Synthesis and biological evaluation of substituted N-[3-(1H-pyrrol-1-yl) methyl]-1,2,5,6-tetrahydropyridin-1-yl]benzamide/benzene Sulfonamides as anti-inflammatory agents

Article abstract of DOI:10.1002/ardp.201300379

The pharmacological activities of tetrahydropyridine (THP) derivatives are dependent on the substituent ring moiety. In this study, we investigate the anti-inflammatory activities of 12 newly synthesized substituted N-[3-(1H-pyrrol-1-yl)methyl]-1,2,5,6-tetrahydrobenzamide/benzene sulfonamides (9a-l) in murine BV-2 microglial cells. All compounds were initially screened for attenuation of nitric oxide (NO) production in lipopolysaccharide (LPS) (1 μg/mL)-activated microglial cells. The data show that only SO ₂-substituted THPs were effective at sub-lethal concentrations (IC₅₀ values of 12.92 μM (9i), 14.64 μM (9j), 19.63 μM (9k)) relative to L-N6-(1-iminoethyl)lysine positive control (IC₅₀ = 3.1 μM). The most potent SO₂-substituted compound (9i) also blocked the LPS-inducible nitric oxide synthase (iNOS) and attenuated the release of several cytokines including IL-1α, IL-10, and IL-6. These findings establish the moderate immuno-modulating effects of SO₂-substituted THP derivatives.

Full text of DOI:10.1002/ardp.201300379

360
Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Full Paper
Synthesis and Biological Evaluation of Substituted N-[3-(1H-
Pyrrol-1-yl)methyl]-1,2,5,6-tetrahydropyridin-1-yl]benzamide/
benzene Sulfonamides as Anti-Inflammatory Agents
Madhavi Gangapuram, Elizabeth Mazzio, Suresh Eyunni, Karam F. A. Soliman, and Kinfe K. Redda
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, USA
The pharmacological activities of tetrahydropyridine (THP) derivatives are dependent on the
substituent ring moiety. In this study, we investigate the anti-inflammatory activities of 12 newly
synthesized substituted N-[3-(1H-pyrrol-1-yl)methyl]-1,2,5,6-tetrahydrobenzamide/benzene sulfon-
amides (9a–l) in murine BV-2 microglial cells. All compounds were initially screened for attenuation
of nitric oxide (NO) production in lipopolysaccharide (LPS) (1 mg/mL)-activated microglial cells. The data
show that only SO2-substituted THPs were effective at sub-lethal concentrations (IC50 values of 12.92 mM
(9i), 14.64 mM (9j), 19.63 mM (9k)) relative to L-N6-(1-iminoethyl)lysine positive control (IC50 ¼ 3.1 mM).
The most potent SO2-substituted compound (9i) also blocked the LPS-inducible nitric oxide synthase
(iNOS) and attenuated the release of several cytokines including IL-1a, IL-10, and IL-6. These findings
establish the moderate immuno-modulating effects of SO2-substituted THP derivatives.
Keywords: Cytokines / Drug design / Microglial cells / Nitric oxide / Tetrahydropyridine
Received: October 16, 2013; Revised: December 2, 2013; Accepted: December 2, 2013
DOI 10.1002/ardp.201300379
Introduction
(monoamine oxidase) [13] or characteristic antifungal [14]/
anti-inflammatory effects [15–17]. In the present study, we
explore the efficacy of synthetic THP derivatives to attenuate
pro-inflammatory molecules in lipopolysaccharide (LPS)-
activated BV-2 murine microglial cells. The activated BV-2
model is frequently used to evaluate the efficacy and potency
of immuno-modulating agents against endotoxic production
of NO, interleukin-6 (IL-6) [18], tumor necrosis factor-alpha
(TNF-alpha), interleukin-1beta (IL-1b), CCL2 or NADPH oxi-
dase [19], contributors to CNS neurodegenerative injury [19,
20]. In this study, we evaluate the anti-inflammatory activities
of synthesized THP derivatives.
Central nervous system (CNS) diseases such as Alzheimer’s
disease (AD) [1], Parkinson’s disease (PD) [2, 3], acute trauma,
stroke [4, 5], amylotrophic lateral sclerosis [6], and hypoxia–
ischemia [7, 8] involve chronic inflammation mediated by
activated astrocytes and microglial cells [9]. While microglial
cells can provide CNS protection against various pathogens,
chronic activation can also contribute to sustained release of
deleterious pro-inflammatory cytokines and neurotoxic sub-
stances such as nitric oxide (NO). Practical use of natural anti-
inflammatory products such as omega-3 polyunsaturated
fatty acids/polyphenolics or synthetic non-steroidal anti-
inflammatory drugs (NSAIDs) is believed to be beneficial in Results
attenuating neurodegenerative decline, if taken over the long
term [10, 11].
New THP derivatives were synthesized using the methods
Tetrahydropyridines (THPs) are known to contain inherent
pharmacological properties [12], such as enzyme inhibition
described [21, 22]. The synthetic routes for the title
compounds are outlined in Scheme 1. The O-mesitylenesul-
fonyl hydroxylamine (MSH) 4 was used to prepare the N-amino
salt as an aminating agent [23]. Substituted THP derivatives
9a–l were obtained by a series of reactions beginning with
commercially available 3-[(1H-pyrrol-1-yl)methyl]pyridine 5,
treated with MSH 4 in dry dichloromethane. This reaction
Correspondence: Dr. Kinfe K. Redda, 410 Foote-Hilyer Administration
Center, Division of Research, Florida A&M University, Tallahassee, FL
32307, USA.
E-mail: kinfe.redda@famu.edu
Fax: þ1-850-4125096
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Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Sulfonamides as Anti-Inflammatory Agents
361
C2H5O
CH3
CH3
CH3
O
N
O
H3C
(i)
H3C
S O
NOH
H3C
S OCl
O
+
C2H5O
O
CH3
CH3
1
2
3
(ii)
H3C
O
H2N
NH2
5, (iii)
N
N
O S
O
CH3
OMes
H3C
6
4
7
(iv)
H
N
N
N
X-R
N
(v)
N
N
X-R
9
8
Scheme 1. Reaction conditions: (i) DMF, Et3N,
0°C, 45 min. (ii) 70% HClO4, p-dioxane, 0°C,
45 min. (iii) 3-[(1H-Pyrrol-1-yl)methyl]pyridine,
CH2Cl2, 0°C, 5 h. (iv) 4-Substituted acyl/sulfonyl
chloride, dry THF, 70°C. (v) NaBH4, abs EtOH,
7 h. R ¼ C6H5, 4-CH3-C6H4, 4-C2H5-C6H4, 4-n-
C4H9-C6H4, 4-OCH3-C6H4, 4-F-C6H4, 4-Cl-C6H4,
4-Br-C6H4, 2-thiophenyl, X ¼ CO, SO2.
H3C
O
OMes
O S
CH3
=
O
H3C
gave N-amino-3-[(1H-pyrrol-1-yl)methyl]pyridinium mesitylene-
sulfonate in good yield. Reaction of the N-amino-
also attenuated production/release of IL-1a, IL-6, and IL-10
determined by ELISarray profiling, but had no effect on TNFa
or G-CSF (Fig. 4). Corroboration by independent enzyme-linked
immunosorbent assays (ELISAs) confirmed the capability of 9i
to attenuate IL-a and IL-6 release (Fig. 5A and B), respectively.
These finding establish the moderate anti-inflammatory
effects of SO2-substituted THPs.
6
pyridinium salt with corresponding substituted acylating
agents such as acylchlorides or sulfonyl chlorides, followed by
treatment with a base, afforded ylides 8 as stable crystalline
solids. Sodium borohydride reduction of 8a–l in absolute
ethanol furnished the title compounds 9a–l in fair-to-good
yields.
All substituted THPs (0.5–1ꢀ00 mM) were screened for dose- Discussion
dependent inhibition of NO2 in LPS (1 mg/mL)-treated BV-2
cells relative to dexamethasone (steroidal anti-inflammatory)
and L-NIL (selective nitric oxide synthase (iNOS) inhibitor)
controls. There were no effects observed for compounds 9a–h
and 9l, with only SO2-substituted THPs capable of mediating
anti-inflammatory effects; including 9i X ¼ SO2 R ¼ H, 9j
X ¼ SO2 R ¼ CH3, and 9k X ¼ SO2 R ¼ OCH3 (Table 1, Fig. 1A–C).
The relative potency of the most effective compound (9i
X ¼ SO2 R ¼ H) to attenuate NO2ꢀ was approximately fourfold
weaker than L-NIL, and displayed an IC50 within a therapeutiꢀc
range; 12.92 mM. The capability of 9i to attenuate NO2
occurred independent of cell toxicity, which was observed at
or above 44.6 mM (Fig. 2A and B) and corresponded to
attenuating iNOS protein expression, where a complete
repression was observed at 26.8 mM 9i (Fig. 3). Compound 9i
Microglial cells are an important part of the CNS immuno-
logical response, also playing a detrimental role in the
development of neurodegenerative disorders including PD,
AD, and stroke [1–7]. Likewise, the activation of microglial
cells can be triggered by disease-specific pathological
components such as hypoxia [24], tau [25], neuromelanin [26],
ammonia [27], heavy metals [11], or misfolded proteins,
concomitant to the extent of neurological injury [28]. Chronic
microglial activation can lead to a continued release of
neurotoxic substances such as pro-inflammatory cytokines
and NO, the latter of which can react with superoxide to
produce peroxynitrite (ONOO–), a potent neurotoxin and
contributor to nitrosative stress evident not only in brain
injury [29] but also in the process of aging [30]. For this reason,
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M. Gangapuram et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Table 1. Evaluation of synthetic THPs on NO2-inhibition in BV-2
LPS-stimulated microglial cells.
Anti-inflammatory properties of substituted
tetrahydropyridines (THPs)
Compound
X
R
LPS-NO2-IC50 (mM)
Dexamethasone
L-N6-(1-iminoethyl)lysine
<1.0
3.16
9i
9j
SO2
SO2
SO2
CO
CO
CO
CO
CO
CO
CO
CO
H
CH3
OCH3
H
CH3
C2H5
OCH3
C4H9
F
12.92
14.64
19.43
>100
>100
>100
>100
>100
>100
>100
>100
9k
9a
9b
9c
9d
9e
9f
9g
9h
Cl
Br
9l
CO
>100
IC50 values were calculated based on regression analysis for data
obtained from mean ꢁ SEM, n ¼ 4 of a minimum of eight
concentrations below 100 mM.
No anti-inflammatory effect.
novel therapeutics with a capacity to attenuate microglial
activation could serve as potential neuro-protective agents.
The data from this study demonstrate that novel synthetic
SO2-substituted THPs were effective in attenuating inflamma-
tory mediators and cytokines in LPS-activated murine BV2
microglia. Future research will be required to determine
safety versus efficacy or application in other inflammatory
models.
In general, the preliminary evaluation of synthetic anti-
inflammatory drugs at early stages requires in vitro screenings
such as that described in this work, which aim to elucidate
structure versus function with respect to attenuation of
immuno-competent parameters including iNOS, TNF-a, IL-6,
IL-1b, NO2ꢀ, and MCP-1 under various inflammatory stimuli
(e.g., amyloid-beta [31, 32], oxygen–glucose deprivation [33], or
LPS). [34, 35] These indicators are commonly used and
represent pathological manifestation of human CNS neuro-
degenerative diseases [36–38]. There is a plethora of natural
plant-derived [39–42] and synthetic drugs described in the
literature with
a capacity to downregulate microglial
Figure 1. (A–C)ꢀThe effect of SO2-substituted tetrahydropyridines
(THPs) on NO2 production by LPS (1 mg/mL)-activated BV-2
microglial cells at 24 h. The data represent NO2ꢀ as % control and
are presented as the mean ꢁ SEM, n ¼ 4. Statistical differences
from the control were evaluated by a one-way ANOVA, followed by
a Tukey post hoc test, ꢂp < 0.05.
activation [10]. More specifically, the ability of a candidate
compound to attenuate IL-6, NO, and iNOS in glial cells often
equates to therapeutic efficacy in diverse in vivo models
[43–45] and involves downregulation of nuclear factor-kappaB
translocation/activation, degradation of IkappaB-alpha,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Sulfonamides as Anti-Inflammatory Agents
363
A
B
50
45
40
35
30
25
20
15
10
5
120%
100%
80%
60%
40%
20%
0%
*
*
*
*
*
*
*
*
0
Cells LPS 4.46 8.93 17.8 26.8 35.7 44.6 53.6
Cells LPS 4.46 8.93 17.8 26.8 35.7 44.6 53.6
µM 9i
µM 9i
Figure 2. (A) Cell viability control for compound 9i in LPS (1 mg/mL)-activated BV-2 microglial cells at 24 h. The data represent viability
% control and are presented as the mean ꢁ SEM, n ¼ 4. Statistical differences from the LPS control were evaluated by a one-way ANOVA,
followed by a Tukey post hoc testꢀ, ꢂp < 0.05. (B) Anti-inflammatory control for compound 9i in LPS (1 mg/mL)-activated BV-2 microglial cells
at 24 h. The data represent NO2 (mM) and are presented as the mean ꢁ SEM, n ¼ 4. Statistical differences from the LPS control were
evaluated by a one-way ANOVA, followed by a Tukey post hoc test, ꢂp < 0.05.
MAPK [33, 35, 46, 47], C/EBPdelta [48], or JAK2-mediated
STAT1 phosphorylation [34, 49], which indirectly controls
the inflammatory process.
In this work, we report that SO2-substituted synthetic THPs
exert moderate anti-inflammatory effects, evidenced by
inhibition of NO2ꢀ, iNOS protein expression, and various
cytokines. Since the potency of these compounds is within the
therapeutic range, research will be required to design THPs
with greater strength, suitable, and safe for long-term use.
AutoSpec M apparatus. The instrument was run at a mass
resolution of 10,000 for the accurate mass analysis, using
perfluorokerosene (PFK) as an internal calibration standard.
Separations by column chromatography were performed on silica
gel (200–425 mesh). All reactions and purification procedures
were monitored by TLC on Whatmann AL SIL g/UV, 250 mm layer
flexible plates, with visualization under UV light.
General procedure A
Synthesis of N-amino-3-[(1H-pyrrol-1-yl)methyl]pyridinium
mesitylenesulfonate (6)
To an ice-cooled solution of 3-[(1H-pyrrol-1-yl)methyl]pyridine (4,
2.94 g, 18.58 mmol) in 25 mL of dry methylene chloride was
added dropwise to O-mesitylenesulfonylhydroxylamine (5, 4.0 g,
18.58 mmol) in 10 mL of dry methylene chloride over 5 min with
stirring. The resulting mixture was stirred at 0°C for 3 h, and at
room temperature for 1 h; excess ether was added and the
suspension was filtered. The precipitate was recrystallized from
ethyl acetate–methanol (5:1 v/v) to give N-amino-3-[(1H-pyrrol-1-yl)-
methyl]pyridinium mesitylenesulfonate (6) in 57.5% yield. 1H
NMR (CDCl3): d 5.20 (s, 2H, –CH2), 6.26 (t, 2H, J ¼ 5.7 Hz, 30H, 40H),
6.70 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 7.39–7.44 (m, 4H, 300H, 400H, 500H,
5H), 7.61–7.564 (dd, 1H, J ¼ 6.0, 1.8 Hz, 4H), 8.152–8.12 (m, 2H,
200H, 600H), 8.68 (s, 1H, 2H), 8.72 (d, 1H, J ¼ 6.0 Hz, 6H).
Experimental
Methods and materials
Hanks balanced salt solution, (4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonic acid) (HEPES), ethanol, dimethyl sulfoxide
(DMSO), 96-well plates, general reagents and supplies were all
purchased from Sigma–Aldrich Co. (St. Louis, MO) and VWR
International (Radnor, PA). Imaging probes were supplied by Life
Technologies (Grand Island, NY). All chemicals and solvents were
purchased from Sigma–Aldrich Chemical Company, Inc. (Mil-
waukee, Wisconsin). The structures of the products described
1
1
were confirmed by IR, H NMR, and elemental analysis data. H
NMR spectra were recorded on Varian Gemini HX 300 MHz
spectrometer. All chemical shifts expressed in parts per million (d,
ppm) are reported relative to tetramethylsilane (TMS) as internal
standard for solution in CDCl3 as a solvent unless otherwise
specified. The IR spectra were run with KBr pellets on Perkin-
Elmer FTIR 1430 spectrometer and are reported in cmꢀ1. Melting
points were determined on a Mel-Temp 3.0 melting point
apparatus and are uncorrected. Elemental analyses were
performed with a Perkin Elmer 2400 CHN elemental analyzer,
by Atlantic Microlab, Inc. (Norcross, GA). C, H, and N values are
within ꢁ0.4% of theoretical values unless otherwise noted. High-
resolution mass spectra analysis was performed with Micromass
General procedure B
Synthesis of 1-(benzoylimino)-3-(1H-pyrrol-1-ylmethyl)-
pyridinium ylide (8a)
The ice-cold solution of N-aminopyridinium salt (6, 2.0 g,
5.36 mmol) in 30 mL of anhydrous tetrahydrofuran (THF)
containing triethylamine was added to benzoyl chloride (7,
1.87 mL, 16.08 mmol) dropwise. The reaction was allowed to
reflux for 12 h at 70°C. After cooling to room temperature, the
reaction was quenched by adding 70 mL of saturated sodium
bicarbonate (NaHCO3) solution. The mixture was shaken
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M. Gangapuram et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Figure 3. Complete inhibition by compound 9i (26 mM) of iNOS protein expression in LPS (1 mg/mL)-activated BV-2 microglial cells at 24 h.
Cells were fixed, permeabilized, and stained with Alexa Fluor1 488/anti-iNOS, N-terminal antibody produced in rabbit and counterstained
with PI. A ¼ untreated cells, B ¼ LPS, C ¼ LPS þ 17.8 mM 9i, and D ¼ LPS þ 26.8 mM 9i.
repeatedly in a 500 mL separatory funnel and allowed to stand for
a few minutes. The solution was extracted with chloroform
(2 ꢃ 100 mL) and dried over anhydrous sodium sulfate, filtered,
and the solvent was removed in vacuo giving the crude product,
which was purified by column chromatography (2.5 cm ꢃ 22 cm)
on silica gel (200–425 mesh) using ethyl acetate/methanol (9:1 v/v)
as eluent. The resultant product 8a was obtained as a light yellow
crystalline solid in 43.9% yield, m.p. 150–151°C. The other
derivatives were similarly prepared and purified. 1H NMR (CDCl3):
d 5.20 (s, 2H, –CH2), 6.26 (t, 2H, J ¼ 5.7 Hz, 30H, C40H), 6.70 (t, 2H,
J ¼ 2.1 Hz, 20H, 50H), 7.39–7.44 (m, 4H, 300H, 400H, 500H, 5H), 7.61–
7.564 (dd, 1H, J ¼ 6.0, 1.8 Hz, 4H), 8.152–8.12 (m, 2H, 200H, 600H),
2.39 (s, 3H, –CH3), 5.22 (s, 2H, –CH2), 6.25 (t, 2H, J ¼ 1.8 Hz, 30H,
40H), 6.69 (t, 2H, J ¼ 1.8 Hz, 20H, 50H), 7.21 (d, 2H, J ¼ 8.1 Hz, 300H,
500H), 7.47 (d, 1H, J ¼ 7.8 Hz, 5H), 7.64 (t, 1H, J ¼ 6.6 Hz, 4H), 8.04 (d,
2H, J ¼ 7.8 Hz, 200H, 600H), 8.69 (s, 1H, 2H), 8.73 (d, 1H, J ¼ 6.6 Hz,
6H). IR (KBr): (n) 1590 (C O) cmꢀ1. Anal. calcd. for C18H17N3O ·
–
–
0.05H2O: C, 73.76; H, 5.93; N, 14.08. Found: C, 73.98; H, 5.86; N,
14.08.
Synthesis of 1-[(4-ethylbenzoyl)imino]-3-(1H-pyrrol-1-
ylmethyl)pyridinium ylide (8c)
The compound 8c was obtained following General Procedure B as
white solid, yield 52.5%, m.p. 212–213°C; 1H NMR (CDCl3): d 1.25
(t, 3H, J ¼ 7.5 Hz, –CH2–CH3), 2.69 (q, 2H, J ¼ 7.8, 7.5 Hz, –CH2–CH3),
5.19 (s, 2H, –CH2), 6.25 (t, 2H, J ¼ 2.1 Hz, 30H, 40H), 6.69 (t, 2H,
J ¼ 2.1 Hz, 20H, 50H), 7.22–7.25 (dd, 2H, J ¼ 6.6, 0.6 Hz, 300H, 500H),
7.39–7.42 (dd, 1H, J ¼ 5.4, 0.6 Hz, 5H), 7.54–7.59 (dd, 1H, J ¼ 6.6,
1.5 Hz, 4H), 8.04–8.06 (dd, 2H, J ¼ 4.2, 2.1 Hz, 200H, 600H), 8.68 (s, 1H,
–
8.68 (s, 1H, 2H), 8.72 (d, 1H, J ¼ 6.0 Hz, 6H). IR (KBr): (n) 1593 (C O)
–
cmꢀ1. Anal. calcd. for C17H15N3O · 0.28H2O: C, 72.60; H, 5.13; N,
14.51. Found: C, 72.31; H, 5.35; N, 14.88.
Synthesis of 1-[(4-methylbenzoyl)imino]-3-(1H-pyrrol-1-
2H), 8.72 (d, 1H, J ¼ 5.4 Hz, 6H). IR (KBr): (n) 1591 (C O) cmꢀ1. Anal.
–
ylmethyl)pyridinium ylide (8b)
–
calcd. for C19H19N3O · 0.15H2O: C, 74.10; H, 5.98; N, 13.55. Found:
C, 74.07; H, 6.22; N, 13.64.
The compound 8b was obtained following General Procedure B as
off-white solid, yield 50.5%, m.p. 188–190°C; 1H NMR (CDCl3): d
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Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Sulfonamides as Anti-Inflammatory Agents
365
140%
Synthesis of 1-[(4-butylbenzoyl)imino]-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8e)
9i
LPS + GM258
Cells
LPS
120%
100%
80%
60%
40%
20%
0%
The compound 8e was obtained following General Procedure B as
off-white color solid, yield 57.3%, m.p. 197–198°C; 1H NMR
(CDCl3): d 0.92 (t, 3H, J ¼ 7.2 Hz, –CH2–CH2–CH2–CH3), 1.36 (q, 2H,
J ¼ 7.8 Hz, –CH2–CH2–CH2–CH3), 1.62 (q, 2H, J ¼ 5.1 Hz,
–CH2–CH2–CH2–CH3), 2.65 (t, 2H, J ¼ 7.8, Hz, –CH2–CH2–CH2–CH3),
5.19 (s, 2H, –CH2), 6.25 (t, 2H, J ¼ 2.1 Hz, 30H, 40H), 6.69 (t, 2H,
J ¼ 2.1 Hz, 20H, 50H), 7.21 (d, 2H, J ¼ 8.1 Hz, 300H, 500H), 7.41 (d, 1H,
J ¼ 7.8 Hz, 5H) 7.54–7.59 (dd, 1H, J ¼ 6.6, 1.5 Hz, 4H), 8.02–8.05 (dd,
2H, J ¼ 4.8, 1.8 Hz, 200H, 600H), 8.68 (s, 1H, 2H), 8.71 (d, 1H,
*
*
*
J ¼ 6.3 Hz, 6H). IR (KBr): (n) 1592 (C O) cmꢀ1. Anal. calcd. for
–
–
C21H23N3O: C, 75.65; H, 6.95; N, 12.60. Found: C, 75.56; H, 6.90; N,
12.40.
IL1a
IL 6
IL10
TNF
G-CSF
Synthesis of 1-[(4-fluorobenzoyl)imino]-3-
Figure 4. Effect of SO2-substituted tetrahydropyridine 9i (26 mM) on (1H-pyrrol-1-ylmethyl)pyridinium ylide (8f)
cytokine release in LPS (1 mg/mL)-stimulated BV-2 microglial cells
at 24 h by multi-analyte ELISArray: MEM-004A – evaluated for: IL-
1a, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17a, IFNg, TNFa, G-CSF,
and GM-CSF. The data represent cytokines released as % LPS
controls for each subset, and are presented as the mean ꢁ STD,
n ¼ 2. Statistical differences from the LPS control were evaluated by
a Student’s t-test, ꢂp < 0.05.
The compound 8f was obtained following General Procedure B as
light yellow color solid, yield 46.8%, m.p. 197–198°C; d 5.21 (s, 2H,
–CH2), 6.26 (s, 2H, 30H, 40H), 6.70 (s, 2H, 20H, 50H), 7.07 (t, 2H,
J ¼ 8.4 Hz, 300H, 500H), 7.45 (d, 1H, J ¼ 8.1 Hz, 5H), 7.61 (t, 1H,
J ¼ 7.8 Hz, 4H), 8.14 (t, 2H, J ¼ 8.1 Hz, 200H, 600H), 8.66 (s, 1H, 2H),
8.71 (d, 1H, J ¼ 5.7 Hz, 6H). IR (KBr): (n) 1604 (C O) cmꢀ1. Anal.
–
–
calcd. for C17H14FN3O: C, 69.14; H, 4.78; N, 14.23. Found: C, 68.95;
H, 4.48; N, 13.88.
Synthesis of 1-[(4-methoxybenzoyl)imino]-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8d)
Synthesis of 1-[(4-chlorobenzoyl)imino]-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8g)
The compound 8d was obtained following General Procedure B as
light yellow solid, yield 48.5%, m.p. 175–177°C; 1H NMR (CDCl3):
d 3.84 (s, 3H, –OCH3), 5.23 (s, 2H, –CH2), 6.26 (t, 2H, J ¼ 1.8 Hz, 30H,
40H), 6.70 (t, 2H, J ¼ 1.8 Hz, 20H, 50H), 6.93 (d, 2H, J ¼ 8.4 Hz, 300H,
500H), 7.49 (d, 1H, J ¼ 8.1 Hz, 5H), 7.65 (t, 1H, J ¼ 6.3 Hz, 4H), 8.13 (d,
2H, J ¼ 8.7 Hz, 200H, 600H), 8.70 (s, 1H, 2H), 8.75 (d, 1H, J ¼ 6.0 Hz,
6H). IR (KBr): (n) 1605 (C¼O) cmꢀ1. Anal. calcd. for C18H17N3O2 ·
0.45H2O: C, 68.87; H, 5.44; N, 13.28. Found: C, 68.53; H, 5.43; N,
13.32.
The compound 8g was obtained following General Procedure B as
light yellow color solid, yield 51.5%, m.p. 197–198°C; d 5.21 (s, 2H,
–CH2), 6.26 (t, 2H, J ¼ 3.0 Hz, 30H, 40H), 6.70 (s, 2H, J ¼ 2.1 Hz, 20H,
50H), 7.37 (d, 2H, J ¼ 8.7 Hz, 300H, 500H), 7.45 (d, 1H, J ¼ 7.5 Hz, 5H),
7.61 (t, 1H, J ¼ 6.3 Hz, 4H), 8.07 (d, 2H, J ¼ 7.8 Hz, 200H, 600H), 8.66ꢀ(s1,
–
1H, 2H), 8.71 (d, 1H, J ¼ 6.0 Hz, 6H). IR (KBr): (n) 1603 (C O) cm
.
–
Anal. calcd. for C17H14ClN3O: C, 65.49; H, 4.53; N, 13.48. Found: C,
65.12; H, 4.25; N, 13.19.
A
B
1600
40
35
30
25
20
15
10
5
1400
1200
1000
800
600
400
200
0
*
*
0
Cells
LPS
Cells
LPS
LPS + 9i
LPS + 9i
Figure 5. (A) Effect of SO2-substituted tetrahydropyridine compound 9i (26 mM) on interleukin-1a release in LPS (1 mg/mL)-stimulated BV-2
microglial cells at 24 h. The data represent interleukin-1a released as (pg/mL) and presented as the mean ꢁ SEM, n ¼ 6. Statistical
differences from the LPS control were evaluated by a Student’s t-test, ꢂp < 0.001. (B) Effect of SO2-substituted tetrahydropyridine compound
9i (26 mM) on interleukin-6 release in LPS (1 mg/mL)-stimulated BV-2 microglial cells at 24 h. The data represent interleukin-6 released as
(pg/mL) and presented as the mean ꢁ SEM, n ¼ 6. Statistical differences from the LPS control were evaluated by a Student’s t-test,
ꢂp < 0.001.
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366
M. Gangapuram et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Synthesis of 1-[(4-bromobenzoyl)imino]-3-
General procedure C
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8h)
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
The compound 8h was obtained following General Procedure B as
brown color solid, yield 56.9%, m.p. 197–198°C; d 5.23 (s, 2H,
–CH2), 6.27 (s, 2H, 30H, 40H), 6.70 (s, 2H, 20H, 5H), 7.47 (d, 2H,
J ¼ 8.7 Hz, 300H, C500-H), 7.54 (d, 1H, J ¼ 7.8 Hz, 5H), 7.64 (t, 1H,
J ¼ 8.7 Hz, 4H), 8.02 (d, 2H, J ¼ 8.1 Hz, 200H, 600H), 8.67 (s, 1H, 2H),
tetrahydropyridin-1-yl]benzamide (9a)
A
solution of 1-(benzoylimino)-3-(1H-pyrrol-1-ylmethyl)pyridi-
nium ylide (8a 0.80 g, 2.55 mmol) in dichloromethane/ethanol
(1:1 v/v, 40 mL) was added dropwise to a stirred suspension of
sodium borohydride (0.38 g, 10.21 mmol) in 20 mL of absolute
ethanol over a period or 30 min. The resulting solution was
stirred for 7 h at 0°C. The excess sodium borohydride was treated
with 50 mL of distilled water and the reaction mixture was
allowed to warm up to room temperature. It was then extracted
with dichloromethane (200 mL) and dried over anhydrous
sodium sulfate. The dichloromethane filtrate was evaporated in
vacuo and the product chromatographed on a column of silica gel
using ethyl acetate/hexane (3:2 v/v) as an eluent. The solid
obtained was further crystallized from dichlormethane/hexane
(3:2 v/v) and furnished 9a as a white solid, 48.9% yield, m.p. 149–
150°C; 1H NMR (CDCl3): d 2.42 (brs, 2H, 5H), 3.28 (t, 2H, J ¼ 5.1 Hz,
6H), 3.51 (s, 2H, 2H), 4.41 (s, 2H, –CH2), 5.68 (m, 1H, 4H, olefinic),
6.14 (t, 2H, J ¼ 1.8 Hz, 30H, 40H), 6.61 (t, 2H, J ¼ 2.1 Hz, 20H, 50H),
6.93 (brs, –NH, D2O exchangeable), 7.38–7.52 (m, 3H, 300H, 400H,
500H), 7.73 (d, 2H, J ¼ 7.2 Hz, 200H, 600H). IR (KBr): (n) 3213 (NH), 1638
8.74 (d, 1H, J ¼ 6.6 Hz, 6H). IR (KBr): (n) 1601 (C O) cmꢀ1. Anal.
–
–
calcd. for C17H14BrN3O: C, 57.32; H, 3.96; N, 11.80. Found: C,
56.95; H, 3.91; N, 11.62.
Synthesis of 1-[(phenylsulfonyl)imino]-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8i)
The compound 8i was obtained following General Procedure B as
1
light yellow crystalline solid in 43.9% yield, m.p. 140–142°C; H
NMR (CDCl3): d 5.20 (s, 2H, –CH2), 6.26 (t, 2H, J ¼ 5.7 Hz, 30H, 40H),
6.70 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 7.34–7.44 (m, 4H, 300H, 400H, 500H,
5H), 7.49–7.50 (dd, 1H, J ¼ 1.8, 1.2 Hz, 4H), 7.66–7.69 (m, 2H, 200H,
600H), 8.16 (s, 1H, 2H), 8.34 (d, 1H, J ¼ 3.0 Hz, 6H). IR (KBr): (n) 1332,
1205 (SO2) cmꢀ1. Anal. calcd. for C16H15N3O2S: C, 61.32; H, 4.82; N,
13.41. Found: C, 61.21; H, 4.74; N, 13.16.
(C O) cmꢀ1; HRMS (EI) m/z [Mþ] calcd. for C17H19N3O, 281.1528;
–
–
Synthesis of 1-([(4-methylphenyl)sulfonyl]imino)-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8j)
found 281.1525. Anal. calcd. for C17H19N3O · 0.05H2O: C, 72.13; H,
6.51; N, 14.85. Found: C, 72.34; H, 6.79; N, 14.89.
The compound 8j was obtained following General Procedure B as
off-white solid in 63.7% yield, m.p. 168–169°C; 1H NMR (CDCl3): d
2.38 (S, 3H, –CH3), 5.11 (s, 2H, –CH2), 6.22 (t, 2H, J ¼ 1.8 Hz, 30H,
40H), 6.56 (t, 2H, J ¼ 1.8 Hz, 20H, 50H), 7.15–7.18 (dd, 2H, J ¼ 8.1,
0.6Hz, 300H, 500H), 7.46–7.48 (dd, 2H, J ¼ 5.2, 1.2 Hz, 200H, 600H), 7.57–
7.60 (dd, 2H, J ¼ 4.8, 1.5 Hz, 5H, 4H), 8.16 (s, 1H, 2H), 8.34 (d, 1H,
J ¼ 2.7 Hz, 6H). IR (KBr): (n) 1286, 1134 (SO2) cmꢀ1. Anal. calcd. for
C17H17N3O2S: C, 62.36; H, 5.23; N, 12.83. Found: C, 62.12; H, 5.13;
N, 12.66.
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-methylbenzamide (9b)
The compound 9b was obtained following General Procedure C as
white color solid, yield 54.5%, m.p. 145–147°C; 1H NMR (CDCl3): d
2.38 (s, 3H, –CH3), 2.35 (brs, 2H, 5H), 3.08 (t, 2H, J ¼ 5.1 Hz, 6H),
3.38 (s, 2H, 2H), 4.39 (s, 2H, –CH2), 5.64 (m, 1H, 4H, olefinic), 6.14 (t,
2H, J ¼ 1.8 Hz, 30H, 40H), 6.62 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.93 (brs,
–NH, D2O exchangeable), 7.23 (d, 2H, J ¼ 8.1 Hz, 300H, 500H), 7.61 (d,
ꢀ1
00
00
–
2H, J ¼ 8.1 Hz, 2 H, 6 H). IR (KBr): (n) 3202 (NH), 1634 (C O) cm
.
–
Synthesis of 1-([(4-methoxyphenyl)sulfonyl]imino)-3-
(1H-pyrrol-1-ylmethyl)pyridinium ylide (8k)
Anal. calcd. for C18H21N3O: C, 73.19; H, 7.17; N, 14.23. Found: C,
73.12; H, 7.40; N, 14.09.
The compound 8k was obtained following General Procedure B as
off-white solid in 60.0% yield, m.p. 153–154°C; 1H NMR (CDCl3): d
3.81 (s, 3H, –OCH3), 5.15 (s, 2H, –CH2), 6.22 (t, 2H, J ¼ 2.4 Hz, 30H,
40H), 6.56 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.83–6.86 (dd, 2H, J ¼ 4.5,
2.1 Hz, 300H, 500H), 7.46–7.48 (dd, 2H, J ¼ 3.9, 0.6 Hz, 200H, 600H),
7.62–7.65 (dd, 2H, J ¼ 4.5, 2.1 Hz, 5H, 4H), 8.18 (s, 1H, 2H), 8.35 (d,
1H, J ¼ 3.0 Hz, 6H). IR (KBr): (n) 1280, 1137 (SO2) cmꢀ1. Anal. calcd.
for C17H17N3O3S · 0.05H2O: C, 59.34; H, 4.86; N, 11.97. Found: C,
59.30; H, 4.98; N, 12.20.
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-ethylbenzamide (9c)
The compound 9c was obtained following General Procedure C as
off-white color solid, yield 61.8%, m.p. 143–145°C; 1H NMR
(CDCl3): d 1.23 (t, 3H, J ¼ 7.8 Hz, –CH2–CH3), 2.35 (brs, 2H, 5H), 2.68
(q, 2H, J ¼ 7.5 Hz, –CH2–CH3), 3.08 (t, 2H, J ¼ 5.1 Hz, 6H), 3.38 (s, 2H,
2H), 4.39 (s, 2H, –CH2), 5.64 (m, 1H, 4H, olefinic), 6.14 (t, 2H,
J ¼ 1.8 Hz, 30H, 40H), 6.62 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.93 (brs, –NH,
D2O exchangeable), 7.23 (d, 2H, J ¼ 8.1 Hz, 300H, 500H), 7.61 (d, 2H,
ꢀ1
00
00
–
J ¼ 8.1 Hz, 2 H, 6 H). IR (KBr): (n) 3183 (NH), 1625 (C O) cm
.
–
Synthesis of 1-(([3-(2-chloro-6-fluorophenyl)-5-methyl-1,2-
oxazol-4-yl]carbonyl)imino)-3-(1H-pyrrol-1-ylmethyl)
pyridinium ylide (8l)
Anal. calcd. for C19H23N3O: C, 73.76; H, 7.49; N, 13.58. Found: C,
73.61; H, 7.52; N, 13.36.
The compound 8l was obtained following General Procedure B as
off white color solid, yield 46.3%, m.p. 149–150°C; 1H NMR
(CDCl3): d 2.81 (s, 3H, –CH3), 5.15 (s, 2H, –CH2), 6.26 (t, 2H,
J ¼ 2.1 Hz, 30H, 40H), 6.66 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 7.03–7.09
(ddd, 1H, J ¼ 8.1, 1.2, 1.5, 0.9 Hz, 300H), 7.23–7.35 (m, 3H, 5H,40’H,
500H), 7.56 (t, 1H, J ¼ 6.6 Hz, 4H), 8.37 (s, 1H, 2H), 8.47 (d, 1H,
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-methoxybenzamide (9d)
The compound 9d was obtained following General Procedure C as
off-white color solid, yield 57.6%, m.p. 151–152°C; 1H NMR
(CDCl3): d 3.81 (s, 3H, –OCH3), 2.36 (brs, 2H, 5H), 3.06 (t, 2H,
J ¼ 5.1 Hz, 6H), 3.34 (s, 2H, 2H), 4.38 (s, 2H, –CH2), 5.62 (m, 1H, 4H,
olefinic), 6.15 (t, 2H, J ¼ 1.8 Hz, 30H, 40H), 6.63 (t, 2H, J ¼ 2.1 Hz, 20H,
50H), 6.91 (brs, –NH, D2O exchangeable), 7.24 (d, 2H, J ¼ 8.1 Hz,
J ¼ 6.0 Hz, 6H). IR (KBr): (n) 1608 (C O) cmꢀ1. Anal. calcd. for
–
–
C21H16ClFN4O2: C, 61.39; H, 3.93; N, 13.64. Found: C, 61.08; H,
3.94; N, 13.28.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
Sulfonamides as Anti-Inflammatory Agents
367
300H, 500H), 7.60 (d, 2H, J ¼ 8.1 Hz, 200H, 600H). IR (KBr): (n) 3193 (NH),
2H, 2H), 4.24 (s, 2H, –CH2), 5.45 (brs, –NH, D2O exchangeable), 5.57
(m, 1H, 4H, olefinic), 6.12 (t, 2H, J ¼ 2.1 Hz, 30H, 40H), 6.50 (t, 2H,
J ¼ 2.1 Hz, 20H, 50H), 7.44–7.49 (m, 3H, 300H, 400H,500H), 7.84 (d, 2H,
J ¼ 8.1 Hz, 200H, 600H). IR (KBr): (n) 3069 (NH), and 1332, 1164 (SO2)
cmꢀ1. Anal. calcd. for C16H19N3O2S: C, 60.54; H, 6.03; N, 13.24.
Found: C, 60.32; H, 5.89; N, 12.98.
1615 (C O) cmꢀ1. Anal. calcd. for C18H21N3O2: C, 69.43; H, 6.80; N,
–
–
13.49. Found: C, 69.28; H, 7.01; N, 13.31.
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-butylbenzamide (9e)
The compound 9e was obtained following General Procedure C as
white color solid, yield 56.3%, m.p. 148–149°C; 1H NMR (CDCl3): d
0.92 (t, 3H, J ¼ 7.2 Hz, –CH2–CH2–CH2–CH3), 1.36 (q, 2H, J ¼ 7.5 Hz,
–CH2–CH2–CH2–CH3), 1.60 (q, 2H, J ¼ 5.1 Hz, –CH2–CH2–CH2–CH3),
2.35 (brs, 2H, 5H), 2.63 (t, 2H, J ¼ 8.1 Hz, –CH2–CH2–CH2–CH3), 3.08
(t, 2H, J ¼ 5.4 Hz, 6H), 3.32 (s, 2H, 2H), 4.39 (s, 2H, –CH2), 5.64 (m,
1H, 4H, olefinic), 6.14 (t, 2H, J ¼ 1.8 Hz, 30H, 40H), 6.62 (t, 2H,
J ¼ 2.1 Hz, 20H, 5H), 6.92 (brs, –NH, D2O exchangeable), 7.23 (d, 2H,
J ¼ 8.4 Hz, 300H, 500H), 7.60 (d, 2H, J ¼ 8.1 Hz, 200H, 600H). IR (KBr): (n)
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-methylbenzenesulfonamide (9j)
The compound 9j was obtained following General Procedure C as
white color flakes, yield 58.5%, m.p. 122–124°C; 1H NMR (CDCl3): d
2.14–2.15 (m, 2H, 5H), 2.73 (t, 2H, J ¼ 6.0 Hz, 6H), 2.91 (s, 2H, 2H),
3.87 (s, 3H, –OCH3), 4.25 (s, 2H, –CH2), 5.40 (brs, –NH, D2O
exchangeable), 5.57 (m, 1H, 4H, olefinic), 6.12 (t, 2H, J ¼ 2.4 Hz,
30H, 40H), 6.50 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.93 (d, 2H, J ¼ 9.0 Hz,
300H, 500H), 7.79 (d, 2H, J ¼ 9.0 Hz, 200H, 600H). IR (KBr): (n) 3065 (NH),
and 1333, 1165 (SO2) cmꢀ1. Anal. calcd. for C17H21N3O2S: C, 61.61;
H, 6.39; N, 12.68. Found: C, 61.48; H, 6.31; N, 12.57.
3192 (NH), 1635 (C O) cmꢀ1. Anal. calcd. for C21H27N3O: C, 74.74;
–
–
H, 8.06; N, 12.45. Found: C, 74.70; H, 8.07; N, 12.26.
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-fluorobenzamide (9f)
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-methoxybenzenesulfonamide
(9k)
The compound 9f was obtained following General Procedure C as
off-white color solid, yield 48.6%, m.p. 152–153°C; 1H NMR
(CDCl3): d 2.36 (brs, 2H, 5H), 3.08 (t, 2H, J ¼ 5.1 Hz, 6H), 3.31 (s, 2H,
2H), 4.39 (s, 2H, –CH2), 5.67 (m, 1H, 4H, olefinic), 6.14 (t, 2H,
J ¼ 1.8 Hz, 30H, 40H), 6.61 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.91 (brs, –NH,
D2O exchangeable), 7.14 (t, 2H, J ¼ 8.4 Hz, 300H, 500H), 7.87 (dd, 2H,
The compound 9k was obtained following General Procedure C as
off-white color solid, yield 65.5%, m.p. 132–134°C; 1H NMR
(CDCl3): d 2.13–2.15 (m, 2H, 5H), 2.42 (s, 3H, –CH3), 2.72 (t, 2H,
J ¼ 6.0 Hz, 6H), 2.92 (s, 2H, 2H), 4.25 (s, 2H, –CH2), 5.37 (brs, –NH,
D2O exchangeable), 5.56 (m, 1H, 4H, olefinic), 6.12 (t, 2H,
J ¼ 2.1 Hz, 30H, 40H), 6.50 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 7.24 (d, 2H,
J ¼ 9.0 Hz, 300H, 500H), 7.74 (d, 2H, J ¼ 8.7 Hz, 200H, 600H). IR (KBr): (n)
ꢀ1
00
00
–
J ¼ 2.1, 8.1 Hz, 2 H, 6 H). IR (KBr): (n) 3190 (NH), 1636 (C O) cm
.
–
Anal. calcd. for C17H18FN3O: C, 68.21; H, 6.06; N, 14.04. Found: C,
67.96; H, 6.01; N, 13.91.
3078 (NH), and 1331, 1163 (SO2) cmꢀ1
. Anal. calcd. for
C
17H21N3O3S: C, 58.77; H, 6.09; N, 12.09. Found: C, 58.57; H,
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-chlorobenzamide (9g)
5.98; N, 11.95.
The compound 9g was obtained following General Procedure C as
off-white color crystalline solid, yield 52.8%, m.p. 147–149°C; 1H
NMR (CDCl3): d 2.54 (brs, 2H, 5H), 3.59 (t, 2H, J ¼ 5.1 Hz, 6H), 3.83 (s,
2H, 2H), 4.43 (s, 2H, –CH2), 5.71 (s, 1H, 4H, olefinic), 6.14 (t, 2H,
J ¼ 2.1 Hz, 30H, 40H), 6.60 (t, 2H, J ¼ 1.8 Hz, 20H, 50H), 6.95 (brs, –NH,
D2O exchangeable), 7.36 (t, 2H, J ¼ 6.9 Hz, 300H, 500H), 7.75 (d, 2H,
Synthesis of 3-(2-chloro-6-fluorophenyl)-5-methyl-N-[3-
((1H-pyrrol-1-yl)methyl)-1,2,5,6-tetrahydropyridin-1-yl]-
1,2-oxazole-4-carboxamide (9l)
The compound 9l was obtained following General Procedure C as
off-white color solid, yield 56.2%, m.p. 188–189°C; 1H NMR
(CDCl3): d 2.10 (brs, 2H, 5H), 2.77 (s, 3H, –CH3), 2.80 (t, 2H,
J ¼ 5.7 Hz, 6H), 3.02 (s, 2H, 2H), 4.29 (s, 2H, –CH2), 5.46 (m, 1H, 4H,
olefinic), 6.14 (t, 2H, J ¼ 1.8 Hz, 30H, 4H), 6.17 (brs, –NH, D2O
exchangeable), 6.55 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 7.17 (t, 1H,
J ¼ 8.4 Hz, 300H), 7.35 (d, 1H, J ¼ 7.5 Hz, 500H), 7.46 (t, 1H, J ¼ 5.7 Hz,
ꢀ1
00
00
–
J ¼ 8.4 Hz, 2 H, 6 H). IR (KBr): (n) 3187 (NH), 1634 (C O) cm
.
–
Anal. calcd. for C17H18ClN3O: C, 64.66; H, 5.75; N, 13.31. Found: C,
64.51; H, 5.69; N, 13.21.
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]-4-bromobenzamide (9h)
4 H). IR (KBr): (n) 3185 (NH), 1634 (C O) cmꢀ1; HRMS (EI) m/z [Mþ]
00
–
–
calcd. for C21H20ClFN4O2 414.1259; found 414.1252. Anal. calcd.
for C21H20ClFN4O2 · 0.35H2O: C, 59.90; H, 4.70; N, 13.02. Found: C,
59.89; H, 4.79; N, 13.02.
The compound 9h was obtained following General Procedure C as
light yellow color solid, yield 60.8%, m.p. 154–155°C; 1H NMR
(CDCl3): d 2.70 (brs, 2H, 5H), 4.01 (t, 2H, J ¼ 5.1 Hz, 6H), 4.23 (s, 2H,
2H), 4.49 (s, 2H, –CH2), 5.76 (m, 1H, 4H, olefinic), 6.15 (t, 2H,
J ¼ 1.8 Hz, 30H, 40H), 6.62 (t, 2H, J ¼ 2.1 Hz, 20H, 50H), 6.91 (brs, –NH,
D2O exchangeable), 7.54 (d, 2H, J ¼ 8.7 Hz, 300H, 500H), 7.75 (d, 2H,
Biological studies
ꢀ1
00
00
–
J ¼ 8.4 Hz, 2 H, 6 H). IR (KBr): (n) 3185 (NH), 1635 (C O) cm
.
–
Cell culture
Anal. calcd. for C17H18BrN3O: C, 56.68; H, 5.04; N, 11.66. Found: C,
56.50; H, 4.95; N, 11.53.
BV-2 microglial cells were kindly provided by Blasi et al. [50]
and cultured in high glucose (4500 mg/mL) DMEM containing
phenol red, 5% FBS, 4 mM L-glutamine, and penicillin/
streptomycin (100 U/0.1 mg/mL). Culture conditions were
maintained (37°C in 5% CO2/atmosphere) and every 2–5 days,
the media was replaced and cells sub-cultured. For experi-
ments, plating media consisted of DMEM (minus phenol red),
Synthesis of N-[3-((1H-pyrrol-1-yl)methyl)-1,2,5,6-
tetrahydropyridin-1-yl]benzenesulfonamide (9i)
The compound 9i was obtained following General Procedure C as
light yellow color solid, yield 54.2%, m.p. 125–127°C; 1H NMR
(CDCl3): d 2.13–2.14 (m, 2H, 5H), 2.72 (t, 2H, J ¼ 6.0 Hz, 6H), 2.91 (s,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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368
M. Gangapuram et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 360–369
5% FBS, penicillin/streptomycin (100 U/0.1 mg/mL) and 3 mM
L-glutamine. All experimental compounds were dissolved in
DMSO (20 mg/mL) and dilutions were prepared in sterile
HBSS þ 5 mM HEPES, adjusted to a pH of 7.4. Activation of BV-
2 cells was established using 1 mg/mL of LPS from Escherichia
coli O111:B4 for 24 h in experimental media, maintaining cell
density at 0.5 ꢃ 106 cells/mL.
sciences, Qiagen, Inc., Valencia, CA), which simultaneously
quantifies 12 pro-inflammatory cytokines including IL-1a, IL-
1b, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17a, IFNg, TNFa, G-CSF, and
GM-CSF. Very briefly, cell supernatants were removed after
24 h and stored at ꢀ20°C. Supernatant from each sample was
thawed and incubated with capture antibodies for 2 h at RT
along with a mixed antigen standard cocktail. After washing,
samples were then re-incubated with biotinylated detection
antibodies for 1 h at RT. After a 2nd washing process, samples
were incubated with avidin–horseradish peroxidase conju-
gate for 30 min, re-washed, and incubated with a development
reagent. After addition of the stop solution, OD was evaluated
on a spectrophotometer at 450 nm with a wavelength
correction at 570 nm. Data was evaluated as % cytokines
production and released of the LPS control.
Cell viability
Cell viability was quantified using resazurin (alamar blue)
indicator dye [51]. A working solution of resazurin was prepared
in sterile phosphate buffered saline (PBS)–phenol red (0.5 mg/
mL) and added (15% v/v) to each sample. Samples were returned
to the incubator for 6–8 h, and reduction of the dye by viable
cells (to resorufin, a fluorescent compound) was quantitatively
analyzed using a microplate fluorometer, model 7620, version
5.02 (Cambridge Technologies, Inc., Watertown, MA) with
settings at 550/580 (excitation/emission) wavelengths. The data
were expressed as percent of live untreated controls.
IL-1a and IL-6
OmniKineTM Murine IL-6 (Catalog # OK-0187) and Murine IL-
1a (Catalog # OK-0181) quantitative “Sandwich” ELISA
(Assay Biotechnology Company, Inc., Sunnyvale, CA) were
used for detection and quantification of IL-6 and IL-1a
produced/released within the range of 62–4000 pg/mL.
Experiments were performed according to the manufac-
turer’s guidelines (Assay Biotech) and quantified using a
spectrophotometer at 450 nm (with correction wavelength
set to 570 nm).
ꢀ
NO2 determination
Quantification of nitrite (NO2ꢀ) was determined by a colorimet-
ric method using the Griess reagent. The Griess reagent was
prepared by mixing an equal volume of 1.0% sulfanilamide in
0.5 N HCl and 0.1% N-(1-naphthyl)-ethylenediamine in deionized
water. The Griess reagent was added directly to the cell
supernatant suspension and incubated under reduced light at
ꢀ
Data analysis
room temperature for 10 min. A standard curve for NO2 was
Statistical analysis was performed using Graph Pad Prism
(version 3.0; Graph Pad Software, Inc., San Diego, CA, USA)
with significance of difference between the groups assessed
using a one-way ANOVA, followed by Tukey post hoc means
comparison test, or Student’s t-test. IC50s were determined by
regression analysis using Origin Software (OriginLab, North-
ampton, MA).
generated from dilutions of sodium nitrite (NaNO2) (1–100 mM)
prepared in plating medium. Controls and blanks were run
simultaneously and subtracted from the final value to eliminate
interference. Samples were analyzed at 550 nm on a UV
microplate spectrophotometer (model 7600, version 5.02,
Cambridge Technologies, Inc.).
iNOS protein expression
This research was supported by the National Center for Research
Resources and the National Institute of Minority Health and Health
Disparities of the National Institutes of Health through Grant No. 8
G12MD007582-28 and the National Institute on Minority Health and
Health Disparities, NIH (1P20 MD006738-01). We would also like to
thank Dr. Equar Taka for providing the BV-2 microglial cell line, which
was received from E. Basli (Department of Experimental Medicine and
Biochemical Sciences, University of Perugia, Italy).
After 24 h treatment, cells were fixed in 4% paraformalde-
hyde/permeabilized in 0.1% Triton X-100 in PBS and incubated
with anti-iNOS, N-terminal antibody produced in rabbit
(Sigma–Aldrich) for 2 h at 37°C. Samples were washed in PBS
and subsequently incubated with anti-rabbit Alexa Fluor1
488 conjugate for 2 h at 37°C. Samples were counterstained
with propidium iodide and photographically observed using a
Nikon TE 900 inverted microscope and a Nikon PCM 2000
confocal microscope. Data acquisition was done using C-
imaging systems confocal PCI-Simple software (Compix, Inc.,
Cranberry Township, PA, USA).
The authors have declared no conflict of interest.
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