7-epi-griffonilide, a new lactone from Bauhinia pentandra:

A new lactone, 7-epi-griffonilide (1), and six known compounds, 2, 3a – 3c, 4a and 4b, were isolated from the leaves of Bauhinia pentandra (Fabaceae). The structures elucidation of 1 and 2 were based on detailed 2D NMR techniques and spectral comparison with related compounds, leading to complete assignment of the H and C NMR spectra.


INTRODUCTION
In the course of our continuing search for natural products from medicinal plants, we have investigated the leaves of Bauhinia pentandra (Bong.) D. Dietr. B. pentandra is widely distribute in Northeast Brazil where is known as "mororó" and used in folk medicine. The genus Bauhinia contains many species of plants with medicinal interest (Silva and Cechinel Filho 2002). It consists of about 300 species, distributed in most tropical countries, including Africa, Asia and America (Cechinel Filho 2009). Previous phytochemical studies with plants from Bauhinia genus report the presence of lactones, flavonoids, terpenoids, steroids, triterpenes, tannins, quinones and alkaloids Cechinel Filho 2002, Maia Neto et al. 2008), while studies from B. pentandra reported the chemical composition of the essential and fatty oils (Duarte-Almeida et al. 2004, Almeida et al. 2015, as well as the isolation of flavonoids (Salatino et al. 1999) and cyanoglucosides (Silva et al. 2013). In this paper, we report the isolation and structure elucidation of lactones (1 and 2), phenylacetic derivatives (3a, 3b and 3c) and a mixture of cyanoglucoside (4a) and glucopyranoside (4b).
Compound 1 is new, and 2, 3a, 3b, 3c, 4a e 4b has not been reported previously in the Bauhinia genus. The structural assignments and relative stereochemistry of 1 and 2 were based on detailed 2D NMR spectroscopy, while 3a, 3b, 3c, 4a e 4b were identified by comparison with NMR spectral data from literature (Wu et al. 1979, Nahar et al. 2005, Kortesniemi et al. 2014). , was further subjected to reversed-phase chromatography, using H 2 O-MeOH (9 : 1) as mobile phase in a isocratic system, flow rate 4.0 mL/min and monitored by HPLC semi-preparative column to yield 1 (15.5 mg, t R 7.15 min) and 2 (10.7 mg, t R 7.67 mn). Another portion of the EtOH extract (12.7 g) was prefractionated by CC on Silica gel eluted successively with hexane, CH 2 Cl 2 , EtOAc and MeOH. F-EtOAc fraction after evaporation of the solvent afforded 448.0 mg. Part of this fraction (72.4 mg) was further chromatographed on Silica gel column using solvents of increasing polarity from CH 2 Cl 2 through EtOAc to MeOH to obtain a solid dark brown viscous consisting of the mixture of compounds 3a, 3b and 3c (13.4 mg), soluble in CHCl 3 . An third portion of the EtOH extract (5.0 g) was dissolved in H 2 O/MeOH (50:50 v/v) and partitioned between hexane (3x100 mL), CH 2 Cl 2 (3x100 mL) and EtOAc (3x100 mL). After evaporation of the organic phases were obtained: 875 mg (F-hexane), 494 mg (F-CH 2 Cl 2 ) and 331 mg (F-EtOAc). F-EtOAc fraction (331 mg) was rechromatographed on Silica gel column eluting, successively with hexano, AcOEt and MeOH. A total of 92 fractions were collected and combined based on their TLC patterns; 200 µL from combined fractions (F 80-92, 190.0 mg), was further subjected to reversed-phase chromatography, using H 2 O-MeOH (9 : 1) as mobile phase in a isocratic system, flow rate 3.0 mL/min and monitored by HPLC semi-preparative C18 column (Phenomenex) to α D -3.11 (c 0.14, MeOH).

GENERAL EXPERIMENTAL PROCEDURES
The FT-IR spectrum showed absorption bands characteristic for OH (ν max 3419 cm -1 ), ester CO (ν max 1733 cm -1 ) and C = C (ν max 1633 cm -1 ).  Table I). The signals at d C 176.7 and 83.5 were used to characterize the presence of a fivemembered lactone ring (Silverstein and Webster 2000). These assignments were consistent with the HREIMS empirical formula, supporting the presence of two hydroxyl groups and five degrees of unsaturation/ring. Thus, NMR data suggested 1 as a bicyclic molecule.

H-and 13 C NMR chemical shifts assignments 1 and 5 (CD 3 OD, 500 MHz). Chemical shifts in d (ppm), coupling constants (J) in Hz (in parenthesis) a .
Position The data were used to postulate the constitutional structure 1a, which was confirmed by correlations exhibited by the HMBC NMR spectrum. Importantly, the olefinic hydrogen H-2 (d H 5.89) showed correlation with C-1 (d C 176.7, 2 J CH ), C-3 (d C 163.3, 2 J CH ) and 3 J CH ) and the H-8 (d H 5.11) with d C C-3 (d C 163.3, 2 J CH ) and CH-2 (d C 113.3, 3 J CH ), in agreement with the presence of an a,b-unsaturated lactone. Further, the olefinic hydrogen H-4 (d H 6.63) revealed correlation with the carbons C-3 (d C 163.3, 2 J CH ) and CH-8 (d C 83.5, 3 J CH ), while the other olefinic H-5 (d H 6.16) showed heteronuclear coupling with C-3 (d C 163.3, 3 J CH ). The latter correlations, in turn, are in agreement with a lactone ring joint with a cyclohexene ring (1a).
The relative stereochemistry of 1a was determined from the coupling constants of relevant hydrogens and from the observed 1 H-1 H NOESY. Thus, the signals corresponding to hydrogens H-6, H-7 and H-8 as broad singlets are consistent with the absence of diaxial interactions (1b). In agreement with these observations, the NOESY spectrum of 1 showed cross-peak assigned to dipolar interaction (spatial proximity) between H-6 and H-8 (1b).
Complete and unambiguous 1 H-and 13 C-NMR chemical shifts assignments of 1 based on 1 H -1 H COSY, 1 H -13 C COSY-n J CH (n=1, HSQC; n=2 and 3, HMBC), and 1 H -1 H NOESY were summarized in Table I Comparative analysis of the { 1 H}-and DEPT 135 o NMR spectra of 2 allowed to recognize signals corresponding six methines [three sp 2 at d C 113.4 (CH-2), 123.4 (CH-4) and 139.8 (CH-5) and three sp 3 oxygenated at d C 68.7 (CH-6), 74.5 (CH-7) and 83.5 (CH-8)] and two sp 2 no hydrogenated at d C 164.5 (C-3) and at d C 176.7 (C-1, conjugated carbonyl) carbon atoms (Table II). Thus, analysis of the 1 H and 13 C NMR spectra data of 2 confirmed that it was closely relate to 1 (and to 5). In fact, the 1 H and 13 C-NMR feature were virtually identical except for the small changes in chemical shifts ( 1 H and 13 C) and more pronounced changes in the multiplicity and the coupling constants in the case of hydrogen atoms (Table II).
The main difference in the 1 H NMR spectrum of 1 and 2 consisted in the signal for H-7, which had changed from a broad singlet at d H 4.49 of 1 to a double doublets at d H 3.64 (J = 10.0 and 4.2 Hz) of 2, indicating diaxial (J = 10.0 Hz) interaction with H-8. The 2D COSY NMR experiment also revealed spin systems compatible with the presence of the HO 6 CH-5 CH= 4 CH-and -O-8 CH-7 CH(OH)-6 CHOH in 2, that were confirmed by 2D HSQC NMR experiment by cross peaks corresponding to heteronuclear interactions involving CH-6 (d C 68.7/d H 4.40), CH-5 (d C 139.8/d H 6.42) and CH-4 (d C 123.4/d H 6.70) and CH-8 (d C 83.5/d H 5.23) and CH-7 (d C 74.5/d H 3.64), summarized in Table II.
The relative stereochemistry of 2 was deduced from their mutual coupling constants of relevant hydrogens and from dipolar interaction revealed by 1 H-1 H NOESY. Unlike 1 was not observed signal nOe between H-6 (d H 4.40) and H-8 (d H 5.23) hydrogen of 2. The diaxial spin-spin interaction between H-7 (d H 3.64) and H-8 (d H 5.23) was de-  .5, 1.9) a Number of hydrogens bound to carbon atoms deduced by comparative analysis of the { 1 H}-and DEPT-13 C NMR spectra. Chemical shifts and coupling constants (J) obtained from 1D 1 H NMR spectrum. The HSQC, HMBC and 1 H-1 H-COSY spectra were also used to 1 H and 13 C chemical shift assignments.