Inhibitory activities against topoisomerase I & II by polyhydroxybenzoyl amide derivatives and their structure-activity relationship

Article abstract of DOI:10.1016/j.bmcl.2004.01.060

o-, m-, p-Phenylenediamines having 2,3,4-trihydroxy, 3,4 dihydroxy, and 4-hydroxybenzoyl moieties were prepared and their inhibitory activities were measured against topoisomerase I and II. More hydroxy groups on two aromatic rings increased the activities. Bis(trihydroxybenzoyl)-o-phenylenediamide showed IC₅₀=0.90 and 0.09 μM against topoisomerase I and II, respectively. Compounds with hydroxy groups protected by acetyl moiety still had the activities. Less hydroxy groups decreased their activities. Benzothiazole derivatives also indicated the activities.

Full text of DOI:10.1016/j.bmcl.2004.01.060

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1669–1672
Inhibitory activities against topoisomerase I & II by
polyhydroxybenzoyl amide derivatives and their
structure–activity relationship
Mohamed Abdel-Aziz,a Kazuya Matsuda,b Masami Otsuka,a Masaru Uyeda,b
Tadashi Okawaraa and Keitarou Suzukib,*
aDepartment of Bioorganic Medicinal Chemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University,
Kumamoto 862-0973, Japan
bDepartment of Pharmaceutical Microbiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University,
Kumamoto 862-0973, Japan
Received 22 November 2003; accepted 21 January 2004
Abstract—o-, m-, p-Phenylenediamines having 2,3,4-trihydroxy, 3,4 dihydroxy, and 4-hydroxybenzoyl moieties were prepared and
their inhibitory activities were measured against topoisomerase I and II. More hydroxy groups on two aromatic rings increased the
activities. Bis(trihydroxybenzoyl)-o-phenylenediamide showed IC50=0.90 and 0.09 mM against topoisomerase I and II, respectively.
Compounds with hydroxy groups protected by acetyl moiety still had the activities. Less hydroxy groups decreased their activities.
Benzothiazole derivatives also indicated the activities.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
phenyl groups on benzofuran-2-one, and one of the
prepared compounds18 having trihydroxyphenyl group
improved topo I inhibition to 3.0 mM (IC50). In this short
communication we prepared o-, m-, p-phenylenediamines
and benzo-thiazoles having more hydroxy groups on
phenyl moiety in order to enhance topo inhibition.
DNA topoisomerases (topo) are ubiquitous enzymes
that perform essential cellular functions involved in
replication, recombination, and packaging and unfold-
ing of DNA in chromatin.1 Topo I and II inhibitors
which have various structures with different side chains
have been reported.2À11 Clinically potent anticancer
agents, topo inhibitors like camptothecin,12 etoposide,13
and doxorubicin14 bind to the cleavable complex
formed between topo and DNA, and keep it from going
back the original DNA. This action is associated with
severe side effects as well as other anticancer agents
targeted at DNA.15,16 Now agents directly inhibiting
topo are urgently being requested. Suzuki et al.17 repor-
ted isoaurostatin isolated from Thermomonospora alba
showed topo I inhibition (IC50=307 mM), and they
proved that this compound directly inhibited topo I. Then
they tried to prepare their modified compounds which
have hydroxyphenyl-, dihydroxyphenyl-, and trihydroxy-
2. Chemistry
Reactions of o-, m-, p-phenylenediamine (1a–c) with
2,3,4-triacetyloxy, 3,4-diacetyloxy, and 4-acetyloxy-
benzoyl chlorides (2a–c) gave the corresponding amides
(3a–i), which were subjected to deacetylation with
hydrazine hydrate yielding the corresponding com-
pounds (4a–i). Benzothiazole derivatives (6a–c) were
also prepared by the same method.
3. Inhibitory activity
Inhibition against relaxation activity of topo was mea-
sured by detecting the conversion of supercoiled
pBR322 DNA to its relaxed form.19,20 As shown in
Table 1, o-compound 4a showed most potent topo I
Keywords: Polyhydroxybenzoyl amide; Topoisomerase inhibitor;
Structure–activity relationship.
* Corresponding author. Tel.: +81-96-371-4327; fax: +81-96-371-
4323; e-mail: keitarou@gpo.kumamoto-u.ac.jp
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.060

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M. Abdel-Aziz et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1669–1672
Scheme 1.
inhibitory activity with IC50 0.9 mM. m- and p-Com-
pounds (4b,c) indicated IC50 1.6 and 1.4 mM, respec-
tively. Their acetyl compounds (3a–c) weakened the
activities showing IC50 4.7, 11.2, and 8.6 mM. Among
3,4-dihydroxy derivatives (4d–f), p-compound 4d
showed the activity IC50 2.6 mM. Their acetyl com-
pounds did not show the activity except p-compound 3e
with IC50 43.2 mM. For monohydroxybenzoyl (4g–i)
and their acetyl (3g–i) derivatives no activity were
observed (Scheme 1).
observed to show IC50 0.09, 0.22, and 0.15 mM, respec-
tively. From these results the following three essential
factors are required for the inhibitory activity. 1: two
trihydroxybenzoyl (galloyl) groups. 2: a hydrogen bond
of carbonyl or enolic hydoxy group with another amide
or nitrogen group. 3: benzene ring.
4. Inhibitory property
Inhibitory properties of 4a were examined as the fol-
lowing four methods.
Dibenzoyl-o-phenylene diamine (4j) and 2,3,4-trihy-
droxy-benzoyl anilide and 2,3,4-trihydroxy-benzoyl
anilide (5) had also no activity. 2-(2,3,4-Trihydroxy-
benzoyl)-2-aminobenzothiazole (6a) and dihydroxy
derivaive (6b) showed IC50 8.2and 16.9 mM, but
monohydroxy derivatives (6c) had no activity. 2-(2,3,4-
Trihydroxy-benzoyl)-2-aminothiazole (7) with no
phenyl group showed weak inhibition. Moreover, topo
II inhibitory activities of compounds (4a–c) were
4.1. Inhibitory manner of 4a against topo I and II
The type of inhibition was determined by Lineweaver–
Burk plots21 of substrate concentrations against the rate
of relaxation of supercoiled pBR322 DNA by topo I
and II in the presence or absence of 4a. As shown in
Figure 1, 4a inhibited the relaxation activities of topo I
and II noncompetitively with respect to pBR322 DNA
exhibiting Ki values of 0.4 mM and 74.1 nM, respectively.
Table 1. Inhibition of topoisomerase I by polyhydroxyphenyl derivatives
Compd
n
R
Position
Topo I
inhibition
(IC50, mM)
3a
3b
3c
4a
4b
4c
3d
3e
3f
4d
4e
4f
3g
3h
3i
4g
4h
4i
4j
5
6a
6b
6c
7
o
m
p
o
m
p
o
m
p
o
m
p
o
m
p
o
m
p
3
3
3
3
3
3
CH3CO
CH3CO
CH3CO
H
3,4,5-
3,4,5-
3,4,5-
3,4,5-
3,4,5-
3,4,5-
3,4-
4.7
11.2
8.6
0.9
1.6
H
H
3CO
3CO
3CO
1.4
2CH
2CH
2CH
2H
2H
2H
1
1
1
1
1
88.0
43.2
>100
3,4-
3,4-
3,4-
3,4-
3,4-
6.4
10.0
2
.6
4-
4-
4-
4-
4-
4-
CH3CO
CH3CO
CH3CO
>100
>100
>100
>100
>100
>100
>100
>100
8.2
H
H
H
1
0
o
H
H
H
3
3,4,5-
23,4-
1
16.9
Figure 1. Lineweaver–Burk plots of pBR322 DNA concentrations
against rate of relaxation by topo I [A] and topo II [B] with (*) and
without 4a (*).
4-
>100
34.4

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The Michaelis constants (Km values) of topo I and II
were 3.7 nM and 10.5 nM, respectively. In view of inhi-
bitory potency (Ki/Km) against DNA relaxation by topo
I and II, 4a was 15-fold potent against topo II than topo
I. From these results, 4a was considered to bind with a
different site from the binding site of the substrate DNA
in the enzyme molecule.
tive to the conformation changes of DNA by
intercalators.24,25 Doxorubicin was used as control of
intercalator at the same concentration. The spectrum of
DNA changed greatly with increasing concentrations of
doxorubicin. On the other hand, the spectral changes by
4a did not occur, therefore, it is clear that 4a has no
ability to intercalate into DNA. Thus, 4a is different
from inhibitors causing DNA damage such as cleavable
complex-forming inhibitors and DNA intercalators.
4.2. Stabilization of topo-cleavable complex by 4a
Topo inhibitors of the cleavable complex-forming type
such as camptothecin and etoposide stabilize the clea-
vable complex (topo–DNA reaction intermediate) and
inhibit the DNA rejoining reaction of topo, which is the
inhibitory mechanism of the inhibitors, therefore the
inhibitors induce nicked or linearized DNA in the clea-
vage assay.22,23 To determine whether 4a is an inhibitor
of the cleavable complex-forming type or not, cleavage
assays were carried out. Camptothecin and etoposide
were used as the controls of cleavable complex-forming
inhibitors against topo I and II, respectively. As shown
in Figure 2[A], camptothecin induced nicked DNA with
increasing concentrations. Unlike camptothecin, 4a
could not induce the nicked DNA even at 100 mM. The
results for the stabilization of topo II-cleavable complex
are shown in Figure 2[B]. Etoposide induced the linear-
ized DNA, but 4a failed to linearize DNA even at 1000
mM. These results suggest that 4a is an inhibitor of the
cleavable-nonforming type. 4a may directly act on topo
I and II molecules in earlier step than the formation of
the topo–DNA complex and inhibit the DNA breaking
and rejoining reactions by the enzymes.
4.4. Effect of 4a on the growth and cell cycle of HeLa cells
The cell growth inhibition of 4a was determined in
HeLa cells by Alamar Blue assay.26 The values of cell
growth inhibition (GI50) of 4a, camptothecin and eto-
poside were 30, 0.6 and 40 mM, respectively. Camp-
tothecin and etoposide promote the accumulation of
damaged DNA by stabilization of cleavable complex in
the cells, therefore the inhibitors arrest the cell cycle
progression. The cell cycle progression was analyzed
with a flow cytometer (Becton Dickinson FACS Cali-
bur) using the ModFit LT which is a software to deter-
mine the percentage of cells in G0/G1, S and G2/M
phases.27 HeLa cells were arrested at S phase and G2/M
phase when cultured with 0.1 mM camptothecin and 2
mM etoposide, respectively. On the other hand, 4a did
not affect on the cell cycle even at an extremely high
concentration (100 mM). The results suggest that the
cytotoxicity of 4a is clearly different from that of
camptothecin and etoposide.
5. Materials and general experimental procedures
4.3. DNA interaction by 4a
o-, m-, p-Phenylenediamines (1a–c) were purchased
from Tokyo Kasei. Polyacetyloxybenzoyl chlorides (2a–
c) were prepared according to Gazit’s method.28
Some topo inhibitors such as doxorubicin and amsa-
crine are DNA intercalators. To determine whether 4a
has the ability to intercalate into DNA strands, CD
(circular dichroism) spectral change of DNA by addi-
tion of 4a was measured, because the spectrum is sensi-
5.1. Bis(polyacetyloxybenzolyl)-o-, m-, p-phenylene-di-
amides (3a–i)
To a solution of 1a–c (0.22g, 2mmol) and triethylamine
(0.70 mL, 5 mmol) in CH2Cl2 (10 mL) was added
dropwise a solution of 2a–b (4 mmol) in CH2Cl2 (10
mL) under cooling with ice and water. The reaction
mixture was allowed to stir for 2h at room temperature.
The CH2Cl2 layer was washed with water (10 mLÂ2),
dried over MgSO4, and evaporated to dryness. The
residue was recrystallized from MeOH. Yield: 68–81%.
5.2. Dipolyhydroxybenzolyl o-, m-, p-phenylenediamines
(4a–i)
Hydrazine hydrate (0.35 g, 7 mmol) was added to a
solution of 3a–i (1 mmol) in CH2Cl2 (10 mL). The
reaction mixture stirred for 2h at room temperature,
washed with water (10 mL), dried over MgSO4, and
evaporated to dryness. The residue was recrystallized
from MeOH. Yield: 61–71%.
Compounds (4j, 5, 6a–c, 7) were prepared by the same
method described above. Structures of each compound
were determined by spectral data and elemental analyses.
Figure 2. Stabilization of topo I [A] and topo II [B]-cleavable com-
plexes by 4a (*), camptothecin (*) and etoposide (~).

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15. Chino, M.; Nishikawa, K.; Yamada, A.; Ohsono, M.;
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