Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
  • C07K5/06156—Dipeptides with the first amino acid being heterocyclic and Trp-amino acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
  • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
  • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

• Serial No. 07/817,039 filed January 7, 1992, and a continuation-in-part of United States patent application Serial No. 07/881,630 filed May 12, 1992.
• Serial No. 07/817,039 is also a continuation-in-part of United States patent application Serial No. 07/747,752 filed August 20, 1991 and United States patent application Serial No. 07/747,752 filed August 20, 1991, both of which are continuations-in-part of United States patent application Serial No. 07/615,798 filed November 21, 1990.
• Serial No. 07/881,630 is a continuation of U. S. patent application Serial No. 07/616,021 filed November 21, 1990.
• the invention relates to synthetic compounds that are inhibitors of matrix metalloproteases, and certain medical applications thereof.
• Collagenase and related enzymes are important in mediating the symptomology of a number of diseases, including rheumatoid arthritis (Mullins, D.E., et al., Biochim Biophys Acta (1983) 695:117-214); the metastasis of tumor cells (ibid., Broadhurst, M.J., et al., EP application 276436 (1987), Reich, R., et al., Cancer Res (1988) 48:3307- 3312); and various ulcerated conditions. Ulcerative conditions can result in the cornea as the result of alkali burns or as a result of infection by Pseudomonas aeruginosa. Acanthamoeba, Herpes simplex and vaccinia viruses. Other conditions characterized by unwanted matrix metalloprotease activity include periodontal disease, epidermolysis bullosa and scleritis.
• inhibitors to this enzyme In view of the involvement of collagenase in a number of disease conditions, attempts have been made to prepare inhibitors to this enzyme. A number of such inhibitors are disclosed in EP applications 126,974 (published 1984) and 159,396 (published 1985) assigned to G.D. Searle. These inhibitors are secondary amines which contain oxo substituents at the 2-position in both substituents bonded to the amino nitrogen.
• Inhibitors have also been disclosed for the related protease, thermolysin. These include hydroxamic peptide derivatives described by Nishino, N., et al., Biochemistry (1979) 18:4340-4347; Nishino, N., et al., Biochemistry (1978) 17:2846-2850. Tryptophan is also known to be therapeutic in various conditions, some of which may involve collagenase (see, for example, JP 57/058626; U.S. 4,698,342; 4,291,048). Also, inhibitors of bacterial collagenases have been disclosed in U.S. 4,558,034.
• the invention compounds add to the repertoire of agents available for the treatment of conditions and diseases which are characterized by unwanted activity by the class of proteins which destroy structural proteins and designated "matrix metalloprotease" herein.
• Angiogenesis is defined as the growth of new blood vessels, in particular, capillaries.
• the ingrowth of such capillaries and ancillary blood vessels is essential for tumor growth and is thus an unwanted physiological response which encourages the spread of malignant tissue and metastases.
• Inhibition of angiogenesis is therefore envisioned as a component of effective treatment of malignancy.
• Neovascularization of the eye is a major cause of blindness.
• proliferative diabetic retinopathy results from diabetes; blindness can also be caused by neovascular glaucoma.
• Inhibition of angiogenesis is useful in treating these conditions also.
• Actinonin is an antibiotic produced by a particular strain of Streptomyces and is a modified peptide structure.
• the methods and compositions of the invention are preferably utilized for preventing or treating certain diseases that have as their underlying cause the activation and/or the expression of unwanted matrix metalloprotease activity.
• diseases include skin disorders, keratoconus, restenosis, wounds, ulcers, particularly of the cornea or mouth, or those disease states that are benefitted by uncontrolled angiogenesis.
• the invention is directed to a method for treating cancer, preferably by inhibiting angiogenesis which facilitates or is required for the growth and spread of cancer throughout a patients body.
• Some members of the class of matrix metalloprotease inhibitors are known in the art; others are described and claimed in U.S. Serial No. 07/747,751, filed 20 August 1991; 07/747,752, filed 20 August 1991; and 07/615,798, filed 21 November 1990, the disclosures of which are incorporated herein by reference.
• Figure 1 shows light microscopic photographs of mouse skin exposed to PdiBu (panel A), or PdiBu and compound 5A (panel B) and stained with hematoxylin and eosin three days latter.
• Figure 2 shows that the protease levels present in mastectomy fluid samples collected on days 1 to 7 after surgery were an average of 0.75 ⁇ 0.06 ⁇ g equivalents of collagenase/ml of wound fluid.
• Figure 3 compares the protease levels present in mastectomy wound fluids collected from closed (collected on different days post operation), open, or chronic wounds. Note that closed wounds exhibited marginal protease activity, while open wound fluid contained an average protease level of 199 ⁇ 59 ⁇ g/ml, and fluids collected from chronic wounds contained an average protease level of 125 ⁇ 95 ⁇ g/ml.
• Figure 4 shows the effect of three protease inhibitors on the protease activity of chronic wound fluid.
• Compound 5A very effectively inhibited proteolytic degradation of Azocoll (approximately 96% of initial proteolytic activity) at final concentrations of 40 ⁇ g/ml (100 ⁇ M) or 4 ⁇ g/ml (10 ⁇ M).
• EDTA a nonspecific inhibitor of metalloproteinases, also effectively reduced protease activity, approximately 96%.
• PMSF a nonspecific inhibitor of serine proteases, reduced proteolytic activity approximately 65% at a concentration of 500 ⁇ M.
• Figure 5 shows the effects of the inhibitors, compound 5A, PMSF and EDTA on protease activity present in open and chronic wounds.
• Compound 5 A and EDTA were very effective inhibitors while PMSF did not significantly reduce the proteolytic activity of the wound fluids.
• Figure 6 shows the effects of compound 5 A, SI 209, ULOOl, MP506, and EDTA on the proteolytic degradation of Azocoll by wound fluids.
• Figure 7 shows the effects of the inhibitors GM6001, GM1339, GM1489 and S1209 on protease activity present in chronic wound fluid.
• the inhibitory compounds of the invention are synthetic inhibitors of mammalian matrix metalloproteases.
• Matrix metalloproteases include without limitation human skin fibroblast collagenase, human skin fibroblast gelatinase, human neutrophil collagenase and gelatinase, and human stromelysin. These are zinc-containing metalloprotease enzymes, as are the angiotensin-converting enzymes and the enkephalinases.
• “mammalian matrix metalloprotease” means any zinc-containing enzyme found in mammalian sources that is capable of catalyzing the breakdown of collagen, gelatin or proteoglycan under suitable assay conditions.
• candidate compounds to inhibit matrix metalloprotease activity can, of course, be tested in the assays described above.
• Isolated matrix metalloprotease enzymes can be used to confirm the inhibiting activity of the invention compounds, or crude extracts which contain the range of enzymes capable of tissue breakdown can be used.
• assay of inhibition activity can be conducted as follows. Inhibitors may be assayed against crude or purified human skin fibroblast collagenase using the synthetic thiol ester substrate at pH 6.5 exactly as described by Kortylewicz & Galardy, J Med Chem (1990) 33:263-273, at a collagenase concentration of 1-2 nM. The candidate inhibitors are tested for their ability to inhibit crude collagenase and gelatinase from human skin fibroblasts, crude collagenase and gelatinase from purulent human neutrophil in this assay. The results may be set forth in terms of Ki, i.e., the calculated dissociation constant for the inhibitor complex with enzyme.
• Ki values for effective inhibitors are ⁇ 500 nM for purified enzyme in this assay.
• excellent inhibitors show Ki values of 10 nM.
• Assays for inhibition of human stromelysin are conducted as described by Teahan, J., et al., Biochemistry (1989) 20:8497-8501.
• the synthetic compounds that are successful in these assays for mammalian matrix metalloprotease inhibition are generally small molecules containing at least one amide bond and have a variety of sidechain substituents. Examples of such compounds known in the art are given, as set forth above, in EP application 423,943, incorporated herein by reference.
• R 3 is H or alkyl (1-4C);
• R 4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene; n is 0, 1 or 2; m is 0 or 1; and
• X is OR 5 or NHR 5 , wherein R 5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); or
• X is an amino acid residue or amide thereof
• X is the residue of a cyclic amine or heterocyclic amine
• R 4 is fused or conjugated unsubstituted or substituted bicycloaryl methylene; n is 0, 1 or 2; m is 0 or 1; and
• X is OR 5 or NHR 5 , wherein R 5 is H or substituted or unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C); or
• X is an amino acid residue or amide thereof; or X is the residue of a cyclic amine or heterocyclic amine; Y is selected from the group consisting of R 7 ONR 6 CONR 6 -, R 6 2 NCONOR 7 -, and
• Alkyl has its conventional meaning as a straight chain, branched chain or cyclic saturated hydrocarbyl residue such as methyl, ethyl, isobutyl, cyclohexyl, t-butyl or the like.
• the alkyl substituents of the invention are of the number of carbons noted which may be substituted with 1 or 2 substituents. Substituents are generally those which do not interfere with the activity of the compound, including hydroxyl, CBZO-, CBZNH-, amino, and the like.
• Aryl refers to aromatic ring systems such as phenyl, naphthyl, pyridyl, quinolyl, indolyl, and the like; aryl alkyl refers to aryl residues linked to the position indicated through an alkyl residue. In all cases the aryl portion may be substituted or unsubstituted.
• "Acyl” refers to a substituent of the formula RCO- wherein R is alkyl or arylalkyl as above-defined. The number of carbons in the acyl group is generally 1-15; however as the acyl substitute is readily hydroxylized in vivo the nature of the group is relatively unimportant.
• Cyclic amines refer to those amines where the nitrogen is part of a heterocyclic ring, such as piperidine, "heterocyclic amines” refer to such heterocycles which contain an additional heteroatom, such as morpholine.
• R 3 are H and methyl, especially H.
• R 4 is a fused or conjugated bicyclo aromatic system linked through a methylene group to the molecule.
• fused or conjugated bicyclo aromatic system is meant a two-ringed system with aromatic character which may, further, contain one or more heteroatoms such as S, N, or O. When a heteroatom such as N is included, the system as it forms a part of formulas 1-4, may contain an acyl protecting group (1-5C) attached to the nitrogen.
• Representative bicyclo fused aromatic systems include naphthyl, indolyl, quinolinyl, and isoquinolinyl.
• Representative conjugated systems include biphenyl, 4-phenylpyrimidyl, 3-phenylpyridyl and the like.
• any available position of the fused or conjugated bicyclic system can be used for attachment through the methylene.
• the fused or conjugated aromatic system may further be substituted by 1-2 alkyl (1-4C) residues and/or hydroxy or any ring nitrogens may be acylated.
• Preferred acylation is acetylation.
• R 4 Preferred embodiments of R 4 include l-(2-methyl naphthyl)methylene; 1-quinolyl methylene; 1-naphthyl methylene; 2-naphthyl methylene; 1-isoquinolyl methylene; 3- isoquinolyl methylene; 3-thionaphthenyl methylene; 3-cumaronyl methylene; 3-(5- methylindolyl)methylene; 3-(5-hydroxyindolyl)methylene; 3-(2- hydroxyindolyl)methylene; biphenyl methylene; and 4-phenylpyrimidyl methylene; and the substituted forms thereof.
• R 4 is 3-indolylmethylene or its N- acylated derivative—i.e., that embodiment wherein the "C-terminal" amino acid is a tryptophan residue or a protected form thereof.
• a preferred configuration at the carbon to which R 4 is bound is that corresponding to L-tryptophan.
• Preferred embodiments of X are those of the formula NHR 5 wherein R 5 is H, substituted or unsubstituted alkyl (1-12C) or aryl alkyl (6-12C). Particularly preferred substitutions on R 5 are a hydroxyl group, or a phenylmethoxycarbamyl (CBZNH-) residue.
• R 5 is H, substituted or unsubstituted alkyl (1-12C) or aryl alkyl (6-12C).
• Particularly preferred substitutions on R 5 are a hydroxyl group, or a phenylmethoxycarbamyl (CBZNH-) residue.
• the compound may be extended by embodiments wherein X is an additional amino acid residue, particularly a glycyl residue, which may also be amidated as described.
• the compounds that are hydroxamates are obtained by converting a carboxylic acid or ester precursor of the formulas
• R is H or alkyl (1-6C) to the corresponding hydroxamates by treating these compounds or their activated forms with hydroxylamine under conditions which effect the conversion.
• the components forming the -NR 3 -CHR 4 COX moiety are readily available in the case of tryptophan and its analogs as esters or amides.
• many analogous fused bicyclo aromatic amino acids are described by Greenstein and Winitz (supra).
• Amino acids corresponding to those wherein R 4 is l-(2-methyl naphthyl)methylene; 1-quinolyl-methylene; 1-naphthyl methylene; 1-isoquinolyl methylene; and 3-isoquinolyl methylene can be prepared from the bicyclo aromatic methylene halides using the acetamido malonic ester synthesis of amino acids, as is well understood in the art.
• the methylene halides themselves can be prepared from their corresponding carboxylic acids by reduction with hthium aluminum hydride and bromination of the resulting alcohol with thionyl bromide.
• the hydroxylamine reagent is formed in situ by mixing the hydroxylamine hydrochloride salt with an excess of KOH in methanol and removing the precipitated potassium chloride by filtration.
• the filtrate is then stirred with the precursor activated carboxylic acid or ester of formula 5 or 6 for several hours at room temperature, and the mixture is then evaporated to dryness under reduced pressure.
• the residue is acidified, then extracted with a suitable organic solvent such as ethyl acetate, the extract washed with aqueous potassium bisulfate and salt, and then dried with a solid drying agent such as anhydrous magnesium sulfate.
• the extract is then again evaporated to dryness and crystallized.
• the substituted forms of the hydroxamate which include -NHOR 7 are synthesized in an analogous manner but substituting H 2 NOR 7 , wherein R 7 is lower alkyl or acyl (1-4C) for hydroxylamine per se.
• the resulting O-alkyl or acyl hydroxamate can then be further alkylated, if desired, to obtain the R 7 ONR 6 - derivative of the carboxylic acid.
• HNR 6 OH may be reacted with the carboxylic acid to obtain the HONR 6 - derivative.
• HNCH 3 OH and H 2 NOCH 3 are commercially available.
• X is other than OH under conditions wherein the condensation to form the amide bond occurs.
• Such conditions typically comprise mixture of the two components in a nonaqueous anhydrous polar aprotic solvent in the presence of base and a condensing agent such as a carbod ⁇ mide.
• the formation of the amide linkage can be catalyzed in the presence of standard dehydration agents such as the carbodiimides, for example dicyclohexyl carbodiimide, or N, N-carbonyl diimidazole.
• the product is then recovered as a mixture of diastereomers of formula 5 or 6. This mixture is preferably used for the conversion to the hydroxamate and one of the resulting diastereomers is crystallized directly from the product mixture. Alternatively, the diastereomers are separated by flash chromatography before conversion to the hydroxamate and recovered separately. This process is less preferred as compared to the process wherein separation of the diastereomers is reserved until the final product is obtained.
• the "A" isomer is defined as that which migrates faster on TLC; the “B” isomer as that which migrates more slowly.
• the "L” form of tryptophan or other amino acid containing a fused bicycloaromatic ring system is used as the residue, and R 1 is H
• the "A” form is that which contains the corresponding configuration at the carbon containing the R 2 substituent (providing that is the only other center of asymmetry) in the final hydroxamate product.
• the "B” isomer contains what would correspond to an "L” configuration at the carbon containing R 2 in the compounds of formula 1.
• the corresponding O- or N-alkyl hydroxylamine is reacted with the methyl ester 4A as performed for unsubstituted hydroxylamine in Example 1.
• the methyl ester 4A can be saponified to its corresponding carboxylic acid and activated with oxalyl chloride or other condensing agent.
• the alkyl hydroxylamine can then be reacted with the activated carboxyUc acid to give the O- or N-substituted hydroxamic acid.
• O- and N-methylhydroxylamine can be purchased from the Aldrich Chemical Company.
• N-alkyl hydroxylamines can be synthesized by conversion of aliphatic aldehydes to their oximes, followed by reduction to the N-alkyl hydroxylamine with borane-pyridine complex in the presence of 6N HC1 (Kawase, M. and Kikugawa, Y.J., Chem Soc, Perkin Trans (1979) 1_:643.
• Other O-alkyl hydroxylamines can be synthesized by the general methods given by Roberts, J.S., "Derivatives of
• R 7 acyl
• a hydroxamic acid of this invention can be acylated with an acid chloride, anhydride, or other acylating agent to give the compounds of this class.
• the derivatized maleic and succinic acid residues required for synthesis of the invention compounds are commercially available. If not, these can readily be prepared, in embodiments wherein R 1 is H or alkyl (1-8C) by reaction of a 2-oxocarboxylic ester of the formula R 2 COCOOR' in a Wittig reaction with an alkyl triphenylphosphoranylidene acetate or ⁇ -triphenylphosphoranylidene alkanoate.
• the methyl acetate or alkanoate is preferred, but any suitable ester can be employed.
• This reaction is conducted in a nonaqueous, nonpolar solvent usually at room temperature.
• R 1 and R 2 taken together are (CH 2 ) p
• the compounds of the invention are prepared analogously to the manner set forth in Reaction Scheme 1, except that the intermediate of the formula ROOCCHR 1 CHR 2 COOH is prepared from the corresponding 1,2-cycloalkane dicarboxylic acid ⁇ i.e., 1,2-cyclopentane dicarboxylic acid anhydride; 1,2-cyclohexane dicarboxylic anhydride or 1,2-cycloheptane dicarboxylic anhydride.
• Preferred compounds of formula (1) or (2) include:
• the compounds are prepared by acylating an ⁇ , ⁇ or ⁇ amino acid, respectively with methyl or ethyl chloroformate, condensing d e resulting amino acid with a protected form of the moiety -NR 3 CHR 4 COX and reacting the resulting carboethoxy "dipeptide" with hydroxylamine or a substituted hydroxylamine as described by Fieser, L.F., et al., "Reagents for Organic Synthesis” (1967) L479 (John Wiley & Sons, New York). This sequence of reactions is shown in Reaction Scheme 1A.
• the ⁇ , ⁇ or ⁇ amino acid is temporarily protected using, for example, carbobenzoxy or tertiary butyloxycarbonyl and coupling it to the carboxy- terminal-protected amino acid moiety containing R 4 .
• the protecting group is then removed by hydrogenolysis or acidolysis as appropriate, and the deprotected ⁇ , ⁇ or ⁇ amino group is reacted with an activated carbonic acid such as carbonyldiimidazole.
• the resultant is then reacted with hydroxylamine or substituted hydroxylamine to obtain the desired product.
• Reaction Scheme 2 In the formula Im-Co-Im, Im represents an imidazole residue.
• Y has the formula R 6 2 NCONOR 7 - and n is 0, 1 or 2.
• R 6 NCONOR 7 - and n is 0, 1 or 2.
• R 7 ONH(CHR 1 ) n CHR 2 COOH.
• R 6 are H
• this intermediate is converted to the desired hydroxyurea by reaction with silicon tetraisocyanate, as described by Fieser and Fieser, "Reagents for Organic Synthesis" (1968) 1:479 (John Wiley & Sons, New York).
• the reaction is conducted with the hydroxyl group protected or substituted by R 7 .
• the resulting hydroxyurea is then coupled to the component of the formula HNR 3 CHR 4 COX to obtain the desired product.
• the amide is first formed and the N-hydroxyl dipeptide is treated with the reagent.
• the ⁇ -N-hydroxyamino acids used as intermediates in the foregoing synthesis can be prepared by a malonic ester synthesis in which diethyl malonate is alkylated twice, one with R 2 -Br and then with benzylchloromethyl ether, for example, for the case wherein R 1 is H.
• the product is saponified, decarboxylated, hydrogenated, and oxidized to give the ⁇ -aldehyde in a manner similar to the synthesis of a homologous aldehyde described by Kortylewicz, Z.P., et al., Biochemistry (1984) 23:2083-2087.
• the desired ⁇ -hydroxyamino acid is then obtained by addition of protected (or alkylated, if R 7 is alkyl or acylated if R7 is acyl) hydroxylamine.
• protected (or alkylated, if R 7 is alkyl or acylated if R7 is acyl) hydroxylamine is then obtained by addition of protected (or alkylated, if R 7 is alkyl or acylated if R7 is acyl) hydroxylamine.
• protected (or alkylated, if R 7 is alkyl or acylated if R7 is acyl) hydroxylamine protected (or alkylated, if R 7 is alkyl or acylated if R7 is acyl) hydroxylamine.
• those compounds wherein Y is of the formula R 6 CONOR 7 - can be prepared by acylation of the corresponding ⁇ , ⁇ or ⁇ N- hydroxy dipeptide.
• the N-hydroxyamino acid can be acylated, followed by condensation to form the amide bond in the compounds of the invention.
• the acylation method is described by, for example, Nishino, N., et al., Biochemistry (1979) 18:4340-4346, cited above.
• the compounds can be prepared by condensing the ylide l,l-dimethoxy-2-(triphenylphosphoranylidene) ethane prepared from triphenylphosphine and l,l-dimethoxy-2-bromoethane with 4- methyl-2-oxopentanoic acid. The product is then hydrogenated to obtain 4,4-dimethoxy- 2-isobutylbutanoic acid which is coupled to the moiety R 3 NHCHR 4 COX to obtain 4,4- dimethoxy-2-isobutylbutanoyl-NR 3 CHR 4 COX.
• a number of diseases are known to be mediated by excess or undesired matrix-destroying metalloprotease activity. These include tumor metastasis, rheumatoid arthritis, skin inflammation, ulcerations, particularly of the cornea or mouth, reaction to infection, and the like.
• Also intended to come within the definition of diseases that can be treated by the invention inhibitors are wounds, preferably chronic dermal wounds.
• the inhibitors of the invention are, however, useful in any ulcerative skin condition, including, for example, decubitus ulcers, ulcers of e mouth, or other conditions where wound healing is slow.
• the compounds of the invention are useful in therapy with regard to conditions involving this unwanted activity.
• the compounds of the instant invention are particularly useful for treating or preventing psoriasis.
• Psoriasis is a common inflammatory skin disease of uncertain etiology, which is characterized by prominent epidermal hype ⁇ lasia, mixed inflammatory infiltrates and vascular alterations.
• the molecular mechanism(s) responsible for epidermal hype ⁇ lasia in psoriasis and other skin disorders remain unresolved.
• various growth factors, cytokines and proto-oncogenes have been implicated in the transduction of growth-promoting signals from the extracellular environment into the epidermal keratinocyte.
• Still another condition responsive to the matrix metalloprotease inhibitors of the invention include restenosis following angioplasty.
• the healthy arterial wall is composted of an outer adventitial layer of fibroblasts, a central medial layer of smooth muscle cells and a luminal intimal layer of endothelial cells.
• one cause of restenosis following balloon angioplasty is the production and release of collagenase by smooth muscle cells that causes degradation of the intima.
• matrix metalloprotease inhibitors of the invention would prevent or inhibit restenosis when administered before or after angioplasty.
• Yet another application of the matrix metalloprotease inhibitors of the invention is the treatment or prevention of cancer, particularly metastatic cancer. Cancer cells migrate from their primary site of origin to remote secondary sites by extravasation into the blood, and subsequent extravasation out of the blood to the target organ. Thus, it would be possible to prevent or eliminate metastasis if extravasation of cancer cells could be controlled. Since a key process in extravasation is the breakdown of the extracellular matrix by enzymes secreted by cancer cells, particularly collagenases, the collagenase inhibitors of the invention have significant applications for the treatment or prevention of cancer.
• the collagenase inhibitors of the invention are useful in any ulcerative skin condition, including, for example, decubitus ulcers, ulcers of the mouth, or other conditions where wound healing is slow. Similar conditions susceptible to treatment by the compounds of the invention include corneal or scleral melting associated with keratomalacia, scleromalacia perforans and connective tissue diseases. An example of the latter is keratoconus which involves thinning and central protuberance of the cornea. Type IV collagenase is thought to be responsible, at least in part, for the disease. Compounds which are synthetic inhibitors of mammalian metalloproteases are useful to inhibit angiogenesis. These compounds can therefore be formulated into pharmaceutical compositions for use in inhibiting angiogenesis in conditions characterized by an unwanted level of such blood vessel growth.
• Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Pubhshing Company, Easton, PA, latest edition.
• the compounds be injected. These conditions include tumor growth and metastasis.
• the compounds can be formulated for injection using excipients conventional for such p pose such as physiological saline, Hank's solution, Ringer's solution, and the like. Injection can be intravenous, intramuscular, intraperitoneal or subcutaneous. Dosage levels are of the order of 0.1 mg/kg of subject to 100 mg/kg of subject, depending, of course, on the nature of the condition, the nature of the subject, the particular embodiment of the invention compounds chosen, and the nature of the formulation and route of administration.
• the compounds of the invention can also be formulated into compositions for transdermal or transmucosal delivery by including agents which effect penetration of these tissues, such as bile salts, fusidic acid derivatives, cholic acid, and the like.
• the compounds can also be used in liposome- based delivery systems and in formulations for topical and oral administration depending on the nature of the condition to be treated. Oral administration is especially advantageous for those compounds wherein the moiety -CONR 3 - is in a modified isosteric form. These compounds resist the hydrolytic action of the digestive tract.
• Oral formulations include syrups, tablets, capsules, and the like, or the compound may be administered in food or juice.
• the inhibitors of the invention can be targeted to specific locations where vascularization occurs by using targeting ligands.
• the inhibitor is conjugated to an antibody or fragment thereof which is immunoreactive with a tumor marker as is generally understood in the preparation of immunotoxins in general.
• the targeting ligand can also be a ligand suitable for a receptor which is present on the tumor. Any targeting ligand which specifically reacts with a marker for the intended target tissue can be used. Methods for coupling the compounds to the targeting ligand are well known and are similar to those described below for coupling to carrier.
• the conjugates are formulated and administered as described above.
• topical administration is preferred.
• direct application to the affected eye may employ a formulation as eyedrops or aerosol.
• the compounds of the invention can also be formulated as gels or ointments, or can be inco ⁇ orated into collagen or a hydrophilic polymer shield.
• the materials can also be inserted as a contact lens or reservoir or as a subconjunctival formulation.
• the compounds of the invention can be administered alone or as mixtures, and the compositions may further include additional drugs or excipients as appropriate for the indication.
• Conditions that benefit from angiogenesis inhibition thus include, generally, cancer, including angiosarcoma, Kaposi's sarcoma, glioblastoma multiforme, hemangioblastoma, including von Hippel-Lindan disease and hemangiopericytoma; eye conditions, such as diabetic retinopathy and neovascular glaucoma; immune system conditions, such as rheumatoid arthritis, angiolymphoid hype ⁇ lasia with eosinophilia; and skin conditions, such as cavernous hemangioma (including Kasabach-Merritt syndrome) and psoriasis.
• TLC solvent systems are as follows: (A) ethyl acetate/methanol (95:5); (B) ethyl acetate/methanol (25:5); (C) ethyl acetate; (D) ethyl acetate/methanol (30:5); (E) ethyl acetate/hexane (1:1); (F) chloroform/me thanol/acetic acid (30:6:2); (G) chloroform methanol/acetic acid (85:10:1).
• Example 1 Preparation of N-[D,L-2-isobutyl-3-(N'- hvdroxycarbonylamido)-propanoyll -tryptophan methylamide
• a suspension of 5 g (0.033 mol) of the sodium salt of 4-methyl-2-oxopentanoic acid and 5.65 g (0.033 mol) of benzyl bromide in 10 ml of anhydrous dimethylformamide was stirred for 4 days at room temperature. After evaporation of the solvent under reduced pressure the residue was diluted to 100 ml with hexane and washed with water (3 x 20 ml) and saturated sodium chloride and dried over anhydrous magnesium sulfate. Evaporation of solvent gave 6.4 g (88% yield) of the benzyl ester of 4-methyl-2-oxopentanoic acid (I) as a colorless oil.
• Isomers 4A and 4B were separated by flash chromatography (silica gel, ethyl acetate).
• the 5A can be crystallized directly from the residue as described above.
• the corresponding compounds of formula 1 are prepared wherein R 1 is H and R 2 is an n-propyl, i-propyl, n-butyl, 2- methylbutyl, and n-octyl, respectively.
• R 1 is H and R 2 is an n-propyl, i-propyl, n-butyl, 2- methylbutyl, and n-octyl, respectively.
• the corresponding compounds of formula 2 wherein R 1 is H and R 2 is as set forth above are obtained.
• the intermediate ester of formula 3 or 4 is deesterified and acylated prior to conversion to the hydroxamate.
• 4A is deesterified with sodium hydroxide in ethanol and then acidified to give N-(L-2-isobutyl-3-carboxypropanoyl)-L-tryptophan methylamide, which is treated with the anhydride of an alkyl (1-4C) carboxylic acid to obtain N-(L-2-isobutyl-3-carboxypropanoyl)-L-((N-acyl)indolyl)-tryptophan methylamide.
• This intermediate is then treated with oxalyl chloride followed by hydroxylamine at low temperature to give the corresponding hydroxamate.
• N-methyl-L-tryptophan methylamide 9 A
• N-r2-isobutyl-3-(N-hvdroxycarbonyl amido)- propanovn-L-3-(2-naphthyl)-alanine methylamide (11 A) N-[D,L-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-3-(2-naphthyl)-alanine l0A was prepared as described in Example 1 from L-3-(2-naphthyl)-alanine methylamide and
• Example 5 Preparation of N-r2-isobutyl-3-(N'-hvdroxycarbonyl amido)-propanoyll-L- tryptophan 2-hvdroxyethylamide (13 A)
• the hydrochloride salt of L-tryptophan 2-hydroxyethylamide was prepared and coupled with 3. as described for the hydrochloride salt of L-tryptophan methylamide in Example 1 except that ,3 was activated with 1,1 '-carbonyldiimidazole for 20 minutes in methylene chloride at room temperature.
• the crude product was a mixture of 0.7 g (67% yield) of the diastereoisomers 12A.B: R f (C) 12A 0.38, R C) 12B 0.19. 12A crystallized from ethyl acetate in 35% yield (0.18 g): mp 161-163°C,
• Example 6 Preparation of N-r2-isobutyl-3-(N'-hvdroxycarbonyl amido)-propanoyll -L-tryptophan amylamide (15 A) The hydrochloride salt of L-tryptophan amylamide was prepared as described in Example 1 for L-tryptophan methylamide and was reacted with 3_ that had been activated with 1,1 '-carbonyldiimidazole for 20 minutes in dichloromethane at room temperature.
• Example 8 Preparation of N-r2-isobutyl-3-(N'-hvdroxycarbonyl amido)-propanov ⁇ ⁇ l-L-trvptophan dodecylamide (19A)
• the reaction of L-tryptophan dodecylamide was prepared in a manner analogous to that described for L-tryptophan methylamide in Example 1. This ester was reacted with 3 , as described in Example 1 to give crude N-[D,L-isobutyl-3-(methoxycarbonyl)- propanol] -L-tryptophan dodecylamide 18A B in 93% yield as a mixture of isomers 19 A and 19B.
• the compound 25A was converted to N-[L-2-isobutyl-3- methoxycarbonylpropanoyl]-L-tryptophan (6-phenylmethoxycarbonylamino-hexyl- l)amide (26A) as follows. A mixture of 0.55 g (1.47 mmol) of 25A and 0.24 g (1.48 mmol) of CDI in 1 mL of 2% dimethylformamide in methylene chloride was stirred for 0.5 hr at room temperature and 0.42 g (1.47 mmol) of 23 was added.
• Example 12 Preparation of N-rcis-2-(N'-hvdroxycarbonyl- amido)-cvclohexylcarbonyll-L-tryptophan methylamide (29A,B)
• a mixture of 2 g (0.013 mol) of cis-l,2-cyclohexane-dicarboxylic anhydride in 15 mL of methanol was refluxed for 5 hr, then evaporated to dryness under reduced pressure to give 2.41 g (100% yield) of cis-2-methoxycarbonyl-cyclohexanecarboxylic acid.
• Example 13 Preparation of N-rtrans-2-(N'-hydroxycarbonyl- amido)-cvclohexylcarbonyl " l-L-tryptophan methylamide (30A,B)
• Example 14 Preparation of N-r2-isobutyl-3-(N'-hvdroxycarbonyl- amido)-propanoyll -L- tryptophan (31 A) 31A was prepared from 25A in Example 10 in a similar manner to the preparation of 5A in Example 1 in 75% yield (128 mg) and isolated as a foam from ethyl acetate: MS-FAB (m/z) (M + + H). A small sample of 31 A recrystallized from ethyl acetate had a melting point of 116-120°C.
• Example 18 Preparation of N-(trans-2-carboxy-cvclohexylcarbonyl)- L-tryptophan methylamide 36
• Example 19 Preparation of N-r2-isobutyl-3-(N'-acetoxycarbonylamido)- propanoyll-L-tryptophan methylamide (37 A) To 97.5 mg (0.25 mmol) of 5A (Example 1) in 0.5 ml of dimethylformamide was added 25.5 mg (0.25 mmol) of acetic anhydride and 37 mg (0.25 mmol) of 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) at room temperature. After standing overnight, the DMF was evaporated under high vacuum and the residue taken up in a mixture of equal volumes of ethyl acetate and 2% potassium bisulfate.
• DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
• Example 22 Inhibition of Angiogenesis
• TPA 12-0-tetradecanoylphorbol-13-acetate
• the phorbol ester, PdiBu (20 nmol in 20 ⁇ l if acetone) was applied to both ears of hairless mice (Hr/Hr) (approximately 1 cm 2 each).
• the test compounds (in total volume of 20 ⁇ l) were then applied to the right ear immediately (15 to 30 min) following PdiBu.
• the left ear of each animal received an equivalent amount (20 ⁇ l) of vehicle.
• Test compounds (and vehicle) were reapplied at 6 and 18 hours following PdiBu. Treatment times were staggered to allow exact time intervals to be obtained.
• animals were anesthetized, and ear thickness values were obtained using a microcaliper.
• the weights of punch biopsies (6mm) were then obtained. Histology was performed on selected samples taken at 30 hours.
• Test compounds included GM6001 (lOmg/ml in ETOH), a negative control, acetohydroxamic acid (AHA; 2mg/ml in ETOH), and fluocinonide (Lidex R ) as a positive control (0.05% in vehicle of alcohol [35%], diisopropyl adipate, citric acid and propylene glycol).
• the controls for this series include: 1) Untreated controls; 2) PdiBu plus vehicle alone (included for each mouse tested); 3) PdiBu plus AHA; 4) PdiBu plus Lidex R as a positive control.
• epidermal hype ⁇ lasia resultsed in more normal appearing epidermal mo ⁇ hology (i.e. reduction of PdiBu-induced parakeratosis, and reduction of irregular basaloid, spinous and granular cell shape and distribution).
• Histologic analysis were performed both on ear and flank samples with similar results; both sites received 20 nmol PdiBu; however, ear received 20 ⁇ l 6001 (200 ⁇ g) over an area of approximately 1 cm 2 , while flank received 50 ⁇ l (500 ⁇ g) over an area of approximately 4 cm 2 .
• Skin samples were prepared using standard histologic methods and stained with hematoxylin/eosin.
• FIG 1 shows typical sections of mouse skin exposed to PdiBu (Panel)
• AHA Control Topical AHA (0.53 ⁇ mol/ear applied at each of three timepoints: 0.25, 6.0, and 24 hours) did not alter the PdiBu-induced increase in ear thickness and biopsy weight:
• Thickness (inch x 10 "3 )
• Lidex R fluocinonide
• GM6001 demonstrated potent anti-inflammatory activity in this standard in vivo model for psoriasis.
• the extent of anti-inflammatory activity was nearly comparable to that observed with Lidex R , the positive control.
• the reduction of PdiBu-induced ear weight was accompanied by similar inhibition of ear thickening.
• Acetohydroxamic acid (AHA) was used as a negative control since it is devoid of inhibitory effects on MMPs. It did not alter PdiBu-induced effects on ear thickness or weight.
• Fluids were collected from 3 types of human wounds termed closed spontaneously healing wounds, open slowly healing wounds, and open chronic wounds.
• the first category were fluids collected from the chest wall of women following mastectomy surgery. Fluids from non-infected wounds that were left open for valid surgical reasons were considered open slowly healing wounds. An occlusive dressing was placed over the wound bed, and fluids were collected after 2-6 hours of occlusion. Finally, fluids were also collected from chronic open wounds by covering the wounds with occlusive dressings. Wounds were considered to be chronic if they were not clinically infected and had been open and not healing for more than 4 weeks.
• the fluids were centrifuged, and the supernatants filtered using a 0.45 u sterile Gelman filter. The filtrates were stored frozen at -80°C until tested for protease activity.
• the effects of four inhibitors on the protease activity present in wound fluids were determined.
• the inhibitors were: compound 5 A, also termed GM 6001 [NHOHCOCH2CH(i-Bu)CO-tryptophan-NHME], GM1339 [NHOHCOCH2CH(i-Bu)CO- tryptophan-NHCHMePh], GM1489 [HOOCCH2CH(i-Bu)CO-tryptophan-NHCHMePh], and S1209 [NHOHCOCH2CH(i-Bu)CO-tyrosine-OMeNHMe].
• inhibitors were compared to certain other inhibitors and these were ULOOl [HSCH2CH(CH2CH(CH3)2)CO-Phe-Ala-NH2] obtained from Peptides International and MP506, obtained from Elastin Products.
• EDTA ethylenediamine tetraacetic acid
• PMSF phenylmethylsulfonyl fluoride
• Stock solutions of the four inhibitors were all prepared at a concentration of 800 ⁇ g/ml. Due to different solubility properties of the inhibitors, different techniques were utilized.
• GM6001 was dissolved in an amount of warmed propylene glycol to give a final concentration of 2.4%, then dissolved in lOmM citrate, pH 5.5, containing 0.05% methyl cellulose.
• the protease substrate, Azocoll was obtained from Sigma Chemical Co ⁇ oration and it is a substrate for collagenase/gelatinase-like metalloproteinase enzymes and general proteases.
• Clostridium histolyticum collagenase (the crude form of the enzyme) was from Worthington Biochemicals.
• General chemicals including TRIS buffer, DMSO, and CaCl 2 were from Sigma Chemical Co ⁇ oration.
• 900 ⁇ l of the Azocoll substrate suspension in buffer (5 ⁇ g/ml in 50 mM TRIS, pH 7.8, ImM CaCl 2 ) was added to 1.5ml microcentrifuge tubes then 50 ⁇ l of inhibitor (or buffer) and 50 ⁇ l of chronic wound fluid (or collagenase standard) were added to the reaction tube.
• the reaction tubes were placed at 37°C in a shaker that inverted the tubes 30 times per minute. After 24 hours of incubation, the reaction tubes were centrifuges at 10,000 X g and the absorbance of the supernatant solution was measured at 520 nm with a Milton-Roy spectrophotometer.
• Proteolysis of Azocoll substrate generates soluble colored fragments from the insoluble Azocoll substrate.
• Wound fluid samples were incubated alone or with die inhibitors. Controls included incubation of the Azocoll substrate with the assay buffer to measure spontaneous degradation of the substrate.
• a standard curve for digestion of the Azocoll substrate was generated by incubation of the Azocoll with crude bacterial collagenase.
• Protease levels were expressed as net ⁇ g of collagenase equivalent per ml of chronic wound fluid. In the figures, certain of the wound fluids are referred to by an individual name. Figure 2 shows the results.
• Mastectomy fluid samples collected on days 1 to 7 after surgery contained low levels of protease activity with an average of 0.75 ⁇ 0.06 ⁇ g equivalents of collagenase/ml of wound fluid.
• figure 3 shows that wound fluids collected from open wounds contained an average protease level of 199 ⁇ 59 ⁇ g/ml of wound fluid, and fluids collected from chronic wounds contained an average protease level of 125 ⁇ 95 ⁇ g/ml. Only one of the thirteen samples of fluids, L. Smith, from chronic or open wounds did not contain measurable levels of Azocoll hydrolysis activity. The protease levels of the remaining twelve samples of open and chronic wounds were all higher than the levels measured in mastectomy fluids and ranged from 2 to 585 ⁇ g/ml wound fluid.
• fluids collected from chronic and open wounds contain very high levels of Azocoll-degrading protease activity compared to fluid collected from mastectomy drains. This suggests that the in vivo environment of open or chronic wounds contains proteases that have the ability to degrade extracellular matrix proteins of wounds.
• GM6001 very effectively inhibited proteolytic degradation of Azocoll (approximately 96% of initial proteolytic activity) by a chronic wound fluid when added at final concentrations of 40 ⁇ g/ml (100 ⁇ M) or 4 ⁇ g/ml (10 ⁇ M).
• GM6001 and EDTA effectively inhibited me protease activity of a chronic wound fluid, but GM6001 was much more potent that EDTA.
• PMSF was not an effective inhibitor except at the highest concentration. This indicates that much of the proteolytic activity present in the chronic wound fluid was due to metalloproteinases. The inhibition observed with high concentration of PMSF was most likely due to nonspecific inhibition of non-serine proteases which has been reported at high concentrations of PMSF (see, for example, Arch. Biochem. Biophys. 124:70). Additional experiments were conducted to ascertain the effects of certain inhibitors on open and chronic wounds, and die results are shown in Figure 5. GM6001 and EDTA were very effective inhibitors while PMSF did not significantly reduce proteolytic activity of the wound fluids.
• GM6001 and EDTA consistently reduced proteolytic activity by 95% in open or chronic wound fluids with high levels of Azocoll-degrading protease activity.
• PMSF did not reduce the levels of proteolytic activity. This supports the general concept that open and chronic wounds consistently have elevated levels of matrix metalloproteinases which can effectively be inhibited by GM6001.
• GM6001 and SI 209 are very effective inhibitors of Azocoll-degrading proteases in a larger series of wound fluids.
• MP506 was a somewhat less effective inhibitor than GM6001 or S1209.
• GM1339, GM1489, and S1209 on the protease activity present in chronic wound fluid.
• different techniques were utilized.
• GM6001 was dissolved in an amount of warmed propylene glycol to give a final concentration of 2.4%, then dissolved in 10 mM citrate, pH 5.5, containing 0.05% metiiyl cellulose.
• S1209 was dissolved in 1% DMSO then dissolved in lOmM citrate, pH 5.5, containing 0.05% methyl cellulose.
• GM1489 was dissolved in propylene glycol (to give a final concentration of 2.4%) then dissolved in 1 mM CaCl 2 , 50 m Tris-Cl, pH 7.8.
• GM1339 was dissolved in propylene glycol (2.4%) metiiyl cellulose (0.05%) and 10 mM citrate, pH 8.
• the chronic wound fluid contained a high level of protease activity with an average of 284 ⁇ 52 ⁇ g collagenase equivalents/ml of wound fluid.
• Addition of GM6001 at 800 ⁇ g/ml reduced the level of protease activity by 84% to 45 ⁇ 1 ⁇ g collagenase equivalents/ml of wound fluid.
• GM6001 lower concentrations of GM6001 resulted in slighdy higher levels of protease activity up to 90 ⁇ 6 ⁇ g collagenase equivalents ml of wound fluid.
• GM1339 and SI 209 also effectively inhibited the protease activity of the chronic wound fluid, with the highest concentration (800 ⁇ g/ml) inhibiting 94% and 70%, respectively (see Table 1).
• the highest level of GM1489 only inhibited 23% of the protease activity, and 13% and 30% increases were measured at the 8 ⁇ g/ml and 0.8 ⁇ g/ml concentrations.
• GM1489 was significantly less potent that GM6001, GM1339, or S1209. and lower concentrations of GM1489 actually increased the level of proteolytic activity of the chronic wound fluid.

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