Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
  • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
  • C07D213/53—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
  • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
  • C07D211/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/81—Amides; Imides
  • C07D213/82—Amides; Imides in position 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
  • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
  • C07D277/04—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic System
  • C07F13/005—Compounds without a metal-carbon linkage
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala

Definitions

• the present invention relates to a dihydropyridine pyridinium salt type of redox, or chemical, delivery system for the site-specific and/or siteenhanced delivery of a radionuclide to the brain and other organs. More particularly, this invention relates to the discovery that a chelating agent capable of chelating with a radionuclide and having a primary, secondary or tertiary amino function can be converted to the corresponding analogue in which said function is replaced with a dihydropyridine pyridinium salt redox system and then complexed with a radionuclide to provide a new radiopharmaceutical that, in its lipoidai dihydropyridine form, penetrates the blood-brain barrier ("BBB”) and allows increased levels of radionuclide concentration in the brain, particularly since oxidation of the dihydropyridine moiety in vivo to the ionic pyridinium salt retards elimination from the brain while elimination from the general circulation is accelerated.
• Radiographic techniques such as scintigraphy, and the like, find application in biological and medical procedures for diagnosis as well as research.
• Scintigraphy involves the use of radiopharmaceuticals; i.e., compounds containing (or labeled with) a radioisotope (i.e. radionuclide) which upon introduction into a mammal become localized in specific organs, tissue, or skeletal material that are sought to be imaged.
• traces, plates, or scintiphotos of the existing distribution of the radionuclide may be made by various radiation detectors known in the art. The observed distribution of the localized radionuclide can then be used to detect the presence of pathological conditions, abnormalities, and the like. Radiopharmaceuticals are thus often referred to as radiodiagnostics.
• radiopharmaceuticals are prepared using target-specific chelating agents which provide a bridge connecting a radionuclide, such as a radioactive metal like technetium-99m, or the like, and a material which will temporarily localize in the organ, tissue, or skeletal material which is to be imaged.
• Typical chelating agents for such purposes a re : polydentate ligands that form a 1:1 or 2:1 ligand: radioactive metal complex; macrocyclic ligands of appropriate ring size and preferably where all coordina ting atoms are in a planar configuration; and bicyclic or polycyclic ligands that can encapsulate the radioactive metal.
• a dihydropyridine pyridinium redox system has now been successfully applied to delivery to the brain of a number of drugs.
• a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the bloodbrain barrier following its systemic administration.
• Subsequent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
• the first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component.
• This approach was first applied to delivering to the brain N-methyl pyridinium-2-carbaldoxime chloride (2-PAM), the active nucleus of which constitutes a quaternary pyridinium salt, by way of the dihydropyridine latentiated prodrug form thereof.
• N-methyl pyridinium-2-carbaldoxime chloride (2-PAM)
• a hyd rophi 1 ic compound (2-PAM) was made lipoidai (i.e. lipophilic) by making its dihydropyridine form (Pro-2-PAM) to enable its penetration through lipoidai barriers.
• a drug [D] is coupled to a quaternary carrier [QC] and the [D-QC] which results is then reduced chemically to the lipoidai dihydro form [D-DHC].
• [D-DHC] is rapidly distributed throughout the body, including the brain.
• the dihydro form [D-DHC] is then in situ oxidized (rate constant, k 1 ) (by the NAD .
• D is the residue of a centrally acting primary, secondary or tertiary amine, and is a radical of the formula
• dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinol ine ring system; m is zero or one; n is zero, one or two; p is zero, one or two, provided that when p is one or two, each R in formula (ii) can be located on either of the two fused rings; q is zero, one, or two, provided that when q is one or two, each R in formula (iii) can be located on either of the two fused rings; and each R is independently selected from the group consisting of halo, C 1 -C 7 alkyl, C 1 -C 7 alkoxy, C 2 -C 8 alkoxycarbonyl, C 2 -C 8 alkanoyloxy, C 1 -C 7 haloalkyl, C 1 -C 7 alkylthi
• the new dihydropyridine analogues described in the preceding paragraph act as a delivery system for the corresponding quaternary compounds in vivo; the quaternary derivatives, which also are chemical inter mediates to the dihydro compounds, are pharmacologically active and are characterized by site-specific and sustained delivery to the brain when administered via the corresponding dihydropyridine form. Nevertheless, a serious need still exists for an effective general method for the site-specific and/or sustained delivery of a desired radionuclide to the brain. It would therefore be desirable to adapt the analogue concept to the radiopharmaceutical area.
• a chemical delivery system based upon a dihydropyridine pyridinium salt type redox system is uniquely well suited for an effective site-specific and/or sustained and/or enhanced delivery of a radionuclide to the brain o r like organ, via novel redox system-containing radiopharmaceuticals, and novel redox system-containing chelating agents and novel redox system-containing precursors thereto, useful in the preparation of said radiopharmaceuticals.
• the present invention thus provides novel redox system-containing chelating agent precursors having the formula
• a chelating agent capable of chelating with a metallic radionuclide, said chelating agent having at least one primary, secondary or tertiary amino functionorial group, said functional group being not essential for the complexing properties of said chelating agent, said residue being characterized by the absence of at least one of said primary, secondary or tertiary amino functional groups from the chelating agent;
• y is 1 or 2; is a radical of the formula
• the present invention provides novel redox system-containing chelating agents having the formula
• i n and y are def i ned as above , and i s a rad ica l of the formul a
• dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroqu inol ine ring system; m is zero or one; n is zero, one or two; p is zero, one or two, provided that when .p is one or two, each R in formula (ii) can be located on either of the two fused rings; q is 2ero, one, or two, provided that when q is one or two, each R in formula (iii) can be located on either of the two fused rings; and each R is independently selected from the group consisting of halo, C 1 -C 7 alkyl, C 1 -C 7 alkoxy, C 2 -C 8 alkoxycarbonyl, C 2 -C 8 alkanoyloxy, C 1 -C 7 haloalkyl, C 1 -C
• the present invention provides, as an effective radionuclide delivery system, novel redox system-containing radiopharmaceutical s of the formula
• (III) is the chelated, or complexed, counterpart of (II), formed by complexing the novel redox system-containing chelating agent of formula (II) with a radioactive metal.
• a radiopharmaceutical of formula (III) is administered, due to its lipoidai nature it readily penetrates the BBB. Oxidation of (III) in vivo affords the corresponding pyridinium salt of the formula
• the present invention does not in fact provide a system for delivery and imaging of previously known radiopharmaceuticals; by the time the present delivery system would no longer be in its "locked in” quaternary form, it would generally no longer be sufficiently radioactive for practical imaging.
• the present invention provides, and indeed requires, an active quaternary form locked in the brain in order to allow effective radionuclide imaging.
• Technetium-99m is a preferred radionuclide for diagnostic purposes 'because of its favorable radiation energy, its relatively short half-life, and the absence of corpuscular radiation, and is preferred for use in the present invention.
• Other radionucl ides that can be used diagnostically herein in a chelated form are cobalt-57, gallium-67, gallium-68, indium-Ill, indium-lllm, and the like.
• non-toxic pharmaceutically acceptable salts as used herein generally includes the non-toxic salts of products of the invention of structures (II) and (III) hereinabove formed with non-toxic, pharmaceutically acceptable inorganic or organic acids of the general formula HX.
• the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pampic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, fumaric, methanesul fonic, toluenesul fonic and the like.
• organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pampic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, fumaric, methanesul fonic, toluene
• halo encompasses fluoro, chVoro, bromo and iodo.
• C 1 -C 7 alkyl includes straight and branched lower alkyl radicals having up to seven carbon atoms. When R, R', R'' and/or R''' are C 1 -C 7 alkyl, they are preferably methyl or ethyl.
• C 1 -C 7 alkoxy includes straight and branched chain lower alkoxy radicals having up to seven carbon atoms. When R is C 1 -C 7 alkoxy, it is preferably methoxy or ethoxy.
• C 2 -C 8 al koxycarbonyl designates straight and branched chain radicals of the formula (C 1 -C 7 a l kyl ) -.
• C 1 -C 7 alkyl group is defined as above.
• R is al koxycarbonyl, it is preferably ethoxycarbonyl or isopropoxycarbonyl.
• C 2 -C 8 alkanoyloxy designates straight and branched chain radicals of the formula
• R is alkanoyloxy, it is preferably acetoxy, pivalyloxy or isobutyryloxy.
• C 1 -C 7 haloalkyl designates straight and branched chain Tower alkyl radicals having up to seven carbon atoms and bearing one or more halo substituents (F, Cl, Br or I), which can be the same or different.
• R is haloalkyl
• the group contains 1 or 2 carbon atoms and bears 1 to 3 halogen substituents, e.g. chloromethyl or trifluoromethyl.
• C 1 -C 7 alkylthio includes straight and branched chain radicals of the type
• C 1 -C 7 alkyl is defined as before.
• R is alkylthio, it is preferably methylthio.
• C 1 -C 7 alkylsulfinyl and " C 1 -C 7 alkylsulfonyl” designate radicals of the formulas
• C 1 -C 7 alkyl is defined as before.
• R is al kylsulfinyl or alkylsulfonyl, methylsulfinyl and methyl sul fonyl are preferred.
• R When R is -CONR'R'', it is preferably -CONH 2 or -C0N(CH 3 ) 2 .
• y is preferably 1; n, m, p or q is preferably one; and R is preferably located in the 3-position in structures (a), (b), (i) or (ii) and in the 4-position in structures (c) or (iii).
• R 1 , R 2 , R 3 and R 4 are each H or C 1 -C 3 alkyl and n' is an Integer of 0 to 3, then the corresponding residue is
• a compound of formula (III) may be administered as the free base or in the form of a non-toxic pharmaceutically acceptable salt thereof, i.e. a salt which can be represented by the formula
• (IV) compound is not necessarily the same as that present in the formula (III) compound. Indeed, the exact identity of the anionic portion of the compound of formula (IV) is immaterial to the in vivo transformation of (III) to (IV).
• the sustained delivery of a radionuclide to the brain in sufficient concentrations for radioimaging can be effected with much lower concentrations in the peripheral circulation and other tissues.
• the present invention of course will allow such imaging of any other organs or glands in which sufficient radioactivity accumulates.
• the quaternary form (IV) which is locked in the brain will be locked in the testes as well.
• the novel radionuclide delivery system of this invention begins with the preparation of the novel redox system-containing chelating agent precursors of formula (I).
• the preparation of those precursors will be tailored to the particular chelating portion and redox portion to be combined, as well as to the presence or absence of other reactive functional groups (amino, mercapto, carboxyl, hydroxy) in either the chelating or redox portion.
• other reactive functional groups amino, mercapto, carboxyl, hydroxy
• Protective groups are well known in the art and include t-butoxycarbonyl for amino groups, N-methyleneacetamido for mercaptans, and N-hydroxysuccinimidyl for carboxyl groups. Acyl or carbonate groups are typically utilized to protect alcohol hydroxyls. When carbonate protecting groups are desired, the step of introducing the protecting groups will involve reacting the alcohol with a halocarbonate of the type ROCOC1 or ROCOBr (formed by reaction of ROH with COCl 2 or COBR 2 ), R typically being lower alkyl.
• any non-toxic redox moiety of structure (a), (b) or (c) hereinabove comprising, containing or includin the pyridinium nucleus, whether or not a part of any larger basic nucleus, and whether substituted or un substituted, the only criterion therefor being capacity for chemical reduction to the corresponding dihydropyridine form , BBB-penetration of and in vivo oxidation of back to the quaternary pyridinium salt redox moiety .
• the ionic pyridinium salt radiopharmaceut ical/redox entity of formula (IV) which results from in vivo oxidation of the dihydropyridine form (III) is prevented from efflux from the brain, while elimination from the general circulation is accelerated.
• Radioimaging of the radionuclide present in the "locked in” formula (IV) quaternary allows observation of the distribution of the localized radionuclide for diagnosis of pathological conditions, abnormalities, etc.
• Schemes 1 (version 1), 5 (versions 1 and 2) and 9 (version 1) above illustrate typical conversion of a carboxylic acid ester group to the corresponding amide (-CONH2); reduction of the amide function to the corresponding amine (-CH 2 NH 2 ); replacement of the -NH 2 group with the desired quaternary function , utilizing a Zincke reagent; and reduction of the resultant quaternary of formula (I) to the corresponding dihydro of formula (II), or conversion of (I) directly to the formula (III) radiopharmaceutical.
• the secondary amino and m ⁇ rcapto groups are regenerated by reacting the protected intermediate with mercuric chloride in an organic solvent such as methanol, conveniently at room temperature, and then decomposing the resulting complex with hydrogen sulfide. See, for example British Patent Specification No. 585,250, which utilizes such a procedure for the production of esters of penicillamine.
• Schemes 2 (version 1), 4 and 10 above illustrate typical conversion of an alcohol (-CH 2 OH), which may be obtained from the corresponding carboxylic acid ester, to the corresponding halide (- C H 2 C 1 o r - C H 2 B r ) ; reaction of the halo derivative with the appropriate pyridine derivative H to afford the desired formula (I) quaternary; and reduction to the corresponding formula (II) dihydro or conversion directly to the corresponding formula (III) radiopharmaceutical.
• Schemes 4 and 10 also illustrate removal of a protecting group immediately after formation of the quaternary, while version 2 of Scheme 2 illustrates introduction and removal of the thiazolidine protecting group discussed above with respect to versions 2 and 3 of Scheme 1, etc.
• Schemes 6, 11 and 12 illustrate yet other methods for lengthening the alkylene chain, the chain here being interrupted by one or more oxygen atoms.
• a -CH 2 OH group is typically converted to the corresponding lithium salt and then reacted ' with an iodoalkanol, e.g. ICH 2 CH 2 OH, to convert the -CH 2 O-Li + group to a -CH 2 OCH 2 CH 2 OH group.
• an iodoalkanol e.g. ICH 2 CH 2 OH
• the processes exemplified by Schemes 1, 3, 5, 7, 8, 9 and 13 include the step of reacting a compound containing an -NH 2 group with a Zincke reagent.
• the Zincke reaction can be used to derive the instant derivatives in which is the residue of a primary amine, or their protected counterparts, directly from the corresponding primary amine/parent chelating agent.
• it is desired to prepare the instant derivatives in which is the residue of a secondary or tertiary amine, or their protected counterparts via the Zincke reaction then one will not use the parent secondary or tertiary amine chelating agent as the starting material but would instead use the corresponding primary amine as the starting material Alternatively, a compound of the formula
• Hal is chloro or bromo and is the residue of the chelating agent as defined hereinbefore or its protected counterpart can be reacted with a pyridine derivative of the formula
• the starting pyridine derivatives are readily available or can be prepared in known manner, e.g. 3-quinolinecarboxamide can be prepared by treating the corresponding acid with ammonia.
• a Zincke reagent When a Zincke reagent is utilized in the reaction sequence, such reagent can be prepared by reacting 1-chloro-2,4-dinitrobenzene with a compound of the formula
• the specific Zincke reagent depicted in Scheme 7 can be prepared by reacting nicotinamide with 1-chloro-2,4-dinitrobenzene. See also Zincke et al, Annalen, 1904, 333, 296; Lettre et al, Annalen, 1953, 579, 123; Keijzer et al, Heterocycle
• the Zincke reagent is reacted with the primary amine, which may be ve ry conveniently employed in the form of its acid addition salt, in the presence of a suitable base, e.g. triethylamine, in an appropriate organic solvent, e.g. methanol, to afford the desired quaternary salt.
• (II) can be conducted at a temperature from about -10°C to room temperature, for a period of time from about
• a large excess of reducing agent is employed, e.g., a 1:5 molar ratio of reducing agent to starting compound of formula (I).
• the process is conducted in the presence of a suitable reducing agent, preferably an alkali metal dithionite such as sodium dithionite or an alkali metal bo rohydride such as sodium borohydride or lithium aluminum borohydride, in a suitable solvent.
• a suitable reducing agent preferably an alkali metal dithionite such as sodium dithionite or an alkali metal bo rohydride such as sodium borohydride or lithium aluminum borohydride.
• Sodium dithionite reduction is conveniently carried out in an aqueous solution; the dihydro product of formula (II) is usually insoluble in water and thus can be readily separated from the reaction medium.
• an organic reaction medium e.g., a lower alkanol such as methanol, an aqueous alkanol or other protic solvent.
• a lower alkanol such as methanol, an aqueous alkanol or other protic solvent.
• the quaternary of formula (I) is reduced in the same reaction mixture as the reduction of the radionuclide (preferably technetium) to an appropriate oxidation state, affording the formula (III) radiopharmaceutical in one step from the formula (I) quaternary. Further details of the one-step reduction are given hereinbelow.
• each R 1 is independently selected from the group consisting of H and C 1 -C 7 alkyl, or an R 1 can
• each R 2 is independently selected from the group consisting of H and C 1 -C 7 alkyl, or an R can be combined with the adjacent such
• each R 3 is independently selected from the group consisting of H and C 1 -C 7 alkyl, alk is a straight or branched lower alkylene group (C 2 -C 8 ) which additionally may contain 1, 2 or 3 nonadjacent oxygen atoms in the chain, and - is as defined with formula (I) hereinabove; X- and t a re as defined with formula (I); and s' is a number which when multiplied by t is equal to one.
• the salts of formula (la) have the partial structure or are position isomers and/or homologs of the first two partial structures shown. It is also preferred that when
• each R 3 is preferably H and alk is preferably a C 1 -C 6 alkylene group, or a C 1 -C 6 alkylene group interrupted by an oxygen atom in the chain; and that when
• alk is preferably a C 1 -C 6 alkylene group, or a C 1 -C 6 alkylene group interrupted by an oxygen atom in the chain.
• Preferred values for - in formula (la) are as given in conjunction with formula (I) hereinabove.
• R 1 and R 2 are as defined with formula (la) and is a radical of the formula
• R 3 and alk are as defined with formula (la) and is as defined with formula (II) hereinabove.
• Preferred compounds of formula (Ila) are the dihydro derivatives corresponding to the preferred compounds of formula (la).
• novel radiopharmaceuticals in which a formula (Ila) compound is chelated with a radioactive metal, especially with technetium.
• a formula (Ila) compound is chelated with a radioactive metal, especially with technetium.
• Especially preferred radiopharmaceuticals have the formula
• R 1 and R 2 are as defined with formula (la) and is a radical of the formula
• R 3 and alk are as defined with formula (la) and is as defined with formula (II) hereinabove; and the corresponding quaternaries, especially those of technetium, "locked in” the brain, which have the formula
• R 1 , R 2 , s', X- and t are as defined with formula (la) and is a radical of the formula
• R 3 and alk are as defined with formula (la) and is as defined with formula (I) hereinabove.
• the preferred complexes of formulas (Ilia) and (IVa) are those which correspond to the preferred derivatives of formulas (la) and (Ila).
• a solution of 8 g of the ester 4 and 7 mL of pyridine in 200 mL of methanol is added dropwise over a two hour period to a solution of 8 g of bisaldehyde 2 in 25 mL of methanol.
• the reaction mixture is cooled in an ice bath after the addition for 1 hour, then is allowed to remafn at room temperature for 1 hour.
• the reaction mixture is then placed in a freezer (-20°C) overnight.
• the solution is concentrated to one-third volume, water is added and the aqueous solution is extracted with cloroform.
• the chloroform extract is washed with saturated aqueous sodium chloride solution and dried over magnesium sulfate.
• a solution of 1.8 g of the amide 7 in 50 mL of dry tetrahydrofuran is added dropwise to a slurry of 1 g of lithium aluminum hydride in 100 mL of dry tetrahydrofuran. The addition takes place over a 30 minute period. The mixture is then heated at the reflux temperature for 20 hours. At the end of that time, the reaction mixture is first cooled and then quenched with saturated Na-K tartrate solution. The aqueous phase is extracted with chloroform. The combined organic phase is then dried over sodium sulfate.
• Methoxyamine hydrochloride (5 g; 0.06 mol) is dissolved in 50 mL of methanol and the solution is neutralized to pH 6 with 1 M methanol ic KOH. The resultant mixture is filtered and to the filtered solution is added 3.2 g (0.03 mol) of 3-pyridinecarboxaldehyde. That mixture is heated at reflux for 4 hours. The methanol is evaporated and the solid is crystallized from a mixture of ethanol and water. There is thus obtained 0-methyl-3-pyridinealdoxime having the structura formula
• amine 8 may be utilized as its hydrobromide salt and the reaction may be conducted in the presence of triethylamine (5-10 mmol).
• triethylamine 5-10 mmol.
• a solution of 0.5 g of 24 and 0.3 g of sodium borohydri in 23 mL of ethanol is stirred at room temperature for 1 hour, then is heated at the reflux temperature for 20 minutes. Then, 10 mL of water are added and the mixture is heated for an additional 10 minutes. The solvent is partially removed by rotary evaporation and the residue is extracted three times with 10 mL portions of chloroform. The chloroform extract is dried over sodium sulfate and the solvent is removed by rotary evaporation. The resultant liquid solidifies on cooling.
• N-(t-butoxycarbonyl), N-(2-merca ⁇ toethyl)glycyl homocysteine thiolactone 67 is prepared as described in Examples 1 and 2 of Byrne et al U.S. Patent No. 4,434,151, and is dissolved (1.0 gram; 3 millimoles) in 25 milliliters of tetrahydrofuran (THF). The resulting solution is then cooled to about 0°C and ethylenediamine (1.8 grams; 30 millimoles) is added to form a new solution. The resulting new solution is maintained for about one hour. The volatile components of the solution are thereafter removed with a rotary evaporator.
• n-Butanol (about 10 milliliters) is added to the "dried" solution components and the liquid components of the resulting composition are again removed by rotary evaporation. The last step is repeated until the vapors remaining in the evaporation vessel do not cause a moistened pH-indicator paper to indicate a basic pH value, thereby also indicating that the ethylendiamine has been substantially removed and that the N-(t-butoxycarbonyl), N-(2-mercaptoethyl)- glycyl N'-(2-aminoethyl)homocysteinamide, i.e. Compound 68 of Scheme 7, so obtained is substantially pure.
• Example 8 The procedure of Example 8 is substantially repeated, except that an equivalent quantity of N-(t- butoxycarbonyl), N-(2-mercaptoethyl)glycyl N'-(2- aminoethyl)homocystei ⁇ e (68) is used in place of the amine 8 and an equivalent quantity of 1-(2,4-dinitro- phenyl)-3-carbamoyl pyridinium chloride (69) is used in place of the Zincke reagent 9. Obtained in this manner is Compound 70 of Scheme 7.
• N-[2-(S-acetamidomethyl)mereaptopropionyl)glycyl homocysteine thiolactone (Compound 77 of Scheme 8), prepared as described in Examples 7 and 9 of Byrne et al U.S. Patent No. 4,434,151, is suspended (1.0 gram; 3 millimoles) in 25 milliliters of THF. The resulting suspension is cooled to a temperature of about 0°C in an ice-water bath, and ethylendiamine (1.8 grams; 30 millimoles) is added to form a new solution.
• Example 8 The procedure of Example 8 is substantially repeated, except that an equivalent quantity of N-[2- (acetamidomethyl)mercaptopropionyl]glycyl N'-(2-amino- ethyl)homocysteinamide (78) is used in place of the amine 8. Obtained in this manner is Compound 79 of Scheme 8.
• Example 17 The general procedure of Example 17 can be repeated to convert Compound 20 to Complex 22; Compound 28 to Complex 30; Compound 36 to Complex 38; Compound 46 to Complex 48; Compound 50 to Complex 52; Compound 61 to Complex 63; Compound 71 to Complex 73; Compound 79 to Complex 81; Compound 89 to Complex 91; Compound 99 to Complex 101; Compound 110 to Complex 112; Compound 118 to Complex 121; and so forth.
• a mi xt u re o f 1 g of the amine 8, 75 mL of acetone and a catalytic amount of p-toluenesulfonic acid is heated at reflux for 24 hours.
• the solvent is removed by rotary evaporation and the residue is taken up in chloroform and treated successively with saturated aqueous sodium bicarbonate solution, aqueous sodium hydroxide sol ution (10%) and saturated aqueous sodium chloride solution.
• the solution is dried over magnesium sulfate. Removal of the solvent leaves a viscous mass.
• Thin layer chromatography (CHC1 3 /methanol, 2:1) indicates two major components having R f values of 0.13 and 0.73.
• Chelating agent precursors, chelating agents and radiopharmaceuticals within the purview of the present invention can also be prepared based on the bifunctional chelating agents of Yokoyama et al U.S. Patent No. 4,287,362.
• R 1 , R 2 , and R 4 are each H or C 1 -C 3 alkyl can be first converted to the corresponding esters
• Another bifunctional chelating agent which can be readily converted to the redox system-containing chelating agent precursors, chelating agents and radiopharmaceuticals of this invention is a compound of the formula
• Amino DTS can be readily converted to the derivatives of the present invention by reacting it with a Zincke reagent of the formula
• R 1 , R 2 , R 3 and R 4 are each H or C 1 -C 3 alkyl and n' is an integer of 0 to 3.
• n' is an integer of 0 to 3.
• An especially preferred chelating agent encompassed by this group is known as amino-PTS, or AEPM, and has the structure
• Amino-PTS can be converted to the derivatives of the present invention via a Zincke reagent, as described supra in connection with amino-.DTS. See, for example, Scheme 15 below.
• One possible structure for 136 is as follows:
• halo derivative can then be reacted with the selected pyridine compound of the formula H , to afford the corresponding quaternary salt of formula (I) herein, which can be converted to the instant derivatives of formulas (II), (III) and (IV) as already described hereinabove.
• Fritzberg U.S. Patent No. 4,444,690 describes an interesting series of 2,3-bis(mercaptoalkanoamido)alkanoic acid chelating agents of the general formula
• X is H or -COOH, and R and R' are H or lower alkyl, and water-soluble salts thereof, used to prepare the corresponding radiopharmaceuticals of the formula
• X is H or -COOH
• R and R' are H or lower alkyl.
• the Fritzberg chelating agents are prepared from the corresponding 2,3-diaminoalkanoic acids by esterification with a lower alkanol containing dry HCl, followed by treating the resultant alkyl ester with a chloroalkanoyl chloride to form the bis(chloroalkanoamide)ester, followed by treating that ester
• Suitable nontoxic pharmaceutically acceptable diluents or vehicles for use with the present complexes of formula (III) will be apparent to those skilled in this art. See, for example, Remington's Pharmaceutical Sciences, 4th Edition (1970). Obviously, the choice of suitable diluents or vehicles will depend upon the exact nature of the particular dosage form selected.
• the dosage ranges for administration of the complexes according to this invention will vary with the size and species of the subject, the objective for which the complex is administered, the particular dosage form employed, and the like, as discussed below.
• the quantity of given dosage form needed to deliver the desired dose of the radiopharmaceutical depends upon the concentration of the complex in any given pharmaceutical composition/dosage form thereof and the radioactivity thereof.
• a 5-50 mg/kg dose of formula (III) radiopharmaceutical injected into the tail vein or carotid vein of rats, due to the "lock in” mechanism will exhibit a ve ry significant difference between brain and peripheral levels of radioactivity, with consequent ready radioimaging of the brain; imaging at approximately 60 to 90 minutes after administration will be most effective, since it will take advantage of this brain/peripheral differential.
• the instant radiopharmaceuticals are generally administered intravenously. Sustained release administration, typically by slow intravenous infusion, will further enhance the site-specificity of the instant redox system. The rate of release of the formula (III) radiopharmaceutical, injected into the tail vein or carotid vein of rats, due to the "lock in” mechanism will exhibit a ve ry significant difference between brain and peripheral levels of radioactivity, with consequent ready radioimaging of the brain; imaging at approximately 60 to 90 minutes after administration will be most effective, since it will take advantage of this brain/peripheral differential.
• the instant radiopharmaceuticals are generally
• radiopharmaceutical from the sustained release system should be comparable to the rate of in vivo oxidation of the dihydro form (III) to the quaternary form (IV) in order to achieve the greatest degree of enhancement of specificity.
• the present invention also provides a process for the manufacture of a diagnostic agent for the visualization of an organ such as the brain.
• the blood-brain barrier penetrating form, formula (III) is admixed with an aqueous buffer medium having a pH value of about 4 to about 8, preferably of about 6.5 to about 7.5, in an effective radioimaging amount.
• Preparation of the radiopharmaceutical can be carried out in the hospital or like location where the patient is found in order to minimize losses of radioactivity caused by the decay of radioactive metal.
• the preparation for visualization is injectable, it must be sterile and pyrogen-free; preferably, it is also isotonic.
• a so-called labeling kit can be provided that permits a simple, rapid and safe labeling of the solution to be injected with the radioactive metal e.g., technetium- 99m.
• Such kits are especially desirable when a shortlived radioisotope such as technetium-99m is used.
• the kit includes a collecting vial for receiving and/or containing an aqueous medium in which the complexing reaction can be effected. Additionally, the kit includes the chelating agent of formula (II) or chelating agent precursor of formula (I) and a pharmacologically acceptable reducing agent for reducing the radioactive element to an appropriate oxidation state for complexing with the chelating agent [and also for reducing the pyridinium moiety to the corresponding dihydropyridine form, when a chelating agent precursor of formula (I) is present].
• the radioactive element is received from a radionuclide generator as an aqueous pertechnetate (T ) solution such as an eluate in isotonic saline, as is well-known in the art.
• T aqueous pertechnetate
• the amount of Tc-99m required to produce a quantity of formula (III) radiopharmaceutical sufficient for diagnostic purposes is generally from 0.01 milliCurie (mCi) to about 500 mCi per mL of 99m- pertechnetate solution.
• the reducing agent for the pertechnetate can be a thiosulfate or dithionite if the reducing reaction is to be carried out in a basic medium, or a tin (II) salt such as SnCl 2 if the reducing reaction is to be carried out in an acid medium.
• a kit for preparing an injectable radiopharmaceutical e.g., for complexing an organ-specific agent labeled with a radioactive metal, includes, in separate containers: (1) a biologically compatible, sterile aqueous medium suitable for complex formation with a radioactive metal, (2) a dihydropyridine pyridinium salt redox system-containing complexing agent of formula (I) or (II) compatible therewith, and (3) a pharmaceutically acceptable reducing agent for the radioactive metal.
• the dihydropyridine pyridinium salt redox moiety may be present in the kit either in its oxidized or its reduced state, as desired.
• the reducing agent for the radioactive metal can be selected to reduce also the oxidized form of the redox moiety, if present, as the radioactive metal is reduced to form the complex preparatory to injection of the radiopharmaceutical into a test animal or a patient.
• a reducing agent capable of reducing both the oxidized form of the redox moiety and the radioactive metal is chosen and the chelating agent precursor of formula (I) is present in the kit.
• the kit comprises, in separate containers (preferebly aspetically and hermetically sealed vials of approximately 5-25 mL volume), (1) a biologically compatible, sterile aqueous medium, (2) a chelating agent precursor of formula (I), and (3) a pharmacologically acceptable reducing agent capable of reducing the chelating agent precursor of formula (I) to a chelating agent of formula (II) and also capable of reducing the radioactive metal to an oxidation state in which it is capable of complexing with the formula (II) chelating agent to form a radiopharmaceutical of formula (III).
• the reducing agent is sodium dithionite; also most preferably, the radioactive metal is technetium.
• the dithionite reduction is preferably carried out in basic medium; this may be accomplished by providing that the aqueous medium (1) above is of basic pH, or by adding an appropriate base (e.g. NaOH, Na 2 CO 3 ) when combining the kit components and the pertechnetate solution.
• the kit could comprise only two separate components: (1) the biologically compatible, sterile aqueous medium of essentially neutral pH containing the chelating agent precursor of formula (I); and (2) the reducing agent e.g. sodium dithionite or (2) the reducing agent together with the base, e.g. sodium dithionite and sodium carbonate.
• Radioactive metal ions are typically not provided with the kit due to the relatively short half-lives of commonly utilized radionucl ides.
• the radionuclide is provided separately as described earlier and admixed with the components of the kit shortly before use, as is known for other radiopharmaceutical delivery systems.
• the pertechnetate solution and the basic aqueous medium may be first combined and then heated, e.g. from 40 to 95°C for 10 to 20 minutes, in the presence of the reducing agent, then cooled to about room temperature or below prior to addition of the formula (I) precursor.
• the technetium will be reduced prior to reduction of the quaternary moiety to the corresponding dihydro form, in which case a substantial portion of the quaternary salt (I) will likely chelate with the reduced technetium to form the quaternary complex (IV) in the reaction mixture as an intermediate to the dihydro complex (III), rather than the quaternary salt (I) being first converted to the dihydro chelating agent (II) and then to the dihydro complex (III).
• the precursor may be present in the initial mixture made from the kit, and it is likely in this instance that the formula (I) quaternary will be first reduced to the formula (II) dihydro, which will then chelate with the reduced technetium to form the complex (III).
• the mixture is mildly basic, e.g. pH 8 to 9, it may be administered as is, after the reduction and chelation have occurred to form the formula (III) radiopharmaceutical, or the pH may be adjusted to about 7.
• the mixture is more strongly basic, e.g. pH 13, it is generally desirable to adjust the pH to a slightly alkaline or neutral value.
• the kit it is preferable for it to contain excess chelating agent precursor (I) or chelating agent (II) with respect to the radionuclide to be complexed therewith, e.g. a 1:2 molar excess.
• the reducing agent is present in a large excess with respect to the chelating agent precursor (I), e.g. 1:5 to 1:10.
• the reducing agent is preferably present in a slight excess with respect to the radionuclide.
• the diagnostic agent is administered to a patient, typically intravenously, with or without further dilution by a carrier vehicle such as physiological saline, phosphate-buffered saline, plasma, or the like.
• a carrier vehicle such as physiological saline, phosphate-buffered saline, plasma, or the like.
• the unit dose to be administered has a radioactivity of about 0.01 milliCurie (mCi) to about 100 milliCuries, preferably about 1 mCi to about 20 mCi.
• the solution to be injected into an adult patient per unit dosage is about 0.01 milliliter (mL) to about 1 milliliter.
• imaging of the organ in vivo can take place after a few minutes. If desired, imaging can also take place hours after the injection, depending upon the half-life of the radioactive material that has been introduced into the patient and upon the amount of such material introduced. Preferably, imaging takes place 60 to 90 minutes after intravenous administration.
• compositions of matter comprising: (1) the residue of a chelating agent having at least one primary, secondary or tertiary amino functional group, said functional group being not essential for the complexing properties of said chelating agent, said residue being characterized by the absence of at least one of said primary, secondary or tertiary amino functional groups, said chelating agent being either (a) capable of chelating with a metallic radionuclide or (b) chelated with a metallic radionuclide; and (2) a dihydropyridine pyridinium salt redox system, which in its oxidized form comprises a radical of the formula wherein n, p, q and R are as defined with formula
• n, p, q, m and R are as defined with formula

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• 1985
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