EP1506221A2 - Promedicaments antitumoraux actives par la fap - Google Patents

Promedicaments antitumoraux actives par la fap

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Publication number
EP1506221A2
EP1506221A2 EP03727438A EP03727438A EP1506221A2 EP 1506221 A2 EP1506221 A2 EP 1506221A2 EP 03727438 A EP03727438 A EP 03727438A EP 03727438 A EP03727438 A EP 03727438A EP 1506221 A2 EP1506221 A2 EP 1506221A2
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EP
European Patent Office
Prior art keywords
formula
xaa
compound
group
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP03727438A
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German (de)
English (en)
Inventor
Erik Patzelt
John Edward Park
Stefan Peters
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Boehringer Ingelheim Pharma GmbH and Co KG
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Boehringer Ingelheim Pharma GmbH and Co KG
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Priority to EP03727438A priority Critical patent/EP1506221A2/fr
Publication of EP1506221A2 publication Critical patent/EP1506221A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

  • the present invention relates to the field of tumor treatment by administration of a prodrug that is converted into a drug at the site of the tumor.
  • the invention relates to prodrugs which are converted into prodrug intermediates by the catalytic action of FAP ⁇ in the environment of a target cell, their manufacture and pharmaceutical use.
  • the human fibroblast activation protein is a M r 95,000 cell surface molecule origi- nally identified with monoclonal antibody (mAb) F19 (Rettig et al. (1988) Proc. Natl. Acad. Sci. USA 85, 3110-3114; Rettig et al. (1993) Cancer Res. 53, 3327-3335).
  • the FAP ⁇ cDNA codes for a type II integral membrane protein with a large extracellular domain, trans- membrane segment, and short cytoplasmic tail (Scanlan et al. (1994) Proc. Natl. Acad. Sci. USA 91, 5657-5661; WO 97/34927).
  • FAP ⁇ shows 48 % amino acid sequence identity to the T-cell activation antigen CD26, also known as dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5), a membrane-bound protein with dipeptidyl peptidase activity (Scanlan et al, loc. cit.).
  • DPPIV dipeptidyl peptidase IV
  • FAP ⁇ has enzymatic activity and is a member of the serine protease family, with serine 624 being critical for enzymatic function (WO 97/34927).
  • FAP ⁇ is selectively expressed in reactive stromal fibroblasts of many histological types of human epithelial cancers, granulation tissue of healing wounds, and malignant cells of certain bone and soft tissue sarcomas. Normal adult tissues are generally devoid of detectable FAP ⁇ , but some fetal rriesenchymal tissues transiently express the molecule. In contrast, most of the common types of epithelial cancers, including >90% of breast, non-small-cell lung, and colorectal carcinomas, contain FAP ⁇ -reactive stromal fibroblasts (Scanlan et al, loc. cit).
  • FAP ⁇ + fibroblasts accompany newly formed tumor blood vessels, forming a distinct cellular compartment interposed between the tumor capillary endothelium and the basal aspect of malignant epithelial cell clusters (Welt et al. (1994) J. Clin. Oncol 12(6), 1193- 1203). While FAP ⁇ + stromal fibroblasts are found in both primary and metastatic carcinomas, the benign and premalignant epithelial lesions tested (Welt et al., loc. cit.), such as fibroadenomas of the breast and colorectal adenomas, only rarely contain FAP ⁇ + stromal cells.
  • Triggering mechanisms can be designed so that the toxic agents synthesized in their prodrug or inactive forms are rendered active when and where required, notably in the cancerous tissues (Panchal (1998) Biochem. Pharmacol. 55, 247-252).
  • Triggering mechanisms may include either exogenous factors such as light or chemicals or endogenous cellular factors, such as enzymes with restricted expression in cancer tissues.
  • ADEPT 'antibody-directed enzyme prodrug therapy'
  • ADC 'antibody-directed catalysis'
  • an antibody directed at a tumor- associated antigen is used to target a specific enzyme to the tumor site.
  • the tumor-located enzyme converts a subsequently administered prodrug into an active cytotoxic agent.
  • the antibody-enzyme conjugate binds to a target antigen on cell membranes or to free antigen in extracellular fluid (ECF).
  • ECF extracellular fluid
  • a time interval between giving the AEC and prodrug allows for the AEC to be cleared from normal tissues so that the prodrug is not activated in the normal tissues or blood.
  • some disadvantages of ADEPT are related to the properties of the AEC (Bagshawe, loc. cit). For example, in humans, only a small fraction of the administered dose of the targeting AEC binds to tumor tissue and the remainder is distributed through body fluids from which it is cleared with significant time delays. Even very low concentrations of unbound enzyme can catalyze enough prodrug to have toxic effects because plasma and normal ECF volumes are much greater than those of tumor ECF.
  • the AEC may also be immunogenic, thus preventing repeat administration, in many instances.
  • N-L-leucyl-doxorubicin prodrug which is capable of being converted into free doxorubicin by the catalytic action of unknown enzymes, and said free doxorubicin being cytotoxic or cytostatic under physiological conditions.
  • N-L-leucyl-doxorubicin possesses improved efficacy in mouse xenograft tumor models, [Breistol et al. (1999) Eur J Cancer 35:1143-1149] as compared to doxorubicin.
  • N-L-leucyl-doxorubicin has comparably poor tolerability, at least in humans, because roughly half of the prodrug is metabolized to free doxorubicin [Canal et al., (1992) Clin. Pharmacol. Ther. 51:249-259] directly in the circulation (whole blood or red blood cells from 107 donors could catalyze this reaction). This results in unspecific systemic exposure to doxorubicin, and therefore tremendously limits the amount of prodrug that can be administered. Myelosuppression was seen in patients and correlated with the AUC values of released doxorubicin, confirming that reduced white blood cell counts were due to generalized release of doxorubicin.
  • the problem underlying the present invention was to provide similar prodrugs with improved stability in whole blood, and presumably, better tolerability in humans.
  • the present invention relates to prodrugs which are capable of being converted into prodrug intermediate of a cytotoxic or cytostatic drug, by the catalytic action of FAP ⁇ , said prodrugs exhibit an oligomeric part comprising up to 9 amino carboxylic acid residues, the amide bond between the C-terminal amino carboxylic acid and the following amino acid thereof is recognized and cleaved by FAP ⁇ in the immediate environment of a target cell, and a cytotoxic or cytostatic part, wherein the N-terminal amino function of the oligomeric part is attached to a capping group (Cg).
  • the prodrug intermediate is cleaved within the target cell by intracellular proteases and kills the target cell or blocks its proliferation.
  • the prodrug is administered to the patient, carried through the blood stream in a stable form, and when in the immediate environment of a target cell, is recognized and cleaved by FAP ⁇ to release said prodrug intermediate.
  • a drug shall mean a chemical compound that may be administered to humans or animals as an aid in the treatment of disease.
  • a drug is an active pharmacological agent.
  • cytotoxic compound shall mean a chemical compound which is toxic to living cells, in particular a drug that destroys or kills cells.
  • cytostatic compound shall mean a compound that suppresses cell growth and multiplication and thus inhibits the pro- liferation of cells.
  • cytotoxic or cytostatic compounds suitable for the present invention are anthracycline derivatives such as doxorubicin, analogs of methotrexate such as methothrexate, pritrexime, trimetrexate or DDMP, melphalan, analogs of cisplatin such as cisplatin, JM216, JM335, bis(platinum) or carboplatin, analogs of purines and pyrimidines ⁇
  • chemotherapeutic agents such as 9- aminocamptothecin, D,L-aminoglutethimide, trimethoprim, pyrimethamine, mitomycin C, mitoxantrone, cyclophosphanamide, 5-fluorouracil, extramustine, podophyllotoxin, bleomycin, epothilone and
  • prodrug shall mean a compound that, on administration, must undergo chemical conversion by metabolic processes before becoming an active pharmacological agent.
  • a prodrug is a precursor of a drug.
  • the prodrug is significantly less cytotoxic or cytostatic than the drug it is converted into upon the catalytic action of FAP ⁇ .
  • the expert knows methods of determining cytotoxicity of a compound, see e.g. example 45 herein, or Mosmann ((1983) J. Immun. Meth. 65, 55-63).
  • the prodrug is at least three times less cytotoxic as compared to the drug in an in vitro assay.
  • a "drug being cytostatic or cytotoxic under physiological conditions” shall mean a chemical compound which is cytostatic or cytotoxic in a living human or animal body, in particular a compound that kills cells or inhibits proliferation of cells within a living human or animal body.
  • a "prodrug having a cleavage site which is recognized by FAP ⁇ ” shall mean a prodrug which can act as a substrate for the enzymatic activity of FAP ⁇ .
  • the enzymatic activity of FAP ⁇ can catalyze cleavage of a covalent amide bond of the prodrug under physiological conditions. By cleavage of this covalent amide bond, the prodrug is converted into the drug, either directly or indirectly via a prodrug intermediate. Indirect activation would be the case if the cleavage product of the FAP ⁇ catalyzed step is not the pharmacologically active agent itself but undergoes a further reaction step, e.g.
  • the cleavage site of the prodrug is specifically recognized by FAP ⁇ , but not by other proteolytic enzymes present in the human or animal body. Also preferably, the cleavage site is specifically recognised by FAP ⁇ , but not by proteolytic enzymes present in human or animal body fluids, especially plasma.
  • the prodrug is stable in plasma, other body fluids, or tissues, in which biologically active FAP ⁇ is not present or detectable.
  • prodrug intermediate as used hereinbefore and hereinbelow defines a compound obtained by the cleavage of the covalent amide bond between the C-terminal amino acid and the following amino acid by the catalytic action of FAP ⁇ of the prodrug according to the invention.
  • the prodrug intermediate is a compound in which one amino acid is attached to a cytotoxic or cytostatic moiety.
  • N-L-leucyl-doxorubicin is used in an in vitro stability assay as a standard.
  • a solution containing human whole blood pretreated to prevent coagulation
  • prodrug at 37°C.
  • Incubations continue until 25% of the standard (N-L-leucyl-doxorubicin prodrug) is converted into free drug (free doxorubicin).
  • the cleavage site should most preferably be specific for FAP ⁇ .
  • the cleavage site comprises a L-proline residue which is linked to the prodrug intermediate.
  • the prodrug intermediate is a single amino acid linked to a cytotoxic or cytostatic drug via an amide bond.
  • An example of a prodrug intermediate is a N-L-leucyl-doxorubicin.
  • An example of this class is a doxorubicin-peptide conjugate.
  • FAP ⁇ may catalyse the cleavage of a peptidic bond between the C-terminal FAP cleavage site and the prodrug intermediate, which is preferably L-leucine, coupled to the cytotoxic or cytostatic compound.
  • Preferred compounds show at least 10% conversion to the prodrug intermediate, under standard conditions listed below. More preferred are compounds that show at least 20% conversion to the prodrug intermediate, under standard conditions. Even more preferred are compounds that show at least 50% conversion to the prodrug intermediate, under standard conditions.
  • standard conditions are defined as follows: Each compound is dissolved in 50 mM Hepes buffer, 150 mM NaCl, pH 7.2, at a final concentration of 5 ⁇ M and incubated with 100 ng CD ⁇ FAP ⁇ (see example 43) for 24 hours at 37 °C. Release of the prodrug intermediate by CD8FAP ⁇ is determined as described in example 5 of WO 00/71571
  • the present invention relates to a compound of formula (I)
  • Xaa 1 each independently represent any genetically encoded amino acid or the N- alkylated derivative thereof, at least one of which being N-terminally linked to
  • Xaa 2 represents any genetically encoded amino acid or the N-alkylated derivative thereof, which is C-terminally linked to Caa
  • Xaa 3 represents an amino acid selected from the group consisting of leucine, phenylalanine, isoleucine, alanine, ⁇ -alanine, glycine, tyrosine, 2- naphthylalanine and serine, or an N-alkylated derivative thereof, which is C- terminally linked to Cyt
  • Caa represents an optionally substituted cyclic amino acid, which is C-terminally linked to Xaa 3
  • Cg represents a capping group, which blocks degradation of the attached oligopeptide moiety in body fluids
  • Cyt represents the residue of a cytotoxic or cytostatic compound
  • m is 0 or an integer from 1 to 6.
  • the capping group serves to protect the prodrug compound from degradation in circulating blood when it is administered to the patient and allows the prodrug to reach the environment of the target cell relatively intact.
  • the stabilizing group protects the prodrug from degradation by proteinases and peptidases present in blood, blood serum, and normal tissue.
  • the capping group caps the N-terminus oligopeptides, and is therefore sometimes referred to as an N-cap or N-block, it serves to ward against exopeptidases to which the prodrug may otherwise be susceptible.
  • the compound is less toxic in vivo than the starting therapeutic agent because the prodrug is not cleaved in blood, heart, brain, bone marrow, in the mucosa and the like. This decrease in toxicity applies, in particular, to the acute effects such as marrow and mucosal toxicity, as well as possible cardiac or neurological toxicity.
  • the capping group is useful in the prodrug of the invention if it serves to protect the prodrug from degradation, especially hydrolysis, when tested by storage of the prodrug compound in human blood at 37 C for 2 hours and results in less than 20%, preferably less than 2%, cleavage of the prodrug by the enzymes present in the human blood under the given assay conditions.
  • Cg represents a capping group of formula R ⁇ CH ⁇ -Z-, in which -Z- represents -CO-, -O-CO-, -NH-CO-, -SO 2 - or a single bond;
  • R 1 is an optionally substituted C * *-C 6 -alkyl, C 3 -C 8 -cycloalkyl, aryl, heterocyclyl or heteroaryl group; and n is 0, 1 or 2.
  • Cg represents a capping selected from the group consisting of succinic acid, diglycolic acid, maleic acid, polyethylene glycol, pyroglutamic acid and glutaric acid; or represents a group of formula (LI)
  • s is an integer of 1 or 2
  • l in particular l
  • t is an integer of 1 or 2, in particular 1.
  • capping groups wherein the group HO-X 1 -(CH 2 ) s - is attached in tthhee tthhee mmeettaa-- oorr/ ⁇ ? ⁇ rr ⁇ --ppoossiittiioonn wwiitthh rreessppeecctt ttoo tthhee ggrroouupp - -(CH 2 ) t -X 2 -.
  • the capping groups selected from the formulae HA and ILB:
  • R and R b together with the interjacent N-C group form an optionally substituted, optionally benzo- or cyclohexano-condensed 3- to 7-membered saturated or unsaturated heterocyclic ring, in which one or two CH 2 groups may also be replaced by NH, O or S.
  • Caa is a prolin group.
  • Xaa 1 and Xaa 2 each independently represent moieties derived from amino carboxylic acids of the formula -[NR 3 -(Y) p -CO]- wherein Y represents CR 4 R 5 and wherein R 3 , R 4 and R 5 each independently represent a hydrogen atom, an optionally substituted C ⁇ -C 6 -alkyl, C 3 - C 8 -cycloalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl group, and p is 1, 2, 3, 4, 5; or Xaa 1 and Xaa 2 each independently represent moieties derived from cyclic amino carboxylic acids of formula
  • R 6 represents C ⁇ -C 6 -alkyl, OH, or NH 2 , q is 0, 1 or 2; and r is 0, 1 or 2.
  • Xaa 1 and Xaa 2 are amino acid moieties, which are each independently selected from glycine (Gly), and the D- or L-forms of alanine (Ala), valine (Val), leucine (Leu), isoleucine ( ⁇ e), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartatic acid (Asp), gluta ic acid (Glu), asparagine (Asn), glutamine (Gin), proline (Pro), 4-hydroxy-proline (Hyp), 5-hydroxy-lysine, norleucine (Nle), 5- hydroxynorleucine (Hyn), 6-hydroxynorleucine, omithine, cyclohexylglycine (Chg
  • Methylalanin N-MeAla
  • N-Methylvaline N-MeVal
  • N-Methylleucine N-MeLeu
  • N- Methylisoleucine N-Melle
  • N-Methylnorleucin N-MeNle
  • N-Methyl-2-aminobutyric acid N-MeAbu
  • N-Methyl-2-aminopentanoic acid N-MeNva
  • the amino acid group Xaa 2 is preferably selected from L-proline, glycine, L-norleucine, L- eyclohexylglycine, L-5-hydroxynorleucine, L-6-hydroxynorleucine, L-5-hydroxylysine, L- arginine, and L-lysine, in particular glycine.
  • the amino acid group Xaa 3 which is directly linked to Cyt is preferably selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and serine, in particular leucine.
  • m is preferably 0 or an integer from 1 to 4, in particular an integer from 1 to 3, most preferably 2.
  • R 3 , R 4 , Cyt, Cg, Xaa 3 , U and N each independently are as defined hereinbefore, or R 4 and R 5 together with the interjacent ⁇ -C group form an optionally substituted, optionally benzo- or cyclohexano-condensed 3- to 7-membered saturated or unsaturated heterocyclic ring.
  • Xaa 1 represents an ⁇ -terminal amino acid selected from the group consisting of Lys, Pro, ⁇ -
  • C Ce-alkyl generally represents a straight-chained or branched hydrocarbon radical having 1 to 6 carbon atoms.
  • C ⁇ -C 6 -alkyl is preferably Ci, more preferably a methylene group.
  • a person of ordinary skill in the chemistry of amino acids and oligopeptides will readily appreciate that certain amino acids may be replaced by other homologous, isosteric and/or isolectronic amino acids wherein the biological activity of the original amino acid or oligopeptide has been conserved upon modification. Certain unnatural and modified natural amino acids may also be utilized to replace the corresponding natural amino acid. Thus, for example, tyrosine may be replaced by 3-iodotyrosine, 2- or 3-methyltyrosine, 3- fluorotyrosine.
  • Heterocyclic ring as used hereinabove and hereinbelow with respect to the group formed by R a and R b together with the interjacent N-C group generally represents a 3 to 7-membered, preferably 4-, 5- or 6-membered non-aromatic heterocyclic ring system, containing one nitrogen atom and optionally 1 or 2 additional heteroatoms selected from the group of nitrogen, oxygen and sulfur, which may be substituted by one or several halogen atoms or d- C 6 -alkyl, C ⁇ -C 6 -alkylamino, di-d-C 6 -alkylamino, C ⁇ -C 6 -alkoxy, thiol, C ⁇ -C 6 -alkylthio, oxo, imino, fomyl, C ⁇ -C 6 -alkoxy carbonyl, amino carbonyl, C 3 -C 8 -cycloalkyl, aryl, or heteroaryl groups, which may be identical to one another or different, and
  • Such heterocyclic rings are preferably azetidine or are derived from a fully or partially hydrogenated pyrrole, pyridine, thiazole, isoxazole, pyrazole, imidazole, indole, benzimidazole, indazole, pyridazine, pyrimidine, pyrazin group.
  • Most preferred are azetidine, pyrrolidine, 3,4-dehydropyrrolidine, piperidine, hexahydro-lH- azepine, octahydroindole, imidazolidine, thiazolidine.
  • the substituents are preferably methyl, ethyl, propyl, 1- methylethyl (isopropyl), butyl, 1-methylpropyl, 2-methylpropyl, 1,1 -dimethyl ethyl, hydroxyl, amino, dimethyl-amino, diethyl-amino, thiol, methyl-thiol, methoxy, ethoxy, -CHO, -COOH, -COOCH 3 , -COOCH 2 CH 3 , or -CONH 2 .
  • Aryl generally represents an aromatic ring system with 6 to 10, preferably 6 carbon atoms which may optionally be substituted by one or several hydroxyl, amino, C ⁇ -C 6 -alkyl-amino, di- C ⁇ -C 6 -alkyl-amino, d-C 6 -alkyl, C ⁇ -C 6 -alkyloxy, thiol, C ⁇ -C 6 -alkyl-thio, -CHO, -COOH, - COOCH 3 , -COOCH 2 CH 3 , -CONH 2 , or halogen substituents, which may be identical to one another or different, and which optionally may be benzocondensed.
  • Aryl substituents may be preferably derived from benzene, preferred examples being phenyl, 2-hydroxy- ⁇ henyl, 3- hydroxy-phenyl, 4-hydroxy-phenyl, 4-amino-phenyl, 2-amino-phenyl, 3-amino-phenyl.
  • the substituents are preferably methyl, ethyl, propyl, 1-methylethyl (iso- propyl), butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, hydroxyl, amino, dime- thyl-amino, diethyl-amino, thiol, methyl-thiol, methoxy, ethoxy, -CHO, -COOH, -COOCH 3 , - COOCH 2 CH 3 , or -CONH 2 .
  • Heteroaryl generally represents a 5 to 10-membered aromatic heterocyclic ring system, containing 1 to 5 heteroatoms selected from the group of nitrogen, oxygen, or sulfur, which may optionally be substituted by one or several hydroxyl, amino, C ⁇ -C 6 -alkyl-amino, di- d- C 6 -alkyl-amino, C ⁇ -C 6 -alkyl, d-C 6 -alkyloxy, thiol, C ⁇ -C 6 -alkyl-thio, -CHO, -COOH, - COOCH 3 , -COOCH 2 CH 3 , -CONH 2 , or halogen substituents, which may be identical to one another or different, and which optionally may be benzocondensed.
  • Heteroaryl substituents may preferably be derived from furane, pyrrole, thiophene, pyridine, thiazole, isoxazole, pyrazole, imidazole, benzofuran, thianaphthene, indole, benzimidazole, indazole, quinoline, pyridazine, pyrimidine, pyrazine, chinazoline, pyrane, purine, adenine, guanine, thymine, cytosine, uracil.
  • the substituents are preferably methyl, ethyl, propyl, 1-methylethyl (isopropyl), butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, hydroxyl, amino, dimethyl-amino, diethyl-amino, thiol, methyl-thiol, methoxy, ethoxy, -CHO, -COOH, - COOCH 3 , -COOCH 2 CH 3 , or -CONH 2 .
  • Residue of a cytotoxic or cytostatic compound means that the compound H 2 N-Cyt', which is released upon cleavage of the amide bond shown in formula (I), is either cytotoxic or cytostatic itself, or may be converted into a cytotoxic or cytostatic compound in a subsequent step.
  • -Cyt' may be a residue of formula -L-Cyt' ' , wherein L is a linker residue derived from a bifunctional molecule, for instance a diamine H 2 N-L'-NH 2 , an amino alcohol H 2 N-L'-OH, for example p-amino-benzyl alcohol (PABOH), an amino carbonate, for ex- ample
  • -Cyt' is of formula -L-Cyt
  • the compound H 2 N-L'- Cyt is generated by the enzymatic cleavage of the amide bond shown in formula (I).
  • the compound H N-L'-Cyt" may be cytotoxic or cytostatic itself or the linker residue cleaved off from Cyt' ' in a subsequent step releasing the cytotoxic or cytostatic agent.
  • the compound H 2 N-L'-Cyt may be hydrolysed under physiological conditions into a compound H 2 N-L'-OH and the cytotoxic or cytostatic compound H-Cyt", which is the active therapeutic agent (In the following, only the term Cyt' is used for both Cyt' and Cyt' ' , and only the term L is used for both L and L', for simplicity).
  • the pharmaceutically acceptable salts of the compounds of the present invention include the conventional non-toxic salts formed from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those from inorganic acids such as hydrochloric acid, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, maleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, oxalictrifluoroacetic and the like.
  • H 2 N-Cyt' is preferably an anthracycline derivative of formula IN, wherein
  • R c represents C ⁇ -C 6 alkyl, d-C 6 hydroxyalkyl or C ⁇ -C 6 alkanoyloxy C ⁇ -C 6 alkyl, in particular methyl, hydroxymethyl, diethoxyacetoxymethyl or butyryloxymethyl;
  • R d represents hydrogen, hydroxy or C ⁇ -C 6 alkoxy, in particular methoxy; one of R e and R f represents a hydrogen atom; and the other represents a hydrogen atom or a hydroxy or tetrahydropyran-2-yloxy (OTHP) group.
  • Cyt' may be derived for example from methotrexate, trimetrexate, pyritrexim, 5,10- dideazatetrahydrofolatepyrimetamine, trimethoprim, 10-propargyl-5,8-dideazafolate2,4- diamino-5 (3 ' ,4' -dichloropheyl)-6-methylpyrimidine, aminoglutethimide, goreserelin, melphalan, chlorambucil, analogs of other chemotherapeutic agents such as 9- aminocamptothecin (for examples see e.g. Burris HA, r. d. and S. M.
  • 9- aminocamptothecin for examples see e.g. Burris HA, r. d. and S. M.
  • Cyt' may also be a biological effector molecule which either directly or indirectly effects destruction of tumor cells, like for example TNF ⁇ .
  • Preferred anthracycline prodrugs are the compounds of formulae (IAl-1) to (IAl-3), wherein
  • the compound of the invention may contain one or more disulfide bonds.
  • cytotoxic or cytostatic compounds which may be used for the present invention has a primary amino function which is available for formation of an amidic bond as shown in formula (I), like doxorubicin.
  • a linker molecule L is not necessary. If a cytostatic or cytotoxic compound does not have such an amino function, such a function may be created in such a compound by way of chemical modification, e.g. by introducing or converting a functional group or attaching a linker molecule to the compound.
  • a linker molecule may also be inserted between the oligomeric part (i.e.
  • the part comprising the amino carboxylic residues) and the cytostatic or cytotoxic part of the compound of the invention to ensure or optimize cleavage of the amide bond between the oligomeric part and the Cytotoxic or cytostatic part.
  • a linker molecule is present, i.e. in compounds containing the structure L- Cyt', the bond between L and Cyt' is preferably an amidic or ester bond.
  • such a linker molecule is hydrolyzed off the cytostatic or cytotoxic compound under physiological conditions after the enzymatic cleavage and thus the free cytostatic or cytotoxic compound is generated.
  • the compound of the invention must have the property of being cleavable upon the catalytic action of FAP ⁇ and, as a direct or indirect consequence of this cleavage, releasing under physiological conditions a cytostatic or cytotoxic compound.
  • the present invention relates to prodrug, which exhibits an oligomeric part comprising up to 9 amino carboxylic acid residues, and a cytotoxic or cytostatic part (Cyt), wherein the N-terminal amino function of the oligomeric part is attached to a capping group (Cg); wherein the amide bond between the C-terminal amino carboxylic acid (Xaa 3 ) and the following amino acid, in particular Caa, thereof is recognized and cleaved by FAP ⁇ in the immediate environment of a target cell to obtain a prodrug intermediate of formula (IN),
  • the oligomeric part comprises two, three, four, five, six, seven, eight or nine amino carboxylic acid residues, more preferably two, three, four or five amino carboxylic residues, in particular five amino acid residues.
  • the compounds of the invention may be synthesized by processes known in the art (E. Wunsch, Synthese von Peptiden, in “Methoden der organischen Chernie”, Houben-Weyl (Eds. E. Muller, O. Bayer), Vol. XV, Part 1 and 2, Georg Thieme Nerlag, Stuttgart, 1974).
  • the peptide, or oligopeptide, sequences in the prodrug conjugates of this invention may be synthesized by the solid phase peptide synthesis (using either t-butyloxycarbonyl (Boc) or 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry) methods or by solution phase synthesis.
  • Boc t-butyloxycarbonyl
  • Fmoc 9- fluorenylmethyloxycarbonyl
  • the general Boc and Fmoc methods are widely used and are described in the following references: Merrifield, J. A. Chem. Soc, 88: 2149 (1963); Bodanszky and Bodanszky, The Practice of Peptide Synthesis, Springer-NerlaQ, Berlin, 7-161 (1994); Stewart, Solid Phase Peptide Synthesis, Pierce Chemical, Rockford, (1984).
  • a peptide of desired length and sequence is synthesized through the stepwise addition of amino acids to a growing chain which is linked to a solid resin.
  • useful Fmoc compatible resins are Wang resin, HMPA-PEGA resin, Rink acid resin, or a hydroxyethyl- photolinker resin.
  • the C-terminus of the peptide chain is covalently linked to a polymeric resin and protected a-amino amino acids were added in a stepwise manner with a coupling reagent.
  • a preferred a-amino protecting group is the Fmoc group, which is stable to coupling conditions and can readily be removed under mild alkyline conditions.
  • the reaction solvents are preferably but not limited to dimethylformamide (DMF), N-methylpyrrolidine (NMP), dichloromethane (DCM), methanol (MeOH), and ethanol (EtOH).
  • Examples of coupling agents are: dicyclohexylcarbodiimide (DCC), diisoprpoylcarbodiimide (DIG), O-(7- Azabenzotriazol-l-yl)-l,l,3,3-tetramethyluroniumhexafluorophosphate (HATU), 2-(lH- benzo1xiazol-l-yl-)-l,l,3,3-tetramethyluroniumhexafluorophosphate (HBTU).
  • N-terminal protecting group Cleavage of the N-terminal protecting group is accomplished in 10-100% piperidine in DMF at 0-40 C, with ambient temperature being preferred.
  • the final Fmoc protecting group is removed using the above N-terminal cleavage procedure.
  • the remaining peptide on resin is cleaved from the resin along with any acid sensitive side chain protecting groups by treating the resin under acidic conditions.
  • an acidic cleavage condition is a mixture of trifluroacetic acid (TFA) in dichloromethane. If the hydroxyethylphotolinker resin is used, the appropriate wavelength for inducing cleavage is X 365 nm ultraviolet light.
  • N-terminus derivatized peptides are conveniently accomplished on solid phase.
  • the capping group (Cg) of choice is coupled next using standard peptide coupling conditions onto the N terminus of the peptide.
  • Cg capping group
  • Boc chemistry either the Merrifield resin or 4-hydroxy- methylphenylacetamidomethyl (PAM) resin is useful.
  • the amino acids are coupled to the growing chain on solid phase by successive additions of coupling agent activated Boc- protected amino acids.
  • Examples of coupling agents are: DCC, DIG, HATU, HBTU.
  • the reaction solvents may be DMF, DCM, MeOH, and NMP.
  • Cleavage of the Boc protecting group is accomplished in 10- 100% TFA in DCM at 0-40 °C, with ambient temperature being preferred.
  • the N-terminus protecting group (usually Boc) is removed as described above.
  • the peptide is removed from the resin using liquid HF or trifluoromethane sulfonic acid in dichloromethane.
  • the peptide intermediate may be made via a solution phase synthesis, utilizing either Boc or Fmoc chemistry.
  • the peptide can be built up by the stepwise assembly in analogy to the solid phase method (in the N-terminal direction or in the C-terminal direction) or through the coupling of two suitably protected dipeptides or a tripeptide with a single amino acid.
  • the capping group When constructing the capping group by solution phase synthesis, the capping group needs to be synthesized by a slightly modified procedure. First the C-terminus of the Fmoc oligopeptide needs to be protected with an acid labile or hydrogenation sensitive protecting group compatible with the selective deprotection of the C-terminus over the capping group. Then the Fmoc protecting group needs to be removed from the oligopeptide to reveal the N- terminus. With the N-terminus deprotected, and the C-terminus protected the oligopeptide is reacted with the activated hemi-ester of the desired Cg. The Cg can be activated using methods for activating amino acids such as DCC or HATU in base and an appropriate solvent.
  • the compounds of formula I, wherein Cg is a capping group of formula II can be synthesized by condensation of the terminal amino function of the oligomeric part of a compound of formula VI with a compound of formula N,
  • X 1 , X 2 , s and t have the meaning given hereinabove, and LG 1 is OH or an activation leaving group, and PG 1 is H or a suitable protection group according to the following reaction scheme: + HN(R 3 )-(Xaa 3 ) m -Xaa 2 -Caa-Xaa 1 -Cyt
  • LG 1 an activation group or leaving group which is suitable to be substituted by an amino group.
  • This activation can be done by conversion of the carboxylic acid into an acid chloride or acid fluoride or by conversion of the carboxylic acid into an activated ester, for instance a N- hydroxysuccinimidyl ester or a pentafluorophenyl ester.
  • Another method of activation is the transformation into a symmetrical or unsymmetrical anhydride.
  • the formation of the amide bonds can be achieved by the use of in situ coupling reagents like benzotriazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) (E. Frerot et al., Tetrahedron, 1991, 47, 259-70), l,r-carbonyldimidazole (CDI) (K. Akaji et al., THL, 35, 1994, 3315-18), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (TBTU) (R.
  • in situ coupling reagents like benzotriazole- 1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) (E. Frerot et al., Tetrahedron, 1991, 47, 259-70), l,r-carbony
  • the compounds of the invention are intended for medical use.
  • these compounds are useful for the treatment of tumors which are associated with stromal fibroblasts that express FAP ⁇ and which are generally not optimally treated with available cytotoxic and/or cytostatic agents.
  • Tumors with this property are, for example, epithelial cancers, such as lung, breast, and colon carcinomas.
  • Tumors, such as bone and soft tissue sarcomas which express FAP ⁇ may also be treated with these compounds.
  • compositions comprising a compound of the present invention and optionally one or more suitable and pharmaceutically acceptable excipients, as exemplified in: Remington: the science and practice of pharmacy. 19th ed. Easton : Mack Publ, 1995.
  • the pharmaceutical compositions may be formulated as solids or solutions. Solid formulations may be for preparation of a solution before injection.
  • the pharmaceutical composition may, for example, be administered to the patient parenterally, especially intravenously, intramuscularly, or intraperitoneally.
  • the pharmaceutical compositions of the invention are solutions for injection.
  • compositions of the invention for parenteral administration comprise sterile, aqueous or nonaqueous solutions, suspensions, or emulsions.
  • a pharmaceutically acceptable solvent or vehicle propylene glycol, polyethylene glycol, mjectable organic esters, for example ethyl oleate, or cyclodextrins may be employed.
  • These compositions can also comprise wetting, emulsifying and/or dispersing agents.
  • the sterilization may be carried out in several ways, for example using a bacteriological filter, by incorporating sterilizing agents in the composition or by irradiation. They may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other sterile injectable medium.
  • the pharmaceutical composition may also comprise adjuvants which are well known in the art (e. g., vitamin C, antioxidant agents, etc.) and capable of being used in combination with the compound of the invention in order to improve and prolong the treatment of the medical condition for which they are administered.
  • Doses for administration to a patient of the compounds according to the invention are generally at least the usual doses of the therapeutic agents known in the field and will be adjusted according to factors like body weight and health status of the patient, nature of the underlying disease, therapeutic window of the compound to be applied, solubility, and the like, as described for example in Bruce A. Chabner and Jerry M. Collins, Cancer Chemotherapy, Lippincott Ed., ISBN 0-397-50900-6 (1990) or they may be adjusted, within the judgment of the treating physician, to accommodate the superior effectiveness of the prodrug formulations or the particular circumstances of the patient being treated.
  • the doses administered hence vary in accordance with the therapeutic agent used for the preparation of the compound according to the invention.
  • the dose will preferably be in the range from 10 rng/m 2 to 2000 mg/m 2 , but also higher or lower doses may be appropriate.
  • a further aspect of the present invention is the use of a compound of the invention in the preparation of a pharmaceutical composition for the treatment of cancer.
  • an aspect of the invention is a method of treatment of cancer, comprising administering an effective amount of a pharmaceutical composition of the invention to a patient.
  • Indications include the treatment of cancer, specifically:
  • Neuroectodermal tumors Peripheral nerve tumors, astrocytomas, melanomas; 5) Mesotheliomas.
  • a further aspect of the invention is a method of treatment of cancer, wherein a prodrug is administered to a patient wherein said prodrug is capable of being converted into a cytotoxic or cytostatic drug by an enzymatic activity, said enzymatic activity being the expression product of cells associated with tumor tissue.
  • said enzymatic activity is the proteolytic activity of FAP ⁇ .
  • One method of administration of the compounds is intravenous infusion.
  • Other possible routes of administration include intraperitoneal (either as a bolus or infusion), intramuscular or intratumoral injection. Where appropriate, direct application may also be possible (for example, lung fibrosis).
  • 1,4-phenylendiacetic acid (4.8 g, 25 mmol) was dissolved in acetonitrile (100 ml) and N,N- dimethylformamide (100 ml) The solution was cooled to 0 °C and N-hydroxysuccinimide (2,9 g, 25 mmol) and diisopropylethylamine (8.6 ml, 50 mmol) were added. The solution was stirred for 30 min at 0 °C. Then a solution of dicyclohexyl carbodiimide(5.2 g, 25 mmol) dissolved in acetonitrile (50 ml) was added dropwise over 1 h. The ice bath was removed an the suspension was stirred over night.
  • the suspension was filtered and the solvent of the filtrate was concentrated under reduced pressure. A residual amount of dicyclohexyl urea was precipitated by addition of diethyl ether and removed by filtration. The filtrate was purified by reversed phase HPLC applying acetonitrile/water gradient. The product fraction was lyophilized to give the product as white crystals (1.1 g, 16 %). The product gave satisfactory analytical data.
  • Aloc-N-MeLeu-Ala-Gly-Pro-Leu-OH (0.45 mmol) and Doxorubicin hydrochloride (0.49 mmol) were dissolved in N,N-dimethylformamide (5 ml).
  • Diisopropylethylamine (153 ⁇ 0.90 mmol) was added and the solution was cooled to 0 °C.
  • HATU O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexfluorophosphate
  • the product was purified by reversed phase HPLC applying acetonitrile/water gradient and the product fraction was lyophilized to give red crystals of H-Pro-Ala-Gly-Pro-Leu-Dox ( 95 %). The product gave satisfactory analytical data.

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Abstract

L'invention porte sur des promédicaments pouvant être convertis en intermédiaires de promédicaments d'un médicament cytotoxique ou cytostatique sous l'action catalytique de la FAPα. Lesdits promédicaments présentent: (i) une partie d'oligomère comportant jusqu'à 9 résidus amino d'acide carboxylique, la liaison amide située entre l'acide carboxylique amino C terminal et son acide aminé prédécesseur étant reconnue et scindée par la FAPα dans l'environnent intermédiaire d'une cellule cible, et (ii) une partie cytotoxique ou cytostatique, la fonction amino N terminale de la partie d'oligomère étant liée à un groupe de coiffage (Cg).
EP03727438A 2002-05-10 2003-05-06 Promedicaments antitumoraux actives par la fap Withdrawn EP1506221A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03727438A EP1506221A2 (fr) 2002-05-10 2003-05-06 Promedicaments antitumoraux actives par la fap

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EP02010552 2002-05-10
EP02010552 2002-05-10
PCT/EP2003/004713 WO2003094972A2 (fr) 2002-05-10 2003-05-06 Promedicaments antitumoraux actives par la fap
EP03727438A EP1506221A2 (fr) 2002-05-10 2003-05-06 Promedicaments antitumoraux actives par la fap

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US20100047170A1 (en) * 2006-01-05 2010-02-25 Denmeade Samuel R Peptide Prodrugs
WO2015192124A1 (fr) 2014-06-13 2015-12-17 Trustees Of Tufts College Agents thérapeutiques activés par fap, et utilisations associées
IL291922A (en) 2014-06-13 2022-06-01 Tufts College Fap-enabled medical substances and related uses
CA3203072A1 (fr) 2020-12-22 2022-06-30 Andrea CASAZZA Composes comprenant une fraction tetrapeptidique
WO2022167664A1 (fr) 2021-02-07 2022-08-11 Cobiores Nv Composés comprenant une fraction tétrapeptidique

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US6613879B1 (en) * 1999-05-14 2003-09-02 Boehringer Ingelheim Pharma Kg FAP-activated anti-tumour compounds
CA2450316A1 (fr) * 2001-06-11 2002-12-19 Medarex, Inc. Composes pour promedicaments actives cd10

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