Details for Patent: 5,877,296
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| Title: | Process for preparing conjugates of methyltrithio antitumor agents |
| Abstract: | This invention describes carrier-drug conjugates prepared from disulfide analogs of the calicheamicin family of potent antitumor antibiotics and their derivatives, as well as similar analogs from related antitumor antibiotics such as the esperamicins. The carrier can be an antibody, growth factor, or steroid which targets an undesired population of cells, such as those of a tumor. Whole protein carriers as well as their antigen-recognizing fragments and their chemically or genetically manipulated counterparts are useful for the targeting portion of the conjugates. This invention includes compounds required for the synthesis of these conjugates, appropriate pharmaceutical compositions of the carrier-drug conjugates, and their method of use. |
| Inventor(s): | Hamann; Philip Ross (Garnerville, NY), Hinman; Lois (N. Tarrytown, NY), Hollander; Irwin (Monsey, NY), Holcomb; Ryan (Glen Rock, NJ), Hallett; William (New City, NY), Tsou; Hwei-Ru (New City, NY), Weiss; Martin J. (Ft. Lee, NJ) |
| Assignee: | American Cyanamid Company (Madison, NJ) |
| Filing Date: | May 26, 1995 |
| Application Number: | 08/452,164 |
| Claims: | 1. A process for preparing the targeted derivatives of formula wherein Z.sup.3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts; Alk.sup.1 and Alk.sup.2 are independently a bond or branched or unbranched (C.sub.1 -C.sub.10) alkylene chain; Sp.sup.1 is a bond, --S--, --O--, --CONH--, --NHCO--, --NR'--, --N(CH.sub.2 CH.sub.2).sub.2 N--, or -X-Ar'-Y-(CH.sub.2).sub.n -Z wherein X, Y, and Z are independently a bond, --NR'--, --S--, or --O--, with the proviso that when n=0, then at least one of Y and Z must be a bond and Ar' is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C.sub.1 -C.sub.5) alkyl, (C.sub.1 -C.sub.4) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR', with the proviso that when Alk.sup.1 ' is a bond, Sp.sup.1 ' is a bond; n is an integer from 0 to 5; R' is a branched or unbranched (C.sub.1 -C.sub.5) chain optionally substituted by one or two groups of --OH, (C.sub.1 -C.sub.4) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, (C.sub.1 -C.sub.3) dialkylamino, or (C.sub.1 -C.sub.3) trialkylammonium-A.sup.- where A.sup.- is a pharmaceutically acceptable anion completing a salt; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.5) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, or COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR' wherein n and R' are as defined above or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene or ##STR16## each naphthylidene or phenothiazine optionally substituted with one, two, three, or four groups of (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.5) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR' wherein n and R' are as defined above, with the proviso that when Ar is naphthylidene, Z.sup.1 is not hydrogen and with the proviso that when Ar is phenothiazine, Sp.sup.1 is a bond only connected to nitrogen; Sp.sup.2 is a bond, --S--, or --O--, with the proviso that when Alk.sup.2 is a bond, Sp.sup.2 is a bond; Z.sup.1 is H, (C.sub.1 -C.sub.5) alkyl, or phenyl optionally substituted with one, two, or three groups of (C.sub.1 -C.sub.5) alkyl, (C.sub.1 -C.sub.4) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR' wherein n and R' are as defined above; z.sup.2 is Q-Sp-S-S-W, wherein W is ##STR17## R.sub.5 is --CH.sub.3, --C.sub.2 H.sub.5, or --CH(CH.sub.3).sub.2 ; X is an iodine or bromine atom; R.sub.5 ' is a hydrogen or the group RCO, wherein R is hydrogen, branched or unbranched (C.sub.1 -C.sub.10) alkyl or (C.sub.1 -C.sub.10) alkylene group, a (C.sub.6 -C.sub.11) aryl group, a (C.sub.6 -C.sub.11) aryl-alkyl (C.sub.1 -C.sub.5) group, or a heteroaryl or heteroaryl- alkyl (C.sub.1 -C.sub.5) group wherein heteroaryl is 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridyl, 2-, 4-, or 5-(N-methylimidizolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, lower (C.sub.1 -C.sub.3) alkoxy, or lower (C.sub.1 -C.sub.5) thioalkoxy groups; Sp is a straight or branched-chain divalent or trivalent (C.sub.1 -C.sub.18) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C.sub.3 -C.sub.18) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C.sub.1 -C.sub.18) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C.sub.1 -C.sub.18) radical or divalent or trivalent (C.sub.2 -C.sub.18) unsaturated alkyl radical, wherein heteroaryl is furyl, thienyl, N-methylpyrrolyl, pyridinyl, N-methylimidazolyl, oxazolyl, pyrimidinyl, quinolyl, isoquinolyl, N-methylcarbazoyl, aminocoumarinyl, or phenazinyl and wherein when Sp is a trivalent radical, Sp may be additionally substituted by lower (C.sub.1 -C.sub.5) dialkylamino, lower (C.sub.1 -C.sub.5) alkoxy, hydroxy, or lower (C.sub.1 -C.sub.5) alkylthio groups; Q is .dbd.NHNCO--, .dbd.NHNCS--, .dbd.NHNCONH--, .dbd.NHNCSNH--, or .dbd.NO--; and m is from about 0.1 to 15; comprising the steps of: (a) reacting H.sub.2 Z.sup.2 with a compound of formula in an alcoholic solvent with a boiling point of less than about 100.degree. C. in the presence of about 5% acetic acid or a carboxylic acid catalyst at about 20.degree. to 70.degree. C. for about 1 to 24 hours, wherein Alk.sup.1 and Alk.sup.2, Sp.sup.1, n, R', Sp.sup.2, Z.sup.1, and Ar are as defined above, to produce an intermediate of formula wherein Alk.sup.1, Sp.sup.1, Ar, Sp.sup.2, Alk.sup.2, Z.sup.1, and Z.sup.2 are as defined above; (b) isolating the intermediate of step (a); (C) reacting the isolated intermediate of step (b) with N-hydroxysuccinimide, 2, 3, 5, 6-tetrafluorophenol, pentafluorophenol, 4-nitrophenol, 2,4-dinitrophenol, or N-hydroxysulfosuccinimide in the presence of DCC, EDCI, or other carbodiimide in an inert organic solvent such as acetonitrile or acetonitrile containing 5-50% DMF to generate the compound wherein Alk.sup.1, Sp.sup.1, Ar, Sp.sup.2, Alk.sup.2, Z.sup.1, and Z.sup.2 are as defined above; and Z.sup.3 is ##STR18## and (d) reacting the compound generated in step (C) of formula with a carrier Z.sup.3, wherein Z.sup.3 is a protein selected from mono- and polyclonal antibodies, their antigen-recognizing fragments, and their chemically or genetically manipulated counterparts, in an aqueous, buffered solution at a pH of between 6.5 and 9.0 and a temperature of 4.degree. to 40.degree. C. for 1 to 48 hours to generate the targeted derivatives of formula defined above. 2. The process of claim 1, wherein Alk.sup.2 and Sp.sup.2 are together a bond and Z.sup.1 is H or (C.sub.1 -C.sub.5) alkyl. 3. The process of claim 2, wherein Sp.sup.1 is a bond, --S--, --O--, --CONH--, --NHCO--, or --NR', with the proviso that when Sp.sup.1 is a bond, Alk.sup.1 is a bond. 4. The process of claim 3, wherein Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.5) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, or COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR' or a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene, each optionally substituted With one, two, three, or four groups of (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.5) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR'. 5. The process of claim 4, wherein a covalent bond to the Z.sup.3 protein is an amide formed from a reaction with the lysine side chains of the Z.sup.3 protein. 6. The process of claim 5, wherein Z.sup.2 is Q-Sp-S-S-W and W is ##STR19## R.sub.5, X, R.sub.5 ', R, and Sp are as defined in claim 1, and Q is .dbd.NHNCO--. 7. The process of claim 6, wherein the alcoholic solvent of step (a) is methanol; the carboxylic acid catalyst of step (a) is 5% acetic acid; the isolated intermediate of step (b) is reacted in step (C) with N-hydroxysuccinimide in the presence of EDCI in acetonitrile; and the aqueous buffered solution of step (d) is phosphate buffer having a pH of 7.4 to 8.0. 8. The process of claim 7, wherein Z.sup.1 is (C.sub.1 -C.sub.5) alkyl. 9. The process of claim 8, wherein Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two, or three groups of (C.sub.1 -C.sub.6) alkyl, (C.sub.1 -C.sub.5) alkoxy, (C.sub.1 -C.sub.4) thioalkoxy, halogen, nitro, or COOR', CONHR', O(CH.sub.2).sub.n COOR', S(CH.sub.2).sub.n COOR', O(CH.sub.2).sub.n CONHR', or S(CH.sub.2).sub.n CONHR'. 10. The process of claim 9, wherein Sp.sup.1 is --O--, Alk.sup.1 is C.sub.4 alkylene, Ar is 1,4-phenylene, and Z.sup.1 is C.sub.1 alkyl. 11. The process of claim 10, wherein Z.sup.3 is antibody h-P67.6, h-CT-M-01, m-CT-M-01, h-A33, m-A33 or anti-Tac. 12. The process of claim 11, wherein Z.sup.2 is calicheamicin gamma dimethyl hydrazide or calicheamicin N-acetyl gamma dimethyl hydrazide. 13. The process of claim 12, wherein Z.sup.3 is antibody h-CT-M-01 and Z.sup.2 is calicheamicin N-acetyl gamma dimethyl hydrazide. 14. The process of claim 12, wherein Z.sup.3 is antibody h-P67.6 and Z.sup.2 is calicheamicin N-acetyl gamma dimethyl hydrazide. 15. The process of claim 4, wherein Sp.sup.1 is --O--or a bond; Alk.sup.1 is a bond or branched or unbranched (C.sub.1 -C.sub.10) alkylene chain, with the proviso that when Alk.sup.1 is a bond, Sp.sup.1 is a bond; Z.sup.1 is (C.sub.1 -C.sub.5) alkyl; and Z.sup.2 is calicheamicin N-acetyl gamma dimethyl hydrazide. |
