Letter pubs.acs.org/OrgLett
cis-Platinum Complex Encapsulated in Self-Assembling Cyclic Peptide Dimers Nuria Rodríguez-Vázquez,† Rebeca García-Fandiño,†,‡ María J. Aldegunde,† José Brea,§ María Isabel Loza,§ Manuel Amorín,† and Juan R. Granja*,† †
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ‡ CIQUP, Department of Chemistry and Biochemistry, Faculdade de Ciencias, Universidade do Porto, 4169-007 Porto, Portugal § Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS) and Department of Pharmacology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain S Supporting Information *
ABSTRACT: A new cyclic peptide dimer that encapsulates cisplatin complexes in its internal cavity is described. The resulting complex showed cytotoxic activity at A2780 ovarian cancer cell lines independent of acquired platinum resistance.
P
Scheme 1. (a) Cyclic Peptide Stacking To Form Dimers (D) or Peptide Nanotubes (SCPN). (b) Model of α,γ-Cyclic Peptide and (c) Interdimer Equilibria Derived from the Different Cross-Strain Interactions Denoted by the Different Colors of the Balls. (d) Retrosynthetic Strategy for the Preparation of CP2
latinum complexes are still among the most widely used anticancer drugs in the chemotherapy of a variety of cancers.1 These drugs have a nearly 100% cure rate for testicular cancer, but their use in the treatment of other cancers is limited by acquired resistance. These compounds have been used for more than 20 years, and some of the side effects can be severe, including damage to the kidneys and nervous system. In addition, the precise mode of action is not well understood.2 Platinum complexes suffer from a variety of deactivation mechanisms before reaching the cell nucleus, and this prevents them from triggering cell apoptosis. In general, these complexes react with nucleophilic components inside the cell, especially sulfur-donor-rich proteins such as human serum albumin3 or metallothioneins and glutathione,4 which sequester platinum complexes. The glutathione−platinum adducts are removed from the cytoplasm by means of export pumps. In fact, overexpression of these pumps has been implicated in resistance to these drugs.2,4 These issues have led to research aimed at finding new analogues or developing efficient drug delivery vectors that would alleviate these toxicity, degradation, and resistance issues. A variety of formulations have been described in recent years, including liposomes,5 polymers and dendrimers,6 macrocycles,7 nanoparticles,8 viruses,9 carbon nanotubes10 and supramolecular cages11 among others, but this problem has not been fully resolved yet.12 Peptides are a versatile tool for the development of new nanobiomaterials for novel biological applications due to their structural variety and modulability.13 Self-assembling cyclic peptide nanotubes (SCPNs) represent a new class of 1D materials with promising properties (Scheme 1).14 SCPNs consist of flat cyclic components that stack on top of each other. In the past few years, we have been working with © 2017 American Chemical Society
peptides that contain cyclic γ-amino acids.15,16 These residues gave rise to improved assembling properties and provided the opportunity to modify the internal characteristics of the assemblages.17 In this paper, we describe the design and Received: March 23, 2017 Published: May 4, 2017 2560
DOI: 10.1021/acs.orglett.7b00871 Org. Lett. 2017, 19, 2560−2563
Letter
Organic Letters synthesis of a new dimer-forming cyclic peptide that can encapsulate platinum complexes within its internal cavity. The resulting complex exhibited in vitro anticancer activity against ovarian cancer cells. The activity was retained against cisplatinresistant ovarian cell lines. Recently, we designed octapeptides that encapsulate metal ions, such as silver(I), through a pyridine moiety projected toward the dimer cavity17b or bipyridines within supramolecular capsules formed by CP−porphyrin hybrids. 18 For Pt encapsulation, we designed a decameric peptide bearing a carboxylate moiety linked to the β-carbon of one of the γresidues. Therefore, protected amino acid 1 was proposed (Scheme 1), and this compound could be obtained from the previously synthesized tetrahydrofuran derivative 2.17a,19 Finally, a self-assembling peptide was also designed to give a single dimer, which ensures the cis-geometry of the Pt complex.15 For this purpose, CP2, characterized by the presence of three consecutive γ-residues, was envisaged to fulfill all of these requirements. The proposed CP can only form the dimer in which the aminocyclohexyl residues (Ach) of each strand are hydrogen bonded. The CP incorporates a nonnatural amino acid, i.e., propargylglycine (Pra), to anchor a PEG chain to increase the water solubility of the peptide by means of Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) reaction.20 The synthesis of amino acid 1 was our first objective (Scheme 2). The treatment of compound 2 with sodium
Scheme 3. Structures of CPs, Dimers (Dn), and the Corresponding Platinum Complexes [Dn⊃Pt(en)] Together with Their Preparation Strategya
a
The arrows shown on Dn represent the interaction observed in NOE experiments.
Scheme 2. Synthesis of CP2
Figure 1. (a) 1H NMR and (b) selected NOESY spectra of D2 (5 mM in CDCl3) and (c) MALDI spectrum confirming the molecular weight of D5⊃Pt(en).
hydride in the presence of methyl bromoacetate provided 3 in 98% yield. Reduction of the azido group by palladium-catalyzed hydrogenation, followed by reaction with 2-nitrobenzenesulfonyl chloride and alkylation of the resulting nosylate using Fukuyama21 conditions provided 4 in 76% yield for the three steps. Hydrolysis of the acetal, followed by oxidation with diacetoxyiodobenzene/TEMPO and esterification, provided compound 1. The nosyl group was removed by the reaction with thiophenol in the presence of potassium carbonate to give compound 5. This compound was coupled with Boc-D-Ala using N-HATU22 to provide dipeptide dp1 in quantitative yield. Finally, this dipeptide and the others shown in Scheme S1 were transformed into CP2, after the basic hydrolysis of CP3 (Scheme 3), following the strategy illustrated in Scheme S1. The peptide CP3 formed the expected dimer D3 (Scheme 3) as confirmed by NMR (Figure 1a) due to the downfield shift of
the amide proton signals, which appear as five doublets. The NOE cross peaks (Figure 1b) between the amide proton of Ala1 and the NH of Ala3 (dark-blue color) and the Hγ of the Ahf residue (cyan color) confirmed the expected dimeric aggregate. Similar NMR results were obtained with the acid derivative CP2 (Figure S1), and these confirmed the formation of the corresponding D2 as a single dimeric structure. The next step was the incorporation of the platinum complex inside the dimer cavity (Scheme 3). Given the partial hydrophobic character of the dimer cavity and its internal diameter, the ethylenediamine complex was selected for this study rather than the diamine or the cyclohexanediamine moiety of the biologically relevant complexes. Reaction of the 2561
DOI: 10.1021/acs.orglett.7b00871 Org. Lett. 2017, 19, 2560−2563
Letter
Organic Letters
see Table 1. The cell viability inhibition was determined using the MTT system.25 The values obtained were compared with those corresponding to cisplatin. Additionally, the ethylenediamino complex 8 was also prepared (Scheme 3) and used also as a reference platinum complex. For the lung and breast cell cancer lines, D6⊃Pt(en) presented low activity at high concentration (100 μM). These values are comparable with those obtained for the sodium salt of cyclic peptide (CP6Na). Complex 8 presented IC50 activities around 50 μM, i.e., between 4 and 7 times less active than cisplatin in the breast and lung cancers. D6⊃Pt(en) was more active in A2780 cell line with an IC50 of 27 μM. The CP without the Pt (CP6Na) was inactive. The simplest oxalic acid derivative 8 was four times (IC50 of 6.9 μM) more active than the CP complex but still nine times less active than cisplatin (0.88 μM). Finally, a cisplatin-resistant ovarian cancer cell line (A2780cis) was also studied.26 Interestingly, the D6⊃Pt(en) complex maintained the activity observed in the nonresistant cell line, while complex 8 and cisplatin showed a significant reduction in potency. Thus, the encapsulation of complex 8 seems to be a useful approach to retain the potency of cytotoxic compounds in the treatment of drug-resistant cancer cell lines. Furthermore, the D6⊃Pt(en) complex did not show any cytotoxic effect when tested in a noncancer cell line (lung fibroblast MRC-5 cell line), while both complex 8 and cisplatin induced cell toxicity, thus suggesting that encapsulation may be a useful approach for treating cisplatin resistant cells without affecting nontumoral cells. In summary, we have prepared a new self-assembling cyclic decapeptide that contains a carboxylic acid group projected toward the internal cavity of the assembly. This peptide forms a dimer that has a cavity whose internal diameter and chemical properties allow the encapsulation of platinum complexes of ciscoordination. The CP-Pt(II) complex was less active than cisplatin in all the cell lines tested, showing cytotoxic activity against A2780 ovarian cancer cells. The complex retains its activity against the cisplatin resistant A2780cis ovarian cell line and does not show any cytotoxicity when tested in a nontumoral cell line. New studies to clarify the observed activity against one specific cell line and to elucidate the mechanism of mammalian cell protection are underway. The extension of this model to nanotube forming peptides might improve delivery and anticancer activity.
sodium salt of CP2 with the dinitro(ethylenediamine) platinum complex 6 (Scheme 3) did not provide the expected complex D2⊃Pt(en) due to the low solubility of the CP in aqueous media. Therefore, a polyethylene glycol oligomer was incorporated in the side chain of the Pra residue. Treatment of CP3 and azido derivative 7 with tetrakis(acetonitrile)copper(I), tris(benzyltriazolylmethyl)amine, and diisopropylethylamine provided CP4 in very good yield.21c,23 The 1H NMR spectrum confirmed the formation of D4 upon PEG incorporation (Figure S2). Hydrolysis under basic conditions followed by treatment of the resulting aqueous solution of the sodium salt (CP5) with 6 at 60 °C provided, after purification on Sephadex, complex D5⊃Pt(en), the molecular weight of which was confirmed by MALDI (Figure 1c). Finally, the trityl group was removed by reaction with 1% TFA in dichloromethane to provide complex D6⊃Pt(en). DFT calculations carried out with the M052X functional24 (see SI for details) provided a model of the dimeric structures D3 and D2⊃Pt(en). In both cases the dimer is slightly distorted and curved (Figure 2), with CO···HN distances in the
Figure 2. Top and lateral views of the computational models of D3 and D2⊃Pt(en) obtained by DFT calculations.
range 1.87−2.06 Å for D3 and 1.86−2.28 Å for D2⊃Pt(en). Two hydrogen bonds between the NH2 moieties of the ethane1,2-diamine and two carbonyl groups pointing out of the cavity are observed in D2⊃Pt(en) and these contribute to the stabilization of the dimeric ensemble (Figure S3). The Pt(en) does not fill the cavity completely, thus allowing the metal complex to flip between the two dimer faces in a way that might facilitate the delivery of metal (Figure S4). Finally, the resulting platinum complex was tested against three different cancer cell lines: human large-cell lung carcinoma line (NCI-H460), human breast adenocarcinoma cell line (MCF-7), and human ovarian cancer cell line (A2780);
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00871.
Table 1. Cytotoxic Activity of the Complexes and Cisplatin at the Five cell lines evaluateda IC50 (μM) complex
MCF-7b
NCI-H460c
8 D6⊃Pt(en) CP6Na cisplatin
47 ± 8.9 >100 (18%) >100 (13%) 13.0 ± 2.8
43 ± 6.3 >100 (15%) >100 (16%) 6.6 ± 0.3
A2780d 6.84 27.1 >100 0.88
± 0.6 ± 1.9 (24%) ± 0.1
A2780-cise
MRC-5f
21.2 ± 2.7 24.5 ± 2.3 >100 (1%) 5.42 ± 0.8
44 ± 6.7 >100 (27%) >100 (1%) 6.12 ± 0.4
a Values represent the mean ± SD of three assays (n = 3). The percentage of inhibition of cell viability observed at the highest concentration tested (100 μM) is shown in parentheses for those compounds that showed low cytotoxic activity. bHuman breast adenocarcinoma cell line. cHuman largecell lung carcinoma cell line. dHuman ovarian cancer cell line. eCisplatin resistant ovarian cancer cell line. fNontumoral lung fibroblast cell line.
2562
DOI: 10.1021/acs.orglett.7b00871 Org. Lett. 2017, 19, 2560−2563
Letter
Organic Letters
■
Synthetic procedures, additional scheme and figures, computational methods, and NMR and MS data (PDF)
(11) Lewis, J. E. M.; Gavey, E. L.; Cameron, S. A.; Crowley, J. D. Chem. Sci. 2012, 3, 778−784. (12) (a) Wang, X.; Guo, Z. Chem. Soc. Rev. 2013, 42, 202−224. (b) Harper, B. W.; Krause-Heuer, A. M.; Grant, M. P.; Manohar, M.; Garbutcheon-Singh, K. B.; Aldrich-Wright, J. R. Chem. - Eur. J. 2010, 16, 7064−7077. (13) (a) Pappas, C. G.; Shafi, R.; Sasselli, I. R.; Siccardi, H.; Wang, T.; Narang, V.; Abzalimov, R.; Wijerathne, N.; Ulijn, R. V. Nat. Nanotechnol. 2016, 11, 960−967. (b) Frederix, P. W. J. M.; Scott, G. G.; Abul-Haija, Y. M.; Kalafatovic, D.; Pappas, C. G.; Javid, N.; Hunt, N. T.; Ulijn, R. V.; Tuttle, T. Nat. Chem. 2015, 7, 30−37. (c) AdlerAbramovich, L.; Gazit, E. Chem. Soc. Rev. 2014, 43, 6881−6893. (14) (a) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988−1011. (b) Brea, R. J.; Reiriz, C.; Granja, J. R. Chem. Soc. Rev. 2010, 39, 1448−1456. (15) (a) Amorín, M.; Castedo, L.; Granja, J. R. J. Am. Chem. Soc. 2003, 125, 2844−2845. (b) Brea, R. J.; Castedo, L.; Granja, J. R. Chem. Commun. 2007, 3267−3269. (c) Amorín, M.; Castedo, L.; Granja, J. R. Chem. - Eur. J. 2005, 11, 6543−6551. (16) (a) Reiriz, C.; Brea, R. J.; Arranz, R.; Carrascosa, J. L.; Garibotti, A.; Manning, B.; Valpuesta, J. M.; Eritja, R.; Castedo, L.; Granja, J. R. J. Am. Chem. Soc. 2009, 131, 11335−11337. (b) Garcia-Fandino, R.; Amorín, M.; Castedo, L.; Granja, J. R. Chem. Sci. 2012, 3, 3280−3285. (c) Montenegro, J.; Vázquez-Vázquez, C.; Kalinin, A.; Geckeler, K. E.; Granja, J. R. J. Am. Chem. Soc. 2014, 136, 2484−2491. (d) Cuerva, M.; García-Fandiño, R.; Vázquez-Vázquez, C.; López-Quintela, M. A.; Montenegro, J.; Granja, J. R. ACS Nano 2015, 9, 10834−10843. (17) (a) Reiriz, C.; Amorín, M.; García-Fandiño, R.; Castedo, L.; Granja, J. R. Org. Biomol. Chem. 2009, 7, 4358−4361. (b) RodríguezVázquez, N.; García-Fandiño, R.; Amorín, M.; Granja, J. R. Chem. Sci. 2016, 7, 183−187. (18) Ozores, H.-L.; Amorín, M.; Granja, J. R. J. Am. Chem. Soc. 2017, 139, 776−784. (19) Rodríguez-Vázquez, N.; Salzinger, S.; Silva, L. F.; Amorín, M.; Granja, J. R. Eur. J. Org. Chem. 2013, 2013, 3477−3493. (20) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (21) (a) Fukuyama, T.; Cheung, M.; Jow, C. K.; Hidai, Y.; Kan, T. Tetrahedron Lett. 1997, 38, 5831−5834. (b) Fuertes, A.; Ozores, H. L.; Amorín, M.; Granja, J. R. Nanoscale 2017, 9, 748−753. (22) El-Faham, A.; Albericio, F. Chem. Rev. 2011, 111, 6557−6602. (23) Picot, S. C.; Mullaney, B. R.; Beer, P. D. Chem. - Eur. J. 2012, 18, 6230−6237. (24) Zhao, Y.; Schultz, N. E.; Truhlar, D. G. J. Chem. Theory Comput. 2006, 2, 364−382. (25) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (26) Behrens, B. C.; Hamilton, T. C.; Masuda, H.; Grotzinger, K. R.; Whang-Pen, J.; Louie, K. G.; Knutsen, T.; McKoy, W. M.; Young, R. C.; Ozols, R. F. Cancer Res. 1987, 47, 414−418.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Juan R. Granja: 0000-0002-5842-7504 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the Spanish Agencia Estatal de Investigació n (AEI), the ERDF (CTQ2013-43264-R, CTQ2016-78423-R, and CTQ2015-74621-JIN), and the Xunta de Galicia and the ERDF (EM2014/011 and Centro singular de investigación de Galicia accreditation 2016-2019, ED431G/09). N.R.-V. thanks the MECD and Gil Davila Foundation for her FPU contract and fellowship, respectively, and R.G.-F. thanks the FCT Investigator Program (Portugal) for a Starting Grant. We also thank the ORFEO-CINCA network and Mineco (CTQ2016-81797-REDC). All calculations were carried out in CESGA.
■
REFERENCES
(1) (a) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. Chem. Rev. 2016, 116, 3436−3486. (b) Johnstone, T. C.; Wilson, J. J.; Lippard, S. J. Inorg. Chem. 2013, 52, 12234−12249. (c) Kelland, L. Nat. Rev. Cancer 2007, 7, 573−584. (2) (a) Dhar, S.; Lippard, S. J. Structural and Mechanistic Studies of Anticancer Platinum Drugs: Uptake, Activation, and the Cellular Response to DNA Binding. In Platinum and Other Heavy Metal Compounds in Cancer Chemotherapy; Bonetti, A., Leone, R., Muggia, F. M., Howell, S. B., Eds.; Humana Press, 2009. (b) Casini, A.; Reedijk, J. Chem. Sci. 2012, 3, 3135−3144. (c) Mezencev, R. Curr. Cancer Drug Targets 2015, 14, 794−816. (3) Ivanov, A. I.; Christodoulou, J.; Parkinson, J. A.; Barnham, K. J.; Tucker, A.; Woodrow, J.; Sadler, P. J. J. Biol. Chem. 1998, 273, 14721− 14730. (4) Rabik, C. A.; Dolan, M. E. Cancer Treat. Rev. 2007, 33, 9−23. (5) (a) Wang, A.; Lin, W.; Liu, D.; He, C. Int. J. Nanomed. 2013, 8, 3309−3319. (b) Khiati, S.; Luvino, D.; Oumzil, K.; Chauffert, B.; Camplo, M.; Barthélémy, P. ACS Nano 2011, 5, 8649−8655. (6) (a) Surnar, B.; Jayakannan, M. Biomacromolecules 2016, 17, 4075−4085. (b) Rademaker-Lakhai, J. M.; Terret, C.; Howell, S. B.; Baud, C. M.; de Boer, R. F.; Pluim, D.; Beijnen, J. H.; Schellens, J. H. M.; Droz, J.-P. Clin. Cancer Res. 2004, 10, 3386−3395. (7) (a) Cao, L.; Hettiarachchi, G.; Briken, V.; Isaacs, L. Angew. Chem., Int. Ed. 2013, 52, 12033−12037. (b) Walker, S.; Oun, R.; McInnes, F. J.; Wheate, N. J. Isr. J. Chem. 2011, 51, 616−624. (c) Krause-Heuer, A. M.; Wheate, N. J.; Tilby, M. J.; Pearson, D. G.; Ottley, C. J.; AldrichWright, J. R. Inorg. Chem. 2008, 47, 6880−6888. (8) (a) Dhar, S.; Daniel, W. L.; Giljohann, D. A.; Mirkin, C. A.; Lippard, S. J. J. Am. Chem. Soc. 2009, 131, 14652−14653. (b) Brown, S. D.; Nativo, P.; Smith, J.-A.; Stirling, D.; Edwards, P. R.; Venugopal, B.; Flint, D. J.; Plumb, J. A.; Graham, D.; Wheate, N. J. J. Am. Chem. Soc. 2010, 132, 4678−4684. (9) Yang, Z.; Wang, X.; Diao, H.; Zhang, J.; Li, H.; Sun, H.; Guo, Z. Chem. Commun. 2007, 3453−3455. (10) (a) Dhar, S.; Liu, Z.; Thomale, J. R.; Dai, H.; Lippard, S. J. J. Am. Chem. Soc. 2008, 130, 11467−11476. (b) Li, J.; Yap, S. Q.; Chin, C. F.; Tian, Q.; Yoong, S. L.; Pastorin, G.; Ang, W. H. Chem. Sci. 2012, 3, 2083−2087. 2563
DOI: 10.1021/acs.orglett.7b00871 Org. Lett. 2017, 19, 2560−2563
