Nitrogen-containing flavonoid analogues as CDK1/cyclin B inhibitors: Synthesis, SAR analysis, and biological activity

Article abstract of DOI:10.1016/j.bmc.2008.06.055

A series of nitrogen-containing flavonoid analogues were designed and synthesized by Mannich reaction, and screened for the inhibitory activities of cyclin-dependent kinases using a FRET-based biochemical assay method. The results showed that C-8 nitrogen-containing baicalein analogues 3a-3f exhibited potent CDK1/Cyclin B inhibitory activities. 5,6,7-Trihydroxy-8-(dimethylaminomethyl)-2-phenyl-4H-chromen-4-one 3a, 5,6,7-trihydroxy-8-(pyrrolid inylmethyl)-2-phenyl-4H-chromen-4-one 3b, and 5,6,7-trihydroxy-8-(piperidinylmethyl)-2-phenyl-4H-chromen-4-one 3c (IC₅₀ 1.05-1.28 μM) were about sixfold more potent than baicalein 2 (IC₅₀ 6.53 μM). 5,6,7-Trihydroxy-8-(morpholinomethyl)-2-phenyl-4H-chromen-4-one 3d, 5,6,7-trihydroxy-8-(thiomorpholinomethy)-2-phenyl-4H-chrom en-4-one 3e, and 5,6,7-trihydroxy-8-(4-methylpiperazinylmethyl)-2-phenyl-4H-chromen-4-one 3f (IC₅₀ 0.27-0.38 μM) were about 20-fold more potent than baicalein, and were at the same level as flavopiridol (IC₅₀ 0.33 μM).

Full text of DOI:10.1016/j.bmc.2008.06.055

Bioorganic & Medicinal Chemistry 16 (2008) 7127–7132
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nitrogen-containing flavonoid analogues as CDK1/cyclin B inhibitors: Synthesis,
SAR analysis, and biological activity
a
b
a
a
a
a
Shixuan Zhang a, , Jigang Ma , Yongming Bao , Puwen Yang , Liang Zou , Kangjian Li , Xiaodan Sun
*
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, Zhongshan Road 158-90, Dalian 116012, PR China
b School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nitrogen-containing flavonoid analogues were designed and synthesized by Mannich reaction,
and screened for the inhibitory activities of cyclin-dependent kinases using a FRET-based biochemical
assay method. The results showed that C-8 nitrogen-containing baicalein analogues 3a–3f exhibited potent
CDK1/Cyclin B inhibitory activities. 5,6,7-Trihydroxy-8-(dimethylaminomethyl)-2-phenyl-4H-chromen-
4-one 3a, 5,6,7-trihydroxy-8-(pyrrolid inylmethyl)-2-phenyl-4H-chromen-4-one 3b, and 5,6,7-trihy-
Received 28 May 2008
Revised 24 June 2008
Accepted 26 June 2008
Available online 3 July 2008
droxy-8-(piperidinylmethyl)-2-phenyl-4H-chromen-4-one 3c (IC50 1.05–1.28
more potent than baicalein 2 (IC50 6.53 M). 5,6,7-Trihydroxy-8-(morpholinomethyl)-2-phenyl-4H-chro-
men-4-one 3d, 5,6,7-trihydroxy-8-(thiomorpholinomethy)-2-phenyl-4H-chrom en-4-one 3e, and 5,6,7-
lM) were about sixfold
Keywords:
Flavonoid analogues
Baicalein
CDK1/Cyclin B inhibitor
SAR
l
trihydroxy-8-(4-methylpiperazinylmethyl)-2-phenyl-4H-chromen-4-one 3f (IC50 0.27–0.38
l
M) were
about 20-fold more potent than baicalein, and were at the same level as flavopiridol (IC50 0.33
lM).
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
spect to ATP.7 It is currently in Phase II clinical trials as an anti-
tumor agent.8
Protein kinases regulate many critical biological mechanisms,
including metabolism and cell growth, proliferation, and differen-
tiation. Aberrations in the activity of the kinases involved in signal
transduction have been linked to many human diseases such as
cancer, diabetes, and inflammation. The discovery of more than
518 kinases encoded by the human genome has spurred develop-
ment of rapid screening techniques for potential drugs against
these enzymes.1
The cyclin-dependent kinases (CDKs) are a prominent family of
protein kinases, which play a key role in cell-cycle progression and
cellular proliferation.2 CDKs are multi-subunit enzymes composed
of at least a catalytic subunit protein kinases (CDK) and a regulatory
subunit (cyclin).3,4 In many tumors, such as melanomas, pancreatic
and esophaegal cancers, endogenous cyclin-dependent kinase
inhibitory proteins (CDKIs) are either absent or mutated. Conse-
quently, selective CDK inhibitors may prove to be effective chemo-
therapeutic agents.5 CDKs have become attractive therapeutic
targets for cancer therapy.6
On other hand, baicalein (5,6,7-trihydorxyflavone), a flavonoid
present in the root of Scutellaria baicalensis Georgi, has been re-
ported to inhibit cell proliferation in several types of cells. It med-
iated G1 and G2 growth arrest accompanied by the down-
regulation of cyclin D2, cyclin A, CDK1, and CDK2, and up-regula-
tion of p15Ink4B, p21CIP1/Waf1, p53, and cyclin E.9 In addition, baica-
lein-induced cell-cycle arrest and apoptosis in human lung
squamous carcinoma CH27 cells through decreasing the expres-
sion and function of CDK4/cyclin B and D.10 The effects of quercet-
inon on proliferation and cell-cycle arrest by modulation of CDK1/
Cyclin B in prostate cancer cells (PC-3) have been reported. Quer-
cetin led to substantial decrease in the expression of CDK1/Cyclin
B1 and phosphorylated pRb while increase in p21. Flowcytometric
analysis showed that quercetin blocks G2-M transition, with
significant induction of apoptosis.11 But, two naturally occurring
bacailein and quercetin have poor CDK inhibitory activity. Both
bacailein and quercetin lack nitrogen-containing ring (D-ring)
present in flavopiridol. Therefore, the introduction of nitrogen
atom to baicalein and quercetin was suggested to increase their
CDKs inhibitory activity. In this paper, sixteen nitrogen-contain-
ing baicalein, quercetin, and chalcone analogues were designed
and prepared through Mannich reaction. Totally 21 flavonoid
analogues were assayed for their CDK1/Cyclin B inhibitory activ-
The semisynthetic flavonoid, flavopiridol (Scheme 1), is de-
rived from rohitukine, which is an alkaloid flavonoid present in
the Indian plant Dysoxylum binectariferum. Flavopiridol could in-
duce cell-cycle arrest at both G1 and G2 phases, and is a potent
inhibitor of CDK1, 2, 4, and 6 in a competitive manner with re-
ity using
a fluorescence-based, coupled-enzyme format bio-
chemical assay screening procedure from Z0-LYTETM. In addition,
the structure–activity relationship (SAR) was summarized
herein.
* Corresponding author. Tel.: +86 411 8899 3891.
E-mail address: zhangsx56@yahoo.com.cn (S. Zhang).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.055

7128
S. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 7127–7132
OH
N
D
OH
OH
O
HO
HO
O
O
HO
O
O
B
HO
A
C
OH
Cl
OH
OH
OH
O
bacailein
Flavopiridol
quercetin
Scheme 1. Chemical structure of flavopiridol, bacailein and quercetin.
pound 4 was prepared by treating baicalin 1 with methanol in
the presence of catalytic amount of concentrated H2SO4. Condensa-
tion of 2-hydroxy-4-methoxy-acetophenone with benzaldehyde in
40% KOH, 50% ethanol solution at room temperature for 20 h pro-
vided compound 5. Compounds 3, 6, and 8 were prepared by Man-
2. Chemistry
Nitrogen-containing flavonoid analogues 3a–3f, 6a–6f and 8a,
8c, 8d, 8f were prepared as shown in Scheme 2. Baicalin 1 was
hydrolyzed with 50% H2SO4 at 95 °C to provide baicalein 2. Com-
HOOC
O
O
O
O
HO
HO
O
O
i)
HO
OH
OH
HO
1
2
OH
iii)
OH
ii)
R1R2N
H3COOC
HO
O
O
O
O
HO
HO
O
OH
OH
HO
4
OH
3a-3f
OH
O
H3CO
OH
H3CO
OH
O
iv)
5
O
v)
H3CO
R1R2N
OH
O
6a-6f
OH
OH
R1R2N
HO
OH
OH
vi)
O
HO
O
OH
OH
7
OH
O
OH
O
8a,8c,8d,8f
NR1R2
(f)
N
N
(b)
(a)
N
N
(c)
N
(d)
O
=
N
(e) S
N
Scheme 2. Reagents and conditions: Synthesis of the flavonoid analogues. (i) 50% H2SO4, 95 °C; (ii) secondary amine, 37% HCHO, CH3OH; (iii) CH3OH, H2SO4; (iv)
benzaldehyde, 40% KOH, EtOH, H2O; (v) secondary amine, 37% HCHO, isopropanol, HCl; (vi) secondary amine, 37% HCHO, DMSO.

S. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 7127–7132
7129
nich reaction using flavonoids, various secondary amines, and
formaldehyde solution in ethanol at 20–60 °C yielding the desired
analogues.
100
80
60
40
20
0
3. Result and discussion
The flavonoid analogues were assayed for their CDK1/Cyclin B
inhibitory activity according to the procedure instruments of Z0-LY-
TETM kinase assay kits. The results (ATP 10
l
M) for flavonoid ana-
logues 2–22 are shown in Table 1. Compounds 3a–3c showed
IC50 values at 1.05, 1.22, and 1.28 M, respectively, and were about
six-fold more potent than baicalein 2 (IC50 6.53 M), or 15-fold
more potent than baicalin 1 (IC50 14.36 M). Compounds 3d–3f
showed IC50 values at 0.28, 0.38, and 0.27 M, (Figs. 1–3), respec-
l
l
l
l
tively, and were about 20-fold more potent than baicalein 2, or
about 50-fold more potent than baicalin 1, at the same level as
flavopiridol (IC50 0.33 lM). It was indicated that the presence of
10
100
1000
the nitrogen on the position C-8 is critical, and introduction of sec-
ond heteroatom on the position C-8 would significantly increase
inhibitory potency. The nitrogen-containing baicalein analogues
were analyzed by HPLC-UV (C18 reversed-phase column, the elu-
tion system consisted of water/methanol/acetonitrile/formic acid
(59:30:10:1, v/v); the flow rate was 1.0 mL/min, and the injection
Concentration (nM)
Figure 2. CDK1/cyclin B inhibitory activity of 5,6,7-trihydroxy-8-(thiomorpholi-
nomethyl)-2-phenyl-4H-chromen-4-one 3e.
volume was 10 lL); the retention time of 3a–3c which have one
heteroatom on the position C-8 was 3c (21.7 min) > 3b
(18.2 min) > 3a (13.4 min); the retention time of 3d–3f which have
two heteroatoms on the position C-8 was 3e (17.0 min) > 3d
(12.0 min) > 3f (10.3 min). The inhibitory potency of 3a–3c was
100
80
60
40
20
0
Table 1
CDK1/cyclin B inhibitory activities of flavonoid analogues
No.
Compounds
IC50
(
lM)
No.
Compounds
IC50 (lM)
1
2
3
4
5
6
7
8
9
Flavopiridol
1
2
3a
3b
3c
3d
3e
3f
4
0.33
14.36
6.53
1.05
1.22
1.28
0.28
0.38
0.27
11.64
12
13
14
15
16
17
18
19
20
21
22
6a
6b
6c
6d
6e
6f
>20
>20
>20
>20
>20
>20
>20
15.74
18.69
>20
>20
7
8a
8c
8d
8f
10
100
1000
10
11
Concentration (nM)
5
>20
Figure 3. CDK1/cyclin B inhibitory activity of 5,6,7-trihydroxy-8-((4-methylpipera-
zin-1-yl)methyl)-2-phenyl-4H-chromen-4-one 3f.
100
3a > 3b > 3c, and the inhibitory potency of 3d–3f were
3f > 3d > 3e; it was indicated that the inhibitory potency will in-
crease accompanied with the rise of hydrophilia of the test
compounds.
According to the X-ray crystal structure of des-chloroavopir-
idol with CDK2,12 the piperidine nitrogen, on the position C-8
partially occupies the adenine amidogen binding region, the
piperidine nitrogen and Asp 145 interaction seems to be very
critical for CDK1/Cyclin B inhibitory activity. Thus, our SAR stud-
ies on C-8 were consistent with the literature above. Baicalins 1
80
60
40
20
0
and 4 showed IC50 values at 14.36, 11.64
were twofold less active compared with baicalein, IC50 values
6.53 M; these indicate that the C-7 hydroxyl is meaningful for
lM, respectively, and
l
the inhibition of kinase activity. The X-ray crystal structure of
des-chloroavopiridol with CDK2 also indicates that the C-5 hy-
droxyl and the C-4 carbonyl are involved in critical hydrogen
bonds with E-81 and L-83 in the adenine binding pocket. Screen-
ing of the commercially available flavonoids showed that at least
one hydroxyl group at either C-3 or C-5 of chromone was neces-
10
100
Concentration (nM)
1000
Figure 1. CDK1/cyclin B inhibitory activity of 5,6,7-trihydroxy-8-(morpholinom-
ethyl)-2-phenyl-4H-chromen-4-one 3d.

7130
S. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 7127–7132
sary for inhibitory activities against CDK2.13 Our results are con-
sistent with the conclusions above, and chalcone analogues 6a–
6f showed no concentration-dependent inhibitory activity, due
to the absence of C-20 and C-50 hydroxyl. Quercetin analogues al-
most have no inhibitory activity. These indicate that the pres-
ence of hydrophilic substituent groups such as hydroxyl group
on the position C-2 would be detrimental to CDK1/Cyclin B
inhibitory potency.
eight concentrations (twofold across)with the highest concentra-
tion of 1 M to determine and prove IC50 values again following
l
the same procedure, respectively. Continuous kinetic monitoring
of enzyme activity was performed on Fluoroskan AscentÒ FL from
thermo scientific (kex 400 nm, kem 445 nm, and kem 520 nm). The
experiment was performed in triplicate and the percent inhibition
of enzyme activity was calculated for all the compounds at each
concentration.
5.2.1. Baicalein (2)
4. Conclusions
To a stirred mixture of baicalin (1) ( from Scutellaria baicalensis
Geoygi. mp 206 °C) (2 g, 7.41 mmol) in H2O (10 ml) was added 50%
concentrated sulfuric acid (50 ml) at 90 °C, and the mixture was
stirred for 10 min and then was poured in ice water (100 ml).
The precipitate that formed was collected by filtration, washed,
and recrystallized from acetone to give the product as yellow solid
(0.36 g, 29.3%). Mp 264–266 °C; MS (API-ES positive) m/z: 271.1
[M+H]+; IR (KBr, cmꢁ1): 3409, 3093, 1659, 1620, 1586; 1H NMR
(DMSO-d6, 400 MHz) d: 12.64 (s, 1H, 5-OH), 10.55 (s, 1H, 7-OH),
8.79 (s, 1H, 6-OH), 8.03–8.05 (t, J = 6.4, 1.6 Hz, 2H, Ar-20, 60-H),
7.52–7.58 (m, 3H, Ar-30,40,50-H), 6.91 (s, 1H, C@CH), 6.60 (s, 1H,
Ar-8-H).
Our SAR studies on flavonoid analogues for their CDK1/Cyclin
B inhibitory activity were suggested as follows: (1) the presence
of the nitrogen on position C-8 is critical, and introduction of
second heteroatom on position C-8 would increase inhibitory
potency. In the same type of substitutes, the inhibitory potency
will increase accompanied with the hydrophilicity of the substi-
tute rising because the nitrogen group on position C-8 partially
occupies the region which the water-solubility substitutes ami-
dogen group of ATP bind with CDK1/Cyclin B. (2) C-5, C-6, C-7
hydroxyl, and C-4 carbonyl are vital for inhibitory activity since
these acidic groups occupy the region of ATP phosphate group
bind with CDK1/Cyclin B. (3) The lipophilic substitutes on
position C-2 are important, because these lipophilic substitutes
occupy lipophilic binding pocket of CDK1/Cyclin B while ATP
does not have these lipophilic substitutes. In other words,
hydrophilic substitutes would be detrimental to inhibitory
potency.
5.2.2. 5,6,7-Trihydroxy-8-(dimethylamino)-2-phenyl-4H-
chromen-4-one (3a)
To the solution of baicalein (2) (0.2 g, 0.74 mmol) in methanol
(14 ml) were added dropwise 37% formaldehyde solution
(0.0741 g, 0.89 mmol) and 33% dimethylamine solution (0.27 g,
2.00 mmol). The mixture was stirred at room temperature for
2 h. The precipitates were collected, filtered, and washed several
times with methanol to get the product as yellow solid (0.41 g,
79.0%). Mp 280–282 °C; MS (API-ES positive) m/z: 328.1 [M+H]+,
350.1 [M+Na]+, 655.3 [2M+H]+, 677.2 [2M+Na]+; IR (KBr, cmꢁ1):
3403, 3093, 2925, 2861, 1637, 1570, 1509; 1H NMR (DMSO-d6,
400 MHz) d: 12.62 (s, 1H, 5-OH), 8.06–8.05 (m, 2H, Ar-20, 60-H),
7.58–7.57 (m, 3H, Ar-30,40,50-H), 6.83 (1H, s, C@CH), 4.23 (s, 2H,
CH2), 2.63 (s, 6H, N(CH3)2).
In summary, three compounds with potent CDK1/Cyclin B ki-
nase inhibitory activity were selected from the series of nitrogen-
containing flavonoid analogues we designed and prepared. Those
compounds were suggested to target the ATP binding site of the ki-
nase as flavopiridol. The CDK1/Cyclin B inhibitory activity of 3d–3f
(IC50 0.27–0.38
lM) was at the same level as flavopiridol (IC50
0.33 M); compound 3f was firstly designed and prepared, which
l
was expected to be novel series of CDK1/Cyclin B inhibitors.
5. Experiments
5.1. Instrument
5.2.3. 5,6,7-Trihydroxy-8-(pyrrolidin-1-ylmethyl)-2-phenyl-4H-
chromen-4-one (3b)
From 5,6,7-trihydroxy-8-((dimethylamino)methyl)-2-phenyl-
4H-chromen-4-one (3a), yellow solid (71%) was determined. Mp
213–215 °C; MS (API-ES negative) m/z: 352.1 [MꢁH]ꢁ; IR (KBr,
cmꢁ1): 3407, 3030, 2975, 2869, 1637, 1580, 1521; 1H NMR
(DMSO-d6, 400 MHz) d: 12.58 (s, 1H, 5-OH), 8.04–8.03(m, 2H, Ar-
20, 60-H), 7.57–7.55 (m, 3H, Ar-30,40,50-H), 6.78 (s, 1H, C@CH), 4.34
(s, 2H, CH2), 3.11 (s, 4H, NCH2), 1.89 (s, 2H, CH2).
IR spectra were obtained as KBr pellets on a Nicolet 50X FT-IR
spectrophotometer. NMR spectra were measured by a Varian INO-
VA-400 spectrometer, with chemical shifts reported in parts per
million (in DMSO, TMS as an internal standard, J values were given
in Hertz). Mass spectra were obtained on a HP1100LC-MSD spec-
trometer. HPLC-UV separations were performed on a Shimadzu
(Kyoto, Japan) C18 reversed-phase column (150 ꢀ 4.6 mm id
5.2.4. 5,6,7-Trihydroxy-8-(piperidin-1-ylmethyl)-2-phenyl-4H-
chromen-4-one (3c)
5łlm). Melting points are uncorrected.
From 5,6,7-trihydroxy-8-((dimethylamino)methyl)-2-phenyl-
4H-chromen-4-one (3a), yellow solid (71.5%) was obtained. Mp
209–211 °C; MS (API-ES positive) m/z: 368.1 [M+H]+, 735.3
[2M+H]+, 757.3 [2M+Na]+; IR (KBr, cmꢁ1): 3400, 3050, 2925,
2861, 1637, 1570, 1509; 1H NMR (DMSO-d6, 400 MHz) d: 12.68
(s, 1H, 5-OH), 8.04–8.02 (m, 2H, Ar-30,40,50-H), 7.57 (m, 3H, Ar-
30,40,50-H), 6.84 (s, 1H, C@CH),4.16 (s, 2H, CH2), 2.84 (s, 4H,
NCH2), 1.62 (s, 4H, CH2),1.48 (s, 2H, CH2).
5.2. Biochemical assay
The CDK1/Cyclin B (Invitrogen Corporation, CA, USA) enzyme
activity was measured by a fluorescence kinetic assay using the
Z0-LYTETM Kinase Assay Kits (Invitrogen Corporation, CA, USA). The
assay was performed at room temperature (23 °C) in 384-well as-
say plate. The total volume was 20
pound, 2.5
L 4ꢀ ATP solution, 5
Cylin B mixture, 5 L development reagent solution, 5
agent). The final concentrations of the assay constituents were
240 ng/ml CDK1/Cylin B, 2 M peptide substrate, and 10 M ATP.
l
l
L (contain 2.5
L 2ꢀ peptide substrate/CDK1/
L stop re-
l
L 4ꢀ test com-
l
l
l
5.2.5. 5,6,7-Trihydroxy-8-(morpholinomethyl)-2-phenyl-4H-
chromen-4-one (3d)
l
l
FromP 5,6,7-trihydroxy-8-((dimethylamino)methyl)-2-phenyl-
4H-chromen-4-one (3a), yellow solid (69.0%) was obtained. Mp
275–277 °C; MS (API-ES positive) m/z: 370.0 [M+H]+; IR (KBr,
cmꢁ1): 3402, 3040, 2936, 2861, 1647, 1560, 1502; 1H NMR
(DMSO-d6, 400 MHz) d: 12.74 (s, 1H, 5-OH) 8.09–8.08 (m, 2H, Ar-
Generally, the compounds were solubilized in 1% DMSO and added
to the reaction mixture at eight concentrations (twofold across)
with the highest concentration of 20
logues. The representative compounds 3d–3f were screened at
lM for the flavonoid ana-

S. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 7127–7132
7131
20,60-H), 7.60 (m, 3H, Ar-30, 40,50-H), 6.95 (s, 1H, C@CH), 3.96 (s, 2H,
CH2), 3.61 (s, 4H, OCH2), 2.62 (s, 4H, NCH2).
3H, Ar-3,4,5-H), 6.87 (s, 1H, Ar-50-H), 4.25 (s, 2H, CH2), 4.00 (s,
3H, CH3), 2.77 (s, 6H, CH3).
5.2.6. 5,6,7-Trihydroxy-8-(thiomorpholinomethyl)-2-phenyl-
4H-chromen-4-one (3e)
5.2.11. (E)-1-(2-Hydroxy-4-methoxy-5-(pyrrolidin-1-
ylmethyl)phenyl)-3-phenylprop-2-en-1-one (6b)
From 5,6,7-trihydroxy-8-((dimethylamino)methyl)-2-phenyl-
4H-chromen-4-one (3a), yellow solid (65%) was obtained. Mp
242–244 °C; MS (API-ES positive) m/z: 386.1 [M+H]+; IR (KBr,
cmꢁ1): 3378, 3050, 2925, 2861, 1637, 1570, 1509; 1H NMR
(DMSO-d6, 400 MHz) d: 12.64 (s, 1H, 5-OH),8.03–8.01 (m, 2H,
Ar-20,60-H), 7.55–7.53 (m, 3H, Ar-30, 40,50-H), 6.88 (s, 1H, C@CH),
3.91 (s, 2H, CH2), 2.81–2.80 (m, 4H, SCH2), 2.62–2.61 (m, 4H,
NCH2).
From (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-methoxy-
phenyl)-3-phenylprop- 2-en-1-one (6a), yellow solid (38.2%) was
obtained. Mp 128–130 °C; MS (API-ES positive) m/z: 338.2
[M+H]+; IR (KBr, cmꢁ1): 3389, 3037, 2925, 1636, 1570, 1498; 1H
NMR (DMSO-d6, 400 MHz) d: 13.35 (s, 1H, OH), 8.62 (s, 1H, Ar-
20-H), 8.17–8.13 (d, 1H, J = 16 Hz, CH@CH), 7.90–7.89 (m, 2H, Ar-
2,6-H), 7.88–7.84 (d, 1H, J = 16 Hz, CH@CH), 7.43 (s, 3H, Ar-3,4,5-
H), 6.64 (s, 1H, Ar-50-H), 4.26 (s, 2H, CH2), 3.95 (s, 3H, OCH3),
2.50 (s, 4H, NCH2), 1.05–1.03 (m, 4H, CH2).
5.2.7. 5,6,7-trihydroxy-8-((4-methylpiperazin-1-yl)methyl)-2-
phenyl-4H-chromen-4-one (3f)
5.2.12. (E)-1-(2-Hydroxy-4-methoxy-5-(piperidin-1-ylmethyl)
phenyl)-3-phenylprop-2-en-1-one (6c)
From 5,6,7-trihydroxy-8-((dimethylamino)methyl)-2-phenyl-
4H-chromen-4-one (3a), yellow solid (72.2%) was obtained. Mp
236–238 °C; MS (API-ES positive) m/z: 383.1 [M+H]+, m/z: 405.1
[M+Na]+, 765.3 [2M+H]+, 787.3 [2M+Na]+; IR (KBr, cmꢁ1): 3402,
3070, 2955, 2841, 1639, 1575, 1503; 1H NMR (DMSO-d6,
400 MHz) d: 12.65 (s, 1H, 5-OH), 8.02–8.01 (m, 2H, Ar-20, 60-H),
7.55–7.53 (m, 3H, Ar-30,40,50-H), 6.87 (s, 1H, C@CH), 3.96 (s, 2H,
CH2), 2.62 (s, 8H, NCH2), 2.13 (s, 3H, NCH3).
From (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-methoxy-
phenyl)-3-phenylprop-2-en-1-one (6a), yellow solid (43.5%) was
obtained. Mp 155–157 °C; MS (API-ES positive) m/z: 352.2
[M+H]+; IR (KBr, cmꢁ1): 3375, 3010, 2974, 1671, 1609, 1474;
1H NMR (DMSO-d6, 400 MHz) d: 13.35 (s, 1H, OH), 8.60 (s, 1H,
Ar-20-H), 8.07–8.03 (d, 1H, J = 16 Hz, CH@CH), 7.90–7.89 (m,
2H, Ar-2,6-H), 7.82–7.78 (d, 1H, J = 16 Hz,CH@CH), 7.43 (s, 3H,
Ar-3,4,5-H), 6.64 (s, 1H, Ar-50-H), 4.17 (s, 2H, CH2), 3.87 (s, 3H,
CH3), 3.26 (s,4H,CH2), 2.62–2.66 (m, 4H, CH2), 0.97–0.98 (m,
2H, CH2).
5.2.8. Baicalin methylate (4)
To the solution of baicalin (5 g, 11.66 mmol) in methanol
(250 ml) was added catalytic amount of sulfuric acid, and the
solution was heated at reflux temperature for 1 h. After filtration,
the filtrate solution was cooled at room temperature, and solvent
was removed under reduced pressure, to give the compound (4)
as yellow powder (5.1 g, 95%). Mp 268–270 °C. MS (API-ES posi-
tive) m/z: 459.1 [M+H]+;1H NMR (DMSO-d6, 400 MHz) d: 12.75
(s, 1H, 5-OH), 10.38 (s, 1H, 40-OH), 8.62 (s, 1H, 6-OH), 8.03–
8.05 (m, 2H, Ar-20,60-H), 7.52–7.58 (m, 3H, Ar-30, 40, 50-H), 6.91
(s, 1H, C@CH2), 6.60 (s, 1H, Ar-8-H), 5.26–5.28 (d, 1H, 100-H),
4.19–4.21 (d, 1H, 500-H), 3.67 (3H, s, –OCH3), 3.36–3.45 (m, 3H,
200,300,400-H).
5.2.13. (E)-1-(2-Hydroxy-4-methoxy-5-(morpholinomethyl)-
phenyl)-3-phenylprop-2-en-1-one (6d)
From (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-methoxy
phenyl)-3-phenylprop- 2-en-1-one (6a). Yellow solid (41.7%) was
obtained. Mp 163–165 °C; MS (API-ES positive) m/z: 354.2
[M+H]+; IR (KBr, cmꢁ1): 3410, 3057, 2935, 1637, 1570, 1500;
1H NMR (DMSO-d6, 400 MHz) d: 13.38 (s, 1H, OH) 8.61 (s,
1H, Ar-20-H), 8.20–8.16 (d, 1H, J = 16 Hz, CH@CH), 7.98–7.97
(m, 2H, Ar-2,6-H), 7.87–7.83 (d, 1H, J = 16 Hz, CH@CH), 7.48–
7.47 (s, 3H, Ar-3,4,5-H), 6.68 (s, 1H, Ar-50-H), 4.28 (s, 2H,
CH2), 3.88 (s, 3H, CH3), 3.35–3.31 (m, 4H, OCH2), 3.11–3.07
(m, 4H, NCH2).
5.2.9. (E)-1-(2-Hydroxy-4-methoxyphenyl)-3-phenylprop-2-en-
1-one (5)
To the solution of 2-hydroxy-4-methoxy-acetophenone (2.0 g,
12.0 mmol) in ethanol (20 ml) was added 40% KOH solution
(30 ml), then was added dropwise benzaldehyde, and the solution
was stirred at room temperature for 20 h. The solution was neu-
tralized to pH 7–8 with hydrochloric acid, and the precipitates
were collected, filtered, and washed several times with methanol
to give a yellow solid (1.79 g, 58%).
5.2.14. (E)-1-(2-Hydroxy-4-methoxy-5-(thiomorpholinomethyl)-
phenyl)-3-phenylprop-2-en-1-one (6e)
From (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-methoxy-
phenyl)-3-phenylprop-2-en-1-one (6a), yellow solid (38.2%) was
obtained. Mp 154–156 °C; MS (API-ES positive) m/z: 370.1
[M+H]+; IR (KBr, cmꢁ1): 3350, 3047, 2933, 1633, 1565,1496; 1H
NMR (DMSO-d6, 400 MHz) d: 13.35 (s, 1H, OH), 8.59 (s, 1H, Ar-20-
H), 8.07–8.03 (d, 1H, J = 16 Hz, CH@CH), 7.90–7.89 (m, 2H, Ar-
2,6-H), 7.86–7.82 (d, 1H, J = 16 Hz, CH@CH), 7.43 (s, 3H, Ar-3,4,5-
H), 6.64 (s, 1H, Ar-50-H), 3.87 (s, 2H, CH2), 3.49 (s, 3H, OCH3),
2.67 (s, 4H, SCH2), 2.60 (s, 2H, NCH2).
5.2.10. (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-
methoxyphenyl)-3-phenylprop-2-en-1-one (6a)
To the solution of (5) (0.5 g,1.95 mmol) were added parafor-
maldehyde (0.088 g, 2. 93 mmol) and 33% dimethylamine solu-
tion (0.27 g, 2.00 mmol) in 10 ml isopropanol, in the presence
of catalytic amount of hydrochloride, and the mixture was stir-
red, and fluxed for 6 h, the mixture was cooled and evaporated
to dryness. 20 ml of 10% acetic acid was added to the solid res-
idue, and the mixture was filtered and the solution was neutral-
ized to pH 7–8 with ammonia; the precipitates were filtered and
washed with water, and the residue was eluted through a silica-
gel column with CH2Cl2/CH3OH (20:1) to give (6a) as yellow so-
lid (0.254 g, 42.0%). Mp 188–190 °C; MS (API-ES positive) m/z:
312.1 [M+H]+; IR (KBr, cmꢁ1): 3425, 3020, 2927, 1643, 1570,
1509; 1H NMR (DMSO-d6, 400 MHz) d: 13.44 (s, 1H, OH), 8.62
(s, 1H, Ar-20-H), 8.10–8.14 (d, 1H, J = 16 Hz, CH@CH), 7.96–7.93
(m, 2H, Ar-2,6-H), 7.85–7.81 (m, 1H, J = 16 Hz, CH@CH), 7.50 (s,
5.2.15. (E)-1-(2-Hydroxy-4-methoxy-5-(4-methylpiperazin-1-
ylmethyl)phenyl)-3-phenylprop-2-en-1-one (6f)
From (E)-1-(5-((dimethylamino)methyl)-2-hydroxy-4-methoxy-
phenyl)-3-phenylprop-2-en-1-one (6a), yellow solid (44.4%) was
obtained. Mp 148–150 °C; MS (API-ES positive) m/z: 367.2
[M+H]+; IR (KBr, cmꢁ1): 3361, 3038, 2783, 1632, 1570, 1498; 1H
NMR (DMSO-d6, 400 MHz) d: 13.38 (s, 1H, OH), 8.51 (s, 1H, Ar-20-
H), 8.20–8.16 (d, 1H, J = 16;Hz, CH@CH), 7.98–7.97 (m, 2H, Ar-
2,6-H), 7.87–7.83 (d, 1H, J = 16 Hz, CH@CH), 7.48–7.47 (s, 3H, Ar-
3,4,5-H), 6.57 (s, 1H, Ar-50-H), 3.86 (s, 2H, CH2), 3.44 (s, 3H,
OCH3), 2.41–2.38 (m, 8H, CH2), 2.13 (s, 3H, NCH3).

7132
S. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 7127–7132
5.2.16. 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(dimethyl-
aminomethyl)-4H-chromen-4-one (8a)
5.2.19. 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-8-((4-methyl-
piperazin-1-yl)methyl)-4H-chromen-4-one (8f)
To the solution of quercetin (7) (from Sophora japonica L. Mp
314 °C) (0.5 g, 1.66 mmol) in DMSO was added 37% formaldehyde
solution (0.161 g, 1.99 mmol)) and 33% dimethylamine solution
(0.27 g, 2.00 mmol). The solution was stirred at room temperature
for 2 h, and the mixture was evaporated to dryness. The resulting
residue was purified by TLC, silica-gel GF254, 1-butanol/water/ace-
tic acid = 4:1:1, yellow solid. MS (API-ES positive) m/z: 360.1
From 2-(3,4-dihydroxyphenyl)-8-((dimethylamino)methyl)-3,5,
7-trihydroxy-4H-chromen-4-one (8a), yellow solid was obtained.
MS (API-ES positive) m/z: 415.2 [M+H]+; 1H NMR (DMSO-d6,
400 MHz) d: 7.70 (d, 1H, Ar-60-H), 7.57 (d, 1H, Ar-20-H ), 6.88 (d,
1H, Ar-50-H), 6.17 (s, 1H, Ar-6-H), 3.96 (s, 2H, CH2), 2.64 (t, 8H,
NCH2), 2.14 (s, 3H, NCH3).
1
[M+H]+; H NMR (DMSO-d6, 400 MHz) d: 7.74 (m, 1H, Ar-60-H),
7.60 (m, 1H, Ar-20-H), 6.92 (m, 1H, Ar-50-H), 6.19 (s, 1H, Ar-6-H),
3.86 (s, 2H, CH2), 2.16 (s, 6H, NCH3).
References and notes
1. Noble, E. M.; Endicott, J. A.; Johnso, L. N. Science 2004, 303, 1800.
2. Liu, T.; Xu, Z. M.; He, Q. J.; Chen, Y. H.; Yang, B.; Hu, Y. Z. Bioorg. Med. Chem. Lett.
2007, 17, 278.
5.2.17. 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(piperidin-
3. Ahn, Y. M.; Vogeti, L.; Liu, C. J.; Santhapuram, K. R.; White, J. M.;
Vasandani, V.; Mitscher, L. A.; Lushington, G. H.; Hanson, P. R.; Powell, D.
R.; Himes, R. H.; Roby, K. F.; Yeb, Q.; Georga, G. I. Bioorg. Med. Chem. Lett.
2007, 14, 702.
4. Kyoung, S. K.; John, S. S.; John, T. S.; Ligang, Q.; Sam, T. C.; Leslie, L.; Yolanda, F.
K.; Raj, N. M.; John, T. H.; David, S. K.; William, G. H.; Barris, S. W.; Janet, G. M.;
Kevin, R. W. J. Med. Chem. 2000, 43, 4126.
5. Murthi, K. K.; Dubay, M.; McClure, C.; Brizuela, L.; Boisclair, M. D.; Worland, P.
J.; Mansuri, M. M.; Pal, K. Bioorg. Med. Chem. Lett. 2000, 10, 1037.
6. Senderowicz, A. M. Cancer Biol. Ther. 2003, 4, 84.
1-ylmethyl)-4H-chromen-4-one (8c)
From
2-(3,4-dihydroxyphenyl)-8-((dimethylamino)methyl)-
3,5,7-trihydroxy-4H-chromen-4-one (8a), yellow solid was ob-
tained. MS (API-ES positive) m/z: 400.2 [M+H]+; 1H NMR (DMSO-
d6, 400 MHz) d: 7.72 (d, 1H, Ar-60-H), 7.61 (d, 1H, Ar-20-H ), 6.92
(d, 1H, Ar-50-H), 6.19 (s, 1H, Ar-6-H), 3.90 (s, 2H, CH2), 2.84 (s,
4H, NCH2), 1.62 (s, 4H, CH2),1.48 (s, 2H, CH2).
7. Senderowicz, A. M. Oncogene 2000, 19, 6600.
8. Zhai, S.; Senderowicz, A. M.; Sausville, E. A.; Figg, W. D. Ann. Pharmacother.
2002, 36, 905.
5.2.18. 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-8-(morpholi-
nomethyl)-4H-chromen-4-one (8d)
9. Hsu, S. L.; Hsieh, Y. C.; Hsieh, W. C.; Chou, C. J. Eur. J. Pharmacol. 2000, 425, 165.
10. Lee, H. Z.; Leung, H. W.; Lai, M. Y.; Wu, C. H. Anticancer Res. 2005, 25, 959.
11. Vijayababu, M. R.; Kanagaraj, P.; Arunkumar, A.; Ilangovan, R.; Aruldhas, M. M.;
Arunakaran, J. J. Cancer Res. Clin. Oncol. 2005, 131, 765.
12. Azevedo, W. F.; Mueller-Dieckmann, H. J.; Schulze-Gahmen, U.; Worland, P. J.;
Sausville, E. A.; Kim, S. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2735.
13. Lee, J.; Park, T.; Jeong, S. W.; Kimb, K. H.; Hong, C. Y. Bioorg. Med. Chem. Lett.
2007, 17, 1284.
From 2-(3,4-dihydroxyphenyl)-8-((dimethylamino)methyl)-3,
5,7-trihydroxy-4H-chromen-4-one (8a), yellow solidwas obtained.
MS (API-ES positive) m/z: 402.1 [M+H]+; 1H NMR (DMSO-d6,
400 MHz)d: 7.74 (d, 1H, Ar-60-H), 7.60 (d, 1H, Ar-20-H), 6.92 (d,
1H, Ar-50-H), 6.19 (s, 1H, Ar-6-H), 3.86 (s, 2H, CH2), 3.17 (s, 4H,
OCH2), 2.51 (s, 4H, NCH2).