Identification and structure activity relationship of novel flavone derivatives that inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2017.03.057

In an effort to identify novel anti-inflammatory compounds, a series of flavone derivatives were synthesized and biologically evaluated for their inhibitory effects on the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), representative pro-inflammatory mediators, in LPS-induced RAW 264.7 cells. Their structure-activity relationship was also investigated. In particular, we found that compound 3g displayed more potent inhibitory activities on PGE₂ production, similar inhibitory activities on NO production and less weak cytotoxicity than luteolin, a natural flavone known as a potent anti-inflammatory agent.

Full text of DOI:10.1016/j.bmcl.2017.03.057

Accepted Manuscript
Identification and structure activity relationship of novel flavone derivatives that
inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells
Ji-Young An, Hwi-Ho Lee, Ji-Sun Shin, Hyung Seok Yoo, Jong Seon Park,
Seung Hwan Son, Sang Won Kim, Jihyun Yu, Jun Lee, Kyung-Tae Lee, Nam-
Jung Kim
PII:
S0960-894X(17)30310-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.057
BMCL 24809
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 December 2016
7 March 2017
22 March 2017
Please cite this article as: An, J-Y., Lee, H-H., Shin, J-S., Yoo, H.S., Park, J.S., Son, S.H., Kim, S.W., Yu, J., Lee,
J., Lee, K-T., Kim, N-J., Identification and structure activity relationship of novel flavone derivatives that inhibit
the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.057
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Graphical Abstract
Leave this area blank for abstract info.
Identification and structure activity
relationship of novel flavone derivatives that
inhibit the production of nitric oxide and
PGE2 in LPS-induced RAW 264.7 cells
Ji-Young An, Hwi-Ho Lee, Ji-Sun Shin, Hyung Seok Yoo, Jong Seon Park, Seung Hwan Son, Sang Won Kim,
Jihyun Yu, Jun Lee, Kyung-Tae Lee*, Nam-Jung Kim*
O
R1: H, CH3, F, Cl, NO2, OMe, OPiv and etc
R : H, -Bu, F, Cl, CF , TMS, OMe, OH, and etc
t
R1
2
3
(R1 = H, R2 = 3',4'- -OH)
O
Flavone 3g
di
R2
PGE2 Production IC50 = 0.99
NO Production IC50 = 6.29
M
M
27 Flavone derivatives

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Identification and structure activity relationship of novel flavone derivatives that
inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells
Ji-Young Ana,†, Hwi-Ho Leeb,†, Ji-Sun Shinb, Hyung Seok Yooa, Jong Seon Parka, Seung Hwan Sona, Sang
a
Won Kim , Jihyun Yu , Jun Lee , Kyung-Tae Lee , Nam-Jung Kim
a
a
b,
a,
∗
∗
a Department of Pharmacy, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea
b Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
In an effort to identify novel anti-inflammatory compounds, a series of flavone derivatives were
synthesized and biologically evaluated for their inhibitory effects on the production of nitric
oxide (NO) and prostaglandin E2 (PGE2), representative pro-inflammatory mediators, in LPS-
induced RAW 264.7 cells. Their structure-activity relationship was also investigated. In
particular, we found that compound 3g displayed more potent inhibitory activities on PGE2
production, similar inhibitory activities on NO production and less weak cytotoxicity than
luteolin, a natural flavone known as a potent anti-inflammatory agent.
Revised
Accepted
Available online
Keywords:
Flavone;
Nitric oxide (NO);
Prostaglandin E2 (PGE2);
Anti-inflammatory;
RAW 264.7 cell;
2009 Elsevier Ltd. All rights reserved.
In the pathological states related to chronic inflammatory
diseases, various pro-inflammatory mediators including
prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-
α), mainly produced from activated immune cells, are
upregulated and orchestrated, leading to the destruction of
normal tissues.1 This symptom generally occurs in auto-immune
disorders. Thus, modulating a series of pro-inflammatory
mediators has been considered as an important strategy to
alleviate auto-immune diseases such as rheumatoid bowel disease
(IBD), rheumatoid arthritis (RA) and multiple sclerosis, which
are accompanied with chronic inflammation. Among pro-
inflammatory mediators, nitric oxide (NO) and PGE2 are some of
the important chemokines for inflammation2, and the inhibitors of
their production have been pursued for the development of novel
anti-inflammatory therapeutics.3
kinase 9 (CDK9) inhibitor, have recently entered into clinical
trials5 or have been used clinically.6 Up to date, anti-
inflammatory activities of flavones have been extensively
investigated.4d,7 However, most of them are focused on natural
products such as luteolin and nobiletin (Fig. 1) while the
investigations of the anti-inflammatory activities of synthetic
flavones have been less pursued.8 In particular, synthesis of anti-
inflammatory flavone derivatives inhibiting NO and PGE2
production9, and their systemic structure-activity relationship
have been rarely reported.
Flavones, a kind of flavonoid, are natural products isolated
from various plants and their synthetic derivatives based on the
backbone of 2-phenylchromene-4-one. Interestingly, some of
them show diverse biological activities4 such as anti-
inflammatory, anti-oxidant and anti-cancer effects as well as
suitable physicochemical properties. Thus, their structures are
considered as privileged. For this reason, a few synthetic flavone
d—er—ivat—ives including flavopiridol, known as a cyclin-dependent
Figure 1. Representative structures of biologically active flavones
∗ Corresponding author. Tel.: +82-2-961-0860; fax: +82-2-961-9580; e-mail: ktlee@khu.ac.kr (K.-T. Lee)
∗ Corresponding author. Tel.: +82-2-961-0580; fax: +82-2-961-9580; e-mail: kimnj@khu.ac.kr (N.-J. Kim)
†These authors contributed equally to this work.

Considering the privileged potential of synthetic flavones and
the insufficient investigation of their anti-inflammatory
properties, synthesis of novel flavones and biological evaluation
of their inhibitory effect on NO and PGE2 production are quite
necessary for providing novel scaffolds as novel anti-
inflammatory therapeutics.
A variety of flavone derivatives were efficiently synthesized
via a concise cascade reaction of dehydrogenation and oxidative
Heck coupling in one-pot Pd(II) catalysis, a synthetic
methodology recently reported.10 As starting materials for the
Pd(II) catalyzed flavone synthesis, chromanones and pinacol
boronic esters were initially prepared from commercial sources
or by a known protocol,12 respectively. Using these starting
materials, most of the desired flavone derivatives such as 3a-d, 3f,
3h-z, and 4 were synthesized via a unified Pd(II)-catalyzed
dehydrogenation and oxidative Heck coupling sequence in a one
pot. Some hydroxy flavones such as 3e and 3g13 were yielded
through additional demethylation of 3d and 3f, respectively, in
the presence of excess pyridinium hydrochloride.
In this study, we synthesized a total of 27 flavone derivatives
via one pot Pd(II) catalysis (Fig.2),10 and evaluated their
inhibitory activities on NO and PGE2 production using a cell-
based assay.11 Based on the biological activities of synthesized
compounds, we also investigated the structure-activity
relationship and thereby identified novel and potent anti-
inflammatory flavone derivatives, which would hopefully
provide a way to develop novel anti-inflammatory drug
candidates.
With these compounds in hand, we attempted to evaluate their
anti-inflammatory activities, particularly focused on the NO and
PGE2 production inhibitory properties. As an initial screening,
we assessed their inhibitory effects on NO production at 20 µM
in LPS-induced Raw 264.7 cells, a murine macrophage cell line
that is commonly used in the screening for the identification of
anti-inflammatory compounds. Cytotoxicities of the synthesized
compounds were also evaluated using an MTT assay in the same
cell line to investigate whether the inhibitory activities on NO
production were caused by their cytotoxic activities. An initial
assay result of the synthesized compounds is summarized in table
1.
Figure 2. Retrosynthesis of novel flavone derivatives
Scheme 1. Synthesis of compound 3a-z and 4. Reagents and conditions: (a) Pd(TFA)2, 5-nitrophenathroline, DMSO, O2, 100 °C, 27-93%; (b) Pyridinium
hydrochloride, 140°C, 50%; (c) Pyridinium hydrochloride, 140°C, 68%.

Table 1. Cytotoxicities and nitric oxide (NO) production inhibitory activities
Compound (20 µM)
Cell viability (%)a
95.39 2.33
101.59 4.51
97.00 1.12
111.45 3.63
96.09 1.77
116.94 5.70
93.56 1.09
107.33 9.57
92.41 4.26
85.85 1.62
87.88 2.92
95.04 2.56
99.10 0.58
98.01 3.58
NO inhibition (%)a
16.33 1.13
15.70 2.03
17.69 1.45
8.76 3.26
Compound (20 µM)
Cell viability (%)a
92.28 1.53
99.76 0.82
90.77 1.43
75.21 3.29
91.39 8.40
116.29 4.46
115.93 1.36
133.45 5.09
101.67 1.26
133.51 12.43
102.62 3.16
108.54 3.96
118.87 2.40
100.01 0.05
NO inhibition (%)a
4.45 1.81
3a
3n
3b
3o
11.57 6.49
11.86 2.76
20.44 2.27
26.11 2.43
17.39 2.83
22.17 2.87
17.74 4.59
28.65 1.58
31.48 9.14
27.20 3.71
37.59 1.21
20.86 2.94
0.00 0.00
3c
3p
3d
3q
3e
37.50 0.80
33.31 2.86
64.32 0.33
20.91 4.08
21.19 4.96
5.07 1.14
3r
3f
3s
3gb
3t
3h
3u
3i
3v
3j
3w
3k
10.72 1.71
17.96 0.49
19.50 1.37
50.39 3.51
3x
3l
3y
3m
3z
Luteolin (5 µM)
LPS (1 µg/mL)
aData are the mean SD (n = 3), b10 µM treatment
As shown in table 1, synthesized compounds generally showed
weak to moderate NO production inhibitory activities at 20 µM
concentration. And all compounds did not exhibit the significant
cytotoxicities at the same concentration. Simple flavone 3a
showed weak inhibitory activity. Interestingly, the flavone
derivatives possessing 4´-hydroxy or 3´,4´-dimethoxy group in
the B ring, 3e and 3f, exhibited relatively moderate NO
production inhibitory activities while the other oxygenated
flavones with 3´-hydroxyl, 3´-methoxy, 4´-methoxy or 2´,4´-
dimethoxy substituents showed less weak activities (3b, 3c, 3d
and 3h). More specifically, 3e with 4´-hydroxy group is slightly
more favorable for the inhibition of NO production than 3f with
3´,4´-dimethoxy substituent. In particular, compound 3g with
3´,4´-dihydroxy moiety showed the most potent activity with
64% NO inhibition, even at 10 µM, which is lower than the other
treatments. Thus, it indicated that flavone 3g has significant anti-
inflammatory potential. Furthermore, these results implied that
the 4´-hydroxy group is essential and that the 3´,4´-disubstitution
is somewhat additionally beneficial for the activity. In the case of
compounds 3i, 3j and 3k, which have electron withdrawing
groups such as halogens on the 4´-position in the B ring, their
biological activities are similar or slightly lower, compared to 3a.
In addition, trimethyl silyl (TMS), alkyl or aryl substituents on
the same positions do not increase the activities (3l, 3m and 3n).
With the compounds that were identified to have moderate to
potent NO production inhibitory activities, we investigated their
anti-inflammatory activities in detail. Prior to the further
experiments, we set 30% NO production inhibition as the cut off
value and selected some flavone derivatives such as 3e, 3f, 3g,
3w and 3y, which exceeded this value. And compound 4
possessing 5,7-dimethoxy and 3´,4´-dihydroxy moieties was
additionally prepared and biologically evaluated. Then, we
treated LPS-stimulated Raw 264.7 cells with increasing
concentrations of the compounds and measured their NO and
PGE2 inhibitory activities by an assay similar to the initial
screening and enzyme-linked immunosorbent assay (ELISA),
followed by determination of their IC50 values, respectively.
Table 2. Cellular toxicities, NO and PGE2 production inhibitory activities of
synthesized flavones 3e-g, 3w, 3y and 4
IC50 (µM)a
Compounds
Cytotoxicity
144.67 1.39
39.68 0.49
45.06 0.92
65.59 0.15
73.94 1.31
> 200
NO production
48.28 1.04
34.54 0.55
6.29 0.19
33.17 0.92
32.50 0.13
34.83 0.26
5.06 0.11
-
PGE2 production
3.77 0.32
6.46 0.21
0.99 0.01
7.43 0.35
3.13 0.36
14.01 0.95
2.87 0.02
6.74 0.71
0.39 0.01
3e
3f
3g
3w
3y
Next, we moved our attention to the investigation of the activity,
depending on the substituents in the A ring. Most of the flavone
derivatives having substituents in the A ring, regardless of their
electronic properties, did not show potent NO inhibitory
activities. Flavone 3v, having 7-pivalyloxy group or the
compounds possessing 5,7-dimethoxy groups, 3w, 3x and 3y,
were moderately active, but their activities are lower than 3g with
3´,4´-dihydroxy moiety.
4
Luteolin
NS398
Ibuprofen
18.95 1.18
-
3309.84 23.59 1483.87 46.03
aData are mean SD. (n = 3)

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The results of these additional assays are summarized in table 2.
Most of the selected compounds, except 3g, displayed a double-
digit micromolar IC50, indicating that they did not have
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significant NO inhibitory activities. However, all of the
compounds showed potent inhibitory effects on PGE2 production
with a range under 10 µM of IC50, and most of them were
relatively more potent than NS398, a reference compound
inhibiting PGE2 production (PGE2 production IC50 = 6.74 µM)
but less potent than ibuprofen, one of the clinically used NSAIDs
(non-steroidal anti-inflammatory drugs). Of the compounds, 3e
and 3y have similar PGE2 inhibitory activities, compared to
luteolin, a potent anti-inflammatory natural flavone (PGE2
production IC50 = 2.87 µM). It is noteworthy that flavone 3g
exhibited the greatest activity on PGE2 inhibition with an IC50 of
0.99 µM, which indicates that it is 3-fold more potent than
luteolin. In addition, 3g showed potent NO inhibitory activity
(NO production IC50 = 6.29 µM), similar or slightly inferior to
the reference compound (NO production IC50 = 5.06 µM). Based
on the result, it was concluded that 3g showed significantly
potent anti-inflammatory activities in LPS-induced Raw 264.7
cells. Given that the 3´, 4´-dihydroxy moiety was commonly
included in the structure of both 3g and luteolin, this catechol
group might be crucial for the desired properties, and this result
was also in accordance with our initial NO screening result. In
particular, all of the assay results including the cell viability test
confirmed that 3g has less cytotoxic property and more potent
anti-inflammatory activities than luteolin, indicating that its
biological activity might not result from death of the cell but
inhibition of pro-inflammatory cytokine biosynthesis. Therefore,
we assumed that compound 3g might be a novel anti-
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inflammatory candidate that could secure the suitable therapeutic
window. However, unlike our expectations, compound 4
possessing both 5,7-dimethoxy and 3´,4´-dihydroxy moiety was
less active than compound 3g in NO and PGE2 production assay,
respectively. Considering that compound 4, and luteolin with
both 5,7-dihydroxy and 3´,4´-dihydroxy moieties, showed lower
activity than compound 3g, it seems that the 5,7-dioxygenation of
3´,4´-dihydroxy flavone might be disfavored for inhibitory
activities on NO and PGE2 production.
7. For reviews see: (a) Manthey, J. A.; Grohmann, K.; Guthrie, N.;
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In summary, a series of novel flavone derivatives were
synthesized and biologically evaluated for their inhibitory effects
on the production of both nitric oxide (NO) and prostaglandin E2
(PGE2) in LPS-induced RAW 264.7 cells, based on a unified and
concise synthetic approach and the cellular assay. In particular,
flavone 3g showed a significantly more potent inhibitory activity
on PGE2 production as well as similar inhibitory activity on NO
production, and a less cytotoxic property, compared to luteolin, a
natural anti-inflammatory flavone, in the cells. These results
indicated that it might be a promising anti-inflammatory agent.
Based on the structure-activity relationship of flavone derivatives,
established in this study, 3´,4´-dihydroxy group in the flavone
skeleton was crucial for the desired biological activities. Further
work in the development of novel anti-inflammatory drugs based
on our study is ongoing.
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B. H.; Shin, J.-S.; An, H. J.; Lee, Y. H.; Yun, Y. D.; Rim, H.-K.;
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Kim, N.-J. Org. Biomol. Chem. 2015, 14, 777.
11. (a) Yun, K. J.; Kim, J. Y.; Kim, J. B.; Lee, K. W.; Jeong, S. Y.;
Park, H. J.; Jung, H. J.; Cho, Y. W.; Yun, K.; Lee, K. T. Int.
Immunopharmacol. 2008, 8, 431; (b) Shin, J. S.; Park, S. J, Ryu S,
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Bioorg. Med. Chem. Lett. 2013, 23, 2571.
Acknowledgments
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF), which was funded by the Ministry of Science, ICT &
Future Planning (NRF- 2016R1A2B4015169).
12. Namikoshi, M.; Nakata, H.; Yamada, H.; Nagai, M.; Saitoh, T.
Chem. Pharm. Bull.1987, 35, 2761.
13. Synthetic procedure and spectral data of compound 3g: To a
flavone 3f10 (230 mg, 0.81 mmol) was added pyridine
hydrochloride under N2 atmosphere. The reaction mixture was
stirred at 140 °C until complete consumption of the starting
material on TLC. Then, the reaction mixture was quenched with
H2O and stirred for 1 hour. The reaction extracted with EtOAc and
dried over MgSO4, filtered, and concentrated under the reduced
References and notes
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pressure. The product was purified via flash column
Supplementary Material
chromatography (MC : MeOH = 10 : 1) to afford 140 mg (68%) of
compound 3g as a yellow powder. 1H-NMR (DMSO, 400 MHz) δ
9.90 (bs, 1H), 9.43 (bs, 1H), 8.03 (dd, 1H, J = 7.8, 1.3 Hz), 7.81
(ddd , 1H, J = 8.4, 7.7, 1.6 Hz), 7.72 (d , 1H, J = 8.2 Hz), 7.50 –
7.44 (m, 3H), 6.93 (d , 1H, J = 9.0 Hz), 6.76 (s, 1H); 13C-NMR
(DMSO, 125 MHz) δ 177.2, 163.7, 156.0, 150.0, 146.2, 134.5,
125.7, 125.2, 123.8, 122.3, 119.3, 118.7, 116.4, 113.8, 105.3; LR-
MS (EI) m/z 254 (M+); HR-MS (EI) calculated for C15H10O4 (M+)
254.0579; found 254.0577.
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
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