A novel structural class of coumarin-chalcone fibrates as PPARα/γ agonists with potent antioxidant activities: Design, synthesis, biological evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.06.033

A series of structurally interesting coumarin-chalcone fibrates were synthesized and evaluated for their PPARα/γ agonist activities and antioxidant activities. Among these compounds, compounds 5a, 5d, and 7a were identified as potent PPARα and γ dual agonists, and their PPARα agonist activities were found to be more potent than that of Fenofibrate. Furthermore, the results of antioxidant investigations revealed that compounds 5d and 6a?6d had greater potency than Trolox with IC₅₀ values ranging from 9.40 μM to 18.63 μM. The structure–activity relationship revealed that the electron-withdrawing nitro group substituted at the C6′ position of the benzopyran moiety increased the PPARα and γ agonist efficacy. Moreover, the presence of a double bond on the benzopyran moiety was essential for PPARα and γ agonist efficacy. The agonist activity of PPARα exhibited by compound 5d was examined by molecular docking studies. Taken together, the results we obtained showed that compound 5d had the potential to be a lead compound for further research.

Full text of DOI:10.1016/j.ejmech.2017.06.033

Accepted Manuscript
A novel structural class of coumarin-chalcone fibrates as PPARα/γ agonists with
potent antioxidant activities: Design, synthesis, biological evaluation and molecular
docking studies
Handong Niu, Wenbao Wang, Jinyan Li, Yu Lei, Yong Zhao, Weixu Yang, Chaoyue
Zhao, Bin Lin, Shaojiang Song, Shaojie Wang
PII:
S0223-5234(17)30479-8
10.1016/j.ejmech.2017.06.033
EJMECH 9528
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 March 2017
Revised Date: 12 May 2017
Accepted Date: 21 June 2017
Please cite this article as: H. Niu, W. Wang, J. Li, Y. Lei, Y. Zhao, W. Yang, C. Zhao, B. Lin, S. Song,
S. Wang, A novel structural class of coumarin-chalcone fibrates as PPARα/γ agonists with potent
antioxidant activities: Design, synthesis, biological evaluation and molecular docking studies, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.033.
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ACCEPTED MANUSCRIPT
A novel structural class of coumarin-chalcone fibrates as PPARα/γ
agonists with potent antioxidant activities: design, synthesis, biological
evaluation and molecular docking studies
Handong Niua,c,1, Wenbao Wangb,1, Jinyan Lia, Yu Leia, Yong Zhaoa, Weixu Yanga, Chaoyue Zhaoa,
Bin Lina, Shaojiang Songb,*, Shaojie Wanga,*
aKey Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang
110016, People’s Republic of China
bDepartment of Natural Products Chemistry, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe
District, Shenyang 110016, People’s Republic of China
cCenter for Drug Evaluation, China Food and Drug Administration, NO.1 of Fuxing Road, Haidian District,
Beijing 100038, People’s Republic of China
*Corresponding author. Tel./fax: +86 24 23986421 (S. J. Wang), +86 24 23986510 (S. J. Song).
E-mail addresses: sjwang_99@163.com (S. J. Wang), songsj99@163.com (S. J. Song).
1 Both authors contributed equally to this work.

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Abstract
A series of structurally interesting coumarin-chalcone fibrates were synthesized and evaluated for their
PPARα/γ agonist activities and antioxidant activities. Among these compounds, compounds 5a, 5d, and 7a were
identified as potent PPARα and γ dual agonists, and their PPARα agonist activities were found to be more potent
than that of Fenofibrate. Furthermore, the results of antioxidant investigations revealed that compounds 5d and
6a−6d had greater potency than Trolox with IC50 values ranging from 9.40 µM to 18.63 µM. The structure–activity
relationship revealed that the electron-withdrawing nitro group substituted at the C6′ position of the benzopyran
moiety increased the PPARα and γ agonist efficacy. Moreover, the presence of a double bond on the benzopyran
moiety was essential for PPARα and γ agonist efficacy. The agonist activity of PPARα exhibited by compound 5d
was examined by molecular docking studies. Taken together, the results we obtained showed that compound 5d
had the potential to be a lead compound for further research.
Keywords: Fibrates; Coumarin-chalcone; PPARα/γ agonists; Antioxidant activity

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1. Introduction
Hypertriglyceridemia and hypercholesterolemia are two major factors associated with
cardiovascular diseases (CVD), which are the leading cause of mortality worldwide [1]. PPARα,
one of the three peroxisome proliferator-activated receptor isoforms (α, γ, and β/δ), is mainly
found in liver, kidney, heart, muscle, and adipose tissue. It plays a critical role in fatty acid
oxidation and lipoprotein metabolism [2,3]. Therefore, PPARα is an important target for the
treatment of dyslipidemia. PPARγ, another very widely investigated PPAR subtype, has been
shown to be an important regulator of target genes involved in glucose and lipid metabolism [4].
In addition, oxidative stress is an important factor in the development of vascular damage [5].
The involvement of reactive oxygen species has been found to be a major causative factor for
peroxidative damage to lipoproteins present in the blood, which is responsible for the initiation
and progression of atherosclerosis [6]. On the basis of the underlying pathophysiological
processes of CVD, therapeutic agents possessing both antihyperlipidemic and antioxidant
activities are an ideal choice for the treatment of CVD and the development of such agents has
been the focus of much research in recent years.
Fibrates, such as Clofibrate, Fenofibrate and Bezafibrate, have been used clinically for many
years for the treatment of dyslipidemia to reduce cardiovascular risk [7]. Although it has been
demonstrated that fibrates exert their effects by activating PPARα [8], most of them show weak
agonist activities and some of them even display severe adverse effects [4,9]. Therefore, the
development of more effective PPARα agonists is urgently needed.
The molecular hybridization approach is one of the most valuable structural modification tools
used for the discovery of ligands. Increasing efforts to discover hybrid drugs resulting from the
combination of pharmacophoric moieties of different known lead compounds have brought new
hope for the treatment of multifactorial diseases in recent years [10]. In our previous work, we
discovered a series of novel non-sugar α-glucosidase inhibitors for the treatment of diabetes by
using this approach [11]. Coumarins and chalcones, two types of naturally occurring and synthetic
compounds, have attracted enormous research attention because of their diverse biological
activities including antioxidant, antihyperglycemic, and antidyslipidemic properties [12,13].

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Shukla et al. reported the hybrid compounds obtained by a combination of the classical fibrate
“headgroup” and chalcone, the results showed that 2-[4-{3-(4-Nitro-phenyl) -acryloyl}-2-methyl-
propionic acid ethyl ester (compound A, Figure 1) displayed marked antidyslipidemic activity [14].
In addition, Sashidhara et al. designed the hybrids of coumarin, chalcone and fibrate, and found
that (E)-Ethyl 8-sec-butyl-6-(3-(4-(1-ethoxy-2-methyl-1-oxopropan-2-yloxy) phenyl)-3-oxoprop-1
-enyl)-2-oxo-2H-chromene-3-carboxylate (compound B, Figure 1) showed significant
hypolipidemic potential [15]. More recently, Sashidhara et al. demonstrated that the
indole-chalcone fibrates had the unique ability to scavenge free radicals and lower blood lipids
[16]. These findings prompted us to investigate the hybrids of coumarin, chalcone and fibrate to
examine their structure–activity relationships in the hope of discovering more potent PPARα/γ
agonists for use as antilipemic agents.
In this paper, as shown in Figure 2, we have designed a novel scaffold that assembled
coumarin, chalcone, and the classical fibrate “head group” by employing the molecular
hybridization approach. Consequently, a series of derivatives with the novel scaffold were
synthesized and evaluated for their PPARα/γ agonist and antioxidant activities. Their
structure–activity relationships were summarized and the mechanism of action that the compounds
exerted their PPARα agonist activities was examined by molecular docking studies.
2. Results and discussion
2.1 Chemistry
The general strategy for the synthesis of all the target compounds is outlined in Scheme 1. The
β-Keto ester 4 was obtained by reacting p-hydroxyacetophenone 1 with ethyl 2-bromoisobutyrate
2 in the presence of K2CO3 in acetonitrile according to the procedure reported in the literature [17],
followed by reacting with diethyl carbonate using sodium hydride [18, 19]. The subsequent
Knoevenagel condensation reaction of β-keto ester 4 with different substituted salicylaldehydes
yielded the desired coumarin-chalcone fibrates 5a−5i [20,21]. Furthermore, compounds 6a−6d
and 7a−7c were prepared from the corresponding precursors by reduction and hydrolysis,
1
respectively [22]. The structures of all the compounds were fully supported by H NMR, 13C

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1
NMR, IR, and ESI-HRMS spectral data. In the H NMR spectra, the H-4 signal of the chromene
ring was observed as a singlet at around 8.20−8.85 ppm for compounds 5a−5i and 7a−7c.
Moreover, the two methyl signals of the target compounds were detected as a singlet, which
ranged from 1.15 to 1.61 ppm.
2.2 In vitro biological activity assay and structure-activity relationships
The PPARα/γ agonist activities of the target compounds were evaluated by a transactivation
assay using firefly luciferase reporter gene technology in HERK293 cells [23,24], and
commercially available PPAR agonists, Fenofibrate and Rosiglitazone, were used as positive
controls for PPARα and γ activity, respectively. The results were summarized in Tables 1−2. In
Table 1, compounds 5a, 5b, 5d, 5e, 6a, 7a, and 7c exerted PARPα/γ activation fold greater than
1.1 at 10 µM. Subsequently, the EC50 values of these compounds were determined.
As shown in Table 2, compound 5a (EC50 = 12.69 µM) and its corresponding acid 7a (EC50 =
11.03 µM) exhibited apparent PPARα agonist activities, which were about 2.4-fold and 2.7-fold
more potent than Fenofibrate (EC50 = 30 µM), respectively. However, when the C6′ position of the
benzopyran moiety was substituted with halogen groups of fluorine (5b) and chlorine (5c), the
PARPα activation activity disappeared completely, these results indicated that the halogen
substitution was unfavorable for our coumarin-chalcone fibrates to display PPARα agonist activity.
Nevertheless, when strong electron-withdrawing nitro group (5d) was introduced to the
corresponding position, the PPARα agonist activity improved remarkably with an EC50 value of
6.99 µM, which was 1.8-fold and 4.3-fold more potent than compound 5a and Fenofibrate (EC50 =
30 µM), respectively. However, when it was reduced to electron-donating amino group (5e)
resulted in the inactivity of PARPα. Meanwhile, when the amino group was replaced with another
two electron-donating groups of methyl (5f) and methoxy (5g), the PPARα agonist activity also
disappeared. In addition, compound 5d possessed stronger PPARα agonist activity than
compounds 5h and 5i with bulky substitution pattern at the benzopyran moiety. These result
showed the C6′ position of the benzopyran moiety substituted with the strong
electron-withdrawing nitro group was more suitable than electron-donating groups, halogens, and

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sterically-hindered groups, which was consistent with previous reports showing that chalcone
fibrate compound A with a nitro group was a very potent antidyslipidemic agent [14].
Furthermore, the PPARγ agonist activities of the new fibrates were also evaluated. Compounds
5a (EC50 = 5.19 µM), 5d (EC50 = 0.91 µM), and 7a (EC50 = 3.67 µM) showed good PPARγ agonist
activities. Compound 5d had a 5.7-fold higher PPARγ agonist activity than 5a. The same structure
activity relationship was also observed between compounds 5a and 5d, which revealed nitro group
was the preferred substitution pattern for the benzopyran moiety. Besides, compound 7a with
unsubstituted benzopyran moiety (EC50 = 3.67 µM) was about 15.5-fold more potent than
compound 7c (EC50 = 56.71 µM). The data obtained also showed that large sterically-hindered
groups were unfavorable substituents.
To increase the molecular flexibility and investigate the effects produced by a change in the
spatial structure of the coumarin-chalcone fibrates, compound 6a was obtained by the reduction of
the double bond on the benzopyran moiety of 5a. To our surprise, the PPARα agonist activity of
5a was lost, which indicated that the double bond of the benzopyran moiety was critical for the
PPARα agonist activity of compound 5a. Sashidhara et al. also confirmed that the α, β-unsaturated
ketone of the chalcone fibrates was essential for their hypolipidemic activities [15].
Taken together, compounds 5a, 5d, and 7a were identified as potent PPARα and γ dual
agonists. The structure-activity relationship suggested that the strong electron-withdrawing nitro
group substituted at the C6′ position of the benzopyran moiety increased the PPARα and γ agonist
efficacy, and the α, β-unsaturated ketone was essential for the coumarin-chalcone fibrates to
display potent PPARα and γ agonistic activity. An explanation for this observation was proposed
based on the molecular docking studies described below.
2.3. Antioxidant activity
To explore whether the target compounds possess antioxidant activity, the synthesized
coumarin-chalcone fibrates were evaluated for their antioxidant activities using the 2,2-azinobis
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+) radical scavenging activity evaluation method
with Trolox as a positive control. The results of ABTS+ inhibition by the target compounds are

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summarized in Table 3. Compounds 5d (IC50 = 18.63 µM) and 5f (IC50 = 8.51 µM) showed higher
ABTS radical scavenging activities than Trolox (IC50 = 24.84 µM). Compound 5f (IC50 = 8.51 µM)
was about 2.2-fold and 2.9-fold more potent than 5d (IC50 = 18.63 µM) and Trolox (IC50 = 24.84
µM), respectively. When comparing compound 5f with compound 5d, we found that strong
electron-withdrawing groups may be unsuitable, as replacing the nitro group (5f) with a methyl
group (5d) led to an increase in antioxidant activity apparently. Moreover, compounds 6a−6d also
exhibited ABTS scavenging abilities with IC50 values of 9.40, 10.35, 17.11, and 14.98 µM,
respectively. It was observed that reduction of the double bond on the benzopyran moiety resulted
in an increase in the antioxidant activities of the coumarin-chalcone fibrates. So, although the
absence of the double bond was detrimental to the PPARα agonist activity, it was beneficial to the
antioxidant activity.
2.3. Molecular Interactions and Docking Simulations.
To gain insight into the interactions between the coumarin-chalcone fibrates and PPARα,
molecular docking simulations were performed using the coumarin-chalcone compounds with
PPARα (PDB code: 2P54 [25]). Fenofibrate was also studied in the same protocol for comparison.
As shown in Fig. 3, the docking results revealed that both Fenofibrate and compound 5d exerted
their PPARα agonist activities in a very similar manner to other agonists as demonstrated in
crystallographic studies [25,26]. The ester functional group formed several hydrogen bonds with
Ser280, Tyr314, His440 and Tyr464. The coumarin-chalcone hybrid moiety interacted with the
hydrophobic pocket formed by Cys275, Met330, Val332, Ile339 and Ile344. This hydrophobic
pocket was large enough to accommodate the 4-clorophenyl group and the nitro-coumarin group.
However, the latter probably formed stronger hydrophobic interactions due to the bulkier
coumarin moiety. More importantly, it also formed an additional hydrogen bond between the nitro
group and the adjacent Thr279. This mode of action explained why both compounds 5a and 5d
were more potent than Fenofibrate and compound 5d was even more potent than 5a due to the
addition of the nitro group on the coumarin moiety. Compound 6a lost its agonist activity on
PPARα following reduction of the double bond in the coumarin moiety. The binding revealed from

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the molecular docking results showed that, due to the spatial change of the molecular shape
following the reduction, compound 6a was not able to fit into the binding pocket in the same
manner as those compounds with an intact double bond in the coumarin moiety. Consequently, the
head group of compound 6a was not able to reach the subpocket formed by Ser280, Tyr314,
His440 and Tyr464 and to form an extensive hydrogen bond network (see Fig. 3), which was
essential for the activation of PPARα. In addition, Compound A and Compound B were also
studied in the same protocol for comparison. The docking results showed that Compound A and
Compound B were shown to bind to PPARα in a very similar manner, as expected (Figure S1, see
supplementary material).
3. Conclusion
In our efforts to design new scaffolds for PPARα agonists, a series of novel coumarin-chalcone
fibrates were designed using the molecular hybridization approach. They were subsequently
synthesized and evaluated for their PPARα agonist and antioxidant activities. Among these
compounds, compounds 5a (EC50 = 12.69 µM), 5d (EC50 = 6.99 µM) and 7a (EC50 = 11.03 µM)
were found to possess significant PPARα agonist activities, and compound 5d had the most potent
PPARα agonist activity and was 4.3-fold more potent than Fenofibrate (EC50 = 30 µM). In
addition, compounds 5a (EC50 = 5.19 µM), 5d (EC50 = 0.91 µM), and 7a (EC50 = 11.03 µM) also
showed PPARγ agonist activities. As a result, compounds 5a, 5d, and 7a were identified as potent
PPARα and γ dual agonists. Furthermore, the antioxidant results revealed that compounds 6a−6d
were more potent than Trolox (IC50 = 24.84 µM) with IC50 values ranging from 9.40 µM to 17.11
µM, and compound 5d also showed good radical scavenging potential (IC50 = 18.63 µM). The
structure–activity relationships revealed that the nitro group substituted at the C6′ position of the
benzopyran moiety improved the agonist activity. Moreover, the presence of a double bond on the
benzopyran moiety was important for PPARα and γ agonist efficacy. Interestingly, the absence of a
double bond on the benzopyran moiety improved the antioxidant activity. The significant PPARα
agonist activity exhibited by compound 5d was explained by molecular docking studies. Therefore,
on the basis of these results, the synthesized coumarin-chalcone fibrates can be considered as

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promising scaffolds for the development of new PPAR agonists, and compound 5d has the
potential to be a lead compound for further research.
4. Experimental section
4.1 Chemistry
All reagents used were commercially available, and some were redistilled under a positive
pressure in a dry nitrogen atmosphere in the presence of an appropriate desiccant when necessary.
Reactions were monitored by thin-layer chromatography (TLC) on glass plates coated with silica
gel with a fluorescent indicator (GF254). Column chromatography was performed on silica gel
1
(160−200 mesh). All the H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz
spectrometer in DMSO-d6 or Acetone-d6. Chemical shifts (δ) were expressed in parts per million
using tetramethylsilane as an internal reference. ESI-MS data were obtained using an Agilent 1100
instrument, and the ESI-HRMS data were obtained using a Bruker microTOF-Q instrument.
Melting points were measured on a YRT-3 apparatus (Tianjin Corp., Tianjin, China). Fenofibrate
and Rosiglitazone were obtained from Sigma-Aldrich.
4.1.1 Synthesis of ethyl 2-(4-acetylphenoxy)-2-methylpropanoate (3)
Ethyl 2-bromoisobutyrate (43 g, 0.22 mol) was added dropwise at 45 oC to a stirred solution of
p-hydroxyacetophenone (27.2 g, 0.2 mol) and K2CO3 (83 g, 0.6 mol) in acetonitrile (250 mL).
Upon completion of the addition, the mixture was heated under reflux for another 4 h. After
filtering the reaction mixture, the filtrate was concentrated in a vacuum. An aqueous solution of
NaOH (1M) and dichloromethane was then added successively to the residue, and the organic
layer was washed with water and then brine and dried over MgSO4, filtered and concentrated
under reduced pressure. Without any purification, compound 3 (24.6 g, 49.2%) was obtained as a
pale yellowish oil.
4.1.2 Synthesis of ethyl 3-{4-[(1-ethoxy-2-methyl-1-oxopropan-2-yl)oxy]phenyl}-3-oxopropanoate
(4)

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A solution of 3 (12.3 g, 0.048 mol) dissolved in tetrahydrofuran (30 mL) was added dropwise
o
at a rate that maintained a reaction temperature < 0 C to a solution containing diethyl carbonate
(22.7 g, 0.192 mol) and sodium hydride (0.096 mol 60% dispersion in mineral oil). The mixture
was stirred at room temperature for 4−5 h. Then it was poured into ice water, acidified with glacial
acetic acid, and extracted with EtOAc (3×20 mL). The EtOAc extract was then dried over
anhydrous MgSO4. After removal of the solvent in vacuo, the crude product was purified by flash
chromatography involving silica gel column chromatography using Petroleum Ether/EtOAc =
30~10:1 as an eluant to afford β-keto Ester 4 as a pale yellowish oil (9.1 g, 58.3%). 1H NMR (400
MHz, DMSO-d6): 7.87 (d, J = 8.9 Hz, 2H, phenyl-H), 6.83 (d, J = 8.9 Hz, 2H, phenyl-H), 4.21 (q,
J = 7.1 Hz, 2H, 2×OCH2), 3.92 (s, 2H,–CO–CH2–CO–), 1.66 (s, 6H, 2×CH3), 1.25 (t, J = 7.1 Hz,
3H, CH3), 1.21 (t, J = 7.1 Hz, 3H, CH3). ESI-MS: m/z 323.2 [M+H]+, 345.2 [M+Na]+.
4.1.3 General procedure for preparation of compounds 5a–5i
To a solution of the β-keto Ester 4 (2.3 g, 0.007 mol) and the corresponding salicylaldehyde
(0.0077 mol) in absolute ethanol (30 mL) was added piperidine-acetic acid in a catalytic amount.
The mixture was refluxed for 2−5 h. After cooling to room temperature, the product was collected
by filtration and washed with ethanol.
4.1.3.1 Ethyl 2-methyl-2-[4-((2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoate (5a)
o
1
White solid, yield 74.1%. m.p. 122.0-122.8 C. H NMR (400 MHz, DMSO-d6): δ 8.37 (s,
1H, chromene-4-H), 7.90 (d, J = 8.9 Hz, 2H, phenyl-H), 7.84 (dd, J = 7.7, 1.3 Hz, 1H,
chromene-5-H), 7.77−7.68 (m, 1H, chromene-7-H), 7.49 (d, J = 8.3 Hz, 1H, chromene-8-H), 7.43
(t, J = 7.5 Hz, 1H, chromene-6-H), 6.86 (d, J = 8.9 Hz, 2H, phenyl-H), 4.17 (q, J = 7.1 Hz, 2H,
OCH2), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ
190.0, 172.8, 160.1, 158.3, 154.2, 144.5, 133.5, 132.0, 129.8, 129.4, 126.9, 125.1, 118.5, 117.3,
116.5, 79.3, 61.6, 25.2, 14.0. ESI-HRMS: m/z [M + Na]+ calcd for C22H20O6Na: 403.1152; found:
403.1154. IR (KBr): cm-1 3432.7, 3113.9, 3078.8, 2982.4, 1730.2, 1656.7, 1595.9, 1573.5, 898.2,
856.3, 761.0.

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4.1.3.2 Ethyl 2-[4-((6-fluoro-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]-2-methylpropanoate (5b)
o
1
White solid, yield 50.2%. m.p.101.4~101.9 C. H NMR (400 MHz, DMSO-d6): δ 8.31 (s,
1H, chromene-4-H), 7.91 (d, J = 8.9 Hz, 2H, phenyl-H), 7.70 (dd, J = 8.4, 3.0 Hz, 1H,
chromene-8-H), 7.61 (td, J = 8.8 Hz, 8.5Hz, 3.0Hz, 1H, chromene-7-H), 7.56 (dd, J = 9.1, 4.5 Hz,
1H, chromene-5-H), 6.86 (d, J = 8.9 Hz, 2H, phenyl-H), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.61 (s,
6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ 190.2, 173.0,
160.5, 158.3, 150.9, 143.8, 143.7, 132.4, 132.4, 129.5, 128.2, 120.9, 119.6, 118.8, 117.6, 117.6,
115.2, 114.9, 79.6, 61.8, 25.5, 14.3. ESI-HRMS: m/z [M + Na]+ calcd for C22H19FO6Na: 421.1058;
found: 421.1055. IR (KBr): cm-1 3433.0, 2982.4, 1728.7, 1651.7, 1596.5, 1490.1, 873.2, 853.1,
824.1, 784.3, 608.2.
4.1.3.3 Ethyl 2-[4-((6-chloro-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]-2-methylpropanoate (5c)
White solid, yield 50.6%. m.p.100.6~101.0 oC. 1H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 1H,
chromene-4-H), 7.95 (d, J = 2.5 Hz, 1H, chromene-5-H), 7.91 (d, J = 8.9 Hz, 2H, phenyl-H), 7.76
(dd, J = 8.9, 2.6 Hz, 1H, chromene-7-H), 7.54 (d, J = 8.9 Hz, 1H, chromene-8-H), 6.86 (d, J = 8.9
Hz, 2H, phenyl-H), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H,
13
CH3). C-NMR (100 MHz, DMSO-d6): δ 190.1, 173.0, 160.6, 158.1, 153.1, 143.8, 133.2, 132.4,
132.4, 129.5, 129.0, 128.9, 128.2, 120.2, 118.8, 117.6, 117.6, 79.6, 61.8, 25.5, 14.3. ESI-HRMS:
m/z [M + Na]+ calcd for C22H19ClO6Na: 437.0762; found: 437.0760. IR (KBr): cm-1 3431.6,
3099.3, 3052.0, 2990.1, 2941.5, 1729.3, 1651.4, 1596.4, 1506.1, 954.7, 836.9, 789.1.
4.1.3.4 Ethyl 2-methyl-2-[4-((6-nitro-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoate (5d)
o
1
Pale yellow powder, yield 50.4%. m.p.126.0~127.0 C. H NMR (400 MHz, DMSO-d6): δ
8.82 (d, J = 2.7 Hz, 1H, chromene-4-H), 8.51 (m, J = 9.1, 2.7 Hz, 2H, chromene-5-H,
chromene-7-H), 7.95 (d, J = 8.9 Hz, 2H, phenyl-H), 7.72 (d, J = 9.1 Hz, 1H, chromene-8-H), 6.87
(d, J = 8.9 Hz, 2H, phenyl-H), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.62 (s, 6H, 2×CH3), 1.15 (t, J =
7.1 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ 189.8, 173.0, 160.7, 158.1, 157.7, 144.1,
143.6, 132.5, 132.5, 129.3, 128.9, 128.0, 125.7, 119.2, 118.4, 117.6, 117.6, 79.6, 61.9, 25.5, 25.5,
14.3. ESI-HRMS: m/z [M + Na]+ calcd for C22H19NO8Na: 448.1003; found: 448.1002. IR (KBr):

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cm-1 3426.8, 3074.4, 2990.4, 2920.3, 2850.7, 1737.0, 1652.3, 1620.5, 1592.3, 1347.8, 1139.5,
913.3.
4.1.3.5 Ethyl 2-methyl-2-[4-((6-amino-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoate (5e)
Yellow powder, yield 36.4%. m.p.121.0~121.9 oC. 1H NMR (400 MHz, DMSO-d6): δ 8.18 (s,
1H, chromene-4-H), 7.85 (d, J = 8.8 Hz, 2H, phenyl-H), 7.19 (d, J = 8.8 Hz, 1H, chromene-8-H),
6.96 (dd, J = 8.9, 2.6 Hz, 1H, chromene-7-H), 6.85 (d, J = 8.7 Hz, 3H, phenyl-H, chromene-5-H),
5.37 (s, 2H, NH2), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 1.60 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H,
13
CH3). C-NMR (100 MHz, DMSO-d6): δ 190.4, 172.6, 159.8, 158.5, 146.0, 145.6, 144.4, 131.7,
131.7, 129.3, 126.4, 120.2, 118.6, 117.1, 117.1, 116.6, 110.7, 79.0, 61.4, 25.0, 25.0, 13.8.
ESI-HRMS: m/z [M + Na]+ calcd for C22H21NO6Na: 418.1261; found: 418.1261. IR (KBr): cm-1
3444.9, 3370.3, 2986.2, 2920.3, 2850.8, 1724.0, 1653.4, 1597.3, 1574.2, 1178.7, 1140.9, 853.2.
4.1.3.6 Ethyl 2-methyl-2-[4-((6-methyl-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoate (5f)
o
1
White solid, yield 73.5%. m.p.92.5~93.0 C. H NMR (400 MHz, DMSO-d6): δ 8.29 (s, 1H,
chromene-4-H), 7.88 (d, J = 8.8 Hz, 2H, phenyl-H), 7.61 (s, 1H, chromene-5-H), 7.55 (d, J = 8.7
Hz, 1H, chromene-7-H), 7.39 (d, J = 8.5 Hz, 1H, chromene-8-H), 6.85 (d, J = 8.9 Hz, 2H,
phenyl-H), 4.17 (q, J = 7.1 Hz, 2H, OCH2), 2.39 (s, 3H, chromene-6-CH3), 1.61 (s, 6H, 2×CH3),
1.15 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ 190.5, 173.1, 160.4, 158.7,
152.6, 144.7, 134.7, 134.6, 132.3, 132.3, 129.7, 129.6, 127.1, 118.5, 117.6, 117.6, 166.6, 79.6,
61.8, 25.5, 20.7, 14.3. ESI-HRMS: m/z [M + Na]+ calcd for C23H22O6Na: 417.1309; found:
4117.1315. IR (KBr): cm-1 3435.6, 3078.3, 2980.9, 2935.8, 1736.6, 1655.9, 1597.8, 1576.0,
1177.4, 1140.0, 856.6, 827.2, 786.6.
4.1.3. Ethyl2-[4-((6,7-dimethoxy-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]-2-methyl-propanoate
(5g)
Yellow powder, yield 77.8%. m.p.165.3~166.2 oC. 1H NMR (400 MHz, DMSO-d6): δ 8.27 (s,
1H, chromene-4-H), 7.83 (d, J = 8.9 Hz, 2H, phenyl-H), 7.39 (s, 1H, chromene-8-H), 7.17 (s, 1H,
chromene-5-H), 6.85 (d, J = 8.9 Hz, 2H, phenyl-H), 4.18 (q, J = 7.1 Hz, 2H, OCH2), 3.91 (s, 3H,

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OCH3), 3.81 (s, 3H, OCH3), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3). ESI-HRMS: m/z
[M + Na]+ calcd for C24H24O8Na: 463.1363; found: 463.1373. IR (KBr): cm-1 3423.6, 2921.4,
2851.2, 1731.0, 1644.3, 1602.1, 1143.2, 961.9, 836.9, 787.8.
4.1.3.8 Ethyl 2-[4-((6,8-di-tert-butyl-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]-2-methylpropan
-oate (5h)
o
1
White solid, yield 65.0%. m.p.99.4~100.4 C. H NMR (400 MHz, DMSO-d6): δ 8.32 (s, 1H,
chromene-4-H), 7.90 (d, J = 8.9 Hz, 2H, phenyl-H), 7.72 (d, J = 2.2 Hz, 1H, chromene-5-H), 7.64
(d, J = 2.3 Hz, 1H, chromene-7-H), 6.87 (d, J = 8.9 Hz, 2H, phenyl-H), 4.18 (q, J = 7.1 Hz, 2H,
OCH2), 1.61 (s, 6H, 2×CH3), 1.49 (s, 9H, CH3), 1.33 (s, 9H, CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3).
ESI-MS: m/z 493.4 [M+H]+, 515.4 [M+Na]+. IR (KBr): cm-1 3431.1, 2919.6, 2850.6, 1722.0,
1140.8, 779.2, 719.2, 619.6.
4.1.3.9 Ethyl 2-methyl-2-[4-((3-oxo-3H-benzo[f]chromen-2-yl)carbonyl)phenoxy]propanoate (5i)
Yellow solid, yield 69.7%. m.p.107.6~108.0 oC. 1H NMR (400 MHz, DMSO-d6): δ 9.17 (s, 1H,
benzo[f]chromene-1-H), 8.61 (d, J = 8.5 Hz, 1H, benzo[f]chromene-6-H), 8.31 (d, J = 9.0 Hz, 1H,
benzo[f]chromene-5-H), 8.11 (d, J = 8.0 Hz, 1H, benzo[f]chromene-7-H), 7.95 (d, J = 8.9 Hz, 2H,
phenyl-H), 7.75 (t, J = 7.1 Hz, 1H, benzo[f]chromene-10-H), 7.66 (t, J = 8.0 Hz, 2H,
benzo[f]chromene-8-H, benzo[f] chromene-9-H), 6.87 (d, J = 8.9 Hz, 2H, phenyl-H), 4.17 (q, J =
7.1 Hz, 2H, OCH2), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3). ESI-MS: m/z 431.1
[M+H]+, 453.2 [M+Na]+. IR (KBr): cm-1 3432.7, 2919.8, 2850.7, 1734.7, 1656.3, 1598.3, 1252.5,
1178.2, 1140.7, 873.4, 847.7, 816.9, 786.7, 748.8.
4.1.4 General procedure for preparation of compounds 6a–6d
To a stirring solution of corresponding coumarin-chalcone fibrate ester derivatives (0.001 mol)
in dry pyridine (10 mL) was added NaBH4 (0.038 g, 0.001 mol). The reaction was stirred at room
temperature for 2 h. Then the reaction mixture was poured into cold 2 M hydrochloric acid (90
mL). The resulting precipitate was collected by filtration, washed with HCl (aq, 2 M), and
recrystallized from methanol.

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4.1.4.1 Ethyl 2-methyl-2-[4-((2-oxochroman-3-yl)carbonyl)phenoxy]propanoate (6a)
White powder, yield 70.7%. m.p.83.9 ~84.3° oC. 1H NMR (400 MHz, Acetone-d6): δ 8.05 (d, J
= 8.9 Hz, 2H, phenyl-H), 7.35−7.27 (m, 2H, chroman-6-H, chroman-7-H), 7.13 (td, J = 7.5, 1.0
Hz, 1H, chroman-5-H), 7.07 (d, J = 8.1 Hz, 1H, chroman-8-H), 6.91 (d, J = 8.9 Hz, 2H, phenyl-H),
5.02 (dd, J = 9.7, 6.5 Hz, 1H, chroman-3-H), 4.21 (q, J = 7.1 Hz, 2H, OCH2), 3.47 (dd, J = 16.2,
9.7 Hz, 1H, chroman-4-H), 3.30 (dd, J = 16.2, 6.5 Hz, 1H, chroman-4-H), 1.64 (s, 6H, 2×CH3),
1.19 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (100 MHz, Acetone-d6): δ 193.8, 173.7, 166.9, 161.3,
152.6, 131.7, 131.7, 130.1, 129.3, 129.0, 125.3, 123.0, 118.2, 118.2, 117.0, 80.1, 62.1, 47.5, 27.7,
25.6, 25.6, 14.2. ESI-HRMS: m/z [M + Na]+ calcd for C22H22O6Na: 405.1309; found: 405.1329.
IR (KBr): cm-1 3440.3, 2986.0, 2920.4, 2850.8, 1752.0, 1734.4, 1670.7, 1175.8, 1141.8, 916.7,
885.2, 850.3, 754.6.
4.1.4.2 Ethyl 2-methyl-2-[4-((6-methyl-2-oxochroman-3-yl)carbonyl)phenoxy]propanoate (6b)
o
1
Yellow solid, yield 75.7%. m.p.77.5~78.0 C. H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J =
8.9 Hz, 2H, phenyl-H), 7.12−7.07 (m, 2H, chroman-5-H, chroman-7-H), 7.00 (d, J = 8.1 Hz, 1H,
chroman-8-H), 6.85 (d, J = 8.9 Hz, 2H, phenyl-H), 5.09 (dd, J = 9.3, 6.8 Hz, 1H, chroman-3-H),
4.17 (q, J = 7.1 Hz, 2H, OCH2), 3.23 (ddd, J = 22.9, 16.2, 9.7 Hz, 2H, chroman-4-H), 2.25 (s, 3H,
chroman-6-CH3), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz, 3H, CH3). ESI-MS: m/z 387.3 [M+H]+,
419.3 [M+Na]+. IR (KBr): cm-1 3440.9, 2992.4, 2921.2, 2852.1, 1748.9, 1735.9, 1667.8, 1175.8,
1141.0, 867.6, 843.5, 816.8.
4.1.4.3 Ethyl 2-[4-((6,7-dimethoxy-2-oxochroman-3-yl)carbonyl)phenoxy]-2-methylpropanoate
(6c)
o
1
Red solid, yield 33.9%. m.p.146.2~146.8 C. H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J =
8.8 Hz, 2H, phenyl-H), 6.90−6.78 (m, 4H, phenyl-H, chroman-5-H, chroman-8-H), 5.07 (t, J = 7.8
Hz, 1H, chroman-3-H), 4.18 (q, J = 7.1 Hz, 2H, OCH2), 3.76 (s, 3H, chroman-6-OCH3), 3.70 (s,
3H, chroman-7-OCH3), 3.26−3.10 (m, 2H, chroman-4-H), 1.61 (s, 6H, 2×CH3), 1.15 (t, J = 7.1 Hz,
3H, CH3). ESI-MS: m/z 443.3 [M+H]+, 465.3 [M+Na]+. IR (KBr): cm-1 3440.2, 2993.5, 2935.8,

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2835.0, 1744.5, 1683.1, 1260.4, 1225.6, 1142.8, 908.0, 846.2.
4.1.4.4 Ethyl 2-methyl-2-[4-((3-oxo-2,3-dihydro-1H-benzo[f]chromene-2-yl)carbonyl)phenoxy]pr-
opanoate (6d)
White solid, yield 60.2%. m.p.156.1~156.7 oC. 1H NMR (400 MHz, DMSO-d6): δ 8.05 (d, J =
8.8 Hz, 2H, phenyl-H), 8.01−7.90 (m, 3H, benzo[f]chromene-7-H, benzo[f]chromene-8-H,
benzo[f]chromene-9-H), 7.58 (t, J = 7.5 Hz, 1H, benzo[f]chromene-6-H), 7.50 (t, J = 7.4 Hz, 1H,
benzo[f]chromene-5-H), 7.35 (d, J = 8.9 Hz, 1H, benzo[f]chromene-10-H), 6.87 (d, J = 8.8 Hz, 2H,
phenyl-H), 5.27 (dd, J = 10.0, 7.0 Hz, 1H, benzo[f]chromene-2-H), 4.17 (q, J = 7.1 Hz, 2H,
OCH2), 3.67 (ddd, J = 26.7, 16.6, 8.6 Hz, 2H, benzo[f]chromene-1-H), 1.61 (s, 6H, 2×CH3), 1.14
(t, J = 7.1 Hz, 3H, CH3). ESI-MS: m/z 433.2 [M+H]+, 455.2 [M+Na]+. IR (KBr): cm-1 3428.4,
2920.2, 2850.9, 2835.0, 1767.3, 1637.3, 1172.7, 1140.0, 813.4, 749.6.
4.1.5 General procedure for preparation of compounds 7a–7c
The corresponding coumarin-chalcone fibrate ester derivatives (0.001 mol) was dissolved in
10 mL ethanol and 2 mL 10% NaOH and the mixture was stirred for 1 h at 50 oC. After cooling to
room temperature, the reaction mixture was poured into water. Next, the pH was adjusted to 3−4
with hydrochloric acid. The mixture was filtered, and the residue was recrystallized from ethanol
to afford products 7a−7c.
4.1.5.1 2-Methyl-2-[4-((2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoic acid (7a)
White powder, yield 31.2%. m.p.151.5~152.0 oC. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s,
1H, COOH), 8.37 (s, 1H, chromene-4-H), 7.91 (d, J = 8.7 Hz, 2H, phenyl-H), 7.84 (d, J = 7.4 Hz,
1H, chromene-5-H), 7.73 (t, J = 7.7 Hz, 1H, chromene-7-H), 7.50 (d, J = 8.3 Hz, 1H,
chromene-8-H), 7.43 (t, J = 7.5 Hz, 1H, chromene-6-H), 6.90 (d, J = 8.7 Hz, 2H, phenyl-H), 1.60
(s, 6H, 2×CH3). 13C-NMR (100 MHz, DMSO-d6): δ 189.9, 174.3, 160.3, 158.1, 154.0, 144.3,
133.3, 131.8, 131.8, 129.6, 128.9, 126.8, 124.8, 118.3, 116.9, 116.9, 116.3, 79.0, 48.6, 25.1, 25.1.
ESI-HRMS: m/z [M + Na]+ calcd for C20H16O6Na: 375.0839; found: 375.0852. IR (KBr): cm-1
3432.7, 2995.7, 2920.2, 2850.9, 1731.7, 1652.9, 1597.7, 1248.2, 1182.3, 1149.1, 897.7, 850.7,

ACCEPTED MANUSCRIPT
765.8.
4.1.5.2 2-Methyl-2-[4-((6-methyl-2-oxo-2H-chromen-3-yl)carbonyl)phenoxy]propanoic acid (7b)
White solid, yield 41.0%. m.p.145.8~146.9 oC. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H,
COOH), 8.29 (s, 1H, chromene-4-H), 7.88 (d, J = 8.8 Hz, 2H, phenyl-H), 7.61 (s, 1H,
chromene-5-H), 7.54 (d, J = 8.5 Hz, 1H, chromene-7-H), 7.39 (d, J = 8.5 Hz, 1H, chromene-8-H),
6.87 (dd, J = 11.4, 8.9 Hz, 2H, phenyl-H), 2.38 (s, 3H, chromene-6-CH3), 1.59 (s, 6H, 2×CH3).
ESI-MS: m/z 367.3 [M+H]+, 389.2 [M+Na]+. IR (KBr): cm-1 3436.0, 2920.0, 2850.9, 1733.2,
1597.1, 1574.7, 1176.0, 1144.8, 968.4, 864.3, 850.0.
4.1.5.3 2-Methyl-2-[4-((3-oxo-3H-benzo[f]chromene-2-yl)carbonyl)phenoxy]propanoic acid (7c)
Yellow powder, yield 24.9%. m.p.100.5~101.5 oC. 1H NMR (400 MHz, DMSO-d6): δ 13.28 (s,
1H, COOH), 9.17 (d, J = 3.1 Hz, 1H, benzo[f]chromene-1-H), 8.61 (d, J = 8.3 Hz, 1H,
benzo[f]chromene-6-H), 8.31 (d, J = 9.1 Hz, 1H, benzo[f]chromene-5-H), 8.10 (d, J = 8.0 Hz, 1H,
benzo[f]chromene-7-H), 7.95 (d, J = 8.8 Hz, 2H, phenyl-H), 7.74 (t, J = 7.6 Hz, 1H,
benzo[f]chromene-10-H), 7.65 (t, J = 8.0 Hz, 2H, benzo[f]chromene-8-H, benzo[f]chromene -9-H),
6.94−6.83 (m, 2H, phenyl-H), 1.62 (s, 6H, 2×CH3). ESI-MS: m/z 403.4 [M+H]+, 425.4 [M+Na]+.
IR (KBr): cm-1 3432.5, 2919.3, 2850.3, 1633.3, 1463.4, 1135.3, 878.1, 838.5, 779.9, 719.3, 618.8.
4.2 In vitro PPARα/γ transactivation assay
Human embryonic kidney cells (HEK293) were grown in Dulbecco’s Modified Eagles’s
Medium (DMEM) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Sigma), 1%
MEM non-essential amino acid (Sigma) and 1% sodium pyruvate MEM 100 mM (Sigma).
o
HEK293 cells were incubated at 37 C in 5% CO2 until they were 70−80% confluent. Then, the
cells were plated in 96-well plates and the culture medium was replaced by fresh medium
without FCS and the transient transfection was then carried out. Cells were transfected with
expression plasmids encoding the fusion protein GAL4-PPARαLBD or GAL4-PPARγLBD (30
ng), reporter plasmid (50 ng), renilla luciferase normalization vector (20 ng), and pGEM carrier

ACCEPTED MANUSCRIPT
DNA (40 ng) to produce a total of 140 ng DNA per well. 8 h after transfection, cells were treated
for 18 h with the indicated ligands. The two luciferase activities were measured using a dual
luciferase assay kit (Promega) on
LuminoskanReader).
a
microplate luminometer (Labsystems Ascent
4.3. ABTS radical cation scavenging activity assay
The antioxidant capacity of compounds 5a–7c was evaluated according to the procedure
reported in the literature [27]. The ABTS radical cation was produced directly by reacting 7 mM
ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 16 h in
the dark at room temperature before use. The ABTS•+ solution was diluted with ethanol to an
absorbance of 0.700ꢀ±ꢀ0.02 at 734ꢀnm. Then, an ethanolic solution (100ꢀ µL) of the samples or
trolox standard at various concentrations (1, 5, 10, 50 and 100ꢀµg/mL) was mixed with 150 µL
diluted ABTS•+ solution. After reaction at room temperature for 30ꢀmin, the absorbance was read
at 734ꢀnm using a Bio-Tek microplate reader. Tests were performed in triplicate. The ABTS+
radical scavenging activities of the samples were expressed in terms of the IC50 (µM).
4.4. Molecular docking
The molecular docking study was performed by AutoDock 4.2.6 [28]. The three dimensional
structure of PPARα was retrieved from the Protein Data Bank (PDB code: 2P54 [25]). The binding
site was located by the position of the native ligand. The 3D structures of Fenofibrate and
compound 5d were built using Discovery Studio 4.5 [29]. Both the protein and the ligands were
prepared by adding polar hydrogen atoms and partial charges with the assistance of
AutoDockTools 1.5.6. Grid points were set to 40 at the x, y, z axes. The conformational search
was carried out using the Lamarckian Genetic Algorithm. All other parameters were set to default
unless stated otherwise. The figures were prepared by UCSF Chimera [30].
Acknowledgements

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The authors are grateful to Wen Li, Aihua Song, and Dr. Ying Peng (Analytical Department of
1
Shenyang Pharmaceutical University, Shenyang, China) for assistance in gathering H-NMR,
13C-NMR, IR, and ESI-HRMS spectroscopy data.
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1. Legends for Fig. 1, Fig. 2, Fig. 3 and Scheme 1
Figure 1. Chemical structures of some representative fibrates.
Figure 2. Schematic diagram depicting the procedure for the design of the target compounds.
Figure 3. Putative binding modes of (a) Fenofibrate in 2D representation (b) Fenofibrate in 3D representation (c)
compound 5d in 2D representation (d) compound 5d in 3D representation from molecular docking. Hydrogen
bond is shown in black dash lines in 2D representation and in green dash lines in 3D representation.
Scheme 1. Reagents and conditions: (a) K2CO3, CH3CN, reflux, 4 h. (b) (EtO)2CO, NaH, THF, 0 oC to rt for 4−5 h.
(c) substituted salicylaldehydes, piperidine, acetic acid, absolute EtOH, reflux, 2−5 h. (d) Fe, Conc. HCl,
EtOH-H2O-DCM, rt, 1h. (e)NaBH4, pyridine, rt, 2 h, then aq HCl. (f) 10% NaOH-EtOH, 50 oC, 1 h.

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2. Graphics for Figure 1, Figure 2, Figure 3 and Scheme 1
Figure 1

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Figure 2

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Figure 3

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Scheme 1

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3. Table
Table 1. In vitro transcription activation fold of compounds (10 µM) on PPARα/γ.
Activation Fold a
Compd.
R1
R2
R3
R4
X
PPARα
PPARγ
5a
H
F
H
H
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
Double bond
Double bond
Double bond
Double bond
Double bond
Double bond
Double bond
Double bond
Double bond
Single bond
Single bond
Single bond
Single bond
Double bond
Double bond
Double bond
-
1.23
1.08
0.88
1.57
1.12
1.07
0.92
1.03
0.91
0.95
0.89
0.80
0.92
1.16
0.97
0.99
1.76
1.44
1.19
0.95
1.49
0.88
1.01
1.01
0.81
0.83
1.31
0.80
0.90
1.05
1.30
1.04
1.30
-
5b
H
5c
Cl
H
H
5d
NO2
NH2
CH3
OCH3
C(CH3)3
H
H
5e
H
H
H
5f
H
5g
OCH3
H
H
5h
C(CH3)3
5i
benzo[f]chromene
6a
H
H
H
H
H
H
6b
CH3
6c
OCH3
OCH3
6d
benzo[f]chromene
7a
H
H
H
H
7b
7c
CH3
H
H
benzo[f]chromene
H
Fenofibrate
-
-
-
-
-
-
Rosiglitazone
-
-
-
-
3.83
a PPAR activation fold obtained with the vehicle control was defined as 1-fold activation. DMSO solution (0.1%)
acted as the vehicle control.

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Table 2. Activity of the tested compounds in vitro transactivation assaya.
transactivation EC50 (µM)
transactivation EC50 (µM)
Compounds
Compounds
PPARα
12.69±2.54
iab
PPARγ
5.19±0.87
iab
PPARα
11.03±1.49
iab
PPARγ
3.67±0.24
56.71±2.12
ndc
5a
5b
5d
5e
6a
7a
7c
6.99±1.26
iab
0.91±0.13
iab
30±2.72
ndc
Fenofibrate
Rosiglitazone
0.04±0.02
iab
39.67±2.18
a Compounds were tested in two separate experiments at ten concentrations ranging from 0.004 to 12.5 µM. b ia =
inactive. c nd = not determined.

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Table 3. Concentration required for 50% scavenging (IC50) of ABTS+ radical scavenging activities of synthesized
compounds (5a−7c).
Compounds ABTS (IC50, µM)
Compounds ABTS (IC50, µM)
>100
>100
9.40±0.31
10.35±0.48
17.11±0.50
14.98±0.52
>100
5a
5b
5c
5d
5e
5f
6a
6b
>100
6c
18.63±1.03
>100
6d
7a
8.51±0.56
>100
>100
7b
>100
5g
5h
7c
Troloxa
>100
24.84±2.84
>100
5i
a Trolox was used as positive control.

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4. Graphical abstract