Synthesis of 4-(3-oxo-3-phenylpropyl)morpholin-4-ium chloride analogues and their inhibitory activities of nitric oxide production in lipopolysaccharide-induced BV2 cells

Article abstract of DOI:10.1016/j.bmcl.2021.127780

Based on our previous report that 3-morpholino-1-phenylpropan-1-one 2, one of the fluoxetine's simplified morpholino analogue, inhibited nitric oxide (NO) production, in this paper, various substituted benzene analogues with morpholine hydrochloride of 2 were synthesized and their inhibitory effects on NO production in lipopolysaccharide (LPS)-induced BV2 cells were tested. Among the synthesized compounds, 2-trifluoromethyl analogue 16n (IC₅₀ = 8.6 μM) showed a significantly higher inhibitory activity than that of the parent compound 2a (IC₅₀ > 50 μM) and suppressed NO production dose-dependently without cytotoxicity. Compound 16n also inhibited iNOS expression in LPS-induced BV2 cells at 2, 10 and 20 μM concentrations. These results suggest that compound 16n inhibited NO production by suppressing the expression of iNOS and can be used as a lead structure for developing new inhibitor of NO production.

Full text of DOI:10.1016/j.bmcl.2021.127780

Bioorg. Med. Chem. Lett. 36 (2021) 127780
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 4-(3-oxo-3-phenylpropyl)morpholin-4-ium chloride analogues
and their inhibitory activities of nitric oxide production in
lipopolysaccharide-induced BV2 cells
Sung-Hwa Yoona, Eunhwa Leea, Duk-Yeon Chob, Hyun Myung Koc, Ha Yeon Baekc,
,
*
Dong-Kug Choib, Eunha Kima, Ju-Young Parkd
a Department of Molecular Science and Technology, Ajou University, Suwon 16499, Republic of Korea
b Department of Biotechnology, Konkuk University, Chungju 27478, Republic of Korea
c Department of Eco-Biological Science, College of Science and Technology, Woosuk University, Jincheon-eup 27841, Republic of Korea
d Molecular Science and Technology Research Center, Ajou University, Suwon 16499, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on our previous report that 3-morpholino-1-phenylpropan-1-one 2, one of the fluoxetine’s simplified
morpholino analogue, inhibited nitric oxide (NO) production, in this paper, various substituted benzene ana-
logues with morpholine hydrochloride of 2 were synthesized and their inhibitory effects on NO production in
lipopolysaccharide (LPS)-induced BV2 cells were tested. Among the synthesized compounds, 2-trifluoromethyl
3-Morpholino-1-phenylpropan-1-one analogues
NO production
BV2 cell
analogue 16n (IC50 = 8.6
μ
M) showed a significantly higher inhibitory activity than that of the parent com-
pound 2a (IC50 > 50
μ
M) and suppressed NO production dose-dependently without cytotoxicity. Compound 16n
also inhibited iNOS expression in LPS-induced BV2 cells at 2, 10 and 20 μM concentrations. These results suggest
that compound 16n inhibited NO production by suppressing the expression of iNOS and can be used as a lead
structure for developing new inhibitor of NO production.
NO (Nitric oxide) plays an important signal molecule involved in the
regulation of physiological and pathophysiological processes in immune
systems, while the excessive accumulation of NO in brain results in
neuronal injury, neuroinflammation and neuronal cell death.1–3 When
microglia become activated by pathogens such as lipopolysaccharides
(LPS), NO is generated by inducible nitric oxide synthases (iNOS).4,5 As
NO production has been widely used as a representative measure of
inflammatory activation of microglia,6 inhibition of NO overproduction
and iNOS overexpression may be a useful drug development strategy for
the treatment of neurodegenerative diseases such as Alzheimer’s disease
and Parkinson’s disease caused by excessive production of NO.7,8
Among various NO inhibitors, fluoxetine which is commonly pre-
scribed for treating a depression has been found to have anti-
inflammatory activities through inhibition of LPS-induced activation
of microglia followed by subsequent release of pro-inflammatory cyto-
kines and NO as well as inhibition of microglial NF-κB (nuclear factor-
kappaB) signaling pathway.9,10 However, previous study reported that
significantly different from the control, but exposure of BV2 cells to
fluoxetine at the concentration of 50 μM results in significantly fewer
viable cells due to toxicity as compared to cells in the control.11
As part of our effort to develop a potent and novel inhibitor of NO
production, we found that a simplified morpholine analogue of fluoxe-
tine,
3-morpholino-1-phenylpropan-1-one
2,
revealed
anti-
inflammatory effect in the LPS-stimulated BV2 microglial cells without
toxicity even at 40
μ
M concentration (Fig. 1).12 With the aim of
improving the potency and elucidating the pharmacophore of com-
pound 2, we have further modified that compound by changing the
position of the carbonyl group, introducing an additional carbonyl
group and introducing various substituents in the benzene ring. This
paper provides details on the synthesis of various analogues of com-
pound 2 and their inhibitory activities on NO production and iNOS
expression on BV2 cells stimulated with LPS.
Keto analogues (6, 8, 11 and 13) which have either different
carbonyl position or additional carbonyl groups in the basic skeleton of
compound 2 were synthesized as depicted in Scheme 1. For the synthesis
cell viability of fluoxetine at low concentration (0.1–10 μM) was not
* Corresponding author at: Department of Molecular Science and Technology Research Center, Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon 16499,
Republic of Korea.
E-mail address: pink1209@ajou.ac.kr (J.-Y. Park).
https://doi.org/10.1016/j.bmcl.2021.127780
Received 7 October 2020; Received in revised form 16 December 2020; Accepted 3 January 2021
Available online 8 January 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S.-H. Yoon et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127780
a HCl salt form. The syntheses of the various substituted benzene ana-
logues (2a and 16b16n) started from commercially available corre-
sponding acetophenones (14a–14m) as depicted in Scheme 2. Mannich
bases (15a–15n) were first prepared from corresponding acetophenones
(14a–14m) by refluxing with paraformaldehyde, N,N-dimethylamine
hydrochloride and sulfuric acid in acetonitrile. After the reaction was
completed, the resulting solution was cooled to room temperature and
then poured into acetone. The precipitate was filtered and washed with
acetone to give the desired compounds. For the synthesis of the amines
(2a and 16b–16n), compounds (15a–15n) were refluxed with excess
morpholine for 30 min to produce the crude products of the
Fig. 1. Structure of fluoxetine 1 and its analogue 3-morpholino-1-phenylpro-
pan-1-one 2.
of 6, benzyl magnesium chloride 3 was reacted with bromoacetyl bro-
mide in THF at ꢀ 78 ◦C, and then the resulting product reacted with
excess morpholine to produce the crude product. Compound 8 was ob-
tained by conversion of the carboxylic acid in 7 into acyl chloride using
oxalyl chloride with catalytic amount of DMF and subsequent reaction
with morpholine at reflux temperature. The alpha diketo compound 11
was prepared from the conversion of 1-phenylpropane-1,2-dione 9 to
the bromide 10 using bromine via alpha halogenation, followed by the
reaction of 10 with excess morpholine. The β-keto amide 13 was syn-
thesized from β-keto ester 12 by refluxing a toluene solution with
morpholine in the presence of DMAP. All final crude products were
purified by silica gel column chromatography (ethyl acetate/hexane) to
afford to pure products in 12.7–82.2% yield.
Table 1
Inhibitory effects of 2 (simplified morpholine analogue of fluoxetine) and its
analogues (6, 8, 11 and 13) on LPS-induced NO production at the concentration
of 20 μM.
Com. Num.
Structure
Inhibition of
Cell viability
(% Control)
NO release (%)
2
31.8 ± 2.3
99.8 ± 2.5
6
8
17.4 ± 4.5
6.2 ± 2.3
100 ± 5.1
99.7 ± 4.7
In previous report, the free amine form of compound 2 was synthe-
sized as an oil. However, in pharmaceutical product development, the
salt form of nitrogen containing compounds has been shown to be more
the stable form, due to the molecule being less susceptible to oxidation
and helps developing products that have longer shelf lives.13 For these
reasons, in this paper, various substituted analogues were synthesized as
11
13
19.2 ± 3.6
20.1 ± 3.3
100 ± 5.9
99.7 ± 5.8
Scheme 1. Synthesis of keto analogues.
Scheme 2. Synthesis of various substituted benzene analogues.
2

S.-H. Yoon et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127780
TNF-
α
and IL-6.14,15 Among these inflammatory molecules, NO pro-
Table 2
Inhibitory effects of 2a (salt form of 2) and its substituted benzene analogues
duction has been widely used as a representative measure of inflam-
matory activation of microglia.6,16 Thus, the inhibitory activities on NO
production and cell viabilities of the synthesized analogues (6, 8, 11, 13
and 16b–16n), compared to the simplified morpholine analogue of
fluoxetine 2 (salt form 2a), are measured and presented in Tables 1 and
2. In order to identify whether the position of the carbonyl group and
additional keto group in structure of 2 can affect activity, four keto
analogues (6, 8, 11 and 13) were first evaluated for the inhibitory ac-
tivities on NO production in BV2 cells. As shown in Table 1, all four
analogues revealed lower inhibitory effects on NO production than
compound 2 while cell viability did not change. These results indicated
that the alpha position of the keto group on the benzene ring moiety is
more favorable for retaining the activity and the additional carbonyl
group did not improve inhibitory activity. Based on this result, the
substituent effect on benzene at the para position was investigated by
introducing an electron withdrawing group such as halogen, nitro and
trifluoromethyl as well as an electron donating group such as amino,
hydroxy, methyl and methoxy group to the basic skeleton of phenyl 3-
morpholinophenylpropane hydrochloride 2a.
(16b-16n) on LPS-induced NO production at the concentration of 20 μM.
Com. Num.
R
Inhibition of
Cell viability
(% Control)
IC50 (μM)
NO release (%)
1
fluoxetine
4-H
Toxic*
2a
29.3 ± 2.7
35.2 ± 2.0
40.3 ± 3.4
41.6 ± 2.9
41.6 ± 1.4
50.3 ± 5.3
73.1 ± 5.9
25.2 ± 3.2
32.8 ± 5.4
36.0 ± 2.2
33.3 ± 2.2
26.5 ± 9.8
44.6 ± 1.0
78.3 ± 0.4
96.7 ± 2.2
95.0 ± 1.0
100 ± 5.6
100 ± 1.7
89.4 ± 6.4
96.1 ± 6.1
98.3 ± 1.6
100 ± 1.9
100 ± 0.8
100 ± 6.3
100 ± 1.9
99.0 ± 1.7
100 ± 3.8
100 ± 1.5
>50
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
4-F
4-Cl
4-Br
4-I
4-CF3
4-NO2
4-NH2
4-N(CH3)2
4-CH3
4-OCH3
4-OH
3-CF3
2-CF3
As shown in Table 2, analogues (16b–16g) possessing electron
withdrawing groups (35.2 ± 2.0 – 73.1 ± 5.9%) generally revealed
better inhibitory activities than analogues (16h–16l) containing elec-
tron donating groups (25.2 ± 3.2 – 36.0 ± 2.2%). Among analogues
possessing electron withdrawing groups (16b–16g), the trifluoromethyl
analogue 16f and the nitro analogue 16g had significantly higher ac-
tivities than the halogen analogues with 50.3 ± 5.3% and 73.1 ± 5.9%,
respectively. These results indicated that a strong electron withdrawing
effect is favorable for better activity in this series, but not high electron
density for retaining the activity.
8.6
*
Inhibition effect of fluoxetine 1 at the concentration of 10 μM was 31.2 ±
0.9%.
corresponding amines, which were purified by chromatography on silica
gel with ethyl acetate/hexane (v/v, 1/1) to give pure products in overall
3.5–50.0% yield. The amino compound 16h was prepared from the
reduction of the nitro group in 16g by hydrogenation with 10% Pd/C in
MeOH.
Although the nitro analogue 16g showed better NO inhibitory
properties in comparison to the trifluoromethyl analogue 16f, further
positioning effects of substituents in benzene ring was investigated with
LPS is used as a strong inflammatory inducer which can stimulate
immune cells to secrete NO and pro-inflammatory cytokines such as
Fig. 2. Effects of 2a and 16n on LPS-induced NO production and cell viability in BV2 cells. BV2 cells were stimulated with LPS (200 ng/mL) or co-treated with LPS
(200 ng/mL) and 2a or 16n at concentrations of 6.25, 12.5, 25 and 50 μM for 24 h. The amounts of NO were determined using the Griess reagent. All the data of each
group are expressed as the mean ± S.E.M. and are representative of results obtained from three independent experiments.
3

S.-H. Yoon et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127780
Fig. 3. Effects of 2a and 16n on iNOS
production in LPS-induced BV2 cells.
BV2 microglial cells were pretreated
with the indicated concentrations of
2a and 16n for 1 h before incubating
with LPS (200 ng/mL) for 6 h (reverse
transcription polymerase chain reac-
tion [RT-PCR]) or 18 h (immunoblot).
Total RNA was prepared for RT-PCR
analysis of iNOS gene expression. Re-
sults are expressed as a ratio of iNOS
and GAPDH (A). Lysates were
analyzed by immunoblotting with
anti-iNOS antibody. Results are
expressed as
a ratio of iNOS and
β-actin (B). GAPDH and β-actin were
used as the internal controls for RT-
PCR analysis and immunoblotting as-
says, respectively. Data are presented
as mean ± S.E.M. (n = 3) for three
independent experiments.
trifluoromethyl analogue 16f instead of nitro analogue 16g because the
presence of a nitro group can result in several toxicity issues including
carcinogenicity, hepatotoxicity and mutagenicity.17 This toxicological
issue generally removes compounds containing nitro groups from
screening collections in drug development.18
understand the mechanism of action of 16n. As shown in Fig. 3, the
unstimulated cells released basal levels of iNOS protein while a signifi-
cant increase of iNOS protein was detected in LPS-stimulated BV2 cells.
When the LPS-treated BV2 cells were treated with 16n at different
concentrations (2, 10 and 20 μM), the densities of iNOS protein in the
When the trifluoromethyl group in analogue 16f was moved from the
para position in the benzene ring to the ortho and meta positions, sur-
prisingly, the NO inhibition of the ortho positioned analogue 16n
exhibited significantly higher inhibition (78.3 ± 0.4% with IC50 value of
cells were decreased in a dose-dependent manner. These results
demonstrated that the inhibitory action of 16n in BV2 cell was related to
modulation of iNOS induction.
In conclusion, a series of simplified morpholine analogue of fluoxe-
tine were synthesized and their inhibitory effects on nitric oxide (NO)
production in LPS-induced BV2 cells were tested. Among the synthe-
8.6 μM) than the meta positioned analogue 16m (44.6 ± 1.0%) and the
para positioned analogue 16f (50.3 ± 5.3%). Analogue 16n exhibited a
NO inhibitory activity in a dose-dependent manner, while parent com-
pound 2a revealed almost the same inhibitory effect of 49% in the range
sized compounds, 2-trifluoromethyl analogue 16n (IC50 = 8.6 μM) was
the most potent and exhibited much higher inhibitory effect than the
parent compound 2a (IC50 > 50 M). Analogue 16n suppressed NO
of 12.5–50
μM without cytotoxicity (Fig. 2).
μ
To clarify whether the inhibitory effects of simplified morpholine
analogue of fluoxetine 2 (salt form 2a) and its analogues (6, 8, 11, 13
and 16b–16m) on NO production were related to their toxicity, the
cytotoxicities of analogues were evaluated by MTT (3-[4,5-dimethylth-
iazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, which is a widely
used test to measure cell viability or drug toxicity.19–21 As shown in
Tables 1 and 2, there was no suppression in the cell viabilities of the BV2
cells after being treated with simplified morpholine analogue of fluox-
etine 2 (salt form 2a) and its analogues (6, 8, 11, 13 and 16b–16m) at
production dose-dependently without cytotoxicity and also inhibited
NO production through the suppression of iNOS expression. Our results
suggested that the proper position of the trifluoromethyl group in the
benzene unit is crucial for high suppression of NO production in this
series and that the ortho positioned analogue 16n could be a good lead
compound for the development of useful therapeutic agents to modulate
inflammatory damage associated with microglia activated by LPS.
Declaration of Competing Interest
the concentrations of 20 μM. This result confirmed that the inhibitory
effects on NO production were due solely to its intrinsic activities rather
than its cytotoxicity.
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.
NO is generated from ι-arginine by three distinct isoforms of nitic
oxide synthases (NOS): neuronal NOS (nNOS), inducible NOS (iNOS)
and endothelial NOS (eNOS).22 While the constitutive eNOS and nNOS
play roles in the nervous and cardiovascular system, iNOS often
expressed at high levels by stimulation of pathogen such as LPS.23 Pre-
vious studies showed that agents with the ability to inhibit iNOS
expression are potentially beneficial in the treatment of disease associ-
ated with an overproduction of NO.11,24 Therefore, the inhibition of NO
overproduction and iNOS overexpression via NF-κB may be a useful drug
target molecule for developing effective therapeutic anti-inflammatory
agent.25,26
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) grant funded
by the Ministry of Education [NRF-2020R1A2B5B02002032 (to S.-H
Yoon and D.-K Choi) and (NRF-2020R1I1A1A01062002 and
2019R1A6A1A11051471 (to J.-Y Park)].
Appendix A. Supplementary data
Since fluoxetine analogues have inhibitory activities on NO pro-
duction in BV2 cell and iNOS expression at the concentration of 20
Supplementary data (Synthetic procedural and data of intermediates
and target compounds 2, 6, 8, 11, 13 and 16b–16n) to this article can be
found online at https://doi.org/10.1016/j.bmcl.2021.127780.
μ
M,9,12 we examined the effect of 16n on iNOS mRNA expression and
protein level using RT-PCR and western blot analysis to further
4

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Bioorganic & Medicinal Chemistry Letters 36 (2021) 127780
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https://doi.org/10.1016/j.bmcl.2012.06.082.
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