Synthesis and biological evaluation of N-(7-indolyl)-3-pyridinesulfonamide derivatives as potent antitumor agents.

Article abstract of DOI:10.1016/S0960-894X(02)00376-1

We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety. E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in nude mice.

Full text of DOI:10.1016/S0960-894X(02)00376-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2097–2100
Synthesis and Biological Evaluation of N-(7-Indolyl)-3-
pyridinesulfonamide Derivatives as Potent Antitumor Agents
Takashi Owa,* Hiroshi Yoshino, Tatsuo Okauchi,y Tadashi Okabe, Yoichi Ozawa,
Naoko Hata Sugi, Kentaro Yoshimatsu, Takeshi Nagasu,
Nozomu Koyanagi and Kyosuke Kitoh
Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
Received 7 March 2002; accepted 16 May 2002
Abstract—We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety.
E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its
tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-
pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in
nude mice. # 2002 Elsevier Science Ltd. All rights reserved.
E7070 (2), a novel sulfonamide antitumor agent, was
discovered from our sulfonamide-focused libraries ori-
ginating in the lead structure 1 (Fig. 1).1ꢀ3 Because of its
promising in vivo efficacy against human tumor xeno-
grafts4 and its unique mechanistic profile,4ꢀ7 E7070
progressed to clinical evaluation. In Phase I studies,8 the
predominant toxicity of E7070 was determined to be
myelosuppression such as neutropenia and thrombocy-
topenia. Some objective responses were observed in
patients with advanced cancers. Phase II trials of this
compound are currently ongoing in Europe and the
United States to assess its clinical efficacy against
several solid tumors.
the G1/S transition eventually led to G2 arrest in
HCT116 human colon cancer cells6 and A549 human
non-small cell lung cancer cells.7 The study using A549
and its E7070-resistant subline A549/ER further clar-
ified that E7070 inhibited pRb phosphorylation,
reduced the protein expression of cyclin A, cyclin B1,
CDK2, and CDC2, and repressed CDK2 activity with
the induction of p53 and p21 proteins only in the par-
ental A549 cells.7 All of these findings indicate that
E7070 belongs to a new class of cell cycle inhibitors.
Given the clinical activity of this interesting small
molecule, it appears to be of particular importance to
continue the drug discovery research on structurally
related compounds. We herein present E7070 analogues
A primary molecular target of E7070 is still unknown,
and thus its precise mechanism of antitumor action has
yet to be fully understood. However, several reports to
date have disclosed that E7070 causes a decrease in the
S phase fraction accompanied by the accumulation of
cells in the G1 phase and/or the G2 phase of the cell
cycle.2ꢀ7 Originally, the compound was found to
increase the G1 proportion of P388 murine leukemia
cells in time- and dose-dependent manners.2,4 There-
after, it was shown that E7070-induced disturbance of
*Corresponding author. Tel.: +81-298-47-5868; fax: +81-298-47-
2037; e-mail: t-owa@hhc.eisai.co.jp
yCurrent address: Department of Applied Chemistry, Kyushu Institute
of Technology, 1-1 Sensuicho, Tobata, Kitakyushu, Fukuoka 804-
8550, Japan.
Figure 1. Flow chart for the discovery of E7070.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00376-1

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T. Owa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2097–2100
containing a 3-pyridinesulfonamide moiety with respect
to their antitumor activities against HCT116 cells both
in vitro and in vivo.
days 1–4). We assessed drug efficacy according to the
following two criteria: Day 9-RTV (relative tumor
volume) and Day 22-T/C(%). The Day 9-RTV value
was calculated as the ratio of average tumor volume on
day 9 to that on day 1. The RTV value <1.0 indicates
that initial tumor volume was reduced by drug treat-
ment. Therefore, it can be used as an index for the
assessment of not only tumor growth suppression but
also tumor regression. The Day 22-T/C value was cal-
culated on day 22 as the ratio of average tumor weight
of the treated group to that of the control group. This is
one of the most widely accepted criteria representing
drug-induced tumor growth suppression. The T/C value
<42 generally indicates in vivo antitumor activity.
The syntheses of the new analogues are outlined in
Scheme 1. Reduction of 3-chloro-7-nitroindole (3) and
3-cyano-7-nitroindole (5) with Fe/NH4Cl and H2/Pd-C
gave the corresponding aminoindoles 4 and 6, respec-
tively.2 Sulfonic acids 7a, 7b,9 and 7c10 were each con-
verted to 8a, 8b, and 8c by using PCl5 and POCl3 under
reflux. These sulfonyl chlorides were coupled with 4 and
6 in the presence of pyridine to afford sulfonamides 9–
14. Amination of 13 and 14 with NH3 in ethanol gave
compounds 15 and 16, respectively. Compound 13 was
further transformed to 17–20 via the reaction with sev-
eral amines and aqueous sodium hydroxide.
After intraperitoneal (ip) drug administration, com-
pounds 12 and 15–18 exhibited potent efficacy based on
the T/C criteria. They, except for 16, also caused a sig-
nificant decrease in tumor volume on day 9. In the ip
treatment, there were no compounds sperior to E7070 in
terms of Day 9-RTV and Day 22-T/C values. As far as
examined, no correlation between in vitro and in vivo
antitumor activities was observed. On this in vivo
model, the 3-cyanoindole derivative 12 showed clear
efficacy accompanying tumor regression whereas its
3-chloroindole analogue 11 was inactive. By contrast,
the efficacy of another 3-cyanoindole derivative 16 was
inferior to that of the corresponding 3-chloroindole
derivative 15, particularly with respect to drug effect
reducing tumor volume. These observations suggest that
the combination of substituents R1 and R2 may be cri-
tical for pharmacokinetic behavior of this series of
compounds.
Compounds 9–12 and 15–20 were initially screened for
in vitro antiproliferative activity against HCT116
human colon cancer cells. Following 72-h continuous
drug exposure, the concentration required for 50% cell
growth inhibition (IC50) was determined by the MTT
colorimetric assay.11 As shown in Table 1, these new
analogues inhibited the proliferation of HCT116 with
IC50 values ranging from 0.19 to 1.3 mM. The in vitro
activities of 9–12 were comparable to that of E7070. 3-
Chloroindole derivatives (9, 11, and 15) and the corre-
sponding 3-cyanoindole derivatives (10, 12, and 16,
respectively) were equally active in this in vitro assay.
All these compounds were next evaluated for in vivo
antitumor activity on the HCT116 xenograft model.
HCT116 cells were implanted subcutaneously into
athymic nude mice and allowed to grow to a size of
about 100 mm3. Subsequently, daily drug administra-
tion (50 mg/kg/day) was conducted for four days (on
Of the compounds found to be active in the ip treat-
ment, 12, 15 (ER-35745), 16, 18, and E7070 were further
Scheme 1.

T. Owa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2097–2100
Table 1. In vitro and in vivo antitumor activities of E7070 and its analogues
2099
Compd
R1
R2
In vitro activity
IC50 (mM)a
Drug
administrationb
In vivo activity
Day 9-RTVc
Day 22-T/C(%)d
Deaths/total
9
H
H
5-CH3
5-CH3
Cl
CN
Cl
0.26
0.23
0.22
0.19
ip
ip
ip
ip
po
ip
po
ip
po
ip
ip
po
ip
3.4
2.6
3.7
0.52
3.4
0.30
0.19
1.5
70
65
82
30
106
26
12
35
75
38
16
55
89
66
23
12
8
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
10
11
12
CN
15 (ER-35745)
16
6-NH2
6-NH2
Cl
0.71
0.64
CN
2.3
17
18
6-NHCH3
6-NHCH(CH3)2
Cl
Cl
0.86
1.3
0.24
0.28
0.90
4.3
19
20
6-NH(CH2)2OH
6-OH
Cl
Cl
0.65
0.80
0.29
ip
po
ip
3.0
2 (E7070)e
0.29
0.10
0.15
iv
aHCT116 human colon cancer cells were treated with each test compound for 72 h. IC50 values were calculated using the least-squares method to
afford the means of three independent assay data. Errors were within ꢃ10% of the reported values.
b50 mg/kg/day of each test compound was given to nude mice (n=5) with HCT116 xenografts following the tumor volume reached about 100 mm3.
Only a vehicle (saline containing 3.5% DMSO and 6.5% Tween 80) was given ip to control mice with the same tumors (n=10). Drug administration
was performed daily for four days (on days 1–4). Administration routes: ip, intraperitoneal; po, per os; iv, intravenous.
c‘(Average tumor volume on day 9)/(average tumor volume on day 1)’ is designated as Day 9-RTV. Day 9-RTV values <1.0 indicate that drug
treatment caused tumor regression. The mean of control Day 9-RTV values was 4.5ꢃ1.1.
d‘(Average tumor weight in treated group on day 22)/(average tumor weight in control group on day 22) ꢁ 100’ is designated as Day 22-T/C.
According to the National Cancer Institute (NCI) activity criteria, Day 22-T/C values <42(%) are judged to be active.
eThe data for ip and iv injections were reported previously.2,4
tested for their oral (po) efficacy. When given orally in a
suspension of methyl cellulose, compounds 12, 16, and
18 proved to be lacking for in vivo antitumor activity
based on the T/C criteria, even though only 18 caused a
marginal reduction in tumor volume on day 9. E7070
has been evaluated in preclinical animal models by
intravenous (iv) administration that is currently used for
clinical trials. In this experiment E7070 was also shown
to be quite active via the po route in addition to ip and
iv routes reported previously.2,4 At the same dose level
of E7070 (50 mg/kg/dayꢁ4), the ip and iv treatments
were considered to be more effective than the po treat-
ment. Importantly, ER-35745 demonstrated notable po
efficacy that was almost comparable to the efficacy of
E7070 administered intravenously. As for the in vivo
antitumor activity of ER-35745, the po treatment was
superior to the ip treatment, suggesting good pharma-
cokinetic profiles of this compound given orally. In fact,
ER-35745 exhibited rapid adsorption (Tmax <1 h) and
remarkable bioavailability (F ꢂ100) in Sprague–Daw-
ley rats given a po 10 mg/kg dose.
common in three different human cancer cell lines,
HCT116-C9 colon carcinoma, MDA-MB-435 breast
carcinoma, and MOLT-4 leukemia.13 These nine genes
are cyclin H, DNA polymerase a subunit, mitochondrial
NAD(P)+-dependent malic enzyme, medium-chain acyl-
CoA dehydrogenase, tropomyosin, calcineurin A1, Rp8
homologue PDCD2, clone 23759, and KIAA0257 (T.
Owa, A. Yokoi, and T. Nagasu, unpublished data). It is
of particular importance that ER-35745 treatment (at
0.8 mMfor 12 h) also repressed more than two-fold the
transcript levels of all nine genes in HCT116-C9 cells.
This strongly suggests that E7070 and ER-35745 share
the same primary mechanism of antitumor action.
Described herein are the synthesis and antitumor activ-
ity of E7070 analogues containing
a
3-pyr-
idinesulfonamide moiety. We have found the 6-amino-
3-pyridinesulfonamide derivative ER-35745 to display
significant oral efficacy against the HCT116 human
colon carcinoma xenograft in nude mice. ER-7070 and
ER-35745 seem to operate by putatively the same
mechanism of action, supported by microarray-based
gene expression analysis.
We have recently focused on gene expression analysis
using Affymetrix high-density oligonucleotide micro-
arrays (HuGene FL arrays) to identify transcriptional
changes closely associated with E7070 efficacy.12 After
12 h of treatment, 0.8 mME7070, a pharmacologically
relevant and a clinically achievable drug concentration,
led to 2-fold or more down-regulation of nine genes in
Acknowledgements
We thank Mr. A. Yokoi, Dr. J. Kuromitsu, and Dr. T.
Kawai for their helpful discussions and assistance with

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T. Owa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2097–2100
the microarray analysis. The pharmacokinetic data for
ER-35745 were kindly provided by Mr. Y. Sawa.
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