RU2804297C2 - Radiopharmaceuticals, radioimaging agents and their use - Google Patents

Abstract

FIELD: radiopharmaceuticals.

SUBSTANCE: invention relates to compounds that can be used as radiopharmaceuticals and radioimaging agents. The following is proposed: a compound of formula (I) or its pharmaceutically acceptable salt or isomer:

(I)

where: X is C₁-C₆ alkyl or a group

;

Y is an unsubstituted C₁-C₁₂ alkylene group; m is either 0, 1 or 2; and n is either 0, 1, or 2; where the connection is coordinated with a metal ion, where the metal ion is selected from the group consisting of ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu and ⁶⁷Cu. Also the following is proposed: individual compounds (options), a composition for radio imaging and a method of radio imaging of a subject.

EFFECT: proposed compounds are useful in radiotherapy and diagnostic imaging.

15 cl, 11 dwg, 2 ex

Description

Field of technology to which the invention relates

The present invention relates to compounds that can be used as radiopharmaceuticals and radioimaging agents that include a radionuclide chelating agent. These coordinated connections are useful in radiation therapy and diagnostic imaging. The invention also relates to methods of diagnosis, prognosis and therapy using non-coordinated and radiolabeled compounds of the invention.

State of the art

Prostate cancer is the leading cause of cancer-related death in men, with mortality rates often attributed to difficulties in detecting and subsequently treating the disease. Prostate-associated tumors often show increased expression of prostate-specific membrane antigen (PSMA), which is an enzyme normally expressed in prostate tissue but is frequently upregulated in some types of prostate cancer. This means that PSMA is a good biomarker or target for imaging, diagnosis, and prognosis. However, because PSMA is also expressed in other tissues, both normal and malignant, there are difficulties in successfully imaging prostate cancer.

Radiolabeled compounds can be used as a radiopharmaceutical or radioimaging agent if the compound can bind sufficiently to the desired site and also deliver a radionuclide to the same site for imaging or therapeutic purposes.

Compounds or ligands containing a urea-based motif, such as the glutamate-substituted urea below, are known to bind to the catalytic site of PSMA with good affinity.

Although compounds having these or similar motifs have been synthesized, problems have been observed with their stability or binding behavior in vivo. In order for a compound to be used in radioimaging or radiotherapy, the compound and the resulting radionuclide-containing complex must be stable in vivo . One known problem with radiolabeled compounds is that the complex formed with the radionuclide is not strong enough and the radionuclide "leaks" from the complex and is not delivered to its intended location. Other problems resulting from radionuclide leakage include diffusion of the radionuclide into undesired locations. This may result in damage to healthy tissue due to the radionuclide. In addition, diffusion of the radionuclide results in lower quality images because the contrast between true binding sites (indicating the location of tumors) and those with unwanted radionuclide is reduced.

Other problems associated with radiolabeled compounds include the possibility of radiolysis, where the radioisotope itself leads to the destruction of the compound and subsequent diffusion of the radionuclide. Radiolysis occurs as a result of the spontaneous decay of a radionuclide with the release of energy leading to the breaking of bonds in the ligand and destruction of the complex. Radiolysis also causes the radionuclide to diffuse into undesired areas, further exacerbating the problems described above.

Since the compound must be administered to a person in need, the compounds must also be non-toxic in nature to the subject. Another problem associated with the use of radiolabeled compounds for diagnostics, imaging and therapy is the problem of binding affinity. If the binding affinity to the target, i.e. PSMA in this case is low, the complex may not achieve binding and may be isolated or may only show limited binding. If the complex is immediately removed from the body, this leads to a decrease in overall effectiveness. However, when a compound exhibits limited excretion and limited binding, the complex may diffuse throughout the subject and lead to the undesirable effects described above. In addition, limited binding of the complex will likely reduce the time available for acceptable tumor imaging or treatment.

There is a need for compounds that can provide the desired binding affinity to tumors associated with prostate cancer and also have the ability to provide the desired imaging properties. There is also a requirement that the compounds be sufficiently stable and not degrade during use.

The essence of the invention

The present invention relates to new compounds that exhibit improved binding affinity to PSMA. The present inventors have discovered that the use of amino acid-substituted urea linked to the macrocyclic sarcophagus through specific linkers provides compounds that bind to PSMA and, when complexed with the radionuclide, provide good/improved imaging properties.

In one aspect, the present invention provides a compound of formula (I) or a salt, complex, isomer, solvate or prodrug thereof:

(I)

Where:

X represents a group selected from H, OH, halogen, cyano, NO ₂ , NH ₂ , optionally substituted C ₁ -C ₁₂ alkyl, optionally substituted amino, optionally substituted amide and optionally substituted aryl;

Y represents an optionally substituted C ₁ -C ₁₂ alkylene group, wherein one or more methylene groups in the alkylene group may be further optionally substituted by a group selected from amido, carbonyl, urea and thiourea;

m is 0, 1 or 2; And

n is 0, 1 or 2.

In one embodiment, the compound or a salt, complex, isomer, solvate or prodrug thereof has the formula:

.

In another embodiment, the compound or a salt, complex, isomer, solvate or prodrug thereof has the formula:

.

In another embodiment, the compound or a salt, complex, isomer, solvate or prodrug thereof has the formula:

.

In another aspect, the present invention relates to a composition comprising a compound according to the above aspect and a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to an aqueous composition for parenteral administration, comprising a compound according to the above aspect; and where the composition further includes ethanol, gentisic acid or a salt thereof and sodium chloride.

In another aspect, the present invention provides a method of treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of a compound according to the above aspect or a composition according to the above aspect.

Brief description of the figures

Fig. 1: PET imaging of LNCaP-bearing NSG mice treated with ⁶⁴ Cu-Sar-PSMA at 30 minutes, 2 hours and 22 hours.

Fig. 2: Graph showing the biodistribution of ⁶⁴ Cu–Sar–PSMA in LNCaP–bearing NSG mice (left), relative to blood levels (right), both after 22 hours.

Fig. 3: PET imaging of LNCaP-carrying NSG mice treated with ⁶⁴ Cu-Sar-PSMA at 1 and 6 hours.

Fig. 4: Graph showing the biodistribution of ⁶⁴ Cu–Sar–PSMA in LNCaP–bearing NSG mice at 1 and 6 hours.

Fig. 5: Graph showing the biodistribution of various radiolabeled complexes in mice bearing LNCaP xenografts, expressed as tumor:kidney uptake ratio at 1 hour.

Fig. 6: Graph showing the preclinical biodistribution of ⁶⁴ Cu–Sar–PSMA and ⁶⁸ Ga–labeled complexes in mice bearing LNCaP xenografts, expressed as tumor:kidney uptake ratio at 1 hour.

Fig. 7: Graph showing the biodistribution of various radiolabeled complexes in mice bearing LNCaP xenografts, expressed as tumor:kidney uptake ratio.

Fig. 8: Structures of PSMA-ligand targets from the prior art.

Fig. 9: HPLC chromatogram of ⁶⁴ Cu–Sar–PSMA ( _RT (retention time): 12.43 min) versus ^nat Cu–Sar–PSMA ( _RT : 12.38 min) with UV detection at 220 nm.

Fig. 10: Radio-HPLC chromatogram ^{of 64} Cu–CoSar(PSMA) ₂ ( _RT : 13.9 min).

Fig. 11: Analytical HPLC chromatogram of CoSar(PSMA) ₂ ( _RT : 10.3 min) with UV detection at 220 nm.

Detailed description

As described and shown herein, the present inventors have discovered that a compound containing an amino acid substituted urea attached to a sarcophagus cell through a linker group can bind to PSMA. Without wishing to be bound by theory, it is believed that the combination of the amino acid-urea moiety, linker, and sarcophagus cage provides the benefits observed and discussed below.

The complexes, as described herein, are radiolabeled with a radionuclide or radioisotope that undergoes spontaneous decay, where these decay byproducts are detected by various methods, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT). The quality of the images obtained, and therefore the reliability of any diagnosis based on these images, depends on the ability of the radiolabeled complex to specifically bind to the site of prostate cancer.

As used herein, the term “sarcophagin” refers to a nitrogen-containing macrocyclic ligand with the formula 3,6,10,13,16,19-hexaazabicyclo[6.6.0]eicosane.

The term "optionally substituted" as used throughout the specification means that a group may or may not be further substituted or fused (to form a fused polycyclic system) with one or more non-hydrogen substituent groups. In some embodiments, the substituent groups are one or more groups independently selected from the group consisting of halogen, =O, =S, –CN, –NO ₂ , –CF ₃ , –OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl , haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkyl pseudo-heteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl , alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl , alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, –C(=O)OH, –C(=O)R ^a , –C(=O)OR ^a , C(=O)NR ^a R ^b , C(=NOH)R ^a , C(=NR ^a )NR ^b R ^c , NR ^a R ^b , NR ^a C(=O)R ^b , NR ^a C(=O)OR ^b , NR ^a C(= O)NR ^b R ^c , NR ^a C(=NR ^b )NR ^c R ^d , NR ^a SO ₂ R ^b , –SR ^a , SO ₂ NR ^a R ^b , –OR ^a _, OC(=O)NR ^a R ^b , OC(=O)R ^a and acyl, where R ^a , R ^b , R ^c and R ^d are each independently selected from the group consisting of H, C ₁ –C ₁₂ alkyl, C ₁ –C ₁₂ haloalkyl, C ₂ –C ₁₂ alkenyl, C ₂ –C ₁₂ alkynyl, C ₂ –C ₁₀ heteroalkyl, C ₃ –C ₁₂ cycloalkyl, C ₃ –C ₁₂ cycloalkenyl, C ₂ –C ₁₂ heterocycloalkyl, C ₂ –C ₁₂ heterocycloalkenyl, C ₆ –C ₁₈ aryl, C ₁ –C ₁₈ heteroaryl and acyl, or any two or more of R ^a , R ^b , R ^c and R ^d , taken together with the atoms to which they are attached, form a heterocyclic ring system with 3 to 12 ring atoms.

In some embodiments, each optional substituent is independently selected from the group consisting of: halogen, =O, =S, –CN, –NO ₂ , –CF ₃ , –OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, –COOH, –SH and acyl.

Examples of particularly suitable optional substituents include F, Cl, Br, I, F, Cl, Br, I, CH ₃ , CH ₂ CH ₃ , OH, OCH ₃ , CF ₃ , OCF ₃ , NO ₂ , NH ₂ , COOH, COOCH ₃ and CN.

"Alkenyl" as a group or part of a group means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, and which may be linear or branched, preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2–6 carbon atoms, in a normal chain. The group may contain multiple double bonds in a normal chain, and the orientation around each is independently E or Z. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, and nonenyl.

"Alkyl" as a group or part of a group refers to a straight or branched chain aliphatic hydrocarbon group, preferably C ₁ -C ₁₂ alkyl, more preferably C ₁ -C ₁₀ alkyl, most preferably C ₁ -C ₆ unless otherwise specified. Examples of suitable straight and branched C ₁ -C ₆ alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexyl and the like.

"Alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched, preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in a normal chain. Typical structures include, but are not limited to, ethynyl and propynyl.

"Aryl" as a group or part of a group means (i) an optionally substituted monocyclic or fused polycyclic aromatic carbocycle (ring structure having ring atoms that are all carbon), preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a _C5-7 cycloalkyl or _C5-7 cycloalkenyl group are condensed together to form a cyclic structure such as tetrahydronaphthyl, indenyl or indanyl. Typically the aryl group is a C ₆ -C ₁₈ aryl group.

"Cycloalkyl" refers to a saturated monocyclic or fused or spiro-polycyclic carbocycle, preferably containing from 3 to 9 carbon atoms in the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise noted. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. The cycloalkyl group is usually a C ₃ -C ₉ cycloalkyl group.

"Halogen" represents chlorine, fluorine, bromine or iodine.

"Heteroalkyl" refers to a straight or branched chain alkyl group, preferably having from 2 to 12 carbon atoms, more preferably from 2 to 6 carbon atoms in a chain in which one or more carbon atoms (and any associated hydrogen atoms), each independently , is substituted with a heteroatom group selected from S, O, P and NR', where R' is selected from the group consisting of H, optionally substituted C ₁ -C ₁₂ alkyl, optionally substituted C ₃ -C ₁₂ cycloalkyl, optionally substituted C ₆ - _C18 aryl and optionally substituted _C1 – _C18 heteroaryl. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides and the like. Examples of heteroalkyl also include _hydroxyC1 – _C6alkyl , C1 _{– C6alkyloxyC1} – _C6alkyl , _aminoC1 – _{C6alkyl , C1} – _{C6alkylaminoC1 – C6alkyl} , and di( _C1 – _C6alkyl )aminoC ₁ –C ₆ alkyl.

"Heteroaryl", either by itself or as part of a group, refers to groups containing an aromatic ring (preferably a 5- or 6-membered aromatic ring) having one or more heteroatoms as ring atoms on the aromatic ring, the remaining ring atoms being are carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulfur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolysine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole , indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazan, phenoxazine, 2–, 3– or 4-pyridyl, 2-, 3-, 4-, 5- or 8-quinolyl, 1-, 3-, 4- or 5-isoquinolinyl, 1-, 2- or 3-indolyl and 2- or 3-thienyl . The heteroaryl group is usually a C ₁ -C ₁₈ heteroaryl group.

As used herein, the term "C ₁ -C ₁₂ alkylene" refers to a straight or branched chain divalent aliphatic hydrocarbon group where the group has from 1 to 12 carbon atoms in the chain.

In one embodiment, X is an optionally substituted C ₁ –C ₁₂ alkyl.

In one embodiment, X is C ₁ –C ₁₂ alkyl.

In one embodiment, X is an optionally substituted C ₁ –C ₃ alkyl.

In one embodiment, X is C ₁ –C ₃ alkyl.

In one embodiment, X is methyl.

In one embodiment, X is CH ₃ .

In one embodiment, X is an optionally substituted amino, for example, –NCH ₃ .

In one embodiment, X is amino.

In one embodiment, X is an optionally substituted amide. As used herein, the term "amide" refers to a functional group consisting of a carbonyl group attached to a nitrogen atom. Therefore, the term "optionally substituted amide" refers to an amide functional group that carries an additional substitution.

In one embodiment, X is an optionally substituted amide, e.g. or

.

In one embodiment, Y is a substituted alkylene group.

In one embodiment, Y is an unsubstituted alkylene group.

In one embodiment, Y is CH ₂ .

In one embodiment, Y is a carbonyl group.

In one embodiment, Y is a substituted C ₁ -C ₁₂ alkylene group, where one or more methylene groups is further substituted by an amido group, for example, .

In one embodiment, n is 1.

In one embodiment, n is 2.

In one embodiment, n is 0.

In one embodiment, m is 1.

In one embodiment, m is 2.

In one embodiment, m is 0.

In one embodiment, the present invention provides a compound of the formula:

.

In another embodiment, the present invention provides a compound of the formula:

,

the two phenylalanine residues are D–Phe to produce the ligand MeCOSar–D–Phe–D–Phe–AOC–Lys–urea–Glu. The inventors have found that the use of phenylalanine residues with specific D-stereochemistry can lead to compounds with improved metabolic stability. In addition, the inventors also determined that the use of two D–Phe residues in the ligand can increase the hydrophobicity of the compounds and the potential pi–pi interactions of the ligand with the binding pocket of the target enzyme.

In another embodiment, the present invention provides a compound of formula

.

In another embodiment, the present invention provides a compound of the formula:

.

This compound includes two linkers and two urea motifs for binding to PSMA. Without wishing to be bound by theory, it would appear that this compound containing two urea motifs may exhibit further improvement in binding affinity to PSMA. It is also believed that this bis derivative, that is, a compound with two linkers and two urea motifs, may provide a better signal-to-noise ratio when used for imaging purposes and compared to the corresponding mono compound with one linker and two urea motifs. The bis compound may also demonstrate further improvement in renal clearance upon administration. This may be due to differences in overall charge and charge separation and distribution when comparing mono and bis compounds.

In another embodiment, the present invention provides a compound of the formula:

.

In another embodiment, the present invention provides a compound of the formula:

.

In another embodiment, the present invention provides a compound of the formula:

.

In one embodiment, the compound is coordinated to a metal ion.

In one embodiment, the metal ion is a Cu, Tc, Gd, Ga, In, Co, Re, Fe, Au, Mg, Ca, Ag, Rh, Pt, Bi, Cr, W, Ni, V, Ir, Zn, Cd, Mn, Ru, Pd, Hg, Ti, Lu, Sc, Zr, Pb, Ac and Y.

In an embodiment, the metal ion is a radionuclide.

In some embodiments, the metal in the metal ion is a radionuclide selected from the group consisting of Cu, Tc, Ga, Co, In, Fe, and Ti. The present compounds have been found to be particularly useful for binding copper ions. In some embodiments, the metal in the metal ion is a radionuclide selected from the group consisting of ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, and ⁶⁷ Cu. In some embodiments, the metal in the metal ion is ⁶⁰ Cu. In some embodiments, the metal in the metal ion is ⁶¹ Cu. In some embodiments, the metal in the metal ion is ⁶² Cu. In some embodiments, the metal in the metal ion is ⁶⁴ Cu. In some embodiments, the metal in the metal ion is ⁶⁷ Cu.

The compound of formula (I) includes a sarcophagin macrocyclic ligand and a Lys-urea-Glu moiety that targets PSMA. The junction also includes intermediate regions that bind the sarcophagin and the PSMA-targeting moiety. In formula (I) they include a propyl linker linked to two amide groups, two phenylalanine residues and an aminooctanoic acid (AOC) group. The propyl linker, phenylalanine residues, and aminooctanoic acid group together act as a spacer group to separate the sarcophagus and the PSMA targeting moiety. It is desirable that there be some degree of separation between the sarcophagus and the PSMA-targeting moiety to ensure that the activities of the two groups do not interfere with each other, but it is also important that the two groups are not so far apart that where the sarcophagus included a bound radionuclide, the radionuclide complex was delivered to the site of action identified by the PSMA-targeting moiety. The PSMA targeting moiety includes a Lys–urea–Glu moiety that has three carboxy functional groups that provide an overall negative charge and contribute to the zinc binding site. The aminooctanoic acid group adjacent to the PSMA targeting moiety is intended to provide a linker group approximately 20 Å in length to share charge between the PSMA targeting moiety and the rest of the molecule. The two D-phenylalanine residues are hydrophobic in nature and allow pi-pi bonding interactions with the active site. These residues also contribute to the metabolic stability of the compound. The propylene group located between the two amide functional groups also serves to provide the required distance between the macrocyclic ligand (which chelates the positively charged Cu ion) and the PSMA targeting moiety. The present inventors have discovered that the compounds of the present invention include various moieties (ie, macrocyclic ligand, linkers, and a PSMA targeting moiety) that together provide a PSMA binding ligand with the required stability and binding affinity.

In an embodiment, the invention relates to compositions comprising a compound as described above together with a pharmaceutically acceptable excipient.

In a further aspect, the invention provides a method of treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of a compound as described above, or a composition thereof.

In an embodiment, the condition is cancer.

In one embodiment, the condition is breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, head and/or neck cancer, or kidney, stomach, pancreatic, brain cancer, and hematologic malignancies, such as lymphoma or leukemia.

In an embodiment, the condition is prostate cancer.

In a further aspect, the invention relates to a method of radioimaging a subject, comprising administering an effective amount of a compound as described above, or a composition thereof.

Ideally, the radiopharmaceutical is retained at the intended target and not at any other sites, and any unbound radiopharmaceutical is cleared from the circulation. This allows images to be obtained with sufficient contrast, which in turn allows for more accurate analysis and diagnosis. To do this, the radiolabeled complex must have the physical and chemical properties where the bound complex remains bound in the desired location for a time sufficient to obtain the required imaging, but any unbound complex must be sufficiently removed from the subject to prevent any background radiation emanating from the subject. unbound complex to interfere with and reduce the contrast of the resulting images.

The compounds of the present invention exhibit a more favorable in vivo distribution profile. In fig. 2 and 4 show that administration of ⁶⁴ Cu–Sar–PSMA to tumor-bearing mice leads to localization of the compound in the tumor, and not in any large organs or blood. Minimizing the binding of the radiolabeled complex to other tissues reduces damage to healthy tissue. The complex shows relatively little accumulation in the bloodstream, which also shows the high binding affinity of the Sar–PSMA complex to tumors expressing PSMA.

Compared to other known radiolabeled complexes (see Fig. 5), such as ⁶⁸ Ga–PSMA–617, ¹⁷⁷ Lu–PSMA–I&T and ⁶⁸ Ga–DOTAGA–ffk(PSMA), the ⁶⁴ Cu–Sar–PSMA complex shows greater uptake in tumors. Additionally, when renal uptake, which refers to the excretion of the compound, is considered, ⁶⁴ Cu–Sar–PSMA shows markedly less accumulation in the kidney compared to other compounds that show similar binding to the tumor site. A similar comparison between the ⁶⁴ Cu–Sar–PSMA complex and various ⁶⁸ Ga complexes is shown in Figure 6, which shows that the use of the ⁶⁴ Cu radionuclide to bind to the tumor is equal to or better than complexes that use the ⁶⁸ Ga radionuclide. In addition, the ⁶⁴ Cu–Sar–PSMA complex shows less accumulation in the kidneys than complexes with the ⁶⁸ Ga radionuclide.

Subsequently, the present inventors discovered that the use of Sar-PSMA ligand and ⁶⁴ Cu radionuclide exhibited better affinity for the tumor site and better renal clearance after administration. These advantages allow for better imaging results, that is, higher affinity for the tumor site provides images with better contrast because the radionuclide is preferentially located at the target site and better removes unbound ligand from the circulation, thereby reducing background accumulation.

These advantages allow for better imaging results, i.e. higher affinity for the tumor site provides images with better contrast as the radionuclide is preferentially located at the target site and better removes unbound ligand from circulation, thereby reducing background accumulation. This allows for improved diagnosis of tumors such as prostate cancer. Increased affinity for the tumor binding site also indicates less diffusion of the radionuclide into other tissues, which improves the quality of the resulting images. In addition, minimizing diffusion of the radionuclide into areas that are not the tumor site means that less radiolabeled complex is required for administration and that any harmful effects of the radioactive complex are localized so that healthy tissue is not affected.

The inventors have discovered that the present compounds can be used as theranostic compounds. The theranostic approach allows the same compound to be used for diagnosis and treatment of indications, providing advantages over using one compound for diagnosis and another compound for treatment. In general, this makes it possible to increase the efficiency of diagnosis and treatment of a specific disease. This differs from traditional methods, where a ligand with a particular isotope may be suitable for diagnosing a disease, but the same combination of ligand and isotope may not be suitable for treating the disease. This then requires that the ligand, the isotope, or both the ligand and the isotope be modified to treat the disease.

In fig. Figure 7 shows the accumulation of radiolabeled complexes in tumors and kidneys over time. When the ⁶⁴ Cu–Sar–PSMA complex is administered, the ratio of tumor-located to tumor-located complex increases to a time of 24 hours. Figure 7 shows that the uptake of the ⁶⁴ Cu–Sar–PSMA complex into tumor sites after 1 hour is greater than that of other radiolabeled complexes, indicating that the ⁶⁴ Cu–Sar–PSMA complex is absorbed faster than the other comparison complexes. In addition, the ratio of the complex in tumors to kidney increases up to 24 hours, which then indicates that the complex has favorable tumor binding affinity. The benefits that flow from this are that since the complex remains bound for a longer period of time, imaging of the subject can be performed during this period of time so that better quality and higher contrast images can be obtained. This allows you to more accurately diagnose the disease.

The ⁶⁴ Cu–Sar–PSMA complex exhibits improved binding affinity compared to other radiolabeled complexes, further indicating that the same complex may be useful for therapy. Since the therapeutic nature of the complex depends on the delivery of the radionuclide to the target site, that is, the tumor, good specificity and affinity for the tumor site is necessary. This allows the radionuclide to deliver the radiotherapeutic effect to the desired location and prevent damage to other tissues. In addition, the ability of the radiolabeled complex to remain associated with the target site provides a long-lasting therapeutic effect, which increases the effectiveness of the treatment method. The present inventors have now shown that the radiolabeled ⁶⁴ Cu–Sar–PSMA complex has sufficient binding specificity and affinity for the target site of PSMA such that administration of a therapeutically effective amount of the radiolabeled complex can provide treatment for prostate cancer.

The present inventors have also now shown that the Sar-PSMA complex, radiolabeled with a copper isotope, can be used for both diagnostic and therapeutic purposes. For example, the radiolabeled ⁶⁴ Cu–Sar–PSMA complex can be used for both diagnostic and therapeutic purposes. The radiolabeled ⁶⁷ Cu–Sar–PSMA complex can also be used for both diagnostic and therapeutic purposes.

The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above compounds, and includes pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) can be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkylsulfonic and arylsulfonic. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. For agents that are solids, those skilled in the art will recognize that the compounds, agents, and salts of the invention may exist in various crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention and the defined formulas.

The term "therapeutically effective amount" or "effective amount" is an amount sufficient to achieve beneficial or desired clinical results. An effective amount may be administered in one or more administrations. An effective amount is usually sufficient to mitigate, improve, stabilize, reverse, delay or delay the progression of the disease state. The radioimaging effective amount is usually sufficient to identify the radionuclide in a subject.

Monitoring a subject for the location of the radiolabeled material typically provides the analyst with information regarding the location of the radiolabeled material and therefore the location of any material targeted by the molecular recognition fragment (such as cancer tissue). The effective amount of the compounds of the invention will depend on a number of factors, and, if necessary, will involve a balance between the amount of radioactivity required to achieve the desired radioimaging effect and the general interest of not exposing the subject (or his tissues or organs) to unnecessary levels of radiation that may be harmful.

The treatment methods of the present invention involve administering a compound of formula (I) which is complexed with a radionuclide. Compounds of formula (I) are capable of delivering a radionuclide to the desired location in the body where its mode of action is required.

The therapeutically effective amount can be readily determined by the attending physician using conventional methods and by observing results obtained under similar circumstances. When determining a therapeutically effective amount, a number of factors should be considered, including, but not limited to, the species of animal, its size, age and general health, the specific disease, the severity of the condition, the patient's response to treatment, the specific radiolabeled compound administered, route of administration, bioavailability of the administered drug, the chosen dosage regimen, the use of other drugs and other relevant circumstances.

In addition, the treatment regimen usually includes a number of cycles of radiation therapy, with these cycles continuing until the condition improves. Again, the optimal number of cycles and the interval between each treatment cycle will depend on a number of factors, such as the severity of the condition being treated, the health (or lack thereof) of the subject being treated, and his response to radiation therapy. In general, the optimal dosage amount and optimal treatment regimen can be readily determined by one skilled in the art using well known methods.

When using the compounds of the invention, they can be administered in any form or mode that makes the compound available for the desired application (imaging or radiotherapy). One skilled in the art of preparing preparations of this type can readily select the appropriate form and route of administration depending on the specific characteristics of the compound selected, the condition being treated, the stage of the condition being treated, and other relevant circumstances. For further information, the inventors refer to Remington's Pharmaceutical Sciences, ^19th edition, Mack Publishing Co. (1995).

The compounds of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, diluent or excipient. The compounds of the present invention, although effective in their own right, are typically formulated and administered in the form of their pharmaceutically acceptable salts since these forms are generally more stable, more readily crystallized, and have increased solubility.

However, these compounds are usually used in the form of pharmaceutical compositions, which are formulated depending on the desired route of administration. The compositions are prepared by methods well known in the art.

The invention in other embodiments provides a pharmaceutical package or kit including one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Such a package or kit may contain at least one container having a unit dose of the agent(s). Conveniently, in kits, unit doses can be provided in sterile vials so that the clinician can use the vials directly, where the vials will have the desired amount and concentration of compound and radionucleotide, which can be mixed before use. Such container(s) may be associated with various written materials, such as instructions for use, or a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, imaging agents, or biological products that reflects the approval of the manufacturing agency. use or sale for human administration.

The compounds of the invention can be used or administered in combination with one or more additional drugs that are anticancer drugs and/or procedures (eg, surgery, radiation therapy) to treat the disorder/diseases. The components can be administered in the same composition or in separate compositions. When administered in separate compositions, the compounds of the invention may be administered sequentially or simultaneously with other drug(s).

In addition to the possibility of administration in combination with one or more additional drugs, which include anti-cancer drugs, the compounds of the invention can be used in combination therapy. In this method, the compounds are usually administered in combination with each other. Thus, one or more compounds of the invention can be administered either simultaneously (as a combination formulation) or sequentially to achieve the desired effect. This is particularly desirable when the therapeutic profile of each compound is different such that the combined effect of the two drugs provides an improved therapeutic outcome.

The pharmaceutical compositions of the present invention for parenteral administration include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be achieved by including various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

If desired, and for more efficient distribution, the compounds can be included in slow release or targeted delivery systems such as polymer matrices, liposomes and microspheres.

Injectable compositions can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injection medium immediately prior to use.

In an embodiment, the present invention relates to an aqueous composition of a compound of formula (I) or a salt thereof:

(I)

Where:

X represents a group selected from H, OH, halogen, cyano, NO ₂ , NH ₂ , optionally substituted C ₁ -C ₁₂ alkyl, optionally substituted amino, optionally substituted amide and optionally substituted aryl;

Y represents an optionally substituted C ₁ -C ₁₂ alkylene group, where one or more methylene groups in the alkylene groups are optionally substituted with a group selected from amide, carbonyl, urea and thiourea;

m is 0, 1 or 2; And

n is 0, 1 or 2;

where the compound of formula (I) forms a complex with a Cu ion;

and where the composition further includes ethanol, gentisic acid or a salt thereof and sodium chloride.

The present inventors have determined that the use of gentisic acid and ethanol in the composition of a compound of formula (I) with a complexing Cu ion can help prevent or minimize radiolysis of the radiolabeled complex.

In the above embodiments, the compositions of the present invention include ethanol as a component. The ethanol used in the composition may be anhydrous ethanol. Alternatively, the ethanol used in the composition may not be subject to drying processes and may be hydrated. The ethanol is preferably pharmaceutical grade ethanol. Ethanol present in the composition may help prevent radiolysis of the radiolabeled complex of Formula (I).

In the above embodiments, the compositions of the present invention also include sodium chloride as a component. The sodium chloride in the compositions of the present invention may be provided as a saline solution. Physiological solution is defined as an aqueous solution of sodium chloride. For example, normal saline is defined as an aqueous solution of sodium chloride at a concentration of 0.9% (w/v). In one embodiment of the present invention, the sodium chloride in the composition is provided in the form of a saline solution.

In the above embodiments, the compositions of the present invention include gentisic acid or pharmaceutically acceptable salts and/or hydrates thereof as a component. Gentisic acid is also known as 2,5-dihydroxybenzoic acid, 5-hydroxysalicylic acid, or hydroquinonecarboxylic acid. Salts of gentisic acid may include sodium salt and sodium salt hydrate. Any reference to gentisic acid may include reference to its salts, where appropriate. The present inventors have found that gentisic acid or a salt thereof in the composition of the present invention can help prevent or minimize radiolysis of the radiolabeled complex of formula (I).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and/or a) excipients or diluents, such as starches, lactose, sucrose, glucose, mannitol and silicic acid , b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants such as glycerin, d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate, e) dissolution retardants such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate, h) absorbents such such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical field. They may optionally include opacifiers and may also be a composition that releases the active ingredient(s) only or preferentially in a specific part of the intestinal tract, optionally in a delayed manner. Examples of potting compositions that can be used include polymeric materials and waxes.

If desired, and for more efficient distribution, the compounds can be included in slow release or targeted delivery systems such as polymer matrices, liposomes and microspheres.

The active compounds may also be in microencapsulated form, if desired, with one or more of the above excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may include inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof.

In addition to inert diluents, oral compositions may also include excipients such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and flavoring agents.

Suspensions, in addition to the active compounds, may include suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters of sorbitan, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and mixtures thereof.

As discussed above, the compounds of the embodiments may be useful for the treatment and/or detection of proliferative diseases. Examples of such cell proliferative diseases or conditions include cancer (including any metastases), psoriasis, and smooth muscle cell proliferative disorders such as restenosis. The compounds of the present invention may be particularly useful for the treatment and/or detection of tumors such as breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, head and/or neck cancer, or kidney, stomach, pancreas cancer glands and brain cancer, as well as hematological malignancies such as lymphoma or leukemia. In addition, the compounds of the present invention may be useful for the treatment and/or detection of a proliferative disease that is resistant to treatment and/or detection by other anticancer drugs; and for the treatment and/or detection of hyperproliferative conditions such as leukemia, psoriasis and restenosis. In other embodiments, the compounds of the present invention may be used to treat and/or detect precancerous conditions or hyperplasia, including familial adenomatous polyposis, adenomatous polyp of the colon, myeloid dysplasia, endometrial dysplasia, endometrial hyperplasia with atypia, cervical dysplasia, vaginal intraepithelial neoplasia , benign prostatic hyperplasia, laryngeal papillomas, actinic and solar keratosis, seborrheic keratosis and keratoacanthoma.

SYNTHESIS OF COMPOUNDS OF THE INVENTION

The agents of the various embodiments can be prepared using reaction routes and synthetic schemes as described below using methods available in the art using readily available starting materials. The preparation of specific compounds of the embodiments is described in detail in the following examples, but one skilled in the art will appreciate that the chemical reactions described can be easily adapted to produce a number of other agents of various embodiments. For example, the synthesis of non-exemplary compounds can be advantageously accomplished by modifications apparent to those skilled in the art, such as by appropriate protection of interfering groups, by substitution with other suitable reagents known in the art, or by making conventional modifications of reaction conditions . A list of suitable protecting groups in organic synthesis can be found in TW Greene's Protective Groups in Organic Synthesis, ^3rd Edition, John Wiley & Sons, 1991. Alternatively, other reactions described herein or known in the art will be found suitable for preparing other compounds of various embodiments. Reagents useful for the synthesis of compounds can be purchased or prepared in accordance with techniques known in the art.

Example 1

Synthesis of Sar–PSMA

Scheme 1 describes the route used to synthesize the Sar–PSMA compound 1.

The MeCOSar–D–Phe–D–Phe–Aoc–Lys–urea–Glu ligand 1, where Aoc=8–aminooctanoic acid (i.e. Sar–PSMA), was prepared by solid phase peptide synthesis. The glutamate–urea–lysine binding motif was synthesized by reacting imidazole-activated and protected glutamic acid with protected L-lysine, which was immobilized on Wang resin. The peptide linker was conjugated to the lysine ε-amine via solid-phase peptide synthesis using the standard Fmoc protocol. Conjugation of the chelator was accomplished by reacting (tBoc) _4–5 MeCOSar with a side chain-protected urea linker on a solid support. Sar-PSMA was cleaved from the resin and simultaneously deprotected (TFA/TIPS/H ₂ O).

Radiolabeling of Sar–PSMA with ⁶⁴ Cu ^II

Sar–PSMA ligand 1 was radiolabeled with ^{64 CuII} at room temperature in aqueous solution (0.1 M _NH4OAc , pH 8, 1–10 nmol Sar–PSMA). Elution from a solid phase extraction cartridge (Phenomenex Strata–X RP 60 mg/mL) yielded ⁶⁴ Cu–Sar–PSMA in >94% yield (ndc) and >97% radiochemical yield (7.95–21.9 GBq/µmol) .

Scheme 1

Synthesis of activated Glu intermediate, 3

Ref: Duspara, PA; Islam, MS; Lough, A. J.; Batey, R.A., Synthesis and reactivity of N–alkyl carbamoylimidazoles: development of N–methyl carbamoylimidazole as a methyl isocyanate equivalent. J Org Chem 2012, 77 (22), 10362–8.

Into a flask containing L–Bis( t Bu)Glu HCl 2 (3.56 g, 12.04 mmol, 1.0 eq.) and carbonyldiimidazole (2.15 g, 13.24 mmol, 1.1 eq.) , a 1:5 DMF/MeCN mixture (50 ml) was added. The reaction mixture was stirred overnight at room temperature. After stirring, the solvent was removed in vacuo and the remaining crude mixture was dissolved and purified by flash chromatography (mobile phase: 7:3:1 petroleum alcohol/chloroform/methanol, Rf: ~0.24, silica/crude weight ratio 30: 1) to obtain the product in the form of a white semi-crystalline powder (2.25 g, yield 52.9%).

Loading Wang resin with Fmoc–Lys(DDiv)–OH

A pre-activated Fmoc–Lys(DDiv)–OH mixture (2.038 g, 3.55 mmol, 3. 0 eq), HCTU (1.33 g, 3.5 mmol, 2.96 eq), DIPEA (1.24 ml, 7.09 mmol, 6 eq), DMAP (43.3 mg, 0.355 mmol , 0.3 eq.) in DMF. The resin was placed on a shaker for 2 hours and left to react. Acetic anhydride (223 μL, 2.36 mmol, 2 eq.) and pyridine (190 μL, 2.36 mmol, 2 eq.) were then added to the reaction mixture to cap the remaining functional groups of the resin and stirred for 30 min. The resin was then filtered and washed with DMF x3, DCM x3, MeOH x2 and Et ₂ O x2, dried and weighed to determine the final resin loading (0.759 mmol/g). The resin loading was determined as follows:

Synthesis of protected KuE on resin, 4

The Sar–PSMA ligand was synthesized from the KuE motif on resin under standard conditions for solid-phase Fmoc peptide synthesis.

General Fmoc Deprotection Protocol

Resin-bound peptide 4 was treated with 20% piperidine in DMF for 5 minutes x3. The resin was then washed successively with DMF x3 and DCM x3.

TNBSA test to confirm combination/deprotection reaction

For each coupling/deprotection step, a qualitative test was performed using the TNBSA (trinitrobenzenesulfonic acid) test. A small fraction of the resin (about 20 beads) was placed in an Eppendorf tube. TNBSA (10 μl 5% solution in DMF) and DIPEA (10 μl 5% solution in DMF) were added and the mixture was stirred for 2 minutes. If no change in color of the resin was observed, then the test indicated the absence of primary amine, while an orange color of the resin indicated the presence of free primary amine.

After deprotection, a solution of Glu-activated intermediate 3 (0.95 g, 2.69 mmol, 2.0 eq) and DIPEA (240 μl, 1.38 mmol, 1.0 eq) in DMF (5 ml). The resin was mixed by hand for 24 hours and washed with DMF x3 and DCM x3. After confirmation of the coupling reaction by TNBSA and digestion/MS test, the DDiv group was deprotected by treatment with 2% hydrazine hydrate in DMF x3 to obtain 5.

General protocol for combining Fmoc amino acids with resin

Fmoc amino acid (3 eq) was activated using HCTU (0.96 eq vs AA) and DIPEA (2 eq vs AA) in DMF. After 5 minutes, the solution was added to the resin and stirred periodically. After 20 min, the resin was filtered and washed successively with DMF x1, DCM x3 and DMF x3 to obtain 6.

Coupling of MeCOSar with PSMA ligand on resin to produce 7

BocMeCOSar (0.464 g, 0.5 mmol, 1.2 eq.) was activated using HCTU (0.207 g), HOBt (67.6 mg) and DIPEA (174 μl) in DMF. After 5 minutes, the solution was added to resin 6 (0.4 mmol) and stirred occasionally. After 24 hours, the resin was filtered and washed successively with DMF x1, DCM x3 and DMF x3.

Resin splitting protocol to produce 1

Resin 7 was washed several times with DCM and then transferred to a 50 ml Falcon tube. 95:2.5:2.5 TFA/TIPS/H ₂ O (15 ml) was added and the resin was stirred at room temperature for 2 hours. The resin was filtered and washed twice with 3 ml TFA. The filtrate was collected and the TFA was evaporated under a stream of N ₂ . The crude peptide was precipitated by adding excess cold Et ₂ O and centrifuged. Et ₂ O was decanted and the process was repeated x3. The precipitated crude peptide was dried, weighed and purified by preparative HPLC.

HPLC Purification

Crude peptide (818 mg) was reduced in 22% MeCN in H ₂ O (6.4 ml) and purified in parts by RP-HPLC (24% isocratic for 60 min) on Kinetex 5 μ 100Å AXIA-packed C18 21.2 ×150 mm semipreparative column at 5 ml/min. Product containing fractions were separated and lyophilized to give Product 1 as a fluffy white powder (58.5 mg, 16.1% based on resin used).

Radiolabeling

An aliquot of ⁶⁴ Cu ^II (30–200 MBq, 0.1M NH ₄ OAc, pH 6) was added to a solution containing Sar–PSMA 1 (5 μg, 4.3 × 10 ^–3 μmol) in MilliQ water, NH ₄ OAc, pH 5 (final concentration: 0.05M), ethanol (10%), and gentisic acid in MilliQ water (final concentration: 0.056%) and pH was measured (pH: 5). The reaction mixture was incubated for 30 min at room temperature. After 30 minutes, an aliquot was analyzed by RP-HPLC to determine the product ⁶⁴ Cu-Sar-PSMA with a radiochemical purity of >98%.

Stability in plasma

A solution of ⁶⁴ Cu–Sar–PSMA in saline (100 μL, ~8.8 MBq, <10% EtOH) was added to 200 μl of fresh human plasma at 37°C and the mixture was incubated at 37°C for 24 hours. After 24 hours, cold acetonitrile (600 μl) was added. Precipitated whey proteins were separated by centrifugation (13,000 rpm) and 300 μl of the supernatant was removed and concentrated by evaporation. The solution was diluted with water (100 μL) and the product was analyzed by RP-HPLC.

Tumor imaging in mice bearing LNCaP tumors

The in vivo biodistribution of ⁶⁴ CuSarPSMA was examined in LNCaP NSG (NOD SCID Gamma) tumor-bearing mice at 1 h, 6 h and 22 h post-injection. At 1 hour, ⁶⁴ CuSarPSMA showed the highest uptake in the kidney, resulting in a low tumor/kidney ratio (Figure 5). However, biodistribution data showed rapid renal clearance and moderate tumor retention at later time points. Despite the moderate retention of ⁶⁴ Cu–Sar–PSMA in the tumor, a significant contrast was observed due to rapid clearance from the circulation and virtually no background accumulation after 6 hours. In addition, low uptake in other PSMA-positive tissues (lungs, spleen) provided high tumor-to-background ratio for ⁶⁴ Cu–Sar–PSMA.

Example 2

Synthesis of CoSar(PSMA) ₂

Synthesis of activated Glu intermediate

Ref: Duspara, PA; Islam, MS; Lough, A. J.; Batey, R.A., Synthesis and reactivity of N–alkyl carbamoylimidazoles: development of N–methyl carbamoylimidazole as a methyl isocyanate equivalent. J Org Chem 2012, 77 (22), 10362–8.

To a flask containing L–Bis(tbu)Glu HCl (3.56 g, 12.04 mmol, 1.0 eq.) and carbonyldiimidazole (2.15 g, 13.24 mmol, 1.1 eq.) was added mixture 1:5 DMF/MeCN (50 ml). The reaction mixture was stirred overnight at room temperature. After stirring, the solvent was removed in vacuo and the remaining crude mixture was dissolved and purified by flash chromatography (mobile phase: 7:3:1 petroleum alcohol/chloroform/methanol, RF: ~0.24, 30:1 mass ratio silica/crude product) to obtain the product as a white semi-crystalline powder (2.25 g, 52.9% yield).

Synthesis of protected urea

To a flask containing H–Lys(Fmoc)–OtBu•HCl (4.84 g, 10.5 mmol, 1.0 eq.) was added the activated Glu intermediate (3.71 g, 10.5 mmol, 1.0 eq. 0 eq.) and DIPEA (1.83 ml, 10.5 mmol, 1 eq.) in DCM (30 ml) and stirred overnight at room temperature. The reaction mixture was washed with water x3, brine and dried over MgSO ₄ , loaded into an 80 g Reveleris HP Silica cartridge and purified using a Biotage Isolera automated flash chromatography purification system (mobile phase: 70:30:2.5 petroleum alcohol/ chloroform/methanol). Fractions were analyzed by TLC (mobile phase: 7:3:1 petroleum alcohol/chloroform/methanol, RF: ~0.30), pooled and solvent removed in vacuo to give the product as a yellow oil, 5.06 g, 68 % exit. ESIMS ⁺ [M+H ⁺ ] m/z 710.386 (experimental), m/z 710.401 (calculated).

Fmoc cleavage of urea

To a flask containing protected urea (5.06 g, 7.13 mmol, 1 eq.), 20% diethylamine in MeCN (100 ml) was added and the reaction mixture was stirred for 7 hours at room temperature. Aliquots were periodically removed for MS analysis to complete the reaction. After 7 hours, diethylamine and MeCN were reduced to 5 mL in vacuo, and an additional 50 mL of MeCN was added to azeotrope diethylamine through a rotary evaporator three times. The reaction mixture was again reduced to 5 ml, 200 ml of 50/50 water/MeCN was added and the reaction mixture was lyophilized. Due to impurities resulting from incomplete removal of the dibenzofulvene moiety, the final product was used without further purification. (Calculated purity 70%) ESIMS ⁺ [M+H ⁺ ] m/z 488.329 (experimental), m/z 488.333 (calculated).

Synthesis of 8–Aoc–ff linker on resin

Linker 8–Aoc–ff was prepared using the following Fmoc protocol below.

General Fmoc Deprotection Protocol

The resin-bound peptide was treated with a solution of 20% piperidine in DMF for 15 minutes x3. The resin was washed successively with DMF x3 and DCM x3.

Protocol for loading Fmoc-amino-octanoic acid (Fmoc-8-Aoc-OH) into 2-CT resin

Fmoc–8–Aoc–OH (5.00 g, 13.1 mmol, 1.75 eq) and DIPEA (2 eq relative to AA) in 80 ml DCM were added to 2-CT resin (7.50 g, 1 mmol/g, 1 eq.) and stirred. After 2 hours, 8 ml of MeOH was added and the resin was stirred for another 30 minutes. The resin was filtered and washed successively with DCM x3, DMF x3, DCM x3, MeOH x2 and Et ₂ O x2 and dried. The resin loading was calculated using the equation below and was 0.628 mmol/g (total 6.16 mmol)

Protocol for combining Fmoc–D–Phe–OH with resin

Fmoc–D–Phe–OH (2 equiv.) was activated with HATU (0.96 equiv. vs. AA) and DIPEA (2 equiv. vs. AA) in NMP. After 5 minutes the solution was added to the resin and stirred for a minimum of 12 hours. The resin was filtered and washed successively with DMF x1, DCM x3 and DMF x3. The coupling reaction was then repeated as described above for at least 12 hours and then filtered and washed successively with DMF x1, DCM x3 and DMF x3.

Resin splitting protocol

The resin was washed several times with DCM and then transferred into two 50 ml Falcon tubes. 5% TFA in DCM (75 ml) was added and the resin was stirred at room temperature for 2 hours. The resin was filtered and washed twice with 15 ml of 5% TFA in DCM. The filtrate was collected and the solvent was reduced under a stream of N ₂ . The crude peptide was redissolved in 50/50 water/MeCN and lyophilized to obtain the crude peptide. The crude peptide was used without further purification.

Trifluoroacetamide protection

To a flask containing the crude peptide linker (1.35 g, 2.98 mmol if pure, 1 eq.) was added ethyl trifluoroacetate (0.532 ml, 4.47 mmol, 1.5 eq.) and DIPEA (1.04 ml, 5 .96 mmol, 2 eq.) in MeOH (10 ml). The reaction mixture was stirred overnight at room temperature and monitored by MS and analytical HPLC to ensure complete conversion of the starting material. MeOH was reduced in vacuo and EtOAc/0.01 M HCl was added to the reaction mixture. The organic layer was separated and washed with 0.01 M HCl x 3 and brine, dried over MgSO ₄ and the solvent was removed in vacuo and dried to give the crude product (0.985 g), which was used without further purification. Orbitrap–MS ⁺ [M+H ⁺ ] m/z 550.253 (experimental), m/z 550.252 (calc.), [2M+H ⁺ ] m/z 1099.498 (experimental), m/z 1099.497 ( calculation).

Urea–linker combination

To a flask containing crude TFA-protected linker (0.985 g, 1.79 mmol, if pure, 1 eq.), HATU (0.608 g, 1.6 mmol, 0.89 eq.) and DIPEA (0.56 ml) were added , 3.2 mmol, 1.79 eq.) in DMF (5 ml) and stirred at room temperature. After 5 minutes, deprotected urea (approximately 1.5 mmol) was added and the reaction was monitored by MS and analytical HPLC overnight. After 24 hours, K ₂ CO ₃ in water was added and the reaction mixture was heated to 60°C overnight and monitored for removal of the trifluoroacetamide protecting group. The reaction mixture was diluted with water (150 ml) and extracted with Et ₂ O x3. The ether fractions were combined and washed with water, 0.01 M HCl x3, and brine and dried over _MgSO4 . The aqueous layer was acidified with 0.1 M HCl and extracted with Et ₂ O, washed with water, 0.01 M HCl and brine and dried over MgSO ₄ . The ether layers were combined, the solvent was removed in vacuo, and the crude product was dissolved in 80% MeCN in water and purified by RP-HPLC (60-77% for 35 minutes) on a Phenomenex Luna 5 μ 100Å C18 21.2 x 250 mm semipreparative column at 8 ml/min. Fractions containing the product were collected and lyophilized to obtain the product as a fluffy white powder (49.6 mg, 98%+ purity). Orbitrap–MS ⁺ [M+H ⁺ ] m/z 923.586 (experimental), m/z 923.585 (calc.), [M+2H ⁺ ] m/z 462.297 (experimental), m/z 462.296 (calc.).

Synthesis (tBoc) _4–5 CoSar-Plus

(tBoc) _4-5 CoSar–Plus can be prepared according to the methods in Ma, MT; Cooper, M.S.; Paul, R.L.; Shaw, K. P.; Karas, J. A.; Scanlon, D.; White, J.M.; Blower, P. J.; Donnelly, PS Inorg Chem 2011 , 50 , 6701.

Synthesis of CoSar–(PSMA) ₂

_4–5 COSar–Plus (25.3 mg, 0.027 mmol, 1 eq.), HATU (20.4 mg, 0.054 mmol, 2 eq.), and DIPEA (18.7 µl, 0.107 mmol, 4 eq.) in NMP (500 µl). The mixture was shaken for 10 minutes to activate and then added to a 2 mL microwave vial containing pure PSMA linker–urea (49.6 mg, 0.054 mmol, 2 eq.) in NMP (400 μL). The reaction mixture was stirred in a microwave reactor at 60°C for 10 minutes, cooled and analyzed by MS and analytical HPLC to show complete consumption of the starting material. 1.8 mL of 72% MeCN in water was added to the reaction material and purified by RP-HPLC (60–90% for 60 min) on a Phenomenex Luna 5 μ 100Å C18 21.2 x 250 mm semipreparative column at 8 mL/min . Fractions containing the product were collected and lyophilized to obtain the protected product as a fluffy white powder (14.0 mg, 5.08 μmol, 18.9% yield). The protected product was dissolved in 95% TFA in water overnight, diluted 50/50 MeCN/water, and lyophilized to give the product as a white powder tris-trifluoroacetate monohydrate salt (12.1 mg, 5.08 µmol) (time retention: 10.3 min, 96.4% purity). Orbitrap–MS ⁺ [M+2H ⁺ ] m/z 1009.065 (experimental), m/z 1009.066 (calc.), [M+3H ⁺ ] m/z 673.046 (experimental), m/z 673.046 ( calculation).

Radiolabeling

An aliquot of ⁶⁴ Cu ^II (100–400 MBq, 0.01 M HCl) was added to a solution containing 0.1 M _NH4OAc , pH 5.5 (500 μL), ethanol (100 μL), MilliQ water (300 μL), and gentisic acid (1.2 mg, 10 mg/ml in MilliQ water) and pH was measured (pH: 5). CoSar–(PSMA) ₂ (20 µg, 8.4 x 10 ^–3 µmol, 1 mg/ml in MilliQ) was added to this solution and the reaction mixture was incubated for 30 min at room temperature. After 30 minutes, an aliquot was analyzed by RP-HPLC to determine the product ⁶⁴ Cu-CoSar(PSMA) ₂ with a radiochemical purity of >97% (retention time: 13.9 min).

In the present specification and in the following claims, unless the context otherwise requires, the word “comprises” and variations such as “includes” and “including” will be understood to mean the inclusion of a specified integer or step or group of integers or steps, but not exclusive of any other integer or stage or group of integers or stages.

Reference herein to any previous publication (or information derived therefrom) or to any known subject matter is not and should not be construed as an endorsement or admission or any form of suggestion that such previous publication (or information derived from it) basis) or the known substance is part of the general knowledge in the field to which this description relates.

Claims (38)

1. A compound of formula (I) or a pharmaceutically acceptable salt or isomer thereof: (I) Where: X is _C1 – _C6 alkyl or group ; Y represents an unsubstituted C ₁ –C ₁₂ alkylene group; m is 0, 1 or 2; And n is 0, 1 or 2; where the compound is coordinated to a metal ion, where the metal ion is selected from the group consisting of ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu and ⁶⁷ Cu. 2. A compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, where: X represents C ₁ –C ₆ alkyl. 3. A compound according to claim 2 or a pharmaceutically acceptable salt or isomer thereof, where: X represents a group . 4. Connection according to any one of paragraphs. 1-3, or a pharmaceutically acceptable salt or isomer thereof, where: m is equal to 1. 5. Connection according to any one of paragraphs. 1-4, or a pharmaceutically acceptable salt or isomer thereof, where: n is 1. 6. A compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, wherein the compound has the formula: . 7. A compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, where the compound has the formula: . 8. A compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, wherein the compound has the formula: 9. A compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, where the compound has the formula: 10. A compound or a pharmaceutically acceptable salt or isomer thereof, where the compound has the formula: where the compound is coordinated to a metal ion, where the metal ion is selected from the group consisting of ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu and ⁶⁷ Cu. 11. A compound or a pharmaceutically acceptable salt or isomer thereof, where the compound has the formula: where the compound is coordinated to a metal ion, where the metal ion is selected from the group consisting of ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu and ⁶⁷ Cu. 12. Composition for radioimaging, including a compound according to any one of paragraphs. 1-11 and a pharmaceutically acceptable excipient. 13. An aqueous composition for parenteral administration, used for radioimaging, including a compound according to any one of claims 1 to 11; where the composition further includes ethanol, gentisic acid or a salt thereof and sodium chloride. 14. Composition according to claim 12 or 13, wherein the compound of formula (I) is . 15. A method for radioimaging a subject, comprising administering an effective amount of a compound according to any one of claims 1 to 11 or a composition according to any one of claims 12 to 14.