Targeted forms of methyltrithio antitumor agents

Abstract: The disclosure describes disulfide analogs of antitumor and antibacterial agents known collectively as the LL-E33288 complex, a method for constructing carrier-drug conjugates from the disulfide analogs and the carrier-drug targeted form conjugates.

Description: SUMMARY OF THE INVENTION The invention is disulfide compounds of the α 1 , α 2 , β 1 , γ, and δ N-acyl derivatives of the LL-33288 complex and derivatives thereof as well as the disulfide compounds of N-acyl derivatives of BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 antibiotics and derivatives thereof prepared by reacting the antibiotic with an unsubstituted or substituted alkyl mercaptan. These disulfide compounds are effective antitumor agents. The invention also describes carrier-drug conjugates of the disulfide analogs prepared from the materials listed above. The carrier (targeting) portion of the conjugate is a mono- or polyclonal antibody, their fragments, chemically or genetically manipulated counterparts, or growth factors or steroids. The invention includes the method of using the carrier-drug conjugates as well as their process of manufacture. DESCRIPTION OF THE DRAWINGS Figure 1 The ultraviolet spectrum of the antitumor antibiotic designated as N-acetyl LL-E33288γ 1 I . Figure 2 The infrared spectrum of the antitumor antibiotic designated as N-acetyl LL-E33288γ 1 I . Figure 3 The proton magnetic resonance spectrum of the antitumor antibiotic designated as N-acetyl LL-E33288γ 1 I . Figure 4 The carbon-13 nuclear magnetic resonance spectrum of the antitumor antibiotic designated as N-acetyl LL-E33288γ 1 I . DETAILED DESCRIPTION OF THE INVENTION The family of antibacterial and antitumor agents, known collectively as the LL-E33288 complex are described and claimed in EPO publication number 0182152A3 published May 28; 1986 and are used to prepare the disulfur antitumor agents which are some of the starting materials for targeted forms of the antitumor agents of our invention. The EPO publication number 0182152A3 describes the LL-E33288 complex, the components thereof, namely, LL-E33288α 1 Br , LL-E33288α 1 I , LL-E33288α 2 Br , LL-E33288α 2 I , LL-E33288β 1 Br , LL-E33288β 1 I , ll-E33288γ 1 Br , LL-E33288γ 1 I and LL-E33288δ 1 I , and methods for their production by aerobic fermentation utilizing a new strain of Micromonospora echinospora ssp calichensis or natural or derived mutants thereof. The EPO publication number 0182152A3 also discloses proposed structures for some of the above named components. These proposed structures are reproduced in Table 1 Additional members of the LL-E33288 complex are described and claimed in EP-A-0 276 485 published August 3, 1988, and are likewise useful for preparing the disulfide derivatives and targeted forms of the antitumor agents of our invention. These latter proposed structures are reproduced in Table 2. Still other members of the LL-E33288 family of antitumor antibiotics are described and claimed in U. S. patent 5,079,233 and also are useful for preparing additional targeted forms of the antitumor agents of our invention. This application describes N-acyl derivatives of several members of the LL-E33288 complex which have been prepared by chemical means. These proposed structures are likewise reproduced in Table 2. Certain other antitumor and antibacterial compounds are useful in our invention, namely: 1) Esperamicin BBM-1675, a novel class of potent antitumor antibiotics. I. Physico-chemical data and partial structure. M. Konishi, et . al ., J. Antibiotics, 38 , 1605 (1985). A new antitumor antibiotic complex, M. Konishi, et . al ., U.K. Patent Application GB 2,141,425A, May 15, 1984. 2) New antitumor antibiotics, FR-900405 and FR-900406.I. Taxonomy of the producing strain. M. Iwami, et . al ., J. Antibiotics 38 , 835 (1985). New antitumor antibiotics FR-900405 and FR-900406.II. Production, isolation, characterization and antitumor activity. S. Kiyoto, et . al ., J. Antibiotics, 38 , 340 (1985). 3) PD 114759 and PD 115028, novel antitumor antibiotics with phenomenal potency. I. Isolation and characterization. R.H. Bunge, et . al ., J. Antibiotics, 37 , 1566 (1984). Biological and biochemical activities of the novel antitumor antibiotic PD 114759 and related derivatives. D.W. Fry et . al ., Investigational New Drugs, 4 , 3 (1986). 4) New antibiotic complex CL-1577A, CL-1577B produced by Streptomyces sp. ATCC 39363. European Patent Application 0,132,082, A2. 5) CL-1577D and CL-1577E Antibiotic antitumor compounds, their production and use. U.S. Patent 4,539,203. 6) CL-1724 Antibiotic compounds, their production and use. U.S. Patent 4,554,162. 7) New antitumor antibiotics BBM-1675-A3 and BBM-1675-A4, obtained by fermentation of actinomadura verrucosospora strains H964-92 (ATCC 39334) or AB27Y (ATCC 39638). U.S. Patent 4,675,187. 8) New N-acetyl-esperamicin A 1 , A 2 and A 1b derivatives with antimicrobial and antitumor activities. European Patent 289,030. Information regarding BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724 is contained in the above citations. The complete structures of esperamicins A 1 , A 2 , and A 1b (the BBM-1675 complex) and their respective N-acetyl derivatives have been reported, and these are included in Tables 1 and 2. The physical characteristics of the other above-named antitumor antibiotics indicate that they all are identical or very similar in structure to the esperamicins, and all contain a methyltrithio functional group. As can be seen from the structures disclosed above, the N-acyl counterparts α 1 , α 2 , β 1 , γ 1 , δ of the LL-E33288 complex, as well as the N-acyl counterparts BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724 antibiotics each contain a methyltrithio group in their structure. It has now been discovered that the reaction of any of the above cited antibiotics with an unsubstituted or substituted alkyl or aryl mercaptan results in displacement of the methyl perthiolate anion from the trisulfide moiety resulting in the formation of a stable disulfide (Scheme I), below. Other compounds on which this transformation works are disclosed in EPO publication number 0313874A2 published May 3, 1989. The compounds of Tables 1 and 2 are transformed with thiol-containing organic molecules according to Scheme I to produce new compositions useful as antibacterial and anticancer agents in their own right, wherein W, R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 2 ,, R 3 ', R 4 ', R 5 ', Ar 1 , Ar 2 and X are as hereinbefore defined in Tables 1 and 2, Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radicals, divalent or trivalent aryl- or heteroarylalkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, or a group chosen from the following: -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen; and when CH 3 -SSS-W is LL-E33288α 1 -Br, α 1 -l, α 2 -Br, a 2 -l, α 3 -Br, α 3 -l, α 4 -Br, β 1 -Br, β 1 -l, β 2 -Br, β 2 -l, γ 1 -Br, γ 1 -l, δ 1 -l, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q cannot be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl. As a preferred embodiment of this invention, disulfides of the formula Q-Sp-SS-W are prepared from the antitumor antibiotics designated LL-E33288 (CH 3 SSS-W) wherein: R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 ; X is an iodine or bromine atom; R 5 is a hydrogen or the group RCO, wherein R is hydrogen, CH 3 , or a mono-substituted aryl group; and Sp and Q are as hereinbefore defined. The transformation of the compounds listed in Tables 1 and 2 to a variety of disulfides can be accomplished with a large variety of thiol-containing organic molecules to yield novel antitumor and antibacterial agents. Moreover, the products from the reaction themselves are amenable to further transformations within the newly-introduced side-chain to produce still more novel compounds with biological activities. It has also been discovered that the displacement of the methyltrithio unit of the compounds listed in Tables 1 and 2 and as depicted in Scheme I can be used to introduce a spacer (Sp), the judicious choice of which enables the introduction of targeting units into the disulfide derivatives compounds of the application. Introduction of targeting units to other disulfide derivatives is disclosed in EPO publication number 0313873A1 published May 3, 1989. Thus, with reference to Scheme I, the compounds of formula CH 3 SSS-W wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 are first reacted with a compound of formula Q-Sp-SH to produce an intermediate wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288α 1 -Br, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, β 2 -Br, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; Q is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , -CON 3 , As long as the product from Scheme contains at least one functional group which can be converted to, or is directly reactive with a targeting unit (Tu), targeted forms of the antitumor antibiotics of the above-named patents and applications can be generated, as shown in Scheme II below: wherein Q, Sp, and W are as hereinbefore defined, Tu is a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from carbohydrate residues, or an amidoalkylthio group; n is an integer of from 1 to 100; Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, -NHCONHN=CH-, -NHCONHNHCH 2 -, -NHCSNHN=CH-, -NHCSNHNHCH 2 -, -ON=CH-, -NH-, -NHCH 2 -, -N=CH-, -CO 2 -, -NHCH 2 CO 2 -, -SS-, and m is 0.1 to 15. As an example, and with reference to Scheme II, above, the 3-mercaptopropionic acid derivative of N-acetyl E-33288γ 1 I (Q=CO 2 H, Sp=-CH 2 CO 2 -), when converted to its activated hydroxysuccinimide form (Q=CO 2 Su, Sp=-CH 2 -CH 2 -) can be used to react with some of the ε-amino groups of lysine residues (e.g., Tu=monoclonal antibody, Y=-NH 2 wherein n=50-100 from available lysine residues), at a pH between 7.0 and 9.5 in aqueous buffered solutions at temperatures between 4 o C to 40 o C to produce targeted forms of the antibiotics attached at random sites along the protein backbone (Tu=monoclonal antibody, Z=-NHCO-, Sp=-CH 2 CH 2 -, m=1-10). Only a fraction of the available lysine residues are substituted in this manner, since high loading is generally not considered compatible with preserving the antibody immunoreactivity. The same randomly-substituted immunoconjugates can also be prepared from the 3-mercaptopropionic acid derivative using other carboxyl group activating agents such as a variety of carbodiimides, or the corresponding acyl azide. Alternatively, a 3-mercaptopropionyl hydrazide derivative of N-acetyl LL-E33288δ 1 I (Q=H 2 NNHCO-, Sp=-CH 2 CH 2 -), when reacted with a periodate-oxidized antibody (Tu=monoclonal antibody, Y=-CHO, n=1-15) as described in U.S. Patent No. 4,671,958 at a pH between 4 and 7, in a buffered aqueous solution at a temperature of between 4 o C and 40 o C, reacts only at the aldehyde functionality (derived from cleavage of vic-diols of carbohydrate residues situated on the Fc portion of the antibodies) to generate monoclonal antibody conjugates containing the drug substituted at specific sites along the backbone of the protein (Tu=monoclonal antibody, Z=-CH=NNHCO-, Sp=-CH 2 CH 2 -, m=0.5-10). In order to block unreacted aldehyde groups on the antibody and thus avoid crosslinking, as well as stabilize the hydrolytically labile Schiff's base linkages, it is preferable (though not essential) to react the latter conjugate first with a compound such as acetyl hydrazide or tyrosine hydrazide, then reduce with sodium cyanoborohydride or sodium borohydride to produce the stabilized constructs of this invention (Tu=monoclonal antibody, Z=-CH 2 NHNHCO-, Sp=-CH 2 CH 2 -, m=0.5-10). Other aldehyde-reactive groups as part of the drug construct are within our invention to generate the products of Scheme II. Such functional groups are preferably, though not limited to, those which react with aldehydes under acidic aqueous conditions. The reactivity of protein lysines under basic conditions is sufficiently great such that their amines compete with the products of Scheme II for available aldehydes of the monoclonal antibody. Alternative aldehyde-reactive groups are, for example, the semicarbazide, the thiosemicarbazide, and the O-substituted hydroxylamine functionalities. Assembly of targeted forms of the compounds listed in Tables 1 and 2 is not restricted to the sequence outlined in Scheme II. The targeting unit (Tu) can be first modified to contain a thiol group, which is then reacted with the compounds of Tables 1 and 2 in accordance with Scheme III below: wherein Tu, Y, Q, Sp, W, n, and m are as hereinbefore defined, and P is hydrogen or 2-(pyridylthio), with the proviso that when Y is a thiol derived from a backbone amino acid residue of Tu, Z-Sp taken together is a covalent bond. As an example, and with references to Scheme III, above, a monoclonal antibody can be reacted with 3-(2-dithiopyridyl)propionic acid hydroxysuccinimide ester to modify the protein through lysine residues (Tu=monoclonal antibody, Y=NH 2 , n=50-100, Q=-CO 2 Su, Sp=-CH 2 -CH 2 -, P=2-pyridylthio). Following reduction with, for example, dithiothreitol, an intermediate is generated (Tu=monoclonal antibody, Z=-NHCO-, Sp=-CH 2 CH 2 -, m=1 to 15) which can be reacted with the compounds of Tables 1 and 2 to generate the subject immunoconjugates. Similarly, 2-iminothiolane can be reacted with a monoclonal antibody to introduce thiol groups onto the surface of the protein directly, without requiring a reduction step (Tu=monoclonal antibody, Z=-NHCO-, Sp=-(CH 2 ) 3 -, m=1 to 15), and this intermediate can be reacted with the compounds of Tables 1 and 2 as before. Alternatively, sulfhydryl groups inherent within the structure of monoclonal antibodies in dimeric form as cystine residues can be used to participate in the reaction of Scheme III directly. Such sulfhydryls are traditionally exposed by a combination of enzymatic digestion and reduction of native monoclonal antibodies (Tu=Fab′ fragment, Z-Sp=bond, Y=SH), but the use of genetically-altered constructs of monoclonal antibodies containing unpaired cystine residues is likewise contemplated. A preferred embodiment of targeted derivatives of this invention is a protein-drug conjugate of the formula: prepared from the class of antitumor antibiotics designated N-acyl LL-E33288 (CH 3 SSS-W) comprising: displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C 2 -C 10 ) radicals or divalent or trivalent (C 2 -C 5 ) arylalkyl or heteroarylalkyl radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; and Q is carboxyl, lower alkyldicarboxyl anhydride, -CO 2 Su, -CONHNH 2 , or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula TU-(Y) n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, or and m is 0.1 to 15. A number of different monoclonal antibodies (MoAbs) are used to exemplify targeting of the methyltrithio anticancer compounds. MoAbs Lym 1 and Lym 2 recognize different antigens on mature B-lymphocytes and their product lymphomas. The production and characteristics of these MoAbs are described by A.L. Epstein, et. al., "Cancer Research" 47 , 830 (1987). MoAb B72.3 targets primarily to carcinomas of the breast and colon, though reactivity with pancreatic, ovarian, and lung carcinomas has also been noted. The antibody has been described by T.L. Klug, et. al., "Int. J. Cancer" 38 , 661 (1986). MoAb CT-M-01, which recognizes primarily breast tumors is described in EPO application 86 401482.4 filed July 3, 1986 and MAC-68 is produced by a sub-clone of the hybridoma which produces CT-M-01, and recognizes both breast and colon carcinomas. Intermediates of the subject compounds useful for, and conjugates with these antibodies, are described in the experimental section. It should not, however, be construed that this patent is limited to or restricted by the aforementioned antibodies. Instead, the methodology is sufficiently general that it can be applied to all antibodies regardless of their class or isotype, their enzymatically-derived fragments, their chemically manipulated and stabilized fragments, as well as their respective chimeric and humanized counterparts. Nor are the targeting units restricted only to monoclonal antibodies. Other proteins, as well as small molecules for which receptors exist on target issues, are within the purview of our discovery as targeting entities. The compounds of this invention designated as Q-Sp-SS-W are active as antibacterial agents. Their in vitro antibacterial activity can be determined against gram-positive and gram-negative bacteria by a standard agar dilution method. Mueller-Hinton agar containing two-fold decreasing concentrations of the antibiotics is poured into petri dishes. The agar surfaces are inoculated with 1 to 5 x 10 4 colony forming units of bacteria by means of a Steers replicating device. The lowest concentration of compound that inhibits growth of a bacterial strain after about 18 hours of incubation at approximately 36 o C is recorded as the minimal inhibitory concentration (MIC) for that strain. The disulfide compounds described above are active antitumor agents. Certain in vivo testing systems and protocols have been developed by the National Cancer Institute for testing compounds to determine their suitability as antineoplastic agents. These have been reported in "Cancer Chemotherapy Reports", Part III, Vol. 3, No. 2 (1972), Geran, et. al. These protocols have established standardized screening tests which are generally followed in the field of testing for antitumor agents. Of these systems, lymphocytic leukemia P388, melanotic melanoma B16, L1210 leukemia and colon 26 adenocarcinoma are particularly significant to the present invention. These neoplasms are utilized for testing as transplantable tumors in mice. Generally, significant anti tumor activity, shown in these protocols by a percentage increase of mean survival times of the treated animals (T) over the control animals (C) is indicative of similar results in human leukemias and solid tumors. Lymphocytic Leukemia P388 Test The animals used were BDF 1 female mice. There were 5 or 6 mice per test group. The tumor transplant was by intraperitoneal injection of 0.5 ml of dilute ascitic fluid containing 1 x 10 6 cells of lymphocytic leukemia P388 on day zero. The test compounds were administered intraperitoneally at a volume of 0.5 ml in sterile, pyrogen free saline on days 1, 5 and 9 (relative to tumor inoculation) at the indicated doses. The mice were weighed and survivors recorded on a regular basis for 30 days. The median survival time and the ratio of survival time for treated (T)/control (C) animals were calculated. Values of T/C greater than or equal to 125 are considered to reflect significant antitumor activity. The results appear in Table 3. Lymphocytic Leukemia P388 Test Compound Dose (mg/kg) Median Survival (Days) T/Cx100 % Propionic acid disulfide of LL-E33288γ 1 -I (from Example 3) 0.005 145 0.0025 125 3-Mercaptopropionyl hydrazide disulfide of N-acetyl LL-E33288γ 1 -I (from Example 4) 0.080 21.0 175 0.040 18.0 150 0.020 15.0 125 Control - 12.0 - 3-Mercaptopropionyl hydrazide disulfide of LL-E33288α 2 -I (from Example 5) 0.010 26.5 230 0.005 21.0 183 0.0025 18.5 161 0.00125 18.5 161 Control - 11.5 - 3-Mercaptobutyryl hydrazide disulfide of LL-E33288γ 1 -I (from Example 7) 0.005 28.5 190 0.0025 23.0 153 0.00125 21.0 140 0.0006 19.5 130 Control - 15.0 - 3-Mercaptoisovaleryl hydrazide disulfide of LL-E33288q 1 -I (from Example 8) 0.010 26.5 252 0.005 18.0 171 0.0025 17.0 162 0.00125 16.0 152 0.0006 13.0 124 0.0003 14.0 133 Control - 10.5 - Melanotic Melanoma B16 Test Animals used can be BDF1 female mice. There are 5 or 6 mice per test group. A one gram portion of melanotic melanoma B16 tumor is homogenized in 10 ml of cold balanced salt solution and a 0.5 ml aliquot of the homogenate is implanted intraperitoneally into each of the test mice. The test compounds are administered intraperitoneally on days 1 through 9 (relative to tumor inoculation) at the indicated doses. The mice are weighed and survivors recorded on a regular basis for 60 days. The median survival time and the ratio of survival time for treated (T)/control (C) animals are calculated. A value of T/C of greater or equal to 125 is considered indicative of significant antitumor activity. The methods of this invention used to produce monoclonal antibody conjugates from the compounds of Tables 1 and 2 and designated as yield constructs which retain good immunoreactivity with target cell lines, as determined by the following in vitro assays: Target Cells All target cells were maintained in RPMI 1640 media supplemented with 5% Fetal Calf Serum (FCS), ITS (Collaborative Research, Cat# 40351), streptomycin (50 µg/ml), penicillin (50 units/ml), gentamycin sulfate (50 µg/ml) and glutamine (.03%). The cells were maintained in a humidified 5% CO 2 incubator at 37 o C. 1. Immunoreactivity Assays Procedure I - Elisa Appropriate target cells were harvested, counted and suspended in Dulbecco's Phosphate Buffered Saline (DPBS) at an optimal concentration for monoclonal antibody (MoAb) being tested. 0.1 ml of cells was aliquoted in each well of a sterile tissue culture polystyrene 96-well plate. The plates were centrifuged for 5 minutes at 1,000 RPM's and the supernatant was flicked off. Plates were air-dried overnight and may be stored at 4 o C for up to 3 months. Non-specific binding sites were blocked by adding 200 µl of 1% gelatin in DPBS per well and incubating the plate for 1 hour at 37 o C in a humid incubator. (All subsequent incubations are done under similar conditions). The plates were washed once with 250 µl of 0.05% TWEEN-20 in DPBS (washing solution) using the automated ELISA washing system from Dynatech (Ultrawash II). Samples to be tested were diluted to make a final concentration of 3 µg/ml MoAb equivalents in 0.1% gelatin-DPBS. Six additional threefold serial dilutions were prepared from each 3 µg/ml sample and 100 µl was added to appropriate wells in triplicate. The bottom row of wells only received 100 µl of 0.1% gelatin as background. Plates were incubated for 45 minutes and then washed three times. Alkaline phosphatase conjugated affinity purified goat anti-mouse immunoglobulins (Cappel Cat# 8611-0231) was diluted 1:125 in 0.1% gelatin and 100 µl was added to each well. Plates were incubated for 45 minutes and then washed three times. 200 µl of p -nitrophenyl phosphate substrate solution (see below) was added to each well. After 45 minutes at room temperature the reaction was stopped by the addition of 50 µl of 3M NaOH. The absorbance of the contents of each well was read at 405 nm in the automated spectrophotometer from Dynatech (EIA Autoreader # EL-310). Substrate Diethanolamine Buffer (10%) 97 ml diethanolamine 800 ml water 0.2 grams NaN 3 100 mg MgCl 2 6H 2 O The reagents were dissolved by continuous stirring and 1M HCl was added until the pH was 9.8. The total volume was made up to 1 liter with water and filter sterilized with a 0.2 µ filter. The buffer was stored in the dark at 4 o C. Immediately before use, p -nitrophenyl phosphate (Sigma, Cat# 104-40) was dissolved in the 10% diethanolamine buffer (must be at room temperature) to give a final concentration of 1 mg/ml. Calculation of O. D. Values The percentage binding of each sample was calculated by the following equation: A-B C-B x 100 = % Binding A = Average O.D. of test sample B = Average O.D. of background C = Average O.D. of 3 µg/ml unmanipulated MoAb control The % binding was plotted on the non-log scale of a semi-log graph and the MoAb concentration was plotted on the log scale. The BD 50 (i.e. dose of antibody needed to give 50% binding) of each test sample was derived from the graph and the amount of retention of immunoreactivity was calculated by the following equation: BD 50 of MoAb control BD 50 of test sample x 100 = % Immunoreactivity retained Procedure 2 - Indirect RIA Appropriate amounts of target cells in 0.2 ml of 10% FCS media were aliquoted into 4 ml polystyrene tubes. Samples to be tested were diluted to a concentration of 2 µg/ml MoAb equivalents in 10% FCS media. Five three-fold serial dilutions were prepared from each 2 µg/ml sample and 0.2 ml was added to each tube in duplicate. Background samples received only cells and media. Cells were incubated at 4 o C for 1 hour, then washed 2 times (all RIA washes were done with a 3 ml volume) with 2% FCS media. 0.05 ml of sheep F(ab') 2 anti-mouse IgG [ 125 I] (DuPont, Cat# NEX 162-0142) containing approximately 500,000 CPM's was added to each tube; cells were incubated an additional hour at 4 o C, washed once with 2% FCS and twice with PBS. 0.5 ml of PBS was added to each tube, cells were vortexed, transferred to clean tubes and counted for 1 minute in a Packard Gamma 500. The % binding of each value was determined and graphed like the preceding ELISA equation, except CPM's were substituted for O.D. units and C = Average CPM's of 1 µg/ml unmanipulated MoAb control. The % immunoreactivity retained of each sample was calculated as previously discussed. Procedure 3 - Direct RIA Appropriate amounts of target cells in 1 ml of 10% FCS media were aliquoted into 4 ml polystyrene test tubes, centrifuged and supernatant was discarded. Samples to be tested were diluted to make a concentration of 200 µg/ml MoAb equivalents in 10% FCS media. Five additional five-fold serial dilutions were prepared from each 200 µg/ml sample and 0.05 ml was added to each tube in duplicate. 0.05 ml of 125 I-MoAb was added to each tube (optimal amount is individually determined for each MoAb and batch). Positive control samples contained cells, media and 125 I-MoAb. Background samples contained non-specific cells, media and 125 I-MoAb. Cells were incubated for 1 hour at 4 o C, washed once with 2% FCS media, twice with PBS, transferred and counted as previously mentioned. The % 125 I-MoAb binding inhibition of each sample was calculated by the following formula: A-B C-B x 100 = % 125 I-MoAb Binding inhibition A = Average CPM's of sample B = Average CPM's of background C = Average CPM's of positive control The plot and % immunoreactivity retained by each sample was calculated as previously discussed except the BD 50 is actually BID 50 (Dose of MoAb needed to give 50% inhibition of the binding of 125 I-MoAb). Notes : 1) Tubes were always vigorously vortexed immediately after the addition of every reagent in the RIA's. 2) An internal control sample equalling 50% of the unmanipulated MoAb control was included in each set of assays to confirm whether each procedure was quantitative in predicting the conjugates' retention of immunoreactivity. The results from these assays are tabulated below in Table 4. Immunoreactivity of MoAb Conjugates Hydrazide of 3-mercaptopropionic acid disulfide of N-acetyl LL-E33288δ 1 I (Example 7) conjugated to: Preparation Immunoreactivity % of unmodified MoAb control Lym 1 82 CT-M-O1 100 B72.3 100 The monoclonal antibody conjugates of this invention are active as anticancer agents. The assay described below for assessing in vitro cytotoxicity shows the dramatic preference of the constructs for target cell lines as opposed to non-target cells, and provides a measure of utility of targeted forms of the compounds compared to their non-targeted counterparts. Cytotoxicity Assays In Vitro Samples to be tested were diluted to a concentration of 0.2 or 0.02 µg/ml of parent drug equivalents (starting concentration is dependent on cell line to be tested and potency of the parent drug). Three or four additional five-fold dilutions were prepared from each original sample dilution and 0.2 ml was added to sterile 15 ml polystyrene tubes. At least one similar conjugate consisting of parent drug and an irrelevant MoAb was included in each assay to determine specificity of the relevant conjugate. 10 5 appropriate target cells in 0.2 ml of 10% FCS media were aliquoted into the tubes and vortexed. In addition, an identical test was performed utilizing irrelevant cells as targets to further confirm specificity of relevant conjugate. MoAb controls received only equivalent amounts of MoAb and positive control samples received only 10% FCS media. Cells were incubated at 37 o C for 7 minutes then washed 4 times with 8 ml of 2% FCS media. 0.1 ml of 10% FCS was added to each tube, cells were vortexed and 0.2 ml was aliquoted to each well of a sterile 96-well polystyrene tissue culture plate. Plates were incubated for 2 days in a humidified 37 o C incubator with 5% CO 2 . One half of the media was removed and replaced with fresh media containing 2 µCi/ml 3 H thymidine (DuPont, NEN, Cat# NET-027). Incubation was continued for 24 hours, cells were frozen, thawed and harvested by a PHD cell harvester (Cambridge Technology, Inc.). Each sample was counted for 1 minute in a Beckman LS 5800 scintillation counter on Channel 1. The % growth inhibition was calculated as follows: Average CPM of test value Average CPM of media control x 100 = % Growth 100 - % Growth = % Inhibition The % inhibition was plotted on the non-log scale of a semi-log graph and the parent drug concentration was plotted on the log scale. The IC 50 (concentration of parent drug needed to give 50% inhibition) of each test sample was derived from the graph and the amount of retention of cytotoxicity was calculated by the following equation: IC 50 of parent drug IC 50 of test sample x 100 = % Cytotoxicity Retained The results from the in vitro cytotoxicity assay are tabulated below in Table 5 In Vitro Cytotoxicity of MoAb Conjugates MoAb Cytotoxicity Hydrazide conjugate prepared using product of Example 3 with: % N-acetyl LL-E33288δ 1 I Lym 1 400 CT-M-01 700 The following assay system was used to measure the in vivo activity of the conjugates of this invention. In vivo tests for antitumor activity on drug-monoclonal antibody conjugates were done using human tumor xenographs in athymic (nude) mice. Burkitt lymphoma (Raji) and myeloma (HS Sultan) cells were harvested from culture flasks and inoculated subcutaneously (≥ 80 x 10 6 Raji cells or 40 x 10 6 HS Sultan cells) into test mice. Solid tumors, ovarian carcinomas (CA73, Ovcar-3), breast carcinoma (MX-1) and colon carcinoma (LS174T) were propagated in athymic mice, removed, cut into 2 mm 3 fragments and implanted subcutaneously into test mice (5-8 fragments per mouse). Drugs, monoclonal antibodies and drug-monoclonal antibody conjugates were administered intraperitoneally once each 3 days for 3 or 5 total injections starting on day 2, 3, 4, 6, 7 or 8 days after tumor implantation. Tumor measurements (the length and width of the tumor) were made by means of a Fowler ultra CAL II electronic caliper each 7 days for 4 to 6 weeks post tumor implantation. Tumor mass in mg was estimated from the formula: Length(mm) x Width(mm) 2 Tumor growth inhibition was calculated for each test group on a percent of control [mean mg of treated (T) divided by mean mg of control (C) x 100]. A T/C value ≤ 42% in groups with ≥ 65% surviving animals is considered necessary to demonstrate activity. The results from this assay appear in Table 6. In Vivo Antitumor Testing Results Dosage(mcg) Tumor Size (T/C)%control S/T MoAb Drug Hydrazide of 3-mercaptopropionic acid disulfide of N-Acetyl LL-E33288δ 1 I conjugated to CT-M-O1 125 1.0 0 6/6 Hydrazide alone - 2.0 71 3/6 Mixture, N-acetyl LL-E33288δ 1 I plus MoAb CT-M-O1 125 1.0 5 1/6 N-acetyl LL-E33288δ 1 I - 1.0 46 2/6 iv treatment against human breast cancer cell line MX-1, three injections starting on day 2 following tumor implantation, measurements given made on day 42 post-implantation Hydrazide of 3-mercaptopropionic acid disulfide of N-acetyl LL-E33288δ 1 I conjugated to Lym 1 37 1.0 11 6/6 Hydrazide alone - 1.0 517 6/6 N-acetyl LL-E33288δ 1 I alone - 0.5 226 6/6 MoAb Lym 1 alone 37 - 178 6/6 iv treatment against human Burkitt lymphoma cell line Raji TC, three injections starting on day 7 after tumor implantation, measurements given made on day 35 post-implantation Hydrazide of 3-mercaptopropionic acid disulfide of N-acetyl LL-E33288δ 1 I conjugated to B72.3 127 2.7 61 6/6 N-acetyl LL-E33288δ 1 I alone - 2.0 32 1/6 MoAb B72.3 alone 127 - 125 6/6 Vincristine (positive control) - 20 23 6/6 iv treatment against human colon cancer cell line LS174T, three injections started on day 8 following tumor implantation, measurements given made on day 21 post-implantation Hydrazide of 3-mercaptopropionic acid disulfide of N-acetyl LL-E33288δ 1 I conjugated to CT-M-O1 112 2.7 40 5/6 N-acetyl LL-E33288δ 1 I alone - 2.0 32 1/6 Vincristine (positive control) - 20 23 6/6 iv treatment against human colon carcinoma line LS174T, three injections started 8 days after tumor implantation, measurements given made on day 21 post-implantation The invention will be further described in conjunction with the following non-limiting examples. Reference Example 1 3-Mercaptopropionic Acid Disulfide of LL-E33288γ 1 I To a solution of 90 mg of LL-E33288γ 1 I in 90 ml of acetonitrile was added 10.6 mg of 3-mercaptopropionic acid in 1 ml of acetonitrile. The solution was vortexed and then stored at -20 o C for 6 days. The solvent was removed in vacuo and the residue chromatographed over 10 ml of silica gel in methylene chloride. The column was developed with 50 ml of methylene chloride, 50 ml of 4% methanol in methylene chloride and finally 100 ml of 8% methanol in methylene chloride. Evaporation of this last fraction gave a residue which was taken up in ethyl acetate with the aid of a little acetone and added dropwise to an excess of hexane. The precipitate was collected and dried, giving 39 mg of the desired product (FABMS, M+H 1394). Reference Example 2 N-Hydroxysuccinimidyl 3-mercaptopropionate disulfide of LL-E33288γ 1 I To a solution of 5 mg of the 3-mercaptopropionic acid disulfide analog of LL-E33288γ 1 I from EPO publication in 0.5 ml of tetrahydrofuran was added 0.45 mg of N-hydroxysuccinimide in 0.1 ml of tetrahydrofuran and then 1.8 mg of dicyclohexylcarbodiimide in 0.2 ml of tetrahydrofuran. The reaction was allowed to stir at room temperature for 4 hours and was then quenched with a large excess of hexanes. The solid was isolated by filtration and dissolved in ethyl acetate. The resulting solution was washed three times with brine, dried with magnesium sulfate, and evaporated to 5 mg of the desired product as a tan powder which was used without further purification. Retention time on reverse phase C 18 HPLC: 15 minutes with 40% acetonitrile/0.1 M aqueous ammonium acetate (starting material: 6.0 minutes). Example 3 3-Mercaptopropionyl hydrazide disulfide of N-acetyl LL-E33288γ 1 I To 10 mg of N-acetyl LL-E33288γ 1 I in 10 ml of acetonitrile at -15 o C was added 1.5 eq of 3-mercaptopropionyl hydrazide in 85 µl acetonitrile. One equivalent of triethylamine was added. The reaction was allowed to stir at 0 o C for two hours and the solvent was then evaporated. The residue was chromatographed on silica gel with a 10-15% methanol-in-chloroform gradient to yield the desired product. FABMS, m/z=1450(M+H); retention time on C 18 reverse phase HPLC:2.5 min. with 50% acetonitrile/0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ 1 I :6.6 min. in the same system). Example 4 3-Mercaptoisovaleryl hydrazide disulfide of N-acetyl LL-E33288γ 1 I To 20 mg of N-acetyl LL-E33288γ 1 I in 15 ml of acetonitrile at -15 o C was added 3 eq of 3-mercaptoisovaleryl hydrazide in 6.2 ml acetonitrile. One equivalent of triethylamine was added. The reaction was allowed to stir at ambient temperature for 2 hours and the solvent was then exaporated. The residue was chromatographed on silica gel with a 10-15% methanol-in-chloroform gradient to yield the desired product. Retention time on C 18 reverse phase HPLC:2.5 min. with 50% acetonitrile/0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ 1 I : 6.6 min. in the same system). Example 5 3-Mercaptobutyryl hydrazide disulfide of N-acetyl LL-E33288γ 1 I To 10 mg of N-acetyl LL-E33288γ 1 I in 7.5 ml off acetonitrile at -15 o C was added 3 eq of 3-mercaptobutyryl hydrazide in 5 ml acetonitrile. One equivalent of triethylamine was added. The reaction was allowed to stir at ambient temperature for 10 hours and the solvent was then evaporated. The residue was chromatographed on silica gel with a 10-15% methanol-in-chloroform gradient to yield the desired product. Retention time on C 18 reverse phase HPLC:7.3 min. with 43% acetonitrile/0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ 1 I :5.6 min. in the same system). Example 15 p-Mercaptodihydrocinnamoyl hydrazide disulfide of N-acetyl LL-E33288γ 1 I To 10 mg of N-acetyl LL-E33288γ 1 I in 7.5 ml of acetonitrile at -15 o C was added 3.0 eq of p-mercaptodihydrocinnamoyl hydrazide in 2.0 ml acetonitrile. One equivalent of triethylamine was added. The reaction was allowed to stir at ambient temperature for 2 hours and the solvent was then evaporated. The residue was chromatographed on silica gel with a 10-15% methanol-in-chloroform gradient to yield the desired product. Retention time on C 18 reverse phase HPLC:7.3 min. with 43% acetonitrile/0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ 1 I :5.6 min. in the same system). Example 16 Non-specific conjugation to proteins The hydroxysuccinimide enter described in Example 3 was covalently attached to antibodies under slightly alkaline conditions. The following is a general procedure used to make the antibody conjugates listed in Table 7. Antibody at a concentration of 3-5 mg/ml in phosphate buffer containing 0.1M sodium chloride, pH 7.5 was reacted with a 5-20-fold molar excess of the product from Example 3 with stirring, at room temperature for from 1-4 hours. The conjugated protein was desalted chromatographically and aggregated protein was separated from monomeric material by gel filtration HPLC. Monomeric fractions were pooled and concentrated. Non-specific conjugates prepared using the product of Example 2 MoAb Drug Loading M/M Lym 1 5.2 B72.3 6.0 B72.3 2.9 Example 8 Site-specific conjugate preparation The general method for attaching hydrazide derivatives of drugs to oxidized antibodies is described in T. J. McKearn, et al., in U.S. Patent No. 4,671,958. The procedure has been applied to preparing antibody conjugates from the products of Examples 4 to 15 with specific modifications as described below. The products from these reactions and their characteristics are summarized in Table 7. (A) Antibody Oxidation Antibody at a concentration of 5 to 10 mg/ml was dialyzed overnight against a 200 fold volume of 50mM sodium acetate buffer, pH 5.5 containing 0.1M sodium chloride (Buffer A). After dialysis, the MoAb was oxidized with 15mM to 200mM periodic acid in 0.2M sodium acetate. The oxidation was allowed to proceed in the dark, with stirring, at 4 o C for 45 minutes after which time the oxidized MoAb was desalted on a ≥5 bed volume Sephadex G-25 column. The degree of oxidation of the antibody was assessed by reaction with p -nitrophenylhydrazine and comparing absorbance of the protein at 280mm vs. p -nitrophenylhydrazine at 395mm. (B) Drug Hydrazide Conjugation The oxidized MoAb was reacted with 10 to 200-fold molar excess of drug hydrazide. The hydrazides were dissolved into dimethylformamide and added to the aqueous solution of MoAb. To avoid precipitation of MoAb, the final volume of dimethylformamide added did not exceed 10% of the total reaction volume. Reaction was allowed to proceed for 3 hours at room temperature, with stirring. To prevent crosslinking of unreacted aldehydes and subsequent aggregation, a blocking agent, acetyl hydrazide was added in 100-fold molar excess three hours after addition of the drug hydrazide. To stabilize the Schiff's base linkage between aldehyde and drug hydrazide (a hydrazone), the product generally was reduced to an alkyl hydrazine by the addition of 10mM sodium cyanoborohydride, allowing the reaction to proceed for one more hour (total conjugation time - 4 hours). The conjugate was chromatographically desalted and exhaustively dialyzed (minimum time 48 hours) into pH 6.5 phosphate buffer for storage and testing. Conjugates were analyzed for the presence of aggregates by gel filtration HPLC and for free drug by reverse phase HPLC. Drug loading was determined spectroscopically using the extinction coefficients of both the antibody and the drug to estimate molar concentrations of drug in conjugates. Hydrazide conjugates prepared from the product of Example 3 Lym 1 2.8 Lym 2 1.4 B72.3 #1 2.1 #2 2.4 CT-M-01 #1 1.6 #2 3.6 #3 2.5 #4 2.4

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, NL, SE A substituted disulfide of formula Q-Sp-SS-W, prepared from a compound of formula CH 3 SSS-W which is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, wherein R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; R is hydrogen or a branched or unbranched alkyl (C 1 -C 10 ) or alkylene (C 1 -C 10 ) group, an aryl or heteroaryl group, or an aryl-alkyl (C 1 -C 5 ) or heteroaryl-alkyl (C 1 -C 5 ) group, all optionally substituted by one or more hydroxy, amino, carboxy,' halo, nitro, lower (C 1 -C 3 ) alkoxy, or lower (C 1 -C 5 ) thioalkoxy groups; Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocyclo-alkyl radicals, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -CSNHNH 2 , -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen, and when CH 3 -SSS-W is LL-E33288 , α 1 -I, α 2 -Br, α 2 -I, , β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q can not be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl. A substituted disulfide of formula Q-Sp-SS-W, according to Claim 1 prepared from a compound of formula CH 3 SSS-W wherein: R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; wherein R is hydrogen, CH 3 , or a mono-substituted aryl group; and Sp and Q are as defined. A carrier-drug conjugate of the formula prepared from a compound or formula (CH 3 SSS-W) wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, wherein W is as hereinbefore described in claim 1 comprising: reacting CH 3 SSS-W with a compound of general formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino , arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288α 1 -Br, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; and Q is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , -CON 3 , to produce an intermediate of formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein the carrier Tu is defined as a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from glycoprotein carbohydrate residues, or an amidoalkylthio group; and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, -NHCONHN=CH-, -NHCONHNHCH 2 -, -NHCSNHN=CH-, -NHCSNHNHCH 2 -, -ON=CH-, -NH-, -NHCH 2 -, -N=CH-, -CO 2 -, -NHCH 2 CO 2 -, -SS-, and m is 0.1 to 15. A protein-drug conjugate according to Claim 3 of the formula prepared from the class of N-acyl derivatives of antitumor antibiotics designated LL-E33288 (CH 3 SSS-W) comprising: displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C 2 -C 10 ) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C 2 -C 5 ) radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; and Q is, or can be subsequently converted to, carboxyl, lower alkyldicarboxylanhydride, -CO 2 Su, -CONHNH 2 , or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, or and m is 0.1 to 15. A process for producing a substituted disulfide of formula Q-Sp-SS-W from a compound of formula CH 3 SSS-W which is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, wherein R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; R is hydrogen or a branched or branched alkyl (C 1 -C 10 ) or alkylene (C 1 -C 10 ) group, an aryl or heteroaryl group, or an aryl-alkyl (C 1 -C 5 ) or heteroaryl-alkyl (C 1 -C 5 ) group, all optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, lower (C 1 -C 3 ) alkoxy, or lover (C 1 -C 5 ) thioalkoxy groups; Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocyclo-alkyl radicals, divalent or trivalent aryl- or hetoroaryl-alkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein it Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxylcarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -CSNHNH 2, -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen; and when CH 3 -SSS-W is LL-E33288 , α 1 -I, α 2 -Br, α 2 -l, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q can not be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl; which comprises reacting a methyl trisulfide antitumor antibiotic from those of the above with an appropriately substituted mercaptan in acetonitrile or a combination of acetonitrile and tetrahydrofuran or acetonitrile and dimethylformamide, at a temperature of between -20 o to about +40 o C, for a period of from one hour to six days, preferably in the presence of one equivalent of a tertiary amine base or a mixture of one equivalent of a tertiary amine base and one equivalent of an organic carboxylic acid. A process for preparing the targeted derivatives of compounds of formula CH 3 SSS-W wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, comprising reacting CH 3 SSS-W with a compound of formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, lower alkyldicarboxyl anhydride, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , or in acetonitrile in the presence of one equivalent of triethylamine or one equivalent of triethylamine and one equivalent of acetic acid at -10 o to -30 o C for 1-48 hours, isolating the intermediate of formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, then reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, or lower alkyldicarboxylic anhydride with a molecule of the formula Tu-(Y) n wherein Tu is a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino or carboxy functionality; n is 1-100, in aqueous buffer at a pH of between 6.5 and 9, at 4 o to 40 o C either directly or in the presence of a water-soluble carbodiimide, to generate the compound wherein Tu, Sp, W, n, and Y are as hereinbefore defined, m is 1-15 and Z is formed from covalent reaction of the groups Q and Y and is -CONH-, -NH-, -N=CH-, or -CO 2 - or reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is a carboxylic acid, with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol, or 4-nitrophenol in the presence of a carboxyl activating agent such as a carbodiimide to generate a compound of formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is with a molecule of formula Tu-(Y) n , where Tu and n are as hereinbefore defined, and Y is a side-chain amino group, in an aqueous buffered solution at a pH between 6.5 and 9, at a temperature of between 4 o and 40 o C, inclusive, to generate compounds of the formula: wherein Tu, Sp, Y, and n are as hereinbefore defined, m is 1-15, and Z is formed from covalent reaction between Q and Y and is defined as -CONH- or reacting a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CONHNH 2 with nitrous acid in agueous acetonitrile to generate a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CON 3 with a compound of formula Tu-(Y) n , wherein Tu, Y, and n are as hereinabove defined to produce a compound of the formula wherein Tu, Z, Sp, W, m, Y, and n are as hereinabove defined; or reacting a compound of formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is hydroxy, with an alpha-haloacetic anhydride to produce a compound wherein Q is α-haloacetyloxy, and reacting the α-haloacetyloxy-Sp-SS-W, or a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is with a molecule of the formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is a side-chain thiol of a protein, or an amidoalkylthio group introduced on an amine of Tu using reagents such as 3-(2-dithiopyridyl)propionic acid hydroxysuccinimide ester followed by reduction with an agent such as dithiothreitol, or an amidoalkylthio group introduced on an amine of Tu using 2-iminothiolane, and n is 1-10, under aqueous buffered conditions at a pH between 4.5 and 7, at a temperature between 4 o and 40 o C, inclusive, to produce a compound of formula: wherein Tu, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y and Z is -SS-, and n is 0.1 to 10; or reacting a compound of the formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is -NH 2 , -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , or -ONH 2 with a molecule of formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is an aldehyde generated from carbohydrate residues on Tu by oxidation in the presence of an alkaline earth periodate, in an aqueous buffer at a pH between 4.0 and 6.5, at 4 o to 40 o C, inclusive, and n is 1 to 20 to generate a compound of formula: wherein Tu, Sp, W, Y, and n are as hereinbefore defined and Z is formed from the covalent reaction of Q and Y and is -ON=CH-, -N=CH-, -CONHN=CH-, -NHCONHN=CH-, or -NHCSNHN=CH-, and m is 0.1 to 15; or treating the compound immediately hereinabove of formula: wherein Tu, Z, Sp, W, Y, n, and m are as immediately hereinabove defined with acetylhydrazine or tyrosine hydrazine in an aqueous buffer at a pH between 4.0 and 6.5, at 4 o to 40 o C, inclusive, to generate a compound of formula: wherein Tu, Z, Sp, W, n, and m are as immediately hereinabove defined and Y is -CH=NNHCOCH 3 or and reacting this compound with sodium cyanoborohydride or sodium borohydride, in an aqueous buffer at a pH of 4.0 to 6.5, at a temperature of 4 o to 40 o C, inclusive, to generate a compound of formula: wherein Tu, Sp, W, m, and n are as hereinabove defined, Z is -NH-CH 2 -, -CONHNHCH 2 -, -NHCONHNHCH 2 -, or -NHCSNHNHCH 2 -, and Y is -CH 2 NHNHCOCH 3 or A product containing a substituted disulfide analog of formula Q-Sp-SS-W, prepared from a compound of formula CH 3 SSS-W which is an N-acyl derivative of LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, for use in bacterial infections therapy wherein R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; R is hydrogen or a branched or unbranched alkyl (C 1 -C 10 ) or alkylene (C 1 -C 10 ) group, an aryl or heteroaryl group, or an aryl-alkyl (C 1 -C 5 ) or heteroaryl-alkyl (C 1 -C 5 ) group, all optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, lower (C 1 -C 3 ) alkoxy, or lower (C 1 -C 5 ) thioalkoxy groups; Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocyclo-alkyl radicals, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein, if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -CSNHNH 2 , -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen and when CH 3 -SSS-W is LL-E33288 , α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, , γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q can not be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl. A product containing a substituted disulfide of formula Q-Sp-SS-W, prepared from a compound of formula CH 3 SSS-W which is an N-acyl derivative of LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, for use in tumor therapy, wherein R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; wherein R is hydrogen or a branched or unbranched alkyl (C 1 -C 10 ) or alkylene (C 1 -C 10 ) group, an aryl or heteroaryl group, or an aryl-alkyl (C 1 -C 5 ) or heteroaryl-alkyl (C 1 -C 5 ) group, all optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, lower (C 1 -C 3 ) alkoxy, or lower (C 1 -C 5 ) thioalkoxy groups; Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocyclo-alkyl radicals, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein, if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lover alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -CSNHNH 2 , -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen; and when CH 3 -SSS-W is LL-E33288 , α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q can not be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl. A product containing a compound prepared from a compound of general formula CH 3 SSS-W, wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 I , α 2 BR , α 2 I , β 1 BR , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, by a process comprising: reacting CH 3 SSS-W with a compound of general formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, a lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288 α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; and Q is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , -CON 3 , to produce an intermediate of formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is defined as a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, growth factors, or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from glycoprotein carbohydrate residues, or an amidoalkylthio group; and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, -NHCONHN=CH-, -NHCONHNHCH 2 -, -NHCSNHN=CH-, -NHCSNHNHCH 2 -, -ON=CH-, -NH-, -NHCH 2 -, -N=CH-, -CO 2 -, -NHCH 2 CO 2 -, -SS-, and m is 0.1 to 15, for use in tumor therapy. A product of a protein-drug conjugate of the formula prepared from the antitumor antibiotic designated N-acetyl LL-E33288γ 1 I (CH 3 SSS-W) having a) ultraviolet spectrum as shown in Figure 1; b) an infrared spectrum as shown in Figure 2; c) a proton magnetic resonance spectrum as shown in Figure 3; and d) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure 4; by a process comprising: displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C 2 -C 5 ) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C 2 -C 5 ) radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; and Q is, or can be subsequently converted to, carboxyl, lower alkyldicarboxylanhydride, -CONHNH 2 , or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, or and m is 0.1 to 15, for use in tumor therapy for in vivo delivery of the conjugate to an antigenic site. Claims for the following Contracting States : ES, GR A process for producing a substituted disulfide of formula Q-Sp-SS-W from a compound of formula CH 3 SSS-W which is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, wherein R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; R is hydrogen or a branched or unbranched alkyl (C 1 -C 10 ) or alkylene (C 1 -C 10 ) group, an aryl or heteroaryl group, or an aryl-alkyl (C 1 -C 5 ) or heteroaryl-alkyl (C 1 -C 5 ) group, all optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, lower (C 1 -C 3 ) alkoxy, or lower (C 1 -C 5 ) thioalkoxy groups; Sp is straight or branched-chain divalent or trivalent (C 1 -C 18 ) radicals, divalent or trivalent aryl or heteroaryl radicals, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocyclo-alkyl radicals, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radicals, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radicals, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, carboxyl, nitrophenyloxylcarbonyl, carboxaldehyde, lower alkoxy, hydroxy, thiol, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -CSNHNH 2 , -NHCSNHNH 2 , or -ONH 2 ; with the provisos that when Sp is ethylidene, Q cannot be hydrogen; and when CH 3 -SSS-W is LL-E33288 , α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and Sp is divalent, Q can not be hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino, or carboxyl; which comprises reacting a methyl trisulfide antitumor antibiotic from those of the above with an appropriately substituted mercaptan in acetonitrile or a combination of acetonitrile and tetrahydrofuran or acetonitrile and dimethylformamide, at a temperature of between -20 o to about +40 o C, for a period of from one hour to six days, preferably in the presence of one equivalent of a tertiary amine base or a mixture of one equivalent of a tertiary amine base and one equivalent of an organic carboxylic acid. The process according to Claim 1 wherein a substituted disulfide of formula Q-Sp-SS-W is prepared from a compound of formula CH 3 SSS-W wherein: R 5 is CH 3 , C 2 H 5 , or (CH 3 ) 2 CH; X is an iodine or bromine atom; wherein R is hydrogen, CH 3 , or a mono-substituted aryl group; and Sp and Q are as defined. A process for preparing the targeted derivatives of compounds of formula CH 3 SSS-W wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, comprising reacting CH 3 SSS-W with a compound of formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288, α1-I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, lower alkyldicarboxyl anhydride, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , or in acetonitrile in the presence of one equivalent of triethylamine or one equivalent of triethylamine and one equivalent of acetic acid at -10 o to -30 o C for 1-48 hours, isolating the intermediate of formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined in claim 1, then reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, or lower alkyldicarboxylic anhydride with a molecule of the formula Tu-(Y) n wherein Tu is a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino or carboxy functionality; n is 1-100, in aqueous buffer at a pH of between 6.5 and 9, at 4 o to 40 o C either directly or in the presence of a water-soluble carbodiimide, to generate the compound wherein Tu, Sp, W, n, and Y are as hereinbefore defined; m is 1-15 and Z is formed from covalent reaction of the groups Q and Y and is -CONH-, -NH-, -N=CH-, or -CO 2 - or reacting the compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is a carboxylic acid, with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol, or 4-nitrophenol in the presence of a carboxyl activating agent such as a carbodiimide to generate a compound of formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is with a molecule of formula Tu-(Y) n , where Tu and n are as hereinbefore defined, and Y is a side-chain amino group, in an aqueous buffered solution at a pH between 6.5 and 9, at a temperature of between 4 o and 40 o C, inclusive, to generate compounds of the formula: wherein Tu, Sp, Y, and n are as hereinbefore defined, m is 1-15, and Z is formed from covalent reaction between Q and Y and is defined as -CONH- or reacting a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CONHNH 2 with nitrous acid in aqueous acetonitrile to generate a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is -CON 3 with a compound of formula Tu-(Y) n , wherein Tu, Y, and n are as hereinabove defined to produce a compound of the formula wherein Tu, Z, Sp, W, m, Y, and n are as hereinabove defined; or reacting a compound of formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is hydroxy, with an alpha-haloacetic anhydride to produce a compound wherein Q is α-haloacetyloxy, and reacting the α-haloacetyloxy-Sp-SS-W, or a compound of formula Q-Sp-SS-W, wherein Sp and W are as hereinbefore defined and Q is with a molecule of the formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is a side-chain thiol of a protein, or an amidoalkylthio group introduced on an amine of Tu using reagents such as 3-(2-dithiopyridyl)propionic acid hydroxysuccinimide ester followed by reduction with an agent such as dithiothreitol, or an amidoalkylthio group introduced on an amine of Tu using 2-iminothiolane, and n is 1-10, under aqueous buffered conditions at a pH between 4.5 and 7, at a temperature between 4 o and 40 o C, inclusive, to produce a compound of formula: wherein Tu, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y and Z is -SS-, and n is 0.1 to 10; or reacting a compound of the formula Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is -NH 2 , -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , or -ONH 2 with a molecule of formula Tu-(Y) n wherein Tu is as hereinbefore defined, Y is an aldehyde generated from carbohydrate residues on Tu by oxidation in the presence of an alkaline earth periodate, in an aqueous buffer at a pH between 4.0 and 6.5, at 4 o to 40 o C, inclusive, and n is 1 to 20 to generate a compound of formula: wherein Tu, Sp, W, Y, and n are as hereinbefore defined and Z is formed from the covalent reaction of Q and Y and is -ON=CH-, -N=CH-, -CONHN=CH-, -NHCONHN=CH-, or -NHCSNHN=CH-, and m is 0.1 to 15; or treating the compound immediately hereinabove of formula: wherein Tu, Z, Sp, W, Y, n, and m are as immediately hereinabove defined with acetylhydrazine or tyrosine hydrazine in an aqueous buffer at a pH between 4.0 and 6.5, at 4 o to 40 o C, inclusive, to generate a compound of formula: wherein Tu, Z, Sp, W, n, and m are as immediately hereinabove defined and Y is -CH=NNHCOCH 3 or and reacting this compound with sodium cyanoborohydride or sodium borohydride, in an aqueous buffer at a pH of 4.0 to 6.5, at a temperature of 4 o to 40 o C, inclusive, to generate a compound of formula: wherein Tu, Sp, W, m, and n are as hereinabove defined, Z is -NH-CH 2 -, -CONHNHCH 2 -, -NHCONHNHCH 2 -, or -NHCSNHNHCH 2 -, and Y is -CH 2 NHNHCOCH 3 or A process for preparing a carrier-drug conjugate of the formula prepared from a compound of formula CH 3 SSS-W wherein CH 3 SSS-W is an N-acyl derivative of an antitumor antibiotic designated as LL-E33288, α 1 I , α 2 Br , α 2 I , β 1 Br , β 1 I , γ 1 Br , γ 1 I , δ 1 I , BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, wherein W is as hereinbefore described in claim 1 comprising: reacting CH 3 SSS-W with a compound of general formula Q-Sp-SH, wherein Sp is a straight or branched-chain divalent or trivalent (C 1 -C 18 ) radical, divalent or trivalent aryl or heteroaryl radical, divalent or trivalent (C 3 -C 18 ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C 1 -C 18 ) radical, divalent or trivalent cycloalkyl- or heterocycloalkyl-alkyl (C 1 -C 18 ) radical or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl radical, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, alkylamino, arylamino heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower alkylthio groups, with the proviso that when CH 3 -SSS-W is the parent antitumor antibiotic LL-E33288α 1 -Br, α 1 -I, α 2 -Br, α 2 -I, β 1 -Br, β 1 -I, γ 1 -Br, γ 1 -I, δ 1 -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp can not be divalent; and Q is, or can be subsequently converted to, halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH 2 , -NHCONHNH 2 , -NHCSNHNH 2 , -ONH 2 , -CON 3 , to produce an intermediate of formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula TU-(Y) n wherein the carrier Tu is defined as a mono- or polyclonal antibody, its fragments, its chemically or genetically manipulated counterparts, or growth factors or steroids; Y is a side-chain amino, carboxy, or thiol group of a protein, an aldehyde derived from glycoprotein carbohydrate residues, or an amidoalkylthio group; and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, -NHCONHN=CH-, -NHCONHNHCH 2 -, -NHCSNHN=CH-, -NHCSNHNHCH 2 -, -ON=CH-, -NH-, -NHCH 2 -, -N=CH-, -CO 2 -, -NHCH 2 CO 2 -, -SS-, and m is 0.1 to 15. A process for preparing a protein-drug conjugate as prepared in Claim 4 of the formula prepared from the class of N-acyl derivatives of antitumor antibiotics designated LL-E33288 (CH 3 SSS-W) comprising: displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C 2 -C 10 ) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C 2 -C 5 ) radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups ; and Q is, or can be subsequently converted to, carboxyl , lower alkyldicarboxylanhydride, -CO 2 Su, -CONHNH 2 , or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, or and m is 0.1 to 15. A process for preparing a protein-drug conjugate of the formula prepared from the antitumor antibiotic designated N-acetyl LL-E33288γ 1 I (CH 3 SSS-W) having a) ultraviolet spectrum as shown in Figure 1; b) an infrared spectrum as shown in Figure 2; c) a proton magnetic resonance spectrum as shown in Figure 3; and d) a carbon-13 nuclear magnetic resonance spectrum as shown in Figure 4; which process comprises displacing the dithiomethyl moiety with a compound of formula Q-Sp-SH, wherein Sp is straight or branched-chain divalent or trivalent (C 2 -C 5 ) radicals or divalent or trivalent aryl- or heteroaryl-alkyl (C 2 -C 5 ) radicals, wherein if Sp is a trivalent radical, it can be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; and Q is, or can be subsequently converted to, carboxyl, lower alkyldicarboxylanhydride, -CONHNH 2 , or to produce an intermediate of general formula Q-Sp-SS-W, wherein Q, Sp, and W are as hereinbefore defined, reacting Q-Sp-SS-W with a molecule of the formula Tu-(Y) n wherein Tu is a monoclonal antibody which exhibits preferential reactivity with a human tumor-associated antigen, Y is a side-chain amino group on the antibody, or an aldehyde generated by oxidation of the carbohydrate groups of the antibody, and n is an integer of from 1 to 100, to produce a compound of the formula: wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z is formed from covalent reaction of the groups Q and Y directly or after subsequent reduction, and Z is -CONH-, -CONHN=CH-, -CONHNHCH 2 -, or and m is 0.1 to 15.

Inventors: ELLESTAD GEORGE A, HAMANN PHILIP R, HINMAN LOIS M, MCGAHREN WILLIAM JAMES, SASSIVER MARTIN LEON, UPESLACIS JANIS, ELLESTAD, GEORGE A., HAMANN, PHILIP R., HINMAN, LOIS M., MCGAHREN, WILLIAM JAMES, SASSIVER, MARTIN LEON, UPESLACIS, JANIS
Applicants: AMERICAN CYANAMID CO, AMERICAN CYANAMID COMPANY
Application year: 1990
Main classification: C07H15
IPC codes: A61K31, A61P35, C07K1, C07K16, A61K39, A61P31, C07K14, A61K47, C07H15