Synthesis and antitumor activity of conjugates of 5-Fluorouracil and emodin

Article abstract of DOI:10.1016/j.ejmech.2011.10.050

A series of conjugates of 5-Fluorouracil (5-FU) and emodin were synthesized by coupling trimethyl emodin with N¹, N³ dialkylated 5-FU. The 5-FU moiety contained various substituents at the N ³-position were linked to the 2

Full text of DOI:10.1016/j.ejmech.2011.10.050

European Journal of Medicinal Chemistry 47 (2012) 255e260
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of conjugates of 5-Fluorouracil and emodin
,
b
,
a
,
,
Li-Ming Zhao a , Li-Ming Zhang b, Jin-Juan Liu c, Li-Jing Wan a b, Yong-Qiang Chen c,
*
Shu-Qing Zhang a b, Zhi-Wei Yan a b, Ji-Hong Jiang c
,
,
,
*
a School of Chemistry and Chemical Engineering, Xuzhou Normal University, Xuzhou 221116, Jiangsu, China
b Jiangsu Key Laboratory of Green Synthetic Chemistry for Functional Materials, Xuzhou Normal University, Xuzhou 221116, Jiangsu, China
c Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Xuzhou Normal University, Xuzhou 221116, Jiangsu, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of conjugates of 5-Fluorouracil (5-FU) and emodin were synthesized by coupling trimethyl
emodin with N1, N3 dialkylated 5-FU. The 5-FU moiety contained various substituents at the N3-position
were linked to the 2-position of trimethyl emodin via a methylene linkage. Their cytotoxicity against
three cancer cell lines and one noncancerous cell were studied. The results revealed that some of
conjugates exhibited better or comparable in vitro antitumor activity to 5-FU and emodin and low
toxicity in the normal cell. The structureeactivity relationship study showed N3-aromatic substituent
was important for their cytotoxic activity.
Received 22 June 2011
Received in revised form
23 October 2011
Accepted 28 October 2011
Available online 6 November 2011
Keywords:
5-FU
Ó 2011 Elsevier Masson SAS. All rights reserved.
Emodin
Synthesis
Antitumor activity
1. Introduction
it has been modified by different ways to synthesize its derivatives
which may improve its therapeutic index [5e8] because of its well-
Despite the fact that some tumors are sensitive enough to be
cured by single drugs, the single drug therapy has fallen short in
treating cancers due to serious toxic side effects and poor selec-
tivity. On the other hand, the development of resistance is
a common problem of cancer chemotherapy, especially when using
a single drug. Nowadays effective cancer chemotherapy often relies
instead on combination therapies [1]. The simultaneous use of two
or more active drugs is common today for the treatment of cancer. If
a combination is synergistic, lower doses can be used to achieve the
same or better efficacy with lower toxicity [2]. In addition to
producing better therapies, chemical combinations also may be
used to study disease and reveal insight into the cellular pathways
at their root. Many combinations, which were named mutual drug,
in clinical use consist of an antimetabolite with one other anti-
neoplastic agent. The two agents may be combined directly or by
means of a linker [3].
known side effects such as short half-life, wide distribution, low
selectivity, and various toxic side effects. Recently chemists have
paid more attention to the conjugates of 5-FU with a wide spectrum
of low- or high-molecular-weight carriers. Some conjugates of 5-FU
have been reported as an approach to develop effective anticancer
nucleoside analogs such as 5-FU-podophyllotoxin [9], 5-FU-cyclo-
triphosphazene [10], 5-FU-cholic acid [11], 5-FU-amino acid ester
[12], and 5-FU-pectin [13], etc (Fig. 1).
Emodin is a naturally occurring anthraquinone present in the
rhubarb as well as the roots and bark of numerous plants of the
genus Rhamnus [14]. Structurally it belongs to the anthraquinone
group along with daunorubicin and mitoxantrone, which have
been in clinical use over 30 years for the treatment of cancers [15].
Attempts to use emodin for the treatment of cancer were mostly
unsuccessful due to the severe side effect, which the treatment kills
healthy cells as well as cancerous ones, hence emodin has been
used as a lead compound for the design and development of anti-
cancer agents. Many emodin derivatives, in which most modifica-
tions were focused on the methyl group and hydroxyl group, have
been synthesized in order to improve efficacy and decrease the
adverse effect potential (Fig. 2) [16e20].
5-Fluorouracil (5-FU) is an antimetabolite of the pyrimidine
analog type, which has been widely used in the treatment of solid
tumors [4]. Although 5-FU has had clinical success as a single agent,
We recently reported a hydroxymethylation of 6,8-O-dimethyl
emodin, which introduced a hydroxymethyl group into the 2-
position of emodin [21]. We therefore considered whether we
* Corresponding authors. Tel.: þ86 516 83403165; fax: þ86 516 83536977.
E-mail addresses: lmzhao@xznu.edu.cn (L.-M. Zhao), jhjiang@xznu.edu.cn
(J.-H. Jiang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.050

256
L.-M. Zhao et al. / European Journal of Medicinal Chemistry 47 (2012) 255e260
Fig. 1. Structures of 5-FU and its conjugates.
might be able to connect emodin with 5-FU by a methylene linkage
at the 2-position to form more effective and less toxic new anti-
tumor compounds. A notable example of this kind was the mutu-
ally beneficial modification of the ampicillin and sulbactam to give
sultamicillin [22]. Survey of literature revealed that till date enough
effort had not been made to connect these moieties as a single
molecular scaffold and identify new candidates that may be value
in designing new antitumor agents. So in this study, we designed
and synthesized a series of new compounds linking emodin with 5-
FU through a methylene to screen their antitumor activity against
different cancer cell lines and noncancerous cells in vitro in order to
get less toxic and selective agents.
9,10-dione 3 was prepared using emodin 1 as starting material via
methylation and modified Marschalk reaction according to the
procedure reported by our group previously [21]. The synthesis of
intermediate 4 involved the methylation of 3 with dimethyl sulfate
in acetone in the presence of anhydrous K2CO3. This reaction yiel-
ded 4 in good yield after chromatographic purification. Chlorina-
tion of 4 with thionyl chloride in CH2Cl2 at room temperature led to
the intermediate 5. The crude product of the reaction was used in
the next step without further purification. Subsequently, alkylation
of 5-FU with intermediate 5 in the presence of K2CO3 and KI gave
N1-substituted 5-FU 6 in good yield. Generally the nucleophilicity
of N3 of 5-FU is higher than that of N1 under the basic condition due
to the higher acidity of the N1eH proton (N1eH pKa 7.95 vs N3eH
pKa 12.4) [23]. Because of the two carbonyls at the 2- and 6-
position of 5-FU, however, the 3-position of 5-FU becomes diffi-
cult to accommodate the bulky group such as the intermediate 5.
Thus the alkylation occurs regioselectively at the 1-position. Baker
et al. observed that the orientation of alkylation in the uracil ring
also depended on the nature of the alkyl halide, that is, allylic
2. Results and discussion
2.1. Chemistry
The synthesis of target conjugates is illustrated in Scheme 1. 1-
Hydroxy-2-(hydroxymethyl)-6,8-dimethoxy-3-methylanthracene-
NR1R2
OH
O
OH
OH
N
O
N
O
O
O
O
NR1R2
HO
HO
O
O
emodin
O
OH
O
O
OH
OH
O
OH
R2R1N
O
R3O
OH
O
OR2
OR1
Fig. 2. Structures of emodin and its derivatives.

L.-M. Zhao et al. / European Journal of Medicinal Chemistry 47 (2012) 255e260
257
OH
O
OH
O
O
OH
O
O
OH
a
b
OH
OH
HO
O
O
O
O
O
O
3
1
2
c
O
O
O
O
O
O
O
O
O
F
e
d
Cl
N
N
NH
O
O
O
O
O
O
O
O
4
6
5
f
O
O
O
O
F
7a. R = CH3
7b. R = CH2CH3
7c. R = CH2CH2CH2CH3
7e. R = CH2CH=C(CH3)2
7g. R = CH2CH2C6H5
7i. R= CH2(2-F-C6H4)
7k. R = CH2(2-CN-C6H4)
7d. R = CH2CH=CHCH3
7f. R = CH2C6H5
R
O
N
7h. R = CH2(3,4-OCH2O-C6H3)
7j. R = CH2(3-F-C6H4)
O
7
Scheme 1. Reagents and conditions: (a) Me2SO4, K2CO3, acetone, reflux, 5 h; (b) Na2S2O4, CH2O (37%), MeOH, NaOH (1 N), 0 ꢀC, 30 min, subsequent H2O2 oxidation; (c) Me2SO4,
K2CO3, acetone, reflux, 16 h; (d) SOCl2, CH2Cl2, r.t., 30 min; (e) 5-FU/K2CO3, KI, DMSO, r.t., 5 h; (f) alkyl halide or benzyl halide, K2CO3, KI, DMSO, r.t., overnight.
halides orienting to the 1-position and alkyl halides to the 3-
position [24]. The regiochemical outcome observed here is
consistent with that reported by Baker and coworkers. A final
alkylation of intermediate 6 and the appropriately alkyl halides or
benzyl halides yielded the target conjugates 7ae7k. This alkylation
was successfully carried out by using the same method for the
synthesis of intermediate 6. It should be noted that the alkylation of
N3 of 5-FU by using chlorocyclohexane under the aforementioned
conditions was unsuccessful, possibly due to the greater steric bulk
of chlorocyclohexane.
8910 cells. While compound 7g exhibited significant cytotoxicity
(IC50 ¼ 11.6 M) in comparison with emodin (IC50 ¼ 16.0 M) and 5-
FU (IC50 ¼ 85.5 M), compounds 7i, 7f, and 7k showed moderate to
M, 45.1 M, 22.6 M respec-
tively). Similarly, compounds 7h, 7g, and 7k displayed moderate to
good cytotoxicity against HepG2 cell line, which was better or
comparative to 5-FU.
m
m
m
good inhibitory effect (IC50 ¼ 91.1
m
m
m
There was a significant difference in term of their cytotoxicity
affected by the type of N3-substituent of 5-FU (aliphatic or
Table 1
2.2. Biological activities
Cytotoxicity of conjugates of emodin derivatives and 5-FU against three tumor cell
lines and one non-tumor cell line.
The in vitro cytotoxicty of the target conjugates 6, 7ae7k were
evaluated over tumor cell lines HO-8910 (human ovarian cancer
cells), SGC-7901 (human gastric cancer cells), and HepG2 (human
liver cancer cells) by the Alamar Blue assay [25], in comparison with
emodin and 5-FU. To assess the selectivity of the tested compounds,
cytotoxicity of some conjugates was further evaluated against one
normal cell line: 293 (human embryonic kidney cells) (Table 1).
As shown in Table 1, some of the conjugates exhibited better
antitumor activity than the positive controls. For instance, conju-
gate 7g was much potent than lead compound emodin and 5-FU
against HO-8910 cells. Conjugates 7f and 7k were much active
than emodin and 5-FU against SGC-7901 cells. Conjugate 7k also
enhanced the inhibitory effect on HepG2 cells in comparison with
5-FU. The combination result suggest that it should be possible that
emodin derivatives can be combined with known anticancer drug
5-FU of low concentration to treat cancer more effectively by
lowering the toxic side effects of 5-FU that comes from the usage of
high concentration.
O
O
O
O
R
O
N
N
O
O
F
Compd.
R
IC50 (m
M)a
HO-8910
SGC-7901
HepG2
293
6
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
>100
>100
>100
>100
>100
>100
45.1
11.6
>100
91.1
>100
>100
>100
>100
>100
>100
39.5
>100
>100
61.0
>100
>100
>100
>100
>100
>100
>100
68.6
>100
N. D.b
N. D.
N. D.
N. D.
N. D.
>100
4.6
30.0
66.6
>100
29.6
42.9
1.3
methyl
ethyl
butyl
crotyl
prenyl
benzyl
phenylethyl
piperonyl
2-fluorobenzyl
3-fluorobenzyl
2-cyanobenzyl
91.0
When these compounds were screened against SGC-7901 cells,
conjugates 7k and 7f were found to be approximately 3 and 1.5-fold
>100
>100
39.9
76.7
13.1
7j
>100
22.6
85.5
>100
21.4
76.0
more active with the IC50 values of 21.4 and 39.5
than emodin (IC50 ¼ 61.3 M). Conjugate 7i displayed essentially
the same value (IC50 ¼ 61.0 M) with the lead compound emodin.
All the three compounds were more active than 5-FU
(IC50 ¼ 76.0 M). The conjugates were then screened against HO-
mM, respectively,
7k
5-FU
emodin
m
16.0
61.3
m
a
IC50 values were calculated from three independent experiments.
N. D., not determined.
b
m

258
L.-M. Zhao et al. / European Journal of Medicinal Chemistry 47 (2012) 255e260
aromatic). In general, compounds having an aromatic substituent
were more potent than the corresponding compounds bearing an
aliphatic substituent. Compounds 6, 7ae7e, which contain only the
aliphatic groups at N3, showed no cytotoxicity against all three
cancer lines. When the substituent was replaced with aromatic
groups, the resulting compounds, 7fe7i, 7k, demonstrated
improvement in cytotoxicity. Furthermore, it seemed that the
increase in the distance between the phenyl ring and uracil frag-
ment led to an increase in antitumor activity, especially for HO-
8910 cell line. For instance, compound 7g with the insertion of an
ethyl group displayed better inhibitory activity against HO-8910
when compared to compounds 7f, 7he7k.
Antitumor drugs may be considered potentially more useful
when they are more active to tumor cell and lower cytotoxicity to
non-tumor cell. Therefore, six compounds: 7fe7k showing good
activity in Alamar Blue assay together with compound 6 were
selected for further evaluation to test their cytotoxicty on normal
cell line. It could be seen from the Table 1 that some of tested
compounds exhibited less toxic than positive controls. The most
notable compound of the conjugates resulted compound 7f, which
showed a good activity against HO-8910, even more potency
(200e300 mesh) for purification of the compounds. Melting points
were measured on an X-4 melting-point apparatus and were
uncorrected. The 1H NMR and 13C NMR spectra were recorded on
Bruker Avance spectrometer at 400 MHz and 100 MHz, respec-
tively. Chemical shift values were reported as d ppm relative to TMS
as internal standard. HRMS were recorded on a Bruker TOF-QⅡ
spectrometer with electrospray ionization (ESI).
4.1.1. 1-Hydroxy-2-(hydroxymethyl)-6,8-dimethoxy-3-methylanthra-
cene-9,10-dione (3)
1-Hydroxy-2-(hydroxymethyl)-6,8-dimethoxy-3-methylanthra-
cene-9,10-dione 3 was synthesized as recently described proce-
dure [21].
4.1.2. 2-(Hydroxymethyl)-1,6,8-trimethoxy-3-methylanthracene-
9,10-dione (4)
To a solution of 3 (656 mg, 2.0 mmol) in acetone (50 mL) was
added anhydrous K2CO3 (1.38 g, 10 mmol) and dimethyl sulfate
(0.38 mL, 4.0 mmol). The mixture was refluxed for 16 h, then
evaporated the solvent, and extracted three times with CH2Cl2. The
organic layer was washed with brine, dried, and concentrated. The
residue was purified by flash column chromatography to afford 4
against SGC-7901 (IC50 ¼ 39.5
m
M) than emodin (IC50 ¼ 61.3
mM)
and 5-FU (IC50 ¼ 76.0 M), but no cytotoxicity against the non-
m
(554 mg, 81%) as yellow solid. 1H NMR (400 MHz, CDCl3)
d 7.84 (s,
tumor cell 293. Similarly, compound 7i exhibited comparable
antitumor activity against HO-8910 and SGC-7901 to 5-FU, but
about 1.5 times less potent than 5-FU on 293. Compound 7k is
1H), 7.34 (d, J ¼ 2.0 Hz,1H), 6.78 (d, J ¼ 2.0 Hz,1H), 4.82 (s, 2 H), 4.02
(s, 3 H), 3.99 (s, 3 H), 3.97 (s, 3 H), 2.53 (s, 3 H).
about
3
times potent than emodin against SGC-7901
M vs IC50 ¼ 61.3 M), but about 23 times less potent
M). Although
4.1.3. 2-(Chloromethyl)-1,6,8-trimethoxy-3-methylanthracene-
9,10-dione (5)
(IC50 ¼ 21.4
m
m
than emodin on 293 (IC50 ¼ 29.6
mM vs IC50 ¼ 1.3
m
A solution of 4 (547 mg, 1.6 mmol) in dry CH2Cl2 (10 mL) was
treated with thionyl chloride (0.4 mL, 5.6 mmol) and stirred for
30 min at room temperature. The solution was evaporated in vacuo
and the residue was used in the next step without further
purification.
emodin showed better antitumor activity against HepG2 and HO-
8910 than compound 7k, no selectivity was reflected between
cancer cells tested and the normal cell (IC50 ¼ 1.3
mM). These results
indicated that emodin derivatives with a methylene-linked nucle-
osides at 2-position enhanced the selectivity in comparison with
the positive control, in spite of the decreased cytotoxic activity of
some compounds against cancerous cells with varying degrees. It is
noteworthy that although the antitumor activity was improved by
increasing the distance between the phenyl ring and the uracil
fragment, the toxicity was increased accordingly as seen from the
4.1.4. 5-Fluoro-1-((1,6,8-trimethoxy-3-methyl-9,10-dioxo-9,10-
dihydroanthracen-2-yl)-methyl)pyrimidine-2,4(1H,3H)-dione (6)
A stirred mixture of 5-FU (156 mg, 1.2 mmol) and anhydrous
K2CO3 (166 mg, 1.2 mmol) in DMSO (14 mL) was heated to 80 ꢀC.
After stirring for 30 min, the mixture was cooled to room temper-
ature. To the solution was added KI (50 mg, 0.3 mmol) and 5
(108 mg, 0.3 mmol). The mixture was stirred for 5 h at room
temperature, then poured into water, and extracted three times
with CH2Cl2. The organic layer was washed with brine, dried, and
concentrated. The residue was purified by flash column chroma-
tography to afford 6 (110 mg, 80.8%) as yellow solid. mp:
IC50 value on 293 of compound 7g (IC50 ¼ 4.6
mM).
3. Conclusions
In summary, we have designed and synthesized twelve conju-
gates of 5-FU and emodin, and evaluated their antitumor activities
against three cancer cell lines and one normal cell. Biological
evaluation revealed that some of conjugates exhibited better or
comparable in vitro antitumor activity to positive controls with low
toxicity in the normal cell. For instance, the conjugate 7f showed
selective cytostatic activity in HO-8910 and SGC-7901 cell lines and
no cytotoxicity to human normal embryonic kidney cell 293.
Compounds 7k were shown to have a broad spectrum of antitumor
activity against the tested three tumor cell lines and much lower
toxic activity toward normal cell compared to emodin. The struc-
tureeactivity relationship study showed N3-aromatic substituent
was important for their cytotoxic activity.
165e166 ꢀC; 1H NMR (400 MHz, DMSO-d6)
1H), 7.94 (d, J ¼ 6.8 Hz, 1H), 7.73 (s, 1H), 7.17 (d, J ¼ 2.4 Hz, 1H), 6.99
(d, J ¼ 2.4 Hz, 1H), 4.90 (s, 2 H), 3.94 (s, 3 H), 3.91 (s, 3 H), 3.79 (s,
3 H), 2.54 (s, 3 H).
d
11.82 (d, J ¼ 5.2 Hz,
4.1.5. General procedure for the preparation of conjugates 7ae7k
A stirred mixture of 6 (136 mg, 0.3 mmol) and anhydrous K2CO3
(124 mg, 0.9 mmol) in DMSO (10 mL) was heated to 80 ꢀC. After
stirring for 30 min, the mixture was cooled to room temperature. To
the solution was added KI (50 mg, 0.3 mmol) and alkyl halides or
benzyl halides. The mixture was stirred overnight at room
temperature, then poured into water, and extracted three times
with CH2Cl2. The organic layer was washed with brine, dried, and
concentrated. The residue was purified by flash column chroma-
tography to afford 7ae7k as yellow solid.
4. Experimental
4.1. Chemistry
Regents and solvents were purchased commercially and used
without further purified, unless otherwise stated. Reactions were
monitored using thin-layer chromatography (TLC) on silica gel
plates. Flash column chromatography was performed on silica gel
4.1.5.1. 5-Fluoro-3-methyl-1-((1,6,8-trimethoxy-3-methyl-9,10-dioxo-
9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(7a). Yield 64.7%; mp: 256e257 ꢀC; 1H NMR (400 MHz, CDCl3)
d
7.90
(s, 1H), 7.34 (d, J ¼ 2.4 Hz, 1H), 7.32 (d, J ¼ 5.6 Hz, 1H), 6.80

L.-M. Zhao et al. / European Journal of Medicinal Chemistry 47 (2012) 255e260
259
(d, J ¼ 2.4 Hz, 1H), 5.09 (s, 2H), 3.99 (s, 6H), 3.96 (s, 3H), 3.38 (s, 3H),
2.55 (s, 3H); 13C NMR (100 MHz, CDCl3)
183.3, 181.0, 164.3, 162.0,
159.7, 156.9 (d, J ¼ 29.1 Hz),150.3,145.3,140.0 (d, J ¼ 233.3 Hz),136.3,
134.6, 133.8, 126.0 (d, J ¼ 33.2 Hz), 125.6, 124.9, 117.8, 105.4, 102.4,
63.1, 56.6, 56.0, 43.7, 28.6, 20.5; HRMS (ESI): 491.1231 for [M þ Na]þ
(calcd 491.1232 for C24H21FN2O7Na).
136.3, 136.1, 134.6, 134.0, 129.1, 128.5, 127.9, 126.0 (d, J ¼ 33.0 Hz),
d
125.6, 124.9, 117.7, 105.3, 102.4, 63.1, 56.6, 56.0, 45.2, 43.2, 20.5;
HRMS (ESI): 567.1573 for [M
C30H25FN2O7Na).
þ
Na]þ (calcd 567.1545 for
4.1.5.7. 5-Fluoro-3-phenethyl-1-((1,6,8-trimethoxy-3-methyl-9,10-
dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (7g). Yield 53.2%; mp: 199e200 ꢀC; 1H NMR (400 MHz,
4.1.5.2. 3-Ethyl-5-fluoro-1-((1,6,8-trimethoxy-3-methyl-9,10-dioxo-
9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(7b). Yield 63.5%; mp: 257e258 ꢀC; 1H NMR (400 MHz, CDCl3)
CDCl3)
d
7.89 (s, 1H), 7.33 (d, J ¼ 2.0 Hz, 1H), 7.30 (d, J ¼ 5.6 Hz, 1H),
7.27 (d, J ¼ 2.0 Hz, 4H), 7.23e7.17 (m, 1H), 6.80 (d, J ¼ 2.0 Hz, 1H),
d
7.91 (s, 1H), 7.35 (d, J ¼ 2.4 Hz, 1H,), 7.29 (d, J ¼ 5.6 Hz, 1H), 6.81 (d,
5.07 (s, 2H), 4.20 (t, J ¼ 8.0 Hz, 2H), 3.99 (s, 6H), 3.97 (s, 3H), 2.93 (t,
J ¼ 2.4 Hz, 1H), 5.09 (s, 2H), 4.04 (q, J ¼ 7.2 Hz, 2H), 4.00 (s, 3H), 3.99
J ¼ 8.0 Hz, 2H), 2.48 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 183.3,
(s, 3H), 3.98 (s, 3H), 2.54 (s, 3H), 1.24 (t, J ¼ 7.2 Hz, 3H); 13C NMR
180.0, 164.3, 162.0, 159.6, 156.8 (d, J ¼ 26.3 Hz), 150.0, 145.4, 140.0
(d, J ¼ 233.8 Hz), 138.0, 136.3, 134.6, 133.9, 129.0, 128.5, 126.6, 126.0
(d, J ¼ 32.9 Hz), 125.6, 124.9, 117.8, 105.4, 102.4, 63.1, 56.6, 56.0, 43.3,
33.5, 29.3, 20.5; HRMS (ESI): 581.1729 for [M þ Na]þ (calcd 581.1701
for C31H27FN2O7Na).
(100 MHz, CDCl3)
d 183.3, 181.0, 164.3, 162.0, 159.6, 156.8 (d,
J ¼ 25.1 Hz), 149.9, 145.3, 140.2 (d, J ¼ 233.6 Hz), 136.3, 134.6, 134.0,
125.9 (d, J ¼ 33.0 Hz), 125.6, 124.9, 117.8, 105.4, 102.4, 63.1, 56.6,
56.0, 43.4, 37.4, 20.4, 12.7; HRMS (ESI): 505.1392 for [M þ Na]þ
(calcd 505.1388 for C25H33FN2O7Na).
4.1.5.8. 3-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-fluoro-1-((1,6,8-trime-
thoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyri-
midine-2,4(1H,3H)-dione (7h). Yield 83.4%; mp: 189e190 ꢀC; 1H
4.1.5.3. 3-Butyl-5-fluoro-1-((1,6,8-trimethoxy-3-methyl-9,10-dioxo-
9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(7c). Yield 84.7%; mp: 214e215 ꢀC; 1H NMR (400 MHz, CDCl3)
NMR (400 MHz, CDCl3)
d
7.89 (s, 1H), 7.35 (d, J ¼ 2.4 Hz, 1H), 7.26 (d,
d
7.91 (s, 1H), 7.35 (d, J ¼ 2.4 Hz, 1H), 7.28 (d, J ¼ 5.6 Hz, 1H), 6.81 (d,
J ¼ 5.6 Hz,1H), 7.01 (s,1H), 7.00 (d, J ¼ 8.4 Hz,1H), 6.81 (d, J ¼ 2.4 Hz,
1H), 6.73 (d, J ¼ 8.0 Hz, 1H), 5.92 (s, 2H), 5.09 (s, 2H), 5.05 (s, 2H),
3.99 (s, 3H), 3.97 (s, 6H), 2.49 (s, 3H); 13C NMR (100 MHz, CDCl3)
J ¼ 2.4 Hz, 1H), 5.10 (s, 2H), 4.00 (s, 6H), 3.98 (s, 3H), 3.97 (t,
J ¼ 8.0 Hz, 2H), 2.53 (s, 3H), 1.65e1.57 (m, 2H), 1.42e1.33 (m, 2H),
0.94 (t, J ¼ 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
183.3, 181.0,
d
183.3, 181.1, 164.3, 162.0, 159.6, 157.0 (d, J ¼ 25.2 Hz), 150.1, 147.6,
164.3, 162.0, 159.6, 156.9 (d, J ¼ 24.9 Hz), 150.1, 145.3, 140.1 (d,
J ¼ 233.5 Hz), 138.9, 136.3, 134.1, 125.9 (d, J ¼ 33.1 Hz), 125.6, 124.9,
117.8, 105.3, 102.4, 63.1, 56.6, 55.9, 43.3, 42.0, 29.5, 20.4, 20.1, 13.7;
147.2, 145.4, 140.1 (d, J ¼ 234.0 Hz), 136.3, 134.6, 134.0, 129.8, 126.1
(d, J ¼ 33.1 Hz), 125.6, 125.0, 123.1, 117.7, 109.9, 108.1, 105.3, 102.4,
101.0, 63.2, 56.6, 56.0, 45.0, 43.2, 20.5; HRMS (ESI): 611.1476 for
[M þ Na]þ (calcd 611.1443 for C31H25FN2O9Na).
HRMS (ESI): 533.1710 for [M
C27H27FN2O7Na).
þ
Na]þ (calcd 533.1701 for
4.1.5.9. 5-Fluoro-3-(2-fluorobenzyl)-1-((1,6,8-trimethoxy-3-methyl-
9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (7i). Yield 33.2%; mp: 233e234 ꢀC; 1H NMR (400 MHz, CDCl3)
4.1.5.4. (E)-3-(But-2-enyl)-5-fluoro-1-((1,6,8-trimethoxy-3-methyl-
9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-
2,4(1H,3H)-dione (7d). Yield 70.4%; mp: 215e216 ꢀC; 1H NMR
d
7.88 (s,1H), 7.33 (d, J ¼ 2.4 Hz,1H), 7.33 (d, J ¼ 5.2 Hz,1H), 7.25e7.21
(400 MHz, CDCl3)
d
7.90 (s, 1H), 7.34 (d, J ¼ 2.4 Hz, 1H), 7.26 (d,
(m, 2H), 7.08e7.02 (m, 2H), 6.80 (d, J ¼ 2.4 Hz,1H), 5.24 (s, 2H), 5.11 (s,
J ¼ 5.6 Hz,1H), 6.80 (d, J ¼ 2.4 Hz,1H), 5.85e5.76 (m,1H), 5.58e5.50
(m,1H), 5.10 (s, 2H), 4.50 (d, J ¼ 6.4 Hz, 2H), 3.99 (s, 6H), 3.97 (s, 3H),
2.52 (s, 3H), 1.67 (d, J ¼ 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3)
2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 2.47 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d
183.3, 181.0, 164.3, 162.0, 160.8 (d, J ¼ 249.9 Hz),
159.6, 156.9 (d, J ¼ 26.4 Hz),149.9,145.4,140.1 (d, J ¼ 234.4 Hz),136.3,
134.6, 134.0, 129.4 (d, J ¼ 3.9 Hz), 129.3 (d, J ¼ 9.3 Hz), 126.2 (d,
J ¼ 32.9 Hz), 125.6, 125.0, 124.1 (d, J ¼ 3.7 Hz), 122.9 (d, J ¼ 14.3 Hz),
117.8, 115.5 (d, J ¼ 21.4 Hz), 105.4, 102.4, 63.2, 56.6, 56.0, 43.1, 39.2,
20.4; HRMS (ESI): 585.1485 for [M þ Na]þ (calcd 585.1451 for
C30H24F2N2O7Na).
d
183.3, 181.0, 164.3, 162.0, 159.6, 156.7 (d, J ¼ 24.9 Hz), 149.9, 145.3,
140.2 (d, J ¼ 234.7 Hz), 136.3, 134.6, 134.0, 131.0, 125.9 (d,
J ¼ 33.1 Hz), 125.6, 125.0, 123.7, 117.8, 105.4, 102.4, 63.1, 56.6, 56.0,
43.5, 43.2, 20.5, 17.7; HRMS (ESI): 531.1582 for [M þ Na]þ (calcd
531.1545 for C27H25FN2O7Na).
4.1.5.5. 5-Fluoro-3-(3-methylbut-2-enyl)-1-((1,6,8-trimethoxy-3-
methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-
2,4(1H,3H)-dione (7e). Yield 80.5%; mp: 260e261 ꢀC; 1H NMR
4.1.5.10. 5-Fluoro-3-(3-fluorobenzyl)-1-((1,6,8-trimethoxy-3-methyl-
9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (7j). Yield 40.1%; mp: 229e230 ꢀC; 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d
7.90 (s, 1H), 7.34 (d, J ¼ 2.4 Hz, 1H), 7.25 (d,
d
7.89 (s, 1H), 7.33 (d, J ¼ 2.4 Hz,1H), 7.31 (d, J ¼ 5.6 Hz,1H), 7.30e7.23
J ¼ 5.6 Hz, 1H), 6.80 (d, J ¼ 2.0 Hz,1H), 5.23 (t, J ¼ 6.8 Hz, 1H), 5.10 (s,
2H), 4.56 (d, J ¼ 7.2 Hz, 2H), 3.99 (s, 3H), 3.97 (s, 6H), 2.52 (s, 3H),
(m, 2H), 7.19e7.16 (m, 1H), 7.00e6.94 (m, 1H), 6.80 (d, J ¼ 2.4 Hz, 1H),
5.13 (s, 2H), 5.10 (s, 2H), 3.99 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 2.49
1.82 (s, 3H), 1.71 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
183.3, 181.0,
(s, 3H); 13C NMR (100 MHz, CDCl3)
d 183.3, 181.0, 164.3, 162.7
164.3, 162.0, 159.6, 156.8 (d, J ¼ 24.9 Hz), 150.0, 145.4, 140.2 (d,
J ¼ 233.6 Hz), 137.9, 136.3, 134.6, 134.1, 125.7 (d, J ¼ 33.1 Hz), 125.6,
124.9, 117.8, 117.7, 105.4, 102.4, 63.1, 56.6, 56.0, 43.2, 40.3, 25.7, 20.5,
18.1; HRMS (ESI): 545.1725 for [M þ Na]þ (calcd 545.1701 for
C28H27FN2O7Na).
(d, J ¼ 244.8 Hz),162.0, 159.6, 156.9 (d, J ¼ 25.3 Hz), 150.1, 145.3, 140.1
(d, J ¼ 234.3 Hz), 138.4 (d, J ¼ 7.4 Hz), 136.3, 134.7, 133.8, 130.0 (d,
J ¼ 8.1 Hz), 126.2 (d, J ¼ 33.1 Hz), 125.6, 125.0, 124.7 (d, J ¼ 2.8 Hz),
117.8, 116.0 (d, J ¼ 21.7 Hz), 114.9 (d, J ¼ 20.8 Hz), 105.3, 102.4, 63.2,
56.6, 56.0, 44.6, 43.2, 20.5; HRMS (ESI): 585.1473 for [M þ Na]þ (calcd
585.1451 for C30H24F2N2O7Na).
4.1.5.6. 3-Benzyl-5-fluoro-1-((1,6,8-trimethoxy-3-methyl-9,10-
dioxo-9,10-dihydroanthracen-2-yl)methyl)pyrimidine-2,4(1H,3H)-
dione (7f). Yield 74.2%; mp: 212e213 ꢀC; 1H NMR (400 MHz, CDCl3)
4.1.5.11. 2-((5-Fluoro-2,6-dioxo-3-((1,6,8-trimethoxy-3-methyl-9,10-
dioxo-9,10-dihydroan thracen-2-yl)methyl)-2,3-dihydropyrimidin-
1(6H)-yl)methyl)benzonitrile (7k). Yield 71.7%; mp: 228e229 ꢀC; 1H
d
7.88 (s, 1H), 7.47 (d, J ¼ 6.8 Hz, 2H), 7.33e7.26 (m, 5H), 6.80 (d,
J ¼ 2.4 Hz, 1H), 5.15 (s, 2H), 5.09 (s, 2H), 3.99 (s, 3H), 3.97 (s, 6H),
2.47 (s, 3H); 13C NMR (100 MHz, CDCl3)
183.2, 180.9, 164.3, 162.0,
159.5, 157.0 (d, J ¼ 25.1 Hz), 150.1, 145.3, 140.1 (d, J ¼ 233.9 Hz),
NMR (400 MHz, CDCl3)
J ¼ 7.6 Hz, 1H), 7.37 (d, J ¼ 5.6 Hz, 1H), 7.37 (t, J ¼ 7.6 Hz, 1H), 7.34 (d,
d
8.00 (s, 1H), 7.67 (d, J ¼ 7.6 Hz, 1H), 7.53 (t,
d
J ¼ 2.4 Hz,1H), 7.26 (d, J ¼ 7.6 Hz,1H), 6.81 (d, J ¼ 2.4 Hz,1H), 5.39 (s,

260
L.-M. Zhao et al. / European Journal of Medicinal Chemistry 47 (2012) 255e260
2H), 5.11 (s, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 2.48 (s, 3H);
13C NMR (100 MHz, CDCl3)
183.3, 180.9, 164.4, 162.0, 159.6, 156.9
Foundation of Jiangsu Province (09KJD150006), and Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
d
(d, J ¼ 25.7 Hz), 150.0, 145.3, 139.9 (d, J ¼ 234.8 Hz), 139.5, 136.3,
134.7, 133.7, 133.1, 133.0, 128.0, 127.3, 126.6 (d, J ¼ 33.0 Hz), 125.6,
125.0, 117.8, 117.2, 112.1, 105.4, 102.4, 63.2, 56.6, 56.0, 43.4, 43.4,
20.5; HRMS (ESI): 592.1524 for [M þ Na]þ (calcd 592.1497 for
C31H24FN3O7Na).
References
[1] A.A. Borisy, P.J. Elliott, N.W. Hurst, M.S. Lee, J. Lehar, E.R. Price, G. Serbedzija,
G.R. Zimmermann, M.A. Foley, B.R. Stockwell, C.T. Keith, Proc. Natl. Acad. Sci.
U. S. A. 100 (2003) 7977e7982.
4.2. Cell culture and cytotoxicity assays
[2] H. Yamada, N. Uchida, R. Maekawa, T. Yoshioka, Cancer Lett. 172 (2001)
17e25.
[3] G.J. Peters, C.L. van der Wilt, C.J.A. van Moorsel, J.R. Kroep, A.M. Bergman,
S.P. Ackland, Pharmacol. Ther. 87 (2000) 227e253.
[4] N.F. Smith, W.D. Figg, A. Sparreboom, Drug Develop. Res. 62 (2004) 233e253.
[5] S.E. Aspland, C. Ballatore, R. Castillo, J. Desharnais, T. Eustaquio, P. Goelet,
Z. Guo, Q. Li, D. Nelson, C. Sun, A.J. Castellino, M.J. Newman, Bioorg. Med.
Chem. Lett. 16 (2006) 5194e5198.
[6] D. Topalis, U. Pradere, V. Roy, C. Caillat, A. Azzouzi, J. Broggi, R. Snoeck,
G. Andrei, J. Lin, S. Eriksson, J.A.C. Alexandre, C. El-Amri, D. Deville-Bonne,
P. Meyer, J. Balzarini, L.A. Agrofoglio, J. Med. Chem. 54 (2011) 222e232.
[7] N. Tzioumaki, E. Tsoukala, S. Manta, C. Kiritsis, J. Balzarini, D. Komiotis, Car-
bohydr. Res. 346 (2011) 328e333.
The HO-8910, SGC-7901, HepG2 and 293 cells were obtained
from Shanghai Institute of Cell Biology, Chinese Academy of
Sciences (Shanghai, China). All cells were maintained in RPMI1640
medium supplemented with 10% fetal bovine serum (FBS), 100 U/
mL penicillin and 100 U/mL streptomycin and incubated under
humidified air with 5% CO2 at 37 ꢀC.
The in vitro cytotoxicity of the target conjugates was deter-
mined by the Alamar Blue analysis with minor modifications.
Briefly, Cells with same number were inoculated into each well in
96-well plates (Costar, Charlotte, NC) with 100
After an overnight incubation, the novel synthesized conjugates in
100 L diluted with the medium were added into the wells for
determine the cytotoxicity. Then the cells were incubated for
another 2 days. Thereafter, 20 L Alamar blue solution was added to
mL culture medium.
[8] H. Chen, J. Zhao, Y. Li, F. Shen, X. Li, Q. Yin, Z. Qin, X. Yan, Y. Wang, P. Zhang,
J. Zhang, Bioorg. Med. Chem. Lett. 21 (2011) 574e576.
[9] S.W. Chen, R. Xiang, J. Liu, X. Tian, Bioorg. Med. Chem. 17 (2009) 3111e3117.
[10] Y.W. Cho, J.R. Lee, S.C. Song, Bioconjug. Chem. 16 (2005) 1529e1535.
[11] S. Qian, J.B. Wu, X.C. Wu, J. Li, Y. Wu, Arch. Pharm. 342 (2009) 513e520.
[12] J. Xiong, H.F. Zhu, Y.J. Zhao, Y.J. Lan, J.W. Jiang, J.J. Yang, S.F. Zhang, Molecules
14 (2009) 3142e3152.
[13] Q.W. Wang, X.Y. Liu, L. Liu, J. Feng, Y.H. Li, Z.J. Guo, Q.B. Mei, Med. Chem. Res.
16 (2008) 370e379.
[14] D.G. Barceloux, Medical Toxicology of Natural Substances: Foods, Fungi,
Medicinal Herbs, Plants, and Venomous Animals. Wiley & Sons, Philadelphia,
PA, 2008, (Chapter 12), pp. 85e86.
m
m
each well after removal of the sample solution and washing with
phosphate-buffered saline (PBS, pH 7.4), and cultured for an addi-
tional 4h. Each conjugates was tested in triplicate wells. The
absorbance was measured using SpectraMax M2 (Molecular
Devices) with a test wavelength of 530 nm and 590 nm. Growth
inhibition rate was calculated by the following formula:
[15] C. Monneret, Eur. J. Med. Chem. 36 (2001) 483e493.
[16] G. Song, H. Liu, W. Zhang, M. Geng, Y. Li, Bioorg. Med. Chem. 18 (2010)
5183e5193.
[17] X. Wang, W. Xu, Bull. Kor. Chem. Soc. 26 (2005) 1923e1924.
[18] M. Koyama, K. Takahashi, T.C. Chou, Z. Darzynkiewicz, J. Kapuscinski,
T.R. Kelly, K.A. Watanabe, J. Med. Chem. 32 (1989) 1594e1599.
[19] J.H. Tan, Q.X. Zhang, Z.S. Huang, Y. Chen, X.D. Wang, L.Q. Gu, J.Y. Wu, Eur. J.
Med. Chem. 41 (2006) 1041e1047.
Growth inhibition rate ¼ [(AControl ꢁ Asample)/
(AControl ꢁ Ablank)] ꢂ 100%
[20] S. Banfi, E. Caruso, M. Gariboldi, S. Alemani, G. Nasini, E. Bombardelli, Synth.
Commun. 38 (2008) 1096e1109.
Acknowledgments
[21] H.S. Jin, L.M. Zhao, Chin. Chem. Lett. 21 (2010) 568e571.
[22] H.A. Friedel, D.M. Campoli-Richards, K.L. Goa, Drugs 37 (1989) 491e522.
[23] S. Boncel, A. Gondela, K. Walczak, Synthesis (2010) 1573e1589.
[24] B.R. Baker, G.D.F. Jackson, J. Pharm. Sci. 54 (1965) 1758e1762.
[25] G.R. Nakayama, M.C. Caton, M.P. Nova, Z. Parandoosh, J. Immunol. Methods
204 (1997) 205e208.
We are thankful for financial support from Science and Tech-
nology Project of Xuzhou (XJ09078), National Natural Science
Foundation of China (No. 30872028), Universities Natural Science