Design and Synthesis of an RGD Peptidomimetic-Paclitaxel Conjugate Targeting α_vβ₃ Integrin for Tumour-Directed Drug Delivery

This article is dedicated to Professor Victor Snieckus on the occasion of his 80^th birthday

Abstract

A 1,2,3-triazole-based RGD peptidomimetic having nanomolar affinity for α_vβ₃ integrin was conjugated to the potent antimitotic paclitaxel via an oxime heterobifunctional linker. The resulting construct maintained nanomolar binding concentration to α_vβ₃ integrin and showed 11-fold selectivity in terms of cytotoxicity towards highly α_vβ₃ expressing U87MG cancer cells relative to non α_vβ₃ expressing MCF7 cells, indicating promising cancer cell targeting capacity.

1 Design

2 Retrosynthesis

3 Synthesis

4 Solid-Phase Receptor Binding Assay

5 Cell Cytotoxicity Assays

6 Metabolic Stability Assays

7 Conclusions

Key words

RGD - peptidomimetic - integrin - paclitaxel - click - cytotoxicity - cancer

• References and Notes

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• 12 Synthesis of 6 To a solution of 7 (as TFA salt, 22 μmol, 20 mg, 1 equiv) in dry DMF (500 μL) Et₃N (66 μmol mmol, 10 μL, 3 equiv), n-Bu₄NOAc (66 μmol, 20 mg, 3 equiv), a catalytic amount of Pd(OAc)₂, and alkyne 8 (0.22 mmol, 18 mg, 10 equiv) were added. The reaction was stirred at 60 °C for 6 h. The mixture was cooled to r.t., diluted with water (4 mL), and then filtered using a 0.20 μm syringe filter. The filtrate was purified by semipreparative RP-HPLC. Analytical RP-HPLC conditions (solvent A = H₂O + 0.1% TFA, solvent B = MeCN, gradient: from 30% B to 50% B in 15 min; flow: 1 mL/min; t _R= 7 min). The product was lyophilized affording 12 mg of a yellow solid (70% yield, isolated as TFA salt): ¹H NMR (400 MHz, CD₃OD): δ = 8.21–8.05 (m, 2 H), 7.73 (d, J = 6.6 Hz, 1 H), 7.51–7.37 (m, 1 H), 6.85 (s, 1 H), 6.80 (dd, J = 6.7, 1.4 Hz, 1 H), 4.62 (t, J = 6.6 Hz, 2 H), 4.29 (dd, J = 8.9, 4.9 Hz, 1 H), 3.82 (dd, J = 13.8, 4.9 Hz, 1 H), 3.74 (t, J = 6.1 Hz, 2 H), 3.52 (dd, J = 13.8, 8.9 Hz, 1 H), 3.42 (t, J = 6.7 Hz, 2 H), 2.68 (t, J = 7.0 Hz, 2 H), 2.43 (s, 3 H), 2.41–2.29 (m, 2 H), 1.93–1.83 (m, 2 H). ¹⁹F NMR (376 MHz, CD₃OD): δ = –63.15 (d, J = 12.7 Hz), –76.96 (s), –110.64 (q, J = 12.7 Hz). ¹³C NMR (101 MHz, CD₃OD): δ = 170.9, 161.4 (d, J = 261.5 Hz), 160.4, 156.7, 152.7, 141.4, 138.2 (d, J = 3.7 Hz) , 134.2, 133.6 (d, J = 10.3 Hz), 126.3, 122.8, 121.9 (q, J = 271.4 Hz), 117.7 (d, J = 22.2 Hz), 114.5, 111.6, 106.2, 64.6, 60.0, 55.4, 45.9, 40.7, 38.6, 30.3, 27.5, 20.5, 15.7. ESI-MS: m/z calcd for C₂₇H₃₀F₄N₇O₆S [M + H]⁺: 655.18; found. 655.2.
• 13 Synthesis of 13 To a suspension of 6 (65 μmol, 50 mg, 1 equiv) and DMP (97.5 μmol, 41 mg, 1.5 equiv) in MeCN (1 mL) DMSO was added dropwise until the solution became clear. The mixture was stirred at r.t. for 6 h and then filtered to remove a white precipitate formed during the reaction. To the filtered solution 4 (195 μmol, 25 mg, 3 equiv) was added, and the mixture was stirred for 1 h at r.t. The product was purified by semipreparative RP-HPLC. Analytical RP-HPLC conditions (solvent A = H₂O + 0.1% TFA, solvent B = MeCN, gradient: from 30% B to 50% B in 15 min; flow: 1 mL/min; t _R = 4.8 min). The product was lyophilized affording 38 mg of a pale yellow solid (60% yield, isolated as bis-TFA salt) containing a mixture of oxime E/Z isomers (3:2). Minor isomer resonances are denoted by an asterisk*: ¹H NMR (400 MHz, CD₃OD): δ = 8.08–8.00 (m, 2 H), 7.63 (d, J = 6.2 Hz, 1 H), 7.49 (t, J = 5.7 Hz, 0.6 H), 7.38–7.28 (m, 1 H), 6.80* (t, J = 5.1 Hz, 0.4 H), 6.69 (s, 1 H), 6.65 (d, J = 6.1 Hz, 1 H), 4.50 (t, J = 6.3 Hz, 2 H), 4.10–3.92 (m, 3 H), 3.78–3.62 (m, 1 H), 3.48–3.34 (m, 1 H), 3.30 (t, J = 6.1 Hz, 2 H), 2.91–2.79 (m, 2 H), 2.67 (t, J = 6.8 Hz, 2 H), 2.62–2.53 (m, 1 H), 2.43 (m, 1 H), 2.29 (s, 3 H), 2.27–2.17 (m, 2 H), 1.72–1.57 (m, 4 H).¹⁹F NMR (376 MHz, CD₃OD): δ = –63.10 (d, J = 12.7 Hz), –76.95, –110.39 to –110.82 (m). ESI-MS: m/z calcd for C₃₁H₃₈F₄N₉O₆S [M + H]⁺: 740.25; found: 740.2.
• 14 Synthesis of 3 To a solution of PTX-2′-succinate-NHS ester 2 (24 μmol, 25 mg, 1 equiv) in DMF (0.5 mL) at r.t., DIPEA (72 μmol, 13 μL, 3 equiv) and 13 (24 μmol, 23 mg, 1 equiv) were added. The reaction mixture was stirred for 5 h at r.t., then filtered and purified by semipreparative RP-HPLC. Analytical RP-HPLC conditions (solvent A = H₂O + 0.1% TFA, solvent B = MeCN, gradient: from 50% B to 1000% B in 15 min, flow: 1 mL/min; t _R= 6.9 min). The product was lyophilized affording 23 mg of a white solid (54% yield, isolated as TFA salt) containing a mixture of oxime E/Z isomers (3:2). Minor isomer resonances are denoted by an asterisk*: ¹H NMR (400 MHz, CD₃OD): δ = 9.03 (d, J = 8.7 Hz, 1 H), 8.29–8.20 (m, 1 H), 8.05–7.98 (m, 4 H), 7.76–7.70 (m, 2 H), 7.62–7.55 (m, 2 H), 7.55–7.26 (m, 11 H), 7.15 (t, J = 7.1 Hz, 1 H), 6.78* (t, J = 5.1 Hz, 0.4 H), 6.73 (s, 1 H), 6.67 (dd, J = 6.6, 1.3 Hz, 1 H), 6.33 (s, 1 H), 5.94 (t, J = 9.0 Hz, 1 H), 5.68 (d, J = 6.6 Hz, 1 H), 5.52 (d, J = 7.2 Hz, 1 H), 5.33 (dd, J = 6.7, 1.2 Hz, 1 H), 4.88 (d, J = 8.1 Hz, 1 H), 4.48 (t, J = 6.0 Hz, 2 H), 4.31–4.13 (m, 2 H), 4.07 (s, 2 H), 3.96 (t, J = 6.3 Hz, 1 H), 3.89 (t, J = 6.3 Hz, 1 H), 3.76–3.66 (m, 2 H), 3.60–3.45 (m, 1 H), 3.45–3.35 (m, 1 H), 3.34–3.25 (m, 2 H), 3.09–2.97 (m, 2 H), 2.72–2.53 (m, 5 H), 2.48–2.34 (m, 4 H), 2.33–2.19 (m, 8 H), 2.10–1.97 (m, 4 H), 1.80 (s, 3 H), 1.75–1.60 (m, 2 H), 1.60–1.37 (m, 8 H), 1.03 (s, 3 H), 1.02 (s, 3 H). ¹⁹F NMR (376 MHz, CD₃OD): δ = –63.06* (d, J = 12.7 Hz), –63.07 (d, J = 12.7 Hz), –77.19 (s), –110.47 to –110.61 (m). ¹³C NMR (101 MHz, CD₃OD): δ = 203.8, 172.4, 172.2, 170.8, 170.1, 169.9, 169.1, 169.0, 166.2, 161.4 (d, J = 260.6 Hz), 160.3, 152.6, 149.3*, 148.6, 141.7, 141.6, 141.0, 138.1 (d, J = 3.7 Hz), 137.0, 134.2, 134.1, 133.6 (d, J = 9.9 Hz), 133.4, 133.2, 131.5, 129.9, 129.8, 128.7, 128.3, 128.1, 127.2, 127.2, 122.5 (d, J = 2.8 Hz), 121.9 (q, J = 272.6 Hz), 117.7 (d, J = 22.1 Hz), 114.6, 105.1, 84.4, 80.8, 78.0, 77.6, 76.0, 75.3, 74.6, 73.2, 72.7, 71.4, 70.9, 70.0, 65.3, 65.2, 57.8, 55.3, 54.0, 46.4, 45.9, 43.1, 40.7, 38.8, 38.6, 36.0, 34.9, 29.8, 29.3, 28.6, 27.7, 27.5, 26.1, 26.0, 25.5, 25.5, 23.9, 21.8, 20.9, 20.6, 19.4, 16.7, 16.2, 13.6, 9.0. ESI-MS: m/z calcd for C₈₂H₉₁F₄N₁₀O₂₂S [M + H]⁺: 1675.59; found: 1675.7.
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• 17 Cell Cytotoxic Assay Cells were seeded in 96-well plates at either 2,500 cells/well (MCF7 cells) or 4,000 cells/well (U87MG cells) and left to grow for 48 h in RPMI containing 10% foetal calf serum (FCS). Stock solutions of compound 1, 3, and paclitaxel (PTX) were prepared in DMSO and diluted to test concentrations in RPMI containing 10% FCS. Cells were exposed to a range of concentrations of test compound for 6 h in triplicate wells. Medium containing the compounds was then removed by aspiration and replaced with fresh RPMI containing 10% FCS. Cells were left to grow for 72 h. The number of cells in each well was then measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Cells convert the MTT reagent into formazan which can be detected at 562 nm in a microplate reader. The concentration of each compound that inhibits cell proliferation by 50% (IC₅₀) was calculated using Graph PAD PRISM. In addition, for each experiment a selectivity ratio was calculated for compound 3 by dividing the IC₅₀ for compound 3 with the paclitaxel IC₅₀. Each IC₅₀value and ratio is the average ± s.e. of 4 independent experiments. A one way ANOVA was performed using IBM SPSS Statistics version 24, to compare the selectivity ratios obtained in the different cell lines.
• 18 Lamidi OF. Sani M. Lazzari P. Zanda M. Fleming IN. J. Cancer Res. Clin. Oncol. 2015; 141: 1575
• 19 In Vitro Stability Assays All assays conducted by Cyprotex Ltd (Macclesfield, UK). Human and Rat Liver Microsomal Stability Assays Briefly: Test compound (3 μM) was incubated with pooled liver microsomes. Test compound was incubated at 5 time points over the course of a 45 min experiment, and the test compound was analysed by LC–MS/MS. Experimental Procedure Pooled human or mouse liver microsomes were purchased from a reputable commercial supplier. Microsomes were stored at –80 °C prior to use. Microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4, and test compound (final substrate concentration 3 μM; final DMSO concentration 0.25%) were pre-incubated at 37 °C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final incubation volume was 50 μL. A minus cofactor control incubation was included for each compound tested where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). Two control compounds were included with each species. All incubations were performed singularly for each test compound. Each compound was incubated for 0, 5, 15, 30, and 45 min. The control (minus NADPH) was incubated for 45 min only. The reactions were stopped by transferring 20 μL of incubate to 60 μL MeOH at the appropriate time points. The termination plates were centrifuged at 2,500 rpm for 20 min at 4 °C to precipitate the protein. Quantitative Analysis Following protein precipitation, the sample supernatants were combined in cassettes of up to 4 compounds, internal standard was added and samples analysed using Cyprotex generic LC–MS/MS conditions.