Structure-activity relationship of indoline-2-carboxylic acid N-(substituted)phenylamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.06.004

Chroman derivatives exhibited potent inhibitory activity of NF-κB. For SAR, the chroman scaffold was modified with an indoline moiety. A series of indoline-2-carboxylic acid N-(substituted)phenylamide derivatives were synthesized to explore their inhibitory activities of NF-κB and they were also evaluated for cytotoxicity against various cancer cell lines. Since intermediates with Boc showed outstanding results, various substituents in place of the Boc group were introduced additionally and these compounds were also evaluated for SAR.

Full text of DOI:10.1016/j.bmcl.2010.06.004

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4620–4623
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of indoline-2-carboxylic acid
N-(substituted)phenylamide derivatives
Jae-Hwan Kwak a, Yoseob Kim a, Hyunjeong Park a, Jae-Yong Jang a, Keun Kuk Lee a, Wonhui Yi a,
Jeong-Ah Kwak a, Song-Gyu Park b, Hwanmook Kim b, Kiho Lee b, Jong Soon Kang b, Sang-Bae Han a,
Bang Yeon Hwang a, Jin Tae Hong a, Jae-Kyung Jung a, Youngsoo Kim a, Jungsook Cho c, Heesoon Lee a,
*
a Department of Pharmacy, Chungbuk National University, Chungbuk 361-763, Republic of Korea
b Bioevaluation Center, KRIBB, Chungbuk 363-883, Republic of Korea
c College of Medicine, Dongguk University, Gyeonbuk 780-714, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Chroman derivatives exhibited potent inhibitory activity of NF-
modified with an indoline moiety. A series of indoline-2-carboxylic acid N-(substituted)phenylamide
derivatives were synthesized to explore their inhibitory activities of NF- B and they were also evaluated
for cytotoxicity against various cancer cell lines. Since intermediates with Boc showed outstanding
results, various substituents in place of the Boc group were introduced additionally and these compounds
were also evaluated for SAR.
j
B. For SAR, the chroman scaffold was
Received 1 April 2010
Revised 28 May 2010
Accepted 1 June 2010
Available online 8 June 2010
j
Keywords:
NF-jB inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
Cytotoxic activity
Indoline-2-carboxamide
Nuclear factor-
j
B, a multifunctional transcription factor, plays
phosphorylated by NIK mediated activation of IKKa and partially
critical roles in physiological and pathological processes such as
immune function, cellular growth, homeostasis, apoptosis, and
degraded to p52/RelB through a ubiquitin-dependent mechanism.
The p52/RelB complex translocates to the nucleus to enhance gene
expression.3
inflammation. NF-
formed by members of the NF-
p50/p105 (NF- B1), and p52/p100 (NF-
In the cytoplasm, NF- B dimer exists in an inactive state and is
associated with I B proteins that mask the nuclear localization sig-
nal (NLS) in NF-
B protein.1,2
There are two major pathways leading to activation of NF-
j
B consists of heterodimers or homodimers
B family, P65 (RelA), RelB, c-Rel,
B2), in mammalian cells.
j
Irregular gene expression though NF-
inflammation, autoimmune diseases, viral infections, and various
cancers. Therefore, it has been suggested that inhibitors of NF-jB
jB is responsible for
j
j
j
j
j
function may be useful both as anti-inflammatory agents and as
antitumor agents.2,7 We recently reported a multiplicity of chro-
jB,
man derivatives having inhibitory activity of NF-j
B.8–11 They were
the canonical (classic) pathway and non-canonical (non-classic)
pathway. The canonical pathway, the major pathway, consists of
designed based on compound KL-1156 (6-hydroxy-7-methoxych-
roman-2-carboxylic acid phenylamide), an inhibitor of transloca-
tion to the nucleus in LPS-stimulated macrophage RAW 264.7
cells.8 In previous studies it was found that the phenolic OH group
I
j
B kinase (IKK), I
IKK is activated by extracellular signals such as tumor necrosis fac-
tor (TNF- ), interlukin 1 (IL-1), lipopolysaccharide (LPS), reactive
jB, and NF-jB (typically p65/p50 heterodimer).
a
a
of the chroman moiety was not required for enhancement of NF-jB
oxygen species (ROS), and a variety of cellular stresses. The acti-
vated IKK phosphorylates the serine residues at positions 32 and
inhibitory activity.10 In chroman derivatives with a short alkyl
group, there are no significant differences between substituent
effects and the inhibition potency.11
36 in I
j
B. The phosphorylation further causes ubiquitination of
B. The acti-
IjB, and then induces proteasome degradation of I
j
To establish a systematic SAR of pyranyl moiety in chroman
compounds, introduction of nitrogen atom together with ring con-
traction, could be considered. Furthermore, an indoline scaffold is
recognized as a privileged pharmacophore,12 which could provide
high water-solubility and exert beneficial pharmacokinetic effects.
vated NF-jB by exposure of the NLS translocates to the nucleus,
where it transcribes genes.3–6
The non-canonical pathway involves NF-jB inducing kinase
(NIK), IKKa, and a p100/RelB heterodimer complex. p100/RelB is
Thus, as part of our ongoing efforts to develop novel NF-jB inhib-
itory agents, we replaced the chroman scaffold with the indoline
* Corresponding author.
moiety. Substituents (H, OH, OCH3, CH3, NO2, Cl, and CF3) on the
E-mail address: medchem@chungbuk.ac.kr (H. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.004

J.-H. Kwak et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4620–4623
4621
N-phenyl ring moiety of target compounds were selected accord-
ing to their electronic and hydrophobic character. The positional
effects of the substituents were also explored by examining com-
pounds with substituents at various positions (2-; 3-; 4-; 3,4-;
3,5-; 2,5-) on the phenyl ring. Herein, we report the syntheses
and structure–activity relationship (SAR).
trifluoroacetic acid (TFA) in dichloromethane to afford the indoline
2 series. A total of twenty-nine compounds of indoline derivatives
were synthesized and evaluated in terms of their inhibitory activ-
ity of NF-jB. In addition, intermediates, N-Boc-indoline deriva-
tives, were evaluated in LPS-stimulated macrophage RAW 264.7
cells (Table 1).
The indoline-2-carboxylic acid N-(substituted)phenyl amide
derivatives were prepared starting from the commercially avail-
able indoline-2-carboxylic acid as depicted in Scheme 1. The indo-
line-2-carboxylic acid was protected with di-tert-butyl dicarbonate
((Boc)2O) to give N-Boc-indoline-2-carboxylic acid. Amidation
between acid 4 and substituted phenylamines 5 using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) in tetrahydrofuran
(THF) was followed by removal of the Boc protecting group with
One compound of indoline-2-carboxamide derivatives (2–2j;
IC50: 12.0
lM) and six compounds (1–1l, 1–1p, 1–2a, 1–2b, 1–2e,
and 1–2j) of N-Boc-indoline-2-carboxamide derivatives (IC50
:
10.1–16.6
(IC50: 40.1
phenyl) amide (1–2b; IC50: 10.1
l
l
M) exhibited more potent activity than KL-1156
M). N-Boc-indoline-2-carboxylic acid N-(3,5-dichloro-
M) showed the best activity.
l
The positional effects of substituents of the indoline moiety on
inhibitory activities are contradictory. Direct correlation was not
R1
H2N
R2
a
b
OH
N
OH
N
Boc
H
O
R3
O
R4
3
4
5
R1
H
R1
R4
R group = H
H
R2
R3
N
R2
R3
Me
N
c
N
Boc
N
H
OH
OMe
Cl
O
O
R4
CF3
NO2
1-1 monosubstituted series
2-1 monosubstituted series
1-2 disubstituted series
2-2 disubstituted series
Scheme 1. General methods to prepare 2 series. Reagents and conditions: (a) Boc2O, NaOH, dioxane, H2O, 2 h, 95%; (b) EDC, THF, 14 h, 40–98%; (c) TFA, DCM, 6 h, 25–98%.
Table 1
Inhibitory effect on LPS-induced NF-
jB transcriptional activity for intermediate 1 series and indoline derivative 2 series
Substituents (R group on phenyl ring)
No.
1 series
% inhibition at 100
No.
2 series
% inhibition at 100
R1
R2
R3
R4
l
M
IC50
(lM)
l
M
IC50 (lM)
Ref.
H
OH
H
H
OCH3
H
H
CH3
H
H
NO2
H
H
CF3
H
H
Cl
H
H
H
H
KL-1156
1–1a
1–1b
1–1c
1–1d
1–1e
1–1f
1–1g
1–1h
1–1i
90
30
93
75
42
76
38
29
68
87
37
63
94
8
5
1
8
4
2
3
3
1
2
6
1
2
40.1
>100
—
58.7
>100
—
>100
>100
62.5
—
>100
—
14.1
—
>100
—
12
KL-1156
2–1a
2–1b
2–1c
2–1d
2–1e
2–1f
2–1g
2–1h
2–1i
90
65
37
30
63
60
64
37
39
29
41
41
49
41
61
99
56
8
6
5
5
3
5
3
9
5
3
8
6
5
4
2
1
5
40.1
71.6
H
H
OH
H
H
OCH3
H
H
CH3
H
H
NO2
H
H
CF3
H
H
Cl
H
Cl
Cl
H
H
H
OH
H
H
OCH3
H
H
CH3
H
H
NO2
H
H
CF3
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
CH3
CH3
H
OCH3
OCH3
CF3
>100
>100
66.2
79.2
—
>100
>100
>100
>100
>100
>100
>100
79.4
46
1–1j
1–1k
1–1l
2–1j
2–1k
2–1l
1–1m
1–1n
1–1o
1–1p
1–1q
1–1r
1–1s
1–2a
1–2b
1–2c
1–2d
1–2e
1–2f
>100
38
>100
>100
50
2–1m
2–1n
2–1o
2–1p
2–1q
2–1r
2–1s
2–2a
2–2b
2–2c
2–2d
2–2e
2–2f
4
79.2
69.7
—
70.7
—
1
—
—
62 10
>100
63
62
72
98
34
87
56
47
15
41
47
96
3
4
3
3
9
2
3
7
8
4
2
3
31
97
—
3
3
>100
12.6
10.1
45.9
—
16.6
50
52.2
—
—
Cl
H
H
CH3
H
H
OCH3
H
H
H
CH3
H
H
OCH3
H
71
64
78
75
65
88
76
—
4
3
9
4
1
1
1
>100
43.3
85
>100
>100
>100
>100
12
CH3
CH3
H
OCH3
OCH3
H
1–2g
1–2h
1–2i
2–2g
2–2h
2–2i
H
H
—
14.9
CF3
1–2j
2–2j
IC50 values are means of the concentration (lM) exhibiting 50% inhibition of LPS-induced NF-jB transcriptional activity.

4622
J.-H. Kwak et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4620–4623
observed based on the electronic and hydrophobic character of the
substituents. The most active compound, 1–2b, was found to be
four times more potent than the reference compound, KL-1156.
of NF-jB similar to that of the reference compound with Boc group
(1–2b). Except for the compound bearing the tosyl group (7f), com-
pounds (7a–e) in the 7 series exhibited inhibitory activity of NF-
NF-
most cell types but also for cancer development in various organs.
Incorrect regulation of NF- B has been related to antitumor activ-
ity.13 For the correlation between the inhibitory activity of NF-
and cytotoxic activity, fifty-eight compounds were evaluated for
cytotoxicity against NCI-H23 (lung) and PC-3 (prostate) cancer cell
lines (Table 2).
j
B is an important factor not only for cell survival signals in
jB. In particular, compounds with 2-(Boc)amino-1-oxypropyl on
indoline exhibited more potent inhibitory activity than compounds
bearing other substituents. The compounds 6e and 7e were found
to be slightly more potent than the reference compound with the
Boc group (1–2b) (Table 3).
The 6 and 7 series compounds were also examined for cytotoxic
activity against ACHN (renal), HCT15 (colon), MM231 (breast),
NUGC-3 (gastric), NCI-H23, and PC-3 cancer cell lines (Table 4).
j
jB
Only eleven compounds showed significant cytotoxicity. Gener-
ally, compounds having NF-
dominant cytotoxicity. However, cytotoxicity was not directly
proportional to NF- B inhibitory activity. Regardless of the substi-
j
B inhibitory activity exhibited pre-
Generally, compounds having inhibitory activity of NF-
cytotoxicity in various cancer cell lines, as stated earlier. Poor
inhibitory activity of NF- B was related to inferior cytotoxic activ-
jB show
j
j
tuent at the nitrogen of the indoline moiety, compounds contain-
ing 3-CF3, 3,4-diCl, 3,5-diCl or 3,5-diCF3 on the phenyl ring
exhibited significant cytotoxicity. In particular, compounds bear-
ing 3,5-diCF3 on the phenyl ring (1–2j and 2–2j; NCI-H23: 1.524
ity. However, compound 6b is an exception, exhibiting cytotoxicity
against ACHN, HCT15, NUGC-3, NCI-H23, and PC-3 cancer cell
lines. Cytotoxicity related inhibitory activity of NF-jB varied
according to cancer cell type. Compound 7b exhibited more potent
and 0.648
comparable cytotoxic activity to doxorubicin (ADR, NCI-H23:
0.178 M; PC-3: 0.317 M).
Since intermediates with Boc showed outstanding inhibitory
activity of NF- B, especially compounds bearing 4-CF3 (1–1p,
IC50: 12.0 M) and 3,5-diCl (1–2b, IC50: 10.1 M) on the phenyl
l
M; PC-3: 0.838 and 0.794
l
M) were found to have
activity than other compounds against all cancer cell lines tested.
l
l
Table 3
Inhibitory effect on LPS-induced NFjB transcriptional activity
j
l
l
R group
4-CF3
IC50
(lM)
3,5-diCl
IC50 (lM)
ring, various substituents (acetyl, benzoyl, 2-(Boc)amino-1-oxy-
propyl, nonanoyl, pivaloyl, and tosyl) instead of the Boc group were
introduced on indoline-2-carboxylic acid N-(4-CF3- and 3,5-diCl-
)phenylamide. 6 series and 7 series compounds were prepared
from compounds 1–1p and 2–2a (Scheme 2). These compounds
were evaluated in LPS-stimulated macrophage RAW 264.7 cells
Acetyl
Nonanoyl
Pivaloyl
Benzoyl
2-(Boc)amino-1-oxypropyl
Tosyl
Ref.
6a
6b
6c
6d
6e
6f
>100
>100
12.7
13.5
7.0
7a
7b
7c
7d
7e
7f
34.1
12.6
14.5
46.5
5.9
>100
—
>100
10.1
—
1–2b
for inhibitory activity of NF-jB.
The IC50 values are means of the concentration (lM) exhibiting 50% inhibition of
LPS-induced NF-jB transcriptional activity.
In compounds with the 4-CF3 substituent on the phenyl ring,
compounds with pivaloyl (6c), benzoyl (6d), and 2-(Boc)amino-1-
oxypropyl (6e) at the nitrogen on indoline show inhibitory activity
Table 4
Table 2
Cytotoxicity against ACHN, HCT15, MM231, NUGC-3, NCI-H23, and PC-3 cancer cell
lines
Cytotoxicity against NCI-H23 and PC-3 cancer cell lines
GI50
(lM)
GI50
MM231
(lM)
NCI-H23
PC-3
ACHN
HCT15
NUGC-3
NCI-H23
PC-3
ADR
0.178
9.316
2.523
6.343
3.207
3.230
3.188
3.390
1.524
4.960
11.604
12.294
0.648
0.317
—
2.794
3.337
2.020
—
4.663
1.676
0.838
4.244
—
ADR
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
0.182
>10
>10
2.177
>10
8.676
2.067
1.73
5.535
3.808
5.491
—
6.512
4.326
>10
0.194
>10
>10
0.202
>10
0.178
>10
0.317
>10
6.37
KL-1156
1–1m
1–1o
1–1p
1–1r
1–2a
1–2b
1–2j
2–1o
2–2a
2–2b
2–2j
7.856
3.452
1.515
6.581
3.348
8.721
7.974
7.842
6.206
>10
7.678
1.619
1.283
3.645
3.463
4.580
4.284
—
3.781
1.892
5.627
3.68
—
8.26
6.992
5.037
>10
>10
1.861
1.611
3.885
4.179
—
6.842
6.255
4.427
6.678
>10
3.573
7.311
4.973
—
6.417
>10
—
>10
>10
6.712
8.968
7.534
2.534
0.794
7f
>10
>10
>10
The GI50 values correspond to the agent’s concentration causing a 50% decrease in
net cell growth.
The GI50 values correspond to the agent’s concentration causing 50% decrease in net
cell growth.
R1
H
R1
R= Acety l(a)
H
N
N
R2
R3
R2
R3
Nonanoyl (b)
N
H
N
R
Pivaloyl (c)
O
O
Benzoyl (d)
2-(Boc)amino-1-oxypropyl (e)
tosyl (f)
R4
R4
6a-f, 7a-f
2-1p : R3 = CF3; R1, R2, R4 = H
2-2a : R2, R4 = Cl; R1, R3 = H
6 series : R3 = CF3; R1, R2, R4 = H
7 series : R2, R4 = Cl; R1, R3 = H
Scheme 2. General methods to prepare 6 and 7 series. Reagents and conditions: (a) Ac2O, THF, 85–90%; (b) nonanoyl chloride, THF, TEA, 60–70%; (c) PivCl, THF, TEA, 55–68%;
(d) Bz2O, THF, 40–57%; (e) Boc-alanin, EDC, THF, 43–50%; (f) TsCl, TEA, THF, 55–60%.

J.-H. Kwak et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4620–4623
4623
In summary, we have described seventy-two indoline-2-carbox-
amide derivatives in relation to NF- B activity. At concentrations
below 20 M, one indoline derivative and twelve compounds with
substituents at the nitrogen of the indoline scaffold showed signifi-
cant activity. Compound 7e showed the highest activity among all
the indoline derivatives. The 2-(Boc)amino-1-oxypropyl group at
the nitrogen of the indoline moiety slightly enhanced the inhibitory
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