Design and synthesis of β-carboline and combretastatin derivatives as anti-neutrophilic inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2021.104846

A series of β-carboline derivatives was synthesized by the Pictet-Spengler reaction with or without the combretastatin skeleton. The structures of these derivatives were elucidated by spectroscopic techniques. All synthesized compounds were evaluated for

Full text of DOI:10.1016/j.bioorg.2021.104846

Bioorganic Chemistry 111 (2021) 104846
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of β-carboline and combretastatin derivatives as
anti-neutrophilic inflammatory agents
,
,
Sunil Kumara b, Yi-Hsuan Wangb c, Po-Jen Chend, Yu-Chia Change, Hemant K. Kashyapf,
Ya-Ching Shena *, Huang-Ping Yug *, Tsong-Long Hwangb
,
,
h,
,c,e,g,h,i,*
a School of Pharmacy, College of Medicine, National Taiwan University, Taipei City 100, Taiwan
b Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan
c Division of Natural Products, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
d Department of Cosmetic Science, Providence University, Taichung 43301, Taiwan
e Research Center for Chinese Herbal Medicine, Graduate Institute of Healthy Industry Technology, College of Human Ecology, Chang Gung University of Science and
Technology, Taoyuan 33303, Taiwan
f Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
g College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
h Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan
i Department of Chemical Engineering, Ming Chi University of Technology, New Taipei City, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of β-carboline derivatives was synthesized by the Pictet-Spengler reaction with or without the com-
bretastatin skeleton. The structures of these derivatives were elucidated by spectroscopic techniques. All syn-
thesized compounds were evaluated for their anti-inflammatory activity in human neutrophils. Among them, two
compounds, NTU-228 and HK-72, showed significant inhibitory effects on N-formyl-Met-Leu-Phe (fMLF)-induced
Organic synthesis
β-carboline
Combretastatin
Neutrophils
superoxide anion generation in human neutrophils with IC50 values of 5.58 ± 0.56 and 2.81 ± 0.07
μM,
Anti-inflammatory agents
p38 MAPK
respectively. Neither NTU-228 nor HK-72 caused cytotoxicity in human neutrophils. NTU-228 inhibited the
phosphorylation of p38 mitogen-activated protein kinase (MAPK) and intracellular Ca2+ levels ([Ca2+]i) in fMLF-
activated human neutrophils. Additionally, HK-72 selectively inhibited the fMLF-induced phosphorylation of p38
and [Ca2+]i in human neutrophils. Molecular docking analysis showed a favorable binding affinity of HK-72
toward p38 MAPK. The proposed synthetic strategy opens up new opportunities for the synthesis of novel po-
tential candidates against neutrophilic inflammation.
Molecular docking
1. Introduction
inflammation. Once neutrophils reach the inflamed sites via adhesion to
endothelial cells and transmigration through blood vessels, they
Inflammation is an important biological process in the human body
in response to harmful stimuli; nevertheless, dysregulated inflammation
contributes to many physiological illnesses and pathological processes,
which can be classified as acute or chronic inflammation and autoim-
mune diseases. A variety of diseases are usually defined as inflammatory
disorders, such as cancers, diabetes, cardiovascular disorders, pancrea-
titis, and arthritis [1–6]. Neutrophils are the most abundant white blood
cells involved in innate immunity against foreign pathogens; however,
overwhelming activation of neutrophils is a critical pathogenic factor
that triggers many inflammatory responses, so-called neutrophilic
generate numerous superoxide anions and associated reactive oxygen
species (ROS), elastase, and neutrophil extracellular traps (NETs),
causing tissue damage and neutrophilic inflammatory diseases,
including acute respiratory distress syndrome, sepsis, psoriasis, or
chronic obstruction pulmonary disease [7–13]. Therefore, targeting
neutrophilic inflammation has been considered a profitable remedy for
inflammatory diseases.
Natural products have been used as traditional herbal medicines to
prevent or treat various diseases for thousands of years [14,15] and also
serve as lead molecules for the development of anti-inflammatory drugs.
* Corresponding authors at: Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan (T.-L. Hwang); College of
Medicine, Chang Gung University, Taoyuan 333, Taiwan (H.-P. Yu); School of Pharmacy, College of Medicine, National Taiwan University, Taipei City 100, Taiwan
(Y.-C. Shen).
E-mail addresses: ycshen@ntu.edu.tw (Y.-C. Shen), yuhp2001@adm.cgmh.org.tw (H.-P. Yu), htl@mail.cgu.edu.tw (T.-L. Hwang).
https://doi.org/10.1016/j.bioorg.2021.104846
Received 27 August 2020; Received in revised form 2 February 2021; Accepted 18 March 2021
Available online 26 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
The anti-inflammatory activities of natural products have been applied
as folk remedies for many inflammatory conditions, such as fever, pain,
migraine, and arthritis [16]. A great diversity of natural product-derived
molecular structures and pharmacological mechanisms provides a
referential significance for the development of new drugs, including
curcumin from turmeric, genistein from soybean, and gingerol from
gingers [17–19]. It has been documented that structural analogs by
molecular modification of functional groups of natural lead compounds
may exhibit more efficient bioactivities or fewer side effects. Therefore,
compounds derived from natural products may provide a possibility to
develop clinically used agents against various diseases [20]. Com-
bretastatins (Fig. 1a) A and B are biosynthetically related stilbene and
dihydrostilbene secondary metabolites isolated from the South African
folk medical tree Combretum caffrum [21–25]. Some combretastatins
have better anti-inflammatory activity than the standard drug ascorbic
acid and diclofenac sodium [26]. 1-Substituted β-carbolines represent a
large group of biologically active natural products. For instance, β-car-
bolines (Fig. 1b, c), canthin-6-ones, and bis-β-carbolines isolated from
Picrasma. quassioides have shown promising anti-inflammatory effects
[27]. Consequently, the total synthesis of these alkaloids has found
widespread interest in the past. Various approaches have been published
for the synthesis of 1-alkyl-, 1-aryl-, and 1-heteroaryl-β-carbolines,
either by building up a heterocyclic ring system starting from trypt-
amine derivatives or by substitution reactions of 1-substituted β-carbo-
lines [28–31].
oxide and DCM. Consecutive Pictet-Spengler reaction in the presence of
TFA, paraformaldehyde, and isopropanol converted NTU-143 to NTU-
236 and NTU-237.
Further, β-carbolines without a combretastatin moiety were syn-
thesized using the Pictet-Spengler reaction. The structures of novel
compounds were established by NMR and mass spectrometry, while
those of others were confirmed by comparison with available data.
These compounds are shown in Schemes 2–5. N-(2-(1H-indol-3-yl)
ethyl)-N-allylprop-2-en-1-amine (HK-4) was synthesized by alkylation
of tryptamine with allyl bromide (Scheme 2) [35].
4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-N,N-dime-
thylbenzenamine (HK-17) was prepared according to a previously
published procedure [33].
The reaction of 3-butyraldehyde with tryptamine under Pictet
Spengler reaction conditions produced HK-27 [36] which was further
oxidized by manganese oxide to form HK-30 (Scheme 2).
HK-28 and 29 were prepared in a ratio of 1:2 by the Pictet Spengler
reaction after BOC protection of tryptamine followed by reaction of allyl
bromide in the presence of a mild base as given in Scheme 3.
HK-46 was synthesized by the reaction of allyl bromide with carba-
zole in the presence of a base and solvent DMF. As shown in Scheme 4,
tryptamine was subjected to the Pictet-Spengler reaction to obtain
compounds HK-47 to HK-52 and HK-71 in – 80–92% yield. Spectral data
of 9-allyl-9H-carbazole (HK-46) [37], 2,3,4,9-tetrahydro-1-(naphthalen-
1-yl)-1H-pyrido[3,4-b]indole (HK-47) [38], 2,3,4,9-tetrahydro-1-
(naphthalen-3-yl)-1H-pyrido[3,4-b]indole (HK-49) [39], 2,3,4,9-tetra-
hydro-1-(4-methoxyphenyl)-1H-pyrido[3,4-b]indole (HK-50) [40], and
4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-2-methoxyphenol
(HK-52) [41] were compared and confirmed with respective references.
As shown in Scheme 5, 5-methoxy tryptamine was used as the
starting material, and the following reactions were similar to those in
Scheme 4. Eventually, we obtained compounds HK-62–68 and HK-72 in
– 70–95% yield. Spectral data of known compounds, such as 4-(2,3,4,9-
tetrahydro-6-methoxy-1H-pyrido[3,4-b]indol-1-yl)-2-methoxyphenol
(HK-63) [42], 2,3,4,9-tetrahydro-6-methoxy-1-(naphthalen-1-yl)-1H-
pyrido[3,4-b]indole (HK-66) [43], 2,3,4,9-tetrahydro-6-methoxy-1-(4-
methoxyphenyl)-1H-pyrido[3,4-b]indole (HK-68) [43], 1-(2-fluo-
rophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (HK-71) [43],
and 1-(2-fluorophenyl)-2,3,4,9-tetrahydro-6-methoxy-1H-pyrido[3,4-b]
indole (HK-72) [43] were compared and confirmed with respective
literature resources.
Hence, the aim of this study was to discover effective β-carboline
with or without combretastatin basic skeletons as anti-inflammatory
agents for the attenuation of neutrophilic inflammation.
2. Results and discussion
2.1. Chemistry
A series of β-carboline molecules with or without the combretastatin
skeleton were synthesized using Knovengial condensation [32], Pictet-
Spengler reaction [33], and oxidation and reduction reactions [34].
These compounds were characterized using NMR and mass spectrom-
etry. The synthetic route to the NTU series compounds is shown in
Scheme 1. Commercially available 3,4,5-trimethoxy phenyl acetic acid
(2) was the starting material for the synthesis. To accomplish our task,
we first synthesized the combretastatin part by reacting isovanillin with
3,4,5-trimethoxy phenyl acetic acid in the presence of trimethylamine
and acetic anhydride. Then, the acid group was converted to alcohol by
valeryl chloride, triethyl amine, and sodium borohydride in THF in 92%
yield. Subsequently, alcohol was oxidized to aldehyde by manganese
oxide with areal oxidation to afford compound 5. Compound 5 with an
aldehyde group was a crucial for the fusion of combretastatin and
β-carbolines core. To synthesize hybrid molecules, compound 5 and
tryptamine were subjected to the Pictet-Spengler reaction in the pres-
ence of TFA and isopropanol to obtain NTU-143 in 60% yield. Further
deacetylation of NTU-143 in the presence of potassium hydroxide in
ethanol gave NTU-142. Tetrahydro-β-carboline (NTU-143) was oxidized
β-carboline (NTU-228 and NTU-229) in the presence of manganese
2.2. Pharmacological activity
Certain β-carboline derivatives with or without combretastatin basic
structures were synthesized and evaluated for their inhibitory effects on
superoxide anion generation and elastase release in N-formyl-L-
methionyl-L-leucyl-L-phenylalanine (fMLF)-activated human neutro-
phils (Table 1). Most compounds exhibited significant inhibitory effects
on superoxide anion generation and elastase release. NTU-236 and HK-
72 showed potent inhibitory effects on fMLF-induced superoxide anion
generation in human neutrophils with IC50 values of 3.72 ± 0.27 and
2.81 ± 0.07 μM, respectively. In addition, NTU-228 inhibited superoxide
Fig. 1. (a) Combretastatin, (b) β-carboline, (c) Tetrahydro-β-carboline.
2

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
Scheme 1. Reagents and conditions: a-Triethyl amine, acetic anhydride, reflux, 12 h; b-valeryl chloride, TEA, THF, rt; NaBH4, THF, 20 ◦C; c- MnO2, CH2Cl2, rt; d-
TFA, isopropanol, reflux, 12 h; e-MnO2, CH2Cl2, 12–48 h; f- KOH, ethanol, rt; g- TFA, paraformaldehyde, isopropanol, reflux, 6 h; h- MnO2, CH2Cl2, rt.
generation in fMLF-activated human neutrophils with IC50 value of 5.58
± 0.56 M. Unlike NTU-236 (10 M), NTU-228 (10 M) and HK-72 (10
respectively (Fig. 8). Taken together, these result suggest that the anti-
inflammatory activities of NTU-228 and HK-72 may be mediated by
p38 MAPK and Ca2+ signaling in human neutrophils and p38 MAPK and
[Ca2+]i may be potential therapeutic targets for the development of anti-
inflammatory agents.
μ
μ
μ
μ
M) had no effect on the viability of human neutrophils, as measured by
lactate dehydrogenase (LDH) release (Figs. 2 and 5). These results sug-
gest that NTU-228 and HK-72 may be more profitable as anti-
inflammatory agents because of their low cytotoxicity.
Neutrophil activation reactions, such as respiratory burst and
degranulation, are mediated by intracellular signaling pathways,
including MAPKs, Akt, and calcium mobilization. Therefore, we further
investigated the molecular mechanisms of NTU-228 and HK-72 in acti-
vated human neutrophils. NTU-228 repressed the p38 MAPK and
intracellular Ca2+ concentration [Ca2+]i in fMLF-activated human neu-
trophils (Figs. 3 and 4). HK-72 also selectively inhibited p38 MAPK and
[Ca2+]i but not Erk, JNK, and Akt in fMLF-activated human neutrophils
(Figs. 6 and 7). Based on molecular docking prediction, HK-72 was
proposed to directly target p38 MAPK via two distinct binding sites with
favorable binding affinity scores of ꢀ 8.4 and ꢀ 8.3 kcal/mol,
2.3. Molecular docking analysis
To determine the binding affinity of HK-72 towards p38 MAPK,
protein-ligand docking, which predicts the molecular interactions be-
tween the targeted receptor and ligand molecule in 3-dimensional space,
was performed. The two most favorable binding modes were defined
with binding affinities of ꢀ 8.4 and ꢀ ꢀ 8.3 kcal/mol in Fig. 8 (a) and (b),
respectively. The values of binding affinity indicate a spontaneous as-
sociation of HK-72 and p38, and two distinct profitable HK-72-targeted
sites in p38 MAPK.
3

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
Scheme 2. Reagents and conditions: a- TFA, isopropanol, N,N-dimethylbenzaldehyde, reflux, 6 h; b- MnO2, pyridine, DCM; c- TFA, isopropanol, N,N-butyral-
dehyde, reflux, 6 h; d- MnO2, pyridine, DCM; e- Allyl bromide, NaHCO3, DMF.
Scheme 3. Reagents and conditions: a-BOC anhydride, NaHCO3; b- Allyl bromide, NaHCO3, DMF, rt; c- TFA, dioxane; d- TFA, isopropanol, N,N-butyraldehyde,
reflux, 6 h.
Scheme 4. Reagents and conditions: a- Allyl bromide, NaHCO3, DMF, rt; b- TFA, isopropanol, substituted-aldehydes, reflux, 5–10 h.
3. Structure – activity relationships (SAR)
β-carboline ring played an important role in the modulation of the anti-
inflammatory activities. In hybrid molecules, removal of the acetate
group from position 3 or addition of an alkoxy group at position 6 led to
enhanced anti-neutrophilic activity. Similarly, the introduction of
seven-membered rings in hybrid molecules enhanced the inhibitory ef-
fect on superoxide generation but did not affect elastase release. More-
over, the presence of a fluorine moiety or the introduction of the allyl
moiety at position N-9 of β-carboline increased the superoxide anion
Comprehensive structure-activity relationship studies of β-carbolines
with combretastatins (Fig. 9) demonstrated that most of the compounds
had moderate effects on superoxide anion generation and elastase
release. However, β-carboline showed the best activity among all the
synthesized compounds. Evaluation of the anti-neutrophilic activities of
1-substituted β-carbolines revealed that the substituent on the
4

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
Scheme 5. Reagents and conditions: a- TFA, isopropanol, substituted-aldehydes, reflux, 5–10 h.
Table 1
Effects of compounds on superoxide anion generation and elastase release in
fMLF/CB-induced human neutrophils.
Compound
Superoxide anion
Elastase
IC50
(μ
M)a
Inh %
IC50
(μ
M)a
Inh %
HK-4
14.13 ± 4.63 *
12.25 ± 5.01 *
20.74 ± 5.12 *
15.80 ± 5.24 *
45.18 ± 1.50 ***
36.49 ± 6.09 **
17.94 ± 6.54
2.37 ± 6.34
HK-17
HK-25
HK-27
HK-28
HK-29
HK-30
HK-46
HK-47
HK-48
HK-49
HK-50
HK-51
HK-52
HK-53
HK-62
HK-63
HK-64
HK-65
HK-66
HK-67
HK-68
HK-71
HK-72
NTU-142
NTU-143
NTU-228
NTU-229
NTU-236
NTU-237
NTU-239
3.71 ± 3.76
ꢀ 5.83 ± 1.42 *
0.92 ± 5.72
ꢀ 7.44 ± 2.92
0.19 ± 1.92
13.60 ± 5.92
31.10 ± 4.01 ***
3.64 ± 3.48
37.64 ± 5.15 **
8.89 ± 0.62 ***
7.16 ± 2.77 *
ꢀ 0.35 ± 3.18
1.07 ± 3.27
8.25±0.56
57.96 ± 1.99 ***
13.46 ± 4.27 *
31.35 ± 5.60 **
28.96 ± 4.69 **
51.76 ± 1.57 ***
16.17 ± 3.66 *
19.77 ± 5.77 *
25.48 ± 6.00 *
28.60 ± 2.30 ***
32.19 ± 1.66 ***
3.19 ± 1.25
5.75 ± 4.19
4.62 ± 6.33
Fig. 2. NTU-228 does not affect viability of human neutrophils. Human neu-
9.38 ± 4.09
trophils were incubated with DMSO (0.1%), NTU-228 (10 μM), or NTU-226 (10
8.64 ± 0.72
14.91 ± 3.70 *
1.18 ± 4.42
μ
M) for 15 min. Cell viability was assessed by determining LDH release in the
cell-free supernatant. The total LDH release was determined by lysing the cells
with 0.1% Triton X-100 for 30 min at 37 ◦C. All data are presented as the mean
± S.E.M. (n = 4).
6.89 ± 3.57
5.38 ± 3.62
3.29 ± 3.27
ꢀ 1.37 ± 1.53
3.51 ± 6.25
activated human neutrophils. Additionally, a molecular docking study
suggested the possibility of an association between HK-72 and p38
MAPK. In the future, these small molecules may serve as lead com-
pounds for the development of potential anti-inflammatory drugs.
12.46 ± 6.32
1.70 ± 6.07
8.33 ± 0.71
2.81 ± 0.07
8.06 ± 1.03
59.51 ± 5.09 ***
63.32 ± 6.08 ***
58.92 ± 5.10 ***
42.04 ± 3.42 ***
81.70 ± 6.64 ***
57.03 ± 3.20 ***
80.24 ± 5.94 ***
35.63 ± 5.60 **
42.59 ± 4.79 ***
2.43 ± 1.01
18.57 ± 3.55 **
2.53 ± 6.55
2.80 ± 4.48
5.58 ± 0.56
7.64 ± 1.56
3.72 ± 0.27
7.28 ± 0.85
63.21 ± 7.72 **
33.63 ± 5.88 **
57.38 ± 2.42 ***
55.03 ± 4.47 ***
39.24 ± 6.43 ***
5. Experimental procedures
8.00 ± 0.60
7.84 ± 0.01
5.1. Chemistry
5.1.1. Experimental apparatus and reagents
Percentage of inhibition (Inh %) at 10 μM concentration. Results are presented
The reagents were purchased from Sigma-Aldrich (MO, USA), Alfa
Aesar (Heysham), and J. T. Baker (Center Valley, PA) and used without
further purification. All yields refer to the separated products after pu-
rification. The products were characterized by spectroscopic data (H
NMR and C NMR spectra). The spectra were measured in DMSO‑d6 re-
lative to TMS (0.00 ppm). Melting points were determined in open
capillaries and were found to be uncorrected. Column chromatography
was performed on silica gel (300–400 mesh), eluting with ethyl acetate
and hexane. TLC was performed on 0.20 mm silica gel 60 F254 plates
(Merck KGaA, Darmstadt, Germany). NMR data were obtained for 1H at
400 MHz and 200 MHz and for 13C at 100 MHz and 50 MHz on a Bruker
Avance 400 spectrometer (Bruker Company, Germany), using TMS as an
internal standard. Chemical shifts were expressed in ppm (parts per
million). High-resolution mass spectral (HRMS) data were acquired on a
Thermo LTQ instrument electrospray mode (ESI). The purity of each
compound (>95%) was determined using a Nexera-i LC-2040C 3D
as mean ± S.E.M. (n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001 compared
with the control (DMSO).
a
Concentration necessary for 50% inhibition (IC50).
inhibitory activity; however, there was no effect on elastase release
activity.
4. Conclusion
Here, we synthesized hybrid molecules containing β-carboline core
with or without a combretastatin basic moiety using Knovengial
condensation, dehydration, Pictet-Spengler reaction, areal oxidation,
and reduction reactions. Furthermore, we demonstrate that NTU-228
and HK-72 potentially inhibit fMLF-induced superoxide anion genera-
tion in human neutrophils. The anti-inflammatory ability of NTU-228
and HK-72 may be mediated via p38 MAPK and [Ca2+]i in fMLF-
5

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
Fig. 3. NTU-228 decreases the phos-
phorylation of p38 MAPK in fMLF-
induced human neutrophils. Human
neutrophils were incubated with DMSO
(0.1%) or NTU-228 (10 μM) for 5 min
and then activated with or without fMLF
(0.1 M). Phosphorylation of (A) p38
μ
MAPK, (B) JNK, (C) Erk, or (D) Akt was
analyzed by immunoblotting with anti-
bodies against phosphorylated or total
protein. Data are normalized to the
corresponding total protein level and
expressed as mean ± S.E.M. **p < 0.01;
***p < 0.001 compared with DMSO +
fMLF (n = 3).
Fig. 4. NTU-228 inhibits Ca2þ mobilization in
fMLF-activated human neutrophils. Fluo-4/
AM-labeled human neutrophils were treated
with DMSO (0.1%) or NTU-228 (10
μ
M) for 5 min
and then activated by fMLF (0.1
μ
M). Mean
fluorescent intensity of [Ca2+
] was detected
upon excitation at 494 and 516i nm with spec-
trofluorometer as representative traces and the
peak and t1/2 of [Ca2+]i. All data are presented as
mean ± S.E.M. *p < 0.05 compared with the
DMSO + fMLF (n = 4).
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S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
5.1.2. (E)-3-(3-acetoxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)
acrylic acid (3)
Isovanilin (1 g, 6.6 mmol) was dissolved in 16 mL of acetic anhydride
followed by addition of 3,4,5-trimethoxy benzoic acid (1.5 g, 6.6 mmol)
and triethylamine (8 mL). The reaction mixture was refluxed for 12 h
under an inert atmosphere. After completion of the reaction, the reac-
tion mixture was cooled to room temperature. Subsequently, water was
added, and the product was extracted with ethyl acetate (25 × 3). The
combined organic layer was dried over anhydrous Mg2SO4 and purified
by silica gel (100–200 mesh) column chromatography using an ethyl
acetate and hexane solvent system (2:1). Product 3 was obtained as a
brown solid (87% yield).
1H NMR (200 MHz, CDCl3), δ 7.83 (s, 1H), 6.99 (d, J = 8.6 Hz, 1H),
6.78 (d, J = 8.6 Hz, 1H), 6.74 (s, 1H), 6.45 (s, 2H), 3.89 (s, 3H), 3.88 (s,
3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 2.24 (s, 3H); 13C NMR (50
MHz, CDCl3) δ 173.5, 169.2, 154.1, 152.8, 141.8, 139.6, 138.1, 131.0,
130.9, 130.1, 127.4, 125.7, 112.3, 106.8, 61.4, 56.5, 56.3, 21.0; HRMS
(ESI): m/z calcd for C21H22O8 [M+H]+ 403.1315, found 403.1395.
Fig. 5. HK-72 does not affect viability of human neutrophils. Human neutro-
phils were incubated with DMSO (0.1%) or HK-72 (10 μM) for 15 min. Cell
viability was assessed by determining LDH release in the cell-free supernatant.
The total LDH release was determined by lysing the cells with 0.1% Triton X-
100 for 30 min at 37 ◦C. All data are presented as the mean ± SEM (n = 3).
5.1.3. (E)-5-(2-(3-ethyl-4,5-dimethoxyphenyl)-3-hydroxyprop-1-en-1-yl)-
2-methoxyphenyl acetate (4)
Liquid Chromatograph (column, Xtimate C18, 4.6 mm × 150 mm, 5
mobile phase, methanol (60%)/H2O (40%); low rate, 1.0 mL/min; UV
wavelength, 254–400 nm; temperature, 25 ◦C; injection volume, 10
L).
μm;
Compound 3 (0.5 g, 1.2 mmol) was dissolved in THF (10 mL), then
valeryl chloride (0.18 mL, 2.2 mmol) and triethylamine (0.1 mL) were
added slowly under an inert atmosphere and stirred at room
μ
Fig. 6. HK-72 decreases the phosphorylation
of p38 MAPK but not JNK, Erk, and Akt in
fMLF-induced human neutrophils. Human
neutrophils were incubated with DMSO
(0.1%) or HK-72 (10
μ
M) for 5 min and then
M).
activated with or without fMLF (0.1
μ
Phosphorylation of (A) p38 MAPK, (B) JNK,
(C) Erk, or (D) Akt was analyzed by immu-
noblotting with antibodies against phos-
phorylated or total protein. Data are
normalized to the corresponding total pro-
tein level and expressed as mean ± S.E.M.
**p < 0.01 compared with DMSO + fMLF (n
= 3).
7

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
Fig. 7. HK-72 inhibits Ca2þ mobilization in fMLF-activated human neutrophils. Fluo-4/AM-labeled human neutrophils were treated with DMSO (0.1%) or HK-
72 (10
μ
M) for 5 min and then activated by fMLF (0.1
μ
M). Mean fluorescent intensity of [Ca2+]i was detected inexcitation at 494 and 516 nm with spectrofluo-
rometer as representative traces and the peak and t1/2 of [Ca2+]i. All data are presented as mean ± S.E.M. *p < 0.05 compared with the DMSO + fMLF (n = 3).
Fig. 8. Docking models of p38 MAPK in complex with HK-72 The binding affinity scores of most favorable binding modes were shown with (a) ꢀ 8.4 and (b) ꢀ 8.3
kcal/mol, respectively.
Fig. 9. Structures of basic skeletons for SAR study: (a) β-carboline with combretastatin (b) β-carboline.
8

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
temperature. After one h, the white precipitate was filtered off and so-
dium borohydride (0.071 g, 1.9 mmol) was added to the filtrate and
stirred for 30 min. Water was added and the solution was neutralized
with dil HCl. Extraction was performed three times with ethyl acetate.
The combined organic layer was dried over anhydrous Mg2SO4 and
purified by silica gel (100–200 mesh) column chromatography using an
ethyl acetate and hexane solvent system (1:1). Product 4 was obtained as
a colorless liquid (92% yield).
(d, J = 3.2 Hz, 1H), 7.17 (s, 1H), 7.12 (dd, J = 7.6 & 7.2 Hz, 1H), 6.98 (d,
J = 8.8 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.54 (s, 1H), 6.51 (s, 2H), 5.22
(s, 1H), 3.68 (s, 3H), 3.65 (s, 3H), 3.52 (s, 6H), 2.06 (s, 3H); 13C NMR
(100 MHz, Acetone-D) δ 168.1, 154.0, 150.8, 140.8, 139.6, 139.1,
138.3, 134.7, 134.0, 130.7, 129.9, 129.7, 129.5, 128.6, 128.1, 123.8,
121.5, 121.4, 119.7, 113.8, 113.4, 112.0, 107.6, 59.9, 55.6, 55.5, 22.4;
HRMS (ESI): m/z calcd for C31H30N2O6 [M+H]+ 527.2104, found
527.2105.
1H NMR (200 MHz, CDCl3), δ 6.92 (d, J = 8.6 Hz, 1H), 6.77 (s, 1H),
6.72 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H), 6.45 (s, 2H), 4.42 (s, 2H), 3.87 (s,
3H), 3.77 (s, 9H), 2.24 (s, 3H); 13C NMR (50 MHz, CDCl3) δ 169.4, 154.1,
150.5, 140.6, 139.5, 137.9, 134.1, 129.7, 128.3, 125.9, 123.8, 112.2,
105.9, 69.0, 61.4, 56.5, 56.2, 21.0; HRMS (ESI): m/z calcd for C22H26O6
[M+H]+ 387.1729, found 387.1445.
5.1.7. (E)-5-(2-(9H-pyrido[3,4-b]indol-1-yl)-2-(3,4,5-trimethoxyphenyl)
vinyl)-2-methoxyphenyl acetate (8)
Yield: 75%; 1H NMR (400 MHz, CDCl3), δ 8.57 (d, J = 4.8 Hz, 1H),
8.13 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 4.8 Hz, 1H), 7.90 (s, NH), 7.50 (dd,
J = 8.4 & 7.6 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.26 (s, 1H), 7.20 (s, 1H),
6.77 (s, 1H), 6.69 (d, J = 8.4 Hz, 1H), 6.52 (m, 3H), 3.84 (s, 3H), 3.73 (s,
6H), 3.65 (s, 3H), 2.22 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.9,
153.3, 150.6, 139.8, 138.3, 137.2, 134.2, 129.9, 128.7, 127.0, 123.8,
121.7, 120.2, 114.3, 112.2, 111.9, 104.9, 60.9, 56.3, 55.8, 20.7; HRMS
(ESI): m/z calcd for C31H28N2O6 [M+H]+ 525.1947, found 525.2041.
5.1.4. (E)-2-methoxy-5-(3-oxo-2-(3,4,5-trimethoxyphenyl)prop-1-en-1-
yl)phenyl acetate (5)
Compound 4 (0.4 g, 1.0 mmol) was dissolved in dichloromethane,
then 1.0 g manganese oxide was added and stirred for 24 h under ni-
trogen. After reaction completion, it was filtered over a celite pad and
purified by silica gel (100–200 mesh) column chromatography using an
ethyl acetate and hexane solvent system (1:1). Product 5 was obtained as
a pale brown liquid (68% yield).
5.1.8. (E)-2-methoxy-5-(2-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
yl)-2-(3,4,5-trimethoxyphenyl)vinyl)phenol (9)
Yield: 55%; 1H NMR (400 MHz, CDCl3), δ 8.25 (s, NH), 7.41 (s, 1H),
7.27 (d, J = 9.6 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H), 6.98–6.89 (m, 6H),
6.28 (s, 2H), 5.40 (s, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 3.73 (s, 6H), 2.97
(m, 2H), 2.44 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 153.7, 147.4,
146.3, 140.3, 138.0, 136.8, 130.9, 128.3, 127.1, 125.8, 123.7, 122.7,
119.6, 117.4, 111.8, 108.6, 108.3, 101.9, 60.9, 60.2, 56.3, 56.0; HRMS
(ESI): m/z calcd for C29H30N2O5 [M+H]+ 487.2155, found 487.2246.
1H NMR (200 MHz, CDCl3), δ 9.69 (s, 1H), 7.26 (s, 1H), 7.14 (d, J =
8.2 Hz, 1H), 6.77 (s, 1H), 6.72 (d, J = 8.6 Hz, 1H), 6.56 (s, 1H), 6.45 (s,
2H), 4.42 (s, 2H), 3.87 (s, 3H), 3.77 (s, 9H), 2.24 (s, 3H); 13C NMR (50
MHz, CDCl3) δ 194.2, 169.2, 154.3, 153.4, 149.7, 140.7, 139.8, 138.2,
131.0, 129.1, 127.2, 125.6, 112.4, 106.3, 61.3, 56.5, 56.4, 21.0; HRMS
(ESI): m/z calcd for C21H22O7 [M+H]+ 387.1366, found 387.1445.
5.1.5. (E)-2-methoxy-5-(2-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-
yl)-2-(3,4,5-trimethoxyphenyl)vinyl)phenyl acetate (6)
5.1.9. (E)-2-methoxy-5-((9,10,11-trimethoxy-1,2,3,13a-tetrahydro-1,7b-
diazabenzo[5,6]cyclohepta[1,2,3-jk]fluoren-13(8H)-ylidene)methyl)
phenyl acetate (10)
Compound 5 (0.63 g, 2.2 mmol) was dissolved in isopropanol (10.0
mL) and tryptamine (0.268 g, 1.7 mmol) was added to the solution. The
reaction mixture was stirred for two min, then 0.5 mL TFA (trifluoro-
acetic acid) was added under an inert atmosphere and refluxed for 10 h.
The reaction progress was monitored by TLC and after completion,
water was added at room temperature and extracted with ethyl acetate
three times. The combined organic layer was dried over anhydrous
Mg2SO4 and evaporated to give a crude product. The crude product was
purified by silica gel (100–200 mesh) column chromatography using an
ethyl acetate and hexane solvent system (5:1). Product 6 was obtained as
a brown solid (60% yield).
Compound 6 (0.2 g, 0.38 mmol) was dissolved in isopropanol (5.0
mL) and then paraformaldehyde (0.045 g) was added to the solution.
The reaction mixture was stirred for two min, then 0.1 mL TFA (tri-
fluoroacetic acid) was added under inert atmosphere and refluxed for 6
h. The reaction progress was monitored by TLC. After completion, water
was added at room temperature and extracted with ethyl acetate three
times. The combined organic layer was dried over anhydrous Mg2SO4
and evaporated to give a crude product. The crude product was purified
by silica gel (100–200 mesh) column chromatography using an ethyl
acetate and hexane solvent system (1:1), and compound 10 and 11 were
obtained.
Yield: 60%; 1H NMR (400 MHz, CDCl3), δ 8.82(s, NH), 7.29 (d, J =
8.0 Hz, 1H), 7.17 (dd, J = 8.0 &7.2 Hz, 1H), 7.10 (dd, J = 7.6 & 7.2 Hz,
1H), 6.78 (dd, J = 8.4 & 2.0 Hz, 1H), 6.64 (s, 1H), 6.63 (d, J = 8.4 Hz,
1H), 6.57 (d, J = 2.0 Hz, 1H), 6.08 (s, 2H), 5.4 (s, 1H), 3.77 (s, 3H), 3.71
(s, 3H), 3.33 (s, 6H), 3.22 (m, 1H), 3.02 (m, 1H), 1.50 (s, 2H), 2.86 (m,
2H), 2.16 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.2, 153.9, 151.1,
139.2, 138.3, 136.5, 133.6, 133.5, 130.5, 128.9, 127.7, 126.9, 123.6,
126.2, 123.6, 123.2, 120.2, 118.6, 111.9, 111.4, 109.0, 106.2, 61.0,
59.6, 55.9, 55.8, 40.6, 29.8, 20.5, 18.3; HRMS (ESI): m/z calcd for
Yield: 60%; 1H NMR (400 MHz, Acetone-D), δ 7.57 (m, 2H), 7.26 (dd,
J = 7.2 & 7.6 Hz, 1H), 7.14 (m, 2H), 7.03 (s, 2H), 6.60 (s, 1H), 6.64 (s,
1H), 6.39 (s, 1H), 5.77 (d, J = 11.2 Hz, 1H), 5.57 (m, 2H), 5.0 (d, J =
16.4 Hz, 1H), 4.5 (d, J = 16.4 Hz, 1H), 3.97 (s, 3H), 3.82 (s, 3H), 3.79 (s,
3H), 3.45 (s, 3H), 3.22–3.18 (m, 4H), 2.17 (s, 3H); 13C NMR (100 MHz,
Acetone-D) δ 168.1, 152.6, 151.3, 149.9, 142.2, 139.9, 138.2, 129.7,
129.1, 128.8, 128.2, 127.9, 126.9, 126.4, 123.6, 122.9, 120.3, 118.7,
118.6, 116.3, 112.4, 110.3, 109.9, 107.3, 71.5, 69.4, 60.2, 60.1, 55.5,
55.0, 21.7, 21.6, 19.6, 19.0, 13.5; HRMS (ESI): m/z calcd for
C
31H32N2O6 [M+H]+ 529.2260, found 529.2346.
C
32H32N2O6 [M+H]+ 541.2260.
5.1.6. (E)-5-(2-(2,9-dihydro-1H-pyrido[3,4-b]indol-1-yl)-2-(3,4,5-
trimethoxyphenyl)vinyl)-2-methoxyphenyl acetate (7)
5.1.10. (E)-2-methoxy-5-((9,10,11-trimethoxy-1,2,3,13a-tetrahydro-
1,7b-diazabenzo[5,6]cyclohepta[1,2,3-jk]fluoren-13(8H)-ylidene)methyl)
phenol (11)
Compound 6 (0.1 g) was dissolved in DCM, and manganese oxide
(1.0 g) and pyridine (2 drops) were added and stirred for 24 h. After
completion of the reaction, it was filtered through celite and then
extracted with water. The organic layer was dried over anhydrous
Mg2SO4 and evaporated. The crude product was purified by silica gel
(100–200 mesh) column chromatography using an ethyl acetate and
hexane solvent system (6:1). Products 7 and 8 were obtained as light
yellow solid (yield, 35% and 40% respectively).
Yield: 40%; 1H NMR (400 MHz, Acetone-D), δ 7.57 (m, 2H), 7.26 (dd,
J = 7.2 & 7.6 Hz, 1H), 7.14 (m, 2H), 6.85 (m, 2H), 6.74 (m, 2H), 6.33 (s,
1H), 5.77 (d, J = 11.2 Hz, 1H), 5.56 (m, 2H), 5.34 (s, 1H), 5.0 (d, J =
16.4 Hz, 1H), 4.5 (d, J = 16.4 Hz, 1H), 3.93 (s, 3H), 3.82 (s, 3H), 3.80 (s,
3H), 3.45 (s, 3H), 3.40–3.20 (m, 4H); 13C NMR (100 MHz, Acetone-D) δ
152.4, 149.9, 147.6, 146.5, 142.2, 138.1, 129.7, 129.0, 126.4, 122.9,
121.0, 120.2, 118.7, 115.8, 111.4, 110.3, 107.5, 71.4, 69.4, 60.2, 60.1,
59.7, 55.5, 55.0, 26.9, 24.9, 22.5, 21.7, 21.6, 19.9, 19.1, 13.6, 13.5;
1H NMR (400 MHz, Acetone-D), δ 9.94 (s, NH), 8.82 (s, 1H), 8.24 (d,
J = 4.8 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 4.8 Hz, 1H), 7.36
9

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
HRMS (ESI): m/z calcd for C30H30N2O5 [M+H]+ 499.2155.
mixture was stirred for two min, and then 0.5 mL TFA (trifluoroacetic
acid) was added under inert atmosphere and refluxed for 10–13 h. The
reaction progress was monitored by TLC. After completion, water was
added at room temperature and extracted with ethyl acetate three times.
The combined organic layer was dried over anhydrous Mg2SO4 and
evaporated to give a crude product. The crude product was purified by
silica gel (100–200 mesh) column chromatography using an ethyl ace-
tate and hexane solvent system. The products HK-46 to HK-52, and HK-
71 were obtained.
5.1.11. 4-(4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)-N,N-
dimethylbenzenamine (HK-25)
Compound HK-17 (0.1 g) was dissolved in DCM (10 mL) and man-
ganese oxide (0.5 g), and then, pyridine (2 drops) were added and stirred
for 24 h. After completion of the reaction, it was filtered through celite
and then extracted with water. The organic layer was dried over anhy-
drous Mg2SO4 and evaporated. The crude product was purified by silica
gel (100–200 mesh) column chromatography using an ethyl acetate and
hexane solvent system (2:1). The product HK-25 was obtained as a light
yellow solid (63% yield).
5.1.15. 2,3,4,9-tetrahydro-1-(3,4,5-trimethoxyphenyl)-1H-pyrido[3,4-b]
indole (HK-48)
1H NMR (400 MHz, Acetone-d), δ 8.03 (d, J = 9.2 Hz, 2H), 7.83 (d, J
= 8.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 8.0 & 8.8 Hz, 1H),
7.25 (t, J = 8.0 & 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 4.10 (m, 2H),
3.33 (m, 2H), 3.18 (s, 6H); HRMS (ESI): m/z calcd for C19H19N3 [M+H]+
290.1579, found 290.1659.
1H NMR (400 MHz, MeOD), δ 7.55 (d, J = 7.6 Hz, 1H), 7.32 (d, J =
8.0 Hz, 1H), 7.16 (t, J = 7.6 & 7.6 Hz, 1H), 7.08 (t, J = 7.6 & 7.6 Hz, 1H),
6.73 (s, 2H), 5.85 (s, 1H), 3.75 (s, 9H), 3.70 (m, 1H), 3.56 (m, 1H), 3.25
(m, 1H), 3.17 (m, 1H); HRMS (ESI): m/z calcd for C20H22N2O3 [M+H]+
339.1630, found 339.1718.
5.1.12. (R)-9-allyl-2,3,4,9-tetrahydro-1-propyl-1H-pyrido[3,4-b]indole
(HK-28) and (S)-9-allyl-2,3,4,9-tetrahydro-1-propyl-1H-pyrido[3,4-b]
indole (HK-29)
5.1.16. 5-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-2-
methoxyphenol (HK-51)
1H NMR (400 MHz, MeOD), δ 7.53 (d, J = 8.0 Hz, 1H), 7.31 (d, J =
7.6 Hz, 1H), 7.16 (t, 1H), 7.06 (t, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.87 (dd,
J = 6.0 & 2.0 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 5.75 (s, 1H), 3.58 (m,
1H), 3.48 (m, 1H), 3.17 (m, 2H); 13C NMR (100 MHz, MeOD) δ 161.8,
161.4, 149.3, 146.9, 136.9, 127.4, 126.4, 125.8, 122.1, 121.1, 120.9,
119.1, 118.2, 117.7, 115.8, 115.3, 111.5, 110.9, 107.3, 60.0, 56.5, 54.9,
40.4, 19.3, 17.9, 12.9; HRMS (ESI): m/z calcd for C18H18N2O2 [M+H]+
295.1368, found 295.1457.
The starting material was dissolved in THF, and then, sodium bi-
carbonate was added and stirred for 15 min. Boc anhydride was added to
the reaction mixture and the stirring continued for a further three h.
After reaction completion, water was added and extracted thrice with
ethyl acetate. The organic layer was vacuum dried to afford compound
3a, which was further subjected to reaction of allyl bromide and sodium
bicarbonate in DMF, resulting in an intermediate 3b. In the next step, the
deprotection of BOC in the presence of TFA and dioxane formed com-
pound 3c.
5.1.17. General procedure for the synthesis of Compounds HK-62 to HK-
68, and HK-72
Compound 3c (0.2 g, 9.9 mmol) was dissolved in isopropanol (10.0
mL), and butyraldehyde (0.072 g, 9.9 mmol) was added to the solution.
TFA (0.5 mL) was added under inert atmosphere and refluxed for 2.5 h.
Subsequently, water was added at room temperature to quench the re-
action and extracted with ethyl acetate (3 × 25 mL). The combined
organic layer was dried over anhydrous Mg2SO4 and evaporated to give
a crude product. The crude product was purified by silica gel (100–200
mesh) column chromatography using an ethyl acetate and hexane sol-
vent system (7:3) to afford compounds HK-28 and HK-29 in yields of
40% and 42%, respectively.
5-Methoxytryptamine (200 mg) was dissolved in isopropanol (10.0
mL), and one equivalent aldehyde was added to the solution. The re-
action mixture was stirred for two min, then 0.5 mL TFA (trifluoroacetic
acid) was added under inert atmosphere and refluxed for 9–11 h. The
reaction progress was monitored by TLC. After completion, water was
added at room temperature and extracted with ethyl acetate three times.
The combined organic layer was dried over anhydrous Mg2SO4 and
evaporated to give a crude product. The crude product was purified by
silica gel (100–200 mesh) column chromatography using an ethyl ace-
tate and hexane solvent system. The products HK-62 to HK-68, and HK-
72 were obtained.
1H NMR (400 MHz, MeOD), δ 7.44 (d, J = 7.6 Hz, 1H), 7.25 (d, J =
8.0 Hz, 1H), 7.13 (m, 1H), 7.03 (m, 1H), 5.91 (m, 1H), 5.09 (d, J = 10.4
Hz, 1H), 4.66 (m, 2H), 3.56–3.47 (m, 2H), 3.23 (m, 2H), 3.04 (m, 2H),
1.94 (m, 2H), 1.53 (m, 2H), 0.97 (m, 3H); 13C NMR (100 MHz, MeOD) δ
138.9, 134.7, 131.5, 127.3, 123.8, 121.0, 119.4, 116.8, 111.1, 107.3,
53.0, 46.8, 39.4, 35.7, 20.1, 19.4, 14.0; HRMS (ESI): m/z calcd for
5.1.18. 2,3,4,9-tetrahydro-1-(4-isopropylphenyl)-6-methoxy-1H-pyrido
[3,4-b]indole (HK-62)
1H NMR (400 MHz, MeOD), δ 7.26–7.23 (m, 3H), 7.07 (d, J = 2.4 Hz,
1H), 6.80 (dd, J = 8.8 & 2.4 Hz, 1H), 6.73 (s, 1H), 6.71 (s, 1H), 5.88 (s,
1H), 3.83 (s, 3H), 3.67 (m, 1H), 3.54 (m, 1H), 3.21 (m, 2H), 2.97 (s, 6H);
HRMS (ESI): m/z calcd for C20H23N3O [M+H]+ 322.1841, found
322.1930.
C
17H22N2 [M+H]+ 255.1783, found 255.1864.
5.1.13. 1-propyl-9H-pyrido[3,4-b]indole (HK-30)
Compound HK-27 (0.1 g, 4.7 mmol) was dissolved in 20 mL of DCM,
and then, manganese oxide (0.5 g) and pyridine (2 drops) were added
and stirred for 20 h. After completion of the reaction, it was filtered
through celite and then extracted with water. The organic layer was
dried over anhydrous Mg2SO4 and evaporated. The crude product was
purified by silica gel (100–200 mesh) column chromatography using an
ethyl acetate and hexane solvent system (2:1). The product HK-30 was
obtained as a yellow solid (75% yield).
5.1.19. 5-(2,3,4,9-tetrahydro-6-methoxy-1H-pyrido[3,4-b]indol-1-yl)-2-
methoxyphenol (HK-64)
1H NMR (400 MHz, MeOD), δ 7.19 (d, J = 8.8 Hz, 1H), 7.02 (m, 2H),
6.88 (dd, J = 8.4 & 2.4 Hz, 1H), 6.81 (m, 2H), 5.75 (s, 1H), 3.86 (s, 3H),
3.84 (s, 3H), 3.54 (m, 1H), 3.49 (m, 1H), 3.12 (m, 2H); 13C NMR (100
MHz, MeOD) δ 155.6, 150.8, 148.4, 133.6, 129.5, 127.9, 127.6, 122.4,
117.3, 113.8, 113.2, 112.9, 108.6, 101.2, 58.2, 56.5, 56.3, 42.0, 19.6;
HRMS (ESI): m/z calcd for C19H20N2O3 [M+Na]+ 347.1474, found
347.1091.
1H NMR (400 MHz, CDCl3), δ 8.36 (d, J = 5.6 Hz, 1H), 8.11 (d, J =
7.6 Hz, 1H), 7.81 (d, J = 5.6 Hz, 1H), 7.54 m, 1H), 3.11 (m, 2H), 2.10
(m, 2H), 1.06 (m, 3H); HRMS (ESI): m/z calcd for C14H14N2 [M+H]+
211.1157, found 211.1237.
5.1.20. 2,3,4,9-tetrahydro-6-methoxy-1-(naphthalen-3-yl)-1H-pyrido
[3,4-b]indole (HK-65)
5.1.14. General procedure for the synthesis of Compounds HK-46 to HK-
52, and HK-71
1H NMR (400 MHz, MeOD), δ 7.91–7.85 (m, 4H), 7.55–7.49 (m, 2H),
7.43 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.09 (s, 1H), 6.78 (dd,
J = 8.8 & 2.4 Hz, 1H), 5.71 (s, 1H), 3.84 (s, 3H), 3.37 (m, 1H), 3.33 (m,
Tryptamine (200 mg) was dissolved in isopropanol (10.0 mL), and
then one equivalent aldehyde was added to the solution. The reaction
10

S. Kumar et al.
Bioorganic Chemistry 111 (2021) 104846
1H), 3.09 (m, 1H), 2.98 (m, 1H); 13C NMR (100 MHz, MeOD) δ 155.6,
136.4, 135.3, 134.9, 133.7, 132.4, 130.3, 130.1, 129.4, 128.9, 128.3,
128.1, 127.8, 127.4, 113.3, 113.1, 109.6, 101.44, 58.9, 56.5, 42.8, 21.3;
HRMS (ESI): m/z calcd for C22H20N2O [M+H]+ 328.1576, found
328.1958.
5.3.5. Cell viability
Cell viability was determined using a commercial lactate dehydro-
genase (LDH) assay kit (Promega, Madison, WI, USA). Human neutro-
phils (6 × 105 cells/mL) were treated with DMSO (0.1%) or test
compounds for 15 min at 37 ◦C. The total LDH release was determined
◦
by lysing the cells with 0.1% Triton X-100 for 30 min at 37 C. After
5.1.21. 2,3,4,9-tetrahydro-6-methoxy-1-(3,4,5-trimethoxyphenyl)-1H-
pyrido[3,4-b]indole (HK-67)
centrifugation, the cell-free supernatant was incubated with LDH assay
reagent for 30 min at 25 ◦C. The absorbance of each well was monitored
at 490 nm using a spectrophotometer (Hitachi, Tokyo, Japan).
1H NMR (400 MHz, MeOD), δ 7.22 (d, J = 8.8 Hz, 1H), 7.05 (d, J =
2.4 Hz, 1H), 6.83 (dd, J = 8.8 & 2.4 Hz, 1H), 6.7 (s, 2H), 5.85 (s, 1H),
4.77 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H) 3.81 (s, 3H), 3.72 (m, 1H), 3.59
(m, 1H), 3.30 (m, 1H), 3.22 (m, 1H); 13C NMR (100 MHz, MeOD) δ
154.2, 153.77, 139.4, 132.2, 129.4, 127.7, 126.2, 112.5, 111.8, 107.3,
106.7, 99.8, 99.6, 78.1, 77.8, 77.5, 59.6, 57.4, 55.3, 54.8, 41.3, 28.7,
28.3, 21.9, 18.1; HRMS (ESI): m/z calcd for C21H24N2O4 [M+H]+
368.1736.
5.3.6. Western blotting
Human neutrophils were treated with DMSO (0.1%) or test com-
◦
pounds at 37 C for 5 min and then activated by fMLF (0.1
μM). The
reactions were stopped by the addition of sample buffer (62.5 mM pH
6.8 Tris-HCl, 5% β-mercaptoethanol, 4% sodium dodecyl sulfate (SDS),
2.5 mM Na3VO4, 0.0125% bromophenol blue, 10 mM di-N-pentyl
◦
phthalate, and 8.75% glycerol) at 100 C for 15 min. The cell lysates
were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and
target proteins were labeled with the corresponding primary antibodies
and peroxidase-conjugated secondary anti-rabbit antibodies. The signals
of labeled proteins were detected using an enhanced chemiluminescence
system (Amersham Biosciences, Piscataway, NJ, USA).
5.2. Molecular docking study
The structure of HK-72 was drawn and optimized using the Avogadro
program [44]. The structure of p38 MAPK in complex with inhibitor 1
was obtained from the Protein Data Bank (PDB; accession code 1kv1)
[45]. AutoDockTools4 [46] was used to prepare the ligand and receptor
and calculate a grid box. A grid map consisting of 70 Å × 70 Å × 70 Å
points around the p38 was used. AutoDock Vina [47] was used to dock
and predict the binding affinity of HK-72 with p38 MAPK.
5.3.7. Statistical analysis
Statistical analysis was carried out using Sigma Plot (version 8.0,
Systat Software, San Jose, CA, USA) [48]. Student’s t test was used to
compare the means, and a p value < 0.05 indicated statistical signifi-
cance. Results are expressed as mean ± SEM.
5.3. Pharmacological study
5.3.1. Human neutrophil isolation
Declaration of Competing Interest
The present study was approved by the Institutional Review Board of
the Chang Gung Memorial Hospital. Informed assent was acquired from
each donor and blood samples were collected using vacutainer heparin
tubes. An equal volume of dextran was added, and neutrophils were
isolated by Ficoll-Hypaque centrifugation. Neutrophils were suspended
in Hank’s balanced salt solution (HBSS) without calcium at 4 ◦C before
use.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by grants from the Chang Gung Memorial
Hospital (BMRP450, CMRPF1J0051~3, and CMRPG3H0811~3), Min-
istry of Education (EMRPD1K0481), and the Ministry of Science and
Technology (MOST 106-2320-B-255-003-MY3 and MOST 108-2320-B-
255-003-MY3), Taiwan. The funder had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
5.3.2. Superoxide anion generation
Superoxide anion generation was measured based on the reduction
of cytochrome c. Human neutrophils (6 × 105 cells/mL) were mixed
with cytochrome c (0.5 mg/mL) in 1 mM CaCl2 for 5 min at 37 ◦C and
then treated with dimethyl sulfoxide (DMSO; 0.1%) or test compounds
for another 5 min. After that, the neutrophils were activated by fMLF
(0.1 μM)/CB (1 μg/mL). The absorbance of each cuvette was detected
continuously for 10 min at 550 nm using a spectrophotometer (U-3010;
Hitachi).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104846.
5.3.3. Elastase release
Human neutrophils (6 × 105 cells/mL) were incubated with elastase
substrate (MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide; 0.1 mM) in 1 mM
◦
CaCl2 for 5 min at 37 C and then treated with DMSO (0.1%) or test
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