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

Abstract

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.

1. Introduction

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 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.

2. Results and Discussion

2.1. 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-(3-dimethylamino-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 ¹H-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 °C), the same spectral data, but opposite specfic rotations of ${\left[\alpha \right]}_D^{18.1}$−10.4 and ${\left[\alpha \right]}_D^{18.1}$+10.4, respectively. In the ¹H-NMR their CH₂SCH₃ fragment methylene protons were observed as multiplets at δ 2.50-2.41 ppm, which overlapped with the signal of the solvent DMSO-d₆. The ¹³C-NMR of 2c and 2d displayed signals at δ 39.7 ppm from the methylene carbon from the CH₂CH(CH₃)₂ moiety which overlapped as well with that of the solvent DMSO-d₆,. The assignment of the above four compounds were further proven by ¹³C-¹H COSY spectra.

2.2. 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-2H-tetrazoliumbromide (MTT) [21] and Sulforhodamine B (SRB) assay methods [22], respectively, with 5-FU and the prodrug FT-207 being used for comparisons (

Table 1. Inhibitory rates (%) against HL-60.

Compounds	Concentration (mol/L)
	10−4	10−5	10−6	10−7	10−8
2a	2.0	8.0	9,6	6.9	8.3
2b	3.7	5.2	6.2	9.7	2.8
2c	33.1	0.1	9.9	9.8	0
2d	27.3	0	1.7	0	10.0
2e-1	22.4	11.2	7.1	4.2	1.2
2e-2	18.5	15.3	3.4	10.5	0
2f-1	3.3	10.8	6.6	12.3	0
2f-2	31.8	9.2	3.7	6.2	0
2g	36.1	10.0	5.7	5.0	2.5
2h	55.7	19.6	23.1	2.3	8.6
2i	2.8	6.7	1.5	8.3	0.6
2j	55.8	12.8	2.7	5.4	5.9
2k-1	29.2	0	3.8	8.2	2.4
2k-2	51.2	15.5	9.8	12.7	8.9
2l	42.4	7.9	5.2	9.9	5.7
2m	65.1	0	12.6	13.0	0.3
2n	11.4	0	0	0	0
2o	22.4	11.2	7.1	4.2	1.2
5-FU	57.4	33.5	0	7.0	10.4
FT-207	0	0	0	0	0

and

Table 2. Inhibitory rates (%) against BEL-7402.

Compounds	Concentration (mol/L)
	10−4	10−5	10−6	10−7	10−8
2a	0	0	0	0	0
2b	9.0	8.1	0	0	1.8
2c	50.0	13.2	5.7	5.2	0
2d	13.2	0	0	0	0
2e-1	41.2	9.7	8.8	8.2	9.1
2e-2	38.4	9.1	8.8	0	5.3
2f-1	17.4	10.5	16.6	14.0	4.6
2f-2	41.2	9.8	0	0	0
2g	36.1	11.1	7.0	7.4	2.4
2h	52.6	15.9	2.4	0.7	0
2i	14.5	8.7	8.0	4.6	11.7
2j	34.0	9.2	4.1	3.5	5.9
2k-1	35.9	0	6.9	3.0	0.7
2k-2	22.4	11.9	7.1	4.2	1.2
2l	36.2	10.2	4.9	0.5	0
2m	71.7	68.3	60.4	43.1	24.3
2n	8.2	4.5	5.2	0	0
2o	9.7	0	8.8	8.2	9.1
5-FU	72.6	53.8	35.0	23.8	16.6
FT-207	58.0	8.1	0	0	0

).

As shown in 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.

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.

3. Experimental

3.1. 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. ¹H-NMR and ¹³C-NMR were recorded on Bruker AVANCE-300 at 300 and 75 MHz, respectively in DMSO-d₆ solutions with TMS as internal standard.

3.2. General procedure for the synthesis of compounds 2a-o

Synthesis of compounds 2a-o was accomplished as shown in Scheme 1. 2-(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) acetic acid (10 mmol), HOBt (10 mmol) and DMF (50 mL) were added to a round-bottom flask, then EDC·HCl (13 mmol), L- or D-amino acid ester hydrochloride (10 mmol), and triethylamine (20 mmol) were added to the above mixture. After 10 h reaction at room temperature with thin layer chromatography (TLC) monitoring, the white solid 5-fluorouracil-1-yl-aceto amino acid esters 2a-o were obtained after filtration, reduced pressure distillation of DMF, and silica gel column chromatography separation.

(S)-Methyl 2-(2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-methylbutanoate (2a). Yield: 68%; m.p. 109-110°C; ¹H-NMR δ: 11.80 (s, 1H, NH of 5-FU), 8.54 (d, 1H, NH, J = 8.1 Hz), 8.01 (d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.39 (d, 2H, NCH₂, J = 16.8 Hz), 4.22 (t, 1H, NCH, J = 7.2 Hz), 3.65 (s, 3H, OCH₃), 2.09-1.98 (m, 1H, CCH, J = 6.6 Hz), 0.89 (d, 3H, CH₃, J = 6.6 Hz), 0.87 (d, 3H, CH₃, J = 6.6 Hz); ¹³C-NMR δ: 171.9, 167.1, 157.7(d, ²J_FC = 25.6 Hz), 149.8, 139.3 (d, ¹J_FC = 226.7 Hz), 131.3 (d, ²J_FC = 33.6 Hz), 57.7, 51.9, 49.5, 30.4, 19.0, 18.3; IR (cm^-1) ν: 3456, 3280, 2969, 1722, 1666, 1560, 1467, 1379, 1227, 1146, 783; MS (ESI) m/z: 300 (M^-); ${\left[\alpha \right]}_D^{10.0}$ -22.0 (c 1.0, DMF).

(R)-Ethyl 2-(2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-methylbutanoate (2b). Yield: 65%; m.p. 136-137 °C; ¹H-NMR δ: 11.83(s, 1H, NH of 5-FU), 8.53 (d, 1H, NH, J = 8.1 Hz), 8.02 (d, 1H, FC=CH, ³J_FH = 6.6 Hz), 4.38 (d, 2H, NCH₂, J = 16.8 Hz), 4.19 (dd, 1H, NCH, J = 8.1, 6.3 Hz), 4.16-4.05 (m, 2H, COOCH₂, J = 7.2 Hz), 2.11-1.98 (m, 1H, CCH), 1.19 (t, 3H, OCH₂CH₃, J = 7.2 Hz), 0.89 (d, 3H, CH₃, J = 6.9 Hz), 0.88 (d, 3H, CH₃, J = 6.6 Hz); ¹³C-NMR δ: 174.0, 169.6, 160.5 (d, ²J_FC = 25.3 Hz), 151.6, 141.2 (d, ¹J_FC = 228.1 Hz), 132.8 (d, ²J_FC = 33.5 Hz), 63.3, 59.9, 51.6; 31.6, 20.0, 19.2, 15.2; IR (cm^-1) ν: 3295, 3253, 2977, 1707, 1552, 1467, 1377, 1225, 1148; MS (ESI) m/z: 314(M^-); ${\left[\alpha \right]}_D^{18.0}$+14.0 (c 0.1, DMF).

(S)-Methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-methylpentanoate (2c). Yield: 75%; m.p. 144-145°C; ¹H-NMR δ: 11.83 (d, 1H, NH of 5-FU, ⁴J_FH = 5.4 Hz), 8.60 (d, 1H, NH, J = 7.8 Hz), 8.02 (d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.32 (s, 2H, NCH₂), 4.35-4.27 (m, 1H, NCH), 3.62 (s, 3H, OCH₃), 1.66-1.59 (m, 1H, CCH), 1.54-1.46 (m, 2H, CCH₂), 0.88 (d, 3H, CH₃, J = 6.3 Hz), 0.83 (d, 3H, CH₃, J = 6.3 Hz); ¹³C-NMR δ 172.9, 167.0, 157.8 (d, ²J_FC = 25.5 Hz), 149.8, 139.4 (d, ¹J_FC = 226.6 Hz), 131.3 (d, ²J_FC = 33.8 Hz), 52.2, 50.5, 49.6, 39.7, 24.3, 22.9, 21.6; IR (cm^-1) ν: 3323, 3046, 2959, 1666, 1543, 1472, 1384, 1244, 1155; MS (ESI) m/z: 314(M^-); ${\left[\alpha \right]}_D^{16.1}$-19.2 (c 1.0, DMF).

(R)-Ethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-methylpentanoate (2d). Yield: 72%; m.p. 146-147°C; ¹H-NMR δ: 11.81(d, 1H, NH of 5-FU, ⁴J_FH = 5.1 Hz), 8.57(d, 1H, NH, J =7.8 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.32(s, 2H, NCH₂), 4.30-4.24(m, 1H, NCH), 4.08(q, 2H, OCH₂, J = 7.2 Hz), 1.67-1.58(m, 1H, CCH), 1.56-1.48(m, 2H, CCH₂), 1.17(t, 3H, OCH₂CH₃, J = 7.2 Hz), 0.89(d, 3H, CH₃, J = 6.3 Hz), 0.84(d, 3H, CH₃, J = 6.3 Hz); ^13C-NMR(75 MHz) δ: 172.3, 166.8, 157.7(d, ²J _FC = 25.2 Hz), 149.7, 139.3(d, ¹J_FC = 226.4 Hz), 131.2 (d, ²J_FC = 33.9 Hz), 60.7, 50.6, 49.5, 39.7, 24.3, 22.8, 21.5, 14.1; IR(KBr, cm^-1) ν: 3328, 2963, 2818, 1690, 1637, 1555, 1473, 1377, 1238, 1196, 1150; MS(ESI) m/z: 328(M^-); ${\left[\alpha \right]}_D^{17.7}$+11.6(c 1.0, DMF).

(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-(methylthio)butanoate (2e-1). Yield: 62%; m.p. 139-140°C; ¹H-NMR(300 MHz) δ: 11.84(s, 1H, NH of 5-FU), 8.64(d, 1H, NH, J = 7.5 Hz), 8.02(d, 1H, FC=CH, ³J = 6.9 Hz), 4.46-4.39(m, 1H, NCH), 4.33(s, 2H, NCH₂), 3.63(s, 3H, OCH₃), 2.50-2.48(m, 2H, CH₂S), 2.02(s,3H, SCH₃), 1.97-1.79(m, 2H, CCH₂); ¹³C-NMR(75 MHz) δ: 172.2, 167.1, 157.8(d, ²J_FC = 25.7 Hz), 149.9, 139.5(d, ¹J_FC = 226.5 Hz), 131.2(d, ²J_FC = 33.8 Hz), 52.3, 51.1, 49.7, 30.9, 29.5, 14.8; IR(KBr, cm^-1) ν: 3345, 3042, 2983, 1687, 1662, 1542, 1474, 1428, 1386, 1233; MS(ESI) m/z: 332(M^-); ${\left[\alpha \right]}_D^{18.1}$-10.4(c 1.0, DMF).

(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-(methylthio)butanoate (2e-2). Yield: 59%; m.p. 138-140 °C; ¹H-NMR(300 MHz) δ: 11.83(s, 1H, NH of 5-FU), 8.63(d, NH, J = 7.6 Hz), 8.02(d, 1H, FC=CH, ³J_FH = 6.0 Hz), 4.43-4.38(m, 1H, NCH), 4.33(s, 2H, NCH₂), 3.64(s, 3H, OCH₃), 2.47-2.43(m, 2H, CH₂S), 2.03(s, 3H, SCH₃), 1.95-1.87(m, 2H, CCH₂); ^13C-NMR(75 MHz) δ: 172.1, 167.1, 157.8(d, ²J_FC = 25.7 Hz), 149.8, 139.5(d, ¹J_FC = 226.7 Hz), 131.2 (d, ²J_FC = 33.7 Hz), 52.3, 51.1, 49.6, 30.9, 29.5, 14.7; IR(KBr, cm^-1) ν: 3348, 3237, 2962, 1725, 1660, 1569, 1478, 1425, 1381, 1345, 1305, 1246, 1162, 794; MS(ESI) m/z: 332(M^-); ${\left[\alpha \right]}_D^{18.1}$+10.4(c 1.0, DMF).

(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxypropanoate (2f-1). Yield: 54%; m.p. 190-191°C; ¹H-NMR(300 MHz) δ: 11.83(d, 1H, NH of 5-FU, ⁴J_HH = 4.5 Hz), 8.63(d, 1H, NH, J = 8.1 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 5.11(t, 1H, OH, J = 5.4 Hz), 4.38(s, 2H, NCH₂), 4.42-4.36(m, 1H, NCH), 3.65(s, 3H, OCH₃), 3.75-3.58(m, 2H, CH₂OH, J = 5.4, 10.8 Hz); ^13C-NMR(75 MHz) δ: 170.9, 167.0, 157.6(d, ²J_FC = 25.7 Hz), 149.8, 139.3(d, ¹J_FC = 226.7 Hz), 131.2(d, ²J_FC = 33.7 Hz), 61.4, 54.8, 52.0, 49.4; IR(KBr, cm^-1) ν: 3459, 3295, 2850, 1713, 1692, 1563, 1467, 1416, 1382, 1277, 1251, 1185, 1071; MS(ESI) m/z: 288(M^-). ${\left[\alpha \right]}_D^{10.7}$-10.0 (c 1.0, DMF).

(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxypropanoate (2f-2). Yield: 55%; m.p. 190-192°C; ¹H-NMR(300 MHz) δ: 11.81(s, 1H, NH of 5-FU), 8.62(d, 1H, NH, J = 7.5 Hz), 8.00(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 5.12(t, 1H, OH, J = 5.4 Hz), 4.37(s, 2H, NCH₂), 4.41-4.35(m, 1H, NCH), 3.63(s, 3H, OCH₃), 3.74-3.56(m, 2H, CH₂OH, J = 5.4, 10.8 Hz); ^13C-NMR(75 MHz) δ: 171.0, 167.0, 157.7(d, ²J_FC = 25.6 Hz), 149.8, 139.3(d, ¹J_FC = 226.6 Hz), 131.4(d, ²J_FC = 33.5 Hz), 61.5, 54.9, 52.1, 49.5; IR(KBr, cm^-1) ν: 3459, 3295, 2850, 1713, 1692, 1563, 1467, 1416, 1382, 1277, 1251, 1185, 1071; MS(ESI) m/z: 288(M^-). ${\left[\alpha \right]}_D^{11.0}$+10.0 (c 1.0, DMF).

(S)-dimethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)succinate (2g). Yield: 65%; m.p. 145-147 °C; ¹H-NMR(300 MHz) δ: 11.85(d, 1H, NH of 5-FU, ⁴J_FH = 5.1 Hz), 8.76(d, 1H, NH, J =7.8 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.6 Hz), 4.68(dd, 1H, NCH, J =7.5, 6.6 Hz), 4.33(s, 2H, NCH₂), 3.64(s, 3H, OCH₃), 3.62(s, 3H, OCH₃), 2.85-2.70(m, 2H, CCH₂, J = 6.6, 7.5, 16.8 Hz); ^13C-NMR(75 MHz) δ: 170.9, 170.5, 166.9, 157.7(d, ²J_FC = 25.7 Hz), 149.8, 139.4(d, ¹J_FC = 228.0 Hz), 131.1 (d, ²J_FC = 33.9 Hz), 52.4, 51.9, 49.5, 48.7, 35.9; IR(KBr, cm^-1) ν: 3348, 3191, 2850, 1755, 1697, 1526, 1430, 1380, 1337, 1216, 1047, 980; MS(ESI) m/z: 330(M^-); ${\left[\alpha \right]}_D^{11.0}$-14.4(c 1.0, DMF).

(R)-diethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)succinate (2h). Yield: 68%; m.p. 116-118 °C; ¹H-NMR(300 MHz) δ: 11.82(d, 1H, NH of 5-FU, ⁴J_FH = 4.8 Hz), 8.71(d, 1H, NH, J = 7.8 Hz), 7.99(d, 1H, FC=CH, ³J_FH = 6.8 Hz), 4.63(dd, 1H, NCH, J = 6.6, 7.5 Hz), 4.33(s, 2H, NCH₂), 4.12-4.03(m, 4H, OCH₂, J = 6.9 Hz), 2.81-2.65(m, 2H, CCH₂, J = 6.6, 16.5 Hz), 1.17(t, 3H, CH₃, J = 6.9 Hz), 1.16(t, 3H, CH₃, J = 6.9 Hz); ^13C-NMR(75 MHz) δ: 170.4, 170.0, 166.9, 157.7(d, ²J_FC = 25.7 Hz), 149.8, 139.4(d, ¹J_FC = 228.3 Hz), 131.2 (d, ²J_FC = 33.8 Hz), 61.2, 60.6, 49.5, 48.9, 36.1, 14.2, 14.1; IR(KBr, cm^-1) ν: 3314, 3217, 2990, 1721, 1691, 1549, 1467, 1378, 1339, 1240, 1167, 1021, 792; MS(ESI) m/z: 358(M^-); ${\left[\alpha \right]}_D^{9.7}$+16.0 (c 1.0, DMF).

(S)-dimethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)pentanedioate (2i). Yield: 42%; m.p. 147-148 °C; ¹H-NMR(300 MHz) δ: 11.85(s, 1H, NH of 5-FU), 8.63(d, 1H, NH, J =7.8 Hz), 8.02(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.32(s, 2H, NCH₂), 4.37-4.34(m, 1H, NCH), 3.63(s, 3H, OCH₃), 3.58(s, 3H, OCH₃), 2.37(t, 2H, CH₂CH₂CO, J = 7.5 Hz), 2.06-1.76(m, 2H, CH₂CH₂CO); ^{13C-NMR(75 MHz)} δ: 172.7, 171.9, 168.0, 157.7(d, ²J_FC = 25.7 Hz), 149.8, 139.4(d, ¹J_FC = 226.7 Hz), 131.1(d, ²J_FC = 33.7 Hz), 52.2, 51.5, 51.3, 49.6, 29.6, 26.4; IR(KBr, cm^-1) ν: 3328, 2964, 1716, 1660, 1541, 1449, 1348, 1261, 800; MS(ESI) m/z: 344(M^-); ${\left[\alpha \right]}_D^{17.8}$-8.39 (c 0.5, DMF).

(R)-diethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)pentanedioate (2j). Yield: 45%; m.p. 108-109 °C; ¹H-NMR(300 MHz) δ: 11.84(s, 1H, NH of 5-FU), 8.61(d, 1H, NH, J = 7.8 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.33(s, 2H, NCH₂), 4.30-4.26(m, 1H, NCH), 4.08(q, 2H, OCH₂CH₃, J = 6.9 Hz), 4.04(q, 2H, OCH₂CH₃, J = 7.2 Hz), 2.36(t, 2H, CH₂CH₂CO, J = 7.5 Hz), 2.03-1.78(m, 2H, CH₂CH₂CO), 1.17(t, 3H, OCH₂CH₃, J = 6.9 Hz), 1.16(t, 3H, OCH₂CH₃, J = 7.2 Hz); ^{13C-NMR(75 MHz)} δ: 172.2, 171.4, 167.0, 157.7(d, ²J_FC = 25.7 Hz), 149.8, 139.4(d, ¹J_FC = 226.9 Hz), 131.2(d, ²J_FC = 33.7 Hz), 60.9, 60.1, 51.5, 49.6, 29.9, 26.5, 14.2, 14.1; IR(KBr, cm^-1) ν: 3304, 3213, 2924, 1725, 1675, 1546, 1468, 1416, 1379, 1250, 1176, 1023; MS(ESI) m/z: 372 (M^-); ${\left[\alpha \right]}_D^{8.7}$+12.40 (c 1.0, DMF).

(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(1H-indol-3-yl)pro-panoate (2k-1). Yield: 50%; m.p. 205-206 °C; ¹H-NMR(300 MHz) δ: 11.84(s, 1H, NH of 5-FU), 10.90(s, 1H, NH of indole), 8.72(d, 1H, NH, J = 7.2 Hz), 7.93(d, 1H, FC=CH, ³J_FH = 6.8 Hz), 7.48(d, 1H, Ar-H, J = 7.5 Hz), 7.33(d, 1H, Ar-H, J = 8.1 Hz), 7.16(d, 1H, =CHN, J = 2.2 Hz), 7.07(t, 1H, Ar-H, J = 8.1, 6.9 Hz), 6.99(t, 1H, Ar-H, J = 7.5, 6.9 Hz), 4.54(dd, 1H, NCH, J = 7.2, 6.6 Hz), 4.33(d, 2H, NCH₂, J = 16.5 Hz), 3.56(s, 3H, OCH₃), 3.19-3.03(m, 2H, CCH₂, J = 7.5, 6.0, 14.4 Hz); ^13C-NMR(75 MHz) δ: 172.2, 166.9, 157.8(d, ²J_FC = 25.7 Hz), 149.9, 139.4(d, ¹J_FC = 228.1 Hz), 136.3, 131.3(d, ²J_FC = 33.8 Hz), 127.3, 124.1, 121.2, 118.7, 118.2, 111.7, 109.2, 53.6, 52.1, 49.5, 27.4; IR(KBr, cm^-1) ν: 3386, 3312, 3069, 2977, 1702, 1545, 1381, 1233, 1062; MS(ESI) m/z: 387(M^-); ${\left[\alpha \right]}_D^{17.9}$+36.4 (c 1.0, DMF).

(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(1H-indol-3-yl)pro-panoate (2k-2). Yield: 53%; m.p. 205-206 °C; ¹H-NMR(300 MHz) δ: 11.84(d, 1H, NH of 5-FU, ⁴J_FH = 5.1 Hz), 10.90(s, 1H, NH of indole), 8.72(d, 1H, NH, J = 7.5 Hz), 7.93(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 7.48(d, 1H, Ar-H, J = 7.8 Hz), 7.34(d,1H, Ar-H, J = 8.1 Hz), 7.16(s, 1H, =CHN), 7.07(t, 1H, Ar-H, J = 7.2, 7.8 Hz), 6.99(t, 1H, Ar-H, J = 7.2 Hz), 4.55(dd, 1H, NCH, J = 6.9, 6.6 Hz), 4.33(d, 2H, NCH₂, J = 16.5 Hz), 3.57(s, 3H, OCH₃), 3.20-3.03(m, 2H, CCH₂, J = 7.5, 6.0, 14.8 Hz); ^13C-NMR(75 MHz) δ: 172.2, 166.9, 157.8 (d, ²J_FC = 25.7 Hz), 149.9, 139.4(d, ¹J_FC = 228.2 Hz), 136.3, 131.3(d, ²J_FC = 33.8 Hz), 127.3, 124.1, 121.2, 118.7, 118.2, 111.7, 109.2, 53.5, 52.1, 49.5, 27.4; IR(KBr, cm^-1) ν: 3392, 3320, 3075, 1732, 1648, 1542, 1446, 1385, 1355, 1248, 1219,1099, 744; MS(ESI) m/z: 387(M^-); ${\left[\alpha \right]}_D^{17.8}$-36.4 (c 1.0, DMF).

(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl) propanoate (2l). Yield: 72%; m.p. 192-193 °C; ¹H-NMR(300 MHz) δ: 11.85(s, 1H, NH of 5-FU), 9.28(s, 1H, OH), 8.68(d, 1H, NH, J = 7.5 Hz), 7.94(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 6.99 (d, 2H, Ar-H, J = 8.4 Hz), 6.66 (d, 2H, Ar-H, J = 8.4 Hz), 4.39(dd, 1H, NCH, J = 6.3, 7.5 Hz), 4.31(d, 2H, NCH₂, J = 16.8 Hz), 3.59(s, 3H, OCH₃), 2.93-2.77(m, 2H, CH₂Ar, J = 6.3, 8.1, 13.8 Hz); ¹³C-NMR(75 MHz) δ: 171.9, 166.9, 157.7(d, ²J_FC = 25.7 Hz), 156.3, 149.8, 139.4(d, ¹J_FC = 226.9 Hz), 131.2(d, ²J_FC = 33.8 Hz), 130.3, 127.0, 115.3, 54.4, 52.0, 49.5, 36.3; IR(KBr, cm^-1) ν: 3271, 1739, 1712, 1661, 1552, 1516, 1451, 1386, 1231, 1164, 778; MS(ESI) m/z: 364(M⁺); ${\left[\alpha \right]}_D^{17.8}$+16.6 (c 1.0, DMF).

(R)-ethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl) propanoate (2m). Yield: 70%; m.p. 156-158 °C; ¹H-NMR(300 MHz) δ: 11.82(s, 1H, NH of 5-FU), 9.23(s, 1H, OH), 8.63(d, 1H, NH, J = 7.5 Hz), 7.94(d, 1H, FC=CH, ³J_FH = 6.6 Hz), 6.99(d, 2H, Ar-H, J = 8.4 Hz), 6.65(d, 2H, Ar-H, J = 8.4 Hz), 4.36(dd, 1H, NCH, J = 7.5, 6.9 Hz), 4.31(d, 2H, NCH₂, J = 16.8 Hz), 4.02(q, 2H, OCH₂, J = 7.2 Hz), 2.89-2.77(m, 2H, CH₂Ar, J = 7.5, 6.0, 13.8 Hz), 1.10(t, 3H, CH₃, J = 7.2 Hz); ^13C-NMR(75 MHz) δ: 171.4, 166.9, 157.7(d, ²J_FC = 25.7 Hz), 156.3, 149.8, 139.4(d, ¹J_FC = 228.1 Hz), 131.2(d, ²J_FC = 33.9 Hz), 130.3, 127.0, 115.3, 60.7, 54.4, 49.5, 36.4, 14.1; IR(KBr, cm^-1) ν: 3336, 3065, 2851, 1691, 1532, 1380, 1340, 1220, 801; MS(ESI) m/z: 378(M^-); ${\left[\alpha \right]}_D^{9.7}$-24.4 (c 1.0, DMF).

(2S,3S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-methylpentanoate (2n). Yield: 70%; m.p. 152-154 °C; ¹H-NMR(300 MHz) δ: 11.82(d, 1H, NH of 5-FU, ⁴J_FH = 5.1 Hz), 8.57(d, 1H, NH, J = 8.1 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.9 Hz), 4.36(d, 2H, NCH₂, J = 16.8 Hz), 4.25(dd, 1H, NCH, J = 8.1, 6.6 Hz), 3.63(s, 3H, OCH₃), 1.77-1.72(m, 1H, CCH), 1.43-1.09(m, 2H, CCH₂), 0.84(t, 3H, CH₂CH₃, J = 7.2 Hz), 0.83(d, 3H, CHCH₃, J = 6.9 Hz); ^13C-NMR(75 MHz) δ: 172.0, 167.1, 157.7(d, ²J_FC = 25.7 Hz), 149.8, 139.3(d, ¹J_FC = 227.9 Hz), 131.4(d, ²J_FC = 33.8 Hz), 56.6, 52.0, 49.5, 36.9, 24.9, 15.6, 11.3; IR(KBr, cm^-1) ν: 3275, 3083, 2968, 1712, 1666, 1571, 1460, 1378, 1340, 1243, 1149, 976, 700; MS(ESI) m/z: 314(M^-); ${\left[\alpha \right]}_D^{17.5}$-3.0 (c 0.5, DMF).

(2S,3S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxybutanoate (2o). Yield: 47%; m.p. 207-208 °C; ¹H-NMR(300 MHz) δ: 11.79(d, 1H, NH of 5-FU, ⁴J_FH = 5.1 Hz), 8.44(d, 1H, NH, J = 8.4 Hz), 8.01(d, 1H, FC=CH, ³J_FH = 6.6 Hz), 5.03(d, 1H, OH, J = 5.1 Hz), 4.42(dd, 2H, NCH₂, J = 6.6, 16.5 Hz), 4.32(dd, 1H, NCH, J = 8.4, 3.3 Hz), 4.15-4.08(m, 1H, CHOH, J = 3.3, 5.1, 6.3 Hz), 1.05(d, 3H, CH₃, J = 6.3 Hz); ¹³C NMR(75 MHz) δ 171.0, 167.4, 157.7(d, ²J_FC = 25.6 Hz), 149.8, 139.3(d, ¹J_FC = 226.6 Hz), 131.4(d, ²J_FC = 33.5 Hz), 66.5, 58.1, 52.0, 49.6, 20.2; IR(KBr, cm^-1) ν: 3484, 3312, 2977, 1718, 1663, 1558, 1376, 1283, 1238, 1140; MS(ESI) m/z: 302(M^-); ${\left[\alpha \right]}_D^{17.6}$-2.0 (c 0.1, DMF).

4. 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.