Synthesis and bioevaluation of 5-fluorouracil derivatives

Article abstract of DOI:10.3390/12112450

A series of six novel 5-fluorouracil derivatives 1-6 were synthesized and their structures confirmed by ¹H- and ¹³C-NMR, MS and elemental analysis. The preliminary in vitro antitumor activities against B16, K562 and CHO cells and the in vivo inhibitions of liver cancer H₂₂ demonstrated that some of these compounds effectively inhibit the growth of tumor cells, but the in vivo trials in mice revealed that the compounds also exhibited serious liver and lung tissue toxicity. The hydrolysis experiments indicated that this type of compound did not readily liberate 5-fluorouracil, as expected.

Full text of DOI:10.3390/12112450

Molecules 2007, 12, 2450-2457
molecules
ISSN 1420-3049
2007 by MDPI
www.mdpi.org/molecules
©
Full Paper
Synthesis and Bioevaluation of 5-Fluorouracil Derivatives
1
, 2
2
2
3
1,
Zhi-Yong Tian , Gang-Jun Du , Song-Qiang Xie , Jin Zhao , Wen-Yuan Gao * and
3
,
Chao-Jie Wang *
1
School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai
District, 300072, Tianjin, P. R. China; Phone: (+86) 22-87401895; E-mail: tzynew@yahoo.com.cn,
2
College of Pharmaceutical Science, Henan University, Jinming District, 475001, Kaifeng, Henan,
P. R. China; Phone: (+86) 0378-3880660; E-mail: xiesq@henu.edu.cn
3
Institute of Natural Products and Medicinal Chemistry, College of Chemistry and Chemical
Engineering, Henan University, Jinming District, 475001, Kaifeng, Henan, P. R. China
*
Authors to whom correspondence should be addressed; E-mail: pharmgao@tju.edu.cn; Phone: (+86)
2-87401895; E-mail: wcjsxq@yahoo.com; Cell Phone: (+86) 13619810550
2
Received: 9 August 2007; in revised form: 9 October 2007 / Accepted: 9 October 2007 / Published: 6
November 2007
Abstract: A series of six novel 5-fluorouracil derivatives 1-6 were synthesized and their
1
13
structures confirmed by H- and C-NMR, MS and elemental analysis. The preliminary in
vitro antitumor activities against B16, K562 and CHO cells and the in vivo inhibitions of
liver cancer H22 demonstrated that some of these compounds effectively inhibit the growth
of tumor cells, but the in vivo trials in mice revealed that the compounds also exhibited
serious liver and lung tissue toxicity. The hydrolysis experiments indicated that this type of
compound did not readily liberate 5-fluorouracil, as expected.
Keywords: 5-Fluorouracil, prodrug, synthesis, bioevaluation
Introduction
-Fluorouracil (5-Fu, 7), first synthesized in 1957 [1], is one of the antitumor agents frequently
5
used for treating solid tumors such as breast, colorectal and gastric cancers [2]. 5-Fu is poorly tumor
selective, and therefore its therapeutic use results in high incidences of bone marrow, gastrointestinal

Molecules 2007, 12
2451
tract and central nervous system toxicity. To tackle these problems, numerous modifications of the 5-
Fu structure have been performed. Thus, a series of 5-Fu prodrugs in which 5-Fu is attached to amino
acids, peptides, phospholipids, and polymers have been reported [3-7]. These N-1 and/or N-3
substituted derivatives have exhibited improved pharmacological and pharmacokinetic properties,
including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity.
Recently, the concept of mutual prodrugs in which two different antineoplastic agents are coupled
directly or by means of a spacer has become well accepted [8-17]. This technique can be used to
overcome many problems including poor solubility or absorption, patient acceptability, drug instability
and toxicity, and especially drug resistance [11]. After the entry of a mutual prodrug into a cancer cell,
the two active components can reach a target simultaneously and are liberated concomitantly, whereas
they might be transported to the same site with different efficacy when administered individually. This
kind of prodrugs can display a broader antitumor spectrum, less drug resistance and less toxicity [18].
Many DNA intercalators have been evaluated as antineoplastic agents in chemotherapy, where they
constitute one of the most important drug classes. These agents are characterized by the presence of
flat chromophores bearing π electron-deficient polyconjugated plane bound to polar groups. Their
antitumor action results from DNA distortion and altered nuclear protein interaction as a consequence
of reversible complex formation. Because the cellular target of 5-Fu is DNA, the combination of 5-Fu
and some antineoplastic DNA binders through an ester bond [8, 14] helps the resulting dual prodrug
reach the target and optimize the efficiency of both 5-Fu and the DNA binders. According to this
strategy, we prepared six novel 5-fluorouracil derivatives and evaluated their antitumor activity both in
vitro and in vivo.
Results and Discussion
Chemistry
The synthetic route towards the target compounds 1-6 is shown in Scheme 1. 5-Fu was reacted with
1
3
formaldehyde to form a mixture of N -hydroxymethylene-5-fluorouracil, N -hydroxymethylene-5-
1
3
fluorouracil and N ,N -dihydroxymethylene-5-fluorouracil. Without separation, the mixture was
directly coupled with the appropriate carboxylic acids to give the target compounds. N,N'-dicyclo-
hexylcarbodiimide (DCC) was the first reagent chosen to facilitate the esterification, but the resulting
1,3-dicyclohexylurea (DCU) byproduct was hard to remove by flash chromatograph and
recrystallization. On the other hand, when N,N'-diisopropylcarbodiimide (DIC) was used, the target
compounds could be purified through flash chromatograph and recrystallization from acetone.
Two kinds of drugs with flat structures were attached to 5-Fu. First, nonsteroidal antiinflammatory
drugs such as naproxen and biphenylacetic acid, which have been reported to inhibit the growth of
cancer cells. In addition, naproxen and biphenylacetic acid can bind with DNA [19]. Second, imides,
which also exhibit efficient DNA-binding properties and antitumor activities on a variety of murine
and human tumor cells [20]. The imides were first converted to the corresponding carboxylic acids
through the reactions of 1,8-naphthalic or phthalic anhydride with relevant amino acids [21], then the
imide moiety was linked to 5-Fu via an (acyloxy)methylene group, known to be easily removable
1
13
[
3,8]. All structures were verified by H-NMR, C-NMR, MS and elemental analysis.

Molecules 2007, 12
2452
Scheme 1. Synthesis of compounds 1-6.
O
O
O
O
O
N
F
F
F
HOH CN
F
F
HCHO HN
60°C, 6h O
2
HOH
CN
RCOOH, DMAP
HN
HN
+
2
+
O
N
O
N
N
O
N
O
H
H
CH
2
OH
CH
2
OH DIC, r. t.
CH OOCR
2
9
10
1-6
7
8
O
H CO
3
N
O
R=
1
2
3
O
O
O
N
O
N
N
O
O
6
4
5
In vitro antitumor activity
All target compounds 1-6 were evaluated for their antitumor activity in vitro against K562, B16,
and CHO cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT)
assay method [6,22]. The reference drug used was 5-fluorouracil. The activity of the samples and the
reference drug was assayed under identical conditions at concentrations of 0.1, 1, 10, 30 and 50 µM.
The lower its IC50 value, the more efficiently a compound kills cancer cells. As shown in Table 1, all
compounds’ in vitro activity was significantly decreased because the N-1 position, the active area of 5-
Fu, was occupied. Compounds 2 and 6 still possessed some moderate to potent inhibition rates on the
three tested cell lines and compounds 3, 4 and 5 exhibited some selectivity against B16 and/or CHO
cells.
Table 1. The in vitro inhibition activity of compound 1-6 against three cell lines.
IC50 (µM)
Sample
K562
0.27
B16
0.23
12.15
36.93
3.72
4.44
3.97
CHO
.35
12.86
>50
5
-Fu
1
0
16.83
43.25
>50
2
3
32.30
16.83
4
30.46
>50
5
7
6
.41
.86
6
14.48
7.35
In vivo antitumor activity
The in vivo antitumor activities of three compounds on mice bearing liver cancer H22 are listed in
Table 2. The T/C ratio is used as an index of the antitumor activity: T/C (%) = (C－T) / C x 100 % (T:

Molecules 2007, 12
2453
Average tumor weight of treated mice; C: Average tumor weight of control mice). As shown in Table
, compounds 2, 5 effectively inhibited tumor growth (ratio of tumor inhibition >40%) although they
2
were less active than 5-Fu. Compound 2 was special because it was ineffective against all three cell
types in vitro but was effective in vivo. We also found that surprisingly all three tested compounds
caused more liver and lung tissue toxicity in mice than 5-Fu. This could happen if the hydrolysis of
compound 2, which contained only two clinically used drugs (5-Fu and naproxen), occurred readily
under physiological conditions, liberating toxic formaldehyde. To verify this the hydrolysis of
compound 2 was monitored by UV spectroscopy at physiological pH, but no 5-Fu was detected, thus
indicating that the (acyloxy)methylene group in target compounds is stable [8], and target compounds
might exert their biological functions as a new kind of “drugs”.
Table 2. The in vivo inhibition activity of compounds 2, 5 and 6 against H liver cancer.
2
2
Sample (T/C, %) Dose (µmol/kg) Weight of tumor (g) Ratio of tumor inhibition
Control
0
1.63±0.31
0.59±0.19
0.95±0.30
0.89±0.53
1.26±0.54
0
5
-Fu
2
150
150
150
150
64.07
41.64
45.17
22.85
5
6
Conclusions
In conclusion, a novel series of 5-fluorouracil derivatives was synthesized and some compounds
were found to be active against tumor cell lines in vitro and in vivo. Three tested compounds caused
more in vivo toxicities on the liver and lung tissues of mice than 5-Fu and were less effective than 5-
Fu. This might be explained by the difficult hydrolysis of target compounds to give free 5-fluorouracil
under physiological pH conditions.
Experimental
General
Melting points were determined on an X-6 hot stage microscope and were uncorrected. Mass
1
13
spectra were obtained on an ESQUIRE-LC instrument. H-NMR spectra and C-NMR were run in
DMSO-d , with TMS as the internal standard, on a Bruker AV-400 instrument operating at 400 MHz.
6
Elemental analysis was performed with a Gmbe VarioEL Elemental analysis instrument.
Synthesis of the target compounds 1-6
Synthesis of compounds 1-6 was accomplished as shown in Scheme 1. 5-Fu (7, 1.3 g, 10 mmol),
formaldehyde (37 wt.%, aq., 1.26 g, 15.5 mmol) and water (10 g) were added to a round-bottom flask,
which was then immersed in water bath at 60°C and agitated for 6 h. The product solution was
concentrated under reduced pressure to give an oily mixture containing 8-10. The oil was dissolved in

Molecules 2007, 12
2454
dry acetonitrile (50 mL), and the corresponding carboxylic acids (10 mmol), DMAP (N,N-
dimethylpyridin-4-amine, 80 mg, 14 mmol) and DIC (2.2 mL, 14 mmol) were added and the mixture
was stirred at room temperature for 48-72 h. The white precipitate was filtered off and the solvent was
removed by evaporation. The residue was dissolved in ethyl acetate (30 mL) and washed by diluted
hydrochloric acid (pH = 3-4), saturated aqueous NaHCO (pH = 7-8) and water. The organic layer was
3
collected, dried over sodium sulphate, filtered and finally concentrated under reduced pressure. The
target compounds were obtained through flash column chromatography: petroleum ether-acetone = 7:3
and recrystalization from acetone [8,14].
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-2-(biphenyl-4-yl)acetate (1). Yield: 31.1%;
1
m. p.: 180.5-182°C; H-NMR δ: 12.00 (s, 1H, NH of 5-Fu), 7.3-8.1 (m, 10H, 9×ArH, CH=CF of 5-
1
3
Fu), 5.58 (s, 2H, CH
2
O), 3.7 (s, 2H, CH
2
C=O); C-NMR δ: 170.89, 157.37 (d, JCCF = 26 Hz), 149.22,
= 34 Hz), 128.89, 127.38,
1
1
4
39.78, 139.41 (d, J = 229 Hz), 138.84, 132.92, 130.00, 129.42 (d, J
CF
CCF
+
2 4
26.61, 126.57, 70.86, 40.22; ESI-MS: m/z 377 (M +Na); Anal. calcd. for C19H15FN O : C, 64.40; H,
.27; N, 7.91; found: C, 64.33; H, 4.29; N, 7.81.
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-2-(7-methoxynaphthalen-2-yl)propanoate
1
(
2). Yield: 60.0%; m. p.: 171.5-172.5°C; H-NMR δ:11.96 (s, 1H, NH of 5-Fu), 7.10-8.1 (m, 7H,
6
1
1
1
xArH, CH=CF of 5-Fu), 5.60 (s, 2H, CH
2
O), 3.92 (s, 3H, CH
3 3
O), 3.84 (q, 1H, J = 6.6 Hz, CH CH),
1
3
.40 (d, J = 7.6 Hz, 3H, CH
3
CH); C-NMR δ: 173.53, 157.28 (d, JCCF = 26 Hz), 157.23, 149.09,
39.34 (d, JCF = 229 Hz), 135.02, 133.33, 129.34 (d, JCCF = 34 Hz), 129.08, 128.34, 127.00, 126.10,
+
25.64, 118.79, 105.70, 70.84, 55.15, 44.13, 18.13; ESI-MS: m/z 395 (M +Na); Anal. calcd. for
19 2 5
C H17FN O : C, 61.29; H, 4.60; N, 7.52; found: C, 61.36; H, 4.33; N, 7.29.
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-3-(1,8-naphthalimido)propanoate
(3).
1
Yield: 20.0%; m. p.: 215.5-217°C; H-NMR δ: 11.93 (s, 1H, NH of 5-Fu), 8.39-8.51 (4H, m, ArH),
8
2
2
.02 (d, 1H, J = 6.0 Hz, CH=CF), 7.83-7.87 (2H, m, ArH), 5.56 (s, 2H, OCH
2
), 4.29 (t, 2H, NCH
2
),
1
3
.74 (t, 2H, CH CO); C-NMR δ: 170.72, 163.3, 157.43 (d, J = 26 Hz), 149.20, 139.34 (d, J =
2
CCF
CF
29 Hz), 134.40, 131.24, 130.68, 129.43 (d, JCCF =34 Hz), 127.31, 127.14, 121.84, 70.42, 35.56, 32.19;
+
ESI-MS : m/z 434 (M +Na); Anal. calcd. for C20
H14FN
3
O
6
·0.30H
2
O: C, 57.64; H, 3.53; N, 10.08;
found: C, 57.71; H, 3.34; N, 9.75.
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-4-(1,8-naphthalimido)butyrate (4). Yield:
1
2
0.0%; m. p.: 233.5-235°C; H-NMR δ: 11.97 (s, 1H, NH of 5-Fu), 8.12-8.43 (m, 4H, ArH), 8.07 (d,
H, J = 6.0 Hz, CH=CF), 7.83-7.85 (m, 2H, ArH), 5.54 (s, 2H, OCH ), 4.06 (t, 2H, NCH ), 2.37 (t, 2H,
CO); C-NMR δ: 172.23, 163.59, 157.46 (d, JCCF = 25 Hz),
1
2
2
1
3
2 2 2
CH CO), 1.84-1.91 (m, 2H, CH CH
1
1
49.27, 139.31 (d, J = 229 Hz), 134.23, 133.96, 131.23, 130.61, 129.58 (d, J
CCF
= 34 Hz), 127.26,
CF
+
22.07, 70.23, 39.11, 30.58, 22.50; ESI-MS: m/z 448 (M + Na); Anal. calcd. for
21 3 6 2
C H16FN O ·0.39H O: C, 58.33; H, 3.91; N, 9.72; found: C, 58.73; H ,3.93; N, 9.26.
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-3-(phthalimido)propanoate (5). Yield:
1
4
6.7%; m. p.: 233-235°C; H-NMR δ: 11.92 (s, 1H, NH of 5-Fu), 8.10 (d, 1H, J = 6.0 Hz, CH=CF),

Molecules 2007, 12
2455
1
3
7
1
.82 (m, 4H, 4×ArH), 5.53 (s, 2H, OCH ), 3.81 (t, 2H, NCH ), 2.72 (t, 2H, CH CO); C-NMR δ:
2 2 2
70.84, 168.04, 157.92 (d, JCCF = 26Hz), 149.69, 139.83 (d, JCF = 229 Hz), 134.90, 131.99, 129.91 (d,
+
J
CCF = 34 Hz), 123.48, 70.93, 33.82, 32.74; ESI-MS: m/z 384 (M +Na); Anal. calcd. for C16
3 6
H12FN O :
C, 53.19; H, 3.35; N, 11.63; found: C, 53.16; H, 3.22; N, 11.37.
(
5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl-4-(phthalimido)butyrate (6). Yield: 71.5%;
1
m. p.: 181.5-184°C; H-NMR δ: 11.99 (s, 1H, NH of 5-Fu), 8.09 (d, 1H, J = 6.0 Hz, CH=CF), 7.76-
7
2 2 2
.84 (m, 4H, 4×ArH), 5.55 (s, 2H, CH O), 3.60 (t, 2H, NCH ), 2.43 (t, 2H, CH CO),1.82-1.86 (m, 2H,
1
3
CH
2
); C-NMR δ: 172.58, 168.29, 157.90 (d, JCCF = 26 Hz), 149.71, 139.83 (d, JCF = 229 Hz), 134.67,
+
1
32.04, 129.96 (d, JCCF = 34 Hz), 123.31, 70.82, 36.89, 30.81, 23.33; ESI- MS: m/z 398 (M + Na);
3 6
Anal. calcd. for C17H14FN O : C, 54.40; H, 3.76; N, 11.20; found: C, 54.54; H, 3.56; N, 11.91.
In vitro antitumor activity experiments
Cell lines (K562, B16, CHO) were obtained from the American Type Culture Collection (ATTC)
and cultured in RPMI1640, supplemented with 10% heat-inactivated fetal calf serum and antibiotics
(
penicillin, 100 units/mL; streptomycin sulfate, 100 μg/mL) at 37°C, in an atmosphere of 95% air and
% CO under humidified condition. All chemicals were purchased from Sigma, unless otherwise
5
2
indicated. RPMI1640 and fetal calf serum (FCS) were purchased from Gibco. Stock solution (10 µM)
of samples was prepared in dimethylsulfoxide (DMSO) and diluted with various concentrations with
serum-free culture medium. The in vitro antitumor activity of compounds 1-6 and 5-Fu were
determined by MTT assay method [10]. Briefly, exponentially growing B16 or CHO cells were seeded
in 96-well plates (5000 to each well) and allowed to attach overnight. After 24 h, the cells were treated
with indicated concentrations of samples for 48 h, and then MTT (100 μL, 1 mg/mL) was added. After
incubation for 4 h at 37°C, the MTT solution was removed and the crystals of viable cells were
dissolved with DMSO (150 μL) in each well; 4000/well exponentially growing K562 were seeded in
9
6-well plates and treated with indicated concentrations of samples for 48 h, and then MTT (10
mg/mL, 10 μL) was added. After incubation for 4 h at 37°C the crystals of viable cells were dissolved
overnight with SDS (sodium dodecylsulfonate, 10%, 100 μL) in each well. The absorbance spectra
were measured on an ELISA Processor II Microplate Reader at a wavelength of 570 nm. The
percentage of cytotoxicity was defined with treated and untreated cell lines. The 50% antitumor
activity dose (IC50) was defined as the concentration of samples that reduced the absorbance of the
treated cells by 50% [22].
In vivo antitumor activity experiments
To evaluate the in vivo antitumor activity of 5-fluorouracil derivatives 2, 5 and 6 mice bearing H22
tumor cells were used. Kunming mice (n = 10) were first injected subcutaneously (s.c.) with liver
5
cancer H cell line (2×10 cells/mL). The mice bearing the liver cancer H tumor cell line were then
2
2
22
injected intraperitoneally (i.p.) with water solutions of compounds 2, 5 or 6 for 8 days. For
comparison, antitumor activities of 5-fluorouracil (5-Fu) also were tested by the same method. A
control group was treated with H22 tumor cells along with the same volume of NaCl (0.9 wt.%, aq.).
The ratio (T/C) obtained by dividing the number of mice treated with 5-fluorouracil derivatives (T)

Molecules 2007, 12
2456
showing tumor inhibition to that of mice in the control groups (C) was used as the antitumor activity
index [22].
Hydrolysis experiments
Ultraviolet and visible spectral measurements were performed with a Varian DMS 300
spectrophotometer. Buffers (trishydroxymethylaminomethane-HCl, Tris-HCl, pH = 7.4) were made in
our laboratory. The UV method is defined as recording the absorbance changes over time for reactions
in which the absorbance of substrate and product differ maximally at a particular wavelength. This
method was used to study the activation of compound 2 [8]. Reactions were performed on aliquots of
buffer solution (3 mL) in a round-bottom flask by addition of compound 2 in chloroform at 25°C for 2
hours. Release of 5-Fu of compound 2 was determined from the absorbance at 266 nm. Initial
-5
concentration of all substrates studied by the UV method was 5× 10 M. No absorbance of 5-Fu was
observed at 266 nm.
Acknowledgements
This work was supported by Henan Natural Science Foundation (0423031800)
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