Synthesis and antiproliferative activities of polymethoxyflavones aminoalkyl and amino acid derivatives

Article abstract of DOI:10.1515/hc-2020-0010

Twelve novel aminoalkyl derivatives 3a-3f, 4a-4f and four novel amino acid derivatives 5a, 5b, 6a and 6b of polymethoxyflavones 1 and 2 were synthesized through regioselective demethylation, etherification, amination, EDCl-mediated amide condensation and

Full text of DOI:10.1515/hc-2020-0010

Heterocycl. Commun. 2020; 26: 68–75
Research Article
Open Access
Li-qiong Ran, Xue-li Li, Man-hui Liu and Qiu-an Wang*
Synthesis and antiproliferative activities of
polymethoxyflavones aminoalkyl and amino
acid derivatives
https://doi.org/10.1515/hc-2020-0010
parent molecule [6-8]. The amino acid derivatives, such as
Received October 20, 2019; accepted February 04, 2020.
tricin-amino acid derivatives, demonstrate favorable cell
permeability and excellent bioavailability [9]. Quercetin-
amino acid derivatives are safe cancer multidrug resis-
tance(MDR)modulators[10,11]. Theresultsofthesestudies
suggested that flavonoid conjugation with aminoalkyl
groups or amino acids was the most promising approach
to enhance its bioactivity and improve bioavailability.
As part of our screening program dedicated to the
search for derivatives of natural flavonoids with antican-
cer properties[12-15], we herein synthesized two series of
PMFs derivatives by attaching aminoakyl or amino acid
moiety through a linking spacer to the free hydroxyl groups
at the C-5 position. Furthermore, their antiproliferative
activities in vitro against four different human cancer cell
lines including Aspc-1 (human pancreatic cancer), SUN-5
Abstract: Twelve novel aminoalkyl derivatives 3a-3f,
4
a-4f and four novel amino acid derivatives 5a, 5b, 6a
and 6b of polymethoxyflavones 1 and 2 were synthesized
through regioselective demethylation, etherification, ami-
nation, EDCl-mediated amide condensation and alkaline
hydrolysis, using tangeretin and nobiletin as starting
materials. Their antiproliferative activities against four
different human cancer cell lines (Aspc-1, SUN5, HepG-2
and HCT116) were evaluated by in vitro CCK-8 assay. The
results show that the majority of the synthetic compounds
exhibited moderate to good antiproliferative activity. In
particular, the antiproliferative activity of compound 5b
against HepG-2 cells (IC 0.057 μM) was equal to the posi-
5
0
tive control drug Staurosporine (IC50 0.0575 μM).
(
human gastric cancer), HepG-2 (human hepatocellular
carcinoma) and HCT-116 (human colon cancer) were eva-
luated by CCK-8 (cell counting kit-8) assay using Stauro-
sporine as the positive control drug.
Keywords: polymethoxyflavones, aminoalkylated deriva-
tives, amino acid derivatives, synthesis, antiproliferative
activity.
Result and discussion
Introduction
The synthesis of novel PMF derivatives 3a-3f, 4a-4f, 5a,
Polymethoxyflavonids (PMFs) are a class of natural pro-
ducts, which widely exist in Citrus plants possessing
high anticancer activity [1-5], however, their bioavailabi-
lity is low. The biological interest to develop structural
analogs of anticancer agents possessing basic nitrogen
atoms seemed highly desirable. It is reported that the
introduction of an aminoalkyl side chain leads to a sig-
nificant increase in biological activity and potency of the
5
b, 6a and 6b was performed according to the reaction
pathways illustrated in Scheme 1. The inexpensive and
easily accessible natural products tangeretin and nobile-
tin were used as the starting materials. Due to the neigh-
boring-group participation of the C-4 carboxyl oxygen,
tangeretin or nobiletin undergo regioselective demethyla-
tion of the C-5 methoxy group after treatment with a Lewis
acids such as boron trichloride or aluminum chloride to
give 5-demethyl tangeretin (1) or 5-demethyl nobiletin (2),
respectively. We obtained compounds 1 and 2 with high
yields of 93% and 90% respectively. PMFs 1 or 2 reacted
with 1,2-diobromoethane in the presence of anhydrous
*
Corresponding author: Qiu-an Wang, College of Chemistry and
Chemical Engineering, Hunan University, Changsha 410082,
China, e-mail: WangQA@hnu.edu.cn
K CO in dry acetone to give compounds 7 or 8. The two
Li-qiong Ran, Xue-li Li and Man-hui Liu, College of Chemistry and
Chemical Engineering, Hunan University, Changsha 410082, China
2
3
latter reacted with various amines in the presence of K CO
2
3
Open Access. © 2019 Ran et al., published by De Gruyter.ꢀ
Attribution alone 4.0 License.
ꢀThis work is licensed under the Creative Commons

L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkylꢁ
ꢁ69
Scheme 1ꢂSynthetic route of aminoalkyl and amino derivatives of polymethoxyflavones
Reagents and conditions: a. AlCl , CH CO C H , reflux, then 5% HCl(aq), reflux; b.BrCH CH Br, K CO , DMF, heat; c. HNR R , K CO , CH CN,
1
2
3
3
2
2
5
2
2
2
3
2
3
3
3
reflux; d. ClCH CO C H , K CO , DMF, heat; e. 10% NaOH (aq.), CH OH, r.t; f. H NCH(R )CO CH , EDCl, DMAP, CH Cl , r.t; g. 10% NaOH (aq),
2
2
2
5
2
3
3
2
2
3
2
2
CH OH, r.t, then 1 M HCl(aq), r.t.
3
and KI, to give compounds 3a-3f and 4a-4f. These com- phenlyamine derivatives 5a, 5b, 6a and 6b were obtained
pounds are extending alkoxyl side chain at the 5-posi- by hydrolysis of 13a, 13b, 14a and 14b. All the new com-
tion, and introducing amine hydrogen bond receptor pounds were purified by recrystallization or chromato-
at the end of the side chain. Compounds 9 and 10 were graphy, and their structures were confirmed by means of
1
13
usually prepared from etherification of the 5-OH group by HNMR, CNMR, and MS spectra.
using methyl chloroacetate in the presence of a base. Two
The antiproliferative activities of the synthesized poly-
key intermediates 5-O-carboxyalkylated polymethoxy methoxyflavones 1 and 2 and their aminoalkyl and amino
flavones 11 and 12 were synthesized by basic hydrolysis of acid derivatives 3a-3f, 4a-4f, 5a, 5b, 6a and 6b against
9
and 10 respectively.
four different human cancer cell lines (Aspc-1, SUN-5,
Two amino acids, glycine (Gly) and phenylalanine HepG-2 and HCT-116) were evaluated by CCK-8 method.
Phe) were attached to the 5-position of the PMFs ring via Based on this procedure, a dose-response curve was
a non-hydrolysable linker. 5-O-carboxyalkylated PMFs obtained for the four cell lines. Each test compound and
1 or 12 were condensed with glycine methyl ester hyd- the concentration that caused 50% cell growth inhibition
(
1
rochloride or phenylalanine methyl ester hydrochloride, (IC , corresponding to the concentration of compound
50
respectively, using 1-ethyl-3-(3-dimethylaminopropyl) car- that inhibition 50% of net cell growth) was determined as
bodiimide hydrochloride (EDCl), N-hydroxybenzotriazole described elsewhere.
(
HOBt) and 4-dimethylaminopyridine (DMAP) as effici-
As shown in Table 1, the majority of the synthetic
ent coupling reagents. The corresponding PMF glycine or compounds exhibited moderate to potent antiproliferative

ꢁ
70ꢁ
L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkyl
a
Table 1 Antiproliferative activities [IC (μM)] of synthetic compounds on four human cancer lines
50
Compound
Aspc-I
>ꢃꢄ
SNUꢃ
HepGꢅ
>ꢃꢄ
HCTꢇꢇꢈ
>ꢃꢄ
ꢀ
2
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
3
3
3
3
3
3
4
4
4
4
4
4
5
5
ꢁ
ꢁ
a
b
c
d
e
f
a
b
c
d
e
f
>ꢃꢄ
>ꢃꢄ
ꢆꢆ.ꢉꢋ±ꢅ.ꢃꢆ
ꢇꢇ.ꢃꢇ±ꢉ.ꢈꢅ
ꢆꢆ.ꢄꢋ±ꢋ.ꢉꢇ
ꢅꢈ.ꢆꢈ±ꢃ.ꢆꢉ
ꢆꢋ.ꢅꢇ±ꢆ.ꢃꢄ
ꢆꢅ.ꢈꢅ±7.ꢆꢉ
>ꢃꢄ
ꢇꢈ.ꢈꢆ±ꢇ.7ꢅ
>ꢃꢄ
ꢆꢊ.ꢃ7±ꢅ.ꢅꢅ
>ꢃꢄ
ꢅꢈ.ꢃꢋ±7.ꢈ7
ꢇꢅ.ꢈꢋ±ꢅ.ꢃꢊ
ꢅꢊ.ꢋꢊ±ꢃ.ꢇꢇ
ꢇ7.ꢊꢄ±ꢆ.ꢆꢊ
>ꢃꢄ
ꢆꢇ.ꢄꢉ±ꢇ.ꢃꢈ
ꢆꢊ.ꢄꢆ±ꢅ.7ꢅ
ꢇꢋ.ꢅꢉ±ꢃ.ꢋꢈ
>ꢃꢄ
ꢅꢆ.ꢄꢇ±ꢆ.ꢈꢇ
>ꢃꢄ
ꢉꢊ.7ꢇ±ꢅ.ꢇꢄ
>ꢃꢄ
ꢉꢃ.ꢈꢄ±ꢆ.ꢅꢄ
ꢆꢉ.ꢊ7±ꢈ.ꢈꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
ꢉꢆ.ꢅꢉ±ꢃ.ꢈꢅ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
ꢉꢅ.ꢃꢊ±ꢅ.ꢉꢆ
>ꢃꢄ
ꢆꢉ.ꢈꢅ±ꢈ.ꢄꢃ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
a
b
a
b
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
ꢄ.ꢄꢃ7±ꢉ.ꢊE-ꢄꢃ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
>ꢃꢄ
Staurosporine
ꢄ.ꢄꢄꢃꢅ±ꢇ.ꢅꢇE-ꢄꢈ
ꢄ.ꢄꢅꢄꢋ±ꢅ.ꢃE-ꢄ7
ꢄ.ꢄꢃ7ꢃ±ꢅ.ꢄꢆE-ꢄꢃ
ꢄ.ꢄꢅ±ꢇ.ꢄE-ꢄꢈ
aData are the mean ± S.D. of three independent experiments performed.
activity. This shows that the introduction of aminoalkyl or was equal to the positive control drug Staurosporine
amino acid moiety at 5-O-position of PMFs 1 or 2 produ- ((IC 0.0575 μM) deserving further investigation.
50
ces the desired effect of increasing the antiproliferation
activities. In particular, compound 5b clearly demonstra-
ted the best results (IC 0.057 μM) in the HepG-2 cell line Experimental
5
0
studied. In fact, by comparing the structures 2 and 5b,
it can be concluded that the presence of phenylalanine Melting points were determined via an XRC-1 apparatus
1
13
amide moiety on 5-position of 5-demethyl nobiletin was and are uncorrected. HNMR and CNMR spectra were
associated with a remarkable increase in the growth inhi- recorded on a Bruker-AV 400 spectrometer at 400 and
bitory effect on HepG-2 cells. The benzyl function group 100MHz, respectively, with TMS as an internal standard
between amido and carboxyl structure has more affinity in CDCl or DMSO-d . Mass spectra (MS) and high-reso-
3
6
to biological cells, so the biological activity is better than lution mass spectra (HRMS) were determined on a VG
Gly derivative. It is suggested that the importance of this Autospec-3000 or Mat 95 XP Spectrometer by the EI or ESI
molecular modification is responsible for the activity method. Column chromatography was carried out on silica
when compared with the corresponding polymethoxy fla- gel 200-300 mesh (Qingdao Ocean Chemical Products of
vones. The clarification of the structural determinants for China). Commercially available AR or chemically pure
the potency would guide the design of novel potent drug reagents were used, and anhydrous solvents were dried
molecules for future development.
and redistilled using standard experimental procedures.
-Demethyl tangeretin (1) and 5-demethyl nobiletin
2) were prepared form tangeretin and noliletin according
to previously published procedures [16].
5
(
Conclusion
Twelve novel aminoalkyl derivatives 3a-3f, 4a-4f and four
novel amino acid derivatives 5a, 5b, 6a and 6b of poly- General procedure for synthesis of bromoethanated poly-
methoxyflavones were synthesized. Their antiprolife- methoxyflavones (7 and 8)
rative activities in vitro results show that the majority of
them exhibited moderate to good antiproliferative activity 1,2-Dibromoethane (11.5 mmol) was added to a solution of
against four different human cancer cell lines (Aspc-1, PMFs 1 or 2 (1.4 mmol) and K CO (14.5 mmol) in acetone
2
3
SUN5, HepG-2 and HCT116). Among them, PMFs amino (50 mL), and the mixture was refluxed for 4h. The pro-
acid derivative 5b against HepG-2 cells (IC 0.057 μM) gress of the reaction was monitored by TLC. After cooling
5
0

L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkylꢁ
ꢁ71
to room temperature, the mixture was filtered, the solvent 3-H), 4.06 (t, J = 6.7 Hz, 2H, 5-OCH ), 4.00, 3.93, 3.85, 3.77
2
was evaporated, and the residue was recrystallized from (s/each, 12H, 4OCH ) , 2.95 (t, J = 6.7 Hz, 2H, CH ), 2.42-2.27
3
2
EtOH to yield compound 7 or 8.
(m, 4H, 2NCH ), 1.46-1.36 (m, 4H, 2CH ), 0.78 (t, J = 7.2 Hz,
2 2
1
3
5
-(2-Bromoethoxy)-4’,6,7,8-tetramethoxyflavone 6H, 2CH ). C NMR (101MHz, CDCl ): δ 177.21, 162.21, 161.02,
3
3
o
1
(
7) yellow solid; 76% yield; m.p. 122-124 C; H NMR (400 151.29, 147.58, 144.05, 137.90, 127.61, 123.72, 114.93, 114.44,
MHz, CDCl ): δ 7.79 (d, J = 8.3 Hz, 2H, 2’-H and 6’-H), 6.94 106.52, 73.20, 61.89, 61.58, 56.77, 55.44, 53.71, 20.41, 11.89.
3
+
(
d, J = 8.3 Hz, 2H, 3’-H and 5’-H), 6.49 (s, 1H, 3-H), 4.27 (t, ESI-MS: m/z 486[M+H] . calc:486.24.
J = 6.5 Hz, 2H, 5-OCH ), 4.03, 3.95 , 3.88, 3.80 (s/each, 12H,
5-(2-Pyrrolidinylethoxy)-4’,6,7,8-tetramethoxy-
2
13
o
4
OCH ), 3.70 (t, J = 6.5 Hz, 2H, CH Br). C NMR (101 MHz, flavone (3c) yellow solid; 87% yield; m.p. 98-100 C;
3
2
1
CDCl ): δ 177.24, 162.36, 161.37, 151.41, 147.77, 146.45, 144.19, H NMR (400 MHz, CDCl ): δ 7.87 (d, J = 8.0 Hz, 2H, 2’-H
3
3
1
38.4, 127.74, 123.66, 114.80, 114.53, 106.56, 74.44, 62.12, and 6’-H), 7.02 (d, J = 8.1 Hz, 2H, 3’-H and 5’-H), 6.57 (s,
1.69, 55.52, 30.13. 1H, 3-H), 4.20 (t, J = 6.0 Hz, 2H, 5-OCH ), 4.10, 4.03, 3.95,
-(2-Bromoethoxy)-3’,4’,6,7,8-pentamethoxyfla- 3.89 (s/each, 12H, 4OCH ), 3.07 (t, J = 6.0 Hz, 2H, CH ), 2.72-
6
2
5
3
2
o
1
13
vone (8) yellow solid; 79% yield; m.p.116-118 C; H NMR 2.63 (m, 4H, 2NCH ), 1.83-1.75 (m, 4H, 2CH ). C NMR (101
2
2
(
400 MHz, CDCl ): δ 7.50 (d, J = 8.4 Hz, 1H, 6’-H), 7.34 (s, 1H, MHz, CDCl ): δ 177.25, 162.26, 161.12, 151.35, 147.70, 147.37,
3 3
’-H), 6.93 (d, J = 8.4 Hz, 1H, 5’-H), 6.53 (s, 1H, 3-H), 4.28 144.17, 138.06, 127.68, 123.79, 114.99, 114.49, 106.58, 73.43,
2
+
(
t, J = 6.5 Hz, 2H, 3-OCH ), 4.04, 3.96, 3.90 (s/each, 15H, 61.94, 61.61, 55.55, 54.38, 23.55. ESI-MS: m/z 478 [M+Na] .
2
13
5
OCH ), 3.71 (t, J = 6.5 Hz, 2H, CH Br). C NMR (101 MHz, calc:478.19.
3
2
CDCl ): δ 177.27, 161.26, 152.02, 151.48, 149.31, 147.77, 146.52,
5-(2-Piperidinylethoxy)-4’,6,7,8-tetramethoxyfla-
3
o
1
1
7
44.23, 138.41, 123.89, 119.71, 114.79, 111.25, 108.57, 106.79, vone (3d) yellow solid; 86% yield; m.p. 106-108 C; H
4.47, 62.11, 61.73, 56.05, 30.08.
NMR (400 MHz, CDCl ): δ 7.78 (d, J = 8.0 Hz, 2H, 2’-H and
3
6
’-H), 6.93 (d, J = 8.0 Hz, 2H, 3’-H and 5’-H), 6.49 (s, 1H,
3
-H), 4.10 (t, J = 6.0 Hz, 2H, 5-OCH ), 4.01, 3.94, 3.86, 3.79
2
General procedure for synthesis of aminalkyl-substitued
polymethoxyflavone derivatives ꢂa-ꢂf and ꢃa-ꢃf
(s/each, 12H, 4OCH ), 2.82 (t, J = 5.9 Hz, 2H, CH ), 2.47-2.41
3
2
(m, 4H, 2NCH ), 1.59-1.46 (m, 4H, 2CH ), 1.36-1.29 (m, 2H,
2
2
1
3
CH ). C NMR (101MHz, CDCl ): δ 177.12, 162.20, 161.05,
2
3
To a suspension of compounds 7 or 8 (0.43 mmol) and 151.24, 147.66, 147.27, 144.04, 138.04, 127.59, 123.63, 114.99,
K CO (7.2 mmol) in acetonitrile (20.0 mL), the amine 114.42, 106.45, 71.60, 66.95, 61.97, 61.84, 61.56, 58.45, 55.41,
2
3
+
(
14.5 mmol) and a catalytic amount of KI (0.07 mmol) were 53.78. ESI-MS: m/z 508 [M+K] . calc:508.21.
added. The resulting mixture was refluxed for 6-12h. After
5-(2-(4-Hydroxypiperidinyl)ethoxy)-4’,6,7,8-tet-
filtering, the resulting filtrate was evaporated to dryness ramethoxyflavone (3e) yellow solid; 77% yield; m.p.
o
1
under reduced pressure. The residue was suspended in 108-110 C; H NMR (400 MHz, CDCl ):δ 7.77 (d, J = 7.7 Hz,
3
water (10.0 mL) and extracted with dichloromethane 2H, 2’-H and 6’-H), 6.92 (d, J = 7.8 Hz, 2H, 3’-H and 5’-H),
(
3×30 mL). The combined organic layers were evaporated 6.48 (s, 1H, 3-H), 4.09 (t, J = 5.2 Hz, 2H, 5-OCH ), 4.01, 3.93,
2
under reduced pressure, and the residue was recrystal- 3.85 , 3.78 (s/each, 12H, 4OCH ), 3.65-3.78 (m, 1H, CH), 2.96-
3
lized from EtOH to yield compounds 3a-3f or 4a-4f.
2.84 (m, 4H, 2NCH ), 2.27(t, J = 5.2 Hz, 2H, NCH ), 1.85-1.76
2
2
1
3
5
-(2-Diethylaminoethoxy)-4’,6,7,8-tetramethoxy- (m, 2H, CH ), 1.62-1.53 (m, 2H, CH ). C NMR (101MHz,
2
2
o
1
flavone (3a) yellow solid; 80% yield; m.p. 110-112 C; H CDCl ): δ 177.36, 162.25, 161.22, 151.35, 147.69, 147.24, 144.07,
3
NMR (400 MHz, CDCl ): δ 7.79 (d, J = 7.8 Hz, 2H, 2’-H and 138.01, 127.68, 123.60, 114.84, 114.45, 106.43, 72.12, 67.43,
3
6
’-H), 6.95 (d, J = 7.8 Hz, 2H, 3’-H and 5’-H), 6.50 (s, 1H, 61.96, 61.62, 57.71, 55.47, 51.31, 34.29, 31.53, 22.61, 14.11.
+
3
-H), 4.13 (t, J = 5.7 Hz, 2H, 5-OCH ), 4.03, 3.95, 3.87, 3.81 ESI-MS: m/z 508 [M+Na] . calc:508.20.
2
(
s/each, 12H, 4OCH ), 3.08 (t, J = 5.7 Hz, 2H, CH ), 2.72
5-(2-(1-Methylhexahydropyrazinyl)ethoxy)-
3
2
(
q, J = 6.8 Hz, 4H, 2NCH ), 1.06 (t, J = 6.9 Hz, 6H, 2CH ). 4’,6,7,8-tetramethoxyflavone (3f) yellow solid; 86%
2
3
13
o
1
C NMR (101 MHz, CDCl ): δ 177.49, 162.37, 161.43, 151.52, yield; m.p. 104-106 C; H NMR (400 MHz, CDCl ): δ 7.78
3
3
1
47.74, 146.96, 144.13, 138.22, 127.74, 123.62, 114.6, 106.48, (d, J = 8.5 Hz, 2H, 2’-H and 6’-H), 6.93 (d, J = 8.5 Hz, 2H,
7
1.89, 61.98, 61.65, 55.52, 52.20, 47.67, 11.29. ESI- MS: m/z 3’-H and 5’-H), 6.49 (s, 1H, 3-H), 4.09 (t, J = 5.7 Hz, 2H,
+
4
58 [M+H] .calc:458.21.
5-OCH ), 4.01, 3.94, 3.86, 3.79 (s/each, 12H, 4OCH ), 2.84
2
3
5
-(2-Dipropylaminoethoxy)-4’,6,7,8-tetrame- (t, J = 5.7 Hz, 2H, CH ), 2.58-2.52 (m, 4H, 2NCH ), 2.41-2.32
2
2
1
3
thoxyflavone (3b): yellow solid; 77% yield; m.p. 88-90 (m, 4H, 2CH ), 2.20 (s, 3H, CH ). C NMR (101MHz, CDCl ):
2
3
3
o
1
C; H NMR (400 MHz, CDCl ): δ 7.77 (d, J = 8.1 Hz, 2H, 2’-H δ 176.1, 161.22, 160.06, 150.27, 146.69, 146.40, 143.10, 137.02,
3
and 6’-H), 6.91 (d, J = 8.2 Hz, 2H, 3’-H and 5’-H), 6.48 (s, 1H, 126.64, 122.76, 114.04, 113.46, 105.55, 71.08, 60.95, 60.61,

ꢁ
L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkyl
7ꢄꢁ
5
6.92, 54.46, 54.13, 52.32, 45.07. ESI-MS: m/z 507 [M+Na]+. 6.50 (s, 1H, 3-H), 4.09 (t, J = 5.3 Hz, 2H, 5-OCH ), 4.02, 3.94,
2
calc:507.22.
3.87 (s/each, 15H, 5OCH ), 3.65-3.61 (m, 1H, CH), 2.90-2.83
3
5
-(2-Diethylaminoethoxy)-3’,4’,6,7,8-pentame- (m, 4H, 2NCH ), 2.27 (t, J = 5.3 Hz, 2H, NCH ), 1.85-1.76 (m,
2
2
1
3
thoxyflavone(4a) yellow solid; 78% yield; m.p. 142-144 2H, CH ), 1.57-1.51 (m, 2H, CH ). C NMR (101MHz, CDCl ):
2
2
3
o
1
C; H NMR (400 MHz, CDCl ): δ 7.48 (d, J = 8.3 Hz, 1H, δ 177.31, 161.07, 151.89, 151.40, 149.20, 147.67, 147.29, 144.10,
3
6
’-H), 7.33 (s, 1H, 2’-H), 6.91 (d, J = 8.3 Hz, 1H, 5’-H), 6.51 137.95, 123.84, 119.63, 114.84, 111.20, 108.48, 106.65, 72.14,
(
s, 1H, 3-H), 4.06 (t, J = 6.3 Hz, 2H, 5-OCH ), 4.02, 3.95, 67.38, 61.91, 61.62, 57.73, 55.98, 51.28, 34.30. ESI-MS: m/z
2
+
3
.91-3.86 (s/each, 15H, 5OCH ), 2.98 (t, J = 6.3 Hz, 2H, CH ), 516 [M+H] . calc:516.22.
3
2
2
.62 (q, J =6.7 Hz, 4H, 2NCH ), 1.01 (t, J = 6.9 Hz, 6H, 2CH ).
5-(2-(1-Methylhexahydropyrazinyl)ethoxy)-
2
3
1
3
C NMR (101MHz, CDCl ): δ 177.28, 160.97, 151.86, 151.40, 3’,4’,6,7,8-pentamethoxyflavone (4f) yellow solid; 85%
3
o
1
1
49.20, 147.68, 147.42, 144.09, 137.91, 123.90, 119.58, 114.89, yield; m.p. 138-140 C; H NMR (400 MHz, CDCl ): δ 7.48 (d,
3
1
1
11.18, 108.44, 106.71, 72.87, 61.90, 61.63, 55.98, 52.37, 47.49, J = 8.4 Hz, 1H, 6’-H), 7.32 (s, 1H, 2’-H), 6.91 (d, J = 8.4 Hz, 1H,
+
1.75. ESI-MS: m/z 488 [M+H] . calc:488.22.
5’-H), 6.50 (s, 1H, 3-H), 4.09 (t, J = 5.3 Hz, 2H, 5-OCH ), 4.02,
2
5
-(2-(Dipropylaminoethoxy)-3’,4’,6,7,8-pentame- 3.95, 3.88 (s/each, 15H, 5OCH ), 2.83 (t, J = 5.3 Hz, 2H, CH ),
3
2
thoxyflavone (4b) yellow solid; 75% yield; m.p. 160-162 2.57-2.49 (m, 4H, 2NCH ), 2.41-2.34 (m, 4H, 2CH ), 2.20 (s,
2
2
o
1
13
C; H NMR (400 MHz, CDCl ): δ 7.46 (d, J = 8.2 Hz, 1H, 3H, CH ). C NMR (101 MHz, CDCl ): δ 177.16, 160.90, 151.84,
3
3
3
6
’-H), 7.31 (s, 1H, 2’-H), 6.90 (d, J = 8.2 Hz, 1H, 5’-H), 6.50 151.31, 149.20, 147.66, 147.42, 144.11, 137.93, 123.93, 119.55,
(
(
(
s, 1H, 3-H), 4.02 (t, J = 6.1 Hz, 2H, 5-OCH ), 4.02, 3.94, 3.86 115.00, 111.17, 108.44, 106.73, 72.04, 61.91, 61.62, 57.92, 55.96,
2
+
s/each, 15H, 5OCH ), 2.94 (t, J = 6.1 Hz, 2H, CH ), 2.41-2.32 55.12, 53.32, 46.07. ESI-MS: m/z 537 [M+Na] . calc: 537.23.
3
2
m, 4H, 2NCH ), 1.40-1.34 (m, 4H, 2CH ), 0.78 (t, J = 6.9
2
2
13
Hz, 6H, 2CH ). C NMR (101MHz, CDCl ): δ 177.16, 160.82,
3
3
1
1
51.78, 151.31, 149.15, 147.64, 147.54, 144.04, 137.79, 123.90, General procedure for synthesis of carboxylic acid
19.51, 114.90, 111.13, 108.36, 106.70, 73.22, 61.89, 61.80, derivatives of polymethoxyflavonoids 11 and 1ꢄ
61.59, 56.74, 56.00, 55.84, 53.64, 20.39, 11.89. ESI-MS: m/z
+
5
16 [M+H] . calc: 516.25.
A mixture of 1 or 2 (1.3 mmol) and potassium carbonate
5
-(2-(Pyrrolidinylethoxy)-3’,4’,6,7,8-pentame- (1.6 mmol) were dissolved in 40 ml DMF and added into a
thoxyflavone (4c) yellow solid; 89% yield; m.p. 124-126 100 ml three-necked round-bottomed flask. The reaction
o
1
o
C; H NMR (400 MHz, CDCl ): δ 7.47 (d, J = 8.3 Hz, 1H, mixture was heated to 40 C, then ethyl chloroacetate (9.07
3
6
’-H), 7.32 (s, 1H, 2’-H), 6.91 (d, J = 8.4 Hz, 1H, 5’-H), 6.50 mmol) was added dropwise into the mixture. The reaction
o
(
(
(
s, 1H, 3-H), 4.13 (t, J = 5.4 Hz, 2H, 5-OCH ), 4.02, 3.95, 3.88 mixture was stirred for 6h at 40 C and then cooled to room
2
s/each, 15H, 5OCH ), 3.01 (t, J = 5.4 Hz, 2H, CH ), 2.67-2.59 temperature. A total of 500 ml of cold water was added to
3
2
13
m, 4H, 2NCH ), 1.76-1.71 (m, 4H, 2CH ). C NMR (101 MHz, the mixture. The solid that separated was filtered, washed
2
2
CDCl ): δ 177.24, 160.97, 151.87, 151.39, 149.21, 147.33, 144.15, with water and dried to obtain 0.48 g of compound 9 or 10.
3
1
7
37.97, 123.91, 119.58, 114.89, 111.19, 108.47, 106.71, 78.98,
A mixture of 9 or 10 (1.13 mmol) was dissolved in 40
+
3.30, 67.76 , 64.67, 54.35, 23.51. ESI-MS: m/z 508 [M+Na] . ml methanol and added into a 100 ml three-necked round-
calc:508.20.
bottomed flask equipped and stirred at room temperature.
5
-(2-(Piperidinylethoxy)-3’,4’,6,7,8-pentame- 10 mL of 10% NaOH aqueous solution was added dropwise
thoxyflavone (4d) yellow solid; 88% yield; m.p. 148-150 into the mixture. The reaction mixture was stirred for 2h
o
1
C; H NMR (400 MHz, CDCl ): δ 7.49 (d, J = 7.3 Hz, 1H, 6’-H), at room temperature and then was concentrated under
3
7
.33 (s, 1H, 2’-H), 6.92 (d, J = 8.1 Hz, 1H, 5’-H), 6.51 (s, 1H, reduced pressure to obtain a white liquid. The pH of the
3
-H), 4.15 (t, J = 5.1 Hz, 2H, 5-OCH ), 4.02,3.95, 3.91,3.86 (s/ mixture was adjusted to be between 2 and 3 using 6 mol/L
2
each, 15H, 5OCH ), 2.95 (t, J = 5.1 Hz, 2H, CH ), 2.64-2.58 (m, hydrochloric acid, The mixture was extracted with dichlo-
3
2
4
H, 2NCH ), 1.60-1.53 (m, 4H, 2CH ), 1.42-1.38 (m, 2H, CH ). romethane, the organic layer was dried over anhydrous
2
2
2
13
C NMR (101MHz, CDCl ): δ 177.37, 161.13, 151.93, 151.47, magnesium sulfate, and concentrated under reduced pres-
3
1
49.23, 147.70, 147.19, 144.11, 138.04, 123.86, 119.65, 114.81, sure to obtain white powder, which was recrystallized with
1
11.20, 108.48, 106.71, 71.75, 61.94, 61.65, 58.36, 56.01, 54.56, ethanol and filtered to obtain 0.38 g compound 11 or 12.
+
2
5.47, 23.89. ESI-MS: m/z 500 [M+H] . calc:500.22.
5-(O-Ethoxyacetyl)-4’,6,7,8-tetramethoxyflavone
o
1
5
-(2-(4-Hydroxypiperidinyl) ethoxy)-3’,4’,6,7,8-pen- (9) yellow solid; 80% yield; m.p.110-112 C; H NMR (400
tamethoxyflavone (4e) yellow solid; 74% yield; m.p. MHz, CDCl ): δ 7.78 (d, J = 8.0 Hz, 2H, 2’-H and 6’-H), 6.94
1
1
3
o
1
95-197 C; H NMR (400 MHz, CDCl ): δ 7.47 (d, J = 8.2 Hz, (d, J = 8.0 Hz, 2H, 3’-H and 5’-H), 6.50 (s, 1H, 3-H), 4.61 (s,
3
H, 6’-H), 7.32 (s, 1H, 2’-H), 6.90 (d, J = 8.4 Hz, 1H, 5’-H), 2H, 3-OCH ), 4.22 (q, J = 6.9 Hz, 2H, CH ), 4.02, 3.95, 3.89,
2
2

L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkylꢁ
ꢁ7ꢂ
1
3
3
.80 (4s, 12H, 4OCH ), 1.25 (t, J = 7.0 Hz, 3H, CH ) ; C NMR
5-(O-Acetylglycine methyl ester)-4’,6,7,8-tetra-
3
3
(
101MHz, CDCl ): δ175.99, 168.00, 161.29, 160.27, 150.28, methoxyflavone (13a) yellow solid; 90% yield; m.p.164-
46.58, 145.02, 142.87, 137.51, 126.68, 122.63, 113.71, 113.48, 166 C; H NMR (400 MHz, CDCl ): δ 10.02 (s, 1H, CONH),
3
o
1
1
1
3
05.53, 70.08, 61.11, 61.03, 60.63, 59.95, 54.47, 13.21.
7.81 (d, J = 8.2 Hz, 2H, 2′-H and 6′-H), 6.96 (d, J = 8.3 Hz,
5
-(O-Ethoxyacetyl)-3’,4’,6,7,8-pentamethoxyfla- 2H, 3′-H and 5′-H), 6.56 (s, 1H, 3-H), 4.74 (s, 2H, 5-OCH ),
2
o
1
vone (10) yellow solid; 78% yield; m.p.112-114 C; H NMR 4.13 (d, J = 5.8 Hz, 2H, CH CO), 4.05, 3.95, 3.82, 3.81 (4s,
2
1
3
(
400 MHz, CDCl ): δ 7.47 (d, J = 8.4 Hz, 1H, 6’-H), 7.31 (s, 1H, 12H, 4OCH ), 3.68 (s, 3H, CH ). C NMR (101MHz, CDCl ): δ
3
3
3
3
2
2
2
’-H), 6.91 (d, J = 8.4 Hz, 1H, 5’-H), 6.51 (s, 1H, 3-H), 4.61 (s, 178.12, 170.22, 170.07, 162.60, 161.88, 152.05, 147.90, 146.84,
H, 5-OCH ), 4.22 (q, J = 6.9 Hz, 2H, CH ), 4.02, 3.95 (2s, 6H, 142.29, 137.79, 127.85, 123.37, 114.64, 112.93, 106.54, 73.65,
2
2
OCH ), 3.89(s, 6H, 2OCH ), 3.87 (s, H, OCH ), 1.24 (t, J = 62.15, 61.68, 61.45, 55.55, 52.15, 40.81.
3
3
3
13
7
.0 Hz, 3H, CH ); C NMR (101MHz, CDCl ): δ 175.95, 167.97,
5-(O-Acetylglycine methyl ester)-3’,4’,6,7,8-penta-
3
3
1
1
60.11, 150.93, 150.33, 148.23, 146.56, 145.07, 142.88, 137.44, methoxyflavone (13b) yellow solid; 87% yield; m.p.140-
o
1
22.83, 118.62, 113.69, 110.21, 107.49, 105.72, 70.09, 61.10, 142 C; H NMR (400 MHz, CDCl ): δ 10.02 (s, 1H, CONH),
3
6
0.95, 60.64, 59.95, 55.06, 54.92, 13.21.
7.48 (d, J = 8.4 Hz, 1H, 6’-H), 7.31 (s, 1H, 2’-H), 6.91 (d, J = 8.5
5
-(O-Carboxymethyl)-4’,6,7,8-tetramethoxyfla- Hz, 1H, 5’-H), 6.55 (s, 1H, 3-H), 4.72 (s, 2H, 5-OCH ), 4.11 (d,
2
o
1
vone (11) yellow solid; 76% yield; m.p.138-140 C; H NMR J = 5.7 Hz, 2H, CH CO), 4.04, 3.94 (2s, 6H, 2OCH ), 3.87 (s,
2
3
1
3
(
400 MHz, CDCl ): δ 13.93 (s, 1H, COOH), 7.83 (d, J = 8.1 6H, 2OCH ), 3.80 (s, 3H, OCH ), 3.67 (s, 3H, CH ). C NMR
3 3 3 3
Hz, 2H, 2’-H and 6’-H), 6.98 (d, J = 8.2 Hz, 2H, 3’-H and (101MHz, CDCl ): δ 177.04, 169.19, 169.06, 160.69, 151.25,
3
5
6
’-H), 6.63 (s, 1H, 3-H), 4.75 (s, 2H, 5-OCH ), 4.07, 3.97 (2s, 151.05, 148.32, 146.83, 145.75, 141.25, 136.67, 122.41, 118.81,
2
13
H, 2OCH ), 3.83 (s, 6H, 2OCH ); C NMR (101 MHz, CDCl ): 111.80, 110.30, 107.52, 105.60, 72.58, 61.03, 60.65, 60.41,
3
3
3
δ 177.75, 169.79, 162.07, 161.92, 151.38, 146.65, 145.38, 141.69, 55.08, 54.92, 51.12, 39.76.
1
6
37.27, 127.07, 121.91, 113.72, 111.09, 105.04, 71.23, 61.21,
5-(O-Acetylphenylalanine methyl)-4’,6,7,8-tetrame-
thoxyflavone (14a) yellow solid; 87% yield; m.p.104-106
+
0.72, 60.36, 54.57. ESI-MS: m/z 455[M+K] . calc: 455.11.
o
1
5
-(O-Carboxymethyl)-3’,4’,6,7,8-pentamethoxyfla-
C; H NMR (400 MHz, CDCl ): δ 9.94 (d, J = 7.8 Hz, 1H,
3
o
1
vone (12) yellow solid; 76% yield; m.p. 212-214 C; H NMR CONH), 7.68 (d, J = 8.6 Hz, 2H, 2’-H and 6’-H), 7.17-6.97 (m,
(
400 MHz, CDCl ): δ 7.53 (d, J = 8.4 Hz, 1H, 6’-H), 7.35 (s, 1H, 5H, C H ), 6.80 (d, J = 8.6 Hz, 2H, 3’-H and 5’-H), 6.46 (s,
3
6
5
2
’-H), 6.95 (d, J = 8.5 Hz, 1H, 5’-H), 6.66 (s, 1H, 3-H), 4.76 (s, 1H, 3-H), 4.76-4.70 (m, 1H, CH), 4.56 (s, 2H, 5-OCH ), 3.94,
2
2
H, 5-OCH ), 4.07, 3.97 (2s, 6H, 2OCH ), 3.91 (s, 6H, 2OCH ), 3.85, 3.67, 3.66 (4s, 12H, 4OCH ), 3.54 (s, 3H), 3.16 (d, J = 9.7
2
3
3
3
13
13
3
.83 (s, H, OCH ); C NMR (101 MHz, CDCl ): δ 178.78, 170.80, Hz, 2H, CH Ar). C NMR (101 MHz, CDCl ): δ 177.54, 172.12,
3
3
2
3
1
62.98, 152.67, 152.47, 149.48, 147.68, 146.44, 142.76, 138.24, 169.41, 162.44, 161.43, 151.75, 147.69, 146.60, 142.04, 137.66,
1
23.12, 120.20, 112.12, 111.36, 108.67, 106.29, 72.26, 62.16, 61.76, 137.18, 129.20, 128.23, 127.65, 126.55, 123.12, 114.50, 112.82,
+
6
1.41, 56.18, 56.03. ESI-MS: m/z 485[M+K] . calc:485.12.
106.44, 73.50, 61.99, 61.52, 61.28, 55.42, 53.76, 52.05, 37.63.
-(O-Acetylphenylalanine methyl)-3’,4’,6,7,8-penta-
methoxyflavone (14b) yellow solid; 84% yield; m.p.110-
5
o
1
General procedure for synthesis of amino acid ester deri- 112 C; H NMR (400 MHz, CDCl ): δ 9.92 (d, J = 7.9 Hz, 1H,
vatives of polymethoxyflavonoids 1ꢂa, 1ꢂb, 1ꢃa and 1ꢃb
3
CONH), 7.52 (d, J = 8.4 Hz, 1H, 6’-H), 7.34 (s, 1H, 2’-H), 7.22-
.06 (m, 5H, C H ), 6.93 (d, J = 8.5 Hz, 1H, 5’-H), 6.59 (s, 1H,
7
6
5
A mixture of 11 or 12 (0.48 mmol), EDCl (0.72 mmol), 3-H), 4.83-4.77 (m, 1H, CH), 4.64 (s, 2H, 5-OCH ), 4.03, 3.95,
2
4
-N,N-dimethylaminopyridine (DMAP, 0.96 mmol) and 3.91, 3.89, 3.76 (5s, 15H, 5OCH ), 3.61 (s, 3H, CH ), 3.21 (d, J
3
3
1
3
glycine methyl ester hydrochloride or L-phenylalanine = 16.2 Hz, 2H, CH Ar). C NMR (101 MHz, CDCl ): δ 176.76,
2
3
methyl ester hydrochloride (0.58 mmol) were dissolved 171.21, 168.36, 160.46, 151.20, 150.95, 148.35, 146.81, 145.86,
in 20 mL dichloromethane, and the reaction was carried 141.19, 136.66, 136.14, 128.25, 127.26, 125.56, 122.58, 118.77,
out at room temperature. The progress of the reaction 111.96, 110.29, 107.54, 105.81, 72.56, 61.03, 60.64, 60.37,
was followed by TLC. After the reaction was completed, 55.10, 54.98, 52.69, 51.13, 36.81.
the solvent was removed, and the residue was poured into
5
0 mL of HCl (1 mol/L), and extracted with CH Cl (3×20
2 2
mL). The organic phases were combined and dried over Synthesis of polymethylflavones amino acid derivatives
anhydrous Na SO . After filtration, the solvent was eva- ꢅa, ꢅb, 6a and 6b
porated to give a crude material. Purification by silica gel
2
4
column chromatography [V (petroleum ether): V (ethyl The solid of 13 or 14 (0.21 mmol) was dissolved in 10 mL
acetate) = 2:1] afforded compounds 13a, 13b, 14a and 14b. methanol, in a 100 mL round bottom flask, and 4 mL of

ꢁ
L-Q. Ran et al.: Synthesis and antiproliferative activities of polymethoxyflavones aminoalkyl
7ꢃꢁ
NaOH (1 mol/L) solution were added dropwise. The reaction 126.89, 123.20, 115.15, 112.84, 106.53, 73.52, 62.39, 61.91, 61.71,
+
was carried out at room temperature and the reaction pro- 55.99, 53.90, 37.24. ESI-MS: m/z 593 [M] . calc:593.19.
gress was tracked by TLC. After the reaction was complete,
the solvent of the reaction solution was removed and water
was added followed by HCl (1 mol/L) to adjust the pH to 2-3,
Antiproliferative Activity
The mixture was then placed in a refrigerator and allowed
The antiproliferative activities of synthetic compounds
to stand for filtration. The filter cake was dried and recrys-
against four human cancer cell lines (Aspc-1, SUN5, HepG-2
tallized from anhydrous C H OH to give a yellow solid.
2
5
and HCT116) were evaluated by CCK-8 assay in vitro using
Staurosporine as the positive control drug. All the cells
were cultured in an RPMI 1640 medium containing 10%
5
-(O-Acetylglycine)-4’,6,7,8-tetramethoxyflavone
o
1
(
5a) yellow solid; 89% yield; m.p. 180-182 C; H NMR (400
MHz, DMSO-d ): δ 9.69 (t, J = 5.6 Hz, 1H, CONH), 7.94 (d,
6
o
FBS, incubated at 37 C in a 5% CO humidified incubator
2
J = 8.5 Hz, 2H, 2’-H and 6’-H), 7.07 (d, J = 8.5 Hz, 2H, 3’-H
to keep the cells growing in the exponential phase. Briefly,
Aspc-1, SUN5, HepG-2 and HCT116 cells in a 100 μL culture
medium were plated into a 96-well plate at 4000-5000
cells per well, respectively, and subsequently cultured in
the RPMI 1640 medium containing 10% FBS, incubated
and 5’-H), 6.79 (s, 1H, 3-H), 4.62 (s, 2H, 5-OCH ), 4.06 (s,
2
3
H, OCH ), 3.98 (d, J = 8.6 Hz, 2H, CH COOH), 3.96, 3.86,
3
2
13
3
.83 (3s, 9H, 3OCH ). C NMR (101MHz, DMSO-d ): δ
3
6
1
77.30, 171.45, 169.27, 162.58, 161.37, 151.85, 147.60, 146.18,
1
42.57, 137.89, 128.25, 123.12, 115.04, 112.95, 106.36, 73.53,
o
at 37 C for 24h prior to drug exposure. The selected com-
+
6
2.33, 61.86, 61.77, 55.92, 40.85; ESI-MS: m/z 473 [M] .
pounds were weighed and dissolved in DMSO and then
diluted with medium to the needed concentrations. Cells
were treated with the final concentrations of 100, 50, 25,
calc:473.13.
5
-(O-Acetylglycine)-3’,4’,6,7,8-tetramethoxyfla-
o
1
vone (5b) yellow solid; 85% yield; m.p.110-112 C; H NMR
1
2.5, 6.25 and 3.125 μmol/L of the tested compounds and
(
(
400 MHz, DMSO-d ): δ 9.71 (t, J = 5.7 Hz, 1H, CONH), 7.63
6
incubated for 48h and then 20μL of 0.5% MTT solution
d, J = 8.5 Hz, 1H, 6’-H), 7.51 (s, 1H, 2’-H), 7.13 (d, J = 8.6 Hz,
was added to each well simultaneously and incubated at
3
1
H, 5’-H), 6.94 (s, 1H, 3-H), 4.63 (s, 2H, 5-OCH ), 4.07, 3.98
2
o
7 C for 4h. The formed formazan crystals were dissolved
(
2s, 6H, 2OCH ), 3.96 (d, J = 5.8 Hz, 2H, CH COOH), 3.87,
3
2
by adding 150 μL of DMSO. The optical density at 570 nm
was determined on a microtiter plate reader. According to
the inhibition ratios, the IC value was obtained.
13
3
.86, 3.84 (3s, 9H, 3OCH ). C NMR (101MHz, DMSO-d ) δ
3
6
1
77.41, 171.40, 169.28, 161.35, 152.41, 151.90, 149.45, 147.64,
50
1
1
46.22, 142.59, 137.84, 123.20, 119.95, 112.95, 112.24, 109.30,
06.62, 73.55, 62.32, 61.90, 61.78, 56.15, 56.09, 40.81. ESI- Conflict of interest: The authors confirm that this article
+
MS:m/z 503 [M] . calc:503.14.
content has no conflict of interest.
5
-(O-Acetylphenylalanine)-4’,6,7,8-tetramethoxy-
Acknowledgments: We thank the Hunan Provincial
Natural Science Foundation of China (NO.2018JJ2034) for
financial support.
flavone (6a) yellow solid; 82% yield; m.p.106-108 oC;
1
H NMR (400 MHz, DMSO-d ): δ 9.74 (d, J = 8.0 Hz, 1H,
6
CONH), 8.04 (d, J = 8.4 Hz, 2H, 2′-H and 6′-H), 7.35-7.16
(
m, 5H,C H ), 7.13 (d, J = 8.5 Hz, 2H, 3′-H and 5′-H), 6.93 (s,
6 5
1
H, 3-H), 4.58 (d, J = 9.0 Hz, 2H, 5-OCH ), 4.07, 3.99, 3.86,
2
References
.80 (4s, 12H, 4OCH ), 3.19 (d, J = 4.8 Hz, 2H, CH Ar). C
3 2
1
3
3
NMR (101 MHz, DMSO-d ): δ 177.48, 173.19, 168.85, 162.67,
6
[1] Miyata Y, Oshitari T, Okuyama Y, Shimada A, Takahashi H,
Natsugari H, et al. Polymethoxyflavones as agents that prevent
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1
1
6
61.48, 151.94, 147.65, 146.29, 142.42, 138.11, 137.85, 129.52,
28.65, 128.41, 126.89, 123.20, 115.15, 112.84, 106.53, 73.52,
+
2.39, 61.91, 61.71, 55.99, 53.90, 37.24. ESI-MS: m/z 563 [M] .,
calc:563.18.
[
2] Guy L, Alexandre M, Sylviane T, Geneviève A, Joëlle D, Thierry C.
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5
-(O-Acetylphenylalanine)-3’,4’,6,7,8-pentame-
thoxyflavone (6b) yellow solid; 82% yield; m.p.106-108
o
1
C; H NMR (400 MHz, DMSO-d ): δ 9.74 (d, J = 8.0 Hz, 1H,
[3] Tetsuta O, Yuji O, Yoshiki M, Hiroshi K, Hideyo T. Hideaki, N.
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CONH), 8.04 (d, J = 8.4 Hz, 2H, 2’-H and 6’-H), 7.35-7.16 (m,
5
H，C H ), 7.13 (d, J = 8.5 Hz, 2H, 3’-H and 5’-H), 6.93 (s, 1H,
6 5
2
011;21:4540–4.
3
-H), 4.58 (d, J = 9.0 Hz, 2H, 5-OCH ), 4.07, 3.99, 3.86, 3.80
2
[
4] Li S, Pan MH, Lai CS, Lo CY, Dushenkov S, Ho CT. Isolation and
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13
(
4s, 12H, 4OCH ), 3.19 (d, J = 4.8 Hz, 2H, CH Ar). C NMR (101
3
2
MHz, DMSO-d ): δ 177.48, 173.19, 168.85, 162.67, 161.48, 151.94,
6
1
47.65, 146.29, 142.42, 138.11, 137.85, 129.52, 128.65, 128.41,

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