Synthesis and Biological Evaluation of Derivatives of Indoline as Highly Potent Antioxidant and Anti-inflammatory Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00001

We describe the preparation and evaluation of novel indoline derivatives with potent antioxidant and anti-inflammatory activities for the treatment of pathological conditions associated with chronic inflammation. The indolines are substituted at position 1 with chains carrying amino, ester, amide, or alcohol groups, and some have additional substituents, Cl, MeO, Me, F, HO, or BnO, on the benzo ring. Concentrations of 1 pM to 1 nM of several compounds protected RAW264.7 macrophages against H₂O₂ induced cytotoxicity and LPS induced elevation of NO, TNF-α, and IL-6. Several derivatives had anti-inflammatory activity at 1/100th of the concentration of unsubstituted indoline. Four compounds with ester, amine, amide, or alcohol side chains injected subcutaneously in mice at a dose of 1 μmol/kg or less, like dexamethasone (5.6 μmol/kg) prevented LPS-induced cytokine elevation in the brain and peripheral tissues. Subcutaneous injection of 100 μmol/kg of these compounds caused no noticeable adverse effects in mice during 3 days of observation.

Full text of DOI:10.1021/acs.jmedchem.8b00001

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Synthesis and Biological Evaluation of Derivatives of Indoline
as Highly Potent Antioxidant and Antiinflammatory Agents
Shani Zeeli, Tehilla Weill, Efrat Finkin-Groner, Corina Bejar, Michal Melamed,
Svetlana Furman, Michael Zhenin, Abraham Nudelman, and Marta Weinstock
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00001 • Publication Date (Web): 22 Apr 2018
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Synthesis and Biological Evaluation of Derivatives of
Indoline as Highly Potent Antioxidant and Anti-
inflammatory Agents
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Shani Zeeli,a,# Tehilla Weill,b,# Efrat FinkinꢀGroner,b Corina Bejar,b
Michal Melamed,b Svetlana Furman,a Michael Zhenin,a Abraham
Nudelman,a,* and Marta Weinstockb,*
aDepartment of Chemistry, Bar Ilan University, Ramat Gan, 5290002, Israel
bInstitute of Drug Research, The Hebrew University of Jerusalem, Jerusalem,
9112102, Israel.
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ABSTRACT: We describe the preparation and evaluation of novel indoline
derivatives with potent antioxidant and antiꢀinflammatory activities for the treatment of
pathological conditions associated with chronic inflammation. The indolines are
substituted at position 1 with chains carrying amino, ester, amide or alcohol groups and
some have additional substituents, Cl, MeO, Me, F, HO or BnO on the benzo ring.
Concentrations of 1pMꢀ1 nM of several compounds protected RAW264.7 macrophages
against H2O2 induced cytotoxicity and LPS induced elevation of NO, TNFꢀα and ILꢀ6.
Several derivatives had antiꢀinflammatory activity at 1/100th of the concentration of
unsubstituted indoline. Four compounds with ester, amine, amide or alcohol side chains
injected subcutaneously in mice at a dose of 1 ꢁmole/kg or less, like dexamethasone (5.6
ꢁmoles/kg) prevented LPSꢀinduced cytokine elevation in the brain and peripheral tissues.
Subcutaneous injection of 100 ꢁmoles/kg of these compounds caused no noticeable
adverse effects in mice during 3 days observation.
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ꢀ INTRODUCTION
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Inflammation is a normal response to infection and injury and involves the
recruitment of the immune system to neutralize invading pathogens, repair injured tissues
and promote wound healing.1 However, during chronic or excessive activation of the
immune system, nitric oxide synthase (iNOS) is stimulated by which nitric oxide (NO)
and proꢀinflammatory cytokines e.g. tumour necrosis factorꢀα (TNFꢀα), interleukin 6 (ILꢀ
6), interleukin 1β (ILꢀ1β) are released.2 In diverse pathological conditions, an increase
also occurs in reactive oxygen species (ROS) including H2O2.3 H2O2 is a freely diffusible
component that can elevate TNFꢀα, and interleukins in macrophages.4,5 Conversely,
chronic infection or the addition of the bacterial pathogen to macrophages is responsible
for the formation of ROS, induced by lipopolysaccharide (LPS).6 Together, these have
been implicated in the etiology of ulcerative colitis,7 Crohn’s disease8 rheumatoid
arthritis,9 diabetes,10 atherosclerosis,11 neuropathic pain,12 psoriasis13 and cancer.14 ROS
and proinflammatory cytokines may also play a role in the etiology of Parkinson’s and
Alzheimer's disease (AD)15ꢀ17 and contribute to ischemic stroke,18 amyotrophic lateral
sclerosis,19 and depression.20
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The most effective TNFꢀα inhibitors in clinical use are monoclonal antibodies that
are given by injection. However they can cause serious adverse effects including
lymphomas, infections (especially reactivation of latent tuberculosis), congestive heart
failure and demyelinating disease. Also, as this antibody treatment is given chronically, it
is inherently immunogenic and there have been reports of allergic reactions, serum
sickness and thromboebolic events21,22 as well the induction of antiꢀdrug antibodies that
23ꢀ25
can reduce treatment efficacy.
While the adverse events may result from blockade
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of the actions of TNFꢀα and the immune system, the allergic reactions and formation of
blocking antibodies are due to the protein nature of the TNFꢀα inhibitors. Therefore, there
is a need for safer, small, nonꢀprotein molecules that do not block the essential activity of
TNFꢀα but inhibit the excessive release occuring in conditions like rheumatoid arthritis
and ulcerative colitis. Several such compounds have recently been described with
thiazole or pyrazolidine cores that inhibit LPS induced TNFꢀα release in macrophages in
the micromolar range.26,27
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Our previously reported carbamate derivatives of indolineꢀ3ꢀpropionic acid esters
and indolineꢀ3ꢀ(3ꢀaminopropyl) have the advantage over the corresponding indoles of
being sufficiently basic to form water soluble acid addition salts. Surprisingly, the
indolines were found to display about a 100 fold higher antioxidant activity than the
indoles, and in addition to inhibiting acetylcholinesterase (AChE) and butyryl
cholinesterase they also showed cytoprotective activity against cytotoxicity induced by
ROS.28 Subsequently, indoline carbamates were also found to reduce the release of TNFꢀ
α and ILꢀ6 from LPSꢀstimulated macrophages at concentrations of 1 nM or less.29 Their
mechanism of action includes inhibition of phosphorylation of the mitogen activated
protein kinase (MAPK) and p38 and reduction of the nuclear translocation of the
transcription factors activator protein 1 (AP1) and nuclear factor κB (NFꢀκB), consequent
to tollꢀ4 receptor stimulation by LPS.30
AChE inhibitors reduce the release of cytokines from LPSꢀactivated macrophages in
the presence of a choline ester by indirect stimulation of α7 nicotinic acetylcholine
31ꢀ34
receptors,
which activates the Jak2/STAT3 pathway.35 By prolonging the action of
acetylcholine, AChE inhibitors also decrease proꢀinflammatory cytokines in various
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animal models of inflammation.32,36,37 Therefore we reasoned that the most potent AChE
inhibitor among the indoline carbamates, 3ꢀ(3ꢀaminopropyl)ꢀindolinꢀ4ꢀylethyl (methyl)
carbamate (1), may combat inflammation in vivo by a combination of these two
mechanisms. However, surprisingly we found that 1 was less effective than 2, (methyl 3ꢀ
(6ꢀ((ethyl(methyl)carbamoyl)oxy)indolinꢀ3ꢀyl)propanoate which did not inhibit AChE at
the dose administered, in preventing inflammation and colon damage in a mouse model
of colitis,38 although the compounds had similar antiꢀinflammatory activity in vitro.
Therefore, we concluded that the carbamate moiety did not improve the antioxidant and
antiꢀinflammatory activity and may even increase the potential for adverse cholinergic
effects.
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Derivatives 1 and 2 that contain a chiral carbon were tested as racemates (Scheme
1).28 In the current study we prepared indoline derivatives lacking both a carbamate
moiety to reduce possible cholinergic side effects, and chiral centers to avoid the need for
enantiomeric separations. Therefore, we synthesized the isomeric analogues having the
side chain linked to the Nꢀ1 of the indoline ring. Switching from structure 3 to structure
7a a double isosteric change took place, in which the NH moiety was replaced by a CH2
group, and the CHꢀCH2, by an NꢀCH2 group. Although, isosteric replacements yield
compounds that are not identical having structural and electronic differences,
nevertheless, if the isosters being considered have similar structural, geometrical, and/or
electronic features they are commonly prepared and compared under the assumption that
the structural changes still enable possible binding of both isosters to the same receptor.
Thus, the novel achiral compounds studied herein all have side chains linked to position
1, based on the isosteric models of compounds 3 and 7a (Figure 1A) and their respective
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electrostatic potential calculations (Figure 1B) that are found to be virtually
superimposable.
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Scheme 1 and Figure 1
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The synthesized indoline derivatives (Table 1) contain side chains substituted with
amino (13 and 15), amido (18), ester (7), acid (7b’) or alcohol (9) groups attached to Nꢀ1
of indoline ring. The antioxidant and antiꢀinflammatory activities of compounds 3 and 23
with methyl propionate or 3ꢀaminopropyl side chains attached to position 3, respectively,
were compared to those in which the analogous side chains were attached to position 1 (7
and 13). Like the indoline derivatives 1 and 3, two of the compounds described below, 7b
and 15a also reduced phosphorylation of p38 MAP kinase and prevented nuclear
translocation of AP1.39
Table 1
ꢀ RESULTS AND DISCUSSION
Chemistry. Indolines substituted with a methyl propionyl chain at position 1 were
prepared from the corresponding indoles 4a-d,f upon treatment with methyl
acrylate/DBU. Reduction of the N-alkylated indoles 5a-d,f with NaBH3CN/AcOH40 or
NaBH4/TFA28 or Et3SiH/TFA41 gave the corresponding indolines 6a-d, f. Alternatively,
6a was obtained in one step from indoline 8a. The final products were isolated as
hydrochlorides 7a-c or pꢀtoluenesulfonate salts (pꢀTSA) 7d, f. Compound 7a did not
undergo hydrolysis at room temperature, in aqueous media for at least 72 h, as
determined by its stability in a D2O solution prepared for NMR determination. Under
more strenuous acidic conditions, 3N HCl at room temperature for 48 h, ester 7b was
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converted into the free acid 7b’. In addition, ester reduction42 of 6a, b followed by
acidification gave the alcohols 9a, b (Scheme 2).
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Scheme 2
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The salts of the Nꢀ(3ꢀaminopropyl) derivatives 13a, g were prepared from the
corresponding indoles 4a, g upon N-alkylation with acrylonitrile,43 followed by initial
reduction of the indolic to the indoline system28,40 and subsequent reduction of the CN44
to the corresponding aminomethyl group. The final products were isolated as pꢀTSA salts
13a, g (Scheme 3).
Scheme 3
An unexpected reaction took place when an attempt was made to debenzylate 12g
under catalytic hydrogenation conditions in a Parr apparatus. Apparently, the
hydrogenator contained traces of acetone, which condensed with the amine to give an
intermediate imine that underwent further reduction and acidification to give N-(3-
isopropylamino)propyl derivative 15i (Scheme 4).
Scheme 4
Other Nꢀ(3ꢀisopropylamino)propyl indolines 15, as well as the corresponding N-(3ꢀ
isopropylpropanamide)indolines 18, were prepared. N-Alkylation of indoles with N-
isopropylacrylamide45 gave amides 16. Reduction of indoles to indolines28,40 and
subsequent acidification led to amides 18. Nꢀ(3ꢀIsopropylamino)propyl indolines 15 were
obtained from 16 by a sequence of reactions involving reduction of the amides to the
amines42 19, followed by indole to indoline reduction28,40 to give 14 and finally,
acidification to give salts 15 (Scheme 5).
Scheme 5
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For comparison with compound 13a, substituted at position 1 with a 3ꢀaminopropyl
group, the analog 23 was prepared substituted at position 3 with the same 3ꢀaminopropyl
group. Amidation of IPA46 gave 20, which underwent two reductive42 steps, followed by
acidification to finally give 23 (Scheme 6).
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Scheme 6
In addition to altering the side chains attached to positions 1 or 3 of the indoline
system, modifications were also made on the substituents attached to the benzo ring.
Following the wellꢀestablished “Operation Scheme” developed by Topliss,47 the
unsubstituted benzo ring (compounds 7a, 9a, 13a, 18a and 23), was initially substituted
with an electron withdrawing chloro (7d, 18d and 18h) or fluoro (18e) groups.
Subsequent replacement of the electron withdrawing groups by electron donating ones
gave the MeO (7b, 7b’, 9b and 15b), BnO (7f and 13g), HO (15i) or Me (7c) derivatives.
The final compounds evaluated for their pharmacological activity are listed in Table 1. A
detailed discussion on SAR of the various analogs is presented below in the
pharmacology section.
Biology.
Effect of indoline derivatives on cell viability. None of the compounds tested
at concentrations ranging from 1 pM to 100 nM had any significant effect on cell
viability (Table 2).
Table 2
Influence of side chain position and that of other substituents on
protection against oxidative stress. H2O2 (100 ꢁM) reduced cell viability in these
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experiments to 65ꢀ72% of that in the vehicle control group. Quercetin, used as positive
control, restored viability to that of vehicle from a concentration of 1 pM (Figure 2).
Prevention by the indoline derivatives of cytotoxicity induced by H2O2 depended on the
nature of the substituent and its position. Compound 13a, with a 3ꢀaminopropyl group at
position 1, restored viability only at a concentration of 10 nM, but this was reduced to 1
pM when the group was at position 3 (23). In contrast, indolineꢀN-methyl propionate (7a)
restored viability of cells at a concentration 10 fold lower than its analog indoline 3ꢀ
methyl propionate (3).
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We are unaware of any studies that investigated the potential antioxidant effect of
unsubstituted indoline (8a). Accordingly, we compared its effect to that of four
derivatives having side chains containing a terminal amino (13a), ester (7a), amide (18a)
or alcohol (9a) group in position 1. Indoline completely protected cells against
cytotoxicity at a concentration of 10 nM but not at lower concentrations (Figure 2).
Compounds 13a or 7a did not differ in their cytoprotective activity from that of indoline
but 3ꢀ(indolinꢀ1ꢀyl)ꢀN-isopropylpropanamide (18a) was much more potent and caused
complete protection at a concentration of 1 pM. On the other hand, the introduction of Nꢀ
(3ꢀhydroxypropyl) (9a) decreased the protective activity of 8a a 10,000ꢀfold (Figure 2).
Figure 2
Table 3 shows the effect on cytoprotective activity of additional substituents at
position 1 on the benzo ring of indoline with 3ꢀaminopropyl, methyl propionate, 3ꢀ(Nꢀ
isopropylpropanamido), or 3ꢀhydroxypropyl groups which depended both on the nature
of the N-side chain and that of the benzoꢀsubstituent. A 7ꢀBnO group in indolineꢀNꢀ(3ꢀ
aminopropyl (13g) reduced protective activity from 1 nM to greater than 100 nM, in
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comparison to 13a. Replacement of the primary NH2 (13a) with a secondary NHꢀiꢀPr
(15a) increased potency 1000ꢀfold, completely inhibiting cytotoxicity at a concentration
of 1 pM. However, addition of 7ꢀHO (15i), or 5ꢀMeO (15b) reduced protective activity to
less than that in the primary amine, 13a. By contrast 5ꢀMeOꢀsubstitution in the propyl
alcohol 9a or propionic ester (7a), which are much less effective protective agents than
15a, increased protective activity 10,000 fold (Figure 3). A 5ꢀCl (7d) substituent in 7a,
was equally effective in increasing activity, while 6ꢀMe (7c) had no effect (Fig. 3 Table
3). Hydrolysis of ester 7b to the acid 7b’, reduced protective activity to that of the
unsubstituted propionate. Addition of 6ꢀF (18e) to the highly active Nꢀ
isopropylpropanamide 18a reduced the activity 10,000ꢀfold (Table 3).
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Figure 3 and Table 3
Summary: (a) The most potent compounds are those that are substituted with 5ꢀ
MeO electron donating group, i.e. compounds 7b and in particular, 9b, which appears to
be much more potent than unsubstituted 9a. The exception includes compounds that carry
an NHꢀiꢀPr group, 15a (ꢀCH2NHꢀiꢀPr) or 18a (ꢀCONHꢀiꢀPr) at the end of the chain and
do not have any substituents on the benzo ring. (b) Addition of substituents to positions 6
or 7, as in compounds 18e (6ꢀF), 15i (7ꢀHO), 13g (7ꢀBnO), decreases cytoprotective
potency. (c) The presence of a terminal secondary amino group, NHꢀiꢀPr (15a) markedly
improves potency in comparison to a primary NH2 group (13a). (d) The least potent
compounds, 9a, 13a, and 7b’ are those with the most polar OH, NH2 and COOH groups
respectively, at the terminal end of the side chain or 6ꢀF (18e) in the N-
isopropylpropanamide, 18a.
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Other measures indicating protection against oxidative stress: lactic
acid dehydrogenase release and caspase 3 activation. The maximal reduction
in lactic acid dehydrogenase (LDH) release induced by any compound did not exceed
16%. However, 1 pM of 7b, 15a and 18a significantly reduced LDH release, indicating
that the compounds were able to improve the integrity of the cell membrane in the
presence of oxidative stress (Figure 4A).
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Oxidative stress can cause apoptosis, mediated by a cascade of proteolytic enzymes
called caspases (cysteinyl aspartateꢀspecific proteases). Caspase 3 is one of a number of
effector caspases involved in this cascade. Compounds 7a, 7b and 15a all reduced
caspase 3 by about 60% at a concentration of 1 pM. Compounds 13a and 13g, which
were among the least effective compounds against cytotoxicity in the MTT assay, were
also significantly less active in reducing caspase 3 (Figure 4B).
Figure 4
Influence of the side chain position and of other substituents on
reduction of nitric oxide and cytokines in LPS activated RAW 246.7
macrophages. The steroid, budesonide was used as a positive control for these
experiments. Because of the large number of compounds that were tested at six different
concentrations, it was necessary to perform many separate experiments. The increase in
NO and cytokines achieved in response to LPS ranged for NO, 1.3ꢀ12.0 ꢁM; TNFꢀα, 1.4ꢀ
13.2 nM; ILꢀ6, 3ꢀ70 nM. Therefore, we expressed the effect of each concentration of
compound as a % of the LPS + medium value obtained in each experiment. Irrespective
of the initial values found for LPS, all the compounds showed a similar reduction in NO
to that of budesonide at concentrations of 1 pM to 1 nM, but budesonide caused a larger
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reduction in TNFꢀα and ILꢀ6 at higher concentrations. The greater efficacy of the steroid
results from its ability to prevent APꢀ1 and NFꢀκB from inducing gene transcription,
whereas the indoline derivatives appear to act further upstream by reducing
phosphorylation of MAPK p38.30 Maximal reductions in NO, TNFꢀα and ILꢀ6 in LPSꢀ
activated macrophages by indoline derivatives with 3ꢀaminopropyl or methyl propionate
side chains in position 1 (13a and 7a), or 3 (23 and 3), were achieved at a concentration
of 100 pM or 1 nM (Figure 5). There were no significant differences among the
compounds.
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Figure 5
3ꢀ(Indolinꢀ1ꢀyl)ꢀN-isopropylpropanamide, 18a, was 1000ꢀfold more potent than
indoline as an antioxidant and was also the most potent antiꢀinflammatory agent,
reducing NO and ILꢀ6 by 50% at a concentration of 100 pM. Compounds 13a and 7a did
not differ from indoline in their antiꢀoxidant activity but caused significantly greater
reductions in NO and TNFꢀα at all concentrations. 3ꢀHydroxypropyl derivative 9a, did
not reduce NO more than indoline 8a at any concentration, but was more effective in
decreasing TNFꢀα and ILꢀ6 at three of the higher concentrations (Figure 6).
Figure 6
The antiꢀinflammatory activity of compounds substituted at position 1 with 3ꢀ
aminopropyl, methyl propionate, 3ꢀ(Nꢀisopropylpropanamido) and 3ꢀhydroxypropyl
groups, and having additional substituents on the benzo ring, depended on: a) their antiꢀ
oxidant activity; b) the measure, cytokine or NO; c) the nature of the N-side chain; and d)
the substituent on the benzo ring. All the 3ꢀaminopropyl derivatives except 15i, showed
similar doseꢀrelated reductions in NO and TNFꢀα. 15i was less active at higher
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concentrations than the other amines in reducing NO and TNFꢀα. This is consistent with
its weaker antiꢀoxidant effect (Figure 7).
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Introduction of 5ꢀCl (7d) or 5ꢀMeO (7b) in 7a did not affect the reduction of NO but
7d caused a greater reduction in TNFꢀα at all concentrations tested (Figure 8). A 6ꢀMe
group in (7c) decreased the antiꢀinflammatory activity of 7a, as did hydrolysis of the ester
to give 7b’, in keeping with its lower protective activity against oxidative stress. In
contrast to the effect on methyl propionate, addition of 5ꢀCl (18d) or 6ꢀF (18e) markedly
reduced antiꢀinflammatory activity of 18a at almost all concentrations. Introduction of 5ꢀ
MeO (9b) in the alcohol derivative 9a also increased antiꢀinflammatory (Figure 9).
Figures 7ꢀ9
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Summary of in vitro studies: The most potent antiꢀinflammatory compounds are also
more effective protectants against cytotoxicity induced by oxidative stress and include
15a, 15b, 18a, 9b, 7a, 7b and 7d. In response to LPS stimulation these compounds may
act by inhibiting the activation of ILꢀ1Rꢀassociated kinaseꢀ1 (IRAKꢀ1) and IRAKꢀ4
thereby preventing nuclear translocation of NFꢀκB, like other antioxidants48 and natural
compounds or their derivatives that have antioxidant activity, although the latter are only
active in the ꢀM range.49 Indoline derivatives can also reduce nuclear translocation of
APꢀ1 by inhibiting the phosphorylation of MAPK p38.30 Introduction of 5ꢀCl (18d) or 6ꢀ
F (18e) in the most potent antioxidant and antiꢀinflammatory compound, 18a reduced
both these activities, as did addition of 7ꢀHO (15i) to 15a or 7ꢀBnO (13g) to 13a.
However, the introduction of 5ꢀCl (7d) to the weaker antioxidant, methyl propionate 7a
increased both activities.
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In vivo studies. Prevention of cytokine elevation induced in mice by
LPS. Representative indolines having a side chain carrying an ester (7a), isopropylamine
(15a), N-isopropylamido (18a) or N-isopropyl alcohol (9b), that showed the highest antiꢀ
inflammatory and antioxidant activity in vitro, were selected for in vivo studies in mice.
Of these, 15a injected sc in doses of 0.1ꢀ10 ꢁmoles/kg produced the most consistent
doseꢀrelated reduction in TNFꢀα and ILꢀ6 after their elevation in plasma (Figure 10) and
tissues (Figures 11 and 12). In keeping with the low concentration that reduced cytokines
in cells, 18a caused a maximal decrease in cytokines in plasma and brain at a dose of 0.1
ꢁmoles/kg. Higher doses were not more, or were less effective. 18a had no effect on ILꢀ6
in the liver, suggesting that it may be metabolized to an inactive compound in that organ.
All four compounds significantly reduced ILꢀ6 in the brain even at the lowest dose
indicating that they may have potential therapeutic benefit in pathological brain
conditions involving oxidative stress and chronic cytokine elevation.
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Figures 10ꢀ12
Acute toxicity of compounds in mice. Compounds 7a, 15a and 18a (100
ꢁmoles/kg) caused no change in food or water intake, body weight or activity of mice
during a period of 72 h observation and could not be distinguished from those given
saline.
ꢀ CONCLUSIONS
We have described the synthesis, antioxidant and antiꢀinflammatory activity in vitro
and in vivo of indoline and its derivatives substituted at position 1 with 3ꢀaminopropyl
[CH2CH2CH2NH2]; (3ꢀisopropylamino)propyl [CH2CH2CH2NHꢀiꢀPr]; methyl propionate
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[CH2CH2COOMe]; 3ꢀ(Nꢀisopropylpropanamido) [CH2CH2CONHꢀiꢀPr]; or 3ꢀhydroxy
propyl [CH2CH2CH2OH] groups. Some of the compounds have one of the following
additional substituents on the benzo ring: 5ꢀCl, 6ꢀCl, 5ꢀMeO, 6ꢀMe, 6ꢀF, 7ꢀHO or 6ꢀBnO,
7ꢀBnO. Indoline completely protected RAW 264.7 macrophages against cytotoxicity
induced by H2O2 at a concentration of 1 nM, but reduced NO, TNFꢀα and ILꢀ6 in LPS
activated macrophages by no more than 20% at concentrations of 100 pMꢀ100 nM. The
esters 7b and 7d; the N-isopropylamide derivatives 18a and 18d and the alcohol 9b, are
more potent antioxidants than indoline. Whether they have similar (15a and 15b), or
greater (7b, 7d, 18a, 18d, and 9b), antiꢀoxidant activity than 8a, the compounds are much
more potent at reducing NO and/or cytokines by 50% at concentrations ranging from 10
pM (18a and 7d), 100 pM (9b) and 1 nM (15a and 15b). Representative compounds
bearing either Nꢀisopropylamide (18a) (0.1 ꢁmole/kg), isopropylamine (15a) methyl
propionate (7a), or propyl alcohol (9b) (1 ꢁmole/kg) substituents reduce the elevation of
TNFꢀα and/or ILꢀ6 in plasma, spleen and brain induced by LPS (10 mg/kg) to a similar
extent to dexamethasone (5.6 ꢁmoles/kg). Compounds 7a, 15a and 9b, but not 18a, also
reduce ILꢀ6 in the liver. 18a may be metabolized to inactive compound(s) in that organ.
The data support our previous findings of the prevention by 7b and 15a (1 ꢁmole/kg)
of liver damage and increase in TNFꢀα and/or ILꢀ6 after injection of galactosamine and
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LPS in mice39. Representative indolines were shown to decrease cytokine release in LPS
28,29
activated macrophages
and in the liver after injection of LPS/galactosamine39 by
inhibiting nuclear translocation of APꢀ1 and NFꢀκB. Although there are no clinical data
on the indoline derivatives to compare with that of TNFꢀα monoclonal antibodies, several
of them reduced inflammation, colon damage, colon levels of TNFꢀα and ILꢀ6 in rat50
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and mouse models38 of ulcerative colitis and in LPS induced lung injury.30 We suggest
that the indoline derivatives, in particular 7a, 15a and 18a, may have therapeutic benefit
in a variety of pathological conditions in which there is an excessive increase in ROS and
proꢀinflammatory cytokines in the peripheral and central nervous systems. Because they
are not proteins and do not block the normal actions of cytokines, but only reduce their
excessive release, they are less likely to cause the adverse immune effects of TNFꢀα
antagonists. The compounds do not appear to cause acute toxicity in mice at 100 times
the active dose. However, it remains to be seen whether they also display a low incidence
of adverse effects after chronic administration in suitable animal disease models or in
humans.
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ꢀ EXPERIMENTAL SECTION
1
General Chemistry Methods. HꢀNMR, 13CꢀNMR spectra were obtained on
Bruker AvanceꢀDPXꢀ300, Avanceꢀ400, AvanceꢀDMXꢀ600 and AvanceꢀIIIꢀ700
spectrometers. Chemical shifts are expressed in ppm downfield from Me4Si (TMS), used
as internal standard, and values are given in δ scale. The "t" is indicative of a multiplet
similar to a triplet with second order characteristics. HRMS and Electron Spray
Ionization (ESI) were obtained on Synapt ESIꢀQꢀTOF, Waters companyꢀUK or Agilent
6545 QꢀTOF LCꢀMS. Progress of the reactions was monitored by TLC on silica gel
(Merck, Art. 5554). Flash chromatographic procedures were carried out on silica gel.
Moisture sensitive reactions were carried out in flameꢀdried vessels. Melting points were
determined on a FisherꢀJohns apparatus. Commercially available compounds were used
without further purification. The nomenclature of the compounds was given according to
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ChemDraw
Ultra
v.
16
(CambridgeSoft).
3ꢀ(3ꢀAminopropyl)ꢀindolinꢀ4ꢀyl
(1),29
ethyl(methyl)carbamate
dihydrochloride
methyl
3ꢀ(6ꢀ
7
8
9
((ethyl(methyl)carbamoyl)oxy)indolinꢀ3ꢀyl)propanoate pꢀtoluenesulfonate (2),28 and
methyl 3ꢀ(indolinꢀ3ꢀyl)propanoate hydrochloride (3), have been reported earlier.51 The
analytical methods used to determine purity were based on 1H and 13C NMR, HRMS, and
elemental analysis for those compounds that were not highly hygroscopic or were
obtained as nonꢀcrystalline oils. The final compounds which were tested biologically
displayed ≥95% purity. The structural model of compounds 3 and 7a were created using
Discovery Studio 4.1. The Electrostatic potentials calculations were carried out with
Gaussian 09 using B3LYP functional and 6ꢀ31G(d) basis set. Avogadro software (1.2.0
version) was used to display electrostatic potential.
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Methyl 3-(Indolin-1-yl)propanoate Hydrochloride, (7a). The hydrochloride
salt 7a prepared by addition of HCl (g) to 6a in dry diethyl ether was isolated as a
1
hygroscopic orange solid in approximately quantitative yield. HꢀNMR (300 MHz,
CD3OD) ppm δ 7.63ꢀ7.60 (m, 1H), 7.54ꢀ7.47 (m, 3H), 4.00 (t, J = 7.5 Hz, 2H), 3.86 (t, J
13
= 6.9 Hz, 2H), 3.74 (s, 3H), 3.37 (t, J = 7.5 Hz, 2H), 2.97 (t, J = 6.9 Hz, 2H; CꢀNMR
(75 MHz, CD3OD) ppm δ 172.10, 141.41, 136.65, 131.77, 129.94, 127.68, 119.88, 55.54,
53.33, 52.84, 30.25, 28.92; Anal. Calcd for C12H16ClNO2 (MW 241.72 g/mol): C, 59.63;
H, 6.67; N, 5.71. Found C, 59.85; H 6.71;1 N 5.83. HRMS: calcd. for C12H15NO2 (M+.;
ESI+) 205.1097, found 205.1101.
Methyl
3-(5-Methoxyindolin-1-yl)propanoate
Hydrochloride,
(7b).
Compound 7b prepared by addition of HCl (g) to 6b in dry diethyl ether was isolated as a
dark red oil in approximately quantitative yield. 1HꢀNMR (300 MHz, CD3OD) ppm δ 7.52
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(d, J = 8.7 Hz, 1H, 7.06ꢀ6.98 (m, 2H), 4.02 (t, J = 7.5 Hz, 2H), 3.84 (s, 3H), 3.81 (t, J =
7.2 Hz, 2H), 3.73 (s, 3H), 3.34 (t, J = 7.5 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H); 13CꢀNMR (75
MHz, CD3OD) ppm δ 172.01, 163.20, 138.55, 133.45, 121.00, 115.89, 112.13, 56.48,
56.05, 53.64, 52.87, 30.20, 29.11. Anal. Calcd for C13H18ClNO3 (MW 271.74 g/mol): C,
57.46; H, 6.68; N, 5.15. Found C, 57.63; H 6.92;1 N 5.38. MS (TOF MS ES+) m/z 236;
HRMS: calcd. for C13H17NO3 (MH+, ESI+) 236.12812, found 236.12731.
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3-(5-Methoxyindolin-1-yl)propanoic Acid Hydrochloride, (7b'). A solution
of 7b in 3N HCl was stirred for 48 h at room temperature and evaporated. The
precipitated 7b’ was isolated as a white hygroscopic solid in approximately quantitative
yield. 1HꢀNMR (300 MHz, CD3OD) ppm δ 7.52 (d, J = 8.7 Hz, 1H), 7.06 (s, 1H), 7.00 (d,
J =8.7 Hz, 1H), 4.02 (t, J = 7.2 Hz, 2H), 3.84 (s, 3H), 3.78 (t, J = 7.2 Hz, 2H), 3.34 (t, J =
6.9 Hz, 2H), 2.93 (t, J = 6.9 Hz, 2H); 13CꢀNMR (75 MHz, CD3OD) ppm δ 173.08, 163.05,
138.47, 133.47, 120.93, 115.82, 112.09, 56.47, 55.94, 53.74, 30.14, 29.07. HRMS: calcd.
for C12H15NO3 (MꢀH)ꢀ 220.09792, found 220.09767.
Methyl 3-(6-Methylindolin-1-yl)propanoate Hydrochloride, (7c). Compound
7c prepared by addition of HCl (g) to 6c in dry diethyl ether was isolated as a yellow oil in
approximately quantitative yield. 1HꢀNMR (300 MHz, CD3OD) ppm δ 7.46 (s, 1H), 7.38ꢀ
7.30 (m, 2H), 4.00 (t, J = 7.5 Hz, 2H), 3.84 (t, J = 7.2 Hz, 2H), 3.70 (s, 3H), 3.30 (t, J =
13
7.5 Hz, 2H), 3.00 (t, J = 6.9 Hz, 2H), 2.40 (s, 3H); CꢀNMR (75 MHz, CD3OD) ppm δ
171.75, 140.93, 140.55, 133.50, 132.68, 127.12, 120.34, 55.53, 53.19, 52.79, 30.16,
28.52, 21.34; HRMS: calcd. for C13H17NO2 (MH+, ESI+) 220.13321, found 220.1327.
Methyl 3-(5-Chloroindolin-1-yl)propanoate p-Toluenesulfonate, (7d).
Compound 7d prepared by addition of pꢀTSA to a solution of 6d in tert-butyl methyl
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ether. The ethereal solution was evaporated to give 7d as a brown oil. HꢀNMR (300
MHz, CD3OD) ppm δ 7.62 (d, J = 8.4 Hz, 4H), 7.53 (d, J = 8.4 Hz, 1H), 7.35ꢀ7.30 (m,
2H), 3.98 (t, J = 7.5 Hz, 2H), 3.79 (t, J = 7.2 Hz, 2H), 3.65 (s, 3H), 3.24 (t, J = 7.5 Hz,
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2H), 2.91 (t, J = 7.2 Hz, 2H), 2.29 (s, 6H); CꢀNMR (75 MHz, CD3OD) ppm δ 171.53,
142.60, 141.73, 139.59, 138.86, 137.05, 129.76, 129.62, 127.39, 126.60, 121.47, 55.77,
53.27, 52.72, 30.03, 28.65, 27.14, 21.32; MS (ES+) m/z 240 (MH+); HRMS: calcd. for
C12H14ClNO2 (M+.; ESI+) 239.07076, found 239.07129.
Methyl 3-(6-(Benzyloxy)indolin-1-yl)propanoate p-Toluenesulfonate, (7f).
Compound 7f prepared by addition of pꢀTSA (1 eq) to 6f (1 eq) in tert-butyl methyl ether
(10 mL) was isolated as a hygroscopic white solid. An Hꢀꢀ>D exchange took place when
7f was dissolved in CD3OD in an NMR tube. This exchange was detected by NMR
already after 5 min, and was shown to take place primarily at position 5, and partly at both
1
positions 5 and 7. HꢀNMR (600 MHz, CD3OD) ppm δ 7.66 (d, J = 8.4 Hz, 4.5H), 7.43
(d, J = 7.2 Hz, 2H), 7.36 (t, J = 7.2 Hz, 2H), 7.33 (s, 1H), 7.32ꢀ7.29 (m, 1H), 7.21 (d, J =
8.4 Hz, 4.5H), 7.15 (s, 0.4H), 5.10 (s, 2H), 3.93 (t, J = 8.2 Hz, 2H), 3.77 (t, J = 8.2 Hz,
13
2H), 3.71 (s, 3H), 3.20 (t, J = 8.2 Hz, 2H), 2.86 (t, J = 8.2 Hz, 2H), 2.35 (s, 7H); Cꢀ
NMR (150 MHz, CD3OD) ppm δ 172.30, 160.69, 143.50, 143.01, 141.75, 138.00, 129.84,
129.62, 129.15, 128.76, 127.88, 127.82, 126.97, 105.67, 71.70, 56.26, 52.91, 52.82,
30.40, 28.28, 21.33; MS (non deuterated product 7f). (EI+) m/z 311.152 (M+.), 312.155
(MH+); HRMS: calcd. for C19H21NO3 (M+.; EI+) 311.1521, found 311.1524 and calcd. for
C19H21NO3Na (MNa+, ESI+) 334.1419, found 334.1422.
3-(Indolin-1-yl)propan-1-ol Hydrochloride, (9a). Compound 9a prepared by
addition of HCl (g) to 9a free base in dry diethyl ether was isolated as a greyish
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hygroscopic solid in approximately quantitative yield. HꢀNMR (600 MHz, CD3OD) ppm
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δ 7.61 (d, J = 7.8 Hz, 1H), 7.53ꢀ7.52 (m, 2H), 7.51ꢀ7.48 (m, 1H), 4.01 (bt, 2H), 3.74 (t, J
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= 6.0 Hz, 2H), 3.67 (bt, 2H), 3.37 (t, J = 7.8 Hz, 2H), 2.02 (quint, J = 7.2 Hz, 2H); Cꢀ
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NMR (150 MHz, CD3OD) ppm δ 141.66, 136.68, 131.79, 129.96, 127.71, 119.97, 60.00,
56.17, 55.50, 29.00, 28.48; MS (ES+) m/z 178.2 (MH+); HRMS: calcd. for C11H15NO
(MH+, ESI+) 178.12264, found 178.12231.
3-(5-Methoxyindolin-1-yl)propan-1-ol Hydrochloride, (9b). Compound 9b
prepared by addition of HCl (g) to 9b free base in dry diethyl ether was isolated as a
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brown hygroscopic solid in approximately quantitative yield. HꢀNMR (700 MHz,
CD3OD, at 275 oK) ppm δ 7.53 (d, J = 9.1 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.97 (dd, J =
9.1, 2.8 Hz, 1H), 4.16 (br, 1H), 3.9ꢀ3.8 (br, 2H), 3.82 (s, 3H), 3.71 (t, J = 6.3 Hz, 2H),
3.45 (br, 1H), 3.36 (br, 1H), 3.27 (br, 1H), 2.07 (br, 1H), 1.98 (br, 1H); 13CꢀNMR (176
o
MHz, CD3OD, 275 K) ppm δ 162.66, 138.35, 133.53, 120.92, 115.542, 111.80, 59.58,
56.37, 55.73, 55.45, 29.03, 28.33. Anal. Calcd for C12H18ClNO2.0.5H2O (MW 252.11
g/mol): C, 57.03; H, 7.58; N, 5.54. Found C, 57.16; H 7.39; N 5.65. MS (ES+) m/z 208
(MH+); HRMS: calcd. for C12H17NO2 (MH+, ESI+) 208.13321, found 208.13375.
3-(Indolin-1-yl)propan-1-amine Hydrochloride, (13a).52 Compound 13a
prepared by addition of HCl to 12a (2 g, 11 mmol) in ether (60 mL), was crystallized from
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DCMꢀether and was isolated as a hygroscopic offꢀwhite solid. HꢀNMR (400 MHz,
CD3OD) ppm δ 7.06ꢀ7.025 (m, 2H), 6.65 (t, J = 7.6 Hz, 1H), 6.58 (d, J = 7.6 Hz, 1H),
3.31 (t, J = 8.2 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H), 3.07 (t, J = 7.6 Hz, 2H), 2.93 (t, J = 8.0
Hz, 2H), 1.99 (quint, J = 7.6 Hz, 2H); 13CꢀNMR (100 MHz, CD3OD) ppm δ 153.57,
131.45, 128.23, 125.38, 119.55, 108.80, 54.48, 48.27, 39.17, 29.41, 26.61. MS (EI+) m/z
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176.137 (M+.), 177.142 (MH+). Anal. Calcd for C11H17ClN2 (MW 212.11 g/mol): C,
62.11; H, 8.06; N, 13.17. Found C, 62.05; H 7.94; N 13.22. MS (EI+) m/z 176.137 (M+.),
177.142 (MH+); HRMS: calcd. for C11H16N2 (M+.; EI+) 176.1313, found 176.1370.
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3-(7-(Benzyloxy)indolin-1-yl)propan-1-amine
di-p-Toluenesulfonate,
(13g). Compound 13g prepared by addition of pꢀTSA to a solution of 12g in tert-butyl
methyl ether, was crystallized from DCMꢀether and was isolated as a hygroscopic white
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solid in approximately quantitative yield. HꢀNMR (300 MHz, CD3OD) ppm δ 7.69ꢀ7.65
(m, 4H), 7.52ꢀ7.33 (m, 6H), 7.20ꢀ7.14 (m, 5H), 7.03 (dd, J = 7.5, 0.6 Hz, 1H), 4.01 (t, J =
7.5 Hz, 2H), 3.60 ("t", J = 7.2 Hz, 2H), 3.35ꢀ3.30 (m, 2H), 2.96 (t, J = 7.5 Hz, 2H), 2.35
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(s, 6H), 2.12ꢀ2.06 (m, 2H); CꢀNMR (75 MHz, CD3OD) ppm δ 151.32, 143.28, 141.85,
138.83, 137.13, 133.91, 129.90, 129.82, 129.55, 129.08, 127.98, 126.86, 119.13, 113.67,
72.12, 66.84, 55.44, 54.01, 37.80, 29.32, 23.78, 21.31, 15.44; HRMS: calcd. for
C18H22N2ONa (M+.; ESI+) 305.1630, found 305.1626.
3-(Indolin-1-yl)-N-isopropylpropan-1-amine
Dihydrochloride,
(15a).
Compound 15a prepared by addition of HCl to a solution of 14a in ether was isolated as a
hygroscopic brown solid. 1HꢀNMR (400 MHz, CD3OD) ppm δ 7.67ꢀ7.65 (m, 1H), 7.54ꢀ
7.48 (m, 3H), 4.06 (t, J = 7.6 Hz, 2H), 3.74 (“t”, J = 8.0 Hz, 2H), 3.46ꢀ3.36 (m, 3H), 3.20
(t, J = 7.6 Hz, 2H), 2.27 (quint, J = 8 Hz, 2H); 13CꢀNMR (100 MHz, CD3OD) ppm δ
141.23, 136.63, 131.75, 129.94, 127.69, 119.97, 55.26, 54.39, 52.32, 43.00, 29.05, 23.15,
19.23, 13.19. Anal. Calcd for C14H24Cl2N2 (MW 291.26 g/mol): C, 57.73; H, 8.32; N,
9.62. Found C, 57.50; H 8.31; N 9.60. HRMS: calcd. for C14H22N2 (MH+, ESI+)
219.18558, found 219.18599.
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N-Isopropyl-3-(5-methoxyindolin-1-yl)propan-1-amine Dihydrochloride,
(15b). Compound 15b prepared by addition of HCl (g) to 14b in dry diethyl ether was
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isolated as a hygroscopic yellow solid in approximately quantitative yield. HꢀNMR (300
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MHz, CDCl3) ppm δ 7.08ꢀ7.05 (m, 1H), 6.91ꢀ6.90 (m, 1H), 6.85ꢀ6.82 (m, 1H), 3.77 (s,
3H), 3.67 (t, J = 7.8, 2H), 3.42ꢀ3.34 (m, 3H), 3.19ꢀ3.11 (m, 4H), 2.12 (quint, J = 7.5 Hz,
2H), 1.35 (d, J = 6.6 Hz, 6H); 13CꢀNMR (75 MHz, CDCl3) ppm δ 159.54, 139.55, 136.10,
116.08, 114.58, 112.34, 56.41, 55.43, 52.41, 52.08, 43.53, 29.41, 24.07, 19.25; MS (ES+)
m/z 249.2 (MH+); HRMS: calcd. for C15H24N2O (MH+, ESI+) 249.19614, found
249.19606.
1-(3-(Isopropylamino)propyl)indolin-7-ol di-p-Toluenesulfonate, (15i).
Compound 15i prepared by addition of pꢀTSA (2 eq) to 14i (1 eq) in tertꢀbutyl methyl
ether, was crystallized from DCMꢀether and was isolated as a hygroscopic solid in
quantitative yield. 1HꢀNMR (700 MHz, CD3OD+D2O) ppm δ 7.69 (d, J = 8.4 Hz, 4H, Hꢀ
20), 7.32 (t, J = 7.7 Hz, 1H), 7.25 (d, J = 8.4 Hz, 4H), 6.94 (dd, J = 7.7, 0.7 Hz, 1H), 6.91
(d, J = 7.7 Hz, 1H), 3.95 (t, J = 7.7 Hz, 2H), 3.66 ("t", J = 7.7 Hz, 2H), 3.37 (septet, J =
6.3 Hz, 1H), 3.34 (t, J = 7.7 Hz, 2H), 3.13 (t, J = 7.7 Hz, 2H), 2.37 (s, 6H), 2.16 (quint, J
= 7.7 Hz, 2H), 1.33 (d, J = 6.3 Hz, 6H); 13CꢀNMR (176 MHz, CD3OD+D2O) ppm δ
149.98, 142.95, 141.99, 138.18, 132.90, 129.92, 127.00, 126.82, 117.74, 116.41, 55.22,
53.35, 52.19, 43.02, 29.31, 23.06, 21.34, 19.20; HRMS: calcd. for C14H22N2O (M+.; EI+)
234.1732, found 234.1736.
3-(Indolin-1-yl)-N-isopropylpropanamide Hydrochloride, (18a). Compound
18a prepared by addition of 3N HCl to 17a in ethyl acetate was crystalized from
MeOH/ether and was isolated as a whiteꢀbrown solid, in approximately quantitative
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yield. Mp 92ꢀ93 oC; 1HꢀNMR (300 MHz, CD3OD) ppm δ 7.64 (d, J = 7.2 Hz, 1H), 7.53ꢀ
7.46 (m, 3H), 4.06 (t, J = 7.2 Hz, 2H), 3.89 (septet, J = 6.6 Hz, 1H), 3.87 (t, J = 6.9 Hz,
2H), 3.39 (t, J = 6.9 Hz, 2H), 2.87 (t, J = 6.6 Hz, 2H), 1.14 (d, J = 6.6 Hz, 6H); 13CꢀNMR
(75 MHz, CD3OD) ppm δ 169.96, 140.59, 136.64, 131.70, 129.66, 127.47, 120.18, 54.96,
53.65, 42.72, 31.22, 28.91, 22.44; MS (ES+) m/z 233 (MH+), 255 (MNa+); HRMS: calcd.
for C14H20N2O (MH+, ESI+) 233.1648, found 233.1647.
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N-Isopropyl-3-(5-methoxyindolin-1-yl)propanamide
Hydrochloride,
(18b). Compound 18b prepared by addition of 3N HCl to 17b in ethyl acetate was
isolated as an orange oil in approximately quantitative yield. 1HꢀNMR (400 MHz, CDCl3)
ppm δ 6.94ꢀ6.91 (m, 1H), 6.71 (d, J = 2.4 Hz, 1H), 6.59 (dd, J = 8.4, 2.4 Hz, 1H), 6.44 (d,
J = 8.4 Hz, 1H), 4.05 (septet, J = 6.8 Hz, 1H), 3.62 (s, 3H), 3.30ꢀ3.20 (m, 4H), 2.90ꢀ2.80
(m, 2H), 2.43 (t, J = 6.8 Hz, 2H), 1.10 (d, J = 6.8 Hz, 6H); 13CꢀNMR (100 MHz, CDCl3)
ppm δ 200.88, 152.91, 145.94, 131.40, 111.59, 107.70, 55.65, 53.57, 46.89, 40.86;
HRMS: calcd. for C14H22N2O2 (MNa+, ESI+) 285.15735, found 285.15824.
3-(5-Chloro-1H-indol-1-yl)-N-isopropylpropanamide
Hydrochloride,
(18d). Compound 18d prepared by addition of 3N HCl to 17d in EtOAc was isolated as a
dark yellow oil in approximately quantitative yield. 1HꢀNMR (300 MHz, CD3OD) ppm δ
7.66ꢀ7.63 (m, 1H), 7.56ꢀ7.50 (m, 2H), 4.08 (t, J = 7.5 Hz, 2H), 3.90 (septet, J = 6.6 Hz,
1H), 3.86 (t, J = 6.9 Hz, 2H), 3.41 (t, J = 7.5 Hz, 2H), 2.83 (t, J = 6.9 Hz, 2H), 1.14 (d, J
= 6.6 Hz, 6H); 13CꢀNMR (75 MHz, CD3OD) ppm δ 170.14, 139.73, 139.25, 137.35,
129.91, 127.685, 121.73, 55.57, 53.94, 42.82, 31.22, 29.03, 22.50; MS (ES+) m/z 267
(MH+), 289 (MNa+); HRMS: calcd. for C14H19ClN2O (MH+, ESI+) 267.12587, found
267.12535.
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3-(6-Fluoroindolin-1-yl)-N-isopropylpropanamide Hydrochloride, (18e).
Compound 18e prepared by addition of HCl (g) to 17e in dry diethyl ether was isolated as
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a hygroscopic whiteꢀpink solid, in approximately quantitative yield. HꢀNMR (400 MHz,
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CD3OD) ppm δ 7.52 (dd, J = 8.4, 5.4 Hz, 1H), 7.46 (dd, J = 8.4, 2.0 Hz, 1H), 7.26 (td, J =
8.8, 2.4 Hz, 1H), 4.06 (t, J = 7.6 Hz, 2H), 3.98 (septet, J = 6.4 Hz, 1H), 3.84 (t, J = 6.8
Hz, 2H), 3.33 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 6.8 Hz, 2H), 1.15 (d, J = 6.8 Hz, 6H); 13Cꢀ
NMR (100 MHz, CD3OD) ppm δ 170.48, 165.03+162.58, 142.71, 132.56,
128.89+128.80, 118.66+118.44, 108.09+107.81, 56.24, 53.90, 42.85, 31.17, 28.49, 22.54;
MS (ES+) m/z 251 (MH+), 273 (MNa+); HRMS: calcd. for C14H19FN2O (MH+, ESI+)
251.15542, found 251.15495.
3-(6-Chloroindolin-1-yl)-N-isopropylpropanamide
p-Toluenesulfonate,
(18h). Compound 18h, prepared by addition of pꢀTSA (1 eq) to 17h (1 eq) in tertꢀbutyl
methyl ether was isolated as a brown oil in 57% yield. 1HꢀNMR (300 MHz, CD3OD) ppm
δ 7.67 (d, J = 8.1 Hz, 2H), 7.58ꢀ7.54 (m, 1H), 7.42ꢀ7.39 (m, 2H), 7.22 (d, J = 8.1 Hz, 2H),
4.00ꢀ3.93 (m, 1H), 3.96 (t, J = 7.5 Hz, 2H), 3.76 (t, J = 6.6 Hz, 2H), 3.26 (t, J = 7.5 Hz,
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2H), 2.72 (t, J = 6.9 Hz, 2H), 2.36 (s, 3H), 1.13 (d, J = 6.6 Hz, 2H); CꢀNMR (75 MHz,
CD3OD) ppm δ 170.66, 143.69, 143.22, 141.84, 135.15, 134.98, 130.58, 129.87, 128.48,
126.86, 119.37, 55.81, 53.47, 55.81, 53.47, 42.76, 31.33, 28.61, 22.49, 21.32; MS (ES+)
m/z 267 (MH+), 289 (MNa+); HRMS: calcd. for C14H19ClN2O (MH+, ESI+) 267.12587,
found 267.12713.
3-(Indolin-3-yl)propan-1-amine di-p-Toluenesulfonate, (23). Compound 23,
prepared by addition of pꢀTSA to a solution of 22 in tert-butyl methyl ether was isolated
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δ 7.05ꢀ7.03 (m, 2H), 6.70 (t, J = 7.5 Hz, 1H), 6.60 (d, J = 7.8Hz, 1H), 6.74 (s, 1H), 2.77
(bs, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.69 (quint, J = 7.2 Hz, 2H);
13CꢀNMR (75 MHz, CDCl3) ppm δ 136.27, 127.11, 121.43, 121.16, 118.40, 118.38,
114.86, 111.08, 61.45, 41.40, 33.36, 29.61, 22.14, 13.86.
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PHARMACOLOGY.
Compounds 7a, 7b, 7b’, 7c, 7d, 9a, 9b, 13a, 13g, 15a, 15b, 15d, 15e, 15i, 18a, 18d,
18e, and 23, were tested as monoꢀ or dihydrochlorides, monoꢀ or diꢀpꢀtoluenesulfonate
salts. All concentrations are expressed as that of the respective salts.
Cell line. The mouse macrophage RAW 264.7 cell line was cultured in Dulbecco's
Modified Eagle's Medium (DMEM), 4500 mg/L Dꢀglucose with 10% fetal calf serum
(FCS), 10000 U/mL penicillin, 100 mg/mL streptomycin and 25 µg/mL Amphotericin B
at 37 °C with 95% air and 5% CO2.
Cell viability. Cell viability was determined using the MTT colorimetric assay. The
assay is based on the ability of mitochondrial dehydrogenases to reduce yellow MTT 3ꢀ
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide to purple insoluble formazan
crystals. RAW 264.7 cells were seeded at a density of 1x104 cells/mL in a 96ꢀwell plate
and incubated for 24 h. MTT stock solution prepared in FCS free medium was added to
each well to a final concentration of 0.5 mg/mL and incubated for 1 h at 37 °C. Media
was removed and the cells were lysed with 100% DMSO to allow the crystals to fully
solubilize. Optical density at 570 nm was determined using a Labsystems plateꢀreader.
Viability of cells treated with different compounds was compared to cells treated with
medium alone.
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Protective activity against oxidative stress. H2O2, a precursor of various
ROS that freely penetrates cell membranes, was chosen as the oxidant reagent for the
following experiments. Compounds were tested for their potential to protect against cell
death induced by H2O2. RAW 264.7 cells were seeded in 96 wellꢀplates at a density of 1 x
104 cells/mL and incubated with different indoline derivatives in concentrations ranging
from 1 pM to 100 nM, for 2 h prior to addition of H2O2 (100 µM). This H2O2
concentration reduced cell viability by 25ꢀ35% relative to that in vehicle treated cells. All
concentrations of each compound were tested in 2ꢀ3 experiments with a minimum of 6
wells per concentration in each experiment.
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LDH activity. An increase in lactate dehydrogenase (LDH) activity is indicative of
cell membrane damage. LDH is an oxidoreductase enzyme that catalyzes the
interconversion of pyruvate and lactate. LDH reduces NAD to NADH, which was
detected using a colorimetric assay (Sigma Aldrich Ltd. Mo USA). Cells were seeded and
incubated with H2O2 (100 µM) as described above. Experiments were performed in
serumꢀfree medium. Culture media were collected at the end of the experiment and
centrifuged. Each 50 ꢁL aliquot was incubated with 50 ꢁL mixed reaction solutions, at
room temperature, for 30 min. Absorbance was measured at 490 nm using a 96ꢀwell plate
reader. Color intensity is proportional to LDH activity.
Caspase 3 activity. The activity of caspase 3 was measured by means of a
luminescent assay (CaspaseꢀGlo 3/7 Assay Promega Ltd). Macrophages were seeded and
pretreated as described above with concentrations of 1 pM and 1 nM of several of the
compounds, 4 h before the addition of H2O2 (100 ꢁM). The medium was aspirated 90
min later, 100 ꢁL of fresh DMEM were added to each well and the plates were incubated
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overnight. CaspaseꢀGloR Reagent (100 ꢁL) was added to each well and the contents were
gently mixed using a plate shaker at 300–500 rpm for 30 sec. The plates were left at room
temperature for 30 minutes and then luminescence of each sample was measured in a
plateꢀreading luminometer (Cytation 3).
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Anti-inflammatory activity in vitro. RAW 264.7 cells were seeded at a density
of 1x105 cells per well in 48ꢀwell culture plates and grown overnight in DMEM
supplemented as indicated above. The wells were washed with PBS (pH 7.4) and the
compounds, prepared in sterile double distilled water, were added to give final
concentrations ranging from 1 pM to 100 nM in medium containing 2% FCS. The cells
o
were incubated for 2 h at 37 C prior to stimulation with LPS, 2.5 µg/mL from
Escherichia coli (SigmaꢀAldrich serotype 0111:B4). Supernatants were harvested after 8
h for measurement of TNFꢀα and after 24 h for ILꢀ6 and nitric oxide (NO) measurement.
NO production was detected by a colorimetric method using Griess reagent (0.2%
naphthylenediamine dihydrochloride, and 2% sulfanilamide in 5% phosphoric acid),
which measures the concentration of nitrite, a stable metabolite produced from NO. TNFꢀ
α and ILꢀ6 protein were detected by a commercial ELISA kit (Biolegend, San Diego, CA,
USA) according to the manufacturer’s instructions. Each concentration of each
compound was tested in twoꢀthree separate experiments in 6 wells per concentration.
Measurement of cytokines in mouse tissues. Male Balb/C mice (7ꢀ8 weeks
old) were injected sc with saline or compounds 7a, 9b, 15a and 18a, (0.1, 1 and 10
µmoles/kg) LPS (10 mg/kg) was injected ip 15 min later. Dexamethasone was used in the
current study as a positive control at a dose of 3 mg/kg (5.6 ꢁmoles/kg). This dose was
shown to cause maximal reduction in lethality and cytokine levels in mice injected with
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LPS. Mice were sacrificed 4 h later for measurements of cytokines in plasma and
spleen as previously described.30 The tissues were rapidly collected, snap frozen in liquid
nitrogen and stored at (ꢀ80 °C) until use. For cytokine measurements, spleens were
weighed and homogenized in PBS solution containing NaCl, (0.8%), NaH2PO4 (0.144%),
KH2PO4 (0.024%) and protease inhibitor cocktail (1%) at a speed of 24,000 rpm and
centrifuged at 14,000 g at 4 ºC for 15 min. Cytokine detection was performed in the
collected supernatants by ELISA as described above. Protein concentrations were
determined by means of a BCA protein assay kit. Under the conditions of the experiment
it was only possible to measure ILꢀ6 in the brain and liver.
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Acute toxicity of compounds in mice. Compounds 7a, 15a and 18a (100
ꢁmoles/kg) or saline (1 mL/100 g) were injected sc in groups of 3 Balb/C male mice
weighing 24ꢀ28 g. The mice were allowed free access to food and water and were
observed for 72 h.
Statistical analyses. The statistical evaluation of the data was performed using
Statistical Package for the Social Science (SPSS) 22. Comparisons were performed by
oneꢀway ANOVA followed by Duncan’s post hoc test. Differences between groups were
considered statistically significant at a level of p<0.05.
ꢀ ASSOCIATED CONTENT
Supporting information
The Supporting Information is available free of charge on the ACS Publications
website at
.
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The supporting information contains experimental procedures for the starting materials
and intermediates, which were not tested biologically. It also contains molecular
formula strings and some data (CVS)
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ꢀ AUTHOR INFORMATION
Corresponding Authors
*A.N. Eꢀmail: nudelman@biu.ac.il; telephone +972.52.393.2671.
*M.W. Eꢀmail: martar@ekmd.huji.ac.il; telephone +972.2.675.8731.
Author Contributions
#equal contributions
Notes
The authors declare no competing financial interest
ꢀ ACKNOWLEDGMENTS
This research was supported by the Ministry of Science, Technology & Space, Israel, and
by the ‘Marcus Center for Medicinal Chemistry’ at Bar Ilan University. We gratefully
acknowledge the performance of all statistical analyses by Mrs. Donna Apelbaum and
technical assistance of Dr. Priyashree Sunita.
ꢀ ABBREVIATIONS USED
AD, Alzheimer's disease; AChE, acetylcholine esterase; AP1, activator protein 1;
CDI, carbonyldiimidazole; CSF Colony Stimulating Factor; Dex, dexamethasone;
DMEM, Dulbecco's modification of Eagle's medium; FCS, Fetal Calf Serum; HBSS,
Hank's Balance Salt Solution; iNOS, inducible nitric oxide synthase; ILꢀ1β, interleukin
1β; ILꢀ6, interleukin 6; IPA, indole propionic acid; LDH, lactic acid dehydrogenase; LPS,
lipopolysaccharide; MAPK, mitogen activated protein kinase; MTT, 3ꢀ(4,5ꢀdimethylꢀ1,3ꢀ
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