Enhancement of the Water Solubility of Flavone Glycosides by

Dec 18, 2012 - especially diosmin, a highly insoluble citroflavonoid prescribed as an oral phlebotropic drug. Disruption of planarity at the aglycone ...
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Enhancement of the Water Solubility of Flavone Glycosides by Disruption of Molecular Planarity of the Aglycone Moiety Guy Lewin,*,† Alexandre Maciuk,† Aurélien Moncomble,‡ and Jean-Paul Cornard‡ †

Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Paris-Sud BIOCIS UMR-8076 CNRS LabEx LERMIT, Avenue J.B. Clément, 92296 Châtenay-Malabry Cedex, France ‡ Laboratoire de Spectrochimie IR et Raman, Université Lille 1 UMR-8516 CNRS, 59655 Villeneuve d’Ascq Cedex, France S Supporting Information *

ABSTRACT: Enhancement of the water solubility by disruption of molecular planarity has recently been reviewed as a feasible approach in small-molecule drug discovery programs. We applied this strategy to some natural flavone glycosides, especially diosmin, a highly insoluble citroflavonoid prescribed as an oral phlebotropic drug. Disruption of planarity at the aglycone moiety by 3-bromination or chlorination afforded 3-bromo- and 3-chlorodiosmin, displaying a dramatic solubility increase compared with the parent compound.

T

he classical approach used to increase water solubility of a drug consists of introducing hydrophilic groups, which results in a concomitant decrease of hydrophobicity, measured by a smaller log P value. However, lowering of oral bioavailability is often observed due to a decrease of membrane permeability related to smaller hydrophobicity. Recently, Ishikawa and Hashimoto reported the improvement in water solubility in small-molecule drug discovery programs by disruption of molecular planarity and symmetry.1 The authors reviewed such an alternative strategy for improving water solubility that does not affect hydrophobicity. One of the examples dealt with a series of aryl hydrocarbon receptor agonists with a naphthoflavone structure. β-Naphthoflavone (1) contains an aryl moiety at C-2 that can rotate around the C-2− C-1′ bond. It was postulated that substitution at C-2′ and/or C6′ would increase the dihedral angle of this bond to explain the enhanced water solubility of compounds 2, 3, and 4.

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RESULTS AND DISCUSSION Diosmin (5) is a citroflavonoid, prepared industrially by dehydrogenation of the corresponding flavanone, hesperidin (6), and is prescribed in some European countries as an oral phlebotropic drug in the treatment of chronic venous insufficiency and hemorrhoidal disease. In a previous study related to Suzuki reactions at C-3 of flavones, we used diosmin to prepare 3-bromodiosmetin (9) via intermediate octacetyldiosmin (7) and its 3-bromo derivative 8 by a three-step sequence of peracetylation in Ac2O−pyridine, 48 h, rt, 3bromination using N-bromosuccinimide (NBS) in dichloromethane (DCM)−pyridine (5:1), 20 h, rt, and concomitant acid hydrolysis of the glycosidic bond and acetyl esters in aqueous HCl, 11 N, 2 h, 55 °C.5 Performing this last step in THF−NaOH(aq), 1 N, 3 h, rt, afforded 3-bromodiosmin (10)

Though water solubility of the four compounds remained weak, compounds 2−4 were 3 to 15 times more soluble than 1. This property was correlated with an increase of the dihedral angle, calculated to be between 17.8° and 70° for 1−4,2 and to a lowering of the melting point via a decrease of the crystal packing energy.3 Moreover, 2−4 displayed increased hydrophobicity compared to 1, as shown by calculated log P (clogP)4 and retention time data on reversed-phase HPLC. Herein, we report a significant enhancement of water solubility of some semisynthetic derivatives of natural flavonoids, especially diosmin (5). © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 2, 2012

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in 82% yield (from 8) by cleavage of only ester bonds. Bromination was also performed in DCM−MeOH (2:1) using 2 equiv of NBS (4 h, rt).6 Under these conditions, compound 11 was obtained in 90% yield as a 43:57 mixture of two diastereoisomeric 3-bromo-2-methoxyflavanones as established by 1H NMR spectroscopy. Similar alkaline hydrolysis of 11 gave 10 in 85% yield by concomitant cleavage of ester bonds and β-elimination of MeOH from the heterocycle. Unexpectedly, a high water solubility was observed for compound 10, while diosmin is known to be highly insoluble. This observation also prompted the preparation of other 3-halogenated analogues from diosmin. The 3-chloro derivative 13 was obtained in the same manner as 10, by replacing NBS with Nchlorosuccinimide (NCS) in the second step [both conditions used for bromination were tested for the halogenation step, but chlorination, leading to 12, was effective only in DCM− pyridine (5:1) (5 equiv of NCS, 80 h, rt)]. 3-Chlorodiosmin (13) was approximately as soluble in water as 10.

3-bromorhoifolin (19) was readily achieved only via the 3bromo-2-methoxyflavanone (18) (direct bromination to the 3bromoflavone in DCM−pyridine was ineffective). As expected from results in the diosmin series, bromo analogues 17 and 19 appeared to possess high water solubility compared to their respective parent compounds. It is noteworthy that similar enhancement of water solubility was not observed in the aglycone series: comparison of the water-insoluble aglycones of diosmin, linarin, and rhoifolin, namely, diosmetin (20), acacetin (21), and apigenin (22), with their respective 3-halo derivatives 9, 23, 26, and 27 did not show any appreciable increase in water solubility. In order to compare physicochemical parameters of the five 3-substituted flavone glycosides, 10, 13, 14, 17, and 19, with those of 3-unsubstituted compounds 5, 15, and 16, we carried out accurate measurements of water solubility, log P, and retention time by RP-HPLC experiments (Table 1). Reversed phase retention time can be correlated to lipophilicity and biological distribution.11 Table 1. Physicochemical Parameters of Diosmin (5), Linarin (15), Rhoifolin (16), and Their 3-Substituted Derivatives compd 5 10 13 14 15 17

We could not study the influence of a 3-fluoro or 3-iodo substituent on water solubility, as efforts to synthesize the required analogues from octacetyldiosmin (7) with Nfluorobenzenesulfonimide7 and iodine−cerium(IV) ammonium nitrate,8 respectively, failed. However, we obtained 3-hydroxydiosmin (14) with the Algar−Flynn−Oyamada (AFO) reaction9 from hesperidin (6), according to the method of Pacheco et al.,10 but 14 was not more water-soluble than diosmin (5). As 3-bromination was easier to carry out than 3-chlorination in the diosmin series, we applied the same functionalization to linarin (3′-deoxydiosmin) (15) and rhoifolin (apigenin 7neohesperidoside) (16), two other very weakly soluble natural 7-O-flavone glycosides, previously obtained from diosmin and naringin, respectively.5 3-Bromolinarin (17) was prepared from 15 by any of the above bromination methods, while synthesis of

16 19 a

water solubility (g/L)a

log P

HPLC retention time (min)b

Diosmin (Diosmetin) Series 0.0012 −0.10 214 +0.61 231 +0.44 0.0056 +0.06 Linarin (Acacetin) Series 0.0046 +0.84 13.8 +1.17 Rhoifolin (Apigenin) Series 0.114 +0.71 >100 +0.93

18.58 20.87 20.57 13.31 36.95 51.51 12.57 18.65

Measured at 20 ± 2 °C. bSee Experimental Section.

The results confirm the dramatic improvement in water solubility observed for 3-bromo and chloro derivatives, especially in the diosmin series with a factor of about 105. Moreover these 3-halo derivatives possess increased hydrophobicity compared with their corresponding parent glycoside, as demonstrated by log P and retention time values. On the contrary, the 3-hydroxy derivative 14 remains almost insoluble in water in spite of a reduced hydrophobicity. To investigate the influence of structure on solubility, quantum chemical calculations were carried out at the DFT B

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level of theory. However, flavone glycosides are conformationally flexible compounds that present many rotatable groups, leading to an excessive number of conformers. Since optimizations of these products require excessive amounts of time, structural analysis of the stable conformations were made with aglycones for the following reasons: (i) the nature of the two glycosyl units (rutinosyl or neohesperidosyl) did not affect the observed trends, (ii) the γ-pyrone-B-ring moiety is suspected to mainly explain the water solubility increase, and (iii) for the derivatives of a parent compound, the glycosyl unit could be considered as a constant modification located away from this γ-pyrone-B-ring moiety. Consequently, the aglycones 20−22 and their 3-substituted derivatives 9 and 23−27 were used as models for diosmin (5), linarin (15), rhoifolin (16), and the synthesized 3-substituted glycosides 10, 13, 14, 17, and 19, respectively (Table 2). First, geometry optimizations were

hydroxy group, is weaker (2.00 Å), due to the strong geometry constraints. To find which major factor (steric hindrance or electronic effects) explains the increase of the dihedral angle in the halogenated compounds, we studied the hypothetical 3mercaptodiosmetin (25) on account of some interesting characteristics of the sulfhydryl functional group for this comparison: first, a size similar to chlorine (van der Waals radii for chlorine and sulfur are 1.75 and 1.80 Å, respectively) and, second, an ability to form hydrogen bonds and electronic characteristics quite close to that of a hydroxy group. Optimization of the structure of 25 clearly shows a similarity with 3-chlorodiosmetin (23): the dihedral angle is 37.6° and the hydrogen bond with the C-5 hydroxy group is 1.72 Å. As this result allowed us to propose that the steric hindrance is the main explanation for the increase of the dihedral angle, we then turned to study the correlation between structure and solubility. Solubility is known to be linked to the energy difference between the solid phase and the liquid phase. This energy difference between a reoptimized planar structure (that allows packing effects in the solid phase) and the fully optimized one (more probable in solution) can be referred as ΔEplan = Eplanar − Eoptimized.14 As expected, a large dihedral angle in the optimized structure of the aglycone (9, 23, 26, 27) leads to a large ΔEplan (Table 2), which is connected to a high water solubility of the corresponding 3-haloflavone glycoside (10, 13, 17, 19): in this case, the rotation from the planar structure to a large dihedral angle releases a large amount of energy during solvation that is linked to better solubility. Inversely, a smaller dihedral angle in the optimized structure of the aglycone (20−22, 24) leads to a smaller ΔEplan, which is connected to low water solubility of the corresponding flavone glycoside (10, 13, 17, 19). Lastly, the slight improvement in water solubility of 3-hydroxydiosmin (14) in relation to diosmin, despite a calculated planar structure of its aglycone 24, may result from the effect of the 3-hydroxy group, moderated, however, by the presence of an additional intramolecular hydrogen bond. In conclusion, this study illustrates the Ishikawa and Hashimoto strategy, which consists in disrupting molecular planarity within a chemical series with the aim of improving water solubility. Our reported results are noteworthy since particularly impressive enhancements in water solubility, in the 105 range, were noted. Such strong enhancement results from the glycosidic nature of the starting compounds, 5, 15, and 16, which are almost insoluble in water (especially diosmin) in spite of a large hydrophilic glycosidic moiety bearing six hydroxy groups. Disruption of the planarity of the aglycone moiety by halogenation at C-3 allows the recovery of high water solubility, more in agreement with the glycosidic nature of the compounds. Therefore, these results unambiguously prove the close relationship of the value of the dihedral angle between the chromone moiety and the B ring, and consequently the conformation of the aglycone part of these glycosides, to the water solubility. Lastly, comparison of 10, 13, and 14, differing in the nature of the C-3 substituent, demonstrates that steric hindrance at C-3 is more significant than electronic factors to increase water solubility: high water solubility is therefore found with halogenated compounds 10 and 13, which are however more lipophilic than diosmin (according to measured log P and retention times in HPLC), while substitution by the hydroxy group leads to 14, which is more polar but only slightly more soluble than diosmin.

Table 2. Selected Geometrical Parameters for Computed Optimized Structures of Aglycones: Properties of their Planar Structures compd

inter-ring angle (deg)

20 9 23 24 25

20.5 44.9 42.6 0.0 37.6

21 26

19.6 44.6

22 27

19.4 44.9

a

H bonds lengths (Å)

ΔEplan (kcal·mol−1)

Diosmetin (Diosmin) Series 1.71 0.18 1.72 2.39 1.72 1.88 1.77; 2.00 0.00 1.72; 2.03 1.41 Acacetin (Linarin) Series 1.71 0.11 1.72 2.34 Apigenin (rhoifolin) series 1.71 0.15 1.72 2.42

nature of the planar structure TSa TS TS minimum TS TS TS TS TS

Transition state connecting the two symmetrical twisted forms.

conducted for model compounds 20−22. As expected, the energy minima structures are not planar.12 The dihedral angle between the chromone moiety and the B ring is close to 20° in the three cases. However, these values are significantly lower than the analogous ca. 45° dihedral angle calculated for biphenyl.13 This difference can be explained by better electron delocalization in flavone, enhanced by the para-substitution of the B ring. A hydrogen bond is present between the C-5 hydroxy group and the C-4 carbonyl group in the three compounds, the O---H distance being 1.71 Å in each case. Geometry optimizations were then carried out for structures of the four 3-haloflavones 9, 23, 26, and 27: while all structures still display the hydrogen bond without any significant change, a strong effect of the 3-substituent on the dihedral angle is observed with calculated values close to 45° for brominated compounds 9, 26, and 27 and 42.6° for 3-chlorodiosmetin (23). A different result was obtained with 3-hydroxydiosmetin (24), for which a planar structure (Cs symmetry) was deduced from computation. For 24, when fixing the angle between the chromone part and the B ring to 20.5° (as in diosmetin), a slightly higher energy (0.2 kcal·mol−1) relative to the planar structure was calculated. This planar structure displays a carbonyl group that is involved in two hydrogen bonds: the first one, with the C-5 hydroxy group, is longer (1.77 Å) than in previous studied aglycones; the second one, with the C-3 C

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Evaporation under vacuum of the remaining n-BuOH as an azeotropic mixture with H2O gave 10 as a pale yellow powder (0.56 g, 82%). Compound 10: [α]D = −53.5 (c 0.2, H2O); UV (EtOH) λmax nm (log ε) 258 (4.39), 274 sh (4.36), 345 (4.16); 1H NMR (DMSO-d6) δ [aglycone moiety] 3.87 (3H, s, OCH3-4′), 6.53 (1H, d, J = 2 Hz, H-6), 6.71 (1H, d, J = 2 Hz, H-8), 7.15 (1H, d, J = 8.5 Hz, H-5′), 7.32−7.33 (2H, m, H-2′ and 6′), 9.5 (1H, br s, OH-3′), 12.4 (1H, s, OH-5); [sugar moiety: inner glucose (″) and terminal rhamnose (‴)] 1.08 (3H, d, J = 6.4 Hz, H-6‴), 3.15 (2H, m, H-4″ and H-4‴), 3.28 (2H, m, H-2″ and H-3″), 3.44 (3H, m, 1H-6″, H-3‴ and H-5‴), 3.61 (1H, m, H-5″), 3.65 (1H, m, H-2‴), 3.81 (1H, m, 1H-6″), 4.54 (1H, s, H-1‴), 5.09 (1H, d, J = 7.2 Hz, H-1″), 4.54, 4.61, 4.70, 5.19 (×2), 5.43 (6H, sugar hydroxyls); 13C NMR (DMSO-d6) δ [aglycone moiety] 55.7 (CH3, OCH3-4′), 94.4 (CH, C-8), 99.9 (CH, C-6), 104.2 (C, C-10), 105.0 (C, C-3), 111.7 (CH, C-5′), 116.2 (CH-2′), 121.5 (CH-6′), 124.0 (C, C-1′), 145.9 (C, C-3′), 150.4 (C, C-4′), 156.5 (C, C-9), 160.5 (C, C-5), 162.7 (C, C-7), 163.1 (C, C-2), 176.7 (C, C-4); [sugar moiety]: inner glucose 66.0 (CH2, C-6″), 69.6 (CH, C-4″), 73.0a (CH, C-2″), 75.6 (CH, C-5″), 76.2a (CH, C-3″), 99.6 (CH, C-1″); terminal rhamnose 17.7 (CH3, C-6‴), 68.2 (CH, C-5‴), 70.2a (CH, C-2‴), 70.7a (CH, C-3‴), 72.0 (CH, C-4‴), 100.5 (CH, C-1‴), ainterchangeable; ESIMS m/z 709−711 [M + Na]+; anal. C 46.69, H 4.77%, calcd for C28H31O15Br (5% H2O), C 46.59, H 4.86%. 3-Chlorooctacetyldiosmin (12). Chlorination of 7 (1.42 g, 1.5 mmol) to compound 12 was performed under the same conditions as previously described for bromination to 8, by using NCS instead of NBS. Chlorination, slower than bromination (80 instead of 20 h), afforded 12 as a yellowish, amorphous powder (1.29 g, 88% yield). Compound 12: 1H NMR (CDCl3) δ [aglycone moiety] 2.33 and 2.44 (6H, 2s, OAc-5 and 3′), 3.91 (3H, s, OMe-4′), 6.69 (1H, d, J = 2 Hz, H-6), 6.89 (1H, d, J = 2 Hz, H-8), 7.08 (1H, d, J = 8.8 Hz, H-5′), 7.58 (1H, d, J = 2.2 Hz, H-2′), 7.77 (1H, dd, J = 8.8 and 2.2 Hz, H-6′); [sugar moiety: inner glucose (″) and terminal rhamnose (‴)] 1.13 (3H, d, J = 6.4 Hz, H-6‴), 1.92−2.06 (18H, 6s, 6 sugar acetyls), 3.66 (1H, H-6″), 3.80 (2H, H-6″and H-5‴), 3.94 (1H, H-5″), 4.67 (1H, s, H-1‴), 4.99 (1H, H-4‴), 5.13−5.28 (5H, H-2″, 3″, 4″, 2‴, and 3‴), 5.27 (1H, H-1″). 3-Chlorodiosmin (13). Saponification of 12 (0.98 g, 1 mmol) to give 13 as a yellow powder (0.565 g, 88%) was done under the same conditions as for the hydrolysis of 8 to 10. Compound 13: [α]D = −53.9 (c 0.2, H2O); UV (EtOH) λmax nm (log ε) 257 (4.33), 271 sh (4.30), 347 (4.14); 1H NMR (DMSO-d6) δ [aglycone moiety] 3.87 (3H, s, OCH3-4′), 6.52 (1H, d, J = 2 Hz, H-6), 6.73 (1H, d, J = 2 Hz, H-8), 7.16 (1H, d, J = 8.5 Hz, H-5′), 7.39−7.40 (2H, m, H-2′ and 6′), 9.5 (1H, br s, OH-3′), 12.3 (1H, s, OH-5); [sugar moiety: inner glucose (″) and terminal rhamnose (‴)] 1.08 (3H, d, J = 6.4 Hz, H6‴), 3.15 (2H, m, H-4″ and H-4‴), 3.28 (2H, m, H-2″ and H-3″), 3.44 (3H, m, 1H-6″, H-3‴ and H-5‴), 3.61 (1H, m, H-5″), 3.65 (1H, m, H-2‴), 3.82 (1H, m, 1H-6″), 4.54 (1H, s, H-1‴), 5.10 (1H, d, J = 7.2 Hz, H-1″), 4.54, 4.61, 4.70, 5.20 (×2), 5.44 (6H, sugar hydroxyls); 13 C NMR (DMSO-d6) δ [aglycone moiety] 55.7 (CH3, OCH3-4′), 94.6 (CH, C-8), 100.0 (CH, C-6), 104.6 (C, C-10), 111.9 (CH, C-5′), 113.6 (C, C-3), 116.0 (CH-2′), 121.5 (CH-6′), 122.6 (C, C-1′), 146.1(C, C-3′), 150.6 (C, C-4′), 156.4 (C, C-9), 160.5 (C, C-5), 161.3 (C, C-2), 163.2 (C, C-7), 176.3 (C, C-4); [sugar moiety]: inner glucose 66.0 (CH2, C-6″), 69.6 (CH, C-4″), 73.0a (CH, C-2″), 75.6 (CH, C-5″), 76.2a (CH, C-3″), 99.7 (CH, C-1″); terminal rhamnose 17.7 (CH3, C-6‴), 68.3 (CH, C-5‴), 70.3a (CH, C-2‴), 70.7a (CH, C3‴), 72.0 (CH, C-4‴), 100.5 (CH, C-1‴), ainterchangeable; ESIMS m/z 665−667 [M + Na]+; anal. C 50.14, H 5.05%, calcd for C28H31O15Cl (4% H2O), C 50.29, H 5.10%. 3-Hydroxydiosmin (14). Aqueous 30% H2O2 (1 mL) was added to a cooled (0 °C) solution of 6 (1 g, 1.64 mmol) in aqueous 2 N NaOH (20 mL). After 24 h at 0 °C, a further aliquot of aqueous H2O2 30% (1 mL) was added and the reaction left for 24 h at 0 °C. The mixture was adjusted to pH 6 with HOAc and kept again for 24 h at 0 °C. Sodium metabisulfite (2.4 g) was added, and the mixture was heated and stirred under reflux for 2 h. The yellow precipitate was recovered by filtration, washed with H2O, and dried with P2O5 under vacuum to afford crude 7-O-rutinosyltamarixetin (14), which was

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Optical Activity PolAAr 32 polarimeter. NMR spectra, including NOESY and 1H−13C (HMQC and HMBC) experiments, were recorded on Bruker AC-300 (300 MHz) or Bruker AM-400 (400 MHz) spectrometers. ESIMS were recorded on an Agilent HP 1100 MSD spectrometer (ESI source). HRMS were obtained from the ILV-UVSQ on a Xevo-Q Tof Waters. HPLC analyses were performed on a Waters system equipped with a 600E controller, a 717 autosampler, and a 996 PDA detector (retention times) or an Agilent HP 1260 Infinity system (solubilities and log P), using a Waters Spherisorb ODS1 5 μm, 4.6 × 100 mm column. Physicochemical Measurements. Chromatographic retention times were measured under isocratic elution using a mobile phase consisting of H2O−MeOH (60:40). Measurements of purities and concentrations for solubilities and log P were done under gradient elution using H2O−MeOH, from 10% to 100% MeOH in 10 min. Detection and integration were performed at wavelengths close to the λmax of the compounds (255 nm for 10, 13, and 14, 267 nm for 17 and 19, 284 nm for 6, and 340 nm for 5, 15, and 16). Quantification was done using calibration curves obtained with compounds dissolved in DMSO. Solubilities were measured following published guidelines.15 Briefly, for poorly soluble compounds, 200 μL of deionized H2O was added to 2 mg of the compound, whereas for highly soluble compounds, 100 μL of deionized H2O was added to 30 mg. Vials were vortexed and left to settle for 2 days, then centrifuged for 15 min at 4000 rpm to allow injection of 1 or 10 μL of supernatant for HPLC analysis. Log P values were determined by adding 500 μL of H2O and 500 μL of octanol to 0.5 mg of compound, which was briefly warmed to 40 °C, vortexed, and left to settle for 1 day. Then 20 μL of each phase was injected in HPLC. Log P determination measurements were done in duplicates. Computational Details. All calculations (for diosmetin, acacetin, apigenin, and their derivatives) were carried out at the DFT level using the Gaussian 09 package.16 The PBE0 exchange−correlation functional was used throughout.17,18 All atoms were described using the 6311+G(d) Pople basis set.19−22 The 6-311++G(d,p) basis set was also used in the case of 3-chlorodiosmetin and 3-hydroxydiosmetin to check the limitation due to the size of the basis set. No significant change in optimized structures was observed, so the smallest basis set was used in the study. Structures were optimized under vacuum without any symmetry constraint. The nature of the stationary points (minima and transition states) was assigned by vibrational analysis (no and one negative eigenvalue in the Hessian matrix, respectively). Planar structures were optimized fixing only the inter-ring dihedral angle to 0° without any symmetry constraint. 3-Bromooctacetyldiosmin (8). Compound 8 was prepared from diosmin (5) in a two-step sequence via octacetyldiosmin (7), as previously described (85% from diosmin).5 3-Bromo-2-methoxyoctacetylhesperidin (11). NBS (0.72 g, 4 mmol) was added to a solution of 7 (1.89 g, 2 mmol) in CH2Cl2− MeOH (2:1) (50 mL). After 4 h at rt, the reaction mixture was taken up in CH2Cl2 and washed with 0.1 M Na2S2O3 (60 mL) and H2O (2 × 80 mL). Standard workup of the organic phase led to a mixture of two diastereoisomers of 11 as a pale yellow, amorphous powder (1.90 g, 90%). Compound 11: 1H NMR (CDCl3) characteristic signals at δ 1.12 and 1.15 (3H, 2d, J = 6.2 Hz, H-6‴ rhamnosyl), 2.33 and 2.38 (6H, 2s, OAc-5 and 3′), 3.07 (3H, s, OMe-2), 3.88 (3H, s, OMe-4′), 4.19 and 4.20 (1H, 2s, H-3), 4.69 and 4.73 (1H, 2s, H-1‴ rhamnosyl), 6.43 and 6.44 (1H, 2d, J = 2.3 Hz, H-6), 6.64 and 6.65 (1H, 2d, J = 2.3 Hz, H-8), 7.04 (1H, d, J = 8.5 Hz, H-5′), 7.29 (1H, d, J = 2.4 Hz, H2′), 7.42 (1H, dd, J = 8.5 and 2.4 Hz, H-6′); ESIMS (+) m/z 1077− 1079 [M + Na]+, 997 [M + Na − HBr]+. 3-Bromodiosmin (10). NaOH, 1 N (50 mL), was added to a solution of 8 (1.02 g, 1 mmol) in THF (30 mL); then the reaction was stirred for 3 h at room temperature. The mixture was diluted with H2O (300 mL), adjusted to pH 6 with 5 N HCl, and then extracted with nBuOH (2 × 100 mL). The organic phase was evaporated to a yellow dried residue, which was dissolved in deionized H2O (30 mL). D

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purified by successive precipitations from MeOH (192 mg, 19%). Compound 14: 1H NMR (DMSO-d6) characteristic signals at δ 3.87 (3H, s, OCH3-4′), 6.52 (1H, d, J = 2 Hz, H-6), 6.73 (1H, d, J = 2 Hz, H-8), 7.16 (1H, d, J = 8.5 Hz, H-5′), 7.39−7.40 (2H, m, H-2′ and 6′), 9.5 (1H, br s, OH-3′), 12.3 (1H, s, OH-5); ESIMS m/z 647 [M + Na]+. 3-Bromolinarin (17). 17 was prepared from linarin (15) (60 mg, 0.1 mmol) under the same conditions as described for 3bromodiosmin (yellow powder, 41 mg, 70%). Compound 17: [α]D = −45.9 (c 0.2, H2O); 1H NMR (DMSO-d6) characteristic signals at δ 1.08 (3H, d, J = 6.4 Hz, H-6‴), 3.86 (3H, s, OCH3-4′), 4.55 (1H, s, H1‴), 5.08 (1H, d, J = 7.2 Hz, H-1″), 6.51 (1H, d, J = 2 Hz, H-6), 6.70 (1H, d, J = 2 Hz, H-8), 7.16 (2H, d, J = 8.5 Hz, H-3′ and 5′), 7.87 (2H, d, J = 8.5 Hz, H-2′ and 6); 13C NMR (DMSO-d6) δ [aglycone moiety] 55.6 (CH3, OCH3-4′), 94.3 (CH, C-8), 99.6a (CH, C-6), 104.2 (C, C-10), 105.3 (C, C-3), 113.9 (CH, C-3′ and C-5′), 124.0 (C, C-1′), 131.4 (CH, C-2′ and C-6′), 156.7 (C, C-9), 161.6 (C, C-5), 162.7b (C, C-4′), 163.2b (C, C-2 and C-7), 176.6 (C, C-4); [sugar moiety]: inner glucose 66.1 (CH2, C-6″), 69.8c (CH, C-4″), 73.0c (CH, C-2″), 75.8 (CH, C-5″), 76.3c (CH, C-3″), 100.2a (CH, C-1″); terminal rhamnose 17.8 (CH3, C-6‴), 68.3 (CH, C-5‴), 70.3 (CH, C2‴), 70.8 (CH, C-3‴), 72.1 (CH, C-4‴), 100.6 (CH, C-1‴), a,b,c interchangeable; ESIMS m/z 693−695 [M + Na]+. 3-Bromo-2-methoxyoctacetylnaringin (18). The mixture of two diastereoisomers (49:51) was prepared from octacetylrhoifolin (100 mg, 0.11 mmol) under conditions described for 3-bromo-2methoxyoctacetylhesperidin (11), but with a longer reaction time (24 h) and was obtained as a pale yellow powder, 87 mg, 77%. Compound 18: 1H NMR (CDCl3) characteristic signals at δ 1.19 and 1.24 (3H, 2d, J = 6.4 Hz, H-6‴ rhamnosyl), 2.35 and 2.40 (6H, 2s, OAc-5 and 3′), 3.09 and 3.14 (3H, 2s, OMe-2), 4.24 and 4.25 (1H, 2s, H-3), 6.48 and 6.49 (1H, 2d, J = 2 Hz, H-6), 6.69 and 6.70 (1H, 2d, J = 2 Hz, H8), 7.23 and 7.26 (2H, d, J = 8.8 Hz, H-3′ and H-5′), 7.62 and 7.66 (2H, 2d, J = 8.8 Hz, H-2′ and H-6′). 3-Bromorhoifolin (19). Compound 19 was prepared from 18 (77 mg, 0.075 mmol) under the same conditions as described for 3bromodiosmin (yellow powder, 38 mg, 77%). Compound 19: 1H NMR (DMSO-d6) δ [aglycone moiety] 6.42 (1H, d, J = 1.8 Hz, H-6), 6.71 (1H, d, J = 1.8 Hz, H-8), 6.95 (2H, d, J = 8.7 Hz, H- 3′ and 5′), 7.74 (2H, d, J = 8.7 Hz, H-2′ and 6′); [characteristic peaks of the sugar moiety: inner glucose (″) and terminal rhamnose (‴)] 1.18 (3H, d, J = 6.2 Hz, H-6‴), 5.13 (1H, s, H-1‴), 5.23 (1H, d, J = 7.2 Hz, H-1″); 13C NMR (DMSO-d6) δ [aglycone moiety] 94.3 (CH, C-8), 99.7 (CH, C6), 104.2 (C, C-10), 104.7 (C, C-3), 115.1 (CH, C-3′ and C-5′), 122.2 (C, C-1′), 131.4 (CH, C-2′ and 6′), 156.6 (C, C-9), 160.4 (C, C-5 and C-4′), 162.8 (C, C-2 and C-7), 176.7 (C, C-4); [sugar moiety]: inner glucose 60.4 (CH2, C-6″), 70.4a (CH, C-4″), 76.2b (CH, C-2″), 77.0b (CH, C-3″), 77.0b (CH, C-5″), 97.7 (CH, C-1″); terminal rhamnose 18.0 (CH3, C-6‴), 68.3 (CH, C-5‴), 69.6a (CH, C-2‴), 70.3a (CH, C3‴), 71.8 (CH, C-4‴), 100.4 (CH, C-1‴), ainterchangeable, binterchangeable; ESIMS m/z 679−681 [M + Na]+.

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ACKNOWLEDGMENTS We thank J.-C. Jullian for NMR spectra registration, K. Leblanc for ESIMS spectra and elemental analysis, and A. Pearson for English corrections of the manuscript.

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REFERENCES

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S Supporting Information *

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H and 13C NMR spectra of diosmin (5) and compounds 10, 13, 17, 19; 1H NMR spectrum of epimeric mixture 11; HRMS data of 10, 13, 17, 19. This material is available free of charge via the Internet at http://pubs.acs.org.

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dx.doi.org/10.1021/np300460a | J. Nat. Prod. XXXX, XXX, XXX−XXX