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Article

PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates

by
Bing Xu
,
Fei Zhou
,
Meng-Meng Yan
,
De-Sheng Cai
,
Wen-Bo Guo
,
Yu-Qin Yang
,
Xiao-Hui Jia
,
Wen-Xi Zhang
,
Tong Li
,
Tao Ma
*,
Peng-Long Wang
* and
Hai-Min Lei
*
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(10), 3251; https://doi.org/10.3390/ijms19103251
Submission received: 20 August 2018 / Revised: 9 October 2018 / Accepted: 14 October 2018 / Published: 19 October 2018
(This article belongs to the Section Biochemistry)
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Versions Notes

Abstract

:
Clinical applications of camptothecin (CPT) have been heavily hindered due to its non-targeted toxicity, active lactone ring instability, and poor water solubility. Targeted drug delivery systems may offer the possibility to overcome the above issues as reported. In this research, a series of prostate-specific membrane antigen (PSMA)-activated CPT prodrugs were designed and synthesized by coupling water-soluble pentapeptide, a PSMA hydrolyzing substrate, to CPT through an appropriate linker. The cytotoxicity of CPT prodrugs was masked temporarily until they were hydrolyzed by the PSMA present within the tumor sites, which restored cytotoxicity. The in vitro selective cytotoxic activities of the prodrugs were evaluated against PSMA-expressing human prostate cancer cells LNCaP-FGC and non-PSMA-expressing cancer cells HepG2, Hela, MCF-7, DU145, PC-3 and normal cells MDCK, LO2 by standard methylthiazol tetrazolium (MTT) assay. Most of the newly synthesized CPT prodrugs showed excellent selective toxicity to PSMA-producing prostate cancer cells LNCaP-FGC with improved water solubility. From among the library, CPT-HT-J-ZL12 showed the best cytotoxic selectivity between the PSMA-expressing and the non-PSMA-expressing cancer cells. For example, the cytotoxicity of CPT-HT-J-ZL12 (IC50 = 1.00 ± 0.20 µM) against LNCaP-FGC (PSMA+) was 40-fold, 40-fold, 21-fold, 5-fold and 40-fold, respectively, higher than that against the non-PSMA-expressing cells HepG2 (IC50 > 40.00 µM), Hela (IC50 > 40.00 µM), MCF-7 (IC50 = 21.68 ± 4.96 µM), DU145 (IC50 = 5.40 ± 1.22 µM), PC-3 (IC50 = 42.96 ± 3.69 µM) cells. Moreover, CPT-HT-J-ZL12 exhibited low cytotoxicity (IC50 > 40 μM) towards MDCK and LO2 cells. The cellular uptake experiment demonstrated the superior PSMA-targeting ability of the CPT-HT-J-ZL12, which was significantly accumulated in LNCaP-FGC (PSMA+), while it was minimized in HepG2 (PSMA−) cells. Further cell apoptosis analyses indicated that it showed a dramatically higher apoptosis-inducing activity in LNCaP-FGC (PSMA+) cells than in HepG2 (PSMA−) cells. Cell cycle analysis indicated that CPT-HT-J-ZL12 could induce cell cycle arrest at the S phase.

Graphical Abstract

1. Introduction

Cancer remains one of the major diseases that severely threatens human life and health. Chemotherapy is still one of the main clinical treatments for cancer, either alone or in combination with other treatment options [1,2]. The low selectivity of conventional chemotherapeutic drugs induces serious adverse events and systemic toxicities [3,4,5,6]. Therefore, it is of great importance to develop targeted anti-cancer drug delivery.
Camptothecin (CPT), a natural alkaloid from Camptotheca acuminate, possesses remarkable antitumor properties against a variety of cancers via inhibition of DNA enzyme topoisomerase I (topo I) [7,8,9]. Even though CPT exhibits potent anticancer activities in vitro and vivo, its therapeutic application is severely hindered due to its poor aqueous solubility and high lipophilicity, active lactone ring instability, and non-targeted toxicity [10,11,12]. Previous research has shown that structural modifications at the 20-OH of camptothecin could enhance the stability of the lactone ring, and prodrugs derivatized at this site have been vigorously pursued [13,14,15,16].
The prostate-specific membrane antigen (PSMA) is a 750-amino acid type II transmembrane peptidase enzyme that is encoded by the folate hydrolase 1 (FOLH1) gene. Previous studies demonstrated that PSMA was highly expressed in prostate cancer cells and in tumor endothelial cells (ECs) of a variety of non-prostatic solid tumor types but was not expressed by ECs in normal tissues. In addition, PSMA is also known as glutamate carboxypeptidase II, N-acetyl-l-aspartyl-l-glutamate peptidase I, and N-acetylaspartylglutamate peptidase. It contains a binuclear zinc site and is active as a glutamate carboxypeptidase, catalyzing the hydrolytic cleavage of α- or γ-linked glutamates from peptides or small molecules [17,18,19,20]. Based on the above, PSMA is becoming one of the current research hotspots and is an excellent target for the anti-angiogenesis targeted therapy [19,20,21,22,23]. The enzymatic activity of PSMA can be exploited for the design of prodrugs, in which an inactive form of the cytotoxic drug is selectively cleaved and thereby activated only at cells that express PSMA within the tumor microenvironment.
In view of the unique carboxypeptidase activity and restricted expression of the PSMA, we accomplished the synthesis of several PSMA-activated camptothecin pentapeptide prodrugs CPT-HT-J-ZLn, by introducing an oriented oligopeptide HT-J (Glu*Glu*Glu*Asp-Glu) (“*” mean the γ-glutamyl linkage; “-” mean the α-glutamyl linkage) to the 20-OH of CPT via carbonate linkers with different lengths, in order to improve their tumor targeting, stability, and aqueous solubility. All newly synthesized compounds were fully characterized by 1H-NMR, 13C-NMR, and HRMS; they were also tested for selective cytotoxic activity against PSMA-expressing human prostate cancer cell LNCaP-FGC and non-PSMA-expressing cells HepG2, Hela, MCF-7, DU145, and PC-3, respectively [24,25,26,27,28] and normal cells MDCK, LO2 by standard MTT assay. Meanwhile, the preliminary anti-tumor mechanisms of the most selectivity compound CPT-HT-J-ZLn were also investigated by fluorescence staining observation and flow cytometric analysis in the present study. In addition, the structure-activity relationships of these derivatives are briefly discussed.

2. Results

2.1. Chemistry

The designed derivatives were prepared following the procedures in Scheme 1, Scheme 2 and Scheme 3. HT-J (Glu*Glu*Glu*Asp-Glu), the pentapeptide used to target PSMA, was synthesized by liquid-phase peptide chemistry starting with Fmoc-Asp(OtBu)-OH (Scheme 1).
The intermediate CPT-A-Ln was subsequently prepared by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)-mediated esterification from CPT. Then CPT-A-Ln was further treated with trifluoroacetic (TFA) in dry dichloromethane (DCM) to remove the Boc-protective group, and the key intermediate CPT-B-Ln was successfully obtained (Scheme 2).
For the synthesis of the PSMA-activated prodrug CPT-HT-J-ZLn, reaction of CPT-B-Ln with HT-J in the presence of EDCI, N,N-Diisopropylethylamine (DIPEA), and 1-hydroxybenzotriazole (HOBT) in DCM afforded the corresponding intermediate CPT-HT-J-Ln, which was further treated with TFA to remove the protective groups, yielding the final prodrug CPT-HT-J-ZLn (Scheme 2). It is worth noting that the tert-butyl cation released in the decomposition of the tert-butyl ester was a powerful electrophile that might react with the substrate. To avoid the formation of undesirable by-products, the nucleophilic scavenger thioanisole was added to TFA.
Previous studies have shown that PSMA is an exopeptidase, and hydrolytic processing of any prodrug would result in a product consisting of a cytotoxin coupled to the acidic amino acids glutamate or aspartate [19]. To better study the PSMA-targeting ability of the prodrug CPT-HT-J-ZL12, the PSMA hydrolysate CPT-D-Ln was prepared as well, according to the procedures in Scheme 3 in this study. In brief, the intermediate CPT-B-Ln underwent coupling reaction with N-Cbz-l-glutamic acid 5-benzyl ester in the presence of EDCI, HOBt, and DIPEA in dry DCM to afford the intermediate CPT-C-GLn, which was further treated with Pd/C (10%) in methanol (MeOH) to give the final compound CPT-D-GLn. The structures of all target derivatives were confirmed by spectral (1H-NMR, 13C-NMR, and HRMS) analysis (Figures S1–S122).

2.2. Cytotoxicity

The cytotoxicities of PSMA-activated prodrug CPT-HT-J-ZLn and PSMA hydrolysate CPT-D-GLn were evaluated by MTT assays on PSMA-expressing (PSMA+) cancer cell LNCaP-FGC, non-PSMA-expressing (PSMA−) cancer cells HepG2, MCF-7, Hela, DU145, and PC-3, and normal cells MDCK, LO2, respectively, using CPT as a positive control. The IC50 values are summarized in Table 1 and Figure 1.
As shown in Table 1 and Figure 1, most of the PSMA-activated prodrug CPT-HT-J-ZLn showed moderate to potent cytotoxicity against all the tested tumor cell lines. However, their cytotoxicity was much lower than the parent compound CPT. The cytotoxicity detection also revealed that most of the prodrug CPT-HT-J-ZLn exhibited better anti-proliferative activities against the PSMA-expressing cell line LNCaP-FGC than the non-PSMA-expressing cell lines HepG2, Hela, MCF-7, DU145, and PC-3.
From among the library, CPT-HT-J-ZL12 showed the most cytotoxic selectivity against the PSMA-expressing and the non-PSMA-expressing cancer cells. For example, the cytotoxicity of CPT-HT-J-ZL12 (IC50 = 1.00 ± 0.20 µM) against LNCaP-FGC (PSMA+) was 40-fold, 40-fold, 21-fold, 5-fold and 40-fold, respectively, higher than that against the non-PSMA-expressing cells HepG2 (IC50 > 40.00 µM), Hela (IC50 > 40.00 µM), MCF-7 (IC50 = 21.68 ± 4.96 µM), DU145 (IC50 = 5.40 ± 1.22 µM), PC-3 (IC50 = 42.96 ± 3.69 µM) cells. Moreover, CPT-HT-J-ZL12 exhibited low cytotoxicity (IC50 > 40.00 µM) towards MDCK and LO2 cells (Figure 2). This was probably because LNCaP-FGC had expression of PSMA, while the others did not. The PSMA present at the membrane of the tumor cells was able to hydrolyze the pentapeptide, and then the subsequent active compound was able to enter the cells and exert the cytotoxic effects. Interestingly, among the non-PSMA-expressing cells, the prodrug CPT-HT-J-ZLn, showed much higher cytotoxicities against MCF-7, DU145, and PC-3 cell lines than other cell lines (Hela and HepG2).
Furthermore, it was observed that the length of the linker chain between CPT and the pentapeptide HT-J affected the cytotoxicity observably. With the extension of the linker chain, the cytotoxicity decreased, as exemplified by CPT-HT-J-ZL2, CPT-HT-J-ZL4 > CPT-HT-J-ZL6, CPT-HT-J-ZL12. The cytotoxicity detection also revealed that most of the PSMA hydrolysate CPT-D-GLn and the intermediate CPT-B-Ln exhibited antiproliferative activities against all tested cancer cells, but their cytotoxic selectivity against the PSMA-expressing and the non-PSMA-expressing cancer cells was lower than the prodrugs CPT-HT-J-ZLn. Structure-activity relationship analysis among CPT-D-GLn and CPT-B-Ln also revealed that the length of the linker chain affected the cytotoxicity observably. With the extension of the linker chain, cytotoxicity was also decreased, as exemplified by CPT-B-L2, CPT-B-L4 > CPT-B-L6, CPT-B-L12; CPT-D-GL2, CPT-D-GL4 > CPT-D-GL6, CPT-D-GL12; therefore, the most selective compound CPT-HT-J-ZL12 was picked out for further studies to investigate its mechanism of growth inhibition on HepG2 and LNCaP-FGC cell lines.

2.3. Computer Simulation of ClogP and the Aqueous Solubility Study

LogP, the logarithm of the octanol/water partition coefficient, is often used as the common quantitative descriptor of lipophilicity [29]. Optimization of solubility and hydrophobicity played an important role in the drug discovery process on account of their being closely associated with the absorption, distribution, metabolism, and excretion (ADME) properties of the compounds [30,31,32]. In this work, the logP values for these derivatives were calculated using the Sybyl-X 2.0 software (Tripos, Certara Inc., St. Louis, MO, USA). As shown in Table 1, all the prodrug CPT-HT-J-ZLn showed lower ClogP than CPT. Meanwhile the aqueous solubility of the prodrug CPT-HT-J-ZLn (19.00 mg/mL in PBS, pH = 7.4), which was much higher than CPT (0.003 mg/mL in PBS, pH = 7.4).

2.4. In Vitro Cellular Uptake

In addition to solubilization, the pentapeptide serves to mask the cytotoxicity by preventing nonspecific uptake of drug into cells due to the presence of negative charged amino acids. Upon cleavage of the prodrug by PSMA within the PSMA-expressing cells, the active compound was able to enter cells. To prove our assumption, the cellular uptake behavior of CPT-HT-J-ZL12 in LNCAP-FGC (PSMA+) and HepG2 (PSMA−) was investigated by confocal laser scanning microscope (CLSM). The nucleus was stained with PI (red) and the fluorescence of CPT was pseudo-colored with blue. As shown in Figure 3, there was almost no blue fluorescence when treated with CPT-HT-J-ZL12 in HepG2 (PSMA−) cells for 1 h and 4 h, demonstrating the poor uptake of CPT-HT-J-ZL12. On the contrary, the blue fluorescence intensity in LNCAP-FGC (PSMA+) for 1 h was stronger, indicating that the active compound was able to enter the cancer cells. In addition, the blue fluorescence in LNCAP-FGC (PSMA+) increased with the incubation time (4 h) (Figure 4 and Figure 5), implying that prolonged treatment facilitated cellular uptake of the subsequent active compound.

2.5. Detection of Apoptosis Using Annexin V-FITC/PI Staining

To further evaluate the selective cytotoxicity on LNCaP-FGC (PSMA+) and HepG2 (PSMA−) after treatment with CPT-HT-J-ZL12, apoptotic rates were analyzed by flow cytometry using an Annexin V-FITC/PI staining. It was observed that the LNCaP-FGC (PSMA+) cell demonstrated a remarkable response to the apoptotic effect of CPT-HT-J-ZL12 in a dose-dependent manner. The apoptotic effect was determined by counting the apoptosis ratios (including the early and late apoptosis ratios). Following different concentrations (0.63, 1.25, 2.50 µM) of CPT-HT-J-ZL12 treatment, the apoptosis ratios increased from 29.90% to 33.30% and 48.90%, respectively (Figure 6). On the contrary, the drug-induced apoptosis for HepG2 (PSMA−) cell demonstrated a totally different trend. The percentage of apoptotic HepG2 (PSMA−) cell was evaluated to be 19.80%, 28.30%, and 30.10% after treatment with 20.00 µM, 40.00 µM and 80.00 µM of CPT-HT-J-ZL12, respectively (Figure 7). These results demonstrated CPT-HT-J-ZL12 showed PSMA-oriented targeting activity.

2.6. Detection of Cell Cycle Using PI Staining

To further study CPT-HT-J-ZL12-induced growth inhibition, the cell cycle changes were evaluated on LNCaP-FGC (PSMA+) using flow cytometric methods. As shown in Figure 8, with the concentration of CPT-HT-J-ZL12 increased from 0.63 to 1.25 to 2.50 µM, the percentage of LNCaP-FGC cells in S phase increased dramatically (from 4.47% to 70.86% to 83.66%), while the cells in G0/G1 phases decreased. This indicated that CPT-HT-J-ZL12 was able to induce cell cycle arrest at the S phase.

3. Discussion

In this article, four novel water-soluble PSMA-activated CPT prodrugs were successfully achieved by coupling pentapeptide HT-J, a PSMA hydrolyzing substrate, to 20-OH of CPT. Their selective cytotoxic activities were determined on several PSMA-positive and PSMA-negative cancer cell and normal cell lines by the standard MTT assay. The results indicated that the prodrug CPT-HT-J-ZLn showed significantly selective cytotoxicity against the PSMA-positive and PSMA-negative cell lines. CPT-HT-J-ZL12, the lead PSMA-activated prodrug identified herein, was ~40-fold more toxic in vitro to PSMA-positive cell LNCaP-FGC (IC50 = 1.00 ± 0.20 µM) compared to PSMA-negative cells HepG2 (IC50 > 40.00 µM) and Hela (IC50 > 40.00 µM). Moreover, CPT-HT-J-ZL12 exhibited low cytotoxicity (IC50 > 40 µM) towards MDCK and LO2 cells. The in vitro cellular uptake study and the cell apoptosis analyses demonstrated the PSMA-oriented targeting of CPT-HT-J-ZL12. The cell cycle analysis indicated that CPT-HT-J-ZL12 could induce cell cycle arrest at the S phase. The PSMA present at the membrane of the PSMA-positive cells was able to hydrolyze the pentapeptide; then the subsequent active compound was able to enter cells and exert the toxic effect. The preclinical results presented here suggested that the attempt to discover tumor-targeting lead compounds by using this PSMA-activated prodrug strategy was viable. Consequently, our original design concept should be perfectly illustrated by the biological evaluations above. In addition, the compound CPT-HT-J-ZL12 was selected for further pharmacodynamics and pharmacokinetic evaluation, including in vivo antitumor activity against LNCaP-FGC human prostate cancer mouse xenograft and MCF-7 human breast cancer xenograft, in vivo pharmacokinetics, in the hope of producing drug candidates for drug development.

4. Materials and Methods

4.1. Chemistry

Materials were obtained from commercial suppliers and used without further purification unless otherwise mentioned. Reactions were monitored by TLC using silica gel-coated aluminum sheets (Qingdao Haiyang Chemical Co., Qingdao, China). All melting points were determined on a micro melting point apparatus and were uncorrected. NMR spectra were recorded on a BRUKER AVANCE 500 and 400 MHz spectrometer (Fällanden, Switzerland) with tetramethylsilane (TMS) as an internal standard; chemical shifts δ were given in ppm and coupling constants J in Hz. HR-MS were acquired using a Thermo Sientific TM LTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY, USA).

4.1.1. General Synthetic Procedure of Condensation Reaction (Method A)

The corresponding intermediate (1 equiv.) was dissolved in dry DCM (20 mL), EDCI (1.5 equiv.) and the corresponding protected amino acid (1.1 equiv.) were added. After addition of HOBT (1.5 equiv.) and DIPEA (2.5 equiv.), the mixture was stirred at 25 °C for 4 h. The reaction mixture was diluted with 50 mL CH2Cl2, then successively washed with water and brine (10 mL each), dried over sodium sulfate and filtered, and the solvent was evaporated. Then the crude product was purified by flash chromatography (silica gel, petroleum ether:ethyl acetate =10:1 to 3:1).

4.1.2. General Synthetic Procedure of Deprotection (Method B)

To a solution of the Fmoc-protected compound (1 equiv.) in dry DMF (20 mL), piperidine (3 equiv.) was added. The mixture was allowed to stir at room temperature for 0.5 h. After completion of the reaction (as monitored by TLC), the solution was evaporated, and the residue was dissolved with ethyl acetate (50 mL). The organic extracts were washed with distilled water (3 × 25 mL) and brine (20 mL), dried over sodium sulfate, filtrated and evaporated. Purification was performed by flash chromatography (silica gel, petroleum ether:ethyl acetate = 3:1 to 0:1).

4.1.3. General Synthetic Procedure of Deprotection (Method C)

Pd/C (10%; 80 mg) was added to a solution of the Cbz-protected compound in 30 mL MeOH. The mixture was stirred at room temperature for 12 h. After completion of the reaction (as monitored by TLC), the solvent was filtered to remove Pd/C. The filtrate was concentrated in vacuum and the residue was purified by flash chromatography (silica gel, dichloromethane:methanol = 40:1 to 10:1).
Characterization of di-tert-butyl ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoyl)-l-glutamate (HT-A). Obtained from Fmoc-l-aspartic acid β-tert-butyl ester by method A as a white powder; m.p. 116.6–116.9 °C; Yield: 85.8%; 1H-NMR (500 MHz, CDCl3): δ 1.44 (m, 27 H, 9 × -CH3), 1.90 (m, 1H), 2.16 (m, 1H), 2.31 (m, 2H), 2.60 (m, 1H), 2.99 (m, 1 H), 4.24 (t, J = 7.1 Hz, 1H), 4.42 (m, 2H), 4.45 (m, 1H), 4.56 (m, 1H), 5.99 (d, J = 8.5 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.60 (m, 2H), 7.76 (d, J = 7.6 Hz, 2H). 13C-NMR (126 MHz, CDCl3): δ 27.8, 28.1, 28.2, 31.4, 37.7, 47.2, 51.2, 52.6, 67.4, 80.7, 82.0, 82.4, 120.1, 125.2, 127.2, 127.9, 141.4, 144.0, 156.1, 170.4, 170.6, 171.5, 172.3; HRMS (ESI) m/z: [M + H]+ 653.3429, calcd. for C36H48N2O9 652.33598.
Characterization of di-tert-butyl ((S)-2-amino-4-(tert-butoxy)-4-oxobutanoyl)-l-glutamate (HT-B). Obtained from HT-A by method B as a white oily liquid; Yield: 87.9%; 1H-NMR (500 MHz, CDCl3): δ 1.43 (s, 9H, 3 × -CH3), 1.44 (s, 9H, 3 × -CH3), 1.46 (s, 9H, 3 × -CH3), 1.89 (m, 2H), 2.29 (m, 2H), 2.58 (dd, J = 16.7, 8.0 Hz, 1H), 2.79 (dd, J = 16.7, 3.7 Hz, 1H), 3.66 (dd, J = 8.0, 3.7 Hz, 1H), 4.45 (td, J = 8.4, 4.9 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H). 13C-NMR (126 MHz, CDCl3): δ 28.1, 28.2, 28.3, 29.8, 31.6, 40.6, 52.1, 52.2, 80.7, 81.4, 82.3, 171.2, 171.3, 172.3, 173.5; HRMS (ESI) m/z: [M + H]+ 431.2751, calcd. for C21H38N2O7 430.26790.
Characterization of di-tert-butyl ((S)-2-((S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamido)-4-(tert-butoxy)-4-oxobutanoyl)-l-glutamate (HT-C). Obtained from HT-B by method A as a white powder; m.p. 106.3–107.3 °C; Yield: 88.1%; 1H-NMR (500 MHz, CDCl3): δ 1.41 (s, 9H, 3 × -CH3), 1.42 (s, 9H, 3 × -CH3), 1.43 (s, 9H, 3 × -CH3), 1.47 (s, 9H, 3 × -CH3), 1.91 (m, 3H), 2.12 (m, 1H), 2.28 (m, 5H), 2.60 (dd, J = 17.1, 6.3 Hz, 1H), 2.91 (dd, J = 17.2, 3.8 Hz, 1H), 4.22 (dd, 1H), 4.40 (m, 3H), 4.78 (d, J = 4.3 Hz, 1H), 5.63 (d, J = 7.7 Hz, 1H), 6.94 (d, J = 7.4 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.3 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.61 (d, J = 7.3 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H). 13C-NMR (126 MHz, CDCl3): δ 27.7, 28.1, 28.2, 28.8, 31.5, 32.4, 37.2, 47.3, 49.4, 52.5, 53.9, 67.1, 80.7, 81.9, 82.3, 82.6, 120.1, 125.3, 127.2, 127.8, 141.4, 143.9, 144.0, 156.3, 170.5, 171.2, 171.6, 171.9, 172.2. HRMS (ESI) m/z: [M + H]+ 838.4416, calcd. for C45H63N3O12 837.44117.
Characterization of di-tert-butyl ((S)-2-((S)-4-amino-5-(tert-butoxy)-5-oxopentanamido)-4-(tert-butoxy)-4-oxobutanoyl)-l-glutamate (HT-D). Obtained from HT-C by method B as a white oily liquid; Yield: 90.4%; 1H-NMR (500 MHz, CDCl3): δ 1.43 (d, 36H, 12 × -CH3), 1.82 (m, 2H), 1.91 (m, 2H), 2.08 (m, 2H), 2.28 (m, 2H), 2.42 (m, 1H), 2.51 (m, 1H), 2.98 (m, 1H), 3.30 (m, 1H), 4.44 (d, J = 4.5 Hz, 1H), 4.80 (d, J = 3.7 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H). 13C-NMR (126 MHz, CDCl3): δ 28.0, 28.1, 28.2, 29.7, 29.8, 31.5, 33.9, 37.1, 49.1, 52.4, 55.4, 80.6, 81.4, 81.8, 82.2, 170.7, 170.8, 171.8, 172.3, 172.7, 174.9. HRMS (ESI) m/z: [M + H]+ 616.3830, calcd. for C30H53N3O10 615.37309.
Characterization of tetra-tert-butyl (5S,10S,15S,18S)-15-(2-(tert-butoxy)-2-oxoethyl)-1-(9H-fluoren-9-yl)-3,8,13,16-tetraoxo-2-oxa-4,9,14,17-tetraazaicosane-5,10,18,20-tetracarboxylate (HT-E). Obtained from HT-D by method B as a white powder; m.p. 91.6–92.2 °C; Yield: 86.7%; 1H-NMR (500 MHz, CDCl3): δ 1.40 (s, 9H, 3 × -CH3), 1.41 (s, 9H, 3 × -CH3), 1.43 (s, 9H, 3 × -CH3), 1.45 (s, 9H, 3 × -CH3), 1.46 (s, 9H, 3 × -CH3), 1.89 (m, 4H), 2.25 (m, 10H), 2.62 (dd, J = 17.2, 6.8 Hz, 1H), 2.84 (dd, J = 17.2, 4.7 Hz, 1H), 4.23 (m, 1H), 4.40 (m, 2H), 4.48 (m, 1H), 4.76 (m, 1H), 5.62 (d, J = 8.2 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.30 (m, 2H), 7.38 (m, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 7.5 Hz, 2H). 13C-NMR (126 MHz, CDCl3): δ 27.7, 28.0, 28.1, 28.2, 28.6, 28.8, 31.5, 32.1, 32.3, 37.2, 42.1, 47.3, 49.5, 52.3, 52.5, 53.9, 67.1, 80.7, 81.8, 82.2, 82.5, 82.6, 120.1, 125.3, 127.2, 127.8, 141.4, 141.5, 143.8, 144.1, 156.5, 170.5, 170.6, 171.2, 171.3, 171.3, 172.0, 172.2, 172.2. HRMS (ESI) m/z: [M + H]+ 1023.5528, calcd. for C54H78N4O15 1022.54637.
Characterization of di-tert-butyl ((S)-2-((S)-4-((S)-4-amino-5-(tert-butoxy)-5-oxopentanamido)-5-(tert-butoxy)-5-oxopentanamido)-4-(tert-butoxy)-4-oxobutanoyl)-l-glutamate (HT-F). Obtained from HT-E by method B as a white oily liquid; Yield: 87.3%; 1H-NMR (500 MHz, CDCl3): δ 1.41 (s, 9H, 3 × -CH3), 1.42 (s, 9H, 3 × -CH3), 1.43 (s, 9H, 3 × -CH3), 1.44 (s, 9H, 3 × -CH3), 1.45 (s, 9H, 3 × -CH3), 1.89 (m, 4H), 2.09 (m, 2H), 2.22 (m, 2H), 2.31 (t, J = 7.3 Hz, 2H), 2.38 (t, J = 7.0 Hz, 2H), 2.61 (dd, J = 17.2, 6.6 Hz, 1H), 2.86 (m, 1H), 3.36 (m, J = 8.4, 4.8 Hz, 1H), 4.42 (m, 2H), 4.76 (m, 1H), 6.89 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H). 13C-NMR (126 MHz, CDCl3): δ 27.7, 28.1, 28.2, 28.5, 30.2, 31.5, 32.5, 32.9, 37.2, 49.5, 52.3, 52.5, 54.5, 80.7, 81.5, 81.8, 82.3, 82.4, 170.5, 170.6, 171.2, 171.4, 172.2, 172.3, 172.8, 174.8. HRMS (ESI) m/z: [M + H]+ 801.4883, calcd. for C39H68N4O13 800.47829.
Characterization of 6-benzyl 11,16,24,26-tetra-tert-butyl (6S,11S,16S,21S,24S)-21-(2-(tert-butoxy)-2-oxoethyl)-2,2-dimethyl-4,9,14,19,22-pentaoxo-3-oxa-5,10,15,20,23-pentaazahexacosane-6,11,16,24,26-pentacarboxylate (HT-I). Obtained from HT-F by method A as a white powder; m.p. 107.0–107.7 °C; Yield: 83.6%; 1H-NMR (500 MHz, CDCl3): δ 1.39 (s, 27H, 9 × -CH3), 1.41 (s, 18H, 6 × -CH3), 1.42 (s, 9H, 3 × -CH3), 1.74 (m, 2H), 2.13 (s, 6H), 2.14 (s, 2H), 2.22 (m, 2H), 2.27 (m, 4H), 2.62 (dd, J = 17.1, 6.9 Hz, 1H), 2.78 (m, J = 17.0, 4.8 Hz, 1H), 4.41 (m, 3H), 4.75 (dd, J = 12.8, 6.8 Hz, 1H), 5.14 (m, 2H), 5.57 (d, J = 8.3 Hz, 1H), 6.52 (m, 1H), 6.86 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.32 (m, 5H). 13C-NMR (126 MHz, CDCl3): δ 27.7, 28.0, 28.1, 28.4, 31.0, 31.5, 31.6, 32.0, 32.2, 37.3, 49.5, 52.0, 52.2, 52.4, 53.1, 67.2, 80.1, 80.6, 81.6, 82.1, 82.4, 128.4, 128.5, 128.7, 135.5, 156.0, 170.6, 170.6, 171.0, 171.1, 171.3, 171.9, 172.1, 172.2, 172.3. HRMS (ESI) m/z: [M + H]+ 1120.6292, calcd. for C56H89N5O18 1119.62026.
Characterization of (7S,10S,15S,20S,25S)-10-(2-(tert-butoxy)-2-oxoethyl)-7,15,20-tris(tert-butoxycarbonyl)-25-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,9,12,17,22-pentaoxo-3-oxa-8,11,16,21-tetraazahexacosan-26-oic acid (HT-J). Obtained from HT-I by method C as a white powder; m.p. 94.3–95.0 °C; Yield: 92.2%; 1H-NMR (500 MHz, CDCl3): δ 1.41 (s, 9H, 3 × -CH3), 1.42 (s, 27H, 9 × -CH3), 1.44 (s, 9H, 3 × -CH3), 1.45 (s, 9H, 3 × -CH3), 1.87 (m, 3H), 2.08 (m, 1H), 2.24 (m, 4H), 2.33 (m, 4H), 2.40 (m, 4H), 2.73 (m, 2H), 4.29 (m, 1H), 4.43 (m, 2H), 4.54 (t, J = 7.3 Hz, 1H), 4.79 (dd, J = 12.7, 7.2 Hz, 1H), 5.56 (d, J = 7.3 Hz, 1H), 6.57 (s, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H). 13C-NMR (126 MHz, CDCl3): δ 27.7, 28.1, 28.2, 28.3, 28.5, 31.2, 31.5, 32.0, 32.4, 37.3, 49.5, 51.7, 52.5, 52.6, 52.8, 80.3, 81.2, 81.8, 82.1, 82.3, 82.6, 156.2, 170.7, 170.8, 171.2, 171.4, 172.2, 172.3, 172.8, 173.0, 174.0. HRMS (ESI) m/z: [M + H]+ 1130.5793, calcd. for C49H83N5O18 1029.57331.

4.1.4. General Synthetic Procedure of the Preparation of PSMA-Activated Prodrug CPT-HT-J-ZLn

General Synthetic Procedure of the Intermediate CPT-A-Ln (Method D)

The compound camptothecin (1 equiv.) was dissolved in dry DCM (25 mL), DMAP (0.1 equiv.) and the corresponding N-boc-amino aliphatic acid (1.2 equiv.) were added. After addition of EDCI (1.5 equiv.), the mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with 50 mL CH2Cl2, then successively washed with water and brine (10 mL each), dried over sodium sulfate and filtered, and the solvent was evaporated. Then the crude product was purified by flash chromatography (silica gel, dichloromethane:methanol = 100:1 to 40:1).

General Synthetic Procedure of the Intermediate CPT-B-Ln (Method E)

To a solution of the Boc-protected compound CPT-B-Ln in dry DCM (30 mL), TFA (3 mL/10 mL DCM) was added. The mixture was stirred in an ice bath for 2 h. After completion of the reaction (as monitored by TLC), the solution was evaporated and the residue was dissolved with DCM (50 mL). The organic extracts were washed with distilled water (3 × 25 mL) and brine (20 mL), dried over sodium sulfate, filtrated and evaporated. Purification was performed by flash chromatography (silica gel, dichloromethane:methanol = 20:1 to 5:1).

General Synthetic Procedure of the Intermediate CPT-HT-J-Ln (Method F)

The corresponding intermediate CPT-B-Ln (1 equiv.) was dissolved in dry DCM (20 mL), EDCI (1.5 equiv.) and HT-J (1.1 equiv.) were added. After addition of HOBT (1.5 equiv.) and DIPEA (2.5 equiv.), the mixture was stirred at 25 °C for 4 h. The reaction mixture was diluted with 50 mL CH2Cl2, then successively washed with water and brine (10 mL each), dried over sodium sulfate and filtered, and the solvent was evaporated. Then the crude product was purified by flash chromatography (silica gel, dichloromethane:methanol = 1:0 to 50:1).

General Synthetic Procedure of the Prodrug CPT-HT-J-ZLn (Method G)

To a solution of the intermediate CPT-HT-J-Ln in 20 mL mixed solution (THF:Thioanisole:H2O = 95:2.5:2.5), the mixture was stirred at room temperature for 2.5 h. After completion of the reaction (as monitored by RP-HPLC), the filtrate was concentrated in vacuum and the residue was purified by PR-HPLC (A = H2O (0.2% TFA), and B = CH3CN (0.2% TFA); solvent gradient: 35–80% B in 25 min).
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(tert-butoxycarbonyl)glycinate (CPT-A-L2). Obtained from CPT by method D as a faint yellow powder; mp > 220 °C; Yield: 65.0%; 1H-NMR (500 MHz, CDCl3): δ 0.98 (t, J = 7.4 Hz, 3H, -CH3), 1.42 (s, 9H, 3 × -CH3), 2.16 (m, 2H), 4.06 (dd, J = 18.0, 4.8 Hz, 1H), 4.21 (dd, J = 18.4, 6.1 Hz, 1H), 5.07 (s, 1H), 5.25 (m, 2H), 5.38 (d, J = 17.1 Hz, 1H), 5.67 (d, J = 17.1 Hz, 1H), 7.30 (s, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.37 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 28.4, 31.8, 42.5, 50.1, 67.2, 80.3, 96.5, 120.2, 128.2, 128.3, 128.5, 129.8, 130.8, 131.3, 145.7, 146.5, 148.9, 152.3, 155.7, 157.4, 167.3, 169.7. HRMS (ESI) m/z: [M + H]+ 506.1917, calcd. for C27H27N3O7 505.18490.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl glycinate (CPT-B-L2). Obtained from CPT-A-L2 by method E as a faint yellow powder; m.p. 194.8–195.2 °C; Yield: 61.2%; 1H-NMR (500 MHz, CD3OD): δ 1.08 (t, J = 7.5 Hz, 3H, -CH3), 2.27 (m, 2H), 4.18 (m, 1H), 4.29 (m, 1H), 5.35 (m, 2H), 5.50 (d, J = 16.7 Hz, 1H), 5.64 (d, J = 16.7 Hz, 1H), 7.44 (s, 1H), 7.1 (t, J = 7.3 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.64 (s, 1H). 13C-NMR (126 MHz, CD3OD): δ 8.0, 31.9, 41.0, 51.6, 67.7, 79.6, 97.7, 120.6, 129.3, 129.8, 130.0, 130.9, 132.1, 133.1, 133.5, 147.4, 147.9, 149.6, 153.4, 159.0, 168.1, 168.6. HRMS (ESI) m/z: [M + H]+ 406.1397, calcd. for C22H19N3O5 405.13247.
Characterization of 14,22,24,9-tetra-tert-butyl 1-((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl) (4S,9S,14S,19S,22S)-19-(2-(tert-butoxy)-2-oxoethyl)-4-((tert-butoxycarbonyl)amino)-3,7,12,17,20-pentaoxo-2,8,13,18,21-pentaazatetracosane-1,9,14,22,24-pentacarboxylate (CPT-HT-J-L2). Obtained from CPT-B-L2 by method F as a faint yellow powder; m.p. 167.4–168.1 °C; Yield: 30.0%; 1H-NMR (500 MHz, CDCl3): δ 0.97 (t, J = 7.5 Hz, 3H, -CH3), 1.26 (s, 2H), 1.35 (s, 9H, 3 × -CH3), 1.37 (s, 9H, 3× -CH3), 1.42 (s, 36H, 12 × -CH3), 1.90 (m, 2H), 2.19 (m, 14H), 2.64 (m, 1H), 2.83 (m, 1H), 4.16 (m, 2H), 4.42 (m, 4H), 4.18 (s, 1H), 5.29 (s, 2H), 5.38 (d, J = 17.4 Hz, 1H), 5.67 (d, J = 17.4 Hz, 1H), 6.67 (m, 1H), 7.11 (m, 1H), 7.33 (m, 4H), 7.67 (t, J = 6.9 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.40 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 27.7, 28.0, 28.1, 28.2, 28.4, 29.4, 31.6, 31.9, 32.5, 37.4, 41.4, 49.4, 50.2, 51.8, 52.3, 52.5, 53.9, 67.2, 79.9, 80.7, 81.6, 82.0, 82.3, 82.5, 82.6, 96.5, 120.3, 128.2, 128.3, 128.6, 129.7, 130.8, 131.4, 145.6, 146.4, 148.8, 152.3, 156.0, 157.4, 167.2, 169.5, 170.9, 171.0, 171.7, 172.3, 172.6, 172.7, 172.9. HRMS (ESI) m/z: [M + H]+ 1417.7008, calcd. for C71H100N8O22 1416.69522.
Characterization of ((S)-4-((S)-4-((S)-4-amino-5-((2-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-5-oxopentanamido)-4-carboxybutanamido)-4-carboxybutanoyl)-l-aspartyl-l-glutamic acid (CPT-HT-J-ZL2). Obtained from CPT-HT-J-L2 by method G as a faint yellow powder; m.p. 179.0–179.5 °C; Yield: 19.9%; 1H-NMR (500 MHz, DMSO): δ 0.93 (t, J = 7.4 Hz, 3H, -CH3), 1.52 (d, J = 6.6 Hz, 2H), 1.76 (s, 3H), 1.96 (s, 6H), 2.16 (s, 6H), 2.26 (m, 5H), 4.15 (m, 4H), 4.58 (m, 2H), 5.27 (s, 1H), 5.35 (s, 1H), 7.18 (s, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 8.06 (d, J = 7.1 Hz, 1H), 8.16 (m, 6H), 8.27 (d, J = 6.6 Hz, 1H), 8.71 (s, 1H), 8.93 (s, 1H). 13C-NMR (101 MHz, DMSO): δ 7.6, 26.2, 27.1, 29.9, 30.4, 31.5, 31.7, 36.2, 36.5, 49.3, 51.3, 51.5, 51.7, 66.3, 76.6, 95.1, 119.0, 127.8, 128.0, 128.7, 128.9, 129.8, 130.5, 131.7, 145.0, 146.1, 147.9, 152.4, 156.5, 167.0, 168.6, 169.0, 171.0, 171.2, 171.4, 171.7, 173.1, 173.3, 173.4, 173.8; HRMS (ESI) m/z: [M + H]+ 1037.3386, calcd. for C46H52N8O20 1036.330.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 4-((tert-butoxycarbonyl)amino)butanoate (CPT-A-L4). Obtained from CPT by method D as a faint yellow powder; m.p. 167.8–170.2 °C; Yield: 46.0%; 1H-NMR (500 MHz, CDCl3): δ 0.98 (t, J = 7.5 Hz, 3H, -CH3), 1.42 (s, 9H, 3 × -CH3), 1.86 (m, 2H), 2.14 (m, 2H), 2.54 (m, 2H), 3.14 (m, 2H), 5.28 (m, 1H), 5.38 (m, 1H), 5.66 (m, 1H), 7.23 (m, 1H), 7.66 (m, 1H), 7.82 (m, 1H), 7.93 (m, 1H), 8.25 (m, 1H), 8.40 (m, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 25.1, 28.5, 31.1, 31.8, 39.6, 50.1, 67.2, 76.0, 79.3, 96.1, 120.3, 128.2, 128.3, 128.6, 129.6, 130.9, 131.5, 146.1, 146.3, 148.8, 152.3, 156.2, 157.5, 167.7, 172.3; HRMS (ESI) m/z: [M + H]+ 534.2231, calcd. for C29H31N3O7 533.21620.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 4-aminobutanoate (CPT-B-L4). Obtained from CPT-A-L4 by method E as a faint yellow powder; mp > 220 °C; Yield: 65.3%; 1H-NMR (500 MHz, DMSO): δ 0.93 (t, J = 7.4 Hz, 3H, -CH3), 1.84 (m, 2H), 2.16 (m, 2H), 2.70 (m, 2H), 2.83 (m, 2H), 5.28 (s, 2H), 5.53 (m, 2H), 7.06 (s, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.86 (d, J = 6.7 Hz, 1H), 8.13 (d, J = 7.5 Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.69 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.7, 22.5, 30.33, 38.0, 50.3, 66.4, 76.1, 94.8, 118.9, 127.8, 128.1, 128.7, 129.0, 129.9, 130.5, 131.7, 145.4, 146.2, 148.0, 152.4, 156.6, 167.4, 171.6; HRMS (ESI) m/z: [M + H]+ 434.1715, calcd. for C24H23N3O5 433.16377.
Characterization of 1,11,16,3-tetra-tert-butyl 26-((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl) (3S,6S,11S,16S,21S)-6-(2-(tert-butoxy)-2-oxoethyl)-21-((tert-butoxycarbonyl)amino)-5,8,13,18,22-pentaoxo-4,7,12,17,23-pentaazahexacosane-1,3,11,16,26-pentacarboxylate (CPT-HT-J-L4). Obtained from CPT-B-L4 by method F as a faint yellow powder; m.p. 129.0–129.6 °C; Yield: 49.3%; 1H-NMR (500 MHz, CDCl3): δ 0.95 (t, J = 7.4 Hz, 3H, -CH3), 1.39 (s, 9H, 3 × -CH3), 1.40 (s, 18H, 6 × -CH3), 1.41 (s, 18H, 6 × -CH3), 1.44 (s, 9H, 3 × -CH3), 1.72 (m, 2H), 1.84 (m, 4H), 2.15 (m, 2H), 2.26 (m, 4H), 2.37 (m, 8H), 2.56 (m, 2H), 2.83 (m, 1H), 3.30 (m, 2H), 3.64 (m, 1H), 4.11 (m, 1H), 4.43 (m, 4H), 4.79 (m, 1H), 5.29 (s, 2H), 5.39 (d, J = 17.2 Hz, 1H), 5.65 (d, J = 17.2 Hz, 1H), 5.79 (m, 1H), 6.38 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.33 (m, 2H), 7.53 (s, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.83 (t, J = 7.4 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.40 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.5, 24.8, 27.6, 28.1, 28.2, 28.4, 28.5, 29.0, 31.3, 31.4, 31.6, 31.9, 32.5, 37.4, 38.7, 49.4, 50.1, 51.7, 52.3, 52.5, 53.2, 53.4, 67.2, 76.0, 79.9, 80.6, 81.6, 81.9, 82.2, 82.4, 96. 4, 120.5, 128.2, 128.3, 128.7, 129.7, 130.9, 131.5, 145.9, 146.2, 148.8, 152.4, 156.2, 157.5, 167.7, 170.9, 171.0, 171.1, 171.5, 172.0, 172.1, 172.2, 172.5, 172.6, 172.7; HRMS (ESI) m/z: [M + H]+ 1445.7339, calcd. for C73H104N8O22 1444.72652.
Characterization of (3S,6S,11S,16S,21S)-21-amino-6-(carboxymethyl)-27-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-5,8,13,18,22,27-hexaoxo-4,7,12,17,23-pentaazaheptacosane-1,3,11,16-tetracarboxylic acid (CPT-HT-J-ZL4). Obtained from CPT-HT-J-L4 by method G as a faint yellow powder; m.p. 189.0–189.5 °C; Yield: 19.9%; 1H-NMR (500 MHz, DMSO): δ 0.92 (t, J = 7.4 Hz, 3H, -CH3), 1.73 (m, 4H), 1.92 (s, 6H), 2.16 (s, 6H), 2.22 (m, 12H), 2.63 (m, 2H), 4.11 (m, 3H), 4.55 (s, 1H), 5.31 (s, 2H), 5.48 (d, J = 16.9 Hz, 1H), 5.52 (d, J = 17.1 Hz, 1H), 7.07 (s, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 7.93 (s, 1H), 8.09 (s, 1H), 8.15 (m, 4H), 8.24 (s, 2H), 8.48 (s, 1H), 8.70 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.6, 24.3, 27.1, 27.2, 27.6, 29.6, 30.3, 30.6, 30.8, 31.5, 31.7, 36.3, 38.1, 50.3, 51.3, 51.4, 51.7, 51.9, 52.1, 66.4, 75.9, 94.7, 118.9, 127.8, 128.0, 128.6, 129.0, 129.9, 130.5, 131.7, 145.4, 146.1, 147.9, 152.4, 156.6, 167.4, 168.5, 170.7, 171.1, 171.5, 171.8, 172.4, 172.9, 173.6. HRMS (ESI) m/z: [M + H]+ 1065.3690, calcd. for C48H56N8O20 1064.36109.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 6-((tert-butoxycarbonyl)amino)hexanoate (CPT-A-L6). Obtained from CPT by method D as a faint yellow powder; m.p. 152.0–152.5 °C; Yield: 55.0%; 1H-NMR (500 MHz, CDCl3): δ 0.97 (t, J = 7.5 Hz, 3H, -CH3), 1.39 (s, 9H, 3 × -CH3), 1.50 (m, 2H), 1.66 (m, 4H), 2.14 (m, 2H), 2.49 (m, 2H), 3.07 (m, 2H), 5.29 (s, 2H), 5.39 (d, J = 17.0 Hz, 1H), 5.67 (d, J = 17.0 Hz, 1H), 7.23 (s, 1H), 7.67 (m, 1H), 7.83 (m, 1H), 7.94 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.40 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.4, 26.2, 28.5, 29.9, 32.0, 33.8, 40.4, 50.1, 67.2, 75.9, 96.3, 120.5, 128.2, 128.3, 128.4, 128.6, 129.6, 130.9, 131.5, 146.2, 146.2, 148.9, 152.4, 157.5, 167.7, 172.7; HRMS (ESI) m/z: [M + H]+ 562.2548, calcd. for C31H35N3O7 561.24750.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 6-aminohexanoate (CPT-B-L6). Obtained from CPT-A-L6 by method E as a faint yellow powder; m.p. 207.5–208.0 °C; Yield: 65.0%; 1H-NMR (500 MHz, CD3OD): δ 1.05 (t, J = 7.4 Hz, 3H, -CH3), 1.48 (m, 2H), 1.70 (m, 4H), 2.23 (m, 2H), 2.62 (m, 2H), 2.91 (m, 2H), 5.27 (s, 2H), 5.47 (d, J = 16.7 Hz, 1H), 5.61 (d, J = 16.7 Hz, 1H), 7.35 (s, 1H), 7.70 (m, 1H), 7.87 (t, J = 7.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.60 (s, 1H). 13C-NMR (126 MHz, CD3OD): δ 8.1, 25.1, 26.6, 28.2, 32.1, 34.2, 40.5, 51.5, 67.7, 77.5, 97.8, 120.6, 129.2, 129.7, 129.7, 130.0, 130.9, 132.0, 133.4, 147.5, 148.4, 149.5, 153.4, 159.0, 169.6, 173.9. HRMS (ESI) m/z: [M + H]+ 462.2030, calcd. for C26H27N3O5 461.19507.
Characterization of 1,11,16,3-tetra-tert-butyl 28-((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl) (3S,6S,11S,16S,21S)-6-(2-(tert-butoxy)-2-oxoethyl)-21-((tert-butoxycarbonyl)amino)-5,8,13,18,22-pentaoxo-4,7,12,17,23-pentaazaoctacosane-1,3,11,16,28-pentacarboxylate (CPT-HT-J-L6). Obtained from CPT-B-L6 by method F as a faint yellow powder; m.p. 138.5–139.0 °C; Yield: 45.2%; 1H-NMR (500 MHz, CDCl3): δ 0.96 (t, J = 7.5 Hz, 3H, -CH3), 1.41 (s, 27H, 9 × -CH3), 1.42 (s, 9H, 3 × -CH3), 1.43 (s, 9H, 3 × -CH3), 1.45 (s, 9H, 3 × -CH3), 1.52 (m, 2H), 1.76 (m, 6H), 2.12 (m, 4H), 2.32 (m, 12H), 2.48 (m, 2H), 2.61 (m, 1H), 2.86 (m, 1H), 3.21 (m, 2H), 4.11 (s, 1H), 4.45 (m, 3H), 4.80 (m, 1H), 5.30 (s, 2H), 5.40 (d, J = 17.2 Hz, 1H), 5.66 (d, J = 17.2 Hz, 1H), 5.84 (d, J = 7.0 Hz, 1H), 6.34 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 7.27 (s, 1H), 7.34 (t, J = 8.3 Hz, 1 H), 7.67 (t, J = 7.4 Hz, 1H), 7.84 (t, J = 7.2 Hz, H), 7.94 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.5 Hz, 1 H), 8.41 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.4, 26.3, 27.6, 27.9, 28.1, 28.2, 28.6, 29.0, 29.2, 31.6, 32.0, 32.1, 32.5, 33.7, 37.4, 39.4, 49.4, 50.1, 51.7, 52.3, 52.6, 53.3, 67.2, 75.8, 79.9, 80.6, 81.6, 81.9, 82.2, 82.5, 96.5, 120.6, 128.3, 128.4, 128.7, 129.5, 131.0, 131.7, 146.1, 148.6, 152.3, 156.2, 157.5, 167.7, 170.9, 171.1, 171.5, 171.7, 172.2, 172.5, 172.7, 172.8. HRMS (ESI) m/z: [M + H]+ 1473.7646, calcd. for C75H108N8O22 1472.75782.
Characterization of (3S,6S,11S,16S,21S)-21-amino-6-(carboxymethyl)-29-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-5,8,13,18,22,29-hexaoxo-4,7,12,17,23-pentaazanonacosane-1,3,11,16-tetracarboxylic acid (CPT-HT-J-ZL6). Obtained from CPT-HT-J-L6 by method G as a faint yellow powder; m.p. 190.0–190.4 °C; Yield: 25.0%; 1H-NMR (500 MHz, DMSO): δ 0.93 (t, J = 7.5 Hz, 3H, -CH3), 1.36 (m, 4H), 1.48 (m, 2H), 1.60 (m, 2H), 1.79 (m, 3H), 1.94 (m, 5H), 2.21 (m, 9H), 2.36 (m, 1H), 2.66 (m, 1H), 3.11 (m, 2H), 3.62(m, 1H), 3.72(s, 1H), 4.20 (m, 3H), 4.60 (m, 1H), 5.31 (s, 2H), 5.49 (m, 2H), 7.06 (s, 1H), 7.72 (t, J = 7.1 Hz, 1H), 7.87 (t, J = 7.7 Hz, 1H), 8.07 (d, J = 6.8 Hz, 1H), 8.15 (m, 5H), 8.33 (m, 2H), 8.70 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.6, 24.0, 25.7, 27.0, 27.7, 28.5, 29.7, 30.0, 30.3, 30.4, 31.6, 31.7, 33.1, 36.2, 38.6, 49.3, 50.3, 51.3, 51.5, 51.7, 51.8, 54.9, 66.4, 75.7, 94.7, 118.9, 127.8, 128.1, 128.6, 129.0, 129.9, 130.5, 131.7, 145.5, 146.0, 147.9, 152.3, 156.6, 163.1, 167.4, 167.9, 170.9, 171.0, 171.2, 171.5, 171.7, 172.1, 172.8, 172.9, 173.1, 173.4, 173.8. HRMS (ESI) m/z: [M + H]+ 1093.4007, calcd. for C50H60N8O20 1092.39239.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 12-((tert-butoxycarbonyl)amino)dodecanoate (CPT-A-L12). Obtained from CPT by method D as a faint yellow powder; m.p. 134.6–140.0 °C; Yield: 67.0%; 1H-NMR (500 MHz, CDCl3): δ 0.96 (t, J = 7.4 Hz, 3H, -CH3), 1.15 (m, 8H), 1.26 (m, 6H), 1.42 (s, 9H, 3 × -CH3), 1.62 (m, 2H), 2.16 (m, 1H), 2.27 (m, 1H), 2.47 (m, 2H), 3.06 (s, 2H), 5.26 (m, 2H), 5.40 (d, J = 17.1 Hz, 1H), 5.66 (d, J = 17.1 Hz, 1H), 7.22 (s, 1H), 7.66 (t, J = 7.3 Hz, 1H), 7.82 (t, J = 7.3 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.39 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.7, 26.9, 28.5, 29.1, 29.3, 29.5, 29.5, 30.1, 32.0, 33.9, 40.7, 50.0, 67.2, 75.7, 79.1, 96.2, 120.5, 128.2, 128.3, 128.3, 128.6, 129.6, 130.8, 131.4, 146.1, 146.2, 148.9, 152.4, 156.1, 157.5, 167.7, 172.9. HRMS (ESI) m/z: [M + H]+ 646.3484, calcd. for C37H47N3O7 645.34140.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 12-aminododecanoate (CPT-B-L12). Obtained from CPT-A-L12 by method E as a faint yellow powder; m.p. 215.5–216.0 °C; Yield: 72.0%; 1H-NMR (500 MHz, CD3OD): δ 1.04 (t, J = 7.4 Hz, 3H, -CH3), 1.23 (m, 8H), 1.33 (m, 6H), 1.63 (m, 4H), 2.23 (m, 2H), 2.54 (m, 2H), 2.89 (m, 2H), 5.27 (s, 2H), 5.47 (d, J = 16.8 Hz, 1H), 5.60 (d, J = 16.8 Hz, 1H), 7.35 (s, 1H), 7.69 (dd, J = 8.0, 7.1 Hz, 1H), 7.86 (t, J = 7.7 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.58 (s, 1H). 13C-NMR (126 MHz, CD3OD): δ 8.0, 25.8, 27.4, 28.6, 30.0, 30.1, 30.3, 30.4, 30.5, 32.2, 34.6, 40.8, 51.5, 67.7, 77.3, 97.9, 120.7, 129.2, 129.7, 129.8, 130.0, 130.8, 131.9, 133.3, 147.5, 148.4, 149.6, 153.4, 159.0, 169.6, 174.1. HRMS (ESI) m/z: [M + H]+ 546.2972, calcd. for C32H39N3O5 545.28897.
Characterization of 1,11,16,3-tetra-tert-butyl 34-((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl) (3S,6S,11S,16S,21S)-6-(2-(tert-butoxy)-2-oxoethyl)-21-((tert-butoxycarbonyl)amino)-5,8,13,18,22-pentaoxo-4,7,12,17,23-pentaazatetratriacontane-1,3,11,16,34-pentacarboxylate (CPT-HT-J-L12). Obtained from CPT-B-L12 by method F as a faint yellow powder; m.p. 121.5–121.9 °C; Yield: 42.0%; 1H-NMR (500 MHz, CDCl3): δ 0.96 (t, J = 7.4 Hz, 3H, -CH3), 1.14 (m, 10H), 1.24 (s, 4H), 1.30 (m, 4H), 1.41 (s, 27H, 9 × -CH3), 1.42 (s, 15H, 5 × -CH3), 1.44 (s, 12H, 4 × -CH3), 1.62 (m, 4H), 1.86 (m, 2H), 2.07 (m, 4H), 2.26 (m, 6H), 2.37 (m, 4H), 2.48 (m, 4H), 2.60 (m, 1H), 2.87 (m, 1H), 3.10 (m, 1H), 3.22 (m, 1H), 4.13 (s, 1H), 4.42 (m, 2H), 4.52 (m, 1H), 4.80 (s, 1H), 5.28 (m, 2H), 5.40 (d, J = 17.0 Hz, 1H), 5.66 (d, J = 17.0 Hz, 1H), 5.87 (d, J = 7.1 Hz, 1H), 6.31 (d, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.37 (t, J = 8.6 Hz, 1H), 7.44 (s, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.40 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.8, 27.0, 27.6, 27.8, 28.1, 28.2, 28.5, 29.0, 29.2, 29.4, 29.5, 29.6, 29.6, 31.5, 32.0, 32.1, 32.5, 33.9, 37.4, 39.8, 49.3, 50.1, 51.7, 52.2, 52.5, 53.3, 67.2, 75.7, 80.0, 80.6, 81.6, 81.9, 82.2, 82.5, 96.2, 120.5, 128.2, 128.3, 128.4, 128.6, 129.7, 130.8, 131.4, 146.1, 146.3, 149.0, 152.5, 156.3, 157.5, 167.7, 170.9, 171.1, 171.5, 171.7, 172.2, 172.5, 172.9, 172.9. HRMS (ESI) m/z: [M + H]+ 1557.8584, calcd. for C81H120N8O22 1556.85172.
Characterization of (3S,6S,11S,16S,21S)-21-amino-6-(carboxymethyl)-35-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-5,8,13,18,22,35-hexaoxo-4,7,12,17,23-pentaazapentatriacontane-1,3,11,16-tetracarboxylic acid (CPT-HT-J-ZL12). Obtained from CPT-HT-J-L12 by method G as a faint yellow powder; m.p. 183.9–184.3 °C; Yield: 24.8%; 1H-NMR (500 MHz, DMSO): δ 0.93 (t, J = 7.4 Hz, 3H, -CH3), 1.12 (m, 10H), 1.26 (m, 5H), 1.37 (m, 4H), 1.45 (m, 1H), 1.54 (m, 3H), 1.80 (m, 2H), 1.93 (m, 4H), 2.22 (m, 7H), 2.35 (m, 2H), 3.10 (m, 2H), 3.30 (m, 1H), 3.40 (m, 1H), 3.59 (s, 1H), 3.62 (s, 1H), 3.72 (s, 1H), 4.17 (m, 3H), 4.57 (m, 1H), 5.31 (s, 2H), 5.50 (m, 2H), 7.05 (s, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 8.15 (m, 6H), 8.32 (m, 2H), 8.71 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.6, 24.0, 24.5, 25.3, 26.0, 26.4, 26.7, 27.0, 27.6, 28.4, 28.7, 28.8, 28.9, 29.7, 30.3, 30.4, 33.2, 38.8, 42.0, 45.8, 50.3, 51.4, 51.6, 52.0, 66.3, 75.6, 94.7, 119.0, 127.8, 128.1, 128.6, 129.0, 129.8, 130.4, 131.6, 145.4, 146.0, 147.9, 152.3, 156.6, 163.1, 167.3, 167.9, 169.3, 171.5, 171.7, 172.1, 172.9, 172.8, 173.4, 173.6, 173.8. HRMS (ESI) m/z: [M + H]+ 1177.4963, calcd. for C81H120N8O22 1176.48629.

4.1.5. General Synthetic Procedure of the Preparation of PSMA Hydrolysate CPT-D-GLn

General Synthetic Procedure of the Intermediate CPT-C-GLn (Method H)

The corresponding intermediate CPT-B-Ln (1 equiv.) was dissolved in dry DCM (20 mL), EDCI (1.5 equiv.) and N-Cbz-l-glutamic acid 5-benzyl ester (1.1 equiv.) were added. After addition of HOBT (1.5 equiv.) and DIPEA (2.5 equiv.), the mixture was stirred at 25 °C for 4 h. The reaction mixture was diluted with 50 mL CH2Cl2, then successively washed with water and brine (10 mL each), dried over sodium sulfate and filtered, and the solvent was evaporated. Then the crude product was purified by flash chromatography (silica gel, dichloromethane:methanol = 1:0 to 50:1).

General Synthetic Procedure of PSMA Hydrolysate CPT-D-GLn (Method I)

The intermediate CPT-C-GLn was dissolved in 20 mL MeOH, Pd/C (10%, 80 mg) was added. The reaction mixture was stirred at room temperature for 12 h and filtered to remove Pd/C. The filtrate was concentrated in vacuum. Purification was performed by flash chromatography (silica gel, dichloromethane:methanol = 20:1 to 5:1).
Characterization of benzyl (S)-4-(((benzyloxy)carbonyl)amino)-5-((2-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-5-oxopentanoate (CPT-C-GL2). Obtained from CPT-B-L2 by method H as a faint yellow powder; m.p. 213.9–214.2 °C; Yield: 50.4%; 1H-NMR (500 MHz, CDCl3): δ 0.97 (t, J = 7.4 Hz, 3H, -CH3), 1.97 (dd, J = 14.3, 7.4 Hz, 1H), 2.14 (m, 2H), 2.26 (dd, J = 14.3, 7.4 Hz, 1H), 2.48 (m, 2H), 4.12 (m, 1H), 4.31 (m, 1H), 4.37 (dd, J = 18.2, 6.1 Hz, 1H), 4.93 (t, J = 14.5 Hz, 2H), 5.02 (t, J = 14.5 Hz, 2H), 5.13 (d, J = 18.9 Hz, 1H), 5.22 (d, J = 18.9 Hz, 1H), 5.37 (d, J = 17.1 Hz, 1H), 5.66 (d, J = 17.1 Hz, 1H), 6.99 (s, 1H), 7.18 (m, 2H), 7.24 (m, 2H), 7.28 (m, 6H), 7.63 (t, J = 7.5 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H). 13C-NMR (126 MHz, CDCl3): 7.7, 28.1, 30.5, 31.8, 41.4, 50.1, 54.1, 66.7, 67.1, 67.2, 96.4, 120.2, 128.1, 128.2, 128.3, 128.4, 128.6, 128.7, 129.7, 130.8, 131.4, 135.7, 136.1, 145.6, 146.4, 148.8, 152.3, 156.4, 157.4, 167.2, 168.9, 171.7, 173.2. HRMS (ESI) m/z: [M + H]+ 759.2653, calcd. for C42H38N4O10 758.25879.
Characterization of (S)-4-amino-5-((2-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-5-oxopentanoic acid (CPT-D-GL2). Obtained from CPT-C-GL2 by method I as a faint yellow powder; Yield: 39.5%; 1H-NMR (500 MHz, DMSO): δ 0.92 (s, 3H), 2.16 (d, J = 6.3 Hz, 2H), 2.42 (dd, J = 16.1, 7.8 Hz, 1H), 2.82 (d, J = 16.1 Hz, 1H), 3.47 (s, 1H), 4.08 (d, J = 17.6 Hz, 1H), 4.19 (d, J = 16.9 Hz, 1H), 5.27 (s, 2H), 5.50 (s, 2H), 7.19 (s, 1H), 7.71 (t, J = 6.5 Hz, 1H), 7.85 (d, J = 6.7 Hz, 1H), 8.11 (d, J = 7.5 Hz, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.67 (s, 1H), 9.02 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 8.0, 28.0, 30.8, 36.4, 40.9, 50.7, 51.2, 66.8, 76.7, 95.9, 119.4, 128.2, 128.4, 129.0, 129.5, 130.2, 130.9, 132.1, 145.6, 146.4, 148.4, 152.8, 157.0, 167.6, 169.4, 171.7. HRMS (ESI) m/z: [M + H]+ 535.1800, calcd. for C27H26N4O8 534.17506.
Characterization of benzyl (S)-4-(((benzyloxy)carbonyl)amino)-5-((4-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-4-oxobutyl)amino)-5-oxopentanoate (CPT-C-GL4). Obtained from CPT-B-L4 by method H as a faint yellow powder; m.p. 118.0–118.7 °C; Yield: 60.0%; 1H-NMR (500 MHz, CDCl3): δ 0.96 (t, J = 7.4 Hz, 3H, -CH3), 1.82 (m, 2H), 1.93 (m, 1H), 2.13 (m, 2H), 2.25 (m, 1H), 2.47 (m, 4H), 3.28 (m, 2H), 4.22 (d, J = 4.8 Hz, 1H), 5.04 (s, 2H), 5.09 (s, 2H), 5.22 (d, J = 18.9 Hz, 1H), 5.27 (d, J = 18.9 Hz, 1H), 5.38 (d, J = 17.1 Hz, 1H), 5.68 (d, J = 17.1 Hz, 1H), 5.76 (d, J = 7.5 Hz, 1H), 6.58 (s, 1H), 7.23 (s, 1H), 7.30 (m, 10H), 7.65 (d, J = 7.5 Hz, 1H), 7.81 (dd, J = 11.3, 4.1 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.36 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.2, 28.2, 30.6, 31.2, 31.9, 38.7, 50.1, 54.4, 66.6, 67.1, 67.3, 76.2, 96.2, 120.2, 128.1, 128.2, 128.3, 128.4, 128.6, 128.7, 129.6, 130.9, 131.5, 135.8, 136.3, 146.0, 146.3, 148.8, 152.4, 156.4, 157.4, 168.0, 171.5, 172.3, 173.2. HRMS (ESI) m/z: [2M + H]+ 1573.7668, calcd. for C44H42N4O10 786.29009.
Characterization of (S)-4-amino-5-((4-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-4-oxobutyl)amino)-5-oxopentanoic acid (CPT-D-GL4). Obtained from CPT-C-GL4 by method I as a faint yellow powder; m.p. 127.5–128.1 °C; Yield: 42.6%; 1H-NMR (500 MHz, CDCl3): δ 0.93 (t, J = 7.4 Hz, 3H, -CH3), 1.69 (m, 2H), 1.74 (m, 1H), 1.88 (m, 1H), 2.16 (m, 2H), 2.23 (t, J = 7.7 Hz, 2H), 2.24 (m, 2H), 3.12 (d, J = 7.1 Hz, 2H), 3.33 (s, 1H), 3.95 (dd, J = 13.9, 8.3 Hz, 1H), 5.02 (s, 1H), 5.03 (s, 1H), 5.31 (m, 2H), 5.48 (d, J = 16.8 Hz, 1H), 5.52 (d, J = 16.8 Hz, 1H), 7.09 (s, 1H), 7.31 (dd, J = 8.5, 4.2 Hz, 1H), 7.36 (s, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.87 (t, J = 7.1 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 8.16 (dd, J = 18.4, 8.2 Hz, 2H), 8.70 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.6, 24.4, 27.2, 30.2, 30.3, 30.7, 37.8, 50.3, 54.1, 65.4, 75.8, 94.8, 118.9, 127.7, 128.0, 128.3, 128.6, 129.0, 129.8, 130.4, 131.6, 145.4, 146.1, 147.9, 152.4, 156.6, 167.3, 171.4, 171.9, 173.9. HRMS (ESI) m/z: [M + H]+ 563.2149, calcd. for C29H30N4O8 562.20636.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 6-((S)-5-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-5-oxopentanamido)hexanoate (CPT-CPT-C-GL6). Obtained from CPT-B-L6 by method H as a faint yellow powder; m.p. 106.3–107.3 °C; Yield: 62.6%; 1H-NMR (400 MHz, CDCl3): δ 0.90 (t, J = 7.4 Hz, 3H, -CH3), 1.27 (m, 2H), 1.42 (m, 2H), 1.50 (m, 2H), 1.83 (m, 2H), 2.14 (m, 6H), 3.04 (m, 2H), 3.63 (s, 1H), 3.67 (s, 1H), 4.19 (m, 1H), 4.33 (m, 2H), 5.30 (d, J = 17.5 Hz, 1H), 5.49(d, J = 17.5 Hz, 1H), 7.32 (dd, J = 17.0, 9.6 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.71 (m, 1H), 7.88 (dd, J = 17.0, 9.6 Hz, 1H), 8.15 (m, 1H), 8.69 (s, 1H). 13C-NMR (101 MHz, CDCl3): 7.7, 24.3, 25.9, 27.8, 28.9, 30.6, 31.9, 33.7, 39.2, 50.1, 52.7, 54.3, 66.6, 67.1, 67.3, 75.9, 96.3, 120.4, 128.1, 128.3, 128.4, 128.7, 129.4, 131.0, 131.6, 135.9, 136.4, 146.1, 146.2, 148.7, 152.4, 157.4, 168.0, 171.1, 172.7, 173.2. HRMS (ESI) m/z: [M + H]+ 815.3279, calcd. for C46H46N4O10 814.32139.
Characterization of (S)-4-amino-5-((6-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-6-oxohexyl)amino)-5-oxopentanoic acid (CPT-D-GL6). Obtained from CPT-C-GL6 by method I as a faint yellow powder; m.p. 114.3–114.9 °C; Yield: 35.9%; 1H-NMR (500 MHz, CDCl3): δ 0.93 (t, J = 7.4 Hz, 3H, -CH3), 1.69 (m, 2H), 1.74 (m, 1H), 1.88 (m, 1H), 2.16 (m, 2H), 2.23 (t, J = 7.7 Hz, 2H), 2.24 (m, 2H), 3.12 (d, J = 7.1 Hz, 2H), 3.33 (s, 1H), 3.95 (dd, J = 13.9, 8.3 Hz, 1H), 5.02 (s, 1H), 5.03 (s, 1H), 5.31 (m, 2H), 5.48 (d, J = 16.8 Hz, 1H), 5.52 (d, J = 16.8 Hz, 1H), 7.09 (s, 1H), 7.31 (dd, J = 8.5, 4.2 Hz, 1H), 7.36 (s, 1H), 7.73 (t, J = 7.1 Hz, 1H), 7.87 (t, J = 7.1 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 8.16 (dd, J = 18.4, 8.2 Hz, 2H), 8.70 (s, 1H). 13C-NMR (101 MHz, DMSO): 7.6, 24.5, 25.4, 25.6, 28.7, 29.3, 30.3, 33.1, 38.3, 53.1, 54.7, 55.9, 65.5, 75.7, 94.8, 118.9, 127.3, 127.8, 128.1, 129.4, 129.9, 130.5, 131.7, 145.5, 146.0, 147.9, 152.3, 156.6, 167.3, 172.1, 173.4, 177.1, 177.4. HRMS (ESI) m/z: [M + H]+ 591.2467, calcd. for C31H34N4O8 590.23766.
Characterization of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl 12-((S)-5-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-5-oxopentanamido)dodecanoate (CPT-C-GL12). Obtained from CPT-B-L12 by method H as a faint yellow powder; m.p. 90.7–91.5 °C; Yield: 48.6%; 1H-NMR (500 MHz, CDCl3): δ 0.97 (t, J = 7.5 Hz, 3H, -CH3), 1.16 (s, 10H), 1.25 (s, 2H), 1.30 (m, 2H), 1.40 (m, 2H), 1.63 (m, 2H), 1.95 (m, 1H), 2.14 (m, 1H), 2.28 (m, 1H), 2.46 (m, 4H), 3.17 (m, 2H), 4.20 (m, 1H), 5.07 (m, 2H), 5.11 (m, 2H), 5.24 (d, J = 19.0 Hz, 1H), 5.29 (d, J = 19.0 Hz, 1H), 5.40 (d, J = 17.0 Hz, 1H), 5.66 (d, J = 17.0 Hz, 1H), 5.71 (s, 1H), 6.24 (s, 1H), 7.23 (s, 1H), 7.32 (s, 6H), 7.32 (s, 4H), 7.66 (t, J = 7.4 Hz, 1H), 7.82 (t, J = 7.4 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.38 (s, 1H). 13C-NMR (126 MHz, CDCl3): δ 7.7, 24.7, 26.9, 28.4, 29.1, 29.3, 29.3, 29.4, 29.5, 29.5, 30.5, 32.0, 33.9, 39.7, 50.0, 54.3, 66.7, 67.1, 67.2, 75.7, 96.2, 120.5, 128.1, 128.2, 128.3, 128.3, 128.4, 128.6, 128.6, 128.7, 129.6, 130.8, 131.4, 135.8, 136.3, 146.1, 146.2, 148.9, 152.4, 156.4, 157.5, 167.7, 171.0, 172.9, 173.3. HRMS (ESI) m/z: [M + H]+ 899.4223, calcd. for C52H58N4O10 898.41529.
Characterization of (S)-4-amino-5-((12-(((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)oxy)-12-oxododecyl)amino)-5-oxopentanoic acid (CPT-D-GL12). Obtained from CPT-C-GL12 by method I as a faint yellow powder; m.p. 180.1–180.9 °C; Yield: 24.2%; 1H-NMR (500 MHz, DMSO): δ 0.91 (t, J = 7.4 Hz, 3H, -CH3), 1.26 (m, 16H), 1.54 (m, 2H), 1.65 (m, 2H), 1.78 (m, 2H), 2.13 (m, 4H), 2.24 (m, 2H), 3.03 (m, 2H), 3.31 (s, 1H), 5.26 (d, 2H), 5.44 (m, 1H), 5.49 (s, 1H), 6.82 (s, 1H), 7.04 (s, 1H), 7.31 (dd, J = 14.7, 7.1 Hz, 1H), 7.46 (d, J = 7.1 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 8.04 (s, 1H), 8.13 (m, 2H), 8.69 (s, 1H). 13C-NMR (126 MHz, DMSO): δ 7.5, 24.5, 26.3, 26.4, 28.4, 28.7, 28.7, 28.8 28.9, 29.0, 29.4, 30.3, 32.3, 33.2, 38.4, 39.0, 39.2, 39.3, 39.5, 39.7, 39.8, 40.0, 50.2, 53.6, 66.3, 75.6, 94.7, 119.0, 127.7, 128.0, 128.6, 128.9, 129.8, 130.4, 131.6, 145.4, 146.0, 147.9, 152.3, 156.6, 165.1, 167.3, 172.1, 172.4, 174.7. HRMS (ESI) m/z: [M + H]+ 675.3390, calcd. for C37H46N4O8 674.33156.

4.2. Bio-Evaluation Methods

4.2.1. Cell Culture

The human prostate cancer cell line (LNCaP-FGC), human prostate cancer cell line (PC-3), human prostate cancer cell line (DU-145), human breast cancer cell line (MCF-7), human hepatocellular carcinoma cell line (HepG2), human cervical cancer cell line (Hela), were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College. The cultures of the cells were maintained as a monolayer in RPMI 1640 or DMEM supplemented with 10% (v/v) heat inactivated fetal bovine serum and 1% (v/v) enicillin/streptomycin (Corning, New York, NY, USA) and incubated at 37 °C in a humidified atmosphere with 5% CO2. The tested compounds were dissolved in DMSO (Sigma, St. Louis, MO, USA) and added at required concentrations to the cell culture.

4.2.2. Cytotoxicity Assay

The cytotoxicity of all the tested compounds was evaluated in vitro via the MTT method against PSMA-expressing LNCaP-FGC cells and non-PSMA-expressing cells HepG2, Hela, MCF-7, DU145, PC-3 cells using CPT as the positive control. Tumor cells growing in the logarithmic phase were seeded in 96-well plates at a density 3 × 103 cells/well and incubated overnight. The following day, cells were then treated with serial dilutions of the tested compounds for 72 h. At the end of this incubation, 20 µL of 5 mg/mL methylthiazol tetrazolium (MTT) was added to each well and incubation proceeded at 37 °C for another 4 h. After the supernatant medium was thrown away, 150 µL dimethylsulphoxide (DMSO) were added to each well and absorbance was measured at 490 nm using a plate reader (BIORAD 550 spectrophotometer, Bio-rad Life Science Development Ltd., Berkeley, CA, USA). Experiments were performed in triplicate and the values were the average of three (n = 3) independent experiments. The concentration of the compound that results in 50% growth inhibition corresponds to the IC50. Tumor cell growth inhibitory rate was calculated using Equation (1):
% inhibition = (1 − Sample group OD/Control group OD) × 100%

4.2.3. Aqueous Solubility Study

The equilibrium solubility of CPT and CPT-HT-J-ZL12 was determined in triplicate in PBS, according to the reference method with some modifications. An excess amount (~5 mg) of the tested compounds were placed into tubes, and 1 mL PBS was added to the tube. Then the tubes were incubated at 37 °C for 24 h in an ermostated oscillator (HZQ-QX, Harbin Donglian Instrument Co., Ltd., Harbin, China). After incubation, samples (0.1 mL) were removed and centrifuged at 10,000× g for 10 min with high speed centrifuge (Sorvall™ Legend™ Micro 21, Thermo Fisher Scientific, Waltham, MA, USA). A 500 µL sample of the supernatant was diluted, and the concentration was measured with HPLC according the method below. CPT and CPT-HT-J-ZL12 were measured by HPLC systems configured with a Waters 2695 separation module and a Waters 2695 ultraviolet (UV) detector with the following conditions: Xbridge C18 column (250 mm × 4.6 mm, 5 mm); temperature, 25 °C; elution flow rate, 1.0 mL/min; detection wavelength, 365 nm. mobile phase, 0.2% b in water (A) and 0.2% formic acid in acetonitrile (B) using a gradient elution of 10–60% B at 0–5.0 min, 60–80% B at 5.0–15.0 min, 80% B at 15.0–17.0 min, and 10% B at 17.1–23.0 min.

4.2.4. In Vitro Cellular Uptake

To evaluate the cellular uptake of the CPT-HT-J-ZL12, LNCaP-FGC (PSMA+) and HepG2 (PSMA−) cells were seeded at a density of 1 × 104 cells/well in 35 mm glass bottom dishes with 1 mL complete 1640 medium [28]. After incubation for 24 h, the culture medium was replaced with 2 mL of fresh medium containing 10 µM CPT-HT-J-ZL12. Then the cells were incubated further for different incubation time periods (1 and 4 h). Following the removal of the culture media, cells were washed with cold PBS, fixed with 70% ethanol for 30 min at 4 °C. Subsequently, the cells were stained with 5 μg/mL propidium iodide (excitation wavelength was 561 nm) for 5 min to visualize nucleus. The cells were observed by a UltraView RS confocal laser scanning microscopy (PerkinElmer, Waltham, MA, USA).

4.2.5. Detection of Apoptosis Using Annexin V-FITC/PI Staining

To determine early apoptosis and secondary necrosis, LNCaP-FGC (PSMA+) and HepG2 (PSMA−) cells were stained with annexin-V FITC apoptosis detection kit (Beijing BioDee Biotech. Co., Ltd., Beijing, China) as per the manufacturer’s instructions. After exposure to different concentrations of CPT-HT-J-ZL12 (0.63, 1.25, 2.50 µM for LNCaP-FGC; 20.00, 40.00, 80.00 µM for HepG2) for 72 h, the cells were collected, washed twice with cold PBS, and centrifuged at 3000× g for 5 min. The resulting pellet was mixed with 200 µL binding buffer of the Annexin V-FITC kit, then 5 µL FITC labeled annexin V was added and mixed gently. After incubation at 4 °C for 10 min in the dark, 5 µL PI was added and mixed gently. Then the cells were immediately analyzed with a flow cytometry.

4.2.6. Detection of Cell Cycle Using PI Staining

The cell cycle was measured using PI staining and flow cytometry analysis according to the instructions of the manufacturer (Cell Cycle and Apoptosis Analysis Kit, Beyotime Biotechnology, Jiangsu, China). LNCaP-FGC cells (2 × 104 cells/mL) in logarithmic growth phase were seeded in 6-well culture plate and incubated for 24 h at 37 °C in incubator with 5% CO2. After exposure to different concentrations of CPT-HT-J-ZL12 (0.63, 1.25, 2.50 μM) for 72 h, cells were harvested and washed twice with cold PBS. Then the cells were centrifuged at 2400× g for 10 min, resuspended in 1 mL 70% cold ethanol and fixed for 12 h at −20 °C. After being washed twice with cold PBS, the cells were incubated with 0.5 mL propidium iodide for 30 min at 37 °C. Detection of the cell cycle was carried out by the Flow Cytometer at 488 nm excitation wavelength

4.2.7. Statistical Analysis

Each experimental value is expressed as the mean ± SD (standard deviation). One-way analysis of variance (ANOVA) was performed to determine the significance between groups; p < 0.05 was considered to be statistically significant.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/19/10/3251/s1.

Author Contributions

Ideas and experiment design: B.X., T.M., P.-L.W., and H.-M.L.; Chemistry and Biology: B.X., F.Z., M.-M.Y., D.-S.C.; Analysis and interpretation of data: W.-B.G., Y.-Q.Y., X.-H.J.; Writing and review of the manuscript: All the authors; Study supervision: H.-M.L., P.-L.W., and T.M.

Funding

This research was funded by the National Natural Science Foundation of China (No. 81173519 and 81603256), the Fundamental Research Funds for the Central Universities (No. 2017-JYB-XS-077), Beijing Key Laboratory for Basic and Development Research on Chinese Medicine (Beijing, 100102) and Young Teachers’ Scientific Research Project of Beijing University of Chinese Medicine (No. 2015-JYB JSMS023).

Acknowledgments

This research was also supported by the Fundamental Research Funds for the Central Universities (BUCM-2019-JCRC002).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Stewart, B.; Wild, C.P. World Cancer Report 2014; International Agency for Research on Cancer: Lyon, France, 2017. [Google Scholar]
  2. Li, Y.; Lin, J.Y.; Ma, J.Y.; Song, L.; Lin, H.R.; Tang, B.W.; Chen, D.Y.; Su, G.H.; Ye, S.F.; Zhu, X.; et al. Methotrexate–Camptothecin prodrug nanoassemblies as a versatile nanoplatform for biomodal imaging-guided self-active targeted and synergistic chemotherapy. ACS Appl. Mater. Interfaces 2017, 9, 34650–34665. [Google Scholar] [CrossRef] [PubMed]
  3. Savard, J.; Ivers, H.; Savard, M.H.; Morin, C.M. Cancer treatments and their side effects are associated with aggravation of insomnia: Results of a longitudinal study. Cancer 2015, 121, 1703–1711. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Sioka, C.; Kyritsis, A.P. Central and peripheral nervous system toxicity of common chemotherapeutic agents. Cancer Chemother. Pharmacol. 2009, 63, 761–767. [Google Scholar] [CrossRef] [PubMed]
  5. Curigliano, G.; Cardinale, D.; Suter, T.; Plataniotis, G.; de Azambuja, E.; Sandri, M.T.; Criscitiello, C.; Goldhirsch, A.; Cipolla, C.; Roila, F.; et al. Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy: ESMO clinical practice guidelines. Ann. Oncol. 2012, 23, 155–166. [Google Scholar] [CrossRef] [PubMed]
  6. Perazella, M.A. Onco-nephrology: Renal toxicities of chemotherapeutic agents. Clin. J. Am. Soc. Nephrol. 2012, 7, 1713–1721. [Google Scholar] [CrossRef] [PubMed]
  7. Pommier, Y. DNA topoisomerase I inhibitors: Chemistry, biology, and interfacial inhibition. Chem. Rev. 2009, 109, 2894–2902. [Google Scholar] [CrossRef] [PubMed]
  8. Pommier, Y.; Leo, E.; Zhang, H.L.; Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 2010, 17, 421–433. [Google Scholar] [CrossRef] [PubMed]
  9. Pommier, Y. Topoisomerase I inhibitors: Camptothecins and beyond. Nat. Rev. Cancer 2006, 6, 789–802. [Google Scholar] [CrossRef] [PubMed]
  10. Liang, X.L.; Gao, C.; Cui, L.G.; Wang, S.M.; Wang, J.R.; Dai, Z.F. Self-Assembly of an amphiphilic janus camptothecin–floxuridine conjugate into liposome-like nanocapsules for more efficacious combination chemotherapy in cancer. Adv. Mater. 2017, 29. [Google Scholar] [CrossRef] [PubMed]
  11. Liu, Y.M.; Chen, X.; Ding, J.M.; Yu, L.; Ma, D.; Ding, J.D. Improved solubility and bioactivity of camptothecin family antitumor drugs with supramolecular encapsulation by water-soluble pillar[6]arene. ACS Omega 2017, 2, 5283–5288. [Google Scholar] [CrossRef] [PubMed]
  12. Sawant, R.R.; Sawant, R.M.; Torchilin, V.P. Mixed PEG–PE/vitamin E tumor-targeted immunomicelles as carriers for poorly soluble anti-cancer drugs: Improved drug solubilization and enhanced in vitro cytotoxicity. Eur. J. Pharm. Biopharm. 2008, 70, 51–57. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Zhao, H.; Lee, C.; Sai, P.; Choe, Y.H.; Boro, M.; Pendri, A.; Guan, S.Y.; Greenwald, R.B. 20-O-acylcamptothecin derivatives: Evidence for lactone stabilization. J. Org. Chem. 2000, 65, 4601–4606. [Google Scholar] [CrossRef] [PubMed]
  14. Li, Q.Y.; Zu, Y.G.; Shi, R.Z.; Yao, L.P. Review camptothecin: Current perspectives. Curr. Med. Chem. 2006, 13, 2021–2039. [Google Scholar] [CrossRef] [PubMed]
  15. Hou, M.; Xue, P.; Gao, Y.E.; Ma, X.; Bai, S.; Kang, Y.; Xu, Z. Gemcitabine–camptothecin conjugates: A hybrid prodrug for controlled drug release and synergistic therapeutics. Biomater. Sci. 2017, 5, 1889–1897. [Google Scholar] [CrossRef] [PubMed]
  16. Henne, W.A.; Doorneweerd, D.D.; Hilgenbrink, A.R.; Kularatne, S.A.; Low, P.S. Synthesis and activity of a folate peptide camptothecin prodrug. Bioorg. Med. Chem. Lett. 2006, 16, 5350–5355. [Google Scholar] [CrossRef] [PubMed]
  17. Ghosh, A.; Heston, W.D. Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. J. Cell. Biochem. 2004, 91, 528–539. [Google Scholar] [CrossRef] [PubMed]
  18. Davis, M.I.; Bennett, M.J.; Thomas, L.M.; Bjorkman, P.J. Crystal structure of prostate-specific membrane antigen, a tumor marker and peptidase. Proc. Natl. Acad. Sci. USA 2005, 102, 5981–5986. [Google Scholar] [CrossRef] [PubMed]
  19. Mhaka, A.; Gady, A.M.; Rosen, D.M.; Lo, K.M.; Gillies, S.D.; Denmeade, S.R. Use of methotrexate-based peptide substrates to characterize the substrate specificity of prostate-specific membrane antigen (PSMA). Cancer Biol. Ther. 2004, 3, 551–558. [Google Scholar] [CrossRef] [PubMed]
  20. Denmeade, S.R.; Mhaka, A.M.; Rosen, D.M.; Brennen, W.N.; Dalrymple, S.; Dach, I.; Olesen, C.; Gurel, B.; Demarzo, A.M.; Wilding, G.; et al. Engineering a prostate-specific membrane antigen–activated tumor endothelial cell prodrug for cancer therapy. Sci. Transl. Med. 2012, 4. [Google Scholar] [CrossRef] [PubMed]
  21. Isaacs, J.T. Prodrug Targeting Based upon the NAALADase Activity to Prostate Specific Membrane Antigen by Prostate Cancer Cells; Johns Hopkins Univ Baltimore Md School of Medicine: Baltimore, MD, USA, 2001. [Google Scholar]
  22. Kularatne, S.A.; Venkatesh, C.; Santhapuram, H.K.; Wang, K.; Vaitilingam, B.; Henne, W.A.; Low, P.S. Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs. J. Med. Chem. 2010, 53, 7767–7777. [Google Scholar] [CrossRef] [PubMed]
  23. Lv, Q.Z.; Yang, J.C.; Zhang, R.S.; Yang, Z.M.; Yang, Z.T.; Wang, Y.J.; Xu, Y.J.; He, Z.G. Prostate-specific membrane antigen targeted therapy of prostate cancer using a DUPA–paclitaxel conjugate. Mol. Pharm. 2018, 15, 1842–1852. [Google Scholar] [CrossRef] [PubMed]
  24. Wullner, U.; Neef, I.; Eller, A.; Kleines, M.; Tur, M.K.; Barth, S. Cell-specific induction of apoptosis by rationally designed bivalent aptamer-siRNA transcripts silencing eukaryotic elongation factor 2. Curr. Cancer Drug Targets 2008, 8, 554–565. [Google Scholar] [CrossRef] [PubMed]
  25. Lee, S.J.; Kim, H.S.; Yu, R.; Lee, K.; Gardner, T.A.; Jung, C.; Jeng, M.H.; Yeung, F.; Cheng, L.; Kao, C. Novel prostate-specific promoter derived from PSA and PSMA enhancers. Mol. Ther. 2002, 6, 415–421. [Google Scholar] [CrossRef] [PubMed]
  26. Uchida, A.; O’Keefe, D.S.; Bacich, D.J.; Molloy, P.L.; Heston, W.D.W. In vivo suicide gene therapy model using a newly discovered prostate-specific membrane antigen promoter/enhancer: A potential alternative approach to androgen deprivation therapy. Urology 2001, 58, 132–139. [Google Scholar] [CrossRef]
  27. Wolf, P.; Gierschner, D.; Bühler, P.; Wetterauer, U.; Elsässer-Beile, U. A recombinant PSMA-specific single-chain immunotoxin has potent and selective toxicity against prostate cancer cells. Cancer Immunol. Immunother. 2006, 55, 1367–1373. [Google Scholar] [CrossRef] [PubMed]
  28. Fan, Z.X.; Liu, G.H.; Li, Y.; Ma, J.Y.; Lin, J.Y.; Guo, F.Q.; Hou, Z.Q.; Xie, L.Y. Self-assembly of the active lactone form of a camptothecin–phospholipid complex for sustained nuclear drug delivery. RSC Adv. 2016, 6, 82949–82960. [Google Scholar] [CrossRef]
  29. Mannhold, R.; Poda, G.I.; Ostermann, C.; Tetko, I.V. Calculation of molecular lipophilicity: State-of-the-art and comparison of log P methods on more than 96,000 compounds. J. Pharm. Sci. 2009, 98, 861–893. [Google Scholar] [CrossRef] [PubMed]
  30. Yamashita, T.; Dohta, Y.; Nakamura, T.; Fukami, T. High-speed solubility screening assay using ultra-performance liquid chromatography/mass spectrometry in drug discovery. J. Chromatogr. A 2008, 1182, 72–76. [Google Scholar] [CrossRef] [PubMed]
  31. Dohta, Y.; Yamashita, T.; Horiike, S.; Nakamura, T.; Fukami, T. A system for LogD screening of 96-well plates using a water-plug aspiration/injection method combined with high-performance liquid chromatography-mass spectrometry. Anal. Chem. 2007, 79, 8312–8315. [Google Scholar] [CrossRef] [PubMed]
  32. Alelyunas, Y.W.; Pelosi-Kilby, L.; Turcotte, P.; Kary, M.B.; Spreena, R.C. A high throughput dried DMSO LogD lipophilicity measurement based on 96-well shake-flask and atmospheric pressure photoionization mass spectrometry detection. J. Chromatogr. A 2010, 1217, 1950–1955. [Google Scholar] [CrossRef] [PubMed]
Ijms 19 03251 sch001 550
Scheme 1. Synthesis of the pentapeptide HT-J. Reagents and Conditions: (a) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (b) Piperidine, dimethylformamide (DMF), 1 h; (c) Wet Pd/C (5%), MeOH, 12 h.
Scheme 1. Synthesis of the pentapeptide HT-J. Reagents and Conditions: (a) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (b) Piperidine, dimethylformamide (DMF), 1 h; (c) Wet Pd/C (5%), MeOH, 12 h.
Ijms 19 03251 sch001
Ijms 19 03251 sch002 550
Scheme 2. Synthesis of the PSMA-activated prodrugs CPT-HT-J-ZLn. Reagents and Conditions: (d) EDCI, DMAP, DCM, 25 °C, 12 h; (e) TFA, DCM, 25 °C, 2 h; (f) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (g) Trifluoroacetic (TFA):Thioanisole:H2O = 95:2.5:2.5, 25 °C, 3 h.
Scheme 2. Synthesis of the PSMA-activated prodrugs CPT-HT-J-ZLn. Reagents and Conditions: (d) EDCI, DMAP, DCM, 25 °C, 12 h; (e) TFA, DCM, 25 °C, 2 h; (f) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (g) Trifluoroacetic (TFA):Thioanisole:H2O = 95:2.5:2.5, 25 °C, 3 h.
Ijms 19 03251 sch002
Ijms 19 03251 sch003 550
Scheme 3. Synthesis of the prostate-specific membrane antigen (PSMA) hydrolysates CPT-D-GLn. Reagents and Conditions: (h) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (i) Wet Pd/C (5%), MeOH, 12 h.
Scheme 3. Synthesis of the prostate-specific membrane antigen (PSMA) hydrolysates CPT-D-GLn. Reagents and Conditions: (h) EDCI, HOBt, DIPEA, DCM, 25 °C, 3 h; (i) Wet Pd/C (5%), MeOH, 12 h.
Ijms 19 03251 sch003
Ijms 19 03251 g001 550
Figure 1. The cytotoxicities of the CPT-HT-J-ZLn and the PSMA hydrolysate CPT-D-GLn to different tumor and normal cells.
Figure 1. The cytotoxicities of the CPT-HT-J-ZLn and the PSMA hydrolysate CPT-D-GLn to different tumor and normal cells.
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Figure 2. The IC50 values of CPT and the prodrug CPT-HT-J-ZL12 on different cell lines. * p < 0.05, *** p < 0.001, vs. LNCaP-FGC cells.
Figure 2. The IC50 values of CPT and the prodrug CPT-HT-J-ZL12 on different cell lines. * p < 0.05, *** p < 0.001, vs. LNCaP-FGC cells.
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Ijms 19 03251 g003 550
Figure 3. Cellular uptake of CPT-HT-J-ZL12 in HepG2 (no expression of PSMA) cells. CLSM images of HepG2 cells incubated with CPT-HT-J-ZL12 (10 μM) for 1 and 4 h. Nuclei were stained by PI (red), the blue color was indicative of CPT. CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500–550 nm. For PI: λex = 561 nm, band-pass filter λ = 575–625 nm.
Figure 3. Cellular uptake of CPT-HT-J-ZL12 in HepG2 (no expression of PSMA) cells. CLSM images of HepG2 cells incubated with CPT-HT-J-ZL12 (10 μM) for 1 and 4 h. Nuclei were stained by PI (red), the blue color was indicative of CPT. CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500–550 nm. For PI: λex = 561 nm, band-pass filter λ = 575–625 nm.
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Ijms 19 03251 g004 550
Figure 4. Cellular uptake of CPT-HT-J-ZL12 in LNCaP-FGC (high expression of PSMA) cells. CLSM images of LNCaP-FGC cells incubated with CPT-HT-J-ZL12 (10 µM) for 1 and 4 h. Nuclei were stained by PI (red); the blue color was indicative of CPT. CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500–550 nm. For PI: λex = 561 nm, band-pass filter λ = 575–625 nm.
Figure 4. Cellular uptake of CPT-HT-J-ZL12 in LNCaP-FGC (high expression of PSMA) cells. CLSM images of LNCaP-FGC cells incubated with CPT-HT-J-ZL12 (10 µM) for 1 and 4 h. Nuclei were stained by PI (red); the blue color was indicative of CPT. CPT: false-color green. For CPT: λex = 405 nm, band-pass filter λ = 500–550 nm. For PI: λex = 561 nm, band-pass filter λ = 575–625 nm.
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Ijms 19 03251 g005 550
Figure 5. Mean fluorescence intensity of CPT-HT-J-ZL12 internalized by LNCaP-FGC (high expression of PSMA) and HepG2 (none expression of PSMA) cells after incubation for 1 and 4 h. * p < 0.05.
Figure 5. Mean fluorescence intensity of CPT-HT-J-ZL12 internalized by LNCaP-FGC (high expression of PSMA) and HepG2 (none expression of PSMA) cells after incubation for 1 and 4 h. * p < 0.05.
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Figure 6. Flow cytometry analysis for apoptosis of LNCaP-FGC cells (high expression of PSMA) induced by CPT-HT-J-ZL12: (a) control group; (b) 0.63 µM; (c) 1.25 µM; and (d) 2.5 µM.
Figure 6. Flow cytometry analysis for apoptosis of LNCaP-FGC cells (high expression of PSMA) induced by CPT-HT-J-ZL12: (a) control group; (b) 0.63 µM; (c) 1.25 µM; and (d) 2.5 µM.
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Ijms 19 03251 g007 550
Figure 7. Flow cytometry analysis for apoptosis of HepG2 (none expression of PSMA) cells induced by CPT-HT-J-ZL12: (a) control group; (b) 20.0 µM; (c) 40.0 µM; and (d) 80.0 µM.
Figure 7. Flow cytometry analysis for apoptosis of HepG2 (none expression of PSMA) cells induced by CPT-HT-J-ZL12: (a) control group; (b) 20.0 µM; (c) 40.0 µM; and (d) 80.0 µM.
Ijms 19 03251 g007
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Figure 8. Effect of CPT-HT-J-ZL12 on cell cycle progression in LNCaP-FGC cells (high expression of PSMA): (a) control group; (b) 0.63 µM; (c) 1.25 µM; and (d) 2.5 µM.
Figure 8. Effect of CPT-HT-J-ZL12 on cell cycle progression in LNCaP-FGC cells (high expression of PSMA): (a) control group; (b) 0.63 µM; (c) 1.25 µM; and (d) 2.5 µM.
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Table
Table 1. The anti-proliferative effects and ClogP values of the CPT-HT-J-ZLn and the prostate-specific membrane antigen (PSMA) hydrolysate CPT-D-GLn.
Table 1. The anti-proliferative effects and ClogP values of the CPT-HT-J-ZLn and the prostate-specific membrane antigen (PSMA) hydrolysate CPT-D-GLn.
CompoundIC50 (µM)CLogP
MCF-7PC-3DU145LNCaP-FGCHepG2HeLaLO2MDCK
CPT0.16 ± 0.100.13 ± 0.090.21 ± 0.090.18 ± 0.175.43 ± 0.812.48 ± 0.800.04 ± 0.010.02 ± 0.010.9
CPT-HT-J-ZL20.11 ± 0.081.00 ± 0.101.16 ± 0.282.04 ± 0.30>40.003.54 ± 2.541.68 ± 0.451.27 ± 0.30−7.45
CPT-HT-J-ZL40.32 ± 0.019.98 ± 2.389.73 ± 3.491.18 ± 0.10>40.00>40.00>40.009.13 ± 2.40−6.86
CPT-HT-J-ZL67.03 ± 3.7640.00 ± 3.3726.28 ± 1.813.13 ± 0.40>40.00>40.00>40.0018.96 ± 3.60−6.60
CPT-HT-J-ZL1221.68 ± 4.9642.96 ± 3.695.40 ± 1.221.00 ± 0.20>40.00>40.00>40.00>40.00−3.42
CPT-B-L24.11 ± 3.095.19 ± 0.300.12 ± 0.090.38 ± 0.10>40.0010.00 ± 2.005.61 ± 1.405.16 ± 1.200.54
CPT-B-L41.58 ± 2.421.00 ± 0.100.25 ± 0.130.37 ± 0.02>40.004.37 ± 0.501.36 ± 0.303.20 ± 2.001.26
CPT-B-L66.76 ± 1.4715.89 ± 1.733.55 ± 0.951.89 ± 0.20>40.0015.0 ± 0.802.71 ± 0.385.44 ± 3.001.76
CPT-B-L125.76 ± 0.866.45 ± 0.427.32 ± 1.431.93 ± 1.03>40.00>40.002.63 ± 0.845.2 ± 0.884.94
CPT-D-GL20.30 ± 0.051.5 ± 0.200.40 ± 0.053.68 ± 0.36>40.00>40.000.16 ± 0.070.26 ± 0.14−2.88
CPT-D-GL44.03 ± 0.3014.93 ± 1.004.89 ± 0.013.68 ± 0.50>40.00>40.000.40 ± 0.150.23 ± 0.10−2.29
CPT-D-GL617.25 ± 3.0012.50 ± 0.803.97 ± 0.168.17 ± 1.00>40.00>40.001.5 ± 0.604.0 ± 0.42−2.03
CPT-D-GL12>40.00>40.007.38 ± 0.202.10 ± 0.68>40.0025.47 ± 1.03.8 ± 0.805.1 ± 1.101.15
CPT-HT-J-L2>40.00>40.00>40.00>40.00>40.00>40.00>40.00>40.004.04
CPT-HT-J-L4>40.00>40.00>40.00>40.00>40.00>40.00>40.00>40.004.62
CPT-HT-J-L6>40.00>40.00>40.00>40.00>40.00>40.00>40.00>40.004.89
CPT-HT-J-L12>40.00>40.00>40.00>40.00>40.00>40.00>40.00>40.008.07

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Xu, B.; Zhou, F.; Yan, M.-M.; Cai, D.-S.; Guo, W.-B.; Yang, Y.-Q.; Jia, X.-H.; Zhang, W.-X.; Li, T.; Ma, T.; et al. PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates. Int. J. Mol. Sci. 2018, 19, 3251. https://doi.org/10.3390/ijms19103251

AMA Style

Xu B, Zhou F, Yan M-M, Cai D-S, Guo W-B, Yang Y-Q, Jia X-H, Zhang W-X, Li T, Ma T, et al. PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates. International Journal of Molecular Sciences. 2018; 19(10):3251. https://doi.org/10.3390/ijms19103251

Chicago/Turabian Style

Xu, Bing, Fei Zhou, Meng-Meng Yan, De-Sheng Cai, Wen-Bo Guo, Yu-Qin Yang, Xiao-Hui Jia, Wen-Xi Zhang, Tong Li, Tao Ma, and et al. 2018. "PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates" International Journal of Molecular Sciences 19, no. 10: 3251. https://doi.org/10.3390/ijms19103251

APA Style

Xu, B., Zhou, F., Yan, M. -M., Cai, D. -S., Guo, W. -B., Yang, Y. -Q., Jia, X. -H., Zhang, W. -X., Li, T., Ma, T., Wang, P. -L., & Lei, H. -M. (2018). PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates. International Journal of Molecular Sciences, 19(10), 3251. https://doi.org/10.3390/ijms19103251

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