Design, synthesis and biological activity of flavonoid derivatives as selective agonists for neuromedin U 2 receptor

Article abstract of DOI:10.1016/j.bmc.2014.08.038

Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents to treat obesity. Based on the structure of rutin, a typi

Full text of DOI:10.1016/j.bmc.2014.08.038

Bioorganic & Medicinal Chemistry 22 (2014) 6117–6123
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activity of flavonoid derivatives
as selective agonists for neuromedin U 2 receptor
Ming-Liang Ma a,b, Ming Li b, Jiao-Jiao Gou a, Tian-Yu Ruan a, Hai-Shan Jin a, Ling-Hong Zhang a,
Liang-Chun Wu a, Xiao-Yan Li a, Ying-He Hu b, Ke Wen c, , Zheng Zhao a,
⇑
⇑
a Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai 200062, China
b Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, East China Normal University, Shanghai 200062, China
c Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai 201210, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and
body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents
to treat obesity. Based on the structure of rutin, a typical flavonoid and one of the NMU2R agonists we
previously identified from an in-house made natural product library, 30 flavonoid derivatives have been
synthesized and screened on a cell-based reporter gene assay. A number of compounds were found to be
selective and highly potent to NMU2R. For example, the EC50 value of compound NRA 4 is very close to
that of NMU, the endogenous peptide ligand of NMU2R. Structure–activity relationship analysis revealed
that a 3-hydroxyl group in ring C and a 20-fluoride group in ring B were essential for this class of com-
pounds to be active against NMU2R.
Received 16 June 2014
Revised 25 August 2014
Accepted 27 August 2014
Available online 8 September 2014
Keywords:
NMU2R
Structure–activity relationship
Flavonoid derivatives
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
potent and selective NMU2R agonist. These results suggest that
NMU regulates food intake and energy expenditure potentially
Neuromedin U (NMU) regulates a variety of physiological
activities in mammals, including stimulation of smooth-muscle
contraction,1 blood pressure regulation,2 ion transport,3,4 local
blood flow control,5 gastric acid secretion and gastric emptying.6
In the central nervous system, NMU plays important roles in the
regulation of feeding behavior7 and energy homeostasis.8 Previous
study has identified two G-protein coupled receptors (GPCRs) for
NMU, NMU1R and NMU2R.7 NMU1R is expressed in a wide range
of peripheral tissues such as intestine, testis, pancreas, uterus, lung
and kidney,7,9 while NMU2R is expressed exclusively in the central
nervous system (CNS), mainly in the paraventricular nucleus (PVN)
and arcuate nucleus of the hypothalamus.9–11 It has been reported
that intracerebroventricular (ICV) injection of NMU decreased the
food intake and body weight in rodents.12 Moreover, ICV injection
of NMU antiserum in rats attenuated the effects of NMU and
increased food intake.13 Recent studies also showed that the
knockout of NMU gene in mice resulted in obesity phenotype
and those mice showed the increase of food intake and
decrease of energy expenditure.14 Deng15 in 2014 reported that
p-synephrine binding to NMU2R with high efficacy and potency;
through the activation of NMU2R in the hypothalamus16,17 and
thus provide a notion that NMU2R is potentially a valuable thera-
peutic target for the development of new therapeutics for treating
obesity.
It is well-established that natural products are excellent
structural sources for biological screening. Previously, by screening
an in-house made natural product library, we have identified a few
natural products which could selectively activate NMU2R in a cell-
based assay. Interestingly, in vivo study demonstrated that the
expressions of NMU2R and NMU were significantly up-regulated
in the hypothalamus of mice at the third or fourth week of the
treatment with one of the compounds.18 Structural analysis
showed that these compounds have similar core structure and
belong to the family of flavonoid glycosides, which are a group of
polyphenolic secondary metabolites present in a wide variety of
plants (Fig. 1).18 However, these compounds generally exhibited
low activity toward NMU2R.18,19 Oriented to the discovery of small
molecular NMU2R agonists, we initiated a medicinal chemistry
program based on the structural modification of the previously
discovered natural product NMU2R agonists, flavonoid glycosides.
By maintaining the core structure of the three ring system, 30
compounds have been synthesized and their biological activities
in NMU2R cell-based assay have been examined. A number of
flavon-3-ol derivatives with strong potency against NMU2R have
the EC50 of p-synephrine is 6.6 lM for the NMU2R, is a highly
⇑
Corresponding authors. Tel.: +86 21 62238005; fax: +86 21 62601953.
E-mail addresses: wenk@sari.ac.cn (K. Wen), zzhao@brain.ecnu.edu.cn (Z. Zhao).
http://dx.doi.org/10.1016/j.bmc.2014.08.038
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
2. Results and discussion
2.1. Chemistry
B
Ring
OH
OH
C
Ring
HO
O
All NRAs (NMU2R Agonists) have been prepared according to
CH3
O
literature procedures by employing 2-hydroxyacetophenone and
commercially available substituted benzaldehydes as starting
materials. Schemes 1–4 illustrate the synthesis leading to the tar-
get structures of flavon-3-ols (NRA 1–17), 3-alkoxy-flavones (NRA
18–20), flavones (NRA 21–25) and flav-3-enes (NRA 26–30).
Detailed synthetic protocols are presented in the experimental
section. NRA 25–30 were synthesized as racemics which bear
one steric center (Schemes 1–4). Table 1 shows the chemical struc-
tures of flavon-3-ols (NRA 1–20) and their EC50 values in a cell-
based NMU2R assay. Screening test revealed that compounds
NRA 21–30 showed no biological activities toward NMU2R. Thus,
these compounds were not listed in Table 1.
O
O
OH
OH
OH
A
O
HO
O
Ring
OH
HO
HO
Figure 1. Chemical structure of rutin.
been identified. To our knowledge, this is the first medicinal
chemistry program aimed at the discovery of NMU2R agonists.
In this report, we showed the biological activities of flavonol
and flavone analogues and discussed the structure–activity rela-
tionship (SAR) of this class of compounds.
R3
R3
R3
R2
R4
R5
R2
R4
R5
OH
R2
O
R4
R5
a/b
c
O
O
O
+
H
R6
R6
O
OH
OR
R6
O
NRA 1-17
NRA 18-20
Scheme 1. Synthesis of NRA 1–20. Reagents and conditions: (a) EtOH/1,4-dioxane, 40% w/v KOH, 0 to rt; (b) 1,4-dioxane/EtOH, 5.4% w/v NaOH, 35% H2O2, 0 to rt, yield:40–
65% (2 steps) (c) RX, K2CO3, DMF, rt, yield: 90–95%.
R3
R3
R6
R3
R6
R2
R4
R5
OH
O
OH
O
R2
O
R4
R5
R2
R4
R5
a
b
O
O
+
H
R6
NRA 21-25
21 R2=F, R3=R4=R5=R6=H
22 R4=OH, R2=R3=R5=R6=H
23 R2=R4=Cl, R3=R5=R6=H
24 R3=OH, R4=OCH3, R2=R5=R6=H
25 R3=OCH3, R4=OH, R2=R5=R6=H
Scheme 2. Synthesis of NRA 21–25. Reagents and conditions: (a) (1) KOH (40%), EtOH, 0 to rt, (2) HCl (6 N); (b) I2, DMSO, reflux, yield: 40–65% (2 steps).
R3
R6
R3
R6
R3
R6
OH
O
OH
O
R2
R4
R5
R2
O
R4
R5
R2
R4
R5
a
b
+
O
H
NRA 26-28
26 R4=OCH3, R2=R3=R5=R6=H
27 R4=N(CH3)2, R2=R3=R5=R6=H
28 R3=OH, R4=OCH3, R2=R5=R6=H
Scheme 3. Synthesis of NRA 26–28. Reagents and conditions: (a) piperidine/EtOH, reflux; (b) NaBH4/THF/EtOH, rt-reflux, yield: 40–50%(2 steps).
R3
R3
R6
R3
R6
R2
R4
R5
OH
O
OH
O
R2
O
R4
R5
R2
R4
R5
b,c
a
O
+
H
R6
OH
R3
R2
R4
R5
O
d
R6
29 R2=F, R3=R4=R5=R6=H
30 R2=R4=Cl, R3=R5=R6=H
NRA 29-30
Scheme 4. Synthesis of NRA 29–30. Reagents and conditions: (a) (1) KOH (40%), EtOH, 0 to rt; (b) NaOAc/ EtOH; (c) NaBH4/THF/EtOHp, reflux-rt; (d) p-TSA, toluene, reflux,
yield: 3 steps, 40% for NRA 29 and 48% for NRA 30.

M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
6119
Table 1
shown in Table 1. The result showed that NRA 1, NRA 2 and NRA
4 exhibited much higher biological activities towards NMU2R than
the original identified natural product, rutin. The dose response
curves of NUM, rutin, NRA 1 and NRA 4 shown in Figure 2. The
EC50 value of NRA 4 was close to that of the endogenous ligand
NMU.
The chemical structures of flavon-3-ols (NRA 1–20) and their EC50 values in a cell-
based NMU2R assay
Compound R2 R3
R4
R5
R6
R
EC50
6.1
1.2 mM
NMU
Rutin
NRA 1
NRA 2
NRA 3
NRA 4
NRA 5
NRA 6
NRA 7
l
M
F
F
F
F
F
F
F
H
H
H
H
H
Cl
H
H
H
H
F
H
Cl
H
H
H
H
H
Cl
Br
OCH3
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
Br
OCH3
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
37.3
31.1
73.0
14.0
lM
lM
lM
lM
2.2.2. Structure–activity relationship
On the basis of the biological activities of the newly synthesized
flavonoid derivatives in the cell-based reporter gene assay, we
have suggested the structure–activity relationship. As shown in
Table 1, NRA 1–5, all exhibit biological activity with EC50 values
253.0
—
lM
—
—
—
—
—
—
—
—
—
—
—
—
NRA 8
NRA 9
ranged from 14.0 lM to 253 lM. The common feature of the five
H
H
compounds is that they all bear a 3-hydroxyl group in the C ring,
and in the B ring, the 20-position is occupied by a fluoride group,
the 60-position remains open. Besides the 20-fluoride substituents
in the B ring, the 60-position of NRA 6 is substituted by a chloride
group, consequently, NRA 6 losses its activity toward NMU2R.
Introducing a 50-fluoride in the B ring of NRA 6, resulted in NRA
7, exhibits no activity. As for NRA 8–17, the C ring is unchanged,
while the 20-fluoride group in the B ring is eliminated, and
positions 30, 40, and 50 of the B ring are substituted by a range of dif-
ferent functional groups. The screening experiments revealed that
NRA 8–17 exhibited no activity. SAR analysis leads to a conclusion
that a 20-fluoride group in the B ring is essential for this class of
compounds (flavon-3-ols, NRA 1–17) to be active toward NMU2R.
By analyzing the structural differences and the corresponding EC50
values of the five biologically active compounds (NRA 1–5), besides
20-fluoride group in the B ring for all five compounds, NRA 1 bears
no substituents in other positions of B ring and showed an EC50
NRA 10
NRA 11
NRA 12
NRA 13
NRA 14
NRA 15
NRA 16
NRA 17
NRA 18
NRA 19
NRA 20
OH
N(CH3)2
Cl
OH
OCH3
OH
OH
OCH3 OH
H
H
H
H
N(Et)2
H
H
CH3
CH2CH2OH
CH2COOC2H5
H
H
H
Br
Br
Br
F
F
47.0
—
l
M
2.2. Biological activity and discussion
2.2.1. Identification of flavonol derivative NMU2R agonists
Using our optimized cell-based reporter gene assay, all newly
synthesized compounds were examined to determine their activi-
ties towards human NMU2R. Several cell lines expressing other
GPCRs, including MC4R, MC3R, M1R, human NMU1R and MRE/
CRE/SRE, were used for specificity assay.
value of 37.3
l
M; NRA 2, with 30-chloride group, NRA 3, with a
40-bromide group, and NRA 4, with a 50-bromide group, showed
EC50 values of 31.1 lM, 73.0 lM and 14.0 lM, respectively. Replac-
In vitro pharmacological analysis indicated that six compounds
could activate NMU2R in a dose response manner, while no
compounds had effects on other GPCRs (see the Supporting
information). The EC50 values of the six active compounds were
ing the 50-Br group in NRA 4 by a methoxyl function, leads to NRA
5, significantly decreased the activity toward NMU2R with an EC50
value of 253 lM. Among the five active compounds, NRA 4 showed
EC50=6.1µM
EC50=1.2 mM
Concentration of NMU (logM)
Concentration of Rutin (logM)
EC50=14.0 µM
EC50=37.3µM
Concentration of NRA 4 (logM)
Concentration of NRA 1 (logM)
Figure 2. Response of NMU2R cell line to NUM, rutin, NRA 1 and NRA 4. Cells were treated with endogenous ligand NMU rutin, NRA 1 and NRA 4, the luciferase activities were
measured. The data were analyzed using four-parameter logistic non-linear regression analysis method and presented as means SEM from three independent experiments.

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M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
the highest activity. Next, through alkylation of the 3-OH group in
ring C of the most active NRA 4, we obtained three 3-alkoxyl-
substituted derivatives of NRA 4, 3-OCH3 (NRA 18), –OCH2CH2OH
(NRA 19), and –OCH2COOCH2CH3 (NRA 20), respectively. As shown
in Table 1, NRA 18 and NRA 20 lost the biological activity, while the
4.1. General procedure for the synthesis of NRAs
All commercially chemicals and solvents are reagent grade and
used without further purification. The chemicals were distilled
prior to use. The reactions were carried out with the use of stan-
dard technique in nitrogen atmosphere. Reactions were monitored
by thin-layer chromatography and visualized with UV light or
iodine vapors. The typical work-up included washing with brine
and drying the organic phase with sodium sulfate before concen-
tration in vacuum. Final compounds were typically purified by
flash chromatography on silica gel or by preparative thin-layer
chromatography. Melting points were determined on a WRS-1A
capillary apparatus and were uncorrected. 1H NMR spectra were
recorded on a Bruker DRX-500 spectrometer with tetramethylsil-
ane as an internal reference in the indicated solvent at room tem-
perature. Chemical shifts were reported in parts per million (ppm,
d units). Coupling constants were reported in units of hertz (Hz).
Mass spectra in the electrospray ionization (ESI) mode were
obtained using a micrOTOF Q mass spectrometer. Purity of all final
compounds was determined by HPLC using a C18 column, and all
compounds were more than 95% pure.
EC50 value of NRA 19 decreased to 47.0 lM from 14.0 lM of its par-
ent NRA 4. NRA 19, with the 3-OH group being replaced by an –
OCH2CH2OH group, remains active toward NMU2R. This may give
a notion that a free –OH in position 3 of the C ring, even as part
of the substituted group, plays a critical role for this class of com-
pounds to be active. Flavones NRA 21–25 and flav-3-enes NRA 26–
30, share the similar three ring scaffold with the flavon-3-ols (NRA
1–20). However, both flavones (NRA 21–25) and flav-3-enes (NRA
26–30), bear no substitution groups in ring C, exhibit no biological
activity toward NMU2R. Direct comparison of the structural differ-
ences between NRA 1 and NRA 21, we can find that the two com-
pounds share the same scaffold in rings A and B, the only structural
difference comes from an 3-OH substituent in ring C (NRA 1 bears
an 3-OH group, while NRA 21 does not). However, the biological
activity data shows that only NRA 1 is active. This result again
demonstrates that a hydroxyl function in the position 3 of ring C
is essential for this class of compounds to be active toward NMU2R.
4.1.1. General procedure for the synthesis of NRA 1–1720,21
A mixture of 2-hydroxyacetophenone (1 equiv) and the corre-
sponding aldehyde (1 equiv) in ethanol (40 mL) and 1,4-dioxane
(20 mL) was cooled to 0–10 °C, 40% w/v KOH (5 equiv) solution
was added dropwise. Then the reaction mixture was stirred at
room temperature and monitored by TLC until the reaction com-
pletion. HCl (10%) was added to neutralize the reaction mixture.
Ethyl acetate (50 mL) was then added, organic layer was separated
and washed with H2O (50 mL) and brine (50 mL), dried over anhy-
drous sodium sulfate and concentrated in vacuum. The residue was
dissolved in 1,4-dioxane (50 mL) and ethanol (30 mL), cooled in an
ice-water bath, NaOH (5.4% w/v) solution (5 equiv), 35% H2O2
(8 equiv) was added dropwise. The reaction mixture was stirred
in an ice bath for 2 h and subsequently at room temperature over-
night, resulting in a yellow suspension. After acidification with 2 M
HCl to neutrality, ethyl acetate (3*50 mL) was used to extract the
product, and the organic layer was washed with H2O (50 mL) and
brine (50 mL), then dried over anhydrous sodium sulfate and con-
centrated in vacuum. The product was purified by column chroma-
tography. The total yields for the two steps were varied from 40%
to 65%.
3. Conclusion
We have successfully synthesized a series of flavon-3-ols, fla-
vone and flav-3-ene derivatives with various substitution groups
aiming at the discovery of potential NMU2R agonists. In vitro study
revealed that NRA 4 exhibited the strongest activity toward
NMU2R in the cell-based assay. SAR analysis revealed that a struc-
tural feature of a hydroxyl group at position 3 of ring C, a 20-fluoride
group in ring B and unsubstituted 60-position in ring B is essential
for this class of compounds to be active toward NMU2R. Besides
bears the above structural feature, the most potent NRA 4 bears a
50-bromide group in the B ring. So far, we mainly discussed the halo
substituent of B ring. We will introduce more functional groups,
such as amino, carboxyl, amide, and trifluoromethyl into the B ring
so that we could gain more insights into better active compounds
and their structure–activity relationships. Now, in our lab we are
researching the effect of some active compounds in animal models,
and we will report the results in the future.
4. Experimental section
4.1.2. 2-(2-Fluorophenyl)-3-hydroxy-4H-chromen-4-one
(NRA 1)
4.0.1. NMU2R cell-based functional assay
Yield 65%; mp: 120.6–121.6 °C; 1H NMR (CDCl3): d 8.29–8.28
(dd, J1 = 1.20, J2 = 8.05, 1H), 7.82–7.79 (t, J1 = 7.35, J2 = 7.40, 1H),
7.73–7.69 (t, J1 = J2 = 7.89, 1H), 7.55 (d, J = 8.45, 1H), 7.53–7.49
(m, 1H), 7.45–7.43 (t, J1 = 7.85, J2 = 7.20, 1H), 7.32–7.30 (t,
J1 = 7.05, J2 = 7.78, 1H), 7.26–7.23 (t, J1 = 9.50, J2 = 8.90, 1H), 6.67
(s, 1H); MS (MꢁH): C15H8FOꢁ3 m/z calcd 255.05, found 255.05.
The functional assay for agonist screening was established as
previously described18 with further optimization. Briefly, human
NMU2R cDNA was cloned using PCR method and the sequence of
the cDNA clone was confirmed. The PCR product was subcloned
into mammalian expression vector pcDNA3.1(+). The stable cell
lines were established via co-transfection of HEK-293 cells with
the reporter gene construct and human NMU2R cDNA. The cells
4.1.3. 2-(3-Chloro-2-fluorophenyl)-3-hydroxy-4H-chromen-4-
one (NRA 2)
were cultured in DMEM (Gibco) containing 600 lg/mL of G418
(Gibco) in a 37 °C incubator under an atmosphere of 5% CO2. The
confluent cells were collected using DMEM with 5% fetal bovine
serum (Gibco), and the transfected cells were then seeded in a den-
sity of 4 ꢀ 104 cells/mL into wells of 96-well plates and incubated
overnight. Forskolin (Sigma) and NMU were applied to the cells as
positive control to evaluate the cell-based functional assay. Ten
microliters of compounds at various concentrations were added
to the wells and incubated at 37 °C for 7 h. One hundred microli-
ters of Bright-Glo™ Luciferase assay reagent (Promega) was added
in each well and the luciferase response was read using Analyst™
HT (Molecular Device).
Yield 55%; mp: 195.8–197.7 °C. 1H NMR (CDCl3): d 8.28 (d,
J = 8.05, 1H), 7.74–7.70 (m, 2H), 7.58–7.54 (m, 2H), 7.46–7.43 (m,
1H), 7.27–7.24 (m, 1H), 6.68 (s, 1H); MS (MꢁH): C15H7ClFOꢁ3 m/z
calcd 289.01, found 289.01.
4.1.4. 2-(4-Bromo-2-fluorophenyl)-3-hydroxy-4H-chromen-4-
one (NRA 3)
Yield 56%; mp: 156.3–161.8oC; 1H NMR (CDCl3): d 8.27 (s, 1H),
7.72–7.67 (m, 2H), 7.53 (d, J = 8.50, 1H), 7.51–7.43 (m, 3H), 6.77 (s,
1H); MS (M+Na+): C15H8BrFO3Na+ m/z calcd 356.95, found 356.95.

M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
6121
4.1.5. 2-(5-Bromo-2-fluorophenyl)-3-hydroxy-4H-chromen-4-
one (NRA 4)
2H), 3.04 (s, 6H); MS (M+Na+): C17H15NO3Na+ m/z calcd 304.09,
found 304.09.
Yield 52%; mp: 216.8–217.6 °C; 1H NMR (CDCl3): d 8.27 (d,
J = 7.95, 1H), 7.94–7.93 (dd, J1 = 2.35, J2 = 6.00, 1H), 7.74–7.71 (t,
J1 = 7.2, J2 = 7.25, 1H), 7.62–7.60 (m, 1H), 7.55 (d, J = 8.50, 1H),
7.46–7.43 (t, J1 = 7.35, J2 = 7.50, 1H), 7.15–7.12 (t, J1 = 9.35,
J2 = 9.20, 1H), 6.71 (s, 1H); MS (M+Na+): C15H8BrFO3Na+ m/z calcd
356.95, found 356.96.
4.1.14. 2-(2,4-Dichlorophenyl)-3-hydroxy-4H-chromen-4-one
(NRA 13)
Yield 59%; mp: 211.9–213.7 °C; 1H NMR (DMSO-d6): d 9.48 (s,
1H), 8.17–8.15 (dd, J1 = 1.25, J2 = 8.00, 1H), 7.85 (d, J = 1.95, 1H),
7.80 (d, J = 1.30, 1H), 7.75 (d, J = 8.30, 1H), 7.66–7.61 (m, 2H),
7.51 (d, J = 7.15, 1H); MS (M+Na+): C15H8Cl2O3Na+ m/z calcd
328.97, found 328.98.
4.1.6. 2-(2-Fluoro-5-methoxyphenyl)-3-hydroxy-4H-chromen-
4-one (NRA 5)
Yield 57%; mp: 187.6–190.5 °C; 1H NMR (CDCl3): d 8.29 (d,
J = 8.00, 1H), 7.73–7.70 (t, J1 = 8.30, J2 = 7.20, 1H), 7.55 (d, J = 8.50,
1H), 7.45–7.42 (t, J1 = 7.60, J2 = 7.50,1H), 7.31–7.29 (m, 1H), 7.17–
7.13 (t, J1 = 9.30, J2 = 9.40, 1H), 7.03–7.01 (m, 1H), 6.72 (br s, 1H);
3.84 (s, 3H); MS (MꢁH): C16H10FOꢁ4 m/z calcd 285.06, found 285.06.
4.1.15. 2-(3,4-Dihydroxyphenyl)-3-hydroxy-4H-chromen-4-one
(NRA 14)
Yield 40%; mp: 274.0–274.8 °C; 1H NMR (DMSO-d6): d 9.57 (s,
1H), 9.28 (d, J = 6.50, 2H), 8.09 (d, J = 8.00, 1H), 7.79–7.71 (m,
2H), 7.70 (d, J = 8.00, 1H), 7.61–7.60 (t, J1 = 1.50, J2 = 8.50, 1H),
7.47–7.44 (t, J1 = 7.50, J2 = 15.00, 1H), 6.90 (d, J = 8.50, 1H); MS
(MꢁH): C15H9Oꢁ5 m/z calcd 269.04, found 269.05.
4.1.7. 2-(2-Chloro-6-fluorophenyl)-3-hydroxy-4H-chromen-4-
one (NRA 6)
Yield 55%; mp: 236.4–238.4 °C. 1H NMR (CDCl3): d 8.32–8.30
(dd, J1 = 1.25, J2 = 8.05, 1H), 7.72–7.70 (m, 1H), 7.53 (d,
J1 = 8.50,1H), 7.47–7.45 (m, 2H), 7.38 (d, J = 7.75, 1H), 7.19–7.16
(t, J1 = 8.55, J2 = 8.58, 1H), 6.58 (s, 1H); MS (MꢁH): C15H7ClFO3ꢁ
m/z calcd 289.01, found 289.00.
4.1.16. 3-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-
chromen-4-one (NRA 15)
Yield 45%; mp: 206.9–207.8 °C; 1H NMR (DMSO-d6): d 9.33 (s,
2H), 8.11–8.09 (dd, J1 = 1.35, J2 = 7.95, 1H), 7.88–7.71 (m, 4H),
7.47–7.44 (m, 1H), 7.10 (d, J = 8.65, 1H), 3.85 (s, 3H); MS (MꢁH):
C
16H11Oꢁ5 m/z calcd 283.06, found 283.06.
4.1.8. 2-(2-Chloro-3,6-difluorophenyl)-3-hydroxy-4H-chromen-
4-one (NRA 7)
4.1.17. 3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-
chromen-4-one (NRA 16)
Yield 42%; mp: 232.8–234.5 °C. 1H NMR (CDCl3): d 8.31 (d,
J = 8.00, 1H), 7.75–7.72 (t, J1 = 8.00, J2 = 7.50, 1H), 7.53 (d, J = 9.00,
1H), 7.48–7.45 (t, J1 = 7.00, J2 = 8.00, 1H), 7.35–7.30 (m, 1H),
7.18–7.13 (m, 1H), 6.53 (s, 1H); MS (M+Na+): C15H7ClF2O3Na+ m/z
calcd 330.99, found 330.99.
Yield 45%; mp: 244.0–244.6 °C; 1H NMR (DMSO-d6): d 9.73 (s,
1H), 9.36 (s, 1H), 8.10 (d, J = 7.75, 1H), 7.83–7.76 (m, 4H), 7.47–
7.44 (m, 1H), 6.96 (d, J = 8.45, 1H), 3.86 (s, 3H); MS (MꢁH):
C
16H11Oꢁ5 m/z calcd 283.06, found 283.07.
4.1.9. 2-(3-Chlorophenyl)-3-hydroxy-4H-chromen-4-one (NRA 8)
Yield 60%; mp: 105.0–106.0 °C; 1H NMR (CDCl3): d 8.27–8.18
(m, 3H), 7.78–7.73 (t, J1 = 8.50, J2 = 7.00, 1H), 7.62 (d, J = 8.50,
1H), 7.50–7.43 (m, 3H), 7.06 (s, 1H); MS (MꢁH): C15H8ClOꢁ3 m/z
calcd 271.01, found 271.01.
4.1.18. 2-(4-(Diethylamino)phenyl)-3-hydroxy-4H-chromen-4-
one (NRA 17)
Yield 56%; mp: 115.6–118.4 °C; 1H NMR (CDCl3-d6): d 8.23 (d,
J = 8.00, 1H), 8.17 (d, J = 9.00, 2H), 7.65–7.63 (m, 1H), 7.39–7.36
(t, J1 = 7.00, J2 = 8.00, 1H), 7.55 (d, J = 8.50, 1H), 6.89 (s, 1H), 6.77
(d, J = 9.00, 2H), 3.47–3.43 (q, J1 = 7.00, J2 = 14.00, 4H), 1.26–1.21
(t, J1 = 7.00, J2 = 7.50, 6H); MS (M+Na+): C19H19NO3Na+ m/z calcd
332.13, found 332.13.
4.1.10. 2-(3-Bromophenyl)-3-hydroxy-4H-chromen-4-one
(NRA 9)
Yield 65%; mp: 252.2–254.0 °C; 1H NMR (DMSO-d6): d 9.55 (s,
1H), 8.13–8.11 (t, J1 = 12.50, J2 = 1.00, 1H), 7.81–7.79 (m, 1H),
7.74 (d, J = 8.50, 1H), 7.68–7.65 (t, J1 = J2 = 8.50, 1H), 7.49–7.46 (t,
J1 = 7.00, J2 = 8.00,2H), 7.38–7.36 (t, J1 = J2 = 8.00, 1H), 6.91–6.89
(m, 1H); MS (MꢁH): C15H8BrO3ꢁ m/z calcd 314.96, found 314.96.
4.2. General procedure for the synthesis of NRA 18–20
A mixture of NRA 4 (1 equiv), potassium carbonate (2.5 equiv)
and the corresponding bromides (1 equiv) in DMF (50 mL) was
stirred at room temperature and the reaction was monitored by
TLC. After the reaction completion, HCl (10%) was employed to
neutralize the reaction mixture and then ethyl acetate (50 mL)
was added, the organic layer was washed with H2O (50 mL) and
brine (50 mL), dried over anhydrous sodium sulfate and concen-
trated in vacuum. The product was purified by column chromatog-
raphy, the yield was in a range of 90ꢂ95%.
4.1.11. 2-(3-Methoxyphenyl)-3-hydroxy-4H-chromen-4-one
(NRA 10)
Yield 55%; mp: 131.4–132.0 °C; 1H NMR (CDCl3): d 8.25 (d,
J = 7.45, 1H), 7.86–7.82 (m, 2H), 7.69 (d, J = 7.05,1H), 7.58 (d,
J = 8.45, 1H), 7.46–7.39 (m, 2H), 7.12 (s, 1H), 7.03–7.01 (m, 1H),
3.90 (s, 3H); MS (MꢁH): C16H11Oꢁ4 m/z calcd 267.07, found 267.08.
4.1.12. 3-Hydroxyl-2-(4-hydroxyphenyl)-4H-chromen-4-one
(NRA 11)
4.2.1. 2-(5-Bromo-2-fluorophenyl)-3-methoxy-4H-chromen-4-
one (NRA 18)
Yield 41%; mp: 275.6–276.4 °C; 1H NMR (DMSO-d6): d 10.11 (s,
1H); 9.32 (s, 1H), 8.12–8.09 (t, J1 = 10.00, J2 = 5.00, 3H), 7.79–7.71
(m, 2H), 7.46–7.43 (t, J1 = 5.00, J2 = 10.00, 1H), 6.95 (d, J = 10.00,
2H), MS (MꢁH): C15H8Oꢁ4 m/z calcd 253.05, found 253.06.
Yield 95%; mp: 111.8–112.9 °C; 1H NMR (CDCl3): d 8.28 (d,
J = 8.00, 1H), 7.78–7.76 (dd, J1 = 2.25, J2 = 5.75, 1H), 7.71–7.68 (t,
J1 = 7.30, J2 = 15.55, 1H), 7.62–7.60 (m, 1H), 7.49 (d, J = 8.45, 1H),
7.44–7.41 (t, J1 = 7.85, J2 = 15.05, 1H), 7.15–7.11 (m, 1H), 3.95 (s,
3H); MS (M+Na+): C16H10BrFO3Na+ m/z calcd 370.97, found 370.98.
4.1.13. 2-(4-(Dimethylamino)phenyl)-3-hydroxy-4H-chromen-
4-one (NRA 12)
4.2.2. 2-(5-Bromo-2-fluorophenyl)-3-(2-hydroxyethoxy)-4H-
chromen-4-one (NRA 19)
Yield 58%; mp: 190.7–191.3 °C. 1H NMR (CDCl3): d 8.23–8.16
(m, 3H), 7.64–7.61 (t, J1 = J2 = 7.50, 1H), 7.53 (d, J = 8.50,1H),
7.37–7.35 (t, J1 = 7.00, J2 = 7.50, 1H), 6.90 (s, 1H), 6.77 (d, J = 8.00,
Yield 90%; mp: 121.1–122.1 °C; 1H NMR (CDCl3): d 8.30 (d,
J = 8.05, 1H), 7.83–7.81 (m, 1H), 7.72 (d, J = 8.35, 1H), 7.65–7.64

6122
M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
(m, 1H), 7.52 (d, J = 8.50, 1H), 7.48–7.45 (t, J1 = 7.35, J2 = 15.10, 1H),
7.17–7.13 (t, J1 = 9.20, J2 = 18.25, 1H), 4.08–4.06 (t, J1 = 4.00,
J2 = 8.15, 2H), 3.80–3.79 (t, J1 = 4.00, J2 = 7.85, 2H); MS (M+Na+):
4.2.9. 2-(4-Hydroxy-3-methoxyphenyl)-4H-chromen-4-one
(NRA 25)
Yield 48%; mp: 191.4–192.1 °C; 1H NMR (CDCl3): d 8.22 (d,
J = 7.85, 1H), 7.69–7.66 (t, J1 = 7.20, J2 = 7.25, 1H), 7.56–7.51 (m,
2H), 7.42–7.37 (m, 2H), 7.05 (d, J = 8.35, 1H), 6.74 (s, 1H), 6.39 (s,
1H), 3.99 (s, 3H); MS (MꢁH): C16H11Oꢁ4 m/z calcd 267.07, found
267.07.
C
17H12BrFO4Na+ m/z calcd 400.98, found 400.98.
4.2.3. Ethyl 2-(2-(5-bromo-2-fluorophenyl)-4-oxo-4H-chromen-
3-yloxy)acetate (NRA 20)
Yield 93%; mp: 79.8–82.1 °C; 1H NMR (CDCl3): d 8.19 (d, J = 8.00,
1H), 7.81–7.80 (m, 1H), 7.64–7.62 (t, J1 = 0.90, J2 = 8.35, 1H), 7.55–
7.54 (t, J1 = 4.35, J2 = 8.75, 1H), 7.44 (d, J = 8.50, 1H), 7.37–7.34 (m,
1H), 7.08–7.04 (t, J1 = 9.30, J2 = 18.35, 1H), 4.89 (s, 2H), 4.16–4.12
(m, 2H), 1.20–1.17 (t, J1 = 7.10, J2 = 14.20, 3H); MS (M+H): C19H15-
BrFO+5 m/z calcd 421.01, found 421.01.
4.3. General procedure for the synthesis of NRA 26–28
2-Hydroxyacetophenone (1 equiv) and the corresponding alde-
hyde 2 (1 equiv) were dissolved in ethanol (30 mL) and piperidine
(5 equiv) was added. The mixture was heated to reflux until the
starting material disappeared (monitored by TLC). The reaction
mixture was then cooled to room temperature and evaporated
the solvent in vacuum. The residue was dissolved in THF (40 mL)
and ethanol (20 mL), and NaBH4 (1 equiv) was slowly added to
the solution. The mixture was heated to reflux and monitored by
TLC until disappearance of the starting material. The product was
purified through column chromatography, the total yield was in
the range of 40ꢂ50%.
4.2.4. General procedure for the synthesis of NRA 21–2522,23
To an ice-cooled 2-hydroxyacetophenone (1 equiv) and the cor-
responding aldehyde (1 equiv) solution in EtOH (70 mL), 40% KOH
(6 equiv) was added. The mixture was stirred under nitrogen atmo-
sphere at ambient temperature until reaction was completed.
Thereafter, the reaction mixture was slowly poured into excess
6 N HC1, extracted with ethyl acetate (50 mL) three times and
washed with H2O and brine. Dried over anhydrous sodium sulfate
and concentrated in vacuum. The crude product 20-hydroxychal-
cone was passed through a fast column and then used in the next
step.
4.3.1. 2-(4-Methoxyphenyl)-2H-chromene (NRA 26)
Yield 50%; mp: 131.5–134.1 °C; 1H NMR (CDCl3): d 7.51 (d,
J = 7.15, 1H), 7.37 (d, J = 8.60, 2H), 7.20 (m, 1H), 7.00–6.97 (t,
J1 = 7.50, J2 = 7.91, 1H), 6.93 (d, J = 8.70, 2H), 6.90–6.87 (t,
J1 = 7.30, J2 = 6.50, 2H), 5.12–5.07 (m, 2H), 3.83 (s, 3H); MS
(M+HCOOꢁ): C17H15Oꢁ4 m/z calcd 283.08, found 283.01.
To
a
solution of the corresponding 20-hydroxychalcone
(1 equiv) in DMSO (20.0 mL), I2 (0.01 equiv) was added. The mix-
ture was heated at reflux for 1 h. Then the reaction mixture was
poured into water and extracted with ethyl acetate (3*25.0 mL).
The organic layer was washed with water and brine, dried over
anhydrous sodium sulfate. The solvent was evaporated in vac-
uum and the residue was purified by column chromatography,
the combined total yield for the two steps ranged from 40% to
65%.
4.3.2. 4-(2H-chromen-2-yl)-N,N-dimethylbenzenamine (NRA 27)
Yield 55%; mp: 76.9–77.7 °C; 1H NMR (CDCl3): d 7.34 (d,
J = 8.0 Hz, 2H), 7.11–7.08 (t, J1 = 7.00, J2 = 8.00, 1H), 7.02 (d,
J = 7.50, 1H), 6.87–6.84 (t, J1 = 7.00, J2 = 7.50, 1H), 6.77–6.72 (m,
3H), 6.55 (d, J = 9.50, 1H), 5.85–5.79 (m, 2H), 2.95 (s, 6H); MS
(M+H): C17H18NO+ m/z calcd 252.14, found 252.14.
4.2.5. 2-(2-Fluorophenyl)-4H-chromen-4-one (NRA 21)
Yield 65%; mp: 100.4–100.7 °C; 1H NMR (CDCl3): d 8.24–8.22
(dd, J1 = 1.60, J2 = 7.95, 1H), 7.93 (d, J = 1.65, 1H), 7.70–7.69 (t,
J1 = 1.20, J2 = 8.25, 1H), 7.54 (d, J = 8.60, 2H), 7.44–7.41 (t,
J1 = 7.80, J2 = 15.05, 1H), 7.34–7.31 (t, J1 = 7.65, J2 = 15.25, 1H),
7.26–7.22 (m, 1H), 6.94 (s, 1H); MS (M+H): C15H10FO+2 m/z calcd
241.07, found 241.07.
4.3.3. 5-(2H-chromen-2-yl)-2-methoxyphenol (NRA 28)
Yield 40%; mp: 187.1–187.7 °C; 1H NMR (CDCl3): d 7.51 (d,
J = 7.05, 1H), 7.22–7.18 (t, J1 = 9.00, J2 = 7.40, 1H), 7.03 (s, 1H),
6.99–6.93 (m, 2H), 6.89–6.86 (m, 2H), 5.65 (s, 1H), 5.11–5.06 (m,
2H), 3.90 (s, 3H); MS (M+Na+): C16H14O3Na+ m/z calcd 277.08,
found 277.09.
4.2.6. 2-(4-Hydroxyphenyl)-4H-chromen-4-one (NRA 22)
Yield 40%; mp: 258.5–262.4 °C; 1H NMR (DMSO-d6): d 10.30 (br
s, 1H), 8.03 (d, J = 8.00, 1H), 7.97 (d, J = 8.50, 2H), 7.83–7.80 (m, 1H),
7.75 (d, J = 8.50, 1H), 7.50–7.47 (t, J1 = 7.00, J2 = 7.50, 1H), 6.94 (d,
J = 8.50, 2H), 6.87 (s, 1H); MS (MꢁH): C15H9Oꢁ3 m/z calcd 237.06,
found 237.06.
4.4. General procedure for the synthesis of NRA 29 and NRA 3024
To an ice-cooled solution of 2-hydroxyacetophenone (1 equiv)
and the corresponding aldehyde (1 equiv) in EtOH (70 mL) was
added 40% KOH (6 equiv). The solution was stirred under nitrogen
atmosphere at ambient temperature until reaction was complete
(monitored by TLC). Thereafter, the reaction mixture was slowly
poured into excess 6 N HC1, extracted with ethyl acetate (50 mL)
three times and washed with H2O and brine, dried over anhydrous
sodium sulfate and concentrated in vacuum. The crude product
20-hydroxychalcone was purified in column chromatography.
The purified product was dissolved in EtOH (40 mL), and
sodium acetate (3.0 equiv) was added. The mixture was refluxed
and monitored by TLC. After the reaction complete, the mixture
was cooled to room temperature, and powdered sodium borohy-
dride (0.5equiv) was added in portions. The mixture was stirred
for additional 1.5 h, and quenched by dropwise addition of acetic
acid. The mixture was then poured into ice–water (100 mL) and
extracted with ethyl acetate (50 mL) three times and washed with
H2O and brine. The organic layer was dried over anhydrous sodium
sulfate and concentrated in vacuum. The crude mid product was
passed through a fast column and subjected to next step.
4.2.7. 2-(2,4-Dichlorophenyl)-4H-chromen-4-one (NRA 23)
Yield 58%; mp: 124.1–125.1 °C; 1H NMR (CDCl3): d 8.29–8.27
(dd, J1 = 1.00, J2 = 8.00, 1H), 7.73–7.72 (t, J1 = 3.00, J2 = 1.00, 1H),
7.63–7.59 (m, 2H), 7.53 (d, J = 9.00, 1H), 7.49–7.42 (m, 2H), 6.67
(s, 1H); MS (M+Na+): C15H8Cl2O2Na+ m/z calcd 312.98, found
312.98.
4.2.8. 2-(3-Hydroxy-4-methoxyphenyl)-4H-chromen-4-one
(NRA 24)
Yield 45%; mp: 192.2–194.9 °C; 1H NMR (DMSO-d6): d 9.48 (s,
1H), 8.05–8.03 (dd, J1 = 1.50, J2 = 7.85, 1H), 7.82–7.80 (dd,
J1 = 1.30, J2 = 6.95, 1H), 7.43 (d, J = 3.35, 1H), 7.59–7.57 (dd,
J1 = 1.75, J2 = 8.50, 1H), 7.50–7.47 (dd, J1 = 7.20, J2 = 12.05, 2H),
7.10 (d, J = 8.60, 1H), 6.83 (s, 1H), 3.87 (s, 3H); MS (MꢁH):
C
16H11Oꢁ4 m/z calcd 267.06, found 267.07.

M.-L. Ma et al. / Bioorg. Med. Chem. 22 (2014) 6117–6123
6123
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1H), 7.18–7.12 (m, 3H), 7.06–7.04 (dd, J1 = 0.15, J2 = 7.50, 1H),
6.94–6.92 (m, 1H), 6.86 (d, J = 8.00, 1H), 6.59–6.57 (dd, J1 = 1.50,
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Yield 48%; 1H NMR (CDCl3): d 7.51 (d, J = 8.40, 1H), 7.39 (d,
J = 2.00, 1H), 7.22–7.20 (dd, J1 = 2.00, J2 = 8.40, 1H), 7.12 (m, 1H),
7.00–6.99 (dd, J1 = 1.30, J2 = 7.40, 1H), 6.88 (m, 1H), 6.80 (d,
J = 8.05, 1H), 6.51 (d, J = 9.05, 1H), 6.30–6.29 (t, J1 = 3.40, J2 = 2.10,
1H), 5.75–5.72 (dd, J1 = 3.35, J2 = 9.85, 1H); MS (M+HCOOꢁ):
C16H11Cl2Oꢁ3 m/z calcd 321.01, found 320.98.
Acknowledgment
This work was supported partially by grants from National Nat-
ural Science Foundation of China (21371177, 81173108, 31171019,
31200820).
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.08.038.
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