Synthesis of Bispidine-Based Prostate-Specific Membrane Antigen-Targeted Conjugate and Initial Investigations

Abstract

Nowadays, PSMA ligands are widely used for radiotheragnostic purposes in prostate cancer. The synthesis of a PSMA-Bisp conjugate was developed and realized with good yield (overall yield ~58% for the last two steps). All newly synthesized compounds were characterized by physicochemical methods: ¹H and ¹³C NMR, HRMS, and LCMS (for biologically tested samples). Subsequently, Bisp1 (diacetate bispidine ligand), Bisp-alkyne (bifunctional derivative of Bisp1), and its conjugate PSMA-Bisp were labeled by ⁶⁴Cu in mild conditions. In vitro studies of the labeled conjugate [⁶⁴Cu]Cu-PSMA-Bisp have shown great stability in model solutions. Finally, [⁶⁴Cu]Cu-PSMA-Bisp was compared to the well-known PSMA-617 conjugate labeled with ⁶⁴Cu and they showed similar stability in excess bovine serum (BVS), and at the same time, labeling PSMA-Bisp with ⁶⁴Cu is characterized by extremely high kinetics in mild conditions, while labeling PSMA-617 with ⁶⁴Cu requires heating (90 °C). Thus, this conjugate can be incredibly promising for nuclear medicine.

1. Introduction

Prostate cancer is one of the most common cancers among the male population [1]. Diagnosis of the disease is possible by using biomarkers such as prostate-specific membrane antigen (PSMA). PSMA is normally found in the prostate epithelium’s secretory cells and is weakly expressed by cells of other organs, such as the kidneys, salivary glands, and small intestine. At the same time, in prostate cancer, overexpression of PSMA is observed in prostate cells. This antigen can be detected not only in affected prostate cells but also in the smallest metastases, which makes it possible to use PSMA as a target for visualization of carcinomas on SPECT or PET/CT [2,3,4,5,6]. Thus, the study of the new prostate-specific radiopharmaceuticals is a relevant and important task.

Among copper isotopes, several ones have the potential to be used in nuclear medicine. ⁶¹Cu (t_1/2 = 3.3 h), ⁶²Cu (t_1/2 = 9.7 min), and ⁶⁴Cu (t_1/2 = 12.7 h) are positron-emitting radionuclides with suitable nuclear properties. The copper isotope ⁶⁷Cu (t_1/2 = 61.8 h) is the longest-lived copper radionuclide, which can also be used as a theragnostic agent: ⁶⁷Cu emits β⁻-particles with an energy of 0.4 to 0.6 MeV, which is ideal for radionuclide therapy of tumors. In addition, ⁶⁷Cu emits two γ-quants with photon energies of 92 and 184 keV that can be used for scintigraphy [7,8].

Among a number of known ligands [9,10,11,12,13] (several examples are listed in Figure 1) for the formation of stable complexes with cations of theragnostic radionuclides, used in nuclear medicine, bispidine ligands have proven themselves to be readily available, as well as being easy to be synthesized and derivatized [14,15,16,17,18]. Both high stability and the inertness of complexes of bispidine ligands with transition metal ions, especially copper cations, are noted [19,20].

2. Materials and Methods

2.1. General

Conventional ¹H and ¹³C NMR spectra were registered on a Bruker Avance 400 spectrometer (Bruker, Billerica, MA, USA) (400 MHz for ¹H and 101 MHz for ¹³C) in CDCl₃ or DMSO-d₆. Preparative column chromatography was performed on an INTERCHIM puriFlash 430 (Interchim, Montlucon, France). For purification and analysis of samples, we used a Shimadzu Prominence LC-20 (Shimadzu, Kyoto, Japan) system with a Phenomenex Luna C18 100A (150 × 4.6 mm) (Phenomenex, Torrance, CA, USA, 2015) column in a column oven at 40 °C and a fraction collector coupled to a single quadrupole mass spectrometer Shimadzu LCMS-2020 (Shimadzu, Kyoto, Japan) with a dual DUIS-ESI-APCI ionization source (Shimadzu, Kyoto, Japan). The mobile phases were 0.1% formic acid in water (A), 10 mM ammonium formate in water (B), and acetonitrile (D). The liquid chromatography (LC) parameters for analysis were a gradient flow of 1 mL/min (0–0.5 min with 5% D, 0.5–10.5 min with 5% to 100% D, 10.5–12 min with 100% D, 12–14.5 min with 100% to 5% D) with optional UV detection for some compounds. The MS parameters were drying gas at 15.0 L/min, nebulizing gas at 1.5 L/min, a desolvation line temperature of 250 °C, a heat block temperature of 400 °C, an interface voltage −3.5 kV, and a corona needle voltage of −3.5 kV. Positive ions (mass range of 250–2000 Da, in some cases 155–2000 Da) and negative ions (mass range: 100–2000 Da) were registered simultaneously. The LC-MS method was used to determine the purity of synthesized ligands and conjugates; the purity of all compounds investigated in vitro and/or in vivo was ≥95%.

High-resolution mass spectra (HR-MS) were registered on an Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, MA, USA) with an ESI ionization source. Compounds with a concentration of 0.1–10 µg/mL (in 1% formic acid in acetonitrile) were directly infused into the ion source with a syringe pump (5 µL/min). We did not use auxiliary or sheath gases; the spray voltage was ±3.5 kV, and the capillary temperature was set to 275 °C. The MS spectra were registered on an Orbitrap analyzer with 480,000 resolution (1 microscan, AGC target value of 1e6, max inject time 1000 ms, averaged on 10 spectra, MS range 100–2000 Da, in some cases 200–4000 Da). We used DMSO and di-iso-octyl phthalate as internal calibration signals (m/z 157.03515 and 413.26623) in positive mode and dodecyl sulfate (m/z 265.14790) in negative mode [21].

All used solvents were purified according to procedures described in [22]. All starting compounds were commercially available reagents or were synthesized (compounds 1 and 4) according to previously published papers [21,23].

2.2. Synthesis

The synthesis and spectral description of compounds 2 and 3 were performed according to the previously published data [24]. Bisp1 [19] and 9-benzylamino-1,5-dimethylbispidine trihydrochloride (S1) [25] were prepared as described previously.

2.2.1. Synthesis of Compound 3

Compound 2 (1 eq, 82 mg, 0.068 mmol), Bisp-Alkyne-tBu₂ (1 eq, 31.5 mg, 0.068 mmol), and CuSO₄·5H₂O (0.4 eq, 7 mg, 27.2 μmol) were dissolved in DMF/H₂O (6 mL/1 mL). The system was purged with argon. Sodium ascorbate (1.2 eq, 16 mg, 81.6 μmol) in H₂O (1 mL) was added to the mixture using a syringe. The resulting solution was stirred for 24 h in an atmosphere of argon, after which EDTA (0.8 eq, 16 mg 54.4 μmol) was added. The mixture was stirred for 3 h. After filtration of the reaction mixture from the precipitate and removing the solvent under reduced pressure, the residue was dissolved in DCM (15 mL) and washed with (1) H₂O (1 × 15 mL), (2) NaHCO₃ (2 × 15 mL), and (3) saturated NaCl solution (1 × 15 mL). The organic fraction was dried over Na₂SO₄; the solvent was removed under reduced pressure. After purification, the residue was purified by column chromatography (Puriflash on a PF-15C18HP-F0012 column (15 μm, 20 g); eluent: H₂O-TFA (0.1%) (90%)/MeCN (10%) => H₂O-TFA (0.1%) (0%)/MeCN (100%) for 20 min after MeCN (100%) for 5 min). Compound 3 was obtained as a salt of ×2 TFA as a white powder (94 mg, 73% yield).

¹H NMR (400 MHz, DMSO-d₆, δ): 0.76 (s, 6H, 10 + 11), 1.40–1.34 (m, 27H, tBu), 1.44 (s, 9H, tBu), 1.47 (s, 9H, tBu), 1.54–1.10 (m, 11H, K2Hb (b) + K2Hd + K2Hg + X3Hb + X3Hd + X3Hg, m + n), 1.62–1.54 (m, 1H, K2Hb (a)), 1.72–1.61 (m, 1H, E1Hb (b)), 1.90–1.80 (m, 1H, E1Hb (a)), 2.00–1.90 (X7Hb), 2.40–2.10 (m, 8H, E1Hg + X3Ham + X3Han + X4Hbmn + X4Hamn), 2.70–2.61 (m, 1H, F5Hb (b)), 3.11–2.84 (m, 9H, F5Hb (a) + F6Hb (a) + F6Hb (b) + X3He (mn) + X7Ha + bispidine (cyclic)), 3.25–3.11 (m, 6H, K2Hemn + bispidine (cyclic)), 3.39–3.30 (m, 2H, bispidine (cyclic)), 3.47 (s, 2H, 12′), 3.81 (s, 2H, 12), 4.06–3.88 (m, 2H, E1Ha + K2Ham + K2Han), 4.14 (d, J = 10.3 Hz, 9), 4.43–4.26 (m, 4H, F5Ha + F6Ha + X7Hg), 4.47 (s, m) and 4.55 (s, n) (2H, X8Ha, m + n), 6.37–6.22 (m, 2H, E1NH + K2NH, m + n), 7.42–7.09 (m, 14H, F5Hd + F5He + F5Hk + F6Hd + F6He + F6Hk + X8Hdn + X8Hdm + X8Hem + X8Hen + X8Hgmn + X8Htmn), 7.70 (t, J = 5.4 Hz, n) and 7.72 (t, J = 5.4 Hz, m) (1H, X7NH, m + n), 7.91 (t, J = 5.4 Hz, n) and 7.94 (t, J = 5.4 Hz, m) (1H, X3NHk, m + n), 8.25–8.16 (br.d, 1H, F6NHmn), 8.33 (d, J = 7.3 Hz, 1H, F5NH), 8.35 (d, J = 10.3 Hz, 1H, X9NH), 8.65 (s, 1H, X9 (4)).

¹³C NMR (100 MHz, DMSO-d₆, δ): 22.3 (K2Cg (m)), 22.4 (K2Cg (n)), 24.6 (X3Cb (n)), 24.7 (X3Cb (m)), 26.2 (X3Cg (n)), 26.3 (X3Cg (m)), 26.7 (K2Cd (n)), 27.57 (E1Cb), 27.61 (tBuE1d), 27.63 (K2Cd (m) + tBuK2), 27.7 (18 + 22 + 26 + tBuE1), 28.7 (X7Cb), 29.0 (X3Cd (n)), 29.1 (X3Cd (m)), 30.5 (X4Cb), 30.7 (X4Ca), 30.9 (E1Cg), 31.8 (K2Cb), 31.9 (X3Ca (m)), 32.3 (X3Ca (n)), 36.5 (X7Ca), 36.7 (F6Cb + X7Cg), 37.0 (F5Cb), 38.6 (X3Ce (n)), 38.7 (X3Ce (m)), 45.2 (K2Ce (n)), 46.8 (K2Ce (m)), 47.1 (X8Ca (m)), 47.8 (series of br. Peaks, DOTAcyclic), 48.3 (series of br. Peaks, DOTAcyclic), 49.6 (X8Ca(n)), 50.8 (series of br. Peaks, DOTAcyclic), 52.2 (E1Ca), 52.9 (K2Ca (m)), 53.0 (K2Ca (n)), 53.3 (br. peak, 15 + 23), 54.4 (19 + F6Ca), 54.6 (br. peak, 2), 55.2 (F5Ca), 79.8 (E1dtBu), 80.30 (K2tBu (n)), 80.4 (K2tBu (m)), 80.6 (E1tBu), 81.3 (17 + 21), 83.4 (21), 111.4 (CF₃), 114.3 (CF₃), 117.2 (CF₃), 120.1 (CF₃), 125.0 (X8Cg (n)), 126.1 (X8Cg (m)), 126.3 (F5Ck), 126.3 (F6Ck + X8Ct (n)), 126.9 (X8Ce (m)), 127.1 (X8Ce (n)), 127.2 (X8Ct (m)), 128.1 (F5Ce), 128.2 (F6Ce), 129.0 (F5Cd + F6Cd), 130.2 (X8Cd (m)), 130.6 (X8Cd (n)), 133.1 (X8Ck (m)), 133.4 (X8Ck (n)), 138.0 (F5Cg), 138.1 (F6Cg), 140.8 (X8Cb (n)), 141.2 (X8Cb (m)), 157.13 (U (n)), 157.15 (U (m)), 157.8 (C(O)TFA), 158.2 (C(O)TFA), 158.5 (C(O)TFA), 158.9 (C(O)TFA), 164.9 (br. peak, 1), 166.0 (br. peak, 20), 169.7 (br. peak, 16 + 24), 170.7 (F6C), 171.1 (F5C), 171.4 (E1Cd), 171.6 (X4C (mn)), 171.9 (E1C), 172.1 (X3C (nm)), 172.19 (K2C (m)), 172.23 (K2C (n)), 172.96 (X4Cg (m)), 173.00 (X4Cg (n)).

LCMS: 100% in positive ion detection mode; 100% in negative ion detection mode.

ESI-MS C₈₆H₁₂₈³⁵ClN₁₃O₁₇: m/z calculated for [M + 2H⁺]²⁺: 825.97; found: 826.65.

HRMS (m/z, ESI): calculated for C₈₆H₁₂₈³⁵ClN₁₃O₁₇ − [M + H⁺]⁺ 1650.9312; found: 1650.9319.

2.2.2. Synthesis of Compound 4 (PSMA-Bisp)

Compound 3 (1 eq, 86 mg, 45.42 μmol) was dissolved in a mixture consisting of TFA/TIPS/H₂O (95%/2.5%/2.5%, V = 4 mL). The mixture was stirred for 4 h. Next, the solvent was removed. The product was precipitated with Et₂O and then washed twice with Et₂O (2 mL). The residue was purified by reversed-phase chromatography (Puriflash PF-15C18AQ-F0012 (15 μ 20 g), eluent: H₂O × TFA (0.1%) (90%)/MeCN (10%) => H₂O × TFA (0.1%) (0%)/MeCN (100%) for 30 min after MeCN (100%) for 5 min). Compound 4 (PSMA-Bisp) was obtained as salt × 2 TFA as a white powder (58 mg, 79% yield).

¹H NMR (400 MHz, DMSO-d₆, δ): 0.76 (s, 6H, 10 + 11), 1.56–1.10 (m, 11H, + K2Hb(b) + K2Hd + K2Hg + X3Hb + X3Hd + X3Hg, m + n), 1.66–1.56 (m, 1H, K2Hb (a)), 1.77–1.66 (m, 1H, E1Hb(b)), 1.99–1.84 (m, 3H, E1Hb (a) + X7Hb), 2.40–2.10 (m, 8H, E1Hg + X3Ham + X4Hamn + X3Han + X4Hbmn), 2.70–2.60 (m, 1H, F5Hb (b)), 3.11–2.82 (m, 9H, F5Hb(a) + F6Hb(a) + F6Hb(b) + X3He(mn) + X7Ha + bispidine(cyclic)), 3.25–3.11 (m, 6H, K2Hemn+ bispidine(cyclic)), 3.40–3.30 (m, 2H, bispidine (cyclic)), 3.43 (s, 2H, 12′), 3.67 (s, 2H, 12), 4.14–3.97 (m, 3H, E1Ha + K2Han + K2Ham + 9), 4.43–4.26 (m, 4H, F5Ha + F6Ha + X7Hg), 4.46 (s, m) and 4.55 (s, n) (2H, X8Ha, m + n), 6.40–6.25 (m, 2H, E1NH + K2NH, m + n), 7.42–7.09 (m, 14H, F5Hd + F5He + F5Hk + F6Hd + F6He + F6Hk + X8Hdm + X8Hdn + X8Hen + X8Hem + X8Hgmn + X8Htmn), 7.76–7.66 (m, 1H, X7NHmn), 7.91 (t, J = 5.4 Hz, n) and 7.93 (t, J = 5.4 Hz, m) (1H, X3NHk, m + n), 8.25–8.16 (br.d, 1H, F6NHmn), 8.32 (d, J = 7.3 Hz, 1H, F5NH), 8.38 (d, J = 10.3 Hz, 1H, X9NH), 8.64 (s, 1H, X9(4)), 12.28 (br.s, 5H, COOH).

¹³C NMR (100 MHz, DMSO-d₆, δ): 19.6 (10 + 11), 22.4 (K2Cg (m)), 22.5 (K2Cg (n)), 24.6 (X3Cb (n)), 24.7 (X3Cb (m)), 26.2 (X3Cg (n)), 26.3 (X3Cg (m)), 26.8 (K2Cd (n)), 27.6 (E1Cb), 27.8 (K2Cd (m)), 29.0 (X3Cd (n)), 29.1 (X3Cd (m)), 29.5 (X7Cb), 30.0 (E1Cg), 30.6 (X4Cb), 30.7 (X4Ca), 31.8 (K2Cb), 31.9 (X3Ca (m)), 32.3 (X3Ca (n)), 35.1 (1 + 5), 35.7 (X7Ca), 36.9 (F6Cb), 37.0 (F5Cb), 38.6 (X3Ce (n)), 38.7 (X3Ce (m)), 45.4 (K2Ce (n)), 46.9 (K2Ce (m)), 47.2 (X8Ca (m)), 47.4 (X7Cg), 49.7 (X8Ca (n)), 51.7 (E1Ca), 52.2 (K2Ca (m)), 52.3 (K2Ca (n)), 54.5 (F6Ca), 54.6 (bispidine (cyclic)), 55.0 (F5Ca), 56.0 (12′ + bispidine(cyclic)), 56.3 (12), 62.8 (9), 125.0 (X8Cg (n)), 126.1 (X8Cg (m)), 126.27 (F5Ck), 126.31 (X8Ct (n)), 126.4 (F6Ck), 126.9 (X8Ce (m)), 127.16 (X8Ce (n)), 127.21 (X8Ct (m)), 127.5 (C4), 128.1 (F5Ce), 128.2 (F6Ce), 129.1 (F6Cd + F5Cd), 130.3 (X8Cd (m)), 130.6 (X8Cd (n)), 133.1 (X8Ck (m)), 133.4 (X8Ck (n)), 138.0 (F5Cg), 138.1 (F6Cg), 140.8 (X8Cb (n)), 141.3 (X8Cb (m)), 142.0 (C3), 157.3 (U), 161.0 (X9C2), 168.2 (C13′), 170.5 (C13), 171.0 (F6C), 171.3 (F5C), 171.6 (X4C (mn)), 172.2 (X3C (nm)), 172.8 (X4Cg (m)), 172.9 (X4Cg (n)), 173.8 (E1Cd), 174.2 (E1C (mn)), 174.53 (K2C (m)), 174.57 (K2C (n)).

LCMS: 97.4% in positive ion detection mode; 100% in negative ion detection mode.

ESI-MS C₆₆H₈₈³⁵ClN₁₃O₁₇: m/z calculated for [M + 2H⁺]²⁺: 685.81; found: 686.4.

HRMS (m/z, ESI): calculated for C₆₆H₈₈³⁵ClN₁₃O₁₇ − [M − 2H⁺]²⁻ 683.7982; found: 683.7986.

2.2.3. Synthesis of Compound Bisp-Alkyne

Bisp-alkyne-t-Bu₂ (1 eq, 20 mg, 43.14 μmol) was dissolved in a mixture consisting of TFA/TIPS/H₂O (95%/2.5%/2.5%, V = 2 mL). The mixture was stirred for 4 h. Next, the solvent was removed. The product was precipitated with Et₂O and washed twice with Et₂O (1 mL). The residue was purified by reversed-phase chromatography (Puriflash PF-15C18AQ-F0012 (15 μ 20 g), eluent: H₂O × TFA (0.1%) (100%)/MeCN (0%) => H₂O × TFA (0.1%) (0%)/MeCN (100%) for 30 min after MeCN (100%) for 5 min). Bisp-alkyne was obtained as salt × 2 TFA as a white powder (20 mg, 82% yield).

¹H NMR (400 MHz, DMSO-d₆, δ): 0.73 (s, 6H, 10 + 11), 2.96–2.86 (m, 2H, bispidine (cyclic)), 3.13–3.03 (m, 2H, bispidine (cyclic)), 3.23–3.13 (m, 4H, bispidine (cyclic)), 3.47 (s, 2H, 12′), 3.67 (s, 2H, 12), 3.88 (d, J = 10.3 Hz, 1H, 9), 4.40 (s, 1H, X9 (4)), 8.83 (d, J = 10.3 Hz, 1H, X9NH), 11.38 (br.s, 2H, COOH).

¹³C NMR (101 MHz, DMSO-d₆, δ): 19.5 (10 + 11), 35.0 (1 + 5), 54.9 (12′), 55.7 (12), 56.2 (bispidine (cyclic)), 62.5 (9), 77.5 (X9(4)), 77.7 (X9 (3)), 152.6 (X9(2)), 168.4 (C3′), 170.3 (13).

LCMS: 100% in positive ion detection mode; 100% in negative ion detection mode.

ESI-MS C₁₆H₂₃N₃O₅: m/z calculated for [M + H⁺]⁺: 338.17; found: 338.25.

HRMS (m/z, ESI): calculated for C₁₆H₂₃N₃O₅ − [M + H⁺]⁺ 338.1710; found: 338.1710.

2.3. Isolation of ⁶⁴Cu

A nickel target of natural isotopic composition was irradiated with a proton flow with an energy of 7–8 MeV at 1–2 μA for 6 h. The target was dissolved in concentrated HCl upon heating with the addition of H₂O₂ to accelerate the dissolution of the metal. The solution was evaporated to dryness and dissolved in 0.01 M HCl. On a Cu-resin sorbent saturated with 0.01 M HCl, ^61,64Cu was separated from ^natNi and ⁵⁵Co; 0.01 M HCl was passed through the chromatographic column until the release of nickel and cobalt ended; then, the copper was washed off with 8 M HCl. The process was monitored using gamma spectrometry using peaks at 931 keV (55Co), 283 keV (⁶¹Cu), and 1346 keV (⁶⁴Cu). The resulting solution was evaporated to dryness and dissolved in 1 mL of 0.1 M HCl.

2.4. Measurement of Radioactivity

Gamma spectrometry was performed using gamma spectrometers GR3818 (Canberra Packard Ind., Schwadorf, Austria) and ORTEC DSPec50 (16013585, ORTEC, Oak Ridge, TN, USA) with a coaxial HPGe-detector GEM-C5060P4-B (56-TP23840B, ORTEC, Oak Ridge, TN, USA).

2.5. Labeling Experiments

Labeling efficiency experiments were performed in sodium acetate buffer solution (0.15 M) with a final pH of 4.5–5.0. The incubation time for each radiolabeling experiment was less than 5 min. To study the labeling efficiency, solutions (300 μL) containing 1 kBq of ⁶⁴Cu, stable Cu(ClO₄) (to reach c(Cu²⁺) = 1 × 10⁻⁸ M) and Bisp1/Bisp-alkyne/PSMA-Bisp with concentrations varying from 1 × 10⁻⁵ M to 1 × 10⁻³ M were equilibrated at room temperature in plastic Eppendorf tubes. The degree of radiochemical conversion (RCC) was measured by thin-layer chromatography (TLC) analysis and gamma spectrometry.

2.6. Thin-Layer Chromatography

For the thin-layer chromatography (TLC), a solvent system involving 10% AcONH₄/CH₃OH 1/1 as the mobile phase and silica gel on aluminum plates (Sigma, Darmstadt, Germany) as the solid phase were used. The solution of free [⁶⁴Cu]Cu²⁺ (blank), labeled complex [⁶⁴Cu]Cu-Bisp1, labeled complex with bifunctional chelator [⁶⁴Cu]Cu-Bisp-alkyne, or labeled conjugate [⁶⁴Cu]Cu-PSMA-Bisp solution containing 100 Bq of ⁶⁴Cu (5–30 μL) was deposited on TLC sheet. The sample distribution on TLC plates after elution was preliminarily visualized by autoradiography using a Perkin Elmer Cyclone Plus Storage Phosphor System and associated OptiQuant™ 5.0software (Figures S2–S4). To quantify the TLC results, plates were cut according to the radiography images and measured by gamma spectrometry.

2.7. In Vitro Evaluation

In order to estimate the stability of [⁶⁴Cu]Cu-PSMA-Bisp in the presence of biologically relevant cations, [⁶⁴Cu]Cu-PSMA-Bisp was prepared in sodium acetate buffer solution (0.15 M) to a final pH of 4.5–5.0 and ligand concentration of 5 × 10⁻⁴ M. It was added to solutions of cations (5 mM Ca²⁺ and Mg²⁺, 0.1 mM Fe³⁺, Zn²⁺, and Cu²⁺). After 1 h of incubation at 37 °C, the bound fraction of radionuclide was determined using TLC and measured via gamma spectrometry: the mixture of complex and salt solution was spotted on a TLC plate and chromatography was performed with 10% AcONH₄/CH₃OH 1/1 as a mobile phase. The sample distribution on TLC plates after elution was preliminarily visualized by autoradiography (Figure S3) and cut according to the radiography images. Radioactivity distribution on the plates was measured by gamma spectrometry.

For serum tests, ⁶⁴Cu blank, [⁶⁴Cu]Cu-Bisp1, [⁶⁴Cu]Cu-Bisp-alkyne, and [⁶⁴Cu]Cu-PSMA-Bisp (300 μL) were prepared in sodium acetate buffer solution (0.15 M) to a final pH of 4.5–5.0 and ligand concentration of 1 × 10⁻³ M. Initial solutions (100 μL) were added to fetal bovine serum (900 μL; HyClone, Cytiva, Logan, USA), mixed, and incubated at 37 °C. An aliquot of the labeled complex–serum mixture of 100 μL was taken at time points of 1, 15, 30, 60, and 180 min, and an aliquot of the labeled conjugate–serum mixture of 100 μL was taken at time points of 5, 10, 35, 60, 300 min and treated with ethanol (300 μL) for protein precipitation. Samples were cooled to 2–4 °C and centrifuged at 4000× g for 5 min. Aliquots (300 μL) of the supernatant were separated and measured using gamma spectrometry. As a reference, initial samples (75 μL) were taken, diluted to 300 μL, and measured. For complexes, in each case, the supernatant was analyzed by TLC and autoradiography: in every case, the R_f of the radioactive compound in the supernatant had the same R_f as the original appropriate complex.

2.8. Cell Lines

22Rv1 and PC-3 cells were received from the MISIS collection of cell lines (less than ten passages from ATCC stock). All cells were cultured in humidified 37 °C incubators with 5% CO₂. All cell lines were tested for the absence of mycoplasma. 22Rv1, androgen-responsive PSMA-positive human prostate carcinoma cells, were cultured in RPMI 1640 media (Gibco, USA) with 10% FBS (Gibco, Waltham, MA, USA), 1 × GlutaMax (Gibco, Waltham, MA, USA), and 1 × Penicillin–Streptomycin (10,000 U/mL, Gibco, Waltham, MA, USA). PC-3, PSMA-negative human prostate cancer cells, were cultured in DMEM/F12 media (Gibco, Waltham, MA, USA) with 10% FBS (Gibco, Waltham, MA, USA), 1 × GlutaMax (Gibco, Waltham, MA, USA), and 1 × Penicillin–Streptomycin (10,000 U/mL, Gibco, Waltham, MA, USA) [26].

2.9. Single-Cell ROS and Cu²⁺ Measurement Using Nanoelectrodes

A detailed description of the fabrication processes of nanoelectrodes for ROS and Cu²⁺ intracellular measurement can be found elsewhere [21,24].

PC-3 (2.0 × 10⁵)/22Rv1 (3.5 × 10⁵) cells were seeded in 35 mm Petri dishes and cultivated under normal conditions for 24 h. Previously, PSMA-Bisp was stoichiometrically mixed with CuCl₂ in a 1:1 molar ratio to obtain a copper complex (PSMA-Bisp-Cu²⁺). Then, the copper complexes (PSMA-Bisp-Cu²⁺) were dissolved in DMSO, diluted in fresh culture medium with <1% FBS 2 mL, and added to Petri dishes. The final concentration of the compounds in the culture medium was IC₅₀ with 1 h of incubation time. Untreated cells were used as a control, which was evaluated at the beginning and at the end of the experiment. The attached cells in Petri dishes were washed three times using Hanks’ Balanced Salt solution to remove the media and traces of complexes. On average, about 10 cells were measured by 2–3 nanoelectrodes in 2 independent Petri dishes for each complex in one independent biological replicate. The intracellular ROS/Cu²⁺ level was determined based on the calibration curve. Results are shown as means ± SEM, where n = 3 (three independent biological replicates). Statistical analyses were conducted using the one-way ANOVA test in Origin 2021 [26].

2.10. Cytotoxicity

Cells were seeded in 96-well plates (Corning, NY, USA) at a concentration of 4000 cells per well for PC-3 culture and 6500 cells per well for the 22Rv1 line. The automated cell counter MOXI was used to calculate the cells. After 1 day, serial dilutions of PSMA-Bisp or its combination with CuCl₂, with corresponding molar ratios of 1/1 or 1/5 in DMSO, and CuCl₂ itself were added to cells. A culture medium with DMSO at concentrations corresponding to that added to the experimental samples was used as a control. The cell medium was used as a negative control, while 30% DMSO diluted in the medium was used as a positive control. Cells were incubated for 72 h at 37 °C and 5% CO₂. Later, the culture medium from each well was removed and 20 μL of MTS reagent (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega, Madison, WI, USA) was added to each well with 100 μL of new culture medium. After 4 h incubation at 37 °C in darkness, the absorbance of the solution was measured at 490 nm wavelength using the Thermo Scientific Multiskan GO spectrometer (Thermo Scientific, Waltham, MA, USA). Cell viability was calculated as the percent compared to cells incubated in the culture medium. The absorbance of the MTS reagent in a culture medium without cells was taken as zero. MTS assay revealed 100% cell death after incubation with 30% DMSO). DMSO at concentrations corresponding to that added to the samples did not cause cell viability to decrease. Experiments were performed in triplicates. Data were analyzed using the t-test in GraphPad Prism 9 software. p values < 0.05 were considered significant [27].

3. Results and Discussion

One of the standard chelating agents for metal ions is DOTA. It allows binding of metal ions such as Ga³⁺, Gd³⁺, In³⁺, Fe²⁺, Co²⁺, Mn²⁺, Ni²⁺ and copper ions (Cu²⁺) [28,29,30,31,32]. For example, recently, a conjugate (1) was synthesized for Lu³⁺ complexation with DOTA as the chelating agent (Scheme 1) [24].

Subsequently, an original synthetic scheme was proposed to produce a conjugate with a bispidine fragment, suitable for binding copper radioisotopes. Given the availability of tert-butyl-protected bispidine derivative Bisp-Alkyne-t-Bu₂, containing an alkyne moiety, it was decided to use the reaction to produce 1,2,3-triazole as a method of coupling these fragments together (Scheme 2).

Since the full exploitation of bispidine-chelating ability requires both nitrogen atoms, linking through position number 9 of cyclic bispidine was chosen. To have orthogonal functionalities at nitrogens and at carbon 9, the introduction of the alkyne moiety at that atom was suggested. The total synthesis route for Bisp-Alkyne-t-Bu₂ is described in the Supplementary Information (Figure S1).

A copper (I)-catalyzed 1,3-dipolar cycloaddition reaction was chosen to prepare conjugate PSMA-Bisp (4) from corresponding tert-butyl protected azide residue (2) and alkyne-containing bispidine Bisp-Alkyne-t-Bu₂ (Scheme 2). The corresponding azide-vector m-chloro-benzyl residue with L-Phe-L-Phe linker was used. Recently, a library of such azides was synthesized and evaluated in vitro. Particularly, azido-residue, applied in this paper, demonstrated IC₅₀ = 38 nM (by PSMA inhibition assay) when evaluated in previous papers [21]. The azide-alkyne cycloaddition reaction is widely used in the synthesis of bioactive organic compounds and is an alternative bioconjugation method for the introduction of a variety of functional fragments. Synthesis of Bisp1 and 9-benzylamino-1,5-dimethylbispidine trihydrochloride (S1) was performed as described in previous publications [19,25]. In the first step, 9-benzylamino-1,5-dimethylbispidine trihydrochloride was introduced to the alkylation reaction with tert-butyl bromoacetate, resulting in compound S2 with 83% yield. Subsequently, thee removal of benzyl groups by hydrogenolysis with 10% Pd/C in a hydrogen atmosphere was carried out. The product (S3) of the reaction was subjected to further acylation by propiolic acid in the presence of dicyclohexylcarbodiimide. The resulting compound Bisp-Alkyne-t-Bu₂ was purified by column chromatography and subjected to further reactions (Scheme 2 and Scheme 3). A schematic representation of the synthesis of Bisp-Alkyne-t-Bu₂ is shown in the Supplementary Information (Figure S1).

The bispidine fragment was selected as a chelator capable of efficient chelation of copper ions, including the ⁶⁴Cu isotope widely used in PET/CT diagnostics [33]. A di-tert-butyl-protected bispidine derivative containing a terminal alkyne group was used as the reagent. After the addition of the chelator, the removal of the protecting tert-butyl groups was carried out with the TFA/TIPS/H₂O mixture. Triisopropylsilane (TIPS) and water are used in the mixture to scavenge cations, resulting in tert-butyl deprotection. As a result, conjugate PSMA-Bisp (4) was obtained with a 79% yield.

As a comparative molecule, additionally, a modified derivative of a bispidine-chelating agent with an alkyne moiety was obtained. For this purpose, the previously used compound Bisp-Alkyne-t-Bu₂ was introduced into the reaction for the removal of tert-butyl protection groups, resulting in compound Bisp-Alkyne with 82% yield (Scheme 3). All newly synthesized conjugates and intermediate compounds were characterized by a set of physicochemical methods: ¹H and ¹³C NMR, and HRMS. The purity of final molecules such as PSMA-Bisp and Bisp alkyne was confirmed by LCMS analysis.

3.1. Labeling Conditions

Labeling with ⁶⁴Cu was carried out in the presence of 0.1 M sodium acetate pH 5 at room temperature (25 °C). Bisp1, Bisp-alkyne, and PSMA-Bisp were labeled with high efficiency at concentrations ˃ 0.1 mM within less than 5 min. According to HPLC and TLC analysis, labeled and stable complexes are chemically identical (Figures S2–S4). Compared to PSMA-617 labeling, higher ligand concentrations are required [34], but labeling Bisp1, Bisp-alkyne, and PSMA-Bisp with ⁶⁴Cu is characterized by a higher rate in mild conditions (

Table 1. Labeling yields for [⁶⁴Cu]Cu-Bisp1, [⁶⁴Cu]Cu-Bisp-alkyne, and conjugate [⁶⁴Cu]Cu-PSMA-Bisp at varying ligand and conjugate concentrations.

c(L), µM	10	20	50	100	200	500	1000
[64Cu]Cu-Bisp1, %	-	19	38	76	96	97	98
[64Cu]Cu-Bisp-alkyne, %	17	31	71	100	100	100	-
[64Cu]Cu-PSMA-Bisp, %	-	-	23	42	-	99	99

) while PSMA-617 can be quantitatively labeled (˃99%) after heating for 5–30 min at 90 °C [34]. The stability of the ligand Bisp1 and its complex with copper cations was studied previously [19].

3.2. Stability

It is important to evaluate the stability of all labeled compounds against transchelation and transmetalation in the presence of relevant metal cations such as Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, and Fe³⁺ and serum proteins. These processes may lead to the release of ⁶⁴Cu in solution, causing the irradiation of non-target tissues and incorrect diagnostics in the case of ⁶⁴Cu-based PET. It was shown that 90–95% were bound by proteins within the first minutes of mixing. All labeled complexes were highly stable (80–100%), both with an excess of microelements and in the medium of serum proteins (

Table 2. The content of intact [⁶⁴Cu]Cu-PSMA-Bisp after 1 h of incubation in a solution with an excess of microelements (%).

% [64Cu]Cu-PSMA-Bisp	Mg2+ 10 mM	Ca2+ 50 mM	Zn2+ 1 mM	Cu2+ 1 mM	Fe3+ 1mM
γ-spectrometry	98 ± 1	98 ± 1	93 ± 1	100 ± 1	100 ± 1

, Figure 2).

3.3. ROS Level and Cytotoxicity Evaluation

ROS measurement for the conjugate was performed to analyze the possible intracellular effects of the conjugate, and Cu²⁺ ion electrochemical assay techniques were performed to potentially evaluate the intracellular delivery of copper ions. We used well-established methods for electrochemical intracellular detection of ROS and Cu²⁺ [35,36]. Before the incubation with cells, PSMA-Bisp was stoichiometrically mixed with CuCl₂ in a 1:1 ratio to obtain a copper complex. According to the data of electrochemical intracellular studies, it was found that after PSMA-Bisp-Cu²⁺ complex exposure, the intracellular ROS level increased by 26% in the PC-3 cell line and by 89% in the 22Rv1 cell line compared to the control. At the same time, the ROS level following incubation with PSMA-Bisp after 1 h showed a significant increase in comparison with both control cell lines (Figure 3A,B). Since the PC-3 cell line lacks PSMA on its membrane surface, the effect of the conjugates is not as significant as for the 22Rv1 cell line (Figure 3A,B). PSMA-Bisp-Cu²⁺ targets PSMA receptors, which are overexpressed on the surface of 22Rv1. When PSMA-Bisp-Cu²⁺ binds to these receptors, it is taken up by the cells more efficiently. This enhanced cellular uptake likely increases the intracellular concentration of PSMA-Bisp-Cu²⁺, facilitating greater ROS generation through its interaction with cellular components.

We also noted that after incubation time, significantly more Cu²⁺ accumulated in the 22Rv1 cell line than in PC-3. This difference in Cu²⁺ accumulation further underscores the varying cellular responses to PSMA-Bisp-Cu²⁺ exposure between the two prostate cancer cell lines, potentially pointing to differences in their transport mechanisms related to copper ions (Figure 3C).

Similar correlations were observed in cytotoxicity experiments. Cytotoxicity probably correlates with the level of copper ions that can enter the cell (Figure 4). Coincubation of the conjugate with copper ions may lead to the formation of a complex that, through binding to PSMA and subsequent endocytosis, can penetrate through the membrane of PSMA+ cells, thus providing an increased content of copper ions inside the cells, which accounts for the main toxic effect on the cells, particularly by the generation of intracellular ROS. In the case of PSMA+ cells, this conjugate is dragged into the cells, and thus we see toxicity to 22Rv1 that is indistinguishable from the copper salts themselves. On the other hand, with the PC-3 cell culture, the complex that binds copper remains outside the cell and cannot be transported inside the cell due to the lack of a receptor, thus reducing the total amount of copper that enters the cell in the form of direct ions.

Also, it may be noted that at concentrations of PSMA-Bisp lower than 200 µM, as used in this experiment, almost complete cell survival is observed; only coincubation of PSMA-Bisp/CuCl₂ (200 µM/1000 µM) results in significant toxicity on PC-3 cells and moderate toxicity on 22Rv1 cells.

4. Conclusions

In this work, we developed and synthesized the first PSMA-targeted bispidine-based conjugate, suitable for Cu²⁺ chelation. All synthesized conjugates and intermediate compounds were characterized by a set of physicochemical methods: ¹H, ¹³C NMR, and HRMS. The purity of final molecules such as PSMA-Bisp and Bisp-alkyne was confirmed by LCMS analysis. Initial physicochemical and in vitro evaluations were carried out. The conjugate PSMA-Bisp demonstrated a good labeling ability with [⁶⁴Cu]-Cu²⁺ ions and was found to be stable against transchelation and transmetalation in the presence of endogenous metal cations such as Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, and Fe³⁺ and serum proteins. The use of the conjugate in in vitro prostate cancer cell models demonstrates high copper ion accumulation on 22Rv1 (PSMA+) cell cultures, which probably leads to excessive ROS formation, as well as increased cytotoxicity when excessive amounts of copper ions are used in the incubation medium, compared to the results on PC-3 (PSMA−) cell culture. Subsequently, an analysis of PSMA-targeted conjugate biodistribution would be performed in further investigations.