Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship. Part 2

Article abstract of DOI:10.1016/j.bmc.2017.05.060

To explore the structure-activity relationships of flavanonols on the inhibition of nitric oxide (NO) production in RAW 264.7 cells, we have prepared a series of synthetic flavanonols. In our previous study, the 2′,3′-dihydroxyphenyl substructure was foun

Full text of DOI:10.1016/j.bmc.2017.05.060

Bioorganic & Medicinal Chemistry xxx (2017) 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. Part 2
a
⇑
Wen-Jun Jiang , Susumu Kitanaka, Tomoko Takamiya, Hiroshi Iijima
School of Pharmacy, Nihon University, 7-7-1 Narashino-dai, Funabashi-shi, Chiba 274-8555, 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 of flavanonols on the inhibition of nitric oxide (NO) pro-
duction in RAW 264.7 cells, we have prepared a series of synthetic flavanonols. In our previous study,
the 20,30-dihydroxyphenyl substructure was found to be the most potent B ring substructure among
the flavanonols having 3,5,7-trihydroxychroman-4-one as the A/C ring. In this study, we examined the
effect of diverse substitutions on the A ring of the 2-(2,3-dihydroxyphenyl)-3-hydroxychroman-4-one
scaffold, i.e., by fixing the B ring to the 20,30-dihydroxyphenyl substructure. Eighteen stereoisomers and
4 racemic mixtures were prepared, and their inhibitory potency on NO production in RAW 264.7 cells
was tested. We observed higher inhibitory activity in the (2R,3R) stereoisomers than in the (2S,3S)
stereoisomers. The presence of a hydroxy or a methoxy group at the 7-postiion enhanced the inhibitory
potency, and the additional substitutions at the 6- or 8-position in the A ring increased potency and stere-
ospecificity. A representative compound, (2R,3R)-20,30,7,8-tetrahydroxyflavanonol 5e, had an IC50 value of
17 mM, whereas its (2S,3S) stereoisomer did not inhibit NO production at all at a concentration of 100 mM.
In this study, it was necessary to determine the absolute configuration of the stereoisomers of the syn-
thesized flavanonols that carry methoxy substitutions in the A ring. The procedure to determine their
absolute configuration by the CD excitation chirality method is also discussed.
Received 6 May 2017
Revised 25 May 2017
Accepted 30 May 2017
Available online xxxx
Keywords:
Flavonoid
Nitric oxide
Macrophage
Structure-activity relationship
Stereoisomer
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
materials from many sources on NO production in RAW 264.7 cells,
which are stimulated by lipopolysaccharide (LPS).23
Flavonoids are well known to have many biological activities,
including anti-inflammatory,1–4 anti-oxidative,5–10 anti-allergic,1,11
anti-microbial,12–14 anti-viral,15–17 anti-diarrheal,5 anti-cancer,2,18–21
and anti-diabetic22 effects. We have been interested in the anti-
inflammatory effects of flavonoids, especially in their effects on
chronic inflammation leading to metabolic disease. Flavonoids,
widely distributed in plants such as vegetables, fruits, teas, and
some herbs, are considered an important dietary material that con-
tributes to the control of chronic inflammation. The production of
nitric oxide (NO) is a primary indicator of macrophage activation.
Indeed, many flavonoids showed inhibitory potency on NO pro-
duction in RAW 264.7 cells. However, the structure-activity rela-
tionship of flavonoids on inhibitory potency against NO
production remained a qualitative one. In 2011, Daikonya et al. iso-
lated flavonoids that carry symmetrically substituted B rings from
Tibetans herbal plants and determined their IC50 values for sup-
pression of NO production.24,25 These structure-activity data suc-
cessfully led us to establish a quantitative structure-activity
relationship (QSAR) model by comparative molecular field analysis
(CoMFA).26 This CoMFA model suggested the importance of the
electrostatic feature of the B ring and prompted us to synthesize
a series of 5,7-dihydroxyflavavonol derivatives that diversify in
their B ring substitutions.27 In the QSAR study, we found that (i)
flavanonols that carry a 20,30-dihydroxyphenyl ring as the B ring
show strong activity, (ii) the inhibition is stereospecific, i.e., the
(2R,3R) stereochemistry is relevant for the activity in macrophage
RAW 264.7 cells, and (iii) the presence of a 40-hydroxy group on
the B ring of flavanonols is unfavorable for such activity. This dis-
advantage of the presence of the 40-hydroxy group on the B ring is
rather unexpected because in natural flavonoids, the 40-position is
NO is produced once the NF-jB complex has been activated by
extracellular stimuli. Thus, chemicals that suppress NO production
in macrophage cells can be expected to be useful suppressors of
inflammation. We have examined the inhibitory effect of herbal
⇑
Corresponding author.
E-mail address: iijima.hiroshi@nihon-u.ac.jp (H. Iijima).
Present address: Jiangsu Yew Pharmaceutical Co., Ltd, QinXin Village, City WuXi,
a
Province JiangSu 214199, China.
http://dx.doi.org/10.1016/j.bmc.2017.05.060
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

2
W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
often substituted by a hydroxy group due to biosynthetic destiny,
and this might be one reason why it is rare to encounter a fla-
vanonol that strongly inhibits NO production in macrophage cells.
In this study, to expand the structure-activity relationship of
flavanonols on the inhibition of NO production, we synthesized fla-
vanonols that carry a 20,30-dihydroxyphenyl substructure as the B
ring, but which have diversity in the A ring.
added to the RAW 264.7 cells left in each well of the 96-well plate
from the above-mentioned NO assay, followed by incubation at
37 °C for 4 h. Absorbance was measured at 570 nm (reference
wavelength: 600 nm).
2.5. Synthesis of MOMO protection acetophenones 1
NaH (1.5 equiv; depending on the number of hydroxyl groups)
in dry THF was slowly added while stirring at 0–5 °C (in an ice-
water bath) to a solution of hydroxyacetophenone (1 equiv) in
dry DMF. When the solution was cooled to 0–5 °C, chloromethyl
methyl ether (1.5 equiv; depending on the number of hydroxyl
groups) was slowly added over a period of 15 min so that the tem-
perature was maintained below 5 °C. The reaction mixture was
then 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 sil-
ica gel column chromatography, eluting with n-hexane and EtOAc
to give compound 1: a colorless oil (80–95% yield).
2. Material and methods
2.1. General experimental procedures
Optical rotations were measured using
a JASCO P-1020
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 a JASCO FT/IR-4200 spectrometer, UV spectra
were recorded on JASCO V-730 spectrometer, and CD spectra were
recorded on JASCO J-600 spectrometer. Melting points were deter-
mined by using AS-ONE ATM-02.
2.2. Separation of stereoisomers by chiral column chromatography
2.6. Synthesis of bis(2-metholmethyl)benzaldehyde 2
All of the synthesized flavanonols were further submitted
K2CO3 (10 equiv) was added while stirring at 0-5 °C (ice-water
bath) to a solution of 2,3-dihydroxylbenzaldehyde (1 equiv) in
dry acetone. When the solution was cooled to 0–5 °C, chloromethyl
methyl ether (3.0 equiv) was slowly added over a period of 15 min
to keep the temperature below 5 °C. The reaction mixture was stir-
red 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 concen-
trated under a vacuum, and the residue was purified by silica gel
column chromatography, eluting with n-hexane and EtOAc to give
compound 2: a colorless oil (90% yield).
to purification by
CHIRALPAK IA, 5
mers using a high-performance liquid chromatography (HPLC)
system (JASCO PU-1580, UV-1575). The elution solvent was
ethanol-n-hexane, the flow rate was 5 mL/min, and the detection
wavelength was 254 nm.
a chiral column chromatograph (DAICEL,
m, 10 mm / ꢀ 250 mm) to isolate the stereoiso-
l
2.3. NO assay
The amount of released nitrite (NO) was quantified by the Griess
method.28 RAW 264.7 cells were cultured in F-12 Ham medium
(Sigma Aldrich, N4888) containing 200 mM L-glutamate (Sigma
1H NMR (600 MHz, CDCl3), d: 3.49 (3H, s, OCH3-3), 3.56 (3H, s,
OCH3-2), 5.21 (2H, s, OCH2-3), 5.23 (2H, s, OCH2-2), 7.12 (1H, t,
J = 7.8 Hz, H-5), 7.38 (1H, dd, J = 7.8, 1.2 Hz, H-4), 7.48 (1H, dd,
J = 7.8, 1.2 Hz, H-6), 10.44 (1H, s, CHO). 13C NMR (150 MHz, CDCl3),
d: 56.5 (OCH3-3), 58.1 (OCH3-2), 95.4 (OCH2-3), 99.9 (OCH2-2),
121.1 (C-4), 122.4 (C-5), 124.7 (C-6), 130.8 (C-1), 150.1 (C-3),
150.3 (C-2), 190.4 (C@O).
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 sus-
pension (1.6 ꢀ 106 cells/mL culture medium) was dispensed in a
well of a 96-well plate (Sumitomo Bakelite, 8096R), and 40 mL of test
compound solution was added. The test compound 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 solution (Sigma Aldrich, L-2880) was
then added to each well. The final concentration of LPS was 100 ng/
mL. After 16 h of incubation in the CO2 incubator, 100 mL of super-
natant 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 1% sulfanilamide solution (in 5 v/v% aqueous
phosphoric acid) was added to each well. A few minutes later, 50 mL
of 0.1% N-1-naphthylethylnediamine (Wako Pure Chemical Inc.,
147-04141) solution was added, and the mixture was 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
control. The concentrations of the test compounds were precisely
2.7. Synthesis of chalcone 3
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-
yellow oil (60–90% yield).
2.8. Synthesis of epoxide 4
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 (95% yield).
determined from the ultraviolet absorption at kmax
.
2.4. Cell viability test
Cell viability was measured using alamarBlueÒ reagent (Bio-Rad
AbD Serotec Ltd.). Ten microliters of alamarBlueÒ solution was
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
isomer: ½a 1D8
ꢁ
ꢂ87.4° (c = 1.0, MeOH), CD(MeOH):
D
e
(nm) +3.37
2.9. Synthesis of flavanonol 5
(320.3), ꢂ3.86 (362.3) (c = 6.0 ꢀ 10ꢂ5).
2.9.4. 20,30,7-Trihydroxyflavanonol 5d
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 Na2SO4.
The filtered EtOAc was evaporated to give a red-yellow oil that
was purified by silica gel column chromatography, eluting with
n-hexane and EtOAc to give 5a–5m (30–55% yield).
Colorless crystal, mp 231–233 °C. UV kmax (MeOH) nm (loge):
277 (4.22), 309 (sh). 1H NMR (600 MHz, acetone-d6) d: 4.77 (1H,
d, J = 12.0 Hz, H-3), 5.59 (1H, d, J = 12.0 Hz, H-2), 6.42 (1H, d,
J = 1.8 Hz, H-8), 6.63 (1H, dd, J = 8.4, 1.8 Hz, H-6), 6.79 (1H, t,
J = 7.8 Hz, H-50), 6.90 (1H, dd, J = 7.8, 1.2 Hz, H-40), 7.08 (1H, dd,
J = 7.8, 1.2 Hz, H-60), 7.75 (1H, d, J = 8.4 Hz, H-5). 13C NMR
(150 MHz, acetone-d6) d: 73.5 (C-3), 79.5 (C-2), 103.7 (C-8), 111.8
(C-6), 113.3 (C-10), 116.1 (C-40), 120.2 (C-60), 120.5 (C-50), 125.0
(C-10), 129.9 (C-5), 145.1 (C-20), 146.1 (C-30), 164.8 (C-9), 165.8
(C-7), 193.3 (C-4). HR-EI-MS: m/z 288.0631 [M]+ (Calcd 288.0634
for C15H12O6).
2.9.1. 20,30-Dihydroxyflavavonol 5a
Colorless crystal, mp 198–200 °C UV kmax (MeOH) nm (loge):
252 (3.90), 286 (3.48), 320 (3.48). 1H NMR (600 MHz, acetone-d6)
d: 4.92 (1H, d, J = 12.0 Hz, H-3), 5.67 (1H, d, J = 12.0 Hz, H-2), 6.79
(1H, t, J = 7.8 Hz, H-50), 6.91 (1H, dd, J = 7.8, 1.8 Hz, H-40), 7.05
(1H, d, J = 8.4 Hz, H-8), 7.10 (1H, dd, J = 7.8, 1.8 Hz, H-60), 7.13
(1H, t, J = 7.8 Hz, H-6), 7.61 (1H, td, J = 7.8, 1.8 Hz, H-7), 7.86 (1H,
dd, J = 7.8, 1.8 Hz, H-5). 13C NMR (150 MHz, acetone-d6) d: 73.8
(C-3), 79.5 (C-2), 116.2 (C-40), 118.8 (C-8), 120.2 (C-60), 120.4 (C-
50), 120.4 (C-10), 122.6 (C-6), 124.8 (C-10), 127.8 (C-5), 137.2 (C-
7), 145.2 (C-20), 146.0 (C-30), 162.7 (C-9), 195.0 (C-4). HR-EI-MS:
m/z 272.0685 [M]+ (Calcd 272.0685 for C15H12O5).
Separation by chiral chromatography: EtOH-n-hexane (35:65
v/v) retention time (2R,3R)-isomer: 5.08 min, (2S,3S)-isomer:
3.92 min. (2R,3R)-isomer: ½a D18
ꢁ
+61.5° (c = 1.0, MeOH), CD (MeOH):
D
e
(nm) ꢂ4.66 (304.0), +2.64 (329.2) (c = 6.0 ꢀ 10ꢂ5). (2S,3S)-iso-
mer: ½a 1D8
ꢁ
ꢂ64.4° (c = 1.0, MeOH), CD (MeOH):
De (nm) +3.82
(304.9), ꢂ3.13 (329.2) (c = 6.0 ꢀ 10ꢂ5).
2.9.5. 20,30,7,8-Tetrahydroxyflavanonol 5e
Separation by chiral chromatography: EtOH-n-hexane (35:65 v/v)
retention time (2R,3R)-isomer: 10.77 min, (2S,3S)-isomer:
Colorless crystal, mp 241–243 °C. UV kmax (MeOH) nm (loge):
7.25 min. (2R,3R)-isomer: ½a D18
ꢁ
+55.8° (c = 1.0, MeOH), CD (MeOH):
292 (4.21). 1H NMR (600 MHz, acetone-d6) d: 4.78 (1H, d,
J = 12.0 Hz, H-3), 5.56 (1H, d, J = 12.0 Hz, H-2), 6.66 (1H, d,
J = 8.4 Hz, H-6), 6.80 (1H, t, J = 8.4 Hz, H-50), 6.90 (1H, dd, J = 8.4,
1.2 Hz, H-40), 7.08 (1H, dd, J = 0.4, 1.2 Hz, H-60), 7.33 (1H, d,
J = 8.4 Hz, H-5). 13C NMR (150 MHz, acetone-d6) d: 73.7 (C-3),
80.0 (C-2), 111.3 (C-6), 113.8 (C-10), 116.1 (C-40), 119.2 (C-5),
120.5 (C-60), 120.5 (C-50), 125.0 (C-10), 133.6 (C-8), 145.1 (C-20),
146.1 (C-30), 151.8 (C-7), 153.3 (C-9), 193.6 (C-4). HR-EI-MS: m/z
304.0583 [M]+ (Calcd 304.0583 for C15H12O7).
D
e
(nm) ꢂ4.77 (309.9),+2.88 (339.9) (c = 6.0 ꢀ 10ꢂ5). (2S,3S)-iso-
mer: ½a 1D8
ꢁ
ꢂ63.4° (c = 1.0, MeOH), CD (MeOH):
De (nm) +4.19
(310.8), ꢂ3.31 (339.9) (c = 6.0 ꢀ 10ꢂ5).
2.9.2. 20,30,5-Trihydroxyflavavonol 5b
Colorless crystal, mp 224–226 °C. UV kmax (MeOH) nm (loge):
276 (3.99), 351 (3.48) 1H NMR (600 MHz, acetone-d6) d: 4.99
(1H, d, J = 11.4 Hz, H-3), 5.69 (1H, d, J = 11.4 Hz, H-2), 6.48 (1H, d,
J = 7.8 Hz, H-6), 6.53 (1H, d, J = 7.8 Hz, H-8), 6.78 (1H, t, J = 7.8 Hz,
H-50), 6.90 (1H, dd, J = 7.8, 1.8 Hz, H-40), 7.06 (1H, dd, J = 7.8,
1.8 Hz, H-60), 7.48 (1H, t, J = 7.8 Hz, H-7). 13C NMR (150 MHz, ace-
tone-d6) d: 73.1 (C-3), 79.3 (C-2), 107.6 (C-10), 108.4 (C-6), 109.9
(C-8), 116.3 (C-40), 120.3 (C-60), 120.4 (C-50), 124.5 (C-10), 139.5
(C-7), 145.3 (C-20), 146.0 (C-30), 162.9 (C-5), 163.0 (C-9), 201.1
(C-4). HR-EI-MS: m/z 288.0635 [M]+ (Calcd 288.0634 for C15H12O6).
Separation by chiral chromatography: EtOH-n-hexane (35:65
v/v) retention time (2R,3R)-isomer: 6.83 min, (2S,3S)-isomer:
Separation by chiral chromatography: EtOH-n-hexane (35:65
v/v) retention time (2R,3R)-isomer: 10.75 min, (2S,3S)-isomer:
12.08 min. (2R,3R)-isomer:
½
a 1D8
+86.1° (c = 1.0, MeOH), CD
ꢁ
(MeOH):
D
e
(nm) ꢂ2.89 (305.9), +1.89 (336.8) (c = 6.0 ꢀ 10ꢂ5).
(2S,3S)-isomer: ½a D18
ꢁ
ꢂ83.1° (c = 1.0, MeOH), CD (MeOH):
De (nm)
+1.84 (306.2), ꢂ2.95 (337.2) (c = 6.0 ꢀ 10ꢂ5).
2.9.6. 20,30-Dihydroxy-6-methoxyflavanonol 5f
Colorless crystal, mp 222-224 °C. UV kmax (MeOH) nm (loge):
252 (sh), 283 (sh), 351 (3.55). 1H NMR (600 MHz, acetone-d6) d:
3.84 (3H, s, OCH3-6), 4.87 (1H, d, J = 12.0 Hz, H-3), 5.61 (1H, d,
J = 12.0 Hz, H-2), 6.79 (1H, t, J = 7.8 Hz, H-50), 6.89 (1H, dd, J = 7.8,
1.2 Hz, H-40), 7.00 (1H, d, J = 9.0 Hz, H-8), 7.09 (1H, dd, J = 7.8,
1.2 Hz, H-60), 7.25 (1H, dd, J = 9.0, 2.4 Hz, H-7), 7.29 (1H, d,
J = 2.4 Hz, H-5). 13C NMR (150 MHz, acetone-d6) d: 56.2 (OCH3-6),
74.1 (C-3), 79.6 (C-2), 108.5 (C-5), 116.1 (C-40), 120.1 (C-8), 120.2
(C-60), 120.3 (C-10), 120.4 (C-50), 125.0 (C-10), 125.7 (C-7), 145.1
(C-20), 146.0 (C-30), 155.4 (C-6), 157.3 (C-9), 194.9 (C-4). HR-EI-
MS: m/z 302.0790 [M]+ (Calcd 302.0790 for C16H14O6).
10.08 min. (2R,3R)-isomer:
½
a 1D8
+67.9° (c = 1.0, MeOH), CD
ꢁ
(MeOH):
D
e
(nm) ꢂ2.39 (316.4), +1.47 (346.7) (c = 6.0 ꢀ 10ꢂ5).
(2S,3S)-isomer: ½a D18
ꢁ
ꢂ66.6° (c = 1.0, MeOH), CD (MeOH):
De
(nm) +1.40 (316.0), ꢂ1.48 (346.7) (c = 6.0 ꢀ 10ꢂ5).
2.9.3. 20,30,6-Trihydroxyflavanonol 5c
Colorless crystal, mp 179–181 °C. UV kmax (MeOH) nm (loge):
255 (sh), 284 (sh), 360 (3.33). 1H NMR (600 MHz, acetone-d6) d:
4.84 (1H, d, J = 12.0 Hz, H-3), 5.58 (1H, d, J = 12.0 Hz, H-2), 6.79
(1H, t, J = 7.8 Hz, H-50), 6.89 (1H, dd, J = 7.8, 1.2 Hz, H-40), 6.92
(1H, d, J = 9.0 Hz, H-8), 7.09 (1H, dd, J = 7.8, 1.2 Hz, H-60), 7.13
(1H, dd, J = 9.0, 3.0 Hz, H-7), 7.26 (1H, d, J = 3.0 Hz, H-5). 13C NMR
(150 MHz, acetone-d6) d: 74.3 (C-3), 79.5 (C-2), 111.4 (C-5), 116.1
(C-40), 119.9 (C-8), 120.2 (C-60), 120.4 (C-50), 120.5 (C-10), 125.2
(C-10), 125.6 (C-7), 145.1 (C-20), 146.1 (C-30), 152.9 (C-6), 156.4
(C-9), 195.1 (C-4). HR-ES-MS: m/z 311.0532 [M]+ (Calcd 311.0532
for C15H12O6Na).
Separation by chiral chromatography: EtOH-n-hexane (40:60
v/v) retention time (2R,3R)-isomer: 9.50 min, (2S,3S)-isomer:
5.87 min. (2R,3R)-isomer:
½
a 2D5
ꢁ
ꢂ186.0° (c = 0.1, MeOH), CD
(MeOH):
D
e
(nm) +13.27 (230.0), ꢂ5.84 (295.3) (c = 6.62 ꢀ 10ꢂ5).
(2S,3S)-isomer: ½a D25
ꢁ
+183.5° (c = 0.1, MeOH), CD (MeOH): De
(nm) ꢂ15.69 (229.6), +45.80 (295.7) (c = 6.62 ꢀ 10ꢂ5).
2.9.7. 20,30-Dihydroxy-7-methoxyflavanonol 5g
Separation by chiral chromatography: EtOH-n-hexane (55:45
v/v) retention time (2R,3R)-isomer: 6.08 min, (2S,3S)-isomer:
Colorless crystal, mp 211–213 °C. UV kmax (MeOH) nm (loge):
274 (4.17), 310 (sh). 1H NMR (600 MHz, acetone-d6) d: 3.89 (3H,
s, OCH3-7), 4.81 (1H, d, J = 12.0 Hz, H-3), 5.62 (1H, d, J = 12.0 Hz,
H-2), 6.54 (1H, d, J = 2.4 Hz, H-8), 6.70 (1H, dd, J = 9.0, 2.4 Hz,
3.92 min. (2R,3R)-isomer: ½a D18
ꢁ
+87.2° (c = 1.0, MeOH), CD (MeOH):
D
e
(nm) ꢂ4.59 (318.3), +3.94 (363.2) (c = 6.0 ꢀ 10ꢂ5). (2S,3S)-
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

4
W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
H-6), 6.80 (1H, t, J = 7.8 Hz, H-50), 6.90 (1H, dd, J = 7.8, 1.2 Hz, H-40),
7.09 (1H, dd, J = 7.8, 1.2 Hz, H-60), 7.78 (1H, d, J = 9.0 Hz, H-5). 13C
NMR (150 MHz, acetone-d6) d: 56.4 (OCH3-7), 73.4 (C-3), 79.7 (C-
2), 101.7 (C-8), 111.3 (C-6), 113.8 (C-10), 116.2 (C-40), 120.2
(C-60), 120.4 (C-50), 124.9 (C-10), 129.5 (C-5), 145.1 (C-20), 146.0
(C-30), 164.8 (C-9), 167.5 (C-7), 193.3 (C-4). HR-EI-MS: m/z
302.0791 [M]+ (Calcd 302.0790 for C16H14O6).
332.0897 [M]+ (Calcd 332.0896 for C17H16O7). The stereoisomers
of 5j could not be separated by chiral chromatography.
2.9.11. 20,30-Dihydroxy-6-methylflavanonol 5k
Colorless crystal, mp 206–208 °C. UV kmax (MeOH) nm (loge):
255 (3.91), 285 (sh), 332 (3.49). 1H NMR (600 MHz, acetone-d6)
d: 2.33 (3H, s, CH3-6), 4.87 (1H, d, J = 12.0 Hz, H-3), 5.62 (1H, d,
J = 12.0 Hz, H-2), 6.79 (1H, t, J = 7.8 Hz, H-50), 6.90 (1H, dd, J = 7.8,
1.2 Hz, H-40), 6.94 (1H, d, J = 9.0 Hz, H-8), 7.09 (1H, dd, J = 7.8,
1.2 Hz, H-60), 7.42 (1H, dd, J = 9.0, 2.4 Hz, H-7), 7.64 (1H, d,
J = 2.4 Hz, H-5). 13C NMR (150 MHz, acetone-d6) d: 20.4 (CH3-6),
74.0 (C-3), 79.4 (C-2), 116.1 (C-40), 118.6 (C-8), 120.0 (C-6), 120.2
(C-60), 120.4 (C-50), 124.9 (C-10), 127.3 (C-5), 132.0 (C-10), 138.2
(C-7), 145.1 (C-20), 146.0 (C-30), 160.8 (C-9), 195.0 (C-4). HR-EI-
MS: m/z 286.0841 [M]+ (Calcd 286.0841 for C16H14O5). The
stereoisomers of 5k could not be separated by chiral
chromatography.
Separation by chiral chromatography: EtOH-n-hexane (50:50
v/v) retention time (2R,3R)-isomer: 7.42 min, (2S,3S)-isomer:
12.83 min. (2R,3R)-isomer:
½
a 2D5
ꢁ
ꢂ86.4° (c = 0.1, MeOH), CD
(MeOH):
D
e
(nm) +6.49 (304.8), ꢂ4.80 (329.8) (c = 6.62 ꢀ 10ꢂ5).
(2S,3S)-isomer: ½a D25
ꢁ
+85.8° (c = 0.1, MeOH), CD (MeOH): De (nm)
ꢂ6.76 (304.8), +4.35 (330.8) (c = 6.62 ꢀ 10ꢂ5).
2.9.8. 20,30-Dihydroxy-6,7-dimethoxyflavanonol 5h
Colorless crystal, mp 245-247 °C. UV kmax (MeOH) nm (loge):
237 (4.35), 276 (4.11), 340 (3.84). 1H NMR (600 MHz, acetone-d6)
d: 3.84 (3H, s, OCH3-7), 3.91 (3H, s, OCH3-6), 4.77 (1H, d,
J = 12.0 Hz, H-3), 5.58 (1H, d, J = 12.0 Hz, H-2), 6.59 (1H, s, H-8),
6.80 (1H, t, J = 7.8 Hz, H-50), 6.89 (1H, dd, J = 7.8, 1.2 Hz, H-40),
7.10 (1H, dd, J = 7.8, 1.2 Hz, H-60), 7.23 (1H, s, H-5). 13C NMR
(150 MHz, acetone-d6) d: 56.5 (OCH3-7), 56.6 (OCH3-6), 73.5 (C-
3), 79.9 (C-2), 101.4 (C-8), 107.7 (C-5), 111.9 (C-10), 116.1 (C-40),
120.2 (C-60), 120.4 (C-50), 125.1 (C-10), 145.1 (C-20), 146.1 (C-30),
146.2 (C-7), 157.9 (C-6), 159.3 (C-9), 193.3 (C-4). HR-EI-MS: m/z
332.0897 [M]+ (Calcd 332.0896 for C17H16O7).
2.9.12. 20,30-Dihydroxy-7-methylflavanonol 5l
Colorless crystal, mp 189–191 °C. UV kmax (MeOH) nm (loge):
255 (3.91), 285 (sh), 332 (3.49). 1H NMR (600 MHz, acetone-d6)
d: 2.37 (3H, s, CH3-7), 4.85 (1H, d, J = 12.6 Hz, H-3), 5.63 (1H, d,
J = 12.6 Hz, H-2), 6.79 (1H, t, J = 7.8 Hz, H-50), 6.86 (1H, s, H-8),
6.90 (1H, dd, J = 7.8, 1.2 Hz, H-40), 6.96 (1H, d, J = 7.8 Hz, H-6),
7.08 (1H, dd, J = 7.8, 1.2 Hz, H-60), 7.74 (1H, d, J = 7.8 Hz, H-5). 13C
NMR (150 MHz, acetone-d6) d: 21.9 (CH3-7), 73.8 (C-3), 79.4 (C-
2), 116.1 (C-40), 118.0 (C-10), 118.8 (C-8), 120.2 (C-60), 120.4 (C-
50), 123.9 (C-6), 124.9 (C-10), 127.7 (C-5), 145.1 (C-20), 146.1 (C-
30), 148.8 (C-7), 162.8 (C-9), 194.5 (C-4). HR-EI-MS: m/z 286.0841
[M]+ (Calcd 286.0841 for C16H14O5). The stereoisomers of 5l could
not be separated by chiral chromatography.
Separation by chiral chromatography: EtOH-n-hexane (70:30
v/v) retention time (2R,3R)-isomer: 8.37 min, (2S,3S)-isomer:
10.75 min. (2R,3R)-isomer:
½
a 2D5
ꢁ
ꢂ87.6° (c = 0.1, MeOH), CD
(MeOH):
D
e
(nm) +5.16 (309.0), ꢂ5.25 (343.1) (c = 6.02 ꢀ 10ꢂ5).
(2S,3S)-isomer: ½a D25
ꢁ
+100.6° (c = 0.1, MeOH), CD (MeOH): De
(nm) ꢂ5.96 (308.6), +5.54 (343.9) (c = 6.02 ꢀ 10ꢂ5).
2.9.13. 20,30-Dihydroxy-6,7-dimethylflavanonol 5m
Colorless crystal, mp 178–180 °C. UV kmax (MeOH) nm (loge):
237 (4.35), 276 (4.11), 340 (3.84). 1H NMR (600 MHz, acetone-d6)
d: 2.26 (3H, s, CH3-7), 2.30 (3H, s, CH3-6), 4.80 (1H, d, J = 12.0 Hz,
H-3), 5.59 (1H, d, J = 12.0 Hz, H-2), 6.80 (1H, t, J = 7.8 Hz, H-50),
6.88 (1H, s, H-8), 6.89 (1H, dd, J = 7.8, 1.2 Hz, H-40), 7.08 (1H, dd,
J = 7.8, 1.2 Hz, H-60), 7.58 (1H, s, H-5). 13C NMR (150 MHz, ace-
tone-d6) d: 18.9 (CH3-7), 20.5 (CH3-6), 73.9 (C-3), 79.4 (C-2),
116.1 (C-40), 118.0 (C-10), 119.3 (C-8), 120.1 (C-60), 120.4 (C-50),
125.1 (C-10), 127.6 (C-5), 131.3 (C-6), 145.0 (C-20), 146.1 (C-30),
147.7 (C-7), 161.1 (C-9), 194.6 (C-4). HR-EI-MS: m/z 300.0998
[M]+ (Calcd 300.0998 for C17H16O5). The stereoisomers of 5m could
not be separated by chiral chromatography.
2.9.9. 20,30,8-Trihydroxy-7-methoxyflavanonol 5i
Colorless crystal, mp 220–222 °C. UV kmax (MeOH) nm (loge):
290 (4.21), 320 (sh). 1H NMR (600 MHz, acetone-d6) d: 3.94 (3H,
s, OCH3-7), 4.80 (1H, d, J = 11.4 Hz, H-3), 5.59 (1H, d, J = 11.4 Hz,
H-2), 6.80 (1H, t, J = 7.8 Hz, H-50), 6.85 (1H, d, J = 9.0 Hz, H-6),
6.89 (1H, dd, J = 7.8, 1.2 Hz, H-40), 7.10 (1H, dd, J = 7.8, 1.2 Hz, H-
60), 7.40 (1H, d, J = 9.0 Hz, H-5). 13C NMR (150 MHz, acetone-d6)
d: 56.8 (OCH3-7), 73.9 (C-3), 79.9 (C-2), 107.2 (C-6), 115.0 (C-10),
116.1 (C-40), 118.6 (C-5), 120.4 (C-60), 120.5 (C-50), 125.0 (C-10),
135.7 (C-8), 145.1 (C-20), 146.1 (C-30), 151.1 (C-9), 154.6 (C-7),
194.1 (C-4). HR-EI-MS: m/z 318.0736 [M]+ (Calcd 318.0740 for
C16H14O7).
Separation by chiral chromatography: EtOH-n-hexane (40:60
2.10. Synthesis of 2-(2,3-dimethoxyphenyl)-6,7-dimethoxy-4-
v/v) retention time (2R,3R)-isomer: 13.0 min, (2S,3S)-isomer:
oxochroman-3-yl 4-chlorobenzoate 7h
11.7 min. (2R,3R)-isomer: ½a D25
ꢂ74.5° (c = 0.1, MeOH), CD (MeOH):
ꢁ
(nm) +2.49 (304.0), ꢂ2.27 (338.5) (c = 6.28 ꢀ 10ꢂ5). (2S,3S)-iso-
Compound 5h (2R,3R) or (2S,3S) (8 mg) was dissolved in 1 mL of
EtOH and stirred in an ice bath until the temperature of the solu-
tion dropped below 4 °C. Then 3 mL of CH2N2-Et2O was added,
and the mixture was kept at 4 °C for 16 h. The organic solution
was removed under a vacuum, and then distilled water was added
to the residue and the mixture was extracted with EtOAc. The
organic layer was combined and dried over Na2SO4. The filtered
EtOAc was evaporated, and the residue was purified by silica gel
column chromatography, eluting with n-hexane and EtOAc to give
white powder 6h (75–80% yield). Compound 6h was dissolved in
1 mL of pyridine, and one drop of 4-chlorobenzoyl chloride was
added, followed by stirring of the mixture solution at room tem-
perature for 18 h. Distilled water was added to the mixture, and
then it was extracted with EtOAc three times, followed by collec-
tion and evaporation of the EtOAc. The residue was purified by
preparative HPLC to give compound 7h (70–80% yield).
D
e
mer: ½a 2D5
ꢁ
+64.1° (c = 0.1, MeOH), CD (MeOH):
D
e
(nm) ꢂ2.70
(305.2), +2.09 (338.9) (c = 6.28 ꢀ 10ꢂ5).
2.9.10. 20,30-Dihydroxy-5,7-dimethoxyflavanonol 5j
Colorless crystal, mp 224–226 °C. UV kmax (MeOH) nm (loge):
285 (4.28), 318 (sh). 1H NMR (600 MHz, acetone-d6) d: 3.87 (3H,
s, OCH3-5), 3.87 (3H, s, OCH3-7), 4.61 (1H, d, J = 12.0 Hz, H-3),
5.50 (1H, d, J = 12.0 Hz, H-2), 6.15 (1H, d, J = 1.8 Hz, H-8), 6.23
(1H, d, J = 3.0 Hz, H-6), 6.79 (1H, t, J = 7.8 Hz, H-50), 6.89 (1H, dd,
J = 7.8, 1.2 Hz, H-40), 7.10 (1H, dd, J = 7.8, 1.2 Hz, H-60). 13C NMR
(150 MHz, acetone-d6) d: 56.3 (OCH3-5), 56.4 (OCH3-7), 73.1 (C-
3), 78.6 (C-2), 93.8 (C-8), 94.5 (C-6), 104.1 (C-10), 116.1 (C-40),
120.0 (C-60), 120.5 (C-50), 125.0 (C-10), 145.0 (C-20), 146.1 (C-30),
163.2 (C-5), 166.0 (C-9), 167.6 (C-7), 191.0 (C-4). HR-EI-MS: m/z
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Colorless crystal, UV kmax (MeOH) nm (log
e
): 205 (4.77), 241
tected with chloromethyl methyl ether (MOMCl) to give com-
pounds 1 (yield 80–95%). Benzaldehydes were also protected
with MOMCl to give compounds 2 (yield 90%). Compounds 1 and
2 were treated with KOH in EtOH to give the intermediates 3 (yield
60–90%), which were then oxidized with H2O2 under alkaline con-
dition to give epoxides 4 (yield 95%). Flavanonols 5a–5m (yield 30–
55%) were then obtained by hydrolysis of the epoxides.9,29
(4.62), 276 (4.25), 338 (3.90). 1H NMR (600 MHz, acetone-d6) d:
3.84 (3H, s, OCH3-30), 3.85 (3H, s, OCH3-7), 3.87 (3H, s, OCH3-20),
3.93 (3H, s, OCH3-6), 6.06 (1H, d, J = 12.0 Hz, H-3), 6.21 (1H, d,
J = 12.0 Hz, H-2), 6.57 (1H, s, H-8), 7.06 (1H, d, J = 8.4 Hz, H-40),
7.14 (1H, t, J = 8.4 Hz, H-50), 7.23 (1H, s, H-5), 7.29 (1H, d, J = 8.4 Hz,
H-60), 7.53 (2H, d, J = 8.4 Hz, H-300, H-500), 7.95 (2H, d, J = 8.4 Hz, H-
13
200, H-600). C NMR (150 MHz, acetone-d6) d: 56.2 (OCH3-30), 56.4
(OCH3-7), 56.7 (OCH3-6), 61.6 (OCH3-20), 74.6 (C-2), 77.5 (C-3),
101.4 (C-8), 107.6 (C-5), 112.4 (C-10), 114.6 (C-40), 120.7 (C-60),
125.1 (C-50), 129.1 (C-10), 129.8 (C-300, C-500), 130.3 (C-100), 132.2 (C-
200, C-600), 140.2 (C-400), 146.4 (C-7), 149.1 (C-20), 153.8 (C-30), 158.1
(C-6), 158.8 (C-9), 164.2 (C-12), 187.2 (C-4). HR-EI-MS: m/z
498.1080 [M]+ (Calcd 498.1081 for C26H23O8Cl). (2R,3R)-isomer:
3.2. Separation of stereoisomers using a chiral column
Nine pairs of stereoisomers (enantiomers) were successfully iso-
lated from the racemic mixtures by using a chiral column. However,
4 racemates (5j, 5k, 5l, and 5m) were inseparable. Thus 5j, 5k, 5l,
and 5m were used as racemic mixtures to measure the NO assay.
CD (MeOH):
D
e
(nm) ꢂ22.51 (207.0), ꢂ13.82 (233.0), +3.26
(248.0), +4.23 (307.0), ꢂ6.89 (344.0) (c = 4.02 ꢀ 10ꢂ5). (2S,3S)-iso-
3.2.1. Determination of absolute configuration of flavanonols
The absolute configurations of the stereoisomers were deter-
mined by 1H NMR and circular dichroism (CD). All of the synthe-
sized compounds showed a coupling constant between H-2 and
H-3 (J2,3) of approximately 12 Hz. Thus, H-2 and H-3 are trans
and diaxial, and therefore, the absolute configuration has to be
either (2R,3R) or (2S,3S).
In the case of naturally occurring flavanonols, the configuration
of the C-2 carbon atom can be reduced by applying a rule pre-
sented by Gaffield:30 a positive Cotton effect at high wavelength
(300–340 nm) indicates the 2R configuration, and a negative Cot-
mer: CD (MeOH):
D
e
(nm) +25.53 (207.0), +13.88 (233.0), ꢂ4.75
(248.0), ꢂ5.97 (307.0), +6.67 (344.0) (c = 4.02 ꢀ 10ꢂ5).
3. Results
3.1. Synthesis of 20,30-dihydroxyflavanonols
The preparation of the 20,30-dihydroxyflavanonols was carried
out as illustrated in Scheme 1. Hydroxyl acetophenones were pro-
Scheme 1. Synthesis of the 20,30-dihydroxyflavanonols. Reagents and conditions: (a) NaH, MOMCl, THF, 0 °C; (b) K2CO3, MOMCl, Me2CO, 0 °C; (c) KOH, EtOH, rt; (d) 30% H2O2,
NaOHaq, MeOH, rt; (e) MeOH-HCl, 55 °C.
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

6
W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
ton effect indicates the 2S configuration. However, the absolute
configuration of the synthetic flavanonols that have methoxy
groups in the A ring (5f, 5g, 5h, and 5i) could not be determined
by this rule because the UV absorption spectra of these compounds
differ from those of natural-type flavanonols (O-functional groups
at the 5- and 7-positions in the A ring); the synthetic compounds
show three absorption maxima at around 240 nm, 280 nm, and
340 nm (Fig. 1-(A)), whereas the natural-type flavanonols show a
maxima at around 290 nm. An example of UV and CD spectra of
a
natural-type flavanonol, 20,30,5,7-tetrahydroxyflavanonol, is
available as supplemental data. To determine the absolute config-
uration of the flavanonols substituted at the 6- and 7-positions by
Fig. 1. (A) CD and UV spectra of 5h and 7h. The thick line corresponds to the (2R,3R)-isomer, and the thin line corresponds to the (2S,3S)-isomer. (B) (2R,3R) stereoisomer of
7h: Newman projection about the C3–C4 bond is shown. Arrows represent two electronic transition moments, the benzoyl substructure of the A ring and the introduced 4-
chlorobenzoyloxy substructure.
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Table 1
O-functional groups, we prepared the 4-chlorobenzoyl ester 7h
Structures and activity of the 20,30-dihydroxyflavanonols (IC50).
from 5h (Scheme 2) and determined its absolute configuration
based on the CD exciton chirality method.31
3.2.2. Synthesis of 2-(2,3-dimethoxyphenyl)-6,7-dimethoxy-4-
oxochroman-3-yl 4-chlorobenzoate 7h
The preparation of each stereoisomer of 2-(2,3-dimethoxyphe-
nyl)-6,7-dimethoxy-4-oxochroman-3-yl 4-chlorobenzoate 7h was
carried out as illustrated in Scheme 2. Each stereoisomer of 5h
was separated by chiral chromatography. Then, the 20,30-dihydroxy
groups of 5h were methoxylated by CH2N2. The product 6h was
reacted with 4-chlorobenzoyl chloride in pyridine to give com-
pound 7h, which corresponds to the original stereoisomer of 5h
(total yield 50–60%).
Compound
Substitution in the A ring
NO production inhibitory
IC50 (mM)
(2R,3R)
(2S,3S)
5a
5b
5c
5d
5e
5f
5g
5h
5i
None
5-OH
6-OH
7-OH
7,8-OH
6-OCH3
7-OCH3
6,7-OCH3
7-OCH3, 8-OH
5,7-OCH3
>100
>100
77.7 0.9
>100
>100
>100
3.2.3. Determination of the absolute configuration of flavanonols
Fig. 1-(A) shows the CD and UV spectra of each stereoisomer of
5h and 7h. Introduction of the 4-chlorobenzoyl moiety of 5h added
an inflection point at 240 nm in the CD spectrum. In the (2R,3R)
stereoisomer of 7h as shown in Fig. 1-(B), two electronic transition
moments, the 4-chlorobenzoyloxy chromophore and the benzoyl
moiety of the A/C ring (the A ring and C4 carbonyl) indicated with
arrows, make a clockwise (positive) rotation. Thus, the positive
Cotton effect at 248 nm and the negative Cotton effect at 233 nm
in the CD spectrum of 7h assign the R-configuration to C-3.
Determination of the stereochemistry of 7h deduced the CD
spectra of each of the stereoisomers of 5h. For flavanonols such
as 5f–5i, which have methoxy-substituted A-rings, the (2R,3R)
stereoisomers showed a negative Cotton effect at a longer wave-
length (340 nm) and a positive Cotton effect at a shorter wave-
length (310 nm).
70.7 7.4
17.0 1.1
>100
>100
>100
>100
24.6 2.0
50.3 1.4
49.7 2.3
Racemic mixture
57.6 2.9
Racemic mixture
45.3 3.1
Racemic mixture
31.8 0.6
Racemic mixture
20.7 2.4
30.3 0.8
>100
88.5 4.9
5j
5k
5l
6-CH3
7-CH3
6,7-CH3
5m
Reference compound: Kaempferol, IC50 = 13.4 0.5 mM.
Cell viability ꢃ 100% in all of the tested compounds.
Data are expressed as the mean S.D. (n = 3).
1.4 mM) and 20,30,8-trihydroxy-7-methoxyflavanonol 5i (93% inhibi-
tion at 100 mM, IC50 = 49.7 2.3 mM) also showed higher inhibitory
potency than the (2S,3S) isomers. The analogues with no substitu-
tion in the A ring, 5a, with 6-hydroxy substitution, 5c, and with
6-methoxy substitution, 5f, are less potent. The magnitude of the
difference in the potency between the stereoisomers roughly corre-
lated with the stereo specificity of inhibition (Fig. 2).
The racemic mixtures of compounds 5j, 5k, 5l, and 5m also
showed strong inhibitory potential against NO production
(IC50 = 57.6 2.9 mM, 45.3 3.1 mM, 31.8 0.6 mM, and 20.7
2.4 mM, respectively). Regrettably, separation of their stereoisomers
could not be done. The stereospecificity of their inhibitory potency
remains to be determined.
3.3. Cell viability
None of the compounds showed cell toxicity at 100 mM.
3.4. Inhibitory potential against NO production in RAW 264.7 cells
The inhibitory activity of the stereoisomers and racemic mix-
tures tested in the RAW 264.7 cells is summarized in Table 1.
(2R,3R)-20,30,7,8-Tetrahydroxyflavanonol 5e showed the highest
inhibitory potential (95% inhibition at 100 mM, IC50 = 17.0 1.1 -
mM). Its inhibitory potential was comparable with the positive con-
trol, kaempferol (IC50 = 13.4 0.5 mM). To our knowledge,
compound 5e is the most potent flavanonol ever reported.
(2R,3R)-20,30,7-Trihydroxyflavanonol 5d also showed comparative
potency (70% inhibition at 100 mM, IC50 = 70.7 7.4 mM). It should
be noted that the (2R,3R) stereoisomer of 5d and 5e exhibited much
higher inhibitory potency than the corresponding (2S,3S) stereoiso-
mer. The (2R,3R) stereoisomers of 20,30-dihydroxy-6,7-
dimethoxyflavanonol 5h (93% inhibition at 100 mM, IC50 = 50.3
4. Discussion
The inhibition rate (%) of the stereoisomers at the concentra-
tion of 100 mM are shown in Fig. 2. Compounds 5a (no substitution
in A ring), 5b (5-OH), 5c (6-OH), and 5g (7-OCH3) showed little
Scheme 2. Synthesis of 2-(2,3-dimethoxyphenyl)-6,7-dimethoxy-4-oxochroman-3-yl 4-chlorobenzoate 7 h. Reagents and conditions: (i) EtOH, CH2N2-Et2O solution, 4 °C; (ii)
4-chlorobenzoyl chloride, pyridine, room temperature.
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060

8
W.-J. Jiang et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
sion of NO production. In fact, none of the compounds
synthesized in this study have yet been isolated from natural
sources or synthesized elsewhere.
5. Conclusion
We prepared a series of 20,30-dihydroxyflavanonols with diver-
sity in the A ring structures. (2R,3R)-20,30,7,8-Tetrahydroxyfla-
vanonol 5e was found to be a potent inhibitor of NO production
(IC50 = 17.1 mM). To our knowledge, this is the most potent fla-
vanonol ever reported.
The (2R,3R) stereoisomers generally showed stronger inhibitory
activity against NO production than the (2S,3S) stereoisomers. For
the studied flavanonols, the stronger the inhibitory potency of an
analogue was, the larger was the difference between the potency
of the (2R,3R) stereoisomer and the (2S,3S) stereoisomer. The struc-
ture–activity relationship obtained in this study suggests the exis-
tence of a biological target that can interact with the flavanonols
and modulate NO production in RAW 264.7 cells.
Fig. 2. Effects of the 20,30-dihydroxyflavanonols on NO production. The vertical axis
shows the inhibition rate of the 20,30-dihydroxyflavanonols on NO production. The
concentration of each specimen was 100 lM. All data are expressed as the mean S.
Acknowledgments
D. (n = 3). Kp: Kaempferol (black bar: reference compound at 100 mM). The striped
bars represent the inhibitory rate of the (2R,3R)-isomers, and the meshed bars
represent that of the (2S,3S)-isomers.
We thank the Watanuki International Scholarship Foundation
and Otsuka Toshimi Scholarship Foundation for supporting the
study and research of author W-J. J.
stereospecificity of the inhibitory effects on NO production. In con-
trast, evident stereospecificity was found in compounds 5d (7-
OH), 5e (7,8-OH), and 5i (7-OCH3, 8-OH). The (2R,3R)-isomer of
5e showed the highest inhibitory potency, whereas the (2S,3S)-
isomer showed a very weak inhibitory rate at 100 mM (0%). Com-
paring the structures of 5d with 5e, introduction of a hydroxyl
group at the 8-position would increase both stereospecificity and
inhibitory activity. Compounds 5c (6-OH) and 5f (6-methoxy)
showed very weak inhibitory activity on NO production. However,
compound 5k (6-CH3, racemic mixture [Table 1]) showed higher
inhibitory potency. Compound 5h (6,7-OCH3) showed stronger
inhibitory potential with enhanced stereospecificity than that of
5f. The presence of a methoxy group at the 7-position of the A ring
would enhance inhibitory potency and stereospecificity. Com-
pound 5g (7-OCH3) showed strong inhibitory potency but little
stereospecificity. Compound 5l (7-methyl), which is a racemic
mixture, also showed high potency (Table 1). The additional sub-
stitution of a hydroxyl group at the 8-position in 5g (7-OCH3)
resulted in clear stereospecificity (5i: 7-OCH3, 8-OH). The sub-
stituent at the 7-position of the A ring is essential for 20,30-dihy-
droxyflavanonols to exhibit inhibitory activity, and the
stereospecificity of the inhibition will increase when an additional
substituent is introduced at the 6- or 8-positions. It should be
noted that compound 5m (6,7-CH3, racemic mixture [Table 1])
exhibited an IC50 value of 20 mM. Considering the high potency
of 5m, its (2R,3R) stereoisomer might be a very potent compound.
The hydrophobicity of the substituents at the 7-position of the A
ring might be important (Table 1: 5d < 5l < 5g, 5h < 5m). Com-
pound 5j (5,7-OCH3, racemic mixture [Table 1]) showed an inhibi-
tion rate of 58% on NO production. Considering that the (2R,3R)
stereoisomer of 20,30,5,7-tetrahydroxyflavanonol, i.e., (2R,3R)-des-
methoxy-5j, showed 70% inhibition at 100 mM,27 substituents at
the 5- and 7-positions would function as hydrogen-bonding
acceptors or molecular volumes.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.05.060.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References
1. Yamamoto Y, Gaynor RB. J Clin Invest. 2001;107:135–142.
2. Cazarolli LH, Zanatta L, Alberton EH, et al. Mini-Rev Med Chem.
2008;8:1429–1440.
3. Osadebe PO, Okoye FB. J Ethnopharmacol. 2003;89:19–24.
4. Rotelli AE, Guardia T, Juárez AO, de la Rocha NE, Pelzer LE. Pharmacol Res.
2003;48:601–606.
5. Schuier M, Sies H, Illek B, Fischer H. J Nutr. 2005;135:2320–2325.
6. Havsteen BH. Pharmacol Ther. 2002;96:67–202.
7. Ng TB, Liu F, Wang ZT. Life Sci. 2000;66:709–723.
8. Weisburger JH. Food Chem Toxicol. 1999;37:943–948.
9. Pandurangan N, Bose C, Banerji A. Bioorg Med Chem Lett. 2011;21:5328–5330.
10. Pietta PG. J Nat Prod. 2000;63:1035–1042.
11. Mlcek J, Jurikova T, Skrovankova S, Sochor J. Molecules. 2016;21.
12. Rahman M, Gray AI. Phytochemistry. 2002;59:73–77.
13. Cushnie TP, Lamb AJ. Int J Antimicrob Agents. 2011;38:99–107.
14. Manner S, Skogman M, Goeres D, Vuorela P, Fallarero A. Int
J Mol Sci.
2013;14:19434–19451.
15. Vlietinck AJ, De Bruyne T, Apers S, Pieters LA. Planta Med. 1998;64:97–109.
16. Kaul TN, Middleton Jr E, Ogra PL. J Med Virol. 1985;15:71–79.
17. Friedman M. Mol Nutr Food Res. 2007;51:116–134.
18. Way TD, Kao MC, Lin JK. FEBS Lett. 2005;579:145–152.
19. Yilmaz Y, Toledo RT. Trends Food Sci Technol. 2004;15:422–433.
20. Moridani MY, Galati G, O’ Brien PJ. Chem Biol Interact. 2002;139:251–264.
21. Sousa RR, Queiroz KC, Souza AC, et al.
2007;22:439–444.
J Enzyme Inhib Med Chem.
22. Asgari S, Naderi GH, Sarrafzadegan N, et al. Pharm Acta Helv. 1999;73:223–236.
23. Kitanaka S. Yakugaku Zasshi. 2016;136:1195–1216.
24. Daikonya A, Kitanaka S. Chem Pharm Bull. 2011;59:1567–1569.
25. Daikonya A, Kitanaka S. Jpn J Food Chem Saf. 2011;18:183–190.
26. Jiang W-J, Daikonya A, Ohkawara M, et al. Bioorg Med Chem. 2017;25:779–788.
27. Jiang W-J, Ishiuchi K, Furukawa M, Takamiya T, Kitanaka S, Iijima H. Bioorg Med
Chem. 2015;23:6922–6929.
28. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.
Anal Biochem. 1982;126:131–138.
29. Jr BR, Fink RC, McMichael MD, Li D, Alberte RS. Phytochemistry.
2009;70:1255–1261.
We observed stronger inhibitory activity in the (2R,3R)
stereoisomers than in the (2S,3S) stereoisomers. The results of this
study indicate that the inhibitory potency of flavanonols relates to
their three-dimensional features such as the existence of a sub-
stituent at the 7-position and an additional substituent in the
neighborhood of the 7-position.
30. Gaffield W. Tetrahedron. 1970;26:4093–4108.
31. Harada N, Nakanishi K. Circular Dichroic Spectroscopy Exciton Coupling in Organic
Stereochemistry. CA: University Science Books; 1983. Oxford: Oxford University
Press.
The existence of a structure-activity relationship strongly sug-
gests the possibility of designing a novel compound for suppres-
Please cite this article in press as: Jiang W.-J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.060