Polypeptide-Based Molecular Platform and Its Docetaxel/Sulfo-Cy5-Containing Conjugate for Targeted Delivery to Prostate Specific Membrane Antigen

A strategy for stereoselective synthesis of molecular platform for targeted delivery of bimodal therapeutic or theranostic agents to the prostate-specific membrane antigen (PSMA) receptor was developed. The proposed platform contains a urea-based, PSMA-targeting Glu-Urea-Lys (EuK) fragment as a vector moiety and tripeptide linker with terminal amide and azide groups for subsequent addition of two different therapeutic and diagnostic agents. The optimal method for this molecular platform synthesis includes (a) solid-phase assembly of the polypeptide linker, (b) coupling of this linker with the vector fragment, (c) attachment of 3-aminopropylazide, and (d) amide and carboxylic groups deprotection. A bimodal theranostic conjugate of the proposed platform with a cytostatic drug (docetaxel) and a fluorescent label (Sulfo-Cy5) was synthesized to demonstrate its possible sequential conjugation with different functional molecules.


Introduction
Prostate cancer (PCa) is one of the most commonly diagnosed men's cancers and remains one of the leading causes of cancer death. In 2018, approximately 1,276,106 new cases and 358,989 suspected deaths were diagnosed worldwide [1,2].
Depending on the stage of the cancer and its severity, various imaging techniques, such as computed tomography (CT), transrectal ultrasound, and relatively recent methods such as magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron

Results and Discussion
Previously, we described therapeutic conjugates of doxorubicin [19] and paclitaxel [20] with PSMA ligands of structurally related types, and it was shown that, for maximum affinity to the receptor, conjugate polypeptide fragments should contain aromatic substituents of different nature in the ζ-NH2 position of Lys-amino acid of the PSMA ligand and the dipeptide fragment Phe(L)-Phe(L) in the linker structure [18,19]. In this article, two functional groups of different nature were introduced into the linker fragment for further stage-by-stage conjugation, with diagnostic and therapeutic moieties at orthogonal conditions to obtain the bimodal theranostic agents. These groups were NH2, which allows attachment of the additional structural fragments using peptide synthesis reactions, and N3, which can be entered into azide-alkyne cycloaddition ( Figure 1).
To obtain the target PSMA ligand with peptide fragments, we developed the synthetic scheme, including the following stages: (1) synthesis of EuK vector 6 with modified urea fragment (Scheme 1), (2)   To demonstrate the possibility of the synthesized bifunctional probe application for the stepwise attachment of diagnostic and therapeutic agents, a double conjugate with docetaxel (DTX) and a fluorescent dye Sulfo-Cyanine5 (Sulfo-Cy5) (Figure 1) was synthesized. This obtained compound was tested for cytotoxic activity and cell staining.

Results and Discussion
Previously, we described therapeutic conjugates of doxorubicin [19] and paclitaxel [20] with PSMA ligands of structurally related types, and it was shown that, for maximum affinity to the receptor, conjugate polypeptide fragments should contain aromatic substituents of different nature in the ζ-NH 2 position of Lys-amino acid of the PSMA ligand and the dipeptide fragment Phe(L)-Phe(L) in the linker structure [18,19]. In this article, two functional groups of different nature were introduced into the linker fragment for further stage-by-stage conjugation, with diagnostic and therapeutic moieties at orthogonal conditions to obtain the bimodal theranostic agents. These groups were NH 2 , which allows attachment of the additional structural fragments using peptide synthesis reactions, and N 3 , which can be entered into azide-alkyne cycloaddition ( Figure 1). To obtain the target PSMA ligand with peptide fragments, we developed the synthetic scheme, including the following stages: (1) synthesis of EuK vector 6 with modified urea fragment (Scheme 1), (2) synthesis of the tripeptide linker using liquid-phase techniques (Scheme 2), (3) alternative synthesis of the tripeptide linker using solid-phase peptide synthesis (SPPS) techniques (Scheme 3), (4) coupling of the vector fragment with the linker with the formation of compound 12 (Schemes 2 and 3), (5) modification of docetaxel with hex-5-ynoic acid giving intermediate 17 (Scheme 4), and (6) click reaction between the compounds 17 and 12 and the subsequent conjugation of the resulting compound with a fluorescent label (Scheme 4).

The Assembly of the Peptide Sequence
The initial stages of the synthesis of the vector fragment 6 (Scheme 1) were realized by previously described methods [21]. Compound 6 was prepared by coupling of succinic anhydrides with compound 5 (Scheme 1); the resulting products contained a free carboxylic group suitable for further addition of the peptide fragment. Tripeptide (Phe(L)-Phe(L)-Lys(L)-(CH2)3-N3) was synthesized from L-phenylalanine (F) and Llysine (K) to obtain highly specific PSMA vectors. Phe(L)-Phe(L) dipeptide fragments in the linker improve the binding to the receptor [18,19]; the dipeptide nature of linkers further improves biodegradability and reduces the unsystematic toxicity of PSMA vectors [22,23]. The coupling of additional lysine amino acid with an azide-containing fragment to the Phe(L)-Phe(L) linker provides the possibility of further modification with therapeutic and diagnostic drugs in orthogonal conditions.

Synthesis of Tripeptide Sequence by Liquid-Phase Technique
The assembly of the peptide sequence was performed in the following manner (Scheme 2): Nα-Fmoc-Nε-Boc-L-lysine was introduced into the reaction with 3-aminopropylazide to obtain compound 7, from which Fmoc was subsequently removed; as a result, the product containing a free amino group 8 was isolated. At the next step, a peptide synthesis between compounds 8 and Fmoc-PhePhe-OH was performed to synthesize the compound 9. The removal of Fmoc protection allowed the desired compound 10 to be obtained as an individual stereoisomer (see Supplementary Information, Figure S4).  The assembly of the peptide sequence was also realized using solid phase peptide synthesis (SPPS) on 2-chlorotrityl chloride resin . This reaction sequence is presented as a classical peptide synthesis scheme: (1) immobilization of N-substituted amino acid on a solid-phase polymer substrate, (2) removal of the protective group, (3) modification of the NH2-group of the amino acid (stages 2 and 3 were repeated to get the desired peptide sequence), and (4) removal of obtained peptide from the polymer substrate [24].
Subsequently, the operations were performed with the necessary amino acids to obtain To obtain conjugate 18 from azide 12 and alkyne 17, we chose the click-reaction of 1,3-dipolar cycloaddition catalyzed by copper(I). This reaction is widely used in synthesizing biologically active organic compounds, in particular, agents against tuberculosis and peptide-carbohydrate conjugates [30]. The complete correlation of signals in NMR spectra of compound 18 was made using 2D NMR spectroscopy (HSQC 1 H-13 C, HMBC 1 H-13 C; see Supplementary Information, Figures S17-S20 , Tables  S3 and S4).
At the next step, the NHS-activated ester of the fluorescent label Sulfo-Cy5 was attached to the free NH2 group of compound 18. Near-infrared fluorescence (NIRF) imaging agents, like Sulfo-Cy5, have high extinction coefficients, large Stokes' shifts, and are able to generate strong fluorescence emission offering the possibility of in vivo cancer diagnosis. Their considerable advantages for in vivo imaging include stronger ligand labeling, signal strength, and tissue absorbance, a wider range of imaging materials for coupling, and less background fluorescence. The far-red cyanine dye Sulfo-Cy5 (λex 640 nm, λem 656 nm), with high detection sensitivity (0.05 vs. 3.15 mM for Indocyanine green (ICG)), tissue penetration (9 vs. 6 mm for ICG), and brightness (quantum yield, 28% vs. 0.3% for ICG) [31], was chosen as the fluorescent label for conjugation with 18.
As a result, the target bimodal conjugate 19 was obtained, and its structure was confirmed by

The Assembly of the Peptide Sequence
The initial stages of the synthesis of the vector fragment 6 (Scheme 1) were realized by previously described methods [21]. Compound 6 was prepared by coupling of succinic anhydrides with compound 5 (Scheme 1); the resulting products contained a free carboxylic group suitable for further addition of the peptide fragment.
Tripeptide (Phe(L)-Phe(L)-Lys(L)-(CH 2 ) 3 -N 3 ) was synthesized from L-phenylalanine (F) and L-lysine (K) to obtain highly specific PSMA vectors. Phe(L)-Phe(L) dipeptide fragments in the linker improve the binding to the receptor [18,19]; the dipeptide nature of linkers further improves biodegradability and reduces the unsystematic toxicity of PSMA vectors [22,23]. The coupling of Molecules 2020, 25, 5784 6 of 21 additional lysine amino acid with an azide-containing fragment to the Phe(L)-Phe(L) linker provides the possibility of further modification with therapeutic and diagnostic drugs in orthogonal conditions.

Synthesis of Tripeptide Sequence by Liquid-Phase Technique
The assembly of the peptide sequence was performed in the following manner (Scheme 2): Nα-Fmoc-Nε-Boc-l-lysine was introduced into the reaction with 3-aminopropylazide to obtain compound 7, from which Fmoc was subsequently removed; as a result, the product containing a free amino group 8 was isolated. At the next step, a peptide synthesis between compounds 8 and Fmoc-PhePhe-OH was performed to synthesize the compound 9. The removal of Fmoc protection allowed the desired compound 10 to be obtained as an individual stereoisomer (see Supplementary Information, Figure S4).

Synthesis of Tripeptide Sequence by SPPS Technique
The assembly of the peptide sequence was also realized using solid phase peptide synthesis (SPPS) on 2-chlorotrityl chloride resin (2-CTC). This reaction sequence is presented as a classical peptide synthesis scheme: (1) immobilization of N-substituted amino acid on a solid-phase polymer substrate, (2) removal of the protective group, (3) modification of the NH 2 -group of the amino acid (stages 2 and 3 were repeated to get the desired peptide sequence), and (4) removal of obtained peptide from the polymer substrate [24].
Subsequently, the operations were performed with the necessary amino acids to obtain compound 15 (Scheme 3).
The 2-CTC resin allows application of the Fmoc SPPS concept and minimizes the adverse reactions. Furthermore, it keeps labile acid functional groups intact, since the amino acid sequence is removed from the polymer substrate under mild conditions (in our case DCM/TFA-99.25%/0.75%, V/V; this system does not affect labile acid actions of the NHBoc and COOtBu groups) [25].

Synthesis of DCL-Modified Tripeptide 12
For the coupling of the vector fragments with peptide sequences by liquid-phase technique vector compound 6 was dissolved in DMF and preactivated using the HOBt/HBTU/DIPEA system for 2 h (Scheme 2). Then, compound 10 was added and the mixture was stirred for 24 h. The reaction product 11 was isolated by column chromatography and further converted to compound 12 (see Section 2.4). All substances were obtained as individual stereoisomers (see Supplementary Information, Figures S7 and S8).
During the SPPS sequence (Scheme 3), vector fragment 6 was attached to tripeptide 15, mounted on 2-CTC. After that, the modified tripeptide was removed from the polymer carrier by treatment with DCM/TFA. As a result, compounds 16 were isolated as individual stereoisomers according to the 1 H NMR, 13 C NMR LCMS, HRMS data (see Supplementary Information, Figures S5 and S6).
Further, 3-aminopropylazide was attached to the free carboxyl group of compounds 16. Based on published data, these reactions may be carried out by one of three possible procedures [26]: Addition of a coupling reagent (carbodiimide, EEDQ (N-Ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline), phosphonium and carbenium salts, trisubstituted phosphates, etc.) and a tertiary amine, if necessary, to a mixture of the acid and the amine nucleophile to be combined; 2.
Addition of the amine nucleophile to a solution of the coupling reagent and the acid only after they were reacted and an activated compound was generated; 3.
Addition of the amine nucleophile to one of the activated forms of the acid (activated ester, acyl azide, anhydrides, etc.) to which it was to be combined.
Considering method 1, it is necessary to note that the activated agent (HBTU) is capable of reacting with N-terminal amino component, leading to a guanidine derivative; this side process may compete for peptide chain elongation. To avoid this side reaction, the preliminary activation of the carboxylic acid component is recommended [27]. We performed method 1 (addition of a coupling reagent and tertiary amine to a mixture of the acid and the amine). Applying this technique to the reaction of compound 16 with 3-aminopropylazide, we obtained the individual stereoisomer of desirable substance 11, as confirmed by NMR spectroscopy (see Supplementary Information, Figure S8).
Taking into account the racemization taking place during the discussed reactions, we concluded that method 2 (adding amine to the activating agent solution, tertiary amine, and acid) was not optimal for stereoselective syntheses of individual stereoisomer of target peptide due to possible intermediate formation of achiral oxazolone intermediate [26]. However, method 2 could be successfully used to obtain compound 11 by the liquid-phase technique (Scheme 2). This is explained by the fact that in the case of the liquid-phase technique, the carboxylic group involved in the formation of a peptide bond is in a vector fragment and does not have a stereocenter in the α-position. Therefore, the possible formation of oxazolone during the reaction does not lead to racemization. NMR spectra of compound 11 obtained by the liquid-phase technique are given in the Supplementary Information ( Figure S7). When using method 3, it should be noted that there is no general method for activated amino acid creation. Also, it is necessary to activate the acid with this method, and then isolate the activated form, which adds an extra stage of synthesis and may lead to undesirable reactions with inappropriate functional groups [26]. For this reason, we did not test method 3 to obtain compound 12.
The next stage of the synthesis was the removal of the protective tert-butyl groups from carboxyl fragments and the Boc group from ε-NH 2 of terminal lysine moiety (Schemes 2 and 3). The deprotection was performed by two methods, i.e., by treating of compound 11 with TFA/DCM or DCM/TFA/TIPS/H 2 O mixture. As a result, target compound 12 was obtained, and its structure was confirmed by HRMSm as well as 1 The data obtained for different methods of vector peptide 12 synthesis are summarized in Table 1. The total yields of the target compounds based on the starting amino acid for linker formation and the starting Boc-Fmoc-protected lysine (Scheme 1) were evaluated. The laboriousness of the syntheses was compared, taking into account the total number of synthetic stages and the number of stages with chromatographic isolation of the target product. Table 1. Comparison of synthetical approaches to obtain target compound 12.

Liquid-Phase Technique (Scheme 2) SPPS Technique (Scheme 3)
Yield based on starting Boc-Fmoc-Lysine 25% 45% Yield based on compound 6 55% 37% The total number of synthesis steps (stages with chromatographic separation) 13 (10) 16 (7) In summary, the liquid-phase technique (Scheme 2) using method 2 (addition of the amine nucleophile to a solution of the coupling reagent and the acid only after they were reacted and generated an activated compound) to create a peptide bond between compound 10 and a vector fragment of ligand 6 was characterized by a maximum yield based on compound 11, but the minimum yield counting of the initial amino acid. The total number of stages using laborious chromatographic isolation was also large.
SPPS technique (Scheme 3) using method 1 (addition of a coupling reagent and tertiary amine to a mixture of the acid and the amine) to create a peptide bond between 16 and 3-aminopropylazide, showed the best yield on the initial amino acid and good yield on compound 6, which seemed to be optimal. Also, this technique showed further advantages over the liquid-phase technique, namely, a less time-consuming process of target platforms obtaining, product isolation simplicity, and the absence of additional purification stages, both for intermediate compounds and the target substance.
However, it should be noted that the liquid-phase technique allows the obtainment of large amounts of target compounds, although it is more laborious and time-consuming than the SPPS approaches. At the same time, obtainment by the SPPS technique may be convenient for the rapid preparation of the libraries of similar compounds, although the reactions proceed with lower yields and require a large excess of amino acids.

Synthesis of the Bimodal Conjugate 19
At the next stage of the work, to demonstrate the possibility of compound 12 use as a molecular platform for bimodal agent preparation, we synthesized its double conjugate with the anticancer drug docetaxel and a fluorescent dye Sulfo-Cy5. Docetaxel is a taxane-derivative diterpenoid and is one of the most widely used anticancer agents in clinical practice today [28]. Analysis of literature data on the effect of modifications of various structural fragments of docetaxel on its activity suggested that the most appropriate strategy for introducing a linker is to form an ester bond with one of the secondary hydroxyl groups [29]. In cells, the ester bond is known to hydrolyze with the extrication of free drug. We carried out the reaction of docetaxel with hex-5-ynoic acid, and the obtained adduct 17 was also introduced into the azide-alkyne cycloaddition with peptide 12. The standard procedure for ester formation in the presence of diisopropylcarbodiimide (DIC) and a catalytic amount of 4-(dimethylamino)pyridine (DMAP) gave compound 17 with reasonable yield (Scheme 4). 2D NMR spectroscopy (HSQC 1 H-13 C, HMBC 1 H-13 C) made it possible to make complete signal correlation in the spectra of compound 17 (Supplementary Information, Figures S13-S16, Tables S1 and S2).
To obtain conjugate 18 from azide 12 and alkyne 17, we chose the click-reaction of 1,3-dipolar cycloaddition catalyzed by copper(I). This reaction is widely used in synthesizing biologically active organic compounds, in particular, agents against tuberculosis and peptide-carbohydrate conjugates [30]. The complete correlation of signals in NMR spectra of compound 18 was made using 2D NMR spectroscopy (HSQC 1 H-13 C, HMBC 1 H-13 C; see Supplementary Information, Figures S17-S20 ,  Tables S3 and S4).
At the next step, the NHS-activated ester of the fluorescent label Sulfo-Cy5 was attached to the free NH 2 group of compound 18. Near-infrared fluorescence (NIRF) imaging agents, like Sulfo-Cy5, have high extinction coefficients, large Stokes' shifts, and are able to generate strong fluorescence emission offering the possibility of in vivo cancer diagnosis. Their considerable advantages for in vivo imaging include stronger ligand labeling, signal strength, and tissue absorbance, a wider range of imaging materials for coupling, and less background fluorescence. The far-red cyanine dye Sulfo-Cy5 (λ ex 640 nm, λ em 656 nm), with high detection sensitivity (0.05 vs. 3.15 mM for Indocyanine green (ICG)), tissue penetration (9 vs. 6 mm for ICG), and brightness (quantum yield, 28% vs. 0.3% for ICG) [31], was chosen as the fluorescent label for conjugation with 18.
As a result, the target bimodal conjugate 19 was obtained, and its structure was confirmed by HRMS, LCMS, and 1 H-NMR data. Moreover, initial biological experiments on the synthesized conjugate interaction with human cells differing in the level of PSMA expression were carried out in order to preliminary estimate its possibility and potential for biomedical application.  We observed a homogeneous diffuse staining of all cells in the LNCaP line and a part of the cell population in 22Rv1 culture after 2 h incubation with PSMA-Cy5. It must be noted that the most intensive fluorescence signal from PSMA-Cy5 conjugate was observed in the perinuclear area. This fact indicated the intracellular localization of the PSMA-Cy5 conjugate. No fluorescence signal from PSMA-Cy5 was detected in cells of PSMA-negative PC-3 line. For compound 19, all LNCaP cells were also positively stained. However, the nature of the staining was different-we revealed the fluorescent signal of conjugate 19 to be mainly point-concentrated, presumably, in cell vesicles. At the same time, less pronounced diffuse staining of the entire cells cytoplasm was found. A similar result was obtained for the 22Rv1 cell line. However, fluorescent signal from conjugate 19 was detected not in all cells of population. Moreover, the presence of point-concentrated localization of compound 19 in these cells was significantly less than in the LNCaP line. Single accumulations of conjugate 19 were identified predominantly in the lamellae of PC-3 cells. Thus, the obtained data demonstrated that the effectiveness of the conjugate 19 selective interaction with PSMA++ LNCaP cells was higher than with PSMA+ 22Rv1 cells. Interaction of compound 19 with PSMA-PC-3 cells was significantly lower than with both investigated PSMA-positive cell lines. Some accumulations of conjugate 19 revealed in PC-3 cells could be due to its nonspecific interaction with the cells and penetration by diffusion presumed for Docetaxel. The mechanism of this taxane penetration inside the cells was well studied only for hepatocytes, while further investigations are required for other cell types [33].

Biological Evaluation
Further, bimodal conjugate 19, as well as its synthetical precursors (conjugate 18, containing docetaxel, but not containing a fluorescent label, peptide vector 12 without imaging or therapeutic agents, and free docetaxel as a comparison substance) were evaluated for in vitro cytotoxicity against two PSMA-positive cell lines-LNCaP and 22Rv1 (Figure 3) [32]. The cargo Docetaxel and conjugate 18 were used as a positive control, whereas compound 12 was used as a negative control. As a result, conjugates 18 and 19 showed good activity against both cell lines with a slightly more pronounced effect on LNCaP cells, where LNCaP IC50 = 100 nM and 200 nM, respectively, as well as 22Rv1 IC50 = 130 nM and >200 nM. Docetaxel by itself caused significant cell death in both cultures, where LNCaP We observed a homogeneous diffuse staining of all cells in the LNCaP line and a part of the cell population in 22Rv1 culture after 2 h incubation with PSMA-Cy5. It must be noted that the most intensive fluorescence signal from PSMA-Cy5 conjugate was observed in the perinuclear area. This fact indicated the intracellular localization of the PSMA-Cy5 conjugate. No fluorescence signal from PSMA-Cy5 was detected in cells of PSMA-negative PC-3 line. For compound 19, all LNCaP cells were also positively stained. However, the nature of the staining was different-we revealed the fluorescent signal of conjugate 19 to be mainly point-concentrated, presumably, in cell vesicles. At the same time, less pronounced diffuse staining of the entire cells cytoplasm was found. A similar result was obtained for the 22Rv1 cell line. However, fluorescent signal from conjugate 19 was detected not in all cells of population. Moreover, the presence of point-concentrated localization of compound 19 in these cells was significantly less than in the LNCaP line. Single accumulations of conjugate 19 were identified predominantly in the lamellae of PC-3 cells. Thus, the obtained data demonstrated that the effectiveness of the conjugate 19 selective interaction with PSMA++ LNCaP cells was higher than with PSMA+ 22Rv1 cells. Interaction of compound 19 with PSMA-PC-3 cells was significantly lower than with both investigated PSMA-positive cell lines. Some accumulations of conjugate 19 revealed in PC-3 cells could be due to its nonspecific interaction with the cells and penetration by diffusion presumed for Docetaxel. The mechanism of this taxane penetration inside the cells was well studied only for hepatocytes, while further investigations are required for other cell types [33].
Further, bimodal conjugate 19, as well as its synthetical precursors (conjugate 18, containing docetaxel, but not containing a fluorescent label, peptide vector 12 without imaging or therapeutic agents, and free docetaxel as a comparison substance) were evaluated for in vitro cytotoxicity against two PSMA-positive cell lines-LNCaP and 22Rv1 (Figure 3) [32]. The cargo Docetaxel and conjugate 18 were used as a positive control, whereas compound 12 was used as a negative control. As a result, conjugates 18 and 19 showed good activity against both cell lines with a slightly more pronounced effect on LNCaP cells, where LNCaP IC 50 = 100 nM and 200 nM, respectively, as well as 22Rv1 IC textsubscript50 = 130 nM and >200 nM. Docetaxel by itself caused significant cell death in both cultures, where LNCaP IC 50 = 1 nM and 22Rv1 IC 50 = 2.1 nM. These data were consistent with the selectivity of the resulting conjugates in relation to cell lines expressing PSMA. The lower toxicity of conjugates 18 and 19 in comparison with free Docetaxel, could apparently be explained by the slow release of the active drug from the conjugate, consistent with previously obtained results [19]. Vector peptide 12, as expected, was not toxic for either of the cell lines.
Molecules 2020, 25, x FOR PEER REVIEW 10 of 22 IC50 = 1 nM and 22Rv1 IC50 = 2.1 nM. These data were consistent with the selectivity of the resulting conjugates in relation to cell lines expressing PSMA. The lower toxicity of conjugates 18 and 19 in comparison with free Docetaxel, could apparently be explained by the slow release of the active drug from the conjugate, consistent with previously obtained results [19]. Vector peptide 12, as expected, was not toxic for either of the cell lines. Based on these data, we can conclude that the designed vector is a perspective conjugate, which demonstrated selectivity and toxicity against PSMA-positive cells and should be further investigated in more detail for targeted drug delivery, at least in PSMA-overexpressed LNCaP cells.

Materials and Methods
All used solvents were purified according to procedures described in [34]. All starting compounds were commercially available reagents. The initial stages of the synthesis of the vector fragment 1-5 (Scheme 1) were made by methods previously developed by our scientific group [21]. Spectral data of the compounds 7 and 8 (Scheme 2) were described in [35]. 1 H NMR was measured using a Bruker Avance spectrometer operating at 400 MHz for 1 H using CDCl3 and DMSO-d6 as solvents. Chemical shifts were reported in δ units to 0.01 ppm precision with coupling constants reported to 0.1 Hz precision using residual solvent as an internal reference. 13 C NMR was measured using a Bruker Avance spectrometer operating at 100 MHz using DMSO-d6 as solvents. Chemical shifts were reported in δ units to 0.1 ppm precision using residual solvent as an internal reference. 2D NMR was measured using an Agilent 600 spectrometer operating at 600 MHz for 1 H and 100 MHz for ( 13 С) using DMSO-d6 as the solvent. As 2D NMR methods were used, such as heteronuclear single quantum coherence spectroscopy 1 H-13 C (gHSQC) and heteronuclear multiple bond correlation 1 H-13 C (gHMBC). NMR spectra were processed and analyzed using Mnova software (Mestrelab Research, Spain). High-resolution mass spectra were recorded on the Orbitrap Elite high-resolution mass spectrometer. Solutions of samples in acetonitrile with 1% formic acid were introduced into the ionization source by electrospray. For the HPLC analysis system with Shimadzu Prominence, an LC-20 column and a convection fraction collector connected with a single quadrupole mass spectrometer Shimadzu LCMS-2020 with dual ionization source DUIS-ESI-APCI were used. The analytical and preparative column was Phenomenex Luna 3u C18 100A. Preparative chromatographic separation of substances was carried out using the INTERCHIM puriFlash 430 chromatograph.
For better interpretation of the NMR spectra of target compound 19, the notation of structural fragments is shown in Figure 4. Based on these data, we can conclude that the designed vector is a perspective conjugate, which demonstrated selectivity and toxicity against PSMA-positive cells and should be further investigated in more detail for targeted drug delivery, at least in PSMA-overexpressed LNCaP cells.

Materials and Methods
All used solvents were purified according to procedures described in [34]. All starting compounds were commercially available reagents. The initial stages of the synthesis of the vector fragment 1-5 (Scheme 1) were made by methods previously developed by our scientific group [21]. Spectral data of the compounds 7 and 8 (Scheme 2) were described in [35]. 1 H NMR was measured using a Bruker Avance spectrometer operating at 400 MHz for 1 H using CDCl 3 and DMSO-d 6 as solvents. Chemical shifts were reported in δ units to 0.01 ppm precision with coupling constants reported to 0.1 Hz precision using residual solvent as an internal reference. 13 C NMR was measured using a Bruker Avance spectrometer operating at 100 MHz using DMSO-d 6 as solvents. Chemical shifts were reported in δ units to 0.1 ppm precision using residual solvent as an internal reference. 2D NMR was measured using an Agilent 600 spectrometer operating at 600 MHz for 1 H and 100 MHz for ( 13 C) using DMSO-d 6 as the solvent. As 2D NMR methods were used, such as heteronuclear single quantum coherence spectroscopy 1 H-13 C (gHSQC) and heteronuclear multiple bond correlation 1 H-13 C (gHMBC). NMR spectra were processed and analyzed using Mnova software (Mestrelab Research, Spain). High-resolution mass spectra were recorded on the Orbitrap Elite high-resolution mass spectrometer. Solutions of samples in acetonitrile with 1% formic acid were introduced into the ionization source by electrospray. For the HPLC analysis system with Shimadzu Prominence, an LC-20 column and a convection fraction collector connected with a single quadrupole mass spectrometer Shimadzu LCMS-2020 with dual ionization source DUIS-ESI-APCI were used. The analytical and preparative column was Phenomenex Luna 3u C18 100A. Preparative chromatographic separation of substances was carried out using the INTERCHIM puriFlash 430 chromatograph.
For better interpretation of the NMR spectra of target compound 19, the notation of structural fragments is shown in Figure 4. Cell Lines: LNCaP, 22Rv1, and PC-3 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
Cell Cultivation: Cells were maintained in RPMI-1640 medium (gibco), supplemented with 10% Fetal Bovine Serum (Sigma), 2 mM L-glutamine, and RPMI vitamin solution (Sigma). Cells were cultured at 37 °C in a humidified incubator (Sanyo) supplied with 5% CO2. Cells were seeded on glass coverslips or in 96-well plates (Corning) at concentrations of 120,000 cells per mL for LNCaP, 200,000 cells per mL for 22Rv1, and 90,000 cells per mL for PC-3 in experiments. The counting of cells was carried out using the automatic cell counter EVE.
Cell Incubation with Conjugates: A day after seeding the cells on glass coverslips, PSMA-Cy5 or fluorescently labeled compound 19 were added in culture medium at a concentration of 30 nM for 2 h. Later, cells were washed with PBS (pH 7.2-7.4) and fixed with 4% formaldehyde (Sigma) (on PBS) for 15 min. Cell nuclei were stained with DAPI (Sigma) for 10 min. Obtained preparations were imaged using an inverted fluorescence microscope EVOS (life technologies, objective PlanFluor 20×/0.45). Further processing of the photos was carried out by ImageJ software.
Cytotoxicity Assay: A day after cell seeding in 96-well plates, serial dilutions of conjugates and Docetaxel in culture medium were added to cells. Cells incubated in culture medium were used as control. DMSO diluted in the cell medium (20%) 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) was added to each well with 100 μL of new culture medium. After 4 h of incubation at 37 °C in darkness, the absorbance of the obtained solution was measured at 490 nm wavelength using the Thermo Scientific Multiskan GO spectrometer. Cell viability was calculated as percent compared to cells incubated in culture medium. MTS assay revealed 100% cell death after incubation with 20% DMSO (data not shown). The absorbance of MTS reagent in culture medium without cells was taken as zero. Experiments were performed in triplicate.
Compound 6. To a solution of compound 5 (1 eq; 725 mg; 1.0 mmol) in 20 mL of DCM, DIPEA (1.4 eq; 244 μL; 1.4 mmol) and succinic anhydride (1.02 eq; 102 mg; 1.02 mmol) were added. The mixture was stirred for 12 h. After that, MeOH (2 eq.) was added and the resulting mixture was stirred for 1 h. Then, the solvent was removed under reduced pressure, and residue was dissolved in DCM and extracted with (1) 0.1 M HCl (2 × 30 mL) and (2) brine (2 × 30 mL). Then, the organic fraction was dried over Na2SO4, and concentrated under reduced pressure to obtain the final compound 6 as a yellow oil (801 mg, yield 97%). Cell Lines: LNCaP, 22Rv1, and PC-3 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
Cell Cultivation: Cells were maintained in RPMI-1640 medium (gibco), supplemented with 10% Fetal Bovine Serum (Sigma), 2 mM L-glutamine, and RPMI vitamin solution (Sigma). Cells were cultured at 37 • C in a humidified incubator (Sanyo) supplied with 5% CO 2 . Cells were seeded on glass coverslips or in 96-well plates (Corning) at concentrations of 120,000 cells per mL for LNCaP, 200,000 cells per mL for 22Rv1, and 90,000 cells per mL for PC-3 in experiments. The counting of cells was carried out using the automatic cell counter EVE.
Cell Incubation with Conjugates: A day after seeding the cells on glass coverslips, PSMA-Cy5 or fluorescently labeled compound 19 were added in culture medium at a concentration of 30 nM for 2 h. Later, cells were washed with PBS (pH 7.2-7.4) and fixed with 4% formaldehyde (Sigma) (on PBS) for 15 min. Cell nuclei were stained with DAPI (Sigma) for 10 min. Obtained preparations were imaged using an inverted fluorescence microscope EVOS (life technologies, objective PlanFluor 20×/0.45). Further processing of the photos was carried out by ImageJ software.
Cytotoxicity Assay: A day after cell seeding in 96-well plates, serial dilutions of conjugates and Docetaxel in culture medium were added to cells. Cells incubated in culture medium were used as control. DMSO diluted in the cell medium (20%) was used as a positive control. Cells were incubated for 72 h at 37 • C and 5% CO 2 . Later, the culture medium from each well was removed and 20 µL of MTS reagent (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega) was added to each well with 100 µL of new culture medium. After 4 h of incubation at 37 • C in darkness, the absorbance of the obtained solution was measured at 490 nm wavelength using the Thermo Scientific Multiskan GO spectrometer. Cell viability was calculated as percent compared to cells incubated in culture medium. MTS assay revealed 100% cell death after incubation with 20% DMSO (data not shown). The absorbance of MTS reagent in culture medium without cells was taken as zero. Experiments were performed in triplicate.
The obtained compounds were characterized by NMR spectroscopy and high-resolution mass spectrometry; complete assignment of signals in the NMR spectra of the compounds 12 and 18 was made using two-dimensional NMR sequences. The reasonable cytotoxicity of vector molecule 12, its conjugate with docetaxel 18, and docetaxel/Sulfo-Cy5 19 against PSMA-expressing cell lines were found during initial in vitro study as well as their selective interaction with cells. However, further in vitro as far as in vivo investigations of the conjugates are required for a more explicit demonstration of their efficacy and selectivity for PSMA-expressing cells and tumors. Anyhow, conjugate 19 can be used as a convenient starting point appropriate for the follow-up structure optimization study.