Novel bifunctional linking compounds, conjugates and methods for their production

PROCESS FOR THE PREPARATION OF BIFUNCTIONAL N-REPLACED HYPAZINS AND PHARMACEUTICAL COMPOSITIONS WHICH CONTAIN THEM

SCOPE OF THIS INVENTION

The present invention relates to novel bifunctional compounds, conjugates containing the compounds and methods for their preparation and use. More specifically, the present invention relates to N-substituted hydrazines that can bind to molecules to target cell populations.

BACKGROUND OF THE INVENTION

Bifunctional compounds which allow the binding of two or more molecules have already been described. For example, bifunctional compounds for binding cytotoxic reagents to molecules to target cell populations are known. Bifunctional compounds should be capable of transporting and releasing these types of cytotoxic reagents in vivo to, for example, provide sufficient levels, i.e. therapeutic levels of reagents in vivo, without damaging the activity of the projected molecules. For certain applications, the formation of a conjugate containing a pH sensitive bond between the reagent and the designed conjugate molecules is desirable, which will provide for the release of the cytotoxic reagent within certain pH ranges.

Reagents especially useful in the treatment of

Cancer is anthracyclines. Anthracyclines are tibiotic compounds which exhibit cytotoxic activity. Studies have shown that anthracyclines can annihilate cells according to a number of different mechanisms including: 1) intercalation of drug molecules in a cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis ; 2) drug production of free radicals which then react with cell macromolecules to cause cell damage or 3) interactions of drug molecule molecules with the cell membrane (Peterson et al., Transport And Storage of Anthracyclines In Experimental Systems And Human Leukemia, in Anthracycline Antibiotics In Cancer Therapy, Muffia et al. (Eds.),: 132 (Martinus Nijhoff Publishers, 1982; and Bachur, Free Radical Damage, id., Pp. 97-102)). Due to their cytotoxic potential, anthracyclines have been used to treat numerous cancers such as leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma and sarcoma (Wiernik, Current Status OF Adriamycin And Daunomycin In Cancer Treatment, in Anthracyclines). : Current Status And New Developments, Crooke et al. (Eds.), Pp. 273-294 (Academic Press, 1980)). Commonly used anthracyclines include adriamycin (ADM, also known as doxorubicin) and daunomycin (DAU, also known as daunorubicin).

While these compounds may be useful in treating neoplasms and other disease states in which a target cell population is to be reduced or eliminated, their therapeutic efficacy is often limited by the dose-dependent toxicity associated with their administration. For example, in the treatment of tumors, the characteristic adverse side effects of these compounds include myelosuppression and cardiotoxicity (Crooke, Goals For Anthracyclines: Analog Development At Bristol Laboratories, Anthracyclines; Current Status And New Developments, supra, p. 11). However, attempts have been made in the treatment of tumors to improve the therapeutic effects of these compounds by binding anthracycline to antibodies directed against tumor associated antigens to form immunoconjugates that selectively release drug into tumor cells. (Hermentin and Seiler, Investigations with monoclonal antibody drug (anthracycline) conjugates, Behring Insti. Mitl., 82 (1988) 197-215). In this way, the drug may be released or projected onto the tumor site and may decrease its toxic side effects on normal body cells. Immunoconjugates consisting of anthracyclines, ADM or DAU linked to monoclonal or polyclonal antibodies to tumor associated antigens are known in the art (e.g., Gallego et al., Preparation Of Four Daunomucin-Monoclonal Antibody 791T / 36 Conjugates With Anti-Tumor Activity, Int. J. Cancer, 33 (1984) 737-744; and Arnon et al., In Vitro And In Vitro Efficacy Of Conjugates Of Daunomycin With Anti-Tumor Antibodies, Immunological Feb., (1982) 5-27).

The most frequently used approaches for binding an anthracycline to an antibody used binding of the anthracycline sugar moiety. For example, the sugar radical was oxidized by treatment with sodium periodate and directly bound to lysine residues of the antibody by forming a Schiff base (Hurwitz et al.

The Covalent Bin-4L ·.

of Daunomycin And Adriamycin To Antibodies, With Retention of Both Drug And Andibody Activities, Cancer Res., 35 (1975) 1175-1181). Alternatively, anthocyclins were bound to antibodies by carbodiimide-mediated binding of the anthracycline amino group to the carboxyl groups of the antibody (Hurwitz et al., Supra), or to an aminoalkyl group (Hurwitz et al., The In vivo effect of the Chemotherapeutic drug-antibody conjugates in two murine experimental tumors systems, Int. J. Cancer, 21 (1978) 747-755). These bonds are not easily hydrolyzable and make controlling anthracycline release difficult. Anthracyclines were also linked to antibodies by crosslinking the sugar of the drug amino group and the amino groups of the antibody with glutaraldehyde (Belles-Isles et al., In Vitro Aetivity of Daunomycin-Anti-AlphaFetoprotein Conjugate On Mouse Hepatoma Cells, Br .

J. Cancer, 41 (1980) 841-42. However, immunoconjugate studies in which the sugar portion of the amino group of the anthracycline molecule was modified by antibody binding indicated a loss of cytotoxic activity of the conjugated drug. (Arnon et al., Supra, pages 7-8). In addition, studies with anthracycline analogues indicated that changes in amino sugars in anthracyclines resulted in a decrease in cytotoxic activity of the drug analog relative to the same family drug (Yamamoto et al., Antitumor Activity of Some Derivatives of Daunomycin At The Amino And Methyl Ketone Functions, J, Med. Chem., 15 (1972) 872-75).

Other immunoconjugates were further prepared in which anthracycline, DAU, was directly bound to an antibody at the carbon 14 (C-14) position of the drug. However, the selective cytotoxic activity of these immunoconjugates against tumor cells was not reproducible and consistently revealed only at a concentration of 20 µg / ml (Gallego et al., Supra).

Japanese Patent Application No. 274658 describes the conjugation of an anthracycline with an antibody via a C-13 acylhydrazone linkage. This conjugation was performed using methods involving antibody derivation and subsequent reaction of these derivatives with anthracycline. These methods are not preferred because the derivation of the antibody involves undesirable non-specific reactions and low anthracyclic ratios are produced. na / antibody. According to the first method, the antibody was treated with carbodiimide in the presence of hydrazine to obtain a hydrazide antibody derivative which is then reacted with anthracycline such that anthracycline directly attaches to the structure of the antibody. However, the resulting immunoconjugates are prone to aggregation of antibody molecules. In addition, because this method requires carboxyl groups which may be limited in number, these immunoconjugates have a low anthracycline / antibody ratio (approximately 1.1 to 1.3). The second method involves reacting the antibody with succinic anhydride to obtain a hemisuccinate derivative of the antibody. This derivative is then reacted with hydrazine to produce a hydrazide derivative antibody which is then first reacted with anthracycline daunomycin. This second approach is imperfect because the reaction of the antibody derivative with hydrazine is non-specific, leading to the production of a mixture of different antibody derivatives in addition to the desired hydrazide derivative. Thus, as indicated in Japanese patent application no. 274 658, the molar ratio between anthracycline and antibody was very low (approximately 1, see Japanese patent application p. 264, column 1). See also European Patent Application, Publication No. 294,294, in which the conjugation of a C-13 hydrazone derivative of an anthracycline to the carbohydrate radical of an antibody is described.

Other anthracycline hydrazones have been described in Tong et al., J. Med. Chem., 21 (1978) 732-37; Smith et al.,

J.Med. Chem., 21 (1978) 280-83; and Brownlee et al., J. Chem Soc., (1986) 659-61 '. See also U.S. Patent 4,112,217, which describes DAU and ADM bishydrazones.

In other studies anthracyclines have been linked to high molecular weight vehicles such as dextran or polyglutamic acid for the purpose of enhancing cytotoxic activity and reducing drug toxicity (Arnon et al., Supra, p. 5 and Hurwitz et al. al., Soluble Macromolecules As Carriers For Daunorubicin, J. Appl. Biochem., 24 (1980) 25-35. These vehicle-linked anthracyclines were also covalently linked to antibodies directed against tumor-associated antigens to form immunoconjugates to specifically design the cytotoxic drug into tumor cells. For example, ADM was ligated to one of these anti-tumor antibodies via a carboxymethyldextranhydrazide bridge whereby the ADM molecule was attached to a carboxymethylantran hydrazide derivative on ADM C-13 earbonyl to form a hydrazone. . Antibody was then ligated to the dextran hydrazide derivative with glutaraldehyde to form an adriamycin-dextran antibody conjugate (Arnon et al., Monoclonal Antibodies As Carriers For Immunotargeting Of Drugs, (Monoclonal Antibodies For Cancer Detection And Therapy, ( Baldwin et al., Eds.), 1985, pp. 365-83 and Hurwitz et al., A Conjugate Of Adriamycin And Monoclonal Antibodies To Thy-1 Antigen inhibits The Human Neuroblastoma Cells In Vitro, Ann. NY Acad. 417 (1983) 125-136).

However, the use of vehicles has some disadvantages. For example, immunoconjugates containing vehicles are quite large in size and are rapidly removed by the reticuloendothelial system in vivo (Dillman et al., Preclinical Trials With Combinations And Conjugates Of T101 Monoclonal Antibody And Doxorubicin, Cancer Res., 46 (1986) 4886 -4891). This rapid removal of vehicle-containing immunoconjugates may not be beneficial for therapeutic purposes because it is possible that the drug will never reach its intended site. In addition, the presence of high molecular weight vehicles has been certified to reduce the binding activity of the conjugate antibody (Embleton et al., Antibody Targeting Of Anti-Cancer Agents, Monoclonal Antibodies For Cancer Detection And Therapy, (Baldwin et al. al., Eds.), 1985 pp. 232-24). Moreover, in studies with tumor cells it has not been possible to prove that immunoconjugates containing high molecular weight vehicles without the ability to localize tumor cells in vivo. (See Ford et al., Localization And Toxicity Study Of A Vindesin-Anti-CEA Conjugate In Patients With Advanced Cancer, Br. J. Cancer, 47 (1983) 35-42, which demonstrates the conjugate localization of antibody-drug directly conjugated to tumor cells in vivo).

Conjugation of anthracyclines to antibodies by the use of specific linkages and vehicles has thus been described. As noted above, the use of these immunoconjugates has distinct disadvantages depending on the specific binding or vehicle used.

Certain conjugates, ligand toxin, have also been described. In U.S. Pat. No. 4,545,985 to Pastan discloses an exotoxin conjugate, which binds Pseudomonas exotoxin (PE) to EGF in a 1: 2 ratio, for use against cells that have numerous EGF receptors. EGF-ricin A and EGF-diphtheria toxin conjugates were also prepared; Caxley et al., Epidermal Growth Facet-Toxin A Chain Conjugates: EGF-Ricin A Is A Potent Toxin While EGF-Diphtheria Fragment A Is Nontoxic ”, Cell, 22 (1980) 563-570 and Shimizu et al., A Cytotoxic Epidermal Growth Factor Cross-Linked To Diohtheria Toxin A-Fragment, FEBS Letters,

118 (No. 2) (1980) 274-78). In addition, Pseudomonas exotoxin fusion proteins were prepared using proteins, polypeptides and growth factors such as TGF-, IL-2, IL-β and CD4 (Pastan et al., Novel Cytotoxic Agents). Fusion Of Growth Factor And Toxin Genes, Fourth Internatl Conference On Monoclonal Antibody Immunoconjugates For Cancer,

P. 36 (March 30 to April 1, 1989); Lorberboum et al., Proc. Natl. Acad. Know. USA, 85 (1988) 1922-1926; Chaudhary et al ,,

Proc. Natl, Acad. Sbi. USA, 84 (1987) 4538-4542; Siegall et al., Proc, Natl. Acad. Know. USA, 85/1988) 9738-9742; and Chaudhary et

Nature, 335 (1988) 369-372). A diphtheria toxin-melanocyte stimulating hormone fusion protein (Murphy et al., Genetic Construction, Expression And Melanoma-Selective Cytotoxicity Of A Toxin-Related Diphtheria-Stimulating Hormone Fusion Protein, Proc. Natl. Acad. Sci. USA, 83/1986) 8258-8262, and U.S. Patent 4,675,382 issued to Murphy). However, ligand conjugates consisting of toxin proteins may be immunogenic in xenogenic hosts.

In addition, anthocyclins such as ADM or DAU have been chemically bound to certain proteins or polypeptide ligands such as transferrin (British Patent Application No. 2116979A) and melanotropin (Varga et al., Melanotropin -Daunomycin Conjugate Shows Receptor-Mediated Cytotoxicity For Cultured Murine Melanoma Cells, Nature, 267 (1977) 56-58). 0 PCT WO 88/00837 describes the binding of EGF via a polymeric carrier to a cytotoxic substance such as DAU, and U.S. Patent Nos. 4,522,750 and 4,590,001 describe transferrin binding to a vinca alkaloid and platinum, respectively.

The cytotoxic drug to be used in the immunoconjugate should be released through a conditional release mechanism, that is, the cytotoxic drug should be released at a specific site, not by gradual, non-site specific hydrolysis. Special immunoconjugate translocation to lysosomes has been hypothesized (deDuve, Lysosomes Revisited, Eur. J. Biochem., 137 (1983) 391-397), which was slightly acidic (pH 5.0 to 5.5) ( Poznansky and Juliano, Biolo

-10ζ gical Approach.es to the Controlled Delivery of Drugs: A Critical Peview,

Pharmacol. Pev., 36/1984) 277-336). The use of acidic drug release conditions has been reported in the development of cisconcitic ADM binders (Shen and Reiser, Cis-Aconity Spacer Between Daunomycin and Macromolecular Carriers:

A Model of pH-Sensitivity Linkage Releasing Drug From a Lysosomotrophic Conjugate, 'Biochem. Biophys / Res. Coromun., 102 (1981)

1048-1054 and Yang and Reisfeld, Doxoruhicin Conjugates With a Monoclonal Antibody Directed to a Human Melanoma-Associated Proteoglycan Suppresses the Growth of Established Tumor Xenografts in Nude Mice, Proc. Natl. Acad. Sci., 85/1988) 1189-1193), and of diphtheria toxin ketyl linkers (Srinivasachar and Neville, New Protein Cross-Linking Reagents That Are Cleaved by Mild Acid, Biochemistry 28 (1989) 2501-2509).

Greenfield and others have recently described the formation of acid-sensitive immunoconjugates containing the acylhydrazine, 3- (2-pyridylditio) propionylhydrazide compound conjugated via an acylhydrazone bridge to the 13-keto position of the anthracycline, and conjugating this anthracycline derivative to an antibody or ligand molecule (Greenfield et al., European Patent Application No. 328 147, published August 16, 1989).

It would be useful to provide additional bifunctional compounds having a structure capable of allowing acid-sensitive bonds between molecules, including targeting molecules and reactive molecules, for use in therapy in vivo.

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SUMMARY OF THIS INVENTION

The present invention provides novel bifunctional compounds that readily conjugate to useful molecules and methods for preparing said bifunctional compounds. The bifunctional compounds contain a pyridinyl dithio or ortho-nitrophenyl dith reactive group. The present invention also provides novel conjugates containing cytotoxic molecules bound to the bifunctional compounds to form cytotoxic molecule derivatives and subsequently conjugates containing the cytotoxic derivative linked to a reactive molecule. with a target cell population that is to be annihilated. This targeting molecule can be a protein, such as an antibody or a ligand, such as bombesin or EGP.

According to one aspect of the present invention, a novel bifunctional compound is synthesized, N- [2-Γ (2-pyridinyl) dithio-y-et-1 / hydrazinecarboxamide (compound 10), and is used to form a semicarfaazone derivative of ADM that contains a semicarbazone bond at the C-13 position of ADM, which functions as the ADM binding site for compound 10.

In another preferred aspect, a novel bifunctional compound is synthesized and used, dihydrazide (2-pyridinyl) dithio N-ethyl N-amino-carbonylcarbonic (compound 11a). to form an ADM carbazone derivative containing an ADM C-13 carbazone bond that functions as an ADM binding site to compound 11a.

In another preferred aspect, a novel bifunctional compound, N - / * 4 - / * (2-pyridinyl) dithio [2-butenyl f-bidrazinecarbotioamide (with -) is synthesized and used.

<img file="PT97639B_D0002.tif" />

12), to form an ADM thio-semi-carbazone derivative containing an ADM thio-semicarbazone bridge at position C-13, which functions as the ADM binding site for compound 12.

In a further preferred aspect, a new bifunctional compound, 2- / (2-pyridinyl) dithio / ethyl hydrazinecarboxylate (compound 13), is synthesized and used to prepare an ADM hydrazone derivative, containing a carboxylate-hydrazone bond at the C-13 position of ADM, which functions as the ADM binding site for compound 13,

In a further preferred aspect, a novel bifunctional compound, N- [2- (2- pyridinyl) dithio / ethyl / hydrazinobenzamide (compound 15) is synthesized, and used to prepare a aryl hydrazone derivative of ADM containing an aryl hydrazone bridge at position C-13 of ADM, which functions as the ADM binding site for compound 15,

According to yet another aspect of the present invention, a number of molecules of the above-mentioned novel anthracycline derivatives are attached to a target cell population of choice. Preferably, the cell-reactive molecule is an antibody, which is a monoclonal antibody. Each anthracycline derivative molecule binds the antibody through the anthracycline-linked bifunctional compound via a semicarbazone, carbazone, thiosemicarbazone, carboxylate hydrazone or arylhydrazone bridge at the C-13 position of the anthracycline molecule to form the novel immunoconjugates of the present invention. A preferred aspect of the present invention involves, for example, the synthesis of a novel adriamycin-derived molecule that is condensed with a thiolated antibody, resulting in the binding of anthracycline to the antibody via the bifunctional compound. The hydrazone bridge formed at position C-13 of the ADM functions as the ADM binding site. Accordingly, a disulfide bond is present in the bifunctional compound through which it binds to the antibody. According to another preferred aspect, the adriamycin derivative (ADM linked to the bifunctional compound) molecule is reduced to form a sulfhydryl group and the resulting derivative is condensed with a maleimide derived antibody. These operations lead to the formation of an immunoconjugate which can. is a Δ-substituted hydrazone bridge as the binding site of the bifunctional compound at the C-13 position of ADM and a bridge thioether in the bifunctional compound through which it is bound to the antibody.

In yet another preferred aspect of the present invention, the novel anthracycline derivatives may be covalently linked to ligands such as bombesin, transferrin or ECE, resulting in the binding of anthracycline to the ligand via a bifunctional compound. As in the other aspects above, anthracycline binds to the bifunctional compound via a hydrazone bridge formed at the C-13 position of anthracycline. Advantageously, the ligand is thiolated prior to binding to the anthracycline derivative.

<img file="PT97639B_D0003.tif" />

but it can also be linked directly to ligands having endogenous free thiol groups.

As is apparent from these aspects, the present invention provides novel bifunctional and anthracycline derivative compounds usable for the preparation of the conjugates of the present invention.

The immunoconjugates of the present invention have an anthracycline / antibody molar ratio of at least 1: 1 to 10: 1, preferably from 4; 1 to 10: 1, and retain

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both antibody activity and cytotoxic drug activity for the annihilation of selected target cells.

The anthracycline-ligand conjugates described herein advantageously have an anthracycline / ligand ratio of at least 1: 1 to 10: 1, preferably from 4: 1 to 10; sensitive to the acid present at the anthracycline binding site to the bifunctional compound of these conjugates? They are also suitable for the release of the active drug under acidic conditions, such as those typically found within a cell, such as in the lysosomal vesicles.

The release of adriamycin by hydrolysis of each of the above derivatives, as a function of pH, showed that the new derivatives exhibit a wide range of release rates under. acid conditions, mimicking the lysosomal medium. These derivatives also demonstrated cytotoxicity as immunoconjugates with monoclonal antibody 5E9 against transferrin receptor.

N-substituted hydrazine bifunctional compounds contain a hydrazine radical and a pyridinyl dithio or ortho-nitrophenyl reactive radical. These novel bifunctional moieties may be used to bind various molecules to form useful conjugates. The molecule to be attached to the hydrazine radical of the bifunctional compound contains a car-? free carbonyl or a group which is derived to contain a carbonyl group, such as a cytotoxic reagent molecule, when the carbonyl group-containing molecule binds to the hydrazine radical of the bifunctional compound, forms a hydrazone bridge, which is a semicarbazone, carbazone, thiosemicarbazone, carboxylate hydrazone or arylhydrazone bridge, as the bifunctional compound of the present invention used to form the conjugate. The molecule that you want to bind S extremi

<img file="PT97639B_D0005.tif" />

The bifunctional compound containing the pyridinylthio or ortho-nitrophenyl radical contains a free sulfhydryl group or a group which can be derived to contain a sulfhydryl group, such as an antibody molecule or a reactive ligand, such as preferably, with the target cell antigens or receptors that are to be annihilated, the pyridyl radical is removed during the reaction of the antibody with the bifunctional compound. The molecule containing a free sulfhydryl group is preferably a cytotoxic reagent molecule, such as an anthracycline capable of annihilating the selected cells. In a preferred aspect, the hydrazone bridge that joins the cytotoxic reagent molecule to the bifunctional = nal compound allows pH-sensitive release of cytotoxic reagent.

Conjugates of the present invention, formed by binding molecules to the bifunctional compounds of the present invention, may be used in pharmaceutical compositions such as those comprising an effective amount of at least one immunoconjugate of the present invention and a pharmaceutically acceptable carrier. The present invention also encompasses methods for the selective release of cytotoxic reagents to a selected target cell population to be eliminated, as well as methods for treating a mammal in a pharmaceutically acceptable manner with an amount

Pharmaceutically effective compositions of the present invention.

Advantageously, the compounds, conjugates, pharmaceutical compositions and methods described herein provide a useful route for the projection of cytotoxic reagents onto a selected cell population, providing preferential annihilation of such target cells in the treatment of diseases such as cancers and other tumors, infections. non-cytocidal viruses and other pathogenic infections and autoimmune disorders.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the structures of the novel adriamycin derivatives of the present invention formed by reacting the multifunctional compounds of the present invention with adriamycin as described in Examples 1-5,^vinfra.

Figure 2 schematically depicts the synthesis of the bifunctional compound N · * · / * (2Tp ± r ± nyl) ^ dithioethyl] hydrazinecarboxamide used to prepare the semicarbazone derivative of adriamycin as described in the Example 1, infra.

Figure 3 is a schematic representation of the synthesis of the bifunctional dihydrazide 77 (7 C 2 -pyridinyl) -dithio-J-ethyl-J-amino-J-carhonyl-J-carbonic compound used to prepare the carbazone derivative of adriamycin as described in Example 2, infra.

Figure 4 schematically indicates the synthesis of the bifunctional compound 4 '(2 = pyridinyl) dithio;⁷-2-butenyl] 7-hydrazinocarbothioamide, used to prepare the deri

Adriamycin thio-semicarbazone as described in Example 3, infra.

Figure 5 schematically indicates the synthesis of the 2- [* (2-pyridinyl) dithio 7-ethyl bifunctional hydrazinecarboxylate compound used to prepare the adriamycin carboxylate hydrazone derivative as described in Example 4, infra.

Figure 6 shows in a schematic manner the synthesis of the bifunctional compound N - / * (2'pyridinyl).-Dithio J 4 ethyl = -4-rhi = · drazinobenzamide, used to prepare the arylhydrazone derivative adriamycin as described in Example 5, infra.

Figure 7 shows schematically the preparation of immunoconjugates according to the present invention using a SPDP thiolated antibody which was reacted with the bifunctional compounds of the present invention as described in Example 7, infra.

Figure 8 schematically indicates the preparation of immunoconjugates according to the present invention using a 2-IT thiolated antibody which was reacted with the bifunctional compounds of the present invention;

Figure 9 schematically indicates the preparation of immunoconjugates according to the present invention having a thioether bond between the antibody and the reduced bifunctional compound using an antibody that was reacted with SMPB to add maleimide groups.

Figure 10 is a graph of time-dependent release of adriamycin following incubation of adriamycin derivatives of the present invention in buffer at pH 4.5 as described in Example 6 below.

Figure 11 is a graph of the time-dependent release of adriamycin following incubation of adriamycin derivatives of the present invention in buffer at pH 5.0 as described in Example 6, infra.

Figure 12 is a graph of release of adriamycin as a function of time after incubation of adriamycin derivatives of the present invention in buffer at pH 7.4 as described in Example 6, infra.

Figure 13 is a graph of the release of adriamycin from the adriamycin carbazone derivative, and a 5E9 immunoconjugate thereof, as a function of time after incubation in buffer at pH 4.5 as described in Example 7; infra.

DETAILED DESCRIPTION OF THE INVENTION

In order to make the present invention more understandable, the following detailed description is provided:

The present invention relates to novel N-substituted N- / 2- (2-pyridinyl) -dithi-7-ethyl-hydrazinecarboxamide (bifunctional) N-substituted hydrazine compounds (compound 10); 2- (2-pyridinyl) -dithio N-ethylamino-7-carbonyl J-carbonic dihydrazide (compound 11a); (2-pyridinyl) dithio 7-2-butenyl

hydrazinocarbothioanide (compound 12); 2- [2- (2-pyridinyl) dithio] 7-ethyl hydrazino acylxylate (Compound 13); and N - [2 - [* (2-pyridinyl) dithio] J-ethyl J⁷'-4-hydrazinobenzamide (compound 15). These compounds are used to form novel N-substituted hydrazone derivatives of cytotoxic reagents such as anthracyclines and to form immunoconjugates when combined.

19- .

to an antibody. The present invention also relates to processes for the preparation of bifunctional compounds, cytotoxic and immunoconjugate derivatives and pharmaceutical compositions and methods for the release of cytotoxic reagents into target cells to treat diseases such as cancers and other tumors. , non-cytocidal viral infections or other pathogenic infections and autoimmune disorders.

Are conjugates made up of at least one molecule of a cytotoxic derivative? bound by one of the bifunctional compounds to at least one reactive molecule with respect to the target cell population. This molecule may be a protein, such as an antibody, preferably a monoclonal antibody, or a ligand, such as bombesin or EGF.

Thus, according to one of the preferred aspects, the new compound, N- / ”2- /^- (2-pyridinyl) dithio J -ilyhydrazinecarboxamide, compound 10, was used to prepare the ADM semicarbazone derivative containing a semicarbazone bridge at ADM position C-13. According to another preferred aspect, the novel dihydrazide 2- / 2- / (2-pyridinyl) dithio N-ethyl N -hydino / N-carbonyl compound was synthesized.

-carbonic compound 11a, and was used to prepare the carbazone derivative of ADM which has a carbazone bridge at position C-13 of the ADM.

In another preferred aspect, hydrazinocarbothioamide, compound 12, was synthesized and used to prepare the thio-sam derivative, icarba, ADM zone having a thio-semicarbazone bridge at the C-13 position of ADM. Still in accordance with another preferred aspect of the present invention, the following

the new one hundred NZ post 4 - / * (2-pyridinyl) dithio J7-2-butenyl J7-20-

<img file="PT97639B_D0006.tif" />

2 - / * (2-pyridinyl) dithio / ethyl ester compound 13, and was used to prepare the ADM hydrazone derivative having a carboxylate hydrazone bridge at position C -13 ADM. Still according to another preferred aspect

-pyridinyl) dithio yetyl-7-4-hydrazinobenzamide, compound 15, to prepare the ADM arylhydrazone derivative having an arylhydrazone bridge at ADM position C-13.

In other aspects, the present invention relates to conjugates containing at least one reactive molecule for a target cell population, such as an antibody or ligand, and at least one cytotoxic molecule that annihilates cells bound to novel bifunctional compounds. of the present invention. Accordingly, in another preferred aspect, the present invention relates to immunoconjugates containing an antibody directed against a target cell population, for example a tumor cell population having the antibody having various anthracycline derivative molecules attached to it. structure. Anthracycline derivative molecules are covalently linked to a thiolated antibody such that a disulfide bridge is formed between each of the drug molecules and an antibody, with the bifunctional compound attached to the anthracycline derivative. by a hydrazone bond at the C-13 position of anthracycline.

More than one drug molecule may be attached to each of the antibody molecules using one bifunctional compound of the present invention per drug molecule. The 4: 1 molar ratio indicates that 4 cyst drug molecules are derived anthracycline) are bound to each antibody.

According to one significant aspect, the anthracycline derivative is reduced to form a sulfhydryl group and this ADM derivative is condensed with a maleimide-modified antibody, forming a thioether bridge between the antibody and anthracycline.

These conjugates allow the release of unmodified, pH-sensitive anthracycline, avoiding structural modifications of the drug which may lead to a reduction in cytotoxicity.

In yet another preferred aspect, the present invention encompasses anthracycline-ligand conjugates consisting of a ligand, such as a polypeptide ligand or a peptide ligand, which reacts with one or more cell surface associated receptors on the cell population. the ligand having at least one molecule of an anthracycline derivative attached to its structure. Anthracycline is covalently bound to the peptide through a bifunctional compound which is attached to anthracycline at the C-13 position of the anthocyclin via a hydrazone bridge. In an alternative aspect, the anthracycline derivative is reduced to form a sulfhydryl group and then the derivative is condensed with a maleimide modified ligand.

The conjugates of the present invention may be prepared step by step by the initial formation of a novel N-substituted hydrazine compound which is used to form a hydrazone derivative of the cytotoxic reagent which is then reacted with a protein or binding, with the appropriate specificity (see Hardy, Purification And Coupling Of Tluorescent Proteins Tor Use In Tlow Cytometry ”, in Handboçk Qff Experimental immunology,

Volume 1; Immunochemistry, DM Weir et al., (Eds.), Pp. 31.4-31.12 (4 Ed., 1986), for discussion of conventional antibody coupling techniques and Varga et al., Supra, for the preparation of ligand conjugates).

The length of the bifunctional compound that binds the cytotoxic reagent to the reactive component of the conjugate cells may vary as the bifunctional compound is linked via one of the hydrazone bridges previously referred to the earbonyl group of the molecule or cytotoxic reagent molecules. (s).

The cytotoxic reagents constituting the conjugates of the present invention may be any molecule containing an carbonyl group. These reagents include, but are not limited to, anthracyclines: adriamycin, daunomycin, detorubicin, carminomycin, idarubicin, epirubicin, esorubicin, 4'-THP-adriamycin, AD-32 and 3'-deamino-3 (3-cyano-4 *). xurrubicin-morpholinyl) (Casazza, Experimental Studies on New Anthracyclines, in Adriamycin: Its' Rolling In Cancer Treatment,

M, Ogawa et al., (Eds.), Pp. 439-52 (Excerpt Medica, 1984)).

It will be appreciated that the cell-reactive molecule to which the cytotoxic reagent is attached to the conjugate via a bifunctional compound may be a molecule that binds or reacts with a cell population to be deleted and which is a sulfhydryl group or which may be modified to contain a sulfhydryl group or a maleimide group. These molecules include, but are not limited to, high molecular weight proteins (generally greater than 10,000 daltons) such as antibodies, lower molecular weight proteins (generally less than 10,000 daltons), polypeptide or peptide bonds and ligands. non-peptidyl.

<img file="PT97639B_D0007.tif" />

The antibodies constituting the immunoconjugates of the present invention may be any antibody that reacts with a specific target cell population to be eliminated or annihilated. Examples of such antibodies include, but are not limited to, antibodies that bind to tumor-associated antigens, such as antigens found on carcinomas, melanomas, lymphomas, bone or soft tissue sarcomas, as well as other tumors, antibodies that bind to virus-associated antigens or other pathogen-associated antigens, and antibodies that bind to abnormal cell surface antigens. These antibodies may be polyclonal or preferably monoclonal and may be produced using techniques well known in the art (DeWeger et al., Eradication Of Murine Lymphoma And Melanoma Cells By Chloramhucil-Antibody Complexes, Immunologic Rev, 62: 29-45 (1982)). ) (and tumor-specific monoclonal antibodies produced and used in conjugates); Yeh et al., Cell Surface Antigens Of Human Melanoma Identified By Monoclonal Antibody-, Proc. Natl. Acad. Sci. 76. (1979) 2927-31, and Brown et al., Structural Characterization of Human Melanoma-Associated Antigen p97 With. Monoclonal Antibodies, J. Immunol ,,

127 No.2) (1981) 539-46 (tumor-specific monoclonal antibodies produced)). For example, human lung carcinoma cell-specific monoclonal antibody L6 or osteogenic sarcoma cell-specific monoclonal antibody 791T / 36 may be used.

In addition, noninvasive antibodies may be used.

Internalizing antibodies or preferably internalizing antibodies. The term antibody, as used in this patent application, includes antigen molecules or fragments.

<img file="PT97639B_D0008.tif" />

intact bodies containing the active antibody binding region, for example Fab or Fab fragments. If monoclonal antibodies are used, the antibodies may be, but are not limited to these rat, human origin or chimera antibodies only.

It will also be appreciated with respect to the present invention that the term ligand encompasses any molecule that specifically binds to a cell surface associated receptor of a target cell population. Preferred ligands which may be used to form the anthracycline-ligand conjugates of the present invention include, but are not limited to, proteins, polypeptides or peptide ligands such as transferrin, epidermal growth factor (EGF), bombesin, gastrin. , gastrin release peptide, platelet-derived growth factor, IL-2, IL-6, TGF-β and TGF-γ3 tumor growth factors, vaccinia growth factor (VGF), insulin and insulin-like growth factors I and II. Other non-peptidyl ligands include steroids, carbohydrates and lectins.

Thus, the targeting molecule that reacts with cells, such as, for example, the antibody or ligand of the conjugates of the present invention, acts to release cytotoxic reagent cells in the specific target cell population with which the antibody is present. or the ligand reacts. For example, an antibody directed against a non-antigen on the surface of tumor cells will bind to these cells and release cytotoxic reagents on these tumor cells, or an antibody directed against a Human Immunodeficiency Virus protein. (HIV) that causes AIDS will release its cytotoxic reagents into HIV-infected cells. Similarly, because tumor cells, such as carcinomas, preferentially express from high density receptor termini, such as the EGF receptor, a ligand such as EGF will bind and release the reagent. cytotoxic in carcinoma cells,

Release of the cytotoxic reagent into or at the site of the particular cell population to which the antibody or ligand reacts results in preferential annihilation of such cells. Thus, it is apparent that the conjugates of the present invention are usable in the treatment of any disease in which a specific cell population that has a cell surface antigen or receptor that allows conjugate binding is desired. The diseases for which the present conjugates are. include, but are not limited to, cancers and other tumors, non-cytocidal viral infections, and other pathogenic infections such as AIDS (AIDS), herpes, CMV (citcmegalovirus, EBV (Epstein Barr Virus) subacute sclerotic pamencephalitis) and rheumatoid arthritis .

Without claiming to limit the present invention by any theory, it is believed. that the antibody or ligand-bound cytotoxic reagent molecules, i.e., in the form of conjugates of the present invention, are released into the target cells to be annihilated by the specificity of the antibody or ligand and may then enter into in the cell through .4

-26 the same endocytic pathway that leads to the internalization of membrane-bound unconjugated antibodies or ligands (Pastan et al., Pathway Q Endocytosis, in Endocytosis, (I. Pastan et al., Eds.) Plenum Press, 1985 pp. 1 Once inside the cell, the endocytic vesicles containing the conjugate fuse with the primary lysosomes to form the secondary lysosomes (Embleton et al., Supra, p. 334). Because cytotoxic molecules bind to the antibody component or the ligand component of the assembly through acid sensitive hydrazone bridges, exposure of the conjugate to the acidic environment of endocytic vesicles and lysosomes results in the release of cytotoxic reagent from the conjugate. In addition, the released reagent is believed to be in the form of a relatively unmodified reagent capable of exerting complete cytotoxic activity. Thus, the acid-sensitive bridge of the conjugate is highly advantageous for the release of cytotoxic reagent into target cells by enhancing the cytotoxicity of the conjugate against such cells.

Alternatively, the hydrazone bridge may be cleaved under acidic and reducing conditions in the immediate external environment or surrounding the target cells, for example at the site of a tumor, with the released drug being absorbed by the tumor cells.

Exemplary are the bifunctional compounds derived from cytotoxic reagents and conjugates of the present invention and methods for their preparation according to the preferred aspects in which anthracycline and adriagicin are used.

In general, carbonyl derivatives of

Adriamycin by treating adriamycin hydrochloride with one of the five bifunctional compounds of the present invention in methanol at room temperature. The addition of catalytic amounts of trifluoroacetic acid (TFA) was found to accelerate the condensation reactions so that the reaction was complete after stirring overnight. Few byproducts were produced in these reactions and purification procedure required only precipitation with acetonitrile. These simplified procedures represent an improvement over those previously reported by Green field et al., Supra, as they are easier to perform, cheaper, faster and provide additional new bifunctional compounds for the conjugation of various molecules.

<img file="PT97639B_D0009.tif" />

According to a first aspect, a new bifunctional compound was prepared by first reacting methoxycarbonylsulfenyl chloride with 2-aminoethanethiol hydrochloride and then with 2-mercaptopyridine to form 2- / (2-pyridinyl hydrochloride). ) -Dithium J7-ethanamine (see Fig. 2). This compound was then reacted with phosgene in the presence of triethylamine (TEA) and then with t-butyl carbazate to form N- [2- (2-pyridinyl) dithio] -ethyl. 7-2- (tert -butoxycarbonyl) hydrazinecarboxamide, Then hydrazinecarboxamide was dissolved in TFA to form N - / * 2 - / * (2-pyridinyl) dithio 7-ethylhydrazinecarboxamide, compound 10 a semicarbazide which was then reacted with adriamycin hydrochloride to form the semicarbazone derivative of ADM, containing a reactive radical, pyridinyl dithio, compound 1, Fig. 1.

In another preferred aspect, a novel bifunctional carbazide compound was prepared (Figure 3). The t-butyl carbazate compound was reacted with triphosgene in the presence of TEA. Then, 2- (2-pyridinyl dithio) ethanamine hydrochloride was added to form dihydrazide 2- [2- (2-pyridinyl) dithio / ethyl / amino] carbonyl-2, 2'-bis- (tert-butoxycarbonyl) carbonic. This intermediate was added to trifluoroacetic acid to form dihydrazide 2- /^- // 2- / (2-pyridinyl) dithio / ethyl / amino J-carbonyl / carbonic compound 11a which was then added to adriamycin hydrochloride to form the carbazone derivative of ADM (compound 2, Figure 1) , which contained a reactive pyridinylthio radical.

In yet another preferred aspect, a novel bifunctional thiosemicarbazide compound was formed (Figure 4). Accordingly, potassium phthalimide was reacted with 1,4-dibromo-2-butene to form 1-bromo-4- (N-phthalimido) -2-butene. This compound was reacted with potassium thio acetate to form 1- (acetylthio) -4- (N-phthalimido) -2-butene, which was then reacted with hydrazine and treated with methoxycarbonylsulfenyl chloride, followed by of 2-mercapto-pyridine to form 1-amino-4- hydrochloride^- (2-pyridinyl) dithio / -2-butene This compound was combined with TEA, followed by di-2-pyridyl thionocarbonate and then t-butyl carbazate was added to form the derivative t N- / 4- / (2-pyridinyl) dithio / -2-butenyl / hydrazinecarbobide amide, compound 12, This compound was dissolved in TFA to give a compound as a gum, which was then reacted with adriamycin hydrochloride and TFA to form the ADM thio semicarbazone derivative which has a binding

Thio-semicarbazone at position C-13 of ADM (compound 3, Figure 1) and which has a reactive pyridinyl dithionic radical.

A further preferred aspect involves the formation of another novel bifunctional compound (Figure 5). Chlorocarbonyl sulfenyl chloride was reacted with 2-mercaptoethanol and 2-mercaptopyridine. An ammonium carbonate solution was added to form 2- (2- (2-pyridinyl) dithio / ethanol) as a colorless oil. Carbonyldiimidazole was added and the mixture was reacted with hydrazine to form 2- (2-pyridinyl) dithioethylethyl hydrazinecarboxylate, compound 13. This compound was reacted with adriamycin hydrochloride and TFA and then with acetonitrile to form the ADM carboxylate-hydrazone derivative (compound 4, Figure 1) which has a carboxylate-hydrazone bridge at position C-13 of ADM and has a reactive pyridinyl dithio radical.

In yet another preferred aspect, a novel bifunctional compound was synthesized (Figure 6). This compound was prepared by reacting 4-hydroxy benzoic acid with di-t-butyl pyrocarbonate to form 4- (N-bochydrazino) benzoic acid. This compound was reacted with N-hydroxysuccinimide and DCC to obtain the N-hydroxysuction cinnamic ester; 4-N-boc- (hydrazino) benzoic acid. This compound was reacted with 2- (2-pyridinyl) dithio / ethanamine hydrochloride and TEA to give Nf 2- (2-pyridinyl) dithio / ethyl-4-N-boc- (hydrazino This compound was then treated with TFA to form N-, 2- (2-pyridinyl) dithio / ethyl / -4-hydrazinobenzamide, compound 15, which was then reacted with hydrochloride. adriamycin to form the aryl hydrazone derivative of ADM (compound 5, Figure 1) which has an arylhydrazone bridge at ADM position C-13 and has a reactive pyridinyl dithionic radical.

The novel N-substituted ADM-substituted hydrazone derivatives referred to above were used to form the conjugates of the present invention. Each of the derivatives was reacted with a monoclonal antibody that had previously been thiolated with SPDP or with 2-IT (2-iminothiolane) as indicated respectively in Figures 7 and 8. The resulting immunoconjugates were ADM molecules conjugated to the monoclonal antibody via the C-13-linked bifunctional compound of each of the ADM molecules via a hydrazone bridge. Bifunctional compounds also contain a disulfide bridge through which each one binds to the antibody.

The bifunctional compounds joining ADM and antibody may be comprised of numerous constituents and linkages, provided that such linkages include an acid-sensitive hydrazone bridge at anthracycline position C-13. The antibody of the preferred aspects was the monoclonal antibody. 5E9.

According to another aspect of the present invention, the novel adriamycin bifunctional compounds are combined to form a derivative which is then reacted with the dithiothreitol reducing agent (DTT) or tributylphosphine to obtain an adriamycin derivative containing a sulfhydryl (-SH) group at the end of the bifunctional compound. This derivative is then reacted with a monoclonal antibody or ligand to which maleimide groups have been attached, for example by reacting the antibody with succinimidyl-4- (p-ma leimidophenyl) butyrate (SMPB), is an immunoconjugate that has a bifunctional compound linked by a hydrazone bridge at the

C-13 of each ADM and having a thioether bond as part of antibody binding (see Figure 9).

Thus, it is evident that the ADM-binding bifunctional compound and the antibody or ligand may be comprised of various constituents and bonds provided that these bonds include a 13-keto hydrazone bridge of ADM and a pyridinyl dithio or ortho group -nitrophenyl dithioid, reactive for antibody binding.

In another aspect, the novel ADM derivatives of the present invention are reacted with a ligand, such as bombesin, EGF or transferrin, and the ligand is first derived to have thiol or maleimide groups. In the case of bombesin, a cysteine ​​residue is introduced at the amino end of the peptide to provide a reactive sulfhydryl group for conjugation with the ADM-derivative. In the case of murine EGF, the SPDP polypeptide is reacted to introduce a reactive sulfhydryl group at the amino end of the molecule for conjugation. In the case of transferrin, the protein is first reacted with 2-IT to introduce reactive thiol groups into the protein structure. In all cases, the thiolated ligand is then reacted with the ADM derivative to form an anthracycline-ligand conjugate according to the present invention, which is a bifunctional compound between the ligand and ADM, finding The bifunctional compound attached to the C-13 position of each anthracycline molecule is via a hydrazone bridge.

Under these conditions, the present invention provides novel anthracycline hydrazone derivatives of the general formula

<img file="PT97639B_D0010.tif" />

<img file="PT97639B_D0011.tif" />

in which

R4 represents an NHCONH group (CH₂)_noSSRg;

NHCONHNHCONH (CH_O) SSR_O;

o η o

NHCSNH (CH_no) CH = CH (CH_no) SSR₀;

m 2 n 8

NHCOO (CH-) SSR_O;

Zn o

NHArCONH (CH_O) SSR_Q?

o η o

NC0NH (CH₉) SH;

NCONHNHCONH (CH₂)_noSH?

NHCSNH (CH₂)_mCH = CH (CH₂)_noSH; NHCOO (CH₂)_noSH; or a group of formula NHArCONH (CH₂)_noSH;

<img file="PT97639B_D0012.tif" />

where Rg represents a group of formula or

<img file="PT97639B_D0013.tif" />

X

X

Wherein X represents a hydrogen or halogen atom or a nitro group; and

<img file="PT97639B_D0014.tif" />

Ar represents a group m and n represent integers from 1 to 10, which may be the same or different;

R₂ represents a group CHg, CHgOH, CHgOCO (CHg) gCHg,

OR CH_noOCOCH (OC-Hp.) „;

Z Ζ O 2

R₃ represents a hydrogen atom or a group

OCHg or OH;

represents an NHg, NHCOCFg, 4-morpholinyl group,

3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine, or 1-cyano-2-methoxyethylamine;

Rg represents a hydrogen atom or an OH or O-THP group; and

Rg represents a hydrogen atom or an OH group. _rwith the proviso that Rg does not represent an OH group when Rg represents an OH or O-THP group,

-34e general formula

<img file="PT97639B_D0015.tif" />

in which

R4 represents an NHCONH group (CH₂)_noSSR_g;

NHCONHNHCONH (CH) SSR;

η o

NHCSNH (CH) CH = CH (CH_) SSR_O;

m 2 η o

NHCOO (CH-) SSR_Q;

η o

NH-Ar-CONH (CH „) SSR_Q ;

z η o

NHCONH (CH 2) SH;

no

NHCONHNHCONH (CHg)_noSH;

NHCSNH (CH_O) CH = CH (CH 3) SH;

m 2 n

NHCOO (CH₂) SH or

NH-Ar-CONH (CH_O) SH 2 n

-35where R_fi represents a group of general formula or

<img file="PT97639B_D0016.tif" />

<img file="PT97639B_D0017.tif" />

XX

<img file="PT97639B_D0018.tif" />

wherein X represents a hydrogen or halogen atom or a NO group;

Ar represents a group m and m represent integers from 1 to 10, which may be the same or different;

R₂ represents a group CHg, CH₂OH, CH₂OCO (CH₂) gCHg OR CH₂OCOCH (OC₂H₅)₂;

represents a hydrogen atom or an OCH group₃ or OH;

r₄ and R₇ each independently represent a hydrogen atom or an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl or substituted aralkyl group; or R4 and R4 together with the nitrogen atom form a tetragonal to heptachronal ring, said ring being optionally substituted; Rg represents a hydrogen atom or an OH or O-THP group; and

R₆ represents a hydrogen atom or an OH group, provided that Rg does not represent an OH group when Rg represents an OH or O-THP group,

The foregoing bifunctional compounds and the novel N-substituted anthracycline hydrazone derivatives are composed of

The anthracycline hydrazone derivatives may be used as novel cytotoxic reagents and also as intermediate compounds in the preparation of the novel conjugates of the present invention.

The anthracycline hydrazone derivatives are exemplified respectively by the adriamycin semicarbazone; car adriamycin bazone; adriamycin thio semicarbazone; adriamycin carboxylate hydrazone and adriamycin aryl hydrazone as described in the preferred aspects set forth herein.

As can be seen from the above formulas, the N-substituted hydrazone derivatives of ADM of the present invention include N-substituted hydrazones of any of several known anthracyclines, such as adriamycin, daunomycin and carminomycin. In addition, the derivatives include N-substituted hydrazones derived at specific sites of the anthracycline structure (e.g. 4'-adriamycin hydrazone and 3'-deamino-3 '- (3-cyano-4-morpholinyl) hydrazone. ) -adriamycin). These latter derivatives can be synthesized by first deriving anthracycline to form a desired analog and then using such an analog to prepare the N-substituted hydrazone derivatives of the present invention. Known anthracycline analogs include those described in U.S. Patent Nos. 4,464,529 and 4,301,277 (anthracycline, 3'-deamino-3 '- (4-morpholinyl) or 3'-deamino-3 analogues '- (3-cyano-4-morpholinyl), U.S. Patent Nos. 4,202,967 and 4,314,054 (anthracycline analogues, 3'-deamino-3' - (1-piperidinyl) or 3'- desamino-3 '- (4-methoxy-1-piperidinyl), U.S. Patent No. 4,250,303

<img file="PT97639B_D0019.tif" />

N-benzyl or β, ou-dibenzyl anthracycline logos), U.S. Patent No. 4,591,637 (N-methoxymethyl or N-cyanomethyl anthracycline analogues and U.S. Patent No. 4,303,785 (acetal analogs). anthracyclines).

Thus, these known anthracycline analogs may be reacted as described above to prepare novel ADM hydrazone derivatives which may then be conjugated to a cell-reactive molecule such as an antibody or a ligand having a desired specificity as described above.

Alternatively, an unmodified N-substituted hydrazone derivative (underivatized) according to the present invention may be produced first, as described, from unmodified anthracycline such as adriamycin, daunomycin or carminomycin, and this new derivative may then be modified to obtain a desired new N-substituted hydrazone. For example, the semicarbazone ADM derivative can be modified to its amino-sugar radical by reductive amination with 2,2'-oxyoxyacetaldehyde using the procedure described in U.S. Patent No. 4,646,529 to obtain the semicarbazone of ADM. 3'-down-3¹- (4-morpholino) -anthracycline. In addition, the hydrazone derivatives may be modified at the position of general formulas I and II as described in U.S. Patent 4,303,785 to obtain acceptable hydrazone derivatives such as hydrazone N -substituted 4'-THP-ADM,

It will be appreciated that in these procedures for the preparation of hydrazones according to the present invention, N-subspecific hydrazones may be used as starting compounds.

Containing other reagents, including various chemotherapeutic agents. And that anthracyclines other than ADM such as daunomycin or carminomycin may be used to obtain new compounds such as N-benzyl daunomycin N-substituted hydrazone or 3'-desamino-3 N-substituted hydrazone¹- (4-morpholinyl) -carminomycin and other compounds using the other derivatives, which are also within the scope of the present invention.

Anthracycline derivatives according to the present invention were evaluated for adriamycin drug release rates at pH values ​​of 4.5, 5.0 and

7.4, a large diversity of release rates has been observed. In addition, the immunoconjugates consisting of the derivatives conjugated to a monoclonal antibody were evaluated for adriamycin release at pH

4.5. Immunoconjugates were tested for toxicity using Daudi cells in the Colony Inhibition assay showing a correlation between the stability of hydrazone derivatives. Immunoconjugates also showed a wide range of release rates and demonstrated antibody-driven cell killing (toxicity) to tumor cells in the colony formation assay.

The N-substituted hydrazine compounds of the present invention provide bifunctional compounds useful for the binding of molecules, such as bleaching and cytotoxic reagents. When used to bind cytotoxic molecules containing a carbonyl group, the bifunctional compounds provide an acid sensitive bond that is cleaved in one of

<img file="PT97639B_D0020.tif" />

pH range is terminated to release the cytotoxic reagent. Anthracycline immunoconjugates according to the present invention appear to be an improvement over the above-mentioned immunoconjugates, in which anthracyclines were directly bound to the antibodies through the amino-sugar portion of anthracycline, as these conjugates bind to the amino-sugar portion often contained lower molar relationships between anthracycline and antibody, were less potent than free ADM and had reduced antibody binding properties (Arnon et al., immunological Rev., 62, Supra; Eurwitz et al., Cancer Res., 35, Supra, and Yamamoto et al., Supra) . In addition, stability studies performed with the immunoconjugates of the present invention indicated that anthracycline is released under acidic conditions similar to those found in a cell medium. Thus, the immunoconjugates of the present invention may release the relatively unmodified drug to be delivered to target cells. Conjugates according to the present invention provide drug release over a wide range of pH values, which may be an advantage for drug delivery.

The bifunctional compounds, immunoconjugates of the present invention and methods for their production are exemplified according to preferred aspects in which anthracycline and adriamycin derivatives are conjugated to the transferrin receptor monoclonal antibody 5E9 .

The present invention also encompasses pharmaceutical compositions, combinations and methods for treating diseases such as cancers, and other tumors, non-cytocidal viral infections or other pathogenic infections and autoimmune diseases. More specifically, the present invention encompasses methods for the treatment of mammalian diseases which consist in administering a pharmaceutically acceptable amount to at least a pharmaceutically acceptable amount to a host mammal. anthracycline-containing conjugate.

As alternative aspects of the methods of the present invention may be mentioned the administration, either simultaneously or sequentially, of a number of different conjugates, i.e. carrying different cytotoxic reagents or different antibodies or ligands, for use in methods of combined chemotherapy.

For example, one aspect of the present invention may involve the use of various anthocyclin immunoconjugates, wherein the antibody component specificity of the conjugate varies, that is, various immunoconjugates are used, each having an antibody which specifically binds to a different antigen or different sites or epitopes of the same antigen present in the cell population of interest. The anthracycline component of these immunoconjugates may be the same or may vary. This may be especially useful in the treatment of certain tumors where the amounts of various antigens on the surface of a tumor are unknown or where the tumor cell population is heterogeneous in antigen expression and is to be assured that it is released. an effective amount of drug on all tumor cells at the tumor site. The use of various conjugates carrying different antigenic or epitope specificities for the tumor increases the likelihood of obtaining sufficient drug at the tumor site. Additionally, this aspect is still important

<img file="PT97639B_D0021.tif" />

To achieve a high degree of specificity for the tumor due to the small likelihood that normal tissues will have all the same tumor-associated antigens (cf. Hellstrom et al., Monoclonal Antibodies to Two Determinants of Melanoma-Antigen p97 Act Synergistically In With Complement-Dependent-Cytotoxicity, J. immunol., 127 (No. 1) (1981)

157-160).

Alternatively, some different immunoconjugates may be used, wherein only the anthracycline component will vary. For example, a particular antibody may be attached to the mycin to form an immunoconjugate and daunomycin may be bound to form a second immunoconjugate. Then, both conjugates may be administered to a host to be treated and localized, due to antibody specificity, at the site of the selected cell population to be deleted. Both drugs will be released on this site. When there is any doubt about drug resistance of a given cell population, such as in the case of a tumor, this aspect of the invention may be important because this method allows for the release of several different drugs into or within cells. target. A further aspect includes the conjugation of more than one type of anthracycline with a given antibody to form an immunoconjugate carrying a variety of different anthracycline molecules along its surface, all of which are linked to the antibody via a hydrazone bridge at the position. 13-keto. Administration of an immunoconjugate according to this procedure results in the release of several different drugs at or within the target cells. In addition, you can use the

The association of anthracycline-ligand conjugates in which the drug can be directed against a cell population carrying a specific antigen as well as a specific receptor for the ligand on its surface. Once again one type of anthracycline or several different drugs may be used in this combination therapy.

The conjugates of the present invention may be administered as pharmaceutical compositions using conventional modes of administration which include, but are not limited to, intravenous, intraperitoneal, oral, intralymphatic administration or direct administration to a population site. selected cell such as a tumor. In the case of immunoconjugates, for in vivo treatment may be useful. conjugates consisting of antibody fragments such as Fab or Ffab fragments. ^or chimera antibodies.

Pharmaceutical compositions of the present invention containing the conjugates may be in various dosage forms which include, but are not limited to, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, solutions or polymeric liquid suspensions, suppositories, microcapsules or microvesicles, liposomes and solutions for injection or infusion. Preferred form depends on the mode of administration and therapeutic application.

A pharmaceutical composition may further contain conventional pharmaceutically acceptable carriers known in the art such as serum proteins, particularly serum albumin, buffering substances such as phosphates, water, salts or electrolytes.

most effective mode of administration and the regimen of

The dosage for the conjugate-containing compositions according to the present invention depends on the severity and course of the disease, the patient's health, the response to treatment and the judgment of the attending physician. As stated above, dosages of conjugates and any accompanying compounds should be established for each patient. However, an effective anthracycline immunoconjugate dose of the present invention may be from about 1 to about 100 mg / m of anthracycline or from about 500 to 5000 mg / m of antibody. An effective dose of anthracycline-ligand conjugates may be from about 1 to about 100 mg / m^z2 anthracycline or from about 1 to about 100 mg / m of ligand.

The following examples are presented for the purpose of making the description of the present invention more understandable.

It will be appreciated that these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

EXAMPLES

Preparation of Bifunctional Compounds and Adriamycin Derivatives (ADM)

Melting points (PF) were determined on the Fisher-Johns melting point apparatus (Medford, MA) and uncorrected. Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker AM 300 apparatus. Infrared (IR) spectra were processed on KBr pellets.

-44 or CHCl3 solutions on a Fourier Transformation Infrared (PE) apparatus (Norwalk, CT), Model 1800. Mass spectra (MS) and high resolution mass spectra (EMER) were obtained, respectively, with Kratos MS25RFA and MS50TC handsets (Manchester, England). Flash chromatography using Woelm silica gel (silica-32-63) (Atlanta, GA). Thin layer chromatography (TLC) was performed on Analtec silica gel GHLF plates (Newark, DE) or RPS-F reverse phase plates, both 250 microns. For routine High Pressure Liquid Chromatography (HPLC), a PE pump, 4 LC serially, an HP 104 6A fluorescence detector and a Phenomenex (Torrance, CA) 1B S11-5C18 column (150 x 4.6 mm). The mobile phase consisted of a 70:30 mixture of phosphate buffer methanol (50 mmol of ammonium phosphate, pH 4.4) at a rate of 1.5 ml / min. To determine the rate of release, the HPLC system consisted of two Model 510 Waters pumps, a Model 712 autosampler, and a Model 680 gradient controller. Chromatography was performed on a Waters C-18 column and a 68:32 mixture of triethylammonium (0.05 M, pH 2.8) and acetonitrile formate buffer was used as the mobile phase respectively. The eluted adriamycin fluorescence was detected using an ABI Model 980 Fluorescence Detector (excitation, 254 nm; 550 nm emission) purchased from Applied Biosystems, Ramsey, NJ. Acetate buffer, pH 4.5 and 5.0; phosphate buffer solution at pH 7.4 was used. Adriamycin hydrochloride was purchased from Sanraku Inc. (Japan). All other chemicals were obtained from commercial sources.

Elemental analyzes were performed in the analysis department of Bristol-Myers Squibb Company, Wallingford, CT and Oneida Research Services.

<img file="PT97639B_D0022.tif" />

EXAMPLE 1

Preparation of Bifunctional TO Compound and Adriamycin Semjcarbazone Derivative (ADM)

In the following example, a method for preparing a bifunctional compound and a semicarbazone derivative of ADM having a semicarbazone bridge at position C-13 of ADM is described. According to this example, N- [2- [2- (2-pyridinyl) dithio] ethyl] 7-hydrazinecarbozamide, compound 10, was prepared according to the reaction sequence indicated in Figure 1. Reacting cysteamine hydrochloride with methoxycarbonylsulfenyl chloride followed by 2-mercaptopyridine gave 2- (2-pyridinyl) dithioethanamine hydrochloride (compound 9, Figure 1). This compound was then reacted with phosgene and t-butyl carbazate followed by trifluoroacetic acid (TFA) to give the desired product (compound 10).

Preparation of 2 - [* (2-Pyridinyl) dithio] -ethanamine Hydrochloride

A methoxycarbonylsulphyl chloride solution was stirred Zumach et al., Agnew Chem. International Edit. (1970) 54-63 J (6.33g, 50 mmol) in HPLC grade methanol (100 mL) under nitrogen and ice-cooling. To this solution was added dropwise a solution of 2-aminoethanethiol hydrochloride (5.7g, 50 mmol) in methanol (50 mL). After the addition was complete, the solution was stirred at room temperature (RT) for 2 hours. Then the solvent was evaporated and the residual oil was crystallized from acetone (100 ml) to give a solid.

-46 (6.9g)

<img file="PT97639B_D0023.tif" />

This solid was dissolved in methanol (100 mL). The solution was cooled on ice, stirred under a nitrogen atmosphere and treated dropwise with a solution of 2-mercaptopyridine (3.82 g, 34 mmol) in methanol (50 mL). The solution was stirred for 1 hour at room temperature, concentrated to a small volume and slowly diluted with acetone until crystallization occurred. After 1 hour in the refrigerator, the solid was collected by filtration and dried to give 2 - / (2-pyridinyl) dithio-ethanamine hydrochloride compound (compound 9). This compound has been described by Field et al., J. Org. Chem., 29 (1964) 1632-1635 and Connor and Schroit, Biochem., 27 (1988) 848-851. The compound had the following characteristics: mp 123-125 ° C (5.8g, 52%). IR (KBr) 2952, 2913, 1610, 1575, 1559, 1451, 1115, 767 cm ”¹. NMR (D₂O). &amp; 8.46, 7.83, 7.34 (d, m, m 4H, Py), 3.37 (t, 2H CH₂ ÇH₂ NH₂), 3.12 (t, 2H, SCHg CH₂). MS (m / e): 187 (corresponds to /'M+H.J7⁺) 170, 152, 142, 112, 104, 76,

Analysis: calculated for ^Ç7^Hno^CLN2^2*¹C, 37.00, H, 5.06,

N, 12.33. Found: C, 36.82; H, 4.99, N, 12.37.

Preparation of N - [2- [2- (2-pyridinyl) dithio] 7-ethyl] J7-2- (tert.butoxycarbonyl) hydrazinecarboxamide

2- (2-Pyridinyl) dithio Z-ethanamine hydrochloride (2.22g, 10 mmol) was suspended in dry methylene chloride (100 mL) and treated with triethylamine (TEA) (5%). 8 ml). This solution was added dropwise to a solution of phosgene (10 ml of a 1.93 M solution in toluene) in ice-cold methylene chloride (200 ml) while stirring. The reaction course followed by TLC and when it was no longer present

<img file="PT97639B_D0024.tif" />

the starting reagent was passed through the mixture for a time. T-Butyl carbazate (1.32 g, 10 mmol) was then added and the mixture was allowed to stir overnight. The solution was washed with water and the solvent was evaporated. The residue was chromatographed on silica using a solvent system consisting of a methylene chloride / methanol (100: 2) mixture. The appropriate fractions were pooled to give 1.74g of N-7 2- [2- (2-pyridinyl) dithio-y-ethyl].^-2- (tert-butoxycarbonyl) hydrazinecarboxamide (compound 9a), in the form of a foam, having the following characteristics; IR (KBr) 3281, 2979, 2932, 1723, 1672, 1577, 1560, 1545, 1448, 1419, 1253, 1161, 762 cm -1.¹. NMR (CDCl 3) &amp; 8.50, 7.56, 7.51, 7.12 (4H, Py), 3.51 (2H, CH₂), 2.89 (2H, CH₂S), 6.84, 6.37, 6.17 (3H, NH), 1.44 (9H), (CHg)₃C), MS (m / e) 345 (corresponds to / 'M + H /⁺) , 317, 289, 245, 213, 178, 134, 112.

Preparation of (2-pyridinyl) dithio-y-ethyl 7-hydrazinecarboxamide

N - / * 2- / (2-pyridinyl) dithio J7-ethyl 7-2- (tert-butoxycarbonyl) hydrazinecarboxamide (570 mg, 1.66 mmol) was dissolved in ice cold TFA (10 ml). The solution was stirred, cooling on ice for 10 minutes and for a further 10 minutes without cooling. Excess TFA was evaporated under reduced pressure as much as possible and the residue was chromatographed on silica gel using a solvent system consisting of a mixture of methylene chloride / itetarei / concentrated ammonium hydroxide (100%). : 5: 0.5). The appropriate fractions were pooled for TLC and the solvent was evaporated to give a crystalline residue (0.42g, quantitative). An analytical sample was prepared.

Recrystallization from isopropyl alcohol (IPA); mp 105-107 ° C. The compound N - [2- [2- (2-pyridinyl) dithio] ethyl] N-hydroxyzecarboxamide (compound 10) was characterized as follows:

IR (KBr) 3336, 3220, 3064, 2949, 2934, 1670, 1623, 1575, 1562, 11-33, 1452, 1369, 1172, 1046, 770 cm¹. NMR (CD₃OD) δ 8.41, 7.78, 7.21 (4H, Py), 3.43 (2H, NCH 3, 2.91 (2H SCHJ. MS (m / e)) 245 (corresponds to / M + H /⁺0, 221, 213, 162, 134, 112,

Analysis: Calculated for ^ 8^Η]_2^Ν4θ®2^: C, 39.32; H, 4.95; N, 22.93; S, 26.24. Found: C, 39.19; H, 4.86; N, 22.48; S, 25.02.

Preparation of Adriamycin Hydrochloride Semi-Carbazone and N- / 2- / (2-pyridinyl) dithio J7-etiT, 7-hydrazinecarboxamide derivative

Compound 10 (0.37 g, 1.5 mmol) in methanol (25 mL) was added to a stirred suspension of adriamycin hydrochloride (0.66 g, 1.14 mmol) in methanol (50 mL). TFA (5 drops) and the mixture was allowed to stir overnight. The clear solution was concentrated and chromatographed on a silica gel C-18 column using a methanol / water (60:40) mixture containing 0.3% ammonium acetate as the solvent system. The appropriate fractions were pooled and the methanol was evaporated as much as possible. The aqueous phase was lyophilized and the residue was dissolved in methanol and added to acetonitrile. The red solid was collected by centrifugation and dried to yield 0.65g (68%). The semicarbazone derivative of ADM was characterized as follows: IR (KBr) 3399, 2976, 2936, 1671, 1618, 1578, 1538, 1417, 1286, 1210, 1117, 1015, 989, 764cm¹. NMR (CD₃OD) is> 8.25, 7.76, 7.62, 7.48, 7.07 (py, ph. Η), 4.95 (anomeric H), 4.63 (CH₂OH) 4.24 (CH₃)H), 3.97 (OCH₃),

-493.5-2.9 (absorption of SSCH, -CH -, CH₂-NH), 1.29 (HC-CH3). MS (m / e) 770 (corresponding to / M + H, 641, 437, 346. EMER: calcd.¹ 770,2166; Found: 770.2157.

<img file="PT97639B_D0025.tif" />

EXAMPLE 2

Preparation of Bifunctional Compound 11a and ADM Carbazone Derivative

The following example describes a process for the preparation of a carbazide bifunctional compound and the ADM carbazone derivative having a carbazone bridge at the C-13 position of ADM. According to example, the reaction, shown in Figure 2 and described in Example 1, between 2- (2- (pyridinyl) dithio] 7-ethanamine hydrochloride and t-butyl carbazate was started with t-butyl carbazate and triphosgene, as shown in Figure 3, to give a bifunctional compound (compound 11a), a carbazide, in which case excess t-butyl carbazate was reacted with phosgene to give the dihydroxide. carbon dioxide.

Preparation of 2- [2- [2- (2-pyridinyl) dithio] dihydrazide JI

-ethyl J-amino j -carbonyl. J-2,2.-Bis (tert-butoxycarbonyl) carbonic

T-Butyl carbazate (0.396g, 3mmol) was dissolved in dry chloroform (10ml). The solution was stirred under nitrogen at room temperature and triethylamine (TEA) (0.6g, 6 mmol) was added. This was followed by the addition of triphosgene (0.296 g, 1 mmol) at one time. A vigorous reaction took place and when it slowed down 2- (2-pyridinyl dithio) -50 hydrochloride was added.

<img file="PT97639B_D0026.tif" />

-ethanamine (0.667 g, mmol) in triethylamine-containing chloroform (TEA) (0.3 g, 3 mmol). The mixture was stirred at room temperature for 1.5 hours and then washed with water (3 mL). x 20 ml), dried and the solvent was evaporated under reduced pressure to give a foam (0.91 g). This product was chromatographed on silica using a solvent chloride mixture. methylene / methanol (100: 2) Fractions were observed by TLC and pooled appropriately, to give 2 - [[[(2-pyridinyl) dithio [ethyl] amino] carbonyl] -2,2'-bis (tert-butoxycarbonyl) carbonic dihydrazide (compound 11). as a foam (0.54 g, 52%). Compound 11 was characterized as follows IV (KBr)

3302, 2980, 2933, 1726, 1683, 1498, 1252, 1160, 1047, 1018, 763cm^-1., NMR (CDCl3).₃) 8.50 7.57, 7.49, 7.10 (d, q, d, t, 4H, Py),

3.52 (t, 2H, SSCH₂) 2.90 (t, 2H CONCH₂), 1.46 ° C (CH₃)₃/ 8.30, 6.50, 6.29 (b, s, s, NH). MS (m / e) 503 (corresponding to M + H / +), 447, 431, 419, 403, 347, 303, 213, 179, 112.

Preparation of Dihydrazide 2 - /// 2- / (2.-pyr.idinyl) dithio /

ethyl / amino / carbonyl / carbonic

Compound 11 (0.34 g, 0.68 mmol) was stirred for 10 minutes with ice-cold trifluoroacetic acid (TFA) (5 mL) and for a further 10 minutes without cooling. TFA was evaporated as much as possible and the residue was chromatographed on silica using a mixture of methylene chloride / methanol / concentrated NH 4 OH (100: 5: 0.5) as a solvent system. . The appropriate fractions were pooled and after birding compound 11a was obtained as a foam.

-51Ç.

hygroscopic (0.2 g of quantitative production). Compound 11a was characterized as follows; IR (film) 3330, 2964,

2929, 1698, 1660, 1576, 1486, 1231, 1045, 759 cm¹. NMR (CDCl3)

8.50 7.56, 7.10 (d, m, m, 4H, Py), 3.52 (q, 2H, CH₂N), 2.91 (t,

2H, CH3 SS), 8.87, 8.85, 4.19, 3.78 (exchangeable protons of D₂O,

NH). MS (m / e) 303 (corresponding to / M + H, 213, 112.

Preparation of the hydrochloride derivative carb.az.one. adriamycin and dihydrazide. 2 - / ^ / ^ 2- /^- (2-pi.rid.inyl.) -Dithio y-et.i.1. j-carbonyl-carbonic amino,

Adriamycin hydrochloride (356 mg, 0.6 mmol) and compound 11a (0.2 g, 0.68 mmol) in methanol (50 mL) containing 2 to 3 drops of trifluoro acetic acid were allowed to stir overnight. (TFA). A clear solution was obtained, indicating by HPLC (solvent system consisting of a methanol / 0.01M ammonium phosphate solution mixture at pH 4.5 at 70.30) that over 90% of adriamycin had been converted. in semicarbazone.

The solvent was therefore evaporated and the residue was chromatographed on a C-18 column using a solvent system consisting of a methanol / water (60; 40) mixture and containing 0.3% by weight. Ammonium acetate The fractions of the reverse phase were analyzed by TLC (same solvent but with 3% ammonium acetate) and / or by HPLC and the adriamycin free fractions were pooled. Most of the methanol was evaporated under reduced pressure, The aqueous solution was lyophilized and the red residue was dissolved in a small volume of methanol. The solution was filtered and added to stirred acetonitrile (1 liter). The clear solution was concentrated to about

From one third of its volume, the solid obtained was collected by centrifugation and dried to give ADM carbazone (with 4) (160 mg). A second collection was obtained by concentrating the solution to 100 ml, diluting with ether and collecting the solid by centrifugation (49% total yield). This carbazone derivative was characterized as follows: IV (KBr):

3346, 2975, 2936, 1711, 1668, 1618, 1578, 1286, 1210, 1083, 1015, 765 cm¹. NMR (CD₃OD) δ 8.43, 7.89, 7.77, 7.52, 7.21, (Py, H phenyl), 5.15 (anomeric H) 4.57 (CH3 OH), 4.25 (CH₃CH), 3.99 (OCH₃), 3.53 (SSCH₂), 3.17 (C-CH₂(ring) 3.05 (CH₂NN =), 2.38 (-CH₂", Ring) 1.29 (CHCH₃). MS (m / e) 828 (corresponding to δ M + H J +), 699, 572, 537, 377, 346, 289, 213,

EXAMPLE 3

Preparation of Bifunctional Compound 12 and ADM thiosepticarbazone derivative

In this example the preparation of the bifunctional compound 12 and the semicarbezone derivative of ADM having a thio semicarbazone bridge at the C-13 position of ADM is described. According to this Example, the semicarbazide thio analog described above in Example 1 (compound 10) was prepared as indicated by the diagram shown in Figure 4. When 2 - / ”(2-pyridinyl) dithio-y-ethanine was used, 2-mercaptopyridine was eliminated, which can be attributed to the nucleophilic increase of the thio-semicarbazide radical in the penultimate step of the product. This problem was overcome using the trans-butene group as shown in Figure 4.

-53Preparation of 1-bromo-4- (N-ftalaffiidoj-2-butene

To a solution of 1,4-dibromo-2-butene (8.4 g, 40 mmol) in dimethylformamide (200 mL) was added potassium phthalimidide (4.62 g 24 mmol) portionwise over 1 hour. . After stirring overnight, the solvent was evaporated and the residue was partitioned between water and methylene chloride. The organic phase was washed several times with water, dried and the solvent was evaporated. The residue was crystallized from 2-propanol to afford the desired product, 1-bromo-4- (N-phthalimido) -2-butene (3.95 g, 50% yield); characterized as follows: mp 101-2 °. IR (KBr) 1775, 1711, 1466, 1436, 1393, 723 cm¹. NMR (CDCl3) δ 7.81, 7.73 (m, m 4H, phenyl), 5.88, 5.81 (m, m 2H, 2 = CH-) 4.30 (d, 2H CH₂-N), 3.90 (d, 2HCH₂Br). MS (m / e) 280 (corresponding to M + HJ⁷*) , 200,.

Analysis: Calculated for C 5.00 Found: C, 52.35;

C, 51.45; H, 3.60; N

4.80 l₂Hio^BrN⁰23H, 3.47, N,

Preparation of 1- (Aeetylthio) -4- (N-phthalimido) -2-butene

A mixture of 1-bromo-4- (N-phthalimido) -2-butene (3.95 g, 14 mmol) and potassium thioacetate (1.77 g, 15.5 mmol) was heated at reflux for 30 minutes. in absolute ethanol (50 ml). The solvent was evaporated and the residue was extracted with methylene chloride. The solvent was evaporated to give a crystalline residue (3.85 g, 99% yield) which was used as it was in the next step. An analytical sample was prepared by crystallization from 2-propanol, mp 69 ° -71 ° C. This compound was characterized as follows: IR (KBr) 1769, 1713, 1688, 1427, 1391, 1114, 958 cm¹NMR (CDCl3) δ 7.80, 7.73 (m, m

-54i-f.

4H Ph), 5.70 (xn, 2H, 2 = CH-), 4.24 (d, 2H, CH 2 N), 3.48 (t, 2H (CH 2 N)), 2.29 (s, 3H, C- (Hg). MS (m / e) 276 (corresponds to fM + H / +), 234, 200.

Analysis: Calculated for C ^^H H gNONOS: C, 61.07; H, 4.76; N, 5.09. Found: C, 61.29; H, 4.82, N, 5.21.

Preparation of 1-amino-4α- (2-pyridinyl) dithio hydrochloride

A solution of 1-acetylthio-4- (N-phthalimido) -2-butene (6.5 g, 23.6 mmol) in absolute ethanol (150 mL) and hydrazide (1.74, 54 mL) was heated under reflux. mmoles). The reaction was followed by TLC analysis and when the starting compounds were no longer present the solution was cooled on ice and treated with 6N hydrochloric acid (10 ml). A large precipitate formed which was identified as phthalhydrazide (NMR, MS) and filtered. The filtrate was concentrated to 10 ml and diluted with water. The solid was filtered off and the filtrate was washed with ether (2 times) and methylene chloride (1 time), filtered through Celite and lyophilized. The solid was dissolved in a small amount of methanol and the solution was filtered through Celite, the solvent was evaporated and the residue was vacuumed overnight. A waxy, hygroscopic product was obtained which had the following characteristics; NMR (DMSO-D 6 O) δ 5.83, 5.60 (m, m 2H, 2 = CH-) 3.40 (d, 2H CHgNHg), 3.16 (d 2H, CHgSH) MS (m / e) 104 (corresponds to M + H / +), 87.70. This waxy product was dissolved in HPLC grade methanol (75 ml). The solution was stirred and treated with methoxycarbonylsulfenyl chloride (3g, 23.7 mmol). After 30 minutes no starting compounds were detected by TLC. The solvent was evaporated.

The residue was redissolved in methanol (75 ml). The solution was stirred and treated with 2-mercaptopyridine (2.7 g, mmol). After 2 hours, the solvent was evaporated and the residue was extracted under high vacuum. The residue was then dissolved in a mixture of 0.01 Me hydrochloric acid and methanol (90:10, 130 ml). The cloudy solution was washed with methylene chloride, filtered through Celite and lyophilized to give 1-amino-4 - ((2-pyridinyl) dithio / -2-butene hydrochloride). form of a highly hygroscopic velvety product © (4g, 68%) having the following characteristics: IR (KBr) 3433, 2959, 2884, 1607, 1576, 1447, 1418, 1118, 767 cm^-1. NMR (D₂O) and> 8.51, 8.13, 8.02, 7.54 (m 4H, Py) 5.87, 572 (m, m 2H, 2 = CH 2) 3.54 (d 2H, CH₂ NH₂) 3.43 (d 2H, CH₂ S). MS (m / e) 213) corresponds to (M + H / +), 196, 112.

Preparation of N- / 4- / (2-pyridinyl) dithio ./-2-b.uteni.l / -hydr.az inocarbothioamide

1-Amino-4- [(2-pyridinyl) dithio] -2-butene hydrochloride (1.5 g, 6 mmol) was suspended in stirring methylene chloride (30 mL). Trifluoroacetic acid (1.46 g, 14.6 mmol) and then di-2-pyridyl thionocarbonate (Kim and Yi), Tetrahedron Lett., 6 (1985) 1661-1664 f (1.4 g, 6 mmol) were added. ). A clear solution was obtained and TLC analysis showed the absence of starting compounds. T-Butyl carbazate (0.8 g, 6 mmol) was added and the solution was stirred for 1 hour. The solution was washed with water and the solvent was evaporated. The residue was chromatographed on silica using a methylene chloride / methanol (100: 2) mixture as the solvent system and rechromatographed again.

<img file="PT97639B_D0027.tif" />

using a hexane / ethyl acetate (75:25) mixture as a solvent system to give a foam which was the N - / ”4 - / * (2-pyridinyl) dithio derivative T-boc 7-2 -butenily-hydrazinocarbotioamide (1.4 g, 58%), which was characterized as follows: IR (KBr) 3238, 2971, 2930, 1718, 1544, 1418,

1156.762 cm¹. NMR (CDCl1)₃/ D₂O) δ 8.43, 7.65, 7.08 (m, m, m 4H, py) 5.57 (m 2H, 2 = CH), 4.12 (D 2H, € H₂ N) 3.45 (d 2H CH₂S),

1.46 (s 9H, 3 CH₃). MS (m / e) 387 (corresponds to M + H 355,

287, 276, 112.

The protected carbothioamide (0.86 g, 2.2 mmol) was dissolved in ice cold trifluoroacetic acid. The solution was kept on ice for 10 minutes (under a nitrogen atmosphere) and for an additional 10 minutes without cooling. Excess acid was evaporated as much as possible under high vacuum and the residue was chromatographed on silica gel using a solvent system consisting of a concentrated methylene chloride / methanol NH 4 OH mixture (100: 5). : 0.5). Appropriate fractions were combined to give compound 12 as a gum (0.39 g; 63% yield) having the following characteristics: IR (film) 3322, 3198, 2974, 1626, 1574 , 1560, 1538, 1448, 1418,

1224,760 cm ”¹. NMR (CDCl1)₃/ D₂O) 8.41, 7.63, 7.11 (mmm 4H,

Py) 5.64 (m 2H, 2 = CH), 4.20 (d 2H, CH₂N) 3.41 (d 2H, CH₂$), MS (m / e) 287 (corresponds to [M + H] +), 225, 221, 144, 112,

Preparation of thio-s.emicarba.zone derivative. of. Adriamycin and N- [4- (2-2-p.ir.id.ini.l.) -ditiohydrochloride. y.-2-b.uten.il Z ~ h.idra.zinocarbotioamide

To a stirred suspension of adriamycin hydrochloride

<img file="PT97639B_D0028.tif" />

(350 mg, 0.6 mmol) in HPLC grade methanol (50 mL) was added a solution of compound 12 (350 mg, 1.2 mmol) in HPLC grade methanol (25 mL). Trifluoroacetic acid (3-4 drops) was added and the mixture was allowed to stir overnight. A clear solution was obtained and no free adriamycin was detected either by HPLC or TLC. The solution was concentrated to a small volume (5 mL) which was added to aoetonitrile (600 mL). A precipitate formed which, after cooling on the crude, was collected by centrifugation and dried under high vacuum (275 mg - 54% yield). The thio semicarbazone derivative had the following characteristics: IR (KBr) 34.18, 2934, 1616, 1578, 1534, 1414, 1284, 1208, 1012, 986 cm -1.¹. NMR (CDCl3) δ 8.30, 7.80, 7.74, 7.52 ((Py, 7H phenyl)), 5.60 (2 = CH) 5.40 (anomeric H) 4.67 ( CH2 OH), 4.22 (CH3 CH), 4.09 (CH3 N), 4.02 (CH2 N), 3.5-1.88 (cluster absorption including -CH3 -SS, -CH2 -CH), 1, 30 (CHg-CH). MS (m / e) 812 (corresponds to M + H 7+}, 701, 683, 669, 572, 554, 540, 536, 522, 504. EMER Calculated for^Ç37^H4₂^N5th θ10 ^ 3^: θ12,2094; Found: 812.2087.

EXAMPLE 4

Preparation of Bifunctional Compound 13 and ADM carboxylate hydrazone derivative

In this example, the preparation of the novel bifunctional compound, compound 13, and the ADM carboxylate-hydrazone derivative is described. The carboxylate-hydrazine bifunctional compound is prepared from mercaptoethanol which has been converted to pyridyldithioethanol as shown in Figure 5. This compound is then converted to an active carbonyl derivative.

-58 condensed with hydrazine.

Preparation of hi.draz.i.no.c.arb.ox.ilate. of .2 -. /. (.2-p.ir.idinyl) dithio / ethyl

To a cooled solution (ΟθΟ of chlorocarbylsulfenyl chloride (1.24g; 9.45mmol) in C ^ C ^ ^) was added dropwise 2-mercaptoethanol (737mg, 9.45mmol). The mixture was stirred for 30 minutes at 0 ° to 15 ° C, cooled to 0 ° C and treated with a solution of 2-mercaptopyridine (1.05g, 9.45 mmol) in CH2 Cl2. (15 ml) The mixture was stirred at 0 ° C for 1 hour and then stirred at room temperature for 16 hours. After addition of a solution of ammonium carbonate (1.0 g in 20 ml of water), the phases were separated and the organic phase was washed with water, dried and concentrated in vacuo to give 2 - (2-pyridinyl dithio) -ethanol (1.75g) as a colorless oil. Carbonyldiimidazole (648 mg, 4 mmol) was added to a solution of 2- (2-pyridinyl dithio) ethanol (714 mg, 3.8 mmol) in EC emC ^ (10 mL). The mixture was stirred for 20 hours and then cooled with ice to -20 ° C and treated with hydrazine (122 mg, 3.8 mmol). The mixture was allowed to stand at -5 ° C for 16 hours and then concentrated under vacuum. The residue was chromatographed on silica gel using a solvent system consisting of a methylene chloride / methanol (100: 1-3) mixture to give compound 13, 2- / (2-pyridinyl) -hydrazinecarboxylate. dithio / ethyl (340 mg; 37% yield) as a colorless oil having the following characteristics: NMR (CDCl 3) &amp; 8.46 (1H), 7.62 (2H),

7.08 (1H), 5.92 (1H), 4.35 (t, 2H), 3.70 (s, ZH), 3.01 (t, 2H),

<img file="PT97639B_D0029.tif" />

1.56 (s, 2H). MS (m / e) 246 (corresponds to M + H /⁺) , 142, 103.

Preparation of the adrianticine hydrochloride hydrazone derivative and 2- (2-pyridinyl) dithio4-ethyl hydrazinecarboxylate

To a suspension of adriamycin hydrochloride (290 mg, 0.5 mmol) in anhydrous methanol (4 mL) was added a solution of compound 13 (170 mg, 6.9 mmol) in methanol (4 mL) and CF₃CO₂H (6 mg) in methanol (1 ml). After stirring for 24 hours, the mixture was concentrated to about 4 mL and acetonitrile (50 mL) was added thereto. The product was isolated by centrifugation. The product was dissolved in water / methanol and then lyophilized to give the adriamycin hydrazone derivative (354 mg, 88% yield) as a dark red solid which had the following characteristics: NMR (CD₃OD) ^ 8.34

<td>(OH)</td><td>, 7.93 (d,</td><td>OH),</td><td>, 7.81 (m, 3H),</td><td>7.54 (d,</td><td>OH),</td><td>, 7.17 (m,</td><td>OH),</td>

<td> 5,49</td><td>(m, 1H),</td><td> 5,19</td><td>(s, 1H) 4.59</td><td>(m, 2H),</td><td> 4,37</td><td>(m, 2H),</td><td>4.25 (m,</td>

<td>OH),</td><td>4.01 (s,</td><td>3H),</td><td>3.63 (m, 1H),</td><td>3.54 (m,</td><td>OH),</td><td>3.10 (t,</td><td>3H),</td>

<td> 2,37</td><td>(m, 2H),</td><td> 2,03</td><td>(m, 1H), 1.89</td><td>(m, 1H),</td><td> 1,29</td><td>(d, 3H).</td><td>In me):</td>

<td> 771</td><td>(correspond</td><td>from to</td><td>/ * M + H J7⁺) , 642</td><td></td><td></td><td></td><td></td>

EXAMPLE 5

Preparation of Bifunctional Compound 15 and ADM ariihydrazone derivative

In this example the method for preparing a novel bifunctional compound, compound 15, and the ADM arylhydrazone derivative having an arylhydrazone bridge at the C-13 position of ADM is described. Compound 15 was prepared using acid

4- (N-Boc-hydrazino) -benzoic, and incorporating the 2- (2-pyridinyl dithio) ethanamine group as shown in Figure 6.

<img file="PT97639B_D0030.tif" />

4- (N-bochydrazino) -benzoic acid

N-Hydrazinobenzoic acid (760 mg, 5 mmol) was dissolved in dioxane (10 mL), water (5 mL) in 1 N NaOH solution (5 mL). Di-t-butyl pyrocarbonate (1.31 g, 6 mmol) was added at 0 ° C and the reaction mixture was stirred at 0 ° C for 1 hour at room temperature for 30 minutes. After this time, the solution was halved and the solution was acidified with 0.5% hydrochloric acid solution and extracted with ethyl acetate. The ethyl acetate solutions were combined, washed with saturated sodium chloride solution and dried over sodium sulfate.

Removal of solvent afforded a slightly brownish solid which was recrystallized from ethyl acetate and hexane (950 mg; 75% yield) and characterized as follows: NMR (CD₃OD) &amp; 7.84 (d, 2H, J = 8.5 Hz), 6.75 (d, 2H, J = 8.5 Hz), 1.4⁸ (s, 9H); IR (KBr) 3316, 1688, 1607, 1298 cm¹.

Preparation of N-2- (2-pyridinyl) dithioic acid. ./- ethoxy-A- (N-bochydrazyl) benzamide

Stirred at room temperature overnight, 4- (N-bochydrazino) benzoic acid (252 mg, 1 mmol), N-hydroxysuc cinimide (115 mg, 1 mmol) and dicyclohexylcarbodiimide (DCC) (247 mg, 1.2 mmol) in β, β-dimethylformamide (DMF) (5 ml). Dicyclourea (DCU) was filtered off and the filtrate was evaporated. The residue was crystallized by the addition of ethyl ether to give 4- (N-bochydrazino) benzoic acid N-hydroxysuccinimyl ester (300 mg). This product (250 mg, 0.72 mmol) and 2- [(2-pyridinyl) dithio] -ethylamine hydrochloride (167 mg, 0.75 mmol) were dissolved in N, N-dimethylformamide (4 mL) . After the addition of trifluoroacetic acid

<img file="PT97639B_D0031.tif" />

(0.125 ml, 0.9 mmol), the mixture was stirred overnight at room temperature. Δ, Ν-dimethylformamide was removed and the residue was chromatographed over SiO₂ (2% MeOH / CH₂Cl₂) giving a foam (217 mg; 52% yield), which had the following characteristics: NMR (CDCl3) δ> 8.37 (d, 1H, J = 5.1 HZ), 8.01 ( t broad, 1H), 7.78 (d, 2H, J = 8.7 Hz), 7.57 (m, 1H), 7.46 (d, 1H, J = 8.1 Hz), 7.09 (m, 1H), 6.84 (d, 2H, J = 8.7 Hz), 7.40 (bs, 1H), 5.92 (bs, 1H), 3.70 (m, 2H) , 2.98 (t, 2H, J = 5.8 Hz), 1.45 (s, 9H); IR (KBr) 3303, 1714, 1610, 1505 cm¹; m / e 421 (M + H), 365, 321, 112, HRMS calculated for^Ç]_9^H25^N4°3^s2 421.1368, found 421.1358.

Preparation of N-2- [2- (2-pyridinyl) dithio] / [ethyl] -4-hydrazinobenzamide

The above compound (200 mg, 0.48 mmol) was treated with trifluoroacetic acid (1.5 mL) at 0 ° C for 1 hour. After this time, the trifluoroacetic acid was evaporated and the residue was triturated with ethyl ether to give approximately 200 mg of N - [2- [2- (2-pyridinyl) dithio] ethyl]. -4-hydrazinobenzamide (compound 15) as an oil, having the following characteristics: NMR (CD 3 OD) δ 8.38 (d, 1H, J = 4.5 Hz), 7.81 (m, 4H), 7.22 (t, 1H, J = 5.8 Hz), 6.97 (d, 2H, J = 8.8 Hz), 3.67 (t, 2H, J = 6.6 Hz) 3.06 (t, 2H, J = 6.6 Hz); IR (film) 3278, 1674, 1613 cm^-1; MS m / e 321 (M + H).

Preparation of the aryl-hi-draz.one derivative of adriamycin and N- [2- (2-pyridinyl dithio) ethyl] -4-hydraz.inobenz.amide

Compound 15 and adriamycin hydrochloride (250 mg, 0.43 mmol) were dissolved in methanol (15 mL) and stirred in the dark for 2 days. The solvent was removed and the residue was chromatographed on a C-18 SiO column.₂, reverse phase.

Elution with a MeOH / H 2 O (2: 1) mixture containing 0.3% NH 4 OAc gave the hydrazone compound 7 as an orange powder (30 mg; 8% yield). which had the following isometric characteristics: NMR (CD9 OD) δ 8.33 (d, 1H, J = 4.8 Hz), 7.85 (d,

<td>OH, J = 7.9</td><td>Hz)</td><td>, 7.74 (t, 1H,</td><td colspan="2">J = 8.0 Hz), 7.66 (m, 4H), 7.41 (d, 1</td>

<td>H, J = 8.5</td><td>Hz),</td><td>7.14 (m, 1H),</td><td>7.02 (d, 2H, J = 8.8 Hz), 5.46</td><td>(s home</td>

<td>(OH),</td><td> 5,16</td><td>(m, 1H) 4.60</td><td>(s, 2H), 4.23 (m, 1H), 3.91</td><td>(s, 3H),</td>

<td>3.62 (m,</td><td>4H),</td><td>3.02 (m, 4H),</td><td>2.61 (m, 1H), 2.38 (m, 1H),</td><td>1.97 (m,</td>

<td>2H), 1.32</td><td>(d,</td><td>3H, J = 6.5 Hz)</td><td>; IR (KBr) 3206, 1708, 1607,</td><td>1578 cm 1;</td>

MS m / e 846 (M + H), 737, 717, HRMS calculated for ^Ç4i^H44^N5 ° x] _^s2 · 846.2479, observed 846.2380.

Elution with a MeOH / 3 O (3: 1) mixture containing 0.3% NH 4 OAc gave the anhydrous derivative as a blue solid (120 mg; 34% yield). NMR (CD 3 OD) &amp; 8.40 (d, 1H,

J = 4.1 Hz), 7.77 (m, 5H), 7.42 (m, 1H), 7.22 (m, 1H), 7.16 (m,

2H), 5.35 (broad s, 1H), 5.26 (m, 1H), 4.65 (s, 2H), 4.00 (m,

1H), 3.93 (s, 3H), 3.67 (t, 2H, J = 6.5 Hz), 3.44 (m, 1H), 3.08 (t, 2H, J = 6.5 Hz), 2.49 (m, 1H), 1.88 (m, 1H), 1.63 (m, 1H), 1.19 (d, 3H, J = 6.5 Hz); MS m / e 828 (M + H) 699, 681, 495; EMER calculated for ^₄^ H₄^ NgO ^ gS₂ 828.2372, observed 828.2300.

EXAMPLE 6

Characterization of ADM Derivatives

Release of ADM from the ADM derivatives of the present invention prepared as described in

Examples 1 to 5 above, for various pH values, comprising:

<img file="PT97639B_D0032.tif" />

Between 4.5 and 7.4 using HPLC analysis. Stock solutions (1 mg / ml) of ADM derivatives in methanol were prepared and aliquots diluted in aqueous buffer solution to pH

4.5, 5.0 and 7.4, to reach final concentrations of approximately 1.6 mmol / ml. Each buffer was incubated at 37 ° C for up to 24 hours and aliquots were analyzed by applying them to an HPLC column to determine the amount of unconjugated ADM. The released product as intact ADM was identified by its retention time on the column and the UV characteristics of the eluted material. Release rates were expressed as a percentage of the maximum amount of ADM as shown in Figures 10 to 12.

As illustrated in these figures, the ADM derivatives of the present invention have a wide range of release rates. The amount of product released from the ADM derivatives increased as pH values ​​decreased from 7 to 4. The derivatives ADMs have an acid sensitive linking group, which results in the release of ADM from the antibody protein. These results are consistent with a semicarbazone, carbazone, thiosemicarbazone, carboxylate hydrazone or arylhydrazone bridge linking ADM to the bifunctional compound.

EXAMPLE 7

Preparation of Anthracycline Immunoclates

In this example, the preparation of anthracycline immunoconjugates according to the present invention is described, wherein

The above-described ADM derivatives (Examples 1 to 5) are conjugated to a monoclonal antibody.

<img file="PT97639B_D0033.tif" />

Preparation of Immunoconjugates that have a Disulfide Bridge in the Bifunctional Compound monoclonal antibody used was 5E9, produced from ATCC hybridoma No. HB21, which is available from the American Type Culture Collection ATCC in Rockville, MD. Monoclonal antibody 5E9 is an IgG-1 antibody, which reacts with the transferrin receptor on all dividing human cells and cross-reacts with the various histological types of cancer cells. Antibody 5E9 was purified from ascites fluid produced in BALB / c mice according to the procedure of Bruck et al., One-Step Purification of Monoclonal Mouse Antibodies From Ascitic Fluid by DEAE-Affigel Blue Chromatography, J '. Immuno1. Methods, 5b (1982) 313-319.

Prior to reacting the ADM derivative with the selected monoclonal antibody, the antibody is thiolated, that is, reactive sulfhydryl groups are introduced into the antibody molecule. The monoclonal antibody (MAb) 5E9 is thiolated using SPDP, essentially as described by Greenfield et al., supra. Briefly, SPDP is added (pierce Chemical Co. IL) (50 mM) dissolved in ethanol to MAb 5E9 (5-10 mg / ml) in phosphate buffered saline (PBS) at pH 7.2 to give a final concentration of 5 to 10 mM . The reaction mixture was incubated for 30 minutes at 30 ° C. Unreacted SPDP was separated from SPDP-derived antibody by filtration chromatography on

-65gel using a PD-10 column (Pharmacia). The pyridinyl dithion reactive groups were removed by reduction with DTT in excess. The reduced antibodies were passed through a PD-10 column and antibodies containing free thiol were used for condensation with the ADM derivatives.

Reactive thiol groups were also introduced into the antibody proteins using 2-IT. Antibody (5-10 mg / ml in 50 mM TEA, 50 mM NaCl, 1 mM EDTA, pH 8.0) was mixed with 2-IT (Pierce Chemical Co., IL) at a final concentration of 5-10 µM mM The reaction was allowed to proceed for 90 minutes at 4 ° C and thiolated antibodies were separated on a PD-10 column equilibrated with 2M NaCl / PBS.

The number of reactive thiol groups incorporated into the antibodies was determined using DTNB (5,5'-dithio-bis (2-nitrobenzoic acid) (E. coli). ⁼14150), according to the procedure described by Ellman, Arch. Biochem. Biophys., 82 (1959) 70-77).

Each of the ADM derivatives was dissolved in dimethyl formamide and added to the reduced thiolated MAb 5E9

SPDP, in PBS. The amount of ADM derivative was equivalent to the number of thiol groups that existed in the antibody. The conjugation reaction was allowed to incubate overnight at 4 ° C.

After this time, the PBS antibody solution was dialyzed to remove the unconjugated adriamycin derivative. The antibody solution was then treated with Bio-Beads SM-2 (Bio-Rad Laboratories, Richmond, CA) overnight. The amount of MAB-bound conjugated anthracycline was determined by absorbance at 495 nm (^AND4g₅ = 8030). The amount of antibody protein was determined by absorbance at 280 nm.

-66 (1 mg / ml = 1.4 OD units). To correct for ADM absorbance overlap at 280 nm, the following formula was used:

<img file="PT97639B_D0034.tif" />

Antibody (mg / ml) = Α ^ θθ - (0.72 XA_4g5 )

1,4

Immunoconjugates were analyzed for the presence of ADM or ADM derivatives using HPLC analysis.

HPLC was performed using a 5 micron C18 IB-SIL droplet Phenomex column. Unconjugated drug, ADM derivatives (i.e. ADM conjugates for each of the bifunctional compounds of the present invention, prepared as described in previous Examples 1 to 5) (0.1%) were applied to the column (0.1%). imoles) or immunoconjugates containing 0.5 to 5 æmoles of equivalent drugs and eluted with methanol and 10 mM ammonium phosphate at pH 4.5 (70:30) at 1.5 ml / min. The immunoconjugates produced did not contain significant amounts (less than 1%) of unconjugated drug as determined by HPLC analysis.

EXAMPLE 8

Characterization of Immunoconjugates

Immunoconjugates, prepared as described above in Example 7, were 13-keto-conjugated ADM molecules with a bifunctional compound that formed a link between ADM and MAb 5E9. In addition, the addition of free thiol groups MAb to the ADM derivative which contained a reactive pyridinyl dithio radical led to the formation of a disulfide bridge in the functional compound joining the ADM to the MAb. Immunoconjugates prepared in accordance with this aspect

globam, although not limited, 5E9-ADM-semicarbazone - / * 3.42 5E9-ADM-carbazone- / 4.37 J⁷; 5E9-ADM-thiosemicarbazone- / 2.51 J⁷; 5E9-ADM-carboxylate hydrazone / 2.35 J; and 5E9-ADM-arylhydraz na- / 2.52}, where the first part of the designation represents the monoclonal antibody used to form the conjugate, the second part represents antibody-bound anthracycline and the numbers in parentheses represent the molar relationship. ADM / antibody in the conjugate to which they refer.

The binding activity of the immunoconjugates of the present invention was determined in a fluorescence binding assay described by Greenfield et al. In In vitro Evaluation of the Immunoconjugates Prepared by Linking Mitomycin C to Monoclonal Antibodies via Polyglutamic Acid Carriers in Antibody Immunoconjugates and Radiopharmaceuticals, (1989) Vol. 2, p.201. Briefly, immunoconjugates were serially diluted in 100 µl of assay media (RPMI 1640 enriched with 10% fetal bovine serum and penicillin / streptomycin, Gibco, Grand Island, NY). Tumor cells (EMC) (ATCC N9 CCL 119) (1 X 10 4 cells) grown on this medium were harvested and washed by centrifugation and then suspended (1 χ 10θ) in medium containing diluted immunoconjugates. . After 1 hour incubation at 4 ° C, the cells were washed and suspended in 100 µl medium containing rat anti-IgG-FITC developed in 1:40 diluted goat (Cappel, Durham, NC) for 1 more hour at 4 ° C. Cells were washed and analyzed using a Coulter Epics V fluorescence cell analysis apparatus. For each assay, diluted MAb was used in a similar manner as described above as an unconjugated positive binding control. These are shown in Table 1.

-68ί percent protein yield (separately obtained), * molar ratios (mole ADM / MAb) and binding expressed as a percentage of original binding.

TABLE 1

<td rowspan="2">5E9 Immunoconjugates</td><td colspan="2">Yield of</td><td rowspan="2">Relations . molars.</td><td rowspan="2">% Original Connection</td>

<td>Proteins .</td><td> (%)</td>

<td>Semicarbazone</td><td> 83</td><td></td><td> 3,42</td><td> 97</td>

<td>Carbazone</td><td> 74</td><td></td><td> 4,37</td><td> 92</td>

<td>Uncle zone</td><td> 61</td><td></td><td> 2,51</td><td> 72</td>

<td>Carboxylate-Hi- drazona</td><td> 78</td><td></td><td> 2,35</td><td> 97</td>

<td>Arylhydrazone</td><td> 86</td><td></td><td> 2,52</td><td> 91</td>

As shown in Table 1, 5E9 immunoconjugates retain more than 90% (except thio semicarbazone) of the original binding activity of the unconjugated 5E9 antibody. This demonstrates that conjugation of ADM derivatives to MAb 5E9 causes loss to a relatively small degree of antibody binding activity. Protein yield indicated that relatively high amounts of protein were conserved throughout the conjugation procedure.

ADM release from Carbazone Immunoconjugate

ADM release rates from the carbazone immunoconjugate of the present invention at pH 4.5, 5.0 and 7.4 were also studied by HPLC analysis as described above in Example 6 for derived from

WMD of the present invention. As shown in Figure 13, the

The rate of release of ADM from the immunoconjugate is essentially the same as indicated in Example 6 for the carbazone derivative of ADM. The amount of material released from the ADM immunoconjugate increased as the pH decreased from 7 to 4. This ADM-containing immunoconjugate has an acid-sensitive linking group that results in the release of ADM from the antibody protein. These results are consistent with the existence of a hydrazone bridge that joins ADM to the bifunctional compound.

The experimental data presented herein indicate that an ADM radical is released from the immunoconjugates of the present invention under physiological conditions, that is, characteristic acid conditions of the lysosomal medium.

Cytotoxic activity of immunoconjugates

The cytotoxicity of the immunoconjugates of the present invention was determined according to in vitro assays using the Soft Agar Colony Formation Assay using Daudi cells (Burtkitt's lymphoma) (phenotype: 5E9⁺, ATCC No. &amp; HB21), as described by Greenfield et al., European Patent Application No. 328,347. supra. Daudi cells were grown in complete medium (RPMI 1640 medium plus 10% fetal calf serum (FCS)). 1 X 10 6 cells in 1 ml medium were exposed for 1.5 hours to serially diluted 5E9-ADM or unconjugated ADM. For each dilution, triplicate determinations were performed. Control cells consisted of similarly treated cells not exposed to drugs. The cells were then washed and resuspended in RPMI 1640 medium containing 15% FBS and 0.3% agarose (Marine Colloid, Rockland, ME). Then 1 ml of the

<img file="PT97639B_D0035.tif" />

cell suspension (1 X 10) over a 0.4% agarose layer in 6-well microtiter plates (Costar, Cambridge, MA). Samples were incubated for 7-10 days at 37 ° C and the resulting colonies stained with 0.5 ml of 1 mg / ml β -iodonitrotetrazolium violet (Sigma Chemical Co., St. Louis, MO). 48 hours Colonies were counted using an Optimax 40-10 image analyzer and inhibition of colony formation was determined by comparing drug-treated or immunoconjugate-treated cells with untreated control cells. The results are shown in Table 2 below as CI_3Q (the concentration required to inhibit colony formation by 50%).

TABLE 2

<td>5E9 immunoconjugate</td><td>^CI5.0</td><td>(Μ) ^&amp;</td>

<td>Semicarbazone</td><td>&gt; 5.1X</td><td> 10’⁷</td>

<td>Carbazone</td><td>4.0X</td><td>IO '⁷</td>

<td>Uncle Semicarbazone</td><td>3.0X</td><td>io '⁷</td>

<td>Carboxylate Hydrazone</td><td>5.9X</td><td>io '⁷</td>

<td>Arylhydrazone</td><td>&gt; 6.4 X</td><td>io^-7</td>

The_M = molar concentration of immunoconjugate required to inhibit colony formation by 50% after 24 hours.

As shown in Table 2, in addition to the release of

ADM, all pH sensitive immunoconjugates have considerable cytotoxic activity in vitro.

EXAMPLE 9

-71Ζ

Preparation of an Anthracycline Conjugated Immuno Containing a Thio-Ether Link

In this example, an alternative means for the preparation of an anthracycline immunoconjugate according to the present invention is described, wherein ADM is conjugated to a monoclonal antibody by one of the ADM derivatives according to the present invention prepared as described above in Examples 1 to 5 and having any of the following five points: semicarbazone, carbazone, thiosemicarbazone, carboxylate hydrazone and aryl hirazone, as a binding to the ADM molecule. In addition, the immunoconjugate has a thioether bond as part of its antibody binding.

MAb 5E9 (2.5 mg in 2.5 ml PBS) was reacted with succinimidyl 4- (p-maleimidophenyl) butyrate SNPB, 54.5 µg in 100 µl tetrahydrofuran) at 30 ° C. ° C for 30 minutes. The pH was adjusted to 6.0 with sodium citrate buffer. The mixture was passed through a PD-10 gel filtration column (soft Phar) to separate maleimide-containing antibody from unreacted products. The ADM derivatives (1 mg) prepared as above were then dissolved in 1 ml MeOH / E 2 O (9: 1) and 0.5 pmole of each derivative was reacted. of ADM with 0.5 æmol tri-n-butyl phosphine in a 4: 1 acetone / water mixture to prepare the reduced form of the ADM derivative. After 10 minutes, 0.1 N sulfur in toluene was added to destroy the remaining phosphine. The reduced ADM derivatives with MAb 5E9 containing maleimide were then mixed. The immunoconjugates thus produced were purified by passing them through a

-72gel PD-10. Where not completely removed, the toluene solvent removal and a layer of organic solvent separates, floating some proteins from the reaction mixture, a gentle blast of air is used to remove the solvent and the denatured proteins by spinning the mixture for 2 minutes at 16,000 Xg. The clear supernatant contains the immunoconjugates and is filtered through gel and analyzed in PBS at a pH of 7.4. The ADM / antibody ratio spectrophotometrically was determined using ^DG280 and ^d4,957 as mentioned above. A characteristic reaction provides immunoconjugates with molar ratios ranging from 3 to 4.

Binding activity and cytotoxic activity of immunoconjugates prepared as described above in this example were assayed as above.

The foregoing examples have demonstrated the preparation of novel N-substituted hydrazine bifunctional compounds, novel ADM N-substituted hydrazone rivals prepared with these bifunctional compounds and novel immunoconjugates in which ADM is conjugated to an antibody by novel acid sensitive binding . The novel bifunctional compounds are readily conjugable to a cytotoxic reagent, ADM and a cell targeting molecule, a monoclonal antibody. Conjugates retained both the binding activity (i.e. the specificity to target a particular cell population) and the cytotoxic activity of the drug and released free and unmodified ADM, under acidic media conditions, characteristic of the cell media. target cells.

Thus, the novel bifunctional immune conjugate compounds of the present invention have shown promise in

<img file="PT97639B_D0036.tif" />

conjugation of molecules, especially useful for providing cytotoxic drugs to a target cell population, for the purpose of preferentially annihilating such cells in the treatment of diseases such as cancers and other tumors, non-cytocidal viral infections and other pathogenic and other infections. in autoimmune disorders.

While several aspects of the present invention have been presented above, it is apparent that their basic description may be altered to provide other aspects in which the bifunctional compounds, cytotoxic reagent derivatives, conjugates and methods of the present invention may be used. .

However, it is to be understood that the scope of the present invention is essentially defined by the appended claims and not by the specifics set forth above by way of example only.