Journal of Natural Products
Article
USA) equipped with a dual-wavelength detector model 2487 adjusted
at 210 and 254 nm. Samples were dried using a Savant Speed Vac Plus
SC210A concentrator. The compounds were visualized by spraying the
TLC plates with 1% vanillin−H2SO4 spray reagent. The reference
standard doxorubicin (purity ≥98.0% pure) was purchased from
Sigma-Aldrich (St. Louis, MO, USA).
Plant Material. The leaves of E. rigida were collected in Guanica,
Puerto Rico, in March 2006. The sample was identified by Mr. F.
Axelrod, and a voucher specimen (MOBOT 3008783) was deposited
in the Herbarium of the Missouri Botanical Garden, St. Louis, MO,
USA.
75:25−0:100 for 20 min, to give compounds 1a (43 mg, 25%) and 3a
(15 mg, 9%).
(2S)-6-Formyl-5,7-dihydroxyflavanone (1a): colorless needles
(CHCl3); mp 160−162 °C; [α]D −31 (c 0.5, MeOH); ECD
(MeOH) λ (θ) 223 (4.44 × 103), 274 (−1.76 × 103), 303 (1.02 ×
1
103) nm; H and 13C NMR spectra in agreement with compound 1;
HRESIMS m/z 285.083 [M + H]+ (calcd for C16H13O5, 285.0685).
(2S)-8-Formyl-5,7-dihydroxyflavanone (3a): colorless needles
1
(CHCl3); mp 160−162 °C; [α]D −31 (c 0.5, MeOH); H and 13C
NMR spectra in agreement with the isolated compound 3; HRESIMS,
m/z 285.0628 [M + H]+ (calcd for C16H13O5, 285.0685).
Synthesis of Compounds 1b and 3b. Application of the same
formylation and separation procedures using 13b28 afforded 1b (43
mg, 25%) and 3b (15 mg, 9%).
Extraction and Isolation. The dried and pulverized leaves of E.
rigida (107 g) were macerated in n-hexane and subsequently extracted
with CH2Cl2 (3 × 600 mL, sonicated for 2 h and then left overnight).
The combined n-hexane and CH2Cl2 extracts were filtered separately
and dried (yields 2.50 and 3.87 g, respectively). A portion of the n-
hexane extract (2 g) was subjected to CPTLC, using a 6 mm custom-
made RP-C18 silica gel disk.34 The sample was dissolved in CH3CN,
and applied on a presaturated (30% MeOH−H2O) disk on a
Chromatotron apparatus, and, after sample application, the disk was
kept in a desiccator. The semidried disk was mounted on the
Chromatotron and was eluted with a step gradient of H2O−MeOH
(from 30% to 0% H2O). Fractions of 60 mL each were collected and
monitored by TLC (silica gel, solvent: toluene−EtOAc, 9:1). Fractions
1−11, eluted with 30% H2O−MeOH, were combined, evaporated to
dryness (560 mg), and subjected to another CPTLC run, using a 4
mm silica gel disk (Analtech). Elution was carried out with n-hexane−
EtOAc mixtures with increasing polarity to afford compounds 1 (3
mg), 3 (3 mg), and 4 (1 mg). A portion of the CH2Cl2 extract (2 g)
was subjected to fractionation on an SPE cartridge filled with 50 g of
C18-silica gel and eluted with a step gradient of H2O−MeOH (from
100% to 0% H2O in MeOH). Fractions of 100 mL each were collected
and monitored by TLC (silica gel, solvent: n-hexane−EtOAc, 7.5:2.5).
Fraction 5 (20 mg), eluted with 70% MeOH−H2O, was chromato-
graphed over preparative HPLC using the gradient mobile phase n-
hexane−EtOAc of increasing polarities to yield 5 (2 mg). Fractions 11
and 12, eluted with 75% MeOH−H2O, were combined and dried (60
mg), then subjected to CPTLC, using a 1 mm silica gel disk, eluted
with CHCl3, to yield 15 subfractions. Subfraction 14 was subjected to
purification by preparative HPLC to afford compound 2 (3 mg).
Fractions 19−22, eluted with 75% MeOH−H2O, were combined,
dried (80 mg), and then subjected to CPTLC, using a 2 mm silica gel
disk, eluted with CHCl3, to yield 16 subfractions. Subfraction 8
afforded compound 6 (1 mg), while subfraction 13 was subjected to
further purification by preparative HPLC to afford compound 7 (5
mg).
(2R)-6-Formyl-5,7-dihydroxyflavanone (1b): colorless needles
(CHCl3); mp 160−162 °C; [α]D +31 (c 0.5, MeOH); ECD
(MeOH) λ (θ) 223 (−7.07 × 103), 274 (2.83 × 103), 303 (−2.26
1
× 103) nm; H and 13C NMR spectra in agreement with the isolated
compound 1; HRESIMS m/z 285.0637 [M + H]+ (calcd for
C16H13O5, 285.0685).
(2R)-8-Formyl 5,7-dihydroxyflavanone (3b): colorless needles
(CHCl3); mp 160−162 °C; [α]D +31 (c 0.5, MeOH); ECD
(MeOH) λ (θ) 223 (−8.27 × 103), 276 (2.27 × 104), 332 (−2.36
1
× 103) nm; H and 13C NMR data in agreement with compound 3;
HRESIMS m/z 285.0637 [M + H]+ (calcd for C16H13O5, 285.0685).
Synthesis of Compounds 8 and 9. Application of the same
formylation and separation procedures using 14 afforded 8 (40 mg,
24%) and 9 (15 mg, 9%).
6-Formyl-5,7-dihydroxyflavone (8): yellow needles; mp 144−146
°C; 1H and 13C NMR data (Table 3); HRESIMS m/z 283.0566 [M +
H]+ (calcd for C16H11O5, 283.0528).
8-Formyl-5,7-dihydroxyflavone (9): yellow needles; mp 144−146
°C; 1H and 13C NMR data (Table 3); HRESIMS m/z 283.0534 [M +
H]+ (calcd for C16H11O5, 283.0528).
Synthesis of 2′,4′,6′-Trihydroxychalcone (10). A solution of
compound 13 (256 mg, 1 mmol) in EtOH (10 mL) was added
dropwise to a stirred solution of 4 M KOH (10 mL) at 0 °C under Ar,
and the reaction mixture was kept at room temperature for 30 min.
The mixture was poured into ice water (10 mL), acidified with 1 M
HCl to pH 3−4, and extracted with EtOAc (3 × 10 mL). The organic
layer was washed with H2O, dried over anhydrous Na2SO4, and
evaporated to dryness The residue was purified by silica gel column
chromatography (n-hexane−EtOAc, 80:20) to give compound 10
(141 mg, 55%) as a yellow, microcrystalline powder; mp 142−143 °C;
HRESIMS m/z 257.0778 [M + H]+ (calcd for C15H13O4, 257.0769).
Synthesis of 3′-Formyl-2′,4′,6′-trihydroxychalcone (11).
Formylation of 10 by the aforementioned procedure yielded 11,
which was purified by silica gel chromatography (n-hexane−EtOAc,
90:10) to afford a yellow powder (30 mg, 34%): 1H NMR (400 MHz,
methanol-d4) δH 10.08 (1H, s, CHO), 8.14 (1H, d, H-α, J = 16.0 Hz),
7.80 (1H, d, H-β, J = 16.0 Hz), 7.66 (2H, m, H-2, H-6), 7.42 (3H, m,
H-3, H-4, H-5), 5.86 (1H, s, H-5′); HRESIMS m/z 285.0646 [M +
H]+ (calcd for C16H13O5, 285.0685).
Synthesis of 2′,4′,6′-Trihydroxydihydrochalcone (12). A
mixture of compound 14 (508 mg, 2 mmol) and 10% Pd−C (1 g)
in EtOH (150 mL) was hydrogenated at 30 atm, for 2 h, at 60 °C.
After filtration, the solvent was evaporated under reduced pressure,
and the residue was chromatographed over silica gel (n-hexane−
EtOAc, 80:20) to afford compounds 9 (230 mg, 45%) and 12 (180
mg, 35%) as yellow, amorphous powders: 1H NMR (400 MHz,
methanol-d4) δH 7.23−7.12 (5H, m, Ar−H), 5.82 (2H, d, H-3′ and H-
5′, J = 1.6 Hz), 3.30 (2H, t, H-α, J = 7.5 Hz), 2.92 (2H, t, H-β, J = 7.5
Hz); HRESIMS m/z 259.0899 [M + H]+ (calcd for C15H15O4,
259.0926).
rac-6-Formyl-5,7-dihydroxyflavanone (1): colorless needles
(CHCl3); mp 160−162 °C; [α]D 0 (c 0.5, MeOH); UV (MeOH)
λmax (log ε) 205 (4.3), 267 (4.5), 331 (3.6) nm; IR νmax (KBr) 2920,
1
1661 cm−1; H and 13C NMR data, Table 1; HRESIMS (+ve) m/z
285.0637 [M + H]+ (calcd for C16H13O5, 285.0685).
2′,6′-Dihydroxy-4′-methoxy-3′-methylchalcone (2): yellow, amor-
1
phous powder (CHCl3); UV (MeOH) λmax (log ε) 325 (4.1) nm; H
and 13C NMR spectral data, see Table 2; HRESIMS (+ve) m/z
285.0667 [M + H]+ (calcd for C16H13O5, 285.0695).
4′,6′-Dihydroxy-2′-methoxy-3′-methyldihydrochalcone (4): yel-
low, microcrystalline powder (CHCl3); mp 205−207 °C; IR νmax
1
(KBr) 2921, 2360, 1661 cm−1; H and 13C NMR data, Table 2.
Synthesis of Compounds 1a and 3a. To a solution of 13a28
(150 mg, 0.59 mmol) and TiCl4 (160 μL, 1.46 mmol) in dry CH2Cl2
(10 mL) at −78 °C was added dropwise dichloromethyl methyl ether
(58 μL, 0.65 mmol) with stirring under N2. After 2 h, the reaction
mixture was warmed to room temperature over 1 h. Aqueous H2SO4
acid (5%, 20 mL) was added to the reaction mixture, and the organic
phase was separated. The aqueous phase was extracted with ether (3 ×
20 mL), the combined organic phase was dried over MgSO4, and the
solvent was removed in vacuo. The residue was chromatographed by
preparative HPLC on silica gel using a mobile phase of n-hexane (A)
and EtOAc (B) in a gradient mode: A:B 100−75:25 for 35 min,
Synthesis of 3′-Formyl-2′,4′,6′-trihydroxydihydrochalcone
(7). Formylation of 12 by the procedure described above followed by
purification by silica gel chromatography (n-hexane−EtOAc, 90:10)
1
yielded 7 (58 mg, 35%) as a yellow powder: H NMR (400 MHz,
methanol-d4) δH 10.09 (1H, s, CHO), 7.63 (5H, m, Ar−H), 5.85 (1H,
G
J. Nat. Prod. XXXX, XXX, XXX−XXX