PSMA TARGETING UREA-BASED LIGANDS FOR PROSTATE CANCER RADIOTHERAPY AND IMAGING
20230201383 · 2023-06-29
Inventors
- Andreas Kjær (FREDERIKSBERG, DK)
- Matthias Manfred HERTH (Malmö, SE)
- Andreas Ingemann JENSEN (Virum, DK)
- Matthias EDER (Heidelberg, DE)
- Ann-Christin EDER (79106, DE)
Cpc classification
A61K51/0497
HUMAN NECESSITIES
A61K51/044
HUMAN NECESSITIES
International classification
Abstract
The present invention provides novel PSMA targeting urea-based ligands that binds to prostate-specific membrane antigen (PSMA) which is expressed 8-to-12-fold higher in prostate cancer cells when compared to healthy tissue. The PSMA targeting urea-based ligands comprises a chelating agent that may comprise a metal and a halogen radioisotope of fluorine, iodine, bromine or astatine. The invention further relates to a method for providing the PSMA targeting urea-based ligands of the invention, to precursors of the PSMA targeting urea-based ligands and to the PSMA targeting urea-based ligands use in radiotherapy, imaging and theranostic.
Claims
1. A PSMA targeting ligand of formula (I) ##STR00058## wherein: A is independently carboxylic acid, sulphonic acid, phosphonic acid, tetrazole or isoxazole; L is selected from the group consisting of urea, thiourea, —NH—(C═O)—O—, —O—(C═O)—NH— or —CH.sub.2—(C═O)—CH.sub.2—; K is selected from the group consisting of —(C═O)—NH—, —CH.sub.2—NH—(C═O)— or ##STR00059## wherein p is independently an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6; q is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; Y is selected from the group consisting of: ##STR00060## wherein Q.sub.1 is —C—R.sub.3 or N, wherein R.sub.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or a radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; M is a chelating agent, that can comprise a metal; n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6; m is an integer selected from the group consisting of 0 and 1; o is an integer selected from the group consisting of 0 and 1; R.sub.1 is —CH—CH.sub.2—Z or —CH—CH.sub.2—Y; wherein Z is selected from the group consisting of: ##STR00061## and Y is selected from the group consisting of: ##STR00062## wherein Q.sub.1 is —C—R.sub.3 or N, wherein R.sub.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; R.sub.2 is —CH—CH.sub.2—Y or —CH.sub.2—X—; wherein X is an aromatic monocyclic or polycyclic ring system having 6 to 14 carbon atoms, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; and Y is selected from the group consisting of: ##STR00063## wherein Q.sub.1 is —C—R.sub.3 or N, wherein R.sub.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; and wherein formula (I) comprises at least one isotope or radioisotope selected from fluorine, iodine, bromine or astatine; and pharmaceutically acceptable salts thereof.
2. A PSMA targeting ligand according to claim 1, having the general formula (Ia): ##STR00064## wherein: A is independently carboxylic acid, sulphonic acid, phosphonic acid, tetrazole or isoxazole; n is an integer selected from the group consisting of 1, 2, 3 and 4; m is an integer selected from the group consisting of 0 and 1; o is an integer selected from the group consisting of 0 and 1; is is selected from the group consisting of: ##STR00065## wherein Q.sub.1 is —C—R.sub.3 or N, wherein R.sub.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; M is a chelating agent, that can comprise a metal; R.sub.1 is —CH—CH.sub.2—Z or —CH—CH.sub.2—Y; wherein Z is selected from the group consisting of: ##STR00066## and Y is selected from the group consisting of: ##STR00067## wherein Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; R.sub.2 is —CH—CH.sub.2—Y or —CH.sub.2—X—; wherein X is an aromatic monocyclic or polycyclic ring system having 6 to 14 carbon atoms, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; and Y is selected from the group consisting of: ##STR00068## wherein Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl; Q.sub.2 is O, S or NH; Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; and wherein formula (I) comprises at least one isotope or radioisotope selected from fluorine, iodine, bromine or astatine; and pharmaceutically acceptable salts thereof.
3. The PSMA targeting ligand according to claim 1, wherein M is selected from the group consisting of: 1,4,7,10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid (DOTA), N,N′-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine N,N′-diacetic acid (HBED-CC), 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid DOTAGA), 4,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclo9.3.1.pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N′-(5-acetyl (hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA); and pharmaceutically acceptable salts thereof.
4. The PSMA targeting ligand according to claim 3, wherein M comprises a metal selected from the group consisting of Y, Lu, Tc, Zr, In, Sm, Re, Cu, Pb, Ac, Bi, Al, Ga, Ho and Sc.
5. The PSMA targeting ligand according to claim 1, wherein R.sub.1 is —CH—CH.sub.2—Y and Hal is selected from the group consisting of .sup.18F, .sup.19F, .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.127I, .sup.211At, .sup.77Br, .sup.80Br, .sup.79Br, and .sup.81Br
6. The PSMA targeting ligand according to claim 1, selected from the group consisting of: ##STR00069## ##STR00070## ##STR00071## ##STR00072## wherein in compound Ii, Ij, Ik, Il, Im and In “I” is an isotope or radioisotope of iodine.
7. A precursor compound of the PSMA targeting ligand according to claim 1 selected from the group comprising the below formulas (II) to (VIII) and the same precursor compounds wherein the Me).sub.3Sn group is replaced with a silyl, boron, iodonium or diazonium group: ##STR00073## ##STR00074##
8. A method for providing the PSMA targeting ligand according to claim 1 comprising Synthesis of a PSMA binding motif Coupling of linkers to the PSMA binding motif, wherein one or more of the precursors of formula (II), (III), (V) and (VII) according to claim 7 is provided Coupling of the PSMA binding motif-linker to a chelator wherein one or more of the precursors of Formula (IV), (VI) and (VIII) according to claim 7 is provided Labeling the PSMA binding motif-linker-chelator with a halogen nuclide or radionuclide.
9. The method according to claim 8, wherein the PSMA binding motif is Lys-urea-Glu.
10. The method according to claim 9 wherein the halogen nuclide is selected from the group consisting of .sup.18F, .sup.19F, .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.127I, .sup.211At, .sup.77Br, .sup.79Br, .sup.80Br, and .sup.81Br.
11. The PSMA targeting ligands of formula (I) according to claim 1 wherein the halogen is selected from the group consisting of .sup.211At, .sup.125I, .sup.123I, .sup.77Br, and .sup.80Br for use in radiotherapy.
12. The PSMA targeting ligands of formula (I) according to claim 1 wherein the halogen is .sup.211At for use in the treatment of cancer, in particular prostate cancer.
13. The PSMA targeting ligands of formula (I) according to claim 1 wherein the halogen is selected from the group consisting of .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.77Br and .sup.80Br for use as a theranostic agent.
14. The use of PSMA targeting ligands of formula (I) according to claim 1 wherein the halogen is selected from the group consisting of .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.77Br and .sup.80Br as an imaging agent.
15. The use of PSMA targeting ligands of formula (I) according to claim 1 wherein the halogen is selected from the group consisting of .sup.19F, .sup.127I, .sup.79Br, and .sup.81Br as test-compounds.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
DETAILED DESCRIPTION OF THE INVENTION
[0037] The PSMA targeting urea-based ligands of the present invention are suitable for use as radiopharmaceuticals, either as imaging agents or for the treatment of prostate cancer, or as theranostic agents.
[0038] The PSMA targeting urea-based ligands of the present invention take advantage of a urea-based binding motif (((S)-5-amino-1-carboxypentyl)carbamoyl)-L-glutamic acid). This motif specifically interacts with the PSMA antigen binding pocket. It contains an urea that forms a coordination complex with a Zn.sup.+2 atom, which is crucial for the binding. Moreover, carboxylic acids also interact with the residues in the vicinity of the binding site, making this scaffold very convenient for PSMA-specific targeting.
[0039] The compounds disclosed herein comprises at least one isotope or radioisotope selected from the halogen group and are suitable for different purposes. The halogen astatine, particularly the radioactive radionuclide .sup.211At, is particularly useful in alpha-particle therapy, whereas .sup.18F and the radionuclides of iodine .sup.125I, .sup.123I, .sup.131I, and .sup.124I, are primarily intended as theranostic companions for the astatine-211 labeled variant. In this sense, .sup.18F and most radioisotopes of iodine are suitable for imaging. Presently, the approach is that patients be first diagnosed using diagnostic imaging, for example with a compound labeled with .sup.18F or radioiodine, and then treated with a modality such as alpha-particle therapy. For this, analogues labeled with radionuclides for imaging are therefore required. For theranostics to work best, the diagnostic variant must have the radionuclide placed in the exact same position as the therapeutic variant, and the rest of the molecule should be identical.
[0040] The bromine radionuclides .sup.77Br and .sup.80Br are primarily relevant for Auger electron radiotherapy and .sup.125I and .sup.123I have also been used for such a purpose. Auger therapy is a form of radiation therapy for the treatment of cancer which relies on a large number of low-energy electrons (emitted by the Auger effect) to damage cancer cells, rather than the high-energy radiation used in traditional radiation therapy. Similar to other forms of radiation therapy, Auger therapy relies on radiation-induced damage to cancer cells (particularly DNA damage) to arrest cell division, stop tumor growth and metastasis and kill cancerous cells. It differs from other types of radiation therapy in that electrons emitted via the Auger electrons are released in large numbers with low kinetic energy.
[0041] Non-radioactive test-compounds corresponding to the radioactive compounds but comprising a non-radioactive isotope of iodine, fluorine and bromine, respectively, can be applied instead of the radioactive variants in order to test the applicability of the compounds. Such test-compounds preferably comprises one of the non-radioactive isotopes .sup.127I, .sup.19F, .sup.79Br or .sup.81Br, respectively, instead of the radioactive variants. There is, however, no non-radioactive isotope for astatine, but .sup.127I can be used as a test-compound instead. For instance, in order to test the impact of influencing the linker region, iodine .sup.127I isotope were provided and tested herein. .sup.127I is a large atom (atomic radius: 198 pm) and similar in size to astatine-211 (atomic radius: 200 pm) and with a highly similar halogen electronic configuration. Accordingly, experiments using the non-radioactive .sup.127I-compound was applied to demonstrate that the linker region can be modified with a large halogen and still display efficient internalization, on par with or better than reported, optimized compounds in clinical use.
[0042] Surprisingly, despite the synthetic modifications in key regions, the binding profile and biodistribution/pharmacokinetics of various of the herein disclosed new compounds are comparable or superior to values observed for PSMA-617 in direct head-to-head comparison.
[0043] This is particularly true for compounds Ii and Im. PSMA-617 is the most clinically applied therapeutic PSMA inhibitor.
[0044] The term “nuclide” comprises both non-radioactive and radioactive nuclides (radionuclides). The compounds of the present invention can comprise either a radioactive or non-radioactive nuclide depending on the intended use. When used herein in relation to specific compounds the terms “nuclide” and “radionuclide” are used to make an indication of whether the final compound is radioactive or not.
[0045] The term “isotope” comprises both non-radioactive and radioactive isotopes (radioisotopes). The compounds of the present invention can comprise either a radioactive or non-radioactive isotope depending on the intended use. When used herein in relation to specific compounds the terms “isotope” and “radioisotope” are used to make an indication of whether the final compound is radioactive or not. The various isotopes of the halogen nuclides iodine, fluorine, bromine and astatine are all well known, and the isotope number will reveal whether the isotope is stable (non-radioactive) or not (radioactive).
[0046] The PSMA targeting urea-based ligands of the present invention have the following general formula (I):
##STR00007##
wherein:
A is independently carboxylic acid, sulphonic acid, phosponic acid, tetrazole or isoxazole;
L is selected from the group consisting of urea, thiourea, —NH—(C═O)—O—, —O—(C═O)—NH— or —CH.sub.2—(C═O)—CH.sub.2—;
K is selected from the group consisting of —(C═O)—NH—, —CH.sub.2—NH—(C═O)— or
##STR00008##
wherein
p is independently an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6;
q is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6;
Y is selected from the group consisting of:
##STR00009##
wherein
Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0047] Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine; M is a chelating agent, that can comprise a metal,
n is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6;
m is an integer selected from the group consisting of 0 and 1;
o is an integer selected from the group consisting of 0 and 1;
R.sub.1 is —CH—CH.sub.2—Z or —CH—CH.sub.2—Y;
wherein Z is selected from the group consisting of:
##STR00010##
and Y is selected from the group consisting of:
##STR00011##
wherein
Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0048] Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine;
R.sub.2 is —CH—CH.sub.2—Y or —CH.sub.2—X—;
wherein X is an aromatic monocyclic or polycyclic ring system having 6 to 14 carbon atoms, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl;
and Y is selected from the group consisting of:
##STR00012##
wherein
Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0049] Hal is a nuclide or radionuclide of the halogen group selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine;
and wherein formula (I) comprises at least one isotope or radioisotope selected from fluorine, iodine, bromine or astatine,
and pharmaceutically acceptable salts thereof.
[0050] In a particular embodiment, the PSMA targeting ligand of formula (I) is selected from the group of compounds of formula (Ia):
##STR00013##
wherein:
A is independently carboxylic acid, sulphonic acid, phosponic acid, tetrazole or isoxazole;
n is an integer selected from the group consisting of 1, 2, 3 and 4;
m is an integer selected from the group consisting of 0 and 1;
o is an integer selected from the group consisting of 0 and 1;
Y is selected from the group consisting of:
##STR00014##
wherein
Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0051] Hal is selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine;
M is a chelating agent that can comprise a metal
R.sub.1 is —CH—CH.sub.2—Z or —CH—CH.sub.2—Y;
wherein Z is selected from the group consisting of:
##STR00015##
and Y is selected from the group consisting of:
##STR00016##
wherein
Q is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0052] Hal (halogen) is selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine;
R.sub.2 is —CH—CH.sub.2—Y or —CH.sub.2—X—;
wherein X is an aromatic monocyclic or polycyclic ring system having 6 to 14 carbon atoms, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl;
and Y is selected from the group consisting of:
##STR00017##
wherein
Q.sub.1 is —C—R.sup.3 or N, wherein R.sup.3 is H or C.sub.1-C.sub.5 alkyl;
Q.SUB.2 .is O, S or NH;
[0053] Hal is selected from the group consisting of isotopes and radioisotopes of fluorine, iodine, bromine or astatine;
and wherein formula (I) comprises at least one isotope or radioisotope selected from fluorine, iodine, bromine or astatine;
and pharmaceutically acceptable salts thereof.
[0054] The chelating agent may be selected from one of the following chelators: [0055] 1,4,7,10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid (DOTA), [0056] N,N′-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine N,N′-diacetic acid (HBED-CC), [0057] 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), [0058] 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), [0059] 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), [0060] 14,7-triazacyclononane phosphinic acid (TRAP), [0061] 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), [0062] 3,6,9,15-tetraazabicyclo9.3.1.pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), [0063] N′-(5-acetyl (hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), [0064] diethylenetriaminepentaacetic acid (DTPA), [0065] trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), [0066] 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), [0067] p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), [0068] 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), [0069] 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), [0070] 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), that can comprise a metal; and pharmaceutically acceptable salts thereof.
[0071] In some embodiments, the chelating agent comprises a metal. In a preferred embodiment, the chelating agent comprises a metal selected from the group consisting of Y, Lu, Tc, Zr, In, Sm, Re, Cu, Pb, Ac, Bi, Al, Ga, Ho and Sc.
[0072] The nuclide or radionuclide (Hal) may be present in the R.sub.1, R.sub.2 and Y groups in Formula (I). The nuclide or radionuclide is selected from the halogen group. This group comprises isotopes and radioisotopes of Fluorine (F), Chlorine (CI), Bromine (Br), Iodine (I) and Astatine (At).
[0073] In a preferred embodiment, the halogen nuclide is a radionuclide selected from a radioisotope of fluorine, a radioisotope of iodine, a radioisotope of bromine or a radioisotope of astatine.
[0074] In another preferred embodiment, the halogen nuclide is a radionuclide selected from the group consisting of .sup.18F, .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.211At, .sup.77Br and .sup.80Br.
[0075] In a particularly preferred embodiment, the halogen nuclide is one of the following radionuclides .sup.123/124/125/131I or .sup.211At.
[0076] In another preferred embodiment, the nuclide is non-radioactive and selected from a non-radioactive isotope of fluorine, iodine or bromine.
[0077] In a more preferred embodiment, the nuclide (Hal) is selected from the group consisting of .sup.127I, .sup.19F, .sup.79Br and .sup.81Br.
[0078] In preferred embodiments, the PSMA targeting ligand according to Formula (I), is one of the following seven compounds:
##STR00018## ##STR00019## ##STR00020##
[0079] In other preferred embodiments, the PSMA targeting ligand according to Formula (I), is one of the following compounds:
##STR00021## ##STR00022##
wherein iodine “I” is selected from .sup.127I, .sup.125I, .sup.123I, .sup.131I, or .sup.124I.
[0080] In a most preferred embodiment, the PSMA targeting ligand according to Formula (I) is Il or Im.
[0081] The PSMA targeting ligands according to formula (I) may be provided by suitable methods known in the art.
[0082] In one aspect, the present invention relates to a method for providing the PSMA targeting ligands according to Formula (I) comprising the steps of: [0083] Synthesis of a PSMA binding motif (BM) [0084] Coupling of linkers to BM [0085] Coupling of BM-linker to a chelator [0086] Labeling the BM-linker-chelator with a halogen nuclide, such as a halogen radionuclide
[0087] In one embodiment, the PSMA binding motif is Lys-urea-Glu (LUG).
[0088] In one embodiment, the chelator is selected from: [0089] 1,4,7,10-tetraazacyclododecane-N,N′,N′,N″-tetraacetic acid (DOTA), [0090] N,N′-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine N,N′-diacetic acid (HBED-CC), [0091] 14,7-triazacyclononane-1,4,7-triacetic acid (NOTA), [0092] 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), [0093] 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), [0094] 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2-carboxyethyl)phosphinic acid-4,7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), [0095] 3,6,9,15-tetraazabicyclo9.3.1.pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), [0096] N′-(5-acetyl (hydroxy)aminopentyl-N-(5-(4-(5-aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl-N-hydroxysuccinamide (DFO), [0097] diethylenetriaminepentaacetic acid (DTPA), [0098] trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), [0099] 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), [0100] p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), [0101] 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), [0102] 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), and [0103] 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA).
[0104] In one embodiment, the halogen radionuclide is selected from an isotope or radioisotope of fluorine, iodine, bromine or astatine.
[0105] In a preferred embodiment, the halogen is a radionuclide being one of the following radioisotopes: .sup.18F, .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.211At, .sup.77Br and .sup.80Br.
[0106] In another preferred embodiment, the halogen is a non-radioactive nuclide selected from one of the following isotopes: .sup.19F, .sup.127I, .sup.79Br and .sup.81Br.
[0107] The present invention also provides (Me).sub.3Sn precursors, silyl precursors, boron-based precursors, iodonium and diazonium salt precursors that can be used to provide the PSMA targeting ligand according to Formula (I).
[0108] These precursors include precursors with and without chelators and have the following structures, here shown for the most preferred (Me).sub.3Sn precursors, but the same structures are applicable for if substituting the (Me).sub.3Sn with silyl, boron, iodonium or diazonium:
##STR00023## ##STR00024## ##STR00025##
[0109] A preferred method comprises the following steps: [0110] Synthesis of a PSMA binding motif (BM) [0111] Coupling of linkers to BM, wherein one or more of the precursors of formula (II), (III), (V) and (VII) is provided [0112] Coupling of BM-linker to a chelator wherein one or more of the precursors of Formula (IV), (VI) and (VIII) is provided [0113] Labeling the BM-linker-chelator with a halogen nuclide, such as a halogen radionuclide.
[0114] In a preferred embodiment, the PSMA binding motif is Lys-urea-Glu (LUG).
[0115] The PSMA targeting ligands of formula (I) such as the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij) (Ik), (Il), (Im) and (In) can be used in radiotherapy, as imaging agents or as both i.e. as theranostic agents.
[0116] Surprisingly, despite the synthetic modifications in key regions, the binding profile and biodistribution/pharmacokinetics of various of the herein disclosed new compounds are comparable or superior to values observed for PSMA-617 in direct head-to-head comparison. This is particularly true for compounds Ii and Im. PSMA-617 is the most clinically applied therapeutic PSMA inhibitor.
[0117] In one aspect, the PSMA targeting ligands of formula (I) are for use in radiotherapy. In a preferred embodiment the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are for use in radiotherapy. In more preferred embodiments, compounds Ii or Im are used in radiotherapy. Preferably, when using ligands of formula (I) for radiotherapy, the halogen isotope is selected from the group consisting of .sup.211At, .sup.125I, .sup.123I, .sup.77Br, and .sup.80Br.
[0118] In another aspect, the PSMA targeting ligands of formula (I) are for use in the treatment of cancer, in particular prostate cancer. In a preferred embodiment, the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are used in the treatment of cancer, in particular prostate cancer. In more preferred embodiments, compounds Ii or Im are used in the treatment of cancer, in particular prostate cancer. Preferably, when using ligands of formula (I) for radiotherapy, the halogen isotope is .sup.211At.
[0119] In yet another aspect, the PSMA targeting ligands of formula (I) are for use as a theranostic agent. In a preferred embodiment, the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij) (Ik), (Il), (Im) and (In) are for use as theranostic agents. In more preferred embodiments, compounds Ii or Im are for use as a theranostic agents. Preferably, when using ligands of formula (I) for theranostic agents, the halogen isotope is selected from the group consisting of .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.77Br and .sup.80Br.
[0120] A further aspect of the invention is the use of PSMA targeting ligands of formula (I) as an imaging agent. In a preferred embodiment, the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are for use as imaging agents. In more preferred embodiments, compounds Ii or Im are for use as imaging agents. Preferably, when using ligands of formula (I) for theranostic agents, the halogen isotope is selected from the group consisting of .sup.125I, .sup.123I, .sup.131I, .sup.124I, .sup.77Br and .sup.80Br.
[0121] A further aspect of the invention is the use of PSMA targeting ligands of formula (I) as non-radioactive test-compounds. In a preferred embodiment, the PSMA targeting ligands of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Il), (Im) and (In) are for use as test-compounds. In more preferred embodiments, compounds Ii or Im are for use test-compound. Preferably, when using ligands of formula (I) for theranostic agents, the halogen isotope is selected from the group consisting of .sup.127I, .sup.19F, .sup.79Br or .sup.81Br.
EXAMPLES
Example 1: General Synthetic Procedures for the Synthesis of DOTA-Bearing PSMA-Targeting Ligands
Glu-NCO.SUP.8.:
[0122] ##STR00026##
[0123] Firstly, di-tert-butyl L-glutamate hydrochloride (6.76 mmol) was suspended in a 1:2 mixture of dichloromethane (DCM) and saturated aqueous NaHCO.sub.3 (72 mL). The mixture was cooled to 0° C. and then triphosgene (3.38 mmol) was added. The reaction mixture was vigorously stirred at 0° C. for 20 minutes, then warmed to room temperature, diluted with DCM and washed with brine (2×30 mL). The organic layers were collected, dried over Na.sub.2SO.sub.4 and concentrated in vacuo, affording the isocyanate Glu-NCO.
PSMA Binding Motive.SUP.8.:
[0124] ##STR00027##
[0125] A solution of Glu-NCO (6.73 mmol) in DCM (16 mL) was added to a mixture of tert-butyl N.sup.6-((benzyloxy)carbonyl)-L-lysinate hydrochloride (7.40 mmol) and dry pyridine (7.40 mmol) in DCM (50 mL). The reaction was stirred at room temperature 18 hours. Afterwards, the reaction was diluted with 10 mL of DCM, washed with 0.1M HCl (5×15 mL) and washed with brine (2×15 mL). The organic layers were collected, dried over sodium sulphate and evaporated in vacuo. Finally, the obtained crude was purified by flash chromatography.
[0126] The Cbz-protected product (3.01 mmol) was mixed with Pd/C (10%) (0.31 mmol) catalyst, dissolved in MeOH (15 mL) and hydrogen gas was bubbled through the mixture overnight. The reaction mixture was filtered through a pad of diatomaceous earth and the solvent was evaporated in vacuo, yielding the PSMA binding motive.
PSMA Binding Motive-Linker:
[0127] ##STR00028##
[0128] The subsequent syntheses of the PSMA-targeting peptidomimetics were carried out with the following general procedure:
[0129] N-Fmoc-amino acid (0.21 mmol) and diisopropylethylamine (DIPEA) (0.51 mmol) were dissolved in dry dimethylformamide (DMF), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (0.31 mmol) was added to the previous solution and stirred for 10 minutes. PSMA binding motive (0.21 mmol) was dissolved in dry DMF and dropwise added to the previous mixture for a total volume of 1 mL. The reaction mixture was stirred at room temperature for 4 to 24 hours depending on when completion was attained.
[0130] Thereafter, the N-terminus was deprotected by adding piperidine (50% relative to DMF) and stirring for 2 hours. Reaction mixture was poured over water and extracted with DCM. Organic layers were dried over Na.sub.2SO.sub.4 and volatiles removed in vacuo. The crude was purified by flash chromatography giving the desired free amine.
PSMA Binding Motive-Linker-Chelator:
[0131] ##STR00029##
[0132] To a PSMA binding motive-linker construct solution (0.2 mmol) in either DCM or DMF (1 mL), trimethylamine (Et.sub.3N) (0.31 mmol), DOTA-mono-NHS-tris(tBu-ester) (0.31 mmol) was added and stirred for 18 hours at room temperature. The subsequent crude mixture was purified by preparative HPLC.
PSMA Binding Motive-Linker-Chelator Radiolabelling:
[0133] A tert-butyl protected PSMA binding motive-linker-chelator construct was dissolved in a solution of Chloramine-T, methanol, .sup.211At and acetic acid. The mixture was stirred and reacted during 30 minutes at room temperature. Afterwards, the mixture was dried by use of a nitrogen stream. The molecule was deprotected by addition of trifluoroacetic acid (TFA) and heating to 60° C. for 30 minutes. Once fully deprotected, the mixture was dried, redissolved in a 50:50 mixture of acetonitrile (MeCN) water and purified by preparative HPLC.
Example 2: Synthesis of PSMA-Targeting Radiopharmaceutical 1
[0134] Using the synthesis methods described in Example I, the following PSMA-targeting ligand was provided:
[0135] Synthesis of PSMA binding motif following the procedures previously described in Example I:
##STR00030##
di-tert-butyl (S)-2-isocyanatopentanedioate
[0136] ##STR00031##
[0137] Di-tert-butyl L-glutamate hydrochloride (2.00 g, 6.76 mmol, 1.0 eq.) was suspended in DCM (24 mL) and sat. NaHCO.sub.3 (aq.) (48 mL). The mixture was cooled to 0° C. and then bis(trichloromethyl) carbonate (triphosgene) (1.00 g, 3.38 mmol, 0.5 eq.) was added (OBS: triphosgene is highly toxic and must be handled with extreme care). The reaction mixture was vigorously stirred at 0° C. for 20 minutes, then allowed to warm to room temperature, diluted with DCM (36 mL) and water (30 mL) and extracted with DCM (1×25 mL). The organic layers were washed with brine (1×20 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo, affording the title compound as a transparent liquid (1.92 g, 6.73 mmol, quantitative).
[0138] .sup.1H-NMR (600 MHz, CDCl.sub.3) δ 3.97 (dd, J=8.6, 4.5 Hz, 1H), 2.40-2.28 (m, 2H), 2.17-2.08 (m, 1H), 1.91 (m, 1H), 1.49 (d, J=0.9 Hz, 9H), 1.44 (d, J=0.9 Hz, 9H). .sup.13C NMR (151 MHz, CDCl.sub.3) δ 171.8, 170.1, 127.4, 83.7, 80.9, 57.5, 31.6, 29.2, 28.2. MS (ESI) m/z: 260.2 [M+3H—CO].sup.+ (hydrolysed product)
tri-tert-butyl (9S,13S)-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane-9,13,15-tricarboxylate
[0139] ##STR00032##
[0140] A solution of di-tert-butyl (S)-2-isocyanatopentanedioate (1.92 g, 6.73 mmol, 1.0 eq.) in dry DCM (16 mL) was added to a mixture of tert-butyl N.sup.6-((benzyloxy)carbonyl)-L-lysinate hydrochloride (2.76 g, 7.40 mmol, 1.1 eq.) and dry pyridine (596 μL, 7.40 mmol, 1.1 eq.) in dry DCM (50 mL). The reaction was stirred at room temperature for 18 hours. Afterwards, the reaction was diluted with DCM (10 mL), washed with 0.1 M HCl.sub.(aq.) (5×15 mL) and brine (15 mL). The organic fraction was collected, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude was purified by CombiFlash (heptane:EtOAc—100:0 to 20:80) to isolate the title compound as a viscous colourless oil (2.93 g, 4.71 mmol, 70%).
[0141] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.37-7.28 (m, 5H), 5.22-5.05 (m, 5H), 4.36-4.29 (m, 2H), 3.22-3.12 (m, 2H), 2.35-2.22 (m, 2H), 2.09-2.02 (m, 1H), 1.87-1.67 (m, 3H), 1.66-1.23 (m, 31H) .sup.13C NMR (151 MHz, CDCl.sub.3) δ 172.6, 172.5, 171.3, 157.1, 156.8, 136.9, 128.6, 128.2, 82.2, 81.8, 80.6, 66.7, 53.4, 53.1, 40.8, 32.8, 32.0, 29.5, 29.1, 28.5, 28.2, 28.1, 22.8, 22.4. MS (ESI) m/z: 622.4 [M+H].sup.+ R.sub.f (40% EtOAc in heptane)=0.42
di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0142] ##STR00033##
[0143] To a flask containing tri-tert-butyl (9S,13S)-3,11-dioxo-1-phenyl-2-oxa-4,10,12-triazapentadecane-9,13,15-tricarboxylate (1.90 g, 3.06 mmol, 1.0 eq.) was added 10 wt. % Pd/C (325 mg, 0.31 mmol, 0.1 eq.) and suspended in MeOH (15 mL). The reaction vessel was purged with nitrogen and hydrogen gas was bubbled through the suspension overnight at atmospheric pressure (balloon). The reaction mixture was filtered over a pad of diatomaceous earth (Celite®) and volatiles removed in vacuo to yield di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate as a viscous oil (1.49 g, 3.05 mmol, 99%).
[0144] .sup.1H NMR (600 MHz, CDCl.sub.3) δ 5.23 (d, J=8.0 Hz, 2H), 4.32 (q, J=7.9, 4.9 Hz, 2H), 2.67 (t, J=6.8 Hz, 2H), 2.37-2.22 (m, 2H), 2.10-2.01 (m, 1H), 1.87-1.72 (m, 1H), 1.65-1.57 (m, 1H), 1.55-1.24 (m, 31H). .sup.13C NMR (151 MHz, CDCl.sub.3) δ 172.7, 172.6, 172.4, 157.0, 82.1, 81.8, 80.7, 53.6, 53.2, 41.9, 33.2, 33.0, 31.8, 28.5, 28.20, 28.16, 28.13, 22.5. MS (ESI) m/z: 488.4 [M++H].sup.+
[0145] Coupling of linkers to the PSMA binding motive and labelling with .sup.211At following the procedures previously described in Example I.
Example 3: Synthesis of PSMA-Targeting Radiopharmaceutical Reference Compounds
2,5-dioxopyrrolidin-1-yl (1r,4r)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylate
[0146] ##STR00034##
[0147] Boc-tranexamic acid (3.00 g, 11.66 mmol) was dissolved in dry THF (100 mL). Thereafter EDC-HCl (3.36 g, 17.48 mmol) and N-hydroxysuccinimide (2.00 g, 17.48 mmol) were added as solids. The mixture was stirred at room temperature and the progress followed by UPLC-MS. The reaction mixture was a cloudy suspension that gradually became a clear solution over 4 hours of stirring. After 72 hours the reaction mixture was diluted with DCM (75 mL), washed with water (3×30 mL), dried over Mg.sub.2SO.sub.4, filtered and solvents evaporated under reduced pressure to yield 2,5-dioxopyrrolidin-1-yl (1r,4r)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylate as a white powder (2.6 g, 7.25 mmol, 63%).
[0148] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.99 (t, J=6.5 Hz, 2H), 2.82 (s, 4H), 2.58 (tt, J=12.2, 3.6 Hz, 1H), 2.17 (dd, J=13.9, 3.6 Hz, 2H), 1.88 (dd, J=13.7, 3.5 Hz, 2H), 1.60 (dd, J=13.0, 3.4 Hz, 2H), 1.55 (d, J=2.8 Hz, 1H), 1.44 (s, 9H), 1.01 (qd, J=13.2, 3.6 Hz, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 170.9, 169.3, 156.2, 79.4, 46.5, 40.8, 37.6, 29.5, 28.5, 28.4, 25.7. MS (ESI) m/z: 255.3 [M+H-Boc].sup.+
General Procedure I: Fmoc Solution-Phase Synthesis of LuG-Linker Molecules
[0149] N-Fmoc-amino acid (Fmoc-AA) (1.0 eq) and DIPEA (2.5 eq) were dissolved in dry DMF (1-3 mL), HATU (1.5 eq) was added to the previous solution and stirred for 15 minutes. The corresponding amine (1.0 eq) was dissolved in dry DMF (1-3 mL) and added to the previous mixture for a total volume of 2-6 mL. The reaction mixture was stirred at room temperature, until completion (5 to 24 hours). Thereafter, the Fmoc-protected N-terminus was deprotected by adding piperidine (50% vol. relative to DMF) and stirred for an additional 2 hours. Then, the reaction mixture was poured over water (10 mL) and extracted with DCM (2×15 mL). The combined organic layers were washed with water (3×10 mL), dried over MgSO.sub.4 and volatiles removed in vacuo. The crude was purified by CombiFlash giving the desired free amine.
General Procedure II: Synthesis of Tranexamic Acid-Peptide Conjugates
[0150] Zwitter ionic alpha-amino acid (1.2 eq.) and Na.sub.2CO.sub.3 (2.0 eq.) were dissolved in 20 mL of a 1:2 mixture of H.sub.2O/1,4-dioxane. Afterwards, a solution of 2,5-dioxopyrrolidin-1-yl (1r,4r)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxylate (1.0 eq.) in 1,4-dioxane (5 mL) was added. The mixture was stirred for 4 hours, where completion was observed by UPLC-MS. Thereafter, the pH of the mixture was adjusted to 3 with 1M HCl.sub.(aq.) and the precipitated product was extracted with EtOAc (3×15 mL), dried over Mg.sub.2SO.sub.4, filtered and solvents evaporated in vacuo to yield the desired compound as a white powder.
General Procedure III: Synthesis of Boc-LuG-Linker Molecules
[0151] Carboxylic acid (1.0 eq) and DIPEA (2.5 eq) were dissolved in dry DMF (1-3 mL), HATU (1.5 eq) was added to the previous solution and stirred for 15 minutes. 12 (1.0 eq) was dissolved in dry DMF (1-3 mL) and added to the previous mixture for a total volume of 2-6 mL. The reaction mixture was stirred at room temperature for 5 to 24 hours depending on when completion was attained (followed by UPLC-MS). Once completed, the mixture was poured into 20 mL of water and extracted with DCM (3×15 mL). The organic fractions were washed once more with water (50 mL) to fully remove the DMF, dried over magnesium sulfate, filtered and solvents removed in vacuo. Crude was purified by CombiFlash (Heptane:EtOAc—100:0 to 0:100 with a gradual increase of 20% EtOAc every 6 minutes). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compounds.
General Procedure IV: Deprotection of Boc-LUG-Linker Molecules
[0152] tu-Boc-protected molecule was dissolved in a 1:1 mixture of TFA:DCM (5 mL). The solution was stirred at room temperature for 2 hours while being monitored by UPLC-MS. Once the deprotection was complete the mixture was evaporated under reduced pressure. Thereafter, the compounds were left under high vacuum for 72 hours to fully remove TFA traces.
General Procedures V and VI: Synthesis of DOTA-Linker-LUG Compounds
[0153] OBS: materials used to handle DOTA molecules are all free from metal, either glass or plastic, to avoid potential undesired chelation.
[0154] V—for Fmoc route: To an LuG-linker solution (1 eq.) in DCM, Et.sub.3N (6 eq.), DOTA-mono-NHS-tris(Su-ester) (1.2 eq.) were added and stirred overnight (˜16 hours). Afterwards the reaction mixture was evaporated under reduced pressure and the resulting crude re-dissolved in 1:1 mixture of TFA/DCM (3 mL) and mechanically shaken for 3 hours. Thereafter, volatiles were removed in vacuo and the oily crude purified by preparatory HPLC. Fractions containing desired product were collected and lyophilized to obtain the compounds as white solids.
[0155] VI—for Boc route: To a LuG-linker solution (1 eq.) in DMF, Et.sub.3N (6 eq.), DOTA-mono-NHS-ester (1.2 eq.) were added and stirred overnight (˜16 hours). Afterwards the reaction mixture was placed under a stream of air until dryness. Thereafter, the oily crude was purified by preparatory HPLC. Fractions containing desired product were collected and lyophilized to obtain the compounds as white solids.
di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0156] ##STR00035##
[0157] di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (500 mg, 1.02 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(naphthalen-2-yl)propanoic acid (Fmoc-3-(2-naphthyl)-L-alanine) (449 mg, 1.02 mmol) as the Fmoc-AA and reacting for 15 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH—100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (352 mg, 0.51 mmol, 50%).
[0158] .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.98 (s, 1H), 7.80-7.73 (m, 3H), 7.67 (s, 1H), 7.52 (t, J=5.9 Hz, 1H), 7.46-7.41 (m, 2H), 7.34 (dd, J=8.4, 1.7 Hz, 1H), 5.95 (s, 1H), 5.88 (d, J=8.1 Hz, 1H), 4.30-4.26 (m, 1H), 4.24-4.16 (m, 1H), 4.14-4.07 (m, 1H), 3.36-3.30 (m, 1H), 3.30-3.23 (m, 1H), 3.13 (dd, J=13.8, 7.8 Hz, 1H), 3.04-2.97 (m, 1H), 2.35-2.25 (m, 2H), 2.08-2.01 (m, 2H), 1.87-1.77 (m, 2H), 1.69-1.61 (m, 1H), 1.57-1.51 (m, 1H), 1.41 (s, 9H), 1.40 (d, J=1.6 Hz, 18H), 1.37-1.31 (m, 1H), 0.92-0.75 (m, 2H). .sup.13C NMR (151 MHz, CDCl.sub.3) δ 173.2, 172.9, 172.6, 157.8, 133.5, 133.3, 132.6, 128.6, 128.4, 127.7, 127.7, 127.3, 126.3, 125.9, 82.1, 81.5, 80.7, 53.3, 53.1, 39.4, 38.9, 31.8, 31.7, 31.6, 28.5, 28.3, 28.1, 28.0, 22.1. MS (ESI) m/z: 685.5 [M+H].sup.+ (10% MeOH in DCM, with tailing)=0.34
di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0159] ##STR00036##
[0160] di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (250 mg, 0.51 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-iodophenyl)propanoic acid (Fmoc-2-iodo-L-phenylalanine) (263 mg, 0.51 mmol) as the Fmoc-AA and reacting for 5 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH—100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a yellowish oil (80 mg, 0.10 mmol, 21%).
[0161] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.83 (d, J=7.9 Hz, 1H), 7.32-7.15 (m, 2H), 6.92 (td, J=7.4, 2.1 Hz, 1H), 5.46-5.34 (m, 2H), 4.31 (m, 3H), 3.67 (m, 1H), 3.49-3.36 (m, 1H), 3.25 (m, 2H), 2.86 (dd, J=14.0, 7.1 Hz, 1H), 2.40-2.20 (m, 2H), 2.11-2.00 (m, 1H), 1.85-1.70 (m, 3H), 1.74-1.57 (m, 4H), 1.56-1.30 (m, 31H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 174.1, 172.58, 172.56, 172.4, 157.2, 141.2, 139.9, 130.83, 130.79, 128.68, 128.63, 128.60, 101.4, 82.0, 81.7, 80.6, 55.9, 53.5, 53.1, 45.6, 38.7, 32.4, 31.8, 29.1, 28.6, 28.2, 22.4. MS (ESI) m/z: 761.3 [M+H].sup.+ R.sub.f (10% MeOH in DCM, with tailing)=0.39
di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0162] ##STR00037##
[0163] di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan yl)carbamoyl)-L-glutamate (180 mg, 0.26 mmol) following general procedure I employing (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)cyclohexane-1-carboxylic acid (Fmoc-tranexamic acid) (100 mg, 0.26 mmol) as the Fmoc-AA and reacting for 15 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH—100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a yellowish oil (85 mg, 0.10 mmol, 40%).
[0164] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.45 (s, 1H), 7.83 (d, J=8.3 Hz, 1H), 7.67-7.61 (m, 2H), 7.54 (d, J=8.1 Hz, 1H), 7.42 (t, J=7.5 Hz, 1H), 7.34 (t, J=7.4 Hz, 1H), 7.18-7.07 (m, 2H), 7.01 (d, 1H), 6.17 (d, J=8.7 Hz, 1H), 5.21-5.16 (m, 1H), 4.68-4.57 (m, 1H), 4.32-4.22 (m, 1H), 3.55-3.41 (m, 2H), 3.20 (dd, J=13.8, 4.1 Hz, 1H), 3.13-2.98 (m, 2H), 2.49-2.30 (m, 3H), 2.24-2.10 (m, 1H), 1.97-1.79 (m, 2H), 1.79-1.68 (m, 2H), 1.57 (s, 8H), 1.46-1.35 (m, 27H), 1.20-1.06 (m, 3H), 0.99-0.60 (m, 4H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 177.26, 174.82, 174.20, 172.52, 157.56, 135.06, 133.35, 132.20, 128.05, 127.85, 127.30, 125.91, 125.36, 82.46, 80.93, 80.39, 54.60, 53.26, 52.28, 50.34, 48.12, 44.63, 40.42, 39.67, 39.45, 32.77, 31.65, 29.98, 29.08, 28.90, 28.21, 28.16, 28.07, 26.90, 25.68, 24.71, 23.42. MS (ESI) m/z: 825.7 [M+H].sup.+ (10% MeOH in DCM, with tailing)=0.28
di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0165] ##STR00038##
[0166] di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-((S)-2-amino-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (75 mg, 0.10 mmol) following general procedure I employing (1r,4r)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)cyclohexane carboxylic acid (Fmoc-tranexamic acid) (38 mg, 0.10 mmol) as the Fmoc-AA and reacting for 7 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH—100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (70 mg, 0.08 mmol, 76%). R.sub.f (10% MeOH in DCM, with tailing)=0.25
[0167] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.74 (dd, J=7.8, 2.7 Hz, 1H), 7.30 (d, J=7.2 Hz, 1H), 7.19 (t, J=7.6 Hz, 1H), 6.87 (td, J=7.6, 1.7 Hz, 1H), 4.87 (q, J=7.8 Hz, 1H), 4.46-4.31 (m, 1H), 4.24-4.13 (m, 1H), 3.44-3.26 (m, 1H), 3.24-3.10 (m, 2H), 3.05 (q, J=7.3 Hz, 3H), 3.00-2.89 (m, 1H), 2.88-2.74 (m, 2H), 2.43-2.24 (m, 2H), 2.21-2.02 (m, 2H), 1.97-1.81 (m, 3H), 1.81-1.60 (m, 5H), 1.60-1.48 (m, 2H), 1.48-1.39 (m, 27H), 1.28-1.10 (m, 1H), 1.07-0.91 (m, 2H). MS (ESI) m/z: 900.2 [M+H].sup.+ (10% MeOH in DCM, with tailing)=0.25
di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4-iodophenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0168] ##STR00039##
[0169] di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4-iodophenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-((S)-2-amino-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (40 mg, 0.58 mmol) following general procedure/employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-iodophenyl)propanoic acid (Fmoc-4-iodo-L-phenylalanine) (30 mg, 0.58 mmol) as the Fmoc-AA and reacting for 17 hours. The crude was purified by CombiFlash, impurities were washed off the column with 100% EtOAc and desired compound was eluted (DCM:MeOH—100:0 to 90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (20 mg, 0.02 mmol, 37%).
[0170] .sup.1H NMR (600 MHz, CDCl.sub.3) δ 7.79-7.68 (m, 4H), 7.65 (d, J=6.9 Hz, 1H), 7.43 (dd, J=6.3, 3.4 Hz, 3H), 7.37 (d, J=7.8 Hz, 2H), 7.34 (s, 1H), 6.68 (d, J=8.0, 4.3 Hz, 1H), 6.01 (s, 1H), 4.81 (s, 1H), 4.40-4.29 (m, 1H), 4.27-4.18 (m, 1H), 3.89-3.66 (m, 1H), 3.35 (dd, J=13.7, 6.0 Hz, 2H), 3.27 (q, J=7.3, 5.5 Hz, 1H), 3.23-3.14 (m, 1H), 3.05-2.95 (m, 1H), 2.90 (dd, J=13.9, 5.6 Hz, 1H), 2.70-2.50 (m, 1H), 2.37-2.29 (m, 2H), 2.12-2.01 (m, 1H), 1.90-1.76 (m, 2H), 1.62 (dt, J=33.8, 9.4 Hz, 3H), 1.46-1.34 (m, 27H), 1.31-1.18 (m, 4H). MS (ESI) m/z: 958.4 [M+H].sup.+ R.sub.f (10% MeOH in DCM, with tailing)=0.27
di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4-iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0171] ##STR00040##
[0172] di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4-iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared from di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (100 mg, 0.12 mmol) following general procedure I employing (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-iodophenyl)propanoic acid (Fmoc-4-iodo-L-alanine) (62 mg, 0.12 mmol) as the Fmoc-AA and reacting for 16 hours. Compound was purified by CombiFlash (Heptane:EtOAc:DCM:MeOH—100:0:0:0 to 0:100:0:0 and then 0:0:100:0 to 0:0:90:10). Fractions containing desired product were combined and volatiles removed under reduced pressure to obtain the compound as a white semisolid (75 mg, 0.07 mmol, 56%).
[0173] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.72-7.53 (m, 5H), 7.44 (t, 1H), 7.39 (t, 1H), 7.21 (t, J=6.1 Hz, 1H), 7.13 (d, J=8.3 Hz, 1H), 6.95 (dd, J=11.7, 8.1 Hz, 2H), 6.12 (d, J=8.5 Hz, 1H), 5.18 (s, 1H), 4.64 (s, 1H), 4.30 (dd, J=9.7, 5.4 Hz, 1H), 3.62-3.38 (m, 4H), 3.22 (dd, J=13.7, 4.3 Hz, 1H), 3.15 (dd, J=13.8, 4.2 Hz, 3H), 3.10-2.99 (m, 4H), 2.98-2.88 (m, 1H), 2.64 (dd, J=13.8, 8.9 Hz, 2H), 2.50-2.32 (m, 4H), 2.27-2.12 (m, 1H), 1.91 (tdd, J=14.8, 8.1, 3.5 Hz, 1H), 1.69 (d, J=40.8 Hz, 2H), 1.59 (s, 8H), 1.46-1.37 (m, 27H), 1.30-1.17 (m, 2H), 1.17-0.95 (m, 1H), 0.93-0.63 (m, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 177.22, 174.96, 173.77, 172.54, 157.58, 137.81, 137.66, 135.08, 133.42, 132.27, 131.50, 131.44, 128.16, 127.96, 127.88, 127.38, 127.07, 126.03, 125.47, 92.19, 82.60, 81.02, 80.48, 56.38, 54.67, 53.34, 52.33, 45.16, 44.52, 40.51, 37.51, 37.07, 31.97, 30.19, 29.65, 29.11, 28.90, 28.29, 28.20, 28.14, 27.96, 26.98, 24.78, 22.78. MS (ESI) m/z: 1098.3 [M+H].sup.+ R.sub.f (10% MeOH in DCM, with tailing)=0.21
(S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4-iodophenyl)propanoic acid
[0174] ##STR00041##
[0175] (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4-iodophenyl)propanoic acid was prepared following general procedure II, employing (S)-2-amino-3-(4-iodophenyl)propanoic acid (4-iodo-L-phenylalanine) as the alpha-amino acid (390 mg, 0.74 mmol, 86%)
[0176] .sup.1H NMR (600 MHz, CD.sub.3OD) δ 7.61 (dt, J=8.3, 1.9 Hz, 2H), 7.01 (dt, J=8.3, 1.8 Hz, 2H), 4.64 (q, J=9.2 Hz, 1H), 3.17 (dd, J=14.0, 5.0 Hz, 1H), 2.93-2.85 (m, 4H), 2.12 (tt, J=12.2, 3.5 Hz, 1H), 1.84-1.74 (m, 5H), 1.67-1.60 (m, 1H), 1.43 (s, 9H), 0.99-0.88 (m, 2H). MS (ESI) m/z: 529.2 [M−H].sup.−
(S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanoic acid
[0177] ##STR00042##
[0178] (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanoic acid was prepared following general procedure II, employing (S)-2-amino-3-(3-iodophenyl)propanoic acid (3-iodo-L-phenylalanine) as the alpha-amino acid (390 mg, 0.74 mmol, 86%)
[0179] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.61 (dt, J=8.3, 1.9 Hz, 2H), 7.01 (dt, J=8.3, 1.8 Hz, 2H), 4.64 (q, J=9.2 Hz, 1H), 3.17 (dd, J=14.0, 5.0 Hz, 1H), 2.93-2.85 (m, 4H), 2.12 (tt, J=12.2, 3.5 Hz, 1H), 1.84-1.74 (m, 5H), 1.67-1.60 (m, 1H), 1.43 (s, 9H), 0.99-0.88 (m, 2H). .sup.13C NMR (101 MHz, MeOD) δ 178.7, 174.4, 158.6, 141.2, 139.5, 136.9, 131.2, 129.7, 94.8, 79.8, 54.3, 46.0, 39.0, 37.8, 30.8, 29.8, 28.8, 26.3. MS (ESI) m/z: 529.3 [M−H].sup.−
(R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanoic acid
[0180] ##STR00043##
[0181] (R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanoic acid was prepared following general procedure II, employing 2-amino-3-(5-iodo-1H-indol-3-yl)propanoic acid as the alpha-amino acid, the compound was purified by CombiFlash (Heptane:EtOAc:AcOH—100:0:0.5 to 15:85:0.5) (100 mg, 0.17 mmol, 46%) R.sub.f (40 EtOAc in heptane, with tailing)=0.21
[0182] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 10.53 (s, 1H), 7.90 (d, J=1.6 Hz, 1H), 7.34 (dd, J=8.5, 1.6 Hz, 1H), 7.16 (d, J=8.5 Hz, 1H), 7.08 (d, J=2.0 Hz, 1H), 4.70 (dd, J=7.8, 4.8 Hz, 1H), 4.11 (q, J=7.1 Hz, 1H), 3.35-3.26 (m, 2H), 3.14 (dd, J=14.7, 7.9 Hz, 1H), 2.87 (d, J=6.7 Hz, 2H), 2.13 (tt, J=12.2, 3.5 Hz, 1H), 1.86-1.73 (m, 3H), 1.74-1.65 (m, 1H), 1.44 (s, 9H), 1.02-0.86 (m, 2H). .sup.13C NMR (101 MHz, CD.sub.3OD) δ 178.7, 175.0, 158.7, 137.1, 131.8, 130.7, 128.4, 125.7, 114.5, 110.9, 82.8, 79.8, 54.6, 46.0, 39.1, 30.9, 30.0, 28.8, 28.2. MS (ESI) m/z: 568.2 [M−H].sup.−
di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4-iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0183] ##STR00044##
[0184] di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4-iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido) (4-iodophenyl)propanoic acid as the carboxylic acid and reacting for 5 hours. Compound was obtained as a white/yellow solid (296 mg, 0.29 mmol, 63%). .sup.1H NMR (400 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 7.56 (d, J=8.0 Hz, 2H), 7.48 (d, J=7.9 Hz, 2H), 6.96-6.86 (m, 2H), 6.67-6.60 (m, 1H), 6.01-5.97 (m, 1H), 5.63-5.57 (m, 1H), 5.48-5.40 (m, 1H), 4.97 (s, 1H), 4.70-4.50 (m, 1H), 4.39-4.17 (m, 2H), 3.28-3.14 (m, 1H), 3.09-2.95 (m, 1H), 2.98-2.86 (m, 4H), 2.89-2.77 (m, 1H), 2.43-2.21 (m, 2H), 2.16-1.98 (m, 1H), 2.05-2.01 (m, 1H), 1.98-1.94 (m, 2H), 1.92-1.68 (m, 1H), 1.81-1.77 (m, 7H), 1.47-1.40 (m, 36H), 1.38-1.29 (m, 1H), 1.28-1.20 (m, 1H), 0.98-0.75 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 176.23, 172.92, 172.70, 172.66, 172.56, 172.50, 171.54, 162.70, 157.66, 157.54, 157.32, 157.28, 156.23, 137.61, 137.44, 136.69, 131.55, 131.44, 92.27, 92.04, 82.57, 82.29, 82.21, 81.82, 81.69, 81.61, 81.09, 80.75, 80.55, 79.18, 77.36, 60.51, 54.35, 54.29, 53.48, 53.26, 53.17, 53.04, 52.43, 46.71, 45.48, 45.14, 44.65, 38.63, 38.18, 36.61, 32.58, 32.00, 31.78, 31.75, 29.98, 29.85, 29.77, 29.44, 29.20, 29.12, 28.86, 28.74, 28.56, 28.45, 28.24, 28.21, 28.15, 22.55, 22.14 MS (ESI) m/z: 1001.5 [M+H].sup.+ R.sub.f (60% EtOAc in heptane)=0.32
di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0185] ##STR00045##
[0186] di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanoic acid as the carboxylic acid and reacting for 24 hours. Compound was obtained as an off-white solid (210 mg, 0.21 mmol, 56%). .sup.1H NMR (400 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 7.59-7.47 (m, 2H), 7.05-6.94 (m, 1H), 6.70-6.62 (m, 1H), 6.03-5.97 (m, 1H), 4.94-4.90 (m, 1H), 4.67-4.55 (m, 2H), 4.35-4.23 (m, 1H), 3.51-3.43 (m, 1H), 3.03-2.85 (m, 4H), 2.81-2.77 (m, 7H), 2.45-2.26 (m, 2H), 2.22-1.98 (m, 1H), 1.92-1.70 (m, 2H), 1.69-1.61 (m, 1H), 1.58-1.54 (m, 6H), 1.50-1.39 (m, 38H), 1.36-1.20 (m, 1H), 0.98-0.84 (m, 1H), 0.84-0.77 (m, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 177.36, 176.09, 175.08, 172.63, 172.58, 172.54, 172.49, 157.56, 157.26, 157.24, 156.15, 140.08, 139.54, 138.47, 138.28, 135.86, 130.32, 130.18, 128.73, 128.41, 94.17, 82.80, 82.22, 82.12, 81.78, 81.63, 81.04, 80.67, 80.48, 79.16, 77.36, 54.73, 53.42, 53.26, 53.16, 52.35, 46.74, 45.51, 44.66, 38.88, 38.73, 37.57, 31.75, 31.73, 30.07, 29.99, 29.88, 29.69, 29.59, 29.21, 29.07, 28.86, 28.55, 28.47, 28.41, 28.24, 28.21, 28.17, 28.15. MS (ESI) m/z: 1001.5 [M+H].sup.+ R.sub.f (60% EtOAc in heptane)=0.38
di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
[0187] ##STR00046##
[0188] di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate was prepared following general procedure III, employing (R)-2-((1r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanoic acid as the carboxylic acid and reacting for 10 hours. Compound was obtained as a white semisolid (80 mg, 0.08 mmol, 45%). .sup.1H NMR (400 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 9.70 (s, 1H), 8.99 (s, 1H), 8.01 (d, J=3.7 Hz, 1H), 7.91 (d, J=1.5 Hz, 1H), 7.44-7.34 (m, 1H), 7.22 (d, J=8.5 Hz, 1H), 7.13 (d, J=8.5 Hz, 1H), 7.02-6.95 (m, 1H), 6.60 (d, J=7.6 Hz, 1H), 6.20 (s, 1H), 5.80 (s, 1H), 5.70 (d, J=8.3 Hz, 1H), 5.42-5.31 (m, 1H), 4.34-4.29 (m, 1H), 4.20-4.12 (m, 1H), 3.16-3.08 (m, 1H), 3.03-2.91 (m, 3H), 2.40-2.27 (m, 2H), 2.10-2.01 (m, 1H), 1.92-1.83 (m, 1H), 1.83-1.80 (m, 2H), 1.79-1.71 (m, 2H), 1.49-1.40 (m, 45H), 1.30-1.21 (m, 3H), 1.21-1.11 (m, 1H), 0.97-0.83 (m, 4H). .sup.13C NMR (101 MHz, CDCl.sub.3, mixture of un-assigned rotamers) δ 176.58, 175.89, 173.80, 172.68, 172.65, 172.59, 171.62, 157.76, 157.63, 156.26, 135.60, 135.41, 130.34, 130.16, 130.06, 127.67, 127.56, 124.80, 124.36, 113.82, 113.56, 110.12, 109.74, 82.97, 82.85, 82.36, 82.33, 81.95, 81.44, 80.90, 80.71, 79.23, 77.36, 60.52, 54.12, 53.90, 53.58, 53.45, 53.31, 52.86, 46.74, 45.22, 44.75, 39.05, 38.76, 37.79, 32.32, 31.87, 31.84, 29.93, 29.75, 29.21, 28.69, 28.57, 28.45, 28.22, 28.16, 28.14, 22.55, 21.16. MS (ESI) m/z: 1040.6 [M+H].sup.+ R.sub.f (60% EtOAc in heptane)=0.33
((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(4-iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate
[0189] ##STR00047##
[0190] ((1S,4r))-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(4-iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(4-iodophenyl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV, as a viscous oil (TFA salt—248 mg, 0.29 mmol, 99%)
[0191] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO) δ 7.96-7.85 (m, 2H), 7.74-7.65 (m, 4H), 7.60 (d, J=7.9 Hz, 2H), 7.02 (d, J=7.9 Hz, 2H), 6.31 (t, J=9.6 Hz, 2H), 4.47-4.36 (m, 1H), 4.14-3.98 (m, 3H), 3.07-2.93 (m, 3H), 2.92-2.84 (m, 1H), 2.76-2.59 (m, 3H), 2.34-2.16 (m, 2H), 2.12-2.00 (m, 1H), 1.98-1.85 (m, 1H), 1.81-1.61 (m, 5H), 1.57-1.42 (m, 1H), 1.41-1.20 (m, 6H), 1.17-1.00 (m, 1H), 0.98-0.81 (m, 2H). .sup.13C NMR (101 MHz, (CD.sub.3).sub.2SO) δ 174.6, 174.5, 174.1, 173.7, 170.8, 157.3, 137.9, 136.6, 131.7, 91.9, 53.3, 52.3, 51.7, 44.3, 43.2, 35.0, 31.7, 29.9, 28.9, 28.8, 28.7, 28.3, 28.0, 27.5, 22.6. MS (ESI) m/z: 732.4 [M−H].sup.+
((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(3-iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate
[0192] ##STR00048##
[0193] ((1S,4r))-4-MS)-1-MS)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino) iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((S)-2-((1r,4S)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(3-iodophenyl)propanamido) oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV, as a transparent oil (TFA salt—116 mg, 0.14 mmol, 94%).sup.1H NMR (400 MHz, D.sub.2O) δ 7.63-7.53 (m, 2H), 7.22 (d, J=7.7 Hz, 1H), 7.08 (t, J=7.8 Hz, 1H), 4.46 (q, J=7.8 Hz, 1H), 4.26 (dd, J=8.9, 5.2 Hz, 1H), 4.22-4.08 (m, 1H), 3.17 (q, J=6.3 Hz, 2H), 3.05-2.89 (m, 3H), 2.89-2.78 (m, 3H), 2.49 (q, J=7.5 Hz, 3H), 2.27-2.08 (m, 2H), 2.02-1.90 (m, 1H), 1.91-1.77 (m, 5H), 1.77-1.70 (m, 2H), 1.66-1.58 (m, 2H), 1.45-1.27 (m, 5H), 1.27-1.13 (m, 2H), 1.12-0.94 (m, 3H). MS (ESI) m/z: 732.4 [M−H].sup.+
((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(5-iodo-1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate
[0194] ##STR00049##
[0195] ((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(5-iodo-1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate was obtained from di-tert-butyl (((S)-1-(tert-butoxy)-6-((R)-2-((1 r,4R)-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-carboxamido)-3-(5-iodo-1H-indol-3-yl)propanamido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate, following general procedure IV. In this case the deprotection cocktail was 1/1 TFA:TIPS:phenol (95:5:5)/DCM. The deprotected compound was purified by preparatory HPLC to obtain the title compound as a transparent oil (TFA salt—23 mg, 0.03 mmol, 34%)
[0196] .sup.1H NMR (600 MHz, CD.sub.3OD-d.sub.4) δ 7.93 (s, 1H), 7.33 (d, J=8.2 Hz, 1H), 7.16 (d, J=8.3 Hz, 1H), 7.09 (d, J=5.8 Hz, 1H), 4.58-4.52 (m, 1H), 4.37-4.28 (m, 1H), 4.23-4.15 (m, 1H), 3.21-3.13 (m, 2H), 3.12-2.99 (m, 2H), 2.83-2.73 (m, 2H), 2.48-2.34 (m, 2H), 2.28-2.18 (m, 1H), 2.16-2.07 (m, 1H), 1.87 (q, J=11.6, 9.3 Hz, 5H), 1.78-1.66 (m, 2H), 1.65-1.51 (m, 2H), 1.48-1.30 (m, 3H), 1.30-1.15 (m, 2H), 1.13-0.99 (m, 2H). .sup.13C NMR (151 MHz, CD.sub.3OD) δ 178.3, 175.1, 174.8, 174.6, 174.6, 160.0, 137.0, 131.6, 130.7, 128.5, 125.9, 114.5, 110.5, 82.8, 55.8, 54.2, 53.5, 46.3, 45.4, 40.0, 36.7, 32.7, 30.9, 30.3, 29.6, 29.4, 29.1, 28.5, 23.9.
(((S)-1-carboxy-5-((S)-2-((1S,4S)-4-(((S)-3-(4-iodophenyl)-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)propanamido)methyl)cyclohexane carboxamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Im)
[0197] ##STR00050##
[0198] The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((1S,4S)-4-(((S)-2-amino-3-(4-iodophenyl)propanamido)methyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate following general procedure V-A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain Im as white powder. (11 mg, 0.01 mmol, 13%).
[0199] .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.88-7.76 (m, 3H), 7.72-7.55 (m, 3H), 7.52-7.41 (m, 1H), 7.41-7.34 (m, 1H), 7.07-6.96 (m, 3H), 4.60-4.52 (m, 1H), 4.52-4.42 (m, 1H), 4.25-4.16 (m, 1H), 4.12-4.01 (m, 1H), 3.57 (s, 8H), 3.29-2.70 (m, 18H), 2.42-2.31 (m, 2H), 2.11-1.99 (m, 1H), 1.87-1.73 (m, 1H), 1.72-1.57 (m, 2H), 1.56-1.41 (m, 2H), 1.40-1.27 (m, 2H), 1.27-1.15 (m, 3H), 1.15-1.03 (m, 1H), 0.78-0.61 (m, 4H).
[0200] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 178.6, 178.51, 176.52, 176.4, 176.2, 175.9, 173.10, 173.07, 172.2, 161.6 (q, J=34.6 Hz), 159.4, 138.5, 138.5, 137.9, 135.9, 134.4, 133.3, 132.80, 132.76, 128.9, 128.8, 128.57, 128.53, 127.2, 126.7, 117.7 (overlapped with CD.sub.3CN signal, q, J=292.9 Hz) 92.6, 55.8, 55.5, 53.8, 53.3, 46.3, 46.1, 45.4, 43.7, 40.8, 39.5, 39.4, 38.6, 38.3, 37.7, 37.5, 31.9, 30.9, 30.3, 30.2, 30.1, 29.71, 29.66, 29.60, 29.5, 29.1, 27.9, 27.6, 25.2, 23.3, 23.1.
(((S)-1-carboxy-5-((S)-2-((S)-3-(4-iodophenyl)-2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)propanamido)-3-(naphthalen-2-yl)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (In)
[0201] ##STR00051##
[0202] The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((S)-2-amino-3-(4-iodophenyl)propanamido)-3-(naphthalen-2-yl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate following general procedure V-A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain In as white powder. (10 mg, 0.01 mmol, 10%).
[0203] .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.83 (q, J=8.3 Hz, 3H), 7.68 (s, 1H), 7.52 (d, J=7.8 Hz, 2H), 7.46 (q, J=7.5 Hz, 2H), 7.36 (dd, J=8.6, 1.8 Hz, 1H), 6.85 (d, J=7.9 Hz, 2H), 4.61 (t, J=7.5 Hz, 1H), 4.58-4.49 (m, 1H), 4.20 (dd, 1H), 4.06 (dd, J=8.7, 4.9 Hz, 1H), 3.85-3.42 (m, 6H), 3.31 (s, 23H), 3.24-3.08 (m, 1H), 3.08-2.83 (m, 2H), 2.67 (dd, J=13.8, 9.4 Hz, 1H), 2.36 (td, J=7.5, 2.3 Hz, 2H), 2.09-2.01 (m, 1H), 1.88-1.77 (m, 1H), 1.64-1.53 (m, 1H), 1.53-1.42 (m, 1H), 1.33-1.18 (m, 2H), 1.18-1.04 (m, 2H).
[0204] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 176.34, 176.18, 175.85, 172.69, 172.06, 161.74, 161.51, 161.28, 161.05, 159.37, 138.51, 138.33, 138.17, 135.55, 134.41, 133.39, 132.76, 129.03, 128.97, 128.79, 128.64, 128.60, 127.27, 126.83, 92.43, 55.64, 55.36, 53.74, 53.33, 39.70, 38.86, 37.62, 32.02, 30.92, 28.94, 27.98, 26.69, 26.16, 24.81, 23.20.
(((S)-1-carboxy-5-((S)-3-(2-iodophenyl)-2-((1r,4S)-4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ii)
[0205] ##STR00052##
[0206] The title compound was obtained from di-tert-butyl (((S)-6-((S)-2-((1r,4S)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(2-iodophenyl)propanamido)-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate following general procedure V-A. Preparatory HPLC purification was carried out using a gradient of 0% to 100% of B over 15 minutes. Fractions containing the desired compound were lyophilised to obtain Ii as white powder. (22 mg, 0.02 mmol, 27%)
[0207] .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.83 (dt, J=7.7, 1.5 Hz, 1H), 7.32-7.26 (m, 1H), 7.26-7.20 (m, 1H), 6.95 (tt, J=7.4, 1.9 Hz, 1H), 4.55-4.49 (m, 1H), 4.23-4.17 (m, 1H), 4.11-4.06 (m, 1H), 3.72 (d, J=90.6 Hz, 5H), 3.29-2.79 (m, 13H), 2.39-2.29 (m, 3H), 2.13-2.01 (m, 1H), 1.88-1.76 (m, 1H), 1.76-1.66 (m, 3H), 1.66-1.59 (m, 1H), 1.59-1.47 (m, 1H), 1.43-1.29 (m, 2H), 1.29-1.12 (m, 4H), 0.93-0.78 (m, 3H).
[0208] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 178.5, 178.4, 176.46, 176.43, 176.3, 175.82, 175.79, 172.54, 172.51, 161.46 (q, J=34.7 Hz), 159.4, 140.8, 140.6, 131.9, 129.8, 129.5, 117.7 (overlapped with CD.sub.3CN signal, q, J=292.9 Hz), 101.4, 62.8, 55.9, 54.2, 54.1, 53.96, 53.93, 53.32, 53.28, 46.4, 45.4, 42.9, 39.6, 39.4, 37.7, 32.1, 32.0, 30.9, 30.34, 30.29, 29.61, 29.58, 29.5, 29.2, 29.1, 28.1, 27.9, 23.4, 23.2.
(((S)-1-carboxy-5-((S)-3-(3-iodophenyl)-2-((1r,4S)-4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ij)
[0209] ##STR00053##
[0210] The title compound was obtained from ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(3-iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V-B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Ij as white powder. (24 mg, 0.02 mmol, 30%)
[0211] .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.59-7.54 (m, 2H), 7.22 (ddt, J=7.8, 2.9, 1.3 Hz, 1H), 7.08-7.02 (m, 1H), 4.45-4.38 (m, 1H), 4.22-4.17 (m, 1H), 4.14-4.07 (m, 1H), 3.89-3.54 (m, 5H), 3.37-2.89 (m, 20H), 2.79 (dd, J=13.8, 9.0 Hz, 1H), 2.37 (ddt, J=8.2, 6.5, 1.6 Hz, 2H), 2.12-2.01 (m, 2H), 1.89-1.77 (m, 1H), 1.76-1.66 (m, 4H), 1.64-1.53 (m, 2H), 1.44-1.32 (m, 3H), 1.32-1.17 (m, 4H), 0.96-0.84 (m, 2H).
[0212] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 178.6, 178.5, 176.50, 176.48, 176.3, 175.88, 175.86, 172.73, 172.70, 161.6 (q, J=34.5 Hz), 159.4, 141.0, 139.2, 136.7, 131.4, 129.8, 117.7 (overlapped with CD.sub.3CN signal, q, J=293.2 Hz), 94.7, 55.9, 55.3, 55.2, 53.93, 53.88, 53.31, 53.28, 46.4, 45.39, 45.37, 39.6, 39.4, 37.9, 37.7, 32.1, 32.0, 30.4, 30.3, 29.79, 29.77, 29.4, 29.2, 29.1, 27.98, 27.94, 23.4, 23.2.
(((S)-1-carboxy-5-((S)-3-(4-iodophenyl)-2-((1r,4S)-4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Ik)
[0213] ##STR00054##
[0214] The title compound was obtained from ((1S,4r)-4-(((S)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(4-iodophenyl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V-B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Ik as white powder. (42 mg, 0.04 mmol, 53%) .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.60 (d, 2H), 6.98 (d, 2H), 4.43-4.34 (m, 1H), 4.20-4.11 (m, 1H), 4.11-4.00 (m, 1H), 3.71-3.39 (m, 8H), 3.31-2.98 (m, 16H), 2.98-2.85 (m, 2H), 2.78 (dd, J=13.5, 8.6 Hz, 1H), 2.39-2.28 (m, 2H), 2.10-1.97 (m, 2H), 1.87-1.74 (m, 1H), 1.73-1.61 (m, 4H), 1.60-1.44 (m, 3H), 1.41-1.27 (m, 1H), 1.27-1.09 (m, 4H), 0.92-0.74 (m, 3H).
[0215] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 178.8, 176.8, 176.7, 176.1, 172.9, 162.1 (q, J=34.2), 159.5, 138.3, 138.1, 132.6, 118.8 (overlapped with CD.sub.3CN signal, q, J=293 Hz), 92.5, 55.8, 55.3, 53.9, 53.3, 46.2, 45.3, 39.6, 37.8, 37.6, 32.0, 30.9, 30.3, 30.2, 29.6, 29.3, 29.1, 27.8, 23.3.
(((S)-1-carboxy-5-((R)-3-(5-iodo-1H-indol-3-yl)-2-((1r,4R)-4-((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)pentyl)carbamoyl)-L-glutamic acid trifluoroacetic acid salt (Il)
[0216] ##STR00055##
[0217] The title compound was obtained from ((1R,4r)-4-(((R)-1-(((S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentyl)amino)-3-(5-iodo-1H-indol-3-yl)-1-oxopropan-2-yl)carbamoyl)cyclohexyl)methanaminium trifluoroacetate following general procedure V-B. Preparatory HPLC purification was carried out using a method consisting of 8 min of 100% A after injection followed by a gradient from 0 to 100% B over 20 min. Fractions containing the desired compound were lyophilised to obtain Il as white powder. (14 mg, 0.01 mmol, 46%) .sup.1H NMR (600 MHz, CD.sub.3CN+10% D.sub.2O) δ 7.91 (s, 1H), 7.36 (dd, J=8.5, 1.7 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 7.08 (d, J=8.3 Hz, 1H), 4.44 (t, J=7.1 Hz, 1H), 4.21 (dt, J=8.9, 4.4 Hz, 1H), 4.07 (dt, J=8.9, 4.9 Hz, 1H), 3.90-3.50 (m, 8H), 3.30-2.96 (m, 18H), 2.37 (td, J=7.0, 6.1, 2.9 Hz, 2H), 2.13-2.02 (m, 3H), 1.89-1.79 (m, 1H), 1.77-1.61 (m, 5H), 1.52 (dtd, J=14.0, 9.2, 5.3 Hz, 1H), 1.46-1.36 (m, 1H), 1.35-1.21 (m, 5H), 1.20-1.11 (m, 2H), 0.96-0.82 (m, 3H).
[0218] .sup.13C NMR (151 MHz, CD.sub.3CN+10% D.sub.2O) δ 178.3, 176.4, 176.2, 175.8, 173.2, 161.4 (q, J=34.7 Hz), 159.5, 136.3, 131.2, 130.5, 128.4, 126.0, 116.7 (overlapped with CD.sub.3CN signal, q, J=292 Hz), 114.8, 110.3, 82.8, 56.0, 55.1, 53.9, 53.3, 46.4, 45.4, 39.6, 39.4, 37.8, 32.2, 32.0, 30.9, 30.4, 30.3, 29.5, 29.1, 29.0, 28.4, 28.0, 27.9, 23.4.
Example 4: Synthesis of PSMA-Targeting Radiopharmaceutical Precursor
di-tert-butyl (((S)-1-(tert-butoxy)-1-oxo-6-((S)-3-(4-(trimethylstannyl)phenyl)-2-((1r,4S)-4-((2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)propanamido)hexan-2-yl)carbamoyl)-L-glutamate
[0219] ##STR00056##
[0220] To an LuG-linker solution (1 eq.) in DCM, Et.sub.3N (6 eq.), DOTA-mono-NHS-tris(tBu-ester) (1.2 eq.) were added and stirred overnight (˜16 hours). Afterwards the reaction mixture was evaporated under reduced pressure.
[0221] One microwave vial was charged with Pd(OAc).sub.2 (1.5 mg, 0.007 mmol) and the crude LuG-linker DOTA-functionalized mixture (7.7 mg, 0.26 mmol). The vial was sealed and purged. Then dry/degassed THF (400 uL) was added to the vial. In another MW vial, hexamethylditin (20.5 uL, 0.099 mmol) was added and the vial purged, thereafter dry/degassed THF was added (300 uL). The hexamehtylditin solution was then added to the Pd(OAc).sub.2 and meCgPPH mixture and the solution stirred for 5 minutes at room temperature.
[0222] Thereafter, fully tBu-protected 4 (48.0 mg, 0.033 mmol) which was previously weighed in a MW vial and dissolved in dry/degassed THF (300 uL) was added to the Pd/meCgPPH/Sn mixture. The vial was then placed on a MW system and heated to 70° C. for 30 min.
[0223] The mixture was dried under a stream of air and re-dissolved in 4 mL 60:40:0.1 (MeCN:H.sub.2O:TFA). The mixture was filtered over a prep HPLC filter and injected into the prepHPLC system in a gradient of 60 to 100% B. which yielded the desired product as a white solid. (12.5 mg, 0.0084 mmol, 25%). MS (ESI) m/z: 747.47 [M+2H].sup.2+
Example 5: Radioastatination and Radiochemical Conversion (RCC)
[0224] Compound Ib was provided in a two-step labelling procedure:
##STR00057##
[0225] The stannane precursor was added to a solution of chloramine-T, methanol, .sup.211At and acetic acid. The mixture was stirred for 30 minutes at room temperature (step 1), followed by drying under a nitrogen stream. The radioastatinated product was deprotected by addition of trifluoroacetic acid (TFA) and heating to 60° C. for 30 minutes (step 2). The radiochemical conversion (% RCC) for this procedure is shown in Table 1:
TABLE-US-00001 TABLE 1 % RCC % RCC % RCC Activity range [radioTLC] [radioTLC] [TLC] (Step 1) (Step 2) (overall) n 5-61 MBq 81 ± 5 91 ± 6 71 ± 7 3
[0226] Thus, the PSMA analogue, provided in a two-step labeling procedure, resulted in a RCC of >60%, which is surprising, since a similar labelling procedure, reported in WO2019/157037 yielded a labelled PSMA analogue ([.sup.211At]VK-02-90) in 12.5% radiochemical yield (RCY, non-decay corrected), over two steps. No detailed data is provided for the method used in WO2019/157037 (one pot procedure, 3 modification), however an RCY of maximum 26% was reported.
[0227] RCC as stated above refers to radiochemical conversion, and is used as a measure of how much of the added activity that is converted to the desired product, as demonstrated by chromatography, typically radio-TLC or radio-HPLC. RCC measurements are made before a potential work-up or purification is carried out. In this way, RCC can be said to measure the efficiency of the chemical radiolabeling reaction. RCY refers to radiochemical yield, and is used as a measure of how much of the added activity ends up as the desired product in a purified form, typically with an associated radiochemical purity (RCP) that says how much of the activity in the purified product is present as the actual desired product. In this sense, RCY reflects both the efficiency of the labeling (RCC) and the efficiency of the work-up procedure, since product may be lost during purification. However, unless a very inefficient type of purification is used, RCY will be similar to RCC, with RCC being slightly higher. In WO2019/157037, purification was done by HPLC, a state-of-the-art, standard procedure. Accordingly, RCY and RCC would be expected to be similar, typically with a difference between the two of about 5-15%. In this sense, the difference between a reported RCY of 26% and an RCC of 71% is substantial and reflects a difference in the efficiency of the radiolabeling reactions themselves. It should be noted that efficient radiochemistry is crucial for commercial use as it limits loss of the radionuclide, makes purification easier, limits radioactive waste, and limits exposure of personnel to radiation.
Example 6: 68Ga-Labeling
[0228] .sup.68Ga [E.sub.β+,max=1.9 MeV (88%), t.sub.1/2=68 min] was eluted from a .sup.68Ge (t.sub.1/2=271 d) generator (ITM AG, Munich, Germany), based on silica gel modified with dodecyl gallate, as [.sup.68Ga]GaCl.sub.3 in 0.1 M HCl and trapped in an SCX cartridge (HyperSep, ThermoFischer). The trapped [.sup.68Ga]Ga.sup.3+ was eluted with 300 μL of a 5 M NaCl/HCl solution generally giving 500-600 MBq of activity and employed in further radiolabellings.
[0229] Radiolabelling of the DOTA-containing peptidomimetics was carried out as follows. 40 uL of [.sup.68Ga]Ga.sup.3+ eluate (˜50-80 MBq) were mixed with 40 μL of 1.0 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 4). If needed, the pH of the solution was adjusted to 3.8-4.2 by addition of 10% NaOH.sub.(aq.). Thereafter, 5 μl (internalization experiment) or 2 μl (PET imaging) of a 1 mM solution of the DOTA-bearing peptide was added and the reaction mixture was heated to 95° C. for 15 min (internalization experiment) or 5 min (PET imaging), respectively. Radiolabelling efficiency was determined by RP-HPLC (5-95% B in 5 minutes—Chromolith RP-18e 100×4.6) and was deemed to be >98% for all the radiolabelled compounds.
Example 7: In Vitro Test of Binding Affinity and Internalisation
[0230] Compounds Ii, Ij, Ik, Il, Im and In was provided using the same method as disclosed in Example 3 and in vitro test of the binding affinity and internalisation of the compounds were examined using the method as described in Benesova et al, 2016.sup.9. The iodine isotope used was .sup.127I.
[0231] For internalisation determinations, a day before the experiment, PSMA(+) LNCaP cells were seeded in a poly-L-lysine coated 24-well plate (10.sup.5 cells per well) and maintained at 37° C. in an atmosphere of 5% CO.sub.2 under supplemented RMPI medium (10% Fetal Calf Serum, 1% sodium pyruvate, 1% FIS). Cells were incubated with 250 μL of radiolabelled compound diluted in RPMI medium (final concentration of [.sup.68Ga]-peptide: 30 nM) and 500 μM of PMPA (2-phosphonomethyl-pentanedioic acid) for the blocked series, for 45 min at 37° C. Cellular uptake was interrupted by washing the cells with ice-cold PBS (3×1 mL). Surface bound radioactivity was removed by incubating twice with 0.5 mL glycine (50 mM, pH=2.8) for 5 min. Thereafter, cells were washed with PBS (1 mL) and lysed employing NaOH (0.3 M) during 10 min. Lysates and surface-bound activity were collected and measured in a gamma-counter (Perkin Elmer 2480, Wizard, Gamma Counter). The cell uptake was calculated as percent of the initially added radioactivity bound to 105 cells [% ID/105 cells].
[0232] For internalisation studies androgen-sensitive human prostate adenocarcinoma cells (LNCaP) highly overexpressing PSMA were incubated with the .sup.68Ga-labeled compounds resulting in specific cell surface binding of all tested compounds (Table 2).
TABLE-US-00002 TABLE 2 Internalisation data*. Specifically cell Specifically surface bound internalised Compound [% IA/10.sup.5 cells].sup.† [% IA/10.sup.5 cells].sup.† PSMA-617 1.5 ± 0.4 0.6 ± 0.2 II 1.1 ± 0.4 0.7 ± 0.1 U 2.3 ± 0.2 0.8 ± 0.3 IK 0.7 ± 0.1 0.6 ± 0.3 IL 1.6 ± 0.4 0.6 ± 0.2 IN 0.4 ± 0.1 0.2 ± 0.1 IM 1.6 ± 0.5 0.9 ± 0.5 *Data are expressed as mean ± SD (n=3), .sup.†68Ga-labeled compounds. Specific cell uptake was determined by blockage using 500 μM 2-PMPA. Values are expressed as % of applied radioactivity (IA) bound to 10.sup.5 cells.
[0233] The results are shown in Table 2. All compounds revealed comparable internalisation properties as PSMA-617, except IN showing a reduced internalised fraction. Especially compound Ij and Im displayed a specific internalization higher than or comparable to PSMA-617, which was surprising, as modifying this amino acid residue in the linker region of PSMA inhibitors can have significant effects on binding and internalisation, as is reported in Benesova et al., 2016.sup.9. Internalisation is the key predictor for successful PSMA therapy.
Example 8: In Vivo Evaluation of the PSMA Targeting Radioligands
[0234] Compounds Ii, Ij, Ik, Il and Im which showed internalisation comparable to PSMA-617 as shown in Example 7 were selected for in vivo evaluation in mice.
[0235] For the experimental tumor models 1×10.sup.7 cells of LNCaP (in 50% Matrigel; Becton Dickinson) were subcutaneously inoculated into the right flank of 7- to 8-week-old male BALB/c nu/nu mice (Janvier). For imaging studies, mice were anesthetized (2% isoflurane) and 0.5 nmol of the .sup.68Ga-labeled compound in 0.9% NaCl (pH 7) were injected into the tail vein. PET imaging was performed with μPET/MRI scanner (BioSpec 3T, Bruker) with a dynamic scan for 60 min. The images were iteratively reconstructed (MLEM 0.5 algorithm, 12 iterations) and were converted to SUV images. Quantification was done using a ROI (region of interest) technique and data in expressed in time activity curves as SUV.sub.body weight. All animal experiments complied with the current laws of the Federal Republic of Germany.
PET Imaging: Tumor Uptake and Pharmacokinetic Profile
[0236]
[0237] The pharmacokinetic properties and tumor targeting properties of the modified compounds were found to be comparable or superior to the parental reference PSMA-617 (see
REFERENCES
[0238] (1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.; Jemal, A. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA. Cancer J. Clin. 2018, 68 (6), 394-424. [0239] (2) Antonarakis, E. S.; Feng, Z.; Trock, B. J.; Humphreys, E. B.; Carducci, M. A.; Partin, A. W.; Walsh, P. C.; Eisenberger, M. A. The Natural History of Metastatic Progression in Men with Prostate-Specific Antigen Recurrence after Radical Prostatectomy: Long-Term Follow-Up. BJU Int. 2012, 109 (1), 32-39. [0240] (3) De Bono, J. S.; Oudard, S.; Ozguroglu, M.; Hansen, S.; MacHiels, J. P.; Kocak, I.; Gravis, G.; Bodrogi, I.; MacKenzie, M. J.; Shen, L.; et al. Prednisone plus Cabazitaxel or Mitoxantrone for Metastatic Castration-Resistant Prostate Cancer Progressing after Docetaxel Treatment: A Randomised Open-Label Trial. Lancet 2010, 376 (9747), 1147-1154. [0241] (4) Israeli, R. S.; Powell, C. T.; Corr, J. G.; Fair, W. R.; Heston, W. D. W. Expression of the Prostate-Specific Membrane Antigen. Cancer Res. 1994, 54 (7), 1807-1811. [0242] (5) Benesova, M.; Schafer, M.; Bauder-Wüst, U.; Afshar-Oromieh, A.; Kratochwil, C.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M. Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer. J. Nucl. Med. 2015, 56 (6), 914-920. [0243] (6) Pomper, M. G.; Mease, R. C.; Chen, Y.; Ray, S.; Zalutsky, M.; Vaidyanathan, G. PSMA TARGETED RADIOHALOGENATED UREAS FOR CANCER RADIOTHERAPY—WO2017070482A2, 2016. [0244] (7) Pomper, M. G.; Mease, R. C.; Kumar, V.; Ray, S.; Zalutsky, M.; Vaidyanathan, G. PSMA TARGETED RADIOHALOGENATED UREA-POLYAMINOCARBOXYLATES FOR CANCER RADIOTHERAPY WO2019157037A1, 2019. [0245] (8) Lowe, P. T.; Dall'Angelo, S.; Fleming, I. N.; Piras, M.; Zanda, M.; O'Hagan, D. Enzymatic Radiosynthesis of a 18 F-Glu-Ureido-Lys Ligand for the Prostate-Specific Membrane Antigen (PSMA). Org. Biomol. Chem. 2019, 17 (6), 1480-1486. [0246] (9) Benešová, M.; Bauder-Wüst, U.; Schäfer, M.; Klika, K. D.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M. Linker Modification Strategies to Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors. J. Med. Chem. 2016, 59 (5), 1761-1775. [0247] (10) Feuerecker B., Tauber, R., Knorr, K. Heck, M., Beheshti, A., Seidl, C., Bruchertseifer, F., Pickhard, A., Gafita, A., Kratochwil, C., Retz, M., Gschwend, J. E., Weber, W. A, D'Alessandria, C., Morgenstern, A., Eiber, M. Activity and Adverse Events of Actinium-225-PSMA-617 in Advanced Metastatic Castration-resistant Prostate Cancer After Failure of Lutetium-177-PSMA. European Urology, 2021, 79 (3), 343-350. [0248] (11) Iley, J., Tolando, R., Constantino, L. Chemical and microsomal oxidation of tertiary amides: regio- and stereoselective aspects. J. Chem. Soc., Perkin Trans. 2, 2001, 1299-1305 [0249] (12) Syrén, P. O. Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation. J. Org. Chem. 2018, 83, 21, 13543-13548