WO2022167087A1 - Dipeptides cytotoxiques renforcés par une peptidase - Google Patents

Dipeptides cytotoxiques renforcés par une peptidase Download PDF

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Publication number
WO2022167087A1
WO2022167087A1 PCT/EP2021/052793 EP2021052793W WO2022167087A1 WO 2022167087 A1 WO2022167087 A1 WO 2022167087A1 EP 2021052793 W EP2021052793 W EP 2021052793W WO 2022167087 A1 WO2022167087 A1 WO 2022167087A1
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compound
phenyl
amino
cancer
ethyl
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PCT/EP2021/052793
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English (en)
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Fredrik Lehmann
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Oncopeptides Ab
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Priority to PCT/EP2021/052793 priority Critical patent/WO2022167087A1/fr
Publication of WO2022167087A1 publication Critical patent/WO2022167087A1/fr

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    • 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
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic

Definitions

  • the present invention relates to compounds that are peptidase enhanced cytotoxics (PEnC), and the use of such compounds in the treatment of diseases such as cancer.
  • PnC peptidase enhanced cytotoxics
  • APN APN-targeted carrier constructs
  • Peptidase Enhanced Cytotoxics are a class of lipophilic peptides that exhibit increased activity via peptidase cleavage and have the potential to treat many cancers.
  • the present inventors have found a new family of Peptidase Enhanced Cytotoxics, which find utility in the treatment of cancers.
  • the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, including a salt of such an ester, amide or carbamate,
  • Ri is H or -Ci-ealkyl
  • R2 is -CI-63 I kyl; -CHz-indolyl; -CHz-phenyl; or -CH2-5-membered heteroaryl comprising 1, 2, 3 or 4 nitrogen or sulphur atoms; wherein -Ci-ealkyl is optionally substituted by -OH; and phenyl is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen; -NH2; -OH; -O-Ci-ealkyl; -N(Ci-ea Ikyl optionally substituted by 1, 2 or 3 halogens; and -NO2; and
  • R3 is H or -C(O)R a ; wherein R a is -Ci-ealkyl or -CH2-phenyl optionally substituted by 1, 2 or 3 halogens.
  • compounds of formula (I) are potent anticancer agents.
  • the present inventors have found using an in vitro cytotoxicity assay that compounds of formula (I) display cytotoxic activity against haematological and solid cancer cells.
  • compounds of formula (I) are particularly effective at inhibiting tumor growth and metastasis in an ex ova chick embryo chorioallantoic membrane (CAM) model of human osteosarcoma.
  • CAM ex ova chick embryo chorioallantoic membrane
  • the invention further provides a pharmaceutical composition comprising a compound according to the invention, together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may optionally comprise an additional therapeutic agent, for example a protease inhibitor (PI), an immunomodulatory drug (I MiD) or an alkylator.
  • PI protease inhibitor
  • I MiD immunomodulatory drug
  • the invention further provides a compound or a pharmaceutical composition according to the invention for use as a medicament.
  • a compound or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of cancer, for example multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.
  • the invention further provides a method of treating a patient which comprises administering a pharmaceutically effective amount of a compound or pharmaceutical composition according to the invention.
  • the invention further provides the use of a compound according to the invention for the manufacture of a medicament for the treatment or prophylaxis of cancer.
  • Figure 1 is a schematic study time line of experimental example 2.
  • Figure 2 shows the mean values ⁇ SEM of tumour weight measured for each MNNG/HOS cell line experiment group.
  • Figure 3 shows the relative amount of metastasis in low CAM for each MNNG/HOS cell line experiment group, compared to negative control (arbitrary value for metastasis is 1).
  • Figure 4 shows the number of dead and surviving embryo for each MNNG/HOS cell line experiment group.
  • Figure 5 is a representative picture of tumors (ex ovo) for the negative control group and the 200 pM Example Compound 1 group of the MNNG/HOS cell line experiment groups.
  • Figure 6 shows the mean values ⁇ SEM of tumour weight measured for each 143B cell line experiment group.
  • Figure 7 shows the relative amount of metastasis in low CAM for each 143B cell line experiment group, compared to negative control (arbitrary value for metastasis is 1).
  • Figure 8 shows the number of dead and surviving embryo for each 143B cell line experiment group.
  • Figure 9 is a representative picture of tumors (ex ovo) for the negative control group and the 200 pM Example Compound 1 group of the 143B cell line experiment groups.
  • the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide or carbamate thereof, including a salt of such an ester, amide or carbamate.
  • the present inventors have found that compounds of formula (I) are surprisingly cytotoxic towards haematological and solid cancer cells. Notably, the present inventors have demonstrated using an ex ova CAM model of human osteosarcoma that compounds of formula (I) are particularly effective at inhibiting growth and metastasis of human osteosarcoma tumors.
  • R 1 is selected from H or -Ci-ealkyl.
  • R 1 is H or -Ci-4alkyl.
  • R 1 may be H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or sec-butyl. More preferably, R 1 is H, ethyl or isopropyl.
  • R 2 is selected from the group consisting of -Ci-ealkyl; -CHz-indolyl; -CHz-phenyl; and -CH2-5-membered heteroaryl comprising 1, 2, 3 or 4 nitrogen or sulphur atoms; wherein -Ci-ealkyl is optionally substituted by -OH; and phenyl is optionally substituted by 1, 2 or 3 substituents independently selected from the group consisting of halogen (for example, F, Cl, Br or I); -NH2; -OH; -O-Ci-ealkyl; -N(Ci-ea Ikyl optionally substituted by 1, 2 or 3 halogens (for example, F, Cl, Br or I); and -NO2.
  • halogen for example, F, Cl, Br or I
  • R 2 may be -Ci-4a Ikyl; -CH2-indolyl; -CH2-phenyl; or - CH2-5-membered heteroaryl comprising 1, 2 or 3 nitrogen or sulphur atoms; wherein -CI-43 I ky I is optionally substituted by -OH; and phenyl is optionally substituted by 1 or 2 substituents independently selected from the group consisting chloro; fluoro; -NH2; -OH; -O-Ci-4alkyl; -N(Ci-4a I ky 1)2 optionally substituted by 1 or 2 chlorine or fluorine atoms; and -NO2.
  • heteroaryl comprising 1, 2, 3 or 4 nitrogen or sulphur atoms means an aromatic cyclic group of carbon atoms wherein one, two, three or four of the carbon atoms is/are replaced by one, two, three or four heteroatoms independently selected from nitrogen and sulphur.
  • 5-membered heteroaryl comprising 1, 2, 3 or 4 nitrogen or sulphur atoms include thiophene, thiazole, isothiazole, pyrrole, pyrroline, pyrazole, pyrazoline, imidazole, imidazoline, triazole, thiadiazole, and tetrazole.
  • R 2 is -CH2-5-membered heteroaryl comprising one nitrogen or sulphur atom; or -CHz-phenyl optionally substituted by 1 or 2 substituents independently selected chloro; fluoro and -O-Ci-4alkyl.
  • R 2 may be -CHz-thiophene; - CHz-pyrrole; -CHz-pyrroline; or -CHz-phenyl optionally substituted by 1 or 2 chlorine or fluorine atoms, or 1 or 2 methoxy groups. More preferably, R 2 is -CHz-thiophene; or -CHz-phenyl optionally substituted by 1 or 2 chlorine or fluorine atoms, or one methoxy group.
  • R 2 may be -CHz-thiophene; methoxybenzyl; fluorobenzyl; chlorobenzyl; difluorobenzyl; or dichlorobenzyl. Even more preferably, R 2 is -CHz-thiophene; 4-methoxybenzyl; 4-fluorobenzyl; 4-chlorobenzyl; 2,4- difluorobenzyl; or 2,4-dichlorobenzyl.
  • R 3 is selected from the group consisting of H and - C(O)R a .
  • R 3 is H or -C(O)R a . More preferably, R 3 is H.
  • R a is -Ci-ealkyl or -CHz-phenyl optionally substituted by 1, 2 or 3 halogens (for example, F, Cl, Br or I).
  • R a is -Ci-4a I kyl or -CHz-phenyl optionally substituted by 1 or 2 chlorine or fluorine atoms.
  • R a may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, phenyl, fluorophenyl, chlorophenyl, difluorophenyl, or dichlorophenyl.
  • R 3 may be -C(O)CH3, - C(O)CH 2 CH3, -C(O)CH2(CH 3 )2, -C(O)CH(CH 3 )CH 2 CH 3 ; -C(O)CH 2 CH2(CH 3 )2; -C(O)- fluorophenyl (i.e. 2-fluorobenzoyl, 3-fluorobenzoyl or 4-fluorobenzoyl) , -C(O)- chlorophenyl (i.e. 2-chlorobenzoyl, 3-chlorobenzoyl or 4-chlorobenzoyl).
  • R 1 is H or -Ci-4a Ikyl
  • R 2 is -CH2-5-membered heteroaryl comprising one nitrogen or sulphur atom, or -CHz-phenyl optionally substituted by 1 or 2 chloro, fluoro or -O-Ci-4alkyl
  • R 3 is
  • the compound of the invention is ethyl (2S)-2-
  • the compound is ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate hydrochloride.
  • the compound of the invention is isopropyl (2S)-
  • the compound of the invention is ethyl (2S)-2- [[(2S)-2-amino-3-[3,5-bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4- chlorophenyl)propanoate:
  • the compound is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-chlorophenyl)propanoate hydrochloride.
  • the compound of the invention is ethyl (2S)-2- [[(2S)-2-amino-3-[3,5-bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4- dichlorophenyl)propanoate:
  • the compound is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride.
  • the compound of the invention is ethyl (2S)-2- [[(2S)-2-amino-3-[3,5-bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4- methoxyphenyl)propanoate:
  • the compound is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-methoxyphenyl)propanoate hydrochloride.
  • the compound of the invention is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-methoxyphenyl)propanoate hydrochloride.
  • the compound of the invention is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-methoxyphenyl)propanoate hydrochloride.
  • the compound is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2-thienyl)propanoate hydrochloride.
  • the compound of the invention is ethyl (2S)-2- [[(2S)-2-amino-3-[3,5-bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-phenyl- propanoate:
  • the compound is ethyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-phenyl-propanoate hydrochloride.
  • the compound of the invention is isopropyl (2S)- 2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4- dichlorophenyl)propanoate:
  • the compound is isopropyl (2S)-2-[[(2S)-2-a mi no-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride.
  • the compound of the invention may comprise an isotope atom.
  • an isotope atom is an atom of an element that is not the most common naturally occurring isotope.
  • Deuterium is a safe and stable isotope of hydrogen.
  • the compound of the invention has a deuterium abundance level greater than the naturally occurring abundance of deuterium. The naturally occurring abundance of deuterium is 0.0156 mol%, wherein mol% is the percentage of the total moles of a sample's hydrogen that is deuterium.
  • a deuterium abundance level greater than the naturally occurring abundance of deuterium may be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol% or 98 mol% deuterium.
  • the compound of the invention has a deuterium abundance level of at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol% or 98 mol% deuterium.
  • the compounds may form esters, amides, carbamates and/or salts.
  • Salts of compounds of the invention which are suitable for use in medicine are those wherein a counterion is pharmaceutically acceptable.
  • salts having non- pharmaceutically acceptable counterions are within the scope of the present invention, for example, for use as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable salts, and physiologically functional derivatives.
  • physiologically functional derivative refers to a chemical derivative of a compound of the invention that has the same physiological function as the compound of the invention, for example, by being convertible in the body thereto.
  • Esters, amides and carbamates are examples of physiologically functional derivatives.
  • Suitable salts according to the invention include those formed with organic or inorganic acids or bases.
  • suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or with organic sulfonic acids, such as (Ci-C4)-a Ikyl- or aryl-sulfonic acids which are unsubstituted or substituted, for example by halogen.
  • Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutical acceptable acid addition salts.
  • Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or a mono-, di- or trihydroxy-lower alkylamine, for example mono-, di- or triethanolamine.
  • Corresponding internal salts may furthermore be formed.
  • Preferred salts of a compound of the present invention include acid addition salts such as those formed from hydrochloric, hydrobromic, acetic, p-toluenesulfonic, tartaric, sulphuric, succinic, phosphoric, oxalic, nitric, methanesulfonic, malic, maleic and citric acid. More preferably, the salt of a compound of the present invention is the hydrochloride salt (i.e. the addition salt formed from hydrochloric acid).
  • Compounds of the invention may have an appropriate group converted to an ester, an amide or a carbamate.
  • typical ester and amide groups formed from an acid group in a compound of the invention include -COOR G , -CONR G 2, -SO 2 OR G , or -SO 2 N(R G ) 2
  • typical ester and amide and carbamate groups formed from an -OH or -NHR G group in the compound of the invention include -OC(O)R G , -NR G C(O)R G , -NR G CO 2 R G , -OSO 2 R G , and -NR G SO 2 R G , where R G is selected from the group consisting of Ci-salkyl, C 2 -salkenyl, C 2 -salkynyl, Cs-scycloalkyl and C3- scycloalkylCi-salkyl, haloCi-salkyl, dihaloCi-salky
  • a compound of the present invention may be in the form of a solvate.
  • Solvates of a compound of the present invention that are suitable for use as a medicament are those wherein the associated solvent is pharmaceutically acceptable.
  • a hydrate is pharmaceutically acceptable solvate.
  • a compound which, upon administration to the recipient, is capable of being converted into a compound of the present invention as described above, or an active metabolite or residue thereof, is known as a "prodrug".
  • a prodrug may, for example, be converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects.
  • Pharmaceutically acceptable prodrugs are described in T.
  • alkyl means both straight and branched chain saturated hydrocarbon groups.
  • alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, pentyl and hexyl groups.
  • unbranched alkyl groups there are preferred methyl, ethyl, n-propyl, iso-propyl, n- butyl groups.
  • branched alkyl groups there may be mentioned t-butyl, i- butyl, 1-ethylpropyl and 1-ethylbutyl groups.
  • cycloalkyl means a saturated group in a ring system.
  • examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • halogen means fluorine, chlorine, bromine or iodine.
  • Fluorine, chlorine and bromine are preferred, and fluorine and chlorine are particularly preferred.
  • the compounds of the invention are peptidase-enhanced cytotoxics (PEnC).
  • PnC peptidase-enhanced cytotoxics
  • the invention also provides a compound according to the invention, or a composition comprising a compound according to the invention together with a pharmaceutically acceptable carrier, for use as a medicament.
  • Compounds, compositions and pharmaceutical compositions according to the invention may be used in the treatment and/or prophylaxis of cancer, reducing tumor growth and/or killing tumor cells.
  • a compound of the invention may be used for curing and/or prolonging the survival of patients afflicted with cancer diseases.
  • the present invention is especially useful in the treatment and/or prophylaxis of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.
  • compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the subject under treatment.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • a compound of the invention may also be presented as a bolus, electuary or paste.
  • Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and
  • compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit dosage or divided dosage containers, for example sealed ampoules and vials.
  • the formulation may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.
  • compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable solutes, diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • suitable non-toxic, parenterally acceptable solutes, diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor.
  • compositions for nasal, aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol.
  • carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol.
  • Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • compositions for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerine or sucrose and acacia.
  • exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).
  • the amount of a compound of the invention which is required to achieve a therapeutic effect will vary with particular route of administration and the characteristics of the subject under treatment, for example the species, age, weight, sex, medical conditions, the particular disease and its severity, and other relevant medical and physical factors.
  • An ordinarily skilled physician can readily determine and administer the effective amount of a compound of the invention required for treatment or prophylaxis of cancer.
  • a compound of the invention may be administered daily, every second or third day, weekly, every second, third or fourth week or even as a high single dose depending on the subject and cancer form to be treated.
  • a compound of the invention, or salt and/or solvate thereof may be administered in an amount of about 1 to 150 mg per administration.
  • a compound of the invention, or salt or solvate thereof may be administered in a single high dose.
  • a single high dose may be about 150 to 800 mg.
  • a compound of the invention may be used as the sole active ingredient in the present invention, it is also possible for it to be used in combination with one or more further therapeutic agent(s), and the use of such combinations provides one preferred embodiment of the invention.
  • Such further therapeutic agents may be agents useful in the treatment or prophylaxis of cancer, or other pharmaceutically active materials. Such agents are known in the art.
  • further therapeutic agents for use in the present invention include steroids (prednisone and dexamethasone), IMiDs (thalidomide, lenalidomide and pomalidomide), Pls (bortezomib and carfilzomib), histone deacetylase (HDAC) inhibitors (panobinostat), a PEnC (e.g.
  • the further therapeutic agent is doxorubicin.
  • the one or more further therapeutic agent(s) may be used simultaneously, sequentially or separately with/from the administration of a compound of the invention. The individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
  • Reagents and conditions a) 2-Mercaptoethanol, Pdz(dba)3, Xantphos, 1,4-Dioxane, 130 °C, 1 h quant; b) Hunig's base, HATU, DMF, 15 min, rt, 72%; c) POCI 3 , DCM, rt, 18 h, 83%.
  • Preparative HPLC was performed on a Gilson system equipped with a UV detector using an XBridge Prep C-18 5 pm OBD, 19 x 50 mm column.
  • Analytical HPLC-MS was performed using an Agilent 1100 series Liquid Chromatograph/Mass Selective Detector (MSD) (Single Quadropole) equipped with an electrospray interface and a UV diode array detector.
  • MSD Liquid Chromatograph/Mass Selective Detector
  • Example Compound 1 Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate hydrochloride
  • Step 1 Preparation of (2S)-3-[3,5-Bis(2-hydroxyethylsulfanyl)phenyl]-2-(tert- butoxycarbonylamino)propanoic acid
  • HATU (323 mg, 0.85 mmol) was added to a mixture of (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid (296 mg, 0.71 mmol), ethyl (2S)-2-amino-3-(4-fluorophenyl)propanoate hydrochloride (211 mg, 0.85 mmol) and Hunig's base (613 pL, 3.54 mmol) in DMF (7 mL). The mixture was stirred at room temperature for 15 minutes where after the solvent was evaporated. The crude residue was flash chromatographed on silica gel (DCM:acetone 5:1). The fractions were concentrated under vacuum. The residue was redissolved in EtOAc and washed with a solution of 0.2 M NaHSC , dried over MgSC and concentrated under vacuum to give the title compound as a white solid (314 mg, 72% yield).
  • Step 3 Preparation of Example Compound 1 (Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5- bis(2-chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate hydrochloride)
  • Example Compound 2 Isopropyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate hydrochloride
  • Step 2 Preparation of Isopropyl (2S)-2-[[(2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)phenyl]-2-(tert-butoxy carbonylamino)propanoyl]amino]-3- (4-fluorophenyl)propanoate
  • HATU 33 mg, 0.09 mmol
  • (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid 30 mg, 0.07 mmol
  • isopropyl (2S)-2-amino-3-(4- fluorophenyl) propanoate-hydrochloride 20 mg, 0.08 mmol
  • Hunig's base 37 pL, 0.22 mmol
  • DMF 0.5 mL
  • Example Compound 3 Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-chlorophenyl)propanoate hydrochloride
  • Step 3 Preparation of Ethyl (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylsulfanyl)phenyl]- 2-(tert-butoxycarbonyl amino)propanoyl]amino]-3-(4-chlorophenyl) propanoate
  • HATU 33 mg, 0.09 mmol
  • (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid 30 mg, 0.07 mmol
  • ethyl (2S)-2-amino-3-(4- chlorophenyl)propanoate-hydrochloride 23 mg, 0.08 mmol
  • Hunig's base 37 pL, 0.22 mmol
  • Step 4 Preparation of Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-chlorophenyl)propanoate hydrochloride
  • Example Compound 4 Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride
  • HATU 33 mg, 0.09 mmol
  • (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid 30 mg, 0.07 mmol
  • ethyl (2S)-2-amino-3-(2,4- dichlorophenyl)propanoate hydrochloride 25 mg, 0.08 mmol
  • Hunig's base 37 pL, 0.22 mmol
  • Step 4 Preparation of Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride
  • HATU 33 mg, 0.09 mmol
  • (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid 30 mg, 0.07 mmol
  • ethyl (2S)-2-amino-3-(4- methoxyphenyl)propanoate hydrochloride 22 mg, 0.08 mmol
  • Hunig's base 37 pL, 0.22 mmol
  • Step 4 Preparation of Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(4-methoxyphenyl)propanoate hydrochloride
  • Example Compound 6 Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2-thienyl)propanoate hydrochloride
  • Step 1 Ethyl (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylsulfanyl)phenyl]-2-(tert- butoxycarbonyl amino)propanoyl]amino]-3-(2-thienyl)propanoate HATU (40 mg, 0.11 mmol) was added to a mixture of (2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)-phenyl]-2-(tert-butoxycarbonylamino)propanoic acid (37 mg, 0.09 mmol) (synthesis described in Example 1), [(lS)-2-ethoxy-2-oxo-l-(2- thienylmethyl)ethyl]ammonium chloride (obtained from commercial sources, 25 mg, 0.11 mmol) and Hunig's base (45 pL, 0.26 mmol) in DMF (0.5 mL).
  • Step 2 Preparation of Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2-thienyl)propanoate hydrochloride
  • Example Compound 7 Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-phenyl-propanoate hydrochloride
  • Step 2 Preparation of Ethyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-phenyl-propanoate-hydrochloride
  • the solvent was concentrated under vacuum and the residue was redissolved in a mixture of DMF (1.5 mL) and water (0.5 mL), and purified by preparative HPLC eluting with 30 to 60% acetonitrile containing 0.1% TFA (3 injections). During purification, pure fractions were taken and immediately chilled on an ice bath; when everything was collected the solvent was removed under freeze drying to give the pure TFA salt (7.5 mg, yield 19.5%).
  • the HCI salt was prepared by dissolving the material in a mixture of acetonitrile (0.6 mL) and water (1.4 mL), and then adding 4 M HCI in dioxane (4.00 M, 0.00725 mL, 2 eq.).
  • Example Compound 8 Isopropyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl]amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride
  • Step 3 Preparation of Isopropyl (2S)-2-[[(2S)-3-[3,5-bis(2- hydroxyethylsulfanyl)phenyl]-2-(tert-butoxycarbonylamino)propanoyl]amino]-3- (2,4-dichlorophenyl)propanoate
  • Step 4 Preparation of Isopropyl (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2- chloroethylsulfanyl)phenyl]propanoyl] amino]-3-(2,4-dichlorophenyl)propanoate hydrochloride
  • the solvent was concentrated under vacuum and the residue was redissolved in a mixture of DMF (1.5 mL) and water (0.5 mL), and purified by preparative HPLC eluting with 40 to 65% acetonitrile containing 0.1% TFA (3 injections). During purification, pure fractions were taken and immediately chilled on an ice bath; when everything was collected the solvent was lyophilized giving the TFA salt.
  • the HCI salt was prepared by dissolving the material in a mixture of acetonitrile (0.6 mL) and water (1.4 mL), and then adding 4 M HCI in dioxane (4.00 M, 0.010 mL, 2 eq.).
  • Example 1 In vitro cytotoxicity protocol - fluorometric microculture cytotoxicity assay (FMCA)
  • RPMI-8226/GFP Multiple Myeloma
  • CCRF-CEM/RFP Leukemia obtained from AntiCancer Inc
  • RPMI-1640 Sigma # R0883
  • 10 % fetal bovine serum Penicillin/Streptomycin (100 U/100 pg/ml)
  • L-glutamine 2mM all from Sigma St Louis, MO, USA.
  • HepG2 human liver cancer cell line obtained from ATCC cultured in Eagle's Minimum Essential Medium (ATCC) supplemented with 10 % fetal bovine serum, Penicillin/Streptomycin (100 U/100 pg/ml) and L-glutamine 2mM (all from Sigma St Louis, MO, USA). HepG2 cells were passaged 2 times/week and used maximally for 20 passages. Cells were seeded in Nunc 384-well assay plates at a density of 1000 cells/well.
  • ATCC Eagle's Minimum Essential Medium
  • Penicillin/Streptomycin 100 U/100 pg/ml
  • L-glutamine 2mM all from Sigma St Louis, MO, USA
  • MCF-7 (breast, adenocarcinoma) obtained from ATCC, cultured in Minimum Essential Medium Eagle (Sigma #M5650) supplemented with 10% fetal bovine serum, Penicillin/Streptomycin (100 U/100 pg/ml), L-glutamine 2mM and ImM Sodium pyruvat (all from Sigma St Louis, MO, USA).
  • FMCA fluorometric microculture cytotoxicity assay
  • Example Compound 1 10 mM stock solution of Example Compound 1 in DMSO was prepared.
  • the compound was added to the cells by acoustic dispensing (Echo 550 from Labcyte Inc, CA, USA) directly from 384-well source plates (Labcyte) containing the 10 mM stock solution or dilutions thereof.
  • the compound was dispensed in dose-response, eight two-fold dilutions in duplicate from top concentrations of 32 or 64 pM, to generate IC50 values.
  • Bortezomib (2 pM) was used as positive control and dose response testing of doxorubicin (top concentration 2 pM) was performed repeatedly to follow assay performance over time.
  • the 384-well plates were centrifuged (200 g, 60 sec), the culture medium was removed by a Biotech ELX washer (Biotek, Winooski, VT, USA) and 70 pl phosphate buffered saline (PBS, Sigma) was added to the wells. This procedure was repeated once.
  • the plates were centrifuged (200 g, 30 sec) and the PBS was removed by the ELX washer after which 50 pL/well of assay buffer (HEPES buffered saline with 0.5 mM MgCb and 0.5 mM CaCb, pH 7.4) was added to the plates using a Multidrop 384 (Thermo Fischer Scientific, NY, USA) and finally 1 pL of fluorescein diacetate (0.5mg/ml in DMSO) was added to each well using the pipetting robot Biomek NX (Beckman Coulter, Fullerton CA, USA).
  • assay buffer HEPBS buffered saline with 0.5 mM MgCb and 0.5 mM CaCb, pH 7.4
  • 1 pL of fluorescein diacetate 0.5mg/ml in DMSO
  • the plates were then incubated for 50 min in a Cytomat (37°C, humidity 95%, 5% CO2, Thermo Fischer Scientific) before being analyzed in a Fluostar Omega (BMG Technologies, Germany) with wavelengths set at ex 485/em 530 nm.
  • Cytotoxicity was assessed after 72 h with cell survival presented as survival index (SI, %) defined as fluorescence in test wells in percent of control cultures with blank values subtracted. Criteria for a successful assay included a signal to noise ratio in control cultures >10, CV ⁇ 30% and a positive control (Bortezomib) SI of ⁇ 5%.
  • the half maximal inhibitory concentration (IC50) was determined from log concentrationeffect curves in Graph Pad Prism (GraphPad software Inc., CA, USA) using non-linear regression analysis.
  • Example Compound 1 is a highly active cytotoxic agent, and is highly active in both haematological and solid cancer cell lines.
  • Example 2 Efficacy study of Melflufen-HCL and Example Compound 1 on osteosarcoma initiated from 143B or MNNG/HOS cell lines
  • the MNNG/HOS cell line (homo sapiens bone osteosarcoma cell line, obtained from ATCC (MNNG/HOS Cl #5 [R-1059-D] (ATCC® CRL-1547TM)) was cultivated in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. On day E9, cells were detached with trypsin, washed with complete medium and suspended in graft medium. An inoculum of lxlO 6 cells was added onto the CAM of each egg. Eggs were then randomized into 8 groups (see Table 4a below).
  • the 143B cell line (homo sapiens bone osteosarcoma cell line; obtained from ATCC (143B (ATCC® CRL-8303TM)) was cultivated in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. On day E9, cells were detached with trypsin, washed with complete medium and suspended in graft medium. An inoculum of 0.5xl0 6 cells was added onto the CAM of each egg. Eggs were then randomized into 8 groups (see Table 4b below).
  • Melflufen-HCL is the hydrochloric acid salt of melphalan flufenamide, and is a peptidase enhanced cytotoxic (PEnC).
  • Melflufen-HCL has the following structure:
  • Doxorubicin is a chemotherapeutic agent known to be used for the treatment of osteosarcoma, and has the following structure:
  • Example Compound 1 was stored at -20 °C
  • Cq is defined as the cycle at which the curvature of the amplification curve is maximal (fractional PCR cycles). Cq is taken in the exponential phase where the qPCR curve is linear. It is the basic result of qPCR: lower Cq values mean higher initial copy numbers of the target gene. When PCR efficiency is 100%, a difference of 1 cycle between two reactions means that there is 2 times more copies (gene) in the reaction which has the lower Cq value than that in the reaction which has the higher Cq value.
  • Statistical analysis was directly done on data from the Bio ⁇
  • Table 5 and Figure 2 present the mean values of the tumor weights for the different 0 MNNG/HOS cell line experimental groups at the end of the study.
  • Figure 5 shows a representative pictures of the tumors (ex ovo).
  • Table 5 Mean value, SD, SEM and p-value of tumor weight (mg) for each
  • Table 6 and Figure 3 present the data analysis of metastasis invasion, measured by qPCR for human Alu sequences in the lower CAM.
  • Table 8 and Figure 6 present the mean values of the tumor weights for the different 143B cell line experimental groups at the end of the study.
  • Figure 9 shows a representative pictures of the tumors (ex ovo).
  • Table 8 Mean value, SD, SEM and p-value of tumor weight (mg) for each 143B cell line experimental group
  • Table 9 and Figure 7 present the data analysis of metastasis invasion, measured by qPCR for human Alu sequences in the lower CAM.
  • Table 10 and Figure 8 present the percentage of dead and surviving embryos at the end of the study (on E18) for all experimental groups. Table 10. Number of dead and surviving embryo for the different experimental groups
  • the aim of this example was to characterize and compare the efficacy of Melflufen- HCL and Example Compound 1 on human osteosarcoma initiated from MNNG/HOS and 143B osteosarcoma cell lines in the model described above. Both compounds were evaluated through their effects on tumor growth and metastasis and their embryonic toxicity.
  • Example Compound 1 showed a significant tumor inhibition effect at the intermediate dose (50 pM) and the higher dose (200 pM) (p ⁇ 0.001 for both doses vs Neg Ctrl), but Melfluflen inhibited the tumor growth only at the higher dose (200 pM) (p ⁇ 0.05).
  • Example Compound 1 showed a significant tumor inhibition effect at the intermediate dose (50 pM) and the higher dose (200 pM) (p ⁇ 0.001 for both doses vs Neg Ctrl), but Melfluflen inhibited the tumor growth only at the higher dose (200pM) (p ⁇ 0.05).
  • Example Compound 1 showed a better performance than Melfluflen at both effective doses in both cell lines: • MNNG/HOS cell line: p ⁇ 0.001 for Example Compound 150 pM vs Melfluflen 50 pM and Example Compound 1 200 pM vs Melfluflen 200 pM.
  • Example Compound 1 For the MNNG/HOS cell line, in terms of metastasis inhibition, there was no significant difference between groups treated with Melflufen and Negative Control, while there was reduction for the groups treated with the higher doses of Example Compound 1 (200 pM). For the 143B cell line, in terms of metastasis inhibition, comparing to the negative control group, only Example Compound 1 compound at 200 pM demonstrated a significant metastasis regression effect.

Abstract

L'invention concerne des composés de formule (I), qui sont des cytotoxines améliorées par peptidase. L'invention concerne en outre des compositions pharmaceutiques comprenant les composés, et l'utilisation des composés ou des compositions pharmaceutiques en tant que médicament, en particulier pour une utilisation dans le traitement ou la prophylaxie de cancers tels que le myélome multiple, l'ostéosarcome, le cancer du sein, le cancer du poumon, le cancer de l'ovaire, la leucémie et le lymphome.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096367A1 (fr) * 2000-06-13 2001-12-20 Oncopeptides Ab Derives de melphalan et leur utilisation comme produits chimiotherapeutiques contre le cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096367A1 (fr) * 2000-06-13 2001-12-20 Oncopeptides Ab Derives de melphalan et leur utilisation comme produits chimiotherapeutiques contre le cancer

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
"Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS
"Design of Prodrugs", 1985, ELSEVIER
BYRN ET AL., PHARM. RES., vol. 12, no. 7, 1995, pages 945 - 954
CANCER LETT, vol. 243, 2006, pages 135 - 43
CANCER SCI, vol. 102, 2011, pages 501 - 8
CURR MED CHEM, vol. 14, 2007, pages 639 - 47
CURR MED CHEM, vol. 15, 2008, pages 2850 - 65
LINDHAGEN ENYGREN PLARSSON R., NAT PROTOC, vol. 3, no. 8, 2008, pages 1364 - 1369
PHARMACOL THER, vol. 83, 1999, pages 67 - 123
TRENDS MOL MED, vol. 14, 2008, pages 361 - 71
V. STELLA: "Prodrugs as Novel Delivery Systems", A. C. S. SYMPOSIUM SERIES, vol. 14, 1976
WANG, Y. J.HANSON, M. A., JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY, 1988, pages 2S

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