Synthesis of novel flavonoid alkaloids as α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.07.055

A series of novel flavonoid alkaloids were synthesized with different flavonoids and attached nitrogen-containing moieties. These new compounds were screened for inhibitory activity of α-glucosidase, among which compound 23 was found to show the lowest IC₅₀ of 4.13 μM. Kinetic analysis indicates that the synthesized compounds 15 and 23 inhibit the enzyme in a non-competitive model with Ki value of 37.8 ± 0.8 μM and 13.2 ± 0.6 μM. Further docking studies suggest that the preferred binding pocket is close to the catalytic center, correlating to the experimental results. Structure activity relationship studies (SAR) indicate that 4′-hyroxyl group and the 4-position carbonyl group in the flavonoid structure are important for this biological activity. Addition of extra hydrogen bonding and hydrophobic groups on ring A would increase the inhibitory activity.

Full text of DOI:10.1016/j.bmc.2017.07.055

Accepted Manuscript
Synthesis of novel flavonoid alkaloids as α-glucosidase inhibitors
Jing Zhen, Yujie Dai, Tom Villani, Daniel Giurleo, James Simon, Qingli Wu
PII:
S0968-0896(17)30874-X
http://dx.doi.org/10.1016/j.bmc.2017.07.055
BMC 13892
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
22 May 2017
27 July 2017
28 July 2017
Please cite this article as: Zhen, J., Dai, Y., Villani, T., Giurleo, D., Simon, J., Wu, Q., Synthesis of novel flavonoid
alkaloids as α-glucosidase inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.07.055
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Synthesis of novel flavonoid alkaloids as α-glucosidase
inhibitors
Jing Zhen1,2, Yujie Dai3, Tom Villani1,2, Daniel Giurleo1,2, James Simon1,2 *, Qingli
Wu1,2 *
1 The New Use Agriculture and Natural Plant Products Program, Department of Plant
Biology, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901, USA
2 Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers
University, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
3 Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science
and Technology, Tianjin 300457, China
*Correspondence authors
Qingli Wu, email: qlwu@sebs.rutgers.edu; Tel: +1 848-932-6238
James E Simon, email: jimsimon@rutgers.edu; Tel: +1 848-932-6239
Address: Department of Plant Biology, Rutgers University, 59 Dudley Road, New
Brunswick, NJ 08901, USA
1

Abstract
A series of novel flavonoid alkaloids were synthesized with different flavonoids and
attached nitrogen-containing moieties. These new compounds were screened for
inhibitory activity of α-glucosidase, among which compound 23 was found to show the
lowest IC50 of 4.13 µM. Kinetic analysis indicates that the synthesized compounds 15 and
23 inhibit the enzyme in a non-competitive model with Ki value of 37.8 0.8 µM and
13.2 0.6 µM. Further docking studies suggest that the preferred binding pocket is close
to the catalytic center, correlating to the experimental results. Structure activity
relationship studies (SAR) indicate that 4’-hyroxyl group and the 4-position carbonyl
group in the flavonoid structure are important for this biological activity. Addition of
extra hydrogen bonding and hydrophobic groups on ring A would increase the inhibitory
activity.
Keywords: Flavonoid alkaloid; Flavonoid; α-Glucosidase inhibitor; Diabetes;
Molecular docking study; SAR
2

1. Introduction
Diabetes is one of the most common chronic diseases with estimated 387 million
patients worldwide and responsible for 4.9 million deaths in 2014 [1]. The number of
diabetes patients is projected to rise to more than 592 million in 2035 [2]. Diabetes is a
group of metabolic diseases characterized by high blood glucose levels. One of the
important therapeutic approaches is to inhibit digesting enzymes such as α-glucosidase,
or α-amylase in the intestine to slow down the digestion and absorption of the sugar and
to suppress post-prandial hyperglycemia [3]. α-Glucosidase hydrolyzes the 1-4 linked α-
glucose residue from the non-reducing side to release a single α-glucose, acting as the
final step in the digestion of dietary carbohydrates. This enzyme α-glucosidase has also
been associated with other diseases such as cancer and viral infection [4, 5]. Its inhibitors,
such as acarbose, miglitol and voglibose, which are carbohydrate mimetics or derivatives,
have already been marketed for the treatment of Type II diabetes mellitus [6]. These drug
molecules can decrease both postprandial hyperglycaemia and hyperinsulinaemia, yet
also have gastrointestinal adverse effects including diarrhea, flatulence and abdominal
discomfort, which limit a long-term compliance to therapy [7]. Developing more efficient
and safer inhibitors for hyperglycemia control in diabetes remains a global heath priority.
With the exception of carbohydrate mimetic inhibitors, flavonoid compounds such as
luteolin, naringenin, also have been reported to act as potential glucosidase inhibitors that
provide protective effects in diabetes therapy both in vitro and in vivo. [8, 9]. However,
their clinical applications for the treatment of diabetes are usually limited by their low
solubility and the lack of selectivity. The introduction of nitrogen moieties may increase
3

the solubility of parent flavonoid compounds, because of its formation of salt with the
addition of acids, such as hydrochloric or trifluoroacetic acid [10]. Structure
modifications on flavonoid compounds could then further increase their selectivity and
efficacy toward specific targets. For example, the introduction of phenylethenyl group at
the position 6 of naringenin greatly increases its inhibition toward cyclooxygenase-1
(COX-1) and shows much improved anticancer activity in vitro and in vivo (Figure 1)
[11]. The flavonoid derivatives containing a nitrogen moiety are referred to as flavonoid
alkaloids. and have for decades been reported to exhibit various biological activities
including anticancer, anti-virus, anti-inflammation and others [12]. The introduction of a
nitrogen moiety to the flavonoid,, such as with flavopiridol (Figure 1), derived from
flavonoid, inhibited cyclin-dependent kinases CDK1 and CDK2 and now serves as an
anti-cancer drug candidate in clinical trials [13]. The introduction of a nitrogen moiety
may also increase the binding affinity with target enzyme and result in lower IC50 values
with higher therapeutic efficacy. Therefore, in this study, we sought to synthesize novel
flavonoid alkaloids that would enhance α-glucosidase inhibitory activities. There are
limited studies that have reported on the structure activity relationship between the
flavonoid derivatives and α-glucosidase and the related inhibitory mechanism. As such,
in this study, we also conducted enzymatic kinetics study and molecular modeling studies
on selected representative compounds with high inhibitory activity and further
investigated their structure activity relationships, which may help drug molecule design
targeting α-glucosidase in the future.
4

Figure 1. The general structure of flavonoids used for derivative synthesis.
In this study, various nitrogen-containing moieties were attached to the four different
members of the flavonoid family, including flavone, flavanone, isoflavone and flavan
(Figure 1). The inhibitory activities of synthesized derivatives were tested on α-
glucosidase produced from yeast Saccharomyces cerevisiae, and the results indicated that
compounds 15 and 23 showed the best inhibitory activity with IC50 value of 23.43 and
4.13 µM, respectively. Their inhibitory kinetics and mechanism were explored with
various concentrations of compounds and substrates. The data analysis was conducted
using Lineweaver–Burk and Dixon plots. Results indicated that compounds 15 and 23
inhibit the enzyme in a non-competitive manner with Ki value of 37.8 0.8 and 13.2
0.6 µM, respectively. Molecular docking studies performed on Autodock vina showed the
chirality at position 2 has limited effect on the binding affinity and docking positions. In
5

the docking study, three locations were identified as the potential binding sites on the
surface of α-glucosidase. Among these three docking sites, the one with highest binding
affinity (location A in Figure 5) correlates very well with both the literatures and our
experimental data in terms of its non-competitive binding mechanism [14]. Binding
mechanism analysis at location A indicates that there are hydrogen bonds and
hydrophobic interactions between synthesized compounds and surrounding amino acid
residue. The aim of this study is to improve the inhibitory activities of flavonoid alkaloids
and explore their inhibitory mechanism, both of which would potentially help for the
future drug development targeting α-glucosidase for the treatment of diabetes.
2. Results and Discussion
2.1. Chemistry
For the synthesis of flavonoid alkaloids 1-8, cyclic imine ∆1-piperideines (1.25 eq),
was added into the flavonoids in solvent methanol and then the mixture was stirred at 80
°C for overnight. For flavonoid alkaloids 9-23, a 37% formaldehyde solution (1.25 eq)
and the corresponding secondary amine (1.25 eq) was added into the flavonoid methanol
solution. The mixture was then stirred and heated for hours until reaction was completed.
TLC was used to monitor the completion of reactions. Afterward, the solvent of each
reaction was separately removed and the residue was applied on silica gel
chromatography for purification using various solvent systems. Due to the very close
chemical properties between positions 6 and 8 of the flavonoid precursors, the site of
alkylation under Mannich reaction varies depending on the solvent, temperature, and
reaction time (Figure 2A). Flavonoids with different chemical properties have distinct
6

regioselectivity toward electrophilic substitution. Chen et al.,reported that if the reaction
is well performed under mild conditions with limited hours, high regioselectivity toward
the position 6 of naringenin was obtained [15]. Nguyen et al. reported that the use of
solvent system H2O /THF (2:1) at 40 °C would yield 99:1 selectivity between positions 6
and 8 [16]. The addition of a base may reverse the reaction and change the ratio of
formed products. Low yield was observed, as compounds 1-8, with the presence of
secondary amine functional group, are very likely to be absorbed on silica gel column
during purification. For the synthesized compounds presented in this study, the structure
1
of each compound was fully elucidated based on H NMR, 13C NMR and 2D NMR.
Substitution at positions 6 or 8 was determined based on published literature and/or the
proton NMR signals. For example, flavonoid chrysin has proton NMR peaks at 6.17 ppm
for H-6 and 6.40 ppm for H-8, in which H-6 is closer to the high field than H-8 with
1
about 0.2 ppm in DMSO-d6. In the H-NMR spectra of synthesized derivatives, for
example, the H-6 signal (5.78 ppm) of compound 5 is still around ~ 0.2 ppm closer to the
high field than the H-8 peak (5.98 ppm) of compound 4. This phenomenon was further
confirmed in our study by HMBC. It was observed there is correlation between H-6
between C-5, C-7 in compound 4 (Figure 2B).
7

A
B
Figure 2. Representative synthetic route of flavonoid alkaloids (A): (a) ∆1-piperideines,
methanol/THF, 80 °C (1-8) or secondary amine, 37% HCHO, methanol (9-23). Structures
of isomers 4, 5 and their HMBC correlation (B).
2.2 Biological activity
All synthesized compounds were screened against α-glucosidase enzyme from yeast
along with flavonoid compound naringenin, which is a known α-glucosidase inhibitor and
used as a positive control. In this study, the measured IC50 of naringenin is 77 μM and
correlates well to the value reported [9]. The concentration of compounds measured
against α-glucosidase range from 3.15 to 806 µM. Some compounds start from relatively
poor concentrations due to the lower solubility. The inhibitory potency of each compound
was evaluated by its IC50 value, which was computed out from the fitting to a sigmoidal
dose-response curve with variable slope. At least three independent experiments were
performed to determine the IC50 values for each compound. Their IC50 values, the
8

indicator of the inhibition strength against α-glucosidase, are summarized in Table 1.
Among these screened compounds, 9, 15, 20 and 23 exhibited strong inhibition against α-
glucosidase with IC50 values of 82.76, 23.43, 65.11 and 4.13 µM, respectively. Their dose-
response curves are shown in Figure 3.
Table 1. Synthesized flavonoid alkaloids and their inhibitory activities
Substituted Groups
Inhibitory activity
(IC50 in µM)
249.4
NO.
Flavonoid Moiety
Alkaloid Moiety
Position
R1
OH
R2
H
R3
H
1
2
6
6
6
6
8
6
6
8
6
6
8
I
OCH3
OH
OH
OH
H
> 500
3
OH
> 500
4
> 500
H
H
-
5
II
> 500
6
OH
-
H
-
H
-
293.3
7
> 500
IV
8
-
-
-
> 500
9
I
OH
H
H
H
-
H
-
82.8
10
11
II
> 500
III
H
-
> 500
12
II
6
H
H
-
217.0
13
14
15
16
17
18
19
20
21
22
I
II
I
6
OH
H
H
H
H
H
H
H
-
202.5
> 500
23.4
6
6
OH
H
H
-
II
I
6
> 500
521.0
> 500
> 500
65.1
6
OH
H
6
II
I
H
H
-
8
6
OH
H
H
H
H
H
-
6
> 500
> 500
II
6 & 8
H
-
23
I
6
OH
H
H
4.1
Note: The general structure of flavonoid moiety is included in Figure 1.
9

Figure 3. α-Glucosidase inhibitory activity of compounds 9, 15, 20 and 23.
The enzyme kinetic assays were performed on compounds 15 and 23 to investigate
their mode of inhibition toward α-glucosidase. In the kinetic studies, a range of
concentrations of test compounds and substrates were used. The data was analyzed using
Lineweaver-Burk and Dixon plots (Figure 4) [17]. The Lineweaver-Burk plot was drawn
by plotting inverse velocity (1/V) as a function of the inverse substrate concentration
(1/[S]), while the Dixon plot was created by plotting inverse velocity (1/V) as a function
of the compound concentration ([I]). Results showed that both compound 15 and 23 are
very close to the non-competitive model inhibitor against α-glucosidase. Their Ki value
shown from Dixon plot, are 37.8 0.8 and 13.2 0.6 µM, respectively.
10

Figure 4. Lineweaver-Burk and Dixon plots of compounds 15 and 23 toward the
inhibition of α-glucosidase. Lineweaver-Burk plot of compound 15 (A); Dixon plot of
compound 15 (B); Lineweaver-Burk plot of compound 23 (C); Dixon plot of compound
23 (D).
2.3 Molecular Modeling
To envisage the binding model between synthesized inhibitor and α-glucosidase, a
molecular docking study was performed. After geometric optimization, both enantiomers
of compounds 15 and 23 were docked into the entire protein molecule. There are mainly
three different docking sites, in which site A has the best correlation with non-
competitive features and highest binding affinity [14]. The other two positions B and C
11

have comparatively less binding affinity and are very close to the glucose ligand binding
pocket, contradicting the non-competitive mode. From the preliminary docking results, it
was observed that the R or S isomers share almost the same binding sites on the protein
through random screening. Their positions are very close when bound with the protein at
the binding site A, suggesting the chirality at position 2 has limited impact on docking
results. Even if site A is not directly responsible for the hydrolysis of carbohydrates, after
binding with small molecules, it could alter protein conformation and interfere indirectly
with the catalytic function (Figure 5). Secondary docking was performed on site A within
smaller area and yielded refined results for binding analysis. The binding site A close to
the catalytic center is formed by amino acid residues ARG 263, ARG 270, HIS 195, GLY
169, ASN 259, TRP 15, LYS 16, LEU 297, SER 298, TRP 343 (Figure 6). There are two
arginine amino acids, ARG 263, ARG 270 at the entrance of the pocket, which are
positively charged at experimental pH 6.8 and may interact with the 4’-hydroxyl group
on flavonoid ring C. The hydrophobic pocket end, formed by two tryptophan amino acids
TRP 15 and TRP 343, is likely to interact with the hydrophobic groups, such as the
tetrahydroisoquinoline group of compound 15 and the ethyl benzoate group of compound
23. Three hydrogen bonds were observed for compound 15: the carbonyl group of
position 4 and the amine on the imidazole of HIS 295 (2.67 Å), phenol group at position
5 and the carbonyl group of ASN 259 (3.08 Å), phenol group at position 7 and the
carboxylic acid group of SER 298 (2.79 Å). For compound 23, there are two hydrogen
bonds: phenol of position 7 and the amine moiety of HIS 195 (3.20 Å), the carbonyl
group of ester group on alkaloid moiety and the amine moiety of LYS 16 (3.02 Å).
12

E
S
I
EI
S
ES
I
ESI
E: Enzyme
E
P
S: Substrate
I: Inhibitor
P: Product
A
B
Figure 5. The discovered binding site A between synthesized compounds 15, 23 and α-
glucosidase (A); the scheme of non-competitive inhibition (B).
Figure 6. The binding model between compounds 15 and 23 and α-glucosidase at
binding site A.
13

2.4 Structure Activity Relationship
In this study, most of the analogues are synthesized from the flavanone naringenin and
the flavone chrysin. Comparing compounds 1 and 2 to that of compounds 4 and 6, it was
expected to find that the hydroxyl group attached at ring position 4’ on ring B is very
essential for inhibitory activity. However, excessive hydroxyl groups at position 3 and 3’
could also decrease inhibitory affinity, as suggested by compound 3. The importance of
4’-hydroxyl group was also suggested by the docking results, in which there may be an
interaction with positively charged arginine groups (ARG 263, ARG 270) at the entrance
of the binding pocket. Most chrysin derivatives without 4’-hydroxyl group have no
inhibitory activities. The flavan derivatives didn’t show activities less than 500 μM,
underlying the importance of C=O at position 4. Further study on more flavan derivatives
are needed to confirm this observation. The introduction of tertiary amine appears to
provide improved inhibitory activity than the secondary amine group as suggested from
the comparison between compound 1 and compound 9. Attachment of hydrophobic
groups at flavonoid ring A yields better activity as compound 15 is more potent than 18;
23 is more potent than 21. The addition of an extra hydrogen bond acceptor at the side
end of the attached moiety may increase the binding with α-glucosidase through
hydrogen bonding. For example, compound 23 has a hydrogen bonding with LYS 16
through the carbonyl group of ester moiety. The proposed structure activity relationships
are illustrated in Figure 7.
14

Figure 7. The proposed structure activity relationships
3. Conclusions
In our study, a collection of flavonoid derivatives was synthesized through Mannich
reaction targeting for the inhibition of α-glucosidase. Most of the compounds synthesized
from flavanone naringenin exhibited potent bioactivities with IC50 values below 500 µM,
in which the most potent compound 23 has IC50 value of 4.13 µM. Enzyme kinetic
investigation suggests the synthesized compounds inhibit enzyme in the non-competitive
manner with Ki value of 37.8
0.8 and 13.2 0.6 µM for compound 15 and 23,
respectively. The molecular docking study suggested synthesized derivatives might bind
to a pocket close the catalytic center and interfere the catalysis process indirectly. The
binding model between α-glucosidase and compounds at location A were carefully
analyzed, in which several hydrogen bonds and hydrophobic interactions were revealed.
Based on tested activities of compounds and SAR, it was found that the hydroxyl group
at position 4’ is essential to exhibit biological activity, and that the introduction of
15

hydrophobic groups and hydrogen acceptors at flavonoid ring A would potentially
increase the binding affinity and yield higher inhibitory activity.
4. Experimental
4.1. General
1H and 13C NMR spectra were performed on Bruker Avance 400 MHz spectrometer
(Billerica, MA). Analytical LC-MS was performed on Hewlett-Packard Agilent 1100
series HPLC-MSD (Agilent Technologies, Waldbronn, Germany) equipped with a
quaternary pump system, a degasser, an auto-sampler, a DAD detector, a MSD trap with
electrospray ion source (ESI). Column chromatography was performed using silica gel
(230 - 400 mesh; Selecto Scientific, Suwanee, GA). All synthetic materials including the
solvents and enzyme α-glucosidase are all purchased from Sigma Aldrich (St. Louis,
MO).
4.2 In vitro α-glucosidase assay
The assay was performed based on a chromogenic reported method with slight
modifications [18]. In brief, 10 µl of enzyme solution (1 U/ml) was diluted with 120 µl of
0.1 M phosphate buffer (pH 6.8). Then 5 µl of the compound solution prepared in DMSO
with various concentrations were added into the enzyme solution. The final concentration
of DMSO is around 2%. The mixture was then incubated at 37°C for 15 min. Afterward
20 µl of 5 mM substrate p-nitrophenyl- α-D-glucopyranoside in phosphate buffer (pH
6.8) were added and incubated for additional 30 min at 37°C. The reaction was quenched
using 80 µl of 0.2 M Na2CO3. Absorption was subsequently measured using UV
16

spectrometer at wavelength of 405 nm. The reaction without enzyme was treated as
blank and each experiment was triplicated. The inhibitory activity was calculated using
the following equation:
Inhibition % = [(control absorption – sample absorption) / control absorption] × 100 %
The IC50 value of each sample was calculated from the fitting of sigmoidal dose-response
curve with variable slope.
4.3. Enzymatic kinetics of α-glucosidase inhibition
Mode of inhibition of synthesized flavonoid alkaloids against yeast α-glucosidase
activity was measured with different concentrations of pNPG (0.625, 1.25, 2.5, 5.0 mM).
Control group has no compound added. Mode of inhibition of each tested compound was
determined by Lineweaver-Burk plot analysis. The Dixon plots were applied to determine
the inhibitory constants with the following equation, where V is the reaction velocity, S is
substrate concentration, Vmax is the maximum enzyme velocity, Km is Michaelis-Menten
constant, I is inhibitor concentration, Ki is inhibition constant and α is the constant that
determines mechanism. In this study, for non-competitive inhibitor, α = 1. If α is very
large, then the mode approaches a competitive model. If α is very small but greater than
zero, the mode is more closer to uncompetitive model [14].
ꢀꢁꢂꢃ
ꢀ =
ꢆ
ꢆ
ꢄꢁꢂ ꢅ1 + ꢈ + ꢃꢂ(1 +ꢂ
)
ꢄꢇ
ꢉꢄꢇ
17

4.4. Molecular Docking
Molecular docking has been commonly used in medicinal chemistry research [19]. The
crystallographic structure of -glucosidase (PDB: 3A4A) was downloaded from the
Protein Data Bank (http://www.rcsb.org/pdb/). All polar hydrogen atoms and charges
were assigned to the receptor using AutoDockTools 1.5.4. Compounds used for docking
study were built using the molecular builder function in MOE 2010.11
(http://www.chemcomp.com/), the energy of each compound was minimized to its local
minima using MMF94X force field to a constant value of 0.05 kcal/mol. Docking
simulation and binding pocket prediction was performed using AutoDock Vina
(http://vina.scripps.edu/) [20]. The protein was held rigid in the docking process. The
inhibitors were allowed to flexible. The initial grid box size was 62 Å× 78 Å×1 72Å in
the x, y, z dimensions. The grid box center was put on x = 23.8, y = -3.834 and z =
19.691 with the protein positioned at the center of the box. The docking method would be
validated using its competitive inhibitor acarbose and glucosidase. The resulting docked
poses were analyzed with AutoDockTools using cluster analysis, PyMOL
(https://www.pymol.org) [21]. Using the established method, control compound acarbose
was docked into the catalytic binding pocket, which correlates well with published
literature [22] and its competitive inhibition mechanism against α-glucosidase.
4.5. Chemical synthesis
4.5.1 General procedure for synthesis of compound 1-8
The flavonoid compound (1 mmol, 1.0 eq) was dissolved in methanol or methanol/
tetrahydrofuran followed by the addition of ∆1-piperideines (124 mg, 1.25 mmol, 1.25
eq). The mixture was then stirred at 80 °C under nitrogen atmosphere for 5 hours. After
18

the reaction, the solvent was removed under reduced pressure and the residue was
purified on silica gel column using dichloromethane/ methanol in gradient to obtain the
desired compound.
4.5.1.1 5, 7-dihydroxy-2-(4-hydroxyphenyl)-6-(piperidin-2-yl)chroman-4-one (1)
Yield: 78 %. White powder. ESI-MS: m/z 356 [M+H]+, 1H NMR (400 MHz, DMSO-d6):
δ 1.50-1.90 (6H, m, H- 4’’, 5’’, 6’’), 2.47 (1H, m, H-3), 2.75 (1H, m, H-3’’), 2.98 (1H, m,
H-3), 3.29 (1H, m, H-3’’), 4.16 (1H, m, H-1’’), 5.20 (1H, m, H-2), 5.33 (1H, s, H-8), 6.78
13
(2H, H-3’, 5’), 7.28 (2H, H-2’, 6’); C NMR (100 MHz, DMSO-d6): δ 22.44, 22.54 (C-
4’’, 5’’), 28.15 (C-6’’), 41.86 (C-3), 43.52 (C-3’’), 52.05 (C-1’’), 77.48 (C-2), 96.91 (C-
10), 98.01 (C-8), 104.58 (C-6), 115.05 (C-2’, 6’), 128.01 (C-3’, 5’), 129.75 (C-1’),
157.40, 160.01, 161.56, 177.67 (C-4’, 5, 7, 9), 191.51 (C-4, C=O).
4.5.1.2 5, 7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-6-(piperidin-2-yl)chroman-4-one
(2)
1
Yield 60%. White solid. ESI-MS: m/z 386 [M+H]+; H NMR (400 MHz, DMSO-d6): δ
1.50-1.90 (6H, m, H- 4’’, 5’’, 6’’), 2.54 (1H, m, H-3), 3.27 (1H, m, H-3’’), 2.92 (1H, m,
H-3), 3.27 (1H, m, H-3’’), 3.76 (1H, s, -OCH3), 4.16 (1H, m, H-1’’), 5.18 (1H, m, H-2),
5.35 (1H, s, H-8), 6.84 (2H, H-5’), 6.91 (2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6):
δ 22.45, 22.54 (C-4’’, 5’’), 28.2 (C-6’’), 41.9 (C-3), 43.5 (C-3’’), 52.0 (C-1’’), 55.7 (-
OCH3), 77.3 (C-2), 96.9 (C-8), 98.5 (C-10), 104.6 (C-6), 112.0 (C-5’), 113.9 (C-2’),
117.3 (C-6’), 132.1 (C-1’), 146.5, 147.6 (C-3’, 4’), 161.5, 160.0, 177.7 (C-5, 7, 9), 191.4
(C-4, C=O).
19

4.5.1.3 2-(3, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-6-(piperidin-2-yl)chroman-4-one (3)
1
Yield: 25 %. Yellow solid. ESI-MS: m/z 388 [M+H]+; H NMR (400 MHz, DMSO-d6)
(mixture of isomers): δ 1.53-1.70 (6H, m, H-4’’, 5’’, 6’’), 2.79 (1H, m, H-3’’), 3.30 (1H,
m, H-3’’), 4.21 (1H, m, H-1’’), 4.27 (1H, d, J = 10 Hz, H-3), 4.75 (1H, d, J = 10 Hz, H-2),
5.40 (1H, s, H-8), 6.84 (1H, m, H-5’), 6.70 (2H, m, H-2’, 6’); 13C NMR (100 MHz,
DMSO-d6): δ 22.41, 27.80, 29.48 (C-4’’, 5’’, 6’’), 43.78 (C-3’’), 51.90 (C-1’’), 82.42 (C-
2), 104.59 (C-6), 97.80 (C-10), 95.95 (C-8), 119.10 (C-2’, 6’), 115.10 (C-5’), 145.51,
160.78 (C-3’, C-4’), 159.98, 161.43 (C-5, 9), 161.32 (C-7), 192.6 (C-4).
4.5.1.4 5,7-dihydroxy-2-phenyl-6-(piperidin-2-yl)-4H-chromen-4-one (4)
Yield: 26 % (together with 14% compound 5). Pale yellow solid. ESI-MS: m/z 338
[M+H]+; 1H NMR (400 MHz, DMSO-d6): δ 1.55-1.85 (6H, m, H-4’’, 5’’, 6’’), 2.75 (1H,
m, H-3’’), 3.31 (1H, m, H-3’’), 4.26 (1H, m, H-1’’), 5.98 (1H, s, H-8), 6.71 (1H, s, H-3),
13
7.55 (3H, m, H-3’, 4’, 5’), 7.99 (2H, m, H-2’, 6’); C NMR (100 MHz, DMSO-d6): δ
22.52, 22.80, 28.12 (C-4’’, 5’’, 6’’), 43.49 (C-3’’), 52.01 (C-1’’), 95.90 (C-8), 99.34 (C-
10), 108.67 (C-8), 125.00 (C-2’, 6’), 130.00 (C-3’, 5’), 131.37 (C-4’), 157.46, 157.28,
161.26 (C-5, 9, 2), 173.88 (C-7), 180.14 (C-4).
4.5.1.5 5, 7-dihydroxy-2-phenyl-8-(piperidin-2-yl)-4H-chromen-4-one (5)
Yield: 14 % (together with 26% compound 4). Yellow solid. ESI-MS: m/z 338 [M+H]+;
1H NMR (400 MHz, DMSO-d6): δ 1.55-1.83 (6H, m, H-4’’, 5’’, 6’’), 2.89 (1H, m, H-3’’),
3.31 (1H, m, H-3’’), 4.48 (1H, m, H-1’’), 5.78 (1H, s, H-6), 6.78 (1H, s, H-3), 7.59 (3H,
m, H-3’, 4’, 5’), 8.00 (2H, m, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6): δ 22.77, 22.98,
28.93 (C-4’’, 5’’, 6’’), 43.58 (C-3’’), 52.69 (C-1’’), 101.57 (C-6), 103.77 (C-10), 104.70
20

(C-8), 125.95 (C-2’, 6’), 129.20 (C-3’, 5’), 131.48 (C-4’), 153.63, 160.67, 161.05 (C-5, 9,
2), 173.14 (C-7), 180.27 (C-4).
4.5.1.6 5, 7-dihydroxy-2-(4-hydroxyphenyl)-6-(piperidin-2-yl)-4H-chromen-4-one (6)
1
Yield: 38 %. Yellow solid. ESI-MS: m/z 354 [M+H]+; H NMR (400 MHz, DMSO-d6):
δ 1.53-1.78 (6H, m, H-4’’, 5’’, 6’’), 2.80 (1H, m, H-3’’), 3.30 (1H, m, H-3’’), 4.25 (1H,
m, H-1’’), 6.07 (1H, s, H-8), 6.58 (1H, s, H-3), 6.89 (2H, m, H-3’, 5’), 7.83 (2H, m, H-2’,
13
6’); C NMR (100 MHz, DMSO-d6): δ 22.61, 22.86, 28.21 (C-4’’, 5’’, 6’’), 43.80 (C-
3’’), 51.95 (C-1’’), 102.11 (C-6), 99.74 (C-10), 95.45 (C-8), 127.98 (C-2’, 6’), 115.86 (C-
3’, 5’), 160.78 (C-4’), 157.43, 157.16, 162.28 (C-5, 9, 2), 171.87 (C-7), 180.56 (C-4).
4.5.1.7 2-(4-hydroxyphenyl)-6-(piperidin-2-yl)chromane-5, 7-diol (7)
Yield: 25 %. White solid. Compounds 7, 8 would be gradually oxidized to purple
1
compounds when exposed to the air. H NMR (400 MHz, DMSO-d6): δ 1.60 (2H, m, H-
5’’ and H-4’’), 1.88 (4H, m, H-3, 4’’, 5’’, 6’’), 2.09 (2H m, H-3, 6’’), 2.62 (2H, m, H-4),
2.90 (1H, m, H-3’’), 3.30 (1H, d, H-3’’), 4.42 (1H, m, H-1’’), 4.88 (1H, m, H-2), 6.03
(1H, d, H-8), 6.80 (2H, d, H-2’, 6’), 7.20 (2H, d, H-3’, 5’); 13C NMR (100 MHz, DMSO-
d6) δ 19.58 (C-4), 21.8, 22.75 (C-4’’, 5’’) 28.56 (C-6’’), 28.54 (C-3), 45.40 (C-3’’) 57.65
(C-1’’), 76.30 (C-2), 95.35 (C-6), 101.78 (C-10), 104.85 (C-8), 114.98 (C-2’, 6’), 127.20
(C-3’, 5’), 157.50, 155.53, 154.10, 153.90 (C-5, 7, 9, 4’).
4.5.1.8 2-(4-hydroxyphenyl)-8-(piperidin-2-yl)chromane-5, 7-diol (8)
1
Yield: 25 %. White solid. H NMR (400 MHz, DMSO-d6): δ 1.60 (2H, m, H-5’’ and H-
4’’), 1.88 (4H, m, H-3, 4’’, 5’’, 6’’), 2.09 (2H m, H-3, 6’’), 2.62 (2H, m, H-4), 2.90 (1H,
21

m, H-3’’), 3.00 (1H, m, N-H), 4.32 (1H, m, H-1’’), 5.00 (1H, m, H-2), 6.25 (1H, d, H-6),
13
6.80 (2H, d, H-2’, 6’), 7.20 (2H, d, H-3’, 5’), C NMR (100 MHz, DMSO-d6) δ 19.58
(C-4), 21.8, 22.75 (C-4’’, 5’’) 28.56 (C-6’’), 28.54 (C-3), 45.40 (C-3’’) 57.65 (C-1’’),
76.30 (C-2), 95.35 (C-6), 101.78 (C-10), 104.80 (C-6), 114.98 (C-2’, 6’), 127.20 (C-3’,
5’), 157.50, 155.53, 154.10, 153.90 (C-5, 7, 9, 4’).
4.5.2 General synthetic procedure of compound (9-23)
The flavonoid compound (1.0 mmol, 1.0 eq) was dissolved in methanol followed by the
addition of 37% formaldehyde solution (97 µl, 1.2 mmol, 1.2 eq) and the corresponding
amine (1.2 mmol, 1.2 eq). The mixture was then stirred at 65°C for various hours. After
the reaction, the solvent was removed under reduced pressure and the residue was
purified on silica gel column using dichloromethane/ methanol in gradient to obtain the
desired compound.
4.5.2.1 5, 7-dihydroxy-2-(4-hydroxyphenyl)-6-(piperidin-1-ylmethyl)chroman-4-one (9)
Yield: 65 %. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 1.29 (2H, H-5’’), 1.40 (4H,
H-4’’, 6’’), 2.44 (1H, H-3, dd, J = 2.8, 17 Hz), 2.49 (4H, H-3’’, 7’’), 3.01 (1H, H-3, dd, J
= 12.6, 17 Hz), 5.17 (1H, H-2, dd, J = 2.8, 12.6 Hz), 5.50 (1H, H-8), 6.62 (2H, H-3’, 5’),
7.13 (2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6): δ 22.77 (C-5’’), 24.50 (C-4’’, 6’’),
41.83 (C-3), 52.27 (C-1’’), 52.32 (C-3’’, 7’’), 78.07 (C-2), 95.86 (C-8), 99.34 (C-10),
99.80 (C-6), 115.09 (C-3’), 128.20 (C-2’), 129.14 (C-1’), 157.60 (C-4’), 160.99, 161.89,
171.60 (C-9, 5, 7), 194.79 (C-4).
4.5.2.2 5, 7-dihydroxy-2-phenyl-6-(piperidin-1-ylmethyl)-4H-chromen-4-one (10)
22

Yield: 21 %. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 1.47 (2H, H-5’’), 1.60 (4H,
H-4’’, 6’’), 2.70 (4H, H-3’’, 7’’), 3.86 (2H, H-1’’), 6.41 (1H, H-8), 6.92 (1H, H-2), 7.57-
7.59 (3H, H-3’, 4’, 5’), 8.05 (2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6): δ 22.82 (C-
5’’), 24.55 (C-4’’, 6’’), 48.54 (C-3’’, 7’’), 52.51 (C-1’’), 94.46 (C-10), 94.85 (C-8),
105.01 (C-6), 105.22 (C-3) 126.26 (C-2’, 6’), 129.11 (C-3’, 5’), 131.28 (C-4’), 162.04
(C-2), 181.67 (C-4).
4.5.2.3 7-hydroxy-3-(4-methoxyphenyl)-8-(piperidin-1-ylmethyl)-4H-chromen-4-one (11)
Yield: 51 %. Pale yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 1.46 (2H, H-5’’), 1.57
(4H, H-4’’, 6’’), 2.58 (4H, H3’’, 7’’), 3.78 (3H, -OCH3), 3.90 (2H, H-1’’) 6.83 (1H, H-6),
13
7.0 (2H, H-3’, 5’), 7.5 (2H, H-2’, 6’), 7.88 (1H, H-5), 8.35 (1H, H-2); C NMR (100
MHz, DMSO-d6): δ 23.24 (C-5’’), 25.26 (C-4’’, 6’’), 52.97 (C-1’’), 53.15 (C-3’’, 7’’),
55.11 (-OCH3), 107.86 (C-8), 113.67 (C-3’, 5’), 115.31 (C-6), 115.92 (C-10), 122.93 (C-
1’), 124.22 (C-3), 125.56 (C-5), 130.05 (C-2’, 6’), 152.68 (C-2), 155.01 (C-9), 158.92 (C-
4’), 163.99 (C-7), 174.64 (C-4).
4.5.2.4 6-((Diethylamino)methyl)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one (12)
1
Yield: 45 % (together with 6, 8 di-substituted side product). Yellow solid. H NMR (400
MHz, DMSO-d6): δ 1.27 (3H, -CH3), 2.61 (4H, H-3’’, 7’’), 3.5 (4H, H-4’’, 6’’), 4.01 (2H,
H-1’’), 4.14 (2H, -OCH2-), 6.30 (1H, H-8), 6.64 (1H, H-3), 7.53 (3H, H-3’, 4’, 5’), 7.80
(2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6): δ 14.77 (-CH3), 43.52 (C-4’’, 6’’), 52.55
(C-1’’), 53.97 (C-3’’, 7’’), 61.82 (-OCH2-), 98.23 (C-8), 100.45 (C-10), 105.03 (C-3),
106.09 (C-6), 126.21, 129.35, 131.66, 131.96 (C-2’, 3’, 4’, 1’), 154.94 (C-9), 155.36 (-
OC=O), 161.94, 163.29, 165.38 (C-2, 5, 7), 182.51 (-C=O).
23

4.5.2.5 5, 7-dihydroxy-2-(4-hydroxyphenyl)-6-(morpholinomethyl)chroman-4-one (13)
1
Yield: 76 %. White solid. H NMR (400 MHz, MeOD-d4): δ 2.59-2.64 (1H, H-3, dd, J =
17.16, 3.1 Hz), 2.72 (4H, H-3’’, 7’’, m), 2.96- 3.04 (1H, H-3, dd, J = 12.68 17.16 Hz),
3.65-3.68 (4H, H-4’’, 5’’, m), 3.79 (2H, H-1’’, s), 5.21-5.25 (1H, H-2, dd, J = 3.1, 12.68
Hz), 6.71 (2H, H-3’, 5’, d, J = 8.6 Hz), 7.19 (2H, H-2’, 6’, d, J = 8.6 Hz), 13C NMR (100
MHz, MeOD-d4): δ 43.86 (C-3), 52.71 (C-1’’), 53.63 (C-3’’, 7’’), 66.90 (C-4’’, 6’’),
80.44 (C-2), 96.73 (C-8), 100.79 (C-10), 102.59 (C-6), 116.38, 116.47 (C-3’, 5’), 129.02
(C-2’ 6’), 131.04 (C-1’), 159.07 (C-4’), 163.43, 164.54 (C-5, 9), 170.07 (C-7), 197.59 (C-
4).
4.5.2.6 5, 7-dihydroxy-6-(morpholinomethyl)-2-phenyl-4H-chromen-4-one (14)
Yield: 46 %. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 2.68 (4H, H-4’’, 6’’), 3.80
(4H, H-3’’, 7’’), 4.01 (2H, H-1’’), 6.31 (1H, H-8), 6.65 (1H, H-3), 7.56 (3H, m, H-3’, 4’
5’), 7.83 (2H, H-2’); 13C NMR (100 MHz, DMSO-d6): δ 53.02 (C-1’’), 54.17 (C-3’’, 7’’),
66.66 (C-4’’, 6’’), 98.00 (C-8), 100.31 (C-3), 104.89 (C-10), 105.97 (C-6), 126.11 (C-2’),
129.23 (C-3’), 131.58 (C-4’), 131.82 (C-1’), 154.89 (C-9), 161.80 (C-2), 163.17 (C-5),
165.35 (C-7), 182.40 (C-4).
4.5.2.7 6-((3, 4-dihydroisoquinolin-2(1H)-yl)methyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)
chroman-4-one (15)
1
Yield: 52 %. White solid. H NMR (400 MHz, MeOD-d4): δ 2.76 (1H, H-3, dd, J = 2.8,
17.2 Hz), 2.97 (4H, -NCH2CH2Ar-, br), 3.08 (1H, H-3, dd, J = 12.8, 17.2 Hz), 3.84 (2H, -
NCH2Ar, br), 3.97 (2H, H-1’’, s), 5.32 (1H, H-2, dd, J = 2.8, 12.8 Hz), 5.97 (1H, H-8, s),
24

6.85 (2H, H-2’, 6’, d, J = 8.8 Hz) 7.00- 7.20 (4H, m, Ar-H), 7.30 (2H, H-3’, 5’, d, J = 8.8
Hz); 13C NMR (100 MHz, MeOD-d4): 28.41 (-CH2-Ar), 43.22 (C-3), 49.96, 52.95, 55.18
(3 × -N-CH2-), 78.78 (C-1), 96.29 (C-8), 100.22 (C-10), 101.89 (C-6), 115.67 (C-3’, 5’),
127.89 (C-2’, 6’), 126.19, 126.62, 126.87, 128.74 (4 × Ar-H), 130.43, 132.62, 133.15 (C-
2 and 2 × Ar-C), 156.47, 161.30, 162.46 (C-5, 9, 4’), 169.27 (C-7), 195.79 (C-4).
4.5.2.8 6-((3, 4-dihydroisoquinolin-2(1H)-yl)methyl)-5, 7-dihydroxy-2-phenyl-4H-
chromen-4-one (16)
1
Yield: 12 % (6, 8 di-substituted product formed). Yellow solid. H NMR (400 MHz,
CDCl3): δ 3.01 (4H, -Ar-CH2-CH2-), 3.89, 4.18 (4H, 2 × -N-CH2-Ar), 6.32 (1H, s, H-8),
6.65 (1H, s, H-2), 7.04-7.19 (4H, m, -Ar-H), 7.50 (3H, m, -Ar-H), 7.85 (2H, m, -Ar-H);
13C NMR (400 MHz, CDCl3): 28.62 (Ar-CH2-), 50.38, 53.56, 55.90 (3 × -N-CH2-), 94.95
(C-8), 100.65 (C-10), 105.00, 106.15 (C-3, 6), 126.32, 126/44, 126.48, 126.86, 127.03,
127.13, 128.97, 129.28, 129.42 (-ArH), 131.98, 132.88, 133.41 (C-1’’ and –Ar-C),
155.04, 161.95, 163.32, 166.16 (C-2, 5, 7, 9), 182.64 (-C=O).
4.5.2.9 5, 7-dihydroxy-6-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-(4-hydroxyphenyl)
chroman-4-one (17)
Yield: 64 %. White solid. 1H NMR (400 MHz, MeOD-d4): δ 2.56- 2.59 (2H, -NCH2-, t, J
= 5.8 Hz), 2.66-2.71 (5H, -N-CH2-CH2-N- and H-3, br), 2.90 (4H, -N-CH2-CH2-N-, br),
3.03 -3.10 (1H, H-3, dd, J = 12.6, 17.1 Hz), 3.67-3.70 (2H, -CH2-OH, t, J = 5.8 Hz), 3.92
(2H, H-1’’, s), 5.29 (1H, H-2, dd, J = 3.1, 12.6 Hz), 5.82 (1H, H-8, s), 6.82-6.84 (2H, H -
13
3’, 5’, d, J = 8.56 Hz), 7.30-7.32 (2H, H-2’, 6’, d, J = 8.56 Hz); C NMR (100 MHz,
MeOD-d4): 43.84 (C-3), 52.65 (C-1’’), 52.84 and 53.22 (-N-CH2-CH2-N-), 59.89 (-N-
25

CH2-), 60.79 (-CH2-OH), 80.19 (C-2), 97.73 (C-8), 100.94 (C-10), 101.63 (C-6), 116.35
(C-3’, 5’), 128.98 (C-2’, 6’), 131.31 (C-1’), 158.97, 163.50, 164.43 (C-5, 9, 4’), 173.55
(C-7), 196.70 (C-4).
4.5.2.10
5,
7-dihydroxy-6-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-phenyl-4H-
chromen-4-one (18)
Yield: 45 %. Yellow solid. 1H NMR (400 MHz, CDCl3): δ 2.57-2.61 (10H, 5 × -CH2-N-),
3.64 (2H, -OCH2-), 3.88 (2H, Ar-CH2-N-), 6.41 (1H, H-8), 6.63 (1H, H-3), 7.52 (3H, H-
3’, 4’, 5’), 7.87 (2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-d6): δ 52.58, 53.27 (-
NCH2CH2N-), 53.88 (C-1’’), 57.98 (-OCH2-), 59.26 (-CH2-N-), 94.69 (C-8), 103.58 (C-
10), 104.42 (C-6), 105.69 (C-3), 126.38 (C-2’, 6’), 129.17 (C-3’, 5’), 131.57 (C-4’),
131.82 (C-1’), 157.36 (C-9), 159.35 (C-5), 163.82 (C-2), 165.97 (C-7), 182.53 (C=O).
4.5.2.11
5,
7-dihydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2-phenyl-4H-
chromen-4-one (19)
Yield: 55 %. Yellow solid. 1H NMR (400 MHz, CDCl3): δ 2.589-2.62 (10H, 5 × -CH2-N-
), 3.64 (2H, -OCH2-), 4.02 (2H, Ar-CH2-N-), 6.30 (1H, H-6), 6.64 (1H, H-3), 7.56 (3H,
H-3’, 4’, 5’), 7.82 (2H, H-2’, 6’); 13C NMR (100 MHz, CDCl3): δ 52.87, 52.68 (-
NCH2CH2N-), 53.88 (C-1’’), 57.97 (-OCH2-), 59.17 (-CH2-N-), 98.46, 100.43, 104.94,
106.05 (C-6, 10, 8, 3), 126.23 (C-2’, 6’), 129.35 (C-3’, 5’), 131.73 (C-4’), 131.95 (C-1’),
154.91 (C-9), 161.83 (C-5), 163.27 (C-2), 165.74 (C-7), 182.54 (C=O).
4.5.2.12 Ethyl-4-((5, 7-dihydroxy-23-(4-hydroxyphenyl)-4-oxochroman-6-yl)methyl)
piperazine-1-carboxylate (20)
26

1
Yield: 64 %. White solid. H NMR (400 MHz, MeOD-d4): δ 1.13-1.18 (3H, m, -CH3),
2.52 (4H, br, -N-CH2-CH2-N-), 2.58-2.63 (1H, H-3, dd, J = 3.0, 17.1 Hz), 2.96- 3.04 (1H,
H-3, dd, J = 12.7, 17.1 Hz), 3.44 (4H, br, -N-CH2-CH2-N-), 3.67 (2H, s, H-1’’), 4.02 (2H,
m, -OCH2-) 5.21-5.28 (1H, H-2, dd, J = 3.0, 12.7 Hz), 5.80 (1H, H-8, s), 6.70-6.72 (2H,
13
H-3’, 5’, d, J = 8.6 Hz), 7.19-7.21 (2H, H-2’, 6’, d, J = 8.6 Hz); C NMR (100 MHz,
MeOD-d4): 14.89 (-CH3), 43.95 (C-3), 44.34 (-N-CH2-CH2-N-), 52.51 (C-1’’), 53.12 (-N-
CH2-CH2-N-), 62.89 (-OCH2-), 80.44 (C-2), 96.61 (C-8), 101.96 (C-6), 116.36 (C-3’, 5’),
129.02 (C-2’, 6’), 131.19 (C-1’’), 157.5, 159.05, 163.15, 164.20 (C-5, 7, 9, 4’), 169.65 (-
N-C=O), 197.74 (-C=O).
4.5.2.13 Ethyl-4-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-6-yl)methyl)piperazine-1-
carboxylate (21)
Yield: 50 %. Yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 1.27 (3H, -CH3), 2.61 (4H,
H-3’’, 7’’), 3.5 (4H, H-4’’, 6’’), 4.01 (2H, H-1’’), 4.14 (2H, -OCH2-), 6.30 (1H, H-8),
6.64 (1H, H-3), 7.53 (3H, H-3’, 4’, 5’), 7.80 (2H, H-2’, 6’); 13C NMR (100 MHz, DMSO-
d6): δ 14.77 (-CH3), 43.52 (C-4’’, 6’’), 52.55 (C-1’’), 53.97 (C-3’’, 7’’), 61.82 (-OCH2-),
98.23 (C-8), 100.45 (C-10), 105.03 (C-3), 106.09 (C-6), 126.21, 129.35, 131.66, 131.96
(C-2’, 3’, 4’, 1’), 154.94 (C-9), 155.36 (-OC=O), 161.94, 163.29, 165.38 (C-2, 5, 7),
182.51 (-C=O).
4.5.2.14
Diethyl-4,
4'-((5,7-dihydroxy-4-oxo-2-phenyl-4H-chromene-6,8-
diyl)bis(methylene))bis(piperazine-1-carboxylate) (22)
Yield: 37 %. Yellow solid. 1H NMR (400 MHz, CDCl3): δ 1.28 (6H, m, 2 × -CH ), 2.62
3
(4H, br, H-3’’, 7’’), 3.56 (4H, br, H-4’’, 6’’), 3.87 (4H, 2 × s, H-1’’), 4.14 (4H, m, 2 × -
27

13
OCH2-), 6.67 (1H, s, H-3), 7.55 (3H, H-3’, 4’, 5’), 7.87 (2H, H-2’, 6’); C NMR (100
MHz, CDCl3): representative peaks: δ 14.52 (-CH3), 43.09, 43.31 (C-4’’, 6’’), 51.63,
52.04 (C-1’’), 54.87 (C-3’’, 7’’), 60.65, 60.74 (-OCH2-), 101.35, 102.53, 104.54, 104.8
(C-6, 8, 10, 3), 126.27, 129.19, 131.04, 131.87 (C-2’, 3’, 4’, 1’), 154.50 (C-9), 154.69 (-
OC=O), 181.68 (-C=O).
4.5.2.15 Ethyl 4-(4-((5, 7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochroman-6-yl)methyl)
piperazin-1-yl)benzoate (23)
Yield: 76%, white solid. 1H NMR (400 MHz, CDCl3): δ 1.22 (3H, t, -CH3), 2.62 (1H, dd,
J = 3.0, 17.1 Hz, H-3), 2.92 (1H, dd, J = 12.8, 17.1 Hz, H-3), 2.60- 3.36 (8H, br, 2 × -N-
CH2-CH2-N-) 3.69 (2H, s, -N-CH2-Ar), 4.20 (2H, q, -CH2-CH3), 5.20 (1H, dd, J = 3.0,
12.8 Hz, H-2), 5.83 (1H, s, H-8), 6.72 (4H, two doublets, J1 = 8.5 Hz, J2 = 8.8 Hz, -ArH),
13
7.17 (2H, d, J = 8.5 Hz, -ArH), 7.78 (2H, d, J = 8.8 Hz, -ArH); C NMR (100 MHz,
CDCl3): 14.39 (-CH3), 43.13 (C-3), 47.58 (C-1’’), 52.12, 53.01 (-N-CH2-CH2-N-), 60.45
(-O-CH2), 78.76 (C-2), 96.01 (C-8), 100.13 (C-10), 102.10 (C-6), 114.14 (-Ar), 115.65
(C-3’, 5’), 121.05 (-C-C=O), 127.89 (C-2’, 6’), 131.20 (-Ar), 137.67 (-Ar-N-), 153.67 (C-
4’), 156.12 (C-9), 161.34 (C-5), 166.57 (C-7), 168.17 (-O-C=O), 195.78 (-C=O).
Acknowledgements
We thank the New Jersey Agricultural Experiment Station, and NIFA Hatch Project
1005685 and NJ12158, the School of Environmental and Biological Sciences at Rutgers
and the New Use Agriculture and National Plant Products Program for partial funding
and support. This work was conducted as part of the dissertation research of Jing Zhen.
28

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