全 文 ：Antitumor Alkaloids Isolated from Tylophora ovata
ZHEN Yue_Ying , HUANG Xue_Shi , YU De_Quan , YU Shi_Shan＊
(Institute of Materia Medica , Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing 100050 , China)
Abstract:　Four phenanthroindolizidine alkaloids , named tylophoridicine A (1), tylophorinine (2),
O_methyl tylophorinidine (3)and tylophorinidine (4), were isolated from the roots of Tylophora ovata
(Lindl.)Hook.ex Steud.Using modern NMR techniques including NOESY and 1H_NMR line broadening ef-
fect experiments , CD spectra and MS analysis as well as chemical methods , their structures were identified as(13aR)_6_hydroxy_3 , 7_dimethoxy_phenanthroindolizidine (1), (13aS , 14R)_14_hydroxy_3 , 6 , 7_trimethoxy_
phenanthro_indolizidine (2), (13aS , 14S)_14_hydroxy_3 , 6 , 7_trimethoxy_phenanthroindolizidine (3), and(13aS ,14S)_6 ,14_dihydroxy_3 , 7_dimethoxy_phenanthroindolizidine (4)respectively.Compound 1 is a new
compound , compounds 2-4 are obtained from this plant for the first time.Compounds 1 , 3 and 4 showed
strong antitumor activities.
Key words:　Tylophora ovata;phenanthroindolizidine alkaloid;tylophoridicine A;tylophorinine;O_methyl
tylophorinidine;tylophorinidine;antitumor
　　Tylophora ovata belongs to Asclepiadaceae.It is a
Chinese traditional toxic medicine.The alkaloid compo-
nents of T .ovata possess very strong antitumor activities
according to literature [ 1] .In order to find some more ef-
fective antitumor agents , we conducted a detailed investi-
gation of the alkaloid components of T.ovata.In this
paper , we describe the isolation and structure elucidation
of a new alkaloid named tylophoridicine A(1), and three
known compounds , viz., tylophorinine (2), O_methyl
tylophorinidine (3)and tylophorinidine (4).In vitro ,
antitumor activities of these compounds were tested.Com-
pounds 1 , 3 and 4 showed strong antitumor activities.
1　Results and Discussion
Tylophoridicine A (1)was isolated as white nee-
dles. The molecular formula was established as
C22H23NO3 by HRMS(M+ at m/ z 349.168 1).The frag-
ment peak at m/ z 280(M+-69)was due to the loss of
dihydropyrrole ring by retro_Diels Alder fission , suggest-
ing the presence of a dihydropyrrole ring.The UV absorp-
tions at 258(4.53), 286(4.39), 311(sh , 3.81)and
343 (2.91)nm were similar to those of phenanthroin-
dolizidine skeleton.After adding 1 mol/L NaOH solution ,
there was a bathochromic shift from 286 and 311(sh)nm
to 297 and 330 nm , showing that a phenolic hydroxyl
group was present.In 1H-NMR(CDCl3)spectrum of 1 ,
the signals at δ3.95 (3H , s)and δ3.94 (3H , s)
showed two methoxyl groups connected with an aromatic
ring.The signals at δ4.53 (1H , d , J =18 Hz)and
3.49(1H , d , J =18 Hz)were assigned to H_9αand H_
9β protons.In addition , five aromatic protons were ob-
served in
1
H_NMR spectrum.The protons at δ8.03(1H , s) and δ7.17 (1H , s) showed single sharp
signals.The other three signals of the aromatic protons
δ7.88(1H , d , J =9 Hz), δ7.17(1H , dd , J =9 , 2
Hz)and δ7.85 (1H , d , J =2 Hz)formed the AMX
system.
These data suggested that compound 1 possessed a
skeleton of phenanthroindolizidine alkaloid with two
methoxyl groups and one phenolic hydroxyl group.The
position of protons was conformed by NOESY(Fig.1).
In CD experiments , there was a positive Cotton ef-
fect , indicating the absolute configuration of 13a as R[ 2] .
On the basis of the above evidence , the structure of 1 was
concluded to be (13aR)_6_hydroxy_3 ,7_dimethoxy_
phenanthroindolizidine.
Compound 2 , an amorphous solid , [ α] 16D -0.2°,
Received:2001-05-31　Accepted:2001-09-14
Supported by the State Key Basic Research and Development Plan of China(G1998051120).
＊Author for correspondence.E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(3):349-353
Fig.1.　NOESY correlation for compound 1.
had a molecular formula of C23H25NO4 , as determined by
1H-NMR , 13C-NMR of its acetate data and EIMS (M+
379).The fragment peak at m/ z 310(M+-69)was due
to the loss of a dihydropyrrole ring by retro_Diels Alder
fission , suggesting the presence of dihydropyrrole ring.
The IR spectrum of 2 showed absorption of the hydroxyl
group at 3 504 cm-1.The UV absorptions at 256(4.66), 285(4.40), 311(sh , 3.88), 338(3.12)nm
were similar to those of phenanthroindolizidine skeleton.
After adding 1 mol/L NaOH solution , there was no
bathochromic shift , showing no phenolic hydroxyl group.
In 1H-NMR spectrum of 2 , existing in form of a dimer[ 1] ,
H_1 to H_8 were in a chiral surroundings , so it gave two
sets of
1
H_NMR data (Table 2).After acetylation , the
dimer was destroyed , its acetate (2a)showed one set of
1H_NMR , 13C_NMR data , rotation was [ α] 25D -22.2°(c
0.27 , CHCl3).In 1H_NMR(CDCl3)spectrum of acetate
2a , the signals atδ4.01(3H , s), δ4.11(3H , s)andδ4.06(3H , s)showed three methoxyl groups connected
with an aromatic ring.The signals atδ4.78(1H , d , J=16.0 Hz)andδ3.62(1H , d , J =16.0Hz)were as-
signed to H_9αand H_9β protons;In addition , five aro-
matic protons were shown in 1H_NMR spectrum.Two sin-
gle sharp signals appeared atδ7.91(1H , s)andδ7.19(1H , s).The other three signals of aromatic protons atδ
7.86(1H , d , J =9.0 Hz), δ7.22 (1H , dd , J =
9.0 , 2.0 Hz)andδ7.90(1H , d , J =2.0 Hz)formed
AMX system.The foregoing evidence suggested that com-
pound 2 possessed a skeleton of phenanthroindolizidine al-
kaloid with three methoxyl groups and one hydroxyl
group.The position of methoxyl and hydroxyl groups and
contribution of protons were conformed by NOESY(Fig.
2).
Fig.2.　NOESY correlations for acetate of compound 2.
The CD spectrum of 2a showed a negative Cotton ef-
fect , indicating that the absolute configuration of H_13a
was S
[ 2] .In the 1H-NMR line broadening effect experi-
ments
[ 3] , 1H-NMR line of H_14 did not broaden at all ,
but H_13a broadened when adding a small quantity of
DCl , which meant the lone pair of electrons of nitrogen
was on the same side withH_13a but not on the same side
with H_14 , suggesting that the absolute configuration of
H_14 was R (not reported before).Therefore , the struc-
ture of 2 was established as(13aS , 14R)_14_hydroxy_3 ,
6 ,7_trimethoxy_phenanthroindolizidine , which was named
as tylophorinine when it was previously isolated
[ 1 ,3 ,5] .
The spectral data of O_methyl tylophorinidine (3)
were similar to those of tylophorinine , but differed in rota-
tion.The fact suggests that O_methyl tylophorinidine has
the same structure as tylophorinine.In CD experiment ,
its negative Cotton effect showed that the absolute configu-
ration of H_13a was S [ 2] .In the 1H-NMR line broadening
effect experiments[ 6] , 1H-NMR lines of H_13a and H_14
did not broaden at all , when adding a small quantity of
DCl , which meant the lone pair of electrons of nitrogen
was not on the same side with H_13a and H_14 , suggest-
ing that the absolute configuration of H_14 was S.With
the above evidence the structure of 3 was elucidated as(13aS , 14S )_14_hydroxy_3 , 6 , 7_trimethoxy_phenan-
throindolizidine , which was named as O_methyl ty-
lophorinidine when it was previously isolated[ 4 , 5] .
Compound 4 was identified as tylophorinidine by
comparing its spectral data (UV , IR , 1H_NMR and
13C_NMR)with literature [ 1 ,4 ,5] .
Antitumor activities of compounds 1 , 3 and 4 were
induced by the method of MTT.The results are shown in
Table 2.Compound 4 showed ED50 values of <5×10-3μg/mL in KB cell , and compounds 1 and 3 showed ED50
values of 0.029 and 0.200 μg/mL respectively in KB
cell.Compounds 1 , 3 and 4 also inhibited A549 cell
growth and the ED50 value is 6.36 , 7.86 and 5.1μg/mL , respectively.
2　Experimental
2.1　General experimental procedures
IR spectra were recorded as KBr discs from the
Perkin_Elmer 683 IR spectrometer;UV spectra were
recorded in MeOH from a Shimadzu UV_240 UV_visible
recording spectrophotometer;1H_NMR and 13C-NMR
spectra as well as 2D NMR experiments were taken with
TMS as internal standard from a Bruker AM 500 FT_NMR
spectrometer.High resolution mass spectra were measured
with an Autospec_ultima TOF (Micromass)spectrometer
and low resolution mass spectra were measured with a VG
ZAB_2f spectrometer.Optical rotations were measured
with the Perkin Elmer 214 polarimeter;CD spectra were
measured with a JOUAN Mark Ⅱ spectropolarimeter.Sil-
ica GF254 for TLC , silica H for low_pressure column
chromatography and silica gel(200-300 mesh)for col-
umn chromatography were purchased from Qingdao Marine
Chemical Company , Qingdao , China.
350　 植物学报　Acta Botanica Sinica　Vol.44　No.3　2002
Table 1　Assignments of absolute stereochemistry to compounds 1-4
Alkaloid CD
H_13a absolute
configuration
1H-NMR line broadening effect
H_13a H_14
H_14 absolute
conf iguration
1 + R
2a - S +(N electrons pai r and H_13a:Cis) -(N electrons pair and H_14:Cis) R
3 - S -(N electrons pair and H_13a:Trans) -(N electrons pair and H_14:Cis) S
4 - S
+, the peak of proton broadened and blunted when adding a small quantity of DCl;-, the peak of proton has no change when adding a small quantity of DCl.
Table 2　Antitumor activity(MTT method)
Compound Cell ED50 (μg/mL)
1 A549 6.36
KB 0.029
3 A549 7.86
KB 0.200
4 A549 5.1
KB <0.005
2.2　Plant materials
Roots of Tylophora ovata (Lindl.)Hook.ex Steud.
were collected in Nov.1996 from Zhuang Autonomous
Region of Guangxi , China.It was harvested and air_dried
at room temperature.The plant was identified by Prof.
LIU Shou_Yang , Department of Pharmacognosy , Guangxi
College of Chinese Traditional Medicine.
2.3　Extraction and isolation
The roots (11.0 kg)were powdered and extracted
Table 3　1H_NMR(500MHz)data for compounds 1-4
Compound 1 2
1 δ7.88(d , J=9 Hz) δ8.40(d, J =9Hz) δ8.41(d , J =9 Hz)
2 δ7.17a (dd , J =9 , 2 Hz) δ7.25(dd , J =9, 2 Hz)
4 δ7.85b (d , J=2 Hz) δ7.74(d, J =2Hz) δ7.73(d , J =2 Hz)
5 δ8.03b s δ7.59 s δ7.60 s
8 δ7.17c ,d s δ6.25 s δ6.28 s
9 δ4.53d (d , J =16 Hz) δ3.50(d , J =16 Hz) δ3.52(d , J =16 Hz)
δ3.49(d , J =16 Hz) δ3.09(d , J =16 Hz) δ3.07(d , J =16 Hz)
11 δ2.30 m , 3.38 m δ2.40 m , 3.32 m
12 δ1.81-1.84 m δ2.02 m , 2.40 m
13 δ1.61 m , 2.12 m δ1.90 m , 2.25 m
13a δ2.36 m δ2.40 m
14 δ2.74 m , 3.03 m δ4.93 brd
OMe_3 δ3.95a s δ4.00
OMe_6 δ4.09
OMe_7 δ3.94c s δ3.83 δ3.84
Compound 2a 3 4
1 δ7.86a (d , J=9 Hz) δ8.44a (d , J =9 Hz) δ8.17a (d , J =9 Hz)
2 δ7.22(dd , J =9 , 2 Hz) δ7.25(dd , J=9 , 2 Hz) δ7.18(dd , J=9 , 2 Hz)
4 δ7.90b ,c (d, J =2Hz) δ7.69b , c (d , J=2 Hz) δ7.83b , c (d , J =2 Hz)
5 δ7.91c s δ7.50c , d s δ8.03c s
8 δ7.19e , f s δ6.01e ,f s δ7.13d s
9 δ4.78e (d , J=16 Hz) δ3.23(d , J =16 Hz) δ4.48(d , J =16 Hz)
δ3.62(d , J =16 Hz) δ2.91f (d , J=16 Hz) δ3.40(d , J =16 Hz)
11 δ2.45 m , 3.53 m δ2.18 m , 3.29 m δ2.30 m , 3.33 m
12 δ1.94 m , 2.02 m δ1.87 m , 2.05 m δ1.77-1.85 m
13 δ1.69 m , 2.08 m δ1.87 m , 2.41 m δ1.77-1.85 m , 2.16 m
13a δ2.71 m δ2.20 m δ2.33 m
14 δ6.71 brd δ4.87a brd δ4.88a brd
OMe_3 δ4.01b δ4.04b s δ3.95b
OMe_6 δ4.11 δ4.08d s
OMe_7 δ4.06d δ3.77e s δ3.94d
Compounds 2 , 3 and 2a in CDCl3 , compounds 1 and 4 in DMSO_d6;a to f mean a cross peak was observed between these signals in the 2D_NOESY spectrum;
Assignments were based upon DEPT , NOESY and HMQC experiments.
ZHEN Yue_Ying et al:Antitumor Alkaloids Isolated from Tylophora ovata 351　
with 95%EtOH at boiling point(4 h , 2 h , 2 h), and
then concentrated to a green_brown gummy residue (1.1
kg).A 0.5%HCl_soluble portions of the residue were
made alkaline (pH 9)with 30%NH4OH solution , and
then extracted repeatedly with CHCl3 until the Dragendorff
test was negative.A crude extract of 35 g total crude al-
kaloids was obtained.
The crude extract (30 g)was chromatographed on
silica gel column eluting successively with CHCl3 and
CHCl3_MeOH gradients.The fractions TO_1 (7.2 g),
TO_2(1.0 g), TO_3 (9.0 g), TO_4 (7.0 g), TO_5(1.0 g), and TO_6(3.0 g)were obtained.
Fraction TO_3 (9.0 g)was further purified using
silica gel column with CHCl3_Acetone (85∶15)to yield
compound 2(1.0 g)and compound 4(1.0 g).
Repeated column chromatography and low pressure
column chromatography of fraction TO_5(7.0 g)over sil-
ica gel and silica gel H column with benzene_ethyl ac-
etate_MeOH (80∶10∶10)gave compound 3(20 mg)and
compound 1(35 mg).
Table 4　13C_NMR(125 MHz)data for compounds 1-4
Compound 1 2a 3 4
1 124.81 128.76 126.78 126.41
2 115.43 115.44 114.70 115.50
3 157.15 157.86 157.60 157.17
4 103.52 105.15 104.22 103.33
5 107.76 104.05 103.13 107.75
6 148.55 149.73 148.75 148.58
7 148.75 149.46 148.56 146.52
8 103.73 103.79 102.63 103.91
9 53.21 53.86 53.22 53.53
11 55.14 54.91 55.73 55.17
12 21.14 21.59 21.62 21.54
13 30.74 25.54 23.91 23.93
13a 59.75 63.59 65.51 64.83
14 32.87 66.78 64.41 63.57
OMe_3 55.50 55.51 55.61 55.51
OMe_6 56.04 55.73
OMe_7 55.50 56.04 55.61 54.81
Ring C 123.11 124.71 123.78 123.60
124.17 124.82 125.04 123.97
124.62 128.76 125.37 124.68
125.43 130.89 128.75 126.35
125.50 - 130.63 128.69
129.65 - - 130.04
The assignment was based upon DEPT , NOESY and HMQC experiments;
Compounds 3 and2a in CDCl3 , compounds 1 and 4 in DMSO_d6;-, signals
overlapped.
2.4　Identification
Tylophoridicine A (1)　Needles (acetone), mp
226-228 ℃, [α] 23D +176.0°(c 0.25 , CHCl3).IR
νKBrmax cm-1:3 531(OH), 1 618 , 1 512 , 1 464.EIMS:
m/ z(ret.)349 (M+ , 28), 280 ([ M+-69] , 100).
UVλMeOHmax (logε):258(4.53), 286(4.39), 311(sh ,
3.81)and 343(2.91)nm.After adding 1 mol/L NaOH
solution , there was a bathochromic shift from 286 , 311(sh)nm to 297 , 330 nm.From HRMS , the molecular
formula is known as C22H23NO3 (M+ 349.168 1 , calcu-
lated molecular weight: 349.167 8). 1H_NMR ,
13C_NMR and NOESY data are given in Tables 3 and 4.
Tylophorinine(2)　White powder (CHCl3), mp
246-248 ℃, [ α] 16D -0.2°(c 0.53 , CHCl3).EIMS
m/z:379(M+ , 13), 310(100), 70(35), 43(13).
UVλMeOHmax (logε):256(4.66), 285(4.40), 311(sh ,
3.88), 338(3.12)nm.After adding 1 mol/L NaOH to
the solution , there was no bathochromic shift.IR νKBrmax
cm-1:3 504 , 3 421 , 3 176 , 1 616 , 1 530 , 1 510 ,
1 471 , 1 419 , 1 257 , 1 232 , 1 203 , 1 165 , 1 036 ,
837.1H-NMR(CDCl3 , 500MHz)data see Table 3(be-
cause of its insolubility , its 13C-NMR data was not ob-
tained).
2a　White powder(in acetone), mp 223-226 ℃,
[ α] 25D -22.2°(c 0.27 , CHCl3).EIMS m/ z:421
(M+ , 2), 361(100), 330(56), 310(84).1H_NMR ,
13C_NMR and NOESY data are given in Tables 3 and 4.
The 2a was synthesized by mixing Ac2O , pyriding and
compound 2 with overnight stirring at room temperature.
O_Methyl tylophorinidine (3)　Crystals (ace-
tone), mp 226 -228 ℃, [ α] 23D +52.3°(c 0.23 ,
CHCl3).IRνKBrmax cm-1:3 197 (OH), 1 616 , 1 512 ,
1 471.EIMS:m/ z (ret.Int.)379 (M+ , 13), 310
([M+-69] , 100), 70(72).UV λMeOHmax (log ε):257(4.68), 284(4.56), 309(sh , 4.06)and 340(3.46)
nm.After adding 1 mol/L NaOH to the solution , there
was no bathochromic shift.From HRMS , the molecular
formula is known as C23H25NO4(M+ 379.174 7 , calcu-
lated molecular weight: 379.178 3). 1H_NMR ,
13C_NMR and NOESY data see Tables 3 and 4.
Tylophorinidine(4)　White powder(CHCl3), mp
216-217 ℃, [ α] 16D +84.8°(c 0.84 , CHCl3).EIMS
m/z:365(M+ , 15), 396(100), 70(20).UV λMeOHmax(log ε):257 (4.52), 285(4.26), 312(sh , 3.76),
341(2.98)nm.After adding 1 mol/L NaOH solution ,
there was a bathochromic shift from:256 (4.45), 295
(4.25), 333(3.77)nm.IRνKBrmax cm-1:3 541 , 3 421 ,
1 616 , 1 537 , 1 508 , 1 462 , 1 421 , 1 259 , 1 230 ,
1 200 , 1 163 , 1 041 , 820.1H_NMR(DMSO_d6 , 500
MHz)and 13C_NMR(DMSO_d6 , 125MHz)data see Ta-
bles 3 and 4.
References:
[ 1] 　Institute of Medical and Pharmaceutical Science of Zhuang
Autonomous Region of Guangxi(广西壮族自治区医药研
究所).Experimental studies on antitumor agents of Ty-
lophora ovata (Lindl.)Hook.ex Steud.Acta Biochim Bio-
phys Sin(生物化学与生物物理学报), 1977 , 9(2):a.
131-138 , b.139-145.[ 2] 　Gellert E , Rudzats R , Craig J C.The absolute configuration
of cryptopleurine and tylocrebrine.Aust J Chem , 1978 ,
31:2095-2097.[ 3] 　Phillipson J D , Tezcan I , Hylands P J.Alkaloids of Ty-
lophora species from Sri lanka.Planta Med , 1974 , 25:
301-309.
352　 植物学报　Acta Botanica Sinica　Vol.44　No.3　2002
[ 4 ] 　Govindachari T R, Viswanathan N.Minor alkaloids of Ty-
lophora asthmatica revised structure of tylophorinidine.
Tetrahedron , 1973 , 29:891-897.[ 5 ] 　Mulchandani N B , Venkatachalam S R.Alkaloids of Per-
gularia pallida.Phytochemistry , 1976 , 15:1561-1563.
[ 6 ] 　He X(贺湘), Lin W_H(林文翰), Xu R_S(徐任生).
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卵叶娃儿藤中的抗癌活性生物碱
甄月英　黄学石　于德泉　庾石山＊
(中国医学科学院中国协和医科大学药物研究所 , 北京 100050)
摘要:　从卵叶娃儿藤(Tylophora ovata (Lindl.)Hook.ex Steud.)根中分离得到 4个菲骈吲哚里西丁类生物碱 ,分别
为 tylophoridicine A(1)、娃儿藤宁(2)、氧甲基娃儿藤定(3)和娃儿藤定(4)。经波谱分析并结合化学方法鉴定 ,其结
构分别为:(13aR)_6_羟基_3 , 7_二甲氧基菲骈吲哚里西丁 、(13aS , 14R)_14_羟基_3 , 6 , 7_三甲氧基_菲骈吲哚里西丁 、
(13aS , 14S)_14_羟基_3 , 6 , 7_三甲氧基_菲骈吲哚里西丁和(13aS , 14S)_6 , 14_二羟基_3 , 7_二甲氧基_菲骈吲哚里西丁。
化合物1 为新化合物 , 化合物 2-4为首次从该植物得到的化合物。经药理筛选 ,化合物 1、3 和 4 显示很强的抗癌
活性。
关键词:　卵叶娃儿藤;菲骈吲哚里西丁类生物碱;tylophoridicine A;娃儿藤宁;氧甲基娃儿藤定;娃儿藤定;抗肿
瘤
中图分类号:R914　　　文献标识码:A　　　文章编号:0577-7496(2002)03-0349-05
收稿日期:2001-05-31　接收日期:2001-09-14
基金项目:国家重点基础研究发展规划项目(G1998051120)。
＊通讯作者。E_mai l:.
(责任编辑:王　葳)
ZHEN Yue_Ying et al:Antitumor Alkaloids Isolated from Tylophora ovata 353