CA2319694A1 - Compound - Google Patents
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- CA2319694A1 CA2319694A1 CA 2319694 CA2319694A CA2319694A1 CA 2319694 A1 CA2319694 A1 CA 2319694A1 CA 2319694 CA2319694 CA 2319694 CA 2319694 A CA2319694 A CA 2319694A CA 2319694 A1 CA2319694 A1 CA 2319694A1
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Abstract
The present invention provides a pharmaceutical compound, or pharmaceutically acceptable salt thereof, for use in medicine, wherein said compound is of formula I
R-Ar-X-Y
I
wherein R is a pharmaceutically active moiety;
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
Further aspects of the invention relate to a process for the preparation of said compound, and the use of such compounds in the palliative and curative treatment of bone disorders.
R-Ar-X-Y
I
wherein R is a pharmaceutically active moiety;
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
Further aspects of the invention relate to a process for the preparation of said compound, and the use of such compounds in the palliative and curative treatment of bone disorders.
Description
I
Compound The present invention relates to a pharmaceutical compound and composition for use in medicine.
It is well known in the art that phosphonate compounds, for example bisphosphonates, have a high affinity for hydroxyapatite crystals and thus tend to localise in vivo in regions of bone metabolism. Moreover, it has also been established that phosphonate compounds are generally low in toxicity.
US 4,880,007 (Amersham International PLC) discloses complexes formed between (a) an amino di- or polyphosphonate; and (b) a paramagnetic metal ion, such as gadolinium (III). Such complexes exhibit calcified tissue seeking properties which I S render them useful as contrast agents for investigating bone metabolism by NMR
scanning.
Similarly, US 5,236,695 (Concat Ltd.) discloses polyphosphonate ligands containing three or more phosphonate groups, combined with paramagnetic metal cations which are administered in the form of pharmacologically acceptable salts. Such compounds are useful as MRI contrast agents which tend to localise in bone tissue without being conjugated to bone-specific biomolecules.
Bisphosphonates have also been used in combination with technetium-99 (Tc-99m).
Indeed, Tc-99m is routinely used with carriers such as methylene bisphosphonate, for imaging in hospitals. Furthermore, US 4,830,847 discloses diphosphonate-derivatised macromolecules, such as proteins, suitable for use as technecium-99m based scanning agents and anticalcification agents. Typically, the scanning agents are prepared by combining Tc-99m in a 3+, 4+ and/or 5+ oxidation state with the disphosphonate-derivatised macromolecule. Also disclosed are pharmaceutical compositions containing such diphosphonate-derivatised macromolecules.
Compound The present invention relates to a pharmaceutical compound and composition for use in medicine.
It is well known in the art that phosphonate compounds, for example bisphosphonates, have a high affinity for hydroxyapatite crystals and thus tend to localise in vivo in regions of bone metabolism. Moreover, it has also been established that phosphonate compounds are generally low in toxicity.
US 4,880,007 (Amersham International PLC) discloses complexes formed between (a) an amino di- or polyphosphonate; and (b) a paramagnetic metal ion, such as gadolinium (III). Such complexes exhibit calcified tissue seeking properties which I S render them useful as contrast agents for investigating bone metabolism by NMR
scanning.
Similarly, US 5,236,695 (Concat Ltd.) discloses polyphosphonate ligands containing three or more phosphonate groups, combined with paramagnetic metal cations which are administered in the form of pharmacologically acceptable salts. Such compounds are useful as MRI contrast agents which tend to localise in bone tissue without being conjugated to bone-specific biomolecules.
Bisphosphonates have also been used in combination with technetium-99 (Tc-99m).
Indeed, Tc-99m is routinely used with carriers such as methylene bisphosphonate, for imaging in hospitals. Furthermore, US 4,830,847 discloses diphosphonate-derivatised macromolecules, such as proteins, suitable for use as technecium-99m based scanning agents and anticalcification agents. Typically, the scanning agents are prepared by combining Tc-99m in a 3+, 4+ and/or 5+ oxidation state with the disphosphonate-derivatised macromolecule. Also disclosed are pharmaceutical compositions containing such diphosphonate-derivatised macromolecules.
The therapeutic applications of bisphosphonate compounds are also well documented in the art. For example, WO 96/39150 (Merck & Co., Inc.) discloses the use of bisphosphonates, such as alendronate, in the prevention or treatment of bone loss associated with rheumatoid arthritis. Similarly, GB 2,331,459 (SPA) discloses an injectable composition for treating skeletal and bone disorders which comprises a bisphosphonate in combination with a benzyl alcohol. Eisenhut et al.. (Appln.
Radiat. Isot., Vol 38, No.7, p535-540) disclose the use of 13~I-labelled benzylidinediphosphonates for the palliative treatment of bone metastases.
Finally, WO 95/11029 (Merck & Co., Inc.) discloses compositions comprising bisphosphonate and growth hormone secretagogues, which are useful for reducing the deleterious effects of osteoporosis in elderly patients.
The present invention seeks to provide improved phosphonate compounds for use in medicine. In particular, the invention seeks to provide pharmaceutical compounds which exhibit improved activity in the palliative and curative treatment of bone disorders, and/or which may also be suitable for use in medical imaging techniques.
Statement of Invention Aspects of the invention are presented in the accompanying claims and in the following description.
In the broadest aspect, the present invention relates to a pharmaceutical compound for use in medicine. The pharmaceutical may be for a therapeutic use and/or a diagnostic use.
More specifically, the present invention provides a pharmaceutical compound, or pharmaceutically acceptable salt thereof, for use in medicine, wherein said compound is of formula I
R-Ar-X-Y
I
Radiat. Isot., Vol 38, No.7, p535-540) disclose the use of 13~I-labelled benzylidinediphosphonates for the palliative treatment of bone metastases.
Finally, WO 95/11029 (Merck & Co., Inc.) discloses compositions comprising bisphosphonate and growth hormone secretagogues, which are useful for reducing the deleterious effects of osteoporosis in elderly patients.
The present invention seeks to provide improved phosphonate compounds for use in medicine. In particular, the invention seeks to provide pharmaceutical compounds which exhibit improved activity in the palliative and curative treatment of bone disorders, and/or which may also be suitable for use in medical imaging techniques.
Statement of Invention Aspects of the invention are presented in the accompanying claims and in the following description.
In the broadest aspect, the present invention relates to a pharmaceutical compound for use in medicine. The pharmaceutical may be for a therapeutic use and/or a diagnostic use.
More specifically, the present invention provides a pharmaceutical compound, or pharmaceutically acceptable salt thereof, for use in medicine, wherein said compound is of formula I
R-Ar-X-Y
I
wherein R is a pharmaceutically active moiety;
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
In a preferred aspect of the invention, Y comprises a geminal bisphosphonate group.
In a further preferred aspect, the invention provides a pharmaceutical compound, or pharmaceutically acceptable salt thereof, of formula II
R-Ar-X~ p03Hz ~z II
wherein Z is H, NH2 or an oxy substituent.
Preferably, Z is H or OH.
The pharmaceutical compound of the present invention comprises a linker group, X.
In a preferred aspect, the linker group of the invention is a substituted or unsubstituted C ~ ~ alkylene group.
In an alternative preferred aspect of the invention, X is a C, _4 amine group, C 1 ~ ether group or a C,_4 thioether group, each of which may be substituted or unsubstituted.
In another preferred aspect of the invention, X is S=O or S02.
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
In a preferred aspect of the invention, Y comprises a geminal bisphosphonate group.
In a further preferred aspect, the invention provides a pharmaceutical compound, or pharmaceutically acceptable salt thereof, of formula II
R-Ar-X~ p03Hz ~z II
wherein Z is H, NH2 or an oxy substituent.
Preferably, Z is H or OH.
The pharmaceutical compound of the present invention comprises a linker group, X.
In a preferred aspect, the linker group of the invention is a substituted or unsubstituted C ~ ~ alkylene group.
In an alternative preferred aspect of the invention, X is a C, _4 amine group, C 1 ~ ether group or a C,_4 thioether group, each of which may be substituted or unsubstituted.
In another preferred aspect of the invention, X is S=O or S02.
Where X is substituted, suitable substituents include one or more groups which do not interfere with the pharmaceutical activity of the compound in question.
Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, and alkyl.
The pharmaceutical compound of the present invention also comprises an aromatic moiety, Ar.
Preferably, the aromatic moiety of the compound is electron deficient.
In a more preferred aspect, the aromatic moiety of the invention is a single aromatic ring. However, other aromatic moieties are also suitable for use in the invention, for example, aromatic moieties comprising more than one aromatic ring, where the aromatic rings may be fused together or joined via one or more suitable spacer groups Examples of aromatic moieties suitable for the present invention include substituted or unsubstituted phenyl, naphthyl, thiophenyl, furyl, pyridyl and pyrrole groups.
Where the aromatic moiety is substituted, suitable substituents include one or more groups which do not interfere with the pharmaceutical activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, and alkyl.
The pharmaceutical compound of the present invention also comprises a pharmaceutically active moiety, R.
In a preferred aspect, R comprises a radiolabel. Examples of radiolabels suitable for use in the present invention include 124h ~25I, ~3iI, 2uAt (an a-emitter), ~g6Re, Tc 99m, and (3-emitting bromine nuclei.
In an alternative preferred aspect, the pharmaceutically active moiety of the invention may comprise a functional group (or ligand) to which a metal ion can be chelated, or is chelated thereto. Species of the former type, i.e. those comprising a functional group to which a metal ion can be chelated, could be potentially useful for complexing any excess radiolabel close to the bone, thereby preventing radiolabel polsomng.
By way of definition, the term "chelate" refers to a complex in which a ligand is coordinated to a metal ion at two or more points, so that there is a ring of atoms including the metal, and where the term "ligand" refers to an ion or molecule that can donate a pair of electrons to said metal ion.
Suitable functional groups or ligands to which a metal ion may be chelated include amine, hydroxy, or carboxylic acid moieties.
In a particularly preferred aspect, the metal ion chelated to the functional group is paramagnetic. In the present context, the term "paramagnetic" refers to metal ions having net orbital or spin magnetic moments that are capable of being aligned in the direction of an applied magnetic field. Such atoms have a positive (but small) susceptibility and a relative permeability slightly in excess of one.
Paramagnetism occurs in all atoms with unpaired electrons, e.g. transition metal ions with unpaired electron shells.
Examples of suitable paramagnetic metals include those of the lanthanide elements with atomic numbers 58 to 70, and those of the transition metals with atomic numbers 21 to 29, 42 and 44. Typical examples include chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praesodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).
The pharmaceutically active moiety, R, may also comprise a paramagnetic component other than a paramagnetic ion, for example, a moiety comprising the group NO'.
Compounds of the invention containing paramagnetic moieties or radiolabels may have applications in the field of medical imaging, especially calcified tissue imaging. In particular, such compounds may be administered to a patient in order to preferentially enhance the NMR image contrast in tissue. By way of definition, the term "NMR" also encompasses magnetic resonance imaging (MRI) in which images of tissue are produced by magnetic resonance techniques.
Thus, in one preferred aspect, the invention provides a pharmaceutical carrier molecule for a radiolabel. In a particularly preferred aspect, the radiolabel is Tc-99m.
The term "calcified tissue" refers to bone, regions of bone metabolism, regions of calcified tumours and other diseased tissues.
In a particularly preferred aspect of the invention, the pharmaceutically active moiety R is attached directly to the aromatic moiety, Ar.
By way of definition, the term "pharmaceutically acceptable salt" includes any salt that has the same general pharmacological properties as the parent species from which it is derived, and which is acceptable from a toxicity view-point.
Typical pharmaceutically acceptable salts include acid addition salts, base salts or solvates or hydrates thereof. A review of suitable salts may be found in Berge et al., J.
Pharm.
Sci., 1977, 66, 1-19.
Suitable acid addition salts are formed from acids which form non-toxic salts.
Examples include hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethane-sulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and examples include alkali metal (sodium and potassium), alkaline earth metal (calcium and magnesium), aluminium, non-toxic heavy metal (zinc, stannous and indium), ammonium and low molecular weight substituted ammonium (mono-, di- and triethanolamine) salts.
Methods for preparing pharmaceutically acceptable salts of compounds of the invention will be familiar to those skilled in the art. Typically, pharmaceutically acceptable salts may be prepared by mixing together a solution of the agent and the desired acid or base, as appropriate. The salt may be recovered by evaporation of the solvent, or by precipitation from solution followed by filtration.
The compound of the present invention may exist in polymorphic form.
The compound of the present invention may contain one or more asymmetric carbon atoms and thus may exist in two or more stereoisomeric forms. The present invention includes the individual stereoisomers of the compound and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Diastereoisomers of compounds of the invention may be separated by conventional techniques such as fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof.
Individual enantiomers of the agent may also be prepared from the corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support. Alternatively, individual enantiomers may be prepared by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
The present invention also includes all suitable isotopic variations of the compound, or pharmaceutically acceptable salts thereof. The term "isotopic variation" as used herein refers to a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention, or pharmaceutically acceptable salts thereof, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, ~3C, 14C, ESN, 1~0, ~gp, 31p~ 32P~ 355 ~sF ~d 3601, respectively. Isotopic variants in which a radioactive isotope is incorporated may have applications in drug and/or substrate tissue distribution studies.
Typical examples preferred for their ease of preparation and detectability include tritium (3H), and carbon-14 (14C) isotopes. Substitution with other isotopes such as deuterium (2H) may afford certain therapeutic advantages resulting from greater metabolic stability, i.e., increased in vivo half life or reduced dosage requirements.
Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures familiar to those skilled in the relevant art using the appropriate isotopic variations of suitable reagents.
It will be appreciated by those skilled in the art that the compound of the present invention may be derived from a prodrug. By way of example, a prodrug includes any entity having one or more protected groups) and which may not possess pharmacological activity per se, but may, in certain instances, be administered (for example orally or parenterally) and thereafter metabolised in the body to form the pharmaceutically active agent of the present invention.
The skilled person in the art will further appreciate that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H.
Bundgaard, Elsevier, 1985 (the disclosured of which is hereby incorporated by reference), may be placed on appropriate functionalities of the compounds. Such prodrugs are also intended to fall within the scope of the present invention.
The present invention also provides a pharmaceutical composition comprising the compound provided by the present invention admixed with a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, and alkyl.
The pharmaceutical compound of the present invention also comprises an aromatic moiety, Ar.
Preferably, the aromatic moiety of the compound is electron deficient.
In a more preferred aspect, the aromatic moiety of the invention is a single aromatic ring. However, other aromatic moieties are also suitable for use in the invention, for example, aromatic moieties comprising more than one aromatic ring, where the aromatic rings may be fused together or joined via one or more suitable spacer groups Examples of aromatic moieties suitable for the present invention include substituted or unsubstituted phenyl, naphthyl, thiophenyl, furyl, pyridyl and pyrrole groups.
Where the aromatic moiety is substituted, suitable substituents include one or more groups which do not interfere with the pharmaceutical activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, and alkyl.
The pharmaceutical compound of the present invention also comprises a pharmaceutically active moiety, R.
In a preferred aspect, R comprises a radiolabel. Examples of radiolabels suitable for use in the present invention include 124h ~25I, ~3iI, 2uAt (an a-emitter), ~g6Re, Tc 99m, and (3-emitting bromine nuclei.
In an alternative preferred aspect, the pharmaceutically active moiety of the invention may comprise a functional group (or ligand) to which a metal ion can be chelated, or is chelated thereto. Species of the former type, i.e. those comprising a functional group to which a metal ion can be chelated, could be potentially useful for complexing any excess radiolabel close to the bone, thereby preventing radiolabel polsomng.
By way of definition, the term "chelate" refers to a complex in which a ligand is coordinated to a metal ion at two or more points, so that there is a ring of atoms including the metal, and where the term "ligand" refers to an ion or molecule that can donate a pair of electrons to said metal ion.
Suitable functional groups or ligands to which a metal ion may be chelated include amine, hydroxy, or carboxylic acid moieties.
In a particularly preferred aspect, the metal ion chelated to the functional group is paramagnetic. In the present context, the term "paramagnetic" refers to metal ions having net orbital or spin magnetic moments that are capable of being aligned in the direction of an applied magnetic field. Such atoms have a positive (but small) susceptibility and a relative permeability slightly in excess of one.
Paramagnetism occurs in all atoms with unpaired electrons, e.g. transition metal ions with unpaired electron shells.
Examples of suitable paramagnetic metals include those of the lanthanide elements with atomic numbers 58 to 70, and those of the transition metals with atomic numbers 21 to 29, 42 and 44. Typical examples include chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praesodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).
The pharmaceutically active moiety, R, may also comprise a paramagnetic component other than a paramagnetic ion, for example, a moiety comprising the group NO'.
Compounds of the invention containing paramagnetic moieties or radiolabels may have applications in the field of medical imaging, especially calcified tissue imaging. In particular, such compounds may be administered to a patient in order to preferentially enhance the NMR image contrast in tissue. By way of definition, the term "NMR" also encompasses magnetic resonance imaging (MRI) in which images of tissue are produced by magnetic resonance techniques.
Thus, in one preferred aspect, the invention provides a pharmaceutical carrier molecule for a radiolabel. In a particularly preferred aspect, the radiolabel is Tc-99m.
The term "calcified tissue" refers to bone, regions of bone metabolism, regions of calcified tumours and other diseased tissues.
In a particularly preferred aspect of the invention, the pharmaceutically active moiety R is attached directly to the aromatic moiety, Ar.
By way of definition, the term "pharmaceutically acceptable salt" includes any salt that has the same general pharmacological properties as the parent species from which it is derived, and which is acceptable from a toxicity view-point.
Typical pharmaceutically acceptable salts include acid addition salts, base salts or solvates or hydrates thereof. A review of suitable salts may be found in Berge et al., J.
Pharm.
Sci., 1977, 66, 1-19.
Suitable acid addition salts are formed from acids which form non-toxic salts.
Examples include hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethane-sulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.
Suitable base salts are formed from bases which form non-toxic salts and examples include alkali metal (sodium and potassium), alkaline earth metal (calcium and magnesium), aluminium, non-toxic heavy metal (zinc, stannous and indium), ammonium and low molecular weight substituted ammonium (mono-, di- and triethanolamine) salts.
Methods for preparing pharmaceutically acceptable salts of compounds of the invention will be familiar to those skilled in the art. Typically, pharmaceutically acceptable salts may be prepared by mixing together a solution of the agent and the desired acid or base, as appropriate. The salt may be recovered by evaporation of the solvent, or by precipitation from solution followed by filtration.
The compound of the present invention may exist in polymorphic form.
The compound of the present invention may contain one or more asymmetric carbon atoms and thus may exist in two or more stereoisomeric forms. The present invention includes the individual stereoisomers of the compound and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
Diastereoisomers of compounds of the invention may be separated by conventional techniques such as fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof.
Individual enantiomers of the agent may also be prepared from the corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support. Alternatively, individual enantiomers may be prepared by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
The present invention also includes all suitable isotopic variations of the compound, or pharmaceutically acceptable salts thereof. The term "isotopic variation" as used herein refers to a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention, or pharmaceutically acceptable salts thereof, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, ~3C, 14C, ESN, 1~0, ~gp, 31p~ 32P~ 355 ~sF ~d 3601, respectively. Isotopic variants in which a radioactive isotope is incorporated may have applications in drug and/or substrate tissue distribution studies.
Typical examples preferred for their ease of preparation and detectability include tritium (3H), and carbon-14 (14C) isotopes. Substitution with other isotopes such as deuterium (2H) may afford certain therapeutic advantages resulting from greater metabolic stability, i.e., increased in vivo half life or reduced dosage requirements.
Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures familiar to those skilled in the relevant art using the appropriate isotopic variations of suitable reagents.
It will be appreciated by those skilled in the art that the compound of the present invention may be derived from a prodrug. By way of example, a prodrug includes any entity having one or more protected groups) and which may not possess pharmacological activity per se, but may, in certain instances, be administered (for example orally or parenterally) and thereafter metabolised in the body to form the pharmaceutically active agent of the present invention.
The skilled person in the art will further appreciate that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H.
Bundgaard, Elsevier, 1985 (the disclosured of which is hereby incorporated by reference), may be placed on appropriate functionalities of the compounds. Such prodrugs are also intended to fall within the scope of the present invention.
The present invention also provides a pharmaceutical composition comprising the compound provided by the present invention admixed with a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).
The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
5 The composition/formulation requirements may vary depending on the delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution.
Alternatively, the composition may be formulated in an injectable form, for 10 parenteral delivery, for example, by an intravenous, intramuscular or subcutaneous route. The formulation may also be formulated so as to be suitable for delivery by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be stable at acid pH, resistant to proteolytic degradation and to the detergent effects of bile.
The pharmaceutical compositions of the invention can be administered topically in the form of a lotion, solution, cream, ointment or dusting powder, or by the use of a skin patch. Alternatively, the compositions can be administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or in the form of a suppository or pessary. Further modes of administration include inhalation, or parenteral injection, for example intravenously, intramuscularly or subcutaneously.
For parenteral administration, the compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example appropriate levels of salts and/or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.
In a preferred aspect, the present invention relates to the use of the compound or composition of the invention in the palliative or curative treatment of bone disorders.
Preferably, the compounds and/or compositions of the invention are used to treat bone disorders that are cancer-related skeletal diseases, for example, skeletal metastases, osteoblastic osteosarcoma or multiple myelomas. However, the compounds and/or compositions of the invention may also be used to treat non-cancerous bone disorders, such as age-related bone loss, rheumatoid-related bone loss, or bone loss related to osteoporosis, disuse or steroid therapy.
The treatment of skeletal metastases is one of the main problems encountered in practical clinical oncology. It is estimated that up to 85 % of all patients with advanced carcinomas to the breast, prostate or lung develop such metastatic conditions (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 349). Up to now, the prognosis for these patients has been poor.
To date, treatment methods in current clinical practice include external radiotherapy, hormone therapy and chemotherapy, although the number of patients achieving complete curation is negligible (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 350). In particular, severe side-effects often limit the applicability of these methods. Consequently, there is a strong need for improved therapeutic methods for slowing down tumour progression and for pain palliation.
Normally, the main method for radiation therapy is external beam irradiation.
However, if there are multiple skeletal metastases present, a more efficient treatment may be the targeted radiotherapy of timorous osseous lesions by means of radioactive compounds with bone affinity. Such regions are characteristic for several types of bone-related diseases, malignant as well as benign. Examples of malignant lesions are skeletal metastases and osteosarcoma, examples of benign lesions are osteoporosis and Paget's disease (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 293). Pathological bone synthesis in skeletal metastases is observed in very large groups of cancer patients who develop metastatic foci originating from different types of soft-tissue tumours.
Furthermore, there is a small but important, clinically difficult niche of patients suffering from metastasised osteoblastic osteosarcoma. In this case, a primitive bone-like substance, osteoid, is produced by the tumour cells themselves.
Although clinically very different from skeletal metastases, the chemistry of these lesions is similar, and they may often be targeted by the same type of bone affinity compounds.
Usually, myelotoxicity sets the limits for the radiation dose that can be administered to the tumour by means of radioactive bone-affinity compounds. Any development which decreases the radiation dose to the bone marrow would therefore substantially improve targeted radiotherapy techniques.
In a preferred aspect, the present invention thus seeks to provide improved bone affinity compounds which expose the bone marrow to substantially lower levels of radiation.
There are two classes of radioactive bone-affinity compounds currently in clinical use. The first and most important class includes ions of the radioisotopes of alkaline earth elements, such as g9Sr2+, which is the most common bone-seeking agent in clinical use (Lewington, V.J., Cancer therapy using bone-seeking isotopes, Physics in Medicine and Biology, 1996; 41: 2031-2032).
The other class of bone-seeking compounds include radioactive bis- or polyphosphonic acids carrying a (3-active radionuclide. The species most commonly used in the clinic are ~g6Re-HEDP (Lewington, V.J., Cancer therapy using bone-s seeking isotopes, Physics in Medicine and Biology, 1996; 41: 2030) and lssSm-EDTMP (Lewington, V.J., Cancer therapy using bone-seeking isotopes, Physics in Medicine and Biology, 1996; 41:2029). The latter has also been applied with success in the palliative treatment of osteoblastic osteosarcoma (Franzuis, C. et al., High Activity Samarium-153-EDTMP Therapy in Unresectable Osteosarcoma, Nuklearmedizin,1999; 38:337-340). However, in contrast to the compounds disclosed in the present invention, none of these nuclides reduce the tumour progression of small tumours, and hence they are not suitable for curative treatment.
(3-emitters are usually classified into three groups according to (3-particle energy and hence range. With regard to physical properties, ~-particle energy and range are parameters which have to be matched to the size of the tumour. The half life and chemical properties, on the other hand, are related to the pharmacokinetics and metabolism of the carrier molecule. Radionuclides with (3 -energy in the range Eavg =
0.08-0.18 MeV (mean range 0.4-0.9 mm) are best suited for treatment of small tumours (Zweit, J., Radionuclides and carrier molecules for therapy, Physics in Medicine and Biology, 1996; 41: 1908-1910). ~31I is the most familiar and the only radionuclide in this group that has been used clinically. Radionuclides with medium ~-energy such as ~s3Sm and ~86Re are less suitable for curative treatment, since the dose distribution will spare small tumours and the tumour on bone marrow ratio is too low. High energy (3 -emitters such as g9Sr are only suited for the palliative and curative treatment of large tumours. There is speculation as to whether the use of a-emitters in combination with (3-emitters would be superior for curative treatment.
However, the short range of a-radiation (40-80 ~,m) would most likely require bonding of the a-emitting carrier molecule to most cancer cells within the tumour.
In an alternative aspect, the invention also relates to the use of the compound of the invention in the preparation of a medicament for the palliative or curative treatment of bone disorders.
As used herein the phrase "preparation of a medicament" includes the use of a compound of the invention directly as the medicament in addition to its use in a screening programme for the identification of further active agents or in any stage of the manufacture of such a medicament.
Such a screening programme may for example include an assay for determining whether a candidate substance is capable of mimicking the activity of a pharmaceutical compound of the present invention.
Another aspect of the invention provides a method of treating a subject in need of the palliative or curative treatment of bone disorders, the method comprising administering to said subject a therapeutically effective amount of the compound or composition of the present invention.
The present invention also provides a process for preparing a compound of the invention, wherein R comprises a radiolabel, said process comprising the following steps:
(i) preparing a phosphonate precursor comprising Ar, X and Y;
(ii) radiolabelling said bisphosphonate precursor.
In a preferred aspect, step (ii) of the above-mentioned process is a deiodosilylation reaction.
For practical purposes, it is desirable to produce targeting radionuclide agents at the site of use, e.g. hospitals.
1$
To date, the demand for convenient labelling chemistry, high stability and favourable biological behaviour has proven difficult to meet with radiohalogenated bisphosphonates. In recent studies, pre-labelled compounds have been connected to bisphosphonic acid functionalities (Fritzberg, A.R. et al., US5202109:
Conjugates for bone imaging and bone cancer therapy; Murud, k. et al., Synthesis, Purification, and in Vitro Stability of 21 ~At- and ~25I-Labeled Amidobisphosphonates;
Nuclear Medicine and Biology, 1999, 26). Favourable results have been obtained with such radioconjugates. However, this strategy requires two reactions involving radioactivity and three purification steps to obtain the final product. When working with therapeutic doses of radioactivity such procedures are inappropriate in view of radiation safety standards. Up to now, no therapeutic experiments have been conducted with such radioconjugated bisphosphonic acids.
The present invention thus provides an improved labelling technique for bisphosphonic acids. More particularly, the invention focuses on precursors in which the label is incorporated in the final stages of the synthesis. The present process is therefore advantageous compared to the preparation of many of the radionucleotide agents currently in clinical use.
More specifically, the present invention employs trialkylarylsilyl precursors.
These substances are easy to synthesise and appear to be very stable; moreover iododesilylation affords the radioiodinated bisphosphonic acids in very high yield.
Dialkylaryltriazene precursors were also investigated, but labelling yields were lower, and the final purification was hampered by the presence of many side products.
From a chemical perspective, it is widely known that non-radioactive bisphosphonic acids may be used as pharmaceuticals for treating bone related disorders.
Moreover, it is known that other moieties linked to bisphosphonic acids may also affect bone affinity. However, it is the bisphosphonic acid functionality that is primarily responsible for the bone affinity of such molecules. This was the motivation for synthesising a series of radioiodinated aromatic bisphosphonic acids with different alpha functionalities and with various linker groups inserted between the aryl group and the bisphosphonic acid moiety (Fig. 1 ).
The synthesis of compound 2g is depicted in Figure 2. The transformation of m-chlorotoluene (2a) to m-trimethylsilyltoluene (2b) by use of magnesium and trimethylchlorosilane in HMPTA is described in the literature (Effenberger, F.
and Habich, D., Liebigs Ann. Chem., 1979, pp. 842-857). However, prolonged heating is required and the work-up uses large amounts of this strongly carcinogenic solvent. A
second way of synthesising m-trimethylsilyltoluene is by reacting m-bromotoluene with molten sodium in toluene (Clark, H. A. et al., J. Am. Chem. Soc., 1951;
73:3798). However, this route is troublesome and hazardous. In contrast, by refluxing m-chlorotoluene with magnesium in THF followed by the addition of trimethylchlorosilane, m-trimethylsilyltoluene can be obtained in 88-90%
yield.
Trimethylsilyltoluene was then brominated with N-bromosuccinimide in carbon tetrachloride as described in the literature (Severson, R. G. et al., J. Am.
Chem. Soc., 1957; 79:6540). The resulting m-trimethylsilylbenzyl bromide (2c) was reacted with the lithium ylide of diethyl methylenephosphonate providing diethyl m-trimethylsilylphenylethylidene-phosphonate (2d) in moderate yield. The phosphonate was then converted to the corresponding lithium ylide using butyllithium and the subsequent reaction with diethyl chlorophosphate gave the tetraethyl bisphosphonate 2e in high yield. Hydrolysis was achieved by trans-esterification to the corresponding tetramethylsilyl ester 2f, followed by the addition of aqueous ethanol. The bisphosphonic acid was isolated as the corresponding disodium salt 2g.
The synthesis of compound 3g is depicted in Figure 3. m-Trimethylsilylbenzyl bromide (2c) was converted to trimethylsilylbenzyl cyanide (3a) with potassium cyanide under phase transfer conditions. Hydrolysis to m-trimethylsilylphenylacetic acid (3b) was then achieved by refluxing the cyanide with sodium hydroxide in methanol. Overall yields in the range of 59-67% were obtained. The acid was then transformed to the acid chloride 3c employing thionyl chloride at ambient temperature. The ultrasound-assisted reaction of the acid chloride with trimethylphosphite afforded a cis/trans stereoisomeric mixture of the enolphosphonate 3d. The second phosphonate moiety was introduced by adding dimethylphosphite to the enolphosphonate under basic conditions. The corresponding tetramethyl bisphosphonate 3e was trans-esterified to the tetratrimethylsilyl ester 3f, which was subsequently hydrolysed by stirring with aqueous ethanol. The bisphosphonic acid was isolated as its disodium salt 3g.
Compounds 2g and 3g were labelled using an iododesilylation reaction (Figure 4).
This was achieved by adding n.c.a. (non carrier added) Na~3lI and N-chlorosuccinimide as an oxidising agent to a solution of the precursor (2g, 3g) in a mixture of acetic acid and trifluoroacetic acid at room temperature. The yields obtained were greater than 95%, as measured by HPLC. The radioiodinated compounds were purified by HPLC and their structures were confirmed by coelution with the corresponding non-radioactive compounds.
Chemical and radiochemical purity is essential for the in vivo application of radiopharmaceuticals in man. Chemical purity demands the isolation of the radiolabelled compounds from all non-radioactive starting compounds and side-products. The bisphosphonates described in the present invention were all labelled with n.c.a. (non carrier added) radioiodine. Purification and confirmation of their structures was achieved by HPLC. The demand for a non-toxic mobile phase along with the complex aqueous chemistry of bisphosphonates made the development of HPLC systems problematic. In particular, bisphosphonates are apparently strongly associated in aqueous solutions and form clusters with a range of sizes all due to a single compound (Wiedmer, W. H. et al., Ultrafiltrability and Chromatographic Properties of Pyrophosphate, 1-Hydroxyethylidene-1,1-Bisphosphonate, Calci~
Tissue Int., 1983; 35:397-400). However, these problems were successfully solved for analytical and purification purposes, resulting in single peaks for each compound, whilst avoiding severe pressure build-up in the HPLC system With regard to biological activity, the success of radionuclide targeting therapy is strongly dependent on the following conditions:
i) choice of radionuclide;
ii) the ability of the targeting compound to home in on the target quickly and in high amounts;
iii) the retention time at the site;
iv) resistance towards degradation;
v) little accumulation in other organs.
Details of the biological activity of the compounds of the invention are discussed further in the Examples section below.
In summary, the biological results clearly indicate that the compounds disclosed herein exhibit superior properties to previously reported radiohalogenated bisphosphonic acids, in terms of bone affinity, selectivity, kinetics and stability in vivo. Moreover, the compounds of the invention are readily available and can be labelled in high yields by simple means from stable precursors With regard to cancer treatment, the compounds of the invention that are labelled with radioiodine have been shown to seek out the desired target quickly, selectively, and in extraordinarily high amounts. In addition, the compounds display resistance to enzymatic dehalogenation.
The present invention will now be described only by way of example and with reference to the accompanying figures, wherein:
Figure 1 shows the structure of bisphosphonate compounds 2h, 3h, 4d and Sx.
Figure 2 shows the reaction scheme for the preparation of compound 2g.
Figure 3 shows the reaction scheme for the preparation of compound 3g.
Figure 4 shows the reaction scheme for radiolabelling compounds 2g and 3g.
Figure 5 shows the biodistribution of intravenously administered compound 2h in balb/c mice (20-25g) after 30 min, 2 hours, 5 hours and 24 hours.
Figure 6 shows a bioscope picture of the spinal cord (balb/c mice) employing compound 2h.
Figure 7 shows the biodistribution of intravenously administered compound 3h in balb/c mice (20-25g) after 30 min, 2 hours, 5 hours and 24 hours.
Figure 8 shows the biodistribution of intravenously administered compound 3h in nude/nude rats 60g) for HPLC purified 3h and crude 3h.
Figure 9 shows a scintillation picture of intravenously administered compound 3h in nude/nude rat (60g) for HPLC purified compound.
Examples m-Trimethylsilyltoluene A solution of m-chlorotoluene (25.3 g, 0.20 mol), 1,2-dibromoethane (2.18 g, 11.6 mmol) and THF (40 cm3) was added to magnesium turnings (5.35 g, 0.22 mol). The mixture was heated under reflux in an atmosphere of argon for 4 h, after which the heating bath was removed and trimethylchlorosilane (23.9 g, 0.22 mol) was added so that gently reflux was maintained. The mixture was stirred for another 30 min and then separated between 5% NaHC03 (100 cm3) and dichloromethane (3 x 100 cm3).
The organic extract was washed with brine and dried (MgS04). The solvent was removed under reduced pressure affording the title compound (29.4 g, 89%) as a 5 pale yellow liquid, which was used without purification in the next reaction. All spectroscopic data were identical to those reported in the literature (Eisenhut et al., ibiclJ.
m-Trimethylsilylbenzylbromide 10 The reaction was carried out as described in the literature (Bijvoet, O.L.M. et al., Bisphosphonates on Bones, Elsevier Sciences B.V., Amsterdam, 1995, pp. 131 and pp. 142) with the following modifications. The reaction mixture was partitioned between water and dichloromethane, the organic extract was washed with brine, dried (MgS04) and the solvent evaporated. The residue was chromatographed on 15 silica gel using hexane as an eluent to give the title compound (96%) as an orange liquid. The product was used without further purification in the next reaction.
m-Trimethvlsilvlbenzylcyanide A mixture of KCN (5.5 g, 84 mmol), m-trimethylsilylbenzylbromide (5.31 g, 21.8 20 mmol), tributylamine (0.11 g, 0.59 mmol) and water (12.5 cm3) was stirred overnight. The product was extracted with dichloromethane (3 x 30 cm3), the extract was filtered through a silica plug (5 g) and the solvent was removed under reduced pressure. The residue was distilled to give the product (2.27 g, 55%) as a colourless liquid.
Bp. 134-136°C (2 mm); V",~(film)/cni ~ 2956, 2255, 1412, 1249, 885 and 867; 8H
(200 MHz; CDC13; Me4Si) 0.31 (9H, s, CH3), 3.76 (2H, s, CH2) and 7.27-7.56(4H, m, Ar); 8~ (50 MHz; CDCl3; Me4Si) 0.3, 24.3, 117.5, 127.7, 127.9, 128.6, 132.0, 132.3 and 141.2; m/z (EI) 189.0972 (M+-C~1H15NSi requires 189.0974).
m-Trimethylsil~phen~lacetic acid To a solution of NaOH (4.0 g, 0.1 mol) in MeOH (20 cm3) m-trimethyl-silylbenzylcyanide (2.84 g, 15.0 mmol) was added and the mixture was heated under reflux for 4 h. The solvent was evaporated and the residue was dissolved in water (50 cm3). The resulting solution was washed with dichloromethane (2 x 20 cm3), the aqueous phase was acidified with phosphoric acid to pH 2 and extracted with dichloromethane (3 x 30 cm3). The extract washed with brine, dried (MgS04) and the solvent evaporated to give the title compound (2.93 g, 94%) as a yellow liquid.
Vm~(fllm)/crri ~ 3600-2600, 1717, 1418, 1254 and 847; 8H (200 MHz; CDC13;
Me4Si) 0.32 (9H, s, CH3), 3.70 (2H, s, CH2) and 7.30-7.53 (4H, m, Ar); 8~ (50 MHz;
CDC13; Me4Si) 0.53, 41.6, 127.8, 129.6, 132.1, 132.3, 134.1, 140.8 and 177.7;
m/z (EI) 208.0918 (M+-C11Hi602Si requires 208.0920).
Dimethyl m-trimethylsilyl~henylacetylphosphonate To m-trimethylsilylphenylacetic acid (1.04 g, 5.0 mmol) was added SOCl2 (0.82 g, 6.9 mmol) and the mixture was stirred overnight. Excess SOC12 was removed by adding toluene (2 cm3) and then evaporating the solvent under reduced pressure. The residue was dissolved in THF (5 cm3) and trimethylphosphite (0.69 g, 5.5 mmol) was added at -20°C. The resulting solution was kept in an ultrasound bath at 0°C for min, after which the solvent was evaporated to leave the phosphonate as a cream coloured semisolid (1.63 g, 5.0 mmol). The product decomposed upon exposure to 25 the atmosphere. An analytical sample was prepared by washing the crude product with hexane and was shown to consist of a mixture of two stereoisomeric enols.
The spectral data represents the major component.
VmaX(fllm)/crri ~ 3600-2600, 1698, 1249 and 1036; 8H (200 MHz; CDC13; Me4Si) 30 0.19 (9H, s, CH3), 3.86 (6H, d , J 14), 6.11 (1H, d, J 13), 7.20-7.78 (4H, m, Ar) and 7.92 (1H, d, J 7); 8(50 MHz; CDC13; Me4Si) 0.68, 54.6, 117.5, 118.1, 128.1, 130.4, 132.9, 133.6, 134.0, 134.9, 138.3, 140.6 and 142.3; 8P(500 MHz; CDC13; H3POa) 16.3; ; m/z (EI) 300.0929 (M+-C,3H21OaPSi requires 300.0947).
Tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) To a solution of dimethyl m-trimethylsilylphenylacetylphosphonate ( 1.63 g, 5.0 mmol) in Et20 ( 10 cm3) was added a solution of dimethylphosphite (0.83 g, 7.5 mmol), dibutylamine (0.32 g, 2.5 mmol) and hexane (10 cm3) at 0°C. A
white solid began to form after a few minutes. After 20 min the mixture was filtrated to give the title compound (1.34 g, 65%) as a white solid. Recrystallization was achieved by adding water (9.5 cm3) to a solution of the title compound (0.50 g) in acetic acid (0.5 cm3) and then leaving the resulting mixture for several days in the refrigerator.
Filtration afforded the bisphosphonate (0.33 g, 66%) as white crystals.
VmaX(neat)/crri ~ 3700-2815, 2958, 1645 and 1251; 8H (200 MHz; CDC13; MeaSi) 0.25 (9H, s, CH3), 3.38 (2H, t, J 14), 3.71-382 (12H, m, OCH3) and 7.26-7.51 (4H, m, Ar); 8~ (50 MHz; CDC13; MeaSi) 0.1, 40.1, 54.9, 73.3, 76.3, 79.3, 127.7, 132.1, 132.4, 133.8, 136.6 and 140.2; bP(500 MHz; CDC13; H3POa; m/z (EI) 410.1080 (M+-CISH2gO~P2Si requires 410.1080).
Disodium m-trimethylsil~phen~l-1-h dy roxy-ethylidenebis phosphonic acid) To tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) (0.82 g, 2.0 mmol) was added trimethylbromosilane (2.45 g, 16 mmol) and the resulting solution was stirred for 1 h. Excess reagent was then removed under reduced pressure and the residue was dissolved in EtOH (75%, 8 cm3) at 0°C and stirred for 0.5 h. The resultant solution was neutralised to pH 8 with aqueous Na2C03 (1.0 M, 3 cm3) and filtrated to give the disodium bis(phosphonic acid) salt (0.75 g, 94%) as a white solid. The product was used without purification in the next reaction.
Tetramethyl m-iodophenyl-1-hydroxy-ethylidenebis(phos~honate) Tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) (0.41 g, 1.0 mmol) was added to a solution of ICl (0.32 g, 2.0 mmol) in acetic acid (2 cm3) and the mixture was stirred for 5 h. The solvent was then evaporated and the residue was extracted with 1:1 hexane:Et20 (2 x 10 cm3). Remaining solvents were removed from the residue under reduced pressure affording the title compound (0.38 g, 81%) as an orange liquid.
8H (200 MHz; CDC13; Me4Si) 3.18 (2H ,t, J 14) 3.68-3.79 (12H, m, OCH3) and 6.95-7.69 (4H, m, Ar); 8~ (50 MHz; CDC13; Me4Si) 39.7, 55.8, 56.3, 73.3, 76.3, 79.3, 94.2, 129.9, 130.8, 136.3, 136.8 and 140.2; 8P(500 MHz; CDC13; H3P04) 19.6.
m-Iodophenyl-1-hydroxy-ethylidenebisl;phosphonic acid) A sample of tetramethyl m-iodophenyl-1-hydroxy-ethylidenebis(phosphonic acid) was prepared by refluxing the parent tetraester in concentrated hydrochloric acid for 3 h. Evaporation to dryness gave the title compound as an orange powder.
m-ji3il]_Iodophenyl -1-hydroxy-ethylidenebis(phosphonic acid) To a solution of Na131I in water (1-2 ~l) was added a solution of N-chlorosuccinimide (0.4 mg, 3 p.mol) in 9:1 TFA:AcOH (10 ~,1) followed by the addition of disodium m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonic acid) (0.1 mg, 0.25 pmol) in TFA (2 pl). The mixture was vortexed and left for min.
Biolo;~ical Testing In order to assess the targeting ability of the radioiodinated bisphosphonic acids described, tissue distributions were carried out in healthy mice.
24 h after injection, the bone uptake of phenylethylidenebisphosphonic acid (2h) was averaging 15 ~ 5 % ID/g (injected dose per gram bone tissue) in femur, with a peak value of 21 ~ 4 % ID/g after 2 h, indicating that the entire molecule is involved in the bone affinity.
Compound 2h contains a hydrogen atom alpha to the bisphosphonate moiety and it is known in the art that hydroxybisphosphonic acids possess a superior bone affinity in general (Bijvoet, O.L.M. et al., ibiclJ. Consequently, the alpha hydrogen in the phenylethylidenebisphosphonic acid was substituted with a hydroxy group. The resulting hydroxybisphosphonic acid 3h showed a two-fold increase in bone affinity compared to the unsubstituted derivative 2h (Fig. 7). 30 min after injection as much as 45 ~ 7 % ID/g was observed in femur. The bone uptake remained high 24 h after injection (38 ~ 12 % ID/g), with a peak value of 46 ~ 4 % ID/g femur after 2 h. The compound cleared rapidly from all other organs, except the kidneys (5 ~ 2 %
ID/g 24 h) and liver (2 ~ 0.7 % ID/g 24 h).
A fair resemblance has been found regarding uptake of bone seeking agents in various specimens (Murud, K. et al., Evaluation of blood clearance, tissue distribution, and bone microdistribution of 21'At and ~25I labeled bisphosphonates in dogs. Eur. J. Nucl. Med., 1999, submitted). However, care must be exercised when comparing different studies, since the age of the animals used is known to greatly affect bone uptake as well as kidney clearance (Hassfjell, S.P. et al., An alpha particle emitting bone seeking agent for targeted radiotherapy, Nucl. Med.
Biol., 1997, in press). To overcome some of these problems an alternative parameter for the targeting ability of each compound was measured, namely % injected dose per gram tissue x animal body weight.
In this respect, the above-described compounds possess a bone uptake in the range 900 to 1100 (% injected dose per gram tissue x animal body weight). In contrast, the most promising radioiodinated compound reported in the literature to date possesses 5 a bone uptake of only slightly greater than 400 (Larsen, R.H. et al., 2uAt-and ~31I-labeled bisphosphonates with high in vivo stability and bone accumulation. J.
Nucl.
Med., 1999, in press). Since radioiodine is known to accumulate to a high degree in the thyroid gland (McNabb, F. M. Anne, Thyroid hormones, Englewood Cliffs, N.J.:
Prentice Hall, 1992, pp. 23-25), uptake in the neck area is a good measurement for 10 the stability of radioiodinated compounds. Indeed, about 0.05% of injected dose remained in the neck 24 h after injection, while values reported previously in the literature for this class of compounds have been in the range of 0.5% and above.
Biodistribution of compound 3h in nude/nude rats 15 Nude/nude rats with a body weight averaging 60 g were used in the biodistribution experiments. The aim of the study was to compare crude 3h with HPLC-purified 3h.
The term "crude compound" refers to the diluted and neutralised labelling reaction mixture described hereinbefore. The compounds were administrated by tail vein injections of 200 ~1 for each animal, using three animals for each sample. 24 h after 20 injection the animals were killed by a lethal injection and the tissue distribution determined.
A significant difference was observed between crude 3h and HPLC purified 3h (Fig.
Alternatively, the composition may be formulated in an injectable form, for 10 parenteral delivery, for example, by an intravenous, intramuscular or subcutaneous route. The formulation may also be formulated so as to be suitable for delivery by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be stable at acid pH, resistant to proteolytic degradation and to the detergent effects of bile.
The pharmaceutical compositions of the invention can be administered topically in the form of a lotion, solution, cream, ointment or dusting powder, or by the use of a skin patch. Alternatively, the compositions can be administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or in the form of a suppository or pessary. Further modes of administration include inhalation, or parenteral injection, for example intravenously, intramuscularly or subcutaneously.
For parenteral administration, the compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example appropriate levels of salts and/or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.
In a preferred aspect, the present invention relates to the use of the compound or composition of the invention in the palliative or curative treatment of bone disorders.
Preferably, the compounds and/or compositions of the invention are used to treat bone disorders that are cancer-related skeletal diseases, for example, skeletal metastases, osteoblastic osteosarcoma or multiple myelomas. However, the compounds and/or compositions of the invention may also be used to treat non-cancerous bone disorders, such as age-related bone loss, rheumatoid-related bone loss, or bone loss related to osteoporosis, disuse or steroid therapy.
The treatment of skeletal metastases is one of the main problems encountered in practical clinical oncology. It is estimated that up to 85 % of all patients with advanced carcinomas to the breast, prostate or lung develop such metastatic conditions (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 349). Up to now, the prognosis for these patients has been poor.
To date, treatment methods in current clinical practice include external radiotherapy, hormone therapy and chemotherapy, although the number of patients achieving complete curation is negligible (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 350). In particular, severe side-effects often limit the applicability of these methods. Consequently, there is a strong need for improved therapeutic methods for slowing down tumour progression and for pain palliation.
Normally, the main method for radiation therapy is external beam irradiation.
However, if there are multiple skeletal metastases present, a more efficient treatment may be the targeted radiotherapy of timorous osseous lesions by means of radioactive compounds with bone affinity. Such regions are characteristic for several types of bone-related diseases, malignant as well as benign. Examples of malignant lesions are skeletal metastases and osteosarcoma, examples of benign lesions are osteoporosis and Paget's disease (Bijvoet, O.L.M., Fleisch, H.A., Bisphosphonate on Bones, Elsevier Science B.V., Amsterdam, 1995; pp. 293). Pathological bone synthesis in skeletal metastases is observed in very large groups of cancer patients who develop metastatic foci originating from different types of soft-tissue tumours.
Furthermore, there is a small but important, clinically difficult niche of patients suffering from metastasised osteoblastic osteosarcoma. In this case, a primitive bone-like substance, osteoid, is produced by the tumour cells themselves.
Although clinically very different from skeletal metastases, the chemistry of these lesions is similar, and they may often be targeted by the same type of bone affinity compounds.
Usually, myelotoxicity sets the limits for the radiation dose that can be administered to the tumour by means of radioactive bone-affinity compounds. Any development which decreases the radiation dose to the bone marrow would therefore substantially improve targeted radiotherapy techniques.
In a preferred aspect, the present invention thus seeks to provide improved bone affinity compounds which expose the bone marrow to substantially lower levels of radiation.
There are two classes of radioactive bone-affinity compounds currently in clinical use. The first and most important class includes ions of the radioisotopes of alkaline earth elements, such as g9Sr2+, which is the most common bone-seeking agent in clinical use (Lewington, V.J., Cancer therapy using bone-seeking isotopes, Physics in Medicine and Biology, 1996; 41: 2031-2032).
The other class of bone-seeking compounds include radioactive bis- or polyphosphonic acids carrying a (3-active radionuclide. The species most commonly used in the clinic are ~g6Re-HEDP (Lewington, V.J., Cancer therapy using bone-s seeking isotopes, Physics in Medicine and Biology, 1996; 41: 2030) and lssSm-EDTMP (Lewington, V.J., Cancer therapy using bone-seeking isotopes, Physics in Medicine and Biology, 1996; 41:2029). The latter has also been applied with success in the palliative treatment of osteoblastic osteosarcoma (Franzuis, C. et al., High Activity Samarium-153-EDTMP Therapy in Unresectable Osteosarcoma, Nuklearmedizin,1999; 38:337-340). However, in contrast to the compounds disclosed in the present invention, none of these nuclides reduce the tumour progression of small tumours, and hence they are not suitable for curative treatment.
(3-emitters are usually classified into three groups according to (3-particle energy and hence range. With regard to physical properties, ~-particle energy and range are parameters which have to be matched to the size of the tumour. The half life and chemical properties, on the other hand, are related to the pharmacokinetics and metabolism of the carrier molecule. Radionuclides with (3 -energy in the range Eavg =
0.08-0.18 MeV (mean range 0.4-0.9 mm) are best suited for treatment of small tumours (Zweit, J., Radionuclides and carrier molecules for therapy, Physics in Medicine and Biology, 1996; 41: 1908-1910). ~31I is the most familiar and the only radionuclide in this group that has been used clinically. Radionuclides with medium ~-energy such as ~s3Sm and ~86Re are less suitable for curative treatment, since the dose distribution will spare small tumours and the tumour on bone marrow ratio is too low. High energy (3 -emitters such as g9Sr are only suited for the palliative and curative treatment of large tumours. There is speculation as to whether the use of a-emitters in combination with (3-emitters would be superior for curative treatment.
However, the short range of a-radiation (40-80 ~,m) would most likely require bonding of the a-emitting carrier molecule to most cancer cells within the tumour.
In an alternative aspect, the invention also relates to the use of the compound of the invention in the preparation of a medicament for the palliative or curative treatment of bone disorders.
As used herein the phrase "preparation of a medicament" includes the use of a compound of the invention directly as the medicament in addition to its use in a screening programme for the identification of further active agents or in any stage of the manufacture of such a medicament.
Such a screening programme may for example include an assay for determining whether a candidate substance is capable of mimicking the activity of a pharmaceutical compound of the present invention.
Another aspect of the invention provides a method of treating a subject in need of the palliative or curative treatment of bone disorders, the method comprising administering to said subject a therapeutically effective amount of the compound or composition of the present invention.
The present invention also provides a process for preparing a compound of the invention, wherein R comprises a radiolabel, said process comprising the following steps:
(i) preparing a phosphonate precursor comprising Ar, X and Y;
(ii) radiolabelling said bisphosphonate precursor.
In a preferred aspect, step (ii) of the above-mentioned process is a deiodosilylation reaction.
For practical purposes, it is desirable to produce targeting radionuclide agents at the site of use, e.g. hospitals.
1$
To date, the demand for convenient labelling chemistry, high stability and favourable biological behaviour has proven difficult to meet with radiohalogenated bisphosphonates. In recent studies, pre-labelled compounds have been connected to bisphosphonic acid functionalities (Fritzberg, A.R. et al., US5202109:
Conjugates for bone imaging and bone cancer therapy; Murud, k. et al., Synthesis, Purification, and in Vitro Stability of 21 ~At- and ~25I-Labeled Amidobisphosphonates;
Nuclear Medicine and Biology, 1999, 26). Favourable results have been obtained with such radioconjugates. However, this strategy requires two reactions involving radioactivity and three purification steps to obtain the final product. When working with therapeutic doses of radioactivity such procedures are inappropriate in view of radiation safety standards. Up to now, no therapeutic experiments have been conducted with such radioconjugated bisphosphonic acids.
The present invention thus provides an improved labelling technique for bisphosphonic acids. More particularly, the invention focuses on precursors in which the label is incorporated in the final stages of the synthesis. The present process is therefore advantageous compared to the preparation of many of the radionucleotide agents currently in clinical use.
More specifically, the present invention employs trialkylarylsilyl precursors.
These substances are easy to synthesise and appear to be very stable; moreover iododesilylation affords the radioiodinated bisphosphonic acids in very high yield.
Dialkylaryltriazene precursors were also investigated, but labelling yields were lower, and the final purification was hampered by the presence of many side products.
From a chemical perspective, it is widely known that non-radioactive bisphosphonic acids may be used as pharmaceuticals for treating bone related disorders.
Moreover, it is known that other moieties linked to bisphosphonic acids may also affect bone affinity. However, it is the bisphosphonic acid functionality that is primarily responsible for the bone affinity of such molecules. This was the motivation for synthesising a series of radioiodinated aromatic bisphosphonic acids with different alpha functionalities and with various linker groups inserted between the aryl group and the bisphosphonic acid moiety (Fig. 1 ).
The synthesis of compound 2g is depicted in Figure 2. The transformation of m-chlorotoluene (2a) to m-trimethylsilyltoluene (2b) by use of magnesium and trimethylchlorosilane in HMPTA is described in the literature (Effenberger, F.
and Habich, D., Liebigs Ann. Chem., 1979, pp. 842-857). However, prolonged heating is required and the work-up uses large amounts of this strongly carcinogenic solvent. A
second way of synthesising m-trimethylsilyltoluene is by reacting m-bromotoluene with molten sodium in toluene (Clark, H. A. et al., J. Am. Chem. Soc., 1951;
73:3798). However, this route is troublesome and hazardous. In contrast, by refluxing m-chlorotoluene with magnesium in THF followed by the addition of trimethylchlorosilane, m-trimethylsilyltoluene can be obtained in 88-90%
yield.
Trimethylsilyltoluene was then brominated with N-bromosuccinimide in carbon tetrachloride as described in the literature (Severson, R. G. et al., J. Am.
Chem. Soc., 1957; 79:6540). The resulting m-trimethylsilylbenzyl bromide (2c) was reacted with the lithium ylide of diethyl methylenephosphonate providing diethyl m-trimethylsilylphenylethylidene-phosphonate (2d) in moderate yield. The phosphonate was then converted to the corresponding lithium ylide using butyllithium and the subsequent reaction with diethyl chlorophosphate gave the tetraethyl bisphosphonate 2e in high yield. Hydrolysis was achieved by trans-esterification to the corresponding tetramethylsilyl ester 2f, followed by the addition of aqueous ethanol. The bisphosphonic acid was isolated as the corresponding disodium salt 2g.
The synthesis of compound 3g is depicted in Figure 3. m-Trimethylsilylbenzyl bromide (2c) was converted to trimethylsilylbenzyl cyanide (3a) with potassium cyanide under phase transfer conditions. Hydrolysis to m-trimethylsilylphenylacetic acid (3b) was then achieved by refluxing the cyanide with sodium hydroxide in methanol. Overall yields in the range of 59-67% were obtained. The acid was then transformed to the acid chloride 3c employing thionyl chloride at ambient temperature. The ultrasound-assisted reaction of the acid chloride with trimethylphosphite afforded a cis/trans stereoisomeric mixture of the enolphosphonate 3d. The second phosphonate moiety was introduced by adding dimethylphosphite to the enolphosphonate under basic conditions. The corresponding tetramethyl bisphosphonate 3e was trans-esterified to the tetratrimethylsilyl ester 3f, which was subsequently hydrolysed by stirring with aqueous ethanol. The bisphosphonic acid was isolated as its disodium salt 3g.
Compounds 2g and 3g were labelled using an iododesilylation reaction (Figure 4).
This was achieved by adding n.c.a. (non carrier added) Na~3lI and N-chlorosuccinimide as an oxidising agent to a solution of the precursor (2g, 3g) in a mixture of acetic acid and trifluoroacetic acid at room temperature. The yields obtained were greater than 95%, as measured by HPLC. The radioiodinated compounds were purified by HPLC and their structures were confirmed by coelution with the corresponding non-radioactive compounds.
Chemical and radiochemical purity is essential for the in vivo application of radiopharmaceuticals in man. Chemical purity demands the isolation of the radiolabelled compounds from all non-radioactive starting compounds and side-products. The bisphosphonates described in the present invention were all labelled with n.c.a. (non carrier added) radioiodine. Purification and confirmation of their structures was achieved by HPLC. The demand for a non-toxic mobile phase along with the complex aqueous chemistry of bisphosphonates made the development of HPLC systems problematic. In particular, bisphosphonates are apparently strongly associated in aqueous solutions and form clusters with a range of sizes all due to a single compound (Wiedmer, W. H. et al., Ultrafiltrability and Chromatographic Properties of Pyrophosphate, 1-Hydroxyethylidene-1,1-Bisphosphonate, Calci~
Tissue Int., 1983; 35:397-400). However, these problems were successfully solved for analytical and purification purposes, resulting in single peaks for each compound, whilst avoiding severe pressure build-up in the HPLC system With regard to biological activity, the success of radionuclide targeting therapy is strongly dependent on the following conditions:
i) choice of radionuclide;
ii) the ability of the targeting compound to home in on the target quickly and in high amounts;
iii) the retention time at the site;
iv) resistance towards degradation;
v) little accumulation in other organs.
Details of the biological activity of the compounds of the invention are discussed further in the Examples section below.
In summary, the biological results clearly indicate that the compounds disclosed herein exhibit superior properties to previously reported radiohalogenated bisphosphonic acids, in terms of bone affinity, selectivity, kinetics and stability in vivo. Moreover, the compounds of the invention are readily available and can be labelled in high yields by simple means from stable precursors With regard to cancer treatment, the compounds of the invention that are labelled with radioiodine have been shown to seek out the desired target quickly, selectively, and in extraordinarily high amounts. In addition, the compounds display resistance to enzymatic dehalogenation.
The present invention will now be described only by way of example and with reference to the accompanying figures, wherein:
Figure 1 shows the structure of bisphosphonate compounds 2h, 3h, 4d and Sx.
Figure 2 shows the reaction scheme for the preparation of compound 2g.
Figure 3 shows the reaction scheme for the preparation of compound 3g.
Figure 4 shows the reaction scheme for radiolabelling compounds 2g and 3g.
Figure 5 shows the biodistribution of intravenously administered compound 2h in balb/c mice (20-25g) after 30 min, 2 hours, 5 hours and 24 hours.
Figure 6 shows a bioscope picture of the spinal cord (balb/c mice) employing compound 2h.
Figure 7 shows the biodistribution of intravenously administered compound 3h in balb/c mice (20-25g) after 30 min, 2 hours, 5 hours and 24 hours.
Figure 8 shows the biodistribution of intravenously administered compound 3h in nude/nude rats 60g) for HPLC purified 3h and crude 3h.
Figure 9 shows a scintillation picture of intravenously administered compound 3h in nude/nude rat (60g) for HPLC purified compound.
Examples m-Trimethylsilyltoluene A solution of m-chlorotoluene (25.3 g, 0.20 mol), 1,2-dibromoethane (2.18 g, 11.6 mmol) and THF (40 cm3) was added to magnesium turnings (5.35 g, 0.22 mol). The mixture was heated under reflux in an atmosphere of argon for 4 h, after which the heating bath was removed and trimethylchlorosilane (23.9 g, 0.22 mol) was added so that gently reflux was maintained. The mixture was stirred for another 30 min and then separated between 5% NaHC03 (100 cm3) and dichloromethane (3 x 100 cm3).
The organic extract was washed with brine and dried (MgS04). The solvent was removed under reduced pressure affording the title compound (29.4 g, 89%) as a 5 pale yellow liquid, which was used without purification in the next reaction. All spectroscopic data were identical to those reported in the literature (Eisenhut et al., ibiclJ.
m-Trimethylsilylbenzylbromide 10 The reaction was carried out as described in the literature (Bijvoet, O.L.M. et al., Bisphosphonates on Bones, Elsevier Sciences B.V., Amsterdam, 1995, pp. 131 and pp. 142) with the following modifications. The reaction mixture was partitioned between water and dichloromethane, the organic extract was washed with brine, dried (MgS04) and the solvent evaporated. The residue was chromatographed on 15 silica gel using hexane as an eluent to give the title compound (96%) as an orange liquid. The product was used without further purification in the next reaction.
m-Trimethvlsilvlbenzylcyanide A mixture of KCN (5.5 g, 84 mmol), m-trimethylsilylbenzylbromide (5.31 g, 21.8 20 mmol), tributylamine (0.11 g, 0.59 mmol) and water (12.5 cm3) was stirred overnight. The product was extracted with dichloromethane (3 x 30 cm3), the extract was filtered through a silica plug (5 g) and the solvent was removed under reduced pressure. The residue was distilled to give the product (2.27 g, 55%) as a colourless liquid.
Bp. 134-136°C (2 mm); V",~(film)/cni ~ 2956, 2255, 1412, 1249, 885 and 867; 8H
(200 MHz; CDC13; Me4Si) 0.31 (9H, s, CH3), 3.76 (2H, s, CH2) and 7.27-7.56(4H, m, Ar); 8~ (50 MHz; CDCl3; Me4Si) 0.3, 24.3, 117.5, 127.7, 127.9, 128.6, 132.0, 132.3 and 141.2; m/z (EI) 189.0972 (M+-C~1H15NSi requires 189.0974).
m-Trimethylsil~phen~lacetic acid To a solution of NaOH (4.0 g, 0.1 mol) in MeOH (20 cm3) m-trimethyl-silylbenzylcyanide (2.84 g, 15.0 mmol) was added and the mixture was heated under reflux for 4 h. The solvent was evaporated and the residue was dissolved in water (50 cm3). The resulting solution was washed with dichloromethane (2 x 20 cm3), the aqueous phase was acidified with phosphoric acid to pH 2 and extracted with dichloromethane (3 x 30 cm3). The extract washed with brine, dried (MgS04) and the solvent evaporated to give the title compound (2.93 g, 94%) as a yellow liquid.
Vm~(fllm)/crri ~ 3600-2600, 1717, 1418, 1254 and 847; 8H (200 MHz; CDC13;
Me4Si) 0.32 (9H, s, CH3), 3.70 (2H, s, CH2) and 7.30-7.53 (4H, m, Ar); 8~ (50 MHz;
CDC13; Me4Si) 0.53, 41.6, 127.8, 129.6, 132.1, 132.3, 134.1, 140.8 and 177.7;
m/z (EI) 208.0918 (M+-C11Hi602Si requires 208.0920).
Dimethyl m-trimethylsilyl~henylacetylphosphonate To m-trimethylsilylphenylacetic acid (1.04 g, 5.0 mmol) was added SOCl2 (0.82 g, 6.9 mmol) and the mixture was stirred overnight. Excess SOC12 was removed by adding toluene (2 cm3) and then evaporating the solvent under reduced pressure. The residue was dissolved in THF (5 cm3) and trimethylphosphite (0.69 g, 5.5 mmol) was added at -20°C. The resulting solution was kept in an ultrasound bath at 0°C for min, after which the solvent was evaporated to leave the phosphonate as a cream coloured semisolid (1.63 g, 5.0 mmol). The product decomposed upon exposure to 25 the atmosphere. An analytical sample was prepared by washing the crude product with hexane and was shown to consist of a mixture of two stereoisomeric enols.
The spectral data represents the major component.
VmaX(fllm)/crri ~ 3600-2600, 1698, 1249 and 1036; 8H (200 MHz; CDC13; Me4Si) 30 0.19 (9H, s, CH3), 3.86 (6H, d , J 14), 6.11 (1H, d, J 13), 7.20-7.78 (4H, m, Ar) and 7.92 (1H, d, J 7); 8(50 MHz; CDC13; Me4Si) 0.68, 54.6, 117.5, 118.1, 128.1, 130.4, 132.9, 133.6, 134.0, 134.9, 138.3, 140.6 and 142.3; 8P(500 MHz; CDC13; H3POa) 16.3; ; m/z (EI) 300.0929 (M+-C,3H21OaPSi requires 300.0947).
Tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) To a solution of dimethyl m-trimethylsilylphenylacetylphosphonate ( 1.63 g, 5.0 mmol) in Et20 ( 10 cm3) was added a solution of dimethylphosphite (0.83 g, 7.5 mmol), dibutylamine (0.32 g, 2.5 mmol) and hexane (10 cm3) at 0°C. A
white solid began to form after a few minutes. After 20 min the mixture was filtrated to give the title compound (1.34 g, 65%) as a white solid. Recrystallization was achieved by adding water (9.5 cm3) to a solution of the title compound (0.50 g) in acetic acid (0.5 cm3) and then leaving the resulting mixture for several days in the refrigerator.
Filtration afforded the bisphosphonate (0.33 g, 66%) as white crystals.
VmaX(neat)/crri ~ 3700-2815, 2958, 1645 and 1251; 8H (200 MHz; CDC13; MeaSi) 0.25 (9H, s, CH3), 3.38 (2H, t, J 14), 3.71-382 (12H, m, OCH3) and 7.26-7.51 (4H, m, Ar); 8~ (50 MHz; CDC13; MeaSi) 0.1, 40.1, 54.9, 73.3, 76.3, 79.3, 127.7, 132.1, 132.4, 133.8, 136.6 and 140.2; bP(500 MHz; CDC13; H3POa; m/z (EI) 410.1080 (M+-CISH2gO~P2Si requires 410.1080).
Disodium m-trimethylsil~phen~l-1-h dy roxy-ethylidenebis phosphonic acid) To tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) (0.82 g, 2.0 mmol) was added trimethylbromosilane (2.45 g, 16 mmol) and the resulting solution was stirred for 1 h. Excess reagent was then removed under reduced pressure and the residue was dissolved in EtOH (75%, 8 cm3) at 0°C and stirred for 0.5 h. The resultant solution was neutralised to pH 8 with aqueous Na2C03 (1.0 M, 3 cm3) and filtrated to give the disodium bis(phosphonic acid) salt (0.75 g, 94%) as a white solid. The product was used without purification in the next reaction.
Tetramethyl m-iodophenyl-1-hydroxy-ethylidenebis(phos~honate) Tetramethyl m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonate) (0.41 g, 1.0 mmol) was added to a solution of ICl (0.32 g, 2.0 mmol) in acetic acid (2 cm3) and the mixture was stirred for 5 h. The solvent was then evaporated and the residue was extracted with 1:1 hexane:Et20 (2 x 10 cm3). Remaining solvents were removed from the residue under reduced pressure affording the title compound (0.38 g, 81%) as an orange liquid.
8H (200 MHz; CDC13; Me4Si) 3.18 (2H ,t, J 14) 3.68-3.79 (12H, m, OCH3) and 6.95-7.69 (4H, m, Ar); 8~ (50 MHz; CDC13; Me4Si) 39.7, 55.8, 56.3, 73.3, 76.3, 79.3, 94.2, 129.9, 130.8, 136.3, 136.8 and 140.2; 8P(500 MHz; CDC13; H3P04) 19.6.
m-Iodophenyl-1-hydroxy-ethylidenebisl;phosphonic acid) A sample of tetramethyl m-iodophenyl-1-hydroxy-ethylidenebis(phosphonic acid) was prepared by refluxing the parent tetraester in concentrated hydrochloric acid for 3 h. Evaporation to dryness gave the title compound as an orange powder.
m-ji3il]_Iodophenyl -1-hydroxy-ethylidenebis(phosphonic acid) To a solution of Na131I in water (1-2 ~l) was added a solution of N-chlorosuccinimide (0.4 mg, 3 p.mol) in 9:1 TFA:AcOH (10 ~,1) followed by the addition of disodium m-trimethylsilylphenyl-1-hydroxy-ethylidenebis(phosphonic acid) (0.1 mg, 0.25 pmol) in TFA (2 pl). The mixture was vortexed and left for min.
Biolo;~ical Testing In order to assess the targeting ability of the radioiodinated bisphosphonic acids described, tissue distributions were carried out in healthy mice.
24 h after injection, the bone uptake of phenylethylidenebisphosphonic acid (2h) was averaging 15 ~ 5 % ID/g (injected dose per gram bone tissue) in femur, with a peak value of 21 ~ 4 % ID/g after 2 h, indicating that the entire molecule is involved in the bone affinity.
Compound 2h contains a hydrogen atom alpha to the bisphosphonate moiety and it is known in the art that hydroxybisphosphonic acids possess a superior bone affinity in general (Bijvoet, O.L.M. et al., ibiclJ. Consequently, the alpha hydrogen in the phenylethylidenebisphosphonic acid was substituted with a hydroxy group. The resulting hydroxybisphosphonic acid 3h showed a two-fold increase in bone affinity compared to the unsubstituted derivative 2h (Fig. 7). 30 min after injection as much as 45 ~ 7 % ID/g was observed in femur. The bone uptake remained high 24 h after injection (38 ~ 12 % ID/g), with a peak value of 46 ~ 4 % ID/g femur after 2 h. The compound cleared rapidly from all other organs, except the kidneys (5 ~ 2 %
ID/g 24 h) and liver (2 ~ 0.7 % ID/g 24 h).
A fair resemblance has been found regarding uptake of bone seeking agents in various specimens (Murud, K. et al., Evaluation of blood clearance, tissue distribution, and bone microdistribution of 21'At and ~25I labeled bisphosphonates in dogs. Eur. J. Nucl. Med., 1999, submitted). However, care must be exercised when comparing different studies, since the age of the animals used is known to greatly affect bone uptake as well as kidney clearance (Hassfjell, S.P. et al., An alpha particle emitting bone seeking agent for targeted radiotherapy, Nucl. Med.
Biol., 1997, in press). To overcome some of these problems an alternative parameter for the targeting ability of each compound was measured, namely % injected dose per gram tissue x animal body weight.
In this respect, the above-described compounds possess a bone uptake in the range 900 to 1100 (% injected dose per gram tissue x animal body weight). In contrast, the most promising radioiodinated compound reported in the literature to date possesses 5 a bone uptake of only slightly greater than 400 (Larsen, R.H. et al., 2uAt-and ~31I-labeled bisphosphonates with high in vivo stability and bone accumulation. J.
Nucl.
Med., 1999, in press). Since radioiodine is known to accumulate to a high degree in the thyroid gland (McNabb, F. M. Anne, Thyroid hormones, Englewood Cliffs, N.J.:
Prentice Hall, 1992, pp. 23-25), uptake in the neck area is a good measurement for 10 the stability of radioiodinated compounds. Indeed, about 0.05% of injected dose remained in the neck 24 h after injection, while values reported previously in the literature for this class of compounds have been in the range of 0.5% and above.
Biodistribution of compound 3h in nude/nude rats 15 Nude/nude rats with a body weight averaging 60 g were used in the biodistribution experiments. The aim of the study was to compare crude 3h with HPLC-purified 3h.
The term "crude compound" refers to the diluted and neutralised labelling reaction mixture described hereinbefore. The compounds were administrated by tail vein injections of 200 ~1 for each animal, using three animals for each sample. 24 h after 20 injection the animals were killed by a lethal injection and the tissue distribution determined.
A significant difference was observed between crude 3h and HPLC purified 3h (Fig.
8). The purified compound possessed a 3-fold higher bone affinity than the crude 25 product. Both samples showed high stability and selectivity although the purified sample was again superior. The bone affinity found for HPLC purified 3h was consistent with the results obtained with balb/C mice. Femur uptake was found to be 1020 %g dose/g. In contrast, the most promising radioiodinated compound reported in the literature to date possesses a bone uptake of only slightly above 400 %g dose/g.
It is interesting to compare the selectivity found in this experiment with the results obtained with balb/c mice. In mice, the highest uptake in non target tissues was found in kidneys. After 24 h the ratio between femur and kidney was found to be 7.
In rats the ratio between femur and kidney was found to be above 45, indicating a remarkable selectivity. A scintillation picture of the HPLC purified compound in nude/nude rat further demonstrated the high selectivity of this compound (Fig.
It is interesting to compare the selectivity found in this experiment with the results obtained with balb/c mice. In mice, the highest uptake in non target tissues was found in kidneys. After 24 h the ratio between femur and kidney was found to be 7.
In rats the ratio between femur and kidney was found to be above 45, indicating a remarkable selectivity. A scintillation picture of the HPLC purified compound in nude/nude rat further demonstrated the high selectivity of this compound (Fig.
9).
To date, the most promising radioiodinated compound reported in the literature possesses ratios between femur uptake and uptake in non target tissues of 3/2 (femurapleen), 7/3 (femur/liver) and 20 (femur/kidneys) after 24 h.
Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.
To date, the most promising radioiodinated compound reported in the literature possesses ratios between femur uptake and uptake in non target tissues of 3/2 (femurapleen), 7/3 (femur/liver) and 20 (femur/kidneys) after 24 h.
Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.
Claims (20)
1. A pharmaceutical compound, or pharmaceutically acceptable salt thereof, for use in medicine, wherein said compound is of formula I
R-Ar-X-Y
I
wherein R is a pharmaceutically active moiety;
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
R-Ar-X-Y
I
wherein R is a pharmaceutically active moiety;
Ar is an aromatic moiety;
X is a linker group; and Y is a moiety comprising two phosphonate groups.
2. A compound according to claim 1 wherein Y comprises a geminal bisphosphonate group.
3. A compound according to claim 1 or claim 2, wherein said compound is of formula II
and wherein Z is H, NH2 or an oxy substituent.
and wherein Z is H, NH2 or an oxy substituent.
4. A compound according to claim 3 wherein Z is H or OH.
5. A compound according to any preceding claim wherein X is a substituted or unsubstituted C1-4 alkylene group.
6. A compound according to any one of claims 1 to 4 wherein X is a C1-4 amine group, C1-4 ether group or a C1-4 thioether group, each of which may be substituted or unsubstituted.
7. A compound according to any one of claims 1 to 4 wherein X is S=O or SO2.
8. A compound according to any preceding claim wherein said pharmaceutically active moiety comprises a radiolabel.
9. A compound according to any preceding claim wherein said pharmaceutically active moiety R is attached directly to said aromatic moiety.
10. A compound according to any preceding claim wherein said aromatic moiety is electron deficient.
11. A compound according to any preceding claim wherein said aromatic moiety is a single aromatic ring.
12. A pharmaceutical composition comprising a compound according to any one of the preceding claims admixed with a pharmaceutically acceptable carrier, diluent, or excipient.
13. A compound according to any one of claims 1 to 11 for use in the palliative or curative treatment of bone disorders.
14. Use of a compound according to any one of claims 1 to 11 in the preparation of a medicament for the palliative or curative treatment of bone disorders.
15. Use according to claim 13 or claim 14 wherein the bone disorder is a cancer-related skeletal disease.
16. A method of treating a subject in need of palliative or curative treatment of bone disorders, the method comprising administering to said subject a therapeutically effective amount of a compound according to any one of claims 1 to 11, or a composition according to claim 12.
17. Use of a compound according to any one of claims 1 to 11 in calcified tissue imaging.
18. A process for preparing a compound according to claim 8, said process comprising the following steps:
(i) preparing a phosphonate precursor comprising Ar, X and Y;
(ii) radiolabelling said phosphonate precursor.
(i) preparing a phosphonate precursor comprising Ar, X and Y;
(ii) radiolabelling said phosphonate precursor.
19. A process according to claim 18 wherein step (ii) is a deiodosilylation reaction.
20. A compound, composition, use or process substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0019377A GB0019377D0 (en) | 2000-08-07 | 2000-08-07 | Compound |
| GB0019377.1 | 2000-08-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2319694A1 true CA2319694A1 (en) | 2002-02-07 |
Family
ID=9897150
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2319694 Abandoned CA2319694A1 (en) | 2000-08-07 | 2000-09-15 | Compound |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2319694A1 (en) |
| GB (1) | GB0019377D0 (en) |
-
2000
- 2000-08-07 GB GB0019377A patent/GB0019377D0/en not_active Ceased
- 2000-09-15 CA CA 2319694 patent/CA2319694A1/en not_active Abandoned
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| GB0019377D0 (en) | 2000-09-27 |
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