CA2044211A1 - Etoposide analogues - Google Patents
Etoposide analoguesInfo
- Publication number
- CA2044211A1 CA2044211A1 CA 2044211 CA2044211A CA2044211A1 CA 2044211 A1 CA2044211 A1 CA 2044211A1 CA 2044211 CA2044211 CA 2044211 CA 2044211 A CA2044211 A CA 2044211A CA 2044211 A1 CA2044211 A1 CA 2044211A1
- Authority
- CA
- Canada
- Prior art keywords
- beta
- demethyl
- och
- compound
- desoxypodophyllotoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical class COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 title abstract description 24
- 150000001875 compounds Chemical class 0.000 claims abstract description 68
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 42
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 20
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 6
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 claims abstract 6
- 125000003118 aryl group Chemical group 0.000 claims description 42
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 229910052760 oxygen Inorganic materials 0.000 claims description 13
- 229910052801 chlorine Inorganic materials 0.000 claims description 12
- 229910052794 bromium Inorganic materials 0.000 claims description 11
- 229910052731 fluorine Inorganic materials 0.000 claims description 11
- 206010028980 Neoplasm Diseases 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 6
- -1 COCH3 Chemical compound 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 5
- 150000004982 aromatic amines Chemical class 0.000 claims description 5
- 125000006237 oxymethylenoxy group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 5
- 125000001424 substituent group Chemical group 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- 230000001747 exhibiting effect Effects 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 125000005842 heteroatom Chemical group 0.000 claims description 2
- 229910052740 iodine Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- XXJGBENTLXFVFI-UHFFFAOYSA-N 1-amino-methylene Chemical compound N[CH2] XXJGBENTLXFVFI-UHFFFAOYSA-N 0.000 claims 3
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 claims 3
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 claims 3
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical compound [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 claims 3
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 claims 3
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims 3
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 101100054666 Streptomyces halstedii sch3 gene Proteins 0.000 claims 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims 1
- 125000005264 aryl amine group Chemical group 0.000 claims 1
- 125000004122 cyclic group Chemical group 0.000 claims 1
- 229960005420 etoposide Drugs 0.000 abstract description 22
- 150000002596 lactones Chemical class 0.000 description 39
- 238000005481 NMR spectroscopy Methods 0.000 description 37
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 36
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 32
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 239000013078 crystal Substances 0.000 description 25
- 108020004414 DNA Proteins 0.000 description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 20
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
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- 125000001931 aliphatic group Chemical group 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 0.000 description 11
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 11
- 229910000042 hydrogen bromide Inorganic materials 0.000 description 10
- 229910052757 nitrogen Inorganic materials 0.000 description 10
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- 239000003814 drug Substances 0.000 description 9
- 229940093499 ethyl acetate Drugs 0.000 description 9
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- 230000005764 inhibitory process Effects 0.000 description 9
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 description 9
- 239000011541 reaction mixture Substances 0.000 description 9
- AYJRCSIUFZENHW-UHFFFAOYSA-L barium carbonate Chemical compound [Ba+2].[O-]C([O-])=O AYJRCSIUFZENHW-UHFFFAOYSA-L 0.000 description 8
- 229960001237 podophyllotoxin Drugs 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 7
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 229940073584 methylene chloride Drugs 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- 101710183280 Topoisomerase Proteins 0.000 description 5
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 5
- 125000001246 bromo group Chemical group Br* 0.000 description 5
- 230000003013 cytotoxicity Effects 0.000 description 5
- 231100000135 cytotoxicity Toxicity 0.000 description 5
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
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- 102000004169 proteins and genes Human genes 0.000 description 4
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- 230000003595 spectral effect Effects 0.000 description 4
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- YJGVMLPVUAXIQN-LGWHJFRWSA-N (5s,5ar,8ar,9r)-5-hydroxy-9-(3,4,5-trimethoxyphenyl)-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[5,6-f][1,3]benzodioxol-8-one Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-LGWHJFRWSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 3
- YVCVYCSAAZQOJI-JHQYFNNDSA-N 4'-demethylepipodophyllotoxin Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YVCVYCSAAZQOJI-JHQYFNNDSA-N 0.000 description 3
- YVCVYCSAAZQOJI-BTINSWFASA-N 4'-demethylpodophyllotoxin Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YVCVYCSAAZQOJI-BTINSWFASA-N 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- 125000001309 chloro group Chemical group Cl* 0.000 description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 3
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- 238000000746 purification Methods 0.000 description 3
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 3
- FOVRGQUEGRCWPD-UHFFFAOYSA-N (5aR)-9t-beta-D-Glucopyranosyloxy-5t-(4-hydroxy-3,5-dimethoxy-phenyl)-(5ar,8at)-5,8,8a,9-tetrahydro-5aH-furo[3',4';6,7]naphtho[2,3-d][1,3]dioxol-6-on Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(OC3C(C(O)C(O)C(CO)O3)O)C3C2C(OC3)=O)=C1 FOVRGQUEGRCWPD-UHFFFAOYSA-N 0.000 description 2
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- QWPGGQLENJCZNL-UHFFFAOYSA-N dichloromethane;ethyl acetate;propan-2-one Chemical compound ClCCl.CC(C)=O.CCOC(C)=O QWPGGQLENJCZNL-UHFFFAOYSA-N 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- PSLIMVZEAPALCD-UHFFFAOYSA-N ethanol;ethoxyethane Chemical compound CCO.CCOCC PSLIMVZEAPALCD-UHFFFAOYSA-N 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- OAMZXMDZZWGPMH-UHFFFAOYSA-N ethyl acetate;toluene Chemical compound CCOC(C)=O.CC1=CC=CC=C1 OAMZXMDZZWGPMH-UHFFFAOYSA-N 0.000 description 1
- 229940093476 ethylene glycol Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 150000004820 halides Chemical group 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- CTRLABGOLIVAIY-UHFFFAOYSA-N oxcarbazepine Chemical compound C1C(=O)C2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 CTRLABGOLIVAIY-UHFFFAOYSA-N 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 208000000649 small cell carcinoma Diseases 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Compounds that are analogs of etoposide and exhibit antitumor activity are disclosed.
The compounds of the present invention have the following formula:
where:
R1 is .beta.-OCH2CH2NH2 .beta.-NHCH(CH3)CH2OH, .beta.-NHCH2CH(CH3)OH, .beta.-Cl, .beta.-Br, .beta.-OH, .alpha.-OH, .beta.-NH2, .alpha.-NH2, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH2OH, .beta.-OCH2CH2OH, , , , , , , , , , , , , , , , , , , , , , or .
R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
Compounds that are analogs of etoposide and exhibit antitumor activity are disclosed.
The compounds of the present invention have the following formula:
where:
R1 is .beta.-OCH2CH2NH2 .beta.-NHCH(CH3)CH2OH, .beta.-NHCH2CH(CH3)OH, .beta.-Cl, .beta.-Br, .beta.-OH, .alpha.-OH, .beta.-NH2, .alpha.-NH2, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH2OH, .beta.-OCH2CH2OH, , , , , , , , , , , , , , , , , , , , , , or .
R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
Description
- ETOPOSI DE ANALOGUES 2 0 4 ~ 2 1 1 This invention relates to compounds that are analogs of etoposide having antitumor activity. This invention also relates to a method for treatinq tumors by administering a S safe and effective amount of the etoposide analog compounds.
BACKGROUND OF THE INVENTION
Podophyllotoxin is a naturally occurring compound extracted from the mandrake plant. Recently two therapeutically useful semi-synthetic glycosides of podopyllotoxin, etoposide (also known as VP-16), shown below, and teniposide (also known as VM-26), have been developed.
R
~0 ~0~0 < ~0 R-CH, ~Etoposide) R- ~ ~Teniposide) ThesQ compounds exhibit therapeutic activity in several human neoplasms, includinq small cell carcinomas of the lung, testicular tumors, Hodgkin~s disease, Papillomavirus, and diffuse histiocytic lymphoma.
It is believed that these drugs block the catalytic activity of DNA topoisomerase II
by stabilizing an enzyme-DNA complex in which the DNA is cleaved and covalently linked to the enzyme. See Chen, G. L., Yang, L., Rowe T. C., Halligan, B.D., Tewey, K., and Liu, L., _. Biol.
20~211 Che~., 259, 13560 (1984); R~ss, W., Rowe, T., Glisson, B., Yalowich, J., and Liu, L., cancer Res., 44, 5857 (1984); Rowe, T., Kuppfer, G., and Ross, W., Biochem. Pharmacol., 34, 2483 (1985), which are all herein specifically incorporated by reference. By way of background, topoisomerases are enzymes which control the topological state of DNA. Type II
topoisomerases catalyze DNA strand passage through transient double strand breaks in the DNA. The resulting change in the linking number of DNA allows these enzymes to mediate DNA
interconversions, such as supercoiling and relaxation of supercoiling, catenation and decatenation, knotting, and unknotting. See Wang, J. C., Annu. Rev. Biochem., 54, 665 (1985) and Maxwell, A., and Gellert, M., Adv. Protein Chem., 38, 69 (1986), which are herein specifically incorporated by reference.
Type II DNA topoisomerase enzy~es have been shown to be involved in a number of vital cellular processes, including DNA replication and transcription, and chromosomal segregation.
These enzymes, therefore, are a critical target for the action of a wide variety of anticancer drugs, including etoposide and teniposid~. The key step leading to cell death may be the capability of these drugs to block the catalytic activity of DNA topoisomerase II, as noted above.
Structure-activity studies have demonstrated a direct correlation between cytotoxicity, DNA breakage, and murine-derived topoisomerase II inhibition activities among the podophyllotoxin analogues. See Minocha, A., and Long, B., Biochem Res. Comm., 122, 165 (1984), which is herein specifically incorporated by reference. The isolation and purification of human type II topoisomerase from lymphocytic leuk~mia cells has provided the means to use this enzyme as a target to investigate the structure-activity relationships among etoposide and related congeners.
It has been shown that the substitution of etoposide's glycosidic moiety by an 4-alkoxy group, as in 4'-demethyl-epipodophyllotoxin ethyl ether, preserves the inhibitory activity of DNA topoisomerase II intact at higher concentrations. See Thurston, L.S., Irie, H., Tani, S., Han, F. S., Liu, Z. C., Cheng, Y.C., and Lee, X. H., J. ~P~L_ÇbSm~, 29, 1547 (1986), which is herein specifically incorporated by reference. However, it has also been shown that a series of 4-acyl congeners are less active, even though some of them possessed potent cytotoxicity. See Thurston, L. S., Imakura, Y., Haruna, M., Li, D. H., Liu, Z. C., Liu , S . Y ., 2~ Cheng, Y. C., and Lee, K. H., J. Med~ Chem., 31, (1988), which is herein specifically incorporated by reference.
Summary of the Inyçntio~
~he present invention provides novel compounds which exhibit antitumor activity. The compounds are analogs of etoposide. More specifically, pre~erred compounds of the present invention are etoposide analogs wherein the glycosidic moiety is replaced by various substituents, such as a 2"-hydroxyethylamino chain, a 2n-methoxyethylamino chain, a 4 " -fluoroanilinyl chain, a chlorine atom, or a bromine atom.
2~4211 present invention. The compounds of the present invention have been shown to inhibit type II
human topoisomerase and also to cause cellular protein-linked DNA breakage and, therefore, may be useful in the treatment of tumors~ The compounds may also be useful in the treatment of papilloma virus.
In accordance with the present invention, there are provided compounds of the formula:
<~
H ~ ~ ~3 wherein R, is B-OCH,CH2NH, B-NHCH(CH,)CH,OH, B-NHCH,CH(CH,)OH, B-Cl, B-Br, B-OH, ~-OH, B-NH~, ~-NH" B-NHCH,CH~OH, ~-NHCH,CHIOH, B-NHCH2CHjCH"
B-NHCH,CH,OCH" B-NHCH2CH=CH" B-NHCH,CH(OH)CH" B-NHCH,CH,CH,OH, B-OCH,CH20H, ~o o~ r r ~0.114~ ~ IN~r ~~ ,B-IIII~ J~llll~ ) C 11 O~
' IIII~CO~C~ J ~ 1111~ ) 2 ~
e l~ ~
11 0 t ,l3~ ~oc~a Rl is H, or Br;
R, is H, or Br;
R, is H, or 8r;
~ is H, or Br; and ~ is H, or -CH,.
The present invention i~ also ~or a process for treatinq tumors in humans and lower animals by administering a safe and effective amount of a compound as described above.
A preferred group of compounds within the present invention exhibit inhibitory activity on human type II DNA topoisomerase to an equal or greater extent than etoposide and are of the formula:
~1 <0~'~
~0 CH~0 0C~
Olî
wherein R, is -NHCH,CH,OH, -NHCH,CH,OCH"
NHCH,CH(OH)CH" NH~H(CH,)CH,OH, Cl, OCH,CH,NH, Il o o 11 ~i~ ) ""~ ) Illl~o~ ~ J ~ ~el~
o~
~l~r ~ ~o ~ ~\o 2~42~1 Particularly preferred compounds include 4'-Demethyl-4B-amino-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin; 4'-Demethyl-4B-[2~-hydroxypropylamino]-4-desoxy-podophyllotoxin; 4'-Demethyl-4B-[l"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin; 4B-Chloro-4-desoxy-podophyllotoxin; 4'-demethyl-4B-Chloro-4-desoxypodophyllotoxin; 4'-Demethyl-4B-[3~'-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4B-[4"-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[2"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3",5"-difluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[4"-chloroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[4"-bromoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[4"-cyanoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~3"-cyanoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t4"-morpholinoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3",4"-methylenedioxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~3",4"-dimethoxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t3"-pyridylamino]-4-20~4211 desoxypodophyllotoxin; and 4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned from the practice of the invention.
Description of The Prefç~ed Embodiments Reference will now be made in detail to the presently preferred embodiments of the invention, which tog~ther with the following examples, serve to explain the principles of the invention.
As noted above, the present invention relates to compounds of the formula:
<~
N,~
wherein R, is B-OCH,CH~H, B-NHCH(CH,)CH,OH, B-NHCH,CH(CH,)OH, B-Cl, B-Br~ B-OH, ~-OH, B-NH"
~-NH" ~-NHCH,CH,OH, B-NHCH,CH,CH3, B-NHCH,CH,OCH"
B-NHCH,CH-CH" B-NHCH,CH(OH)CH" B-NHCH,CH,CH,OH, or -OCH,CH,OH, 2 ~ 2 ~ 1 p ~
R, is H, or Br;
R, is H, or Br;
R, is H, or Br;
Rs is H, or Br; and R~ is H, or -CH,.
As noted above, a preferred group of compounds within the present invention exhibit inhibitory activity on human type II DNA
topoisomerase to an equal or greater extent as etoposide and are of the formula:
<0~
C H ~ 0~0 C H
whereln R, is NHCH,CH,OH, NHCH,CH,OCH"
NHCH~CH(OH)CH" NHCH(CH,)CH,OH, Cl, OCH,CH,NH"
1~0 011 f IIN~3 j #N~ ~~011 ~ 1111~ ) #II~C 1~
`l ~"~r ~ #1~3t~ O or 2~4~
Another preferred group of compounds under the present invention will possess a 4'-demethyl-4~-substituted anilinyl 4-desoxypodophyllotoxin wherein the compound possesses the following formula.
'~i ~
~O~
c~,o 1, oc~
wherein Ar is an aryla~ine as in the formula below, - N~
wherein R, i8 H, OH, F, Cl, Br, CO,CH" CO,C2H"
CN, NO" NH" N(CH,)" OCH" CH,OH, CH" CF"
CH,CH,OH, COCH" CH2NH,;
R, is H, OH, F, Cl, Br, CO,CH" CO,C,H" CN, NO"
NH" N~CH,)" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" CHOHCH" SCH" CH" CO,CH,;
R, is H, ON, F, Cl, Br, I, CO,CH" CO,C,H" CN, NO"
NH2, N(CH,~" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" ~ . , ~ , N~CH,CH,OH)" CH,;
20~211 R, is H, F, Cl, OH, OCH" CO,CH" CO2C2H" CH3, CF" NO2, NH" C l;
R, is H, F, Cl, CH" CF" OH, OCH" NO2;
R, and R, are OCH,O or OCH,CH,O.
The preferred compou~ds of the present invention can also be more generally described as a compound of the formula:
<0~
CH~OJ~OCH~
0~
wherein R is a flat aromatic group which may contain a heteroatom or alternatively may contain electron donating substituents at the 3"
or 4" position of the aromatic ring.
Specifically the substituents may be oxygen containing groups, or the aromatic group may be pyridine.
The compounds of the present invention are analogs of etoposide wherein the glycosidic moiety has been replaced. Compounds exhibiting potent inhibition of human DNA topoisomerase II
result from replacing the glycosidic moiety with a 2~-hydroxyethylamino chain, a 2"-methoxyethylamino chain, or a substituted arylamine at the C-4B position. Inhibitory activity can also be increased by substituting the glycosidic moiety by chlorine, bromine, or an amino group at the C-4B position. It is believed that the stereochemistry of the 4B-substituents plays an important role in 204~211 determininq inhibitory potency. In general, B-isomers exhibit greater activity than the corresponding ~-isomers. Another factor affecting the potency is the length of the S substituent group at the C-4 position and the substitution on that group. This distance factor is different for halide and hydroxy substituted arylamines. For the hydroxy substituted arylamine, the meta position showed the most potency, while in the halogens, para substitution showed the most potency. A second potency factor regarding the halogen substituted aryl amines is the size of the halogen.
Fluorine, the smallest has the most potency, lS while Iodine, the largest has the least. In addition, substitution of bromine at either one or more of the R" R" R" and ~ positions will result in compounds having significant inhibitory activity. ~ can be varied by substituting hydrogen with a methyl group.
These modifications will produce changes in inhibitory activity which can be readily determined by assays known in the prior art through the exercise of routine skill in light of the teachings contained herein.
The compounds of the present invention were tested for their degree of inhibitory activity on human type II DNA topoisomerase, their effect on the formation of protein-linked DNA breakage, and their cytotoxicity. The inhibitory activity for compounds of the present invention correlated with the ability of the compounds to cause DNA strand breakage.
However, the ~n vitro cytotoxicity of the compounds tested did not appear to correlate with the enzyme inhibitory activity and DNA
strand break activity. The results of the tests 2~211 on some of the compounds of the present invention are shown in Tables I and III. For a description of the assays used with respect to the compounds listed in Tables I and III see Thurston, L.S., Irie, H., Tani, S., Han, F. s., Liu , Z . C ., Cheng, Y.c., and Lee, K. H., Antitumor Agents 78. Inhibition of Human DNA
Topoisomerase II by Podophyllotoxin and ~-Peltatin Analogues, 3. Med. Chem. 29, 1547 (1986), and the references cited therein.
Tab}es I, and III illustrate the inhibitory activity, DNA strand breakage ability, as well as the cytotoxicity of etoposide and some of the compounds of the present invention. As shown in Tables I and III, the inhibitory activity of 4'-Demethyl-4B-amino-4-desoxypodophyllotoxin (Example 3), 4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotixin (Example 5), 4'-Demethyl-4B-t2"-hydroxypropylamino]-4-desoxypodophyllotoxin (Example 10), and 4'-Demethyl-4B-tln-methyl-2~-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 11), 4'-Demethyl-4B-t2n-aminoethoxy]-4-desoxypodophyllotoxin (Example 13); 4'-Demethyl-4B-t3n-hydroXyanilinyl~-4-desoxypodophyllotoxin ~Example 33), 4'-Demethyl-4B-t2~-hydroxyanilinyl~-4-desoxypodophyllotoxin (Example 34), 4'-Demethyl-4~-[4"-hydroxyanilinyl~-4-desoxypodophyllotoxin (Example 35), 4'-Demethyl-4~-t3"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 26); 4'-Demethyl-4~-t4"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 28); 4'-Demethyl-4~-t4"-chloroanilinyl]-4-desoxypodophyllotoxin (Example 38); 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin (Example 19); 4'-Demethyl-4~-t4 N -cyanoanilinyl]-4-desoxypodophyllotoxin 2~21 ~
(Example 20~; 4'-Demethyl-4~-~3~-cyanoanilinyl]-4-desoxypodophyllotoxin (Example 21); 4~-Deme.hyl-4~-[4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin (Example 22); 4'-Demethyl-4~-[4~'-morpholinoanilinyl]-4-desoxypodophyllotoxin (Example 23); 4~-Demethyl-4B-~3~4~-methylenedioxyanilinyl]-4-desoxypodophyllotoxin (Example 24); 4'-Demethyl-4~-t3 n, 4 " -dimethoxyanilinyl~-4-desoxypodophyllotoxin (Example 25); 4~-Demethyl-4,~-t3"-pyridylamino]-4-desoxypodophyllotoxin (Example 30); 4'-Demethyl-4~-t2"-pyridylamino]-4-desoxypodophyllotoxin (Example 31); and 4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin (Example 32) equals or exceeds that of etoposide. In addition, as shown in Tables I and III, the DNA strand breakage abilities of 4'-Demethyl-4B-t2"-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 5), 4'-Demethyl-4B-t2"-methoxyethylamino]-4-desoxypodophyllotoxin (Example 8), 4'-Demethyl-4B-t2"-hydroxypropylamino]-4-desoxy-podophyllotoxin (Example 10), and 4'-Demethyl-4B-t1"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 11), 4'-Demethyl-4B-~2"-aminoethoxy]-4-desoxypodophyllotoxin (Example 13);4~-Demethyl-4B-~3-hydroxyanilinyl]-4-dexoypodophyllotoxin (Exa~ple 33), 4'-Demethyl-4B-t2-hydroxyanilinyl]-4-desoxypodophyllotoXin (Example 34), 4'-Demethyl-4B-t4-hydroxyanilinyl]-4-desoxypodophyllotoxin (Example 35), 4'-Demethyl-4~-t3"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 26); 4'-Demethyl-4~-[2"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 27); 4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin 20~42t~
S (Example 28j; 4'-Demethyl-4~-[3",5"-difluoroanilinyl]-4-desoxypodophyllotoxin (Example 29); 4'-Demethyl-4~-anilinyl-4-desoxypod~phyllotoxin (Example 19); 4'-Demethyl-4B-[4"-cyanoanilinyl~-4-desoxypodophyllotoxin (Example 20); 4'-De~ethyl-4~-t3"-cyanoanilinyl~-4-desoxypodophyllotoxin (Example 21); 4'-Dem~thyl-4~-~4"-ethoxycarbonylanilinyl~-4-desoxypodophyllotoxin ~Example 22); 4~-Demethyl-4~-~4"-morpholinoanilinyl]-4-desoxypodophyllOtOXin (Example 23); 4'-Demethyl-4~-~3",4~-methylenedioxyanilinyl]-4-desoxypodophyllotoxin (Example 24); 4'-Demethyl-4~-~3",4"-dimethoxyanilinyl]-4-desoxypodophyllotoxin (Example 25); 4'-Demethyl-20 4~ _ [ 3 n -pyridylamino]-4-desoxypodophyllotoxin ~Example 30); 4'-Demethyl-4~-t3"-quinolinylamino]-4-desoxypodophyllotoxin (Example 32); and 4'-Demethyl-4~-[4~-bromoanilinyl]-4-desoxypodophyllotoxin (Example - 25 40) greatly exceeds that of etoposide.
Table II compares the relative DNA
topoisomerase II inhibitory activity of several compounds of the present invention with etoposide. As shown in Table II, the compounds test-d exhibited inhibitory activity exceoding that of etopo-ide by two to eight times.
Preparation of compounds within the 5COp- of the present invention appear in the following examples.
~A~ilE_L
Preparation of 4'-Demethylepipodophyllotoxin.
5 g. (12.1 mmol) of podophyllotoxin were dissolved in 75 ml of anhydrous CH,Cl,. Dry hydrogen bromide gas was then bubbled through the solution to saturation. The reaction 2 ~ 2 1 1 mixture was then capped and allowed to stand at room temperature for 48 hours. Removal of the solvent yielded a residue which was then treated with 25 ml of water, 50 ml of acetone and 5 g.
of BaC0" and refluxed for one hour. The reaction ~ixture was extracted with chloroform and chromatographed on a silica gel column. The product was obtained by elution with chloroform-methanol (30:1) and recrystallized from CH,Cl,/ethylether to give 2.5 g. (52t) of 4'-Demethylepipodophyllotoxin. Spectral data agreed with that described by Kuhn, M., Keller-Julsen, C., and von Wartburg, A., ~çlv. Chi~-aç~, 52, 944 (1969), which is herein specifically incorporated by reference.
(See Scheme 1) - EX~PL~ 2 Preparation of 4'-Demethylpodophyllotoxin.
4'-Demethylpodophyllotoxin was obtained using the silica gel column of Example 1 by further elution with chloroform-methanol (30:1).
The product obtained by elution was crystallized from acetone in 5% (0.5 g) yield. Spectral data agreed with that dQscribod by Kuhn, M., and von 25 Wartburg, A., H~ly~_Ch1m~ Acta, 52, 948 (1969) (hereinafter Kuhn and von Wartburg), which is herein specifically incorporated by reference.
~AMpL~ 3 Preparation of 4'-DQmothyl-4B-amino-4-desoxy-podophyllotoxin. (Scheme II) A. Preparation of 4'-0-Carbobenzoxyepipodo-phyllotoxin.
A solution of 2 g. of 4'-Demethy-lepipodophyllotoxin (5 mmol) in 200 ml of anhydrous dichloromethane was cooled in an ice 2 ~ 2 ~ 1 bath, and treated with 2.5 ml of triethylamine ~18 mmol), and 2.5 ml of carbobenzoxychloride (17.5 mmol). The reaction mixture was stirred at room temperature for 2 hours after which time 100 ml of water was added. The organic layer was dried using MgS0" concentrated, and purified using silica gel column chromatography. The product was obtained upon elution with chloroform and recrystallized from chloroformtethanol to give 2.4 g. (89%).
Spectral data agreed with that described by Kuhn and von Wartburg.
B. Preparation of 4'-0-Carbobenzoxy-4-epia~idopodophyllotoxin.
A solution of 3 g. (5.6 mmol) of 4'-0-Carbobenzoxy-4-epipodophyllotoxin (the product of Example 3) in 100 ml of anhydrous methylene chloride was cooled in an ice bath and treated successively with 1.5 ml (10.8 mmol) of triethylamine, and 1.2 ml (15.5 mmol) of methanesulfonylchloride. The ice bath was then removed and the reaction mixture was stirred at room temperature for one hour. This mixture was then evaporated in vacuo to dryness, and 40 ml of anhydrous DMF was added along with 3 g~ ~46 mmol) of sodium azide. The reaction mixture was stirred overnight at room temperature and then partitioned between water (100 ml) and ethylacetate. The organic layer was washed with water, dried using MgS0" and concentrated to yield a crude residue, which was checked by TLC
and NMR analyses to be a mixture of 4~- and 4B-azido isomers (ca.1:3). Crystallization from chloroform/ethanol provided the pure B-isomer 4'-0-Carbobenzoxy-4-epiazidopodophyllotoxin (2.3 g, 73%) having the following properties:
17 2~
mp.202-204 C; MS, m/z 559 (~), 424, and 382; IR
(KBr) 29SO, 2900, 2100 (azide), 1770 (carbonate C=O), 1745 (lactone C=O), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~ 7.40 (m, 5H, cbz aromatic), 6.82 (s, lH, 5-H), 6.58 (s, lH, 8-H), 6.27 (s, 2H, 2'.6'-H), 6.03 (ABq, J=2.4 Hz, 2H, O-CH,-O), 5.25 (s, 2H, OCH2Ph), 4.77 (d, J=4 Hz, lH, 4-H), 4.65 (d, J-5 Hz, lH, l-H), 4.31 (d, J-9 Hz, 2H, 11, ll'-H), 3.66 (8, 6H, 3',5'-OCH,), 3.2(dd, J=5,14 Hz, lH, 2-H), and 2.90 (m,lH, 3-H); Anal. (C"H"O,N, 1/2 H,O), C.H.
C. Preparation of 4'-Demethyl-4B-amino-4-desoxy-podophyllotoxin.
500 mg of 10% palladium on carbon was added to a solution of the crude 4'-demethyl-4-azidopodophyllotoxin (2.3g, 4.1 mmol), obtained according to steps A and B, and 200 ml of ethylacetate. This mixture was shaken under 40 psi of hydrogen for four hours. The reaction mixture was then filtered over celite and the filtrate evaporated in vacuo. The residue was chromatographed on a silica gel column and eluted first with a chloroform/ethylacetate (2:1) solvent system to remove the non-polar products. Further elution wlth a chloroform/methanol (19:1) mobile phase ylelded 0.85g, (52%) of the desired product. The product wa~ then crystallized from methylene chloride/ethylether and had the following properties: mp 132-135 C; MS m/z 399 (~); IR
(KBr) 3360 (OH), 3290 (primary amine), 2900 (aliphatic C-H), 1745 (lactone), 1590 (aromatic C-H) cm l;'H NMR (CDCl,) ~ 6.81 (s, lH,5-H), 6.49 (s, lH, 8-H~, 6.30 (s, 2H, 2~,6~-H), 5.96 (ABq, 3s J-l Hz, 2H, OCH,O), 5.3 (s, lH, OH, D,O
exchangeable), 4.55 (d, J=5.2 Hz, lH, l-H), 4.28 2~21~
(d, J--9.5 Hz, 2H, 11,11'-H), 4.17 (d, J=4.1 Hz, lH, 4-H) 3.77 (s, 6H, 3',5'-OCH,), 3.28 (dd, J=5.2,14 Hz, lH, 2-H), and 2.85 (m, lH, 3-H), Anal. (Cl,HI,O~NH2Q), C.H.
ExA~LE-~
Preparation of 4'-Demethyl-4~-amino-4-desoxy-podophyl lotoxin .
BACKGROUND OF THE INVENTION
Podophyllotoxin is a naturally occurring compound extracted from the mandrake plant. Recently two therapeutically useful semi-synthetic glycosides of podopyllotoxin, etoposide (also known as VP-16), shown below, and teniposide (also known as VM-26), have been developed.
R
~0 ~0~0 < ~0 R-CH, ~Etoposide) R- ~ ~Teniposide) ThesQ compounds exhibit therapeutic activity in several human neoplasms, includinq small cell carcinomas of the lung, testicular tumors, Hodgkin~s disease, Papillomavirus, and diffuse histiocytic lymphoma.
It is believed that these drugs block the catalytic activity of DNA topoisomerase II
by stabilizing an enzyme-DNA complex in which the DNA is cleaved and covalently linked to the enzyme. See Chen, G. L., Yang, L., Rowe T. C., Halligan, B.D., Tewey, K., and Liu, L., _. Biol.
20~211 Che~., 259, 13560 (1984); R~ss, W., Rowe, T., Glisson, B., Yalowich, J., and Liu, L., cancer Res., 44, 5857 (1984); Rowe, T., Kuppfer, G., and Ross, W., Biochem. Pharmacol., 34, 2483 (1985), which are all herein specifically incorporated by reference. By way of background, topoisomerases are enzymes which control the topological state of DNA. Type II
topoisomerases catalyze DNA strand passage through transient double strand breaks in the DNA. The resulting change in the linking number of DNA allows these enzymes to mediate DNA
interconversions, such as supercoiling and relaxation of supercoiling, catenation and decatenation, knotting, and unknotting. See Wang, J. C., Annu. Rev. Biochem., 54, 665 (1985) and Maxwell, A., and Gellert, M., Adv. Protein Chem., 38, 69 (1986), which are herein specifically incorporated by reference.
Type II DNA topoisomerase enzy~es have been shown to be involved in a number of vital cellular processes, including DNA replication and transcription, and chromosomal segregation.
These enzymes, therefore, are a critical target for the action of a wide variety of anticancer drugs, including etoposide and teniposid~. The key step leading to cell death may be the capability of these drugs to block the catalytic activity of DNA topoisomerase II, as noted above.
Structure-activity studies have demonstrated a direct correlation between cytotoxicity, DNA breakage, and murine-derived topoisomerase II inhibition activities among the podophyllotoxin analogues. See Minocha, A., and Long, B., Biochem Res. Comm., 122, 165 (1984), which is herein specifically incorporated by reference. The isolation and purification of human type II topoisomerase from lymphocytic leuk~mia cells has provided the means to use this enzyme as a target to investigate the structure-activity relationships among etoposide and related congeners.
It has been shown that the substitution of etoposide's glycosidic moiety by an 4-alkoxy group, as in 4'-demethyl-epipodophyllotoxin ethyl ether, preserves the inhibitory activity of DNA topoisomerase II intact at higher concentrations. See Thurston, L.S., Irie, H., Tani, S., Han, F. S., Liu, Z. C., Cheng, Y.C., and Lee, X. H., J. ~P~L_ÇbSm~, 29, 1547 (1986), which is herein specifically incorporated by reference. However, it has also been shown that a series of 4-acyl congeners are less active, even though some of them possessed potent cytotoxicity. See Thurston, L. S., Imakura, Y., Haruna, M., Li, D. H., Liu, Z. C., Liu , S . Y ., 2~ Cheng, Y. C., and Lee, K. H., J. Med~ Chem., 31, (1988), which is herein specifically incorporated by reference.
Summary of the Inyçntio~
~he present invention provides novel compounds which exhibit antitumor activity. The compounds are analogs of etoposide. More specifically, pre~erred compounds of the present invention are etoposide analogs wherein the glycosidic moiety is replaced by various substituents, such as a 2"-hydroxyethylamino chain, a 2n-methoxyethylamino chain, a 4 " -fluoroanilinyl chain, a chlorine atom, or a bromine atom.
2~4211 present invention. The compounds of the present invention have been shown to inhibit type II
human topoisomerase and also to cause cellular protein-linked DNA breakage and, therefore, may be useful in the treatment of tumors~ The compounds may also be useful in the treatment of papilloma virus.
In accordance with the present invention, there are provided compounds of the formula:
<~
H ~ ~ ~3 wherein R, is B-OCH,CH2NH, B-NHCH(CH,)CH,OH, B-NHCH,CH(CH,)OH, B-Cl, B-Br, B-OH, ~-OH, B-NH~, ~-NH" B-NHCH,CH~OH, ~-NHCH,CHIOH, B-NHCH2CHjCH"
B-NHCH,CH,OCH" B-NHCH2CH=CH" B-NHCH,CH(OH)CH" B-NHCH,CH,CH,OH, B-OCH,CH20H, ~o o~ r r ~0.114~ ~ IN~r ~~ ,B-IIII~ J~llll~ ) C 11 O~
' IIII~CO~C~ J ~ 1111~ ) 2 ~
e l~ ~
11 0 t ,l3~ ~oc~a Rl is H, or Br;
R, is H, or Br;
R, is H, or 8r;
~ is H, or Br; and ~ is H, or -CH,.
The present invention i~ also ~or a process for treatinq tumors in humans and lower animals by administering a safe and effective amount of a compound as described above.
A preferred group of compounds within the present invention exhibit inhibitory activity on human type II DNA topoisomerase to an equal or greater extent than etoposide and are of the formula:
~1 <0~'~
~0 CH~0 0C~
Olî
wherein R, is -NHCH,CH,OH, -NHCH,CH,OCH"
NHCH,CH(OH)CH" NH~H(CH,)CH,OH, Cl, OCH,CH,NH, Il o o 11 ~i~ ) ""~ ) Illl~o~ ~ J ~ ~el~
o~
~l~r ~ ~o ~ ~\o 2~42~1 Particularly preferred compounds include 4'-Demethyl-4B-amino-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin; 4'-Demethyl-4B-[2~-hydroxypropylamino]-4-desoxy-podophyllotoxin; 4'-Demethyl-4B-[l"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin; 4B-Chloro-4-desoxy-podophyllotoxin; 4'-demethyl-4B-Chloro-4-desoxypodophyllotoxin; 4'-Demethyl-4B-[3~'-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4B-[4"-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[2"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3",5"-difluoroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[4"-chloroanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[4"-bromoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[4"-cyanoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~3"-cyanoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t4"-morpholinoanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-[3",4"-methylenedioxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-~3",4"-dimethoxyanilinyl]-4-desoxypodophyllotoxin; 4'-Demethyl-4~-t3"-pyridylamino]-4-20~4211 desoxypodophyllotoxin; and 4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned from the practice of the invention.
Description of The Prefç~ed Embodiments Reference will now be made in detail to the presently preferred embodiments of the invention, which tog~ther with the following examples, serve to explain the principles of the invention.
As noted above, the present invention relates to compounds of the formula:
<~
N,~
wherein R, is B-OCH,CH~H, B-NHCH(CH,)CH,OH, B-NHCH,CH(CH,)OH, B-Cl, B-Br~ B-OH, ~-OH, B-NH"
~-NH" ~-NHCH,CH,OH, B-NHCH,CH,CH3, B-NHCH,CH,OCH"
B-NHCH,CH-CH" B-NHCH,CH(OH)CH" B-NHCH,CH,CH,OH, or -OCH,CH,OH, 2 ~ 2 ~ 1 p ~
R, is H, or Br;
R, is H, or Br;
R, is H, or Br;
Rs is H, or Br; and R~ is H, or -CH,.
As noted above, a preferred group of compounds within the present invention exhibit inhibitory activity on human type II DNA
topoisomerase to an equal or greater extent as etoposide and are of the formula:
<0~
C H ~ 0~0 C H
whereln R, is NHCH,CH,OH, NHCH,CH,OCH"
NHCH~CH(OH)CH" NHCH(CH,)CH,OH, Cl, OCH,CH,NH"
1~0 011 f IIN~3 j #N~ ~~011 ~ 1111~ ) #II~C 1~
`l ~"~r ~ #1~3t~ O or 2~4~
Another preferred group of compounds under the present invention will possess a 4'-demethyl-4~-substituted anilinyl 4-desoxypodophyllotoxin wherein the compound possesses the following formula.
'~i ~
~O~
c~,o 1, oc~
wherein Ar is an aryla~ine as in the formula below, - N~
wherein R, i8 H, OH, F, Cl, Br, CO,CH" CO,C2H"
CN, NO" NH" N(CH,)" OCH" CH,OH, CH" CF"
CH,CH,OH, COCH" CH2NH,;
R, is H, OH, F, Cl, Br, CO,CH" CO,C,H" CN, NO"
NH" N~CH,)" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" CHOHCH" SCH" CH" CO,CH,;
R, is H, ON, F, Cl, Br, I, CO,CH" CO,C,H" CN, NO"
NH2, N(CH,~" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" ~ . , ~ , N~CH,CH,OH)" CH,;
20~211 R, is H, F, Cl, OH, OCH" CO,CH" CO2C2H" CH3, CF" NO2, NH" C l;
R, is H, F, Cl, CH" CF" OH, OCH" NO2;
R, and R, are OCH,O or OCH,CH,O.
The preferred compou~ds of the present invention can also be more generally described as a compound of the formula:
<0~
CH~OJ~OCH~
0~
wherein R is a flat aromatic group which may contain a heteroatom or alternatively may contain electron donating substituents at the 3"
or 4" position of the aromatic ring.
Specifically the substituents may be oxygen containing groups, or the aromatic group may be pyridine.
The compounds of the present invention are analogs of etoposide wherein the glycosidic moiety has been replaced. Compounds exhibiting potent inhibition of human DNA topoisomerase II
result from replacing the glycosidic moiety with a 2~-hydroxyethylamino chain, a 2"-methoxyethylamino chain, or a substituted arylamine at the C-4B position. Inhibitory activity can also be increased by substituting the glycosidic moiety by chlorine, bromine, or an amino group at the C-4B position. It is believed that the stereochemistry of the 4B-substituents plays an important role in 204~211 determininq inhibitory potency. In general, B-isomers exhibit greater activity than the corresponding ~-isomers. Another factor affecting the potency is the length of the S substituent group at the C-4 position and the substitution on that group. This distance factor is different for halide and hydroxy substituted arylamines. For the hydroxy substituted arylamine, the meta position showed the most potency, while in the halogens, para substitution showed the most potency. A second potency factor regarding the halogen substituted aryl amines is the size of the halogen.
Fluorine, the smallest has the most potency, lS while Iodine, the largest has the least. In addition, substitution of bromine at either one or more of the R" R" R" and ~ positions will result in compounds having significant inhibitory activity. ~ can be varied by substituting hydrogen with a methyl group.
These modifications will produce changes in inhibitory activity which can be readily determined by assays known in the prior art through the exercise of routine skill in light of the teachings contained herein.
The compounds of the present invention were tested for their degree of inhibitory activity on human type II DNA topoisomerase, their effect on the formation of protein-linked DNA breakage, and their cytotoxicity. The inhibitory activity for compounds of the present invention correlated with the ability of the compounds to cause DNA strand breakage.
However, the ~n vitro cytotoxicity of the compounds tested did not appear to correlate with the enzyme inhibitory activity and DNA
strand break activity. The results of the tests 2~211 on some of the compounds of the present invention are shown in Tables I and III. For a description of the assays used with respect to the compounds listed in Tables I and III see Thurston, L.S., Irie, H., Tani, S., Han, F. s., Liu , Z . C ., Cheng, Y.c., and Lee, K. H., Antitumor Agents 78. Inhibition of Human DNA
Topoisomerase II by Podophyllotoxin and ~-Peltatin Analogues, 3. Med. Chem. 29, 1547 (1986), and the references cited therein.
Tab}es I, and III illustrate the inhibitory activity, DNA strand breakage ability, as well as the cytotoxicity of etoposide and some of the compounds of the present invention. As shown in Tables I and III, the inhibitory activity of 4'-Demethyl-4B-amino-4-desoxypodophyllotoxin (Example 3), 4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotixin (Example 5), 4'-Demethyl-4B-t2"-hydroxypropylamino]-4-desoxypodophyllotoxin (Example 10), and 4'-Demethyl-4B-tln-methyl-2~-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 11), 4'-Demethyl-4B-t2n-aminoethoxy]-4-desoxypodophyllotoxin (Example 13); 4'-Demethyl-4B-t3n-hydroXyanilinyl~-4-desoxypodophyllotoxin ~Example 33), 4'-Demethyl-4B-t2~-hydroxyanilinyl~-4-desoxypodophyllotoxin (Example 34), 4'-Demethyl-4~-[4"-hydroxyanilinyl~-4-desoxypodophyllotoxin (Example 35), 4'-Demethyl-4~-t3"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 26); 4'-Demethyl-4~-t4"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 28); 4'-Demethyl-4~-t4"-chloroanilinyl]-4-desoxypodophyllotoxin (Example 38); 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin (Example 19); 4'-Demethyl-4~-t4 N -cyanoanilinyl]-4-desoxypodophyllotoxin 2~21 ~
(Example 20~; 4'-Demethyl-4~-~3~-cyanoanilinyl]-4-desoxypodophyllotoxin (Example 21); 4~-Deme.hyl-4~-[4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin (Example 22); 4'-Demethyl-4~-[4~'-morpholinoanilinyl]-4-desoxypodophyllotoxin (Example 23); 4~-Demethyl-4B-~3~4~-methylenedioxyanilinyl]-4-desoxypodophyllotoxin (Example 24); 4'-Demethyl-4~-t3 n, 4 " -dimethoxyanilinyl~-4-desoxypodophyllotoxin (Example 25); 4~-Demethyl-4,~-t3"-pyridylamino]-4-desoxypodophyllotoxin (Example 30); 4'-Demethyl-4~-t2"-pyridylamino]-4-desoxypodophyllotoxin (Example 31); and 4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin (Example 32) equals or exceeds that of etoposide. In addition, as shown in Tables I and III, the DNA strand breakage abilities of 4'-Demethyl-4B-t2"-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 5), 4'-Demethyl-4B-t2"-methoxyethylamino]-4-desoxypodophyllotoxin (Example 8), 4'-Demethyl-4B-t2"-hydroxypropylamino]-4-desoxy-podophyllotoxin (Example 10), and 4'-Demethyl-4B-t1"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin (Example 11), 4'-Demethyl-4B-~2"-aminoethoxy]-4-desoxypodophyllotoxin (Example 13);4~-Demethyl-4B-~3-hydroxyanilinyl]-4-dexoypodophyllotoxin (Exa~ple 33), 4'-Demethyl-4B-t2-hydroxyanilinyl]-4-desoxypodophyllotoXin (Example 34), 4'-Demethyl-4B-t4-hydroxyanilinyl]-4-desoxypodophyllotoxin (Example 35), 4'-Demethyl-4~-t3"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 26); 4'-Demethyl-4~-[2"-fluoroanilinyl]-4-desoxypodophyllotoxin (Example 27); 4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin 20~42t~
S (Example 28j; 4'-Demethyl-4~-[3",5"-difluoroanilinyl]-4-desoxypodophyllotoxin (Example 29); 4'-Demethyl-4~-anilinyl-4-desoxypod~phyllotoxin (Example 19); 4'-Demethyl-4B-[4"-cyanoanilinyl~-4-desoxypodophyllotoxin (Example 20); 4'-De~ethyl-4~-t3"-cyanoanilinyl~-4-desoxypodophyllotoxin (Example 21); 4'-Dem~thyl-4~-~4"-ethoxycarbonylanilinyl~-4-desoxypodophyllotoxin ~Example 22); 4~-Demethyl-4~-~4"-morpholinoanilinyl]-4-desoxypodophyllOtOXin (Example 23); 4'-Demethyl-4~-~3",4~-methylenedioxyanilinyl]-4-desoxypodophyllotoxin (Example 24); 4'-Demethyl-4~-~3",4"-dimethoxyanilinyl]-4-desoxypodophyllotoxin (Example 25); 4'-Demethyl-20 4~ _ [ 3 n -pyridylamino]-4-desoxypodophyllotoxin ~Example 30); 4'-Demethyl-4~-t3"-quinolinylamino]-4-desoxypodophyllotoxin (Example 32); and 4'-Demethyl-4~-[4~-bromoanilinyl]-4-desoxypodophyllotoxin (Example - 25 40) greatly exceeds that of etoposide.
Table II compares the relative DNA
topoisomerase II inhibitory activity of several compounds of the present invention with etoposide. As shown in Table II, the compounds test-d exhibited inhibitory activity exceoding that of etopo-ide by two to eight times.
Preparation of compounds within the 5COp- of the present invention appear in the following examples.
~A~ilE_L
Preparation of 4'-Demethylepipodophyllotoxin.
5 g. (12.1 mmol) of podophyllotoxin were dissolved in 75 ml of anhydrous CH,Cl,. Dry hydrogen bromide gas was then bubbled through the solution to saturation. The reaction 2 ~ 2 1 1 mixture was then capped and allowed to stand at room temperature for 48 hours. Removal of the solvent yielded a residue which was then treated with 25 ml of water, 50 ml of acetone and 5 g.
of BaC0" and refluxed for one hour. The reaction ~ixture was extracted with chloroform and chromatographed on a silica gel column. The product was obtained by elution with chloroform-methanol (30:1) and recrystallized from CH,Cl,/ethylether to give 2.5 g. (52t) of 4'-Demethylepipodophyllotoxin. Spectral data agreed with that described by Kuhn, M., Keller-Julsen, C., and von Wartburg, A., ~çlv. Chi~-aç~, 52, 944 (1969), which is herein specifically incorporated by reference.
(See Scheme 1) - EX~PL~ 2 Preparation of 4'-Demethylpodophyllotoxin.
4'-Demethylpodophyllotoxin was obtained using the silica gel column of Example 1 by further elution with chloroform-methanol (30:1).
The product obtained by elution was crystallized from acetone in 5% (0.5 g) yield. Spectral data agreed with that dQscribod by Kuhn, M., and von 25 Wartburg, A., H~ly~_Ch1m~ Acta, 52, 948 (1969) (hereinafter Kuhn and von Wartburg), which is herein specifically incorporated by reference.
~AMpL~ 3 Preparation of 4'-DQmothyl-4B-amino-4-desoxy-podophyllotoxin. (Scheme II) A. Preparation of 4'-0-Carbobenzoxyepipodo-phyllotoxin.
A solution of 2 g. of 4'-Demethy-lepipodophyllotoxin (5 mmol) in 200 ml of anhydrous dichloromethane was cooled in an ice 2 ~ 2 ~ 1 bath, and treated with 2.5 ml of triethylamine ~18 mmol), and 2.5 ml of carbobenzoxychloride (17.5 mmol). The reaction mixture was stirred at room temperature for 2 hours after which time 100 ml of water was added. The organic layer was dried using MgS0" concentrated, and purified using silica gel column chromatography. The product was obtained upon elution with chloroform and recrystallized from chloroformtethanol to give 2.4 g. (89%).
Spectral data agreed with that described by Kuhn and von Wartburg.
B. Preparation of 4'-0-Carbobenzoxy-4-epia~idopodophyllotoxin.
A solution of 3 g. (5.6 mmol) of 4'-0-Carbobenzoxy-4-epipodophyllotoxin (the product of Example 3) in 100 ml of anhydrous methylene chloride was cooled in an ice bath and treated successively with 1.5 ml (10.8 mmol) of triethylamine, and 1.2 ml (15.5 mmol) of methanesulfonylchloride. The ice bath was then removed and the reaction mixture was stirred at room temperature for one hour. This mixture was then evaporated in vacuo to dryness, and 40 ml of anhydrous DMF was added along with 3 g~ ~46 mmol) of sodium azide. The reaction mixture was stirred overnight at room temperature and then partitioned between water (100 ml) and ethylacetate. The organic layer was washed with water, dried using MgS0" and concentrated to yield a crude residue, which was checked by TLC
and NMR analyses to be a mixture of 4~- and 4B-azido isomers (ca.1:3). Crystallization from chloroform/ethanol provided the pure B-isomer 4'-0-Carbobenzoxy-4-epiazidopodophyllotoxin (2.3 g, 73%) having the following properties:
17 2~
mp.202-204 C; MS, m/z 559 (~), 424, and 382; IR
(KBr) 29SO, 2900, 2100 (azide), 1770 (carbonate C=O), 1745 (lactone C=O), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~ 7.40 (m, 5H, cbz aromatic), 6.82 (s, lH, 5-H), 6.58 (s, lH, 8-H), 6.27 (s, 2H, 2'.6'-H), 6.03 (ABq, J=2.4 Hz, 2H, O-CH,-O), 5.25 (s, 2H, OCH2Ph), 4.77 (d, J=4 Hz, lH, 4-H), 4.65 (d, J-5 Hz, lH, l-H), 4.31 (d, J-9 Hz, 2H, 11, ll'-H), 3.66 (8, 6H, 3',5'-OCH,), 3.2(dd, J=5,14 Hz, lH, 2-H), and 2.90 (m,lH, 3-H); Anal. (C"H"O,N, 1/2 H,O), C.H.
C. Preparation of 4'-Demethyl-4B-amino-4-desoxy-podophyllotoxin.
500 mg of 10% palladium on carbon was added to a solution of the crude 4'-demethyl-4-azidopodophyllotoxin (2.3g, 4.1 mmol), obtained according to steps A and B, and 200 ml of ethylacetate. This mixture was shaken under 40 psi of hydrogen for four hours. The reaction mixture was then filtered over celite and the filtrate evaporated in vacuo. The residue was chromatographed on a silica gel column and eluted first with a chloroform/ethylacetate (2:1) solvent system to remove the non-polar products. Further elution wlth a chloroform/methanol (19:1) mobile phase ylelded 0.85g, (52%) of the desired product. The product wa~ then crystallized from methylene chloride/ethylether and had the following properties: mp 132-135 C; MS m/z 399 (~); IR
(KBr) 3360 (OH), 3290 (primary amine), 2900 (aliphatic C-H), 1745 (lactone), 1590 (aromatic C-H) cm l;'H NMR (CDCl,) ~ 6.81 (s, lH,5-H), 6.49 (s, lH, 8-H~, 6.30 (s, 2H, 2~,6~-H), 5.96 (ABq, 3s J-l Hz, 2H, OCH,O), 5.3 (s, lH, OH, D,O
exchangeable), 4.55 (d, J=5.2 Hz, lH, l-H), 4.28 2~21~
(d, J--9.5 Hz, 2H, 11,11'-H), 4.17 (d, J=4.1 Hz, lH, 4-H) 3.77 (s, 6H, 3',5'-OCH,), 3.28 (dd, J=5.2,14 Hz, lH, 2-H), and 2.85 (m, lH, 3-H), Anal. (Cl,HI,O~NH2Q), C.H.
ExA~LE-~
Preparation of 4'-Demethyl-4~-amino-4-desoxy-podophyl lotoxin .
4'-Demethyl-4~-amino-4-desoxy-podophyllotoxin was obtained from the column used in Example 3 by further elution with a chloroform/methanol (19:1) mobile phase. The pure product (0.34g, 20%) was crystallized from methylene chloride/ethylether and had the following properties: mp 133-135 C; MS m/z 399 (~); IR (KBr) 3360 (OH), 3295 (NH,), 2900 (aliphatic C-H), 1743 (lactone), 1590 (aromatic C-H) cm l;'H NMR (CDCl,) ~ 7.14 (s, lH, 5-H), 6.54 (s, lH, 8-H), 6.20 (s, 2H, 2',6'-H), 6.00 (ABq, J=l Hz, 2H, OCH,O), 4.63 (d, J-5.1 Hz, lH, l-H), 4.61 (d, J=9.0 Hz, lH, ll~-H), 4.07 (dd, J=9.0,10.4 Hz, lH, llB-H), 3.83 (d, J=10.3 Hz, lH, 4-H), 3.81 (s, 6H, 3',5'-OCH,), 2.85 (dd, J=5.1,14.1 Hz, lH, 2-H), and 2.57(m, lH, 3-H);
Anal. (C"~,O,N H,O), C.H.
~
Preparation o~ 4-alkylamino-4-desoxy-podophyllotoxins. (Scheme III) The 4-alkylamino-4-desoxy-podophyllotoxins specified in Examples 5-12 were prepared according to the following procedure.
A solution of podophyllotoxin (5 g, 12.1 mmol) in 50 ml of anhydrous methylenechloride was kept at room temperature and dry hydrogen bromide gas was bubbled through the solution until saturation was achieved. The flask was then 20~2~
capped and allowed to stand for 48 hours after which time dry nitrogen was bubbled through the solution to drive off excess HBr. Then 2 g. of anhydrous BaCO, and 2 ml of the appropriate amine were added. Vigorous evolution of gas was observed. The mixtur~ was allowed to stand for 5 hours at room temperaturs after which the reaction mixture was filtered, washed with water, dried, and purified via column chromatography. Yields ranged from 5-10%. The products obtained in these examples had the characteristics listed below.
EXAMP~ 5 4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin.
Amorphous powder from CH,Cl,-ether : mp 120 C; MS m/z 443 (M-~; IR (KBr) 3420 (NH,OH), 2900 (aliphatic C-H~, 1755 (lactone), 1600, and 1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ~ 6.82 20 (s, lH, 5-H), 6.49 (s, lH, 8-H), 6.29 (s, 2H, 2',6'-H), 5.97 (ABq, J=1.0,4.4 Hz, 2H, OCH,O), 4.57 (d, J35.0 Hz, lH, 1-H), 4.35 (m, 2H, 11,11'-H), 3.93 (d, J=4.0 Hz, lH, 4-H), 3.79 (s, 6H, 3',5'-OCH,), 3.76 (m, 2H, 2"-H), 3.3 (dd, 25 J=5.0,13.5 Hz, lH, 2-H), 3.09 (m. lH, 3-H), and 2.75 (m, 2H, 1"-H); Anal. (C7,H"O,N l/2 H70), C.H.
4'-Demethyl-4~-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin.
Crystals rom CH,Cl7-ether : mp 230-234 C; MS m/z 443 (M-); IR (KBr) 3425 (NH,OH), 2900 (aliphatic C H), 1753 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CD,OD) ~ 6.83 ~s, lH, 5-H), 6.47 (s, lH, 8-H), 6.38 (s, 2H, 35 2',6'-H), 5.g2 (ABq, J-1.0,14.3 Hz, 2H, OCH70), 2~4~
4.40 (s, lH, ll-H), 4.25 (s, lH, ll'-H), 3.75 (s, 6H, 3',5'- OCH,), 3.65 (m, 4H, 1 ",2"-H), 3.56 (m, lH, l-H), 3.49 (dd, J=6.1,11.3 Hz, lH, 4-H), 2.87 (ddd, J-5.1, S.3, 13.5, lH, 3-H), and 2.67 (dd, J=6.1, 8.2, lH, 2-H); Anal.
(C2,H"O~N-l/2 H2O), C.H.
EX~MPLE 7 4'-Demethyl-4B-propylamino-4-desoxypodophyl lotoxin.
Crystals from CH,Cl,-ether: mp 153-156C; MS m/z 441 (M+); IR (KBr) 3470 (OH), 3320 (NH), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm~'; 'H NMR (CDCl,) ~ 6.77 (s, lH, 5-H), 6.47 (s, lH, 8-H), 6.28 (s, 2H, 2',6'-H), 5.95 (ABq, J=1.2,5.0 Hz, 2H, OCH,O), 4.30 (d, J=5.0 Hz, lH, l-H), 4.30 (d, J=4.0 Hz, lH, 4-H), 4.28(m, 2H, 11,11'-H), 3.78(8, 6H, 3',5'-OCH3), 3.30 (dd, J=5.0, 13.9 Hz, lH, 2-H~, 2.83 (m, 2H, 1"-H), 2.52 (m, lH, 3-H), 1.55~m, 2H, 2"-H), and 0.95 (t, J=7.6 Hz, 3H, 3"-H); Anal. (C"H2,O,N
1/2H,O), C.H.
EX~MpLE 8 4'-Demethyl-4B-[2"-methoxyethylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl,-ether : mp 202-204 C; MS m/z 457 (~); IR (KBr) 3440 (OH, NH), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~ 6.80 (5, lH, 5-H), 6.44 (s, lH, 8-H), 6.25 (s, 2H, 2',6'-H), 5.92 (ABq, J=1.0,5.0 Hz, 2H, OCH,O), 4.50 (d, J=5.4 Hz, lH, 1-H), 4.28 (m, 2H, ll,11'-H), 3.88 (d, J=4.0 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.52 (m, 2H, 2"-H), 3.37 (s, 3H, 3"-H), 3.38 (dd, J=14.4,5.4 Hz, lH, 2-H), 3.05 (m, lh, 3-H), and 2.75 (m, 2H, l"~H); Anal. (C"H"O,N 1/4 H,O), C.H.
2~2~ ~
EX~MPL~:_9 4'-Demethyl-4B-allylamino-4-desoxypodophyllo-toxin.
Amorphous powder from CH2Cl,-ether: mp 225-228 C; MS m/z 439 (M-); IR (KBr) 3340 (OH, NH), 2885 (aliphatic C-H~, 174~ (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~
6.77 (s, lh, 5-H), 6.49 (s, lH, 8-H), 6.28 (s, 2H, 2',6'-H), 5.95 (ABq, J=1.0, 4.5 Hz, 2H, OCH20), 5.90 (m, lH, 2"-H), 5.4 (m, lH, N-H, D20 exchangeable), 5.22 (dd, J=4.0, 17.5 Hz, 2H, 3"-H), 4.53 (d, J=5.5 Hz, lH, l-H), 4.30 (m, 2H, 11,11'-H), 3.88 (d, J=3.6 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.30 (dd, J=5.4, 14.4 Hz, lH, 2-15 H), 3.30 (m, lH, 1"-H), and 2.80 (m, lH, 3-H);
Anal. (C"H2,0,N 2.2 H20), C.H.
EXAM~eLE 1~
4'-Demethyl 4B-[2"-hydroxypropylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl2-ether: mp 145-150 C; MS m/z 457 (M-); IR (KBr) 3330 (OH, NH), 2890 Saliphatic C-H), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ô 6.83 (c, lh, 5-H), 6.47 (~, lH, 8-H), 6.27 (s, 2H, 25 2',6'-H), 5.95 (ABq, J~1.0, 6.3 Hz, 2H, OCH~O), 5.40 (m, lh, N-H, D~O exchangeable ), 4.54 (d, J34.6 Hz, lHI, 1-H), 4.30 (m, 2H, 11,11'-H), 3.85 (~, lH, 2"-11), 3.85 (d, J--3.8 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.25 (dd, J=4.6, 13.8 Hz, 30 lH, 2-H), 2.85 (dd, J=6.8, 12.5 Hz, lH, l"-H), 2.82 (m, lh, 3-H), 2.63 (dd, J=3.8, 12.5 Hz, lH, 1'l-H), and 1.20 (d, J=6.3 Hz, 3H, 3"-H); Anal.
(C2,H"O,N î/2 H~O), C.H.
2~211 4'-Demethyl-4~-[1"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin.
Amorphous powder from CH2Cll-ether : mp 220-225O C; MS m/z 457 (~);'H NMR (CDCl,) ~ 6.89 (s, lH, 5-H), 6.47 (s, lH, 8-H), 6.27 (s, 2H, 2',6'-H3, 5.95 (ABq, J=1.0, 4.5 Hz, 2H, OCH,O), 5.40 (m, lH, N-H, D,O exchangeable), 4.52 (d, J=4.8 Hz, lH, l-H), 4.30 ~d, J-9.0 Hz, 2H, 11,11'-H), 4.00 (d, J=4.0 Hz, lH, 4-H), 3.74 (s, 6H, 3',5'-OCH,), 3.50 (m, 2H, 2"-H), 3.22 (dd, J=4.8, 13.5 Hz, lH, 2-H), 2.85 (m, lH, 3-H), 2.82 (m, lH, 1"-H)m and 1.05 (d, J=6.3 Hz, 3H, 1'-CH,); Anal. (C,~H"O,N 1/2 H,O), C.H.
E~AM~LE 12 4'-Demethyl-4B-[3"-hydroxypropylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl,-ether : mp 193-196 C; MS m/z 457 (~); IR (KBr) 3460 (OH) 3320 (NH), 2900 (aliphatic C-H), 1740 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMX (CDCl,) ~ 6.75 (s, lH, 5-H), 6.46 (8, lH, 8-H), 6.24 (s, 2H, 2',6'-H), 5.94 (ABq, J-1.0,4.4 Hz, 2H, OCHaO), 4.52 (d, J=5.3 Hz, lH, l-H), 4.33 ~dd, J-7.9,8.0 Hz, lH, ll-H), 4.23 ~dd, J~8.0,10.7 Hz, ll'-H), 3.78 ~d, J~4.0 Hz, lH, 4-H), 3.73 (o, 6H, 3',5'-OCH,), 3.72 (t, 2H, 3"-H), 3.21 ~dd, J=5.3,14.0 Hz, lH, 2-H), 3.11 (dd, J=5.9,11.4 Hz, lH, l"-H), 2.64 (ddd, J-14.0,7.0,11.0 Hz, lH, ll'-H) and 1.75 (m, 2H, 2"-H); Anal. (C,~H"O,N 3/4 H,O), C.H.
Preparation of 4'-Demethyl-4B(2"-aminoethoxy)-4-desoxypodophyllotoxin. ~Scheme I~') 2~21~
~3 4'-Demethyl-4B(2l'-aminoethoxy)-4-desoxypodophyllotoxi~ was prepared according to the following procedure.
A. 4'-D~methyl-4B-(2"-bromoethoxy)-4-desoxy-podophyllotoxin.
Podophyllotoxin (500 mg) was suspendsd in anhydrous dichloromet~ane (15 ml~. Dry hydrogen bromide gas was bubbled through the mixture until saturation was achieved. The flask was then capped and allowed to stand at room temperature for 48 h. After bubbling nitrogen gas through the solution to drive off excess hydrogen bromide gas, barium carbonate (1.50 g) and 2-bromoethanol (1 ml) were added and stirred at room temperature for 10 h. The mixture was diluted with dichloromethane, filtered and evaporated to dryness. The syrupy residue was purified by silica gel column chromatography eluting with chloroform-acetone ~30 : 1 v/v). For further purification the product was chromatographed on silica gel column using toluene-ethylacetate (5 : 1 v/v) as an eluant. Yield (218 mg). mp 194-195C. 'H-NMR
(CDC1,): 6 6.79 (lH, S, 5-H), 6.56 (lH, S, 8-H), 6.25 (2H, S, 2', 6'-H), 6.00-5.97 (each d, J= 1.2 Hz, OCHzO), 5.40 (H, S, 4-OH), 4.88 (lH, dd, J= 7.60, 8.34 Hz, 11-H), 4.61 (lH, d, J= 5.2 Hz, l-H), 4.50 (2H, ~), 4.04 (lH, m), 3.80 (lH, m), 3.77 (6H, S, 2 x OCH,), 3.47 (2H, m), 3.42 (lH, dd, J- 5.39, 14.08 Hz, 2-H), 2.90 (lH, m, 3-H) Anal. Calcd. for C"H2,BrO, : C, S4.45; H, 4.57.
Found: C, 54.35; H, 4.60.
~. 4'-Demethyl-4B-(2"-azidoethoxy)-4-desoxy-podophyllctoxin.
2~2~
The mixture of 4' demethyl-4~-(2-bromoethyl~ epipodophyllotoxin (157 mg) and sodium azide (150 mg) in N,N-dimethylformamide (6 ml) was stirred for 10 h at room temperature.
Pouring the reaction mixture into water and stirring gave a white precipitate, which was collected by filtration and dried in the air.
Recryst llization from chloroform-ether gave pure product (120 mg). mp 215-217C IR (CHCl,) cm': 3538 (OH), 2205 (N,), 1770 (lactone), 1615 (aromatic C=C) Anal. Calcd. for C~,H~,N,O,: C, 58.84; H, 4.93; N, 8.95.
Found: C, 58.78; H, 4.98; N, 9.28.
C. 4'-Demethyl-4B(2"-aminoethoxy)-4-desoxy-podoplyllotoxin.
A mixture of 4'-Demethyl-4B-(2"-azidoethyl)epipodophyllotoxin (108 mg) and 10 %
palladium in carbon (55 mg) in ethylacetate (20 ml~ was stirred under a hydrogen atmosphere for 5 h. After the removal of the catalyst by filtration, the filtrate was evaporated to dryness under reduced pressure. The crude product was purified by silica column chromatography eluting with chloroform-methanol (5 : 1 v/v) to obtain pure material ~82 mg) having the Eollowing characteristics.
mp 143-145C
Anal. Cald. for C,~H"NO, H,O: C, 58.72; H, 6.00;
N, 2.97.
Found: C, 58.74; H, 5.98; N, 2.97.
Preparation of 4'-Demethyl-4B-(2"-hydroxy-ethoxy)-4-desoxypodophyllotoxin. (Scheme V) Podophyllotoxin (200 mg) was suspended in anhydrous dichloromethane (15 ml). Dry 2~2~1 hydrogen bromide gas was bubbled through the mixture until saturation was achieved. The flask was then capped and allowed to stand at room temperature for 48 h. After bubbling nitrogen gas through the solution to drive off excess hydrogen bromide gas, anhydrous barium carbonate (500 mg) and ethyleneglycol (500 mg) were added and stirred at room temperature for 10 h. The mixture was diluted with dichloromethane, filtered, washed with water and the organic layer was dried over anhydrous MgSO,.
The removal of solvent gave a syrup, which was purified by silica gel column chromatography eluting with chloroform-acetone (30 : 1 v/v) to obtain pure product (80 mg). The resulting product had the following properties:
'H-NMR (CDC1,): ~ 6.82 (lH, S, 5-H), 6.51 (lH, S, 8-H), 6.23 (2H, S, 2', 6'-H), 5.95 (2H, S,OCH~O), 5.52 (lH, S, OH), 4.70 4.15 (m), 3.72 (s, 6H, 3',5'-OCH,).
.
Preparation of 4'-demethyl-4~-Chloro-4-desoxypodophyllotoxin. (Scheme VI) Methylsulfide (0.3 ml) and N-chloro-succinimide (60 mg, 0.45 mmole) were added at 0C to a solution of 4'-demethylpodophyllotoxin (100 mg, 0.25 mmole) in methylene chloride ~15 ml). The mixture was otirred for 5 h at 0C
under a nitrogen atmosphere. After the removal of the volatile reagents by evaporation in vacuo, the residue was purified by silica gel chromatography eluting with methylene chloride and acetone to obtain the pure product (82 mg, 78.5%)~ The product had the following properties:
IR(CHCl,) cm':3540(0H), 1770(1actone) 2~42~1 'H-NMR(CDCl3) S:6.57 (s, lH, 8-H), 6.53 (s, lH, 5-H), ~.30 (s, 2H, 2'6'-H), 6.02, 5.98 (each d, 2H, J=1.3 Hz, OCH~O), 5.42 (s, lH, OH), 4.62 (d, lH, J=5.2 Hz, l-H), 4.53 (m, 2H, ll-CH7), 4.45 (d, lH, J-3.3 Hz, 4-H), 3.80 (s, 6H, 3', 5'-OCH,), 3.41 (dd, lH, J-5.3, 14 Hz, 2-H), 2.86 (m, lH, 3-H) EXAMPLES 16. 17, AND 18 Preparation of 4, 5, 8, 2 '-tetrabromo-4 B-desoxypodophyllotoxin, 5, 2', 6'-tribromo-4B-desoxypodophyllotoxi~, and 5, 8, 2',6'-tetrabromo-4B-desoxypodophyllotoxin.(Scheme VII) The compounds of Examples 16,17, and 18 were prepared according to the following procedure.
To a solution of podophyllotoxin (200 mg, 0.54 mMol) in CHC1, (7 ml) was added bromine (0.5 ml, 9.70 mMol). After stirring for 2 h. at room temperature, the mixture was poured into ice-water, and extracted with CHC1" followed by washing with 5% sodium hydrosulfite to remove excess bromine. The organic layer was dried over anhydrous magnesium sufate, filtered, and concentrated ~ vacuo to yield a crude product.
Thi8 was purified by use of preparative TLC
(CHCl,-acetone 15:1) to obtain the compounds of Example 16, 17, and 18.
4, 5, 8, 2'-tetrabromo-4B-desoxypodophyllotoxin: 16 mg; NMR (CDC1,)~ 6.21 (d, J-1.0 Hz, 2H, OCH,O), 5.85 (s, lH, 6'-H), 5.74 (d, J=3.2 Hz, lH, 4-H), 5.45 (d, J=6.4 Hz, lH, 1-H), 4.49 (~, 2H, 11,11'-H), 3.95 (s, 3H, 3'-OCH,), 3.92 (s, 3H, 4'-OCH,), 3.68 (s, 3H, 5'-OCH,), 3.44 (dd, J=6.4, 14.4 Hz, lH, 2-H) and 20~4211 3 . 35 (m, lH, 3-H); IR (CHC1,) no OH barld, 1785 ( lactone) cm ' .
EXAMp~ Z
5, 2', 6'-tribromo-4B-desoxypodophyllotoxin: 60 mg: NMR (CDC1,)6 6.33 (s, lH, 8-H), 6.04 (d, J=l.OHz, 2H, OCH,O), 5.72 (d, J=9.5 Hz, lH, 1-H), 5.25 lbr.s, lH, 4-H), 4.40 (m, 2H, 11, ll'-H), 4.95 (B, 3H, 3'-OCH3), 3.94 (s, 3H, 5'-OCH,), 3.81 (s, 3H, 4'-OCH,), 3.70 (m, lH, 3-H), and 3.55 (dd, J=9.5, 15.0 Hz, lH, 2-H: IR (CHC1,) 3600 (OH), 1776 (lactone) C~' .
~X~pL~ 18~
5, 8, 2', 6'-tetrabromo-4B-lS desoxydophyllotoxin: 16 mg; NMR (CDCl,)~ 6.14 (d, J=2.2, 2H, OCH2O), 5.70 (d, J=7.6, lH, 1-H), 5.21 (d, J=2.8 Hz, lH, 4-H), 4.40 (m, 2H, 11/11'-H 3.93 (s, 6H, 3'and 5'-OCH,), 3.78 (8, 3H, 4'-OCH,), 3.65 (m, lH, 3-H) and 3.49 (dd, . 20 J=9.5, 15 Hz, lH, 2-H); IR (CHC1,) 3600 (OH), 1776 (lactone) cm'.
16, R,~R,=R,~R,-Br, R,=H
17, R,-OH, R,-R,-R,~Br, R,=H
18, R,-OH, Rl=R,-R,=R,=Br Ex~eh~ 19 - 41 Preparation of 4'demethyl-4~-(arylaminoS-4-desoxypodophyllotoxins (19-41).
(Scheme VIII) The 4'-demethyl-4~-(arylamino)-4-deoxypodopohyllotoxins specified in examples 19 - 41 were prepared according to the following procedure:
~42~1 A solution of 4'-demethyl~
epipodophyllotoxin (10 g, 24 mmol) in 250 ml of dry dichloromethane was kept at 0C, and dry hydrogen bromidP gas was bubbled through the solution. After 30 min., nitrogen was bubbled through the solution to drive off excess hydrogen bromide. The solution was then evaporated in vacuum to dryness by means of azeotropic distillation with benzen~.
The desired product (11.5 g) was obtained and then used for the next step reaction without any further purification.
Spectral data agreed with that described by M. Xuhn and A. Von Wartburg. Helv.
Chim. Acta, 52, 944 (1969).
A solution containing 4'-demethyl-4~-bromo-4-deoxypodophyllotoxin (300 mg, 0.65 mmol), barium carbonate (153 mg, 0.78 mmol) and the appropriate arylamines (0.78 mmol) in 7 ml of dry 1, 2-dichloroethane was stirred overnight at room temperature. The reaction mixture was filtered, diluted with ethyl acetate, washed with water, dried and puri~ied via column chromatography. The products (19-41) obtained in the examples had the characteristics listed below.
~XAMPLE 19 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin.
Crystals from methanol; mp 172-173, [~]"D-120 (C-1.0, CHCI,); IR (RBr) 3500 (OH), 3360 (NH), 2900 (aliphatic C-H), 1755 (lactone), 1595, 1500 and 1475 (aromatic c=c) cm': 'H NMR
CDCI, ~ 7.22 (t, J=7.5 Hz, 2H, 3", 5"-H), 6.80 (m, 2H, 4" -H and 5-H), 6.50 (m, 3H, 2"-H, 6"-H
and 8-H), 6.33 (s, 2H, 2', 6'-H), 5.97 (ABa, J=1.3, 3.6 Hz, OCH,O), 5.42 (s, lH, exchangeable, 2~421~
4'-O~), 4.68 (br, lH, 4-H), 4.60 (d, J=4.9 ~z, 1-H), 4.38 (t, J=8.4 Hz, lH, 11-H), 4.01 (t, J=8.4 Hz, 1~, 11-H), 3.85 (br, lH, exchangeable, NH), 3.79 (s, 6H, 3', 5'-OCH,), 3.16 (dd, J=5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H~.
Anal. (C2,H"NO7), C.H.N.
EXAMPLE_~0 4'-Demethyl-4~-[4"-cyanoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 187-189, [~]2~D-1~50 (C=1.0, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2890 (aliphatic C-H), 2210 (lactone), 1600~ 1510 and 1475 (aromatic C=C) cm': 'H NMR
CDCI, ~ 7.50 (d, J-8.7 Hz, 2H, 3", 5"-H), 6.74 15 (s, lH, 5-H), 6.57 (d, J=8.7 Hz, 2H, 2", 6"-H), 6.55 (s, lH, 8-H), 6.32 (s, 2H, 2', 6'-H), 5.99 (AB~, J=1.2, 8.3 Hz, 2H, OCH20), 5.44 (8, lH, exchangeable, 4'-OH), 4.78 (~, lH, exchangeable, NH), 4.63 (d, J=4.2 Hz, lH, 4-H), 4.36 (m, 2H, 20 ll-H), 3.85 (m, lH, 1-H), 3.79 (s, 6H, 3', 5'-OCH,), 3.09 (dd, lH, 2-H), 3.05 (m, lH, 3-H).
Anal. (C2,H24NO7), C.H.N.
EXA~LE_2L
4'-Demethyl-4~-[3"-cyanoanilinyl]-4-desoxypodophyllotoXin.
Cry~tals from methanol; mp 191-192, t~]2'D-117 (C~0.33, CHCI,); IR (KBr) 3450 (OH), 3360 (NH), 2900 (aliphatic C-H), 2225 (CN), 1750 (lactone), 1595, 1500 and 1450 (aromatic C=C) cm~
30 ': 'H NMR CDCI, ~ 7.31 (t, J27.6 Hz, 5"-H), 7.07 (d, J=7.6 Hz, 4"-H), 6.80 (d, 2H, 2"-H and 6"-H), 6.74 (s, lH, 5-H), 6.55 (s, lH, 8-H), 6.33 (s, 2H, 2', 6'-H), 6.00 (d, J-7.0 Hz, 2H, OCH20), 5.48 (s, lH exchangeable, 4'-OH), 4.69 (d, Ja3.8 Hz, lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 2-H), 4.41 2~21~
(t, J=8.s Hz, lH, 11-H), 3.92 (t, J=8.5 Hz, lH, 11-H), 3.~1 (s, 6H, 3', 5'-OCH,), 3.14-3.00 tm, 2H, 2-H and 3-H).
Anal. (C,~H2~N2O~)-1/2 H,O, C.H.N.
4'-Demethyl-4~-[4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 270-27i, [~2'D-145 (C=0.33, CHCI,); IR (KBr) 3500 (OH), 10 3370 (NH), 2940 (aliphatic C-H), 1762 (lactone~, 1695 (ester), 1610, 1520 and 1480 (aromatic C=C~
cm-': 'H NMR CDCI, ~ 7.92 (d, J=8.8 Hz, 2H, 3", 5"-H), 6.77 (s, lH, 5-H), 6.55 (d, J=8.8 Hz, 2H, 2", 6'~-H), 6.54 (s, lH, 8-H), 6.33 (s, 2H, 2', lS 6'-H), 5.99 (AB~, J=l.1, 8.2 Hz, 2H, OCH,O), 5.44 (s, lH, exchangeable, 4' OH), 4.78 (d, J=3.3 Hz, lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 1-H), 4.40 ~m, 2H, 4-H and 11-H), 4.37 (g, J=7.1 Hz, 2H, CO2CH2CH,), 4.32 (d, J-7.1, lH, exchangeable, NH), 20 3.92 (t, J=7.5 Hz, lH, ll-H), 3.80 (s, 6H, 3', 5'-OCH,), 3.10 (dd, lH, 2-H), 3.08 (m, lH, 3-H), 1.38 (t, J=7.1 Hz, 3H, CO,CH~L).
Anal. (C~oH2~0~), C.H.N.
4~-Demethyl-4~-~4"-morpholinoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 235-237, t~]"D-129 (C=l, CHCI,); IR (K~r) 3500 (OH), 3300 (NH), 2880 (aliphatlc C-H), 1755 (lactone), 30 16~0, 1510 and 1475 (aromatic C=C) cm-': 'H NMR
CDCI, ~ 6.86 (d, J=9.5 Hz, 2H, 3", 5"-H), 6.76 (s, lH, 5-H), 6.52 (br, 3H, 8-H and 2", 6"-H), 6.35 (s, 2H, 2', 6'-H), 5.96 (d, J=66.7 Hz, 2H, OCH,O), 5.44 (s, lH, exchangeable, 4~-OH), 4.61 (m, 2H, 4-H and 1-H), 4.37 (t, J=7.0 Hz, lH, 11-H), 4.08 (t, J=7.0 Hz, lH, ll-H), 3.82 (br, 4H, 2 1 ~
~ , 3.80 ~s, 6H, 3', 5'-3CH,), 3.22-2.90 (m, 2H, 2-H, 3-H and 4H, ~ ~;
Anal. (C,,H,IN,O,), C.H.N.
4'-Demethyl-4~-[3", 4"-(methylenedioxy)anilinyl~-4-desoxypodophyllotoxin.
Crystals from methanol; mp 247-2497, [~]2~D-126 (C=1, CHCI,); IR (KBr) 3500 (OH), 3340 10 (NH), 2900 (aliphatic C-H), 1752 (lactone), 1605, 1496 and 1475 (aromatic C=C) cm~': 'H NMR
CDCI" ~ 6.76 (s, lH, 5-H), 6.68 (d, J=8.1 Hz, lH, 5n-H), 6.52 (s, lH, 8-H), 6.33 (s, 2H, 2', 6'-H), 6.17 (d, J31.2 Hz, lH, 2"-H), 5.96 (q, 15 J=1.2, 8.1 Hz, 3H, 6"H and OCH,O), 5.90 (s, 2H, 7"-H), 5.43 (s, lH exchangeable, 4'-OH3, 4.59 (d, J=4.9 Hz, lH, 4-H), 4.56 (d, J=3.9 Hz, lH, l-H), 4 . 37 (t, lH, 11-H), 4.05 (t, lH, 11-H), 3.79 (s, 6H, 3', 5'-OCH,), 3.15 (dd, lH, 2-H), 20 2.95 (m, lH, 3-H).
Anal. (C2,H"NO~), C.H.N.
EXAMP~E 25 4'-Demethyl-4~-[3", 4"-dimethoxyanilinyl]~4-desoxypodophyllotoxin.
Cry~tals from methanol; mp 233-~34 tdec); t~]2'D-118D (C=1, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2920 (aliphatic C-H), 1770 (lactone), 1605, and 1505 (aromatic C=C) cm': 'H
NMR CDCI" ~ 6.78 (s, lH, 5-H), 6.75 (d, J=8.5 30 Hz, lH, 5"-H), 6.53 (s, lH, 8-H), 6.34 (s, 2H, 2', 6'-H), 6.17 (s, lH, 2"-H), 6.05 (d, J=8.5 Hz, lH, 6"-H), 5.96 (d, J=2.4 Hz, 2H, OCH2O), 5.43 (s, exchangeable, 4'-OH), 4.60 (d, 2H, 4-H
and l-H), 4.38 (t, J=8.3 Hz, lH, 11-H), 4.05 2~4~2~1 (t, J=8.3 Hz, 1, ll-H), 3.83 (s, 3H, 4"-OCH,), 3.81 (s, 3H, 3"-OCH3), 3.80 (s, 6H, 3', 5'-OCH,), 3.18 (dd, J=5.0, 14.0 Hz, lH, 2-H), 2.96 (m, lH, 3-H).
Anal. (Cl,H"NOq), C.H.N.
EXA~PLE 26 4'-Demethyl-4~-[3"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from methanol; mp 201-203C
10 (dec.);[~]2'D-132 tc = 1, CHCl,); IR (KBr) 3500 (OH), 3360 (NH), 2900 (aliphatic C-H), 1750 (lactone), 1605, 1500 1475 (aromatic C=C) cm-';
'HNMR (CDCl,) ~ 7.15 (t, J = 7.4 Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.53 (s, lH, 8-H), 6.49 (dd, H = 1.2, 7.4 Hz, lH, 4"-H), 6.40 (s, 2H, 2', 6'-H), 6.32 (d, J = 1.2 Hz, lH, 2"-H), 6.24 (dd, J
= 1.2, 7.4 Hz, lH, 6l'-H), 5.97 (ABq, J = 1.2, 7.9 Hz, 2H OCH~O), 5.44 (s, lH, exchangeable, 4'-OH), 4.67 (s, lH, exchangeable, NH), 4.63 (d, J
= 4.0 Hz, lH, 4-H), 4.59 (d, J = 5.0 Hz, lH, 1-H), 4.39 (t, J = 8.5 Hz, lH, 11-H) 3.98 (t, J =
Anal. (C"~,O,N H,O), C.H.
~
Preparation o~ 4-alkylamino-4-desoxy-podophyllotoxins. (Scheme III) The 4-alkylamino-4-desoxy-podophyllotoxins specified in Examples 5-12 were prepared according to the following procedure.
A solution of podophyllotoxin (5 g, 12.1 mmol) in 50 ml of anhydrous methylenechloride was kept at room temperature and dry hydrogen bromide gas was bubbled through the solution until saturation was achieved. The flask was then 20~2~
capped and allowed to stand for 48 hours after which time dry nitrogen was bubbled through the solution to drive off excess HBr. Then 2 g. of anhydrous BaCO, and 2 ml of the appropriate amine were added. Vigorous evolution of gas was observed. The mixtur~ was allowed to stand for 5 hours at room temperaturs after which the reaction mixture was filtered, washed with water, dried, and purified via column chromatography. Yields ranged from 5-10%. The products obtained in these examples had the characteristics listed below.
EXAMP~ 5 4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin.
Amorphous powder from CH,Cl,-ether : mp 120 C; MS m/z 443 (M-~; IR (KBr) 3420 (NH,OH), 2900 (aliphatic C-H~, 1755 (lactone), 1600, and 1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ~ 6.82 20 (s, lH, 5-H), 6.49 (s, lH, 8-H), 6.29 (s, 2H, 2',6'-H), 5.97 (ABq, J=1.0,4.4 Hz, 2H, OCH,O), 4.57 (d, J35.0 Hz, lH, 1-H), 4.35 (m, 2H, 11,11'-H), 3.93 (d, J=4.0 Hz, lH, 4-H), 3.79 (s, 6H, 3',5'-OCH,), 3.76 (m, 2H, 2"-H), 3.3 (dd, 25 J=5.0,13.5 Hz, lH, 2-H), 3.09 (m. lH, 3-H), and 2.75 (m, 2H, 1"-H); Anal. (C7,H"O,N l/2 H70), C.H.
4'-Demethyl-4~-[2"-hydroxyethylamino]-4-desoxy-podophyllotoxin.
Crystals rom CH,Cl7-ether : mp 230-234 C; MS m/z 443 (M-); IR (KBr) 3425 (NH,OH), 2900 (aliphatic C H), 1753 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CD,OD) ~ 6.83 ~s, lH, 5-H), 6.47 (s, lH, 8-H), 6.38 (s, 2H, 35 2',6'-H), 5.g2 (ABq, J-1.0,14.3 Hz, 2H, OCH70), 2~4~
4.40 (s, lH, ll-H), 4.25 (s, lH, ll'-H), 3.75 (s, 6H, 3',5'- OCH,), 3.65 (m, 4H, 1 ",2"-H), 3.56 (m, lH, l-H), 3.49 (dd, J=6.1,11.3 Hz, lH, 4-H), 2.87 (ddd, J-5.1, S.3, 13.5, lH, 3-H), and 2.67 (dd, J=6.1, 8.2, lH, 2-H); Anal.
(C2,H"O~N-l/2 H2O), C.H.
EX~MPLE 7 4'-Demethyl-4B-propylamino-4-desoxypodophyl lotoxin.
Crystals from CH,Cl,-ether: mp 153-156C; MS m/z 441 (M+); IR (KBr) 3470 (OH), 3320 (NH), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm~'; 'H NMR (CDCl,) ~ 6.77 (s, lH, 5-H), 6.47 (s, lH, 8-H), 6.28 (s, 2H, 2',6'-H), 5.95 (ABq, J=1.2,5.0 Hz, 2H, OCH,O), 4.30 (d, J=5.0 Hz, lH, l-H), 4.30 (d, J=4.0 Hz, lH, 4-H), 4.28(m, 2H, 11,11'-H), 3.78(8, 6H, 3',5'-OCH3), 3.30 (dd, J=5.0, 13.9 Hz, lH, 2-H~, 2.83 (m, 2H, 1"-H), 2.52 (m, lH, 3-H), 1.55~m, 2H, 2"-H), and 0.95 (t, J=7.6 Hz, 3H, 3"-H); Anal. (C"H2,O,N
1/2H,O), C.H.
EX~MpLE 8 4'-Demethyl-4B-[2"-methoxyethylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl,-ether : mp 202-204 C; MS m/z 457 (~); IR (KBr) 3440 (OH, NH), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~ 6.80 (5, lH, 5-H), 6.44 (s, lH, 8-H), 6.25 (s, 2H, 2',6'-H), 5.92 (ABq, J=1.0,5.0 Hz, 2H, OCH,O), 4.50 (d, J=5.4 Hz, lH, 1-H), 4.28 (m, 2H, ll,11'-H), 3.88 (d, J=4.0 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.52 (m, 2H, 2"-H), 3.37 (s, 3H, 3"-H), 3.38 (dd, J=14.4,5.4 Hz, lH, 2-H), 3.05 (m, lh, 3-H), and 2.75 (m, 2H, l"~H); Anal. (C"H"O,N 1/4 H,O), C.H.
2~2~ ~
EX~MPL~:_9 4'-Demethyl-4B-allylamino-4-desoxypodophyllo-toxin.
Amorphous powder from CH2Cl,-ether: mp 225-228 C; MS m/z 439 (M-); IR (KBr) 3340 (OH, NH), 2885 (aliphatic C-H~, 174~ (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~
6.77 (s, lh, 5-H), 6.49 (s, lH, 8-H), 6.28 (s, 2H, 2',6'-H), 5.95 (ABq, J=1.0, 4.5 Hz, 2H, OCH20), 5.90 (m, lH, 2"-H), 5.4 (m, lH, N-H, D20 exchangeable), 5.22 (dd, J=4.0, 17.5 Hz, 2H, 3"-H), 4.53 (d, J=5.5 Hz, lH, l-H), 4.30 (m, 2H, 11,11'-H), 3.88 (d, J=3.6 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.30 (dd, J=5.4, 14.4 Hz, lH, 2-15 H), 3.30 (m, lH, 1"-H), and 2.80 (m, lH, 3-H);
Anal. (C"H2,0,N 2.2 H20), C.H.
EXAM~eLE 1~
4'-Demethyl 4B-[2"-hydroxypropylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl2-ether: mp 145-150 C; MS m/z 457 (M-); IR (KBr) 3330 (OH, NH), 2890 Saliphatic C-H), 1750 (lactone), 1600, and 1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ô 6.83 (c, lh, 5-H), 6.47 (~, lH, 8-H), 6.27 (s, 2H, 25 2',6'-H), 5.95 (ABq, J~1.0, 6.3 Hz, 2H, OCH~O), 5.40 (m, lh, N-H, D~O exchangeable ), 4.54 (d, J34.6 Hz, lHI, 1-H), 4.30 (m, 2H, 11,11'-H), 3.85 (~, lH, 2"-11), 3.85 (d, J--3.8 Hz, lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.25 (dd, J=4.6, 13.8 Hz, 30 lH, 2-H), 2.85 (dd, J=6.8, 12.5 Hz, lH, l"-H), 2.82 (m, lh, 3-H), 2.63 (dd, J=3.8, 12.5 Hz, lH, 1'l-H), and 1.20 (d, J=6.3 Hz, 3H, 3"-H); Anal.
(C2,H"O,N î/2 H~O), C.H.
2~211 4'-Demethyl-4~-[1"-methyl-2"-hydroxyethylamino]-4-desoxypodophyllotoxin.
Amorphous powder from CH2Cll-ether : mp 220-225O C; MS m/z 457 (~);'H NMR (CDCl,) ~ 6.89 (s, lH, 5-H), 6.47 (s, lH, 8-H), 6.27 (s, 2H, 2',6'-H3, 5.95 (ABq, J=1.0, 4.5 Hz, 2H, OCH,O), 5.40 (m, lH, N-H, D,O exchangeable), 4.52 (d, J=4.8 Hz, lH, l-H), 4.30 ~d, J-9.0 Hz, 2H, 11,11'-H), 4.00 (d, J=4.0 Hz, lH, 4-H), 3.74 (s, 6H, 3',5'-OCH,), 3.50 (m, 2H, 2"-H), 3.22 (dd, J=4.8, 13.5 Hz, lH, 2-H), 2.85 (m, lH, 3-H), 2.82 (m, lH, 1"-H)m and 1.05 (d, J=6.3 Hz, 3H, 1'-CH,); Anal. (C,~H"O,N 1/2 H,O), C.H.
E~AM~LE 12 4'-Demethyl-4B-[3"-hydroxypropylamino]-4-desoxy-podophyllotoxin.
Crystals from CH,Cl,-ether : mp 193-196 C; MS m/z 457 (~); IR (KBr) 3460 (OH) 3320 (NH), 2900 (aliphatic C-H), 1740 (lactone), 1600, and 1475 (aromatic C=C) cm l;'H NMX (CDCl,) ~ 6.75 (s, lH, 5-H), 6.46 (8, lH, 8-H), 6.24 (s, 2H, 2',6'-H), 5.94 (ABq, J-1.0,4.4 Hz, 2H, OCHaO), 4.52 (d, J=5.3 Hz, lH, l-H), 4.33 ~dd, J-7.9,8.0 Hz, lH, ll-H), 4.23 ~dd, J~8.0,10.7 Hz, ll'-H), 3.78 ~d, J~4.0 Hz, lH, 4-H), 3.73 (o, 6H, 3',5'-OCH,), 3.72 (t, 2H, 3"-H), 3.21 ~dd, J=5.3,14.0 Hz, lH, 2-H), 3.11 (dd, J=5.9,11.4 Hz, lH, l"-H), 2.64 (ddd, J-14.0,7.0,11.0 Hz, lH, ll'-H) and 1.75 (m, 2H, 2"-H); Anal. (C,~H"O,N 3/4 H,O), C.H.
Preparation of 4'-Demethyl-4B(2"-aminoethoxy)-4-desoxypodophyllotoxin. ~Scheme I~') 2~21~
~3 4'-Demethyl-4B(2l'-aminoethoxy)-4-desoxypodophyllotoxi~ was prepared according to the following procedure.
A. 4'-D~methyl-4B-(2"-bromoethoxy)-4-desoxy-podophyllotoxin.
Podophyllotoxin (500 mg) was suspendsd in anhydrous dichloromet~ane (15 ml~. Dry hydrogen bromide gas was bubbled through the mixture until saturation was achieved. The flask was then capped and allowed to stand at room temperature for 48 h. After bubbling nitrogen gas through the solution to drive off excess hydrogen bromide gas, barium carbonate (1.50 g) and 2-bromoethanol (1 ml) were added and stirred at room temperature for 10 h. The mixture was diluted with dichloromethane, filtered and evaporated to dryness. The syrupy residue was purified by silica gel column chromatography eluting with chloroform-acetone ~30 : 1 v/v). For further purification the product was chromatographed on silica gel column using toluene-ethylacetate (5 : 1 v/v) as an eluant. Yield (218 mg). mp 194-195C. 'H-NMR
(CDC1,): 6 6.79 (lH, S, 5-H), 6.56 (lH, S, 8-H), 6.25 (2H, S, 2', 6'-H), 6.00-5.97 (each d, J= 1.2 Hz, OCHzO), 5.40 (H, S, 4-OH), 4.88 (lH, dd, J= 7.60, 8.34 Hz, 11-H), 4.61 (lH, d, J= 5.2 Hz, l-H), 4.50 (2H, ~), 4.04 (lH, m), 3.80 (lH, m), 3.77 (6H, S, 2 x OCH,), 3.47 (2H, m), 3.42 (lH, dd, J- 5.39, 14.08 Hz, 2-H), 2.90 (lH, m, 3-H) Anal. Calcd. for C"H2,BrO, : C, S4.45; H, 4.57.
Found: C, 54.35; H, 4.60.
~. 4'-Demethyl-4B-(2"-azidoethoxy)-4-desoxy-podophyllctoxin.
2~2~
The mixture of 4' demethyl-4~-(2-bromoethyl~ epipodophyllotoxin (157 mg) and sodium azide (150 mg) in N,N-dimethylformamide (6 ml) was stirred for 10 h at room temperature.
Pouring the reaction mixture into water and stirring gave a white precipitate, which was collected by filtration and dried in the air.
Recryst llization from chloroform-ether gave pure product (120 mg). mp 215-217C IR (CHCl,) cm': 3538 (OH), 2205 (N,), 1770 (lactone), 1615 (aromatic C=C) Anal. Calcd. for C~,H~,N,O,: C, 58.84; H, 4.93; N, 8.95.
Found: C, 58.78; H, 4.98; N, 9.28.
C. 4'-Demethyl-4B(2"-aminoethoxy)-4-desoxy-podoplyllotoxin.
A mixture of 4'-Demethyl-4B-(2"-azidoethyl)epipodophyllotoxin (108 mg) and 10 %
palladium in carbon (55 mg) in ethylacetate (20 ml~ was stirred under a hydrogen atmosphere for 5 h. After the removal of the catalyst by filtration, the filtrate was evaporated to dryness under reduced pressure. The crude product was purified by silica column chromatography eluting with chloroform-methanol (5 : 1 v/v) to obtain pure material ~82 mg) having the Eollowing characteristics.
mp 143-145C
Anal. Cald. for C,~H"NO, H,O: C, 58.72; H, 6.00;
N, 2.97.
Found: C, 58.74; H, 5.98; N, 2.97.
Preparation of 4'-Demethyl-4B-(2"-hydroxy-ethoxy)-4-desoxypodophyllotoxin. (Scheme V) Podophyllotoxin (200 mg) was suspended in anhydrous dichloromethane (15 ml). Dry 2~2~1 hydrogen bromide gas was bubbled through the mixture until saturation was achieved. The flask was then capped and allowed to stand at room temperature for 48 h. After bubbling nitrogen gas through the solution to drive off excess hydrogen bromide gas, anhydrous barium carbonate (500 mg) and ethyleneglycol (500 mg) were added and stirred at room temperature for 10 h. The mixture was diluted with dichloromethane, filtered, washed with water and the organic layer was dried over anhydrous MgSO,.
The removal of solvent gave a syrup, which was purified by silica gel column chromatography eluting with chloroform-acetone (30 : 1 v/v) to obtain pure product (80 mg). The resulting product had the following properties:
'H-NMR (CDC1,): ~ 6.82 (lH, S, 5-H), 6.51 (lH, S, 8-H), 6.23 (2H, S, 2', 6'-H), 5.95 (2H, S,OCH~O), 5.52 (lH, S, OH), 4.70 4.15 (m), 3.72 (s, 6H, 3',5'-OCH,).
.
Preparation of 4'-demethyl-4~-Chloro-4-desoxypodophyllotoxin. (Scheme VI) Methylsulfide (0.3 ml) and N-chloro-succinimide (60 mg, 0.45 mmole) were added at 0C to a solution of 4'-demethylpodophyllotoxin (100 mg, 0.25 mmole) in methylene chloride ~15 ml). The mixture was otirred for 5 h at 0C
under a nitrogen atmosphere. After the removal of the volatile reagents by evaporation in vacuo, the residue was purified by silica gel chromatography eluting with methylene chloride and acetone to obtain the pure product (82 mg, 78.5%)~ The product had the following properties:
IR(CHCl,) cm':3540(0H), 1770(1actone) 2~42~1 'H-NMR(CDCl3) S:6.57 (s, lH, 8-H), 6.53 (s, lH, 5-H), ~.30 (s, 2H, 2'6'-H), 6.02, 5.98 (each d, 2H, J=1.3 Hz, OCH~O), 5.42 (s, lH, OH), 4.62 (d, lH, J=5.2 Hz, l-H), 4.53 (m, 2H, ll-CH7), 4.45 (d, lH, J-3.3 Hz, 4-H), 3.80 (s, 6H, 3', 5'-OCH,), 3.41 (dd, lH, J-5.3, 14 Hz, 2-H), 2.86 (m, lH, 3-H) EXAMPLES 16. 17, AND 18 Preparation of 4, 5, 8, 2 '-tetrabromo-4 B-desoxypodophyllotoxin, 5, 2', 6'-tribromo-4B-desoxypodophyllotoxi~, and 5, 8, 2',6'-tetrabromo-4B-desoxypodophyllotoxin.(Scheme VII) The compounds of Examples 16,17, and 18 were prepared according to the following procedure.
To a solution of podophyllotoxin (200 mg, 0.54 mMol) in CHC1, (7 ml) was added bromine (0.5 ml, 9.70 mMol). After stirring for 2 h. at room temperature, the mixture was poured into ice-water, and extracted with CHC1" followed by washing with 5% sodium hydrosulfite to remove excess bromine. The organic layer was dried over anhydrous magnesium sufate, filtered, and concentrated ~ vacuo to yield a crude product.
Thi8 was purified by use of preparative TLC
(CHCl,-acetone 15:1) to obtain the compounds of Example 16, 17, and 18.
4, 5, 8, 2'-tetrabromo-4B-desoxypodophyllotoxin: 16 mg; NMR (CDC1,)~ 6.21 (d, J-1.0 Hz, 2H, OCH,O), 5.85 (s, lH, 6'-H), 5.74 (d, J=3.2 Hz, lH, 4-H), 5.45 (d, J=6.4 Hz, lH, 1-H), 4.49 (~, 2H, 11,11'-H), 3.95 (s, 3H, 3'-OCH,), 3.92 (s, 3H, 4'-OCH,), 3.68 (s, 3H, 5'-OCH,), 3.44 (dd, J=6.4, 14.4 Hz, lH, 2-H) and 20~4211 3 . 35 (m, lH, 3-H); IR (CHC1,) no OH barld, 1785 ( lactone) cm ' .
EXAMp~ Z
5, 2', 6'-tribromo-4B-desoxypodophyllotoxin: 60 mg: NMR (CDC1,)6 6.33 (s, lH, 8-H), 6.04 (d, J=l.OHz, 2H, OCH,O), 5.72 (d, J=9.5 Hz, lH, 1-H), 5.25 lbr.s, lH, 4-H), 4.40 (m, 2H, 11, ll'-H), 4.95 (B, 3H, 3'-OCH3), 3.94 (s, 3H, 5'-OCH,), 3.81 (s, 3H, 4'-OCH,), 3.70 (m, lH, 3-H), and 3.55 (dd, J=9.5, 15.0 Hz, lH, 2-H: IR (CHC1,) 3600 (OH), 1776 (lactone) C~' .
~X~pL~ 18~
5, 8, 2', 6'-tetrabromo-4B-lS desoxydophyllotoxin: 16 mg; NMR (CDCl,)~ 6.14 (d, J=2.2, 2H, OCH2O), 5.70 (d, J=7.6, lH, 1-H), 5.21 (d, J=2.8 Hz, lH, 4-H), 4.40 (m, 2H, 11/11'-H 3.93 (s, 6H, 3'and 5'-OCH,), 3.78 (8, 3H, 4'-OCH,), 3.65 (m, lH, 3-H) and 3.49 (dd, . 20 J=9.5, 15 Hz, lH, 2-H); IR (CHC1,) 3600 (OH), 1776 (lactone) cm'.
16, R,~R,=R,~R,-Br, R,=H
17, R,-OH, R,-R,-R,~Br, R,=H
18, R,-OH, Rl=R,-R,=R,=Br Ex~eh~ 19 - 41 Preparation of 4'demethyl-4~-(arylaminoS-4-desoxypodophyllotoxins (19-41).
(Scheme VIII) The 4'-demethyl-4~-(arylamino)-4-deoxypodopohyllotoxins specified in examples 19 - 41 were prepared according to the following procedure:
~42~1 A solution of 4'-demethyl~
epipodophyllotoxin (10 g, 24 mmol) in 250 ml of dry dichloromethane was kept at 0C, and dry hydrogen bromidP gas was bubbled through the solution. After 30 min., nitrogen was bubbled through the solution to drive off excess hydrogen bromide. The solution was then evaporated in vacuum to dryness by means of azeotropic distillation with benzen~.
The desired product (11.5 g) was obtained and then used for the next step reaction without any further purification.
Spectral data agreed with that described by M. Xuhn and A. Von Wartburg. Helv.
Chim. Acta, 52, 944 (1969).
A solution containing 4'-demethyl-4~-bromo-4-deoxypodophyllotoxin (300 mg, 0.65 mmol), barium carbonate (153 mg, 0.78 mmol) and the appropriate arylamines (0.78 mmol) in 7 ml of dry 1, 2-dichloroethane was stirred overnight at room temperature. The reaction mixture was filtered, diluted with ethyl acetate, washed with water, dried and puri~ied via column chromatography. The products (19-41) obtained in the examples had the characteristics listed below.
~XAMPLE 19 4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin.
Crystals from methanol; mp 172-173, [~]"D-120 (C-1.0, CHCI,); IR (RBr) 3500 (OH), 3360 (NH), 2900 (aliphatic C-H), 1755 (lactone), 1595, 1500 and 1475 (aromatic c=c) cm': 'H NMR
CDCI, ~ 7.22 (t, J=7.5 Hz, 2H, 3", 5"-H), 6.80 (m, 2H, 4" -H and 5-H), 6.50 (m, 3H, 2"-H, 6"-H
and 8-H), 6.33 (s, 2H, 2', 6'-H), 5.97 (ABa, J=1.3, 3.6 Hz, OCH,O), 5.42 (s, lH, exchangeable, 2~421~
4'-O~), 4.68 (br, lH, 4-H), 4.60 (d, J=4.9 ~z, 1-H), 4.38 (t, J=8.4 Hz, lH, 11-H), 4.01 (t, J=8.4 Hz, 1~, 11-H), 3.85 (br, lH, exchangeable, NH), 3.79 (s, 6H, 3', 5'-OCH,), 3.16 (dd, J=5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H~.
Anal. (C2,H"NO7), C.H.N.
EXAMPLE_~0 4'-Demethyl-4~-[4"-cyanoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 187-189, [~]2~D-1~50 (C=1.0, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2890 (aliphatic C-H), 2210 (lactone), 1600~ 1510 and 1475 (aromatic C=C) cm': 'H NMR
CDCI, ~ 7.50 (d, J-8.7 Hz, 2H, 3", 5"-H), 6.74 15 (s, lH, 5-H), 6.57 (d, J=8.7 Hz, 2H, 2", 6"-H), 6.55 (s, lH, 8-H), 6.32 (s, 2H, 2', 6'-H), 5.99 (AB~, J=1.2, 8.3 Hz, 2H, OCH20), 5.44 (8, lH, exchangeable, 4'-OH), 4.78 (~, lH, exchangeable, NH), 4.63 (d, J=4.2 Hz, lH, 4-H), 4.36 (m, 2H, 20 ll-H), 3.85 (m, lH, 1-H), 3.79 (s, 6H, 3', 5'-OCH,), 3.09 (dd, lH, 2-H), 3.05 (m, lH, 3-H).
Anal. (C2,H24NO7), C.H.N.
EXA~LE_2L
4'-Demethyl-4~-[3"-cyanoanilinyl]-4-desoxypodophyllotoXin.
Cry~tals from methanol; mp 191-192, t~]2'D-117 (C~0.33, CHCI,); IR (KBr) 3450 (OH), 3360 (NH), 2900 (aliphatic C-H), 2225 (CN), 1750 (lactone), 1595, 1500 and 1450 (aromatic C=C) cm~
30 ': 'H NMR CDCI, ~ 7.31 (t, J27.6 Hz, 5"-H), 7.07 (d, J=7.6 Hz, 4"-H), 6.80 (d, 2H, 2"-H and 6"-H), 6.74 (s, lH, 5-H), 6.55 (s, lH, 8-H), 6.33 (s, 2H, 2', 6'-H), 6.00 (d, J-7.0 Hz, 2H, OCH20), 5.48 (s, lH exchangeable, 4'-OH), 4.69 (d, Ja3.8 Hz, lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 2-H), 4.41 2~21~
(t, J=8.s Hz, lH, 11-H), 3.92 (t, J=8.5 Hz, lH, 11-H), 3.~1 (s, 6H, 3', 5'-OCH,), 3.14-3.00 tm, 2H, 2-H and 3-H).
Anal. (C,~H2~N2O~)-1/2 H,O, C.H.N.
4'-Demethyl-4~-[4"-ethoxycarbonylanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 270-27i, [~2'D-145 (C=0.33, CHCI,); IR (KBr) 3500 (OH), 10 3370 (NH), 2940 (aliphatic C-H), 1762 (lactone~, 1695 (ester), 1610, 1520 and 1480 (aromatic C=C~
cm-': 'H NMR CDCI, ~ 7.92 (d, J=8.8 Hz, 2H, 3", 5"-H), 6.77 (s, lH, 5-H), 6.55 (d, J=8.8 Hz, 2H, 2", 6'~-H), 6.54 (s, lH, 8-H), 6.33 (s, 2H, 2', lS 6'-H), 5.99 (AB~, J=l.1, 8.2 Hz, 2H, OCH,O), 5.44 (s, lH, exchangeable, 4' OH), 4.78 (d, J=3.3 Hz, lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 1-H), 4.40 ~m, 2H, 4-H and 11-H), 4.37 (g, J=7.1 Hz, 2H, CO2CH2CH,), 4.32 (d, J-7.1, lH, exchangeable, NH), 20 3.92 (t, J=7.5 Hz, lH, ll-H), 3.80 (s, 6H, 3', 5'-OCH,), 3.10 (dd, lH, 2-H), 3.08 (m, lH, 3-H), 1.38 (t, J=7.1 Hz, 3H, CO,CH~L).
Anal. (C~oH2~0~), C.H.N.
4~-Demethyl-4~-~4"-morpholinoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 235-237, t~]"D-129 (C=l, CHCI,); IR (K~r) 3500 (OH), 3300 (NH), 2880 (aliphatlc C-H), 1755 (lactone), 30 16~0, 1510 and 1475 (aromatic C=C) cm-': 'H NMR
CDCI, ~ 6.86 (d, J=9.5 Hz, 2H, 3", 5"-H), 6.76 (s, lH, 5-H), 6.52 (br, 3H, 8-H and 2", 6"-H), 6.35 (s, 2H, 2', 6'-H), 5.96 (d, J=66.7 Hz, 2H, OCH,O), 5.44 (s, lH, exchangeable, 4~-OH), 4.61 (m, 2H, 4-H and 1-H), 4.37 (t, J=7.0 Hz, lH, 11-H), 4.08 (t, J=7.0 Hz, lH, ll-H), 3.82 (br, 4H, 2 1 ~
~ , 3.80 ~s, 6H, 3', 5'-3CH,), 3.22-2.90 (m, 2H, 2-H, 3-H and 4H, ~ ~;
Anal. (C,,H,IN,O,), C.H.N.
4'-Demethyl-4~-[3", 4"-(methylenedioxy)anilinyl~-4-desoxypodophyllotoxin.
Crystals from methanol; mp 247-2497, [~]2~D-126 (C=1, CHCI,); IR (KBr) 3500 (OH), 3340 10 (NH), 2900 (aliphatic C-H), 1752 (lactone), 1605, 1496 and 1475 (aromatic C=C) cm~': 'H NMR
CDCI" ~ 6.76 (s, lH, 5-H), 6.68 (d, J=8.1 Hz, lH, 5n-H), 6.52 (s, lH, 8-H), 6.33 (s, 2H, 2', 6'-H), 6.17 (d, J31.2 Hz, lH, 2"-H), 5.96 (q, 15 J=1.2, 8.1 Hz, 3H, 6"H and OCH,O), 5.90 (s, 2H, 7"-H), 5.43 (s, lH exchangeable, 4'-OH3, 4.59 (d, J=4.9 Hz, lH, 4-H), 4.56 (d, J=3.9 Hz, lH, l-H), 4 . 37 (t, lH, 11-H), 4.05 (t, lH, 11-H), 3.79 (s, 6H, 3', 5'-OCH,), 3.15 (dd, lH, 2-H), 20 2.95 (m, lH, 3-H).
Anal. (C2,H"NO~), C.H.N.
EXAMP~E 25 4'-Demethyl-4~-[3", 4"-dimethoxyanilinyl]~4-desoxypodophyllotoxin.
Cry~tals from methanol; mp 233-~34 tdec); t~]2'D-118D (C=1, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2920 (aliphatic C-H), 1770 (lactone), 1605, and 1505 (aromatic C=C) cm': 'H
NMR CDCI" ~ 6.78 (s, lH, 5-H), 6.75 (d, J=8.5 30 Hz, lH, 5"-H), 6.53 (s, lH, 8-H), 6.34 (s, 2H, 2', 6'-H), 6.17 (s, lH, 2"-H), 6.05 (d, J=8.5 Hz, lH, 6"-H), 5.96 (d, J=2.4 Hz, 2H, OCH2O), 5.43 (s, exchangeable, 4'-OH), 4.60 (d, 2H, 4-H
and l-H), 4.38 (t, J=8.3 Hz, lH, 11-H), 4.05 2~4~2~1 (t, J=8.3 Hz, 1, ll-H), 3.83 (s, 3H, 4"-OCH,), 3.81 (s, 3H, 3"-OCH3), 3.80 (s, 6H, 3', 5'-OCH,), 3.18 (dd, J=5.0, 14.0 Hz, lH, 2-H), 2.96 (m, lH, 3-H).
Anal. (Cl,H"NOq), C.H.N.
EXA~PLE 26 4'-Demethyl-4~-[3"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from methanol; mp 201-203C
10 (dec.);[~]2'D-132 tc = 1, CHCl,); IR (KBr) 3500 (OH), 3360 (NH), 2900 (aliphatic C-H), 1750 (lactone), 1605, 1500 1475 (aromatic C=C) cm-';
'HNMR (CDCl,) ~ 7.15 (t, J = 7.4 Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.53 (s, lH, 8-H), 6.49 (dd, H = 1.2, 7.4 Hz, lH, 4"-H), 6.40 (s, 2H, 2', 6'-H), 6.32 (d, J = 1.2 Hz, lH, 2"-H), 6.24 (dd, J
= 1.2, 7.4 Hz, lH, 6l'-H), 5.97 (ABq, J = 1.2, 7.9 Hz, 2H OCH~O), 5.44 (s, lH, exchangeable, 4'-OH), 4.67 (s, lH, exchangeable, NH), 4.63 (d, J
= 4.0 Hz, lH, 4-H), 4.59 (d, J = 5.0 Hz, lH, 1-H), 4.39 (t, J = 8.5 Hz, lH, 11-H) 3.98 (t, J =
8.5 Hz, lH, 11-H), 3.79 (8, 6H, 3',5'-OCH,), 3.11 (dd, J = 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal. calcd for Cl7H"FNO7:C,65.71;H, 4.90; N, 25 2.84. Found: C, 66.81; H, 4.94; N, 2.79.
~Z~
4'-Demethyl-4~-t2"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from methanol; mp 197-198C;[~'D-128 (c = 0.25, CHCl,); IR (KBr) 3500 (OH), 4480 (NH), 2890 (aliphatic C-H), 1755 (lactone), 1610, 1505 and (aromatic C=C) cm~'; 'H
NMR (CDCl,) ~ 7.04 (m, 2H, 3",6"-H), 6.76 (s, lH, 5H), 6.72 (m, lH 5"-H), 6.60 (t, J = 7.2 35 Hz, lH, 4"-H), 6.54 (s, lH, 8-H), 6.34 (s, 2H, 2~2~1 1 2',6'-H), 5.97 (d, J = 7.3 Hz, 2H, OCH,O), C.46 (s, lH, exchangeable, 4'-OH), 4.69 (d, J = 4.2 Hz, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, 1-H), 4.38 tt, J = 8.2 Hz, lH, 11-H) 4.10 (t, lH, exchan~eable, NH), 3.82 (t, J = 8.2 Hz, lH, 11-H) 3.79 (s, 6H, 3',5'-OCH,), 3.15 (dd, J = 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal.
calcd for C"H,4FNO,:C,65.71;H, 4.90; N, 2.84.
Found: C, 66.80; H, 4.95; N, 2.84.
4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 176-177C;[]~'D-100 (c - 0.8, CHCl,); IR (KBr) 3540 15 (OH), 3420 (NH), 2900 (aliphatic C-H), 1740 (lactone), 1610, 1500 1480 (aromatic C=C) cm~';
'HNMR (CDCl,) ~ 6.94 (t, J = 6.7, 2-H, 3",5",-H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8H), 6.49 (q, J = 2.2, 6.2 Hz, 2H, 2",6"-H), 6.33 (s, 2H, 2',6'-H), 5.96 (ABq, J = 1.2, 7.5 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.60 (d, 2H, 4-H and l-H), 4.37 (t, J s 7.5 Hz, lH, ll-H) 3.99 (t, J = 7.5 Hz, lH, ll-H), 3.79 (æ, 6H, 3',5'-OCH,), 3.73 (br, lH, exchangeable, NH), 25 3.13 (t, J ~ 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal. C~7H,~FNO7), C.H.N.
4'-Demethyl-4~-t3", 5"-difluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 180-183C;~]"D-132 (c = 0.33, CHCl,); IR (KBr) 3500 (OH), 3370 (NH), 2890 (aliphatic C-H), 1750 (lactone), 1620, 1590, 1500 and 1475 (aromatic C=C) cm~~; 'HNMR (CDCl,) ~ 6.75 (s, lH, 5-H), 6.54 (s, lH, 8-H), 6.32 (s, 2H, 2',6'-H), 6.23 (m, 2~
lH, 4l'-H), 6.07 (m, 2H, 2",6"-H), 5.98 (ABq, J =
1.3, 9.o Hz, 2H OCH2O), 5.45 (s, lH, exchangeable, 4'-OH), 4.61 (m, 2H, 4-H and 1-H), 4.39 (t, J = 8.5 Hz, lH, 11 H) 4.10 (d, J -6.1 Hz, lH, lH, exchangeable, NH), 3.85 (t, J =
8.5 Hz, lH, ll-H), 3.81 (s, 6H, 3',5'-OCH,), 3.08 (dd, J = 4.8, 14.1 Hz, lH, 2-H), 3.02 (m, lH, 3-H). Anal. C2,H2,NF2OI). C.H.N.
EXAM~LE 30 4'-Demethyl-4~-[3"-pyridylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 179-181 (dec); [~]2~D-99o (C=0.33, CHCI,); IR (KBr~ 3500 (OH), 3350 (NH), 2900 (aliphatic C-H), 1765 (lactone), 1575, 1500 and 1470 (aromatic C=C~N) cm': 'H NMR CDCI, ~ 8.08 (d, J=5.5 Hz, lH, 6"-H), 8.02 (br, lH, 2"-H), 7.16 (m, lH, 5"-H), 6.85 (dd, lH, 4"-H), 6.75 (g, lH, 5-H), 6.55 (s, lH, 8-H), 6.32 (s, 2H, 2', 6'H), 5.98 (AB~, J=1.3, 7.3 Hz, 2H, OCH2O), 4.65 (d, J=4.9 Hz, lH, 4-H), 4.60 (m, lH, 1-H), 4.20 (t, J=8.2 Hz, lH, ll-H), 3.90 (m, 2H, 11-H and NH), 3.80 (s, 6H, 3', 5'-OCH,), 3.18 (dd, J=5.0, 14.1 Hz, lH, 2-H), 3.03 (m, lH, 3-H).
Anal. (C,~H~,N,O,) 1/2 H~O, C.H.N.
4'-Demethyl-4~-~2"-pyridylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 215-218 (dec); [a~ -82 (C=0.33, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2950 (aliphatic C-H), 1760 (lactone), 1690, 1645, 1600 and 1460 (aromatic C=C=N) cm~': 'H NMR CDCI, ~ 8.11 (d, lH, 4"-H), 7.45 (m, lH, 4"-H), 6.81 (s, lH, 5-H), 6.67 (m, lH, 5"-H), 6.55 (s, lH, 8-H), 6.45 (d, lH, 3"-20~42~l1 H), 6.34 (s, 2H, 2', 6'-H3, 5.97 (AB~, J-1.3, 6.7 Hz, 2H, OCH,O), 5.43 (br, lH, exchangeable, 4'-OH), 5.35 (m, lH, exchangeable, NH), 4.60 ~d, J=4.2 Hz, lH, 4-H), 4.24 (m, 2H, l-H and llNH) 3.85 (m, lH, llH), 3.78 (s, 6H, 3', S'-OCH,), 3.05 (m, 2H, 2-H and 3-H).
Anal. (C26H,~N,O~), 1/2 H,O, C.H.N.
4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol-ether; mp 243-246 (dec); [~]"n~l79 (C=0.5, CHCI,); IR (RBr) 3460 (OH~, 3380 (NH), 2900 (aliphatic C-H), 1775 (lactone), 1605, 1510 and 1480 (aromatic C=C=N) cm-': 'H NMR CDCI, ~ 8.46 (d, J=2.9 Hz, 2"-H), 7.97 (m, lH, 4"-H), 7.65 (m, lH, 7"-H), 7.48 (m, 2H, 5", 6"-H), 6.g9 (d, J=2.9 Hz, 8'-H), 6.76 (s, lH, 5-H), 6.57 (5, lH, 8-H), 6.35 (s, 2H, 2', 6'-H), 5.99 (AB~, J=1.1, 8.0 Hz, 2H, OCH,O), 5.48 (s, lHj exchangeable, 4'-OH), 4.78 (d, J=4.8 Hz, lH, l-H), 4.45 (t, lH, ll-H), 4.23 (d, lH, exchangeable, NH), 3.99 (t, lH, ll-H), 3.81 (s, 6H, 3', 5'-OCH,), 3.15 (m, 2H, 2-H and 3-H).
Anal. (C,oH2,N,O7) 1/2~,0, C.H.N.
~a~LE_~l 4'-Demethyl-4B-[3 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous powder from ether: mp 163-166C; IR (KBr) 3480 (OH), 3380 (NH), 2900 (aliphatic CH), 1750 (lactone), 1590, 1475 (aromatic C=C) cm-';'H NMR (CDCl,) ~
7.05(t,J=8Hz,lH,5"-H), 6.78 (s,lH,5-H), 6.52(s,1H,8-H), 6.33(s,2H,2',6'-H), 6.24 (dd,J=2.2, 8Hz,lH,4"-H), 6.15(dd,J=1.7, 2 ~ 1 8H~,lH,6"-H), 6.07(t,J=2.2Hz,1H,2"-H), 5.97(d,J=4.4Hz,2H, OCH,O), 5 . 43(s, exchangeabl e), 4 . 82 (s, excha~geable), 4.65 (d,J=3.9Hz,lH,4-H), 4.58(d,J=4.8Hz,lH,l-H), 4.37(t,J=8.7Hz,lH,11-H), 4.0(t,J=8.7Hz,lH,ll-H), 3.79(s,6H,3'5'-OCH,), 3.1(dd, J=4.8, 14.1 Hz, lH, 2-H), 2.98(m,1H,3-H); MS, m/z=491 (m+). Anal. Calcd for C"H2,NO,.H20: C,63.65jH,5.30. Found: C,63.35;
H,5.44.
EXAMPLE_34 4'-Demethyl-4~-[2 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous crystals from ether: mp 175C; IR (KBr) 3360 (OH, NH), 2900 (aliphatic C-H), 1750 (lactone, 1600, 1475 (aromatic C=C) cm-';'H NMR (CDCI,) ~ 6.88(t,J=7.4H2,lH,4"-H), 6.78(s,1H,5-H), 6.65(m,2H,3",6"-H), 6.5(m,2H,8-H,5"-H), 6.35(s,2H,2'6'-H), 5.96(AB~
J=1.2Hz,3.5Hz,2H,OCH,O), 5.44(s.exchangeable), 5.10(s, exchangeable), 4.67(d,J=4Hz,lH,4-H), 4.61(d,J=4.8Hz,lH,l-H), 4.38(t,J=8.5Hz,lH,ll-H), 3.98(t,J=8.5Hz,lH,ll-H), 3.79(s,6H,3',5'-OCH,), 3.24 (dd,J=4.8,14Hz,lH,2-H), 3.02(m,1H,3-H), MS, m/z-491(m+). Anal. Calcd for C,7H"NO,:
C,65.99; H,5.09. Found: C, 65.85; H,5.18.
EXAMPLE ~
4'-Demethyl-4B-~4 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous powder from ether mp 162-165C;IR
(RBr) 3525 (OH), 3345 (NH), 3010 (aromatic CH), 2900 (aliphatic CH), 1745 (lactone), 1600, 1475 (aromatic C=C) cm~';'H NMR (DMSO d~, D,O exchange) ~ 6.69~s,1H,5-H), 6.55(s,4H,2",3",5",6"-H), 6.48(s,1H,8-H), 6.23(s,2H,2'6'-H), 37 2~2~
5.94~d,J=9.7Hz,2H,O-cH2-O), 4.68(d,J=4.3Hz, lH, 4 H), 4.46(d,J=5.4HZ,lH,l-H), 4.29(t,J=7.6,1H,ll-H), 3.76(t,J=7.6Hz,lH,ll-H), 3.61(s,6H,3',5'-OCH,), 3.28(dd,J=5.4,15.8Hz,lH,2~H), 2.95(m,1H,3-H).
ExAMpLE ~
4'~Demethyl-4B-[2"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 253-255C;[~]"D-90 (c = 1.0, CHCl,); IR (KBr) 3500 (OH), 3450 (NH), 2895 (aliphatic CH), 1751 (lactone), 1590, 1500 1472 (aromatic C=C) cm~';
'H NMR (CDCl,) 7.31 (dd, J = 1.4, 7.9 Hz, lH, 3"-H), 7.18 (t, J = 8.8, Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.73 (t, J = 9.0 Hz, 4l'-H) 6.58 (d, J
= 8.2 Hz, lH, 6"-H), 6.54 (s, lH, 8-H), 6.35 (s, 2H, 2',6'-H), 5.98 (ABq, J = 1.2, 4.2 Hz, 2H
OCOCH,O), 5.44 (s, lH, exchangeahle, 4'-OH), 4.73 (t, J = 4.9 Hz, lH, 4-H) 4.64 (d, J - 4.9 Hz, lH, l-H), 4.49 (d, J = 6.0 Hz, lH, exchangeable, NH), 4.36 (t, J = 8.3 Hz, lH, ll-H), 3.91 (t, J
= 8.3, Hz, lH, ll-H), 3.80 (s, 6H, 3,'5'-OCH,~, 3.17 (dd, J = 4.8, 14.0 Hz, lH, 2-H), 3.04 ~m, lH, 3-H). Anal. C"H"CINO, C.H.N.
EXA~ Z
4'--Demethyl-4~-[3"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 174-176C;[~]"D-112 (C = 1.0, CHCl,); IR (KBr) 30 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1752 (lactone), 1580, and 1452 (aromatic C=C) cm~'; 'H
NMR (CDCl,) 7.12 (t, J = 8.1, Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.74 (dd, J = 1.0, 8.1 Hz, lH 4"-H), 6.53 (br, 2H, 8-H and 2"-H), 6.42 (dd, J =
35 1.6, 6.5 Hz, lH, 6"-H), 6.33 (s, 2H, 2',6'-H), 2~2~
5.97 (ABq, J 5 1.0~ 8.7 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.66 (br, lH, 4-H), 4.59 (d, J - 4.8 Hz, lH, 1-H), 4.39 (t, J = 7.7 Hz, lH, ll-H), 3.99 (t, J = 7.7 Hz, lH, 11-H) 3.96 (br, lH, exchangeable, NH~, 3.79 ts, 6H, 3,'5'-OCH,), 3.11 (dd, J - 5.8, 14.0 Hz, H, 2-H), 3.01 (m, lH, 3-H). Anal. C"H2,CINO,) C.H.N.
4'-Demethyl-4B-~4"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 25~-255Cj[~]"D-125 (c = 0.75 CHCl,); IR (KBr) 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1758 (lactone), 1605, 1590 and 1475 (aromatic C=C) cm-15 ; H NMR (CDCl,) 8 7.17 (d, J - 8.7, Hz, 2H, 3",5"-H), 6.74 (5, lH, 5-H), 6.53 (s, lH, 8-H), 6.48 (d, J = 8.7 Hz, 2H, 2",6"-H), 6.32 (s, 2'6'-H), 5.96 (ABq, J = 1.0, 6.8 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.63 (d, J =
4.2 Hz, lH, 4-H), 4.59 (d, J = 4.9 Hz, lH, 1-H), 4.38 (t, J 2 8.0 Hz, lH, ll-H) 3.96 (t,J =
8.0 Hz, lH, ll-H), 3.79 (s, 6H 3,'5'-OCH,), 3.12 (dd, J = 4.9, 14.1 Hz, H, 2-H), 2.99 (m, lH, 3-H). Anal. C"Hl,CINOT) C.H.N.
~ EL~
4'-Demethyl-4B-t3"-bromoanilinyl~-4-desoxypodophyllotoxin.
Crystals from methanol/ether; mp 177-179C;t~]"D-105 (c = 1, CHCl,); IR (RBr) 3450 (OH), 3340 (NH), 2900 (aliphatic CH), 1740 (lactone), 1590, 1500 and 1475 (aromatic C=C) cm~
'; 'H NMR (CDCl,) 8 7.07 (t, J = 8.0, Hz, lH, 5"-H), 6.90 (dd, J = 0.9, 7.9 Hz, lH, 4"-H), 6.75 (s, lH, 5-H), 6.70 (br, lH, 2"-H), 6.53 (s, lH, 8-H), 6.47 (dd, J = 1.7, 8.3 Hz, lH 6"-H), 6.33 2~2~:~
(s, 2H, 2',6'-H), 5.97 (dd, J = 1.2, 9.3 Hæ, 2H, OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.65 (d, J = 4.2 Hz, lH, 4-H), 4.6Q (d, J = 4.8 Hz, lH, 1-H), 4.39 (t, J = 7.3 Hz, lH, 11-H) 3.96 (t, J = 7.3 Hz, lH, 11-H), 3.90 (d, J = 6.2, Hz, lH, exchangeable, NH), 3.R0 (s, 6H, 3,'5'-OCH,), 3.10 (dd, J = 4.9, 14.0 Hz, lH, 2-H), 3.02 (m, lH, 3-H). Anal. C"H"BrNO7) C.H.N.
4'-Demethyl~4~-[4"-bromoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ethanol; mp 227-230C,[~]"D-110 (c = 0.5, CHCl,); IR (KBr) 3500 (OH), 3330 (NH), 2900 (aliphatic CH), 1755 (lactone), 1605, 1590 and 1475 (aromatic C-C) cm~
'; 'H NMR (CDCl,) ~ 7.30 (d, J = 8.9, Hz, 2H, 3",5"-H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8-H), 6.44 (d, J = 8.9 Hz, 2H, 2",6"-H), 6.32 (s, 2H, 2',6'-H), 5.98 (A8q, J = 1.3, 8.3 Hz, 2H, OCH~O), 5-42 (5, lH, exchangeable, 4'-OH), 4.62 (m, 2H, 4-H and 1-H), 4.36 (t, J = 8.5 Hz, lH, 11-H), 3.95 (t, 8.5 Hz, 11-H), 3.86 (d, J = 708, lH, exchan~eable, NH), 3.73 (s, 6H, 3,'5'-OCH,), 3.11 (dd-, J s 4.8, 14.1 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal. C~H,4BrNO,) C.H.N.
EX~MP~E 41 4'-De~ethyl-4B-t4"-iodoanilinyl]-4-desoxypodophyllotoxin.
Cry~tals from ethanol; mp 198-200C(dec.);~]"D-111 (c = 0.5, CHCl,); IR (KBr) 3540 (OH), 3420 (NH), 2900 (aliphatic CH), 1770 (lactone), 1610, 1585 and 1480 (aromatic C=C) cm~
'; 'H NMR (CDCl,) ~ 7.46 (d, J = 8.8 Hz, 2H, 2",6"-H), 6.32 (s, 2H, 2',6' H), 5.59 (ABq, J =
1.1, 8.9 Hz, 2H, OCHiO), 5.44 (s, lH, 20~2~
exchangeabl~, 4'-OH), 4.62 (~, lH, 4-H), 4.58 td, J = 4.9 Hz, lH, 1-H), 4.34 (t, J = 8.5 Hz, lH, l1-H), 3.94 (m, 2H, 11-H and NH), 3.78 (s, 6H, 3~,5~-OCH,), 3.09 (dd, J = 4.9, 14.1 Hz, lH, 2-H), 2.99 (m, lH, 3-H). Anal. C"H"BrNO,) C.H.N.
EXAMP~ 42 - 44 (Scheme IX) Podophyllotoxin (500mg, 1.2 mmol) was dissolved in dry dichloromethane (lOml) and kept at 0C. Hydrogen bromide gas was introduced into the solution for 45 min., after which time the solvent was evaporated in vacuo, anhydrous tetrahydrofuran (15 ml), anhydrous barium carbonate (474 mg, 2.4 mmol) and the appropriate hydroxyaniline ~142mg, 1.3 mmol) was added. The mixture stood at room temperature overnight, and then was filtered and concentrated. The crude product was purified using column chromatography tsilica gel 45 g with dichloromethane-acetone-ethyl acetate 100:5:5 as an eluant). Theproducts (42-44) obtained in the examples had the characteristic~ listed below.
~Z
4B-~2"-hydroxyanilinyl]-4-de60xypodophyllotoxin, Amorphous crystals from ether: mp 145-148C; IR (KBr) 3480 (OH), 3410 (NH), 2900 (aliphatic CH), 1760 (lactone), 1580, 1475 (aromatic C=C) cm'; H NMR (CDCl,) ~ 6.90 (t,J =
6.6 Hz, lH, 4"-H), 6.78 (s, lH, 5-H), 6.65 (~, 2H, 3",6"-H), 6.53 (m, 2H,8-H, 5"-H), 6.34 (s, 2H 2',6'H), 5.96 (ABq, J = 1.0, 3.5 Hz,2H, OCH,O), 5.02 (s, lH, exchangeable, 2"-OH), 4.68 (m, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, l-H), 2~2~ ~
4.38 (t, J = 8.6 Hz, lH, ll-H), 4.33 (m, lH, exchangeable, NH), 4.00 (t, J = 8.6 Hz, lH, llH), 3.82 (s, 3H, 4'-OCH,), 3.76 (s, 6H, 3',5'-OCH,), 3.25 ~dd, J = 5.1, 14.0 Hz, lH, 2-H), 3.05 (m, lH, 3-H). MS, m/z = 505 (m+). Anal.
(C"H"NOl 3/2 H2O) C.H.
4B-[3"-hydroxyanilinyl]~-Desoxypodophyllotoxin.
Amorphous powder from ether, mp 148-150C; IR(RBr) 3370 (OH,NH), 2900 (aliphatic CH), 1760 (lactone), 158S, 1475 (aromatic C=C) cm~'; 'H NMR (CDCl,) ~ 7.05 (t, J = 8.0 Hz, lH, 5"-H), 6.77 (8, lH, 5H), 6.52 (s, lH, 8-H), 6.32 (s, 2H, 2' 6'-H), 6.25 (dd, J = 2.2, 8.0 Hz, lH, 4"-H), 6.14 (dd, J = 2.2, 8.0 Hz, lH, 6"-H), 6.05 (t = 2.2 Hz, lH, 2"-H), 5.96 (ABq, J = 1.3, 3.8 Hz, 2H OCH,O), 4.64 (d, J = 3.9 lH, 4-H), 4.49 (d, J = 5.0, Hz, lH, l-H), 4.4 (t, J = 8.7 Hz, lH, 11-H), 4.03 (t, J = 8.7 Hz, lH, 11-H), 3.81 (s, 3H, 4'0CH,), 3.76 (s, 6H, 3',5'-OCH,), 3.18 (s, 6H, 3',5'-OCH,), 3.18 (dd, J = 5.0 Hz, 14.0 Hz, lH,2-H), 3.02 (m, lH, 3-H); MS, m/z =
505 (m+). Anal. (C,tH"N~, H,O) C.H.
~XAX~J~ 44 4B-[4"-hydroxyanilinyl]-4-de~oxypodophyllotoxin.
Crystals from chloroform; mp 145-150C;
IR(KBr) 3310 (OH,NH), 3010 (aromatic CH), 2900 (aliphatic CH), 1730 (latone), 1575, 1475 (aromatic C=H) cm-'; 'H NMR (CDCl,) D,O exchange) 6.75 (d, J = 8.3 Hz, 3H, 5-H, 3",5"-H), 6.53 (s, lH, 8H), 6.45 (d, J - 8.3 Hz, 2H, 2",6"-H), 6.23 (s, 2H 2'6'-H), 5.95 (ABq, J = 1.0, 4.0 Hz, 2H
OCH,O), 4.60 (d, J = 4.2 Hz, lH, 4-H), 4.57 (d, J
= 4.6 Hz, lH, 1-H), 4.38 (t, J = 6.0 Hz, lH, 11-42 2 a~42 H), 4.05 (t, J = 6.0 Hz, lH, llH) 3.83 (s, 3H, 4-OCH,3, 3.75 (s, 6H, 3',5'-OCH,), 3.18 (dd, J =
4.6 Hz, 14.0 Hz, lH,2-H), 3.0 (m, lH, 3-H).
Anal. (C,~H,7NO, 1/2 H~O) C.H.
Isolation of Human DNA Topç~somerase II.
Human DNA topoiso~erase II was isolated from peripheral blast cells of a patient with acute leukemia. The isolation procedure is described in Thurston, L., Imakura, Y., Haruna, M., Li, Z. C., Liu, S. Y., and Lee, K. H., J.
Med. Chem., 31, COMPLETE (1988) and is a partial combination of the procedure described in Goto, T., Laiapia, P. and Wang, J., J. Biol Chem~, 259, 10422 (1984) and Halligan, B., Edwards, X., and Liu, L., 2l_~inL-_She~., 260, 2475 (1985) which are herein specifically incorporated by reference.
Preparations of Druas.
Drugs were dissolved in Me,SO at a concentration of 20 mM as the stock solution and diluted before use with water to the desired concentration of each druq.
DNA TQpoiso~e~ase II Assay.
The P4 unknotting reaction was a modification Or the procedure described by Hseih, T., ~ iol~ m~, 258, 8413 ~1985), which i8 herein specifically incorporated by reference.
The reaction mixture (20 ~L), which contained 50 mM HEPES, pH 7.0, 50 mM KCI, 100 mM
NaCl, 0.1 mM EDTA, 10 mM MgCl" 1.0 mM ATP, 50 ~g/mL bovine serum albumin, 0.4 ~g P4 knotted DNA, and enzyme, was incubated with or without drugs.
2 ~
The reaction mixture was incubated at 37 C for 30 min and terminated by adding 5.0 ~1 of a stop solution 52~ sodium dodecyl sulfate, 20% glycerol, 0. 05~ bromophenol blue). These samples were loaded onto a 1% agarose gel and electrophoresed at 55 V overnight with an electrophoresis buffer that contained 90 mM
Tris-boric acid, pH 8.3, and 2.5 mM EDTA. At completion, the gel was stained in 0.5 ~g/mL of ethidium bromide. Then a photograph was taken of the DNA bands visualized with fluorescence induced by a long-wavelength W lamp. The data reported in Table 1 reflect a lOo ~M drug concentration.
K-SpS Precipi~ation Assay_or P~otein-DNA
CompLç~çs.
The intracellular formation of covalent topoisomerase II-DNA complexes was quantitated using the potassium SDS precipitation assay, a procedure adapted from the method described in Rowe, T.C., Chen, G. L., ~siang, Y. H., and Liu, L., Cancer RÇs., 46, 2021 (1986) (hereinafter Rowe et al.), which is herein specifically incorporated by reference. KB ATCC cells were prelabeled with 0.05 mCi/ml 14C-thymidine ~specific activity 50.5 mCi/mmol) for 18 hr. A
~inal concentration of 5xlO' cells/sample were treated with 10 ~M of the drugs at 37 C for 1 hr and proceeded according to the procedure described by Rowe et al. to detect the protein-linked DNA levels.
It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the 20~42~1 modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.
TABLE I
BIOLOGICAL EVALUATION OF 4'-DEMETHYL-4-ALKYLAMINO-PODOPHYLLOTOXIN ANALOGUES
R
< C~
O
o H3CO~OCH3 OH DNAC~LLULAR
TOPO-PROTEIN-CYTO- ISOMERASE II DNA
TOXICITY- AC~IVITYCOMPLEX
COMPOUND R ED,~KB(~g/ml) % INHIBITION~ FORMATIO~%
.
Etoposlde O ~ 2a +++ 100.0 Example:
1 B-OH 0.34 ++ 42.2 2 ~-OH 0.045 + 3.3 3 B-NH2 1.0 ++++36.4 4 ~-NH, 0.42 ~ 8.0 ~-NHC~,CH,OH 1.6 ++++121.4 6 ~-NHCH,CH,OH 710.0 - 0.0 7 B-NHCH,CH,CH, <0.4 ++ 69.7 8 B-NHCH,CH,OCH, >4.0 +++110.8 9 ~-NHCH,CH~CH, 3.4 +++ 84.1 B-NHCH,CH(OH)CH, 3.6 ++++ 167.2 11 8-NHCH(CH,)CH,OH 2.3 ++++ 161.7 12 B-NHCH,CH,CH,OH 4.0 ++ 89.2 13 B-OCH,CH,NH, 0.1 ++++300.0 14 B-OCH,CH,OH 0.7 ++ 50.0 ' ED,o is the concentration of drug which affords 50%
reduction in cell number after 3 days incubation.
+, ++, +++, ++++ and - denote for 25%, 50%, 75~, >75%, and 0% inhibition.
' Relative activities of cellular protein-DNA complex formation in KB ATCC tissue culture cells measured at 10 ~M drug concentration as compared the complex formed by 10 ~M of etoposide.
20~211 TABLE II
COMPOUNDS WITH POTENT TOPOISOMERASE II
INHIBITORY ACTIVITY
DNA
TOPOISOMERASE DNA TOPOISOMERASE
II
INHIBITORY ACTIVITY' COMPOUNDS (RELATIVE POTENCY) .. .. _ Etoposide 1.0 2.0 - 6.0 16 8.0 17 2.0 18 2.0 '~e o~
0.
OH
15 R~Cl 16 R,~R,=R,-R,=Br,R,-H
17 R,-OH, R,~R,-R,-Br,R,-H
18 R,~OH, R,-R,-R4=R,~Br Several different concentrations of tested compounds were employed for the determination of their potency. The relative potency with respect to etoposide as expressed in Table I was the relative concentration of compounds tested to achieve the same degree inhibition by etoposide in the range of 25 to 400 ~M.
2~21~
q7 Table ILE~
~iological Evaluation of 4-Demethyl-4B(arylamino)-4-desoxy Podopyllotoxin It~
<0~O
O
CH~O ~ OC~
OH
Compound R, DNA Cellular Cytotoxicity Topoisomerase II Protein-DNA IDso ~
Activity Complex (uM) % Inhi~ition Formation Etoposide +++ 100 0.2 19 ~ ~ +++ 243 0.71 20 ~ +++~ 211 0.64 21 ~ ~ +++ 137 0.6g 22 ~ ~ eo~c~ +++ 207 <0.10 23 ~ +++ 140 0.66 24 .~? ++++ 164 <1 0 oc~
~ ++ 180 <1.0 ~6 ~ +++ 158 0.23 2n~4~
Compound R, DNA Cellular Cytotoxicity Topoisomerase II Protein-DNA ID50 K~
Activity Complex (uM) % Inhibition Formation 27 ~ ~ ++ 121 0.25 28 ~f ++++ 213 0.24 29 ~ ~ ++ 115 1.08 f ~ +++ 148 0.24 31 ~ ~ ++ 97 0.71 >~
32 ~ ++ 123 <1.0 o~l 33 ~ ++++ 290 0.45 34 ~ ~ ++++ 151 4.54 35~ ~ o~ +++ 211 2.26 tl 36 ~ + 32 2.34 2~4211 Compound R, DNA Cellular cytotoxicity Topoisomerase II Protein-DNA ID, Activity Complex (uM)' % Inhibition~ FormationC
37 ~ ~ ++ 51 2.29 38 ~ ~ cl +++ 99 0.22 ~1 ,--39 ~ ~ ~ + 62 2.36 ~ ' +~ 179 0.22 41 ~ + 6~ 0.34 ' ID,o is the concentration of drug which affords 50% reduction in cell number ater 3 days incubation.
~ Activities of cellular protein-DNA complex formation in KB
ATCC tissue culture cells relative to Etoposide.
' +,++,+++,++++ denote 0-24%,25-49%,50-74%, and 275% inhibition respectively.
204~211 Tabl~ IV
Biological Evaiuation 014~ (arylamino)-4-desoxypodophyl~otoxins <~0 O
H3CO~OCH3 OcH3 Compound R DNA Cellubr Cytoloxicily Topoisolnerase l l prolein DNA 1~5O KB (~lM) Activity Compl~x Formatbn ~ Inhibitbn Etoposlde ~ 100 0.2 OH , 42 NH~ ~ 6 4.11 OH
43 NH~ ~ 37 0.31 44 ~ 21 0.31 2~421~
Scherre I
OH ~ ~
<~ ~ H~O X~0 H3CO~ocH3 H3COJ~OCH3 H3CO~OCH~
~2) fl . r~ OH
Scherne 11 OH SO,CH, CBz - Cl ~ CH3S02C1 ~
<~.""( <O~V ~
O _ O
H3COJ~OCH~ H3CO~OCH3 OCBZ Oc82 N;~N3 < ~O 2, < ~O
H3CO~oCH3 H3CO~OcH3 ~3) R ~ B-NH2 ~) R ~ NH2 20~211 Scheme 111 NHR
RNH2 < ~
O
HaCO OCH3 (S 12) Scheme IV
OCH2CH2Elr OCH2,CH2N3 HOCH2CH2Br ~1~ NaN3 Il <0~ "~ ~ ~
H3COJ~oCH3 H3COJ~oCH3 OH OH
~V~ ~) OcH2cH2NH2 H2 <~
H3CO~OCH3 OH
(13) 2~4~2~
SCheme V
OCH2CH2C~I
HOCH2cH20H < ~
o .."( o H3CO~ oc~3 OH
Schen~ Vl a o~o<O~o a - o - ' ~
H3CO~OCH3 ~ 15 ) Schem~ Vll Br2 < ~
RJ ' O ~ 16 ) RlnR2~R3~Rs~B~, R~.H
R~ ~, Rs ( 17 ) R~.OH, R2.R~.Rs.Br, R3-H
H3CO~OCH3 ~1B ) R~.OH, R2.~3.R~.Rs..Br 20~23 ~
Schernl~ Vlil I~HAr H8r NH2A~ O ~
11 <~ 0 ~ O
H3CO J~ OCH3 ( 1~1 41 ) Schen~ IX
Br NHAr HBr<~,~ NH2Ar < ~O
. ""~ O "'~( H3CO~ocH3 H3COJ~H3 (42-~4)
~Z~
4'-Demethyl-4~-t2"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from methanol; mp 197-198C;[~'D-128 (c = 0.25, CHCl,); IR (KBr) 3500 (OH), 4480 (NH), 2890 (aliphatic C-H), 1755 (lactone), 1610, 1505 and (aromatic C=C) cm~'; 'H
NMR (CDCl,) ~ 7.04 (m, 2H, 3",6"-H), 6.76 (s, lH, 5H), 6.72 (m, lH 5"-H), 6.60 (t, J = 7.2 35 Hz, lH, 4"-H), 6.54 (s, lH, 8-H), 6.34 (s, 2H, 2~2~1 1 2',6'-H), 5.97 (d, J = 7.3 Hz, 2H, OCH,O), C.46 (s, lH, exchangeable, 4'-OH), 4.69 (d, J = 4.2 Hz, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, 1-H), 4.38 tt, J = 8.2 Hz, lH, 11-H) 4.10 (t, lH, exchan~eable, NH), 3.82 (t, J = 8.2 Hz, lH, 11-H) 3.79 (s, 6H, 3',5'-OCH,), 3.15 (dd, J = 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal.
calcd for C"H,4FNO,:C,65.71;H, 4.90; N, 2.84.
Found: C, 66.80; H, 4.95; N, 2.84.
4'-Demethyl-4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 176-177C;[]~'D-100 (c - 0.8, CHCl,); IR (KBr) 3540 15 (OH), 3420 (NH), 2900 (aliphatic C-H), 1740 (lactone), 1610, 1500 1480 (aromatic C=C) cm~';
'HNMR (CDCl,) ~ 6.94 (t, J = 6.7, 2-H, 3",5",-H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8H), 6.49 (q, J = 2.2, 6.2 Hz, 2H, 2",6"-H), 6.33 (s, 2H, 2',6'-H), 5.96 (ABq, J = 1.2, 7.5 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.60 (d, 2H, 4-H and l-H), 4.37 (t, J s 7.5 Hz, lH, ll-H) 3.99 (t, J = 7.5 Hz, lH, ll-H), 3.79 (æ, 6H, 3',5'-OCH,), 3.73 (br, lH, exchangeable, NH), 25 3.13 (t, J ~ 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal. C~7H,~FNO7), C.H.N.
4'-Demethyl-4~-t3", 5"-difluoroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 180-183C;~]"D-132 (c = 0.33, CHCl,); IR (KBr) 3500 (OH), 3370 (NH), 2890 (aliphatic C-H), 1750 (lactone), 1620, 1590, 1500 and 1475 (aromatic C=C) cm~~; 'HNMR (CDCl,) ~ 6.75 (s, lH, 5-H), 6.54 (s, lH, 8-H), 6.32 (s, 2H, 2',6'-H), 6.23 (m, 2~
lH, 4l'-H), 6.07 (m, 2H, 2",6"-H), 5.98 (ABq, J =
1.3, 9.o Hz, 2H OCH2O), 5.45 (s, lH, exchangeable, 4'-OH), 4.61 (m, 2H, 4-H and 1-H), 4.39 (t, J = 8.5 Hz, lH, 11 H) 4.10 (d, J -6.1 Hz, lH, lH, exchangeable, NH), 3.85 (t, J =
8.5 Hz, lH, ll-H), 3.81 (s, 6H, 3',5'-OCH,), 3.08 (dd, J = 4.8, 14.1 Hz, lH, 2-H), 3.02 (m, lH, 3-H). Anal. C2,H2,NF2OI). C.H.N.
EXAM~LE 30 4'-Demethyl-4~-[3"-pyridylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 179-181 (dec); [~]2~D-99o (C=0.33, CHCI,); IR (KBr~ 3500 (OH), 3350 (NH), 2900 (aliphatic C-H), 1765 (lactone), 1575, 1500 and 1470 (aromatic C=C~N) cm': 'H NMR CDCI, ~ 8.08 (d, J=5.5 Hz, lH, 6"-H), 8.02 (br, lH, 2"-H), 7.16 (m, lH, 5"-H), 6.85 (dd, lH, 4"-H), 6.75 (g, lH, 5-H), 6.55 (s, lH, 8-H), 6.32 (s, 2H, 2', 6'H), 5.98 (AB~, J=1.3, 7.3 Hz, 2H, OCH2O), 4.65 (d, J=4.9 Hz, lH, 4-H), 4.60 (m, lH, 1-H), 4.20 (t, J=8.2 Hz, lH, ll-H), 3.90 (m, 2H, 11-H and NH), 3.80 (s, 6H, 3', 5'-OCH,), 3.18 (dd, J=5.0, 14.1 Hz, lH, 2-H), 3.03 (m, lH, 3-H).
Anal. (C,~H~,N,O,) 1/2 H~O, C.H.N.
4'-Demethyl-4~-~2"-pyridylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol; mp 215-218 (dec); [a~ -82 (C=0.33, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2950 (aliphatic C-H), 1760 (lactone), 1690, 1645, 1600 and 1460 (aromatic C=C=N) cm~': 'H NMR CDCI, ~ 8.11 (d, lH, 4"-H), 7.45 (m, lH, 4"-H), 6.81 (s, lH, 5-H), 6.67 (m, lH, 5"-H), 6.55 (s, lH, 8-H), 6.45 (d, lH, 3"-20~42~l1 H), 6.34 (s, 2H, 2', 6'-H3, 5.97 (AB~, J-1.3, 6.7 Hz, 2H, OCH,O), 5.43 (br, lH, exchangeable, 4'-OH), 5.35 (m, lH, exchangeable, NH), 4.60 ~d, J=4.2 Hz, lH, 4-H), 4.24 (m, 2H, l-H and llNH) 3.85 (m, lH, llH), 3.78 (s, 6H, 3', S'-OCH,), 3.05 (m, 2H, 2-H and 3-H).
Anal. (C26H,~N,O~), 1/2 H,O, C.H.N.
4'-Demethyl-4~-[3"-quinolinylamino]-4-desoxypodophyllotoxin.
Crystals from ethanol-ether; mp 243-246 (dec); [~]"n~l79 (C=0.5, CHCI,); IR (RBr) 3460 (OH~, 3380 (NH), 2900 (aliphatic C-H), 1775 (lactone), 1605, 1510 and 1480 (aromatic C=C=N) cm-': 'H NMR CDCI, ~ 8.46 (d, J=2.9 Hz, 2"-H), 7.97 (m, lH, 4"-H), 7.65 (m, lH, 7"-H), 7.48 (m, 2H, 5", 6"-H), 6.g9 (d, J=2.9 Hz, 8'-H), 6.76 (s, lH, 5-H), 6.57 (5, lH, 8-H), 6.35 (s, 2H, 2', 6'-H), 5.99 (AB~, J=1.1, 8.0 Hz, 2H, OCH,O), 5.48 (s, lHj exchangeable, 4'-OH), 4.78 (d, J=4.8 Hz, lH, l-H), 4.45 (t, lH, ll-H), 4.23 (d, lH, exchangeable, NH), 3.99 (t, lH, ll-H), 3.81 (s, 6H, 3', 5'-OCH,), 3.15 (m, 2H, 2-H and 3-H).
Anal. (C,oH2,N,O7) 1/2~,0, C.H.N.
~a~LE_~l 4'-Demethyl-4B-[3 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous powder from ether: mp 163-166C; IR (KBr) 3480 (OH), 3380 (NH), 2900 (aliphatic CH), 1750 (lactone), 1590, 1475 (aromatic C=C) cm-';'H NMR (CDCl,) ~
7.05(t,J=8Hz,lH,5"-H), 6.78 (s,lH,5-H), 6.52(s,1H,8-H), 6.33(s,2H,2',6'-H), 6.24 (dd,J=2.2, 8Hz,lH,4"-H), 6.15(dd,J=1.7, 2 ~ 1 8H~,lH,6"-H), 6.07(t,J=2.2Hz,1H,2"-H), 5.97(d,J=4.4Hz,2H, OCH,O), 5 . 43(s, exchangeabl e), 4 . 82 (s, excha~geable), 4.65 (d,J=3.9Hz,lH,4-H), 4.58(d,J=4.8Hz,lH,l-H), 4.37(t,J=8.7Hz,lH,11-H), 4.0(t,J=8.7Hz,lH,ll-H), 3.79(s,6H,3'5'-OCH,), 3.1(dd, J=4.8, 14.1 Hz, lH, 2-H), 2.98(m,1H,3-H); MS, m/z=491 (m+). Anal. Calcd for C"H2,NO,.H20: C,63.65jH,5.30. Found: C,63.35;
H,5.44.
EXAMPLE_34 4'-Demethyl-4~-[2 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous crystals from ether: mp 175C; IR (KBr) 3360 (OH, NH), 2900 (aliphatic C-H), 1750 (lactone, 1600, 1475 (aromatic C=C) cm-';'H NMR (CDCI,) ~ 6.88(t,J=7.4H2,lH,4"-H), 6.78(s,1H,5-H), 6.65(m,2H,3",6"-H), 6.5(m,2H,8-H,5"-H), 6.35(s,2H,2'6'-H), 5.96(AB~
J=1.2Hz,3.5Hz,2H,OCH,O), 5.44(s.exchangeable), 5.10(s, exchangeable), 4.67(d,J=4Hz,lH,4-H), 4.61(d,J=4.8Hz,lH,l-H), 4.38(t,J=8.5Hz,lH,ll-H), 3.98(t,J=8.5Hz,lH,ll-H), 3.79(s,6H,3',5'-OCH,), 3.24 (dd,J=4.8,14Hz,lH,2-H), 3.02(m,1H,3-H), MS, m/z-491(m+). Anal. Calcd for C,7H"NO,:
C,65.99; H,5.09. Found: C, 65.85; H,5.18.
EXAMPLE ~
4'-Demethyl-4B-~4 " -hydroxyanilinyl]-4-desoxypodophyllotoxin.
Amorphous powder from ether mp 162-165C;IR
(RBr) 3525 (OH), 3345 (NH), 3010 (aromatic CH), 2900 (aliphatic CH), 1745 (lactone), 1600, 1475 (aromatic C=C) cm~';'H NMR (DMSO d~, D,O exchange) ~ 6.69~s,1H,5-H), 6.55(s,4H,2",3",5",6"-H), 6.48(s,1H,8-H), 6.23(s,2H,2'6'-H), 37 2~2~
5.94~d,J=9.7Hz,2H,O-cH2-O), 4.68(d,J=4.3Hz, lH, 4 H), 4.46(d,J=5.4HZ,lH,l-H), 4.29(t,J=7.6,1H,ll-H), 3.76(t,J=7.6Hz,lH,ll-H), 3.61(s,6H,3',5'-OCH,), 3.28(dd,J=5.4,15.8Hz,lH,2~H), 2.95(m,1H,3-H).
ExAMpLE ~
4'~Demethyl-4B-[2"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 253-255C;[~]"D-90 (c = 1.0, CHCl,); IR (KBr) 3500 (OH), 3450 (NH), 2895 (aliphatic CH), 1751 (lactone), 1590, 1500 1472 (aromatic C=C) cm~';
'H NMR (CDCl,) 7.31 (dd, J = 1.4, 7.9 Hz, lH, 3"-H), 7.18 (t, J = 8.8, Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.73 (t, J = 9.0 Hz, 4l'-H) 6.58 (d, J
= 8.2 Hz, lH, 6"-H), 6.54 (s, lH, 8-H), 6.35 (s, 2H, 2',6'-H), 5.98 (ABq, J = 1.2, 4.2 Hz, 2H
OCOCH,O), 5.44 (s, lH, exchangeahle, 4'-OH), 4.73 (t, J = 4.9 Hz, lH, 4-H) 4.64 (d, J - 4.9 Hz, lH, l-H), 4.49 (d, J = 6.0 Hz, lH, exchangeable, NH), 4.36 (t, J = 8.3 Hz, lH, ll-H), 3.91 (t, J
= 8.3, Hz, lH, ll-H), 3.80 (s, 6H, 3,'5'-OCH,~, 3.17 (dd, J = 4.8, 14.0 Hz, lH, 2-H), 3.04 ~m, lH, 3-H). Anal. C"H"CINO, C.H.N.
EXA~ Z
4'--Demethyl-4~-[3"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 174-176C;[~]"D-112 (C = 1.0, CHCl,); IR (KBr) 30 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1752 (lactone), 1580, and 1452 (aromatic C=C) cm~'; 'H
NMR (CDCl,) 7.12 (t, J = 8.1, Hz, lH, 5"-H), 6.76 (s, lH, 5-H), 6.74 (dd, J = 1.0, 8.1 Hz, lH 4"-H), 6.53 (br, 2H, 8-H and 2"-H), 6.42 (dd, J =
35 1.6, 6.5 Hz, lH, 6"-H), 6.33 (s, 2H, 2',6'-H), 2~2~
5.97 (ABq, J 5 1.0~ 8.7 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.66 (br, lH, 4-H), 4.59 (d, J - 4.8 Hz, lH, 1-H), 4.39 (t, J = 7.7 Hz, lH, ll-H), 3.99 (t, J = 7.7 Hz, lH, 11-H) 3.96 (br, lH, exchangeable, NH~, 3.79 ts, 6H, 3,'5'-OCH,), 3.11 (dd, J - 5.8, 14.0 Hz, H, 2-H), 3.01 (m, lH, 3-H). Anal. C"H2,CINO,) C.H.N.
4'-Demethyl-4B-~4"-chloroanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp 25~-255Cj[~]"D-125 (c = 0.75 CHCl,); IR (KBr) 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1758 (lactone), 1605, 1590 and 1475 (aromatic C=C) cm-15 ; H NMR (CDCl,) 8 7.17 (d, J - 8.7, Hz, 2H, 3",5"-H), 6.74 (5, lH, 5-H), 6.53 (s, lH, 8-H), 6.48 (d, J = 8.7 Hz, 2H, 2",6"-H), 6.32 (s, 2'6'-H), 5.96 (ABq, J = 1.0, 6.8 Hz, 2H OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.63 (d, J =
4.2 Hz, lH, 4-H), 4.59 (d, J = 4.9 Hz, lH, 1-H), 4.38 (t, J 2 8.0 Hz, lH, ll-H) 3.96 (t,J =
8.0 Hz, lH, ll-H), 3.79 (s, 6H 3,'5'-OCH,), 3.12 (dd, J = 4.9, 14.1 Hz, H, 2-H), 2.99 (m, lH, 3-H). Anal. C"Hl,CINOT) C.H.N.
~ EL~
4'-Demethyl-4B-t3"-bromoanilinyl~-4-desoxypodophyllotoxin.
Crystals from methanol/ether; mp 177-179C;t~]"D-105 (c = 1, CHCl,); IR (RBr) 3450 (OH), 3340 (NH), 2900 (aliphatic CH), 1740 (lactone), 1590, 1500 and 1475 (aromatic C=C) cm~
'; 'H NMR (CDCl,) 8 7.07 (t, J = 8.0, Hz, lH, 5"-H), 6.90 (dd, J = 0.9, 7.9 Hz, lH, 4"-H), 6.75 (s, lH, 5-H), 6.70 (br, lH, 2"-H), 6.53 (s, lH, 8-H), 6.47 (dd, J = 1.7, 8.3 Hz, lH 6"-H), 6.33 2~2~:~
(s, 2H, 2',6'-H), 5.97 (dd, J = 1.2, 9.3 Hæ, 2H, OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.65 (d, J = 4.2 Hz, lH, 4-H), 4.6Q (d, J = 4.8 Hz, lH, 1-H), 4.39 (t, J = 7.3 Hz, lH, 11-H) 3.96 (t, J = 7.3 Hz, lH, 11-H), 3.90 (d, J = 6.2, Hz, lH, exchangeable, NH), 3.R0 (s, 6H, 3,'5'-OCH,), 3.10 (dd, J = 4.9, 14.0 Hz, lH, 2-H), 3.02 (m, lH, 3-H). Anal. C"H"BrNO7) C.H.N.
4'-Demethyl~4~-[4"-bromoanilinyl]-4-desoxypodophyllotoxin.
Crystals from ethyl acetate/ethanol; mp 227-230C,[~]"D-110 (c = 0.5, CHCl,); IR (KBr) 3500 (OH), 3330 (NH), 2900 (aliphatic CH), 1755 (lactone), 1605, 1590 and 1475 (aromatic C-C) cm~
'; 'H NMR (CDCl,) ~ 7.30 (d, J = 8.9, Hz, 2H, 3",5"-H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8-H), 6.44 (d, J = 8.9 Hz, 2H, 2",6"-H), 6.32 (s, 2H, 2',6'-H), 5.98 (A8q, J = 1.3, 8.3 Hz, 2H, OCH~O), 5-42 (5, lH, exchangeable, 4'-OH), 4.62 (m, 2H, 4-H and 1-H), 4.36 (t, J = 8.5 Hz, lH, 11-H), 3.95 (t, 8.5 Hz, 11-H), 3.86 (d, J = 708, lH, exchan~eable, NH), 3.73 (s, 6H, 3,'5'-OCH,), 3.11 (dd-, J s 4.8, 14.1 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal. C~H,4BrNO,) C.H.N.
EX~MP~E 41 4'-De~ethyl-4B-t4"-iodoanilinyl]-4-desoxypodophyllotoxin.
Cry~tals from ethanol; mp 198-200C(dec.);~]"D-111 (c = 0.5, CHCl,); IR (KBr) 3540 (OH), 3420 (NH), 2900 (aliphatic CH), 1770 (lactone), 1610, 1585 and 1480 (aromatic C=C) cm~
'; 'H NMR (CDCl,) ~ 7.46 (d, J = 8.8 Hz, 2H, 2",6"-H), 6.32 (s, 2H, 2',6' H), 5.59 (ABq, J =
1.1, 8.9 Hz, 2H, OCHiO), 5.44 (s, lH, 20~2~
exchangeabl~, 4'-OH), 4.62 (~, lH, 4-H), 4.58 td, J = 4.9 Hz, lH, 1-H), 4.34 (t, J = 8.5 Hz, lH, l1-H), 3.94 (m, 2H, 11-H and NH), 3.78 (s, 6H, 3~,5~-OCH,), 3.09 (dd, J = 4.9, 14.1 Hz, lH, 2-H), 2.99 (m, lH, 3-H). Anal. C"H"BrNO,) C.H.N.
EXAMP~ 42 - 44 (Scheme IX) Podophyllotoxin (500mg, 1.2 mmol) was dissolved in dry dichloromethane (lOml) and kept at 0C. Hydrogen bromide gas was introduced into the solution for 45 min., after which time the solvent was evaporated in vacuo, anhydrous tetrahydrofuran (15 ml), anhydrous barium carbonate (474 mg, 2.4 mmol) and the appropriate hydroxyaniline ~142mg, 1.3 mmol) was added. The mixture stood at room temperature overnight, and then was filtered and concentrated. The crude product was purified using column chromatography tsilica gel 45 g with dichloromethane-acetone-ethyl acetate 100:5:5 as an eluant). Theproducts (42-44) obtained in the examples had the characteristic~ listed below.
~Z
4B-~2"-hydroxyanilinyl]-4-de60xypodophyllotoxin, Amorphous crystals from ether: mp 145-148C; IR (KBr) 3480 (OH), 3410 (NH), 2900 (aliphatic CH), 1760 (lactone), 1580, 1475 (aromatic C=C) cm'; H NMR (CDCl,) ~ 6.90 (t,J =
6.6 Hz, lH, 4"-H), 6.78 (s, lH, 5-H), 6.65 (~, 2H, 3",6"-H), 6.53 (m, 2H,8-H, 5"-H), 6.34 (s, 2H 2',6'H), 5.96 (ABq, J = 1.0, 3.5 Hz,2H, OCH,O), 5.02 (s, lH, exchangeable, 2"-OH), 4.68 (m, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, l-H), 2~2~ ~
4.38 (t, J = 8.6 Hz, lH, ll-H), 4.33 (m, lH, exchangeable, NH), 4.00 (t, J = 8.6 Hz, lH, llH), 3.82 (s, 3H, 4'-OCH,), 3.76 (s, 6H, 3',5'-OCH,), 3.25 ~dd, J = 5.1, 14.0 Hz, lH, 2-H), 3.05 (m, lH, 3-H). MS, m/z = 505 (m+). Anal.
(C"H"NOl 3/2 H2O) C.H.
4B-[3"-hydroxyanilinyl]~-Desoxypodophyllotoxin.
Amorphous powder from ether, mp 148-150C; IR(RBr) 3370 (OH,NH), 2900 (aliphatic CH), 1760 (lactone), 158S, 1475 (aromatic C=C) cm~'; 'H NMR (CDCl,) ~ 7.05 (t, J = 8.0 Hz, lH, 5"-H), 6.77 (8, lH, 5H), 6.52 (s, lH, 8-H), 6.32 (s, 2H, 2' 6'-H), 6.25 (dd, J = 2.2, 8.0 Hz, lH, 4"-H), 6.14 (dd, J = 2.2, 8.0 Hz, lH, 6"-H), 6.05 (t = 2.2 Hz, lH, 2"-H), 5.96 (ABq, J = 1.3, 3.8 Hz, 2H OCH,O), 4.64 (d, J = 3.9 lH, 4-H), 4.49 (d, J = 5.0, Hz, lH, l-H), 4.4 (t, J = 8.7 Hz, lH, 11-H), 4.03 (t, J = 8.7 Hz, lH, 11-H), 3.81 (s, 3H, 4'0CH,), 3.76 (s, 6H, 3',5'-OCH,), 3.18 (s, 6H, 3',5'-OCH,), 3.18 (dd, J = 5.0 Hz, 14.0 Hz, lH,2-H), 3.02 (m, lH, 3-H); MS, m/z =
505 (m+). Anal. (C,tH"N~, H,O) C.H.
~XAX~J~ 44 4B-[4"-hydroxyanilinyl]-4-de~oxypodophyllotoxin.
Crystals from chloroform; mp 145-150C;
IR(KBr) 3310 (OH,NH), 3010 (aromatic CH), 2900 (aliphatic CH), 1730 (latone), 1575, 1475 (aromatic C=H) cm-'; 'H NMR (CDCl,) D,O exchange) 6.75 (d, J = 8.3 Hz, 3H, 5-H, 3",5"-H), 6.53 (s, lH, 8H), 6.45 (d, J - 8.3 Hz, 2H, 2",6"-H), 6.23 (s, 2H 2'6'-H), 5.95 (ABq, J = 1.0, 4.0 Hz, 2H
OCH,O), 4.60 (d, J = 4.2 Hz, lH, 4-H), 4.57 (d, J
= 4.6 Hz, lH, 1-H), 4.38 (t, J = 6.0 Hz, lH, 11-42 2 a~42 H), 4.05 (t, J = 6.0 Hz, lH, llH) 3.83 (s, 3H, 4-OCH,3, 3.75 (s, 6H, 3',5'-OCH,), 3.18 (dd, J =
4.6 Hz, 14.0 Hz, lH,2-H), 3.0 (m, lH, 3-H).
Anal. (C,~H,7NO, 1/2 H~O) C.H.
Isolation of Human DNA Topç~somerase II.
Human DNA topoiso~erase II was isolated from peripheral blast cells of a patient with acute leukemia. The isolation procedure is described in Thurston, L., Imakura, Y., Haruna, M., Li, Z. C., Liu, S. Y., and Lee, K. H., J.
Med. Chem., 31, COMPLETE (1988) and is a partial combination of the procedure described in Goto, T., Laiapia, P. and Wang, J., J. Biol Chem~, 259, 10422 (1984) and Halligan, B., Edwards, X., and Liu, L., 2l_~inL-_She~., 260, 2475 (1985) which are herein specifically incorporated by reference.
Preparations of Druas.
Drugs were dissolved in Me,SO at a concentration of 20 mM as the stock solution and diluted before use with water to the desired concentration of each druq.
DNA TQpoiso~e~ase II Assay.
The P4 unknotting reaction was a modification Or the procedure described by Hseih, T., ~ iol~ m~, 258, 8413 ~1985), which i8 herein specifically incorporated by reference.
The reaction mixture (20 ~L), which contained 50 mM HEPES, pH 7.0, 50 mM KCI, 100 mM
NaCl, 0.1 mM EDTA, 10 mM MgCl" 1.0 mM ATP, 50 ~g/mL bovine serum albumin, 0.4 ~g P4 knotted DNA, and enzyme, was incubated with or without drugs.
2 ~
The reaction mixture was incubated at 37 C for 30 min and terminated by adding 5.0 ~1 of a stop solution 52~ sodium dodecyl sulfate, 20% glycerol, 0. 05~ bromophenol blue). These samples were loaded onto a 1% agarose gel and electrophoresed at 55 V overnight with an electrophoresis buffer that contained 90 mM
Tris-boric acid, pH 8.3, and 2.5 mM EDTA. At completion, the gel was stained in 0.5 ~g/mL of ethidium bromide. Then a photograph was taken of the DNA bands visualized with fluorescence induced by a long-wavelength W lamp. The data reported in Table 1 reflect a lOo ~M drug concentration.
K-SpS Precipi~ation Assay_or P~otein-DNA
CompLç~çs.
The intracellular formation of covalent topoisomerase II-DNA complexes was quantitated using the potassium SDS precipitation assay, a procedure adapted from the method described in Rowe, T.C., Chen, G. L., ~siang, Y. H., and Liu, L., Cancer RÇs., 46, 2021 (1986) (hereinafter Rowe et al.), which is herein specifically incorporated by reference. KB ATCC cells were prelabeled with 0.05 mCi/ml 14C-thymidine ~specific activity 50.5 mCi/mmol) for 18 hr. A
~inal concentration of 5xlO' cells/sample were treated with 10 ~M of the drugs at 37 C for 1 hr and proceeded according to the procedure described by Rowe et al. to detect the protein-linked DNA levels.
It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the 20~42~1 modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.
TABLE I
BIOLOGICAL EVALUATION OF 4'-DEMETHYL-4-ALKYLAMINO-PODOPHYLLOTOXIN ANALOGUES
R
< C~
O
o H3CO~OCH3 OH DNAC~LLULAR
TOPO-PROTEIN-CYTO- ISOMERASE II DNA
TOXICITY- AC~IVITYCOMPLEX
COMPOUND R ED,~KB(~g/ml) % INHIBITION~ FORMATIO~%
.
Etoposlde O ~ 2a +++ 100.0 Example:
1 B-OH 0.34 ++ 42.2 2 ~-OH 0.045 + 3.3 3 B-NH2 1.0 ++++36.4 4 ~-NH, 0.42 ~ 8.0 ~-NHC~,CH,OH 1.6 ++++121.4 6 ~-NHCH,CH,OH 710.0 - 0.0 7 B-NHCH,CH,CH, <0.4 ++ 69.7 8 B-NHCH,CH,OCH, >4.0 +++110.8 9 ~-NHCH,CH~CH, 3.4 +++ 84.1 B-NHCH,CH(OH)CH, 3.6 ++++ 167.2 11 8-NHCH(CH,)CH,OH 2.3 ++++ 161.7 12 B-NHCH,CH,CH,OH 4.0 ++ 89.2 13 B-OCH,CH,NH, 0.1 ++++300.0 14 B-OCH,CH,OH 0.7 ++ 50.0 ' ED,o is the concentration of drug which affords 50%
reduction in cell number after 3 days incubation.
+, ++, +++, ++++ and - denote for 25%, 50%, 75~, >75%, and 0% inhibition.
' Relative activities of cellular protein-DNA complex formation in KB ATCC tissue culture cells measured at 10 ~M drug concentration as compared the complex formed by 10 ~M of etoposide.
20~211 TABLE II
COMPOUNDS WITH POTENT TOPOISOMERASE II
INHIBITORY ACTIVITY
DNA
TOPOISOMERASE DNA TOPOISOMERASE
II
INHIBITORY ACTIVITY' COMPOUNDS (RELATIVE POTENCY) .. .. _ Etoposide 1.0 2.0 - 6.0 16 8.0 17 2.0 18 2.0 '~e o~
0.
OH
15 R~Cl 16 R,~R,=R,-R,=Br,R,-H
17 R,-OH, R,~R,-R,-Br,R,-H
18 R,~OH, R,-R,-R4=R,~Br Several different concentrations of tested compounds were employed for the determination of their potency. The relative potency with respect to etoposide as expressed in Table I was the relative concentration of compounds tested to achieve the same degree inhibition by etoposide in the range of 25 to 400 ~M.
2~21~
q7 Table ILE~
~iological Evaluation of 4-Demethyl-4B(arylamino)-4-desoxy Podopyllotoxin It~
<0~O
O
CH~O ~ OC~
OH
Compound R, DNA Cellular Cytotoxicity Topoisomerase II Protein-DNA IDso ~
Activity Complex (uM) % Inhi~ition Formation Etoposide +++ 100 0.2 19 ~ ~ +++ 243 0.71 20 ~ +++~ 211 0.64 21 ~ ~ +++ 137 0.6g 22 ~ ~ eo~c~ +++ 207 <0.10 23 ~ +++ 140 0.66 24 .~? ++++ 164 <1 0 oc~
~ ++ 180 <1.0 ~6 ~ +++ 158 0.23 2n~4~
Compound R, DNA Cellular Cytotoxicity Topoisomerase II Protein-DNA ID50 K~
Activity Complex (uM) % Inhibition Formation 27 ~ ~ ++ 121 0.25 28 ~f ++++ 213 0.24 29 ~ ~ ++ 115 1.08 f ~ +++ 148 0.24 31 ~ ~ ++ 97 0.71 >~
32 ~ ++ 123 <1.0 o~l 33 ~ ++++ 290 0.45 34 ~ ~ ++++ 151 4.54 35~ ~ o~ +++ 211 2.26 tl 36 ~ + 32 2.34 2~4211 Compound R, DNA Cellular cytotoxicity Topoisomerase II Protein-DNA ID, Activity Complex (uM)' % Inhibition~ FormationC
37 ~ ~ ++ 51 2.29 38 ~ ~ cl +++ 99 0.22 ~1 ,--39 ~ ~ ~ + 62 2.36 ~ ' +~ 179 0.22 41 ~ + 6~ 0.34 ' ID,o is the concentration of drug which affords 50% reduction in cell number ater 3 days incubation.
~ Activities of cellular protein-DNA complex formation in KB
ATCC tissue culture cells relative to Etoposide.
' +,++,+++,++++ denote 0-24%,25-49%,50-74%, and 275% inhibition respectively.
204~211 Tabl~ IV
Biological Evaiuation 014~ (arylamino)-4-desoxypodophyl~otoxins <~0 O
H3CO~OCH3 OcH3 Compound R DNA Cellubr Cytoloxicily Topoisolnerase l l prolein DNA 1~5O KB (~lM) Activity Compl~x Formatbn ~ Inhibitbn Etoposlde ~ 100 0.2 OH , 42 NH~ ~ 6 4.11 OH
43 NH~ ~ 37 0.31 44 ~ 21 0.31 2~421~
Scherre I
OH ~ ~
<~ ~ H~O X~0 H3CO~ocH3 H3COJ~OCH3 H3CO~OCH~
~2) fl . r~ OH
Scherne 11 OH SO,CH, CBz - Cl ~ CH3S02C1 ~
<~.""( <O~V ~
O _ O
H3COJ~OCH~ H3CO~OCH3 OCBZ Oc82 N;~N3 < ~O 2, < ~O
H3CO~oCH3 H3CO~OcH3 ~3) R ~ B-NH2 ~) R ~ NH2 20~211 Scheme 111 NHR
RNH2 < ~
O
HaCO OCH3 (S 12) Scheme IV
OCH2CH2Elr OCH2,CH2N3 HOCH2CH2Br ~1~ NaN3 Il <0~ "~ ~ ~
H3COJ~oCH3 H3COJ~oCH3 OH OH
~V~ ~) OcH2cH2NH2 H2 <~
H3CO~OCH3 OH
(13) 2~4~2~
SCheme V
OCH2CH2C~I
HOCH2cH20H < ~
o .."( o H3CO~ oc~3 OH
Schen~ Vl a o~o<O~o a - o - ' ~
H3CO~OCH3 ~ 15 ) Schem~ Vll Br2 < ~
RJ ' O ~ 16 ) RlnR2~R3~Rs~B~, R~.H
R~ ~, Rs ( 17 ) R~.OH, R2.R~.Rs.Br, R3-H
H3CO~OCH3 ~1B ) R~.OH, R2.~3.R~.Rs..Br 20~23 ~
Schernl~ Vlil I~HAr H8r NH2A~ O ~
11 <~ 0 ~ O
H3CO J~ OCH3 ( 1~1 41 ) Schen~ IX
Br NHAr HBr<~,~ NH2Ar < ~O
. ""~ O "'~( H3CO~ocH3 H3COJ~H3 (42-~4)
Claims (25)
1. A compound having the formula:
where:
R1 is .beta.-OCH2CH2NH2 .beta.-NHCH(CH3)CH2OH, .beta.-NHCH2CH(CH3)OH, .beta.-Cl, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3 .beta.-NHCH2CH2CH2OH; or .beta.-OCH2CH2OH;
wherein R1 is , , , R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
where:
R1 is .beta.-OCH2CH2NH2 .beta.-NHCH(CH3)CH2OH, .beta.-NHCH2CH(CH3)OH, .beta.-Cl, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3 .beta.-NHCH2CH2CH2OH; or .beta.-OCH2CH2OH;
wherein R1 is , , , R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
2. A compound having the formula:
where:
R1 is -NHCH2CH2OH, -NHCH2CH2OC3, -NHCH2CH(OH)CH2, NHCH(CH3)CH2OH, or Cl.
where:
R1 is -NHCH2CH2OH, -NHCH2CH2OC3, -NHCH2CH(OH)CH2, NHCH(CH3)CH2OH, or Cl.
3. A compound having the formula:
where R1 is -OCH2CH2H3 or -NHCH2CH2OH
where R1 is -OCH2CH2H3 or -NHCH2CH2OH
4. A compound having the formula:
where R1 is -NHCCH3HCH2OH, -NHCH2CCH3HOH, or NHR?, wherein R? is , , .
where R1 is -NHCCH3HCH2OH, -NHCH2CCH3HOH, or NHR?, wherein R? is , , .
5. A compound having the formula:
where R1 is -NCH2CH2OH.
where R1 is -NCH2CH2OH.
6. A compound having the formula:
wherein R is Cl.
wherein R is Cl.
7. A compound having the formula:
where R1 is Cl;
R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3
where R1 is Cl;
R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3
8. A compound having the formula:?
where R is .beta.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH2OH, .beta.-OCH2CH2OH or .beta.-OCH2CH2NH3.
where R is .beta.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH2OH, .beta.-OCH2CH2OH or .beta.-OCH2CH2NH3.
9. A process for treating tumors in humans and lower animals by administering a safe and effective amount of a compound according to claim 1.
10. A pharmaceutical composition comprising a compound having antitumor activity of the formula:
where:
R1 is .beta.--OCH2CH2NH2, .beta.-NHCCH3HCH2OH, .beta.-NHCH2CCH3HOH, .beta.-NHR2, .beta.-Cl, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH3OH, .beta.-OCH2CH2OH;
wherein R1 is , , R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
where:
R1 is .beta.--OCH2CH2NH2, .beta.-NHCCH3HCH2OH, .beta.-NHCH2CCH3HOH, .beta.-NHR2, .beta.-Cl, .beta.-NHCH2CH2OH, .alpha.-NHCH2CH2OH, .beta.-NHCH2CH2CH3, .beta.-NHCH2CH2OCH3, .beta.-NHCH2CH=CH2, .beta.-NHCH2CH(OH)CH3, .beta.-NHCH2CH2CH3OH, .beta.-OCH2CH2OH;
wherein R1 is , , R2 is H, or Br;
R3 is H, or Br;
R4 is H, or Br;
R5 is H, or Br; and R6 is H, or -CH3.
11. A compound according to claim 1 exhibiting antitumor activity.
12. A compound of the formula:
where R1 is a substituted or unsubstituted arylamine.
where R1 is a substituted or unsubstituted arylamine.
13. A compound according to claim 12 where R1 is .
14. A compound according to Claim 12 where R1 is selected from:
, , , , , , , , , , , , , , , , , , , , or .
, , , , , , , , , , , , , , , , , , , , or .
15. A compound of the formula:
Where R1 is , , .
and R2 is CH3.
Where R1 is , , .
and R2 is CH3.
16. A process for treating tumors in humans and lower animals by administering a safe and effective amount of a compound according to claim 12.
17. A compound according to claim 12 exhibiting antitumor activity.
18. A pharmaceutical composition comprising a compound having the formula:
where R is selected from:
, , , , , , , , , , , , , , , , , , , , , or .
where R is selected from:
, , , , , , , , , , , , , , , , , , , , , or .
19. A compound of the formula:
wherein Ar is an arylamine as in the formula:
wherein R1 is H, OH, F, Cl, Br, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2;
R2 is H, OH, F, Cl, Br, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2, CHOHCH3, SCH3, CH3, CO2CH3;
R3 is H, OH, F, Cl, Br, I, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2, , , N(CH2CH2OH)3, CH3;
R4 is H, F, Cl, OH, OCH3, CO2CH3, CO2C2H3, CH3, CF3, NO2, NH2, Cl;
R5 is H, F, Cl, CH3, CF3, OH, OCH3, NO2;
R3 and R4 are OCH2O or OCH2CH2O.
wherein Ar is an arylamine as in the formula:
wherein R1 is H, OH, F, Cl, Br, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2;
R2 is H, OH, F, Cl, Br, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2, CHOHCH3, SCH3, CH3, CO2CH3;
R3 is H, OH, F, Cl, Br, I, CO2CH3, CO2C2H3, CN, NO2, NH2, N(CH3)2, OCH3, CH2OH, CH3, CF3, CH2CH2OH, COCH3, CH2NH2, , , N(CH2CH2OH)3, CH3;
R4 is H, F, Cl, OH, OCH3, CO2CH3, CO2C2H3, CH3, CF3, NO2, NH2, Cl;
R5 is H, F, Cl, CH3, CF3, OH, OCH3, NO2;
R3 and R4 are OCH2O or OCH2CH2O.
20. A compound of the formula:
wherein R1 is a flat aromatic ring system, and said ring system contains a heteroatom or is substituted with an electron donating group at the 3 or 4 position on the ring.
wherein R1 is a flat aromatic ring system, and said ring system contains a heteroatom or is substituted with an electron donating group at the 3 or 4 position on the ring.
21. A compound according to Claim 20 wherein the electron donating substituent at the 3 or 4 position is oxygen.
22. A compound according to Claim 20 wherein the flat aromatic ring system is pyridine.
23. A process for treating tumors in humans and lower animals by administering a safe and effective amount of a compound according to claim 18.
24. A process for treating tumors in humans and lower animals by administering a safe and effective amount of a compound according to claim 19.
25. A compound having the formula:
wherein R1 is selected from .beta.-Br, .beta.-OH, .alpha.-OH, B-NH2 and .alpha.-NH2;
R2, R3, R4, R5 are each independently selected from H or Br, wherein at least one of R2, R3, R4, or R5 is Br; and R6, is selected from H or CH3.
wherein R1 is selected from .beta.-Br, .beta.-OH, .alpha.-OH, B-NH2 and .alpha.-NH2;
R2, R3, R4, R5 are each independently selected from H or Br, wherein at least one of R2, R3, R4, or R5 is Br; and R6, is selected from H or CH3.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2044211 CA2044211A1 (en) | 1991-06-10 | 1991-06-10 | Etoposide analogues |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2044211 CA2044211A1 (en) | 1991-06-10 | 1991-06-10 | Etoposide analogues |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2044211A1 true CA2044211A1 (en) | 1992-12-11 |
Family
ID=4147774
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2044211 Abandoned CA2044211A1 (en) | 1991-06-10 | 1991-06-10 | Etoposide analogues |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA2044211A1 (en) |
-
1991
- 1991-06-10 CA CA 2044211 patent/CA2044211A1/en not_active Abandoned
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