Article pubs.acs.org/Biomac

Supramolecular Glycodendrimer-Based Hybrid Drugs Marco Paolino,*,† Hartmut Komber,‡ Laura Mennuni,§ Gianfranco Caselli,§ Dietmar Appelhans,‡ Brigitte Voit,‡ and Andrea Cappelli† †

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy ‡ Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany § Rottapharm Biotech S.r.l., Via Valosa di Sopra 3, 20900 Monza, Italy ABSTRACT: Specific noncovalent interactions are commonly used by nature to modulate numerous processes including cell recognition, viral adhesion, and transmembrane communications. Here we report on the design, synthesis, and preliminary characterization of new supramolecular glycodendrimer-based hybrid drugs based on adamantyl-modified glycodendrimers of third, fourth, or fifth generation (mPPI-G3-AdaB, mPPI-G4-AdaB, and mPPI-G5-AdaB) and a new heterobifunctional ligand. This component was tailored to bind through noncovalent interactions both the multimeric natural 5-HT3 receptor (through an optimized arylpiperazine pharmacophore) and the adamantyl groups located on the glycodendrimer surfaces (through a β-cyclodextrin residue) giving rise to biorelevant supramolecular constructs.

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INTRODUCTION In contrast to traditional chemistry, based on strong and irreversible covalent bonds, supramolecular chemistry exploits weak and reversible noncovalent interactions to create complex functional structures.1,2 Multiple supramolecular interactions are involved in many natural processes to generate exact structural forms of biological macromolecules (proteins, enzymes, receptors, DNA) or to ensure specific functions in living organisms (e.g., DNA replication, catalysis by enzyme− substrate recognition, transmembrane transductions mediated by ligands).1,3 These specific processes, meticulously carried out in living systems, had always been source of study and inspiration for chemists. With the advent of supramolecular chemistry, ever more efficient supramolecular systems with applications in the fields of nanotechnology, catalysis, medicine, and pharmaceuticals have been achieved.4,5 Different types of interactions have been exploited to build supramolecular systems and great importance was attributed to the specific interactions generated through donor−acceptor hydrogen bonds,6,7 π-stacking,8−10 molecular recognition, or inclusion complexes.11−14 Dendrimers are macromolecules that are extensively studied in the context of supramolecular chemistry because their exact shape and size allow precise functional nanoscale systems to be obtained.15 They are frequently employed as nanoscale containers able to noncovalently accommodate small molecules either inside of the dendritic scaffold or on their surface16 or as nanoscaffolds for the covalent conjugation of functional molecules.17,18 In recent years, our research team has developed interesting glycodendrimers based on poly(propyleneimine) (PPI) den© 2014 American Chemical Society

drimers as core scaffolds decorated on their surface with maltose or maltotriose molecules. The glycoshell on the dendrimer surface significantly decreases the cell toxicity of PPI dendrimers19−23 and makes them excellent candidates for the development of drug carriers.24−26 Moreover, owing to the high propensity to establish hydrogen bonds on the outer shell, dense shell maltosemodified PPI dendrimers can effectively interfere with prion proteins and Alzheimer’s amyloid Aβ(1−40) peptide formation and could be considered as potential antiamyloidogenic agents.21−23,27−29 Recently, we reported a supramolecular study focused on the interaction between dendrimers with a hybrid surface (maltose−adamantane residues) and β-cyclodextrin (β-CD) in monomeric and polymeric form.30 These studies showed the ability of β-CD to effectively capture the adamantyl groups placed on the relatively congested surfaces of lower generation glycodendrimers generating a dynamic dense sugar shell. The supramolecular behavior of our adamantyl-modified glycodendrimers taken together with their water solubility and high biocompatibility led us to envision their use as hosts in potentially bioactive supramolecular systems. The 5-HT3 receptor (5-HT3R) is a ligand-gated ion channel (LGIC) permeable to sodium, potassium, and calcium ions composed of five subunits in different arrangements. The homopentameric form (composed of 5-HT3A subunits only) is a functional receptor showing some differences with respect to Received: July 20, 2014 Revised: September 30, 2014 Published: October 6, 2014 3985

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out with a Bruker IFS66 spectrometer equipped with a heatable Golden Gate Diamond ATR-Unit (SPECAC). Synthesis. 3-Methyl-2-(4-methylpiperazin-1-yl)-N-(31-oxo3,6,9,12,15,18,21,24,27-nonaoxa-30,32-diazapentatriacont-34ynyl)quinoline-4-carboxamide (4). A mixture of amine 339 (0.17 g, 0.23 mmol) in CHCl3 (10 mL) with 4-nitrophenyl prop-2ynylcarbamate40 (0.052 g, 0.23 mmol) and TEA (1.0 mL) was stirred at room temperature under a nitrogen atmosphere for 24 h. The reaction mixture was then concentrated under reduced pressure, and the resulting residue was purified by flash chromatography with ethyl acetate−TEA−methanol (7:2:1) as the eluent to give pure 4 as a yellow oil (0.12 g, yield 65%). 1H NMR (400 MHz, CDCl3): 2.16 (t, 4 J = 2.3, 1H, H21), 2.38 (s, 3H, H5′), 2.41 (s, 3H, H11), 2.62 (m, 4H, H3′), 3.32 (m, 6H, H2′, H17), 3.4−3.7 (34H, H15, H16), 3.75 (m, 4H, H13, H14), 3.95 (dd, 4J = 2.4, 3J = 7.9, 2H, H19), 5.51 and 5.54 (2 t, 2H, -NH-CO-NH-), 6.73 (t, 1H, -NH-CO-), 7.35 (t, 1H, H6), 7.55 (t, 1H, H7), 7.68 (d, 1H, H5), 7.83 (d, 1H, H8). 1H NMR (400 MHz, DMSOd6): 2.29 (s, 3H, H5′), 2.30 (s, 3H, H11), 2.57 (m, 4H, H3′), 3.02 (t, 1H, 4J = 2.2, H21), 3.14 (q, 2H, H17), 3.22 (m, 4H, H2′), 3.37 (t, 2H, H16; overlaps with H2O), 3.45−3.58 (34H, H15, H13), 3.60 (m, 2H, H14), 3.77 (dd, 4J = 2.2, 3J = 5.6, 2H, H19), 6.00 (t, 1H, (OEG)-NHCO-NH-), 6.25 (t, 1H, -NH-CO-NH-CH2-), 7.39 (t, 1H, H6), 7.59 (t, 1H, H7), 7.62 (d, 1H, H5), 7.75 (d, 1H, H8), 8.66 (t, 1H, -NH-CO-). 13 C NMR (100 MHz, DMSO-d6): 15.4, 28.8, 38.7, 39.6, 45.7, 49.3, 54.6, 68.9, 69.6, 69.8, 70.1, 72.5, 82.6, 120.7, 121.9, 124.6, 124.7, 127.3, 128.8, 145.0, 145.1, 157.5, 160.7, 167.0. MS(ESI): m/z 827 (M + Na+).

the heteropentameric form obtained by the coexpression of the 5-HT3B subunits together with 5-HT3A.31,32 Pharmaceutical industry has produced a large number of monovalent 5-HT3R antagonists showing remarkable efficacy in preventing acute chemotherapy-induced nausea and vomiting (CINV). Moreover, a potential usefulness has been suggested for these compounds in peripheral inflammatory diseases (such as asthma, rheumatoid diseases and irritable bowel syndrome) and central nervous system (CNS) disorders, including Alzheimer’s disease (AD).33,34 Indeed, 5-HT3R antagonists improved acetylcholine (ACh) release in cortical tissue by reducing the tonic inhibitory effect of 5-HT, and this facilitation of cholinergic functions could account for the preclinical and clinical efficacy of ondansetron in ameliorating age-related memory impairment.35,36 Our major goals in the exploration of the 5-HT3R started from the development of arylpiperazine derivatives related to quipazine showing high affinity and different intrinsic efficacies.37,38 Subsequently, on the basis of the optimized arylpiperazine moiety MPQC (methyl-piperazinyl-quinolinecarboxamide), we have designed multivalent ligands differing in their assembly. In particular, we developed homobivalent and homotetravalent ligands capable of highlighting the multivalent nature of the 5-HT3R39,40 and heterobivalent ligands bearing tacrine (an acetylcholinesterase inhibitor) that showed both high 5-HT3R affinity and potent acetylcholinesterase (AChE) inhibitory activity (Figure 1). These compounds, possessing the dual activity, could be considered interesting candidates for restoring the normal cholinergic tone in AD patients.41 In the present work, we introduce the design and construction of supramolecular glycodendrimer-based hybrid drugs constituted from two components: (1) a poly(propyleneimine) dendrimer in which the terminal amino groups have been appropriately substituted with maltose molecules and adamantyl residues and (2) a bioactive molecule appropriately designed to interact with both the 5-HT3R and the adamantyl groups of the dendrimeric macromolecule. We expected that the combination of the interesting features shown by glycodendrimers (i.e., water solubility, high biocompatibility, and intrinsic pharmacological activity) with the affinity of the new heterobifunctional ligand for the natural 5-HT3 receptor in a conjugate could give rise to potentially biorelevant supramolecular constructs.

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Synthesis of the Heterobifunctional Ligand 1. In a microwave tube, a mixture of β-CD-N3 (synthesized as reported in ref 42; 0.10 g, 0.086 mmol) in DMF (5.0 mL) containing propargyl urea 4 (0.070 g, 0.087 mmol), CuBr (0.006 g, 0.042 mmol), and DIPEA (7.3 μL, 0.042 mmol) was exposed to microwave irradiation into a CEM Discover instrument for 75 min (5 × 15 min, T = 85 °C, W = 200). The reaction mixture was filtered, and the resulting clear solution was concentrated under reduced pressure. The resulting oily residue was dissolved in water (1.0 mL) and purified by reverse phase chromatography (ISOLUTE C-18 cartridge, Biotage) using a water− ethanol (1:1) mixture as the eluent. After concentration under reduced pressure, the residue was redissolved in water and precipitated with acetone to obtain a whitish solid (0.051 g, yield 30%). 1H NMR (400 MHz, DMSO-d6): 2.26 (s, 3H, H5′), 2.30 (s, 3H, H11), 2.54 (m, 4H, H3′), 2.88 and 3.10 (2H, H6CD of the glucose unit bonded to C1′CD), 3.17 (q, 2H, H17), 3.20 (m, 4H, H2′), 3.2−3.8 (H2′CD-H4′CD, H2CDH6CD, H13-H16; overlaps with H2O), 3.96 (m, 1H, H5′CD), 4.21 (d, 2H, H19), 4.3−4.6 (CH2-OH), 4.54 and 4.85 (2H, H6′CD), 4.7−5.1 (7H, H1CD, H1′CD), 5.6−5.9 (CH-OH), 6.04 (t, 1H, (OEG)-NH-CO-NH-), 6.35 (t, 1H, -NH-CO-NH-CH2-(triazole)), 7.39 (t, 1H, H6), 7.59 (t, 1H, H7), 7.62 (d, 1H, H5), 7.75 (d, 1H, H8), 7.82(s, 1H, H21), 8.65 (t, 1H, -NH-CO-). 13C NMR (400 MHz, DMSO-d6): 15.4, 35.1, 38.8, 39.5, 46.0, 49.4, 50.4, 54.8, 59.1, 60.1, 68.9, 69.7, 69.9, 70.1, 72.2, 72.3, 72.5, 72.8, 73.1, 81.2, 81.5, 83.5, 102.0, 102.3, 120.7, 121.9, 124.2, 124.6, 124.7, 127.6, 128.8, 144.9, 145.0, 158.0, 160.8, 167.1. MS(ESI): m/z 982.4 (M + 2H+). Synthesis of the Adamantyl-Modified Glycodendrimers. The synthesis of the adamantyl-modified glycodendrimer mPPI-G3-AdaB

EXPERIMENTAL SECTION

Materials and Methods. All chemicals used were of reagent grade. Third, fourth, and fifth generation poly(propyleneimine) dendrimers PPI-G3 (M = 1686.8 g/mol), PPI-G4 (M = 3513.9 g/ mol), and PPI-G5 (M = 7168.1 g/mol) were used as received from Symo-Chem (Eindhoven, The Netherlands) as DAB-Am-16, DABAm-32, and DAB-Am-64, respectively. Membrane tubes (ZelluTransRoth VSerie with 1000 MWCO, Carl Roth GmbH&Co, Karlsruhe/ Germany) for dialysis were used after washing with deionized water. Yields refer to purified products and are not optimized. Merck silica gel 60 (230−400 mesh) or aluminum oxide 90 active neutral (70−230 mesh) was used for column chromatography. Merck TLC plates, silica gel 60 F254, were used for TLC. NMR spectra were recorded with a Bruker DRX-400 AVANCE, or Bruker DRX-500 AVANCE spectrometer in CDCl3 (δ(1H) = 7.25 ppm), DMSO-d6 (δ(1H) = 2.50 ppm; δ(13C) = 39.6 ppm), and D2O.30 The chemical shift values are expressed in ppm and the coupling constants (J) in Hz. An Agilent 1100 LC/MSD operating with an electrospray source was used in mass spectrometry experiments. Laser-induced liquid bead ionization/ desorption mass spectrometry (LILBID-MS) measurements were carried out as described in ref 25. The IR investigations were carried 3986

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conversion (25%). 1H NMR (500 MHz, CDCl3): 1.39 (a), 1.45−1.7 (d, g, j), 1.65 (z), 1.96 (x), 2.04 (y), 2.3−2.55 (b, c, e, f, h, i), 2.72 (k in I), 3.13 (k in II), 4.7−5.3 (br, NH bonded to Ada), 5.3−6.0 ppm (two broad overlapping signals at 5.5 and 5.8 ppm; NH bonded to PPI-G3). IR: 3310 (NH2), 2903, 2848, 2800 (CH, CH2), 1636 (CO), 1558 cm−1 (N−H, NH2). NMR data of fully adamantyl-substituted PPI-G3 dendrimers were reported by Baars et al. in ref 43. Second Step. PPI-G3-AdaB (0.19 g, 0.079 mmol), D-(+)-maltose monohydrate (6.85 g, 19.0 mmol), and 8 M borane−pyridine complex solution (4.76 mL, 38.1 mmol) were taken up in a 0.1 M sodium borate buffer (30 mL). The reaction mixture was stirred at 50 °C for 7 days. The crude reaction mixture was purified by dialysis against deionized water for 3 days. Then, the water was eliminated by freezedrying to obtain mPPI-G3-AdaB as a white cottony material (0.46 g, yield 90%). 1H NMR (500 MHz, D2O): 1.2−1.9 (a, d, g, j), 1.57 (z), 1.81 (x), 1.95 (y), 2.2−3.2 (b, c, e, f, h, i, k, 1′), 3.2−4.3 (2−6, 2′-6′), 4.8−5.2 (1). IR: 3326 (OH), 2905, 2849 (CH, CH2), 1639 (CO), 1559 (N−H), 1021 cm−1 (C−O). LILBID MS: C420H820N34O244 (10251 g/mol−1 relating to 24 maltose units and 4 adamantyl urea moieties connected to PPI-G3); m/z = top of the peak of about 10823 (M−). Noncovalent Synthesis of mPPI-G3-AdaB/1 and mPPI-G4AdaB/1 Supramolecular Complexes. mPPI-G3-AdaB (10 mg, 9.2 × 10−4 mmol) or mPPI-G4-AdaB (10 mg, 5.2 × 10−4 mmol) were dissolved in Millipore water, and ligand 1 (at 1:1 molar ratio with respect to Ada/β-CD residues) was added. Then, the solvent was slowly evaporated with a gentle nitrogen flux. The solid residues were kept under vacuum until constant weight to give two samples of the supramolecular complexes as off-white materials (mPPI-G3-AdaB/1 complex: 17.3 mg, calculated M = 18683 g/mol, and mPPI-G4-AdaB/ 1 complex 18.2 mg, calculated Mw = 34908 g/mol). Dynamic Light Scattering (DLS). To characterize the particle size and size distribution of mPPI-Gx-AdaB samples and their complexes with ligand 1, dynamic light scattering (DLS) measurements were carried out at 25 °C at a fixed angle of 173° using the Nano Zetasizer (Malvern), equipped with a He−Ne laser (4 mW) and

was carried out by following the two step general procedure previously used in ref 30 for the synthesis of the glycodendrimers mPPI-G4AdaB and mPPI-G5-AdaB.

First Step. PPI-G3 dendrimer (0.20 g, 0.12 mmol) was dissolved in 30 mL of chloroform, and the solution was degassed in argon atmosphere for 1 h. Then, 1-adamantyl isocyanate (0.085 g, 0.48 mmol) was added, and the resulting mixture was stirred at room temperature for 24 h in argon atmosphere. The solvent was removed under reduced pressure to obtain the desired intermediate material PPI-G3-AdaB as a colorless oil (0.22 g, yield 92%). The number of adamantyl groups was determined on the basis of the intensities of the signals at 2.72 ppm (CH2 near to unreacted amino groups) and at 3.13 ppm (CH2 near to reacted amino groups) taking into account 16 terminal NH2 groups for the precursor macromolecule PPI-G3. A value of 4.4 for the adamantyl groups was determined (27% conversion of amino groups) in good agreement with the intended

Figure 1. Rational design of the heterobifunctional ligand 1 based on the arylpiperazine moiety (bioactive group) and β-CD (functional group). 3987

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Scheme 1. Synthesis of Heterobifunctional Ligand 1a

a

Reagents: (i) TFA; (ii) CHCl3, TEA, 4-nitrophenyl prop-2-ynylcarbamate; (iii) DMF, CuBr, DIPEA, MW (200 W, 85 °C). Incubation was stopped by rapid filtration under vacuum through Unifilter GF/B glass fiber filter plates, presoaked in Hepes buffer, 50 mM, pH 7.4, containing 0.1% polyethylenimine, by means of a cell harvester. Filters were immediately rinsed three times with cold buffer and dried for 2 h at room temperature; then 0.2 mL of MicroScint-50 (PerkinElmer) was added, and after at least 2 h stabilization period, radioactivity was determined. Competition experiments were analyzed by nonlinear regression fitting using GraphPhad software (version 6 for Windows), in order to obtain the IC50 value (the concentration of unlabeled drug that caused 50% inhibition of specific binding).

a digital autocorrelator. The particle size distribution was determined using a multimodal peak analysis by intensity, volume, and number. The DLS measurements were made at concentrations of about 1 × 10−3 M in β-CD unit and successive additions of appropriate glycodendrimers were made to obtain the ratio β-CD/Ada = 1. The same concentration in Ada residues was used for the DLS measurements of the glycodendrimers. The investigated solutions were filtered directly through 0.8 mm filters and measured after 24 h. In Vitro Binding Assays. Male Wistar rats (Harlan, Italy) weighing 275−300 g were used. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/ UE). Animals were sacrificed by decapitation, brains were rapidly removed, and cerebral cortical tissues and hippocampi were dissected and used for binding assay preparation according to Nelson al. ref 44, with slight modification. The cerebral membrane preparation was finally suspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 50 mM, pH 7.4, just before the binding assay was performed, in order to obtain 0.5 mg of protein/sample. [3H]BRL43694 (granisetron; s.a. 81 Ci/mmol; PerkinElmer Life Science Products) binding, at the concentration corresponding to the Kd value, was assayed in polystyrene multiwell plate (24 well) for 30 min at 25 °C in final incubation volumes of 1.0 mL. The specific binding of the tritiated ligand was defined as the difference between the binding in the absence (total binding) and in the presence of 30 μM unlabeled 5HT (nonspecific binding). It represented in an average 70% of the total binding.

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RESULTS AND DISCUSSION Design and Synthesis of the Bioactive β-Cyclodextrin Based Guest 1. Our long-lasting interest in the 5-HT3R has led to the development of numerous (monovalent and multivalent) arylpiperazine derivatives showing nanomolar affinity and provided a considerable amount of information on the possible ligand−receptor interactions. In particular, SAR studies interfaced with docking studies have shown the receptor’s ability to accommodate the arylpiperazine moiety inside the serotonin binding site through specific interactions involving the charged head of the piperazine moiety, the nearby amide NH, and the ligand aromatic moiety. Furthermore, the introduction of long oligo(ethylene glycol) (OEG) spacers attached to the amide nitrogen of the MPQC portion is compatible with the interaction with the 5-HT3R binding site. 3988

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Scheme 2. Synthesis of the Adamantyl-Modified Glycodendrimer Hostsa

a

Reagents: (i) 1-adamantyl isocyanate, CHCl3; (ii) maltose, BH3·Pyr, 0.1 M sodium borate solution.

of β-CD molecules, which dynamically reconstructs the sugar shell. On the basis of these considerations, the introduction of adamantyl residues (needed to obtain specific interactions with guest 1) into the maltose dense shell was carried out at 25% in order to retain a large excess of hydrophilic (maltose) terminal groups on the dendrimeric surface with respect to lipophilic (adamantane) residues and to get a relatively high number of possible interactions between hosts and guests. The introduction of adamantyl groups and sugar molecules on the periphery of the PPI dendrimers was carried out in two reaction steps (Scheme 2).25,30 In the first step, the adamantyl groups were introduced by the addition of 1-adamantyl isocyanate (25% of the primary amino groups) to the appropriate dendrimeric solution (PPIG3, PPI-G4, or PPI-G5 in chloroform) to obtain the partially modified dendrimers PPI-G3-AdaB, PPI-G4-AdaB, and PPIG5-AdaB bearing about 25% of the free amino groups substituted with adamantyl urea moieties. In the second step, the adamantyl modified dendrimers were reacted in a 0.1 M aqueous solution of sodium borate with an excess of maltose in the presence of borane−pyridine complex as a reducing agent to substitute the remaining free amino groups of the dendrimer surface with maltose groups (Table 1). DLS analysis performed on water dispersions of the adamantyl-modified dendrimers (Table 1) showed dimensions in the same range previously observed and a relatively low propensity to aggregation.30 Supramolecular Interaction between the Bioactive βCyclodextrin Based Guest 1 and the AdamantylModified Glycodendrimers. The host−guest interaction between β-cyclodextrin based compound 1 and adamantyl groups present on the surface of the glycosylated dendrimers was studied by 1H NMR spectroscopy to prove the possibility to assemble supramolecular complexes in water. In a previous work, we have demonstrated the host−guest interaction between β-CD and adamantyl-modified glycodendrimers bearing different oligosaccharide open and dense shells by 1H NMR studies.30 In particular, at the same β-CD/Ada ratio the accessibility of Ada moieties decreases with increasing the dendrimer generation and the number of Ada units on the

This allowed the design of bifunctional ligands potentially able to allocate the (chemofunctional or bioactive) groups placed at the other end of the spacers outside the receptor surface.39,40 In order to prove definitely this latter assumption, we designed heterobifunctional ligand 1 (Figure 1) consisting of the bioactive portion MPQC linked by means of a 10 unit OEG spacer to a β-CD unit capable of ensuring the supramolecular interaction with the adamantyl-modified glycodendrimers outside of the 5-HT3 receptor. Heterobivalent ligand 1 was synthesized starting from propargyl derivative 4, which was reacted with 6I-azido-βcyclodextrin (β-CD-N3)42 by exploiting the Cu(I)-catalyzed azide−alkyne 1,3-dipolar cycloaddition (CuAAC) click chemistry reaction (Scheme 1).45−47 The propargyl group of compound 4 was introduced starting from compound 2, which after deprotection with TFA (to obtain the free amine 3) was reacted with 4-nitrophenyl prop-2-ynylcarbamate.40 DLS measurements performed at room temperature on water dispersions containing 1 (concentration ca. 0.001 M, data not shown) revealed a detectable self-aggregation liability for this compound probably related to the presence of a potential guest moiety (the arylpiperazine)48 and a host moiety (the cyclodextrin) in the same molecule. We assume that the presence of the OEG spacer allows the formation of intermolecular homoinclusion complexes showing quite defined architectures and dimensions (ca. 80 and 320 nm). Design and Synthesis of the Adamantyl-Modified Glycodendrimer Hosts. The dendrimeric hosts were designed trying to keep as much as possible unaltered the physicochemical properties conferred by the high presence of maltose molecules in the outer shell.19−29 In particular, glycodendrimers show high water solubility, low cytotoxicity, absence of charge on the surface, poor aggregation tendency, and the ability to establish interactions with natural macromolecules through multiple hydrogen bonds. These properties can be altered by the introduction of other terminal groups that can create imperfections in the maltose-dense shell. However, as previously observed, a low number of adamantyl (Ada) groups in the maltose-dense shell causes only a slight alteration of the macromolecular properties that can be restored by the complexation of the hydrophobic residues in the internal cavity 3989

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Table 1. Characteristics of Adamantyl-Modified Dendrimers number of sugar units

compound PPI-G3AdaB PPI-G4AdaB30 PPI-G5AdaB30 mPPI-G3AdaB mPPI-G4AdaB30 mPPI-G5AdaB30

number of adamantyl groups (degree of derivatization, %)

Mn (g/mol)

davg (nm)f

4.4 (27)c

2467d

g

8.3 (26)c

4985d

g

17.9 (28)c

10341d

g

theora exptb

4 (25)b

24

24

10823e

4.2

8 (25)b

48

44

19188e

4.6

15 (23)b

98

98

41806e

5.7

a

Calculated assuming 100% conversion of remaining NH groups with sugar. bCalculated from LILBID mass spectrometry data. cCalculated from 1H NMR signal integrals; estimated relative error ±5%. d Calculated from Mn of PPI-G3, PPI-G4, and PPI-G5 and degree of substitution. em/z value of center of the peak obtained by LILBID mass spectroscopy. fAverage size distribution measured by DLS analysis. gNot determined.

surface. However, these two factors hinder but do not completely prevent the complexation of Ada units by β-CD. Besides proof of spatial neighborhood of β-CD ring and Ada unit in the inclusion complex by NOESY or ROESY measurements, the complex formation results also in characteristic downfield shifts for the methylene and methine proton signals of the adamantyl group when located in the hydrophobic cavity of the β-CD. The observed chemical shift effect can be related to the molar fraction of complexed Ada units.13,49,50 In the present study, the β-cyclodextrin based compound 1 was dissolved in D2O (1 mM) and increasing amounts of the glycodendrimers mPPI-G3-AdaB, mPPI-G4-AdaB, and mPPIG5-AdaB were added from solutions with 10 mM in Ada units. The first experiment with a 5-fold β-CD excess gives the highest degree of Ada complexation and thus the largest chemical shift effects. The chemical shift changes with decreasing β-CD/Ada ratio were followed to obtain information about the equilibrium between complex and free species. In order to estimate the interaction between the β-CD moiety of 1 and the adamantyl residues of the three glycodendrimers, the chemical shift of the CH protons of the Ada unit was evaluated at different β-CD/Ada ratios (δexp). The signal of these protons showed larger chemical shift change compared with methylene proton signals of Ada and did not show significant overlap with PPI signals. The molar fraction of complexed Ada units, MFAda, can be calculated according to equation MFAda = (δexp − δ0)/Δδmax where δ0 is the chemical shift determined in absence of β-cyclodextrin based 1 (δ0 = 1.953 ppm for mPPI-G3-AdaB, 1.954 ppm for mPPI-G4AdaB, and 1.947 ppm for mPPI-G5-AdaB) and Δδmax is the methine proton chemical shift at full complexation (MF = 1). This value (Δδmax = +0.147 ppm) was taken from our comprehensive study on β-CD−Ada host−guest interactions on the surface of adamantyl-modified dendrimers.30 Figure 2 depicts the complexation of the Ada moieties of the three glycodendrimers mPPI-Gx-AdaB (x = 3, 4, or 5) at different βCD/Ada ratios.

Figure 2. Molar fraction of complexed Ada units of mPPI-G3-AdaB (■, with ligand 1), mPPI-G4-AdaB (▲, with ligand 1), and mPPIG5-AdaB (●, with β-CD) in dependency on the molar ratio β-CD/ Ada.

The curves reveal significant differences in complexation behavior. In agreement with our previous studies, the β-CD complexation ability is reduced with increasing PPI generation due to steric hindrance on the surface, but it is never completely prevented. A high portion of surface Ada units can be accessed by the β-CD moiety of 1 for mPPI-G3-AdaB and mPPI-G4-AdaB. Thus, at a 3.33-fold excess of βcyclodextrin based compound 1, the molar fraction of complexed Ada moieties is 83% for mPPI-G3-AdaB and 56% for mPPI-G4-AdaB, which are promising values. The corresponding values at a 1:1 ratio are 59% and 31%, respectively. In contrast, the Ada units of mPPI-G5-AdaB can be accessed only to 12% with the β-CD ring of the compound 1 (data not shown) and 25% using free β-CD at 3.33-fold excess (Figure 2). These values remain more or less the same at 1:1 ratio (10% and 22% for complexation with βcyclodextrin derivative 1 and free β-CD respectively). This behavior confirms the stability of the β-CD−Ada complex but its slight increase with significant β-CD excess is in accordance with limited accessibility of most surface Ada units. With increasing dendrimer generation, the shell density increases51 and the Ada residues are strongly packaged on the surface, or even poorly exposed for the complexation. This behavior is in accordance with results of Reinhoudt et al. showing that in contrast to adamantyl-terminated PPI-G1, PPI-G2, PPI-G3, and PPI-G4, in adamantyl-terminated PPI-G5 not all 64 Ada groups at the surface can be complexed by β-CD for steric reasons.52 Summarizing, the complexation behavior of the glycodendrimers reflects the competition of strong host−guest interaction of β-CD and Ada on one side and hindered accessibility of Ada units within the maltosylated PPI surface by the bulky β-CD ring of 1 on the other side. A proper choice of 3990

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Figure 3. Illustration of the supramolecular complex generated by the dense shell glycodendrimer mPPI-G4-AdaB and the bioactive β-CD based guest 1.

the β-CD/Ada ratio allows generation of supramolecular complexes (Figure 3) in equilibrium with a balanced content of free ligands and macromolecules. The results of DLS experiments performed on water dispersions of heterobifunctional ligand 1 containing increasing amounts of the adamantyl-modified dendrimers confirmed the assumed complexation behavior because the presence of the adamantyl-modified dendrimers is capable of disrupting the self-aggregates of 1 with the formation of objects showing dimensions slightly larger (4.47 and 5.36 nm for mPPI-G3AdaB and mPPI-G4-AdaB at 1:1 β-CD/Ada ratio, respectively) than those of the adamantyl-modified dendrimers. Interaction of the Bioactive β-Cyclodextrin Based Guest 1 with the Natural 5-HT3 Receptor. The affinity of βcyclodextrin based guest 1 and of the precursor compounds 2 and 4 for the serotonin 5-HT3R was measured by means of displacement studies performed with radiolabeled granisetron specifically bound to the 5-HT3R in rat cortical membranes (see Experimental Section for details), and the results are summarized in Table 2. The results of the binding studies show that compound 1 is capable of interacting with the 5-HT3 binding site exhibiting an

IC50 value in the nanomolar range (43 nM). The value is an apparent IC50 calculated from the nominal concentration of heterobifunctional ligand 1 by neglecting its self-aggregation equilibrium, which is assumed to be based on a relatively weak interaction compared with that of the arylpiperazine moiety with the 5-HT3 receptor binding site. This value is very similar to the affinity shown by intermediates 2 and 4 having the same OEG spacer but smaller terminal groups [i.e., NH-Boc end group in the case of compound 2 (IC50 = 136 nM) and a propargyl ureidic group in compound 4 (IC50 = 49 nM)]. The homogeneity in the IC50 values suggests a good tolerance to very bulky terminal groups. On the basis of its large size, β-CD moiety can be assumed to hardly enter inside the receptor. Therefore, we assumed that ligand 1 places the β-CD portion outside the receptor and interacts with the binding site of serotonin through the MPQC moiety. In order to study the interaction of the heterobifunctional ligand 1 with the 5-HT 3 R in the presence of the glycodendrimers, two supramolecular complexes with a stoichiometric ratio (Ada/β-CD residues) of 1:1 were evaluated in binding studies. The results of NMR studies (see above) supported the existence of a highly dynamic equilibrium between the free (or self-aggregated) and complexed forms of ligand 1, which, in principle, can interact through the arylpiperazine moiety with the 5-HT3R binding site. Since the characterization of the complexation equilibrium in the conditions used in the binding studies was considered to be a rather difficult task, the results are expressed in Table 2 as apparent IC50 values calculated from the nominal concentration of the complex by neglecting the complexation−decomplexation equilibrium. The binding studies showed apparent IC50 values (16 nM for mPPI-G3-AdaB/1 and 15 nM for mPPIG4-AdaB/1) in the same nanomolar range of that shown by ligand 1. These results confirmed the strong interaction of heterobifunctional ligand 1 with the 5-HT3R binding site even in the presence of macromolecular hosts, which did not seem to interfere with the ligand−receptor interaction. Moreover, glycodendrimers do not show significant interactions with the receptor as confirmed by the binding results in the absence of ligand 1 (Table 2). No clear experimental evidence was obtained to support a multivalent interaction of supramolecular complexes with the 5-

Table 2. 5-HT3R Binding Affinities compound 1 2 4 mPPI-G3-AdaB/1 complex mPPI-G4-AdaB/1 complex mPPI-G3-AdaB mPPI-G4-AdaB

IC50 (nM)a 43 136 49 16 15 >1000 >1000

± ± ± ± ±

2b 23 9 3c 3c

Each value is the mean ± SEM of three determinations and represents the concentration producing half the maximum inhibition of [3H]granisetron (final concentration 1 nM) specific binding to rat cortical membranes. bThe value is an apparent IC50 calculated from the nominal concentration of heterobifunctional ligand 1 by neglecting its self-aggregation equilibrium. cThe value is an apparent IC50 calculated from the nominal concentration of the complex by neglecting the highly dynamic equilibrium between the free and complexed forms of heterobifunctional ligand 1. a

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Figure 4. Illustration of the possible interactions between the 5-HT3R and ligand 1 released (monovalent interaction) or complexed (statistical effect) by mPPI-G4-AdaB/1.

further refinements in order to improve the interaction between host and guest.

HT3 receptor. The slight change in the apparent IC50 values of the ligand 1 in the presence of the glycodendrimer hosts can be attributed to the presence of free ligand 1 generated from the complexation−decomplexation equilibrium, which can produce concentrations of 1 higher than the nominal one. However, in the absence of conclusive information on the potential supramolecular active species, a binding mechanism was assumed to be characterized by a moderate translocalization of the supramolecular complex close to the 5-HT3 receptor that might increase the local concentration of the supramolecular glycodendrimer-based hybrid drug in the cerebral compartment (Figure 4).

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

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The authors declare no competing financial interest.

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CONCLUSIONS In conclusion, by exploiting the specific supramolecular interaction between adamantane and β-cyclodextrin, we have built bioactive supramolecular systems consisting of two components: an adamantyl-modified glycodendrimer (host) and a bioactive β-CD based guest 1 capable of interacting with both the macromolecular host and the natural 5-HT3R. Water solubility, biocompatibility, neutral surface charge, and poor aggregation tendency are some of the properties that make glycodendrimers excellent candidates for application in the pharmaceutical field as drug carriers and diagnostic agents. In addition, they showed a pharmacological potential in interfering with Aβ(1−40) and Aβ(1−42) amyloid peptide formation leading us to consider them as potential antiAlzheimer’s disease agents. On the other hand, 5-HT3R antagonists have been proposed to play a potential clinical role in Alzheimer’s disease treatment because they improve ACh release in cortical tissue by reducing the tonic inhibitory effect of 5-HT and the density of 5-HT3R is not altered in AD patients. Thus, the supramolecular system described in the present paper configures a new way to conceive supramolecular hybrid drugs, where the system components can show intrinsic and synergistic (i.e., different targets within the same pathology) activities. Obviously, the present system will be subject to

ACKNOWLEDGMENTS Thanks are due to Italian MIUR for financial support. Mr. A. Korwitz (IPF Dresden) for assistance in the NMR measurements, Mrs. A. Caspari (IPF Dresden) for DLS measurements, and Dr. M. Cernescu (Goethe-Universität Frankfurt a.M.) for carrying out LILBID-MS are also acknowledged.

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ABBREVIATIONS 5-HT3R, 5-HT3 receptor; PPI, poly(propyleneimine); TLC, thin layer chromatography; AD, Alzheimer’s disease; β-CD, βcyclodextrin; LGIC, ligand-gated ion channel; MPQC, methylpiperazinyl-quinoline-carboxamide; OEG, oligo(ethylene glycol); β-CD-N3, 6I-azido-β-cyclodextrin; TFA, trifluoroacetic acid

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REFERENCES

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