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Synthesis and antitumor activity of spin labeled derivatives of podophyllotoxin
life sciences, Vol. 60, No. 8, pp. 511417, 1997 CopyTigbt 0 1997 E!kdr science Inc. Printed in the WA. All rights rcmxwd 0024-32m/w 517.00 + .al
PII SOO24-3205(%)00689-3
ELSEVIER
SYNTHESIS AND ANTITUMOR DERIVATIVES Xuan Tian’, Yan-guang
ACTIVITY Oii SPIN LABELED
OF PODOPHYLLOTOXIN
Wa.ng*.Ming-gui Yang’ and Yao-zu Chen’,‘*
‘State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R.China; *Department of Chemistry, Zhejiang University, Hangzhou 3 10027, P.R.China (Received in final form December 2,19!X)
Summary Three new nitroxyl labeled derivatives of podophyllotoxin 4-6 have been synthesized and evaluated for their antitumor activity in vitro. Compounds 4-6 showed significant inhibitory activity against human nasopharyngeal carcinoma KB,lung cancer AS49 and stomach carcinoma SGC-7901 cells, as well as mouse leukemia L12 10 and P388 cells. Compounds 4 and 5 exhibited comparable or superior activity to clinically used etoposide (VP- 162) in their inhibition of these ceil lines. Key Word: podophyllotoxin, nitroxyl, spin labeling, antitumor
Semisynthetic analogues of the naturally occurring podophyllotoxin (1) have drawn much renewed interest in recent years as a result of the development of etoposide (VP-162) and teniposide (VM-26J) as anticancer drugs ( 1,2). It is believed that such analogues of 4’demethylepipodophyllotoxin exert their antitumor activity through stabilization of a cleavable complex between DNA and type II DNA topoisomerase. This leads ultimately to inhibition of DNA catenation activity and produces single and double strand breaks (3,4). In our previous studies (%lO),we found that a number of nitroxyl spin labeled derivatives of podophyllotoxin had significant antitumor activity with marked decrease in toxicity compared with the parent compounds 1 and 2. A series of spin labeled derivatives of thio-TEPA (I l-13) nitrosourea (14),rubomycin (15) were also reported to have superior pharmacological properties to their parent compounds. In view of these results, we are interested in further studies in the spin labeled podophyllotoxin series. Therefore, we have synthesized and tested in vitro for antitumor activity three new nitroxyl labeled analouges of 4’-demethylepipodophyllotoxin with structural features shown in formulae g-6. Materials and Methods Spin labeled derivatives of podophyllotoxin 4-6 were prepared as shown in Scheme 2. Podophyllotoxin (.I); isolated from a Chinese medicmal herb Podonhvllum emodi Wall var. Chinesis Sprague, was used as starting material. 4-Bromo-4’-demethylepipodophyllotoxin (1) and
512
Antitumor Activity of Podophyllototi
Vol. 60, No. 8, 1997
_?. R=Me
OMe Me0
HNR
Scheme 1
epipodophyllotoxin
(8) were synthesized by our previous procedure (5,6).
Epipodophyllotoxin (8) was treated with HN1 in the presence of BF,.OEt, to yield 4@azido4’-demethylepipodophyllotoxin (9) as the major product,which was accompanied by the C-4 isomer product, 4c+azido-4’-demethylepipodophyllotoxin. To a solution of 8 (5.Og,l3mmol) in CH$& (175ml) and HN3 (13mmol) in benzene was added dropwise BFr.OEt, (2.25ml) at -15 ‘C. and the mixture was stirred at -15 “C for lh. After being quenched with pyridine (2.25ml), the solution was washed with water and dried over Na2S0+ Evaporation of the solvent gave a solid, which was checked by TLC and NMR analyses to be a mixture of 4a- and 4j3-azido isomers (ca.l:S).Crystallization from acetoneimethol provided the pure 4P-isomer 9. ‘HNMR (CDCI?) 6 6.82 (s,lH,H-5) 6,58(s,lH,H-8) 6.“” ’ L/ (s,2H,H-2’,6’),6.01 jd,2H,OCH#), 5.47 (s,lH,4’-OII), 4.81 (d, J=4,0Hz,lH,H-4) 4.63 (d.J=4,4Hz,lH,H-1), 4.32 (d,J=9.5Hz,2H,H-11) 3,87(s,hH,3’.5’-OCH3),
513
Antitumor Activity of Podophyllotoxin
Vol. 60, No. 8, 1997
Br
OH
I
HBr 1
NH2 I
HN3
H2
*
G%Pd/C
BF3-0Et2
1
0
c NO -
I!2 4
DCC
O=C:=N
-
c
0
OMe
10
N6
-5
12
11
S=C=N
Me0
OMe
Me0
HOOC
)
Ncj
lo
,fj
c A.3 Scheme 2 3.17 (dd, 3=4.4.12.6Hz.
lH, H-2), 2.93 (m, 1H. H-3)ppm.
Further reduction of ii@-azido-4’-demethyl-epipodophyllotoxin (9) led to 4p-amino-4’in EtOAc (lOOmI) was To a solution of 9 (5.Og, 1lmmol) demethyl-epipodophylln!oxin (U).
514
Antitumor Activity of Podophyllotoxin
TABLE I.
Analytical Data of Compounds d-6,9 and lo.
Compound Number
m.p.
Yield
( “C)
z4
!8?-188
(%) 72
Molecular Formula C;iH;;‘N;(‘,9 (579.62)
5
183-184
66
6
189-200”
67
9
213-214”
91
CZIHI~N~ (425.39)
212-214
68
CZIHZINO~ (399.40)
I.0 B
Decomposition
4” 5”
C~OHUN& (580.61) C3 IHMN@~S (612.71)
9 lo
64.24 63.93 62.06
6.09 6.10 5.90
62.20 60.77 60.83 59.29 59.15 63.15 63.02
6.03 6.25 6.32 4.50 4.52 5.30 5.35
IR(KBr) (cm-‘) \ , 3374(NH, OH), 1775(lactone), 15 10 and 1485(aromatic C=C), 3381 (NH, OH), 1777(lactone), 15 15 and 1483(aromatic C=C),
MS m/z 1674(NHCO), 1380(NO) 162 R(NHC0) 1369(NO)
581(20),580(25),579(33), 550( 12),383(30),282( 100) 581(14), 580(12), 549(9), 383(6),279( 100) 614(79),613(15),612(53), 578(53),552(52),383(100) 425(34),383(5),307(42), 154( 100) 399( 100),383(89),354(28), 154(32)
ESR (1 x lOAM in EtOH): 3 lines, aN=15-16G, AHr,=2.2-2.8G, g,,=2.0060.
TABLE III. Biological Evaluation Compound Number
N 4.83 5.06 7.24 7.34 6.86 7.01 9.88 9.80 3.51 3.50
Spectral Data of Compounds 4-6,9 and _lJ.
3325(NH, OH), 177l(lactone), 162’7(NHCO) 15 11 and 1484(aromatic C=C), 1370(NO) 3420(0H), 2100(azide), 1765( lactone),l598, 1508 and 1484(aromatic C=C), 930(OCHz0) 3440-33OO(OH, NH& 1745(lactone), 1605, 1500 and 1480(aromatic C=C), 931(OCH20)
6”
Cal&Found(%) C H
point
TABLE Il. Compound Number
Vol. 60, No. 8, 1997
of Spin Labeled Derivatives of Podophyllotoxins
KBh L1210” 2 0.20 0.27 4 0.028 0.18 5 0.026 0.24 _< 6 0.22 0.68 Concentration which cawes 50% inhibition Results obtained after 721~. Results obtained after 24h.
ID50(uM)” P388” A549h SGC-7901 h 0.012 0.44 0.32 0.016 0.50 0.21 0.0085 0.32 0.38 0.024 1.4 0.46 of the growth of each cell line
Vol. 60, No. 8, 1997
Antitumor Activityof Podophyllotoxb
515
added 10% palladium on carbon (l.Og). This mixture was shaked under 40 psi of H2 for 16h. The reaction mixture was filtered through celite and the filterate evaporated in vacua. Crystallization from MeOH gave 3.lg of lo. ‘HNMR (CD(&) 6 6.81 (s,lH, H-5), 6.49(s,lH, H-8), 6.30 (s,2H, H2’,6’),5.96 (d,2H,0CH20), 5.06 (s,lH,4’-OH), 4.55 (d, J= 5.2Hz,lH,H-I), 4.28 (d,J=9.5Hz,2H,H1 I), 4.20(d.J=4.lHz.lH.H-4). 3.7’7<~.6H.3’.5’-OCH~). 3.28 (dd. J-5.2. 14Hz, 1H. H-2). 2.85 (m, 1H. H-3)ppm. Spin labeled compound 4 was synthesized by direct condensation of carboxylic acid u(l6) and 4P-amino-4’-demethyl-epipodophyllotoxin (10) in the presence of DCC To a solution of lo (0.2Og,O.5mmol) and 1 (O.lOg, O.Smmol) in lOm1 CH2ClZ was added DCC (O.lOg,0.5mmol).The reaction mixture was stirred at room temperature for 6h. After 2 drops of acetic acid was added, the mixture was filtered, washed with water, dried over anhydrous Na2S04, and chromatographed on a silica gel column and eluted with CH2C12-Me2C0 (20:1) to give an orange solid compound 4. The reaction of 4P-amine-4’-demethyl-epipodophyllotoxin (l0) and isocyanate 12. (17) resulted in spin labeled compound 5. To a solution of lO(0.40g,lmmol) in 20 ml of anhydrous CH$& was added dropwise nitroxyl 12 (O.l8g,lmmol) in 10 ml of anhydrous CH& The mixture was stirred at room temperature for lh, then washed with water,dried over anhydrous MgS04, and chromatographed through silica gel using EtOAc as eluent to give an orange solid compound 5. Compound 6 was synthesized by the reaction of lo with isothiocyanide 13 (18). The solution of lo (0,40g,lmmol) and isothiocyanide 12 (0.2lg,lmmol) in 20 ml of dried CH&N was refluxed for 3h. The solvent was removed in vacua. The residue was chromatographed on a silica gel column and eluted with CH2ClZ-Me&O (5: 1) to yield an orange solid compound 6. All melting points were taken on a X4 melting point apparatus and uncorrected. ‘HNMR spectra were obtained by using a Bruker AM 400 spectrometer, all chemical shifts were reported in ppm from TMS. ESR spectra were obtained with a Bruker ER-200D-SRC X-band spectrometer. Mass spectral analyses were taken on a V.G. ZAB-HS instrument at 70 eV with a direct inlet system. Elemental analyses were determined on a Carlo Elba 1106 instrument. IR spectra were recorded on a NIC-5DX spectrophotometer. The in vitro biological activity of compounds 4-6 was performed by Beijing Institute of Materia Medica, Chinese Academy of Medicine, and Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Drugs were dissolved diluted before use with Hz0 RPM1 1640 medium (Nissui penicillin G (100 W/ml) and
in Me#O at a concentration of 20mM as the stock solution and to the desired concentration of each drug. The cells were grown in Pharmaceutical Co., Ltd, Tokyo, Japan) with 10% fetal calf serum, streptomycin (lOOpg/ml) at 37 “C under a humidified 95% air + 5%
CO* atmosphere. Assays for the cytotoxicity in KB, A549, SC<-7901, I,1210 and P388 cells were carried out according tc the published procedure (8, 20, 21). The data were recorded as the concentration (PM) which causes 50% inhibition of the growth of each cell line.
516
AntitumorActivity of Podophyllobxin
Vol. 60, No. 8, 1997
Result and Discussion Synthesized compounds $5, 9 and lo were characterized by m.p., ‘HNMR, MS and IR spectral analyses, as well as elemental analysis. The physical properties of these new compounds are show-nin TABLE I and II. The data on -in vitro biological evaluation of spin labeled derivatives of podophyllotoxin 4-6 tested against human nasopharyngeal carcinoma KB,lung cancer A549 and stomach carcinoma SGC-7901 cells as well as mouse leukemia L1210 and P388 cells are shown in TABLE III. As illustrated in TABLE III, compounds 3-5 showed significant inhibitory activity against these cell lines. Compounds 4 and 5 were found to have comparable or superior activity to clinically used etoposide (VP-l& 2). The results demonstrated that the glycosidic moiety of VP-16 is not so essential for the antitumor activity and considerable simplification in the sugar structure might be permitted. The results also indicated that the nitroxyl radical moiety can exhibit a beneficially modifying effect on the pharmacological properties of podophyllotoxin (1) and VP-16(z) as shown in our previous studies (5,6,9). We believe that spin labeling of drugs is a promising direction in antitumor chemotherapy not only because they exhibit superior pharmacological properties to the parent compounds but also because they can be monitored by ESR in pharmacological experiments (5,19). Acknowledgment The authors thank Beijing Institute of Materia Medica, Chinese Academy of Medicine, and Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for cytotoxicity assay. This work was financially supported by the National Natural Science Foundation of China. References I. JARDINE, Podophvllotoxins _ Anticancer Agents Based on Natural p-2 Products Models 319-351, Academic Press,Inc., New York (1980). 2. H.F. STAHELIN, A. VON WARTBURG, Cancer Res. 2 5-15(1991). 3. G. CHEN, Y. LIU, T. ROWE, B. HALLIGAN, J. TEUEY and L. LIU, J.Biol.Chem. 259 13560-13568 (1984). 4. J. LOIKE, S. HORTWITZ, Biochem. 15 5443-5449 (1986). 5. Y.Z. CHEN, C.J. ZHANG and X. TIAN, Scientia Sinica 30B 1070-1079 (1987). 6. Y.Z. CHEN, Y.G. WANG, J.X. LI, X. TIAN, Z.P. JIA, P.Y. ZHANG, Life Sci. 45 25692575 (1989). 7. Y.Z. CHEN, Y.G. WANG, J.X. LI, X. TIAN, Current Sci. 59 517-518 (1990). 8. Z.P. JIA,P.Y. ZHANG,Z.D. LIANG, Y.G. WANG, Y.Z. CHEN, J.X. LI, X. TIAN, Acta Pharmacologica Sinica 11549-553 (1990). 9. J.Z. WANG, X. TIAN, H. TSUMURA, Anti-Cancer Drug Design 8 193-202 (1993). 10 X.Q. HE, P.Y. ZHANG, X. TIAN, Acta Pharmacologica Sinica 12 276-279 (1992). 11 G. SOSNOVSKY and S.W. LI, Life Sci., 36,1473-1477 (1985). 12. G. SOSNOVSKY, N. 0. MAHESWARA RAO and S. W. LI, J.Med.Chem. 29 2225-2230 (1986). 13. G. SOSNOVSKY, Pure and Appl. Chem. 62 289-294 (1990). 14. G. SOSNOVSKY, S.W. LI and N.U. MAHESWARA RAO, Z. Naturforsh. 42c 921-931 1.
Vol. 60, No. 8, 1997
15. 16. 17. 18. 19. 20. 2 I.
Antitumor Activity of Podophyllotoxin
(1987). N.M. EMANUE L, N.P. KONOVALOVA, R.F. DJACHKOVSKAYA and L. K. DENISOVA, Antibiotiki 22 8 11-8 15 (1982). T.L. LAWRENCE, Can. J. Chem. 52.3381-3383 (1974). H.O. HANKOVSZKY, K. HIDEG and J. TIGYI, Acta Chim. Acad. Sci. Hung., Tomus. 98 339-345 (1978). H.O. HANKOVSZKY, K. HIDEG and L. LEX, Synthesis 92 147-149 (1981). P.L. GUTIERREZ, B.E. COHEN, G. SOSNOVSKY, T.A. DAVIS and M.J. BGORIN, Cancer Chemother. Pharmacol. 15.185-191 (1985). P.J. FERGUSON, M.H. FISHER, J. STEPHENSON, D.H. LI, B.S. ZHOU and Y .C. CHENG, Cancer Res. 48 5956-5965 (1988). Z.P. 514 P.Y. ZHANG and Z.D. LIANG, Chin. J. Pharmacol. Toxicol. 5 47-49 (1991).
517
PII SOO24-3205(%)00689-3
ELSEVIER
SYNTHESIS AND ANTITUMOR DERIVATIVES Xuan Tian’, Yan-guang
ACTIVITY Oii SPIN LABELED
OF PODOPHYLLOTOXIN
Wa.ng*.Ming-gui Yang’ and Yao-zu Chen’,‘*
‘State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R.China; *Department of Chemistry, Zhejiang University, Hangzhou 3 10027, P.R.China (Received in final form December 2,19!X)
Summary Three new nitroxyl labeled derivatives of podophyllotoxin 4-6 have been synthesized and evaluated for their antitumor activity in vitro. Compounds 4-6 showed significant inhibitory activity against human nasopharyngeal carcinoma KB,lung cancer AS49 and stomach carcinoma SGC-7901 cells, as well as mouse leukemia L12 10 and P388 cells. Compounds 4 and 5 exhibited comparable or superior activity to clinically used etoposide (VP- 162) in their inhibition of these ceil lines. Key Word: podophyllotoxin, nitroxyl, spin labeling, antitumor
Semisynthetic analogues of the naturally occurring podophyllotoxin (1) have drawn much renewed interest in recent years as a result of the development of etoposide (VP-162) and teniposide (VM-26J) as anticancer drugs ( 1,2). It is believed that such analogues of 4’demethylepipodophyllotoxin exert their antitumor activity through stabilization of a cleavable complex between DNA and type II DNA topoisomerase. This leads ultimately to inhibition of DNA catenation activity and produces single and double strand breaks (3,4). In our previous studies (%lO),we found that a number of nitroxyl spin labeled derivatives of podophyllotoxin had significant antitumor activity with marked decrease in toxicity compared with the parent compounds 1 and 2. A series of spin labeled derivatives of thio-TEPA (I l-13) nitrosourea (14),rubomycin (15) were also reported to have superior pharmacological properties to their parent compounds. In view of these results, we are interested in further studies in the spin labeled podophyllotoxin series. Therefore, we have synthesized and tested in vitro for antitumor activity three new nitroxyl labeled analouges of 4’-demethylepipodophyllotoxin with structural features shown in formulae g-6. Materials and Methods Spin labeled derivatives of podophyllotoxin 4-6 were prepared as shown in Scheme 2. Podophyllotoxin (.I); isolated from a Chinese medicmal herb Podonhvllum emodi Wall var. Chinesis Sprague, was used as starting material. 4-Bromo-4’-demethylepipodophyllotoxin (1) and
512
Antitumor Activity of Podophyllototi
Vol. 60, No. 8, 1997
_?. R=Me
OMe Me0
HNR
Scheme 1
epipodophyllotoxin
(8) were synthesized by our previous procedure (5,6).
Epipodophyllotoxin (8) was treated with HN1 in the presence of BF,.OEt, to yield 4@azido4’-demethylepipodophyllotoxin (9) as the major product,which was accompanied by the C-4 isomer product, 4c+azido-4’-demethylepipodophyllotoxin. To a solution of 8 (5.Og,l3mmol) in CH$& (175ml) and HN3 (13mmol) in benzene was added dropwise BFr.OEt, (2.25ml) at -15 ‘C. and the mixture was stirred at -15 “C for lh. After being quenched with pyridine (2.25ml), the solution was washed with water and dried over Na2S0+ Evaporation of the solvent gave a solid, which was checked by TLC and NMR analyses to be a mixture of 4a- and 4j3-azido isomers (ca.l:S).Crystallization from acetoneimethol provided the pure 4P-isomer 9. ‘HNMR (CDCI?) 6 6.82 (s,lH,H-5) 6,58(s,lH,H-8) 6.“” ’ L/ (s,2H,H-2’,6’),6.01 jd,2H,OCH#), 5.47 (s,lH,4’-OII), 4.81 (d, J=4,0Hz,lH,H-4) 4.63 (d.J=4,4Hz,lH,H-1), 4.32 (d,J=9.5Hz,2H,H-11) 3,87(s,hH,3’.5’-OCH3),
513
Antitumor Activity of Podophyllotoxin
Vol. 60, No. 8, 1997
Br
OH
I
HBr 1
NH2 I
HN3
H2
*
G%Pd/C
BF3-0Et2
1
0
c NO -
I!2 4
DCC
O=C:=N
-
c
0
OMe
10
N6
-5
12
11
S=C=N
Me0
OMe
Me0
HOOC
)
Ncj
lo
,fj
c A.3 Scheme 2 3.17 (dd, 3=4.4.12.6Hz.
lH, H-2), 2.93 (m, 1H. H-3)ppm.
Further reduction of ii@-azido-4’-demethyl-epipodophyllotoxin (9) led to 4p-amino-4’in EtOAc (lOOmI) was To a solution of 9 (5.Og, 1lmmol) demethyl-epipodophylln!oxin (U).
514
Antitumor Activity of Podophyllotoxin
TABLE I.
Analytical Data of Compounds d-6,9 and lo.
Compound Number
m.p.
Yield
( “C)
z4
!8?-188
(%) 72
Molecular Formula C;iH;;‘N;(‘,9 (579.62)
5
183-184
66
6
189-200”
67
9
213-214”
91
CZIHI~N~ (425.39)
212-214
68
CZIHZINO~ (399.40)
I.0 B
Decomposition
4” 5”
C~OHUN& (580.61) C3 IHMN@~S (612.71)
9 lo
64.24 63.93 62.06
6.09 6.10 5.90
62.20 60.77 60.83 59.29 59.15 63.15 63.02
6.03 6.25 6.32 4.50 4.52 5.30 5.35
IR(KBr) (cm-‘) \ , 3374(NH, OH), 1775(lactone), 15 10 and 1485(aromatic C=C), 3381 (NH, OH), 1777(lactone), 15 15 and 1483(aromatic C=C),
MS m/z 1674(NHCO), 1380(NO) 162 R(NHC0) 1369(NO)
581(20),580(25),579(33), 550( 12),383(30),282( 100) 581(14), 580(12), 549(9), 383(6),279( 100) 614(79),613(15),612(53), 578(53),552(52),383(100) 425(34),383(5),307(42), 154( 100) 399( 100),383(89),354(28), 154(32)
ESR (1 x lOAM in EtOH): 3 lines, aN=15-16G, AHr,=2.2-2.8G, g,,=2.0060.
TABLE III. Biological Evaluation Compound Number
N 4.83 5.06 7.24 7.34 6.86 7.01 9.88 9.80 3.51 3.50
Spectral Data of Compounds 4-6,9 and _lJ.
3325(NH, OH), 177l(lactone), 162’7(NHCO) 15 11 and 1484(aromatic C=C), 1370(NO) 3420(0H), 2100(azide), 1765( lactone),l598, 1508 and 1484(aromatic C=C), 930(OCHz0) 3440-33OO(OH, NH& 1745(lactone), 1605, 1500 and 1480(aromatic C=C), 931(OCH20)
6”
Cal&Found(%) C H
point
TABLE Il. Compound Number
Vol. 60, No. 8, 1997
of Spin Labeled Derivatives of Podophyllotoxins
KBh L1210” 2 0.20 0.27 4 0.028 0.18 5 0.026 0.24 _< 6 0.22 0.68 Concentration which cawes 50% inhibition Results obtained after 721~. Results obtained after 24h.
ID50(uM)” P388” A549h SGC-7901 h 0.012 0.44 0.32 0.016 0.50 0.21 0.0085 0.32 0.38 0.024 1.4 0.46 of the growth of each cell line
Vol. 60, No. 8, 1997
Antitumor Activityof Podophyllotoxb
515
added 10% palladium on carbon (l.Og). This mixture was shaked under 40 psi of H2 for 16h. The reaction mixture was filtered through celite and the filterate evaporated in vacua. Crystallization from MeOH gave 3.lg of lo. ‘HNMR (CD(&) 6 6.81 (s,lH, H-5), 6.49(s,lH, H-8), 6.30 (s,2H, H2’,6’),5.96 (d,2H,0CH20), 5.06 (s,lH,4’-OH), 4.55 (d, J= 5.2Hz,lH,H-I), 4.28 (d,J=9.5Hz,2H,H1 I), 4.20(d.J=4.lHz.lH.H-4). 3.7’7<~.6H.3’.5’-OCH~). 3.28 (dd. J-5.2. 14Hz, 1H. H-2). 2.85 (m, 1H. H-3)ppm. Spin labeled compound 4 was synthesized by direct condensation of carboxylic acid u(l6) and 4P-amino-4’-demethyl-epipodophyllotoxin (10) in the presence of DCC To a solution of lo (0.2Og,O.5mmol) and 1 (O.lOg, O.Smmol) in lOm1 CH2ClZ was added DCC (O.lOg,0.5mmol).The reaction mixture was stirred at room temperature for 6h. After 2 drops of acetic acid was added, the mixture was filtered, washed with water, dried over anhydrous Na2S04, and chromatographed on a silica gel column and eluted with CH2C12-Me2C0 (20:1) to give an orange solid compound 4. The reaction of 4P-amine-4’-demethyl-epipodophyllotoxin (l0) and isocyanate 12. (17) resulted in spin labeled compound 5. To a solution of lO(0.40g,lmmol) in 20 ml of anhydrous CH$& was added dropwise nitroxyl 12 (O.l8g,lmmol) in 10 ml of anhydrous CH& The mixture was stirred at room temperature for lh, then washed with water,dried over anhydrous MgS04, and chromatographed through silica gel using EtOAc as eluent to give an orange solid compound 5. Compound 6 was synthesized by the reaction of lo with isothiocyanide 13 (18). The solution of lo (0,40g,lmmol) and isothiocyanide 12 (0.2lg,lmmol) in 20 ml of dried CH&N was refluxed for 3h. The solvent was removed in vacua. The residue was chromatographed on a silica gel column and eluted with CH2ClZ-Me&O (5: 1) to yield an orange solid compound 6. All melting points were taken on a X4 melting point apparatus and uncorrected. ‘HNMR spectra were obtained by using a Bruker AM 400 spectrometer, all chemical shifts were reported in ppm from TMS. ESR spectra were obtained with a Bruker ER-200D-SRC X-band spectrometer. Mass spectral analyses were taken on a V.G. ZAB-HS instrument at 70 eV with a direct inlet system. Elemental analyses were determined on a Carlo Elba 1106 instrument. IR spectra were recorded on a NIC-5DX spectrophotometer. The in vitro biological activity of compounds 4-6 was performed by Beijing Institute of Materia Medica, Chinese Academy of Medicine, and Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Drugs were dissolved diluted before use with Hz0 RPM1 1640 medium (Nissui penicillin G (100 W/ml) and
in Me#O at a concentration of 20mM as the stock solution and to the desired concentration of each drug. The cells were grown in Pharmaceutical Co., Ltd, Tokyo, Japan) with 10% fetal calf serum, streptomycin (lOOpg/ml) at 37 “C under a humidified 95% air + 5%
CO* atmosphere. Assays for the cytotoxicity in KB, A549, SC<-7901, I,1210 and P388 cells were carried out according tc the published procedure (8, 20, 21). The data were recorded as the concentration (PM) which causes 50% inhibition of the growth of each cell line.
516
AntitumorActivity of Podophyllobxin
Vol. 60, No. 8, 1997
Result and Discussion Synthesized compounds $5, 9 and lo were characterized by m.p., ‘HNMR, MS and IR spectral analyses, as well as elemental analysis. The physical properties of these new compounds are show-nin TABLE I and II. The data on -in vitro biological evaluation of spin labeled derivatives of podophyllotoxin 4-6 tested against human nasopharyngeal carcinoma KB,lung cancer A549 and stomach carcinoma SGC-7901 cells as well as mouse leukemia L1210 and P388 cells are shown in TABLE III. As illustrated in TABLE III, compounds 3-5 showed significant inhibitory activity against these cell lines. Compounds 4 and 5 were found to have comparable or superior activity to clinically used etoposide (VP-l& 2). The results demonstrated that the glycosidic moiety of VP-16 is not so essential for the antitumor activity and considerable simplification in the sugar structure might be permitted. The results also indicated that the nitroxyl radical moiety can exhibit a beneficially modifying effect on the pharmacological properties of podophyllotoxin (1) and VP-16(z) as shown in our previous studies (5,6,9). We believe that spin labeling of drugs is a promising direction in antitumor chemotherapy not only because they exhibit superior pharmacological properties to the parent compounds but also because they can be monitored by ESR in pharmacological experiments (5,19). Acknowledgment The authors thank Beijing Institute of Materia Medica, Chinese Academy of Medicine, and Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for cytotoxicity assay. This work was financially supported by the National Natural Science Foundation of China. References I. JARDINE, Podophvllotoxins _ Anticancer Agents Based on Natural p-2 Products Models 319-351, Academic Press,Inc., New York (1980). 2. H.F. STAHELIN, A. VON WARTBURG, Cancer Res. 2 5-15(1991). 3. G. CHEN, Y. LIU, T. ROWE, B. HALLIGAN, J. TEUEY and L. LIU, J.Biol.Chem. 259 13560-13568 (1984). 4. J. LOIKE, S. HORTWITZ, Biochem. 15 5443-5449 (1986). 5. Y.Z. CHEN, C.J. ZHANG and X. TIAN, Scientia Sinica 30B 1070-1079 (1987). 6. Y.Z. CHEN, Y.G. WANG, J.X. LI, X. TIAN, Z.P. JIA, P.Y. ZHANG, Life Sci. 45 25692575 (1989). 7. Y.Z. CHEN, Y.G. WANG, J.X. LI, X. TIAN, Current Sci. 59 517-518 (1990). 8. Z.P. JIA,P.Y. ZHANG,Z.D. LIANG, Y.G. WANG, Y.Z. CHEN, J.X. LI, X. TIAN, Acta Pharmacologica Sinica 11549-553 (1990). 9. J.Z. WANG, X. TIAN, H. TSUMURA, Anti-Cancer Drug Design 8 193-202 (1993). 10 X.Q. HE, P.Y. ZHANG, X. TIAN, Acta Pharmacologica Sinica 12 276-279 (1992). 11 G. SOSNOVSKY and S.W. LI, Life Sci., 36,1473-1477 (1985). 12. G. SOSNOVSKY, N. 0. MAHESWARA RAO and S. W. LI, J.Med.Chem. 29 2225-2230 (1986). 13. G. SOSNOVSKY, Pure and Appl. Chem. 62 289-294 (1990). 14. G. SOSNOVSKY, S.W. LI and N.U. MAHESWARA RAO, Z. Naturforsh. 42c 921-931 1.
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15. 16. 17. 18. 19. 20. 2 I.
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(1987). N.M. EMANUE L, N.P. KONOVALOVA, R.F. DJACHKOVSKAYA and L. K. DENISOVA, Antibiotiki 22 8 11-8 15 (1982). T.L. LAWRENCE, Can. J. Chem. 52.3381-3383 (1974). H.O. HANKOVSZKY, K. HIDEG and J. TIGYI, Acta Chim. Acad. Sci. Hung., Tomus. 98 339-345 (1978). H.O. HANKOVSZKY, K. HIDEG and L. LEX, Synthesis 92 147-149 (1981). P.L. GUTIERREZ, B.E. COHEN, G. SOSNOVSKY, T.A. DAVIS and M.J. BGORIN, Cancer Chemother. Pharmacol. 15.185-191 (1985). P.J. FERGUSON, M.H. FISHER, J. STEPHENSON, D.H. LI, B.S. ZHOU and Y .C. CHENG, Cancer Res. 48 5956-5965 (1988). Z.P. 514 P.Y. ZHANG and Z.D. LIANG, Chin. J. Pharmacol. Toxicol. 5 47-49 (1991).
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