Design and synthesis of novel camptothecin/5-fluorouracil conjugates as cytotoxic agents

Article abstract of DOI:10.1080/14786411003792181

In an effort to overcome several limitations associated with the synthesis of camptothecin (CPT), seven conjugates (10a-10g) composed of CPT and a 5-fluorouracil derivative joined by suitable dipeptide linkages were synthesised, and their cytotoxic activi

Full text of DOI:10.1080/14786411003792181

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Design and synthesis of novel
camptothecin/5-fluorouracil conjugates
as cytotoxic agents
Ying-Qian Liu a b , Wei Dai b , Liu Yang c & Hong-Yu Li a b
a MOE Key Laboratory of Arid and Grassland Ecology , School of
Life Sciences, Lanzhou University , Lanzhou 730000, P.R. China
b School of Pharmacy, Lanzhou University , Lanzhou 730000, P.R.
China
c School of Environmental and Municipal Engineering, Lanzhou
Jiaotong University , Lanzhou 730000, P.R. China
Published online: 30 Mar 2011.
To cite this article: Ying-Qian Liu , Wei Dai , Liu Yang & Hong-Yu Li (2011) Design and synthesis
of novel camptothecin/5-fluorouracil conjugates as cytotoxic agents, Natural Product Research:
Formerly Natural Product Letters, 25:19, 1817-1826, DOI: 10.1080/14786411003792181
To link to this article: http://dx.doi.org/10.1080/14786411003792181
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Natural Product Research
Vol. 25, No. 19, November 2011, 1817–1826
Design and synthesis of novel camptothecin/5-fluorouracil conjugates
as cytotoxic agents
Ying-Qian Liuab, Wei Daib, Liu Yangc and Hong-Yu Liab
*
aMOE Key Laboratory of Arid and Grassland Ecology, School of Life Sciences,
Lanzhou University, Lanzhou 730000, P.R. China; School of Pharmacy, Lanzhou
b
c
University, Lanzhou 730000, P.R. China; School of Environmental and Municipal
Engineering, Lanzhou Jiaotong University, Lanzhou 730000, P.R. China
(Received 4 November 2009; final version received 19 March 2010)
In an effort to overcome several limitations associated with the synthesis
of camptothecin (CPT), seven conjugates (10a–10g) composed of CPT and
a 5-fluorouracil derivative joined by suitable dipeptide linkages were
synthesised, and their cytotoxic activity against four human tumour cell
lines as well as an in vitro pharmacokinetic determination of their lactone
stability were studied. Among these compounds, most tested conjugates
showed cytotoxic activities comparable or superior to CPT-11 (2), but they
were less potent when compared with CPT (1). Interestingly, all of the
compounds showed selective inhibitory activities against BGC-823, with
IC50 values lower than 0.1 mmol, which is more potent than CPT-11 (2).
Also, the in vitro pharmacokinetic determination of the lactone levels of the
representative compound 10b showed that the biological life span of their
lactone forms in human and mouse plasma were significantly increased
when compared with their mother compound CPT (1).
Keywords: camptothecin; 5-fluorouracil; antineoplastic properties;
conjugates
1. Introduction
A challenging research focus at present in cancer therapy is the discovery of
molecules that could be selective for tumour cells and that could be characterised
by reduced undesirable effects. Among the various strategies to improve the
therapeutic index of different drugs, conjugation of cytotoxic drug components
has proven to be a promising approach to enhance the activity and selectivity of
some monomeric leads by forming bivalent heterodimers (Liu, Yang, & Tian, 2007;
Tietze, Bell, & Chandrasekhar, 2003). This concept is now accepted as an effective
strategy for designing ligands, inhibitors and other drugs that influence biological
systems. On the basis of this theory, some interesting results have been reported
by our group as well as others in recent years (Chen, Xiang, Liu, & Tian 2009; Liu,
Liu, Tian, & Yang, 2008; Ohtsu, Nakanishi, Bastow, F. Lee, & K. Lee, 2003;
Shi et al., 2001; Tatsuzaki et al., 2007). In our previous study, we explored the
*Corresponding author. Emails: lihy@lzu.edu.cn; yqliu@lzu.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/14786411003792181
http://www.tandfonline.com

1818
Y.-Q. Liu et al.
R3
N
R2
R1
O
O
N
O
N
F
HN
N
O
O
O
O
N
H
OH
N
O
OH
5-Fluorouracil (4)
O
R3=CH2CH3
R2=H,
,
Irinotecan (2):
N
R1=
Camptothecin (1)
O
Topotecan (3): R1=OH, R2=(CH3)2NHCH2, R3=H
Figure 1. Structures of CPT (1), irinotecan (2), topotecan (3) and 5-fluorouracil (4).
syntheses and evaluation of heterodimer conjugates by the combination of two
kinds of antitumour drugs through various linkages, which led to a large
increase in potency over their monomeric counterparts (Chen et al., 2009; Liu
et al., 2007, 2008).
Camptothecin (CPT) (1) is a topoisomerase I-targeting cytotoxic alkaloid
with significant antineoplastic activity (Liew
& Yang, 2008; Slichenmyer,
Rowinsky, Donehower, & Kaufmann, 1993; Takimoto, Wright, & Arbuck, 1998).
Hydrophilisation of camptothecin molecules results in the identification and
development of topotecan (2) and irinotecan (3) (Figure 1), which have been
clinically proven for use in the treatment of ovarian and colon cancers, respectively
(Li, Zu, Shi, & Yao, 2006; Lorence & Nessler, 2004). However, as an important class
of anticancer compounds, both poor water-solubility and lactone instability appear
to be the predominant reason for the difficulty in clinically developing CPTs and
therefore, these compounds are suitable for incorporation as parent drugs in a
prodrug approach (Driver & Yang, 2005; Du, 2003; Verma & Hansch, 2009). Using
the prodrug concept, the water solubility of the most active CPTs can be adequately
enhanced so as to allow parenteral administration in an aqueous medium. The
acylation of the 20-hydroxyl group may also render the lactone more stable towards
ring opening. This may increase the availability of an active drug that contains the
intact lactone.
Antimetabolite 5-fluorouracil (5-FU) (4) is one of the major anticancer agents
used clinically for the treatment of stomach, colorectal, head and neck cancers
(Campos, Domınguez, Gallo, & Espinosa, 2000). But 5-FU is poorly tumour
´
selective, so its therapy causes high incidences of toxicity in the bone marrow,
gastrointestinal tract, central nervous system and skin, which has prompted efforts
in the development of derivatives aiming at reducing the adverse effects of 5-FU.
Among the various attempts to optimise the efficacy of 5-FU, one of the main
strategies that potentiates its antitumour activity is its chemical and biochemical
modulation, by combination with other cytotoxic agents or with biochemical
modulators (Longley, Harkin, & Johnston, 2003; Peters et al., 2000). Also,
considerable data from clinical studies have shown that the combined use of CPT-
related analogues and 5-FU resulted in higher response rates than for either agent
alone, and simultaneously circumvented some faults, such as decreasing the amount
of repair needed for CPT-induced DNA damage and increasing the induction of

Natural Product Research
1819
apoptosis, or increasing DNA adduct formation in cancer cells (Guichard,
Hennebelk, Bugat, & Cana, 1998; Peters et al., 2000).
Based on the abovementioned information, the conjugation of 5-FU and CPT
with dipeptide spacers might provide new classes of antitumour drug candidates with
improved tumour selectivity, efficiency and safety.
2. Results and discussion
2.1. Chemistry
The reaction of 5-FU (4) with chloroacetic acid in the presence of potassium
hydroxide gave the corresponding 5-fluorouracil-1-acetic acid (5) in an almost
excellent yield (Liu, Yang, & Tian, 2006), which upon treatment with p-nitrophenol
using N,N-dicyclohexylcarbodiimide (DCC) afforded activated ester 6. Sequential
treatment of 6 with a series of L-amino acids in alkaline DMF–H2O solvent produced
the corresponding [(5-fluorouracil-1-yl)acetyl]-L-amino acids 7a–7g (Scheme 1)
(Chen, Tian, & Tu, 2004).
CPT (1) was isolated from a Chinese medicinal plant, C. acuminata, and it served
as the starting material for the preparation of all the derivatives. The conversion
of the isolated available CPT to the CPT-20-ester of N-Boc-glycine derivative 8
in the presence of DCC and 4-dimethylaminopyridine (DMAP) as catalysts was
accomplished by a modified version of Greenwald’s method (Greenwald et al., 1998),
followed by the removal of the N-Boc group of 8 with TFA in CH2Cl2 (1 : 1),
forming the TFA salt 9. The desired compounds 10a–10g were achieved by treating
the TFA salt 9 with the corresponding [(5-fluorouracil-1-yl)acetyl]-L-amino acids
7a–7g using 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride
(EDCI) as the coupling agent (Scheme 2). The desired compounds 10a–10g were
purified by standard flash chromatography on silica gel and characterised by m.p.,
1
IR, H-NMR and HRMS analyses. The analytical and spectral data are illustrated
in Tables 1 and 2, respectively.
O
O
F
O
F
F
HN
HN
p-NO2C6H5OH
ClCH2COOH
HN
O
N
O
10%KOH, reflux
N
DCC/DMF, rt, stir
O
N
H
NO2
O
OH
O
O
5
6
4
RCH(NH2)COOH,
5% NaOH
O
F
HN
DMF-H2O, stir
O
O
N
H
N
OH
a. R = H
b. R = CH3
c. R = CH2Ph
e. R = CH2CH(CH3)2
d. R = CH(CH3)2
f. R = CH2SCH3
O
R
7a–7g
g. R = CH(OH)CH3
Scheme 1. Synthesis of [(5-fluorouracil-1-yl)acetyl]-L-amino acids 7a–7g.

1820
Y.-Q. Liu et al.
O
O
O
N
50%TFA/CH Cl
2
N
Boc-NHCH CO
H
2
2
2
N
N
N
r t
N
DCC/DMAP,
CH Cl , rt
O
O
O
O
2
2
O
O
O
O
OH
2TFA
NH
NH
Boc
O
O
1
8
9
7a–7g
O
O
N
EDCI/HOBt/DMF,stir
N
F
N
HN
O
O
O
a. R = H
b. R = CH3
d. R = CH(CH3)2
O
H
N
c. R = CH2Ph
O
e. R = CH2CH(CH3)2
g. R = CH(OH)CH3
f. R = CH2SCH3
N
H
O
O
R
10a–10g
Scheme 2. Synthesis of the target compounds 10a–10g.
Table 1. Analytical data of compounds 10a–10g.
20
½ꢀꢁD (ꢀ)
Yield m.p.
Compound (%)
(ꢀC) (c ¼ 0.5, DMF)
HRMS (m/z)
10a
10b
10c
10d
10e
10f
10g
73 186–188
ꢂ79
Calcd for C30H25N6O9F: 633.1740 [M þ H]þ;
found: 633.1740 [M þ H]þ
69 189–191
ꢂ80
ꢂ71
ꢂ90
ꢂ77
ꢂ67
ꢂ75
Calcd for C31H27N6O9F: 647.1896 [M þ H]þ;
found: 647.1896 [M þ H]þ
56 190–192
52 186–188
45 176–178
68 192–194
62 192–194
Calcd for C37H31N6O9F: 745.209 [M þ Na]þ;
found: 745.2014 [M þ Na]þ
Calcd for C33H31N6O9F: 675.2209 [M þ H]þ;
found: 675.2219 [M þ H]þ
Calcd for C34H33N6O9F: 689.2366 [M þ H]þ;
found: 689.2366 [M þ H]þ
Calcd for C33H31N6O9SF: 729.1749 [M þ Na]þ;
found: 729.1738 [M þ Na]þ
Calcd for C32H29N6O10F: 699.1821 [M þ Na]þ;
found: 699.1825 [M þ Na]þ
2.2. Cytotoxic activities
The cytotoxicities of compounds 10a–10g were measured on four different human
cancer cell lines (SGC-7901, BGC-823, A-549 and HePG-2) using an in vitro MTT
assay. Irinotecan (2) and CPT (1) were used as reference compounds. The results are
listed in Table 3.
As shown in Table 3, all the conjugates were less potent than 1 in the cytotoxic
assay, which is almost a common feature of prodrugs, whereas most of the
compounds exhibited cytotoxic activities comparable or superior to irinotecan (2).
Significantly, the compounds were more sensitive against the BGC-823 cell line than
against the other cell lines, which is consistent with the clinical behaviour of CPT
derivatives. For the BGC-823 cell line, all the compounds showed high inhibitory
activities with IC50 values lower than 0.1 mM, which was more potent than

Natural Product Research
1821
Table 2. Spectral data of compounds 10a–10g.
Compound
IR KBr, ꢁmax (cmꢂ1
)
1H-NMR: (DMSO-d6) ꢂ (ppm)
10a
3463 (NH), 3073, 1619,
1595, 1559 (ArH),
1762, 1702, 1664
(C ¼ O), 123 6(C–F),
1184, 115 5(ester link-
age –O–C)
0.89 (t, 3H, H-18), 2.13 (q, 2H, H-19), 4.04 and
4.23 (2d, 4H, 2 ꢃ CH2–Gly-Gly), 4.29 (s, 2H, 5-
FU–N–CH2), 5.47 (s, 2H, H-17), 5.26 (s, 2H,
H-5), 7.14 (s, 1H, H-14), 7.70 (t, 1H, H-11),
7.84 (s, 5-FU-ring–H-6), 7.935 (t, 1H, H-10),
8.123(d, 1H, H-12), 8.460 (d, 1H, H-9), 8.661
(s, 1H, 1H, H-7), 11.80 (s, 1H, 5-FU-ring–NH)
10b
3359 (NH), 3067, 1600,
1558 (ArH), 1749,
1698, 1662 (C ¼ O),
1237 (C–F), 1187,
1158 (ester linkage
–O–C)
0.92 (t, 3H, H-18), 1.24 (d, 3H, L-alanine–CH3),
2.15 (q, 2H, H-19), 4.05 (d, 2H, CH2–Gly),
4.25(m, 1H, L-alanine–CH), 4.35 (s, 2H, 5-FU–
N–CH2), 5.31 (s, 2H, H-5), 5.50 (s, 2H, H-17),
7.17 (s, 1H, H-14), 7.73 (t, 1H, H-11), 7.85 (s,
1H, 5-FU-ring–H-6), 7.89 (t, 1H, H-10), 8.15
(d, 1H, H-12), 8.48 (d, 1H, H-9), 8.71 (s, 1H,
H-7), 11.80 (s, 1H, 5-FU-ring–NH)
10c
3293 (NH), 3065, 1598,
1559 (ArH), 1750,
1699, 1662 (C ¼ O),
1237(C–F), 1188,
1161 (ester linkage
–O–C)
0.92 (t, 3H, H-18), 2.15 (q, 2H, H-19), 3.03 (m,
2H, L-phenylalanine–CH2), 4.08 (d, 2H, CH2–
Gly), 4.35 (s, 2H, 5-FU–N–CH2), 4.56 (m, 1H,
L-phenylalanine–CH), 5.30 (s, 2H, H-5), 5.51
(s, 2H, H-17), 7.19 (s, 1H, H-14), 7.23 (m, 5H,
L-phenylalanine–ArH), 7.73 (t, 1H, H-11), 7.85
(s, 1H, 5-FU-ring–H-6), 7.88 (t, 1H, H-10), 8.15
(d, 1H, H-12), 8.54 (d, 1H, H-9), 8.70 (s, 1H,
H-7), 11.77 (s, 1H, 5-FU-ring–NH)
10d
3311 (NH), 3067, 1599,
1557 (ArH), 1751,
1699, 1661 (C ¼ O),
1236 (C–F), 1186,
1156 (ester linkage
–O–C)
0.83 (dd, 6H, L-valine–CH(CH3)2), 0.92 (t, 3H,
H-18), 1.97 (m, H, L-valine–CH(CH3)2), 2.15
(q, 2H, H-19), 4.14 (d, 2H, CH2–Gly), 4.36 (s,
2H, 5-FU–N–CH2), 4.42 (m, 1H, L-valine–CH),
5.31 (s, 2H, H-5), 5.50 (s, 2H, H-17), 7.17
(s, 1H, H-14), 7.73 (t, 1H, H-11), 7.87(s, 1H,
5-FU-ring–H-6), 7.89 (t, 1H, H-10), 8.16 (d,
1H, H-12), 8.60 (d, 1H, H-9), 8.71 (s, 1H, H-7),
11.78 (s, 1H, 5-FU-ring–NH)
10e
3291 (NH), 3069, 1598,
1557 (ArH), 1751,
1700, 1661 (C ¼ O),
1237 (C–F), 1188,
1160 (ester linkage
–O–C)
0.81 (m, 6H, 2 ꢃ L-leucine–CH3), 0.91 (t, 3H,
H-18), 1.48 (m, 2H, L-leucine–CH2), 1.59 (m,
1H, L-leucine–CH(CH3)2), 2.13 (q, 2H, H-19),
4.06 (d, 2H, CH2–Gly), 4.19 (m, 1H, L-leucine–
ꢀ-CH), 4.37 (s, 2H, 5-FU–N–CH2), 5.31 (s, 2H,
H-17), 5.50 (s, 2H, H-5), 7.17 (s, 1H, H-14),
7.73 (t, 1H, H-11), 7.97 (s, 1H, 5-FU-ring–H-6),
8.17 (t, 1H, H-10), 8.40 (d, 1H, H-9), 8.59
(d, 1H, H-12), 8.71 (s, 1H, H-7), 11.79 (s, 1H,
5-FU-ring–NH)
10f
3307 (NH), 3066, 1599,
1541 (ArH),1752,
1702, 1660 (C ¼ O),
1236 (C–F), 1180,
1154 (ester linkage
–O–C)
0.91 (t, 3H, H-18), 1.92 (s, 3H, L-methionine–
CH3), 2.13 (m, 3H, L-methionine–CH2SCH3
and H-19), 2.41 (m, 2H, L-methionine–
CH2CH2SCH3), 4.09 (d, 2H, CH2–Gly), 4.30
(m, 1H, L-methionine–ꢀ-CH), 4.38 (s, 2H,
5-FU–N–CH2), 5.29 (s, 2H, H-17), 5.48 (s, 2H,
H-5), 7.15 (s, 1H, H-14), 7.72 (t, 1H, H-11),
7.94 (s, 1H, 5-FU-ring–H-6), 8.15 (t, 1H, H-10),
(Continued )

1822
Y.-Q. Liu et al.
Table 2. Continued.
Compound
IR KBr, ꢁmax (cmꢂ1
)
1H-NMR: (DMSO-d6) ꢂ (ppm)
8.44 (d, 1H, H-9), 8.54 (d, 1H, H-12), 8.68
(s, 1H, H-7), 11.78 (s, 1H, 5-FU-ring–NH)
0.91 (t, 3H, H-18), 1.05 (m, 3H, L-threonine–
CH(OH)CH3), 2.17 (q, 2H, H-19), 4.07 (d, 2H,
CH2–Gly), 4.12 (m, 1H, L-threonine–
CH(OH)CH3), 4.19 (m, 1H, L-threonine–ꢀ-
CH), 4.28 (s, 2H, 5-FU–N–CH2), 5.50 (s, 2H,
H-17), 5.31 (s, 2H, H-5), 7.19 (s, 1H, H-14),
7.75 (t, 1H, H-11), 7.87 (s, 1H, 5-FU-ring–H-6),
7.95 (t, 1H, H-10), 8.18 (d, 1H, H-12), 8.38
(d, 1H, H-9), 8.71 (s, 1H, H-7), 11.79 (s, 1H,
5-FU-ring–NH)
10g
3421, 3308 (NH, OH),
3072, 1589, 1562
(ArH), 1755, 1699,
1659 (C ¼ O), 1238
(C–F), 1182, 1162
(esterlinkage –O–C)
Table 3. In vitro cytotoxicity of compounds 10a–10g against
human tumour cell lines (IC50 values).
Cytotoxic activities (IC50, mM)
Compound
SGC-7901
BGC-823
A-549
HePG-2
10a
10b
10c
10d
10e
10f
10g
1
3.10
2.35
4.62
2.92
2.38
5.99
8.01
0.625
7.60
0.089
0.045
0.033
0.067
0.041
0.074
0.058
0.030
1.720
2.45
0.45
0.57
0.38
1.43
5.21
5.37
0.091
1.17
4.41
3.29
2.35
1.21
8.48
5.44
4.63
8.75
19.96
2
irinotecan. The different cytotoxic activity range of compounds 10a–10g indicated
that the substituents of dipeptide linkages obviously affected the selectivity as well as
the activity against the four tested cancer cell lines. This investigation further
highlighted that the constitution of the dipeptide spacers has a major impact on the
cytotoxic activity of such analogues. Hence, a systemic, predictable correlation could
be made between the nature of dipeptides and anticancer activities.
2.3. In vitro determination of lactone levels in human and mouse plasma for
1 and 10b
The result of the in vitro determination of lactone levels in human and mouse plasma
buffers for 10b and CPT (1) based on the method described in the literature
(He et al., 2004) are shown in Table 4.
As shown in Table 4, the percentage of lactone of CPT (1) in human blood is 28.4
after 4 h, 10.9 after 8 h and 3.8 after 24 h. In other words, the active form of 1 is
significantly decreased in a relatively shorter time period after oral administration.

Natural Product Research
1823
Table 4. Comparison between the values of the percentage of lactone of the representive
prodrug 10b and 1 in human and mouse plasma.
Time (h)
0
2
4
6
8
24
Human plasma
Percentage of lactone for 10b
Percentage of lactone for 1
100.0
100.0
81.9
61.4
70.3
28.4
54.3
19.4
42.5
10.9
10.5
3.8
Mouse plasma
Percentage of lactone for 10b
Percentage of lactone for 1
100.0
100.0
83.8
55.1
80.7
40.5
73.1
24.5
52.6
14.6
17.9
5.3
This is in contrast to what is observed in mice, for which the active drug form (i.e. the
closed lactone form) lasts for a relatively longer time period. For example, the closed
lactone form of 1 in mouse plasma is still 40.5% even after 4 h. The closed lactone
form of 10b in human plasma is much more stable than its mother compound 1.
For example, 70.3% of 10b is still detected as the closed lactone form even after 4 h.
CPT (1), in terms of lactone level, shows a difference between mouse and human
plasma. Similarly, this kind of difference is also observed for prodrug 10b. From
the results, it is further evident that the biological life span of the lactone forms
of these compounds in human and mouse plasma significantly increased when
compared with their mother compound CPT (1). Meanwhile, the cleavage of 10b
in both mouse and human plasma was monitored by HPLC, which clearly showed
that compound 10b was cleaved to afford mainly its parental compound of CPT
in both mouse and human plasma, and the cleavage of 10b is the result of
the combination of chemical and enzymatic effects in both mouse and human
plasma. This was also the case in phosphate buffer at pH 7.4.
Another important aspect which has to be addressed is that these compounds
would be cleaved to release active CPT and 5-FU in both mouse and human plasma.
Such an assumption was supported by employing the HPLC experimental method,
which simultaneously implied that these conjugates might act as mutual prodrugs
and exert their cytotoxicities by hydrolysing to their parent compounds CPT and
5-FU, and they could have synergetic effects in biological systems.
3. Experimental
The values of melting points were taken on a Kofler melting point apparatus
and were uncorrected; IR spectra were obtained on a NIC-5DX spectrophotometer;
mass spectral analysis was performed on a ZAB-HS and Bruker Daltonics
APEXII49e instrument. Optical rotations were determined on a Perkin Elmer 341
spectropolarimeter. NMR spectra were recorded on a Bruker AM-400 spectrometer
at 400 MHz using TMS as the reference (Bruker Company, USA). The starting CPT
was isolated from the Chinese medicinal plant Camptotheca acuminata and was
purified before being used. The [(5-fluorouracil-1-yl)acetyl]-L-amino acids 7a–7g

1824
Y.-Q. Liu et al.
used for the experiments were prepared by following a modified previous procedure
(Chen et al., 2004; Liu et al., 2006).
3.1. Synthesis of CPT-20-glycinate TFA salt 9
t-Boc-glycine (377 mg, 2.1 mmol) was dissolved in 200 mL of anhydrous dichlor-
omethane at room temperature, and to this solution CPT (250 mg, 0.72 mmol), DCC
(444 mg, 2.1 mmol) and DMAP (87 mg, 0.72 mmol) at 0ꢀC were added. The reaction
mixture was stirred for 1 h and subsequently left overnight at room temperature.
The solution was washed with 0.1N HCl, dried, and evaporated under reduced
pressure to yield a white solid, which was recrystallised from methanol to give
CPT-20-ester of t-Boc-glycine (8), The t-Boc protection group was removed by
dissolving it in a mixture of methylene chloride (15 mL) and TFA (15 mL) and
stirring at room temperature for 1 h. The solvent was removed and the solid was
recrystallised from methylene chloride and ether to give CPT-20-glycinate TFA salt
9. The spectral data for 9 are identical to those reported by Greenwald (1998).
3.2. General procedure for synthesis of target compounds (10a–10g)
[(5-fluorouracil-1-yl)acetyl]-L-amino acids 7a–7g (0.047 mol) were dissolved in 50 mL
of anhydrous DMF and cooled to 0ꢀC. 1-Hydroxybenzotriazole (HOBt) (0.07 mol)
and 0.056 mol of N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI) were added, and the mixture was stirred for 30 min at 0ꢀC. Subsequently,
0.039 mol of CPT-20-glycinate TFA salt 9 and finally 24.3 mL N-ethyl diisopropy-
lamine were added. The mixture was stirred for 16 h at ambient temperature.
The solvent was removed under reduced pressure and the residue was separated
by flask-column chromatography (gradient elution with mixtures of chloroform–
methanol) on silica gel and was monitored by TLC. The synthesised target
1
compounds 10a–10g were characterised by m.p., IR, H-NMR and high-resolution
mass spectrometry (HRMS) analyses. Their analytical and spectral data are given in
Tables 1 and 2, respectively.
3.3. Cytotoxicity assays
Cytotoxicity assays were performed on four different human cancer cell lines
(SGC-7901, BGC-823, A-549 and HePG-2). Cells (6000–10,000) in 100 mL culture
medium per well were seeded into 96-well microtest plates (Falcon, CA). Cells were
treated in triplicate with a gradient concentration of test compounds and incubated
at 37ꢀC for 48 h (or 72 h). A microculture tetrazolium (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, MTT; Sigma, St. Louis, MO) assay was per-
formed to measure the cytotoxic effects. The drug concentration required for 50%
growth inhibition (IC50) of tumour cells was determined from the dose–response
curve.
3.4. In vitro determination of lactone levels in human and mouse plasma for
1 and 10b
The test compounds (0.2 mL, 100 mg mLꢂ1) in acetonitrile were added to 0.8 mL
pre-incubated human and mouse plasma, respectively. The mixture was incubated

Natural Product Research
1825
at 37ꢀC, and 100 mL aliquots were taken at time points 2, 4, 6, 8 and 24 h. To
precipitate plasma protein, 400 mL of acetonitrile (ꢂ20ꢀC) was added, vortexed
for 20 s and centrifuged at 10,000 rpm for 5 min. The supernatant was transferred
to a glass vial and stored at ꢂ20ꢀC immediately until HPLC analysis was complete.
HPLC (HP 1100) analysis: 20 mL of solution obtained as abovementioned was
injected onto a C18 column (Zobax SB, 4.6 ꢃ 150 mm) and chromatographed with
methanol/water/0.1% acetic acid as mobile phase. CPT (1) and 10b were detected
(detector: DAB) at 254 nm. The percentage of lactone was determined by the ratio
of the lactone levels measured at different time points to the lactone levels measured
at the starting point (t ¼ 0 h).
4. Conclusions
In summary, we synthesised seven CPT/5-FU conjugates joined by dipeptide
linkages, based on the effective combination principle. The compounds selected
for the in vitro determination of lactone levels showed that their biological life span
in human and mouse plasma was much longer than that of their parent compound
CPT. Meanwhile, most compounds exhibited potent cytotoxic activity against
tumour cell replication. Interestingly, all the compounds were selectively active
against BGC-823, with IC50 values lower than 0.1 mmol. These results are
encouraging and suggest that the design and synthesis of these compounds will be
beneficial for the therapeutic values of CPT analogues; the approach should be
applicable for other antitumour agents, and it is worthwhile to explore the
antitumour potential of these and similar types of compounds. In-depth mechanistic
studies and the development of new CPT/5-FU conjugates are actively underway in
our laboratory.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(nos. 30800720 and 30670070); the Post-Doctor Research Foundation (no. 20090450142);
the Interdisciplinary Innovation Research Foundation for Young Scholars, Lanzhou
University (no. LZUJC2007018); the Key Scientific and Technological Project Fund of
Gansu Province (no. 2GS064-A43-019-02); and the International Cooperation Fund of Gansu
Province (no. 0708WCGA150).
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