Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase and NFκB inhibitors

Article abstract of DOI:10.1039/c2ob26456k

Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NFκB could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and cancer. During the course of evaluating the biological potential of a commercial compound library, 2-phenylindole (1) displayed inhibitory activity against nitrite production and NFκB with IC₅₀ values of 38.1 ± 1.8 and 25.4 ± 2.1 μM, respectively. Based on this lead, synthesis and systematic optimization have been undertaken in an effort to find novel and more potent nitric oxide synthase and NFκB inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and 2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and 3-oxime derivatives 2-7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited the strongest nitrite inhibitory activities (IC₅₀ = 4.4 ± 0.5 and 4.8 ± 0.4 μM, respectively); as well as NFκB inhibition (IC₅₀ = 6.9 ± 0.8 and 8.5 ± 2.0 μM, respectively). In addition, the 6′-MeO-naphthalen-2′-yl indole derivative 10at displayed excellent inhibitory activity against NFκB with an IC₅₀ value of 0.6 ± 0.2 μM.

Full text of DOI:10.1039/c2ob26456k

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PAPER
Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase
and NFκB inhibitors†
Xufen Yu, Eun-Jung Park, Tamara P. Kondratyuk, John M. Pezzuto and Dianqing Sun*
Received 26th July 2012, Accepted 13th September 2012
DOI: 10.1039/c2ob26456k
Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NFκB
could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and
cancer. During the course of evaluating the biological potential of a commercial compound library,
2-phenylindole (1) displayed inhibitory activity against nitrite production and NFκB with IC50 values of
38.1 1.8 and 25.4 2.1 μM, respectively. Based on this lead, synthesis and systematic optimization
have been undertaken in an effort to find novel and more potent nitric oxide synthase and NFκB
inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and
2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and
3-oxime derivatives 2–7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized
from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium
catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological
evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited
the strongest nitrite inhibitory activities (IC50 = 4.4 0.5 and 4.8 0.4 μM, respectively); as well as
NFκB inhibition (IC50 = 6.9 0.8 and 8.5 2.0 μM, respectively). In addition, the 6′-MeO-naphthalen-
2′-yl indole derivative 10at displayed excellent inhibitory activity against NFκB with an IC50 value of
0.6 0.2 μM.
deal of effort has been expended for the development of novel
and advanced synthetic methods for the preparation of indole
Introduction
The indole heterocyclic ring constitutes an important structural
scaffold in natural products and pharmaceutical molecules
(Fig. 1).1–3 Among them, indolyl alkaloids represent a distinct
class of bioactive molecules in chemical and medicinal fields.
As examples, the alkaloid reserpine was developed as one of the
first drugs for the treatment of central nervous system (CNS) and
cardiovascular system (CVS)-related diseases,4,5 and vincristine,
a member of the Vinca alkaloids and a mitotic inhibitor, was dis-
covered in the 1960s and clinically used for cancer chemo-
therapy.4 Since then, indole chemistry has received increasing
interest in diverse drug discovery programs. Derivatives have
been developed as antiinflammatory,6–8 antidepression,2 anti-
hypertensive,9,10 and antiosteoporosis11 agents, among others.
In addition, indole-based molecules have been used as molecular
probes and pharmacological tools.12–14 In this regard, a great
and related chemical derivatives, such as using transition metal-
catalyzed cross coupling reactions,15 multi-component reac-
tions,16 microwave-assisted organic synthesis (MAOS),17 and
solid-phase organic synthesis (SPOS).18,19
It is generally agreed that chronic inflammation is an
important contributing factor in the process of carcinogenesis.20
Various transcriptional factors, including nuclear factor-κB
(NFκB), interferon regulatory factor-1 (IRF-1), signal transducer
and activator of transcription-1α (STAT-1α), activating protein-1
(AP-1), T-cell factor 4 (TCF-4), forkhead (Drosophila) homolog
rhabdomyosarcoma-like 1 (FKHRL1), nuclear receptors, gluco-
corticoid receptor-α, and -β, and estrogen receptor-α and -β,
have been reported to regulate inducible nitric oxide synthase
(iNOS) expression.21 Nitric oxide (NO) is produced from L-argi-
nine by NOS.22 This substance serves as a cellular signaling
molecule and may be generated as an inflammatory response due
to cellular stress and bacterial infection.23 However, overproduc-
tion can lead to tumor promotion and progression,24 so NOS
inhibitors have therapeutic potential for the prevention and treat-
ment of inflammation and cancer.
Department of Pharmaceutical Sciences, College of Pharmacy,
University of Hawai’i at Hilo, Hilo, HI, USA.
E-mail: dianqing@hawaii.edu; Fax: +1 808 933 2974;
Tel: +1 808 933 2960
†Electronic supplementary information (ESI) available: Copies of 1H,
13C NMR spectra, and HPLC chromatographs of target compounds. See
DOI: 10.1039/c2ob26456k
In addition, in part due to facilitating iNOS expression, NFκB
has been recognized for a dominant role in inflammation and
This journal is © The Royal Society of Chemistry 2012
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Fig. 1 Selected examples of drug molecules with an indole scaffold.
cancer.25–27 Inactivation of the NFκB pathway can decrease the
probability of tumor initiation and progression, and activate
apoptosis and decrease cell proliferation.28–34 Accordingly,
investigations have been conducted to discover inhibitors of
NFκB with potential as promising anticancer and cancer chemo-
prevention agents.35
With lipopolysaccharide (LPS)-stimulated RAW 264.7 cells,
NFκB binds to the promoter region of the iNOS gene and plays
an important role in iNOS expression and NO production.36
Thus, this cell line can be used as a model for studying inhibi-
tory activity. In our ongoing effort of developing antiinflamma-
tory and cancer preventive agents, we have employed natural
product-inspired and high throughput screening approaches.37
Recently, during the course of evaluating the biological potential
of a commercial compound library, 2-phenylindole 1 was identifi-
ed as an inhibitor of nitrite production and NFκB with modest
IC50 values of 38.1 1.8 and 25.4 2.1 μM, respectively.
Thus far, no phenylindole derivatives have been described in
the literature as inhibitors of NO production and NFκB regu-
lation. Accordingly, on the basis of this 2-phenylindole lead, we
sought to synthesize a diverse array of indole derivatives by sys-
tematically modifying the functional groups on the 2-phenyl-
indole motif using one-pot palladium-catalyzed Sonogashira-
type alkynylation and base-assisted cycloaddition. Here we
report the synthesis and biological evaluation of 2-phenylindole
derivatives.38
Scheme 1 Chemical derivatizations of 1.
In contrast, when benzyl, propargyl, and prenyl bromides were
used as alkylating agents, lower yields were obtained due to
the formation of their corresponding 1,3-disubstituted indole
derivatives (2b′, 2d′, and 2e′). The reaction of 1 with (Boc)2O
proceeded smoothly and afforded N-Boc-2-phenylindole (2f) in
81% yield.41 For comparison, the reduction of 1 was studied to
examine if the aromatic indole system plays an important role in
its biological activity. The 2-phenylindoline (3) was afforded in
72% yield under NaBH3CN in acetic acid (Scheme 1).42
Based on our initial screening data, compared with 1, the
2-phenylindole-3-carboxaldehyde 4 showed much less inhibitory
activity in the nitrite and NFκB assays. Then we chose to syn-
thesize 3-substituted 2-phenylindole derivatives to evaluate the
effects of different substituents in the 3-position of the 2-phenyl-
indole scaffold. Chemical transformations based on 4 were
undertaken to generate a series of 3-functionalized derivatives,
such as oxime and cyano derivatives (Scheme 2). Several substi-
tuted 2-phenyl-1H-indole-3-carboxaldehyde oximes 5 and 6a–b
were then prepared by reacting 4 and the hydrochloride salt of
hydroxylamine, methoxylamine, and O-benzylhydroxylamine,
respectively. All reactions proceeded smoothly to produce the
corresponding oxime-derivatives in excellent yields.43,44 Under
ultrasound irradiation for 2 h with Cu(OAc)2 as the catalyst, the
3-cyano-2-phenylindole 7 could be obtained from 5 in 84%
yield.45
Results and discussion
Chemistry
Our investigation started with chemical derivatizations of
2-phenylindole 1. N-Alkylation of 1 took place in moderate to
high yields on treatment with NaH and DMF or THF at room
temperature (Scheme 1).39 Deprotonation of 1 with NaH as the
base, followed by reaction with MeI40 or MOMCl, afforded the
N-alkylation derivatives 2a or 2c in 94% yields, respectively.
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were carried out at a compound concentration of 50 μM. If a
compound displayed over 60% inhibition, further evaluations
were then performed for the determination of IC50 values.
Nitrite inhibitory activity. As summarized in Table 2, the syn-
thetic 2-phenylindole 10aa showed comparable inhibitory
activity (IC50 = 24.7 μM) to commercial sample 1 (IC50
=
38.1 μM). To further evaluate the influence of the N-substituents,
the alkylation derivatives of 2-phenylindole (2a–2e) and the
N-Boc derivative 2f were synthesized and tested. Compound 2d
(IC50 = 44.3 μM) with a propargyl substituent and compound 2f
(IC50 = 34.2 μM) with a N-Boc group showed similar moderate
inhibitory effects against nitrite production. However, the inhibi-
tory effects of 2d and 2f were accompanied by cytotoxicity with
IC50 values of 46.8 and 38.2 μM, respectively. Other alkylated
indole products (2a–2c and 2e) were mainly inactive and exhi-
bited less than 50% inhibition at a concentration of 50 μM.
These data indicate an alkylation substituent at the N1 position
may have a detrimental effect on NO inhibitory activity.
Compound 3, the reduced variant of 1, was essentially in-
active, indicating the importance of retaining the intact aromatic
indole scaffold. From the 3-oxime series of 2-phenylindole, it
was observed that 2-phenyl-1H-indole-3-carboxaldehyde oxime
5 showed the best inhibitory activity (IC50 = 4.4 μM). Com-
pound 6a, with the N-methoxyl oxime functionality, exhibited a
weaker response (IC50 = 22.5 μM) in comparison with 5. The
bulky benzyl substituted product 6b was less effective. On the
other hand, compound 7, with a 3-cyano group, exhibited inhibi-
tory activity comparable with compound 5 (IC50 = 4.8 μM).
These data suggest that both the size and electronic character of
the substituent at the 3-position of 2-phenylindole have an effect
on the inhibitory activity.
When different functional substituents were introduced to
investigate the influence of the 2-phenyl group on activity, vari-
able results were obtained. Compared with 2-phenylindole 10aa,
compound 10ab (2′-Me, IC50 = 2.8 μM) exhibited much more
potent activity, whereas compound 10ac was essentially inactive
(4′-Me, 39.0% at 50 μM). However, 10ab was also cytotoxic,
yielding a cytotoxic IC50 value of 32.9 μM. This effect is likely
due to the steric hindrance of the 2′-Me group and subsequent
conformational change of the 2-phenylindole moiety.
We also investigated the effect of halogen atoms attached to
the phenyl group. Derivative 10ad, bearing two chloro atoms,
showed moderate inhibitory activity (IC50 = 45.0 μM). A
reduction in activity was obtained by introducing two fluoro
atoms (10ae, 23.5% inhibition at 50 μM). Interestingly, the inter-
mediate 10ae′ (IC50 = 4.6 μM) showed much better inhibitory
activity than the final cycloaddition product 10ae. Further
improved activity was observed with compound 10af (4′-F
group; IC50 = 17.9 μM). In contrast, the corresponding 4′-Br
derivative (10ak) showed less activity (42.4% at 50 μM). Other
groups, such as methoxyl (10ag–10ai), trifluoromethoxyl (10aj)
Scheme 2 Chemical transformations of 4.
To further expand the chemical diversity of 2-phenylindole
derivatives, a focused 2-phenylindole library 10 was next
synthesized employing a one-pot, two-step, Sonogashira-type
alkynylation and base-assisted cycloaddition from an array of
commercially available 2-iodoanilines 8 and terminal alkynes 9
(Table 1).46 The reaction was monitored by HPLC to make sure
that the diaryl alkyne intermediate was transformed into the
final cyclized indole product. Based on the optimized procedure,
the scope and generality of Sonogashira-type alkynylation and
base-assisted cycloaddition were examined.
As shown in Table 1, in general, good functional group com-
patibility was observed among the terminal alkynes tested. Both
electron-donating and electron-withdrawing substituents at the
para, meta, or ortho positions of the 2-aryl ring were tolerated,
producing the corresponding indole products 10aa–10at in
moderate to good yields from 2-iodoaniline 8a (R1 = H)
(entries 1–21, Table 1). Importantly, all the Sonogashira-type
alkynylation reactions occurred smoothly within 1 h. With
regarding to base-assisted cycloaddition, phenylacetylene
with electron-withdrawing groups reacted faster than those with
electron-donating groups. Unfortunately, 3-ethynylbenzoic acid
was found to be a poor substrate under these conditions (entry
14). Overall, terminal alkynes with heteroaromatic ring systems,
such as pyridine and thiophene moieties, also reacted well, pro-
viding the corresponding indole derivatives in moderate yields
(entries 18 and 20). However, the reaction of 2-ethynylpyridine
was totally repressed compared with 3-ethynylpyridine, possibly
due to the chelation of the 2′-nitrogen atom and the alkyne group
with the palladium catalyst (entry 19).47 In contrast to the aro-
matic alkynes, when aliphatic terminal alkynes, such as ethynyl-
cyclohexane and ethynylcyclopentane were introduced in the
reaction, no pure products could be isolated (data not shown).
Meanwhile, the scope of substrate 8 was also briefly surveyed;
different substituted 2-iodoanilines proved to be reactive under
the same conditions (entries 22–28). It is important to note that
electron-withdrawing groups on the substrates significantly
facilitate the reaction, which is consistent with a previous
report.46 For example, with the nitro or nitrile groups, the reac-
tion proceeded to completion even at room temperature and the
final products were obtained in high yields (entries 22, 23, and
26). The 5-amino derivative 10ha was also synthesized by
reduction of 5-nitro-2-phenyl-1H-indole 10ba in 67% yield,
employing Pd/C (10%) as catalyst under a hydrogen atmosphere
(entry 29).48
and long alkyl chain (10al) were also tested; 10ag (IC50
=
29.6 μM) with 3′,5′-dimethoxyl groups demonstrated the most
potent activity in this series. The mono-substituted methoxyl
derivative (10ah, 4′-OMe, 48.6% inhibition at 50 μM; 10ai,
3′-OMe, IC50 = 49.7 μM) showed much weaker inhibitory activity.
By appending an amine group in the benzene ring, compound
10an with a 3′-amino group (IC50 = 30.1 μM) produced better
Biological activities
All target compounds were tested as inhibitors of NO production
and as the regulators of NFκB activity. Initial determinations
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Table 1 Synthesis of indole derivatives 10 from 2-iodoanilines 8 and terminal alkynes 9a
Entry
1
R1
H
R2
H
Product
Yieldb (%)
70
2
H
2′-Me
67
3
4
H
H
4′-Me
68
59
3′,4′-Dichloro
5
H
H
3′,5′-Difluoro
3′,5′-Difluoro
70
95
6c
7
8
H
H
4′-F
66
67
3′,5′-Dimethoxy
9
H
H
4′-OMe
3′-OMe
59
70
10
11
12
13
H
H
H
4′-OCF3
4′-Br
71
56
61
4′-Pentyl
14
15
H
H
3′-COOH
3′-NH2
0
60
16
17
H
H
4′-NH2
52
40
4′-NMe2
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Table 1 (Contd.)
Entry
18
R1
H
R2
Product
Yieldb (%)
60
3′-Pyridyl
19
20
21
H
H
H
2′-Pyridyl
0
3′-Thiophenyl
66
77
6′-Methoxy-naphthalen-2′-yl
22
23
5-NO2
5-NO2
H
79
84
2′-Me
24
5-Cl
H
64
25
26
27
28
29d
6-Cl
H
H
H
H
H
59
84
83
25
67
5-CN
5-Br
6-Me
5-NH2
a Conditions: 0.5 mmol of substrate 8, 0.75 mmol of substrate 9, 3 mol% of PdCl2(PPh3)2, 3 mol% of CuI, 1.5 equiv. of NHEt2, 10 equiv. of NaOH,
and 3 mL of DMA. b Isolated yield after column chromatography on silica gel. c Isolated alkynylation intermediate. d 5-Amino-2-phenyl-1H-indole
10ha was synthesized via reduction of 5-nitro-2-phenyl-1H-indole 10ba.
inhibition of NO production than compound 10ao with a
4′-amino group. No improved activity could be achieved when
the amino group was substituted with two methyl groups (10ap,
28.8% inhibition at 50 μM). Changing the phenyl into a hetero-
aromatic group, the thiophen-3-yl indole derivative showed
greater activity (10as, IC50 = 20.4 μM) than the pyridine moiety
(10aq, 46.6% at 50 μM).
and its corresponding reduced variant, 5-NH2-2-phenylindole
(10ha), showed good inhibitory activities with IC50 values of
11.8 and 21.1 μM, respectively. Compounds 10bb–10ga with
Cl, CN, Br and Me substituents on the indole system were only
marginally active.
For direct comparison, 2-phenylbenzimidazole 11 was assessed
in this assay. Only marginal activity was observed (13.8% at
50 μM), which indicates that the replacement of the pyrrole ring
with the imidazole ring is detrimental for inhibitory activity.
Finally, the effect of substituents on the indole moiety was
studied. It was observed that only 5-NO2-2-phenylindole (10ba)
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Table 2 Evaluation of 2-arylindole derivatives for potential to inhibit iNOS activity and regulate NFκBa
Nitrite assay
% Inhibition
NFκB assay
Compound
% Survivalb
IC50c (μM)
Cytotoxic IC50c (μM)
% Inhibition
% Survivalb
IC50c (μM)
1
2a
2b
2b′
2c
2d
2e
2e′
2f
3
4
5
6a
6b
7
60.4 2.2
33.6 4.9
0.0
87.3 3.9
74.3 6.2
84.6 2.0
101.0 6.1
67.3 6.2
35.8 3.2
75.6 4.7
89.0 3.4
27.6 2.4
102.7 7.4
109.8 6.7
90.8 2.4
48.3 7.8
59.2 7.7
82.1 4.1
66.7 4.0
31.5 2.7
57.9 9.5
67.4 2.8
86.5 9.4
30.8 0.6
69.7 3.7
109.5 2.4
59.8 4.0
92.8 6.4
105.7 3.5
69.3 2.7
131.8 12.6
112.5 6.0
58.7 2.9
60.9 4.0
107.0 7.0
90.6 2.6
58.0 7.7
81.7 3.4
89.2 9.5
77.8 2.9
74.0 11.9
78.1 3.8
91.8 8.7
76.0 4.5
100.2 6.7
97.7 5.6
38.1 1.8
72.3 5.0
0.0
0.0
113.1 2.8
105.5 12.3
96.3 8.9
124.1 6.2
106.7 2.8
105.2 5.3
113.3 5.6
104.6 11.2
107.1 16.3
87.9 8.8
25.4 2.1
0.0
0.0
33.8 4.9
96.9 1.4
33.1 3.1
59.2 2.7
92.4 4.0
35.0 7.0
30.6 2.1
97.7 0.7
97.2 2.4
85.3 1.2
70.7 3.5
79.0 2.0
97.5 0.4
39.0 2.5
55.8 2.7
23.5 4.6
97.4 1.2
58.0 5.9
62.6 3.4
48.6 6.9
50.7 0.9
31.2 5.7
42.4 3.5
31.9 6.8
70.8 4.3
86.1 1.2
28.8 1.5
46.6 3.4
66.3 4.6
13.9 3.6
66.1 3.8
45.9 3.0
49.3 2.1
36.3 2.6
45.6 2.5
42.4 4.0
45.1 1.6
67.4 3.3
13.8 6.4
21.5 3.3
46.2 2.1
47.0 2.7
60.9 1.1
0.0
65.0 4.1
36.2 11.4
73.6 2.6
48.8 8.0
50.2 3.9
70.2 3.8
76.3 8.2
0.0
96.6 0.6
68.2 6.3
62.3 8.3
31.5 4.3
77.9 1.4
56.4 2.3
81.3 1.9
85.5 3.0
79.7 8.8
82.3 0.8
76.1 4.1
72.0 1.9
81.7 4.2
95.9 0.2
12.3 4.4
35.6 3.1
94.4 1.6
92.8 4.0
60.6 3.2
2.3 0.9
91.5 1.2
50.6 3.8
92.2 2.7
97.3 1.7
78.4 12.1
73.6 3.1
44.3 2.1
46.8 1.9
38.2 5.0
47.2 0.0
34.2 1.5
42.3 2.8
NDd
139.2 14.3
105.5 10.3
51.8 23.0
4.4 0.5
22.5 1.2
34.6 2.4
4.8 0.4
24.7 2.4
2.8 0.3
6.9 0.8
48.0 5.1
32.9 2.5
42.9 5.1
66.1 18.8
91.4 2.1
79.8 5.9
121.1 4.1
86.6 3.9
115.1 2.1
107.9 10.0
121.3 1.9
84.3 8.8
66.2 4.1
85.0 2.9
65.8 15.0
74.8 6.6
86.7 11.2
86.6 12.1
70.7 10.0
59.7 2.6
74.6 11.0
71.4 5.5
76.0 16.6
93.5 2.8
68.2 11.1
119.2 8.6
112.6 12.1
89.4 15.7
72.4 6.2
86.1 6.8
84.2 12.0
73.8 19.6
86.2 18.0
8.5 2.0
15.4 3.3
10aa
10ab
10ac
10ad
10ae
10ae′e
10af
10ag
10ah
10ai
10aj
10ak
10al
10an
10ao
10ap
10aq
10as
10at
10ba
10bb
10ca
10da
10ea
10fa
10ga
10ha
11f
1.5 0.3
40.2 3.5
48.3 1.5
45.0 2.5
4.6 0.6
17.9 3.1
29.6 3.8
16.5 3.7
1.4 0.1
5.8 1.1
6.5 0.1
12.5 2.0
18.1 5.0
12.8 2.0
9.0 3.3
1.5 0.7
49.7 0.2
30.1 2.1
21.9 1.5
20.4 1.8
11.8 0.9
0.6 0.2
1.1 0.1
NDd
9.8 2.3
2.4 0.3
0.9 0.4
24.6 3.6
11.2 0.3
3.8 0.6
21.1 5.7
22.1 0.2
Positive controlg
a Concentration for screening: 50 μM. b Percentage of cell survival in comparison with vehicle-treated controls. c IC50 (μM): the half maximal
inhibitory concentration (μM); if not listed, the IC50 values were >50 μM. d ND indicates the response was not dose-dependent. e 10ae′: 2-[(3,5-
difluorophenyl)ethynyl]aniline (the intermediate of 10ae). f 2-Phenylbenzimidazole 11 was purchased from Sigma-Aldrich. g IC50 values of control
inhibitors in these assays; L-NG-monomethyl arginine for the nitrite assay and N-tosyl-L-phenylalanine-chloromethyl ketone for the NFκB assay.
NFκB inhibitory activity. In the NFκB assay, 2-phenylindole
10aa displayed moderate inhibitory activity (IC50 = 15.4 μM),
whereas the commercial sample 1 exhibited an IC50 value of
25.4 μM. From the N-alkylation and N-Boc series, all com-
pounds (2a–f) were inactive at a concentration of 50 μM, except
substituents on the 3-position of 2-phenylindole scaffold may
enhance the inhibitory effect.
In terms of different substituents on the 2-phenyl ring, most
compounds tested showed significant NFκB inhibitory activity.
The substitution pattern also influenced activity. For instance,
compound 10ac bearing a 4′-Me group produced potent activity
(IC50 = 1.5 μM), while compound 10ab with a 2′-Me group was
inactive. Introducing a mono-halogen atom into the benzene ring
proved to be beneficial for activity compared with compound
10aa. For example, compound 10af with a 4′-F and 10ak with a
4′-Br on the benzene ring showed slightly improved activities
(10af, IC50 = 16.5 μM; 10ak, IC50 = 12.5 μM). In contrast, both
compounds 10ad with two chloro atoms and 10ae with two
the diprenyl substituted 2-phenylindole derivative (2e′, IC50
=
42.3 μM). In the oxime series (5 and 6a–b), only compound 5
with a N-hydroxyl group exhibited good NFκB inhibitory
activity (IC50 = 6.9 μM). These data demonstrated the impor-
tance of the free N-hydroxyl group. Similar to the pattern
observed in the nitrite assay, compound 7 with a cyano group in
the 3-position also showed stronger inhibitory activity (IC50
=
8.5 μM). Taken together, it appears that small and polar
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fluoro atoms showed a significant decrease in NFκB inhibition,
with IC50 values of 40.2 and 48.3 μM, respectively. For compari-
son, the intermediate 10ae′ was also included in the assay and it
displayed lower activity than the final cycloaddition product
10ae.
As presented in Table 2, it appears the methoxy group affected
the NFκB inhibition in a manner similar to halogen atoms. Com-
pound 10ag, bearing 3′,5′-dimethoxy on the phenyl ring, was
inactive and only exhibited 56.4% inhibition at 50 μM. In con-
trast, when one methoxy group is attached, both compounds
10ah (4′-OMe) and 10ai (3′-OMe) showed much more potent
inhibitory activities, with IC50 values of 1.4 and 5.8 μM, respecti-
vely. The most potent inhibitor obtained was compound 10at
(IC50 = 0.6 μM), bearing one methoxy group on the naphthalene
moiety. This was probably due to the enhanced and favorable
lipophilicity profile.
Compared with the free amino-substituted compounds 10an
(3′-NH2) and 10ao (4′-NH2), compound 10ap exhibited higher
inhibition of NFκB with an IC50 of 1.5 μM. Less than 40% inhi-
bition at 50 μM was produced when the phenyl group was
replaced with heteroaromatic rings, including the 3-pyridyl and
3-thiophenyl moieties. For substituents on the indole scaffold,
compounds 10ba with a 5-NO2 group and 10ga with a 6-Me
group demonstrated significant inhibitory activities with IC50
values of 1.1 and 0.9 μM, respectively. For the halogen substi-
tuted compounds, 10da and 10fa showed intermediate activities
with IC50 values of 9.8 and 2.4 μM, respectively. In addition,
2-phenylbenzimidazole 11 mediated an inhibitory response with
an IC50 value of 11.2 μM.
Fig. 2 The correlation between inhibitory activities of eleven com-
pounds (closed diamond) in nitrite and NFκB assays. Trend analysis
using Microsoft Office Excel 2007: R2 = 0.76. 10ai with an IC50 value
of 49.7 μM (round number as 50 μM), is excluded.
fibroblasts via different mechanisms.50 In the final analysis,
further studies are required to clarify underlying mechanisms.
In considering compounds mediating superior activity in one
of our assay systems, cleavage of the pyrrole ring in 10ae led to
loss of NFκB inhibitory activity but improved inhibition of
nitrite production. The location and number of fluoro atoms at
the 2-phenyl ring might be crucial for the dual activities. Com-
pound 10ae with the 3′,5′-difluoro substituents showed NFκB
inhibition while 10af with the 4′-F substituent exerted dual inhi-
bition. Similarly, the location and number of methoxy (–OMe)
groups affected the inhibitory activities (10ah, 10ai vs. 10ag).
Modifications of the oxime functionality (vN–OH) in 5 with
a methyl or a benzyl group resulted in loss of NFκB activities,
suggesting the crucial role of the free oxime functionality in phe-
nylindoles for NFκB inhibition. Also, the introduction of the
amino group (–NH2) at the 3′, 4′, or 5-position of the 2-phenyl-
indole moiety (10an, 10ao, and 10ha) showed dual activities,
while the NMe2-substituted compound (10ap) lost inhibitory
activity in nitrite production. Both hydroxyl (–OH) and amino
(–NH2) groups can be involved in hydrogen bonding, therefore,
the inhibitory effects might be associated with hydrogen bonds
with these compounds. A preliminary SAR is summarized in
Fig. 3.
Correlation between NOS and NFκB and preliminary SAR
considerations. Several factors can affect the production of NO
including the cellular level of L-arginine as an iNOS substrate
and the enzymatic activity or expression level of iNOS.49 In the
assay employed for this investigation, increased production of
NO results mainly from iNOS expression following stimulation
with LPS. One potential mechanism of the inhibitory effect
mediated by a test compound is inhibition of NFκB activity, one
of the transcription factors related to iNOS expression.21 In
support of this suggestion, among the compounds tested with
IC50 values under 50 μM in both the nitrite and NFκB assays
(1, 2e′, 5, 7, 10aa, 10ad, 10af, 10an, 10ao, 10ba, and 10ha),
linear regression analysis revealed a correlation coefficient of
R² = 0.76 (Fig. 2).
However, a lack of correlation with other compounds is not
surprising. First of all, the NFκB and nitrite assays were estab-
lished using different experimental conditions, e.g., different
stimuli (TNFα vs. LPS) with different signaling pathways (TNF
receptor vs. Toll-like receptor 4) in different cell lines (HEK293
vs. RAW 264.7). Compounds exerting inhibitory effects on
nitrite production without NFκB inhibition might suppress other
signaling pathways such as JAK2/STAT1 and MAPK. On the
other hand, compounds showing only NFκB inhibition might
inhibit the TNF receptor directly. In addition, despite structural
similarities through sharing the phenylindole moiety, distinctly
different mechanisms of actions may apply. For instance, it has
been reported that resveratrol and an analog 4,4′-dihydroxy-
trans-stilbene mediate an antiproliferative response in human
Conclusions
In conclusion, a series of 2-phenylindole derivatives has been
synthesized. The reactions employing one-pot palladium-
catalyzed Sonogashira-type alkynylation and base-assisted cyclo-
addition feature broad substrate scope. This indole-based
chemical library was subsequently evaluated for inhibition of
nitrite production and NFκB activity. The 2-phenylindole-3-
carboxaldehyde oxime 5 showed the most potent inhibition
of nitrite production (IC50 = 4.4 μM) among this series of
This journal is © The Royal Society of Chemistry 2012
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was added dropwise to the reaction mixture. Upon disappearance
of the starting material as indicated by TLC (1 h), the reaction
mixture was poured into ice water and extracted with EtOAc (3 ×
10 mL). The organic layer was combined and dried over Na2SO4
and the solvent was removed in vacuo. The crude material was
purified by flash column chromatography on silica gel (hexane/
EtOAc = 95/5) to yield 97.2 mg (94%) of 2-phenyl-N-methyl-
indole (2a) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.57
(d, J = 8.0 Hz, 1H), 7.43 (d, J = 6.8 Hz, 2H), 7.37 (t, J = 7.6 Hz,
2H), 7.32–7.28 (m, 2H), 7.17 (t, J = 8.0 Hz, 1H), 7.07 (t, J = 8.0
Hz, 1H), 6.49 (s, 1H), 3.63 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 141.6, 138.5, 132.9, 129.4, 128.5, 128.1, 127.9, 121.7,
120.6, 119.9, 109.7, 101.7, 31.2; ESI-MS: calc. for C15H13N
[M + H]+: 208.1, found: 208.1. HPLC purity: 98.7% (254 nm),
tR: 7.47 min; 97.6% (220 nm), tR: 7.46 min.
Fig. 3 Preliminary SAR of 2-phenylindole derivatives.
compounds without appreciable cytotoxicity (90.8 2.4% survi-
val at the highest concentration of 50 μM tested). Stronger inhi-
bition of NO production was observed with the 2′-tolyl indole
derivative 10ab (IC50 = 2.8 μM). Although notable cytotoxicity
was observed at 50 μM as 31.5 2.7% cell survival with an IC50
value of 32.9 μM, the cytotoxic effect of 10ab did not affect the
inhibitory activity (e.g., the cell survival was 94.5% at the IC80
concentration, 9.3 μM). With NFκB, most 2-phenylindole
derivatives exhibited good to excellent inhibitory activities,
especially the 6′-MeO-naphthalen-2′-yl indole derivative 10at
(IC50 = 0.6 μM). It is also important to note that compounds 5
and 7 inhibit both NOS and NFκB with IC50 values ranging
from 4.4–8.5 μM. Based on these promising results and existing
SAR, further medicinal chemistry optimization is warranted to
identify more potent and less toxic 2-arylindole dual NOS/NFκB
inhibitors with antiinflammatory and anticancer therapeutic
potential.
2-Phenyl-N-benzylindole 2b. White solid; yield 64%; 1H
NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.44–7.36 (m, 5H),
7.28–7.22 (m, 3H), 7.17–7.15 (m, 3H), 7.03–7.02 (m, 2H),
6.67–6.65 (m, 1H), 5.36 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 141.9, 138.3, 138.0, 132.8, 129.3, 128.8, 128.6, 128.4, 128.1,
127.2, 126.1, 122.0, 120.6, 120.3, 110.6, 102.4, 47.8; ESI-MS:
calc. for C21H17N [M + H]+: 284.1, found: 284.2. HPLC purity:
100% (254 nm), tR: 7.83 min; 100% (220 nm), tR: 7.82 min.
2-Phenyl-3-benzyl-N-benzylindole 2b′. Yellow solid; yield
1
28%; H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.0 Hz, 1H),
7.35 (t, J = 3.2 Hz, 3H), 7.30–7.28 (m, 2H), 7.23–7.11 (m,
10H), 7.06 (t, J = 6.8 Hz, 1H), 6.95 (d, J = 6.8 Hz, 2H), 5.25 (s,
2H), 4.09 (s, 2H); 13C NMR (400 MHz, CDCl3) δ 141.9, 138.9,
138.4, 137.0, 131.7, 130.5, 128.6, 128.4, 128.2, 127.0, 126.1,
125.6, 121.9, 119.6, 112.2, 110.3, 47.6, 30.7; ESI-MS: calc. for
C28H23N [M + H]+: 374.2, found: 374.3. HPLC purity: 100%
(254 nm), tR: 8.16 min; 100% (220 nm), tR: 8.15 min.
Experimental section
Chemistry
All reagents and anhydrous solvents were purchased from com-
mercial sources and were used without further purification. All
reactions were monitored either by thin-layer chromatography
(TLC) or by analytical high performance liquid chromatography
(HPLC) to detect the completion of reactions. Hydrogenation
reactions were performed employing a domnick hunter NITROX
UHP-60H hydrogen generator, USA. Compounds were purified
by flash column chromatography on silica gel using a Biotage
2-Phenyl-N-methoxymethylindole 2c. White solid; yield 94%;
1H NMR (400 MHz, CDCl3) δ 7.60 (t, J = 7.2 Hz, 3H), 7.49 (d,
J = 8.0 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 7.2 Hz,
1H), 7.24 (t, J = 7.2 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.59 (s,
1H), 5.37 (s, 2H), 3.25 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 141.7, 138.3, 132.4, 129.4, 128.5, 128.3, 128.1, 122.3, 120.7,
120.5, 110.2, 103.4, 74.7, 55.8; ESI-MS: calc. for C16H15NO
[M + H]+: 238.1, found: 238.2. HPLC purity: 100% (254 nm),
tR: 7.37 min; 100% (220 nm), tR: 7.37 min.
1
Isolera One system and a Biotage SNAP cartridge. H and 13C
NMR spectra were obtained on a Bruker Avance DRX-400
instrument with chemical shifts (δ, ppm) determined using TMS
as internal standard. Coupling constants (J) are in hertz (Hz).
ESI mass spectra in either positive or negative mode were pro-
vided by Varian 500-MS IT Mass Spectrometer. The purity of
compounds was determined by analytical HPLC using a Gemini,
3 μm, C18, 110 Å column (50 mm × 4.6 mm, Phenomenex) and
a flow rate of 1.0 mL min−1. Gradient conditions: solvent A
(0.1% trifluoroacetic acid in water) and solvent B (acetonitrile):
0–2.00 min 100% A, 2.00–7.00 min 0–100% B (linear gradient),
7.00–8.00 min 100% B, UV detection at 254 and 220 nm.
1
2-Phenyl-N-propargylindole 2d. White solid; yield 70%; H
NMR (400 MHz, CDCl3) δ 7.59 (dd, J = 8.0 and 7.2 Hz, 3H),
7.45 (dd, J = 8.8 and 8.4 Hz, 3H), 7.37 (t, J = 7.2 Hz, 1H), 7.27
(t, J = 8.0 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.57 (s, 1H), 4.76
(d, J = 2.4 Hz, 2H), 2.30 (t, J = 2.4 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 140.9, 137.6, 132.2, 129.2, 128.7, 128.3,
128.1, 122.1, 120.7, 120.5, 110.0, 102.5, 78.9, 72.7, 34.0;
ESI-MS: calc. for C17H13N [M + H]+: 232.1, found: 232.1.
HPLC purity: 100% (254 nm), tR: 7.40 min; 100% (220 nm), tR:
7.40 min.
Representative procedure for the alkylation of 2-phenylindoles
(2a–e). A solution of 2-phenylindole (96.5 mg, 0.5 mmol) in
DMF (1 mL) was added dropwise to a slurry of NaH (24 mg,
0.6 mmol, 60% dispersed in mineral oil) in DMF (2 mL) at
0 °C. After stirring for 1 h, neat iodomethane (71 mg, 0.5 mmol)
2-Phenyl-N-prenylindole 2e. Oil; yield 8%; 1H NMR
(400 MHz, CDCl3) δ 7.62 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 7.6
Hz, 2H), 7.45 (t, J = 7.2 Hz, 1H), 7.40–7.29 (m, 3H), 7.23 (t,
J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.54 (s, 1H),
5.32–5.30 (m, 1H), 4.71 (d, J = 6.0 Hz, 2H), 1.69 (s, 3H), 1.33
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(s, 3H); 13C NMR (100 MHz, CDCl3) δ 141.3, 137.6, 134.2,
133.0, 129.5, 128.4, 128.2, 127.8, 121.5, 121.1, 120.5, 119.8,
110.2, 101.7, 42.6, 25.5, 17.9; ESI-MS: calc. for C19H19N [M +
H]+: 262.2, found: 262.2. HPLC purity: 76.4% (254 nm), tR:
7.97 min; 81.1% (220 nm), tR: 7.97 min; with the remaining of 2e′.
Synthesis of 2-phenylindole-3-carboxaldehyde oxime 5.
Hydroxylamine hydrochloride (104 mg, 1.5 mmol) was added to
a solution of 2-phenylindole-3-carboxaldehyde 4 (221 mg,
1 mmol) and sodium hydroxide (60 mg, 1.5 mmol) in methanol
(5 mL). The reaction mixture was stirred at room temperature
until no starting material was detected by TLC and HPLC analy-
sis (3 h). The reaction mixture was evaporated in vacuo and
EtOAc was added to the residue. The solution was washed with
water, and the organic layer was dried over Na2SO4. The solvent
was removed under reduced pressure and the residue was
purified by flash column chromatography on silica gel to give
the 2-phenylindole-3-carboxaldehyde oxime 5 (197.2 mg, 83%)
2-Phenyl-3-prenyl-N-prenylindole 2e′. Oil; yield 6%; 1H
NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.0 Hz, 1H), 7.44 (t, J =
7.2 Hz, 2H), 7.38 (t, J = 6.8 Hz, 3H), 7.30 (d, J = 8.0 Hz, 1H),
7.21 (t, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 5.29 (t, J =
7.2 Hz, 1H), 5.20 (t, J = 6.4 Hz, 1H), 4.56 (d, J = 6.4 Hz, 1H),
3.35 (d, J = 6.8 Hz, 1H), 1.65 (s, 6H), 1.52 (d, J = 4.0 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ 137.3, 136.4, 133.9, 132.3,
130.9, 130.3, 128.2, 127.9, 127.8, 124.4, 121.5, 121.1, 119.2,
119.1, 112.8, 109.8, 42.3, 25.6, 25.5, 23.9, 17.74, 17.71;
ESI-MS: calc. for C24H27N [M + H]+: 330.5, found: 330.3.
HPLC purity: 85.1% (254 nm), tR: 8.46 min; 86.4% (220 nm),
tR: 8.46 min; with the remaining of 2e.
1
as a white solid. H NMR (400 MHz, d6-DMSO) δ 11.75 (s,
1H), 10.73 (s, 1H), 8.30 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.62
(t, J = 8.8 Hz, 2H), 7.56 (t, J = 7.6 Hz, 2H), 7.49–7.44 (m, 3H),
7.22 (t, J = 7.2 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H); 13C NMR
(100 MHz, d6-DMSO) δ 144.4, 139.7, 136.4, 131.4, 129.0,
128.6, 125.6, 122.8, 121.9, 120.5, 111.5, 105.9; ESI-MS: calc.
for C15H13N2O [M + H]+: 237.1, found: 237.1. HPLC purity:
100% (254 nm), tR: 6.49 min; 100% (220 nm), tR: 6.49 min.
Synthesis of 2-phenyl-N-Boc-indole (2f). Under nitrogen
atmosphere, (Boc)2O (d 0.95 g mL−1, 0.125 mL, 0.55 mmol)
was added to a solution of 2-phenylindole (96.5 mg, 0.5 mmol)
and 4-(N,N-dimethylamino)pyridine (DMAP) (1.8 mg,
0.015 mmol) in dry acetonitrile (3 mL) at room temperature. The
solution was stirred at room temperature for 24 h, and then was
evaporated under reduced pressure. After water was added, the
resulting mixture was extracted twice with EtOAc (2 × 10 mL).
The combined organic layer was washed with brine, dried with
Na2SO4, and evaporated under reduced pressure. The residue
was purified with flash column chromatography on silica gel
(EtOAc/hexane = 1/50) to give the pure product 2f (118.4 mg,
Representative procedure for the preparation of oximes 6a
and 6b. Methoxylamine hydrochloride (50 mg, 0.6 mmol,
1.2 equiv.) was suspended in 1 mL of absolute ethanol, and
anhydrous pyridine (158 mg, 2 mmol, 4.0 equiv.) was added
quickly dropwise. Then 2-phenylindole-3-carboxaldehyde 4
(110.5 mg, 0.5 mmol) was added and the reaction was stirred at
room temperature for 4 h. The ethanol was removed and the
residue was redissolved in dichloromethane and washed with
water. The organic layer was dried with Na2SO4, filtered and
evaporated to give a crude oil, which was purified by flash
chromatography to afford a clear oil as product 6a in 91% yield.
2-Phenyl-1H-indole-3-carboxaldehyde O-methyl oxime 6a: 1H
NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.38–8.36 (m, 2H),
7.46–7.44 (m, 5H), 7.37–7.36 (m, 1H), 7.30–7.28 (m, 2H), 4.06
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 145.2, 140.6, 136.1,
131.2, 129.1, 128.9, 128.8, 125.9, 123.7, 122.9, 121.6, 111.0,
106.6, 61.9; ESI-MS: calc. for C16H14N2O [M + H]+: 251.3,
found: 251.2. HPLC purity: 100% (254 nm), tR: 7.19 min; 100%
(220 nm), tR: 7.19 min.
1
81%) as a colorless solid. H NMR (400 MHz, CDCl3) δ 8.22
(d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.42–7.30 (m,
6H), 7.24 (t, J = 7.2 Hz, 1H), 6.54 (s, 1H), 1.29 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ 150.2, 140.5, 137.4, 135.0, 129.2,
128.7, 127.7, 127.5, 124.2, 122.9, 120.4, 115.2, 109.9, 83.3,
27.5; ESI-MS: calc. for C19H19NO2 [M − H]−: 292.1, found:
292.3. HPLC purity: 100% (254 nm), tR: 7.98 min; 100%
(220 nm), tR: 7.97 min.
Typical procedure for the reduction of 2-phenylindole to
2-phenylindoline 3. To a solution of 2-phenylindole (96.5 mg,
0.5 mmol) in AcOH (2 mL) was added NaBH3CN (189 mg,
3 mmol), and the resulting mixture was stirred until no starting
material could be detected by TLC analysis (24 h). Then, 6 mL
of H2O was added and additional NaOH pellets were added until
pH > 12, the resulting solution was extracted with Et2O (3 ×
10 mL). The organic phases were combined, dried over Na2SO4
and the solvent was evaporated under reduced pressure to give
the residue, which was purified by flash column chromatography
on silica gel to afford the pure product 2-phenylindoline 3
(70.5 mg, 72%). Oil; 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J =
7.2 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.6 Hz, 1H),
7.07–7.03 (m, 2H), 6.71 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 7.6 Hz,
1H), 4.91 (t, J = 8.8 Hz, 1H), 3.41 (dd, J = 8.8 and 15.6 Hz,
1H), 2.96 (dd, J = 8.8 and 15.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 150.9, 144.6, 128.5, 128.0, 127.5, 127.3, 126.2, 124.5,
118.8, 108.8, 63.4, 39.5; ESI-MS: calc. for C14H13N [M + H]+:
196.1, found: 196.2. HPLC purity: 100% (254 nm), tR:
5.68 min; 100% (220 nm), tR: 5.68 min.
2-Phenyl-1H-indole-3-carboxaldehyde
O-benzyl
oxime
6b. Oil, yield: 89%; 1H NMR (400 MHz, CDCl3) δ 8.40 (s,
1H), 8.28 (s, 1H), 8.18 (s, 1H), 7.46 (d, J = 7.2 Hz, 2H),
7.33–7.28 (m, 7H), 7.28–7.22 (m, 4H), 5.23 (s, 2H); 13C NMR
(100 MHz, CDCl3) δ 145.6, 140.7, 137.9, 136.0, 131.2, 129.0,
128.9, 128.7, 128.5, 127.9, 126.0, 123.6, 123.0, 121.6, 111.0,
106.6, 76.3; ESI-MS: calc. for C22H18N2O [M + H]+: 327.4,
found: 327.2. HPLC purity: 100% (254 nm), tR: 7.63 min; 100%
(220 nm), tR: 7.63 min.
Synthesis of 2-phenylindole-3-carbonitrile 7. 2-Phenylindole-
3-carboxaldehyde oxime 5 (38.7 mg, 0.16 mmol), Cu(OAc)2
(1.5 mg, 5 mol%) and acetonitrile (1.0 mL) were added to a
10 mL tube. After being capped, the reaction mixture was under
continuous ultrasound irradiation for 2 h. Following sonication,
the reaction mixture was concentrated in vacuo and the residue
was purified by flash column chromatography on silica gel
(EtOAc/hexane = 1/8) to afford 2-phenylindole-3-carbonitrile 7
(29.3 mg, 84%) as a white solid. 1H NMR (400 MHz,
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d6-DMSO) δ 12.59 (s, 1H), 7.98 (d, J = 7.6 Hz, 2H), 7.65–7.60
(m, 3H), 7.58–7.52 (m, 2H), 7.34–7.24 (m, 2H); 13C NMR
(100 MHz, d6-DMSO) δ 144.7, 135.5, 129.9, 129.3, 128.2,
127.0, 123.9, 122.0, 118.3, 116.9, 112.6, 81.4; ESI-MS: calc. for
C15H10N2 [M + H]+: 219.1, found: 219.1. HPLC purity: 95.3%
(254 nm), tR: 6.87 min; 99.9% (220 nm), tR: 6.87 min.
262.0. HPLC purity: 100% (254 nm), tR: 7.63 min; 100%
(220 nm), tR: 7.64 min.
2-(3,5-Difluorophenyl)-1H-indole 10ae. White solid; yield:
1
70%; H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.62 (d, J =
7.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.25–7.21 (m, 1H),
7.16–7.12 (m, 3H), 6.83 (d, J = 1.2 Hz, 1H), 6.76–6.72 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 164.8, 164.7, 162.3, 162.2,
137.0, 135.5, 128.9, 123.3, 121.1, 120.7, 111.1, 108.0, 107.9,
107.8, 107.7, 103.0, 102.8, 102.5, 101.8; ESI-MS: calc. for
C14H9F2N [M − H]−: 228.1, found: 228.2. HPLC purity: 100%
(254 nm), tR: 7.29 min; 100% (220 nm), tR: 7.30 min.
Representative procedure for the synthesis of functionalized
2-phenylindole derivatives 10. A mixture of 2-iodoaniline
(109.5 mg, 0.5 mmol), phenylacetylene (76.5 mg, 0.75 mmol),
PdCl2(PPh3)2 (10.5 mg, 3 mol%), CuI (2.85 mg, 3 mol%) in
DMA (3 mL) was stirred in a sealed tube at room temperature
until no starting material was detected by HPLC (1 h). NaOH
(200 mg, 5 mmol) was added and the reaction temperature was
raised to 140 °C for several hours (the completion of the cycliza-
tion was monitored by HPLC). The reaction was cooled to room
temperature, and EtOAc and water were added. The separated
aqueous phase was extracted with EtOAc (3 × 10 mL) and the
combined organic layers were washed with water (2 × 20 mL)
and dried over Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by silica gel column
chromatography (EtOAc/hexane = 1/20) to afford 2-phenyl-1H-
indole 10aa (67.5 mg, 70%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ 8.31 (s, 1H), 7.61 (d, J = 7.2 Hz, 3H), 7.40
(t, J = 7.2 Hz, 2H), 7.35 (dd, J = 0.8 and 8.4 Hz, 1H), 7.29 (t,
J = 7.2 Hz, 1H), 7.21–7.18 (m, 1H), 7.16–7.09 (m, 1H), 6.80 (d,
J = 0.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 137.9, 136.8,
132.4, 129.3, 128.9, 127.7, 126.1, 122.3, 120.6, 120.2, 110.9,
99.9; ESI-MS: calc. for C14H11N [M − H]−: 192.1, found:
192.3. HPLC purity: 100% (254 nm), tR: 7.12 min; 100%
(220 nm), tR: 7.11 min.
2-((3,5-Difluorophenyl)ethynyl)aniline
10ae′. Brown oil;
1
yield: 95%; H NMR (400 MHz, CDCl3) δ 7.34 (t, J = 8.0 Hz,
1H), 7.18–7.14 (m, 1H), 7.04–6.99 (m, 2H), 6.81–6.70 (m, 3H),
4.20 (s, br, 2H); 13C NMR (100 MHz, CDCl3) δ 164.0, 163.9,
161.6, 161.5, 148.1, 132.4, 120.5, 126.1, 118.1, 114.6, 114.5,
114.3, 114.2, 106.9, 104.6, 104.3, 104.1, 92.53, 92.49, 92.46,
88.1; ESI-MS: calc. for C14H9F2N [M + H]+: 230.1, found:
230.2. HPLC purity: 100% (254 nm), tR: 7.19 min; 100%
(220 nm), tR: 7.19 min.
2-(4-Fluorophenyl)-1H-indole 10af. White solid; yield: 66%;
1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.65 (d, J = 7.6 Hz,
1H), 7.62–7.58 (m, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.23 (t, J =
7.6 Hz, 1H), 7.18–7.12 (m, 3H), 6.76 (d, J = 2.0 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 163.9, 161.4, 137.2, 137.1, 129.5,
129.0, 128.9, 127.1, 127.0, 122.6, 120.8, 120.6, 116.3, 116.1,
111.1, 100.2; ESI-MS: calc. for C14H10FN [M + H]+: 212.2,
found: 212.2. HPLC purity: 100% (254 nm), tR: 7.14 min; 100%
(220 nm), tR: 7.14 min.
1
2-o-Tolyl-1H-indole 10ab. Brown solid; yield: 67%; H NMR
2-(3,5-Dimethoxyphenyl)-1H-indole 10ag. White solid; yield:
70%; H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.61 (d, J =
1
(400 MHz, CDCl3) δ 8.14 (s, 1H), 7.64 (d, J = 7.6 Hz, 1H),
7.48–7.45 (m, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.32–7.27 (m, 3H),
7.23–7.20 (m, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.61 (d, J = 1.2 Hz,
1H), 2.51 (d, J = 1.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 137.5, 136.23, 136.18, 132.7, 131.1, 129.1, 128.9, 128.0,
126.2, 122.1, 120.6, 120.1, 110.6, 103.0, 21.2; ESI-MS: calc. for
C15H13N [M − H]−: 206.1, found: 206.3. HPLC purity: 100%
(254 nm), tR: 7.23 min; 100% (220 nm), tR: 7.22 min.
7.2 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.23–7.09 (m, 2H), 6.80
(s, 3H), 6.44 (s, 1H), 3.84 (s, 6H); 13C NMR (100 MHz, CDCl3)
δ 161.3, 137.8, 136.7, 134.3, 129.1, 122.4, 120.7, 120.2,
110.9, 103.6, 100.3, 99.7, 55.4; ESI-MS: calc. for C16H15O2N
[M + H]+: 254.1, found: 254.2. HPLC purity: 100% (254 nm),
tR: 7.15 min; 100% (220 nm), tR: 7.14 min.
2-(4-Methoxyphenyl)-1H-indole 10ah. Brown solid; yield:
1
1
59%; H NMR (400 MHz, d6-DMSO) δ 11.39 (s, 1H), 7.77 (d,
2-p-Tolyl-1H-indole 10ac. Yellow solid; yield: 68%; H NMR
(400 MHz, CDCl3) δ 8.24 (s, 1H), 7.62–7.59 (m, 1H), 7.50 (dd,
J = 2.0 and 6.8 Hz, 2H), 7.34 (dd, J = 2.0 and 6.8 Hz, 1H), 7.22
(dd, J = 1.2 and 7.2 Hz, 2H), 7.19–7.16 (m, 1H), 7.15–7.08 (m,
1H), 6.77–6.76 (m, 1H), 2.37 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 138.2, 137.8, 136.8, 129.8, 129.7, 129.5, 125.2, 122.3,
120.7, 120.3, 111.0, 99.5, 21.4; ESI-MS: calc. for C15H13N
[M + H]+: 208.1, found: 208.2. HPLC purity: 100% (254 nm),
tR: 7.29 min; 100% (220 nm), tR: 7.28 min.
J = 8.8 Hz, 2H), 7.48 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 8.0 Hz,
1H), 7.05–6.97 (m, 4H), 6.73 (d, J = 2.0 Hz, 1H), 3.79 (s, 3H);
13C NMR (100 MHz, d6-DMSO) δ 159.0, 138.0, 137.1, 129.0,
126.6, 125.1, 121.3, 119.9, 119.5, 114.6, 111.3, 97.6, 55.4;
ESI-MS: calc. for C15H13ON [M + H]+: 224.1, found: 224.2.
HPLC purity: 97.8% (254 nm), tR: 7.06 min; 100% (220 nm),
tR: 7.06 min.
2-(3-Methoxyphenyl)-1H-indole 10ai. White solid; yield:
1
2-(3,4-Dichlorophenyl)-1H-indole 10ad. White solid; yield:
70%; H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.60 (d, J =
1
59%; H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.56 (d, J =
7.6 Hz, 1H), 7.32–7.29 (m, 2H), 7.18–7.08 (m, 4H), 6.82 (dd,
J = 2.0 and 7.6 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H), 3.81 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 160.2, 137.9, 136.9, 133.9,
130.2, 129.3, 122.5, 120.8, 120.4, 117.8, 113.2, 111.1, 111.0,
100.3, 55.4; ESI-MS: calc. for C15H13ON [M + H]+: 224.1,
found: 224.2. HPLC purity: 100% (254 nm), tR: 7.11 min; 100%
(220 nm), tR: 7.10 min.
2.0 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H),
7.30 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 2.0 and 8.0 Hz, 1H), 7.12
(t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.69 (d, J = 1.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 137.0, 135.3, 133.1, 132.3,
131.3, 130.9, 129.0, 126.7, 124.2, 123.0, 120.9, 120.6, 111.1,
101.2; ESI-MS: calc. for C14H9Cl2N [M + H]+: 262.0, found:
8844 | Org. Biomol. Chem., 2012, 10, 8835–8847
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2-(4-(Trifluoromethoxy)phenyl)-1H-indole 10aj. White solid;
yield: 71%; 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.65 (t,
J = 9.2 Hz, 3H), 7.39 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz,
2H), 7.23 (t, J = 7.2 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 6.81 (d,
J = 1.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 148.6, 137.0,
136.5, 131.2, 129.8, 129.1, 126.4, 122.7, 121.6, 120.8, 120.5,
111.0, 100.7; ESI-MS: calc. for C15H10F3ON [M + H]+: 276.2,
found: 276.2. HPLC purity: 100% (254 nm), tR: 7.49 min; 100%
(220 nm), tR: 7.49 min.
HPLC purity: 100% (254 nm), tR: 5.73 min; 100% (220 nm), tR:
5.72 min.
1
2-(Pyridin-3-yl)-1H-indole 10aq. White solid; yield: 60%; H
NMR (400 MHz, CDCl3) δ 9.12 (s, 1H), 8.95 (d, J = 1.6 Hz,
1H), 8.51 (d, J = 4.4 Hz, 1H), 7.92–7.64 (m, 1H), 7.62 (d, J =
7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.30 (q, J = 4.8 Hz, 1H),
7.23–7.17 (m, 1H), 7.12 (t, J = 8.0 Hz, 1H), 6.85 (s, 1H); 13C
NMR (100 MHz, CDCl3) δ 148.4, 146.4, 137.6, 134.7, 132.7,
129.2, 128.9, 124.0, 123.1, 121.0, 120.7, 111.3, 101.5; ESI-MS:
calc. for C13H10N2 [M + H]+: 195.2, found: 195.2. HPLC
purity: 98.6% (254 nm), tR: 5.28 min; 98.2% (220 nm), tR:
5.27 min.
2-(4-Bromophenyl)-1H-indole 10ak. Yellow solid; yield:
1
56%; H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.62 (d, J =
7.6 Hz, 1H), 7.54 (dd, J = 1.6 and 6.4 Hz, 2H), 7.49 (dd, J = 1.6
and 6.4 Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz,
1H), 7.12 (t, J = 7.2 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 137.1, 136.8, 132.3, 131.5, 129.3,
126.7, 122.9, 121.7, 120.9, 120.6, 111.1, 100.7; ESI-MS: calc.
for C14H10BrN [M + H, 81Br]+: 274.0, found: 274.1; calc. for
C14H10BrN [M + H, 79Br]+: 272.0, found: 272.1. HPLC purity:
98.9% (254 nm), tR: 7.42 min; 96.6% (220 nm), tR: 7.42 min.
2-(Thiophen-3-yl)-1H-indole 10as. Yellow solid; yield: 66%;
1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.58 (d, J = 8.0 Hz,
1H), 7.38 (s, 3H), 7.36–7.32 (m, 1H), 7.17 (dt, J = 1.2 and 8.0
Hz, 1H), 7.09 (dt, J = 0.8 and 8.0 Hz, 1H), 6.67 (d, J = 1.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 136.7, 134.4, 134.2, 129.4,
126.8, 125.9, 122.5, 120.8, 120.5, 119.3, 110.8, 100.2; ESI-MS:
calc. for C12H9NS [M + H]+: 200.3, found: 200.1. HPLC purity:
100% (254 nm), tR: 6.99 min; 100% (220 nm), tR: 6.99 min.
2-(4-Pentylphenyl)-1H-indole 10al. Yellow solid; yield: 61%;
1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.60 (d, J = 7.6 Hz,
1H), 7.55 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.23 (d,
J = 8.0 Hz, 2H), 7.17 (t, J = 8.0 Hz, 1H), 7.10 (t, J = 8.0 Hz,
1H), 6.77 (d, J = 1.2 Hz, 1H), 2.63 (t, J = 7.6 Hz, 2H),
2-(6-Methoxynaphthalen-2-yl)-1H-indole 10at. White solid;
yield 77%; 1H NMR (400 MHz, d6-DMSO) δ 11.62 (s, 1H),
8.28 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.87 (t, J = 8.0 Hz, 2H),
7.54 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.32 (d, J =
2.8 Hz, 1H), 7.20 (d, J = 2.8 and 8.0 Hz, 1H), 7.10 (t, J = 8.0
Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.96 (d, J = 1.2 Hz, 1H), 3.88
(s, 3H); 13C NMR (100 MHz, d6-DMSO) δ 157.6, 138.0, 137.4,
133.8, 129.7, 129.0, 128.8, 127.6, 127.5, 124.4, 123.0, 121.7,
120.2, 119.6, 119.4, 111.4, 106.3, 99.0, 55.4; ESI-MS: calc. for
C19H15NO [M + H]+: 274.3, found: 274.3. HPLC purity: 100%
(254 nm), tR: 7.45 min; 100% (220 nm), tR: 7.45 min.
1.66–1.60 (m, 2H), 1.36–1.32 (m, 4H), 0.90 (t, 6.8 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) δ 142.8, 138.2, 136.8, 129.9, 129.4,
129.1, 125.2, 122.2, 120.6, 120.2, 110.9, 99.5, 35.7, 31.6, 31.1,
22.6, 14.1; ESI-MS: calc. for C19H21N [M + H]+: 264.2, found:
264.4. HPLC purity: 100% (254 nm), tR: 7.99 min; 100%
(220 nm), tR: 7.97 min.
3-(1H-Indol-2-yl)aniline 10an. White solid; yield: 60%; 1H
NMR (400 MHz, d6-DMSO) δ 11.37 (s, 1H), 7.50 (d, J = 8.0
Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.13–7.02 (m, 2H), 7.00–6.96
(m, 3H), 6.70 (t, J = 1.2 Hz, 1H), 6.56–6.53 (m, 1H), 5.15 (s,
2H); 13C NMR (100 MHz, d6-DMSO) δ 149.0, 138.7, 136.9,
132.8, 129.4, 128.6, 121.2, 119.8, 119.2, 113.5, 113.1, 111.2,
110.5, 97.9; ESI-MS: calc. for C14H12N2 [M + H]+: 209.2,
found: 209.2. HPLC purity: 100% (254 nm), tR: 5.49 min; 100%
(220 nm), tR: 5.49 min.
5-Nitro-2-phenyl-1H-indole 10ba. Yellow solid; yield: 58%;
1H NMR (400 MHz, d6-DMSO) δ 12.30 (s, 1H), 8.52 (s, 1H),
8.00 (dd, J = 1.2 and 8.8 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.55
(d, J = 9.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.6 Hz,
1H), 7.16 (s, 1H); 13C NMR (100 MHz, d6-DMSO) δ 141.6,
141.1, 140.4, 131.1, 129.2, 128.6, 128.0, 125.5, 117.1, 111.8,
101.0; ESI-MS: calc. for C14H10N2O2 [M + H]+: 239.2, found:
239.2. HPLC purity: 100% (254 nm), tR: 7.11 min; 100%
(220 nm), tR: 7.11 min.
4-(1H-Indol-2-yl)aniline 10ao. Brown solid; yield: 52%; 1H
NMR (400 MHz, d6-DMSO) δ 11.19 (s, 1H), 7.52 (d, J = 8.0
Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H),
7.00–6.93 (m, 2H), 6.63 (d, J = 8.4 Hz, 2H), 6.58 (s, 1H), 5.31
(s, 2H); 13C NMR (100 MHz, d6-DMSO) δ 148.5, 139.2, 136.7,
129.1, 126.1, 120.3, 119.9, 119.2, 119.0, 114.0, 110.8, 95.5;
ESI-MS: calc. for C14H12N2 [M + H]+: 209.2, found: 209.2.
HPLC purity: 100% (254 nm), tR: 5.51 min; 100% (220 nm), tR:
5.51 min.
5-Nitro-2-o-toyl-1H-indole 10bb. Yellow solid; yield: 84%; 1H
NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.59 (d, J = 1.6 Hz,
1H), 8.11 (dd, J = 2.4 and 9.2 Hz, 1H), 7.45 (dd, J = 6.4 and
9.2 Hz, 2H), 7.34–7.29 (m, 3H), 6.76 (s, 1H), 2.50 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 142.1, 140.8, 139.0, 136.3, 131.3,
128.9, 128.8, 128.2, 126.3, 117.7, 117.6, 110.7, 104.7, 20.9;
ESI-MS: calc. for C15H12N2O2 [M + H]+: 253.1, found: 253.1.
HPLC purity: 100% (254 nm), tR: 7.25 min; 100% (220 nm), tR:
7.25 min.
4-(1H-Indol-2-yl)-N,N-dimethylaniline 10ap. Yellow solid;
1
yield: 40%; H NMR (400 MHz, d6-DMSO) δ 11.26 (s, 1H),
7.66 (d, J = 7.2 Hz, 2H), 7.44 (d, J = 7.6 Hz, 1H), 7.34 (d, J =
7.6 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H),
6.77 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 1.6 Hz, 1H), 2.93 (s, 6H);
13C NMR (100 MHz, d6-DMSO) δ 149.9, 138.8, 136.9, 129.2,
126.1, 120.6, 120.2, 119.4, 119.2, 112.5, 111.0, 96.1, 40.1;
ESI-MS: calc. for C16H16N2 [M + H]+: 237.3, found: 237.2.
5-Chloro-2-phenyl-1H-indole 10ca. White solid; yield: 64%;
1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.60 (d, J = 8.0 Hz,
2H), 7.56 (d, J = 1.6 Hz, 1H), 7.42 (t, J = 8.0 Hz, 2H), 7.32 (t,
J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.11 (dd, J = 2.0 and
8.4 Hz, 1H), 6.72 (d, J = 1.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 139.3, 135.1, 131.8, 130.3, 129.1, 128.1, 125.8, 125.2,
This journal is © The Royal Society of Chemistry 2012
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Biological assays
122.5, 119.9, 111.8, 99.5; ESI-MS: calc. for C14H10ClN
[M + H]+: 228.1, found: 228.2. HPLC purity: 100% (254 nm),
tR: 7.40 min; 100% (220 nm), tR: 7.39 min.
Measurement of the production of NO in LPS-stimulated
RAW 264.7 murine macrophage cells (nitrite assay). The level
of NO in the cultured media was estimated by measuring the
level of nitrite due to the instability of NO and its subsequent
conversion to nitrite. The nitrite assay was performed as pre-
viously described.51 Briefly, RAW 264.7 cells were incubated in
96-well culture plates at 37 °C, 5% CO2 in a humidified air incu-
bator for 24 h. Then cells were treated with serially diluted com-
pounds for 15 min, followed by treatment with or without LPS
(1 μg mL−1) for an additional 20 h. After the incubation, nitrite
released in the cultured media was measured using Griess regent
[1 : 1 mixture (v/v) of 1% sulfanilamide in 5% H3PO4 and 0.1%
N-(1-naphthyl)ethylenediamine dihydrochloride solution], and
absorbance was measured at 540 nm. The concentration of nitrite
was calculated using a standard curve created with known con-
centrations of sodium nitrite. Under the same experimental con-
ditions, sulforhodamine B (SRB) assay was performed to
evaluate the cytotoxic effect of tested compounds on RAW
264.7 cells. After transferring 100 μL of the cultured media,
cells were fixed with 10% trichloroacetic acid, and stained with
0.4% SRB solution in 1% acetic acid. The protein-bound SRB
was dissolved in 10 mM Tris base solution, and the absorbance
was measured at 515 nm. Percentage of cell survival was calcu-
lated in comparison with the LPS-treated control.52 Initial
screening was performed at a concentration of 50 μM. Com-
pounds which exhibited over 50% inhibition of nitrite pro-
duction, and under 50% cell survival, were considered as active
and cytotoxic, respectively. Only active inhibitors of nitrite pro-
duction lacking appreciable cytotoxicity were examined in
greater detail to determine whether the inhibitory effect was
derived from a false positive cytotoxic effect, and to compare the
relative potency of the compounds (SAR).
6-Chloro-2-phenyl-1H-indole 10da. Yellow solid; yield: 64%;
1H NMR (400 MHz, d6-DMSO) δ 11.71 (s, 1H), 7.83 (d, J =
7.6 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.48–7.42 (m, 3H), 7.33
(t, J = 7.6 Hz, 1H), 7.01 (dd, J = 2.0 and 7.6 Hz, 1H), 6.91 (d,
J = 1.6 Hz, 1H); 13C NMR (100 MHz, d6-DMSO) δ 138.9,
137.6, 131.8, 129.1, 127.9, 127.5, 126.2, 125.2, 121.5, 119.9,
110.9, 98.9; ESI-MS: calc. for C14H10ClN [M + H]+: 228.1,
found: 228.2. HPLC purity: 96.8% (254 nm), tR: 7.38 min;
96.4% (220 nm), tR: 7.38 min.
5-Carbonitrile-2-phenyl-1H-indole 10ea. White solid; yield:
1
84%; H NMR (400 MHz, d6-DMSO) δ 12.14 (s, 1H), 8.05 (s,
1H), 7.88 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.47 (t,
J = 8.0 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.6 Hz,
1H), 7.04 (s, 1H); 13C NMR (100 MHz, d6-DMSO) δ 140.4,
138.9, 131.2, 129.1, 128.5, 128.4, 125.6, 125.5, 124.4, 120.8,
112.6, 101.5, 99.4; ESI-MS: calc. for C15H10N2 [M + H]+:
219.2, found: 219.2. HPLC purity: 100% (254 nm), tR:
6.95 min; 100% (220 nm), tR: 6.95 min.
5-Bromo-2-phenyl-1H-indole 10fa. White solid; yield: 83%;
1H NMR (400 MHz, d6-DMSO) δ 11.78 (s, 1H), 7.85 (d, J = 8.0
Hz, 2H), 7.71 (s, 1H), 7.47 (t, J = 8.0 Hz, 2H), 7.36 (t, J = 7.6
Hz, 2H), 7.20 (d, J = 8.8 Hz, 1H), 6.88 (s, 1H); 13C NMR
(100 MHz, d6-DMSO) δ 139.2, 135.8, 131.7, 130.5, 129.0,
127.9, 125.2, 124.0, 122.2, 113.3, 111.9, 98.3; ESI-MS: calc. for
C14H10BrN [M + H, 81Br]+: 274.2, found: 274.1; C14H10BrN
[M + H, 79Br]+: 272.2, found: 272.2. HPLC purity: 100%
(254 nm), tR: 7.47 min; 100% (220 nm), tR: 7.46 min.
NFκB luciferase assay. Human embryonic kidney cells 293
were used to monitor any changes occurring along the NFκB
pathway. This cell line contains chromosomal integration of a
luciferase reporter construct regulated by the NFκB response
element. Transcription factors can bind to the response element
when stimulated by certain agents, allowing transcription of the
luciferase gene. Following an incubation period of 6 h with
TNFα and test compounds, cells were analyzed for luciferase
activity using the Luc assay system from Promega Corporation
(Madison, WI). Results were expressed as a percentage, relative
to control (TNFα-treated with DMSO) samples, and dose-
response curves were constructed for the determination of IC50
values, which were generated from the results of five serial
dilutions of test compounds and were the mean of two different
experiments.53 N-Tosyl-L-phenylalanyl chloromethyl ketone
(TPCK) was used as a positive control (IC50 = 3.8 0.6 μM).
6-Methyl-2-phenyl-1H-indole 10ga. White solid; yield: 25%;
1H NMR (400 MHz, d6-DMSO) δ 11.36 (s, 1H), 7.83 (d, J =
7.6 Hz, 2H), 7.46–7.40 (m, 3H), 7.29 (t, J = 7.6 Hz, 1H), 7.19
(s, 1H), 6.83 (s, 2H), 2.40 (s, 3H); 13C NMR (100 MHz,
d6-DMSO) δ 137.6, 137.0, 132.4, 130.7, 128.9, 127.2, 126.5,
124.8, 121.2, 119.8, 111.1, 98.6, 21.5; ESI-MS: calc. for
C15H13N [M + H]+: 208.3, found: 208.2. HPLC purity: 98.8%
(254 nm), tR: 7.28 min; 100% (220 nm), tR: 7.28 min.
Synthesis of 5-amino-2-phenylindole 10ha. A mixture of
5-nitro-2-phenylindole 10ba (33 mg, 0.13 mmol) and 10% Pd/C
(1.5 mg) in 2.5 mL of absolute ethanol was hydrogenated at
room temperature. The reaction was detected by HPLC and was
found to be completed after 4 h. The catalyst was filtered off and
the solvent was evaporated. The residue was purified by flash
column chromatography to afford 18 mg (67%) of 5-amino-
2-phenylindole 10ha as a brown solid. 1H NMR (400 MHz,
CDCl3) δ 8.16 (s, 1H), 7.62 (d, J = 7.2 Hz, 2H), 7.40 (t, J =
7.2 Hz, 2H), 7.32–7.27 (m, 1H), 7.22 (s, 1H), 6.92 (d, J = 2.0
Hz, 1H), 6.66–6.63 (m, 2H), 3.2 (br, 2H); 13C NMR (100 MHz,
CDCl3) δ 140.0, 138.5, 132.6, 131.9, 130.3, 129.0, 127.6, 125.1,
113.3, 111.5, 105.5, 99.2; ESI-MS: calc. for C14H12N2
[M + H]+: 209.2, found: 209.2. HPLC purity: 99.2% (254 nm),
tR: 5.32 min; 99.1% (220 nm), tR: 5.30 min.
Acknowledgements
This project was supported in part by grants from the National
Center for Research Resources (5P20RR016467-11) and the
National
Institute
of
General
Medical
Sciences
(8P20GM103466-11) from the National Institutes of Health. The
content is solely the responsibility of the authors and do not
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29 M. Hinz, D. Krappmann, A. Eichten, A. Heder, C. Scheidereit and
M. Strauss, Mol. Cell. Biol., 1999, 19, 2690–2698.
30 M. Karin, Y. Cao, F. R. Greten and Z.-W. Li, Nat. Rev. Cancer, 2002, 2,
301–310.
necessarily represent the official views of the National Institutes
of Health. We also thank UH Hilo CoP for start-up funds.
31 D. J. Van Antwerp, S. J. Martin, T. Kafri, D. R. Green and I. M. Verma,
Science, 1996, 274, 787–789.
Notes and references
1 Indole and its Derivatives, in Science of Synthesis: Houben-Weyl Methods
of Molecular Transformation, ed. E. J. Thomas, Thieme, Stuttgart, 2000.
2 A. J. Kochanowska-Karamyan and M. T. Hamann, Chem. Rev., 2010,
110, 4489–4497.
32 C.-Y. Wang, J. C. Cusack, R. Liu and A. S. Baldwin, Nat. Med., 1999, 5,
412–417.
33 C.-Y. Wang, M. W. Mayo and A. S. Baldwin, Science, 1996, 274, 784–
787.
3 W. Gul and M. T. Hamann, Life Sci., 2005, 78, 442–453.
4 M. Bandini and A. Eichholzer, Angew. Chem., Int. Ed., 2009, 48, 9608–
9644.
5 F.-E. Chen and J. Huang, Chem. Rev., 2005, 105, 4671–4706.
6 R. Ambros, S. Von Angerer and W. Wiegrebe, Arch. Pharm., 1988, 321,
481–486.
34 D. C. Guttridge, C. Albanese, J. Y. Reuther, R. G. Pestell and
A. S. Baldwin, Mol. Cell. Biol., 1999, 19, 5785–5799.
35 S. Luqman and J. M. Pezzuto, Phytother. Res., 2010, 24, 949–963.
36 C. E. P. Goldring, R. Narayanan, P. Lagadec and J. F. Jeannin, Biochem.
Biophys. Res. Commun., 1995, 209, 73–79.
37 L. Shen, E.-J. Park, T. P. Kondratyuk, D. Guendisch, L. Marler,
J. M. Pezzuto, A. D. Wright and D. Sun, Bioorg. Med. Chem., 2011, 19,
6182–6195.
7 G. Winters, N. Di Mola, M. Berti and V. Arioli, Farmaco, 1979, 34, 507–
517.
8 A. R. Saundane, S. H. Ranganath, G. Prayagraj, K. Rudresh and
N. D. Satyanarayana, Orient. J. Chem., 1998, 14, 251–254.
9 S. Grasso, C. Molica, A. M. Monforte, P. Monforte, M. Zappala,
M. T. Monforte and A. Trovato, Farmaco, 1995, 50, 113–117.
10 B. Scheiper, H. Matter, H. Steinhagen, U. Stilz, Z. Böcskei, V. Fleury and
G. McCort, Bioorg. Med. Chem. Lett., 2010, 20, 6268–6272.
11 C. R. Hopkins, S. V. O’Neil, M. C. Laufersweiler, Y. Wang, M. Pokross,
M. Mekel, A. Evdokimov, R. Walter, M. Kontoyianni, M. E. Petrey,
G. Sabatakos, J. T. Roesgen, E. Richardson and T. P. Demuth Jr., Bioorg.
Med. Chem. Lett., 2006, 16, 5659–5663.
38 Part of this work has been presented at the following ACS meeting, see:
X. Yu, E.-J. Park, T. P. Kondratyuk, J. M. Pezzuto and D. Sun, in 243rd
ACS National Meeting & Exposition, American Chemical Society, San
Diego, CA, United States, March 25–29, 2012, pp. MEDI-101.
39 C. A. Merlic, Y. You, D. M. McInnes, A. L. Zechman, M. M. Miller and
Q. Deng, Tetrahedron, 2001, 57, 5199–5212.
40 Z. Shi, S. Ding, Y. Cui and N. Jiao, Angew. Chem., Int. Ed., 2009, 48,
7895–7898.
41 R. Kuwano and M. Kashiwabara, Org. Lett., 2006, 8, 2653–2655.
42 V. Gotor-Fernández, P. Fernández-Torres and V. Gotor, Tetrahedron:
Asymmetry, 2006, 17, 2558–2564.
43 R. Alonso, A. Caballero, P. J. Campos and M. A. Rodríguez, Tetrahedron,
2010, 66, 8828–8831.
44 C. J. MacNevin, R. L. Moore and D. C. Liotta, J. Org. Chem., 2008, 73,
1264–1269.
45 N. Jiang and A. J. Ragauskas, Tetrahedron Lett., 2010, 51, 4479–
4481.
12 E. Dolušić, M. Kowalczyk, V. Magnus, G. Sandberg and J. Normanly,
Bioconjugate Chem., 2001, 12, 152–162.
13 T. A. Hill, C. P. Gordon, A. B. McGeachie, B. Venn-Brown, L. R. Odell,
N. Chau, A. Quan, A. Mariana, J. A. Sakoff, M. Chircop, P. J. Robinson
and A. McCluskey, J. Med. Chem., 2009, 52, 3762–3773.
14 D. Sugden, H. Pickering, M.-T. Teh and P. J. Garratt, Biol. Cell, 1997, 89,
531–537.
15 R. Vicente, Org. Biomol. Chem., 2011, 9, 6469–6480.
16 M. Shiri, Chem. Rev., 2012, 112, 3508–3549.
46 R. Sanz, V. Guilarte and A. Pérez, Tetrahedron Lett., 2009, 50, 4423–
4426.
17 S. A. Patil, R. Patil and D. D. Miller, Curr. Med. Chem., 2011, 18, 615–
637.
18 S. A. Patil, R. Patil and D. D. Miller, Curr. Med. Chem., 2009, 16, 2531–
2565.
19 S. Bräse, C. Gil and K. Knepper, Bioorg. Med. Chem., 2002, 10, 2415–
2437.
20 J. K. Kundu and Y.-J. Surh, Mutat. Res., Rev. Mutat. Res., 2008, 659,
15–30.
47 T. W. Lyons and M. S. Sanford, Chem. Rev., 2010, 110, 1147–1169.
48 S. Taliani, E. Da Pozzo, M. Bellandi, S. Bendinelli, I. Pugliesi,
F. Simorini, C. La Motta, S. Salerno, A. M. Marini, F. Da Settimo,
B. Cosimelli, G. Greco, E. Novellino and C. Martini, J. Med. Chem.,
2010, 53, 4085–4093.
49 C. V. Suschek, O. Schnorr and V. Kolb-Bachofen, Curr. Mol. Med., 2004,
4, 763–775.
50 M. Savio, T. Coppa, L. Bianchi, V. Vannini, G. Maga, L. Forti,
O. Cazzalini, M. C. Lazzè, P. Perucca, E. Prosperi and L. A. Stivala,
Int. J. Biochem. Cell Biol., 2009, 41, 2493–2502.
21 A. Pautz, J. Art, S. Hahn, S. Nowag, C. Voss and H. Kleinert, Nitric
Oxide, 2010, 23, 75–93.
22 W. K. Alderton, C. E. Cooper and R. G. Knowles, Biochem. J., 2001,
357, 593–615.
23 L. J. Hofseth, Cancer Lett., 2008, 268, 10–30.
24 F. Aktan, Life Sci., 2004, 75, 639–653.
25 T. D. Gilmore, Oncogene, 1999, 18, 6842–6844.
26 C. R. Gélinas and B. Rayet, Oncogene, 1999, 18, 6938.
27 M. Karin, Cold Spring Harbor Perspect. Biol., 2009, 1, a000141.
28 A. A. Beg and D. Baltimore, Science, 1996, 274, 782–784.
51 J. Hoshino, E.-J. Park, T. P. Kondratyuk, L. Marler, J. M. Pezzuto,
R. B. van Breemen, S. Mo, Y. Li and M. Cushman, J. Med. Chem., 2010,
53, 5033–5043.
52 S. K. Lee, B. Cui, R. R. Mehta, A. D. Kinghorn and J. M. Pezzuto,
Chem.-Biol. Interact., 1998, 115, 215–228.
53 T. P. Kondratyuk, E.-J. Park, R. Yu, R. B. van Breemen, R. N. Asolkar,
B. T. Murphy, W. Fenical and J. M. Pezzuto, Mar. Drugs, 2012, 10, 451–
464.
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