Synthesis and biological evaluation of pyranoisoflavone derivatives as anti-inflammatory agents

Article abstract of DOI:10.1016/j.fitote.2014.06.002

In this paper, barbigerone (1a) and its twenty-seven related structural analogues were synthesized via complementary synthetic routes and their anti-inflammatory effects on the expression of TNF-α in LPS-stimulated splenocytes were evaluated. Among these compounds, 1a, 1d, 1f and 1g were found to remarkably inhibit TNF-α production. Furthermore, 1g showed the most potent and dose-dependent manner inhibitory effect on TNF-α release, with better IC₅₀ value (3.58 μM) than barbigerone (8.46 μM). Oral administration of 1g at 20 mg/kg/day for two weeks obviously demonstrated protective effect in adjuvant-induced arthritis models as evaluated by clinical score of paws, and histological examination of joint tissues from rats. Mechanism studies on mRNA and protein level suggested that 1g inhibited the TNF-α production via depressing TNF-α converting enzyme (TACE) mRNA expression. In conclusion, these data show 1g with potential therapeutic effects as an anti-inflammatory agent.

Full text of DOI:10.1016/j.fitote.2014.06.002

Synthesis and Biological Evaluation of Pyranoisoflavone Derivatives as
Anti-Inflammatory Agents
Zhe Wei, Youzhe Yang, Caifeng Xie, Chunyan Li, Guangcheng Wang,
Liang Ma, Mingli Xiang, Jian Sun, Yuquan Wei, Lijuan Chen
PII:
DOI:
Reference:
S0367-326X(14)00155-5
doi: 10.1016/j.fitote.2014.06.002
FITOTE 2961
To appear in:
Fitoterapia
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Accepted date:
7 April 2014
30 May 2014
2 June 2014
Please cite this article as: Wei Zhe, Yang Youzhe, Xie Caifeng, Li Chunyan, Wang
Guangcheng, Ma Liang, Xiang Mingli, Sun Jian, Wei Yuquan, Chen Lijuan, Synthesis
and Biological Evaluation of Pyranoisoflavone Derivatives as Anti-Inflammatory Agents,
Fitoterapia (2014), doi: 10.1016/j.fitote.2014.06.002
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ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of Pyranoisoflavone
Derivatives as Anti-Inflammatory Agents
Zhe Wei,† Youzhe Yang,†, ‡ Caifeng Xie,† Chunyan Li,† Guangcheng Wang,† Liang Ma,† Mingli
Xiang,† Jian Sun,*, ‡ Yuquan Wei† and Lijuan Chen*, †
†State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan
University, Chengdu 610041, P. R. China
‡Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041, P. R. China
Corresponding Author
*Address correspondence and reprint requests to Prof. Lijuan Chen, State Key Laboratory of
Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road
4, Gaopeng Street, Chengdu, 610041, P. R. China. Tel.: +86-28-85164063. Fax: +86-28-85164060.
E-mail: chenlijuan125@163.com.
Additional corresponding author Prof. Jian Sun, No.9 Section 4, Renmin Nan Road, Chengdu,
Sichuan, P.R. China. Tel.: +86-28-82890803. E-mail: sunjian@cib.ac.cn.
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Abstract
In this paper, barbigerone (1a) and its twenty-seven related structural analogues were synthesized
via complementary synthetic routes and evaluated their anti-inflammatory effects on the expression
of TNF-α in LPS-stimulated splenocytes. Among these compounds, 1a, 1d, 1f and 1g were found to
remarkably inhibit TNF-α production. Furthermore, 1g showed the most potent and dose-dependent
manner inhibitory effect on TNF-α release, with better IC50 vaule (3.58 μM) than barbigerone (8.46
μM). Oral administration of 1g at 20 mg/Kg/day for two weeks demonstrated obviously protective
effect in adjuvant-induced arthritis models as evaluated by clinical score of paws, and histological
examination of joint tissues from rats. Mechanism studies on mRNA and protein level suggested
that 1g inhibited the TNF-α production via depressing TNF-α converting enzyme (TACE) mRNA
expression. In conclusion, these data show 1g with potential therapeutic effects as an
anti-inflammatory agent.
Key Words: Tumor necrosis factor α, inflammatory, pyranoisoflavone, TACE mRNA
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1. Introduction
Inflammation is a primary response of an organism to various injurious stimuli, and the typical
syndromes of inflammation are characterized by heat, redness, pain, even loss of function, and so
on.[1] Cytokines are directly implicated in the processes of inflammation, such as interleukin 1β
(IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). TNF-α, a pro-inflammatory
cytokine synthesized as a membrane-bound protein which can be cleaved by proteases to soluble
form, is commonly released by macrophages and lymphocytes, and plays a very important role in
the inflammatory cascades through activating nuclear factor-κB (NF-κB).[2, 3] Over expression of
cytokines is responsible for a number of inflammatory pathological conditions such as asthma,
colitis, hepatitis and rheumatoid arthritis (RA).
Rheumatoid arthritis, an autoimmune disease, is a synovial inflammatory disease of unknown
etiology that occurs in about 1% population of the world, people of any age can be affected.[4-6] As
a debilitating disease, RA causes chronic inflammation and involves the destruction of joints of the
hands and feet, and it also causes inflammation of the tissue around the joints, as well as in other
organs of the body.[7, 8] It has been proposed that the initiating event in RA involves an infectious
agent or other environmental exposure.[9] Over the past decades, significant advances in
understanding the pathogenesis of RA have led to the identification of novel therapeutic targets.[10]
Cytokines are directly implicated in the processes of RA. Accordingly, cytokines modulation alters
the outcomes in many rodent models of arthritis.[11] Hence, inhibition of TNF-α production is one
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of the crucial aspects for the disease control and prognosis improvement, and now has been targeted
in the standard treatment of patients with RA.[12, 13]
Barbigerone (1a, Figure 1), a naturally occurring pyranoisoflavone, was first isolated from the
seeds of Tephrosia barbigeria and can be obtained from other plants.[14-17] Previous studies
discovered that barbigerone exhibited various pharmacological properties, such as anti-plasmodia,
antioxidant and 15-lipoxygenase inhibitory activity.[18, 19] Recently, barbigerone was also found
to inhibit the proliferation of several cancer cell lines.[16, 20, 21]
Figure 1. Structure of barbigerone and some typical bioactive pyranochalcones
In spite of the increasing interest in biological profiles of barbigerone, little attention has been
paid to its structure-activity relationship (SAR) study. The first total synthesis of barbigerone
presented by Pathak, however, was inefficient and low-overall-yielding (less than 4%).[22] On the
other hand, isolation of barbigerone from natural sources was also proven in our hands to be
inefficient and difficult to achieve enough amounts for in-depth biological and medicinal
studies.[23] In our previous work, we have developed a concise and efficient route for the synthesis
of barbigerone, which also proves to be viable for the synthesis of its several unnatural
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analogues.[24] Herein, we report the complementary synthesis, SAR and mechanism study of
barbigerone and its structural analogues as anti-inflammatory agents for the first time.
2. Results and discussion
2.1 Chemistry
In order to explore the SAR of barbigerone, its related structural analogues were prepared
according to the complementary synthetic routes outlined in the Schemes 1-6. All of the target
compounds were synthesized from simple and commercial available starting materials.
The synthesis of 7-hydroxy-isoflavones by using ‘one-pot method’ described by Singh and Pratap
was involved in the synthesis of target compounds 1b and 1c.[25] Briefly, resorcinol 2 reacted with
phenylacetic acid to generate intermediate deoxybenzoins 3 in the presence of anhydrous Lewis
acid ZnCl2. Based on Vilsmeier-Haack reaction, the isoflavone ring closing to form
7-hydroxy-isoflavone 4 was finished in reasonable yield. Treatment of 4b and 4c with
1,1-diethoxy-3-methylbut-2-ene to construct pyranoid ring to afford 1b and 1c proceed smoothly
according to the known pyranoid formation method with the over-all yield of 24% and 30%,
respectively (as shown in Scheme 1).[26]
Scheme 1 ‘One-pot’ synthesis of 1b and 1c
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Reagents and conditions: (a) anhy. ZnCl2, 2-phenylacetic acsid derivatives, 120 °C; (b) dry DMF,
BF3•OEt2, 0 °C, MeSO2Cl, 50-110 °C, 3 h; (c) 1,1-diethoxy-3-methylbut-2-ene, 3-picoline(cat.
amount), dry xylene, reflux, 12h.
The preparation of some special 2-phenylacetic acid needs several steps from simple starting
material, ‘one-pot method’ is not fit for the synthesis of barbigerone,[27] the second method for the
synthesis of barbigerone and main related analogues was designed and carried out as we described
before.[24] As outlined in Scheme 2, our synthetic work began with Friedel-Crafts acylation of
resorcinol 2 with acetic anhydride under the catalytic effect of BF3•Et2O, which afforded
1-(2,4-dihydroxyphenyl)ethanone 5 in 79% yield. Intermediate 6a was produced from 5 and
1,1-dimethoxy-3-methylbut-2-ene with 65% yield, which was then subjected to ‘Aldol reactions’
with different aldehydes under basic condition in methanol to afford the key intermediate chalcones
7a, d-k with moderate to good yields. To synthesize intermediates 8, the oxidation-rearrangement
reaction of 7 was examined under different conditions. TTN (Thallium(III) nitrate trihydrate)
proved to be superior to other oxidants such as DIB (Iodosobenzene diacetate) and HTIB
(Hydroxy(tosyloxy)iodobenzene), which drove the reaction to completion under mild conditions in
12 hours. However, the oxidation-rearrangement of 7i-k with unprotected hydroxy group and
electron-withdrawing group in the right phenyl ring was proven to be inefficient. After careful
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work-up and purification, the crude products 8a, d-h were directly treated with 1N HCl in methanol
under reflux, construction of the main isoflavone skeleton was finished and the target compounds
1a, d-h were obtained with yields ranging from 64% to 78% (based on chalcones).
Scheme 2 Synthesis of 1a, d-f based on ‘oxidation-rearrangement reaction’
Reagents and conditions: (a) anhy. AlCl3, AcCl, dry CS2, reflux, 12 h; (b)
1,1-diethoxy-3-methylbut-2-ene, 3-picoline(cat. amount), dry xylene, reflux, 12h; (c) aq. 40%
NaOH, various aldehydes, MeOH, 0 °C-r.t., 12-36 h; (d) Thallium(III) nitrate trihydrate(TTN),
MeOH, r.t. overnight; (e) 1N HCl, MeOH, reflux, 6 h.
Treatment of 6a with DMF-DMA under reflux, intermediate 9 was produced, which was then
transformed to target compound 1i and another key intermediate 10 by reacting with 1N HCl or
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iodine in methanol.[28] Target compound 1j was synthesized by coupling 10 to
4-(trifluoromethyl)phenylboronic acid smoothly with 82% yield by using Pd/C as catalyst (as
shown in Scheme 3).[29]
Scheme 3 Synthesis of 1i and 1j
Reagents and conditions: (a) DMF-DMA, reflux, 6 h; (b) 1N HCl, MeOH; (c) I2, MeOH, r.t.,
overnight; (d) 4-(trifluoromethyl)phenylboronic acid, 10% Pd/C (cat. amount), K2CO3, MeCN:H2O
= 1:1, reflux, overnight.
Several special target compounds were also prepared in order to investigate the importance of
pyranoid ring according to the Schemes 4-6. By using the same method as the synthesis of 1j
described above, target compound 1k was prepared from 5 with the yield of 73% (last step).
Scheme 4 Synthesis of 1k
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Reagents and conditions: (a) DMF-DMA, reflux, 6 h; then I2, MeOH, r.t., overnight; (b)
4-hydroxyphenylboronic acid, 10% Pd/C (cat. amount), K2CO3, MeCN:H2O = 1:1, reflux,
overnight; (c) 1,1-diethoxy-3-methylbut-2-ene, 3-picoline(cat. amount), dry xylene, reflux, 12h.
Target compound 1l and 1m without pyranoid ring were prepared from 4b and 13 by treating
them with 1-bromo-3-methylbut-2-ene with good yields under mild condition, 68% and 72%,
respectively.[30]
Scheme 5 Synthesis of 1l and 1m
Reagents and conditions: 1-bromo-3-methylbut-2-ene, K2CO3, acetone, reflux, overnight.
Intermediates 7-amino isoflavone 16 and 6-amino isoflavone 19 were prepared from compounds
14 and 17 by using the same method as the synthesis of 7-hydroxy isoflavone 4. By using
silicotungstic acid as catalyst, intermediates 16 and 19 reacted with acetone under reflux for
overnight afforded target compounds 1n and 1o with acceptable yield, 52% and 62%,
respectively.[31]
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Scheme 6 General route for the synthesis of aza-isoflavone 1n and 1o
Reagents and conditions: (a) 2-phenylacetic acid, anhy. AlCl3, dry CS2, reflux, 12 h; (b) dry DMF,
BF3•OEt2, 0 °C, MeSO2Cl, 50-110 °C, 3 h; then 6N HCl, MeOH, reflux, 6 h; (c) acetone,
silicotungstic acid (cat. amount), reflux, 12 h.
2.2 Inhibitory effect of TNF-α production in vitro and SAR analysis
Activated inflammatory cells play crucial roles in the initiation and maintenance of inflammation.
LPS promotes the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, in
many cell types especially in lymphocytes and macrophages. Reduction of TNF-α production could
serve as a therapy for inflammatory diseases. Barbigerone and its twenty-seven derivatives were
evaluated for their anti-inflammatory activity of inhibiting the TNF-α release in LPS-stimulated
mouse splenocyte. The result was shown in Figure 2, dexamethasone (DEX), a steroidal
anti-inflammatory drug, was chosen as a positive control. Among these compounds, 1a, 1d, 1f and
1g effectively suppressed LPS-induced TNF-α production at 10 μM (the inhibitory rates were more
than 65.0%) and closed to positive control DEX. 1g showed the most potent inhibitory effect on
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LPS-induced TNF-α production and its inhibitory rates reached 78.3%, compared to the LPS
control.
Figure 2. Inhibitory effects on TNF-α production at a concentration of 10 μM in spleen cells. The
amount of TNF-α with LPS-treated group was set as 100.0% and the vehicle control as 0.0%.
Inhibition (%) = [LPS(OD450) - compounds(OD450)]/[LPS(OD450) - control(OD450)] × 100%.
Values (means ± SD) were combined from three independent experiments (* P < 0.05, ** P < 0.01,
***P < 0.001).
Compared with 1b, inhibitory potency decreased dramatically when substituent of
electron-withdrawing group was added (para-trifluoromethyl for 1j) on the right phenyl ring.
However, the addition of electron-donating group obviously showed positive effect on inhibitory
potency (different types of methoxy for compounds 1a, c-h). Compound 1d with meta-methoxy on
the right phenyl ring showed a little better potency than that of compound 1e with para-methoxy.
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Addition of methoxy groups helped increasing the potency (78.3% for 1g, 76.0% for 1a and 61.9%
for 1h were better than 52.3% for 1b). A conclusion that dimethoxy on position C2 and C5 of right
phenyl ring was the dominant factor for maintaining and enhancing the inhibitory potential could be
drawn. These results also indicated that the properties of inhibitory potency may contribute to the
bulk and electron density of the right aromatic ring. Taking some natural pyranochalcones possess
anti-inflammation property into consideration (Figure 1),[32, 33] chalcones 7a, d-k, precursors of
isoflavones, were also tested for their TNF-α production inhibitory effect. However, chalcones 7
showed week to moderate inhibitory effect, and all of them were not as good as the corresponding
target compounds. For example, 66.1% for 1d was better than that of 25.0% for 7d, 78.3% for 1g
was better than 39.4% for 7g. This indicated that chromone structure was very important for their
activity against TNF-α production. Removal of pyranoid ring on the left phenyl ring clearly had
negative effect on inhibitory potency (53.2% for 1b vs 27.7% for 4b, 60.4% for 1e vs 34.4% for
13). The presence of prenyl seemed to reduce the capacity of 7-hydroxyl isoflavones 4b (27.7% vs
7.7% for 1l) and 13 (34.4% vs 16.5% for 1m). On the other hand, the migration of pyranoid ring
from the left phenyl ring to the right phenyl ring or replacement of pyranoid ring with quinolin ring
also reduced the inhibitory activity dramatically (60.4% for 1e vs -14.3% for 1k, 52.3% for 1b vs
21.6% for 1n or 28% for 1o). These results suggested that pyranoid ring on the left phenyl ring was
very important for the activity and could not be modified.
Furthermore, compounds 1a, 1d, 1f and 1g were chosen to investigate both the inhibitory activity
against TNF-α production and the cytotoxicity in spleen cells at different concentrations. As
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exhibited in Table 1, 1g and 1a presented remarkable inhibitory effect on TNF-α production, with
IC50 of 3.58 and 8.46 μM respectively. To check whether the inhibitory effect against TNF-α
production was related to cell viability, a MTT assay was also adopted. As the result shown, the two
compounds exerted little cytotoxicity in splenocyte (without or with LPS, IC50 > 70.00 μM).
Table 1. IC50 values on TNF-α production and cytotoxicity in mouse spleen cells.
cytotoxicity IC50 (μM)
TNF-α IC50 (μM) without LPS
with LPS
CPD
1a
8.46
> 10
> 10
3.58
82.14
85.99
-
-
-
-
1d
1f
77.78
74.39
1g
*Cytotoxicity = [1 - Compounds(OD570)/Control(OD570)] × 100%.
2.3 Anti-arthritic effects of 1g on AIA models
Adjuvant-induced arthritis (AIA) is a form of chronic arthritis and commonly used to test agents
for anti-inflammatory activity. As the most potent target compound 1g was chosen to examine the
ability of anti-inflammation on AIA rats. AIA Lewis rats were treated orally with 1g at a dose of 10
mg/kg/day and 20 mg/kg/day following the appearance of first AIA signs. Results showed that the
severity of AIA was dose-dependently improved in 1g treated animal models (Figure 3).
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Figure 3. Anti-arthritic effects of 1g on AIA models. 1g was orally administered once daily at a
dose of 10 mg/kg and 20 mg/kg. (A) Clinical disease activity score of 1g. The result were expressed
as the means (n = 5; ***P < 0.001). (B) Ankle circumference after adjuvant injection. The result
were expressed as the means (n = 5; ***P < 0.001). (C) Macroscopic observations of the ankles on
day 30. (D) Representative histopathologies of the knee joints stained with H&E and Safranin O
fast green (× 40).
The time course of adjuvant arthritic development, expressed as arthritic scores and ankle
circumference, were significantly reduced in the therapeutic process (Figure 3A and 3B). This
effect was further confirmed with macroscopic observations of the ankles and joint histology.
Compared to the vehicle control groups, 1g significantly reduced the paw edema of rats after 17
days continuous treatment at a dose of 20 mg/kg/day (Figure 3C). Histological evaluation by H&E
and Safranin O staining were carried out from vehicle and 1g treated mice too. A destroyed joint
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space with large amounts of inflammatory cell infiltration and cartilage destruction were observed
in AIA vehicle groups (showed as arrows in Figure 3D). Treatment of 1g at a dose of 20 mg/kg/day
led to significantly and obviously suppression of these histopathological changes with few signs of
inflammation and cartilage was almost completely preserved in histological state of joints (Figure
3D).
To explore whether 1g had the ability of reducing pro-inflammatory cytokines expression, joint
tissues were collected and the relative mRNA levels of TNF-α, IL-1β and IL-6 were measured by
RT-PCR. Treatment with 1g significantly reduced the levels of these pro-inflammatory cytokines in
the joint tissues (Figure 4A). To define whether 1g had a systemic effect, quantity of TNF-α, IL-1β
and IL-6 in collected serum samples as described above were measured by ELISA. Treatment with
1g showed a significant effect on the systemic level of these cytokines (Figure 4B, 4C and 4D).
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Figure 4. Inhibitory effects on the mRNA levels of TNF-α, IL-1β and IL-6 in joint tissues of animal
vehicle. The inflammatory cytokine mRNA levels in joint tissues were measured by RT-PCR (A).
Treatment with 1g significantly inhibited expression of inflammatory cytokine. Serum samples on
day 30 were measured for the quantity of TNF-α (B), IL-1β (C) and IL-6 (D). Values (means ± SD,
n = 5 for each group) were from one representative experiment of three independent experiments
(*P < 0.05; **P < 0.01; ***P < 0.001).
2.4 Mechanism study of 1g
Since compound 1g significantly suppressed LPS-triggered TNF-α production on splenocytes, we
also investigated whether 1g inhibited LPS-induced TNF-α production in Raw 264.7 cells. As
shown in Figure 5A, 1g reduced LPS-induced TNF-α production in Raw 264.7 cells in a
dose-dependent manner.
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Figure 5. (A) RAW 264.7 were treated in the presence or absence of different dose of 1g culturing
with LPS (1 μg/ml). Supernatants at 2, 4, 8, 12, 24h post LPS addition were collected. Data (means
± SD) combined from three independent experiment (**P < 0.01). (B) Luciferase assay was
performed in the presence or absence of 1g (10 μM) as described in Materials and Methods. Values
(means ± SD) combined from three independent experiments (**P < 0.01).
To clarify the underlying molecular mechanisms against TNF-α production of 1g, we tested
whether 1g affected TNF-α mRNA level in vitro at first. RAW 264.7 cells were treated in the
presence or absence of 1g followed by LPS stimulation, and cells were collected for the detection of
TNF-α mRNA level by RT-PCR analysis. Result showed that 1g almost had no effect on the
transcription of TNF-α mRNA (Figure 6). Then we decided to examine whether 1g affected the
TNF-α production at protein level. TNF-α-luciferase transgenic mouse splenocytes were collected
and luciferase activity was measured as described previously to detect the intracellular TNF-α
expression. Interestingly, results indicated that treatment with 1g had no clearly effects on the
intracellular protein level of TNF-α (Figure 5B). However, to some extent, it even enhanced the
intracellular protein level after 4h LPS-induction. Our results showed that 1g had the ability of
inhibition of TNF-α secretion, but could not inhibit the TNF-α expression at mRNA and protein
level. We supposed that this compound may influence the processing of pro-TNF-α to a soluble
form. Consequently, we chose the most important TNF-α converting enzyme (TACE) for further
study. As we all known, TACE is a membrane-bound disintegrin metalloproteinase that processes
the membrane-associated cytokine pro-TNF-α to a soluble form. According to the result showed in
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Figure 6, TACE mRNA is abundantly expressed in RAW264.7 cells stimulated by LPS. The
1g-treated group than in LPS-treated group decreased expression, and as the dose increased
gradually, TACE mRNA expression decreased. High dose group was significantly reduced
compared with the other three groups expression. The result showed that 1g decreased TACE
mRNA level in RAW 264.7 cells in a dose-dependent manner.1g decreased TACE mRNA level in
RAW 264.7 cells in a dose-dependent manner. So, we could draw a conclusion that 1g inhibited
TNF-α production through depressing the TACE mRNA expression.
Figure 6. 1g inhibited TNF-α production through effects on TACE mRNA level. The TNF-α and
TACE mRNA levels in RAW 264.7 were measured by RT-PCR as described before.
3. CONCLUSION
Inflammation is a response of host to inside and outside stimuli. TNF-α, a pro-inflammation
cytokine mainly produced by macrophages and lymphocytes, plays a very important role in the
inflammatory cascades, and inhibition of TNF-α production serves as an effective therapy for
chronic inflammatory disease, such as rheumatoid arthritis.
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In this study, barbigerone and its 27 related structural analogues were designed, synthesized, and
evaluated for their pharmacological activity. At 10 μM, barbigerone (1a) and 1g significantly
reduced the production of TNF-α, with IC50 of 8.46 and 3.58 μM, respectively. Both of the
compounds also exerted low cytotoxicity (without or with LPS, IC50 > 70 μM). SAR analysis
indicated that: (1) pyranoid ring on the left phenyl ring and chromone structure of isoflavone are
very important for the inhibitory activity on TNF-α production; (2) the bulk and electron density of
right phenyl ring shows even more regularity with respect to the appropriate typical activity, and
suitable electron-donating substitutions on C2 and C5 are the dominant factor for maintaining or
enhancing the activity. Furthermore, oral administration of 1g, at a dose of 20 mg/Kg/day for about
two weeks, obviously led to improve the inflammatory response such as significantly reduction of
footpad swelling and the improvement of pathology from joints of AIA rats. Mechanism studies
suggested that 1g inhibits the TNF-α production by means of depressing TACE mRNA expression.
In summary, above results indicate that 1g might be a potential agent for the treatment of chronic
inflammatory diseases.
4. Experimental
4.1 Chemistry methods. All the reagents and solvents of analytical grade were purchased from
commercial and used without further purification. Reactions were monitored by thin-layer
chromatography (TLC) on silica gel 60 F-254 thin-layer plates and spots of desired products were
located by UV lamp and Iodine. Intermediates and target compounds were purified by silica gel
column. The purities of all compounds in biological assays were higher than 95%, which were
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confirmed by HPLC analysis with a phodediode array detector (Waters, Milford, MA, USA) and
the chromatographic column was an atlantis C18 (150 mm × 4.6 mm, I.D. 5 μm, Waters, Milford,
Ireland). Samples were supplied as 0.1 mg/ml in methanol with 10 μl injected on a partial loop fill
at a flow rate of 1 ml/min for 20 min. Melting points were measured on X-4 digital microscopic
1
melting point apparatus and are not correct. H spectra were recorded on Bruker AV-600, 400 and
13
300 MHz, C spectra were record on Bruker AV-150 and 100 MHz. MS (low resolution) spectra
were measured by Waters Quattro Premier XE and HRMS (high resolution) spectra were measured
by Bruker Daltonics micrOTOF-Q II Electrospray Ionization Mass Spectrometer and Waters
Q-TOF Premier mass spectrometer utilizing electrospray ionization (ESI) (Micromass, Manchester,
UK).
General experimental procedure for the synthesis of 4b-c. To the molten anhydrous ZnCl2
(3.3 mmol), phenylacetic acid (3.6 mmol) was added with vigorous stirring and heating at about
120-130 °C followed by the slow addition of resorcinol 2 (3 mmol). After completion of the
reaction according to TLC monitor, the mixture was cooled to room temperature, followed by the
addition of dry DMF (2 ml), and BF3•OEt2 (12 mmol) at 0 °C. To this mixture was added a solution
of MeSO2Cl (9 mmol) in 5ml DMF at 50 °C and then the temperature was raised to 110 °C and
stirred for about 2 hours. After completion, the reaction mixture was cooled and poured into ice
water. The separated oil was extracted with ethyl acetate, and the organic layer was combined,
washed with brine, and dried by MgSO4, then the solvent was removed. The residue was
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recrystallized from methanol. 7-hydroxy-isoflavones 4b and 4c were obtained as white needle
solids.
General experimental procedure for the synthesis of 1b-c. Under N2 atmosphere,
intermediates 4b or 4c (0.5 mmol) was dissolved into 10 ml dried toluene, then catalytic 3-picoline
was added, followed by the addition of 1,1-diethoxy-3-methyl-2-butene (1 mmol), stirred for a
while and then the mixture was refluxed at 130 °C for about 18 hours. After the disappearance of
reactant according to TLC, the solvent was evaporated and the residue was chromatographed over
silica gel using petroleum/ethyl acetate = 3/1 as eluent, 1b and 1c were obtained with the yield of
24% and 30% in two steps, respectively.
3-phenyl-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1b): white powder, over-all
yield 24%; HPLC: 99.4%; 1H NMR (CDCl3, 600 MHz) δ (ppm) = 1.55 (s, 6H), 5.77 (d, J = 12.0
Hz, 1H), 6.87 (d, J = 12.0 Hz, 1H), 6.91 (d, J = 12.0 Hz, 1H), 7.42-7.61(m, 5H), 8.01 (s, 1H), 8.12
(d, J = 12.0 Hz, 1H). MS (ESI), m/z: 305.07 [M + H]+. NMR spectra match the reference.[34]
3-(1’,3’-dioxolphenyl-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1c): white
powder, over-all yield 30%; HPLC: 98.4%; 1H NMR (CDCl3, 600 MHz) δ (ppm) = 1.50 (s, 6H),
5.72 (d, J = 12.0 Hz, 1H), 5.99 (s, 2H), 6.81 (d, J = 12.0 Hz, 1H), 6.86 (t, J = 6.0 Hz, 2H), 6.96 (d, J
= 6.0 Hz, 1H), 7.10 (s, 1H), 7.92 (s, 1H), 8.06 (d, J = 12.0 Hz, 1H). MS (ESI), m/z: 349.12 [M +
H]+. NMR spectra match the reference.[35]
21

ACCEPTED MANUSCRIPT
General experimental procedure for the synthesis of 7a, d-k and 1a, d-h. Compound 6a (2
mmol) was dissolved into 10 ml methanol and cooled to 0 °C, a solution of 40% NaOH (3.5 ml)
was added to the previous mixture and stirred at the temperature for about 30 minutes, then
different substituted aldehydes (2.4 mmol) was added slowly. The mixture was allowed raise to
room temperature and stirred for about 24 to 36 hours. After the disappearance of 6a, the mixture
was acidified with 2N HCl (about 10 ml), chalcones 7a, d-k came into appearance as yellow
precipitates. The precipitates were collected and recrystallized from methanol, high purity of 7a,
d-k were obtained with the yield ranging from 64%-78%. To the stirred suspension of chalcones 7a,
d-h (0.5 mmol) in methanol (5 ml) were added thallium (III) nitrate (0.75 mmol) carefully, and
stirred continued for overnight at room temperature. After the disappearances of chalcones, enough
volume of saturated sodium sulfite (about 30 ml) was added, and stirred for about 1 hour, then the
mixture was extracted with ethyl acetate for three times. The organic phase were combined and
dried with MgSO4, then the solvent were removed in vacuo and the stick yellowish oil was purified
by silica gel chromatography using DCM as eluent. The purified products were dissolved into
methanol (15 ml) and treated with 2N HCl (3 ml), and the mixture was refluxed for about 3 hours,
as the time went by, white or pale yellow solid precipitated. After completion of the reagents, the
precipitates were collected and recrystallized from methanol, target compounds 1a, d-h were
obtained with yields of 64%-78% (based on chalcones).
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-on
e (7a): yellowish powder, yield 69%; HPLC: 99.5%.[24]
22

ACCEPTED MANUSCRIPT
Barbigerone (1a): pale yellowish powder, yield 76%; HPLC: 99.6%.[24]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3-methoxyphenyl)prop-2-en-1-one
(7d): yellowish powder, yield 72%; HPLC: 99.5%.[24]
3-(3’-methoxyphenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1d): white
powder, yield 78%; HPLC: 96.1%.[24]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-methoxyphenyl)prop-2-en-1-one
(7e): yellowish powder, yield 69%; HPLC: 99.6%.[24]
3-(4’-methoxyphenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1e): white
solid, yield 78%; HPLC: 98.8%.[24]
(E)-3-(3,4-dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one
(7f): yellowish powder, yield 73%; HPLC: 99.7%; 1H NMR (CDCl3, 300 MHz) δ (ppm) = 1.47 (s,
6H), 3.94 (s, 3H), 3.96 (s, 3H), 5.59 (d, J = 12.0 Hz, 1H), 6.38 (d, J = 9.0 Hz, 1H), 6.75 (d, J = 12.0
Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 7.15 (d, J = 1.5 Hz, 1H), 7.22 (d, J = 1.5 Hz, 1H), 7.42 (d, J =
15.4 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 15.4 Hz, 1H), 13.79 (s, 1H). MS (ESI), m/z:
367.17 [M + H]+. NMR spectra match the reference.[33]
3-(3’,4’-dimethoxyphenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1f):
off-white solid, yield 64%; HPLC: 98.3%; 1H NMR (CDCl3, 600 MHz) δ (ppm) = 1.50 (s, 6H),
3.91 (s, 3H), 3.93 (s, 3H), 5.72 (d, J = 12.0 Hz, 1H), 6.81 (d, J = 12.0 Hz, 1H), 6.87 (d, J = 6.0 Hz,
23

ACCEPTED MANUSCRIPT
1H), 6.92 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 7.21 (s, 1H), 7.96 (s, 1H), 8.02 (d, J = 6.0
Hz, 1H). MS (ESI), m/z: 365.22 [M + H]+. NMR spectra match the reference.[36]
(E)-3-(2,5-dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one
(7g): yellowish powder, yield 77%; HPLC: 98.3%.[24]
3-(2’,5’-dimethoxyphenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1g): pale
white solid, yield 68%; HPLC: 98.4%.[24]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-on
e (7h): yellowish powder, yield 81%; HPLC: 99.4%.[24]
3-(3’,4’,5’-trimethoxyphenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1h):
pale white solid, yield 71%; HPLC: 99.6%.[24]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(3-hydroxyphenyl)prop-2-en-1-one
(7i): yellowish powder, yield 76%; HPLC: 99.6%; 1H NMR (CDCl3, 300 MHz) δ (ppm) = 1.47 (s,
6H), 5.60 (d, J = 12.0 Hz, 1H), 6.39 (d, J = 6.0 Hz, 1H), 6.76 (d, J = 12.0 Hz, 1H), 6.89 (t, J = 6.0
Hz, 1H), 7.11 (s, 1H), 7.22 (d, J = 12.0 Hz, 1H), 7.28-7.30 (t, J = 12.0, 6.0 Hz, 1H), 7.53 (d, J = 6.0
Hz, 1H), 7.71 (d, J = 6.0, 1H), 7.80 (d, J = 18.0 Hz, 1H), 13.62 (s, 1H). MS (ESI), m/z: 323.19 [M +
H]+. NMR spectra match the reference.[37]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (7j):
yellowish soild, yield 72%; HPLC: 99.7%; 1H NMR (CDCl3, 300 MHz) δ (ppm) = 1.47 (s, 6H),
24

ACCEPTED MANUSCRIPT
5.59 (d, J = 6.0 Hz, 1H), 6.38 (d, J = 3.0 Hz, 1H), 6.76 (d, J = 6.0 Hz, 1H), 6.88 (d, J = 6.0 Hz, 1H),
7.09 (d, J = 6.0 Hz, 1H), 7.42-7.46 (dd, J = 6.0, 3.0 Hz, 1H), 7.57 (d, J = 3.0 Hz, 1H), 7.70-7.72
(dd, J = 3.0, 3.0 Hz, 1H), 7.72-7.86 (dd, J = 6.0, 3.0 Hz, 1H), 13.75 (d, J = 3.0 Hz, 1H). MS (ESI),
m/z: 323.17 [M + H]+. NMR spectra match the reference.[38]
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-(trifluoromethyl)phenyl)prop-2-en-1-
one (7k): yellow powder, yield 43%; HPLC: 97.0%; 1H NMR (CDCl3, 400 MHz) δ (ppm) = 1.48
(s, 6H), 5.61 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 12.0 Hz, 1H), 7.62 (d, J =
16.0 Hz, 1H), 7.67-7.75 (m, 5H), 7.86 (d, J = 16.0 Hz, 1H), 13.52 (s, 1H). HRMS (ESI) exact mass
calcd. for (C21H17F3O3-H)- requires m/z 373.1130, found m/z 373.1054.
Synthesis of 8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1i): under N2
atmosphere, compound 6a (3 mmol) was dissolved into 3 ml DMF-DMA and refluxed for about 3
hours, after completion of the reaction and cooled down, 10 ml ethanol was added to the mixture,
yellowish needles (9) came into appearance. Collected and dried in vacuo, 579 mg 9 was obtained.
At room temperature, 9 (0.5 mmol) was dissolved into 3 ml methanol followed by the addition of 3
ml 2N HCl. The mixture was refluxed for about 6 hours. The acidic solution was neutralized by 20
ml saturated Na2CO3 solution. Extracted by ethyl acetate for 3 times, combined and dried with
MgSO4, the solvent was removed under reduced pressure. The residue was purified by silica gel
column, using petroleum/ethyl acetate = 4/1 as eluent, and 79 mg yellowish solid was obtained,
yield 69%; HPLC: 99.2%; 1H NMR (CDCl3, 400 MHz) δ (ppm) = 1.49 (s, 6H), 5.71 (d, J = 8.0 Hz,
1H), 6.27 (d, J = 4.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 4.0 Hz,
25

ACCEPTED MANUSCRIPT
1H), 7.97 (d, J = 8.0 Hz, 1H). MS (ESI), m/z: 228.04 [M + H]+. NMR spectra match the
reference.[34]
Synthesis of
3-(4’-(trifluoromethyl)phenyl)-8,8-dimethyl-4-oxo-4H,8H-benzo[1,2-b:3,4-b’]dipyran (1j): 400
mg (1.46 mmol) compound 9 was dissolved into 10 ml methanol, then 444.4 mg (1.75 mmol) I2 was
added to the mixture and stirred at room temperature for overnight. After disappearance of the
reactant, 30 ml saturated Na2SO3 solution was added, extracted with ethyl acetate for 3 times, the
organic phase was combined and dried with MgSO4, the solvent was removed under reduced
pressure. The residue was purified by silica gel column, and 272 mg compound 10 as off-white
powder was obtained. 70.8 mg (0.2 mmol) compound 10 and 57 mg (0.3 mmol)
4-(trifluoromethyl)phenylboronic acid were dissolved into 3 ml acetonitrile and 3 ml water,
followed by the addition of catalytic amount 10% Pd/C and 41 mg (0.3 mmol) K2CO3. The mixture
was refluxed for about 6 hours. After the completion of the reaction, 20 ml ethyl acetate and 30 ml
water was added to the reaction mixture, the organic phase was collected and dried with MgSO4, the
solvent was removed under reduced pressure. The residue was purified by silica gel column, using
petroleum/ethyl acetate = 3/1 as eluent, and 49.3 mg white solid was obtained, clarified as titled
compound, yield 66%; HPLC: 98.7%; 1H NMR (CDCl3, 400 MHz) δ (ppm) = 1.51 (s, 6H), 5.74 (d,
J = 8.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 7.69 (s, 4H), 8.01 (s, 1H), 7.16
(s, 1H), 8.07 (d, J = 8.0 Hz, 1H). HRMS (ESI) exact mass calcd. for (C21H15F3O3-H)- requires m/z
371.0973, found m/z 371.0895.
26

ACCEPTED MANUSCRIPT
Synthesis of 7-methoxy-2',2'-dimethyl-2'H,4H-3,6'-bichromen-4-one (1k): Starting from
compound 5, the synthetic methods of 1j and 1b-c were used for the synthesis of 1k, yield 50.2%
(final step); HPLC: 98.8%; 1H NMR (DMSO-d6, 600 MHz) δ (ppm) = 1.26 (s, 6H), 3.89 (s, 3H),
6.80 (d, J = 6.0 Hz, 2H), 7.07 (m, 1H), 7.13 (d, J = 6.0 Hz, 1H), 7.38 (d, J = 6.0 Hz, 2H), 8.02 (d, J
= 6.0 Hz, 1H), 8.35 (s, 1H), 9.50 (s, 1H). MS (ESI), m/z: 335.12 [M + H]+. NMR spectra match the
reference.[39]
Synthesis of 7-(3-methylbut-2-enyloxy)-3-phenyl-4H-chromen-4-one (1l): compound 4b (0.5
mmol) was dissolved into 10 ml acetone, 138 mg K2CO3 was added and followed by the addition of
1-bromo-3-methylbut-2-ene (0.6 mmol, 71 μl), the mixture was refluxed for about 3 hours. Filtrated
and the solvent was removed, the residue was purified by silica gel column, using petroleum/ethyl
acetate = 6/1 as eluent, 96 mg white solid was obtained and then clarified as titled compound, yield
69%; HPLC: 99.1%; 1H NMR (CDCl3, 400 MHz) δ (ppm) = 1.70 (s, 3H), 1.74 (s, 3H), 4.53 (d, J =
4.0 Hz, 2H), 5.42 (t, J = 8.0, 8.0 Hz, 1H), 6.77 (s, 1H), 6.91 (d, J = 8.0, 1H), 7.28 (t, J = 4.0, 8.0 Hz,
1H), 7.34 (t, J = 8.0, 8.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H).
MS (ESI), m/z: 329.11 [M + Na]+. NMR spectra match the reference.[40]
Synthesis of 3-(4-methoxyphenyl)-7-(3-methylbut-2-enyloxy)-4H-chromen-4-one (1m):
according to the synthetic method as 1l, 112 mg white solid was obtained from 13, which was then
clarified as titled compound, yield 72%; HPLC: 98.4%; 1H NMR (CDCl3, 400 MHz) δ (ppm) =
1.71 (s, 3H), 1.75 (s, 3H), 3.76 (s, 3H), 4.54 (d, J = 8.0 Hz, 2H), 5.43 (t, J = 4.0, 8.0 Hz, 1H), 6.78
27

ACCEPTED MANUSCRIPT
(s, 1H), 6.88 (s, 1H), 6.91 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.83 (s, 1H), 8.12 (d, J = 8.0
Hz, 1H). MS (ESI), m/z: 359.12 [M + Na]+. NMR spectra match the reference.[41]
7-amino-3-phenyl-4H-chromen-4-one (16): By using the same method as the synthesis of
7-hydroxy-isoflavones 4, to the molten anhydrous ZnCl2 (3.3 mmol), phenylacetic acid (3.6 mmol)
was added with vigorous stirring and heating at about 120-130 °C followed by the slow addition of
N-(3-methoxyphenyl)acetamide (3 mmol). After completion according to TLC, the reaction mixture
was cooled to room temperature, followed by the addition of dry dimethylformamide (DMF) (2 ml),
and BF3•OEt2 (12 mmol) at 0 °C. To this mixture was added a solution of methanesulfonyl chloride
(MeSO2Cl, 9 mmol) in 5ml DMF at 50 °C and then the temperature was raised to about 110 °C for
about 2 hours. After completion, the reaction mixture was cooled and poured into ice water. The
separated oil was extracted with ethyl acetate for 3 times, and the organic layer washed with brine,
combined and dried with MgSO4, then the solvent was removed. The residue was purified by silica
gel column, 286 mg N-(5-methoxy-2-(2-phenylacetyl)phenyl)acetamide (pale yellowish powder,
discarded) and 158 mg N-(4-oxo-3-phenyl-4H-chromen-7-yl)acetamide as pale brown powder were
obtained, which was then dissolved into 5 ml MeOH, followed by the addition of 6N HCl (3 ml),
the mixture was refluxed for about 6 hours, then neutralized by enough volume saturated K2CO3
solution, extracted by ethyl acetate for 3 times, combined and dried, the organic phase was removed
under reduced pressure. The residue was recrystallized from methanol, 106 mg titled compound
was obtained as pale brown solid, yield 78% (last step); HPLC: 99.2%; 1H NMR (DMSO-d6, 400
MHz) δ (ppm) = 6.32 (s, 2H), 6.52 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 7.35-7.43 (m, 3H), 7.55 (d, J =
28

ACCEPTED MANUSCRIPT
8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H). MS (ESI), m/z: 238.10 [M + H]+. NMR spectra
match the reference.[42]
8,8,10-trimethyl-3-phenyl-7,8-dihydro-4H-pyrano[2,3-f]quinolin-4-one (1n): 90 mg
compound 16 was dissolved into 10 ml acetone, followed by the addition of catalytic amount
silicotungstic acid, the mixture was refluxed for about 12 hours, filtered and removed of the solvent,
the residue was purified by silica gel column, using petroleum/ethyl acetate = 1/1 as eluent, 66 mg
compound 1n was obtained as pale brown solid, yield 52%; HPLC: 99.1%; 1H NMR (CDCl3, 300
MHz) δ (ppm) = 1.34 (s, 6H), 2.32 (d, J = 1.5 Hz, 3H), 5.32 (d, J = 1.5 Hz, 1H), 7.36-7.45 (m, 3H),
7.57 (t, J = 1.5, 7.5 Hz, 2H), 7.91 (s, 1H), 8.00 (d, J = 1.5 Hz, 1H). HRMS (ESI) exact mass calcd.
for (C21H19NO2+H)+ requires m/z 318.1416, found m/z 318.1487.
6-amino-3-phenyl-4H-chromen-4-one (19): Starting from N-(4-methoxyphenyl)acetamide (3
mmol), by using the same method as the synthesis of compound 16, 462 mg titled compound was
obtained as pale brown powder, yield 65%; HPLC: 99.1%; 1H NMR (DMSO-d6, 400 MHz) δ (ppm)
= 5.52 (s, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 7.37-7.45 (m, 4H), 7.58 (d, J = 8.0 Hz, 2H),
8.04 (s, 1H). MS (ESI), m/z: 238.08 [M + H]+. NMR spectra match the reference.[42]
8,8,10-trimethyl-2-phenyl-7,8-dihydro-1H-pyrano[3,2-f]quinolin-1-one (1o): Starting from
compound 19 (90 mg), and by using the same method as the synthesis of 1n, 79 mg titled
compound was obtained as pale brown powder, yield 63%; HPLC: 97.7%; 1H NMR (CDCl3, 300
MHz) δ (ppm) = 1.29 (s, 6H), 2.04 (d, J = 1.5 Hz, 3H), 5.48 (d, J = 1.5 Hz, 1H), 6.79-6.81 (m, 1H),
29