JPH0338294B2 - - Google Patents
Info
- Publication number
- JPH0338294B2 JPH0338294B2 JP55066352A JP6635280A JPH0338294B2 JP H0338294 B2 JPH0338294 B2 JP H0338294B2 JP 55066352 A JP55066352 A JP 55066352A JP 6635280 A JP6635280 A JP 6635280A JP H0338294 B2 JPH0338294 B2 JP H0338294B2
- Authority
- JP
- Japan
- Prior art keywords
- formula
- group
- polymer
- same
- solution
- 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.)
- Expired - Lifetime
Links
- 229920000642 polymer Polymers 0.000 claims description 94
- -1 2-nitrophenylthio group Chemical group 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 25
- 231100000433 cytotoxic Toxicity 0.000 claims description 21
- 230000001472 cytotoxic effect Effects 0.000 claims description 21
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 16
- 229920013730 reactive polymer Polymers 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 230000000259 anti-tumor effect Effects 0.000 claims description 13
- 125000003277 amino group Chemical group 0.000 claims description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 12
- 125000001841 imino group Chemical group [H]N=* 0.000 claims description 8
- 238000006116 polymerization reaction Methods 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 125000000217 alkyl group Chemical group 0.000 claims description 7
- 239000007795 chemical reaction product Substances 0.000 claims description 7
- 125000002947 alkylene group Chemical group 0.000 claims description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims description 6
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- 125000004434 sulfur atom Chemical group 0.000 claims description 6
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 claims description 5
- 239000000470 constituent Substances 0.000 claims description 5
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 claims description 5
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 229940100198 alkylating agent Drugs 0.000 claims description 2
- 239000002168 alkylating agent Substances 0.000 claims description 2
- 230000000340 anti-metabolite Effects 0.000 claims description 2
- 229940100197 antimetabolite Drugs 0.000 claims description 2
- 239000002256 antimetabolite Substances 0.000 claims description 2
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 230000003115 biocidal effect Effects 0.000 claims description 2
- 229940126062 Compound A Drugs 0.000 claims 1
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 68
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 35
- 238000006243 chemical reaction Methods 0.000 description 35
- 239000011347 resin Substances 0.000 description 29
- 229920005989 resin Polymers 0.000 description 29
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 28
- 239000000203 mixture Substances 0.000 description 25
- 238000000034 method Methods 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 229920002643 polyglutamic acid Polymers 0.000 description 19
- 239000002246 antineoplastic agent Substances 0.000 description 18
- 239000002244 precipitate Substances 0.000 description 17
- 239000011734 sodium Substances 0.000 description 17
- 239000007983 Tris buffer Substances 0.000 description 16
- 229910052708 sodium Inorganic materials 0.000 description 16
- 159000000000 sodium salts Chemical class 0.000 description 16
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 16
- 239000012299 nitrogen atmosphere Substances 0.000 description 14
- 229960001924 melphalan Drugs 0.000 description 13
- 239000006185 dispersion Substances 0.000 description 12
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 11
- 239000011541 reaction mixture Substances 0.000 description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 9
- 229960003767 alanine Drugs 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 9
- BGGHCRNCRWQABU-JTQLQIEISA-N (2s)-2-amino-5-oxo-5-phenylmethoxypentanoic acid Chemical compound OC(=O)[C@@H](N)CCC(=O)OCC1=CC=CC=C1 BGGHCRNCRWQABU-JTQLQIEISA-N 0.000 description 8
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 8
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 7
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 229920001577 copolymer Polymers 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 6
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 6
- 229930195714 L-glutamate Natural products 0.000 description 6
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 6
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 6
- 229940041181 antineoplastic drug Drugs 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 231100000599 cytotoxic agent Toxicity 0.000 description 6
- 239000002619 cytotoxin Substances 0.000 description 6
- 101710112752 Cytotoxin Proteins 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 239000012300 argon atmosphere Substances 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 4
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 229910052786 argon Inorganic materials 0.000 description 4
- 230000008033 biological extinction Effects 0.000 description 4
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 125000004035 thiopropyl group Chemical group [H]SC([H])([H])C([H])([H])C([H])([H])* 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 229920000298 Cellophane Polymers 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-L L-glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 229940098779 methanesulfonic acid Drugs 0.000 description 3
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 229960005190 phenylalanine Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
- ATCFYQUZTYQTJN-AXDSSHIGSA-N (2s)-2-amino-4-benzylpentanedioic acid Chemical compound OC(=O)[C@@H](N)CC(C(O)=O)CC1=CC=CC=C1 ATCFYQUZTYQTJN-AXDSSHIGSA-N 0.000 description 2
- RRHTVPZYUQSUFT-UHFFFAOYSA-N 2-(propyldisulfanyl)ethanamine Chemical compound CCCSSCCN RRHTVPZYUQSUFT-UHFFFAOYSA-N 0.000 description 2
- MERLDGDYUMSLAY-UHFFFAOYSA-N 4-[(4-aminophenyl)disulfanyl]aniline Chemical compound C1=CC(N)=CC=C1SSC1=CC=C(N)C=C1 MERLDGDYUMSLAY-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical class [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 2
- 229940099500 cystamine Drugs 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- 239000012456 homogeneous solution Substances 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 239000003999 initiator Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 108700024573 poly-gamma-benzyl-L-glutamate Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- HFZKKJHBHCZXTQ-JTQLQIEISA-N (4s)-4-azaniumyl-5-oxo-5-phenylmethoxypentanoate Chemical compound OC(=O)CC[C@H](N)C(=O)OCC1=CC=CC=C1 HFZKKJHBHCZXTQ-JTQLQIEISA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- KTWIATYFXLGCQQ-UHFFFAOYSA-N 2-[(4-carboxypyridin-2-yl)disulfanyl]pyridine-4-carboxylic acid Chemical compound OC(=O)C1=CC=NC(SSC=2N=CC=C(C=2)C(O)=O)=C1 KTWIATYFXLGCQQ-UHFFFAOYSA-N 0.000 description 1
- UFULAYFCSOUIOV-XSCORUHJSA-N 2-aminoethanethiol Chemical group NCC[35SH] UFULAYFCSOUIOV-XSCORUHJSA-N 0.000 description 1
- 125000001340 2-chloroethyl group Chemical group [H]C([H])(Cl)C([H])([H])* 0.000 description 1
- ABFYEILPZWAIBN-UHFFFAOYSA-N 3-(iminomethylideneamino)-n,n-dimethylpropan-1-amine;hydrochloride Chemical compound Cl.CN(C)CCCN=C=N ABFYEILPZWAIBN-UHFFFAOYSA-N 0.000 description 1
- UHBAPGWWRFVTFS-UHFFFAOYSA-N 4,4'-dipyridyl disulfide Chemical compound C=1C=NC=CC=1SSC1=CC=NC=C1 UHBAPGWWRFVTFS-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-YDKYIBAVSA-N 4-amino-1-[(2r,3s,4r,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-YDKYIBAVSA-N 0.000 description 1
- WCDSVWRUXWCYFN-UHFFFAOYSA-N 4-aminobenzenethiol Chemical group NC1=CC=C(S)C=C1 WCDSVWRUXWCYFN-UHFFFAOYSA-N 0.000 description 1
- NUDZXWNVRWSFQI-UHFFFAOYSA-N 4-nitro-2-[(4-nitropyridin-2-yl)disulfanyl]pyridine Chemical compound [O-][N+](=O)C1=CC=NC(SSC=2N=CC=C(C=2)[N+]([O-])=O)=C1 NUDZXWNVRWSFQI-UHFFFAOYSA-N 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-L 5-[(3-carboxylato-4-nitrophenyl)disulfanyl]-2-nitrobenzoate Chemical compound C1=C([N+]([O-])=O)C(C(=O)[O-])=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C([O-])=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-L 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- GUGHGUXZJWAIAS-QQYBVWGSSA-N Daunorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 GUGHGUXZJWAIAS-QQYBVWGSSA-N 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 102100021587 Embryonic testis differentiation protein homolog A Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000898120 Homo sapiens Embryonic testis differentiation protein homolog A Proteins 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- RXPFHUMNDYEGBN-VIFPVBQESA-N benzyl (2r)-2-amino-3-sulfanylpropanoate Chemical group SC[C@H](N)C(=O)OCC1=CC=CC=C1 RXPFHUMNDYEGBN-VIFPVBQESA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000006229 carbon black Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000004581 coalescence Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 229960005215 dichloroacetic acid Drugs 0.000 description 1
- HCUYBXPSSCRKRF-UHFFFAOYSA-N diphosgene Chemical compound ClC(=O)OC(Cl)(Cl)Cl HCUYBXPSSCRKRF-UHFFFAOYSA-N 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical group [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 150000004986 phenylenediamines Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- ISEQANYLRYIGLF-UHFFFAOYSA-N s-(1h-benzimidazole-2-carbonylsulfanyl) 1h-benzimidazole-2-carbothioate Chemical compound C1=CC=C2NC(C(SSC(=O)C=3NC4=CC=CC=C4N=3)=O)=NC2=C1 ISEQANYLRYIGLF-UHFFFAOYSA-N 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical class [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- Polyamides (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は、細胞毒性物質を結合した反応性重合
体及びその製造法に関する。更に詳しくは、本発
明は、腫瘍細胞等の標的物に結合能を有する抗腫
瘍抗体等と、細胞毒物を結合して得られる標的指
向型制ガン剤（抗腫瘍剤）等において、細胞毒物
として用いることのできる反応性重合体及びその
製造法に関するものである。
従来、抗腫瘍抗体に細胞毒物を結合して抗腫瘍
剤を製造することは公知である。
例えば、特開昭51−61640号には、抗腫瘍免疫
グロブリンと分子中にアミノ基又はカルボキシル
基を有する制ガン剤を水溶性カルボジイミドの存
在下に反応させ、免疫グロブリン１分子当り５〜
15分子の制ガン剤がアミド結合で結合された抗腫
瘍剤を得たことが開示されている。また、特開昭
51−144723号には、抗腫瘍抗体として抗腫瘍免疫
グロブリンのFab′2量体を用い、これの遊離アミ
ノ基に、過ヨウ素酸ナトリウムで酸化された制ガ
ン剤（例えば、ダウノマイシン）を結合させシツ
フ塩基とし、次いで水素化ホウ素ナトリウムによ
り生成結合を安定化させることにより、Fab′2量
体と制ガン剤からなる抗腫瘍剤を得たことが開示
されている。更に、特開昭51−126281号には、抗
腫瘍免疫グロブリンと、１分子当り制ガン剤を５
〜500分子共有結合しているポリマー担体（例え
ば、ポリダルタミン酸）を、アミド結合によつて
結合させて抗腫瘍剤を得たことが開示されてい
る。
これらの方法で得られた抗腫瘍剤は、腫瘍細胞
と選択的に結合し腫瘍細胞に毒性を発揮すること
が期待されるものであり、非常に興味のある薬剤
である。しかしながら、これらの公知の方法及び
それによつて得られた抗腫瘍剤の最大の欠点は、
抗腫瘍抗体と細胞毒物（制ガン剤又は制ガン剤を
結合したポリマー担体）との結合がアミド結合に
よつて、即ち抗腫瘍抗体中の遊離のアミノ基又は
カルボキシル基を介した結合によつて行なわれて
いるという点である。免疫グロブリンはその抗原
認識部位にも多数のアミノ基やカルボキシル基を
有している。従つて、抗腫瘍免疫グロブリンに細
胞毒物をアミド結合によつて結合させる場合に
は、抗腫瘍免疫グロブリンの抗原認識部位にも細
胞毒物が結合することになり、その結果、得られ
た抗腫瘍剤は最早腫瘍細胞に対する結合能を全く
失うかあるいは低下せしめられるという問題が生
じるのである。また、特開昭51−61640号記載の
方法では、抗体分子内及び分子間でもアミド結合
が形成される。更に、特開昭51−126281号記載の
方法では、抗体分子内及びポリグルタミン酸の分
子内、あるいは同種分子間でもアミド結合が形成
される。そして、これらの望ましくないアミド結
合の形成の結果、得られる抗腫瘍剤はその性能が
低下し、更に腫瘍の治療に用いるのが不適当な高
分子量物質を含むという問題も生じるのである。
本発明者らはかかる先行技術の欠点を解決する
ために鋭意研究を行なつた結果、免疫グロブリン
の限られた位置に存在するジスルフイド結合を利
用して細胞毒物を結合させれば、前記欠点のない
抗腫瘍剤が得られることを見い出した。そして本
発明は、かかる目的のために、あるいは又その他
の標的指向型薬剤を製造するために最適に使用で
きる細胞毒物を提供するものである。
即ち、本発明は、細胞毒性物質を結合した式
〔〕で表わされる重合度（ｎ＋ｐ＋ｑ）が10〜
1500の反応性重合体である。
〔式〔〕において、Ｘは水素原子又は２−ピリ
ジルチオ基、４−ピリジルチオ基、３−カルボキ
シ−４−ニトロフエニルチオ基、４−カルボキシ
−２−ピリジルチオ基、Ｎ−オキシ−２−ピリジ
ルチオ基、２−ニトロフエニルチオ基、４−ニト
ロ−２−ピリジルチオ基、２−ベンゾチアゾイル
チオ基、２−ベンゾイミダゾイルチオ基及びＮ−
フエニリアミノ−N′−フエニルイミノメチルチ
オ基から成る群から選ばれた隣りの硫黄原子と共
に活性ジスルフイド結合を形成しうる基を表わ
す。Ｙは分子中にアミノ基又はイミノ基を含む抗
腫瘍性のアルキル化剤、代謝拮抗剤又は抗生物質
のアミノ基又はイミノ基反応残基を表わす。Ｚは
水素原子又は１価の陽イオンを表わす。Ｗは置換
または非置換のアルキレン又はフエニレン基を表
わす。R1は水素原子又は炭素数１〜４のアルキ
ル基を表わす。R2はα−アミノ酸のα位側鎖
（但しカルボキシル基を有する基は除く）を表わ
す。ｎ、ｐ及びｑは重合体中の各構成単位の数を
表わすが、構成単位の配列はランダムであり、
ｎ、ｐ、ｑに係る各構成単位の全体に占める割合
は、それぞれ５〜50％、40〜95％、０〜10％であ
る。ｍは１−４の整数を表わす。〕
式〔〕のＸは、水素原子又は隣りの硫黄原子
と共に活性ジスルフイド結合を形成しうる基を表
わすが、後者としては、例えば、２−ピリジルチ
オ基（
The present invention relates to a reactive polymer bound to a cytotoxic substance and a method for producing the same. More specifically, the present invention is directed to use as a cytotoxin in a target-directed anticancer drug (anti-tumor drug) obtained by combining an anti-tumor antibody or the like that has the ability to bind to a target such as a tumor cell with a cytotoxin. The present invention relates to reactive polymers that can be used in a variety of ways, and methods for producing the same. Conventionally, it has been known to produce an antitumor agent by binding a cytotoxin to an antitumor antibody. For example, in JP-A No. 51-61640, an antitumor immunoglobulin is reacted with an anticancer drug having an amino group or a carboxyl group in the molecule in the presence of water-soluble carbodiimide.
It is disclosed that an antitumor agent was obtained in which 15 molecules of an anticancer agent were linked with an amide bond. Also, Tokukai Akira
No. 51-144723 uses a Fab' dimer of antitumor immunoglobulin as an antitumor antibody, and binds an anticancer drug (e.g., daunomycin) oxidized with sodium periodate to the free amino group of the Fab′ dimer, and then binds it to a Schiff base. It is disclosed that an antitumor agent consisting of a Fab' dimer and an anticancer agent was obtained by stabilizing the resulting bond with sodium borohydride. Furthermore, JP-A-51-126281 discloses that anti-tumor immunoglobulin and anti-cancer drugs are contained at 50% per molecule.
It is disclosed that an antitumor agent was obtained by linking a polymer carrier (eg, polydaltamic acid) with ~500 molecules covalently bonded through an amide bond. The antitumor agents obtained by these methods are expected to selectively bind to tumor cells and exhibit toxicity to tumor cells, and are therefore very interesting agents. However, the biggest drawback of these known methods and the antitumor agents obtained by them is that
The binding between the anti-tumor antibody and the cytotoxic substance (anticancer drug or polymer carrier bound to the anti-tumor antibody) is carried out through an amide bond, that is, through a free amino group or carboxyl group in the anti-tumor antibody. That is the point. Immunoglobulins also have many amino groups and carboxyl groups in their antigen recognition sites. Therefore, when a cytotoxin is bound to an antitumor immunoglobulin through an amide bond, the cytotoxin also binds to the antigen recognition site of the antitumor immunoglobulin, and as a result, the resulting antitumor agent The problem arises that the cells no longer have the ability to bind to tumor cells completely or are reduced. Furthermore, in the method described in JP-A-51-61640, amide bonds are formed both within and between antibody molecules. Furthermore, in the method described in JP-A-51-126281, amide bonds are formed within the antibody molecule, within the polyglutamic acid molecule, or between molecules of the same type. As a result of the formation of these undesirable amide bonds, the performance of the resulting antitumor agent is reduced, and there is also the problem that it contains high molecular weight substances that are unsuitable for use in tumor treatment. The present inventors have conducted extensive research to solve the drawbacks of the prior art, and have found that if cytotoxic substances are bound using disulfide bonds that exist in limited positions in immunoglobulins, the above-mentioned drawbacks can be overcome. It was discovered that an antitumor agent can be obtained. The present invention provides cytotoxins that can be optimally used for such purposes or for the production of other targeted drugs. That is, in the present invention, the degree of polymerization (n+p+q) expressed by the formula [] in which the cytotoxic substance is bound is 10 to
1500 reactive polymer. In [Formula [], , 2-nitrophenylthio group, 4-nitro-2-pyridylthio group, 2-benzothiazoylthio group, 2-benzimidazoylthio group and N-
Represents a group capable of forming an active disulfide bond with an adjacent sulfur atom selected from the group consisting of phenyliamino-N'-phenyliminomethylthio groups. Y represents an amino group- or imino-reactive residue of an antitumor alkylating agent, antimetabolite, or antibiotic containing an amino group or imino group in the molecule. Z represents a hydrogen atom or a monovalent cation. W represents a substituted or unsubstituted alkylene or phenylene group. R 1 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R 2 represents the α-position side chain of the α-amino acid (excluding groups having a carboxyl group). n, p and q represent the number of each structural unit in the polymer, but the arrangement of the structural units is random,
The proportions of each of the constituent units n, p, and q in the whole are 5 to 50%, 40 to 95%, and 0 to 10%, respectively. m represents an integer of 1-4. ] In the formula [], X represents a hydrogen atom or a group capable of forming an active disulfide bond with an adjacent sulfur atom, and the latter includes, for example, a 2-pyridylthio group (
【式】）、４−ピリジルチオ基 （[Formula]), 4-pyridylthio group (
【式】）、３−カルボキシ−４−ニト ロフエニルチオ基（[Formula]), 3-carboxy-4-nito lophenylthio group (
【式】）、４
−カルボキシ−２−ピリジルチオ基
（[Formula]), 4-carboxy-2-pyridylthio group (
【式】）、Ｎ−オキシ−２−ピ リジルチオ基（[Formula]), N-oxy-2-p Lysylthio group (
【式】）、２−ニトロフ エニルチオ基（[Formula]), 2-nitroph enylthio group (
【式】）、４−ニトロ −２−ピリジルチオ基（[Formula]), 4-nitro -2-pyridylthio group (
【式】）、
２−ベンゾチアゾイルチオ基
（[Formula]), 2-benzothiazoylthio group (
【式】）、２−ベンゾイミダゾイ ルチオ基（[Formula]), 2-benzimidazoi Ruthio group (
【式】）及びＮ−フエ
ニルアミノ−N′−フエニルイミノメチルチオ基
（[Formula]) and N-phenylamino-N'-phenyliminomethylthio group (
【式】）がある。
Ｙは分子中にアミノ基又はイミノ基を含む細胞
毒性物質のアミノ基又はイミノ基反応残基を表わ
す。本発明における細胞毒性物質とは、そのまま
の状態で細胞に毒性を発揮する物質、あるいはそ
のままでは毒性を発揮しないが、生体内で細胞に
毒性を発揮し得る物質に転換し得る物質をいう。
これらの例としては、
Ｐ−〔Ｎ，Ｎ−ビス（２−クロロエチル）〕フエ
ニレンジアミン
Ｐ−〔ビス（２−クロロエチル）アミノ〕Ｌ−
フエニルアラニン（メルフアラン）
２−アミノ−Ｎ−〔Ｐ−ビス（２−クロロエチ
ル）アミノ〕フエニル−３−ヒドロキシプロピオ
ンアミド
１−（β−Ｄ−アラビノフラノシル）シトシン
またはそのモノホスフエート
１−〔5′−（２−アミノエチルホスホリル）−β
−Ｄ−アラビノフラノシル〕シトシン
２−アミノ−Ｎ−〔Ｐ−ビス（２−クロロエチ
ル）アミノ〕フエニル−３−ヒドロキシ−２−ヒ
ドロキシメチルプロピオンアミド
メトトレキセート
アクチノマイシンＤ
マイトマイシンＣ
Ｘ＝Ｈ、ダウノマイシン
Ｘ＝OH、アドリアマイシン
等が挙げられる。
Ｚは水素原子又は１価の陽イオン、例えば
Na+、K+、NH4 +である。
Ｗは２価の有機基を表わし、本発明の反応性重
合体を得る過程で何ら反応に関与しない不活性な
基である限り特に限定されない。これらの基とし
ては、例えば、２−アミノエタンチオール残基
（−CH2CH2−）の如き直鎖の、あるいはシステ
インベンジルエステル残基
（[Formula]). Y represents an amino group- or imino-reactive residue of a cytotoxic substance containing an amino group or imino group in the molecule. A cytotoxic substance in the present invention refers to a substance that exhibits toxicity to cells as it is, or a substance that does not exhibit toxicity as it is but can be converted into a substance capable of exhibiting toxicity to cells in vivo.
Examples of these are: P-[N,N-bis(2-chloroethyl)]phenylenediamine P-[bis(2-chloroethyl)amino]L-
Phenylalanine (Melphalan) 2-Amino-N-[P-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide 1-(β-D-arabinofuranosyl)cytosine or its monophosphate 1-[5'-(2-aminoethylphosphoryl)-β
-D-arabinofuranosyl]cytosine 2-Amino-N-[P-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide Methotrexate Actinomycin D Mitomycin C Examples include X=H, daunomycin, X=OH, and adriamycin. Z is a hydrogen atom or a monovalent cation, e.g.
They are Na + , K + , and NH 4 + . W represents a divalent organic group, and is not particularly limited as long as it is an inert group that does not participate in any reaction during the process of obtaining the reactive polymer of the present invention. These groups include, for example, straight chain residues such as 2-aminoethanethiol residue (-CH 2 CH 2 -), or cysteine benzyl ester residue (
【式】）やホモシステインベ ンジルエステル残基（[Formula]) and homocysteine ester residue (
【式】）
の如き側鎖を有するアルキレン基、４−アミノチ
オフエノール残基（[Formula]) Alkylene group having a side chain such as 4-aminothiophenol residue (
【式】）の如き置換
基を有しない、あるいは置換基を有するフエニレ
ン基が挙げられるが、炭素数１〜４のアルキレン
基が特に好ましい。
R1は水素原子又は炭素数１〜４のアルキル基
であるが、好ましいのは水素原子である。
R2はα−アミノ酸のα−位側鎖（但しカルボ
キシル基を有する基は除く）であり、例えばα−
アミノ酸がグリシンの場合はR2＝Ｈ、アラニン
の場合はR2＝CH3、フエニルアラニンの場合は
Examples include phenylene groups having no substituents or having substituents such as [Formula]), and alkylene groups having 1 to 4 carbon atoms are particularly preferred. R 1 is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, preferably a hydrogen atom. R 2 is the α-position side chain of the α-amino acid (excluding groups having a carboxyl group), for example, α-
When the amino acid is glycine, R 2 = H; when the amino acid is alanine, R 2 = CH 3 ; when the amino acid is phenylalanine,
【式】セリンの場合はR2＝
CH2OHである。式〔〕の反応性重合体におい
て、かかるα−アミノ酸からなる単位は、細胞毒
性物質との結合には何ら関与しないが、反応性重
合体の脂溶性や水溶性、あるいは細胞膜との親和
性等を調節するのに役立つものである。従つて、
脂溶性や水溶性の調節が格別に必要ない場合に
は、かかるα―アミノ酸単位を含有しないものの
方（式〔〕においてｑ＝０）が実用的に有利で
ある。
ｍは１〜４の整数を表わすが、好ましいのはｍ
が１又は２の場合である。なお、式〔〕で表わ
される反応性重合体としては、例えば、ｍ＝１の
ものとｍ＝２のものが混在している様な重合体も
含む。
ｎは細胞毒性物質が結合した構成単位の数を表
わし、ｐは細胞毒性物質が結合していない構成単
位の数を表わし、ｑは側鎖にカルボキシル基を有
しないα−アミノ酸単位（側鎖は修飾されていて
もよい）の数を表わすが、これらの単位の重合体
中での配列状態は任意である。即ち、式〔〕に
おいては、便宜上ブロツク重合体の如く表わして
あるが、これに限られるものではなく、通常の方
法で得られるものはランダム配列の重合体であ
る。ｎ＝５〜1500、好ましくは10〜500であり、
ｐ＝０〜1500、好ましくは０〜500であり、ｑ＝
０〜1500、好ましくはｑ＝０〜500である。
本発明の式〔〕で表わされる反応性重合体の
うち、Ｘが水素原子であるもの、即ち下記式〔
−ａ〕で表わされる反応性重合体を製造する方法
について２つの方法を説明する。
〔式〔−ａ〕において、Ｙ、Ｚ、Ｗ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。〕
第１番目の方法は、分子中にジスルフイド結合
を含有する式〔〕で表わされる重合体に、
〔式〔〕において、R3はアルキル基、アラル
キル基又はアリール基を表わす。Ｚ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。〕
前述した如き、分子中にアミノ基又はイミノ基
を含む細胞毒性物質を反応させ、次いで反応生成
物を、例えばチオール化合物や水素化ホウ素化合
物と反応させて、得られた反応生成物中のジスル
フイド結合を還元的に切断する方法である。
第２番目の方法は、分子中にジスルフイド結合
を含有する式〔〕で表わされる重合体に、
〔式〔〕において、Ｚ、Ｗ、R1、R2、ｍ、ｎ、
ｐ及びｑの定義は式〔〕の場合と同じ。W′は
Ｗと同一又は異なる、置換または非置換のアルキ
レン又はフエニレン基を表わす。R1′はR1と同一
又は異なり、水素原子又は炭素数１〜４のアルキ
ル基を表わす。n′、p′及びq′はそれぞれｎ、ｐ及
びｑに対応し重合体中の各構成単位の数を表わす
が、構成単位の配列はランダムであり、ｎとn′、
ｐとp′、ｑとq′に係る各構成単位の全体に占める
割合は、それぞれ５〜50％、40〜95％、０〜10％
である。〕
前述した如き、分子中にアミノ基又はイミノ基
を含む細胞毒性物質を反応させ、次いで反応生成
物をチオール化合物や水素化ホウ素化合物と反応
させて、得られた反応生成物中のジスルフイド結
合を還元的に切断する方法である。
前記式〔〕又は式〔〕で表わされる分子中
にジスルフイド結合を有する重合体と、分子中に
アミノ基又はイミノ基を含む細胞毒性物質との反
応は、通常、水又はジメチルホルムアミドやジメ
チルスルホキシド等の有機溶剤を反応溶媒とする
均一反応系で行なわれる。反応に際しては、例え
ば１−エチル−３−（３−ジメチルアミノプロピ
ル）カルボジイミド塩酸塩やジシクロヘキシルカ
ルボジイミドで重合体中のカルボキシル基を活性
化してもよく、あるいはカルボキシル基を混合酸
無水物の形に活性化しておいてもよい。反応温度
は−40〜100℃、反応時間は10分〜10日間が適当
である。用いられる重合体と細胞毒性物質との割
合は、所望されるｎの数によつて異なるが、通常
重合体中の−COOZ基の５〜200％に相当する量
が適当である。
かくして得られた細胞毒性物質を結合された重
合体とチオール化合物との反応は、通常、水又は
ジメチルホルムアミドやジメチルスルホキシド等
の有機溶剤を反応溶媒とする均一反応系で行なわ
れる。適当なチオール化合物としては、例えば、
ジチオスレイトール、２−メルカプトエタノール
がある。チオール化合物は、重合体中のジスルフ
イド結合に対し１〜100倍モル量用いられる。反
応温度は−５〜70℃、反応時間は５分〜10日間が
好ましい。水素化ホウ素化合物、例えば、水素化
ホウ素ナトリウム、水素化ホウ素カリウム、水素
化ホウ素カルシウムを用いる場合には、重合体と
の反応は通常、水溶液中で行なわれる。
なお、前記式〔〕又は〔〕で表わされる化
合物の製造法は後述の参考例でも詳述するが、そ
の概略を示すと以下の通りである。例えば、グル
タミン酸ベンジルエステル（γ−ベンジル−Ｌ−
グルタミン酸）にホスゲンを作用させてγ−ベン
ジル−Ｌ−グルタメートＮ−カルボン酸無水物を
得、これを、例えばｎ−プロピル２−アミノエチ
ルジスルフイド（CH3CH2CH2S−
SCH2CH2NH2）を用いて重合させ重合体とし、
この重合体を酸分解又はアルカリ分解すると式
〔〕の化合物が得られる。無水物を、例えばシ
スタミン（H2NCH2CH2SSCH2CH2NH2）を用
いて重合させ重合体とし、この重合体を酸分解又
はアルカリ分解すると式〔〕の化合物が得られ
る。
また、例えば、γ−ベンジル−Ｌ−グルタメー
トＮ−カルボン酸無水物と、Ｌ−アラニンにホス
ゲンを作用させて得られたＬ−アラニンＮ−カル
ボン酸無水物の混合物（例えばモル比95：５）
を、４−アミノフエニルジスルフイド
（[Formula] In the case of serine, R 2 = CH 2 OH. In the reactive polymer of formula [], the unit consisting of such α-amino acids does not participate in any way in binding with cytotoxic substances, but it affects the fat solubility and water solubility of the reactive polymer, or the affinity with cell membranes, etc. It is useful for adjusting. Therefore,
If there is no particular need to adjust fat solubility or water solubility, it is practically advantageous to use one that does not contain such an α-amino acid unit (q=0 in formula []). m represents an integer from 1 to 4, preferably m
is 1 or 2. The reactive polymer represented by the formula [] also includes, for example, a polymer in which m=1 and m=2. n represents the number of structural units to which a cytotoxic substance is bound, p represents the number of structural units to which a cytotoxic substance is not bound, and q is an α-amino acid unit that does not have a carboxyl group in its side chain (the side chain is (which may be modified), but the arrangement of these units in the polymer is arbitrary. That is, in the formula [], for convenience, it is expressed as a block polymer, but it is not limited to this, and polymers obtained by ordinary methods are randomly arranged polymers. n=5 to 1500, preferably 10 to 500,
p=0-1500, preferably 0-500, q=
0 to 1500, preferably q=0 to 500. Among the reactive polymers represented by the formula [] of the present invention, those in which X is a hydrogen atom, that is, the following formula [
Two methods for producing the reactive polymer represented by -a] will be explained. [In formula [-a], Y, Z, W, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] The first method is to add a polymer represented by the formula [ ] containing a disulfide bond in the molecule, In [Formula []], R 3 represents an alkyl group, an aralkyl group, or an aryl group. Z, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] As mentioned above, by reacting a cytotoxic substance containing an amino group or an imino group in its molecule, and then reacting the reaction product with, for example, a thiol compound or a borohydride compound, disulfide in the obtained reaction product can be removed. This is a method of reductively breaking bonds. The second method is to use a polymer represented by the formula [] containing a disulfide bond in the molecule, In [formula], Z, W, R 1 , R 2 , m, n,
The definitions of p and q are the same as in formula []. W' represents a substituted or unsubstituted alkylene or phenylene group, which is the same as or different from W. R 1 ' is the same as or different from R 1 and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. n', p' and q' correspond to n, p and q, respectively, and represent the number of each structural unit in the polymer, but the arrangement of the structural units is random, and n and n',
The proportions of each constituent unit related to p and p′, q and q′ in the whole are 5 to 50%, 40 to 95%, and 0 to 10%, respectively.
It is. ] As mentioned above, a cytotoxic substance containing an amino group or an imino group in the molecule is reacted, and the reaction product is then reacted with a thiol compound or a borohydride compound to remove the disulfide bond in the resulting reaction product. This is a reductive cutting method. The reaction between a polymer having a disulfide bond in the molecule represented by the above formula [] or formula [] and a cytotoxic substance containing an amino group or imino group in the molecule is usually carried out using water, dimethylformamide, dimethylsulfoxide, etc. The reaction is carried out in a homogeneous reaction system using an organic solvent as the reaction solvent. During the reaction, the carboxyl groups in the polymer may be activated with, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide, or the carboxyl groups may be activated in the form of a mixed acid anhydride. You may also leave it as Appropriate reaction temperature is -40 to 100°C and reaction time is 10 minutes to 10 days. The ratio of the polymer to the cytotoxic substance used varies depending on the desired number of n, but it is generally appropriate to use an amount corresponding to 5 to 200% of the -COOZ groups in the polymer. The reaction between the cytotoxic substance-bound polymer thus obtained and the thiol compound is usually carried out in a homogeneous reaction system using water or an organic solvent such as dimethylformamide or dimethyl sulfoxide as the reaction solvent. Suitable thiol compounds include, for example:
These include dithiothreitol and 2-mercaptoethanol. The thiol compound is used in an amount of 1 to 100 times the mole of disulfide bonds in the polymer. The reaction temperature is preferably -5 to 70°C, and the reaction time is preferably 5 minutes to 10 days. When using borohydride compounds such as sodium borohydride, potassium borohydride, calcium borohydride, the reaction with the polymer is usually carried out in an aqueous solution. The method for producing the compound represented by the formula [] or [] will be described in detail in Reference Examples below, but the outline thereof is as follows. For example, glutamic acid benzyl ester (γ-benzyl-L-
Glutamic acid) is reacted with phosgene to obtain γ-benzyl-L-glutamate N-carboxylic acid anhydride, which is converted into, for example, n-propyl 2-aminoethyl disulfide (CH 3 CH 2 CH 2 S-
SCH 2 CH 2 NH 2 ) to make a polymer,
When this polymer is decomposed with an acid or alkali, a compound of formula [] is obtained. The anhydride is polymerized using, for example, cystamine (H 2 NCH 2 CH 2 SSCH 2 CH 2 NH 2 ) to form a polymer, and this polymer is decomposed with an acid or alkali to obtain a compound of the formula []. Also, for example, a mixture of γ-benzyl-L-glutamate N-carboxylic anhydride and L-alanine N-carboxylic anhydride obtained by reacting phosgene with L-alanine (e.g., molar ratio 95:5).
, 4-aminophenyl disulfide (
【式】）を用い
て共重合させて重合体とし、この共重合体を酸分
解又はアルカリ分解すると式〔〕の化合物が得
られる。
本発明の式〔〕で表わされる反応性重合体の
うち、Ｘが隣りの硫黄原子と共に活性ジスルフイ
ド結合を形成しうる基であるもの、即ち下記式
〔−ｂ〕で表わされる反応性重合体を製造する
方法について説明する。
〔式〔−ｂ〕において、X′は隣りの硫黄原子
と共に活性ジスルフイド結合を活性ジスルフイド
化合物の反応残基を表わし、式〔〕の水素原子
を除いたＸと同じである。Ｙ、Ｚ、Ｗ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。〕
その方法は、前記式〔−ａ〕で表わされる分
子中に細胞毒性物質を結合しかつチオール基を含
有する重合体と、活性ジスルフイド化合物を反応
させる方法である。活性ジスルフイド化合物とし
ては、例えば、２−ピリジルジスルフイド
（[Formula]) is copolymerized to obtain a polymer, and this copolymer is decomposed with an acid or alkali to obtain a compound of the formula []. Among the reactive polymers represented by the formula [] of the present invention, those in which X is a group capable of forming an active disulfide bond with the adjacent sulfur atom, that is, the reactive polymers represented by the following formula [-b] The manufacturing method will be explained. In [formula [-b], X' represents a reactive residue of an active disulfide compound that forms an active disulfide bond together with the adjacent sulfur atom, and is the same as X in formula [] except for the hydrogen atom. Y, Z, W, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] The method is a method in which a polymer having a cytotoxic substance bonded to the molecule represented by the above formula [-a] and containing a thiol group is reacted with an active disulfide compound. Examples of active disulfide compounds include 2-pyridyl disulfide (
【式】）、４−ピリジルジ スルフイド（[Formula]), 4-pyridyldi Sulfide (
【式】）、 ５，5′−ジチオビス（２−ニトロ安息香酸） （【formula】), 5,5'-dithiobis(2-nitrobenzoic acid) (
【式】）、
４−カルボキシ−２−ピリジルジスルフイド
（
）、Ｎ−オキシ−２−ピリジルジスルフイド
（[Formula]), 4-carboxy-2-pyridyl disulfide ( ), N-oxy-2-pyridyl disulfide (
【式】）、２−ニトロフエニ ルジスルフイド （[Formula]), 2-nitropheni Ludisulfide (
【式】）、４−ニトロ
−２−ピリジルジスルフイド
（[Formula]), 4-nitro-2-pyridyl disulfide (
【式】）、２− ベンゾチアゾイルジスルフイド （[Formula]), 2- Benzothiazoyl disulfide (
【式】）、２
−ベンゾイミダゾイルジスルフイド
（[Formula]), 2-benzimidazoyl disulfide (
無水テトラヒドロフラン120ml中に、γ−ベン
ジル−Ｌ−グルタミン酸10.0ｇを加え分散液を調
製した。これとは別に窒素雰囲気下、クロロギ酸
トリクロロメチル20mlを、カーボンブラツク10.0
ｇ上に徐々に70分かけて滴下しホスゲンを発生さ
せた。発生したホスゲンは、前記γ−ベンジル−
Ｌ−グルタミン酸の分散液に窒素雰囲気下で吸込
んだ。70分後に分散液は淡黄色透明液となつたの
で、ホスゲンを止め、その後窒素を1.5時間吸込
んで未反応のホスゲンを除去した。得られた透明
液から、窒素気流下減圧して溶媒を留去した
（140mmHg、27℃）。
残査に無水ｎ−ヘキサン150mlを加えて溶解し、
その後氷浴上で５分間撹拌したところ白色固体が
析出した。この固体を窒素雰囲気下に酢酸エチル
−ｎ−ヘキサン（無水）の系で２回再沈澱により
精製し、吸引ろ取後減圧乾燥して、γ−ベンジル
−Ｌ−グルタメートＮ−カルボン酸無水物（下記
構造式を有する）
7.75ｇを白色固体として得た。このものの融点
は94.0〜94.5℃（分解）であり、収率は69.8％で
あつた。
参考例 ２
〔Ｌ−グルタメートの重合体の合成(1)（式
〔〕の重合体）〕
γ−ベンジル−Ｌ−グルタメートＮ−カルボン
酸無水物7.75ｇを乾燥１，４−ジオキサン185ml
に窒素雰囲気下、撹拌しながら溶解した。かくし
て得られた溶液に、95mgのシスタミンを10mlの乾
燥ジオキサンに溶解して得られた溶液を添加混合
し、窒素雰囲気下に室温で24時間撹拌して重合反
応を行なわせた。反応後、反応混合物を４のイ
ソプロピルエーテル中に撹拌しながら加え、生成
した重合体を沈澱させた。沈澱した白色の重合体
を取し減圧下に乾燥したところ、収量は6.19ｇ
で収率は95.9％であつた。得られた重合体の平均
分子量を粘度法（ジクロル酢酸、25.0℃）で求め
たところ47300であつた。（P.Dotyら、J.Am.
Chem.Soc.、78巻、947頁、1956年参照）。得られ
た重合体は、用いた原料と開始剤及び反応機構か
ら、下記式のポリ−γ−ベンジル−Ｌ−グルタメ
ートを主体とするものであることが合理的に推定
され、また赤外吸収スペクトルによつても確認さ
れた。
上記で得られたポリ−γ−ベンジル−Ｌ−グル
タメートの3.11ｇを、トリフルオロ酢酸25.0mlと
アニソール4.5mlの混合液に溶解した。かくして
得られた溶液に、25.0mlのメタンスルホン酸を加
えて窒素雰囲気下氷浴上で20分間撹拌し、その後
室温で30分間撹拌して、γ−ベンジルエステルの
酸分解反応を行なつた。反応後、反応混合物を
450mlのイソプロピルエーテル中に撹拌しながら
加え、重合体を沈澱させた。沈澱した白色の重合
体を吸引取し、50mlの水に懸濁させた。これに
飽和重そう水約60mlを添加混合し、室温で30分間
撹拌してカルボキシル基の中和反応を行ない、均
一溶液を得た。その後、得られた反応液をセルロ
ースチユーブを用いて純水に対して４℃で３日間
透析し、次いで凍結乾燥して1.91ｇの白色固体を
得た。得られた固体を赤外吸収スペクトルで調べ
たところベンジルエステルの吸収は消失してお
り、かつカルボキシル基がナトリウム塩になつて
いることが確認された。ポリ−Ｌ−グルタメート
のナトリウム塩としての収率は89.3％であつた。
また、粘度法（食塩−燐酸バツフアー混合液、イ
オン強度0.11及び1.01、25.5℃）により求めた平
均分子量は29200であつた。（R.B.Hawkinsら、
Macromolecules、５巻、294頁、1972年参照）。
得られた重合体は、前記式〔〕において、Ｚ＝
Na、R1＝R1′＝Ｈ、Ｗ＝W′＝−CH2CH2−、ｌ
＝l′＝２、ｍ＝２、ｑ＝q′＝０の場合に相当する
下記式のポリ−Ｌ−グルタメートのナトリウム塩
を主体とするものである。
参考例 ３
〔Ｌ−グルタメートの重合体の合成(2)（式
〔〕の重合体）〕
γ−ベンジル−Ｌ−グルタメートＮ−カルボン
酸無水物5.50ｇを乾燥１，４−ジオキサン150ml
に窒素雰囲気下、撹拌しながら溶解した。かくし
て得られた溶液に、ｎ−プロピル２−アミノエチ
ルジスルフイド142mgを乾燥ジオキサン10mlに溶
解して得られた溶液を加え、窒素雰囲気下に室温
で40時間撹拌して重合反応を行なわせた。反応
後、反応混合物を４のイソプロピルエーテル中
に撹拌しつつ加え、生成した重合体を沈澱させ
た。沈澱を取し、減圧下に乾燥したところ、収
量は4.41ｇで収率は96.3％であつた。
次いで、得られた重合体4.00ｇを、トリフルオ
ロ酢酸35mlとアニソール5.0mlの混合液に溶解し、
さらに35mlのメタンスルホン酸を加えて窒素雰囲
気下氷冷下で30分間、室温下で30分間撹拌するこ
とにより、γ−ベンジルエステルを酸分解した。
反応終了後、反応混合物を、540mlのイソプロピ
ルエーテル中に撹拌しつつ加え、重合体を沈澱せ
しめ取した。これを5.0％重そう水100mlに溶解
して、カルボキシル基の中和反応を行ない、次い
で、反応液をセルロースチユーブを用いて、純水
に対して４℃で３日間透析した。得られた溶液を
凍結乾燥したところ、ナトリウムポリ−Ｌ−グル
タメート2.33ｇ（収率77.4％）が吸湿性綿状固体
として得られた。生成物の赤外吸収スペクトル
に、ベンジルエステルの吸収は見られず、カルボ
キシル基がナトリウム塩になつていることが確認
された。平均分子量は、前記と同様な方法で測定
したところ16700であつた。得られた重合体は、
前記式〔〕において、Ｚ＝Na、R1＝Ｈ、ｍ＝
２、R3＝−CH2CH2CH3、Ｗ＝−CH2CH2−、ｑ
＝０の場合に相当する下記式のポリ−Ｌ−グルタ
メートのナトリウム塩を主体とするものである。
参考例 ４
〔Ｌ−グルタメートとＬ−アラニンの共重合体
の合成（式〔〕の重合体）〕
γ−ベンジル−Ｌ−グルタメートＮ−カルボン
酸無水物10.0ｇとＬ−アラニンＮ−カルボン酸無
水物0.23ｇを、乾燥１，４−ジオキサン280mlに
窒素雰囲気下に加え、撹拌して溶解した。かくし
て得られた溶液に、198mgの４−アミノフエニル
ジスルフイドを10mlの乾燥ジオキサンに溶解して
得られた溶液を添加混合し、窒素雰囲気下に室温
で24時間撹拌して重合反応を行なわせた。反応
後、反応混合物を４のイソプロピルエーテル中
に撹拌しつつ加え、生成した重合体を沈澱物とし
た。重合体の沈澱を取し、減圧下に乾燥し、
8.21ｇを得た。収率は97％であつた。
得られた重合体は、用いた原料、開始剤、及び
反応機構から、下記のγ−ベンジル−Ｌ−グルタ
メートとＬ−アラニンの共重合体であることが合
理的に推定され、又、赤外吸収スペクトルによつ
ても確認された。
かくして得られた共重合体の4.0ｇを、トリフ
ルオロ酢酸30mlとアニソール5.0mlの混合液に溶
解した。かくして得られた溶液に、30mlのメタン
スルホン酸を加えて、窒素雰囲気下氷浴上で20分
間撹拌し、その後室温で30分間撹拌して、γ−ベ
ンジルエステルの酸分解反応を行なつた。反応
後、反応混合物を600mlのイソプロピルエーテル
中に撹拌しながら加え、重合体を沈澱させた。沈
澱した白色の重合体を吸引取し、65mlの水に懸
濁させた。これに飽和重そう水約80mlを添加混合
し、室温で30分間撹拌してカルボキシル基の中和
反応を行なうことにより均一溶液とした。得られ
た溶液をセルロースチユーブを用いて純水に対し
て４℃で３日間透析し、次いで凍結乾燥して2.45
ｇの白色固体を得た。得られた固体を赤外吸収ス
ペクトルで調べたところ、ベンジルエステルの吸
収は消失しており、かつカルボキシル基がナトリ
ウム塩になつていることが確認された。ナトリウ
ム塩としての収率は88％であつた。
得られた重合体は、前記式〔〕においてＺ＝
Na、R1＝R1′＝Ｈ、
A dispersion was prepared by adding 10.0 g of γ-benzyl-L-glutamic acid to 120 ml of anhydrous tetrahydrofuran. Separately, under a nitrogen atmosphere, add 20 ml of trichloromethyl chloroformate to carbon black 10.0
was gradually dropped over 70 minutes to generate phosgene. The generated phosgene is the γ-benzyl-
The dispersion of L-glutamic acid was sucked under a nitrogen atmosphere. After 70 minutes, the dispersion became a pale yellow transparent liquid, so the phosgene was stopped, and then nitrogen was sucked in for 1.5 hours to remove unreacted phosgene. The solvent was distilled off from the resulting transparent liquid under reduced pressure under a nitrogen stream (140 mmHg, 27°C). Add 150 ml of anhydrous n-hexane to the residue and dissolve it.
Thereafter, the mixture was stirred for 5 minutes on an ice bath, and a white solid was precipitated. This solid was purified by reprecipitation twice in a system of ethyl acetate-n-hexane (anhydrous) under a nitrogen atmosphere, collected by suction filtration, and dried under reduced pressure to produce γ-benzyl-L-glutamate N-carboxylic anhydride ( It has the following structural formula) Obtained 7.75 g as a white solid. The melting point of this product was 94.0-94.5°C (decomposition), and the yield was 69.8%. Reference Example 2 [Synthesis of L-glutamate polymer (1) (polymer of formula [])] 7.75 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride was mixed with 185 ml of dried 1,4-dioxane.
The mixture was dissolved in a nitrogen atmosphere with stirring. A solution obtained by dissolving 95 mg of cystamine in 10 ml of dry dioxane was added to and mixed with the solution thus obtained, and the mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours to carry out a polymerization reaction. After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitated white polymer was taken and dried under reduced pressure, the yield was 6.19g.
The yield was 95.9%. The average molecular weight of the obtained polymer was determined by the viscosity method (dichloroacetic acid, 25.0°C) and was found to be 47,300. (P. Doty et al., J. Am.
Chem.Soc., vol. 78, p. 947, 1956). It is reasonably estimated that the obtained polymer is mainly composed of poly-γ-benzyl-L-glutamate of the following formula from the raw materials and initiator used and the reaction mechanism, and the infrared absorption spectrum It was also confirmed by. 3.11 g of the poly-γ-benzyl-L-glutamate obtained above was dissolved in a mixed solution of 25.0 ml of trifluoroacetic acid and 4.5 ml of anisole. To the thus obtained solution, 25.0 ml of methanesulfonic acid was added, and the mixture was stirred for 20 minutes on an ice bath under a nitrogen atmosphere, and then stirred for 30 minutes at room temperature to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture
The polymer was precipitated by stirring into 450 ml of isopropyl ether. The precipitated white polymer was suctioned off and suspended in 50 ml of water. Approximately 60 ml of saturated deuterated water was added and mixed to this, and the mixture was stirred at room temperature for 30 minutes to carry out a carboxyl group neutralization reaction to obtain a homogeneous solution. Thereafter, the obtained reaction solution was dialyzed against pure water at 4° C. for 3 days using a cellulose tube, and then freeze-dried to obtain 1.91 g of white solid. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield of poly-L-glutamate as sodium salt was 89.3%.
Further, the average molecular weight determined by the viscosity method (salt-phosphoric acid buffer mixture, ionic strength of 0.11 and 1.01, 25.5°C) was 29,200. (R.B.Hawkins et al.
Macromolecules, vol. 5, p. 294, 1972).
In the above formula [], the obtained polymer has Z=
Na, R 1 = R 1 ′=H, W=W′=−CH 2 CH 2 −, l
It is mainly composed of a sodium salt of poly-L-glutamate of the following formula, which corresponds to the case where =l'=2, m=2, and q=q'=0. Reference Example 3 [Synthesis of L-glutamate polymer (2) (polymer of formula [])] 5.50 g of γ-benzyl-L-glutamate N-carboxylic acid anhydride was dried in 150 ml of 1,4-dioxane.
The mixture was dissolved in a nitrogen atmosphere with stirring. A solution obtained by dissolving 142 mg of n-propyl 2-aminoethyl disulfide in 10 ml of dry dioxane was added to the solution thus obtained, and the mixture was stirred at room temperature under a nitrogen atmosphere for 40 hours to carry out a polymerization reaction. . After the reaction, the reaction mixture was added to the isopropyl ether of 4 with stirring to precipitate the produced polymer. When the precipitate was collected and dried under reduced pressure, the yield was 4.41 g, with a yield of 96.3%. Next, 4.00 g of the obtained polymer was dissolved in a mixed solution of 35 ml of trifluoroacetic acid and 5.0 ml of anisole.
Further, 35 ml of methanesulfonic acid was added, and the mixture was stirred for 30 minutes under ice-cooling under a nitrogen atmosphere and for 30 minutes at room temperature, thereby acid decomposing the γ-benzyl ester.
After the reaction was completed, the reaction mixture was added to 540 ml of isopropyl ether with stirring, and the polymer was precipitated and collected. This was dissolved in 100 ml of 5.0% heavy sour water to carry out a carboxyl group neutralization reaction, and then the reaction solution was dialyzed against pure water at 4° C. for 3 days using a cellulose tube. The resulting solution was freeze-dried to yield 2.33 g (77.4% yield) of sodium poly-L-glutamate as a hygroscopic flocculent solid. No benzyl ester absorption was observed in the infrared absorption spectrum of the product, confirming that the carboxyl group was converted into a sodium salt. The average molecular weight was 16,700 when measured using the same method as above. The obtained polymer is
In the above formula [], Z=Na, R 1 =H, m=
2, R 3 = -CH 2 CH 2 CH 3 , W = -CH 2 CH 2 -, q
It is mainly composed of a sodium salt of poly-L-glutamate of the following formula, which corresponds to the case where =0. Reference Example 4 [Synthesis of copolymer of L-glutamate and L-alanine (polymer of formula [])] 10.0 g of γ-benzyl-L-glutamate N-carboxylic anhydride and L-alanine N-carboxylic anhydride 0.23 g of the compound was added to 280 ml of dry 1,4-dioxane under a nitrogen atmosphere and dissolved with stirring. To the thus obtained solution, a solution obtained by dissolving 198 mg of 4-aminophenyl disulfide in 10 ml of dry dioxane was added and mixed, and the mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours to perform a polymerization reaction. I set it. After the reaction, the reaction mixture was added to the isopropyl ether in step 4 with stirring, and the resulting polymer was precipitated. The polymer precipitate was collected and dried under reduced pressure.
8.21g was obtained. The yield was 97%. The obtained polymer is reasonably estimated to be a copolymer of γ-benzyl-L-glutamate and L-alanine as described below from the raw materials, initiator, and reaction mechanism used, and it is also It was also confirmed by absorption spectrum. 4.0 g of the thus obtained copolymer was dissolved in a mixed solution of 30 ml of trifluoroacetic acid and 5.0 ml of anisole. To the thus obtained solution, 30 ml of methanesulfonic acid was added, and the mixture was stirred for 20 minutes on an ice bath under a nitrogen atmosphere, and then stirred for 30 minutes at room temperature to carry out an acid decomposition reaction of the γ-benzyl ester. After the reaction, the reaction mixture was added to 600 ml of isopropyl ether with stirring to precipitate the polymer. The precipitated white polymer was suctioned off and suspended in 65 ml of water. Approximately 80 ml of saturated deuterated water was added and mixed to this, and the mixture was stirred at room temperature for 30 minutes to carry out a neutralization reaction of carboxyl groups, thereby forming a homogeneous solution. The resulting solution was dialyzed against pure water for 3 days at 4°C using a cellulose tube, and then lyophilized to 2.45
g of white solid was obtained. When the obtained solid was examined by infrared absorption spectrum, it was confirmed that the absorption of benzyl ester had disappeared and that the carboxyl group had become a sodium salt. The yield as sodium salt was 88%. The obtained polymer has Z= in the above formula []
Na, R 1 = R 1 ′ = H,
一方、Ｌ−グルタメートのナトリウム塩単位
のうちＬ−グルタメート−PDM結合体単位に
変つた割合をｘとすると、重合体中のＬ−グル
タメート単位の分子量Ｍは、Ｍ＝151×（１−
ｘ）＋344xで表わされ、従つてPDM残基のモ
ル濃度Ｃは、下記式で計算される。
Ｃ＝測定に用いた重合体の重量（ｇ）／Ｍ×
ｘ×1000／100＝4.075×10-3／151×（１−ｘ）＋344x
×ｘ×1000／100
……(b)
前記(a)式と(b)式よりｘを求めるとｘ＝0.212
となる。即ち、重合体中のＬ−グルタメートの
ナトリウム塩単位のうち21.2％がＬ−グルタメ
ート−PDM結合体単位に変つていると結論さ
れる。
また、重合体の平均分子量MWは下記式で計
算され、約37000と結論される。
MW＝193×Ｍ＝193×｛151×（１−ｘ）＋344
x｝≒37000
(3) ポリ−Ｌ−グルタメートのナトリウム塩−
PDM結合体中のジスルフイド結合の定量
（DTNB法）：
前記(1)で得られたポリ−Ｌ−グルタメートの
ナトリウム塩−PDM結合体14.8mg（4.00×10-7
モル）を0.1Mトリス・塩酸−１ｍＭ EDTA
溶液（PH8.50）500μに溶解し、これに0.1M
ジチオスレイトール水溶液50μを加えて、50
℃で２時間反応させた（ジスルフイド結合が切
断され−SH基となる）。その後、反応液に５，
5′−ジチオ−ビス（２−ニトロ安息香酸）
（DTNB）の0.25Mアセトン溶液50μを加え
て反応を行ない（−SH基が−STNB基に変換
する）、その後反応混合物をセフアデツクスＧ
−50（フアイン）カラムクロマトグラフイーに
付し、0.1M燐酸バツフアー−１ｍＭ EDTA
溶液（PH6.90）で溶出して、低分子化合物を除
き重合体のみを取り出した。得られた重合体溶
液にジチオスレイトールを加えて、重合体の末
端−STNB基よりTNB陰イオンを遊離せしめ
た（重合体は再び末端がSH基となる）。かくし
て得られた重合体溶液中の遊離したTNB陰イ
オンの吸光度（412nｍ）を実測し、前記(2)の
式(a)からTNB陰イオンの濃度を求めたところ
5.98×10-7モルであつた（式(a)においてｌ＝1.0
cm、ε＝1.36×104（TNB陰イオンの吸光係数）
である）。遊離したTNB陰イオンのモル数は、
重合体中の末端−STNB基のモル数に等しく、
従つて、また、これはジスルフイド結合を有す
る重合体のモル数の２倍に相当する。従つてポ
リ−Ｌ−グルタメートのナトリウム塩−PDM
結合体（4.00×10-7モル）中のジスルフイド結
合の含有率は
5.98×10-7／4.00×10-7×１／２×100＝74.8％
と計算される。即ち、約25％は分子中にジスル
フイド結合を含有しないものが含まれているこ
とがわかる。
(4) 末端SH−ポリ−Ｌ−グルタメート−PDM結
合体の製造（式〔−ａ〕の化合物）：
ポリ−Ｌ−グルタメートのナトリウム塩−
PDM結合体（PDM結合率21.2％）181.1mg
（4.89×10-6モル）を6.0mlの0.1Mトリス・塩酸
−１ｍＭ EDTA溶液（PH8.48）に溶解し、こ
れに0.02Mのジチオスレイトールを含む2.0ml
の0.1Mトリス・塩酸−１ｍＭ ETDA溶液
（PH8.45）を加え、反応系を密封し、50℃で100
分間反応させた。（この反応で、重合体中のジ
スルフイド結合が還元的に切断され、分子末端
にSH基を有するより低分子の重合体が２分子
生成する。）
次に、反応混合物に、活性化チオプロピルセ
フアロース6B樹脂（フアルマシア社製、樹脂
中に官能基として
On the other hand, if x is the proportion of L-glutamate sodium salt units converted to L-glutamate-PDM conjugate units, then the molecular weight M of L-glutamate units in the polymer is M = 151 x (1-
x)+344x, and therefore the molar concentration C of PDM residues is calculated by the following formula. C=weight of polymer used for measurement (g)/M×
x×1000/100=4.075× 10-3 /151×(1-x)+344x
×××1000/100
...(b) Calculating x from equations (a) and (b) above, x = 0.212
becomes. That is, it is concluded that 21.2% of the sodium salt units of L-glutamate in the polymer have been converted to L-glutamate-PDM conjugate units. Furthermore, the average molecular weight MW of the polymer is calculated using the following formula and concluded to be approximately 37,000. MW=193×M=193×{151×(1-x)+344
x}≒37000 (3) Sodium salt of poly-L-glutamate
Quantification of disulfide bonds in PDM conjugate (DTNB method): 14.8 mg (4.00×10 -7
mol) in 0.1M Tris-HCl-1mM EDTA
Dissolve in 500μ solution (PH8.50) and add 0.1M to this
Add 50μ of dithiothreitol aqueous solution and
The reaction was carried out at ℃ for 2 hours (the disulfide bond is cleaved to form a -SH group). After that, add 5,
5'-dithio-bis(2-nitrobenzoic acid)
The reaction was carried out by adding 50μ of a 0.25M acetone solution of (DTNB) (-SH group is converted to -STNB group), and then the reaction mixture was mixed with Sephadex G
-50 (fine) column chromatography, 0.1M phosphate buffer -1mM EDTA
It was eluted with a solution (PH6.90) to remove low molecular weight compounds and take out only the polymer. Dithiothreitol was added to the obtained polymer solution to release the TNB anion from the -STNB group at the end of the polymer (the end of the polymer becomes an SH group again). The absorbance (412 nm) of the free TNB anion in the polymer solution thus obtained was actually measured, and the concentration of the TNB anion was determined from equation (a) in (2) above.
It was 5.98×10 -7 mol (l=1.0 in formula (a)
cm, ε=1.36×10 4 (extinction coefficient of TNB anion)
). The number of moles of free TNB anion is
equal to the number of moles of terminal -STNB groups in the polymer,
This also therefore corresponds to twice the number of moles of polymer with disulfide bonds. Therefore, the sodium salt of poly-L-glutamate - PDM
The content of disulfide bonds in the conjugate (4.00 x 10 -7 mol) is calculated as 5.98 x 10 -7 /4.00 x 10 -7 x 1/2 x 100 = 74.8%. That is, it can be seen that about 25% of the molecules contain molecules that do not contain disulfide bonds. (4) Production of terminal SH-poly-L-glutamate-PDM conjugate (compound of formula [-a]): Sodium salt of poly-L-glutamate -
PDM conjugate (PDM binding rate 21.2%) 181.1mg
(4.89 × 10 -6 mol) was dissolved in 6.0 ml of 0.1 M Tris/HCl-1 mM EDTA solution (PH 8.48), and 2.0 ml containing 0.02 M dithiothreitol was added.
Add 0.1M Tris-HCl-1mM ETDA solution (PH8.45), seal the reaction system, and incubate at 50℃ for 100℃.
Allowed to react for minutes. (In this reaction, the disulfide bonds in the polymer are reductively cleaved, producing two molecules of a lower molecular weight polymer having an SH group at the end of the molecule.) Next, activated thiopropylacetate is added to the reaction mixture. Allose 6B resin (manufactured by Pharmacia, as a functional group in the resin)
【式】基を有す
る）8.0ｇ（乾燥重量）を0.25M燐酸ナトリウ
ムバツフアー（PH6.86）100mlに分散した分散
液を加え、一夜撹拌することによつて、分子末
端にSH基を有する重合体を樹脂に吸着させた。
（重合体は、樹脂−Ｓ−Ｓ−重合体の形で樹脂
に吸着される。）その後重合体を吸着した樹脂
を別し、0.25M燐酸ナトリウムバツフアー
（PH6.86）800mlで十分に洗浄した。（樹脂に吸
着されなかつた、即ち分子中にSH基を有しな
い本発明の副反応生成物は、この操作で除かれ
る。）洗浄した樹脂は30mlの0.1Mトリス・塩酸
−１ｍＭ EDTA溶液（PH8.5）に分散させ、
これに62mgのジチオスレイトールを加え、反応
系を密封して一夜撹拌し、重合体を再生させ
た。（この操作で、樹脂に吸着されていた重合
体は、樹脂から脱着し末端にSH基を有する重
合体として再生する。）その後、樹脂を別し
0.25M燐酸ナトリウムバツフアー（PH6.86）
400mlで洗浄し、液と洗液を合わせ、これに
塩酸を加えてPH４とすると、末端にSH基を有
する重合体の沈澱が生成した。沈澱は遠心分離
によつて採取し、0.1M酢酸ナトリウム−１ｍ
Ｍ EDTA溶液（PH3.96）6.2ml中に分散させ、
アルゴンを吹き込み密栓して冷却（４℃）下に
保存した。
得られた、末端にSH基を有する重合体の量
は、DTNB法による末端基定量によると4.86×
10-6モルであつた。またアセチル化PDMの
ε275nm＝1.62×104を適用して測定したPDM残
基の量は108.4×10-6モルであつた。従つて、
本結合体は、１分子中に１個の末端SH基と平
均22.3個（108.4／4.86）のPDM残基を含有し
ている。また、本結合体中のＬ−グルタメート
単位の総数は22.3／0.212＝105となるので、そ
の平均分子量は105（0.212×344＋0.788×151）
≒20000と計算される。
実施例 ２
本実施例は、末端に活性ジスルフイド結合を有
する重合体、末端Ｓ−TNB−ポリ−Ｌ−グルタ
メートのナトリウム塩−PDM結合体の製造例を
示す（式〔−ｂ〕の化合物）。
実施例１の(1)で得られたポリ−Ｌ−グルタメー
トのナトリウム塩−PDM結合体148mgを、0.1M
トリス・塩酸−１ｍＭ EDTA溶液（PH8.5）4.0
mlに溶解し、これに0.02Mのジチオスレイトール
を含む2.0mlの0.1Mトリス・塩酸−１ｍＭ
EDTA溶液をアルゴン気流下に加え、40℃で３
時間反応させた。
次に、反応混合物に、活性化チオプロピルセフ
アロース6B樹脂20ml（湿潤容積）を0.1M燐酸ナ
トリウムバツフアー−１ｍＭ EDTA溶液（PH
7.0）40mlに分散した分散液を加え、反応系をア
ルゴン置換して一夜撹拌し、分子末端にSH基を
有する重合体を樹脂に吸着させた。その後重合体
を吸着した樹脂を別し、0.1Mトリス・塩酸−
１ｍＭ EDTA溶液（PH8.0）337mlで十分に洗浄
した。洗浄した樹脂は40mlの0.1Mトリス・塩酸
−１ｍＭ EDTA溶液（PH8.5）に分散させ、こ
れに624mgの２−メルカプトエタノールを加え、
アルゴン気流下に３時間撹拌し、末端にSH基を
有する重合体を再生させた。その後、樹脂を別
して、0.01Mの２−メルカプトエタノールを含む
0.1Mトリス・塩酸−１ｍＭ EDTA溶液（PH
8.5）400mlで洗浄した。液と洗液を合わせ、こ
れを塩酸でPH４とし末端にSH基を有する重合体
を沈澱させ、４℃に１時間放置した後、遠心分離
操作により上清を除き更に沈澱を蒸留水で洗い、
重合体の沈澱の分散液を得た。
かくして得られた末端にSH基を有する重合体
の分散液に、５，5′−ジチオ−ビス（２−ニトロ
安息香酸）（DTNB）31mgを1N燐酸ナトリウム
−１ｍＭ EDTA溶液（PH8.0）30mlに溶解して
得られた溶液を加えて重合体を溶解しつつ反応せ
しめ、その後混合液を0.1N食塩溶液に対して４
℃で５日間透析した。回収液50.5mlを、セロフア
ン透析膜を使用して減圧による限外過に付し、
液量を1.05mlとした。これに0.15mlの0.1M燐酸ナ
トリウム−１ｍＭ EDTA溶液（PH7.0）を加え、
末端Ｓ−TNB−ポリ−Ｌ−グルタメートのナト
リウム塩−PDM結合体の0.01M燐酸ナトリウム
−0.1ｍＭ EDTA溶液（PH7.0）1.20mlを得た。
得られた、末端に−STNB基を有する重合体
の量は、DTNB法による末端基定量によると1.85
×10-6モルであつた。またアセチル化PDMの
ε275nm＝1.62×104を適用して測定したPDM残基
の量は53.47×10-6モルであつた。従つて、本結
合体は、１分子中に１個の末端−STNB基と平
均28.9個のPDM残基を含有している。また、本
結合体の平均分子量は約26000と計算された。
実施例 ３
(1) ポリ−Ｌ−グルタメートのナトリウム塩−ダ
ウノマイシン結合体の製造：
前記参考例２で得られたポリ−Ｌ−グルタメ
ートのナトリウム塩を主体とする重合体44.7mg
を５mlの水に溶解し、更にこれに１−エチル−
３−（３−ジメチルアミノプロピル）カルボジ
イミド塩酸塩（EDC）84.4mgを溶解させた。次
いで、得られた水溶液に、ダウノマイシン塩酸
塩25mgを10mlの水に溶解して得られた水溶液を
添加混合し、室温で１時間撹拌した。その後、
反応液をセロフアン透析膜を用いて0.9％食塩
水溶液に対して４℃で３日間透析した。回収液
11.0mlに1.0Mトリス・塩酸−１ｍＭ EDTA
溶液（PH8.5）1.22mlを加えることにより、ポ
リ−Ｌ−グルタメートのナトリウム塩−ダウノ
マイシン結合体の0.1Mトリス・塩酸−0.1ｍＭ
EDTA溶液（PH8.5）12.22mlを得た。
ダウノマイシンのＮ−アシル体の吸収
（485nｍ）の測定から、実施例１の(2)と同様な
方法で（但しε＝12000として）、前記の如くし
て得られたポリ−Ｌ−グルタメートのナトリウ
ム塩−ダウノマイシン結合体中のＬ−グルタメ
ート−ダウノマイシン結合単位量を求めたとこ
ろ25.6μモルであつた。
(2) 末端Ｓ−TNB−ポリ−Ｌ−グルタメートの
ナトリウム塩−ダウノマイシン結合体の製造
（式〔−ｂ〕の化合物）：
前記(1)で得られたポリ−Ｌ−グルタメートの
ナトリウム塩−ダウノマイシン結合体の0.1M
トリス・塩酸−0.1ｍＭ EDTA溶液（PH8.5）
12.20mlに、0.02Mのジチオスレイトールを含
む0.1Mトリス・塩酸−１ｍＭ EDTA溶液
（PH8.5）1.15mlを加え、アルゴン雰囲気下45℃
で１時間加温の後、室温下一夜放置した。これ
に、活性化チオプロピルセフアロース6B樹脂
3.83ｇを0.25M燐酸ナトリウム−１ｍＭ
EDTA溶液（PH6.85）40mlに分散した分散液を
加え、アルゴン雰囲気下26時間ゆるやかに撹拌
した後、樹脂を別し、0.05Mトリス・塩酸−
0.5ｍＭ EDTA溶液（PH8.0）で洗浄した。
次に樹脂を0.1Mトリス・塩酸−１ｍＭ
EDTA溶液（PH8.5）40mlに分散し、これに２
−メルカプトエタノール359mgを加え、アルゴ
ン雰囲気下ゆるやかに15時間撹拌した。反応後
樹脂を別し、10ｍＭの２−メルカプトエタノ
ールを含む0.05Mトリス・塩酸−0.5ｍＭ
EDTA溶液（PH8.0）50mlで洗浄し、液と洗
液をあわせて塩酸でPH4.0とし、冷却して沈澱
（末端SH−ポリ−Ｌ−グルタメートのナトリウ
ム塩−ダウノマイシン結合体）を生成せしめ
た。
沈澱を遠心分離し、水で３回洗つた後、５，
5′−ジチオ−ビス（２−ニトロ安息香酸）
（DTNB）11.64mgを1N−燐酸ナトリウム−１
ｍＭ EDTA溶液（PH8.0）５mlに溶解した溶
液を加え、沈澱を溶かすと同時に反応を行なわ
せ、末端のSH基を活性ジスルフイド基（末端
Ｓ−TNB基）に変換させた。生成した末端Ｓ
−TNB−ポリ−Ｌ−グルタメートのナトリウ
ム塩−ダウノマイシン結合体を精製するため
に、上記反応混合物を、0.05N燐酸ナトリウム
−0.5ｍＭ EDTA（PH6.90）で平衡化されたセ
フアデツクスＧ−25（フアイン）カラムクロマ
トグラフイーにかけ、3.93mlずつ30個のフラク
シヨンに分けた。ダウノマイシンのＮ−アシル
体の吸収（485nｍ）及びジチオスレイトール
添加による412nｍの吸光度増加量
（A dispersion of 8.0 g (dry weight) of [formula] having a group in 100 ml of 0.25 M sodium phosphate buffer (PH6.86) was added and stirred overnight. The coalescence was adsorbed onto the resin.
(The polymer is adsorbed to the resin in the form of resin-S-S-polymer.) The resin adsorbed with the polymer is then separated and thoroughly washed with 800 ml of 0.25M sodium phosphate buffer (PH6.86). did. (Side reaction products of the present invention that were not adsorbed to the resin, that is, do not have SH groups in their molecules, are removed by this operation.) The washed resin was mixed with 30 ml of 0.1M Tris/HCl-1mM EDTA solution (PH8 .5) dispersed in
To this was added 62 mg of dithiothreitol, and the reaction system was sealed and stirred overnight to regenerate the polymer. (With this operation, the polymer adsorbed to the resin is desorbed from the resin and regenerated as a polymer with an SH group at the end.) After that, the resin is separated.
0.25M sodium phosphate buffer (PH6.86)
After washing with 400 ml, the liquid and the washing liquid were combined, and hydrochloric acid was added to the mixture to adjust the pH to 4, resulting in the formation of a precipitate of a polymer having an SH group at the end. The precipitate was collected by centrifugation and diluted with 0.1M sodium acetate-1m
Dispersed in 6.2 ml of M EDTA solution (PH3.96),
Argon was blown into the container, the container was tightly stoppered, and the container was stored under cooling (4° C.). According to the terminal group determination using the DTNB method, the amount of the obtained polymer having an SH group at the end was 4.86×
It was 10 -6 mol. Further, the amount of PDM residues measured by applying ε 275 nm =1.62×10 4 of acetylated PDM was 108.4×10 −6 mol. Therefore,
This conjugate contains one terminal SH group and an average of 22.3 (108.4/4.86) PDM residues in one molecule. Also, the total number of L-glutamate units in this conjugate is 22.3/0.212 = 105, so its average molecular weight is 105 (0.212 x 344 + 0.788 x 151).
It is calculated as ≒20000. Example 2 This example shows an example of the production of a polymer having an active disulfide bond at the terminal, a sodium salt of terminal S-TNB-poly-L-glutamate-PDM conjugate (compound of formula [-b]). 148 mg of the poly-L-glutamate sodium salt-PDM conjugate obtained in (1) of Example 1 was added to 0.1 M
Tris/HCl-1mM EDTA solution (PH8.5) 4.0
ml of 0.1M Tris-HCl - 1mM containing 0.02M dithiothreitol.
Add EDTA solution under argon stream and incubate at 40℃ for 3
Allowed time to react. Next, 20 ml (wet volume) of activated thiopropyl sepharose 6B resin was added to the reaction mixture in 0.1 M sodium phosphate buffer-1 mM EDTA solution (PH
7.0) 40 ml of the dispersion was added, the reaction system was purged with argon, and stirred overnight to adsorb the polymer having an SH group at the molecular end to the resin. After that, the resin that adsorbed the polymer was separated, and 0.1M Tris/hydrochloric acid was added.
It was thoroughly washed with 337 ml of 1 mM EDTA solution (PH8.0). The washed resin was dispersed in 40ml of 0.1M Tris/HCl-1mM EDTA solution (PH8.5), and 624mg of 2-mercaptoethanol was added to this.
The mixture was stirred for 3 hours under an argon stream to regenerate the polymer having an SH group at the end. Then, separate the resin and add 0.01M 2-mercaptoethanol.
0.1M Tris/HCl-1mM EDTA solution (PH
8.5) Washed with 400ml. The solution and washing solution were combined, the pH was adjusted to 4 with hydrochloric acid, a polymer having an SH group at the end was precipitated, and after being left at 4°C for 1 hour, the supernatant was removed by centrifugation, and the precipitate was further washed with distilled water.
A dispersion of polymer precipitate was obtained. 31 mg of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was added to the thus obtained dispersion of the polymer having an SH group at the terminal and 30 ml of 1N sodium phosphate-1mM EDTA solution (PH8.0). The solution obtained by dissolving the polymer was added to react while dissolving the polymer, and then the mixture was diluted with 0.1N salt solution at 4%
Dialysis was performed at ℃ for 5 days. 50.5 ml of the recovered liquid was subjected to ultrafiltration under reduced pressure using a cellophane dialysis membrane,
The liquid volume was 1.05ml. Add 0.15ml of 0.1M sodium phosphate-1mM EDTA solution (PH7.0) to this,
1.20 ml of a 0.01M sodium phosphate-0.1mM EDTA solution (PH7.0) of the terminal S-TNB-poly-L-glutamate sodium salt-PDM conjugate was obtained. The amount of the obtained polymer having a -STNB group at the end was 1.85 according to end group determination using the DTNB method.
It was ×10 -6 mol. Further, the amount of PDM residues measured by applying ε 275 nm =1.62×10 4 of acetylated PDM was 53.47×10 −6 mol. Therefore, the present conjugate contains one terminal -STNB group and an average of 28.9 PDM residues in one molecule. Furthermore, the average molecular weight of this conjugate was calculated to be approximately 26,000. Example 3 (1) Production of poly-L-glutamate sodium salt-daunomycin conjugate: 44.7 mg of the polymer mainly composed of the poly-L-glutamate sodium salt obtained in Reference Example 2 above.
was dissolved in 5 ml of water, and 1-ethyl-
84.4 mg of 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was dissolved. Next, an aqueous solution obtained by dissolving 25 mg of daunomycin hydrochloride in 10 ml of water was added to and mixed with the obtained aqueous solution, and the mixture was stirred at room temperature for 1 hour. after that,
The reaction solution was dialyzed against a 0.9% saline solution at 4° C. for 3 days using a cellophane dialysis membrane. Recovery liquid
11.0ml 1.0M Tris-HCl - 1mM EDTA
Sodium salt of poly-L-glutamate-daunomycin conjugate by adding 1.22 ml of solution (PH8.5) in 0.1 M Tris-HCl-0.1 mM
12.22 ml of EDTA solution (PH8.5) was obtained. From the measurement of the absorption (485 nm) of the N-acyl form of daunomycin, the sodium of poly-L-glutamate obtained as described above was determined in the same manner as in Example 1 (2) (however, with ε = 12000). The amount of L-glutamate-daunomycin binding units in the salt-daunomycin conjugate was determined to be 25.6 μmol. (2) Production of terminal S-TNB-sodium salt of poly-L-glutamate-daunomycin conjugate (compound of formula [-b]): Sodium salt of poly-L-glutamate obtained in (1) above-daunomycin 0.1M of conjugate
Tris/HCl-0.1mM EDTA solution (PH8.5)
Add 1.15 ml of 0.1 M Tris-HCl-1 mM EDTA solution (PH8.5) containing 0.02 M dithiothreitol to 12.20 ml, and heat at 45°C under an argon atmosphere.
After heating for 1 hour, the mixture was left at room temperature overnight. This is combined with activated thiopropyl sepharose 6B resin.
3.83g of 0.25M sodium phosphate - 1mM
The dispersion was added to 40 ml of EDTA solution (PH6.85), stirred gently for 26 hours under an argon atmosphere, and then the resin was separated and mixed with 0.05 M Tris/hydrochloric acid.
Washed with 0.5mM EDTA solution (PH8.0). Next, the resin was mixed with 0.1M Tris/HCl-1mM
Disperse in 40ml of EDTA solution (PH8.5) and add 2
- 359 mg of mercaptoethanol was added, and the mixture was gently stirred for 15 hours under an argon atmosphere. After the reaction, separate the resin and add 0.05M Tris/HCl containing 10mM 2-mercaptoethanol to 0.5mM
Washed with 50 ml of EDTA solution (PH8.0), combined the solution and washing solution, adjusted to pH4.0 with hydrochloric acid, and cooled to form a precipitate (terminal SH-poly-L-glutamate sodium salt-daunomycin conjugate). Ta. After centrifuging the precipitate and washing it three times with water, 5.
5'-dithio-bis(2-nitrobenzoic acid)
(DTNB) 11.64mg 1N-sodium phosphate-1
A solution dissolved in 5 ml of mM EDTA solution (PH 8.0) was added to dissolve the precipitate, and at the same time a reaction was carried out to convert the terminal SH group into an active disulfide group (terminal S-TNB group). The generated terminal S
To purify the -TNB-poly-L-glutamate sodium salt-daunomycin conjugate, the above reaction mixture was mixed with Sephadex G-25 (Fine) equilibrated with 0.05N sodium phosphate-0.5mM EDTA (PH6.90). ) The mixture was subjected to column chromatography and divided into 30 fractions of 3.93 ml each. Absorption of N-acyl form of daunomycin (485 nm) and increase in absorbance at 412 nm due to addition of dithiothreitol (
に入れて計算すると0.178ｍモルであつた。
(2) 末端SH−ポリ−Ｌ−グルタメート−ara−
Ｃ結合体の製造（式〔−ａ〕の化合物）：
前記(1)で得られたポリ−Ｌ−グルタメートの
ナトリウム塩−ara−Ｃ結合体の溶液2.8mlを、
17.2mlの0.1Mトリス・塩酸−１ｍＭ EDTA
溶液（PH8.5）と混合し、全体を20mlとした。
これにジチオスレイトール7.7mgを加え45℃で
３時間反応させ、重合体中のジスルフイド結合
を切断し末端SH基を生成させた。次いで反応
液を塩酸でPH3.8とし、重合体を沈澱させ、生
じた沈澱は遠心分離により分離し希塩酸で３回
洗浄した。かくして得られた重合体の沈澱を、
活性化チオプロピルセフアロース6B樹脂3.0ml
（湿潤体積）を0.1M燐酸ナトリウム−１ｍＭ
EDTA溶液（PH7.8）７mlに分散して得られた
分散液10ml中に溶解し、アルゴン雰囲気下に４
℃で５時間ゆるやかに撹拌し、末端SH基を有
する重合体を樹脂に吸着させた。その後、樹脂
を別し、0.05Mトリス・塩酸−0.5ｍＭ
EDTA溶液（PH8.0）50mlで洗浄した。そして、
次に樹脂を0.1Mトリス・塩酸−１ｍＭ
EDTA溶液（PH8.0）10ml中に分散し、これに
ジチオスレイトール92.4mgを加え、アルゴン雰
囲気下に４℃で５時間ゆるやかに撹拌し、末端
SH基を再生させた。樹脂を別し、0.05Mト
リス・塩酸−0.5ｍＭ EDTA溶液（PH8.0）30
mlで洗浄した。液と洗液を合わせて、これに
塩酸を加えPHを4.0とし、冷却することによつ
て沈澱（末端SH−ポリ−Ｌ−グルタメート−
ara−Ｃ結合体）を析出させた。沈澱は遠心分
離により分離し、希塩酸で３回洗浄した後、
0.1M酢酸ナトリウムバツフアー−１ｍＭ
EDTA溶液（PH3.5）中に分散させ（分散液と
して2.0ml）４℃で保存した。
かくして得られた末端SH−ポリ−Ｌ−グル
タメート−ara−Ｃ結合体中の−SH量の
DTNB法による測定量は0.63μモルであつた。
また結合したara−Ｃの量は（ara−ＣのN4位
アシル体のε299nm＝8000として測定）13.5μモル
であつた。従つて、本結合体は、１分子中に１
個の末端SH基と平均21.4個のara−Ｃ残基を含
有している。
実施例 ６
(1) Ｌ−グルタメートとＬ−アラニン共重合体の
ナトリウム塩−メルフアラン結合体の製造：
前記参考例４で得られたＬ−グルタメートと
Ｌ−アラニン共重合体のナトリウム塩を主体と
する重合体450mgを10mlの水に溶解し、これに、
１−エチル−３−（３−ジメチルアミノプロピ
ル）カルボジイミド塩酸塩（EDC、縮合剤）
66.3mgを５mlの水に溶解して得られた水溶液を
加えた。かくして得られた溶液に、制ガン剤と
して公知のＰ−〔ビス（２−クロロエチル）ア
ミノ〕Ｌ−フエニルアラニン（メルフアラン）
88.5mgを20mlの水に溶解して得られた水溶液
を、10分間かかつて滴下し、その反応液を室温
で一夜撹拌した。その後、反応液に塩酸を添加
し、PHを4.0に調節したところ、白色沈澱を生
じた。これを遠心分離し、希塩酸で洗浄した。
次いで、得られた白色固体を0.1Nカセイソー
ダ水溶液20mlに溶解し、セロフアン透析チユー
ブを用いて純水に対して４℃で３日間透析し
た。透析物を凍結乾燥して、Ｌ−グルタメート
−Ｌ−アラニン共重合体ナトリウム塩−メルフ
アラン結合体の綿状固体484mgを得た。
得られた生成物の水溶液の紫外線吸収スペク
トルは258.5nｍと302.0nｍに最大吸収を示すこ
とより、メルフアランの一級アミノ基が、共重
合体中のＬ−グルタミン酸残基中のカルボキシ
ル基とアミド結合を形成して、重合体に結合し
ていることが判明した。便宜上、ポリマー結合
メルフアランの分子吸光系数が、メルフアラン
の分子吸光系数（ε258.5nm＝1.37×104）と同一
とみなして測定した結果、上記綿状固体484mg
に含有されるメルフアランの量は0.263ミリモ
ルであつた。
(2) 末端SH−ポリ−Ｌ−グルタメート−Ｌ−ア
ラニン−メルフアラン結合体の製造（式〔−
ａ〕の化合物）：
前記(1)で得られたポリ−Ｌ−グルタメート−
Ｌ−アラニン−メルフアラン結合体の固体400
mgを、15mlの0.1Mトリス・塩酸−１ｍＭ
EDTA溶液（PH8.5）に溶解し、これにジチオ
スレイトール12.8mgを加え、反応系を密閉して
45℃で３時間反応させて重合体中のジスルフイ
ド結合を切断し、末端SH基を生成させた。次
いで反応液を1N塩酸でPH3.4とし、重合体を沈
澱させ、生じた沈澱を遠心分離により分離し、
希塩酸で３回洗浄した。かくして得られた重合
体の沈澱を0.1N NaOH溶液30mlに溶解し、得
られた溶液を、活性化チオプロピルセフアロー
ス6B樹脂46ml（湿潤体積）を0.1M燐酸ナトリ
ウム−１ｍＭ EDTA溶液（PH7.5）50mlに分
散して得られた分散液に加えた後、窒素雰囲気
下に室温下ゆるやかに撹拌し、末端SH基を有
する重合体を樹脂に吸着させた。樹脂を別
し、0.1Mトリス・塩酸−１ｍＭ EDTA溶液
（PH8.0）1000mlで洗浄した。次に、樹脂を
0.1Mトリス・塩酸−１ｍＭ EDTA溶液（PH
8.5）70ml中に分散し、これにジチオスレイト
ール770mgを加え、窒素雰囲気下に一夜ゆるや
かに撹拌し、末端SH基を再生させた。樹脂を
別し、0.05Mトリス・塩酸−0.5ｍＭ
EDTA溶液（PH8.5）300mlで洗浄した。液
と、洗液を合わせ、1N塩酸を加えてPHを3.5と
し、冷却することによつて、末端SH−ポリ−
Ｌ−グルタメート−Ｌ−アラニン−メルフアラ
ン結合体を析出させた。沈澱を遠心分離により
分離し、希塩酸で３回洗浄したのち、0.1M酢
酸ナトリウムバツフアー１ｍＭ EDTA（PH
3.0）10ml中に分散させ４℃で保存した。
かくして得られた末端SH−ポリ−Ｌ−グル
タメート−Ｌ−アラニン−メルフアラン結合体
中の−SH基量のDTNB法による測定量は9.1×
10-6モルであつた。また、結合したメルフアラ
ンの量は結合体残基のε258.5nm＝1.37×104とし
て吸光度より測定した結果、92.1×10-6モルで
あつた。従つて、本結合体は１分子中に１個の
末端SH基と平均10.1個のメルフアラン残基を
含有している。
When calculated by adding , it was 0.178 mmol. (2) Terminal SH-poly-L-glutamate-ara-
Production of C conjugate (compound of formula [-a]): 2.8 ml of the solution of the poly-L-glutamate sodium salt-ara-C conjugate obtained in (1) above,
17.2ml 0.1M Tris-HCl - 1mM EDTA
solution (PH8.5) to make a total volume of 20 ml.
7.7 mg of dithiothreitol was added to this and reacted at 45°C for 3 hours to cleave the disulfide bonds in the polymer and generate terminal SH groups. Next, the reaction solution was adjusted to pH 3.8 with hydrochloric acid to precipitate the polymer, and the resulting precipitate was separated by centrifugation and washed three times with diluted hydrochloric acid. The polymer precipitate thus obtained was
Activated thiopropyl sepharose 6B resin 3.0ml
(wet volume) 0.1M sodium phosphate - 1mM
Dissolved in 10 ml of dispersion obtained by dispersing in 7 ml of EDTA solution (PH 7.8), and incubated for 4 hours under an argon atmosphere.
The mixture was gently stirred at ℃ for 5 hours, and the polymer having a terminal SH group was adsorbed onto the resin. Then, separate the resin and add 0.05M Tris/HCl - 0.5mM
Washed with 50 ml of EDTA solution (PH8.0). and,
Next, the resin was mixed with 0.1M Tris/HCl-1mM
It was dispersed in 10 ml of EDTA solution (PH8.0), 92.4 mg of dithiothreitol was added thereto, and the mixture was gently stirred at 4°C for 5 hours under an argon atmosphere.
The SH group was regenerated. Separate the resin and add 0.05M Tris/HCl-0.5mM EDTA solution (PH8.0) 30
Washed with ml. The solution and washing solution were combined, hydrochloric acid was added to the mixture to adjust the pH to 4.0, and the mixture was cooled to form a precipitate (terminated SH-poly-L-glutamate).
ara-C conjugate) was precipitated. The precipitate was separated by centrifugation, washed three times with diluted hydrochloric acid,
0.1M sodium acetate buffer - 1mM
It was dispersed in an EDTA solution (PH3.5) (2.0 ml as a dispersion) and stored at 4°C. The amount of -SH in the terminal SH-poly-L-glutamate-ara-C conjugate thus obtained
The amount measured by the DTNB method was 0.63 μmol.
The amount of bound ara-C was 13.5 μmol (measured assuming that ε 299nm of the N4 -acyl form of ara-C was 8000). Therefore, this conjugate contains 1 in 1 molecule.
It contains terminal SH groups and an average of 21.4 ara-C residues. Example 6 (1) Production of sodium salt of L-glutamate and L-alanine copolymer-melphalan conjugate: Mainly the sodium salt of L-glutamate and L-alanine copolymer obtained in Reference Example 4 above. Dissolve 450 mg of the polymer in 10 ml of water, and add
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, condensing agent)
An aqueous solution obtained by dissolving 66.3 mg in 5 ml of water was added. P-[bis(2-chloroethyl)amino]L-phenylalanine (Melphalan), which is known as an anticancer agent, is added to the solution thus obtained.
An aqueous solution obtained by dissolving 88.5 mg in 20 ml of water was added dropwise over a period of 10 minutes, and the reaction was stirred overnight at room temperature. Thereafter, hydrochloric acid was added to the reaction solution to adjust the pH to 4.0, and a white precipitate was produced. This was centrifuged and washed with dilute hydrochloric acid.
Next, the obtained white solid was dissolved in 20 ml of 0.1N caustic soda aqueous solution and dialyzed against pure water at 4°C for 3 days using a cellophane dialysis tube. The dialysate was freeze-dried to obtain 484 mg of a flocculent solid of L-glutamate-L-alanine copolymer sodium salt-melphalan conjugate. The ultraviolet absorption spectrum of the aqueous solution of the obtained product shows maximum absorption at 258.5 nm and 302.0 nm, indicating that the primary amino group of melphalan binds the carboxyl group and amide bond in the L-glutamic acid residue in the copolymer. was found to form and bind to the polymer. For convenience, the molecular extinction number of polymer-bound melphalan was assumed to be the same as that of melphalan (ε 258.5 nm = 1.37 × 10 4 ), and the result was 484 mg of the flocculent solid.
The amount of melphalan contained in the sample was 0.263 mmol. (2) Production of terminal SH-poly-L-glutamate-L-alanine-melphalan conjugate (formula [-
Compound a): Poly-L-glutamate obtained in (1) above
L-alanine-melphalan conjugate solid 400
mg to 15ml of 0.1M Tris-HCl - 1mM
Dissolve in EDTA solution (PH8.5), add 12.8 mg of dithiothreitol, and seal the reaction system.
The reaction was carried out at 45°C for 3 hours to cleave disulfide bonds in the polymer and generate terminal SH groups. Next, the reaction solution was adjusted to pH 3.4 with 1N hydrochloric acid to precipitate the polymer, and the resulting precipitate was separated by centrifugation.
Washed three times with diluted hydrochloric acid. The polymer precipitate thus obtained was dissolved in 30 ml of 0.1 N NaOH solution, and the resulting solution was mixed with 46 ml (wet volume) of activated thiopropyl sepharose 6B resin in 0.1 M sodium phosphate-1 mM EDTA solution (PH 7.5). ) After adding it to the obtained dispersion liquid, it was stirred gently at room temperature under a nitrogen atmosphere, and the polymer having a terminal SH group was adsorbed onto the resin. The resin was separated and washed with 1000 ml of 0.1M Tris/HCl-1mM EDTA solution (PH8.0). Next, add the resin
0.1M Tris/HCl-1mM EDTA solution (PH
8.5) 770 mg of dithiothreitol was added to the dispersion in 70 ml, and the mixture was gently stirred overnight under a nitrogen atmosphere to regenerate the terminal SH group. Separate the resin and add 0.05M Tris/HCl - 0.5mM
Washed with 300 ml of EDTA solution (PH8.5). The solution and washing solution were combined, 1N hydrochloric acid was added to adjust the pH to 3.5, and by cooling, the terminal SH-poly-
L-glutamate-L-alanine-melphalan conjugate was precipitated. The precipitate was separated by centrifugation, washed three times with diluted hydrochloric acid, and then mixed with 0.1M sodium acetate buffer and 1mM EDTA (PH
3.0) Dispersed in 10 ml and stored at 4°C. The amount of -SH groups in the thus obtained terminal SH-poly-L-glutamate-L-alanine-melphalan conjugate measured by the DTNB method was 9.1×
It was 10 -6 mol. Further, the amount of bound melphalan was 92.1×10 -6 mol, as measured by absorbance assuming ε 258.5 nm of the bound residue = 1.37×10 4 . Therefore, this conjugate contains one terminal SH group and an average of 10.1 melphalan residues in one molecule.
Claims (1)
重合度（ｎ＋ｐ＋ｑ）が10〜1500の反応性重合
体。 ［式〔〕において、Ｘは水素原子又は２−ピリ
ジルチオ基、４−ピリジルチオ基、３−カルボキ
シ−４−ニトロフエニルチオ基、４−カルボキシ
−２−ピリジルチオ基、Ｎ−オキシ−２−ピリジ
ルチオ基、２−ニトロフエニルチオ基、４−ニト
ロ−２−ピリジルチオ基、２−ベンゾチアゾイル
チオ基、２−ベンゾイミダゾイルチオ基及びＮ−
フエニルアミノ−N′−フエニルイミノメチルチ
オ基からなる群から選ばれた隣の硫黄原子と共に
活性ジスルフイド結合を形成しうる基を表す。Ｙ
は分子中にアミノ基又はイミノ基を含む抗腫瘍性
のアルキル化剤、代謝拮抗剤又は抗生物質のアミ
ノ基又はイミノ基反応残基を表す。Ｚは水素原子
又は１価の陽イオンを表す。Ｗは置換又は非置換
のアルキレン又はフエニレン基を表す。R1は水
素原子又は炭素数１〜４のアルキル基を表す。
R2はα−アミノ酸のα位側鎖（但しカルボキシ
ル基を有する基は除く）を表す。ｎ、ｐ及びｑは
重合体中の各構成単位の数を表すが、構成単位の
配列はランダムであり、ｎ、ｐ、ｑに係る各構成
単位の全体に占める割合は、それぞれ５〜50％、
40〜95％、０〜10％である。ｍは１〜４の整数を
表す。］ ２ 分子中にジスルフイド結合を含有する式
〔〕で表される重合度（ｎ＋ｐ＋ｑ）が10〜
1500の重合体に、 ［式中〔〕において、R3はアルキル基、アラ
ルキル基又はアリール基を表す。Ｚ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。］ 分子中にアミノ基又はイミノ基を含む細胞毒性
物質を反応させ、次いで反応生成物のジスルフイ
ド結合を還元的に切断することを特徴とする、細
胞毒性物質を結合した式〔−ａ〕で表される反
応性重合体の製造法。 ［式〔−ａ〕において、Ｙ、Ｚ、Ｗ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。］ ３ 分子中に細胞毒性物質を結合しかつチオール
基を含有する式〔−ａ〕で表される重合度（ｎ
＋ｐ＋ｑ）が10〜1500の重合体と、 ［式〔−ａ〕において、Ｙ、Ｚ、Ｗ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。］ 活性ジスルフイド化合物を反応させることを特
徴とする、細胞毒性物質を結合した式〔−ｂ〕
で表される反応性重合体の製造法。 ［式〔−ｂ〕において、X′は隣の硫黄原子と
共に活性ジスルフイド結合を形成しうる活性ジス
ルフイド化合物の反応残基を表す。Ｙ、Ｚ、Ｗ、
R1、R2、ｍ、ｎ、ｐ及びｑの定義は式〔〕の
場合と同じ。］ ４ 分子中にジスルフイド結合を含有する式
〔〕で表される重合度（ｎ＋ｐ＋ｑ＋n′＋p′＋
q′）が20〜3000の重合体に、 ［式〔〕において、Ｚ、Ｗ、R1、R2、ｍ、ｎ、
ｐ及びｑの定義は式〔〕の場合と同じ。W′は
Ｗと同一又は異なる、置換又は非置換のアルキレ
ン又はフエニレン基を表す。R1′はR1と同一又は
異なり、水素原子又は炭素数１〜４のアルキル基
を表す。n′、p′及びq′はそれぞれｎ、ｐ及びｑに
対応し重合体中の各構成単位の数を表すが、構成
単位の配列はランダムであり、ｎとn′、ｐとp′、
ｑとq′に係る各構成単位の全体に占める割合は、
それぞれ５〜50％、40〜95％、０〜10％である。］ 分子中にアミノ基又はイミノ基を含む細胞毒性
物質を反応させ、次いで反応生成物のジスルフイ
ド結合を還元的に切断することを特徴とする、細
胞毒性物質を結合した式〔−ａ〕で表される反
応性重合体の製造法。 ［式〔−ａ〕において、Ｙ、Ｚ、Ｗ、R1、R2、
ｍ、ｎ、ｐ及びｑの定義は式〔〕の場合と同
じ。］[Scope of Claims] 1. A reactive polymer having a degree of polymerization (n+p+q) of 10 to 1500, represented by the formula [], in which a cytotoxic substance is bound. In [Formula [], , 2-nitrophenylthio group, 4-nitro-2-pyridylthio group, 2-benzothiazoylthio group, 2-benzimidazoylthio group and N-
Represents a group selected from the group consisting of phenylamino-N'-phenyliminomethylthio groups that can form an active disulfide bond with an adjacent sulfur atom. Y
represents an amino group- or imino-reactive residue of an antitumor alkylating agent, antimetabolite, or antibiotic containing an amino group or imino group in the molecule. Z represents a hydrogen atom or a monovalent cation. W represents a substituted or unsubstituted alkylene or phenylene group. R 1 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
R 2 represents the α-position side chain of the α-amino acid (excluding groups having a carboxyl group). n, p, and q represent the number of each structural unit in the polymer, but the arrangement of the structural units is random, and the proportion of each structural unit related to n, p, and q in the total is 5 to 50%. ,
40-95%, 0-10%. m represents an integer of 1 to 4. ] 2 Polymerization degree (n + p + q) represented by the formula [] containing a disulfide bond in the molecule is 10 ~
1500 polymers, [In the formula [], R 3 represents an alkyl group, an aralkyl group, or an aryl group. Z, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] A cytotoxic substance-bound compound represented by the formula [-a] characterized by reacting a cytotoxic substance containing an amino group or an imino group in the molecule, and then reductively cleaving the disulfide bond of the reaction product. A method for producing reactive polymers. [In formula [-a], Y, Z, W, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] 3 Polymerization degree (n
+p+q) is a polymer of 10 to 1500, [In formula [-a], Y, Z, W, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ] A formula combining a cytotoxic substance [-b] characterized by reacting an active disulfide compound
A method for producing a reactive polymer represented by [In formula [-b], X' represents a reactive residue of an active disulfide compound capable of forming an active disulfide bond with the adjacent sulfur atom. Y, Z, W,
The definitions of R 1 , R 2 , m, n, p and q are the same as in formula []. ] 4 Polymerization degree (n+p+q+n′+p′+
For polymers with q′) of 20 to 3000, [In formula [], Z, W, R 1 , R 2 , m, n,
The definitions of p and q are the same as in formula []. W' represents a substituted or unsubstituted alkylene or phenylene group, which is the same as or different from W. R 1 ' is the same as or different from R 1 and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. n', p' and q' correspond to n, p and q, respectively, and represent the number of each constituent unit in the polymer, but the arrangement of the constituent units is random, and n and n', p and p',
The proportion of each constituent unit related to q and q′ in the whole is
They are 5-50%, 40-95%, and 0-10%, respectively. ] A cytotoxic substance-bound compound represented by the formula [-a] characterized by reacting a cytotoxic substance containing an amino group or an imino group in the molecule, and then reductively cleaving the disulfide bond of the reaction product. A method for producing reactive polymers. [In formula [-a], Y, Z, W, R 1 , R 2 ,
The definitions of m, n, p and q are the same as in formula []. ]
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6635280A JPS579724A (en) | 1980-05-21 | 1980-05-21 | Reactive polymer containing bonded cytotoxic substance and its preparation |
| DE8181302118T DE3175151D1 (en) | 1980-05-21 | 1981-05-13 | Reactive polymer and process for the preparation thereof |
| EP81302118A EP0040506B1 (en) | 1980-05-21 | 1981-05-13 | Reactive polymer and process for the preparation thereof |
| US06/265,924 US4385169A (en) | 1980-05-21 | 1981-05-21 | Reactive alpha amino acid polymer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6635280A JPS579724A (en) | 1980-05-21 | 1980-05-21 | Reactive polymer containing bonded cytotoxic substance and its preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS579724A JPS579724A (en) | 1982-01-19 |
| JPH0338294B2 true JPH0338294B2 (en) | 1991-06-10 |
Family
ID=13313372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6635280A Granted JPS579724A (en) | 1980-05-21 | 1980-05-21 | Reactive polymer containing bonded cytotoxic substance and its preparation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS579724A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59116232A (en) * | 1982-12-24 | 1984-07-05 | Teijin Ltd | Cytotoxic complex and its production method |
| FR2604092B1 (en) * | 1986-09-19 | 1990-04-13 | Immunotech Sa | IMMUNOREACTIVES FOR TARGETING ANIMAL CELLS FOR VISUALIZATION OR DESTRUCTION IN VIVO |
-
1980
- 1980-05-21 JP JP6635280A patent/JPS579724A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS579724A (en) | 1982-01-19 |
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