Synthesis and in vitro evaluation of anti-inflammatory activity of ester and amine derivatives of indoline in RAW 264.7 and peritoneal macrophages

Article abstract of DOI:10.1016/j.bmcl.2014.03.081

A prolonged increase in pro-inflammatory cytokines, TNF-α and IL-6 occurs in inflammatory diseases. Although existing therapies like steroids and TNF-α antagonists are effective they may cause serious adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethylamino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were previously shown to inhibit acetylcholinesterase with IC_50s ranging from 0.4 to 55 μM and to prevent cytotoxicity induced by reactive oxygen species in a concentration range of 100 pM-1 μM. Compounds 25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF-α and IL-6 at concentrations of 1-10 pM in LPS-activated RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was associated with an increase in IκBα degradation and a decrease in the phosphorylation of p38 but not that of ERK. Conclusion: Indoline derivatives substituted at position 3 with chains carrying ester or amino groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.

Full text of DOI:10.1016/j.bmcl.2014.03.081

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2283–2287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of anti-inflammatory activity of
ester and amine derivatives of indoline in RAW 264.7 and peritoneal
macrophages
Svetlana Furman a, Elinor Nissim-Bardugo a, Shani Zeeli a, Michal Weitman a, Abraham Nudelman a,
Efrat Finkin-Groner b, Dorit Moradov b, Helena Shifrin b, Donna Schorer-Apelbaum b, Marta Weinstock b,
⇑
a Department of Chemistry, Bar Ilan University, Ramat Gan, Israel
b Institute of Drug Research, The Hebrew University of Jerusalem, Ein Kerem, Jerusalem 91120, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
A prolonged increase in pro-inflammatory cytokines, TNF-
a
and IL-6 occurs in inflammatory diseases.
Received 4 March 2014
Revised 23 March 2014
Accepted 25 March 2014
Available online 2 April 2014
Although existing therapies like steroids and TNF-
a antagonists are effective they may cause serious
adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel
derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethyl-
amino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were pre-
viously shown to inhibit acetylcholinesterase with IC50s ranging from 0.4 to 55
cytotoxicity induced by reactive oxygen species in a concentration range of 100 pM–1
25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF- and IL-6 at concentrations of 1–10 pM in LPS-activated
RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was asso-
ciated with an increase in I degradation and a decrease in the phosphorylation of p38 but not that
l
M and to prevent
Keywords:
Acetylcholinesterase inhibition
IL-6
Nitric oxide
p38
l
M. Compounds
a
jBa
of ERK. Conclusion: Indoline derivatives substituted at position 3 with chains carrying ester or amino
groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.
Ó 2014 Elsevier Ltd. All rights reserved.
TNF-a
Inflammation is a normal response to infection and injury and
involves the recruitment of immune systems to neutralize invad-
ing pathogens, repair injured tissues and promote wound healing.1
Chronic or excessive activation of the immune system is associated
with an increase reactive oxygen species (ROS), prolonged activa-
tion of inducible NO synthase (iNOS) and of the release of pro-
inflammatory cytokines.2,3 This may increase susceptibility to
infections and cause ulcerative colitis (UC), rheumatoid arthritis
(RA),4 diabetes,5 and cancer.6 Drugs that block the action of the
excessive activity of cytokines and ROS without interfering with
their normal function.
Indole-3-propionic acid (IPA) is a metabolite of 5-hydroxytryp-
tamine that acts as an oxygen radical scavenger15 and can protect
cells against oxidative stress.16 We found that the introduction of
an OH into the 4 or 6 position of IPA increases the antioxidant abil-
ity which is further enhanced by its conversion to a carbamate.17
However, the carbamate derivatives of IPA are much less water sol-
uble than their corresponding indolines which were also more
effective as oxygen radical scavengers and in conferring cytopro-
tection against oxidative stress.17 The naturally occurring indole,
melatonin has an amido side chain and has both antioxidant18,19
and anti-inflammatory activity in vitro20 and in vivo.21
cytokine, tumor necrosis factor-a (TNF-a) have proved to be very
effective in the treatment of UC and RA.7–9 However, their chronic
use may increase the risk of serious adverse effects,10–14 and has
resulted in the search for other therapies that prevent the
Recognition of conserved products unique to micro-organisms
such as lipopolysaccharide (LPS) occurs through activation of cell
surface receptors–toll-like receptors (TLRs). This initiates a cascade
of intracellular responses culminating in the release of NO and
cytokines.22 In the current study, we describe the evaluation of
the potential anti-inflammatory activity of several carbamate
derivatives of indoline propionic esters previously described.17
We also synthesized novel indoline carbamates with a propionic
ester, 2-aminoethyl, 3-aminopropyl, 2-(dimethylamino)ethyl or
Abbreviations: AD, Alzheimer’s disease; TLRs, toll-like receptors; IPA, indole-3-
propionic acid; iNOS, inducible NO synthase; DMEM, Dulbecco’s modification of
Eagle’s medium; FCS, fetal calf serum; MTT, 3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-
diphenyl-2H-tetrazole-3-ium bromide; PMSF, phenyl methyl sulfonyl fluoride; RA,
rheumatoid arthritis; ROS, reactive oxygen species; UC, ulcerative colitis; AP-1,
activator protein 1; MAPKs, mitogen activated protein kinases; ERK, extracellular
signal-regulated kinase; JNK, c-jun NH2-terminal kinase.
⇑
Corresponding author. Tel.: +972 2 675 8731; fax: +972 2 675 7478.
E-mail address: martar@ekmd.huji.ac.il (M. Weinstock).
http://dx.doi.org/10.1016/j.bmcl.2014.03.081
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2284
S. Furman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2283–2287
Table 1
3-(dimethylamino)propyl group in the 3 or 1 positions and evalu-
ated their anti-inflammatory activity in macrophages.
Indolic and indolinic derivatives
The syntheses of the various novel indole and indoline deriva-
tives and their intermediates are described in Scheme 1. The final
derivatives (some of which were described earlier)17 were evalu-
ated for anti-inflammatory activity as described below. Table 1
lists the novel compounds and those that were synthesized previ-
ously.17 Starting materials 1 and 6 are commercially available and
some of the early intermediates have been reported in the
literature. In our previous paper17 most of the studied derivatives
possessed side chains that carried carboxylate functional groups,
whereas latter compounds have side chains substituted with ami-
no groups. All the indolinic compounds, which were evaluated for
their biological activity, were isolated as the corresponding acid
addition salts.
Compound
X
Position of X
1
2
3
4
5
6
7
8
HO
HO
AcO
BnO
AcO
BnO
HO
HO
4
6
4
6
4
6
4
6
9–29
(Et,Me)NCOO
4 or 6
In each pair of compounds 9–29 the one with the odd number has the carbamate at
position 4 and the one with the even number has the carbamate at position 6.
were subsequently converted in the hydrochlorides 21 and 22.
Compounds 23–29 were described earlier17 and are incorporated
into Scheme 1 since some of their biological activities are described
herein.
The 2-dimethylaminoethyl-substituted indolines 9 and 10 were
prepared by reduction of the corresponding oxamides 5 and 6,23
which were debenzylated to give indoles 7 and 8, that were then
carbamoylated24 and reduced to indolines with Et3SiH/TFA.25,26 It
should be noted that in the course of the indole-to-indoline reduc-
tions carried out in TFA, both here as well as in the preparation of
compounds 17 and 18, small amounts of side-products having a
CF3CH2-group attached to the ring-nitrogen were also
obtained.27,28 These minor side-products, which are not shown in
Scheme 1 or in Table 1, were removed in the course of the purifi-
cation of the indolines. The intermediates 13 and 14 were prepared
NO release was measured (as NaNO2) in RAW 264.7 macro-
phages activated by LPS (5
Ltd),32 and cytokines were measured in peritoneal macrophages
activated by LPS 1
g/mL.33 Figure 1 shows the concentration of
each of the indoline derivatives that maximally reduced NO in
RAW 264.7 macrophages and TNF- and IL-6 in peritoneal macro-
lg/mL) from Escherichia coli (Sigma
l
a
phages, in comparison with the steroid budesonide and the natu-
rally-occurring indole melatonin. Higher concentrations than
those shown were either not more, or even less effective. In RAW
264.7 macrophages all the compounds were 10–1000 times more
potent than budesonide and some were at least 100 times more
potent than melatonin in reducing NO. Irrespective of the position
of the side chain, compounds with a carbamate in position 4 (9, 17,
25) tended to be more effective than those with the carbamate in
position 6, (10, 18, 26) except compound 21 which was less effec-
tive than compound 22 (Fig. 1A). Substitution of position 1 with
by
a Knoevenagel condensation between the corresponding
aldehydes 11 and 12 and cyanoacetic acid.29 The acrylonitrile
30
side-chain was reduced by catalytic hydrogenation over PtO2 to
give indoles 15 and 16, which were further reduced with Et3SiH/
TFA,25,26 to give the indolines 17 and 18 initially isolated as TFA
salts that were subsequently converted into the corresponding
hydrochlorides. The N-alkylated indoles 19 and 20 obtained from
1 and 4 upon reaction with Me2NCH2CH2CH2Cl/NaH,31 were re-
duced with Et3SiH/TFA,26 to give the TFA salts of the indolines, that
Scheme 1. Synthesis of indolines and their synthetic intermediates. Reagents and conditions: (a) (COCl)2; (b) Me2NH; (c) LiAlH4; (d) H2/Pd/C 10%; (e) (Me,Et)NCOCl; (f)
Et3SiH, TFA; (g) HCl; (h) POCl3/DMF; (i) HOOCCH2CN; (j) H2/PtO2/EtOH; (k) ClCH2CH2NMe2ÁHCl/NaH/DMF; (l) CH2@CH-COOMe/DBU/CH3CN; (m) NaBH3CN/AcOH or NaBH4/
TFA; (n) HOOCCH2COOMe/Py/piperidine; (o) 10% Pd/C/HCO2NH4; (p) MeNO2/NH4OAc; (q) NaBH4/silica gel/n-PrOH.

S. Furman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2283–2287
2285
(A)
(B)
(C)
Figure 1. Reduction by novel indoline derivatives of NO and cytokines induced by LPS in macrophages. Data show the concentrations of compounds expressed in terms of the
salt that caused the greatest reduction in NO or cytokines. The results are presented as the mean SEM from 6 measurements per compound in three independent
experiments. Statistical differences between data from cells treated with LPS alone and those with compounds were analyzed by Analysis of Variance using SPSS statistical
package version 19. A p value of <0.05 was considered to be significant. Each bar represents the mean SEM. (A) Compounds were administered 0.5 h prior to exposure to LPS
(5 lg/mL) and measurements of NO (as NaNO2) were made in RAW 264.7 cells after 24 h. Budesonide (B) was used as a positive control for these and subsequent experiments.
M = melatonin. (B) TNF-a-Measurements were made in peritoneal macrophages 6 h after addition of LPS. (C) IL-6-Measurements were made in peritoneal macrophages 24 h
after addition of LPS. Significantly different from LPS alone, ⁄p <0.05; ⁄⁄p <0.01.
the propionic ester resulted in a reduction in activity, particularly if
IL-6 at higher concentrations than those that inhibited TNF-a. The
the carbamate was in position 6 (compound 24).
most potent inhibitors among the indoline carbamates had a 3-
aminopropyl or propionic ester in position 3 and a carbamate in
position 4 (17, 25). Substitution of the propionic ester in position
1, (compound 23) resulted in a complete loss of inhibitory activity
against IL-6. Compounds 9 and 10 with a (2-dimethylamino)ethyl-
group at position 3, or compounds 21 and 22 having a (3-dimeth-
ylamino)propyl-group at position 1 were much less active or inac-
tive as inhibitors of IL-6 (Fig. 1C). Our data differ from those
described by Liu et al.,34 who found that many of the prenylated
mono-carbonyl derivatives of curcumin were effective in reducing
In peritoneal macrophages, the most potent inhibitors of TNF-
a
were those with a 3-aminopropyl or propionic ester substituent,
(17, 18, 25, 26) with the carbamate either in position 4 or 6. Attach-
ing the propionic ester substituent to position 1 resulted in a sig-
nificant reduction in activity (23, 24). Compounds 21 and 22
with a 3-(dimethyl amino) propyl side chain attached to position
1 were less potent than compounds 17 and 18 with a 3-aminopro-
pyl or 25 and 26 with a propionic ester group at position 3 (Fig. 1B).
Budesonide reduced IL-6 at much lower concentrations than
those affecting NO or TNF-
a, but the indoline derivatives inhibited
IL-6 but not TNF-a in RAW 264.7 macrophages. Since blockade of

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S. Furman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2283–2287
Table 2
TNF-a is considered to be the most effective treatment of RA and
UC,7–9 the indoline derivatives may be more efficacious than those
of curcumin in treating chronic inflammatory conditions. Treat-
ment of RAW 264.7 and peritoneal macrophages with all the com-
Inhibitory activity of acetyl and butyrylcholinesterase by indoline carbamates
Compound
IC50 AChE (
lM)
IC50 BuChE (lM)
9
10
17
18
21
22
23
24
25a
26a
29a
1.3 0.09
21.3 0.8
1.8 0.2
36.0 1.4
3.7 0.04
48.0 1.1
5.6 0.3
0.31 0.03
2.20 0.03
1.20 0.04
4.7 0.2
0.90 0.08
4.27 0.18
0.70 0.13
6.78 0.03
0.30 0.04
3.9 0.2
pounds in concentrations ranging from 10 pM to 10 lM had no
effect on cell viability as measured by the 3-(4,5-dimethyl-1,3-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazole-3-ium bromide (MTT)
assay35 (data not shown).
Binding of LPS to TLR4 in macrophages involves the activation
of multiple signal transduction pathways, including mitogen acti-
vated protein kinases (MAPKs) such as p38, extracellular signal-
regulated kinase (ERK) or c-jun NH2-terminal kinase (JNK) which
activate the transcription factor activator protein 1 (AP-1) leading
to the production of pro-inflammatory entities.36 Another pathway
activated by LPS via TLR4 involves the phosphorylation of nuclear
377 10
7.40 0.03
55.2 0.1
0.40 0.04
0.20 0.01
a
Compounds described previously in Yanovsky et al. (2012).
factor
degradation of I
the p50/p65 complex, resulting in nuclear translocation of NF-
B.37 The latter binds to specific regulated sequences in the DNA,
thus controlling gene transcription, stimulation of iNOS to form
NO and release of TNF-
and IL-6.38 Pretreatment of LPS-stimu-
lated peritoneal macrophages with compound 25 (100 pM), one
of the most potent inhibitors of NO, TNF- and IL-6, inhibited the
j
B-light polypeptide gene enhancer inhibitor (I
jB) and the
basic group with the peripheral anionic site situated at the en-
trance to the narrow gorge of the enzyme which could improve
the alignment of the molecule to the catalytic site situated near
its base, as previously suggested.17 For all pairs of compounds,
those with a carbamate in position 4 were 13–67 times more po-
tent as ChE inhibitors than their analogs with the carbamate in po-
sition 6, irrespective of the nature of the side chain. The reduced
activity of all the compounds having the carbamate at position 6
compared to those with a carbamate at position 4, may stem from
a hindered alignment of the former molecules to the active site of
the AChE. Although the indolines with the carbamate in position 6
were also less potent than those with the carbamate in position 4
as BuChE inhibitors, the difference between them was much less
than for inhibition of AChE. This observation may be explained
by the much wider gorge found in BuChE than in AChE enabling
easier access for the compound to the active site. The concentra-
tions of the compounds that inhibited NO and cytokine release
from macrophages were at least 4–5 orders of magnitude lower
than those inhibiting AChE and BuChE. It is therefore very unlikely
that the indoline carbamates will cause any cholinergic adverse ef-
fects when administered for the treatment of inflammatory
conditions.
jBa
that exposes nuclear localization signals on
j
a
a
phosphorylation of p38 but not that of ERK (Fig. 2) or JNK (not
shown) assessed as described in.39 Compound 25 also reduced
the degradation of I
the nuclear translocation of NF-
j
B
a
, which would be expected to decrease
B and explains how the com-
j
pound reduces NO and cytokines in cells activated by LPS.
It has been suggested that neuroinflammation occurs in Alzhei-
mer’s disease (AD).40 Although the acetylcholinesterase (AChE)
inhibitor, donepezil currently used for treatment of this condition
was shown to exhibit anti-inflammatory activity in cells, this was
seen at much higher concentrations than those that inhibit AChE,41
making it unlikely that it contributes to the therapeutic effect of
the drug. The indoline-3-propionic esters containing a carbamate
moiety were previously shown to inhibit both AChE and butyrylch-
olinesterase (BuChE).17 It was therefore of interest to see whether
the novel compounds with an amino group in the side chain also
inhibited AChE and BuChE at similar or lower concentrations than
those which showed anti-inflammatory activity. AChE and BuChE
inhibitory activity was assessed as previously described17 and is
depicted in Table 2 as IC50s (concentrations required to reduce en-
zyme activity by 50%). Compounds 9, 17, 21 and 29 with amino
groups and carbamates at position 4 were found to be more potent
inhibitors of AChE than compounds 25 and 26 with a propionic es-
ter. The increased activity may result from the interaction of the
Summary: This study describes for the first time the anti-inflam-
matory activity of a series of novel carbamate derivatives of indo-
line-3- or -1 substituted with propionic esters, primary and tertiary
amines in LPS activated macrophages. Several of the indoline
derivatives are able to reduce NO, TNF-a and IL-6 proteins at con-
centrations of 1–10 pM, which are 1000 to 100-fold lower than
those of the steroid budesonide. Although the compounds are also
AChE inhibitors, their anti-inflammatory activity occurs at much
Figure 2. Effect of compound 25 on the phosphorylation of p38 and ERK and I
compound 25 (0.1 nM) for 50 min then activated with LPS (1
Each bar represents the mean STD from 3–5 measurements. Compound 25 significantly reduced phosphorylation of p38, had no effect on phosphorylation of ERK and
significantly reduced the degradation of I
. Significantly different from LPS alone ⁄p <0.05.
jB
a
degradation in peritoneal macrophages. Peritoneal macrophages were pre-treated with
lg/mL). Cell lysates were subjected to immunoblot analysis using antibodies against p38, p-p38 ERK and p-ERK.
jBa

S. Furman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2283–2287
2287
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lower concentrations than those that inhibit this enzyme and is
mediated by the reduction in phosphorylation of p38 and increase
in IjBa degradation. In addition to their potential anti-inflamma-
19. Reiter, R. J.; Manchester, L. C.; Tan, D. X. Curr. Neuropharmacol. 2010, 8, 194.
20. Xia, M. Z.; Liang, Y. L.; Wang, H.; Chen, X.; Huang, Y. Y.; Zhang, Z. H.; Chen, Y. H.;
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Yuan, M. C.; Hsieh, C. J.; Huang, C. Y.; Wang, M. H.; Chiu, C. H.; Hsieh, T. C.; Wu,
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tory activity, several of the indoline carbamate esters and amines
can prevent cytotoxicity induced by oxidative stress in cardiomyo-
cytes and neuronal cells.17 This suggests that the compounds may
have a beneficial effect in neurodegenerative conditions such as AD
and Parkinson’s disease in which chronic inflammation and oxida-
tive stress occur, as well as in RA, diabetes type 2 and UC. Further
studies are in progress to see whether the compounds can produce
beneficial effects in animal models of pathological conditions in
which excess activity of cytokines and oxidative–nitrative stress
play a leading role.
Acknowledgment
This work was partially supported by the ‘Marcus Center for
Medicinal Chemistry’ at Bar Ilan University. S.F. and E.B.-N. have
contributed equally to this wok.
Supplementary data
32. For NO release measurements, RAW 264.7 cells (2 Â 105 cells/mL) were seeded
in a 48-wells plate and incubated overnight in complete DMEM containing
4.5 g/L glucose, 0.2 mM
L-glutamate, 1% v/v penicillin/streptomycin/
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
03.081.
amphotericin B, 1% v/v sodium pyruvate, 1% v/v Modified Eagle’s Medium
(MEM), 1% non-essential amino acids solution and 10% v/v fetal calf serum
(FCS). Compounds were added to the cells and after 30 min the medium was
removed and fresh medium containing FCS and LPS (5 lg/mL) was added and
the cells maintained at 37 °C for 24 h. Supernatants were collected after 24 h
(based on preliminary experiments which showed these to be the optimal time
points). The release of NO into the medium was determined indirectly by
measurement of nitrite levels using Griess reagent reaction.
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5 min. Cell pellets were incubated for 30 min with shaking on ice in lysis buffer
containing NaF (1 mM), Na3VO4, (1 mM) b-glycerophosphate (1 mM), sodium
pyrophosphate (2.5 mM), protease inhibitor cocktail (1%) and PMSF (0.5%) all
dissolved in RIPA buffer. Samples were then centrifuged at 13,000 rpm at 4 °C
for 5 min and supernatants were collected and stored at (À80 °C) until use.
Protein concentration was determined by means of BCA protein assay kit.
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polyacrylamide gels with 4.5% SDS stacking gel. Samples were electro-
transferred onto nitrocellulose membranes. Blots were probed with
antibodies against
I
j
Ba
,
JNK, phosphorylated JNK (p-JNK), ERK,
phosphorylated ERK (p-ERK), p38, phosphorylated p38 (p-p38) and tubulin
(Santa Cruz). An appropriate IR-dye was used as a secondary antibody and
reactive bands were visualized using the Infrared imaging system. Blots were
quantified using TINA software. Results were presented as optical density of
IjBa divided by the optical density of tubulin and of the phosphorylated form
of the protein divided by the density of the non-phosphorylated form.
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