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WO1998035982A1 - Composes destines a etre utilises dans une therapie par promedicament enzymatique dirige contre un anticorps - Google Patents

Composes destines a etre utilises dans une therapie par promedicament enzymatique dirige contre un anticorps Download PDF

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Publication number
WO1998035982A1
WO1998035982A1 PCT/GB1998/000413 GB9800413W WO9835982A1 WO 1998035982 A1 WO1998035982 A1 WO 1998035982A1 GB 9800413 W GB9800413 W GB 9800413W WO 9835982 A1 WO9835982 A1 WO 9835982A1
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Prior art keywords
alkyl
amino
bis
benzoyl
compound
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PCT/GB1998/000413
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English (en)
Inventor
David Huw Davies
Robert Ian Dowell
Peter Robert Marsham
Janet Elizabeth Pease
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Syngenta Ltd
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Zeneca Ltd
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Priority to AU60004/98A priority Critical patent/AU6000498A/en
Publication of WO1998035982A1 publication Critical patent/WO1998035982A1/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/52—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • 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
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • 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
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • 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
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • 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
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to prodrug and drug compounds for use in ADEPT (antibody directed enzyme prodrug therapy) systems comprising mutant CPB (carboxypeptidase B) enzymes.
  • ADEPT antibody directed enzyme prodrug therapy
  • CPB carboxypeptidase B
  • the invention also relates to methods of manufacturing the compounds, pharmaceutical compositions and methods of treating diseases, especially cancer.
  • ADEPT is one approach to overcome the problem.
  • ADEPT uses a tumour selective antibody conjugated (e.g. by gene fusion) to an enzyme.
  • the conjugate is administered to the patient (usually intravenously), allowed to localise at the tumour site(s) and clear from the general circulation.
  • a prodrug is administered to the patient which is converted by the enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an amplification effect is produced.
  • tumour cells not displaying the antigen recognised by the antibody tumors usually display microheterogeneity
  • a suitable enzyme mutation is a polarity change in its active site such that it turns over a prodrug with a complementary polarity; the prodrug not being significantly turned over by the unmutated host enzyme.
  • the natural host enzyme recognises its natural substrate by an ion pair interaction and this interaction is reversed in the design of mutated enzyme and complementary prodrug.
  • Point mutations will be referred to as follows: natural amino acid (using the 1 letter nomenclature) , position, new amino acid. For example "D253K” means that at position 253 of mature active HCPB an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one enzyme will be shown between square brackets with individual mutations separated by commas.
  • the present invention relates to the discovery of a new class of prodrugs for use in ADEPT systems based on human CPB.
  • A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C ]-4 alkyl or C !-4 alkoxy;
  • X is a direct bond, CH or oxygen; Y and Y are independently selected from chloro, bromo, iodo, -0-S0 2 -C 1-4 alkyl and -O-
  • R is hydrogen, fluoro, chloro, bromo, C !-4 alkyl or C 1-4 alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 remaining substituent values are hydrogen;
  • R is C 1-6 alkyl, hydroxyC ]-6 alkyl, phenylC ⁇ -6 alkyl, C 1- alkoxy C] -4 alkyl, phenylC ⁇ -4 alkoxyCj -4 alkyl, C 1-4 alkylthioC 1- alkyl, phenylC].
  • A is optionally substituted phenyl
  • X is oxygen
  • Y 1 and Y2 are independently selected from chloro, bromo or iodo
  • R is C 1-6 alkyl.
  • the carboxypeptidase activatable prodrug is of Formula I
  • A is optionally substituted with upto 4 substituents independently selected from fluoro, chloro, bromo, C 1-4 alkyl or C 1-4 alkoxy;
  • X is a direct bond, CH 2 or oxygen;
  • V is NH or O;
  • r is 0-2, provided that when V is O then r is zero;
  • Y and Y are independently selected from chloro, bromo, iodo, -0-S0 2 -Ci -4 alkyl and -O-
  • W is COOH or lH-l,2,3,4-tetrazol-5-yl
  • R 1 is hydrogen, fluoro, chloro, bromo, C 1-4 alkyl or C )-4 alkoxy; p is 0-4 wherein values of R may be the same or different and when p is less than 4 remaining substituent values are hydrogen;
  • R is C 1-6 alkyl, hydroxyC 1-6 alkyl, phenylC 1-6 alkyl, C ⁇ -4 alkoxyC 1-4 alkyl, phenylC I-4 alkoxyC 1-4 alkyl, C ) -4 alkylthioC] -4 alkyl, phenylC 1-4 alkylthioC 1-4 alkyl or carbamoylC ] -4 alkyl; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, i -vivo hydrolysable ester or solvate thereof with the proviso that the following compounds and salts thereof are excluded, N-[N-(4- ⁇ 4-[N,N- bis-(2-chloroethyl)-amino]-3-methyl-phenoxy ⁇ -benzoyl)-L-alanyl]-L-glutamic acid and
  • W is COOH.
  • X is oxygen.
  • Y 1 and Y2 are independently selected from chloro, bromo or iodo.
  • R2 is Preferably V is oxygen.
  • r is 1.
  • A is optionally substituted phenyl.
  • Preferred compounds which are prodrugs are any one of the following compounds or a pharmaceutically acceptable salt thereof: (N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid;
  • the compound (N-[4-(4-[N,N-bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L- alanyl)-L-glutamic acid or a pharmaceutically acceptable salt thereof is especially preferred.
  • Preferred compounds which are drugs are any one of the following compounds or a pharmaceutically acceptable salt thereof:
  • alkyl includes both straight-chain and branched-chain alkyl groups.
  • references to individual alkyl groups such as “propyl” are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as “isopropyl” are specific for the branched-chain version only.
  • An analogous convention applies to other generic terms.
  • Any phenyl ring in R is optionally mono- or di-substituted with substituents independently selected from C ⁇ _4alkyl, halogen, OH, C ⁇ _4alkoxy, C ⁇ _4alkanoyl,
  • C i _4alkanoyloxy amino, C ⁇ _4alkylamino, di(C ⁇ _4alkyl)amino, C ⁇ alkanoylamino, nitro, cyano, carboxy, carbamoyl, C ⁇ _4alkoxycarbonyl, thiol, Cj ⁇ alkylsulfanyl, C ⁇ _4alkylsulfinyl, C]_4alkylsulfonyl, C]_4alkylsulfonamido, carbamoylC 1-4 alkyl, N-(monoC 1-4 alkyl)- carbamoylC 1-4 alkyl, N-(diC 1-4 alkyl)carbamoyl-C ⁇ _ 4 alkyl, hydroxyC 1-4 alkyl and C 1-4 alkoxyC 1-4 alkyl.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • BOC refers to tert-butyl-O-C(O)-.
  • Examples of C ⁇ -6 alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl, tert-butyl and pentyl; examples of Cj. 4 alkyl include methyl, ethyl, propyl, isopropyl, sec-butyl and tert- butyl; examples of C 1-4 alkoxy include methoxy, ethoxy and propoxy; examples of
  • Cj_4alkanoyl include formyl, acetyl and propionyl; examples of Cj_4alkanoyloxy include acetyloxy and propionyloxy; examples of C 1-4 alkylamino include methylamino, ethylamino, propylamino, isopropylamino, sec-butylamino and tert-butylamino; examples of di- (C ⁇ -4 alkyl)amino include di-methylamino, di-ethylamino and N-ethyl-N-methylamino; examples of C ⁇ _4alkanoylamino include acetamido and propionylamino; examples of C ⁇ -4 alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl and propoxy carbonyl; examples of Cj_4alkylsulfanyl include methylsulfanyl, ethylsulfanyl, propyl
  • X is a direct bond, CH 2 or oxygen;
  • V is NH or O; r is 0-2, provided that when V is O then r is zero;
  • Y and Y are independently selected from chloro, bromo, iodo, -O-S0 2 -C 1-4 alkyl and -O- SO 2 -phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from
  • R 1 is hydrogen, fluoro, chloro, bromo, C ⁇ -4 alkyl or C )-4 alkoxy; p is 0-4 wherein values of R may be the same or different;
  • R is C ]-6 alkyl, hydroxyC 1-6 alkyl, phenylC ⁇ -6 alkyl, C 1- alkoxyC I-4 alkyl, phenylC 1- alkoxyC ]-4 alkyl, C 1-4 alkylthioC 1-4 alkyl, phenylC 1-4 alkylthioC 1-4 alkyl or carbamoylC ⁇ -4 alkyl ; or an enantiomer, diastereoisomer, pharmaceutically acceptable salt, in-vivo hydrolysable ester or solvate thereof with the proviso that
  • N-[N-(4- ⁇ 4-[bis-(2-chloroethyl)-amino]-phenoxy ⁇ -benzoyl)-L-alanine]-L-glutamic acid are excluded.
  • N-(4- ⁇ 4- [bis-(2-chloroethyl)-amino] -3 -methyl-phenoxy ⁇ -benzoyl)-L- alanine and N-(4- ⁇ 4-[bis-(2-chloroethyl)-amino]-phenoxy ⁇ -benzoyl)-L-alanine are excluded.
  • the excluded compounds relate to one of our earlier patent filings, International Patent Application WO 97/07769, published 6-Mar-97. General, preferred and specific values for variable groups are as set out for the corresponding compounds of Formula I.
  • the compounds of Formula la are mustard drugs obtainable by hydrolysis of the terminal amino acid from a corresponding carboxypeptidase activatable prodrug such as the terminal amino acid (or analogue thereof) from the right hand side of a compound of Formula I or I' by a reversed polarity CPB enzyme.
  • reversed polarity CPB enzymes include any one of [D253KJHCPB, [G251T.D253K] HCPB and
  • [A248S,G251T,D253K]HCPB The drug compounds may also be obtained by conventional chemical synthesis. General, preferred and specific values for variable groups are as set out for the corresponding compounds of Formula I.
  • A is preferably
  • A is most preferably unsubstituted but if A is substituted then A is preferably mono- or di- substituted, especially mono-substituted.
  • Preferred substituents on A are fluoro, C 1-4 alkyl or C]- alkoxy; more preferably fluoro or C ⁇ -4 alkyl; more preferably fluoro or methyl.
  • a preferred value for V is NH. When V is NH then r is preferably 1.
  • Preferred values for X are a direct bond or -O- and X is especially -0-.
  • Y and Y are preferably independently selected from chloro, bromo and iodo, more preferably bromo and iodo and especially bromo. Y and Y are preferably the same.
  • R is hydrogen or C 1-4 alkyl; more preferably hydrogen or methyl and especially hydrogen. If R 1 takes a value other than hydrogen then p is preferably 1 or 2, especially 1.
  • R is preferably C 1-6 alkyl or phenylC 1-6 alkyl optionally mono or di substituted on phenyl with hydroxy; more preferably C ] -6 alkyl or phenylC 1-6 alkyl optionally mono substituted on phenyl with hydroxy; more preferably methyl, phenylmethyl or 4-hydroxyphenylmethyl; and especially R 2 is methyl.
  • the compounds of the present invention possess chiral centres and the present invention covers all stereoisomers and mixtures thereof. It is preferred that the chiral centres of the "dipeptide moiety" in Formula I have the configuration indicated in the Formula below which will generally give L-stereochemistry in the corresponding amino acid.
  • Preferred individualised compounds of the invention are as follows: (N-[4-(4-[N,N-Bis-(2-bromoethyl)amino]phenoxy)-benzoyl]-L-alanyl)-L-glutamic acid ;
  • Compounds of the invention may form salts which are within the ambit of the invention.
  • Pharmaceutically acceptable salts are preferred although other salts may be useful in, for example, isolating or purifying compounds.
  • a suitable pharmaceutically- acceptable salt of the invention when the compound contains an acidic moiety is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a pharmaceutically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
  • Solvates for example hydrates, are also within the ambit of the invention and may be prepared by generally known methods.
  • An in vivo hydrolysable ester of a compound of the Formula I containing carboxy group is, for example, a pharmaceutically-acceptable ester which is hydrolysed in the human or animal body to produce the parent acid.
  • Suitable pharmaceutically-acceptable esters for carboxy include C] -6 alkoxymethyl esters for example methoxymethyl, C 1-6 alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C 3-8 cycloalkoxycarbonyloxyC ⁇ -6 alkyl esters for example 1-cyclohexylcarbonyloxyethyl; l,3-dioxolen-2-onylmethyl esters for example 5-methyl-l,3-dioxolen-2-onylmethyl; and C 1-6 alkoxycarbonyloxy ethyl esters for example 1 -methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention.
  • a pharmaceutical composition comprising a compound as defined in Formula I, I' or Formula la or an individual end product compound described herein together with a pharmaceutically acceptable diluent or carrier.
  • a preferred pharmaceutical composition is one suitable for parenteral administration such as for example a solution, preferably sterile and suitable for injection or infusion.
  • the compound may be supplied in dry form, such as freeze-dried, ready for solution in a suitable solvent such as for example an aqueous buffer or water.
  • compositions of the invention may preferably be in a form suitable for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, or intramuscular dosing or as a suppository for rectal dosing).
  • parenteral administration for example as a sterile aqueous or oily solution for intravenous, subcutaneous, or intramuscular dosing or as a suppository for rectal dosing.
  • the compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
  • compositions in the form of a sterile injectable aqueous or oily suspension may be formulated according to known procedures using one or more appropriate dispersing or wetting agents and suspending agents.
  • a sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent.
  • Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable excipients include, for example, cocoa butter and polyethylene glycols.
  • the amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration.
  • a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.
  • Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.
  • the size of the dose for therapeutic or prophylactic purposes of a compound of the Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula I are useful in treating tumours.
  • LoVo tumour cells (10 ) (ECACC No: 87060101) are injected subcutaneously on the flank of athymic nude mice.
  • the tumour xenograf s are 4-5 mm in diameter antibody-mutant-CPB enzyme (for example as described in International Patent Application WO 96/20011) is administered i.v. at a dose of 2.5 mg/kg (500U CPG2 activity /kg).
  • prodrug (3 X 70 mg/kg) is administered i.p. as 3 bolus doses, hourly, over a two hour period.
  • Tumour regression and tumour growth delays are measured.
  • Typical conjugate doses of 0.25-10 mg/kg in combination with prodrug (3 X 50-90 mg/kg) may be tested in this model.
  • Using 3 doses of prodrug over 2hr is preferred to a single dose.
  • a clinical dose of conjugate would generally be in range 0.025-1.0 mg/kg, and more preferably 0.5-1.0 mg/kg.
  • Prodrug would be administered once the conjugate had localised to the tumour and cleared from blood and normal tissues (conjugate level in plasma ⁇ 1 ⁇ g/ml).
  • this is likely to be after 48-96 hr following conjugate administration.
  • prodrug would preferably be administered 24- 72 h after conjugate.
  • the dose of prodrug would generally be administered intravenously as 3 bolus doses, hourly, over a two hour period.
  • anti-tumour activity in patients would generally be expected with a total dose of 15-150 mg/kg.
  • standard dose escalation studies of prodrug would be used to define the optimal therapeutic dose.
  • Compounds of this invention may be useful in combination with known anti-cancer and cytotoxic agents. If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate.
  • Drug compounds of Formula la are useful as cytotoxic agents in their own right for example by direct application (for example by injection) into tumours. Such compounds are also useful in the context of being produced in situ by a suitable ADEPT conjugate from a corresponding prodrug.
  • Prodrugs of Formula I may also exhibit anti-tumour activity in their own right when administered in vivo; the anti-tumour activity thereof is expected to be enhanced when used in conjunction with an antibody-enzyme conjugate in ADEPT.
  • a method of treating tumours by administering an effective amount of a prodrug compound of Formula I or a pharmaceutically-acceptable salt thereof, to a mammal in need of such treatment, the mammal having a tumour localised ADEPT conjugate for prodrug activation at the tumour.
  • an ADEPT product containing a conjugate and a prodrug compound of Formula I or a pharmaceutically- acceptable salt thereof as a combined preparation for sequential use in treatment of tumours.
  • a method for the 5 delivery of a cytotoxic drug to a site which comprises administering to a host a first component, which first component comprises an antibody or fragment thereof capable of binding a given antigen, the antibody or fragment thereof being conjugated to a mutant CPB enzyme (preferably mutant human pancreatic CPB) capable of converting a compound of Formula I or pharmaceutically acceptable salt thereof into a cytotoxic drug; followed by
  • a second component which second component comprises a compound of Formula I or a pharmaceutically acceptable salt thereof convertible under the influence of the enzyme to the cytotoxic drug.
  • the components are delivered in effective amounts.
  • the preferred host is human.
  • the enzyme is [A248S,G251T,D253K]HCPB and the antibody is preferably humanised CDR grafted
  • the site to which the cytotoxic drug is to be delivered is preferably tumour cells which will generally be present in a tumour-bearing mammalian host such as a human.
  • the antibody or antibody fragment moiety of the conjugate directs the conjugate to the site of the tumour and binds the conjugate to the tumour cells.
  • the second component may be administered to the host. It is highly desirable to substantially eliminate unbound conjugate from the host before administration of the second component,
  • tumours of interest include carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, prostate, stomach, cervix, thyroid and skin.
  • CEA positive tumours are particularly preferred such as for example CEA positive colorectal
  • Antibody may be obtained from a hybridoma using standard techniques known in the art such as documented in Fenge C, Fraune E & Schuegerl K in "Production of Biologicals from Animal Cells in Culture” (Spier RE, Griffiths JR & Meignier B, eds) Butterworth-Heinemann, 1991, 262-265 and Anderson BL & Gruenberg ML in "Commercial Production of Monoclonal Antibodies” (Seaver S, ed), Marcel Dekker, 1987, 175-195.
  • the cells may require re-cloning from time to time by limiting dilution in order to maintain good levels of antibody production.
  • Antibodies useful in ADEPT have been described as follows. Antibody BW 431/26 was described in Haisma, H.J. et al., Cancer Immunol. Immunother., 34: 343-348 (1992). Antibodies L6, 96.5, and 1F5 were described in European Patent 302 473. Antibody 16.88 was described in International Patent Application WO90/07929. Antibody B72.3 was described in European Patent No. 392 745. Antibody CEM231 was described in European Patent No. 382 411.
  • Antibodies HMFG-1 and HMFG-11 react with a mucin-like glycoprotein molecule on milk fat globule membranes and may be used to target breast and ovarian cancers.
  • Antibody SM3 (Chemicon International Ltd, London, United Kingdom) reacts with core protein of mucin and may be used to target breast and ovarian cancer.
  • Antibodies 85A12 (Unipath Ltd, Basingstoke, Hants, United Kingdom) and ZCEA1 ( Pierce Chemical Company, Chester, United Kingdom) react with tumour antigen CEA.
  • Antibody PR4D1 (Serotec, Oxford, United Kingdom) reacts with a colon tumour associated antigen.
  • Antibody E29 (Dako Ltd, High Wycombe, United Kingdom) reacts with epithelial membrane antigen.
  • Antibody C242 is available from CANAG Diagnostics, Gothenberg, Sweden.
  • antibodies useful in ADEPT are poorly internalised by the tumour cells they recognise. This allows the targeted prodrug-activating enzyme to be resident on the cell surface and thus generate active drug at the tumour site from circulating prodrug.
  • a matched two component system designed for use in a host in which the components comprise: (i) a substantially non-immunogenic first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of CPB enzyme capable of converting a prodrug of Formula I into an antineoplastic drug and; (ii) a second component that is a prodrug of Formula I convertible under the influence of the enzyme to the antineoplastic drug, the prodrug not being significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme.
  • the prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme
  • the prodrug does not give undue untargeted toxicity problems on administration to the host.
  • substantially non-immunogenic means that the first component can be administered to the host on more than one occasion without causing significant host immune response as would be seen with for example the use of a mouse antibody linked to a bacterial enzyme in a human host.
  • the mutated enzyme is based on an enzyme from the same species as the host for which the system is intended for use but the mutated enzyme may be based on a host enzyme from a different species as long as the structure of the enzyme is sufficiently conserved between species so as not to create undue immunogenicity problems. More preferably the mutated enzyme is any one of [D253K]HCPB, [G251T,D253K] HCPB and [A248S,G251T,D253K]HCPB. [A248S,G251T,D253K]HCPB is especially preferred.
  • the targeting moiety is an antibody, especially an antibody fragment such as for example F(ab')2- Linkage to enzyme for conjugate synthesis may be effected by any suitable method such as for example use of heterobifunctional reagents as cross-linkers or preferably by gene fusion.
  • Antibody may be from the same host (eg use of mouse antibody in mice) or the antibody may be manipulated such that it is not significantly recognised as foreign in the chosen host (eg use of chimeric, CDR grafted or veneered mouse antibodies in humans).
  • the first component is a fusion protein between an anti-CEA antibody and a reversed polarity human CPB enzyme.
  • a preferred anti-CEA antibody is the antibody obtainable from hybridoma 806.077 deposited as ECACC deposit no. 96022936.
  • Hybridoma 806.077 antibody was deposited at the European Collection of Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom on 29th February 1996 under accession no. 96022936 in accordance with the Budapest Treaty. Humanisation of antibody 806.077 and production of fusion proteins thereof has been described in International Patent Application WO 97/42329, Zeneca Limited, published 13-Nov-97.
  • the constant region domains may be for example human IgA, IgE, IgG or IgM domains. Human IgG2 and 3 (especially IgG2) are preferred but IgG 1 and 4 isotypes may also be used. Human antibodies per se may also be used such as those generated in mice engineered to produce human antibodies. (Fishwald et al. in Nature Biotechnology (1996), ⁇ 4, 845-851).
  • a matched two component system designed for use in a host in which the components comprise: (i) a substantially non-immunogenic first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of CPB enzyme capable of converting a prodrug of the invention into an antineoplastic drug and;
  • the enzyme is [A248S,G251T,D253K]HCPB and the targeting moiety is humanised CDR grafted 806.077 antibody.
  • the compounds of the Formula I and la are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
  • the present invention provides a process for preparing a compound of the Formula I or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof which process comprises a) deprotecting a compound of the Formula II:
  • Pr and Pr independently represent hydrogen or carboxy protecting groups which may be the same or different, other variable groups are as hereinbefore defined, and wherein any other functional group is optionally protected with the proviso there is at least one protecting group and optionally, if desired, forming a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.
  • deprotection step a) is preferred.
  • Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question, and may be introduced by conventional methods. Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule. Specific examples of protecting groups are given below for the sake of convenience, in which "lower” signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
  • a carboxyl protecting group may be the residue of an ester-forming aliphatic or arylalkyl alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
  • carboxy protecting groups include straight or branched chain (l-12C)alkyl groups (eg isopropyl, t ⁇ butyl); lower alkoxy lower alkyl groups (eg methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (eg acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (eg 1-methoxycarbonyloxyethyl, 1 -ethoxycarbonyloxy ethyl); aryl lower alkyl groups (eg benzyl, p-methoxybenzyl, o ⁇ nitrobenzyl, r nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (eg trimethylsilyl and t ⁇ butyldimethylsilyl); tri(lower alkyl)sily
  • Methods particularly appropriate for the removal of carboxyl protecting groups include for example metal-catalysed hydrogenolysis or acid-, base-, or enzymically-catalysed hydrolysis. It should be noted however that base catalysed hydrolysis is not suitable for mustard compounds due to potential damage thereof in the presence of base.
  • hydroxyl protecting groups include lower alkyl groups (eg t-butyl), lower alkenyl groups (eg allyl); lower alkanoyl groups (eg acetyl); lower alkoxycarbonyl groups (eg t-butoxycarbonyl); lower alkenyloxycarbonyl groups (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (eg trimethylsilyl, tibutyldimethylsilyl) and aryl lower alkyl (eg benzyl) groups.
  • lower alkyl groups eg t-butyl
  • lower alkenyl groups eg allyl
  • lower alkoxycarbonyl groups eg t-butoxycarbonyl
  • amino protecting groups include formyl, aralkyl groups (eg benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (eg t- butoxycarbonyl); lower alkenyloxycarbonyl (eg allyloxycarbonyl); aryl lower alkoxycarbonyl groups (eg benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (eg trimethylsilyl and t-butyldimethylsilyl); alkylidene (eg methylidene); benzylidene and substituted benzylidene groups.
  • aralkyl groups eg benzyl and substitute
  • Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis, for groups such as p-nitrobenzyloxycarbonyl, hydrogenation and for groups such as o-nitrobenzyloxycarbonyl, photolytically.
  • the compound of Formula II may be prepared by reacting a compound of Formula III
  • Formula II where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula III by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence of I 2 , CBr 4 or CCl 4 .
  • the compound of Formula III may be prepared by reacting a compound of Formula V
  • Formula V with ethylene oxide under suitable conditions.
  • Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature.
  • the compound of Formula V may be prepared by hydrogenating a compound of Formula VI
  • Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and an organic solvent such as for example ethyl acetate at a non-extreme temperature.
  • the compound of Formula VI may be prepared by reacting a compound of Formula VII
  • Formula VIII under suitable amide bond forming conditions.
  • a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°.
  • an organic solvent preferably an anhydrous polar aprotic organic solvent
  • Compounds of Formulas VII and VIII are either commercially available or readily synthesised from known starting materials using standard techniques.
  • the compound of Formula Ila may be prepared by reacting a compound of Formula Ilia
  • Y a and Y b independently represent -SO 2 -C 1-4 alkyl and -S0 2 -phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C 1-4 alkyl, halo, cyano or nitro and other variable groups are as hereinbefore defined, under suitable conditions (for example using a polar aprotic organic solvent and a base at a non-extreme temperature) to give a compound of Formula Ila where Y and Y represent -O-SO 2 -C 1-4 alkyl and -O-S0 2 -phenyl wherein phenyl is optionally substituted with upto 3 substituents selected from C ⁇ -4 alkyl, halo, cyano or nitro; and optionally,
  • Formula Ila where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula Ilia by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence of I 2 , CBr 4 or CCl 4 .
  • the compound of Formula Ilia may be prepared by reacting a compound of Formula
  • Formula Va with ethylene oxide under suitable conditions. Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature.
  • the compound of Formula Va may be prepared by hydrogenating a compound of
  • Formula Via under suitable conditions. Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and an organic solvent such as for example ethyl acetate at a non-extreme temperature.
  • a catalyst such as palladium on carbon
  • an organic solvent such as for example ethyl acetate at a non-extreme temperature.
  • the compound of Formula Via may be prepared by reacting a compound of Formula
  • COOPr 2 Formula Villa under suitable amide bond forming conditions.
  • a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°.
  • an organic solvent preferably an anhydrous polar aprotic organic solvent
  • Formula XIV wherein Wa represents a carboxyl group in protected form or a tetrazol-5-yl group, under suitable amide bond forming conditions.
  • a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region -10 to 40°, typically ambient temperature of about 20°.
  • Suitable reaction conditions include use of hydrogen in the presence of a catalyst such as palladium on carbon at ambient temperature.
  • Formula X where Y and Y represent chloro, bromo or iodo may also be prepared directly from compounds of Formula XI by reaction with a phosphine in the presence of a chloro, bromo or iodo reagent. Suitable examples include use of triphenylphosphine in the presence ofI 2 , CBr 4 or CCl 4 .
  • Formula XII with ethylene oxide under suitable conditions. Suitable conditions include the presence of an aqueous protic solvent at a non-extreme temperature.
  • Compounds of Formula XII can be prepared by hydrogenating compounds of Formula XIII
  • Suitable conditions include hydrogen in the presence of a catalyst such as palladium on carbon, and a polar aprotic organic solvent at a non-extreme temperature.
  • HCPB human carboxypeptidase B preferably pancreatic
  • the starting material was made from 4-phenoxybenzoic acid and L-alanyl-L-glutamic acid di- tert-butyl ester as described in Example 3.
  • End product b was made using analogous methodology.
  • step B Product from step B (4.5 g) was dissolved in ethyl acetate (100 ml) and 10% Platinum on carbon (50% moist with water, 0.9 g) added. The mixture was stirred under an atmosphere of hydrogen for 3 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated to dryness. The residue was chromatographed on silica gel, eluting with 1.1 hexane/ethyl acetate to give (N-[4-(4-aminophenoxy)-benzoyl]-L-alanyl)-L- glutamic acid dibenzyl ester as an oil 3.0 g (71%).
  • step C Product from step C (3.0 g) was dissolved in 1 : 1 acetic acid/water (60 ml) and ethylene oxide (3 g) passed in during 1 h. The mixture was allowed to stand at RT for 18 h, evaporated to dryness, the residue treated with sodium hydrogen carbonate solution (150 ml) and extracted twice with ethyl acetate. The combined organic extracts were washed with water and evaporated to dryness.
  • step E The product from step D (2 g) in dichloromethane (40 ml) was cooled to -10° under an inert atmosphere and triethylamine (1.6 ml) added. To this mixture was added a solution of methanesulphonic anhydride (2.06 g) in dichloromethane (5 ml) keeping the temperature at -10° to -5°. The reaction was held at this temperature for a further lh, washed twice with ice- cold water, dried, and evaporated to an oil. This oil was dissolved in DMF (25 ml) and lithium bromide (2.4 g) was added.
  • step C) The product from step B (645 mg) was dissolved in ethyl acetate (35 ml) and 10% Palladium on carbon (50% moist with water, 65 mg) was added. The mixture was stirred under an atmosphere of hydrogen until uptake ceased. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate was evaporated to dryness. The residue was chromatographed eluting with 1.1 hexane/ethyl acetate to give di-tert-butyl N-[4-(4- aminophenyl)-benzoyl]-L-alanyl-L-glutamate (506 mg).
  • step C Product from step C (470mg) was dissolved in 1 : 1 acetic acid/water (50 ml) and ethylene oxide (2.8 g) passed in during lh. The mixture was allowed to stand at RT for 18 h. After evaporation to a small volume the residue was treated with sodium hydrogen carbonate solution (150 ml) and extracted twice with ethyl acetate. The combined organic extracts were washed with water and evaporated to dryness.
  • the starting material was prepared as follows.
  • N-[4-[4-[N,N-Bis-(2-hydroxyethyl)amino]phenyl]-benzoyl]-L-alanyl-L-glutamic acid di- tert-butyl ester was prepared using analogous methodology with that set out in Example 3, steps B-D.
  • the starting material was prepared as follows.
  • Triphenylphosphine (1.22 g), imidazole (0.32 g) and iodine (1.22 g) were dissolved in dry dichloromethane (20 ml).
  • a solution of N-[4-(4-[N,N-Bis-(2- hydroxyethyl)amino]phenyl)-benzoyl]-L-alanine tert-butyl ester (0.5g) in dichloromethane (20 ml) was added to this solution.
  • the reaction mixture was stirred at RT for 18 h under nitrogen, filtered to remove imidazole hydroiodide and the filtrate was evaporated.
  • step E The product of step E was reacted with ethylene oxide (as described in the corresponding step in Example 3) to give di-tert-butyl N-[4-(4-[N,N-Bis-(2- hydroxyethyl)amino]benzyl)benzoyl]-L-alanyl-L-glutamate.
  • Triethylamine (870 ⁇ l) was added to a stirred solution of 4-carboxy-4'- ethoxycarbonyldiphenylmethane (A Wallon et al, Chem. Ber. 1990, 123, 375) (1.70 g) in t- butanol (20 ml). Diphenylphosphoryl azide (1.77 ml) was added and the reaction mixture was stirred for 4 h under reflux. The cooled reaction mixture was partitioned between ethyl acetate and water. The ethyl acetate solution was filtered through diatomaceous earth to remove a fine white precipitate and evaporated to dryness.
  • step B The product from step B (1.15 g) was dissolved in HCl in ethyl acetate(3M,12 ml). A white precipitate slowly formed. After 3 h the suspension was evaporated to give ethyl 4-(4- aminobenzyl)benzoate as a white solid.
  • step D The white solid from step C was dissolved in acetic acid (25 ml) and water (5 ml) and cooled to 0°. Ethylene oxide (ca. 10 g) was condensed into the solution which was kept overnight at RT under a dry ice/isopropanol reflux condenser. The reaction mixture was evaporated to dryness and the residue was partitioned between ethyl acetate (2 x 30 ml) and water (20 ml) with the addition of solid NaHCO 3 until the aqueous phase had pH >7.0. The ethyl acetate solution was washed with brine, dried and evaporated.
  • step D The product from step D (2.05 g) was stirred in a mixture of EtOH (16 ml) and 2N aqueous NaOH (16.5 ml) to give a clear solution which was kept overnight.
  • EtOH was removed by rotary evaporation and the aqueous residue was acidified (cone. HCl) to pH 3.0.
  • the white solid precipitate was filtered off, washed with water and vacuum dried to give 4-(4-[N,N-bis-(2-hydroxyethyl)amino]benzyl)benzoic acid (Compound 5): 228 mg.
  • K m and k cat were assessed for convenience, assessment of K m and k cat was performed using prodrug analogues (des mustard compounds) because these enzyme parameters are believed to be affected only by the "dipeptide moiety" of the prodrugs.
  • Purified mutant [A248S,G251T,D253K]HCPB enzyme and native human CPB were assayed for their ability to cleave glutamic acid from a glutamic acid prodrug analogue (compound without a mustard moiety). Cleavage liberates a mono-carboxylic acid compound from the di-carboxylic acid (glutamic acid containing) prodrug analogue. Conversion of "glutamic acid prodrug analogues" to the "drug analogues" was measured using a HPLC based assay.
  • Prodrug analogue was diluted in the range 1-0.003 mM in 0.025 M Tris-HCL buffer, pH 7.5. Where necessary prodrug samples were adjusted to pH 7.5 with 0.1M NaOH. [A248S,G251T,D253K]HCPB or native HCPB, both at a final concentration of 2- 0.005 ⁇ g/ml, were added to the prodrug analogues (500 ⁇ l reaction volume prewarmed to 37° for 2 min) to start the reaction. Samples were incubated for 15-30 minutes at 37°. The reaction was terminated by the addition of 500 ⁇ l 98.8 % MeCN, 0.2% TFA and the samples placed on ice. The amount of product (drug analogue) produced was then quantified by HPLC.
  • the compound of Reference Example 1 showed substrate recognition and turnover, K m of 0.18 mM and a k cat of 56 sec "1 , by [A248S,G251T,D253K]HCPB enzyme together with desired lack of turnover by native HCPB.
  • the differential cytotoxicity to tumour cells of the glutamic acid prodrug of Example 1 and corresponding des glutamate drug of Example 2 has been demonstrated by the following means. LoVo colorectal tumour cells were incubated with prodrug or drug over a final concentration range of 5 X 10 "4 to 5 X 10 "8 M in 96- well (2,500 cells/well) microtitre plates for 1 h at 37°. The cells were then washed and incubated for a further three days at 37°C. After washing to remove dead cells, TCA was added and the amount of cellular protein adhering to the plates was assessed by addition of SRB dye as described by P. Skehan et al, J. Natl. Cancer Inst. 82, 1107 (1990). Potency of the compounds was assessed by the concentration required to inhibit cell growth by 50% (IC50).
  • Example 10 Anti-tumour activity of prodrugs and humanised antibody-mutant HCPB fusion protein in xenografted mice.
  • anti-tumour efficacy of suitable prodrugs and humanised anti-CEA antibody-mutant HCPB fusion protein can be demonstrated in the following model.
  • LoVo colorectal tumour cells (ECACC no. 87060101) (1 X 10 7 ) are injected s.c. into athymic nude mice.
  • the conjugate is administered i.v. at doses between 10-100 mg/kg.
  • the prodrug is administered either i.v or i.p. to the mice in doses ranging between 10-1000 mg/kg either as a single or multiple doses.
  • the time interval between conjugate administration and prodrug administration can be optimised as required.
  • residual conjugate levels could be monitored to allow for clearance of conjugate from the bloodstream and normal tissues whilst allowing tumour localised conjugate to be optimised relative thereto for maximal therapeutic effect.
  • Another example would be to set up experiments using a series of intervals and simply select the interval giving the best therapeutic effect.
  • a typical dose of antibody-enzyme is 30 mg followed 3 days later by prodrug.
  • the starting material was prepared as follows.
  • step D Product from step D (12.8 g) was dissolved in dichloromethane (100 ml) and triethylamine (12.7 ml) added. The reaction was cooled to -10 ° and then methane sulphonyl chloride (5.9 ml) in dichloromethane (50 ml) added under an inert atmosphere over 6 min. The reaction was then allowed to warm to RT over 4.5 h with stirring. The solution was then diluted with dichloromethane (100 ml) and washed with water and brine.
  • step F Product from step F (12.8 g) was dissolved in ethyl acetate (150 ml) and 10% palladium on carbon (50 % moist with water, 10 g) added. The mixture was stirred under an atmosphere of hydrogen for 15 h. The catalyst was removed by filtration through a pad of diatomaceous earth and the filtrate evaporated. The resulting solid was triturated with 20 % diethyl ether/hexane to give 4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoic acid as a white powder, 8.2g (80%).
  • step H Product from step H (2.3 g) was dissolved in HCl in ethyl acetate (3 M, 20 ml) and stirred for 4 h. The solution was evaporated to dryness and then azeotroped with toluene to give dibenzyl N-(L-tert-leucyl)-L-glutamate hydrochloride, 2 g.
  • step G (0.3 g) was dissolved in DMF (20 ml) and product from step I (0.429 g) added, followed by HOBT (0.12 g), EDCI (0.17 g) and N-methylmorpholine (0.19 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the resulting residue was chromatographed on silica gel eluting with 1.1 hexane/ethyl acetate to give the desired starting material as an oil, 0.44 g (62%).
  • Example 1 but using 3,5-dimethyl-4-(4-nitrophenoxy)benzoic acid in place of 4-(4- nitrophenoxy)benzoic acid.
  • the 3,5-dimethyl-4-(4-nitrophenoxy)benzoic acid was prepared as described by Rarick, Brewster and Daines J.A.C.S. 55 (1993), 1289-90 but using 3,5- dimethyl-4-hydroxybenzoic acid in place of 4-hydroxybenzoic acid.
  • the starting material was prepared as follows.
  • B) 4-(2-methyl-4-nitrophenoxy)benzoic acid was prepared as described by Rarick, Brewster and Daines J.A.C.S. 55 (1993 ) 1289-90 but using 5-fluoro-2-nitrotoluene in place of 4-fluoro-l -nitrobenzene.
  • the starting material was prepared as follows.
  • the starting material was made using an analogous procedure to that described in the previous example but using alanine tertbutyl ester hydrochloride in place of dibenzyl L- alanyl-L-glutamate hydrochloride and lithium chloride in place of lithium bromide in the final step.
  • the starting material was prepared as follows.
  • B) 4-(4-nitro-3-methoxyphenoxy)-3,5-dimethylbenzoic acid was prepared as described by Rarick et al J.A.C.S. 55 (1993) 1289-90 but using 4-fluoro-2-methoxy-l -nitrobenzene in place of 4-fluoro-l -nitrobenzene and 4-hydroxy-3,5-dimethylbenzoic acid in place of 4- hydroxybenzoic acid.
  • the starting material was prepared using analogous methodology to that described in Example 1 but using dibenzyl L-leucyl-L-glutamate (Loukas, Spyros; Varoucha, Dido; Zioudrou, Christine; Streaty, Richard A.; Klee, Werner A; Biochemistry (1983), 22(19),
  • the starting material was prepared using analogous methodology to that described in Example 2 but using L-leucine tertbutyl ester at step B thereof and using the following bromination methodology. Bromine (1.8 ml) was added to a solution of triphenylphosphine (8.9 g) and imidazole
  • the organic layer was washed twice with sodium bicarbonate solution, water, 0.5 M citric acid, water and saturated brine solution. The organic layer was dried and the solvent removed in vacuo. The residue formed was chromatographed on silica gel eluting with 3 % ethyl acetate/methylene chloride and then 5 % ethyl acetate/methylene chloride to give the desired starting material, 48 mg (18 %).
  • step C Product from step C (7.5 g) was dissolved in dry DMF and lithium bromide (20.28 g) was added. The mixture was heated and stirred at 100 ° under nitrogen for 6 hours, the temperature reduced to 60 ° and the mixture stirred overnight. The DMF was removed in vacuo and the residue was dissolved in water and extracted twice with ethyl acetate. The organics were washed twice with water and saturated brine then evaporated in vacuo.
  • step D The crude product from step D (185 mg) was dissolved in DMF (20 ml) and added to the dipotassium salt of 4-hydroxybenzoic acid. The mixture was heated to 140 ° with stirring under nitrogen for 3.5 hours. The reaction mixture was cooled and poured into water (150 ml), acidified with 2M hydrochloric acid, extracted into ethyl acetate (150ml), washed twice with water, brine, dried and the solvent removed in vacuo. The solid was triturated with hot water and filtered to give 1 -benzoyl-4-nitronaphthalene, 227 mg (43%).
  • step G Product from step G (0.95 g) was dissolved in acetic acid (20 ml) and the mixture was stirred whilst adding water (15 ml). Ethylene oxide was passed through the mixture until 3.5 g had been added. The mixture was then stirred overnight and the solvent was removed in vacuo. The residue was partitioned between saturated sodium bicarbonate solution and ethyl acetate The organic layer was washed twice with water, brine, dried and the solvent removed in vacuo.
  • step H Product from step H (360 mg) was dissolved in methylene chloride (8 ml) and was stirred in an ice/salt bath at 0°. Triethylamine (310 ⁇ l) was added and then methanesulphonyl chloride (141 ⁇ l) was added dropwise over 3-4 minutes, keeping the temperature below 5°. After addition was complete the mixture was stirred at 0° for 5 minutes and then for lhour at room temperature. The mixture was diluted with methylene chloride and washed in turn with ice cold water, ice cold sodium bicarbonate solution and saturated brine.
  • Trifluoroacetic acid (5 ml) was added to a solution of starting material di-tert-butyl ⁇ N-[4-(4-[N,N-bis(2-chloroethyl)amino]-3-methylphenoxy)benzoyl]-L-alanyl ⁇ -L-aspartate (310 mg, 0.46 mmol) in dichloromethane (5 ml). The solution was kept for 18 h at ambient temperature and evaporated to dryness. The residue was triturated with 1 :1 diethyl ether/isohexane to yield a white solid. The solvent was decanted off and the solid was vacuum dried to yield the desired end product, 335 mg.
  • the reaction mixture was diluted with ethyl acetate (50 ml) and washed with 10 % citric acid, aqueous sodium bicarbonate solution and brine. The solution was dried and evaporated to a gum which was purified by chromatography eluting with 0-15% ethanol/dichloromethane to yield ditertbutyl Z-L-alanyl-L-aspartate as an almost colourless gum. 1.85 g.
  • N-methylmorpholine 125 ⁇ l, 1.13 mmol
  • 1- hydroxybenzotriazole hydrate 20 mg
  • the resulting solution was washed with water, dried and evaporated to dryness.
  • the crude product was purified by chromatography on silica, eluting with 0-3 % ethanol/dichloromethane to give the desired starting material as a white foam, 320 mg.
  • the starting material was prepared as follows.
  • step B Product from step B was converted to di-tert-butyl N-[4-(4-[N,N-bis-(2- hydroxyethyl)amino]-2-methylphenyl)-benzoyl]-L-alanyl-L-glutamate using analogous methodology to that described in Example 1 up to step D thereof but using 4-( 4-nitro2- methylphenyl ) benzoic acid in place of 4-( 4-nitrophenoxy ) benzoic acid.
  • step C Product from step C (600 mg, 1.36 mM) was dissolved in dry dichloromethane (20 ml) and triphenylphosphine (1.422 g, 5.42 mM), imidazole (369 mg, 5.42 mM) and carbon tetrabromide (1.779 g, 5.42 mM), each being added in 1 portion to this solution. The reaction was stirred at room temperature for 4 hours and the solution evaporated. The crude mixture was purified by chromatography on silica eluting with 15 % ethyl acetate/isohexane to give the desired starting material (552mg) as a gum.
  • the desired end product was prepared in an analogous manner to the methodology described in Example 31 except that L-alanyl-L-glutamic acid di-tert-butyl ester was replaced with L-alanine tert-butyl ester.
  • the starting material was prepared as follows. B)-G) These steps were carried out as described in Example 12. H) N-Boc-O3-methyl-L-serine (0.5 g, Bachem) was dissolved in DMF (20 ml) and dibenzyl L-glutamate (1.3 g) added, followed by HOBT (0.34 g), EDCI (0.48 g) and N- methylmorpholine (0.28 ml). The mixture was stirred at RT under an inert atmosphere for 15 h. The mixture was evaporated to dryness and the residue partitioned between 1 M citric acid and ethyl acetate.
  • Reagents a) H 2 N-CH(R)-C0 2 -t-Bu, EDCI.HCl, HOBT, Et3N, b) H2, 30%Pd/C.
  • EtOAc c) Ethylene oxide/ aq HOAc, d) (MeSO 2 ) 2 O, CH 2 C1 2 , Et 3 N, e) LiBr, DMF, f) TFA, CH 2 C1 2 g) P(Ph) 3 /I 2 /IMIDAZOLE h) TMSI
  • Reagents - a) Alanine Benzyl ester, EDCI, DMF; b) 10%Pd/C, H 2 ; c) Ala-Glu-dibenzyl ester, EDCI, DMF.

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Abstract

On décrit des composés de formule (I) dans laquelle, A représente phényle ou naphtyle facultativement substitué; X représente une liaison directe, CH2 ou oxygène; V représente NH ou O; r représente un entier compris entre 0 et 2 à condition que lorsque V représente O, r représente alors zéro; Y1 et Y2 sont indépendamment sélectionnés parmi chloro, bromo, iodo, -O-SO¿2?-C1-4alkyle et -O-SO2-phényl; W représente COOH or 1H-1,2,3,4-tétrazol-5-yle; R?1¿ représente hydrogène, fluoro, chloro, bromo, C¿1-4?alkyle ou C1-4alcoxy; p représente un entier compris entre 0 et 4, les valeurs de R?1¿ pouvant être identiques ou différentes et lorsque p est inférieur à 4, les valeurs de substituant restant représentent hydrogène; R2 représente C¿1-6?alkyle, hydroxyC1-6alkyle, phénylC1-6alkyle, C1-4alcoxyC1-4alkyle, phénylC1-4alcoxyC1-4alkyle, C1-4alkylthio1-4alkyle, phénylC1-4alkylthioC1-4alkyle ou carbamoylC1-4alkyle ou un énantiomère, un diastéréoisomère, un sel pharmaceutiquement acceptable, un ester ou un solvate de ce dernier hydrolysable in vivo. Ces composés sont utiles pour le traitement du cancer à l'aide d'une thérapie par promédicament enzymatique dirigé contre un anticorps et plus particulièrement en tant que promédicaments pouvant être activés par la carboxypeptidase B.
PCT/GB1998/000413 1997-02-15 1998-02-10 Composes destines a etre utilises dans une therapie par promedicament enzymatique dirige contre un anticorps Ceased WO1998035982A1 (fr)

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AU60004/98A AU6000498A (en) 1997-02-15 1998-02-10 Compounds for use in adept

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GBGB9703201.5A GB9703201D0 (en) 1997-02-15 1997-02-15 Chemical compounds
GB9703201.5 1997-02-15

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6420396B1 (en) * 1998-12-16 2002-07-16 Beiersdorf Ag Biphenyl and biphenyl-analogous compounds as integrin antagonists
WO2002100431A1 (fr) * 2001-06-08 2002-12-19 The Forth Military Medical University Kit pharmaceutique comprenant une proteine de fusion carboxypeptidase humaine dirigee contre un anticorps a chaine simple de proteine plasmatique seminale et promedicament
US6521663B2 (en) 2000-10-06 2003-02-18 3-Dimensional Pharmaceuticals, Inc. Aminoguanidinyl- and Alkoxyguanidinyl-substituted phenyl acetamides as protease inhibitors
US6677360B2 (en) 1998-12-16 2004-01-13 Bayer Aktiengesellschaft Biphenyl and biphenyl-analogous compounds as integrin antagonists
EP2514402A4 (fr) * 2009-12-16 2014-05-28 Pola Chem Ind Inc Agent prophylactique ou améliorant pour la pigmentation
TWI477287B (zh) * 2010-12-21 2015-03-21 Pola Chem Ind Inc 絲胺酸衍生物及製造色素沉澱之預防或改善劑之用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013095A2 (fr) * 1993-11-12 1995-05-18 The Wellcome Foundation Limited Therapie
WO1996020011A1 (fr) * 1994-12-23 1996-07-04 Zeneca Limited Composes chimiques
WO1997007769A2 (fr) * 1995-08-16 1997-03-06 Zeneca Limited Composes chimiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013095A2 (fr) * 1993-11-12 1995-05-18 The Wellcome Foundation Limited Therapie
WO1996020011A1 (fr) * 1994-12-23 1996-07-04 Zeneca Limited Composes chimiques
WO1997007769A2 (fr) * 1995-08-16 1997-03-06 Zeneca Limited Composes chimiques

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6420396B1 (en) * 1998-12-16 2002-07-16 Beiersdorf Ag Biphenyl and biphenyl-analogous compounds as integrin antagonists
US6677360B2 (en) 1998-12-16 2004-01-13 Bayer Aktiengesellschaft Biphenyl and biphenyl-analogous compounds as integrin antagonists
US7094911B2 (en) 1998-12-16 2006-08-22 Bayer Aktiengesellschaft Biphenyl and biphenyl-analogous compounds as integrin antagonists
US6521663B2 (en) 2000-10-06 2003-02-18 3-Dimensional Pharmaceuticals, Inc. Aminoguanidinyl- and Alkoxyguanidinyl-substituted phenyl acetamides as protease inhibitors
US6900231B2 (en) 2000-10-06 2005-05-31 3-Dimensional Pharmaceuticals, Inc. Aminopyridyl-substituted phenyl acetamides as protease inhibitors
WO2002100431A1 (fr) * 2001-06-08 2002-12-19 The Forth Military Medical University Kit pharmaceutique comprenant une proteine de fusion carboxypeptidase humaine dirigee contre un anticorps a chaine simple de proteine plasmatique seminale et promedicament
EP2514402A4 (fr) * 2009-12-16 2014-05-28 Pola Chem Ind Inc Agent prophylactique ou améliorant pour la pigmentation
AU2010331250B2 (en) * 2009-12-16 2015-07-30 Pola Chemical Industries Inc. Preventing or ameliorating agent for pigmentation
US9414998B2 (en) 2009-12-16 2016-08-16 Pola Chemical Industries Inc. Preventing or ameliorating agent for pigmentation
KR101877575B1 (ko) * 2009-12-16 2018-07-12 포라 가세이 고교 가부시키가이샤 색소 침착 예방 또는 개선제
TWI477287B (zh) * 2010-12-21 2015-03-21 Pola Chem Ind Inc 絲胺酸衍生物及製造色素沉澱之預防或改善劑之用途

Also Published As

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GB9703201D0 (en) 1997-04-02
ZA981222B (en) 1998-08-17
AU6000498A (en) 1998-09-08

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