Bavachinin analogues as agonists of pan-peroxisome proliferator-activated receptors

Article abstract of DOI:10.1007/s00044-018-2197-6

Peroxisome proliferator-activated receptors (PPARs) agonists contribute to the regulation of glucose, lipid, and cholesterol metabolism and have emerged as key targets to treat metabolic syndrome. In our previous study, the natural compound bavachinin was found to have pan-PPAR agonist activity. In this study, five isoflavones, three isoflavanones, and five scaffold-hopping analogues of bavachinin were designed, synthesised, and evaluated through reporter gene assays for pan-PPAR agonist activity. The analogue 2-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-2,3-dihydroquinolin-4(1H)-one (21) was identified as a pan-PPAR agonist, exhibiting substantially higher PPAR α/β agonist activity and equal PPAR-γ agonist activity than does bavachinin.

Full text of DOI:10.1007/s00044-018-2197-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2197-6
ORIGINAL RESEARCH
Bavachinin analogues as agonists of pan-peroxisome proliferator-
activated receptors
Jingyu Yi1,2 Guoxin Du1 Yuanyuan Zhao1 Liuqiang Zhang1 Bo Li3 Weiliang Zhu3 Cheng Huang1 Yiming Li1
Fujiang Guo1
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Received: 27 February 2018 / Accepted: 25 May 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Peroxisome proliferator-activated receptors (PPARs) agonists contribute to the regulation of glucose, lipid, and cholesterol
metabolism and have emerged as key targets to treat metabolic syndrome. In our previous study, the natural compound
bavachinin was found to have pan-PPAR agonist activity. In this study, five isoflavones, three isoflavanones, and five
scaffold-hopping analogues of bavachinin were designed, synthesised, and evaluated through reporter gene assays for pan-
PPAR agonist activity. The analogue 2-(4-hydroxyphenyl)-6-isopentenyl-7-methoxy-2,3-dihydroquinolin-4(1H)-one (21)
was identified as a pan-PPAR agonist, exhibiting substantially higher PPAR α/β agonist activity and equal PPAR-γ agonist
activity than does bavachinin.
●
●
●
Keywords Bavachinin Metabolic syndrome PPARs Reporter gene assaysThese authors contributed equally: Jingyu Yi, Guoxin
Du.
Introduction
increases with age, with certain ethnic groups being particu-
larly affected (Malan-Müller et al. 2016). The current therapy
for MS requires multiple medications, including anti-
hypertensives, insulin sensitisers, and cholesterol-lowering
agents, which increase the side effects, drug−drug interac-
tions, and economic costs (Beltrán-Sánchez et al. 2013).
Peroxisome proliferator-activated receptors (PPARs)
belong to a family of ligand-activated nuclear receptors and
consist of three subtypes: PPAR-α, PPAR-β/δ, and PPAR-γ.
PPAR-α is mainly found in hepatocytes, smooth muscle
cells, and vascular endothelial cells and improves lipid
metabolism. Fibrate-derived PPAR-α agonists (clofibrate
and bezafibrate) are used in the treatment of dyslipidaemia
(Fu et al. 2003). PPAR-γ contributes to lipid and glucose
metabolism in various cells, including adipocytes. PPAR-γ
agonists (rosiglitazone and pioglitazone) are used in clinical
practice to treat diabetes (Weidner et al. 2012). PPAR-β/δ is
distributed throughout the body and is involved in lipid
metabolism, fatty acid oxidation, and energy dissipation
(Cairns 2004). PPARs contribute to the regulation of glu-
cose, lipid, and cholesterol metabolism and have therefore
emerged as key targets for treating MSs such as diabetes,
dyslipidaemia and atherosclerosis. There is a strong medical
need for the development of efficient pan-PPAR agonists,
because they have the ability to activate all three PPAR
isoforms. Some research programmes have aimed to
Metabolic syndrome (MS) is a multiplex risk factor for car-
diovascular disease and type 2 diabetes as well as abdominal
obesity, dyslipidaemia, insulin resistance, and elevated blood
pressure (Grundy 2016). In the United States, approximately
25% of the adult population has MS, and the prevalence
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2197-6) contains supplementary
material, which is available to authorized users.
* Yiming Li
ymlius@163.com
* Fujiang Guo
gfj@shutcm.edu.cn
1
School of Pharmacy, Shanghai University of Traditional Chinese
Medicine, 1200 Cailun Road, Shanghai 201203, China
2
Pharmacy Department, Zhejiang Hospital, 12 Lingyin Road,
Hangzhou 310013, China
3
Key Laboratory of Receptor Research & Drug Discovery and
Design Center, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China

Medicinal Chemistry Research
Bavachinin analogues 6–13 were generated through the
synthetic route outlined in Scheme 1. Commercially avail-
able paeonol 1 was treated with prenyl bromide in acetone
in the presence of potassium carbonate to yield 2. The
Claisen rearrangement was carried out in N,N-dimethyl-
formamide to obtain the intermediate 3 (Liu et al. 2010).
Intermediate 4 was obtained through the condensation
reaction between 3 with N,N-dimethylformamide dimethy-
lacetal. Subsequently, 3-iodochromone 5 was obtained
through a cascade reaction involving one-pot, two-step ring
closure and iodination through the addition of I2 in
methanol (Mutai et al. 2015). Some derivatives of prenyl
isoflavones (6, 10, 11) were synthesised from 5 and various
phenylboronic acids (PBA) through a Suzuki coupling
reaction, which employed poly(ethylene glycol) 6000 (PEG
6000) as the ligand, along with palladium diacetate and
sodium carbonate in methanol (Biegasiewicz et al. 2014).
Isoflavanone 11 was reduced to produce 12 in anhydrous
tethrahydrofun (THF) using lithium triisobutylhydroborate
as a reducing agent (Wang and Xu 2017). However, this
reduction reaction was not feasible on the substrate 3-
iodochromone 5, without the B-ring, and resulted in the
unexpected formation of compound 13. In addition, a
MOM-mask was necessary for the synthesis of isoflavanone
9 for the phenol group.
OH
B
O
O
C
A
O
Fig. 1 Chemical structure of bavachinin
develop agonists that combine the therapeutic effects of
both PPAR-γ, PPAR-α, and PPAR-β selective agonists
(Pourcet et al. 2006).
Bavachinin (BVC, Fig. 1) is a naturally occurring fla-
vanone isolated from the seeds of Psoralea corylifolia (Ma
et al. 2016). It exhibits various biological characteristics,
including antiangiogenic, antitumour, antiallergic, and
antibacterial activity (Du et al. 2015). Our previous study
showed that natural bavachinin exhibits potential PPAR
agonist activity, which when combined with the PPAR-γ
agonist rosiglitazone and PPAR-α agonist fibrates, can also
be used to lower glucose and triacylglycerol levels in db/db
mice (Feng et al. 2016). However, further in vitro metabolic
studies showed that its metabolism is faster than previously
thought and that its biological availability is lower than
expected (Xie et al. 2016).
C1 position N-substituted analogues 22 and 23 were
prepared through the synthesis route outlined in Scheme 2.
The p-methoxybenzyl (PMB) group was introduced to the
phenolic hydroxy of 4-hydroxy-benzaldehyde 14 to form
15. Subsequently, 17 was prepared from 4-bromo-3-
methoxyaniline 16 through an improved Friedel–Crafts
reaction (Gim et al. 2014). Compound 15 was treated with
17 in ethyl alcohol in the presence of NaOH, yielding the
intermediate 18. Regioselective cyclisation of 18 through
reflux with antimony trichloride in acetonitrile yielded the
corresponding flavanone, intermediate 19. Compound 20
was synthesised from 19 and 3,3-dimethylallylboronic acid
pinacol ester through a Suzuki coupling reaction using Pd
(dppf)2Cl2 as a catalyst. Subsequently, p-toluenesulfonic
acid was used successfully for deprotection of the PMB
group to obtain 21. Oxidising the corresponding 20 and 21
with iodine in dimethyl sulfoxide generated 23 and 22,
respectively.
The C3 position N-substituted analogue 32 was synthe-
sised through the method outlined in Scheme 3. 25 was
prepared through the esterification of 4-methoxysalicylic
acid 24. The intermediate 26 was synthesised from 25
through a bromination substitution reaction (Gisch et al.
2007). The ρ-sulfonyl benzene sulfonyl (Ts) group was
introduced to the phenolic hydroxy of the intermediate 26 to
form 27 in the presence of triethanolamine (TEA). Then, 28
was prepared using 27 through a Suzuki coupling reaction
with 3,3-dimethylallylboronic acid pinacol ester (Farmer
In our previous work, we studied the structure-activity
relationship of bavachinin and screened potential PPAR-γ
agonists. A series of bavachinin analogues with systematic
modifications at the A, B, and C rings was designed, syn-
thesised, and subjected to in vitro bioevaluation as PPAR-γ
agonists (Du et al. 2017). This paper introduces our efforts
to further the study of the structure-activity relationship of
bavachinin.
Results and discussion
Chemistry
Bavachinin was used as a lead compound for designing
analogues. Because some isoflavones were identified as
potent pan-PPAR agonists (Matin et al. 2013) and several
isoflavones isolated from the seeds of Psoralea corylifolia
along with bavachinin also exhibited PPAR γ agonist
activity (Ma et al. 2016), some isoflavones and iso-
flavanones that maintained an unchanged A-ring structure
were designed in Scheme 1 to determine whether the B-ring
linked site played a role in pan-PPAR agonist activity.
Some scaffold-hopping analogues of the C-ring were
designed in Schemes 2–4 under the assumption that the
introduction of nitrogen elements could improve pan-PPAR
agonist activity.

Medicinal Chemistry Research
Scheme 1 Reagents and
conditions: a Prenyl bromide,
K2CO3, acetone, reflux, 5 h; b
PhNEt2, reflux, 4 h; c DMF-
DMA, reflux, 15 h; d I2, MeOH,
r.t., 24 h; e Na2CO3, Pd(OAc)2,
PEG 6000, 50 °C, 2 h; f
CH3OCH2Cl, K2CO3, acetone,
rt, 2 h; g Lithium
triisobutylhydroborate, THF,
−78 °C, 2 h; h 3N-HCl, MeOH,
reflux, 25 min
et al. 2012). After the alkaline hydrolysis of 28 in sodium
hydroxide (NaOH) solution, salicylic acid analogue 29 was
formed. Compound 30 was synthesised through the con-
densation of 29 in the presence of HATU ((1-[bis(dime-
thylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate) (Jin et al. 2017). Cyclisation
of 15 and 30 using piperidine yielded 31. Tetra-n-buty-
lammonium fluoride was used successfully for the depro-
tection of the TBS group to obtain the end product, 32.
C1, 3 position N-substituted analogue 40 was synthe-
sised through the route outlined in Scheme 4. Intermediate
37 was prepared using 2-amino-4-methoxybenzoic acid 33
as a raw material through esterification, bromination,
Suzuki coupling reaction, and alkaline hydrolysis under the
reaction conditions used in Scheme 3. Through the cycli-
sation of 37 in the presence of triphosgene, 38 was obtained
(Wehle et al. 2016), and 39 was prepared from 38 through
aminolysis (Wang et al. 2015). End-product 40 was syn-
thesised through the condensation of 39 in the presence of
ρ-toluenesulfonic acid.
Furthermore, we retested the selected analogues at doses
of 0.1, 0.3, 1, 3, 10, 30, and 100 μM using the same assay to
obtain the effective concentration (EC50) of the selected
analogues for in vitro PPAR α, PPAR-β, and PPAR-γ
activity separately. The results are summarised in Table 1.
In the PPAR-α agonist activity test, analogues 7 and 10
(EC50=28.57 and 38.67 μM, respectively) decreased the
PPAR-α agonist activity, whereas analogue 21 (EC50
=
22.28 μM) exhibited a substantially higher activity com-
pared with that of bavachinin (EC50 = 12.46 μM). Introdu-
cing an N atom at C1 position resulted in greater PPAR-α
agonist activity than found with bavachinin. Analogues 7
and 40 (EC50 = 0.71 and 0.84 μM, respectively) exhibited
less PPAR-β agonist activity relative to bavachinin. Ana-
logue 21 (EC50 = 3.54 μM) significantly increased the
PPAR-β agonist activity compared with bavachinin (EC50
= 10.80 μM). Introducing a N atom at the C1 position likely
produces greater PPAR-β agonist activity than that doing so
at the C3 position. Analogue 40 (EC50 = 0.40 μM) mark-
edly decreased PPAR-γ agonist activity, whereas analogue
21 (EC50 = 65.33 μM) showed approximately equal PPAR-
PPAR agonist activity
γ agonist activity when compared with bavachinin (EC50
14.46 μM).
=
To identify new pan-PPAR modulators, reporter gene
assays were conducted to analyse the effects of analogues
on the PPAR α/β/γ ligand binding domain (LBD) (Del Bas
et al. 2012). Commercially available PPAR-α, PPAR-β, and
PPAR-γ agonists, namely fenofibric acid, GW7647, and
pioglitazone, were used as positive controls for PPAR α/β/γ
activity. In total, 13 synthesised analogues of bavachinin
were analysed. The results are summarised in Table 1.
Analogues 7, 10, and 21 (25 μM) exhibited potential PPAR-
α agonist activity. Analogues 7, 21, and 40 (25 μM)
exhibited potential PPAR-β agonist activity. Analogues 21,
40 (25 μM) exhibited potential PPAR-γ agonist activity.
Conclusions
With bavachinin as a lead compound, some isoflavones and
isoflavanones maintained an unchanged A-ring structure (6,
7, 8, 9, 10, 11, 12), and some scaffold-hopping analogues of
the C-ring (21, 22, 23, 32, 40) were designed and synthe-
sised. Changing the analogues into isoflavones or iso-
flavanones reduced pan-PPAR agonist activity when
compared with bavachinin. The position at which the N
element is introduced is crucial. The C3 and C1, 3 position

Medicinal Chemistry Research
Scheme 2 Reagents and
conditions: a PMBCl, DMF,
K2CO3, rt, 16 h; b BCl3, AlCl3,
CH3CN, 70 °C, 16 h; c NaOH,
EtOH, 60 °C,12 h; d SbCl3,
CH3CN, reflux, 6 h; e Pd(dppf)
2Cl2, Cs2CO3, DMF, 70 °C,12 h;
f Ts-OH, THF/MeOH, 50 °C,
15 h; g I2, DMSO, 75 °C, 1 h
(of C-ring) N-substituted analogues 32 and 40 reduced pan-
PPAR agonist activity, whereas C1 position N-substituted
analogue 21 enhanced it. The results suggest that the
introduction of nitrogen element in C1 position basically
not affected pan-PPAR agonist activity of BVC. Analogue
21 is a potent pan-PPAR agonist that exhibits substantially
higher PPAR α/β agonist activity and equal PPAR-γ agonist
activity when compared with bavachinin.
containing 10% FBS for 24 h, cells were cotransfected with
the expression plasmids pCMX-Gal-mPPAR-α (or PPAR-β,
PPAR-γ) LBD, Gal4 reporter vector MH100 × 4-TK-Luc
and pREP7 reporter by using FuGENE-HD (Roche, Swit-
zerland). The transfection system contained 2 mg of Gal-
mPPAR-γ LBD, Gal4 reporter vector MH100 × 4-TK Luc,
0.3 mg of pREP7 (Renilla luciferase) reporter plasmids, and
8 μL FuGENE-HD per mL of DMEM. Renilla luciferase
activity was used to normalise the transfection efficiencies.
For the primary screen, the compounds (25 μM) and the
PPAR-α, PPAR-β, and PPAR-γ agonists, fenofibric acid,
GW7647, and pioglitazone (20 μM), were added to the
medium and incubated for another 24 h, after which the
cells were lysed and harvested. The cell lysate was mixed
with luciferin solution and luminescence was measured with
the FB 12 Luminometer (Berthold Detection Systems
GmbH, Pforzheim, Germany). To further winnow our pri-
mary screen, we retested the positive compounds at doses of
0.1, 0.3, 1, 3, 10, 30, and 100 μM using the same assay. All
experiments were performed in triplicate.
Materials and methods
All starting materials were obtained from commercial sup-
pliers and used without further purification. The 1H and 13
C
spectra were taken on Bruker Avance III 500 or 400 or
1
300 MHz for H NMR, and 125, or 100 or 75 MHz for 13C
NMR, with tetramethylsilane (TMS) as the internal stan-
dardand. 13C NMR spectra were recorded with complete
proton decoupling. The ESI-MS was recorded on Finnigan
LCQ/DECA. The HRMS were obtained from Mcromass Q-
TOF Ultima (ESI). Silica gel F254 was used in analytical
thin-layer chromatography (TLC), and silica gel was used in
column chromatography; visualizations were accomplished
with UV light (254 nm).
Experimental section for synthesis
Preparation of 2′-hydroxyl-4′-methoxyl-5′-
isopentenylacetophenone (3)
PPAR transactivation assay
To a solution of paeonol 1 (4.15 g, 25.0 mmol) and anhy-
drous potassium carbonate (12.10 g, 87.5 mmol) in acetone
(60 mL), prenyl bromide (3.70 g, 25.0 mmol) was added.
Subsequently, the reaction mixture was stirred at reflux for
5 h. The reaction mixture was quenched with deionized
water and extracted with ethyl acetate. The organic layers
Reporter gene assays were performed using the Dual-
Luciferase Reporter Assay System (Promega, Madison, WI,
USA). In brief, 293 T cells were seeded in 48-well plates at
4000 cells per well. After being cultured at 37 °C in Dul-
becco’s Modified Eagle’s Medium (DMEM) (Gibco)

Medicinal Chemistry Research
Scheme 3 Reagents and
conditions: a H2SO4, CH3OH,
reflux, 48 h; b Br2, DCM, rt, 3 h;
c p-TsCl, TEA, DCM, rt 3 h; d
Pd(dppf)2Cl2, Cs2CO3, DMF,
70 °C, 15 h; e NaOH, THF/
CH3OH/H2O, 50 °C, 16 h; f
NH3/THF, HATU, DIPEA,
DCM, rt, 16 h; g piperidine,
toluene, 100 °C, 16 h; h TBAF,
DCM, 0 °C, 15 min
were combined, washed with saturated sodium chloride
solution, dried over sodium sulfate, filtrated, and evaporated
under vacuum to produce crude 2. The crude product 2 was
used for the next reaction step directly without further
purification.
7.2 Hz, 1 H), 3.85 (s, 3 H), 3.25 (d, J = 7.2 Hz, 2 H), 3.15 (s,
3 H), 2.94 (s, 3 H), 1.73 (s, 3 H).
Preparation of 3-iodo-7-methoxy-6-isopentenyl-4H-
chromen-4-one (5)
Product 2 was dissolved in diethylaniline (40 mL), and
the solution was stirred at reflux under an inert atmosphere
for 4 h. After cooling, the reaction mixture was quenched
with 2N aqueous HCl and extracted with ethyl acetate. The
organic layers were combined, washed with deionized water
and saturated sodium chloride solution, dried over sodium
sulfate, filtrated, and evaporated under vacuum. The residue
was purified through silica gel column chromatography
with petroleum ether/ethyl acetate to afford a yellow oil,
4 (2.76 g, 10.0 mmol) and iodine were diluted with anhy-
drous methanol (40 mL), and the reaction mixture was
stirred at room temperature for 24 h. The reaction mixture
was quenched with saturated sodium thiosulfate and
extracted with ethyl acetate. The organic layers were com-
bined, washed with saturated sodium chloride solution,
dried over sodium sulfate, filtrated, and evaporated under
vacuum. The residue was purified by silica gel column
chromatography with petroleum ether/ethyl acetate to afford
1
designated as 3 (1.97 g, 33.7%). 3: H NMR (400 MHz,
1
CDCl3) δ 12.74 (s, 1H), 7.42 (s, 1H), 6.41 (s, 1H), 5.27 (t, J
= 7.3 Hz, 1H), 3.88 (s, 3H), 3.24 (d, J = 7.3 Hz, 2H), 2.56
(s, 3H), 1.78 (s, 3H), 1.73 (s, 3 H).
a yellow solid 5 (1.4 g, 37.8%). 5: H NMR (400 MHz,
CDCl3) δ 8.20 (s, 1H), 7.95 (s, 1H), 6.78 (s, 1H), 5.29 (m,
1H), 3.93 (s, 3H), 3.35 (d, J = 7.1 Hz, 2H), 1.75 (s, 3H),
1.70 (s, 3H).
Preparation of 1-(2′-hydroxy-4′-methoxy-5′-isopentenyl)-3-
(dimethylamino)-2E-propen-1-one (4)
General method for Suzuki coupling
3 (2.34 g, 10.0 mmol) was diluted with DMF/DMA
(5.31 mL, 40.0 mmol), and the reaction mixture was stirred
at reflux under an inert atmosphere for 15 h. After cooling,
the reaction mixture was quenched with deionized water
and extracted with ethyl acetate. The organic layers were
combined, washed with saturated sodium chloride solution,
dried over sodium sulfate, filtrated, and evaporated under
vacuum to afford a yellow needle 4 (2.80 g, 96.9%). 4: H
NMR (400 MHz, CDCl3) δ 7.85 (d, J = 12.2 Hz, 1H), 7.41
(s, 1H), 6.41 (s, 1H), 5.69 (d, J = 12.2 Hz, 1H), 5.29 (t, J =
A round bottom flask was charged with methanol (54 mL)
and Na2CO3 (1.8 g, 16.7 mmol) and allowed to stir. PEG
6000 (49.8 g) was ground to fine consistency and added to
the flask with Pd(OAc)2 (163.0 mg, 0.73 mol). The reaction
mixture was then warmed to 50 °C in an oil bath. Once the
mixture turned black, 5 and phenylboronic acids were
added and left to stir for 2 h. After the reaction, the resulting
mixture was emptied into a Büchner funnel, and the filtrate
was quenched with deionized water and extracted with ethyl
acetate. The organic layers were combined, washed with
1

Medicinal Chemistry Research
Scheme 4 Reagents and
conditions: a CH3OH, H2SO4,
reflux, 24 h; b Br2, DCM, rt,
20 h; c Pd(dppf)2Cl2, Cs2CO3,
DMF, 70 °C, 24 h; d NaOH,
CH3OH, THF, H2O, 60 °C, 12 h;
e Triphosgene, THF, rt, 2 h; f
(NH4)2CO3, 1,4-dioxane, 60 °C,
7 h; g TsOH, DMF, rt, 2 h
saturated sodium chloride solution, dried over sodium sul-
fate, filtrated, and evaporated under vacuum. The residue
was purified by silica gel column chromatography to afford
the target product 6, 10, 11.
and extracted with ethyl acetate. The organic layers were
combined, washed with saturated sodium chloride solution,
dried over sodium sulfate, filtrated, and evaporated under
vacuum. The residue was purified by silica gel column
chromatography with petroleum ether/ethyl acetate to afford
1
Preparation of 3-(4-hydroxyphenyl)-6-isopentenyl-7-
methoxy-4H-chromen-4-one (6)
white powder 7 (40.0 mg, 81.0%). 7: H NMR (300 MHz,
CDCl3) δ 8.02 (s, 1H), 7.90 (s, 1H), 7.50 (d, J = 8.7 Hz,
2H), 7.10 (d, J = 8.7 Hz, 2H), 6.80 (s, 1H), 5.35 – 5.30 (m,
1H), 5.21 (s, 2H), 3.93 (s, 3H), 3.49 (s, 3H), 3.37 (d, J =
7.3 Hz, 2H), 1.75 (s, 3H), 1.71 (s, 3H).
5 (370.0 mg, 1.0 mmol) and 4-hydroxyphenylboronic acid
(206.9 mg, 1.5 mmol) were added to the mixture of Pd
(OAc)2 solution (10.8 mL). 6 (200.0 mg, 60.0%) was pur-
ified by silica gel column chromatography with dichlor-
Preparation of 7-methoxy-3-(4-(methoxymethoxy)phenyl)-
6-(isopentenyl)chroman-4-one (8)
1
omethane/methanol. 6: H NMR (500 MHz, DMSO-d6) δ
9.55 (s, 1 H), 8.36 (s, 1H), 7.81 (s, 1H), 7.40 (d, J = 8.4 Hz,
2H), 7.14 (s, 1H), 6.82 (d, J = 8.4 Hz, 2H), 5.30 (t, J =
6.6 Hz, 1H), 3.94 (s, 3H), 3.32 (d, 2H), 1.73 (s, 3H), 1.69 (s,
3H). 13C NMR (125MHz, DMSO-d6) δ 175.0, 162.0, 157.6,
156.5, 153.3, 133.2, 130.4×2, 128.7, 125.1, 124.1, 122.9,
121.9, 117.4, 115.4×2, 99.3, 56.8, 28.1, 26.0, 18.0; HR-
ESI-MS m/z: Anal. Calcd. for C21H21O4 337.1441; found
337.1434 [M + H]+.
7 (36.0 mg, 0.1 mmol) was diluted with anhydrous THF
(1 mL), and the reaction mixture was cooled to –78 °C under
an inert atmosphere for 3 h, and a solution of a solution of
1.0 M L-selectride in THF (0.24 mL) was added slowly via
syringe. The reaction mixture was stirred at –78 °C for 2 h and
quenched slowly by the addition of MeOH, then 1.0 M HCl.
After cooling, the reaction mixture was extracted with ethyl
acetate. The organic layers were combined, washed with
saturated sodium chloride solution, dried over sodium sulfate,
filtrated, and evaporated under vacuum. The residue was
purified by silica gel column chromatography with petroleum
ether/ethyl acetate to afford a white powder 8 (13 mg, 34.0%).
Preparation of 7-methoxy-3-(4-(methoxymethoxy)phenyl)-
6-(isopentenyl)-4H-chromen-4-one (7)
To a solution of hydroxylbenzaldehydes 6 (43.6 mg,
0.13 mmol) and anhydrous potassium carbonate (72.0 mg,
0.52 mmol) in acetone (3 mL) was added chloromethyl
methyl ether (25.2 mg, 0.32 mmol) slowly. After that, the
reaction mixture was stirred at room temperature for 2 h.
The reaction mixture was quenched with deionized water
1
8: H NMR (300 MHz, CDCl3) δ 7.69 (s, 1H), 7.20 (d, J =
8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.39 (s, 1H), 5.26 (t, J
= 7.4 Hz, 1H), 5.15 (s, 2H), 4.60 (dd, J = 6.6, 3.1Hz, 2H),
3.87 (s, 3H), 3.83 (d, J = 7.3 Hz, 1H), 3.46 (s, 3H), 3.23 (d, J
= 7.2 Hz, 2H), 1.72 (s, 3H), 1.68 (s, 3H).

Medicinal Chemistry Research
Table 1 In vitro cell-based
PPAR transactivation activity
Compounds
PPARα
% maxa
PPAR β
PPAR γ
EC50 (μM)
% max
EC50(μM)
% max
EC50(μM)
(Mean SD) (Mean SD) (Mean SD) (Mean SD) (Mean SD) (Mean SD)
Fenofibric acid 151.29
10.82 0.73
–
–
–
–
–
6.02
GW0742
–
–
–
–
322.99
10.56
0.11 0.03
–
Pioglitazone
Bavachinin
–
–
315.77
11.60
0.07 0.02
14.46 4.25
ND
100.00
12.64
12.46 0.76 100.00
6.62
10.80 0.61 100.00
15.99
5
33.45 1.71 NDb
15.32 1.28 ND
44.40 5.10 ND
4.64 0.38
6
22.74 1.48 ND
15.69 2.22 ND
14.63 1.26 ND
7
62.31 6.82 28.57 3.01 61.77 7.90 0.71 0.26
8
38.32 3.85 ND
49.51 3.68 ND
21.25 9.39 ND
25.93 2.95 ND
8.79 1.89
8.13 1.22
ND
ND
9
10
62.46 5.61 38.67 2.70 47.03
13.20
ND
21.40 6.83 ND
11
12
21
42.63 6.46 ND
31.52 1.68 ND
12.50 1.08 ND
11.57 1.03 ND
2.21 0.25
8.76 0.15
ND
ND
114.73
9.09
22.28 2.35 97.51
3.54 1.49
98.83
20.89
65.33
28.64
15.57
22
23
53.20
12.16
ND
28.82
11.92
ND
ND
31.16 1.42 ND
4.63 1.55 ND
14.41 1.91 ND
15.18 0.54 ND
25.98
15.70
32
40
34.54 2.13 ND
45.00 4.85 ND
19.66 3.31 ND
84.85
12.48
0.84 0.22
50.22
11.30
0.40 0.01
The concentration of positive control fenofibric acid, GW0742 and pioglitazone were 20 μM, and the test
analogues of bavachinin were used at 25 μM
aRelative maximal activity normalized by that of bavachinin
bNot determined
Preparation of 7-methoxy-3-(4-hydroxyphenyl)-6-
(isopentenyl)chroman-4-one (9)
126.3, 126.1, 124.0, 121.1, 114.6×2, 113.2, 97.8, 71.4, 54.5,
50.5, 26.7, 24.0, 15.9. HR-ESI-MS m/z: Anal. Calcd. for
C21H21O4 337.1445; found 337.1445 [M + H]+.
To a solution of 8 (10.0 mg, 0.026 mmol) in methanol (1 mL)
was added 3N aqueous HCl (0.5 mL). Then the reaction
mixture was stirred at reflux for 25 min. After cooling, the
reaction mixture was quenched with deionized water and
extracted with ethyl acetate. The organic layers were com-
bined, washed with saturated sodium chloride solution, dried
over sodium sulfate, filtrated, and evaporated under vacuum.
The crude material was purified by silica gel column chro-
matography with petroleum ether/ethyl acetate to afford 9
Preparation of 3-(3-fluoro-4-hydroxyphenyl)-6-isopentenyl-
7-methoxy-4H-chromen-4-one (10)
5 (185.0 mg, 0.5 mmol) and (3-fluoro-4-hydroxyphenyl)
boronic acid (116.9 mg, 0.75 mmol) were added to the
mixture of Pd(OAc)2 solution (10.8 mL). 10 (29.0 mg, 16%)
was purified by silica gel column chromatography with
dichloromethane/methanol. 10: 1H NMR (500 MHz,
CD3OD) δ 8.18 (d, J = 1.7 Hz, 1H), 7.89 (s, 1H), 7.32 (dd,
J = 12.4, 2.0 Hz, 1H), 7.17–7.11 (m, 1H), 7.02 (s, 1H), 6.96
(t, J = 8.7 Hz, 1H), 5.37–5.26 (m, 1H), 3.96 (s, 3H), 3.36
(d, J = 7.3 Hz, 2H), 1.76 (s, 3H), 1.71 (s, 3H); HR-ESI-MS
m/z: Anal. Calcd. for C21H20FO4 335.1340; found 335.1345
[M + H]+.
1
(7.0 mg, 79.7%). 9: H NMR (500 MHz, CD3OD) δ 7.58 (s,
1H), 7.07 (d, J = 8.5 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 6.51
(s, 1H), 5.25 (tt, J = 7.3, 1.4 Hz, 1H), 4.60–4.52 (m, 2H), 3.88
(s, 3H), 3.83 (dd, J = 8.0, 5.4 Hz, 1H), 3.21 (d, J = 7.3 Hz,
2H), 1.73 (s, 3H), 1.68 (s, 3H); 13C NMR (125 MHz,
CD3OD) δ 192.4, 163.9, 162.4, 156.1, 132.0, 128.8 × 2,

Medicinal Chemistry Research
Preparation of 3-(4-Cyanophenyl)-6-isopentenyl-7-
methoxy-4H-chromen-4-one (11)
temperature 16 h. It was diluted with water and extracted
with ethyl acetate. The organic layers were combined,
washed with saturated sodium chloride solution, dried over
sodium sulfate, filtrated, and evaporated under vacuum to
afford white solid 15 (6.2 g, 85.3%).
5 (185.0 mg, 0.5 mmol) and 4-cyanophenylboronic acid
(109.5 mg, 0.8 mmol) were added to the mixture of Pd
(OAc)2 solution (10.8 mL). 11 (60.0 mg, 34.0%) was pur-
ified by silica gel column chromatography with petroleum
Preparation of 1-(2-amino-5-bromo-4-methoxyphenyl)-
ethanone (17)
1
ether/ethyl acetate. 11: H NMR (500 MHz, CDCl3) δ 8.02
(s, 1H), 7.99 (s, 1H), 7.72 (s, 4H), 6.83 (s, 1H), 5.32 (t, J =
7.3 Hz, 1H), 3.96 (s, 3H), 3.38 (d, J = 7.3 Hz, 2H), 1.76 (s,
3H), 1.72 (s, 3H) 13C NMR (125 MHz, CDCl3) δ 174.8,
162.4, 156.6, 152.9, 137.1, 133.7, 132.1 × 2, 130.0,
129.5×2, 125.9, 123.7, 121.0, 118.8, 117.6, 111.6, 98.0,
56.0, 28.2, 25.8, 17.8; HR-ESI-MS m/z: Anal. Calcd. for
C22H20NO3 346.1438; found 346.1443 [M + H]+.
To a solution of 4-bromo-3-methoxyaniline pe="Bold">16
(6 g, 30.0 mmol) in dichloromethane (30 mL) was added
boron trichloride (1 N, 33.0 mL, 33.0 mmol) dropwise over
30 min at 0 °C under N2. Acetonitrile (3.2 mL, 60.0 mmol)
and aluminium chloride (4.4 g, 33.0 mmol) was added and
the mixture was stirred at 0 °C under N2 for 30 min and the
stirred at 70 °C for 16 h. The resulting mixture was cooled
to 0 °C and 2 N HCl was added to the solution and the
mixture was stirred at 70 °C for 1 h. It was cooled to RT and
extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over Na2SO4 and filtered. It
was then concentrated and the residue was purified by silica
gel column chromatography eluting with cyclohexane/EA
Preparation of 4-(7-methoxy-6-(isopentenyl)-4-
oxochroman-3-yl)benzonitrile (12)
The procedure was the same as described in 1.2.8. 11
(20.7 mg, 0.06 mmol) was diluted with anhydrous THF
(2 mL), and a solution of a solution of 1.0 M L-selectride in
THF (0.15 mL) was added slowly, affording a white powder
1
(50:1) to give brown solid 17 (3.7 g, 50.7%). 17: H NMR
(300 MHz, CDCl3) δ 7.85 (s, 1 H), 6.48 (s, 2 H), 6.10 (s,
1
12 (15 mg, 72.0%). 12: H NMR (500 MHz, CDCl3) δ7.67
(s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H),
6.40 (s, 1H), 5.27–5.22 (m, 1H), 4.72–4.54 (m, 2H),
3.98–3.91 (m, 1H), 3.88 (s, 3H), 3.23 (d, J = 7.2 Hz), 1.73
(s, 3H), 1.68 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 189.3,
164.4, 162.1, 141.1, 133.2, 132.4 × 2, 129.3 × 2, 127.7,
125.5, 121.4, 118.6, 113.6, 111.6, 98.5, 71.1, 55.8, 51.7,
27.7, 25.8, 17.7. HR-ESI-MS m/z: Anal. Calcd. for
C22H20NO3 346.1449; found 346.1446 [M-H]−.
1 H), 3.89 (s, 3 H), 2.53 (s, 3 H).
Preparation of compound 18
To a solution of compound 15 (1.7 g, 6.5 mmol) in EtOH
(20 mL) were added compound 17 (1.2 g, 5.0 mmol) and
NaOH (0.8 g, 20.0 mmol). The mixture was was stirred at
60 °C for 12 h. It was cooled to RT and filtered. The filter
cake was washed with EtOH (3 mL) and dried to give
1
Preparation of 7-methoxy-6-isopentenyl-chroman-4-one (13)
yellow solid compound 18 (2.2 g, 93.2%). 18: H NMR
(300 MHz, CDCl3) δ 8.00 (s, 1 H), 7.70 (d, J = 15.3 Hz,
1 H), 7.59 (d, J = 8.4 Hz, 2H), 7.44–7.33 (m, 3H), 6.96 (dd,
J = 21.1, 8.3 Hz, 4H), 6.59 (s, 2H), 6.13 (s, 1H), 5.04 (s,
2H), 3.89 (s, 3H), 3.82 (s, 3H).
The procedure was the same as described in 1.2.8. 5 (185 mg,
0.5 mmol) was diluted with anhydrous THF (3 mL), and a
solution of a solution of 1.0 M L-selectride in THF (1.2 mL)
was added slowly, affording a pale-yellow solid 13 (40 mg,
32.5%). 13: 1H NMR (500 MHz, CDCl3) δ 7.64 (s, 1H), 6.36
(s, 1H), 5.26 (t, J = 7.2 Hz, 1H), 4.49 (t, J = 6.4 Hz, 2H), 3.85
(s, 3H), 3.22 (d, J = 7.3 Hz, 2H), 2.73 (t, J = 6.4Hz, 2H), 1.73
(s, 3H), 1.69 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 190.6,
163.9, 162.4, 132.9, 127.2, 124.7, 121.7, 114.4, 98.5, 67.4,
55.7, 37.4, 27.7, 25.8, 17.7; HR-ESI-MS m/z: Anal. Calcd. for
C15H19O3 247.1329; found 247.1330 [M + H]+.
Preparation of 6-bromo-7-methoxy-2-(4-((4-
methoxybenzyl)oxy)phenyl)-2,3-dihydroquinolin-4(1H)-one
(19)
To a solution of compound 18 (933.0 mg, 2.00 mmol) in
CH3CN (15 mL) was added SbCl3 (136.8 mg, 0.60 mmol).
The mixture was stirred at reflux for 6 h. The filter cake was
washed with CH3CN and dried to give yellow solid com-
pound 19 (495.0 mg, 53.3%). 19: 1H NMR (400 MHz,
CDCl3) δ 8.04 (s, 1H), 7.35 (dd, J = 8.5, 5.7 Hz, 4H), 6.95
(dd, J = 22.2, 8.7 Hz, 4H), 6.13 (s, 1H), 5.00 (s, 2H), 4.67
(dd, J = 13.6, 3.9 Hz, 1H), 4.54 (s, 1H), 3.87 (s, 3H), 3.82
(s, 3H), 2.81 (dd, J = 16.3, 13.5 Hz, 1H), 2.69 (dd, J = 16.4,
3.8 Hz, 1H).
Preparation of 4-((4-methoxybenzyl)oxy) benzaldehyde (15)
To
a mixture of 4-hydroxy-benzaldehyd 14 (3.7 g,
30.0 mmol) and K2CO3 (12.4 g, 90.0 mmol) in DMF
(40 mL) was added 4-methoxybenzylchloride (3.5 g,
30.0 mmol) portionwise. The mixture was stirred at room

Medicinal Chemistry Research
Preparation of 7-methoxy-2-(4-((4-methoxybenzyl)oxy)
phenyl)-6-(isopentenyl)-2,3-dihydroquinolin-4(1H)-one (20)
reaction mixture was stirred at 75 °C for 1 h. After cooling,
the reaction mixture was quenched with a saturated solution
of Na2S2O3 and extracted with ethyl acetate. The organic
layers were combined, washed with brine. It was dried over
Na2SO4 and filtered. The filtrate was concentrated and the
residue was purified by silica gel column chromatography
eluting with CH2Cl2/MeOH (uv254, 20:1) to give pale-
yellow powder 22 (7.0 mg, 34.8%). 22: 1H NMR
(500 MHz, CD3OD) δ 7.96 (s, 1H), 7.64 (d, J = 8.7 Hz,
2H), 7.12 (s, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.45 (s, 1), 5.35
(t, J = 7.3 Hz, 1H), 3.97 (s, 3H), 3.36–3.29 (m, 2H), 1.77 (s,
3H), 1.74 (s, 3H); 13C NMR (125 MHz, CD3OD) δ 178.3,
161.4, 159.9, 151.2, 140.9, 132.7, 128.8, 128.4, 124.7,
124.2, 121.4, 118.0, 115.6, 105.5, 97.0, 54.8, 27.9, 24.6,
16.4; HR-ESI-MS m/z: Anal. Calcd. for C21H22NO3
336.1594; found 336.1589 [M + H]+.
To a solution of compound 19 (468.0 mg, 1.0 mmol) in
DMF (15 mL) were added 4,4,5,5-tetramethyl-2-(3-
methylbut-2-enyl)-1,3,2-dioxaborolane
(0.59 mL,
2.5 mmol), Pd(dppf)2Cl2 (245 mg, 0.3 mmol) and Cs2CO3
(977.5 mg, 3.0 mmol). The mixture was stirred at 70 °C for
12 h with the protection of N2. It was cooled to RT and
diluted with water and extracted with ethyl acetate. The
combined organic layers were washed with brine. It was
dried over Na2SO4 and filtered. The filtrate was con-
centrated and the residue was purified by silica gel column
chromatography eluting with cyclohexane/EA (20:1) to
give pale-yellow powder 20 (198.0 mg, 43.0%). 20: 1H
NMR (500 MHz, CDCl3) δ 7.63 (s, 1H), 7.35 (dd, J = 8.6,
4.2 Hz, 4H), 6.94 (dd, J = 24.0, 8.6 Hz, 4H), 6.07 (s, 1H),
5.26 (t, J = 7.3 Hz, 1H), 4.99 (s, 2H), 4.64 (dd, J = 13.8,
3.5 Hz, 1H), 4.41 (s, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.19 (d,
J = 7.3 Hz, 2H), 2.78 (dd, J = 16.1, 13.9 Hz, 1H), 2.65 (dd,
J = 16.2, 2.7 Hz, 1H), 1.72 (s, 3H), 1.69 (s, 3H).
Preparation of 7-methoxy-2-(4-((4-methoxybenzyl)oxy)
phenyl)-6-(isopentenyl)quinolin-4(1H)-one (23)
To a solution of compound 20 (45.7 mg, 0.10 mmol) in
DMSO (5 mL) was added iodine (12.6 mg, 0.05 mmol). The
reaction mixture was stirred at 75 °C for 1 h. After cooling,
the reaction mixture was quenched with a saturated solution
of Na2S2O3 and extracted with ethyl acetate. The organic
layers were combined, washed with brine. It was dried over
Na2SO4 and filtered. The residue was evaporated under
vacuum without purify, affording a pale-yellow powder 23
Preparation of 2-(4-hydroxyphenyl)-6-isopentenyl-7-
methoxy-2,3-dihydroquinolin-4(1H)-one (21)
To a solution of compound 20 (110.0 mg, 0.25 mmol) in
THF (2 mL) and MeOH (2 mL) was added 4-
methylbenzenesulfonic acid monohydrate (215.0 mg,
1.25 mmol). The mixture was stirred at 50 °C for 15 h. The
mixture was cooled to room temperature and diluted with
water and extracted with ethyl acetate. The combined
organic layers were washed with brine. It was dried over
Na2SO4 and filtered. The filtrate was concentrated and the
residue was purified by silica gel column chromatography
eluting with CH2Cl2/MeOH (100:1) to give pale-yellow
powder 21 (18.0 mg, 0.05 mmol) and CH2Cl2/MeOH
1
(21.0 mg, 46.2%). 23: H NMR (500 MHz, DMSO-d6) δ
7.80 (m, J = 8.8 Hz, 3H), 7.41 (d, J = 8.5 Hz, 2H), 7.27 (s,
1H), 7.20 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.43
(s, 1H), 5.30 (t, J = 7.1 Hz, 1H), 5.13 (s, 2H), 3.92 (s, 3H),
3.76 (s, 3H), 3.34 (d, J = 7.3 Hz, 2H), 1.73 (s, 3H), 1.69 (s,
3H); 13C NMR (125 MHz, DMSO-d6) δ 160.5, 160.3,
159.1, 149.7, 132.4, 129.6, 128.8, 128.7, 128.5, 127.7,
125.8, 124.0, 121.7, 117.4, 115.3, 113.9, 105.20, 98.39,
69.2, 55.8, 55.1, 27.9, 25.6, 17.6; ESI-MS (m/z): 456.2 [M
+ H]+.
1
(50:1). 21: H NMR (500 MHz, CD3OD) δ 7.47 (s, 1H),
7.31 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 6.31 (s,
1H), 5.29–5.22 (m, 1H), 4.60 (dd, J = 13.4, 4.1 Hz, 1H),
3.84 (s, 3H), 3.17 (d, J = 7.3 Hz, 2H), 2.76 (dd, J = 16.2,
13.4 Hz, 1H), 2.58 (dd, J = 16.2, 4.1Hz, 1H), 1.75 (s, 3H),
1.70 (s, 3H); 13C NMR (125 MHz, CD3OD) δ 193.6, 164.3,
156.9, 154.3, 132.3, 131.8, 127.5 × 2, 126.7, 122.2, 120.8,
114.9 × 2, 111.2, 95.8, 57.5, 54.6, 45.5, 27.2, 24.5, 16.3;
HR-ESI-MS m/z: Anal. Calcd. for C21H24NO3 338.1751;
found 338.1761 [M + H]+.
Preparation of methyl-2-hydroxy-4-methoxybenzoate (25)
To a solution of 4-methoxysalicylic acid 24 (4.0 g,
23.8 mmol) in MeOH (40 mL) was added H2SO4 (6 mL).
The mixture was stirred at reflux for 48 h. It was then
concentrated and the residue was diluted with water
(50 mL), the pH of the mixture was adjusted to 5~6 with
K2CO3 and the aqueous solution of was extracted with
DCM (40 mL × 3). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4 and filtered.
The residue was purified by silica gel column chromato-
graphy eluting with PE/EA to give pale-yellow oil 25 (4.3 g,
99.2%). 25: ESI-MS (m/z): 183.1 [M + H]+.
Preparation of 2-(4-hydroxyphenyl)-7-methoxy-6-
(isopentenyl)quinolin-4(1H)-one (22)
To a solution of compound 21 (20.0 mg, 0.06 mmol) in
DMSO (2 mL) was added iodine (7.6 mg, 0.03 mmol). The

Medicinal Chemistry Research
Preparation of methyl 5-bromo-2-hydroxy-4-
methoxybenzoate (26)
Preparation of 2-hydroxy-4-methoxy-5-(isopentenyl)
benzamide (30)
To a solution of compound 25 (4.0 g, 22.0 mmol) in DCM
(40 mL) was added Br2 (3.9 g, 24.2 mmol) dropwise over
10 min at 0 °C. The mixture was stirred at 0 °C or 30 min
and then stirred at room temperature for 3 h. It was con-
centrated and the residue was purified by silica gel column
chromatography eluting with PE/EA to give white solid 26
(5.0 g, 81.14%). 26: ESI-MS (m/z): 260.9 [M + H]+.
To a solution of compound 29 (0.54 g, 2.28 mmol) in DCM
(6 mL) were added HATU (1.30 g, 3.43 mmol), DIPEA
(0.88 g, 6.86 mmol) and NH3/THF (1 N, 22.8 mL,
22.8 mmol). The mixture was stirred at room temperature
for 16 h. It was diluted with water (20 mL) and extracted
with EA (40 mL × 3). The combined organic layers were
washed with brine (80 mL). It was dried over Na2SO4 and
filtered. The filtrate was concentrated and the residue was
purified by prep-TLC to give yellow solid 30 (0.25 g,
46.49%). 30: ESI-MS (m/z): 236.1 [M + H]+.
Preparation of methyl 5-bromo-4-methoxy-2-(tosyloxy)
benzoate (27)
To a solution of compound 26 (5.0 g, 19.2 mmol) in DCM
(50 mL) were added 4-methylbenzene-1-sulfonyl chloride
(5.1 g, 26.9 mmol) and TEA (7.8 g, 76.9 mmol). The mix-
ture was stirred at room temperature for 3 h. It was con-
centrated and the residue was purified by silica gel column
chromatography eluting with PE/EA to give pale-yellow
solid 27 (7.5 g, 94.3 %). 27: ESI-MS (m/z): 415.0 [M + H]
Preparation of compound 31
A mixture of compound 30 (0.54 g, 1.06 mmol), compound
15 (0.40 g, 1.69 mmol) and piperidine (0.02 g, 0.21 mmol)
in toluene (5 mL) was stirred at 100 °C for 16 h. The mix-
ture was cooled to RT and concentrated. The residue was
purified by prep-TLC to give pale-yellow solid 31 (0.07 g,
14.52%). 31: ESI-MS (m/z): 454.2 [M + H]+.
+
.
Preparation of methyl 4-methoxy-5-(isopentenyl)-2-
(tosyloxy) benzoate (28)
Preparation of compound 32
To a solution of compound 31 (70 mg, 0.15 mmol) in DCM
(5 mL) was added TBAF (81 mg, 0.31 mmol) at 0 °C for
15 min. It was then diluted with water (20 mL) and
extracted with DCM (15 mL × 3). The combined organic
layers were washed with brine (30 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by prep-
TLC to give compound yellow solid 32 (20 mg, 38.19%).
32: 1H NMR (400 MHz, CD3OD-CDCl3) 7.62 (s, 1 H),
7.47 – 7.38 (d, J = 8.6 Hz, 2 H), 6.88 (d, J = 8.6 Hz, 2 H),
6.44 (s, 1 H), 6.08 (s, 1 H), 5.30 – 5.21 (m, 1 H), 3.82 (s,
3 H), 3.24 (d, J = 7.3 Hz, 2 H), 1.72 (s, 3 H), 1.68 (s, 3 H);
13C NMR (100 MHz, CD3OD-CDCl3) δ 165.3, 162.9,
158.7, 158.1, 132.9, 128.6×2, 127.8, 126.9, 125.3, 121.7,
115.5 × 2, 109.4, 98.3, 85.7, 55.6, 27.7, 25.5, 17.5. ESI-MS
(m/z): 340.3 [M + H]+; HR-ESI-MS m/z: Anal. Calcd. for
C20H22NO4 340.1543; found 340.1536 [M + H]+.
To a solution of compound 27 (2.7 g, 6.52 mmol) in DMF
(20 mL) were added 4,4,5,5-tetramethyl-2-(3-methylbut-2-
enyl)-1,3,2-dioxaborolane (1.7 g, 8.47 mmol), Pd(dppf)2Cl2
(0.5 g, 0.65 mmol) and Cs2CO3 (4.2 g, 13.04 mmol). The
mixture was stirred at 70 °C for 15 h with the protection of
N2. It was cooled to room temperature and diluted with water
(50 mL) and extracted with EA (50 mL × 3). The combined
organic layers were washed with brine (80 mL × 3). It was
dried over Na2SO4 and filtered. The filtrate was concentrated
and the residue was purified by silica gel column chromato-
graphy eluting with PE/EA to give pale-yellow oil 28 (1.0 g,
38.02%). 28: ESI-MS (m/z): 405.1 [M + H]+.
Preparation of 2-hydroxy-4-methoxy-5-(isopentenyl)
benzoic acid (29)
To a solution of compound 28 (1.0 g, 2.5 mmol) in THF
(24 mL), MeOH (8 mL) and H2O (8 mL) was added NaOH
(0.5 g, 12.4 mmol). The mixture was stirred at 50 °C for
16 h. It was cooled to room temperature and concentrated.
The residue was dissolved in H2O (15 mL). The pH of the
mixture was adjusted to 5~6 with 1 N HCl solution. It was
then extracted with EA (25 mL × 3). The combined organic
layers were washed with brine (80 mL). It was dried over
Na2SO4 and filtered. The filtrate was concentrated to give
white solid 29 (0.54 g, 92.44%). 29: ESI-MS (m/z): 237.0
[M + H]+.
Preparation of 2-amino-4-methoxybenzoate (34)
To a solution of 2-amino-4-methoxybenzoic acid 33 (5.0 g,
0.03 mol) in methanol (50 mL) was added strong sulphuric
acid (9 mL). The solution was stirred at reflux for 24 h. It
was cooled to room temperature and the pH of the mixture
was adjusted to 5~6 with saturated sodium carbonate
solution. The solution was extracted with EA (100 mL*2).
The combined organic layers were washed with brine
(50 mL), dried over Na2SO4, filtered and concentrated,
1
affording white powder 34 (4.11 g, 75.92%). 34: H NMR

Medicinal Chemistry Research
1
(400 MHz, CDCl3) 7.81 (d, J = 8.9 Hz, 1 H), 6.25 (dd, J =
9.0, 2.5 Hz, 1H), 6.13 (d, J = 2.4 Hz, 1H), 5.80 (s, 2H), 3.86
(s, 3H), 3.81 (s, 3H).
83.66%). 37: H NMR (400 MHz, DMSO-d6) 7.38 (s, 1H),
6.26 (s, 1H), 5.22–5.14 (m, 1H), 3.74 (s, 3H), 3.06 (d, J =
7.4 Hz, 2H), 1.68 (s, 3H), 1.63 (s, 3H). ESI-MS (m/z): 234.1
[M-H]−.
Preparation of 2-amino-5-bromo-4-methoxybenzoate (35)
Preparation of compound 38
To a solution of compound 34 (4.0 g, 0.02 mol) in DCM
(200 mL) was added bromine (1.36 mL, 0.02 mol). The
mixture was stirred at room temperature for 20 h. It was
diluted with saturated sodium hydrogen sulfite (50 mL) and
the aqueous solution was extracted with DCM. The com-
bined organic layers were washed with brine, dried over
Na2SO4 and filtered. The residue was purified by silica gel
column chromatography eluting with PE/EA to give white
To a solution of compound 37 (300 mg, 1.28 mmol) in THF
(15 mL) were added triphosgene (378 mg, 1.28 mmol). The
solution was stirred at room temperature for 2 h. The reac-
tion solution was diluted with water (15 mL) and extracted
with ethyl acetate (15 mL × 2). The combined organic layers
were washed with brine (15 mL), dried over Na2SO4, fil-
tered and concentrated, affording yellow solid 38 (320 mg,
96.05%). 38: 1H NMR (400 MHz, DMSO-d6) 11.66 (s, 1H),
7.57 (s, 1H), 6.60 (s, 1H), 5.23 (t, J = 7.4 Hz, 1H), 3.88 (s,
3H), 3.22 (d, J = 7.4 Hz, 2 H), 1.71 (s, 3H), 1.65 (s, 3H).
ESI-MS (m/z): 260.1 [M-H]−.
1
powder 35 (4.71 g, 82.06%). 35: H NMR δ (400 MHz,
CDCl3) 8.04 (s, 1H), 6.14 (s, 1H), 5.87 (s, 2H), 3.89 (s, 3H),
3.87 (s, 3H).
Preparation of 2-amino-4-methoxy-5-(isopentenyl)benzoate
(36)
Preparation of 2-amino-4-methoxy-5-(isopentenyl)
benzamide (39)
To a solution of compound 35 (500 mg, 1.92 mmol) in
DMF (10 mL) were added 4,4,5,5-tetramethyl-2-(3-
To a solution of compound 38 (320 mg, 1.22 mmol) in 1,4-
dioxane (15 mL) were added ammonium carbonate
(1176 mg, 12.24 mmoL). The solution was stirred at 60 °C
for 7 h. The reaction solution was diluted with water
(15 mL) and extracted with ethyl acetate (15 mL × 2). The
combined organic layers were washed with brine (15 mL),
dried over Na2SO4, filtered and concentrated, affording
pale-yellow solid 39 (240 mg, 83.64%). 39: 1H NMR
(400 MHz, DMSO-d6) 7.55 (s, 1H), 7.30 (s, 1H), 6.74 (s,
1H), 6.60 (s, 2H), 6.22 (s, 1H), 5.21 (t, J = 7.3 Hz, 1H),
3.71 (s, 3H), 3.06 (d, J = 7.2 Hz, 2H), 1.66 (s, 3H), 1.65 (s,
3H). ESI-MS (m/z): 235.0 [M + H]+.
methylbut-2-enyl)-1,3,2-dioxaborolane
(452 mg,
2.31 mmol), Pd(dppf)2Cl2 (140 mg, 0.19 mmol) and
Cs2CO3 (1253 mg, 3.85 mmol). The mixture was stirred at
70 °C for 24 h with the protection of N2. It was cooled to RT
and diluted with water and extracted with ethyl acetate. The
combined organic layers were washed with brine. It was
dried over Na2SO4 and filtered. The filtrate was con-
centrated and the residue was purified by silica gel column
chromatography eluting with cyclohexane/EA (uv254,
20:1) to give pale-yellow powder 36 (198.0 mg, 43.0%). 36:
1H NMR (500 MHz, CDCl3) δ 7.63 (s, 1H), 7.35 (dd, J =
8.6, 4.2 Hz, 4H), 6.94 (dd, J = 24.0, 8.6Hz, 4H), 6.07 (s,
1H), 5.26 (t, J = 7.3 Hz, 1H), 4.99 (s, 2H), 4.64 (dd, J =
13.8, 3.5 Hz, 1H), 4.41 (s, 1H), 3.81 (s, 3H), 3.80 (s, 3H),
3.19 (d, J = 7.3 Hz, 2H), 2.78 (dd, J = 16.1, 13.9 Hz, 1H),
2.65 (dd, J = 16.2, 2.7 Hz, 1H), 1.72 (s, 3H), 1.69 (s, 3H).
Preparation of 2-(4-hydroxyphenyl)-7-methoxy-6-
(isopentenyl)-2,3-dihydroquinazolin-4(1H)-one (40)
To a solution of compound 39 (100 mg, 0.43 mmol) and p-
hydroxybenzaldehyde (104 mg, 0.85 mmol) in DMF
(20 mL) were stirred and added p-toluenesulfonic acid
(16 mg, 0.085 mmol). The solution was stirred at room
temperature for 2 h. The reaction solution was diluted with
water (20 mL) and extracted with ethyl acetate (20 mL×2).
The combined organic layers were washed with brine
(15 mL), dried over Na2SO4, filtered. The filtrate was con-
centrated and the residue was purified by silica gel column
chromatography eluting with PE/EA to give white solid 40
(100 mg, 69.24%). 40: 1H NMR (400 MHz, DMSO-d6)
9.48 (s, 1H), 7.83 (s, 1H), 7.31 (s, 1H), 7.27 (d, J = 8.3 Hz,
2H), 6.80 (s, 1H), 6.74 (d, J = 8.2 Hz, 2H), 6.27 (s, 1H),
5.58 (s, 1H), 5.20 (t, J = 7.5 Hz, 1H), 3.71 (s, 3H), 3.09 (d,
J = 7.4 Hz, 2H), 1.68 (s, 3H), 1.63 (s, 3H). 13C NMR δ
Preparation of 2-amino-4-methoxy-5-(isopentenyl)benzoic
acid (37)
To a solution of compound 36 (380 mg, 1.52 mmol) in
mixed solution of THF (15 mL), methanol (5 mL) and
deionized water (5 mL) were added NaOH (305 mg,
7.62 mmol). The solution was stirred at 60 °C for 12 h.
After removing the THF and methanol by reduced pressure
distillation, the pH of the mixture was adjusted to 4 with 1 N
hydrochloric acid solution and the aqueous solution was
extracted with EA (20 mL*2). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, fil-
tered and concentrated, affording yellow solid 37 (300 mg,

Medicinal Chemistry Research
(150 MHz, DMSO-d6) 164.4, 161.6, 158.0, 148.7, 132.4,
131.7, 128.6, 128.2, 123.3, 119.2, 115.4, 107.9, 96.3, 67.2,
55.7, 27.7, 26.1, 18.0. ESI-MS (m/z): 337.3 [M-H]−. HR-
ESI-MS m/z: Anal. Calcd. for C20H21N2O3 337.1552; found
337.1544 [M + H]+.
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Acknowledgements This work was supported by the Excellent Aca-
demic Leaders Program of Shanghai (16XD1403500), the Shanghai
Science and Technology Committee funding (16401902000) and the
program of Shanghai E-Research Institute of Bioactive Constituents in
Traditional Chinese Medicine.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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