Dual-mode 18F-labeled theranostic compounds and uses thereof

Abstract

Compound or molecular complex. The compound or molecular complex may be a metal chelator configured for chelation with a radioactive or non-radioactive isotope; 19 F / 18 It comprises a trifluoroborate (BF3)-containing moiety or precursor thereof configured for F exchange, and optionally a cell targeting domain.

Description

[Technical field]

[0001] The present invention relates to compounds / conjugates for imaging and / or therapy. In particular, the present invention relates to 18 It concerns dual-mode compounds / complexes configured for imaging when F-labeled and for therapeutic use when chelated with a radiometal. [Background technology]

[0002] PET imaging is playing an increasingly important role in non-invasive clinical diagnostics. 1~7 Compared with MRI or SPECT, PET offers a much higher sensitivity for investigating biodistribution and clearance. 10 and dynamic spatiotemporal resolution. 11、12 . 18 F-deoxyglucose (FDG) and 18 F-thymidine (FLT) 13 provides a picture based on the increased metabolism that characterizes most, but not all, cancers. 4 Cancer subtyping distinguishes pathologically distinct cell types and assesses the presence of specific molecular targets 4、14~19 are increasingly differentiated by their 20~23 Clinical peptide tracers and drugs include octreotate. 24~28 , Bombesin 29 , and RGD 30~32 , Leuprorelin™ and Sandostatin™ 33、34 Includes:

[0003] Radioactive metals (e.g. 68 Ga, 64 Cu, 99m Tc) chelation provides ease of labeling, but 18 F-fluoride offers scalability at a lower cost. Therefore, 18 F-fluoride is preferred, 18 F-fluoride decays cleanly (>97% β+); 68It can be produced with high isotopic purity at a much lower cost than Ga (>1 Ci for $400). 35 , with optimal imaging properties 36、37 The short half-life minimizes radiation dose, while the high specific activity allows the tracer to meet microdosing requirements. 38 Nevertheless, its short half-life (109.8 min) also poses challenges for labeling peptides. 18 Previous work on F-labeled organotrifluoroborates (RBFs) has now been carried out as well as radiometal chelation. 18 Facilitates F labeling 39 Specific 18 F-RBF3 peptide shows high tumor uptake 40 A distinct advantage of radiometallated peptides is that they generally exhibit high tumor uptake. 41 .

[0004] Nevertheless, radiotoxic metallopeptides are used to treat certain cancers for which few therapeutic options exist. For example, octreotate-chelators are used to treat pancreatic cancer. 90 Complexed with Y 42 Several peptides have emerged as targeting agents for radiotherapy. 43、44 Nevertheless, not all patients respond to such treatment. Ideally, patients should be imaged with the same peptide before treatment. However, PET imaging of radiotherapeutic peptides requires the use of a pair of isotopes, one for imaging and the other for treatment (e.g. 86 Y / 90 Y, 64 Cu / 67 Cu, 203 Pb / 212 Limited to metals containing Pb 45、46 Unfortunately, production of diagnostic PET metals is limited and expensive. To make matters worse, some radioactive metals, e.g. 177 For Lu, there are no isotopes readily available or useful for PET (β+ emitting 167There is one report of PET using Lu.) Typically, different metals are used for imaging.

[0005] Thus, current practice creates significant problems when one radiometal is used for imaging and a different radiometal is used for therapy. 111 In (for imaging) and 90 Y (therapeutic). Significant differences in uptake indicate that TATE releases the same isotope, i.e., β+. 86 We came to the conclusion that it should be evaluated at Y. 45、46 .unfortunately, 86 Y is very expensive, 18 Lower resolution than F 47 Similar problems also exist in SPECT imaging. 111 In 、67 Ga, 177 Lu, and 90 The Y-DOTA-TATE chelates all exhibit different affinities, making it difficult to correlate images between different metallopeptides, as the radiometal affects both affinity and imaging signal. 43、48 Similar differences were seen with bombesin-NOTA, 68 Ga-chelate is 111 It shows a much lower affinity than the In-chelate, 1.2 nM vs. 23 pM, respectively. 49 Variations in affinity hinder image correlation and prediction of radiotherapy uptake. Furthermore, there are only a few examples where two metal isotopes can be identified to provide theranostic pairs of isotopically substituted entities. Thus, there is an unmet need in the field of theranostic dual-function PET imaging tracer / radiotherapeutic agents that would facilitate improved treatment planning based on PET imaging results.

[0006] No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Summary of the Invention

[0007] Various embodiments relate to compounds or molecular complexes, which include a metal chelator configured for chelation with a radioactive or non-radioactive metal isotope, and 19 F / 18 a trifluoroborate (BF3)-containing moiety configured for F exchange; or 18 Contains boronate precursors that can be converted to F-labeled trifluoroborates.

[0008] Various embodiments relate to compounds or molecular complexes, which include a cell targeting domain, a metal chelator configured for chelation with a metal radioisotope or a non-radioactive metal isotope, and 19 F / 18 a trifluoroborate (BF3)-containing moiety configured for F exchange; or 18 The cell targeting domain comprises a boronate precursor capable of being converted to F-labeled trifluoroborate. In some such embodiments, the cell targeting domain can comprise a peptide, polypeptide or protein, a peptidomimetic, or a nucleic acid aptamer, a macrocycle, a steroid, or a small molecule, and the cell targeting domain specifically binds to a cell marker. In other such embodiments, the cell targeting domain comprises LLP2A, PSMA-617, TATE, or the peptide D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2. In other such embodiments, the cell targeting domain comprises (i) an antibody or antibody derivative or fragment that specifically binds to an antigen of a target cell, or (ii) a protein domain that specifically binds to an antibody or antibody derivative or fragment that specifically binds to an antigen.

[0009] In some embodiments, the metal chelator can be linked to the cell targeting domain by a linker that contains a BF3-containing moiety. The linker can include multiple BF3-containing moieties. The linker can be a peptide linker. The linker can include Lys(AMBF3).

[0010] In some embodiments, the metal chelator compound or molecular complex is unchelated or chelated with a non-radioactive metal isotope and the BF3-containing moiety is 18 It is labeled F.

[0011] In some embodiments, the metal chelator is chelated with a radioactive metal isotope and the BF3-containing moiety 19 It is labeled F.

[0012] In some embodiments, the metal chelator is chelated with a radioactive metal isotope and the BF3-containing moiety 18 It is labeled F.

[0013] In some embodiments, the radioactive metal isotope is an alpha emitter, a beta emitter, or an Auger emitter.

[0014] In some embodiments, the metal chelator is selected from DOTA and derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogs which may be selected from CHX-A″-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcofazine and sarcofazine derivatives which may be selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; H2-macropa or derivatives thereof; H2dedpa, H4octapa, H4py4pa, H4Py pa, H2azapa, H5decapa, and other picolinic acid derivatives; CP256; PCTA; DOTP; HEHA; C-NETA; C-NE3TA; HBED; SHBED; BCPA; CP256; YM103; cyclam; DiamSar; desferrioxamine (DFO) and DFO derivatives; H6phospa; trithiol chelate; mercaptoacetyl; hydrazinonicotinamide; dimercaptosuccinic acid; 1,2-ethylenediylbis-L-cysteine ​​diethyl ester; methylenediphosphonate; hexamethylpropyleneamine oxime; and hexakis(methoxyisobutylisonitrile); a chelator selected from the group consisting of porphyrin, chlorin, texafrin, and phthalocyanine. In some embodiments, the metal chelator is selected from DOTA and DOTA derivatives. In some embodiments, the metal chelator is DOTA.

[0015] In some embodiments, the BF3-containing moiety is

[0016] [ka] or a group shown in Table 3 or 4, where each R is independently a C1 to C5 straight chain or branched alkyl group.

[0017] In some embodiments, the compound is selected from the group consisting of DOTA-AMBF3-PEG2-LLP2A, PSMA-617-LysAMBF3-DOTA, DOTA-Lys(AMBF3)-TATE, and DOTA-Lys-AMBF3-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2.

[0018] In some embodiments, the compound or molecular complex further comprises a fluorophore or other light-emitting moiety.

[0019] In some embodiments, the compound or molecular complex comprises Lys(AMBF3).

[0020] In some embodiments, the compound comprises DOTA-Bn-NH-Lys-(AMBF3).

[0021] In some embodiments, where the compound or molecular conjugate comprises both a BF3-containing moiety and a cell targeting domain, the compound or molecular conjugate comprises: 18 The compounds or molecular conjugates are for use in imaging a subject to identify the presence of cellular markers of a disease or condition using F-labeled compounds or molecular conjugates, and in treating a disease or condition using compounds or molecular conjugates chelated with a therapeutic radioisotope.

[0022] In some embodiments where the compound or molecular complex is a compound that includes a BF3-containing moiety and does not include a cell targeting domain, the compound or molecular complex is for use in combination with a bispecific antibody to image and / or treat a disease or condition in a subject, the bispecific antibody being specific for (i) a cell marker for the disease or condition, and (ii) a metal chelator. In some such embodiments, the compound or molecular complex may be for administration to a subject as a complex with the bispecific antibody. In other such embodiments, the bispecific antibody may be for administration to a subject prior to administration of the compound or molecular complex to the subject in a pre-targeting step in which the bispecific antibody binds to the cell marker.

[0023] The features of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows a schematic diagram of the various relative configurations of functional domains of a particular dual-mode PET imaging radiotherapeutic agent.

[0025] [Diagram 2] FIG. 1 shows the chemical structures of DOTA-AMBF3-PEG2-LLP2A (also called LLP2A-LysAMBF3-DOTA) and PSMA-617-LysAMBF3-DOTA.

[0026] [Diagram 3] FIG. 1 shows maximum intensity projection images (1 hour after injection) of F-18 labeled LLP2A-Lys-AMBF3-DOTA in mice bearing B16F10 melanoma xenografts without (A) and with (B) co-injection of non-radioactive LLP2A-Lys-AMBF3-DOTA (100 μg).

[0027] [Figure 4] Maximum intensity projection images (1 hour after injection) of F-18 labeled PSMA-617-Lys-AMBF3-DOTA in mice bearing LNCaP prostate cancer xenografts without (A) and with (B) co-injection of DCFPyL (0.5 mg).

[0028] [Diagram 5]Representative HPLC chromatograms of purified 18F radiotracer, 7, formulated in (1:9) EtOH:0.9% saline (v / v) (after preparative HPLC purification and C18 Sep-Pak elution) are shown, a) radiochromatogram of 7 showing 98.5% radiochemical purity, 1.1% 18F- (tR=2.30 min) and 0.4% impurities (tR=8.26 min) at tR=14.97 min using HPLC method B, and b) absorbance chromatograms of 7 alone (tR=14.44 min) and co-injection with 0.1 nmol of cold precursor 6 (tR=14.49 min).

[0029] [Figure 6] PET images at 1 h pi of 7 alone (a and b, 4–6 MBq injection) and from co-injection of 7 (4–6 MBq injection) with blocking agent 1 (200 μg) (c and d) are shown in mice bearing B16-F10 tumors (arrows), scale bars are 0–20% ID / g.

[0030] [Figure 7] Dynamic PET scans (5 s to 52.5 min pi, 28 time points) of [18F]6 (5 MBq injection) in B16-F10 tumor-bearing mice are shown.

[0031] [Figure 8] The 1H NMR (acetone-d6, 300 MHz, room temperature) spectrum of 1 is shown.

[0032] [Figure 9] The ESI-MS(+) spectrum of LLP2A-PEG2-AMBF3-Fmoc (3) is shown, calculated for C76H98BF3N14O13 as 1483.5 m / z, and found to be [M+Na]+ = 1506.8 m / z, [M+K]+ = 1520.9 m / z.

[0033] [Figure 10]The ESI-MS(+) spectrum of AMBF3-PEG2-LLP2A-NH2(4) is shown, calculated for C61H88BF3N14O11 as 1261.3 m / z, and found to be [M+H]+=1262.6 m / z, [M+Na]+=1284.6 m / z.

[0034] [Figure 11] The ESI-MS(+) spectrum of DOTA-AMBF3-PEG2-LLP2A (6) is shown; calculated for C77H114BF3N18O18 is 1647.6 m / z, and found to be [M+H]+ = 1648.9 m / z, [M+Na]+ = 1670.9 m / z, [M+K]+ = 1687.0 m / z.

[0035] [Figure 12] FIG. 1 shows an analytical HPLC chromatogram (λAbs=241 nm) of 6 (tR=8.9 min) after semi-preparative HPLC purification (>95% purity) performed using HPLC method A.

[0036] [Figure 13] FIG. 1 shows the standard curve obtained using HPLC method C, which was used to characterize [18F]6 (after HPLC purification and formulation) based on the UV-vis absorption of 6 (λ=257 nm).

[0037] [Figure 14] In vitro binding saturation assay of 18F radiotracer [18F]6 using VLA-4 expressing B16-F10 cells (each replicate, error bars indicate ±SD) was shown with a calculated Kd value of 6.9 ± 0.59 nM (n = 3).

[0038] [Figure 15] The 13C{1H}NMR (acetone-d6, 75 MHz, room temperature) spectrum of 1 is shown.

[0039] [Figure 16]The ESI-MS(+) spectrum of 1 is shown, with the calculated C12H23BNO2 + = 224.1 m / z and the observed value [M]+ = 224.6 m / z.

[0040] [Figure 17] The 1H NMR of 2 (acetone-d6, 300 MHz, room temperature) is shown.

[0041] [Figure 18] The 13C{1H}NMR (acetone-d6, 75 MHz, room temperature) spectrum of 2 is shown.

[0042] [Figure 19] The 19F{1H}NMR (acetone-d6, 282 MHz, room temperature) spectrum of 2 is shown.

[0043] [Figure 20] The ESI-MS(+) spectrum of 2 is shown, calculated for C6H11BF3N as 165.0 m / z, and found to be [M+Na]+ = 188.4 m / z.

[0044] [Figure 21] The 1H NMR (CDCl3, 300 MHz, room temperature) spectrum of 3 is shown.

[0045] [Figure 22] The 13C{1H}NMR (CDCl3, 75 MHz, room temperature) spectrum of 3 is shown.

[0046] [Figure 23] The ESI-MS(-) spectrum of 3 is shown, calculated for C21H22N4O4 as 394.4 m / z, with the observed values ​​of [MH]- = 393.4 m / z and [2M-H]- = 787.7 m / z.

[0047] [Figure 24] The 1H NMR (CD3CN, 300 MHz, room temperature) spectrum of 4 is shown.

[0048] [Diagram 25] The 13C{1H}NMR (CD3CN, 75 MHz, room temperature) spectrum of 4 is shown.

[0049] [Figure 26] The 19F{1H}NMR (CD3CN, 282 MHz, room temperature) spectrum of 4 is shown.

[0050] [Figure 27] The ESI-MS(-) spectrum of 4 is shown; calculated for C27H33BF3N5O4: 559.4 m / z, found: [MH]-=558.5 m / z and [M+I]-=686.4 m / z.

[0051] [Figure 28] The 1H NMR (CD3CN, 300 MHz, room temperature) spectrum of 5 is shown.

[0052] [Figure 29] The 13C{1H}NMR (CD3CN, 75 MHz, room temperature) spectrum of 5 is shown.

[0053] [Diagram 30] Figure 1 shows the C DEPT-135 NMR (CDCN, 75 MHz, room temperature) spectrum of 5.

[0054] [Diagram 31] The 19F{1H}NMR (CD3CN, 282 MHz, room temperature) spectrum of 5 is shown.

[0055] [Diagram 32] The ESI-MS(+) spectrum of 5 is shown; calculated for C31H36BF3N6O6 is 656.5 m / z, found to be [M+Na]+ = 679.8 m / z.

[0056] [Diagram 33]The HRMS spectrum of 5 is shown, calculated for C31H36BF3N6O6 as 656.4702 m / z, and the observed value was [M+Na]+ = 679.2618 m / z.

[0057] [Diagram 34] The 1H NMR (CD3CN, 300 MHz, room temperature) spectrum of Fmoc-LysAMBF3-OH is shown.

[0058] [Diagram 35] The 13C{1H}NMR (CD3CN, 75 MHz, room temperature) spectrum of Fmoc-LysAMBF3-OH is shown.

[0059] [Diagram 36] The 19F{1H}NMR (CD3CN, 282 MHz, room temperature) spectrum of Fmoc-LysAMBF3-OH is shown.

[0060] [Figure 37] The ESI-MS(-) spectrum of Fmoc-Lys(AMBF3)-OH is shown, calculated for C27H33BF3N5O4 as 559.4 m / z, and found to be [MH]- = 558.5 m / z and [M+I]- = 686.4 m / z.

[0061] [Figure 38] Figure 1 shows structural characterization of Fmoc-LysAMBF-O-NHS by 1H NMR (CD3CN, 300 MHz, room temperature).

[0062] [Figure 39] Structural characterization of Fmoc-LysAMBF-O-NHS by 13C{1H}NMR (CD3CN, 75 MHz, room temperature).

[0063] [Diagram 40] The 19F{1H}NMR (CD3CN, 282 MHz, room temperature) spectrum of Fmoc-LysAMBF3-O-NHS is shown.

[0064] [Diagram 41] The ESI-MS(+) spectrum of Fmoc-LysAMBF3-O-NHS is shown, calculated as 656.5 m / z for C31H36BF3N6O6, and the observed value was [M+Na]+ = 679.8 m / z.

[0065] [Diagram 42] The ESI-MS(+) spectrum of PSMA-617-NH2 is shown, calculated for C41H61N5O9 as 767.97 m / z, and the observed value was [M+H]+ = 768.7 m / z.

[0066] [Diagram 43] The ESI-MS(+) spectrum of PSMA-617-LysAMBF3-Fmoc is shown; calculated for C68H92BF3N10O12 is 1309.35 m / z, found to be [MF]+=1290.1 ​​m / z, [M+Na]+=1332.0 m / z.

[0067] [Diagram 44] The ESI-MS(+) spectrum of PSMA-617-LysAMBF3-NH2 is shown; calculated for C53H82BF3N10O10 is 1087.10 m / z, and found to be [MF]+=1068.1 m / z, [M+H]+=1088.1 m / z, [M+Na]+=1110.1 m / z.

[0068] [Diagram 45] The ESI-MS(+) spectrum of PSMA-617-LysAMBF3-DOTA is shown, calculated for C61H93BF3N13O17 as 1360.31 m / z, and found to be [M-3F-2H]+ = 1302.1 m / z, [M+H]+ = 1362.1 m / z.

[0069] [Diagram 46]The ESI-MS(+) spectrum of PSMA-617-LysAMBF3-DOTA(Cu) is shown; calculated for C61H90BCuF3N10O10 as 1422.8 m / z, and found to be [M-3F-2H]+=1362.8 m / z, [MF]+=1402.8 m / z, [M+H]+=1423.8 m / z, [M+Na]+=1444.8 m / z.

[0070] [Figure 47] The DOTA / AMBF3 conjugated RM2 peptide (11) is shown.

[0071] [Figure 48] The DOTA / AMBF3 conjugated BK peptide (9) is shown.

[0072] [Figure 49] Common protecting groups used in solid phase peptide synthesis are shown.

[0073] [Figure 50] 4 shows the HPLC trace of DOTA-Lys(AMBF3)-BK (compound 9) at 229 nm.

[0074] [Figure 51] A MALDI-TOF of the digested peptide (10) is shown.

[0075] [Figure 52] The characteristic isotope pattern of the digested peptide (10) is shown.

[0076] [Diagram 53] MALDI-TOF of DOTA-Lys(AMBF3)-BK(9) is shown.

[0077] [Figure 54] The characteristic isotope pattern of DOTA-Lys(AMBF3)-BK(9) is shown.

[0078] [Figure 55] Figure 52 shows the proposed deboronation product of DOTA-Lys(AMBF3)-BK (10), predicted to result from deboronation of peptide 9 during the cleavage procedure (Scheme 6v). The accurate mass is 1784.03. Figure 52 shows the MALDI-TOF of peptide 10, showing the predicted isotopic pattern at [M+Na+]+=1807.02.

[0079] [Figure 56] MALDI-TOF of DOTA-Lys(AMBF3)-RM2(11) is shown.

[0080] [Figure 57] The structure of DOTA-((L)-Lys-ε-1,2,3 triazole-N,N-dimethyl-ammoniomethyl-trifluoroborate)-4-amino-1-carboxymethyl-piperidine-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu-COOH is shown.

[0081] [Figure 58] The HPLC trace (229 nm) of crude compound 9 is shown.

[0082] [Figure 59] 1 shows the MALDI-TOF spectrum of compound 9.

[0083] [Figure 60] The ESI-MS(+) spectrum of DOTA-Lys(AMBF3)-TATE is shown, calculated as 1811.8 m / z for C79H114BF3N20O21S2, and the observed value was [M+2Na]+ = 1855.1 m / z. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] As used herein, the terms "comprising," "having," "including," and "containing" and grammatical variations thereof are inclusive or open ended and do not exclude additional unrecited elements and / or method steps, even if the features / components defined as part thereof consist or consist essentially of the particular features / components. The term "consisting essentially of," when used herein in connection with a compound, composition, use, or method, indicates that additional elements and / or method steps may be present, but that these additions do not substantially affect the manner in which the recited compound, composition, method, or use functions. The term "consisting of," when used herein in connection with a feature of a compound, composition, use, or method, excludes the presence of additional elements and / or method steps in that feature. A compound, composition, use, or method described herein as including particular elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically mentioned. Uses or methods described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically mentioned.

[0085] Reference to an element by the indefinite article "a" does not exclude the possibility of a plurality of elements being present, unless the context clearly requires that there is only one of the element. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0086] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range, including all whole numbers, all integers, and, where appropriate, all decimal intermediates (e.g., 1 to 5 can include 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.).

[0087] Unless otherwise specified, "specific embodiments," "various embodiments," "embodiments," and similar terms include the particular feature(s) described for that embodiment, either alone or in combination with any other embodiment or embodiment described herein, whether or not other embodiments are directly or indirectly referenced, and whether or not that feature or embodiment is described in the context of a method, product, use, composition, compound, etc.

[0088] As used herein, the terms "treat," "treatment," "therapeutic," and the like include amelioration of symptoms, reduction of disease progression, improved prognosis, and reduction of recurrence.

[0089] As used herein, the term "diagnostic agent" includes an "imaging agent." Thus, a "diagnostic radionuclide" includes a radionuclide suitable for use in an imaging agent.

[0090] The term "subject" refers to an animal (e.g., a mammal or a non-mammal). The subject may be a human or a non-human primate. The subject may be a laboratory mammal (e.g., a mouse, a rat, a rabbit, a hamster, etc.). The subject may be an agricultural animal (e.g., a horse, a sheep, a cow, a pig, a camel, etc.) or a livestock animal (e.g., a dog, a cat, etc.). In some embodiments, the subject is a human.

[0091] The compounds disclosed herein may also include their free base forms, solvates, salts or pharma- ceutically acceptable salts.Unless otherwise specified or indicated, the compounds claimed and described herein are meant to include all racemic mixtures and all individual enantiomers or combinations thereof, whether or not expressly represented herein.

[0092] The compounds disclosed herein may be shown as having one or more charged groups, may be shown with the ionizable groups in an uncharged (e.g., protonated) state, or may be shown without a formal charge designation. As will be understood by those skilled in the art, the ionization state of a particular group in a compound (e.g., but not limited to, CO2H, PO3H2, SO2H, SO3H, SO4H, OPO3H2, etc.) depends, among other things, on the pKa of the group and the pH of its location. For example, but not limited to, carboxylic acid groups (i.e., COOH) are generally understood to be deprotonated (and negatively charged) at neutral pH and most physiological pH values, unless the protonation state is stabilized. Similarly, OSO3H (i.e., SO4H), SO2H, SO3H, OPO3H2 (i.e., PO4H2), and PO3H groups are generally deprotonated (and negatively charged) at neutral and physiological pH values.

[0093] As used herein, the terms "salt" and "solvate" have their usual meaning in chemistry. Thus, when a compound is a salt or solvate, it is associated with an appropriate counterion. Methods of preparing salts or exchanging counterions are well known in the art. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of an appropriate base (e.g., but not limited to, Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, etc.) or by reacting the free base forms of these compounds with a stoichiometric amount of an appropriate acid. Such reactions are generally carried out in water or in an organic solvent, or in a mixture of the two. The counterion can be changed, for example, by ion exchange techniques such as ion exchange chromatography. All zwitterions, salts, solvates, and counterions are intended unless a particular form is specifically indicated.

[0094] In certain embodiments, the salt or counterion may be pharma- ceutically acceptable for administration to a subject. As used herein, "pharma- ceutically acceptable" means suitable for in vivo use in a subject, including but not necessarily limited to therapeutic use, including diagnostic use. More generally, with respect to any pharmaceutical composition disclosed herein, non-limiting examples of suitable excipients include any suitable buffer, stabilizer, salt, antioxidant, complexing agent, isotonicity agent, cryoprotectant, cryoprotectant, suspending agent, emulsifying agent, antimicrobial agent, preservative, chelating agent, binder, surfactant, wetting agent, non-aqueous vehicle, such as fixed oil, or polymer for sustained or extended release. See, for example, Berge et al., 1977 (J. Pharm Sci. 66:1-19) or Remington-The Science and Practice of Pharmacy, 21st Edition (Gennaro et al., eds., Lippincott Williams & Wilkins, Inc., Philadelphia). Each of these is incorporated by reference in its entirety.

[0095] As used herein, the expression "Cy-Cz" refers to a compound in which y and z are integers (e.g., C1-C 15, C1~C 30 , C1~C 100 etc.), it refers to the number of carbons in the compound, R group or substituent (e.g., in an alkyl, alkenyl or alkynyl group) or, if the expression is further defined as having or may have one or more heteroatoms, it refers to the number of carbons plus heteroatoms. In the latter case, the formula "Xy-Xz" can be used (e.g., X3-X 15 etc.), where y and z are integers referring to the number of carbons plus heteroatoms. The heteroatoms can include any, some, or all possible heteroatoms. For example, in some embodiments, the heteroatoms are selected from N, O, S, P, and Se. In some embodiments, the heteroatoms are selected from N, O, S, and P. Such embodiments are non-limiting.

[0096] Unless otherwise specified, the term "alkyl" refers to an alkane lacking a hydrogen atom, and includes any one or more of straight chain alkyl, branched alkyl, acyclic alkyl, cycloalkyl, including monocyclic and polycyclic cycloalkyl (e.g., fused rings, multiple non-fused rings, or combinations thereof), and / or unsubstituted or substituted. For example, an alkyl may be both branched and cyclic. If not specified, the size of the alkyl is that which would be reasonable to one of ordinary skill in the art. For example, but not limited to, if not specified, the size of the alkyl is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more carbons in length, according to the general knowledge of one of ordinary skill in the art. As used herein, the term "alkylenyl" refers to the divalent analog of an alkyl group.

[0097] The term "straight-chain" as used herein in the context of an alkyl group of a compound may be used as commonly understood by one of ordinary skill in the art and generally refers to a chemical entity that includes a backbone or main chain that is not divided into two or more continuous chains. Non-limiting examples of straight-chain alkyls include methyl, ethyl, n-propyl, and n-butyl.

[0098] As used herein, the term "branched" may be used as commonly understood by those of skill in the art and generally refers to a chemical entity that includes a backbone or main chain that splits into two or more continuous chains. The portion of the backbone or main chain that splits in two or more directions may be linear, cyclic, or any combination thereof. Non-limiting examples of branched alkyl groups include tert-butyl and isopropyl.

[0099] C1~C 20 Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1,2-dimethylpropyl, 2-ethylpropyl, 1-methyl-2-ethylpropyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1,2-triethylpropyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, Examples may include 1,3-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, sec-hexyl, t-hexyl, n-heptyl, i-heptyl, sec-heptyl, t-heptyl, n-octyl, i-octyl, sec-octyl, t-octyl, n-nonyl, i-nonyl, sec-nonyl, t-nonyl, n-decyl, i-decyl, sec-decyl, t-decyl, cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, cycloheptanyl, cyclooctanyl, cyclononanyl, cyclodecanyl, and the like. Thus, unless otherwise specified, C1-C 20 Alkylenyl includes, but is not limited to, all divalent analogs of the above saturated alkyl groups.

[0100] As used herein, the terms "alkenyl" and "alkynyl" refer to alkenes and alkynes, respectively, that are devoid of a hydrogen atom and can include linear, branched and / or cyclic groups, which may be unsubstituted or substituted. 20Non-limiting examples of alkenyl groups can include vinyl, allyl, isopropenyl, l-propen-2-yl, 1-buten-l-yl, l-buten-2-yl, l-buten-3-yl, 2-buten-l-yl, 2-buten-2-yl, octenyl, decenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononanenyl, cyclodecanenyl, and the like. Thus, unless otherwise specified, C1-C 20 Alkenylenyl includes, but is not limited to, all divalent analogs of the above alkenyl groups. 20 Non-limiting examples of alkynyl groups can include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like. Thus, unless otherwise specified, C1-C 20 Alkynylenyl includes, but is not limited to, all divalent analogs of the above alkynyl groups.

[0101] Non-limiting examples of non-aromatic heterocyclic groups include aziridinyl, azetidinyl, diazetidinyl, pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, imidazolinyl, pyrazolidinyl, imidazolidinyl, phthalimidyl, succinimidyl, oxiranyl, tetrahydropyranyl, oxetanyl, dioxanyl, thietanyl, thiepinyl, morpholinyl, oxathiolanyl, and the like. Unless further specified, "aryl" groups include both single aromatic rings as well as fused rings containing at least one aromatic ring. C3-C 20 Non-limiting examples of aryl groups include phenyl (Ph), pentalenyl, indenyl, naphthyl and azulenyl. 20Non-limiting examples of aromatic heterocyclic groups include pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, acridinyl, indolyl, isoindolyl, indolizinyl, purinyl, carbazolyl, indazolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, phenanthridinyl, phenazinyl, phenanthrolinyl, perimidinyl, furyl, dibenzofuryl, xanthenyl, benzofuryl, thiophenyl, thianthrenyl, benzothiophenyl, phosphorinyl, phosphinolinyl, phosphindolyl, thiazolyl, oxazolyl, isoxazolyl, and the like.

[0102] As used herein, the term "substituted" is used as commonly understood by those skilled in the art, and generally refers to a compound or chemical entity in which one chemical group is replaced with a different chemical group. Unless otherwise specified, a substituted alkyl is an alkyl in which one or more hydrogen atoms are each independently replaced with a non-hydrogen atom. For example, chloromethyl is a non-limiting example of a substituted alkyl, more specifically an example of a substituted methyl. Aminoethyl is another non-limiting example of a substituted alkyl, more specifically an example of a substituted ethyl. Unless otherwise specified, a substituted compound or group (e.g., alkyl, aryl, etc.) can be substituted with any chemical group reasonable to one skilled in the art. For example, but not limited to, a hydrogen bonded to a carbon or heteroatom (e.g., N) may be substituted with a halide (e.g., F, I, Br, Cl), amine, amide, oxo, hydroxyl, thiol, phosphate, phosphonate, sulfate, SOH, SOH, alkyl, aryl, ketone, carboxaldehyde, carboxylate, carboxamide, nitrile, monohalomethyl, dihalomethyl, or trihalomethyl.

[0103] As used herein, the term "unsubstituted" is used as commonly understood by one of ordinary skill in the art. Non-limiting examples of unsubstituted alkyl include methyl, ethyl, tert-butyl, pentyl, etc. The phrase "optionally substituted" is used interchangeably with the phrase "unsubstituted or substituted."

[0104] In the structures provided herein, hydrogen may or may not be shown. In some embodiments, hydrogen (shown or implied) is replaced by protium (i.e., 1 H), deuterium (i.e. 2 H) or 1 H and 2 It may also be a combination of H. 1 H 2 Methods for exchanging with H are well known in the art. In the case of solvent exchangeable hydrogen, 1 H and 2 The exchange of H occurs readily without a catalyst in the presence of a suitable deuterium source. The use of acid, base or metal catalysts, combined with high temperature and pressure conditions, promotes the exchange of non-exchangeable hydrogen atoms, typically resulting in the exchange of all hydrogen atoms in a molecule. 1 H 2 Can be exchanged for H.

[0105] The wavy line "" shown through a bond or at the end of a chemical formula (e.g., in the groups listed in Tables 3 and 4) [ka] The " symbol is intended to define the R group on one side of the wavy line without changing the definition of the structure on the other side of the line. When an R group is attached to more than one side (e.g., R 2), atoms shown outside the wavy lines are intended to clarify the orientation of the R group. Thus, only the atoms between the two wavy lines constitute the definition of the R group. When atoms are not shown outside the wavy lines, or when a chemical group is shown without a wavy line but has bonds on multiple sides (e.g., -C(O)NH-, etc.), the chemical group is to be read from left to right, consistent with the orientation of the formula to which the group pertains, unless another orientation is clearly intended (e.g., the formula -R a -R b -R c -, R as -C(O)NH- b The definition of -R a -NHC(O)-R c -R, not - a -C(O)NH-R c - should be incorporated into the formula.

[0106] The present disclosure relates to compounds, complexes or molecular compositions that are radioactive and non-radioactive isotopes, as well as isotopic substitutes. When an isotopic substitute is identified as containing a particular isotope, it is understood that the compound / complex / composition may in fact be obtained in a mixture of isotopic substitutes that greatly favor the identified isotope. For example, hot-F or 18 Preparations of compounds / complexes / compositions identified as F isotope substitutes may in fact be 19 Conversely, it may contain a minimal amount of hot-M or radioactive metallated isotopes (e.g., 177 Preparations of compounds / complexes / compositions identified as Lu) may be prepared using the corresponding non-radioactive isotopic substitutes (e.g. 174 Lu, nat Lu) may be contained in a minimum amount.

[0107] The term "Xaa" refers to an amino acid residue in a peptide chain or otherwise part of a compound. An amino acid has both an amino group and a carboxylic acid group, either or both of which can be used for covalent attachment. Upon attachment to the remainder of the compound, the amino and / or carboxylic acid groups may be converted to an amide or other structure, for example, the carboxylic acid group of a first amino acid is converted to an amide (i.e., a peptide bond) when attached to the amino group of a second amino acid. Thus, Xaa can be represented by the formula -N(R a )R b C(O)—, where R a and R b is an R group. a is typically hydrogen or methyl, or R a and R bmay form a cyclic structure. The amino acid residues of the peptide may include typical peptide (amide) bonds and may further include bonds between a side chain functional group and a side chain or main chain functional group of another amino acid. For example, the side chain carboxylate of one amino acid residue in the peptide (e.g., Asp, Glu, etc.) may be bonded to the amine of another amino acid residue in the peptide (e.g., Dap, Dab, Orn, Lys). Further details are provided below. Unless otherwise indicated, "Xaa" may be any amino acid, including proteinogenic or non-proteinogenic amino acids.Non-limiting examples of non-proteinogenic amino acids are provided in Table 1 and include D-amino acids, including but not limited to the D-forms of the following amino acids: ornithine (Orn), 3-(1-naphthyl)alanine (Nal), 3-(2-naphthyl)alanine (2-Nal), α-aminobutyric acid, norvaline, norleucine (Nle), homonorleucine, beta-(1,2,3-triazol-4-yl)-L-alanine, 1,2,4-triazol-3-alanine, Phe(4-F), ... -Cl), Phe(4-Br), Phe(4-I), Phe(4-NH2), Phe(4-NO2), homoarginine (hArg), 2-amino-4-guanidinobutyric acid (Agb), 2-amino-3-guanidinopropionic acid (Agp), β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, 10-aminodecanoic acid, 2-aminooctanoic acid, 2-amino-3-(anthracene- 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(pyren-1-yl)propanoic acid, Trp(5-Br), Trp(5-OCH3), Trp(6-F), Trp(5-OH) or Trp(CHO), 2-aminoadipic acid (2-Aad), 3-aminoadipic acid (3-Aad), propargylglycine (Pra), homopropargylglycine (Hpg), beta-homopropargylglycine (Bpg), 2,3-diazotidine (Diazolyl) Aminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), azidolysine (Lys(N3)), azidoornithine (Orn(N3)), 2-amino-4-azidobutanoic acid Dab(N3), Dap(N3), 2-(5'-azidopentyl)alanine, 2-(6'-azidohexyl)alanine, 4-amino-1-carboxymethyl-piperidine (Pip), 4-(2-aminoethyl)-1-carboxymethyl-piperazine (Acp), and tranexamic acid. When not specified as an L- or D-amino acid, an amino acid is to be understood as an L-amino acid.

[0108] [Table 1-1] [Table 1-2]

[0109] As used herein, the term "molecular composition" is understood to mean a molecular complex in which two or more molecules are held together by non-covalent interactions (e.g., hydrophobic, ionic, electrostatic, etc.), such as a multi-chain protein.

[0110] Various aspects of the invention relate to compounds or molecular complexes, Metal chelators configured to chelate radioisotopes or non-radioisotopes or non-metals; · 19 F / 18 A trifluoroborate (BF3)-containing moiety configured for radiofluorination by F exchange, or 18 A boronate precursor capable of being converted to an F-labeled trifluoroborate, and optionally Contains a cell targeting domain.

[0111] Such compounds / conjugates are dual-modal in that they are suitable for imaging or radiotherapy, or can be used for both applications. For example, the compounds / conjugates can be 18 When F-labeled ("hot-F"), it may be used as an imaging / diagnostic agent, or when chelated with a therapeutic radioactive metal isotope ("hot-M"), it may be used as a therapeutic agent. This offers the advantage of using the same compound for imaging and therapy, i.e., chemically identical to radiotherapeutic agents (optimized for therapy with hot-M). 18 F-labeled companion diagnostics (optimized for imaging with hot-F) are provided.

[0112] In some embodiments, the metal chelator is unchelated or chelated with a non-radioactive metal isotope ("cold-M"). As used herein, both unchelated and chelated with a non-radioactive metal isotope are considered to be "cold-M". The BF3-containing moiety of such embodiments is 18 When F-labeled, the hot-F / cold-M compounds / complexes are useful as imaging or diagnostic agents without any adverse effects from radioactive metal isotopes.

[0113] If imaging reveals that the subject is a candidate for therapeutic treatment, the same compound / complex can be administered (as either cold-F / hot-M or hot-F / hot-M). Thus, the hot-F / cold-M compound / complex is useful as a companion diagnostic to the hot-M therapeutic. It is contemplated that in other cases, unchelated compounds / complexes can be used equally well, and it is recognized that in other embodiments, alternative metal cations may be used in place of the hot metal cation. For example, in some embodiments, radioactive Zn 2+ Non-radioactive Zn, even if not commonly used therapeutically or diagnostically 2+ Thus, in some embodiments, the metal chelator is chelated with a radioisotope and the BF3-containing moiety is 19 In another embodiment, the metal chelator is chelated with a radioisotope and the BF3-containing moiety is also 18 Labelled with F.

[0114] The terms "BF3" and "BF3" (i.e., the subscript "3") have the same meaning and are used interchangeably in this application.

[0115] The term "cell targeting domain" (also referred to as "cell antigen targeting module") has a broad meaning and includes any compound or complex that specifically binds to a cell marker, such as, but not limited to, peptides, polypeptides, proteins, peptidomimetics, nucleic acids, steroids, aptamers, affibodies, minibodies, vitamins, small molecules, macrocycles, and the like. The term "cell marker" includes, but is not limited to, cell surface antigens, such as cluster of differentiation (CD) molecules. Unless otherwise indicated, it is understood that the term "antigen" as used herein does not necessarily require an immune response to be elicited by binding of the cell targeting domain to the cell marker (or antigen), and various embodiments require only specific binding to the cell marker under physiological conditions (e.g., in vivo). In some embodiments, the cell targeting domain targets a human cell marker or a human CD molecule. A variety of cell targeting domains in each of the above categories have been produced, and many are commercially available. For example, antibodies have been generated against a wide range of human CD molecules.

[0116] As used herein, the phrase "specifically binds" refers to a preferred association (e.g., formation of a non-covalent complex) as opposed to background association with a heterogeneous population of proteins and / or other macromolecules. Thus, under specified conditions (e.g., immunoassay conditions), a cell targeting domain "specifically binds" to a cell marker when it associates at least twice the background level of association with other macromolecules present in a sample (in vitro) or organism (in vivo). A variety of immunoassay formats or other binding assays can be used to select a cell targeting domain that specifically binds to a particular target marker. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically bind to proteins. In some embodiments, the cell targeting domain produces a binding signal that is at least two-fold over background signal, and in some cases at least 10-fold to 100-fold over background. Unless otherwise specified, the association of the cell targeting domain with the target marker generally occurs at about 10 -4 M~10-15 The equilibrium dissociation constant of M (K D In some embodiments, the association is about 10 -4 In some embodiments, the association is less than about 10 -5 In some embodiments, the association is less than about 10 -6 In some embodiments, the association is less than about 10 -7 In some embodiments, the association is less than about 10 -8 In some embodiments, the association is less than about 10 -9 In some embodiments, the association is less than about 10 -10 In some embodiments, the association is less than about 10 -11 In some embodiments, the association is less than about 10 -12 In some embodiments, the association is less than about 10 -13 In some embodiments, the association is less than about 10 -14 The equilibrium dissociation constant is less than M. Any method known in the art can be used to measure the equilibrium dissociation constant.

[0117] In some embodiments, the cell targeting domain is a peptide, polypeptide, or protein. Peptides and polypeptides can be synthesized using standard methods, non-limiting examples of which are described in more detail below. Proteins can be prepared using standard molecular biology methods.

[0118] A variety of peptides, polypeptides and peptidomimetics are known that specifically bind to target cell markers. Non-limiting examples include LLP2A, PSMA-617, TATE, bombesin or derivatives (e.g., D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, etc.), RGD, cholecystokinin. LLP2A targets the transcellular very late antigen 4 (VLA-4). PSMA-617 targets the prostate specific membrane antigen (PSMA). TATE targets the somatostatin receptor. Bombesin and its derivatives target the gastrin releasing peptide receptor (GRPR). Bradykinin (BK) and its derivatives target the bradykinin 1 receptor. Any other peptide ligand of the receptor can be used.

[0119] In some embodiments, the cell targeting domain is a peptidomimetic. Peptidomimetics can be produced by replacing peptide / polypeptide groups with non-peptide groups. For example, but not limited to, peptide bond amide (-C(O)-NH-) can be replaced with pseudopeptide bond (-CH2-NH-), carbon-carbon bond (-CH2-CH2-) or urea bond (-NH-C(O)-NH-) to increase the peptide half-life in vivo.

[0120] Various types of polypeptide / protein domains are known that can specifically target cell markers. In some embodiments, the cell targeting domain is or comprises an antibody that specifically binds to the cell marker, or an antibody derivative / fragment that includes an antibody variable domain. In the case of antibodies and antibody derivatives, the cell marker is considered to be an antigen that contains an epitope that is specifically bound by the antibody variable domain. In general, an "epitope" may be a peptide, protein, nucleic acid, carbohydrate, polysaccharide, lipid, organic compound, etc., as well as complexes thereof that form contacts with an antibody variable domain. An epitope may be continuous or discontinuous. The region of an epitope that contacts an antibody variable domain is typically about 4-10 nm 2Methods for linking an antibody or antibody derivative / fragment, with or without a linker, to the remainder of a compound / conjugate are known. For example, a compound has been linked to an antibody at its N-terminus, C-terminus, cysteine ​​residue, lysine residue, or elsewhere by chemical or enzymatic conjugation (e.g., as is well documented for antibody-drug conjugates).

[0121] The heavy chain of an antibody consists of a variable domain (V H ) and multiple constant domains (e.g., C H 1 , C H 2 and C H 3 The light chain of an antibody is composed of a variable domain (V L ) and the constant domain (C L ) is composed of V L and V H Each of the six CDRs each contain three complementarity determining regions (CDRs) as well as framework regions. All six CDRs can contribute to epitope binding, although their relative contributions vary, and in certain cases, not all six CDRs are required for binding. For example, CDR3 of the heavy chain tends to contribute disproportionately to epitope binding. In addition, single domain antibodies, nanobodies, etc., having only three CDRs are known (e.g., single domain antibodies obtained or derived from the heavy chain variable domain of dromedary, camel, llama, alpaca, shark or similar animals, or engineered from the heavy chain of a conventional antibody, including but not limited to human and murine antibodies). Unless otherwise specified, the phrase "antibody variable domain" as used herein refers to a V H , V H and V LThe term "antibody variable domain" refers to any protein, including both single domain antibodies, nanobodies, or any antibody-derived protein that properly arranges the CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs) necessary for specific binding to an epitope portion of an antigen. Methods for producing proteins that contain antibody variable domains that bind to a target epitope are known and include (but are not limited to) isolation of antibodies from immunized animals, modification of whole antibodies, de novo synthesis using recombinant DNA methods or solid phase peptide / polypeptide synthesis, or isolation of antibodies selected from display libraries, etc.

[0122] In some embodiments, the cell targeting domain comprises an antibody. The antibody may be of any species or may be chimeric or artificial or genetically engineered. For example, but not limited to, the antibody may be non-human (e.g., camelids such as camel, dromedary, alpaca, llama, etc., cartilaginous fish such as shark, mouse, rat, monkey or other), primatized, humanized or fully human. A chimeric antibody comprises amino acid sequences from multiple species, e.g., human and non-human or two non-human species. Methods for humanizing or primatizing a non-human antibody are well known in the art, e.g., by replacing the non-human (or non-primate) constant domains with the constant domains of a human antibody (creating a chimeric antibody) or by replacing one or more (e.g., one, two, three, four, five or six) of the CDRs of a human (or primate) antibody with a non-human antibody. An antibody may be composed of two heavy chains and two light chains. The antibody may be a single chain antibody, having a heavy chain and a light chain separated by a linker. The antibody may be a heavy chain only antibody (e.g., a camel, dromedary, alpaca, llama, or shark antibody lacking light chains, or a human heavy chain). The antibody may be a single domain antibody (sdAb).

[0123] In some embodiments, the cell targeting domain comprises an antibody derivative. As used herein, the term "antibody derivative" includes antibody fragments that retain antigen-binding function, as well as artificial antibodies. Antibody derivatives include antibody variable domains. Antigen-binding fragments include V L and V HBoth or V L Not including V H In some embodiments, the antibody derivative comprises a Fab, a Fab', a F(ab')2, a scFv (i.e., a single chain Fv), a scFv-Fc, a sdAb, a minibody, a nanobody, a bibody, or a triabody.

[0124] In some embodiments, the antibody or antibody derivative is an IgA, IgM, IgG, IgE, IgD, sdAb, Fab, Fab', F(ab')2, scFv, scFv-Fc, minibody, nanobody, bispecific antibody, or trispecific antibody. In some embodiments, the antibody is an IgG antibody.

[0125] Many antibodies have K values ​​in the low micromolar to nanomolar range. D values, whereas high affinity antibodies have low nanomolar K D A very high affinity antibody has a K value of picomolar D In some embodiments, the antibody or antibody derivative has a K value of less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1 nM. D In some embodiments, the antibody or antibody derivative binds to a cellular antigen with a K D (10 -10 M~10 -12 M) can bind to a binding substrate. Such binding affinity can be obtained by screening libraries by selecting for specific and high affinity to a desired target using known display techniques such as mRNA display, phage display, ribosome display, and yeast display, and in some cases by affinity maturation methods.

[0126] In some embodiments, the cell targeting domain is a nucleic acid aptamer that specifically binds to a cell marker. Aptamers are single-stranded oligonucleotides (DNA or RNA) that can bind to a wide range of cell targets. Aptamers can be engineered to bind to desired targets with high affinity, e.g., sub-nanomolar K, using a variety of methods, such as selection from random libraries and sequence optimization. D Aptamers can be synthesized by standard oligonucleotide synthesis methods / apparatus and attached to the remainder of the compound / complex using chemical conjugation, with or without a linker.

[0127] In some embodiments, the cell targeting domain may be used in a pre-targeting context, where it is first conjugated to a reactive group that reacts with the chelator-BF3 either before or after injection.For example, but not limited to, an antibody or aptamer is conjugated to cyclooctene.It is injected and allowed to associate with the target, and then the same animal or patient is injected with the chelator-BF3 linked to tetrazine, which reacts in vivo with the cyclooctene conjugate via N2-extrusion cycloaddition.In other cases, the N2-extrusion cycloaddition is performed before injection.

[0128] The metal chelator may be any suitable chelator for binding radiometals. Many suitable radiometal chelators are known, for example as summarized in Price and Orvig, Chem. Soc. Rev., 2014, 43, 260-290, and a wide variety of metal chelators are commercially available (e.g., from Macrocyclics™) or described in the literature, too numerous to list here.

[0129] In some embodiments, the metal chelator may be, but is not limited to, DOTA and derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogs which may be selected from CHX-A″-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcofagin and sarcofagin derivatives which may be selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; H2-macropa or derivatives thereof; H2dedpa, H4octap a, H4py4pa, H4Pypa, H2azapa, H5decapa, and other picolinic acid derivatives; CP256; PCTA; DOTP; HEHA; C-NETA; C-NE3TA; HBED; SHBED; BCPA; CP256; YM103; cyclam; DiamSar; desferrioxamine (DFO) and DFO derivatives; H6phospa; trithiol chelate; mercaptoacetyl; hydrazinonicotinamide; dimercaptosuccinic acid; 1,2-ethylenediylbis-L-cysteine ​​diethyl ester; methylenediphosphonate; hexamethylpropyleneamine oxime; and hexakis(methoxyisobutylisonitrile); porphyrin, chlorin, texafrin, phthalocyanine. In some embodiments, the metal chelator is DOTA or a DOTA derivative. In particular, one of skill in the art may substitute any of the chelating agents listed herein with another chelating agent known in the art.

[0130] Illustrative non-limiting examples of metal chelators and exemplary radiometals that can be chelated by these chelators are shown in Table 2. In alternative embodiments, the metal chelator is or includes a metal chelator selected in Table 2.

[0131] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0132] In some embodiments, the metal chelator is non-chelated (ie, non-metallated).

[0133] In some embodiments, the metal chelator is chelated / complexed to a metal, either a radioactive metal isotope (radiometal) or a non-radioactive metal isotope. In some embodiments, the chelated metal is non-radioactive. In some embodiments, the chelated metal is a radioactive metal. In some embodiments, the radioactive metal is a therapeutic radiometal, meaning that it is radiotoxic (also referred to herein as a "radiotoxin"). In some embodiments, the radioactive metal is a therapeutic alpha emitter. In some embodiments, the radioactive metal is a beta emitter. In some embodiments, the radioactive metal is an Auger emitter. In some embodiments, the therapeutic radiometal is 64 Cu, 67 Ga, 111 In, 177 Lu, 117m Sn, 165 Er, 90 Y, 227 Th, 225 Ac, 213 Bi, 212 Bi, 211 As, 212 Pb, 47 Sc, 166 Ho, 188 Re, 186 Re, 149Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr or 114m In.

[0134] In some embodiments, the radiometal is 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga, 111 In, 44 Sc, 86 Y, 89 Zr, 90 Nb, 177 Lu, 117m Sn, 165 Er, 90 Y, 227 Th, 225 Ac, 213 Bi, 212 Bi, 72 As, 77 As, 211 At, 203 Pb, 212 Pb, 47 Sc, 166 Ho, 188 Re, 186 Re, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr, 114m In, 94m Tc, 99m Tc, 149 Tb, 152 Tb, 155 Tb or 161 In another embodiment, the radiometal, radionuclide-bound metal, or radionuclide-bound metal-containing moiety or prosthetic group is 68 Ga, 61 Cu, 64 Cu, 67 Cu, 67 Ga,111 In, 44 Sc, 86 Y, 177 Lu, 90 Y, 225 Ac, 213 Bi or 212 In some embodiments, the chelator is a chelator of Table 2 and the chelated radiometal / radionuclide is a radionuclide shown in Table 2 as the binding agent of the chelator.

[0135] In some embodiments, the chelator is DOTA or 177 Lu, 111 In, 213 Bi, 68 Ga, 67 Ga, 203 Pb, 212 Pb, 44 Sc, 47 Sc, 90 Y, 86 Y, 225 Ac, 117m Sn, 153 Sm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 165 Er, 213 Bi, 224 Ra, 212 Bi, 212 Pb, 225 Ac, 227 Th, 223 Ra, 47 Sc, 64 Cu or 67 Its Cu-chelated derivatives, 225 H2-MACROPA conjugated with Ac, 227 Th-chelated Me-3,2-HOPO, 225 Ac, 227 Th or 177 H4py4pa chelated with Lu, 177 H4pypa chelated with Lu, 68 NODAGA chelated with Ga; 111 In-chelated DTPA, or 89 This is DFO chelated with Zr.

[0136] In some embodiments, the chelator is selected from the group consisting of TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid), HBED (N,N ’ -Bis(2-hydroxybenzyl)-ethylenediamine-N,N ’ -diacetic acid), 2,3-HOPO (3-hydroxypyridin-2-one), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), DFO (desferrioxamine), DTPA (diethylenetriaminepentaacetic acid), OCTAPA (N,N ’ -Bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N ’ -diacetic acid) or another picolinic acid derivative.

[0137] In some embodiments, the chelating agent is selected from the group consisting of mercaptoacetyl, hydrazinonicotinamide, dimercaptosuccinic acid, 1,2-ethylenediylbis-L-cysteine ​​diethyl ester, methylene diphosphonate, hexamethylpropyleneamine oxime, and hexakis(methoxyisobutylisonitrile). 99m Tc, 94m Tc, 186 Re, or 188A chelating agent for radiolabeling with Re. In some embodiments, the chelating agent is mercaptoacetyl, hydrazinonicotinamide, dimercaptosuccinic acid, 1,2-ethylenediylbis-L-cysteine ​​diethyl ester, methylenediphosphonate, hexamethylpropyleneamine oxime, or hexakis(methoxyisobutylisonitrile). In some of these embodiments, the chelating agent is bound by a radiometal. In some such embodiments, the radiometal is 99m Tc, 94m Tc, 186 Re, or 188 Re.

[0138] In some embodiments, the chelator is 72 As or 77 A chelator capable of binding As, such as a trithiol chelate. In some embodiments, the chelator is a trithiol chelate. In some embodiments, the chelator is 72 In some embodiments, the chelator is 77 It is conjugated to As.

[0139] Non-radioactive metals suitable for chelation to the above metal chelators, e.g. 89 Y, 174 Lu, 208 Pb and the like are well known and include the known stable or metastable isotopes commonly known in the transition metals, lanthanides and actinides as typically used by those skilled in the art of metal chelation.

[0140] The BF3-containing moiety (also called a "BF3-containing prosthetic group") is 18 F / 19 It may be any such group capable of F-exchange radiolabeling.

[0141] In some embodiments, the BF3-containing moiety [ka] and R a and R b Each of R is independently a C1 to C5 straight chain or branched alkyl group. a is methyl. In some embodiments, R a is ethyl. In some embodiments, R a is propyl. In some embodiments, R a is isopropyl. In some embodiments, R a is n-butyl. In some embodiments, R b is methyl. In some embodiments, R b is ethyl. In some embodiments, R b is propyl. In some embodiments, R b is isopropyl. In some embodiments, R b is n-butyl. In some embodiments, R a and R b are identical. In some embodiments, R a and R b is different.

[0142] In some embodiments, the BF3-containing moiety is a group as shown in Table 3 (below), e.g., each R (if present) in a pyridine substituted with a -OR, -SR, -NR-, -NHR, or -NR2 group is independently a C1-C5 straight chain or branched alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is isopropyl. In some embodiments, R is n-butyl. In some embodiments, the BF3-containing moiety is [ka] It is.

[0143] [Table 3-1] [Table 3-2]

[0144] In some embodiments, the BF3-containing moiety is a group shown in Table 4 (below), e.g., each R (if present) in a pyridine substituted with an -OR, -SR, -NR-, -NHR, or -NR2 group is independently a C1-C5 straight chain or branched alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is isopropyl.

[0145] [Table 4-1] [Table 4-2]

[0146] In some embodiments, the BF3-containing moiety is 18 In some embodiments, one fluorine in the BF3-containing moiety is 18 F. In some embodiments, all three fluorines are 18 F. In some embodiments, all three fluorines in the BF3-containing moiety are 19 It's F.

[0147] In some embodiments, the compound or complex comprises multiple BF3-containing moieties, each of which may be the same or different, or a combination thereof. Some embodiments comprise two BF3-containing moieties, each of which is independently selected from those listed above. Some embodiments comprise three BF3-containing moieties, each of which is independently selected from those listed above. Some embodiments comprise four BF3-containing moieties, each of which is independently selected from those listed above. In some embodiments, the BF3-containing moiety is attached to a linker located between the metal chelator and the cell targeting domain.

[0148] By isotope exchange 18 F labeling may be the preferred method of labeling, but it is understood that conversion of other sp2 / sp3 hybridized boronate species may be advantageous in certain cases. Examples of such boronates include pinacolates, di-, tri-, tetra-arylated pinacolates, neopentyldiolates, catecholates, diolates in general, MIDA-boronates, anthranilamide-based complexes, and in some cases borohydrides. Such boronate complexes are known or readily known to those skilled in the art of organoboron chemistry, and are of interest in some cases when such complexes can be readily converted to the corresponding trifluoroborates. Thus, in some embodiments, the compounds / complexes contain fluorine-containing ... 18 It contains a boronate precursor that can be converted to an F-labeled trifluoroborate.

[0149] The compound or molecular complex may further comprise a linker. For example, but not limited to, the metal chelator may be linked to the cell targeting domain by a linker containing a BF3-containing moiety. In some embodiments, the linker comprises two (or more) BF3-containing moieties. However, the components of the compound / complex may have any configuration. For example, the BF3-containing moiety(s) may be directly attached to the metal chelator and / or the cell targeting domain. A non-limiting example of a linker is a peptide linker.

[0150] The linker may be, for example, but is not limited to, an amino acid linker, a peptide linker, a polyethylene glycol (PEG) linker, an alkylene linker (e.g., C1-C 30 ), ether, ester, thioether, disulfide, thioester, amide, carbamate, ureido, phosphodiester. In some embodiments, the linker is or includes -N(H)-(CH2)nC(O)-, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15.

[0151] In some embodiments, the linker is a linear or branched peptide linker (Xaa 1 ) n wherein n is 1 to 8, and each Xaa 1 are the same or different, or a combination thereof. In some embodiments, the peptide linker is a linear peptide linker. In some embodiments, the peptide linker is a branched peptide linker. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, each Xaa 1are independently selected from the proteinogenic and non-proteinogenic amino acids listed in Table 1. In some embodiments, each peptide backbone amino group in the linker is optionally methylated.

[0152] In some embodiments, the linker comprises Lys(AMBF3). In some embodiments, the linker comprises (but is not limited to) a PEG linker or a peptide linker (Xaa 1 ) 1~8 In some embodiments, the linker is -N(H)-C(R 1 R 2 R 3 ) Amino acid residue Xaa defined as (H)-C(O)- 2 wherein R 1 is a C1-C5 alkylenyl group, R 2 teeth [ka] or [ka] and R 3 teeth [ka] or a group shown in Table 1 or Table 2, where each R is independently a C1-C5 straight chain or branched alkyl group. In some embodiments, R 1 is -CH-. In some embodiments, R 1 is -(CH2)2-. In some embodiments, R 1 is -(CH2)3-. In some embodiments, R 1 is -(CH2)4-. In some embodiments, R 1 is -(CH2)5-. In some embodiments, R 2 teeth, [ka] In some embodiments, R 2 teeth, [ka] In some embodiments, R 3 teeth, [ka] where R a and R b Each of is independently a C1 to C5 straight or branched chain alkyl group. a is methyl. In some embodiments, R a is ethyl. In some embodiments, R a is propyl. In some embodiments, R a is isopropyl. In some embodiments, R a is n-butyl. In some embodiments, R b is methyl. In some embodiments, R b is ethyl. In some embodiments, R b is propyl. In some embodiments, R b is isopropyl. In some embodiments, R b is n-butyl. In some embodiments, R a and R b are identical. In some embodiments, R a and R b are different. In some embodiments, R 3 teeth, [ka] In some embodiments, R 3 teeth, [ka] In some embodiments, the linker comprises two Xaa, each independently as defined above. 2 The residues are the same or different.

[0153] In some embodiments, the metal chelator is attached to the linker or cell targeting domain by forming an amide bond (between an amino group and a carboxylic acid group) or a 1,2,3-triazole (reaction between an azide and an alkyne), or by reaction between a maleimide and a thiol group.

[0154] In some embodiments, the chelator is attached to the N-terminus of the peptide / polypeptide / protein cellular targeting domain (with or without a linker) and the BF3-containing moiety (or precursor) is attached to the C-terminus of the cellular targeting domain (with or without a linker). In some embodiments, the chelator is attached to the C-terminus of the peptide / polypeptide / protein cellular targeting domain (with or without a linker) and the BF3-containing moiety (or precursor) is attached to the N-terminus of the cellular targeting domain (with or without a linker). In some embodiments, the chelator is attached to the N-terminus of the peptide / polypeptide / protein cellular targeting domain (with or without a linker) and the BF3-containing moiety (or precursor) is attached to a side chain of the cellular targeting domain (with or without a linker). In some embodiments, the chelator is attached to the C-terminus of the peptide / polypeptide / protein cellular targeting domain (with or without a linker) and the BF3-containing moiety (or precursor) is attached to a side chain of the cellular targeting domain (with or without a linker). In some embodiments, both the chelator and the BF3-containing moiety are attached to separate side chains of the peptide / polypeptide / protein cellular targeting domain (with or without a linker). In some embodiments, the chelator is attached to the N-terminus or C-terminus of the peptide / polypeptide / protein cellular targeting domain via a linker and the BF3-containing moiety (or precursor) is attached to the linker. In some embodiments, the chelator is attached to the side chain of the peptide / polypeptide / protein cellular targeting domain via a linker and the BF3-containing moiety (or precursor) is attached to the linker. For illustrative purposes, FIG. 1 shows various non-limiting configurations of compounds or complexes.

[0155] In some embodiments, the compound or molecular complex has formula I, or is a salt or solvate of formula I: [chelator]-[linker]-[cell targeting domain] (I), wherein the chelator is any chelator described above, the cell-targeting domain is any cell-targeting domain described above, and the linker comprises one or more of any BF3-containing moieties independently selected from those described above. In some embodiments, the linker is any peptide linker defined above.

[0156] In some embodiments, the compound or molecular complex is or comprises DOTA-AMBF3-PEG2-LLP2A.

[0157] In some embodiments, the compound or molecular complex is or comprises PSMA-617-LysAMBF3-DOTA.

[0158] In some embodiments, the compound or molecular complex is or comprises DOTA-Lys(AMBF3)-TATE.

[0159] In some embodiments the compound or molecular complex is or comprises DOTA-Lys-AMBF3-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2.

[0160] In some embodiments, the composition comprises [chelator]-[linker]-BF3.

[0161] In some embodiments, the compound or molecular complex further comprises a fluorophore or other light-emitting moiety, such as (but not limited to) Cy3, Cy5, Cy7, other near-IR dyes, or fluorescein.

[0162] Various embodiments of the compounds or molecular complexes are suitable for imaging and radiotherapy and include a) a cellular antigen targeting module, b) 18The present invention relates to a dual-mode compound or molecular composition comprising a) a BF3-containing moiety / prosthetic group that is readily labeled with F, and b) a radiotoxin-containing moiety that is readily compounded with a radiotherapeutic or non-radioactive isotope. As noted above, in the diagnostic imaging mode / format, the BF3 moiety contains at least one "hot" radioactive fluorine (i.e., 18 F), and the radiotoxin-containing moiety contains a non-radioactive ("cold") isotope / metal atom (or none at all). Thus, in some embodiments, the BF3-containing moiety / prosthetic group is 18 In targeted radiotherapy modes / forms, the BF3 moiety contains a "cold" fluorine (i.e., 19 F), whereas the radiotoxin-containing moiety contains a radioactive / radiotoxic "hot" isotope / metal atom (suitably useful for radiotherapy). Thus, in some embodiments, the BF3-containing moiety / prosthetic group is 19 The F-labeled, radiotoxin-containing moiety contains a non-radioactive isotope or does not contain a radioactive isotope. Thus, such compounds comprise useful theranostic pairs, thereby enabling diagnostic imaging modalities (i.e. 18 F) for disease detection / diagnosis, and for targeted radiotherapy mode (i.e. 18 Disease treatment / therapy is carried out by subsequent use of the same composition in two different radioisotope / therapeutic regimes (without F and with radiotherapeutic isotopes), the former constituting a "companion diagnostic" for the latter.

[0163] As noted above, in various embodiments, the functional domains of the dual-mode (imaging / radiotherapy) compounds of the invention can be separated by a variety of linkers / intervening groups of various sizes / lengths known to those of skill in the art. In some embodiments, the cellular antigen binding module, the BF3-containing prosthetic group, and the radiotoxin-containing moiety can be arranged relative to one another in a number of different configurations to obtain the desired properties of theranostic pairs (imaging and targeted radiotherapy companion diagnostics of the same chemical composition). Figure 1 shows a non-limiting representation of some configurations of dual-mode PET imaging agents / radiotherapeutic agents.

[0164] In some embodiments, the cellular antigen binding module can include i) a peptide / polypeptide, ii) a non-peptide / non-protein ligand capable of binding to a targeted cellular antigen, iii) an antibody or antibody fragment, iv) an scFv domain / fragment, v) a nucleic acid aptamer, or vi) a bispecific antibody or fragment thereof. Thus, modification with both trifluoroborate and a chelator can be applied to non-peptide small molecules, drugs, or other compositions or larger molecules with diverse properties, such as antibodies, aptamers, etc. In some embodiments, the targeted cellular antigen is an integrin known as transcellular very late antigen 4 (VLA-4), and the compound is or comprises DOTA-AMBF3-PEG2-LLP2A. In some embodiments, the targeted cellular antigen / protein is prostate specific membrane antigen (PSMA), and the compound is or comprises PSMA-617-LysAMBF3-DOTA. In some embodiments, the targeted cellular antigen is a somatostatin receptor (i.e., SSTR), and the compound is or comprises DOTA-Lys(AMBF3)-TATE. In some embodiments, the targeted cellular antigen is a gastrin releasing peptide receptor (GRPR) and the compound is or comprises DOTA-Lys(AMBF3)-RM2 (DOTA-Lys-AMBF3-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2). In some embodiments, the targeted cellular antigen is a bradykinin receptor (e.g., bradykinin-1 receptor, B1R) and the compound is or comprises DOTA-Lys(AMBF3)-BK. In some embodiments, 18 BF3-containing moieties / prosthetic groups that are readily labeled with F are as disclosed in the following patent applications WO 2005 / 077967, WO 2009 / 012596A1, and WO 2014 / 134716, each of which claims priority to U.S. Patent Application No. 61 / 775,280, which is incorporated by reference in its entirety.

[0165] In some embodiments, the radiotoxin chelating moiety can include, but is not limited to, a metal ion chelator (e.g., DOTA, NOTA, NODAGA, Octapa, Macropa, etc.). In other embodiments, the chelator can be used to chelate metals for fluorescent applications, MRI applications, and other applications involving metals (therapeutic, diagnostic, or radiation) in a manner that uses the metal and chelator for applications that may be considered bimodal or multimodal. In some embodiments, the therapeutic radiotoxin / metal can include, but is not limited to, 177 Lu or 210 Bi or 212 Pb. Other suitable radioactive metals that may be useful in practicing the therapeutic modes of the present invention are known or available. In some embodiments, the chelator is used without a metal or with a non-radioactive metal, such as Zn 2+ , Ca 2+ , Ni 2+ , Gd 3+ and the like. Such compounds may be useful for imaging (e.g., PET imaging). In some embodiments, the compounds are 18 F and radioactive metals (e.g. 225 Such compounds may contain both 225 With the knowledge that no stable isotopes of Ac exist, 225 It can be used for imaging (e.g., PET) to identify patients who will respond to targeted therapy with Ac. 225 While holding Ac 19 F isotope substitutes are used. In some embodiments, one or more trifluoroborates can be linked to a tracer that includes at least one chelating agent. Thus, in some embodiments, the compound / composition includes two or more trifluoroborate groups. For imaging applications where radioactive metals are also desired, limited amounts of 18 It is also possible to prepare dual-labeled radiotracers using F-fluoride.

[0166] The compounds presented herein incorporate peptides that may be synthesized by any of a variety of methods established in the art, including, but not limited to, solution and solid phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) and / or t-butyloxycarbonyl (Boc) chemistry, and / or other synthetic approaches.

[0167] Solid phase peptide synthesis methods and techniques are well established in the art. For example, peptides can be synthesized by sequentially incorporating the desired amino acid residues one at a time. In such methods, peptide synthesis is typically initiated by coupling the C-terminal amino acid of the peptide of interest to a suitable resin. Prior to this, the reactive side chain and alpha amino group of the amino acid are protected from reaction by suitable protecting groups, allowing only the alpha carboxyl group to react with functional groups such as amine groups, hydroxyl groups, or halogenated alkyl groups on the solid support. Following coupling of the C-terminal amino acid to the support, the protecting group on the side chain of the amino acid and / or the alpha amino group are selectively removed to allow coupling of the next amino acid of interest. This process is repeated until the desired peptide is fully synthesized, at which point the peptide can be cleaved from the support and purified. A non-limiting example of an instrument for solid phase peptide synthesis is the Aapptec Endeavor90 peptide synthesizer.

[0168] To allow the coupling of further amino acids, the Fmoc protecting group can be removed from the amino acids on the solid support under mildly basic conditions, for example piperidine in DMF (20-50% v / v). The amino acid to be added must also be activated for coupling (e.g., with the alpha carboxylate). Non-limiting examples of activating reagents include, but are not limited to, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). Racemization is minimized by using triazoles such as 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt). Coupling can be carried out in the presence of a suitable base such as N,N-diisopropylethylamine (DIPEA / DIEA). For longer peptides or if desired, peptide synthesis and ligation can be used.

[0169] Apart from the formation of a typical peptide bond to extend a peptide, the peptide can be extended in a branched manner by coupling to side chain functional groups (e.g., carboxylic acid or amino groups), either side chain to side chain, or side chain to backbone amino or carboxylate. Coupling to amino acid side chains can be performed by known methods and can be performed on-resin or off-resin. Non-limiting examples include the formation of amides between an amino acid side chain containing a carboxyl group (e.g., Asp, D-Asp, Glu, D-Glu, etc.) and an amino acid side chain containing an amino group (e.g., Lys, D-Lys, Orn, D-Orn, Dab, D-Dab, Dap, D-Dap, etc.) or the peptide N-terminus, the formation of amides between an amino acid side chain containing an amino group (e.g., Lys, D-Lys, Orn, D-Orn, Dab, D-Dab, Dap, D-Dap, etc.) and either an amino acid side chain containing a carboxyl group (e.g., Asp, D-Asp, Glu, D-Glu, etc.) or the peptide C-terminus, and the formation of 1,2,3-triazoles between an amino acid side chain containing an azide group (e.g., Lys(N3), D-Lys(N3), etc.) and an alkyne group (e.g., Pra, D-Pra, etc.) via click chemistry. Although the protecting groups on the appropriate functional groups must be selectively removed prior to amide bond formation, the reaction between alkynes and azide groups via a click reaction to form 1,2,3-triazoles does not require selective deprotection. Non-limiting examples of selectively removable protecting groups include 2-phenylisopropyl ester (O-2-PhiPr) (e.g., on Asp / Glu) and 4-methyltrityl (Mtt), allyloxycarbonyl (alloc), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) ​​(e.g., on Lys / Orn / Dab / Dap). O-2-PhiPr and Mtt protecting groups can be selectively deprotected under mild acidic conditions such as 2.5% trifluoroacetic acid (TFA) in DCM.The Alloc protecting group can be selectively deprotected using tetrakis(triphenylphosphine)palladium(0) and phenylsilane in DCM. The Dde and ivDde protecting groups can be selectively deprotected using 2-5% hydrazine in DMF. The deprotected side chains of Asp / Glu (L- or D-form) and Lys / Orn / Dab / Dap (L- or D-form) can then be coupled, for example by using the coupling reaction conditions described above.

[0170] The peptide backbone amide may be N-methylated (i.e., alpha aminomethylated). This can be achieved by directly using Fmoc-N-methylated amino acids during peptide synthesis. Alternatively, N-methylation may be performed under Mitsunobu conditions. First, the free primary amine groups are protected using 4-nitrobenzenesulfonyl chloride (Ns-Cl) and 2,4,6-trimethylpyridine (collidine) solution in NMP. N-methylation can then be achieved in the presence of triphenylphosphine, diisopropyl azodicarboxylate (DIAD) and methanol. N-deprotection can then be performed using mercaptoethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in NMP. HATU, HOAt and DIEA can be used to couple the protected amino acid to the N-methylated alpha amino group.

[0171] Coupling between the linker and different components of the compounds / conjugates of the invention may require the formation of thioether (-S-) or ether (-O-) bonds, which can be achieved either in solid phase or solution phase. For example, the formation of thioether (-S-) bonds can be achieved by coupling between a thiol-containing compound (such as a thiol group on a cysteine ​​side chain) and an alkyl halide (such as 3-(Fmoc-amino)propyl bromide) in a suitable solvent (such as N,N-dimethylformamide) in the presence of a base (such as N,N-diisopropylethylamine). The formation of an ether (-O-) bond can be achieved by the Mitsunobu reaction between an alcohol (such as a hydroxyl group on a serine or threonine side chain) and a phenol group (such as a tyrosine side chain) in the presence of triphenylphosphine and diisopropyl adicarboxylate (DIAD) in an aprotic solvent (such as 1,4-dioxane). When the reaction is carried out in solution phase, the reactants used are preferably in an equivalent molar ratio (1 to 1), and the desired product can be purified by flash column chromatography or high performance liquid chromatography (HPLC). When the reaction is carried out in solid phase, i.e., one reactant is bound to a solid phase, the other reactant is usually used in excess (3 or more equivalents of the reactant bound to the solid phase). After the reaction, excess unreacted reactants and reagents can be removed by sequentially washing the solid phase (resin) with a combination of solvents, such as, for example, N,N-dimethylformamide, methanol, and dichloromethane.

[0172] Non-peptide moieties (e.g., radiolabeled groups, albumin binding groups and / or linkers) can be coupled to the peptide N-terminus while the peptide is attached to the solid support. This is easier if the non-peptide moiety contains an activated carboxylate (and optionally protected groups) so that coupling can be performed to the resin. For example, but not limited to, a bifunctional chelator such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tris(tert-butyl ester) can be activated in the presence of N-hydroxysuccinimide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC) for coupling to the peptide. Alternatively, non-peptide moieties can be incorporated into the compound via copper-catalyzed click reaction under either solution or solid-phase conditions. Copper-catalyzed click reaction is well established in the art. For example, 2-azidoacetic acid is first activated by NHS and DCC and coupled to the peptide. The alkyne-containing non-peptide moiety is then reacted with Cu in water and organic solvents such as acetonitrile (ACN) and DMF. 2+ and in the presence of sodium ascorbate, it can be clicked onto an azide-containing peptide.

[0173] The synthesis of radioactive metal chelators is well known, and many chelators are commercially available (e.g., Sigma-Aldrich™ / MilliporeSigma™, etc.). Protocols for the conjugation of radioactive metals to chelators are also well known (see, e.g., Example 1 below).

[0174] In general, BF3-containing motifs can be attached to linkers via click chemistry by forming a 1,2,3-triazole ring between a BF3-containing azide (or alkynyl) group and an alkynyl (or azide) group on the linker, or by forming an amide bond between a BF3-containing carboxylate and an amino group on the linker. To generate a BF3-containing azide, alkyne, or carboxylate, a boronic ester-containing azide, alkyne, or carboxylate is first prepared, followed by conversion of the boronic ester to BF3 in a mixture of HCl, DMF, and KHF2. In the case of alkylBF3, a boronic ester-containing azide, alkyne, or carboxylate can be prepared by coupling a boronic ester-containing alkyl halide (such as iodomethylboronic acid pinacol ester) with an amine-containing azide, alkyne, or carboxylate (e.g., N,N-dimethylpropargylamine). In the case of aryl BF3, the boronic ester can be prepared by Suzuki coupling using an aryl halide (iodine or bromide) and bis(pinacolato)diboron.

[0175] 18 F- 19 of BF3-containing moieties via F isotope exchange reaction 18 F fluorination can be accomplished following previously published procedures (Liu et al., Nat Protoc 2015 10:1423-1432, incorporated by reference in its entirety). In general, approximately 100 nmol of BF3-containing compound is dissolved in a mixture of 15 μl of pyridazine-HCl buffer (pH=2.0-2.5, 1 M), 15 μl of DMF, and 1 μl of 7.5 mM KHF2 aqueous solution. 18 F-fluoride solution (60 μl in saline) is added to the reaction mixture and the resulting solution is heated for 20 min at 80° C. At the end of the reaction, the desired product can be purified by solid phase extraction or inverse high performance liquid chromatography (HPLC) using a mixture of water and acetonitrile as the mobile phase.

[0176] When the peptide is fully synthesized on the solid support, the desired peptide / compound can be cleaved from the solid support using appropriate reagents such as TFA, triisopropylsilane (TIS) and water. Side chain protecting groups such as Boc, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), trityl (Trt) and tert-butyl (tBu) are simultaneously removed (i.e., deprotected). The crude peptide / compound can be precipitated and recovered from the solution by adding cold ether followed by centrifugation. Purification and characterization of the peptide can be performed by standard separation techniques such as high performance liquid chromatography (HPLC) based on the size, charge and polarity of the peptide. The identity of the purified peptide can be confirmed by mass spectrometry or other similar approaches.

[0177] Exemplary synthetic schemes are provided in the Examples.

[0178] As noted above, the compounds / conjugates disclosed herein are dual-modal in that they are suitable for imaging or radiotherapy, or can be used for both applications. For example, the compounds / conjugates can be 18 When F-labeled ("hot-F"), they may be used as imaging / diagnostic agents, or when chelated with therapeutic radioactive metal isotopes ("hot-M"), they may be used as therapeutic agents.

[0179] Thus, in some embodiments, the metal chelator is hot-F / cold-M, i.e. 18F labeled (preferred for PET) and either unchelated or chelated with a non-radioactive metal isotope. Such embodiments are useful as imaging or diagnostic agents without the adverse effects from radioactive metal isotopes. If imaging reveals that the subject is a candidate for therapeutic treatment, the same compound / complex can be administered (either as cold-F / hot-M or hot-F / hot-M). Thus, hot-F / cold-M compounds / complexes are useful as companion diagnostics to hot-M therapeutics. Thus, 18 F is a non-radioactive metal isotope, e.g. 89 Y, 174 Lu, 208 Used to image binding compounds / complexes chelated to Pb. Patients who show positive images with hot-F / cold-M isotopes can be treated with radiotoxic isotopes containing cold-F / hot-M.

[0180] BF3-containing partial group is 18 When F labeled (i.e., hot-F), the use of the compounds / conjugates to prepare radiolabeled tracers for imaging tissues expressing the cell markers / antigens in a subject is disclosed. A method of imaging tissues expressing the cell markers / antigens in a subject is also disclosed, which comprises administering to the subject a composition comprising the compounds / conjugates and a suitable excipient, and imaging the tissue of the subject using, for example, PET or SPECT. If the tissue is a diseased tissue, targeted therapy using the hot-M version can be selected to treat the subject.

[0181] When the metal chelator is chelated to a therapeutic radioisotope, the use of the compound / conjugate (or a pharmaceutical composition thereof) is disclosed for treating a subject with a condition or disease associated with expression of a cellular marker / antigen. Accordingly, there is provided the use of the compound in the preparation of a medicament for treating a subject with a condition or disease associated with expression of a cellular marker / antigen. Also disclosed is a method of treating a condition or disease in a subject, the method comprising administering to the subject a composition comprising the compound / conjugate and a pharma- ceutically acceptable excipient.

[0182] Various embodiments of the compound or molecular complex include 18 Compounds or molecular conjugates labeled with F can be used to image a subject to identify the presence of cellular markers of a disease or condition, and compounds or molecular conjugates chelated with therapeutic radioisotopes can be used to treat a disease or condition. Thus, 18 administering to a subject an F-labeled compound or a molecular complex of theranostic pair; 18 Methods are disclosed that include imaging a subject using a F-labeled compound or molecular conjugate to identify the presence of a cellular marker for the disease or condition, and treating the disease or condition by administering a radioactive metallated compound or molecular conjugate of the theranostic pair. In some embodiments, the method / use comprises: 18 F / 19 and performing a F isotope exchange reaction to prepare a radiofluorinated compound / complex. In some embodiments, the method / use further comprises performing a chelation reaction to prepare a radiometallated compound / complex. In some embodiments, the compound / complex used in the diagnostic step is metallated with a non-therapeutic isotopic substitute of the therapeutic metal chelated to the compound / complex used in the therapeutic step.

[0183] For example, but not limited to, the cell targeting domain may be LLP2A and the tissue to be imaged may be a VLA-4 expressing tissue. For example, but not limited to, the cell targeting domain may be LLP2A and the condition or disease may be a VLA-4 expressing condition or disease such as multiple myeloma, leukemia and other hematological malignancies, as well as melanoma, multiple sclerosis, asthma, Crohn's disease, inflammatory bowel disease, etc. For example, but not limited to, the compound may be DOTA-AMBF3-PEG2-LLP2A.

[0184] For example, but not limited to, the cell targeting domain may be PSMA-617 and the tissue to be imaged may be a PSMA-expressing tissue. For example, but not limited to, the cell targeting domain may be PSMA-617 and the disease may be a PSMA-expressing cancer. For example, but not limited to, the compound may be PSMA-617-LysAMBF3-DOTA. PSMA expression has been detected in various cancers (e.g., Rowe et al., 2015, Annals of Nuclear Medicine 29:877-882; Sathekge et al., 2015, Eur J Nucl Med Mol Imaging, 42:1482-1483; Verburg et al., 2015, Eur J Nucl Med Mol Imaging, 42:1622-1623; and Pyka et al., J Nucl Med 2015 Nov. 19, jnumed.115.164442). Thus, without limitation, the PSMA-expressing cancer may be prostate cancer, renal cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, brain cancer, melanoma, neuroendocrine tumors, ovarian cancer, or sarcoma. In some embodiments, the cancer is prostate cancer.

[0185] For example, but not limited to, the cell targeting domain may be TATE and the tissue to be imaged may be a somatostatin receptor expressing tissue. For example, but not limited to, the cell targeting domain may be TATE and the condition or disease may be a somatostatin receptor expressing condition or disease, such as neuroendocrine tumors, breast cancer, small cell lung cancer, lymphoma, meningioma, pituitary adenoma and pancreatic cancer. For example, but not limited to, the compound may be DOTA-Lys(AMBF3)-TATE.

[0186] For example, but not limited to, the cell targeting domain may be bombesin or a bombesin derivative (e.g., D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) and the tissue to be imaged may be a GRPR expressing tissue. For example, but not limited to, the cell targeting domain may be bombesin or a bombesin derivative (e.g., D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) and the condition or disease may be a GRPR expressing condition or disease. Aberrant or ectopic GRPR expression has been detected in a variety of conditions and diseases, including psychiatric / neurological disorders, inflammatory diseases and cancer. Thus, but not limited to, the GRPR expressing condition or disease may be a psychiatric disorder, a neurological disorder, an inflammatory disease, prostate cancer, lung cancer, head and neck cancer, colon cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, breast cancer, glioma or neuroblastoma. In some embodiments, the cancer is prostate cancer. For example, but not limited to, the compound may be DOTA-Lys(AMBF3)-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2.

[0187] Uncited references JPEG0007659268000024.jpg98167 JPEG0007659268000025.jpg224167 JPEG0007659268000026.jpg223167 JPEG0007659268000027.jpg62167

[0188] The invention is further illustrated in the following examples.

[0189] example

[0190] 18 The F-labeled companion diagnostic agent is chemically identical to the radiotherapeutic agent. 18 Since F is preferred, hot-F / cold-M isotopes for imaging can be prepared, which serve as companion diagnostics to cold-F / hot-M isotopes that can be used therapeutically. 18 F can be used to synthesize non-radioactive metal isotopes, e.g. 89 Y, 174 Lu, 208 Pb-chelated peptides can be imaged, and patients who image positive with the hot-F / cold-M isotope substitute can then be treated with a radiotoxic isotope substitute containing cold-F / hot-M.

[0191] For treatment, radioactive toxic metals (e.g. 90 Y or 177 Lu 50、51 For pre-treatment imaging, e.g. for SPECT, 111 For In or PET 64 Different diagnostic metals are usually used, such as Cu. This practice creates problems in correlating images with potential treatment outcomes. For example, DOTA-TATE is used for SPECT imaging. 111 Labeled with In and then used for treatment 90 Y is labeled, but there are clear differences in uptake. 86 Using the same tracer as Y is expensive and not readily available 45、46 .therefore, 18For labeling with F-fluoride, there are advantages to peptides that contain both a metal chelator, such as DOTA, as well as a pendant organotrifluoroborate. Finally, the chelator can be used for metal chelation to either radiotoxic metals for therapeutic use, or non-radioactive metals that can be used as substitutes for radiotoxins (i.e., radiotoxic metals), or for other purposes including altering the PKPC of F-18 labeled tracers, but compounds / complexes (tracers) with both a chelator and a BF3 prosthetic group can be excellent PET imaging agents.

[0192] First, two compositions were prepared based on PSMA-targeted urea and the integrin-targeted peptidic ligand LLP2A (see FIG. 2). Surprisingly, isotope exchange afforded 18 When labeled with F-fluoride, these nonmetallated compositions provide excellent tumor uptake values ​​with exceptional tumor:nontumor ratios (see Tables 5, 6, 7 and Figures 3 and 4).

[0193] Cell culture method

[0194] B16F10 melanoma cell line (Mus musculus) was purchased from ATTC (CRL-6475). The cell line was confirmed to be pathogen-free by the IMPACT1 Mouse Profile Test (IDEXX BioResearch). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, StemCell Technologies) supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a humidified incubator containing 5% CO2. Cells grown to approximately 90% confluence were washed with sterile 1x PBS (pH 7.4) and then trypsinized.

[0195] The LNCaP cell line was obtained from ATCC (LNCaP clone FGC, CRL-1740). The cell line was established from a metastatic site in the left supraclavicular lymph node of a human prostate adenocarcinoma. Cells were cultured in PRMI 1640 medium supplemented with 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37 °C in a humidified incubator containing 5% CO2. Cells grown to 80-90% confluence were then washed with sterile phosphate-buffered saline (1x PBS pH 7.4) and trypsinized.

[0196] In vivo biodistribution and PET / CT imaging studies of F-18 LLP2A-Lys-AMBF3-DOTA

[0197] All animal experiments were performed in accordance with guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee at the University of British Columbia. Mice were housed under pathogen-free conditions and maintained on a 12-h light-dark cycle at the Animal Resource Centre, BC Cancer Research Centre, Vancouver, Canada. PET imaging and biodistribution studies were performed using male C57BL / 6J mice. For tumor implantation, mice were anesthetized by inhalation with 2% isoflurane in 2.0 L / min oxygen and injected with 1 × 10 6 B16F10 cells were implanted subcutaneously on the right dorsum at the level of the forelimb. When the tumors grew to reach a diameter of 8-10 mm in 8-10 days, the mice were imaged or used for biodistribution studies.

[0198] PET / CT imaging studies were performed on a microPET / CT scanner (Inveon, Siemens). Briefly, for static PET scans, each tumor-bearing mouse was injected with 4–6 MBq of F-18-labeled LLP2A-Lys-AMBF3-DOTA via the caudal lateral tail vein under isoflurane sedation. For blocking studies, mice were co-injected with 100 μg of non-radioactive LLP2A-Lys-AMBF3-DOTA. After injection, mice were allowed to recover and roam freely in their cages. After 50 min, mice were sedated again and placed in the scanner. A baseline CT scan was obtained for localization and attenuation correction. This was followed by a 10-min static PET scan. Mice were kept warm by a heating pad during acquisition. Mice were euthanized using CO2 inhalation after static PET imaging, followed by biodistribution. PET images were reconstructed using an ordered subset expectation maximization and maximum a posteriori probability algorithm (OSEM3D / MAP) with 2 OSEM3D iterations followed by 18 MAP iterations at a required resolution of 1.5 mm.

[0199] Mice for biodistribution studies only were anesthetized by 2% isoflurane inhalation and injected with 1–2 MBq of F-18-labeled LLP2A-Lys-AMBF3-DOTA. For blocking studies, 100 μg of non-radioactive LLP2A-Lys-AMBF3-DOTA was co-injected with the radioactive compound. After injection, mice were allowed to recover and roam freely in their cages, and euthanized 1 h later by CO2 inhalation. Blood was immediately drawn, and organs of interest were harvested, rinsed in 1x PBS (pH 7.4), and blotted dry. Each organ was weighed, and radioactivity in the collected tissue was measured using a WIZARD 2480 (PerkinElmer), normalized to the injected dose using a standard curve, and expressed as a percentage of the injected dose per gram of tissue (%ID / g).

[0200] In vivo biodistribution and PET / CT imaging studies of F-18 PSMA-Lys-AMBF3-DOTA

[0201] Imaging and biodistribution experiments were performed using NODSCID 1L2RγKO male mice. Mice were maintained and experiments were performed in accordance with guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee at the University of British Columbia. Mice were anesthetized by inhalation with 2% isoflurane in oxygen and 1 × 10 7 LNCaP cells were implanted subcutaneously. When tumors had grown to reach a diameter of 5-8 mm over a period of 5-6 weeks, mice were imaged or used for biodistribution studies.

[0202] PET imaging experiments were performed using a Siemens Inveon (Erlanger, Germany) microPET / CT scanner. Each tumor-bearing mouse was injected with 6–8 MBq of F-18-labeled PSMA-Lys-AMBF3-DOTA via the tail vein under anesthesia (2% isoflurane in oxygen). For blocking, the mouse was co-injected with non-radioactive DCFPyL (0.5 mg). The mouse was allowed to recover and roam freely in its cage. After 50 min, the mouse was again sedated with 2% isoflurane in oxygen inhalation and placed in the scanner. A 10-min CT scan was first performed for localization and attenuation correction after segmentation to reconstruct the PET images. Then, 10 min of static PET imaging was performed to determine uptake in the tumor and other organs. The mouse was kept warm by a heating pad during acquisition.

[0203] For biodistribution studies, mice were injected with radioactive tracer as described above. For blocking, mice were co-injected with non-radioactive DCFPyL (0.5 mg). After 1 h, mice were anesthetized by 2% isoflurane inhalation and euthanized by CO2 inhalation. Blood was immediately drawn from the heart and organs / tissues of interest were harvested. Harvested organs / tissues were weighed and counted using an automated gamma counter. Uptake in each organ / tissue was normalized to the injected dose using a standard curve and expressed as %ID / g.

[0204] Example 1: DOTA conjugation for VLA-4 targeted PET imaging of melanoma 18 Tailored biodistribution of F-LLP2A trifluoroborate radiotracer

[0205] 1.1 Summary

[0206] The very late transcellular antigen 4 (VLA-4) is associated with tumor metastasis, drug resistance, and is overexpressed by many cancers. Previous reports have shown that chelated LLP2A conjugates 64 Cu and 68 More recently, using Ga, 18 F]RBF3 radioactive prosthetic group was used 18 We have shown that PET imaging of VLA-4-expressing melanoma tumors using F has been successful. However, these previous 18 [F]RBF3-PEG2-LLP2A derivatives showed moderate tumor uptake values ​​and significant accumulation in the GI tract. To address this, we designed novel RBF3-LLP2A bioconjugates with appended DOTA moieties that increase tumor uptake and decrease GI accumulation. Methods: Herein, we 18 We describe the synthesis of a modified LLP2A-PEG-NH2 (1) conjugate equipped with F-trifluoroborate radioactive prosthetic group, AMBF3, and DOTA moieties. The precursor DOTA-AMBF3-PEG2-LLP2A (6) was radiolabeled by isotope exchange and purified by semi-preparative HPLC and C18 cartridge elution. Male C57BL / 6J mice bearing B16-F10 melanoma tumors overexpressing the VLA-4 target were used to perform static and dynamic PET scans, biodistribution studies, and [ 18 Using a blocking combination by co-injecting excess 1 relative to DOTA-[F]6, 18 F]AMBF3-PEG2-LLP2A([ 18 F]6) was evaluated. Results: Precursor 6 was synthesized, 18F-labeling was performed to obtain [ 18 F]6 formulation was obtained. 18 In vivo static PET imaging of [F]6 showed clear tumor visualization, and biodistribution studies showed tumor uptake was 9.46 ± 2.19% of the injected dose per gram of tissue (%ID / g), with high tumor:muscle and tumor:blood contrast ratios of approximately 8 and 10, respectively. Blocking enhanced the expression of [F]6 against VLA-4 in tumors and bone marrow. 18 The specificity of [F]6 was confirmed. Dynamic PET showed that the 18 F]6 clearance and intestinal accumulation were previously investigated [ 18 F]RBF3-PEG2-LLP2A, but splenic uptake was enhanced to levels similar to previously reported LLP2A-chelator radiotracers. 18 We highlight [F]6 as a promising VLA-4 radiotracer and demonstrate how the biodistribution of the LLP2A radiotracer can be rerouted from the GI tract to the spleen and bladder.

[0207] 1.2 Introduction

[0208] The transcellular very late antigen 4 (VLA-4) receptor is overexpressed in several cancers (1-15) and correlates with tumor metastasis (11, 16-18) and resistance to chemotherapy (19). It therefore represents a versatile biomarker for imaging with the high-affinity peptidomimetic ligand LLP2A. Since its initial development by Lam et al. (20), the LLP2A pharmacophore has been extensively used in NIR fluorescence imaging (21), 111 In(22) and 99m Single-photon emission computed tomography (SPECT) using Tc(23), and 68 Ga and 64 In vivo VLA-4 targeting has been extensively validated by Cu-based positron emission tomography (PET) ( 23 – 27 ). 18There has been only one report of PET imaging with F, in which LLP2A-PEG-NH2 (1) was attached to one of two radioactive prosthetic groups, ammonium dimethyl-trifluoroborate (AMBF3) and N-pyridinyl-para-trifluoroborate (N-Pyr-p-BF3), and then photoinduced PET imaging in a single step. 18 We successfully F-labeled and performed PET imaging in mice bearing B16-F10 melanoma tumors (28). One hour after injection (pi), these two 18 Tumor uptake values ​​for [F]RBF3-PEG2-LLP2A were moderate (2.8 and 4.4%ID / g), whereas high intestinal accumulation (approximately 52%ID / g) was observed.

[0209] In contrast, LLP2A conjugates to chelated radiometals consistently show tumor uptake values ​​in the range of 10-15% ID / g at 1-2 h pi using the same mouse melanoma tumor model (23-27). Nevertheless, radiometallated LLP2A-chelator conjugates are consistently sequestered in the spleen due to partial clearance by the reticuloendothelial system (RES). As previously mentioned [ 18 Since F]RBF3-PEG2-LLP2A exhibited hydrophobic properties resulting in suboptimal imaging, we hypothesized that incorporation of the hydrophilic chelator DOTA into the RBF3-PEG2-LLP2A bioconjugate would favor renal clearance. Thus, to test this hypothesis, we attached 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the nearly identical AMBF3-PEG2-LLP2A precursor scaffold. For this purpose, we also designed a novel amino acid, Fmoc-Lys-(AMBF3), which was grafted and deprotected to allow further addition of DOTA moieties. The resulting conjugate DOTA-[ 18 F]AMBF3-LLP2A(6) 18 F / 191 was labeled by F isotope exchange (IEX) (28, 29) and investigated by in vivo PET imaging (static and dynamic scans), biodistribution studies, and VLA-4 blocking studies using 1 as a validated blocking agent. To the best of our knowledge, this report is the first to report the use of any DOTA-chelator appended with 1 to tailor biodistribution to favor renal clearance. 18 This represents the first investigation of an F-labeled peptide radiotracer.

[0210] 1.3 Materials and Methods.

[0211] Synthesis of DOTA-AMBF3-PEG2-LLP2A (6). The LLP2A peptide mimetic was synthesized on solid phase as previously reported (20), using O-bis-(aminoethyl)ethylene glycol trityl resin to anchor the PEG2 spacer and -NH2 conjugation handle as previously reported (28, 30). LLP2A-PEG2-NH2 (1) (4.2 mg, 4.46 μmol, 1 equiv.) was dissolved in 200 μL (1:19) DIPEA:DMF (v / v) and then used to dissolve Fmoc-Lys-(AMBF3)-NHS (2) (5.85 mg, 8.91 μmol, 2 equiv.), the synthesis of which is shown in the Supplementary Information (Scheme 2). The conjugation proceeded for 2 h at room temperature. The mixture was concentrated (~50 μL-100 μL) by speed-vac, precipitated with 1.0 mL of Et2O, and centrifuged. The supernatant was removed and the product was redissolved in 50 μL of DMF. The described Et2O precipitation / centrifugation method was repeated and the final pellet was dried by speed-vac. These methods afforded 6.5 mg (~4.3 μmol) of product in near quantitative yield of LLP2A-PEG2-AMBF3-Fmoc (3). ESI-MS (+): C 76 H 98 BF3N 14 O 13 The calculated m / z value for is 1483.5, and the measured value is [M+Na] + =1506.8m / z, [M+2Na-H] += 1528.8 m / z (Figure 9). TLC: [(1:19) NH4OH:EtOH, v / v], R f = 0.46 (visible at 254 nm). Intermediate 3 (1.5 mg, 1 μmol, 1 equiv) was dissolved in 200 μL (1:4) piperidine:DMF (v / v) and Fmoc removal was achieved within 2 h at room temperature. The mixture was concentrated and subjected to the described Et2O precipitation / centrifugation method twice. The final pellet was dried by speed-vac to give approx. 1.3 mg (approx. 1 μmol) of AMBF3-PEG2-LLP2A-NH2 (4) in quantitative yield. ESI-MS (+): C 61 H 88 BF3N 14 O 11 The calculated m / z value is 1261.3, and the measured value is [M+H] + =1262.6m / z, [M+Na] + = 1284.6 m / z (Figure 10). TLC: [(1:19) NH4OH:EtOH, v / v], R f = 0.18 (visible at 254 nm, stained with ninhydrin). Intermediate 4 (1.8 mg, 1.4 μmol, 1 equiv.) was dissolved in 100 μL (1:19) DIPEA:DMF (v / v) and conjugated to DOTA-NHS (5) (1.6 mg, 2.1 μmol, 1.5 equiv.) within 2 h at room temperature. The mixture was concentrated and subjected to the described Et2O precipitation / centrifugation method twice. The final pellet was dried by speed-vac to give 2.4 mg (~1.4 μmol) of crude DOTA-AMBF3-PEG2-LLP2A (6). The above procedure was repeated to give 42.1 mg of crude 6 for HPLC purification. These combined samples were dissolved in 1.0 mL (1:1) 0.1% formic acid in MeCN:0.1% formic acid in H2O (v / v) and purified by HPLC method A. The product (6) was eluted with t R The mixture was collected at 0.5°C for 8.9 min, diluted with an equal volume of HO, frozen on dry ice, and lyophilized. These methods afforded 3.1 mg (1.9 μmol) of purified (>95% purity) radioactive tracer precursor 6 (characterized by ESI-MS, FIG. 11 and HPLC, FIG. 12) in 7.4% yield. ESI-MS (+): C 77 H 114 BF3N18 O 18 The calculated m / z value is 1647.6, and the measured value is [M+H] + =1648.9m / z, [M+Na] + =1670.9m / z, [M+K] + = 1687.0 m / z. TLC: [(1:19) NH4OH:MeOH], R f = 0.67 (visible at 254 nm, stained light blue with bromocresol green).

[0212] Scheme 2: Synthesis scheme of Fmoc-Lys(AMBF3)-O-NHS. [ka]

[0213] ESI-MS Characterization. Mass spectra were acquired using a Waters ZQ spectrometer with either MeOH or CH3CN as the mobile phase.

[0214] HPLC methods and characterization. Precursor 6 was purified by HPLC method A using an Agilent 1100 HPLC (autosampler unit and multichannel PDA detector) from Chromatographic Specialties. A Knauer Smartline Pump 100 and a Bioscan radiation detector were used to purify precursor 6 by HPLC method B. 18 The purpose of the experiment was to purify F radiotracer 7. An Agilent Technologies 1200 HPLC (single channel 1200 series PDA detector and Bioscan radiodetector coupled by an Agilent Interface 35900E) was used to generate standard curves for precursor 6 and for QC of the seven formulations prior to animals by HPLC method C.

[0215] HPLC Method A: Purification and Analysis of 6. Agilent Eclipse XDB-C18, 9.4 mm x 250 mm, 5 μm column, Solvent A = MeCN with 0.1% formic acid (v / v) and Solvent B = HO with 0.1% formic acid (v / v), flow rate = 2.0 mL / min, absorbance channels = 257 nm (purification) and 241 nm (analysis). Gradient: i) 25% to 80% solvent A for 15 min, ii) 80% to 100% solvent A for 1 min, iii) 100% solvent A for 6 min, iv) 100% to 25% solvent A for 1 min, v) 25% solvent A for 6 min.

[0216] HPLC method B:[ 18 [F] Purification of 6. Phenomenex Luna C18-100A, 10 mm x 250 mm, 5 μM column, solvent A = HO with 0.1% TFA (v / v) and solvent B = MeOH with 0.1% TFA (v / v), flow rate = 4.5 mL / min, absorbance channel = 257 nm. Initially, an isocratic mixture containing 40% solvent B (v / v) was used for 12 min, then switched to 100% solvent B (v / v) for another 12 min. 18 F radioactive tracer [ 18 [F]6 was collected in approximately 21 to 23 minutes.

[0217] HPLC method C:[ 18 Analysis of [F]6. Phenomenex Jupiter C18-300A, 4.6 mm x 250 mm, 10 μm column, solvent A = HO with 0.1% TFA (v / v), solvent B = MeCN with 0.1% TFA (v / v), flow rate = 2.0 mL / min, absorbance channel = 257 nm. Gradient: i) 0% solvent B for 4 min, ii) 0% to 40% solvent B for 2 min, iii) 40% solvent B for 4 min, iv) 40% to 80% solvent B for 4 min, v) 80% solvent B for 11 min, vi) 80% to 0% solvent B for 3 min, and vii) 0% solvent B for 4 min.

[0218] Synthesis of Fmoc-Lys-AMBF3-NHS

[0219] Chemicals, solvents, hardware. 3-Dimethylamino-1-propyne and trifluoromethylsulfonyl anhydride were purchased from Sigma-Aldrich. Iodomethylboronic acid pinacol ester was purchased from Frontier Scientific. Potassium difluoride (KHF2) was purchased from Acros Organics. Sodium azide was purchased from Honeywell Riedel-de Haen. Fmoc-Lys-OH was purchased from Novabiochem. Copper sulfate, ascorbic acid, sodium hydroxide, and potassium carbonate were purchased from Fisher Scientific. N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide were purchased from Alfa Aesar. Flash chromatography was performed using silica gel (230-400 mesh) purchased from Silicycle. Thin layer chromatography (TLC) was performed using silica gel 60 F purchased from EMD Chemicals. 254 So I went. 1 H, 19 F and 13 C NMR spectra were recorded on a Bruker AV300 instrument at room temperature. Deuterated solvents acetonitrile-d3, chloroform-d and acetone-d6 were purchased from Sigma-Aldrich. Electrospray ionization mass spectrometry (ESI-MS) was performed using a Micromass LCT instrument. HRMS was performed using a Waters-Micromass LCT equipped with a time-of-flight (TOF) detector.

[0220] N-Propargyl-N,N-dimethylammoniomethylboronyl pinacolatoate. Ar (g)A flame-dried round-bottom flask was charged with 1.5 mL of 3-dimethylamino-1-propyne (13.8 mmol, 1.01 equiv.) and dissolved in 28 mL of Et2O. A 3.67 g sample of iodomethylboronic acid pinacol ester (13.7 mmol, 1 equiv.) was added dropwise and the reaction was stirred at room temperature for 30 min. The product precipitated as a white solid and was collected by vacuum filtration. The crude product was washed with cold Et2O. The crude product was transferred to a pre-weighed scintillation vial and dried in vacuum. This afforded 3.4 g (9.81 mmol) of 1 as a pale beige powder in 72% yield.

number

[0221] N-Propargyl-N,N-dimethylammoniomethyl-trifluoroborate. 3M KHF 2(水溶液) and 4M HCl (水溶液) A stock solution of the following was prepared: Compound 1 (1.87 g, 5.33 mmol, 1 equiv.) was placed in a 50 mL Falcon tube containing 10 mL of CH3CN. A 9 mL volume of 3 M KHF was added. 2(水溶液) (27 mmol, 5.1 equiv.) and 8 mL of 4 M HCl (水溶液) (32 mmol, 6 equiv.) was added and the solution was stirred with heating at 45° C. for 2 h. The solution was then diluted with concentrated NH4OH (水溶液) The crude product was neutralized to pH 7 with 100 mL of ethyl acetate to form a biphasic layer of CH3CN and aqueous layers. The top layer (organic CH3CN layer) was collected in a 250 mL round bottom flask and the aqueous layer was washed with CH3CN (3 × 10 mL). The crude product was dried by rotary evaporation. A 20 mL volume of acetone was used to further precipitate salts from the crude product sample and the mixture was filtered using a glass sintered funnel. The filtrate was collected in a round bottom flask, concentrated by rotary evaporation and further dried in vacuum. The crude product was then washed with 20 mL of diethyl ether and then 20 mL of chloroform to dissolve any remaining excess pinacol. The crude sample was dissolved in a minimum of acetone and subjected to column chromatography (silica gel, 43-60 μm, 230-400 mesh, ca. 40 g, isocratic acetone) and TLC (acetone, Rf = 0.41, stained with I2) while monitoring the eluted fractions. Fractions containing purified 2 were pooled, concentrated by rotary evaporation, and further dried in vacuum. The solid product was collected in a pre-weighed vial and dried in vacuum. This gave 911 mg (5.52 mmol) of 2 as a pale orange solid in quantitative yield.

number

[0222] Fmoc-Lys(N3)-OH. Trifluoromethylsulfonyl azide was used for diazotransfer of the primary amine of Fmoc-Lys-OH. A 2.94 g sample of NaN3 (45.2 mmol, 8.63 equiv.) was placed in a round bottom flask and dissolved in 20 mL of (2:3) H2O:CHCl2 (v / v). While the mixture was stirred, 1.02 mL of trifluoromethanesulfonic anhydride (Tf2O) (6 mmol, 1.15 equiv.) was added dropwise over 30 min. The reaction was stirred in an ice-water bath at approximately 0 °C for 5 h. The organic layer was extracted with CHCl2 (2 × 20 mL) followed by washing the collected organic layer with saturated NaHCO 3(水溶液) (20 mL). Trifluoromethylsulfonyl azide was dissolved in the collected organic layer as described. A 1.93 g sample of Fmoc-Lys-OH (5.24 mmol, 1 equiv.) was placed in a round bottom flask containing 2.32 g K2CO3 (16.8 mmol, 3 equiv.) and 15 mg Cu(II)SO4 (94.0 μmol, 0.39 mol%) and dissolved in 15 mL (1:1) MeOH:H2O (v / v). The organic layer containing trifluoromethanesulfonyl azide was added dropwise over 30 min. The solution was stirred at room temperature for 21 h and the reaction was diluted with 60 mL of 2.5 M HCl. (水溶液)The mixture was quenched with 50 mL of CH2Cl2. The organic layer was extracted with CHCl2 (3 × 20 mL) and washed with brine (2 × 50 mL). The collected organic layer was dried over anhydrous Na2SO4 and salts were removed by vacuum filtration. The collected solution was concentrated by rotary evaporation. Column chromatography (silica gel, 43-60 μm, 230-400 mesh, ca. 80 g, solvent gradient of (0.5:99.5) MeOH:CH2Cl2 (v / v) to (1:99) MeOH:CH2Cl2 (v / v)) was performed using TLC ((1:9) MeOH:CH2Cl2 (v / v), R f Elution was monitored by HPLC (HPLC-MS / HPLC-MS / MS ...

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[0223] Fmoc-Lys(AMBF3)-OH. A fresh stock solution of 1 M sodium ascorbate was prepared by dissolving 1.98 g of sodium ascorbate (0.01 mol) in 10 mL of DI HO. Then, 1 M Cu(II)SO 4(水溶液) A stock solution of was prepared by dissolving 1.60 g of anhydrous Cu(II)SO4 (0.01 mol) in 10 mL of DI HO. A 414.6 mg sample of compound 2 (2.513 mmol, 5.24 equiv.) was placed in a round bottom flask and dissolved in 2.5 mL of (3:2) CH3CN:HO (v / v). A 1.5 mL volume of 1 M Cu(II)SO 4(水溶液)(1.5 mmol, 3.1 equiv) was added first, followed by 3 mL of 1 M sodium ascorbate (3 mmol, 6.2 equiv). A 189 mg sample of compound 3 (479 μmol, 1 equiv) was then added to the solution. 101 mg of K2CO3 (732 μmol, 1.5 equiv) was then added to neutralize the solution to about pH 7, then stirred at 45 °C for 16 h. The mixture was vacuum filtered to remove precipitate, and the filtrate was concentrated by rotary evaporation. The dried crude product was resuspended in (1:1) MeOH:CHCl2 (v / v) (5 × 10 mL), vacuum filtered to remove precipitate, and the filtrate was dried by rotary evaporation. The dried crude product was then resuspended in (5:95) MeOH:CHCl2 (v / v) (5 × 10 mL), vacuum filtered to remove precipitate, and the filtrate was dried again by rotary evaporation. Column chromatography (silica gel, 230-400 mesh, ca. 50 g, solvent: (1:4:95) AcOH:MeOH:CH2Cl2 (v / v / v)) was performed. f Elution fractions were monitored by HPLC (pH 7.0, visible at 254 nm, stained with I2). Fractions containing pure 4 were pooled, concentrated by rotary evaporation, and further dried in vacuum to give 192 mg (343 μmol) of 4 as a dark yellow oil in 71% yield.

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[0224] Fmoc-Lys(AMBF3)-O-NHS. A 163.3 mg sample of 4 (291.7 μmol, 1 equiv.) was added to a round-bottom flask and dissolved in 8 mL of (1:1) CHCl2:CHCN (v / v). A 305 mg sample of DCC (1.48 mmol, 5.07 equiv.) was added to the solution, followed by 170 mg of NHS (1.48 mmol, 5.07 equiv.) and the solution was stirred at room temperature for 21 h. The reaction mixture was vacuum filtered using a sintered funnel and the collected filtrate was concentrated by rotary evaporation. Column chromatography (column diameter = 0.5 cm, silica gel (230-400 mesh), approximately 10 g, gradient of CH2Cl2 to (1:1) CH2Cl2:CH3CN (v / v) increasing with 10% CH3CN (v / v) per 50 mL) was performed using TLC (1:1) CH2Cl2:CH3CN (v / v), R f Elution fractions were monitored by HPLC (HPLC-MS / HPLC-MS / MS ...

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[0225] The structural characterization of Lys-AMBF3 is shown in the accompanying Figures 15 to 33.

[0226] Saturation binding assay. In vitro binding saturation assays were performed on B16-F10 cells according to published procedures (n = 3) (28). Cells were grown to near confluence on 24-well poly-D-lysine plates. Growth medium was removed and reaction medium (RPMI, 1% BSA, 100 U / mL penicillin / streptomycin) was added and incubated at 37 °C for at least 1 h. Increasing concentrations (5 pM–20 nM) of [ 18 [F]6 was added to the cells and incubated for 1 h at 25 °C with gentle agitation. Nonspecific binding was determined by simultaneous addition of [ 18The dissociation constant (K) was determined by repeating the incubation described with [F]6. After incubation, cells were washed twice with ice-cold PBS and harvested after incubation with trypsin, and radioactivity was measured using a WIZARD 2480 gamma counter (PerkinElmer). Values ​​from the nonspecific binding assay were subtracted from the respective values ​​from the specific binding assay. d 's) were determined using GraphPad PRISM 7 with a one-site specific binding model.

[0227] [ 18 Radiolabeling of DOTA-AMBF-PEG-LLP2A (6) with [F]fluoride anion. Precursor 6 (80 nmol) was dissolved in 15 μL of 1 M pyridazine-HCl (pH = 2), 10 μL of DMF, 15 μL of MeOH, and 4 μL of 5 mM KHF. 2(水溶液) (50mol% 19 F - carrier) and the final pH was about 2.0 (Scheme 1). 18 F / H2 18 O (approximately 1 Ci) was passed through an activated 9 mg QMA cartridge (preconditioned with 1.7 mL of brine, then rinsed with 2.5 mL of water and dried with 3 mL of air). 18 F was captured (>95% efficiency) and then eluted (>90% efficiency) into a septum-sealed reaction vessel containing the precursor using approximately 80-100 μL of 0.9% saline. The solution was heated at 82-84 °C for 5 min and then vacuum-sealed for 15 min. 2 mL of 40 mM NH4HCO 2(水溶液) After quenching with 100 mL of 1000 mM KOH (pH = 6.8), the solution was purified by semi-preparative HPLC using two successive isocratic solvent conditions (HPLC method B). 18 The [F]6 was collected and diluted directly into 50 mL of H2O. A small amount of He was added to the container. (g) The resulting solution was passed through a Sep-Pak Light C18 cartridge (pre-washed successively with 9 mL EtOH, 9 mL HO, and 10 mL air) by applying a pressure of 100 MPa. 18[F]6 was dissolved in a septum-sealed vial containing 0.5 mL of (9:1) EtOH:0.9% saline (v / v) and finally formulated with 4 mL of PBS to prepare [F]6 in (1:9) EtOH:PBS (v / v) at pH ∼7 for animal injection. 18 F]6 was obtained. 18 All purified preparations of [F]6 were characterized by analytical HPLC (Method C) to quantitate radiochemical purity, radiochemical yield and molar activity based on a standard curve (Figure 13) prior to animal injection.

[0228] Scheme 1.4 and [ 18 Synthesis of precursor 6 to generate F]6. [ka]

[0229] DOTA-[ 18 F]AMBF3-PEG2-LLP2A([ 18 F]6) PET imaging. B16-F10 cells (ATTC, CRL-6475) were cultured in DMEM containing 10% FBS (v / v), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C and 5% CO 2(g) All animal experiments were performed in accordance with the guidelines of the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia. Male C57BL / 6J mice were anesthetized with 2% isoflurane and cultured at 1 × 10 6 B16-F10 cells were injected subcutaneously into the right shoulder. Tumors were allowed to grow until they reached a diameter of 7–9 mm. PET and CT imaging studies used a microPET / CT scanner (Inveon, Siemens) as previously reported (28, 31–33). For static PET scans (10 min recording), mice were first injected with 4–6 MBq ([ 18Each subject was injected with 100 μg of 1 and subjected to a baseline CT scan (recorded for 10 min) before PET imaging. For static PET images with competitive VLA-4 blocking, 200 μg of 1 was injected with 4–6 MBq of [F]6. 18 The rats were co-injected with [F]6 and both baseline CT and PET images were obtained at 1 hour p.i. using the methods described. Dynamic PET scans were performed under anesthesia as described, with 4–6 MBq of [F]6. 18 The study consisted of injections of [F], a baseline CT scan, and PET scans recorded from 5 s to 52.5 min p.i. (28 time points). After each static PET imaging study, the mice were finally 2(g) Mice were euthanized using inhalation and their organs were harvested for biodistribution measurements.

[0230] DOTA-[ 18 F]AMBF3-PEG2-LLP2A([ 18 Mice were anesthetized using 2% isoflurane and administered 2–3 MBq of [F] via the tail vein. 18 For competitive VLA-4 blocking studies, 200 μg of 1 was injected with 2–3 MBq of [F]6. 18 At 1 h p.i., mice were co-injected with CO 2(g) Mice were euthanized by inhalation, blood was collected, and organs were excised. After rinsing and drying the samples, organs were weighed and radioactivity was recorded using a Wallac WIZARD 2 gamma counter (PerkinElmer) and values ​​were expressed as %ID / g for each organ. Two-tailed ANOVA Sidak's multiple comparison test (GraphPad Prism) was used to calculate the mean [ 18 The statistical significance between the biodistribution of [F]6 alone and the competitive blocking of VLA-4 by co-injection of 1 was evaluated.

[0231] Further details are provided in Lepage et al., ChemBioChem 10.1002 / cbic.201900632.

[0232] 1.4 Results

[0233] Synthesis and VLA-4 affinity of DOTA-AMBF3-PEG2-LLP2A (6). The LLP2A peptidomimetic was synthesized as previously described by others (20) with a modification reported by our laboratory (PEG2-NH2 incorporation upon resin cleavage) (28). The terminal primary amine of intermediate 1 was coupled to Fmoc-protected lysine-AMBF3 conjugate 2 via NHS chemistry in quantitative yield. Intermediate 3 was then subjected to efficient Fmoc removal (intermediate 4) and coupled to unprotected DOTA-NHS (5) under basic conditions. These procedures were repeated to give a substantial crude sample (42.1 mg) of 6 for HPLC purification. This strategy allowed for subsequent synthesis of the lysine-AMBF3 conjugate 2 in quantitative yield. 18 3.1 mg (1.9 μmol) of lyophilized precursor 6 was obtained with high purity (>95% by HPLC) for F radiolabeling. The K d The value is calculated (Figure 14), and other [ 18 K found for the [F]RBF3-PEG2-LLP2A conjugate d was at least 5-fold higher than the values ​​(Figure 8).

[0234] DOTA-[ 18 F]AMBF3-PEG2-LLP2A([ 18 Radiosynthesis of [F]6). Precursor 6 (80 nmol) was radioisotope-treated in acidic aqueous pyridazine buffer via IEX in 71.8 ± 1.0 min (± SD). 18 Radiolabeled with [F] fluoride (in the formulation) 18 F / H2 18 O delivery, n=4). 18 After confirming the identity of the samples as F-labeled derivatives (Figure 5b), [ 18 The mean radiochemical purity of [F]6 was calculated to be 95.9 ± 1.8% (± SD) (Figure 5a). 18 The average radiochemical yield of [F]6 was 4.8 ± 2.9%, and the average molar activity (A m ) was calculated to be 131.72±50.32 GBq / μmol (3.56±1.36 Ci / μmol).

[0235] DOTA-[ 18 F]AMBF3-PEG2-LLP2A([ 18 F]6) PET imaging.

[0236] [ 18 Static PET imaging of F6 showed preferential tracer uptake in the tumor, spleen, kidney, and bone marrow (Figures 6a and 6b) of mice bearing B16-F10 tumors at 1 hour pi (n=2). 18 Blocking studies involving co-injection of [F]6 with 1 (n=2) reduced tracer uptake in tumors and bone marrow to near baseline levels, while activity remained evident in the spleen (Fig. 6c and d). All images showed substantial activity in the bladder and [F]6 via the kidney. 18 Renal clearance of [F]6 was confirmed.

[0237] Dynamic PET scans can be used to measure the 18 The results showed early perfusion of [F]6 and first pass through the liver within 4 min pi (Figure 7). The radiotracer showed efficient uptake into the tumor within 1.9 min pi and stable accumulation in the tumor by 52.5 min pi. VLA-4 targeting was also observed by radiotracer accumulation in the bone marrow. These scans also demonstrated the presence of [F]6 in the intestine during the scan period. 18 The early presence and retention of [F]6 in the intestine was identified. 18 Dynamic PET scans also reproduced the high-contrast static PET images, as [F]6 accumulation remained low in background muscle tissue.

[0238] Biodistribution studies showed that in the spleen 18 High accumulation of [F]6 was evident and was substantially taken up in the lungs and intestine at 1 hour pi (Table 5). Specific VLA-4 binding was also observed in the tumor and bone marrow. 18Co-injection of 1 significantly reduced the uptake of IFN-γ in the spleen (88% lower, p<0.0001), intestine (43% lower, p<0.05), lung (85% lower, p<0.0001), bone and bone marrow (78% lower, p<0.0001), and tumor (75% lower, p<0.0001). 18 The contrast ratio (Table 6) was approximately 7-fold higher in the background muscle and 9-fold higher in the blood pool compared to the [F]6 accumulation. 18 F]6 showed relatively high tumor uptake. VLA-4 blocking was 18 F]6 reduced the contrast of these tumors versus muscle (92% lower, p<0.0001) and tumor versus blood (85% lower, p<0.0001), but the contrast of tumor versus kidney and tumor versus bone and bone marrow was not significantly affected (p>0.05).

[0239] [Table 5]

[0240] [Table 6]

[0241] 1.5 Discussion

[0242] 18 We previously generated F-labeled LLP2A radiotracers, each of which was coupled to two structurally related but relatively hydrophobic [ 18 [F]RBF3-PEG2-LLP2A tracer was labeled by isotope exchange. Although several approaches exist to increase the polarity of radioactive tracers (e.g., adding glutamate and increasing the length of the PEG linker), the present disclosure presents a novel approach of introducing a DOTA moiety as a means to increase hydrophilicity and thereby modulate the clearance of the tracer. 18High intestinal uptake was previously observed for the [F]RBF3-PEG2-LLP2A conjugate. To test the LLP2A class of ligands as a peptidic scaffold, compound 6 was designed by appending DOTA to an otherwise nearly identical LLP2A conjugate, minimizing any differences in the DOTA moiety (see comparison of structures in Figure 8). The synthesis of DOTA-modified LLP2A derivative 6 proceeded in an efficient stepwise manner, with near quantitative yields for most chemical reactions. The relatively low recovery of 6 in the final step was attributed to sample loss as a result of stringent HPLC purification conditions. However, the recovered material was of high purity and showed excellent solubility in all of the described compounds. 18 Sufficient quantities were obtained for F radiolabeling and in vivo studies.

[0243] [ 18 Radiosynthesis of [F]6 under aqueous conditions, as previously demonstrated for the AMBF3 radioactive prosthetic group and other trifluoroborates evaluated by our laboratory. 18 The radiosynthesis time, radiochemical yield, purity and molar activity were suitable for in vivo studies and were comparable to previous [ 18 F]AMBF3-PEG2-LLP2A and [ 18 F]N-Pyr-p-BF3-PEG2-LLP2A (28). The yield was relatively low, but [ 18 Sufficient activity of [F]6, 0.37-1.85 GBq (10-50 mCi), was consistently obtained for each in vivo study (n>6). Since both the labeling and recovery methods were not optimized, we 18 We believe that these yields can be increased by using alternative solutions (e.g., buffered aqueous isotonic ethanol solutions) during HPLC purification for the direct formulation and recovery of [F]6. This is due to the fact that the 18 This circumvents the need for C18 cartridge isolation of [F]6, as this hydrophilic tracer was likely lost during the initial capture step.

[0244] A standard mouse melanoma model, B16-F10, expressing the target VLA-4 was imaged.18 At 1 hour pi, [F]6 showed preferential accumulation in the spleen, tumor, and bone marrow. 18 F]6 uptake and corresponding tumor-to-muscle and tumor-to-blood ratios were measured in the same tumor model at similar time points (1-2 h pi). 64 Cu]Cu-CB-TE1A1P-PEG4-LLP2A, [ 64 Cu]Cu-CB-TE2A-LLP2A, 64 Cu]Cu-NODAGA-PEG4-LLP2A and [ 68 Blockade of VLA-4 in tumors and bone marrow was similar to that observed with [Ga]Ga-NODAGA-PEG4-LLP2A (23, 25-27). 18 This confirms the specificity of VLA-4 in B16-F10 tumors and is consistent with the well-known VLA-4 expression in hematopoietic stem cells found in bone marrow (2,9,10,34-37). 18 Although high accumulation of [F]6 is undesirable, 64 Cu and 68 In Ga-labeled LLP2A derivatives, trapping of the LLP2A radiotracer in the RES has been observed (23, 25-27). Our results, as well as all previously reported in vivo studies using LLP2A radiotracers (22, 24, 26, 38-40), also show that [ 18 It was confirmed that the clearance pathway of [F6] was via the kidneys and bladder.

[0245] [ 18 F]6 is 64 Cu-labeled LLP2A derivatives were labeled with higher molar activity than previously reported (24, 26, 27). 18 Although the high molar activity may contribute to the high-contrast static PET images of [F]6, two previously reported [F6-binding domains] that showed 4.6- and 23-fold higher in vitro binding affinity for VLA-4 were not significantly different from those of [F]6-binding domains. 18 Similar high molar activities were achieved for the [F]RBF3-PEG2-LLP2A tracer, however, in both prior examples, tumor uptake values ​​were 2-3 fold lower than those presented here.

[0246] Notably, the only notable difference between 6 and the preceding AMBF3-PEG2-LLP2A conjugate is the DOTA moiety (Figure 8), which we suggest is the main cause of the higher tumor uptake value and better renal clearance. In contrast, the DOTA moiety was significantly higher than that of the previously reported AMBF3-PEG2-LLP2A conjugate (Figure 8). 18 [F]RBF3-PEG2-LLP2A 18 F]6, these derivatives therefore showed approximately 3-5 fold lower retention in the spleen and approximately 11 fold higher accumulation in the GI tract. In general, these results, along with others, support the addition of a more hydrophilic moiety (previously using phosphate and carboxylate groups; here DOTA) as a means to influence the clearance pathway (i.e., renal vs. hepatobiliary) of LLP2A bioconjugates. Unlike other chemical functional groups that have been positioned to facilitate renal clearance, DOTA is able to selectively bind a variety of non-radioactive metals (e.g., Zn 2+ , Ca 2+ ) can be chelated to 18 This could provide the added advantage that the effects on in vivo PK / PC could be further investigated via F-PET imaging. Furthermore, companion dual isotope "hot-cold / cold-hot" isotope-substituted theranostics (e.g., 18 F / 174 Lu and 19 F / 177 The possibility of developing a new fluorine-containing ...

[0247] This example shows 18 While F labeling proceeds by isotopic exchange at high molar activity, 18We show that tumor uptake of F-labeled RBF3-PEG2-LLP2A can be enhanced by using hydrophilic metal-free DOTA, which can be introduced by a simple synthetic approach. In vivo PET imaging and biodistribution studies demonstrate that the DOTA moiety diverts radiotracer accumulation from the GI tract to the RES, particularly accumulating in the spleen. We demonstrate a significantly improved method for VLA-4 targeted imaging. 18 Having presented a F-labeled LLP2A radiotracer, the appended DOTA appears to function as a novel and useful group for modulating tracer clearance. 18 There is also potential for use with F-labeled radioactive prosthetic groups.

[0248] Example 2: PSMA-617-LysAMBF3-DOTA

[0249] AMBF3 and Fmoc-LysN3-OH were prepared according to reported procedures. Perrin et al., Angew. Chem. Int. Ed. 2014, 53, 11876-11880. Nakahara et al., Tetrahedron Lett. 2008, 49, 5492-5494. [ka]

[0250] I. Fmoc-LysAMBF3-OH [ka]

[0251] A fresh stock solution of 1 M sodium ascorbate was prepared by dissolving 1.98 g of sodium ascorbate (0.01 mol) in 10 mL of deionized water. Then, 1 M Cu(II)SO 4(水溶液)A stock solution of was prepared by dissolving 1.60 g of anhydrous Cu(II)SO4 (0.01 mol) in 10 mL of deionized water. AMBF3 (414.6 mg, 2.513 mmol, 5.24 equiv.) was placed in a flask and dissolved in (3:2) MeCN:HO (v / v) (2.5 mL). A 1.5 mL volume of 1 M Cu(II)SO 4(水溶液) (1.5 mmol, 3.1 equiv) was added first, followed by 3 mL of 1 M sodium ascorbate (3 mmol, 6.2 equiv). Fmoc-LysN3-OH (189.1 mg, 0.479 mmol, 1 equiv) was then added to the solution. K2CO3 (101.1 mg, 0.732 mmol, 1.5 equiv) was then added to neutralize the solution to pH 7. The solution was stirred at 45 °C for 16 h. The mixture was vacuum filtered to remove the precipitate, and the filtrate was concentrated by rotary evaporation. The dried crude product was resuspended in (1:1) MeOH:DCM (v / v) (5 × 10 mL), vacuum filtered to remove the precipitate, and the filtrate was dried by rotary evaporation. The dried crude product was then resuspended in (5:95) MeOH:DCM (v / v) (5 × 10 mL), vacuum filtered to remove the precipitate, and the filtrate was dried again by rotary evaporation. Column chromatography (silica gel, 230-400 mesh, 50 g, solvent: (1:4:95) AcOH:MeOH:DCM (v / v)) was used to determine the elution state of the product. f Elution was monitored by HPLC (pH 7.0, visible under UV lamp at 254 nm, stained with I2 / silica). Pure fractions were concentrated by rotary evaporation and further dried in vacuum to give Fmoc-LysAMBF3-OH (192.0 mg, 0.343 mmol) as a dark yellow oil in 71% yield.

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[0252] II. Fmoc-LysAMBF3-O-NHS [ka]

[0253] Fmoc-LysAMBF3-OH (163.3 mg, 295.5 μmol, 1 equiv.) was added to a flask and dissolved in 8 mL of (1:1) DCM:MeCN (v / v). Dicyclohexylcarbodiimide (DCC) (305 mg, 1.48 mmol, 5 equiv.) was added to the solution, followed by N-hydroxysuccinimide (NHS) (170 mg, 1.48 mmol, 5 equiv.). The solution was stirred at room temperature for 21 h. The reaction mixture was vacuum filtered using a sintered funnel, and the collected filtrate was concentrated on a rotary evaporator. Column chromatography (column diameter = 0.5 cm, silica gel (230-400 mesh), 10 g, gradient of DCM to (1:1) DCM:MeCN (v / v) increasing with 10% CH3CN (v / v) per 50 mL) was performed using TLC (1:1 DCM:CH3CN, R of the product f Elution was monitored by HPLC (visible under UV lamp at 254 nm, stained with I2 / silica). Pure fractions were pooled, concentrated on a rotary evaporator, and further dried in vacuum to give Fmoc-LysAMBF3-O-NHS (118.3 mg, 180.2 mmol) as a yellow oil in 61% yield.

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[0254] III. PSMA-617-NH2

[0255] PSMA-617-Fmoc on resin was synthesized by SPPS according to the procedure reported in Eder et al., J. Nucl. Med. 2015, 56, 914-920. [ka]

[0256] PSMA-617-Fmoc on resin (45.6 mg, 15.1 μmol) was placed in an Eppendorf tube. DMF (400 μL) was added and the resin was allowed to swell for 30 min by placing the sample on a rotisserie shaker. The mixture was centrifuged and the supernatant was removed. (1:4) piperidine:DMF (200 μL) was added to the resin and the mixture was agitated on a rotisserie shaker at room temperature for 30 min. The mixture was then centrifuged and the supernatant was removed. DMF (3×200 μL) was used to wash the resin by vortexing, centrifugation, and removal of the supernatant. (3:7) HFIP:DCM (200 μL) was added to cleave the product from the resin and the mixture was agitated on a rotisserie shaker for 30 min. The product mixture was filtered through a 1 mL pipette filter tip to remove the resin and the filtrate was collected in a separate Eppendorf tube. The mixture was concentrated by gentle air blowing until the volume was about 50-100 μL. Cold diethyl ether (1 mL) was then added to triturate the product. The product was vortexed and centrifuged. The supernatant was then removed and the product was dissolved in 50 μL of DMF. Diethyl ether (1 mL) was added for further washing, and the mixture was vortexed and centrifuged. The supernatant was removed and the mixture was dried by speed-vac to give the expected PSMA-617-NH2 in quantitative yield (11.6 mg, 15.1 μmol). ESI-MS (+): C 41 H 61 The calculated m / z for N5O9 is 767.97, and the observed value is [M+H] + = 768.7 m / z. TLC (2:98) MeOH, R f =0.29, visible under a 254 nm UV lamp. See Figure 42 for data.

[0257] IV. PSMA-617-LysAMBF3-NH2 [ka]

[0258] PSMA-617-NH2 (12.2 mg, 9.3 μmol, 1 equiv) in an Eppendorf tube was dissolved in (1:19) DIPEA:DMF (200 μL) and transferred to another Eppendorf tube containing Fmoc-LysAMBF3-O-NHS (25.2 mg, 38.4 μmol, 4.1 equiv). The mixture was stirred at room temperature for 2 h using a rotisserie stirrer. The mixture was concentrated by speed-vac until the volume was approximately 50 to 100 μL. Cold diethyl ether (1 mL) was added to precipitate the product. The mixture was then vortexed and centrifuged to remove the supernatant. A minimal amount of DMF (50 μL) was added to the product to dissolve the product, and diethyl ether (1 mL) was added to wash the crude product. The mixture was vortexed and centrifuged to remove the supernatant. The product was then dried by speed-vac. MeCN (500 μL) was then added to dissolve and remove excess Fmoc-LysAMBF3-O-NHS. The mixture was centrifuged and the supernatant containing excess reagent was removed. The product was dried again in a speed-vac. A small sample of the product PSMA-617-LysAMBF3-Fmoc was dissolved in MeOH for MS. ESI-MS (+): C 68 H 92 BF3N 10 O 12 The calculated m / z value is 1309.35, and the measured value is [MF] + =1290.1m / z, [M+Na] + = 1332.0 m / z. TLC (1:19 NH4OH:EtOH, product Rf = 0.42, visible at 254 nm). See Figure 43.

[0259] PSMA-617-LysAMBF3-Fmoc was then dissolved in (1:4) piperidine:DMF (200 μL) to remove Fmoc. The reaction was carried out at room temperature for 30 min by mixing in a rotisserie stirrer. The mixture was concentrated using a speed-vac until the volume was approximately 50-100 μL. Cold diethyl ether (1 mL) was added to precipitate the product. This was then vortexed and centrifuged to remove the supernatant. A minimal amount of DMF (50 μL) was added to the product to dissolve it, and the diethyl ether wash was performed again. The mixture was vortexed and centrifuged. The supernatant was removed and the product was then dried by speed-vac to obtain a crude mixture containing PSMA-617-LysAMBF3-NH2. ESI-MS (+):C 53 H 82 BF3N 10 O 10 The calculated m / z value is 1087.10, and the measured value is [MF] + =1068.1m / z, [M+H] + =1088.1m / z, [M+Na] + = 1110.1 m / z. TLC (1:19 NH4OH:MeOH, R f =0.23, visible under UV lamp at 254 nm, stained with ninhydrin). See Figure 44.

[0260] V.PSMA-617-LysAMBF3-DOTA [ka]

[0261] DOTA-NHS·HPF6·TFA (20.2 mg, 26.2 μmol, 2.8 equiv.) was weighed and added to the crude PSMA-617-LysAMBF3-NH2 (max 9.3 μmol, 1 equiv.) in an Eppendorf tube. (1:19) DIPEA:DMF (v / v) was added to the mixture and stirred at room temperature for 2 h using a rotisserie stirrer. The mixture was concentrated by speed-vac to a minimum volume (50–100 μL) and the mixture was precipitated by diethyl ether (1 mL). The mixture was vortexed and centrifuged. The supernatant was removed and then the product was dried by speed-vac. The tert-butyl was deprotected by adding (95:2.5:2.5) TFA:TIPS:H2O (v / v / v) (200 μL) and the mixture was stirred at room temperature for 1 h. The mixture was then concentrated by gentle air blowing until the volume was 50-100 μL. Diethyl ether (1 mL) was added to precipitate the product, and the mixture was vortexed and centrifuged. The supernatant was removed, and the mixture was then dried by speed-vac to give a crude residue (33 mg) containing PSMA-617-LysAMBF3-DOTA. ESI-MS (+): C 61 H 93 BF3N 13 O 17 The calculated m / z value for is 1360.31, and the measured value is [M-3F-2H] + =1302.1m / z, [M+H] + = 1362.1 m / z. See Figure 45.

[0262] The crude residue (33 mg) was dissolved in (1:1) MeCN:HO (+0.1% formic acid) (1 mL) and purified by HPLC (column Agilent Eclipse XDB-C18, 5 μm, 9.4 mm × 250 mm, flow rate 2 mL / min, UV-vis detector at 276 nm, gradient 15% to 65% MeCN:HO (+0.1% formic acid) over 19 min). The peak was collected at 9.25 min. The product was collected in a 50 mL Falcon tube and frozen on dry ice. Once completely frozen, the collected fractions were lyophilized to give pure PSMA-617-LysAMBF3-DOTA (2.5 mg, 1.8 μmol, 19%).

[0263] VI.PSMA-617-LysAMBF3-DOTA(Cu) [ka]

[0264] PSMA-617-LysAMBF3-DOTA(Cu) was synthesized by treating PSMA-617-LysAMBF3-DOTA (44.2 mg, 31.1 μmol) with 0.1 M CuCl2-NaOAc (1.55 mL, pH=4) with MeCN (1.5 mL). The mixture was placed on a hot plate at 65 °C for 30 min. The mixture was then concentrated using a Speed-vac to give a crude residue containing the expected copper chelate. ESI-MS (+): C 61 H 90 BCuF3N 10 O 10 The calculated m / z value for is 1422.8, and the measured value is [M-3F-2H] + =1362.8m / z, [MF] + =1402.8m / z, [M+H] + =1423.8m / z, [M+Na] + =1444.8m / z.

[0265] The crude residue (44.2 mg) was dissolved in (1:1) MeCN:HO (+0.1% formic acid) (1 mL) and purified by HPLC (column Agilent Eclipse XDB-C18, 5 μm, 9.4 mm × 250 mm, flow rate 2 mL / min, UV-vis detector at 276 nm, gradient 15% to 40% MeCN:HO (+0.1% formic acid) over 23 min). The product was collected in a 50 mL falcon tube and frozen on dry ice. Once completely frozen, the collected fractions were lyophilized to give pure PSMA-617-LysAMBF3-DOTA(Cu) (8.5 mg, 6.0 μmol, 19%). See Figure 46.

[0266] VII. PSMA-617-LysAMBF3-DOTA or PSMA-617-LysAMBF3-DOTA(Cu) 18 F-label.

[0267] 80 nmol 19 F-PSMA-617-LysAMBF3-DOTA or 19 F-PSMA-617-LysAMBF3-DOTA(Cu) was resuspended in aqueous pyridazine-HCl buffer (15 μL, 1 M, pH=2), DMF (15 μL) and aqueous KHF2 (4 μL, 5 mM) in a polypropylene tube. 18 MeV protons were introduced into the H2 18 After collision with O, the ions were captured on an anion exchange column (9 mg, QMA, chloride form), but no carrier was added. 18 No F-fluoride was obtained. 18 F-fluoride was eluted into the reaction tube with saline (100 μL). The reaction mixture was heated at 80° C. under vacuum for 20 min and diluted with 40 mM aqueous ammonium formate (2 mL). The solution was purified by HPLC using a semi-preparative column and eluted with (30:70) MeCN / water (+0.1% TFA) (v / v) at a flow rate of 4.5 mL / min. Retention times were approximately 10 min for both chelated and unchelated tracers. The decay-corrected radiochemical yields were 18 F-PSMA-617-LysAMBF3-DOTA or 18 For F-PSMA-617-LysAMBF3-DOTA(Cu), it was 1.0 ± 0.3% (n = 3) or 2.7 ± 0.7% (n = 3), respectively. Radiochemical purity of >99% was achieved for both labeled tracers as determined by radioactive HPLC. Specific activity was measured using an analytical HPLC system. It was calculated by dividing the injected radioactivity (1.5-3 mCi) in the final product solution by the mass in the injected solution. The mass of the injected product was estimated by comparing the UV absorbance obtained from the injection to a previously prepared standard curve. 18 F-PSMA-617-LysAMBF3-DOTA or 18The specific activity of F-PSMA-617-LysAMBF3-DOTA(Cu) was 3.7±2.5 Ci / μmol (n=3) or 4.8±2.2 Ci / μmol (n=3), respectively.

[0268] result

[0269] Table 7 shows the biodistribution of F-18 labeled PSMA-617-Lys-AMBF3-DOTA in mice bearing PSMA-expressing LNCaP prostate cancer xenografts (1 hour after injection). The structure of PSMA-617-LysAMBF3-DOTA is shown in Figure 2. Figure 4 shows maximum intensity projection PET images of F-18 labeled PSMA-617-Lys-AMBF3-DOTA in mice bearing LNCaP prostate cancer xenografts (1 hour after injection).

[0270] [Table 7]

[0271] References for Example 2 JPEG0007659268000047.jpg184167 JPEG0007659268000048.jpg223167 JPEG0007659268000049.jpg224167 JPEG0007659268000050.jpg55167

[0272] Examples 3 and 4: Synthesis and characterization of DOTA-Lys(AMBF3)-RM2 (DOTA-Lys-AMBF3-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) and DOTA-Lys(AMBF3)-BK

[0273] Results and Discussion

[0274] Synthesis of DTPA [ka]

[0275] Diethylenepentaacetic acid (DTPA) is a compound that converts radioactive metals (e.g. 64 It is an acidic chelating agent used in the synthesis of both therapeutic and diagnostic agents as a chaperone for Cu. 25 Because this chelator has five arms available for conjugation to peptides, the arms themselves must be protected in a design unique to this application. For our purposes, one free arm was desired for solid-phase chemistry, while the other four arms were to be protected by acid-labile protecting groups (compound 4). Final cleavage of the peptide from the resin with acidic TFA then allowed removal of the protecting groups. This design concept led to the synthetic scheme 3, which was adopted from the literature. 26、27 .

[0276] Scheme 3. Synthesis scheme of DTPA. [ka]

[0277] The synthesis of compound 4 was carried out by mixing commercially available ethanolamine with 2 equivalents of t-butyl bromoacetate under basic conditions. After extraction with diethyl ether followed by washing with sodium bicarbonate and brine, the oily residue was purified on a silica column. Approximately 3 g of compound 1 was purified in 71% yield, so the purification was suitable for scale-up. 1 H-NMR showed high purity with negligible signals from water and hexane (see Appendix). Compound 1 was then converted to 2 by treatment of the tertiary amino alcohol with NBS / PPh3 in DCM under anhydrous conditions and purified in 71% yield. Upon completion of the reaction, the triphenylphosphine oxide by-product was removed by filtering the crude reaction mixture through a short silica column, and the desired product was then purified by a second silica column using 5:1 hexane / diethyl ether. The purity of this compound was 11 H-NMR confirmed the product, and ESI-MS showed the expected bromine isotope pattern (data not shown).

[0278] At this point, the appropriate glycine ester was double alkylated with 2 to give the DTPA derivative (3). We initially chose the methyl ester because we envisaged that the methyl group could be easily removed by saponification without affecting the tert-butyl protecting groups of the other four arms. To this end, the synthesis of 3a was carried out in a phosphate-buffered aqueous acetonitrile solution made from sodium phosphate salts. Unfortunately, the solubility of phosphate salts was very low and the salts often precipitated when the ambient temperature was reduced. Therefore, mono- and di-potassium salts of phosphate salts were used instead to make the buffer and the pH was adjusted to 8 by adding aqueous NaOH or HCl. This highly concentrated solution formed a two-phase system when mixed with acetonitrile. After 24 hours, the mixture was filtered, the organic layer removed and evaporated. To remove the remaining inorganic salts, the amorphous oil was dissolved in CHCl3 and refiltered. Chromatographic purification of this compound was problematic due to the three tertiary amino groups that could be easily protonated on acidic silica. Unfortunately, complete purification was still not possible even when the silica was pretreated with triethylamine and eluted with 1% triethylamine. Nevertheless, we decided to treat the impure 3a with 1 equivalent of aqueous LiOH to effect saponification of the methyl ester (4). After TLC showed the reaction was complete, the product was extracted into diethyl ether. Mass spectrometry confirmed the presence of the mono-deprotected DTPA adduct, with [M+Na + ] signal (data not shown). Saponification resulted in the formation of some of the desired material, but the reaction was neither clean nor high yielding. We hypothesized that the saponification conditions precluded purification of the product, and indeed a literature search showed that the synthesis of monomethyltetra-tert-butyl protected DTPA (3a) had not been reported. Using a benzyl protected glycine ester instead, palladium catalyzed hydrogenation afforded the desired product (3b). 26、28 .

[0279] To synthesize 3b, glycine benzyl ester was first isolated from the salt of benzyl glycinate p-toluenesulfonate using ether / carbonate extraction and used immediately. This was reacted with compound 2 under similar conditions (see above) to give benzyl protected DTPA in 27% crude yield. Again, due to the nature of the functional groups, chromatographic purification proved difficult and could not be pursued exhaustively. Other purification methods (e.g., acidic / basic / neutral alumina, C18 reverse phase, distillation) were not investigated. Rather, crude 3b was deprotected by palladium catalyzed hydrogenation in MeOH overnight in 81% yield. Once complete, the Pd / C solids were removed by filtering the mixture through a sintered glass funnel. Interestingly, compound 4 was 1 No further purification was required, as confirmed by H-NMR (see Appendix). The overall yield was 11.4% over four steps.

[0280] Synthesis of novel AMBF3

[0281] At this point in the project, our focus shifted from the synthesis of the DTPA chelator (4) to the construction of novel trifluoroborates that could be incorporated into any peptide chain. Dimethylammoniomethyltrifluoroborate (AMBF3) (7) was chosen as a precursor due to its high hydrolytic stability, ease of preparation, and availability in copper-assisted azide-alkyne cycloaddition reactions. 19 Indeed, even at very dilute conditions (~5 mM) at pH 7.5, compound 7 lost free fluoride at a very slow first-order rate, as shown in Figure 4. 19 This in vitro stability, combined with the ease of synthesis of compound 7, makes the AMBF3 structure a unique target for PET imaging agents.

[0282] Compound 6 was obtained from the equimolar reaction of commercially available iodomethylboronate pinacol ester with N,N-dimethylpropargylamine in diethyl ether (see Scheme 4). Rapid precipitation of the salt followed by filtration afforded the NMR-pure product in >90% yield (see Appendix). Fluorination with KHF2 at pH 2 afforded compound 7 (see Scheme 4). Free fluoride was removed by passing through a short silica plug eluting with 20% ACN / EtOH. 1 H-NMR shows δ=1.15 ppm, indicating pinacoldiol impurity. In addition, fluoride salt, KBF4 (-150 ppm) 19 Reaction yields were often 200%-300% due to contaminations such as KBF4 (as seen by F-NMR), dissolved silicates, and other uncharacterized inorganic impurities. To remedy some of these issues, the crude solid was dissolved in diethyl ether and refiltered. The resulting white powder contained almost no KBF4 except for the still unremoved KBF4 impurity. 1 H-NMR and 19 F-NMR was pure.

[0283] Scheme 4. Synthesis scheme of AMBF3(7). [ka]

[0284] An enlightening discussion of the electronic properties of alkyltrifluoroborates is given in the study of compounds 6 and 7. 1It can be made on the basis of H-NMR spectra. All NMR experiments were carried out in DO, since this was the only solvent in which the two compounds were soluble and did not interfere with the signals of interest. The signals of the methylene protons between the ammonium group and the boron shift significantly upon fluorination. Specifically, the 2H singlet in 6 is at δ=3.09 ppm, whereas in compound 7 the signal is present at δ=2.55 ppm (see Appendix). It is clear that the boron in compound 7 has a formal negative charge that should provide a more shielding NMR environment, whereas the presence of three fluorine atoms provides a very electron-withdrawing (and therefore unshielding) environment for the methylene protons. This opposing functionality actually manifests itself as electron shielding (electron donating), since an upfield shift is observed in both DO and MeOD (data not shown). In addition to the change in chemical shift of the methylene signal, its appearance also changes, from an intense singlet to a broad singlet or possibly multiplet. Coupling to boron-11 can be ruled out as this was not seen in the spectrum of compound 6. This could therefore be explained by long-range coupling to fluorine-19 or due to some conformational inflexibility within the NMR timescale. Verification of either of these theories was not pursued exhaustively.

[0285] Compounds 7 and 8 19 Regarding F-NMR, 11 B- 19 The 1:1:1:1 quartet expected from F coupling is not observed at high resolution. 11 B has a nuclear spin of 3 / 2, but relaxes relatively quickly, so that the splitting pattern can be difficult to visualize in NMR experiments using standard pulse programs. 29~31 What can be seen is a fairly broad signal at -140 ppm. As mentioned before, washing compound 7 with diethyl ether does not remove the tetrafluoroborate impurity, and this signal appears at -150 ppm in compound 7. However, the modified AMBF3 residue obtained in the next step does not have a signal at -150 ppm. 11It should be noted that the B-NMR analysis did not provide any additional information since the peaks were very broad (5 ppm) and no splitting pattern was obtained (data not shown).

[0286] The azide partner in the click reaction took the form of a modified lysine residue (5). As shown in Scheme 5, this short synthetic protocol allows the use of standard solid-phase peptide synthesis to obtain a potentially versatile peptide building block that can be used to incorporate the required trifluoroborate functional group. This is an important difference, since the aryl trifluoroborates previously used by Perrin et al. could only be appended to peptides after they had been cleaved from the solid support and HPLC purified. In the past two years, several publications have described this technique, focusing primarily on the copper-catalyzed click reaction between the azide derivative of RGD and the alkyne-ArBF3. 18、32~35 The synthesis and purification of these peptides presented difficulties because the alkyne-substituted aryl trifluoroborates were unstable at the low pH required for resin cleavage in SPPS. In fact, even the use of a 0.1% TFA / HO solvent system for HPLC resulted in decomposition of the aryl trifluoroborates. 19 The discussion in section 2.3 outlines the stability of the two peptides conjugated with the novel lysine-AMBF3. To synthesize 5, a mixture of NaN3 and Tf2O in DCM was added to Fmoc-Lys-OH. The mechanism of this reaction proceeds via a trifluoromethanesulfonyl azide (TFA) intermediate, which acts as an electrophile for the ε-amino group on lysine. This secondary reaction intermediate is subsequently attacked at the ε-carbon by the recently released azide anion to give Fmoc-Lys(N3)-OH (5) in 86% yield.

[0287] Scheme 5. Synthesis scheme for Lys(AMBF3)(8). [ka]

[0288] First described by Sharpless et al. in 2001, the click reaction is defined as any reaction that is "modular, broad in scope, high-yielding, stereospecific, and produces only harmless by-products." 36 Under the ever-growing umbrella of click chemistry, the most widely used example is the copper-catalyzed azide-alkyne cycloaddition developed by Rolf Huisgen in the early 60s. 37 Using this chemistry, compound 8 was synthesized using alkyne compound 7 and azide compound 5. The synthesis of 8 was difficult due to problems with optimizing reaction conditions and reproducibility (Table 8).

[0289] [Table 8]

[0290] Sodium ascorbate was used to reduce copper in situ from Cu(II) to Cu(I) for initial attachment of the alkyne substrate. The solvent for all reactions was approximately 3:2 ACN / H2O. This ratio was adjusted as necessary to maintain a monophasic system. All reactions were carried out overnight at 45 °C. The reducing agent, 1 M sodium ascorbate, was freshly prepared. Fmoc-Lys-(N3)-OH was reacted with 1 M NaOH in 9:1 DCM / MeOH. f = 0.37, whereas compound 8 has R f=0.15 and close to baseline, so the progress of the reaction was monitored by TLC. Reactions 1 and 2 did not significantly convert starting material to product over 24 hours (UV visualization). A similar problem occurred with reaction 3, which was not neutralized by 1M sodium bicarbonate. Reaction 4 was neutralized and further column purified in a manner similar to reactions 1-3. Purification was very slow in the solvent system, and the product eluted over many fractions. The crude mixture of reaction 5 was dissolved in DCM and washed with brine (3 x 5 mL) to remove inorganic salts. The organic extract was more amenable to column purification, but the overall reaction yield was still low. For reaction 6, a simple DCM extraction of the crude reaction mixture was used to remove inorganic salts from the organic layer. The organic extract was then purified by silica gel column chromatography (yield was 65%). Unfortunately, upon scale-up, the method was poorly reproducible, with yields of reactions 7 and 8 (2x scale and 4x scale) being less than 45% and 10%, respectively. Further insight is needed to understand why this click reaction is not compatible with gram-scale conditions. The overall yield of compound 8 was calculated to be 56% over four steps (convergent synthesis).

[0291] Recently, a "dry-down" method has been proposed to drive the reaction to completion by both concentrating the mixture and degassing the solution to promote a reducing environment for sodium ascorbate. Reactions 9-11 were carried out using increasing equivalents of copper and ascorbate. The reactions were not purified, but visual inspection of the TLC plates indicated that there was no discernible improvement with the dry-down method. However, after 24 hours, reactions 9 and 10 were found to have significantly more starting material remaining compared to 11. This suggests that the use of a stoichiometric, or even excess, of copper is essential for this reaction.

[0292] Solid Phase Peptide Synthesis

[0293] Recently, two peptides were synthesized that incorporate a Lys (AMBF3) residue into their sequence. The first peptide, DOTA- and AMBF3-conjugated RM2 (DOTA-Lys-AMBF3-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), was generated using an automated peptide synthesizer and purified by HPLC by Zhibo Liu (Figure 47). 19 The resin-bound peptide RM2 was provided to us by the Lin Lab at the BC Cancer Association (BCCA). RM2 is a bombesin antagonist with high affinity for the gastrin-releasing peptide receptor (GRPr) found in prostate, breast, and GI tract tumors. 20、38~42 The following discussion focuses on the synthesis of a second peptide, the bradykinin-derived AMBF3 conjugate (Figure 48). Scheme 6 was first used for the synthesis of the RM2 conjugate and was subsequently adapted for manual solid-phase peptide synthesis.

[0294] Naturally occurring bradykinin and its analogs, collectively known as kinins, are present in the body as signals for pain and normal physiological regulation. 43 These nonapeptides have two types of receptors, B1 and B2. 44 The latter are more prevalent than intracellular B1 receptors and bind normal nonamers and similar kinin analogues. Conversely, B1 receptors bind only kinins that are one amino acid shorter, specifically [des-Arg 9 ]-BK 44 The main enzyme that breaks down BK in the body is angiotensin I-converting enzyme (ACE), which cleaves it at the 7-8 and 5-6 bonds. 45 The role of BK receptors in cancer therapy is evidenced by the fact that B2 receptors are not attractive drug targets because they are highly concentrated throughout the body. Rather, B1 receptors are promising drug targets because they are specific to cancer sites, although they become more prevalent during increased inflammation and tumor growth. 45 For this reason, we have synthesized Lys(AMBF3) as a B1 receptor-specific [des-Arg 9] was chosen to be conjugated to a bradykinin analogue.

[0295] The design of these peptides was based on several factors. First, as mentioned in section 1.5, these naturally lipophilic peptides have been shown to accumulate in the liver and intestine in PET scans of mice. 21 The addition of pharmacokinetic modifiers, such as the chelator DOTA, helps reduce hepatobiliary clearance and improves image contrast. 22、23 However, this is not the only purpose for attaching chelators. As the names suggest, chelating functionalities such as DOTA and DTPA can be used to deliver chelates as therapeutic agents for disease or, like 18F, as diagnostic imaging agents (e.g., 68 Certain metals (e.g., Ga) can act either as 90 Y or 186 Helps chelate Re 25、46、47 This relatively new field of medicine, coined theranostics by Funkhouser in 2002, holds great promise because targeted treatments make it possible to both see and attack disease. 47、48 In our architecture, AMBF3 is 18 It is hypothesized that DOTA-RM2 can function as a diagnostic when combined with F-PET imaging, and that metals chelated to DOTA can serve as a potential therapeutic against cancer. Both of these functions rely on high tumor uptake, which makes the cationic linker between Lys(AMBF3) and the native peptide crucial. Mansi et al. showed that 4-amino-1-carboxymethyl-piperidine, when conjugated to the peptide and protonated at physiological pH, increases the K of DOTA-RM2. d showed that it is possible to reduce 20 .

[0296] Scheme 6. Synthesis scheme for DOTA-Lys(AMBF3)-BK peptide (9). (i) Standard Fmoc synthesis. Procedure performed in the Lin Lab at BCCA. The resin coupling BK used in step (ii) was provided by Collaborative Research and consisted of: (ii) 20% piperidine / DMF, RT, 3×5 min, followed by Fmoc-4-amino-1-carboxymethyl-piperidine, HBTU, DIPEA, DMF, RT, 2 h; (iii) 20% piperidine / DMF, RT, 3×5 min, followed by Fmoc-L-Lys-AMBF3-OH(8), HBTU, DIPEA, DMF, RT, 24 h; (iv) 20% piperidine / DMF, RT, 3×5 min, followed by DOTA-tri-t-butyl-ester, NHS, DCC, DCM, RT, 24 h; (v) 95:2.5:2.5 TFA / HO / TIPS with 30 mM KHF2. [ka]

[0297] Scheme 6 shows the manual solid-phase peptide synthesis protocol used to generate peptide 9. The solid support is a hydroxymethylphenyl-functionalized resin (Wang resin), which was coupled to Fmoc-protected modified bradykinin with BCCA by the Lin lab. To obtain higher in vivo stability, the BK analog used in this synthesis is Hyp 3 and Cha 5 are modified at positions 3 and 5 using, respectively (Figure 48). These modifications alter the effectiveness of ACE at these positions, which should increase the half-life of the peptide and provide superior PET imaging. 49The lysine and arginine residues are protected by Boc (tert-butoxycarbonyl) and Pbf (2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl) groups, respectively. These groups are acid labile and therefore only detach upon final TFA resin cleavage. As native bradykinin does not contain a terminal leucine residue, a terminal leucine residue was added to convert the agonist BK to its antagonist form. All reactions are carried out in 3 mL spin columns. Deprotection conditions consisted of three successive washes of 2.5 mL of 20% piperidine / DMF for 5 min each. After washing the resin with DMF and DCM, a cationic spacer was attached in the first coupling reaction (Scheme 6ii) and was carried out in DMF using HBTU as the activating agent. The Kaiser (ninhydrin) test was used after each deprotection / coupling reaction (Scheme 6ii-6iv) to test for completion. Briefly, this color test could rapidly distinguish between protected and unprotected peptides by the presence of primary amino groups. 50 Although this is a semi-quantitative analytical method (as visual inspection of the beads alone cannot accurately distinguish how much of the protected amine has been deprotected), the use of a positive control allowed one to determine how long the beads should be heated. The test involved taking a few beads of resin and treating them with ninhydrin, phenol in ethanol, and an aqueous solution of KCN in pyridine. One test tube contained Rink amide resin with a free amino group (positive control), and the other contained the resin with the peptide in question. Upon heating, a dark blue color was expected for the free terminal amine, but no color change was observed for the Fmoc-protected peptide.

[0298] The next coupling reaction (Scheme 6iii) used similar conditions, but was mixed for 24 hours to ensure the maximum possible yield, as compound 8 is much more valuable than the cationic linker. Coupling of DOTA-tri-t-butyl-ester (Scheme 6iv) was first attempted using NHS / DCC in DMF for 24 hours. After the reaction period, a significant blue color was still observed by Kaiser test. Similar results were seen after another 24 hours. Thus, the solvent was switched to DCM, and satisfactorily, the Kaiser test became negative after 48 hours. HBTU has been shown to work with DOTA for the synthesis of small peptides, but previous experience with RM2 and BK analogs has shown that the synthesis of NHS esters of DOTA by DCC coupling is a more efficient method. 19、51 .

[0299] Difficulties arose when attempting to cleave the peptide from the resin using 95% TFA in water. HPLC traces of the cleaved peptides showed multiple peaks at 10-20 min, and no product could be confirmed by MALDI-TOF analysis (data not shown). It was hypothesized that either AMBF3 had decomposed upon cleavage of the sequence from the resin, or that TFA had potentially cleaved the peptide at multiple positions. This was disappointing, as the aim of this new technique was hydrolytic stability. Nevertheless, it was important to realise that these were extremely harsh conditions and therefore likely would not allow for any trifluoroborate to be present. To solve this problem, it was suggested that an excess of free fluoride should be added to the cleavage mixture to drive the equilibrium towards the trifluoroborate rather than towards the boronic acid. This was based on kinetic studies performed by Ting et al., shown in Scheme 4. In light of this reasoning, 30 mM KHF2 was added to 95:2.5:2.5 TFA / H2O / TIPS and the cleavage reaction was run for up to 2 h. The solution was filtered, collected, evaporated and triturated with MeOH / diethyl ether. The precipitated solid was dissolved in 50% aqueous acetonitrile and purified by HPLC as shown in Figure 50 (see Methods and Materials for detailed HPLC protocol). Peaks at 10-11 min (peak 1), 11.1-12 min (peak 2), 14-14.8 min (peak 3) and 14.9-15.7 min (peak 4) were collected and analyzed by MALDI-TOF. Only the first two peaks provided useful characterization data (Figures 51-54).

[0300] HPLC purification of the bradykinin derivatives has proven difficult as both peaks are essentially similar with broad retention times. This may be due to the quality of the synthetic procedure, but similar HPLC traces have been observed previously for other BK analogues. 19A possible explanation is that the BK peptide tends to have multiple conformations due to the Pro and Hyp residues at positions 3 / 4 and 8, respectively. Therefore, even chemically pure peptides may elute in broad peaks. As seen in Figure 50, peaks 1 and 2 elute separately, and unfortunately the separation was not completely effective, since the compound of peak 1 (m / z=1807) can be found in the MALDI-TOF of peak 2 and vice versa. The identity of peak 1 is believed to be the deboronation product of peptide 9 (Figure 55). The predicted isotope pattern of the structure matches very well with the observed MALDI-TOF spectrum (Figure 52).

[0301] Figure 53 shows the MALDI-TOF of peak 2, which is the desired product DOTA-Lys(AMBF3)-BK. Unfortunately, as a result of the difficult HPLC purification, the spectrum shows that there is a small amount of the deboronylated product plus some further uncharacterized impurities. This is thought to be specific to bradykinin, and with the RM2 peptide mentioned above, the HPLC trace is clean and the MALDI-TOF spectrum matches very well (Figure 56).

[0302] The yield of peptide 9 was only 5.5%, which can be explained by several reasons. First, the overall scale of the solid-phase synthesis was very low, since only 16.5 μmol of protected BK was used. Moreover, as mentioned above, HPLC purification of the product was hindered by other peaks with similar retention times as the desired peptide. Finally, deboronation of DOTA-Lys(AMBF3)-BK is partly responsible for the low yield. Nevertheless, it is gratifying to see that MADLI-TOF analysis confirmed the success of this method even for synthetically challenging peptides such as bradykinin.

[0303] The MALDI-TOF of peptide 11 was + ] +It shows a single peak at 1959.1. This pure spectrum demonstrates the relative ease of synthesis of RM2 compared to BK (peptide 9).

[0304] Based on the above discussion, the following question arises: is this technology specific to AMBF3 or can it also be used for aryl trifluoroborates, which can be conjugated to amino acid residues and incorporated into peptides by solid-phase synthesis? The observations presented herein suggest that it is much easier to synthesize these peptides using the Lys(AMBF3) functionality than using ArBF3. The ArBF3 functionality can only be conjugated after SPPS and purification of the clickable peptide, since the cleavage and purification conditions destroy the trifluoroborate. 21 This is due to the increased hydrolytic stability of the trifluoroborate when adjacent to an ammonium group, compared to the aryltrifluoroborate congeners (Figures 3 and 4). 19 Furthermore, AMBF3 has the inherent advantage of being less stable in vivo, resulting in less uptake of free fluoride into bone. Although only two peptides have been synthesized with the novel AMBF3, we plan to investigate several more peptides in due course.

[0305] conclusion

[0306] The aim of this project is to develop novel aryl trifluoroborates 18 The aim of this study was to improve several of the problems associated with F-PET imaging agents. Specifically, their high lipophilicity and incompatibility with solid-phase peptide synthesis resulted in poor image quality and limited synthetic versatility, respectively. To this end, protected metal chelators were targeted for conjugation to peptides to increase polarity. Tetra-t-butyl-protected DTPA (4) was synthesized over four steps in an overall yield of 11%. Problems with the purification of compounds 3a and 3b resulted in unsatisfactory yields.

[0307] A novel alkyltrifluoroborate (8) was synthesized as a result of the click reaction of AMBF3 (7) with modified lysine-azide amino acid (5). Based on previous kinetic studies showing the improved in vitro stability of AMBF3 against hydrolysis, it was hypothesized that this residue would be compatible with solid-phase peptide synthesis of small peptides with high tumor affinity. 16 A bradykinin antagonist was selected as a substrate for the addition of Lys(AMBF3) residues. The peptide was also coupled with DOTA (a metal chelator) and a cationic ammonium linker, which are known to be essential for low lipophilicity and high tumor uptake, respectively. 20 The peptides were purified by HPLC and characterized by MALDI-TOF analysis.

[0308] The strength of this new technique is its high versatility in the synthesis of trifluoroborate peptide analogues by solid-phase peptide synthesis. AMBF3 is also hydrolytically stable, which is essentially useful for reducing the incorporation of free fluoride into bone. This study 18 F-labeled peptides can be coupled to therapeutic metals chelated to DOTA, enabling dual-modal theranostic studies.

[0309] experiment

[0310] All commercial chemicals were purchased from Sigma-Aldrich, Fischer Scientific, Alfa-Aesar, Oakwood Chemicals, or Combi-Block and used without further purification. Solvents were purchased from Fischer Scientific or Sigma-Aldrich and used without further purification. Dry THF was obtained by distillation over sodium metal using benzophenone as an indicator. DMF was dried by storing the solvent over activated 4A molecular sieves for at least 24 hours before use. Deuterated solvents were purchased from Cambridge Isotope Laboratories.1 H-NMR data were collected on a 300 MHz Bruker Avance Spectrometer and all chemical shifts are reported in ppm on the δ scale with the solvent signal as the reference point: δ 7.26 for CDCl3, δ 1.94 for CD3CN, δ 4.79 for D2O, δ 5.32 for CD2Cl2, and δ 3.31 for CD3OD. Multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), or broad (br) singlet. Mass spectra were acquired using a Waters ZQ GC-MS coupled with a Waters 2695 HPLC for sample injection. The HPLC silica gel used for flash column chromatography was SiliaFlash F60 (230-400 mesh) silica gel from SiliCycle. HPLC purification was performed using an Agilent 1100 series system via an Agilent Eclipse XDB-C18 column. Detection was by UV absorbance at 229 nm. The gradient program was as follows: solvent A was acetonitrile, solvent B was 0.1% TFA / H2O, 0%-5% A from 0-2 min, 5%-20% A from 2-15 min, 20%-35% A from 15-16 min, 35%-100% A from 16-18 min, 100%-5% A from 18-19 min, flow rate was 3 mL / min, and column temperature was 50-51 °C. The synthetic procedure, adapted from the literature, is described below by reference.

[0311] N,N-bis[(tert-butyloxycarbonyl)methyl]-2-aminoethanol (1) 26 .

[0312] In a flame-dried round-bottom flask, a mixture of tert-butyl bromoacetate (5.00 mL, 33.9 mmol, 2.5 equiv), anhydrous KHCO3 (3.45 g, 34.5 mmol, 2.5 equiv) and DMF (22 mL) was cooled to 0 °C. A solution of ethanolamine (0.826 mL, 13.7 mmol, 1 equiv) in DMF (1.68 mL) was added dropwise to the flask over 5 min under inert atmosphere and stirred for 30 min. The reaction was stirred at room temperature for an additional 24 h. The mixture was then filtered and to the filtrate was added diethyl ether (25 mL) and saturated NaHCO3 (17 mL). The aqueous layer was then extracted with diethyl ether (3 x 10 mL) and the combined organic extracts were washed with brine (3 x 10 mL). After drying over MgSO4, the organic mixture was filtered and evaporated under reduced pressure to give a viscous oil, which was purified by flash chromatography on a silica column (petroleum ether / diethyl ether, 2:1, R f =0.17).

number

[0313] N,N-bis[tert-butyloxycarbonyl)methyl]-2-bromoethylamine (2) 26

[0314] In a flame-dried round-bottom flask, 1 (2.77 g, 9.51 mmol, 1 equiv.) was dissolved in DCM (15 mL). To this solution was added triphenylphosphine (2.17 g, 8.27 mmol, 0.8 equiv.) and the mixture was cooled to 0° C. Then, N-bromosuccinimide (1.47 g, 8.27 mmol, 0.8 equiv.) was added slowly over 5 min. The colorless mixture was stirred at 0° C. for 90 min with little observable color change. The mixture was evaporated under reduced pressure to give a pale pink solid. To this was added diethyl ether (28 mL) and the resulting white precipitate was filtered. The filtrate was evaporated again under reduced pressure to give a yellow oil, which was loaded onto a short silica column and eluted with diethyl ether. The crude eluate was evaporated again and finally subjected to flash chromatography on a silica column (hexane / diethyl ether, 5:1, Rf =0.65).

number

[0315] N,N,N'',N''-Tetrakis[(tert-butyloxycarbonyl)methyl]-N'-[(methyloxycarbonyl)methyl]diethylenetriamine (3a) 52

[0316] A solution of glycine methyl ester hydrochloride (32.5 mg, 259 μmol, 1 equiv.) and 2 (187 mg, 533 μmol, 2.1 equiv.) in acetonitrile (2 mL) was made. Phosphate buffer (1 mL, 2 M, pH 8), made by mixing K2HPO4 (3.26 g, 18.8 mmol) and KH2PO4 (167 mg, 1.23 mmol) in HO (10 mL), was then added and the reaction was stirred at room temperature for 24 h. The reaction mixture was filtered and the filtrate was transferred to a separatory funnel. The organic layer was removed from the aqueous layer and dried over MgSO4. Evaporation under reduced pressure gave a yellow oil, which was purified by column chromatography (CHCl3 / MeOH / NEt3 150:3:1, R f =0.3). This solvent system, among others that were tried (e.g., Hex / Ether / NEt3, Hex / EtOAc / NEt3), was unable to isolate the target compound in high purity due to the nature of the acidic and basic functional groups as well as solubility issues.

[0317] N,N,N'',N''-Tetrakis[(tert-butyloxycarbonyl)methyl]-N'-[(benzyloxycarbonyl)methyl]diethylenetriamine (3b) 52、27

[0318] Benzyl glycinate p-toluenesulfonate (200 mg, 592 μmol, 1 equiv.) was added to diethyl ether (4 mL) and aqueous Na2CO3 (189 mg, 1.8 mmol, 3 equiv., 4 mL of solution) was added. After stirring at room temperature for 30 min, the ether layer was separated, dried over MgSO4, and evaporated under reduced pressure to give benzyl glycine ester (89 mg, 540 μmol). This intermediate was used without further purification towards the target compound 3b. Phosphate buffer (1 mL, 2 M, pH=8) was added to benzyl glycine ester (60 mg, 364 μmol, 1 equiv.) and 2 (280 mg, 795 μmol, 2.2 equiv.) dissolved in acetonitrile (2 mL). The biphasic reaction was stirred at room temperature for 24 h. The precipitated salts were filtered off and the organic layer was isolated, dried over MgSO4, and evaporated under reduced pressure. The semi-solid residue was then dissolved in chloroform, filtered and evaporated again. The resulting yellow oil was purified by flash chromatography (CHCl3 / MeOH / NEt3 150:3:1, R f = 0.35). As with 3a, complete purification by silica chromatography was difficult due to the nature of the basic functional groups.

[0319] N,N,N'',N''-Tetrakis[(tert-butyloxycarbonyl)methyl]-N'-[acetic acid]diethylenetriamine (4) 52、27

[0320] 3b (76 mg, 108 μmol, 1 equiv) was dissolved in methanol (1 mL) and 10% Pd / C (approximately 20 mg, 50% wet) was added. The reaction was stirred under a hydrogen atmosphere (2 large balloons) at room temperature for 24 h. The reaction was filtered through a sintered glass funnel and evaporated under reduced pressure to give a viscous yellow oil.

number

[0321] Fmoc-(L)-Lys(N3)-OH(5) 24

[0322] At 0 °C, NaN3 (7.3 g, 113 mmol) was added to a mixture of H2O (20 mL) and DCM (30 mL). To this, Tf2O (3.8 mL, 22.45 mmol) was added dropwise over 30 min. The reaction was stirred at 0 °C for 5 h and then extracted with DCM (2 × 50 mL). The organic layers were combined, washed with a saturated aqueous solution of Na2CO3 (1 × 50 mL) and used without further purification. A mixture of K2CO3 (5.6 g, 39.5 mmol), CuSO4·5H2O (ca. 25 mg) and Fmoc-(L)-Lys-OH (4.78 g, 13 mmol) in 1:1 MeOH / H2O (33 mL) was then generated. To this, the organic extract was added dropwise over 30 min and the mixture was further stirred at room temperature overnight. The reaction was quenched using 2.5 N HCl (150 mL) and then the product was extracted with DCM (3 x 50 mL), washed with brine (2 x 50 mL) and dried over anhydrous Na2SO4. The mixture was filtered and evaporated under reduced pressure to give a crude oil which was immediately loaded onto a silica column for flash chromatography (MeOH / DCM gradient 0.5:99.5 to 1:99).

number

[0323] N-Propargyl-N,N-dimethyl-ammoniomethyl-boronyl pinacolatoate (6) 53

[0324] In a flame-dried round-bottom flask, N,N-dimethylpropargylamine (0.67 mL, 6.27 mmol, 1 equiv) was added to diethyl ether (28 mL) and the reaction was heated to 45° C. Iodomethyl pinacol boronate (1.144 mL, 6.27 mmol, 1 equiv) was added dropwise to the solution. Immediately after the addition the mixture became cloudy followed by a white solid precipitating out of solution. The reaction was stirred for an additional 15 min and the product was filtered and washed with cold diethyl ether. The residue was dried under high vacuum to give a flaky white solid.

number

[0325] N-Propargyl-N,N-dimethyl-ammoniomethyl-trifluoroborate (7)

[0326] 6 (1 g, 4.46 mmol, 1 equiv.) was dissolved in acetonitrile (12 mL) and fluorinated by the addition of aqueous KHF2 (3 M, 4.25 mL, 12.8 mmol, 2.8 equiv.) and HCl (4 M, 4.25 mL, 17 mmol, 3.8 equiv.) at 45 °C for 2 h. The clear orange solution was quenched by the addition of concentrated aqueous NH4OH (ca. 1.5 mL) to pH 7. The mixture was transferred to a larger round-bottom flask and 20% acetonitrile / ethanol solution (100 mL) was added. Silica (40 mL) was placed directly into the solution and the mixture was stirred for 20 min. The previously bright yellow solution lost a great deal of color and turned into a clear, colorless mixture. The mixture was loaded onto a short silica column, which was placed in a sintered glass funnel and eluted with 20% acetonitrile / ethanol. After evaporation under reduced pressure followed by drying under high vacuum, a pale yellow solid was isolated. This was washed and filtered with cold diethyl ether (20 mL) to produce the NMR-pure zwitterion. Iodide (observable by ESI-MS) and KBF4 ( 19 In addition to the cleavage observed in the F-NMR, the yield of the product was greater than quantitative due to excess salt remaining in the product. For further reactions, the yield of the synthesis of 7 was assumed to be quantitative.

number

[0327] Fmoc-(L)-Lys-ε-1,2,3-triazole-N,N-dimethyl-ammoniomethyl-trifluoroborate [Fmoc-Lys(AMBF3)] (8)

[0328] A mixture of aqueous CuSO4 (1M, 0.75 mL, 0.75 mmol) and sodium ascorbate (1M, 1.5 mL, 1.5 mmol) was added to 7 (250 mg, ca. 0.75 mmol, ca. 3 equiv.) dissolved in acetonitrile / water 3:2 (2.5 mL). 5 (100 mg, 0.25 mmol, 1 equiv.) was dissolved in a minimum amount of acetonitrile and added dropwise to the mixture and stirred at 45 °C overnight. It was common for some Cu salt or ascorbate salt to precipitate throughout the reaction. The reaction was monitored by silica TLC and starting material disappeared over the course of 20-24 h reaction time. Reproducibility was not always achieved upon scale-up and often more catalyst, reducing agent, or alkyne had to be added to drive the reaction to completion. The precipitate was filtered and the reaction mixture was evaporated under reduced pressure. The dark red residue was dissolved in 1:1 MeOH / DCM, filtered, and evaporated again. This process was repeated again to remove all excess salts and insoluble impurities using 5:95 MeOH / DCM. The final crude oil was completely dissolved in 5:95 MeOH / DCM and then loaded onto a silica column and eluted by flash chromatography using a gradient system of MeOH / DCM (5%-10%).

number

[0329] DOTA-((L)-Lys-ε-1,2,3-triazole-N,N-dimethyl-ammoniomethyl-trifluoroborate)-4-amino-1-carboxymethyl-piperidine-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu-COOH (9)

[0330] The linear modified peptide was prepared using standard Fmoc solid-phase peptide synthesis chemistry on Wang (hydroxymethylphenyl) resin. The unnatural form of bradykinin (Fmoc-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu-COOH) was preloaded onto the resin and used as a scaffold for attaching the cationic spacer, Lys(AMBF3) residue and DOTA chelator. The resin loading value was precalculated at 0.44 mmol / g. The dried resin (37.5 mg, 16.7 μmol) was added to a 3 mL spin column and allowed to swell in DMF (2.5 mL) for 2 h before the first coupling reaction. The DMF was filtered off and the Fmoc was removed using 20% ​​piperidine / DMF (3 × 2.5 mL, 5 min each). The resin was washed with DMF (3 × 2.5 mL), DCM (3 × 2.5 mL), and DMF (3 × 2.5 mL). Fmoc-4-amino-1-carboxymethyl-piperidine (25 mg, 65 μmol, 4 equiv), HBTU (25 mg, 65 μmol, 4 equiv), and DIPEA (11.4 μL, 65 μmol, 4 equiv) were dissolved in DMF (2.5 mL) and added to a capped spin column. The reaction was shaken at room temperature for 2 h. The solution was filtered through a spin column and washed with DMF (3×2.5 mL), DCM (3×2.5 mL), and DMF (3×2.5 mL) before the next coupling reaction. 8 (14 mg, 25 μmol, 1.5 equiv), HBTU (10 mg, 25 μmol, 1.5 equiv), and DIPEA (5.6 μL, 32.5 μmol, 2 equiv) were dissolved in DMF (2.5 mL) and added to a capped spin column. The reaction was shaken at room temperature overnight. Again, the solution was filtered through a spin column and washed with DMF (3×2.5 mL), DCM (3×2.5 mL), and DMF (3×2.5 mL) before the next coupling reaction. DOTA-tri-t-butyl-ester (24 mg, 41.6 μmol, 2.5 equiv), NHS (4.6 mg, 41.6 μmol, 2.5 equiv) and DCC (8.5 mg, 41.6 μmol, 2.5 equiv) were dissolved in DCM (2.5 mL) and added to the capped spin column. The reaction was shaken at room temperature for 48 h.As this final appendage did not have a protecting Fmoc group, the peptide was cleaved from the resin using a cocktail (2.5 mL) made from stock solutions TFA (9.5 mL), HO (0.25 mL), TIPS (0.25 mL) and 30 mM KHF2 (10 μL of 3M KHF2). The mixture was shaken for 2 h at room temperature, filtered and evaporated under reduced pressure. The residue was triturated with 1:11 MeOH / Et2O (24 mL) at 0 °C to give a white solid. The mixture was divided equally between 24 1.5 mL Eppendorf tubes, centrifuged and the diethyl ether supernatant was removed. Diethyl ether (1 mL) was added again to each tube, centrifuged and the supernatant was removed. The remaining solid was recovered with 1:1 HO / acetonitrile (2 mL total) and purified by HPLC (conditions as above).

[0331] References for Examples 3 and 4 JPEG0007659268000064.jpg45167 JPEG0007659268000065.jpg223167 JPEG0007659268000066.jpg218167

[0332] Example 5: DOTA-Lys(AMBF3)-TATE and DOTA-Bn-NH-Lys-(AMBF3)-NHFmoc.

[0333] OctreoTATE [ka]

[0334] 19.9 mg of resin containing TATE (4.6 μmol, 1 equiv.) was swollen in DMF for 30 min. DMF was removed by centrifugation and removing the supernatant. 6 mM thallium(III) trifluoroacetate in DMF (713 μL, 4.28 μmol, 0.93 equiv.) was added and the reaction was stirred at room temperature for 8 h. The resin was centrifuged and the supernatant removed. DMF (200 μL) was used to wash three times. (500 μL) DCM was then added and centrifuged. The supernatant was removed and the sample was dried by speed vac. A small sample of the resin was scooped with a micropipette tip and placed in a separate Eppendorf tube and the product was cleaved using (3:7) HFIP:DCM (100 μL) for 10 min at room temperature. The mixture was filtered using a filter pipette tip and the filtrate was collected in another Eppendorf tube. The mixture was dried by gentle air blowing and diethyl ether was added to triturate the product. The mixture was centrifuged and the supernatant was removed. MeOH (200 μL) was added to dissolve the product for MS. ESI-MS (+): C 101 H 127 N 11 O 15 S2Si2 = 1855.5 m / z, and the measured value is [M-Trt+2H] + =1615.8m / z, [M-TBS-tBu] + =1684.9m / z. [ka]

[0335] (1:4) piperidine:DMF (200 μL) was added to the resin and the mixture was stirred at room temperature for 1 h. The mixture was centrifuged and the supernatant removed. DMF (3×200 μL) was added to wash the resin. The mixture was vortexed and centrifuged. The supernatant was removed and the mixture was dried by speed vac. A small sample of the resin was scooped with a micropipette tip into another Eppendorf tube and the product was cleaved using (3:7) HFIP:DCM (100 μL) for 10 min at room temperature. The mixture was filtered using a filter pipette tip and the filtrate was collected in another Eppendorf tube. The mixture was dried by gentle air blowing and diethyl ether was added to triturate the product. The mixture was centrifuged and the supernatant removed. MeOH (200 μL) was added to dissolve the product for MS. ESI-MS (+):C 86 H 117 N 11 O 13 The calculated m / z value for S2Si2 is 1633.2, and the measured value is [M] + =1663.0, [M+2CH3CN+2H] + =1390.7m / z. [ka]

[0336] Fmoc-Lys(AMBF3)-NHS (15.1 mg, 23.0 μmol, 5 equiv.) was weighed and dissolved in 200 μl (1:19) DIPEA:DMF. The solution was then added to the resin, mixed, and stirred at room temperature for 2 h. The mixture was centrifuged and the supernatant removed. DMF (3×200 μL) was added to wash the resin. The mixture was vortexed and centrifuged. The supernatant was removed and the mixture was dried by speed vac. A small sample of the resin was scooped with a micropipette tip and placed in a separate Eppendorf tube, and the product was cleaved using (3:7) HFIP:DCM (100 μL) for 10 min at room temperature. The mixture was filtered using a filter pipette tip, and the filtrate was collected in another Eppendorf tube. The mixture was dried by gentle air blowing, and diethyl ether was added to triturate the product. The mixture was centrifuged and the supernatant removed. MeOH (200 μL) was added to dissolve the product for MS. ESI-MS (+): C 113 H 148 BF3N 16 O 16 The calculated m / z for S2Si2 is 2173.03, and the measured value is [M+CH3CN+Na] + =2236.5m / z. [ka]

[0337] (1:4) piperidine:DMF (200 μL) was added to the resin and the mixture was stirred at room temperature for 1 h. The mixture was centrifuged and the supernatant removed. (3×200 μL) DMF was added to wash the resin. (200 μL) DCM was added and centrifuged. The DCM was removed and the resin was dried by speed vac. A small sample of the resin was scooped with a micropipette tip into another Eppendorf tube and the product was cleaved using (3:7) HFIP:DCM (100 μL) for 10 min at room temperature. The mixture was filtered using a filter pipette tip and the filtrate was collected in another Eppendorf tube. The mixture was dried by gentle air blowing and diethyl ether was added to triturate the product. The mixture was centrifuged and the supernatant removed. MeOH (200 μL) was added to dissolve the product for MS. ESI-MS (+):C 98 H 138 BF3N 16 O 14 The calculated m / z for S2Si2 is 1952.3, and the measured value is [M+Na] + =1975.4m / z. [ka]

[0338] DOTA-NHS·HPF6·TFA (8.75 mg, 11.5 μmol, 2.5 equiv) was dissolved in (1:19) DIPEA:DMF (200 μL) and the mixture was added to the resin along with H2N-Lys(AMBF3)-TATE and stirred at room temperature for 2 h. The resin was washed with DMF (3 × 200 μL). The mixture was vortexed and centrifuged. The supernatant was removed. The resin was dried by speed vac. A small sample of the resin was scooped with a micropipette tip and placed in a separate Eppendorf tube and the product was cleaved using (3:7) HFIP:DCM (100 μL) for 10 min at room temperature. The mixture was filtered using a filter pipette tip and the filtrate was collected in a separate Eppendorf tube. The mixture was dried by gentle air blowing and diethyl ether was added to triturate the product. The mixture was centrifuged and the supernatant was removed. MeOH (200 μL) was added to dissolve the product for MS. ESI-MS (+): C 114 H 164 BF3N 20 O 21 The calculated m / z value for S2Si2 is 2337.1, and the measured value is [M+K] + =2375.4m / z. [ka]

[0339] (95:2.5:2.5) TFA:TIPS:H2O (200 μL) was added to the resin to perform universal deprotection and cleave DOTA-Lys(AMBF3)-TATE from the resin. The resin was stirred at room temperature for 30 min. The filtrate was collected using a pipette tip filter to remove the resin. The filtrate was concentrated by gentle air blowing to a volume of approximately 50-100 μL. Diethyl ether (1 mL) was added to triturate the product, and the mixture was vortexed and centrifuged. The supernatant was removed and the mixture was dried by speed vac. The yield was 3.2 mg. ESI-MS (+):C 79 H 114 BF3N 20 O 21The calculated m / z for S2 is 1811.8, and the observed value is [M+2Na] + =1855.1m / z.

[0340] Synthesis of DOTA-Bn-NH-Lys-(AMBF3)-NHFmoc: [ka]

[0341] DOTA-Bn-NH2 (10 mg, 0.015 mmol, 1 equiv) in an Eppendorf tube was dissolved in (2:18) DIPEA:DMF (200 μL) and transferred to another Eppendorf tube of Fmoc-Lys(AMBF3)-NHS (14 mg, 0.025 mmol, 1.5 equiv). The mixture was stirred at room temperature for 2 hours using a rotisserie stirrer. 1 mL of cold diethyl ether was then added to precipitate the product. It was then vortexed and centrifuged to remove the supernatant. A minimal amount of DMF (approximately 50 μL) was added to the product to redissolve the product, and diethyl ether (1 mL) was added to rewash the crude product. The mixture was vortexed and centrifuged to remove the supernatant. The product was then dried, and MeCN (approximately 500 μL) was then added to redissolve and remove the excess Fmoc-Lys(AMBF3)-NHS. The mixture was centrifuged and the supernatant containing excess reagent was removed. The product was again dried in a speed vac. A small sample of the product was dissolved in MeOH for MS. ESI-MS(+):C 50 H 66 BF3N 10 O 11 The calculated m / z value is 1050.94, and the measured value is [M+H] + = 1051.7 m / z, [TLC (1:19 NH4OH:EtOH, product Rf = 0.2, visible at 254 nm).

[0342] It is further understood by those skilled in the art that the above DOTA-Lys(AMBF3) can be further conjugated to fluorescent molecules.Such an example is provided below as one of many examples that can be considered by those skilled in the art of multimodal imaging applications.Such an example will not be limited to DOTA, Cy7 or LysAMBF3 that are found to be practical. [ka]

[0343] The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that numerous variations and modifications can be made without departing from the scope of the present invention. The scope of the present invention should not be limited by the preferred embodiments described in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[0344] PRIORITY APPLICATION. This application claims priority to U.S. Patent Application No. 62 / 781,584, filed December 18, 2018, which is incorporated by reference in its entirety. One embodiment of the present invention will be described below, but the present invention is not limited thereto. [Invention 1] a compound or molecular complex configured for chelating a radioactive or non-radioactive metal isotope; and 19 F / 18 Trifluoroborate (BF) configured for F exchange 3 ) containing part or 18 and a boronate precursor capable of being converted to an F-labeled trifluoroborate. [Invention 2] A compound or molecular conjugate, the compound or molecular conjugate comprising a cell targeting domain and a metal chelator configured for chelating a radioactive or non-radioactive metal isotope; 19 F / 18 Trifluoroborate (BF) configured for F exchange 3 ) containing part or 18 and a boronate precursor capable of being converted to an F-labeled trifluoroborate. [Invention 3] The compound or molecular complex of invention 2, wherein the cell targeting domain comprises a peptide, polypeptide, protein, peptidomimetic, nucleic acid aptamer, macrocycle, steroid, or small molecule, and the cell targeting domain specifically binds to a cell marker. [Invention 4] The cell targeting domain is selected from the group consisting of LLP2A, PSMA-617, TATE, and the peptide D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 The compound or molecular complex according to claim 3, [Invention 5] 5. The compound or molecular complex according to any one of claims 2 to 4, wherein the cell targeting domain comprises (i) an antibody or an antibody derivative or fragment which specifically binds to an antigen on a target cell, or (ii) a protein domain which specifically binds to an antibody or an antibody derivative or fragment which specifically binds to the antigen. [Invention 6] The metal chelating agent is 3 6. The compound or molecular complex according to any one of claims 2 to 5, linked to the cell targeting domain by a linker containing a containing moiety. [Invention 7] The linker may be a plurality of BF 3 7. A compound or molecular complex according to claim 6, comprising a containing moiety. [Invention 8] 8. The compound or molecular complex according to claim 6 or 7, wherein the linker is a peptide linker. [Invention 9] The linker is Lys(AMBF 3 9. The compound or molecular complex according to claim 8, comprising: [Invention 10] The metal chelator is unchelated or chelated with a non-radioactive metal isotope, and the BF 3 The contained part 18 10. The compound or molecular complex according to any one of Inventions 1 to 9, which is F-labeled. [Invention 11] The metal chelator is chelated with a radioactive metal isotope, and the BF 3 The contained part 19 10. The compound or molecular complex according to any one of Inventions 1 to 9, which is F-labeled. [Invention 12] The metal chelator is chelated with a radioactive metal isotope, and the BF 3 The contained part 18 10. The compound or molecular complex according to any one of Inventions 1 to 9, which is F-labeled. [Invention 13] 13. The compound or molecular complex according to claim 11 or 12, wherein the radioactive metal isotope is an alpha emitter, a beta emitter or an Auger emitter. [Invention 14] The metal chelator may be selected from DOTA and derivatives; DOTAGA; NOTA; NODAGA; NODASA; CB-DO2A; 3p-C-DEPA; TCMC; DO3A; DTPA and DTPA analogs which may be selected from CHX-A″-DTPA and 1B4M-DTPA; TETA; NOPO; Me-3,2-HOPO; CB-TE1A1P; CB-TE2P; MM-TE2A; DM-TE2A; sarcofazine and sarcofazine derivatives which may be selected from SarAr, SarAr-NCS, diamSar, AmBaSar, and BaBaSar; TRAP; AAZTA; DATA and DATA derivatives; H2-macropa or derivatives thereof; H2dedpa, H4octapa, H4py4pa, H4Pypa, H2azapa, H5d A compound or molecular complex according to any one of Inventions 1 to 13, which is a chelating agent selected from the group consisting of ecapa, and other picolinic acid derivatives; CP256; PCTA; DOTP; HEHA; C-NETA; C-NE3TA; HBED; SHBED; BCPA; CP256; YM103; cyclam; DiamSar; desferrioxamine (DFO) and DFO derivatives; H6phospa; trithiol chelate; mercaptoacetyl; hydrazinonicotinamide; dimercaptosuccinic acid; 1,2-ethylenediylbis-L-cysteine ​​diethyl ester; methylene diphosphonate; hexamethylpropyleneamine oxime; and hexakis(methoxyisobutylisonitrile); porphyrin, chlorin, texafrin, and phthalocyanine. [Invention 15] 14. The compound or molecular complex according to any one of claims 1 to 13, wherein the metal chelating agent is selected from DOTA and DOTA derivatives. [Invention 16] BF 3 The contained part [C1] JPEG0007659268000075.jpg1726 or a group shown in Table 3 or 4, wherein each R is independently C 1 ~C 5 16. The compound or molecular complex according to any one of Inventions 1 to 15, which is a straight-chain or branched alkyl group. [Invention 17] DOTA-AMBF3-PEG2-LLP2A; PSMA-617-LysAMBF3-DOTA; DOTA-Lys(AMBF3)-TATE; and DOTA-Lys-AMBF3-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 The compound or molecular complex according to claim 2, selected from the group consisting of: [Invention 18] 18. The compound or molecular conjugate according to any one of claims 1 to 17, wherein said compound or molecular conjugate further comprises a fluorophore or other light-emitting moiety. [Invention 19] Lys(AMBF 3 19. The compound or molecular complex according to claim 1 or 18, which is a compound comprising: [Invention 20] 20. The compound or molecular complex according to claim 1, 18 or 19, wherein the metal chelating agent is DOTA. [Invention 21] The compound is DOTA-Bn-NH-Lys-(AMBF 3 21. The compound or molecular complex according to claim 20, comprising: [Invention 22] A compound or molecular complex according to any one of claims 2 to 18, The compound or molecular complex is 3 comprising both a containing moiety and said cell targeting domain, 18 imaging a subject using the F-labeled compound or molecular complex to identify the presence of a cellular marker for the disease or condition; treating said disease or condition using said compound or molecular complex chelated with a therapeutic radioisotope; 23. A compound or molecular complex for use in [Invention 23] A compound or molecular complex according to any one of claims 1 or 6 to 22 for use in combination with a bispecific antibody for imaging and / or treating a disease or condition in a subject, said compound or molecular complex comprising the BF 3 a compound or molecular complex comprising a containing moiety, the compound or molecular complex not comprising said cell targeting domain, wherein said bispecific antibody is specific for (i) a cell marker for said disease or condition and (ii) said metal chelator. [Invention 24] 24. The compound or molecular complex according to claim 23, wherein the compound or molecular complex is for administration to the subject as a complex with the bispecific antibody. [Invention 25] 24. The compound or molecular complex according to claim 23, wherein the bispecific antibody is for administration to the subject prior to administration of the compound or molecular complex to the subject in a pre-targeting step in which the bispecific antibody binds to the cell marker.

Claims

1. a) a cellular antigen binding module that targets a very late transcellular antigen 4 (VLA-4), a prostate specific membrane antigen (PSMA), a somatostatin receptor, a gastrin releasing peptide receptor (GRPR), or a bradykinin receptor; b) at least one metal chelating agent selected from DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid), Octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), and Macropa (N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6); c) Dimethylammoniomethyltrifluoroborate (AMBF ３ ) including trifluoroborates (BF ３ )-containing moiety; A compound comprising:

2. 2. The compound of claim 1, wherein the cellular antigen binding module comprises a peptide, a polypeptide, a non-peptide / non-protein ligand capable of binding to a targeted cellular antigen, or an antibody or antibody fragment.

3. The cellular antigen binding module is selected from the group consisting of LLP2A, PSMA-617, TATE, and the peptide D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH ２ 2. The compound of claim 1 comprising:

4. The compound of claim 1 comprising a linker.

5. The linker is ３ BF including ３ The compound of claim 4 comprising a containing moiety.

6. The AMBF ３ BF including ３ The containing moiety is Lys-ε-1,2,3 triazole-N,N-dimethyl-ammoniomethyl-trifluoroborate (Lys(AMBF ３ 6. The compound according to claim 5, wherein

7. The BF ３ The containing portion has at least one １８ 7. The compound of claim 1, comprising F, wherein the at least one metal chelator is unchelated or chelated with a non-radioactive metal isotope.

8. The BF ３ The contained part １８ The compound according to any one of claims 1 to 6, which is not labeled with F and wherein the at least one metal chelator is unchelated or chelated with a non-radioactive metal isotope.

9. The BF ３ The containing portion has at least one １８ 7. The compound of claim 1, comprising F, wherein the at least one metal chelator is chelated with a radioactive metal isotope.

10. 【Chemical 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 and 【Chemistry 5】 A compound selected from the group consisting of:

11. The compound of claim 10 which is chelated with a radioactive metal isotope.

12. At least one １８ 12. The compound of claim 10 or 11, comprising F.

13. A compound according to any one of claims 1 to 6 for use in the treatment of a disease or for use in radiotherapy, the metal chelator is chelated with a radioactive metal isotope; compound.

14. A compound according to claim 11 or claim 12 which refers to claim 11 for use in the treatment of a disease or for use in radiation therapy.

15. 15. The compound of claim 13 or 14, wherein the disease is cancer.

16. 16. The compound of claim 15, wherein the cancer is melanoma or prostate cancer.

17. A compound according to any one of claims 1 to 6 and 10 to 12 for use in a method for in vivo diagnostic imaging, comprising the BF ３ The containing portion has at least one １８ A compound comprising F.

18. A compound according to any one of claims 1 to 6 and 10 to 12 for use in a method for ex vivo diagnostic imaging, comprising the BF ３ The containing portion has at least one １８ A compound comprising F.

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