WO2016065145A2 - Psma targeted reversed carbamates and methods of use thereof - Google Patents

Psma targeted reversed carbamates and methods of use thereof Download PDF

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WO2016065145A2
WO2016065145A2 PCT/US2015/056914 US2015056914W WO2016065145A2 WO 2016065145 A2 WO2016065145 A2 WO 2016065145A2 US 2015056914 W US2015056914 W US 2015056914W WO 2016065145 A2 WO2016065145 A2 WO 2016065145A2
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group
imaging
compound
psma
cancer
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PCT/US2015/056914
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French (fr)
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WO2016065145A3 (en
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Martin G. Pomper
Ronnie MEASE
Sangeeta Ray
Ying Chen
Xing Yang
Georgio ATTARDO
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The Johns Hopkins University
Avid Radiopharmaceuticals
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Publication of WO2016065145A3 publication Critical patent/WO2016065145A3/en

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    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
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    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
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    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
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    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
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    • C07C275/06—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton
    • C07C275/16—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to acyclic carbon atoms of an acyclic and saturated carbon skeleton being further substituted by carboxyl groups
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    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
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    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
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    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules

Definitions

  • PSMA Prostate-specific membrane antigen
  • PSMA is among the most intensively targeted biomarkers for imaging metastatic prostate cancer.
  • PSMA is a zinc- dependent metallopeptidase that catalyzes the hydrolysis of a series of N- acylpolygammaglutamate derivatives (Mesters, 2006; Barinka, 2004; Pinto, 1996). It is expressed within certain normal tissues (Ghosh, 2004), but transitions to abundant plasma membrane expression in the epithelium of most prostate cancer and within other solid tumor neovasculature (Rajasekaran, 2005). PSMA membrane expression is associated with metastasis (Chang, 2001), castration resistance (Bander, 2005) and progression of prostate cancer (Perner, 2007).
  • FIG.1 those potent scaffolds share common features, namely; a) a pentanedioic acid as a glutamate mimic to fit within the S1’ binding pocket of the PSMA active site; and b) a zinc-binding group to interact with the catalytic zinc atom at the PSMA active site.
  • a substituent (R) can reside either within the S1 binding pocket or within a void in the protein that extends to the surface.
  • Several of these have been translated to phase 0-1 clinical trials, where they have enabled visualization of both primary and metastatic bone and soft-tissue lesions due to prostate cancer.
  • PSMA-binding carbamate The only PSMA-binding carbamate reported is gly-amino-pentanedioic acid 1 (Wang, 2013) displayed low binding affinity to PSMA, most likely due to the absence of productive binding within the S1 pocket, similar to ureido compound 8.
  • gly-amino-pentanedioic acid 1 Wang, 2013
  • a new class of potent PSMA inhibitors based on the reversed carbamate scaffold to maintain glutamate and S1 pocket side chain geometry and for putative binding to zinc have been reported.
  • Reversed carbamate scaffolds may complement the existing urea and other scaffolds upon which inhibitors, imaging and therapeutic agents targeting PSMA have been based.
  • fluorescent-linker-reversed carbamate based PSMA inhibitors have also been investigated.
  • Targeted fluorescent PSMA binding compounds may find utility in fluorescence guided surgery and biopsy of PSMA positive tumors and tissues, the former providing visual confirmation of complete removal of PSMA containing tissue.
  • reversed carbamate conjugates of photosensitizing dyes also provide PSMA targeted photodynamic therapy agents.
  • Z is tetrazole or CO 2 Q;
  • Q is H or a protecting group;
  • t is an integer selected from the group consisting of 1, 2, 3, 4;
  • p 2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p 2 is 2 or 3, each R 1 is the same or different;
  • p 1, p 3 , and p 4 are each independently 0 or 1;
  • m 1 and m 2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
  • V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,– NRC(S)–, and–OC(O)–;
  • A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which can optionally comprise one or more radioactive isotope suitable for imaging and /or radiotherapy, or a photosensitizing dye suitable for imaging and/or photodynamic therapy.
  • the presently disclosed subject matter provides a method for imaging one or more prostate-specific membrane antigen (PSMA)-expressing tumors or cells, the method comprising contacting the one or more tumors or cells with an effective amount of a compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), and making an image, wherein the compound of formula (I) further comprises a radioactive isotope or a photosensitizing dye suitable for imaging.
  • PSMA prostate-specific membrane antigen
  • the presently disclosed subject matter provides a method for treating or preventing a disease or condition associate with one or more PSMA expressing tumors or cells, the method comprising administering at least one compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), to a subject in an amount effective to treat or prevent the disease or condition wherein the compound of formula (I) further comprises a radioactive isotope suitable for radiotherapy or a photosensitizing dye suitable for photodynamic therapy.
  • FIG.1 shows scaffolds available for synthesis of small-molecule PSMA inhibitors; those potent scaffolds share common features, namely; a) a pentanedioic acid (green) as a glutamate mimic to fit within the S1’ binding pocket of the active site; and b) a zinc-binding group (blue) to interact with the catalytic zinc atom at the PSMA active site (blue); a substituent (R) can reside either within the S1 binding pocket or within a void in the protein that extends to the surface;
  • FIG.2A and FIG.2B show (FIG.2A) urea- and carbamate-based PSMA binding compounds; (FIG.2B) binding affinities of urea- and carbamate-based PSMA binding compounds and compounds of the presently disclosed subject matter;
  • FIG.3A and FIG.3B show (FIG.3A) the lysine- reversed carbamate scaffold used to design compounds of the presently disclosed subject matter: oxypentanedioic acid (OPA) corresponding to a "reversed" carbamate scaffold; (FIG.3B) general structure of OPA, high binding affinity structure of OPA and versatile intermediate of OPA for functionalization;
  • OPA oxypentanedioic acid
  • FIG.3B general structure of OPA, high binding affinity structure of OPA and versatile intermediate of OPA for functionalization
  • FIG.4 is a synthesis scheme for the presently disclosed reversed carbamates
  • FIG.5 shows preparative Radio-HPLC chromatograms of 2-[18F]fluoro-4- bromo-benzaldehyde [18F]29b; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 54/46/0.1 water/acetonitrile/TFA, 4 mL/m; double radioactive peak at 18- 19.5 min is due to saturation of the radioactivity detector;
  • FIG.6 shows preparative Radio-HPLC chromatogram of 2-[ 18 F]fluoro-4-iodo- benzaldehyde [ 18 F]29c; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 54/46/0.1 water/acetonitrile/TFA, 4 mL/m; double radioactive peak at 21-23 minutes is due to saturation of the radioactivity detector;
  • FIG.7 shows preparative Radio-HPLC chromatograms of N-succinimidyl 2- [ 18 F]fluoro-4-bromobenzoate [ 18 F]30b; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 50/50/0.1 water/acetonitrile/TFA, 4 mL/m;
  • FIG.8 shows preparative Radio-HPLC chromatogram of N-succinimidyl 2- [ 18 F]fluoro-4-iodobenzoate [ 18 F]30c; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 50/50/0.1 water/acetonitrile/TFA, 4 mL/m;
  • FIG.9 shows preparative Radio-HPLC chromatogram of (S)-2-((((S)-5-(4- bromo-2-[ 18 F]fluorobenzamido)-1-carboxypentyl)carbamoyl)oxy)pentanedioic acid [ 18 F]23 ([ 18 F]XY-52); 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 4 mL/m;
  • FIG.10 shows quality control analytical radio-HPLC chromatogram of purified (S)-2-((((S)-5-(4-bromo-2-[ 18 F]fluorobenzamido)-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid [ 18 F]23 ([ 18 F]XY-52); 4.6 X 150 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 1 mL/m;
  • FIG.11 shows preparative Radio-HPLC chromatogram of (S)-2-((((S)-5-(4- iodo-2-[ 18 F]fluorobenzamido)-1-carboxypentyl)carbamoyl)oxy)pentanedioic acid [ 18 F]24 ([ 18 F]XY-47);10 X 250 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 4 mL/m;
  • FIG.12 show quality control analytical radio-HPLC chromatogram of purified (S)-2-((((S)-5-(4-iodo-2-[ 18 F]fluorobenzamido)-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid [ 18 F]24 ([ 18 F]XY-47); 4.6 X 150 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 1 mL/m;
  • FIG.13A, FIG.13B, FIG.13C, and FIG.13D show docking of 9 (YC-I-26), 12 (XY-20), and 13 (XY-48) to PSMA;
  • FIG.13A urea 9 (YC-I-26);
  • FIG.13B Lys-NPA carbamate 12 (XY-20);
  • FIG.13C Lys-OPA reversed carbamate 13 (XY- 48);
  • FIG.13D overlay of 9 (YC-I-26) (red), 12 (XY-20) (blue) and 13 (XY-48) (green);
  • FIG.14A and FIG.14B show: (FIG.14A) maximum intensity projections (MIPs) after PET imaging with the [ 18 F]13 ([ 18 F]XY-48) compound in male NOD- SCID mice; no blocking study was done in this instance; and (FIG.14B) standardized uptake values generated from the images;
  • FIG.15A, FIG.15B, FIG.15C, and FIG.15D show sequential Micro-PET (coronal view) images of [ 18 F]13 ([ 18 F]XY-48) in a male SCID mouse containing PSMA+ PC-3 PIP and PSMA- PC-3 flu tumor xenografts at (FIG.15A) 10 minutes, (FIG.15B) 30 minutes , (FIG.15C) 60 minutes and (FIG.15D) 120 minutes after injection;
  • FIG.16 shows maximum intensity projections (MIPs) after PET imaging with the [ 18 F]23 ([ 18 F]XY-52 and [ 18 F]24 ([ 18 F]XY-47) compounds in male NOD-SCID mice;
  • FIG.17 shows TLC analysis of Metabolism of [ 125 I]31 ([ 125 I]XY-26) and [ 125 I]32 ([ 125 I]XY-57) in PC 3 PIP (PSMA+) and PC3 flu (PSMA-) cells;
  • FIG.18 shows PSMA urea-binding scaffolds upon which radiohalogenated PSMA binding radiotherapeutics can be built: the lysine-glutamate urea, cysteine- glutamate urea, and glutamate-glutamate urea;
  • FIG.19 shows a glutamate-OPA reversed carbamate and a cysteine-OPA reversed carbamate scaffolds used to design compounds of the presently disclosed subject matter
  • FIG.20 shows the cysteine-glutamate urea scaffolds used for PSMA binding and imaging for over 10 years: C-11 labeled DCMC, F-18 labeled DCFBC both for PET imaging with the latter currently in use in patients, and I-125 labeled DCIBC for SPECT imaging and or radiotherapy;
  • FIG.21 shows two specific examples of IRDye800CW-linker-ureas
  • FIG.22A and FIG.22B show (FIG.22A) the structure of compound YC-XIX- 33
  • FIG.22B shows (FIG.22A) the structure of compound YC-XIX- 33
  • FIG.22B images of two mice bearing PSMA+PC3 PIP tumor after injection of YC-XIX-33 (1nmole in PBS) into the tail vein at 10 min, 30 min, 1h, 2h, 4h, and 24h using the Pearl Impulse Imager (excitation at 785 nm and emission at 800 nm).
  • the presently disclosed subject matter provides“reversed” carbamate based scaffolds that have high binding affinity to PSMA. These scaffolds can be radiolabeled and used to image cells and tumors that express PSMA. Some advantages to the presently disclosed methods include, but are not limited to, lower renal uptake of the presently disclosed compounds compared with existing urea-based PSMA-targeted radiotracers, as well as more rapid renal clearance as compared to compounds known in the art.
  • the presently disclosed“reversed” carbamate based scaffolds complement existing urea and other scaffolds upon which inhibitors, imaging, and therapeutic agents targeting PSMA have been based.
  • Versatile intermediates for“reversed” carbamate scaffolds can be functionalized in one or two steps toward PET imaging agents.
  • the presently disclosed“reversed” carbamate based scaffolds are conjugated with near infrared dye molecules and their use in imaging PSMA and PSMA targeted photodynamic therapy agents also are provided.
  • Z is tetrazole or CO 2 Q;
  • Q is H or a protecting group;
  • t is an integer selected from the group consisting of 1, 2, 3, 4;
  • p 2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p 2 is 2 or 3, each R 1 is the same or different;
  • p 1, p 3 , and p 4 are each independently 0 or 1;
  • m 1 and m 2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
  • each R 1 is independently H, C 1 -C 6 alkyl, C 2 -C 12 aryl or C 4 -C 16 alkylaryl
  • R 2 and R 3 are each independently H and CO 2 R 4 , wherein R 4 is selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 12 aryl, and C 4 -C 16 alkylaryl, wherein when one of R 2 or R 3 is CO 2 R 4 , then the other is H
  • V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–
  • A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which can optionally comprise one or more radioactive isotope suitable for imaging and /or radio
  • photosensitizing dye suitable for imaging and/or photodynamic therapy.
  • the compound of formula (I) is selected from the group consisting of:
  • L is a linker that can be present or absent, and has the following general structure:
  • L is selected from the group consisting of:
  • A has the following general structure:
  • the compound of formula (I) is selected from the group consisting of:
  • the compound of formula (I) is:
  • the photosensitizing dye suitable for imaging and/or photodynamic therapy is a fluorescent dye moiety which emits in the visible or near infrared spectrum, wherein the fluorescent dye moiety comprises carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660,
  • AlexaFluor680 AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
  • the fluorescent dye moiety is selected from the
  • the compound of formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the presently disclosed subject matter provides a method for imaging one or more prostate-specific membrane antigen (PSMA)- expressing tumors or cells, the method comprising contacting the one or more tumors or cells with an effective amount of a compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic) and making an image, the compound of formula (I) comprising:
  • Z is tetrazole or CO 2 Q;
  • Q is H or a protecting group;
  • t is an integer selected from the group consisting of 1, 2, 3, 4;
  • p 2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p 2 is 2 or 3, each R 1 is the same or different;
  • p 1, p 3 , and p 4 are each independently 0 or 1;
  • m 1 and m 2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
  • each R 1 is independently H, C 1 -C 6 alkyl, C 2 -C 12 aryl or C 4 -C 16 alkylaryl;
  • R 2 and R 3 are each independently H and CO 2 R 4 , wherein R 4 is selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 12 aryl, and C 4 -C 16 alkylaryl, wherein when one of R 2 or R 3 is CO 2 R 4 , then the other is H;
  • V is selected from the group consisting of– C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–;
  • A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for imaging, or a photosensitizing dye suitable for imaging.
  • Contacting means any action which results in at least one compound comprising the imaging agent of the presently disclosed subject matter physically contacting at least one PSMA-expressing tumor or cell. Contacting can include exposing the cell(s) or tumor(s) to the compound in an amount sufficient to result in contact of at least one compound with at least one cell or tumor.
  • the presently disclosed methods may include one or more radioactive isotopes capable of emitting radiation suitable for detection with PET or SPECT or a photosensitizing dye suitable for fluorescent optical imaging.
  • the image is made using PET the image is made using positron emission tomography (PET) and the radiohalogen is selected from the group consisting of 18 F and 124 I.
  • PET positron emission tomography
  • the image is made using Single-photon emission computed tomography (SPECT) and the radiohalogen is selected from the group consisting of 77 Br, 131 I, 125 I, and 123 I.
  • SPECT Single-photon emission computed tomography
  • the image is made using florescent optical imaging and the photosensitizing dye suitable for imaging is selected from the group consisting of: ;
  • the one or more PSMA-expressing tumors or cells is selected from the group consisting of: a prostate tumor or cell, a metastasized prostate tumor or cell, a lung tumor or cell, a renal tumor or cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor or cell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor or cell, a germ cell, a pheochromocytoma, an esophageal tumor or cell, a stomach tumor or cell, and combinations thereof.
  • the one or more PSMA-expressing tumors or cells is a prostate tumor or cell.
  • the one or more PSMA-expressing tumors or cells is in vitro, in vivo, or ex vivo.
  • the method can be practiced in vitro or ex vivo by introducing, and preferably mixing, the compound and cell(s) or tumor(s) in a controlled environment, such as a culture dish or tube.
  • the method can be practiced in vivo, in which case contacting means exposing at least one cell or tumor in a subject to at least one compound of the presently disclosed subject matter, such as administering the compound to a subject via any suitable route.
  • the one or more PSMA-expressing tumors or cells is present in a subject.
  • the subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term“subject.”
  • a“subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal (non-human) subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like.
  • mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; cap
  • an animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a“subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease.
  • the terms “subject” and“patient” are used interchangeably herein.
  • the subject is human. In other embodiments, the subject is non-human.
  • a detectably effective amount of the imaging agent of the presently disclosed methods is administered to a subject.
  • a detectably effective amount is defined as an amount sufficient to yield an acceptable image using equipment which is available for clinical use.
  • a detectably effective amount of the imaging agent may be administered in more than one injection.
  • the detectably effective amount of the imaging agent can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, the dosimetry, and instrument and film- related factors. Optimization of such factors is well within the level of skill in the art.
  • the“effective amount” of an active agent refers to the amount necessary to elicit the desired biological response.
  • the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the target tissue, and the like.
  • the compound comprising the imaging agent prefferably to localize to the tumor or cell quickly after administration so as to minimize any side effects to the subject. Accordingly, in some embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 60 minutes to about 240 minutes of administration and, in some embodiments, about 60 minutes. In other embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 30 minutes of administration. In still other embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 10 minutes of administration.
  • the compounds of the presently disclosed subject matter are excreted from tissues of the body quickly to prevent prolonged exposure to the radiation of the radiolabeled compound administered to the patient.
  • compounds of the presently disclosed subject matter are eliminated from the body in less than about 24 hours. More preferably, compounds of the presently disclosed subject matter are eliminated from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes.
  • the presently disclosed methods comprise clearance of the compound comprising the imaging agent from the tumor or cell in the subject. At least one advantage of the presently disclosed methods is that, in some embodiments, there is more rapid clearance of the compound comprising the imaging agent from the kidneys than from the tumor of the subject.
  • the compound comprising the photosensitizing dye suitable for imaging is visible at about 4 hours after injection and presents the brightest signal at about 24 hours after injection.
  • the presently disclosed methods use compounds that are stable in vivo such that substantially all, e.g., more than about 50%, 60%, 70%, 80%, or more preferably 90% of the injected compound is not metabolized by the body prior to excretion.
  • the compound comprising the imaging agent is stable in vivo.
  • A“tumor,” as used herein, refers to all neoplastic cell growth and
  • the tumor cells express PSMA, such as prostate tumor cells or metastasized prostate tumor cells.
  • a tumor may be treated by targeting adjacent or nearby cells which express PSMA.
  • vascular cells undergoing angiogenesis associated with a tumor may be targeted.
  • Essentially all solid tumors express PSMA in the neovasculture. Therefore, methods of the presently disclosed subject matter can be used to image nearly all solid tumors including, but not limited to, lung, renal cell, glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germ cell, pheochromocytoma, esophageal, and stomach tumors.
  • certain benign lesions and tissues including, but not limited to, endometrium, schwannoma and Barrett's esophagus, can be imaged according to the presently disclosed methods.
  • the presently disclosed subject matter provides a method for treating or preventing a disease or condition associate with one or more PSMA expressing tumors or cells, the method comprising administering at least one compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), to a subject in an amount effective to treat or prevent the disease or condition, the compound of formula (I) comprising:
  • Z is tetrazole or CO 2 Q;
  • Q is H or a protecting group;
  • t is an integer selected from the group consisting of 1, 2, 3, 4;
  • p 2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p 2 is 2 or 3, each R 1 is the same or different;
  • p 1, p 3 , and p 4 are each independently 0 or 1;
  • m 1 and m 2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
  • each R 1 is independently H, C 1 -C 6 alkyl, C 2 -C 12 aryl or C 4 -C 16 alkylaryl;
  • R 2 and R 3 are each independently H and CO 2 R 4 , wherein R 4 is selected from the group consisting of H, C 1 -C 6 alkyl, C 2 -C 12 aryl, and C 4 -C 16 alkylaryl, wherein when one of R 2 or R 3 is CO 2 R 4 , then the other is H;
  • V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–;
  • A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for or radiotherapy, or a photosensitizing dye suitable for
  • the radiohalogen suitable for radiotherapy is selected from the group consisting of 80m Br, 77 Br, 125 I, 123 I, 131 I and 211 At.
  • the photosensitizing dye suitable for photodynamic therapy is selected from the group consisting of ;
  • the disease or condition is a prostate cancer, renal cancer, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer,
  • the disease or condition is prostate cancer. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the cancer or the tumor neovasculature.
  • A“cancer” in a subject refers to the presence of cells possessing
  • cancer cells will be in the form of a tumor; such cells may exist locally within a subject, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • a cancer can include, but is not limited to, renal cancer, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, and adenomas.
  • the disease or condition is prostate cancer.
  • a detectably effective amount of the therapeutic agent of the presently disclosed methods is administered to a subject.
  • substituted refers to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • substituent may be either the same or different at every position.
  • the substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
  • R groups such as groups R 1 , R 2 , and the like, or variables, such as “m” and“n”
  • R 1 and R 2 can be substituted alkyls, or R 1 can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • a when used in reference to a group of substituents herein, mean at least one.
  • a compound is substituted with“an” alkyl or aryl, the compound is optionally substituted with at least one alkyl and/or at least one aryl.
  • the group may be referred to as“R-substituted.” Where a moiety is R- substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.
  • a named“R” or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein.
  • certain representative“R” groups as set forth above are defined below.
  • hydrocarbon refers to any chemical group comprising hydrogen and carbon.
  • the hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions.
  • the hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
  • Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, acyclic or cyclic hydrocarbon group, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent groups, having the number of carbon atoms designated (i.e., C 1 -C 10 means one to ten carbons).
  • alkyl refers to C 1-20 inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n- undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.
  • “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • “alkyl” refers, in particular, to C 1-8 straight-chain alkyls.
  • “alkyl” refers, in particular, to C 1-8 branched-chain alkyls.
  • alkyl groups are C 1 -C 6 alkyl groups or C 1 -C 4 alkyl groups.
  • C 1 -C 6 alkyl as used herein means straight-chain, branched, or cyclic C 1 -C 6 hydrocarbons which are completely saturated and hybrids thereof, such as (cycloalkyl)alkyl.
  • C 1 -C 6 alkyl substituents include methyl (Me), ethyl (Et), propyl (including n-propyl (n-Pr, n Pr), iso-propyl (i-Pr, 1 Pr), and cyclopropyl (c- Pr, 0 Pr)), butyl (including n-butyl (n-Bu, n Bu), iso-butyl (i-Bu, 1 Bu), sec-butyl (s-Bu, s Bu), tert-butyl (t-Bu, 1 Bu), or cyclobutyl (c-Bu, 0 Bu)), and so forth.
  • Alkyl groups can optionally be substituted (a“substituted alkyl”) with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • alkyl chain There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as“alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon group, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule.
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(O)R’, - C(O)NR’, -NR’R”, -OR’, -SR, and/or -SO 2 R’.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -NR’R or the like, it will be understood that the terms heteroalkyl and -NR’R” are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R” or the like.
  • (cycloalkyl)alkyl cycloalkyl, and alkyl are as defined above, and the point of attachment is on the alkyl group. This term encompasses, but is not limited to, cyclopropylmethyl, cyclopentylmethyl, and cyclohexylmethyl.
  • the alkyl group may be substituted or unsubstituted.
  • Cyclic and“cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene.
  • cyclic alkyl chain There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group.
  • Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.
  • cycloheteroalkyl or“heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10- member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), and optionally can include one or more double bonds.
  • N nitrogen
  • O oxygen
  • S sulfur
  • P phosphorus
  • Si silicon
  • the cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings.
  • Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • heterocylic refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7- membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring.
  • Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
  • cycloalkyl and“heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and“heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • cycloalkylene and“heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.
  • cycloalkylalkyl refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above.
  • alkyl group also as defined above.
  • examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • Alkyl groups which are limited to hydrocarbon groups are termed“homoalkyl.”
  • alkenyl refers to a monovalent group derived from a C 1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1- methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, and butadienyl.
  • cycloalkenyl refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • alkynyl refers to a monovalent group derived from a straight or branched C 1-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond.
  • Examples of“alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl, and allenyl groups, and the like.
  • alkylene by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally unsaturated and/or substituted with one or more“alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • Exemplary alkylene groups include methylene (–CH 2 –); ethylene (–CH 2 –CH 2 –); propylene (–(CH 2 ) 3 —);
  • An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure.
  • A“lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • heteroalkylene by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH 2 - CH 2 -S- CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroalkylene groups heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like).
  • no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and–R’OC(O)-.
  • aryl means, unless otherwise stated, an aromatic hydrocarbon substituent that can be a single ring or multiple rings (such as from 1 to 3 rings), which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1- pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, ind
  • the term“aryl” when used in combination with other terms includes both aryl and heteroaryl rings as defined above.
  • the terms“arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like).
  • haloaryl is meant to cover only aryls substituted with one or more halogens.
  • heteroalkyl where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g.“3 to 7 membered”), the term“member” refers to a carbon or heteroatom.
  • alkylaryl includes alkyl groups, as defined above, substituted by aryl groups, as defined above.
  • the aryl group may be connected at any point on the alkyl group.
  • C 4 -C 16 alkylaryl includes alkylaryl groups having a total of 4 to 16 carbon atoms, counting the carbon atoms on the alkyl group and aryl group together. Examples of alkylaryl groups include but are not limited to benzyl (phenylmethyl), phenyl ethyl, and naphthylmethyl.
  • the alkylaryl group may be substituted or unsubstituted. Substituents are not counted towards the total number of atoms in the alkylaryl group, so long as the total atoms in the substituent(s) are not larger than the alkylaryl group.
  • a ring structure for example, but not limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure.
  • the presence or absence of the R group and number of R groups is determined by the value of the variable“n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group.
  • the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
  • a dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
  • a substituent bearing a broken bond such as the example shown below, means that the substituent is directly bonded to the molecule at the indicated position. No additional methylene (CH 2 ) groups are implied.
  • the symbol ( ) denotes the point of attachment of a moiety of the molecule.
  • R’, R”, R’” and R” each may independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • an“alkoxy” group is an alkyl attached to the remainder of the molecule through a divalent oxygen.
  • each of the R groups is independently selected as are each R’, R”, R’” and R”” groups when more than one of these groups is present.
  • R’ and R are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring.
  • -NR’R is meant to include, but not be limited to, 1- pyrrolidinyl and 4- morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and - CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like).
  • haloalkyl e.g., -CF 3 and - CH 2 CF 3
  • acyl e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3 , and the like.
  • Two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR’) q -U-, wherein T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR’- or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the
  • acyl specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
  • alkoxyl or“alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl–O–) or unsaturated (i.e., alkenyl–O– and alkynyl–O–) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,”“alkenyl,” and“alkynyl” are as previously described and can include C 1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.
  • alkoxyalkyl refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
  • Aryloxyl refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl.
  • aryloxyl as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
  • Alkyl refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl.
  • exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • Alkyloxyl refers to an aralkyl-O– group wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxyl group is benzyloxyl.
  • Alkoxycarbonyl refers to an alkyl-O-CO— group.
  • alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O-CO– group.
  • aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-O-CO– group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an amide group of the formula–CONH 2 .
  • Alkylcarbamoyl refers to a R’RN–CO– group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and/or substituted alkyl as previously described.
  • Dialkylcarbamoyl refers to a R’RN–CO– group wherein each of R and R’ is independently alkyl and/or substituted alkyl as previously described.
  • carbonyldioxyl refers to a carbonate group of the formula–O—CO—OR.
  • “Acyloxyl” refers to an acyl-O– group wherein acyl is as previously described.
  • the term“amino” refers to the–NH 2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • acylamino and“alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • an“aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
  • alkylamino refers to a group having the structure–NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure–NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups.
  • trialkylamino refers to a group having the structure–NR’R”R”’, wherein R’, R”, and R’” are each independently selected from the group consisting of alkyl groups. Additionally, R’, R”, and/or R’” taken together may optionally be–(CH 2 ) k – where k is an integer from 2 to 6.
  • Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
  • the amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl–S–) or unsaturated (i.e., alkenyl–S– and alkynyl–S–) group attached to the parent molecular moiety through a sulfur atom.
  • thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • “Acylamino” refers to an acyl-NH– group wherein acyl is as previously described.“Aroylamino” refers to an aroyl-NH– group wherein aroyl is as previously described.
  • “carboxyl” refers to the–COOH group. Such groups also are referred to herein as a“carboxylic acid” moiety.
  • halo refers to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.
  • hydroxyl refers to the–OH group.
  • hydroxyalkyl refers to an alkyl group substituted with an–OH group.
  • oxo as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.
  • thio refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • thiohydroxyl or thiol refers to a group of the formula –SH.
  • ureido refers to a urea group of the formula–NH—CO—NH 2 .
  • a“substituent group,” as used herein, includes a functional group selected from one or more of the following moieties, which are defined herein:
  • A“lower substituent” or“lower substituent group,” as used herein means a group selected from all of the substituents described hereinabove for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 5 - C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
  • A“size-limited substituent” or“size-limited substituent group,” as used herein means a group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 4 -C 8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
  • Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and/or isolate.
  • the present disclosure is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • racemates racemic forms
  • Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
  • Cis and trans geometric isomers of the compounds of the presently disclosed subject matter are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral (enantiomeric and diastereomeric), and racemic forms, as well as all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
  • the compounds herein described may have one or more charged atoms.
  • the compounds may be zwitterionic, but may be neutral overall.
  • Other embodiments may have one or more charged groups, depending on the pH and other factors.
  • the compound may be associated with a suitable counter-ion.
  • salts or exchange counter-ions can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid.
  • Counter-ions may be changed, for example, by ion-exchange techniques such as ion- exchange chromatography. All zwitterions, salts and counter-ions are intended, unless the counter-ion or salt is specifically indicated.
  • the salt or counter-ion may be pharmaceutically acceptable, for administration to a subject. Pharmaceutically acceptable salts are discussed later.
  • a "protecting group” is a chemical substituent which can be selectively removed by readily available reagents which do not attack the regenerated functional group or other functional groups in the molecule.
  • Suitable protecting groups are known in the art and continue to be developed. Suitable protecting groups may be found, for example in Wutz et al. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protection of the carboxyl group, as described by Wutz et al. (pages 533-643), are used in certain embodiments. In some embodiments, the protecting group is removable by treatment with acid. Specific examples of protecting groups include but are not limited to, benzyl, p-methoxybenzyl (PMB), tertiary butyl ( t Bu),
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or I4 C-enriched carbon are within the scope of this disclosure.
  • the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure may exist as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts is meant to include salts of active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein.
  • Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,
  • (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures), or teoclate may be prepared by methods known to those skilled in art.
  • Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20 th ed.) Lippincott, Williams & Wilkins (2000).
  • base addition salts such as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like, see, for example, Berge et al,“Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1- 19).
  • Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
  • the present disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure.
  • prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present disclosure when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • compositions iii.
  • a pharmaceutical composition comprises an effective amount (e.g., a or detectably effective amount) of a compound described hereinabove.
  • a presently disclosed composition can be formulated as a pharmaceutical composition, which comprises a presently disclosed compound and pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • pharmaceutically acceptable carriers and other components of pharmaceutical compositions see, e.g., Remington's Pharmaceutical Sciences, 18 l ed., Mack Publishing Company, 1990.
  • suitable pharmaceutical carriers include, e.g., water (including sterile and/or deionized water), suitable buffers (such as PBS), physiological saline, cell culture medium (such as DMEM), artificial cerebral spinal fluid, or the like.
  • compositions of the presently disclosed subject matter will depend, in part, upon the particular agent that is employed, and the chosen route of administration. Accordingly, there is a wide variety of suitable formulations of compositions of the presently disclosed subject matter.
  • compositions can be in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for animal (e.g. human) subjects, each unit containing a predetermined quantity of a presently disclosed agent, alone or in combination with other therapeutic agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired effective amount or effective concentration of the agent in the individual patient.
  • the dose of a presently disclosed composition, administered to an animal, particularly a human, in the context of the presently disclosed subject matter should be sufficient to produce at least a detectable amount of a diagnostic response in the individual over a reasonable time frame.
  • the dose used to achieve a desired effect will be determined by a variety of factors, including the potency of the particular agent being administered, the pharmacodynamics associated with the agent in the host, the severity of the disease state of infected individuals, other medications being administered to the subject, and the like.
  • the size of the dose also will be determined by the existence of any adverse side effects that may accompany the particular agent, or composition thereof, employed. It is generally desirable, whenever possible, to keep adverse side effects to a minimum.
  • the dose of the biologically active material will vary; suitable amounts for each particular agent will be evident to a skilled worker.
  • a “pharmaceutically acceptable carrier” refers to a biocompatible solution, having due regard to sterility, p[Eta], isotonicity, stability, and the like and can include any and all solvents, diluents (including sterile saline, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other aqueous buffer solutions), dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like.
  • the pharmaceutically acceptable carrier also can contain stabilizers, preservatives, antioxidants, or other additives, which are well known to one of skill in the art, or other vehicles as known in the art.
  • A“cancer” in an animal refers to the presence of cells possessing
  • cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells.
  • control is meant a standard or reference condition.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, organ, organism, or subject.
  • administering refers to contacting a subject with a presently disclosed agent.
  • delivery device is meant any device that provides for the release of an imaging agent.
  • exemplary delivery devices include tablets and pills, described below, as well as syringes, osmotic pumps, indwelling catheters, delayed-release and sustained-release biomaterials.
  • the terms“a,”“an,” and“the” refer to“one or more” when used in this application, including the claims.
  • reference to“a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
  • the terms“comprise,” “comprises,” and“comprising” are used in a non-exclusive sense, except where the context requires otherwise.
  • the term“include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
  • the term“about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • Radiolabeled urea-based low-molecular weight inhibitors of the prostate-specific membrane antigen (PSMA) are under intense investigation as imaging and therapeutic agents for prostate and other cancers.
  • PSMA prostate-specific membrane antigen
  • a new class of potent PSMA inhibitors based on the reversed carbamate scaffold has been reported, to maintain glutamate and S1 pocket side chain geometry and for putative binding to zinc.
  • Reversed carbamates contain the oxy-pentanedioic acid moiety (OPA) (FIG.3A).
  • 18 F-labeled inhibitors of PSMA based on reversed carbamate scaffolds have been synthetized.
  • OPA 4-Bromo-2-[ 18 F]fluorobenzoyl-lysine-oxy-pentanedioic acid
  • OPA 4-iodo-2-[ 18 F]fluorobenzoyl-lysine OPA reversed carbamate [ 18 F]24 (
  • 1,1’- Carbonyldiimidazole, iodomethane, N-hydroxysuccinimide, (diacetoxyiodo)benzene, triethylsilane (Et 3 SiH), diisopropylethylamine (DIEA) and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). 4-bromo-2-nitro-benzaldehyde was purchased from Combi-Block (San Diego, CA).
  • N-Succinimidyl 4-bromo/iodo-2-fluorobenzoate 4-bromo-2-fluorobenzoic acid (or 4-iodo-2-fluorobenzoic acid) 1 mmol and N-hydroxysuccinimide 125 mg (1.08 mmol) were dissolved in 2 mL dry DMF. To the solution, N,N- dicyclohexylcarbodiimide 170 ⁇ L (1.10 mmol) was added and the reaction was kept at room temperature overnight. After a flash column chromatography with ethyl acetate/hexane, 1:1, the N-succinimidyl 4-bromo/4-iodo-2-fluorobenzoates were obtained as white solids.
  • N-Succinimidyl 4-bromo-2-fluorobenzoate 1 H-NMR (500 MHz, CDCl 3 ): ⁇ 7.97-7.94 (m, 1H), 7.48-7.44 (m, 2H), 2.92 (s, 4H).
  • N-Succinimidyl 4-iodo-2- fluorobenzoate 1 H-NMR (500 MHz, CDCl 3 ): ⁇ 7.78-7.75 (m, 1H), 7.68-7.64 (m, 2H), 2.92 (s, 4H).
  • the PSMA inhibitory activity was determined using a modification of the fluorescence-based Amplex Red Glutamic Acid Assay (Life Technologies, Grand Island, NY)(Kozikowski, 2004). Briefly, lysates of LNCaP cell extracts (25 ⁇ L) were incubated with the inhibitor (12.5 ⁇ L) in the presence of 4 ⁇ M N-acetylaspartylglutamate (NAAG) (12.5 ⁇ L) for 120 min. The amount of the glutamate released by NAAG hydrolysis was measured by incubating with a working solution (50 ⁇ L) of the Amplex Red Glutamic Acid Kit for 60 min.
  • NAAG N-acetylaspartylglutamate
  • Enzyme inhibitory constants were generated using the Cheng-Prusoff conversion (Cheng, 1973). Assays were performed in triplicate. Data analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, California).
  • PC3 PIP PC3 PIP
  • PC3 flu PC3 flu
  • PC3 PIP PSMA-expressing
  • PC3 flu PCa cell lines
  • RPMI 1640 medium Corning Cellgro, Manassas, VA
  • FBS fetal bovine serum
  • Penicillin-Streptomycin Farm Cellgro, Manassas, VA
  • PC-3 PIP cells were grown under 20 ⁇ g/mL of puromycin selection in the growthmedium to maintain PSMA expression. All cell cultures were maintained in an atmosphere containing 5 % carbon dioxide (CO 2 ), at 37.0 °C in a humidified incubator. Animal studies were carried out in full compliance with the regulations of the Johns Hopkins Animal Care and Use Committee.
  • mice Six- to eight- week-old male, non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice (Johns Hopkins Immune Compromised Core) were implanted subcutaneously (sc) with PC3 PIP (PSMA+) and PC3 flu (PSMA-) cells (1 x 106 in 100 ⁇ L of HBSS (Corning Cellgro, Manassas, VA) at the forward right and left flanks, respectively. Mice were imaged or used in ex vivo biodistribution assays when the xenografts reached 5 to 7 mm in diameter.
  • NOD non-obese diabetic
  • SCID severe-combined immunodeficient mice
  • N- succinimidyl-4-[ 125 I]iodobenzoate was prepared by a modification of the method of Dekker et al (Dekker et al., 2015).
  • N-succinimidyl-4- [ 125 I]iodobenzoate eluted at 14 min. This was diluted with 20 mL water, loaded onto an activated Waters C18 Sep-Pak Plus cartridge, washed with 10 mL water, dried with a stream of nitrogen for 2min, then eluted with 2mL methylene chloride through a Na 2 SO 4 drying cartridge.
  • the methylene chloride solution of N-succinimidyl-4- [ 125 I]iodobenzoate (5.9 mCi) was stored at 0-2 °C. The methylene chloride solution was then evaporated to dryness under a stream of nitrogen and to this was added a solution of 22 (2 mg/200 ⁇ L DMSO). To this solution is added 5 ⁇ L
  • 18 F fluoride was produced by a General Electric PET trace biomedical cyclotron (GE HealthCare) using 18 MeV proton bombardment on an 18 O- H 2 O target and trapped on a Chromafix 30-PS-HCO3 QMA cartridge. The cartridge was eluted with 0.5 mL of a solution of potassium bicarbonate (4.5 mg/0.5 mL) into a 3 mL Wheaton reaction vial.
  • GE HealthCare General Electric PET trace biomedical cyclotron
  • the cartridge was eluted with 0.5 mL of a solution of potassium bicarbonate (4.5 mg/0.5 mL) into a 3 mL Wheaton reaction vial.
  • the product HPLC fraction was diluted with water to a volume of 45 mL and loaded onto an Oasis HLB Sep-Pak Cartridge, washed with 5 mL water, then dried by passing Argon through the cartridge for two minutes.
  • a sodium sulfate drying tube was then added to the end of the Oasis Sep-Pak and the aldehyde eluted with 2 mL methylene chloride into another Wheaton reaction vial.
  • the methylene chloride is evaporated under a stream of Argon and the residue was dissolve in 0.5mL acetonitrile. To this is added 50 mg N-hydroxysuccinimide and 28 mg
  • N-succinimidyl-4-bromo-2-[ 18 F]fluorobenzoate 30b eluted at 12 min
  • N-succinimidyl-4-iodo-2-[ 18 F]fluorobenzoate 30c eluted at 14 min. (FIG.7 and FIG.8) .
  • the product HPLC fraction was diluted with water to a volume of 45 mL and loaded onto a Waters C18 Sep-Pak Plus Cartridge, washed with 5 mL water, then dried by passing Argon through it for two minutes.
  • [ 18 F]24 ([ 18 F]XY-47). Specific activity ranged from 37,00-177,600 GBq/mmol (1000-4800 Ci/mmol) ) for [ 18 F]23 ([ 18 F]XY-52) and 31,080-92,500 GBq/mmol (840- 2500 Ci/mmol) for [ 18 F]24 ([ 18 F]XY-47).
  • PET images were acquired at 10 min, 30 min, 1 h, 2 h and 4 h p.i. as a pseudodynamic scan, i.e., a sequence of successive whole-body images were acquired in two bed positions. The dwell time at each bed position was 10 min for a total scan time of 20 minutes. An energy window of 250– 700 keV was used. Images were reconstructed using the FORE/2D-OSEM method (one iteration, 16 subsets) and included correction for radioactive decay, scanner dead time, and scattered radiation.
  • mice bearing PSMA+ PC-3 PIP and PSMA- PC-3 flu xenografts were injected via the tail vein with 740 kBq (20 ⁇ Ci) of [ 18 F]23 ([ 18 F]XY-52) or [ 18 F]24 ([ 18 F]XY-47) in 200 ⁇ L of saline.
  • mice were sacrificed by cervical dislocation and the blood immediately collected by cardiac puncture.
  • the heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large intestines, urinary bladder, PSMA+ PC-3 PIP and PSMA- PC-3 flu tumors were collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/ LKBNuclear, Inc., Mt. Waverly, Vic.
  • the % ID/g was calculated by comparison with samples of a dilution of a standard dose. All measurements were corrected for decay. Data are expressed as mean ⁇ standard deviation (SD).
  • PC-3 PIP (PSMA+) and PC-3 flu (PSMA-) cells were cultured as previously described (Banerjee et al., 2014). 300,000 PIP or flu cells were seeded into three wells each of a 6 well plate using RPMI 1640 + 10 % fetal bovine serum + 1 % Penicillin-Streptomycin (Corning Cellgro, Manassas, VA) and were grown to 80 % confluency.
  • the culture medium was refreshed and 50 ⁇ Ci (1.35 kBq) of [ 125 I]31 ([ 125 I]XY-26) or [ 125 I]32 ([ 125 I]XY-57) was added to both a PIP- and flu-containing well.
  • the plate was returned to the incubator (humidified 37 °C, 5 % CO 2 ) for 30 minutes.
  • the medium was then carefully removed and saved for counting in a LKB Wallac 1282 Compugamma gamma counter (Mount Waverly, Vic, Australia).
  • the cells were washed twice with ambient temperature PBS, pH 7.4 followed by the addition of ddH 2 O to lyse the cells.
  • Lysis took place over 30 minutes inside the incubator. The lysates were then collected and counted using the gamma counter. Equal amounts of radioactivity from the supernatant and lysates were spotted onto silica gel 60 RP-18 F254S glass TLC plates (EMD Millipore Corp., Billerica, MA) and the plates were developed using a mobile phase consisting of 55 % acetonitrile, 45 % water and 0.1 % trifluoroacetic acid. The TLC plate was dried and exposed to Kodak Biomax x-ray film (Fisher Scientific) prior to digitizing using the MCID Core package (Interfocus Imaging, Cambridge, UK).
  • OPA reversed carbamate 13 (XY-48) was prepared from (S)-dimethyl-2- hydroxypentanedioate 19, for which the corresponding tert-butyl ester was not easily accessible (Scheme 1). After conversion of 19 to the N-imidazolecarbamate, 20 was first transformed to its imidazolium salt via treatment with iodomethane and then reacted with N ⁇ -Boc-lysine-tert-butyl ester to afford 21. Attempts to couple N ⁇ -boc- lysine-tert-butyl ester and 20 directly failed to give the desired product, presumably due to side reactions involving the methyl esters. After two deprotection steps amine 22 was conjugated with N-succinimidyl-4-fluorobenzoate to give OPA- reversed carbamate 13 (XY-48).
  • iodinated reversed carbamate 32 (XY- 57) has been prepared by reacting N-succinimidyl-4-iodobenzoate with 22 (Scheme 1).
  • the 4-iodo-benzoyl group would require an [ 18 F]fluoro substituent in the activated ortho position.
  • OPA reversed carbamates 23 (XY-52) and 24 (XY-47) were generated by reacting 22 with N- succinimidyl-4-bromo/iodo-2-fluorobenzoate (Scheme 1).
  • ureido analogs 26 and 27 were also synthesized from urea 25 (Scheme 1) (Chen, 2012; Maresca, 2009).
  • PSMA inhibitory activities of the prepared compounds were measured using a modification of the fluorescence-based Amplex Red Glutamic Acid assay (Kozikowski, 2004).
  • Carbamates 31 (XY-26) and reversed carbamate 32 (XY-57) inhibited PSMA at K i 0.9 nM and 0.04 nM, respectively.
  • Lys-N-H (1.92 ⁇ ) slightly closer to the carbonyl oxygen than Glu-N-H (2.03 ⁇ ) (FIG.13A).
  • Lys-OPA carbamate 13 (XY-48), with the Glu- N-H changed to O resulting in the loss of a hydrogen bond, retained nearly all of the features of 9 (YC-I-26), when binding to PSMA (FIG.13B), and the Lys-N-H distance to G518 slightly increased 1.95 ⁇ , with the carbamate shifting slightly towards the zinc (FIG.13B vs. FIG.13A). This resulted in a 30-fold reduction in binding affinity compared to 9 (YC-I-26).
  • [ 18 F]29b was converted to its N-hydroxysuccinimide ester [ 18 F]30b by treating with (diacetoxyiodo)benzene in the presence of N-hydroxysuccinimide in acetonitrile (Glaser, 2009). After conjugating the F-18 labeled NHS ester with Lys-OPA reversed carbamate precursor 22, [ 18 F]23 ([ 18 F]XY-52) was obtained in a non-decay corrected radiochemical yield of 5 % and specific radioactivity ranging from 37,000-177,600 GBq/mmole (1,000- 4,800 Ci/mmole).
  • mice bearing PSMA+ PC-3-PIP and PSMA- PC-3-flu xenografts were assessed for their tumor uptake and pharmacokinetics in mice bearing PSMA+ PC-3-PIP and PSMA- PC-3-flu xenografts by ex vivo biodistribution and or micro-PET imaging.
  • Table 2 shows the percent injected dose per gram of tissue in selected organs for [ 18 F]23 ([ 18 F]XY-52).
  • Compound [ 18 F]23 ([ 18 F]XY-52) demonstrated high uptake in the PC-3 PIP tumor, wherein the uptake of radioactivity reached a maximum of 90% ID/g at 2 h post-injection.
  • the PSMA- PC3 flu tumor showed no specific uptake.
  • the distribution within non-target tissues was generally low, except for kidney, liver and spleen.
  • the PC-3 PIP:muscle and PC-3 PIP:blood ratios were 93 and 13 at 0.5 h, 208 and 40 at 1 h, 643 and 148 at 2 h, 792 and 240 at 4 h, respectively.
  • Table 2 Biodistribution of 18 F 23 18 F XY-52
  • bPIP:tissue ratio To determine if the radiolabeled [ 18 F]13 ([ 18 F]-XY-48), was capable of being imaged, male nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice with PSMA-expressing (PC3-PIP) and PSMA-negative (PC3-flu) tumors were injected with the radiolabeled compound and imaged by positron emission tomography–computed tomography (PET-CT; compound structure of [ 18 F]13
  • FIG.14A shows maximum intensity projections (MIPs) after PET imaging with the [ 18 F]13 ([ 18 F]XY-48), compound. Standardized uptake values were generated from the images (FIG.14B). These data showed significantly higher levels of binding of radiolabeled [ 18 F]13 ([ 18 F]XY-48), to the PSMA-expressing PC3-PIP tumors as compared to the PSMA- negative PC3-flu tumors.
  • Micro-PET (coronal view) imaging of [ 18 F]13 ([ 18 F]XY-48) showed a high and prolonged PC-3 PIP tumor and kidney uptake than [ 18 F]12 ([ 18 F]XY-20) and is pronounced of ureido compound [ 18 F]9 ([ 18 F]YC-I-26) (Chen, 2008).
  • NPA carbamates may be subject to PSMA specific metabolism and may be the reason for their rapid clearance from PSMA expressing tumors and organs. Discussion
  • [ 18 F]DCFBC (Cho, 2012), and [ 68 Ga]DOTA-DUPA-Pep (Reske, 2013)) showed persistent blood pool occupancy but low salivary gland uptake.
  • [ 18 F]DCFPyL (Szabo, 2015), [ 124 I]-MIP-1095 (Zechmann, 2014), and [ 68 Ga]DKFZ- 11(Afshar-Oromieh, 2012;Afshar-Oromieh, 2013) showed rapid clearance from the blood but high uptake in salivary and lacrimal glands. The latter has also been observed in radiopharmaceutical therapy studies using [ 177 Lu]-DKFZ-617
  • the OPA scaffold may be most useful for radiotherapy with alpha, beta, or Auger emitters when labeled with radiohalogenated ( 211 At, 131 I, 125 I, 123 I, 80m Br, or 77 Br) benzoyl groups where the S1 and auxillary hydrophobic pockets are fully utilized.
  • radiohalogenated 211 At, 131 I, 125 I, 123 I, 80m Br, or 77 Br
  • [ 18 F]23 ([ 18 F]XY-52) and [ 18 F]24 ([ 18 F]XY-47) reported here provide low overall radiochemical yields and are cumbersome to perform, the initial goal was only to produce enough radiolabeled material for preliminary testing of the reversed carbamates in vivo. Improved radiochemistry will be required for [ 18 F]23 ([ 18 F]XY- 52) and [ 18 F]24 ([ 18 F]XY-47) to achieve their full potential as PSMA imaging agents, and is under way.
  • Binding affinity data of representative compounds of formula (I) are provided herein below in Table 4.
  • the most potent compounds contain a fluorine atom at R 1 and a large halogen, e.g., either Br or I, or a phenyl ring at the 4-position of the arylbenzoyl moiety, i.e., R 2 and are annotated with an "*".
  • DCFBC is N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-fluorobenzyl-l-cysteine:
  • ⁇ ⁇ DCIBC is N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-iodobenzyl-l-cysteine
  • ⁇ ⁇ DCFPyL is 2-(3- ⁇ 1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl ⁇ - ureido)-pentanedioic acid:
  • Radiohalogenated Lysine, Glutamate and Cysteine-OPA carbamate compounds for Imaging and Cancer Radiotherapy
  • the development of low molecular weight radiotherapeutic agents is different from developing radiopharmaceuticals for imaging in that longer tumor residence times are required for the former.
  • Many radionuclides primarily ⁇ - and alpha emitters, have been investigated for targeted radioimmunotherapy and include both radiohalogens and radiometals.
  • the studies presented in the disclosed subject matter have been focused on radiohalogens, 125 I, 123 I, 131 I, 211 At, 77 Br (Table 6), with several specific examples of appropriate ways of introducing them into PSMA-targeting molecules.
  • Several of these radionuclides ( 123 I, 131 I, 77 Br) also emit imaging gamma rays providing both imaging and radiotherapeutic applications. Table 6.
  • radiohalogens are covalently bound to the targeting moiety and unlike large chelated radiometals are small enough that the entire radiolabeled PSMA inhibitor can fit within the PSMA binding cavity thereby retaining the high binding affinity.
  • the same radiolabeled prosthetic groups can be conjugated to linker- inhibitor conjugates to move the radiolabeled portion of the inhibitor to the exterior of the PSMA protein.
  • Radiohalogenated PSMA binding radiotherapeutics can be built upon multiple PSMA binding scaffolds.
  • the urea based scaffolds include: the lysine-glutamate urea, cysteine-glutamate urea, and glutamate-glutamate urea (FIG.18).
  • PSMA inhibitors built upon novel lysine- reversed carbamate scaffold (OPA) have been disclosed in example 1 of the present application, and cysteine -OPA carbamate and glutamate-–OPA carbamate scaffolds, as shown FIG.19, have been envisioned.
  • the lead compound in the lysine-glutamate ureas is YC-I-27.
  • Glutamate- glutamate ureas have been used by others to conjugate bulky radiometal chelating agents, fluorescent molecule, and chemotherapeutics (Kularatne et al., 2009;
  • Lysine glutamate ureas and glutamate-glutamate ureas linker conjugates have been used to attach bulky radiometal chelates or fluorescent molecules for PSMA specific imaging and radiotherapy, with a focus on the use of polyethylene glycol (PEG) and lysine-suberate linkers (Banerjee et al., 2008; Chen et al., 2012).
  • the new PSNA binding scaffolds described herein can also be used to prepare radiolabeled linker conjugates.
  • a route to the synthesis of a lysine-suberate-lysine-OPA carbamate and its use in preparing radiohalogenated linker OPA carbamates is shown in Scheme 8. Scheme 8.
  • PSMA binding ureas conjugated to fluorescent molecules via various linkers for imaging PSMA expressing tumors and tissues have been previously described.
  • PDT photodynamic therapy
  • Two specific examples of IR-Dye-800-CW-linker- ureas are shown in FIG.21.
  • PSMA inhibitors built upon lysine-carbamate scaffolds (OPA and NPA) including F-18 labeled analogs have been disclosed in the present application, and the F-18 labeled NPA and OPA compounds demonstrated selective uptake in PSMA positive tumor mouse xenografts.
  • YC-XIX-33 To a solution of 4 (0.5 mg, 0.75 ⁇ mol) in DMSO (0.1 mL) was added N,N-diisopropylethylamine (0.005 mL, 0.029 mmol), followed by the NHS ester of IRDye800CW (0.5mg, 0.43 ⁇ mol). After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC to afford YC-XIX-33 (0.6 mg, 90 %).
  • ESI-Mass calcd for C 69 H 94 N 5 O 27 S 4 [M+H] + 1552.5, found: 1552.5. h m 1
  • mice Animal imaging. YC-XIX-33 (1 nmole in PBS) was injected via the tail vein into two 4-6 week old male athymic nude mice containing PSMA+ PC3 PIP and PSMA- PC3 flu tumor xenografts on opposite flanks. The mice were imaged using the Pearl Impulse Imager (excitation at 785 nm and emission at 800 nm) at 10 min, 30 min, 1h, 2h, 4h, and 24h. One mouse was sacrificed at 4 hours post injection and the other at 24h. Tumor and organs were removed, placed in a Petri dish and reimaged. All images were scaled to the same maximum intensity for direct comparison. Results
  • PSMA prostate specific membrane antigen
  • PSMA anti-prostate-specific membrane antigen
  • PSMA prostate specific membrane antigen
  • PSMA prostate-specific membrane antigen
  • Makarasen, A., Nishikawa, T., Isobe, M. Synthesis of four lysine-linked cereulide analogues showing ionophoric activity towards potassium cations as lead compounds for emetic toxin detection by immunoassays. Synthesis 2009, 2184-2004;

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Abstract

"Reversed" carbamate based scaffolds that have high binding affinity to PSMA are disclosed. These scaffolds can be radiolabeled and used for imaging cells and tumors that express PSMA or for cancer radiotherapy. These compounds also can comprise a fluorescent dye and be used for imaging cells and tumors that express PSMA or for photodynamic therapy.

Description

PSMA TARGETED REVERSED CARBAMATES AND
METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.
62/067,185, filed, October 22, 2014, which is incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under R01 CA134675 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND
Prostate-specific membrane antigen (PSMA) is among the most intensively targeted biomarkers for imaging metastatic prostate cancer. PSMA is a zinc- dependent metallopeptidase that catalyzes the hydrolysis of a series of N- acylpolygammaglutamate derivatives (Mesters, 2006; Barinka, 2004; Pinto, 1996). It is expressed within certain normal tissues (Ghosh, 2004), but transitions to abundant plasma membrane expression in the epithelium of most prostate cancer and within other solid tumor neovasculature (Rajasekaran, 2005). PSMA membrane expression is associated with metastasis (Chang, 2001), castration resistance (Bander, 2005) and progression of prostate cancer (Perner, 2007).
Several different scaffolds are available for synthesis of small-molecule PSMA inhibitors, and have been reviewed (Byun, 2009; Tsukamoto, 2007; Zhou, 2005). As shown in FIG.1 those potent scaffolds share common features, namely; a) a pentanedioic acid as a glutamate mimic to fit within the S1’ binding pocket of the PSMA active site; and b) a zinc-binding group to interact with the catalytic zinc atom at the PSMA active site. A substituent (R) can reside either within the S1 binding pocket or within a void in the protein that extends to the surface. Scaffolds composed of phosphonates or phosphinates,(Jackson, 1996; Jackson, 2000), phosphoramidates (Maung, 2004) and ureas (Kozikowski, 2001; Kozikowski, 2004; Maung, 2004) of general structures 1-3, as well as thiol 4 (Majer, 2003; Stoermer, 2012) and hydroxamate 5 (Stoermer, 2003) have been reported as effective zinc binding groups for PSMA inhibition. However, the presence of a zinc binding moiety and a glutamate mimic residing in the S1’ pocket are themselves not sufficient for high binding as demonstrated by gly-urea-glu compound 8 (FIG.2A and FIG.2B) (Wang, 2013). Of the reported PSMA binding scaffolds, urea-based inhibitors, first introduced by Kozikowski in 2001 (Kozikowski, 2001) for inhibition of glutamate carboxypeptidase II within the central nervous system, have been utilized the most for targeting PSMA due to their high binding affinity and synthetic simplicity
(Kozikowski, 2001; Kozikowski, 2001; Foss, 2012; Mease, 2013; Vargas, 2015; Chen, 200; Barinka, 2008). A variety of low-molecular-weight compounds based on the various scaffolds discussed above, primarily the ureas, have been labeled with radionuclides for positron emission tomography (PET) and single photon emission computed tomography (SPECT), namely, 125/124I, 99mTc, 111In, 18F, 11C, 68Ga, 64Cu, and 86Y, and have demonstrated PSMA-targeted imaging of prostate cancer in experimental models (Foss, 2012; Mease, 2013; Vargas, 2015; Banerjee, 2008;
Banerjee, 2015; Banerjee, 2010; Banerjee, 2014; Chen, 2008; Chen, 2011; Eder, 2012; Foss, 2005; Hillier, 2013; Kularatne, 2009; Lapi, 2009; Maresca, 2009; Mease, 2008; Ray Banerjee, 2013; Weineisen, 2014). Several of these have been translated to phase 0-1 clinical trials, where they have enabled visualization of both primary and metastatic bone and soft-tissue lesions due to prostate cancer. (Afshar-Oromieh, 2015; Afshar-Oromieh, 2012; Afshar-Oromieh, 2013; Afshar-Oromieh, 2014; Barrett, 2013;Cho, 2012; Rowe, 2015; Szabo, 2015; Vallabhajosula, 2014).
However, clinical imaging studies also exhibited considerable uptake in non- target PSMA-expressing tissues such as the salivary glands and kidneys, bringing to light potential dose-limiting off-target effects, particularly for radiotherapeutic analogs. Additional PSMA-binding scaffolds that might preserve the positive imaging characteristics of the ureido scaffolds but clear from the non-target organs were sought. The "reversed" carbamate scaffold has been chosen because it would retain the overall geometry of the existing inhibitors, differing only with an O for NH substitution, which eliminates a potential hydrogen bonding donor group present in the ureas. The only PSMA-binding carbamate reported is gly-amino-pentanedioic acid 1 (Wang, 2013) displayed low binding affinity to PSMA, most likely due to the absence of productive binding within the S1 pocket, similar to ureido compound 8. Herein a new class of potent PSMA inhibitors based on the reversed carbamate scaffold to maintain glutamate and S1 pocket side chain geometry and for putative binding to zinc have been reported. Reversed carbamate scaffolds may complement the existing urea and other scaffolds upon which inhibitors, imaging and therapeutic agents targeting PSMA have been based.
Because of the favorable pharmacokinetic profile of this class of compounds, i.e., low nonspecific binding, lack of metabolism in vivo and reasonable tumor residence times, it has been reasoned that reversed carbamate-based agents could also be used for molecular radiotherapy. This will be in analogy with radioimmunotherapy (RIT), which has proved remarkably successful in the treatment of lymphoma with two commercial products routinely integrated into clinical practice. However, (RIT) is fraught with difficulties inherent in the use of radiolabeled antibodies for imaging, including prolonged circulation times, unpredictable biological effects and the occasional need for pre-targeting strategies. Furthermore, antibodies may have less access to tumor than low molecular weight agents, which can be manipulated pharmacologically. Therefore a need remains for low molecular weight compounds with high binding affinity to PSMA for the imaging and radiotherapy of tumors.
Further, for the aforementioned reasons, fluorescent-linker-reversed carbamate based PSMA inhibitors have also been investigated. Targeted fluorescent PSMA binding compounds may find utility in fluorescence guided surgery and biopsy of PSMA positive tumors and tissues, the former providing visual confirmation of complete removal of PSMA containing tissue. Moreover, reversed carbamate conjugates of photosensitizing dyes also provide PSMA targeted photodynamic therapy agents. SUMMARY
In some aspects, the presently disclosed subject matter provides a compound of formula (I):
Figure imgf000004_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order; Z is tetrazole or CO2Q; Q is H or a protecting group; t is an integer selected from the group consisting of 1, 2, 3, 4; p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different; p1, p3, and p4 are each independently 0 or 1; m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and–NR–C(=O)–;
W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,– C(O)–,–NRC(O)–,–NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,– OC(O)–,–C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q– , wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole group that can be resent or absent and is selected from the group consisting of
Figure imgf000005_0001
; each R1 is independently H, C1-C6 alkyl, C2- C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H;
V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,– NRC(S)–, and–OC(O)–; A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which can optionally comprise one or more radioactive isotope suitable for imaging and /or radiotherapy, or a photosensitizing dye suitable for imaging and/or photodynamic therapy.
In other aspects, the presently disclosed subject matter provides a method for imaging one or more prostate-specific membrane antigen (PSMA)-expressing tumors or cells, the method comprising contacting the one or more tumors or cells with an effective amount of a compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), and making an image, wherein the compound of formula (I) further comprises a radioactive isotope or a photosensitizing dye suitable for imaging.
In yet other aspects, the presently disclosed subject matter provides a method for treating or preventing a disease or condition associate with one or more PSMA expressing tumors or cells, the method comprising administering at least one compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), to a subject in an amount effective to treat or prevent the disease or condition wherein the compound of formula (I) further comprises a radioactive isotope suitable for radiotherapy or a photosensitizing dye suitable for photodynamic therapy.
Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:
FIG.1 shows scaffolds available for synthesis of small-molecule PSMA inhibitors; those potent scaffolds share common features, namely; a) a pentanedioic acid (green) as a glutamate mimic to fit within the S1’ binding pocket of the active site; and b) a zinc-binding group (blue) to interact with the catalytic zinc atom at the PSMA active site (blue); a substituent (R) can reside either within the S1 binding pocket or within a void in the protein that extends to the surface;
FIG.2A and FIG.2B show (FIG.2A) urea- and carbamate-based PSMA binding compounds; (FIG.2B) binding affinities of urea- and carbamate-based PSMA binding compounds and compounds of the presently disclosed subject matter;
FIG.3A and FIG.3B show (FIG.3A) the lysine- reversed carbamate scaffold used to design compounds of the presently disclosed subject matter: oxypentanedioic acid (OPA) corresponding to a "reversed" carbamate scaffold; (FIG.3B) general structure of OPA, high binding affinity structure of OPA and versatile intermediate of OPA for functionalization;
FIG.4 is a synthesis scheme for the presently disclosed reversed carbamates; FIG.5 shows preparative Radio-HPLC chromatograms of 2-[18F]fluoro-4- bromo-benzaldehyde [18F]29b; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 54/46/0.1 water/acetonitrile/TFA, 4 mL/m; double radioactive peak at 18- 19.5 min is due to saturation of the radioactivity detector;
FIG.6 shows preparative Radio-HPLC chromatogram of 2-[18F]fluoro-4-iodo- benzaldehyde [18F]29c; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 54/46/0.1 water/acetonitrile/TFA, 4 mL/m; double radioactive peak at 21-23 minutes is due to saturation of the radioactivity detector;
FIG.7 shows preparative Radio-HPLC chromatograms of N-succinimidyl 2- [18F]fluoro-4-bromobenzoate [18F]30b; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 50/50/0.1 water/acetonitrile/TFA, 4 mL/m;
FIG.8 shows preparative Radio-HPLC chromatogram of N-succinimidyl 2- [18F]fluoro-4-iodobenzoate [18F]30c; 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 50/50/0.1 water/acetonitrile/TFA, 4 mL/m;
FIG.9 shows preparative Radio-HPLC chromatogram of (S)-2-((((S)-5-(4- bromo-2-[18F]fluorobenzamido)-1-carboxypentyl)carbamoyl)oxy)pentanedioic acid [18F]23 ([18F]XY-52); 10 X 250 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 4 mL/m;
FIG.10 shows quality control analytical radio-HPLC chromatogram of purified (S)-2-((((S)-5-(4-bromo-2-[18F]fluorobenzamido)-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid [18F]23 ([18F]XY-52); 4.6 X 150 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 1 mL/m;
FIG.11 shows preparative Radio-HPLC chromatogram of (S)-2-((((S)-5-(4- iodo-2-[18F]fluorobenzamido)-1-carboxypentyl)carbamoyl)oxy)pentanedioic acid [18F]24 ([18F]XY-47);10 X 250 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 4 mL/m;
FIG.12 show quality control analytical radio-HPLC chromatogram of purified (S)-2-((((S)-5-(4-iodo-2-[18F]fluorobenzamido)-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid [18F]24 ([18F]XY-47); 4.6 X 150 mm, 10 micron Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 1 mL/m;
FIG.13A, FIG.13B, FIG.13C, and FIG.13D show docking of 9 (YC-I-26), 12 (XY-20), and 13 (XY-48) to PSMA; (FIG.13A) urea 9 (YC-I-26); (FIG.13B) Lys-NPA carbamate 12 (XY-20); (FIG.13C) Lys-OPA reversed carbamate 13 (XY- 48); (FIG.13D) overlay of 9 (YC-I-26) (red), 12 (XY-20) (blue) and 13 (XY-48) (green);
FIG.14A and FIG.14B show: (FIG.14A) maximum intensity projections (MIPs) after PET imaging with the [18F]13 ([18F]XY-48) compound in male NOD- SCID mice; no blocking study was done in this instance; and (FIG.14B) standardized uptake values generated from the images;
FIG.15A, FIG.15B, FIG.15C, and FIG.15D show sequential Micro-PET (coronal view) images of [18F]13 ([18F]XY-48) in a male SCID mouse containing PSMA+ PC-3 PIP and PSMA- PC-3 flu tumor xenografts at (FIG.15A) 10 minutes, (FIG.15B) 30 minutes , (FIG.15C) 60 minutes and (FIG.15D) 120 minutes after injection;
FIG.16 shows maximum intensity projections (MIPs) after PET imaging with the [18F]23 ([18F]XY-52 and [18F]24 ([18F]XY-47) compounds in male NOD-SCID mice;
FIG.17 shows TLC analysis of Metabolism of [125I]31 ([125I]XY-26) and [125I]32 ([125I]XY-57) in PC 3 PIP (PSMA+) and PC3 flu (PSMA-) cells;
FIG.18 shows PSMA urea-binding scaffolds upon which radiohalogenated PSMA binding radiotherapeutics can be built: the lysine-glutamate urea, cysteine- glutamate urea, and glutamate-glutamate urea;
FIG.19 shows a glutamate-OPA reversed carbamate and a cysteine-OPA reversed carbamate scaffolds used to design compounds of the presently disclosed subject matter;
FIG.20 shows the cysteine-glutamate urea scaffolds used for PSMA binding and imaging for over 10 years: C-11 labeled DCMC, F-18 labeled DCFBC both for PET imaging with the latter currently in use in patients, and I-125 labeled DCIBC for SPECT imaging and or radiotherapy;
FIG.21 shows two specific examples of IRDye800CW-linker-ureas; and FIG.22A and FIG.22B show (FIG.22A) the structure of compound YC-XIX- 33; and (FIG.22B) images of two mice bearing PSMA+PC3 PIP tumor after injection of YC-XIX-33 (1nmole in PBS) into the tail vein at 10 min, 30 min, 1h, 2h, 4h, and 24h using the Pearl Impulse Imager (excitation at 785 nm and emission at 800 nm).
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. DETAILED DESCRIPTION
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Examples and Figures, in which some, but not all embodiments of the presently disclosed subject matter are illustrated. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Examples and Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. I. PSMA TARGETED REVERSED CARBAMATES AND METHODS OF USE THEREOF
The presently disclosed subject matter provides“reversed” carbamate based scaffolds that have high binding affinity to PSMA. These scaffolds can be radiolabeled and used to image cells and tumors that express PSMA. Some advantages to the presently disclosed methods include, but are not limited to, lower renal uptake of the presently disclosed compounds compared with existing urea-based PSMA-targeted radiotracers, as well as more rapid renal clearance as compared to compounds known in the art.
In some embodiments, the presently disclosed“reversed” carbamate based scaffolds complement existing urea and other scaffolds upon which inhibitors, imaging, and therapeutic agents targeting PSMA have been based. Versatile intermediates for“reversed” carbamate scaffolds can be functionalized in one or two steps toward PET imaging agents. In other embodiments, the presently disclosed“reversed” carbamate based scaffolds are conjugated with near infrared dye molecules and their use in imaging PSMA and PSMA targeted photodynamic therapy agents also are provided.
A. Compounds of Formula (I)
Accordingly, in some embodiments, the presently disclosed subject matter provides a compound of formula (I):
Figure imgf000010_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order; Z is tetrazole or CO2Q; Q is H or a protecting group; t is an integer selected from the group consisting of 1, 2, 3, 4; p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different; p1, p3, and p4 are each independently 0 or 1; m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and–NR–C(=O)–; W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,–C(O)–,–NRC(O)–,– NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,–OC(O)–,–C(O)NR–,– NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q–, wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; each R is independently H, C1- C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a t i l b present
or absent and is selected from the group consisting of
Figure imgf000010_0002
and
Figure imgf000010_0003
; each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H; V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–; A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which can optionally comprise one or more radioactive isotope suitable for imaging and /or radiotherapy, or a
photosensitizing dye suitable for imaging and/or photodynamic therapy.
In particular embodiments, the compound of formula (I) is selected from the group consisting of:
Figure imgf000011_0001
wherein L is a linker that can be present or absent, and has the following general structure:
Figure imgf000011_0002
wherein p1, p2, p3, m1, m2, Tz, W2, R, R1, R2, R3, V and A are defined as hereinabove.
Suitable linkers are disclosed in U.S. Patent Application Publication No. US2011/0064657 A1, for "Labeled Inhibitors of Prostate Specific Membrane Antigen (PSMA), Biological Evaluation, and Use as Imaging Agents," published March 17, 2011, to Pomper et al., and U.S. Patent Application Publication No. US2012/0009121 A1, for "PSMA-Targeting Compounds and Uses Thereof," published January 12, 2012, to Pomper et al, each of which is incorporated by reference in its entirety. In yet more particular embodiments, L is selected from the group consisting of:
Figure imgf000012_0001
In some embodiments, A has the following general structure:
Figure imgf000012_0002
wherein:
X is CH or N; and R5, R6, and R7 are each independently selected from the group consisting of H, halogen, alkoxyl, alkythioether, substituted and unsubstituted aryl, CH2-NH- C(=NH)-NH2, NH-(C=O)-R8, wherein R8 is alkyl, -(C=O)-NR9R10, wherein R9 and R10 are each independently selected from the group consisting of H and alkyl, -X1- (CH2)q-Ph-X2, -X1-(CH2)q-X2, -X1-(CH2)q-NH-C(=O)-Ph-X2, wherein each X1 is independently O or S, each q is independently an integer selected from the group consisting of 1, 2, 3, and 4, each Ph is phenyl, and each X2 is halogen.
In particular embodiments, the compound of formula (I) is selected from the group consisting of:
Figure imgf000013_0001
. In yet more particular embodiments, the compound of formula (I) is:
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
;
Figure imgf000018_0001
In certain embodiments, the photosensitizing dye suitable for imaging and/or photodynamic therapy is a fluorescent dye moiety which emits in the visible or near infrared spectrum, wherein the fluorescent dye moiety comprises carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660,
AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
In particular embodiments, the fluorescent dye moiety is selected from the
group consisting of:
Figure imgf000019_0001
;
Figure imgf000019_0002
;
;
Figure imgf000020_0001
In more specific embodiments, the compound of formula (I) is
Figure imgf000021_0001
B. Methods of Using Compounds of Formula (I) for Imaging a PSMA-expressing Tumor or Cell
In some embodiments, the presently disclosed subject matter provides a method for imaging one or more prostate-specific membrane antigen (PSMA)- expressing tumors or cells, the method comprising contacting the one or more tumors or cells with an effective amount of a compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic) and making an image, the compound of formula (I) comprising:
Figure imgf000021_0002
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order; Z is tetrazole or CO2Q; Q is H or a protecting group; t is an integer selected from the group consisting of 1, 2, 3, 4; p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different; p1, p3, and p4 are each independently 0 or 1; m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and–NR–C(=O)–; W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,–C(O)–,– NRC(O)–,–NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,–OC(O)–,– C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q–, wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole group that can be resent or absent and is selected from the group consisting of
Figure imgf000022_0001
each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H; V is selected from the group consisting of– C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–; A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for imaging, or a photosensitizing dye suitable for imaging.
"Contacting" means any action which results in at least one compound comprising the imaging agent of the presently disclosed subject matter physically contacting at least one PSMA-expressing tumor or cell. Contacting can include exposing the cell(s) or tumor(s) to the compound in an amount sufficient to result in contact of at least one compound with at least one cell or tumor.
By“making an image”, it is meant using PET, SPECT or florescent optical imaging to form an image of a cell, tissue, tumor, part of body, and the like. The presently disclosed methods may include one or more radioactive isotopes capable of emitting radiation suitable for detection with PET or SPECT or a photosensitizing dye suitable for fluorescent optical imaging.
In some embodiments, the image is made using PET the image is made using positron emission tomography (PET) and the radiohalogen is selected from the group consisting of 18F and 124I.
In some other embodiments, the image is made using Single-photon emission computed tomography (SPECT) and the radiohalogen is selected from the group consisting of 77Br, 131I, 125I, and 123I.
In yet some other embodiments, the image is made using florescent optical imaging and the photosensitizing dye suitable for imaging is selected from the group consisting of:
Figure imgf000023_0001
;
Figure imgf000024_0001
In certain embodiments, the one or more PSMA-expressing tumors or cells is selected from the group consisting of: a prostate tumor or cell, a metastasized prostate tumor or cell, a lung tumor or cell, a renal tumor or cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor or cell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor or cell, a germ cell, a pheochromocytoma, an esophageal tumor or cell, a stomach tumor or cell, and combinations thereof. In yet more certain embodiments, the one or more PSMA-expressing tumors or cells is a prostate tumor or cell.
In some embodiments, the one or more PSMA-expressing tumors or cells is in vitro, in vivo, or ex vivo. The method can be practiced in vitro or ex vivo by introducing, and preferably mixing, the compound and cell(s) or tumor(s) in a controlled environment, such as a culture dish or tube. The method can be practiced in vivo, in which case contacting means exposing at least one cell or tumor in a subject to at least one compound of the presently disclosed subject matter, such as administering the compound to a subject via any suitable route.
In particular embodiments, the one or more PSMA-expressing tumors or cells is present in a subject. The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a“subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal (non-human) subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a“subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and“patient” are used interchangeably herein. In some embodiments, the subject is human. In other embodiments, the subject is non-human.
In some embodiments, a detectably effective amount of the imaging agent of the presently disclosed methods is administered to a subject. In accordance with the presently disclosed subject matter, "a detectably effective amount" of the imaging agent is defined as an amount sufficient to yield an acceptable image using equipment which is available for clinical use. A detectably effective amount of the imaging agent may be administered in more than one injection. The detectably effective amount of the imaging agent can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, the dosimetry, and instrument and film- related factors. Optimization of such factors is well within the level of skill in the art.
In general, the“effective amount” of an active agent refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the target tissue, and the like.
It is preferable to have the compound comprising the imaging agent to localize to the tumor or cell quickly after administration so as to minimize any side effects to the subject. Accordingly, in some embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 60 minutes to about 240 minutes of administration and, in some embodiments, about 60 minutes. In other embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 30 minutes of administration. In still other embodiments, the compound comprising the imaging agent substantially localizes to the tumor or cell within about 10 minutes of administration.
It is also preferable that the compounds of the presently disclosed subject matter are excreted from tissues of the body quickly to prevent prolonged exposure to the radiation of the radiolabeled compound administered to the patient. Typically compounds of the presently disclosed subject matter are eliminated from the body in less than about 24 hours. More preferably, compounds of the presently disclosed subject matter are eliminated from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes.
In some embodiments, the presently disclosed methods comprise clearance of the compound comprising the imaging agent from the tumor or cell in the subject. At least one advantage of the presently disclosed methods is that, in some embodiments, there is more rapid clearance of the compound comprising the imaging agent from the kidneys than from the tumor of the subject.
In other embodiments, for example for fluorescence guided surgery and biopsy of PSMA positive tumors and tissues the compound comprising the photosensitizing dye suitable for imaging is visible at about 4 hours after injection and presents the brightest signal at about 24 hours after injection. In some embodiments, the presently disclosed methods use compounds that are stable in vivo such that substantially all, e.g., more than about 50%, 60%, 70%, 80%, or more preferably 90% of the injected compound is not metabolized by the body prior to excretion. In other embodiments, the compound comprising the imaging agent is stable in vivo.
A“tumor,” as used herein, refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
In some embodiments, the tumor cells express PSMA, such as prostate tumor cells or metastasized prostate tumor cells. In other embodiments, a tumor may be treated by targeting adjacent or nearby cells which express PSMA. For example, vascular cells undergoing angiogenesis associated with a tumor may be targeted. Essentially all solid tumors express PSMA in the neovasculture. Therefore, methods of the presently disclosed subject matter can be used to image nearly all solid tumors including, but not limited to, lung, renal cell, glioblastoma, pancreas, bladder, sarcoma, melanoma, breast, colon, germ cell, pheochromocytoma, esophageal, and stomach tumors. Also, certain benign lesions and tissues including, but not limited to, endometrium, schwannoma and Barrett's esophagus, can be imaged according to the presently disclosed methods.
C. Methods of Using Compounds of Formula (I) for Treating a disease or condition associated with one or more PSMA-expressing Tumor or Cell
In some embodiments, the presently disclosed subject matter provides a method for treating or preventing a disease or condition associate with one or more PSMA expressing tumors or cells, the method comprising administering at least one compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), to a subject in an amount effective to treat or prevent the disease or condition, the compound of formula (I) comprising:
Figure imgf000027_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order; Z is tetrazole or CO2Q; Q is H or a protecting group; t is an integer selected from the group consisting of 1, 2, 3, 4; p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different; p1, p3, and p4 are each independently 0 or 1; m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and–NR–C(=O)–; W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,–C(O)–,– NRC(O)–,–NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,–OC(O)–,– C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q–, wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole group that can be resent or absent and is selected from the group consisting of
Figure imgf000028_0001
;
each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H; V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,–NRC(S)–, and–OC(O)–; A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for or radiotherapy, or a photosensitizing dye suitable for photodynamic therapy.
In further embodiments, the radiohalogen suitable for radiotherapy is selected from the group consisting of 80mBr, 77Br, 125I, 123I, 131I and 211At. In yet other embodiment, wherein the photosensitizing dye suitable for photodynamic therapy is selected from the group consisting of
Figure imgf000029_0001
;
Figure imgf000030_0001
In specific embodiments, the disease or condition is a prostate cancer, renal cancer, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer,
leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, adenomas, and tumor neovasculature. In more specific embodiments, the disease or condition is prostate cancer. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the cancer or the tumor neovasculature.
A“cancer” in a subject refers to the presence of cells possessing
characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within a subject, or circulate in the blood stream as independent cells, for example, leukemic cells.
A cancer can include, but is not limited to, renal cancer, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, and adenomas. In more specific embodiments, the disease or condition is prostate cancer. In some embodiments, a detectably effective amount of the therapeutic agent of the presently disclosed methods is administered to a subject.
D. Definitions
i. Chemical Definitions
While the following terms in relation to compounds of formula (I) are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.
The terms substituted, whether preceded by the term“optionally” or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
Where substituent groups or linking groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-; -C(=O)O- is equivalent to -OC(=O)-; - OC(=O)NR- is equivalent to - NRC(=O)O-, and the like.
As used herein, where an internal substituent is flanked by bonds (for example, -NRC(O)-) the order of the atoms is fixed, the orientation of the group may not be reversed, and is inserted into a structure in the orientation presented. In other words -NRC(O)- is not the same as -C(O)NR-. As used herein the term C(O) (for example -NRC(O)-) is used to indicate a carbonyl (C=O) group, where the oxygen is bonded to the carbon by a double bond.
When the term“independently selected” is used, the substituents being referred to (e.g., R groups, such as groups R1, R2, and the like, or variables, such as “m” and“n”), can be identical or different. For example, both R1 and R2 can be substituted alkyls, or R1 can be hydrogen and R2 can be a substituted alkyl, and the like.
The terms“a,”“an,” or“a(n),” when used in reference to a group of substituents herein, mean at least one. For example, where a compound is substituted with“an” alkyl or aryl, the compound is optionally substituted with at least one alkyl and/or at least one aryl. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as“R-substituted.” Where a moiety is R- substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.
A named“R” or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative“R” groups as set forth above are defined below.
Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.
The term hydrocarbon, as used herein, refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
The term“alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, acyclic or cyclic hydrocarbon group, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent groups, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). In particular embodiments, the term“alkyl” refers to C1-20 inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
Representative saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n- undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.
“Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.“Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.“Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments,“alkyl” refers, in particular, to C1-8 straight-chain alkyls. In other embodiments,“alkyl” refers, in particular, to C1-8 branched-chain alkyls.
In certain embodiments, alkyl groups are C1-C6 alkyl groups or C1-C4 alkyl groups. The term "C1-C6 alkyl" as used herein means straight-chain, branched, or cyclic C1-C6 hydrocarbons which are completely saturated and hybrids thereof, such as (cycloalkyl)alkyl. Examples of C1-C6 alkyl substituents include methyl (Me), ethyl (Et), propyl (including n-propyl (n-Pr, nPr), iso-propyl (i-Pr, 1Pr), and cyclopropyl (c- Pr, 0Pr)), butyl (including n-butyl (n-Bu, nBu), iso-butyl (i-Bu, 1Bu), sec-butyl (s-Bu, sBu), tert-butyl (t-Bu, 1Bu), or cyclobutyl (c-Bu, 0Bu)), and so forth.
Alkyl groups can optionally be substituted (a“substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term“alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as“alkylaminoalkyl”), or aryl.
Thus, as used herein, the term“substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
The term“heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon group, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2- N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH25-S(O)-CH3, -CH2-CH2-S(O)2-CH3, - CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, 0-CH3, -0- CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.
As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(O)R’, - C(O)NR’, -NR’R”, -OR’, -SR, and/or -SO2R’. Where“heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR’R or the like, it will be understood that the terms heteroalkyl and -NR’R” are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R” or the like.
In the term "(cycloalkyl)alkyl", cycloalkyl, and alkyl are as defined above, and the point of attachment is on the alkyl group. This term encompasses, but is not limited to, cyclopropylmethyl, cyclopentylmethyl, and cyclohexylmethyl. The alkyl group may be substituted or unsubstituted.
“Cyclic” and“cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.
The terms“cycloheteroalkyl” or“heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10- member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), and optionally can include one or more double bonds.
The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings. Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7- membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
The terms“cycloalkyl” and“heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and“heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene” and“heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.
The term“cycloalkylalkyl,” as used herein, refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. Alkyl groups which are limited to hydrocarbon groups are termed“homoalkyl.”
More particularly, the term“alkenyl” as used herein refers to a monovalent group derived from a C1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1- methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, and butadienyl.
The term“cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
The term“alkynyl” as used herein refers to a monovalent group derived from a straight or branched C1-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of“alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl, and allenyl groups, and the like.
The term“alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more“alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (–CH2–); ethylene (–CH2–CH2–); propylene (–(CH2)3–);
cyclohexylene (–C6H10–);–CH=CH–CH=CH–;–CH=CH–CH2–; -CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, -CH2CH2CH(CH2CH2CH3)CH2-,–(CH2)q–N(R)– (CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (–O–CH2–O–); and ethylenedioxyl (–O– (CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A“lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The term“heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2- CH2-S- CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and–R’OC(O)-.
The term“aryl” means, unless otherwise stated, an aromatic hydrocarbon substituent that can be a single ring or multiple rings (such as from 1 to 3 rings), which are fused together or linked covalently.
The term“heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1- pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, indazolyl, 1-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms“arylene” and“heteroarylene” refer to the divalent forms of aryl and heteroaryl, respectively.
For brevity, the term“aryl” when used in combination with other terms (e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms“arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like). The term“haloaryl,” however, as used herein, is meant to cover only aryls substituted with one or more halogens.
Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g.“3 to 7 membered”), the term“member” refers to a carbon or heteroatom.
As used herein, the term "alkylaryl" includes alkyl groups, as defined above, substituted by aryl groups, as defined above. The aryl group may be connected at any point on the alkyl group. The term C4-C16 alkylaryl includes alkylaryl groups having a total of 4 to 16 carbon atoms, counting the carbon atoms on the alkyl group and aryl group together. Examples of alkylaryl groups include but are not limited to benzyl (phenylmethyl), phenyl ethyl, and naphthylmethyl. The alkylaryl group may be substituted or unsubstituted. Substituents are not counted towards the total number of atoms in the alkylaryl group, so long as the total atoms in the substituent(s) are not larger than the alkylaryl group.
Further, a structure represented enerall b the formula:
Figure imgf000039_0001
as used herein refers to a ring structure, for example, but not limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable“n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
Figure imgf000040_0001
and the like.
A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
A substituent bearing a broken bond, such as the example shown below, means that the substituent is directly bonded to the molecule at the indicated position. No additional methylene (CH2) groups are implied. The symbol ( ) denotes the point of attachment of a moiety of the molecule.
Figure imgf000040_0002
Substituents bearing two broken bonds, such as the example shown below, means that the orientation of the atoms is as-indicated, left to right and should be inserted into a molecule in the orientation shown. No additional methylene (CH2) groups are implied unless specifically indicated.
Figure imgf000040_0003
When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being“absent,” the named atom is replaced by a direct bond.
Each of above terms (e.g. ,“alkyl,”“heteroalkyl,”“cycloalkyl, and “heterocycloalkyl”,“aryl,”“heteroaryl,”“phosphonate,” and“sulfonate” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.
Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl monovalent and divalent derivative groups (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’,-C(O)NR’R”, -OC(O)NR’R”, - NR”C(O)R’, -NR’-C(O)NR”R’”, -NR”C(O)OR’, -NR-C(NR’R”)=NR’”, -S(O)R’, - S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and -NO2 in a number ranging from zero to (2m’+l), where m’ is the total number of carbon atoms in such groups. R’, R”, R’” and R”” each may independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. As used herein, an“alkoxy” group is an alkyl attached to the remainder of the molecule through a divalent oxygen. When a compound of the disclosure includes more than one R group, for example, each of the R groups is independently selected as are each R’, R”, R’” and R”” groups when more than one of these groups is present. When R’ and R” are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR’R” is meant to include, but not be limited to, 1- pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and - CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).
Similar to the substituents described for alkyl groups above, exemplary substituents for aryl and heteroaryl groups (as well as their divalent derivatives) are varied and are selected from, for example: halogen, -OR’, -NR’R”, -SR’, -halogen, - SiR’R”R’”, -OC(O)R’, -C(O)R’, -CO2R’, -C(O)NR’R”, -OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R’”, -NR”C(O)OR’, -NR-C(NR’R”R’”)=NR””, -NR- C(NR’R”)=NR’” -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and -NO2, -R’, - N3, -CH(Ph)2, fluoro(C1-C4)alkoxo, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on aromatic ring system; and where R’, R”, R’” and R”” may be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the disclosure includes more than one R group, for example, each of the R groups is independently selected as are each R’, R”, R’” and R”” groups when more than one of these groups is present.
Two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR’)q-U-, wherein T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR’- or a single bond, and r is an integer of from 1 to 4.
One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of aryl or heteroaryl ring may optionally be replaced with a substituent of the
formula -(CRR’)s-X’- (C”R’”)d-, where s and d are independently integers of from 0 to 3, and X’ is -O-, -NR’-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR’-. The substituents R, R’, R” and R’” may be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
As used herein, the term“acyl” refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
The terms“alkoxyl” or“alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl–O–) or unsaturated (i.e., alkenyl–O– and alkynyl–O–) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,”“alkenyl,” and“alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.
The term“alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
“Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term“aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
“Aralkyloxyl” refers to an aralkyl-O– group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.
“Alkoxycarbonyl” refers to an alkyl-O-CO– group. Exemplary
alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
“Aryloxycarbonyl” refers to an aryl-O-CO– group. Exemplary
aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
“Aralkoxycarbonyl” refers to an aralkyl-O-CO– group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
“Carbamoyl” refers to an amide group of the formula–CONH2.
“Alkylcarbamoyl” refers to a R’RN–CO– group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and/or substituted alkyl as previously described. “Dialkylcarbamoyl” refers to a R’RN–CO– group wherein each of R and R’ is independently alkyl and/or substituted alkyl as previously described.
The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula–O—CO—OR.
“Acyloxyl” refers to an acyl-O– group wherein acyl is as previously described. The term“amino” refers to the–NH2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms “acylamino” and“alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
An“aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure–NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure–NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure–NR’R”R”’, wherein R’, R”, and R’” are each independently selected from the group consisting of alkyl groups. Additionally, R’, R”, and/or R’” taken together may optionally be–(CH2)k– where k is an integer from 2 to 6.
Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
The amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl–S–) or unsaturated (i.e., alkenyl–S– and alkynyl–S–) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like. “Acylamino” refers to an acyl-NH– group wherein acyl is as previously described.“Aroylamino” refers to an aroyl-NH– group wherein aroyl is as previously described.
The term“carbonyl” refers to the–(C=O)– group.
The term“carboxyl” refers to the–COOH group. Such groups also are referred to herein as a“carboxylic acid” moiety.
The terms“halo,”“halide,” or“halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C1-C4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.
The term“hydroxyl” refers to the–OH group. The term“hydroxyalkyl” refers to an alkyl group substituted with an–OH group.
The term“mercapto” refers to the–SH group.
The term“oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.
The term“nitro” refers to the–NO2 group.
The term“thio” refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
The term“sulfate” refers to the–SO4 group.
The term thiohydroxyl or thiol, as used herein, refers to a group of the formula –SH.
The term ureido refers to a urea group of the formula–NH—CO—NH2. Unless otherwise explicitly defined, a“substituent group,” as used herein, includes a functional group selected from one or more of the following moieties, which are defined herein:
(A) -OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from:
(i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from:
(a) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, substituted with at least one substituent selected from oxo, -OH, -NH2, -SH, -CN, - CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl. A“lower substituent” or“lower substituent group,” as used herein means a group selected from all of the substituents described hereinabove for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5- C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
A“size-limited substituent” or“size-limited substituent group,” as used herein means a group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or
unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.
It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms (racemates), by asymmetric synthesis, or by synthesis from optically active starting materials. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column. Many geometric isomers of olefins, C=N double bonds, and the like also can be present in the compounds described herein, and all such stable isomers are contemplated in the presently disclosed subject matter. Cis and trans geometric isomers of the compounds of the presently disclosed subject matter are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral (enantiomeric and diastereomeric), and racemic forms, as well as all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
The compounds herein described may have one or more charged atoms. For example, the compounds may be zwitterionic, but may be neutral overall. Other embodiments may have one or more charged groups, depending on the pH and other factors. In these embodiments, the compound may be associated with a suitable counter-ion. It is well known in the art how to prepare salts or exchange counter-ions. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Counter-ions may be changed, for example, by ion-exchange techniques such as ion- exchange chromatography. All zwitterions, salts and counter-ions are intended, unless the counter-ion or salt is specifically indicated. In certain embodiments, the salt or counter-ion may be pharmaceutically acceptable, for administration to a subject. Pharmaceutically acceptable salts are discussed later.
As used herein, a "protecting group" is a chemical substituent which can be selectively removed by readily available reagents which do not attack the regenerated functional group or other functional groups in the molecule. Suitable protecting groups are known in the art and continue to be developed. Suitable protecting groups may be found, for example in Wutz et al. ("Greene's Protective Groups in Organic Synthesis, Fourth Edition," Wiley-Interscience, 2007). Protecting groups for protection of the carboxyl group, as described by Wutz et al. (pages 533-643), are used in certain embodiments. In some embodiments, the protecting group is removable by treatment with acid. Specific examples of protecting groups include but are not limited to, benzyl, p-methoxybenzyl (PMB), tertiary butyl (tBu),
methoxymethyl (MOM), methoxyethoxymethyl (MEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), benzyloxymethyl (BOM), trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), and triphenylmethyl (trityl, Tr). Persons skilled in the art will recognize appropriate situations in which protecting groups are required and will be able to select an appropriate protecting group for use in a particular circumstance.
Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13C- or I4C-enriched carbon are within the scope of this disclosure.
The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
ii. Pharmaceutical Salts
The compounds of the present disclosure may exist as pharmaceutically acceptable salts. The term“pharmaceutically acceptable salts” is meant to include salts of active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrates, (e.g. (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures), or teoclate. These salts may be prepared by methods known to those skilled in art. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).
Also included are base addition salts such as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like, see, for example, Berge et al,“Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1- 19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
In addition to salt forms, the present disclosure provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. Additionally, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present disclosure when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
iii. Pharmaceutical Compositions
The compounds disclosed herein can be formulated into various compositions, for use in diagnostic and imaging methods. Generally, a pharmaceutical composition comprises an effective amount (e.g., a or detectably effective amount) of a compound described hereinabove.
A presently disclosed composition can be formulated as a pharmaceutical composition, which comprises a presently disclosed compound and pharmaceutically acceptable carrier. By a "pharmaceutically acceptable carrier" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. For a discussion of pharmaceutically acceptable carriers and other components of pharmaceutical compositions, see, e.g., Remington's Pharmaceutical Sciences, 18l ed., Mack Publishing Company, 1990. Some suitable pharmaceutical carriers will be evident to a skilled worker and include, e.g., water (including sterile and/or deionized water), suitable buffers (such as PBS), physiological saline, cell culture medium (such as DMEM), artificial cerebral spinal fluid, or the like.
One skilled in the art will appreciate that the particular formulation will depend, in part, upon the particular agent that is employed, and the chosen route of administration. Accordingly, there is a wide variety of suitable formulations of compositions of the presently disclosed subject matter.
One skilled in the art will appreciate that a suitable or appropriate formulation can be selected, adapted or developed based upon the particular application at hand. Dosages for presently disclosed compositions can be in unit dosage form. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for animal (e.g. human) subjects, each unit containing a predetermined quantity of a presently disclosed agent, alone or in combination with other therapeutic agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired effective amount or effective concentration of the agent in the individual patient.
The dose of a presently disclosed composition, administered to an animal, particularly a human, in the context of the presently disclosed subject matter should be sufficient to produce at least a detectable amount of a diagnostic response in the individual over a reasonable time frame. The dose used to achieve a desired effect will be determined by a variety of factors, including the potency of the particular agent being administered, the pharmacodynamics associated with the agent in the host, the severity of the disease state of infected individuals, other medications being administered to the subject, and the like. The size of the dose also will be determined by the existence of any adverse side effects that may accompany the particular agent, or composition thereof, employed. It is generally desirable, whenever possible, to keep adverse side effects to a minimum. The dose of the biologically active material will vary; suitable amounts for each particular agent will be evident to a skilled worker.
A "pharmaceutically acceptable carrier" refers to a biocompatible solution, having due regard to sterility, p[Eta], isotonicity, stability, and the like and can include any and all solvents, diluents (including sterile saline, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other aqueous buffer solutions), dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and the like. The pharmaceutically acceptable carrier also can contain stabilizers, preservatives, antioxidants, or other additives, which are well known to one of skill in the art, or other vehicles as known in the art.
iv. General Definitions
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Particular definitions are provided herein for clarity. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.
A“cancer” in an animal refers to the presence of cells possessing
characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells.
By "control" is meant a standard or reference condition.
By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, organ, organism, or subject.
The term "administering" as used herein refers to contacting a subject with a presently disclosed agent.
By "delivery device" is meant any device that provides for the release of an imaging agent. Exemplary delivery devices include tablets and pills, described below, as well as syringes, osmotic pumps, indwelling catheters, delayed-release and sustained-release biomaterials. Following long-standing patent law convention, the terms“a,”“an,” and“the” refer to“one or more” when used in this application, including the claims. Thus, for example, reference to“a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms“comprise,” “comprises,” and“comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term“include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term“about” even though the term“about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term“about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term“about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range. EXAMPLES
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods. EXAMPLE 1
Synthesis and Evaluation of Reversed Carbamates Based Agents for Imaging of
PSMA and Cancer Radiotherapy
Overview
Radiolabeled urea-based low-molecular weight inhibitors of the prostate- specific membrane antigen (PSMA) are under intense investigation as imaging and therapeutic agents for prostate and other cancers. Here a new class of potent PSMA inhibitors based on the reversed carbamate scaffold has been reported, to maintain glutamate and S1 pocket side chain geometry and for putative binding to zinc.
Reversed carbamates contain the oxy-pentanedioic acid moiety (OPA) (FIG.3A).
In an effort to provide agents with less non-target organ uptake than the ureas, 18F-labeled inhibitors of PSMA based on reversed carbamate scaffolds have been synthetized. 4-Bromo-2-[18F]fluorobenzoyl-lysine-oxy-pentanedioic acid (OPA) reversed carbamate [18F]23 ([18F]XY-52) and 4-iodo-2-[18F]fluorobenzoyl-lysine OPA reversed carbamate [18F]24 ([18F]XY-47) in particular exhibited high target- selective uptake in PSMA+ PC3 PIP tumor xenografts, with tumor-to-kidney ratios > 1 by 4 h post-injection, an important benchmark. Because of its high tumor uptake (90% injected dose per gram of tissue at 2 h post-injection) and high tumor-to-organ ratios, [18F]23 ([18F]XY-52) is promising for clinical translation. Prolonged tumor- specific uptake demonstrated by [18F]24 ([18F]XY-47), which did not reach equilibrium during the 4 h study period, suggests carbamates as alternative scaffolds for mitigating renal dose for radiotherapeutics. Material and Methods
General procedures. Solvents and chemicals purchased from commercial sources were of analytical grade or better and used without further purification. Tert- butyl-1,3-diisopropylisourea, L-glutamic acid di-tert-butyl ester, Nε -Boc-lysine-tert- butyl ester hydrochloride, 1-hydroxybenzotriazole monohydrate and 2-(1H- benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Chem Impex International Inc. (Wooddale, IL). 1,1’- Carbonyldiimidazole, iodomethane, N-hydroxysuccinimide, (diacetoxyiodo)benzene, triethylsilane (Et3SiH), diisopropylethylamine (DIEA) and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). 4-bromo-2-nitro-benzaldehyde was purchased from Combi-Block (San Diego, CA). (S)-6-((tert-butoxycarbonyl)amino)- 2-hydroxyhexanoic acid (Makarasen, 2009) 4-iodo-2-nitro-benzaldehyde (Litosh, 2009) and (S)-dimethyl 2-hydroxypentanedioate (Winkler, 2013) were synthesized by previously reported procedures. Analytical thin-layer chromatography (TLC) was performed using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates and visualized by ultraviolet light (254 nm), I2 and 1 % ninhydrin in EtOH. Flash chromatography was performed using silica gel (MP SiliTech 32-63 D 60Å) purchased from Bodman (Aston, PA). All in vitro PSMA binding studies were performed in triplicate. 1H NMR spectra were recorded on a Bruker Ultrashield™ 400 MHz or 500 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield by reference to proton resonances resulting from incomplete deuteration of the NMR solvent. Low resolution ESI mass spectra were obtained on a Bruker Daltonics Esquire 3000 Plus spectrometer. High resolution mass spectra were obtained at the University of Notre Dame Mass Spectrometry & Proteomics Facility, Notre Dame, IN, using ESI either by direct infusion on a Bruker microTOF-II or by LC elution via an ultra-high pressure Dionex RSLC with C18 column coupled to a Bruker microTOF-Q II. HPLC purification was performed using a Phenomenex C18 Luna 10 × 250 mm2 column on Agilent Technologies 1260 Infinity Preparative HPLC System. HPLC purification of [18F] labeled compounds were performed on a Varian Prostar System (Palo Alto, CA), equipped with a Varian ProStar 325 UV-Vis variable wavelength detector and a Bioscan Flow-count in-line Radioactivity detector, all controlled by Galaxie software. The specific radioactivity was calculated as the ratio of the radioactivity eluting at the retention time of product during the preparative HPLC purification to the mass corresponding to the area under the curve of the UV absorption.
Synthesis and Characterization of Reversed Carbamates.
N-Succinimidyl 4-bromo/iodo-2-fluorobenzoate: 4-bromo-2-fluorobenzoic acid (or 4-iodo-2-fluorobenzoic acid) 1 mmol and N-hydroxysuccinimide 125 mg (1.08 mmol) were dissolved in 2 mL dry DMF. To the solution, N,N- dicyclohexylcarbodiimide 170 µL (1.10 mmol) was added and the reaction was kept at room temperature overnight. After a flash column chromatography with ethyl acetate/hexane, 1:1, the N-succinimidyl 4-bromo/4-iodo-2-fluorobenzoates were obtained as white solids. TLC: silica gel, 1:1 ethylacetate:hexane. N-succinimidyl 4- bromo-2-fluorobenzoate (205mg) was obtained in a yield of 65%; Rf = 0.6. N- succinimidyl 4-iodo-2-fluorobenzoate (330mg) was obtained in a yield of 52%; Rf = 0.6. N-Succinimidyl 4-bromo-2-fluorobenzoate: 1H-NMR (500 MHz, CDCl3): δ 7.97-7.94 (m, 1H), 7.48-7.44 (m, 2H), 2.92 (s, 4H). N-Succinimidyl 4-iodo-2- fluorobenzoate: 1H-NMR (500 MHz, CDCl3): δ 7.78-7.75 (m, 1H), 7.68-7.64 (m, 2H), 2.92 (s, 4H).
(S)-dimethyl 2-((1H-imidazole-1-carbonyl)oxy)pentanedioate 20: (S)- dimethyl 2-hydroxypentanedioate 19 (220 mg, 1.25 mmol) and 1,1’- carbonyldiimidazole (300 mg, 1.85 mmol) were mixed in 5 mL anhydrous methylene chloride. The reaction was kept at room temperature for 1 h. (S)-dimethyl 2-((1H- imidazole-1-carbonyl)oxy)pentanedioate (290 mg was obtained by silica gel flash column chromatography (Hexane:Ethyl acetate 1:2, Rf 0.4). The yield is 85%.1H NMR (400 MHz, CDCl3): δ 8.20(s, 1H), 7.49(s, 1H), 7.12(s, 1H), 5.35(dd, J1 = 8.0 Hz, J2 = 4.8 Hz, 1H), 3.83(s, 3H), 3.72(s, 3H), 2.58-2.34(m, 4H).13C NMR (125 MHz, CDCl3) δ 172.3, 168.7, 148.0, 137.2, 131.0, 117.2, 74.4, 53.0, 29.4, 26.1. MS: Calculated for [C11H15N2O6]+, 271.0925 [M+H]+; Found 271.0932.
(10S,14S)-10-tert-butyl 14,16-dimethyl 2,2-dimethyl-4,12-dioxo-3,13- dioxa-5,11-diazahexadecane-10,14,16-tricarboxylate 21: (S)-dimethyl 2-((1H- imidazole-1-carbonyl)oxy)pentanedioate 20 (110 mg, 0.41 mmol) was dissolved in 2 mL MeCN and 1mL MeI. The mixture was sealed and heated at 55oC for 3 h. The solvent was removed under vacuum. Then, a mixture of Nε-Boc-lysine-tert-butyl ester hydrochloride (275 mg, 0.81 mmol) and triethylamine (0.5 mL) in 5 mL DMSO was added. The reaction was kept under room temperature overnight. (10S,14S)-10-tert- butyl 14,16-dimethyl 2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,11-diazahexadecane- 10,14,16-tricarboxylate was obtained by flash column chromatography (Hexane:Ethyl acetate 1:2, Rf 0.5). The yield is 150mg (75 %).1 H NMR (400MHz δ 5.49(br, 1H), 5.08-5.06(m, 1H), 4.70(br, 1H), 3.80(s, 3H), 3.73(s, 3H) 3.16-3.13(m, 2H), 2.54- 2.48(m, 2H), 2.28-2.17(m, 2H), 1.89-1.86(m, 1H), 1.68-1.64(m, 1H), 1.63-1.41(m, 22H).13C NMR (125 MHz, CDCl3) δ 172.8, 171.3, 170.8, 156.1, 154.9, 82.3, 79.1, 74.6, 71.6, 54.3, 52.4, 51.8, 40.2, 32.5, 29.5, 28.5, 28.0, 26.5, 22.2. MS: Calculated for [C23H41N2O10]+, 505.2756 [M+H]+; Found 505.2735.
(S)-2-((((S)-5-amino-1-carboxypentyl)carbamoyl)oxy)pentanedioic acid 22: (10S,14S)-10-tert-butyl 14,16-dimethyl 2,2-dimethyl-4,12-dioxo-3,13-dioxa-5,11- diazahexadecane-10,14,16-tricarboxylate(21) (150 mg, 0.30 mmol) was treated with a 6 mL solution of TFA/methylene chloride(1/1) for 2 h. The solvent was removed under vacuum and the remaining material was redissolved in 5 mL THF/H2O (1/1). To the solution, LiOH monohydrate (40 mg , 1 mmol) was added and the reaction was stirred at room temperature for 4 hours. The final product was purified by HPLC in 80 mg, yield 65%.1H NMR (400 MHz, D2O): δ 4.82-4.77(m, 1H), 4.11-4.07(m, 1H), 2.90(t, J = 7.2 Hz, 2H), 2.62-2.42(m, 2H), 2.14-2.00(m, 2H), 1.87-1.80(m, 1H), 1.70- 1.60(m, 3H), 1.41-1.35(m, 2H).13C NMR (125 MHz, D2O): δ 177.1, 175.9, 174.6, 157.1, 72.3, 53.7, 39.2, 30.2, 29.3, 26.1, 21.9. MS: Calculated for [C12H21N2O8]+, 321.1292 [M+H]+; Found 321.1318. HPLC (10 × 250 mm Phenomenix Luna C18 column, mobile phase 97/3/0.1% water/acetonitrile/TFA, flow 10 mL/min). 22 eluted at 8 min.
(S)-2-((((S)-1-carboxy-5-(4-fluorobenzamido)pentyl)carbamoyl)oxy) pentanedioic acid 13 (XY-48). (S)-2-((((S)-5-amino-1-carboxypentyl) carbamoyl) oxy) pentanedioic acid TFA salt 22 (10 mg, 0.024 mmol) was dissolved in 1 mL DMSO and 20 µL triethylamine. To the solution, N-succinimidyl 4-fluorobenzoate (11.5 mg, 0.045 mmol) was added. The resulting solution was kept at room temperature for 2 hours. After the solvent removed under vacuum, 9 mg product was obtained after HPLC purification. Yield is 89 %. 1H NMR (500 MHz, D2O/CD3CN 1:1): δ 7.77- 7.73 (m, 2H), 7.17-7.13(m, 2H), 4.82-4.80(m, 1H), 4.06-4.04(m, 1H), 3.29-3.26(m, 2H), 2.42-2.36(m, 2H), 2.10-2.06(m, 1H), 2.00-1.95(m, 1H), 1.80-1.76(m, 1H), 1.67- 1.63(m, 1H), 1.54(m, 2H), 1.38(m, 2H), 13C NMR (125 MHz, D2O/CD3CN 1:1): δ 178.4, 177.8, 175.8, 170.6, 168.3/166.4(coupled with F), 159.1, 133.4,
132.6/132.5(coupled with F), 118.3/118.2(coupled with F), 74.5, 56.7, 42.2, 33.5, 32.0, 31.0, 28.9, 25.4. MS: Calculated for [C19H23FN2NaO9]+, 465.1280 [M+Na]+; Found 465.1306. HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 75/25/0.1% water/acetonitrile/TFA, flow 4 mL/min). 13 (XY-48) eluted at 11.5 min.
(S)-2-((((S)-5-(4-bromo-2-fluorobenzamido)-1-carboxypentyl)
carbamoyl)oxy)pentanedioic acid 23 (XY-52). (S)-2-((((S)-5-amino-1- carboxypentyl)carbamoyl)oxy) pentanedioic acid TFA salt 22. (10 mg, 0.024 mmol) was dissolved in 1 mL DMSO and 20 µL triethylamine. To the solution, N- succinimidyl 4-bromo-2-fluorobenzoate (14 mg, 0.045 mmol) was added. The resulting solution was kept at room temperature for 2 hours. After the solvent removed under vacuum, and HPLC purification, 8 mg product was obtained. Yield is 64 %.1H NMR (500 MHz, D2O/CD3CN 1:1): δ 7.55(t, J = 8.2 Hz, 1H), 7.43-7.41(m, 2H), 4.82-4.80(m, 1H), 4.06-4.03(m, 1H), 3.29(t, J = 6.6 Hz, 2H), 2.43-2.36(m, 2H), 2.10-2.05(m, 1H), 2.01-1.95(m, 1H), 1.85-1.76(m, 1H), 1.67-1.63(m, 1H), 1.55- 1.50(m, 2H), 1.40-1.36(m, 2H).13C NMR (125 MHz, D2O/CD3CN 1:1): δ 176.2, 175.5, 173.6, 165.1, 160.9/158.9(coupled with F), 156.7, 132.1, 128.5, 125.8, 122.3/122.2(coupled with F), 120.3/120.1(coupled with F), 72.2, 54.3, 39.9, 31.1, 29.7, 28.5, 26.5, 22.9. MS: Calculated for [C19H22BrFN2NaO9]+, 543.0385 [M+Na]+; Found 543.0372. HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 70/30/0.1% water/acetonitrile/TFA, flow 4 mL/min). 23 (XY-52) eluted at 12.9 min.
(S)-2-((((S)-1-carboxy-5-(2-fluoro-4-iodobenzamido pentyl)
carbamoyl)oxy)pentanedioic acid 24 (XY-47). (S)-2-((((S)-5-amino-1- carboxypentyl) carbamoyl) oxy) pentanedioic acid TFA salt 22. (10 mg, 0.024 mmol) was dissolved in 1 mL DMSO and 20 uL triethylamine. To the solution, N- succinimidyl 4-iodo-2-fluorobenzoate (15 mg, 0.045 mmol) was added. The resulting solution was kept at room temperature for 2 hours. After the solvent removed under vacuum, and HPLC purification 7 mg was obtained . Yield is 51 %.1H NMR (500 MHz, D2O/CD3CN 1:1): δ 7.63-7.59(m, 2H), 7.39(t, J = 8.0 Hz, 1H), 4.82-4.79(m, 1H), 4.06-4.03(m, 1H), 3.28(t, J = 6.8 Hz, 2H), 2.46-2.34(m, 2H), 2.12-2.05(m, 1H), 2.01-1.95(m, 1H), 1.82-1.75(m, 1H), 1.69-1.61(m, 1H), 1.58-1.48(m, 2H), 1.42- 1.34(m, 2H).13C NMR (125 MHz, D2O/CD3CN 1:1): δ 176.2, 175.5, 173.5, 165.2, 160.6/158.6(coupled with F), 156.8, 134.6, 132.1, 126.1/125.9 (coupled with F), 123.0/122.9(coupled with F), 97.5/97.4 (coupled with F), 72.2, 54.4, 40.0, 31.2, 29.7, 28.7, 26.6, 23.0. MS: Calculated for [C19H23IFN2O9]+, 569.0427 [M+H]+; found 569.0438. HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 70/30/0.1% water/acetonitrile/TFA, flow 4 mL/min). 24 (XY-47) eluted at 17.0 min. (S)-2-(3-((S)-5-(4-bromo-2-fluorobenzamido)-1-carboxypentyl)ureido) pentanedioic acid 26 (XY- 58). (S)-2-(3-((S)-5-amino-1- carboxypentyl)ureido)pentanedioic acid TFA salt 25 (10 mg, 0.024 mmol) was dissolved in 1 mL DMSO and 20 uL triethylamine. To the solution, N-succinimidyl 4- bromo-2-fluorobenzoate (14 mg, 0.045 mmol) was added. The resulting solution was kept at room temperature for 2 hours. After the solvent removed under vacuum, 9 mg was obtained after HPLC purification. Yield is 71 %. 1H NMR (500 MHz,
D2O/CD3CN 1:1): δ 7.58-7.54(m, 1H), 7.46-7.40(m, 2H), 4.16-4.09(m, 2H), 3.28(t, J = 6.8 Hz, 2H), 2.35(t, J = 7.5 Hz, 2H), 2.06-1.99(m, 1H), 1.85-1.71(m, 2H), 1.66- 1.50(m, 3H), 1.40-1.34(m, 2H) 13C NMR (125 MHz, D2O/CD3CN 1:1): δ 176.6, 176.5, 175.7, 165.2, 160.9/158.9(coupled with F), 159.0, 132.0, 128.5,
125.8/125.7(coupled with F), 122.3/122.2(coupled with F), 120.3/120.1(coupled with F), 53.4, 52.7, 39.9, 31.4, 30.4, 28.6, 27.1, 22.9. MS: Calculated for
[C19H24BrFN3O8]+, 520.0725 [M+H]+; Found 520.0762. HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 75/25/0.1% water/acetonitrile/TFA, flow 10 mL/min). 26 (XY- 58) eluted at 7.2 min.
(S)-2-(3-((S)-5-(4-iodo-2-fluorobenzamido)-1-carboxypentyl)ureido) pentanedioic acid 27 (XY-44). (S)-2-(3-((S)-5-amino-1- carboxypentyl)ureido)pentanedioic acid TFA salt 25 (10 mg, 0.031 mmol) was dissolved in 1 mL DMSO and 20 uL triethylamine. To the solution, N-succinimidyl 4-iodo-2-fluorobenzoate (14 mg, 0.045 mmol) was added. The resulting solution was kept at room temperature for 2 hours. After the solvent removed under vacuum, 11 mg of 27 was obtained after HPLC purification. Yield is 81 %. 1H NMR (500 MHz, D2O/CD3CN 1:1): δ, 7.63-7.60(m, 2H), 7.38(t, J = 7.8 Hz, 1H), 4.14- 4.07( m, 2H), 3.27(t, J = 6.9 Hz, 2H), 2.34(t, J = 7.6 Hz, 2H), 2.05-1.96(m, 1H), 1.85-1.72(m, 2H), 1.65-1.49(m, 3H), 1.38-1.33(m, 2H), 13C NMR (125 MHz, D2O/CD3CN 1:1): δ 176.7, 176.0, 165.2, 160.5/158.5(coupled with F), 159.1, 134.6, 132.0, 126.1/125.9(coupled with F), 122.9/122.8(coupled with F), 97.5, 53.6, 53.0, 40.0, 31.6, 30.5, 28.7, 27.4, 23.0. MS: Calculated for [C19H23IFN3NaO8]+, 590.0406 [M+Na]+; Found 590.0406. HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 75/25/0.1% water/acetonitrile/TFA, flow 10 mL/min). 27 eluted at 8.5 min. Scheme 1.
Figure imgf000060_0001
(a) carbonyldiimidazole, CH2Cl2, rt, 1h, (b) MeI, MeCN, 55oC, 3h (c) N-Boc-lysine-tert-butyl ester hydrochloride, Et3N, DMSO, rt, overnight, (d) TFA/CH2Cl2(1/1), rt, 2 h (e) LiOH, THF/H2O (1/1), rt, 4h,
(f) N-succinimidyl-4-fluorobenzoate or N-succinimidyl-4[18F]fluorobenzoate, Et3N, DMSO, rt, 2h
(g) N-succinimidyl-4-[125I]iodobenzoate or N-succinimidyl-4-iodobenzoate, diisopropylethylamine, DMSO, rt, 1h; (h) N-succinimidyl 4-bromo-2-fluorobenzoate, Et3N, DMSO, rt, 2h; (i) N-succinimidyl 4-iodo-2-fluorobenzoate, Et3N, DMSO, rt, 2h; NAALADase Assay. The PSMA inhibitory activity was determined using a modification of the fluorescence-based Amplex Red Glutamic Acid Assay (Life Technologies, Grand Island, NY)(Kozikowski, 2004). Briefly, lysates of LNCaP cell extracts (25 µL) were incubated with the inhibitor (12.5 µL) in the presence of 4 µM N-acetylaspartylglutamate (NAAG) (12.5 µL) for 120 min. The amount of the glutamate released by NAAG hydrolysis was measured by incubating with a working solution (50 µL) of the Amplex Red Glutamic Acid Kit for 60 min. Fluorescence was measured with a VICTOR3V multilabel plate reader (Perkin Elmer Inc., Waltham, MA) with excitation at 490 nm and emission at 642 nm. Inhibition curves were determined using semi-log plots and IC50 values were determined at the
concentration at which enzyme activity was inhibited by 50 %. Enzyme inhibitory constants (Ki values) were generated using the Cheng-Prusoff conversion (Cheng, 1973). Assays were performed in triplicate. Data analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, California).
Cell Lines and Mouse Models. Sublines of the androgen-independent PC3 human prostate cancer cell line, originally derived from an advanced androgen independent bone metastasis, were used. These sublines have been modified to express high (PC3 PIP) or possess low (PC3 flu) levels of PSMA, and were generously provided by Dr. Warren Heston (Cleveland Clinic). PSMA-expressing (PC3 PIP), non-expressing (PC3 flu) PCa cell lines, were grown in RPMI 1640 medium (Corning Cellgro, Manassas, VA) containing 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St.Louis, MO) and 1 % Penicillin-Streptomycin (Corning Cellgro, Manassas, VA). PC-3 PIP cells were grown under 20 µg/mL of puromycin selection in the growthmedium to maintain PSMA expression. All cell cultures were maintained in an atmosphere containing 5 % carbon dioxide (CO2), at 37.0 °C in a humidified incubator. Animal studies were carried out in full compliance with the regulations of the Johns Hopkins Animal Care and Use Committee. Six- to eight- week-old male, non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice (Johns Hopkins Immune Compromised Core) were implanted subcutaneously (sc) with PC3 PIP (PSMA+) and PC3 flu (PSMA-) cells (1 x 106 in 100 μL of HBSS (Corning Cellgro, Manassas, VA) at the forward right and left flanks, respectively. Mice were imaged or used in ex vivo biodistribution assays when the xenografts reached 5 to 7 mm in diameter.
Radiosynthesis.
(S)-2-((((S)-1-carboxy-5-(4-[18F]fluorobenzamido)pentyl)carbamoyl)oxy) pentanedioic acid [18F]13 ([18F]XY-48). [18F]SFB was prepared according to a literature procedure (J. Label. Comp. Radiopharm, 2008, 51, 68-71). A methylene chloride solution of [18F]SFB was evaporated under a stream of argon gas. To this residue was added 3 mg of (S)-2-((((S)-5-amino-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid 22 in 200 µL dry DMF and 5 µL triethylamine. This was heated for 10 min at 50 °C in a 40 W microwave (Resonance Instruments), cooled to room temperature, acidified with trifluoroacetic acid, diluted with water, and injected onto a radio-HPLC (10-mm × 250-mm Phenomenix Luna C18 column, mobile phase 75/25/0.1% water/acetonitrile/TFA, flow 4 mL/min). 13 (XY-48) eluted at 11.5 min. The product HPLC fraction was collected, neutralized with sodium bicarbonate, concentrated under vacuum, and dissolved in sterile saline for injection. The non-decayed corrected radiochemical yield from [18F]SFB was 5 %.
Scheme 2.
Figure imgf000062_0001
(S)-2-(((((S)-1-carboxy-5-(4-[125I]iodobenzamido)pentyl)
carbamoyl)oxy)pentanedioic acid [125I]32 ([125I]XY-57). Referring to Scheme 3, N- succinimidyl-4-[125I]iodobenzoate was prepared by a modification of the method of Dekker et al (Dekker et al., 2015). In particular, to a solution of 0.1 mg N- succinimidyl-4-tributylstannylbenzoate (Dekker et al., 2015) in 100 µL methanol was added 2 µL glacial acetic acid, 7.3mCi of Na[125I] (Perkin Elmer, Billerica, MA), and 5 µL of solution of N-chlorosuccinimide in methanol (10mg N-chlorosuccinide in 1.5 mL methanol). This was allowed to stand at room temperature for 20 min, then diluted with 200 µL methanol and injected onto a semi-preparative-HPLC (10 X 250 mm, 10 micron, Phenomenex Luna C18 column, 55/45/0.1
water/acetonitrile/trifluoroacetic acid, flow = 4 mL/m). N-succinimidyl-4- [125I]iodobenzoate eluted at 14 min. This was diluted with 20 mL water, loaded onto an activated Waters C18 Sep-Pak Plus cartridge, washed with 10 mL water, dried with a stream of nitrogen for 2min, then eluted with 2mL methylene chloride through a Na2SO4 drying cartridge. The methylene chloride solution of N-succinimidyl-4- [125I]iodobenzoate (5.9 mCi) was stored at 0-2 °C. The methylene chloride solution was then evaporated to dryness under a stream of nitrogen and to this was added a solution of 22 (2 mg/200µL DMSO). To this solution is added 5 µL
diisopropylethylamine. Reaction is shaken and allowed to stand at room temperature for one hour. The reaction is then acidified by the addition of 20 µL TFA and diluted with 1mL water. This is injected onto a semi-preparative-HPLC (10 X 250mm, 10 micron, Phenomenex Luna C18 column, 72/28/0.1 water/acetonitrile/trifluoroacetic acid, flow = 4mL/m). Retention time of [125I]32 ([18F]XY-57) were 12 min and 11 min respectively. Product fraction was diluted with 40 mL water, loaded onto an activated Waters C18 Sep-Pak Plus cartridge, washed with 10 mL water, dried with a stream of nitrogen for 2min, then eluted with 2 mL ethanol. Ethanol solution was concentrated under a stream of nitrogen until dryness and reconstituted in buffer for in-vitro assay. Starting with 2.0 mCi and 2.2 mCi of N-succinimidyl-4- [125I]iodobenzoate, 1.8 and 2.0 mCi of [125I]32 ([18F]XY-57) was prepared. Scheme 3.
Figure imgf000063_0001
(S)-2-((((S)-5-(4-bromo-2-[18F]fluorobenzamido)-1-carboxypentyl) carbamoyl)oxy)pentanedioic acid [18F]23 ([18F]XY-52) and (S)-2-((((S)-1-carboxy- 5-(2-[18F]fluoro-4-iodobenzamido pentyl) carbamoyl)oxy)pentanedioic acid
[18F]24 ([18F]XY-47). 18F fluoride was produced by a General Electric PET trace biomedical cyclotron (GE HealthCare) using 18 MeV proton bombardment on an 18O- H2O target and trapped on a Chromafix 30-PS-HCO3 QMA cartridge. The cartridge was eluted with 0.5 mL of a solution of potassium bicarbonate (4.5 mg/0.5 mL) into a 3 mL Wheaton reaction vial. To this was added 15-18 mg 4,7,13,16,21,24-hexaoxa- 1,10-diazabicyclo[8.8.8]hexacosane (K2.2.2) in 1 mL of acetonitrile and heated to 100 °C under a stream of Argon gas to dryness. Further drying was accomplished by azeotropic distillation using 3 × 0.5 mL additions of acetonitrile. The vial is cooled to room temperature and a solution of 8-16 mg 4-halo-2-nitrobenzaldehyde (28b) in 250 µL DMSO is added and heated at 120 °C for 20 min then cooled to room temperature and diluted to a volume of two mL with 25 % acetonitrile/water for purification by radio-HPLC (10 × 250 mm Phenomenex Luna C18 column, 54/46/0.1
water/acetonitrile/TFA, 4 mL/m). 4-bromo-2-[18F]fluorobenzaldehyde ([18F]29b) eluted at 18.5 min. (FIG.5) , results are given in Table 1, and 4-iodo-2- [18F]fluorobenzaldehyde ([18F]29c) eluted at 23 min (FIG.6). Table 1. Radiofluorination Conditions for 4-bromo-2-[18F]fluorobenzaldehyde
([18F]29b)
Figure imgf000064_0001
a non-decay corrected microwave
The product HPLC fraction was diluted with water to a volume of 45 mL and loaded onto an Oasis HLB Sep-Pak Cartridge, washed with 5 mL water, then dried by passing Argon through the cartridge for two minutes. A sodium sulfate drying tube was then added to the end of the Oasis Sep-Pak and the aldehyde eluted with 2 mL methylene chloride into another Wheaton reaction vial. The methylene chloride is evaporated under a stream of Argon and the residue was dissolve in 0.5mL acetonitrile. To this is added 50 mg N-hydroxysuccinimide and 28 mg
(diacetoxyiodo)benzene. Vial is capped, shaken, and allowed to stand at room temperature for 15 min, then diluted with 1/1 acetonitrile/water for injection on to the radio-HPLC (10- × 250-mm Phenomenex Luna C18 column, 50/50/0.1
water/acetonitrile/TFA, 4mL/m). N-succinimidyl-4-bromo-2-[18F]fluorobenzoate 30b eluted at 12 min and N-succinimidyl-4-iodo-2-[18F]fluorobenzoate 30c eluted at 14 min. (FIG.7 and FIG.8) . The product HPLC fraction was diluted with water to a volume of 45 mL and loaded onto a Waters C18 Sep-Pak Plus Cartridge, washed with 5 mL water, then dried by passing Argon through it for two minutes. A sodium sulfate drying tube was then added to the end of the C18 Sep-Pak Plus cartridge and the ester was eluted with 2 mL methylene chloride into another Wheaton reaction vial. The methylene chloride is evaporated under a stream of Argon. To the residue was added a solution of 3 mg (S)-2-((((S)-5-amino-1- carboxypentyl)carbamoyl)oxy)pentanedioic acid in 200 µL DMSO and 5µL diisopropylethylamine. Vial was capped, shaken, and heated at 50 °C for 10 min then allowed to cool to room temperature. Reaction was acidified by the addition of 20µL TFA and diluted with water for purification by HPLC (10- × 250-mm Phenomenex Luna C18 column, 70/30/0.1 water/acetonitrile/TFA, 4mL/m). [18F]23 ([18F]XY-52) eluted at 13 min. (FIG.9) and [18F]24 ([18F]XY-47) eluted at 17 min. (FIG.11). Non- decay corrected radiochemical yield from 18F fluoride was 5 % for each. Non-decay corrected radiochemical yield from 18F fluoride was 5 % for [18F] 35 and 3% for
[18F]24 ([18F]XY-47). Specific activity ranged from 37,00-177,600 GBq/mmol (1000-4800 Ci/mmol) ) for [18F]23 ([18F]XY-52) and 31,080-92,500 GBq/mmol (840- 2500 Ci/mmol) for [18F]24 ([18F]XY-47).
Scheme 4.
Figure imgf000065_0001
Imaging study. Dynamic and whole-body PET and CT images were acquired on an eXplore VISTA small-animal PET (GE Healthcare) and an X-SPECT small SPECT/CT system (Gamma Medica Ideas), respectively. For imaging studies, anesthesia was induced with 3% and maintained under 1.5 % isoflurane (v/v) in oxygen. PET or PET-CT imaging studies were performed on NOD/SCID mice bearing PSMA+ PC-3 PIP and PSMA- PC-3 flu tumors. Following intravenous injection of [18F]13 ([18F]XY-48), [18F] 23 ([18F]XY-52) or [18F] 24 ([18F]XY-47), PET images were acquired at 10 min, 30 min, 1 h, 2 h and 4 h p.i. as a pseudodynamic scan, i.e., a sequence of successive whole-body images were acquired in two bed positions. The dwell time at each bed position was 10 min for a total scan time of 20 minutes. An energy window of 250– 700 keV was used. Images were reconstructed using the FORE/2D-OSEM method (one iteration, 16 subsets) and included correction for radioactive decay, scanner dead time, and scattered radiation.
Biodistribution. Mice bearing PSMA+ PC-3 PIP and PSMA- PC-3 flu xenografts (Chang et al., 1999) were injected via the tail vein with 740 kBq (20 μCi) of [18F]23 ([18F]XY-52) or [18F]24 ([18F]XY-47) in 200 μL of saline. At various time points post-injection, mice were sacrificed by cervical dislocation and the blood immediately collected by cardiac puncture. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large intestines, urinary bladder, PSMA+ PC-3 PIP and PSMA- PC-3 flu tumors were collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/ LKBNuclear, Inc., Mt. Waverly, Vic.
Australia). The % ID/g was calculated by comparison with samples of a dilution of a standard dose. All measurements were corrected for decay. Data are expressed as mean ± standard deviation (SD).
Molecular Modeling. All molecular modeling experiments were performed using Discovery Studio 4.0 developed by Accelrys, Inc. (San Diego, CA).
Protein and Ligand Structure Preparation. The X-ray structure of PSMA co- crystallized with the competitive inhibitor DCIBzL (PDB: 3D7H) was downloaded from the protein data bank (RCSB, http://www.rcsb.org/pdb/home/home.do). The water molecules were removed while the co-crystallized ligand was used as a template to sketch the compounds using the Sketch Molecules module in Discovery Studio.
In Situ Ligand Minimization. Each ligand was minimized while binding to its target protein using the in situ ligand minimization module with the following parameters: CHARMmas an input force field, minimization algorithm as smart minimizer, maximum minimization steps equal to 1000, minimization with RMS gradient equal to 0.001 Å, and minimization energy change set equal to zero. After the minimization protocol was executed, the in situ minimized ligands were stripped of their nonpolar hydrogens to simplify the overall view. The protein was depicted in the form of a light gray line ribbon. The bound ligand is depicted as a stick with atoms color-coded according to element: carbon (gray), nitrogen (blue), and oxygen (red) (FIG.13A and FIG.13B).
In vitro stability studies. PC-3 PIP (PSMA+) and PC-3 flu (PSMA-) cells were cultured as previously described (Banerjee et al., 2014). 300,000 PIP or flu cells were seeded into three wells each of a 6 well plate using RPMI 1640 + 10 % fetal bovine serum + 1 % Penicillin-Streptomycin (Corning Cellgro, Manassas, VA) and were grown to 80 % confluency. At the time of assay, the culture medium was refreshed and 50 µCi (1.35 kBq) of [125I]31 ([125I]XY-26) or [125I]32 ([125I]XY-57) was added to both a PIP- and flu-containing well. After radiotracer addition, the plate was returned to the incubator (humidified 37 °C, 5 % CO2) for 30 minutes. The medium was then carefully removed and saved for counting in a LKB Wallac 1282 Compugamma gamma counter (Mount Waverly, Vic, Australia). The cells were washed twice with ambient temperature PBS, pH 7.4 followed by the addition of ddH2O to lyse the cells. Lysis took place over 30 minutes inside the incubator. The lysates were then collected and counted using the gamma counter. Equal amounts of radioactivity from the supernatant and lysates were spotted onto silica gel 60 RP-18 F254S glass TLC plates (EMD Millipore Corp., Billerica, MA) and the plates were developed using a mobile phase consisting of 55 % acetonitrile, 45 % water and 0.1 % trifluoroacetic acid. The TLC plate was dried and exposed to Kodak Biomax x-ray film (Fisher Scientific) prior to digitizing using the MCID Core package (Interfocus Imaging, Cambridge, UK). Standards solutions of [125I]31 ([125I]XY-26) and [125I]32 ([125I]XY-57) had Rf values of 0.8. Intracellular and extracellular metabolites of [125I]31 ([125I]XY-26) in PC-3 PIP cells had an Rf value of approximately 0.44. Results
Chemistry. Among the potent urea-based PSMA binding ligands, 4- fluorobenzoyl- and 4-iodobenzoyl-lys-glu urea, compounds 9 (YC-I-26) and 10 (YC- I-27) respectively, have produced some of the highest affinities reported (FIG.2A and FIG.2B) (Chen, 2008). Based on these ureas, OPA-carbamate 13 (XY-48) (Scheme 1) was the initial target compound. It has been reasoned that the 4-fluorobenzoyl side-chain would provide high affinity and specificity by utilizing the S1 binding pocket and be amenable to radiolabeling with 18F.
OPA reversed carbamate 13 (XY-48) was prepared from (S)-dimethyl-2- hydroxypentanedioate 19, for which the corresponding tert-butyl ester was not easily accessible (Scheme 1). After conversion of 19 to the N-imidazolecarbamate, 20 was first transformed to its imidazolium salt via treatment with iodomethane and then reacted with Nε-Boc-lysine-tert-butyl ester to afford 21. Attempts to couple Nε-boc- lysine-tert-butyl ester and 20 directly failed to give the desired product, presumably due to side reactions involving the methyl esters. After two deprotection steps amine 22 was conjugated with N-succinimidyl-4-fluorobenzoate to give OPA- reversed carbamate 13 (XY-48).
X-ray co-crystal studies with bound urea-based ligands such as 10 have demonstrated a unique cation-π interaction with PSMA, with the benzoyl group of the ligand fully inserted into the“arginine patch” of the PSMA S1 binding pocket and an adjacent hydrophobic subpocket that can accommodate the para-iodo substituent of 10.{Barinka, 2008). That additional interaction likely accounted for the increase in binding affinity of 10 compared to 9 (Chen, 2008; Barinka, 2008). To increase the binding affinities of the reversed carbamates, iodinated reversed carbamate 32 (XY- 57) has been prepared by reacting N-succinimidyl-4-iodobenzoate with 22 (Scheme 1). For PET imaging with 18F, the 4-iodo-benzoyl group would require an [18F]fluoro substituent in the activated ortho position. With that in mind OPA reversed carbamates 23 (XY-52) and 24 (XY-47) were generated by reacting 22 with N- succinimidyl-4-bromo/iodo-2-fluorobenzoate (Scheme 1). For comparison, the corresponding ureido analogs 26 and 27 were also synthesized from urea 25 (Scheme 1) (Chen, 2012; Maresca, 2009). In vitro PSMA binding. The PSMA inhibitory activities of the prepared compounds were measured using a modification of the fluorescence-based Amplex Red Glutamic Acid assay (Kozikowski, 2004). Carbamates 12 (XY-20) and reversed carbamate 13 (XY-48) inhibited PSMA at Ki = 42 nM and 9.2 nM, respectively, significantly less potent than the corresponding ureido analog 9 (XY-I-26), (Ki = 0.25nM) (Chen, 2008). Carbamates 31 (XY-26) and reversed carbamate 32 (XY-57) inhibited PSMA at Ki = 0.9 nM and 0.04 nM, respectively. Reversed carbamates 23 (XY-52) and 24 (XY-47) demonstrated potent inhibition of PSMA, with Ki = 0.11 nM and 0.21 nM, respectively, while the corresponding ureas 26 (XY-58) and 27 (XY-44) inhibited PSMA with Ki = 0.04 and 0.02 nM, respectively.
PSMA Inhibitor Docking Studies. Urea 9 (XY-I-26), NPA-carbamate 12 (XY- 20) and OPA-reversed carbamate 13 (XY-48) were subjected to in situ ligand minimization docking experiments based on the reported X-ray crystal structure of PSMA known as 3D7H (Barinka, 2008). The results are shown in FIG.13A and FIG. 13B. The urea N-H in 9 (YC-I-26) binds to the carbonyl oxygen of G518
asymmetrically, with the Lys-N-H (1.92 Å) slightly closer to the carbonyl oxygen than Glu-N-H (2.03 Å) (FIG.13A). Lys-OPA carbamate 13 (XY-48), with the Glu- N-H changed to O resulting in the loss of a hydrogen bond, retained nearly all of the features of 9 (YC-I-26), when binding to PSMA (FIG.13B), and the Lys-N-H distance to G518 slightly increased 1.95Å, with the carbamate shifting slightly towards the zinc (FIG.13B vs. FIG.13A). This resulted in a 30-fold reduction in binding affinity compared to 9 (YC-I-26).
Radiochemistry. Reversed carbamate [18F]13 ([18F]XY-48) was prepared by reacting N-succinimidyl-4-[18F]fluorobenzoate (Tang, 2008), [18F]SFB, with 18
(Scheme 2). 125I labeled analog 32 (XY-57) was prepared likewise using N- succinimidyl-4-[125I]iodobenzoate (Zalutsky, 1987) (Scheme 3). To synthesize
[18F]23 ([18F]XY-52) and[18F]24 ([18F]XY-47), the conjugation of N-succinimidyl-4- bromo/iodo-2-[18F]fluorobenzoate ([18F]30b and 30c) with precursor 22 was envisioned (Scheme 4). Compared with the synthesis of 4-[18F]fluorobenzoate and 2- [18F]fluorobenzoate derivatives, the synthesis of 4-bromo/iodo-2-[18F]fluorobenzoate derivatives has inherent challenges. The bromo/iodo group could potentially serve as a leaving group and be displaced by [18F]fluoride, producing a radiofluorinated side product. The only related example reported previously was the synthesis of 4-chloro- 2-[18F]fluorobenzaldehyde 29a from 4-chloro-2-nitrobenzaldehyde 28a (Scheme 4) in a 60% radiochemical yield using Kryptofix 2.2.2 and K2CO3 in DMSO at 140oC for 20 min (Ai-Darwich, 1996). As the first step in the synthesis of [18F]23 ([18F]XY-52) and[18F]24 ([18F]XY-47), these reaction conditions were applied to the radiosynthesis of 4-bromo- and 4-iodo-2-[18F]fluorobenzaldehyde 29b and 29c, starting from 4- bromo- and 4-iodo-2-nitrobenzaldehyde, 28b and 28c respectively (Scheme 4).
Compared with the reported success of [18F]29a, the highest yield that could be achieved for [18F]29b was only 15% non-decay corrected yield (Table 1, entries 1 to 8). No [18F]29c was produced under these conditions. Under these conditions decomposition of precursors 28b and 28c was observed by high performance liquid chromatography (HPLC). Decomposition was reduced using a milder base, KHCO3, enabling the radiosynthesis of aldehyde [18F]29b and [18F]29c. The complete radiosynthesis of [18F]23 and [18F]24 is shown in Scheme 4. [18F]29b was converted to its N-hydroxysuccinimide ester [18F]30b by treating with (diacetoxyiodo)benzene in the presence of N-hydroxysuccinimide in acetonitrile (Glaser, 2009). After conjugating the F-18 labeled NHS ester with Lys-OPA reversed carbamate precursor 22, [18F]23 ([18F]XY-52) was obtained in a non-decay corrected radiochemical yield of 5 % and specific radioactivity ranging from 37,000-177,600 GBq/mmole (1,000- 4,800 Ci/mmole). Compound [18F]24 ([18F]XY-47), was prepared in the same fashion (3 % non-decay corrected yield; specific radioactivity ranging between 31,080-92,500 GBz/mmole (840-2,500 Ci/mmole). During the radiosynthesis each intermediate was purified by semi-preparative radio-HPLC, concentrated on a Sep-Pak® C-18 cartridge, and eluted with methylene chloride for the next step. The total synthesis time was 3.5 hours.
Biodistribution and imaging. Compounds [18F]13 ([18F]XY-48), [18F]23
([18F]XY-52 and [18F]24 ([18F]XY-47) were assessed for their tumor uptake and pharmacokinetics in mice bearing PSMA+ PC-3-PIP and PSMA- PC-3-flu xenografts by ex vivo biodistribution and or micro-PET imaging.
Table 2 shows the percent injected dose per gram of tissue in selected organs for [18F]23 ([18F]XY-52). Compound [18F]23 ([18F]XY-52) demonstrated high uptake in the PC-3 PIP tumor, wherein the uptake of radioactivity reached a maximum of 90% ID/g at 2 h post-injection. On the contrary, the PSMA- PC3 flu tumor showed no specific uptake. The distribution within non-target tissues was generally low, except for kidney, liver and spleen. Significant renal uptake was observed, which can be attributed to the expression of PSMA in the proximal renal tubule with concurrent renal excretion.(Silver, 1997;Slusher, 1992) Radioactivity cleared from the kidneys and by 4 h was less than the level in PC3-PIP tumor. Other than kidney, the highest non-specific uptake was observed in liver and spleen at 30 min, both of which demonstrated rapid clearance of radiotracer, reaching minimal levels by 4 h. Low radiotracer uptake within bone indicated lack of significant de- fluorination in vivo. The PC-3 PIP:muscle and PC-3 PIP:blood ratios were 93 and 13 at 0.5 h, 208 and 40 at 1 h, 643 and 148 at 2 h, 792 and 240 at 4 h, respectively. Table 2. Biodistribution of 18F 23 18F XY-52
Figure imgf000071_0001
aValues expressed as percent injected dose per gram, N = 4 for all tissues bPIP:tissue ratio The biodistribution of [18F] 24 ([18F]XY-47) is shown in Table 3. Blood, other organs and normal tissues displayed relatively low uptake and rapid clearance.
Kidneys showed high initial uptake with clearance beginning after 2 h and was less than the uptake in PC-3PIP tumor by 4 h. The PC-3 PIP: muscle and PC-3 PIP :blood ratios were 47 and 5.6 at 0.5 h, 100 and 20 at 1 h, 250 and 66 at 2h, 743 and 156 at 4 h, respectively. Table 3. Biodistribution of 18F 24 18F XY-47
Figure imgf000072_0001
aValues expressed as percent injected dose per gram. N = 4
bPIP:tissue ratio To determine if the radiolabeled [18F]13 ([18F]-XY-48), was capable of being imaged, male nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice with PSMA-expressing (PC3-PIP) and PSMA-negative (PC3-flu) tumors were injected with the radiolabeled compound and imaged by positron emission tomography–computed tomography (PET-CT; compound structure of [18F]13
([18F]XY-48), shown in scheme 2).
FIG.14A shows maximum intensity projections (MIPs) after PET imaging with the [18F]13 ([18F]XY-48), compound. Standardized uptake values were generated from the images (FIG.14B). These data showed significantly higher levels of binding of radiolabeled [18F]13 ([18F]XY-48), to the PSMA-expressing PC3-PIP tumors as compared to the PSMA- negative PC3-flu tumors. Micro-PET (coronal view) imaging of [18F]13 ([18F]XY-48) (FIG.15A through FIG.15D) showed a high and prolonged PC-3 PIP tumor and kidney uptake than [18F]12 ([18F]XY-20) and is reminiscent of ureido compound [18F]9 ([18F]YC-I-26) (Chen, 2008).
Whole body micro-PET imaging was also performed for [18F]23 ([18F]XY-52) and [18F]24 ([18F]XY-47). Intense and selective radiotracer uptake could be seen in PC3-PIP tumors beginning at 30 min post-injection (FIG.16). The imaging studies are in concordant with the biodistribution results, the kidneys also had significant uptake. Besides PC-3 PIP tumor, kidneys and bladder, no significant non-specific organ uptake was observed.
In vitro stability studies. Because NPA carbamate [18F]12 ([18F]XY-20) (FIG. 2A) and OPA reversed carbamate [18F]13 ([18F]XY-48) exhibited such differing pharmacokinetics (FIG.2B), the stability of NPA carbamate [125I]31 ([125I]XY-26) and OPA reversed carbamate [125I]32 ([125I]XY-57) in-vitro have been tested. The radioiodinated compounds were utilized for the convenience of the long half-life of I- 125. Polar radiometabolite(s) in extracellular and intracellular fluid were only seen for NPA [125I]31 ([125I]XY-26) in PC-3 PIP cells (FIG.17). This suggests that NPA carbamates may be subject to PSMA specific metabolism and may be the reason for their rapid clearance from PSMA expressing tumors and organs. Discussion
Recent clinical PET imaging using either 18F, 124I or 68Ga labeled urea based PSMA inhibitors have produced high quality PET images allowing the detection of both primary and metastatic lesions, some of which were undetectable by
conventional imaging. Although all agents exhibited renal uptake, some agents (Ex:
[18F]DCFBC (Cho, 2012), and [68Ga]DOTA-DUPA-Pep (Reske, 2013)) showed persistent blood pool occupancy but low salivary gland uptake. On the other hand, [18F]DCFPyL (Szabo, 2015), [124I]-MIP-1095 (Zechmann, 2014), and [68Ga]DKFZ- 11(Afshar-Oromieh, 2012;Afshar-Oromieh, 2013) showed rapid clearance from the blood but high uptake in salivary and lacrimal glands. The latter has also been observed in radiopharmaceutical therapy studies using [177Lu]-DKFZ-617
(Kratochwil, 2015) and [131I]-MIP-1095 (Zechmann, 2014). Absorbed dose estimates for [131I]-MIP-1095 identified the salivary glands, lower large intestinal wall, and kidneys as dose-limiting organs (Zechmann, 2014). Therefore, new scaffolds that would preserve the positive distribution characteristics of the urea-based agents but provide better clearance from non-target organs were sought. Reversed carbamate- based agents, as demonstrated here, have retained potency and also rapidly cleared from normal tissues. However [18F]13 ([18F]XY-48) had high and prolonged tumor and kidney uptake. In addition, in situ ligand minimization docking experiments showed that compound 13 (XY-48) possesses a similar position with regards to the zinc molecule and G518 in the binding site.
Higher potency Lys-OPA carbamates 23 (XY-52) (Ki = 0.11 nM) and 24 (XY- 47) (Ki = 0.21 nM) were obtained by utilization of 4-bromo/iodo-2-fluorobenzoyl groups which increase PSMA binding presumably through interaction with the arginine patch and the auxillary hydrophobic subpocket (Barinka, 2008). Compounds 23 (XY-52) and 24 (XY-47) bind with less than a 10-fold drop in potency compared with the corresponding ureas, 26 (XY-58) (Ki = 0.036 nM) and 27 (XY-44) (Ki = 0.023 nM). This suggests that utilization of the S1 binding site (arginine patch) and the auxillary hydrophobic pocket can partially offset the loss of one hydrogen bond in the OPA carbamates compared to ureas.
The biodistribution and imaging data demonstrated specific binding of Lys- OPA carbamates [18F]23 ([18F]XY-52) and [18F]24 ([18F]XY-47) to PSMA. Little bone uptake suggested the stability of the [18F]fluorine on the 4-bromo/iodo-2- [18F]fluorobenzoyl group. For [18F]25 the initial PSMA+ PC3 PIP tumor uptake at 30 min was 63 % ID/g, increasing to 90 % ID/g at 2 h and then dropped slightly to 74 % ID/g at 4 h. In the case of [18F]24 ([18F XY-47), the initial PSMA+ PC3 PIP tumor uptake observed was 45 % ID/g, increasing to 97 % ID/g by 4 h. Both radiotracers showed little uptake in the isogenic, PSMA- PC3 flu tumors. For blood and normal organs, the highest non-specific uptake appeared in liver and spleen, but this radioactivity cleared rapidly resulting in increasing tumor/normal organ ratios.
Although [18F]23 ([18F]XY-52) and [18F]24 ([18F XY-47) still possess considerable kidney uptake, their tumor/kidney ratios are approximately 1 at 2-4 hours and importantly, are 4-10 times higher than the ratios we previously reported for ureas [18F]9 ([18F]YC-I-26) and [125I]10 ([125I]YC-I-27). Interestingly, [18F]23 ([18F]XY- 52) and [18F]24 are more potent than our 2nd generation F-18 labeled PSMA inhibitor [18F]DCFPyL (Ki = 1.1nM) currently in clinical trials; however, in PC-3 PIP tumor bearing mice [18F]DCFPyL demonstrated higher tumor:kidney ratios (>2 at 2h) (Chen, 2011). Clinical imaging studies will be necessary to determine if OPA carbamates offer any improvements over [18F]DCFPyL for imaging. The OPA scaffold may be most useful for radiotherapy with alpha, beta, or Auger emitters when labeled with radiohalogenated (211At, 131I, 125I, 123I, 80mBr, or 77Br) benzoyl groups where the S1 and auxillary hydrophobic pockets are fully utilized.
The N-succinimidyl 4-bromo/iodo-2-[18F]fluorobenzoates [18F]30b and
[18F]30c utilized in the synthesis of [18F]23 ([18F]XY-52) and [18F]24 ([18F]XY-47) are new radiofluorinated prosthetic groups. Even though the radiosyntheses of
[18F]23 ([18F]XY-52) and [18F]24 ([18F]XY-47) reported here provide low overall radiochemical yields and are cumbersome to perform, the initial goal was only to produce enough radiolabeled material for preliminary testing of the reversed carbamates in vivo. Improved radiochemistry will be required for [18F]23 ([18F]XY- 52) and [18F]24 ([18F]XY-47) to achieve their full potential as PSMA imaging agents, and is under way.
Herein the carbamate scaffold for preparation of new and potent inhibitors of PSMA has been described. By utilizing the affinity-enhancing interactions of the arginine patch and auxillary hydrophobic subpocket, high affinity OPA carbamates [18F]23 ([18F]XY-52) and [18F]24 ([18F]XY-47) demonstrated high target-selective uptakes in PSMA+ PC-3 PIP tumor xenografts. These agents produced higher PSMA+ PC-3 PIP:kidney ratios compared to similar ureas such as [125I]10 ([125I]YC- I-27) and [18F]9 ([18F]YC-I-26), suggesting this alternative scaffold for development of new agents with improved dosimetry, particularly for radiotherapeutics. Because of its faster normal organ clearance [18F]23 ([18F]XY-52) will be particularly suitable for clinical translation for PSMA PET imaging once the radiosynthesis is improved. EXAMPLE 2
Binding Affinity Data of Representative Compounds of Formula (I) Binding affinity data of representative compounds of formula (I) are provided herein below in Table 4.
Figure imgf000076_0001
Figure imgf000077_0002
Some other additional data, including the binding affinity Ki, distribution coefficient CLogD, and PSA, of representative compounds of Formula (I) are provided in Table 5. In these exemplary, non-limiting embodiments, the most potent compounds contain a fluorine atom at R1 and a large halogen, e.g., either Br or I, or a phenyl ring at the 4-position of the arylbenzoyl moiety, i.e., R2 and are annotated with an "*".
Figure imgf000077_0001
†DCFBC is N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-fluorobenzyl-l-cysteine:
Figure imgf000078_0001
††DCIBC is N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-iodobenzyl-l-cysteine
Figure imgf000078_0002
†††DCFPyL is 2-(3-{1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl}- ureido)-pentanedioic acid:
Figure imgf000078_0003
.
††††C8 is
Figure imgf000078_0004
EXAMPLE 3
Synthesis and use of Radiohalogenated Lysine, Glutamate and Cysteine-OPA carbamate compounds for Imaging and Cancer Radiotherapy The development of low molecular weight radiotherapeutic agents is different from developing radiopharmaceuticals for imaging in that longer tumor residence times are required for the former. Many radionuclides, primarily β- and alpha emitters, have been investigated for targeted radioimmunotherapy and include both radiohalogens and radiometals. The studies presented in the disclosed subject matter have been focused on radiohalogens, 125I, 123I, 131I, 211At, 77Br (Table 6), with several specific examples of appropriate ways of introducing them into PSMA-targeting molecules. Several of these radionuclides (123I, 131I, 77Br) also emit imaging gamma rays providing both imaging and radiotherapeutic applications. Table 6. Therapeutic Radionuclides
Figure imgf000079_0001
These radiohalogens are covalently bound to the targeting moiety and unlike large chelated radiometals are small enough that the entire radiolabeled PSMA inhibitor can fit within the PSMA binding cavity thereby retaining the high binding affinity. The same radiolabeled prosthetic groups can be conjugated to linker- inhibitor conjugates to move the radiolabeled portion of the inhibitor to the exterior of the PSMA protein.
Radiohalogenated PSMA binding radiotherapeutics can be built upon multiple PSMA binding scaffolds. The urea based scaffolds include: the lysine-glutamate urea, cysteine-glutamate urea, and glutamate-glutamate urea (FIG.18). PSMA inhibitors built upon novel lysine- reversed carbamate scaffold (OPA) have been disclosed in example 1 of the present application, and cysteine -OPA carbamate and glutamate-–OPA carbamate scaffolds, as shown FIG.19, have been envisioned.
These compounds are based on extensive structure-activity relationships– not merely of the imaging precursors or PSMA binding compounds, but on actual imaging agents already synthesized and tested in vivo– as well as on molecular modeling.
The lead compound in the lysine-glutamate ureas is YC-I-27. This iodinated compound has high and prolonged tumor uptake and a very high PSMA binding affinity Ki = 0.01 nM (Chen et al., 2008). It has also been co-crystalized with PSMA. This has shown that the bulky iodo-phenyl moiety is accommodated by a hydrophobic auxillary sub-pocket extending beyond the normal binding pocket and the additional hydrophobic-hydrophobic interactions accounts for the high binding affinity (Barinka et al., 2008). Table Xsummarizes the halogen containing compounds that have been prepared to date and demonstrates the high binding affinity for the halogenated compounds using the new lysine -NPA reversed carbamate scaffold. It also shows the increase in binding affinity for the iodinated and brominated OPA analogs compared to the smaller fluorinated OPA analogs (XY-60 and XY-57 vs. XY-48). The radiosynthesis of [18F]XY-47 and [18F]XY-52 have been reported in example 1 of the present application, both of which exhibited high uptake in PSMA positive mouse tumor xenografts and rapid normal organ clearance by imaging and excised tissue counting comparable to the urea [18F]DCFPyL which is currently being evaluated in a clinical trial for imaging metastatic prostate cancer. This demonstrates the utility of the new Lysine-OPA scaffold and suggests that they can be applied to PSMA targeted radiotherapy of prostate cancer. This also suggests that cysteine-OPA scaffold as well as glutamate-OPA carbamate scaffold will also produce PSMA targeted
radiotherapeutic agents.
Synthesis of Radiohalogenated Lysine– OPA carbamate compounds. Parent lysine– OPA carbamate scaffolds were reported in example 1 of the present application, and can be utilized, as shown in Scheme 5, to prepare radiohalogened lysine–OPS carbamates utilizing known stannane containing prosthetic groups (Garg, et al., 1991; Vaidyanathan G, and Zalutsky MR, 2007; Talanov et al., 2006).
h m
Figure imgf000081_0001
Synthesis of Radiohalogenated Glutamate-OPA carbamate compounds. Glutamate- glutamate ureas have been used by others to conjugate bulky radiometal chelating agents, fluorescent molecule, and chemotherapeutics (Kularatne et al., 2009;
International Patent Applications Nos. PCT/US2008/073375, PCT/US2009/061067, and PCT/US2011/026238). These compounds are too large to utilize the non- pharmacophore binding pocket and must utilize a void region to extend outside of the protein. A synthetic route to an analogous radiohalogenated glutamate-OPA carbamate is shown in Scheme 6. Scheme 6.
Figure imgf000082_0001
Scheme 2.
Synthesis of Radiohalogenated Cysteine-OPA carbamate compound. The cysteine-glutamate urea scaffold has been utilized by us for PSMA binding and imaging for over 10 years starting with C-11 labeled DCMC (Pomper et al., 2002; Foss et al., 2005), continuing with F-18 labeled DCFBC (Mease et al., 2008; Cho et al., 2012) both for PET imaging with the latter currently in use in patients, and I-125 labeled DCIBC (Dusich, 2008) for SPECT imaging and or radiotherapy (FIG.20). A synthetic route to analogous radiohalogenated cysteine-OPA carbamates is shown in Scheme 7. Scheme 7
Figure imgf000083_0001
Homologs of the cysteine-OPA carbamates may have even higher binding affinity because the extended alkyl chain will permit a deeper penetration of the 4- halobenzyl group into the non-pharmacophore binding pocket. These compounds can be prepared analogously using Scheme 7 starting with commercial Nα-Fmoc-S-trityl- L-homocysteine and L-5-[S-trityl]-[N-9-fluorenylmethyloxycarbonyl]- mercaptonorvaline.
Lysine glutamate ureas and glutamate-glutamate ureas linker conjugates have been used to attach bulky radiometal chelates or fluorescent molecules for PSMA specific imaging and radiotherapy, with a focus on the use of polyethylene glycol (PEG) and lysine-suberate linkers (Banerjee et al., 2008; Chen et al., 2012). The new PSNA binding scaffolds described herein can also be used to prepare radiolabeled linker conjugates. A route to the synthesis of a lysine-suberate-lysine-OPA carbamate and its use in preparing radiohalogenated linker OPA carbamates is shown in Scheme 8. Scheme 8.
Figure imgf000084_0001
EXAMPLE 4
Synthesis and Use of YC-XIX-33 and Related Agents for PSMA Targeted Imaging
Guided Surgery and Photodynamic Therapy
Overview
The preparation and use of PSMA binding ureas conjugated to fluorescent molecules via various linkers for imaging PSMA expressing tumors and tissues (Chen et al., 2009; Banerjee et al., 2011; Chen et al., 2012) and photodynamic therapy (PDT) have been previously described. Two specific examples of IR-Dye-800-CW-linker- ureas are shown in FIG.21. PSMA inhibitors built upon lysine-carbamate scaffolds (OPA and NPA) including F-18 labeled analogs have been disclosed in the present application, and the F-18 labeled NPA and OPA compounds demonstrated selective uptake in PSMA positive tumor mouse xenografts. Herein the synthesis and use of YC-XIX-33, an IR-Dye-800-CW-PEG-linked OPA for imaging PSMA positive tumors and tissues as an example is disclosed. Various dyes and linkers previously disclosed in Patent Application No. PCT/US2010/028020 for use with ureas can be attached to the OPA scaffolds to provide novel optical agents for imaging prostate cancer. Material and methods
2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5- azaicosan-20-oate, 2. To a solution of 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5- azaicosan-20-oic acid, 1 (0.10 g, 0.27 mmol, Quanta Biodesign) in CH2Cl2 (1 mL) was added N-hydroxysuccinimide (0.032 g, 0.278 mmol), followed by the N,N′- dicyclohexylcarbodiimide (0.056 g, 0.271 mmol). After stirring overnight at room temperature, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to afford 2 (0.12 g, 95 %).1H NMR (500 MHz, CDCl3) δ 5.12 (bs, 1H), 3.85 (t, J = 6.5 Hz, 2H), 3.66 (m, 12H), 3.54 (m, 2H), 3.30 (m, 2H), 2.91 (t, J = 6.5 Hz, 2H), 2.82 (s, 4H), 1.48 (s, 9H).
26S,30S)-2,2-dimethyl-4,20,28-trioxo-3,8,11,14,17,29-hexaoxa-5,21,27- triazadotriacontane-26,30,32-tricarboxylic acid, 3. To a solution of 5 (0.023 g, 0.055 mmol) in DMSO (0.1 mL) was added N,N-diisopropylethylamine (0.05 mL, 0.286 mmol), followed by 2 (0.026 g, 0.056 mmol). After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC to afford 3 (0.024 g, 65%).
(21S,25S)-21,25,27-tricarboxy-15,23-dioxo-3,6,9,12,24-pentaoxa-16,22- diazaheptacosan-1-amine, trifluoroacetic acid salt, 4. A solution of TFA/H2O (95:5, 0.3 mL) was added to 3 (0.024 g, 0.036 mmol). The reaction mixture was stirred for 2 h at room temperature and then concentrated under reduced pressure to afford 4 (0.023 g, 96 %).1H NMR (500 MHz, D2O) δ 4.81 (m, 1H), 4.01 (m, 1H), 3.61 (m, 4H), 3.54 (m, 12H), 3.06 (m, 4H), 2.41 (m, 2H), 2.36 (m, 2H), 2.08 (m, 1H), 2.00 (m, 1H), 1.74 (m, 1H), 1.60 (m, 1H), 1.39 (m, 2H), 1.28 (m, 2H). ESI-Mass calculated for C23H42N3O13 [M]+ 568.3, found: 568.3.
YC-XIX-33. To a solution of 4 (0.5 mg, 0.75 µmol) in DMSO (0.1 mL) was added N,N-diisopropylethylamine (0.005 mL, 0.029 mmol), followed by the NHS ester of IRDye800CW (0.5mg, 0.43 µmol). After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC to afford YC-XIX-33 (0.6 mg, 90 %). ESI-Mass calcd for C69H94N5O27S4 [M+H]+ 1552.5, found: 1552.5. h m 1
Figure imgf000086_0001
Animal imaging. YC-XIX-33 (1 nmole in PBS) was injected via the tail vein into two 4-6 week old male athymic nude mice containing PSMA+ PC3 PIP and PSMA- PC3 flu tumor xenografts on opposite flanks. The mice were imaged using the Pearl Impulse Imager (excitation at 785 nm and emission at 800 nm) at 10 min, 30 min, 1h, 2h, 4h, and 24h. One mouse was sacrificed at 4 hours post injection and the other at 24h. Tumor and organs were removed, placed in a Petri dish and reimaged. All images were scaled to the same maximum intensity for direct comparison. Results
In vivo and ex-vivo imaging of YC-XIX-33 were performed on mice containing PSMA+ PC3 PIP and PSMA- PC3 flu tumor xenografts on opposite flanks as shown on FIG.22B. Specific uptake of YC-XIX-33 in PSMA+ PC3 PIP tumor xenografts as early as 4 hours post-injection is observed. By 24 hours the PSMA+ PC3 PIP tumor displays the brightest signal. REFERENCES
All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein. International PCT Patent Application Publication No. PCT/US2008/073375 to Low PS, Kularatne SA. for PSMA binding ligand-linker conjugates and methods for using, published 2009/02/26 (WO 2009/026177A1);
International PCT Patent Application Publication No. PCT/US2009/061067 to Low PS, Kularatne SA. for PSMA binding ligand-linker conjugates and methods for using, published 2010/04/22 (WO 2010/045598 A2);
International PCT Patent Application Publication No. PCT/US2011/026238 to Low PS, Chelvam V, Kim Y. for PSMA binding ligand-linker conjugates and methods for using, published 2011/09/01 (WO 2011/106639 A1);
International PCT Patent Application Publication No. PCT/US03/00680 to Pomper MG, Zhang J, Kozikowski AP, Musachio JL. for Imaging agents and methods of imaging NAALADase of PSMA, published 2003/07/24 (WO 03/60523 A1);
International PCT Patent Application Publication No. PCT/US2008/007947 to Pomper, M.G., Ray, S., Mease, R.C., Foss, C. for Labeled inhibitors of prostate specific membrane antigen (PSMA), biological evaluation, and use as imaging agents, published 2008/12/31 (WO 2009/002529 A2);
International PCT Patent Application Publication No. PCT/US2009/052456 to Pomper MG, Mease RC, Chen Y. for Preparation of glutamic acid heterodimer prostate-specific membrane antigen PSMA binding agents for therapeutic and imaging use, published 2010/02/04 (WO 2010/014933 A2);
International PCT Patent Application Publication No. PCT/US2010/028020 to Pomper, Martin G.; Mease, Ronnie C.; Ray, Sangeeta; Chen, Ying. for PSMA- targeting compounds and uses thereof, published 2010/09/23 (WO 2010/108125 A2);
International PCT Patent Application Publication No. PCT/US2012/067162 to Pomper, Martin G. Mease, Ronnie C., Ray, Sangeeta, Shallal, Hassan for
Homomultivalent and heteromultivalent inhibitors of prostate specific membrane antigen (PSMA) and uses thereof, published 2013/06/06 (WO2013/082338 A1)
International PCT Patent Application Publication No. PCT/US2011/026238 to Low P.S., Chelvam V., Kim Y. for PSMA binding ligand-linker conjugates and methods for using, published 2011/09/01 (WO2011/106639);
International PCT Patent Application Publication No. PCT/US2009/061067 to Low P.S., Chelvam V., Kim Y. for PSMA binding ligand-linker conjugates and methods for using, published 2010/04/22 (WO 2010/045598 A2)
International PCT Patent Application Publication No. PCT/US2008/073375 to Low P.S., Chelvam V., Kim Y. for PSMA binding ligand-linker conjugates and methods for using, published 209/02/26 (WO 2009/026177 A1);
U.S. Patent Application Publication No. US 2004/0054190 A1 to Pomper MG, Zhang J, Kozikowski AP, Musachio JL. for Imaging agents and methods of imaging NAALADase of PSMA, Licensed to: Molecular Insight Pharmaceuticals, Inc. published 2004/03/18 (10/340,864);
U.S. Patent Application Publication No. US 2008/0193381 A1 to Babich JW, Zimmerman CN, Maresca KP. for Heterodimers of glutamic acid, published
2008/08/14 (11/936,659); U.S. Patent Application Publication No. US 2013/0034494 A1 to Babich JW, Zimmerman C, Joyal JL, Lu G. for Radiolabeled prostate specific membrane antigen inhibitors, published 2013/02/07 (13/566,674);Afshar-Oromieh, A.; Haberkorn, U.; Eder, M.; Eisenhut, M.; Zechmann, C. M., [68Ga]Gallium-labelled PSMA ligand as superior PET tracer for the diagnosis of prostate cancer: comparison with 18F-FECH. European journal of nuclear medicine and molecular imaging 2012, 39 (6), 1085-6;
Afshar-Oromieh, A.; Malcher, A.; Eder, M.; Eisenhut, M.; Linhart, H. G.; Hadaschik, B. A.; Holland-Letz, T.; Giesel, F. L.; Kratochwil, C.; Haufe, S.;
Haberkorn, U.; Zechmann, C. M., PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions. European journal of nuclear medicine and molecular imaging 2013, 40 (4), 486-95;
Afshar-Oromieh, A.; Haberkorn, U.; Hadaschik, B.; Habl, G.; Eder, M.; Eisenhut, M.; Schlemmer, H. P.; Roethke, M. C., PET/MRI with a 68Ga-PSMA ligand for the detection of prostate cancer. European journal of nuclear medicine and molecular imaging 2013, 40 (10), 1629-30;
Afshar-Oromieh, A.; Zechmann, C. M.; Malcher, A.; Eder, M.; Eisenhut, M.; Linhart, H. G.; Holland-Letz, T.; Hadaschik, B. A.; Giesel, F. L.; Debus, J.;
Haberkorn, U., Comparison of PET imaging with a (68)Ga-labelled PSMA ligand and (18)F-choline-based PET/CT for the diagnosis of recurrent prostate cancer. European journal of nuclear medicine and molecular imaging 2014, 41 (1), 11-20;
Afshar-Oromieh, A.; Avtzi, E.; Giesel, F. L.; Holland-Letz, T.; Linhart, H. G.; Eder, M.; Eisenhut, M.; Boxler, S.; Hadaschik, B. A.; Kratochwil, C.; Weichert, W.; Kopka, K.; Debus, J.; Haberkorn, U., The diagnostic value of PET/CT imaging with the (68)Ga-labelled PSMA ligand HBED-CC in the diagnosis of recurrent prostate cancer. European journal of nuclear medicine and molecular imaging 2015, 42 (2), 197-209;
Ai-Darwich, M. J., Plenevaux, A., Lemair3e, C., Fiore, G.D., Christianes, L., Comar, D., Luxen, A. , Enantioselective synthesis of no-carrieradded (S)-4-chloro-2- [18F]fluorophenylalanine and (S)-a-methyl)-4-chloro-2-[18F]fluorophenylalanine. J. Fluor. Chem.1996, 80, 117-124;
Bander, N. H.; Milowsky, M. I.; Nanus, D. M.; Kostakoglu, L.;
Vallabhajosula, S.; Goldsmith, S. J., Phase I trial of 177lutetium-labeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2005, 23 (21), 4591-601;
Banerjee SR, Foss CA, Castanares M, Mease RC, Byun Y, Fox JJ, Hilton J, Lupold S, Kozikowski AP, Pomper MG. Synthesis and evaluation of technetium- 99m- and rhenium-labeled inhibitors of the prostate-specific membrane antigen (PSMA). J Med Chem 2008; 51:4504-4517;
Banerjee SR, Pullambhatla M, Byun Y, Nimmagadda S, Foss CA, Green G, Fox JJ, Lupold SE, Mease RC, Pomper MG. Sequential SPECT and optical imaging of experimental models of prostate cancer with a dual modality inhibitor of the prostate-specific membrane antigen. Angew Chem Int Ed Engl 2011; 50:9167-70.
Banerjee SR, Pullambhatla M, Byun Y, Nimmagadda S, Green G, Fox JJ, Horti A, Mease RC, Pomper MG. 68Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J Med Chem 2010, 53:5333- 5341;
Banerjee, S.R, Pullambhatla, M., Foss, C.A., Nimmagadda, S. et al.64Cu- labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer. Journal of medicinal chemistry, 2014, 57(6), 2657-69;
Banerjee, S. R.; Foss, C. A.; Pullambhatla, M.; Wang, Y.; Srinivasan, S.; Hobbs, R.; Baidoo, K. E.; Brechbiel, M. W.; Nimmagadda, S.; Mease, R. C.; Sgouros, G.; Pomper, M. G., Preclinical Evaluation of 86Y-Labeled Inhibitors of Prostate- Specific Membrane Antigen for Dosimetry Estimates. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2015;
Barinka C, Byun Y, Dusich CL, Ray S, Chen Y, Castanares M, Kozikowski AP, Mease RC, Pomper MG, Lubkowski J. Interactions between Human Glutamate Carboxypeptidase II and Urea-based Inhibitors: Structural Characterization. J Med Chem, 2008; 51:7737-7743;
Barinka, C.; Sacha, P.; Sklenar, J.; Man, P.; Bezouska, K.; Slusher, B. S.; Konvalinka, J., Identification of the N-glycosylation sites on glutamate
carboxypeptidase II necessary for proteolytic activity. Protein science : a publication of the Protein Society 2004, 13 (6), 1627-35;
Barrett, J. A.; Coleman, R. E.; Goldsmith, S. J.; Vallabhajosula, S.; Petry, N. A.; Cho, S.; Armor, T.; Stubbs, J. B.; Maresca, K. P.; Stabin, M. G.; Joyal, J. L.; Eckelman, W. C.; Babich, J. W., First-in-Man Evaluation of 2 High-Affinity PSMA- Avid Small Molecules for Imaging Prostate Cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2013, 54 (3), 380-7;
Byun, Y., Mease, R.C., Lupold, S., Pomper, M.G., Drug Design of Zinc- Enzyme Inhibitors. Functional Structureal and Disease Applications. In Recent Developments of Diagnostic and Therapeutic Agents Targeting Glutamate
Carboxypeptidase II (GCPII) Supuran, C. T., Winum, J.Y., Ed. John Wiley & Sons: Hoboken, 2009; pp 881-910;
Chang, S. S.; Reuter, V. E.; Heston, W. D.; Bander, N. H.; Grauer, L. S.; Gaudin, P. B., Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature. Cancer Res 1999, 59 (13), 3192-8;
Chang, S. S.; Reuter, V. E.; Heston, W. D.; Gaudin, P. B., Comparison of anti- prostate-specific membrane antigen antibodies and other immunomarkers in metastatic prostate carcinoma. Urology 2001, 57 (6), 1179-83;
Cheng, Y.; Prusoff, W. H., Relationship between the inhibition constate (K1) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem Pharmcol 1973, 22, 3099-3108;
Chen, Y.; Foss, C. A.; Byun, Y.; Nimmagadda, S.; Pullambhatla, M.; Fox, J. J.; Castanares, M.; Lupold, S. E.; Babich, J. W.; Mease, R. C.; Pomper, M. G., Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)-Based Ureas as Imaging Agents for Prostate Cancer. Journal of medicinal chemistry 2008, 51 (24), 7933-7943;
Chen Y, Dhara S, Banerjee S, Byun Y, Pullambhatla M, Mease RC, Pomper MG. A low molecular weight PSMA-based fluorescent imaging agent for cancer. Biochem. Biophys Res. Commun.390: 624-629 (2009);
Chen, Y.; Pullambhatla, M.; Banerjee, S. R.; Byun, Y.; Stathis, M.; Rojas, C.; Slusher, B. S.; Mease, R. C.; Pomper, M. G., Synthesis and biological evaluation of low molecular weight fluorescent imaging agents for the prostate-specific membrane antigen. Bioconjugate chemistry 2012, 23 (12), 2377-85;
Chen, Y.; Pullambhatla, M.; Foss, C. A.; Byun, Y.; Nimmagadda, S.;
Senthamizhchelvan, S.; Sgouros, G.; Mease, R. C.; Pomper, M. G., 2-(3-{1-Carboxy- 5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pen tanedioic acid, [18F]DCFPyL, a PSMA-based PET imaging agent for prostate cancer. Clin Cancer Res 2011, 17 (24), 7645-53;
Cho SY, Gage KL, Mease RC, et al. Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low molecular weight inhibitor of PSMA, in patients with metastatic prostate cancer. J. Nucl. Med.2012; 53:1883-1891.
Dekker B, Keen H, Shaw D, Disley L, Hastings D, Hadfield J, Reader A, Allan D, Julyan P, Watson A, Zweit J. Fundtional comparison of annexin V analogues labeled indirectly and directly with iodine-124. Nuclear Medicine and Biology 2005; 32:403-413;
Dusich CL. Imaging prostate cancer: Design, synthesis and biological evaluation of optical and radioactive prostate-specific membrane antigen (PSMA) inhibitors. PhD Thesis Johns Hopkins University 2008;
Eder, M.; Schafer, M.; Bauder-Wust, U.; Hull, W. E.; Wangler, C.; Mier, W.; Haberkorn, U.; Eisenhut, M., 68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjugate chemistry 2012, 23 (4), 688-97;
Foss CA, Mease RC, Fan H, Wang Y, Ravert HT, Dannals RF, Olszewski RT, Heston WD, Kozikowski AP, Pomper MG. Radiolabeled small molecule ligands for prostate-specific membrane antigen: in vivo imaging in experimental models of prostate cancer. Clin Cancer Res 2005; 11:4022-4028;
Foss, C. A.; Mease, R. C.; Cho, S. Y.; Kim, H. J.; Pomper, M. G., GCPII imaging and cancer. Current medicinal chemistry 2012, 19 (9), 1346-59;
Garg S, Garg PK, Zalutsky MR. N-succinimidyl 5-(trialkylstannyl)-3- pyridinecarboxylates: a new class of reagents for protein radioiodination.
Bioconjugate Chem.1991; 2:50–56;
Ghosh, A.; Heston, W. D., Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. Journal of cellular biochemistry 2004, 91 (3), 528-39;
Glaser, M., Arstad, E., Luthra, S.K., Robins, E.G., Two-step radiosynthesis of [18F]N-succinimidyl-4-fluorobenzoate ([18F]SFB. J. Label. Compd. Radiopharm. 2009, 52, 327-330;
Hillier SM, Maresca KP, Femia FJ, Marquis JC, Foss CA, Nguyen N, Zimmerman CN, Barrett JA, Eckelman WC, Pomper MG, Joyal JL, Babich JW. Preclinical Evaluation of Novel Glutamate-Urea-Lysine Analogues That Target Prostate-Specific Membrane Antigen as Molecular Imaging Pharmaceuticals for Prostate Cancer. Cancer Res 2009; 69: 6932-40;
Hillier, S. M.; Maresca, K. P.; Lu, G.; Merkin, R. D.; Marquis, J. C.;
Zimmerman, C. N.; Eckelman, W. C.; Joyal, J. L.; Babich, J. W., 99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2013, 54 (8), 1369-76;
Jackson, P. F.; Cole, D. C.; Slusher, B. S.; Stetz, S. L.; Ross, L. E.; Donzanti, B. A.; Trainor, D. A., Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase. Journal of medicinal chemistry 1996, 39 (2), 619-22;
Jackson, P. F.; Tays, K. L.; Maclin, K. M.; Ko, Y. S.; Li, W.; Vitharana, D.; Tsukamoto, T.; Stoermer, D.; Lu, X. C.; Wozniak, K.; Slusher, B. S., Design and pharmacological activity of phosphinic acid based NAALADase inhibitors. Journal of medicinal chemistry 2001, 44 (24), 4170-5;Kabalka, G. W.; Mereddy, A. R., A facile no-carrier-added radioiodination procedure suitable for radiolabeling kits. Nuclear medicine and biology 2004, 31 (7), 935-8;
Kratochwil, C.; Giesel, F. L.; Eder, M.; Afshar-Oromieh, A.; Benesova, M.; Mier, W.; Kopka, K.; Haberkorn, U., [Lu]Lutetium-labelled PSMA ligand-induced remission in a patient with metastatic prostate cancer. European journal of nuclear medicine and molecular imaging 2015;
Kularatne SA, Zhou Z, Yang J, Post CB, Low PS. Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen (PSMA)-targeted-99mTc radioimaging agents. Molecular Pharmaceutics 2009; 6(3) 790-800;
Kurth M, Pelegrin A, Rose, K, Offord RE, Pochon S, Mach J-P, Buchegger F. Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor. J. Med. Chem.1993; 36:1255-1261;
Kozikowski, A. P.; Nan, F.; Conti, P.; Zhang, J.; Ramadan, E.; Bzdega, T.; Wroblewska, B.; Neale, J. H.; Pshenichkin, S.; Wroblewski, J. T., Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase). Journal of medicinal chemistry 2001, 44 (3), 298-301; Kozikowski, A. P.; Zhang, J.; Nan, F.; Petukhov, P. A.; Grajkowska, E.;
Wroblewski, J. T.; Yamamoto, T.; Bzdega, T.; Wroblewska, B.; Neale, J. H., Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents. Journal of medicinal chemistry 2004, 47 (7), 1729-38;
Lapi, S. E.; Wahnishe, H.; Pham, D.; Wu, L. Y.; Nedrow-Byers, J. R.; Liu, T.; Vejdani, K.; VanBrocklin, H. F.; Berkman, C. E.; Jones, E. F., Assessment of an 18F- labeled phosphoramidate peptidomimetic as a new prostate-specific membrane antigen-targeted imaging agent for prostate cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2009, 50 (12), 2042-8;
Litosh, V. A. H., M.N.; Stupi, B.P.; Wu, W.; Metzker, M.L. Nucleotides and nucleosides and methods for their use in DNA sequencing.2009;
Majer, P.; Jackson, P. F.; Delahanty, G.; Grella, B. S.; Ko, Y. S.; Li, W.; Liu, Q.; Maclin, K. M.; Polakova, J.; Shaffer, K. A.; Stoermer, D.; Vitharana, D.; Wang, E. Y.; Zakrzewski, A.; Rojas, C.; Slusher, B. S.; Wozniak, K. M.; Burak, E.;
Limsakun, T.; Tsukamoto, T., Synthesis and biological evaluation of thiol-based inhibitors of glutamate carboxypeptidase II: discovery of an orally active GCP II inhibitor. Journal of medicinal chemistry 2003, 46 (10), 1989-96;
Makarasen, A., Nishikawa, T., Isobe, M., Synthesis of four lysine-linked cereulide analogues showing ionophoric activity towards potassium cations as lead compounds for emetic toxin detection by immunoassays. Synthesis 2009, 2184-2004;
Maresca, K. P.; Hillier, S. M.; Femia, F. J.; Keith, D.; Barone, C.; Joyal, J. L.; Zimmerman, C. N.; Kozikowski, A. P.; Barrett, J. A.; Eckelman, W. C.; Babich, J. W., A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer. Journal of medicinal chemistry 2009, 52 (2), 347-57;
Mease RC, Dusich CL, Foss CA, Ravert HT, Dannals RF, Seidel J, Prideaux A, Fox JJ, Sgouros G, Kozikowski AP, Pomper MG. Synthesis and in vivo evaluation of N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl-L-cysteine,
[18F]DCFBC: a new imaging probe for prostate cancer. Clin Cancer Res 2008; 14:3036-3043;
Mease, R. C.; Foss, C. A.; Pomper, M. G., PET imaging in prostate cancer: focus on prostate-specific membrane antigen. Current topics in medicinal chemistry 2013, 13 (8), 951-62; Mesters, J. R.; Barinka, C.; Li, W.; Tsukamoto, T.; Majer, P.; Slusher, B. S.; Konvalinka, J.; Hilgenfeld, R., Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. Embo J 2006, 25 (6), 1375-84;
Perner, S.; Hofer, M. D.; Kim, R.; Shah, R. B.; Li, H.; Moller, P.; Hautmann, R. E.; Gschwend, J. E.; Kuefer, R.; Rubin, M. A., Prostate-specific membrane antigen expression as a predictor of prostate cancer progression. Human pathology 2007, 38 (5), 696-701;
Pinto, J. T.; Suffoletto, B. P.; Berzin, T. M.; Qiao, C. H.; Lin, S.; Tong, W. P.; May, F.; Mukherjee, B.; Heston, W. D., Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells. Clin Cancer Res 1996, 2 (9), 1445-51;
Maung, J.; Mallari, J. P.; Girtsman, T. A.; Wu, L. Y.; Rowley, J. A.; Santiago, N. M.; Brunelle, A. N.; Berkman, C. E., Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates.
Bioorganic & medicinal chemistry 2004, 12 (18), 4969-79;
Pomper MG, Musachio JL, Zhang J, Zhou Y, Scheffel U, Hilton J, Maini A, Dannals RF, Wong DF, Kozikowski AP. 11C-MCG: Synthesis, uptake selectivity and primate PET of a probe for glutamate carboxypeptidase II (NAALADase.) Mol Imaging 2002; 1:96-101;
Rajasekaran, A. K.; Anilkumar, G.; Christiansen, J. J., Is prostate-specific membrane antigen a multifunctional protein? American journal of physiology. Cell physiology 2005, 288 (5), C975-81;
Ray Banerjee, S.; Pullambhatla, M.; Foss, C. A.; Falk, A.; Byun, Y.;
Nimmagadda, S.; Mease, R. C.; Pomper, M. G., Effect of chelators on the pharmacokinetics of (99m)Tc-labeled imaging agents for the prostate-specific membrane antigen (PSMA). Journal of medicinal chemistry 2013, 56 (15), 6108-21;
Reske, S. N.; Winter, G.; Baur, B.; Machulla, H. J.; Kull, T., Comment on Afshar-Oromieh et al.: PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions. European journal of nuclear medicine and molecular imaging 2013, 40 (6), 969-70;
Rowe, S. P. G., K.L.; Macura, K.; Guner, G.; Faraj, S.F.; Munari, E.;
Rodriquez, R.; Han, M.; Blackford, A.; Netto, G.; Lodge, M.; Mease, R.C.; Pomper, M.G., Cho, S.Y., PSMA-based low molecular weight 18F-DCFBC PET/CT for detection and characterization of primary prostate cancer. J. Nucl. Med.2015, In Press;
Silver, D. A.; Pellicer, I.; Fair, W. R.; Heston, W. D.; Cordon-Cardo, C., Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res 1997, 3 (1), 81-5;
Slusher, B. S.; Tsai, G.; Yoo, G.; Coyle, J. T., Immunocytochemical localization of the N-acetyl-aspartyl-glutamate (NAAG) hydrolyzing enzyme N- acetylated alpha-linked acidic dipeptidase (NAALADase). The Journal of comparative neurology 1992, 315 (2), 217-29;
Stoermer, D.; Vitharana, D.; Hin, N.; Delahanty, G.; Duvall, B.; Ferraris, D. V.; Grella, B. S.; Hoover, R.; Rojas, C.; Shanholtz, M. K.; Smith, K. P.; Stathis, M.; Wu, Y.; Wozniak, K. M.; Slusher, B. S.; Tsukamoto, T., Design, synthesis, and pharmacological evaluation of glutamate carboxypeptidase II (GCPII) inhibitors based on thioalkylbenzoic acid scaffolds. Journal of medicinal chemistry 2012, 55 (12), 5922-32;
Stoermer, D.; Liu, Q.; Hall, M. R.; Flanary, J. M.; Thomas, A. G.; Rojas, C.; Slusher, B. S.; Tsukamoto, T., Synthesis and biological evaluation of hydroxamate- Based inhibitors of glutamate carboxypeptidase II. Bioorganic & medicinal chemistry letters 2003, 13 (13), 2097-100;
Szabo, Z.; Mena, E.; Rowe, S. P.; Plyku, D.; Nidal, R.; Eisenberger, M. A.; Antonarakis, E. S.; Fan, H.; Dannals, R. F.; Chen, Y.; Mease, R. C.; Vranesic, M.; Bhatnagar, A.; Sgouros, G.; Cho, S. Y.; Pomper, M. G., Initial Evaluation of
[F]DCFPyL for Prostate-Specific Membrane Antigen (PSMA)-Targeted PET Imaging of Prostate Cancer. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging 2015;
Tsukamoto, T.; Wozniak, K. M.; Slusher, B. S., Progress in the discovery and development of glutamate carboxypeptidase II inhibitors. Drug discovery today 2007, 12 (17-18), 767-76
Tang, G., Zeng, W., Yu, M., Kabalka, G. Facile synthesis of N-succinimidyl- 4-[`F]fluorobenzoate ([18F]SFB for protein labeling. J Label Comp Radiopharm, 2008, 51, 68-71;
Talanov, VS, Garmestani K, Regino CAS, Milenic DE, Plascjak PS, Waldmann TA, Brechbiel MW. Preparation and in vivo evaluation of a novel stabilized linker for 211At labeling of protein. Nucl. Med. Biol.2006; 33:469-480;
Vaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4- guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides. Nat Protoc 2007; 2:282-286;
Vallabhajosula, S.; Nikolopoulou, A.; Babich, J. W.; Osborne, J. R.; Tagawa, S. T.; Lipai, I.; Solnes, L.; Maresca, K. P.; Armor, T.; Joyal, J. L.; Crummet, R.; Stubbs, J. B.; Goldsmith, S. J., 99mTc-labeled small-molecule inhibitors of prostate- specific membrane antigen: pharmacokinetics and biodistribution studies in healthy subjects and patients with metastatic prostate cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2014, 55 (11), 1791-8;
Vargas, H. A.; Grimm, J.; O, F. D.; Sala, E.; Hricak, H., Molecular imaging of prostate cancer: translating molecular biology approaches into the clinical realm. European radiology 2015
Wang, H.; Byun, Y.; Barinka, C.; Pullambhatla, M.; Bhang, H. E.; Fox, J. J.; Lubkowski, J.; Mease, R. C.; Pomper, M. G., Bioisosterism of urea-based GCPII inhibitors: Synthesis and structure-activity relationship studies. Bioorganic & medicinal chemistry letters 2010, 20 (1), 392-7;
Wang, E. K., H.C.; Szardenings, A.K., Liu, C,; Walsh, J.C.; Chen, G.; Sinha, A.; Kasi, D.; Gangadharmath, U.B.; Yu, C.; Zhang, W.; Zhao, T.; Mocharla, V.P. PSMA Imaging Agents.28.02.2013, 2013;
Weineisen, M. S., J.; Schottelius, M.; Schwaiger, M.; Wester, H-J. , Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI 2014, 4 (63);
Winkler, J. W.; Uddin, J.; Serhan, C. N.; Petasis, N. A., Stereocontrolled total synthesis of the potent anti-inflammatory and pro-resolving lipid mediator resolvin D3 and its aspirin-triggered 17R-epimer. Organic letters 2013, 15 (7), 1424-7;
Zalutsky, M. R.; Narula, A. S., A method for the radiohalogenation of proteins resulting in decreased thyroid uptake of radioiodine. Int J Rad Appl Instrum Part A 1987, 38 (1051-1055);
Zechmann, C. M.; Afshar-Oromieh, A.; Armor, T.; Stubbs, J. B.; Mier, W.; Hadaschik, B.; Joyal, J.; Kopka, K.; Debus, J.; Babich, J. W.; Haberkorn, U., Radiation dosimetry and first therapy results with a (124)I/ (131)I-labeled small molecule (MIP-1095) targeting PSMA for prostate cancer therapy. European journal of nuclear medicine and molecular imaging 2014, 41 (7), 1280-92;
Zhong, C.; Zhao, X.; Sarva, J.; Kozikowski, A.; Neale, J. H.; Lyeth, B. G., NAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in rats. J Neurotrauma 2005, 22 (2), 266-76;
Zhou J, Neale JH, Pomper MG, Kozikowski AP. NAAG Peptidase inhibitors and their potential for diagnosis and therapy. Nat Rev Drug Discovery 2005; 4:1015- 1026. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED: 1. A compound of formula (I):
Figure imgf000099_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order;
Z is tetrazole or CO2Q;
Q is H or a protecting group;
t is an integer selected from the group consisting of 1, 2, 3, 4;
p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different;
p1, p3, and p4 are each independently 0 or 1;
m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and– NR–C(=O)–;
W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,– C(O)–,–NRC(O)–,–NR'C(O)NR–,–NRC(S)NR'2–,–NRC(O)O–,–OC(O)NR–,– OC(O)–,–C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q– , wherein q is selected from the group consisting of 1,
2,
3,
4, 5, 6, 7, and 8;
each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole rou that can be resent or absent and is selected from the
group consisting
Figure imgf000099_0002
;
each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R5 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H; V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,– NRC(S)–, and–OC(O)–.
A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which can optionally comprise one or more radioactive isotope suitable for imaging and /or radiotherapy, and a photosensitizing dye suitable for imaging and/or photodynamic therapy. 2. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of:
Figure imgf000100_0001
wherein L is a linker that can be present or absent, and has the following general structure:
Figure imgf000100_0002
wherein p1, p2, p3, m1, m2, Tz, W2, R, R1, R2, R3, V and A are defined as hereinabove. 3. The compound of claim 1, wherein L is selected from the group consisting of:
Figure imgf000101_0001
4. The compound of claim 1, wherein A has the following general structure:
Figure imgf000101_0002
;
wherein:
X is CH or N; and R5, R6, and R7 are each independently selected from the group consisting of H, halogen, alkoxyl, alkythioether, substituted and unsubstituted aryl, CH2-NH- C(=NH)-NH2, NH-(C=O)-R8, wherein R8 is alkyl, -(C=O)-NR9R10, wherein R9 and R10 are each independently selected from the group consisting of H and alkyl, -X1- (CH2)q-Ph-X2, -X1-(CH2)q-X2, -X1-(CH2)q-NH-C(=O)-Ph-X2, wherein each X1 is independently O or S, each q is independently an integer selected from the group consisting of 1, 2, 3, and 4, each Ph is phenyl, and each X2 is halogen.
5. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of:
.
Figure imgf000102_0001
6. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of:
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
7. The compound of claim 1, wherein the photosensitizing dye suitable for imaging and/or photodynamic therapy is a fluorescent dye moiety which emits in the visible or near infrared spectrum, wherein the fluorescent dye moiety comprises carbocyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron- dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS; and wherein R1 or R2 can optionally be a radioactive isotope suitable for imaging or radiotherapy or optionally substituted with said radioisotope.
8. The compound of claim 7, wherein the fluorescent dye moiety is selected from the group consisting of:
Figure imgf000108_0001
Figure imgf000109_0001
9. The compound of claim 7, wherein the compound is
Figure imgf000110_0002
10. A method for imaging one or more prostate-specific membrane antigen (PSMA) tumors or cells, the method comprising contacting the one or more tumors or cells with an effective amount of a compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), and making an image, the compound of formula (I) comprising:
Figure imgf000110_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order;
Z is tetrazole or CO2Q;
Q is H or a protecting group;
t is an integer selected from the group consisting of 1, 2, 3, 4;
p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different;
p1, p3, and p4 are each independently 0 or 1;
m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and– NR–C(=O)–; W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,– C(O)–,–NRC(O)–,–NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,– OC(O)–,–C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q– , wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8;
each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole group that can be present or absent and is selected from the
group consisting of
Figure imgf000111_0001
each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H;
V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,– NRC(S)–, and–OC(O)–.
A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for imaging, or a photosensitizing dye suitable for imaging.
11. The method of claim 10, wherein the image is made using positron emission tomography (PET) and the radiohalogen is selected from the group consisting of 18F and 124I.
12. The method of claim 10, wherein the image is made using Single- photon emission computed tomography (SPECT) and the radiohalogen is selected from the group consisting of 77Br, 131I, 125I, and 123I.
13. The method of claim 10, wherein the image is made using florescent optical imaging and the photosensitizing dye suitable for imaging is selected from the group consisting of:
Figure imgf000112_0001
Figure imgf000113_0001
14. The method of claim 10, wherein the one or more PSMA-expressing tumors or cells is selected from the group consisting of: a prostate tumor or cell, a metastasized prostate tumor or cell, a lung tumor or cell, a renal tumor or cell, a glioblastoma, a pancreatic tumor or cell, a bladder tumor or cell, a sarcoma, a melanoma, a breast tumor or cell, a colon tumor or cell, a germ cell, a
pheochromocytoma, an esophageal tumor or cell, a stomach tumor or cell, and combinations thereof.
15. The method of claim 10, wherein the one or more PSMA-expressing tumors or cells is a prostate tumor or cell.
16. The method of claim 10, wherein the one or more PSMA-expressing tumors or cells is in vitro, in vivo, or ex vivo.
17. The method of claim 10, wherein the one or more PSMA-expressing tumors or cells is present in a subject.
18. The method of claim 10, wherein the compound of formula (I) comprising the imaging agent substantially localizes to the tumor or cell within about 60 minutes of administration.
19. The method of claim 10, wherein the compound of formula (I) comprising the radioisotope suitable for imaging substantially localizes to the tumor or cell within about 30 minutes of administration.
20. The method of claim 10, wherein the compound of formula (I) comprising the radioisotope suitable for imaging substantially localizes to the tumor or cell within about 10 minutes of administration.
21. The method of claim 10, wherein the compound of formula (I) comprising the radioisotope suitable for imaging is cleared from the tumor or cell in the subject.
22. The method of claim 21, wherein the compound of formula (I) comprising the radioisotope suitable for imaging is cleared more rapidly from a subject's kidneys than from a tumor of the subject.
23. The method of claim 10, wherein the compound comprising the photosensitizing dye suitable for imaging is visible at about 4 hours after injection.
24. The method of claim 10, wherein the compound comprising the photosensitizing dye suitable for imaging presents the brightest signal at about 24 hours after injection.
25. A method for treating or preventing a disease or condition associated with one or more PSMA expressing tumors or cells, the method comprising administering at least one compound of formula (I), including compounds of formula (Ia), (Ib), and (Ic), to a subject in an amount effective to treat or prevent the disease or condition, the compound of formula (I) comprising:
Figure imgf000115_0001
wherein the subunits associate with elements p1, p2, p3 and p4 may be in any order;
Z is tetrazole or CO2Q;
Q is H or a protecting group;
t is an integer selected from the group consisting of 1, 2, 3, 4;
p2 is an integer selected from the group consisting of 0, 1, 2, and 3, and when p2 is 2 or 3, each R1 is the same or different;
p1, p3, and p4 are each independently 0 or 1;
m1 and m2 are each an integer independently selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;
W1 is selected from the group consisting of a bond,–S–,–C(=O)–NR–, and– NR–C(=O)–;
W2 is selected from the group consisting of a bond, -S-, -CH2-C(=O)-NR-,– C(O)–,–NRC(O)–,–NRC(O)NR–,–NRC(S)NR–,–NRC(O)O–,–OC(O)NR–,– OC(O)–,–C(O)NR–,–NR–C(O)–,–C(O)O–,–(O–CH2–CH2)q– and–(CH2-CH2-O)q– , wherein q is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8;
each R is independently H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl; Tz is a triazole group that can be present or absent and is selected from the group consisting of
Figure imgf000115_0002
each R1 is independently H, C1-C6 alkyl, C2-C12 aryl or C4-C16 alkylaryl; R2 and R3 are each independently H and CO2R4, wherein R4 is selected from the group consisting of H, C1-C6 alkyl, C2-C12 aryl, and C4-C16 alkylaryl, wherein when one of R2 or R3 is CO2R4, then the other is H;
V is selected from the group consisting of–C(O)–, -C(S)-,–NRC(O)–,– NRC(S)–, and–OC(O)–.
A is selected from the group consisting of naphthyl, biphenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, each of which comprising one or more radioactive isotope suitable for radiotherapy, or a photosensitizing dye suitable for photodynamic therapy.
26. The method of claim 25, wherein the radiohalogen suitable for radiotherapy is selected from the group consisting of 80mBr, 77Br, 125I, 123I, 131I and 211At.
27. The method of claim 25, wherein the photosensitizing dye suitable for photodynamic therapy is selected from the group consisting of
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
28. The method of claim 25, wherein the disease or condition is a prostate cancer, renal cancer, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, adenomas, and tumor neovasculature.
29. The method of claim 25, wherein the disease or condition is prostate cancer.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170015684A1 (en) * 2014-02-28 2017-01-19 Stella Pharma Corporation Method for producing 4-borono-l-phenylalanine having 18f atom introduced thereinto, and precursor of 4-borono-l-phenylalanine having 18f atom introduced thereinto
WO2018005625A1 (en) * 2016-06-28 2018-01-04 Cornell University 18f-labeled triazole containing psma inhibitors
WO2018091043A1 (en) * 2016-11-18 2018-05-24 Abx Advanced Biochemical Compounds Gmbh Precursors for radiofluorination
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US10815200B2 (en) 2015-09-30 2020-10-27 Deutsches Krebsforschungszentrum 18F—tagged inhibitors of prostate specific membrane antigen (PSMA) and their use as imaging agents for prostate cancer
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Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130116404A1 (en) 2011-11-08 2013-05-09 Case Western Reserve University Targeted non-invasive imaging probes of egfr expressing cells
EP4374924A2 (en) 2013-10-18 2024-05-29 Novartis AG Labeled inhibitors of prostate specific membrane antigen (psma), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer
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MX2019012017A (en) 2017-04-07 2020-02-12 Juno Therapeutics Inc Engineered cells expressing prostate-specific membrane antigen (psma) or a modified form thereof and related methods.
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JP7131499B2 (en) * 2018-08-09 2022-09-06 信越化学工業株式会社 Resist material and pattern forming method
KR20200043115A (en) * 2018-10-17 2020-04-27 고려대학교 세종산학협력단 Prostate-specific membrane antigen inhibitors and pharmaceutical composition for diagnosis and treatment of prostate cancer
AU2020304640A1 (en) * 2019-06-26 2022-01-27 Lunella Biotech, Inc. Carbocyanine compounds for targeting mitochondria and eradicating cancer stem cells
US20220273826A1 (en) * 2019-07-02 2022-09-01 Novartis Ag Prostate specific membrane antigen (psma) ligands and uses thereof
CN112898172B (en) * 2019-12-04 2022-05-31 中国科学院大连化学物理研究所 Synthesis method of amphiphilic functional group compound capable of being enzymolyzed by carboxypeptidase
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Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7109299B1 (en) * 1999-12-16 2006-09-19 Affymax, Inc. Peptides and compounds that bind to the IL-5 receptor
ATE443260T1 (en) 2002-01-10 2009-10-15 Univ Johns Hopkins CONTRAST AGENTS AND METHOD FOR IMAGING NAALDASE OR PSMA
CN1826331A (en) * 2003-04-03 2006-08-30 塞马福尔药业公司 Pi-3 kinase inhibitor prodrugs
PL2097111T3 (en) 2006-11-08 2016-01-29 Molecular Insight Pharm Inc Heterodimers of glutamic acid
EP3466928A1 (en) 2007-06-26 2019-04-10 The Johns Hopkins University Labeled inhibitors of prostate-specific membrane antigen (psma), biological evaluation, and use as imaging agents
PL2187965T3 (en) 2007-08-17 2020-05-18 Purdue Research Foundation Psma binding ligand-linker conjugates and methods for using
BRPI0915173B8 (en) 2008-06-11 2021-05-25 Agilent Technologies Inc nucleotide and nucleoside compounds and method of sequencing a target nucleic acid
HUE059436T2 (en) * 2008-08-01 2022-11-28 Univ Johns Hopkins Psma-binding agents and uses thereof
WO2010045598A2 (en) 2008-10-17 2010-04-22 Purdue Research Foundation Psma binding ligand-linker conjugates and methods for using
US9056841B2 (en) * 2009-03-19 2015-06-16 The Johns Hopkins University PSMA-targeting compounds and uses thereof
US20120322741A1 (en) * 2010-02-25 2012-12-20 Purdue Research Foundation Psma binding ligand-linker conjugates and methods for using
WO2013022797A1 (en) 2011-08-05 2013-02-14 Molecular Insight Pharmaceuticals Radiolabeled prostate specific membrane antigen inhibitors
US10011632B2 (en) 2011-08-22 2018-07-03 Siemens Medical Solutions Usa, Inc. PSMA imaging agents
CN113149921A (en) 2011-11-30 2021-07-23 约翰霍普金斯大学 Homogenic and xenogenic multivalent inhibitors of Prostate Specific Membrane Antigen (PSMA) and uses thereof
WO2016065145A2 (en) 2014-10-22 2016-04-28 The Johns Hopkins University Psma targeted reversed carbamates and methods of use thereof

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