Design, Synthesis, and Biological Evaluation of Novel cRGD

Article pubs.acs.org/bc

Design, Synthesis, and Biological Evaluation of Novel cRGD− Paclitaxel Conjugates for Integrin-Assisted Drug Delivery Michael Pilkington-Miksa,† Daniela Arosio,‡ Lucia Battistini,§ Laura Belvisi,# Marilenia De Matteo,† Francesca Vasile,† Paola Burreddu,∥ Paola Carta,▲ Gloria Rassu,∥ Paola Perego,⊥ Nives Carenini,⊥ Franco Zunino,⊥ Michelandrea De Cesare,⊥ Vittoria Castiglioni,□,¶ Eugenio Scanziani,□,¶ Carlo Scolastico,† Giovanni Casiraghi,§ Franca Zanardi,*,§ and Leonardo Manzoni*,‡ †

Centro Interdipartimentale Studi Biomolecolari e Applicazioni Industriali, Università degli Studi di Milano, Via Fantoli 16/15, I-20138 Milano, Italy ‡ Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Via Golgi 19, I-20133 Milano, Italy § Dipartimento Farmaceutico, Università degli Studi di Parma, Parco Area delle Scienze 27A, I-43124 Parma, Italy # Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, I-20133 Milano, Italy ∥ Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Traversa La Crucca 3, I-07100 Li Punti, Sassari, Italy ▲ Porto Conte Ricerche Srl, I-07041 Tramariglio Alghero, Sassari, Italy ⊥ Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42, I-20133 Milano, Italy □ Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria (DIPAV), Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Via Celoria 10, I-20133 Milano, Italy ¶ Mouse and Animal Pathology Laboratory, Fondazione Filarete, Viale Ortles 22/4, I-20139 Milano, Italy S Supporting Information *

ABSTRACT: The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrinrecognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent αVβ3 binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.

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INTRODUCTION

use of available drugs, in particular, cytotoxic agents. Impressive advances in the molecular and biological characterization of human tumors have led to the identification of specific alterations exploitable for tumor targeting. In this context, strong interest for integrins has recently emerged.2 Various

The curative potential of clinically available antitumor agents is limited by a number of factors which may include tumor-related factors (e.g., activation of drug resistance mechanisms by tumor cells), drug-related factors (e.g., inadequate intratumor concentration of the drug), tumor microenvironment interactions (e.g., hypoxia, microvesicle release), and individual features of the patients (i.e., genetic polymorphisms).1 Thus, an impressive effort is still required in an attempt to optimize the © 2012 American Chemical Society

Received: April 5, 2012 Revised: May 21, 2012 Published: July 9, 2012 1610

dx.doi.org/10.1021/bc300164t | Bioconjugate Chem. 2012, 23, 1610−1622

Bioconjugate Chemistry

Article

therapeutics where the RGD-homing function is coupled to the effector moiety under the shape of either conventional covalent conjugates10,20−29 or nanosized molecular/supramolecular entities.10,20,21,30−34 Among the chemotherapeutics which were incorporated in these RGD-conjugates, the microtubuleinterfering agent paclitaxel (PTX, 1, Scheme 1) has often been selected, in an attempt to overcome the inherent drawbacks such as low aqueous solubility, short half-life, poor bioavailability, and systemic toxicityall features resulting in a poor therapeutic index. The use of paclitaxel is of specific interest in this approach, because taxanes are known to have antiangiogenic effects.35 Quite surprisingly, however, the broad spectrum of tumor-addressing integrin ligands to be exploited in conjugated entities was limited to linear RGD-embedded peptides or no more than a couple of cyclic peptide candidates, namely, c(RGDfK), c(RGDyK), or E-[c(RGDfK)2], which represent the conjugable versions of Cilengitide.10,27−29 In the structural design of novel, potentially bioactive conjugates, the judicious calibration of both the targeting device and the appropriate linker is crucial for the successful delivery of a given cytotoxic drug. Along this line, we present here nine novel molecular conjugates where the common PTX cytotoxic cargo is covalently attached to either cyclic AbaRGD or AmproRGD integrin-recognizing matrices via structurally diverse connections. Preeminent goals of the study were as follows: (1) to assay the feasibility of the chemistry involved by means of simple and efficient conjugation reactions under gentle and faithful conditions; (2) to assess the structural integrity/purity of the targets; (3) to evaluate by biological tests in vitro the mutual influence of the Aba/AmproRGD, PTX, and linker modules on the αVβ3/αVβ5 targeting capabilities, solubility, and tumor cell sensitivity; and (4) to assay via in vivo biological studies the antitumor activity of a selected candidate in order to gain insight into possible structural amendments and future development of the proposed conjugates.

studies have indicated that integrins promote cell attachment and migration on the surrounding extracellular matrix and mediate cell interactions during several pathological processes.2 Indeed, the available evidence supports a role for integrins in proliferation, migration, and survival of tumor cells, as well as in additional processes that favor tumor progression such as angiogenesis and inflammation.2 In particular, specific integrin receptors, i.e., the αVβ3 and αVβ5 families, have been elected as compelling molecular indicators of tumor angiogenesis and tumor progression, invasion, and metastasis, since they are overexpressed on proliferating endothelial cells as well as on various tumor cells including breast,3,4 glioma,5 melanoma,6,7 prostate,8 and ovarian carcinoma.9 A large number of specific, highly potent αVβ3- and αVβ5addressed small molecule ligands have been designed and implemented so far, which contain or mimic the key receptorrecognizing RGD (Arg-Gly-Asp) tripeptide motif;10,11 these include the landmark cyclic pentapeptide c(RGDf[N-Me]V) (Cilengitide, I)12 (Chart 1), the azabicycloalkane-based cyclic RGD semipeptide II (AbaRGD),13 and the 4-aminoprolinebased cyclic RGD semipeptide III (AmproRGD).14 Chart 1High-Affinity αVβ3-Targeting Cyclic RGD Ligandsa

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EXPERIMENTAL PROCEDURES Chemistry. General. All chemicals and solvents were of reagent grade and were used without further purification. Solvents were dried by standard procedures and reactions requiring anhydrous conditions were performed under nitrogen or argon atmosphere. 1H and 13C NMR spectra were recorded at 300 K on Bruker AVANCE-400 or Bruker AVANCE-600 MHz spectrometers. Chemical shifts δ are expressed in ppm relative to internal Me4Si as the standard reference. Mass spectra were obtained with an ESI apparatus Bruker Esquire 3000 plus. Thin-layer chromatography (TLC) was carried out with precoated Merck F254 silica-gel plates. Flash chromatography (FC) was carried out with Macherey-Nagel silica gel 60 (230−400 mesh). Reverse-phase column chromatography was carried out with the Biotage SP1 or SP4 systems using the Biotage 25+M C18 cartridges. Unless otherwise stated, semipreparative HPLC was carried out on a Waters SymmetryPrep C18-7 μm 7.8 × 300 mm column or a Waters Atlantis C18 OBD 5 μm 19 mm × 10 cm or a Supelco Ascentis RPAmide 5 μm 21.1 mm × 15 cm; gradient from 100% H2O (+ 0.1% TFA) to 75% H2O (+ 0.1% TFA)/25% MeCN (+ 0.1% TFA) over 30 min. Representative Procedure A. Preparation of C2′ Paclitaxel Ester 7. To Paclitaxel 1 (PTX, 50 mg, 0.059 mmol, 1.0 equiv), 4-pentynoic acid 2 (6.32 mg, 0.0644 mmol, 1.1 equiv), and a catalytic quantity of N,N-dimethylaminopyridine (DMAP) in

aa

Ref 12. bRef 13. cRef 14.

Apart from the preeminent use of small-molecule integrin antagonists as antiangiogenic and antitumor agents per se,10,11,15−18 such motifs may be exploited with even more success as active targeting tools, provided that appropriate cytotoxic and/or imaging cargos are consigned to them.10,19−21 Since the pioneering work by Arap and Pasqualini,22 where effective delivery of the antitumor agent doxorubicin to human breast cancer xenografts in nude mice was given in charge of a small RGD-doxo conjugate, several reports have appeared concerning the synthesis and biology of actively targeted 1611



Article

Scheme 1. Synthesis of Activated Taxane Modules 7−11

11 was recovered as a white solid in 95% yield. For characterization data of compound 11, see Supporting Information. Preparation of Cyclic AbaRGD (Compounds 12 and 13) and AmproRGD (Compounds 14−16 and 18) Modules. Cyclic AbaRGD compounds 12 and 13 were prepared according to a reported procedure.13 Cyclic AmproRGD compounds 14−16 were prepared according to a reported procedure.14 Compound 18. Azidopeptide 14 (280 mg, 0.55 mmol, 2.2 equiv) and dialkyne 1736 (93 mg, 0.25 mmol, 1.0 equiv), dissolved in 20 mL of tert-butanol/water (1:1), were sequentially treated with 0.3 M solution of copper(II) acetate (333 μL, 0.1 mmol, 0.4 equiv) and 0.9 M solution of sodium Lascorbate (222 μL, 0.2 mmol, 0.8 equiv). The resulting heterogeneous mixture was sonicated for 1 h and then stirred at room temperature for additional 4 h. After reaction completion, the mixture was lyophilized and the residue was purified by flash chromatography (EtOAc/MeOH, 85:15 to 75:25) affording a protected dimeric peptide (250 mg, 72%) as a glassy solid. This protected intermediate (250 mg, 0.18 mmol, 1.0 equiv) was treated with a solution of TFA/TIS/water 95:2.5:2.5 (16 mL) at room temperature. After 5 h, the solvent was evaporated under vacuum and the residue dissolved in water (15 mL) and thoroughly washed with Et2O (4×). The aqueous phase was concentrated under vacuum furnishing 240 mg (95%) of dimeric amine 18 as a trifluoroacetate salt, which was used as such in the following coupling step. For characterization data of compound 18, see Supporting Information. Representative Procedure C. Preparation of TriazolylLinked Conjugate 19. To azido-cRGD compound 12 (33.8 mg, 0.059 mmol, 1.0 equiv) and PTX ester 7 (54.7 mg, 0.059 mmol, 1.0 equiv) in tert-butanol/water (6:4, 3 mL) was added 0.9 M sodium L-ascorbate (28.39 μL, 0.026 mmol, 0.4 equiv). To this solution was then added 0.3 M copper(II) acetate (42.6 μL, 0.013 mmol, 0.2 equiv) and the resulting mixture was left to

dry dichloromethane (1.0 mL), diisopropylcarbodiimide (DIC, 13.6 μL, 11.08 mg, 0.088 mmol, 1.5 equiv) was added. The resulting solution was left to stir under argon at room temperature for 20 h. The reaction mixture was then concentrated in vacuo to give a yellow solid. This was purified by reverse-phase chromatography eluting with water and acetonitrile to give a white solid in 99% yield. For characterization data of compound 7, see Supporting Information. Compounds 8 and 9 were obtained following the above procedure as white solid in 99% and 94% yield, respectively. Paclitaxel Ester 8. The title compound was obtained from Paclitaxel 1 and undec-10-ynoic acid 3 following representative procedure A. After reverse-phase chromatography, compound 8 was recovered as a white solid in 99% yield. For characterization data of compound 8, see Supporting Information. Paclitaxel Ester 9. The title compound was obtained from Paclitaxel 1 and alkyne linker 4 (preparation reported in the Supporting Information) following representative procedure A. After reverse-phase chromatography, compound 9 was recovered as a white solid in 94% yield. For characterization data of compound 9, see Supporting Information. Representative Procedure B. Preparation of C2′ Paclitaxel Ester 10. To PTX 1 (50 mg, 0.059 mmol, 1.0 equiv) and diglycolic anhydride 5 (8.6 mg, 0.074 mmol, 1.25 equiv) in dry dichloromethane (1.0 mL), pyridine (0.474 mL, 463 mg, 5.855 mmol, 100 equiv) and a catalytic quantity of DMAP were added. The resulting solution was left to stir under argon for 3 h at 0 °C and then left to warm to room temperature. After 20 h, the reaction mixture was concentrated in vacuo; the resulting solid was dissolved in water/acetonitrile/acetic acid (3 mL, 1:1:1) and purified by reverse-phase chromatography eluting with water and acetonitrile to give a white solid in 99% yield. For characterization data of compound 10, see Supporting Information. Paclitaxel Ester 11. The title compound was obtained from PTX 1 and succinic anhydride 6 following representative procedure B. After reverse-phase chromatography, compound 1612



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compound 26 was recovered as a white solid in 40% yield. For characterization data of compound 26, see Supporting Information. Amide-Linked Conjugate 27. The title compound was obtained from amino-cRGD 13 and PTX ester 11 following representative procedure D. After RP-HPLC purification, compound 27 was recovered as a white solid in 62% yield. For characterization data of compound 27, see Supporting Information. Biology. Solid-Phase Receptor Binding Assay. Purified αvβ3 and αvβ5 receptors (Chemicon International, Inc., Temecula, CA, USA) were diluted to 0.5 μg/mL in coating buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MnCl2, 2 mM CaCl2, and 1 mM MgCl2. An aliquot of diluted receptors (100 μL/well) was added to 96-well microtiter plates (NUNC MW 96F Medisorp Straight) and incubated overnight at 4 °C. The plates were then incubated with blocking solution (coating buffer plus 1% bovine serum albumin) for an additional 2 h at room temperature to block nonspecific binding, followed by 3 h incubation at room temperature with various concentrations (10−5−10−12 M) of test compounds in the presence of biotinylated vitronectin (1 μg/mL). Biotinylation was performed using an EZ-Link SulfoNHS-Biotinylation kit (Pierce, Rockford, IL, USA). After washing, the plates were incubated for 1 h at room temperature with biotinylated streptavidin−peroxidase complex (Amersham Biosciences, Uppsala, Sweden) followed by 30 min incubation with 100 μL/well Substrate Reagent Solution (R&D Systems, Minneapolis, MN) before stopping the reaction with the addition of 50 μL/well 2 N H2SO4. Absorbance at 415 nm was read in a SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments, Inc.). Each data point represents the average of triplicate wells; data analysis was carried out by nonlinear regression analysis with GraphPad Prism 5.0 software. Each experiment was repeated in triplicate. Drugs. For in vitro studies, PTX and compounds under investigation were dissolved in dimethylsulfoxide (DMSO) and then added to culture medium. DMSO concentration in medium never exceeded 0.5%. For in vivo studies, PTX was dissolved in a mixture of ethanol and cremophor ELP (50 + 50%) and kept at 4 °C. At treatment, the drug was diluted in 90% of cold saline after magnetic stirring and administered i.v. keeping the vial in ice. Compound 21 was dissolved and administered like PTX at room temperature. Cell Lines and Growth Conditions. The human ovarian carcinoma IGROV-1 cell line,37 the cisplatin-resistant IGROV1/Pt1 subline38 and the human large cell lung H460 carcinoma cell line (ATCC, HTB-177) were cultured in RPMI-1640 medium; the human osteosarcoma U2-OS cell line (ATCC, HTB-96) was grown in McCoy’s 5A medium. Medium was supplemented with 10% fetal calf serum. Cell Sensitivity to Drugs. The cell sensitivity to antitumor agents was measured by using the growth-inhibition assay based on cell counting. Cells were seeded in duplicates into 6-well plates and exposed to drug 24 h later. The compounds were dissolved in DMSO at 3.3 mM. Before cell treatment, the compounds were further diluted in DMSO. Five microliters of 200× solutions were used for treating cells in a volume of 1 mL of culture medium. The final DMSO concentration was not toxic. After 72 h of drug incubation, cells were harvested for counting with a cell counter. IC50 is defined as the drug concentration producing 50% decrease of cell growth. At least three independent experiments were performed.

stir at room temperature and monitored by LC-MS. When the reaction reached completion (2−4 h), the solution was frozen in liquid nitrogen and then freeze−dried. The solid recovered from freeze−drying was purified by RP-HPLC eluting with water + 0.1% formic acid and acetonitrile + 0.1% formic acid. The purified product was then freeze−dried to give a white solid in 75% yield. For characterization data of compound 19, see Supporting Information. Triazolyl-Linked Conjugate 20. The title compound was obtained from azido-cRGD 12 and PTX ester 8 following representative procedure C. After RP-HPLC purification, compound 20 was recovered as a white solid in 59% yield. For characterization data of compound 20, see Supporting Information. Triazolyl-Linked Conjugate 21. The title compound was obtained from azido-cRGD 12 and PTX ester 9 following representative procedure C. After RP-HPLC purification, compound 21 was recovered as a white solid in 46% yield. For characterization data of compound 21, see Supporting Information. Triazolyl-Linked Conjugate 22. The title compound was obtained from azido-cRGD 14 and PTX ester 7 following representative procedure C. After RP-HPLC purification, compound 22 was recovered as a white solid in 57% yield. For characterization data of compound 22, see Supporting Information. Triazolyl-Linked Conjugate 23. The title compound was obtained from azido-cRGD 15 and PTX ester 7 following representative procedure C. After RP-HPLC purification, compound 23 was recovered as a white solid in 48% yield. For characterization data of compound 23, see Supporting Information. Representative Procedure D. Preparation of Amide-Linked Conjugate 24. To PTX-ester 10 (122 mg, 0.126 mmol, 1.0 equiv) and N-hydroxysulfosuccinimide sodium salt (34.1 mg, 0.157 mmol, 1.25 equiv) in dry N,N-dimethylformamide (DMF, 4.0 mL) was added diisopropylcarbodiimide (DIC, 29.2 μL, 23.8 mg, 0.188 mmol, 1.5 equiv). The resulting mixture was left to stir under argon at room temperature for 24 h. The reaction was then concentrated in vacuo to give an offwhite solid. To this solid was added amino-cRGD 13 (108 mg, 0.126 mmol, 1.0 equiv) in acetonitrile (2.0 mL) and pH 7.0 phosphate buffer (PBS, 0.5 M, 1.0 mL), and the resulting mixture was rapidly cooled to 0 °C and then sonicated to favor dissolution. The reaction mixture was then left to stir at 0 °C for 10 h after which time it was allowed to warm to room temperature. After 18 h stirring, dioxane/water (1:1, 10 mL) was added and the resulting solution was freeze−dried. The solid recovered from freeze−drying was purified by RP-HPLC eluting with water + 0.1% formic acid and acetonitrile + 0.1% formic acid. The purified product was then freeze−dried to give a white solid in 43% yield. Compounds 25−27 were obtained following the above procedure as white solid in 45%, 40%, and 62%, respectively. Amide-Linked Conjugate 25. The title compound was obtained from amino-cRGD 16 and PTX ester 10 following representative procedure D. After RP-HPLC purification, compound 25 was recovered as a white solid in 45% yield. For characterization data of compound 25, see Supporting Information. Amide-Linked Conjugate 26. The title compound was obtained from amino-cRGD 18 and PTX ester 10 following representative procedure D. After RP-HPLC purification, 1613



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Scheme 2. Structures of Monomeric AbaRGD and AmproRGD Modules 12−16 and Synthesis of Dimeric AmproRGD Module 18

formula: LCK = (T − C)/(3.32 × DT), where T and C are the times (days) to reach a tumor volume in treated and control mice, respectively, and DT is the doubling time of control tumors. Tumor DT (days) was obtained from semilog best-fit curves of mean tumor volumes in untreated control mice plotted against time. The toxicity of the drug treatment was determined as body weight loss and lethal toxicity. Student's t test (two-tailed) was used for statistical comparison of tumor volumes in mice. Immunohistochemistry. Tumor xenografts and adjacent tissues were excised and formalin fixed and paraffin embedded. Four micrometer sections from each tumor xenograft were routinely stained with Hematoxylin-Eosin (HE) and evaluated under a light microscope. Mitoses were evaluated in 3 randomly selected 400× fields within the bulk of the xenograft, avoiding areas of necrosis and hemorrhage. The total number of mitoses and the mean value for each sample were evaluated. Furthermore, mitoses were differentiated as “normal” and “aberrant”, the latter class characterized by both small, condensed hyperchromatic nuclei and large cells composed of nuclear envelope around individual clusters of mis-segregated chromosomes (mitotic catastrophe), and the ratio between these two classes was evaluated. The analysis of mitoses was performed in a blind fashion. Statistical analysis of the obtained data was carried out with the Kruskal−Wallis test followed by Dunn’s multiple comparison test using GraphPad Prism (GraphPad Software, Inc.).

Analysis of Integrin Levels. The expression of integrins was measured by flow cytometry, following optimization of antibody concentration. Exponentially growing cells were harvested and incubated for 30 min at 4 °C with anti-human αvβ3 or αvβ5 antibodies or isotypic controls (Millipore, Temecula, CA; Chemicon International). Cells were then washed and samples were immediately used for flow cytometric analysis (FACScan, Becton-Dickinson). Integrin levels were expressed as the ratio between the mean fluorescence intensity obtained in cells incubated with anti-integrin antibodies and that of cells incubated with isotypic control (Table 2). Antitumor Activity Studies. All experiments of antitumor activity were carried out using female athymic Swiss nude mice, 8−10 weeks old (Charles River, Calco, Italy). Mice were maintained in laminar flow rooms keeping temperature and humidity constant. Mice had free access to food and water. Experiments were approved by the Ethics Committee for Animal Experimentation of the Istituto Nazionale Tumori of Milan according to institutional guidelines. The IGROV-1/Pt1 human tumor xenograft derived from cultures of the ovarian carcinoma cell line38 was used. Exponentially growing cells were s.c. injected into the right flank of athymic nude mice, and the tumor line was achieved by serial s.c. passages of fragments of regrowing tumors into healthy mice. Groups of five mice bearing bilateral s.c. tumors were employed. Tumor fragments were implanted on day 0 and tumor growth was followed by biweekly measurements of tumor diameters with a Vernier caliper. Tumor volume (TV) was calculated according to the formula: TV (mm3) = d2 × D/2 where d and D are the shortest and the longest diameter, respectively. Drugs were delivered i.v. into the lateral tail vein using a 26G needle equipped syringe. Drugs were administered in a volume of 10 mL/kg of body weight every 4 days for 4 times (q4d × 4). Treatment started three days after tumor implant, when tumors were just palpable. The efficacy of the drug treatment was assessed as tumor volume inhibition percentage (TVI%) in treated versus control mice, calculated as follows: TVI% = 100 − (mean TV treated/ mean TV control × 100) and log cell kill, evaluated by the

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RESULTS Chemistry. In engineering versatile, covalent connections of the building modules (PTX, RGD-semipeptide, and linker), mainly two requisites had to be satisfied. First, these linkages had to be easy to make and robust enough to guarantee the structural integrity of the whole construct until contacting and entering the cell compartment in such a way to fully exploit the specific targeting capabilities and the target-mediated cell internalization elicited by the RGD moiety. Second, connection to PTX, chosen at its C2′−OH position, has to be scissile 1614



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Scheme 3. Synthesis of Triazolyl-Linked Paclitaxel-AbaRGD and Paclitaxel-AmproRGD Conjugates 19−23

(hopefully inside the cell) to allow complete exercise of the cytotoxic function.39 Accordingly, either a triazole-forming Hüisgen 1,3-dipolar cycloaddition reaction (“click reaction”)40 or popular amide conjugation was judged a viable solution, considering their intrinsic robustness, easy execution, and compatibility of the experimental conditions with the exposed functional groups within the moieties. Synthesis of Activated Taxane Modules. The first step in the synthesis of the conjugates involved preparation of various C2′-esters of PTX 7−11 (Scheme 1) bearing either alkyne or carboxylic acid terminating moieties for subsequent triazolyl/ amide conjugation. For 7−9, two commercially available, diversely sized alkynoic acids 2 and 3, along with an ad hoc prepared short PEG acid 4 (for preparation, see Supporting Information), were regioselectively coupled to PTX at the free C2′-position. Standard DIC/DMAP chemistry in dichloromethane was effective for preparing the corresponding esters 7−9 in 94−99% isolated yields. Notably, no parasite esterification at the unprotected C7-hydroxyl of the taxane was observed. For carboxylic acids 10 and 11, two commercially available cyclic acid anhydrides, namely, glycolic anhydride 5 and succinic anhydride 6, were used and coupled to the cytotoxic drug 1 without any byproducts under conventional esterification conditions. Synthesis of Cyclic AbaRGD and AmproRGD Modules. As disclosed above, a key point of innovation of the present work with respect to the reported studies in the field is the selection of the αVβ3-integrin targeting unit. A first class of integrin ligands (compounds 12 and 13 in Scheme 2) features a fifteenmembered cyclopentapeptide where a 5,6-fused azabicycloalkane (Aba) amino acid moiety is tethered to the amino/acid terminals of the Arg-Gly-Asp tripeptide sequence. A second, different category of integrin ligands is represented by compounds 14, 15, 16, and 18 (Scheme 2), all characterized by a fourteen-membered cyclotetrapeptide where a 4-aminoproline scaffold (Ampro) is grafted onto the Arg-Gly-Asp moiety. Structurally related to the known, high-affinity ligands

II and III (Chart 1), these RGD peptides have the notable presence of exocyclic free azide or amine functional groups prone to conjugation with external partners. Compounds 12−16 were synthesized from the respective amino acid building blocks in 30−44% yields, as reported.13,14 Another point of interest was the multipresentation of the ligands, since it is known that in many cases close correlation exists between the “RGD-valency” of the construct and the targeting capability enhancement.41−43 To this end, compound 18 was built, where a rigid phenolic core is twofold linked to the AmproRGD unit through robust triazolyl connections, while leaving a free amino function ready for further conjugation. Thus, ad hoc-prepared bis-alkyne 17, in turn obtained from 3,5-dihydroxy methyl benzoate and 1,2-diaminoethane according to a reported procedure,36 was reacted with azide 14 (2 mol equiv) under copper-catalyzed Hüisgen 1,3dipolar cycloaddition conditions40 to furnish, after acidic removal of the Boc protection, dimeric amine 18 in 68% yield for the two steps. Of note, RGD-cyclopeptides 12−16 and 18 were recovered as fully deprotected, >95% pure, and homogeneous materials as trifluoroacetate salts after semipreparative HPLC and proved completely water-soluble (25 °C, pH 7). Synthesis of Triazolyl-Linked Paclitaxel-AbaRGD and Paclitaxel-AmproRGD Conjugates. With alkyne-ending taxane esters 7−9 and azide-terminating cycloRGD-peptides 12, 14, and 15 in hand, the next task was to couple these modules to create the projected triazolyl-linked conjugates. The regioselective copper-catalyzed Hüisgen 1,3-dipolar cycloaddition between an azide and a terminal alkyne, often referred to as “click reaction”,40 has been reported to proceed in very good yields in t-butanol/water, a solvent mixture that appeared ideal for dissolution of both lipophilic PTX and water-soluble cyclopeptides. Thus, the “click” reactions of completely deprotected azides 12, 14, or 15 with taxane alkynes 7, 8, or 9 proceeded fairly rapidly giving the corresponding triazolyl conjugates 19−23 in 46−75% yields (Scheme 3). 1615



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Scheme 4. Synthesis of Amide-Linked Paclitaxel-AbaRGD and Paclitaxel-AmproRGD Conjugates 24−27

common AbaRGD unit linked to the taxane by different diglycolic vs succinic acid spacers. Biology. Receptor Binding Assay. The ability of the nine PTX-AbaRGD and PTX-AmproRGD conjugates 19−27 to bind to human, isolated αVβ3 and αVβ5 integrin receptors in vitro was evaluated by competitive displacement assays using biotinylated vitronectin (Table 1). To better estimate the impact of taxane-linker conjugation on the binding capability of the RGD portions, the data were compared to those of unconjugated parent RGD counterparts 12−16 and to known high-affinity binders c(RGDfV), Aba-based compound II, and Ampro-based derivative III (see Chart 1). Triazolyl conjugates 19−23 showed variable results. In particular, AbaRGD derivatives exhibited some decrease (8-fold for 19 and 7-fold for 21) or complete abrogation (for 20) of αVβ3-binding affinity with respect to the parent azide counterpart 12, while AmproRGD compounds 22 and 23 showed substantially unaltered, if not improved, one-digit nanomolar affinity and αVβ3/αVβ5 selectivity (as compared to azides 14 and 15). For diglycolic amide-linked conjugates 24 and 25, a nanomolar binding capability was observed in both cases: the former displayed a 30-fold IC50 decrease with untouched αVβ3/ αVβ5 selectivity as compared to the parent compound 13, while the latter exhibited a comparable IC50 value and 4-fold increased αVβ3/αVβ5 selectivity as compared to the parent compound 16. Succinic ester-linked Aba-conjugate 27 exhibited dual affinity for both receptors similar to triazolyl derivative 19; compound 26, bearing a 2-fold AmproRGD repeat, showed notable 0.23 nM αVβ3 binding capability, in line with the behavior observed for similar multimeric presentations.14,41−43 In this case, again, remarkable selectivity amplification was observed.

Structurally speaking, these conjugates differed from each other by the nature of the integrin-ligand moiety (e.g., AbaRGD 19−21 vs AmproRGD 22−23), by the distance of the taxane unit from the triazole (e.g., 19 vs 20 vs 21) and by the distance of the RGD unit from the triazole (e.g., 22 vs 23). Synthesis of Amide-Linked Paclitaxel-AbaRGD and Paclitaxel-AmproRGD Conjugates. For the amide coupling between primary amine RGD ligands of types 13, 16, and 18 and carboxylic acid taxanes 10 and 11, we had to face and solve some inherent, exquisitely chemical problems such as (1) the use of solvent mixtures compatible with both reacting partners; (2) the minimization of undesired side reactions between the free functional groups of the cyclopeptide and the taxane moiety, which is normally susceptible to attacks from nucleophiles; (3) the annihilation of the possibly competitive acylation at the RGD guanidinium group; and (4) acceleration of the amidation reaction as compared to unwanted competitive hydrolytic paths. Following indications given by Staros44 and Anjaneyulu,45 optimum conditions were found using the carbodiimide DIC and N-hydroxysulfosuccinimide (NHSS) activating couple in organic−aqueous media at nearly neutral pH at low temperature. In practice, as outlined in Scheme 4, DMF solutions of carboxylic acids 10 or 11 were reacted with DIC and NHSS to furnish the corresponding hydrophilic N-hydroxysulfosuccinimidyl ester intermediates. Subsequent addition of the amine component (13, 16, or 18) in a pH 7.0 phosphate buffered water/acetonitrile (1:1) solvent mixture at 0 °C ensured preparation of the respective amide conjugates 24−27 in 40− 62% yields. From a structural viewpoint, amides 24 and 25 share a common diglycolic ester link but differ from each other in their targeting RGD unit, while compound 26 is the dimeric version of its counterpart 25. Finally, compounds 24 and 27 exhibit a 1616



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Table 1. Solid-Phase Receptor Binding Assaya

IC50 (nM) ± SD αVβ3

compound 19 20 21 22 23 24 25 26 27 12 13 14 15 16 c(RGDfV) II III vitronectin

266 >10−5 220.0 4.3 2.3 35.6 8.9 0.23 237 32.6 1077 6.0 6.4 6.1 3.2 53.7 2.4 47.1

αVβ5

± 75 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13.2 1.6 2.0 9.7 0.9 0.012 89 15.7 624 1.5 2.4 1.6 1.3 17.3 1.0 10.0

330 >10−5 805 187 82 344 1909 725.0 219 98.3 11300 1208 38 315 7.5 205 38 13.7

± 54 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

309 65 16 196 351 234.4 51 51.4 1480 373 20 137 4.8 33.5 11 6.2

a IC50 values were calculated as the concentration of compound required for 50% inhibition of biotinylated vitronectin binding to human, isolated αVβ3 and αVβ5 integrin receptors. High-affinity integrin binders c(RGDfV), II and III, were used as positive controls.

As a sole exception, compound 20 exhibited a completely annihilated binding affinity, possibly due to the presence of a long, flexible hydrocarbon linker chain, which would allow folding of the cytotoxic unit onto the RGD portion, thereby suppressing its integrin recognizing capability. In Vitro Cell Sensitivity Assays. The ability of PTX conjugates 19−27 to inhibit growth of tumor cells was assessed using a growth inhibition assay (72 h exposure) in which the effect of free PTX 1 was also examined. Cell sensitivity to the different conjugates was evaluated using cell lines characterized

Overall, the presence of a common taxane unit and chemically different linkers covalently attached to the integrin-recognizing RGD units led to appreciable results, with satisfactory to excellent αVβ3 binding capabilities (in the 266−0.23 nM range) which were not generally compromized by the added cargo. It appears that interposition of a flexible chain (e.g., ethylene) between the RGD moiety and the linker is favorable for affinity (e.g., 22, 23, 25, and 26); also, the presence of the amide-attached diglycolic unit seems to be beneficial to the overall binding capability (e.g., 24, 25, and 26). 1617



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22 and 23, displaying very good in vitro αVβ3 affinity, the cell sensitivity of the different cell lines was variable, reaching in some cases interesting results. Conjugates 24 and 25, bearing diglycolic amide linkers, show nonflattened one-to-two-digit nanomolar IC50 values for all cell lines; interestingly enough, treatment with compound 24 inhibited proliferation of ovarian carcinoma IGROV-1 cells at a lower IC50 as compared to free PTX (1.04 nM vs 23.4 nM) and this could be due to an additive cytotoxic effect deriving from both PTX and the RGD unit itself. Compound 26, bearing two RGDs per PTX, represents a dimeric version of 25 and displays superior in vitro αvβ3-targeting capabilities. Accordingly, 26 shows a 2-fold cytotoxic activity as compared to 25, supporting the hypothesis of active directioning and αVβ3-mediated endocytosis. Unconjugated integrin ligands 13 and 16 do not show any sign of cytotoxicity in the present assay. In Vivo Antitumor Activity. Due to the good growth inhibition capability (slightly improved in comparison to PTX) displayed in vitro by compound 21, it was selected to be further examined for in vivo studies. When mice were xenografted s.c. with IGROV-1/Pt1, a significant inhibition of tumor growth was observed following 21 administration. The compound was administered i.v. (36 mg/kg) every 4 days for 4 times. Under these conditions, the TVI (tumor volume inhibition) was 98% (Figure 1, Table 4, P < 0.05 vs PTX-treated mice). PTX used at the same treatment conditions displayed a lower antitumor effect. An improved antitumor activity of 21 was also indicated by the LCK (log cell kill) value. Histological analysis highlighted an exceedingly higher number of mitoses in the group treated with compound 21, compared to other groups (Figure 2). The majority of the mitoses observed in the group treated with compound 21 and in the group treated with PTX were aberrant, an observation consistent with the mechanism of action of the drug.46−48 Examples are provided in Figure 3.

by a variable expression of αvβ3 and αvβ5 integrins. In U2-OS cells, the basal expression of αvβ5 was more marked than that of all other cell systems, whereas the cell line displaying the highest level of expression of αvβ3 was the cisplatin-resistant IGROV-1/Pt1 subline (Table 2). For this reason, the IGROVTable 2. Cytofluorimetric Analysis of αVβ3 and αVβ5 Levels in Cell Lines of Different Tumor Types relative mean fluorescence intensitya cell line H460 U2-OS IGROV-1 IGROV-1/Pt1

αVβ3 1.1 2.43 2.70 13.27

± ± ± ±

αVβ5 0.5 0.3 0.6 4.9

3.36 27.3 4.33 2.8

± ± ± ±

1.1 1.4 1.7 0.3

The value is the ratio between mean fluorescence intensity of cells incubated with primary antibody and that of cells incubated with isotypic control. a

1/Pt1 cell model was employed for subsequent in vivo studies. As detailed in Table 3, all conjugate compounds 19−27 showed an antiproliferative effect which varies from low nanomolar to micromolar IC50 values, indirectly supporting a possible cleavage and release of the PTX drug from the conjugate in the 72 h exposure time.39 Triazolyl-linked compound 20, which does not possess appreciable binding affinity toward the αVβ3/ αVβ5 integrins, may be regarded as a nondirecting control; the IC50 values of 20 are the highest in the series along all tested tumor cell lines supporting the belief of a close correlation between overall antiproliferative activity and αV β3 /αV β5 recognition capability. Residual, poor activity could be ascribed to aspecific delivery of the drug. Compound 19, a shortened homologue of 20, is endowed with improved targeting ability, and accordingly, the IC50 values appear appreciably improved, though still far from action of the free drug. Derivative 21, which presents a robust triazole ring connected to ethylene glycol units by an amide function, shows flattened, lownanomolar IC50 values that are comparable, if not superior, to those of free PTX; and this could be imputable either to excellent active targeting and αVβ3-mediated internalization or, simply, to premature and complete release of the cytotoxic cargo during the 72 h exposure. For robust triazolyl compounds

■

DISCUSSION In the present study, we report the synthesis and biological evaluation of nine PTX conjugates characterized by structural novelty due to Aba-based or Ampro-based RGD moieties which are joined as C2′ PTX esters through variable triazolyl- or amide-connecting moieties. A number of molecules for which

Table 3. Cell Sensitivity of Cells with Variable Expression of Integrins to cRGD Ligand Conjugatesa IC50 (nM) ± SD compound 19 20 21 22 23 24 25 26 27 PTX 13 16

U2-OSb 107 244 2.0 nd 28.0 10.1 nd nd 6.36 3.4 >4000 >50 200

± 27 ± 20 ± 1.1 ± 6.7 ± 3.3

± 0.18 ± 0.5

IGROV-1c 541 2870 1.6 277 333.4 1.04 43.1 28.0 81.0 23.4 22 300 >16 700

± ± ± ± ± ± ± ± ± ±

334 822 0.9 180.1 200 0.47 6.8 2.2 6.8 8.2

IGROV-1/Pt1d 140 219 1.6 11.7 73.3 10.1 3.4 1.6 5.8 2.2 >30 000 >16 700

± ± ± ± ± ± ± ± ± ±

53 63 1.1 6.2 6.7 4.7 2.4 0.5 0.5 0.8

H460e 58 236 9.5 nd nd 13.1 nd nd 6.9 5.4 >4000 nd

± 27 ± 79 ± 2.2

± 2.7

± 1.4 ± 1.3

a Cell sensitivity was evaluated by growth inhibition assays based on cell counting after 72 h exposure. IC50 is defined as the drug concentration inhibiting cell growth by 50%. bHuman osteosarcoma. cHuman ovarian adenocarcinoma. dCisplatin resistant human ovarian adenocarcinoma. e Human large cell lung carcinoma.

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Figure 1. Antitumor activity studies of compound 21. Efficacy of i.v. 21 (●) and paclitaxel (▲) administered at 36 mg/kg every fourth day four times on the ovarian carcinoma IGROV-1/Pt1 xenografted s.c. in athymic nude mice. Solvent was injected for the control group (○). Each point represents the mean tumor volume from 9 to 11 tumors. Bars represent SD.

Table 4. Efficacy of i.v. 21 and PTX (36 mg/kg q4d×4) on the Ovarian Carcinoma IGROV-1/Pt1 Xenografted s.c. in Athymic Nude Mice compound

TVI%a

LCKb

BWL%c

Toxd

21 PTX

98 81

2 0.7

7 5

0/5 0/5

a

Tumor volume inhibition % in treated over control mice assessed 7 days after last treatment. bGross log10 cell kill to reach 700 mm3 of tumor volume. cBody weight loss % induced by treatment; the highest change is reported. dDead/treated mice.

Figure 2. Comparison between normal (■) and aberrant (□) mitoses in different groups of treatment. Mitoses were evaluated in 3 randomly selected 400× fields using quadruplicate samples. The reported numbers correspond to the total number of normal/aberrant mitoses in analyzed group 1, control group; 2, group treated with ligand 13; 3, group treated with compound 21; 4, group treated with PTX. Group treated with compound 21 has the highest number of mitoses and in the meanwhile the highest number of aberrant mitoses (p < 0.05).

Figure 3. Randomly selected fields of (A) G1 sample, characterized by up to two “normal” mitoses (hematoxylin and eosin; Bar, 50 μm); (B) sample from mice treated with compound 21. Note the central aberrant mitosis, characterized by mis-segregated chromosomes within a nuclear envelope (mitotic catastrophe), surrounded by a large number of small condensed hyperchromatin nuclei, both examples of “atypical” mitoses (hematoxylin and eosin; Bar, 50 μm); (C) G4 sample. In the center, there is an aberrant mitosis, characterized by mis-segregated chromosomes surrounded by a nuclear envelope (mitotic catastrophe) (hematoxylin and eosin; Bar, 50 μm).

conjugation did not negatively influence binding to integrins was obtained. Such molecules were also endowed with marked cytotoxic activity in the range of that observed for PTX. In fact, cell sensitivity assays toward αVβ3/αVβ5-overexpressing human tumor cell lines support that PTX preserves or improves its cytotoxic activity when delivered through the covalent conjugates 19−27, thus indirectly supporting its release as a free drug.39 Whether it is prematurely released outside the cell 1619



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moieties which are joined as C2′ PTX esters through variable triazolyl- or amide-connecting moieties. The main goals of the work have been accomplished. The synthesis proved viable and faithful, efficiently furnishing the nine conjugates in 38−74% yields from the taxane and respective RGD starting modules. Purification and complete structural characterization of the final products guaranteed reliability during subsequent biological tests. Preservation of high in vitro αVβ3-receptor affinity observed for almost all conjugate candidates in the series as compared to free RGD-counterparts demonstrated that the cytotoxic cargo and/or the linker unit may comfortably coexist with the RGD moiety without damaging the exquisite αVβ3selective targeting capability. In vitro growth inhibition assays on a panel of αVβ3/αVβ5-overexpressing human tumor cell lines showed remarkable cytotoxic activity. Finally, in vivo evaluation of conjugate 21 confirmed the effectiveness of this compound to inhibit tumor growth, displaying an enhanced activity in comparison to that of free Paclitaxel. This result is further strengthened by the fact that the effective quantity of Paclitaxel administered to the animals treated with compound 21 is almost half the given doses. Favorable indications of the present work incite further investigations (e.g., evaluation of metabolic stability, toxicity, and actual drug release localization) to be performed on selected candidates in order to gain insight into optimal structural compromise between selective targeting ability, incell drug release capability, and overall solubility/permeability in human tissues.

and subsequently internalized through cell membrane diffusion or it is actively targeted and released inside the cell following integrin-mediated endocytosis, it is hard to definitely conclude here. Observation of nonflattened and nonhomogeneous data in the tested assay (with exception of compound 21), which are closely related to the corresponding affinity toward the receptors, favors the second hypothesis. Though the details of the release process remain to be defined, it would seem likely that the PTX-AbaRGD and PTXAmproRGD junction is a well-done marriage where the partners, more than simply tolerant, cooperate with each other. Last, during in vivo assays performed on compound 21, outstanding control of tumor growth (TVI 98% for 21 vs 81% for PTX at 25 day) in a model overexpressing αVβ3 was achieved at well-tolerated doses of the conjugate (36 mg/kg). This result is even more impressive if we consider that the effective amount of PTX administered to the animals treated with compound 21 is 17 mg/kg (the MW being almost double). Since tumors from mice treated with compound 21 had the highest number of mitoses and the major part of them were atypical, it can be speculated that neoplastic cells treated with compound 21 enter mitosis but fail to replicate and incur in mitotic arrest, as already described in the current literature for “spindle poisons” such as PTX. As mentioned in the Introduction, a few studies exist where cyclopeptide RGD moietiesnamely, dimeric E-[c(RGDfK) 2 ] or E-[c(RGDyK)2]were covalently conjugated to PTX in its 2′OH position through a succinate spacer.27−29 In a first contribution, Chen and Neamati28 tested their PTX-RGD construct against metastatic breast cancer cells (MDA-MB-438) showing a cell proliferation inhibition comparable to that observed for paclitaxel and a slightly decreased integrin binding affinity with respect to the unconjugated counterpart. In vivo and ex vivo data of the same conjugate were reported shortly afterward29 and its antitumor therapeutic effect compared to simple PTX/RGD combinations. In this case, although the tritiated conjugate showed higher tumor uptake and longer retention of PTX in the MDA-MB-438 tumor than PTX alone, the absolute tumor uptake value was still rather low. The E[c(RGDfK)2]-PTX conjugate was also evaluated by SatchiFainaro and Kratz et al.27 demonstrating that in a standard 72 h assay it inhibited the proliferation of HUVECs in a similar manner as free PTX (IC50 0.4 nM) suggesting complete hydrolysis of the succinate ester under the tested conditions. Furthermore, in vivo studies in a OVCAR-3 xenograft model demonstrated no antitumor efficacy. Here, the connection of robust semipeptidic and highly αVβ3selective RGD ligands to PTX through chemically diverse tethers gave rise to constitutionally and geometrically diverse conjugate products whose varied physical and chemical properties were anticipated to significantly impact the biological response. Indeed, the diversified, yet interesting in vitro cytotoxic activity as well as the outstanding in vivo antitumor response obtained in this work seem unique in the field; and this strengthens the notion that the Aba- and Ampro-based RGD semipeptides may be conveniently added in the list of the integrin-targeting moieties for the construction of effective and selective antitumor and anti-angiogenesis agents.

■

ASSOCIATED CONTENT

S Supporting Information *

Characterization data, HPLC traces, and copies of 1H NMR spectra of RGD conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Leonardo Manzoni: Tel. (+39) 02 50314064, Fax (+39) 02 50314072, e-mail: [email protected]. Franca Zanardi: Tel. (+39) 0521 905067, Fax (+39) 0521 905006, e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by Associazione Italiana per la Ricerca sul Cancro (Milano, Italy), Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR, PRIN 2008), and Fondazione Banco di Sardegna. Thanks are due to the Centro Interdipartimentale Misure “G. Casnati” (Università degli Studi di Parma) for instrumental facilities, and to “Regione Autonoma della Sardegna” for research fellowships to P.B. and P.C. (PO Sardegna FSE 2007-2013, L.R. 7/2007 “Promozione della ricerca scientifica e dell’innovazione tecnologica in Sardegna”).

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REFERENCES

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CONCLUSION The study focused on the synthesis and biological evaluation of nine PTX-conjugates compounds 19−27, whose structural novelty is represented by Aba-based or Ampro-based RGD 1620



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Design, Synthesis, and Biological Evaluation of Novel cRGD

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