Microtropins JP : 6 ’-O-( 2 ” S , 3 ” R )-2 ”-Ethyl-2 ” , 3 ”-Dihydroxybutyrates of Phenolic Alcohol-D-Glucopyranosides from the Branches of Microtropis japonica

From the branches of Microtropis japonica (Celastraceae), seven phenolic alcohol glucosides, named microtropins J-P (1-7), were isolated. The 6-position of glucose was esterified with 2-ethyl-2,3-dihydroxybutyric acid. Microtropin K (2) was hydrolyzed under a mild basic condition to give methyl (2S,3R)-2-ethyl-2,3-dihydroxybutyrate, whose absolute structure was determined by the comparison of NMR data and the optical rotation value with that reported.


Results and Discussion
Seven new 2-ethyl-2,3-dihydroxybutyrates of various phenolic glucosides, named microtropins J-P (1-7), were isolated from the 1-BuOH-soluble fraction of a MeOH extract of the branches of M. japonica by a combination of various separation procedures.Their structures were elucidated from spectroscopic evidence.Microtropin J (1), [] D 24 -59.8, was isolated as an amorphous powder and its elemental composition was determined to be C 19 H 26 O 11 by high-resolution (HR)-electrospray ionization (ESI) mass spectrometry (MS).The IR spectrum exhibited absorption bands assignable to hydroxy (3380 cm -1 ), ester carbonyl (1730 cm -1 ), carboxylic acid (1705 cm -1 ), aromatic ring (1606, and 1511 cm -1 ), phenolic alcohol (1239 cm -1 ), and aliphatic alcohols (1073 cm -1 ).The UV absorption band at 245 nm also supported the presence of an aromatic ring.In the 13 C-NMR spectra, six signals assignable to 2-ethyl-2,3-dihydroxybutyrate were observed together with ones assignable to glucose, which had a substituent at the 6-position.The 1H-NMR spectrum of the aglycone moiety comprised seven signals, which included one for a para-substituted aromatic ring with a carboxyl functional group.Acid hydrolysis liberated Dglucose, which was identified by HPLC analysis with a chiral detector.In the heteronuclear multiple bond correlation spectrum (HMBC), the anomeric proton (δ H 5.03) showed a correlation peak with C-4 (δ C 162.7), and H-6' (δ H 4.21 and 4.65) with the carbonyl signal at δ C 176.2.Therefore, the structure of microtropin J (1) was elucidated to be p-hydroxybenzoic acid O--D-glucopyranoside 6'-O-2"-ethyl-2",3"-dihydroxybutyrate, as shown in Figure 1.The absolute configuration of the acyl moiety was expected to be the same (2"S,3"R) as that of microtropin A [4].
Microtropin K (2), [] D 24 -50.6, was isolated as an amorphous powder and its elemental composition was determined to be C 20 H 28 O 12 by HR-ESI-MS.The IR and UV spectra showed similar absorption bands to those of 1, and in the NMR spectra, a methoxy signal [δ H 3.90 (3H, s) on δ C 56.8] was observed.The AA'BB' type coupled four protons observed in 1 were replaced by three aromatic protons coupled in an ABX system.In the HMBC spectrum, one of the aromatic protons [H-2, δ H 7.625 (s)] showed correlation peaks with C-1 (δ C 126.4), C-4 (δ C 151.8), and C-7 (δ C 169.5), and the anomeric proton (δ H 5.03) with C-4.From the above evidence, the structure of aglycone was determined to be vanillic acid and the overall structure is shown as 2 in Figure 1.Since microtropin K (2) was isolated in a good quantity, it was hydrolyzed under a mild alkaline condition to give vanllic acid -D-glucopyranoside (2a) [5] and methyl 2ethyl-(2S,3R)-dihydroxybutyrate (2b) [4].
Microtropin L (3), [] D 23 -31.6, was isolated an amorphous powder and its elemental composition was determined to be C 21 H 30 O 12 by HR-ESI-MS.Spectroscopic data were almost superimposable on those of 2, except for the presence of an ester methoxy group (δ H 3.89 on δ C 52.6), and the molecular weight was 14 mass units larger than that of 2, which accounted for a labile hydrogen atom being replaced by a methyl group.Therefore, the structure of 3 was elucidated to be ester of 2, and it may be an artifact produced during the extraction and isolation procedures.
Microtropin M (4), [] D 25 -96.7, was isolated as an amorphous powder and its elemental composition was determined to be C 20 H 30 O 12 by HR-ESI-MS.The IR spectrum exhibited a strong absorption band for a carbonyl functional group and two methoxy signals were observed in the NMR spectra (δ H 3.73 on δ C 61.1 and 3.80 on δ C 56.5).Judging from the NMR data, the aromatic ring had two meta-coupled protons [δ H 6.27 (d, J = 2.7 Hz) and 6.32 (d, J = 2.7 Hz)] and thus was suggested to have unsymmetrically substituted benzene, that is, four electro-negative functional groups are substituted at the 1, 3, 4 and 5 positions.The relatively deshielded methoxy signal (δ C 61.1) was implied that it was located between the substituents.The HMBC correlations from H-1' (δ H 4.77) to δ C 155.6 (C-1), those from H-2 and H-6 to δ C 133.6, to which the methoxy signal at δ H 3.73 on δ C 61.1 also correlated, and those from H-2 and H-6 to C-6 and C-2 respectively were observed (Figure 2).Therefore, the structure of aglycone was expected to be 4,5-dimethoxybenzene-1,3-diol 1-O-β-D-glucopyranoside, which was also supported by the NOESY spectrum in which the anomeric proton showed significant correlation cross peaks with H-2 and H-6.The structure was finally elucidated to be 4 shown in Figure 1.
Figure 2.Microtropin N (5), [] D 25 -11.5, was isolated as an amorphous powder and its elemental composition was determined to be C 19 H 28 O 11 .In the NMR spectra, one methoxy carbon and three protons coupled in an ABX system were observed in the aglycone region.Other NMR spectral data for the aglycone were essentially the same as those of isotachioside isolated from Isotachis japonica [6].Therefore, the structure of 5 was elucidated to be isotachioside 6'-O-(2"S,3"R)-2"-ethyl-2",3"-dihydroxybutyrate, as shown in one methoxy signal and three protons couple in an ABX system, one doublet methyl and two oxymethines were observed.The methyl protons were coupled with one of the oxymethine protons and the two oxymethine protons were coupled with each other.Thus, the structure of the aglycone moiety was expected to be 4-hydroxy-2-methoxy or 4-hydroxy-3-methoxyphenylpropane-7,8-diol.In the HMBC spectrum, H-2 and 6 (δ H 7.03 and 6.87, respectively) showed correlation peaks with C-7 (δ C 79.9) as well as C-4, to which also the anomeric proton (δ H 4.87) was correlated.The key correlation from H-5 (δ H 7.10) to C-3 (δ C 151.0) established the structure of the aglycone to be 4-hydroxy-3-methoxy-phenylpropane-7,8-diol.Therefore, the structure of 6 was elucidated to be as shown in Figure 1.From spectroscopic data, microtropin P ( 7) was expected to be a similar compound to 6.Although the 13 C-NMR data of both compounds were almost superimposable (Table 1), they were separated in the same HPLC runs and exhibited significantly different retention times.The stereochemistry of both side chains has not fully been clarified yet, but, judging from the fairly large coupling constants of H-7 and H-8 (6.7 Hz for 6 and 6.9 Hz for 7) in the 1 H-NMR, their aglycones are in a threo-form, accordingly the stereochemistry of the diol-part must be enantiomers, each other [7].

General Experimental Procedures
Optical rotations were measured on a JASCO P-1030 digital polarimeter.IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively.stationary and mobile phases, respectively.Five-gram fractions were collected and numbered according to their order of elution with the mobile phase.HPLC was performed on an ODS column (Inertsil; ODS-3, GL Science, Tokyo, Japan; Φ = 6 mm, L = 250 mm, 1.6 mL/min), and the eluate was monitored with a UV detector at 254 nm, and a refractive index monitor.

Extraction and Isolation
Air-dried branches of M. japonica (13.0 kg) were extracted three times with MeOH (30 L × 3) at room temperature for one week and then concentrated to 3 L in vacuo.The concentrated extract was washed with n-hexane (3 L, 53.8 g), and then the MeOH layer was concentrated to a gummy mass.The latter was suspended in water (3 L) and then extracted with EtOAc (3 L) to give 103 g of an EtOAc-soluble fraction.The aqueous layer was extracted with 1-BuOH (3 L) to give a 1-BuOH-soluble fraction (40.9 g), and the remaining water-layer was concentrated to furnish 107 g of a water-soluble fraction.The 1-BuOH-soluble fraction (39.9 g) was sub- The residue (1.17 g out of 1.27 g) in fractions 39 -47 obtained on silica gel CC was separated by ODS open CC and the residue (351 mg) in fractions 121 -144 was applied to DCCC.The residue (86.6 mg out of 279 mg) in fractions 30 -46 was purified by HPLC (H 2 O-MeOH, 4:1) to yield 14.2 mg of 1 and 21.0 mg of 2 from the peaks at 50 min and 65 min, respectively.