Synthesis and biological evaluation of novel conjugates of camptothecin and 5-flurouracil as cytotoxic agents

Article abstract of DOI:10.1590/S0103-50532011000200017

A series of novel conjugates of camptothecin and 5-fluorouracil were first synthesized and their cytotoxic activities against two human tumor cell lines (SGC-7901 and A-549) as well as in vitro pharmacokinetic determination of lactone stability were studi

Full text of DOI:10.1590/S0103-50532011000200017

J. Braz. Chem. Soc., Vol. 22, No. 2, 308-318, 2011.
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Article
A
Synthesis and Biological Evaluation of Novel Conjugates of Camptothecin and
5-Flurouracil as Cytotoxic Agents
Liu Yang,a Chun-Yan Zhaob and Ying-Qian Liu*,b
aEnvironmental and Municipal Engineering School, Lanzhou Jiaotong University, Lanzhou 730000,
People’s Republic of China
bSchool of Pharmacy, Lanzhou University, Lanzhou 730000, People’s Republic of China
Uma série de novos conjugados de camptotecina e 5-fluorouracil foi sintetizada pela primeira
vez e suas atividades citotóxicas contra duas linhagens de células humanas tumorais (SGC-7901
e A-549) assim como a determinação farmacocinética in vitro da estabilidade de lactona foram
investigadas. Dentre estes compostos, a maioria apresentou atividades citotóxicas comparáveis
ou superiores a 2, mas menos potentes quando comparados a 1. Em particular, os conjugados
10b e 10d foram altamente ativos contra A-549 com valores de IC50 de 0,45 e 0,38 mmol L-1,
respectivamente. Além disto, a determinação farmacocinética in vitro dos níveis de lactona do
composto representativo 10b mostraram que o tempo de vida biológico de suas formas de lactona
em plasma humano e de rato aumentaram significativamente quando comparados com o composto-
mãe 1. O método de relações quantitativas de estrutura-atividade (QSAR) foi então aplicado para
o desenvolvimento de modelos lineares para prever as atividades citotóxicas destes derivados que
ainda não foram sintetizados ou testados experimentalmente. Além disto, ‘docking’ molecular
foi utilizado para esclarecer o modo de ligação destes derivados a DNA topoisomerase humana I.
Ligações de hidrogênio importantes foram observadas entre estes derivados e seu receptor. Os
resultados de modelagem molecular e estudos QSAR podem ser usados para guiar o design de
novos conjugados com maior atividade antitumoral.
A series of novel conjugates of camptothecin and 5-fluorouracil were first synthesized and their
cytotoxic activities against two human tumor cell lines (SGC-7901 and A-549) as well as in vitro
pharmacokinetic determination of lactone stability were studied. Among these compounds, most
tested conjugates showed comparable or superior cytotoxic activities to 2, but less potent compared
with 1. Particularly, conjugates 10b and 10d were highly active against A-549 with IC50 values of
0.45 and 0.38 µmol L-1, respectively. Also, the in vitro pharmacokinetic determination of lactone
levels of representative compound 10b showed that the biological life span of their lactone forms
in human and mouse plasma significantly increased compared with their mother compound 1.
Quantitative structure-activity relationship (QSAR) method was then applied for developing linear
models to predict the cytotoxic activities of these derivatives that have not yet been synthesized
or experimentally tested. In addition, molecular docking was used to clarify the binding mode of
these derivatives to human DNA topoisomerase I. The important hydrogen-bonding interactions
were observed between these derivatives and their receptor. The results from molecular modeling
and QSAR study can guide the design of novel conjugates with higher antitumor activity.
Keywords: camptothecin, 5-fluorouracil, cytotoxic agents, conjugates
Introduction
drug selectivity, 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.1,2 This concept is now accepted as
an effective strategy for designing ligands, inhibitors, and
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.3-7 In our prior study,
A challenging research focus in current cancer therapy
is the discovery of molecules that could be selective for
tumor cells and that could be characterized by reduced
undesirable effects. Among various strategies to improve
*e-mail: yqliu@lzu.edu.cn

Vol. 22, No. 2, 2011
Yang et al.
309
we explored the syntheses and evaluation of heterodimer
conjugates by combination of an antimetabolite together
with one or more different anticancer agents through various
linkages, which has led to a large increase in potency over
their monomeric counterparts.2,3,7
such novel conjugates and the effect of different dipeptide
spacer linkers at the C-20-position.
Experimental
Camptothecin (CPT) (1) is a topoisomerase I targeting
cytotoxic alkaloid with significant antineoplastic activity.8-10
The therapeutic use of unmodified CPT was however
suspended because of high toxicity stemming in part from
instability of the active lactone form due to preferential
binding of the carboxylate to serum albumin and problems
with delivery due to poor water solubility. Included in
approaches to improving the biological profile of CPTs are
the development of prodrugs for preferential cellular uptake
into tumor cells, enhanced solubility and lipophilicity, and
stabilization of the camptothecin E-ring lactone. These
efforts have led to the food and drug administration (FDA)
approval of topotecan (2) and irinotecan (3) for colon and
ovarian cancers treatment, respectively, and to the synthesis
of several novel CPT derivatives that are currently in various
stages of clinical trials.11-13
Melting points were taken on a Kofler melting point
apparatus and are uncorrected, infrared (IR) spectra were
obtained on NIC-5DX spectrophotometer, mass spectral
analysis was performed on a ZAB-HS and Bruker Daltonics
APEXII49e instrument. Optical rotations were determined
on PerkineElmer Model 341 spectropolarimeter. Nuclear
magnetic resonance (NMR) spectra were recorded
on a Bruker AM-400 spectrometer at 400 MHz using
tetramethylsilane (TMS) as reference (Bruker Company,
USA).ThestartingcamptothecinwasisolatedfromaChinese
medicinal plant C. acuminata and was purified before
being used. The [(5-fluorouracil-1-yl)acetyl]-L-amino
acids 7a-7g used for the experiments were prepared by
following a modified previous procedure.18,19
Synthesis of camptothecin-20-glycinate TFA salt (9)
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.14 But 5-FU
is poorly selective toward tumor, so its therapy causes high
incidences of toxicity in the bone marrow, gastrointestinal
tract,centralnervesystemandskin.Tooptimizetheefficacyof
5-FU, it is often administrated by continuous infusion as well
as in combination with other cytotoxic or with biochemical
modulators.15,16 Moreover, the considerable data from
clinical studies showed that the combined use of CPT-related
analogues and 5-FU resulted in higher response rates than
for either agent alone and simultaneously circumvent some
faults and decreased repair of camptothecin-induced DNA
damage and increased induction of apoptosis, or increased
DNA adduct formation in cancer cells.16,17
t-Boc-glycine (377 mg, 2.1 mmol) was dissolved
in 200 mL of anhydrous dichloromethane at room
temperature, and to this solution were added CPT (250 mg,
0.72 mmol), dicyclohexylcarbodiimide (DCC) (444 mg,
2.1 mmol) and 4-dimethylaminopyridine (DMAP) (87 mg,
0.72 mmol) at 0 oC. The reaction mixture was stirred for
1 h and subsequently allowed to room temperature and
left overnight. The solution was washed with 0.1 mol L-1
hydrochloric acid (HCl), dried and evaporated under
reduced pressure to yield a white solid, which was
recrystallized from methanol to give camptothecin-20-ester
of t-Boc-glycine 8, The t-Boc protection group was removed
by dissolving in a mixture of methylene chloride (CH2Cl2)
(15 mL) and TFA (trifluoroacetic acid) (15 mL) and stirred
at room temperature for 1 h. Solvent was removed and
the solid was recrystallized from CH2Cl2 and ether to give
Herein, the aim of our work was to investigate the
cytotoxicity, stability of the lactone ring and SAR studies of
R3
N
R2
R1
O
O
F
O
N
N
HN
N
O
O
O
O
N
H
O
OH
N
OH
5-Fluorouracil (4)
O–
,
R3 = CH CH
2 3
2
R1 =
R = H,
Irinotecan (2):
N
Camptothecin (1)
O
Topotecan (3): R1 = OH, R2 = (CH3)2NHCH2, R3 = H
Figure 1. Structures of camptothecin (1), irinotecan (2), topotecan (3) and 5-fluorouracil (4).

310
Synthesis and Biological Evaluation of Novel Conjugates of Camptothecin and 5-Flurouracil
J. Braz. Chem. Soc.
camptothecin-20-glycinate TFA salt 9. Spectral data for 9
4.35 (2H, s, 5-Fu-N-CH2), 5.31 (2H, s, H-5), 5.50 (2H,
s, H-17), 7.17 (1H, s, H-14), 7.73 (1H, t, H-11), 7.85
(1H, s, 5-Fu-ring-H-6), 7.89 (1H, t, H-10), 8.15 (1H, d,
H-12), 8.48 (1H, d, H-9), 8.71 (1H, s, H-7), 11.80 (1H, s,
5-Fu-ring-NH); 13C NMR (DMSO-d6) d 168.62, 167.17,
166.93, 157.56, 157.30, 156.42, 152.23, 149.54, 147.81,
145.95,144.93, 138.15 and 140.42 (5-Fu-ring-5-C, JCF
228 Hz), 131.47, 130.84, 130.50, 130.32, 129.62, 128.83,
128.42, 127.88 and 127.60 (5-Fu-ring-C-6, JCF 28 Hz),
118.91, 95.08, 76.33, 66.28, 50.10, 49.37, 40.26, 30.37,
7.42. HRMS (m/z) calculated for C31H27N6O9F: 647.1896
[M+ H]+. Found: 647.1896.
are identical to the reported by Greenwald.20
General procedure for synthesis of target compounds
(10a-10g)
[(5-Fluorouracil-1-yl)acetyl]-L-amino acids 7a-7g
(0.047 mol) are dissolved in 50 mL of anhydrous
dimethylformamide (DMF) and cooled to 0 °C. 1-Hydroxy-
benzotriazole(HOBt)(0.07mol)and(0.056 mol)ofN-ethyl-
N-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI) are added, and the mixture is stirred for 30 min at 0
°C. Subsequently, (0.039mol)ofcamptothecin-20-glycinate
TFA salt 9 and finally 24.3 mL N-ethyl diisopropylamine
are added. The mixture is stirred for 16 h at room
temperature. The solvent was removed under reduced
pressure and the residue was separated by flash-column
chromatography (gradient elution with mixtures of
chloroform-methanol) on silica gel and monitored by TLC.
Synthesized target compounds 10a-10g were characterized
by mp, IR, 1H NMR, 13C NMRand HRMS analyses.
Spectral data of compound 10c
20
Yield: 56%; mp 190-192 oC; [α]D –71o (c 0.5,
DMF); IR (KBr) nmax/cm-1 3293 (NH), 3065, 1598, 1559
(ArH), 1750, 1699, 1662 (C=O), 1237 (C-F), 1188,
1161 (ester linkage-O-C); 1H NMR (DMSO-d6 400 MHz)
d 0.92 (3H, t, H-18), 2.15 (2H, q, H-19), 3.03 (2H, m,
L-phenylalanine-CH2), 4.08 (2H, d, CH2-Gly), 4.35 (2H,
s, 5-Fu-N-CH2), 4.56 (1H, m, L-phenylalanine-CH), 5.30
(2H, s, H-5), 5.51 (2H, s, H-17), 7.19 (1H, s, H-14), 7.23
(m, 5H, L-phenylalanine-ArH), 7.73 (1H, t, H-11), 7.85
(1H, s, 5-Fu-ring-H-6), 7.88 (1H, t, H-10), 8.15 (1H, d,
H-12), 8.54 (1H, d, H-9), 8.70 (1H, s, H-7), 11.77 (1H, s,
5-Fu-ring-NH); 13C NMR (DMSO-d6) d 171.25, 168.77,
166.91, 166.35, 157.49, 157.23, 156.45, 152.29, 149.50,
147.83, 145.95, 144.98, 137.92, 137.45, 131.50, 130.91,
130.58, 130.35, 129.69, 129.08, 128.87, 128.46, 127.98,
127.90, 127.64, 126.20, 118.94, 95.12, 76.27, 66.28, 53.85,
50.13, 49.13, 40.30, 37.65, 30.39, 7.43. HRMS (m/z)
calculated for C37H31N6O9F: 745.209 [M+ Na]+. Found:
745.2014.
Spectral data of compound 10a
Yield: 73%; mp: 186-188oC; [α]D20 –79o (c 0.5, DMF);
IR (KBr) nmax/cm-1 463(NH), 3073, 1619, 1595, 1559
(ArH), 1762, 1702, 1664 (C=O), 1236 (C-F), 1184, 1155
1
(ester linkage-O-C); H NMR: (DMSO-d6, 400 MHz) d
0.89 (3H, t, H-18), 2.13 (2H, q, H-19), 4.04 and 4.23
(4H, 2d, 2×CH2-Gly-Gly), 4.29 (2H, s, 5-Fu-N-CH2),
5.47 (2H, s, H-17), 5.26 (2H, s, H-5), 7.14 (1H, s, H-14),
7.70 (1H, t, H-11), 7.84 (1H, s, 5-Fu-ring-H-6), 7.94 (1H,
t, H-10), 8.12 (d, 1H, H-12), 8.46 (d, 1H, H-9), 8.66 (s,
1H, 1H, H-7), 11.80 (s, 1H, 5-Fu-ring-NH); 13C NMR
(DMSO-d6, 400 MHz) d 169.05, 168.81, 166.95, 157.58,
157.33, 156.45, 152.23, 149.66, 147.82, 145.92, 144.99,
138.07 and 140.36 (5-Fu-ring-5-C, JCF 229 Hz), 131.52,
131.02, 130.69, 130.04, 129.64, 128.85,128.46, 127.90
and 127.64 (5-Fu-ring-C-6, JCF 26 Hz), 118.96, 95.11,
76.27, 66.31, 50.11, 49.45, 41.77, 30.25, 7.45. HRMS
(m/z) calculated for C30H25N6O9F: 633.1740 [M+ H]+.
Found: 633.1740.
Spectral data of compound 10d
Yield: 52%; mp 186-188oC; [α]D20 –90o (c 0.5, DMF);
IR (KBr) nmax/cm-1 3311(NH), 3067, 1599, 1557 (ArH),
1751, 1699, 1661 (C=O), 1236 (C-F), 1186, 1156 (ester
1
linkage-O-C); H NMR (DMSO-d6 400 MHz) d 0.83
(6H, dd, L-valine-CH(CH3)2), 0.92 (3H, t, H-18), 1.97
(1H, m, L-valine-CH(CH3)2), 2.15 (2H, q, H-19), 4.14
(2H, d, CH2-Gly), 4.36 (2H, s, 5-Fu-N-CH2), 4.42 (1H,
m, L-valine-CH), 5.31 (2H, s, H-5), 5.50 (2H, s, H-17),
7.17 (1H, s, H-14), 7.73 (1H, t, H-11), 7.87 (1H, s, 5-Fu-
ring-H-6), 7.89 (1H, t, H-10), 8.16 (1H, d, H-12), 8.60 (1H,
d, H-9), 8.71 (1H, s, H-7), 11.78 (1H, s, 5-Fu-ring-NH);
13C NMR (DMSO-d6) d 171.89, 169.56, 167.25, 158.30,
158.05, 157.15, 152.87, 150.31, 148.49, 146.63, 145.78,
140.93 and 138.66 (5-Fu-ring-5-C, JCF 227 Hz), 132.20,
Spectral data of compound 10b
Yield: 69%; mp 189-191 oC; [α]D20 –80o (c 0.5,
DMF); IR (KBr) nmax/cm-1 3359 (NH), 3067, 1600, 1558
(ArH), 1749, 1698, 1662 (C=O),1237 (C-F),1187, 1158
(ester linkage-O-C); 1H NMR (DMSO-d6) d 0.92 (3H, t,
H-18), 1.24 (3H, d, L-Alanine-CH3), 2.15 (2H, q, H-19),
4.05 (2H, d, CH2-Gly), 4.25 (1H, m, L-alanine-CH),

Vol. 22, No. 2, 2011
Yang et al.
311
131.99, 131.66, 131.07,130.26, 129.15, 129.11, 128.57 and
128.33 (5-Fu-ring-C-6, JCF 24 Hz), 119.61, 109.99, 95.58,
76.96, 66.99, 56.73, 50.80, 31.39, 31.20, 19.18, 18.62,
8.18. HRMS (m/z) calculated for C33H31N6O9F: 675.2209
[M+ H]+. Found: 675.2219.
Spectral data of compound 10g
Yield: 62%; mp 192-194oC; [α]D20 –75o (c 0.5, DMF);
IR (KBr) nmax/cm-1 3421, 3308 (NH,OH), 3072, 1589,
1562 (ArH), 1755, 1699, 1659 (C=O), 1238 (C-F), 1182,
1162 (esterlinkage-O-C); 1H NMR (DMSO-d6 400 MHz) d
0.91 (3H, t, H-18),1.05 (3H, m, L-threonine-CH(OH)CH3),
2.17 (2H, q, H-19), 4.07 (2H, d, CH2-Gly), 4.12 (1H, m,
L-threonine-CH(OH)CH3), 4.19 (m, 1H, L-threonine-α-
CH), 4.28 (2H, s, 5-Fu-N-CH2), 5.50 (2H, s, H-17), 5.31
(2H, s, H-5), 7.19 (1H, s, H-14), 7.75 (1H, t, H-11), 7.87
(1H, s, 5-Fu-ring-H-6), 7.95 (1H, t, H-10), 8.18 (1H, d,
H-12), 8.38 (1H, d, H-9), 8.71 (1H, s, H-7), 11.79 (1H, s,
5-Fu-ring-NH); 13C NMR (DMSO-d6) d 170.30, 168.81,
166.96, 166.79, 157.56, 157.30, 156.46, 152.28, 149.61,
147.82, 145.93, 144.99, 137.95 and 140.22 (5-Fu-ring-
5-C, JCF 227 Hz), 131.54, 131.22, 130.88, 130.38, 129.68,
128.87, 128.47, 127.91 and 127.66 (5-Fu-ring-C-6, JCF
25 Hz), 118.96, 95.20, 76.23, 66.52, 58.24, 50.14, 49.47,
40.43, 30.50, 19.81, 7.45. HRMS (m/z) calculated for
C32H29N6O10F: 699.1821 [M+Na]+. Found: 699.1825.
Spectral data of compound 10e
Yield: 45%; mp 176-178 oC; [α]D20–77o (c 0.5, DMF);
IR (KBr) nmax/cm-1 3291(NH), 3069, 1598, 1557 (ArH),
1751, 1700, 1661 (C=O), 1237 (C-F), 1188, 1160 (ester
1
linkage-O-C); H NMR (DMSO-d6 400 MHz) d 0.81
(6H, m, 2×L-leucine-CH3), 0.91 (3H, t, H-18), 1.48 (2H,
m, L-leucine-CH2), 1.59 (1H, m, L-leucine-CH(CH3)2),
2.13 (2H, q, H-19), 4.06 (2H, d, CH2-Gly), 4.19 (1H, m,
L-leucine-α-CH), 4.37 (2H, s, 5-Fu-N-CH2), 5.31 (2H, s,
H-17), 5.50 (2H, s, H-5), 7.17 (1H, s, H-14), 7.73 (1H, t,
H-11), 7.97 (1H, s, 5-Fu-ring-H-6), 8.17 (1H, t, H-10), 8.40
(1H, d, H-9), 8.59 (1H, d, H-12), 8.71 (1H, s, H-7),11.79
(1H, s, 5-Fu-ring-NH); 13C NMR (DMSO-d6) d 169.53,
167.67, 167.04, 158.29, 158.04, 157.17, 152.96, 150.32,
148.55, 146.68, 145.80, 140.98 and 138.71 (5-Fu-ring-
5-C, JCF 227 Hz), 132.23, 131.92, 131.59, 131.07, 130.36,
129.61, 129.17, 128.62 and 128.36 (5-Fu-ring-C-6, JCF
26 Hz), 119.62, 95.79, 76.98, 66.99, 56.72, 51.58, 50.16,
41.91, 31.16, 24.72, 23.58, 22.22, 19.20, 8.20. HRMS (m/z)
calculated for C34H33N6O9F: 689.2366 [M+H]+. Found:
689.2366.
Cytotoxicity assays
Cytotoxicity assays are performed on human gastric
tumor SGC-7901 and human lung carcinomaA-549. Cells
(6000-10.000) in 100 µL culture medium per well were
seeded into 96 well microtest plates (Falcon, CA). Cells
were treated in triplicate with gradient concentration of test
compounds and incubated at 37 oC for 48 h (or 72 h). The
microculture [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide, MTT (Sigma, St. Louis, MO) assay
was performed to measure the cytotoxic effects. The drug
concentration required for 50% growth inhibition (IC50) of
tumor cells was determined from the dose-response curve.
Spectral data of compound 10f
Yield: 68%; mp 192-194 oC; [α]D20–67o (c 0.5, DMF);
IR (KBr) nmax/cm-1 3307 (NH), 3066, 1599, 1541 (ArH),
1752, 1702, 1660 (C=O), 1236 (C-F), 1180, 1154 (ester
1
linkage-O-C); H NMR (DMSO-d6 400 MHz) d 0.91
(3H, t, H-18), 1.92 (3H, s, L-methionine-CH3), 2.13 (3H,
m, L-methionine-CH2SCH3 and H-19), 2.41 (2H, m,
L-methionine-CH2CH2SCH3), 4.09 (2H, d, CH2-Gly), 4.30
(1H, m, L-methionine-α-CH), 4.38 (2H, s, 5-Fu-N-CH2),
5.29 (2H, s, H-17), 5.48 (2H, s, H-5), 7.15 (1H, s, H-14),
7.72 (1H, t, H-11), 7.94(1H, s, 5-Fu-ring-H-6), 8.15 (1H,
t, H-10), 8.44(1H, d, H-9), 8.54 (1H, d, H-12), 8.68 (1H,
s, H-7),11.78 (1H, s, 5-Fu-ring-NH);13C NMR (DMSO-d6)
d 171.32, 168.80, 166.91, 166.57, 157.56, 157.30, 156.44,
152.23, 149.63, 147.82, 145.94, 144.99, 140.28 and 138.01
((5-Fu-ring-5-C, JCF 217 Hz), 131.49, 131.15, 130.81,
130.35, 129.64, 128.873 and 128.61 (5-Fu-ring-C-6,
JCF 26 Hz),127.89, 127.63, 118.92, 95.07, 76.28, 66.27,
51.73, 50.111, 49.52, 40.34, 32.09, 30.43, 29.35, 14.47,
7.46. HRMS (m/z) calculated for C33H31N6O9SF: 729.1749
[M+Na]+. Found: 729.1738.
In vitro determination of lactone levels in human and mouse
plasma for 1 and 10b
To 0.8 mL pre-incubated human and mouse plasma,
test compounds (0.2 mL, 100 µg mL-1) in acetonitrile were
added. The mixture was incubated at 37 °C, and 100 µL
aliquots were taken at 2, 4, 6, 8 and 24 h. To precipitate
plasma protein, 400 µL acetonitrile (-20 °C) were added,
vortexed for 20 s, and centrifuged at 10.000 rpm for 5 min.
Supernatant was transferred to a glass vial and stored
at -20 °C immediately until high performance liquid
chromatography(HPLC)analysis. HPLC(HP1100)analysis:
20 µL of solution obtained above were injected onto a C-18
column (Zobax SB, 4.6×150 mm) and chromatographed
with methanol:water 0.1% acetic acid as mobile phase.

312
Synthesis and Biological Evaluation of Novel Conjugates of Camptothecin and 5-Flurouracil
J. Braz. Chem. Soc.
Camptothecin and 10b were detected (detector: DAB) at
254 nm. The percent of lactone was determined by the ratio
of lactone levels measured at different time points to the
lactone levels measured at starting point (t = 0 h).
was employed to select the optimal subset from original
calculated descriptors. The step involves correlation of the
given property with (i) the top descriptor in the above list
with each of the remaining descriptors and (ii) the next one
with each of the remaining descriptors, etc. The best pairs,
as evidenced by the highest F-values in the two-parameter
correlations, are chosen. Then, as shown above, three
descriptors were finally obtained. Thus, the linear functions
can be developed based on the three descriptors using
multiple linear regression module of Codessa software.
Computational details
General
The crystal structure for complex CPT-DNA-Topo I
(3.0 Å resolution, Rcryst = 0.244) was obtained from the
Brookheaven protein databank with PDB code 1T8I. All
docking calculations were performed with discovery studio
2.1 software package and QSAR models were performed
with Codessa software. All performances were completed
on Pentium IV computer.
Results and Discussion
Reaction of 5-fluorouracil 4 with chloroacetic
acid in the presence of potassium hydroxide gave the
corresponding 5-fluorouracil-1-acetic acid 5 in almost
excellent yields,18 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).19 Camptothecin 1 was isolated
from a Chinese medicinal plant Camptotheca acuminata
and served as the starting material for the preparations of
all the derivatives. Conversion of the isolated available
camptothecin to camptothecin-20-ester of N-Boc-glycine
derivative8inthepresenceofN,N-dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP) as catalyst
was accomplished by a modified version of Greenwald’s
method,20 followed by 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 obtained by treating the
TFA salt 9 with the corresponding [(5-fluorouracil-1-yl)
acetyl]-L-amino acids 7a-7g using EDCI as the coupling
agent (Scheme 2). The desired compounds 10a-10g were
purified by standard flash chromatography on silica gel and
characterized by mp, IR, 1H NMR, 13C NMR and HRMS
analyses.
Docking analysis
The protein atoms were then typed using the CharMm
force field. Ligand conformations were randomly generated
and energy minimized using the CharMm force field. We
set the maximum number of conformations generated to
300 (MaxConfs 300) with a 20 kcal mol-1 energy window
to generate lowest energy conformers per ligand. The
maximum number of conformations was chosen to ensure
adequate coverage of conformational space. For protein,
the binding site can be found using as volume of selected
ligand from protein complex with a value of 9 Å distance
for the site opening based on binding site module. Then,
LibDock procedure was applied to position conformation of
these compounds correctly in the active site. The procedure
was performed using LibDock module. The binding results
could be displayed by scoring ligand poses and several
scoring functions can be used for measuring the goodness of
a docking study to find a top ranked pose for ligands. In this
study, relatively energy, absolute energy and Libdock score
can be obtained and the latter was used as the final criteria.
QSAR study
Cytotoxic activities
The structures of the compounds were drawn with
the ISIS DRAW 2.3 program. The final geometries were
obtained with the semi-empirical PM3 method in the
HYPERCHEM program. All calculations were carried
out at restricted Hartree-Fock level with no configuration
interaction. The molecular structures were optimized
using the Polak-Ribiere algorithm until the root-mean-
square gradient was 0.001, the resulted geometry was
transferred into software Codessa, which can calculate 470
descriptors for each compound. Then, heuristic method
The cytotoxicities of compounds 10a-10g were
measured on two different human cancer cell lines (human
gastric tumor SGC-7901 and human lung carcinomaA-549)
using MTT assay in vitro. Irinotecan (2) and CPT (1) were
used as reference compounds. The IC50 values are shown
in Table 1.
As it can be seen in Table 1, against the two cell lines,
all of the conjugates were less potent than 1 in cytotoxic
assay, which was almost the common feature of prodrugs,
and whereas most of compounds exhibited comparable

Vol. 22, No. 2, 2011
Yang et al.
313
O
N
O
N
F
O
F
F
HN
HN
p-NO2C6H5OH
ClCH2COOH
HN
O
O
10% KOH, reflux
DCC/DMF, r.t., 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
d. R = CH(CH3)2
f. R = CH2SCH3
c. R = CH2Ph
e. R = CH2CH(CH3)2
g. R = CH(OH)CH3
O
R
7a-7g
Scheme 1. Synthesis of [(5-fluorouracil-1-yl)acetyl]-L-amino acids 7a-7g.
O
O
O
N
N
50%TFA/CH2Cl2
Boc-NHCH2CO2H
N
N
N
r.t.
N
DCC/DMAP,
CH2Cl2, r.t.
O
O
O
O
O
O
O
O
OH
TFA
NH2
NH
Boc
O
O
1
8
9
7a-7g
O
O
N
EDCI/HOBt/DMF, stir
N
F
N
HN
O
O
a. R = H
b. R = CH3
d. R = CH(CH3)2
f. R = CH2SCH3
O
H
c. R = CH2Ph
N
O
O
e. R = CH2CH(CH3)2
g. R = CH(OH)CH3
N
H
O
O
R
10a-10g
Scheme 2. Synthesis of the target compounds 10a-10g.
Table 1. In vitro cytotoxicity assay against SGC-7901andA-549 cell lines
or superior cytotoxic activities to irinotecan 2. Their
cytotoxic activity IC50 values were in the range of several
micromole. In particular, compounds 10b, 10c and 10d
exhibited more potent cytotoxicities than irinotecan 2
againstA-549 cell line with IC50 value of < 1 µmol L-1. The
different cytotoxic activity range of compounds 10a-10g
indicated that substituent of dipeptides linkages obviously
affected the activity profiles of this compound class and this
difference could be ascribed to a combination of factors,
like nature of the substituent (which may depend on the
size of substituent, electronic characteristics of substituent,
and other factors) or by a different interaction at the site.As
seen with compounds 10f and 10g, inclusion of moieties
Compound
Cytotoxic activities (IC50, µmol L-1)
SGC-7901
3.10
A-549
2.45
0.45
0.57
0.38
1.43
5.21
5.37
0.091
1.17
10a
10b
10c
10d
10e
10f
10g
1
2.35
4.62
2.92
2.38
5.99
8.01
0.625
7.60
2

314
Synthesis and Biological Evaluation of Novel Conjugates of Camptothecin and 5-Flurouracil
J. Braz. Chem. Soc.
containing hydroxyl and methylthio groups in the dipeptide
side chains decreased the activity significantly, this decrease
may be caused by hydrogen bonding between the nitrogen
or oxygen atoms and the enzyme/DNA might cause the loss
of activity, or the relatively polar moieties might impede an
important hydrophobic interaction between the molecules
and topoisomerase I/DNA. This investigation also further
highlighted the fact that the constitution of the dipeptide
spacers has a major impact on cytotoxic activity of such
analogues. Hence, a systemic, predictable correlation could
be made between the nature of dipeptides and anticancer
activities. Further pharmacological and toxicological
evaluation of these promising compounds is in progress.
The detailed explanation of SAR would be discussed in the
following QSAR studies.
Molecular docking
Human DNA topoisomerase I (Topo I) is molecular
target of CPT derivatives. These compounds can bind to
a transient Topo I-DNA covalent complex, leading to an
accumulation of DNA strand breaks upon replication,
ultimately causing cell death.21 Thus, we were interested in
understanding the interactions of camptothecin derivatives
with topoisomerase I by investigating the binding behavior
of the two compounds 10b and 10g, which can be treated
as the representation for these derivatives based on
their cytotoxicities on two different human cancer cell
lines listed in Table 1. In the present paper, a ligand-
receptor docking studies were carried out to explore the
probable binding modes within the active site of protein
topoisomerase I.
Figure 2. Base pairs forming stack interaction between Topoisomerase
I and compounds 10b and 10g. Amino acid residues are colored purple.
Compounds 10b and 10g are represented by green and yellow color
respectively. The backbone of DNA is expressed as white arrows and the
base pairs as ladders.
The crystal structure of topoisomerase I (code: 1T8I)
was extracted from Protein Data Bank. The hydrogen
atoms, missing atoms and residues were added to complete
the protein chain. Compounds 10b and 10g were minimized
and the lowest energy conformation of each ligand was then
located. The whole docking studies were performed using
Libdock method based on discovery studio 2.1 software in
our molecular modeling process. As shown in Figure 2, it
can be seen that the two compounds can intercalate at the
site of DNA cleavage with base-stacking interactions with
both the -1 (upstream) and +1 (downstream) base pair.
The orientations of 10b and 10g in the active site cavity
are perpendicular to the main axis of the DNA while the
direction of camptothecin moiety is reverse. Previous studies
indicated that hydrogen-bonding interaction between ligand
and receptor always played key roles during the formation
of drug-DNA-enzyme ternary complex.22,23 As shown in
Figure 3, ternary complex of compound 10b-DNA-enzyme
formed four hydrogen bonds, while the 10g-DNA-enzyme
Figure 3. Stereoview of hydrogen bonds (marked in green dotted lines)
formed between topoisomerase I and the compounds 10b (A) and 10g
(B). The nucleotides forming hydrogen-bonds are colored blue and the
residues are colored carmine.

Vol. 22, No. 2, 2011
Yang et al.
315
ternary complex formed two H-bonds, respectively. For the
complex with compound 10b (Figure 3A), camptothecin
and 5-fluorouracil moieties formed H-bonds with amino
ARG364 and ASN722, in addition, dipeptides linkage
moiety produced other two H-bonds with nucleotides.
For the complex with compound 10g (Figure 3B), the two
H-bonds formed between the dipeptides linkage moiety
with amino acid residues, THR718 and ARG364. As
we know, the more hydrogen bond interactions always
indicated stronger binding affinity. Thus, compound 10b
has higher potency to form the hydrogen bonding with
protein compared with compound 10g.
From above it can be seen that amino acid residue
ARG364 is the necessary amino acid when hydrogen bond
formed for both compounds 10b and 10g. Furthermore, it
can be deduced that amino acid residue ARG364 is likely
to play a key role for the process of compounds binding
to topoisomerase I.
A large pool of 470 descriptors, including constitutional,
topologicalandquantumchemistrypropertiesforcompounds
10a-10g was calculated using Codessa software. After
correlation analysis of the descriptors, some of them
were removed to eliminate redundant information. Three
descriptors were then obtained to develop the predictive
model and their physical-chemical meanings were listed in
Table 2. Thus, in the present paper, the cytotoxic activities
against human gastric tumor cell SGC-7901and human lung
carcinoma A-549 were regarded as the dependent variable
and the selected descriptors were treated as independent
variable for constructing the linear regression function 1
and 2 with statistical results squared correlation coefficient
(R2), and squared correlation coefficient for leave-one-out
cross-validation R2cv. In addition, the predicted cytotoxicity
activities based on QSAR models were shown in Table 3
and Figure 4.
For cytotoxic activity against human gastric tumor cell
SGC-7901 (equation 1):
QSAR study
IC50(SGC-7901) = 2.77×104 + 3.86×105MaxREHN
2.01×101FNSA2 + 5.37×10-2HASA2
–
To gain insight into the structural and biological activities
of these derivatives, QSAR (quantitative structure-activity
relationships) analysis has been widely accepted to account
for the various types of activities of the molecule and they are
found useful in predicting the activities of other derivatives
hitherto unknown. In the present paper, heuristic method
(HM)24 available in the framework of the Codessa program
was undertaken to perform a complete search for the best
multilinear correlations with a multitude of descriptors.
n = 7, R2 = 0.9989, R2cv = 0.9893, F = 598.82
(1)
For cytotoxic activity against human gastric tumor cell
A-549 (equation 2):
IC50(a-549) = –7.03×103 + 1.69×10-1HASA1
1.42×103MinEEC-N – 2.05×101ABIC2
+
n = 7, R2 = 0.9975, R2cv = 0.9903, F = 403.54
(2)
Table 2. Physical-chemical meanings for selected descriptors
Dependent variable
IC50 (SGC-7901)
Independent variable
MaxPCN
Chemical-physical meaning
Max partial charge for a N atom [Zefirov’s PC]
FNSA-2 Fractional PNSA (PNSA-2/TMSA) [Zefirov’s PC]
HASA-2 [Quantum-Chemical PC]
FNSA2
HASA2
IC50(A-549)
HASA1
HASA-1 [Zefirov’s PC]
MinEEC-N
ABIC2
Min exchange energy for a C-N bond
Average Bonding Information content (order 2)
Table 3. Experimental and predicted activities
Compounds
Experimental activity
Predicted activity
IC50 (SGC-7901)
IC50 (SGC-7901)
IC50(A-549)
2.45
IC50(A-549)
2.53
10a
10b
10c
10d
10e
10f
3.10
2.35
4.62
2.92
2.38
5.99
8.01
3.07
2.43
4.56
2.84
2.47
5.96
8.06
0.45
0.45
0.57
0.57
0.38
0.36
1.43
1.40
5.21
5.18
10g
5.37
5.36

316
Synthesis and Biological Evaluation of Novel Conjugates of Camptothecin and 5-Flurouracil
J. Braz. Chem. Soc.
Among the selected descriptors which were used to
can convey information about the specific atom types
(such as N, O) or encode the hydrogen-bonding ability
of a molecule. In addition, as we know, hydrogen bonds
are formed when a hydrogen atom is shared between two
molecules. A hydrogen atom covalently attached to a very
electronegative atom (N, O, or P) shares its partial positive
charge with a second electronegative atom (N, O, or P).
Thus, for the parameter “Max partial charge for N atom”,
it is also related with the ability of hydrogen-bonding.
Then, from inspection of predictive model, it indicated
that the hydrogen bonding interactions may be the most
relevant factor controlling the binding behavior and the
cytotoxicity activity. Considering the good statistical
results, the development QSAR model is suitable to do a
good prediction for cytotoxicity activity and it can give us
some insight into the factors that govern the cytotoxicity
activity and binding behavior with topoisomerase I.
build QSAR model, FNSA2, HASA2 and HASA1 were
all related with charged partial surface area (CPSA)
descriptors, which were originally designed to capture
information about the features of molecules responsible
for polar intermolecular interactions in terms of functional
group portions.By combining the atomic surface areas
and charges for specific atom types, CPSA descriptors
In vitro determination of lactone levels in human and mouse
plasma for 1 and 10b
The results of in vitro determination, based on literature
method,25 of lactone levels in human and mouse plasma
buffer for 10b and CPT (1) are shown in Table 4.
As shown in Table 4, the percent 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 relative short time period
after oral administration. This is the opposite to what is
observed in mice, in which the active drug form (i.e., the
closed lactone form) lasts for a relative long time period.
For example, the lactone form of 1 in mouse plasma is still
40.5% even after 4 h. The 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 lactone
form even after 4 h. CPT 1, in terms of lactone level, shows
a difference between mouse and human. Similarly, this
kind of difference is also observed for prodrug 10b. These
results further show that the biological life span of lactone
Figure 4. Plot of calculated versus experimental cytotoxicity activity
against human gastric tumor SGC-7901(A) and human lung carcinoma
A-549 (B).
Table 4. Comparison of percent lactone of the representative prodrug 10b and 1 in human and mouse plasmas
time (h)
0
2
4
6
8
24
Human plasma
lactone for 10b (%)
lactone for CPT (%)
Mouse plasma
100.0
100.0
81.9
61.4
70.3
28.4
54.3
19.4
42.5
10.9
10.5
3.8
lactone for 10b (%)
100.0
100.0
83.8
55.1
80.7
40.5
73.1
24.5
52.6
14.6
17.9
5.3
lactone for CPT (%)

Vol. 22, No. 2, 2011
Yang et al.
317
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 plasmas was monitored by HPLC that
clearly showed that compound 10b was cleaved to afford
mainly its parental compound of CPT in both mouse and
human plasmas. The cleavage of 10b is the result of the
combination of chemical and enzymatic effects in both
mouse and human plasmas. It was also the case in pH 7.4
phosphate buffer.
Another important aspect, which has to be addressed,
is that these compounds would be cleaved to release active
camptothecin and 5-fluorouracil in both mouse and human
plasma and such an assumption was supported by employing
HPLC chromatography experimental method, which
simultaneously implied that these conjugates might act as
mutual prodrugs and exert their cytotoxicities by hydrolyzing
to the parent compounds camptothecin and 5-fluorouracil,
and they could have synergetic effects in biological systems.
agents, and it is worthwhile to explore the antitumor
potential of these and similar types of compounds. In-
depth mechanistic studies and the development of new
camptothecin/5-fluorouracil conjugates are actively
underway in our laboratory.
Acknowledgments
We thank for NeroTrident Technology Limited
Company for providing Discovery Studio software. This
work was financially supported by the National Natural
Science Foundation of China (30800720); the Key
Laboratory of Environmental Chemistry and Ecotoxicology
Fund of Chinese Academy of Sciencer (KF2008-17); the
Post-Doctor Research Foundation (20090450142); the
Interdisciplinary Innovation Research Foundation For
Young Scholars, Lanzhou University (LZUJC2007018).
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