Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2015.09.042

To explore the structure-activity relationships on the inhibitory activity of flavanonols against nitric oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common 3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to investigate the structure-activity relationship. We also succeeded in isolating stereoisomers from 16 of the flavanonols using chiral column chromatography. The inhibitory effects of these compounds on NO production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were activated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in (2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker. We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of NO production by flavanonols is stereospecific and cannot simply be explained by their radical scavenging activity. We propose the possible existence of a 'target' molecule for flavanonols which is involved in the production and/or regulation of NO in RAW 264.7 cells.

Full text of DOI:10.1016/j.bmc.2015.09.042

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Stereospecific inhibition of nitric oxide production in macrophage
cells by flavanonols: Synthesis and the structure–activity relationship
Wen-Jun Jiang a, Kan’ichiro Ishiuchi a,b, Megumi Furukawa a, Tomoko Takamiya a, Susumu Kitanaka a,
Hiroshi Iijima a,
⇑
a School of Pharmacy, Nihon University, 7-7-1 Narashino-dai, Funabashi-shi, Chiba 274-8555, Japan
b Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya-shi, Aichi 467-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore the structure–activity relationships on the inhibitory activity of flavanonols against nitric
oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common
3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to inves-
tigate the structure–activity relationship. We also succeeded in isolating stereoisomers from 16 of the fla-
vanonols using chiral column chromatography. The inhibitory effects of these compounds on NO
production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were acti-
vated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in
(2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker.
We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhy-
drazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The
radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of
NO production by flavanonols is stereospecific and cannot simply be explained by their radical scaveng-
ing activity. We propose the possible existence of a ‘target’ molecule for flavanonols which is involved in
the production and/or regulation of NO in RAW 264.7 cells.
Received 13 August 2015
Revised 25 September 2015
Accepted 26 September 2015
Available online xxxx
Keywords:
Flavonoid
Synthesis
Stereoisomer
Nitric oxide
Anti-oxidation
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
production of NO by macrophages contributes to numerous
pathological processes. In particular, when NO reacts with super-
Flavonoids are widely distributed in plants. Humans ingest
them from fruits, vegetables, teas, dietary supplements and other
sources. Some flavonoids are known to show anti-allergic,1 anti-in-
flammatory,1–4 anti-microbial,5–7 anti-viral,8–10 anti-cancer,2,11
anti-diarrheal,12 antitumor,13–15 anti-diabetic,16 and antioxi-
dant12,17–20 activities. The anti-inflammatory and antioxidant func-
tions of flavonoids from many plants have often reported. These
two activities are the most important and versatile functions of
flavonoids.
oxide anion radicals, it produces peroxynitrite anions, which are
strongly reactive. Aside from this direct action, the production of
NO is one of the primary indicators of macrophage activation.
Therefore, compounds that suppress NO production might have
therapeutic value.
Oxidative stress arises from disturbances in the balance
between the production of reactive oxygen species (ROS) and the
antioxidant defenses. ROS can be beneficial as a means of attacking
pathogens;21 however, excessive ROS activity, which may occur
when the balance of the ROS and antioxidant potential is disturbed,
has a self-destructive effect. Animals ingest antioxidants through
many foods, including fruits and vegetables. Recent research2
shows that flavonoids are the predominant source of antioxidants
in the animal body.
In the inflammation processes, the expression of cytokines or
mediators such as tumor necrosis factor-
interleukin-6, as well as nitric oxide synthase are induced in
immune cells by lipopolysaccharide (LPS) and interferon- . They
a, interleukin-1b and
c
play a critical causative role in rheumatoid arthritis, asthma, and
atherosclerosis. Nitric oxide (NO) plays an important physiological
role as a defense molecule in the immune system, while the excess
Numerous structure–activity relationship studies have
investigated the NO production inhibitory activity22,23 and the
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activ-
ity24,25 of flavonoids. In the present study, we aimed to synthesize
flavanonols with an identical chroman scaffold in their A and C
⇑
Corresponding author. Tel./fax: +81 47 465 6470.
E-mail address: iijima.hiroshi@nihon-u.ac.jp (H. Iijima).
http://dx.doi.org/10.1016/j.bmc.2015.09.042
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042

2
W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
medium (Sigma Aldrich, N4888) containing 200 mM L-glutamate
The structure and activity of the flavanonols
(Sigma Aldrich, G7513), penicillin (100 U/mL)—streptomycin
(0.1 mg/mL) (Sigma Aldrich, P4333) and immobilized fetal bovine
serum (10 v/v %) (Biowest, S1780). One hundred fifty microliters
of cell suspension (1.6 ꢀ 106 cell/mL culture medium) was dis-
pensed in a well of a 96-well plate (Sumitomo Bakelite, #8096R)
and 40 lL of test compound solution was added. The test com-
pound solution was prepared by diluting the DMSO solution of
the flavanonols by a ratio of 1:100 with culture medium. The cells
were incubated for 2 h at 37 °C in a CO2 incubator. Cells adhered to
the culture well during this process. Ten microliters of LPS (Sigma
Aldrich, #L-2880) solution was then added to each well. The final
concentration of LPS was 100 ng/mL. After 16 h of incubation,
100 lL of supernatant medium was transferred to another plate.
The remaining cells were submitted to a cell viability test as
described in Section 2.4. Fifty microliters of sulfanilamide solution
Compound Substitution in the B
ring
NO-production
inhibitory % at
DPPH radical
scavenging EC50
100
lM
(lM)
(2R,3R) (2S,3S) (2R,3R) (2S,3S)
AG
Gallic acid
72
10–12
5
None
<10
40
35
<10
<10
70
<10
<15
<15
<10
<10
45
>100
>100
>100
>100
>100
12.5
>100
>100
>100
>100
>100
13.5
5a
5b
5c
5d
5e
5f
5g
5h
5i
20-OH
30-OH
40-OH
(50 mg of sulfanilamide dissolved in a mixture of 250
phoric acid and 5 mL of water) was added to each well. A few min-
utes later, 50 L of 0.1% N-1-naphthylethylnediamine (Wako Pure
lL of phos-
20,60-OH
20,30-OH
20,40-OH
20,50-OH
30,40-OH
30,50-OH
20-OCH3
l
Chemical Inc., 147-04141) solution was added and incubated at
room temperature in the dark for 10 min. Absorbance at 540 nm
(reference wavelength: 655 nm) was then measured using a micro-
plate reader (BioRad Model 3550). Aminoguanidine hydrochloride
(Wako Pure Chemical Inc., 328-26432) was used as a positive con-
trol. The concentrations of the test compounds were precisely
determined from ultraviolet absorption at kmax using a UV spec-
trometer (JASCO V-730).
<10
30
<10
<10
Racemic
<10
<15
<10
<10
>100
24.5
>100
25.4
13.9
12.6
>100
Racemic
>100
5j
mixture <10
mixture >100
5k
5l
5m
30-OCH3
40-OCH3
20,60-OCH3
<10
<10
<10
<10
>100
>100
>100
>100
Racemic
mixture <10
Racemic
mixture >100
5n
5o
5p
5q
20,30-OCH3
20,40-OCH3
20,50-OCH3
30,40-OCH3
<10
<10
<10
Racemic
mixture <10
<10 <10
<10
<10
<10
>100
>100
>100
Racemic
mixture >100
>100 >100
>100
>100
>100
2.4. Cell viability test
Cell viability was measured using AlamarBlueÒ reagent (Bio-
Rad AbD Serotec Ltd.). Ten microliters of AlamarBlueÒ solution
was added to the RAW 264.7 cells left in each well of the 96-well
plate from the above-mentioned NO assay, which was then incu-
bated at 37 °C for 4 h. Absorbance was measured at 570 nm (refer-
ence wavelength: 655 nm).
5r
30,50-OCH3
rings but with alterations to the structure of their B ring and in
their stereochemistry (Table 1). The synthesized flavanonols were
used to evaluate the effects of the structure–activity relationship
on the inhibition of NO production in RAW 264.7 cells and on DPPH
radical scavenging ability.
2.5. DPPH radical scavenging assay27
One hundred twenty microliters of ethanol-buffer solution
(ethanol-0.5 M sodium acetate buffer pH 5.6, 105:15) was added
to each well of a 96-well plate (Sumitomo Bakelite, #8096R). Forty
microliters of compound solution (in ethanol) was added. Next,
2. Material and methods
2.1. General experimental procedures
40 lL of DPPH solution (1,1-diphenyl-2-picrylhydrazyl, Tokyo
Optical rotations were measured using
a
JASCO P-1020
Chemical Industry. D4313, 0.5 mM ethanol solution) was added.
The plate was shaken on a shaker for 1 min and kept in the dark
at room temperature for 30 min. Absorbance was measured at
517 nm (reference wavelength: 655 nm). Gallic acid monohydrate
(Wako Pure Chemical Inc., 077-06092) was used as the standard
polarimeter. The 1H NMR (600 MHz) and 13C NMR (150 MHz) spec-
tra were recorded on a JEOL JNM-ECX600 spectrometer. Mass spec-
tra were obtained on a JEOL GCmate mass spectrometer. IR spectra
were recorded on JASCO FT/IR-4200 spectrometer. UV spectra were
recorded on JASCO V-730 spectrometer. CD spectra were recorded
on JASCO J-600 spectrometer. Melting points were determined by
using AS-ONE ATM-02.
compound (EC50 = 10–12 lM).
2.6. Synthesis of 2,4,6-trimomoacetophenone 1
2.2. The separation of enantiomers by a chiral column
NaH (4.5 equiv) in dry DMF was slowly added while stirring at
0–5 °C (in an ice-water bath) to a solution of 2,4,6-trihydroxyace-
tophenone (1 equiv) in dry DMF. When the solution was cooled
to 0–5 °C, chloromethyl methyl ether (4.5 equiv) was slowly added
over a period of 15 min so that the temperature was maintained at
less than 5 °C. The reaction mixture was stirred at room tempera-
ture for another 30 min, quenched by the addition of cold distilled
water and extracted with EtOAc. The combined organic layer was
washed with distilled water and brine and then dried over Na2SO4.
The filtered organic layer was concentrated under a vacuum and
the residue was purified by silica gel column chromatography elut-
ing with n-hexane and EtOAc to give compound 1: a colorless oil
(80–95% yield).
All of the synthesized flavanonols were further submitted to
purification by a chiral column (DAICEL, CHIRALPAK, IA, 5 lm,
10 / mm ꢀ 250 mm) to isolate the enantiomers using a high per-
formance liquid chromatography system (JASCO PU-1580, UV-
1575). The elution solvent was ethanol–hexane and the flow rate
was 5 mL/min. Detection wavelength was 254 nm.
2.3. NO assay
The amount of released nitrite (NO) was quantified by the
Griess method26. RAW 264.7 cells were cultured in F-12 Ham
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W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
1H NMR (600 MHz, CDCl3), d: 2.47 (3H, s, C(@O)CH3), 3.44 (6H, s,
OCH3-2, OCH3-6), 3.45 (3H, s, OCH3-4), 5.12 (6H, d, J = 2.4 Hz,
OCH2-2, OCH2-4, OCH2-6), 6.49 (2H, s, H-3, H-5). 13C NMR
(150 MHz, CDCl3), d: 32.5 (C(@O)CH3), 56.1 (OCH3-4), 56.3
(OCH3-2, OCH3-6), 94.4 (OCH2-4), 94.7 (OCH2-2, OCH2-6), 97.0
(C-3, C-5), 116.8 (C-1), 155.1 (C-2, C-6), 159.4 (C-4), 201.4 (C@O).
HR-EI-MS: m/z 300.1209 [M]+ (Calcd 300.1209 for C14H20O7).
Separation by chiral chromatography: EtOH–n-hexane (20:80 v/
v) Retention time (2R,3R)-isomer: 9.08 min, (2S,3S)-isomer:
7.75 min.
23
(2R,3R)-isomer: [
a]
+12.8° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ5.66 (287.6),+2.03 (327.2) (c = 7.35 ꢀ 10ꢁ5). (2S,3S)-isomer:
23
[a
]
ꢁ12.2° (c = 1.0, MeOH), CD (MeOH):
De (nm)+5.21 (289.0),
D
ꢁ1.94 (326.0) (c = 7.35 ꢀ 10ꢁ5).
2.7. Synthesis of MOMO protection benzaldehyde 2
2.10.2. 20,3,5,7-Tetrahydroxy flavanonol (5a)
1H NMR (600 MHz, acetone-d6) d: 4.89 (1H, d, J = 12.0 Hz, H-3),
5.62 (1H, d, J = 12.0 Hz, H-2), 5.96 (1H, d, J = 2.4 Hz, H-6), 6.00 (1H,
d, J = 2.4 Hz, H-8), 6.45 (1H, dd, J = 8.4, 8.4 Hz, H-50), 6.93 (1H, d,
J = 8.4 Hz, H-30), 7.24 (1H, ddd, J = 8.4, 8.4, 2.4 Hz, H-40), 7.53 (1H,
dd, J = 8.4, 2.4 Hz, H-60). 13C NMR (150 MHz, acetone-d6) d: 72.4
(C-3), 79.2 (C-2), 96.1 (C-6), 97.1 (C-8), 101.7 (C-10), 116.9 (C-50),
120.6 (C-30), 124.3 (C-10), 129.7 (C-60), 130.8 (40-C), 156.8 (C-20),
164.5 (C-5), 165.2 (C-9), 167.9 (C-7), 198.4 (C-4). HR-EI-MS: m/z
288.0632 [M]+ (Calcd 288.0634 for C15H12O6).
K2CO3 (10 equiv) was added with stirring at 0–5 °C (ice-water
bath) to a solution of hydroxylbenzaldehyde (1 equiv) in dry ace-
tone, When the solution was cooled to 0–5 °C chloromethyl methyl
ether (1.5 equiv; dependent on the number of the hydroxyl groups)
was slowly added over a period of 15 min to keep the temperature
under 5 °C. The reaction mixture was stirred at room temperature
for another 30 min, quenched by the addition of cold distilled
water and extracted with EtOAc. The combined organic layer was
washed with distilled water and brine and then dried over Na2SO4.
The filtered organic layer was concentrated under a vacuum and
the residue was purified by silica gel column chromatography elut-
ing with n-hexane and EtOAc to give compound 2: a colorless or
light yellow oil (80–95% yield).
Separation by chiral chromatography: EtOH–n-hexane (20:80 v/
v) Retention time (2R,3R)-isomer: 9.67 min, (2S,3S)-isomer:
7.88 min.
12
(2R,3R)-isomer: [
a
]
+80.1° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ2.98 (284.9), +1.67 (325.8) (c = 6.94 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ72.2° (c = 1.0, MeOH), CD (MeOH):
De (nm) +3.02 (284.1),
2.8. Synthesis of chalcone 3
ꢁ1.70 (324.1) (c = 6.94 ꢀ 10ꢁ5).
KOH (3 equiv) ethanol solution was added to a solution of 1
(1 equiv) in EtOH. Then 2 (1 equiv) was added to the reaction mix-
ture solution and stirred at room temperature for 3 h. Distilled
water was added and extracted with EtOAc and the combined
organic layer was washed with distilled water and brine and dried
over Na2SO4. The filtered organic layer was concentrated under a
vacuum and the residue was purified by silica gel column chro-
matography eluting with n-hexane and EtOAc to give 3: a light yel-
low oil (60–90% yield).
2.10.3. 3,30,5,7-Tetrahydroxy flavanonol (5b)
1H NMR (600 MHz, acetone-d6) d: 4.63 (1H, d, J = 11.4 Hz, H-3),
5.12 (1H, d, J = 11.4 Hz, H-2), 5.98 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H,
d, J = 1.8 Hz, H-8), 6.89 (1H, dd, J = 8.4, 3.0 Hz, H-40), 7.06 (1H, d,
J = 8.4 Hz, H-60), 7.07 (1H, d, J = 3.0 Hz, H-20), 7.25 (1H, dd, J = 8.4,
8.4 Hz, H-50). 13C NMR (150 MHz, acetone-d6) d: 73.3 (C-3), 84.4
(C-2), 96.2 (C-6), 97.3 (C-8), 101.6 (C-10), 115.8 (C-20), 116.6 (C-
40), 120.0 (C-60), 130.3 (C-50), 139.8 (C-10), 158.3 (C-30), 164.1 (C-
5), 165.1 (C-9), 167.9 (C-7), 198.0 (C-4). HR-EI-MS: m/z 288.0632
[M]+ (Calcd 288.0634 for C15H12O6).
2.9. Synthesis of epoxide 4
Separation by chiral chromatography: EtOH–n-hexane (50:50 v/
v) Retention time (2R,3R)-isomer: 5.30 min, (2S,3S)-isomer:
H2O2 (30%) and aqueous 2 M NaOH were added to a methanol
solution of chalcone 3, and the mixture was stirred for 3 h at room
temperature. Methanol was removed under a vacuum. Distilled
water was added to the resultant aqueous suspension and
extracted with EtOAc. The combined organic layer was dried over
Na2SO4. The organic layer was concentrated under a vacuum to
give compound 4: a colorless oil (85–95% yield; in the cases of
4m and 4o, the yield was 50–60%).
3.88 min.
12
(2R,3R)-isomer: [
a
]
+9.4° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ5.19 (289.5), +1.57 (327.4) (c = 6.94 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ7.3° (c = 1.0, MeOH), CD (MeOH):
De (nm) +5.36 (291.1),
ꢁ1.80 (326.7) (c = 6.94 ꢀ 10ꢁ5).
2.10.4. 3,40,5,7-Tetrahydroxy flavanonol (5c)
1H NMR (600 MHz, acetone-d6) d: 4.66 (1H, d, J = 11.4 Hz, H-3),
5.09 (1H, d, J = 11.4 Hz, H-2), 5.95 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H,
d, J = 1.8 Hz, H-8), 6.90 (2H, d, J = 8.4 Hz, H-30, H-50), 7.42 (d, 2H,
J = 8.4 Hz, H-20, H-60). 13C NMR (150 MHz, acetone-d6) d: 73.2 (C-
3), 84.4 (C-2), 96.1 (C-6), 97.2 (C-8), 101.6 (C-10), 116.0 (C-30, C-
50), 129.2 (C-10), 130.4 (C-20, C-60), 158.9 (C-40), 164.3 (C-5), 165.1
(C-9), 167.9 (C-7), 198.4 (C-4). HR-EI-MS: m/z 288.0627 [M]+ (Calcd
288.0634 for C15H12O6).
2.10. Synthesis of flavanonols
Epoxide 4 was dissolved in HCl-MeOH (1 M), and stirred at
55 °C for 25 min. MeOH was removed under a vacuum. Distilled
water was added to the residue and the mixture was extracted
with EtOAc. The organic layer was combined and dried over Na2-
SO4. The filtered EtOAc was evaporated to give red-yellow oil
which was purified by silica gel column chromatography eluting
with n-hexane and EtOAc to give 5, and 5a–5r (30–55% yield).
Separation by chiral chromatography: EtOH–n-hexane (30:70 v/
v) Retention time (2R,3R)-isomer: 7.55 min, (2S,3S)-isomer:
6.10 min.
12
(2R,3R)-isomer: [
a]
+23.3° (c = 1.0, MeOH), CD (MeOH): De
D
2.10.1. 3,5,7-Trihydroxy flavanonol (5)
(nm) ꢁ6.48 (289.4), +1.77 (327.4) (c = 6.94 ꢀ 10ꢁ5). (2S,3S)-isomer:
1H NMR (600 MHz, acetone-d6), d: 4.68 (1H, d, J = 12.0 Hz, H-3),
5.19 (1H, d, J = 12.0 Hz, H-2), 5.98 (1H, d, J = 2.4 Hz, H-6), 6.01 (1H,
d, J = 2.4 Hz, H-8), 7.44 (1H, m, H-40), 7.44 (2H, m, H-30, H-50), 7.63
(2H, m, H-20, H-60). 13C NMR (150 MHz, acetone-d6), d: 73.3 (C-3),
84.5 (C-2), 96.2 (C-6), 97.3 (C-8), 101.6 (C-10), 128.9 (C-20, C-60),
129.2 (C-30, C-50), 129.7 (C-40), 138.4 (C-10), 164.1 (C-5), 165.1 (C-
9), 168.0 (C-7), 198.1 (C-4). HR-EI-MS: m/z 272.0684 [M]+ (Calcd
272.0685 for C15H12O5).
[a
]
D
ꢁ20.5° (c = 1.0, MeOH), CD (MeOH):
De (nm) +6.75 (289.7),
12
ꢁ1.94 (326.7) (c = 6.94 ꢀ 10ꢁ5).
2.10.5. 20,3,5,60,7-Pentahydroxy flavanonol (5d)
1H NMR (600 MHz, acetone-d6) d: 5.52 (1H, d, J = 12.0 Hz, H-3),
5.81 (1H, d, J = 12.0 Hz, H-2), 5.92 (1H, d, J = 2.4 Hz, H-6), 5.96 (1H,
d, J = 2.4 Hz, H-8), 6.46 (2H, d, J = 8.4 Hz, H-30, H-50), 7.03 (1H, t,
J = 8.4 Hz, H-40). 13C NMR (150 MHz, acetone-d6) d: 70.4 (C-3),
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042

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W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
76.9 (C-2), 95.9 (C-8), 96.8 (C-6), 101.7 (C-10), 108.3 (C-30, C-50),
110.7 (C-10), 131.2 (C-40), 158.9 (C-20, C-60), 165.2 (C-9), 165.3 (C-
5), 167.6 (C-7), 199.8 (C-4). HR-EI-MS: m/z 304.0582 [M]+ (Calcd
304.0583 for C15H12O7).
Separation by chiral chromatography: EtOH–n-hexane (20:80 v/v)
Retention time (2R,3R)-isomer: 9.80 min, (2S,3S)-isomer:
2.10.9. 3,30,40,5,7-Pentahydroxy flavanonol (5h)
1H NMR (600 MHz, acetone-d6) d: 4.61 (1H, d, J = 11.4 Hz, H-3),
5.02 (1H, d, J = 11.4 Hz, H-2), 5.95 (1H, d, J = 1.8 Hz, H-6), 5.99 (1H,
d, J = 1.8 Hz, H-8), 6.86 (1H, d, J = 8.4 Hz, H-50), 6.92 (1H, dd, J = 8.4,
1.8 Hz, H-60), 7.07 (1H, d, J = 1.8 Hz, H-20). 13C NMR (150 MHz, ace-
tone-d6) d: 73.2 (C-3), 84.6 (C-2), 96.1 (C-6), 97.1 (C-8), 101.6 (C-
10), 115.8 (C-50), 116.0 (C-20), 120.9 (C-60), 129.9 (C-10), 145.8 (C-
30), 146.7 (C-40), 164.2 (C-5), 165.1 (C-9), 167.9 (C-7), 198.3 (C-4).
HR-EI-MS: m/z 304.0584 [M]+ (Calcd 304.0583 for C15H12O7).
Separation by chiral chromatography: EtOH–n-hexane (50:50 v/
v) Retention time (2R,3R)-isomer: 5.1 min, (2S,3S)-isomer:
10.18 min.
12
(2R,3R)-isomer: [
a
]
+102.2° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ0.50 (298.6), +1.69 (327.4) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ97.3° (c = 1.0, MeOH), CD (MeOH):
De (nm) +0.45 (297.8),
ꢁ1.63 (324.2) (c = 6.58 ꢀ 10ꢁ5).
4.25 min.
12
(2R,3R)-isomer: [
a]
+17.8° (c = 1.0, MeOH), CD (MeOH): De
D
2.10.6. 20,3,30,5,7-Pentahydroxy flavanonol (5e)
(nm) ꢁ6.95 (294.2), +1.93 (327.2) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
1H NMR (600 MHz, acetone-d6) d: 4.86 (1H, d, J = 11.4 Hz, H-3),
5.61 (1H, d, J = 11.4 Hz, H-2), 5.96 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H,
d, J = 1.8 Hz, H-8), 6.78 (1H, dd, J = 7.8, 7.8 Hz, H-50), 6.89 (1H, dd,
J = 7.8, 1.8 Hz, H-40), 7.03 (1H, dd, J = 7.8, 1.8 Hz, H-60). 13C NMR
(150 MHz, acetone-d6) d: 72.5 (C-3), 79.2 (C-2), 96.1 (C-6), 97.1
(C-8), 101.7 (C-10), 116.2 (C-40), 120.2 (C-60), 120.4 (C-50), 124.6
(C-10), 145.2 (C-20), 146.0 (C-30), 164.5 (C-5), 165.2 (C-9), 167.9
(C-7), 198.3 (C-4). HR-EI-MS: m/z 304.0583 [M]+ (Calcd 304.0583
for C15H12O7).
12
[a]
ꢁ15.7° (c = 1.0, MeOH), CD (MeOH):
De (nm) +7.15 (294.1),
D
ꢁ1.95 (329.9) (c = 6.58 ꢀ 10ꢁ5).
2.10.10. 3,30,5,50,7-Pentahydroxy flavanonol (5i)
1H NMR (600 MHz, acetone-d6) d: 4.57 (1H, d, J = 11.4 Hz, H-3),
5.02 (1H, d, J = 11.4 Hz, H-2), 5.97 (1H, d, J = 1.8 Hz, H-6), 5.99 (1H,
d, J = 1.8 Hz, H-8), 6.38 (1H, t, J = 1.8 Hz, H-40), 6.56 (2H, d,
J = 1.8 Hz, H-20, H-60). 13C NMR (150 MHz, acetone-d6) d: 73.3 (C-
3), 84.6 (C-2), 96.1 (C-6), 97.2 (C-8), 101.6 (C-10), 103.8 (C-40),
107.4 (C-20, C-60), 140.4 (C-10), 159.4 (C-30, C-50), 164.1 (C-5),
165.1 (C-9), 168.0 (C-7), 198.0 (C-4). HR-EI-MS: m/z 304.0580
[M]+ (Calcd 304.0583 for C15H12O7).
Separation by chiral chromatography: EtOH–n-hexane (40:60 v/
v) Retention time (2R,3R)-isomer: 5.83 min, (2S,3S)-isomer:
5.03 min.
12
(2R,3R)-isomer: [
a
]
+71.5° (c = 1.0, MeOH), CD (MeOH): De
D
Separation by chiral chromatography: EtOH–n-hexane (40:60 v/
v) Retention time (2R,3R)-isomer: 6.50 min, (2S,3S)-isomer:
(nm) ꢁ3.12 (283.0), +1.48 (325.0) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ64.6° (c = 1.0, MeOH), CD (MeOH):
De (nm) +2.88 (284.5),
4.50 min.
ꢁ1.44 (324.2) (c = 6.58 ꢀ 10ꢁ5).
12
(2R,3R)-isomer: [
a]
+10.9° (c = 0.5, MeOH), CD (MeOH): De
D
(nm) ꢁ4.32 (289.0), +1.68 (327.8) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
2.10.7. 20,3,40,5,7-Pentahydroxy flavanonol (5f)
[a]
ꢁ9.0° (c = 0.5, MeOH), CD (MeOH):
De (nm) +4.53 (289.8),
D
ꢁ1.86 (326.9) (c = 6.58 ꢀ 10ꢁ5).
1H NMR (600 MHz, acetone-d6) d: 4.87 (1H, d, J = 11.4 Hz, H-3),
5.49 (1H, d, J = 11.4 Hz, H-2), 5.93 (1H, d, J = 1.8 Hz, H-6), 5.98 (1H,
d, J = 1.8 Hz, H-8), 6.43 (1H, dd, J = 8.4, 3.0 Hz, H-50), 6.47 (1H, d,
J = 3.0 Hz, H-30), 7.32 (1H, dd, J = 8.4, 3.0 Hz, H-60). 13C NMR
(150 MHz, acetone-d6) d: 72.2 (C-3), 79.3 (C-2), 96.0 (C-6), 97.0
(C-8), 101.7 (C-10), 103.7 (C-30), 107.9 (C-50), 115.5 (C-10), 130.8
(C-60), 158.1 (C-20), 159.9 (C-40), 164.7 (C-5), 165.1 (C-9), 167.8
(C-7), 198.8 (C-4). HR-EI-MS: m/z 304.0584 [M]+ (Calcd 304.0583
for C15H12O7).
2.10.11. 3,5,7-Trihydroxy-20-methoxy flavanonol (5j)
Colorless powder, mp 218–220 °C; IR (KBr) mmax
: 3444,
1643 cmꢁ1; UV kmax (MeOH) nm (log ): 290 (4.16), 320 (sh); 1H
e
NMR (600 MHz, acetone-d6) d: 3.86 (3H, s, OCH3-20), 4.86 (1H, d,
J = 10.8 Hz, H-3), 5.62 (1H, d, J = 10.8 Hz, H-2), 5.94 (1H, d,
J = 1.8 Hz, H-6), 6.00 (1H, d, J = 1.8 Hz, H-8), 7.04 (1H, ddd, J = 8.4,
8.4, 1.2 Hz, H-50), 7.08 (1H, d, J = 8.4 Hz, H-30), 7.39 (1H, ddd,
J = 8.4, 8.4, 1.2 Hz, H-40), 7.58 (1H, dd, J = 8.4, 1.2 Hz, H-60). 13C
NMR (150 MHz, acetone-d6) d: 56.2 (OCH3-20), 72.3 (C-3), 78.7
(C-2), 96.0 (C-6), 97.2 (C-8), 101.8 (C-10), 112.3 (C-30), 121.4 (C-
50), 125.9 (C-10), 129.7 (C-60), 131.1 (C-40), 159.2 (C-20), 164.5 (C-
5), 165.2 (C-9), 167.9 (C-7), 198.5 (C-4). HR-EI-MS: m/z 302.0789
[M]+ (Calcd 302.0790 for C16H14O6). The enantiomers of 5j could
not be separated using chiral chromatography.
Separation by chiral chromatography: EtOH–n-hexane (25:75 v/v)
Retention time (2R,3R)-isomer: 9.03 min, (2S,3S)-isomer: 7.25 min.
12
(2R,3R)-isomer: [
a
]
D
+84.3° (c = 1.0, MeOH), CD (MeOH): De
(nm) ꢁ4.05 (292.2), +1.70 (328.3) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
ꢁ80.4° (c = 1.0, MeOH), CD (MeOH):
De (nm) +4.11 (293.2),
D
ꢁ1.63 (326.7) (c = 6.58 ꢀ 10ꢁ5).
2.10.8. 20,3,5,50,7-Pentahydroxy flavanonol (5g)
2.10.12. 3,5,7-Trihydroxy-30-methoxy flavanonol (5k)
1H NMR (600 MHz, acetone-d6) d: 4.79 (1H, d, J = 11.4 Hz, H-3),
5.57 (1H, d, J = 11.4 Hz, H-2), 5.96 (1H, d, J = 1.8 Hz, H-6), 5.99 (1H,
d, J = 1.8 Hz, H-8), 6.72 (1H, dd, J = 8.4, 3.0 Hz, H-40), 6.79 (1H, d,
J = 8.4 Hz, H-30), 6.99 (1H, d, J = 3.0 Hz, H-60). 13C NMR (150 MHz,
acetone-d6) d: 72.6 (C-3), 79.1 (C-2), 96.1 (C-6), 97.1 (C-8), 101.6
(C-10), 115.6 (C-60), 117.4 (C-40), 117.7 (C-30), 124.9 (C-10), 149.5
(C-20), 151.4 (C-50), 164.4 (C-5), 165.2 (C-9), 167.9 (C-7), 198.1
(C-4). HR-EI-MS: m/z 304.0584 [M]+ (Calcd 304.0583 for C15H12O7).
Separation by chiral chromatography: EtOH–n-hexane (30:70 v/
v) Retention time (2R,3R)-isomer: 7.37 min, (2S,3S)-isomer:
Colorless powder, mp 203–205 °C; IR (KBr) mmax
: 3435,
1637 cmꢁ1; UV kmax (MeOH) nm (log ): 291 (4.15), 318 (sh); 1H
e
NMR (600 MHz, acetone-d6) d: 3.83 (3H, s, OCH3-30), 4.68 (1H, d,
J = 12.0 Hz, H-3), 5.16 (1H, d, J = 12.0 Hz, H-2), 5.98 (1H, d,
J = 1.8 Hz, H-6), 6.01 (1H, d, J = 1.8 Hz, H-8), 6.97 (1H, dd, J = 7.8,
1.2 Hz, H-40), 7.16 (1H, d, J = 7.8 Hz, H-60), 7.18 (1H, d, J = 1.8 Hz,
H-20), 7.35 (1H, dd, J = 7.8, 7.8 Hz, H-50). 13C NMR (150 MHz, ace-
tone-d6) d: 55.7 (OCH3-30), 73.2 (C-6), 84.4 (C-2), 96.2 (C-6), 97.3
(C-8), 101.6 (C-10), 114.6 (C-20), 115.1 (C-40), 121.1 (C-60), 130.3
(C-50), 139.9 (C-10), 160.7 (C-30), 164.1 (C-5), 165.1 (C-9), 168.0
(C-7), 198.0 (C-4). HR-EI-MS: m/z 302.0790 [M]+ (Calcd 302.0790
for C16H14O6).
5.62 min.
12
(2R,3R)-isomer: [
a
]
+48.2° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ3.88 (287.3), +1.8 (317.2) (c = 6.58 ꢀ 10ꢁ5). (2S,3S)-isomer:
Separation by chiral chromatography: EtOH–n-hexane (70:30 v/
v) Retention time (2R,3R)-isomer: 5.17 min, (2S,3S)-isomer:
3.95 min.
12
[a
]
D
ꢁ45.6° (c = 1.0, MeOH), CD (MeOH):
De (nm) +3.99 (287.3),
ꢁ1.87 (318.2) (c = 6.58 ꢀ 10ꢁ5).
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042

W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
12
(2R,3R)-isomer: [
a
]
+4.7° (c = 1.0, MeOH), CD (MeOH):
D
e
(OCH3-40), 56.2 (OCH3-20), 72.1 (C-3), 78.8 (C-2), 96.0 (C-6), 97.0
(C-8), 99.3 (C-50), 101.6 (C-10), 105.9 (C-30), 118.1 (C-10), 130.7
(C-60), 160.4 (C-40), 162.6 (C-20), 164.6 (C-5), 165.1 (C-9), 167.9
(C-7), 198.7 (C-4). HR-EI-MS: m/z 332.0897 [M]+ (Calcd 332.0896
for C17H16O7).
D
(nm) ꢁ6.52 (288.7), +2.08 (327.1) (c = 6.62 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[
a
]
ꢁ5.5° (c = 1.0, MeOH), CD (MeOH):
De (nm) +6.54 (289.6),
D
ꢁ2.01 (324.2) (c = 6.62 ꢀ 10ꢁ5).
2.10.13. 3,5,7-Trihydroxy-40-methoxy flavanonol (5l)
Separation by chiral chromatography: EtOH–n-hexane (20:80 v/v)
Retention time (2R,3R)-isomer: 11.50 min, (2S,3S)-isomer:
1H NMR (600 MHz, acetone-d6) d: 3.83 (3H, s, OCH3-40), d 4.67
(1H, d, J = 11.2 Hz, H-3), 5.13 (1H, d, J = 11.2 Hz, H-2), 5.96 (1H, d,
J = 1.8 Hz, H-6), 6.00 (1H, d, J = 1.8 Hz, H-8), 7.00 (2H, d,
J = 9.0 Hz, H-30, H-50), 7.52 (2H, d, J = 9.0 Hz, H-20, H-60). 13C NMR
(150 MHz, acetone-d6) d: 54.8 (OCH3-40), 72.3 (C-3), 83.3 (C-2),
95.2 (C-6), 96.3 (C-8), 100.7 (C-10), 113.7 (C-30, C-50), 129.4 (C-20,
C-60), 129.5 (C-10), 160.3 (C-40), 163.3 (C-5), 164.2 (C-9), 167.0
(C-7), 197.4 (C-4). HR-EI-MS: m/z 302.0788 [M]+ (Calcd 302.0790
for C16H14O6).
10.50 min.
12
(2R,3R)-isomer: [
a]
+77.7° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ4.84 (292.3), +1.70 (326.7) (c = 6.06 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ75.0° (c = 1.0, MeOH), CD (MeOH):
De (nm) +4.28 (291.5),
ꢁ1.70 (324.7) (c = 6.06 ꢀ 10ꢁ5).
2.10.17. 3,5,7-Trihydroxy-20,50-dimethoxy flavanonol (5p)
Colorless powder, mp 223–225 °C; IR (KBr) mmax
1638 cmꢁ1; UV kmax (MeOH) nm (log ): 293 (4.13), 320 (sh); 1H
:
3428,
Separation by chiral chromatography: EtOH–n-hexane (25:75 v/v)
Retention time (2R,3R)-isomer: 10.22 min, (2S,3S)-isomer:
e
NMR (600 MHz, acetone-d6) d: 3.78 (3H, s, OCH3-20), 3.80 (3H, s,
OCH3-50), 4.86 (1H, d, J = 12.0 Hz, H-3), 5.60 (1H, d, J = 12.0 Hz, H-
2), 5.95 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H, d, J = 1.8 Hz, H-8), 6.95
(1H, dd, J = 9.0, 2.4 Hz, H-40), 7.02 (1H, d, J = 9.0 Hz, H-30), 7.19
(1H, d, J = 2.4 Hz, H-60). 13C NMR (150 MHz, acetone-d6) d: 56.0
(OCH3-20), 57.0 (OCH3-50), 72.4 (C-3), 78.6 (C-2), 96.1 (C-6), 97.2
(C-8), 101.7 (C-10), 113.8 (C-30), 115.5 (C-60), 115.6 (C-40), 127.0
(C-10), 153.3 (C-20), 154.8 (C-50), 164.4 (C-5), 165.2 (C-9), 167.9
(C-7), 198.4 (C-4). HR-EI-MS: m/z 332.0901 [M]+ (Calcd 332.0896
for C17H16O7).
8.58 min.
12
(2R,3R)-isomer: [
a
]
+15.6° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ7.68 (288.4), +2.26 (327.4) (c = 6.62 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[
a]
ꢁ21.0° (c = 1.0, MeOH), CD (MeOH):
De (nm) +8.02 (288.8),
D
ꢁ2.23 (326.7) (c = 6.62 ꢀ 10ꢁ5).
2.10.14. 3,5,7-Trihydroxy-20,60-dimethoxy flavanonol (5m)
Colorless powder, mp 268–270 °C; IR (KBr) mmax
1637 cmꢁ1; UV kmax (MeOH) nm (log ): 288 (4.22), 320 (sh); 1H
:
3478,
e
NMR (600 MHz, acetone-d6) d: 3.83 (6H, s, OCH3-20, OCH3-60),
5.40 (1H, d, J = 12.6 Hz, H-3), 5.81 (1H, d, J = 12.6 Hz, H-2), 5.90
(1H, d, J = 2.4 Hz, H-6), 5.97 (1H, d, J = 2.4 Hz, H-8), 6.72 (1H, d,
J = 8.4 Hz, H-30, H-50), 7.36 (1H, t, J = 8.4 Hz, H-40). 13C NMR
(150 MHz, acetone-d6) d: 56.4 (OCH3-20, OCH3-60), 70.4 (C-3), 76.3
(C-2), 95.9 (C-6), 96.8 (C-8), 101.6 (C-10), 105.5 (C-30, C-50), 113.2
(C-10), 131.9 (C-40), 161.0 (C-20, C-60), 165.2 (C-5), 165.3 (C-9),
167.5 (C-7), 199.6 (C-4). HR-EI-MS: m/z 332.0895 [M]+ (Calcd
332.0896 for C17H16O7). The Enantiomers of 5m could not be sep-
arated using chiral chromatography.
Separation by chiral chromatography: EtOH–n-hexane (50:50 v/
v) Retention time (2R,3R)-isomer: 5.00 min, (2S,3S)-isomer:
4.30 min.
12
(2R,3R)-isomer: [
a]
+46.7° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ4.64 (286.6), +1.86 (325.2) (c = 6.06 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ47.4° (c = 1.0, MeOH), CD (MeOH):
De (nm) +4.43 (288.8),
ꢁ1.84 (325.9) (c = 6.06 ꢀ 10ꢁ5).
2.10.18. 3,5,7-Trihydroxy-30,40-dimethoxy flavanonol (5q)
1H NMR (600 MHz, acetone-d6) d: 3.84 (3H, s, OCH3-40), 3.85
(3H, s, OCH3-30), 4.71 (1H, d, J = 12.0 Hz, H-3), 5.11 (1H, d,
J = 12.0 Hz, H-2), 5.96 (1H, d, J = 1.8 Hz, H-6), 6.00 (1H, d,
J = 1.8 Hz, H-8), 6.99 (1H, d, J = 1.8 Hz, H-50), 7.11 (1H, dd, J = 8.4,
1.8 Hz, H-60), 7.22 (1H, d, J = 1.8 Hz, H-20). 13C NMR (150 MHz, ace-
tone-d6) d: 56.2 (OCH3-40), 56.3 (OCH3-30), 73.2 (C-3), 84.6 (C-2),
96.1 (C-6), 97.2 (C-8), 101.6 (C-10), 112.4 (C-50), 112.6 (C-20),
121.8 (C-60), 130.7 (C-10), 150.3 (C-30), 151.0 (C-40), 164.2 (C-5),
165.1 (C-9), 167.9 (C-7), 198.3 (C-4). HR-EI-MS: m/z 332.0896
[M]+ (Calcd 332.0896 for C17H16O7). The enantiomers of 5q could
not be separated using chiral chromatography.
2.10.15. 3,5,7-Trihydroxy-20,30-dimethoxy flavanonol (5n)
Colorless powder, mp 233–235 °C; IR (KBr) mmax
: 3432,
1632 cmꢁ1; UV kmax (MeOH) nm (log ): 290 (4.33), 320 (sh); 1H
e
NMR (600 MHz, acetone-d6) d: 3.83 (3H, s, OCH3-30), 3.89 (3H, s,
OCH3-20), 4.81 (1H, d, J = 12.0 Hz, H-3), 5.58 (1H, d, J = 12.0 Hz, H-
2), 5.95 (1H, d, J = 1.8 Hz, H-6), 6.01 (1H, d, J = 1.8 Hz, H-8), 7.09
(1H, dd, J = 7.8, 1.8 Hz, H-60), 7.16 (1H, dd, J = 7.8, 7.8 Hz, H-50),
7.22 (1H, dd, J = 7.8, 1.8 Hz, H-40). 13C NMR (150 MHz, acetone-
d6) d: 56.3 (OCH3-20), 61.4 (OCH3-30), 72.4 (C-3), 78.9 (C-2), 96.1
(C-6), 97.2 (C-8), 101.6 (C-10), 114.1 (C-60), 120.9 (C-50), 124.9
(C-40), 131.6 (C-10), 149.3 (C-20), 154.0 (C-30), 164.3 (C-5), 165.2
(C-9), 167.9 (C-7), 198.4 (C-4). HR-EI-MS: m/z 332.0897 [M]+ (Calcd
332.0896 for C17H16O7).
2.10.19. 3,5,7-Trihydroxy 30,50-dimethoxy flavanonol (5r)
Colorless powder, mp 191–193 °C; IR (KBr) mmax
: 3474,
1644 cmꢁ1; UV kmax (MeOH) nm (log ): 289 (4.14), 318 (sh); 1H
e
Separation by chiral chromatography: EtOH–n-hexane (25:75 v/
v) Retention time (2R,3R)-isomer: 7.13 min, (2S,3S)-isomer:
NMR (600 MHz, acetone-d6) d: 3.81 (6H, s, OCH3-30, OCH3-50),
4.67 (1H, d, J = 12.0 Hz, H-3), 5.11 (1H, d, J = 12.0 Hz, H-2), 5.98
(1H, d, J = 2.4 Hz, H-6), 6.01 (1H, d, J = 2.4 Hz, H-8), 6.52 (1H, dd,
J = 2.4, 2.4 Hz, H-40), 6.77 (2H, d, J = 2.4 Hz, H-20, H-60). 13C NMR
(150 MHz, acetone-d6) d: 55.8 (OCH3-30, OCH3-50), 73.2 (C-3),
84.5 (C-2), 96.2 (C-6), 97.3 (C-8), 101.2 (C-40), 101.6 (C-10), 107.0
(C-20, C-60), 140.5 (C-10), 161.8 (C-30, C-50), 164.0 (C-5), 165.1 (C-
9), 167.9 (C-7), 198.0 (C-4). HR-EI-MS: m/z 332.0901 [M]+ (Calcd
332.0896 for C17H16O7).
6.05 min.
12
(2R,3R)-isomer: [
a
]
+61.6° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ2.76 (290.7), +1.35 (323.2) (c = 6.06 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[
a]
D
ꢁ73° (c = 1.0, MeOH), CD (MeOH):
De (nm) +2.75 (290.9),
ꢁ1.41 (324.6) (c = 6.06 ꢀ 10ꢁ5).
2.10.16. 3,5,7-Trihydroxy-20,40-dimethoxy flavanonol (5o)
Colorless powder, mp 203–205 °C; IR (KBr) mmax
1639 cmꢁ1; UV kmax (MeOH) nm (log ): 289 (4.19), 318 (sh); 1H
:
3478,
Separation by chiral chromatography: EtOH–n-hexane (80:20 v/
v) Retention time (2R,3R)-isomer: 6.83 min, (2S,3S)-isomer:
e
NMR (600 MHz, acetone-d6) d: 3.83 (3H, s, OCH3-20), 3.83 (3H, s,
OCH3-40), 4.85 (1H, d, J = 12.0 Hz, H-3), 5.51 (1H, d, J = 12.0 Hz, H-
2), 5.91 (1H, d, J = 2.4 Hz, H-6), 5.98 (1H, d, J = 2.4 Hz, H-8), 6.59
(1H, d, J = 2.4 Hz, H-30), 6.61 (1H, dd, J = 8.4, 2.4 Hz, H-50), 7.47
(1H, d, J = 8.4 Hz, H-60). 13C NMR (150 MHz, acetone-d6) d: 55.8
4.17 min.
12
(2R,3R)-isomer: [
a
]
+0.8° (c = 1.0, MeOH), CD (MeOH): De
D
(nm) ꢁ5.84 (292.0), +1.98 (328.3) (c = 6.06 ꢀ 10ꢁ5). (2S,3S)-isomer:
12
[a
]
D
ꢁ0.8° (c = 1.0, MeOH), CD (MeOH):
De (nm) +5.46 (290.1),
ꢁ1.84 (324.7) (c = 6.06 ꢀ 10ꢁ5).
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042

6
W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3.2. The separation of enantiomers using a chiral column and
3. Results
determination of absolute configuration of flavanonols
3.1. The synthesis of flavanonols
We separated 32 enantiomers (Table 1) from the racemic mix-
tures using chiral column chromatography. The separation of com-
pounds 5j, 5m and 5q was not satisfactory. Thus 5j, 5m and 5q
were used as racemic mixtures.
The absolute configurations of the stereoisomers were deter-
mined by circular dichroism (CD) and the coupling constant (J)
between H-2 and H-3 measured by 1H NMR. Positive Cotton effect
around 320 nm and negative Cotton effect around 290 nm indi-
cates that the C-2 is in R configuration28,29. The coupling constant
of 10–12 Hz indicates that H-2 and H-3 are in trans configuration.
As far as the flavanonols that were synthesized in this study are
The preparation of flavanonols was carried out as illustrated in
Scheme 1. 2,4,6-Trihydroxyacetophenone was protected with an
excessive amount of chloromethyl methyl ether (MOMCl) in the
presence of an excessive amount of NaH suspended in DMF, while
cooling in an ice-water bath, to give compound 1. The hydroxyl-
benzaldehydes were protected with an excessive amount of
MOMCl in the presence of an excessive amount of K2CO3 in ace-
tone, while cooling in an ice-water bath, to give compound 2. Com-
pounds 1 and 2 were treated with KOH in ethanol at room
temperature for 3 h to give compound 3. Compound 3 was epoxi-
dized with H2O2 under alkaline conditions (NaOH aqueous) in
ethanol to give compound 4. Finally compound 5 was obtained
by treating compound 4 with HCl in methanol at 55 °C. All of the
compounds were characterized by 1H NMR, 13C NMR, and mass
spectroscopy.
concerned, the optical rotation ([a]D) of (2R,3R)-isomer was
positive.
3.3. The inhibitory potential against NO production in RAW
264.7 cells
We prepared 19 flavanonols with a common 3,5,7-trihydroxy-
chroman scaffold. The compounds differed in the substitution of
the B ring. Stereoisomers were separated from 16 of the com-
pounds. Thus 32 stereochemically pure compounds and 3 racemic
mixtures were tested by examining their inhibitory potential
against NO production in RAW 264.7 cells, and evaluating their
DPPH radical scavenging potential.
The (2R,3R)-isomer of 5e, with a 20,30-dihydroxyl substitution in
the B ring, indicated the highest inhibitory potential of the 35 spec-
imens (70% inhibition at 100 lM). Its inhibitory potential was com-
parable with the positive control, aminoguanidine HCl (AG). The
(2R,3R)-isomers of 5a and 5b, with 20- and 30-hydroxyl substitu-
tions in the B ring, respectively, showed weaker but significant
Scheme 1. Synthesis of flavanonols. Reagents and conditions: (a) NaH, MOMCl, DMF, 0 °C; (b) K2CO3, MOMCl, Me2CO, rt; (c) KOH, EtOH, rt; (d) 30% H2O2, NaOH aq, MeOH, rt;
(e) MeOH-HCl, 55 °C.
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W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Figure 1. The effects of the flavanonols on the NO production (A) and the cell viability of RAW 264.7 cells (B). 5e: 20,3,30,5,7-Pentahydroxy flavanonol, 5a: 20,3,5,7-
tetrahydroxy flavanonol, 5b: 3,30,5,7-tetrahydroxy flavanonol, 5g: 20,3,5,50,7-pentahydroxy flavanonol, AG: aminoguanidine hydrochloride. The final concentration of the
tested specimen including AG was 100 lM. All data are expressed as the mean S.D (n = 3).
inhibitory activity (40% and 35% inhibition at 100
(2R,3R)-isomer of 5g, with 20,50-dihydroxyl substitution, also
showed weaker inhibitory potential (30% inhibition at 100 M).
Their enantiomers (i.e., the (2S,3S)-isomers) indicated lower or
very weak potential (Fig. 1A and Table 1). The inhibitory activity
l
M). The
from nature is significantly smaller than other flavonoids. More-
over there was no report that stereoisomers of flavonoids have dif-
ferent effects on NO production inhibitory activity. Therefore in
order to explore wider chemical space to look for bioactive flavo-
noids, we synthesized flavonoids which structures are unnatural,
rare and new.
l
of the other compounds was not detectable at 100 lM. The pres-
ence of the hydroxyl group at the 20- and 30-positions of the B ring
is important for the inhibition of NO production. The methylation
of the 20-hydroxyl group (5j), 30-hydroxyl group (5k) and both
groups together (5n) resulted in a loss of activity, suggesting the
importance of the 20- and 30- hydroxyl groups for the inhibitory
activity. The substitution at the 40-position lowered the inhibitory
potential (compounds 5c, 5f and 5h). The potent flavanonols were
The (2R,3R)-isomers of 5e, 5a, 5b and 5g were found to be
potent flavanonols for the inhibition of NO production (Fig. 1A).
Flavanonols (2R,3R)-5e and (2R,3R)-5g were also found to have
radical scavenging potential (Table 1). However, (2R,3R)-5a and
(2R,3R)-5b which exhibited inhibitory activity on NO production
did not exhibit radical scavenging potential. On the other hand,
(2R,3R)- and (2S,3S)-isomers of 5h both of which demonstrated
strong radical scavenging potential, did not inhibit the NO
production.
As expected the radical scavenging potential was not stereospe-
cific. Our results support the long-speculated hypothesis that the
radical scavenging potential of flavanonols might simply be
explained by the oxidation–reduction potential of the B ring moi-
ety. Our study supports this hypothesis and confirms that it is also
valid for unnatural flavonoids.
not found to be toxic to RAW 264.7 cells at 100 lM. No significant
difference was found in the toxicity of the stereoisomers (Fig. 1B).
The configuration of all of the potent compounds was (2R,3R).
The inhibition of NO production by the flavanonols was stereospe-
cific. It should be noted that most flavanonols from natural sources
have the (2R,3R) configuration30
.
3.4. Radical scavenging potential
In contrast, the inhibitory potential of NO production in RAW
264.7 cells was stereospecific and irrelevant to the radical scaveng-
ing potential. This suggests that flavanonols are stereospecifically
recognized by RAW 264.7 cells and that active flavanonols would
bind to a biological target that plays a role in the production
and/or regulation of NO.
Table 1 shows the results of a radical scavenging potential
(EC50) assay of the flavanonols. The most potent compounds were
the (2R,3R)- and (2S,3S)-isomers of 20,30-dihydroxyl analogue 5e,
and 30,40-dihydroxyl analogue 5h. The EC50 value of the (2R,3R)-iso-
mer of 20,30-dihydroxy flavanonol 5e was found to be 12.5
lM,
while that of the (2S,3S)-isomer of 5e was 13.2
isomer of 5h, called dihydroquercetin, is often used as a standard
lM. The (2R,3R)-
5. Conclusion
specimen for DPPH assays. Dihydroquercetin and its enantiomer
We prepared 35 flavanonols with an identical chroman scaffold
in the A and C rings but with differences in their B ring structure
and stereochemistry. Nineteen of the synthesized flavanonols were
new compounds that have not been isolated from nature. We car-
ried out an NO production inhibitory assay and a DPPH radical
scavenging assay. The NO production inhibitory activity was stere-
ospecific and the (2R,3R)-configuration was required for this activ-
ity to occur. Existence of 20- and 30-hydroxy groups in the B ring
was important for the inhibition of NO production. The flavanonols
with ortho- or para-hydroquinone moiety in their B ring exhibited
strong radical scavenging potential. No correlation was found
between the inhibition of NO production and the radical scaveng-
ing activity. Taken together the inhibitory effect of flavanonols
against NO production cannot solely be explained by anti-oxidative
potential. An existence of a biological target could be proposed to
explain the inhibitory effect.
(2S,3S)-5h had EC50 values of 13.9 and 12.6
l
M, respectively.
The activity of 20,50-dihydroxy analogue 5g (25
lM) was half
that of 5e and 5h. Again, both the (2R,3R)- and (2S,3S)-isomers of
5g were found to have equivalent radical scavenging potential
(EC50: 24.5 lM and 25.4 lM, respectively).
Radical scavenging activity was not detected in any compounds
other than 5e, 5g and 5h. In short, the flavanonols with ortho- or
para-hydroquinone moiety in the B ring showed radical scavenging
activity. The stereochemistry was irrelevant to the radical scaveng-
ing potential.
4. Discussion
Many studies have been carried out about the anti-inflamma-
tory activity of flavonoids using RAW 264.7 cells [e.g., 22–25].
Analyses of the complied results suggest that, in general, flavo-
noids having C2-C3 double bond show stronger NO production
inhibitory activity as well as DPPH radical scavenging activity than
C2-C3 is single bond. In our knowledge, no flavanonol has been rec-
ognized as a strong inhibitor for NO-production in RAW 264.7 cells.
This may be due to the fact that number of flavanonols isolated
Acknowledgments
We thank the Otsuka Toshimi Scholarship Foundation and
Watanuki International Scholarship Foundation for supporting
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042

8
W.-J. Jiang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
13. Way, T. D.; Kao, M. C.; Lin, J. K. FEBS Lett. 2005, 579, 145.
14. Yilmaz, Y.; Toledo, R. T. Trends Food Sci. Technol. 2004, 15, 422.
15. Moridani, M. Y.; Galati, G.; O’Brien, P. J. Chem. Biol. Interact. 2002, 139, 251.
16. Asgari, S.; Naderi, G. H.; Sarrafzadegan, N.; Ghassemi, N.; Boshtam, M.; Rafie,
M.; Arefian, A. Pharm. Acta Helv. 1999, 73, 223.
the study and research of author W.-J.J. Part of this work was sup-
ported by JSPS KAKENHI Grant Number 24590028 (S.K.).
Supplementary data
17. Havsteen, B. H. Pharmacol. Ther. 2002, 96, 67.
18. Ng, T. B.; Liu, F.; Wang, Z. T. Life Sci. 2000, 66, 709.
19. Weisburger, J. H. Food Chem. Toxicol. 1999, 37, 943.
20. Pandurangan, N.; Bose, C.; Banerji, A. Bioorg. Med. Chem. Lett. 2011, 21, 5328.
21. Segal, A. W. Annu. Rev. Immunol. 2005, 9, 197.
22. Matsuda, H.; Morikawa, T.; Ando, S.; Toguchida, I.; Yoshikawa, M. Bioorg. Med.
Chem. 1995, 2003, 11.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.042.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
23. Daikonya, A.; Kitanaka, S. Chem. Pharm. Bull. 2011, 59, 1567.
24. Andrei, I. K.; Igor, A. S.; Nina, G. D.; Liliya, N. K.; Mark, T. Q. Bioorg. Med. Chem.
2007, 15, 1749.
References and notes
25. Sreeparna, D.; Indrani, M.; Shaikh, B.; Md, N. A.; Kunal, R. Bioorg. Med. Chem.
Lett. 2014, 24, 5050.
26. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
27. Sharma, O. P.; Tej, K. B. Food Chem. 2009, 113, 1202.
28. Nonaka, G.; Goto, Y.; Kinjo, J.; Nohara, T.; Nishioka, I. Chem. Pharm. Bull. 1987,
35, 1105.
29. Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy-exciton Coupling in
Organic Stereochemistry; University Science Books: California, U.S.A., 1983.
30. Structural search by SciFinder (American Chemical Society; https://
scifinder.cas.org) indicated that 994 flavanonols having (2R,3R) configuration
were known, while number of the flavanonols having (2S,3S) configuration was
67. In the reviews by Kazi, A. et al. (World Journal of Pharmaceutical research
2015, 4, 560) and by Sareedenchai, V. et al. (Biochem. Syst. Ecol. 2010, 38, 93), all
flavanonols listed have (2R,3R) configuration. Takahashi, H. et al. (Chem. Pharm.
Bull. 1984, 32, 4852) also stated the dominancy of (2R,3R) configuration among
naturally occurring flavanonol.
1. Yamamoto, Y.; Gaynor, R. B. J. Clin. Invest. 2001, 107, 135.
2. Cazarolli, L. H.; Zanatta, L.; Alberton, E. H.; Figueiredo, M. S.; Folador, P.;
Damazio, R. G.; Pissolatti, M. G.; Silva, F. R. Mini-Rev. Med. Chem. 2008, 8, 1429.
3. Osadebe, P. O.; Okoye, F. B. J. Ethnopharmacol. 2003, 89, 19.
4. Rotelli, A. E.; Guardia, T.; Juárez, A. O.; de la Rocha, N. E.; Pelzer, L. E. Pharmacol.
Res. 2003, 48, 601.
5. Rahman, M.; Gray, A. I. Phytochemistry 2002, 59, 73.
6. Cushnie, T. P.; Lamb, A. J. Int. J. Antimicrob. Agents 2011, 38, 99.
7. Manner, S.; Skogman, M.; Goeres, D.; Vuorela, P.; Fallarero, A. Int. J. Mol. Sci.
2013, 14, 19434.
8. Vlietinck, A. J.; de Bruyne, T.; Apers, S.; Pieters, L. A. Planta Med. 1998, 64, 97.
9. Kaul, T. N.; Middleton, E., Jr.; Ogra, P. L. J. Med. Virol. 1985, 15, 71.
10. Friedman, M. Mol. Nutr. Food Res. 2007, 51, 116.
11. Sousa, R. R.; Queiroz, K. C.; Souza, A. C.; Gurgueira, S. A.; Augusto, A. C.;
Miranda, M. A.; Peppelenbosch, M. P.; Ferreira, C. V.; Aoyama, H. J. Enzyme
Inhib. Med. Chem. 2007, 22, 439.
12. Schuier, M.; Sies, H.; Illek, B.; Fischer, H. J. Nutr. 2005, 135, 2320.
Please cite this article in press as: Jiang, W.-J.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.042