Linear Trifluoroethylamine RGD Peptidomimetics: Stereoselective Synthesis and Integrin αvβ3 Affinity

Monica Piras; Ian N. Fleming; William T. A. Harrison; Matteo Zanda

doi:10.1055/s-0032-1317557

Synlett, Table of Contents

Synlett 2012; 23(20): 2899-2902
DOI: 10.1055/s-0032-1317557

letter

Linear Trifluoroethylamine RGD Peptidomimetics: Stereoselective Synthesis and Integrin α_vβ₃ Affinity

Monica Piras

^aKosterlitz Centre for Therapeutics, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK Email: m.zanda@abdn.ac.uk

,

Ian N. Fleming

^bAberdeen Biomedical Imaging Centre, School of Medicine and Dentistry, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK

,

William T. A. Harrison

^cDepartment of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, Scotland, UK

,

Matteo Zanda*

^aKosterlitz Centre for Therapeutics, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK Email: m.zanda@abdn.ac.uk

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Abstract

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Abstract

The Arg-Gly-Asp (RGD) tripeptide is the smallest recognition sequence for the α_vβ₃ integrin receptor. A number of nonpeptide analogues of the RGD linear sequence, some of them having improved pharmacological properties, have been proposed in the last two decades. In this study, the stereogenic trifluoroethylamine function was employed as peptide bond surrogate for the development of a new class of RGD peptide mimics. In fact, the trifluoroethylamine group was shown to behave as a peptide-bond surrogate maintaining the H-bond donor capacity of the NH peptide group and a CH(CF₃)NH backbone angle close to the native peptide bond 120°. The trifluoroethylamine function is also known to display high metabolic stability and in some cases it positively affects ligand–receptor interactions. Two linear Ψ[CH(CF₃)NH]Gly-RGD diastereoisomers were synthesized with good stereocontrol and submitted to biological assays. A remarkable loss in α_vβ₃ integrin affinity was observed for both compounds, suggesting that the bulky CF₃ moiety is not well tolerated within the α_vβ₃ binding pocket.

Key words

trifluoroethylamine - peptidomimetics - RGD - integrins

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References

References and Notes
1 Ruoslahti E. J. Clin. Invest. 1991; 87: 1

2a Jin H, Varner J. Brit. J. Cancer 2004; 90: 561
2b Cai W, Chen X. Anticancer Agents Med. Chem. 2006; 6: 407

3 Ruoslahti E, Pierschbacher MD. Science 1987; 238: 491
4 Mas-Moruno C, Rechenmacher F, Kessler H. Anticancer Agents Med. Chem. 2010; 10: 753
5 Haubner R, Finsinger D, Kessler H. Angew. Chem., Int. Ed. Engl. 1997; 36: 1374
6 Sulyok GA, Gibson C, Goodman SL, Hölzemann G, Wiesner M, Kessler H. J. Med. Chem. 2001; 44: 1938

7a Sani M, Volonterio A, Zanda M. ChemMedChem 2007; 2: 1693
7b Meanwell NA. J. Med. Chem. 2011; 54: 2529
7c Tkachenko AN, Radchenko DS, Mykhailiuk PK, Shishkin OV, Tolmachev AA, Komarov IV. Synthesis 2012; 44: 903

8 Black WC, Bayly CI, Davis DE, Desmarais S, Falgueyret JP, Léger S, Li CS, Massé F, McKay DJ, Palmer JT, Percival MD, Robichaud J, Tsou N, Zamboni R. Bioorg. Med. Chem. Lett. 2005; 15: 4741
9 Gauthier JY, Chauret N, Cromlish W, Desmarais S, Duong LT, Falgueyret JP, Kimmel DB, Lamontagne S, Léger S, LeRiche T, Li CS, Massé F, McKay DJ, Nicoll-Griffith DA, Oballa RM, Palmer JT, Percival MD, Riendeau D, Robichaud J, Rodan GA, Rodan SB, Seto C, Thérien M, Truong VL, Venuti MC, Wesolowski G, Young RN, Zamboni R, Black WC. Bioorg. Med. Chem. Lett. 2008; 18: 923
10 Molteni M, Consonni R, Giovenzana T, Malpezzi L, Zanda M. J. Fluorine Chem. 2006; 127: 901
11 Molteni M, Bellucci MC, Bigotti S, Mazzini S, Volonterio A, Zanda M. Org. Biomol. Chem. 2009; 7: 2286
12 Aza-Michael Reaction of 1 with 2 To a stirred solution of 2 (1 mmol, 141 mg) and 1 (0.67 mmol, 189 mg) in dry toluene (10 ml) at r.t., DIPEA (0.74 mmol, 126 μL) was added. After 30 min at r.t. the mixture was concentrated, dissolved in EtOAc, and washed with 1 M HCl. The organic layers were dried over anhyd Na₂SO₄ and concentrated at reduced pressure. The crude was purified by flash chromatography (hexane–Et₂O = 9:1) affording 166 mg (64% yield) of the two pure diastereoisomers 3b and 3a in a 5:1 ratio. Compound 3b (major diastereoisomer): ¹H NMR (400 MHz, CDCl₃): δ = 4.72–4.62 (m, 2 H), 4.10–4.01 (m, 1 H), 3.70–3.62 (m, 1 H), 2.70–2.63 (dd, J = 16.2, 4.3 Hz, 1 H), 2.59–2.53 (dd, J = 16.2, 7.5 Hz, 1 H), 2.44–2.37 (m, 1 H), 1.45 (s, 18 H). ¹³C NMR (100 MHz, CDCl₃): δ = 172.26, 170.00, 124.70 (q, J = 284.3 Hz), 82.70, 81.72, 74.55, 58.09 (q, J = 30.1 Hz), 58.04, 40.15, 28.14, 28.04. ¹⁹F NMR (376 MHz, CDCl₃): δ = –75.31 (d, J = 7.2 Hz). ESI-MS: m/z calcd for C₁₅H₂₅F₃N₂O₆ [M + H]⁺: 387.2; found: 387.2. Compound 3a (minor diastereoisomer): ¹H NMR (400 MHz, CDCl₃): δ = 4.58 (dd, J = 13.2, 4.2 Hz, 1 H), 4.40 (dd, J = 13.2, 9.1 Hz, 1 H), 4.20–4.10 (m, 1 H), 3.68–3.76 (m, 1 H), 2.59 (dd, J = 16.5, 4.1 Hz, 1 H), 2.45 (dd, J = 16.5, 7.9 Hz, 1 H), 2.53–2.59 (m, 1 H), 1.46 (s, 9 H), 1.45 (s, 9 H). ¹³C NMR (100 MHz, CDCl₃): δ = 171.72, 169.97, 124.99 (q, J = 285.2 Hz), 82.66, 81.57, 74.63, 58.08 (q, J = 29.1 Hz), 58.09 (q, J = 30.1 Hz), 57.01, 40.40, 28.17, 28.01. ¹⁹F NMR (376 MHz, CDCl₃): δ = –74.33 (d, J = 6.9 Hz). ESI-MS: m/z calcd for C₁₅H₂₅F₃N₂O₆ [M + H]⁺: 386.2; found: 386.2.
13 CCDC 899221 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
14 Itoh J, Fuchibe K, Akiyama T. Angew. Chem. Int. Ed. 2008; 48: 4016
15 Aschi M, Lucente G, Mazza F, Mollica A, Morera E, Nalli M, Paglialunga Paradisi M. Org. Biomol. Chem. 2003; 1: 1980
16 RGD Mimic 8 ¹H NMR (400 MHz, D₂O): δ = 4.80–4.78 (m, 1 H), 4.31–4.28 (m, 1 H), 3.94 (t, J = 5.7 Hz, 1 H), 3.72–3.60 (m, 2 H), 3.51 (dd, J = 13.9, 6.4 Hz, 1 H), 3.22 (t, J = 6.7 Hz, 2 H), 2.50 (s, 3 H), 1.92–1.83 (m, 1 H), 1.78–1.57 (m, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 175.69, 174.65, 174.4, 162.94 (q, J = 35.4 Hz), 156.73, 125.29 (q, J = 282.4 Hz), 116.32 (q, J = 291.6 Hz), 57.49 (q, J = 27.5 Hz), 56.56, 53.56, 40.44, 37.64, 36.97, 28.04, 24.38, 21.67. ¹⁹F NMR (376 MHz, D₂O): δ = –74.15 (d, J = 7.2 Hz), –75.58 (s). ESI-MS: m/z calcd for C₁₅H₂₅F₃N₆O₆ [M + H]⁺: 443.2; found: 443.2. RGD Mimic 9 ¹H NMR (400 MHz, D₂O): δ = 4.80–4.78 (m, 1 H), 4.28–4.25 (m, 1 H), 4.04 (t, J = 6.1 Hz, 1 H), 3.71–3.60 (m, 2 H), 3.55–3.50 (m, 1 H), 3.21 (t, J = 6.7 Hz, 2 H), 2.93 (dd, J = 17.1, 5.3 Hz, 1 H), 2.83 (dd, J = 17.1, 6.9 Hz, 1 H), 2.04 (s, 3 H), 1.89–1.80 (m, 1 H), 1.77–1.55 (m, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 175.13, 174.4, 162.92 (q, J = 35.5 Hz), 156.72, 125.08 (q, J = 283.0 Hz), 116.30 (q, J = 291.7 Hz), 57.26 (q, J = 27.9 Hz), 55.97, 53.59, 40.41, 37.17, 36.72, 28, 24.36, 21.62. ¹⁹F NMR (376 MHz, D₂O): δ = –73.92 (d, J = 7.1 Hz), –75.59 (s). ESI-MS: m/z calcd for C₁₅H₂₅F₃N₆O₆ [M + H]⁺: 443.2; found: 443.2.
17 Isolated α_vβ₃ Binding Assay Inhibitory effects of test molecules were quantified by measuring their effects on the interaction between immobilized α_vβ₃ integrin (Millipore) and biotinylated CRGDfK-(PEG)2 (Pepnet), essentially as described (reference: Mould AP, Current Protocols in Cell Biology, 2011, Chapter 9, Unit 9.4; Analysing Integrin-Dependent Adhesion). The 96-well microtiter plates were coated with purified α_vβ₃ integrin (0.5 μg/mL in PBS, pH 7.2) overnight at 4 °C, then blocked with 5% (w/v) bovine serum albumin (BSA) in 25 mM Tris, pH 7.4 and 150 mM NaCl for 1 h. Integrin was incubated with biotinylated CRGDfK-(PEG)2 (100 nM) in binding buffer (0.1% BSA, 25 mM Tris, pH 7.4, 150 mM NaCl, and 1 mM MnCl₂) for 3 h at r.t. in the presence or absence of test molecules. After 3 washes with binding buffer, wells were incubated for 30 min at r.t. with ExtraAvidin-peroxidase reagent (Sigma) diluted 1:1000 in binding buffer. Wells were washed four times with binding buffer and bound biotinylated ligand detected using 3′3′5′5-tetramethylbenzine (Sigma) as chromogen. Assay was stopped with 50 μL 1 M HCl and absorbance at A450 measured in a microtiter plate reader. Biotin-CRGDfK-(PEG)2 binding in the absence of test compounds was defined as 100% of signal. Binding to blocked wells in the absence of integrin was defined as 0%. Signal-to-noise ratio was >10. Concentrations of test compounds required for 50% inhibition of signal (IC₅₀ values) were calculated using GraphPad Prism. Assays were performed in triplicate. The linear heptapeptide (Gly-Arg-Gly-Asp-Ser-Pro-Lys) was included as external reference and IC₅₀ values for test compounds were normalized to the reference IC₅₀ to allow comparisons between different experiments, as described in: Haubner R, Wester HJ, Reuning U, Senekowitsch-Schmidtke R, Diefenbach B, Kessler H, Stocklin G, Schwaiger M. J. Nucl. Med. 1999; 40: 1061
18 McCusker CF, Kocienski PJ, Boyle FT, Schätzlein AG. Bioorg. Med. Chem. Lett. 2002; 12: 547
19 Dijkgraaf I, Kruijtzer JA. W, Liu S, Soede AC, Oyen WJ. G, Corstens FH. M, Liskamp RM. J, Boerman OC. Eur. J. Nucl. Med. 2007; 34: 267
20 Hautanen A, Gailit J, Mann DM, Ruoslahti E. J. Biol. Chem. 1989; 264: 1437