1968
Bioconjugate Chem. 2008, 19, 1968–1971
N,N,N′,N′-Tetramethylchloroformamidinium Hexafluorophosphate (TCFH), a Powerful Coupling Reagent for Bioconjugation ´ ngela Torres,‡ Pilar Calvo,§ Miriam Royo,‡ and Fernando Albericio*,†,|,∆ Judit Tulla-Puche,†,* A Institute for Research in Biomedicine, Combinatorial Chemistry Unit, and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain, PharmaMar, S.A., 28770-Colmenar Viejo, Spain, and Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain. Received June 14, 2008; Revised Manuscript Received August 29, 2008
Prodrugs are increasingly used as delivery vehicles for pharmaceutical agents that present solubility and/or pharmacokinetic/metabolic issues. In the course of the development of prodrugs for the antitumoral agent thiocoraline, standard coupling reagents and procedures failed to provide the desired target derivatives because of the lack of reactivity of its quinolinic alcohol. In contrast, the use of N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate (TCFH) resulted in good yields of ester prodrugs of thiocoraline and could be applicable to other drugs with similar limitations.
The development of numerous drugs currently in the pipeline of pharmaceutical companies is being hampered by their poor solubility and/or pharmacokinetic/metabolic properties. A way to overcome this limitation is the use of prodrugs (1), which are chemically modified drugs that remain inactive while being delivered, but that, having reached the target site, release the active drug in response to a change in pH or to the presence of certain enzymes (2). The use of prodrugs has increased enormously over recent years, which is reflected by the fact that 15% of the pharmaceutical agents approved in 2001 and 2002 were prodrugs (3). The design of prodrugs is limited in each case by the functional groups amenable to derivatization in the parent drug. Thus, we can find hydroxyls, carboxylic acids, amines, phosphate, and carbonyl groups susceptible to modification to esters, carbonates, carbamates, amides, phosphates, and oximes (4). Of these, esters are the most common type of prodrug, because their synthesis is usually straightforward, and the presence of many types of esterases in the body makes the ester bond highly labile (5). This linkage is used, for instance, in PGA1-Paclitaxel (Xyotax), currently under phase To whom correspondence should be addressed: E-mail: albericio@ irbbarcelona.org;
[email protected]. Phone: +34 93 4037088. Fax: +34 93 4037126. † Institute for Research in Biomedicine, Barcelona Science Park. ‡ Combinatorial Chemistry Unit, Barcelona Science Park. § PharmaMar, S.A. | Department of Organic Chemistry, University of Barcelona. ∆ CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park. 1 Abbreviations: BOP-Cl, bis(2-oxo-3-oxazolidinyl)phosphinic chloride; CTC, chlorotrityl chloride (Barlos) resin; DAST, (diethylamino) sulfur trifluoride; DIEA, N,N-diisopropylethylamine; DIPCDI, N,N′diisopropylcarbodiimide; DMAP, 4-dimethylaminopyridine; HOBt, 1-hydroxybenzotriazole; HOAt, 1-hydroxy-7-azabenzotriazole(3-hydroxy-3H-1,2,3-triazolo-[4,5-b]pyridine); MSNT, 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole; NMI, N-methyl imidazole; PEG, poly(ethylene glycol); PGA, poly(glutamic acid); PS, polystyrene; TBTU, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium tetrafluoroborate 3-oxide; TCFH, N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate; TFFH, N,N,N′,N′-tetramethylfluoroformamidinium hexafluorophosphate; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMUCCl, N-[chloro(dimethylamino)methylene]-N-methylmethanaminium chloride; Trt, trityl.
Figure 1. Structure of thiocoraline.
III clinical trials (6, 7). PEGylation (8, 9) has also frequently been used for drug conjugation, since it greatly enhances water solubility and decreases immunogenicity (10). PEGs have been introduced into many drugs, such as paclitaxel, camptothecin, doxorubicin, or interferon, among others, which have undergone clinical trials or are on the market. One drug that presents poor solubility is thiocoraline (Figure 1), a potent antitumoral agent (10-9 M) isolated from marine Actynomycetes. Thiocoraline (11, 12) has a highly hydrophobic structure, where the only polar groups are the two quinolinic hydroxyls and a few amides. To develop prodrugs, we focused on anchoring solubilizing groups to the quinolinic hydroxyl group. Nevertheless, this functionality proved to be unreactive, and esterification with common coupling reagents resulted in low yields or full recovery of starting material. To optimize conditions, we examined acetylation with several coupling systems (Table 1). For the first five entries, only the starting material was recovered. Thus, in situ activation of AcOH by formation of the corresponding fluoride (TFFH 1, DAST 2) (13, 14), “the mixed anhydride” (BOP-Cl, 3) (15), or by using Ac2O in the presence of pyridine (4), failed in rendering the desired derivative. The same results were obtained by previously activating resin-bound HOBt in polystyrene (16, 17) with Ac2O and pyridine (5). Next, formation of the ester bond with carbodiimide/DMAP with and without activating agent (HOAt) was examined (6, 7). In these conditions, some conversion was observed, although the recovery of starting material was considerable. Similar results were obtained when using a uronium salt such as TBTU (8) or the sulfonate MSNT (9) (18),
10.1021/bc8002327 CCC: $40.75 2008 American Chemical Society Published on Web 09/20/2008
Communications
Bioconjugate Chem., Vol. 19, No. 10, 2008 1969
Table 1. Coupling Systems entry 1 2 3 4 5 6 7 8 9 10 11 12
coupling system AcOH/TFFH/DMAP/Et3N/CH2Cl2 AcOH/DAST/anh. CH2Cl2 AcOH/BOP-Cl/DIEA/CH2Cl2 Ac2O/pyridine/CH2Cl2 Ac2O/PS-HOBt/pyridine AcOH/DIPCDI/DMAP/CH2Cl2 AcOH/DIPCDI/HOAt/DMAP/CH2Cl2 AcOH/TBTU/HOAt/DIEA/DMF AcOH/MSNT/NMI/DIEA/CH2Cl2 AcOH/TMUC-Cl/DIEA/CH2Cl2 AcOH/TCFH/HOAt/DIEA/DMF AcOH/TCFH/HOAt/DIEA/CH2Cl2
which is routinely used for esterification. In the latter case, the presence of several byproducts was observed, probably caused by the large excess of DIEA and NMI applied for this type of coupling. Reaction with AcCl (10 equiv), DIEA (10 equiv), and DMAP (0.1 equiv) in CH2Cl2 (10) gave good yields, thus obtaining only the bis derivative with 76% yield. In view of these results and to avoid the previous preparation of each single acid chloride, reagents that allow in situ formation of acid chlorides were studied. In the case of TMUC-Cl (19), no product was recovered. However, the use of N,N,N′,N′-tetramethylchloroformamidinium hexafluorophosphate (TCFH) resulted in high conversions to the bis and mono derivatives. Here, we present TCFH (Figure 2) as a powerful derivatizing coupling reagent for the syntheses of ester prodrugs. The best results for obtaining the bis derivative were achieved using AcOH/TCFH/HOAt/DIEA (10 equiv/10 equiv/10 equiv/ 30 equiv) in CH2Cl2, for 2 h at 25 °C (as the ester is not stable to basic treatment, products were directly purified by RP-HPLC). Under these conditions, a range of percentages of the mono and bis derivatives were obtained. Longer reaction times resulted in hydrolysis of the ester with more recovery of starting material.
Figure 2. Structure of TCFH. Scheme 1. Esterification of Thiocoraline on Solid Phase
The application of microwave irradiation for this reaction was also studied in CH2Cl2 (MW 50) and DMF (MW 25). However, while in the first case no improvement was noticed, the use of DMF caused degradation of thiocoraline. Another way to drive reactions to completion is the application of solid-phase synthesis, since large excesses of reagent can be used, which can later be washed away easily, thereby avoiding tedious purifications. Thus, thiocoraline was loaded on 2-CTC resin (20) through the quinolinic alcohol with DIEA in CH2Cl2 (Scheme 1). Next, the remaining free quinolinic alcohol was reacted with TCFH/HOAt/DIEA in CH2Cl2 for 2 h and then the resin was cleaved with TFA-CH2Cl2 (1:99). However, under these conditions, low yields of product were obtained. Thus, subsequent derivatizations were carried out in solution. After optimization of the coupling conditions, a number of carboxylic acids were tested (Table 2). These included PEG acids of various sizes, protected amino acids, aliphatic acids, aliphatic acids with a protected thiol at the terminus, tertiary amine-containing acids, and diacids. For most of these, mixtures of bis and mono derivatives were obtained. The ratio of these two products depended on the acid used. Thus, in the case of octanoic acid (11), for instance, the bis derivative was obtained as a major product, while the mini PEG (3) gave mainly the mono derivative. For 14-16 (tertiary amines and protected Cys), thiocoraline degradation was observed, whereas for succinic acid (17), no reaction occurred. All products were purified by semipreparative HPLC. Attempts to Cleave the Fmoc and Boc Groups from Protected Conjugates To improve solubility, compounds with free amino functionalities were pursued. Thus, coupling with protected amines was performed followed by deprotection of the corresponding Boc and Fmoc groups. In this regard, BocLys(Boc)-OH (8) was coupled to thiocoraline, thus obtaining the corresponding mono and bis derivatives. Treatment of the products with 25% TFA in CH2Cl2 or 1 M HCl in dioxane/ CH2Cl2 resulted in recovery of the starting thiocoraline. Under milder reaction conditions (AcCl, MeOH), monitoring of the reaction by HPLC showed the disappearance of the protected compound and appearance of plain thiocoraline. The same
1970 Bioconjugate Chem., Vol. 19, No. 10, 2008 Table 2. Derivatization of Thiocoraline Using TCFH/HOAt/DIEA
* Sum of PEGytaded products.
results were obtained with the derivative Boc-Glu(tBu)-Othiocoraline (9), and Boc-Lys(Boc)-Pro-O-thiocoraline (not shown), which was synthesized with the aim of keeping the free amino groups further away from thiocoraline. Attempts were also made to remove the Fmoc group of several conjugates both in solution (from 10) and on solid-phase, since in the latter case, cleavage of the Fmoc group is easier. As explained, thiocoraline was loaded on 2-CTC resin, and Fmoc-β-Ala-OH or Fmoc-6-aminocaproic acid was coupled. The Fmoc group was then removed with piperidine-DMF (1:4), using only two treatments of 1 min. Under these conditions, only thiocoraline was recovered. These results suggest that free amino groups attack the newly formed ester bond, even when using longer chains. To obtain free thiols that could be conjugated to gold nanoparticles, protected thiols were used and then deprotected. However, as described above, complex mixtures were obtained when using protected Cys (15-16). In contrast, when a longchain aliphatic carboxylic acid bearing an S-Trt protected thiol
Communications Table 3. In Vitro Results
group (13) was used, the reaction gave the mono derivative, and the Trt group was cleanly removed using TFA-TISCH2Cl2 (10:2.5:87.5). The final product was purified by reversed-phase HPLC, albeit purification proved difficult because of the hydrophobicity of the compound. These results suggest that PEG and aliphatic chains can be used for thiocoraline prodrug formation, but that other functionalities such as free carboxylic acids, tertiary amines, free amines, or short thiol alkyl chains are not tolerated. Biological Activity. The products obtained were tested in three cell lines: breast, nonsmall cell lung (NCSLC), and colon (Table 3). In Vitro results showed that, for most of the derivatives, the activity is maintained, or decreases slightly, thereby suggesting that the ester bond is rapidly cleaved before bisintercalation to DNA. In the case of PEG 2000 and 5000, the decrease in activity can be attributed to the high increase in molecular weight of the resulting prodrug. In conclusion, using TCFH as a coupling reagent for the esterification of thiocoraline, we prepared a number of conjugates including PEGylated compounds with higher solubility. These derivatives were assayed for biological activity in three cell lines and gave, in most cases, results comparable to the parent natural product. On the basis of our findings, we conclude that the coupling system TCFH/HOAt/DIEA could be applied
Communications
for the syntheses of ester prodrugs of molecules bearing groups of low reactivity.
ACKNOWLEDGMENT This study was partially supported by CICYT (CTQ200603794/BQU), the ISCIII (CIBER, nanomedicine), CENIT (Nanomedicine), PharmaMar S. A., the Institute for Research in Biomedicine, and the Barcelona Science Park. J.T.-P. is a Juan de la Cierva fellow (MEC). Supporting Information Available: Experimental procedures and characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED (1) Stella, V. J., and Nti-Addae, K. W. (2007) Prodrug strategies to overcome poor water solubility. AdV. Drug DeliVery ReV. 59, 677–694. (2) Liederer, B. M., and Borchardt, R. T. (2006) Enzymes involved in the bioconversion of ester-based prodrugs. J. Pharm. Sci. 95, 1177–1195. (3) Stella, V. J. (2004) Prodrugs as therapeutics. Expert Opin. Ther/ Pat. 14, 277–280. (4) Routio, J., Kumpulainen, H., Heimbach, T., Oliyai, R., Oh, D., Ja¨rvinen, T., and Savolainen, J. (2008) Prodrugs: design and clinical applications. Nat. ReV. 7, 255–270. (5) Beaumont, K., Webster, R., Gardner, I., and Dack, K. (2003) Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: challenges to the discovery scientist. Curr. Drug Metab. 4, 461–485. (6) Li, C., Yu, D. F., Newman, R. A., Cabral, F., Stephens, L. C., Hunter, N., Milas, L., and Wallace, S. (1998) Complete regression well-established tumors using a novel water soluble poly(Lglutamic acid)-paclitaxel conjugate. Cancer Res. 58, 2404–2409. (7) Greco, F., and Vicent, M. J. (2008) Polymer-drug conjugates: current status and future trends. Front. Biosci. 13, 2744–2756. (8) Abuchowski, A., McCoy, J. R., Palczuk, N. C., van Es, T., and Davis, F. F. (1977) Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252, 3582–3586. (9) Veronese, F. M., and Pasut, G. (2005) PEGylation, successful approach to drug delivery. Drug DiscoVery Today 10, 1451– 1458. (10) Greenwald, R. B., Choe, Y. H., McGuire, J., and Conover, C. D. (2003) Effective drug delivery by PEGylated drug conjugates. AdV. Drug DeliVeryV. 55, 217–250.
Bioconjugate Chem., Vol. 19, No. 10, 2008 1971 (11) Romero, F., Espliego, F., Pe´rez Baz, J., Garcı´a de Quesada, T., Gravalos, D., De la Calle, F., and Ferna´ndez-Puentes, J. L. (1997) Thiocoraline, a new depsipeptide with antitumor activity produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation, and biological activities. J. Antibiot. 50, 734– 737. (12) Pe´rez Baz, J., Can˜edo, L. M., Ferna´ndez Puentes, J. L., and Silva Elipe, M. V. (1997) Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine Micromonospora. II. Physico-chemical properties and structure determination. J. Antibiot. 50, 738–741. (13) Carpino, L. A., and El-Faham, A. (1995) Tetramethylfluoroformamidinium hexafluorophosphate: a rapid-acting peptide coupling reagent for solution and solid phase peptide synthesis. J. Am. Chem. Soc. 117, 5401–5402. (14) Kaduk, C., Wenschuh, H., Beyerman, M., Forner, K., Carpino, L. A., and Bienert, M. (1996) Synthesis of Fmoc-amino acid fluorides via DAST, an alternative fluorinating agent. Lett. Pept. Sci. 2, 285–288. (15) Diago-Meseguer, J., Palomo-Coll, A. L., Ferna´ndez-Lizarbe, J. R., and Zugaza-Bilbao, A. (1980) A new reagent for activating carboxyl groups: preparation and reactions of N,N-bis[2-oxo-3oxazolidinyl]phosphorodiamidic chloride. Synthesis 547–551. (16) (a) Kalir, R., Warshawsky, A., Fridkin, M., and Patchornik, A. (1975) New useful reagents for peptide synthesis. Insoluble active esters of polystyrene-bound 1-hydroxybenzotriazole. Eur. J. Biochem. 59, 55–61. (b) Mokotoff, M., and Patchornik, A. (1983) Synthesis of the C-terminal half of thymosin R1 by the polymeric reagent method. Int. J. Pept. Protein Res. 21, 145– 154. (17) Pop, I. E., Deprez, B. P., and Tartar, A. L. (1997) Versatile acylation of N-Nucleophiles using a new polymer-supported 1-hydroxybenzotriazole derivative. J. Org. Chem. 62, 2594–2603. (18) Blankemeyer-Menge, B., Nimtz, M., and Frank, R. (1990) An efficient method for anchoring Fmoc-amino acids to hydroxyl-functionalized solid supports. Tetrahedron Lett. 31, 1701– 1704. (19) Vendrell, M., Ventura, R., Ewenson, A., Royo, M., and Albericio, F. (2005) N-[Chloro(dimethylamino)methylene]-Nmethylmethanaminium chloride (TMUC Cl), the reagent of choice for the solid-phase synthesis of anilides. Tetrahedron Lett. 46, 5383–5386. (20) Barlos, K., Gatos, D., Kallitsis, J., Papaphotiu, G., Sotiriu, P., Yao, W., and Schaefer, W. (1989) Preparation of protected peptide fragments using triphenylmethyl resins. Tetrahedron Lett. 30, 3943–3946. BC8002327
