new unusual pregnane glycosides with antiproliferative activity from< i> solenostemma argel

Steroids 70 (2005) 594–603

New unusual pregnane glycosides with antiproliferative activityfromSolenostemma argel

Alberto Plazaa, Angela Perronea, Maria Luisa Balestrierib,Francesca Feliceb, Ciro Balestrierib, Arafa I. Hamedc,

Cosimo Pizzaa,∗, Sonia Piacentea

a Dipartimento di Scienze Farmaceutiche, Universit`a degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano, SA, Italyb Dipartimento di Biochimica e Biofisica, Seconda Universit´a degli Studi di Napoli, Napoli 80138, Italy

c Faculty of Science, South Valley University, Aswan 81528, Egypt

Received 14 December 2004; received in revised form 16 February 2005; accepted 22 February 2005Available online 8 June 2005

Abstract

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Seven new 15-keto pregnane glycosides, namely Stemmosides E–K, were isolated fromSolenostemma argel. Stemmosides E–J are chcterized by the occurrence of an uncommon 14� proton configuration while stemmosides E and F possess in addition a rare enolic f

n C-16. On the other hand, stemmosides G–J display an unusual C-17� side chain. Their structures were established by ESI-MS andxperiments. Moreover, the effect of these compounds on the VEGF-induced in Kaposi’s sarcoma cell proliferation was teste

ndicated that all the compounds reduced the cell proliferation in a dose dependent manner.2005 Elsevier Inc. All rights reserved.

eywords: Solenostemma argel; Pregnane glycosides; 15-Keto;cisCD junction; Cell proliferation; Kaposi’s sarcoma

. Introduction

Plants belonging to the family Asclepiadaceae are fre-uently used in traditional medicine and have been reported

o be rich in steroidal glycosides[1,2]. Solenostemma argelayne (Asclepiadaceae) is an Egyptian wild perennial erecthrub growing in the eastern desert and along the Nile inouth Egypt[3], whose leaves are commonly used in tra-itional medicine as a purgative, antipyretic, expectorant,ntispasmodic and in cases of bile congestion[4]. Previoustudies have reported the occurrence of monoterpene glyco-ides[5], pregnane derivatives[6] and pregnane glycosidesncluding stemmosides A and B[5,7] and acylated phenoliclycosides in the leaves[8] while 14,15-secopregnane gly-osides[9] and pregnane glycosides namely stemmosides Cnd D have been isolated from the pericarps[10].

∗ Corresponding author. Tel.: +39 089 962813; fax: +39 089 962828.E-mail address:[email protected] (C. Pizza).

In our on going research of new bioactive compoundS. argel, here we report the occurrence of five new unu15-keto pregnane glycosides namely stemmosides E–I1–5)from the leaves and two new 15-keto pregnane glycosstemmoside J and K (6, 7), along with the known compound stemmoside A, from the pericarps. Their structwere elucidated by extensive spectroscopic methods ining 1D- (1H and 13C) and 2D-NMR experiments (DQCOSY, HSQC, HMBC, ROESY and HOHAHA) as wellESI-MS analysis. Compounds1–6 are characterized by thoccurrence of an uncommon 14� proton configuration whilcompounds1 and2 possess in addition a rare enolic fution in C-16. Apparently,S. argel is the only plant fromwhich pregnanes possessing a 15-keto,cis CD ring junc-tion have been isolated[10]. A few other naturally occurrinsteroids showing a 15-keto,cisCD ring junction have beeisolated only from marine sponges[11–15]. On the othehand, to the author’s knowledge compounds3–6 along withstemmoside C and D are the only natural compounds w

039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
oi:10.1016/j.steroids.2005.02.019

A. Plaza et al. / Steroids 70 (2005) 594–603 595

display an unusual C-17� side chain and no substitution inC-12.

In addition, since pregnanes and their glycosides haveshown to possess antitumor and cytotoxic activities[16–19]and on the basis of the novelty of the structure features ofstemmoside1–8 we examined whether these compounds,along with the previously isolated stemmosides C and D[10], can be useful in the control of tumor cell proliferation.Kaposi’s sarcoma (KS) cells were used as a model to test theanti-proliferative properties of these compounds. Unlike themore indolent endemic and sporadic form of KS, this dis-ease is usually aggressive and unpredictable in individualsinfected by HIV-1, and the neoplasm is the cause of signifi-cant morbidity and occasional mortality. KS is usually treatedwith cytostatic drugs, cytokine or radiotherapy to control cellproliferation[20], thus the identification of new moleculesof pharmacological interest could be of clinical relevance forthe development of novel therapeutic strategies.

2. Experimental

2.1. General

Optical rotations were measured on a Jasco DIP 1000polarimeter. IR measurements were obtained on a BrukerIFS-48 spectrometer. Exact masses were measured by a Voy-a osterC stedl . Am ca andd romA nsinI per-f eters wasa plesw byu -i ubele pec-t iredi mit-t tionalP ationi hasec C,H ireda iconG ol-u H-20( -m l-u m;C tions

were carried out on a Waters 590 system equipped with aWaters R401 refractive index detector, a Waters XTerra PrepMSC18 column, and a U6K injector. TLC was performed onsilica gel F254 (Merck) plates, and reagent grade chemicals(Carlo Erba) were used throughout.

2.2. Plant material

Fresh samples ofS. argelleaves and pericarps were col-lected at Allaqi (south-east of Aswan, Egypt) in May 2002and identified by one of the authors (A.I.H.).

2.3. Extraction and isolation

The dried pericarps (1.5 kg) extracted with EtOH 80%yielding 254 g of extract while the dried leaves (100 g) wereextracted with EtOH 70% yielding 20 g of extract. Part ofthe leaves extract (2.5× 2 g) was fractionated on SephadexLH-20 (100 cm× 5 cm) using MeOH as the mobile phase.Sixty-seven fractions (8 mL) were obtained. The fractionscontaining pregnane glycosides (frs. 13–17, 1.0176 g) werechromatographed by MPLC on Si gel with a gradient (flowrate 3.0 mL/min) of chloroform/methanol (from 100:0 to 9:1,stepwise) as eluent to afford 1660 fractions (8 mL) moni-tored by TLC. Fractions 1133–1156 (48.5 mg) were chro-matographed by RP-HPLC on a Waters (�-Bondapak C )c asm d(o LCou rate2F phedb( bilept asfic asm s( ft adexL se.N nsc werec , onau rate27

2

U1 -

ger DE mass spectrometer (Applied Biosystems, Fity, CA, USA). Samples were analyzed by matrix assi

aser desorption ionization (MALDI) mass spectrometryixture of analyte solution and�-ciano-4-hydroxycinnamicid (Sigma) was applied to the metallic sample plateried. Mass calibration was performed with the ions fCTH (fragment 18–39) at 2465.1989 Da and Angiote

II at 931.5154 Da as internal standard. ESI-MS wasormed on a Finnigan LC-Q Deca Ion Trap mass spectromcanned from 150 to 1200 Da. The mass spectral datacquired and processed using Xcalibur software. Samere dissolved in MeOH and infused in the ESI sourcesing a syringe pump at a flow rate of 3�L/min. The cap

llary voltage was 5 V, the spray voltage 5 kV and the tens offset 50 V. The capillary temperature was 220◦C. NMRxperiments were performed on a Bruker DRX-600 srometer at 300 K. All the 2D-NMR spectra were acqun CD3OD in the phase-sensitive mode with the transer set at the solvent resonance and TPPI (Time Proporhase Increment) used to achieve frequency discrimin

n the�1 dimension. The standard pulse sequence and pycling were used for DQF-COSY, 2D-TOCSY, HSQMBC and ROESY spectra. The spectra were acqut 600 MHz. The NMR data were processed on a Silraphic Indigo2 Workstation using UXNMR software. Cmn chromatography was performed over Sephadex LPharmacia), MPLC was carried out on a Buchi 688 chroatography pump and Buchi B-685 borosilicate glass comn (230 mm× 26 mm). Silica gel 60 (0.040–0.063 marlo Erba) was used as column material. HPLC separa

18olumn (300 mm× 7.8 mm) using methanol/water (78:22)obile phase (flow rate 2.5 mL/min) to yield compoun4

2.2 mg,tR = 57.1 min),5 (1.5 mg,tR = 67.2 min) along withne fraction A (6.0 mg) that was finally purified by RP-HPn a Waters (�-Bondapak C18) column (300 mm× 7.8 mm)sing methanol/water (75:25) as mobile phase (flow.5 mL/min) to yield compound1 (2.6 mg, tR = 54.1 min).ractions 1157–1191 (66.6 mg) were chromatogray RP-HPLC on a Waters (�-Bondapak C18) column300 mm× 7.8 mm) using methanol/water (78:22) as mohase (flow rate 2.5 mL/min) to yield compound3 (2.7 mg,

R = 39.3 min) along with one fraction B (7.8 mg) that wnally purified by RP-HPLC on a Waters (�-Bondapak C18)olumn (300 mm× 7.8 mm) using methanol/water (75:25)obile phase (flow rate 2.5 mL/min) to yield compound2

3.0 mg,tR = 42.0 min) and8 (1.3 mg,tR = 38.0 min). Part ohe pericarp extract (2.2 g) was fractionated on SephH-20 (100 cm× 5 cm) using MeOH as the mobile phainety-five fractions (8 mL) were obtained. The fractioontaining pregnane glycosides (frs. 29–33, 375 mg)hromatographed by HPLC (Refractive index detector)Waters (XTerra Prep MSC18) column (300 mm× 7.8 mm)sing methanol/water (74:26) as mobile phase (flow.5 mL/min) to yield compound6(2.6 mg,tR = 52.0 min), and(2.3 mg,tR = 32.7 min).

.3.1. Stemmoside E (1)White amorphous powder; [α]24

D −15.4◦ (c 0.2, MeOH);V (MeOH) λmax 244 nm; logε 3.84; IR (KBr) νmax 3451,719, 1650, 1070 cm−1; 1H NMR (CD3OD, 600 MHz) agly

596 A. Plaza et al. / Steroids 70 (2005) 594–603

Table 113C NMR Data of the Aglycone Moieties of Compounds1, 3, 5, and7 inCD3OD

Position 1 3 5 7

1 37.7 37.9 37.8 38.22 30.2 30.4 29.8 30.03 79.0 79.1 78.5 78.54 39.6 39.8 35.5 35.55 141.6 140.8 45.2 44.96 123.6 123.6 29.6 29.67 30.1 28.2 29.6 24.78 31.0 29.5 33.6 39.99 43.7 44.9 48.0 50.1

10 39.8 37.9 36.2 36.511 21.4 22.1 21.8 21.312 31.9 30.9 30.7 30.113 43.3 42.9 42.4 46.214 54.5 58.2 59.5 82.915 206.7 220.4 216.7 218.016 152.2 71.8 72.2 40.217 152.9 53.1 51.6 43.718 25.5 21.9 21.9 15.519 17.8 19.8 11.9 12.120 18.7 16.3 16.3 22.621 12.5 13.7 13.1 13.4

cone moietyδ 5.44 (1H, dd,J= 3.2, 2.4 Hz, H-6), 3.49 (1H, m,H-3), 3.05 (1H, ddd,J= 13.5, 15.8, 3.2 Hz, H-7�), 2.31 (1H,m, H-20a), 2.28 (1H, m, H-20b), 2.06 (1H, m, H-8), 1.98(1H, dd J= 15.8, 2.4 Hz, H-7�), 1.93 (1H, m, H-14), 1.24(3H, s, Me-18), 1.18 (3H, t,J= 7.0 Hz, Me-21), 1.05 (3H,s, Me-19);13C NMR (CD3OD, 150 MHz), aglycone moietyseeTable 1; 1H NMR (CD3OD, 600 MHz) and13C NMR(CD3OD, 150 MHz), sugar portion seeTable 2; ESI-MSm/z 1091 [M+ Na]+, 929 [M+ Na-162]+, 785 [M+ Na-162-144]+, 641 [M+ Na-162-144-144]+; HR-MALDI-MS m/z1091.5779 [M+ Na]+, calcd. for C55H88O20Na, 1091.5767.

2.3.2. Stemmoside F (2)White amorphous powder; [α]24

D −19.2◦ (c 0.2, MeOH);λmax 244 nm; logε 3.84, IR (KBr) νmax 3457, 1715, 1643,1070; UV (MeOH) λmax 243 nm; log ε 3.84; 1H NMR(CD3OD, 600 MHz) and13C NMR (CD3OD, 150 MHz),aglycone moiety superimposable on those reported for com-pound 1; 1H NMR (CD3OD, 600 MHz) and13C NMR(CD3OD, 150 MHz), sugar portion seeTable 2; ESI-MSm/z1077 [M+ Na]+, 915 [M+ Na–162]+, 771 [M+ Na–162-144]+, 641 [M+ Na–162-144-130]+; HR-MALDI-MS m/z1077.5601 [M+ Na]+, calcd. for C54H86O20Na, 1077.5610.

2.3.3. Stemmoside G (3)White amorphous powder; [α]24

D −9.8◦ (c 0.2, MeOH);IR (KBr) νmax 3415, 2945, 1730, 1465, 1098, 1059 cm−1;1H NMR (CD3OD, 600 MHz) aglycone moietyδ 5.42 (1H,dd, J= 3.5, 2.4 Hz, H-6), 4.05 (1H, d,J= 9.7 Hz, H-16),3.55 (1H, m, H-3), 3.09 (1H, ddd,J= 13.5, 15.7, 3.5 Hz,H-7�), 2.36 (1H, m, H-14), 1.92 (1H, m, H-8), 1.87 (1H,dd, J= 9.7, 1.6 Hz, H-17), 1.80 (1H, dd,J= 15.7, 2.6 Hz,H-7�), 1.20 (3H, s, Me-18), 1.07 (3H, s, Me-21), 1.00 (3H,s, Me-19);13C NMR (CD3OD, 150 MHz), aglycone moietyseeTable 1; 1H NMR (CD3OD, 600 MHz) and13C NMR(CD3OD, 150 MHz), sugar portion seeTable 2; ESI-MSm/z1079 [M+ Na]+, 917 [M+ Na-162]+, 773 [M+ Na–162-144]+, 643 [M+ Na–162–144-130]+; HR-MALDI-MSm/z 1090.5875 [M+ Na]+, calcd. for C54H88O20Na,1090.5860.

2.3.4. Stemmoside H (4)White amorphous powder; [α]24

D −4.7◦ (c 0.2, MeOH);IR (KBr) νmax 3410, 2940, 1735, 1461, 1098, 1059 cm−1;1H NMR (CD3OD, 600 MHz) aglycone moietyδ 5.51 (1H,d,J= 10.1 Hz, H-16), 5.42 (1H, dd,J= 3.2, 2.4 Hz, H-6), 2.99(1H, ddd,J= 13.5, 15.5, 3.2 Hz, H-7�), 2.38 (1H, m, H-14),2.14 (3H, s, COMe), 1.93 (1H, m, H-8), 1.81 (1H, dd,J= 15.5,2.4 Hz, H-7�), 1.22 (3H, s, Me-18), 1.01 (3H, s, Me-19), 0.92( 13

a C-4 (C-9 8.7( e-12(m1M1

2

I61 s,C -

3H, t, J= 7.0 Hz, Me-21); C NMR (CD3OD, 150 MHz),glycone moietyδ 37.9 (C-1), 30.2 (C-2), 78.8 (C-3), 39.7 (), 140.3 (C-5), 123.2 (C-6), 27.9 (C-7), 29.5 (C-8), 44.6), 36.6 (C-10), 21.7 (C-11), 30.1 (C-12), 42.3 (C-13), 5C-14), 214.6 (C-15), 72.3 (C-16), 51.5 (C-17), 21.4 (M8), 19.6 (Me-19), 16.2 (C-20), 13.2 (Me-21), 171.9 (COMe),0.3 (COMe); 1H NMR (CD3OD, 600 MHz) and13C NMRCD3OD, 150 MHz), sugar portion seeTable 2; ESI-MS/z 1135 [M+ Na]+, 973 [M+ Na-162]+, 829 [M+ Na-62-144]+, 685 [M+ Na–162–144-144]+; HR-MALDI-S m/z 1135.6036 [M+ Na]+, calcd. for C57H92O21Na,135.6029.

.3.5. Stemmoside I (5)White amorphous powder; [α]24

D −8.0◦ (c 0.2, MeOH);R (KBr) νmax 3413, 2947, 1745 cm−1; 1H NMR (CD3OD,00 MHz) aglycone moietyδ 5.48 (1H, d,J= 10.1 Hz, H-6), 2.38 (1H, m, H-7�), 2.37 (1H, m, H-14), 2.14 (3H,OMe), 2.04 (1H, dd,J= 10.1, 1.7, H-17), 1.29 (1H, m, H


Table 213C and1H NMR of the Sugar Portions of Compounds1 and2 in CD3OD

Position 1, chain A 2, chain B

δC δH (J in Hz) δC δH (J in Hz)

�-d-CymI �-d-CymI

1 97.0 4.89 dd (9.2, 2.0) 96.9 4.90 dd (9.2, 2.0)2 36.4 2.10 m 36.8 2.08 m

1.59 m 1.57 m3 78.3 3.87 br m 78.3 3.88 br m4 83.7 3.26 dd (9.5, 3.0) 83.7 3.25 dd (9.5, 3.0)5 69.7 3.85 dq (9.5, 6.1) 69.6 3.85 dq (9.5, 6.1)6 18.1 1.22 d (6.1) 18.3 1.22 d (6.1)OMe 58.2 3.46 s 58.1 3.46 s

�-d-CymII �-d-CymII

1 101.0 4.82 dd (9.6, 2.0) 100.9 4.83 dd (9.6, 2.0)2 36.1 2.17 m 36.2 2.17 m

1.63 m 1.62 m3 78.3 3.87 br m 78.3 3.88 br m4 83.7 3.26 dd (9.5, 3.0) 83.7 3.30 dd (9.5, 3.0)5 69.4 3.85 dq (9.5, 6.1) 69.6 3.85 dq (9.5, 6.1)6 18.2 1.25 d (6.1) 18.3 1.25 d (6.1)OMe 58.2 3.46 s 58.1 3.45 s

�-d-OleI �-d-Can

1 102.5 4.62 dd (9.6, 2.0) 102.1 4.67 dd (9.6, 2.0)2 37.6 2.35 ddd (13.0, 4.0, 2.0) 39.4 2.23 ddd (13.0, 4.0, 2.0)

1.48 ddd (13.0, 9.6, 9.0) 1.52 ddd (13.0, 9.6, 9.0)3 80.1 3.43 ddd (9.5, 9.0, 4.0) 70.5 3.63 ddd (9.5, 9.0, 4.0)4 84.1 3.21 dd (9.5, 9.5) 88.5 3.04 dd (9.5, 9.5)5 72.1 3.38 dq (9.5, 6.1) 71.5 3.39 dq (9.5, 6.1)6 18.4 1.32 d (6.1) 17.9 1.32 d (6.1)OMe 58.2 3.46 s

�-d-OleII �-d-OleI

1 100.9 4.73 dd (9.6, 2.0) 102.1 4.67 dd (9.6, 2.0)2 37.8 2.38 ddd (13.0, 4.0, 2.0) 37.4 2.43 ddd (13.0, 4.0, 2.0)

1.43 ddd (13.0, 9.6, 9.0) 1.49 ddd (13.0, 9.6, 9.0)3 80.1 3.43 ddd (9.5, 9.0, 4.0) 79.7 3.49 ddd (9.5, 9.0, 4.0)4 83.7 3.31 dd (9.5, 9.5) 83.2 3.37 dd (9.5, 9.5)5 72.5 3.42 dq (9.5, 6.1) 72.7 3.55 dq (9.5, 6.1)6 18.4 1.44 d (6.1) 18.1 1.43 d (6.1)OMe 58.0 3.50 s 58.0 3.51 s

�-d-Glc �-d-Glc

1 103.9 4.48 d (7.9) 103.9 4.49 d (7.9)2 75.4 3.21 dd (9.0, 7.9) 75.3 3.21 dd (9.0, 7.9)3 77.9 3.38 dd (9.0, 9.0) 77.9 3.37 dd (9.0, 9.0)4 71.6 3.28 dd (9.0, 9.0) 71.5 3.27 dd (9.0, 9.0)5 78.1 3.28 dd (9.0, 9.0) 78.1 3.28 dd (9.0, 9.0)6 62.8 3.89 dd (12.0, 2.5) 62.7 3.90 dd (12.0, 2.5)

3.68 dd (12.0, 4.5) 3.67 dd (12.0, 4.5)

7�), 1.18 (3H, s, Me-18), 0.91 (3H, t,J= 7.0 Hz, Me-21), 0.82(3H, s, Me-19);13C NMR (CD3OD, 150 MHz), aglyconemoiety seeTable 1; 1H NMR (CD3OD, 600 MHz) and13CNMR (CD3OD, 150 MHz), sugar portion seeTable 2; ESI-MS m/z 1137 [M+ Na]+, 975 [M+ Na-162]+, 831 [M+ Na-162-144]+, 687 [M+ Na-162-144-144]+; HR-MALDI-MSm/z 1137.6190 [M+ Na]+, calcd. for C57H94O21Na,1137.6185.

2.3.6. Stemmoside J (6)White amorphous powder; [α]24

D −11.5◦ (c 0.2, MeOH);IR (KBr) νmax 3423, 2950, 1743;1H NMR (CD3OD,600 MHz) and13C NMR (CD3OD, 150 MHz), aglyconemoiety superimposable on those reported for compound5;1H NMR (CD3OD, 600 MHz) and13C NMR (CD3OD,150 MHz), sugar portion seeTable 2; ESI-MS m/z 1123[M+ Na]+, 961 [M+ Na-162]+, 817 [M+ Na-162-144]+, 687


[M+ Na-162-144-130]+; HR-MALDI-MS m/z 1123.6036[M+ Na]+, calcd. for C56H92O21Na, 1123.6029.

2.3.7. Stemmoside K (7)White amorphous powder; [α]24

D +0.6◦ (c 0.2, MeOH);IR (KBr) νmax 3431, 2921, 1758 cm−1; 1H NMR (CD3OD,600 MHz) δ 2.58 (1H, dd,J= 18.0, 9.0 Hz, H-16�), 2.15(1H, m, H-17), 2.25 (1H, m, H-7�), 1.85 (1H, m, H-7�),1.76 (1H, dd,J= 18.0, 9.0 Hz, H-16�), 1.04 (3H, s, Me-18), 0.96 (3H, s, Me-21), 0.82 (3H, s, Me-19);13C NMR(CD3OD, 150 MHz), aglycone moiety seeTable 1; 1H NMR(CD3OD, 600 MHz) and13C NMR (CD3OD, 150 MHz),sugar portion seeTable 2; ESI-MS m/z 1081 [M+ Na]+,919 [M+ Na-162]+, 775 [M+ Na-162-144]+, 645 [M+ Na-162-144-130]+; HR-MALDI-MS m/z1081.5927 [M+ Na]+,calcd. for C54H90O20Na, 1081.5923.

2;

I( ,4

5 1),2 e-1( -5),2 0),2 7.6( 19),2s ,

J= 9.0, 7.5, H-2glc), 3.37 (1H, t,J= 9.0, H-3glc), 3.21 (1H,t, J= 9.0, H-4glc), 3.28 (1H, ddd,J= 9.0, 4.5, 2.5, H-5glc),3.59 (1H, dd,J= 12.0, 4.5, H-6aglc), 3.91 (1H, dd,J= 12.0,2.5, H-6bglc); 13C NMR (CD3OD, 150 MHz) sugar moietyδ 101.4 (C-1glc), 75.1 (C-2glc), 77.4 (C-3glc), 71.9 (C-4glc),78.2 (C-5glc), 63.0 (C-6glc); ESI-MSm/z531 [M+ Na]+, 369[M+ Na-162]+; HR-MALDI-MS m/z 531.25855 [M+ Na]+,calcd. for C27H40O9Na, 531.2594.

2.4. Cell cultures and treatments

KS immortalized cells were cultured in 25-cm2 flasks con-taining RPMI-1640 media supplemented with 10% fetal calfserum (Gibco BRL, Poland) at 37◦C in a humidified chamberwith 5% CO2. Cells were grown in 24 well-plates at a celldensity of 5× 103/well and were cultured for 12 h in RPMI-1 nts.S ndD anda ively,t s1 dia.T than0 than9 ontrolw sedt cells.

ith2 ulare SA)ih

celln ssay( elln rmedo XTTp to

.3.8. Stemmoside A (8)White amorphous powder; [α]24

D −13.0◦ (c0.49, MeOH)R (KBr) νmax 3405, 1706, 1375, 810 cm−1; 1H NMRCD3OD, 600 MHz) aglycone moietyδ 5.74 (1H, s, H-4).89 (1H, dd,J= 9.3, 6.3 Hz, H-15), 2.76 (1H, dd,J= 9.2,

.7 Hz, H-17), 2.46 (1H, m, H-16a), 2.29 (3H, s, Me-2

.04 (1H, m, H-16b), 1.25 (3H, s, Me-19), 1.07 (3H, s, M8); 13C NMR (CD3OD, 150 MHz) aglycone moietyδ 36.6C-1), 34.5 (C-2), 202.0 (C-3), 123.8 (C-4), 175.5 (C9.4 (C-6), 32.3 (C-7), 42.1 (C-8), 49.2 (C-9), 40.5 (C-11.2 (C-11), 39.4 (C-12), 48.9 (C-13), 83.5 (C-14), 7C-15), 30.5 (C-16), 60.9 (C-17), 16.4 (C-18), 18.1 (C-16.0 (C-20), 31.4 (C-21);1H NMR (CD3OD, 600 MHz)ugar moietyδ 4.31 (1H, d,J= 7.5, H-1glc), 3.19 (1H, dd

640 media without 10% fetal calf serum before treatmetock solutions of compounds1–8, and stemmosides C awere prepared in DMSO at a concentration of 5 mM

fterward diluted to the required concentration. Successhe cells were pre-treated for 1 h at 37◦C with compound–8, stemmosides C or D in serum-free RPMI-1640 mehe final DMSO concentration in the media was less.1% and after treatments the cells viability was higher5%, as assessed by trypan blue dye exclusion. In the cells, medium, with DMSO at the same concentration u

o dissolve the tested compounds, only was added to the

To stimulate proliferation, cells were washed twice wmL of HBSS-10 mM Hepes and treated with vascndothelial growth factor 50 ng/mL (VEGF, Peprotech, U

n HBSS-10 mM Hepes for 48 h at 37◦C in 5% CO2 andumidified air atmosphere.

Cell proliferation was determined by two methods,umber count using Trypan blue exclusion and XTT aXTT Kit I, Boehringer Mannheim Corp., Germany). Cumber count was expressed as cells/mL and was perfonly on viable cells based on trypan dye test. For theroliferation assay, 0.5 mL of XTT mixture was added


each well. After 1 h, only 0.2 mL of the solution was usedto measure the spectrophotometrical absorbance at 490 nm.The XTT method was also used as assay to determine thecytotoxic effect of the tested compounds.

Data are expressed as means± S.E.M. from at least threeindependent experiments in duplicates. Statistical analysiswas performed by Student’st-test. Statistical significance wasset atP< 0.05.

3. Results and discussion

3.1. Structural elucidation

The methanol extract of the leaves ofS. argelwas fraction-ated on Sephadex LH-20 and then by MPLC on Si gel and RPHPLC, as described in Section2, to give five new pregnaneglycosides (1–5). The methanol extract of the dried pericarpsof S. argelwas fractionated on Sephadex LH-20 and then byRP HPLC to afford two new pregnane glycosides (6 and7)along with the known compound stemmoside A (8).

Stemmoside E (1): Compound1 showed a major ionpeak at m/z 1091 [M+ Na]+ and significant fragmentsat m/z 929 [M+ Na-162]+, m/z 785 [M+ Na-162-144]+,m/z 641 [M+ Na-162-144-144]+ in the positive ESI-MS.Its molecular formula was unequivocally established tob1 ).Ie pec-t p-t dk g-n and3t ot ap eoN d beu rtic-u oft f twoo ico e(o -m fromt tonso rre-l nr 9.8( imi-l thep cesa

17) establishing the connectivity between the rings B andC. Finally, the HMBC correlations between the proton atδ

1.18 (Me-21) and the carbon resonances atδ 152.9 (C-17)andδ 18.7 (C-20); the protons atδ 2.31 (H-20a) andδ 2.28(H-20b) and the carbon resonances atδ 43.3 (C-13),δ 152.2(C-16),δ 152.9 (C-17), andδ 1.18 (Me-21); and the protonat δ 1.93 (H-14) and the carbon resonance atδ 43.3 (C-13)andδ 206.7 (C-15), allowed us to deduce that the pregnaneskeleton of1 was characterized by the occurrence of a rareenolic function in C-16, and a ketone function in C-15. Fur-ther evidence of the placement of the ketone in position 15was provided by the deshielded chemical shift of the protonresonance of H-7� (� 3.05) which was ascribable to a neigh-boring C-15 ketone effect[12]. The relative configurationof H-14 was established by the chemical shifts of H-7� andMe-18. It has been described that the deshielding effect of a15-ketone functionality on 7� or 7� proton depends on theC-14 configuration. The 7� proton resonates at low field intransCD 15-keto steroids, while incisCD 15-keto steroids,the 7� proton resonates at low field. Moreover, it was notedthattransCD 15-keto steroids display in the1H NMR spec-trum the signal for Me-18 singlet in the range 0.70–1.00 ppm,whereas the corresponding signal incisCD 15-keto steroidsranges between 1.10 and 1.30 ppm[21]. In compound1, thesignal atδ 3.05 displayed two large coupling constants, thefirst one, 18.0 Hz related to the geminal proton at 2.06 ppm,w itionw elds .

t bletm1m ass22 d,J hes oft ll thei inedf Ys ira fromt hep4 )a nsC e� vityi m,w(((

e C55H88O20 by HR-MALDI mass spectrometry (m/z091.5779 [M+ Na]+, calcd. for C55H88O20Na, 1091.5767

R absorptions at 3451, 1719, and 1650 cm−1 implied thexistence of hydroxyl, carbonyl and olefinic groups, resively. The UV spectrum of1 showed a maximum absorion at 244 nm, which is characteristic for�,�-unsaturateetones. The13C NMR spectrum of1 showed 54 carbon sials, 21 of them corresponding to a pregnane skeleton3 corresponding to a sugar portion. The1H NMR spec-

rum of the aglycone portion of1 showed signals for twertiary methyl groups atδ 1.05 (3H, s) and 1.24 (3H, s),rimary methyl signal atδ 1.18 (3H, t,J= 7.0 Hz), and onlefinic proton atδ 5.44 (1H, dd,J= 3.2, 2.4 Hz). The13CMR chemical shifts of all the protonated carbons coulnambiguously assigned by the HSQC spectrum. In palar, the analysis of the13C NMR spectrum on the basis

he HSQC correlations clearly showed the occurrence olefinic quaternary carbons (δ 141.6 and 152.9), one olefinxygenated quaternary carbon (δ 152.2), one olefinic methinδ 123.6), one secondary oxygenated carbon (δ 79.0), andne carbonyl carbon (δ 206.7) (seeTable 1). The three sixembered rings of a pregnane skeleton were established

he analysis of the strong HMBC correlations of the prof the angular methyls Me-18 and Me-19. Long-range co

ations from the proton signal atδ 1.05 (Me-19) to the carboesonances atδ 37.7 (C-1), 141.6 (C-5), 43.7 (C-9) and 3C-10), revealed the connectivity of the A and B rings. Sarly, the HMBC spectrum indicated correlations betweenroton signals atδ 1.24 (Me-18) and the carbon resonant δ 31.9 (C-12), 43.3 (C-13), 54.5 (C-14), andδ 152.9 (C-

hile the second one, 13.5 Hz revealed a 1,2 diaxial posith respect to H-8. This allowed us to assign this low fiignal to H-7�, thus corroborating thecisCD arrangement

Moreover, the sugar portion of compound1 showed inhe1H NMR spectrum signals corresponding to four douethyls atδ 1.44 (3H, d,J= 6.1 Hz), 1.32 (3H, d,J= 6.1 Hz),.25 (3H, d,J= 6.1 Hz) and 1.22 (3H, d,J= 6.1 Hz), fourethoxy groups atδ 3.50 (3H, s) and 3.46 (9H, s), as well

ignals for five anomeric protons atδ 4.89 (1H, dd,J= 9.2,.0 Hz), 4.82 (1H, dd,J= 9.6, 2.0 Hz), 4.73 (1H, dd,J= 9.6,.0 Hz), 4.62 (1H, dd,J= 9.6, 2.0 Hz) and 4.48 (1H,= 7.9 Hz) (seeTable 2). All these data indicated that tugar chain of compound1 consisted of five sugars, fourhem being 2,6-dideoxy sugars. The chemical shifts of andividual protons of the five sugar units were ascertarom a combination of 2D-HOHAHA and DQF-COSpectral analysis, and the13C NMR chemical shifts of thettached carbons could be assigned unambiguously

he HSQC spectrum (seeTable 2). These data showed tresence of two�-d-cymaropyranosyl units (δ 4.89 and.82), two �-d-oleandropyranosyl units (δ 4.73 and 4.62nd one�-d-glucopyranosyl unit (δ 4.48). Glycosidatiohifts were observed for C-4cymI (δ 83.7), C-4cymII (δ 83.7),-4oleI (δ 84.1) and C-4oleII (δ 83.7) suggesting that th-d-glucopyranosyl was a terminal unit. Direct connecti

nformation was obtained from the HMBC spectruhich showed the following key correlation peaks:δH 4.89

H-1cymI) andδC 79.0 (C-3),δH 4.82 (H-1cymII) andδC 83.7C-4cymI), δH 4.62 (H-1oleI) andδC 83.7 (C-4cymII), δH 4.73H-1oleII) and δC 84.1 (C-4oleI), δH 3.31 (H-4oleII) and δC


103.9 (C-1glc), andδH 4.48 (H-1glc) andδC 83.7 (C-4oleII).It is worthwhile to note that these results are in accordancewith the fact that C-1 of�-d-cymarose characteristicallyresonates upfield (∼97.0 ppm) when linked at C-3 of theaglycone in opposition to the resonance at∼101.0 ppmwhen it is linked to the hydroxyl group of a different sugar[22]. Thus, the structure of compound1 was established as3�,16-dihydroxy-14H�-pregn-5,16-dien-15-one-3-O-�-d-glucopyranosyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)-�-d-cymaropyranoside.

np tsam .T d tob1 ),w .A ata( fc icalir heN ,d 9(N -p Ae inal� lu it(O thea reso-n s3gd�

Stemmoside G (3): The HR-MALDI mass spectrum (m/z1090.5875 [M+ Na]+, calcd. for C54H88O20Na, 1090.5860)of compound3supported a molecular formula of C54H88O20.It showed a major ion peak atm/z 1079 [M+ Na]+ and sig-nificant fragments atm/z 917 [M+ Na-162]+, m/z 773[M+ Na-162-144]+, m/z 643 [M+ Na-162-144-130]+ in thepositive ESI-MS. The13C NMR spectrum of3 was verysimilar to that of stemmoside D, previously isolated from thepericarps ofS. argel[10], only differing by the absence ofthe carbon signals atδ 171.9 andδ 20.3 corresponding to the

ao asa ntlys1 redt 5.51(A theD stedt nceo 16.T hedbCa ntly,a ntumm dfp ereast didn tonsbr3 e,3po�

Stemmoside F (2): Compound2 showed a major ioeak at m/z 1077 [M+ Na]+ and significant fragment m/z 915 [M+ Na-162]+, m/z 771 [M+ Na-162-144]+,/z 641 [M+ Na-162-144-130]+ in the positive ESI-MShe molecular formula was unequivocally establishee C54H86O20 by HR-MALDI mass spectrometry (m/z077.5601 [M+ Na]+, calcd. for C54H86O20Na, 1077.5610hich was confirmed by data from the13C NMR spectrumdetailed comparison of the aglycone portion NMR d

1H, 13C, HSQC, HMBC, DQF-COSY, 2D-HOHAHA) oompounds1 and2 revealed that the aglycone was ident

n the two compounds (see Section2). Additionally for 2,esonances for the anomeric protons were observed in t1HMR spectra at� 4.90 (1H, dd,J= 9.2, 2.0 Hz), 4.83 (1Hd, J= 9.6, 2.0 Hz), 4.67 (2H, dd,J= 9.6, 2.0 Hz), and 4.4d, J= 7.9 Hz). Complete assignments of the1H and 13CMR signals of the sugar portion (seeTable 2) were accomlished by HSQC, HMBC, DQF-COSY and 2D-HOHAHxperiments which led to the identification of one term-d-glucopyranosyl unit (δ 4.49), two�-d-cymaropyranosynits (δ 4.90 and 4.83), one�-d-canaropyranosyl unδ 4.67), and one�-d-oleandropyranosyl unit (δ 4.67).n the basis of the HMBC correlations betweennomeric protons signals and the substituted carbonances of each sugar unit, compound2 was established a�,16-dihydroxy-14H�-pregn-5,16-dien-15-one-3-O-�-d-lucopyranosyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�--canaropyranosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)--d-cymaropyranoside.

cetyl group in C-16. Additionally, in the1H NMR spectrumf 3 the signal atδ 2.14 observed for stemmoside D wbsent, while the signals of H-16 and H-17 were significahifted to upfield values atδ 4.05 (1H, d,J= 9.7 Hz) andδ.87 (1H, dd,J= 9.7, 1.6 Hz), respectively, when compa

o those of stemmoside D where they resonated at1H, d,J= 10.1 Hz) and 2.08 (1H, dd,J= 10.1, 1.7 Hz)[10].ll these evidences, and the correlations observed inQF-COSY, HSQC and HMBC experiments sugge

hat 3 differed from stemmoside D only by the presef a hydroxyl group instead of an acetyl group in C-he�-orientation of H-14, H-16 and H-17 were establisy comparison of the chemical shifts of C-12 (δ 30.9),-14 (δ 58.2) and C-20 (δ 16.3), and3JHH value of H.16nd H-17 (9.7 Hz), to those of stemmoside D. Recemethodology based on the combined use of quaechanical calculations of1H, 13C andJ values was use

or the characterization of stemmosides C and D[10]. Theroposed procedure has been shown to be crucial wh

he analysis of NMR 2D-NOESY and ROESY spectraot allow to unambiguously establish the position of proelonging to ring D[10]. Once again, the H-14� configu-ation was corroborated by the downfield value of H-7� (δ.09) and Me-18 value (δ 1.20)[21]. Based on this evidenc

was established as 3�,16�-dihydroxy-14H�,17H�-regn-5-en-15-one-3-O-�-d-glucopyranosyl-(1→ 4)-�-d-leandropyranosyl-(1→ 4)-�-d-canaropyranosyl-(1→ 4)--d-cymaropyranosyl-(1→ 4)-�-d-cymaropyranoside.


Stemmoside H (4): Compound4 showed a major ionpeak at m/z 1135 [M+ Na]+ and significant fragmentsat m/z 973 [M+ Na-162]+, m/z 829 [M+ Na-162-144]+,m/z 685 [M+ Na-162-144-144]+ in the positive ESI-MS.Its molecular formula was unequivocally establishedto be C57H92O21 by HR-MALDI mass spectrometry(m/z 1135.6036 [M+ Na]+, calcd. for C57H92O21Na,1135.6029). The1H and 13C NMR spectra of4 closelyresembled to those of stemmoside D[10] only differingby the replacement of the�-d-canaropyranosyl unit bya �-d-oleandropyranosyl unit (see Section2). All theNMR data were assigned on the basis of the HSQC,DQF-COSY, 2D-HOHAHA and HMBC correlations. Thus,the structure of4 was deduced to be 16�-acetoxy-3�-hydroxy-14H�,17H�-pregn-5-en-15-one-3-O-�-d-glucopy-ranosyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-olean-dropyranosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)-�-d-cymaropyranoside.

Stemmoside I (5), stemmoside J (6), and stemmoside K(7): The HR-MALDI mass spectrum of compounds5 (m/z1137.6190 [M+ Na]+, calcd. for C57H94O21Na, 1137.6185),6 (m/z 1123.6036 [M+ Na]+, calcd. for C56H92O21Na,1123.6029), and7 (m/z 1081.5927 [M+ Na]+, calcd. forC54H90O20Na, 1081.5923) supported a molecular formula ofC57H94O21, C56H92O21, and C54H90O20, respectively. It wasapparent from the NMR data (1H, 13C, HSQC, HMBC, DQF-Cca ps ofS thed , thesd -( -np3npa s3npa

SI-M Ab er aveso

3

dia-t ella itro[ F,

and hepatocyte growth factor (HGF) have been postulatedto be involved in angiogenesis[26]. In particular, the pro-duction of autocrine growth factors by KS, which includesVEGF, dictates the progression of the lesion and are likelyto play a central role in the development and progression ofKS [27]. In the present study, we modeled VEGF-inducedKS cell proliferation to test the anti-proliferative propertiesof the compounds1–8, and stemmosides C and D.

Before studying the effect of these compounds on the cellproliferation we tested their cytotoxic concentration by theXTT and trypan blue dye assays. Results indicated that com-pounds1, 3, 5–8, and stemmosides C and D did not show anycytotoxic activity up to a concentration of 20�M, whereascompounds2 and4 start inducing a noticeable reduction ofthe cell viability (< 70%) at higher concentrations than 1�Mand 0.1�M, respectively.

Therefore, we next performed a dose dependence study totest the effect on the KS cell proliferation in a concentrationrange 0.1–20�M for compounds1, 3, 5–8, and stemmosidesC and D, and in a concentration range 0.001–1�M for com-pounds2 and4.

Compounds1, 3, 5–8, and stemmosides C and D reducedthe VEGF-induced KS cell proliferation in a dose-dependentmanner and the highest reduction occurred at concentrationof 10�M. In particular, the dose dependence effect of stem-moside D (SmD) is shown inFig. 1.

ts theVo m-ma

ltsi tiono -p the 8 h

F sideD tionso L).T nderS ente

OSY, 2D-HOHAHA) of compounds5, 6, and7 that theseompounds only differed from compound4, stemmoside Dnd stemmoside C, previously isolated from the pericar. argel[10], only in the nature of the aglycone whereouble bond between C-5 and C-6 was lacking. Thustructure of5 was determined to be 16�-acetoxy-3�-hy-roxy-14H�,17H�-pregn-15-one-3-O-�-d-glucopyranosyl1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-oleandropyraosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)-�-d-cymaro-yranoside, the structure of6 was deduced as 16�-acetoxy-�-hydroxy-14H�,17H�-pregn-15-one-3-O-�-d-glucopyra-osyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-canaro-yranosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)-�-d-cym-ropyranoside, and the structure of7 was established a�,14�-dihydroxy-17H�-pregn-15-one-3-O-�-d-glucopyra-osyl-(1→ 4)-�-d-oleandropyranosyl-(1→ 4)-�-d-canaro-yranosyl-(1→ 4)-�-d-cymaropyranosyl-(1→ 4)-�-d-cym-ropyranoside.

Compound8showed a major ion peak in the positive ES atm/z531 [M+ Na]+. It was identified as stemmosidey comparison of its1H and 13C NMR spectra with thoseported by Hamed for the same compound from the lef S. argel[7].

.2. Anti-proliferative activity

KS cells produce and respond to various soluble meors; in particular, IL-1, IL-6, VEGF, and GM-CSF, as ws Tat-protein are known to stimulate KS cell growth in v

23–25]. Furthermore, fibroblast growth factor (FGF), VEG

The results summarized inTable 3, clearly indicates thatemmoside D was the most effective (65% inhibition ofEGF-induced cell proliferation) at 10�M. The inhibitionf the VEGF-induced cell proliferation was 45.3% for steoside C and ranged from 24.4–39.2% for compounds1, 3nd5–8.

In the case of compounds2and4,the dose response resundicates that the highest reduction of the cell proliferaccurred at 1�M for compound2 (27.5 %), while for comound4 (21.5%) at 0.1�M. Moreover, the treatment wiach of the compounds for 1 h followed by incubation for 4

ig. 1. Graphical representation for anti-proliferative activity of stemmo(SmD). KS cells were pre-treated with or without different concentra

f stemmoside D (SmD) followed by stimulation with VEGF (50 ng/mhe cell proliferation was determined with XTT assay as described uection2. Data represent the means± S.E.M. from at least three independxperiments in duplicates.


Fig. 2. Effect of stemmoside D (SmD) on the VEGF-induced KS cell proliferation. Relative absorbance at 490 nm of the XTT cell proliferation assay andrepresentatives photographs of untreated cells (control), cells treated with VEGF (50 ng/mL) or with stemmoside D (SmD, 10�M) alone, and of cells pre-treated with SmD (10�M) followed by stimulation with VEGF (50 ng/mL). Data represent the means± S.E.M. from at least three independent experiments induplicates. Statistical analysis was performed by Student’st-test. Statistical significance was set atP< 0.05.

without VEGF did not have any significant effect on the KScell proliferation when compared to untreated cells (control).Fig. 2 shows the effect of SmD (10�M) on the VEGF-induced cell proliferation. In particular, the absorbance at490 nm increased from 1.5 in control cells to 3.2 in VEGF-

Table 3Effect of stemmosides on the VEGF-induced cell proliferation

Compound Cell proliferation inhibition (%)

Stemmoside E (1) 24.4± 0.5Stemmoside F (2) 27.5± 0.2Stemmoside G (3) 24.3± 1.1Stemmoside H (4) 21.5± 0.9Stemmoside I (5) 22.5± 1.1Stemmoside J (6) 38.5± 0.8Stemmoside K (7) 39.5± 1.1Stemmoside A (8) 39.2± 0.9Stemmoside C 45.3± 0.5Stemmoside D 65.3± 0.5

KS cells were pre-treated with or without compounds1, 3, 5–8, and stem-mosides C and D (10�M), 2 (1�M), or 4 (0.1�M) followed by stimulationwith VEGF (50 ng/mL). The cell proliferation was determined with XTTassay as described under Section2. Data represent the means± S.E.M. fromat least three independent experiments in duplicates.

treated cells, and returned to near control value when cellswere pre-treated with SmD (10�M) before VEGF stimula-tion.

In the present study, we observed that pregnane glycosidesisolated fromS. argelstart inducing cell death at concentra-tion values over 20�M (compounds1, 3, 5–8, and stem-mosides C and D) or at concentration higher than 1�M or0.1�M (compounds2 and4, respectively). In order to obtainmore information about the cytotoxicity of these unusualcompounds, and as a part of our on going research on antipro-liferative constituents of medicinal plants, we are investigat-ing their possible effect on the process of cell death, eithernecrotic or apoptotic.

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new unusual pregnane glycosides with antiproliferative activity from< i> solenostemma argel

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