Synthesis and Cytotoxicity Evaluation of Tyrosine-5 Fluorinated Analogues of RA-VII, An Antitumor Bicyclic Hexapeptide

Abstract

RA-VII analogues fluorinated at each ε-position of Tyr-5 were designed. The synthesis of these peptide analogues commenced with the preparation of atropisomeric fluorocycloisodityrosines from protected 3-fluoro-L-tyrosyl-3-boronyl-L-tyrosine mediated by copper(II) acetate and 4-(dimethylamino)pyridine. After N-methylation, the tetrapeptide segment was coupled with the N-terminus of each fluorocycloisodityrosine to afford a hexapeptide. After removal of the C- and N-terminal protecting groups, each peptide was subjected to macrocyclization to produce an analogue. The analogue with a β-oriented fluorine atom was equipotent to RA-VII with regard to cytotoxicity toward human mammary carcinoma MCF-7 cells, and showed 2.1-fold and 1.4-fold lower cytotoxicities than RA-VII toward human promyelocytic leukemia HL-60 and human colorectal cancer HCT-116 cells, respectively, whereas the analogue with an α-oriented fluorine atom showed 7.7-fold, 6.0-fold, and 14-fold lower cytotoxicities than RA-VII toward those cells, respectively.

Key words

RA-VII analogues - peptides - macrocyclic peptides - medicinal chemistry - cytotoxicity - Rubia cordifolia

Publication History

Received: 30 April 2023

Accepted after revision: 24 May 2023

Article published online:
10 July 2023

• References and Notes

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• 23 12a and 12b To a solution of 9 (16.0 mg, 0.0299 mmol) in CH₂Cl₂ (30 mL) were added DMAP (43.9 mg, 0.359 mmol) and powdered 4 Å MS (200 mg), and the mixture was stirred at rt for 30 min. Cu(OAc)₂ (65.2 mg, 0.359 mmol) was added and the resultant mixture was stirred at rt for 72 h. The mixture was then centrifuged and the supernatant was transferred to an InertSep PRS column (2 g/2 mL) and eluted with 1:1 EtOAc–CHCl₃ (30 mL). The eluate was concentrated under reduced pressure, and the residue was separated by HPLC (H₂O–MeOH, 50:50) to afford 12a and 12b as colorless amorphous solids. 12a Yield: 5.8 mg (40%); [α]_D ²⁵ –31.3 (c 0.41, MeOH). IR (film): 3312, 1764, 1710, 1664, 1518, 1501, 1260, 1166 cm^–1. ¹H NMR (500 MHz, CDCl₃) δ = 1.46 (s, 9 H), 2.82 (overlapped, 1 H), 2.82 (dd, J = 16.5, 1.6 Hz, 1 H), 2.88 (dd, J = 16.5, 8.0 Hz, 1 H), 3.40 (dd, J = 13.2, 4.8 Hz, 1 H), 3.56 (s, 3 H), 3.92 (s, 3 H), 4.21 (m, 1 H), 4.41 (m, 1 H), 5.28 (br s, 1 H), 5.32 (br s, 1 H), 5.92 (br s, 1 H), 6.60 (dd, J = 8.2, 1.9 Hz, 1 H), 6.77 (d, J = 8.2 Hz, 1 H), 7.06–7.18 (m, 3 H). ¹³C NMR (125 MHz, CDCl₃) δ = 28.2 (CH₃, 3 C), 34.4 (CH₂), 38.9 (CH₂), 52.2 (CH₃), 53.8 (CH), 56.2 (CH₃), 57.3 (CH), 81.0 (C), 112.3 (CH), 114.8 (CH), 119.1 (CH), 122.6 (CH), 126.8 (CH), 127.1 (CH), 129.0 (C), 136.0 (C), 144.8 (C), 147.2 (C), 150.9 (C), 154.9 (C), 156.4 (CF), 170.4 (C), 170.6 (C). HR-ESI-MS: m/z [M + Na]⁺ calcd for C₂₅H₂₉FN₂NaO₇; 511.1856; found: 511.1857. 12b Yield: 2.4 mg (16%); [α]_D ²⁵ +38.7 (c 0.21, MeOH). IR (film): 3352, 1751, 1716, 1670, 1519, 1503, 1261, 1164 cm^–1. ¹H NMR (500 MHz, CDCl₃) δ = 1.49 (s, 9 H), 2.76–2.90 (m, 3 H), 3.45 (dd, J = 13.4, 4.8 Hz, 1 H), 3.65 (s, 3 H), 3.94 (s, 3 H), 4.22 (m, 1 H), 4.58 (m, 1 H), 4.96 (d, J = 8.7 Hz, 1 H), 5.28 (s, 1 H), 6.03 (brd, J = 6.4 Hz, 1 H), 6.66 (dd, J = 8.2, 1.8 Hz, 1 H), 6.80 (d, J = 8.2 Hz, 1 H), 7.04 (d, J = 8.3 Hz, 1 H), 7.08 (d, J = 11.1 Hz, 1 H), 7.17 (t, J = 8.0 Hz, 1 H). ¹³C NMR (125 MHz, CDCl₃) δ = 28.3 (CH₃, 3 C), 34.8 (CH₂), 38.5 (CH₂), 52.4 (CH₃), 53.9 (CH), 56.3 (CH₃), 56.5 (CH), 81.4 (C), 112.3 (CH), 113.4 (CH), 120.5 (CH), 122.6 (CH), 124.6 (CH), 125.8 (CH), 130.0 (C), 135.9 (C), 144.9 (C), 147.1 (C), 150.7 (C), 154.7 (C), 156.7 (CF), 170.6 (C), 171.3 (C). HR-ESI-MS: m/z [M + Na]⁺ calcd for C₂₅H₂₉FN₂NaO₇; 511.1856; found: 511.1857.
• 24 4a and 4b A LiOOH solution [LiOH·H₂O (3.9 mg, 0.093 mmol) dissolved in 35% aq H₂O₂ (0.1 mL) and MeOH (0.2 mL)] was slowly added to a cooled (0 °C) solution of 15a (14.0 mg, 0.0152 mmol) in MeOH (0.1 mL), and the resulting mixture was stirred at 0 °C for 48 h. A 10% aq solution of citric acid (1 mL) and 5% aq NaHSO₃ (0.5 mL) were added at 0 °C, and the resulting mixture was stirred for 15 min. The mixture was then extracted with EtOAc (3 × 1.5 mL), and the combined organic layers were washed with sat. aq NaCl (1 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was dissolved in CHCl₃ (2 mL) and treated by slow addition of TFA (2 mL) at 0 °C, and the resultant solution was stirred at 0 °C for 30 min and then at rt for 2 h. Volatiles were removed under reduced pressure, and the residue was passed through a Bond Elut DEA column (500 mg/3 mL) using MeOH (30 mL) as the eluent. The eluate was concentrated to dryness under reduced pressure, and the residue was dissolved in DMF (12 mL). Et₃N (21 μL, 0.15 mmol) and DPPA (6.5 μL, 0.030 mmol) were added at 0 °C, and the solution was stirred under argon at 0 °C for 4 d. The solvent was removed under reduced pressure, and the residue was dissolved in CHCl₃ (4 mL). The solution was washed sequentially with sat. aq NaHCO₃ (1 mL) and sat. aq NaCl (1 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by HPLC (H₂O–MeCN, 55:45) to give 4a as a colorless amorphous solid; yield: 3.7 mg (31%); [α]_D ²⁵ –213 (c 0.17, MeOH). IR (film): 3387, 1661, 1633, 1514 cm^–1. ¹H NMR (600 MHz, CDCl₃) (major conformer): δ = 1.12 (d, J = 6.7 Hz, 3 H), 1.31 (d, J = 7.0 Hz, 3 H), 1.36 (d, J = 6.9 Hz, 3 H), 2.61 (dd, J = 11.4, 3.0 Hz, 1 H), 2.69 (s, 3 H), 2.85 (s, 3 H), 3.01 (dd, J = 17.8, 4.0 Hz, 1 H), 3.09 (dd, J = 17.8, 11.9 Hz, 1 H), 3.12 (s, 3 H), 3.36 (dd, J = 14.1, 10.9 Hz, 1 H), 3.39 (dd, J = 14.1, 4.8 Hz, 1 H), 3.58 (dd, J = 10.9, 4.8 Hz, 1 H), 3.65 (t, J = 11.4 Hz, 1 H), 3.79 (s, 3 H), 3.95 (s, 3 H), 4.35 (quintet, J = 6.9 Hz, 1 H), 4.58 (dd, J = 11.9, 4.0 Hz, 1 H), 4.61 (d, J = 1.8 Hz, 1 H), 4.75 (dq, J = 7.7, 6.7 Hz, 1 H), 4.85 (dq, J = 8.5, 6.9 Hz, 1 H), 5.41 (dd, J = 11.4, 3.0 Hz, 1 H), 6.05 (d, J = 8.5 Hz, 1 H), 6.41 (d, J = 6.7 Hz, 1 H), 6.65 (dd, J = 8.3, 1.8 Hz, 1 H), 6.69 (d, J = 7.7 Hz, 1 H), 6.837 (app d, J = 8.6 Hz, 2 H), 6.843 (d, J = 8.3 Hz, 1 H), 7.05 (app d, J = 8.6 Hz, 2 H), 7.06 (dd, J = 11.2, 2.0 Hz, 1 H), 7.18 (dd, J = 8.4, 2.0 Hz, 1 H), 7.28 (t, J = 8.2 Hz, 1 H). ¹³C NMR (150 MHz, CDCl₃) (major conformer): δ = 16.7 (CH₃), 18.5 (CH₃), 20.7 (CH₃), 29.4 (CH₃), 30.5 (CH₃), 32.7 (CH₂), 35.8 (CH₂), 37.0 (CH₂), 39.7 (CH₃), 44.6 (CH), 46.4 (CH), 47.9 (CH), 54.1 (CH), 55.3 (CH₃), 56.4 (CH₃), 57.4 (CH), 68.4 (CH), 112.3 (CH), 113.0 (CH), 114.1 (CH, 2C), 120.6 (CH), 122.0 (CH), 125.9 (CH), 127.8 (CH), 128.1 (C), 130.3 (CH, 2C), 130.7 (C), 137.6 (C), 145.1 (C), 147.1 (C), 151.8 (C), 156.1 (C), 158.4 (C), 168.0 (C), 169.4 (C), 170.6 (C), 171.8 (C), 172.2 (C), 172.4 (C). ¹⁹F NMR (471 MHz, CDCl₃) (major conformer): δ = –129.8 (dd, J = 11.2, 8.0 Hz). HR-ESI-MS: m/z [M + H]⁺ calcd for C₄₁H₅₀FN₆O₉: 789.3623; found: 789.3621. 4b Prepared from 15b (9.6 mg, 0.0104 mmol) by using the same procedure as that described for the synthesis of 4a as a colorless amorphous solid; yield: 2.4 mg (29%); [α]_D ²⁵ –203 (c 0.12, MeOH). IR (film): 3387, 1666, 1633, 1514 cm^–1. ¹H NMR (600 MHz, CDCl₃) (major conformer): δ = 1.13 (d, J = 6.7 Hz, 3 H), 1.32 (d, J = 6.9 Hz, 3 H), 1.36 (d, J = 6.9 Hz, 3 H), 2.61 (dd, J = 11.4, 3.2 Hz, 1 H), 2.71 (s, 3 H), 2.86 (s, 3 H), 2.99 (dd, J = 18.2, 3.6 Hz, 1 H), 3.12 (s, 3 H), 3.15 (dd, J = 18.2, 12.1 Hz, 1 H), 3.36 (dd, J = 14.0, 10.9 Hz, 1 H), 3.39 (dd, J = 14.0, 4.9 Hz, 1 H), 3.58 (dd, J = 10.9, 4.9 Hz, 1 H), 3.66 (t, J = 11.4 Hz, 1 H), 3.79 (s, 3 H), 3.94 (s, 3 H), 4.35 (quintet, J = 6.8 Hz, 1 H), 4.56 (d, J = 2.0 Hz, 1 H), 4.59 (dd, J = 12.1, 3.6 Hz, 1 H), 4.74 (dq, J = 7.7, 6.7 Hz, 1 H), 4.87 (dq, J = 8.5, 6.9 Hz, 1 H), 5.49 (dd, J = 11.4, 3.2 Hz, 1 H), 6.02 (d, J = 8.5 Hz, 1 H), 6.44 (d, J = 6.6 Hz, 1 H), 6.64 (dd, J = 8.3, 2.0 Hz, 1 H), 6.69 (d, J = 7.7 Hz, 1 H), 6.84 (app. d, J = 8.6 Hz, 2 H), 6.84 (overlapped, 1 H), 6.95 (t, J = 8.1 Hz, 1 H), 7.05 (app. d, J = 8.6 Hz, 2 H), 7.06 (dd, J = 8.1, 1.8 Hz, 1 H), 7.24 (dd, J = 11.7, 1.8 Hz, 1 H). ¹³C NMR (150 MHz, CDCl₃) (major conformer): δ = 16.7 (CH₃), 18.5 (CH₃), 20.7 (CH₃), 29.3 (CH₃), 30.5 (CH₃), 32.7 (CH₂), 35.4 (CH₂), 37.0 (CH₂), 39.8 (CH₃), 44.6 (CH), 46.4 (CH), 47.9 (CH), 54.0 (CH), 55.3 (CH₃), 56.3 (CH₃), 57.6 (CH), 68.4 (CH), 111.8 (CH), 112.7 (CH), 114.1 (CH, 2C), 118.8 (CH), 121.8 (CH), 126.4 (CH), 128.1 (CH), 128.6 (C), 130.3 (CH, 2C), 130.7 (C), 137.2 (C), 144.7 (C), 146.7 (C), 151.8 (C), 158.0 (C), 158.4 (C), 168.0 (C), 169.2 (C), 170.5 (C), 171.9 (C), 172.1 (C), 172.4 (C). ¹⁹F NMR (471 MHz, CDCl₃) (major conformer): δ = –132.9 (t, J = 9.2 Hz). HR-ESI-MS: m/z [M + H]⁺ calcd for C₄₁H₅₀FN₆O₉: 789.3623; found: 789.3618.
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