US 2003.0185793A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0185793 A1 Kratz (43) Pub. Date: Oct. 2, 2003

(54) THERAPEUTIC AND DIAGNOSTIC LIGAND (30) Foreign Application Priority Data SYSTEMS COMPRISING TRANSPORT MOLECULE BINDING PROPERTIES AND Mar. 13, 2000 (DE)...... 1OO12120.9 MEDICAMENTS CONTAINING THE SAME Publication Classification (76) Inventor: Felix Kratz, Ihringen (DE) (51) Int. Cl." ...... A61K 38/19; A61K 38/14; A61K 38/16 Correspondence Address: (52) U.S. Cl...... 424/85.1; 514/2; 514/6; 514/8; WEINGARTEN, SCHURGIN, GAGNEBIN & 514/27; 514/44; 530/350; 530/351; LEBOVICI LLP 530/322; 530/391.1 TEN POST OFFICE SQUARE BOSTON, MA 02109 (US) (57) ABSTRACT The invention relates to transport molecule binding ligand (21) Appl. No.: 10/221,544 compounds which comprise a therapeutically and/or diag nostically active Substance and a carrier molecule-affine (22) PCT Filed: Mar. 13, 2001 Substance with a high association constant to the carrier molecule. The invention also relates to medicaments con (86) PCT No.: PCT/EP01/02833 taining these ligand compounds and to diagnostic kits. US 2003/0185793 A1 Oct. 2, 2003

THERAPEUTIC AND DIAGNOSTIC LIGAND action (for example, as a cytostatic agent and as an anti SYSTEMS COMPRISING TRANSPORT rheumatic agent etc.). The term "diagnostically active Sub MOLECULE BINDING PROPERTIES AND stance' means that the respective Substance is detectable, MEDCAMENTS CONTAINING THE SAME preferably also quantifiable, in the organism or parts thereof, Such as for example cells and/or fluids, Such as for example DESCRIPTION Serum, by means of Suitable chemical and/or physical mea Surement methods. 0001. The present invention relates to carrier molecule binding ligand compounds comprising a therapeutically 0009. The carrier-molecule-affinitive substance in the and/or diagnostically active Substance and a carrier-mol carrier-molecule-binding ligand compound according to the ecule-affinitive Substance with a high asSociation constant invention has no covalent interaction with the carrier mol relative to the carrier molecule, as well as pharmaceutical ecule, i.e., the carrier-molecule-affinitive Substance binds to products and diagnostic kits containing these ligand com the carrier molecule on the basis of interactions of physical pounds. nature Such as electroStatic interactions, hydrogen bonds, van der Waals bonds, and/or hydrophobic interactions. 0002. A number of transport proteins for lipids, hor Moreover, with regard to the Specificity of the carrier mones, metabolites, pharmaceutical drugs, and Vitamins molecule-affinitive Substance (in respect) to the carrier mol circulate in the bloodstream. Examples of Such transport ecule, it is crucial that the equilibrium constant of the proteins are albumin, haptoglobin, prealbumin, low-density asSociation reaction between the carrier molecule and the lipoprotein (LDL), retinol-binding protein, transcortin, Vita carrier-molecule-affinitive Substance be Sufficiently high, min D-binding protein, or transcobalamin. The affinity which according to the invention corresponds to a value of between the transport protein and its ligand is described in >10M (log K-3), preferably at least 10'M' (log K at particular by means of the association constant KA. least 4), more preferably >10 M' (log Ka-5), even more 0003. It is known that a number of compounds, such as preferably >107M (log K-7) at most preferably >108 for example dyes, porphyrins, organic acids or pharmaceu M (log KA>8). In the ligand compound according to the tical drugs, enter into a distinct noncovalent, i.e., physical invention, the therapeutically and/or diagnostically active interaction with certain transport proteins in the blood Substance may be bound directly to the carrier-molecule Stream, whereby a Selectivity exists for certain transport affinitive substance. In a preferred embodiment of the ligand proteins, for example for albumin. compound according to the invention, the linkage between the therapeutically and/or diagnostically active Substance 0004 For a number of blood proteins it is further known and the carrier-molecule-affinitive Substance either is not that they accumulate in pathogenic tissue, for example in cleavable or this linkage is pH-dependent and/or enzymati malignant or inflamed tissue. For example, Such accumula cally cleavable, within the body. tion has been demonstrated for the Serum protein albumin (Kratz, F., Beyer U. (1998) Serum proteins as drug carriers 0010. In a further preferred embodiment of the ligand of anticancer agents, a review. Drug Delivery, 5, 1-19). compound according to the invention, the therapeutically and/or diagnostically active Substance and the carrier-mol 0005 The technical problem underlying the present ecule-affinitive Substance are bound to each other through a invention is to therapeutically and diagnostically utilize the Spacer molecule. Moreover, as already explained above for above-indicated carrier-molecule-binding properties, Such direct linkage between the therapeutically and/or diagnosti as for example binding to transport proteins in the blood cally active Substance and the carrier-molecule-affinitive Stream due to distinct physical interactions, and to provide Substance, the Spacer molecule and/or the linkage between a new ligand System with carrier-molecule-binding proper the therapeutically and/or diagnostically active Substance ties and pharmaceutical products containing these carrier and the Spacer molecule and/or the linkage of the carrier molecule-binding compounds. molecule-affinitive Substance and the Spacer molecule either 0006 The solution to this technical problem is achieved is uncleavable or the Spacer molecule and/or the indicated by means of the embodiments of the present invention as linkages can be pH-dependent and/or enzymatically cleav characterized in the claims. able, within the body. 0007. In particular, a carrier-molecule-binding ligand 0011. According to a further preferred embodiment, the compound is provided that comprises at least one therapeu direct linkage between the therapeutically and/or diagnosti tically or pharmaceutically and/or diagnostically active Sub cally active Substance and the carrier-molecule-affinitive stance and at least one carrier-molecule-affinitive Substance, Substance or the Spacer molecule and/or the linkage between having an association or binding constant KA relative to the the therapeutically and/or diagnostically active Substance carrier molecule of >10 M', preferably >10 M', even and the Spacer molecule and/or the linkage between the more preferably >107 M', most preferably >108 MT', carrier-molecule-affinitive Substance and the Spacer mol through a noncovalent bond. ecule contains at least one acid-labile bond. Examples of Such acid-labile bonds are ester, acetal, ketal, imine, hydra 0008. The term “therapeutically or pharmaceutically Zone, carboxylhydraZone, or SulfonylhydraZone compounds. active Substance' means that the respective Substance, either itself or after it is metabolized in the respective organism, 0012. According to a further embodiment of the ligand has a pharmacological effect, and thus the term also com compound according to the invention, either the direct prises the derivatives resulting from these conversions. Of linkage between the therapeutically and/or diagnostically course, the therapeutically active Substance can have a single active Substance and the carrier-molecule-affinitive Sub pharmacological spectrum of action (for example, only as a stance or the Spacer molecule and/or the linkage between the cytostatic agent) or a broad pharmacological spectrum of therapeutically and/or diagnostically active Substance and US 2003/0185793 A1 Oct. 2, 2003 the Spacer molecule and/or the linkage between the carrier 0016. The term “carrier molecule” comprises both natu molecule-affinitive Substance and the Spacer molecule con ral and Synthetic molecules that are Suitable for transporting tains at least one peptide bond. The peptide bond preferably ligand compounds according to the invention, for example is located within a peptide Sequence which contains at least in body fluids such as blood serum. Suitable transport one protease cleavage Sequence. Hence at least one peptide molecules are naturally occurring or Synthetic macromol bond can be realized by addition of a peptide Sequence into ecules, for example polyethylene glycol (PEG) or dextran, the direct linkage between the therapeutically and/or diag and biological macromolecules Such as proteins. Preferred nostically active Substance and the carrier-molecule-affini proteins that are Suitable as carrier molecules are Selected tive Substance or into the Spacer molecule and/or into the from the group consisting of Serum proteins. Albumin is an linkage between the therapeutically and/or diagnostically example of a preferred Suitable Serum protein. Further active Substance and the Spacer molecule and/or into the linkage between the carrier-molecule-affinitive Substance transport proteins Suitable as carrier molecules are transfer and the Spacer molecule, i.e., the respective linkage is a rin, haptoglobin, prealbumin, low-density lipoprotein peptide bond, and preferably consists of about 1 to 30 amino (LDL), retinol-binding protein, transcortin, as well as Vita acids. The peptide Sequence is preferably tailored to the min D-binding protein or transcobalamin. Substrate Specificity of certain endogenous enzymes or enzymes that occur in microorganisms or are formed there 0017 Suitable transport molecules are therefore naturally from. As a result, the peptide Sequence or a portion of this occurring or Synthetic macromolecules, for example Sequence is recognized by enzymes within the body and the polysaccharides, polypeptides, polyalcohols, polyamines, peptide is cleaved. dendrimers, copolymers, polyethylene glycol (PEG) or functionalized polyethylene glycols, and biological macro 0013 The enzymes are, for example, proteases and pep tidases, for example matrix metalloproteases (MMP1-20), molecules Such as proteins. cysteine proteases (for example cathepsin B, D, L, H), 0018. According to a preferred embodiment, the thera aspartyl proteases (for example cathepsin D), Serine pro teases (for example plasmin, “tissue-type' plasminogen peutically and/or diagnostically active Substance is a cyto activator (tPA), “urokinase-type' plasminogen activator), Static agent, a cytokine, an immunosuppreSSant, an antirheu kallikrein or kallikrein-like proteases (for example prostate matic, an antiinflammatory, an antibiotic, an analgesic, a Specific antigen), that in diseases Such as rheumatoid arthri Virostatic or an antimycotic. Especially Suitable cytostatics tis or cancer are formed to an increased extent or are for the ligand compound of the present invention are the activated, which leads to excessive tissue decomposition, to N-nitroSoureas Such as nimustine, the anthracyclines doxo inflammations, and to metastasizing. Target enzymes are in rubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone particular MMP-2, MMP-3 and MMP-9, cathepsins B, D, H and ametantrone as well as related derivatives, the alkylating and L, tissue-type plasminogen activator (tPA) and uroki agents chlorambucil, bendamustine, melphalan and oxaza nase-type plasminogen activator as well as prostate-specific phosphorines as well as related derivatives, the antimetabo antigen, which have been identified as key enzymes in lites, for example purine antagonists or pyrimidine antago inflammatory and malignant diseases and participate in the indicated pathological processes as proteases (Vassalli, J., nists and folic acid antagonistS Such as methotrexate, Pepper, M. S. (1994), Nature 370, 14-15; Brown, P. D. 5-fluorouracil, 5'-deoxy-5-fluorouridine, gemcitabines and 1995), Advan. Enzyme Regul. 35, 291-301; Schmitt M., et thioguanine as well as related derivatives, the taxaneSpacli al., J. Obstetrics & Gynaecology, 21, 151-65, 1995, T. T. Lah taxel and docetaxel as well as related derivatives, the camp et al. (1998), Biol. Chem. 379, 125-301). tothecines topotecan, irinotecan, 9-aminocamptothecine and camptothecine as well as related derivatives, the podophyl 0.014. In a further embodiment of the ligand compound lotoxin derivatives etoposide, teniposide and mitopodozide according to the invention, either the direct linkage between the therapeutically and/or diagnostically active Substance as well as related derivatives, the Vinca alkaloids vinblastine, and the carrier-molecule-affinitive Substance or the Spacer Vincristine, Vindesline, and Vinorelbine as well as related molecule and/or the linkage between the therapeutically derivatives, calicheamicins, maytansinoids, epithilones Such and/or diagnostically active Substance and the Spacer mol as epithilone A and B and related derivatives, and platinu ecule and/or the linkage between the carrier-molecule-af m(II) complex compounds in the cis configuration, of gen finitive Substance and the Spacer molecule contains at least eral formulas I to XIV: one bond that is enzymatically cleavable but consists not of a peptide bond. Examples are carbamate bonds, for which the active Substance or a derivative of the active Substance Formula I is released by cleavage with disease-specific enzymes, for example, glutathione S transferases, glucuronidases, galac N. c. tosidases. M Pt(No.

0.015 All three types of bonds-acid-labile bond, peptide Xt bond, enzymatically cleavable bond that does not contain a peptide bond-ensure that the therapeutically and/or diagnos Formula II tically active Substance or a corresponding active derivative is cleaved at the extracellular and/or intracellular Site of action and the Substance can exert its pharmaceutical and/or diagnostic effect. US 2003/0185793 A1 Oct. 2, 2003

-continued -continued Formula III Formula XIII NH y O 2 P-c / HN3 p NH2 X HN > Pt Yo-cXO-x Formula IV O O O-C H3NN / -Pt X HN Y-c Formula XIV O O Formula V N y - C O

| N N p-g (Dr.(O-C XO-x ( N Dr.( X O O-C

O Formula VI 0019 wherein X is the spacer molecule and/or the trans O port molecule-affinitive Substance. O-C 0020 Particularly suitable cytokines in ligand com H3NN / pounds of the present invention are, for example, interleukin HN -Pt Y, X 2, interferon C-2a, interferon B-2b, interferon C-1a, inter Formula VII feron B-1b, interferon Y-1b and related derivatives. The O cytokines used are, for example, genetically engineered | pharmaceutical products. N p-g 0021 Particularly suitable immunosuppressants in ligand ( DP X compounds of the present invention are, for example, N Y, cyclosporin A, FK506 (tacrolimus) and related derivatives. NH2 N 0022 Particularly suitable antirheumatics in conjugates of the present invention are, for example, methotrexate, ( N O NH2 SulfaSalazine, chloroquine and related derivatives. Formula IX 0023 Particularly suitable antiinflammatories and/or analgesics in ligand compounds of the present invention are, H3NN /N for example, Salicylic acid derivatives, Such as acetylsali Pt cylic acid and related derivatives, pharmaceutical drug H.N1 Y-1-x derivatives having an acetic acid or propionic acid group Formula X Such as diclofenac or indomethacin or ibuprofen or naproxen, and aminophenol derivatives Such as paraceta OS mol. (Dr.( C 0024 Particularly suitable antimycotics in ligand com N1 Y-1-x pounds of the present invention are, for example, amphot Formula XI ericin B and related derivatives. O 0025 Preferred virostatics in ligand compounds of the O-C H3NN / present invention are, for example, nucleoside analogS Such -Pt as aciclovir, ganciclovir, idoxuridine, ribavirin, Vidaribine, HN Y, / \ zidovudine, didanosine and 2',3'-dideoxycytidine (ddC) and SS related compounds, as well as amantadine. Formula XII 0026 Preferred antibiotics in the ligand compound O according to the invention are Sulfonamides, for example Sulfanilamide, Sulfacarbamide and Sulfametoxydiazine and N p-c related derivatives, penicillins, for example 6-aminopenicil (DP lanic acid, penicillin G, as well as penicillin V and related N Y, / \ derivatives, isoxazoyl penicillins Such as oxacillin, cloxacil SC lin and flucloxacillin as well as related derivatives, C-Sub Stituted benzylpenicillins Such as amplicillin, carbenicillin, US 2003/0185793 A1 Oct. 2, 2003 pivampicillin, amoxicillin and related derivatives, acylami are hydrophilic groups Such as Sulfonic acid (including Salts nopenicillins, for example meZlocillin, azlocillin, piperacil thereof with alkali metals or alkaline earth metals), carboxyl lin, apalcillin and related derivatives, amidino penicillins, (including salts thereof with alkali metals or alkaline earth for example mecillinam, a typical B-lactams Such as imipe metals), amino, aminoalkyl, and hydroxy groups. nam and aztreonam, cephalosporins, for example cefalexin, cefradine, cefaclor, cefadroxil, cefixime, cefpodoxime, cefa 0029 Water solubility of the ligand compounds accord Zolin, cefazedone, cefuroxime, cefamandol, cefotiam, ing to the invention can also be achieved or improved by the cefoxitin, cefotetan, cefinetazole, latamoxef, cefotaxime, carrier-molecule-affinitive Substance itself having one or ceftriaxone, ceftizoxime, cefinenoXime, ceftazidime, cefSu more water-Soluble groups, for example a Sulfonic acid lodin and cefoperaZone as well as related derivatives, tetra (including salts thereof with alkali metals or alkaline earth cyclines Such as tetracycline, chlortetracycline, Oxytetracy metals), carboxylic acid group (including Salts thereof with cline, demeclocycline, rollitetracycline, doxycycline, alkali metals or alkaline earth metals), amino, aminoalkyl, minocycline and related derivatives, chloramphenicols Such and/or hydroxy groups, or by introducing Such groups in as chloramphenicol and thiamphenicol as well as related Synthesis Steps. derivatives, gyrase inhibitors, for example nalixidic acid, pipemidic acid, norfloxacin, ofloxacin, ciprofloxacin and 0030 Preferred diagnostically active substances of the enoxacin as well as related derivatives, and antituberculous ligand compound according to the invention contain, for agents Such as isoniazid and related derivatives. example, one or more radionuclides, ligands comprising one 0.027 Of course, a single therapeutic and/or diagnostic or more radionuclides, preferably ligands complexing Such Species (for example, a therapeutic agent with a cytostatic as radionuclides, one or more positron emitters, one or more the therapeutically active Substance) or different therapeutic NMR contrast media, one or more fluorescent compound(s), and/or diagnostic species (for example, Several different or one or more contrast media in the near IR region. cytostatics or a cytostatic and an antirheumatic etc. as the therapeutically active Substance) can be present bonded in 0031. The ligand compound according to the invention, the ligand compound according to the invention per mol. containing at least one therapeutically and/or diagnostically active Substance (WS), at least one carrier-molecule-affini 0028. In a further preferred embodiment of the ligand tive Substance (TAS), and optionally a spacer molecule compound according to the invention, the Spacer molecule (SM), can be prepared according to one of the following comprises a Substituted or unsubstituted, branched-chain or general descriptions, depending on the functional group unbranched-chain aliphatic alkyl residue and/or at least one present. Substituted or unsubstituted aryl residue. The aliphatic alkyl residue preferably contains 1 to 20 carbon atoms, which can 0032. Therapeutically and/or diagnostically active sub be partially replaced by oxygen or nitrogen atoms, for stances for the ligand compounds according to the invention example to increase the water Solubility, wherein Such that have one HOOC group can, for example, be derivatized residues are preferably derived from an oligoethylene oxide as follows:

O COOH + HO-smHTAs s (ws –-o-si TAS

'optional or oligopropylene-oxide chain. Particularly Suitable residues 0033 Esterification is carried out in this case by proce that are derived from oligoethylene oxide or oligopropylene dures well known in the prior art. oxide chains comprise, for example, diethylene glycol, 0034). It is possible in addition to convert the HOOC triethylene glycol, tetraethylene glycol, pentaethylene gly group to a hydrazide group, for example by reaction with col, hexaethylene glycol, heptaethylene glycol, and octaeth tert-alkylcarbazates followed by cleavage with acids (see ylene glycol chains as well as analogous oligopropylene DE-A-196 36 889), and to react the compound having a glycol chains. A preferred aryl residue is an unsubstituted or hydrazide group with a group containing a carbonyl com Substituted phenyl residue, in which likewise one or more ponent, consisting of the transport molecule-affinitive Sub carbon atoms can be replaced by heteroatoms. Preferred stance and the Spacer molecule, as described inter alia in Substituents of the aliphatic alkyl residue or the aryl residue DE-A-19636 889:

O O O R C-NH-NH + R–HS HAs) s (ws –-N-N= SM.' TAS US 2003/0185793 A1 Oct. 2, 2003

-continued R = H, alkyl, phenyl, substituted phenyl 3: optional.

0035. Therapeutically and/or diagnostically active sub 0040. The conversion to the carboxyhydrazone, Sulfonyl stances for the ligand compounds according to the invention hydrazone, hydrazone, or imine derivatives is carried out in that have one HN group can, for example, be derivatized as follows: this case by procedures well known in the prior art.

Nii, R--MHAs)=(ws-N=-sMO R HAs R = H, alkyl, phenyl, substituted phenyl 'optional

0.036 The reaction to form the imine derivatives is car 0041 Ligand compounds according to the invention that ried out in this case by procedures well known in the prior contain a peptide bond can, for example, be prepared by art. reacting a peptide, consisting of 2 to about 30 amino acids, 0037. Therapeutically and/or diagnostically active sub with a carrier-molecule-affinitive compound, So that a car stances for the ligand compounds according to the-invention rier-molecule-affinitive compound is introduced directly or that have one HO-group can, for example, be derivatized through a Spacer molecule at the N-terminal end of the as follows: peptide.

on to--sO HAs) - (ws-o-HsuO Has 3: optional.

0.038 Esterification is carried out in this case by proce 0042. The peptide derivatives obtained in this way, dures well known in the prior art. together with the therapeutic agent and/or diagnostic agent 0.039 Therapeutically and/or diagnostically active sub or derivatives thereof having an HN- or HO-group, can stances for the ligand compounds according to the invention be converted to the corresponding ligand compounds that have one carbonyl component can, for example, be according to the invention in the presence of a condensation derivatized as follows: agent Such as for example N,N'-dicyclohexylcarbodiimide

Z. O Z. O (ws – =o -- is-ni- SM.' TAS l=s-N-Hsi" TAS Z. O Z. O =o -- is-ni- SM.' TAS = -Ni--st TAS

Z. =o + H-N-NH-SMHTAs s (ws---si-sy TAS Z. Z.

... al --- TAS Z = chemical group of the therapeutically and/or diagnostically active substance * optional. . US 2003/0185793 A1 Oct. 2, 2003

(DCC), N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide Ser-Tyr-Tyr-Ser-Gly- are known that are cleaved between P1 metho-p-toluenesulfonate (CMC), (benzotriazol-1-yloxy)- and P1" by PSA (Yang et al., J. Peptide Res. 54. 444-448, trispyrrolidinophosphonium hexafluorophosphate (pyBOP) 1999; Denmeade et al., Cancer Res. 57, 4924-4930, 1997; O O-benzotriazole-N,N,N',N'-tetramethyluronium Coombs et al., Chemistry & Biology 5, 475-488, 1998). hexafluorophosphate, and optionally with addition of a hydroxy compound Such as for example N-hydroxySuccin 0048. Furthermore, Substrate-specific dipeptides are imide, a water Soluble N-hydroxySuccinimide Such as for known for cathepsin with the Sequence -Arg-Arg-, -Phe example N-hydroxySuccinimide-3-sulfonic acid Sodium Salt Lys-, Gly-Phe-Leu-Gly, Gly-Phe-Ala-Leu or Ala-Leu-Ala or 1-hydroxybenzotriazole, and/or in the presence of a base Leu (Werle, B., Ebert, E., Klein, W., Spiess, E. (1995), Biol. Such as for example N-methylmorpholine or triethylamine: Chem. Hoppe-Seyler 376, 157-164; Ulricht, B., Spiess, E.,

O WS NH2 + HOOC-peptide SMHTAS) = (wsHNi---Hsu HAs) O

al NH-C WS NH2 + HOOC-peptide-TAS) re O WS OH -- HOOC-peptide SMHTAs) = - (ws HO--Peptide SMHTAS) O WS OH -- HOOC-peptide-TAS) o 'optional

0043. The substrate specificity of the target enzymes, Schwartz-Albiez, R., Ebert, W. (1995), Biol. Chem. Hoppe Such as for example MMP-2, MMP-3, MMP-9, cathepsin B, Seyler 376, 404-414). D, L and H, is well known (Netzel-Arnett et al. (1993), 0049. The peptide sequence containing the peptide cleav Biochemistry 32, 6427-6432, Shuja, S., Sheahan, K., Mur age site or predetermined breaking point relevant for the name, M. J. (1991), Int. J. Cancer 49,341-346, Lah, T. T., target enzyme can also be constructed So that the peptide Kos, J. (1998), Biol. Chem. 379,125-130). cleavage Site is multiply repeated, Such as for example by:

-Gly-Pro-Leu-Gly--IIe-Ala-Gly-Gln-Gly-Pro-Leu-Gly--Ile-Ala-Gly-Gln or -Phe-Lys-Phe-Lys-Phe-Lys-Phe-Lys-Phe-Lys-Phe-Lys

0044) For example, octapeptides (P-P) have been iden 0050 or a repeating peptide sequence can be integrated tified for MMP-2 and MMP-9 (see Table 1) which simulate that increases the distance between the thiol-binding groups the cleavage Sequence of the collagen chain, and are par and the relevant peptide cleavage Sites, Such as for example ticularly efficiently cleaved by MMP-2 and MMP-9: by:

TABLE 1. 0051) -(Gly)-Phe-Lys-Phe-Lys -Peptide 0052 with preferably n=2 to 20, more preferably ns 12, P. P. P., P, P, P', P, P. 0053 or by an oxyethylene glycol unit as a water-soluble Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln component Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln 0054 -(O-CH-CH-)-Phe-Lys-Phe-Lys Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg 0055) with preferably n=1 to 8. 0056 An important feature of this embodiment of the ligand compound according to the invention is the fact that 0045 (Netzel-Arnett et al., Biochemistry 32, 1993, 6427 the peptide cleavage Site relevant for the respective target 6432) enzyme occurs at least once in an oligopeptide consisting of 0046) The peptides are enzymatically cleaved exclusively about 1 to 30 amino acids. at the P-P'-bond. 0057 The oligopeptides indicated above are representa 0047 Substrate-specific hexapeptide sequences (P4-P2) tive examples for the enzymatically cleavable bond in the are known for prostate-specific antigen (PSA), i.e., -His ligand compounds according to the invention. The proteases Ser-Ser-Lys-Leu-Gln-, ASn-Ser-Ser-Tyr-Phe-Gln- or -Ser indicated above are examples of disease-associated US 2003/0185793 A1 Oct. 2, 2003 enzymes. The therapeutic preparation can be independently binding behavior is maintained as compared with the used for other disease-associated proteases. unmodified compound with the carrier molecule. AS an 0.058. Therapeutic or diagnostic derivatives of the ligand example, a dye used as a carrier-molecule-affinitive Sub compounds according to the invention that contain a cytok stance, for example an azo dye, may be derivatized by the ine can, for example, be prepared by reacting the cytokine above-indicated chemical modification, for example by with a Spacer molecule-linked carrier-molecule-affinitive reduction of the azo group or by replacement of the azo Substance which has a carboxylic acid or an activated group by a C-C single bond or a C-C double bond, in carboxylic acid group: Such a way that it is no longer colored.

O

NH2 + R-C SM.' TAS --- cytokine-NH--SM TAS

O O NaOS R = HO-, N-O- N-O-

O O 3: optional.

0059. If the spacer molecule has an N-hydroxysuccinim 0061 Examples of carrier-molecule-affinitive substances ide ester group (N-hydroxySuccinimide or the Sodium Salt of in the ligand compound according to the invention are N-hydroxysuccinimide-3-sulfonic acid), then it is directly Selected from the following groups: reacted with the cytokine. The reaction of cytokine with a 0062) naphthalene and naphthyl derivatives: for example Spacer-linked carrier-molecule-affinitive Substance having a carboxylic acid group, to form the corresponding carrier 0063) 1,4,5,8-NAPHTHALENETETRACARBOXY molecule-affinitive cytokine derivatives, is carried out in the LIC ACID, NAPHTHOL, presence of a condensation agent Such as for example 0064) 1,1'-(1.5-NAPHTHALENEDIYL)BIS(3-CY N,N'-dicyclohexylcarbodiimide (DCC) or N-cyclohexyl-N'- CLOHEXYLUREA), (2-morpholinoethyl)-carbodiimide-metho-p-toluene 0065) 4-ACETAMIDO-5-HYDROXY-2.7-NAPH sulfonate (CMC), and optionally with addition of N-hydrox THALENE DISULFONIC ACID, ySuccinimide or the Sodium Salt of N-hydroxySuccinimide 3-Sulfonic acid. Cytokines derivatized in this way are 0066) 2-(2-HYDROXY-1-NAPHTHYLMETHYL purified, -for example, using Size-exclusion chromatogra ENEAMINO)BENZOIC ACID, phy. The conversions described above are well known in the 0067) 9-FLUORENYLIDENEAMINO N-(1-NAPH prior art (for example, see Bioconjugate Techniques, G. T. THYL)CARBAMATE, Hermanson, Academic Press, 1996). 0068. 2-(N-(1-NAPHTHYL)CARBAMOYL)CY 0060 Preferred carrier-molecule-affinitive substances in CLOHEXANECARBOXYLIC ACID, the ligand compound according to the invention are Selected 0069) 1,1'-(1.5-NAPHTHALENEDIYL)BIS(3-(3-PY from the group consisting of phthalocyanines, coumarins, RIDYLMETHYL)UREA), flavonoids, tetracyclines, naphthalenes, aryl- and heteroaryl carboxylic acids, lipids and fatty acids, for example long 0070) 1,1'-(1.5-NAPHTHALENEDIYL)BIS(3-BEN chain fatty acids Such as Co-Co fatty acids, cyclic or linear ZYLUREA), tetrapyrroles and organometallic compounds thereof, for 0071) 2,2'-OXYBIS(ACETIC ACID (2-HYDROXY example porphyrins and protoporphyrins (for example, 1-NAPHTHYLMETHYLENE)HYDRAZIDE), bilirubin and derivatives thereof, hematin and derivatives thereof, aromatic acid derivatives Substituted with 2-5 halo 0072 ADIPIC ACID BIS(1-NAPHTHYLMETHYL gen atoms (Cl, Br or 1) Such as iophenoxic acid, organic ENE)HYDRAZIDE), dyes, for example Evans blue and bromcreSol dyes Such as 0073) 3-(N-PHENYLCARBAMOYL)-2-NAPH bromcreSol green and bromcreSol purple, and the tryptophan THYLN-(5-CHLORO-2-METHOXYPHENYL)CAR and thyroxine analog compounds as well as derivatives of BAMATE, the above-indicated classes of compounds. Furthermore, the organic dyes used as carrier-molecule-affinitive Substances 0074 6-BENZOYL-2-NAPHTHYL PHOSPHATE, can be chemically modified or derivatized before or after SODIUM SALT, binding to the therapeutically and/or diagnostically active 0075). L-ARGININE 4-METHOXY-BETA-NAPH Substance or to the Spacer molecule, whereby however the THYLAMIDE HYDROCHLORIDE, US 2003/0185793 A1 Oct. 2, 2003

0076) 1-AMINO-8-NAPHTHOL-2,4-DISULFONIC 0097 2-HYDROXY-3-NAPHTHOIC ACID 2-AMI ACID, NOANTHRAQUINONYLAMIDE PHOSPHATE, 0077 FMOC-D-2-NAPHTHYLALANINE, LYS 0098 2-HYDROXY-3-NAPHTHOIC ACID 2-AMI ALA 4-METHOXY-BETA-NAPHTHYLAMIDE NOAZOBENZANILIDE PHOSPHATE, DIHYDROCHLORIDE, 0099) 3-(5-(2-CARBOXYETHANESULFONYL 0078) 4-(1,8-DIHYDROXY-3,6-DISULFO-2-NAPH )NAPHTHALENE-1-SULFONYL) PROPIONIC THYLAZO)-SALICYLIC ACID, ACID, 0079) 1,1'-HEXAMETHYLENEBIS(3-(1-NAPHTH 01.00) 4-(4-CL-PHENYLIMINO)-ME)-3-HO YLMETHYL)UREA, NAPHTHALENE-2-CARBOXYLIC ACID (2-MEO PH)-AMIDE, 0080 CARBOBENZYLOXYTRYPTOPHAN BETA NAPHTHYLAMIDE, 01.01 4-HYDROXY-7-METHOXY-1-(3-METHOXY PHENYL)-NAPHTHALENE-2-CARBOXYLIC 0081) 3-(N-(2-METHOXYPHENYL)CARBAM ACID, OYL)-2-NAPHTHYL N-(5-CHLORO-2-METHOX YPHENYL)CARBAMATE, 0102) 3,6-DISULFO-NAPHTHALENE-1,8-DICAR BOXYLIC ACID, L-ALA-ALA-L-PHE-ALPHA 0082) 2-(1-NAPHTHYL)-2-PHENYLACETIC ACID, NAPHTHYLAMIDE, 8-(2-CARBOXYETHYL)THIO-1-NAPHTHOIC ACID, 0103 CBZ-L-TH R-L-VAL-BETA-NAPHTHYLA MIDE, CBZ-GLY-L-VAL-BETA-NAPHTHYLA 0083) 3-1-(2-CARBOXYETHYL)-2-OXO-1,2-DI MIDE, HYDROACENAPHTHYLEN-1-YLPROPANOIC ACID, 3-(1-NAPHTHYLAMINO)CARBONYLBI 0104 CBZ-ILE-L-ALA-BETA-NAPHTHYLAM CYCLO2.2.1]-HEPT-5-ENE-2-CARBOXYLIC IDE, CBZ-L-ALA-THR-BETA-NAPHTHYLAMIDE, ACID, 01.05) DISODIUM 4-(BENZOYLAMINO)-5-ME 0084) N"N"-DIL1-(2-NAPHTHYL)ETHYLIDENE) THYL-2,7-NAPHTHALENEDISULFONATE, CARBONIC ACID DIHYDRAZIDE, 01.06 NAPHTHALENE-14,8-TRICARBOXYLIC 0085) 4-4-(2-NAPHTHYL)-1,3-THIAZOL-2-YL ACID, AMINO-4-OXOBUTANOIC ACID, 01.07 2-[2-(1-NAPHTHYL)ACETYLAMINO) SUCCINIC ACID, 0086) FMOC-L-2-NAPHTHYLALANINE, 1,4,5,8- NAPHTHALENETETRACARBOXYLIC ACID 01.08 (10-HO-10-PH-4H-8-THIA-7, 10A-DIAZA HYDRATE, PENTALENO(1.2-A)NAPHTHALEN-9-YL)ACETIC ACID, 0087 BIS(2-NAPHTHYL) N,N'-(4-METHYL-1,3- PHENYLENE)BISCARBAMATE, 01.09) 1-(NAPHTHALENE-2-CARBONYL)-3- NAPHTHALEN-2-YLUREA, 0088 8-HYDROXY-5-(1-NAPHTHYLAZO)-2- NAPHTHALENESULFONIC ACID, SODIUMSALT, 0110 1-(3-METHOXY-PHENYL)-3-(NAPHTHA LENE-1-CARBONYL)THIOUREA, (S)-N-(1-(1- 0089 N1,N'3-DI(1-NAPHTHYLMETHYLIDENE)- NAPHTHYL)ETHYL)SUCCINAMIC ACID, 2-BUTYLPROPANEDIOIC HYDRAZIDE, 0111 L-LYSYL-L-ALANINE-4-METHOXY-BETA 0090 (+/-)-1,1'-BINAPHTHYL-2,2'-DICARBOXY NAPHTHYLAMIDE HYDROBROMIDE, LIC ACID, 0112) 5-((2-(T-BOC)-GAMMA-GLUTAMYLAMI 0091 (+/-)-2,2'-DIMETHOXY-1,1'-BINAPHTHYL NOETHYL)AMINO)NAPHTHALENE-1-SUL 3,3'-DICARBOXAMIDE, FONIC ACID, 0092] 1-HYDROXY-4-(1-PHENYL-1H-TETRA 0113 8-(2-(ACETYLOXY)METHYLPHENYL) ZOL-5-YL-THIO)-2-NAPHTHOIC ACID, 3,3'- THIO)-1-NAPHTHOIC ACID, DIPHENYL-(1,1)BINAPHTHALENYL-2,2'-DICAR 0114) 2-(9H-FLUOREN-9-YLMETHOXY)CAR BOXYLIC ACID, BONYLAMINO)-3-(2-NAPHTHYL)PROPANOIC 0093) 2,6-DIMETHOXY-4-OXO-3,5-DIOXA-PHOS ACID, PHA-CYCLOHEPTA(2,1-A,3,4- 0115) I-NAPHTHYLACETYLSPERMINE TRIHY A)DINAPHTHALEN-4-OL, DROCHLORIDE, 0094) 4-((BR-4-ME-PHENYLIMINO)-ME)-3-HO 0116) 4-(2-ETHOXY-2-OXOETHYL)THIONAPH NAPHTHALENE-2-CARBOXYLIC ACID (2-MEO THALENE-1,8-DICARBOXYLIC ACID, FMOC-(R)- PH)AMIDE, 3-AMINO-4-(2-NAPHTHYL)BUTYRIC ACID, 2,2'- O095 8-(2-(METHYLAMINO)CARBONYLPHE BIOUINONYL DICARBOXYLIC ACID; NYLTHIO)-1-NAPHTHOIC ACID, 0.117) benzophenone derivatives: for example 0096) N-GLUTARYL-L-PHENYLALANINE-BETA 0118 BENZOPHENONE 2,4'-DICARBOXYLIC NAPHTHYLAMIDE, ACID, US 2003/0185793 A1 Oct. 2, 2003

0119) 3,3'44'-BENZOPHENONE TETRACAR 0.143 quinoline and isoquinoline derivatives: for BOXYLIC ACID, example 0120) 2,3,4-BENZOPHENONE TRICARBOXYLIC 0144) N,N'-BIS(8-QUINOLYLOXYCARBONYL)-4- ACID, METHYL-1,3-PHENYLENEDIAMINE, 3,3'-BIS(2- 0121) 2,2'-DIHYDROXY-4,4'-DIMETHOXY-5-SUL (2-QUINOLYL)VINYL)AZOBENZENE, FOBENZOPHENONE, 0145) 8-SULFO-2,4-QUINOLINEDICARBOXYLIC ACID, 0122) 4-N-2-(ACETAMIDO)ETHYL-N-METHY LAMINO-2'-CARBOXY-2-HYDROXYBEN 0146) 2-4-(ACETYLAMINO)PHENYL-7-METH ZOPHENONE; YLOUINOLINE-4-CARBOXYLIC ACID, 0123 phthalimide and isophthalimide derivatives and 0147 3-(1,3-DIOXO-1H,3H-BENZO(DE)ISO phthalic acid and isophthalic acid derivatives: for example QUINOLIN-2-YL)BENZOIC ACID, DISODIUM 3-METHYL-4-(QUINOLIN-2-YLMETHYLENE O124) 5-SULFOISOPHTHALIC ACID MONOSO )PENT-2-ENEDIOATE, DIUM SALT, 0148) 2-(5-CARBOXY-PENTYL)-3-PH-1-PR-5,6,7, 0125 2-PHENYL-3-PHTHALIMIDOQUINOLINE 8-TETRAHYDRO-ISOQUINOLINIUM, 4-CARBOXYLIC ACID, 0149) 1-(1H-INDOL-3-YL)-1H-ISOQUINOLINE-2- 0126 2-PHTHALIMIDOGLUTARIC ACID, CARBOXYLIC ACID PHENYLAMIDE, O127 2.5-BIS(2-HYDROXYETHYLAMINO)T. O150 1-ET-6-F-4-OXO-7-(4-(2-OXO-PR)-PIPER EREPHTHALIC ACID, AZIN-1-YL)-2H-QUINOLINE-3-CARBOXYLIC ACID, 0128 2- and/or 4-SULFOTEREPHTHALIC ACID, 0151. N-ETHYL-3-(PYRIDIN-2-YL)-4-(QUINO 0129 5-SULFOISOPHTHALIC ACID, LIN-4-YL)PYRAZOLE-1-CARBOXAMIDE, 0130 2.5-BIS(N-(2,5-XYLYL)CARBAMOYL 0152 2-4-(5-CHLORO-2-QUINOLYL)OXYPHE )TEREPHTHALIC ACID, NOXY PROPANOIC ACID, 0131) 4-(2-CARBOXYPHENYL)THIOJISOPH 0153) N2-(2-(3-CYANO-4-METHYL-2- THALIC ACID, QUINOLYL)THIOPHENYL-2-FURAMIDE, 0132) 4-(4-ACETYL-3-HYDROXY-2-PROPYLPHE 0154) 3-(1-1,1'-BIPHENYL-4-YL-1H-1,2,3,4-TET. NOXY)ISOPHTHALIC ACID, 2.4.6-TRIMETHYLA RAAZOL-5-YL)QUINOLIN-4(1H)-ONE, FMOC NILINE SALT, BETA-(2-QUINOLYL)-ALA-OH, 0133) 4-(N-(4-METHOXYPHENYL)CARBAM 0.155) N-BENZYL-2-(4-METHYL-5-QUINOLIN-6- OYL)ISOPHTHALIC ACID, YL-4H-1,2,4-TRIAZOL-3-YL)THIOJACETAMIDE, 0134) PHTHALIC ACID MONO-((2-CHLOROPHE 0156 FMOC-ALA(3'-QUINOYL)-OH, NYL)PHENYLMETHYL) ESTER, 015.7 FMOC-(S)-2-TETRAHYDROISOQCUINO 0135) 4-(4-(3,4-DIMETHYLPHENYLSULFANYL LINE ACETAMIDE; )BENZENESULFONYL)PHTHALIC ACID, 0158 anthraquinone derivatives: for example 0136) 4-(4-(4-CARBOXYPHENOXY)-BEN 0159) 1.8-BIS(BENZAMIDO)ANTHRAQUINONE, ZOYL)PHTHALIC ACID, 0160 N,N'-1,5-ANTHRAQUINONYLENE DIAN 0137) 4-(4-CHLORO-BENZOYL)PHTHALIC ACID, THRANILIC ACID; 0138 5-(5-(4-HYDROXYPHENYL)-3-PHENYL-4, 0.161 anthracene derivatives: for example 5-DIHYDROPYRAZOL-1-YL)ISOPHTHALIC 0162 2-(7-OXO-7H-BENZODE)ANTHRACEN-3- ACID, YLTHIO)ACETIC ACID, 0139 5-(5-(4-DIMETHYLAMINO-PH)-3-PHENYL 4.5-DIHYDRO-PYRAZOL-1-YL)ISOPHTHALIC 0163 9,10-DIHYDRO-9,10-DIOXO-2,3-AN ACID, THRACENEDICARBOXYLIC ACID, O140 PHTHALIC ACID MONO-(BIPHENYL-4-YL 0164) 9-HYDROXYIMINO-9,10-DIHYDRO-AN PHENYLMETHYL) ESTER, PHTHALIC ACID. THRACENE-1-CARBOXYLIC ACID; MONO-(1-METHYL-2.2-DIPHENYLETHYL) 0.165 phenanthrene and phenanthroline derivatives: for ESTER, example 0141 4.5-BIS-(4-AMINOPHENOXY)PHTHALIC 0166 1,10-PHENANTHROLINE-2,9-DICARBOXY ACID, LIC ACID, 0142. 4,5-DI(4-METHOXYPHENOXY)PHTHALIC 0167) 4-P-TOLYL-1,10-PHENANTHROLINE-29 ACID; DICARBOXYLIC ACID, US 2003/0185793 A1 Oct. 2, 2003

0168 1,2,3,4-TETRAHYDRO-PHENANTHRENE-1, 0188 2,6-BIS(4-ACETAMIDOBENZYLIDENE)-1- 2-DICARBOXYLIC ACID, CYCLOHEXANONE, 01.69 benzylidene derivatives: for example 0189 N-BENZOYLANTHRANILIC ACID (4-HY 0170) 2,2'-OXYDIACETIC ACID BIS((4-ACETAMI DROXY-3-METHOXYBENZYLIDENE)HY DOBENZYLIDENE) HYDRAZIDE), DRAZIDE, 0171 2-(FURFURYLUREIDO)ACETIC ACID 0.190) 4-(3-(3-CHLOROPHENYL)UREIDO)BEN N2-(4-HYDROXY-3-METHOXYBENZYLIDENE) ZOIC ACID (3-ETHOXY-4-HYDROXY-BEN HYDRAZIDE, ZYLIDENE)HYDRAZIDE, 0172 2-(3-(3,4-DICHLOROPHENYL)URE 0191) 4-(3-(3-CHLOROPHENYL)UREIDO)BEN IDO)ACETIC ACID (5-BROMO-2-HYDROXYBEN ZOIC ACID (4-ETHOXY-3-METHOXYBEN ZYLIDENE) HYDRAZIDE, ZYLIDENE)HYDRAZIDE, 0173 2-(3-(2-ETHOXYPHENYL)UREIDO)ACETIC 0192] 4-(3-(3-CHLOROPHENYL)UREIDO)BEN ACID (3,4-DIMETHOXYBENZYLIDENE) ZOIC ACID (2-CHLORO-4-(DIMETHYLAMI HYDRAZIDE, NO)BENZYLIDENE)HYDRAZIDE, 0174) 2-(3-(4-METHOXYPHENYL)UREIDO)ACE 0193 4-(3-(3-CHLOROPHENYL)UREIDO)BEN TIC ACID (2,3,4-TRIMETHOXYBENZYLIDENE ZOIC ACID (3.4-DICHLOROBENZYLIDENE)HY )HYDRAZIDE, DRAZIDE, 0175) 4-(3-(2-FLUOROPHENYL)UREIDO)BEN 0194 4-FLUOROBENZYL N-(4-METHOXYBEN ZOIC ACID (3.4-DICHLOROBENZYLIDENE)HY ZYLIDENE)-(ANILINOCARBONYL)AMINO) DRAZIDE, METHANEHYDRAZONO, 0176) 4-(3-(2-FLUOROPHENYL)UREIDO)BEN 0.195 2-((2-HO-BENZYLIDENE)-AMINO)-5-(N'-(2- ZOIC ACID (4-METHYLBENZYLIDENE)HY HO-BENZYLIDENE)-GUANIDINO)PENTANOIC DRAZIDE, ACID, 0177) 4-(3-(2-FLUOROPHENYL)UREIDO)BEN 0196) 2,3-BIS-(3-METHOXYBENZYLIDENE)SUC ZOIC ACID (4-METHOXYBENZYLIDENE)HY CINIC ACID, DRAZIDE, 0197) 2,3-BIS-(3,4-DIMETHOXYBENZYLIDENE 0178) 4-(3-(2-FLUOROPHENYL)UREIDO)BEN )SUCCINIC ACID, ZOIC ACID (4-HYDROXY-3-METHOXYBEN ZYLIDENE)HYDRAZIDE, 0198 2,3-BIS-(4-CHLOROBENZYLIDENE)SUC CINIC ACID, 0179) 4-(3-(2-FLUOROPHENYL)UREIDO)BEN ZOIC ACID (4-ACETAMIDOBENZYLIDENE)HY 0199 2,3-DIBENZYLIDENE SUCCINIC ACID, DRAZIDE, 0200 BENZYLIDENE CBZ-NEURAMINIC ACID, 0180 4-(3-(2-FLUOROPHENYL)UREIDO)BEN ZOIC ACID (3.4-DIMETHOXYBENZYLIDENE 0201 3-HYDROXY-2-((2-HYDROXYBENZYLIDE )HYDRAZIDE, NE)AMINO)-3-PHENYLPROPIONIC ACID, 0181 2-(2-CHLOROBENZAMIDO)BENZOIC 0202) 3-HO-3-(4-((2-HO-BENZYLIDENE)- ACID (2-CHLORO-4-(DIMETHYLAMINO)BEN AMINO)-PH)-2-PHENYLACETYLAMINOPROPI ZYLIDENE)HYDRAZIDE, ONIC ACID; 0182] 2-(3-(3-CHLOROPHENYL)UREIDO)ACETIC 0203 diphenyl and biphenyl derivatives: for example ACID (4-BUTOXYBENZYLIDENE)HYDRAZIDE, 0204) 3-CHLORO-4-BIPHENYLYLN-(2,5- 0183 2-(3-(6-METHYL-2-PYRIDYL)URE DIMETHOXYPHENYL)CARBAMATE, IDO)ACETIC ACID (3,4-DIMETHOXYBEN 0205) 2'-BENZOYL-2-BIPHENYLCARBOXYLIC ZYLIDENE)HYDRAZIDE, ACID, 0184] 2-(3-(2-ETHOXYPHENYL)UREIDO)ACETIC ACID (2-HYDROXYBENZYLIDENE)HY 0206. 2'-(4-FLUOROANILINO)CARBONYL DRAZIDE, 0207 (1,1'-BIPHENYL-2-CARBOXYLIC ACID, 0185. 2-(3-(2-ETHOXYPHENYL)UREIDO)ACETIC 0208) 3-(2,4,6-TRIMETHYLBENZOYL)-2-BIPHE ACID (4-ACETAMIDOBENZ-YLIDENE)HY NYLCARBOXYLIC ACID, DRAZIDE, 0209. 2'-BENZYLCARBAMOYLBIPHENYL-2- 0186 N1-(4-CHLOROBENZYLIDENE)-3-(5-(2,3- CARBOXYLIC ACID, DICHLOROPHENYL)-2H-1,2,3,4-TETRAAZOL-2- YL), 0210 3-CHLORO-3'-METHYLBIPHENYL-2,2'-DI 0187 N1-BENZYLIDENE-2-3-(4-METHYL-1,3- CARBOXYLIC ACID, THIAZOL-2-YL)METHYL-4-OXO-3,4-DIHYDRO 0211) 2-HO-5-(4-HO-BIPHENYL-4-YLAZO)BEN PHTHAL, ZOIC ACID, US 2003/0185793 A1 Oct. 2, 2003

0212 2-(1,1'-BIPHENYL-4-YLAMINO)CARBO 0235 2-((5-BROMO-1,3-DIOXO-1,3-DIHYDRO NYLBENZOIC ACID, ISOINDOL-2-YLMETHYL)AMINO)BENZOIC 0213 3-(1-1,1'-BIPHENYL-4-YL-1H-1,2,3,4-TET. ACID, RAAZOL-5-YL)QUINOLIN-4(1H)-ONE, 2-(3-1, 0236 3-(2-(2-CARBOXYBENZOYLAMINO)ET. 1'-BIPHENYL-4-YL-3-OXOPROPYLIDENE HYL)-5-CHLORO-1H-INDOLE-2-CARBOXYLIC )AMINOIOXY-N-(5-IODO-2- ACID, PYRIDINYL)ACETIC ACID, 0237) 3-PHENYL-PYRROLO(2.1.5-CD)INDOLIZ 0214) 4,4'-DI-BOC-DIAMINOBIPHENYL-2,2'-DI INE-1,2-DICARBOXYLIC ACID, CARBOXYLIC ACID, 0238 (RS)-FMOC-1,3-DIHYDRO-2H-ISOINDOLE 0215 2,3,2'-BIPHENYLTRICARBOXYLIC ACID, CARBOXYLIC ACID, 0216) 3-CHLORO-4-BIPHENYLYLN-(2,5- 0239) 1-(1H-INDOL-3-YL)-1H-ISOQUINOLINE-2- DIMETHOXYPHENYL)CARBAMATE, CARBOXYLIC ACID PHENYLAMIDE, 0217 4,4'-BIS(3-(3-PYRIDYL)UREIDO)DIPHE 0240 N'-2-[4-(DIMETHYLAMINO)PHENYLME NYLMETHANE, THYLENE-3-PHENYL-1H-INDOLE-2-CARBOHY DRAZIDE, 0218 4,4'-BIS(3-METHYL-3-PHENYLUREIDO )DIPHENYLMETHANE, 0241 INDOLE-3-ACETYL-DL-TRYPTOPHAN, 0242 2-(1,3-DIOXO-1,3-DIHYDRO-2H-ISOIN 0219) 4,4'-METHYLENEBIS(1,3-DIPHENYL-1- DOL-2-YL)-5-OXO-5-(4-TOLUIDINO)PENTANOIC ETHYLUREA), ACID, 0220) 2,2'-CARBOXYDIPHENYLSULFONE, 2,3- 0243) 4-(2-TERT-BUTYL-(1H)-INDOL-5- DIPHENYLSUCCINIC ACID, YL)AMINO)-1-(2-CHLOROPHENYL)CARBO 0221) 1,3-DIPHENYL-1-(2-MORPHOLINO-1-CY NYLPIPERIDINE, CLOPENTEN-1-YLCARBONYL)UREA, 0244) 2-(2-CHLOROBENZOYL)AMINO-N-2- 0222 2-BENZOYLAMINO-3-(DIPHENYLPHOS (1H-INDOL-3-YL)ETHYLBENZAMIDE, PHINOYL)-3-PHENYL-ACRYLIC ACID, MESO-2, 0245) 5-(4-CHLOROANILINO)-2-(1,3-DIOXO-1,3- 3-DIPHENYLSUCCINIC ACID, DIHYDRO-2H-ISOINDOL-2-YL)-5-OXOPEN 0223) 4,5-DIPHENYL-2,3-DIHYDRO-1H-PYRA TANOIC ACID, ZOLO3,4-CIPYRIDAZIN-3-ONE, 0246 CARBOBENZYLOXY-L-METHIONYL-L- 0224) 2-HYDROXY-2.2-DIPHENYLACETIC ACID TRYPTOPHAN, (1-(4-BROMOPHENYL)ETHYLIDENE)HY O247 CARBOBENZYLOXY-L-PHENYLALANYL DRAZIDE, L-TRYPTOPHANAMIDE, 0225 2.5-DIPHENYLFURAN-3,4-DICARBOXY 0248) N-ACETYL-5-BENZYLOXY-DL-TRYP LIC ACID, TOPHAN, CARBOBENZYLOXYTRYPTOPHAN 0226) 1-(2,3-DIPHENYLACRYLOYL)-3-(4-METH BETA-NAPHTHYLAMIDE, OXYPHENYL)-THIOUREA, N1-2-(1,3-DIPHE 0249) CARBOBENZYLOXYNORVALYLTRYP NYL-1H-4-PYRAZOLYL)-1-(HYDRAZINOCAR TOPHANAMIDE, L-NORLEUCYL-L-TRYP BONYL)VINYL-2,4-DICHLOROBENZOIC ACID, TOPHAN, 0227 N1-1-(BENZYLAMINO)CARBONYL-2-(1, 0250) CARBOBENZYLOXY-L-TRYPTOPHYL-L- 3-DIPHENYL-1H-PYRAZOL-4-YL)VINYLBEN PHENYLALANINAMIDE, CARBOBENZYLOXY ZAMIDE, L-TRYPTOPHYL-L-LEUCINAMIDE, 0228) N2-ACETYL-O6-(DIPHENYLCARBAM 0251 CARBOBENZYLOXY-L-TRYPTOPHYLGLY OYL)GUANINE; CYLGLYCINE METHYL ESTER, 0229) tryptophan and indole derivatives: for example 0252) N-BETA-FMOC-L-BETA-HOMOTRYP 0230) 5-BENZYLOXYINDOLE-3-ACETIC ACID, TOPHAN 0231 6-BENZAMIDO-N-(2-(3-INDOLYL)ETHYL 0253) indane derivatives: for example )HEXANAMIDE, N-(3-INDOLYLACETYL)-DL 0254) 4-SULFOBENZOIC ACID (5-INDANYLM ASPARTIC ACID, ETHYLENE)HYDRAZIDE, 0232 INDOLE-3-ACETYL-L-ASPARTIC ACID, 0255 PHENYLINDANE DICARBOXYLIC ACID, 0233 4-(3-INDOLYLMETHYLAMINO)BENZOIC 0256 GLUTARIC ACID (5-INDANYLMETHYL ACID, ENE)HYDRAZIDE, 0234 2-(2,2-BIS-(1H-INDOL-3-YL)-ETHYL)PHE 0257) MALONIC ACID (5-INDANYLMETHYL NYLAMINE, ENE)HYDRAZIDE,

US 2003/0185793 A1 Oct. 2, 2003 20

4-se 4 = ligand -$ therapeutic or diagnostic agent

C spacer molecule F bond not cleavable in the body.

4-O-is pH-dependent O NH-$ enzymatically cleavable in the body C3 week, a C transport protein

For the example of Evans blue and bromcresol, which both have a high affinity for albumin, the structure of the ligand system according to the invention is schematically represented as: SB = predetermined breaking point, acid-labile or enzymatically cleavable active substance

S8 SO, Na US 2003/0185793 A1 Oct. 2, 2003 21

0461 For the example of Evans blue and bromcresol, medium at the Site of action. Furthermore, the ligand com which both have a high affinity for albumin, the structure of pound according to the invention has the advantage that for the ligand System according to the invention is Schematically preparation of the conjugate with the carrier molecule, the represented as: carrier molecule is not covalently modified Since the inter

SB = predetermined breaking point, acid-labile or enzymatically cleavable active substance

SONa ( ) NHSB NH ( ) SONa

NEN

CH CH general structure of a protein-affinitive ligand system with Evans blue O

Br Br

CH CH C Br

O 2 OH NH Br active substance wc12 N general structure of a protein-affinitive ligand system with bromcresol green

0462 Surprisingly, by providing the ligand compound action between the carrier-molecule-affinitive Substance and according to the invention, carrier-molecule-affinitive Sub the carrier molecule is of a physical nature. In preparation of stances Such as, for example, the above-indicated ligands of Such conjugates, therefore, a large number of process Steps transport proteins that enter into a Strong noncovalent inter that are usually necessary are cancelled, which results in action there with in the bloodstream, can be used therapeu considerable Savings of time and material and thus consid tically and/or diagnostically by covalently bonding thera erable cost reduction. This is also the case because the ligand peutically and/or diagnostically active Substances to Such compound does not have to be linked to the carrier molecule carrier-molecule-affinitive Substances. Since Such ligand eX Vivo, but they can be joined together at the Site of action. Systems bind to the carrier molecule, for example a transport protein, via the carrier-molecule-affinitive ligand, for 0464) A further embodiment of the present invention thus example, a protein-affinitive ligand, the therapeutic or diag relates to an adduct or a conjugate or a complex of a carrier nostic agents bound to the ligand in this way are transported molecule and the ligand compound as defined above. to their target Site or their target cells or to pathogenic tissue, 0465 Preferred carrier molecules in the adduct according Such as for example tissue having malignant -degenerated to the invention are as defined above. cells. According to the invention, an acid-labile or enzy matically cleavable bond between the active Substance and 0466 A further embodiment of the present invention the protein-affinitive ligand or spacer ensures that the active relates to a proceSS for preparation of an adduct as defined Substance is released at the intracellular or extracellular site above, comprising the Steps: of action. 0467 (i) Preparation of the ligand compound defined 0463 Thus, the ligand compound according to the inven above and tion provides a novel and Superior prodrug concept, Since the 0468 (ii) Bringing the ligand compound into contact ligand compound according to the invention is transported to with the carrier molecule. the site of action via the carrier-molecule-affinitive Sub stance, which enters into a strong physical interaction with 0469 Bringing them into contact preferably comprises the carrier molecule, for example a Serum protein Such as the Step of oral administration of the ligand compound albumin, and accumulates at that site. The ligand compound and/or the Step of injection of the ligand compound into an according to the invention also has excellent Solubility in the organism, more preferably into the bloodstream. AS US 2003/0185793 A1 Oct. 2, 2003 22 explained above, this approach makes it possible by the fact able vehicle and/or excipient and/or diluent. The pharma that the carrier molecule, for example albumin, does not ceutical product according to the invention can preferably be have to be isolated, and moreover, further material and time used to treat cancers, autoimmune disorders, acute or Savings are achieved, because a Synthesis Step outside the chronic inflammatory diseases that are caused by viruses or organism is avoided. microorganisms, Such as for example bacteria and/or fungi. 0470. After synthesis of the therapeutically and/or diag 0476 A further embodiment of the present invention nostically active Substance, an injectable pharmaceutical relates to a diagnostic kit containing a ligand compound as preparation containing the therapeutically or diagnostically defined above and/or an adduct as defined above. The active Substance is prepared in a Suitable liquid vehicle. The diagnostic kit according to the invention can be used pref therapeutically and/or diagnostically active Substance gen erably to detect the above defined diseases and/or to detect erally is present as a Solid Substance or as a Solution, wherein carrier molecules and/or to determine their distribution in the usual vehicles and/or pharmaceutical excipients can be the body. added Such as polySorbates, glucose, lactose, mannose, 0477 The therapeutic and/or diagnostic ligand systems mannitol, citric acid, trometamol, triethanolamine or ami described above represent formulations of the therapeutic noacetic acid. The injectable pharmaceutical preparation and/or diagnostic agents that, because of their affinity for must be prepared in Such a way that the therapeutically or certain carrier molecules, decisively alter and improve the diagnostically active Substance is not deactivated, cleaved, pharmacokinetic profile of the therapeutic or diagnostic or hydrolyzed by dissolving in the injectable liquid vehicle. agents. After the ligand compound has been brought into Furthermore, it must be ensured that the acid-labile bond in contact with the respective carrier molecule, for example a the pharmacologically active Substance, which for example transport protein Such as albumin, within a body fluid or is an ester, acetal, ketal, imine, hydraZone, carboxylhydra even outside the body, it binds to the carrier molecule due to Zone, or Sulfonylhydrazone bond, is not hydrolyzed. Gen physical interactions, in order to be present as a transport erally, the pH value is in the range from pH 5.0 to 9.0, form So that the therapeutic and/or diagnostic agent con preferably from pH 6.0 to pH 8.0. tained in the ligand compound is transported to the target Site 0471. The liquid vehicles used are approximately iso and/or released in a dosed form. tonic buffers, for example phosphate, acetate, or citrate buffers, such as for example 0.004 M sodium phosphate, 0478. The following examples explain the present inven 0.15 M NaCl-pH 6.0–7.0 or 0.01 M sodium acetate, 0.15 M tion in more detail. NaCl-pH 5.0-6.5. The liquid vehicle used may also be an isotonic sodium chloride solution. The buffers may contain EXAMPLES conventional vehicles and/or excipients to ensure that they are isotonic, Such as for example polySorbates, glucose, 0479 All compounds were characterized by H and 'C lactose, mannose, mannitol, citric acid, trometamol, trietha NMR. silica gel 60, 0.06 mm-0.1 mm was used for column nolamine or aminoacetic acid. chromatography. 0472. The solubility of the therapeutically or diagnosti 0480 Preparation of Carrier-Molecule-Affinitive Ligand cally active Substance in the injectable liquid vehicle may be Compound According to the Invention BC-DOX1 improved by means of pharmaceutical Solvents Such as for 0481 Bromcresol green sulfonic acid hydrazide was example ethanol, isopropanol, 1,2-propylene glycol, glyc reacted with the cytostatic agent doxorubicin to form the erol, macrogols, polyethylene glycols or polyethylene corresponding Sulfonic acid hydrazone derivative (abbrevi oxides or by means of a Solubilizer, for example, Tween, Cremophor or polyvinylpyrrollidone. For this purpose, the ated as BC-DOX1 in the following): therapeutically or diagnostically active Substance is dis Br Br Solved in either the pharmaceutical Solvent and Solubilizer, respectively, and then diluted with Saline buffer, or a liquid vehicle, containing the Saline buffer and at least one phar maceutical Solvent and Solubilizer, respectively, is used to CH CH directly dissolve the pharmacologically active Substance. The concentration of the pharmaceutical Solvent and Solu C Br bilizer, repectively, in this case does not exceed the quanti ties specified by the Pharmaceutical-Products Act. 0473. The process of dissolving the therapeutically or OH diagnostically active Substance in the liquid vehicle is gen erally completed within a few minutes, So that an injectable Br pharmaceutical preparation can be provided for a patient at the patients bedside. 0474. In a further embodiment, the process according to the invention comprises the further Step of preparation of the carrier molecule, and the ligand compound is brought into contact with the carrier molecule eX Vivo. In this way, if necessary, the Selectivity of the ligand compound for a carrier molecule, for example a transport protein Such as H3CO O HO O albumin or a transport protein that occurs in Small amounts in the blood, Such as for example transcortin, can be improved. 0475 A further embodiment of the present invention relates to a pharmaceutical product containing a ligand compound as defined above and/or an adduct as defined above, and optionally at least one pharmaceutically accept US 2003/0185793 A1 Oct. 2, 2003

0482 Bromcresol green sulfonic acid hydrazide in this is removed under vacuum and the preparation is treated case was prepared in two steps, as explained below. with 10 ml dry ether, so that after filtering out the solid 0483 1. Reaction of bromcresol green sulfonic acid and washing with n-hexane, 180 mg bromcreSol green Sodium salt with N,N'-dicyclohexylcarbodiimide and a Sulfonic acid hydrazide trifluoroacetate Salt is obtained. ten-fold excess of tert-butylcarbazate in absolute tet 0488 Synthesis of BC-DOX-1: rahydrofuran (THF). The product was isolated on silica 0489) 30 mg (0.05 mmol) doxorubicin and 123 mg (0.15 gel. THF:hexane=3:2. mmol) bromcresol green Sulfonic acid hydrazide trifluoro 0484 2. Cleavage of the BOC protecting group with acetate Salt and 10 ultrifluoroacetic acid are dissolved in 10 trifluoroacetic acid and precipitation of the hydrazide ml absolute ethanol and stirred for 24 hours in the dark at with ether. room temperature. Then, it is concentrated to ~5 ml under

O

Br Br Br

DCC, t-Butylcarbazate CH CH s CH CH C Br C Br

O O 2 OH 2 OH ONa Br N H Br

Br Br Br Br

CH3 CH CF3COOH CH3 CH

C Br Br

O O 21 OH 21 OH t Br Br t NH2 BOC CF3COOH

0485 Synthesis of Bromcresol Green Sulfonic Acid vacuum, and the product is brought to cloudiness with 50 ml Hydrazide: anhydrous ethyl acetate and then set aside at +5 C. for 16 h. The precipitate is obtained by centrifugation. Then, it is 0486 4.32 g (6 mmol) bromcresol green sulfonic acid washed twice with 6 ml ethyl acetate and, after drying under sodium salt and 7.93 g (60 mmol) tert-butylcarbazate are high vacuum, 45 mg of product is obtained. Rf=0.16 dissolved in 50 ml dry tetrahydrofuran and mixed with 1.36 (reversed phase, 50% CHCN/50% KHPO pH 7.0, +2 g/1 g (6.6 mmol) N,N'-dicyclohexylcarbodiimide (DCC) dis heptanesulfonic acid) solved in 20 ml dry tetrahydrofuran. The reaction mixture is stirred for 16 hat room temperature, filtered and the solvent 0490 Binding Behavior with Human Serum Albumin is removed under vacuum. The oily residue is purified by 0491 Bromcresol green or bromcresol green sulfonic column chromatography (silica gel, tetrahydrofuran:hex acid have binding constants relative to human Serum albu ane=3:2). After drying under high vacuum; 300 mg bro min (HSA) of log KA->10", and even after incubation times mcreSol green Sulfonic acid tert-butylcarbazate ester is of only a few seconds with HSA (mole ratio 1:1 for obtained; Rf-value (tetrahydrofuran : hexane=3:2)=0.35. physiological concentrations) cannot be isolated by size 0487 200 mg bromcresol green sulfonic acid tert-butyl exclusion chromatography, for example with SephadeX(R) carbazate ester is dissolved in 1.0 ml trifluoroacetic acid and G-25, but, in contrast to free doxorubicin, elute together with Stirred for 1 h at room temperature. The trifluoroacetic acid HSA US 2003/0185793 A1 Oct. 2, 2003 24

0492 Furthermore, bromcresol green or bromcresol 0497 Preparation of Crocein Orange G Acid Chloride green sulfonic acid do not exhibit high affinity for other plasma proteins. When bromcreSol green Sulfonic acid is 0498) 10 g (28.5 mmol) crocein orange G (sodium salt) incubated with transferrin, bromcreSol green Sulfonic acid and 20 g (96.0 mmol) phosphorus pentachloride are heated could be almost completely recovered by size-exclusion as the solid powder for 45 min under reflux at 150° C. After chromatography with SephadeXOR G-25. cooling, the reaction mixture is taken up in 150 ml dry diethyl ether, filtered and precipitated with 250 ml dry 0493. In the incubation experiments described above, the n-hexane. The preparation is stored for 12 hat -20°C. Then, sulfonic acid hydrazone derivative BC-DOX1 exhibits the precipitate is filtered out and washed four times with 60 behavior like that of bromcreSol green or bromcreSol green ml n-hexane each. After the precipitate is dried under high Sulfonic acid. Vacuum, 3.2 g is obtained as an orange powder. Rf=0.48 (tetrahydrofuran) 0494 Thus the carrier-molecule-affinitive ligand com pound BC-DOX1 according to the invention displays a very 0499 Preparation of Crocein Orange G Acid Hydrazide high binding constant relative to the transport protein human Serum albumin that Surprisingly is Several orders of magni 0500 3.65 g (10.5 mmol) crocein orange acid chloride is tude greater than that of free doxorubicin. Thus BC-DOX1, dissolved in 10 ml dry tetrahydrofuran. 2.09 g (15.8 mmol) after being brought into contact with HSA or after injection tert-butylcarbazate, dissolved in 15 ml dry tetrahydrofuran, into the bloodstream, is tightly bound to that blood protein. is added with Stirring. Then, at room temperature 1.46 ml Since BC-DOX1 in addition has a sulfonylhydrazone bond triethylamine, dissolved in 5 ml dry tetrahydrofuran, is as an acid-labile bond between the carrier-molecule-affini added and it is stirred for 16 h. The preparation is filtered and tive Substance and the cytostatic agent, in the acid environ the solvent is drawn off under vacuum. The oily residue is ment of tumor tissue or in the acid intracellular compart taken up in 50 ml ethyl acetate and extracted once with 100 ments of the tumor cell, doxorubicin is released as the active ml 0.01 NHCl and twice with 75 ml HO each. The organic Substance So it can exert its therapeutic effect there. phase is dried over NaSO and the solvent is drawn off under Vacuum. The residue is purified by column chroma 0495 Preparation of Doxorubicin Derivatives with Albu tography (silica gel, ethyl acetate:n-hexane=1:2). After dry min-Binding Ligands (Crocein Orange, Stearic Acid ing under high vacuum, 2.45 g crocein orange acid tert Hydrazide) butylcarbazate is obtained as a red oil; R =0.5 (ethyl 0496 Preparation of Doxorubicin Derivative of Crocein acetate:n-hexane=1:2). Orange Sulfononic Acid Hydrazide (DOXO-CROC) 0501) 2.0 g crocein orange acid tert-butylcarbazate is dissolved in 4.0 ml trifluoroacetic acid at room temperature, and the mixture is stirred for 30 min. The trifluoroacetic acid is removed under high vacuum, So that 1.9 g crocein orange acid hydrazide (trifluoracetate salt) is obtained; R=0.25

OH (ethyl acetate:n-hexane=1:2). 0502. 200 mg (0.35 mmol) doxorubicin, 790 mg (1.73 mmol) crocein orange acid hydrazide (trifluoracetate salt) and 60 ultrifluoroacetic acid are dissolved in 40 ml absolute methanol and Stirred overnight in the dark at room tempera ture. Then, the mixture is filtered, the Solvent is removed under vacuum down to a volume of about 30 ml, and the product is precipitated with 80 ml acetonitrile (HPLC grade). The mixture is stored for 12 hat -20° C. Then, the precipitate is centrifuged off and washed with acetonitrile until the Supernatant is colorleSS. Finally, it is washed once with 10 ml diethyl ether and, after drying under high vacuum, 250 mg of product is obtained. Rf=0.14 (reversed phase, 50% CHCN/50% KHPO, pH 7.0+2 g/l heptane Sulfonic acid) 0503 Preparation of Doxorubicin Hydrazone Derivative of Crocein Orange and HeXaethylene Glycol as Spacer (DOXO-CROC-HEXA) US 2003/0185793 A1 Oct. 2, 2003 25

0504 Synthesis of Aminohexaethylene Glycol t-butyl carbazate Spacer (4) 0505 Synthesis Route:

Ho-Nuo-1Nuo-1Nuo-1N-O1N-O1N-OH US 2003/0185793 A1 Oct. 2, 2003 26

-continued O I sin-ir-ir-ir-ir-ir-ir-s-s-AO

0506 Synthesis for Step 1 (1) After drying under high vacuum, 10.2 g is obtained as an oil. 0507 51.05 g (175.4 mmol) hexaethylene glycol is dis Rf=0.23 (ethyl acetate:methanol=8:1) solved in 90 ml absolute THF and mixed with 211 mg (1.8 0513) Synthesis for Step 4 (4) mmol) potassium tert-butylate. 9.2 ml acrylic acid tert-butyl ester, dissolved in 15 ml dry THF, is added dropwise with 0514) 10.2 g (20.7 mmol) 3 is dissolved in 100 ml Stirring at room temperature over a period of 30 min. The absolute methanol and 1.4 g 10% palladium on activated preparation is stirred for 16 h at room temperature. The charcoal is added in portions under nitrogen. Then 5.4 g reaction mixture is neutralized, with Stirring, with 2 ml of 1 NHCl. The solvent is removed under vacuum, and the oily (82.9 mmol) ammonium formate, dissolved in 60 ml abso residue is taken up in 50 ml of Saturated NaCl solution and lute methanol, is added and is stirred for 12 h at 50 C. The extracted three times with 50 ml dichloromethane. The reaction mixture is filtered through Cellite and the solvent is organic phases are combined, dried over NaSO, filtered removed under vacuum. The oily residue is purified by and the Solvent is removed under vacuum. The oily residue column chromatography (silica gel; methanol--1% triethy is purified by column chromatography (silica gel; ethyl lamine). acetate:methanol=4:1). After drying under high Vacuum, 20.25 g is obtained as a light yellowish oil. Rf=0.63 (ethyl 0515. After drying under high vacuum, 8.53 g is obtained acetate: methanol=4:1) as an oil. 0508) Synthesis for Step 2 (2) 0516 Rf=0.22 (methanol--1% triethylamine) 0509. 20.25 g (49.4 mmol) of 1 is dissolved in 60 ml of 0517) Synthesis of Hexaethylene Glycol Hydrazide absolute THF. 5.73 ml (74.1 mmol) methanesulfonyl chlo Derivative of Crocein Orange (5): ride followed by 10.3 ml triethylamine (74.1 mmol) are added to this Solution with Stirring at room temperature. The 0518) 120 mg (0.24 mmol) 4 and 84 mg (0.24 mmol) mixture is stirred for 16 hat room temperature. The reaction crocein orange acid chloride are dissolved in 2 ml dry mixture is filtered and the Solvent is removed under vacuum. tetrahydrofuran and then mixed with 68 pi triethylamine. After drying under high vacuum, 26.98 g is obtained as a The mixture is stirred for another 2.5 h. The Solvent is light brownish oil, which is dissolved in 90 ml of dry removed under vacuum. After drying under vacuum, 200 mg acetonitrile and mixed with 7.19 g (110.6 mmol) sodium is obtained as a light yellowish oil, which is mixed with 400 azide. The reaction mixture is stirred for 36 h under reflux. All trifluoroacetic acid and stirred for 10 min. The trifluoro The reaction mixture is filtered and the solvent is removed acetic acid is removed under Vacuum and the oily residue is under vacuum. After drying under high vacuum, 23.06 g is purified by column chromatography (silica gel; ethyl obtained as a light brownish oil, which is dissolved in 30 ml acetate:methanol=2:1). After drying under high vacuum, dichloromethane. 32.5 ml trifluoroacetic acid is added drop 137 mg of product (trifluoroacetate salt) is obtained as a light wise with Stirring at room temperature over a period of 10 yellowish oil; Rf=0.3 (ethyl acetate/methanol=2:1). min. The mixture is stirred for 2 h. The Solvent and the trifluoroacetic acid are evaporated under high vacuum. The 0519 Synthesis of DOXO-CROC-HEXA: oily residue is purified by column chromatography (silica gel; ethyl acetate:methanol=6:1+0.5% acetic acid). After 0520 25 mg (0.04 mmol) doxorubicin and 104.5 mg drying under high Vacuum, 16.1 g is obtained as an oil. (0.15 mmol) crocein orange hexaethylene glycol hydrazide (trifluoroacetate salt) (5) are dissolved in 5 ml absolute 0510) Rf=0.2 (ethyl acetate:methanol=6:1+0.5% acetic methanol and stirred for 24 hours in the dark at room acid) temperature. Then, the product is precipitated with 75 ml 0511 Synthesis for Step 3 (3) ethyl acetate (HPLC grade) and centrifuged for 10 minutes at +4 C. Then, it is washed twice with 10 ml ethyl acetate. 0512 14.03 g (37.21 mmol) 2 and 5.41 g tert-butylcar After drying under high vacuum, 35 mg of product is bazate are dissolved in 420 ml apure dichloromethane and obtained. Rf=0.11 (reversed phase, 50% CHCN/50% then 6.34 ml diisopropylcarbodiimide is added dropwise KHPO, pH 7.0, +2 g/l heptanesulfonic acid) over a 2 min period. The mixture is stirred for 4 h. The reaction mixture is filtered and the Solvent is evaporated 0521 Preparation of Doxorubicin Hydrazone Derivative under vacuum. The oily residue is purified by column of 2,3,5-triiodobenzoic Acid and Hexaethylene Glycol as chromatography (Silica gel, ethyl acetate:methanol=12:1). Spacer (DOXO-TIB-HEXA) US 2003/0185793 A1 Oct. 2, 2003 27

I

NH

O 1n 10-N-O1 N1 1a1 O I I 1N1 1N1 s 1n 10

O OH N -- COOOS"OH OCH O OH O O CH

HO NH, HCI)

0522 Synthesis of the Hexaethylene Glycol Hydrazide acetic acid is removed under vacuum, So that 150 mg of the Derivative of 2,3,5-triiodobenzoic Acid (6): hexaethylene glycol hydrazide derivative of 2,3,5-triiodo benzoic acid is obtained as an oil, which is directly used in 0523) 124 mg (0.26 mmol) 4, 132.8 mg (0.26 mmol) the following. 2,3,5-triiodobenzoic acid and a spatula-tipful of dimethy laminopyridine are dissolved in 3.0 ml dry tetrahydrofuran. 0524) Synthesis of DOXO-TIB-HEXA: The mixture is cooled down to 0° C. and mixed with 40 ul 0525) 30 mg (0.06 mmol) doxorubicin and 150 mg (0.15 diisopropylcarbodiimide. The reaction mixture is stirred for mmol) triiodobenzoic acid hexaethylene glycol hydrazide another 12 h, protected from light. The solvent is removed (trifluoroacetate salt) are dissolved in 6 ml absolute metha under vacuum. The oily residue is purified by column nol and Stirred for 24 hours in the dark at room temperature. chromatography (silica gel, tetrahydrofuran:hexane=8:1). Then the product is precipitated with 60 ml ethyl acetate After drying under high vacuum, 195 mg of product (hexa (HPLC grade) and centrifuged for 10 minutes at +4 C. ethylene glycol tert-butylcarbazate derivative of 2,3,5-tri Then, it is washed twice with 6 ml ethyl acetate, and after iodobenzoic acid) is obtained as a light yellowish oil; drying under high vacuum, 42 mg of product is obtained. Rf=0.46 (tetrahydrofuran:hexane=8:1). 160 mg of the hexa Rf=0.06 (reversed phase, 50% CHCN/50% KHPO pH ethylene glycol tert-butylcarbazate derivative of 2,3,5-tri 7.0, +2 g/l heptaneSulfonic acid). iodobenzoic acid is dissolved in 500 ul trifluoroacetic acid 0526 Preparation of Doxorubicin Hydrazone Derivative and stirred for 30 min at room temperature. The trifluoro of Stearic Acid Hydrazide (DOXO-SAH)

NH, HCI) US 2003/0185793 A1 Oct. 2, 2003 28

0527 Preparation of Stearic Acid Hydrazide 0535 Furthermore, the albumin-binding ligand com 0528 Stearic acid tert-butylcarbazate: 6.06 g (20 mmol) pounds of doxorubicin, after incubation times of only a few Stearic acid chloride is dissolved in 10 ml dry THF. 2.77ml seconds with HSA or blood plasma (molar ratio 1:1, pH (20 mmol) triethylamine and 2.91 g (22 mmol) tert-butyl 7.0–7.5), cannot be isolated by size-exclusion chromatogra carbazate, dissolved in 10 ml THF, are added dropwise with phy, for example with SephadeX(R) G-25, but, in contrast to Stirring at room temperature over a 10 min period, and the free doxorubicin, elute together with HSA. mixture is stirred for 30 min. The reaction mixture is filtered 0536 Furthermore, the albumin-binding ligand com and the Solvent is removed under vacuum. The oily residue pounds of doxorubicin do not exhibit high affinity for other is taken up in 50 ml ethyl acetate and extracted twice with plasma proteins: If they are incubated with transferrin, the 70 ml HO. The organic phase is dried over NaSO and albumin-binding ligand compounds of doxorubicin are filtered, and the Solvent is evaporated under Vacuum. The almost completely recovered by size-exclusion chromatog oily residue is dissolved in 50 ml warm ethanol and stored raphy with Sephadex(R) G-25. for 12 h at +4 C. The white precipitate is filtered and 0537 Thus, the carrier-molecule-affinitive ligand com washed twice with 30 ml ethanol each. After drying under pounds of doxorubicin according to the invention display a high vacuum, 3.6 g Stearic acid tert-butylcarbazate is very high binding constant relative to the transport protein obtained; Rf=0.9 (ethyl acetate/n-hexane 2/1). human Serum albumin, which Surprisingly is Several orders 0529 Stearic acid hydrazide: 1.8 g. (4.52 mmol) stearic of magnitude greater than that of free doxorubicin. Thus, the acid tert-butylcarbazate is dissolved in 3 ml trifluoracetic albumin-binding ligand compounds of doxorubicin, after acid, stirred for 15 min and the solvent is removed under being brought into contact with HSA or after injection into vacuum. The mixture is treated with 30 ml dry ether and the the bloodstream, have a strong interaction with this blood precipitate formed is filtered out and washed twice with 50 protein. Since the albumin-binding ligand compounds of ml dry diethyl ether. After drying under high vacuum, 1.03 doxorubicin have a hydrazone bond as an acid-labile bond g stearic acid hydrazide is obtained; Rf=0.66 (ethyl acetate/ between the carrier-molecule-affinitive Substance and the methanol 1/1) cytostatic agent, in the acid environment of tumor tissue or in the acid intracellular compartments of the tumor cell, 0530 Synthesis of DOXO-SAH: doxorubicin can be released as the active Substance So it can 0531 50 mg (0.09 mmol) doxorubicin hydrochloride and exert its therapeutic effect there. 42 mg (0.1 mmol) Stearic acid hydrazide trifluoroacetate are dissolved in 30 ml methanol (HPLC grade) and stirred for 18 0538. The antitumor effect of DOXO-CROC was studied hours in the dark at room temperature. Then, the product is in a melanoma xenograft model (MV-3). The data are precipitated with 40 ml acetonitrile (HPLC grade). The Summarized in Table 3 below: precipitate is centrifuged for 10 minutes at room tempera ture, then washed twice with 30 ml acetonitrile and once TABLE 3 with 10 ml diethyl ether, and after drying under high Number of Treatment Mor Optimum. T/C- vacuum, 60 mg of product is obtained; Rf=0.15 (reversed Substance nude mice scheme tality value (%)* phase, 50% CHCN/50% 20 mM. KHPO pH 7.0, +2 g/1 NaCl 8 3x (Day 10, 17, heptanesulfonic acid). 24) DOXO-CROC 8 3 x 16 mg/kg 37 0532 Binding Behavior with Human Serum Albumin i.p. Tween 0533. A Scatchard plot is used to determine the binding (Day 10, 17, constants relative to human serum albumin (HSA from the 24) Dessau company, Germany) at pH 7.4 by means of equilib *Ratio of tumor sizes in treated group compared to control group rium dialysis (equilibrium dialyzer from Spectrum, Inc., USA; dialysis membrane: cut-off MW 10000) for the pre 0539 Treatment with DOXO-CROC was tolerated very pared doxorubicin derivatives, all including an acid-labile well: The decrease in body weight during treatment with hydrazone bond as the predetermined breaking point, and DOXO-CROC was only -3%; growth of the subcutaneously for doxorubicin. The results are Summarized in Table 2 growing tumors was able to be inhibited by approximately below. more than 60% compared with the control. TABLE 2 Substance K relative to HSA 1. Carrier molecule-binding ligand compound, compris ing at least one therapeutically and/or diagnostically active Doxorubicin 2.9 x 103 2.7 x 103* DOXO-CROC 6.4 x 10° Substance and at least one carrier-molecule-affinitive Sub DOXO-CROC-HEXA 7.2 x 107 stance which has an association constant K relative to a DOXO-TIB-HEXA 5.5 x 10 carrier molecule greater than 10 M' via a noncovalent DOXO-SAH 6.1 x 10° bond, wherein the linkage between the therapeutically and/ * Literature value (Demant et al., Biochemical Pharmacology 55, 27-32, or diagnostically active Substance and the carrier-molecule 1998) affinitive Substance is pH-dependent and/or is enzymatically cleavable within the body. 0534. The albumin-binding ligand compounds of doxo 2. Ligand compound according to claim 1, wherein the rubicin have HSA binding constants that are 2-3 orders of therapeutically and/or diagnostically active Substance and magnitude higher than the binding constants for doxorubi the carrier-molecule-affinitive Substance are bound to each cin. other through a Spacer molecule. US 2003/0185793 A1 Oct. 2, 2003 29

3. Ligand compound according to claim 2, wherein the isoquinolines, anthraquinones, anthracenes, phenanthrenes, Spacer molecule and/or the linkage between the therapeuti phenanthrolines, benzylidenes, diphenyls and biphenyls, cally and/or diagnostically active Substance and the Spacer indoles, indanes, hippuric acids, imidazoles, benzimida molecule and/or the linkage between the carrier-molecule Zoles, quinoxalines, pyridines, pyrimidines, piperidines, Sar affinitive Substance and the Spacer molecule is pH-depen cosines, oxazoles, oxadiazoles, isoxazoles, pyrazoles, triaz dent and/or enzymatically cleavable within the body. oles and tetrazoles, benzothiazoles, triazines, morpholines, 4. Ligand compound according to any one of claims 1 to chromenes, trisubstituted, tetrasubstituted, and pentasubsti 3, wherein the linkage and/or the Spacer molecule contains tuted ethanoic and propanoic acids, Stilbenes and tryptophan at least one peptide bond. analog and thyroXin analog compounds and derivatives 5. Ligand compound according to claim 4, wherein the thereof, as well as dipeptides, tripeptides, tetrapeptides, peptide bond is located within a peptide Sequence which pentapeptides, and hexapeptides. contains at least one protease cleavage Sequence. 15. Adduct of a carrier molecule and the ligand compound 6. Ligand compound according to any one of claims 1 to according to any one of claims 1 to 14. 5, wherein the linkage and/or the Spacer molecule contains 16. Adduct according to claim 15, wherein the carrier at least one acid-labile bond. molecule is a protein. 7. Ligand compound according to any one of claims 1 to 17. Adduct according to claim 16, wherein the protein is 6, wherein the carrier molecule is a protein. Selected from the group consisting of Serum proteins. 8. Ligand compound according to claim 7, wherein the 18. Adduct according to claim 17, wherein the serum protein is Selected from the group consisting of Serum protein is albumin. proteins. 19. Pharmaceutical product containing the ligand com 9. Ligand compound according to claim 8, wherein the pound according to any one of claims 1 to 14 and/or the Serum protein is albumin. adduct according to any one of claims 15 to 18 and option 10. Ligand compound according to any one of claims 1 to ally at least one pharmaceutically acceptable vehicle and/or 9, wherein the therapeutically active Substance is a cytostatic excipient and/or diluent. agent, a cytokine, an immunosuppreSSant, an antirheumatic, 20. Pharmaceutical product according to claim 19 for an antiinflammatory, an antibiotic, an analgesic, a Virostatic treatment of cancers, autoimmune disorders, acute or or an antimycotic agent. chronic inflammatory diseases and diseases that are caused 11. Ligand compound according to claim 10, wherein the by viruses and/or microorganisms. cytostatic agent is Selected from the group consisting of 21. Diagnostic kit containing the ligand compound anthracyclines, N-nitroSoureas, alkylating agents, purine or pyrimidine antagonists, folic acid antagonists, taxanes, according to any one of claims 1 to 14 and/or the adduct camptothecines, podophyllotoxin derivatives, Vinca alka according to any one of claims 15 to 18. loids, calicheamicins, maytansinoids, epithilones or platinu 22. Diagnostic kit according to claim 21 for detection of m(II) complexes in the cis configuration. cancers, autoimmune diseases, acute or chronic inflamma 12. Ligand compound according to any one of claims 1 to tory diseases and diseases that are caused by viruses and/or 9, wherein the diagnostically active Substance contains one microorganisms, and/or for detection of the carrier molecule or more radionuclides, one or more ligands comprising and/or its distribution in the body. radionuclides, one or more positron emitters, one or more 23. Method for preparing the adduct according to any one NMR contrast media, one or more fluorescent compound(s), of claims 15 to 18, comprising the Steps: or one or more contrast media in the near IR region. (i) Preparation of the ligand compound according to any 13. Ligand compound according to any one of claims 1 to one of claims 1 to 14 and 12, wherein the Spacer molecule comprises a Substituted or unsubstituted, branched-chain or unbranched-chain aliphatic (ii) Bringing the ligand compound into contact with the alkyl residue with 1 to 20 carbon atoms, which can be carrier molecule. partially replaced by Oxygen or nitrogen atoms, and/or at 24. Method according to claim 23, wherein bringing the least one Substituted or unsubstituted aryl residue. components into contact comprises the Step of oral admin 14. Ligand compound according to any one of claims 1 to istration and/or the Step of injection of the ligand compound 13, wherein the carrier-molecule-affinitive Substance is into an organism. Selected from the group consisting of phthalocyanines, cou 25. Method according to claim 24, wherein injection marins, flavonoids, tetracyclines, naphthalenes, aryl- and occurs into the bloodstream. heteroarylcarboxylic acids, long-chain fatty acids, pyrroles, 26. Method according to claim 25, which comprises the dipyrroles and tripyrroles, cyclic and linear tetrapyrroles and further Step of providing of the carrier molecule, and organometallic compounds thereof, aromatic acid deriva wherein bringing the components into contact occurS eX tives substituted with 2-5 halogen atoms (C1, Br or I), WVO. organic dyes, benzophenones, phthalimides and isophthal imides, phthalic acids and isophthalic acids, quinolines,