Synthesis and Antitumor Activity of Amino Acid Ester Derivatives Containing 5-Fluorouracil

A series of amino acid ester derivatives containing 5-fluorouracil were synthesized using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxybenzotriazole (HOBt) as a coupling agent. The structures of the products were assigned by NMR, MS, IR etc. The in vitro antitumor activity tests against leukaemia HL-60 and liver cancer BEL-7402 indicated that (R)-ethyl 2-(2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl) propanoate showed more inhibitory effect against BEL-7402 than 5-FU.


5-Fluorouracil (5-FU)
is an antimetabolite of the pyrimidine analogue type, which is frequently used for treating solid tumors, such as colorectal, gastric tract, and liver carcinomas [1][2][3]. However, the clinical applications of 5-FU are greatly limited by its short plasma half-life, poor tumor affinity, myelosuppression, and strong intestinal toxicity. Consequently, numerous research efforts have focused on the discovery of suitable carrier-linked prodrugs, in which 5-FU is conjugated with a wide spectrum of low-or high-molecular-weight carriers including glucose, peptides, and biodegradable polymers such as polysaccharides, liposomes, etc [4][5][6][7][8][9][10]. In general prodrug systems the drug is bound

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to the carrier through a spacer that incorporates a predetermined breaking point that allows the bound drug to be released at the cellular target site. Therefore, the optimization physicochemical properties of a carrier, the modification of the carrier with 5-FU to preserve the targeting properties of the carrier and ensure a controlled release of 5-FU inside or outside the tumor cells are the critical aspects of 5-FU prodrug design [3,11].
Peptides play an important role in human metabolism. Some peptide derivatives of 5-FU have been reported as an approach to develop chemotherapeutic agents with improved physicochemical and biological characteristics [4,12,13], and we also have previously reported some peptide derivatives of 5-FU [14][15][16]. In continuation of the research, we now describe our studies on the synthesis and assessment of some amino acid ester derivatives containing 5-FU with the aim of finding appropriate biodegradable linkages.

Chemistry
The synthetic route to the target compounds 2a-o is shown in Scheme 1 and Figure 1. The starting material 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) acetic acid (or 5-fluorouracil-1-yl acetic acid) (1) could be easily prepared by carboxymethylation of 5-fluorouracil according to the literature [17]. Treatment of compound 1 with a series of amino acid esters using 1-ethyl-3-(3dimethylamino-propyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxybenzotriazole (HOBt) as a coupling agent yielded a series of 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-aceto amino acid ester derivatives 2. HOBt was reported as a racemisation suppressant in peptide coupling reactions with carbodiimide coupling reagents [18][19][20].    The purity and structures of compounds 2a-o were established on the basis of their melting points, specific rotations and spectral data, which were in full agreement with the proposed molecular structures. The 1 H-NMR spectra of all compounds showed doublets at 7.93-8.02 ppm, which corresponded to the coupling of fluorine and hydrogen signals in the FC=CH moieties. Compounds 2e-1 and 2e-2, for example, almost have the same melting point (139-140 ºC), the same spectral data, fragment methylene protons were observed as multiplets at δ 2.50-2.41 ppm, which overlapped with the signal of the solvent DMSO-d 6 . The 13 C-NMR of 2c and 2d displayed signals at δ 39.7 ppm from the methylene carbon from the CH 2 CH(CH 3 ) 2 moiety which overlapped as well with that of the solvent DMSO-d 6 ,. The assignment of the above four compounds were further proven by 13 C-1 H COSY spectra.

In vitro antitumor activity
All target compounds 2a-o were evaluated for their in vitro antitumor activity against the HL-60 leukaemia and BEL-7402 liver cancer cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazoliumbromide (MTT) [21] and Sulforhodamine B (SRB) assay methods [22], respectively, with 5-FU and the prodrug FT-207 being used for comparisons (Tables 1 and 2). Table 1, all compounds' in vitro inhibition rates against HL-60 were significantly lower than that of 5-FU, except for the R-type compounds 2h, 2j, 2k-2 and 2m, which exhibited equivalent inhibitory effect as 5-FU at 10 -4 mol/L concentration, but the activity decreased rapidly when the concentration declined. The results indicate that these compounds were less sensitive to HL-60 at lower concentrations when the N-1 position of 5-FU was occupied. Table 1. Inhibitory rates (%) against HL-60.

Compounds
Concentration (mol/L) 10 -4 10 -5 10 -6 10 -7 10 -8 In Table 2, almost all the compounds showed less sensitivity to BEL-7402, except 2m, which showed more potent inhibitory effect than 5-FU. The reason maybe was the R-conformation of 2m with a moderately rigid stereo structure, being composed of the pyrimidine ring and the phenyl ring, so it could release 5-FU sufficiently, while other compounds showed either more flexible configurations (such as 2a-e), or a more rigid structure such as the case of 2k [23]. The different inhibition against HL-60 and BEL-7402 between R-type and S-type compounds suggested the complexity of the antitumor mechanism.

General
Melting points of synthesized compounds were determined on a Digital Melting Point Appatatus X-4 and were uncorrected. Mass spectra were obtained on a DECAX-30000 LCQ DecaXP Plus instrument. IR spectra were recorded (in KBr) on a Bruker EQUINOX 55. 1 H-NMR and 13 C-NMR were recorded on Bruker AVANCE-300 at 300 and 75 MHz, respectively in DMSO-d 6 solutions with TMS as internal standard.

Conclusions
A serials of amino acid ester derivatives containing 5-fluorouracil were synthesized by EDC/HOBt method and characterized. The in vitro antitumor activity tests indicated that the synthesized compounds had less inhibition rates against HL-60 and BEL-7402 than 5-FU except compound 2m, which showed more potent inhibitory effect against BEL-7402 than 5-FU. This might be explained by the R configuration of compound 2m with the moderate rigid framework composed of pyrimidine ring and hydroxyphenyl ring, which may be easily to give 5-fluorouracil.