Mannich bases of scutellarein as thrombin-inhibitors: Design, synthesis, biological activity and solubility

Article abstract of DOI:10.1016/j.bmc.2012.10.015

Two series of 8-aminomethylated derivatives were prepared by Mannich reaction of scutellarein (2) with appropriate aliphatic amines, alicyclic amines and formaldehyde. All the compounds were tested for their thrombin inhibition activity through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). The antioxidant activities of these target products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay method and the solubility were assessed by ultraviolet (UV). The results showed that morpholinyl aminomethylene substituent derivative (3d) demonstrated stronger anticoagulant activity, better water solubility and good antioxidant activity compared with scutellarein (2), which warrants further development as a agent for ischemic cerebrovascular disease treatment.

Full text of DOI:10.1016/j.bmc.2012.10.015

Bioorganic & Medicinal Chemistry 20 (2012) 6919–6923
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mannich bases of scutellarein as thrombin-inhibitors: Design, synthesis,
biological activity and solubility
Nian-Guang Li a,b, Shu-Lin Song a, Min-Zhe Shen a, Yu-Ping Tang a, , Zhi-Hao Shi a,c, Hao Tang a,
⇑
Qian-Ping Shi a,b, Yi-Fan Fu a, Jian-Ao Duan a,
⇑
a Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210046, China
b Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing 210046, China
c Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of 8-aminomethylated derivatives were prepared by Mannich reaction of scutellarein (2) with
appropriate aliphatic amines, alicyclic amines and formaldehyde. All the compounds were tested for their
thrombin inhibition activity through the analyzation of prothrombin time (PT), activated partial throm-
boplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). The antioxidant activities of these target
products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)
hydrazyl (DPPH) assay using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT)
assay method and the solubility were assessed by ultraviolet (UV). The results showed that morpholinyl
aminomethylene substituent derivative (3d) demonstrated stronger anticoagulant activity, better water
solubility and good antioxidant activity compared with scutellarein (2), which warrants further develop-
ment as a agent for ischemic cerebrovascular disease treatment.
Received 31 August 2012
Revised 17 October 2012
Accepted 17 October 2012
Available online 23 October 2012
Keywords:
Ischemic cerebrovascular disease
Thrombin
Antioxidant
Solubility
Ó 2012 Elsevier Ltd. All rights reserved.
Scutellarin
Scutellarein
1. Introduction
On this basis, some researchers have recently proposed and
searched some natural product with anticoagulant capacity and
Nowadays, ischemic cerebrovascular disease is a common and
frequently-occurring disease that seriously endangers human
health. It is one of the leading causes of death and disability world-
wide.1 Increasing evidence suggests a critical role of thrombin in
ischemic cerebrovascular disease,2 thrombin is generated in re-
sponse to vascular injury, it acts as a multifunctional serine protease
and catalyzes the proteolytic cleavage of the soluble plasma-protein
fibrinogen to form insoluble fibrin leading to clot formation. In addi-
tion, thrombin also serves as a potent platelet agonist and amplifies
its own generation by feedback activation of several steps in the
coagulation cascade.3 Secondly, oxidative stresses are major causes
of ischemic cerebrovascular disease,4,5 reactive oxygen species
(ROS) including the superoxide anion radical (O2ꢀÅ), hydroxyl radi-
cal (ÅOH), hydrogen peroxide (H2O2), singlet oxygen (1O2), and nitric
oxide (NOÅ) are constantly generated by various physiological func-
tions in the human body.6 The excessive production of ROS may
result in increased levels of low-density lipoprotein (LDL), oxidative
modification of LDL, and an impairment of endothelial derived
relaxing factor (EDRF, nitric oxide, NO)-mediated bioactions.7
antioxidant activity for the treatment of cerebrovascular disease.8
Scutellarin(40,5,6-trihydroxyflavone-7-glucuronide) (Fig. 1), the
major anti-oxidant constituent in breviscapine extracted from Chi-
nese herb of Erigeron breviscapus (vant.) Hand.–Mazz., showed the
effectiveness on dilating blood vessels, improving microcirculation,
increasing cerebral blood flow, and inhibiting platelet aggregation
since the 1970s.9 In addition, it has been clinically used to treat
acute cerebral infarction and paralysis induced by cerebrovascular
diseases such as hypertension, cerebral thrombosis, cerebral
haemorrhage in China since 1984.10
However, scutellarin has low water-solubility,11 and the bio-
availability of scutellarin was very low with the absolute bioavail-
ability in Beagle dog administered orally was rarely 0.4%.12
Interestingly, some researchers found that scutellarin was mainly
OH
OH
OH
O
O
HO
HO
O
O
O
HO
HO
O
O
OH
HO
OH
OH
⇑
Corresponding authors. Tel./fax: +86 25 85811916.
E-mail addresses: yupingtang@njutcm.edu.cn (Y.-P. Tang), dja@njutcm.edu.cn
Scutellarein (2)
Scutellarin (1)
(J.-A. Duan).
Figure 1. Chemical structures of scutellarin (1) and scutellarein (2).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.015

6920
N.-G. Li et al. / Bioorg. Med. Chem. 20 (2012) 6919–6923
absorbed in the form of its hydrolyzed product scutellarein (2)
(Fig. 1) by intestinal,13 furthermore, in the clinical trials,12 a large
amount of scutellarein (2) was found in urine and plasma after oral
administration of breviscapine, indicating that breviscapine was
firstly hydrolyzed into aglycone when reaching colon and was then
absorbed as scutellarein (2) as the real bioactive components in the
body. Pharmacodynamics confirmed that scutellarein (2) had bet-
ter protective effect than scutellarin (1) in rat cerebral ischemia,14
scutellarein (2) can prevent thrombosis and platelet aggregation,
and improve the characteristics of hemorheology in rats.15 In our
previous studies, we found that scutellarein (2) had stronger scav-
enging capacities toward DPPH, ABTS+Å, ÅOH free radicals than scu-
tellarin (1), and had better protective effect on H2O2-induced
cytotoxicity in PC12 cells.16,17 These results suggested that scutel-
larein (2) can be a promising lead compound for the discovery of
potent agent with thrombin-inhibitory and antioxidant activities
for the treatment of ischemic cerebrovascular disease.
Firstly, our docking studies of scutellarein (2) with thrombin
(2R2M)18 showed that the B ring and C ring in ligand could interact
well with S1 pocket and S2 pocket, respectively (Fig. 2), however, A
ring partly interact with the S3 pocket in thrombin, so the inhibi-
tory activity could be improved if a side chain is introduced at po-
sition 8 in the A ring by the Mannich reaction. Furthermore,
Mannich bases have been associated with increased bioactivity.19
The presence of a Mannich base group in many natural products
may increase biological potency due to the greater number of
molecular sites for electrophilic attack by cellular constituents, as
well as due to the cascade effect of preferential chemosensitiza-
tion. Aminoalkylation of aromatic substrates by the Mannich reac-
tion has considerable importance for the synthesis and
modification of biologically active compounds. This technique pro-
vides convenient access to many useful synthetic building blocks,
because the resulting amino group can be easily converted to var-
ious functionalities, particularly, to quaternary ammonium salts to
increase water solubility.
Herein, we describe the synthesis and in vitro biological evalu-
ation of Mannich bases of scutellarein with electrophilic substitu-
tion at C-8, together with a discussion of structure–activity
relationships. The thrombin inhibition activity of all the new ana-
logs were evaluated through the analyzation of prothrombin time
(PT), activated partial thromboplastin time (APTT), thrombin time
(TT) and fibrinogen (FIB). The antioxidant activities of these target
products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical
2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay using
3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide
(MTT) assay method and the water solubility were assessed by
ultraviolet (UV).
2. Results and discussion
2.1. Chemistry
In general, Mannich bases of scutellarein derivatives 3a–3g
were synthesized via a two-step procedure as described in Scheme
1. According to our previous procedure,16,17 the scutellarein (2)
could be obtained from the hydrolysis of scutellarin (1) by reflux-
ing it with 6 N HCl in 90% ethanol under the N2 protection. Then
our strategy for the synthesis of the targeted analogues was
achieved by the Mannich reaction of the scutellarein (2) with form-
aldehyde in the presence of the secondary amines in methonal
(Scheme 1). The classical conditions of the Mannich reaction for
the hydroxyl compounds are based on the ratio of substrate, amine
and formaldehyde in alcohol with prolonged heating.20 In our case,
scutellarein (2), formaldehyde and secondary amines were in
1:1:1.1 ratio, respectively, and stirred at 30 °C for 30 min–24 h to
afford the C8-aminomethylated derivatives. The structures of the
resulting Mannich bases were confirmed by 1H NMR and mass
analysis. The 1H NMR spectra of compounds 3a–3g clearly indi-
cated the absence of the signal at d 6.57 for H-8 proton of the flav-
anoid ring system. It should be noted that in all cases the
electrophilic substitution occurred solely in ring A even at the large
excess of the reagent and under more severe conditions.21
S1
S3
S2
2.2. Biological activity
Mannich reaction
2.2.1. Anti-thrombic activity
Because the thrombin inhibition activity can be assessed by
assaying the prolongation of the plasma clotting time of TT, APTT,
INR increasement of PT, and reduction of FIB content according to
our previous studies,18 so the thrombin time of different com-
pounds was investigated for TT, PT, APTT and FIB. The results were
shown in Table 1.
There were two series of Mannich bases of scutellarein, one ser-
ies was aliphatic amine (methyl, ethyl and isopropyl group) sub-
stituents (3a–3c), and the other series was alicyclic amine
(morpholinyl or piperzinyl ring) substituents (3d–3g). As shown
in Table 1, both series showed stronger thrombin inhibition activ-
ity compared with scutellarein (2). In the series of aliphatic amine
substituents Mannich bases, the most active compound was ethyl
amine Mannich base (3b), this compound significantly prolonged
TT and APTT, increased PT and decreased FIB content compared
to scutellarein (1). When the ethyl group changed into methyl
S3
S1
R
OH
B
8
O
C
HO
HO
A
3
O
OH
S2
Figure 2. The strategy for the design of compound 3.

N.-G. Li et al. / Bioorg. Med. Chem. 20 (2012) 6919–6923
6921
OH
OH
OH
R
OH
O
O
HO
HO
O
O
O
HO
HO
O
O
HO
HO
O
O
a
b
OH
HO
OH
OH
OH
2
3
1
3d. R=Morpholinyl (C4H8NO)
3a. R=Dimethyl amino (C2H6N)
3b. R=Diethyl amino (C4H10N)
3c. R=Diisopropyl amino (C6H14N)
3e. R=N-methyl piperzinyl (C5H11N2)
3f. R=N-ethyl piperzinyl (C6H13N2)
3g. R=N-n-butyl piperzinyl (C8H17N2)
Scheme 1. Reagents and conditions: (a) Concentrated hydrochloric acid, EtOH, N2, 120 °C, 17.0%; (b) MeOH, formaldehyde–water (37%), amine, 30 °C, 30.7–41.3%.
Table 1
Effect of Mannich bases of scutellarein on thrombin time
Compd. (25
lM)
Plasma coagulation parameters
TT (S)
APTT(s)
PT(s)
FIB(g/L)
2
10.25 0.24
12.58 0.42
12.63 0.35
11.75 0.80
14.48 0.48
14.40 0.14
14.13 0.19
13.45 0.37
33.36 2.47
34.23 2.34
34.62 4.45
34.52 4.22
35.48 4.26
35.42 3.38
35.15 2.51
35.01 3.58
7.34 0.30
7.45 0.42
7.55 0.39
7.47 0.27
7.84 0.31
7.71 0.19
7.41 0.27
7.40 0.37
5.96 0.64
5.56 0.36
5.36 0.26
5.54 0.52
5.26 0.31
5.46 0.46
5.54 0.20
5.56 0.33
3a
3b
3c
3d
3e
3f
3g
Data represent mean S.D. n = 4.
group or isopropyl group, the thrombin inhibition activity de-
creased, such as in the case of 3a, it shortened TT and APTT, de-
creased PT and increased FIB compared to 3b. This result
revealed that the presence of ethyl aminomethylene group substi-
tution at C8 position was very important in showing thrombin
inhibition activity. In the series of alicyclic amine Mannich bases,
our data indicated that morpholinyl aminomethylene substituent
(3d) showed the most active thrombin inhibitory activity among
all the synthesized Mannich bases of scutellarein derivatives, when
the morpholinyl group changed into piperzinyl group, the throm-
bin inhibition activity decreased, for example, 3e shortened TT
and APTT, decreased PT and increased FIB compared to 3d. One
interesting phenomenon for the piperzinyl ring substituent was
that the thrombin inhibition activity decreased as the atom num-
bers of the substituent in the piperzinyl ring became more, for in-
stance, the n-butyl piperazinyl substituent 3g shortened TT and
APTT, decreased PT and increased FIB compared to the methyl sub-
stituent 3e.
Figure 3. 3d docked into the pockets of thrombin (2R2M).
In the thrombin inhibition activity tests, 3d showed the stron-
gest inhibitory activity on thrombin, so 3d was selected for the
subsequent molecular docking experiment with thrombin
(2R2M) (Fig. 3). The thrombin docking showed that the morpholi-
nyl aminomethylene substituent at C7 in 3d occupied the deep S3
pocket (composed of Asn131, Leu132, Iie209, Gly258, Glu259,
Ser256, Try257) of thrombin. As displayed in Figure 4, ligand 3d
formed three hydrogen bonds with the active site residues of
2R2M in the binding mode, and the active site residues were
Asp229, Ala230 and Tyr83. These results confirmed our strategy
for designing Mannich bases of scutellarein as thrombin-inhibitors.
Figure 4. 3d docked into residues of 2R2M, hydrogen bonding interactions are
shown as red dashed lines.
yellow and was monitored spectrophotometrically. The IC50 value
is defined as the concentration of sample that causes 50% loss of
the radical.
The data obtained was depicted in Table 2. Although these com-
pounds showed no more antioxidant activity than scutellarein (2),
they still exhibited some interesting inhibition of oxidative activ-
ity. From the results obtained in the DPPH assay, we can infer that,
compounds 3a–3c, where the C8 position on the A-ring was occu-
pied by aliphatic aminomethylene groups, showed a higher antiox-
idant activity than the compounds 3d–3e where the C8 position on
the A-ring was occupied by alicyclic aminomethylene groups, such
2.2.2. Antioxidants
Antioxidants protect the aging brain against oxidative damage
associated with pathological changes of ischemic cerebrovascular
disease. So the in vitro antioxidant activity22 of scutellarein deriv-
atives was evaluated by DPPH23 radical-scavenging activity assays
using MTT assay method. DPPH assay measured the hydrogen-
donating ability of antioxidants to convert the stable DPPH free
radical into 1,1-diphenyl-2-picrylhydrazine.24 The reaction was
accompanied by a change in color from deep-yellow to light-
as in the case of 3a and 3g, where the IC50s were 26.46
lM and
32.70 M, respectively. Among all the synthesized Mannich bases
l

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N.-G. Li et al. / Bioorg. Med. Chem. 20 (2012) 6919–6923
Table 2
specified. Air- and moisture-sensitive liquids and solutions were
In vitro antioxidant activity in DPPH (IC50 in
solubility in water of Mannich derivatives
l
M) radical-scavenging assay and the
transferred via syringe or stainless steel cannula. Organic solutions
were concentrated by rotary evaporation below 45 °C at approxi-
mately 20 mm Hg. All non-aqueous reactions were carried out un-
der anhydrous conditions using flame-dried glassware within an
argon atmosphere in dry and freshly distilled solvents, unless
otherwise noted. Reactions were monitored by thin-layer chroma-
tography (TLC) carried out on 0.15ꢁ0.20 mm Yantai silica gel
plates (RSGF 254) using UV light as the visualizing agent. Chroma-
tography was performed on Qingdao silica gel (160ꢁ200 mesh)
using petroleum ether (60ꢁ90) and ethyl acetate as the eluanting
solvent. The melting points (Mp) were measured on a WRS-1B
apparatus and were not corrected. 1H NMR spectra were obtained
using a Bruker AV-300 (300 MHz) and AV-500 (500 MHz). Chemi-
cal shifts were recorded in ppm downfield from tetramethylsilane.
J values were given in Hz. Abbreviations used were s (singlet), d
(doublet), t (triplet), q (quartet), b (broad), and m (multiplet).
a
Compd. Antioxidant antivity IC50
(l
M)
Water solubility
a
Solubility
(
l
M) Fold increaseb
2
24.04
26.46
24.96
30.08
30.89
32.47
32.44
32.70
6.95
8.58
7.71
7.97
11.62
7.28
7.07
9.11
1.0
3a
3b
3c
3d
3e
3f
1.23
1.11
1.15
1.67
1.05
1.02
1.31
3g
a
Values are the means of at least two independent determinations; errors are
within 20%.
b
Fold increase in water solubility relative to scutellarein (2).
of scutellarein, the most active compound was 3b, with its IC50 for
DPPH was 24.96 lM.
ESI-MS spectra were recorded on
spectrometer.
a Waters Synapt HDMS
2.2.3. Solubility
The aqueous solubility of the synthesized Mannich bases of scu-
tellarein has been determined using UV spectrophotometer.25–27
As presented in Table 2, the water solubitily of the scutellarein
derivatives has been greatly increased compared to scutellarein
(2). The best soluble compound was morpholinyl aminomethylene
4.2. Synthesis of Mannich bases of scutellarein
4.2.1. Synthesis of scutellarien (2)
To a stirring mixture of 1 (10.0 g, 20.6 mmol) and concentrated
hydrochloric acid (120 mL) in ethanol (120 mL) was added water
(10 mL), the reaction mixture was refluxed under a N2 atmosphere
for 36 h. After cooled down to the room temperature, the mixture
was poured into water. The solid obtained was filtered followed by
silica gel column chromatographic purification of the residue using
50% ethyl acetate in petroleum ether afforded the compound 2 in
17.0% yield as yellow solid, mp 160–162 °C. 1H NMR (DMSO-d6,
substituent (3d) with its solubility in water was 11.62 lg/ml,
which was increased 1.67-fold compared to that of scutellarein
(2). Among the aliphatic amine substituents Mannich bases of scu-
tellarein, methyl amine (3a), ethyl amine (3b) and isopropyl amine
(3c) exhibited remarkable increases in solubility and showed 1.23-
fold, 1.11-fold and 1.15-fold, respectively. Among the alicyclic
amine Mannich bases of scutellarein 3d–3g, introduction of methyl
piperzinyl ring (3e) and ethyl piperzinyl ring (3f) showed similar
solubility to that of scutellarein (2), with their values were
0
300 MHz) d: 12.78 (s, 1H, C5-OH), 10.44 (s, 1H, C4 -OH), 10.29 (s,
1H, C7-OH), 8.71 (s, 1H, C6-OH), 7.90 (d, J = 8.8 Hz, 2H, C2 C6 -H),
0
0
0
0
6.91 (d, J = 8.8 Hz, 2H, C3 C5 -H), 6.74 (s, 1H, C3-H), 6.57 (s, 1H, C8-
H). ESI-MS: m/z 287 [M+H]+.
7.28 lg/ml and 7.07 lg/ml, respectively. Interestingly, replace-
ment of methyl and ethyl group in the piperzinyl ring by the larger
n-butyl group (3g) further increased experimental solubility about
1.31-fold compared to that of scutellarein (2).
4.2.2. General procedure for the preparation of Mannich bases
of scutellarein 3a–3g
To a solution of scutellarein (2) (0.35 mmol) in MeOH (7.5 mL)
was added 37% formaldehyde–water (27.5 lL, 0.35 mmol,
3. Conclusion
1.0 equiv), after vigorous stirring at 30 °C for 30 min, different
amines (0.39 mmol, 1.1 equiv) were added and the mixture was
stirred for 30 min–24 h after the reaction was completed. The
resulting mixture was evaporated under vacuo and the crude prod-
uct was purified by silica gel column chromatography (20% MeOH
in CH2Cl2) to yield the desired Mannich bases of scutellarein 3a–3g
(yields 30.7–41.3%).
Scutellarin has been clinically used to treat acute cerebral
infarction and paralysis induced by cerebrovascular diseases such
as hypertension, cerebral thrombosis, cerebral haemorrhage in
China for many years,10 and it showed the effectiveness on dilating
blood vessels, improving microcirculation, increasing cerebral
blood flow, and inhibiting platelet aggregation.9 However, scutell-
arin has pharmacokinetic problems such as low solubility in water
and fast metabolism which results in its low bioavailability. In this
study, based on the metabolic mechanism of scutellarin in vivo, we
took scutellarien (2) as a promising lead compound, designed and
synthesized new Mannich base derivatives of scutellarein such as
aliphatic amine substituents (3a–3c) and alicyclic amine substitu-
ents (3d–3g) to improve biological activities of scutellarien (2). In
particular, morpholinyl aminomethylene substituent (3d), demon-
strated stronger anticoagulant activity, better water solubility and
good antioxidant activity compared with scutellarein (2), which
warrants further development as a agent for ischemic cerebrovas-
cular disease treatment.
4.2.2.1. 8-(N,N-Dimethyl)-methyl-amino-5,6,7,40-tetrahydroxyf-
lavone (3a).
Yellow solid, 32.6% yield, mp 216–218 °C. 1H
NMR (DMSO-d6, 500 MHz) d: 13.11 (s, 1H, C5-OH), 10.25 (s, 1H,
0
0
0
C4 -OH), 8.05 (s, 1H, C6-OH), 7.95 (d, J = 9.0 Hz, 2H, C2 -H, C6 -H),
0
0
6.93 (d, J = 9.0 Hz, 2H, C3 -H, C5 -H), 6.96 (s, 1H, C3-H), 4.13 (s, 2H,
CH2), 2.56 (s, 6H, 2 ꢂ CH3). ESI-MS: m/z 344 [M+H]+.
4.2.2.2. 8-(N,N-Diethyl)-methyl-amino-5,6,7,40-tetrahydroxyf-
lavone (3b).
NMR (DMSO-d6, 500 MHz) d: 12.75 (s, 1H, C5-OH), 7.91 (d,
Yellow solid, 41.3% yield, mp 206–207 °C. 1H
0
0
0
0
J = 9.0 Hz, 2H, C2 -H, C6 -H), 6.94 (d, J = 9.0 Hz, 2H, C3 -H, C5 -H),
6.68 (s, 1H, C3-H), 4.28 (s, 2H, CH2), 2.90–2.97 (q, 4H, 2 ꢂ CH2),
1.17–1.22 (t, 6H, 2 ꢂ CH3). ESI-MS: m/z 372 [M+H]+.
4. Experimental
4.1. General methods
4.2.2.3. 8-(N,N-Diisopropylamine)-methyl-amino-5,6,7,40-tetra-
hydroxyflavone (3c).
Yellow solid, 39.6% yield, mp 223–
Reagents and solvents were purchased from commercial
sources and used without further purification unless otherwise
225 °C. 1H NMR (DMSO-d6, 500 MHz) d: 12.78 (s, 1H, C5-OH),
0
0
0
7.95 (d, J = 8.5 Hz, 2H, C2 -H, C6 -H), 6.93 (d, J = 8.5 Hz, 2H, C3 -H,

N.-G. Li et al. / Bioorg. Med. Chem. 20 (2012) 6919–6923
6923
0
C5 -H), 6.88 (s, 1H, C3-H), 4.28 (s, 2H, CH2), 2.30–2.34 (m, 2H,
was monitored by UV absorption spectrophotometry at the wave-
length of maximum absorbance (334 nm) from the whole spec-
trum using a multicomponent exploitation method. Each tested
2 ꢂ CH), 1.19–1.23 (d, 12H, 4 ꢂ CH3). ESI-MS: m/z 400 [M+H]+.
4.2.2.4. 8-(Morpholine base)-methyl-amino-5,6,7,40-tetrahydr-
compound (300
of the tested compounds had concentrations ranging from 3
mL to 12 g/mL. Different concentration solutions of each com-
l
g) was dissolved in 25 mL CH3OH. The solutions
oxyflavone (3d).
Yellow solid, 32.7% yield, mp 208–210 °C.
lg/
1H NMR (DMSO-d6, 500 MHz) d: 12.89 (s, 1H, C5-OH), 10.29 (s,
l
0
0
0
1H, C4 -OH), 7.94 (d, J = 9.0 Hz, 2H, C2 -H, C6 -H), 6.95 (d,
pound were determined by UV scanning, and the absorbances were
obtained. The results showed a good linear relationship, then all
the standard curves were completed. Each tested compound
0
0
J = 9.0 Hz, 2H, C3 -H, C5 -H), 6.75 (s, 1H, C3-H), 3.94 (s, 2H, CH2),
3.58–3.63 (t, 4H, 2 ꢂ CH2), 2.52–2.63 (t, 4H, 2 ꢂ CH2). ESI-MS: m/
z 386 [M+H]+.
(120 lg) was ultrasound dissolved in 10 mL pure water for 1 h at
room temperature. The solutions were stranded for 30 min and
centrifuged at the speed of 30000 r/min. The aqueous solution of
each compound was determined by UV scanning, and the absor-
bances were obtained. Then through the analyzation of standard
curve, all the compounds in the water solubility were obtained.
4.2.2.5. 8-(N-Methyl piperazine base)-methyl-amino-5,6,7,40-
tetrahydroxyflavone (3e).
Yellow solid, 35.8% yield, mp 215–
217 °C. 1H NMR (DMSO-d6, 500 MHz) d: 12.86 (s, 1H, C5-OH), 10.28
0
0
0
(s, 1H, C4 -OH), 7.92 (d, J = 8.5 Hz, 2H, C2 -H, C6 -H), 6.93 (d,
0
0
J = 8.5 Hz, 2H, C3 -H, C5 -H), 6.73 (s, 1H, C3-H), 3.95 (s, 2H, CH2),
2.60–2.64 (t, 4H, 2 ꢂ CH2), 2.46–2.50 (t, 4H, 2 ꢂ CH2), 1.22 (s, 3H,
CH3). ESI-MS: m/z 399 [M+H]+.
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (No. 81001382, 81274058), the Program for New
Century Excellent Talents by the Ministry of Education (NCET-
09-0163), 333 High-level Talents Training Project Funded by
Jiangsu Province, Six Talents Project Funded by Jiangsu Province
(2011-D-078), Program for Outstanding Scientific and Technologi-
cal Innovation Team of Jiangsu Higher Education (2009), National
Key Technology R&D Program (2008BAI51B01), Key Research
Project in Basic Science of Jiangsu College and University (NO.
07KJA36024, 10KJA360039), Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education Insti-
tutions (ysxk-2010), and Construction Project for Jiangsu Engineer-
ing Center of Innovative Drug from Blood-conditioning TCM
Formulae.
4.2.2.6. 8-(N-Ethyl piperazine base)-methyl-amino-5,6,7,40-tet-
rahydroxyflavone (3f).
Yellow solid, 38.4% yield, mp 221–
223 °C. 1H NMR (DMSO-d6, 500 MHz) d: 12.83 (s, 1H, C5-OH),
0
0
0
7.93 (d, J = 8.7 Hz, 2H, C2 -H, C6 -H), 6.95 (d, J = 8.7 Hz, 2H, C3 -H,
0
C5 -H), 6.73 (s, 1H, C3-H), 4.02 (s, 2H, CH2), 2.66–2.70 (t, 4H,
2 ꢂ CH2), 2.40–2.44 (t, 4H, 2 ꢂ CH2), 2.33–2.38 (q, 2H, CH2), 0.97–
1.02 (t, 3H, CH3). ESI-MS: m/z 413 [M+H]+.
4.2.2.7. 8-(N-Butyl piperazine base)-methyl-amino-5,6,7,40-tet-
rahydroxyflavone (3g).
Yellow solid, 34.3% yield, mp 217–
219 °C. 1H NMR (DMSO-d6, 500 MHz) d: 12.87 (s, 1H, C5-OH),
0
0
0
10.26 (s, 1H, C4 -OH), 7.93 (d, J = 8.5 Hz, 2H, C2 -H, C6 -H), 6.94 (d,
0
0
J = 8.5 Hz, 2H, C3 -H, C5 -H,), 6.73 (s, 1H, C3-H), 4.01 (s, 2H, CH2),
2.63–2.67 (m, 4H, 2 ꢂ CH2), 2.30–2.42 (m, 4H, 2 ꢂ CH2), 1.38–
1.41 (t, 2H, CH2), 1.23–1.31 (m, 4H, 2 ꢂ CH2), 0.86–0.89 (t, 3H,
CH3). ESI-MS: m/z 441 [M+H]+.
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