Design, synthesis and biological evaluation of piperazino-enaminones as novel suppressants of pro-Inflammatory cytokines

Article abstract of DOI:10.1016/j.bmc.2018.06.003

Infection triggers the release of pro-inflammatory cytokines (TNF-alpha and IL-6). Over-production, however, cause tissue injury seen in severe asthma. The ability of enaminone E121 to reduce pro-inflammatory cytokines in our laboratory encouraged further examination of its structural scaffold. Piperazino-enaminones were designed by incorporating n-arylpiperazine motif into the aromatic enaminone. Four possible modifications were explored systematically. Synthesis was accomplished by amination of the corresponding methyl/ethyl 2,4-dioxo-6-(substituted)cyclohexane-carboxylate. Sixteen novel compounds were synthesized. Biological activity was tested in J774 macrophages stimulated with lipopolysaccharides. The release of cytokines was measured via ELISA. Four compounds significantly suppressed TNF-alpha and IL-6 release in dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2018.06.003

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Piperazino-Enaminones as Nov-
el Suppressants of Pro-Inflammatory Cytokines
Ola M. Ghoneim, Ashley Bill, Jyothi Dhuguru, Doreen E. Szollosi, Ivan O.
Edafiogho
PII:
S0968-0896(18)30701-6
https://doi.org/10.1016/j.bmc.2018.06.003
BMC 14395
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 April 2018
25 May 2018
3 June 2018
Please cite this article as: Ghoneim, O.M., Bill, A., Dhuguru, J., Szollosi, D.E., Edafiogho, I.O., Design, Synthesis
and Biological Evaluation of Piperazino-Enaminones as Novel Suppressants of Pro-Inflammatory Cytokines,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.003
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Design, Synthesis and Biological Evaluation of
Piperazino-Enaminones as Novel Suppressants of
Pro-Inflammatory Cytokines
Ola M. Ghoneim, Ashley Bill, Jyothi Dhuguru, Doreen E. Szollosi, Ivan O. Edafiogho

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Design, Synthesis and Biological Evaluation of Piperazino-Enaminones as Novel
Suppressants of Pro-Inflammatory Cytokines
Ola M. Ghoneim , Ashley Bill , Jyothi Dhuguru , Doreen E. Szollosi , and Ivan O. Edafiogho
a*
a
a,b
a
a
a
Department of Pharmaceutical Sciences, School of Pharmacy, University of Saint Joseph, Hartford, CT 06103, USA
b
Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01655, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Infection triggers the release of pro-inflammatory cytokines (TNF-alpha and IL-6). Over-
production, however, cause tissue injury seen in severe asthma. The ability of enaminone E121 to
reduce pro-inflammatory cytokines in our laboratory encouraged further examination of its
structural scaffold. Piperazino-enaminones were designed by incorporating n-arylpiperazine motif
into the aromatic enaminone. Four possible modifications were explored systematically. Synthesis
was accomplished by amination of the corresponding methyl/ethyl 2,4-dioxo-6-
Received in revised form
Accepted
Available online
(
substituted)cyclohexane-carboxylate. . Sixteen novel compounds were synthesized. Biological
activity was tested in J774 macrophages stimulated with lipopolysaccharides. The release of
cytokines was measured via ELISA. Four compounds significantly suppressed TNF-alpha and
IL-6 release in dose-dependent manner.
Keywords:
Inflammation
Design
2
018 Elsevier Ltd. All rights reserved.
Synthesis
Pro-inflammatory
Cytokines
———
Corresponding author: Ola Ghoneim, Associate professor at University of Saint Joseph-School of Pharmacy, oghoneim@usj.edu
*
3

inflammatory E121 can play in the management of severe
asthma encouraged our research team to explore its unique
chemical structural scaffold and its involvement in
suppressinginflammation.
1
1
. Introduction
.1 Inflammation and pro-inflammatory cytokines
In severe asthma, the development of persistent airway
hyperresponsiveness results from the release of pro-
inflammatory cytokines from infiltrating immune cells,
including macrophages . Under normal conditions,
macrophages release pro-inflammatory cytokines such as
Tumor Necrosis Factor Alpha (TNF-alpha), Interleukin-6 (IL-
1
.3 JODI series
The rationale for designing the piperazino-enaminones (JODI
series) was based on adding N-alkylated piperazine to the
aromatic side of the enaminone scaffold. The N-alkylated
piperazine motif was reported to increase the chemokine
receptor (CCR2) antagonistic activity of other anti-
1
6
), Interleukin-1 (IL-1), and nitric oxide (NO) in the presence
of pathogen-associated products such as lipopolysaccharide
LPS) or cytokines such as interferon-gamma (IFN). While
inflammatory series and reduce the unwanted cardiac hERG
17, 18
activity by ten-fold.
(
In addition, the piperazino group has
these cytokines are important for the normal development of
an immune response, in the case of autoimmunity, they are
often overproduced and can lead to the development of tissue
been reported to be beneficial for the water solubility and
metabolic stability for several drug classes, for instance,
19
epoxide hydrolase inhibitors. Accordingly, we hypothesized
1-3
and organ injury
.
that the piperazino-enaminone series could potentially act
with dual functionality as a cytokine inhibitor and chemokine
receptor antagonist with better drug-like molecule properties.
Attaching the piperazine ring from the N-1 directly to the
existing aromatic ring will have a slight or no effect on the
While chronic asthma and airway hyperresponsiveness can
usually be managed with combination therapies of inhaled
4
,5
glucocorticoids
resistance can be a problem in severe asthma sufferers
and bronchodilators, glucocorticoid
6
-8
20
overall pK
a
or the basicity of the molecule. However,
Additionally, the list of possible adverse effects resulting from
glucocorticoid use is long, and can include immunodeficiency
and increased risk of infection, osteoporosis, hyperglycemia,
incorporating the N-alkylated piperazine will play a role in
2
attenuating the LogP of this series.
1
4
weight gain, fluid retention, and insomnia.
Compounds JODI-15 and -17 were first synthesized because
of their closeness in structure to E121. As seen from Figure 1,
E121 carries a methyl group as R , an ethyl ester as R , and a
1
.2 Enaminone E121
1
2
Previously synthesized in our laboratory, the enaminone E121
(Figure 1) represents the prototype for our series. The
chlorophenyl in the para position to the aniline ring amino
group as R . Both JODI-15 and -17 (Figure 1 and Table 1)
3
enaminone scaffold consists of a cyclic alkene substituted on
one side with a secondary amine group, and on the other side
carry methyl groups in their R area, methyl and ethyl ester
1
9
with a ketone in a fixed trans configuration. This particular
groups in their R
methylpiperazine in the ortho position as R
2
area respectively, and an added N-
. Both JODI-15
3
arrangement is unique for all enaminones, and accounts for
their abbreviated chemical name.
and -17 were able to reduce TNF-alpha and IL-6 released by
LPS- stimulated macrophages, but their effects were not as
efficacious when compared to E121. At the 1 mM
concentration, JODI-15 and -17 were able to inhibit TNF-
alpha release after LPS stimulation by approximately 47%
and 53%, respectively, compared to E121 inhibition of 70%
at the same concentration. Similarly, they were able to
inhibit IL-6 release after LPS stimulation by approximately
68% and 72%, respectively, compared to E121 inhibition of
We became very interested in the biological evaluation of
E121 because it has been shown to decrease inflammation via
the suppression of TNF-alpha and IL-10 release, as well as
other cytokines from LPS-activated human peripheral blood
1
0
mononuclear cells (PBMCs). Moreover, E121 has been
shown to decrease airway hyperresponsiveness and immune
cell infiltrate in an ovalbumin-induced mouse model of
1
0
14
allergic asthma. E121 has also been suggested to have anti-
96% at the same concentration. The fact that the added N-
1
1
tussive and bronchodilation activity. Furthermore, it has
been reported that E121 is capable of reducing the enhanced
tumor necrosis receptor-associated factor 2 (TRAF-2) in the
methylpiperazino group is in the ortho position to the aniline
ring might play a role in explaining their biological
responses. The bulkiness of the N-methylpiperazine group in
close proximity to the amine group (Figure 1) might have
cause steric hindrance, by which it limits the access of this
amino group to potential binding regions in the binding
pocket.
1
2
colon of dextran sulfate sodium treated mice. TRAF-2 acts
as an adaptor that transmits signals through the TNF receptor
13-14
superfamily, and IL-1.
This indicates that the anti-
inflammatory property of E121 may be in part mediated
through modulating the activity of pro-inflammatory mediator
signaling.
In the case of JODI-18 and JODI-19 (Figure 1 and table 1),
two factors were embedded at the same time. The R1 is now
a phenyl ring, which adds lipophilicity to the molecule and
increases the partition coefficient (LogP) to 4.02 and 4.39 as
compared to their methyl counterparts (JODI-15 and -17),
which were 2.65 and 3.02 respectively. Another major difference
is the position of the N-methylpiperazin-1-yl group. The
added N-methylpiperazine is now the farthest away (para
position) from the aniline amino group, mimicking the para
chloro position of the original E121 compound. Therefore,
In our laboratory, E121 was also able to suppress the release
of pro-inflammatory cytokines TNF-alpha and IL-6, and
moderately decreased total nitric oxide in an LPS-activated
1
4, 15
macrophage cell line.
common glucocorticoid), our preliminary studies using RU-
86 as a glucocorticoid receptor antagonist indicated that
E121 does not require the glucocorticoid receptor in order to
In comparison to dexamethasone (a
4
1
6
exert its anti-inflammatory effects. The role that the anti-
4

steric hindrance is likely not a factor here. The results from
the JODI-18 and -19 pair were encouraging, in the TNF-
alpha and IL-6 assays. At the 1mM concentration, JODI-18
and JODI-19 were able to inhibit TNF-alpha release after
LPS stimulation by approximately 80% and 100 %
respectively, compared to E121 inhibition of 70% at the same
corresponding halo-nitrobenzene in dimethylsulfoxide at
80°C followed by reducing the corresponding nitro analogs
using palladium catalyst and polymethylhydrosiloxane
(scheme 1).
Scheme 1: Synthesis of JODI and JOAB series
1
4
concentration. Similarly, JODI-18 and -19 were able to
inhibit IL-6 release after LPS stimulation by approximately
7
E121 inhibition of only 20 % at the same concentration.
0% and 98% respectively at only 0.25 mM compared to
1
4
A
similar trend was observed when compared to
dexamethasone. At 1 mM, JODI-18 and -19 were able to
inhibit the TNF-alpha release after LPS stimulation by 82%
and 100 % respectively, compared to dexamethasone
inhibition of 62% at the same concentration. The inhibition
of IL-6 release was even more encouraging with 98% and
1
00% inhibition by JODI-18 and -19 respectively at only 0.5
mM compared to dexamethasone inhibition of 40% at the
same concentration. It is hypothesized that the added
aromaticity in both JODI-18 and -19 might play a role in
adding a possible Van der Waals bond with potential aromatic
amino acids at the binding pocket of their target. Moreover,
the added lipophilicity of the JODI-19 compound might also
play a significant role in explaining its superior mechanism of
action among the other members of the JODIfamily.
The aniline was then refluxed in ethanol and/or isopropanol
for 6-24 h with the corresponding beta diketones; E115,
E164, E201, and E203 (previously synthesized in our
22,23
laboratory by Dr. Edafiogho).
All reactions were
monitored by thin layer chromatography using
dichloromethane: methanol 90/10. Upon completion,
reaction mixtures were filtered, and the mother liquor was
evaporated under reduced pressure. The resulting crude
product was adsorbed on silica gel and purified by
automated flash chromatography (hexanes: ethyl acetate:
methanol 80/15/5) to yield pale yellow to bright yellow
solids with variable yields (Table 1).
Figure 1: Chemical structures of E121 (left) and JODI/JOAB
series (right)
We synthesized a total of sixteen novel compounds from
JODI and JOAB series (Table 1). With respect to R
novel compounds were synthesized where R is a CH
similar to the original enaminone E121). The yield for
compounds containing R , as CH group comprises a wide
1
, eight
1
3
group
(
1
3
range from 18%-80%). The reaction solvent plays a crucial
role on the overall reaction yield. For instance, JOAB-44
was prepared using isopropanol or ethanol, 98%. The yield
from the ethanol was significantly higher (see JOAB-44a
and 44b), and therefore, ethanol was the solvent of choice
for the remaining reactions. On the other hand, eight novel
compounds were synthesized with the phenyl group as R1
(encouraged by the positive results of JODI-19). The yield
ranged from 15-95%. It should be noted that JODI-19 was
re-synthesized under the name of JOAB-40.
1
.4 JOAB series
To identify the structural components of the newly
synthesized JODI series that are required for their
corresponding biological activity, a systematic thorough
structure activity relationship was proposed. The JOAB
series explores 4 different modifications on the enaminone
scaffold, while keeping the enaminone ring intact. The four
1 2 3
modifications are R , R , R , and the position of the N-
alkylated piperazine (Figure 1). We hypothesized that by
introducing the structural modifications, one at a time, we
will shed the light on the pharmacophoric elements of this
piperazino-enaminone series. The detailed chemistry and
biological evaluation of JODI and JOAB series are presented
herein.
2
With respect to the ester at R , eight compounds belong to
the methyl ester, and eight compounds belong to the ethyl
ester series. No difference was observed in the reaction yield
of these two series (Table 1). Regarding the substitution of
the N-1 of the piperazine ring, we explored two different
alkyl groups, methyl and ethyl to examine the substituent
effect by elongating the chain on the piperazine ring on the
biological activity. Shorter reaction times and higher yields
2
2
. Results
.1 Chemistry
The chemical synthesis of all JODI and JOAB series utilizes
the corresponding 2(3 or 4)-[4- methyl(ethyl) piperazine]
aniline which was either purchased commercially from
Sigma-Aldrich or synthesized in our laboratory by first
combining the N-methyl(ethyl)piperazine with the
3
were observed when R was an ethyl group (see compounds
JOAB 52, 54, 56, and 58).
Reaction time and yields were also affected by the position
of the piperazine ring with respect to the aniline amino
5

group. Four compounds were synthesized having the
piperazine ring in the ortho position (JODI-15, JODI-17,
JOAB-68, JOAB-70; Table 1), and four compounds with the
piperazine ring in the meta position (JOAB-48, JOAB-62,
JOAB-64, JOAB-66; Table 1). Eight compounds were
synthesized with the piperazine ring in the para position
JOAB-60.
Figure 2: Effect of JODI-19/JOAB-40 and analogs on the release
of TNF-alpha in LPS-stimulated macrophages
(
with either a methyl or an ethyl substitution). The starting
materials required for synthesizing an N-ethylpiperazine in
the meta and ortho position were not commercially
available, so we did not pursue it further.
2
.2 Biological Characterization
Experiments to explore the effects of the compounds in
reducing TNF-alpha and IL-6 in LPS-stimulated macrophages
were conducted on the JOAB series (similar to E121 and
JODI series) in an effort to examine the structural components
responsible for the biological activity. In these experiments,
one chemical modification was analyzed at a time. In the first
round of testing, JODI-19/JOAB-40 was tested with its
analogs JOAB-52 (R is ethyl), JOAB-54 (R is methyl) and
3 1
JOAB-66 (meta position of the piperazine ring).
Subsequently, JODI-18 was tested with its analogs JOAB-58
Figure 2 illustrates the ability of JODI-19/JOAB-40 and its
analogs (JOAB-52, JOAB-54, and JOAB-66) to reduce TNF-
alpha in LPS-stimulated macrophages. At the 0.25 mM
concentration, JOAB-40 was the best in its series, reducing
52% of the TNF-alpha, followed by JOAB-52 at 62%. Results
from JOAB-54 and 66 were a little inconsistent, creating the
highest amount of error, but did not create significant
differences in the TNF-alpha release after LPS stimulation.
Their activity becomes more apparent at higher concentration
(>0.5 mM).
3 1
(R is ethyl), JOAB-60 (R is methyl) and JOAB-64 (meta
position of the piperazine ring), and JOAB-68 (ortho position
of the piperazine ring). Two compounds were further
examined when we changed two factors at a time from the
original parent compounds JODI-19 and JODI-18. Tested in
that category, is JOAB-54, which represents the phenyl analog
of JOAB-52. Similarly, JOAB-62 is the meta analog of
Table 1: Physicochemical properties of new piperazino enaminones
Compound
Mol.
Wt.
R
1
R
2
R
3
Piperazine
Position
Ortho
%
Yield time (h)
Reaction Log P Melting
Point
______
JODI-15
JODI-17
JODI-18
357.45 CH3
371.48 CH3
CH3
CH3
CH3
CH3
CH3
CH3
33
66
70
91
18
12
8
2.65
3.02
4.02
4.39
3.07
C H
2
Ortho
Para
Para
Para
______
136-139
158-161
152-154
5
419.52 C H
6
CH3
8
5
JODI-19 (JOAB-40) 433.54 C H5
6
C H
2
8
5
JOAB-44
371.47 CH3
371.47 CH3
C H
2
10
6
5
a
a
a
JOAB-44
JOAB-48
3
C H
CH3
Meta
Para
29
95
30
80
33
35
47
84
79
78
15
5
3.05
4.77
3.45
3.07
4.39
2.70
2.67
3.99
4.37
3.97
4.35
_______
126-130
135-138
141-143
145-147
154-158
141-143
131-132
108-112
82-85
2
5
5
5
JOAB-52
JOAB-54
JOAB-56
JOAB-58
JOAB-60
JOAB-62
JOAB-64
JOAB-66
JOAB-68
JOAB-70
447.57 C H
6
C H
2
C H
2
6
5
5
5
5
5
385.50 CH3
371.47 CH3
C H
C H
Para
6
2
2
CH3
CH3
CH3
CH3
CH3
C H
2
Para
6
433.54 C H
6
C H
Para
6
5
2
357.45 CH3
357.45 CH3
CH3
CH3
CH3
CH3
CH3
Para
6
Meta
Meta
Meta
Ortho
Ortho
6
419.51 C H
6
24
8.5
17
10
5
5
5
5
433.54 C H
6
C H
2
5
419.51 C H
6
CH3
433.54 C H
6
C H
2
CH3
a
109-111
5
a
All reaction were conducted in 95% ethanol except when noted was conducted in isopropanol
6

The ability of JODI-19/JOAB-40 and its analogs (JOAB-52,
JOAB-54, and JOAB-66) to reduce IL-6 in LPS-stimulated
macrophages is illustrated (Figure 3). Similar to the TNF-
alpha results, both JOAB-40 and JOAB-52 were able to
reduce IL-6 in LPS-stimulated macrophages at 0.25 mM with
Figure 5 illustrates the ability of JODI-18 and its analogs to
reduce IL-6 in LPS-stimulated macrophages. All JODI
analogs tested in this assay performed significantly better than
JODI-18 itself. At 0.25mM, JODI-18 was able to reduce Il-6
by only 9%, followed by JOAB-60 at 53%, JOAB-64 at 72%,
and JOAB-58 at 86%.
6
4% and 62% respectively. No significant differences
between the two compounds were seen at 0.5 mM, and 1 mM
concentration. On the other hand, if we compare JOAB-54 to
both JOAB-40 and JOAB-52, one can notice that the ability of
the compound to reduce IL-6 was significantly lower in
JOAB-54 than its counterpart JOAB-40 and JOAB-52, which
is also similar with their results at the TNF-alpha assay
Figure 5: Effect of JODI-18 and analogs on the release of IL-6 in
LPS-stimulated macrophages
(
6
Figure 2). At 0.25 mM, JOAB-54 was able to only reduce IL-
by 25% compared to 62-64% reduction with JOAB-52 and
JOAB-40 at the same concentration. At 0.25 mM, JOAB-66
was only able to reduce IL-6 by 35% compared to JOAB-52
and JOAB-40 (62-64%) at the same concenetration.
Figure 3: Effect of JODI-19/JOAB-40 and analogs on the release
of IL-6 in LPS-stimulated macrophages
3
. Discussion
Results from the JODI-19/JOAB-40 series in both TNF-alpha
and IL-6 assays indicates that there is no significant difference
between JOAB-40 (R = methyl) and JOAB-52 (R
both assays, suggesting that the size of R
3
3
= ethyl) in
might not be a
3
determining factor on the ability of the compound to reduce
TNF-alpha or IL-6 after LPS-stimulation at the tested
concentrations (i.e the target binding site might be able to
tolerate a bigger alkyl group in that position). This becomes
more apparent in the JODI-18 series and its analogs, where
JOAB-58 actually performed consistently better than JODI-18
in both TNF-alpha and IL-6 assays (65%, and 86% reduction,
respectively).
Figure 4 illustrates the ability of JODI-18 and its analogs
(
JOAB-58, JOAB-60, JOAB-62, JOAB-64, and JOAB-68) to
reduce TNF-alpha in LPS-stimulated macrophages. At the
.25 mM concentration, JODI-18 resulted in only 30%
0
1
Size and lipophilicity of R seems to play a bigger role. The
reduction of TNF-alpha, followed by JOAB-62 at 40%
reduction, then JOAB-64 at 53% reduction. The best
compound of that series was JOAB-58 at 65% reduction of
TNF-alpha release. The lowest performing compound in this
phenyl ring in position R seems to be more favorable than the
1
methyl counterpart, which was observed earlier when we
compared the JODI series to E121. The same rule also seems
to also be applicable in the JOAB series. If we compare
JOAB-54 to both JOAB-40 and JOAB-52, one can notice that
the ability of the compound to reduce IL-6 was significantly
assay was JOAB-60.
A
noteworthy observation for
compounds JOAB-58 and JOAB-64 is that their ability to
reduce TNF alpha release increases significantly with the
increase in their concentrations from 0.25 mM to 0.5 mM,
unlike JODI-18 itself.
lower when R
(
1
is a methyl group (JOAB-54) vs phenyl group
JOAB-40 and JOAB-52), which is also similar to their results
in the TNF-alpha assay (Figure 2). At 0.25 mM, JOAB-54
was able to only reduce IL-6 by 25% compared to 62-64%
reduction with JOAB-52 and JOAB-40 at the same
concentration. A similar trend is noticeable with the JODI-18
series as well, where JOAB-58, and JOAB-64 perform at a
much higher level in reducing TNF-alpha or IL-6 as opposed
Figure 4: Effect of JODI-18 and analogs on the release of TNF-
alpha in LPS-stimulated macrophages
1
to their R methyl counterparts (JOAB-60 and JOAB-62),
The effect of the piperazine position on the structure activity
relationship of the piperazino-enaminone series seems to be
less consistent. When examining the JOAB series, JOAB-66,
with piperazine ring in the meta position, at 0.25mM is able to
reduce IL-6 significantly less than its counterparts JOAB-40
and JOAB-52, with piperazine ring in the para position. In the
case of TNF-alpha, activity of JOAB-66 is not significantly
different than JOAB-40 and 52. On the other hand, JODI-64
seems to reduce both TNF-alpha and IL-6 significantly better
7

than its para position counterpart JODI-18. The same
observation occurs with JOAB-62 when compared to JOAB-
Reaction mixture was stirred for 10 min at room temperature
after the addition of PMHS. The crude compound was filtered
on a celite column, washed with water, and extracted with
diethyl ether, and evaporated in vacuum, and purified by
automated flash chromatography (hexanes: ethyl acetate:
methanol 60/20/20). The resulted product was obtained in 72-
6
0. These results might suggest that the ability of the
piperazino-enaminone to reduce TNF-alpha or IL-6 does not
depend on the position of the piperazine ring on the
enaminone ring.
85% yield. 3-(4-methylpiperazino)aniline (4c) and 4-(4-
ethylpiperazino) aniline (4d) were used from commercial
sources.
Another important property of the piperazino-enaminone
series is their wide range of lipophilicity with calculated LogP
between 2.70-4.77. The lowest LogP for the series are JODI-
1
5 and JOAB- 60 (with Log P=2.70) and the highest LogP is
Synthesis of methyl 6-methyl-4-((2-(4methylpiperazin-1-yl)
phenyl)amino)-2-oxocyclohex-3-ene-1-carboxylate (JODI-
15)
JOAB-52 (4.77), followed by JOAB-58 and JOAB-40 (4.39).
Since these three compounds belong to the active members of
both series at both assays, one can propose that high
lipophilicity of this series play a major role on their ability to
interact with their target site(s).
To 0.7 g of 2(4-methylpiperazino)aniline (4a; 0.00365 mol)
in a dry round bottom flask was added 20 mL of 98% ethanol.
The mixture was stirred at room temperature till complete
dissolution. To this 0.92 g (0.005 mol) of methyl 2-methyl-
It is also worthy to mention that this class of compounds were
synthesized by non-stereoselective methods, and evaluated as
mixtures of stereoisomers. Resolution of previous enaminones
has not been yet successful. Lastly, reduction of IL-6 seems
to be more significant than reduction for TNF-alpha in both
series. This might be indicative of an effect on a differential
4
,6-dioxocyclohexanecarboxylate
(E115;
previously
22
synthesized in our laboratory) The resulting reaction
mixture was refluxed for 8 hours, and monitored by thin
layer chromatography (dichloromethane: methanol 90/10).
After the reaction was completed, the resulting crude was
adsorbed on silica gel and purified by automated flash
chromatography (hexanes: ethyl acetate: methanol 80/15/5)
24
signaling mechanism or
a transcription factor, which
preferentially promotes IL-6 over TNF-alpha. This remains to
be determined in future experiments.
1
to yield a yellow solid (0.58 g; 33% yield). H-NMR (300
MHz, d- CDCl
2
3
): δ (ppm) 2.503 (s, 6H), 2.513-2.668 (m,
H), 2.708-2.779 (m, 8H), 2.907-2.922 (m, 2H), 2.994 (s, 3
H), 3.025-3.141 (m, 2H), 5.769 (s, 1H), 6.715-6.761 (t, 2H),
6.914-6.991 (m, 2H). HRMS (ESI): m/z, Calcd. for
4
. Experimental
1
H-NMR spectra were recorded with
a Varian 300
+
C H N O [M+H] : 358.2120, found 358.2200
spectrometer using tetramethylsilane (TMS) as the internal
standard in CDCl unless otherwise specified. High resolution
20
27
3
3
3
mass spectra (HRMS) were recorded on Quadrupole-Time-of-
Flight (QStar Elite-AB Sciex) Mass Spectrometer. Melting
points were measured on a Mel-Temp-1101D micro melting
point apparatus. All chemicals and solvents used were of
reagent grade without further purification or dried. All the
reactions were monitored by thin-layer chromatography
Synthesis of ethyl 6-methyl-4-((2-(4-methylpiperazin-1-yl)
phenyl)amino)-2-oxocyclohex-3- ene-1-carboxylate (JODI-
1
7)
To 0.7 g of 2(4-methylpiperazino)aniline (4a; 0.00365 mol)
in a dry round bottom flask, was added 20 mL of98% ethanol.
The mixture was stirred at room temperature till complete
dissolution. To this 1.0 g (0.005 mol) of ethyl 2-methyl-4,6-
dioxocyclohexanecarboxylate (E164; previously synthesized
(TLC) under a UV lamp at 254 nm. Column chromatography
separations were performed on Biotage-Isolera 1.
2
3
in our laboratory) was then added. The resulting mixture was
refluxed for hours and monitored by thin layer
Synthesis of 2(4)-(4-methylpiperazino)aniline (4a and 4b)
To 3 g of the corresponding halonitrobenzene in a round
bottom flask, 10 mL of dimethylsulfoxide was added and
stirred for 5 min followed by the addition of 4.4 g (1.5 eq) of
potassium carbonate and 4.5 g (2 eq) of N-methylpiperazine.
The reaction was refluxed for 5 hours, monitored by thin layer
chromatography, and upon completion, the reaction mixture
was quenched with water, and extracted with
dichloromethane. It was dried over anhydrous magnesium
sulfate, and evaporated under vacuum. The resulting crude
compound was purified using dichloromethane: methanol
8
chromatography (dichloromethane: methanol 90/10). After
the reaction was completed, the resulting crude was adsorbed
on silica gel and purified by automated flash chromatography
(hexanes: ethyl acetate: methanol 80/15/5) to yield a yellow
powder (1.2 g; 66% yield). H-NMR (300 MHz, d- CDCl
1
3
):
δ (ppm) 1.226-1.325 (q, 3H), 2.388 (s, 3H), 2.495-2.548 (m,
2
3
2
3
H), 2.650-2.724 (m, 8H), 2.897-2.900 (m, 2H), 2.911 (s,
H), 5.777 (s, 1H), 6.712-6.758 (t, 2H), 6.909-7.016 (m,
+
H). HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
72.2276, found 372.2434
2
1
29
3
3
9
methylpiperazine)nitrobenzene (3a and 3b). Immediately
0/10 to yield bright orange solid (59% yield) of the 2(4)-(4-
2
5
Synthesis of methyl 5-((4-(4-methylpiperazin-1-yl)phenyl)
amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-2-
carboxylate (JODI-18)
To 0.32 g of 4(4-methylpiperazino)aniline (4b; 0.00167
mol) in a dry round bottom flask, was added 10 mL of 98%
ethanol. The mixture was stirred at room temperature till
complete dissolution. To this 0.33 g (0.00134 mol) of methyl
after, the orange solids were dissolved in tetrahydrofuran
(
THF) under inert atmosphere. To this solution was added, 0.1
eq of Pd(OAc) dissolved in THF, followed by a solution of
2
degassed solution of 0.5 eq potassium fluoride dissolved in
minimum amount of de-ionized water. The resulting reaction
mixture was cooled and stirred at 0-5 ˚C for 15-20 min
followed by the drop wise addition of 0.5 eq of
polymethylhydrosiloxane (PMHS). Reaction mixture turned
black upon the addition of PMHS and the TLC monitoring
was required immediately following the addition of PMHS.
2
,4-dioxo-6-phenylcyclohexanecarboxylate
(E201;
22
previously synthesized in our laboratory) was then added.
The resulting mixture was refluxed for 8 hours, and
8

+
monitored by thin layer chromatography (dichloromethane:
methanol 90/10). After the reaction was completed, the
precipitate was filtered, washed with methanol and
evaporated under reduced pressure. The resulting crude was
adsorbed on silica gel and purified by automated flash
chromatography (hexanes: ethyl acetate: methanol 80/15/5)
C H N O [M+H] : 372.2276, found 372.2251.
21 29 3 3
Synthesis of ethyl 6-methyl-4-((3-(4-methylpiperazin-1-yl)
phenyl)amino)-2-oxocyclohex-3-ene-1-carboxylate (JOAB-
4
8)
To a 0.320 g of 3-(4-methylpiperazino)aniline (4c; 0.00167
mol) in dry round bottom flask was added 10 mL of 98%
ethanol. The mixture was stirred at room temperature till
complete dissolution. To this 0.265 g of ethyl 2,4-dioxo-6-
methylcyclohexanecarboylate (E164; previously synthesized
in our lab) was added. The reaction mixture was refluxed
for about 5 hours. The reaction was monitored by thin layer
chromatography (dichloromethane: methanol 85/15). After 5
hours, the heat was turned off and reaction continued to stir
overnight. After reaction was completed, the reaction was
adsorbed on a silica gel and purified by automated flash
chromatography (dichloromethane: methanol 85/15) to yield
1
to yield a yellow powder (0.39 g; 70% yield). H-NMR (300
MHz, d- CDCl
3
): δ (ppm) 2.350 (s, 3H), 2.576-2.767 (t,
H), 3.178-3.340 (t, 4H), 3.424 (s, 3H), 3.554-3.672 (m,
H), 5.433 (s, 1H), 6.820-6.848 (d, 2H), 6.997-7.026 (d,
4
2
2
13
2
3
H), 7.208-7.374 (m, 5H), 7.435 (s, Br, NH). C-NMR (300
MHz, CDCl3) δ (ppm) 36.0, 42.9, 46.0, 48.8, 51.8, 54.8,
5
1
9.1, 76.6, 77.1, 77.5, 97.2, 116.3, 125.7, 127.1, 127.3,
28.6, 128.7, 128.8, 129.3, 141.1, 149.4, 162.6, 170.9, 191.4.
+
HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
29
2
5
3
3
4
20.2276, found 420.2324.
1
Synthesis of ethyl 5-((4-(4-methylpiperazin-1-yl)phenyl)
amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-2-
carboxylate (JODI-19) aka JOAB-40
a bright yellow semi-solid (0.145g, 29 %). H-NMR (300
3
MHz, d- CDCl ): δ (ppm) 1.103-1.116 (d, 3H), 1.262-1.348
(t, 3H), 2.357-2.383 (s, 3H), 2.554-2.588 (t, 4H), 3.073-
To 0.32 g of 4(4-methylpiperazino)aniline (4b; 0.00167
mol) in a dry round bottom flask, was added 10 mL of 98%
ethanol. The mixture was stirred at room temperature till
complete dissolution. To this 0.35 g (0.00134 mol) of ethyl
3
6
.220 (t, 4H), 4.218-4.289 (q, 2H), 5.586 (s, 1H), 6.609-
.635 (t, 1H), 6.668-6.682 (d, 2H), 7.188-7.242 (d, 1H).
+
HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
2
1
29
3
3
3
72.2276, found 372.2251.
2
,4-dioxo-6-phenylcyclo-hexanecarboxylate
(E203;
2
3
previously synthesized in our laboratory) was then added.
The resulting mixture was refluxed for 8 hours, and
monitored by thin layer chromatography (dichloromethane:
methanol 90/10). After the reaction was completed, the
resulting crude was adsorbed on silica gel and purified by
automated flash chromatography (hexanes: ethyl acetate:
Synthesis of ethyl 5-((4-(4-ethylpiperazin-1-yl)phenyl)
amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-2-carbox-
ylate (JOAB-52)
To a 0.342 g of 4-(4-ethyl-piperazine-1yl)phenylamine
(
4d; 0.00167 mol) in a dry round bottom flask was added 10
mL of 98% ethanol. The mixture was stirred at room
temperature till complete dissolution. Then 0.350 g of ethyl
methanol 80/15/5) to yield a yellow powder (0.52 g; 91%
1
yield). H-NMR (300 MHz, d- CDCl
3
): δ (ppm) 0.856-
2
,4-dioxo-6-phenylcyclohexanecarboxylate
(E164;
2
3
0
(
1
.903(t, 3H), 2.338 (s, 3H), 2.579-2.789 (t, 4H), 3.159-3.345
t, 4H), 3.526-3.620 (m, 2H), 3.827-3.850 (q, 2H), 5.413 (s,
H), 6.790-6.818 (d, 2H), 6.977-7.005(d, 2H), 7.149-7.276
previously synthesized in our lab) was added. The reaction
mixture was refluxed for about 6 hours. The reaction was
monitored by thin layer chromatography (dichloromethane:
methanol 85/15). After 6 hours, the heat was turned off and
reaction continued to stir overnight. After reaction was
completed, the reaction was adsorbed on a silica gel and
1
3
(
(
m, 5H), 7.826 (s, NH). C-NMR (300 MHz, CDCl3) δ
ppm) 13.9, 36.0, 43.1, 45.8, 48.7, 54.8, 59.2, 60.5, 76.7,
7
1
7.1, 77.6, 96.9, 116.3, 125.5, 127.2, 128.6, 129.6, 141.0,
49.2, 162.9, 170.4, 191.6. HRMS (ESI): m/z, Calcd. for
purified
by
automated
flash
chromatography
(dichloromethane: methanol 75/25) to yield a dark orange
+
C H N O [M+H] : 434.2433, found 434.2435.
31
2
6
3
3
1
3
solid (0.7059g, 94.9%). H-NMR (300 MHz, d- CDCl ): δ
Synthesis of ethyl 6-methyl-4-((4-(4-methylpiperazin-1-yl)
phenyl)amino)-2-oxocyclohex-3-ene-1-carboxylate (JOAB-
(ppm) 0.992-1.040 (t, 3H), 1.115-1.163 (t, 3H), 2.474-2.498
(q, 2H), 2.600-2.633 (t, 4H), 3.203-3.237 (t, 4H), 3.237 (d,
1H), 3.622- 3.663 (m, 1H), 4.008-4.037 (q, 2H), 5.482 (s,
1H), 6.876-6.906 (d, 2H), 7.052-7.082 (d, 2H), 7.297-7.326
4
4)
To a 0.320 g of 4-(4-methylpiperazino)aniline (4b;
.00167 mol) in dry round bottom flask was added 10 mL of
+
0
(m, 5H). HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
3
2
7
33
3
isopropyl alcohol (JOAB-44) or 98% ethanol (JOAB-44a).
The mixture was stirred at room temperature till complete
4
48.2589, found 448.2635
dissolution. To this 0.265
methylcyclohexanecarboylate (E164; previously synthesized
g of ethyl 2,4-dioxo-6-
Synthesis of ethyl 4-((4-(4-ethylpiperazin-1-yl)phenyl)
amino)-6-methyl-2-oxocyclohex-3-ene-1-carboxylate
2
3
in our lab) was added. The reaction mixture was refluxed
for about 6-10 hours. The reaction was monitored by thin
layer chromatography (dichloromethane: methanol 90/10).
The reaction continued to stir at room temperature overnight.
After reaction was completed, the reaction was absorbed on
a silica gel and purified by automated flash chromatography
(
JOAB-54)
To a 0.342 g of 4-(4-ethylpiperazine-1-yl)phenylamine
4d; 0.00167 mol) in a dry round bottom flask was added 10
mL 98% ethanol. Then 0.265 g of ethyl 2,4-dioxo-6-
methylcyclohexanecarboxylate (E164; previously
(
2
3
synthesized in our lab) was added. The reaction mixture
was refluxed for about 6 hours and a total of 5 ml additional
ethanol was added. The reaction was monitored by thin layer
chromatography (dichloromethane: methanol 85/15). After 6
hours, the heat was turned off and reaction continued to stir
at room temperature overnight. After reaction was
completed, the reaction was adsorbed on a silica gel and
(
(
dichloromethane: methanol 75/25) to yield an orange solid
0.0157g, 3%[44a], 0.0907g, 18% [44b]). H-NMR (300
1
MHz, d- CDCl
(
(
3
): δ (ppm) 1.065-1.129 (d, 3H), 1.244-1.291
t, 3H), 2.338-2.360 (s, 3H), 2.426-2.595 (t, 4H),3.164-3.197
t, 4H), 4.182-4.254 (q, 2H), 5.372 (s, 1H), 6.839-6.869 (d,
H), 7.008-7.038 (d, 2H). HRMS (ESI): m/z, Calcd. for
2
9

2
2
purified
(
by
dichloromethane: methanol 75/25) to yield a yellow semi-
automated
flash
chromatography
synthesized in our lab) was added. The reaction was
refluxed for about 6 hours and a total of 3 ml of additional
ethanol was added. The reaction was monitored by thin layer
chromatography (dichloromethane: methanol 85/15). After 6
hours, the heat was turned off and reaction continued to stir
overnight. After reaction was completed, the reaction was
adsorbed on a silica gel and purified by automated flash
chromatography (dichloromethane: methanol 75/25) to yield
1
solid (54) (0.154g, 30%). H-NMR (300 MHz, d-CDCl
3
): δ
ppm) 1.099-1.176 (t, 3H), 1.276-1.355 (t, 3H), 1.951 (d,
(
3
H), 2.392-2.547 (q, 2H), 2.498-2.641 (t, 4H), 3.211-3.244
(
t, 4H), 4.239-4.287 (q, 2H), 5.403 (s, 1H), 6.873-6.903 (d,
H), 7.039-7.069 (d, 2H). HRMS (ESI): m/z, Calcd. for
2
C H N O [M+H] : 386.2433, found 386.2564.
+
2
2
31
3
3
1
a brown and yellow solid (0.018g, 35.3% yield). H-NMR
Synthesis of methyl 4-((4-(4-ethylpiperazin-1-yl)phenyl)
amino)-6-methyl-2-oxocyclohex-3-ene-1-carboxylate
(300 MHz, d-CDCl ): δ(ppm) 1.056-1.078 (d, 3H), 2.378 (s,
3
3H), 2.627-2.660 (t, 4H), 3.028-3.064 (d, 1H), 3.193-3.229
(t, 4H), 3.746 (s, 3H), 5.381 (s, 1H), 6.870-6.881 (d, 2H),
7.005-7.016 (d, 2H). HRMS (ESI): m/z, Calcd. for
(
JOAB-56)
To a 0.342 g of 4-(4-ethyl-piperazin-1-yl)phenylamine
4d; 0.00167 mol) in a dry round bottom flask was added 10
+
(
C H N O [M+H] : 358.2120, found 358.2200.
27
20
3
3
mL of 98% ethanol. The mixture was stirred at room
temperature till complete dissolution. To this 0.247 g of
methyl 2-methyl-4,6-dioxocyclohexanecarboxylate (E115;
Synthesis of methyl 6-methyl-4-((3-(4-methylpiperazin-1-
yl)phenyl)amino)-2-oxocyclohex-3-ene-1-carboxylate
2
2
previously synthesized in our lab) was then added. The
reaction mixture was refluxed for 6 hours and a total of 5 ml
of additional ethanol was added. The reaction was monitored
by thin layer chromatography (dichloromethane: methanol
(
JOAB-62)
To a 0.320 g of 3(4-methyl-piperazino)aniline (4c;
.00167 mol) in a dry round bottom flask was added 10 mL
of 98% ethanol. Then 0.247 g of methyl 2,4-dioxo-6-
methylcyclohexanecarboxylate (E115; previously
0
8
5/15). After 6 hours, the heat was turned off and reaction
continued to stir at room temperature overnight. After
reactionwas completed, the reaction was adsorbed on a silica
gel and purified by automated flash chromatography
22
synthesized in our lab) was added. The reaction was
refluxed for about 6 hours and a total of 7 ml of additional
ethanol was added to the round bottom flask. The reaction
(
(
dichloromethane: methanol 75/25) to yield a yellow solid
1
0.4 g; 80.3%yield). H-NMR (300 MHz, d-CDCl ): δ(ppm)
was
monitored
by thin
layer chromatography
3
(dichloromethane: methanol 85/15). After 6 hours, the heat
was turned off and reaction continued to stir overnight.
After reaction was completed, the reaction was adsorbed on
a silica gel and purified by automated flash chromatography
(
orange semi-solid (0.226g, 47%). H-NMR (300 MHz, d-
1
2
4
7
.020-1.079 (t, 3H), 1.100-1.256 (d, 3H), 2.452-2.501 (q, 2H),
.525-2.644 (t, 4H), 3.047-3.119 (m, 1H), 3.170-3.236 (t,
H), 3.756 (s, 3H), 5.387 (s, 1H), 6.859-6.889 (d, 2H),
.026-7.055 (d, 2H). HRMS (ESI): m/z, Calcd. for
dichloromethane: methanol 75/25) to yield a yellow and
1
+
C H N O [M+H] : 372.2276, found 372.2434.
29
21
3
3
CDCl ): δ (ppm) 1.032-1.093 (d, 3H), 2.450 (s, 3H), 2.594-
3
Synthesis
yl)phenyl)amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-
-carboxylate (JOAB-58)
To a 0.342 g of 4-(4-ethyl-piperazin-1-yl)phenylamine (4d;
of
methyl
5-((4-(4-ethylpiperazin-1-
2.627 (t, 4H), 3.177-3.210 (t, 4H), 3.828 (s, 3H), 5.563 (s,
1H), 6.198-6.253 (m, 1H), 6.333-6.339 & 6.361-6.367 (dd,
1H), 6.610-6.674 (m,1H), 7.008-7.061 (t, 1H). HRMS
2
+
(ESI): m/z, Calcd. for C H N O [M+H] : 358.2120, found
3
2
0
27
3
0
9
.00167 mol) in a dry round bottom flask was added 10 mL of
8% ethanol. Then 0.314 of methyl 2,4-dioxo-6-
358.2200.
g
phenylcyclohexanecarboxylate (E201; previously synthesized
Synthesis of methyl 5-((3-(4-methylpiperazin-1-yl)
phenyl)amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]- 2-
carboxylate (JOAB-64)
22
in our lab) was added. The reaction was refluxed for about 6
hours and a total of 3 ml of additional ethanol was added. The
reaction was monitored by thin layer chromatography
To a 0.320 g of 3(4-methyl-piperazino)aniline (4c;
0.00167 mol) in a dry round bottom flask was added 10 mL
of 98% ethanol. The mixture was stirred at room
temperature till complete dissolution. A 0.33 g (0.00134
mol) of methyl 2,4-dioxo-6-phenylcyclohexanecarboxylate
(
dichloromethane: methanol 85/15). After 6 hours, the heat
was turned off and reaction continued to stir overnight. After
reaction was completed, the reaction was adsorbed on a silica
gel and purified by automated flash chromatography
2
2
(
solid (0.189g, 33%). H-NMR (300 MHz, d-CDCl3):
dichloromethane: methanol 75/25) to yield a dark orange
1
(E201; previously synthesized in our laboratory) was then
added. The reaction mixture was refluxed temperature for
12 hours and a total of 15 ml of additional ethanol was
added. The reaction was monitored by thin layer
chromatography (dichloromethane: methanol 85/15). After
12 hours, the heat was turned off and reaction continued to
stirred overnight. After reaction was completed, the reaction
was adsorbed on a silica gel and purified by automated flash
chromatography (dichloromethane: methanol 75/25) to yield
δ
(
(
1
ppm)1.133-1.258 (t, 3H), 2.523-2.571 (q, 2H), 2.617-2.684
t, 4H), 3.219-3.469 (t, 4H), 3.252-3.550 (s, 3H), 5.476 (s,
H), 6.872-6.902 (d, 2H), 7.050-7.080 (2d, H), 7.263-7.328
+
(
m, 5H). HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
26 31 3 3
4
34.2433, found 434.2435.
Synthesis of methyl 6-methyl-4-((4-(4-methylpiperazin-1-
yl)phenyl)amino)-2-oxocyclohex-3-ene-1-carboxylate
1
a yellow solid (0.48 g;85.7% yield). H-NMR (300 MHz, d-
CDCl ): δ (ppm) 2.360 (s, 3H), 2.570-2.639 (t, 4H), 2.789-
3
(JOAB-60)
To a 0.320 g of 4(4-methylpiperazino)aniline (4b; 0.00167
2
3
6
.881 (m, 1H), 3.180-3.213(t, 4H), 3.442-3.470 (d, 1H),
.521 (s, 3H), 3.606-3.737 (m, 2H), 5.642 (s, 1H), 6.625-
.760 (m, 4H), 7.180-7.291 (m, 5H). HRMS (ESI): m/z,
mol) in a dry round bottom flask was added 10 mL of 98%
of methyl 2,4-dioxo-6-
(E115; previously
ethanol. Then 0.247
methylcyclohexanecarboxylate
g
+
Calcd. for C H N O [M+H] : 420.2276, found 420.2324.
25 27
3
3
10

2
.630-2.707 (t, 4H), 2.912-2.929 (t, 4H), 3.255-3.270 (d, 1H),
Synthesis of ethyl 5-((3-(4-methylpiperazin-1-yl)phenyl)
amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-2-
carboxylate (JOAB-66)
4.019-4.089 (q, 2H), 5.921 (s, 1H), 5.640 (s, 1H), 6.904 (s, br,
NH,) 7.037-7.060 (m, 2H), 7.117-7.127 (m, 3H). 7.206-7.401
+
(m, 5H). HRMS (ESI): m/z, Calcd. for C H N O [M+H] :
434.2433, found 434.2632.
2
6
31
3
3
To a 0.320 g of 3(4-methyl-piperazino)aniline (4c;
0
.00167 mol) in a dry round bottom flask was added 10 mL
of 98% ethanol. Then 0.495 g of ethyl 2,4-dioxo-6-
phenylcyclohexanecarboxylate (E203; previously
Cell Culture and Stimulation
23
synthesized in our laboratory) was added. The reaction
was refluxed for 8.5 hours and total of 5 ml of additional
ethanol was added. The reaction was monitored by thin
layer chromatography (dichloromethane: methanol 85/15).
After the reaction has refluxed, the heat was turned off and
reaction continued to stir overnight. After reaction was
completed, the reaction was adsorbed on a silica gel and
An adherent murine macrophage cell line, J774.2 was
purchased (Sigma Aldrich, St. Louis, MO) and grown in
75cm cell culture flasks in Dulbecco’s Modified Eagle
Medium supplemented with 5% Fetal Bovine Serum and 1%
4
Penicillin/Streptomycin. Approximately 2x10 cells were
plated in individual wells of a 96-well plate. Cells were
stimulated with LPS from Escherichia coli 026:B6 (Sigma
Aldrich) at a final concentration of 5µg/mL. Tested
compounds were added to wells at varying concentrations,
from 0.25 mM to 1 mM. The total well volume for
stimulation was 200 µL and the plates were incubated for 24
2
purified
by
automated
flash
chromatography
dichloromethane: methanol 75/25) to yield a yellow solid
(
(
1
0.463g, 79% yield). H-NMR (300 MHz, d-CDCl : δ (ppm)
3
0
3
2
7
.916-1.001 (t, 3H), 2.388 (s, 3H), 2.617-2.648 (t, 4H),
.190-3.223 (d, 4H), 3.592-3.707 (d, 2H), 3.916-4.009 (q,
H), 5.640 (s, 1H), 6.639-6.745 (m, 3H), 6.912 (s, br, NH,),
hours at 37 ˚C and 5% CO2.
ELISA
.171-7.255 (m, 5H). HRMS (ESI): m/z, Calcd. for
+
Cell supernatants collected after 24 hour stimulation assays
were analyzed for TNF-alpha and IL-6 levels. Commercial
enzyme-linked immunosorbent assay kits were used (Mouse
ELISA Ready- SET-Go! to detect TNF-alpha, and IL-6,
Thermo Fisher, Agawam, MA). ELISAs were performed in
C H N O [M+H] : 434.2433, found 434.2632.
31
26
3
3
Synthesis of methyl 5-((2-(4-methylpiperazin-1-yl)phenyl)
amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-2-
carboxylate (JOAB-68)
To a 0.320 g of 2-(4-methyl-piperazino)aniline (4a; 0.00167
mol) in a round dry bottom flask was added 10 mL of 98%
9
microplate reader (Synergy HT, BioTek, Winooski, VT) with
6 well plates, and plates were read at 450 nm in a
TM
Gen5 All-In-One Microplate Reader Software.
ethanol. Then 0.495
phenylcyclohexanecarboxylate (E203; previously synthesized
g
of methyl 2,4-dioxo-6-
2
3
Statistical Analysis
in our laboratory) was added. The reaction was refluxed for
7 hours and an additional 7 ml of ethanol was added to
Data collected from ELISA assays are expressed as mean
pg/ml ± standard error of the mean and were analyzed using
SPSS 25.0 software. Comparisons between experimental
treatments (n=3-4 replicates per condition) were analyzed
using the Independent samples t-test. Statistical significance
was defined as p<0.05.
1
reaction mixture. The reaction was monitored by thin layer
chromatography (dichloromethane: methanol 85/15). After
the reaction has refluxed, the heat was turned off and reaction
continued to stir overnight. After reaction was completed, the
reaction was adsorbed on a silica gel and purified by
automated flash chromatography (dichloromethane: methanol
1
5/25) to yield a dark yellow solid (0.4379g, 78% yield). H-
5. Conclusion
7
NMR (300 MHz, d-CDCl : δ (ppm) 2.486 (s, 3H), 2.865-
3
In summary, we have synthesized a novel series of
compounds (JODI and JOAB) to investigate their anti-
inflammatory activity. Our data suggests that the JODI and
JOAB compounds are potentially useful anti-inflammatory
agents by acting as suppressants of pro-inflammatory
cytokines. JOAB-40, JOAB-52, and JOAB-58 appear to be
the most potentially useful novel anti-inflammatory agents
in our series. While we believe the most potent JODI
compounds have a more profound suppressive effect on IL-
6, ongoing experiments to be published suggest that these
compounds are also able to suppress other pro-inflammatory
cytokines besides TNF-α and IL-6, as well as chemokines.
2
(
2
.889 (t, 4H), 3.066-3.097 (t, 4H), 3.521 (s, 3H), 3.660-3.758
m, 2H), 5.853 (s, 1H), 6.728-6.775 (t, 2H), 6.941-7.074 (m,
H), 7.203-7.380 (m, 5H). HRMS (ESI): m/z, Calcd. for
+
C H N O [M+H] : 420.2276, found 420.2324.
3
25
29
3
Synthesis
of
ethyl
5-((2-(4-methylpiperazin-1-yl)
phenyl)amino)-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-2-
carboxylate (JOAB-70)
To a 0.320 g of 2-(4-methyl-piperazino)aniline (4a; 0.00167
mol) in a round dry bottom flask was added 10 mL of 98%
Ethanol. Then 0.495
phenylcyclohexanecarboxylate (E203; previously synthesized
g
of Ethyl 2,4-dioxo-6-
2
3
in our laboratory) was added. The reaction was refluxed for
0 hours and an additional 12 ml of ethanol was added. The
reaction was monitored by Thin Layer chromatography
Dichloromethane: Methanol 85/15). After the reaction has
Future studies will further elucidate mechanism of action of
these compounds in order to advance them as possible novel
therapeutics.
1
(
refluxed, the heat was turned off and reaction continued to stir
overnight. After reaction was completed, the reaction was
absorbed on a silica gel and purified by automated flash
chromatography (Dichloromethane: Methanol 75/25) to yield
a bright yellow solid (0.0881g, 15.2% yield). H-NMR (300
MHz, d-CDCl3: δ (ppm) 1.021-1.068 (t, 3H), 2.408 (s, 3H),
COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they have no competing interests
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