Folic Acid Derivatization of FdUMP[10] Enhances Cytotoxicity toward 5

Jinqian Liu,† Carol Kolar, Terrence A. Lawson, and William H. Gmeiner*,‡. Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska...
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J. Org. Chem. 2001, 66, 5655-5663

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Targeted Drug Delivery to Chemoresistant Cells: Folic Acid Derivatization of FdUMP[10] Enhances Cytotoxicity toward 5-FU-Resistant Human Colorectal Tumor Cells Jinqian Liu,† Carol Kolar, Terrence A. Lawson, and William H. Gmeiner*,‡ Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 [email protected] Received December 4, 2000

Current chemotherapy protocols that include fluoropyrimidines, such as 5-fluorouracil (5-FU), are limited by the development of chemoresistance during the course of treatment. Our laboratory has developed a novel class of fluoropyrimidines, FdUMP[N], that are oligodeoxynucleotides (ODNs) composed of some number, N, of 5-fluoro-2′-deoxyuridine-5′-O-monphosphate (FdUMP) nucleotides. Novel synthetic procedures are described that permit conjugation of folic acid to the 5′-OH of FdUMP[10] via a phosphodiester linkage using automated synthesis. The synthetic methods developed are generally applicable for ODN conjugation with folic acid. The folic acid conjugate FA-FdUMP[10] showed improved cytotoxicity toward human colorectal tumor cells (H630), and 5-FU-resistant colorectal tumor cells (H630-10). Enhanced cytotoxicity was observed for FAFdUMP[10] relative to nonconjugated FdUMP[10] for cells grown under folate-restricted conditions, consistent with cellular uptake being, in part, receptor-mediated. Folate receptor R (FRR) mRNA was shown by RT-PCR to be overexpressed 26.3-fold in 5-FU-resistant H630-10 cells relative to H630 cells. Thus, FA-FdUMP[N] may prove useful for the treatment of 5-FU-resistant malignancies. Introduction The development of novel chemotherapeutic drugs is required to treat malignancies that are refractory to currently available chemotherapeutic regimens.1 In many cases, the malignancy initially responds to chemotherapy, but prolonged treatment results in selection of resistant cells that clonally expand and render the tumor chemoresistant.2-4 In the case of chemotherapy with fluorinated pyrimidines, such as 5-fluorouracil (5-FU), resistance generally arises due to overexpression of thymidylate synthase (TS).5 Our laboratory has developed a novel class of fluorinated pyrimidines, FdUMP[N], that are oligodeoxyribonucleotides (ODN) composed of N 5-fluoro2′-deoxyuridine-5′-O-monophosphate (FdUMP) nucleotides (Figure 1).6,7 We have shown that FdUMP[10] enters cells in multimeric form and is considerably more cytotoxic toward human colorectal tumor cells than 5-FU.6 In the present paper, we describe synthetic * To whom correspondence should be addressed. Phone: (336) 7166216. Fax: (336) 716-7671. † Present address: Tularik Inc., South San Francisco, CA 94080. ‡ Present address: Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157. (1) Kroep, J. R.; Pinedo, H. M.; van Groeningen, C. J.; Peters, G. J. Ann. Oncol. 1999, 10s4, 234-238. (2) Cascinu, S.; Aschele, C.; Barni, S.; Debernardis, D.; Baldo, C.; Tunesi, G.; Catalano, V.; Staccioli, M. P.; Brenna, A.; Muretto, P.; Catalano, G. Clin. Cancer Res. 1999, 5, 1996-1999. (3) Kitchens, M. E.; Forsthoefel, A. M.; Barbour, K. W., Spencer, H. T.; Berger, F. G. Mol. Pharm. 1999, 56, 1063-1070. (4) Kornman, M.; Danenberg, K. D.; Arber, N.; Beger, H. G.; Danenberg, P. V.; Korc, M. Cancer Res. 1999, 59, 3505-3511. (5) Copur, S.; Aiba, K.; Drake, J. C.; Allegra, C. J.; Chu, E. Biochem. Pharmacol. 1995, 49, 1419-1426. (6) Liu, J.; Skradis, A.; Kolar, C.; Kolath, J.; Anderson, J.; Lawson, T.; Talmadge, J.; Gmeiner, W. H. Nucl. Nuct. 1999, 18, 1789-1802. (7) Liu, J.; Kolath, J.; Anderson, J.; Kolar, C.; Lawson, T.; Talmadge, J.; Gmeiner, W. H. Antisense Nucl. Acid Drug Des. 1999, 9, 481-485.

Figure 1. Structure of FA-FdUMP[10] (1). FdUMP[10] is an oligodeoxyribonucleotide 10mer in which 5-fluorouracil is the only nucleobase. The synthesis and cytotoxic properties of FdUMP[10] have been described previously. Folic acid was conjugated to the 5′-hydroxyl of FdUMP[10] via a phosphodiester linkage using automated methods.

methodology for covalent attachment of folic acid8,9 (vitamin B9, an essential nutrient in the human diet) to FdUMP[10] and show that the resulting conjugate (FA-

10.1021/jo005757n CCC: $20.00 © 2001 American Chemical Society Published on Web 08/01/2001

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FdUMP[10], Figure 1) is more cytotoxic than the nonconjugated multimer and much more cytotoxic than 5-FU to H630-10 cells. H630-10 cells are relatively resistant to 5-FU due to overexpression of TS.5 In this paper, we also show that H630-10 cells overexpress FRR, one of two proteins principally responsible for intracellular transport of folates, and a protein that has high affinity for folic acid. TS is the enzyme responsible for production of dTMP (thymidylate), one of the four deoxyribonucleotides required for DNA synthesis and cell division.10,11 TS is also the principal molecular target of several anticancer drugs, most notably 5-fluorouracil (5-FU).12 TS inhibition results in dTMP depletion, and the occurrence of thymineless cell death.13,14 Santi and co-workers showed that the 5-FU metabolite FdUMP covalently bound TS and inhibited TS catalytic activity.15,16 The mechanism of TS catalysis involves one carbon atom transfer from N,5N10methylene tetrahydrofolate, which serves as a cofactor in the reaction, to C5 of dUMP. TS catalysis thus requires ternary complex formation involving enzyme, substrate, and the reduced folate cofactor. Mechanism-based TS inhibitors structurally resemble either the nucleotide substrate, dUMP (e.g., FdUMP), or the reduced folate cofactor (e.g., tomudex) of the TS catalyzed methylene transfer reaction.17-20 Identification of TS inhibition as the principal target of fluoropyrimidine chemotherapy resulted in the design of clinical protocols designed to maximize TS inhibition upon administration of fluoropyrimidines by malignant cells. The requirement for a reduced folate cofactor in the TS catalyzed methylene transfer to dUMP led to clinical trials that proved co-administration of N5-formyltetrahydrofolate (leucovorin) with 5-FU resulted in improved efficacy relative to single-agent 5-FU.21 Co-administration of leucovorin with 5-FU resulted in increased formation of stable ternary enzymatic complexes and to more efficient inhibition of TS.22-24 Although response rates with 5-FU/leucovorin were improved relative to singleagent 5-FU, overall response rates for most tumors (8) Sirotnak, F. M.; Tolner, B. Annu. Rev. Nutr. 1999, 19, 91-122. (9) Scott, J. M.; Weir, D. G. J. Cardiovasc. Risk 1998, 5, 223-227. (10) Landis, D. M.; Gerlach, J. L.; Adman, E. T.; Loeb, L. A. Nucleic Acids Res. 1999, 27, 3702-3711. (11) Anderson, A. C.; O’Neil, R. H.; DeLano, W. L.; Stroud, R. M. Biochemistry 1999, 38, 13829-13836. (12) Ferguson, P. J.; Collins, O.; Dean, N. M.; DeMoor, J.; Li, C. S.; Vincent, M. D.; Koropatnick, J. Br. J. Pharm. 1999, 58, 973-981. (13) Houghton, J. A.; Harwood: F. G.; Houghton, P. J. Cancer Res. 1994, 54, 4967-4973. (14) Tillman, D. M.; Petak, I.; Houghton, J. A. Clin. Canc. Res. 1999, 5, 425-430. (15) Santi, D. V.; McHenry, C. S. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 1855-1857. (16) Ullman, B.; Lee, M.; Martin, D. W., Jr.; Santi, D. V. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 980-983. (17) Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson, W.; Aherne, G. W.; Hardcastle, A.; Boyle, F. T. Br. J. Cancer 1995, 71: 914-924. (18) Marsham, P. R.; Wardleworth, J. M.; Boyle, F. T.; Hennequin, L. F.; Kimbell, R.; Brown, M.; Jackman, A. L. J. Med. Chem. 1999, 42, 3809-3820. (19) Costi, P. M.; Rinalsi, M.; Tondi, D.; Pecorari, P.; Barlocco, D.; Ghelli, S.; Stroud, R. M.; Santi, D. V.; Stout, T. J.; Musiu, C.; Marangiu, E. M.; Pani, A.; Congiu, D.; Loi, G. A.; LaColla, P. J. Med. Chem. 1999, 42, 2112-2124. (20) Collins, J. M.; Klecker, R. W.; Katki, A. G. Clin. Cancer Res. 1945, 5, 1976-1981. (21) Grogan, L.; Sotos, G. A.; Allegra, C. J. Oncology 1993, 7, 6372. (22) Weckbecker, G. Pharmac. Ther. 1991, 50, 367-424. (23) Pratt, W. B.; Ruddon, R. W.; Ensminger, W. D. The Anticancer Drugs; Oxford University Press: Oxford, 1994.

Liu et al.

treated with fluoropyrimidines remained unacceptably low ( 10) for investigating mechanistic issues. One of the significant advantages of FdUMP[N], relative to monomeric fluoropyrimidines, is the possibility of targeting the multimer specifically to malignant cells through chemical conjugation. The improved cytotoxicity of FdUMP[10] that has been covalently modified with folic acid (FA-FdUMP[10], Figure 1) toward 5-FU-resistant human colorectal tumor cells (H630-10) is the subject of the present paper. The requirement for reduced folate in the catalytic activity of TS led us to investigate the expression levels of proteins that function in folate transport in cell lines resistant to 5-FU as a consequence of TS overexpression. Thus, while elevated TS expression in tumor cells is a likely genetic consequence during cellular adaptation to medium containing TS-inhibitory drugs, the expression levels of other genes are also likely to be altered during the adaptation process.27 Since reduced folates are required for ternary enzymatic complex formation, adaptation of malignant cells to medium containing TSinhibitory compounds is also likely to affect expression of proteins involved in folate transport. Folates are transported into cells by two distinct mechanisms. The (24) Shapiro, J. D.; Harold, N.; Takimoto, C.; Hamilton, J. M.; Vaughn, D.; Chen, A.; Steinberg, S. M.; Liewehr, D.; Allegra, C.; Monahan, B.; Lash, A.; Grollman, F.; Flemming, D.; Behan, K.; Johnston, P. G.; Haller, D.; Quinn, M.; Morrison, G.; Grem, J. L. Clin. Cancer Res. 1945, 5, 2399-2408. (25) Wolmark, N.; Rockette, H.; Fisher, B.; Wickerham, D. L.; Redmond, C.; Fisher, E. R.; Jones, J.; Mamounas, E. P.; Ore, L.; Petrelli, N. J.; Spurr, C. L.; Dimitrov, N.; Romond, E. H.; Sutherland, C. M.; Kardinal, C. G.; Defusco, P. A.; Jochimsen, P. J. Clin. Oncol. 1993, 11, 1879-1887. (26) van Trieste, B.; Pinedo, H. M.; van Hensbergen, Y.; Smid, K.; Telleman, F.; Schoenmakers, P. S.; van der Wilt, C. L.; van Laar, J. A.; Noordhuis, P.; Jansen, G.; Peters, G. J. Clin. Cancer Res. 1945, 5, 643-654. (27) Ju, J.; Pedersen-Lane, J.; Maley, F.; Chu, E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3769-3774.

Improved Cytotoxicity of FA-FdUMP[10] to H630-10 Cells

more widespread of these mechanisms involves the reduced folate carrier (RFC), while the second folate transport process utilizes FRR.28,29 FRR expression is normally restricted to certain cells of the placenta, the choroid plexus (brain), and the proximal tubule cells in the kidney.30 High levels of FRR are, however, observed in malignant tissues of epithelial origin, particularly ovarian carcinoma (80% nonmucinous adenocarcinomas and 100% adenomas), while very low levels are expressed by normal tissues apart from those mentioned above.31 The adaptation of malignant cells to an environment rich in TS-inhibitory drugs may be expected to alter the expression of proteins involved in folate transport due to the requirement for a reduced folate cofactor during TS catalysis. In the present study, the expression of TS, FRR, and RFC1 mRNAs in human colorectal tumor (H630, H630-10) cell lines was examined using RT-PCR. Adaptation of H630 cells to medium containing 10 µM 5-FU (H630-10 cells) resulted in moderate elevation of TS mRNA expression (2.3-fold), but substantial elevation of FRR mRNA (26.3-fold). The correlated overexpression of FRR and TS in human colorectal tumor cells presents an opportunity for selective delivery of FdUMP[N] into 5-FU-resistant cells, provided appropriate conjugation strategies can be developed to covalently attach folic acid to FdUMP[N] and provided the resulting conjugate showed enhanced uptake into the resistant cells. Conjugation with folic acid has been used to improve the cellular uptake properties of various other macromolecular drugs, including antisense oligodeoxynucleotides (ODNs).32-36 In the present paper, we describe novel synthetic chemistry for attaching folic acid to FdUMP[10] via a phosphodiester linkage (Figure 2). Further, we show that the resulting FAFdUMP[10] conjugate is superior to underivatized FdUMP[10], and far superior to monomeric 5-FU, as a cytotoxic drug toward H630-10 cells. The studies described are indicative of a general approach to targeting 5-FU-resistant tumor cells with folic acid-conjugated FdUMP[N]. This approach may be useful for treatment of chemoresistant malignancies. Results and Discussion Synthesis of FA-FdUMP[10]. The procedures used successfully for synthesis of FA-FdUMP[10] are outlined in Figure 2. The overall strategy is straightforward in conception: to convert the γ-carboxylate of the glutamic acid functionality of folic acid into a reactive phosphoramidite and to couple this to the 5′-hydroxyl of FdUMP[10] using standard procedures for oligodeoxynucleotide (28) Lewis, C. M.; Smith, A. K.; Kamen, B. A. Cancer Res. 1998, 58, 2952-2956. (29) Sirotnak, F. M.; Goutas, L. J.; Jacobsen, D. M.; Mines, L. S.; Barrueco, J. R.; Gaumont, Y.; Kisliuk, R. L. Biochem. Pharmacol. 1987, 36, 1659-1667. (30) Kamen, B. Nat. Canc. Inst. Monogr. 1987, 5, 37-39. (31) Ross, J. F.; Chaudhuri, P. K.; Ratnam, M. Cancer 1994, 73, 2432-2443. (32) Leamon, C. P.; Low, P. S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5572-5576. (33) Coney, L. R.; Mezzanzanica, D.; Sanborn, D.; Casalini, P.; Colnaghi, M. I.; Zurawski, V. R. Cancer Res. 1994, 54: 2448-2455. (34) Leamon, C. P.; Low, P. S. J. Biol. Chem. 1992, 267, 2496624971. (35) Leamon, C. P.; Pastan, I.; Low, P. S. J. Biol. Chem. 1993, 268, 24847-24854. (36) Wang, S.; Lee, R. J.; Cauchon, G.; Gorenstein, D. G.; Low, P. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92: 3318-3322.

J. Org. Chem., Vol. 66, No. 17, 2001 5657 Scheme 1a

a Reagents and conditions: (a) bromoethanol, K CO ; (b) 4,4′2 3 dimethoxytrityl chloride, pyridine in DMSO, room temperature, 12 h; (c) cyanoethyl N,N′-diisopropyl phosphonamidic chloride.

(ODN) synthesis. The chemical nature of folic acid, however, made implementation of this strategy challenging. In particular, appropriate protection/deprotection chemistry had to be employed to permit solubility in organic solvents, and appropriate linker chemistry had to be developed to permit formation of a reactive phosphoramidite. Our initial strategy for preparing FA-FdUMP[10] involved esterification of the R and γ carboxylates of folic acid with 2-bromoethanol, with subsequent conversion of one of the primary hydroxyls generated into a reactive phosphoramidite (Scheme 1). The bis-esterification product 3 formed readily and was characterized by NMR and MS. Further reaction of one hyroxyl of 3 with 1 equiv of 4,4′-dimethoxytrityl chloride proceeded to yield compound 4 (as a mixture of regioisomers), and the unreacted hydroxyl was then reacted with cyanoethyl N,N-diisopropylphosphonamidic chloride to yield the phosphoramidite, 5. Compound 5, however, was only sparingly soluble in the organic solvents used for ODN synthesis such as acetonitrile, dichloromethane, or their mixtures. These properties made 5 not only difficult to purify but also difficult to use for automated ODN synthesis. Direct coupling of unpurified 5 to FdUMP[10] in DMF failed to yield detectable amounts of FA-FdUMP[10]. The requirement that the folic acid phosphoramidite product be soluble in solvents suitable for automated ODN synthesis led to investigation of protecting groups for the pteridine 2-amino and 4-carbonyl functionalities, as well as the glutamyl carboxylate groups, to enhance the lipophilicity of the protected form of folic acid. Folic acid is nominally a water-soluble vitamin (vitamin B9); however, folic acid is not soluble in water at the high

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Figure 2. Depiction of the convergent synthesis of FA-FdUMP[10]. Folic acid (vitamin B9) was protected at the 2-amino and 4-carbonyl functionalities of the pteridine ring with amidine and ether protecting groups, respectively. Both the R and γ carboxylates of the glutamic acid moiety were also protected with 2-acetyloxyethylamine. The carboxylates were hemi-deprotected to yield a single primary hydroxyl group that was converted to a reactive phosphoramidite and coupled to the 5′-hydroxyl of FdUMP[10] while still on the solid support using automated synthetic methods.

concentrations required for synthetic chemistry (∼1 M). Folic acid is also not soluble in most common organic solvents such as methanol and acetonitrile. Although soluble in warm DMF solution, folic acid will precipitate from this solvent, as well, upon cooling. To develop an efficient method for synthesis of the phosphoramidite of folic acid, the glutamyl carboxylates were protected with 2-acetyloxyethylamine hydrochloride to yield compound 7 (Scheme 2). This protection strategy also generated two hydroxyls, one of which was required for subsequent synthesis of the phosphoramidite. Unfortunately, addition of ethanolamine hydrochloride to acetyl chloride followed by heating the mixture to a reflux failed to yield 2-acetyloxyethylamine 6, as expected. Increasing the reaction time (4 h vs 1 h) and recrystallization of the putative product yielded only starting materials. It was

observed that ethanolamine hydrochloride did not dissolve in refluxing acetyl chloride, and the lack of solubility is the probable cause for the lack of reactivity. Addition of a small amount of either acetic acid or water improved the solubility of ethanolamine hydrochloride in refluxing acetyl chloride and allowed the reaction to proceed smoothly and in high yields. Amidation of the glutamyl carboxylates was carried out in aqueous solution with 2 equiv (per carboxylate) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (DEC). Although literature precedent suggested use of a 3-fold molar excess of DEC (per carboxylate), the use of 2 equiv resulted in efficient conversion to the diamide 7. The diamide 7 (Scheme 2) could be used in subsequent protection reactions after removal of reaction byproducts accomplished first by removal of reaction solvents (water,

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Scheme 2a

a Reagents and conditions: (a) compound 6, pyridine, DEC, 12 h, room temperature; (b) N,N-dimethylformamide diethyl diacetal, room temperature, 12 h; (c) p-nitrophenylethyl alcohol, HMPA, Mitsunobu conditions.

pyridine) under reduced pressure, followed by redissolving the product in DMF, and precipitating the product by addition of water. The main byproduct present is 1-[3(dimethylamino)propyl]-3-ethylurea. Protection of the 2-amino functionality of 7 with N,Ndimethylformamide diethyl diacetal occurred smoothly at room temperature to yield 8 (Scheme 2). Protection of the 4-carbonyl group of 8 was then carried out under Mitsunobu reaction conditions.37-39 Triphenylphosphine and diethyl azodicarboxylate (DEAD) were used to generate the active intermediate, p-nitrophenethyl phosphonium salt, under argon. Due to the complicated nature of this reaction, a mixture of several products always resulted (at least nine new spots were observed by TLC). There were three main products in this mixture evident in TLC plates developed in dichloromethane/methanol (9: 1). Increasing the reaction temperature enhanced the yield of the product migrating the fastest on TLC while lowering the reaction temperature increased the yield of the product with the second-fastest migration on TLC. The three main spots were separated as a mixture from the unreacted p-nitrophenethanol and the unreacted/ decomposed folic acid. Further separation of the mixture was achieved by column chromotography on silica gel using a acetone/dichloromethane/methanol solvent system. The product with the second-fastest migration on TLC was purified by column chromotography on silica gel using a methanol/dichloromethane/acetone (1:6:3) solvent system. Analyses from NMR, MS, and elemental analysis confirmed the identity of the fully protected folic acid structure 9. The fully protected folic acid 9 is soluble in the solvents used normally for automated ODN synthesis. (37) Yamamoto, H.; Hanaya, T.; Torigoe, K.; Pleiderer, W. Chemistry and Biology of Pteridines and Folates; Plenum Press: New York, 1993. (38) Mitsunobu, O. Synthesis 1981, 1-28. (39) Gao, X.; Gaffney, B. L.; Hadden, S.; Jones, R. A. J. Org. Chem. 1986, 51, 755-758.

Figure 3. Projected synthetic route for converting the protected form of folic acid, 9, to a reactive phosphoramidite, 12, suitable for conjugation to an oligodeoxyribonucleotide using automated synthetic methods. The route was not successful due to the lack of reactivity for the primary hydroxyl groups of compound 10.

Preparation of a reactive phosphoramidite of folic acid from 9 requires removal of the acetyl groups from the protected glutamyl carboxylates (Figure 3). The deprotection of the acetyl group to yield compound 10 was accomplished using dilute methanolic aminoethanol. Deprotection reactions always yielded impurities in addition to the desired product, with simultaneous removal of the amidine protecting group of the 2-amino functionality, in particular, being problematic. The desired product 10 was purified by column chromotography, and its structure was confirmed by NMR, MS, and elemental analysis. Unfortunately, the two hydroxyls of 10 were relatively inert, and very low yields of product occurred upon addition of 4,4′-dimethoxytrityl chloride to solutions of 10 either in DMSO with K2CO3 or in pyridine under refluxing conditions (Figure 3). The chemical inertness

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J. Org. Chem., Vol. 66, No. 17, 2001 Scheme 3a

Liu et al. Table 1. Relative Levels of MRNA Expression in H630 and H630-10 Cells H630a H630-10 H630 (-folic acid)c H630-10 (-folic acid)

FRR

TS

RFC1

1.0 26.3 4.5 28.9

1.0 2.3 1.6 1.1

1.0 NDb ND ND

a mRNA expression levels were quantified relative to expression in the H630 cell line. b No RFC1 expression was detected in these cell lines. c Cells were grown under conditions of folate deprivation as explained in the Experimental Section.

a Reagents and conditions: (a) ethanolamine, methanol, room temperature, 12 h; (b) diisopropylethylamine, cyanoethyl N,N′diisopropylphosphonamidic chloride, THF/methylene chloride; (c) standard automated ODN synthesis conditions.

of the two hydroxyls was confirmed by the failure of 10 to react with ethyl iodide, even under refluxing condition. Fortunately, one of the hydrolysis byproducts of 9 was found to react with cyanoethyl N,N-diisopropylphosphonamidic chloride. Separation of the reactive compound 14 by column chromotography on silica gel and NMR, MS, and elemental analysis allowed identification of the structure of the reactive compound as the monohydrolyzed product 14 (Scheme 3). Attempts to improve the yield of the monohydroxylation product by performing the hydrolysis with different ratios of methanolic ethanolamine were unsuccessful. The conversion of 14 to a phosphoramidite, however, proceeded smoothly with yields of 15 in excess of 50% based on 14. Attempts to establish 15 as a single regioisomer, or as a mixture of R and γ isomers, using NMR were not successful. The coupling of FdUMP[10] with the phosphoramidite of folic acid 15 was carried out following the standard procedures for ODN synthesis (Scheme 3). Coupling was initiated after synthesis of FdUMP[10] was complete. FdUMP[10] was synthesized as previously described. FdUMP[10] remained attached to the solid support during the coupling procedure, and the column was washed with acetonitrile. Compound 15 and imidazole were dissolved in 1:1 dichloromethane/tetrahydrofuran solution, and this solution was brought into the column

to initiate coupling. The coupling reaction was allowed to proceed for 2 min at room temperature and was followed by flushing of the column with acetonitrile. The column was purged with dry argon and then oxidized with I2/THF in aqueous solution to the phosphodiester. The product was cleaved from the column with ammonium hydroxide at room temperature for 1.5 h, dried, and purified by HPLC. Deprotection of the folic acid protecting groups of FA-FdUMP[10] was achieved by incubation of the protected FA-FdUMP[10] in 450 µL of 0.5 M DBU/DMF solution for 4 h,40-42 followed by neutralization with 50 µL of acetic acid (to pH ∼5.5), and cooled to room temperature. The deprotected FA-FdUMP[10] was purified by HPLC. 1H NMR was used to confirm the FA-FdUMP[10] structure. TS, FR-r, RFC-1 Expression. Elevated TS expression may arise as a consequence of cellular adaptation to medium containing TS-inhibitory drugs such as the 5-FU metabolite FdUMP. To determine what changes in expression of folate transport proteins occurred concomitantly with elevated TS expression during cellular adaptation to TS-inhibitory compounds, we measured the expression of TS, FRR, and RFC1 mRNA levels in H630 and H630-10 cells. Expression of mRNA was quantified by comparison with β-actin mRNA expression in each cell line. The results are summarized in Table 1. TS mRNA expression in H630-10 cells was elevated slightly more than 2-fold relative to parental H630 cells. H630-10 cells have been adapted to grow in medium containing 10 µM 5-FU, and resistance to 5-FU is due to overexpression of TS. H630-10 cells also have significant alterations in the expression of folate transport proteins relative to H630 cells. In particular, FRR mRNA expression was dramatically increased (26.3-fold), while RFC1 expression was not detected. RFC1 mRNA expression was, however, detected in H630 cells. Thus, adaptation of H630 cells to growth in medium containing 5-FU resulted in elevated expression of TS, highly elevated expression of FRR, and substantially decreased expression of RFC1. Cytotoxicity of FA-FdUMP[10]. The cytotoxicity of FA-FdUMP[10], FdUMP[10], and 5-FU was evaluated in two human colorectal tumor cell lines (H630 and H63010) to determine if conjugation with folic acid improved the cytotoxicity of FdUMP[10]. Both cell lines were grown in folate-deprived medium as described in the Experimental Section. Folate deprivation sensitized these cell lines to each of the three fluoropyrimidines evaluated, but the sensitizing effects of folate deprivation were particularly evident in cells exposed to FA-FdUMP[10] (40) Trichtinger, T.; Charubala, R.; Pleiderer, W. Tetrahedron Lett. 1983, 24, 711-714. (41) Xu, Y.; Swann, P. A. Nucl. Acids Res. 1990, 14, 4061-4065. (42) Li, B.; Reese, C. B.; Swann, P. F. Biohemistry 1987, 26, 10861093.

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Table 2. IC50 Values (M) for 5-FU, FdUMP[10], and FA-FdUMP[10] FA-FdUMP[10] 5-FU H630 H630-10 a

10-7

4.80 × 3.30 × 10-5

(-)

FAa 10-11

5.40 × 1.80 × 10-10

(+)

FdUMP[10] FAb 10-11

8.90 × 5.80 × 10-10

(-)FAa 10-10

6.60 × 4.30 × 10-9

(+)FAb 6.30 × 10-10 5.50 × 10-9

Cells were maintained under conditions of folate deprivation. b Drug effects measured with 1 × 10-9 M folic acid in medium.

and FdUMP[10]. The results are summarized in Table 2. Toward H630 cells, FdUMP[10] was about 1000-fold more cytotoxic than 5-FU, and the enhancement was independent of the presence of folic acid in the medium. FA-FdUMP[10] was about 10-fold more cytotoxic toward H630 cells than FdUMP[10] or about 10000-fold more cytotoxic than 5-FU. FA-FdUMP[10] was slightly less cytotoxic in the presence of 10-9 M folic acid than in folate-free medium. The three fluoropyrimidines each showed similar cytotoxicity profiles toward H630-10 cells as toward H630 cells, with a few subtle differences. H630-10 cells were adapted for growth in medium containing 10 µM 5-FU, and 5-FU was about 100-fold less cytotoxic toward H63010 cells than toward H630 cells. FdUMP[10] was about 10000-fold more cytotoxic toward H630-10 cells than 5-FU, or about 10-fold increased relative to the 1000-fold increased cytotoxicity observed in H630 cells. Thus, while H630-10 cells were ∼100-fold resistant to 5-FU, H63010 cells were only ∼10-fold resistant to FdUMP[10] (and FA-FdUMP[10]; see below). As was the case in H630 cells, the cytotoxicity of FdUMP[10] toward H630-10 cells did not depend on the presence of folic acid in the culture medium (10-9 M). The relative cytotoxicity of FAFdUMP[10] toward H630-10 cells was similar to that observed in H630 cells. FA-FdUMP[10] was about 10fold more cytotoxic than FdUMP[10], or about 100,000fold more cytotoxic than 5-FU toward H630 cells. The enhanced cytotoxicity of FA-FdUMP[10] was dependent on the presence of folic acid in the medium, although not dramatically so (∼3-fold). Thus, while FA-FdUMP[10] was ∼10-fold more cytoxic than FdUMP[10] toward H630-10 cells in the presence of 10-9 M folic acid, FAFdUMP[10] was ∼25-fold more cytotoxic than FdUMP[10] toward H630-10 cells under conditions of folate deprivation. The sensitivity of FA-FdUMP[10] to the presence of folic acid in the culture medium is consistent with folic acid competing with FA-FdUMP[10] for cellular uptake, and consistent with the hypothesis that FA-FdUMP[10] can be directed to 5-FU-resistant tumors. The magnitude of the effect was diminished somewhat from what might have been expected based on the degree of overexpression of FRR mRNA in H63010 cells relative to H630 cells (∼25-fold) and was similar to the level of TS mRNA overexpression in these cells (∼3-fold). Summary The synthesis of a reactive phosphoramidite suitable for covalent attachment of folic acid to FdUMP[10] has been described. Although conceptually straightforward, the synthetic target was technically challenging due to low solubility in solvents compatible with automated ODN synthesis and the occurrence of unexpectedly low reactivity of the ethanolamine derivatives of the glutamyl carboxylates. A strategy was devised that overcame these difficulties. The strategy consisted of the following steps

(Schemes 2 and 3): (1) amidation of the glutamyl carboxylates of folic acid with 2-acetyloxyethylamine in aqueous solution in a DEC-catalyzed reaction to yield the diamide 7; (2) protection of the 2-amino functionality of 7 with N,N-dimethylformamide diethyl diacetal to yield 8; (3) protection of the 4-carbonyl group of 8 under Mitsunobu reaction conditions to yield 9; (4) hemideprotection of the acetoxy functionality of 9 to yield 14; and (5) preparation of the reactive phosphoramidite of 14, followed by automated ODN synthesis and deprotection. The strategy is generally applicable to automated ODN synthesis, regardless of sequence. The target compound 1, FA-FdUMP[10] showed enhanced cytotoxicity toward 5-FU-resistant human colorectal cancer cell lines in a folate-dependent manner. The enhanced cytotoxicity of FA-FdUMP[10] was 183000-fold greater relative to 5-FU in H630-10 cells and ∼25-fold better than unconjugated FdUMP[10] toward H630-10 cells in folatefree medium. The improved cytotoxicity of FA-FdUMP[10], relative to FdUMP[10], is accounted for, in part, by the overexpression of FRR in H630-10 cells relative to H630 cells facilitating the uptake of FA-FdUMP[10].

Experimental Section Synthetic Chemistry. All chemicals used were of reagent grade. The progress of chemical reactions was monitored by analytical TLC using silica gel (Whatman AL Sil G/UV analytic sheets) coated aluminum-backed sheets. Preparative separations were performed by column chromatography on silica gel (70-230 mesh; Aldrich). Solvent systems used for analytical and preparative separations were as follows: solvent 1, CH2Cl2/MeOH/NH4OH (5:4:1, v/v/v); solvent 2, MeOH/CH2Cl2 (1: 9, v/v); solvent 3, MeOH/CH2Cl2/acetone (1:10:6, v/v/v); solvent 4, MeOH/CH2Cl2/acetone (1:6:3, v/v/v); solvent 5, CH2Cl2/ triethylamine (9:1, v/v); solvent 6, CH2Cl2/triethylamine/MeOH (8:1:1, v/v); solvent 7, MeOH/CH2Cl2/acetic acid (2:8:0.25, v/v/v). 1H NMR spectra were recorded on a Varian 300 or UNITY 500 MHz spectrometer and referenced to internal TMS in the samples. Mass spectrometry data were collected by FAB at the Midwest Center for Mass Spectrometry at the University of NebraskasLincoln. Elemental analyses were conducted by Galbraith Laboratories. Unless otherwise noted, all chemicals were purchased from Aldrich. 2-Acetyloxyethylamine (6). A 10.0 g portion of 2-hydroxyethylamine hydrochloride was placed in a 500 mL flask, followed by addition of 7.5 mL of acetic acid and 30 mL of acetyl chloride. The reactants were stirred, and occurrence of reaction was evident by evolution of heat. The reaction mixture was cooled by immersing the flask in ice-water, as needed. The reaction was stirred at room temperature overnight, and the excess acetyl chloride and acetic acid were removed under vacuum. The product was crystallized from absolute ethanol, and crystals were collected and dried with heat (70-80 °C) to collect pure 6 (12.35 g, yield 86.25%): 1H NMR (300 MHz, DMSO-d6) δ 8.34 (s, 3H, NH3+), 4.20 (t, J ) 5.2 Hz, 2 H, CH2), 3.03 (t, J ) 5.3 Hz, 2 H, CH2), 2.04 (s, 3H,CH3). N1,N5-Bis[2-(acetyloxy)ethyl]-N2-[4-[[[2-amino-4-oxo1,4-dihydropteridin-6-yl]methyl]amino]benzoyl]glutamamide (7). Folic acid dihydrate (1.50 g, 3.14 mmol) was mixed together with pyridine (7 mL) and distilled H2O (10 mL) in a 50 mL flask. The reaction mixture was heated (∼115 °C) until the folic acid was completely dissolved. The reaction mixture

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was maintained at this temperature, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (DEC) (1.20 g, 6.28 mmol) was added, followed by slow addition of the mixed solution of 6 (0.88 g, 6.30 mmol) and a second portion of DEC (1.20 g, 6.28 mmol) dissolved in 10 mL of water. The reaction was stirred at room temperature overnight, forming a gel-like material. Most of the pyridine and distilled H2O were removed under reduced pressure, and the gel was redissolved in DMF following which the DMF was removed under reduced pressure. The solid was dissolved in CH2Cl2 and the desired product precipitated by addition of distilled H2O and collected by filtration. The filtrate was dried under vacuum over P2O5 to yield compound 7 (1.89 g, 98.4%). The product was purified on silica gel using solvent 5 (Rf ) 0.64): MS (FAB) m/z 612.5 (M+ + 1), 527.4 (M+ - COCH3(×2) + 1); 1H NMR (300 MHz, DMSOd6) δ 8.67 (8.66) (s, 7-CH), 8.05 (8.02) (t, NH), 7.68 (7.66) (d, J ) 6.8 Hz, aromatic H × 2), 7.38 (br, NH), 6.66 (6.63) (d, J ) 7.0 Hz, aromatic H × 2), 4.51 (s, CH2), 4.30 (m, RH × 1), 3.96 (m, 4H, CH2 × 2), 3.01 (m, 4H, CH2 × 2), 2.30-2.00 (m, 4H, βand γ CH2), 1.93(s, CH2). Anal. Calcd for C27H33N9O8‚2.5H2O‚ 0.5(C2H5)3N: C, 50.93; H, 6.50; N, 18.81. Found: C, 50.71; H, 6.56; N, 18.91. N1,N5-Bis[2-(acetyloxy)ethyl]-N2-[4-[[[2-[[(dimethylamino)methylidene]amino]-4-oxo-1,4-dihydropteridin-6-yl]methyl]amino]benzoyl]glutamamide (8). Compound 6 (1.89 g, 3.09 mmol) was dissolved in 20 mL of DMF in a 50 mL flask. To the reaction was added 1.60 mL of N,N-dimethylformamide diethyl diacetal (F ) 0.859 g/mL, 9.34 mmol), and the reaction was stirred at room temperature overnight. Solvent was removed under reduced pressure to yield compound 8. The product was purified by column chromatography on silica gel using solvent 6 (CH2Cl2/MeOH/triethylamine 8/1/1 v/v/v) and gradually increasing the percent MeOH and triethylamine to CH2Cl2/MeOH/triethylamine 2:1:1 v/v/v) to yield 1.6 g (50.96%) of compound 8: Rf ) 0.54 (solvent 2); MS (FAB) m/v 667.1 (M + H)+, 610.1 (M - (CH3)2NCH + H)+; 1H NMR (500 MHz, DMSO-d6) δ 8.79 (8.72) (s, 7-CH), 8.00 (7.98) (t, NH), 7.69 (7.66) (d, J ) 8.6 Hz, aromatic H × 2), 7.00 (t, J ) 6.0 Hz, NH), 6.66 (6.63) (d, J ) 8.7 Hz, aromatic H × 2), 4.54 (s, CH2), 4.52 (s, CH2), 4.30 (m, RH × 1), 4.10 (t, J ) 5.3 Hz, CH2), 3.98 (m, 4H, CH2 × 2), 3.50-3.01 (m, CH3 × 2, CH2), 2.25-1.97 (m, 4H, β- and γ CH2), 1.96 (s, CH3). Anal. Calcd for C30H38N10O8‚ 2.25C6H15N‚4H2O: C, 54.05; H, 8.33; N, 17.75. Found: C, 54.30; H, 8.18; N, 17.65. N1,N5-Bis[2-(acetyloxy)ethyl]-N2-[4-[[[2-[[(dimethylamino)methylidene]amino]-4-[2-(4-nitrophenyl)ethoxy]pteridin-6-yl]methyl]amino]benzoyl]glutamamide (9). The mixture obtained in the last step could be used without further purification. DMF was removed under reduced pressure, and the mixture was redissolved in 50 mL of HMPA. The solution was heated (T < 120 °C) under reduced pressure to remove the last traces of DMF remaining in the solution. The final volume of the reaction mixture was ∼40 mL. After the mixture was cooled to room temperature, argon was introduced into the solution for 10 min, 2.34 g PPh3 (8.92 mmol), 1.49 g p-nitrophenethyl alcohol (8.91 mmol), and 4.23 mL diethyl azodicarboxylate (F ) 1.106 g/mL, 8.06 mmol) were added, and the reaction was stirred at room temperature overnight. HMPA was removed under reduced pressure, and the solid was redissolved in dichloromethane. The desired product (9) was partially purified by column chromatography on silica gel with chloroform/methanol (9:1, v/v), Rf 0.52-0.61. Further separation of the mixture with acetone/dichloromethane/ methanol (6:10:1, v/v/v) resulted in pure 9 (0.53 g): Rf ) 0.22 (solvent 3); yield 20.81% (from folic acid); MS (FAB) 816.2 (M+ + 1), 838.3 (M+ + Na+); 1H NMR (500 MHz, DMSO-d6) δ 8.73 (8.59) (s, 7-CH), 8.15 (8.13) (d, J ) 8.5 Hz, 2H), 7.97 (7.96) (d, J ) 5.5 Hz, NH), 7.67 (7.64) (d, J ) 8.50 Hz, 2H), 7.49 (7.47) (d, J ) 8.5 Hz, 2H), 6.99 (t, J ) 6.7 Hz, NH), 6.65 (6.63) (d, J ) 8.5 Hz, 2H, CH), 6.82 (d, J ) 8.6 Hz, 2H), 4.54 (d, J ) 5.5 Hz, CH2), 4.48 (t, J ) 7.0 Hz, 2H), 4.29 (m, 1H), 3.98 (m, 2H), 3.40-2.90 (m, 8H), 2.15-1.83 (m, 7H). Anal. Calcd for C38H45N11O10‚0.5CH3OH‚H2O: C, 54.41; H, 5.82; N, 18.13. Found: C, 54.79; H, 5.51; N, 17.68.

Liu et al. N 1 ,N 5 -Bis[2-(hydroxy)ethyl]-N 2 -[4-[[[2-[[(dimethylamino)methylidene]amino]-4-[2-(4-nitrophenyl)ethoxy]pteridin-6-yl]methyl]amino]benzoyl]glutamamide (10). A 1.0 g portion of 9 (1.22 mmol) was dissolved in 10 mL of methanol and 1 mL of ethanolamine (F ) 1.102 g/mL, 18.04 mmol), and the solution was stirred at room temperature overnight. Solvent was removed under reduced pressure, and the product was purified by column chromatography on silica gel using chloroform/methanol (10:3) to yield compound 10a: Rf ) 0.10 (solvent 2); yield 21.71%; MS (FAB) 677.2 (M+ + 1), 444.2 (M+ - glutamine diamide + 1); 1H NMR (300 MHz, DMSO-d6) δ 8.67 (s, C8-H), 8.21 (d, J ) 8.8 Hz, aromatic 2H), 8.04 (d, J ) 7.8 Hz, NH), 7.79 (t, J ) 5.13 Hz, NH), 7.64 (d, J ) 8.8 Hz, aromatic 2H), 6.95 (t, NH), 6.65 (d, J ) 8.9 Hz, aromatic 2H), 4.65 (q, CH2), 4.50 (d, J ) 6.4 Hz, CH2), 4.29 (m, RH), 3.35 (m, CH2), 3.12 (m, CH2), 2.12-1.80 (m, CH2). 10a was stirred overnight at room temperature in 6 mL of DMF and 1 mL of N,N-dimethylformamide diethyl diacetal and purified again by chromatography using a silica gel column to yield compound 10 (0.17 g): yield 86.7%; Rf ) 0.41 (solvent 2); MS (FAB) 732.2 (M+ + 1). 1H NMR (500 MHz, DMSO-d6) δ 8.69 (s, C8-H), 8.25 (d, J ) 8.8 Hz, aromatic 2H), 8.09 (d, J ) 7.8 Hz, NH), 7.69 (d, J ) 8.8 Hz, aromatic 2H), 7.00 (t, NH), 6.64 (d, J ) 8.9 Hz, aromatic 2H), 4.50 (q, CH2), 4.32 (d, J ) 6.4 Hz, CH2), 3.35 (m, CH2), 3.19 (m, CH2), 2.652.64 (m, CH2). Anal. Calcd for C34H41N11O8‚CH3OH‚H2O: C, 53.75; H, 6.07; N, 19.70. Found: C, 53.37; H, 5.90; N, 20.20. N1,N5-[2-(Acetyloxy)ethyl]-[2-(hydroxy)ethyl]-N2-[4[[[2-[[(dimethylamino)methylidene]amino]-4-[2-(4-nitrophenyl)ethoxy]pteridin-6-yl]methyl]amino]benzoyl]glutamamide (14). A 1.0 g portion of 9 (1.22 mmol) was dissolved in 7 mL of CH2Cl2/methanol (5:2, v/v) solution, to which was added three 2.5 mL portions of 2-hydroxyethylamine/methanol (1.36:100, v/v) over 8 h with stirring. The solution was stirred for 3 days, following which the solvent was removed under reduced pressure, and the solid was redissolved in 5 mL of DMF. N,N-dimethylformamide diethyl diacetal (1 mL) was added to the solution, which was stirred overnight at room temperature. DMF was removed under reduced pressure, and the product was purified as an uncharacterized mixture of Rand γ-regioisomers by chromatography on silica gel using (sequentially) CH2Cl2/methanol (96:4, v/v) 200 mL, (94:6) 200 mL, (90:10) 200 mL, and (85:15) 200 mL. The products obtained were unreacted 9 (0.35 g, 35%), dihydroxy 10 (0.30 g, 33.4%), and 14 (0.25 g, Rf ) 0.15-0.20 (solvent 4), yield 26.3%): MS (FAB) 774.25 (M+ + 1), 716.24 ((M+ + 1) - CH3CO2 + 1); 1H NMR (500 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.75(8.61) (s, 1H), 8.15 (d, J ) 7.5 Hz, 2H), 7.92 (m, 1H), 7.76 (s, 1H), 7.67 (d, J ) 9.0 Hz, 2H), 7.50 (d, J ) 8.0 Hz, 2H), 6.94 (t, J ) 5.5 Hz, 2H), 6.67 (d, J ) 9.0 Hz, 2H), 4.55 (s, 1H), 4.50 (t, J ) 7.0 Hz, 1H), 3.98 (m,1H), 3.61 (3.55) (s, 1H), 3.37 (m, 1H), 3.29 (s, 6H), 3.24-3.07 (m, 6H), 2.17 (m, 1H), 1.97 (s, 3H), 1.90-1.80 (m, 1H). Anal. Calcd for C36H43N11O9‚0.5H2O: C, 55.22; H, 5.67; N, 19.68. Found: C, 55.16; H, 5.65; N, 19.51. N1,N5-[2-(Acetyloxy)ethyl]-[2-(hydroxy)ethyl]-N2-[4[[[2-[[(dimethylamino)methylidene]amino]-4-[2-(4-nitrophenyl)ethoxy]pteridin-6-yl]methyl]amino]benzoyl]glutamamide N5,N1-(2-Cyano)ethyl Diisopropylphosphoramidite (15). Diisopropylethylamine (0.15 mL, 0.86 mmol), THF/CH2Cl2 (1:1) (5 mL), and cyanoethyl N,N-diisopropylphosphonamidic chloride (70 µL, F ) 1.1061 g/mL, 0.31 mmol) were mixed together with 0.16 g (0.20 mmol) of 14 dissolved in 5 mL of THF/CH2Cl2 (1:1) in a 50 mL flask. The mixture was stirred at room temperature for 4 h and quenched by addition of 25 mL of EtOAc. The solution was washed with saturated NaCl (aq), and the solvent was removed under reduced pressure. The product, an uncharacterized mixture of R- and γ-regioisomers, was purified by chromatography on silica gel using CH2Cl2/triethylamine (9:1, v/v) to obtain 0.11 g: Rf ) 0.16 (0.21) (solvent 5); yield 54.7%; 31P NMR (DMSO, 85% phosphoric acid as reference at 0 ppm) δ 152.56, 152.41; MS (FAB neg. ion) 973.6 (M - H)+; 1H NMR (500 MHz, DMSOd6) δ 7.57 (d, J ) 7.0 Hz, 2H), 7.43 (d, J ) 8.5 Hz, 2H), 7.18 (d, J ) 7.0 Hz, 2H), 7.03 (t, J ) 7.0 Hz, 1H), 6.78 (d, J ) 8.5, 2H), 4.64 (m, 2H), 4.18 (d, J ) 3.0 Hz, 1H), 4.14 (d, J ) 3.0

Improved Cytotoxicity of FA-FdUMP[10] to H630-10 Cells Hz, 1H), 3.40-3.04 (m, 12H), 2.39 (q, J ) 6.5 Hz, 2H), 2.28 (m, 1H), 2.12 (m, 1H), 2.01 (m, 1H), 1.32 (bs, 1H), 1.06 (s, 12H), 0.99-0.88 (m, 2H). Anal. Calcd for C45H60N13O10P‚1.25Et3N‚ 6H2O: C, 53.17; H, 7.57; N, 16.51. Found: C, 53.27; H, 7.11; N, 16.36. Synthesis of Folic Acid-FdUMP[10] (1). FdUMP[10] was prepared as previously described.6 Compound 15 was manually coupled to the 5′-OH of FdUMP[10] while the ODN was still attached to the solid support. Compound 15 was dissolved in 0.75 mL of acetonitrile/dichloromethane (1:1) and allowed to couple for 10 min at room temperature. The intermediate phosphoramidite was oxidized to the phosphodiester with I2/THF, and the coupled product (FA-FdUMP[10]) was cleaved from the CPG support by treatment with ammonium hydroxide at room temperature for 1.5 h. Solvent was then removed under reduced pressure. The 4-(p-nitro)phenylethyl group was then removed from the pteridine carbonyl oxygen of FA-FdUMP[10] by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF solution (50 µL/450 µL of DMF) at room temperature for 4 h. The deprotection reaction went to completion on the basis of analytical HPLC. The solution was then added to 50 µL of acetic acid to neutralize unreacted DBU, and the FA-FdUMP[10] product was purified by HPLC using a Waters 600E HPLC system controller and a Waters µBondpak C18 (7.8 × 300 mm) column (Milford, MA). Material was eluted using mixtures of 0.1 M triethylammonium acetate (solvent A) and 80% aqueous acetonitrile (solvent B). Solvent B was linearly increased from 6% to 12% over 10 min, increased from 12% to 26% over 40 min, and finally increased from 26% to 35% over 5 min. The retention time for FA-FdUMP[10] under these conditions was 45 min, while FdUMP[10] lacking the folic acid eluted at 32 min. Cell Culture. Investigations of the correlation between TS and folate transport protein (FRR and RFC1) mRNA expression levels were conducted using H630 (human colorectal tumor) and H630-10 cell lines.5 The H630-10 cell line was derived from H630 by adaptation to growth in media containing 10 µM 5-FU. H630 cells were cultured using RPMI 1640 (Life Technologies, Inc., Grand Island, NY) containing Lglutamine and supplemented with 10% Nu-serum IV. All culture media were supplemented with penicillin (100 units/ mL) and streptomycin (0.1 mg/mL) (Sigma). Cells were grown at 37 °C in an air atmosphere containing 5% CO2. Culturing conditions for H630-10 cells were identical to those described for H630 cells with the exception of containing a 10 µM concentration of 5-FU. Cells in the logarithmic growth phase (∼3 days after passage) were analyzed for TS and FRR mRNA expression levels. H630 and H630-10 cell lines deprived of folic acid were developed from H630 and H630-10 cells by complete folic acid deprivation for 18 days by using dialyzed RPMI1640 medium and 9% FBS (dialyzed). The cells were then grown in dialyzed media with minimum folic acid (10-9 M) and passaged at least four additional times prior to analysis of drug effects using a clonogenic assay.

J. Org. Chem., Vol. 66, No. 17, 2001 5663 TS and FRR Expression. Total RNA was isolated using the acid guanidinium phenol chloroform method. cDNA was synthesized by reverse transcriptase (M-MLV RT; Gibco BRL) from total RNA using a random primer (TaKaRa) for 1 h at 37 °C and was stored at -20 °C until needed. Separate PCR amplification of TS, FRR, and RFC1, as well as for β-actin, was done in serial dilutions of each cDNA solution. Transcription of the PCR amplified cDNA to RNA was done using T7 RNA polymerase in the presence of [32P]CTP. Polyacrylamide gel electrophoresis (PAGE) was performed on a 2% agarose gel for each transcription reaction, and quantitation of the RNA product was achieved by measuring the radioactivity of the band in the phosphorimager (Eppley Institute). The linear range of amplification of the gene of interest, and the reference gene, were found by plotting the amount of radioactivity in the RNA bands against the volumes of the cDNA solution added to the PCR reaction. The ratio between the slopes of the linear amplification regions of the gene of interest, and the reference gene (β-actin), were determined for each sample. Primers used for PCR amplification of the TS gene were the same as previously described.31 Drugs. The concentration of FA-FdUMP[10] solutions was determined by measuring the absorbance at 363 nm (363 ) 13 600 M-1 cm-1). Solutions of folic acid were prepared from a 0.052 M stock solution obtained by dissolving 1.0 g of folic acid (1 equiv) and 0.35 g of NaHCO3 (2 equiv) in sterilized PBS (final volume to 40 mL). The FdUMP[10] solution concentrations were determined from absorbance measurements at 260 nm using the conversion 33 µg/ODU. 5-Fluorouracil solution (Sigma) was prepared by dissolving 102 mg of 5-FU in 10 mL of PBS solution. Clonogenic Assay. About 1000 viable H630 or H630-10 cells suspended in 10 mL of RPMI 1640 medium with 9% NU serum IV were grown at 37 °C in a humidified 5% CO2-air atmosphere in 100 × 20 mm plastic Petri dishes. After 48 h, the medium was removed and replaced with 10 mL of fresh medium containing either 5-FU, FdUMP[10], or FA-FdUMP[10]. After 72 h, the medium containing drugs was removed and replaced by fresh medium. Plates were incubated for 2-3 weeks with fresh medium added, if necessary, after which time the medium was removed and the clones were identified by staining with 0.1% methylene blue in 70% ethanol aqueous solution. The number of surviving colonies was counted and calculated as a percent of controls. Each study was repeated twice.

Acknowledgment. This work was supported by NIH-NCI 60612 (W.H.G.) and NIH-NCI CA-36727. We thank John W. Jost, Executive Director of IUPAC, and Warren H. Powell (Columbus, OH) for help with nomenclature for novel folic acid derivatives. JO005757N