FUNCTIONALIZED BISAMINOTHIOL DERIVATIVES, COMPLEXES WITH THESE BISAMINOTHIOL DERIVATIVES AND USE OF SAID COMPLEXES AS DIAGNOSTICS AND THERAPEUTICS
20250319213 · 2025-10-16
Assignee
- ABX ADVANCED BIOCHEMICAL COMPOUNDS – BIOMEDIZINISCHE FORSCHUNGSREAGENZIEN GMBH (Radeberg, DE)
- HELMHOLTZ-ZENTRUM DRESDEN-ROSSENDORF E.V. (Dresden, DE)
Inventors
- Christian LIS (Dresden, DE)
- Alexander HOEPPING (Dresden, DE)
- Hans-Joachim Lankau (Weinböhla, DE)
- Christoph MEYER (Radeberg, DE)
- Desna JOSEPH (Albuquerque, NM, US)
- Christiane SCHULTZE (Dresden, DE)
- Steffen FISCHER (Dresden, DE)
- Friedrich-Alexander LUDWIG (Dresden, DE)
- Martin ULLRICH (Dresden, DE)
- Wiebke SIHVER (Dresden, DE)
Cpc classification
C07C323/25
CHEMISTRY; METALLURGY
A61K51/0478
HUMAN NECESSITIES
International classification
Abstract
A compound of general formula I
##STR00001## where A is a chelator selected from the group of
##STR00002## k is independently at each occurrence 0, 1, or 2; m is independently at each occurrence 1, 2, 3, 4 or 5; n is independently at each occurrence 0, 1, 2 or 3; p is independently at each occurrence 1, 2 or 3; q is independently at each occurrence 1, 2 or 3; u is independently at each occurrence 0 or 1; X and Y are substituted or unsubstituted amino acids; L is a bifunctional linker selected from group of
##STR00003## where v, x, and y are independently of each other 0, 1, 2, or 3 and z is 0, 1, 2, 3, 4 or 5; and R is H, methyl or ethyl.
Claims
1-15. (canceled)
16. A compound of general formula I ##STR00052## wherein A is a chelator selected from the group consisting of ##STR00053## k is independently at each occurrence 0, 1, or 2; m is independently at each occurrence 1, 2, 3, 4 or 5; n is independently at each occurrence 0, 1, 2 or 3; p is independently at each occurrence 1, 2 or 3; q is independently at each occurrence 1, 2 or 3; u is independently at each occurrence 0 or 1; X and Y are substituted or unsubstituted amino acids; L is a bifunctional linker selected from group, consisting of ##STR00054## wherein v, x and y are independently of each other 0, 1, 2, or 3 and z is 0, 1, 2, 3, 4 or 5; and R is H, methyl or ethyl.
17. The compound of general formula I according to claim 16, wherein the amino acids are substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine and substituted or unsubstituted serine.
18. The compound of general formula I according to claim 16, wherein the amino acids are substituted or unsubstituted phenylalanine or substituted or unsubstituted glutamic acid.
19. The compound of general formula I according to claim 16, wherein L is L1 or L2.
20. The compound of general formula I according to claim 16, wherein A is A1 or A2.
21. The compound of general formula I according to claim 16, wherein k is 1, m is 3, n is 2, p is 1 or 2, q is 1 or 2 and u is 1, X and Y independently are substituted or unsubstituted phenylalanine or glutamic acid; L is L1 with v=1 or L2 with x and y=1, and A is A1 or A2.
22. The compound according to claim 16, wherein the compound of general formula I is selected from the group consisting of ##STR00055## ##STR00056## ##STR00057##
23. A complex of a compound according to claim 16 as ligands and a metal.
24. The complex according to claim 23, wherein the metal is an isotope selected from the group consisting of .sup.99mTc, .sup.99Tc, .sup.94mTc, .sup.186Re, .sup.188Re, .sup.64Cu and .sup.67Cu.
25. A complex according to claim 23 for use as a medicinal drug.
26. A complex according to claim 23 for use as a medicinal drug for the diagnosis and treatment of diseases in which PSMA is involved.
27. The complex according to claim 25, wherein the medicinal drug is a radiopharmaceutical for nuclear medical imaging or radioligand therapy.
28. A medicinal drug containing a complex according to claim 23 or a pharmaceutically acceptable salt thereof.
29. A method for preparing a complex according to claim 23, wherein a compound according to claim 16 is contacted with the metal.
30. A method according to claim 29, wherein the compound is contacted with the metal at a reaction temperature in the range of from 20 to 100 C. at ambient pressure.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0071] In the following, the invention is explained in more detail with the help of examples not intended to limit the invention with respect to the drawings. Here
[0072]
[0073]
[0074]
[0075]
[0076]
DETAILED DESCRIPTION
Examples
General Method for the Synthesis of Compounds 1, 2, 4, 5, and 6
[0077] Compounds 1, 2, 4, 5, and 6 were synthesized via solid phase peptide synthesis (SPPS) on 2-chlorotrityl resin.
[0078] Analysis of the synthesized molecules was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Ascentis Express C18, 1504.6 mm; Supelco, Germany) with a linear A-B gradient (5% B to 100% B in 10 min) at a flow rate of 1.5 mL/min (analysis). Purification was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Gemini-NX C18, 25050 mm; Phenomenex, Germany) with a linear A-B gradient at a flow rate of 100 mL/min. Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in ACN.
[0079] The HPLC system (Dionex Ultimate 3000; Thermo-Fisher, Germany) was equipped with a UV detector. UV absorbance was measured at 200, 210 and 230 nm. Mass spectrometry was performed with a LC-MS System (Dionex 3000, ThermoFisher, Germany).
Examples 1 to 4
Synthesis of Intermediates
[0080] To prepare compounds 1 to 6 the following intermediates have been prepared.
Example 1
Synthesis of Intermediate 101: Glu-CO-Im ((di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate))
[0081] H-Glu (OtBu)-OtBu (29.59 g, 1 eq. 100 mmol) was dissolved in 400 ml DCM. Triethylamine (25.3 g, 2.5 eq., 250 mmol) was added slowly. Carbonyldiimidazole (17.84 g, 1.1 eq., 110 mmol) was added in small portions. Reaction was left agitating for 4 hours. The solution was washed with water, NaHCO.sub.3 and brine. The organic phase was dried over Na.sub.2SO.sub.4 and evaporated in vacuo to give the target compound 101 as an oil.
Example 2
Synthesis of Intermediate 103: N-[2-(4-Methoxy-benzylsulfanyl)-ethyI]-N-{2-[4-methoxy-benzylsulfanyl)-ethylamino]-ethyl}-propan-1,3-diamine
##STR00039##
[0082] The synthesis of intermediate 103 was performed analogously to WO 2012/022812 A1.
Step 1:2-(4-Methoxy-benzylsulfany)-ethylamine (Compound 104)
[0083] Sodium (4.5 g, 196 mmol) was added to vigorously stirred methanol (150 ml, dry). When the sodium is completely dissolved, 2-aminoethanethiol hydrochloride (10.8 g 95.0 mmol) was added. After that, p-methoxybenzylchloride (14.9 g, 95.5 mmol) was added via a dropping funnel. The mixture was heated under reflux at 70 C. for 30 minutes. Subsequently the mixture was cooled to room temperature. The solid was removed by filtration and the filter cake washed with methanol (3 times with 25 ml). The organic extracts were combined and volatiles removed under reduced pressure. This residue was re-dissolved in DCM (75 ml), extracted with water (75 ml3), dried (MgS04), filtered and solvent removed to leave compound 104 a colorless oil. Yield 18.5 g (99%).
Step 2: N-[(4-Methoxy-benzylsulfanyl)-ethyl]-2-chloroacetamide (Compound 105)
[0084] Chloroacetyl chloride (4.24 ml, 53.2 mmol) in dry DCM (50 ml) was added dropwise over 90 minutes with stirring to an ice bath cooled (0 C.) solution of 2-(4-methoxy-benzylsulfanyl)-ethylamine 104 (9.4 g, 47.5 mmol) and triethylamine (8.0 ml) in dry DCM (200 ml). After addition the cooling bath was removed and stirring continued for 60 minutes. The solution was extracted with water (2250 ml), dried (MgSO4), filtered and solvent evaporated under reduced pressure to leave compound 105 as a colored solid. Yield 12.93 g (99%).
Step 3: Methyl 3-[(4-methoxy-benzylsufanyl)-ethylamino]-propanoate (Compound 106)
[0085] Methyl acrylate (4.46 ml 49.2 mmol) in methanol (10 ml) was added to a stirred solution of 2-(4-methoxy-benzylsulfanyl)-ethylamine 104 (8.85 g, 48.3 mmol) in methanol (50 ml). The colorless solution was allowed to stir at r.t. for 6 hours. Volatiles were removed by rotary evaporation to leave compound 106 as colorless viscous oil. Yield 1.35 g (97%).
Step 4:3-[2-(4-Methoxybenzylsulfanyl)-ethylamino]-propanamide (Compound 107)
[0086] Methyl 3-[(4-Methoxy-benzylsufanyl)-ethylamino]propanoate 106 (10.65 g 37.6 mmol), methanol (120 ml) and ammonia solution (200 ml) were stirred at r.t. for 24 hours. Volatiles were removed under reduced pressure to give compound 107 as an almost white solid. Yield 9.98 g (99%).
Step 5: 3-([2-(4-Methoxy-benzylsulfany)-ethyl]-{[2-(4-methoxy-benzylsulfanyl)ethylcarbamoyl]-methyl}-amino)-propionamide (Compound 108)
[0087] 3-[2-(4-Methoxybenzylsulfanyl)-ethylamino] propenamide 106 (10.33 g, 38.5 mmol), N-[(4-Methoxy-benzylsulfanyl)-ethyl]-2-chloroacetamide 105 (10.54 g, 38.5 mmol), triethylamine (6.5 ml) and acetonitrile (80 ml) were heated at 70 C. overnight. Subsequently the mixture was cooled to room temperature. The solvent was removed under reduced pressure to give a brown residue which was purified over silica eluting with DCM/Methanol 20:1 to yield compound 108 as a colorless oil (yield 8.3 g, 43%).
Step 6: N-[2-(4-Methoxy-benzylsulfanyl)-ethyl]-N-{2-[4-methoxy-benzylsulfanyl)-ethylamino]-ethyl}-propan-1,3-diamine (Compound 109)
[0088] 1.0M borane in THF (102 ml, 102 mmol) was added via a syringe under argon atmosphere to 3-([2-(4-Methoxy-benzylsulfanyl)-ethyl]-{[2-(4-methoxybenzylsulfanyl)-ethylcarbamoyl]-methyl}-amino)-propionamide 108 (3.8 g, 7.5 mmol). The resulting colorless solution was heated under reflux at 70 C. overnight. After cooling to r.t. water (40 ml) was added dropwise. The solvent was removed under reduced pressure to leave a waxy solid which was diluted with HCl (0.5 N, 400 ml). The mixture was heated under reflux at 100 C. for 3 hours. After cooling to r.t. sodium hydroxide was added until a pH 10-11 was obtained. This mixture was extracted with DCM (4200 ml) and the organic fractions were combined, dried (MgSO4) and filtered. The solvent was evaporated to leave a waxy solid, which was purified over silica eluting with DCM/Methanol/NH.sub.4OH=9:1:0.1 to yield compound 109 as a colorless oil. Yield 1.29 mg (36%)
Example 3
Synthesis of Intermediate 4: N-Boc-N-(5-carboethoxypentyl-N,N-bis-(2-(4-methoxy-benzylthio)-2-methylpropyl)-ethylenediamine (Compound 110)
##STR00040##
[0089] The synthesis of intermediate 110 was performed analogously to U.S. Pat. No. 5,776,428 A.
Step 1:2,2-Dithiobis(2-methylpropanal) (Compound 111)
[0090] To 2-Methylpropanal (28.8 g, 0.4 mol) in carbon tetrachloride (30 ml) was added sulfur monochloride (27 g, 0.2 mol). The reaction mixture was stirred at 50 C. for 16 hours. After cooling to room temperature, the volatiles were evaporated in vacuo and residue was purified by distillation in vacuo (bp at 0.5 Torr: 98-102 C.) to yield 22.7 g (55%) of 2,2-dithiobis(2-methylpropanal) 111.
Step 2:3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiadiazecine (Compound 112)
[0091] To a stirred solution of 2,2-dithiobis(2-methylpropanal) 111 (5 g) in chloroform (20 mL) were added 1.8 g of ethylenediamine. The reaction mixture was stirred at room temperature for 2 hours. After the removal of the solvent the residue was triturate with water until crystals began to form. The white crystals were collected by filtration and washed with ethanol to yield 4.8 g (86%) of 3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiadiazecine 112.
Step 3: N,N-Bis(2-mercapto-2-methylpropyl)ethylene diamine (Compound 113)
[0092] To 4.1 g (18 mmol) of 3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiadiazecine 112, dissolved in 70 ml of dry THF were added with stirring (argon atmosphere) 1.35 g of LiAlH.sub.4 (0.36 mmol). The reaction mixture was refluxed for 4 hours and subsequently hydrolyzed by the cautious addition of saturated NaK-tartrate solution (20 ml), and then diethyl ether (100 ml) were added. The sludge was separated by decanting or filtration (Celite) and washed well with ether. The solvent was removed in vacuo to yield 0.8 g (20%) of the compound 113.
Step 4: N,N-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 114)
[0093] A solution of N,N-bis(2-mercapto-2-methylpropyl)ethylene diamine 113 (1.1 g, 4.65 mmol) in methanol (50 mL) was cooled in ice/water bath and then saturated with gaseous ammonia over 30 min. To this was added 4-methoxybenzyl chloride (1.9 g, 12.3 mmol). The reaction was allowed to warm to room temperature overnight with stirring under argon. Methanol was evaporated in vacuo and the residue was then partitioned between diethyl ether (50 mL) and 0.5M KOH (40 mL). The aqueous layer was further extracted with diethyl ether (225 mL). The combined organic layers were washed with NaCl solution and concentrated in vacuo to give a clear colorless oil. The oil was dissolved in diethyl ether (200 mL) and then acidified with 4.0M HCl in dioxane. The white precipitate was collected by filtration and washed with diethyl ether. The HCl salts were partitioned between 1M KOH (30 mL) and ethyl acetate (30 mL). The aqueous layer was extracted with ethyl acetate (230 mL) and the combined organic layers were washed with NaCl solution, dried over Na.sub.2SO.sub.4 and concentrated to give the pure compound 114 (free base) as a light-yellow oil (0.99 g. 45% yield).
Step 5: N-(5-carboethoxypentyl-N,N-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 115)
[0094] To N,N-Bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine 114 (921 mg, 1.93 mmol) in acetonitrile (15 mL) was added K.sub.2CO.sub.3 (270 mg) followed by ethyl 5-bromovalerate (807 mg). The reaction was stirred at reflux overnight and subsequently concentrated in vacuo. The residue was partitioned between ethyl acetate (50 mL) and 0.5M KOH (50 mL). The aqueous layer was extracted with ethyl acetate (250 mL) and the combined organic layers were washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and concentrated to give a yellow oil, which was purified over silica eluting with DCM/Methanol=20:1 to yield the desired compound 115 as a yellowish oil. Yield 635 mg (54%)
Step 6: N-Boc-N-(5-carboethoxypentyl-N,N-bis-(2-(4-methoxybenzylthio)-2-methyl-propyl)-ethylenediamine (Compound 116)
[0095] To N-(5-carboethoxypentyl)-N,N-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine 115 (635 mg 1.05 mmol) in THF (40 mL) was added water (30 mL) and 1M KOH (2.5 mL, 2.5 mmol). The homogeneous solution was refluxed overnight. The solution was then cooled to room temperature and the THF was removed in vacuo. The residue was diluted with 50 mL water and the pH was adjusted to 2-3 by addition of 1M HCl. The solution was extracted with ethyl acetate (350 mL). The combined organic layers were washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give 575 mg of a crude intermediate.
[0096] The crude intermediate was dissolved in ACN (50 mL) and Boc.sub.2O (326 mg) followed by triethylamine (0.280 mL) were added. The homogenous solution was stirred at room temperature overnight under argon. The solution was then concentrated in vacuo and subsequently partitioned between ethyl acetate (25 mL) and 1M KH.sub.2PO.sub.4 (25 mL). The organic layer was washed with 5% citric acid (225 mL) and brine (25 mL), dried over Na.sub.2SO.sub.4 and concentrated to give a yellow oil (890 mg). Purification by column chromatography over silica eluting with DCM/Methanol=20:1 yielded compound 116 as a yellowish oil. Yield 455 mg (64%).
Example 4
Synthesis of Compound 5 [ABX 474]
[0097] The synthesis of the inventive compound 5
##STR00041##
is shown in Scheme EX-1.
##STR00042## ##STR00043##
In Scheme EX-1, letter a) indicates the use of 20% Piperidine in DMF; letter b) the use of Fmoc-Glu(OtBu)-OH, HATU, HOAt, DIPEA, and DMF; letter c) the use of Intermediate 110, PyBOP, DIPEA, and DMF; letter d) the use 2% N.sub.2H.sub.2.Math.H.sub.2O in DMF; letter e) the use of Sub-NHS, DIPEA, and DMF; letter f) the use of TSTU, DIPEA and DMF; letter g) the use of Fmoc-Lys-OtBu, DIPEA, and DMF; letter h) the use of Glu-CO-Im, NMM, and DMF; letter i) the use of TFA:TIS:H.sub.2O:EDT (92.5:2.5:2.5:2.5); and letter j) the use of 10% TFMSA.
[0098] For synthesizing of compound 501, Fmoc-D-Lys(Dde)-OH was loaded to 2-CTC resin (in Schema EX-1, the resin is represent by a circle) and Fmoc was deprotected with 20% piperidine in DMF. Then, for synthesizing of compounds 502 and 503, Fmoc-Glu(OtBu)-OH (2 eq.) was activated with HATU (2 eq.), HOAt (2 eq.) and DI-PEA (5,6 eq.) in DMF and added to the resin. The reaction mixture was agitated for 2 h at room temperature (r.t.). Fmoc-deprotection was performed with 20% piperidine in DMF. For synthesizing compound 504, intermediate 110 was conjugated to the peptide sequence using PyBOP (2 eq.) and DIPEA (2 eq.) in DMF for 2 h at r.t. Dde-deprotection was conducted using 2% hydrazine monohydrate in DMF. Afterwards, for synthesizing of compound 505, suberic acid-mono-NHS ester (2 eq.) and DIPEA (2 eq.) were dissolved in DMF (1 mL) and allowed to react with the resin bound peptide for 2 h at r.t. Then, for synthesizing of compound 506, the free carboxylic acid was treated with TSTU (2 eq.) and DIPEA (2 eq.) in DMF (10 mL/g resin) at r.t. for 1 h. After the formation of NHS ester, the resin bound peptide was treated with Fmoc-Lys-OtBu (2 eq.) and DIPEA (2 eq.) in DMF (2 mL) for 2 h at r.t. Fmoc was cleaved using 20% piperidine in DMF and then the free amine was treated with Glu-CO-Im (Intermediate 101, 3 eq.), and NMM (3 eq.) in DMF for 2 h at r.t. After final coupling, the resin was washed with DMF (35 mL), DCM (35 mL), IPA (35 mL), and Et.sub.2O (35 mL). For synthesizing of compound 5, cleavage from resin and final deprotection was performed by treatment with TFA/TIS/EDT/water (v/v/v/v; 92.5/2.5/2.5/2.5) at 0 C. and subsequent dropwise addition of 10% TFMSA.
[0099] The crude peptide was precipitated from ice-cold diethyl ether and purified via RP-HPLC. After RP-HPLC purification, compound 4 [ABX 474] (TFA salt) was obtained as a white to off white solid (isolated yield 38%). Calculated monoisotopic mass (C.sub.51H.sub.89N.sub.9O.sub.18S.sub.2): 1179.58; found: m/z=1180.5 [M+H].sup.+, 590.79 [M+2H].sup.2+.
Example 5
Synthesis of Compound 6 [ABX 490]
[0100] The synthesis of the inventive compound 6
##STR00044##
is shown in Scheme EX-2.
##STR00045## ##STR00046## ##STR00047##
In Scheme EX-2, letter a) indicates the use of 20% Piperidine in DMF; letter b) the use of Sub-NHS, DIPEA, and DMF; letter c) the use of TSTU, DIPEA and DMF and a reaction time of 1 h; letter d) the use of Fmoc-Lys-OtBu, DIPEA, and DMF; letter e) the use of Glu-CO-Im, NMM, and DMF; letter f) the use of 2% N.sub.2H.sub.2.Math.H.sub.2O in DMF; letter g) the use of Fmoc-Glu(OtBu)-OH, PyBop, HOBt, DIPEA, and DMF; letter h) the use of succinic anhydride, DIPEA, and DMF; letter i) the use of intermediate 103, DIPEA, and DMF; letter j) the use of TFA:TIS:H.sub.2O:EDT (92.5:2.5:2.5:2.5); and letter k) the use of 10% TFMSA.
[0101] For synthesizing compound 601, Dde-D-Lys(Fmoc)-OH was loaded to the 2-CTC resin (in Schema EX-2, the resin is represent by a circle) and Fmoc was deprotected with 20% piperidine in DMF. Afterwards, for synthesizing compound 602, suberic acid-mono-NHS ester (2 eq.) and DIPEA (2 eq.) were dissolved in DMF (1 mL) and allowed to react with resin bound peptide for 2 h at r.t. Then, for synthesizing compound 603, the free carboxylic acid was treated with TSTU (2 eq.) and DIPEA (2 eq.) dissolved in DMF (2 mL) and reacted for 1 h at r.t. After the formation of NHS ester, resin bound peptide was treated with Fmoc-Lys-OtBu (2 eq.) and DIPEA (2 eq.) in DMF (1 mL) for 2 h at r.t. For synthesizing compound 604, Fmoc was cleaved using 20% piperidine in DMF and then the free amine was treated with Glu-CO-Im 101 (3 eq.), and NMM (3 eq.) in DMF for 2 h at r.t. Dde-deprotection was conducted using 2% hydrazine monohydrate in DMF. Afterwards, for synthesizing compound 605, Fmoc-Glu(OtBu)-OH (2 eq.) was activated with PyBOP (2 eq.), HOBt (2 eq.) and DI-PEA (2 eq.) in DMF and added to the resin. The reaction mixture was agitated for 2 h at r.t. For synthesizing compound 606, Fmoc was deprotected with 20% piperidine in DMF. Succinic anhydride (2 eq.) and DIPEA (2 eq.) dissolved in DMF (2 mL) was added to resin bound peptide and agitated for 2 h at r.t. Then the free carboxylic acid was activated with TSTU (2 eq.) and DIPEA (2 eq.) dissolved in DMF (2 mL) and reacted for 1 h at r.t. After the formation of NHS ester, resin bound peptide was treated with intermediate 103 (1 eq.) and DIPEA (2 eq.) in DMF (2 mL) for 2 h to synthesize compound 607. After completion of the reaction, resin was washed with DMF (35 mL), DCM (35 mL), IPA (35 mL), and Et.sub.2O (35 mL). For synthesizing compound 6, cleavage from resin and final deprotection was performed by treatment with TFA/TIS/EDT/water (v/v/v/v; 92.5/2.5/2.5/2.5) at 0 C. and subsequent dropwise addition of 10% TFMSA.
[0102] The crude peptide was precipitated from ice-cold diethyl ether and purified via preparative RP-HPLC. After RP-HPLC purification, compound 6 [ABX 490] (TFA salt) was obtained as a white to off white solid (yield: 12%). Calculated monoisotopic mass (C.sub.49H.sub.84N.sub.10O.sub.19S.sub.2): 1180.54; found: m/z=1181.38 [M+H].sup.+, 591.27 [M+2H].sup.2+.
Example 6
Synthesis of Compound 1 [ABX 408]
[0103] Inventive compound 1
##STR00048##
was synthesized by means of the procedure described in Example 4 apart from the use of Fmoc-L-Lys(Dde)-OH instead of Fmoc-D-Lys(Dde)-OH and Fmoc-Phe-OH instead of Fmoc-Glu(OtBu)-OH. The crude peptide was purified via preparative RP-HPLC whereby compound 4 was obtained. Calculated monoisotopic mass (C.sub.59H.sub.93N.sub.9O.sub.14S.sub.2): 1215.63; found: m/z=1214.73 [MH].sup..
Example 7
Synthesis of Compound 2 [ABX 451]
[0104] Inventive compound 2
##STR00049##
was synthesized by means of the procedure described in Example 4 apart from the use of Fmoc-Phe-OH instead of Fmoc-Glu(OtBu)-OH. The crude peptide was purified via preparative RP-HPLC. Calculated monoisotopic mass (C.sub.59H.sub.93N.sub.9O.sub.14S.sub.2): 1215.63; found: m/z=1216.70 [M+H].sup.+.
Comparative Example 1
Synthesis of Compound 3 [ABX 455]
[0105] Comparative compound 3
##STR00050##
was synthesized by means of the procedure described in Example 4 apart from the omission of Fmoc-Glu(OtBu)-OH. The crude peptide was purified via preparative RP-HPLC. Calculated monoisotopic mass (C.sub.41H.sub.75N.sub.7O.sub.12S.sub.2): 921.49; found: m/z=922.45 [M+H].sup.+.
Example 8
Synthesis of Compound 4 [ABX 456]
[0106] Inventive compound 4
##STR00051##
was synthesized by means of the procedure described in Example 4 apart from the use of Fmoc-D-Phe-OH and Fmoc-D-Tyr(3I)-OH instead of Fmoc-Glu(OtBu)-OH. The crude peptide was purified via preparative RP-HPLC. Calculated monoisotopic mass (C.sub.59H.sub.92IN.sub.9O.sub.15S.sub.2): 1357.52; found: m/z=1356.70 [MH].sup..
Example 9
Synthesis of .SUP.nat.Re-Complexes
[0107] The compounds 1, 3 and 5 were used to synthesize complexes with naturally abundant Re isotopes (.sup.natRe). These synthesized complexes can be used as PSMA ligands.
[0108] 0.06 mmol of the corresponding compound 1, 3 or 5 were dissolved in 2 ml of methanol. Subsequently 1 ml of a 1 N NaOAc solution and 0.06 mmol oxotrichloro[(dimethylsulfide)-triphenylphosphine oxide]-rhenium (V) were added. The reaction mixture was stirred at room temperature overnight. The crude product was precipitated with ice-cold diethyl ether and purified via preparative RP-HPLC. Table Ex-1 shows the results of the LC-MS analysis of the synthesized rhenium complexes.
TABLE-US-00001 TABLE EX-1 calculated monoisotopic mass found .sup.natReO-1 1415.55 m/z = 1416.53 [M + H].sup.+ [=.sup.natReO-ABX408] .sup.natReO-3 1121.42 m/z = 1122.58 [M + H].sup.+ [=.sup.natReO-ABX455] .sup.natReO-5 1379.50 m/z = 690.92 [M + 2H].sup.2+, [=.sup.natReO-ABX474] 1380.08 [M + H].sup.+
[0109] The .sup.nat-Re-complexes of the compounds 1, 3 and 5 can be used as standards for the radiosynthesis of .sup.186Re and .sup.188Re-labeled PSMA-ligands. Analogously, the .sup.nat-Re-complexes of the compounds 2, 4 and 6 can be used as standards for the radiosynthesis of .sup.186Re and .sup.188Re-labeled PSMA-ligands.
Example 10
Radiolabeling of Compounds 1 to 6 with .sup.99mTc
[0110] The compounds 1 to 6 were used to synthesize complexes with .sup.99mTc. These synthesized complexes can be used as PSMA radioligands.
[0111] The corresponding compound 1, 2, 3, 4, 5 or 6 (50 g), mannitol (1 mg), and calcium heptagluconate (10 g) were stirred in 1-1.5 mL sodium pertechnetate/saline solution (from commercially available .sup.99Mo/.sup.99mTc generator, 0.2-4 GBq) at r.t. for 10 min. After addition of stannous chloride (1 g in 100 L of 0.01 N HCl; solution saturated with helium) the solution was kept at r.t. for 20 min and finally at 80 C. for another 20 min. After cooling down, the product was ready for further use. The product is a complex of .sup.99mTc and the corresponding compound 1, 2, 3, 4, 5 or 6. These complexes were provided as a solution of the respective complexes.
[0112] Quality control of the product was performed by radio-reversed-phase-high-performance liquid chromatography (radio-RP-HPLC) using a Poroshell 120 EC-C18 column (3.5 m, 1003 mm; Agilent Technologies Deutschland, Waldbronn, Germany) and a solvent system consisting of water (0.1% TFA) and ACN (0.1% TFA). Analyses were performed using two different gradient elution methods: a) 0-0.5 min 0%, 0.5-3 min 0-100%, 3-5 min 100%, 5-8 min 0% ACN (0.1% TFA); flow rate: 0-0.5 min 0.4 mL/min, 0.5-8 min 0.7 mL/min and b) 0-1.5 min 0%, 1.5-10 min 0-100%, 10-12 min 100%, 12-15 min 0% ACN (0.1% TFA); flow rate: 0.7 mL/min, as well as isocratic elution methods using a particular percentage of ACN (0.1% TFA) appropriate for the respective radioligand. In addition, for determination of possible .sup.99mTc/.sup.99Tc-colloid formation, radio-thin layer chromatography (radio-TLC) was performed on silica gel TLC sheets (POLYGRAM SIL G UV254, Macherey-Nagel, Dren, Germany) using methanol/ammoniumacetate (2 M) 1:1. Table 2 shows the radiochemical purity and the retention time t.sub.R of the obtained complexes.
TABLE-US-00002 TABLE EX-2 Radiochemical Radioligand purity* t.sub.R* [.sup.99mTc]TcO-1 92.2% 3.64 min [=[.sup.99mTc]TcO-ABX408] [.sup.99mTc]TcO-2 93.0% 3.45 min [=[.sup.99mTc]TcO-ABX451] [.sup.99mTc]TcO-3 93.6% 3.34 min [=[.sup.99mTc]TcO-ABX455] [.sup.99mTc]TcO-4 94.5% 3.46 min [=[.sup.99mTc]TcO-456] [.sup.99mTc]TcO-5 96.7% 4.16 min .sup.99m[= Tc]TcO-ABX474] [.sup.99mTc]TcO-6 94.3% 3.65 min [=[.sup.99mTc]TcO-ABX490] *determined by radio-RP-HPLC, method a)
Example 11
Determination of log D Values
[0113] Log D values were determined for different media by the shake-flask method (Andrs, A.; Ross, M.; Rfols, C.; Bosch, E.; Espinosa, S.; Segarra, V.; Huerta, J. M. Setup and validation of shake-flask procedures for the determination of partition coefficients (log D) from low drug amounts. Eur. J. Pharm. Sci. 2015, 76, 181-191.). In preparation, octanol was saturated with each of the used buffer solutions and vice versa. To 3 mL of octanol, together with 3 mL of buffer solution 200-300 L of the .sup.99mTc-labeled radioligand (15-25 MBq, samples in triplicate) were added and shaken vigorously for 30 min. After centrifugation at 9,000 rpm for 15 min, samples were taken from both separated phases and measured by gamma-counter. Log D values were calculated as the ratio of radioactivity determined in octanol and aqueous phase. Table EX-3 shows the determined Log D values of the .sup.99mTc complexes in Na-phosphate, TRIS buffer and PBS.
TABLE-US-00003 TABLE EX-3 LogD (shake-flask, octanol/medium) TRIS Na-phosphate* buffer 0.1M PBS 0.1M Radioligand pH 7.4 pH 7.4 pH 7.4 [.sup.99mTc]TcO-1 2.18 0.002 2.18 0.01 1.62 0.01 [=[.sup.99mTc]TcO-ABX408] [.sup.99mTc]TcO-2 2.08 0.01 2.16 0.02 2.16 0.03 [=[.sup.99mTc]TcO-ABX451] [.sup.99mTc]TcO-3 1.93 0.01 2.08 0.02 2.07 0.01 [=[.sup.99mTc]TcO-ABX455] [.sup.99mTc]TcO-5 2.41 0.08 1.98 0.032 2.52 0.03 [=[.sup.99mTc]TcO-ABX474] *4.4 mM Na.sub.2HPO.sub.4, 2.2 mM NaH.sub.2PO.sub.4
Example 12
Stability Studies
[0114] The stability of the complexes of .sup.99mTc with the compounds 1 to 6 were determined in different media.
[0115] In brief, the freshly produced .sup.99mTc-labeled radioligand solution (10-20 MBq, 20-100 L) was added to the respective medium (200-400 L), vortexed quickly, and shook gently at the respective temperature mentioned below. At certain time points, samples were taken, diluted, and measured by radio-RP-HPLC. Hereby, investigation of plasma samples included protein precipitation with the 4-fold volume of an ice-cold mixture of methanol/water (4/1, v/v), intensive shaking (5 min) and centrifugation (14,000 rpm, 10 min), before inspection of the supernatant. In addition, the recovery of extracted activity was calculated after activity measurement of supernatant and residue by a gamma counter.
TABLE-US-00004 TABLE EX-4 Product stability at room temperature Fraction of radioligand* at indicated time point after preparation (without addition of any media) Radioligand 0 h 2 h 4 h 6 h [.sup.99mTc]TcO-3 96% 93% 92% 91% [=[.sup.99mTc]TcO-ABX455] [.sup.99mTc]TcO-5 94% 91% 95% 94% [=[.sup.99mTc]TcO-ABX474] [.sup.99mTc]TcO-6 89% 87% 88% 85% [=[.sup.99mTc]TcO-ABX490] *determined by radio-RP-HPLC, method a
TABLE-US-00005 TABLE EX-5 Product stability in DPBS and physiological saline Fraction of radioligand* at indicated time point in DPBS.sup.# at 25 C. Radioligand 1 h 2 h 4 h 20 h [.sup.99mTc]TcO-1 101.5 0.6% 99.5 0.8% 100.2 1.0% 93.5 1.8% [=[.sup.99mTc]TcO-ABX408] [.sup.99mTc]TcO-2 89.9 2.3% 97.7 2.3% 80.7 12.0% 89.2 2.7% [=[.sup.99mTc]TcO-ABX451] [.sup.99mTc]TcO-3 98.2 1.9% 97.3 3.6% 98.4 1.0% 89.1 7.4% [=[.sup.99mTc]TcO-ABX455] [.sup.99mTc]TcO-4 88.4 14.8% 100 1.8% 105.5% 98.0 1.0% [=[.sup.99mTc]TcO-ABX456] [.sup.99mTc]TcO-5 101.8 0.03% 100.6 0.3% 100.3 1.0% 96.7 2.1% [=[.sup.99mTc]TcO-ABX474] [.sup.99mTc]TcO-6 75.9 0.2% 74.4 0.1% 72.3 0.9%.sup.& 18.7% [=[.sup.99mTc]TcO-ABX490] *indicated as mean SD (n = 2-4), determined by radio-RP-HPLC, method a) as described in Example 10, and normalized to the fraction of radioligand determined immediately after preparation .sup.#DPBS (Dulbecco's phosphate-buffered saline, modified: +Ca.sup.2+, +Mg.sup.2+) .sup.&after 6 h instead of 4 h
TABLE-US-00006 TABLE EX-6 Product stability in mouse plasma (in vitro) Fraction of radioligand* Radioligand after 6 h in mouse plasma at 37 C. [.sup.99mTc]TcO-1 96.8 0.3% [=[.sup.99mTc]TcO-ABX408] [.sup.99mTc]TcO-2 98.3 1.1% [=[.sup.99mTc]TcO-ABX451] [.sup.99mTc]TcO-3 98.5 0.8% [=[.sup.99mTc]TcO-ABX455] [.sup.99mTc]TcO-5 96.7 1.3% [=[.sup.99mTc]TcO-ABX474] *indicated as mean SD (n = 3-4), determined by radio-RP-HPLC, method a) as described in Example 10 and normalized to the fraction of radioligand determined immediately after preparation; Recovery of activity after plasma precipitation: 93.3 0.6% (mean SD; n = 9)
TABLE-US-00007 TABLE EX-7 Product stability in human plasma (in vitro) Fraction of radioligand* at indicated timepoint in human plasma at 37 C. Radioligand 0 h 1 h 2 h 4 h 6 h [.sup.99mTc]TcO-5 95.8% 91.1 91.2 91.1 91.7 [=.sup.99mTc]TcO- 0.6% 0.2% 0.8% 0.3% ABX474] *indicated as mean SD (n = 5), determined by radio-RP-HPLC, method a) as described in Example 10; Recovery of activity after plasma precipitation: 92.4% 0.9 (mean SD; n = 20)
Example 11
In Vitro Assays
[0116] Example 11 describes in vitro assays which were performed to determine the properties of the compounds 1, 2, 3, 4, 5 and 6. For this reason, complexes of compound 1, 2, 3, 4, 5 or 6 with .sup.99mTc as described in Example 10 and complexes of compound 1, 2, 3, 4, 5 or 6 with .sup.natRe as described in Example 9 were prepared.
a) Cell Culture
[0117] Competition, saturation and internalization assays were assessed using the high PSMA expressing human prostate carcinoma cell line LNCaP (ATCC CRL-1740). The cells were grown as monolayer at 37 C. in a humidified atmosphere comprising 5% CO2 and 95% air in RPMI medium including 10% FCS (Merck KGAA, Germany). After washing the confluent cells twice with phosphate buffered saline (PBS) and detaching them with trypsin/EDTA (0.05%/0.02%), the cells were suspended in medium and counted (Casy TT, Omni Life Science, Germany).
b) .SUP.68.Ga-Labeling of PSMA-11
[0118] A .sup.68Ga generator was purchased from iThemba LABS (Republic of South Africa). PSMA-11 (2-4 g=2.11-4.21 nmol) was labeled with .sup.68Ga (100 to 200 MBq) in a mixture of ammonium acetate (2 M) and HCl at a pH of 4.5. The reaction mixture was incubated for 10 min at 90 C. Quality control of radiolabeled PSMA-11 was performed using high-performance liquid chromatography (HPLC) with a C-18 reversed-phase column (semi-preparative Zorbax 300SB-C18, 9.4250 mm 5 m; Agilent Technologies, USA). The radiochemical yield was >97% for [.sup.68Ga]Ga-PSMA-11 at molar activities between 30 and 60 GBq/mol.
c) Determination of the Competitive and the Direct Binding Affinity (Competition and Saturation)
[0119] For competition monolayer of LNCaP cells were seeded, 110.sup.5 cells/well in 24-well plates two days before the assay. On the day of competition, after aspirating the medium, 100 L of PBS and, for competition 100 L of different concentrations of the test compound in PBS (10.sup.12 to 10.sup.6 M) were pipetted into the wells, simultaneously to 400 L of medium including 1 nM of [.sup.68Ga]Ga-PSMA-11.
[0120] For saturation monolayer of LNCaP cells were seeded, 510.sup.4 cells/well in 48-well plates two days before the assay. On the day of saturation, after aspirating the medium, 160 L of medium (for .sup.99mTc-labeled radioligand with D-mannitol (1 mg/mL)) were pipetted per well, for non-specific binding samples including 100 M 2-PMPA. After preincubation for 5 min, 40 L activity ([.sup.68Ga]Ga-PSMA-11 or .sup.99mTc-labeled radioligand solution) were added (total volume/well: 200 L). Eight concentrations ranged between 0.3 and 40 nM.
[0121] After 1 hour of incubation at 37 C. both for competition and saturation samples, the supernatants were aspirated and the cells washed twice with cold PBS. The cell lawn was lysed with 500 L NaOH/SDS (0.1 M/1%) by shaking for 3 to 5 minutes. After transferring the lysates into measuring tubes, the activity of the samples was measured in a gamma counter (2480 Automatic Gamma Counter Wizard 2, Perkin Elmer, USA).
[0122] The half maximal inhibitory concentrations (IC.sub.50) and the dissociations constants (K.sub.d) were calculated by fitting the data using a nonlinear curve-fitting program (GraphPad Prism 9). With the K.sub.d value of [.sup.68Ga]Ga-PSMA-11 on LNCaP and the known concentration of [.sup.68Ga]Ga-PSMA-11 in the competition assay the inhibition constants (K.sub.i) was also determined with the curve-fitting program. The K.sub.i values of the .sup.natRe-complexes and the K.sub.d values of [.sup.99mTc]TcO-complexes are shown in table EX-8.
TABLE-US-00008 TABLE EX-8 K.sub.i [nM]* of .sup.natRe-Com- K.sub.d [nM]* of [.sup.99mTc]TcO- compound plex complexes 1 [ABX408] 4.8 3.4 15.5 3.2 2 [ABX451] n.d. 32.1 24.3 3 [ABX455] 7.3 1.6 15.8 9.1 4 [ABX456] n.d. 4.7 3.2 5 [ABX474] 9.3 0.9 7.2 1.7 6 [ABX490] n.d. 7.3 .sup.natGa-PSMA-11 8.5 0.9 12.4 2.1 *mean SEM; n.d. = not determined
d) Determination of the Internalization
[0123] For internalization LNCaP and PC3 cells were seeded, 110.sup.5 cells/well in 24-well plates two days before the assay. On the day of internalization, after aspirating the medium, 100 L of PBS and, for non-specific binding samples, 100 L 2-PMPA (100 M) were pipetted into the wells, followed by 400 L medium including activity. The concentration of the .sup.99mTc-labeled radioligands was 25 nM. After 1 h of incubation at 37 C. and adjacent well plates at 4 C., the supernatant was removed and the cells washed with cold PBS. Surface-bound activity was stripped with 4 C. cold acid-wash buffer (0.2 M glycine, pH 2.8) for 5 min. The acid wash buffer was transferred from the plate wells to measuring tubes, as was the PBS buffer after washing once (binding on the surface). Cytosolic activity was determined after treatment with cell lysis buffer (0.1 M NaOH/1% SDS). Cell surface and cytosolic activity were measured separately in a gamma counter. Determination of protein content from cell lysate was performed using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, USA) at an absorbance of 280 nm. The results are shown in Table EX-9.
TABLE-US-00009 TABLE EX-9 [.sup.99mTc]TcO-3 [.sup.99mTc]TcO-4 [.sup.99mTc]TcO-5 [.sup.99mTc]TcO-6 [=[.sup.99mTc]TcO- [.sup.99mTc]TcO- [=[.sup.99mTc]TcO- [=[.sup.99mTc]TcO- .sup.68Ga- % AD/mg Protein ABX455] ABX456 ABX474] ABX490] PSMA11 Binding*, 1.8 0.3 2.9 0.5 3.2 1.0 4.7 0.2 0.8 0.2 37 C. Internalization*, 1.0 0.3 1.9 0.9 2.8 0.8 2.9 0.2 0.5 0.2 37 C. Binding*, 2.1 1.2 2.2 0.1 2.5 0.3 2.5 0.4 0.3 0.4 4 C. Internalization*, 0.7 0.2 0.2 0.0 0.2 0.0 0.2 0.0 0.1 0.0 4 C. % Internalization, 35.8 39.2 46.6 38.5 40.3 37 C. *mean SEM
[0124] Compared to comparative compound 3 the addition of the substituted or unsubstituted amino acids X and Y leads to an increase of the internalized % AD/mg protein.
Example 12
SPECT/CT and PET/CT Imaging
[0125] All animal experiments were carried out according to the guidelines of the German Regulations for Animal Welfare and have been approved by the local Ethical Committee for Animal Experiments.
[0126] A prostate cancer xenograft model was generated via subcutaneous injection of human LNCaP cells into the right shoulder of 8-12 week-old male nude mice (Rj:NMRI-Foxn1.sup.nu/nu, Janvier Labs, Le Genest-Saint-Isle, France). Imaging studies were performed when tumors reached a diameter of >6 mm. General anesthesia was induced and maintained with inhalation of 10% (v/v) desflurane in 30/10% (v/v) oxygen/air. During anesthesia, animals were continuously warmed at 37 C.
[0127] Small animal single-photon emission computed tomography (SPECT) was performed using the nanoSPECT/CT scanner (Mediso Medical Imaging Systems) equipped with an APT63 aperture consisting of four M3 multi-pinhole collimators. Each animal received 30 MBq of .sup.99mTc-labeled compounds delivered in 0.2 mL of Dulbecco's phosphate-buffered saline as a single intravenous injection through a tail vein catheter. Photon emission was recorded using frame times of 60 s (1 h scan: 40-70 min), 90 s (4 h scan, 3.5-4.5 h), and 320 s (20 h scan, 18.5-21.5 h), and binned simultaneously within the 20% energy window of the 140.5 keV photopeak. With each SPECT scan, a corresponding CT image was recorded and used for anatomical referencing and attenuation correction. SPECT images were reconstructed using the Tera-Tomo three-dimensional (3D) algorithm at normal range with a voxel size of 0.4 mm and applying corrections for scatter attenuation, and decay.
[0128] Small animal positron emission tomography (PET) was performed using the nanoPET/CT scanner (Mediso Medical Imaging Systems). Each animal received 10 MBq of the radiolabeled reference compound [.sup.68Ga]Ga-PSMA-11 delivered in Dulbecco's phosphate-buffered saline as a single intravenous injection through a tail vein catheter. Emission of the 511 keV annihilation photons was recorded continuously at a coincidence mode of 1:5 for 60 min following radiotracer injection. With each PET scan, a corresponding CT image was recorded and used for anatomical referencing and attenuation correction. Three-dimensional list mode data were binned using the 400-600 keV energy window. PET images of the 40-60 min time frame were reconstructed using the Tera-Tomo three-dimensional (3D) algorithm with a voxel size of 0.4 mm and applying corrections for random events, scatter, attenuation, and decay.
[0129] All images were post-processed and analyzed using ROVER (ABX) and displayed as maximum intensity projections at indicated scaling. Three-dimensional VOIs were created applying fixed threshold for delineation of tumor (30%), muscle (0%); and kidneys (39%). Standardized uptake values (SUV=[MBq detected activity/mL tissue]/[MBq injected activity/g body weight], mL/g) were determined and reported as max SUV (VOI-maximum). Time-activity curves were generated and further analyzed using Prism (GraphPad Software, San Diego CA, USA).
[0130] The tumor-uptake of [.sup.99mTc]-labeled radioligands in LNCaP tumor bearing mice is shown in
[0131]
[0132]
[0133]
[0134] [.sup.99mTc]TcO-5[=[.sup.99mTc]TcO-ABX474] displayed similar uptake in tumors and lower uptake in kidneys compared to reference compounds along with a higher contrast compared to [.sup.99mTc]TcO-PSMA-I&S and higher image resolution compared to [.sup.68Ga]Ga-PSMA-11 (see
[0135] It is shown in
LIST OF ABBREVIATIONS
[0136] Ac acetate [0137] ACN acetonitrile [0138] AD applied dose [0139] Boc tert-butoxycarbonyl [0140] Boc.sub.2O di-tert-butyl dicarbonate [0141] Bp boiling point [0142] CT computed tomography [0143] 2-CTC 2-chlorotrityl chloride [0144] DCM dichloromethane [0145] Dde N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) [0146] DIPEA N,N-diisopropylethylamine [0147] DMF dimethylformamide [0148] DPBS Dulbecco's phosphate-buffered saline [0149] EDT 1,2-ethanedithiol [0150] EDTA ethylenediaminetetraacetic acid [0151] Et.sub.2O diethyl ether [0152] FCS fetal calf serum [0153] Fmoc fluorenylmethoxycarbonyl [0154] HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate [0155] HOAt 1-hydroxy-7-azabenzotriazole [0156] HYNIC hydrazinonicotinic acid [0157] IPA 2-propanol [0158] LNCaP Lymph Node Carcinoma of the Prostate, a human prostate cancer cell line [0159] log D distribution coefficient [0160] NaOAc sodium acetate [0161] NMM 4-methylmorpholine [0162] NHS N-hydroxysuccinimide [0163] PBS phosphate buffered saline [0164] PC3 a human prostate cancer cell line [0165] PET positron emission tomography [0166] 2-PMPA 2-(phosphonomethyl) pentanedioic acid [0167] PSMA prostate-specific membrane antigen [0168] PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [0169] RLT radioligand therapy [0170] r.t. room temperature [0171] RP-HPLC reversed-phase high-performance liquid chromatography [0172] RPMI RPMI is a growth medium for cell culture [0173] RT radiotracer [0174] SDS sodium dodecyl sulfate [0175] SEM standard error of the mean [0176] SPECT single-photon emission computed tomography [0177] SPPS solid phase peptide synthesis [0178] Sub suberic acid [0179] SUV standardized uptake value [0180] tBu tert-butyl [0181] TFA trifluoroacetic acid [0182] TFMSA trifluoromethanesulfonic acid [0183] THF tetrahydrofuran [0184] TIS triisopropylsilane [0185] TRIS tris(hydroxymethyl)aminomethane [0186] TSTU O(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate [0187] VOI volume of interest