Therapeutic and diagnostic agents for cancer
11639373 · 2023-05-02
Assignee
Inventors
- Theresa Osl (Garching bei München, DE)
- Hans-Jürgen Wester (Ilmmünster, DE)
- Margret Schottelius (Munich, DE)
Cpc classification
A61K47/65
HUMAN NECESSITIES
A61K51/088
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
C07K7/64
CHEMISTRY; METALLURGY
International classification
A61K38/12
HUMAN NECESSITIES
A61K51/08
HUMAN NECESSITIES
C07K7/64
CHEMISTRY; METALLURGY
A61K47/65
HUMAN NECESSITIES
Abstract
The present disclosure relates to imaging and endoradiotherapy of diseases involving chemokine receptor 4 (CXCR4). Provided are compounds which bind or inhibit CXCR4 and furthermore carry at least one moiety which is amenable to labeling. Provided are also medical uses of such compounds.
Claims
1. A compound of the following formula (I) ##STR00065## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is an alkanediyl chain; R.sup.2 is a group of formula (II): ##STR00066## or a group of formula (IV): ##STR00067## which is linked to the remainder of the compound with the bond marked by the dashed line, and wherein R.sup.A is H or alkyl; R.sup.B is substituted alkyl, which substituted alkyl is substituted with at least one group selected from —NH.sub.2 and the guanidino group —NH—C(═NH)—NH.sub.2; R.sup.C is H or optionally substituted alkyl, with one or more optional substituents being selected from —NH.sub.2, —NH—C(═NH)—NH.sub.2, —COOH, —CONH.sub.2, —OH, —SH, —S—CH.sub.3, and 5- to 10-membered carbocycle or 5- to 10-membered heterocycle containing oxygen, nitrogen and/or sulfur as heteroatom(s), wherein R.sup.C may be further substituted with or may comprise a cytotoxic moiety; p is 0, 1 or 2; q is 0, 1 or 2; p+q is 0, 1 or 2; m is 0 or 1; R.sup.D is H or forms a 5 or 6-membered heterocycle together with the adjacent nitrogen which heterocycle also includes a part of R.sup.E; R.sup.E is a group which comprises at least one of the following: (i) a chelating moiety, (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, (iii) a moiety carrying a covalently bound radioisotope, or a precursor suitable to be labeled with such radioisotopes, (iv) a cytotoxic moiety, and (v) a fluorescent moiety; and R.sup.3 is H or I.
2. The compound or salt of claim 1, wherein the compound has the formula (Ia): ##STR00068## and wherein R.sup.2 and R.sup.3 are defined as in claim 1.
3. The compound or salt of claim 1, wherein the compound has the formula (Ib): ##STR00069## and wherein R.sup.2 and R.sup.3 are defined as in claim 1.
4. The compound or salt of claim 1, wherein R.sup.B is substituted linear C1-C6 alkyl, which is substituted at its terminal carbon with one group selected from —NH.sub.2 and —NH—C(═NH)—NH.sub.2.
5. The compound or salt of claim 1, wherein R.sup.2 has the formula (IIa) ##STR00070## wherein R.sup.A, R.sup.B, R.sup.D and R.sup.E are defined as in claim 1.
6. The compound or salt of claim 1, wherein R.sup.2 has a formula selected from formulae (IIb) and (IId): ##STR00071## wherein R.sup.A, R.sup.B, R.sup.C, p, q and m are defined as in claim 1, and R.sup.E1 is a group which comprises at least one of the following: (i) a chelating moiety, (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, (iii) a moiety carrying a covalently bound radioisotope, or a precursor suitable to be labeled with such a radioisotope, (iv) a cytotoxic moiety, and (v) a fluorescent moiety.
7. The compound or salt of claim 1, wherein R.sup.E is selected from (i) a group which comprises a chelating moiety, and (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, and wherein the chelating moiety comprises at least one of a macrocyclic ring structure with 8 to 20 ring atoms of which 2 or more are selected from oxygen atoms, sulfur atoms and nitrogen atoms; and an acyclic, open chain chelating structure with 8 to 20 main chain atoms of which 2 or more are heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms.
8. The compound or salt of claim 1, wherein R.sup.2 has a formula selected from formulae (IIf) to (IIh): ##STR00072## wherein R.sup.A, R.sup.B, are defined as in claim 1, and R.sup.E2 is selected from (i) a residue of a chelating agent comprising a carboxyl group and (ii) a chelate formed by a residue of a chelating agent comprising a carboxyl group with a chelated radioactive or non-radioactive cation, said residue being obtainable by forming an amide bond from the carboxyl group of the chelating agent and the nitrogen atom to which R.sup.E2 is attached.
9. A pharmaceutical composition comprising or consisting of a compound or salt of claim 1 and an excipient.
10. A method of diagnosing, preventing, or treating a disease or disorder in a patient in need thereof, comprising administering to the patient a compound or salt of claim 1.
11. The method of claim 10, wherein the method further comprises the use of nuclear medicine, nuclear molecular imaging, optical imaging, or targeted endoradiotherapy.
12. The method of claim 10, wherein the method is further defined as treating or preventing a disease or disorder associated with increased expression of chemokine receptors subtype 4 (CXCR4).
13. The method of claim 10, wherein the method is further defined as diagnosing cancer or cardiovascular disease.
14. The method of claim 12, wherein the disease is cancer or a lymphoproliferative disease.
15. The method of claim 10, wherein the disease or disorder is cardiovascular disease, AIDS, or an inflammatory disorder.
16. A compound of the following formula (I) ##STR00073## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is an alkanediyl chain; R.sup.2 is a group of formula (III): ##STR00074## which is linked to the remainder of the compound with the bond marked by the dashed line, and wherein R.sup.A is H or alkyl; R.sup.B is substituted alkyl, which substituted alkyl is substituted with at least one group selected from —NH.sub.2 and the guanidino group —NH—C(═NH)—NH.sub.2; R.sup.C is H or optionally substituted alkyl, with one or more optional substituents being selected from —NH.sub.2, -NH—C(═NH)—NH.sub.2, —COOH, —CONH.sub.2, —OH, —SH, —S—CH.sub.3, and 5- to 10-membered carbocycle or 5- to 10-membered heterocycle containing oxygen, nitrogen and/or sulfur as heteroatom(s), wherein R.sup.C may be further substituted with or may comprise a cytotoxic moiety; p is 0, 1 or 2; q is 0, 1 or 2; p+q is 0, 1 or 2; m is 0 or 1; R.sup.D is H or forms a 5 or 6-membered heterocycle together with the adjacent nitrogen which heterocycle also includes a part of R.sup.E; R.sup.E is a group which comprises at least one of the following: (i) a chelating moiety, (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, (iii) a moiety carrying a covalently bound radioisotope, or a precursor suitable to be labeled with such radioisotopes, (iv) a cytotoxic moiety, and (v) a fluorescent moiety; and R.sup.3 is H or I.
17. The compound or salt of claim 16, wherein the compound has the formula (Ia): ##STR00075## and wherein R.sup.2 and R.sup.3 are defined as in claim 16.
18. The compound or salt of claim 16, wherein the compound has the formula (Ib): ##STR00076## and wherein R.sup.2 and R.sup.3 are defined as in claim 16.
19. The compound or salt of claim 16, wherein R.sup.B is substituted linear C1-6 alkyl, which is substituted at its terminal carbon with one group selected from —NH.sub.2 and —NH—C(═NH)—NH.sub.2.
20. The compound or salt of claim 16, wherein R.sup.2 has the formula (IIIa) ##STR00077## wherein R.sup.A, R.sup.B, R.sup.D and R.sup.E are defined as in claim 16.
21. The compound or salt of claim 16, wherein R.sup.E is selected from (i) a group which comprises a chelating moiety, and (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, and wherein the chelating moiety comprises at least one of a macrocyclic ring structure with 8 to 20 ring atoms of which 2 or more are selected from oxygen atoms, sulfur atoms and nitrogen atoms; and an acyclic, open chain chelating structure with 8 to 20 main chain atoms of which 2 or more are heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms.
22. A pharmaceutical composition comprising or consisting of a compound or salt of claim 16 and an excipient.
23. A method of diagnosing, preventing, or treating a disease or disorder in a patient in need thereof, comprising administering to the patient a compound or salt of claim 16.
24. The method of claim 23, wherein the method further comprises the use of nuclear medicine, nuclear molecular imaging, optical imaging, or targeted endoradiotherapy.
25. The method of claim 23, wherein the method is further defined as treating or preventing a disease or disorder associated with increased expression of chemokine receptors subtype 4 (CXCR4).
26. The method of claim 23, wherein the method is further defined as diagnosing cancer or cardiovascular disease.
27. The method of claim 25, wherein the disease is cancer or a lymphoproliferative disease.
28. The method of claim 23, wherein the disease or disorder is cardiovascular disease, AIDS, or an inflammatory disorder.
Description
(1) The figures show.
(2)
(3)
(4)
(5)
(6)
(7)
(8) The examples illustrate the invention.
EXAMPLE 1
(9) Materials and Methods
(10) 1. Chemicals and Instrumentation
(11) Solvents were purchased from Aldrich, Fluka, Merck and Prolabo, reagents from Aldrich, Fluka, Sigma, Merck, Acros and Lancaster in the quality “for synthesis”, “per analysis” or “HPLC grade” and used without purification. Amino acids, derivatives for protecting groups as well as coupling reagents were purchased from Iris Biotech (Marktredwitz, Germany), Bachem (Bubendorf, Switzerland) or Carbolution (Saarbrücken, Germany). All reaction sensitive to oxygen or water were carried out in an argon atmosphere.
(12) Solid phase peptide synthesis was carried out in plastic syringes (VWR) which were equipped with a filter. Reaction and wash solutions were sucked in the syringe, for mixing, the syringes were shaken manually or using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). For loading of the first amino acid, the required equivalents (eq.) were calculated based on the theoretical loading capacity provided by the supplier.
(13) Thin layer chromatography (TLC) for reaction control and the determination of R.sub.f-values was done on aluminium foils coated with Silica 60, F254 (Merck). Peak detection was done under UV-light (254 nm) or after staining with Mostain-solution, respectively.
(14) Preparative flash chromatogaphy was performed with 50- to 100-fold mass excess Silica 60 (particle size 0.040-0.063 mm, Merck) applying 1-1.5 bar overpressure.
(15) Analytical and semipreparative reversed phase high performance liquid chromatography (HPLC) was performed using the following devices:
(16) a) Sykam: gradient HPLC System (Sykam GmbH, Eresing, Germany), 206 PHD UV-Vis detector (Linear™ Instruments Corporation, Reno, USA), Winnie 32 software. Columns: a) Nucleosil 100 C18 (5 μm, 125×4.0 mm) (CS Chromatographie Services GmbH, Langerwehe, Germany), analytical, and b) Multospher 100 RP 18-5 (250×20 mm) (CS), semipreparative. For radioactivity detection, the outlet of the UV-photometer was connected to a Nal(TI) well-type scintillation counter from EG&G Ortec (MOnchen, Germany). b) Shimadzu: Prominence Gradient HPLC System (Shimadzu, Duisburg, Germany).
(17) As eluents, mixtures of H.sub.2O (solvent A) and acetonitrile (solvent B) containing 0.1 vol-% TFA were used. Different, linear gradient profiles in 15-30 min (analytical) and 15-30 min (semipreparative) were applied. Flow rates were 1 mL/min (analytical) and 8-9 mL/min (semipreparative), respectively. UV-detection was carried out at 220 and 254 nm.
(18) Electrospray ionization mass spectrometry, ESI-MS was performed using
(19) a) a device from Finnigan (Typ LCQ in combination with the HPLC-system Hewlett Packard HP 1100). Columns: a) YMC Hydrosphere C18 (120 Å, 3 μm, 125 mm×2.1 mm), flow rate: 0.55 mL/min; b) YMC-UltraHT-Hydrosphere C18 (120 Å, 2 μm, 50 mm×2.0 mm), flow rate: 0.75 mL/min; c) YMC Octyl C8 (120 Å, 5 μm, 250 mm×2.1 mm), flow rate: 0.35 mL/min. As eluents, mixtures of H.sub.2O and acetonitrile containing 0.1 vol-% formic acid were used for different linear gradients (7 min, 15 min, 40 min). or b) a Varian 500-MS IT mass spectrometer (Agilent Technologies, Santa Clara, USA).
(20) Fluorescence Microscopy experiments were carried out using a BioRevo BZ9000 Fluorescence Microscope (Keyence, Osaka, Japan).
(21) 2. Synthesis
(22) 2.1. General Procedures
(23) GP1. Loading of Tritylchloridpolystyrene (TCP) Resin
(24) Peptide synthesis was carried out using TCP-resin (0.9 mmol/g) following standard Fmoc-strategy. Fmoc-Xaa-OH (1.2 eq.) were attached to the TCP resin with DIEA (2.5 eq.) in anhydrous DCM (0.8 mL/g resin) at room temperature for I h. The remaining trityl chloride groups were capped by addition of 1 mL/g(resin) of a solution of MeOH, DIEA (5:1; vzv) for 15 min. The resin was filtered and washed 5 times with DCM and 3 times with MeOH. The loading capacity was determined by weight after drying the resin under vacuum and ranged from 0.4-0.9 mmol/g.
(25) GP2. Fmoc Deprotection
(26) The resin-bound Fmoc peptide was treated with 20% piperidine in NMP (v/v) for 10 minutes and a second time for 5 minutes. The resin was washed 5 times with NMP.
(27) GP3. N-Methylation Under Mitsunobu Conditions A solution of triphenylphosphine (5 eq.), DIAD (5 eq.) and MeOH (10 eq.) in dry THF (I mL/g resin) was added to the resin bound Ns protected peptides and shaken at room temperature for 10 minutes. The resin was filtered off, and washed 3 times with dry THF and 3 times with NMP.
(28) GP4. HATU/HOAt Coupling
(29) A solution of Fmoc-Xaa-OH, HATU (2 eq.), HOAt (2 eq.), DIPEA (4 eq.) in NMP (1 mL/g resin) was added to the resin bound peptides and shaken for 3 hours at room temperature and w ashed 5 times with NMP.
(30) GP5. On-Resin Ns Deprotection
(31) For Ns deprotection, the resin-bound Ns-peptides were stirred in a solution of inercaptoethanol (10 eq.) and DBU (5 eq.) in NMP (I mL/g resin) for 5 minutes. The deprotection procedure was repeated one more time and the resin was washed 5 times with NMP.
(32) GP6. Peptide Cleavage from Resin
(33) For complete cleavage from the resin the peptides were treated three times with a mixture of acetic acid/2,2,2-trifluoroethanol/DCM (3/1/6, v/v/v) at room temperature for half an hour and the solvents were evaporated under reduced pressure.
(34) GP7. Peptide Backbone Cyclization
(35) To a solution of peptide in DMF (I mM peptide concentration) and NaHCO.sub.3 (5 eq.), DPPA (3 eq.) was added at RT and stirred over night or until no linear peptide could be observed by ESI-MS. The solvent was evaporated to a small volume under reduced pressure and the peptides precipitated in saturated NaCl solution and washed two times in HPLC grade water.
(36) GP8. Removal of Dde Protecting Group
(37) Dde-protection was carried out using 2% hydrazine in DMF at room temperature. After 30 min, deprotected peptides were precipitated using water (Pbf/tBu/Boc-protected peptides) or diethyl ether (deprotected peptides) and dried in a desiccator before further functionalization.
(38) GP9. Removal of Acid Labile Side Chain Protectinq Groups
(39) Cyclized peptides were stirred in a solution of TFA, water and TIPS (95:2.5:2.5; v:v:v) at room temperature for one hour or until no more protected peptide could be observed by ESI-MS and subsequently precipitated in diethyl ether, washed twice with diethyl ether and dried.
(40) GP10. N-Methylation Under Mitsonobu Conditions
(41) A solution of triphenylphosphine (5 eq.), DIAD (5 eq.) and MeOH (10 eq.) in dry THF (1 ml/g resin) was added to the resin-bound o-Ns protected peptides and shaken for 10 min at room temperature. The resin was filtered off, and washed 3 times with dry THE and 3 times with NMP.
(42) GP11. Conjugation of Free Amino Function with Unprotected DOTA
(43) DOTA (4 eq.), NHS (5 eq.) and EDCI (5 eq.) are dissolved in water, and DIPEA (8 eq.) are added. After 15 min, the respective peptide (1 eq.) is added in an equal volume of water. Progress of the coupling reaction is monitored using RP-HPLC. Upon completion of the reaction, the solvents are evaporated in vacuo. The residue is resuspended in methanol, the suspension is centrifuged, and the product dissolved in the methanolic supernatant is precipitated using diethyl ether, dried and purified using preparative RP-HPLC.
(44) GP12. Preparation of .sup.natGa-, .sup.natLu-, .sup.natBi- and .sup.natY-DOTA Reference Compounds
(45) For the preparation of the .sup.natGa- and .sup.natBi-complexes, equal volumes of a 2 mM solution of Ga(NO.sub.3).sub.3 or Bi(OAc).sub.3 in 1 M NaOAc buffer and a 2 mM aqueous solution of the respective peptide are mixed and heated to 95° C. for 30 min for .sup.natGa- and 15 min RT for .sup.natBi-complexation. The corresponding .sup.natLu and .sup.natY complexes are prepared by adding a 2.5-molar excess of the respective metal chloride dissolved in water to the peptide. Upon heating to 95° C. for 30 min, formation of the respective metal complexes is confirmed using RP-HPLC and ESI-MS.
(46) GP13. Solution Phase Coniuaation of Peptide Linker and the Cyclicpentapeptide Scaffold
(47) A solution of Fmoc-Linker (1.5 eq.), TBTU (1.5 eq), HOBt (1.5 eq.) and DIPEA (3 eq.) in DMF was added to a solution of D-Orn-Dde-deprotected peptide in DMF (1 eq) and stirred for 90 min at RT. The product was then precipitated in saturated NaCl solution and washed two times in HPLC grade water.
(48) GP14. Removal of Acid Labile Side Chain Protecting Groups
(49) The side chain protected peptide was stirred in a solution of TFA, water and TIPS (95:2.5:2.5; v:v:v) at RT for one hour or until no more protected peptide could be observed by ESI-MS and precipitated in diethyl ether and washed two more times.
(50) 2.2. Synthesis of Cyclic Pentapeptide Analogs R and R1
(51) CPCR4 (R)
(52) CPCR4 (c[yorn′RNaIG] (R) was prepared as described previously.sup.1. Briefly, TCP-resin is loaded with Fmoc-Gly-OH according to GP1 and the linear peptide H-d-Orn-R(Pbf)-Nal-G is synthesized according to standard Fmoc-procedure (GP2 and GP4, respectively). The peptide is subsequently methylated according to GP10 and Fmoc-d-Tyr(OtBu)-OH (GP4) is coupled to the Ns-deprotected peptide. After deprotection (GP2), cleavage from the resin (GP6) and backbone cyclization (GP7) are carried out. Removal of the Dde-protecting group (GP8) is followed by precipitation of the crude peptide in sat. aq. NaCl-solution and lyophilization from a .sup.tBuOH/H.sub.2O-solution. R (450 mg, 0.64 mmol, 64%) is obtained as yellowish powder (purity>90%).
(53) c[yorn′RNaIG) (R)
(54) ##STR00025##
(55) HPLC (10-50% 15 min): t.sub.R=9.7 min. MS (ESI): m/z=702.4 [M+H].sup.+.
(56) Iodo-CPCR4 (R1)
(57) Synthesis of the unlabeled reference compound iodo-CPCR4.3 was carried out using N-iodosuccinimide (NIS) in acetonitrile/water.sup.2 Briefly, CPCR4 was dissolved in a 1:1 (v/v) mixture of acetonitrile and water to yield a 9 mM solution, and 0.45 eq NIS were added. Upon completion of the reaction (5-10 min at RT), iodo-CPCR4 (R1) was isolated using semipreparative RP-HPLC.
(58) c[iyorn′RNaIG) (R1)
(59) ##STR00026##
(60) HPLC (15-55% B in 15 min): t.sub.R=8 min; MS (ESI): m/z=829.4 [M+H].sup.+.
(61) FC131 (R2)
(62) The synthesis of FC131 (R2) was performed according to the protocol described for peptide R.
(63) c[yRRNaIG] (R2)
(64) ##STR00027##
(65) HPLC (15-55% B in 15 min): t.sub.R=8 min; MS (ESI): m/z=829.4 [M+H].sup.+.
(66) 2.3. Synthesis of linking units (L1-L5) for conjugation with the D-Orn-side chain of R or R1 TCP-resin is loaded with Fmoc-ABS-OH or (Fmoc-AMBS-OH) according to GP1 and the linear peptide chains are constructed according to standard Fmoc-procedure (GP2 and GP4, respectively). After cleavage from the resin (GP6) as the Fmoc-protected derivative, the peptide linking units are used without further purification.
(67) Fmoc-dDap(Boc)-Gly-ABS (L1)
(68) Resin bound 4-(Fmoc-amino)benzoic acid (0.224 mmol, 1.0 eq.) were allowed to pre swell for 30 min in DMF. 3.0 eq. Fmoc-Gly-OH and 1.5 eq. of Fmoc-D-Dap(Boc)-OH were coupled according to GP2 and GP4, respectively). The linear peptide was cleaved from the resin (GP6), precipitated in diethyl ether and freeze-dried overnight to give the linking unit L1.
(69) Fmoc-dDap(Boc)-Gly-ABS (R)-4-(2-(2,3-diaminopropanamido)acetamido)benzoic acid (L1)
(70) ##STR00028##
(71) HPLC (10-90% B in 15 min): t.sub.R=12.0 min; MS (ESI): m/z=502.5 [M+H-(Boc)].sup.+.
(72) Fmoc-dLys(Boc)-Gly-ABS (L2)
(73) Resin bound 4-(Fmoc-amino)benzoic acid (0.224 mmol, 1.0 eq.) were allowed to pre swell for 30 min in DMF. 3.0 eq. Fmoc-Gly-OH and 1.5 eq. of Fmoc-D-Lys(Boc)-OH were coupled according to GP2 and GP4, respectively). The linear peptide was cleaved from the resin (GP6), precipitated in diethyl ether and freeze-dried overnight to give the linking unit L2.
(74) Fmoc-dLys(Boc)-Gly-ABS (L2)
(75) ##STR00029##
(76) HPLC (10-90% B in 15 min): t.sub.R=13.0 min; MS (ESI): m/z=545.4 [M+H-(Boc)].sup.+.
(77) Fmoc-dArq(Pbf)-Gly-ABS (L3)
(78) Resin bound 4-(Fmoc-amino)benzoic acid (0.224 mmol, 1.0 eq.) were allowed to pre swell for 30 min in DMF. 3.0 eq. Fmoc-Gly-OH and 1.5 eq. of Fmoc-D-Arg(Pbf)-OH were coupled according to GP2 and GP4, respectively). The linear peptide was cleaved from the resin (GP6), precipitated in diethyl ether and freeze-dried overnight to give the linking unit.
(79) Fmoc-dArg(Pbf)-Gly-ABS (L3)
(80) ##STR00030##
(81) HPLC (10-90% B in 15 min): t.sub.R=12.6 min; MS (ESI): m/z=573.5 [M+H-(Pbf)].sup.+.
(82) Fmoc-dArg(Pbf)-dAla-ABS (L4)
(83) Resin bound 4-(Fmoc-amino)benzoic acid (0.224 mmol, 1.0 eq.) were allowed to pre swell for 30 min in DMF. 3.0 eq. Fmoc-D-Ala-OH and 1.5 eq. of Fmoc-D-Arg(Pbf)-OH were coupled according to GP2 and GP4, respectively). The linear peptide was cleaved from the resin (GP6), precipitated in diethyl ether and freeze-dried overnight to give the linking unit.
(84) Fmoc-dArg(Pbf)-dAla-ABS (L4)
(85) ##STR00031##
(86) HPLC (10-90% B in 15 min): t.sub.R=11.6 min; MS (ESI): m/z=587.5 [M+H-(Pbf)].sup.+.
(87) Fmoc-dArq(Pbf)-dAla-AMBS (L5)
(88) Resin bound 4-(Fmoc-amino)methylbenzoic acid (0.224 mmol, 1.0 eq.) were allowed to pre swell for 30 min in DMF. 3.0 eq. Fmoc-D-Ala-OH and 1.5 eq. of Fmoc-D-Arg(Pbf)-OH were coupled according to GP2 and GP4, respectively). The linear peptide was cleaved from the resin (GP6), precipitated in diethyl ether and freeze-dried overnight to give the linking unit.
(89) Fmoc-dArg(Pbf)-dAla-AMBS (L5)
(90) ##STR00032##
(91) HPLC (10-90% B in 15 min): t.sub.R=11.6 min; MS (ESI): m/z=587.5 [M+H-(Pbf)].sup.+.
(92) 2.4. Synthetic Description of the Individual Compounds (P1-P6)
(93) TCP-resin is loaded with Fmoc-Gly-OH according to GPI and the linear peptide H-d-Ala-R(Pbf)-Nal-G is synthesized according to standard Fmoc-procedure (GP2 and GP4, respectively). The peptide is subsequently alkylated with Dde-aminohexanol according to GP10 and Fmoc-d-Tyr(OtBu)-OH (GP4) is coupled to the Ns-deprotected peptide. After deprotection (GP2), cleavage from the resin (GP6) and backbone cyclization (GP7) are carried out. Removal of the Dde-protecting group (GP8) is followed by precipitation of the crude peptide in sat. aq. NaCk-solution and lyophilization from a ACN/H.sub.2O-solution. PO (132 mg, 126 μM, 31%) is obtained as yellowish powder (purity>90%). The respective scaffold R or R1 was conjugated with linking units (L1-L5) according to GP13. Subsequent condensation of the chelator is performed according to GP11.
(94) cyclo[i-yorn′(DOTA-d-(4-amino)benzoyl-)RNaIG (P1)
(95) Synthesis of the respective Dde-deprotected, cyclic peptide R1 was carried out according to the general procedures outlined above. Fmoc-ABS and Fmoc-DAsp(tBu) was coupled according to GP4 and GP2. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(96) cyclo[i-yorn′(DOTA-d-(4-amino)benzoyl-)RNaIG (P1)
(97) ##STR00033##
(98) HPLC (25-55% B in 15 min): t.sub.R=10.1 min; MS (ESI): m/z=1448.8 [M+H].sup.+, 725.3 [M+H+H].sup.2+.
(99) cyclo[i-yorn′(DOTA-dapG-(4-amino)benzoyl-)RNaIG (P2):
(100) R1 was coupled to L1 according to the general procedures GP13. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(101) cyclo[i-yorn′(DOTA-dapG-(4-amino)benzoyl-)RNaIG (P2):
(102) ##STR00034##
(103) HPLC (25-65% B in 15 min): t.sub.R=11.9 min; MS (ESI):m/z=1476.8 [M+H].sup.+, 739.3 [M+H+H].sup.2+.
(104) cyclo[i-yorn′(DOTA-kG-(4-amino)benzoyl-)RNaIG (P3):
(105) R1 or R was coupled to L2 according to the general procedures GP13. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(106) cyclo[i-yorn′(DOTA-kG-(4-amino)benzoyl-)RNaIG (P3):
(107) ##STR00035##
(108) cyclo[i-yorn′(DOTA-kG-(4-amino)benzoyl-)RNaIG (P3b):
(109) ##STR00036##
(110) HPLC (25-55% R in 15 min): t.sub.R=12.8 min: HPLC (15-55% B in 15 min): t.sub.R=7.9 min: MS MS (ESI): m/z=1519.1 [M+H].sup.+, 760.4 (ESI): m/z=1393.2 [M+H].sup.+. [M+H+H].sup.2+.
(111) cyclo[i-yorn′(DOTA-rG-(4-amino)benzoyl-)RNaIG (P4):
(112) R1/R was coupled to L3 according to the general procedures GP13. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(113) ##STR00037##
(114) cyclo[i-yorn′(DOTA-ra-(4-amino)benzoyl-)RNaIG (P5):
(115) R1/R was coupled to L4 according to the general procedures GP13. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(116) ##STR00038##
(117) cyclo[i-yorn′(DOTA-ra-(4-amino)methylbenzoyl-)RNaIG (P6):
(118) R1/R was coupled to L5 according to the general procedures GP13. Upon Fmoc-deprotection using 20% piperidine in DMF (GP2), the peptide was purified using preparative RP-HPLC and DOTA was conjugated according to GP11. Again, the peptide was purified using preparative RP-HPLC.
(119) cyclo[-yorn′(DOTA-ra-(4-amino)methylbenzoyl-)RNaIG (P6b):
(120) ##STR00039##
(121) HPLC (15-55% B in 15 min): t.sub.R=8.0 min; MS
(122) (ESI): m/z=1435.1 [M+H].sup.+, 717.3 [M+H+H].sup.2+.
(123) .sup.natGa-compounds: .sup.natGa.sup.III-chelate formation was achieved using the protocol GP12. The resulting 1 mM aqueous solutions of the respective .sup.natGa-complexes were diluted (serial dilution 10.sup.−4 to 10.sup.−11 M in Hanks salt solution (HBSS) with 1% BSA) and used in the in vitro IC.sub.50 studies without further processing.
(124) cyclo[i-yorn′([.sup.natGa]DOTA-dapG-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P2): HPLC (25% to 65% B in 15 min): t.sub.R=11.4 min; K′=5.3; Calculated monoisotopic mass (C.sub.64H.sub.84GalN.sub.17O.sub.16): 1542.46, found by ESI-MS: m/z=1544.8 [M+H].sup.+, 772.4 [M+2H].sup.2+.
(125) cyclo[yorn′([.sup.natGa]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P3b): HPLC (15% to 55% B in 15 min): t.sub.R=7.6 min; K′=3.0; Calculated monoisotopic mass (C.sub.67H.sub.91GaN.sub.17O.sub.16): 1458.61, found by ESI-MS: m/z=1459.8 [M+H].sup.+, 730.2 [M+2H].sup.2+.
(126) cyclo[i-yorn′([.sup.natGa]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P3): HPLC (25% to 55% B in 15 min): t.sub.R=12.9 min; K′=6.1; Calculated monoisotopic mass (C.sub.67H.sub.90GalN.sub.17O.sub.16): 1584.51, found by ESI-MS: m/z=1585.0 [M+H].sup.+, 1608.9 [M+Na].sup.+, 793.7 [M+2H].sup.2+.
(127) cyclo[yorn′([.sup.natGa]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P4b): HPLC (15% to 55% B in 15 min): t.sub.R=7.7 min; K′=3.2; Calculated monoisotopic mass (C.sub.67H.sub.91GaN.sub.19O.sub.16): 1468.61, found by ESI-MS: m/z=745.3 [M+2H].sup.2+.
(128) cyclo[i-yorn′([.sup.natGa]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P4): HPLC (25% to 55% B in 15 min): t.sub.R=12.6 min; K′=6.8; Calculated monoisotopic mass (C.sub.67H.sub.90GalN.sub.19O.sub.16): 1612.51, found by ESI-MS: m/z=1614.9 [M+H].sup.+, 808.0 [M+2H].sup.2+.
(129) cyclo[yorn′([.sup.natGa]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P5b): HPLC (15% to 55% B in 15 min): t.sub.R=6.8 min; K′=3.5; Calculated monoisotopic mass (C.sub.68H.sub.93GaN.sub.19O.sub.16): 1500.63, found by ESI-MS: m/z=751.5 [M+2H].sup.2+, 787.2 [M+2K].sup.2+.
(130) cyclo[i-yorn′([.sup.natGa]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natGa]P5): HPLC (25% to 55% B in 15 min): t.sub.R=7.9 min; K′=3.9; Calculated monoisotopic mass (C.sub.68H.sub.92GalN.sub.19O.sub.16): 1626.53, found by ESI-MS: m/z=1626.8 [M+H].sup.+, 815.0 [M+2H].sup.2+.
(131) cyclo[i-yorn′([.sup.natGa]DOTA-ra-(4-amino)methylbenzoyl-)RNaIG ([.sup.natGa]P6b): HPLC (25% to 55% B in 15 min): t.sub.R=7.9 min; K′=3.9; Calculated monoisotopic mass (C.sub.68H.sub.92GalN.sub.19O.sub.16): 1626.53, found by ESI-MS: m/z=1626.8 [M+H].sup.+, 815.0 [M+2H].sup.2+.
(132) .sup.natLu-compounds: .sup.natLu.sup.III-chelate formation was achieved using the protocol GP12. The resulting 1 mM aqueous solutions of the respective .sup.natLu-complexes were diluted (serial dilution 10.sup.−4 to 10.sup.11 M in HBSS with 1% BSA) and used in the in vitro IC.sub.50 studies without further processing.
(133) cyclo[i-yorn′([.sup.natLu]DOTA-d-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P1): HPLC (35% to 65% B in 15 min): t.sub.R=8.0 min; K′=3.4; Calculated monoisotopic mass (C.sub.63H.sub.79ILuN.sub.15O.sub.17): 1619.42, found by ESI-MS: m/z=1620.9 [M+H].sup.+, 1,642.8 [M+Na].sup.+, 811.2 [M+2H].sup.2+, 822.0 [M+H+Na].sup.2+.
(134) cyclo[i-yorn′([.sup.natLu]DOTA-dapG-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P2): HPLC (25% to 65% B in 15 min): t.sub.R=10.8 min; K′=9.8; Calculated monoisotopic mass (C.sub.64H.sub.83ILuN.sub.17O.sub.16): 1647.47, found by ESI-MS: m/z=1648.9 [M+H].sup.+, 825.2 [M+2H].sup.2+, 860.5 [M+2K].sup.2+.
(135) cyclo[yorn′([.sup.natLu]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P3b): HPLC (15% to 55% B in 15 min): t.sub.R=7.5 min; K′=4.0; Calculated monoisotopic mass (C.sub.67H.sub.90LuN.sub.17O.sub.16): 1563.62, found by ESI-MS: m/z=1565.4 [M+H].sup.+.
(136) cyclo[i-yorn′([.sup.natLu]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P3): HPLC (25% to 55% B in 15 min): t.sub.R=13.1 min; K′=7.7; Calculated monoisotopic mass (C.sub.67H.sub.89ILuN.sub.17O.sub.16): 1689.51, found by ESI-MS: m/z=1691.8 [M+H].sup.+, 1712.7 [M+Na].sup.+, 846.4 [M+2H].sup.2+, 857.1 [M+H+Na].sup.2+.
(137) cyclo[yorn′([.sup.natLu]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P4b): HPLC (15% to 55% B in 15 min): t.sub.R=7.2 min; K′=4.1; Calculated monoisotopic mass (C.sub.67H.sub.90LuN.sub.19O.sub.16): 1591.62, found by ESI-MS: m/z=1592.5 [M+H].sup.+, 1631.0 [M+K].sup.+.
(138) cyclo[i-yorn′([.sup.natLu]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P4): HPLC (25% to 55% B in 15 min): t.sub.R=13.4 min; K′=7.9; Calculated monoisotopic mass (C.sub.67H.sub.89IN.sub.19O.sub.16): 1717.52, found by ESI-MS: m/z=1719.9 [M+H].sup.+, 860.5 [M+2H].sup.2+, 871.1 [M+H+Na].sup.2+, 895.7 [M+2K].sup.2+.
(139) cyclo[yorn′([.sup.natLu]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P5b): HPLC (15% to 55% B in 15 min): t.sub.R=7.5 min; K′=4.0; Calculated monoisotopic mass (C.sub.68H.sub.92LuN.sub.19O.sub.16): 1605.64, found by ESI-MS: m/z=1732.8 [M+H].sup.+, 867.3 [M+2H].sup.2+, 902.6 [M+2K].sup.2+.
(140) cyclo[i-yorn′([.sup.natLu]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natLu]P5): HPLC (25% to 55% B in 15 min): t.sub.R=6.5 min; K′=2.3; Calculated monoisotopic mass (C.sub.68H.sub.91LuN.sub.19O.sub.16): 1731.53, found by ESI-MS: m/z=1732.8 [M+H]+, 867.3 [M+2H].sup.2+, 902.6 [M+2K].sup.2+.
(141) The .sup.natY-complexes were prepared as described in GP12. After cooling, the .sup.natY.sup.III-chelate formation was confirmed using HPLC and MS. The resulting 1 mM aqueous solutions of the respective .sup.natY-complexes were diluted (serial dilution 10.sup.−4 to 10.sup.11 M in HBSS with 1% BSA) and used in the in vitro IC.sub.50 studies without further processing.
(142) cyclo[i-yorn′([.sup.natY]DOTA-dapG-(4-amino)benzoyl-)RNaIG ([.sup.natY]P2): HPLC (25% to 65% B in 15 min): t.sub.R=11.5 min; K′=6.1; Calculated monoisotopic mass (C.sub.64H.sub.83IN.sub.17O.sub.16Y): 1561.43, found by ESI-MS: m/z=1562.7 [M+H].sup.+, 782.3 [M+2H].sup.2+, 793.0 [M+H+Na].sup.2+
(143) cyclo[yorn′([.sup.natY]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natY]P3b): HPLC (15% to 55% B in 15 min): t.sub.R=7.8 min; K′=2.7; Calculated monoisotopic mass (C.sub.67H.sub.90N.sub.17O.sub.16Y): 1477.58, found by ESI-MS: m/z=1478.9 [M+H].sup.+, 740.4 [M+2H].sup.2+.
(144) cyclo[i-yorn′([.sup.natY]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natY]P3): HPLC (25% to 55% B in 15 min): t.sub.R=12.9 min; K′=7.6; Calculated monoisotopic mass (C.sub.67H.sub.89IN.sub.17O.sub.16Y): 1603.48, found by ESI-MS: m/z=1605.7 [M+H].sup.+, 1626.6 [M+Na].sup.+, 803.4 [M+2H].sup.2+, 814.1 [M+H+Na].sup.2+.
(145) cyclo[yorn′([.sup.natY]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natY]P4b): HPLC (15% to 55% B in 15 min): t.sub.R=8.0 min; K′=2.8; Calculated monoisotopic mass (C.sub.67IHON.sub.19O.sub.16Y): 1505.59, found by ESI-MS: m/z=1506.2 [M+H].sup.+, 754.3 [M+2H].sup.2+.
(146) cyclo[i-yorn′([.sup.natY]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natY]P4): HPLC (25% to 55% B in 15 min): t.sub.R=13.3 min; K′=7.9; Calculated monoisotopic mass (C.sub.67H.sub.89IN.sub.19O.sub.16Y): 1631.48, found by ESI-MS: m/z=1632.9 [M+H].sup.+, 817.4 [M+2H].sup.2+, 828.1 [M+H+Na].sup.2+, 852.7 [M+2K].sup.2+.
(147) cyclo[yorn′([.sup.natY]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natY]P5b): HPLC (15% to 55% B in 15 min): t.sub.R=7.0 min; K′=3.6; Calculated monoisotopic mass (C.sub.68H.sub.92N.sub.19O.sub.16Y): 1519.60, found by ESI-MS: m/z=761.0 [M+2H].sup.2+.
(148) cyclo[i-yorn′([.sup.natY]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natY]P5): HPLC (25% to 55% B in 15 min): t.sub.R=9.6 min; K′=2.8; Calculated monoisotopic mass (C.sub.68H.sub.91IN.sub.19O.sub.16Y): 1645.50, found by ESI-MS: m/z=1646.9 [M+H].sup.+, 824.1 [M+2H].sup.2+, 859.5 [M+2K].sup.2+.
(149) Bismuth complexation was performed using the protocol described in GP12. Formation of the .sup.natBi.sup.III-chelate was confirmed using HPLC and ESI-MS. The resulting 1 mM aqueous solutions of the respective .sup.natBi-complexes were diluted (serial dilution 10.sup.−4 to 10.sup.−10 M in HBSS with 1% BSA) and used in the in vitro IC.sub.50 studies without further processing.
(150) cyclo[i-yorn′([.sup.natBi]DOTA-dapG-(4-amino)benzoyl-)RNaIG ([.sup.natBi]P2): HPLC (15% to 55% B in 15 min): t.sub.R=8.9 min; K′=3.9; Calculated monoisotopic mass (C.sub.64H.sub.83BiIN.sub.17O.sub.16): 1681.51, found by ESI-MS: m/z=1682.2 [M+H].sup.+, 839.8 [M+2H].sup.2+.
(151) cyclo[i-yorn′([.sup.natBi]DOTA-kG-(4-amino)benzoyl-)RNaIG ([.sup.natBi]P3): HPLC (15% to 55% B in 15 min): t.sub.R=8.9 min; K′=3.3; Calculated monoisotopic mass (C.sub.67H.sub.89BiN.sub.17O.sub.16): 1724.56, found by ESI-MS: m/z=1726.2 [M+H].sup.+, 863.2 [M+2H].sup.2+.
(152) cyclo[i-yorn′([.sup.natBi]DOTA-rG-(4-amino)benzoyl-)RNaIG ([.sup.natBi]P4): HPLC (15% to 55% B in 15 min): t.sub.R=9.0 min; K′=3.7; Calculated monoisotopic mass (C.sub.67H.sub.89BiN.sub.19O.sub.16): 1751.56, found by ESI-MS: m/z=1752.3 [M+H].sup.+.
(153) cyclo[i-yorn′([.sup.natBi]DOTA-ra-(4-amino)benzoyl-)RNaIG ([.sup.natBi]P5): HPLC (15% to 55% B in 15 min): t.sub.R=9.1 min; K′=4.0; Calculated monoisotopic mass (C.sub.68H.sub.91BiIN.sub.19O.sub.16): 1765.57, found by ESI-MS: m/z=1767.5 [M+H].sup.+, 885.1 [M+2H].sup.2+.
(154) 2.5. Utilization of the Novel Linking Unit and Synthetic Description Thereof (F1-F4)
(155) 2.5.1 Synthesis of .sup.18F-Labeling Precursors for Conjugation with the D-Orn-Side Chain of R or R1
(156) TCP-resin is loaded with Fmoc-ABS-OH according to GP1 and the linear peptide chains are constructed according to standard Fmoc-procedure (GP2 and GP4, respectively). The Fmoc-deprotected N-terminus was converted to the respective azide according to a previously published procedure (Goddard-Borger and Stick, An efficient, inexpensive, and shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide hydrochloride. Organic letters 2007, 9 (19), 3797-3800). Briefly, imidazole-1-sulfonyl azide hydrochloride (1.5 eq.), CuSO.sub.4 and DIPEA were added to the resin bound peptide at 4° C. Cooling was continued for 2 h and the resin bound peptide was shaken overnight at RT. The peptide was cleaved from the resin according to GP6 and freeze-dried overnight. N-propargyl-N,N-dimethyl-ammoniomethylboronylpinacolate (1.5 eq.) was combined with KHF.sub.2 (3 M solution in water, 2.6 eq.) and 3.6 eq. HCl (4 M solution in water) and heated to 45° C. for 2 h. After addition of 1.2 mL of NH.sub.40H (1 M in water) to adjust the pH, the solution was added to the azide-peptide. The mixture was heated to 55° C. for 15 h and purified using preparative HPLC. The purified fragment was coupled to R or R1, respectively employing HOBt, TBTU and DIPEA under standard conditions (GP4).
(157) N-propargyl-N,N-dimethyl-ammoniomethylboronylpinacolate: o
(158) ##STR00040##
(159) The synthesis was performed according to a published procedure with small modifications (Liu et al., Kit-like 18 F-labeling of RGD-19 F-Arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging. Nuclear medicine and biology 2013, 40 (6), 841-849). Briefly, a dry round bottom flask was loaded with 53.8 μL (0.5 mmol, 1.0 eq.) of N,N-dimethylpropargylamine and 3 mL of dry DCM under a nitrogen atmosphere. 80.1 μL (0.5 mmol, 1.0 eq.) of iodomethyl-boronylpinacolate was added dropwise at RT. On stirring, the solution became cloudy and the white precipitated was filtered of after 2 h of vigorously stirring at 0° C. The precipitate was washed with ice cold diethyl ether two times and used without further purification. 104 mg (0.45 mmol, 93%) were collected as a white solid. Calculated monoisotopic mass (C.sub.12H.sub.23BNO.sub.2.sup.+): 224.18, found by ESI-MS: m/z=224.2 [M].sup.+.
(160) .sup.1H-NMR (400 MHz [Bruker], CD.sub.3CN): δ [ppm]=1.31 (s, 12H), 3.17 (s, 6H), 3.21 (t, 2H), 3.23 (s, 1H), 4.22 (d, 2H). .sup.13C-NMR (101 MHz [Bruker], CD.sub.3CN): δ [ppm]=24.89.
(161) ##STR00041##
(162) imidazole-1-sulfonyl azide hydrochloride: The synthesis was performed according to a published protocol (Hansen et al., Simple and efficient solid-phase preparation of azido-peptides. Organic letters 2012, 14 (9), 2330-2333). Briefly, sulfonylchloride (1.62 mL, 20.0 mmol, 1.0 eq.) was added drop-wise to an ice-cooled suspension of NaN.sub.3(1.3 g, 20.0 mmol, 1.0 eq.) in MeCN (20 mL) and the mixture was stirred overnight at RT. While stirring vigorously, imidazole (2.5 g, 38.0 mmol, 2.0 eq.) was added carefully to the ice-cooled solution and stirred for 3 h at RT. The solution was diluted with EtOAc (40 mL) and washed with water (2×40 mL) and saturated aqueous NaHCO.sub.3 (2×20 mL), dried over MgSO.sub.4 and filtered. A fresh solution of HCl in EtOH (obtained through drop-wise addition of AcCl (10.0 mL) to ice-cooled dry ethanol (25 mL)) was added slowly to the filtrate while stirring at 0° C. The crystallized product was filtered off on ice and the white crystals were washed with ice cold EtOAc to yield colorless needles (2.0 g, 9.5 mmol, 48%). The compound was used without further purification and stored at −20° C.
(163) 2.5.2 Synthetic Description of the Individual Compounds (F1-F4)
cyclo[i-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)4-amino)benzoyl-)RNaIG] (F1) (Reference Compound)
(164) R1 was coupled with AMBF.sub.3-ABS-OH according to the general procedures GP4. The peptide was purified using preparative RP-HPLC.
cyclo[i-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)4-amino)benzoyl-)RNaIG] (F1)
(165) ##STR00042##
(166) HPLC (25-65% B in 15 min): t.sub.R=10.3 min; MS (ESI): m/z=1138.6 [M+H].sup.+.
cyclo[i-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)-vG-4-amino)benzoyl-)RNaIG] (F2) (Reference Compound)
(167) R1 was coupled with AMBF.sub.3-vG-ABS-OH according to the general procedures GP4. The peptide was purified using preparative RP-HPLC.
(168) ##STR00043##
cyclo[i-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)-rG-4-amino)benzoyl-)RNaIG] (F3)
(169) R1 was coupled with AMBF.sub.3-rG-ABS-OH according to the general procedures GP4. The peptide was purified using preparative RP-HPLC.
(170) ##STR00044##
cyclo[-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)-rG-4-amino)benzoyl-)RNaIG] (F3b)
(171) R was coupled with AMBF.sub.3-rG-ABS-OH according to the general procedures GP4. The peptide was purified using preparative RP-HPLC.
(172) ##STR00045##
cyclo[i-yorn′(AMBF.SUB.3.(methyl(1H-1,2,3-triazol-4-yl)-kG-4-amino)benzoyl-)RNaIG] (F4)
(173) R1 was coupled with AMBF.sub.3-rG-ABS-OH according to the general procedures GP4. The peptide was purified using preparative RP-HPLC.
(174) ##STR00046##
(175) 3. Radiolabeling
(176) 3.1. Radioiodination
(177) All peptides were radioiodinated using the lodoGen® method. Briefly, 100-200 μg of peptide were dissolved in 5-10 μL of DMSO. This solution was diluted with 0.5 mL TRIS iodination buffer (25 mM Tris.HCl, 0.4 M NaCl, pH 7.5) and transferred to an Eppendorf reaction tube coated with 150 μg of lodoGen®. Upon addition of [.sup.125I]Nal (18-20 MBq, Hartmann Analytic, Braunschweig, Germany) or [.sup.123l]Nal (220 MBq, GE Healthcare, Braunschweig, Germany), the reaction vessel was briefly vortexed and the labeling reaction was allowed to proceed for 15 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of the labeled products from unlabeled precursor was achieved using gradient RP-HPLC. For in vitro binding studies, the HPLC product fraction was used as such and diluted to the required concentration using the respective assay medium. For biodistribution experiments, the respective product fraction was diluted with water and passed onto a SepPak Plus C-18 cartridge (Waters, Eschborn, Germany). The cartridge was washed with water, and the immobilized radiopeptide was then eluted using 1 ml of acetonitrile. The solvent was removed by bubbling an argon stream through the radioligand solution at 90° C. for 20 min. The radioiodinated peptides were then reconstituted to an activity concentration of app. 1 MBq/100 μL using PBS and were used as such for the in vivo animal study.
(178) 3.2. .sup.68Ga-Labeling
(179) .sup.68Ga was obtained by elution of a .sup.68Ge/.sup.68Ga generator with SnO.sub.2 matrix (iTHEMBA LABS, South Africa) with 1 M HCl (5.5 mL) and immobilized on a strong cationic exchanger cartridge (SCX—Chromafix, size M, Macherey-Nagel, Duren, Germany).
(180) For animal studies, .sup.68Ga-pentixafor was prepared on a Gallelut.sup.+ system in analogy to a previously published .sup.68Ga labeling procedure (Notni, et al., TRAP, a powerful and versatile framework for gallium-68 radiopharmaceuticals. Chemistry 2011, 17 (52), 14718-22.) (SCINTOMICS GmbH, Germany). Briefly, .sup.68Ga-generator eluate fractions (1.25 mL, 600-800 MBq, buffered to pH 3.3 with 900 μL of a solution of 14.4 g HEPES in 12 mL water) were reacted with 3.5 nmol of the respective DOTA-peptide (P1 to P5) for 5 min. The radiochemical purity was always >99% as confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Addition of 1 mL PBS and concentration in vacuo to 1 mL total volume yielded solvent-free formulations with specific activities ranging from 100-150 MBq/nmol.
(181) 3.3. .sup.177Lu-Labeling
(182) For .sup.177Lu-labeling, the respective DOTA-peptides (P1 to P5) were dissolved in water to yield a 100 μM solution. Of this solution, the required volume was added to .sup.177LuC1.sub.3 in 0.04 M HCl (itg Isotope Technologies Garching, Garching, Germany; activity concentration: 370 MBq/500 μl) to achieve a peptide-to-.sup.177Lu-activity ratio of 0.75 nmol peptide per 25 MBq .sup.177LuCl.sub.3. To this mixture, 10 μL of 1 M NH.sub.40Ac was added, together with water to yield 100 μL total reaction volume. The solution was heated to 95° C. for 30 min. Upon cooling, the radiochemical purity was determined using thin layer chromatography (TLC) (usually >98%). For in vitro and in vivo studies, the reaction mixture was diluted with PBS to the desired activity concentration and used as such for the experiments.
(183) 4. Determination of Lipophilicity
(184) To a solution of app. 0.5 MBq of radiolabeled peptide in 500 μL of PBS (pH 7.4), 500 μL of octanol were added (n=6). Vials were vortexed vigorously for 3 min. To achieve quantitative phase separation, the vials were centrifuged at 14,600×g for 6 min in a Biofuge 15 (Heraeus Sepatech, Osterode, Germany). The activity concentrations in 100 μL-samples of both the aqueous and the organic phase were measured in a γ-counter. Both the partition coefficient Po.sub.w, which is defined as the molar concentration ratio of a single species A between octanol and an aqueous phase at equilibrium, and log Po.sub.w, which is an important parameter used to characterize lipophilicity of a compound, were calculated.
(185) 5. In Vitro Evaluation
(186) For in vitro experiments, the following cell lines were used: Jurkat human T-cell leukemia cells, Ep-Myc1080 mouse B-cell lymphoma cells and Chem-1. Jurkat cells were cultivated in RPMI 1640 medium (Biochrom, Germany) containing 10% fetal calf serum (FCS) (Biochrom, Germany). The human CXCR4 expressing cell-line Chemicon's Wild-Type (Chem-1) was cultured in DMEM medium (Biochrom, Germany) supplemented with 10% FCS, 1% non-essential amino acids (Biochrom, Germany) and 1% HEPES (1M). The murine CXCR4 expressing cell line Ep-Myc1080 (Donnou et al., Murine models of B-cell lymphomas: promising tools for designing cancer therapies. Adv Hematol. 2012; 2012:701704) was grown in RPMI 1640 medium supplemented with 20% FCS, 1% non-essential amino acids (Biochrom, Germany) and 0.1% 2-Mercaptoethanol (Sigma-Aldrich, Germany). All cell lines were cultured at 37° C. in a humidified atmosphere with 5% CO.sub.2 and passaged two to three times a week, depending on denseness of the cells.
(187) Receptor Affinity Assays
(188) Competition studies addressing the human CXCR4 were performed using CXCR4 positive Jurkat human T-cell leukemia cells and .sup.125I-FC131 (.sup.125I-2), addressing the murine CXCR4, Ep-Myc1080 mouse B-cell lymphoma cells and .sup.125I-CPCR4.3 (Demmer et al. A Conformationally Frozen Peptoid Boosts CXCR4 Affinity and Anti-HIV Activity. Angewandte Chemie International Edition. 2012; 51:8110-8113) as the radioligand were utilized as described previously (Poschenrieder et al., The influence of different metal-chelate conjugates of pentixafor on the CXCR4 affinity. EJNMMI research. 2016; 6:1-8).
(189) Internalization and Externalization Studies and Log P.sub.(octanol/PBS)
(190) Internalization and cell efflux studies of the respective ligands were performed using a previously published protocol (Poschenrieder et al., First 18F-Labeled Pentixafor-Based Imaging Agent for PET Imaging of CXCR4 Expression In Vivo. 2016). The distribution coefficients of the respective .sup.68Ga- and .sup.17Lu-labeled peptides in octanol and PBS buffer were determined applying the shake flask method as described previously (Weineisen et al., Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI Research. 2014; 1:1-15).
(191) 6. In Vivo Experiments
(192) Animal Model
(193) All animal experiments were conducted in accordance with general animal welfare regulations in Germany (Deutsches Tierschutzgesetz, approval #55.2-1-54-2532 71-13). The human B-lymphoblast cell line Daudi was suspended (v/v) 1:1 in serum-free medium and Matrigel (BD Biosciences, Germany) and approximately 10.sup.7 cells in 200 μL were inoculated subcutaneously on the right shoulder of 6 to 8 weeks old CB-17 SCID mice (Charles River, Germany). Tumors were grown for 2 to 4 weeks to reach 8 to 10 mm in diameter.
(194) Biodistribution
(195) .sup.177Lu-labeled CXCR4 ligands (approximately 5-10 MBq, 0.06 to 0.1 nmol) were injected into the tail vein of anesthetized animals. 6 h and 48 h after intravenous injection of approximately 0.15 mL of the radiolabeled peptide, the animals (n=4 for every time point) were sacrificed. The tissues and organs were weighted directly after preparation and the radioactivity was counted in a γ-counter. The % ID/g of each tissue was calculated and corrected with the activity found in the tail.
(196) μPET Imaging
(197) Imaging studies were performed at a Siemens Inveon small animal PET, followed by data analysis using the Inveon Research Workplace software. The animals were anesthetized with isoflurane and injected via the tail vein with 10 to 15 MBq (0.1 nmol) of tracer. Dynamic imaging was performed after on-bed injection for 1.5 h. Static images were recorded at 1 h p.i. with an acquisition time of 15 min. For competition experiments, animals were coinjected with 50 μg of AMD3100. Images were reconstructed using 3D ordered-subsets expectation maximum (OSEM3D) algorithm without scanner and attenuation correction.
EXAMPLE 2
(198) Chemistry
(199) The fully deprotected, cyclic CPCR4 binding scaffold (cyclo[D-Tyr-NMe-D-Orn-Arg-Nal-Gly](1), see
(200) Radiochemistry
(201) Preparation of .sup.68Ga-labeled peptides (5 nmol) was performed with a fully automated module (Scintomics, Germany) in high yields and specific activities of 100 to 120 GBq/μmol. .sup.177Lu-labeled peptides were obtained after incubation of the respective DOTA-peptide (0.75 nmol) with 20 MBq of .sup.177LuCl.sub.3 in 1.0 M NH.sub.40Ac buffer (calculated to be 10% of total reaction volume) and heating to 95° C. for 30 min, which yielded in specific activities of 26 GBq/μmol.
EXAMPLE 3
(202) CXCR4 Binding Affinity
(203) The binding affinities (IC.sub.50) of the respective .sup.natLu and .sup.natGa derivatives towards the human and murine CXCR4 (Table 1a) were determined in a competitive binding assay using Jurkat human T-cell leukemia cells or Ep-Myc1080 mouse B-cell lymphoma cells (4.0 and 2.0*10.sup.5 cells/well, 2 h, RT) and .sup.125I-2 or .sup.125I-CPCR4.3 (0.1 nM) as the radioligand. To be able to assess the effect of structural modifications in the SAR study on CXCR4 binding affinity, data for .sup.natGa-pentixafor (.sup.natGa-3) and .sup.natLu-pentixather (.sup.natLu-4) are also included in Table 1a.
(204) TABLE-US-00001 TABLE 1a Structural modifications of synthesized compounds and binding affinity to human and murine CXCR4 (IC.sub.50) of .sup.natLu and .sup.natGa-complexes (see FIG. 1). IC.sub.50 [nM] for IC.sub.50 [nM] for mIC.sub.50[nM] .sup.natLu-complexes .sup.natGa-complexes .sup.natLu complexes and human and human and murine R.sub.1 Xaa.sub.1 Xaa.sub.2 R.sub.2 CXCR4 CXCR4 CXCR4 2 — — — — 13.1 ± 5.1 119 ± 69 3 H —.sup.[a] — DOTA 41 ± 12 24.8 ± 2.5.sup.[a] >1000 4 I —.sup.[a] — DOTA 14.6 ± 1.0 6.1 ± 1.5 567 ± 62 5 I — — DOTA 12.5 ± 3.2 282 ± 90 — 6 I — — DOTAGA 28.3 ± 9 14.4 ± 0.3 — 7 I Gly — DOTA 5.9 ± 0.3 7.9 ± 1.1 — 8 I Gly — DOTAGA 38.8 ± 1.3 47.4 ± 8.1 — P1 I D-Asp — DOTA 106 ± 10 — — P2 I Gly D-Dap.sup.[b] DOTA 3.5 ± 0.3 3.6 ± 0.7 — P3b H Gly D-Lys DOTA 8.0 ± 3.1 8.9 ± 3.8 — P3 I Gly D-Lys DOTA 3.6 ± 1.1 2.4 ± 0.1 61.4 ± 17 P4b H Gly D-Arg DOTA 5.4 ± 1.6 9.7 ± 2.8 — P4 I Gly D-Arg DOTA 2.1 ± 0.3 1.4 ± 0.2 37.1 ± 2.9 P5b H D-Ala D-Arg DOTA 1.5 ± 0.1 0.4 ± 0.1 — P5 I D-Ala D-Arg DOTA 1.7 ± 0.6 2.6 ± 1.0 48.5 ± 0.5 Binding assays were performed using Jurkat cells (400,000/well) and ([.sup.125I]FC131) (c = 0.1 nM) as the radioligand for hCXCR4 and Eμ-Myc1080 mouse B-cell lymphoma cells and [.sup.125I]CPCR4.3 as radioligand. Cells were incubated in HBSS (1% BSA) at RT for 2 h. Data are expressed as mean ± SD (n = 3). .sup.[a]4-aminobenzoic acid spacer is substituted by 4-aminomethylbenzoic acid (see FIG. 1 (5-16), .sup.[b](R)-2,3-diaminopropanoic acid.
(205) As demonstrated by six-fold enhanced binding affinities towards hCXCR4 (7 vs 8), the chelator DOTA is preferred over DOTAGA and an additional glycine in the linker beneficially contributes to the binding affinity (12.5±3.2 nM for .sup.natLu-5 vs. 5.9±0.3 nM for .sup.natLu-7, respectively). Supplemental insertion of cationic amino acids in the linker further improves the binding affinities almost two-fold with 3.5±0.3 nM for .sup.natLu-P2. Mutation of the cationic amino acid at that position in combination with a final optimization step wherein glycine was substituted with D-alanine (P5), led to an additional almost two-fold increase in affinity compared to P2 (1.7±0.6 nM for .sup.natLu-P5). Finally, the optimized “linking unit” also allowed the utilization of the non-iodinated scaffold (1), which surprisingly also resulted in enhanced binding affinity (1.5±0.1 nM and 0.4±0.1 nM for .sup.natLu-P5b and .sup.natGa-P5b, respectively). Additionally, .sup.natLu-P4 and .sup.natLu-P5 showed dramatically improved binding affinities towards the murine receptor with 37.1±2.9 nM for .sup.natLu-P4 and 48.5±0.5 nM for .sup.natLu-P5 in comparison to 567±62 nM for .sup.natLu-4.
(206) Further affinity data for P1, P2, P3, P3b, P4, P4b, P5 and P5b and further compounds are given in Tables 1b to 1d below.
(207) TABLE-US-00002 TABLE 1b Binding affinities (IC.sub.50 in nM) of novel CXCR4 ligands to human CXCR4 (hCXCR4). Affinities were determined using Jurkat human T-cell leukemia cells (400.000 cells/sample) and [.sup.125I]FC-131 as the radioligand. Each experiment was performed in triplicate, and results are means ± SD from three separate experiments. IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] c(iodoyorn′(R.sup.1)RNalG) to to to to R.sup.1 = com- hCXCR4 hCXCR4 hCXCR4 hCXCR4 Metal complexes pound [.sup.natGa] [.sup.natLu] [.sup.natY] [.sup.natBi]
(208) TABLE-US-00003 TABLE 1c Binding affinities (IC.sub.50 in nM) of novel CXCR4 ligands to mouse CXCR4 (mCXCR4). Affinities were determined using Eμ-Myc1080 mouse B-cell lymphoma (200.000 cells/sample), and [.sup.125I]CPCR4.3.sup.8 as the radioligand. Each experiment was performed in triplicate, and results are means ± SD from three separate experiments. c(iodoyorn′(R.sup.1)RNalG) IC.sub.50 [nM] to R.sup.1═ compound mCXCR4 12 = P3 37.1 ± 2.9 14 = P4 48.5 ± 0.5 16 = P5 61.4 ± 17
(209) TABLE-US-00004 TABLE 1d cyclo[tyr-N(Me)orn(S)-Arg-Nal-Gly] IC.sub.50 [nM] to S = orn side chain modification compound hCXCR4 logP .sup.19F-labeled precursor (FBOA-analog)
(210) Internalization and Externalization Studies
(211) The internalization and cell efflux kinetics of the most affine peptides .sup.177Lu-P5b and .sup.177Lu-P5 were determined using hCXCR4.sup.+ Chem_1 cells (1.25×10.sup.5 cells/well) at 37° C. (
(212) Determination of Lipophilicity
(213) Lipophilicity was measured using the shake flask method. The logarithm of the partition coefficient P, where P is the ratio of activity distribution of the respective .sup.68Ga/.sup.177Lu-labeled peptide in n-octanol and PBS are listed in Table 2. The novel peptides all showed enhanced hydrophilicity. Among all tested compounds, .sup.68Ga-15 was the most hydrophilic compound.
(214) TABLE-US-00005 TABLE 2 Lipophilicity of the radiolabeled ligands (logP(o/w); distribution coefficient in n-octanol/PBS) CXCR4 ligand logP.sub.(o/w) .sup.68Ga-3 −2.90 ± 0.08 .sup.68Ga-P5b −3.58 ± 0.06 .sup.68Ga-P5 −3.29 ± 0.02 .sup.177Lu-4 −1.80 ± 0.20 .sup.117Lu-P5b −2.96 ± 0.13 .sup.177Lu-P5 −2.75 ± 0.04 Data are expressed as mean ± SD (n = 6).
(215) Biodistribution Studies
(216) Comparative biodistribution data for .sup.177Lu-P5b, .sup.177Lu-P5 and .sup.177Lu-4 (each 0.1 to 0.2 nmol) 6 h and 48 h after injection in Daudi lymphoma-bearing SCID mice (n=4) are summarized in
(217) Small-Animal PET Imaging
(218) As demonstrated with the PET images in
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