Synthesis and Evaluation of Folate-Based Chlorambucil Delivery

Article pubs.acs.org/bc

Synthesis and Evaluation of Folate-Based Chlorambucil Delivery Systems for Tumor-Targeted Chemotherapy Annalisa Guaragna,*,†,‡ Angela Chiaviello,§,‡ Concetta Paolella,† Daniele D’Alonzo,† Giuseppe Palumbo,§ and Giovanni Palumbo† †

Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cinthia, 4 I-80126 Napoli, Italy Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università di Napoli Federico II, via S. Pansini, 5 I-80131 Napoli, Italy

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ABSTRACT: The development of tumor-targeting drug delivery systems, able to selectively transport cytotoxic agents into the tumor site by exploiting subtle morphological and physiological differences between healthy and malignant cells, currently stands as one of the most attractive anticancer strategies used to overcome the selectivity problems of conventional chemotherapy. Owing to frequent overexpression of folate receptors (FRs) on the surface of malignant cells, conjugation of cytotoxic agents to folic acid (FA) via suitable linkers have demonstrated to enhance selective drug delivery to the tumor site. Herein, the chemical synthesis and biological evaluation of two novel folate-conjugates bearing the anticancer agent chlorambucil (CLB) tethered to either an aminoether (4,7,10-trioxa-1,13-tridecanediamine) or a pseudo-β-dipeptide (β-Ala-ED-β-Ala) linker is reported. The two drug delivery systems have been prepared in high overall yields (54% and 34%) through straightforward and versatile synthetic routes. Evaluation of cell specificity was examined using three leukemic cell lines, undifferentiated U937 (not overexpressing FRs, FR−), TPA-differentiated U937 (overexpressing FRs, FR+), and TK6 (FR+) cells. Both conjugates exhibited high specificity only to FR+ cells (particularly TK6), demonstrating comparable antitumor activity to CLB in its free form. These data confirm the reliability of folate-based drug delivery systems for targeted antitumor therapy; likewise, they lay the foundations for the development of other folate-conjugates with antitumor potential.

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missiles” or “molecular Trojan horses”) have been deeply investigated over the last years.2 A tumor-targeting drug delivery system typically consists of a tumor recognition moiety and a cytotoxic warhead connected directly, or through a suitable linker, to form a conjugate which is inactive and sufficiently stable while in circulation; then, once selectively internalized into the cancer cell, cleavage of the conjugate restores the active cytotoxic warhead. The devised mechanism enables the conjugate to exhibit a strongly selective antitumor effect; however, to efficiently deliver the drug to the tumor site, it is important to have a detailed knowledge of the specific morphological and physiological differences distinguishing malignant from healthy tissues. Rapidly dividing cancer cells require various nutrients and vitamins to maintain their rapid growth. Therefore, those receptors involved in the uptake of the essential vitamins for cell growth are often overexpressed on the surface of malignant cells. This key finding has allowed identification of useful biomarkers either for the imaging and detection of tumor cells or for the delivery of

INTRODUCTION With the exception of coronary and heart diseases, cancer remains the major cause of death in the Western world. Despite significant progress in the development of anticancer technologies (involving tumor detection, prevention, surgery, and chemotherapeutic treatments), there is still no efficient cure for patients with malignant diseases. One of the major drawbacks in anticancer chemotherapy is the lack of selectivity of cytotoxic drugs, which usually do not accurately discriminate between healthy and malignant cells, leading to systemic toxicity. To limit the onset of severe side effects, anticancer chemotherapeutics are often given at suboptimal doses, thus hampering their ability to reach tumor-destroying drug concentrations while prompting, after prolonged treatments, the development of resistance phenomena. Therefore, the construction of innovative tumor-specific drug delivery systems is urgently needed. Several approaches1 aimed at improving selectivity of anticancer agents are currently being pursued: they are mostly based on subtle biochemical differences discriminating healthy from malignant cells. Among such approaches, those involving conjugation of the drug to tumor-specific ligands (the so-called “guided molecular © 2011 American Chemical Society

Received: August 12, 2011 Revised: October 31, 2011 Published: November 28, 2011 84

dx.doi.org/10.1021/bc200410d | Bioconjugate Chem. 2012, 23, 84−96

Bioconjugate Chemistry

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Figure 1. FR-mediated endocytosis and trafficking.

Chart 1. Tumor-Targeting Drug-Delivery Systems 1 and 2

cytotoxic agents to tumors.3 Among such receptors, those devoted to the transport of vitamin B12,4 biotin,5,6 and riboflavin7 have been quite recently introduced as methods for targeted drug delivery. On the other hand, it has long been recognized that folate receptors (FRs) are excellent biomarkers to this end.8−10 Folic acid (FA) is the natural vitamin B9 (Mr = 441): it is required by all living cells, especially those rapidly replicating, because it is necessary for the de novo nucleotide biosynthesis and one-carbon metabolism. It is a stable, inexpensive, and generally poorly immunogenic chemical endowed with a remarkable affinity for FRs (Kd ∼ 0.1−1.0

nmol/L). FRs are glycosyl-phosphatidylinositol (GPI)-anchored 38 kDa membrane glycoproteins11 which exist in three main isoforms,12 namely, FR-α, FR-β, and FR-γ. Among them, FR-α is often up-regulated on cell surface of a wide variety of human carcinomas, including ovary, brain, kidney, breast, colon, and lung malignancies; but it is more rarely expressed on most normal cells. FR-β is expressed on activated macrophages and on the surface of malignant cells of hematopoietic origin. Since FA enters cells by receptor-mediated endocytosis (Figure 1), a striking consequence is that those FRs expressed by cancer cells can be exploited to selectively convey specific anticancer drugs.7 85



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spectrometers. MS spectra were recorded on a Voyager DEPRO MALDI-TOF mass spectrometer. Biology. Cell Lines. The p53-null U937 human leukemic monocyte lymphoma and the p53-positive isogenic human lymphoblast TK6 cell lines were obtained from American Type Culture Collection (Rockville, MD). Both cells lines grew in Dulbecco’s Modified Eagle Medium, 2 mM L-glutamine, 100 μg/mL streptomycin, 100 units/mL penicillin, and 10% Foetal Calf Serum (FCS). All media and cell culture reagents were purchased from Life Technologies (San Giuliano Milanese, Italy). Cell Differentiation. Differentiation of the promyelocytic cell line U937 cells into macrophages was achieved according to Sordet et al. 20 by treating cells with 20 nM 12-Otetradecanoylphorbol-13-acetate (TPA) for 24 h. At the end of this time, the differentiation process was considered complete as indicated by the massive attachment of cells to the plate. TPA was obtained by Sigma Aldrich. A 100 μM stock solution was prepared in 100% DMSO. CLB and FA-CLB Conjugates Stock Solutions. CLB was dissolved in 100% DMSO to obtain a concentrated (75 mM) stock solution. Before measurements, appropriate aliquots of this solution were diluted with Dulbecco’s complete medium to the desired concentration. Stock solutions of the same concentration (75 mM) of folate-trioxa-CLB and folate-(βAla-ED-βAla)-CLB stock solutions were (both) also obtained by dissolving the powders in 100% DMSO under continuous stirring at room temperature overnight in the dark. These concentrated solutions were stored at 4 °C wrapped in aluminum foil. Before use, appropriate amounts of each stock solution were diluted with Dulbecco’s complete medium to the desired concentration. Metabolic Activity. Metabolic activity was assayed by using the Cell Proliferation Kit II (XTT, Roche, Milan Italy). The assay is based on cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by metabolic active cells.21 Therefore, this conversion only occurs in viable cells. Normally 1 × 104 cells/well were seeded into 96-well plates and incubated for 24, 48, and 72 h with CLB, folate-trioxa-CLB, or folate-(βAla-ED-βAla)-CLB at established concentrations and then analyzed in triplicate. Stability of FA-CLB Conjugates in DMSO Solutions. To assess the time-stability of CLB-folate derivatives stock solutions, we evaluated their biological effects on cell metabolic activity immediately after their preparation or after several weeks of storage. The changes in cytotoxic activity on U937 and TK6 cells of freshly prepared or stored (5 weeks) stock solutions (both 75 mM) were evaluated by comparing the residual metabolic activity of these cells (XTT assay) following 48 h treatment with scalar doses (normally 0−50 μM) of each compound. As concentrated solutions are normally more stable than the diluted ones, the check of stability and full retention of cytotoxic activity was also performed starting from 100× more diluted stock solutions (i.e., 0.75 mM in 33% DMSO). Even in this case, the assays were performed immediately after their preparation or after several weeks of storage at 4 °C (5 weeks). Stability of FA-CLB Conjugates in Human Serum. To test the stability of FA-conjugates in human serum, we have diluted appropriate amounts of stock solutions of folate-trioxa-CLB or folate-(βAla-ED-βAla)-CLB in human serum so that the final concentrations of the drugs were in both cases 0.5 mmol/L. These CLB-containing solutions, used as drug sources, were then incubated at 37 °C for 12 h and finally used to treat

Tumor-targeting FA conjugates covalently linked via folate’s γ-carboxyl moiety maintain a high affinity for the FRs, and the mechanism of cellular uptake of folate conjugates by FRs is as effective as that displayed by FA in its free form.13 Despite their molecular complexity, folate-conjugates can still enter cells by FR-mediated endocytosis11 and move through many organelles supplying transported materials to cell cytoplasm. The drug is then released in the endosomes/lysosomes mainly by enzymatic cleavage (owing to the acidic environment occurring in lysosomes, pH-dependent drug release has also been extensively considered).1,8 Due to recycling of the unligated FR back to the cell surface, the uptake process can be reiterated, allowing continuous supply of folate-linked drugs into the cell (Figure 1). Over the years, the intriguing potential of folate conjugates in targeted anticancer chemotherapy has been witnessed by the number of preclinical and clinical studies14−17 of several drug delivery systems obtained varying both the cytotoxic warhead18 (depending on the type of target tumor cells) and the biostable linker.1,19 Our approach to the synthesis of folate conjugates has been focused on the construction of molecular systems bearing two novelty elements: on one hand, we have chosen the potent anticancer agent chlorambucil (CLB) as cytotoxic warhead, to evaluate its efficacy in folate-based antitumor chemotherapy. On the other hand, we have considered the introduction of β-amino acids in the spacer, to study their potential to work as biostable linkers in these drug delivery systems. Based on these aims, we conceived the synthesis and biological evaluation of the two antitumor systems 1 and 2 (Chart 1), where FA is connected through suitable linkers (aminoether and pseudo β-peptide spacers) to the cytotoxic agent CLB. While the presence of FA ensures appropriate selectivity to tumor cells to the resulting conjugates, CLB is amenable to being released at intracellular level since the amide linkage originated from the COOH terminus of CLB and the amino function of each linker should be easily cleaved by lysosomal proteases. The results reported below are evidence for how our molecular biosystems are characterized by the ability to specifically recognize, target, and kill tumor cells expressing FRs.

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EXPERIMENTAL PROCEDURES Materials and Methods. Chemistry. All moisture-sensitive reactions were performed under nitrogen atmosphere using oven-dried glassware. Solvents were dried over standard drying agents and freshly distilled prior to use. Triethylamine (TEA) and N,N-diisopropylethylamine (DIPEA) were redistilled from NaOH. N,N′-Dicyclohexylcarbodiimide (DCC) and folic acid (FA) were purchased from Sigma-Aldrich Inc. Chlorambucil (CLB), dimethyl sulfoxide (DMSO, anhydrous), and pyridine (Py, anhydrous) were used as purchased (Fluka Chemical Co.) without further purification. Reactions were monitored by TLC (precoated silica gel plate F254, Merck). Column chromatography: Merck Kieselgel 60 (70−230 mesh); flash chromatography: Merck Kieselgel 60 (230−400 mesh). Purity of all reaction intermediates and target molecules (>95%) was established by combustion analyses, which were performed by using a CHNS analyzer. Structure determination of all new compounds was carried out by NMR and MS analysis. 1H and 13 C NMR spectra were recorded with the following instruments: Varian Gemini (200 MHz), Varian Gemini (300 MHz), Bruker DRX (400 MHz), and Varian Inova (500 MHz) 86



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data for comparisons between two means. Statistical significance was defined as *, p < 0.01; **, p < 0.001; ***, p < 0.0001. Synthetic Procedures and Characterization Data for Intermediates and Conjugates. Compound 4. A stirring solution of 4,7,10-trioxa-1,13-tridecanediamine (3, 2.0 g, 9.1 mmol) in anhydrous CH3OH (152.0 mL) was treated with Boc2O (2.0 g, 9.1 mmol) and TEA (2.9 mL, 21.0 mmol). The reaction mixture was left at reflux for 16 h. The solvent was removed under reduced pressure and the resulting yellow oil was purified by silica gel chromatography (CHCl3/CH3OH/ NH4OH = 89:10:1) to give pure 4 (2.5 g, 86% yield). Oily; 1H NMR (300 MHz, CD3OD) δ: 1.44 (s, 9H), 1,73−1,78 (m, 2H), 1.90−1.96 (m, 2H), 2.03 (bs, 2H), 2.90 (t, 2H, J = 6.7 Hz), 3.12 (t, 2H, J = 6.0 Hz), 3.52 (t, 2H, J = 6.0 Hz), 3.56− 3.68 (m, 10H), 5.1 (bs, 1H). 13C NMR (100 MHz, CDCl3) ppm: 28.2, 29.3, 32.7, 38.3, 39.3, 69.2, 69.8, 69.9, 70.2, 70.3 78.2, 156.2. MALDI-TOF MS: m/z 320.23 (calcd); 321.20 [M +H]+ (found). Anal. Calcd for C15H32N2O5: C, 56.23; H, 10.07; N, 8.74. Found: C, 56.40; H, 10.03; N, 8.71. Compound 7. To a stirring solution of FA (5, 0.25 g, 0.56 mmol) in an anhydrous DMF:Py (5:1, 24 mL) solution, DCC (0.70 g, 3.4 mmol) was added in one portion. The reaction mixture was kept in an ultrasound bath in the dark for 30 min. Then, the resulting suspension was quickly filtered over a sintered funnel and the precipitate washed with a minimum amount of anhydrous DMF:Py solution. The N-Boc-4,7,10trioxa-1,13-tridecanediamine (4) (0.18 g, 0.6 mmol) was then added to the filtrate. The resulting reaction mixture was further stirred in the dark for 16 h. Afterward, it was poured dropwise into a stirred solution of cold diethyl ether/acetone (70:30): a yellow precipitate was formed and collected on a sintered glass funnel. After washing several times with acetone and ether, the material was dried to give 7 as a yellow powder (0.35 g, 85% yield) which was used in the next step without further purification. Mp 209−210 °C; 1H NMR (200 MHz, DMSO-d6) δ: 1.34 (s, 9H), 1.49−1.65 (m, 4H), 1.80−2.05 (m, 2H), 2.12− 2.34 (m, 2H), 2.86 (dd, 2H, J = 6.5, 12.4 Hz), 3.05 (bt, 2H, J = 6.1 Hz), 3.25−3.40 (m, 4H), 3.41−3.50 (m, 8H), 4.20−4.38 (m, 1H), 4.46 (bd, 2H, J = 5.4 Hz), 6.61 (bd, 2H, J = 7.6 Hz), 6.75−6.98 (m, 3H), 7.63 (bd, 2H, J = 7.6 Hz), 7.76−7.86 (m, 1H), 7.90 (bd, 1H, J = 8.4 Hz), 8.62 (bs, 1H), 11.4 (s, 1H). 13C NMR (100 MHz, DMSO-d6): ppm 29.4, 30.0, 30.4, 31.2, 36.6, 37.9, 46.7, 52.9, 68.8, 70.4, 70.5, 78.1, 111.1, 122.2, 128.7, 129.7, 149.3, 151.5, 154.5, 156.3, 156.7, 161.7, 166.9, 172.4, 174.6, 174.9. MALDI-TOF MS: m/z 743.36 (calcd); 766.20 [M+Na] + , 782.20 [M+K] + (found). Anal. Calcd for C34H49N9O10: C, 54.90; H, 6.64; N, 16.95. Found: C, 55.04; H, 6.62; N, 16.89. Compound 8. TFA (1.7 mL) was added to 7 (250 mg, 0.33 mmol) at 0 °C and under magnetic stirring. The resulting mixture was stirred at room temperature for 2 h, then TFA was evaporated under vacuum and the resulting residue was dissolved in a small amount of anhydrous DMF. Pyridine was added dropwise until complete formation of a yellow precipitate, which was collected by filtration, washed with Et2O and dried under vacuum to yield amine 8 (0.42 g, 99% yield). Yellow powder, Mp 205−207 °C; 1H NMR (500 MHz, CD3OD) δ: 1.44−1.62 (m, 2H), 1.70−1.80 (m, 2H), 1.82− 2.00 (m, 2H), 2.05−2.17 (m, 2H), 2.84 (bt, 2H, J = 7.4 Hz), 2.98−3.07 (m, 2H), 3.18−3.38 (m, 2H), 3.40−3.57 (m, 10H), 4.20−4.37 (m, 1H), 4.46 (d, 2H, J = 5.8 Hz), 6.63 (d, 2H, J = 7.4 Hz), 6.80−6.98 (m, 3H), 7.65 (bd, 2H, J = 7.4 Hz), 7.85

undifferentiated U937 and TK6 cells. Analysis of cell metabolic activity by XTT assay was performed 48 h later. In these experiments, CLB in free (control) or conjugated forms was 30 μM. IC50 Assessment. The IC50 estimations of CLB and FAconjugates were done using a point to point method according to Aloyz and co-workers,22 except for the fact that the XTT assay was used in place of the MTT assay. IC50 values were derived using linear interpolation from dose response curves in which mean viability values were plotted against the drug concentrations. Straight lines between consecutive points were drawn and the IC50 calculated where this line crosses the value representing the 50% of residual viability. In particular, IC50 determinations were performed on TK6 or U937 cells incubated for 24, 48, and 72 h. Competition Assay. In order to show that FA and FAconjugates compete for a single class of receptors, we preincubated about 104 either receptor positive TPA-differentiated and TK6 cells or receptor negative cells (U937) with 1.5 mM FA. After one hour in this condition, we added to each cell line conjugates 1 and 2 and free CLB (all 30 μM). At the end of this incubation (72 h), the XTT assay was performed as indicated before. Flow Cytometry. Undifferentiated or TPA-activated U937 cells and TK6 cells were incubated individually for 48 h with CLB or with its folate conjugated forms (30 μM). After incubation, cells were washed twice with 1 mL phosphate saline buffer pH 7.4 (PBS), and resuspended and fixed in 70% ethanol. Before analysis, fixed cells were washed, centrifuged, and resuspended in 1 mL PBS containing 1 μg RNase and 100 μg propidium iodide.23 Samples were stored in the dark for 20 min at room temperature before final readings. The cellular orange fluorescence of propidium iodide was detected in a linear scale using a CyAn ADP Flow Cytometer (DAKOCytomation, Ely, UK) and analyzed by using ModFit/LT software (Verity Software, Topsham, ME). About 30 000 events (i.e., fluorescence readings, corresponding to not less than 20 000 cells) were recorded for each sample. Electrophoresis and Western Blot Analysis. Western blot analysis was always performed on cells treated for 48 h with 30 μM CLB or CLB conjugates. Typically, total cellular protein extracts were obtained by lysing cells in 50 mM Tris (pH 7.5), 100 mM NaCl, 1% NP40, 0.1% Triton X 100, 2 mM EDTA, 10 μg/mL aprotinin, and 100 μg/mL phenylmethylsulfonyl fluoride. Protein concentration was routinely measured with the Bio-Rad protein assay. Polyacrylamide gels (10% or 15%) were prepared essentially as described by Laemmli.24 Molecular weight standards were from New England Biolabs (Beverly, MA, USA). Proteins separated on polyacrylamide gels were blotted onto nitrocellulose filters (Hybond-C pure, Amersham Italia, Milan, Italy). Filters were washed and stained with specific primary antibodies and then with secondary antisera conjugated with horseradish peroxidase (Bio-Rad; diluted 1:2000). Filters were developed using an electro-chemiluminescent Western blotting detection reagent (Amersham Italia, Milan, Italy); profiles were acquired and grossly quantified by scanning with a Discover Pharmacia scanner equipped with a Sun Spark Classic Workstation. The anti-p53 (DO1), anti-Bax (P-19), and anti-Clusterin (N-18) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antitubulin (MCA77G) was from Serotec (Kidlington, UK). Statistical Analysis. All data are expressed as mean ± SD. Significance was assessed by the Student’s t test for unpaired 87



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Scheme 1. Synthesis of Conjugate 1

(bd, 1H, J = 8.0 Hz), 8.65 (s, 1H), 11.38 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): ppm 27.2, 29.3, 30.6, 35.6, 36.9, 46.2, 53.8, 67.4, 68.0, 69.5, 69.6, 69.7, 111.2, 121.4, 128.3, 129.4, 148.3, 151.0, 153.3, 158.5, 160.8, 166.5, 172.1, 174.4. MALDITOF MS: m/z 643.31 (calcd); 666.40 [M+Na]+, 682.34 [M +K]+ (found). Anal. Calcd for C29H41N9O8: C, 54.11; H, 6.42; N, 19.58. Found: C, 54.29; H, 6.40; N, 19.50. Compound 1. To a stirring solution of CLB (0.46 g, 1.5 mmol) in anhydrous DMSO (5.0 mL), DIPEA (0.26 mL, 1.5 mmol), 8 (0.32 g, 0.5 mmol) and PyBOP (0.78 g, 1.5 mmol) were sequentially added. The resulting mixture was stirred for 16 h at room temperature. Then, it was poured dropwise into a stirred solution of cold diethyl ether/acetone (70:30): a yellow precipitate was formed and collected on a sintered glass funnel. After washing several times with acetone, ether, and finally chloroform, the material was dried to give 1 as a brownish yellow solid (0.38 g, 75% yield). 1H NMR (500 MHz, DMSOd6) δ: 1.48−1.63 (m, 4H), 1.63−1.78 (m, 2H), 1.79−1.98 (m, 2H), 2.05 (appt, 2H, J = 7.3 Hz), 2.10−2.22 (m, 2H), 2.44 (appt, 2H, J = 7.3 Hz), 3.03−3.18 (m, 2H), 3.15−3.50 (m, 14H), 3.78 (bs, 4H), 4.23−4.40 (m, 1H), 4.52 (bd, 2H, J = 4.1 Hz), 6.63−6.76 (m, 4H), 6.83−6.98 (m, 2H), 7.02 (d, 2H, J = 7.0 Hz), 7.65 (t, 1H, J = 8.0 Hz), 7.76−7.87 (m, 2H), 7.93 (bd, 1H, J = 6.6 Hz), 8.02−8.23 (m, 1H), 8.63 (s, 1H), 11.43 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): ppm 27.0, 29.4, 30.6, 32.0, 33.8, 34.8, 35.6, 35.9, 40.4, 45.8, 52.2, 53.6, 58.5, 67.3, 68.0, 69.5, 69.7, 111.1, 111.9, 121.4, 127.9, 128.9, 129.5, 138.4, 144.4, 148.4, 150.7, 153.4, 155.1, 159.0, 160.9, 166.3, 171.8, 173.8, 174.0. MALDI-TOF MS: m/z 928.38 (calcd); 951.03

[M+Na] + , 967.04 [M+K] + (found). Anal. Calcd for C43H58Cl2N10O9: C, 55.54; H, 6.29; Cl, 7.63; N, 15.06. Found: C, 55.70; H, 6.27; Cl, 7.60; N, 15.00. Compound 12. Conjugate 12 was obtained (82% yield) under the same conditions reported for the synthesis of compound 7 from FA and amine 11.25 Yellow solid, Mp 207− 209 °C; 1H NMR (200 MHz, DMSO-d6) δ: 1.38 (s, 9H), 1.82−2.01 (m, 2H), 2.08−2.32 (m, 6H), 2.98−3.18 (m, 4H), 3.21−3.42 (m, 4H), 4.22−4.38 (m, 1H), 4.52 (bs, 2H), 6.62 (d, 2H, J = 7.5 Hz), 6.68−6.78 (m, 1H), 6.82−7.05 (m, 3H), 7.65 (d, 2H, J = 7.5 Hz), 7.81 (bs, 2H), 7.91−8.02 (m, 1H), 8.69 (s, 1H), 11.42 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): ppm 28.3, 30.7, 31.8, 35.4, 36.0, 36.6, 39.0, 39.1, 46.0, 51.8, 77.6, 111.2, 121.4, 128.0, 129.0, 148.5, 148.7, 150.8, 153.7, 155.5, 156.6, 160.9, 166.4, 170.5, 171.7, 173.8, 174.1. MALDI-TOF MS: m/z 725.32 (calcd); 748.66 [M+Na]+, 764.68 [M+K]+ (found). Anal. Calcd for C32H43N11O9: C, 52.96; H, 5.97; N, 21.23. Found: C, 52.84; H, 5.96; N, 21.31. Compound 13. Folate conjugate 13 was obtained (82% yield) under the same conditions reported for the preparation of compound 8. Yellow solid, Mp 204−207 °C; 1H NMR (500 MHz) (DMSO-d6) δ: 1.79−2.15 (m, 2H), 2.18−2.30 (m, 6H), 2.99−3.32 (m, 8H), 4.38 (bs, 1H), 4.50 (bd, 2H, J = 5.0 Hz), 4.52 (bs, 2H), 6.58 (d, 2H, J = 7.3 Hz), 6.78−7.02 (m, 4H), 7.62 (d, 2H, J = 7.3 Hz), 7.78−7.98 (m, 1H), 8.02−8.08 (m, 1H), 8.65 (s, 1H), 11.40 (bs, 1H). 13C NMR (100 MHz, DMSO-d6): ppm 31.6, 32.1, 36.0, 36.8, 37.0, 38.8, 39.1, 46.5, 52.4, 111.7, 121.4, 128.0, 129.6, 148.2, 148.4, 151.8, 156.0, 157.5, 161.6, 167.1, 171.0, 172.6, 173.6, 174.0. MALDI-TOF 88



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MS: m/z 625.45 (calcd); 648.49 [M+Na]+, 664.45 [M+K]+ (found). Anal. Calcd for C27H35N11O7: C, 51.83; H, 5.64; N, 24.63. Found: C, 51.96; H, 5.62; N, 24.57. Compound 2. Folate-CLB conjugate 2 was obtained (79% yield) under the same conditions reported for the synthesis of compound 1. Brownish purple solid, Mp 220 °C (dec.); 1H NMR (400 MHz) (DMSO-d6) δ: 1.65−1.69 (m, 2H), 1.79− 1.98 (m, 2H), 2.03 (appt, 2H, J = 7.2 Hz), 2.14−2.26 (m, 5H), 2.30 (t, 1H, J = 6.4 Hz), 2.32−2.42 (m, 2H), 2.97−3.18 (m, 4H), 3.19−3.37 (m, 4H), 3.60 (t, 2H, J = 6.3 Hz), 3.67 (s, 2H), 4.23−4.38 (m, 1H), 4.48 (bd, 2H, J = 5.9 Hz), 6.57−6.69 (m, 4H), 6.78−7.02 (m, 4H), 7.58−7.62 (d, 2H, J = 7.3 Hz), 7.82− 7.94 (m, 2H), 7.98 (t, 1H, J = 7.6 Hz), 8.10 (d, 1H, J = 7.8 Hz), 8.60 (bs, 1H), 11.41 (bs, 1H). 13C NMR (100 MHz, DMSOd6): ppm 26.4, 30.9, 31.8, 33.5, 35.2, 35.6, 35.7, 39.0, 39.1, 40.1, 46.2, 52.6, 53.8, 111.5, 112.2, 121.7, 128.3, 129.4, 129.6, 148.9, 149.0, 151.0, 153.8, 154.1, 156.9, 161.1, 166.7, 169.9, 170.8, 172.0, 174.2. MALDI-TOF MS: m/z 910.34 (calcd); 933.11 [M+Na] + , 949.13 [M+K] + (found). Anal. Calcd for C41H52Cl2N12O8: C, 54.01; H, 5.75; Cl, 7.78; N, 18.43. Found: C, 54.19; H, 5.73; Cl, 7.75; N, 18.35.

established use of polyether linkers devoted to the same purposes,26 on the other hand preparation of a β-amino acidbased linker such as that leading to conjugate 2 beats a completely unexplored path. In this case, we conceived to replace linkers composed by natural α-amino acids, which are amenable to proteolytic degradation in vivo, with those containing the more stable yet biocompatible β-amino acids.27 From a synthetic standpoint, even though preparation of 1 and 2 can be in principle carried out by a straightforward route comprising two subsequent coupling reactions between the linkers, FA and CLB, some problems regarding all FAcontaining reaction intermediates must be pointed out. The main hurdle in the synthesis of FA derivatives is represented by the very poor solubility of all FA-containing products in all organic solvents (except DMSO). This made, on one hand, most reaction intermediates to be unsuitable for purification on common solid chromatographic supports. For this reason, precipitation was always preferred to purify reaction products by traces of unreacted reagents and other byproducts. On the other hand, solubility problems often resulted in low reaction rates, both in the coupling reaction of FA with various nucleophiles and in the following synthetic steps, thus leading to low yields and, after prolonged reaction times, to the formation of a complex mixture of byproducts. Therefore, accurate tuning of reaction conditions was considered to avoid contamination of reaction products by undesired compounds and thus to ensure efficient purification of desired folate conjugates. This was particularly crucial for the conjugation step with FA, since the incidental presence of any trace of unreacted FA during in vitro assays could hamper the subsequent binding between folate conjugates and FRs through competitive interactions. Synthesis of the CLB-Delivery System 1. Preparation of conjugate 1 was carried out starting from 4,7,10-trioxa-1,13tridecanediamine (3), a PEG bis-amine (NH2−PEG13) as the linker unit (Scheme 1). Use of this spacer offers a 2-fold advantage: (a) it is biocompatible and hydrophilic as the most commonly employed polymeric NH 2 PEG linkers (NH2PEG2000, NH2PEG1000, etc.) and (b) it is very inexpensive if compared to other NH2PEG linkers. N-Bocmonoprotection of amine 3 was achieved by treatment with Boc2O in MeOH under refluxing conditions (86% yield). The resulting carbamate 4 was then subjected to various reaction conditions to enable conjugation to FA. After early unsatisfactory attempts using standard synthetic procedures (DCC/HOBT, DCC/NHS), amide 7 was successfully obtained by means of the activated folate anhydride 6, in turn prepared in situ by treatment of 5 with dicyclohexylcarbodiimide (DCC) in a 5:1 mixture of DMF/pyridine for 30 min in an ultrasonic bath. Accordingly, once the white crystals of DCU were formed in the reaction vessel (indicating the reaction progress toward 6), amine 4 (1 equiv) was added, smoothly leading to amide 7. The concentrated DMF solution containing the reaction product was dropwise added to a cold mixture of acetone and diethyl ether (30:70 v/v ratio), from which desired 7 was isolated by precipitation (85% yield). N-Boc group removal of conjugate 7 under common reaction conditions (TFA) quantitatively released amine 8. The latter was hence coupled with CLB (9): while treatment with DCC was not convenient, due to the difficulty to remove the resulting DCU from the reaction mixture, on the other hand reaction with PyBOP/ DIPEA was revealed to be more effective, easily affording

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RESULTS Chemistry. Synthesis of conjugates 1 and 2 is shown in Schemes 1 and 2. We chose to attach the FA and CLB units to

Scheme 2. Synthesis of Conjugate 2

the distal ends of two linkers of sufficient length such that basic requirements for interaction with FRs were maintained. It must be preliminarily noted that, while choice of an aminoether linker (leading to conjugate 1) relies on an extensively 89



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Figure 2. Metabolic activity of FR−-U937 (left) and FR+-TK6 cells (right) treated with increasing doses of CLB at different incubation times.

specific cell lines used in this work. To this purpose, we incubated undifferentiated U937 and TK6 cells for 48 and 72 h with CLB at concentrations ranging from 0 to 60 μM. As shown in Figure 2, metabolic activity of both cell lines was inhibited as drug concentrations and incubation times were increased. In all cases, however, changes in cell metabolic activity were less pronounced at CLB concentrations on the order of 30 μM. Cytotoxicity Studies with CLB Conjugates 1 and 2. A first comparison between efficacy of CLB and our FA-conjugates was obtained by observing the presence/absence of statistically significant changes in metabolic activities of undifferentiated U937 and TK6 cells incubated for 24, 48, and 72 h with media containing 30 μM of CLB (to which both cell lines should presumably respond in a similar manner) or 30 μM FAconjugates 1 and 2 (to which receptor positive cells should preferentially respond). As shown in Figure 3 (upper panel), metabolic activity of undifferentiated U937 cells treated with

desired conjugate 1 as a brownish yellow precipitate (75% yield). Synthesis of the CLB-Delivery System 2. Conjugate 2 was prepared by a similar synthetic route, employing a pseudoβ-dipeptide, composed by two units of β-alanine (β-Ala) bridged by an ethylenediamine (ED) moiety. The presence of ED is required to obtain two terminal amino functions in the linker. As shown in Scheme 2, N-Boc-β-Ala-ED-β-Ala (11) was obtained in satisfactory overall yield (65%) from 10 by a previously reported synthetic protocol.25 Coupling of 11 with the in situ formed anhydride 6 under the above-described conditions (DCC) led to amide 12 in good yield (82%). The N-Boc group was then removed with TFA at 0 °C, and the obtained amine 13 was finally coupled with CLB (9) using PyBOP/DIPEA in anhydrous DMSO, to give conjugate 2 (as a brownish purple solid) which was isolated as described above (79% yield). The presence in FA of two carboxyl groups, namely, α and γ, poses a problem since both can act as a joint for covalent attachment. Indeed, it has been extensively demonstrated that the FA unit of a drug delivery system linked via its γ-carboxyl group retains a strong affinity toward its receptor, whereas the corresponding α-carboxyl derivatives are not readily recognized.8 For this reason, it was essential to establish the relative amounts of α- and γ-carboxyl-linked conjugates in compounds 1 and 2. As confirmed by 1H and COSY NMR experiments, under our conditions an almost exclusive γ-conjugation was found for both conjugates 1 and 2, whereas the amounts of the corresponding α-regioisomers were negligible (100 >100 68 32 64 27

>100 >100 68 32 62 21

32.8 32.4 19.8 17.1 13.6 13.9

48 72

Another proof of a direct involvement of FRs in cell response was obtained by studying the effect of FA-conjugates 1 and 2 on TPA-activated U937 cells. Previous observations demonstrated that, upon differentiation, these cells acquired a macrophage-like phenotype, including FR expression. As expected, these cells, originally unaffected by the action of FA-CLB conjugates (except when the incubation with conjugate 2 was prolonged to 72 h) become fully responsive. Interestingly, both conjugates were manifestly selective toward FR+ cells, since free FA (50 fold molar excess) was demonstrated to efficiently compete in the binding to the FRs, while under the same conditions, FA was ineffective in 93



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ACKNOWLEDGMENTS H NMR, 13C NMR, and MS experiments were performed at the “Centro Interdipartimentale di Metodologie ChimicoFisiche” (CIMCF), University of Napoli “Federico II”. This work was supported by a grant to GP by MIUR, Rome, Italy (Project code E61J10000200001). The authors wish to thank Dr. L. Campanella and Dr. C. Selleri for helpful discussions on tumor-targeted chemotherapy.

U937 cells that do not express the receptor (Figure 4). Overall, these data highlight the selectivity of our FA-CLB conjugates in targeting FR+ cells, as responsiveness (and unresponsiveness) of cells to conjugates 1 and 2 was strictly dependent on the FR expression on the cell membrane. Interestingly, cell cycle data of Figure 6a definitely demonstrated that FR− cells did not significantly modify their profiles when incubated with conjugates 1 and 2. The Sub-G1 and G2 fractions did not appear to be quantitatively changed except when cells were incubated with pure CLB. On the contrary, definite and meaningful alterations in the cell cycle profiles were present in prodrug-treated FR+ cells. Indeed, treatment of these cells with both conjugates caused a significant enrichment in the number of cells staying in G2 and Sub-G1 phases (Figure 6b and c). The presence of the latter large fractions suggests that FR+ cells treated for more than 24 h with the two conjugates (and both FR+ and FR− cells treated with unconjugated free CLB) were progressively prompted to apoptosis. This hypothesis has been definitely strengthened by Western blot experiments that enlightened significant enhancement in the expressions of the pro-apoptotic proteins BAX, Clusterin, and also p53, as the TK6 cells are concerned.

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CONCLUSIONS

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AUTHOR INFORMATION

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1

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ABBREVIATIONS LIST: β-Ala, β-alanine; CLB, chlorambucil; CLL, chronic lymphocytic leukemia; ED, ethylenediamine; FA, folic acid; FR, folate receptor; FR+, expressing FRs; FR−, not expressing FRs; GPI, glycosyl-phosphatidylinositol; PEG, polyethylene glycol; TPA, 12-O-tetra-decanoyl-phorbol-13-acetate

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REFERENCES

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We have successfully reported the chemical synthesis and biological evaluation of two tumor-targeted drug delivery systems 1 and 2, which bear an FA unit as the tumor recognition moiety and the CLB as the cytotoxic agent, the two bioactive units having been jointed by aminoether (4,7,10trioxadecane-1,13-diamine) and pseudo-β-dipeptide (β-Ala-EDβ-Ala) linkers. Preparation of the two molecular systems has been devised by a straightforward and efficient route (34−54% o.y.) based on 4−7 steps starting from amines 3 and 10. When evaluated using both FR− (undifferentiated U937) and FR+ (TK6 and TPA-differentiated U937) leukemic cells, both conjugates exhibited activity only against FR+ cells (particularly TK6), exerting antitumor effects which are comparable to that displayed by CLB in its free form. Our in vitro results, then, show that 1 and 2 specifically bind to FR+ cells, that this binding is significantly higher than in FR− cells, and that the interaction is inhibited by free FA. Given the promising results described in this paper, further research, aimed at developing other structurally related folatebased drug delivery systems, is advisible. It must be emphasized that, to the best of our knowledge, use of β-amino acids has never been taken into consideration to this end. For this reason, the design of other β-amino acid-based linkers is currently in progress, with the scope of enhancing the pharmacokinetic profiles of the corresponding conjugates, thus leading to targeted drug delivery systems endowed with improved antitumor properties.

Corresponding Author *Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cinthia, 4 I-80126 Napoli, Italy. Phone/ Fax: +39 081 674119; E-mail: [email protected]. Author Contributions ‡ These authors contributed equally to this work. 94



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Synthesis and Evaluation of Folate-Based Chlorambucil Delivery

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