2286
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