Antiproliferative cardenolides from Pentopetia...

Indian Journal of Experimental Biology

Vol. 48, March 2010, pp. 248-257

Antiproliferative cardenolides from Pentopetia androsaemifolia Decne.

from the Madagascar rain forest†

aEba Adou,

bJames S Miller

‡,

cFidisoa Ratovoson,

cChris Birkinshaw,

dRabodo Andriantsiferana,

dVincent E Rasamison &

aDavid G I Kingston

1

aDepartment of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA bMissouri Botanical Garden, PO Box 299, St. Louis, Missouri 63166-0299,

cMissouri Botanical Garden, BP 3391, Antananarivo 101, Madagascar, dCentre National d’Application des Recherches Pharmaceutiques, BP 702, Antananarivo 101, Madagascar

Received 4 November 2009; revised 11 December 2009

Plant natural products have historically been very important to drug discovery and development, particularly in the

anticancer field. This is illustrated by a discussion of the structures and activities of camptothecin and its analogues,

paclitaxel (Taxol®), the vinca alkaloids vinblastine and vincristine, and podophyllotoxin and its analogues. A description of

the isolation of one new and three known cardenolides from the Madagascar plant Pentopetia androsaemifolia is then

provided as an example of this approach to drug discovery. The paper concludes with a brief discussion of betulinic acid, an

old compound which is being developed into an anticancer and anti-HIV agent, and ipomoeassin F, an interesting

antiproliferative compound isolated from a plant collected in Suriname.

Keywords: Biodiversity, Cardenolides, Drug discovery, Pentopetia androsaemifolia

Over the centuries, people have been living in close

association with the environment and relying on its flora

and fauna as a source of food and medicine. As a result,

many societies have their own rich plant pharmacopeias.

In developing countries, due to economic factors, nearly

80% of the population still depends on the use of plant

extracts as a source of medicine.

Natural products also play an important role in the

health care system in developed countries. The

isolation of the analgesic morphine from the opium

poppy, Papaver somniferum, in 1816 led to the

development of many highly effective pain relievers1.

Discovery of penicillin from the filamentous fungus,

Penicillium notatum, by Fleming in 1929 had a great

impact on the investigation of nature as a source of new

bioactive agents2. Natural products can also be used as

starting materials for semisynthetic drugs. The main

examples are plant steroids, which led to the

manufacture of oral contraceptives and other steroidal

hormones. Almost every pharmacological class of drugs

contains a natural product or natural product analogue3.

The investigation of higher plants has led to the

discovery of many new drugs. So far only a small

portion of higher plants has been investigated.

Consequently, plants still remain a large reservoir of

useful chemical compounds not only as drugs, but

also as templates for synthetic analogues.

Efforts to find anticancer agents from higher plants

were launched by the US National Cancer Institute

(NCI) in 1957. So far plants have been a proven

source of useful antitumour substances. Many of the

useful and curative anticancer drugs are derived from

natural product sources4. Since the initiation of the

program by NCI, more than 35,000 plant species have

been investigated. Investigations of plants by various

groups have produced the discovery of anticancer

drugs such as vincristine, vinblastine, taxol, indicine-

N-oxide, etoposide and its analogues, camptothecin

and its analogues, etc.

Camptothecin (1), isolated from Camptotheca

acuminata5, is too insoluble for drug use, but its

______________ 1Correspondent author

Telephone: +1-540-231-6570

E-mail: [email protected]

‡ Present address:

The New York Botanical Garden, Bronx, NY 10458-5126

___________ †Biodiversity Conservation and Drug Discovery in Madagascar,

Part 41. For Part 40, see Hou Y, Cao S, Brodie P J, Miller J S,

Birkinshaw C, Andrianjafy M N, Andriantsiferana R, Rasamison V

E, TenDyke K, Shen Y, Suh E M & Kingston D G I, Euphane

triterpenoids of Cassipourea lanceolata from the Madagascar rain

forest, Phytochemistry, 71 (2010);

doi:10.1016/j.phytochem. 2009.12.009

ADOU et al.: ANTIPROLIFERATIVE CARDENOLIDES FROM PENTOPETIA ANDROSAEMIFOLIA

249

analogues topotecan and irinotecan (Fig. 1) are used

to treat gastric, rectal, colon, and bladder cancers.6

They act on both DNA and RNA synthesis by

inhibiting the enzyme topoisomerase I, which results

in protein-linked breakdown of DNA.7-9

Taxol (paclitaxel) (3), isolated from Taxus

brevifolia10

(Fig. 2), is used in the treatment of

ovarian and breast cancers11

. It binds to the β-tubulin

subunit of microtubules and stabilizes the microtubule

to normal disassembly12

. This results in mitotic block

and ultimately in cell death by apoptosis13-14

. Its

chemistry and biological activity have already been

reviewed15-17.

Vinblastine (4) and vincristine (5), isolated from

Catharanthus roseus (Fig. 3), are used to treat

leukemia, bladder and testicular cancers18

. Their mode

of action is to bind to tubulin and stop its

polymerization into microtubules, thus blocking cell

division19-20

.

Etoposide (6) and its thiophene analogue,

teniposide (7) are semisynthetic derivatives of the

natural product epipodophyllotoxin (8) (Fig. 4), and

are used clinically to treat small-cell lung cancer,

testicular cancer, lymphomas and other cancers21

.

They inhibit the enzyme DNA topoisomerase II and

cause DNA cleavage22-23

.

In view of this history of successful anticancer drug

development from natural products, it is reasonable to

believe that more antitumor compounds still exist in

nature, and that they can be discovered with

appropriate strategies, resources and effort.

The first step in the discovery of new natural

product drugs from plants is the selection of the plant

species to be investigated. Since tropical rain forests

in developing countries account for a large portion of

the world’s available plant biomass, these are

important sources of new and potentially important

drugs. Due to deforestation for economic and other

reasons, the rain forests with their valuable

ecosystems are disappearing rapidly. Since drug

discovery from botanical sources requires the

screening of many extracts, the loss of plants and

marine organisms is thus a significant barrier to drug

discovery.

The International Cooperative Biodiversity Group

(ICBG) program started in 1992 to promote

biodiversity conservation, drug discovery and

economic development in developing countries. The

objective of this program is to discover new drugs and

to share any benefit with host countries if a drug is

discovered from their botanical sources. The ICBG

program at Virginia Polytechnic Institute and State

University currently works in Madagascar, and the

Fig. 1—Structures of camptothecin and its analogues. (compound 1, 2a, 2b)

Fig. 3—Structures of vinblastine and vincristine. (compound 4, 5)

Fig. 2—Structure of taxol (paclitaxel; compound 3).

INDIAN J EXP BIOL, MARCH 2010

250

isolation of some cardenolides from Pentopetia

androsaemifolia Decne is described as an example of

this approach to drug discovery.

Materials and Methods

General experimental procedures—Optical

rotations were measured with a Perkin-Elmer Model

241 polarimeter. The NMR spectra were obtained on

either a JEOL Eclipse 500 spectrometer or on a Inova

400 spectrometer. The mass spectra were obtained on

a JEOL JMS-HX-110 instrument. A flash

chromatograph from Biotage Inc. was used for flash

chromatography. HPLC was performed on a

Shimadzu LC-10AT instrument using a Varian

Dynamax C18 column (250 × 10 mm). C-18 SPE

columns were obtained from Supelco.

Cytotoxicity bioassays—The A2780 human ovarian

cancer cell line cytotoxicity assay was performed at

Virginia Polytechnic Institute and State University as

previously described24

.

Plant material—Roots, stems and leaves of

Pentopetia androsaemifolia Decne (Asclepiadaceae)

were collected by F. Ratovoson and his assistants

from secondary forest at an elevation of 800 m in the

vicinity of Vohimena, Madagascar, on 08 December,

2001. The collection coordinates were 17°22'16"S

048°37'55"E. The plant was identified and

authenticated by F. Ratovoson and was assigned

collector number 616. It was a lianescent shrub with

yellow flowers and a white latex, and inflorescenses

in false umbels. Its vernacular name in Malagasy is

Tandrokapora. Voucher specimens have been

deposited at herbaria of the Centre National

d’Application des Recherches Pharmaceutiques,

Madagascar (CNARP); the Parc Botanique et

Zoologique de Tsimbazaza, Madagascar (TAN); the

Missouri Botanical Garden, St. Louis, Missouri (MO);

and the Muséum National d’Histoires Naturelles,

Paris, France (P).

Extraction and isolation—The roots and stem of

the dried plant material were extracted with EtOH to

yield extracts MG 1228 and 1229. Extraction of 460 g

of roots and 467 g of stems gave 18.7 and 19.3 g of

extract, respectively, of which 4.7 g and 4.5 g were

made available for this work. Combined extracts MG

1228 and 1229 from Pentopetia androsaemifolia (2 g)

were partitioned between hexane and MeOH:H2O,

60:40, and the latter extract was diluted to

MeOH:H2O, 50:50, and extracted with CH2Cl2. All

the resulting fractions were evaporated to dryness and

tested for their biological activity. The CH2Cl2 and

MeOH fractions were the most active with IC50 values

of 0.4 µg/ml and 0.4 µg/ml, respectively. The MeOH

fraction was then partitioned between BuOH and

water, and tested for their activity. The BuOH fraction

was the only active fraction. Thin layer

chromatography (TLC) revealed that both the CH2Cl2

and BuOH fractions contained almost the same

constituents. Therefore, they were combined and

purified through reversed phase preparative HPLC to

yield compounds 9 12 (Figs 5-9).

Compound 9 (Fig. 5)White amorphous powder

(15 mg); 1H NMR (500 MHz, CD3OD) selected

chemical shifts; δ 0.88 (s, 3H, H3-18), 0.92 (s, 3H, H3-

19), 2.83 (dd, 1H, J = 9.0, 6.0 Hz, H-17), 4.91 (dd,

1H, J = 19, 1.3 Hz, H-21), 5.02 (dd, 1H, J = 19, 1.2

Hz, H-21), 5.89 (brs, 1H, H-22). 13

C NMR (500 MHz,

C5D5N) (Table 1); HRFABMS m/z found 881.4145

[M + Na]; calcd for C42H66NaO18 881.4147.

Compound 10 (Fig. 6) White amorphous powder

(8 mg); 1H NMR (500 MHz, CD3OD); selected

chemical shift δ 0.87 (s, 3H, H3-18), 0.93 (s, 3H, H3-

19).δ 2.83 (dd, 1H, J = 9.0, 6.0 Hz, H-17), 4.33 (dd, J

= 9.4, 2.0 Hz, 1′-H), 4.92 (dd, 1H, J = 19, 1.3 Hz, H-

21), 5.02 (dd, 1H, J = 19, 1.2 Hz, H-21), 5.89 (brs,

1H, H-22).13

C NMR (500 MHz, C5D5N), (Table 1);

HRFABMS m/z found 719.3614 [M + Na]; calcd for

C36H56NaO13 719.3618.

Compound 11 (Fig. 8)White amorphous powder

(7.5 mg); 1H NMR (500 MHz, C5H5N) selected

Fig. 4—Structures of podophyllotoxin analogues. (compound 6, 7, 8)


251

chemical shifts; δ 0.86 (s, 3H, H3-19), and 0.98 (s,

3H, H3-18), 2.75 (dd, 1H, J = 9.0, 6.0 Hz, H-17), 4.98

(dd, 1H, J = 19.7, 1.6 Hz, H-21), 5.30 (dd, 1H, J =

19.7, 1.2 Hz, H-21), 6.02 (br s, 1H, H-22). Sugar

moiety: δ 5.07 (d, 1H, J = 7.8 Hz, H-1′), 2.30 (ddd,

2H, J = 14.2, 3.2, 2.1 Hz, H2-2′), 1.90 (ddd, 2H, J =

12.5, 12.0, 9.5 Hz, H2-2′), 4.04 (brd, 1H, J = 3 Hz, H-

3′), 3.61 (br d, 1H, J = 5.0 Hz, H-4′), 4.12 (dq, 1H, J

= 10, 6.0 Hz, H-5′), 1.61 (d, 3H, J = 6 Hz, H-6′), 3.41

(s, 3H, 3′-O-CH3), 4.65 (d, 1H, J = 7.8 Hz, H-1′′),

4.37 (m, 1H, H-2′′), 3.50 (dd, 1H J = 9.9, 3.2 Hz, H-

3′′), 4.25 (dd, 1H, J = 4.5, 2.5 Hz, H-4′′), 3.69 (d, 1H,

J = 6.4 Hz, H-5′′), 1.54 (d, 3H, J = 6.2 Hz, H-6′′),

3.64 (s, 3H, 3′′-O-CH3), 5.16 (d, 1H, J = 9.4 Hz, H-

1′′′), 3.92 (t, 1H, J = 7.8 Hz, H-2′′′), 4.18 (t, 1H, J = 8

Hz, H-3′′′), 3.93 (m, 1H, H-4′′′), 4.08 (m, 1H, H-5′′′),

4.56 (br d, 2H, J = 10.2 Hz, H-6′′′), 4.25(br d, 2H, J =

10 Hz, H-6′′′). 13

C NMR (C5D5N, Table 2);

HRFABMS m/z found 863.4401 [M + Na]; calcd for

C43H68NaO16 863.4405.

Fig. 5—Structure of compound 9.


Fig. 7—HMBC correlations of compound 11.


252

Results

Pentopetia androsaemifolia Decne belongs to the

family Asclepiadaceae. No previous chemical studies

of this plant have been reported. Roots, stems and

leaves of this plant were collected in Madagascar as a

part of the ICBG program in order to investigate their

potential anticancer activity, and extracts of the roots

and stems were found to have antiproliferative

activity against the A2780 ovarian cancer cell line.

These extracts were, thus, selected for detailed

examination.

The EtOH extract of the roots and stems of

Pentopetia androsaemifolia was subjected to solvent

partitioning between hexane and aqueous MeOH, and

the aqueous MeOH fraction was then diluted with

H2O and extracted with CH2Cl2. The three resulting

fractions were evaporated under reduced pressure and

tested for their biological activities against the A2780

ovarian cancer cell line. The CH2Cl2 and aqueous

MeOH fractions were both active fractions. The

aqueous MeOH fraction was then partitioned between

BuOH and water, and both fractions tested for their

activity. The BuOH fraction was the only active

fraction from this partition. Thin layer

chromatography (TLC) revealed that both the active

CH2Cl2 and BuOH fractions contained similar

constituents. They were, therefore, combined and

purified through reversed phase preparative HPLC

using MeOH/H2O (6/4) as mobile phase to yield three

known cardenolide glycosides (9, 10, and 11) and one

new cardenolide glycoside 12.

Compound 9 was isolated as a white amorphous

powder, and its HRFABMS indicated a molecular

formula of C42H66O18. Its 1H NMR spectrum revealed

two singlets at δ 0.88 (s, 3H, H3-18), and 0.92 (s, 3H,

H3-19). Diagnostic peaks were also observed at δ 2.83

(dd, 1H, J = 9.0, 6.0 Hz, H-17), δ 4.91 (dd, 1H,

J = 19, 1.3 Hz, H-21), δ 5.02 (dd, 1H, J = 19, 1.2 Hz,

Fig. 8—Structure of compound 11 showing fragments a and b.



253

Table 213C NMR and 1H NMR spectra of compound 11

δH δC

a Litb

Aglycon

1 30.8 29.9

2 27.3 27.0

3 73.4 72.9

4 30.6 31.0

5 37.0 37.0

6 27.0 27.2

7 21.5 21.6

8 41.9 42.0

9 35.8 35.8

10 35.5 35.6

11 22.0 22.0

12 39.8 39.9

13 50.1 50.2

14 84.6 84.6

15 33.1 33.2

16 27.0 27.4

17 2.75 (dd, 1H, J = 9.0, 6.0 Hz) 51.4 51.5

18 0.98 (s, 3H) 16.2 16.2

19 0.86 (s, 3H) 23.8 24.2

20 176.0 175.9

21 4.98 (dd, 1H, J = 19.7, 1.6 Hz), 5.30

(dd, 1H, J = 19.7, 1.2 Hz)

73.7 73.7

22 6.02 (brs, 1H) 117.6 117.7

23 174.5 174.5

Sugar I

1' 5.07 (d, 1H, J = 7.8 Hz) 96.6 97.0

2' 2.30 (ddd, 2H, J = 14.2, 3.2, 2.1 Hz),

1.90 (ddd, 2H, J = 12.5, 12.0, 9.5 Hz)

36.8 36.1

3' 4.04 (brd, 1H, J = 3 Hz) 77.9 77.8

4' 3.61 (br d, 1H, J = 5.0 Hz) 83.6 82.8

5' 4.12 (dq, 1H, J = 10, 6.0 Hz) 69.4 69.2

6' 1.61 (d, 3H, J = 6 Hz) 18.8 18.3

3'-O-CH3 3.41 (s, 3H) 58.3 58.3

Sugar II

1'' 4.65 (d, 1H, J = 7.8 Hz) 106.6 103.4

2'' 4.37 (m, 1H) 71.4 71.2

3'' 3.50 (dd, 1H J = 9.9, 3.2 Hz) 85.3 85.5

4'' 4.25 (dd, 1H, J = 4.5, 2.5 Hz) 76.7 76.6

5'' 3.69 (d, 1H, J = 6.4 Hz) 70.6 70.5

6'' 1.54 (d, 3H, J = 6.2 Hz) 17.6 17.7

3''-OCH3 3.64 (s, 3H) 59.0 58.9

Sugar III

1''' 5.16 (d, 1H, J = 9.4 Hz) 105.6 105.4

2''' 3.92 (t, 1H, J = 7.8 Hz) 75.9 76.0

3''' 4.18 (t, 1H, J = 8 Hz) 78.3 78.5

4''' 3.93 (m, 1H) 71.7 71.9

5''' 4.08 (m, 1H) 78.6 78.3

6''' 4.56 (br d, 2H, J = 10.2 Hz), 4.25 (br

d, 2H, J = 10 Hz)

63.1 63.1

a In C5D5N b Aglycone and sugars II and III compared to β-D-glucosyl- β-D-

digitaloside27; sugar I compared to sugar I of compound 923

Table 1 13C NMR spectra of compounds 9, 10, and 12

9a Lit data 10a,b Lit. data26 12c Lit.data29

Aglycon

1 25.8 25.6 25.9 24.9 25.4 25.4

2 26.2 26.0 26.3 25.3 26.2 26.1

3 75.1 74.7 75.3 74.9 75.3 75.3

4 35.2 34.9 35.2 34.3 34.2 34.6

5 73.5 73.5 73.6 72.7 73.5 73.6

6 35.1 34.9 35.4 34.3 33.8 34.1

7 24.2 23.9 24.2 23.3 23.7 23.6

8 40.8 40.6 40.8 39.9 40.9 40.8

9 39.1 38.8 39.1 38.1 40.1 39.2

10 41.1 40.8 41.1 40.1 40.7 40.7

11 21.9 21.7 21.9 20.9 21.6 21.5

12 39.8 39.6 39.8 38.8 39.20 40.1

13 49.9 49.7 49.8 48.9 49.4 49.4

14 84.5 84.4 84.5 83.6 85.6 85.5

15 32.9 32.7 33.0 32.01 33.0 33.0

16 27.1 26.9 27.1 26.2 26.8 26.8

17 51.1 50.9 51.2 50.2 50.7 50.7

18 16.0 15.9 16.0 15.1 15.7 15.7

19 17.1 16.8 17.1 16.1 16.8 16.7

20 175.8 175.9 175.8 174.9 174.2 174.5

21 73.5 73.5 73.6 72.7 73.4 73.4

22 117.6 117.2 117.7 116.6 117.8 117.7

23 174.5 174.5 174.5 173.5 174.3 174.4

Sugar I

1′ 97.3 97.0 97.2 96.3 96.4 96.4

2′ 36.5 36.1 36.3 35.5 34.6 33.8

3′ 77.9 77.8 78.3 77.2 77.3 77.3

4′ 83.2 82.8 82.8 81.8 72.3 72.3

5′ 69.5 69.2 69.4 68.4 70.9 70.9

6′ 18.5 18.3 18.6 17.6 18.2 18.2

3′-OCH3 58.5 58.3 58.4 57.5 57.4 57.3

Sugar II

1″ 105.6 105.1 106.5 105.3

2″ 75.2 74.7 75.3 74.2

3″ 77.9 77.9 78.4 77.7

4″ 71.8 71.3 71.7 70.7

5″ 76.8 76.5 77.8 76.8

6″ 70.3 70.3 62.9 61.9

Sugar III

1″′ 106.5 106.0

2″′ 75.9 75.4

3″′ 78.4 78.0

4″′ 71.6 71.4

5″′ 78.5 78.0

6″′ 62.7 62.3

a δ values in C5D5N; b The literature data were obtained using

TMS as reference, while the experimental data were obtained

using the C-4 carbon of pyridine at 135.91 ppm as the reference.

Since the chemical shift of this carbon is sensitive to conditions

has been cited as high as 138.7 ppm (Crews, P.; Rodriguez, J.;

Jaspars, M.; Organic Structure Analysis Oxford University Press,

New York, 1998, p 88), the systematic deviation of the

experimental results from the literature data by about 1.0 ppm is

most probably due to a variation in the chemical shift of the

pyridine reference signal. c δ in CDCl3.


254

H-21) and δ 5.89 (brs, 1H, H-22). These peaks,

coupled with the two singlets (2 × CH3), suggested

that compound 9 could be a cardenolide, since these

chemical shifts are characteristic of the aglycon

portion of cardenolides. The 13

C NMR spectrum of

compound 9 showed two peaks at δ 177.24 for C-23

and at δ 178.37 for C-20; these signals are indicative

of the α,β-unsaturated γ-lactone unit of cardenolides.

The 1H NMR spectrum had signals for three anomeric

protons at δ 4.35 (d, J = 8 Hz, 1H), δ 4.34 (d, J = 7.8

Hz, 1H) and δ 4.95 (dd, J = 9.4, 2.0 Hz, 1H)

indicating the presence of three sugars. This

conclusion was confirmed by its 13

C NMR spectrum,

which contained signals for three anomeric carbons at

δ 98.19, 105.20, and 106.23. The presence of a

doublet at δ 1.28 (d, J = 6 Hz, 3H), a doublet of

doublets of doublets at δ 2.15 (ddd, J = 14.0, 3.2, 2.1

Hz) and a singlet at δ 3.45 (s, 3H) representing one

methoxy group indicated that one of the sugars could

be cymarose. The final structure and the connectivity

of the three sugars were confirmed by the COSY and

HMBC correlations. The stereochemistries of the

sugars were determined using ROESY, 1D-TOCSY

and by measurement of the different coupling

constants of the sugars. The NMR data for compound

9 combined with its elemental composition indicated

that it was most probably a cardenolide glycoside of

the periplogenin class previous isolated from Biondia

hemsleyana. A comparison of its NMR data with

the literature data of this glycoside25

indicated

that compound 9 is periplogenin-3-O-[β-D-

glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-β-

D-cymaropyranoside] (Table 1). It had an IC50 of 0.22

µg/ml against the A2780 cell line.

Compound 10 was also isolated as a white

amorphous powder and its HRFABMS indicated a

molecular formula of C36H56O13. Its NMR data were

similar to those of compound 9, except that it had

signals for only two anomeric protons at δ 4.33 (d, J =

8.0 Hz, 1H) and δ 4.95 (dd, J = 9.4, 2.0 Hz, 1H) in its 1H NMR spectrum, and for only two anomeric

carbons at δ 98.14 and 106.23 in the 13

C NMR

spectrum. These observations suggested that

compound 10 had two sugars. The final structure and

the connectivity of the two sugars were also

confirmed by COSY and HMQC correlations and the

HMBC correlations. The stereochemistries of the

sugars were determined using ROESY, 1D-TOCSY

and by measuring proton-proton coupling constants of

the sugars. The NMR data for compound 10,

combined with its elemental composition indicated that

it was most probably a cardenolide glycoside of the

periplogenin class. A comparison of its NMR data with

the literature data of periplogenin-β-D-glucopyranosyl-

β-D-cymaropyranoside]26

indicated that compound 10

was the known periplogenin-3-O-[β-D-glucopyranosyl-

(1→4)-β-D-cymaropyranoside]. It had an IC50 of 0.24

µg/ml against the A2780 cell line.


amorphous powder and its HRFABMS indicated a

molecular formula of C43H68O16. Its 1H NMR

spectrum in C5H5N revealed two singlets at δ 0.86 (s,

3H, H3-19), and 0.98 (s, 3H, H3-18), and additional

diagnostic peaks at δ 2.75 (dd, 1H, J = 9.0, 6.0 Hz, H-

17), δ 4.98 (dd, 1H, J = 19.7, 1.6 Hz, H-21), δ 5.30

(dd, 1H, J = 19.7, 1.2 Hz, H-21) and δ 6.02 (brs, 1H,

H-22). These peaks taken together suggested that

compound 11 could also be a cardenolide. The

aglycone of this cardenolide lacked a hydroxyl group

at the C-5 position, based on its HMBC correlations

(Fig. 7). Its NMR data were obtained in both CD3OD

and C5H5N. In C5H5N, the signals for protons H3-18

and H3-19 switched position. Its 13

C NMR spectrum

also showed signals at δ 174.52 for C-23 and at δ

175.93 for C 20 which are indicative of the α,β-

unsaturated γ-lactone unit of cardenolides (Table 2).

Its 1H NMR spectrum had signals for three

anomeric protons at δ 4.65 (d, H-1″, J = 7.8 Hz, 1H),

δ 5.07 (d, H-1′″ J = 7.8 Hz, 1H) and δ 5.16 (dd, H-1′,

J = 9.4, 2.0 Hz, 1H) indicating the presence of three

sugars. This conclusion was confirmed by its 13

C

NMR spectrum, which had signals for three anomeric

carbons at δ 96.61 (C-1′), 105.65 (C-1′″), and 106.65

(C-1″). The 1H NMR spectrum also showed a doublet

at δ 1.56 (d, H-6′″ J = 6.2 Hz, 3H), a doublet at δ 1.62

(d, H-6′, 6.2 Hz, 3H), a doublet of doublets of

doublets at δ 2.30 (ddd, J = 14.0, 3.2, 2.1 Hz) and two

singlets at δ 3.41 (s, 3H) and 3.63 (s, 3H) for two

methoxy groups. The final structure and the

connectivity of the sugars were confirmed by its

COSY and HMQC spectra and by HMBC correlations

depicted in Fig. 7. The stereochemistries of the sugars

were determined using ROESY, 1D-TOCSY and by

comparison of proton-proton coupling constants with

those previously determined for β-D-glycosyl-β-D-

digitaloside27

and the 13

C NMR chemical shifts of the

sugar moiety part b to those of the cannogenin-β-D-

glycosyl-β-D-digitaloside isolated from Apocynum

cannabinum.27

Part a of the sugar moiety is identical

to the same unit in compounds 9 and 10 (Table 2).


255

The 13

C NMR chemical shifts of the aglycone

part were identical to those of digitoxigenin

3-O-[ β-D-glucopyranosyl- (1→4) -2′-O-acetyl-α-L-

thevetopyranoside28

. The NMR data for compound 11

combined with its elemental composition indicated

that it was a cardenolide glycoside of the

digitoxigenin class. It was determined to be the new

cardenolide glycoside, digitoxigenin-β-D-glycosyl-β-

D-digitaloside-β-D-cymaropyranoside. It had an IC50

of 0.22 µg/ml against the A2780 cell line.


amorphous substance. Its HRFABMS indicated a

molecular formula of C30H35O8. Its 1H NMR spectrum

revealed two singlets at δ 0.88 (s 3H, H3-18) and δ

0.94 (s, 3H, H3-19), a doublet at δ 1.27 (d, 3H, J =

6.1, H3-6′) and a doublet of doublets at δ 2.78 (dd, 1H,

J = 9.3, 5.6, H-17). It had one methoxy group at δ

3.43 (s, 3H), and one anomeric proton at δ 4.78 (dd,

1H, J = 9.9, 2.1, H-1′). The presence of a single sugar

was confirmed by its 13

C NMR and by signal at δ

96.44, two doublets of doublets at δ 4.98 (dd, 1H, J =

18.2, 1.3 Hz, H-21) and δ 4.81 (dd, 1H, J = 18.2, 1.8

Hz, H-21), and a singlet at δ 5.88 (brs, 1H, H-22) in

its 1H NMR spectrum. Based on its

13C NMR data and

its HMBC correlations, compound 12 had the same

aglycone as compounds 9 and 10. The NMR data for

compound 12 combined with its elemental

composition indicated that it was most probably a

cardenolide glycoside of the periplogenin class. A

comparison of its NMR data with the literature data of

the cardenolide glycoside periplocymarin29

indicated

that compound 12 was periplocymarin. It had an IC50

of 0.4 µg/ml against the A2780 cell line.

Discussion As noted before, natural products continue to be an

excellent source of bioactive compounds. However,

one of the challenges facing natural products research

is that of the reisolation of known compounds. The

present finding illustrates this problem, as three of the

four bioactive compounds isolated turned out to be

known compounds. In addition, all the compounds

were from a well-known class of compounds, and are

thus not likely to be viable drug candidates.

Even though this work did not show leads to new

drugs, it should be noted that even well-known

compounds can prove to have important activities.

This is illustrated by the case of the well-known

triterpenoid betulinic acid (12), which has been

derivatized to prepare compounds with anti-HIV30

and

cancer preventive31

activity. The issue of compound

supply, which can be a problem for non-microbial

natural products, can often be solved by synthetic

chemistry. An example of this is the recent synthesis

of ipomoeassin F (13)32

(Fig. 10), which is an

interesting lead compound isolated from Ipomoea

squamosa as a part of our earlier ICBG program in

Suriname33-34

. The future of natural products research,

thus, continues to be bright, provided that modern

isolation and structure elucidation methods are

coupled with good biological assays and strong

synthetic chemistry.

Acknowledgement This project was supported by the Fogarty

International Center, the National Cancer Institute, the

National Science Foundation, the National Heart

Lung and Blood Institute, the National Institute of

Mental Health, the Office of Dietary Supplements,

and the Office of the Director of NIH, under

Cooperative Agreement U01 TW00313 with the

International Cooperative Biodiversity Groups. This

support is gratefully acknowledged. We thank S.

Randrianasolo, A. Rakotondrafara, A. Razakanirina, T.

Fig. 10—Structure of compounds 13 and 14.


256

Randriamboavonjy, and P. Andriamamonjisoa for

assistance with the recollection of the plant, B.

Bebout for obtaining the mass spectra and T. Glass

for assistance with the NMR spectra. Field work

essential for this project was conducted under a

collaborative agreement between the Missouri

Botanical Garden and the Parc Botanique et

Zoologique de Tsimbazaza and a multilateral

agreement between the ICBG partners, including the

Centre National d’Applications des Recherches

Pharmaceutiques. We gratefully acknowledge

courtesies extended by the Government of

Madagascar (Ministère des Eaux et Forêts).

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