Design, Synthesis, and Biological Evaluation of Novel 3-Aminomethylindole Derivatives as Potential Multifunctional Anti-Inflammatory and Neurotrophic Agents

Article abstract of DOI:10.1021/acschemneuro.1c00079

The development of multifunctional molecules that are able to simultaneously interact with several pathological components has been considered as a solution to treat the complex pathologies of neurodegenerative diseases. Herein, a series of aminomethylindole derivatives were synthesized, and evaluation of their application for antineuroinflammation and promoting neurite outgrowth was disclosed. Our initial screening showed that most of the compounds potently inhibited lipopolysaccharide (LPS)-stimulated production of NO in microglial cells and potentiated the action of NGF to promote neurite outgrowth of PC12 cells. Interestingly, with outstanding NO/TNF-α production inhibition and neurite outgrowth-promoting activities, compounds 8c and 8g were capable of rescuing cells after injury by H2O2. Their antineuroinflammatory effects were associated with the downregulation of the LPS-induced expression of the inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Western blotting and immunofluorescence assay results indicated that the mechanism of their antineuroinflammatory actions involved suppression of the MAPK/NF-κB signal pathways. Further studies revealed that another important reason for the high comprehensive antineuroinflammatory activity was the anti-COX-2 capabilities of the compounds. All these results suggest that the potential biochemical multifunctional profiles of the aminomethylindole derivatives provide a new sight for the treatment of neurodegenerative diseases.

Full text of DOI:10.1021/acschemneuro.1c00079

pubs.acs.org/chemneuro
Research Article
Design, Synthesis, and Biological Evaluation of Novel
3‑Aminomethylindole Derivatives as Potential Multifunctional Anti-
Inflammatory and Neurotrophic Agents
Wei-Wei Wang,† Ting Liu,† Yu-Meng Lv, Wu-Yang Zhang, Zhi-Gang Liu, Jin-Ming Gao,* and Ding Li*
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ABSTRACT: The development of multifunctional molecules that are able
to simultaneously interact with several pathological components has been
considered as a solution to treat the complex pathologies of neuro-
degenerative diseases. Herein, a series of aminomethylindole derivatives
were synthesized, and evaluation of their application for antineuroin-
flammation and promoting neurite outgrowth was disclosed. Our initial
screening showed that most of the compounds potently inhibited
lipopolysaccharide (LPS)-stimulated production of NO in microglial cells
and potentiated the action of NGF to promote neurite outgrowth of PC12
cells. Interestingly, with outstanding NO/TNF-α production inhibition and
neurite outgrowth-promoting activities, compounds 8c and 8g were capable
of rescuing cells after injury by H2O2. Their antineuroinflammatory effects
were associated with the downregulation of the LPS-induced expression of
the inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Western blotting and
immunofluorescence assay results indicated that the mechanism of their antineuroinflammatory actions involved suppression of the
MAPK/NF-κB signal pathways. Further studies revealed that another important reason for the high comprehensive
antineuroinflammatory activity was the anti-COX-2 capabilities of the compounds. All these results suggest that the potential
biochemical multifunctional profiles of the aminomethylindole derivatives provide a new sight for the treatment of
neurodegenerative diseases.
KEYWORDS: Neurodegenerative diseases, anti-inflammatory, neurite outgrowth, indole, COX-2, multifunction
that reduced neurotrophic support is a significant factor in the
pathogenesis of neurodegenerative diseases.10 Therefore,
neurotrophins are attractive candidates for therapeutic agents
in chronic neurodegenerative diseases and acute injuries
including trauma and strokes.11 Unfortunately, therapeutic
application of neurotrophins is severely restricted by their
undesirable apoptotic effect through interactions with the
p75NTR receptor and poor penetration of the blood−brain
barrier (BBB).12 Thus, there is obviously a need for
identification of small molecules that can mimic the neuro-
trophic action as an alternative therapy approach.9,13
1. INTRODUCTION
Neuroinflammation is indicated as one of the principal causes
of the pathogenesis of several acute and chronic neurological
disorders, such as Alzheimer’s disease (AD) and Parkinson’s
disease (PD).1,2 The characteristics of neuroinflammation are
attributed to the activation of microglia, astrocytes in the brain
parenchyma, and the increase in inflammatory factors, such as
chemokines, cytokines, and ROS/NOS, among others.3,4
Another significant characteristic is the interaction between
glial cells and neurons; neurons upon an injury or a stimuli
release factors that when perceived by glial cells can induce
neuronal protection or neuroinflammation.4,5 However, the
aberrant activation of microglia leads to extent of neuro-
inflammation and thereby triggers neuronal damage and the
progression of neurodegenerative disease. Therefore, neuro-
inflammation in microglia might serve as an attractive
therapeutic target to moderate the progression of inflamma-
tory-mediated neurodegenerative diseases.6,7
In recent years, multifunctional compounds that are able to
simultaneously interact with several pathological components
have been considered as a solution to treat the complex
Received: February 9, 2021
Accepted: March 29, 2021
Published: April 22, 2021
Neurotrophins, such as nerve growth factor (NGF), are a
family of secreted proteins that play vital roles in promoting
neural survival, regeneration, and differentiation throughout an
individual’s entire lifetime.8,9 Cumulative evidence suggests
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Scheme 1. Synthesis of the 3-((Benzylamino)methyl)-1H-indole Derivatives 2 and 5
pathologies of neurodegenerative diseases.14,15 Discovery of
Scheme 2. Synthesis of the 3-((Benzylamino)methyl)-1H-
antineuroinflammatory agents in combination with the
induction of neurite outgrowth that promote neuronal survival,
differentiation, and regeneration or potentiation of the actions
of NGF might represent potential strategies for neuro-
degenerative disease treatment.16,17 Indole and its derivatives
participate in a wide spectrum of biological activities, such as
antimicrobial, anti-Alzheimer’s disease, anti-inflammatory,
antidepressant, antitumor, antioxidant, antiparkinsonian dis-
eases, antimigraine activities.18,19 Indole derivatives have
therefore captured the attention of organic synthetic chemists.
Many anti-inflammatory agents containing indole scaffolds
have been reported.18,19 For example, acemetacin and etodolac
are nonsteroidal anti-cyclooxygenase (anti-COX) inhibitors
that are used in the treatment of osteoarthritis and rheumatoid
arthritis.19 However, few aminomethylindole derivatives have
been reported for neuroinflammation or neurite outgrowth.
Herein, we designed and synthesized three series of amino-
methylindole derivatives. The structure−activity relationship
studies on anti-inflammatory and neurotrophic effects were
conducted, followed by the primary mechanisms of the active
compounds. Taken together, all the data suggest that the
multifunctional effects of the compounds provide a new insight
for the treatment of neurodegenerative diseases.
indole Derivatives 7 and 8
Scheme 3. Synthesis of the 1H-Indole-5-carboxamide
Derivative 9
2.2. Inhibition of LPS-Induced NO Production in BV-2
Microglia Cells. Nitric oxide (NO), as a main signal molecule
released in quantity by activated microglial, plays a vital role in
neuroinflammation. Inhibition of NO overproduction is
considered to be a significant indicator of antineuroinflamma-
tion.23 Thus, a cytotoxicity assay was conducted using the
MTT method, and the effect of the synthesized compounds on
NO production in lipopolysaccharide (LPS)-induced BV-2
cells was evaluated using the Griess reagent. As shown in Table
1, cell viability did not change significantly after treatment with
10 μM concentrations of each compound, apart from 5g, 5h,
and 5j. More importantly, lots of compounds were found to
possess good inhibitory activities and IC50 values of the test
compounds, with an inhibition rate of more than 80% at 10
μM. Obviously, the inhibition effect of the compounds on NO
production was divergent to a large extent, and it was
dramatically influenced by different substituents on C5, N9,
and C10. The primary structure−activity relationships on C5,
N9, and the 4′-position of the phenyl ring for R1 are described
as follows:
2. RESULTS AND DISCUSSION
2.1. Chemistry. The synthesis route for the 3-amino-
methylindole derivative (5) is shown in Scheme 1. By use of
the 5-bromoindole (1) as a raw material, compound 2 (5-
cyano-1H-indole) was obtained by reaction with cuprous
cyanide and a catalytic amount of KI in N-methylpyrrolidinone
under N2 (g).20 The substituted indoles (1, 2) were submitted
to a Vilsmeier−Haack reaction to furnish aldehydes (3, 4) in
position 3 with 85−92% yields.21 Reductive amination of 1H-
indole-3-carbaldehydes (3, 4) with the relevant substituted
phenylmethanamine and sodium borohydride produced the
target compound (5). The methanol/DCM/THF (1:2:1, v/v/
v) solvent combination was used as a way to accelerate the
reaction and achieved 60−90% yields.
In parallel, the N-methyl-1-phenylmethanamine intermedi-
ate (7) was obtained from reductive amination of the relevant
benzaldehyde (6) with the basified methylamine hydrochloride
in a methanol solution. The substituted indoles 1 and 2 then
underwent a Mannich reaction with the intermediate (7)22 and
formaldehyde aqueous in 1,4-dioxane/glacial acetic acid
solution to give the target compound (8) (Scheme 2).
Finally, as depicted in Scheme 3, hydrolysis of the relevant 5-
cyano-1H-indole derivative 5 or 8 using hydrogen peroxide as
an oxidant yielded the primary amide (9).
(i) When the hydrogen on N9 was not methylated, the
activity was determined by comprehensive factors of the
substituents on C5 and the 4′-position of the phenyl ring. In
general, unsubstituted phenyl and 4′-methoxyphenyl for R1 did
not produce a high inhibitory potency, whether the substituent
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Table 1. Inhibitory Effects of Compounds 5a−5l, 8a−8l, 9a−k and the Control Curcumin on NO Production in LPS-Activated
BV-2 Cells
a
IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated BV-2 cells.
b
Cell viability for LPS-activated BV-2 cells after treatment with 10 μM of each compound was assessed by MTT assay and was expressed as a
c
percentage (%). Inhibition rate of each compound at 10 μM that inhibited NO production in LPS-activated BV-2 cells.
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Figure 1. Effects of the prepared compounds on the NGF-induced neurite outgrowth in PC12 cells at 10 μM. Data are the mean standard error
of three independent experiments: *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the NGF-induced group.
on C5 was a bromine, a cyano group, or a carbamoyl group. In
the 5 series, with Br for C5 and Cl on the 4′-position of the
phenyl ring, compound 5c (IC50 = 1.41 0.39 μM) had the
best effect on the inhibition of NO production. Compared to
5c, a dramatic decrease in activity was seen by the introduction
of a cyano group on C5 (compound 5h, inhibition rate = 63.29
2.85% at 10 μM). In contrast, when the substituent for R1
was 4′-allyloxyphenyl and the Br group (compound 5f) was
switched to a cyano group (compound 5l, IC50 = 3.22 0.26
μM), a significantly improved inhibitory activity was observed.
In particular, compound 9e was the only one with relatively
good activity in series 9, but it showed a lower activity than the
parent compound 5l. This indicated that the hydrolysis of the
relevant 5-cyano-1H-indole derivatives led to a significant
reduction of the inhibitory potency. This occurrence was
further verified from the screening results in Table 1.
by the compounds, that did not exert detectable cytotoxicity,
are shown in Figure 1. Notably, compounds 5f, 8c, 8g, 8k, 9c,
9e, 9f, and 9h greatly induced the formation and growth of
neurites and profoundly increased the length of neurites in the
presence of NGF (10 ng/mL). For instance, the percentage of
neurite-bearing cells reached 13.2 0.3% and 17.6 1.3% for
8c and 8g, respectively. These values were significantly higher
than those obtained from cells treated with 10 ng/mL NGF
alone (8.1 0.8%). The expression of the neuronal biomarker
β-tubulin in 8c- and 8g-treated PC12 cells was also determined
by the immunofluorescence assay experiments. As shown in
Figure 2, 8c and 8g effectively enhanced the effect of NGF on
β-tubulin induction and promoted neuronal survival and
differentiation. These results indicated that these compounds
could enhance the activity of NGF on neuritogenesis.
(ii) Clearly, methylation of N9 in series 8 provided an
impressive inhibitory potency. Many of these compounds
showed high inhibitory activities with IC50 values of 1.11−1.97
μM, such as 8a−8g, 8l. When the substituent on C5 was a
bromine, it seemed that different groups at the 4′-position of
the phenyl ring for R1 did not weaken the powerful activity. In
contrast, when the substituent on C5 was a cyano group, the
inhibitory potency order was Cl > NO2 > OCF3
>
OCH2CHCH2 > OCH3 > H, followed by compounds 8l, 8g,
8j, 8i, 8h, and 8k. Similarly, it was revealed that hydrolysis of
the cyano group quenched the inhibitory action.
Taking the data together, methylation of N9 was proved to
be critical for the inhibition potency. On the other hand, the
substituent on C5 was the second most important factor in the
inhibition of NO production. Obviously, different combina-
tions of substitutions on C5 and C10 showed significantly
different inhibitory activities. Among them, compounds 5c, 5d,
5i, 8a−8g, and 8l yielded the corresponding IC50 values less
than 2 μM, which were superior to that of the positive control
drug curcumin (IC50 = 2.04 0.17 μM).
2.3. Effect of Compounds on the Neurite Outgrowth
of PC12 Cells. The rat pheochromocytoma cell line PC12 is
widely used as a cell culture model for neuronal differentiation
and survival.24 The neuritogenic activities of the compounds
were examined using PC12 cells. The cells were incubated with
the compounds at a concentration of 10 μM in combination
with NGF (10 ng/mL). After 48 h of treatment, the cell
viability was analyzed using an MTT assay, and the neurite-
bearing cells were photographed under a light microscope and
enumerated. The percentages of neurite-bearing cells induced
Figure 2. Immunofluorescence staining of neuronal biomarker β-
tubulin. The morphological characteristics were observed under an
OLYMPUS fluorescence microscope microscopy (20×): (a) control,
(b) NGF (10 ng/mL), (c) 8c (10 μM) + NGF (10 ng/mL), and (d)
8g (10 μM) + NGF (10 ng/mL).
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The pharmacological effect can be dramatically affected by
multiple substituted groups and the type of the substituent. In
contrast to the detrimental effect on the inhibition of LPS-
induced NO production, the carbamoyl on the C5 of the
indole ring potently induced neurite outgrowth in PC12. For
the case in which R2 was H, compound 9c with OCF3 on the
4′-position of the phenyl ring for R1 was the best for the
neurite outgrowth of PC12 cells, followed by compound 9f
with CH3 for R2 and NO2 on the 4′-position of the phenyl ring.
Comparison of the bioactivity of compound 5f showed that
changing the bromine to a cyano group (5l) and the
methylation of N9 (8e and 8i) exhibited a significantly lower
activity and even cytotoxicity. However, 8g with methylation of
N9 showed a higher activity than compound 5k. On the basis
of the above-mentioned analysis, it was suggested that the type
of substituent on the C5 of the indole ring could endow the
final structure with an excellent neuritogenic activity potential,
but methylation of the N9 was not critical for the
neuritogenesis activity of the compounds.
In view of the comprehensive evaluation of the NO
inhibition assay and neurite outgrowth assay, with outstanding
biochemical activities, compounds 8c and 8g were selected for
further tests to explore their effects on the protection against
H2O2 and the production of inflammatory cytokine and to
further dissect the underlying mechanisms.
2.4. Effects of Compounds on Protection against
H2O2. Oxidative stress is a remarkable pathological indicator of
neurodegenerative diseases. Cells of the nervous system are
particularly vulnerable to oxidative stress-mediated damage.25
H2O2 is used extensively as an inducer of oxidative stress in
vitro because its cellular action and pathophysiological roles
have been well studied.26 The neuroprotective activities of
compounds 8c and 8g against H2O2-induced oxidative stress
were evaluated on PC12 cells at different concentrations. As
shown in Figure 3, after incubation for 24 h, the cell viability of
stimulated inducible nitric oxide synthase (iNOS) and the
levels of iNOS, cyclooxygenase-2 (COX-2), and tumor
necrosis factor α (TNF-α), as principal proinflammatory
mediators, also increased in the activated microglia.23 Western
blotting analysis and the enzyme-linked immunosorbent assay
(ELISA) assay were employed to determine whether
compounds 8c and 8g could further downregulate the
expression of iNOS, COX-2, and TNF-α. As illustrated in
Figure 4, the production of NO, iNOS, COX-2, and TNF-α
markedly increased compared to that of the control group with
LPS treatment (1 μg/mL). Compounds 8c and 8g not only
specifically inhibited LPS-induced NO production but also
suppressed the expressions of TNF-α, iNOS, and COX-2 in
activated BV-2 cells in a concentration-dependent manner.
Thus, the results indicated that compounds 8c and 8g were
antineuroinflammatory agents.
2.6. Effects of Compounds on MAPK Signal Pathway.
It has been reported that the mitogen-activated protein kinase
(MAPK) pathway regulates the production of proinflammatory
mediators.27,28 To confirm that the anti-inflammatory activities
of 8c and 8g occurred through the regulation of the MAPK
signaling transduction pathway, Western blotting experiments
were conducted. As shown in Figure 5, the phosphorylation of
ERK and JNK increased in the LPS-stimulated BV-2 cells. The
significant reductions of the phosphorylated ERK and JNK
were observed in both the 2 and 10 μM 8c treatments.
However, 8g could significantly reduce the phosphorylation of
ERK at a concentration of only 10 μM and slightly inhibited
the phosphorylation of JNK. The phosphorylation of p38 was
not affected by compounds 8c and 8g (data not shown). These
results indicated that the MAPK signal pathway is involved in
the antineuroinflammatory action of the 3-aminomethylindole
derivatives.
2.7. Effects of Compounds on NF-κB Signal Pathway.
The transcription factor NF-κB is a crucial transcriptional
factor in the regulation of iNOS, COX-2, and TNF-α
expression.27,28 To investigate the possible anti-inflammatory
mechanisms of 8c and 8g further, the effects of the compounds
on the NF-κB pathway were examined. As shown in Figure 6,
the levels of phosphorylated NF-κB p65 were clearly increased
by the LPS treatment. Compound 8c significantly attenuated
the phosphorylation of p65 in the LPS stimulated BV-2 cell
nucleus at 10 μM, while 8g significantly suppressed the
phosphorylation of p65 in the nucleus at 2 and 10 μM.
Evidently, 8g was more efficient than 8c in blocking the NF-κB
pathway. These findings were confirmed by immunofluor-
escence assay experiments (Figure 7). The results demon-
strated that compounds 8c and 8g downregulated proin-
flammatory mediators by blocking the NF-κB pathway.
2.8. COX-2 Inhibition Assay. Targeting cyclooxygenase 2
(COX-2) is an excellent strategy for developing nonsteroidal
anti-inflammatory drugs (NSAIDs) to treat inflammation-
related disorders.23,29 To test the COX-2 inhibitory effect of
the synthesized compounds, COX-2 inhibitor screening assay
was performed using commercial assay kits and celecoxib as a
control. Fortuitously, the preliminary screening results
suggested that 16 compounds with low IC50 values for
inhibition of the NO production exhibited different inhibitory
activities on the COX-2 at 50 μM, as shown in Figure 8A.
Some of the compounds were endowed with good identified
inhibitory powers, as evidenced by the impressive inhibition
rates of compounds 5c (61.8%), 5l (74.3%), 8b (60.8%), 8c
(71.7%), and 8f (81.2%). As representatives, the IC50 values of
Figure 3. Neuroprotective effect of 8c and 8g on H2O2-induced PC12
cell death. Data are the mean standard error of three independent
experiments: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the
H2O2-treated group.
the PC12 cells exposed to 500 μM H2O2 was decreased to
56.9%. Both compounds 8c and 8g were capable of rescuing
the cells after injury. The neuroprotective effect of 8c was
observed at concentrations of 5, 10, and 20 μM. The most
potent concentration was 10 μM, and the cell viability was
79.9%. Compound 8g could protect H2O2-induced cell
damage in a dose-dependent manner.
2.5. Effects of Compounds on LPS-Induced TNF-α
Production in BV-2 Cells and the Expression of iNOS
and COX-2. A high concentration of NO is produced by the
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Figure 4. Anti-inflammatory effect of 8c and 8g in LPS-induced BV-2 cells. (A, B) Effects of 8c and 8g on the production of nitric oxide (NO) and
tumor necrosis factor α (TNF-α) in LPS-induced BV-2 cells. (C) Effects of 8c and 8g on the expression of iNOS and COX-2. (D) Densitometric
analyses of the iNOS and COX-2. Cells were treated with compounds for 24 h. Data are the mean
standard error of three independent
experiments: ###p < 0.001 vs untreated controls; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the LPS-treated group.
activity with an IC50 value of 23.96
4.99 μM, which was
better than that of 8g (IC50 = 54.85 7.38 μM). Given that 8c
and 8g could suppress the expression of COX-2 in activated
BV-2 cells, moderate anti-COX-2 capabilities of the com-
pounds may also be an important reason for their high
comprehensive antineuroinflammatory activities.
3. CONCLUSION
In summary, three series of aminomethylindole derivatives
were designed and synthesized. Several biological assays were
conducted, which proved that the aminomethylindole
derivatives can act as multifunctional agents to treat neuro-
inflammation and mimic the neurotrophic action. In the
inhibition of NO production assay, most of the compounds
exhibited diverse inhibitory effects with IC50 values ranging
from 1.11 to greater than 10 μM. Among these, 11 compounds
showed significantly better inhibition of NO production than
curcumin. In the neurite outgrowth assay, eight compounds
greatly induced the formation and growth of neurites and
profoundly increased the length of the neurites in the presence
of NGF (10 ng/mL). The detailed structure−activity relation-
ships suggested that the methylation of the N9 was critical for
the NO production inhibition potency, and the type of
substituent on the C5 of indole ring could endow the final
structure with an excellent neuritogenic activity potential. On
the basis of the comprehensive NO production inhibition and
neurite outgrowth-promoting activities, compounds 8c and 8g
were chosen for further studies. ELISA assay and Western
blotting analyses further showed that 8c and 8g significantly
suppressed the expression of TNF-α and downregulated the
expressions iNOS and COX-2 in activated BV-2 cells in a
concentration-dependent manner. In addition, the immuno-
fluorescence assay reconfirmed that 8c and 8g could potentiate
the actions of NGF. Additionally, both compounds 8c and 8g
Figure 5. 8c and 8g attenuate the phosphorylation of ERK and JNK
in LPS-induced BV-2 cells: (A) levels of ERK and JNK expressed as
changes relative to the control group; (B, C) densitometric analyses of
ERK and JNK. Cells were treated with compounds for 1 h. Data are
the mean standard error of three independent experiments: *p <
0.05, **p < 0.01 compared to the LPS-treated group.
the compounds 8c and 8g were further determined (Figure 8B
and Figure 8C). Compound 8c showed moderate anti-COX-2
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Figure 6. 8c and 8g downregulate proinflammatory mediators by blocking the NF-κB pathway. (A) Levels of NF-κB p65 in the nucleus and
cytoplasm are expressed as a change relative to the control group. (B) Densitometric analyses of of the NF-κB p65 nuclear/cytoplasm. Cells were
treated with compounds for 1 h. Data are the mean standard error of three independent experiments: ##p < 0.01 vs untreated controls; *p < 0.05,
compared to the LPS-treated group.
Figure 7. Immunofluorescence staining detected the distribution of NF-κB p65 in both the nuclei and cytoplasm by some colocalization of
receptors (p65, red) with the nuclei (DAPI, blue).
BBB permeability (BBB+) and druglikeness parameters were then
synthesized.
were capable of rescuing cells after injury by H2O2.
Furthermore, the Western blotting and immunofluorescence
assay indicated that the mechanism of their antineuroin-
flammatory action involved suppression of the MAPK/NF-κB
signal pathways. The COX-2 inhibition assay suggested that
another important reason for the high comprehensive
antineuroinflammatory activity was the anti-COX-2 capabilities
of the compounds. In conclusion, the 3-aminomethylindole
derivatives were proven to be effective antineuroinflammatory,
neuroprotective, and neurotrophic agents. These findings
opened up the new road for the treatment of neuro-
degenerative diseases.
All of the chemical reagents and solvents were purchased from
commercial suppliers and used without further purification unless
stated otherwise. The melting points were measured on an X-4
apparatus (Beijing Tech Instrument Co., Beijing, P. R. China) and
were uncorrected. The 1H and 13C NMR spectra were recorded on an
Avance III 500 MHz instrument (Bruker, Karlsruhe, Germany).
Chemical shifts (δ values) and coupling constants (J values) are given
in parts per million and hertz, respectively. High resolution ESI-MS
was performed on an AB SCIEX TripleTOF 5600+ LC/MS/MS
system. All reactions were monitored by thin-layer chromatography
(TLC), using precoated silica gel plates and ultraviolet light
visualization. Crude products were purified by column chromatog-
raphy using silica gel (200−300 mesh, Qingdao Marine Chemical
Ltd., P. R. China).
4. METHODS
4.1.2. Synthesis of 5-Cyano-1H-indole (2). A mixture of 5-
bromoindole (1, 10.0 g, 0.051 mol), cuprous cyanide (7.9 g, 0.078
mol), and N-methylpyrrolidinone (vacuum distilled prior to use, 44
mL) was charged in a round-bottom flask under N2 (g) and stirred at
155 °C for 6 h. After being naturally cooled to room temperature, the
reaction system was quenched by the addition of cold water (20 mL)
and filtered with diatomite. A solid precipitate was washed with 5%
4.1. Chemistry. 4.1.1. General. Carrying the lead drug candidates
through the blood−brain barrier (BBB) is a major challenge, and it is
a deciding factor for the development of novel neurotherapeutics.30
The BBB permeability and druglikeness parameters of all designed
compounds were predicted using admetSAR:31 a wide-ranging source
for calculation of chemical ADMET properties of the compounds
(Table S1, Supporting Information). Only the compounds with good
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Figure 8. Effects of compounds on COX-2 activities. (A) Inhibition rate of the compounds at 50 μM. (B, C) IC50 values of 8c and 8g were
calculated by the sigmoidal dose−response equation. Data are the mean standard error of three independent experiments.
ammonium hydroxide until the aqueous layer was colorless, and then
it was sequentially extracted with chloroform (20 mL) three times.
The combined organic layers were washed with water and saturated
aqueous sodium chloride and dried with MgSO4. The resulting crude
would further be purified with column chromatography (silica gel,
petroleum ether (PE):ethyl acetate (EA), 2:1 v/v) to give 5.8 g (80%)
of 5-cyano-1H-indole (2) as a white solid (yield 99.59%). Mp: 106.1−
mmol, 1.2 equiv) and freshly activated molecular sieves (4 Å, 0.2 g)
were added and the mixture was stirred under nitrogen at room
temperature for 8−24 h. After imine formation, solvent was removed
in vacuo and the crude material was dissolved in methanol.
Approximately 34 mg of NaBH4(0.9 mmol, 2 equiv) was added
under an ice bath, and the solution was stirred at 20 °C for 16 h.32
The completion of the reaction was monitored with the help of TLC.
After the completion of the reaction, solvent was distilled off under a
reduced pressure, and the residue was dissolved in 4 mL of ethyl
acetate. The organic layer was then washed with saturated aqueous
NaHCO3, dried over MgSO4, and concentrated in vacuo. The crude
product was purified using flash chromatography with dichloro-
methane/methanol as eluents. The reaction mixture was worked up
according to the general procedure and purified by column
chromatography on silica gel to afford the compounds 5a−5l.
N-Benzyl-1-(5-bromo-1H-indol-3-yl)methanamine (5a). Yellow
solid; yield 90.05%. Mp: 81.2−83.0 °C. 1H NMR (500 MHz,
MeOD): δ 7.62 (d, J = 1.5 Hz, 1H), 7.27−7.11 (m, 8H), 3.78 (s, 2H),
3.68 (s, 2H). 13C NMR (125 MHz, MeOD): δ 140.29, 136.50,
130.03, 129.52, 129.39, 128.12, 126.17, 125.16, 121.86, 113.90,
113.37, 113.09, 53.50, 43.75. LRMS (ESI), m/z calcd for C16H15BrN2,
[M + H]+ 316.21, found 316.90.
1-(5-Bromo-1H-indol-3-yl)-N-(4-methoxybenzyl)methanamine
(5b). White solid; yield 88.05%. Mp: 122.1−123.5 °C. 1H NMR (500
MHz, acetone): δ 10.25 (s, 1H), 7.85 (s, 1H), 7.35 (d, J = 8.6 Hz,
1H), 7.30 (d, J = 8.5 Hz, 3H), 7.20 (dd, J = 8.6, 1.4 Hz, 1H), 6.87 (d,
J = 8.5 Hz, 2H), 3.92 (s, 2H), 3.77 (d, J = 3.9 Hz, 5H), 2.67 (s, 1H).
13C NMR (125 MHz, acetone): δ 159.45, 136.37, 133.73, 129.99,
125.54, 125.38, 124.56, 122.45, 115.07, 114.24, 113.79, 112.22, 55.30,
52.99, 44.49. LRMS (ESI), m/z calcd for C17H17BrN2O, [M + H]+
346.24, found 346.97.
1
108.0 °C. H NMR (500 MHz, CDCl3): δ 8.59 (s, 1H), 8.01−7.98
(m, 1H), 7.48−7.41 (m, 2H), 7.36−7.33 (m, 1H), 6.65−6.63 (m,
1H). LRMS (ESI), m/z calcd for C9H6rN2, [2 M + Na]+ 307.10,
found 307.88.
4.1.3. General Procedure for Synthesis of 1H-Indole-3-carbalde-
hyde Precursor (3, 4). Phosphorus oxychloride (POCl3,1.2 mL, 12.75
mmol, 2.5 equiv) was added dropwise to previously cooled DMF (8.0
mL at 0−5 °C) and stirred for 20 min. A solution of the relevant
indole derivate (1, 2, 5.1 mmol, 1 equiv) in DMF (20 mL) was added
dropwise to the DMF−POCl3 mixture. The mixture was then warmed
to room temperature and stirred at 30 °C for 3 h. The reaction
mixture was diluted with ice−water (50 mL), neutralized with 20%
NaOH to a pH of 8−9, and stirred under reflux conditions overnight.
The reactor was cooled down in an ice−water bath until solid
precipitation appeared to have ceased. The precipitate in the mixture
was then filtered off and purified by recrystallization from a 20%
ethanol aqueous solution to give the compounds 3 and 4.
Bromo-1H-indole-3-carbaldehyde (3). Crystalline powder; yield
1
90.81%. Mp: 204.1−205.2 °C. H NMR (500 MHz, MeOD): δ 9.88
(s, 1H), 8.30 (s, 1H), 8.11 (s, 1H), 7.41−7.36 (m, 2H). LRMS (ESI),
m/z calcd for C9H6BrNO, [M + H]+ 223.97; found 224.01.
3-Formyl-1H-indole-5-carbonitrile (4). Off-white crystalline pow-
der; yield 92.19%. Mp: 248.3−249.8 °C. 1H NMR (500 MHz,
MeOD): δ 9.97 (s, 1H), 8.54 (s, 1H), 8.28 (s, 1H), 7.66−7.55 (m,
2H). LRMS (ESI), m/z calcd for C10H6N2O, [M + H]+ 171.06, found
171.10.
1-(5-Bromo-1H-indol-3-yl)-N-(4-chlorobenzyl)methylamine (5c).
White solid; yield 88.25%. Mp: 131.1−132.6 °C. 1H NMR (500
MHz, CDCl3): δ 8.08 (s, 1H), 7.75 (s, 1H), 7.25 (d, J = 13.5 Hz,
5H), 7.19 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 3.89 (s, 2H), 3.79 (s,
2H). 13C NMR (126 MHz, CDCl3): δ 138.97, 135.16, 132.79, 129.69,
129.03, 128.67, 125.21, 123.81, 121.90, 114.90, 113.03, 112.73, 52.79,
4.1.4. General Procedure for Synthesis of 3-((Benzylamino)-
methyl)-1H-indole Derivative 5. First, relevant 1H-indole-3-carbal-
dehyde (3−4, 0.45 mmol, 1 equiv) was dissolved in 4 mL of 1:2:1
methanol/DCM/THF. Next, 4-substituted phenylmethanamine (0.54
1600
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Research Article
44.09. HRMS (ESI), m/z calcd for C16H14BrClN2, [M + H]+
349.0102 (76.40%), 351.0080 (100.00%), found 349.0098
(76.08%), 351.0075(100.00%).
113.99, 113.51, 102.62, 56.75, 49.85. LRMS (ESI), m/z calcd for
C17H14N4O2, [2 M + Na]+ 635.66, found 635.02.
3-((((4-(Allyloxy)benzyl)amino)methyl)-1H-indole-5-carbonitrile
1
(5l). Yellow solid; yield 56.23%. Mp: 40.0−41.0 °C. H NMR (500
1-(5-Bromo-1H-indol-3-yl)-N-(cyclohexylmethyl)methanamine
1
MHz, CDCl3): δ 9.50 (s, 1H), 7.99 (s, 1H), 7.37 (d, J = 5.7 Hz, 2H),
7.29 (d, J = 8.5 Hz, 2H), 7.26 (s, 1H), 6.92 (dd, J = 13.2, 8.6 Hz, 2H),
6.08 (m, 1H), 5.45 (d, J = 17.3 Hz, 1H), 5.32 (d, J = 10.5 Hz, 1H),
4.55 (t, J = 6.6 Hz, 2H), 4.01 (s, 2H), 3.86 (s, 2H). 13C NMR (125
MHz, CDCl3): δ 158.03, 138.16, 133.32, 131.24, 129.73, 126.98,
125.44, 124.91, 124.83, 120.96, 117.80, 114.87, 112.29, 102.36, 68.94,
52.54, 43.26. HRMS (ESI), m/z calcd for C20H19N3O, [M + H]+
318.1601, found 318.1600.
(5d). Yellow solid; yield 70.25%. Mp: 57.0−58.5 °C. H NMR (500
MHz, CDCl3): δ 8.79 (s, 1H), 7.79 (s, 1H), 7.28 (dd, J = 8.8, 1.2 Hz,
1H), 7.19 (d, J = 8.6 Hz, 1H), 7.09 (s, 1H), 3.94 (s, 2H), 2.59 (d, J =
6.3 Hz, 2H), 2.08 (d, J = 6.2 Hz, 1H), 1.77 (dd, J = 24.6, 12.9 Hz,
4H), 1.25 (ddd, J = 31.8, 22.2, 14.2 Hz, 5H), 0.96 (dd, J = 24.1, 11.9
Hz, 2H). 13C NMR (125 MHz, CDCl3): δ 135.10, 129.01, 124.91,
124.03, 121.52, 114.59, 112.81, 112.79, 56.58, 44.86, 37.89, 31.62,
26.76, 26.16. HRMS (ESI), m/z calcd for C16H21BrN2, [M + H]+
321.0961 (100.00%), 323.0940 (98.94%); found 321.0955
(100.00%), 323.0934 (97.62%).
4.1.5. General Procedure for Synthesis of the N-Methyl-1-
phenylmethanamine Intermediate 7. A solution of methylamine
hydrochloride (2.16 mmol, 1.2 equiv) in 2 mL of methanol was
basified with 1.2 equiv of triethylamine for 30 min, and then the
relevant aldehyde (1.8 mmol, 1 equiv) was added. The resulting
solution was stirred at room temperature under the protection of inert
gas for 24 h. After imine formation, the N-methyl-1-(4-substituted
phenyl)methanamine intermediate 7 was prepared with yields of 92−
95% according to the general procedure provided in section 4.1.4.
4.1.6. General Procedure for Synthesis of the 3-
((Benzylaminomethyl)methyl)-1H-indole Derivative 8. A mixture
of the indole derivative (1, 2, 0.562 mmol, 1 equiv), formaldehyde
(65.5 μL, 0.675 mmol, 1.2 equiv), 1,4-dioxane solution (2 mL), glacial
acetic acid (0.8 mL), and the relevant N-methyl-1-phenylmethan-
amine intermediate 7 (1.2 equiv) was charged in a Schlenk tube under
N2 (g) and stirred at room temperature for 12 h. Once completion of
reaction was obtained, as indicated by TLC, the reaction was
quenched by saturated aqueous Na2CO3 and the product was
extracted by dichloromethane. The organic layer was washed with
distilled water and dried over MgSO4, and solvent of the organic
phase was evaporated under reduced pressure. The crude product was
purified using flash chromatography with dichloromethane/methanol
as eluents. The reaction mixture was worked up according to the
general procedure and purified by column chromatography on silica
gel to afford the compounds 8a−8l.
1-(5-Bromo-1H-indol-3-yl)-N-(4-(trifluoromethoxy)benzyl)-
methanamine (5e). White solid; yield 78.25%. Mp: 107.8−109.2 °C.
1H NMR (500 MHz, CDCl3) δ 8.26 (s, 1H), 7.69 (d, J = 1.4 Hz, 1H),
7.28 (d, J = 8.5 Hz, 2H), 7.17 (dd, J = 8.7, 1.8 Hz, 1H), 7.08 (d, J =
8.3 Hz, 3H), 6.99 (s, 1H), 3.85 (s, 2H), 3.76 (s, 2H). 13C NMR (125
MHz, CDCl3): δ 148.33, 139.09, 135.15, 129.62, 128.98, 125.13,
123.93, 121.77, 121.66, 121.06, 119.62, 114.57, 112.95, 112.78, 52.70,
44.13. HRMS (ESI), m/z calcd for C17H14BrF3N2O, [M + H]+
399.0310 (100.00%), 401.0294 (99.34%), found 399.0302 (100.00%),
401.0285 (97.86%).
1-(5-Bromo-1H-indol-3-yl)-N-(4-(allyloxy)benzyl)methanamine
1
(5f). Yellow oil; yield 65.20%. H NMR (500 MHz, CDCl3): δ 8.24
(s, 1H), 7.71 (s, 1H), 7.31 (d, J = 4.8 Hz, 2H), 7.29 (d, J = 1.4 Hz,
1H), 7.24 (s, 1H), 7.19 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.11 (m,
1H), 5.46 (d, J = 17.2 Hz, 1H), 5.33 (t, J = 8.7 Hz, 1H), 4.59 (d, J =
5.3 Hz, 2H), 3.80 (s, 2H), 3.76 (s, 2H). 13C NMR (125 MHz,
CDCl3): δ 135.12, 133.56, 130.48, 129.65, 125.02, 124.79, 122.68,
117.76, 115.12, 114.69, 112.92, 112.55, 69.03, 68.11, 48.81. LRMS
(ESI), m/z calcd for C19H19BrN2O, [M + H]+ 372.28, found 372.90.
3-((Benzylamino)methyl)-1H-indole-5-carbonitrile (5g). Yellow
1
oil; yield 66.16%. H NMR (500 MHz, MeOD): δ 8.27 (s, 1H),
7.70 (d, J = 8.6 Hz, 1H), 7.54 (s, 1H), 7.47−7.21 (m, 6H), 4.39 (s,
2H), 4.17 (s, 2H). 13C NMR (125 MHz, CDCl3, MeOD): δ 138.66,
138.24, 128.89, 128.78, 127.87, 127.03, 126.59, 124.75, 124.45,
121.18, 113.09, 112.74, 101.84, 52.79, 42.93. LRMS (ESI), m/z calcd
for C17H15N3, [M + H]+ 262.33; found 262.07.
1-(5-Bromo-1H-indol-3-yl)-N-benzyl-N-methylmethylamine (8a).
White solid; yield 63.06%. Mp: 122.3−124.2 °C. 1H NMR (500
MHz, CDCl3): δ 8.07 (s, 1H), 7.84 (s, 1H), 7.38−7.31 (m, 4H),
7.28−7.24 (m, 2H), 7.22 (d, J = 8.6 Hz, 1H), 7.14 (s, 1H), 3.67 (s,
2H), 3.55 (s, 2H), 2.22 (s, 3H). 13C NMR (125 MHz, MeOD): δ
139.79, 136.71, 130.95, 130.58, 129.30, 128.25, 127.14, 125.14,
122.63, 113.87, 113.09, 112.25, 62.59, 52.82, 42.41. HRMS (ESI), m/
z calcd for C17H17BrN2, [M + H]+ 329.0648 (100.00%), 331.0627
(99.14%), found 329.0648 (100.00%), 331.0630 (95.43%).
3-((((4-Chlorobenzyl)amino)methyl)-1H-indole-5-carbonitrile
1
(5h). Yellow oil; yield 66.62%. H NMR (500 MHz, CDCl3): δ 8.88
(s, 1H), 8.03 (d, J = 15.1 Hz, 1H), 7.40−7.36 (m, 3H), 7.28 (d, J =
3.7 Hz, 3H), 7.23 (d, J = 8.4 Hz, 1H), 3.96 (s, 2H), 3.82 (s, 2H). 13C
NMR (125 MHz, CDCl3): δ 138.62, 138.25, 132.87, 129.64, 128.68,
127.06, 125.27, 125.09, 124.86, 120.97, 115.79, 112.24, 102.48, 52.84,
43.98. LRMS (ESI), m/z calcd for C17H14ClN3, [M + H]+ 296.77,
found 296.03.
1-(5-Bromo-1H-indol-3-yl)-N-(4-methoxybenzyl)-N-methylme-
1
thylamine (8b). Yellow solid; yield 72.18%. Mp: 98.5−100.2 °C. H
NMR (500 MHz, CDCl3) δ 8.37 (s, 1H), 7.78 (d, J = 1.0 Hz, 1H),
7.26 (d, J = 8.5 Hz, 2H), 7.22 (dd, J = 8.7, 1.7 Hz, 1H), 7.14 (d, J =
8.6 Hz, 1H), 7.05 (d, J = 1.4 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 3.79
(s, 3H), 3.63 (s, 2H), 3.49 (s, 2H), 2.19 (s, 3H). 13C NMR (125
MHz, CDCl3): δ 158.83, 135.09, 131.24, 130.44, 129.72, 124.90,
124.85, 122.38, 113.82, 113.27, 112.87, 112.62, 61.32, 55.40, 52.23,
42.25. HRMS (ESI), m/z calcd for C18H19BrN2O, [M + H]+
359.0752 (100.00%), 361.0733 (99.57%), found 359.0743
(100.00%), 361.0725 (97.39%).
3-((((4-(Trifluoromethoxy)benzyl)amino)methyl)-1H-indole-5-
1
carbonitrile (5i). White solid; yield 78.16%. Mp: 99.4−101.1 °C. H
NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.00 (s, 1H), 7.37−7.33 (m,
2H), 7.33−7.28 (m, 2H), 7.18 (s, 1H), 7.10 (d, J = 8.3 Hz, 2H), 3.92
(s, 2H), 3.79 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 148.35,
139.06, 138.18, 129.56, 127.13, 125.35, 125.29, 124.58, 121.13,
120.85, 116.23, 112.16, 102.92, 52.89, 44.23. HRMS (ESI), m/z calcd
for C18H14F3N3O, [M + H]+ 346.1161, found 346.1158.
3-((((4-Methoxybenzyl)amino)methyl-1H-indole-5-carbonitrile
(5j). Yellow oil; yield 85.35%. 1H NMR (500 MHz, CDCl3): δ 7.94 (s,
1H), 7.34 (q, J = 8.5 Hz, 1H), 7.26−7.18 (m, 4H), 6.86 (dd, J = 12.0,
8.6 Hz, 3H), 3.79 (dd, J = 9.3, 6.9 Hz, 7H). 13C NMR (125 MHz,
CDCl3): δ 159.03, 138.19, 134.25, 129.73, 129.30, 128.63, 125.34,
124.95, 124.90, 120.93, 114.12, 114.06, 112.26, 102.58, 55.38, 45.51,
43.34. LRMS (ESI), m/z calcd for C18H17N3O, [M + H]+ 292.35,
found 292.09.
3-((((4-Nitrobenzyl)amino)methyl)-1H-indole-5-carbonitrile (5k).
Yellow solid; yield 76.14%. Mp: 108.2−109.7 °C. 1H NMR (500
MHz, MeOD): δ 8.05 (d, J = 0.8 Hz, 2H), 7.46 (d, J = 0.5 Hz, 1H),
7.44 (s, 1H), 7.37−7.34 (m, 4H), 4.77 (s, 4H). 13C NMR (125 MHz,
MeOD): δ 140.12, 128.06, 127.10, 125.76, 125.34, 121.89, 117.68,
1-(5-Bromo-1H-indol-3-yl)-N-(4-nitrobenzyl-N-methylmethyl-
1
amine (8c). Yellow solid; yield 72.26%. Mp: 131.1−133.0 °C. H
NMR (500 MHz, CDCl3): δ 8.32 (s, 1H), 8.09 (dd, J = 11.1, 5.6 Hz,
2H), 7.67 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.25−7.22 (m, 1H), 7.20
(d, J = 8.6 Hz, 1H), 7.01 (d, J = 1.6 Hz, 1H), 3.87 (s, 2H), 3.46 (s,
2H), 2.92 (s, 3H). 13C NMR (125 MHz, DMSO): δ 147.90, 146.45,
135.07, 129.57, 129.27, 126.26, 123.44, 123.26, 121.28, 113.41,
111.11, 59.82, 52.19, 41.73. HRMS (ESI), m/z calcd for
C17H16BrN3O2, [M + H]+ 374.0495 (100.00%), 376.0478 (97.3%),
found 374.0486 (100.00%), 376.0467 (97.27%).
1-(5-Bromo-1H-indol-3-yl)-N-(4-(trifluoromethoxy)benzyl)-N-
methylmethylamine (8d). White solid; yield 65.04%. Mp: 96.6−97.7
1
°C. H NMR (500 MHz, MeOD): δ 8.29 (s, 1H), 7.70 (dd, J = 8.6,
1601
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1.4 Hz, 1H), 7.56 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6
Hz, 2H), 6.92 (d, J = 8.6 Hz,2H), 4.39 (s, 2H), 4.14 (s, 2H), 3.75 (s,
3H). 13C NMR (125 MHz, MeOD): δ 140.70, 133.28, 132.29,
130.53, 128.40, 127.26, 125.46, 123.59, 120.77, 116.27, 113.42,
108.19, 56.58, 51.98, 43.37. HRMS (ESI), m/z calcd for
C18H16BrF3N2O, [M + H]+ 413.0471 (100.00%), 415.0452
(99.56%), found 413.0473 (100.00%), 415.0459 (97.42%).
112.05, 102.58, 62.11, 52.49, 42.51. LRMS (ESI), m/z calcd for
C18H17N3, [M + H]+ 276.36, found 276.11.
3-((((4-Chlorobenzyl)(methyl)amino)methyl)-1H-indole-5-car-
1
bonitrile (8l). White solid; yield 70.62%. Mp: 105.5−107.2 °C. H
NMR (500 MHz, CDCl3): δ 8.51 (s, 1H), 8.09 (s, 1H), 7.42−7.37
(m, 2H), 7.29−7.22 (m, 5H), 3.68 (s, 2H), 3.48 (s, 2H), 2.17 (s, 3H).
13C NMR (125 MHz, CDCl3): δ 138.27, 137.81, 132.85, 130.36,
128.56, 127.65, 125.81, 125.47, 125.19, 121.01, 114.88, 112.12,
102.69, 61.26, 52.75, 42.41. HRMS (ESI), m/z calcd for C18H16ClN3,
[M + H]+ 310.1105, found 310.1106.
1-(5-Bromo-1H-indol-3-yl)-N-(4-(allyloxy)benzyl)-N-methylme-
1
thylamine (8e). White solid; yield 60.52%. Mp: 79.5−81.5 °C. H
NMR (500 MHz, CDCl3): δ 8.26 (s, 1H), 7.79 (d, J = 1.4 Hz, 1H),
7.24 (dd, J = 12.2, 5.1 Hz, 3H), 7.17 (d, J = 8.6 Hz, 1H), 7.09 (d, J =
1.9 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.10−6.01 (m, 1H), 5.41 (dd,
= 17.3, 1.4 Hz, 1H), 5.29−5.25 (m, 1H), 4.53 (d, J = 5.3 Hz, 2H),
3.63 (s, 2H), 3.48 (s, 2H), 2.19 (s, 3H). 13C NMR (125 MHz,
CDCl3): δ 157.88, 135.11, 133.56, 131.49, 130.40, 129.72, 124.96,
124.78, 122.44, 117.72, 114.67, 113.41, 112.92, 112.59, 69.02, 61.34,
52.31, 42.28. HRMS (ESI), m/z calcd for C20H21BrN2O, [M + H]+
385.0910 (99.94%), 387.0890 (100.00%), found 385.0915 (98.81%),
387.0896 (100.00%).
4.1.7. General Procedure for Synthesis of the 1H-Indole-5-
carboxamide Derivative 9. Hydrogen peroxide and 20% NaOH were
added dropwise to a solution of the relevant 5-cyano-1H-indole
derivative 8 or 9 (30 mg), respectively, in 200 μL of methanol/THF
(3:17, v/v) at −5 °C.33 The mixture was warmed to room
temperature and stirred at 25 °C for 24 h. After completion of the
reaction, it was then diluted with distilled water and extracted with
dichloromethane. The dichloromethane extracts were washed with
saturated brine, dried over anhydrous MgSO4, concentrated in vacuo,
and purified by column chromatography on silica gel to afford the
target compounds 9a−9k.
1-(5-Bromo-1H-indol-3-yl)-N-(4-chlorobenzyl)-N-methylmethyl-
amine (8f). White solid; yield 78.32%. Mp: 121.2−123.0 °C. 1H
NMR (500 MHz, CDCl3): δ 8.20 (s, 1H), 7.88 (s, 1H), 7.33 (d, J =
15.0 Hz, 5H), 7.27 (d, J = 8.6 Hz, 1H), 7.18 (s, 1H), 3.72 (s, 2H),
3.55 (s, 2H), 2.25 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 137.83,
135.12, 132.82, 130.50, 129.63, 128.54, 125.12, 124.76, 122.45,
113.10, 113.02, 112.63, 61.06, 52.68, 42.31. HRMS (ESI), m/z calcd
for C17H16BrClN2, [M + H]+ 363.0258 (76.27%), 365.0238
(100.00%), found 363.0259 (77.13%), 365.0239 (100.00%).
3-((Methyl(4-nitrobenzyl)amino)methyl)-1H-indole-5-carboni-
3-((Benzylamino)methyl)-1H-indole-5-carboxamide (9a). Yellow
solid; yield 52.86%. Mp: 199.1−201.0 °C. 1H NMR (500 MHz,
MeOD): δ 8.27 (s, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.54 (s, 1H), 7.47−
7.35 (m, 6H), 4.39 (s, 2H), 4.17 (s, 2H). 13C NMR (125 MHz,
MeOD): δ 173.60, 140.00, 133.71, 130.82, 130.30, 130.19, 129.49,
127.72, 126.50, 122.75, 120.01, 112.63, 108.11, 54.78, 43.03. LRMS
(ESI), m/z calcd for C17H17N3O, [M + H]+ 280.34, found 280.05.
3-((((4-Chlorobenzyl)amino)methyl)-1H-indole-5-carboxamide
1
1
(9b). Yellow oil; yield 51.56%. H NMR (500 MHz, MeOD): δ 8.30
trile (8g). Yellow oil; yield 61.22%. H NMR (500 MHz, CDCl3): δ
(s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.56 (s, 1H), 7.47−7.42 (m, 3H),
7.40 (d, J = 8.4 Hz, 2H), 4.42 (s, 2H), 4.19 (s, 2H). 13C NMR (125
MHz, MeOD): δ 173.57, 139.98, 136.37, 132.62, 132.17, 130.26,
129.72, 127.68, 126.52, 124.29, 122.79, 120.03, 112.65, 54.78, 43.11.
LRMS (ESI), m/z calcd for C17H16ClN3O, [M + H]+ 314.79; found
314.01.
8.64 (s, 1H), 8.17−8.12 (m, 3H), 7.49 (d, J = 8.4 Hz, 2H), 7.44 (s,
2H), 7.29 (s, 1H), 3.76 (s, 2H), 3.61 (s, 2H), 2.23 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 147.41, 147.20, 138.26, 129.46, 127.54, 125.69,
125.53, 125.32, 123.68, 120.92, 114.65, 112.22, 102.88, 61.08, 53.25,
42.65. HRMS (ESI), m/z calcd for C18H16N4O2, [M + H]+ 321.1346,
found 321.1352.
3-((((4-(Trifluoromethoxy)benzyl)amino)methyl)-1H-indole-5-
carboxamide (9c). White solid; yield 51.40%. Mp: 88.5−90.0 °C. 1H
NMR (500 MHz, MeOD): δ 8.28 (s, 1H), 7.71 (dd, J = 8.6, 1.3 Hz,
1H), 7.49 (d, J = 8.5 Hz, 2H), 7.46−7.41 (m, 2H), 7.27 (d, J = 8.1
Hz, 2H), 4.17 (s, 2H), 3.99 (s, 2H). 13C NMR (125 MHz, MeOD): δ
173.85, 150.13, 140.13, 137.35, 131.88, 127.90, 127.64, 125.83,
122.92, 122.44, 122.20, 120.13, 112.43, 112.34, 52.15, 43.85. LRMS
(ESI), m/z calcd for C18H16F3N3O2, [M + H]+ 364.34, found 364.02.
3-((((4-Methoxybenzyl)amino)methyl)-1H-indole-5-carboxamide
3-((((4-Methoxybenzyl)(methyl)amino)methyl)-1H-indole-5-car-
bonitrile (8h). White extract; yield 62.12%. 1H NMR (500 MHz,
MeOD): δ 9.19 (s, 1H), 7.86 (s, 1H), 7.24−7.18 (m, 2H), 7.11 (d, J
= 8.1 Hz, 3H), 6.73 (d, J = 8.5 Hz, 2H), 3.64 (s, 4H), 3.56 (s, 2H),
3.38 (s, 2H), 2.06 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 158.97,
138.27, 130.53, 130.30, 127.65, 126.21, 125.45, 124.82, 121.09,
113.86, 113.66, 112.24, 102.24, 61.19, 55.36, 51.94, 41.96. LRMS
(ESI), m/z calcd for C19H19N3O, [2 M + K]+ 649.56, found 649.70.
3-((((4-(Allyloxy)benzyl)(methyl)amino)methyl)-1H-indole-5-car-
bonitrile (8i). White extract; yield 61.42%. 1H NMR (500 MHz,
CDCl3): δ 8.48 (s, 1H), 8.07 (s, 1H), 7.40 (q, J = 8.5 Hz, 2H), 7.25
(d, J = 8.7 Hz, 3H), 6.90 (d, J = 8.5 Hz, 2H), 6.06 (m 1H), 5.42 (dd, J
= 17.2, 1.3 Hz, 1H), 5.28 (d, J = 10.8 Hz, 1H), 4.54 (d, J = 5.3 Hz,
1H), 4.12 (d, J = 7.1 Hz, 1H), 3.68 (s, 2H), 3.50 (s, 2H), 2.20 (s,
3H). 13C NMR (125 MHz, CDCl3): δ 158.03, 138.16, 133.32, 131.24,
129.73, 126.98, 125.44, 124.91, 124.83, 120.96, 117.80, 114.87,
112.29, 102.36, 68.94, 65.01, 52.54, 43.26. HRMS (ESI), m/z calcd
for C21H21N3O, [M + H]+ 332.1757, found 332.1752.
1
(9d). Yellow oil; yield 53.34%. H NMR (500 MHz, MeOD): δ 8.27
(s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.56 (s, 1H), 7.46 (d, J = 8.6 Hz,
1H), 7.35 (dd, J = 15.8, 8.5 Hz, 2H), 6.94 (dd, J = 8.5, 3.3 Hz, 2H),
4.41 (s, 2H), 4.15 (s, 2H), 3.77 (s, 3H). 13C NMR (125 MHz,
MeOD): δ 173.56, 162.05, 139.97, 132.50, 131.51, 129.75, 127.66,
126.59, 124.64, 124.21, 122.79, 119.99, 115.56, 112.67, 107.39, 55.83,
54.78, 42.60. LRMS (ESI), m/z calcd for C18H19N3O2, [M + H]+
310.37, found 310.03.
3-((((4-(Allyloxy)benzyl)amino)methyl)-1H-indole-5-carboxa-
1
mide (9e). White oil; yield 50.02%. H NMR (500 MHz, MeOD): δ
3-((Methyl(4-(trifluoromethoxy)benzyl)amino)methyl)-1H-in-
dole-5-carbonitrile (8j). White oil; yield 62.05%. 1H NMR (500
MHz, CDCl3): δ 8.56 (s, 1H), 8.13 (s, 1H), 7.42 (s, 2H), 7.35 (d, J =
8.5 Hz, 2H), 7.25 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 8.1 Hz, 2H), 3.71
(s, 2H), 3.52 (s, 2H), 2.20 (s, 3H). 13C NMR (125 MHz, CDCl3): δ
148.32, 138.28, 138.17, 130.22, 127.62, 125.80, 125.47, 125.18,
121.04, 120.93, 114.88, 112.14, 102.63, 61.14, 52.91, 42.45. HRMS
(ESI), m/z calcd for C19H16F3N3O, [M + H]+ 360.1318, found
360.1317.
3-((Benzyl(methyl)amino)methyl)-1H-indole-5-carbonitrile (8k).
White solid; yield 76.85%. Mp: 134.1−136.0 °C. 1H NMR (500
MHz, CDCl3): δ 8.57 (s, 1H), 8.06 (s, 1H), 7.38 (dd, J = 8.1, 4.7 Hz,
2H), 7.35−7.32 (m, 4H), 7.25 (m, 2H), 3.68 (s, 2H), 3.54 (s, 2H),
2.21 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 139.21, 138.22, 129.18,
128.45, 127.68, 127.25, 125.86, 125.46, 125.10, 121.06, 114.97,
8.05 (s, 1H), 7.54 (t, J = 4.1 Hz, 2H), 7.43 (d, J = 8.5 Hz, 1H), 7.33
(d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.05 (m, 1H), 5.39 (dd,
J = 17.3, 1.3 Hz, 1H), 5.25 (dd, J = 10.6, 1.0 Hz, 1H), 4.56 (d, J = 5.0
Hz, 2H), 4.19 (s, 2H), 3.96 (s, 2H). 13C NMR (125 MHz, MeOD): δ
171.32, 160.20, 139.81, 134.78, 131.73, 129.86, 129.19, 128.11,
125.65, 125.39, 121.67, 117.56, 116.05, 113.80, 111.22, 69.80, 52.15,
42.87. HRMS (ESI), m/z calcd for C20H21N3O2, [M + H]+ 336.1706,
found 336.1710.
3-((Methyl(4-nitrobenzyl)amino)methyl)-1H-indole-5-carboxa-
mide (9f). Yellow oil; yield 50.62%. 1H NMR (500 MHz, DMSO): δ
11.17 (s, 1H), 8.28 (s, 1H), 8.17 (d, J = 8.6 Hz, 2H), 7.82 (s, 1H),
7.64 (dd, J = 18.0, 8.5 Hz, 3H), 7.35 (d, J = 8.5 Hz, 2H), 7.09 (s, 1H),
3.75 (s, 2H), 3.64 (s, 2H), 2.14 (s, 3H). 13C NMR (125 MHz,
DMSO): δ 169.09, 146.44, 138.72, 137.94, 129.54, 126.97, 125.87,
124.92, 123.27, 120.78, 119.52, 112.38, 110.68, 59.83, 59.73, 52.33,
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41.77. LRMS (ESI), m/z calcd for C18H18N4O3, [M + H]+ 339.37,
found 339.10.
4.2.4. Measurement of TNFα with ELISA Kits. BV-2 cells were
seeded in 6-well plates with a density of 2 × 104 cells/well. After 24 h,
cells were treated with 8c and 8g with concentrations of 2 and 10 μM,
respectively, in the absence or presence of LPS (1 μg/mL) for 24 h.
Supernatant from the culture was collected to determine the
concentration of TNF-α with the ELISA kits according to the
protocol described in the kit instructions.
4.2.5. Western Blotting. BV-2 cells were harvested and suspended
in RIPA lysis buffer containing protease and phosphatase inhibitor
cocktails. The concentrations of protein were determined by a BCA
protein assay kit. Equal amounts of proteins were electrophoresed in
10% SDS−PAGE and then transferred to a polyvinylidene difluoride
(PVDF) Western blot membrane, which were then blocked using 5%
(W/V) skim milk in TBST (Tris-buffered saline with 0.1% Tween 20)
for 2 h at room temperature. The membranes were then incubated
with specific primary antibodies at 4 °C overnight. After washing
three times with TBST, the membranes were incubated with
corresponding secondary antibodies at 4 °C for 2 h. Finally,
immunoreactive signals were detected using the ChemiDoc Touch
imaging system (Bio-Rad Laboratories).
4.2.6. Measurement of Neurite Outgrowth in PC-12 Cells. PC-12
cells were seeded into 24-well plates coated with PLL at a density of
1.5 × 104 cells/mL. After 24 h, the medium was replaced in
differentiation medium containing 2% HS and 1% FBS for 16 h and
then treated with the tested compounds and NGF in combination for
48 h. NGF (10 ng/mL) was used as a positive control. The PC12 cells
were then photographed using optical microscopy. The cells bearing
neurites with sizes greater than or equal to the length of the cell body
were determined to be a positive cell. The cells in five random
different random views were counted per well.9
3-(((4-Methoxybenzyl)(methyl)amino)methyl)-1H-indole-5-car-
1
boxamide (9g). Yellow solid; yield 55.32%. Mp: 69.8−71.5 °C. H
NMR (500 MHz, MeOD): δ 8.23 (s, 1H), 7.68 (dd, J = 8.6, 1.3 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 7.35−7.29 (m, 1H), 7.25 (d, J = 8.5
Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 3.83 (s, 2H), 3.78 (s, 3H), 3.58 (s,
2H), 2.24 (s, 3H). 13C NMR (125 MHz, MeOD): δ 174.01, 160.64,
140.11, 132.02, 130.66, 128.79, 127.75, 125.57, 122.18, 120.69,
114.79, 112.92, 112.16, 61.72, 55.68, 52.62, 41.91. LRMS (ESI), m/z
calcd for C19H21N3O2, [M + H]+ 324.40, found 324.07.
3-(((4-(Allyloxy)benzyl)(methyl)amino)methyl)-1H-indole-5-car-
1
boxamide (9h). Yellow solid; yield 50.14%. Mp: 89.1−91.0 °C. H
NMR (500 MHz, MeOD): δ 8.23 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H),
7.60 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 6.98
(d, J = 8.4 Hz, 2H), 6.02 (m, 1H), 5.36 (dd, J = 17.3, 1.4 Hz, 1H),
5.22 (dd, J = 10.6, 1.2 Hz, 1H), 4.54 (d, J = 5.1 Hz, 2H), 4.45 (s, 2H),
4.20 (s, 2H), 2.63 (s, 3H). 13C NMR (125 MHz, MeOD): δ 173.48,
161.18, 140.02, 134.57, 133.43, 130.87, 128.32, 126.90, 122.87,
120.04, 117.74, 116.39, 112.80, 69.87, 60.16, 51.88, 39.50. LRMS
(ESI), m/z calcd for C21H23N3O2, [M + H]+ 350.43, found 350.06.
3-((Methyl(4-(trifluoromethoxy)benzyl)amino)methyl)-1H-in-
dole-5-carboxamide (9i). Yellow solid; yield 56.07%. Mp: 61.5−63.0
1
°C. H NMR (500 MHz, MeOD): δ 8.31 (d, J = 1.0 Hz, 1H), 7.69
(dd, J = 8.6, 1.6 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.6 Hz,
1H), 7.38 (s, 1H), 7.24 (d, J = 8.1 Hz, 2H), 3.94 (s,2H), 3.72 (s, 2H),
2.30 (s, 3H). 13C NMR (125 MHz, MeOD): δ 173.96, 149.93,
140.18, 132.39, 128.71, 128.03, 125.69, 122.25, 121.97, 120.78,
112.23, 61.16, 53.21, 41.80. LRMS (ESI), m/z calcd for
C19H18F3N3O2, [M + H]+ 378.37, found 378.07.
3-((Benzyl(methyl)amino)methyl)-1H-indole-5-carboxamide
1
4.2.7. Immunofluorescence. PC12 cells (1.5 × 104 cells/mL) were
cultured on the climbing slices coated with PLL in 24-well plates.
After replacement with medium containing 2% HS and 1% FBS for 16
h, the cells were treated with 8c and 8g with concentrations of 10 μM
in the presence of NGF (10 ng/mL) for 48 h. NGF (10 ng/mL) was
used as a positive control. The cells were fixed with cold 4%
paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100
for 30 min, and then blocked with 10% normal goat serum for 2 h at
room temperature. After incubating with a primary antibody specific
to β-tubulin (1:50) overnight at 4 °C, the cells were washed with
TBST and incubated with the secondary antibody labeled with Alexa
Fluor 488 diluted to 1:100 in TBST at 37 °C for 2 h. Following
washing, the nuclei were stained by 4′,6-diamidino-2-phenylindole
(DAPI) and the climbing slices were washed and sealed. Images were
acquired via the fluorescence microscope (OLYMPUS, Japan) with
excitation/emission wavelengths of 490 nm/540 nm for the Alexa
Fluor-488 and 360 nm/450 nm for the DAPI. The immunofluor-
escence assay of the NF-κB p65 was as described above.
4.2.8. COX-2 Inhibition Assay. The COX-2 inhibitory activities of
the compounds were measured using human COX-2 inhibitory
screening assay kit (Beyotime, catalog no. S0168) according to the
manufacturer’s instructions. The final concentrations of the
compounds were 3.125, 6.25, 12.5, 25, 50, and 100 μM. The
absorbance was measured in a microplate reader (Bioteck Synergy
HTX, USA) at 405 nm. IC50 values were calculated by the sigmoidal
dose−response equation (variable slope) (GraphPad software). All of
the experiments were repeated three times.
(9j). White solid; yield 58.08%. Mp: 96.0−98.0 °C. H NMR (500
MHz, MeOD): δ 8.27 (s, 1H), 7.71 (dd, J = 8.6, 1.4 Hz, 1H), 7.46−
7.42 (m, 2H), 7.36 (dt, J = 15.6, 7.4 Hz, 5H), 4.04 (s, 2H), 3.83 (s,
2H), 2.38 (s, 3H). 13C NMR (125 MHz, MeOD): δ 173.83, 140.08,
131.13, 129.67, 129.19, 128.71, 128.64, 125.94, 122.39, 120.51,
112.36, 110.95, 61.80, 52.67, 41.34. LRMS (ESI), m/z calcd for
C18H19N3O, [M + H]+ 294.37, found 294.02.
3-(((4-Chlorobenzyl)(methyl)amino)methyl)-1H-indole-5-car-
1
boxamide (9k). White solid; yield 59.35%. Mp: 160.3−162.0 °C. H
NMR (500 MHz, MeOD): δ 8.29 (s, 1H), 7.71 (dd, J = 8.6, 1.4 Hz,
1H), 7.46 (d, J = 8.9 Hz, 2H), 7.41−7.37 (m, 4H), 4.14 (s, 2H), 3.90
(s, 2H), 2.43 (s, 3H). 13C NMR (125 MHz, MeOD): δ 173.57,
139.98, 136.37, 132.62, 132.17, 130.26, 129.72, 127.68, 126.52,
124.29, 122.79, 120.03, 112.65, 51.00, 43.11, 41.08. LRMS (ESI), m/z
calcd for C18H19N3O, [M + H]+ 328.81, found 328.11.
4.2. Biological Assays. 4.2.1. Determination of NO Production.
BV-2 cells were seeded in 96-well plates with a density of 2 × 104
cells/well. After 24 h, cells were treated with or without LPS (1 μg/
mL) in the absence or presence of compounds at 10 μM for 24 h. NO
production in the supernatant was assessed by an NO assay kit based
on the Griess reaction.34 Nitrite concentrations in the cell culture
medium were calculated from a prepared sodium nitrite standard
curve as follows: NO inhibition (%) = 1 − (sample OD − control
OD)/(LPS OD − control OD) × 100%.
4.2.2. Cell Viability Assay. BV-2 cells were seeded into a 96-well
plate with a density of 2 × 104 cells/well. After 24 h, cells were
incubated with the compounds under investigation (10 μM) for 24 h.
Next, MTT (10 μL, 0.5 mg/mL) was added to each well, and then the
cells were maintained at 37 °C for 4 h. After this, the medium was
removed, 150 μL of DMSO was added, and the absorbance was
measured at 570 nm using a microplate absorbance reader.
4.2.3. Determination of Neuroprotection against H2O2. PC12
cells were seeded in a 96-well plate at 1.5 × 104 cells/well, incubated
for 24 h, and subsequently treated with compounds 8c and 8g (5, 10,
and 20 μM) for 2 h. The cells were then treated with H2O2 (500 μM)
for 24 h. After this, MTT (2.5 mg/mL) was added and incubated for
another 4 h at 37 °C. The medium was removed, 150 μL of DMSO
was added, and the optical density of absorbance was measured at 570
nm.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00079.
Predicted BBB permeability and drug-like parameters of
compounds 5a−9k and spectroscopic data of com-
pounds 2−4, 5a−9k (PDF)
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Corresponding Authors
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Jin-Ming Gao − Shaanxi Key Laboratory of Natural Products
& Chemical Biology, College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, P. R. China;
orcid.org/0000-0003-4801-6514; Email: jinminggao@
nwsuaf.edu.cn
Ding Li − Shaanxi Key Laboratory of Natural Products &
Chemical Biology, College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, P. R. China;
orcid.org/0000-0003-3130-6099; Phone: +86-29-
87092335; Email: jbolid@nwsuaf.edu.cn; Fax: +86-29-
87092335
Authors
Wei-Wei Wang − Shaanxi Key Laboratory of Natural
Products & Chemical Biology, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling 712100, P.
R. China
Ting Liu − Shaanxi Key Laboratory of Natural Products &
Chemical Biology, College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, P. R. China
Yu-Meng Lv − Shaanxi Key Laboratory of Natural Products
& Chemical Biology, College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, P. R. China
Wu-Yang Zhang − Shaanxi Key Laboratory of Natural
Products & Chemical Biology, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling 712100, P.
R. China
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Zhi-Gang Liu − Shaanxi Key Laboratory of Natural Products
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Northwest A&F University, Yangling 712100, P. R. China
Complete contact information is available at:
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Author Contributions
W.-W.W. conducted the biological study and performed the
experiments. T.L., Y.-M.L., and W.-Y.Z. synthesized the
compounds. Z.-G.L. contributed to data analysis and
interpretation. W.-W.W., J.-M.G., and D.L. conceived and
designed the experiments. W.-W.W., T.L., and D.L. wrote the
manuscript and revised the manuscript. All authors discussed
the results and implications and commented on the manuscript
at all stages.
Author Contributions
†W.-W.W. and T.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Natural
Science Foundation of China (21502152) and the Natural
Science Foundation of Shaanxi Province (2019JM-334). The
authors thank Life Science Research Core Services at
Northwest A&F University (Luqi Li and Junmin Li) for the
ESI-HRMS data.
REFERENCES
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Research Article
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https://doi.org/10.1021/acschemneuro.1c00079
ACS Chem. Neurosci. 2021, 12, 1593−1605