First synthesis and in vitro biological assessment of isosideroxylin, 6,8-dimethylgenistein and their analogues as nitric oxide production inhibition agents

Article abstract of DOI:10.1016/j.cclet.2016.12.041

A modular and efficient synthesis of the biologically significant C-methylisoflavones isosideroxylin (1), 6,8-dimethylgenistein (2) and their analogues (3–8) is established for the first time. The synthesis is realized in 7-8 steps in overall yields of 16%-24% from commercially inexpensive phloroglucinol and features a high yielding Vilsmeier–Haack reaction, Friedel-Crafts acylation, Gammill's protocol and Suzuki coupling as the pivotal transformations. Next, these compounds evaluated for their inhibitory potency on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated RAW-264.7 cells as an indicator of anti-inflammatory activity. The results showed that all the compounds decreased NO production in a dose-dependent manner without marked cytotoxicity and IC₅₀ values are found in the range of 10.17–33.88?μmol/L. Of note, compounds 3 followed by 1, 7 and 8 show comparable inhibitory activity with positive control (N-monomethyl-L-arginine, L-NMMA).

Full text of DOI:10.1016/j.cclet.2016.12.041

Accepted Manuscript
Title: First synthesis and in vitro biological assessment of
isosideroxylin, 6,8-dimethylgenistein and their analogues as
nitric oxide production inhibition agents
Author: <ce:author id="aut0005"
author-id="S100184171730027X-
9
3b7a4dbf35d8eded5392619d29542d9"> Jong-Woon
Jung<ce:author id="aut0010"
author-id="S100184171730027X-
5604bdb6af8dbea53cd6d559220cf0f7"> Kongara
Damodar<ce:author id="aut0015"
author-id="S100184171730027X-
9
fb913d1dbd4919edbe2f59e717f88ee"> Jin-Kyung
Kim<ce:author id="aut0020"
author-id="S100184171730027X-
6
9d740812ee195bf96178623a4d72543"> Jong-Gab
Jun
PII:
S1001-8417(17)30027-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2016.12.041
CCLET 3954
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
31-10-2016
6-12-2016
14-12-2016
Please cite this article as: Jong-Woon Jung, Kongara Damodar, Jin-Kyung Kim, Jong-
Gab Jun, First synthesis and in vitro biological assessment of isosideroxylin, 6,8-
dimethylgenistein and their analogues as nitric oxide production inhibition agents,
Chinese Chemical Letters http://dx.doi.org/10.1016/j.cclet.2016.12.041
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Original article
First synthesis and in vitro biological assessment of isosideroxylin, 6,8-
dimethylgenistein and their analogues as nitric oxide production inhibition agents
a
a
b
a 
Jong-Woon Jung , Kongara Damodar , Jin-Kyung Kim , Jong-Gab Jun
a
Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 24252, South Korea
Department of Biomedical Science, College of Natural Science, Catholic University of Daegu, Gyeungsan-Si 38430, South Korea
b

Corresponding author.
E-mail address: jgjun@hallym.ac.kr
—
——

Graphical abstract
A modular and efficient first synthesis of the biologically active C-methylisoflavones isosideroxylin (1), 6,8-dimethylgenistein
(
2) and their analogues (3-8) is achieved in 7-8 steps with overall yields of 16%-24%. Next, in their nitric oxide (NO)
production inhibition assay in lipopolysaccharide-induced RAW 264.7 macrophages as an indicator of anti-inflammatory
activity, all compounds showed moderate to good inhibitory activity in a concentration-dependent manner without notable
cytotoxicity and IC50 values are found in the range from 10.17 μmol/L to 33.88 μmol/L.
ABSTRACT
Available online
A modular and efficient synthesis of the biologically significant C-methylisoflavones isosideroxylin
(1), 6,8-dimethylgenistein (2) and their analogues (3-8) is established for the first time. The synthesis is realized in 7-8 steps in overall yields of 16%-
2
4% from commercially inexpensive phloroglucinol and features a high yielding Vilsmeier–Haack reaction, Friedel-Crafts acylation, Gammill’s
protocol and Suzuki coupling as the pivotal transformations. Next, these compounds evaluated for their inhibitory potency on the production of nitric
oxide (NO) in lipopolysaccharide (LPS)-activated RAW-264.7 cells as an indicator of anti-inflammatory activity. The results show that all the
compounds decreased NO production in a dose-dependent manner without marked cytotoxicity and IC50 values are found in the range of 10.17-33.88
μmol/L. Of note, compounds 3 followed by 1, 7 and 8 show comparable inhibitory activity with positive control (N-monomethyl-L-arginine, L-
NMMA).
Keywords: Isosideroxylin, 6,8-Dimethylgenistein, Vilsmeier–Haack reaction, Suzuki coupling, Nitric oxide (NO), Anti-inflammatory
1. Introduction
Inflammation is an early protective reaction of a host, initiated after infection and/or injury. This complex but highly coordinated
process results in the increased production of various soluble mediators including chemokines, cytokines, free radicals (such as nitric
oxide (NO)), and eicosanoids (prostaglandins) by resident cells (that is, tissue macrophages, lymphocytes, fibroblasts, endothelial cells
and mast cells) in the injured or infected tissue [1]. Recently, inflammatory related diseases treatments are mainly associated with
interrupting the synthesis or action of these mediators. NO, a multifunctional gaseous free radical, is one of the key signaling molecules
that is synthesized from the amino acid L-arginine by nitric oxide synthase (endothelial-NOS, neuronal-NOS and inducible-NOS). It
regulates various physiological and pathophysiological processes and its role in the pathogenesis of inflammation tightly depends upon
its concentration [2]. Prolonged and overproduction of NO could potentially result in tissue damage and activation of proinflammatory
mediators associated with acute and chronic inflammations such as rheumatoid arthritis, asthma, diabetes, stroke, cancer and
neurodegenerative disorders [3]. Hence, suppression of overproduction of NO represents a beneficial therapeutic strategy.
Nonsteroidal anti-inflammatory drugs (NSAIDs) which act on cyclooxygenase (COX-1 and -2), classical steroidal anti-inflammatory
drugs (SAIDs), antihistamines and selective COX-2 inhibitors (COXIBs) are generally used to treat a wide spectrum of inflammatory
diseases. Although these small-molecule inhibitors have provided some relief to patients suffering from pain and inflammation, they
are not without their shortcomings. Therefore, there is a need for the identification and development of safe, effective and novel anti-
inflammatory agents.
Isoflavones, as members of the flavonoid family, are important bioactive secondary metabolites of plants, chiefly, found in legumes
(Fabaceae) such as soya, chick pea and fenugreek. Generally, these compounds exist as their glycosides. Their beneficial health effects
are known for a long time. Nowadays, soy products/soy isoflavones (such as genistein, daidzein and glycitein) are very common in
human diet for their various presumed health benefits [4]. The broad spectrum biological activities of isoflavones include anti-
inflammatory [5], antioxidant [6], anticancer [7], antiviral [8], antifungal [9], antibacterial [10], anticataracts [11] and antifertility [12].
Apart from this, some isoflavones are investigated as kinase inhibitors [13]. They are also effective in human obesity and have a
positive influence on plasma cholesterol [14]. Low rates of prostate and breast cancers incidents among Asians when compared to

people in the Western world could be related to the difference in consumption of isoflavones in Asian diets (15-47 mg/day) compared
with Western diets (0.15-1.7 mg/day) [15]. In recent decades, research on isoflavone and related compounds has been received
considerable attention around the globe due to their ample occurrence in nature, accessible structural modifications, effortless synthesis
and multifarious biological activities.
As part of our continuing research efforts focused on the synthesis and biological evaluation of bioactive natural products and their
analogues as potential NO inhibitors [16], herein, we describe the first synthesis of naturally occurring C-methylisoflavones
isosideroxylin (1), 6,8-dimethylgenistein (2) and their derivatives (3-8) and preliminary investigation of their NO inhibitory potency in
lipopolysaccharide (LPS) macrophage cell line RAW264.7 as an indicator of anti-inflammatory effect. Compound 1 was isolated from
Leiophyllum buxifolium and displayed a selective antiproliferative effect against MDAMB- 231 cells with an IC50 value of 7.0 μmol/L
and also showed weak inhibition against the MCF-7 cells [17]. Compound 2 was isolated from Henriettella fascicularis and showed
significant competitive binding to estrogen receptor-β with an IC50 value of 0.88 μmol/L and moderate antiestrogenic activity with
cultured Ishikawa cells [18].
2. Results and discussion
2.1 Chemistry
Our retrosynthetic analysis of isoflavones 1-8 is outlined in Scheme 1. We envisioned a selective or complete O-demethylation of 3,
-13 which in turn obtained by a Suzuki coupling between 3-iodochromone 14 and corresponding boronic acids results the target
9
compounds. Compound 14 can be easily accessed from a fully functionalized acetophenone, 15 by Gammill’s protocol [19] which
involves the condensation of 15 with DMF-DMA followed by iodine-mediated ring closure of the resulting enaminoketone. Compound
15 can be conveniently prepared from phloroglucinol (16) by 4 step synthetic sequence comprising Vilsmeier–Haack reaction,
reduction, methylation and Friedel-Crafts acylation.
Thus, our synthetic approach commenced with the Vilsmeier–Haack reaction of phloroglucinol (16) (Scheme 2). Treatment of 16
with 2 equiv. in situ generated Vilsmeier reagent from DMF and POCl afforded dialdehyde 17 in 87% yield. Facile reduction of two
3
carbonyl groups gave compound 18 using a Clemmensen reduction type condition. Reaction of 17 with 3 equiv. sodium
cyanoborohydride/ HCl (3 mol/L) system in THF and methyl orange as an indicator offered 18 in almost quantitative yield (98%).
Previously, two methods reported to prepare 18, however, these processes were plagued by low yields, harsh conditions and usage of
toxic reagent [20]. When we tried the one-pot reaction between 18 and 2-(4-hydroxyphenyl)acetic acid (19) followed by treatment with
PCl
or Ac
To circumvent this detrimental effect of the hydroxy groups, 18 was converted as 20 using dimethyl sulfate. Now, compound 20 was
subjected to Friedel-Crafts acylation using AcCl and AlCl . Reaction in diethyl ether and THF solvents gave acetylated product 21 in
1% and 76%, respectively, whereas with Ac O/BF ·Et O in solvent-free condition the desired 2'-hydroxyacetophenone 15 was
5
/DMF as a model reaction, compound 2 was obtained in low yield (14%) [21]. Next, Friedel-Crafts acylation over 18 using AcCl
2
O in presence of boron trifluoride diethyl etherate (BF ·Et O) or AlCl led to only the O-acetylation product as major compound.
3
2
3
3
5
2
3
2
obtained in 75% yield. Subsequently, compound 15 was applied to the Gammill’s method to generate iodochromone 14 via
enaminoketone 22 formation. Condensation of 15 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) gave 22 in 92% yield.
Iodine-mediated tandem cyclization and iodination of the enaminoketone 22 in CHCl
With the requisite precursor 14 in hand, next we executed the Suzuki coupling to achieve 1-8. Treatment of 14 with 1.5 equiv.
boronic acids 23-28, 1.5 equiv. Cs CO and 0.1 equiv. Pd(PPh Cl in sealed tube at 75 ºC led to the formation of isoflavones 3 and 9-
3 in 69%-85% yields. Finally, we were delighted to observe that the selective ortho-demethylation of 3 and 9-13 using 1.0 mol/L
3
produced the 3-iodochromone 14 in 67% yield.
2
3
3
)
2
2
1
boron trichloride solution in CH
2
2
Cl at 0 ºC for 1 h gave isosideroxylin (1) and analogues 4-8 in 94%-99% yields.
Another natural isoflavone, 6,8-dimethylgenistein (2) was obtained from 3 by complete O-demethylation using 1.0 mol/L boron
1
13
tribromide solution in CH
2
Cl . All the products 1-8 were structurally confirmed by their spectral data ( H NMR & C NMR and MS).
2
2
.2. Pharmacology
The synthesized isoflavonoids 1-8 were examined for their capacity to inhibit the production of NO in murine RAW 264.7
macrophage cells stimulated with LPS. As we know that NO is a highly reactive molecule with very short half-life (<1 s in blood) in
−
−
the presence of O
2
and other scavenging molecules and hence difficult to quantify [22]. Nitrite (NO
2
) and nitrate (NO ), the stable
3
oxidation end products of NO, are measured spectrophotometrically by employing the acidic Griess reagent as an index of systemic
G
NO production. Cell viability was determined by the MTT assay and N -monomethyl-L-arginine acetate (L-NMMA) [23] was
employed as positive control. Initially, cells were pretreated with compounds 1-8 at 0.1, 1, 10, 25, 50 and 100 µmol/L concentrations
for 12 h followed by activation with LPS (500 ng/mL) for an additional 18 h. However, significant NO inhibition activity changes were
observed at 1 µmol/L to 10 µmol/L. At 25, 50 and 100 µmol/L concentrations, compounds 1-8 exhibited almost same level activity.
Hence, we discussed the activity at 1 µmol/L and 10 µmol/L concentrations only. As shown in Table 1, all the isoflavonoids (1-8) had
a concentration- dependent inhibition on NO production. At the lowest concentration (1 µmol/L), compound 2 (80.53 ± 3.07) followed
by compounds 3 (86.79 ± 1.58) followed by 4 (88.78 ± 2.49) released the lower amount of NO that means the better NO inhibition
potency compared to the positive control, L-NMMA (88.88 ± 9.32). At the highest concentration (10 µmol/L), compound 3 (51.58 ±
2
5
.27) followed by compounds 1 (66.34 ± 3.46) and 6 (67.69 ± 5.33) released the comparable amount of NO with L-NMMA (40.95 ±
.98).

Next, the cytotoxicity of isoflavones against RAW 264.7 cells viability was analyzed to ensure that cell death was not responsible
for the decreased NO production. Incubation with 1 µmol/L and 10 µmol/L concentrations of isoflavones for 24 h did not cause any
significant viability changes compared to the control group which indicates that the compounds exerted no cytotoxic effect and do not
affect normal cell growth (Table 1). The IC50 values of 1-8 were evaluated by using GraphPad Prism 4.0 software and the values were
13.2, 10.17, 20.09, 20.89, 33.88, 21.93, 13.21 and 14.67 µmol/L, respectively compared to L-NMMA (7.82 µmol/L). Overproduction
of NO is due to the iNOS high expression, hence, we studied the ability of compounds 1-8 to modulate the LPS-induced iNOS
expression. As shown in Fig. 2, expression was markedly attenuated in RAW 264.7 macrophages by pretreatment with 3 which was
also correlated with the NO measurement (Table 1). Therefore, the reduced expression of iNOS due to this compound exposure was
responsible for the significant inhibition of NO production. Based on these pharmacological results, it can be concluded that compound
3
which strongly inhibited LPS-stimulated NO production without significant cytotoxicity could be deserved as a promising NO
production inhibitor agent for further investigation.
3. Conclusion
This work highlights the first concise and efficient synthesis of naturally occurring bioactive C-methylisoflavones isosideroxylin (1),
,8-dimethylgenistein (2) and their derivatives (3-8) in 7-8 steps with an overall yields of 16-24% from commercially available
6
precursors. In this strategy Vilsmeier–Haack reaction, Friedel-Crafts acylation, Gammill’s protocol and Suzuki coupling reactions were
applied as the key steps to realize the synthesis. Subsequently, compounds 1-8 were assayed for their potential to inhibit the NO
production in LPS-induced RAW 264.7 macrophage cells. All the samples tested reduced NO production in a concentration-dependent
manner and did not show obvious cytotoxic effect at the highest concentration (10 µmol/L) leading to the effective inhibition with IC50
values ranged from 10.17 to 33.88 μmol/L. Of note, compounds 3 (IC50 = 10.17 µmol/L) followed by 1 (IC50 = 13.2 µmol/L), 7 (IC50
3.21 µmol/L) and 8 (IC50 = 14.67 µmol/L) showed comparable inhibitory activity with L-NMMA (IC50 = 7.82 µmol/L), used as
positive control. This modular approach could also be used for the synthesis of other C-methylisoflavone analogues.
=
1
4. Experimental
All chemicals were obtained from commercial suppliers and were used without further purification unless stated otherwise. All
solvents used for reactions were freshly distilled from proper dehydrating agents under nitrogen atmosphere. All solvents used for
1
chromatography were purchased and directly used without further purification. H NMR spectra were recorded on a Varian Mercury-
1
3
3
00 MHz FT-NMR and 75 MHz for C, with the chemical shift (δ) reported in parts per million (ppm) downfield relative to TMS and
the coupling constants (J) quoted in Hz. Peak splitting patterns were abbreviated as s (singlet), d (doublet), t (triplet), q (quartet), dd
doublet of doublet) and m (multiplet) and CDCl /CD OD/acetone-d /DMSO-d was used as a solvent and an internal standard. Low
resolution mass spectra (electron ionization, EI) were recorded using Agilent-5977E spectrometer. High resolution mass spectra
electron ionization, EI) were recorded on a JMS-700 (JEOL, Japan) spectrometer. Melting points were measured on a MEL-TEMP II
(
3
3
6
6
(
apparatus and were uncorrected. Thin-layer chromatography (TLC) was performed on DC-Plastikfolien 60, F254 (Merck, layer
thickness 0.2 mm) plastic-backed silica gel plates and visualized by UV light (254 nm) or staining with p-anisaldehyde and
phosphomolybdic acid (PMA) stain. Chromatographic purification was carried out using Kieselgel 60 (60-120 mesh, Merck).
2.1 General procedure for Suzuki coupling
To a 15 mL sealed tube equipped with a magnetic stir bar were added the iodo compound 14 (0.07 g, 0.2 mmol, 1.0 equiv.), boronic
acid (23-28) (1.5 equiv.), Cs
for 2 min. Pd(PPh Cl (0.01 g, 0.02 mmol, 0.1 equiv.) was added to the mixture and degassed for another 2 min. The reaction was
then stirred at 75 ºC for 12 h. After cooling to room temperature, water (10 mL) was added and extracted with ether (3 × 15 mL). The
combined organic layer was washed with H O (3 × 10 mL), brine (2 × 10 mL), dried over anhyd. Na SO and concentrated in vacuo.
The crude was purified by column chromatography (EtOAc/hexane=1/5-1/3) to afford the pure isoflavone product (3, 9-13).
2
CO
3
(0.10 g, 0.3 mmol, 1.5 equiv.), and N,N-dimethylformamide (2 mL) and the mixture was degassed
3
)
2
2
2
2
4
2.2 General procedure for ortho-demethylation of isoflavone using boron trichloride
o
To a stirred solution of isoflavone (3, 9-13) (0.12 mmol, 1.0 equiv.) in anhydrous CH
2
Cl (3 mL) at 0 C was added boron trichloride
2
(
0.6 mL, 1.0 mol/L in CH
excess reagent was quenched with MeOH (1 mL) and extracted with CH
10 mL), brine (2 × 10 mL), dried over anhyd. Na SO and concentrated in vacuo. The crude was purified by column chromatography
2
Cl
2
, 5.0 equiv.) dropwise under nitrogen atmosphere and stirred for 1 h. After completion of the reaction,
2
Cl (2 × 15 mL). Combined organic layer washed with H O (2
2
2
×
2
4
(EtOAc/hexane=1/5-1/3) to afford the pure product (1, 4-8).
Physical and spectroscopic characterization data of the compounds and experimental procedures described in this article were given
in Supporting information.
Acknowledgment
This research was financially supported by industrial co-work program with SunChem (SunKyung Chemical Co., South Korea).

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Fig. 1. Structures of isosideroxylin (1), 6,8-dimethylgenistein (2) and their analogues (3-8).
0
.08
1
1.6
1.18
1.24
1.74
1.26
1.44
1.20
1.04
0.70
iNO
β-actin
MED
LPS
1
3
2
4
5
6
7
8
NMMA
Compound (10 μmol/L)
Fig. 2. Effects of compounds 1-8 on iNOS expression (Western blot).

Scheme 1. Retrosynthetic analysis of 1-8.

Scheme 2. Synthesis of isosideroxylin (1), 6,8-dimethylgenistein (2) and their analogues (3-8).

Table 1
NO production inhibitory effects of isosideroxylin (1), 6,8-dimethylgenistein (2) and their analogues (3-8).
a,b
a
Compd.
NO Production
Proliferation
IC50 (µmol/L)
1
µmol/L
10 µmol/L
1 µmol/L
10 µmol/L
Medium
7.80 ± 2.71
7.80 ± 2.71
100.0 ± 8.75
100.0 ± 8.75
LPS
1
2
3
4
5
6
7
8
100.0 ± 6.15
99.05 ± 3.29
80.53 ± 3.07*
86.79 ± 1.58
88.78 ± 2.49
92.20 ± 10.72
94.10 ± 0.86
98.82 ± 2.06
100.0 ± 8.69
88.88 ± 9.32
100.0 ± 6.15
66.34 ± 3.46*
68.00 ± 1.12**
51.58 ± 2.27**
73.26 ± 5.59*
90.12 ± 9.05
67.69 ± 5.33*
68.21 ± 5.17*
72.55 ± 4.95*
40.95 ± 5.98***
124.3 ± 6.58
107.6 ± 4.99
121.1 ± 9.33
121.8 ± 12.60
109.6 ± 12.81
118.4 ± 11.96
114.7 ± 8.02
114.7 ± 8.12
102.2 ± 5.66
110.0 ± 0.99
99.49 ± 3.97
104.6 ± 7.28
101.5 ± 7.69
108.4 ± 6.21
93.18± 2.93
109.8 ± 6.00
105.0 ± 6.34
100.7 ± 3.84
13.20
20.09
10.17
20.89
33.88
21.93
13.21
14.67
7.82
L-NMMA
a
The results are reported as mean value ± SEM for n = 3. Statistical significance is based on the difference when compared with LPS-treated groups (*P <
.05, **P < 0.01, ***P < 0.001).
Inhibition is based on LPS.
0
b