KR0159767B1 - Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group, targeted forms and preparation thereof - Google Patents

Abstract

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Description

Substituted disulfide which is an anti-tumor antimicrobial agent prepared from a compound having a methyl-trithio group, a desired form thereof and a method for producing the same.

Figure 1: Ultraviolet spectrum of anti-tumor antibiotic named N-acetyl LL-E33288δ ₁ ^I.

Figure 2: Infrared spectrum of anti-tumor antibiotic named N-acetyl LL-E33288δ ₁ ^I.

Figure 3: Proton magnetic resonance spectra of anti-tumor antibiotics named N-acetyl LL-E33288δ ₁ ^I.

Figure 4: Carbon-13 nuclear magnetic resonance spectra of anti-tumor antibiotics named N-acetyl LL-E33288δ ₁ ^I.

FIG. 5: Ultraviolet Spectrum of Antitumor Antibiotic Named as Iodine LL-E33288 Pseudoaglycone.

Figure 6: Infrared spectrum of antitumor antibiotic named iodine LL-E33288 pseudoaglycone.

Figure 7: Proton magnetic resonance spectra of anti-tumor antibiotics named as iodine LL-E33288 pseudoaglycone.

Figure 8: Carbon-13 magnetic resonance spectra of anti-tumor antibiotics named as iodine LL-E33288 pseudoaglycones.

The present invention unsubstitutes or disulfide compounds of BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 antibiotics or antibiotics. As well as their derivatives prepared by reacting with substituted alkyl mercaptans, as well as disulfide compounds of the α₁, α₂, α₃, α₄, β₁, β₂, γ, δ, and pseudoaglycon components and their derivatives of the LL-E33288 complex . These disulfide compounds are effective antitumor agents.

The invention also describes carrier- weak conjugates of disulfide analogues prepared from the materials described above. Mono- or polyclonal antibodies, fragments thereof, chemically or genetically engineered counterparts, or growth applications or steroids of the carrier (target) portion of the conjugate. The method of producing the carrier-drug conjugates of the present invention as well as the method of use thereof are included.

The antimicrobial and antitumor family, collectively known as the LL-E33288 complex, is described and claimed in EPO Publication No. 0182152A3, published May 28, 1986, and several starting materials for antitumor agents that are the target forms of the present invention. Used to prepare phosphorus disulfur antitumor agents.

EPO Publication No. 0182152A3 discloses LL-E33288 complex, their components, ie, LL-E33288α ₁ ^BR , LL-E33288α ₁ ^I , LL-E33288α ₂ ^BR , LL-E33288α ₂ ^I , LL-E33288α ₃ ^BR , LL-E33288α ₃ ^I , LL-E33288α ₄ ^BR , LL-E33288β ₁ ^BR , LL-E33288β ₁ ^I , LL-E33288β ₂ ^BR , LL-E33288β ₂ ^I , LL-E33288γ ₁ ^BR , LL-E33288γ ₁ ^I , and LL-E33288δ ₁ ^I and We describe a novel strain, Mytromonospora echinospora ssp calichensis or their preparation by aerobic fermentation using their natural or induced mutants.

EPO Publication 018152A3 also describes the proposed structure for some of the components named above. These proposed structures are shown in Table 1.

Additional types of LL-E33288 complexes are described and claimed in EPO Publication No. 027485A2, published August 3, 1988, which are likewise useful for preparing the disulfide derivatives of the invention and antitumor agents of the desired form. This application describes a series of LL-E33288 bromo- and iodine- pseudoaglycones that have been prepared by chemical means. The application also describes dihydro derivatives obtainable from the antitumor antibiotics named above by reducing the sodium borohydride with a hydroxyl group at C ₁₁ with a hydroxyl group. These latter proposed structures are described in Table 2.

Another class of anti-tumor antibiotics, the LL-E33288 family, are described and claimed in the co-pending application (30,553-01), which are also useful for preparing additional target forms that are antitumor agents of the present invention. The present application describes several types of N-acyl derivatives of the LL-E33288 complex that have been prepared by chemical means. Likewise these proposed structures are described in Table 2.

Certain other antitumor antimicrobial compounds are useful in this invention.

Namely: 1) Esperamicin BBM-1675, a novel potential antitumor antibiotic species, I. Physico-chemical data and partial structure. Konishi party, Jay. Antibiotics, 38, 1605 (1985). New Antitumor Antibiotic Complex, M. Cornish Group, British Patent Application No. 2,141,425A, May 15, 1984.

2) new antitumor antibiotics, FR-900405 and FR-900406. I. Production Strain Classification. M. Iwami party, Jay. Antibiotics 38, 835 (1985). New antitumor antibiotics FR-900405 and FR-900406. II. Production, separation, characterization and anti-tumor action. s. Kiyoto and Jay. Antibiotics, 38, 340 (1985).

3) PD 114759 and 115028, novel anti-tumor antinews with great efficacy. I. Separation and Characterization. Aar. H. Bunge party, Jay. Antibiotics, 1566 (1984). Biological and biochemical action of the novel antitumor antibiotic PD 114759 and related derivatives. Dee. W. Fry, Investigational New Drugs, 4, 3 (1986).

4) Streptomyces sp. Novel Antibiotic Complexes CL-1577A, CL-1577B, Produced by ATCC 39363, European Patent Application No. 0,132,082, A2

5) CL-1577D and CL-1577E antibiotic anti-tumor compounds, their production and use. U.S. Patent 4,539,203.

6) CL-1724 antibiotic compounds, their production and use. United States Patent No. 4,554,162.

7) New antitumor antibiotics BBM-1675-A3 and BBM-1675-A4, obtained by fermentation of actinomadura verrucosospora strain H964-92 (ATCC 39334) or AB27Y (ATCC 39638). United States Patent No. 4,675,187.

8) Novel N-acetyl-esperamicin A ′, A₂, and A1b derivatives with antimicrobial and antitumor activity. EP 289,030

All information regarding BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724 included in the above citation is herein incorporated by reference. Quote from

The complete structures of esperamicin A ′, A ₂ and A _1b (BBM-1675 complex) and their respective N-acetyl derivatives have been reported, which are included in Tables 1 and 2. The physical properties of the other antitumor antibiotics named above indicate that they are identical or very similar in structure to esperamines, and all contain methyltrithio functional groups.

As can be seen from the structures described above, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724 anti-matter and their N In addition to the acyl counterparts, the α₁, α₂, α₃, α₄, β₁, β₂, γ ₁ , δ and pseudoaglycone components, their dihydro and N-acyl counterparts of the LL-E33288 complex are each methyl in their structure. It contains a trithio group.

Now, any of the antibiotics cited above can be reacted with unsubstituted or substituted alkyl or aryl mercaptans to replace the methyl perthiolate anion from the trisulfide moiety, which results in the formation of the following stable disulfides (Scheme I). did. Other compounds in which such translocations occur are described in EPO Publication No. 0313874A2, published May 3, 1989.

The compounds of Tables 1 and 2 are inverted with thiol-containing organic molecules according to Scheme I, resulting in novel compositions that are useful as antimicrobial anticancer agents. Where W, R, R₁, R₂, R₃, R₄, R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₂ ', R ₃ ', R ₄ ', R ₅ ' Ar₁, Ar₂ and X are As defined above in Tables 1 and 2, Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, divalent or _trivalent (C ₃ -C ₁₈ ') cyclo Alkyl or heterocycloalkyl, 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 divalent or trivalent (C ₂ -C ₁₈ ) unsaturated alkyl radical, where Sp is a trivalent radical, which is optionally substituted with amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio group Can be; Q is hydrogen, halogen, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, pepperidino, pyrrolidino, piperazino, cartboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, Hydroxy, thiol, lower alkyldicarboxyl or a group selected from -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH ₂ , or -ONH ₂ ; Provided that when Sp is ethylidene, Q cannot be hydrogen; CH ₃ -SSS-W is LL-E33288α ₁ -Br, α ₁ -1, α₂-Br, α₂-I,

α ₃ -Br, α ₃ -I, α ₁ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, When BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E or CL-1724 and Sp is valent, Q is hydrogen, halogen, It may not be amino, alkylamino, dialkylamino, arylamino, heteroallylamino, pyrrididino, pyrrolidino, pyrerazino or carboxyl.

In a preferred embodiment of the invention, the disulfide of the general formula Q-Sp-SS-W is prepared from an anti-tumor antibiotic named LL-E33288 (CH ₃ SSS-W). Where W is

Or H;

Or H; And R ₅ is DH ₃ , C ₂ H ₅ or (CH ₃ ) ₂ ; X is an iodine or bromine atom; R ₅ is hydrogen or an RCO group wherein R is hydrogen, CH ₃ or a mono-substituted aryl group; Sp and Q are as defined above.

The translocation of the compounds listed in Tables 1 and 2 to various disulfides can be performed with a wide variety of thiol-containing organic molecules, resulting in novel antitumor and antibacterial agents. In addition, the products from those reactions can be further translocated within the separately introduced side chains to produce novel compounds with biological action.

The compounds of the present invention are useful for assessing new ways of anticancer action at the cellular level in addition to their use as antitumor and antibacterial agents. Naturally derived antibiotics described by the above-cited applications and patents do not contain chromogenic units strong enough that their intracellular localization can be adequately described. Double-stranded DNA destruction and upstream when natural trithiometal derivatives react with thiol-containing molecules that additionally contain chromophoric units that absorb or emit light within the full-spectrum spectral region not detected by cell chromophores. Biochemical tools useful for studying cell localization of compounds can be generated. Similarly, the introduction of radioisotopes by this reaction is also within the scope of the present invention.

Thus, for example, when E-33288 γ ₁ -I reacts with 7- (2-thiomethylamino) -4-methylcoumarin, a derivative is obtained that strongly fluoresces upon irradiation with ultraviolet light, which causes the compound to Allow to be located within the compartment.

In this embodiment of the invention, the compounds of Tables 1 and 2 are transferred to thiol-containing organic molecules according to Scheme I, producing novel compositions that are additionally useful as molecular probes. Where W, R, R₁, R₂, R₃, R₄, R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₂ ', R ₃ ', R ₄ ', R ₅ ', Ar₁, Ar₂ and X are As defined above in Tables 1 and 2, Sp is a straight or branched divalent (C ₁ -C ₁₈ ) radical, a divalent aryl or heteroaryl radical, a divalent (C ₃ -C ₁₈ ) cycloalkyl or heterocycloalkyl radical, divalent Aryl or heteroaryl-alkyl (C ₁ -C ₁₈ ) radicals, divalent cycloalkyl or heterocycloalkyl-alkyl (C ₁ -C ₁₈ ) radicals and divalent (C ₂ -C ₁₈ ) unsaturated alkyl radicals; Q is a ³ H, ¹⁴ C, ³⁵ S or ³² P radioisotope as part of an unsubstituted or substituted fluorescent nucleus or structure of dibenzooxazine, rhodamine, carbostyryl, coumarin, fluorescein, or acridine It is a substituent containing.

Now, substitutions of the methyltrithio units of the compounds as depicted in Scheme I, and listed in Tables 1 and 2, can be used to introduce the spacer (Sp), and this wise choice allows the target units to be incorporated into the disulfide derivative compounds herein. It has been found that it can be introduced. The introduction of the target unit into other disulfide derivatives is described in EPO Publication No. 0313873A1, published May 3, 1989. Thus, in Scheme I, the general formula CH3SSS-W (where CH3SSS-W is LL-E33288 α)_One ^Br, α_One ^I, α₂ ^Br, α₂ ^I, α₃ ^Br, α₃ ^I, α₄ ^Br, β_One ^Br, B_One ^I, β₂ ^Brβ₂ ^I, γ_One ^Br, γ_One ^I, δ_One ^I, Iodo or bromo pseudoaglycones, their dihydro or N-acetyl counterparts, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D , CL-1577E, CL-1724 or an anti-tumor antibiotic named as their N-acetyl counterpart) are first reacted with a compound of the general formula Q-Sp-SH to react intermediate Q-Sp-SS-W. Create

Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, a divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocycloalkyl radical, divalent or _trivalent Aryl- or heteroalkyl-alkyl (C ₁ -C ₁₈ ) radicals, divalent or trivalent cycloalkyl- or heterocyclo alkyl-alkyl (C ₁ -C ₁₈ ) radicals and divalent or trivalent (C ₂ -C ₁₈ ) unsaturated alkyl radicals ego; Provided that if Sp is a trivalent radical, it may be optionally substituted with amino, alkyl amino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups, and CH3-SSS-W Parent antitumor antibiotics LL-E33288 α ₁ -Br, α ₁ -1, α₂-Br, α₂-I, α ₃ -Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B Sp, if it is, CL-1577D, CL-1577E or CL-1724 itself, Sp cannot be divalent; Sp

And W is as shown in Tables 1 and 2 above, Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH ₂ , -ONH ₂ , -CON ₃ ,

Or continue to switch to them.

As long as the product from the scheme contains at least one functional group that can be directly reacted with or converted to the target unit (Tu), the target forms of the antitumor antibiotics of the above applications and patents are shown in Table II below. Can be generated as

Wherein Q, Sp and W are as defined above and Tu is a mono- or polyclonal antibody, a fragment thereof, a chemically or genetically processed counterpart thereof, or a growth factor or a steroid; Y is an aldehyde or amidoalkylthiogo derived from a thiol group, carbohydrate moiety of a side chain amino, carboxy, or protein; n is an integer from 1 to 100; Z is formed after subsequent reduction or directly from the covalent reaction of Q and Y groups, -CONH-, -CONHN = CH-, -CONHNHCH _2- , -NHCONHN = CH _1- , -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 example, in Figure II above, the 3-methcaptorropionic acid derivative of N-acetyl E-33288γ ₁ ^I (Q = -CO ₂ H, Sp = -CH ₂ CH _2- ) has its active hydroxysuccinimide When converted to form (Q = CO ₂ Su, Sp = -CH ₂ -CH _2- ), it is a lysine residue (eg Tu = monoclonal antibody, Y at pH 7.0 to 9.5 in aqueous buffer at 4 ° C. to 40 ° C.). = -NH ₂ where n = 50 to 100 from commercially available lysine residues), used to react with some of the ε-amino groups, the desired form of antibiotic bound to random sites along the protein backbone (Tu = single Clone antibody, Z = -NHCO-, Sp = -CH ₂ CH _2- , m = 1-10). Only a fraction of commercially available lysine residues is substituted by this method. This is because the load is usually considered unsuitable for maintaining antibody immune reactivity. The same randomly-substituted immune conjugates can also be prepared from 3-methcaptopropionic acid derivatives or corresponding acylazides using other carboxyl activators such as various carbodiimides. On the contrary, N-acetyl, 3-mergatopropionyl hydrazide derivatives (Q = H ₂ NNHCO-, Sp = -CH ₂ CH _2- ) of LL-E33288δ ₁ ^I in buffered aqueous solutions at 4 ° C. to 40 ° C. When reacted with periodate-oxidized antibodies (Tu = monoclonal antibody, Y = -CHO, n = 1-15) at pH 4-7 as described in US Pat. No. 4,671,958, it is only an aldehyde functional site ( A monoclonal antibody conjugate (Tu = monoclonal antibody, Z) containing drugs substituted at specific sites along the protein backbone, reacting only with vic-diol cleavage of carbohydrate residues located on the Fc portion of the antibody = -CH = NNHCO-, Sp = -CH ₂ CH _2- , m = 0.5-10). The latter conjugate is first reacted with acetyl hydrazide or tyrosine hydrazide, and then sodium cyanoborohydra to not only stabilize hydrolytically labile Schiff base bonds, but also block unreacted aldehyde groups on the antibody to prevent crosslinking. Reduction with id or sodium borohydride to produce a stabilized structure of the invention (Tu = monoclonal antibody, Z = -CH ₂ NHNHCO-, Sp = -CH ₂ CH _2- , m = 0.5-10) Not required). Other aldehyde-reactive groups as part of the drug constituent are within the scope of the present invention to produce the product of Scheme II. Such functional groups are preferably, but not limited to, those that react with aldehydes under acidic aqueous conditions. The reactivity of protein lysines under base conditions is large enough that their amines compete with the products of Scheme II for the useful aldehydes of monoclonal antibodies. Alternative aldehyde-reactive groups are, for example, semicarb azide, thiosemicarb azide and O-substituted hydroxylamine functional groups.

The collection of target forms of the compounds listed in Tables 1 and 2 is not limited to the sequences summarized in Figure II. The target unit (Tu) may first be modified to contain a thiol group and then react with the compounds of Tables 1 and 2 according to Scheme III below.

Wherein, Tu, Y, Q, Sp, W, n and m are as defined above and P is hydrogen or 2- (pyridylthio) provided that Y is a thiol derived from the backbone amino acid residues of Tu, Z-Sp taken together is a covalent bond.

For example, in Scheme III, monoclonal antibodies can be reacted with 3- (2-dithiopyridyl) propionic acid hydroxysucciimide esters to modify the protein via lysine residues. (Tu = monoclonal antibody, Y = NH _2, n = 50-100, q = -CO ₂ Su, Sp = -CH ₂ -CH ₂ , P = 2-pyridylthio). For example, in the following reduction with dithiothreitol, an intermediate that reacts with the compounds of Tables 1 and 2 to produce the subject's immune conjugate (Tu = monoclonal antibody, Z = -NHCO-, Sp = CH ₂ CH _2- , m = 1 to 15). Similarly, 2-aminothiolane reacts with monoclonal antibodies to introduce thiol groups directly on the protein surface without reducing steps (Tu = monoclonal antibody, Z = -NHCO-, Sp =-(CH ₂ ) _3- , m = 1 to 15), and the intermediate may react with the compounds of Tables 1 and 2 previously. Alternatively, the sulfhydryl groups inherent in the monoclonal antibody structure in dimeric form as cystine residues can be used to directly participate in the reaction of Scheme III. The sulfhydryls are typically exposed by enzymatic digestion and reduction of native monoclonal antibodies (Tu = Fab 'fragments, Z-Sp = binding, Y = SH), but contain monoclonal unpaired cystine residues. The use of genetically-modified structures of antibodies is likewise contemplated.

Preferred embodiments for the target derivatives of the invention are those wherein the dithiomethyl moiety is represented by the general formula Q-Sp-SH, wherein Sp is a straight or branched chain divalent or trivalent (C ₂ -C ₁₀ ) radical or divalent or trivalent (C ₂ -C ₅ ) arylalkyl or hetero arylalkyl radicals, provided that when Sp is a trivalent radical, it can be further substituted by amino, hetero arylamino, hydroxy or thiol groups; Q is carboxyl, lower alkyl Dicarboxylic anhydride, -CO ₂ Su, -CONHNH ₂ or

To an intermediate of the general formula Q-Sp-SS-W, wherein Q, Sp and W are as defined above, and Q-Sp-SS-W is represented by the general formula Tu- ( Y) n (where Tu is a monoclonal antibody that shows preferential reactivity with human tumor-associated antigens, Y is an aldehyde produced by oxidation of the side chain amino group on the antibody or the carbohydrate group of the antibody, and n is an integer 1 to 100), followed by reaction with a molecule to give a compound of

(Wherein Tu, Y, Sp, W and n are as defined above, Z is formed after subsequent reduction or directly Q is formed from the covalent reaction of the Y group, which is -CONH-, -CONHN = CH-,- CONHNHCH ₂ -or

m is from 0.1 to 15), which is a protein-drug conjugate of the general formula prepared from an anti-tumor antibiotic named LL-E33288 (CH ₃ SSS-W) by a method consisting of:

Many different monoclonal antibodies (MoAbs) are used to illustrate the targeting of methyltrithio anticancer compounds. MoAbs Lym 1 and Lym 2 recognize different antigens on mature β-lymphocytes and their product lymphomas. The production and characterization of these MoAbs is shown in A. L. As described in Epstein's Cancer Research (47,830 (1987)) Reactivity with pancreatic, ovarian and lung cancers has also been reported, but MoAb B72.3 is primarily related to breast and colon cancer.

The antibody is T. L. Klug Int. J. Cancer (38, 661 (1986). MoAb CT-M-01, which primarily recognizes breast tumors, is described in EPO application 86 401482.4, filed Jul. 3, 1986, and MAC-68 is CT- Intermediates herein and conjugates with these antibodies, which are produced by sub-clone of hybridomas producing M-01, recognize both breast and colon cancer. However, this patent should not be construed as being limited or constrained by the antibody, but instead their individual or individualized type as well as their type or isotype, their enzymatically-derived fragments, their Regardless of the chemically processed and stabilized fragments, the method is sufficiently general that it can be applied to any antibody, and the target unit is not limited to only monoclonal antibodies. As well as small molecules for which receptors other protein material also lies within the scope of the invention as a target substance.

The compounds of the present invention named Q-Sp-SS-W are effective as antibacterial agents. Their in vitro antibacterial action can also be measured for Gram-positive and Gram-negative bacteria by standard agar dilution methods. Mueller-Hinton agar containing antibiotics with 2x decrease in concentration was poured into a Petri dish. Using a Steers replication device, agar surfaces were inoculated with 1-5 × 10 colonies to form bacterial units.

The lowest concentration of compound that inhibited the growth of bacterial strains after about 18 hours after inoculation at about 36 ° C. is recorded as the minimum inhibitory concentration (MIC) for that strain.

The disulfide compounds described above are active antitumor agents.

To measure the suitability of test compounds as antineoplastic agents, specific in vivo test systems and protocols have been developed by the National Cancer Institute. These have been reported in Cancer Chemotherapy Reports, Part III, Vol. 3, No. 2 (1972, Geran et al.) These protocols are usually standardized for screening in antitumor trials. Among these systems, lymphocytic leukemia P388, melanoma melanoma B16, L1210 leukemia and colon 26 adenocarcinoma are particularly meaningful in the present invention, these neoplasms are used for testing as transplantable tumors in mice. In contrast, the distinct anti-tumor activity shown in these protocols with an increase in the percent survival time of treated animals (T) on control standard animals (C) shows similar results in human leukemia and solid tumors.

(Lymphocytic leukemia P388 test)

The animals used were BDF1 female mice. There are 5 or 6 mice per test group. On the day of reference, tumors were transplanted by intraperitoneal injection of 0.5 ml of an ascitic fluid containing 1 × 10 ⁶ of lymphocytic leukemia p388 cells. Test compounds were administered intraperitoneally on a 1.5 and 9 day (in proportion to tumor inoculation) at 0.5 ml volume in sterile, pyrogen-free saline at the indicated doses. Mice were weighed and survivors were recorded on a constant basis for 30 days. Tumor survival time and survival time ratios for treated (T) / control (C) animals were calculated. If the T / C value is 125 or higher, it is considered to exhibit significant antitumor activity. The results are shown in Table 3.

Melanoma melanoma B16 test

The animal used may be a BDF1 female mouse. There are 5 or 6 mice per test group. A portion of 1 g of melanoma melanoma B16 tumor is homogenized in 10 ml of cold equilibrium salt solution and a 0.5 ml aliquot of homogenate is implanted intraperitoneally in each test mouse. Test compounds are administered intraperitoneally on days 1-9 (in proportion to tumor inoculation) at the indicated doses. Mice were weighed and surviving mice were recorded for 60 days on a constant basis. Median survival time and survival time ratios for treated (T) / control (C) animals were calculated. A T / C value of 125 or above is believed to indicate significant antitumor activity.

The method of the present invention used to generate monoclonal antibody conjugates, represented as, produces constructs that retain good immunoreactivity with the target cell line, as measured in the following in vitro assays:

[Red blood cells]

All red blood cells were collected in 5% fetal calf serum (FCS), ITS (Collaborative Research, Cat # 40351), streptomycin (50 ug / ml), penicillin (50 units / ml), gentamicin sulfate (50ug / ml And RPMI 1640 medium supplemented with glutamine (.03%). The cells were kept in a 5% CO ₂ incubator at 37 ° C.

I. Immune Reactivity Analysis

[Method I-Elisa]

Appropriate target cells were harvested and counted and suspended in Dulbecco phosphate buffered saline (DPBS) at the optimal concentration for the monoclonal antibody (MoAb) tested. 0.1 ml of cells were aliquoted into each well of sterile tissue culture polystyrene 96-well plates. The plate was centrifuged at 1,000 RPM for 5 minutes and the supernatant was shaken off. The plates may be air dried overnight and stored at 4 ° C. for up to 3 months. 200 μl of 1% gelatin in DPBS per wall was added and the plate was incubated for 1 hour at 37 ° C. in a wet thermostat (all subsequent incubations were done under similar conditions) to block non-specific binding sites. The plate was washed once with 250 μl of 0.05% TWEEN-20 (wash solution) in DPBS using an automated ELISA washing system (Ultrawash II) from Dynatech. The sample to be tested was diluted to a final concentration of 3 μl / ml MoAb equivalent in 0.1% gelatin-DPBS. Six additional triple serial dilutions were prepared from each 3 μl / ml sample and 100 μl was added to the appropriate wells. Only the bottom row of wells received 100 μl of 0.1% gelatin as the background. Plates were incubated for 45 minutes and washed three times. Affinity with Alkaline Phosphatase Purified goat anti-mouse immunoglobulin (Cappel Cat # 8611-0231) was diluted 1: 125 in 0.1% gelatin and 100 μl was added to each well. The plate was incubated for 45 minutes and washed three times. 200 μl of p-nitrophenyl phosphate support solution (see below) was added to each well. After 45 minutes at room temperature, 50 μl of 3M NaOH was added to stop the reaction. The absorbance of each well component was read at 405 nm with an automatic spectrophotometer from Dinatech (EIA Autoreader # EL-310).

Lipid diethanolamine buffer (10%)

97 ml diethanolamine

800 ml water

0.2g NaN3

100mg MgCl2 6H2O

The reaction mass was dissolved by stirring continuously and 1M HCI was added until the pH was 9.8. The total volume was 1 liter with water and sterilized with a 0.2 μ filter. The buffer was stored at 4 ° C. in the dark. Immediately before use, p-nitrophenyl phosphate (Sigma, Cat # 104-40) was dissolved (must be at room temperature) in 10% diethanolamine buffer to obtain a final concentration of 1 mg / ml.

[O.D. Value calculation]

The percent binding of each sample was calculated by the following equation:

A = average O.D of test samples

B = average O.D. in the background.

C = mean O, D of 3 μl / ml unengineered MoAb control.

% Binding was plotted on a non-log scale of semi-log graph and MoAb concentration was plotted on a log scale. The BD ₅₀ of each test sample (ie the antibody dose required to confer 50% binding) was derived from the graph and the retention of immune reactivity was calculated by the following equation:

[Method 2-Indirect RIA]

Appropriate amounts of erythrocytes in 0.2 ml of 10% FCS medium were aliquoted into 4 ml polystyrene tubes. The sample to be tested was diluted to a concentration of 2 μl / ml MoAb equivalents of 10% FCS medium. Five triplicate serial dilutions were prepared from each 2 μl / ml sample and 0.2 ml was added to each tube. Background samples received only cells and media. Cells were incubated at 4 ° C. for 1 hour and then washed twice with 2% FCS medium (all RIA washes were done in 3 ml volumes). 0.05 ml of sheep F (ab ') 2 anti-mouse IgG (Dupont, Cat # NEX 162-0142) containing about 500,000 CPM was added to each tube; The cells were further incubated at 4 ° C., washed once with 2% FCS and twice with PBS. 0.5 ml of FBS was added to the tube and the cells were vortexed and transferred to a clean tube, which was counted for 1 minute at Packard Gamma 500.

CPM's O.D. The percent binding of each value was measured and graphed according to the ELISA equation described above, except that the units were substituted in units and C = averaged CPM of 1 μl / ml of the unmodified MoAb control. Before discussing, the% retained immunoreactivity of each sample was calculated.

[Method 3-Direct RIA]

Appropriate amounts of target cells in 1 ml of 10% FCS medium were aliquoted into 4 ml polystyrene test tubes, centrifuged, and the supernatant discarded. The sample to be tested was diluted to a concentration of 200 μl / ml MoAb equivalent in 10% FCS medium.

Five additional 5-fold serial dilutions were prepared from each 200 μl / ml sample and 0.05 ml was added to each tube. 0.05 ml 125 I-MoAb was added to each tube (optimally measured for each MoAb and batch individually). Positive control samples included cells, media and ¹²⁵ I-MoAbs. Cells were incubated for 1 hour at 4 ° C., washed once with 2% FCS medium, washed twice with FCS, metastasized and numbered as mentioned above.

% ¹²⁵ I-MoAb binding inhibition of each sample was calculated by the following formula.

A = mean CPM of the sample

B = average CPM of background

C = mean CPM of the positive control

The% immune reactivity and plots retained by each sample were calculated as discussed above except that BD ₅₀ was actually BID ₅₀ (the required MoAb dose given 50% inhibition of ¹²⁵ I-MoAb binding).

Notes:

1) The tubes were always vigorously vortexed after the addition of each reactant in the RIA.

2) Internal control samples equivalent to 50% of the unengineered MoAb control were included in each set of assays to confirm that each method was quantitative in illustrating immunoreactive conjugate retention. The results from these analyzes are shown in Table 4 below.

The monoclonal antibody conjugates of the present invention are effective as anticancer agents. The assay described below for assessing cytotoxicity in vitro shows the preference of the construct for the target cell line in contrast to the non-target cells and the degree of utility of the target form of the compound compared to the non-target counterpart of the compound. To provide.

Cytotoxicity assay

[In vitro]

Samples to be tested were diluted to a parent equivalent of 0.2 or 0.02 μl / ml. (Starting concentration depends on the cell line to be tested and the potency of the parent.) Three or four additional 5-fold dilutions were added to each initial sample. Prepared from the diluent and added 0.2 ml to a sterile 15 ml polystyrene tube. At least one similar conjugate consisting of the parent drug and the inappropriate MoAb was included in each assay to determine the specificity of the appropriate conjugate. Appropriate red blood cells in 0.2 ml of 10% FCS medium 10 Was aliquoted into tubes and vortexed. In addition, the same test was performed using inappropriate cells as targets to further confirm the specificity of the appropriate conjugate. The MoAb control received only the same amount of MoAb and the positive control sample received only 10% FCS media.

Cells were incubated at 37 ° C. for 7 minutes and washed 4 times with 8 ml of 2% FCS medium. 0.1 ml of 10% FCS was added to the tubules, vortex the cells, and each well of a sterile 96-well polystyrene tissue culture plate. 0.2 ml was aliquoted.

Plates were incubated for 2 days in a 37 ° C. thermostat moistened with 5% CO. Medium ½ removed, 2 μCi / ml Replaced with fresh medium containing H Tamidine (Dupont, NEN, Cat # NET-027). Continued incubation for 24 hours, cells were frozen and thawed and harvested with a PHD cell harvester (Cambridge Technology, Inc.).

Each sample was counted for 1 minute on channel 1 in a Beckman LS 5800 scintillation counter.

% Growth inhibition was calculated as follows;

Percent inhibition was plotted on a non-log scale of semi-log graph and mother concentration was plotted on the scale. IC ₅₀ (parent concentration required to confer 50% inhibition) of each test sample was derived from the graphs and the cytotoxicity retention was calculated by the following equation;

The results from in vitro cytotoxicity assays are shown in Table 5 below.

The following assay system was used to measure the in vivo action of the conjugates of the invention.

In vivo tests were performed on anti-tumor activity on drug-monoclonal drug conjugates using human tumor xenographs in unconscious (as is) mice.

Burkitt lymphoma (Raji) and myeloma (HS sultanate) cells were harvested from the culture flask and subcutaneously (≥ 80 x 10). Raji cells or 40 x 10 HS Sultan Cells) were cultured in test mice. Solid tumors, ovarian cancer (CA73, Ovcar-3), breast cancer (MX-1) and colon cancer (LS174T) are propagated, removed, and 2 mm in unconscious mice. Cut into fragments and implanted subcutaneously (5-8 fragments per mouse) into test mice.

The weakly-monoclonal antibody and the weakly-monoclonal antibody conjugates were administered intraperitoneally with a total of three or five injections, once every three days, starting 2,3,4,6,7 or 8 post tumor implantation. Tumor species (tumor length and width) were measured with a Flowerer ultra CAL II electronic caliper each 7 days for 4-6 weeks post tumor implantation. The mass mass (mg) was calculated from the following formula:

Tumor growth inhibition was calculated for each test group on the control percent. [Mg of mean of group (T) treated; Mean mg × 100 of control (C) group] T / C values ≦ 42% in the group with ≧ 65% of surviving animals are considered necessary to demonstrate action.

The results from the analysis are shown in Table 6.

Intraperitoneal treatment of human vikit lymphoma cell line Raji TC, starting 3 injections 7 days post tumor implantation, measured 28 days post transplant

Intraperitoneal treatment of human breast cancer cell line MX01, 3 injections started 2 days post tumor implantation, measured 35 days post transplant

Intraperitoneal treatment of human vikit lymphoma cell line Raji TC, starting 3 injections 7 days after tumor implantation, measured 35 days post transplant

Intraperitoneal treatment of human vikit lymphoma cell line Raji TC, starting 3 injections 7 days after tumor implantation, measured 35 days post transplant

Intravenously treated human colon cancer cell line LS174T, started 3 injections 8 days after tumor transplantation, measured 21 days after transplantation

Treatment intravenously for human colon cancer system LS174T, starting 3 injections 8 days after tumor transplantation. Measured 21 days after transplant

The invention will be further described with the following bar-limiting examples.

Example 1

[LL-E33288γ 3-mercaptopropropionic disulfide]

LL-E33288γ in 90 ml of acetonitrile To a 90 mg solution was added 10.6 mg of 3-mercaptopropionic acid in 1 ml of acetonitrile. The solution was vortexed and stored at -20 ° C for 6 days. The solvent was removed in vacuo and the residue was chromatographed on 10 ml of methylene chloride silica gel. 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. The last fraction was evaporated to give a residue, which was taken up in ethyl acetate with a small amount of acetone and added directly to excess hexane. The precipitate was collected and dried to give 39 mg of the desired product (FABMS, M + H 1394).

Example 2

[LL-E33288γ Of p-nitrophenyl 3-mercaptopropionic acid disulfide (A) 3-mercaptopropionic acid]

Conventional 3-mercaptopropionic acid in methylene chloride containing a catalytic amount of concentrated sulfuric acid was treated with isobutylene for 20 minutes. The solution was then treated with 1N sodium bicarbonate solution and the methylene chloride solution was dried using anhydrous magnesium sulfate. The solution was then evaporated to give a colorless mobile liquid and the NMR and mass spectral data shown were for S-t-butylmercaptopropionic acid, t-butyl ester.

An aliquot of the ester was refluxed with 6N hydrochloric acid in dioxane for 2.5 hours. The solvent was evaporated, ethyl acetate was added and the solution extracted with sodium carbonate. The sodium monohydrate extract was treated with 6N hydrochloric acid until the pH of the suspension reached 2.0.

The suspension was then extracted with ethylate and the extract was dried over anhydrous magnesium sulfate and the solvent was evaporated to give a colorless liquid, according to the 'H NMR and mass spectrum data shown, which was S-t-butylmercaptopropionic acid.

Treatment with equimolar amounts of p-nitrophenol and dicyclohexyl carbodiimide in tetrahydrofuran for 4 hours converted to the compound p-nitrophenyl ester. Filtration removes the dicyclohexyl urea byproduct, the filtrate is evaporated to give an oil, which is purified by passing over neutral silica gel using a solvent system hexane: methylene chloride (50:50). Pure p-nitrophenyl ester derivative was a faint yellow, transfer oil.

Free mercaptan was identified by the following procedure. S-t-butyl mercaptopropionic acid p-nitrophenyl ester was dissolved in trifluoroacetic acid and a slight molar excess (10%) of mercuric acetate mercuric chloride was added. The mixture was stirred for 30 minutes, then trifluoroacetic acid was evaporated and the residue was taken up in dimethyloformamide. The solution was treated with hydrogen sulfide gas for 15 minutes, then black mercury sulfide ions were filtered off, and the filtrate was evaporated under reduced pressure to remove 99% or less of dimethylformamide. Results A slightly brown transfer liquid was purified on neutral silica gel using hexanes: methylene chloride (50:50). The main component contains a small amount of t-butylmercapto derivative It was shown by H NMR. Two vertical Perkin-Elmer Pecospheres using a gradient system of 37.5 / 62.5 to 47.5 / 52.5 of acetonitrile and 0.1 M ammonium acetate buffer (acetic acid) at pH6.5 for 12 minutes Analytical HPLC on C18 columns [4.6 x 33 mm and 4.6 x 83 mm] shows that the product is slightly polar with 88% p-nitrophenyl ester of 3-mercaptopropionic acid and 10% p-nitrophenyl ester of St-butylmercaptopropionic acid Im appeared. There was also a small amount of free p-nitrophenol.

(B) LL-E33288γ Reaction of with 3-mercaptopropionic acid with p-nitrophenyl ester

LL-E33288γ The 100 mg portion was dissolved in 50 ml of acetonitrile. To this was added a 25.7 mg solution of p-nitrophenyl ester of 3-mercaptopropionic acid in 1 ml of acetonitrile. The reaction was placed at -20 ° C for 48 hours. HPLC indicated the reaction was complete. The solution was evaporated to dryness and the residue was taken up in 4-5 ml of ethyl acetate using sonication to give a solution. The mixture was filtered and the filtrate was dropped into 45 ml of stirred hexane. The resulting pale yellow solid was collected and dried under reduced pressure As confirmed by H NMR, LL-E33288γ 93 mg of p-nitrophenyl esters of the propionic acid derivatives of were obtained. [M + H] ions were found to be m / z = 1515 by FABMS.

C. Reverse phase HPLC phase retention time: 18 minutes with 50% acetonitrile / 0.1 M aqueous ammonium acetate (LL-E33288δ) 8.0 minutes ester hydrolysis product: 1.5 minutes)

Example 3

[LL-E33288γ N-Hydroxysuccinimidyl 3-mercaptopropionate

Disulfide]

LL-E33288γ from EPO publication in 0.5 ml tetrahydrofuran 0.45 mg of N-hydroxysuccinimide in 0.1 ml of tetrahydrofuran was added to a 5 mg solution of 3-mercaptopropionic acid disulfide-like compound of hexane, followed by addition of 1.8 mg of dicyclohexyl carbodiimide in 0.2 ml of terahydrofuran. did. The reaction was stirred for 4 hours at room temperature and then quenched with excess hexane. The solid was isolated by filtration and dissolved in ethyl acetate. The resulting solution was washed three times with brine, dried over magnesium sulfate and evaporated to give 5 mg of the desired product as a tan powder which was used without further purification. Inversion phase CHPLC phase retention time: 15 minutes with 40% acetonitrile / 0.1M aqueous ammonium acetate (starting material: 6.0 minutes).

Example 4

[LL-E33288γ And 3-mercaptoproonyl hydrazide disulfide]

To 5.4 ml (3eg) of anhydrous hydrazine in 100 ml of tetrahydrofuran refluxed under argon was added dropwise 9.2 ml (83 mmol) of methyl 3-mercaptopropionate in 50 ml of tetrahydrofuran for 2 hours. The solution was refluxed for another 2 hours, evaporated, diluted and evaporated twice from 300 ml of toluene. The product was applied to a silica gel plug with 5% ethyl acetate / chloroform and eluted from the plug with 20% methanol / chloroform. The resulting 3-mercaptopropionyl hydrazide was a faint pink oil that solidified when cooled but melted at room temperature

LL-E33288γ in 50 ml of acetonitrile at -15 ° C To 50 mg was added 6.6 mg of 3-mercaptopropionyl hydrazide in 1 ml of tetrahydrofuran. One equivalent of triethylamine or one equivalent of triethylamine and one equivalent of acetic acid were added. The reaction was stirred for 1 hour at 0 ° C. and the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-chloroform gradient to afford 26 mg of the desired product. FABMS, m / z = 1408 (M + H); Inversion phase CHPLC phase retention time: 5.0 min in 41% acetonitrile / 0.1 M aqueous ammonium acetate.

Example 5

[LL-E33288γ N-[(4-methyl-coumarin-7-ylamino] acetyl] cysteine hydrazide disulfide]

A mixture of 1.0 g (5.7 mmol) of 4-methyl-7aminocoumarin, 3.0 ml (5 eq) of ethyl bromoacetate, 90 mg (0.1 eq) of sodium iodide and 30 ml of dimethylformamide was heated at 80 ° C. for 5 hours under argon. . The mixture was cooled, diluted with ethyl ether, washed three times with 50% brine, dried over magnesium sulfate and evaporated to dryness. The crude product was dissolved in chloroform containing 1% ethyl acetate and filtered through a silica gel plug. Recrystallization from diethyl ether containing traces of chloroform gave pure ethyl N-[(4-methyl-coumarin-7-yl) amino] acetate.

To 1.96 g (7.5 mmol) of the ester in 15 ml of methanol and 15 ml of tatrahydrofuran were added 10 ml of 1N aqueous sodium hydroxide. After 30 minutes, 4 ml of 10% aqueous hydrochloric acid was added. The organic solvent was triturated and the resulting crystalline product was filtered off, washed with cold methanol and then with ether. The material was dissolved in 20 ml of tetrahydrofuran and 4 ml of dimethylformamide, dicyclohexyl carbonyldiimidazole (1.3 g, 2.2eg) was added and the reaction stirred for 15 minutes. Cysteine ethyl ester hydrogen chloride (1.6 g, 2.5eg) and triethylamine (1.2 ml) were then added. After 3 hours, the reaction was diluted with ethyl ether containing 5% methylene chloride and washed once with 10% aqueous hydrochloric acid and twice with brine. After drying over magnesium sulfate and evaporation of the solvent, it was dissolved in chloroform containing a small amount of ethanol, and excess ether was added to crystallize the crude product. The crystals were filtered and dried to give pure N-[[(4-methyl-coumarin-7-yl) amino] acetyl] cysteine ethyl ester.

A mixture of 5 ml of chloroform, 20 ml of methanol and 0.4 ml of hydrazine hydrate was heated to reflux under argon. To this was added 550 mg of N-[[4-methyl-coumarin-7-yl) amino] acetyl] cysteine ethyl ester. After refluxing for 9 hours, the mixture was cooled and the solid product was filtered off, washed with chloroform and then with ethyl ether. The crude product (which includes thiols and disilphide) was dissolved in dimethylformamide and triethylamine containing dithiolthritol. After 30 minutes, the product was precipitated with excess ethyl ether and collected by filtration. Recrystallized from degassed acetonitrile containing dithiothritol and traces of triethyl amine, the material was further purified to pure N-[[4-methyl-coumarin-7-yl) amino] acetyl] cysteine hydrazide Got.

LL-E33288γ in 12 ml of acetonitrile at 0 ° C. To 12 mg was added 4 mg of N-[[(4-methyl-coumarin-7-yl) amino] acetyl] cysteine hydrazide in 1.2 ml of dimethylformamide. After stirring overnight, 2 mg of N-[[(4-methyl-coumarin-7-yl) amino] acetyl] cysteine hydrazide was added to 0.6 ml of dimethylformamide. The reaction was stirred at 0 ° C. for 3 days and filtered. Acetonitrile was evaporated and the resulting dimethylformamide solution was diluted with excess 1: 1 hexane / ether. The product was isolated by filtration and further purified by chromatography on silica gel with a gradient of 15-20% methanol in gloform to afford 3 mg of the desired product. Inversion phase CHPLC phase retention time: 3.5 min using 45% acetonitrile / 0.1M aqueous ammonium acetate LL-E33288δ 15.5 minutes on the same system.

Example 6

[LL-E33288α 3-mercaptoropioyl hydrazide disulfide]

LL-E33288α in 9 ml of acetonitrile at -15 ° C To 10 mg was added 6.6 mg of 3-mercaptoropioyl hydrazide in 1 ml of acetonitrile. One equivalent of triethylamine or one equivalent of triethylamine and one equivalent of acetic acid were added. The reaction was stirred for 1 hour at 0 ° C. and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. FABMS. m / g-1251 (M + H); Reverse phase C18 HPLC phase residence time: 2.1 min with 45% acetonitrile / 0.1 M aqueous ammonium acetate system (LL-E33288α) : 5.7 minutes with the same system)

Example 7

[N-acetyl LL-E33288γ 3-mercaptoropionyl hydrazide disulfide]

N-acetyl LL-E33288γ in 10 ml of acetonitrile at -15 ° C To 10 mg was added 1.5eg of 3-mercaptoropioyl hydrazide in 85 μl of acetonitrile. One equivalent of triethylabin was added. The reaction was stirred for 2 hours at 0 ° C. and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. FABMS, m / z = 1450 (M + H): C inverted phase HPLC retention time: 50% acetonitrile / 0.05M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ : 6.6 minutes with the same system)

Example 8

[LL-E33288α] 3-mercaptoropionyl hydrazide disulfide]

LL-E33288α in 10 ml of acetonitrile at -15 ° C To 10 mg was added 1.5eq of 3-mercaptoropionyl hydrazide in 1 ml of acetonitrile. One equivalent of triethylamine was added. The reaction was stirred for 1 hour at 0 ° C. and then the solvent was evaporated. The residue was chromatographed on silica gel with 5-15% methanol in chloroform gradient to afford the desired product. FABMS, m / z = 1248 (M + H); C. Inversion phase HPLC retention time: 2.6 min with 58% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (LL-E33288α) : 7.5 minutes with same system)

Example 9

[3-mercaptopropionyl hydrazide disulfide of iodine LL-E33288 pseudoaglycone]

1.5eq of 3-mercaptopropionyl hydrazide in 1 ml of acetonitrile was added to 10 mg of iodine LL-E33288 pseudoacrylone in 9 ml of acetonitrile at -15 ° C. One equivalent of triethylamine was added as a catalyst. The reaction was stirred for 1 hour at 0 ° C. and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. FABMS, m / z = 1091 (M + H); C. Inverse phase HPLC phase retention time: 2.8 min with 50% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (iodine LL-E33288 pseudoaglycone: 7.9 min with same system)

Example 10

[LL-E33288γ 3-mercaptoropionyl hydrazide disulfide]

18 ml (0.26 mol) of thioacetic acid was added to 17.2 g (0.2 mol) of crotonic acids. The mixture was heated at reflux under argon for 6 h. Excess thioacetic acid was removed under an aspirator vacuum and the residual oil was dissolved in 100 ml of anhydrous ethanol containing 200 [mu] l of concentrated sulfuric acid. The reaction was refluxed for 10 hours and then the volume was reduced under aspirator vacuum. Hexane was added and the resulting solution was washed successively with two portions of saturated sodium bicarbonate and one portion of water. The solution was then dried over magnesium sulfate, filtered and the volume reduced to an oil. The crude product was dissolved in 250 ml of methanol containing 12 ml of hydrazine and the resulting mixture was refluxed under argon for 10 hours. The reaction mixture was reduced to volume and then quickly distilled with Kugelrohr and crystallized from a mixture of chloroform-hexanes to give 3-mercaptobutyryl hydrazide.

LL-E33288γ in 5 ml of acetonitrile at -15 ° C To 5 mg was added 1.5eq of 3-mercaptoropionyl hydrazide in 1 ml of acetonitrile. One equivalent of triethylamine was added. The reaction was stirred for 1 hour at 0 ° C. and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol in chloroform gradient to afford the desired product. FABMS, m / e = 1422 (M + H); C. Inverse phase HPLC retention time: 3.5 min with 43% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (LL-E33288γI: 13.4 min with the same system)

Example 11

[LL-E33288γ 3-mercaptoisovaleryl hydrazide disulfide]

9 ml (0.13 mol) of thioacetic acid was added to 10 g (0.1 mol) of 3, 3- dimethyl acrylic acid. The mixture was heated at reflux in an argon column for 6 hours. Excess thioacetic acid was removed under an aspirator vacuum and the resulting oil was dissolved in 100 ml of anhydrous ethanol containing 200 [mu] l of concentrated sulfuric acid. The reaction was refluxed for 34 h and 16 ml of hydrazine were added. The resulting mixture was refluxed for 24 h under argon. The volume of the reaction mixture was reduced and then dissolved in brine and saturated sodium bicarbonate. The product was extracted with several volumes of chloroform. The combined chloroform layer was dried over magnesium sulfate, filtered and the volume reduced to an oil. The oil was purified by flash chromatography with methanol-chloroform gradient and crystallized from chloroform-hexane to afford 3-mercaptoisovaleryl hydrazide.

LL-E33288γ in 5 ml of acetonitrile at -15 ° C To 15 mg, 1.5 eq of 3-mercaptoisovaleryl hydrazide in 100 μl of acetonitrile was added. One equivalent of triethylamine was added as a catalyst. The reaction was stirred for 3 hours at ambient temperature before the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol in chloroform gradient to afford the desired product. FABMS, m / z = 1436 (M + H); C. Inversion phase HPLC retention time: 3.9 min with 43% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (LL-E33288γ) : 13.4 minutes with the same system)

Example 12

[LL-E33288γ P-mercaptodihydrocinnamoyl hydrazide disulfide]

To 500 mg (2.75 mmol) of p-mercaptodihydrocinnamic acid was added 15 ml of methanol containing one drop of concentrated sulfuric acid. The reaction was refluxed for 5 hours and then cooled to ambient temperature. The reaction was stirred at 0 ° C. for 10 hours and then the solvent was evaporated. Hydrazine (1.5 ml) was added and the resulting mixture was refluxed under argon for 2 hours and then stirred at ambient temperature for 10 hours. A 200 mg portion of dithiothritol was added to reduce any disulfide present and the reaction mixture was cooled to -15 ° C. The resulting crystals were filtered, washed with a mixture of ether and methanol, and then dried in a vacuum oven (50/5 micron / 10 hours) to obtain p-mercaptodihydro cinnamoyl hydrazide.

LL-E33288γ in 25 ml of acetonitrile at -15 ° C To 25 mg was added 1.5eq of p-mercaptodihydrocinnamoyl hydrazide in 1 ml of acetonitrile. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. FABMS, m / z = 1484 (M + H); C. Inverted phase HPLC retention time: 43% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (LL-E33288γI: 13.4 min in the same system)

Example 13

[N-acetyl LL-E33288γ 3-mercaptoisovaleryl hydrazide disulfide]

LL-E33288γ in 15 ml of acetonitrile at -15 ° C To 20 mg was added 3-eq 3-mercaptoisovaleryl hydrazide in 6.2 ml of acetonitrile. One equivalent of triethylamine was added. The reaction was stirred at ambient temperature for 2 hours and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. C. Inverted phase HPLC retention time: 2.5 min with 50% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γI: 6.6 min with the same system)

Example 14

[N-acetyl LL-E33288γ 3-mercaptobutyryl hydrazide disulfide]

N-acetyl LL-E33288γ in 7.5 ml of acetonitrile at -15 ° C To 10 mg was added 3eq 3-mercaptobutyryl hydrazide in 5 ml of acetonitrile. One equivalent of triethylamine was added. The reaction was stirred at ambient temperature for 10 hours and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. C. Inversion phase HPLC retention time: 7.3 min with 43% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ : 5.6 minutes using the same system

Example 15

[N-acetyl LL-E33288γ P-mercaptodihydro cinnamoyl hydrazide disulfide]

N-acetyl LL-E33288γ in 7.5 ml of acetonitrile at -15 ° C To 10 mg was added 3.0eq of 3-mercaptodihydro cinnamoyl hydrazide in 2.0 ml of acetonitrile. One equivalent of triethylamine was added. The reaction was stirred at ambient temperature for 2 hours and then the solvent was evaporated. The residue was chromatographed on silica gel with 10-15% methanol-in-chloroform gradient to afford the desired product. Retention time on CHPLC: 7.3 min with 43% acetonitrile / 0.05 M aqueous ammonium dihydrogen phosphate (N-acetyl LL-E33288γ : 5.6 minutes using the same system

Example 16

[Non-Specific Conjugation to Proteins]

The hydroxysuccinimide ester described in Example 3 was covalently bound to the antibody under some alkaline conditions. The following is a general method used to make the antibody conjugates listed in Table 7. The antibody (pH 7.5) was reacted with 5-20 fold molar excess of product from Example 3 by stirring at room temperature for 1-4 hours at a concentration of 3-5 mg / ml in 0.1M sodium chloride phosphate buffer. Conjugated protein was chromatographically desalted and gel-aggregated HPLC separated the aggregated protein from the monofactor-expressing material. Monofactor-expressing fragments were pooled and concentrated.

Example 17

Site-Specific Conjugate Preparation

A general method of binding hydrazide derivative drugs to oxidized antibodies is described in US Pat. No. 671,958. Described by McCeron's group. The method is used to prepare antibody aggregates from the products of Examples 4-15 with specific alterations as described below. The products from these reactions and their properties are summarized in Table 7.

(A) Antibody Oxidation Antibodies at a concentration of 5-10 mg / ml were dialyzed overnight against 50 mM sodium acetate buffer, pH 5.5, 200-fold volume containing 0.1 M sodium chloride. After dialysis, MoAb was oxidized with 15 mM to 200 mM periodic acid in 0.2 M sodium acetate. After oxidizing in the dark with agitation at 4 ° C. for 45 min, the oxidized MoAb was desalted on a ≧ 5 adsorbent volume Sephadex G-25 column. The degree of oxidation of the antibody was analyzed by reaction with P-nitrophenylhydrazine, and the protein absorbance at 280 mm versus the absorbance of p-nitrophenylhydrazine at 395 mm.

(B) drug hydrazide conjugation

The oxidized MoAb was reacted with 10 to 200 fold molar excess of about hydrazide. Hydrazide was dissolved in dimethylformamide and added to an aqueous solution of MoAb. To avoid precipitation of MoAb, the final volume of added dimethylformamide should not exceed 10% of the total reaction volume. The reaction was continued for 3 hours at room temperature while stirring. To prevent crosslinking and subsequent aggregation of unreacted aldehydes, a shielding agent, acetyl hydrazide, was added in 100-fold molar excess three hours after the addition of weak hydrazide. To stabilize the Schiff base bonds between aldehydes and weak hydrazides (hydrazones), the product was prepared by adding 10 mM sodium cyanoborohydride, allowing the reaction to proceed for an additional hour (total assembly time-4 hours). Reduced to alkyl hydrazine. The aggregates were chromatographically desalted and dialyzed (minimum time 48 hours) with pH 6.5 phosphate buffer for storage and testing.

The aggregates were analyzed for the presence of aggregates by Kell filtration HPLC and the aggregates were analyzed for free drug by reversed phase HPLC. About dropping was spectroscopically measured using the absorbance coefficients of the antibody and bivalent to determine the molar concentration of the drug in the conjugate.

Claims (11)

Prepared from compounds of the general formula CH ₃ SSS-W, LL-E33288α ₁ -Br, α _1- I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, iodine or bromo pseudoaglycones, their dehydro or N- Represented as acyl counterparts, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724, or their N-acetyl counterparts. , Substituted disulfides of the general formula Q-Sp-SS-W: Where W is

ego; R ₁ is

Or CH ₃ ; R ₂ is

Or H; R ₃ is

Or H; R ₄ is

Or H; R ₆ or R ₇ is H or

ego; R ₅ is CH ₃ , C ₂ H ₅ or (CH ₃ ) ₂ CH: R ₈ is OH and R ₉ is H, or R ₈ and R ₉ together are a carbonyl group: X is an iodine or bromine atom: R _{5 'it} is hydrogen or a group RCO, where R is hydrogen or one or more hydroxy, amino, carboxy, halo, nitro, lower (C ₁ -C ₃₎ alkoxy, or lower (C ₁ -C ₅₎ or substituted thioalkoxy Unsubstituted branched or unbranched alkyl (C ₁ -C ₁₀ ) or alkylene (C ₁ -C ₁₀ ) groups, aryl groups or heteroaryl groups, or aryl-alkyl (C ₁ -C ₅ ) groups or heteroaryl -Alkyl (C ₁ -C ₅ ) group; Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, a divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent or _trivalent aryl Or is a heteroaryl-alkyl (C ₁ -C ₁₈ ) radical, divalent or trivalent cycloalkyl- or hetero cycloalkyl-alkyl (C ₁ -C ₁₈ ) or divalent trivalent (C ₂ -C ₁₈ ) unsaturated alkyl radical, if If Sp is a trivalent radical, it may be additionally substituted by amino, alkylamino, arylamino, hetero arylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups and Q is hydrogen, halo Ken, amino, alkylthio group, dialkylamino, arylamino, heteroarylamino, pyreridino, pyrrolidino, pyrerazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxy, Thiol, that Alkyl dicarboxylic _{acids, -CONHNH 2, -NHCONHNH 2, -CSNHNH} 2, -NHCSNHNH 2, or -ONH _2, and; Provided that when Sp is ethylidine, Q cannot be hydrogen and CH3-SSS-W is LL-E33288α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR- 900405, FR-900406, PR114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 and when Sp is divalent, Q is hydrogen, halogen, amino, alkylamino, di Alkylamino, arylamino, heteroarylamino, piperidino, pyrrolidino, piperazino or carboxyl.) The substituted disulfide of general formula Q-Sp-SS-W according to claim 1, which is prepared from a compound of general formula CH ₃ SSS-W: Where W is

ego; R ₁ is

ego; R ₂ is

Or H; R ₄ is

Or H; R ₅ is CH ₃ , C ₂ H ₅ or (CH ₃ ) ₂ CH: X is an iodine or bromine atom; R _{5 '} is hydrogen or an RCO group, wherein R is hydrogen, CH ₃ or a mono-substituted aryl group; Sp and Q are as defined above) CH ₃ SSS-W is represented by the 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- C18} ) cycloalkyl or heterocycloalkyl radical, divalent or trivalent aryl- or heteroaryl-alkyl (C ₁ -C ₁₈ ) radicals, divalent or trivalent cycloalkyl- or hetero cycloalkyl-alkyl (C ₁ -C ₁₈ ) radicals , Or a divalent trivalent (C ₂ -C ₁₈ ) unsaturated alkyl radical, and if Sp is a trivalent radical, it is amino, alkylamino, arylamino, hetero arylamino, carboxyl, lower alkoxy, hydroxy, thiol, or lower Additionally substituted by an alkylthio group, provided that CH3-SSS-W is the parent antitumor antibiotic LL-E33288α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α _3- Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675 , FR-900405, FR-900406, PR114759, PR11 When 5028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724 itself, Sp cannot be divalent; Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, Hydroxy, thiol, α-haloacetyloxy, lower alkyldicarboxyl, -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH ₂ , -ONH ₂ , -CON ₃ ,

Or later converted to them to form intermediates of the general formula Q-Sp-SS-W, wherein Q Sp and W are as defined above, and Q-Sp-SS. -W is represented by the formula Tu- (Y) n, wherein the carrier Tu is a single- or polyclonal antibody, a fragment thereof, a chemically or genetically engineered counterpart thereof, or a growth factor or a steroid; Y is a side chain amino , A carboxy, or a thiol group of a protein, an aldehyde derived from a glycoprotein carbohydrate moiety, or an amidoalkylthio group; n is an integer of 1 to 100).

Wherein Tu, Y, Sp, W and n are as defined above and Z is formed after subsequent reduction or directly from the covalent reaction of Q and Y groups, -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-,

, M is 0.1 to 15), and LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycones, their dihydro or N-acyl counterparts, BBM-1675, Formula CH3SSS-, an anti-tumor antibiotic represented by FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl counterparts. Carrier-drug cojugates of the general formula prepared from the compound of W:

(W is as defined above) The dithiomethyl moiety of claim 3, wherein the dithiomethyl moiety is represented by the formula Q-Sp-SH wherein Sp is a straight or branched chain divalent or trivalent (C ₂ -C ₁₀ ) radical or a divalent or trivalent aryl- C ₂ -C ₅ ) radical, and if Sp is a trivalent radical, it may be additionally substituted by amino, heteroarylamino, hydroxy, or thiol groups; Q is carboxyl, lower alkyldicarboxylic anhydride , -CONHNH ₂ or

Or later converted to them to form intermediates of the general formula Q-Sp-SS-W, wherein Q, Sp, and W are as defined above, and Q-Sp. -SS-W is represented by the general formula Tu- (Y) n, wherein Tu is a monoclonal antibody that shows preferential reactivity with human tumor-associated antigens, and Y is a side chain amino group on the antibody, or a carbohydrate of the antibody. Is an aldehyde produced by oxidation of a group, n is an integer from 1 to 100) and a compound of the general formula

Wherein, Tu, Y, Sp, W and n are as defined above, Z is formed after subsequent reduction or directly from the covalent reaction of Q and Y groups, -CONH-, CONHN = CH-, -CONHNHCH ₂ -or

, M is 0.1 to 15), a protein-drug conjugate of the general formula prepared from an anti-tumor antibiotic represented by LL-E33288 (CH3SSS-W):

Acetonitrile or a mixture of acetonitrile and tetrahydrofuran or aceto at -20 to 40 ° C. for 1 hour to 6 days in the presence of 1 equivalent of tertiary amine base or 1 equivalent of amine base and 1 equivalent of organic carboxylic acid In a mixture of nitrile and dimethylformamide, LL-E33288α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ -I, α ₄ -Br, β _1- Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, iodine or bromo pseudoaglycones, their dehydro or N-acyl counterparts , Methyl trisulfide antitumor from BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl counterparts LL-E33288α1-Br, α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ , consisting of reacting antibiotics with appropriately substituted mercaptans -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ _1- I, δ ₁ -I, iodine or bromo pseudoaglycones, their dehydro or N-acyl counterparts, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL- Process for producing substituted disulfides of the general formula Q-Sp-SS-W from compounds of the general formula CH ₃ SSS-W, represented as 1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl counterparts . Where W is

ego; R1 is

Or CH 3; R2 is

Or H; R3 is

Or H; R1 is

Or H; R6 or R7 is H or And R ₅ is CH ₃ , C ₂ H ₅ or (CH ₃ ) ₂ CH: R ₈ is OH and R ₉ is H, or R ₈ and R ₉ together are a carbonyl group: X is an iodine or bromine atom: R _{5 ′} is a hydrogen or RCO group, where R is hydrogen or one or more hydroxy, amino, carboxy, halo, nitro, lower (C ₁ -C ₃ ) alkoxy, or lower (C ₁ -C ₅ ) thioalkoxy groups Substituted or unsubstituted branched or unbranched alkyl (C ₁ -C ₁₀ ) or alkylene (C ₁ -C ₁₀ ) groups, aryl groups or heteroaryl groups, or aryl-alkyl (C ₁ -C ₅ ) groups or Heteroaryl-alkyl (C ₁ -C ₅ ) group; Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, a divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent or _trivalent aryl Or is a heteroaryl-alkyl (C ₁ -C ₁₈ ) radical, a divalent or trivalent cycloalkyl- or hetero cycloalkyl-alkyl (C ₁ -C ₁₈ ) radical, or a divalent trivalent (C ₂ -C ₁₈ ) unsaturated alkyl radical If Sp is a trivalent radical, it may be additionally substituted by amino, alkylamino, arylamino, hetero arylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups and Q is hydrogen , Haloken, amino, alkylthio group, dialkylamino, arylamino, heteroarylamino, pyreridino, pyrrolidino, pyrerazino, carboxyl, nitrophenyloxycarbonyl, carboxaldehyde, lower alkoxy, hydroxide Roxy, Ol, lower alkyl _{dicarboxylate, -CONHNH 2, -NHCONHNH 2, -CSNHNH} 2, -NHCSNHNH 2, or -ONH _2, and; Provided that when Sp is ethylidine, Q cannot be hydrogen and CH ₃ -SSS-W is LL-E33288α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR -900405, FR-900406, PR114759, PR115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724, and when Sp is divalent, Q is hydrogen, halogen, amino, alkylamino, It may not be dialkylamino, arylamino, heteroarylamino, pepperidino, pyrrolidino, pepperazino or carboxyl. CH ₃ SSS-W is represented by the general formula Q-Sp-SH in acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours. Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent Or trivalent aryl- or heteroaryl-acyl (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 ) It is an unsaturated alkyl radical, and if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups; CH ₃ -SSS-W is the parent antitumor term Biomass LL-E33288 α ₁ -Br, α ₁ -I, α ₂ -Br, α ₂ -I, α ₃ -Br, α ₃ -I, α ₄ -Br, β ₁ -Br, β ₁ -I, β ₂ -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL- When 1577D, CL-1577E, or CL-1724 itself, Sp cannot be divalent; Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy or lower alkyldicarboxylic anhydride). React with the compound, The intermediate of formula Q-Sp-SS-W (where Q, Sp and W are as defined above) is separated and then an aqueous buffer having a pH of 6.5 to 9 in the presence of water-soluble carbodiimide or directly at 4 to 40 ° C. Within, the general formula Q-Sp-SS-W, wherein Sp and W are as defined above and Q is halogen, amino, alkylamino, carboxyl, carboxaldehyde, hydroxy or lower alkyldicarboxylic anhydride Is a compound of the formula Tu- (Y) n, wherein Tu is a monoclonal or polyclonal antibody, fragment thereof, chemically or genetically engineered counterpart or growth factor or steroid; Y is branched chain amino Or a carboxyl functional group; n is 1 to 100).

Wherein, Tu, Sp, W, n and Y are as defined above, m is 1 to 15, Z is formed from the covalent reaction of Q and Y groups,

Antitumor antibiotic LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycones, their dihydro or N-cetyl counterparts, BBM-1675, FR-900405, FR- 900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 A method for preparing the following target derivatives of the general formula CH ₃ SSS-W compounds which are their N-acetyl counterparts.

In acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours, CH3SSS-W is represented by the general formula Q-Sp-SH , Sp is a straight or branched 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-acyl (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 And if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups, provided that CH is ₃ -SSS-W is the parent antitumor antibiotic Quality _{LL-E33288α 1 -Br, α 1} -I, α 2 -Br, α 2 -I, α 3 -Br, α 3 -I, α 4 -Br, β 1 -Br, β 1 -I, β 2 -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D , Sp-1577E, or CL-1724 itself, Sp cannot be divalent; Q is carboxyl, and reacts with a compound of formula Q-Sp-SS-W, wherein Q, Sp and W Is separated from the intermediate, and then in the presence of a carboxyl activator such as carbodiimide, the formula Q-Sp-SS-W (where Sp and W are as defined above and Q is a carboxylic acid Is reacted with N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol or 4-nitrophenol to yield the general formula Q-Sp-SS-W, wherein , Sp and W are as defined above, Q is

or

To a compound of the general formula Tu- (Y) n, wherein Tu and n are as defined above and Y is a side chain amino group. Reacted with a molecule of

Wherein, Tu, Sp, Y and n are as defined above, m is 1 to 15 and Z is formed from a covalent reaction between Q and Y, defined as -CONH-. Antitumor antibiotics LL-E33288 α ₁ - ^Br , α ₁ - ^I , α ₂ - ^Br , α ₂ - ^I , α ₃ - ^Br , α ₃ - ^I , α ₄ - ^Br , β ₁ - ^Br , β ₁ - ^I , β ₂ - ^Br , β ₂ - ^I , γ ₁ - ^Br , γ ₁ - ^I , δ ₁ - ^I , iodine or bromo pseudoaglycones, their dihydro or N-acetyl counterparts, BBM- 1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or the following general formula CH ₃ SSS-W compounds which are their N-acetyl counterparts Method for preparing the target derivative.

CH ₃ SSS-W is represented by the general formula Q-Sp-SH in acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours. Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent Or trivalent aryl- or heteroaryl-acyl (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 ) It is an unsaturated alkyl radical, and if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups; However, CH ₃ -SSS-W is the parent antitumor wherein Material _{LL-E33288α 1 -Br, α 1} -I, α 2 -Br, α 2 -I, α 3 -Br, α 3 -I, α 4 -Br, β 1 -Br, β 1 -I, β 2 -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D When Sp, CL-1577E, or CL-1724 itself, Sp cannot be divalent; Q is -CONHNH ₂ , and reacts with a compound of formula Q-Sp-SS-W, wherein Q, Sp and The intermediate of W is as defined above), and then in aqueous acetonitrile, a compound of formula Q-Sp-SS-W, wherein Sp and W are as defined above and Q is -CONHNH ₂ Is reacted with nitrous acid to generate a compound of the general formula Q-Sp-SS-W, wherein Sp and W are as defined above and Q is -CON ₃ , which is a general formula Tu- (Y) n Reacted with a compound of the formula wherein Tu, Y and n are as defined above, wherein the compound of formula:

Wherein, Tu, Z, Sp, W, m, Y and n are as defined above), antitumor antibiotic LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycone, Their dihydro or N-acetyl counterparts, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl To prepare the following target derivatives of the corresponding general formula CH ₃ SSS-W compounds:

CH ₃ SSS-W is represented by the general formula Q-Sp-SH in acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours. Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent Or trivalent aryl- or heteroaryl-acyl (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 ) It is an unsaturated alkyl radical, and if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups; However, CH ₃ -SSS-W is the parent antitumor wherein Material _{LL-E33288α 1 -Br, α 1} -I, α 2 -Br, α 2 -I, α 3 -Br, α 3 -I, α 4 -Br, β 1 -Br, β 1 -I, β 2 -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D When Sp, CL-1577E, or CL-1724 itself, Sp cannot be divalent; Q is

or

And an intermediate of the general formula Q-Sp-SS-W (where Q, Sp and W are as defined above), and then the general formula Q-Sp-SS-W (where Sp and W are as defined above and Q is hydroxy) to react with α-haloacetic anhydride to produce a compound wherein Q is α-haloacetyloxy, α-halo acetyloxy-Sp-SS- W or the general formula Q-Sp-SS-W, where Sp and W are as defined above and Q is

or

The compound of formula Tu- (Y) n (wherein Tu is as defined above and Y is protein side chain thiol, or 3- () under aqueous buffer conditions at pH 4-7 at 4-40 ° C. Amido alkylthio groups or 2-iminothiolanes introduced on amines of Tu using reagents such as 2-thiolpyridyl) propionic acid hydroxysuccinamide esters and then reduced with agents such as dithiothreitol An amidoalkylthio group introduced onto an amine of which n is 1 to 10), followed by reaction with a molecule of the general formula:

Wherein, Tu, Sp, W and n are as defined above and Z is formed from the covalent reaction of Q and Y groups,

or

, M is 0.1 to 10), antitumor antibiotic LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycones, their dihydro or N-acetyl counterparts, BBM-1675 The following targets of the general formula CH ₃ SSS-W compounds which are FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl counterparts How to prepare derivatives:

CH ₃ SSS-W is represented by the general formula Q-Sp-SH in acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours. Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent Or trivalent aryl- or heteroaryl-acyl (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 ) It is an unsaturated alkyl radical, and if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups; However, CH ₃ -SSS-W is the parent antitumor wherein Material _{LL-E33288α 1 -Br, α 1} -I, α 2 -Br, α 2 -I, α 3 -Br, α 3 -I, α 4 -Br, β 1 -Br, β 1 -I, β 2 -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D , Sp-1577E, or CL-1724 itself, Sp cannot be divalent; Q is -CONHNH2, -NHCONHNH ₂ , -NHCSNHNH ₂ or -ONH ₂ ), and After separating the intermediate of the general formula Q-Sp-SS-W (where Q, Sp and W are as defined above), the general formula Q-Sp-SS-W (where Sp and W are as defined above , Q is -NH ₂ , -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH ₂ or -ONH ₂ , a compound of the general formula Tu- (Y) n where Tu is as defined above and Y is 4 to 40 Is an aldehyde produced from a carbohydrate moiety on Tu by oxidation in the presence of alkaline earth periodate in an aqueous buffer at pH 4.0 to 6.5, wherein n is from 1 to 20), Compounds of the general formula

Wherein Tu, Sp, W, Y and n are as defined above and Z is formed from the covalent reaction of Q and Y groups, -ON = CH-, -N = CH-, -CONHN = CH- , -NHCONHN = CH-, or -NHCSNHN = CH-, and m is 1 to 15), antitumor antibiotic LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycone, Their dihydro or N-acetyl counterparts, BBM-1675, FR-900405, FR-900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or their N-acetyl To prepare the following target derivatives of the corresponding general formula CH ₃ SSS-W compounds:

CH ₃ SSS-W is represented by the general formula Q-Sp-SH in acetonitrile in the presence of 1 equivalent of triethylamine or 1 equivalent of triethylamine and 1 equivalent of acetic acid at -10 to -30 ° C for 1 to 48 hours. Wherein Sp is a straight or branched divalent or trivalent (C ₁ -C ₁₈ ) radical, divalent or _trivalent aryl or heteroaryl radical, a divalent or _trivalent (C ₃ -C ₁₈ ) cycloalkyl or heterocyclo-alkyl radical, divalent Or trivalent aryl- or heteroaryl-acyl (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 ) It is an unsaturated alkyl radical, and if Sp is a trivalent radical, it may be further substituted by amino, alkylamino, arylamino, heteroarylamino, carboxyl, lower alkoxy, hydroxy, thiol or lower alkylthio groups; However, CH ₃ -SSS-W is the parent antitumor wherein Material _{LL-E33288α 1 -Br, α 1} -I, α 2 -Br, α 2 -I, α 3 -Br, α 3 -I, α 4 -Br, β 1 -Br, β 1 -I, β 2 -Br, β ₂ -I, γ ₁ -Br, γ ₁ -I, δ ₁ -I, BBM-1675, FR-900405, FR-900406, PD114759, PR115028, CL-1577A, CL-1577B, CL-1577D , Sp-1577E, or CL-1724 itself, Sp cannot be divalent; Q is -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH _2, or -ONH ₂ ), and a general formula Q- After separating the intermediate of Sp-SS-W (where Q, Sp and W are as defined above), the general formula Q-Sp-SS-W (where Sp and W are as defined above and Q is- The compound of NH ₂ , -CONHNH ₂ , -NHCONHNH ₂ , -NHCSNHNH ₂ or -ONH ₂ is a compound of the formula Tu- (Y) n where Tu is as defined above and Y is pH4.0 at 4 to 40 ° C. Aldehydes produced from carbohydrate residues on Tu by oxidation in the presence of alkaline earth periodate in an aqueous buffer of from about 6.5 to about 6.5; n is 1 to 20) to react with a compound of the general formula:

Wherein Tu, Sp, W, Y and n are as defined above and Z is formed from the covalent reaction of Q and Y groups, -ON = CH-, -N = CH-, -CONHN = CH- , -NHCONHN = CH-, or -NHCSNHN = CH-, and m is 1 to 15; Compounds of the following general formulas mentioned above in aqueous buffers having a pH of 4.0 to 6.5 at 4 to 40 ° C:

Wherein Tu, Z, Sp, W, Y, n and m are as defined immediately above, are immediately treated with acetyl hydrazine or tyrosine hydrazine to give compounds of the general formula:

(Wherein Tu, Z, Sp, W, n and m are as previously defined and Y is CH = NNHCOCH ₃ or

And reacting the compound with sodium cyanoborohydride or sodium borohydride in an aqueous buffer at pH 4.0-6.5 at 4-40 ° C. to yield a compound of the general formula:

Wherein Tu, Sp, W, m and n are as defined above, Z is -NH-CH _2- , -CONHNHCH _2- , -NHCONHNCH _2- , or -NHCSNHNHCH _2- , and Y is -CH ₂ NHNHCOCH ₃ or

Antitumor antibiotic LL-E33288 α ₁ ^BR , α ₁ ^I , α ₂ ^BR , α ₂ ^I , α ₃ ^BR , α ₃ ^I , α ₄ ^BR , β ₁ ^BR , β ₁ ^I , β ₂ ^BR , β ₂ ^I , γ ₁ ^BR , γ ₁ ^I , δ ₁ ^I iodine or bromo pseudoaglycones, their dihydro or N-acetyl counterparts, BBM-1675, FR-900405, FR- 900406, PD114759, PD115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E, CL-1724 or a process for preparing the following target derivatives of the general formula CH ₃ SSS-W compounds which are their N-acetyl counterparts:

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