2-[N1-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate: A potent antitumor agent

Article abstract of DOI:10.1016/S0960-894X(01)00157-3

2-[N¹-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate has been prepared by reaction of chlorambucil with sulfadiazine derivative. Schiff's base has been used as the protective group of the aromatic amine in the synthesis. It can be completely removed by the irradiation of 365 nm UV light at room temperature. The title compound exhibits a high antitumor activity with a therapeutic index (TI) of 47.55 which is twice that of chlorambucil's (TI: 22.84).

Full text of DOI:10.1016/S0960-894X(01)00157-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1099–1103
2-[N1-2-Pyrimidyl-aminobenzenesulfonamido]
Ethyl 4-Bis(2-chloroethyl) Aminophenyl Butyrate:
A Potent Antitumor Agent
Zhaohua Huang,a Genjin Yang,b Zhaoliang Lina and Junlian Huanga,*
aThe Key Laboratory of Molecular Engineering of Polymers, Education Ministry of China,
Department of Macromolecular Science, Fudan University, Shanghai 200433, China
bSchool of Pharmacy, The Second Military Medical University, Shanghai 200433, China
Received 6 December 2000; accepted 20 January 2001
Abstract—2-[N1-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate has been prepared by
reaction of chlorambucil with sulfadiazine derivative. Schiff’s base has been used as the protective group of the aromatic amine in
the synthesis. It can be completely removed by the irradiation of 365 nm UV light at room temperature. The title compound exhi-
bits a high antitumor activity with a therapeutic index (TI) of 47.55 which is twice that of chlorambucil’s (TI: 22.84). # 2001
Elsevier Science Ltd. All rights reserved.
In the long history of drug discovery, an interesting
phenomenon has been noted that compounds with the
same structural feature show diverse biological activ-
ities. For instance, the sulfonamides with different
substituted groups display a wide variety of pharmaco-
logical activities such as antibacterial, insulin-releasing
antidiabetic, carbonic anhydrase inhibitory, high-ceiling
diuretic, and antithyroid.1 Recently more papers have
reported several kinds of antitumor agents possessing
the feature structure of sulfonamide.2ꢀ5 Itis well known
that sulfadiazine is a useful antibacterial drug with the
typical sulfonamide structure. It has been shown to
concentrate selectively in the Yoshida sarcoma.6 This
finding has aroused considerable interest and great
efforts have been made to design new antitumor agents
by combining sulfadiazine and antitumor agents in one
compound. Generally, there are two reactive sites for
the modification of sulfadiazine, one is the aromatic
amine, the other is sulfonamide. Heretofore, the main
work of modified sulfadiazine has been focused on the
aromatic amine due to its relatively high reaction
activity.7ꢀ11
all these compounds lost selectivity for the tumors that
concentrate sulfadiazine. Further work in our group
shows that if the polymers, such as polyethylene oxide,
were used as matrix, and the sulfadiazine and antitumor
drugs were fastened on its two ends, then the selectivity
was recovered, although the mechanism of selectivity is
still unclear.12ꢀ16 Inspired by these results, we believe
that more potent antitumor agents may be prepared by
combining sulfadiazine and a conventional antitumor
agentsuch as chlorambucil (CBL) in one molecule
through an appropriate way. Thus, we have designed
and synthesized compound 12, which is the combination
of sulfadiazine and CBL.
In the design of 12 (Scheme 3), we considered three
factors. First, instead of the aromatic amine, sulfona-
mide has been chosen as the reaction site for modifica-
tion since those modified on the aromatic amine lost
selectivity toward tumor cells. Second, sulfadiazine
should be connected with CBL in an appropriate way
such that both the selectivity of sulfadiazine toward
tumor cells and the antitumor activity of CBL are not
destroyed. Third, CBL and 10 were connected by an ester
bond, so that free CBL can be released in the body.
As shown in Scheme 1, 14C labeled sulfadiazine mustard
has been synthesized by Nguyen,10 and sulfadiazine-
acrylamide and its polymer by Abel et al.11 Unfortunately,
To prepare 12, 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 acidic or alkaline conditions and can be
*Corresponding author. Tel.:+86-21-65643049; fax: +86-21-656-
40293; e-mail: jhuang@fudan.edu.cn
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00157-3

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Z. Huang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1099–1103
easily removed by catalytic hydrogenation. However, as
shown in Scheme 2, this synthetic route led to 5 instead
of the desired 12. Compound 1 was prepared by react-
ing sulfadiazine with benzyl chloroformate. Treatment
of 1 with aqueous sodium hydroxide led to compound
2. The reaction of 2 with 2-bromoethanol in DMF
afforded 3. Compound 4 was obtained by the coupling
of 3 with CBL in the presence of DCC at room tem-
perature. The hydrogenation of 4 was investigated
under several kinds of reaction conditions using differ-
enthydrogen donors, such as hydrogen gas, cyclohex-
ene and ammonium formate. Unfortunately, the
pyrimidine ring was hydrogenated before the hydro-
genation of the Cbz group under all these conditions.
When using cyclohexene as hydrogen donor, the pro-
ductwas mainly 5. But, when hydrogen gas or ammo-
nium formate was used as hydrogen donor, the product
was a mixture of 5 and its deprotected compound. Thus,
the target molecule 12 cannotbe prepared by htis
method.
Since 12 is our target compound, we developed another
synthetic route for it, which is shown in Scheme 3. In this
route, firstly acetyl was used as the protective group of the
aromatic amine, then 7 and 8 were synthesized according
Scheme 1. 14C labeled sulfadiazine derivatives.
Scheme 2. Reagents and conditions: (a) benzyl chloroformate, pyridine, 0 ꢁC, 8 h; (b) NaOH (aq, 1.0 equiv); (c) DMF, BrCH2CH2OH (1.0 equiv),
80 ꢁC, 8 h; (d) chlorambucil (1.0 equiv), DCC (1.1 equiv), pyridine, rt, 48 h; (e) CHCl3, cyclohexene (excessive), 10% Pd–C, reflux, 18 h.

Z. Huang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1099–1103
1101
to the synthetic procedures for 2 and 3, respectively.
And the acetyl group was removed by refluxing 8 in HCl
(1.2 M) for about1 h. The reaction of 9 with benzalde-
hyde afforded Schiff’s base protected compound 10.
Compound 11 was prepared according to the procedure
of 4. Irradiation of 11 with 365 nm UV light at rt
afforded 12 in high yield (98%), which exhibits great
advantage over the conventional deprotection method.
Compound 12 was obtained only in 43% yield when 11
was hydrolyzed in dilute hydrochloric acid. In this
route, two different protective groups had been applied,
the acetyl and the Schiff’s base. Acetyl as well as Cbz is
stable under all the reaction conditions, but can not be
removed without destroying the target molecule. Schiff’s
base can be easily removed by the irradiation of UV
light that won’t destroy the target molecule (12).
Therefore, after the preparation of 8, the acetyl group
was replaced by the Schiff’s base, which is unstable in
the preceding reactions, but stable in the following
reactions. All the new compounds have been fully
characterized by IR, NMR, MS and elemental analysis.17
TA1 mice (propagated at the animal supply center of
the Shanghai Institute of Pharmaceutical Industry), 6–8
weeks age, weighing 18–20 g of either sex were used for
the biological assay. They were maintained under con-
trolled temperature and humidity with sterile bedding
and food and water ad libitum. For the assessment of
the acute toxicity, compounds were injected ipꢂ1 into
the TA1 mice at five different dose levels on day 0. Then
Scheme 3. Reagents and conditions: (a) acetyl chloride, pyridine, 0 ꢁC, 2 h; (b) NaOH (aq, 1.0 equiv); (c) DMF, BrCH2CH2OH (1.0 equiv), 80 ꢁC,
8 h; (d) HCl (1.2 M), reflux, 1 h; (e) benzaldehyde, 100 ꢁC, 6 h; (f) chlorambucil (1.0 equiv), DCC (1.1 equiv), pyridine, rt, 48 h; (g) acetone, 365 nm
UV light, rt, 1 h.

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Z. Huang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1099–1103
Table 1. In vivo antitumor activity of CBLd and 12 againstmurine S-180 sarcoma
Drug
Dose (mg/kg)
Schedule
Mice In.a/Fi.b
Body wt. In./Fi.(g)
Tumor wt. XꢃSD (g)
Inhibition (%)
P
CBL
CBL
CBL
CBL
12
12
12
12
6.8
5
2.5
1.3
13
9.6
6.5
4.8
3.3
2.5
0.2c
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
ipꢂ7qd
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
0.50ꢃ0.14
0.59ꢃ0.16
1.04ꢃ0.16
1.39ꢃ0.22
0.41ꢃ0.14
0.51ꢃ0.11
0.57ꢃ0.10
0.81ꢃ0.21
1.09ꢃ0.20
1.15ꢃ0.23
2.21ꢃ0.22
77.38
73.30
52.94
37.10
81.45
76.92
74.21
63.35
50.68
47.96
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
10/9
10/10
10/10
10/10
10/10
20/20
12
12
CONTROL
aInitial stage of experiment.
bFinal stage of experiment.
cmL/mouse.
dChlorambucil.
Table 2. Antitumor activity comparison of 12 with CBL at dose of
equal toxicity
Table 3. Antitumor activity comparison of 12 with CBL at dose of
equal molarity
Drug
LD50ꢂ1/10
LD50ꢂ1/20
LD50ꢂ1/40
Drug
0.022
(mmol/kg)
0.016
(mmol/kg)
0.008
(mmol/kg)
0.004
(mmol/kg)
12b
CBL
81.5%
73.3%
74.2%
52.9%
50.7%
37.1%
12
CBL
81.5%
77.4%
76.9%
73.3%
63.4%
52.9%
47.9%
37.1%
the behavior and the death distribution of the test mice
were recorded. LD50 was calculated by using the Bliss
method. The in vivo antitumor activity was tested
against the mouse solid tumor S-180 cell line which was
maintained by intraperitoneal passage at weekly inter-
vals in male TA1 mice. Results are expressed as the
meanꢃSD. The significance of difference between
groups and/or drugs was assessed by using Student’s
t-test. P<0.05 was taken as significant.
from the data in Table 1 is 47.55, which is about the
twice of CBL’s (TI: 22.84, calculated from the data
obtained in the same test system as 12). Thus, 12 is
much safer and more useful than its mother compound
CBL when used as antitumor agents.
In summary, we have synthesized 12 as a potent anti-
tumor agent by combining sulfadiazine and CBL in one
molecule through an ester bond. 12 is more potent and
safer than its mother compound CBL. This class of tar-
geting agents may be further developed to form candi-
date drugs, which may have advantages over the
currently available anticancer agents.
According to the LD50 values of 12 (130.76 mg/kg ip in
mice, 0.225 mmol/kg) and CBL (49.56 mg/kg ip in mice,
0.163 mmol/kg), it can be concluded that the acute
toxicity of 12 is lower than that of its mother compound
CBL. The results of in vivo antitumor activity of 12 and
CBL againstmurine S-180 sarcoma are listed in Table 1.
The antitumor activity comparison of 12 with CBL was
listed in Table 2 (at equal toxicity) and Table 3 (at equal
molarity), respectively.
References and Notes
1. Maren, T. H. Annu. Rev. Pharmacol. Toxicol. 1976, 16, 309.
2. Toth, J. E.; Grindey, G. B.; Ehlhardt, W. J., et al. J. Med.
Chem. 1997, 40, 1018.
The data in Tables 2 and 3 indicate that 12 is more
potent than CBL when the tested mice were adminis-
tered at doses of either equal toxicity or equal molarity,
especially atrelaitvely low dose range. This effectmay
be partly attributed to the targeting action of 12 that
may lead to a relatively high drug concentration in the
tumor cells. Since the active moiety of 12 is still CBL
which demonstrated a strong dose–effect relationship
mainly atlow dose range as shown in Table 1, the inhi-
bition increased by 12 atrelaitvely low dose is more
obvious than that at high dose. However, compared
with sulfadiazine, the concentration effect of 12 isn’t
obvious as we expected. It may due to the hydrolysis of
the ester bond in the body that some free CBL had
already been released before the concentration of 12 in
tumor cells. The TI (therapeutic index) of 12 calculated
3. Medina, J. C.; Roche, D.; Shan, B., etal. Bioorg. Med.
Chem. Lett. 1999, 9, 1843.
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17. Selected data for compound 12: mp: 97–98 ꢁC; IR nmax
(KBr, cmꢀ1) 3469, 3348 (NH2), 1735 (C¼O), 1593, 1546, 1514
(phenyl), 1272 (SO2N); 1H NMR (300 MHz, CDCl3) d 1.81
(m, 2H, CH2CH2CH2), 2.23 (t, 2H, J=7.2 Hz,
O¼CCH2CH2CH2), 2.53 (t, 2H, J=7.2 Hz, CH2CH2CH2Ph),
3.60 (t, 4H, J=6.0 Hz, N(CH2CH2Cl)2), 3.70 (t, 4H,
J=6.0 Hz, N(CH2CH2Cl)2), 4.25 (t, 2H, J=4.5 Hz,
NCH2CH2O), 4.43 (t, 2H, J=4.5 Hz, NCH2CH2O), 6.46 (dd,
1H, J=2.4 Hz, J=4.2 Hz, pyrimidinyl), 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) d 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.