absolute configuration assignment to tetralones and chroman-4-ones can often lead to wrong assignments (Kurtan et al. 2012; Mazzeo et al., 2014). In the case of 1, the 1HNMR coupling constants in CDCl3 (Table 1) reveal an axial-equatorial relationship between the two H-2 protons and H-3 and then an axial position for the aryl substituent at C-3. This conformation, probably stabilized by intramolecular H-bonds between the phenolic groups and the carbonyl, is unexpected for isoflavanones, usually displaying equatorial C-3 substituents (Galeffi et al., 1997; Yenesew et al., 2000). On the contrary, 1HNMR spectrum of 1 in methanol-d4 (Figure S25, Supplementary Material) shows the presence of a trans-diaxial relationship between H-2ax and H-3 (J=11.0 Hz), thus revealing a preferred equatorial conformation for the C-3 aryl substituent. Clearly, methanol breaks the intramolecular hydrogen bonding, disfavoring the axial arrangement. As reported above, for the same absolute configuration, an opposite conformation of the C-3 substituent should lead to an opposite sign of the n-π* band in the ECD spectrum. On the contrary, the experimental ECD spectrum of 1 in both methanol (Figure 2) and an aprotic solvent such as acetonitrile (Figure S26, Supplementary Material) appears quite similar, displaying bands of the same sign and position. The 16

UV spectrum of 1 recorded in methanol in the 190-360 nm range (Figure 2) shows several broad and intense absorption bands. The most intense ones being that at 220 nm, which can be allied to the 1B transition, that at 270 nm, which can be ascribed mainly to the 1La transition, and the two broad bands centered at about 290 and 305 nm which could be allied to both aromatic 1Lb and carbonyl n-π* transitions. The ECD spectrum in methanol in the same wavelength range (Figure 2) shows four distinct, albeit weak, Cotton effects: a negative one in the 190-230 nm range, a positive one in the 230-280 nm range, a weak negative one in the 280-300 nm range, and a positive band in the 300-340 nm range The complex conformational behavior of 1 rises serious concern upon the possibility to obtain reliable results by applying the above mentioned empirical rule for isoflavanones. Therefore, we decided to resort to a computational approach (Autschbach, 2012) for ECD spectra analysis, an approach which has demonstrated high reliability for absolute configuration assignment to structurally complex natural products (Li et al., 2010; Superchi et al., 2017). Assignment of the absolute configuration of 1 was then carried out by ab initio computational analysis of its ECD spectrum. At first, a computational conformational analysis for 1 was carried out at DFT/B3LYP/TZVP level in both methanol and acetonitrile as solvent, providing in both cases six populated conformers at room temperature (Figure 3 and Table S1 in Supplementary Material). The axial and equatorial conformers are almost equally populated. In fact, in methanol the most abundant axial one 1b, accounts for 45% of the overall conformers population, while the following two most populated conformers 1a and 1d, both equatorial, accounted for 22% and 26% of the conformers population. The conformers structures and populations are quite similar in acetonitrile (Table S1, Supplementary Material). As inferred from the conformers structure, the axial one 1b is indeed stabilized by intramolecular H-bonds between the phenolic groups of the aryl moieties and the carbonyl of the saturated heterocycle. The UV and ECD spectra of the (3S) enantiomer of 1 were then calculated in both solvents at TDDFT/CAM-B3LYP/6-311G(d,p) level on previously found conformers and Boltzmann averaged over conformers populations. As inferred from Figure 2 17

the theoretical ECD spectrum for (S)-1 in methanol well reproduces in position and intensity the main ECD bands of the experimental spectrum but with opposite sign. Therefore, the absolute configuration of 1 is (3R), opposite to the one chosen for calculations. A good reproduction of the ECD spectrum also in acetonitrile (Figure S26 in Supplementary Material) confirmed such assignment. This result shows that the assignment of the absolute configuration to isoflavanones cannot be carried out reliably by applying simple empirical ECD correlations. In fact, the real conformational situation in solution cannot be simply deduced by NMR analysis, which shows only the signal allied to main conformer but does not reveal the full conformers population. Moreover, the n-π* transition cannot be simply associated to the lowest energy ECD band. In fact, the analysis of the Molecular Orbitals allied to the main spectral electronic transitions reveals that in the main axial conformer 1b the lowest energy band is due to the contribution of several aromatic -* transitions and not to the expected carbonyl n-* transition which, on the contrary, is blue shifted at 280 nm. Moreover, in the equatorial conformers 1a and 1c, the n-* transition, albeit not blue shifted, is not correlated with the sign of the lowest energy band. It follows that even when empirical methods provide correct absolute configuration assignments such agreement can be mainly fortuitous. The absolute configuration to compounds 2 and 5 was then assigned by simple comparison of their ECD spectra with that of (R)-1. Both isoflavanones 2 and 5 display almost the same chromophoric system than 1 and show also very similar ECD spectra, albeit lower in intensity. In both ECD spectra of 2 and 5 are clearly visible the negative 210 nm Cotton effect and the positive one at 270 nm, while in the spectrum of 5 the 310 positive band is missing (Figure S27 and S28, Supplementary Material). Therefore, also for 2 and 5 the (R) absolute configuration at C-3 can be confidently assigned. Since racemization of isoflavanones often occurs also under mild conditions (Dewick, 1982), it is possible that during extraction and purification lowering of 2 and 5 optical purity occurred, giving rise to a lower chiroptical response.

18

Fig. 2. Experimental UV (solid blue line) and ECD (solid red line) spectra of 1 in methanol. Computed UV (dashed blue line) and ECD (dashed red line) spectra of (S)-1.

Fig. 3. Most stable conformers of (3S)-1 calculated at the DFT/B3LYP/TZVP level of theory in methanol. 3.2 Antimycobacterial activity All the pure isolated compounds, except lupinifolinol due to the small amount, were tested first against Mtb H37Rv (Table 3). Lupinifolin (4) and cajanone (5) were the most active 19

antimycobacterial compounds with a minimal inhibitory concentration (MIC) of 31.25 and 62.5 µg/mL (76.93 μM and 147.94 μM), respectively, with equal MBC, while the new compounds 1, 2 and 3 showed a MIC of 62.5 µg/mL (148.64 μM), and compounds 1 and 2 resulted bactericidal at 125 µg/mL 297.29 μM). Compounds 1-3 and 5 equalled the inhibitory activity of the dichloromethane crude extract of R. precatoria root (62.5 µg/mL, 148.64 μM), while 4 had two-fold more inhibitory and four-fold more bactericidal activity than the original crude extract. Previous studies reported the activity of 4 and 6 against Mtb H37Ra with MICs of 12.5 and 25 µg/mL (30.77 and 59.18 μM, respectively) (Sutthivaiyakit et al., 2009). The anti-Mtb activities of 1-3 and 5 are reported here for the first time, while 4 has not been tested previously against the virulent Mtb strain. These results suggest that the basic flavanone structure seems to be responsible of its antimycobacterial activity, and the prenylation present in compounds 4 and 5, especially the one linked to the A ring, may play an essential role in increasing it. In addition, compounds were tested against Msm where the most active compounds were 1 and 5 with MIC of 125 µg/mL (297.29 µM), and MBC of 250 (594.58 µM) and 125 µg/mL (297.29 µM) respectively; while 2 and 4 had a MIC of 250 µg/mL (594.58 µM) (Table 3).

Table 3. Activity of flavonoids isolated from Rhynchosia precatoria DC against Mycobacterium tuberculosis H37Rv (ATCC 27294) and Mycobacterium smegmatis (ATCC 700084). M. tuberculosis H37Rv (ATCC 27294)

Precatorin A (1) Precatorin B (2) Precatorin C (3) Lupinifolin (4) Cajanone (5) DCM root extract Gentamicin

MIC99 µg/mL (μM)a

MBC µg/mL (μM)

62.5 (148.64) 62.5 (148.64) 62.5 (149.36) 31.25 (76.93) 62.5 (147.94)

125 (297.29) 125 (297.29) ND 31.25 (76.93) 62.5 (147.94)

62.5

125

ND

ND

M. smegmatis (ATCC 700084) MIC90 MBC µg/mL (μM)b µg/mL (μM)b 125 (297.29) 250 (594.58) 250 (594.58) NB NI NB 250 (615.49) NB 125 (295.87) 125 (295.87) 250 250 ≤0.125 (0.26)

2 (4.18)

Rifampin 0.0625 (0.07) 0.25 (0.30) Determined by the Microplate Alamar Blue Assay (MABA). MIC 99: minimal concentration that inhibits the growth of 99% of the bacterial inoculum tested. bDetermined by fluorometric Resazurin Microplate Assay (fREMA). MIC90: is the lowest drug, extract or antibiotic concentration tested that had relative fluorescent units (RFU) lower than those presented by a 10% a

20

growth control; MBC: minimal bactericidal concentration; NI: No inhibitory activity at the maximum concentration evaluated (250 µg/ml); NB: no bactericidal at the maximum concentration evaluated (250 µg/ml); ND: not determined.

Furthermore, synergistic antimycobacterial activity was evaluated against Msm by fREMA, using compounds 1, 2, 4 and 5 alone and in combinations at one-half, one-fourth and one-eighth of their MIC, establishing that lupinifolin (4) had a synergistic effect with precatorin A (1), precatorin B (2) and cajanone (5) up to the combination containing 1/2 MIC of each compound; also precatorin B (2) and cajanone (5) showed this effect in the same conditions (Table 4; Figure 4a). The synergistic effect among lupinifolin (4) and cajanone (5) is very remarkable since the bacterial growth was reduced to only 1.32%, and the X/Y quotient was 0.03 (Figure 4b); very similar results were obtained with the combination of precatorin A (1) and lupinifolin (4). X/Y quotient analysis for combinations of 1/4 and 1/8 of each compound resulted only in additive activity. The synergistic antimycobacterial activity of these compounds have not been reported previously. Table 4. Synergistic activity of compounds from R. precatoria root against M. smegmatis ATCC 700084 determined by X/Y quotient analysis 1/2 MICa 1/4 MIC Precatorin A (1) + Precatorin B (2) 0.76 0.95 Precatorin A (1) + Lupinifolin (4) 0.68 0.04 Precatorin A (1) + Cajanone (5) 0.78 0.63 Precatorin B (2) + Cajanone (5) 0.64 0.14 Precatorin B (2) + Lupinifolin (4) 0.64 0.11 Lupinifolin (4) + Cajanone (5) 0.67 0.03 a Figures in bold shows synergistic effect.

1/8 MIC 0.87 0.84 0.69 0.86 0.75 0.59

On the other hand, compounds 1-5 cytotoxicity was tested towards murine macrophages RAW 264.7 as a model of the host cells, and Vero cell line a model toxicity to mammalian cells commonly used in the antimycobacterial research (Katsuno et al., 2015). IC50 values ranged from 13.73 to 160.52 µM and SI’s toward Mtb and Msm ranged from 0.04 to 1.08, respectively (Table 5), which means that these compounds possessed very low selectivity and cannot be used for drug therapy as they are. However, they could be used as templates to produce synthetic analogs with particular hydroxyl substitutions described on antimycobacterial flavonoids by previous structureactivity relationship studies (Yadav et al., 2013) in order to increase their activity and selectivity. 21

Table 5. Cytotoxicity and selectivity index (SI) of compounds against RAW 264.7 and Vero cells RAW 264.7

Vero

IC50 (μM)

SI (Mtb)

SI (Msm)

IC50 (μM)

SI (Mtb)

SI (Msm)

Precatorin A (1)

46.98 ± 4.7

0.32

0.16

160.52 ± 6.6

1.08

0.54

Precatorin B (2)

32.53 ± 8.8

0.22

0.55

83.35 ± 6.6

0.56

0.14

Precatorin C (3)

37.9 ± 0.3

0.25

ND

ND

ND

ND

Lupinifolin (4)

26.49 ± 0.6

0.34

0.04

22.89 ± 2.0

0.30

0.04

Cajanone (5)

13.73 ± 0.1

0.11

0.05

43.36 ± 4.1

0.29

0.15

IC50: 50% inhibitory concentration, values (mean ± SD, n = 3); ND: not determined.

X/Y quotient analysis suggests that the antimycobacterial effect of the dichloromethane crude extract of R. precatoria root occurs by a synergistic effect between lupinifolin (4) and precatorin A (1), B (2) and/or cajanone (5), caused by at least two different mechanisms of action. It is possible that the lupinifolin mycobactericidal activity is caused by disrupting bacterial cell membrane as it was described for Staphylococcus aureus (Yusook et al., 2016). Flavonoids also have been reported to inhibit the Mtb proteasome activity (Zheng et al., 2014) and the mycolic-acid-producing fatty acid synthase II (FAS-II) (Brown et al., 2007). However, more studies in order to determine the antimycobacterial mechanism of action of these flavonoids are needed. Molecular biology approaches on the expression of mycobacterial genes as groEL1 (modulates synthesis of mycolates), mmpL11 (contributes to mycobacterial cell wall biosynthesis) and lsr2 (involved in the mycolyl-diacylglycerol lipids synthesis), just to mention some may be performed in the near future (Chen et al., 2009; Ojha et al., 2005; Pacheco et al., 2013).

22

a) 100

% Growth

80 60 40 20

10 Pr ec at or 1/ in 2 Pr A ec at or in 1/ 2 B Lu 1/ pi 2P ni f re ol ca 1/ in 2 to C rin 1/ aj 2P an A re +1 on ca /2 e Lu to rin 1/ pi 2P n B ifo re + lin ca 1/ to 2C rin aj 1/ an B 2L +1 on up /2 e Lu in ifo pi ni lin f ol + in 1/ 2C aj an on e

1/ 2

C

on

tr

C

ol

on

1:

tr

ol

0

b) 100

X/Y= 0.03

% Growth

80 60

Y= 47.12%

51.59%

40

X= 1.32%

20

aj an on e

e

1/ 2C

C aj an on 1/ 2L u

pi ni fo lin

+

1/ 2

lin in ifo Lu p 1/ 2

on tr ol 1: 10 C

C

on tr ol

0

Fig. 4. a) Synergism determination by fREMA. Compounds were tested alone and in combination at 1/2 of their MIC against M. smegmatis. Control indicates bacterial growth free of testing compounds; control 1:10 represents the 10% of the bacterial population tested; 1/2 represents the half of compounds MIC against M. smegmatis. b) The value of the X/Y quotient denotes a synergistic combination among 1/2 MIC of lupinifolin and 1/2 MIC of cajanone.

4. Conclusion We isolated two new isoflavanones (1, 2) and one new isoflavone (3) with a weak antimycobacterial activity. The (3R) absolute configuration was assigned to 1 by computational analysis of its ECD spectrum and to 2 and 5 by similarity of their ECD spectra with that of 1. The unreliability of the empirical rules employed so far for assignment of absolute configuration to isoflavanones was also revealed. We are also reporting for first time activity against a virulent strain of M. tuberculosis for compounds 4 and 5. Antimycobacterial effect of the dichloromethane crude extract of R. precatoria root occurs by a synergy among it compounds. 23

ASSOCIATED CONTENT Supplementary Material. Purification details, NMR and ECD spectra of the new compounds (1-3) can be found in the online version at

Notes The authors declare no competing financial interest.

Author contribution

MCM conceived the experimental chemical aspect of the project. MCM and MC supervised the chemical work. EWCA and GG performed the chemical work. AGE conceived experimental biological aspect of the project and analysed data. EWCA performed the biological work. CIEP and JLC supervised the experimental biological work. SS conceived and supervised the assignment of absolute configuration by ECD analysis. SV performed the experimental and computational ECD analysis. RERZ conceived and performed the cytotoxicity tests. MCM, EWCA, AGE and SS wrote the manuscript. All the authors revised the manuscript and approved it in the final form.

24

ACKNOWLEDGMENT The authors acknowledge financial support from the National Commission of Science and Technology of Mexico (CONACYT) for funding via grant CONACYT PDCPN 2013-01 215469 and 344972 for E.W. Coronado-Aceves and thank Prof. Jesús Sánchez-Escalante for the identification of the R. precatoria specimen. Thanks to Segura-Salinas E. and Silva-Miranda M. for technical support in MABA assay. Proofreading Service is acknowledged for writing assistance.

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