Synthesis and antitumor activity of amino acid ester derivatives containing 5-fluorouracil

Article abstract of DOI:10.3390/molecules14093142

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 assi

Full text of DOI:10.3390/molecules14093142

Molecules 2009, 14, 3142-3152; doi:10.3390/molecules14093142
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antitumor Activity of Amino Acid Ester
Derivatives Containing 5-Fluorouracil
Jing Xiong 1,2,*, Hai-Feng Zhu 1, Ya-Juan Zhao 1, Yun-Jun Lan 1, Ji-Wang Jiang 1,
Jing-Jing Yang 2 and Shu-Feng Zhang 2,*
1
College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325000, China
2
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
* Author to whom correspondence should be addressed; E-mails: xiongjing@wzu.edu.cn (J.X.),
Zhangshf@chem.dlut.edu.cn (S.-F.Z.); Tel.: +86-13738390567.
Received: 15 July 2009; in revised form: 18 August 2009 / Accepted: 20 August 2009 /
Published: 25 August 2009
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.
Keywords: 5-fluorouracil; amino acid ester; antitumor activity
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 [1-3]. 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 [4-10]. In general prodrug systems the drug is bound

Molecules 2009, 14
3143
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 [14-16]. 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 [18-20].
Scheme 1. Synthesis of 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-aceto
amino acid ester derivatives 2.
COOR2
F
R1
C
H
NH2
OH
N
N
EDC/DMF
TEA
O
N
O
+
HN
O
N
OH
1
(HOBt)
F
COOR2
R1
O
O
CH3
CH3
H
H
.
O
N
O
CH2C N CH
+
HCl
+ HOBt
C2H5 N C NH(CH2)3N
HN
2

Molecules 2009, 14
3144
Figure 1. Structural formulae of compounds 2.
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
1
structures. The 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,
18.1
D
18.1 -10.4 and
[
α
]
+10.4, respectively. In the H-NMR their
1
but opposite specfic rotations of
[
α
]
D
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 (Tables 1 and 2).

Molecules 2009, 14
3145
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.
Table 1. Inhibitory rates (%) against HL-60.
Concentration (mol/L)
Compounds
10-4
2.0
10-5
8.0
5.2
0.1
0
10-6
9,6
6.2
9.9
1.7
7.1
3.4
6.6
3.7
5.7
23.1
1.5
2.7
3.8
9.8
5.2
12.6
0
10-7
6.9
9.7
9.8
0
10-8
8.3
2.8
0
10.0
1.2
0
2a
2b
2c
3.7
33.1
27.3
22.4
18.5
3.3
31.8
36.1
55.7
2.8
2d
2e-1
2e-2
2f-1
2f-2
2g
2h
2i
11.2
15.3
10.8
9.2
10.0
19.6
6.7
12.8
0
4.2
10.5
12.3
6.2
5.0
2.3
8.3
5.4
8.2
12.7
9.9
13.0
0
0
0
2.5
8.6
0.6
5.9
2.4
8.9
5.7
0.3
0
2j
55.8
29.2
51.2
42.4
65.1
11.4
22.4
57.4
0
2k-1
2k-2
2l
2m
2n
2o
5-FU
FT-207
15.5
7.9
0
0
11.2
33.5
0
7.1
0
0
4.2
7.0
0
1.2
10.4
0
Table 2. Inhibitory rates (%) against BEL-7402.
Concentration (mol/L)
Compounds
10-4
0
9.0
10-5
0
8.1
13.2
0
10-6
0
10-7
0
10-8
0
1.8
0
2a
2b
2c
0
5.7
0
0
5.2
0
50.0
13.2
41.2
38.4
17.4
41.2
36.1
52.6
14.5
34.0
35.9
22.4
36.2
71.7
8.2
2d
0
2e-1
2e-2
2f-1
2f-2
2g
2h
2i
9.7
9.1
10.5
9.8
11.1
15.9
8.7
9.2
0
11.9
10.2
68.3
4.5
0
8.8
8.8
16.6
0
8.2
0
14.0
0
7.4
0.7
4.6
3.5
3.0
4.2
0.5
43.1
0
9.1
5.3
4.6
0
2.4
0
11.7
5.9
0.7
1.2
0
24.3
0
7.0
2.4
8.0
4.1
6.9
7.1
4.9
60.4
5.2
8.8
35.0
0
2j
2k-1
2k-2
2l
2m
2n
2o
5-FU
FT-207
9.7
72.6
58.0
8.2
23.8
0
9.1
16.6
0
53.8
8.1

Molecules 2009, 14
3146
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
1
instrument. IR spectra were recorded (in KBr) on a Bruker EQUINOX 55. H-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
1
(2a). Yield: 68%; m.p. 109-110ºC; H-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, 3JFH = 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),
13
2
0.87 (d, 3H, CH3, J = 6.6 Hz); C-NMR δ: 171.9, 167.1, 157.7(d, JFC = 25.6 Hz), 149.8, 139.3 (d,
1JFC = 226.7 Hz), 131.3 (d, 2JFC = 33.6 Hz), 57.7, 51.9, 49.5, 30.4, 19.0, 18.3; IR (cm-1) ν: 3456, 3280,
10.0
D
2969, 1722, 1666, 1560, 1467, 1379, 1227, 1146, 783; MS (ESI) m/z: 300 (M-);
[α
]
-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).
1
Yield: 65%; m.p. 136-137 ºC; H-NMR δ: 11.83(s, 1H, NH of 5-FU), 8.53 (d, 1H, NH, J = 8.1 Hz),
3
8.02 (d, 1H, FC=CH, JFH = 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,
13
J = 7.2 Hz), 0.89 (d, 3H, CH3, J = 6.9 Hz), 0.88 (d, 3H, CH3, J = 6.6 Hz); C-NMR δ: 174.0, 169.6,
160.5 (d, 2JFC = 25.3 Hz), 151.6, 141.2 (d, 1JFC = 228.1 Hz), 132.8 (d, 2JFC = 33.5 Hz), 63.3, 59.9, 51.6;

Molecules 2009, 14
31.6, 20.0, 19.2, 15.2; IR (cm-1) ν: 3295, 3253, 2977, 1707, 1552, 1467, 1377, 1225, 1148; MS (ESI)
3147
m/z: 314(M-);
[
α
]
18.0 +14.0 (c 0.1, DMF).
D
(S)-Methyl
2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-methylpentanoate
(2c). Yield: 75%; m.p. 144-145ºC; 1H-NMR δ: 11.83 (d, 1H, NH of 5-FU, 4JFH = 5.4 Hz), 8.60 (d, 1H,
3
NH, J = 7.8 Hz), 8.02 (d, 1H, FC=CH, JFH = 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),
13
2
0.83 (d, 3H, CH3, J = 6.3 Hz); C-NMR δ 172.9, 167.0, 157.8 (d, JFC = 25.5 Hz), 149.8, 139.4 (d,
1JFC = 226.6 Hz), 131.3 (d, 2JFC = 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-);
[
α
]
16.1 -19.2 (c 1.0, DMF).
D
(R)-Ethyl
2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-methylpentanoate
1
4
(2d). Yield: 72%; m.p. 146-147ºC; H-NMR δ: 11.81(d, 1H, NH of 5-FU, JFH = 5.1 Hz), 8.57(d, 1H,
3
NH, J =7.8 Hz), 8.01(d, 1H, FC=CH, JFH = 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, 1JFC = 226.4 Hz), 131.2 (d, 2JFC = 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-);
[
α
]
17.7 +11.6(c 1.0, DMF).
D
(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-(methylthio)butanoate
1
(2e-1). Yield: 62%; m.p. 139-140ºC; H-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)
2
1
δ: 172.2, 167.1, 157.8(d, JFC = 25.7 Hz), 149.9, 139.5(d, JFC = 226.5 Hz), 131.2(d, 2JFC = 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-);
[
α
18.1 -10.4(c 1.0, DMF).
D
]
(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-4-(methylthio)butanoate
1
(2e-2). Yield: 59%; m.p. 138-140 ºC; H-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, 3JFH = 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)
2
δ: 172.1, 167.1, 157.8(d, 2JFC = 25.7 Hz), 149.8, 139.5(d, 1JFC = 226.7 Hz), 131.2 (d, JFC = 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-);
[
α
]
18.1 +10.4(c 1.0, DMF).
D
(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxypropanoate
(2f-1). Yield: 54%; m.p. 190-191ºC; 1H-NMR(300 MHz) δ: 11.83(d, 1H, NH of 5-FU, 4JHH = 4.5 Hz),
8.63(d, 1H, NH, J = 8.1 Hz), 8.01(d, 1H, FC=CH, 3JFH = 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, JFC = 25.7 Hz), 149.8, 139.3(d, JFC = 226.7 Hz),
2
1

Molecules 2009, 14
3148
131.2(d, 2JFC = 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-). 10.7 -10.0 (c 1.0, DMF).
α
[ ]
D
(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxypropanoate
1
(2f-2). Yield: 55%; m.p. 190-192ºC; H-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, 3JFH = 6.9 Hz), 5.12(t, 1H, OH, J = 5.4 Hz), 4.37(s, 2H, NCH2),
13
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); C-
NMR(75 MHz) δ: 171.0, 167.0, 157.7(d, 2JFC = 25.6 Hz), 149.8, 139.3(d, 1JFC = 226.6 Hz), 131.4(d,
2JFC = 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-).
[
α
]
11.0 +10.0 (c 1.0, DMF).
D
(S)-dimethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)succinate (2g). Yield:
1
4
65%; m.p. 145-147 ºC; H-NMR(300 MHz) δ: 11.85(d, 1H, NH of 5-FU, JFH = 5.1 Hz), 8.76(d, 1H,
3
NH, J =7.8 Hz), 8.01(d, 1H, FC=CH, JFH = 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-
2
1
NMR(75 MHz) δ: 170.9, 170.5, 166.9, 157.7(d, JFC = 25.7 Hz), 149.8, 139.4(d, JFC = 228.0 Hz),
131.1 (d, 2JFC = 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-);
[
α
]
11.0 -14.4(c 1.0, DMF).
D
(R)-diethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)succinate (2h). Yield:
1
4
68%; m.p. 116-118 ºC; H-NMR(300 MHz) δ: 11.82(d, 1H, NH of 5-FU, JFH = 4.8 Hz), 8.71(d, 1H,
NH, J = 7.8 Hz), 7.99(d, 1H, FC=CH, 3JFH = 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,
1
2
2JFC = 25.7 Hz), 149.8, 139.4(d, JFC = 228.3 Hz), 131.2 (d, JFC = 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-);
[
α
9.7 +16.0 (c 1.0, DMF).
D
]
(S)-dimethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)pentanedioate (2i).
1
Yield: 42%; m.p. 147-148 ºC; H-NMR(300 MHz) δ: 11.85(s, 1H, NH of 5-FU), 8.63(d, 1H, NH,
3
J =7.8 Hz), 8.02(d, 1H, FC=CH, JFH = 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, 2JFC = 25.7 Hz), 149.8, 139.4(d, 1JFC = 226.7 Hz),
2
131.1(d, JFC = 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-);
[
α
]
17.8 -8.39 (c 0.5, DMF).
D
(R)-diethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)pentanedioate (2j).
1
Yield: 45%; m.p. 108-109 ºC; H-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, 3JFH = 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);

Molecules 2009, 14
3149
13C-NMR(75 MHz) δ: 172.2, 171.4, 167.0, 157.7(d, 2JFC = 25.7 Hz), 149.8, 139.4(d, 1JFC = 226.9 Hz),
2
131.2(d, JFC = 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-);
[
α
]
8.7 +12.40 (c
D
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-
1
panoate (2k-1). Yield: 50%; m.p. 205-206 ºC; H-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, 3JFH = 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, 2JFC = 25.7 Hz), 149.9, 139.4(d, 1JFC = 228.1 Hz), 136.3, 131.3(d, 2JFC
= 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-);
[
α
]
17.9 +36.4 (c 1.0,
D
DMF).
(R)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(1H-indol-3-yl)pro-
1
panoate (2k-2). Yield: 53%; m.p. 205-206 ºC; H-NMR(300 MHz) δ: 11.84(d, 1H, NH of 5-FU,
4JFH = 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,
3JFH = 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, 2JFC = 25.7 Hz), 149.9, 139.4(d, 1JFC = 228.2 Hz), 136.3,
2
131.3(d, JFC = 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-);
[
α
]
17.8 -36.4 (c 1.0, DMF).
D
(S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl)
1
propanoate (2l). Yield: 72%; m.p. 192-193 ºC; H-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, 3JFH = 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, 2JFC = 25.7 Hz), 156.3, 149.8, 139.4(d, 1JFC = 226.9 Hz), 131.2(d, 2JFC = 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+);
[α
]
17.8 +16.6 (c 1.0, DMF).
D
(R)-ethyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl)
1
propanoate (2m). Yield: 70%; m.p. 156-158 ºC; H-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, 3JFH = 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,

Molecules 2009, 14
3H, CH3, J = 7.2 Hz); 13C-NMR(75 MHz) δ: 171.4, 166.9, 157.7(d, JFC = 25.7 Hz), 156.3, 149.8,
3150
2
1
2
139.4(d, JFC = 228.1 Hz), 131.2(d, JFC = 33.9 Hz), 130.3, 127.0, 115.3, 60.7, 54.4, 49.5, 36.4, 14.1;
9.7
IR(KBr, cm-1) ν: 3336, 3065, 2851, 1691, 1532, 1380, 1340, 1220, 801; MS(ESI) m/z: 378(M-);
[
α
]
-
D
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
1
4
(2n). Yield: 70%; m.p. 152-154 ºC; H-NMR(300 MHz) δ: 11.82(d, 1H, NH of 5-FU, JFH = 5.1 Hz),
3
8.57(d, 1H, NH, J = 8.1 Hz), 8.01(d, 1H, FC=CH, JFH = 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:
2
1
2
172.0, 167.1, 157.7(d, JFC = 25.7 Hz), 149.8, 139.3(d, JFC = 227.9 Hz), 131.4(d, JFC = 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-);
[
α
]
17.5 -3.0 (c 0.5, DMF).
D
(2S,3S)-methyl 2-(2-5-(fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-hydroxybutanoate
1
4
(2o). Yield: 47%; m.p. 207-208 ºC; H-NMR(300 MHz) δ: 11.79(d, 1H, NH of 5-FU, JFH = 5.1 Hz),
3
8.44(d, 1H, NH, J = 8.4 Hz), 8.01(d, 1H, FC=CH, JFH = 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,
1
2
2JFC = 25.6 Hz), 149.8, 139.3(d, JFC = 226.6 Hz), 131.4(d, JFC = 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-);
[
α
17.6 -2.0 (c 0.1, DMF).
D
]
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.
Acknowledgements
We thank the National Centre for Drug Screening, Shanghai, China, for evaluation of the in vitro
antitumor activities. This research was supported by Zhejiang Provincial Technology Project
Foundation (No. 2008C21034) and Wenzhou Technology Project Foundation (No. S20060029).
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Sample Availability: Samples of the compounds are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).