A novel kind of antitumour drugs using sulfonamide as parent compound

Article abstract of DOI:10.1016/S0223-5234(01)01285-5

To obtain potent antitumour agents with low toxicity, sulfonamide derivatives containing 5-flurouracil and nitrogen mustard, respectively are designed and synthesised. 1-(3-(4-Acetylaminobenzenesulfonamido)-3-oxopropyl)-5-fluoropyrimidine-2, 4-dione (4) was obtained by the coupling of p-acetamidobenzenesulfonamide with 3-(5-fluorouracil-1-yl) propionic acid. The hydrolysis of 4 led to 1-(3-(4-aminobenzenesulfonamido)-3-oxopropyl)-5-fluoropyrimidine-2,4-dione (5). Treatment of p-acetamidobenzenesulfonyl chloride with bis(2-chloroethyl) amine led to 4-acetylamino-N,N-bis(2-chloroethyl)benzenesulfonamide (6). Subsequent hydrolysis of 6 in hydrochloric acid led to 4-amino-N,N-bis(2-chloroethyl)benzenesulfonamide hydrochloride (7). Two different synthetic route were investigated in the synthesis of 2-[N¹-2-pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate (12b). Carbobenzyloxy was proved to be unsuitable for the protection of the aromatic amino group of sulfadiazine since the pyrimidine ring was also hydrogenated at the last step of the first route under the deprotection condition. In another route, acetyl was firstly used as the protective group, then it was replaced by the Schiff's base. The reaction of chlorambucil with 2-[N¹-2-pyrimidinyl-(p-acetyl)aminobenzenesulfonamido] ethanol (10b) afforded 2-[N¹-2-pyrimidinyl-(p-benzylidene)aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate (11b). Compound 12b was obtained by the hydrolysis of 11b. The acute toxicity and antitumour activity of 5, 7 and 12b have been investigated in mice. Compound 12b exhibited high antitumour activity and low toxicity with a therapeutic index (TI) of 47.55.

Full text of DOI:10.1016/S0223-5234(01)01285-5

Eur. J. Med. Chem. 36 (2001) 863–872
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01285-5/FLA
Original article
A novel kind of antitumour drugs using sulfonamide as parent compound
Zhaohua Huanga,b, Zhaoliang Lina,b, Junlian Huanga,b,
*
aThe Key Laboratory of Molecular Engineering of Polymers, Ministry of Education, People’s Republic of China
bDepartment of Macromolecular Science, Fudan University, Shanghai 200433, People’s Republic of China
Received 6 September 2001; revised 17 October 2001; accepted 17 October 2001
Abstract – To obtain potent antitumour agents with low toxicity, sulfonamide derivatives containing 5-flurouracil and nitrogen
mustard, respectively are designed and synthesised. 1-(3-(4-Acetylaminobenzenesulfonamido)-3-oxopropyl)-5-fluoropyrimidine-2,4-
dione (4) was obtained by the coupling of p-acetamidobenzenesulfonamide with 3-(5-fluorouracil-1-yl) propionic acid. The
hydrolysis of 4 led to 1-(3-(4-aminobenzenesulfonamido)-3-oxopropyl)-5-fluoropyrimidine-2,4-dione (5). Treatment of p-acetami-
dobenzenesulfonyl chloride with bis(2-chloroethyl) amine led to 4-acetylamino-N,N-bis(2-chloroethyl)benzenesulfonamide (6).
Subsequent hydrolysis of 6 in hydrochloric acid led to 4-amino-N,N-bis(2-chloroethyl)benzenesulfonamide hydrochloride (7). Two
different synthetic route were investigated in the synthesis of 2-[N1-2-pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl)
aminophenyl butyrate (12b). Carbobenzyloxy was proved to be unsuitable for the protection of the aromatic amino group of
sulfadiazine since the pyrimidine ring was also hydrogenated at the last step of the first route under the deprotection condition. In
another route, acetyl was firstly used as the protective group, then it was replaced by the Schiff’s base. The reaction of chlorambucil
with 2-[N1-2-pyrimidinyl-(p-acetyl)aminobenzenesulfonamido] ethanol (10b) afforded 2-[N1-2-pyrimidinyl-(p-benzylidene)-
aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate (11b). Compound 12b was obtained by the hydrolysis of
11b. The acute toxicity and antitumour activity of 5, 7 and 12b have been investigated in mice. Compound 12b exhibited high
´
antitumour activity and low toxicity with a therapeutic index (TI) of 47.55. © 2001 Editions scientifiques et me´dicales Elsevier SAS
sulfonamide / antitumour activity / targeting / nitrogen mustard
1. Introduction
agents in one compound. Heretofore, the main work
of modified sulfadiazine has been focused on the
Conventional cancer chemotherapy is highly inade-
aromatic amine due to its relatively high reaction
quate as a result of the lack of selectivity between
activity [3–7]. As shown in figure 1, 14C labelled
cancer cells and normal cells. This calls for novel
sulfadiazine mustard has been synthesised by Nguyen
cancer therapies for selectively targeting cancers with-
et al. [6], and sulfadiazine acrylamide and its polymer
out toxicity to normal tissues. Sulfapyrazine has been
by Abel et al. [7].
shown to concentrate selectively in the Walker car-
The surprising finding that all these compounds
cinoma growing in rats [1]. Similarly sulfadiazine was
were only taken up poorly by those tumours which
found concentrated in the Yoshida sarcoma [2]. These
concentrate sulfadiazine led to a reconsideration of
findings have aroused considerable interest and great
the drug design strategy. Unfortunately, there was not
efforts have been made to design new antitumour
any successful report about the application of the
agents by combining sulfadiazine and antitumour
targeting function of sulfadiazine. In 1996, Huang
suggested a new idea to design polymeric medicine
using sulfadiazine as targeting compound [8]. The
work in Huang’s group showed if polyethyleneoxide
(Mn=2000) were used as matrix, and the sulfadi-
* Correspondence and reprints
E-mail addresses: huang zhaohua@yahoo.com (Z. Huang),
–
jlhuang@fudan.edu.cn (J. Huang).

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Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
2. Chemistry
azine was attached on its end at the site of sulfon-
amido group rather than the aromatic amino group,
then the selectivity was recovered although the mech-
anism is still unclear [8–13]. Recently, more papers
have reported several types of antitumour agents pos-
sessing the feature structure of sulfonamide [14–24].
Therefore, the sulfonamide moiety appears to be a
crucial functionality to interact with cellular targets.
From this point of view, we have synthesised several
sulfonamide derivatives containing 5-FU and nitro-
gen mustard moiety respectively, in order to get po-
tent antitumour drugs with low toxicity.
As shown in figure 2, compound 2 was prepared
from 5-FU and ethyl acrylate by the method of Ouchi
and Yuyama [25], m.p. 186–187 °C (lit., 194–
195 °C). The structure of 2 was corroborated by the
1H-NMR analyses. Compound 4 was obtained by the
coupling of p-acetamidobenzenesulfonamide (3) with
compound 2 in the presence of DCC and DMAP at
room temperature. The unreacted 3 was removed by
adjusting the pH value of the solution since 3 precipi-
tated completely at pH of 5.5 while most of 4 was still
remained in the solution. Hydrolysis of 4 in aqueous
sodium hydroxide under given conditions afforded
compound 5. The synthetic route of compound 7 was
shown in figure 3. Treatment of p-acetamidobenzene-
sulfonyl chloride with bis(2-chloroethyl) amine led to
compound 6. Subsequent hydrolysis of 6 in hydro-
chloric acid led to 7.
To synthesis our target compound 12b (figure 5), it
is important to choose an appropriate protective
group for the aromatic amine of sulfadiazine because
of its relatively high reactivity. We first selected Cbz
(carbobenzyloxy) as the protective group since it is
relatively stable at acidic or alkaline conditions and
can be easily removed by catalytic hydrogenation.
However, as shown in figure 4, this synthetic route led
to 12a instead of the desired 12b. Compound 8a was
prepared by reacting sulfadiazine with benzyl chloro-
formate. Treatment of 8a with aqueous sodium hy-
droxide led to compound 9a. The reaction of 9a with
2-bromoethanol in DMF afforded 10a. Compound
11a was obtained by the coupling of 10a with CBL
(chlorambucil) in the presence of DCC at room tem-
perature. The hydrogenation of 11a had been investi-
gated under several kinds of reaction conditions using
different hydrogen donors, such as hydrogen gas,
cyclohexene and ammonium formate. Unfortunately,
the pyrimidine ring was hydrogenated before the hy-
drogenation of the Cbz group under all these condi-
tions. When using cyclohexene as hydrogen donor,
the product was mainly 12a. But, when hydrogen gas
or ammonium formate was used as hydrogen donor,
the product was a mixture of 12a and its deprotected
compound. Thus, 12b cannot be prepared by this
method.
Figure 1. 14C labelled sulfadiazine derivatives.
Figure 2. Reagents and conditions: (a) Na (catalytic amount),
ethyl acrylate, ethanol, reflux, 56 h; (b) HCl (1.2 M), reflux, 1
h; (c) 2 (1.0 equivalent), DCC (1.1 equivalent), DMAP (0.1
equivalent), pyridine, r.t., 48 h; (d) NaOH (2.5 M), 70–74 °C,
0.5 h.
Since 12b is our target compound, we had devel-
oped another synthetic route for it, which is shown in
figure 5. In this route, first acetyl was used as the
protective group of the aromatic amine, then 9b and

Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
865
target molecule (12b). Therefore, after the preparation
of 10b, the acetyl group was replaced by the Schiff’s
base, which is unstable under the preceding reaction
conditions, but relatively stable under the subsequent
reaction conditions.
3. Results and discussion
As we mentioned above, the aim of our work is to
get potent antitumour drugs with low toxicity by
combining conventional antitumour drugs and sufon-
amides in one molecule. With this in mind, we had
designed 5 and 7 at first, which contain the simplest
sufonamide structure. The acute toxicity of 5 and 7
were investigated in mice and the results were listed in
table I. As we had expected, 5 showed very low
toxicity that no sign of toxicity was observed even
when the mice were treated with a dose as high as 2 g
kg−1 by the intraperitoneal route.
Unfortunately, the antitumour activity of 5 was
reduced markedly by comparison with the mother
compound 5-FU. The results of in vivo antitumour
activity of 5 and 7 against murine S-180 sarcoma were
listed in table II. The low cytotoxicity of 5 may be
attributed to the high stability of 2 that free 5-FU
cannot be easily released. The LD50 of 7 is 281.28 mg
Figure 3. Reagents and conditions: (a) Et3N (1.2 equivalent),
pyridine, 0–40 °C, 3 h; (b) HCl (6 M), reflux, 40 min.
10b were synthesised according to the synthetic proce-
dures for 9a and 10a, respectively. And the acetyl
group was removed by refluxing 10a in HCl (1.2 M)
for about 1 h. The reaction of 13 with benzaldehyde
afforded Schiff’s base protected compound 14. Com-
pound 11b was prepared according to the procedure
of 11a. Compound 12b was obtained by the hydroly-
sis of 11b in hydrochloric acid at room temperature.
In this route, two different protective groups were
applied, the acetyl and the Schiff’s base. Acetyl as
well as Cbz is stable under all the reaction conditions,
but cannot be removed without destroying the target
molecule. Schiff’s base can be removed under mild
condition that would not break the ester bond in the
Figure 4. Reagents and conditions: (a) benzyl chloroformate, pyridine, 0 °C, 8 h; (b) NaOH (aq., 1.0 equivalent); (c) DMF,
BrCH2CH2OH (1.0 equivalent), 80 °C, 8 h; (d) chlorambucil (1.0 equivalent), DCC (1.1 equivalent), pyridine, r.t., 48 h; (e) CHCl3,
cyclohexene (excessive), 10% PdꢀC, reflux, 18 h.

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Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
mg kg−1) only exhibited 19% inhibition while nor-N-
mustard showed 43.43% inhibition.
Despite the disappointing results, we still believe
that desirable antitumour agents may be obtained.
Thus, we have designed compound 12b, which is
composed of sulfadiazine and CBL (chlorambucil). In
the design of 12b, three points had been considered.
First, instead of the simplest sulfonamide, sufadiazine
was selected as the targeting moiety, which had been
proved to concentrate in the Yoshida sarcoma [2].
Second, sulfadiazine should be connected with CBL
in an appropriate way that both the selectivity of
sulfadiazine toward tumour cells and the antitumour
activity of CBL are reserved. Third, CBL and 14
should be connected by an appropriate bond, so that
free CBL can be released in body, preferably in the
tumour cells.
According to the LD50 values of 12b (130.76 mg
kg−1 ip in mice, 0.225 mmol kg−1) and CBL (49.56 mg
kg−1 ip in mice, 0.163 mmol kg−1), it can be con-
cluded that the acute toxicity of 12b is lower than that
of its mother compound CBL. The results of in vivo
antitumour activity of 12b and CBL against murine
S-180 sarcoma were listed in table III. And the antitu-
mour activity comparison of 12b with CBL was listed
in table IV (at equal toxicity) and table V (at equal
molarity), respectively.
Figure 5. Reagents and conditions: (a) acetyl chloride, pyridine,
The data in tables III and IV indicate that 12b is
more potent than CBL at dose of either equal toxicity
or equal molarity. It is also noted that this effect is
more obvious at relatively low dose range. This effect
may be partly attributed to the targeting action of 12b
that may lead to a relatively high drug concentration
in the tumour cells. Since the active moiety of 12b is
still CBL which demonstrated strong dose–effect rela-
tionship mainly at low dose range as shown in table I,
the inhibition increased by 12b at relatively low dose
is more obvious than that at high dose. However,
compared with sulfadiazine, the concentration effect
of 12b is not obvious as we expected. It may due to
the hydrolysis of the ester bond in body that some
free CBL had already been released before the con-
centration of 12b in tumour cells. The TI (therapeutic
index) of 12b figured out from the data is 47.55,
which is about the twice of CBL’s (TI: 22.84). Thus,
12b is much safer than its mother compound CBL
when used as antitumour agents.
0 °C,
2 h; (b) NaOH (aq., 1.0 equivalent); (c) DMF,
BrCH2CH2OH (1.0 equivalent), 80 °C, 8 h; (d) HCl (1.2 M),
reflux, 1 h; (e) benzaldehyde, 100 °C, 6 h; (f) chlorambucil (1.0
equivalent), DCC (1.1 equiv.), pyridine, r.t., 48 h; (g) HCl (1
M), r.t., 10 h.
Table I. LD50 of the test compounds (ip in mice).
a
Compound
LD50 (mg kg−1
)
5
\2000 b
281.28 (253.45–312.17)
130.76 (118.53–144.28)
49.56 (44.82–54.83)
7
12b
CBL
a PB0.05.
b No sign of toxicity was observed.
kg−1 i.p. in mice, which is less toxic than the highly
toxic N-methyl-mustard (LD50, 1.1 mg kg−1), and
nor-N-mustard (LD50, 100 mg kg−1) [26]. The potent
doses of 7 (inhibition>30%, P<0.05) for the S-180
were 60 and 30 mg kg−1, respectively. However, com-
pared with nor-N-mustard at equal molar dose, 7 (6
The rationale of the accumulation of sulfonamides
in tumours is still not very clear. It was ever thought
to be related to the difference in pH between tumours

Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
867
Table II. In vivo antitumour activity of (5) and (7) against murine S-180 sarcoma.
Drug
Dose
Schedule
Mice
Body weight
In./Fi. (g)
Tumour
WT. X9SD (g)
Inhibition
(%)
P
(mg kg−1
)
In. a/Fi. b
Control
0.2 c
500
100
35
ip×7qd
ip×7qd
ip×7qd
ip×7qd
20/20
10/10
10/10
10/10
19.7/26.5
19.7/24.9
19.9/25.7
19.9/23.9
2.9890.31
1.9790.23
2.3790.31
1.5790.19
–
–
5
5
33.89
20.45
47.32
B0.01
B0.05
B0.01
5-Fu
Control
0.2 c
60
30
6
ip×7qd
ip×7qd
ip×7qd
ip×7qd
20/20
10/10
10/10
10/10
20.3/26.5
20.5/25.1
20.1/25.7
20.3/25.9
2.2190.29
1.1790.23
1.3790.34
1.7990.28
–
–
7
7
7
47.06
38.01
19.00
B0.01
B0.01
B0.05
NNM d
3
ip×7qd
10/10
20.3/24.8
1.2590.31
43.43
B0.01
a Initial stage of experiment.
b Final stage of experiment.
c mL/mouse.
d Nor-nitrogen mustard.
Table III. In vivo antitumour activity of CBL d and (12b) against murine S-180 sarcoma.
Drug
Dose
Schedule
Mice
Body weight
In./Fi. (g)
Tumour
WT. X9SD (g)
Inhibition
(%)
P
(mg kg−1
)
In. a/Fi. b
CBL
CBL
CBL
CBL
12b
6.8
5
2.5
1.3
13
9.6
6.5
4.8
3.3
2.5
0.2 c
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
ip×7qd
10/9
10/9
10/10
10/10
10/9
19.8/18.4
19.7/19.1
20.0/21.3
19.9/23.4
19.8/18.0
19.6/18.3
19.8/19.5
19.8/20.7
19.6/22.5
19.9/23.4
20.1/25.8
0.5090.14
0.5990.16
1.0490.16
1.3990.22
0.4190.14
0.5190.11
0.5790.10
0.8190.21
1.0990.20
1.1590.23
2.2190.22
77.38
73.30
52.94
37.10
81.45
76.92
74.21
63.35
50.68
47.96
B0.01
B0.01
B0.01
B0.01
B0.01
B0.01
B0.01
B0.01
B0.01
B0.01
12b
10/9
12b
10/10
10/10
10/10
10/10
20/20
12b
12b
12b
Control
a Initial stage of experiment.
b Final stage of experiment.
c mL/mouse.
d Chlorambucil.
and the normal tissue [3]. It is well known, the sulfon-
amide group of sulfadiazine is on acid, it can react
with sodium hydroxide to form the salt. On the other
hand, the aromatic amine of sulfadiazine is on basic,
it is easy to react with acidic compound. As we
mentioned above, the early unsuccessful investiga-
tions about the sulfadiazine all focused on the modifi-
cation of the aromatic amino group [3–7], the basic
aromatic amine was reacted and the acidic sulfon-
amide was remained, it led to the loss of selectivity
toward tumour cells. In our continuous study of this
kind of drugs, it was noticed that the aromatic amino
group of sulfadiazine should be kept unsubstituted or
the selectivity will be lost [8, 11]. It may be explained
by this way that the tumour cells on acid show
selective affinity toward the basic aromatic amine
rather than the acidic sulfonamide.
Table IV. Antitumour activity comparison of 12b with CBL at
dose of equal toxicity.
Drug
LD50×1/10
(%)
LD50×1/20
(%)
LD50×1/50
(%)
12b
CBL
81.5
73.3
74.2
52.9
50.7
37.1

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Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
Table V. Antitumour activity comparison of 12b with CBL at dose of equal molarity.
Drug
0.022 mmol kg−1 (%)
0.016 mmol kg−1 (%)
0.008 mmol kg−1 (%)
0.004 mmol kg−1 (%)
12b
CBL
81.5
77.4
76.9
73.3
63.4
52.9
47.9
37.1
4. Conclusions
and DMAP (1.23 g, 0.01 mol). The mixture was stirred
under nitrogen atmosphere at room temperature (r.t.) for
48 h. Glacial acetic acid (5 mL) was then added to
destroy the excessive DCC and the precipitate was
filtered off. The filtrate was concentrate to 1/6 of its
original volume, then poured into 250 mL water with
vigorous stirring. The crude product obtained by suction
filtration was dissolved in dilute aq. NaOH, and the pH
value of the solution was adjusted to about 5.5 by dilute
HCl. Then the precipitate was filtered off, and the pH
value of the filtrate was adjusted to about 1.5. The white
precipitate was collected and rinsed with water for sev-
eral times, then dried to constant weight. The yield was
53.9%. m.p. 252–253 °C; IR wmax (KBr, cm−1) 3429
In conclusion, we have synthesised three new com-
pounds (5, 7, 12b) by combining sulfonamides and
conventional anticancer agents in one molecule. In-
stead of 12b, 12a was obtained when using carboben-
zyloxy as protective group. Compound 12b
demonstrates high antitumour activity and is more
potent and safer than its mother compound CBL. The
concentration of sulfonamides in tumour cells may be
related to the basicity of the aromatic amino group
and the acidity of the tumour cells. This class of
targeting agents may be further developed to form
candidate drugs, which may have advantages over the
currently available anticancer agents.
1
(NH), 1707 (CꢁO), 1593, 1538, 1479 (phenyl); H-NMR
(300 MHz, DMSO-d6) l 2.09 (s, 3H, CH3CꢁO), 2.63 (t,
2H, J=6.6 Hz, NCH2CH2CONH), 3.73 (t, 2H, J=6.6
Hz, NCH2CH2CONH), 7.78 (d, 2H, J=9 Hz, phenyl),
7.83 (d, 2H, J=9 Hz, phenyl), 7.86 (d, 1H, J=6.9 Hz,
6-H of 5-Fu), 10.42 (s, 1H, CH3CONH), 11.78 (d, 1H,
J=5.1 Hz, CONH of 5-Fu), 12.18 (s, 1H, SO2NH); MS
(EI) m/z (RI) 399 ([M+H]+, 3), 257 (58), 198 (56), 130
(43), 109 (100), 43 (94). Anal. C15H15FN4O6S (C, H, N,
S).
5. Experimental
5.1. Chemistry
Melting points were determined with a Thiele appara-
tus in open capillary tubes and are uncorrected. IR
spectra were measured on a Magna-550 FTIR spectrom-
1
eter. H-NMR spectra were recorded on a Bruker MSL-
300 spectrometer with tetramethylsilane (TMS) as the
internal standard and are reported as parts-per-million
(ppm). The following abbreviations are used: singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), broad
(br), relative intensity (RI). Mass spectra were taken with
HP5989A. Elemental analyses for C, H, N and S were
determined on a Perkin–Elmer 2400 Series II. Analysis
indicated by the symbols of the elements were within
0.4% of the theoretical values. Analytical thin layer
chromatography was performed on precoated silica gel
plates (GF-254). Sulfadiazine (SD) was purchased from
Shanghai Sanwei Pharmaceutical Ltd. Co. and used
directly. Solvents and reagents were purified by standard
methods as necessary.
5.1.2. 1-(3-(4-Aminobenzenesulfonamido)-3-
oxopropyl)-5-fluoropyrimidine-2,4-dione (5)
The mixture of 4 (10 g, 25 mmol) and aq. sodium
hydroxide (2.5 M, 100 mL) in a 250-mL round-bottom
flask was stirred at 70–74 °C for 30 min. After the
solution was cooled to the r.t., the pH value of it was
adjusted to 3.5–4.0 by HCl. The precipitate was collected
and rinsed with water for several times. The dry product
was obtained in 78% yield. m.p. 237–238 °C; IR wmax
(KBr, cm−1) 3459, 3360 (NH2), 1722, 1692 (CꢁO), 1593
1
(phenyl); H-NMR (300 MHz, DMSO-d6) l 2.58 (t, 2H,
J=6.6 Hz, NCH2CH2CONH), 3.73 (t, 2H, J=6.6 Hz,
NCH2CH2CONH), 6.16 (s, 2H, NH2), 6.60(d, 2H, J=9
Hz, phenyl), 7.52 (d, 2H, J=9 Hz, phenyl), 7.86 (d, 1H,
J=6.6 Hz, 6-H of 5-Fu), 11.76 (d, 1H, J=5.1 Hz,
CONH of 5-Fu), 11.81 (s, 1H, SO2NH); MS (EI) m/z
(RI) 356 [M+, 7], 156 (78), 130 (40), 109 (100), 100 (87),
44 (48). Anal. C13H13FN4O5S (C, H, N, S).
5.1.1. 1-(3-(4-Acetylaminobenzenesulfonamido)-3-
oxopropyl)-5-fluoropyrimidine-2,4-dione (4)
To a solution of 2 (20.2 g, 0.1 mol) and 3 (21.4 g, 0.1
mol) in DMF (180 mL), was added DCC (22 g, 0.11 mol)

Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
869
5.1.3. 4-Acetylamino-N,N-bis(2-chloroethyl)-
benzenesulfonamide (6)
7.04 (t, 1H, J=4.8 Hz, pyrimidinyl), 7.39 (m, 5H,
phenyl), 7.62 (d, 2H, J=7.2 Hz, phenyl), 7.90 (d, 2H,
J=7.2 Hz, phenyl), 8.50 (d, 2H, J=4.8 Hz, pyrim-
idinyl), 10.26 (s, 1H, ArNHCO), 11.69 (br s, 1H,
SO2NH); MS (EI) m/z (RI) 385 ([M+H]+, 8), 319 (82),
211 (100), 185 (29), 108 (12), 91 (77), 79 (14). Anal.
C18H16N4O4S (C, H, N, S).
The solution of triethylamine (9.6 mL, 82.7 mmol) in
pyridine (65 mL) was added dropwise into bis(2-
chloroethyl) amine hydrochloride (12.44 g, 69.7 mmol)
at 0 °C with stirring. Then p-acetamidobenzenesulfonyl
chloride (17.9 g, 76.7 mmol) was added to the mixture
by small portions to keep the reaction temperature not
higher than 15 °C. After the addition, the mixture was
stirred at 38–42 °C for 3 h, then poured into 550 mL
water with stirring. The filter cake was washed with
water, and dried in vacuo. White crystal was obtained
by recrystallisation from toluene in 85% yield. m.p.
116–118 °C; IR wmax (KBr, cm−1) 3306 (NH2), 1677
(CꢁO), 1598, 1546 (phenyl), 1335 (SO2N); 1H-NMR
(300 MHz, DMSO-d6) l 2.23 (s, 3H, CH3CO), 3.47 (t,
4H, J=6.6 Hz, NCH2CH2Cl), 3.68 (t, 4H, J=6.6 Hz,
NCH2CH2Cl), 7.70 (d, 2H, J=5.7 Hz, phenyl), 7.74 (s,
1H, CONH), 7.72 (d, 2H, J=5.7 Hz, phenyl); MS (EI)
m/z (RI) 339 [M+, 22], 289 (64), 198 (100), 134 (27), 43
(30). Anal. C12H16Cl2N2O3S (C, H, N, S).
5.1.6. N1-2-Pyrimidyl-(p-benzyloxycarbonyl)amino-
benzenesulfonamide sodium salt (9a)
To an aq. solution of sodium hydroxide (0.2 g in 200
mL), 8a (19.2 g, 0.05 mol) was added, the solution was
stirred and gently warmed until all of 8a was dissolved
and the pH of the solution dropped to about 8. After
filtration the filtrate was condensed by rotary evapora-
tion to a paste. The residue was purified by recrystallisa-
tion in 95% EtOH, and 9a (17.3 g, 85%) was obtained as
a white solid. IR wmax (KBr, cm−1) 1701 (CꢁO), 1603
(phenyl), 1532 (phenyl), 1236 (SO2N); 1H-NMR (300
MHz, DMSO-d6) l 5.17 (s, 2H, ArCH2O), 6.39 (t, 1H,
J=4.8 Hz, pyrimidinyl), 7.39 (m, 5H, phenyl), 7.62 (d,
2H, J=7.2 Hz, phenyl), 7.90 (d, 2H, J=7.2 Hz,
phenyl), 8.50 (d, 2H, J=4.8 Hz, pyrimidinyl), 10.26 (s,
1H, ArNHCO).
5.1.4. 4-Amino-N,N-bis(2-chloroethyl)benzene-
sulfonamide hydrochloride (7)
The mixture of 6 (10 g, 29.5 mmol) and hydrochloric
acid (6 M, 100 mL) was refluxed for 40 min. Then it was
cooled to r.t. and kept for 10 h. The crystal was
obtained in 92% yield. IR wmax (KBr, cm−1) 3380, 2618
5.1.7. N1-2-Pyrimidyl-(p-acetyl)aminobenzene-
sulfonamide sodium salt (9b)
According to the procedure for the synthesis of 9a, 9b
was prepared in 80% yield. IR wmax (KBr, cm−1) 3361
(NH), 1665 (CꢁO), 1588, 1532, 1445 (phenyl), 1236
1
(ArNH3Cl), 1593, 1498 (phenyl), 1341(SO2N); H-NMR
(300 MHz, DMSO-d6) l 3.39 (t, 4H, J=6.9 Hz,
NCH2CH2Cl), 3.70 (t, 4H, J=6.9 Hz, NCH2CH2Cl),
5.83 (br, s, 3H, NH3), 6.88 (d, 2H, J=8.7 Hz, phenyl),
7.59 (d, 2H, J=8.7 Hz, phenyl); MS (EI) m/z (RI) 296
[M−HCl, 8], 247 (26), 156 (100), 108 (27), 92 (36), 65
(19). Anal. C10H15Cl3N2O2S (C, H, N, S).
1
(SO2N); H-NMR (300 MHz, DMSO-d6) l 2.04 (s, 3H,
CH3CꢁO), 6.37 (t, 1H, J=4.8 Hz, pyrimidinyl), 7.52 (d,
2H, J=8.7 Hz, phenyl), 7.68 (d, 2H, J=8.7 Hz,
phenyl), 8.08 (d, 2H, J=4.8 Hz, pyrimidyl), 10.05 (s,
1H, ArNHCꢁO).
5.1.5. N1-2-Pyrimidyl-(p-benzyloxycarbonyl)amino-
benzenesulfonamide (8a)
5.1.8. 2-[N1-2-Pyrimidinyl-(p-benzyloxycarbonyl)amino-
benzenesulfonamido] ethanol (10a)
To a stirred solution of sulfadiazine (25 g, 0.1 mol) in
pyridine (300 mL) was added dropwise benzyl chloro-
formate (20.5 g, 0.12 mol) at 0–5 °C. The reaction
mixture was stirred for 8 h at r.t., then the solution was
concentrated to about 40 mL and poured into excessive
water. The precipitate was washed with 1 M HCl and
water successively, then dried to constant weight. Re-
crystallisation from acetonitrile yielded the title com-
pound 8a (32.6 g, 85%) as a white solid. m.p.
219–220 °C; IR wmax (KBr, cm−1) 3399 (NH), 1732
To a solution of 9a (4.07 g, 10 mmol) in DMF (30
mL), was added 2-bromoethanol (1.25 g, 10 mmol).
Then the solution was heated to 80 °C with stirring and
maintained at this temperature for 8 h. The reaction
mixture was concentrated to about 10 mL and poured
into 200 mL water. Recrystallisation of the precipitate
from acetonitrile yielded 10a (2.1 g, 43%) as a white
solid. m.p. 200–201 °C; IR wmax (KBr, cm−1) 3489
(OH), 3305 (NH), 1737 (CꢁO), 1607, 1588, 1503
1
(phenyl), 1206 (SO2N); H-NMR (300 MHz, DMSO-d6)
1
(CꢁO), 1588 (phenyl), 1507 (phenyl), 1225 (SO2N); H-
NMR (300 MHz, DMSO-d6) l 5.17 (s, 2H, ArCH2O),
l 3.73 (t, 2H, J=4.5 Hz, CH2CH2OH), 4.12 (t, 2H,
J=4.5 Hz, CH2CH2OH), 5.04 (t, 1H, J=4.5 Hz,

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Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
CH2CH2OH), 5.16 (s, 2H, ArCH2O), 6.80 (dd, 1H,
J=2.4 Hz, J=4.2 Hz, pyrimidinyl), 7.39 (m, 5H,
phenyl), 7.53 (d, 2H, J=9 Hz, phenyl), 7.70 (d, 2H,
J=9 Hz, phenyl), 8.27 (dd, 1H, J=2.4 Hz, J=4.2 Hz,
pyrimidinyl), 8.59 (q, 1H, J=2.4 Hz, pyrimidinyl),
10.10 (s, 1H, ArNHCO); MS (EI) m/z (RI) 429 ([M+
H]+, 1), 333 (17), 108 (82), 91 (100), 79 (77). Anal.
C20H20N4O5S (C, H, N, S).
idyl), 9.08 (s, 1H, ArNHCꢁO). Anal. C34H37Cl2N5O6S
(C, H, N, S).
5.1.11. 2-[N1-2-(1,4,5,6-Tetrahydropyrimidyl)-
(p-benzyloxycarbonyl) aminobenzenesulfonamido] ethyl
4-bis(2-chloroethyl) aminophenyl butyrate (12a)
To a stirred suspension of 11a (2 g, 2.8 mmol) and
10% PdꢀC (2 g) in chloroform (60 mL), cyclohexene (5
mL) was added under nitrogen. The reaction mixture
was refluxed for 18 h. Then the catalyst was removed by
filtration. The filtrate was concentrated under reduced
pressure, and the residue was purified by column chro-
matography over silica gel (dichloromethane–chloro-
form, 1:1) to give 12a (1.8 g, 90%) as a yellow solid.
5.1.9. 2-[N1-2-Pyrimidinyl-(p-acetyl)aminobenzene-
sulfonamido] ethanol (10b)
According to the procedure for the synthesis of 10a,
10b was prepared in the yield of 41%. White solid, m.p.
239–240 °C; IR wmax (KBr, cm−1) 3379 (br, OH, NH),
1697 (CꢁO), 1587, 1545, 1514 (phenyl), 1263 (SO2N);
1
m.p. 96–98 °C; H-NMR (300 MHz, acetone-d6) l 1.69
1H-NMR (300 MHz, DMSO-d6)
l 2.08 (s, 3H,
(m, 2H, CH2CH2CH2), 1.87 (m, 2H, CH2CH2CH2), 2.31
(t, 2H, J=7.2 Hz, OꢁCCH2CH2CH2), 2.56 (t, 2H,
J=7.2 Hz, CH2CH2CH2Ph), 2.87 (m, 4H, NCH2CH2),
3.62 (t, 2H, J=2.4 Hz, NCH2CH2O), 3.78 (m, 8H,
N(CH2CH2Cl)2), 4.22 (t, 2H, J=2.4 Hz, NCH2CH2O),
5.25 (s, 2H, PhCH2O), 6.77 (d, 2H, J=7.5 Hz, phenyl),
7.12 (d, 2H, J=7.5 Hz, phenyl), 7.44 (m, 5H, phenyl),
7.75 (d, 2H, J=7.2 Hz, phenyl), 7.79 (s, 1H, NH), 7.83
(dd, 2H, J=7.2 Hz, phenyl), 9.14 (s, 1H, ArNHCꢁO);
MS (EI) m/z (RI) 718 [M+, 4], 305 (9), 303 (13), 256
(37), 254 (100), 118 (20), 91 (22), 79 (19). Anal.
C34H41Cl2N5O6S (C, H, N, S).
CH3CꢁO), 3.75 (t, 2H, J=4.5 Hz, CH2CH2OH), 4.12 (t,
2H, J=4.5 Hz, CH2CH2OH), 5.05 (t, 1H, J=4.8 Hz,
CH2CH2OH), 6.80 (dd, 1H, J=2.4 Hz, J=4.2 Hz,
pyrimidinyl), 7.64 (d, 2H, J=7.2 Hz, phenyl), 7.78 (d,
2H, J=7.2 Hz, phenyl), 8.29 (dd, 1H, J=2.4 Hz,
J=4.2 Hz, pyrimidyl), 8.60 (q, 1H, J=2.4 Hz, pyrim-
idyl), 10.19 (s, 1H, ArNHCꢁO); MS (EI) m/z (RI) 337
([M+H]+, 23), 214 (29), 198 (78), 172 (96), 156 (71), 110
(100), 97 (72), 43 (95). Anal. C14H16N4O4S (C, H, N, S).
5.1.10. 2-[N1-2-Pyrimidyl-(p-benzyloxycarbonyl)
aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl)
aminophenyl butyrate (11a)
To a stirred solution of chlorambucil (0.3 g, 1 mmol)
and 10a (0.43 g, 1 mmol) in anhydrous pyridine (5 mL)
was added DCC (0.22 g, 1.05 mmol) and catalytic
amount of DMAP. The reaction was maintained at r.t.
for 24 h before the precipitate was filtered off. Then the
filtrate was poured into excessive water (80 mL). The
precipitate was purified by recrystallization from ace-
tone–ethanol (1:3/v:v). 11a (0.46 g, 65%) was obtained
5.1.12. 2-(N1-2-Pyrimidinyl-aminobenzenesulfonamido)
ethanol (13)
In a 150 mL round-bottom flask provided with reflux
condenser, a mixture of 10b (10 g, 30 mmol) and
hydrochloric acid (1.2 M, 60 mL) was refluxed for about
1 h. When the solution obtained was cooled to r.t., aq.
NaOH was added to neutralise the solution. The
precipitate was isolated and washed with water, then
dried in vacuo to yield 13 (7.8 g, 88%) as an orange
solid. m.p. 224–226 °C; IR wmax (KBr, cm−1) 3448
(OH), 3351, 3249 (NH2), 1599, 1507, (phenyl), 1241
(SO2N); lH (300 MHz, DMSO-d6) 3.73 (t, 2H, J=4.5
Hz, CH2CH2OH), 4.07 (t, 2H, J=4.5 Hz, CH2CH2OH),
5.02 (t, 1H, J=5.4 Hz, CH2CH2OH), 5.66 (br s, 2H,
NH2), 6.51 (d, 2H, J=8.7 Hz, phenyl), 6.73 (dd, 1H,
J=2.4 Hz, J=4.2 Hz, pyrimidinyl), 7.51 (d, 2H, J=8.7
Hz, phenyl), 8.23 (dd, 1H, J=2.4 Hz, J=4.2 Hz,
pyrimidyl), 8.58 (q, 1H, J=2.4 Hz, pyrimidyl); MS (EI)
m/z (RI) 295 ([M+H]+, 15), 172 (60), 156 (100), 108
(29), 108 (83), 92 (81), 65 (68). Anal. C12H14N4O3S (C,
H, N, S).
as a white solid. m.p. 173–174 °C; IR wmax (KBr, cm−1
)
3348 (NH), 1735 (CꢁO), 1593, 1546, 1514 (phenyl), 1272
(SO2N); 1H-NMR (300 MHz, acetone-d6) l 1.85 (m,
2H, CH2CH2CH2), 2.32 (t, 2H, J=7.2 Hz,
OꢁCCH2CH2CH2), 2.51 (t, 2H, J=7.2 Hz, CH2CH2-
CH2Ph), 3.81 (m, 8H, N(CH2CH2Cl)2), 4.47 (t, 2H,
J=2.4 Hz, NCH2CH2O), 4.55 (t, 2H, J=2.4 Hz,
NCH2CH2O), 5.23 (s, 2H, PhCH2O), 6.76 (d, 2H, J=
8.7 Hz, phenyl), 6.79 (dd, 1H, J=2.4 Hz, J=4.2 Hz,
pyrimidinyl), 7.06 (d, 2H, J=8.7 Hz, phenyl), 7.46 (m,
5H, phenyl), 7.68 (d, 2H, J=6.6 Hz, phenyl), 7.95 (d,
2H, J=6.6 Hz, phenyl), 8.34 (dd, 1H, J=2.4 Hz,
J=4.2 Hz, pyrimidyl), 8.59 (q, 1H, J=2.4 Hz, pyrim-

Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
871
1
5.1.13. 2-[N1-2-Pyrimidinyl-(p-benzylidene)amino-
benzenesulfonamido] ethanol (14)
1272 (SO2N); H-NMR (300 MHz, CDCl3) l 1.81 (m,
2H, CH2CH2CH2), 2.23 (t, 2H, J=7.2 Hz,
OꢁCCH2CH2CH2), 2.53 (t, 2H, J=7.2 Hz,
To 40 mL freshly distilled benzaldehyde in 100 mL
round-bottom flask, was added 13 (10 g, 34 mmol).
Then the mixture was heated to about 100 °C, and
maintained at this temperature under nitrogen atmo-
sphere for about 6 h until the mixture became a clear
solution. The solution was cooled to r.t., and kept at
refrigerator for 3 h. The crude product collected by
suction filter was washed with small portion of cold
EtOH for twice, then dried in vacuo to give 14 (10.8 g,
83%) as a yellow solid. m.p. 186–187 °C; IR wmax (KBr,
cm−1) 3428 (OH), 1624 (CHꢁNAr), 1599, 1547, 1512
CH2CH2CH2Ph),
N(CH2CH2Cl)2),
3.60
3.70
(t,
(t,
4H,
4H,
J=6.0
J=6.0
Hz,
Hz,
N(CH2CH2Cl)2), 4.25 (t, 2H, J=4.5 Hz, NCH2CH2O),
4.43 (t, 2H, J=4.5 Hz, NCH2CH2O), 5.64 (br s, 2H,
NH2), 6.46 (dd, 1H, J=2.4 Hz, J=4.2 Hz, pyrim-
idinyl), 6.64 (dd, 4H, J=8.4 Hz, J=2.4 Hz, phenyl),
7.00 (d, 2H, J=6.9 Hz, phenyl), 7.66 (dd, 1H, J=2.4
Hz, J=4.2 Hz, pyrimidyl),7.81 (d, 2H, J=6.9 Hz,
phenyl), 8.58 (q, 1H, J=2.4 Hz, pyrimidyl); 13C-NMR
(75 MHz, CDCl3) l 26.27, 33.07, 33.71, 40.56, 52.93,
53.51, 60.14, 106.85, 112.19, 113.51, 129.28, 129.57,
130.11, 131.68, 144.38, 149.57, 154.06, 164.08, 172.71.
Anal. C26H31Cl2N5O4S (C, H, N, S).
1
(phenyl), 1266 (SO2N); H-NMR (300 MHz, DMSO-d6)
l 3.77 (t, 2H, CH2CH2OH), 4.15 (t, 2H, CH2CH2OH),
5.06 (t, 1H, CH2CH2OH), 6.84 (dd, 1H, pyrimidinyl),
7.30 (d, 2H, phenyl), 7.58 (m, 3H, phenyl), 7.89 (d, 2H,
phenyl), 7.95 (dd, 2H, phenyl), 8.32 (dd, 1H, pyrimidyl),
8.61 (dd, 1H, pyrimidyl), 8.63 (s, 1H, ArCHꢁN); MS
(EI) m/z (RI) 382 [M+, 6], 348 (19), 279 (46), 260 (100),
244 (38), 196 (82), 180 (85), 152 (55), 109 (31). Anal.
C19H18N4O3S (C, H, N, S).
5.2. Biological studies
5.2.1. Animal studies
TA1 mice (propagated at the animal supply centre of
the Shanghai Institute of Pharmaceutical Industry), 6–8
weeks age, weighing 18–20 g of either sex were used.
They were maintained under controlled temperature and
humidity with sterile bedding and food and water ad
libitum.
5.1.14. 2-[N1-2-Pyrimidinyl-(p-benzylidene)amino-
benzenesulfonamido] ethyl 4-bis(2-chloroethyl)
aminophenyl butyrate (11b)
According to the procedure for the synthesis of 11a,
11b was prepared in the yield of 65%. m.p. 141–142 °C;
1H-NMR (300 MHz, acetone-d6) l 1.83 (m, 2H,
CH2CH2CH2), 2.32 (t, 2H, J=7.2 Hz, OꢁCCH2-
CH2CH2), 2.50 (t, 2H, J=7.2 Hz, CH2CH2CH2Ph),
3.80 (m, 8H, N(CH2CH2Cl)2), 4.51 (t, 2H, J=2.1 Hz,
NCH2CH2O), 4.59 (t, 2H, J=2.1 Hz, NCH2CH2O),
6.74 (d, 2H, J=8.7 Hz, phenyl), 6.79 (dd, 1H, J=2.4
Hz, J=4.2 Hz, pyrimidinyl), 7.04 (d, 2H, J=8.7 Hz,
phenyl), 7.32 (d, 2H, J=8.7 Hz, phenyl), 7.59 (m, 3H,
phenyl), 8.02 (d, 2H, J=3 Hz, phenyl), 8.07 (d, 2H,
J=3 Hz, phenyl), 8.38 (dd, 1H, J=2.4 Hz, J=4.2 Hz,
pyrimidyl), 8.60 (s, 1H, ArCHꢁNAr), 8.62 (q, 1H, J=
2.4 Hz, pyrimidyl). Anal. C33H35Cl2N5O4S (C, H, N, S).
5.2.2. Toxicity assessment
For the assessment of the acute toxicity, compounds
were injected ip×1 into the TA1 mice at five different
dose levels on day 0. Then the behaviour and the death
distribution of the test mice were recorded. The highest
death rate appeared on day 1 and the condition of the
survivals was good after 2 weeks. LD50 was calculated
by using the Bliss method.
5.2.3. In vivo antitumour activity
The mouse solid tumour S-180 cell line was main-
tained by intraperitoneal passage at weekly intervals in
male TA1 mice. In experiments with solid S-180 cells,
1×106 cells in 0.2 mL were injected intraperitoneal into
the right axilla of TA1 mice (10 mice each group) on
day 0. The test compound was suspended in 0.5%
sodium carboxymethyl cellulose containing 3.5% Tween
80 and was administered intraperitoneal with a consecu-
tive (qd×7) schedule at various doses from day 1.
Control mice were given saline alone with the same
schedule. The mice were weighed twice a week to moni-
tor the toxic effects. The mice were killed on day 14, and
5.1.15. 2-[N1-2-Pyrimidyl-aminobenzenesulfonamido]
ethyl 4-bis(2-chloroethyl) aminophenyl butyrate (12b)
A mixture of hydrochloric acid (1 M, 10 mL) and 11b
(1 g) was stirred at r.t. for 10 h, then was neutralised by
aq. sodium hydroxide (1 M). The precipitate was sepa-
rated by filtration and rinsed with water, then dried in
vacuo. Compound 12b (0.43 g, 53%) was obtained as a
white solid. m.p. 97–98 °C; IR wmax (KBr, cm−1) 3469,
3348 (NH2), 1735 (CꢁO), 1593, 1546, 1514 (phenyl),

872
Z. Huang et al. / European Journal of Medicinal Chemistry 36 (2001) 863–872
the tumours were removed and weighed. The antitu-
mour activity was evaluated as the percentage of the
tumour weight inhibition (TWI) compared with the
mean tumour weight in the control group, according to
the following formula.
[9] Chen S., Huang Z.H., Huang J.L., Eur. Polym. J. 36 (2000)
1703–1710.
[10] Huang J.L., Wang H.Y., Tian X.Y., J. Polym. Sci. Part A:
Polym. Chem. 34 (1996) 1933–1940.
[11] Huang J.L., Chen S., Tan L.S., Wang H.Y., Sci. China (Series
B) 41 (1998) 54–57.
[12] Huang Z.H., Chen S., Huang J.L., J. Appl. Polym. Sci. 73
TWI%=(1−T/C)×100%
(1999) 1379–1385.
[13] Chen S., Studies on Synthesis and Properties of Polymeric
Targeting Antitumor Medicines Containing 5-FU and Nitrogen
Mustard., Ph.D. dissertation Fudan University, Shanghai,
1998.
T and C represent the mean tumour weight in the test
group and the control group, respectively.
[14] Supuran C.T., Scozzafava A., Eur. J. Med. Chem. 35 (2000)
5.2.4. Statistical analyses
867–874.
Results are expressed as the mean SD. The signifi-
cance of difference between groups and/or drugs was
assessed by using Student’s t-test. P<0.05 was taken as
significant.
[15] Scozzafava A., Supuran C.T., Bioorg. Med. Chem. Lett. 10
(2000) 1117–1120.
[16] Supuran C.T., Briganti F., Tilli S., et al., Bioorg. Med. Chem.
Lett. 9 (2001) 703–714.
[17] Owa T., Okauchi T., Yoshimatsu K., et al., Bioorg. Med.
Chem. Lett. 10 (2000) 1223–1226.
[18] Supuran C.T., Scozzafava A., J. Enzyme Inhib. 15 (2000)
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