Urea-based prostate specific membrane antigen (PSMA) inhibitors for imaging and therapy

The present invention relates to compounds according to Formula I and Formula IV. These compounds display very good binding affinities to the PSMA binding sites. They can be labeled with [68Ga]GaCl3 with high yields and excellent radiochemical purity. The present invention also relates to pharmaceutical compositions comprising a pharmaceutical acceptable carrier and a compound of Formula I or Formula IV, or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

This invention is in the field of radiolabeled imaging and radioactive therapy agents. In particular, derivatives of urea based prostate-specific membrane antigen (PSMA) inhibitors are disclosed. Derivatives with a chelating moiety are capable of chelating a radioactive metal. Compounds containing a novel phenoxy linker were also prepared, and the linker attaches a chelating moiety or a radioactive group with an urea based PSMA targeting moiety.

Prostate-specific membrane antigen is a highly specific prostate epithelial cell membrane antigen. It is a type II transmembrane protein consisting of a short NH2-terminal cytoplasmic domain, hydrophobic transmembrane region, and a large extracellular domain. This is a transmembrane enzyme with overlapping carboxypeptidase enzyme activities similar to (a) glutamate carboxypeptidase II (GCPII, E.C.3.17.21), a zinc-dependent metallopeptidase, and (b) folylpolyglutamate synthetase (FPGS). The extracellular portion of the peptide sequence exists as a dimer and shows a strong binding to glutamate and glutamate related structures (brain related PSMA), its natural substrates are N-acetyl-aspartylglutamate and folyl-poly-γ-glutamates (prostate related PSMA) (Scheme 1).

PSMA is highly expressed in various tumors, including prostate cancer. Often, PSMA expression increases in higher-grade cancers and metastatic diseases. In the vast majority of neovasculature in solid tumors, there is high expression of PSMA, but not in normal vasculature. This makes PSMA a suitable target for cancer detection and therapy. Prostascint® (In-111 Capromab pendetide) developed by Cytogen was the first antibody of PSMA approved for clinical use. This antibody only recognizes the intracellular epitope on PSMA, which is associated with dead or necrotic cells commonly found in lymph nodes. Prostascint® is not useful for imaging living tumor cells because of its lack of cell penetration. SPECT (single photon emission computer tomography) imaging of this agent exhibits prolonged background activity and an unfavorable signal to background ratio even at 4 days post injection.

A specific antibody targeting the extracellular portion of PSMA, J591, has been reported and shown to have improved PSMA targeting properties. This antibody has been radiolabeled with various isotopes,89Zr,111In,177Lu, etc., for imaging and radiotherapy. J591 is an antibody against the extracellular epitope of PSMA, and it is targeting the PSMA binding sites on the membrane of tumor cells. Its in vivo retention and circulation time is relatively long, thus contributing to a prolonged waiting period to reach optimal imaging. An isotope with a longer physical half-life is essential for this purpose, therefore89Zr, a positron-emitting isotope with a physical half-life of 78.4 hours, is more appropriate. [89Zr]J591 bound strongly to PSMA, and clinical studies in humans suggested that it is useful for defining the tumor location by PET imaging.

A number of small molecule-based PSMA imaging agents have been reported in the literature. Different PSMA-targeting core structures have been employed, including: 2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid (GPI), 2-(3-mercaptopropyl)pentane-dioic acid (2-PMPA), phosphoramidates, and urea (Glu-NH—CO—NH-Lys(Ahx)), originally reported in 2000 (Scheme 2). See e.g. US2004054190; Kozikowski A P, et al.,J. Med. Chem.47:1729-38 (2004). Based on these binding core structures, many of the PSMA inhibitors were reported to be highly selective and potent. After labeling with different isotopes, they can be employed for in vivo imaging (SPECT or PET).

Several potential PSMA-targeted imaging agents using urea based ligand systems (Glu-NH—CO—NH or Glu-NH—CO—NH-Lys(Ahx)), including SPECT imaging agents: [123I]MIP-1072, [123I]MIP-1095 [49-51], [99mTc]MIP-1404, and [99mTc]Tc-MIP-1405 (Scheme 3), have entered into clinical trials. Results of phase II clinical studies suggest that these SPECT PSMA imaging agents are suitable for the diagnosis of prostate and other related solid tumors.

Several11C and18F labeled PET imaging agents targeting PSMA have also been reported (Scheme 4). Again, these are derivatives of Glu-NH—CO—NH— or Glu-NH—CO—NH-Lys(Ahx), such as [11C](S)-2-[3-((R)-1-carboxy-2-methylsulfanyl-ethyl)-ureido]-pentanedioic acid,11C-MCG, Two fluorinated version of PSMA-targeting agents, [18F]DCFBC: N—[N—[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[18F]-fluorobenzyl-L-cysteine, and [18F]DCFPyL: 2-(3-(1-carboxy-5-[(6-[18]fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid, have been reported. Both agents showed promising results in imaging patients with metastatic prostate cancer. The preparation of11C and18F labeled PSMA imaging agents require a near-by cyclotron, because the physical half-life is 20 min and 110 min, respectively. As an alternative,68Ga can be used for PET imaging in a laboratory setting without a near-by cyclotron.

In the past few years, [68Ga]Glu-NH—CO—NH-Lys(Ahx)-HBED-CC (monomer, [68Ga]1a) and its dimer, [68Ga](Glu-NH—CO—NH-Lys(Ahx))2-HBED-CC were successfully prepared and showed high PSMA binding (Scheme 5). Although both [68Ga]Glu-NH—CO—NH-Lys(Ahx)-HBED-CC (monomer) and [68Ga](Glu-NH—CO—NH-Lys(Ahx))2-HBED-CC (dimer) exhibited comparable preclinical data, currently, the most popular PSMA/PET imaging agent that has been successfully applied in humans is [68Ga]Glu-NH—CO—NH-Lys(Ahx)-HBED-CC. See Eder M, et al.,Bioconjug. Chem.23:688-97 (2012).

Recently PSMA-617 and DOTAGA-(yl)-fk(sub-KuE) (I&T) were reported (Scheme 6). These two compounds contain different linkers between the chelating moiety and the urea based PSMA targeting moiety. These linkers have various amino acid residues. These PET tracers appear to provide useful diagnostic information in humans. A comparison of PET imaging using [68Ga]Ga-PSMA-HBED-CC and [18F]DCFPyL, in prostate cancer patients has been reported. Additional imaging agents with structure modifications in the linker regions have been reported to have improved tumor targeting properties and pharmacokinetics. See US Published Appl. No. 2016/0228587.

A need continued to exist to further improve the Glu-NH—CO—NH-Lys(Ahx)-HBED-CC amide derivatives as PSMA inhibitor for in vivo imaging and radiation therapy.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

X is selected from the group consisting of:

R7is hydrogen or a (C1-C6) alkanoyl group; and

R8is hydrogen or an α-position substituent of an amino acid,

In another embodiment, the invention relates to a compound according to Formula II:

wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

X is selected from the group consisting of:

R8is hydrogen or an α-position substituent of an amino acid; and

M is a metal selected from the group consisting of44Sc,47Sc,67Ga,68Ga,72As,99mTc,111In,90Y,97Ru,62Cu,64Cu,52Fe,52mMn,140La,175Yb,153Sm,166Ho,149Pm,177Lu,142Pr,159Gd,213Bi,149Pm,67Cu,111Ag,199Au,161Tb,203Pb, and51Cr,

In another embodiment, the invention relates to a compound according to Formula III:

wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

R1and R2are independently hydrogen or a carboxylic acid protecting group;

M is a chelating metal selected from the group consisting of44Sc,47Sc,68Ga,99mTc,111In,90Y,153Sm,166Ho,177Lu,159Gd,213Bi,149Pm,161Tb,203Pb, and51Cr.

In one embodiment, the invention relates to a compound according to Formula IV:

or a pharmaceutically acceptable salt thereof,
wherein

Z is a chelating moiety, ora group having the structure:

wherein Y10is CH or N;L is a bond or a divalent linking moiety comprising 1 to 6 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;R* is a positron emitting radioactive isotope;R20is selected from the group consisting of alkyl, alkoxyl, halide, haloalkyl, and CN;p is an integer from 0 to 4, wherein when p is greater than 1, each R20is the same or different;

W is a PSMA-targeting ligand;

A4is a bond or a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

G is O, S, or NR3;

R1is hydrogen or a carboxylic acid protecting group;

R19is selected from the group consisting of alkyl, alkoxyl, halide, haloalkyl, and CN;

m is an integer from 1 to 6; and

o is an integer from 0 to 4, wherein when o is greater than 1, each R19is the same or different.

In one embodiment, the invention relates to a method for imaging in a subject, comprising administering a radiolabeled compound disclosed herein to the subject; and obtaining an image of the subject or a portion of the subject. In another embodiment, the method for imaging comprises obtaining an image with a device that is capable of detecting positron emission. Additionally, the invention relates to methods of making a compound of Formula I, Formula II, and Formula IV.

DETAILED DESCRIPTION OF THE INVENTION

An attractive and versatile approach in obtaining radiopharmaceuticals for PET/CT is the use of a68Ge/68Ga generator to produce68Ga (T1/2=68 min) PET imaging agents. There are several advantages for using68Ga for PET imaging: (1) It is a short-lived positron emitter (half-life 68 min, β+). (2) A68Ge/68Ga generator readily produces68Ga in a laboratory setting without a nearby cyclotron. (3) The parent,68Ge, has a physical half-life of 270 days, providing a useful life of 6 to 12 months. (4) There are several commercial vendors now supplying this generator for clinical practice on a routine basis. (5) The coordination chemistry for Ga(III) is highly flexible and large number of Ga chelates with varying stability constants and metal chelating selectivity have been reported; It has been demonstrated that68Ga radiopharmaceuticals target various tissues or physiological processes for cancer diagnosis. (6) An important factor to consider is that the emitting β+energy for18F and68Ga is 0.63 MeV and 1.90 MeV, respectively. However, despite the difference in the β+energy,18F and68Ga radiopharmaceuticals give similar spatial resolution, sensitivity, image contrast, and activity recovery coefficients in human tissue, and they produce comparable clinical images in humans. These factors listed above lend themselves in support of developing68Ga radiopharmaceuticals for clinical diagnosis.

In the past two decades there are many reports on using68Ga labeled small molecules and peptides for imaging various tumors. Among them [68Ga]DOTA-TOC, [68Ga]DOTA-TATE, and [68Ga]DOTA-NOC are the most commonly employed agents for the detection of neuroendocrine tumors (NET) expressing somatostatin receptors. Additional chelates for making68Ga agents, such as NOTA, HBED-CC, TRAP, and many other polyaza carboxylic acids have been reported (Scheme 7). The improved chelates, such as NOTA, NODAGA, and NOTGA, will have the advantage of forming stable68Ga labeled complexes at room temperature (i.e. stable in vitro and in vivo), which simplifies preparation and makes it more suitable in a clinical setting. It was previously reported that the stability constants (log Kd) for Ga-HBED, Ga-NOTA, and Ga-DOTA were 39, 31, and 21, respectively.

68Ga labeled agents provide an alternative approach to producing generator-based PET imaging agents without the need for a nearby cyclotron. Several different versions of68Ga labeled PSMA imaging agents have recently been reported. Chelating groups for complexing Ga(III), including DOTA, triazacyclononane-triphosphinate, 1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid] (NOPO), H2CHXdedpa (cyclohexyl-1,2-[[6-carboxy-pyridin-2-yl]-methylamino]ethane), and (5S,8S,22S,26S)-1-amino-5,8-dibenzyl-4,7,10,19,24-pentaoxo-3,6,9,18,23,25-hexaazaoctacosane-22,26,28-tri-carboxylic acid trifluoroacetate (CHX-A″-DTPA-DUPA-Pep) were reported. All of the Ga-PSMA tagged complexes showed high affinity binding and effective targeting of PSMA expressing tumor models in vitro. However, only limited preclinical data was available for these68Ga labeled agents.

New amide derivatives 1b-g (Scheme 8) were prepared. Of particular interest and novelty is the ligand 1g, in which both HBED (for chelating Ga(III)) and DOTA (for chelating other radioactive metal for radiation therapy) moieties are included in one molecule. This approach allows the use of one ligand to label different types of radioactive metals for multiple applications. Additionally, di-pyridyl derivatives 2 and 3, and mono-pyridyl derivatives, 4a and 4b, were also prepared.

Successful PET/CT imaging studies of tumor targeting prostate-specific membrane antigen (PSMA) using68Ga labeled Glu-NH—CO—NH-Lys(Ahx)-HBED-CC, [68Ga]1a, has demonstrated great potential for clinical in diagnosis of prostate cancer; and successful imaging studies using [68Ga]1a, in humans have been widely reported. Five different series of Glu-NH—CO—NH-Lys(Ahx) amide derivatives have been prepared including HBED-CC derivative containing amino acids, 2-glucosamine and DOTA (1b-g), di-pyridyl derivatives (2 and 3) and mono-pyridyl derivatives (4a and 4b) (Scheme 8). The “cold” ligands, 1b-g, 2, 3, 4a and 4b displayed very good binding affinities (IC50=3-35 nM) to the PSMA binding sites. These new ligands, 1b-g, 2, 3, 4a and 4b were labeled with [68Ga]GaCl3with high yields and excellent radiochemical purity. Results of in vivo biodistribution studies in mice after an i.v. injection of [68Ga]1b-g, 4a and 4b suggested that they are specifically localized in tissues express the PSMA sites. So, [68Ga]1b-g, 4a and 4b are useful as imaging agents for detecting PSMA expression in tumor tissues. The DOTA containing derivative, 1g, can also be separately labeled with177Lu,90Y and213Bi for radiation therapy of PSMA expressing tumors.

Compounds with a novel phenoxy linker were prepared. This series of PSMA inhibitors including the sub-structure of an urea based PSMA targeting moiety and a novel linker were tested by in vitro binding, tumor cell uptake as well as in vivo biodistribution studies. These PSMA inhibitors showed equal or better binding affinity than [68Ga]1a. The novel PSMA inhibitors can have a chelating moiety, such as compounds 5a, 5a′, and 5b; or they can have a radioactive group, such as compounds 5c, 5d, 5e, and 5f (Scheme 9).

In one embodiment, the invention relates to a compound according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

X is selected from the group consisting of:

R7is hydrogen or a (C1-C6) alkanoyl group; and

R8is hydrogen or an α-position substituent of an amino acid,provided that X is not X1when A1, A2, and B are CH2and Y1, Y2, Y3, Y4, Y5, and Y6are CH.

In another embodiment, the invention relates to a compound according to Formula II:

wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

X is selected from the group consisting of:

R8is hydrogen or an α-position substituent of an amino acid; and

M is a metal selected from the group consisting of44Sc,47Sc,67Ga,68Ga,99mTc,72As,111In,90Y,97Ru,62Cu,64Cu,52Fe,52mMn,140La,175Yb,153Sm,166Ho,149Pm,177Lu,142Pr,159Gd,213Bi,149Pm,67Cu,111Ag,199Au,161Tb,203Pb,51Cr,

In another embodiment, the invention relates to a compound according to Formula III:

wherein

A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

B is CR4R5;

R1and R2are independently hydrogen or a carboxylic acid protecting group;

M is a chelating metal selected from the group consisting of44Sc,47Sc,68Ga,99mTc,111In,90Y,153Sm,166Ho,177Lu,159Gd,213Bi,149Pm,161Tb,203Pb, and51Cr.

In certain embodiments, the compounds of the present invention are represented by generalized Formulae I, II, and III and the attendant definitions, wherein A1and A2are independently a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—. In another embodiment, A1and A2are independently a divalent linking moiety comprising a C1-C10alkylene group wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—. In another embodiment, A1and A2are independently (CH2)n, wherein n is an integer from 0 to 6. In another embodiment, A1and A2are independently (CH2)n, wherein n is 1, 2, or 3. In another embodiment, A1and A2are CH2. Useful examples of the divalent linking moiety include —CH2—, —CH2CH2—, —CH2CH2CH2—, —OCH2—, —OCH2CH2—, —OCH2CH2CH2—, —NHCH2—, —NHCH2CH2—, —NHCH2CH2CH2—, —COCH2—, —COCH2CH2—, and —COCH2CH2CH2.

In certain embodiments, the compounds of the present invention are represented by generalized Formulae I and II and the attendant definitions, wherein X is selected from the group consisting of:

In another embodiment, X is a carboxylic acid group or its derivative (X1). In another embodiment, X contains glucosamine group or its derivative (X2). In another embodiment, X contains an amino acid residue or its derivative (X3), including glycine, aspartic acid, glutamic acid. In another group, X contains a DOTA moiety (X4).

Useful R6, R9, and R10groups include a methyl ester, a t-butyl ester, a benzyl ester, and an allyl ester.

In one embodiment, the invention relates to a compound according to Formula IV:

or a pharmaceutically acceptable salt thereof,
wherein

Z is a chelating moiety, ora group having the structure:

wherein Y10is CH or N;L is a bond or a divalent linking moiety comprising 1 to 6 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;R* is a positron emitting radioactive isotope;R20is selected from the group consisting of alkyl, alkoxyl, halide, haloalkyl, and CN;p is an integer from 0 to 4, wherein when p is greater than 1, each R20is the same or different;

W is a PSMA-targeting ligand;

A4is a bond or a divalent linking moiety comprising 1 to 10 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—;

G is O, S, or NR3;

R1is hydrogen or a carboxylic acid protecting group;

R19is selected from the group consisting of alkyl, alkoxyl, halide, haloalkyl, and CN;

m is an integer from 1 to 6; and

o is an integer from 0 to 4, wherein when o is greater than 1, each R19is the same or different.

In one embodiment, the invention relates to a compound according to Formula IV-a:

or a pharmaceutically acceptable salt thereof,
wherein R17is aryl; and wherein A4, Z, and W are as defined herein.

Positron emitting radioactive isotopes are known in the art, and they can be, for example,11C,18F,123I,125I, and131I.

PSMA-targeting ligands are known in the art and they refer to groups that can bind to PSMA. PSMA-targeting ligands can be urea-based ligand systems discussed herein.

In some embodiments, the PSMA-targeting ligand W has the structure:

wherein R20and R21are each independently an amino acid residue linked via an amino group thereof to the adjacent —C(O)— group.

In some embodiments, W has the structure:

wherein R2is hydrogen or a carboxylic acid protecting group, r is an integer from 1 to 6, and s is an integer from 1 to 4. In one embodiment, W has the structure:

In certain embodiments, the compounds of the present invention are represented by generalized Formulae IV and IV-a, and the attendant definitions.

In some embodiments, L is a bond or a divalent linking moiety comprising 1 to 6 carbon atoms in a chain, a ring, or a combination thereof, wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—. In some embodiment, L is a bond. In another embodiment, L is a divalent linking moiety comprising a C1-C6alkylene group wherein at least one carbon atom is optionally replaced with O, —NR3—, or —C(O)—. In some embodiments, L is (CH2)n, —(OCH2CH2)n—, —(NHCH2CH2)n—, or —C(O)(CH2)n—, wherein n is 1, 2, or 3. In another embodiment, L is —OCH2CH2—. Other useful examples of the divalent linking moiety include —CH2—, —CH2CH2—, —CH2CH2CH2—, —OCH2CH2CH2—, —NHCH2CH2—, —NHCH2CH2CH2—, —COCH2—, —COCH2CH2—, and —COCH2CH2CH2—.

In some embodiments, R17is an aryl. In one embodiment, R17is optionally substituted phenyl. In another embodiment, R17is optionally substituted naphthyl.

In some embodiments, the invention relates to a complex comprising a compound according to Formula IV chelated to a metal M wherein Z is a chelating moiety. In some embodiments, the metal M is selected from the group consisting of44Sc,47Sc,203Pb,67Ga,68Ga,72As,99mTc,111In,90Y,97Ru,62Cu,64Cu,52Fe,52mMn,140La,175Yb,153Sm,166Ho,149Pm,177Lu,142Pr,159Gd,213Bi,67Cu,111Ag,199Au,161Tb, and51Cr,99mTc.

In some embodiments, the complex has the structure:

In one embodiment, the invention relates to methods of making a compound of Formulae I, II, and III.

In one embodiment, the present invention provides pharmaceutical compositions comprising a pharmaceutical acceptable carrier and a compound of Formulae I, II, III, and IV. The present invention also provides pharmaceutical compositions comprising a pharmaceutical acceptable carrier and a pharmaceutically acceptable salt of a compound of Formula I. In certain embodiments, the pharmaceutical composition will comprise the reaction precursors necessary generate the compound or salt according to Formula I or subformula thereof upon combination with a radiolabeled precursor.

In one embodiment, the present invention provides a kit formulation, comprising a sterile container containing a compound of Formula I or Formula IV or a pharmaceutically acceptable isotonic solution for i.v. injection thereof, and instructions for diagnostic imaging (for example,68Ga) and radiation therapy (for example,90Y) use.

The present invention also provides for methods of in vivo imaging, comprising administering an effective amount of a radiometal complex or a radioactive compound disclosed herein to a subject, and detecting the pattern of radioactivity of the complex or compound in the subject. In one embodiment, the invention relates to a method for imaging in a subject, comprising administering a radiolabeled compound disclosed herein to the subject; and obtaining an image of the subject or a portion of the subject. In another embodiment, the method for imaging comprises obtaining an image with a device that is capable of detecting positron emission.

The present invention also provides for methods of in vivo imaging, comprising administering an effective amount of a radiometal complex or a radioactive compound disclosed herein to a subject, and detecting the pattern of radioactivity of the complex or compound in said subject.

Typical subjects to which compounds of the invention may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g. livestock such as cattle, sheep, goats, cows, swine and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. Additionally, for in vitro applications, such as in vitro diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use such as mammalian, particularly primate such as human, blood, urine or tissue samples, or blood urine or tissue samples of the animals mentioned for veterinary applications.

Radiopharmaceuticals in accordance with this invention can be positron emitting gallium-68 complexes which, when used in conjunction with a68Ge/68Ga parent/daughter radionuclide generator system, will allow PET imaging studies, avoiding the expense associated with operation of an in-house cyclotron for radionuclide production.

The complexes are formulated into aqueous solutions suitable for intravenous administration using standard techniques for preparation of parenteral diagnostics. An aqueous solution of the present complexes can be sterilized, for example, by passage through a commercially available 0.2 micron filter. The complexes are typically administered intravenously in an amount effective to provide tissue concentrations of the radionuclide complex sufficient to provide the requisite photon (gamma/positron) flux for imaging the tissue. The dosage level for any given complex of this invention to achieve acceptable tissue imaging depends on its particular biodistribution and the sensitivity of the tissue imaging equipment. Effective dosage levels can be ascertained by routine experimentation. They typically range from about 5 to about 30 millicuries. Where the complexes are gallium-68 complexes for PET imaging of myocardial tissue, adequate photon fluxes can be obtained by intravenous administration of from about 5 to about 30 millicuries of the complex.

The term “amino acid” used herein include both naturally occurring amino acids and unnatural amino acids. Naturally occurring amino acid refers to amino acids that are known to be used for forming the basic constituents of proteins, including alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof. Examples of unnatural amino acids include: an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid other than proline.

The term “alkanoyl” used herein refers to the following structure:

The term “cycloalkyl” used herein includes saturated ring groups, having the specified number of carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Cycloalkyl groups typically will have 3 to about 12 ring members. In one embodiment, the cycloalkyl has one or two rings. In another embodiment, the cycloalkyl is a C3-C8cycloalkyl. In another embodiment, the cycloalkyl is a C3-7cycloalkyl. In another embodiment, the cycloalkyl is a C3-6cycloalkyl. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, and adamantyl.

The term “heterocycloalkyl” used herein refers to saturated heterocyclic alkyl groups.

Suitable carboxylic acid protecting group are well known and include, for example, any suitable carboxylic acid protecting group disclosed in Wuts, P. G. M. & Greene, T. W.,Greene's Protective Groups in Organic Synthesis,4rd Ed., pp. 16-430 (J. Wiley & Sons, 2007), herein incorporated by reference in its entirety. Those skilled in the art will be familiar with the selection, attachment, and cleavage of protecting groups and will appreciate that many different protective groups are known in the art, the suitability of one protective group or another being dependent on the particular synthetic scheme planned. Suitable carboxylic acid protecting group include, for example, the methyl esters, t-butyl esters, benzyl esters, and allyl esters.

Materials and Methods

General

All reagents and solvents were purchased commercially (Aldrich, Acros, or Alfa Inc.) and were used without further purification, unless otherwise indicated. Solvents were dried through a molecular sieve system (Pure Solve Solvent Purification System; Innovative Technology, Inc.).1H and13C NMR spectra were recorded on a Bruker Avance spectrometer at 400 MHz and 100 MHz, respectively, and referenced to NMR solvents as indicated. Chemical shifts are reported in ppm (δ), with a coupling constant, J, in Hz. The multiplicity is defined by singlet (s), doublet (d), triplet (t), broad (br), and multiplet (m). High-resolution mass spectrometry (HRMS) data was obtained with an Agilent (Santa Clara, Calif.) G3250AA LC/MSD TOF system. Thin-layer chromatography (TLC) analyses were performed using Merck (Darmstadt, Germany) silica gel 60 F254plates. Generally, crude compounds were purified by flash column chromatography (FC) packed with silica gel (Aldrich). High performance liquid chromatography (HPLC) was performed on an Agilent 1100 series system. A gamma counter (Cobra II auto-gamma counter, Perkin-Elmer) measured68Ga radioactivity. Reactions of non-radioactive chemical compounds were monitored by thin-layer chromatography (TLC) analysis with pre-coated plates of silica gel 60 F254. An aqueous solution of [68Ga]GaCl3was obtained from a68Ge/68Ga generator (Radiomedix Inc.). Solid-phase extraction cartridges (SEP Pak® Light QMA, Oasis® HLB 3cc) were obtained from Waters (Milford, Mass., USA).

Synthesis of example compounds, 1a-g, 2, 3, 4a-b, and 5a-f containing Glu-NH—CO—NH-Lys(Ahx)-HBED-CC group, were prepared by reactions described in the following sections. It is noted that [68Ga]1a, (commonly referred to as PSMA-11) is a known PSMA imaging agent, and it is presented as a positive control for binding to PSMA.

Previously reported synthesis of Glu-NH—CO—NH-Lys(Ahx)-HBED-CC (monmer, 1a) and (Glu-NH—CO—NH-Lys(Ahx))2-HBED-CC (dimer) employed a Fe-complex of HBED-CC as the intermediate. The reaction scheme was not very efficient, a new scheme without the use of Fe(III) HBED-CC complex was devised (Scheme 10).

Methyl 3-(4-hydroxyphenyl)propanoate (A) was prepared by O-methylation (esterification) of carboxylic acids in good yield (84%). The methyl ester was treated with MgCl2and paraformaldehyde to give salicylaldehyde, B, in excellent yield (90%). Condensation of salicylaldehyde with ethylenediamine produced Schiff base without further purification. The corresponding secondary amine, 7, was obtained from the Schiff base after the reduction reaction with Sodium Borohydride in 69% yield. The secondary amines were condensed with excess amount of tert-butyl bromoacetate to afford 8 in 87% yield. The methyl ester group of compound, 8 was selectively removed by NaOH hydrolysis to give acid, 9, in 96% yield. Subsequent HOBt/EDCI promoted coupling reaction with tert-butyl 2-(3-((S)-6-(6-aminohexanamido)-1-tert-butoxy-1-oxohexan-2-yl)ureido)pentanedioate (10) produced protected Glu-NH—CO—NH-Lys(Ahx)-HBED-CC (11a). The intermediates 11b-g were synthesized by coupling reaction of 11a with corresponding amino acids, in 22-63% yield. The tert-butyl protection group was then removed to give 1a-g in 26-79% yield. The precursor, 1a-g employed as the starting material for labeling, and subsequently forming complex with GaCl3afforded “cold compound” [natGa]1a-g.

Scheme 12 and 13 outline the synthetic strategy applied to efficiently produce compound 2 and 3. The key intermediate, 20, was successfully prepared through a 9 steps reaction (Scheme 11). Subsequently, HOBt/EDCI promoted coupling reaction with 10 produced protected Glu-NH—CO—NH-Lys(Ahx)-HPyED-CC monomer (21) and dimer (22), followed by a simple acidic de-protection to give final compound 2 and 3.

For the synthesis of the other pyridinyl derivatives linked via amide bonds, the intermediates 28 (Scheme 14) and 35 (Scheme 16) were readily prepared according to similar method. The methyl ester was converted to carboxylic acid by treating with NaOH, which on coupling reaction with 10 provided Glu-NH—CO—NH-Lys(Ahx)-HBE-HPyED-CC (29) and Glu-NH—CO—NH-Lys(Ahx)-HPyED-HBED-CC (36). The protection group was easily removed in the presence of TFA to give 4a and 4b.

NaH (60% in mineral oil, 211 mg, 7 mmol) was placed in a two-neck flask and washed with hexane. 20 mL DMF was added to form a suspension. A solution of 5-hydroxy-2-bromopyridine (779 mg, 3.5 mmol) in 10 mL DMF was added dropwise at 0° C. After stirring at rt for 30 min, the mixture was cooled to 0° C., and benzyl bromide (898 mg, 5.25 mmol) was added dropwise, and the reaction mixture was stirred at rt overnight. The mixture was then poured into 50 mL cold sat. NH4Cl, and extracted with DCM (50 mL×2). The organic layer was washed with H2O (30 mL) and brine (30 mL), dried over Na2SO4, concentrated and purified by FC (EtOAc/hexane=2/8) to give 1.08 g clear oil 14 (yield: 99%).

A mixture of 14 (4.67 g, 21.6 mmol), methyl acrylate (7.39 g, 43.2 mmol), potassium carbonate (K2CO3, 7.45 g, 54 mmol), tetrabutylammonium bromide (13.9 g, 43.2 mmol), and palladium acetate (Pd(OAc)2, 263.5 mg, 1.08 mmol) in 100 mL DMF was deoxygenated by purging into nitrogen for 15 min and then heated at 120° C. for overnight. The mixture was cooled to RT, diluted with 300 mL EtOAc and washed with H2O (80 mL×2) as well as brine (80 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (EtOAc/hexane=2/8) to give 3.86 g colorless oil 15 (yield: 66.2%): HRMS (ESI) calculated for C16H16NO3 (M+H+), 270.1130; found, 270.1109.

A mixture of 15 (3.86 g, 14.3 mmol) and Pd/C (500 mg) in 50 mL MeOH was stirred at rt under H2for 4 h. The resulting mixture was filtered and the filtrate was concentrated to give 2.6 g colorless oil 16 (yield: 100%): HRMS (ESI) calculated for C9H12NO3(M+H+), 182.0817; found, 182.0740.

To a solution of 16 (480 mg, 2.7 mmol) in 15 mL H2O was added NaOH (216 mg, 5.4 mmol) and paraformaldehyde (486 mg, 16.2 mmol). After stirring at 90° C. for 6 h, the mixture was cooled with ice-bath. The pH was adjusted to 7 with 1 N HCl. The solvent was removed in vacuo. 20 mL DMF was then added to the residue, followed by iodomethane (2.3 g, 16.2 mmol) and sodium bicarbonate (1.36 g, 16.2 mmol). After stirred at rt for overnight, the mixture was then poured into 100 mL EtOAc and washed with H2O (30 mL×2) as well as brine (30 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 440 mg white solid 17 (yield: 76.9%): HRMS (ESI) calculated for C10H14NO4(M+H+), 212.0923; found, 212.0933.

To a solution of 17 (190 mg, 0.90 mmol) in 5 mL chloroform was added phosphorus tribromide (121 mg, 0.45 mmol) dropwise under ice-bath. The mixture was warmed to rt and maintained for 3 h. The resulting mixture was then cooled to 0° C. DIPEA (462 mg, 3.58 mmol) was added followed by 13 (102.5 mg, 0.356 mmol). The ice-bath was then removed. The mixture was stirred at rt overnight. The solvent was removed in vacuo and the residue was purified by FC (DCM/MeOH/NH4OH=85/15/1.5) to give 140 mg colorless oil 18 (yield: 58.3%): HRMS (ESI) calculated for C34H51N4O10(M+H+), 675.3605; found, 675.3545.

To a solution of 18 (140 mg, 0.21 mmol) in 5 mL DMF was added 4-methoxybenzyl (130 mg, 0.83 mmol) and NaH (33.2 mg, 0.83 mmol) at 0° C. The mixture was the warmed to rt and maintained for 6 h. The resulting mixture was then poured into 30 mL EtOAc and washed with H2O (10 mL×2) as well as brine (10 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 61.8 mg white solid 19 (yield: 32.6%): HRMS (ESI) calculated for C50H67N4O12(M+H+), 915.4755; found, 915.4689.

To a solution of 19 (61.8 mg, 0.068 mmol) in 2 mL MeOH was added 2 mL NaOH (1 N). After stirred at rt for 4 h, 1 N HCl was added to the mixture till pH=4-5. The resulting mixture was then extracted with EtOAc (20 mL×3). The organic layer was collected, washed with brine (20 mL), dried by Na2SO4and filtered. The filtrate was concentrated to give 47.7 mg white solid 20 (yield: 79.1%): HRMS (ESI) calculated for C48H63N4O12(M+H+), 887.4442; found, 887.4342.

To a solution of 13 (1.7 g, 5.9 mmol) in 75 mL EtOH and 75 mL toluene was added 5 (885 mg, 4.92 mmol) and paraformaldehyde (1.06 g, 35.3 mmol) at rt. The mixture was heated under reflux for overnight. The mixture was concentrated, and the residue was purified by flash chromatography (FC) (EtOAc/hexane=2/8) to give 850 mg colorless oil 23 (yield: 36%): HRMS (ESI) calculated for C25H41N2O7(M+H+), 481.2914; found, 481.2963.

A mixture of 14 (5.56 g, 17.8 mmol), tert-butyl acrylate (4.58 g, 35.7 mmol), potassium carbonate (K2CO3, 4.92 g, 35.7 mmol), tetrabutylammonium bromide (11.5 g, 35.7 mmol), and palladium acetate (Pd(OAc)2, 217 mg, 0.89 mmol) in 75 mL DMF was deoxygenated by purging into nitrogen for 15 min and then heated at 120° C. for overnight. The mixture was cooled to RT, diluted with 250 mL EtOAc and washed with H2O (60 mL×2) as well as brine (60 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (EtOAc/hexane=2/8) to give 2.15 g colorless oil 24 (yield: 38.7%): HRMS (ESI) calculated for C19H22NO3(M+H+), 312.1600; found, 312.1672.

A mixture of 24 (2.15 g, 6.89 mmol) and Pd/C (430 mg) in 50 mL MeOH was stirred at rt under H2for 4 h. The resulting mixture was filtered and the filtrate was concentrated to give 1.54 g colorless oil 25 (yield: 100%): HRMS (ESI) calculated for C12H18NO3(M+H+), 224.1287; found, 224.1208.

To a solution of 25 (2.15 g, 9.6 mmol) in 50 mL H2O was added NaOH (422 mg, 10.56 mmol) and paraformaldehyde (1.73 g, 57.6 mmol). After stirring at 90° C. for 3 h, the mixture was cooled with ice-bath. The pH was adjusted to 7 with 1 N HCl. The solvent was removed in vacuo. The residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 1.3 g white solid 26 (yield: 53.3%): HRMS (ESI) calculated for C13H20NO4(M+H+), 254.1392; found, 254.1436.

To a solution of 26 (153 mg, 0.6 mmol) in 5 mL chloroform was added phosphorus tribromide (81.4 mg, 0.3 mmol) dropwise under ice-bath. The mixture was warmed to rt and maintained for 3 h. The resulting mixture was then cooled to 0° C. DIPEA (384 mg, 3 mmol) was added followed by 23 (241 mg, 0.5 mmol). The ice-bath was then removed. The mixture was stirred at rt overnight. The solvent was removed in vacuo and the residue was purified by FC (DCM/MeOH/NH4OH=85/15/1.5) to give 25 mg colorless oil 27 (yield: 7%): HRMS (ESI) calculated for C38H58N3O10(M+H+), 716.4122; found, 716.4169.

To a solution of 27 (23 mg, 0.032 mmol) in 2 mL DMF was added 4-methoxybenzyl (17.4 mg, 0.064 mmol) and Cs2CO3(20.93 mg, 0.064 mmol) at 0° C. The mixture was the warmed to rt and maintained for 4 h. The resulting mixture was then poured into 30 mL EtOAc and washed with H2O (10 mL×2) as well as brine (10 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 25 mg clear oil 28 (yield: 81.5%): HRMS (ESI) calculated for C54H74N3O12(M+H+), 956.5272; found, 956.5240.

To a solution of 28 (25 mg, 0.026 mmol) in 1 mL MeOH was added 1 mL NaOH (1 N). After stirred at rt for 4 h, 1 N HCl was added to the mixture till pH=4-5. The resulting mixture was then extracted with EtOAc (10 mL×3). The organic layer was collected, washed with brine (10 mL), dried by Na2SO4and filtered. The filtrate was concentrated to give 23 mg white solid. 2 mL DMF was then added to the residue, followed by Glu-NH—CO—NH-Lys(Ahx)-NH2(10, 19.1 mg, 0.032 mmol), N,N′-dicyclohexylcarbodiimide (EDCI, 7.57 mg, 0.040 mol), N-Hydroxybenzotrizole (HOBt, 6.7 mg, 0.040 mmol), and

To a solution of 32 (565 mg, 2.54 mmol) in 30 mL EtOH was added 13 (880 mg, 3 mmol) and paraformaldehyde (762 mg, 25.4 mmol) at rt. The mixture was heated under reflux for 6 h. The mixture was concentrated, and the residue was purified by flash chromatography (FC) (DCM/MeOH/NH4OH=90/9/1) to give 900 mg colorless oil 33 (yield: 67.7%): HRMS (ESI) calculated for C28H47O7(M+H+), 523.3383; found, 523.3484.

To a solution of 17 (150 mg, 0.71 mmol) in 5 mL chloroform was added phosphorus tribromide (95.6 mg, 0.35 mmol) dropwise under ice-bath. The mixture was warmed to rt and maintained for 3 h. The resulting mixture was then cooled to 0° C. DIPEA (547 mg, 4.24 mmol) was added followed by 33 (295 mg, 0.57 mmol). The ice-bath was then removed. The mixture was stirred at rt overnight. The solvent was removed in vacuo and the residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 120 mg colorless oil 34 (yield: 29.6%): HRMS (ESI) calculated for C38H58N3O10(M+H+), 716.4122; found, 716.4241.

To a solution of 34 (120 mg, 0.17 mmol) in 5 mL DMF was added 4-methoxybenzyl (105 mg, 0.67 mmol) and Cs2CO3(217.8 mg, 0.67 mmol) at 0° C. The mixture was the warmed to rt and maintained for 6 h. The resulting mixture was then poured into 30 mL EtOAc and washed with H2O (10 mL×2) as well as brine (10 mL). The organic layer was dried by Na2SO4and filtered. The filtrate was concentrated, and the residue was purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 80 mg colorless oil 35 (yield: 49.8%): HRMS (ESI) calculated for C54H74N3O12(M+H+), 956.5272; found, 956.5322.

To a solution of 35 (80 mg, 0.084 mmol) in 2 mL MeOH was added 2 mL NaOH (1 N). After stirred at rt for 4 h, 1 N HCl was added to the mixture till pH=4-5. The resulting mixture was then extracted with EtOAc (20 mL×3). The organic layer was collected, washed with brine (20 mL), dried by Na2SO4and filtered. The filtrate was concentrated to give 65 mg white solid. 4 mL DMF was then added to the residue, followed by Glu-NH—CO—NH-Lys(Ahx)-NH2(10, 41.4 mg, 0.069 mmol), N,N′-dicyclohexylcarbodiimide (EDCI, 19.7 mg, 0.104 mol), N-Hydroxybenzotrizole (HOBt, 17.5 mg, 0.104 mmol), and DIPEA (26.7 mg, 0.207 mmol). After stirred at rt overnight, the mixture was diluted with EtOAc (30 mL), washed with H2O (10×2 mL) and brine (10 mL), dried over Na2SO4, concentrated and purified by FC (DCM/MeOH/NH4OH=90/9/1) to give 20 mg clear oil 36 (yield: 15.6%): HRMS (ESI) calculated for C83H126N7O19(M+H+), 1524.9108; found, 1524.9266.

Gallium-68 eluted in 0.05 N HCl solution was obtained from a68Ge/68Ga generator (iTG, Germany). To prepare the new ligands with HBED-PSMA derivatives as precursors for68Ga labeling, stock solutions of 1 mg in 1 mL 0.1 N NaOAc were prepared and used for each radiolabelling study. Labeling of68Ga was performed after adding68Ga solution and 2N NaOAc solution to ligands. Optimal reaction parameters were determined through various pH levels (2-7) and at a ligand concentration ranging from 0.6-3.0 μM. For in vivo studies, a higher amount of radioactivity of68Ga labeled agents was needed. The labeling was performed in aq. NaOAc buffer (120 μL, 2.0 M) by adding a ligand solution (20 μL, 1 mg/mL) to68Ga solution (4 mL in 0.05 N HCl). The final pH of the solution was 4.10.

Influence of other metal ions on labeling of [68Ga]1a-g, 2, 3 and 4a-b was tested by performing the optimized labeling reaction in the presence of various potential metal contaminants, such as Zn+2, Fe+3, Cu+2and Sn+2. Labeling was performed in aq. NaOAc buffer (30 μL, 0.2 M) by combining the ligand solution (5 μL, 0.1 mg/mL),68Ga solution (100 μL in 0.05 N HCl) and 15 μL of stock solution of the respective metal chloride necessary to obtain the desired final contaminant concentration.

Radiolabeling yields were determined after maintaining the reaction mixture at room temperature for 10 min. Radiochemical yields for [68Ga]1a-g, 2, 3 and 4a-b, were determined by HPLC. The HPLC system was developed using a Gemini C18 column (solvent A: MeOH; solvent B: 0.1% TFA in water) with the gradient: 0-6 min (0-100% A); flow rate 2 mL/min. The68Ga complexation of all ligands, [68Ga]1a-g, 2, 3 and 4a-b, resulted in high radiochemical yields of 90-99% after 10 min reaction time at room temperature. As a consequence, radiotracers were subsequently used for in vitro and in vivo experiments without further purification.

A proper metal ion, such as Lu(III) chloride, can be identified for selective radiolabeling of the DOTA moiety of compound 1g based on difference in the metal's complexing capability and stability constants for metal complexes with DOTA and HBED. The conditions for the selective radiolabeling can be routinely optimized under a similar reaction condition as described above for68Ga(III), except that the reception required heating the reaction mixture of177Lu(III) and the ligand, 1g, at 95° C. for 30 min. The reaction for making [177Lu]1g proceeded smoothly with an excellent radiochemical yield (>99%).

Preparation of the intermediate compound 43 was based on the following chemical reactions (Scheme 18).

Preparation of compound 5a was based on the following chemical reactions (Scheme 19).

Preparation of compound 5b was based on the following chemical reactions (Scheme 20).

0.5-1 mL eluant in 0.05 M HCl of68Ge/68Ga generator (ITG) and 25 μL 2 NaOAc were added and mixed with the precursor 5b (2-4 nmol) and incubated at 60° C. After 10 min, labeling efficiency and radiochemical purity were determined using Radio-HPLC. Radiochemical purity of68Ga-labelled conjugate was ≥98%. Therefore, the tracer was diluted and used in vitro and in vivo experiments without further purification. Specific activities of the68Ga-labeled PSMA inhibitors were 500 to 1000 Ci/mmol. Analytical reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a Luna C18 (5 μm, 150×4.6 mm) column using an Agilent gradient HPLC System. The [68Ga]P16-093 was eluted applying different gradients of 0.1% (v/v) trifluoroacetic acid (TFA) in H2O and 0.1% TFA (v/v) in MeOH at a constant flow of 2 mL/min (0-6 min, from 100% H2O with 0.1% TFA to 100% MeOH with 0.1% TFA and then back to 100% H2O with 0.1% TFA 6-8 min). The radiolabeling yields were consistently >90% and radiochemical purity >98%.

Compounds 5c and 5d were prepared based on the following chemical reactions (Scheme 21).

Compound 5e was prepared based on the following chemical reactions (Scheme 22).

Synthesis of Compound 49

Synthesis of Compound 5e

Radiolabeling of 5e can be produced by the scheme describe below (Scheme 23).

Compound 5f was prepared based on the following chemical reactions (Scheme 24). Radiolabeling of 5f can be performed by known methods.

68Ga Labeling of 5a and 5b

The68Ge/68Ga-generator (ITG, Germany) was eluted with 4 mL of 0.01N HCl. Typically, 2 nmol of 5a or 5b was added to a mixture of 25 μL 2 N NaOAc and 500 μL [68Ga]GaCl3eluate. The pH of the labelling solution was adjusted using various strength of NaOAc solution. The reaction mixture was incubated for 10 min at 90° C. for 5a and at room temperature for 5b. The radiochemical purity (RCP) was determined via analytical RP-HPLC.

Labeling with [68Ga]GaCl3typically yields more than 97% radiochemical purity both 5a and 5b. The effects of ligand amount, time, pH and temperature on labeling were tested.

5b was labeled quantitatively with [68Ga]GaCl3in the condition of pH 3.2˜4.6, as low as 2 nmole of ligand and longer than 4 min at room temperature. For the labeling of 5a, heating at 70-90° C. for 5 min was needed.

Biological Evaluation

In Vitro Competitive Binding Assay to Determine IC50to PSMA

In order to determine the binding affinity, in vitro competitive binding assays were performed. The LNCaP cells were incubated with 150,000 cpm of [125I]MIP-1095 in the presence of 10 different concentrations of competing drugs. After incubation at 37° C. for 1 h, the bound and free radioactivity were separated by vacuum filtration through GF/B filter paper using a Brandel M-24R cell harvester followed by washing twice. Non-specific binding was defined with 10 μM PMPA. The cell bound radioactivity was measured with a gamma counter, 2470 Wizard2(Perkin-Elmer, IL). The IC50values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software).

The PSMA binding affinities were determined in a competitive binding assay using LNCaP human prostate carcinoma cells and the known high affinity PSMA ligand, [125I]MIP-1095 as the radioligand. The IC50values for the metal-free PSMA-inhibiting ligands and known PSMA inhibitors are summarized in Table 1. Data are expressed as mean±SD (n=4).

Compound 5b has a little improved affinity to PSMA-11 with IC50values of 11.6±5.2 nM and 16.6±2.4 nM, respectively. Known PSMA inhibitors, ZJ-43 and 2-PMPA showed much lower binding affinities than compound 5b. Introduction of gallium into 5b did not cause a change in inhibitory activity of compound 5b, demonstrating higher binding affinity to PSMA comparable to the unchelated compound.

In Vitro Binding Signals of68Ga Labeled Ligands

To compare the binding affinity and specificity of [68Ga]labeled ligands, cell binding studies with hot ligands were performed. 100 μL of freshly harvested PSMA cells (3 different cell numbers: 4×105, 2×105, 1×105) were incubated with 100 μL hot ligand and 50 μL PBS for TB or 50 μL 1a (PSMA11) (10 μM) for non-specific binding (NSB). After incubation at 37° C. for 60 min, the cell bound fractions were collected using a cell harvester (Brandel, MD). After washing twice with 5 mL ice-cold washing buffer, the cell-bound radioactivity was measured with a gamma counter (Wizard, Perkin Elmer).

All tracers ([68Ga]1a-g, 2, 3, 4a-b and 5a-b) showed specific binding to LNCaP tumor homogenates (Table 2). However, [68Ga]2 and [68Ga]3 showed high nonspecific binding and lower specific binding. The specific bindings of [68Ga]1b-g, [68Ga]4a, [68Ga]4b and [68Ga]5b were comparable to that of the known compound, [68Ga]1a (PSMA11). The results suggest that these new HBED-PSMA derivatives may be useful imaging agents for PSMA expressing tumors.

Cell Uptake Comparisons

Cell uptake studies were performed using PSMA expressing LNCaP cells. Cells were grown in 6 well plates for 2 days. After incubation with68Ga-labeled ligands for 1 hr at 37° C., media were removed. After washing twice with 3 mL PBS buffer, cells were lysed with 0.1 N NaOH. Lysed cells were wiped with filter paper and radioactivity in filter paper was measured with a gamma counter.

As shown inFIG. 1, most of the tracers, [68Ga]1b-g, and 4a-b, showed better or comparable cell uptakes to [68Ga]1a. The LNCaP cells over express PSMA receptor binding sites, the level of binding, % uptake/well, was an indicator of PSMA binding, the higher the better. [68Ga]1a, a known PSMA imaging agent (PSMA-11), was used as a control. It was found that ([68Ga]1b-g and 4a-b displayed excellent uptake comparable or better than that of [68Ga]1a. However, [68Ga]2 and 3, (indicated by arrows) the di-pyridyl derivatives, showed low cell uptakes, suggesting that these two ligand displayed the least binding under the assaying conditions. It is likely that [68Ga]2 and 3 are not stable in the test media.

In Vitro Autoradiography of LNCaP Tumor and Mouse Kidney Sections

LNCaP tumor and mouse kidneys were cut at 20 μm on a cryostat, thaw-mounted onto slides. Slides are incubated with radiotracers (3 μCi/ml) in PBS for 30 min and washed with PBS twice for 3 min each. After drying, the slides put into a plate for exposure for 30 min. Images were acquired with Typhoon FLA 7000 (GE Healthcare).

To validate the PSMA binding, in vitro autoradiography studies using LNCaP tumor and mouse kidney sections were carried out. Autoradiography studies demonstrated all radioligands have good binding to LNCaP tumors and kidneys. Incubation with 2-PMPA, a known PSMA inhibitor, blocked radiotracers' binding to tumor and kidney. These data confirm that all tracers ([68Ga]1a-g, 2, 3 and 4a-b) bind to PSMA in prostate tumors and PSMA expressed in kidneys.

FIGS. 2A-2Kshow in vitro autoradiography of LNCaP tumor (left side) and mouse kidney sections (right side). The new [68Ga]1b-g, 2, 3 and 4a-b, target compounds, displayed high binding to PSMA expressed in LNCaP tumors and mouse kidneys. These new PSMA target compounds, display high uptake in the sections. [68Ga]1a (PSMA-11) was used as a control.

Small Animal Imaging with a microPET

Male athymic mice (CD-1 nude, 5-6 weeks old) were obtained from Charles River, and were allowed to acclimatize at the vivarium for 1 week prior to implanting tumors. Mice were provided with food and water ad libitum. LNCaP tumors were induced on the left shoulder by sub-cutaneous (s.c.) injection of 5.0×106cells in a 200 μL cell suspension of a 1:1 v/v mixture of media with reconstituted basement membrane (BD Matrigel™, Collaborative Biomedical Products Inc., Bedford, Mass.). Similarly, PC-3 tumors were induced on the right shoulder by s.c. injection of 2.0×106cells. Palpable LNCaP tumors developed after a period of 4-5 weeks.

Dynamic small animal PET (APET) imaging studies of LNCaP (left shoulder) and PC-3 (right shoulder) tumor bearing nude mouse were performed with [68Ga]1a and [68Ga]4a. PET imaging studies were performed on a Phillips Mosaic small animal PET scanner, which has an imaging field of view of 11.5 cm. Under isoflurane anesthesia (1-2%, 1 L/min oxygen), the tumor-bearing nude mouse was injected with 0.5 mCi activity by an intravenous injection into the lateral tail vein. Data acquisition began at 30 min after the injection. Dynamic scans were conducted over a period of 1 h (5 min/frame; image voxel size 0.5 mm3). Mouse was visually monitored for breathing, and a heating pad was used to maintain body temperature throughout the entire procedure. Images were reconstructed and a region of interest (ROI) analysis was performed using AMIDE software (http://amide.sourceforge.net/).

Representative animal PET images of LNCaP xenograft mice between 60 to 75 min after i.v. injection of [68Ga]1a, and [68Ga]4a are shown inFIGS. 3A-3F. Only LNCaP tumor was clearly visualized with all tracers with good tumor-to-background contrasts. PSMA negative tumor, PC-3 did not show any uptakes of radiotracers. The results showed that tumor xenografts, in which high expression of PSMA (LNCaP tumor), showed the highest uptake and retention. These agents also exhibited high kidney uptake and predominant renal excretion.FIGS. 3A-3Fshow sagittal, transaxial and coronal sections of APET images of nude mouse with LNCaP tumor at left shoulder and PC-3 tumor at right shoulder between 60 to 75 min post i.v. injection of [68Ga]1a (FIGS. 3A-3C) and [68Ga]4a (FIG. 3D-3F). The data confirmed that the PSMA positive tumor on the left shoulder displayed high uptake and retention at 60 min post i.v. injection.

Cell Binding and Internalization

The cellular uptake and internalization kinetics of the [68Ga]1a, [68Ga]5a and [68Ga]5b were determined using PSMA-expressing LNCaP cells. Furthermore, to be able to discriminate between total cellular activity (sum of membrane-associated and internalized activity) and internalized activity, all incubations were followed by a washing step with mild acid at 4° C. to remove specifically cell-surface bound radioligand by displacement.

LNCaP cells (in 6-well plates in triplicates) were incubated in RPMI-1640 medium with [68Ga]1a, [68Ga]5a or [68Ga]5b for 0-2 h at 37° C. At the indicated time, the medium was removed and the cells were washed twice and then incubated with a mild acid buffer (50 mM glycine, 150 mM NaCl, pH 3.0) at 4° C. for 5 min. The supernatant (containing cell surface-bound radioactivity) was pooled and the cell pellet (containing internalized radioactivity) was collected with filter paper and then radioactivity in supernatant and cell pellet was counted on a gamma counter.

LNCaP cells were incubated with [68Ga]1a, [68Ga]5a and [68Ga]5b for up to 2 h at 37° C. to determine whether the compound is internalized by endocytosis. The cells were then washed with a mild acid buffer to remove extracellularly bound compound. The cell surface binding and the acid-insensitive binding, or internalized compound, to LNCaP cells are shown in Table 3. The cellular binding and internalization of [68Ga]1a, [68Ga]5a and [68Ga]5b showed a time-dependent increase over the time and reached plateau between 60 and 90 min. The internalized activity of [68Ga]5b was much higher than those of [68Ga]1a and [68Ga]5a.

FIG. 4shows the kinetics of [68Ga]5b uptakes in PSMA expressing LNCaP cells. Non specific binding (NSB) was evaluated by blocking with 20 μM PMPA. Specificity of cell uptake (SB) was calculated by subtracting the respective signals resulting from PMPA blocking. Values are expressed as % of applied radioactivity bound to 106cells. The data clearly suggested that the non specific binding (NSB) was extremely low and the binding of [68Ga]5b to the cells was contribution from the specific binding to PSMA.

Biodistribution of [68Ga]Labeled Ligand in PSMA Positive Tumor Bearing Nude Mice

In a dish contained 5×106cells of LNCaP in 50% Matrigel (Becton Dickinson, Heidelberg, Germany) were subcutaneously inoculated into the left shoulder of male 5- to 6-week-old CD-1 nu/nu mice (Charles River Laboratories). The tumors were allowed to grow for 8 weeks until approximately 0.5 cm3in size.

The68Ga-radiolabeled compounds were injected via tail vein (25 μCi per mouse; 0.1-0.2 nmol). At 1 h after injection, the animals were sacrificed. Organs of interest were dissected and weighed. The radioactivity was measured with a gamma counter and calculated as % ID/g.

[68Ga]5b showed the high tumor and kidney uptake. In addition, [68Ga]5b was cleared in other organs much better than [68Ga]1a. Although [68Ga]5a demonstrated lower tumor uptake, but its kidney retention was the lowest, which is desirable for a therapeutic drug. For example, [177Lu]5a can be used as a therapeutic drug.

Biodistribution of [68Ga] Radiotracers in Normal Mice

Normal CD-1 male mice were injected via the tail vein with 35 μCi of [68Ga] radiotracers (0.2 nmole of ligand). Each four mice were sacrificed by cervical dislocation at 2, 30, 60 and 120 min p.i. All organs were removed and blood was also collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (Wizard, Perkin Elmer). The % ID/g was calculated by comparison with samples of a standard dilution of the initial dose. All measurements were corrected for decay.

The kidneys and spleen are the most prominent organs in the biodistribution, because PSMA is expressed naturally in kidneys and spleen in mouse and because [68Ga]5b is also excreted through kidneys. The tracer was cleared quickly and well except kidneys and spleen. No significant tracer activity is seen in other tissue.

Additional biodistribution studies for [177Lu]1g were performed in normal mice. The lutetium-177 is an isotope with a longer half-life (T1/2, 6.73 days) and weak beta emission for radiotherapy. Initial uptakes in the kidneys, as an indicator for PSMA binding, were comparable to that of [68Ga]1g, suggesting that the Lu-DOTA has no effect on the tumor targeting. The results suggested that both68Ga and177Lu can be used to label 1g, and [68Ga]1g and [′77Lu]1g will retain a high tumor PSMA targeting.

Small Animal microPET Imaging in Tumor Bearing Nude Mice

To illustrate the usefulness of [68Ga]5b as a PET tracer for PSMA imaging, microPET studies with tumor bearing nude mice were performed. This study was performed in a small animal imaging facility Male CD-1-nu/nu mice were implanted subcutaneously with 5×106 LNCaP cells and PC-3 cells. When the tumors reached approximately 5-10 mm in diameter, the mice were used for microPET imaging. Mice bearing LNCaP tumor and PC-3 tumors were injected via the tail vein with ˜0.5 mCi of [68Ga]5b. Imaging studies were carried out under general anesthesia of the animals, induced with inhalation of 10% and maintained with inhalation of 6.5% isoflurane in 30% oxygen/air. Animals were positioned prone in the scanner. Whole body scan was performed for 15 min from 60 min post-injection of radiotracers. PET images were generated using the AMIDE software. MicroPET images obtained in LNCaP and PC-3 tumor xenografts from 60 min to 75 min after injection of [68Ga]5b are shown inFIGS. 5A-5C.

FIGS. 5A, 5B, and 5Cshow microPET images of tumor (LNCaP PSMA+ and PC-3 PSMA−) bearing mice between 60 min to 75 min after injection of [68Ga]5b.

Intense [68Ga]5b uptake was seen only in the kidneys, bladder and PSMA positive LNCaP tumor. PSMA negative PC-3 tumors did not show any uptakes of [68Ga]5b. The intense renal uptake was partially due to specific binding of the radiotracer to proximal renal tubules as well as to excretion of this hydrophilic compound.

Blocking of [68Ga]5b with 2-PMPA was performed in the same mouse. CD-1 nu/nu mouse bearing LNCaP and PC-3 tumor xenografts was injected with [68Ga]5b alone or with 2-PMPA (2 mg/kg), a structurally unrelated PSMA inhibitor, to demonstrate that binding to LNCaP tumors was specific to PSMA.

Representative animal PET images of LNCaP xenograft mice between 60 to 75 min after i.v. injection of [68Ga]1a, and [68Ga]5b are shown inFIGS. 5A-5C. Only LNCaP tumor was clearly visualized with all tracers with good tumor-to-background contrasts. PSMA negative tumor, PC-3 did not show any uptakes of radiotracers. The results showed that tumor xenografts, in which high expression of PSMA (LNCaP tumor), showed the highest uptake and retention. These agents also exhibited high kidney uptake and predominant renal excretion.FIGS. 5A-5Cshow sagittal, transaxial and coronal sections of APET images of nude mouse with LNCaP tumor at left shoulder and PC-3 tumor at right shoulder between 60 to 75 min post i.v. injection of [68Ga]1a and [68Ga]5b. The data confirmed that the PSMA positive tumor on the left shoulder displayed high uptake and retention at 60 min post i.v. injection. Uptake was high in PSMA expressing kidney and LNCaP tumor xenograft. Also evident was renal excretion through the bladder. [68Ga]5b localized to the PSMA-expressing LNCaP tumor, but not to the PSMA-deficient PC-3 tumor. Further, binding was abolished in the LNCaP tumor and kidney tissue when a 2 mg/kg dose of 2-PMPA was co-injected, indicating that binding was indeed saturable and specific to PSMA. These results clearly indicate that [68Ga]5b is suitable as a tracer for PSMA imaging in prostate cancer with PET.

REFERENCES

Abbreviations