Novel Hybrid Molecules of Epinastine and Mefenamic Acid for Bioactive Assessment as Potential Anti-inflammatory Agents

Article abstract of DOI:10.1002/bkcs.11990

A set of novel epinastine (1) and mefenamic acid (2) hybrids (3–7) tethered via amide bond were designed and synthesized with the artistry of molecular hybridization. Then their inhibitory effect on nitric oxide (NO) production in LPS-induced RAW-264.7 ma

Full text of DOI:10.1002/bkcs.11990

Article
DOI: 10.1002/bkcs.11990
BULLETIN OF THE
J. M. Lee et al.
KOREAN CHEMICAL SOCIETY
Novel Hybrid Molecules of Epinastine and Mefenamic Acid for
Bioactive Assessment as Potential Anti-inflammatory Agents
†
†
‡
†
§
Ju Mi Lee, Kongara Damodar, Yeontaek Lee, Hyeong Ryeol Woo, Hong Won Suh,
‡,
†,
*
*
Sung Ho Jeon, and Jeong Tae Lee
†Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 24252,
Republic of Korea. *E-mail: jtshl@hallym.ac.kr
‡
Department of Life Science and Multidisciplinary Genome Institute, Hallym University, Chuncheon
2
4252, Republic of Korea. *E-mail: sjeon@hallym.ac.kr
§
Department of Pharmacology and Institute of Natural Medicine, College of Medicine, Hallym
University, Chuncheon 24252, Republic of Korea
Received December 18, 2019, Accepted January 31, 2020
A set of novel epinastine (1) and mefenamic acid (2) hybrids (3–7) tethered via amide bond were designed and
synthesized with the artistry of molecular hybridization. Then their inhibitory effect on nitric oxide
(
(
NO) production in LPS-induced RAW-264.7 macrophages and the expression levels of prostaglandin
PG) synthesis regulatory genes were evaluated in vitro. All the hybrids were found to have remarkable NO pro-
duction inhibitory potential with IC50 values ranging in between 27.69 ꢀ 3.06 and 40.19 ꢀ 5.52 μM without
notable cytotoxicity (CC50 ≥ 100 μM) except 6. Comparing the inhibitory effects, cytotoxicity and in vitro effi-
cacy index (iEI), 4 (IC50 = 32.09 ꢀ 4.04 μM; iEI = 6.23) and 7 (IC = 27.69 ꢀ 3.06 μM; iEI = 6.40) were bet-
50
ter suited than other hybrids as well as their parent compounds EH and MA. This outcome was further
harmonized with the reduced microsomal prostaglandin E synthase-1 expression and unaltered housekeeping
COX-1 expression by hybrids 4 and 7 treatment. Altogether, our findings signify that hybrids 4 and 7 may serve
as platforms for continued investigations for the development of more efficient anti-inflammatory agents.
Keywords: Epinastine, Mefenamic acid, Hybrid molecules, Anti-inflammatory, Nitric oxide (NO),
in vitro efficacy index (iEI), PGs synthetic pathway
Introduction
inhibitor by limiting the discharge of inflammatory mediators
like histamine, eosinophil, and platelet-activating factor.
6
Inflammation refers to our body’s protective activity during
fighting against adverse situations, such as infections, injuries,
Mefenamic acid (2, 2-[(2,3-dimethyphenyl)-amino] benzoic
acid), an N-phenylanthranilic acid derivative, belongs to the
“fenamate” family of NSAIDs with marked anti-inflammatory
1,2
and foreign organisms, in pursuit to restore itself. Different
molecules like prostaglandins (PGs), nitric oxide (NO), cyto-
kines, vasoactive amines, and leukotrienes (LTs) can elicit an
inflammatory response. In an acute inflammation, the response
ends in a short period, but it lingers in chronic inflammation,
which could lead to a range of conditions. Many effective
medicines including nonsteroidal anti-inflammatory drugs
7
and analgesic, antipyretic activity. Additionally, its in vitro
8
antiproliferative effect on cancer cells, antimetastatic effect
and antimitotic effect also have been previously reported. His-
torical uses of 2 include premenstrual syndrome, menorrhagia,
pain syndromes (such as primary dysmenorrhea, gynecologi-
cal and obstetrical pain, musculoskeletal injury, osteoarthritis
and dental pain), pediatric use, asthma, urticaria, and
(NSAIDs), steroids, and antihistamines exist to treat the pain
7
Alzheimer’s disease. Nevertheless, similar to other NSAIDs,
and inflammation; however, none of these are outside of their
2
2
also exhibits general toxicities. Hence, derivatization of
may allow better drugs with less toxic profile. Recently,
3
pitfalls. Hence, synthesis and screening directed to explore
novel anti-inflammatory drugs with high potency as well as
low adverse events have great importance.
The ophthalmic second-generation antihistamine, epinastine
molecular hybridization concept, which offers the combining
of two pharmacophores usually through a linker into a single
chemical entity, has received increasing interest in the quest of
1
, has been employed for the prevention of the signs and symp-
9–11
new drug discovery.
Inspired by this perception, we
4
toms related to allergic conjunctivitis. It has a faster onset of
action without any CNS or cardiac complications compared to
oral antihistamines. Reduction in itching and vasodilation
intended that amalgamation of 2 with 1 using amino acids as
linkers may offer synergistic anti-inflammatory effect. Amino
acids/peptides linkers allow facile release of the pharm-
5
12
were resulted by the competitive binding of 1 to histamine
receptor sites. Furthermore, 1 acts as mast cells degranulation
acophores upon cleavage by specific intracellular proteases.
Additionally, their safety profiles, achieving desirable
Bull. Korean Chem. Soc. 2020
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open column chromatographic purifications, silica gel
6
0 (230–400 mesh size; Merck, Darmstadt, Germany) was uti-
lized. The preparations of 12–15 were performed as described
15
previously.
N-(9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5-a]azepin-
-yl)-2-((2,3-dimethylphenyl) amino)benzamide (3).
Cooled solution of 2 (0.13 g, 0.52 mmol) in tetrahydrofuran
3 mL) was treated with EDCI (0.09 mL, 0.52 mmol)
3
(
followed by HOBt (0.07 g, 0.52 mmol) under argon atmo-
sphere and stirred for 20 min. 1 (0.10 g, 0.35 mmol) in tetrahy-
drofuran (3 mL) was treated with DIPEA (0.24 mL,
ꢁ
1
.40 mmol), stirred for 20 min at 25 C and added to 2 con-
taining reaction mixture. The resultant mixture was stirred at
5 C for 6 h. The reaction solution was diluted with water
ꢁ
2
(
(
Figure 1. Structures of compounds 1, 2 and their hybrid com-
pounds (3–7).
10 mL) and the whole was extracted with ethyl acetate
3 × 20 mL). The combined organic phase was washed by
deionized water (2 × 20 mL) and sat. NaCl solution (20 mL)
successively, dried over sodium sulfate, and concentrated in
vacuo. Purification of the resulting residue by column chroma-
tography (petroleum ether/ethyl acetate, v/v, 2:1) gave
pharmacokinetic profiles and improved bioavailability have
1
3,14
15
made them as ideal linkers.
Continuing on this strategy,
herein, we have reported the design, synthesis of novel anti-
inflammatory hybrids (3–7) (Figure 1) of 1 and 2 tethered via
amide bond and evaluation of their in vitro NO production
inhibition potential and cytokine activity. Hybrid 3 is having
no linker between 1 and 2, whereas, 4–7 are linked by aliphatic
amino acids glycine, β-alanine, γ-aminobutyric acid (GABA)
and 5-aminovaleric acid, respectively. In a nutshell, amine
group of 1 and carboxy group of 2 were separated by 3, 4,
3
(0.09 g, 57.3%) as pale yellow solid. R = 0.58 (petroleum
f
1
ether/ethyl acetate = 2:1). H NMR (400 MHz, CDCl ) δ 1.79
3
(s, 3H), 2.26 (s, 3H), 3.51 (d, 1H, J = 14.4 Hz), 3.75 (t, 1H,
J = 9.6 Hz), 4.29 (t, 1H, J = 9.4 Hz), 4.64 (d, 1H, J = 14.1 Hz),
5.21 (t, 1H, J = 10.1 Hz), 6.55 (d, 1H, J = 8.4 Hz), 6.59 (t, 1H,
J = 7.7 Hz), 6.79 (t, 1H, J = 7.7 Hz), 6.92–7.04 (m, 5H), 7.10
(t, 1H, J = 8.6 Hz), 7.21 (t, 2H, J = 5.2 Hz), 7.25–7.27 (m, 2H),
7.46 (dd, 1H, J = 7.9 Hz, J = 1.0 Hz), 8.13 (dd, 1H, J = 8.0 Hz,
13
5, and 6 bonds (atoms) in 4–7 hybrids, respectively, which will
J = 1.7 Hz), 9.29 (s, 1H), 10.19 (s, 1H); C NMR (100 MHz,
give varying degree of flexibility to hybrids.
CDCl ) δ 179.1, 162.6, 149.3, 140.0, 139.3, 137.6, 136.6,
3
1
36.0, 135.9, 133.3, 132.8, 132.4, 130.6, 128.3, 128.0, 127.9,
Experimental
127.9, 127.6, 127.5, 127.3, 126.2, 125.7, 124.0, 119.1, 115.7,
+
113.6, 61.1, 50.6, 39.0, 20.7, 13.9; EI-MS m/z 472 ([M] ,
Commercially available materials, reagents, and solvents were
acquired from the commercial suppliers and used without fur-
base), 352, 194, 118; HRMS (EI) m/z calcd for C H N O:
472.2263 ([M] ), found: 472.2262.
tert-Butyl (5-((9,13b-dihydro-1H-dibenzo[c,f]imidazo
31 28 4
+
1
ther purification. Proton nuclear magnetic resonance ( H NMR
1
3
13
4
{
00 MHz) spectra and proton decoupled carbon C ( C
[1,5-a]azepin-3-yl)amino)-5-oxopentyl)carbamate (15).
1
H}) NMR (100 MHz) spectra were obtained at 300 K on a
Yield: 98.1%; white solid. R = 0.40 (petroleum ether/ethyl
f
1
JNM-ECZRS FT-NMR (JEOL Ltd., Tokyo, Japan) and deu-
terated chloroform was used as a solvent (Appendix S1).
Chemical shifts were expressed in δ parts per million (ppm)
relative to tetramethylsilane (TMS, δ 0.00 ppm) or the solvent
acetate = 1:6). H NMR (400 MHz, CDCl ) δ: 1.44–1.52 (m,
3
11H), 1.58–1.66 (m, 2H), 2.34 (qt, 2H, J = 15.3, 7.4 Hz), 3.10
(q, 2H, J = 7.2 Hz), 3.51 (d, 1H, J = 14.1 Hz), 3.64 (t, 1H,
J = 10.4 Hz), 4.19 (t, 1H, J = 9.3 Hz), 4.62 (br d, 2H,
1
resonance (chloroform‑d, δ 7.26 ppm for H NMR and δ
J = 14.0 Hz), 5.11 (t, 1H, J = 10.4 Hz), 6.98–7.49 (m, 8H),
1
3
13
7
7.0 ppm for C NMR). The following pattern was utilized
9.09 (br s, 1H); C NMR (100 MHz, CDCl ) δ: 187.8, 162.7,
3
1
for H NMR spectra: chemical shift (multiplicity [singlet = s],
doublet = d], [triplet = t], [quartet = q], [doublet of dou-
blet = dd] and [multiplet = m], integration, coupling constants
J] quoted in Hz). Low-resolution mass spectra [MS (EI)] and
156.1, 139.0, 136.9, 135.8, 135.8, 130.4, 128.4, 127.9, 127.7,
127.6, 127.6, 127.5, 127.3, 79.0, 61.3, 49.9, 40.5, 40.3, 39.1,
[
+
+
29.6, 28.5, 23.1; MS (EI ) m/z (%): 448 ([M] ), 276 (base),
250, 129, 99.
[
high-resolution mass spectra [HRMS (EI)] analyses were
acquired on a JMS-700 (JEOL Ltd.) spectrometer at the central
laboratory of Kangwon National University. Routine monitor-
ing of reactions was performed by thin-layer chromatography
using Merck silica gel plates 60 F254. The spots visualization
was carried out by ultraviolet light (λ = 254 nm) and/or an
appropriate reagent (7% ethanolic phosphomolybdic acid,
p-anisaldehyde solution in H SO followed by heating. For
N-(2-((9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5-a]azepin-
3-yl)amino)-2-oxoethyl)-2-((2,3-dimethylphenyl)amino)
benzamide (4). According to the similar procedure used to
compound 3, the reaction of 2 (0.22 g, 0.93 mmol) with Boc-
free compound of 12 (0.25 g, 0.62 mmol) in the presence of
EDCI (0.16 mL, 0.93 mmol), HOBt (0.13 g, 0.93 mmol) and
DIPEA (0.43 mL, 2.48 mmol) for 15 h afforded, after purifica-
tion by column chromatography (petroleum ether/ethyl
2
4
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2

Novel Hybrid Molecules of Epinastine and Mefenamic Acid for Bioactive
BULLETIN OF THE
Article
Assessment as Potential Anti-inflammatory Agents
KOREAN CHEMICAL SOCIETY
acetate, v/v, 1:1), compound 4 (0.16 g, 49.5%) as a white solid.
135.8, 131.8, 130.7, 130.5, 128.4, 128.0, 127.9, 127.8, 127.8,
127.6, 127.6, 127.4, 125.7, 125.3, 120.6, 117.3, 116.6, 114.6,
61.2, 50.1, 39.7, 39.0, 38.5, 24.8, 20.8, 14.0; EI-MS m/z
1
R = 0.20 (petroleum ether/ethyl acetate = 1:1). H NMR
f
(
400 MHz, CDCl ) δ 2.17 (s, 3H), 2.29 (s, 3H), 3.52 (d, 1H,
3
+
J = 14.5 Hz), 3.69 (dd, 1H, J = 9.9 Hz, J = 11.2 Hz), 4.07 (qd,
557 ([M] ), 308 (base), 250, 224, 195, 179; HRMS (EI) m/z
+
2
1
6
8
H, J = 4.1 Hz, J = 18.9 Hz), 4.24 (t, 1H, J = 9.6 Hz), 4.57 (d,
H, J = 14.2 Hz), 5.19 (t, 1H, J = 10.4 Hz), 6.52–6.56 (m, 1H),
.86–7.05 (m, 4H), 7.09–7.37 (m, 10H), 7.49–7.51 (m, 1H),
calcd for C H N O : 557.2791 ([M] ), found: 557.2790.
3
5 35 5 2
Benzyl 5-((tert-butoxycarbonyl)amino)pentanoate (16).
K CO (0.29 g, 2.07 mmol) was added to a stirred solution of
2
3
13
ꢁ
.98 (br s, 1H), 9.54 (br s, 1H); C NMR (100 MHz, CDCl3)
11 (0.30 g, 1.38 mmol) in NMP (4 mL) at 25 C. Benzyl bro-
mide (0.16 mL, 1.38 mmol) was slowly added to it and stirred
for 5 h before it was quenched by cold water (10 mL). The
reaction mixture was extracted with ethyl acetate (3 × 20 mL),
the combined organic layers were sequentially washed with
sat. NaHCO3 solution (2 × 20 mL), deionized water
(2 × 20 mL) and sat. NaCl solution (20 mL), dried over
Na SO , and concentrated. Purification of the crude material
δ 180.8, 168.9, 162.4, 147.6, 139.7, 139.4, 138.0, 136.2,
1
1
1
5
35.7, 135.5, 132.0, 131.2, 130.6, 128.6, 128.1, 128.0, 127.9,
27.8, 127.7, 127.7, 127.5, 125.7, 125.6, 121.2, 116.5, 116.1,
14.6, 61.1, 50.3, 46.4, 39.0, 20.7, 14.0; EI-MS m/z
+
29 ([M] ), 280 (base), 249, 223, 194; HRMS (EI) m/z calcd
+
for C H N O : 529.2478 ([M] ), found: 529.2478.
33 31 5 2
N-(3-((9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5-a]
azepin-3-yl)amino)-3-oxopropyl)-2-((2,3-dimethylphenyl)
amino)benzamide (5). According to the similar procedure
used to compound 3, the reaction of 2 (0.17 g, 0.72 mmol)
with Boc-free compound of 13 (0.2 g, 0.48 mmol) in the
presence of EDCI (0.13 mL, 0.72 mmol), HOBt (0.1 g,
2
4
by column chromatography (petroleum ether/ethyl acetate,
v/v, 6:1) gave 16 (0.4 g, 96.4%) as colorless liquid. R = 0.1
f
1
(petroleum ether/ethyl acetate = 6:1). H NMR (400 MHz,
CDCl ) δ 1.44 (s, 9H), 1.47–1.54 (m, 2H), 1.64–1.71 (m, 2H),
3
2.39 (t, 2H, J = 7.4 Hz), 3.12 (q, 2H, J = 6.7 Hz), 4.55 (s, 1H),
5.12 (s, 2H), 7.31–7.40 (m, 5H).
0.72 mmol) and DIPEA (0.33 mL, 1.92 mmol) for 23 h
afforded, after purification by column chromatography (petro-
leum ether/ethyl acetate, v/v, 1:1), compound 5 (0.23 g,
Benzyl 5-(2-((2,3-dimethylphenyl)amino)benzamido)pen-
tanoate (17). Dropwise addition of trifluoroacetic acid
(2.34 mL, 30.6 mmol) to 16 (0.94 g, 3.06 mmol) in anhydrous
methylene chloride (24 mL) was took place under argon at
8
7.1%) as a white foam solid. R = 0.15 (petroleum ether/ethyl
f
1
acetate = 1:1). H NMR (400 MHz, CDCl ) δ 2.18 (s, 3H),
3
ꢁ
ꢁ
2
(
3
.30 (s, 3H), 2.57 (t, 2H, J = 5.6 Hz), 3.40–3.48 (m, 1H), 3.53
d, 1H, J = 14.4 Hz), 3.66 (dd, 1H, J = 9.5 Hz, J = 11.8 Hz),
.74–3.81 (m, 1H), 4.21 (t, 1H, J = 9.5 Hz), 4.61 (d, 1H,
J = 14.2 Hz), 5.15 (dd, 1H, J = 9.3 Hz, J = 11.6 Hz), 6.52 (td,
H, J = 1.2 Hz, J = 7.7 Hz), 6.76 (dd, 1H, J = 1.7 Hz,
J = 8.0 Hz), 6.88–7.06 (m, 4H), 7.13–7.31 (m, 8H), 7.45–7.51
0 C and then it was heated to 25 C and stirred for 3 h before
it was concentrated under vacuum. The remaining traces
trifluoroacetic acid was eliminated from the crude residue by
repetitive evaporation after diluting the crude with methylene
chloride to give the free amine quantitatively, which was used
in the next step without further purification. According to the
similar procedure used to compound 3, the reaction of
2 (1.11 g, 4.58 mmol) with the above obtained Boc-free com-
pound of 16 (0.93 g, 3.06 mmol) in the presence of EDCI
(0.65 mL, 3.67 mmol), HOBt (0.62 g, 4.58 mmol) and DIPEA
(2.13 mL, 12.23 mmol) for 12 h afforded, after purification
by column chromatography (petroleum ether/ethyl acetate,
1
1
3
(m, 2H), 9.09 (br s, 1H), 9.36 (br s, 1H); C NMR (100 MHz,
CDCl ) δ 185.8, 169.2, 162.6, 147.0, 139.8, 139.1, 138.0,
3
1
1
1
36.4, 135.6, 135.6, 131.7, 130.7, 130.5, 128.6, 128.1, 128.0,
27.8, 127.7, 127.5, 127.4, 127.3, 125.7, 125.3, 120.6, 117.4,
16.7, 114.5, 61.2, 50.2, 39.7, 39.0, 35.8, 20.8, 14.0; EI-MS
+
m/z 543 ([M] ), 249, 276 (base), 249, 223, 194; HRMS (EI) m/z
calcd for C H N O : 543.2634 ([M] ), found: 543.2630.
+
v/v, 3:1), compound 17 (0.91 g, 69.0%) as colorless liquid.
34 33 5 2
1
N-(4-((9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5-a]
azepin-3-yl)amino)-4-oxobutyl)-2-((2,3-dimethylphenyl)
amino)benzamide (6). According to the similar procedure
used to compound 3, the reaction of 2 (0.13 g, 0.56 mmol)
with Boc-free compound of 14 (0.16 g, 0.37 mmol) in the
presence of EDCI (0.1 mL, 0.56 mmol), HOBt (0.08 g,
R = 0.32 (petroleum ether/ethyl acetate = 3:1). H NMR
f
(400 MHz, CDCl ) δ 1.62–1.77 (m, 4H), 2.19 (s, 3H), 2.31
3
(s, 3H), 2.42 (t, 2H, J = 7.1 Hz), 3.42 (q, 2H, J = 6.8 Hz), 5.12
(s, 2H), 6.32 (br t, 1H, J = 5.1 Hz), 6.65–6.69 (m, 1H),
6.91–6.94 (m, 2H), 7.05 (t, 1H, J = 7.7 Hz), 7.15–7.21 (m,
13
2H), 7.29–7.40 (m, 6H), 9.21 (br s, 1H); C NMR (100 MHz,
0.56 mmol) and DIPEA (0.26 mL, 1.48 mmol) for 17 h
CDCl ) δ 173.5, 169.9, 147.2, 139.7, 138.1, 136.0, 132.3,
3
afforded, after purification by column chromatography (petro-
leum ether/ethyl acetate, v/v, 1:2), compound 6 (0.14 g,
130.9, 128.7, 128.4, 128.3, 127.4, 125.8, 125.6, 120.9, 117.1,
116.8, 115.0, 66.4, 39.4, 33.8, 29.1, 22.2, 20.8, 14.0; EI-MS
m/z 430 ([M] , base), 304, 223, 208, 91.
+
6
7.4%) as a white foam solid. R = 0.30 (petroleum ether/ethyl
f
1
acetate = 1:2). H NMR (400 MHz, CDCl ) δ 1.90–1.97 (m,
5-(2-((2,3-Dimethylphenyl)amino)benzamido)pentanoic
acid (18). Pd/C (10%, 0.06 g) was added to a stirred solution
of 17 (0.81 g, 1.88 mmol) in methanol (10 mL) and stirred at
3
2H), 2.20 (s, 3H), 2.31 (s, 3H), 2.42–2.52 (m, 2H), 3.38–3.51
(m, 3H), 3.65 (dd, 1H, J = 9.9 Hz, J = 11.0 Hz), 4.20 (t, 1H,
ꢁ
J = 9.5 Hz), 4.56 (d, 1H, J = 14.2 Hz), 5.10 (t, 1H,
J = 10.4 Hz), 6.61 (t, 1H, J = 7.4 Hz), 6.91–6.98 (m, 3H), 7.05
25 C under hydrogen for 4 h. The reaction solution was fil-
®
tered through Celite , washed with methanol (10 mL) and
(
9
t, 1H, J = 7.6 Hz), 7.11–7.31 (m, 10H), 7.40–7.42 (m, 1H),
.07 (br s, 1H), 9.39 (br s, 1H); C NMR (100 MHz, CDCl3)
concentrated under vacuum. The crude mixture was purified
13
by column chromatography (methylene chloride/methanol,
1
δ 187.8, 169.7, 162.6, 147.1, 139.8, 139.2, 138.1, 136.5,
v/v, 97:3) gave 17 (0.4 g, 62.1%) as off-white foam solid. H
Bull. Korean Chem. Soc. 2020
© 2020 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Novel Hybrid Molecules of Epinastine and Mefenamic Acid for Bioactive
BULLETIN OF THE
Article
Assessment as Potential Anti-inflammatory Agents
KOREAN CHEMICAL SOCIETY
NMR (400 MHz, CDCl ) δ 1.65–1.79 (m, 4H), 2.19 (s, 3H),
2H), 3.37–3.47 (m, 3H), 3.64 (dd, 1H, J = 9.5 Hz, J = 11.5 Hz),
4.18 (t, 1H, J = 9.5 Hz), 4.56 (d, 1H, J = 14.2 Hz), 5.09 (dd, 1H,
J = 9.3 Hz, J = 11.5 Hz), 6.64–6.68 (m, 1H), 6.84 (br t, 1H,
J = 5.4 Hz), 6.91–6.98 (m, 3H), 7.05 (t, 1H, J = 7.7 Hz),
7.14–7.27 (m, 8H), 7.39–7.45 (m, 2H), 9.07 (br s, 1H), 9.33
3
2
6
2
.32 (s, 3H), 2.43 (t, 2H, J = 7.0 Hz), 3.46 (q, 2H, J = 6.6 Hz),
.28 (br t, 1H, J = 5.7 Hz), 6.66–6.70 (m, 1H), 6.90–6.95 (m,
H), 7.06 (t, 1H, J = 7.7 Hz), 7.14–7.22 (m, 2H), 7.38–7.40
1
3
(m, 1H), 9.15 (br s, 1H); C NMR (100 MHz, CDCl ) δ
3
13
1
1
1
78.7, 170.0, 147.2, 139.7, 138.2, 132.4, 131.0, 127.4, 125.9,
25.7, 121.0, 117.1, 116.9, 115.1, 39.4, 33.5, 29.1, 22.0, 20.8,
4.0; EI-MS m/z 340 ([M] , base), 223, 208, 194, 180.
(br s, 1H); C NMR (100 MHz, CDCl ) δ 187.9, 169.8, 162.7,
3
147.1, 139.8, 139.1, 138.1, 136.7, 135.8, 131.9, 130.7, 130.5,
128.4, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 125.7, 125.4,
+
1
20.6, 117.4, 116.7, 114.7, 61.3, 50.0, 40.1, 39.9, 39.0, 28.4,
+
N-(5-((9,13b-Dihydro-1H-dibenzo[c,f]imidazo[1,5-a]
azepin-3-yl)amino)-5-oxopentyl)-2-((2,3-dimethylphenyl)
amino)benzamide (7). According to the similar procedure
used to compound 3, the reaction of 18 (0.35 g, 1.03 mmol)
with 1 (0.32 g, 1.13 mmol) in the presence of EDCI (0.2 mL,
23.4, 20.8, 14.0; EI-MS m/z 571 ([M] ), 322, 249, 223, 194
(base); HRMS (EI) m/z calcd for C H N O : 571.2947
36 37 5 2
+
([M] ), found: 571.2950.
1.13 mmol), HOBt (0.15 g, 1.13 mmol) and DIPEA (0.72 mL,
Pharmacology
4.11 mmol) for 16 h afforded, after purification by column
chromatography (petroleum ether/ethyl acetate, v/v, 1:1), com-
Cell Culture and Cell Viability Assay. RAW264.7 mac-
rophage cells were acquired from Korea Cell line Bank
(Seoul, Republic of Korea) and grown in Dulbecco’s
modified Eagle’s medium (Gibco, Carlsbad, CA, USA)
pound 7 (0.51 g, 86.5%) as a white foam. R = 0.19 (petroleum
f
1
ether/ethyl acetate = 1:2). H NMR (400 MHz, CDCl3)
δ 1.64–1.77 (m, 4H), 2.20 (s, 3H), 2.31 (s, 3H), 2.33–2.50 (m,
Scheme 1. Synthesis of 1 conjugates 3–7. (a) EDCI/HOBt, DIPEA, THF, 0 C—rt., 6–16 h; (b) 8/9/10/11, EDCI/HOBt, DIPEA,
ꢁ
ꢁ
2 2
Cl , rt., 1 h; (ii) EDCI/HOBt, DIPEA, THF, 0 C—rt., 12–20 h; (d) benzyl bro-
DMF/THF/ (1/2–1/6), 0 C—rt., 45–65 h; (c) (i) TFA, CH
mide, K CO , NMP, rt., 5 h; (e) H , Pd/C, MeOH, rt., 4 h.
2
3
2
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Novel Hybrid Molecules of Epinastine and Mefenamic Acid for Bioactive
BULLETIN OF THE
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Assessment as Potential Anti-inflammatory Agents
KOREAN CHEMICAL SOCIETY
supplemented with 2 mM glutamine, 100 units/mL
Table 1. NO production inhibitory effects of 1, 2 and their hybrid
compounds (3–7) .
a
penicillin–streptomycin and 10% fetal bovine serum
4
(
Gibco). Cells (5 × 10 cells/well) were cultured on 96-well
Compound IC50 (μM)
CC50 (μM)
iEI (CC50/IC50)
plates and treated with serial dilutions of testing materials
1
2
3
4
5
6
7
a
51.62 ꢀ 1.56
132.41 ꢀ 4.62
2.24
5.08
5.55
6.23
3.51
2.20
6.40
for 24 h. Cell viability was measured by MTT assay using
CellTiter 96® AQueous assay kit (Promega, Fitchburg,
39.05 ꢀ 2.22 198.21 ꢀ 18.89
36.03 ꢀ 1.80
32.09 ꢀ 4.04
≥ 200.00
≥ 200.00
USA). After removing medium and wash cells with phos-
phate buffered saline (PBS), MTS tetrazolium was added to
30.97 ꢀ 6.03 109.00 ꢀ 21.93
ꢁ
the plates and incubated at 37 C for 1 h. Absorbance was
40.19 ꢀ 5.52
27.69 ꢀ 3.06 177.39 ꢀ 22.16
The findings are stated as average score ꢀ SEM for n = 3.
88.74 ꢀ 12.71
measured at 490 nm utilizing a Microplate reader (Thermo
Scientific, Waltham, USA).
Nitrite Assay. Lipopolysaccharide (LPS) derived from
Escherichia coli (serotype 0111:B4) was received from
Sigma-Aldrich (St. Louis, MO, USA). RAW264.7 cells
were treated with LPS (100 ng/mL) and various amounts of
group of 11 with benzyl bromide (BnBr) in the presence of
potassium carbonate in N-methyl-2-pyrrolidone (NMP) at
ambient temperature afforded 16 in 96.4% yield which fur-
ther underwent Boc-deprotection and subsequent coupling
with 2 to give compound 17 in 69% yield. Benzyl group of
17 was removed under hydrogenolysis condition to produce
18. Finally, EDCI/HOBt-promoted coupling of 1 with 18
proceeded efficiently to furnish 7 in 86.5% yield.
compounds (25–100 μM) for 24 h. The amount of nitrite
−
(
NO ) in the culture supernatant was quantified by Griess
2
Reagent System (Promega Corp).
Reverse Transcriptase-Polymerase Chain Reaction.
Total RNA from RAW264.7 cells was garnered with TRIzol
reagent (Invitrogen, Waltham, MA, USA). The relative amount
of given mRNA was measured by RT-PCR using amfiRivert
cDNA Synthesis Platinum Master Mix (GenDEPOT, Baker,
TX, USA). The sequences of the sense- and antisense-strand
primers used for PCR amplification were inducible nitric oxide
Bioactivity. All the molecules (1–7) were examined for
possible anti-inflammatory activity. The CC50 of 1–7 was
above 100 μM except for 6 (CC50 = 88.74 ꢀ 12.71 μM).
All the hybrids significantly reduced NO production in
LPS-stimulated macrophages (Table 1). More importantly,
7 followed by 4 appeared to be more effective with IC50
values 27.69 ꢀ 3.06 μM and 32.09 ꢀ 4.04 μM, respec-
tively, than other hybrids and parent compounds EH and
0
synthase (iNOS), 5 -GCTACCACATTGAAGAAGCTGGTG-
0
0
0
3
5
, 5 -CCATAGGAAAAGACTGCACCGAAG-3 ; mPGES-1,
0
0
0
-TTACAGGAGTGACCCAGATGTGGA-3 , 5 -CCGGG
TAGAACTCTAAAGCATCGA-3 ; cyclooxygenase-2 (COX-2),
-GTCTCTCAATGAGTACCGCAAACG-3 , 5 -CTACC
ATGGTCTCCCCAAAGATAG-3 and COX-1, 5 -TGTCA
TCAAGG AGTCCCGAGAGAT-3 , 5 -CAACGAGGAAA
TGTATCCAGGCGA-3 . As a control, glyceraldehyde-
0
0
0 0
5
0
0
16
0
0
MA. The in vitro efficacy index (iEI), the ratio of toxicity
(CC )/anti-inflammatory potency (IC ), values are also
0
50
50
impressive for 4 (iEI = 6.23) and 7 (iEI = 6.40); hence,
these two hybrids were selected to further investigate their
inhibitory role on the signaling pathway of the inflamma-
tory response. NO is produced by inducible nitric oxide
synthase (iNOS) expressed in tissue macrophage under
3
-phosphate dehydrogenase (GAPDH) gene was also ampli-
0
0
fied using 5 -GACATCAAGAAGGTGGTGAAGCAG-3 ,
0
0
5
-CCCTGTTGCTGTAGCCGTATTCAT-3 .
Results and Discussion
Chemistry. The synthetic path for the hybrid molecules
(
3–7) of 1 and 2 is outlined in Scheme 1. At first, 1 was
0
coupled to
2
with N-(3-dimethylaminopropyl)-N -
ethylcarbodiimide (EDCI)/1-hydroxybenzotriazole hydrate
HOBt) and N,N-diisopropylethylamine (DIPEA) in tetra-
(
hydrofuran at ambient temperature for 6 h to obtain the
hybrid 3 in 57.3% yield. Next, spacer attachment to 1 was
conducted by EDCI/HOBt-mediated reaction with Boc-
glycine (8), Boc-β-alanine (9), Boc-GABA (10) and
5-(Boc-amino)valeric acid (11) to obtain Boc-protected
intermediates 12–15 in 72.8–95.3% yields, respectively.
Eventually, trifluoroacetic acid mediated Boc-deprotection
of 12–14 in methylene chloride followed by coupling with
Figure 2. Inhibitory effect of 1, 2, 4, and 7 on LPS-mediated
inflammatory gene expression in murine macrophages.
RAW264.7 cells were stimulated with LPS (100 ng/mL) and the
indicated concentrations of compounds were treated for 24 h. after
endpoint RT-PCR, gene expression levels of iNOS, mPGES-1,
COX-2 and COX-1 were analyzed by 1.5% agarose gel electro-
phoresis. GAPDH was used as a control.
2
favored the desired hybrids 4–6 in 49.5–87.1% yields.
Unfortunately, synthesis of 7 from 15 and 2 gave very poor
yield (13.8%). An alternative effort was made to achieve
the hybrid 7 in a more efficient way. Protection of carboxy
Bull. Korean Chem. Soc. 2020
© 2020 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5

Novel Hybrid Molecules of Epinastine and Mefenamic Acid for Bioactive
BULLETIN OF THE
Article
Assessment as Potential Anti-inflammatory Agents
KOREAN CHEMICAL SOCIETY
inflammatory conditions. As shown in Figure 2, com-
pounds 4 and 7 reduced LPS-induced iNOS gene expres-
sion in a dose-proportional mode. This reduction was much
higher than the starting materials 1 and 2. Compounds
E synthase-1 (mPGES-1) by compounds 4 and 7 without
altering COX-1 levels. On the whole, hybrids 4 and 7 could
be reviewed as lead molecules for the future anti-
inflammatory drug development.
4
and 7 also reduced gene expression of microsomal prosta-
Acknowledgments.
This work was supported by the
glandin E synthase-1 (mPGES-1), which synthesizes a
potent inflammatory mediator, prostaglandin E2 (PGE2)
from prostaglandin H2 (PGH ). However, there was no
change in the expression of COX-2, an upstream mediator
of PGs synthesis.
National Research Foundation of Korea (NRF) grant funded
by the Korea government (MOE) (2017R1D1A1A02018831,
J. T. Lee) and by the Hallym University Leading Research
Group Support Program of 2017 (HRF-LGR-2017-0004).
2
Traditional NSAIDs nonselectively inhibit constitutive
COX-1 and inducible COX-2, which transform arachidonic
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
acid to PGH . NSAIDs bind directly to COX to block
2
enzyme activity, and other anti-inflammatory regulators,
such as flavonoids, down-regulate gene expression of COX
17
through inhibition of signal transduction pathways.
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1
1
1
1
7
4
(IC50 = 27.69 ꢀ 3.06 μM; iEI = 6.40) followed by
(IC50 = 32.09 ꢀ 4.04 μM; iEI = 6.23) exerted the best
inhibitory effect among the hybrids as well as their parent
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with the subdued expression of microsomal prostaglandin
Bull. Korean Chem. Soc. 2020
© 2020 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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