Identification of indole scaffold-based dual inhibitors of NOD1 and NOD2

Article abstract of DOI:10.1016/j.bmc.2016.08.044

NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect. To gain additional insight into the structure–activity relationships of NOD1 inhibitors, a series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds 4 and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. The results obtained have enabled a deeper understanding of the structural requirements for NOD1 and NOD2 inhibition.

Full text of DOI:10.1016/j.bmc.2016.08.044

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of indole scaffold-based dual inhibitors of NOD1
and NOD2
Kaja Ke cˇ ek Plešec, Dunja Urban cˇ i cˇ , Martina Gobec, Aleksandra Pekošak, Tihomir Tomaši cˇ , Marko Anderluh,
⇑
ˇ
Irena Mlinari cˇ -Raš cˇ an, Ziga Jakopin
Faculty of Pharmacy, University of Ljubljana, Ašker cˇ eva 7, SI-1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial
role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has
also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target
in this respect. To gain additional insight into the structure–activity relationships of NOD1 inhibitors, a
series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory
activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds
Received 25 May 2016
Revised 22 August 2016
Accepted 23 August 2016
Available online xxxx
Keywords:
NOD1 inhibitor
NOD2 inhibitor
NF-jB activation
Scaffold hopping
4
and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory
activity on both targets in the low micromolar range. The results obtained have enabled a deeper under-
standing of the structural requirements for NOD1 and NOD2 inhibition.
Ó 2016 Elsevier Ltd. All rights reserved.
Bioisosteric replacement
Immunomodulation
1
. Introduction
NOD1 and NOD2 exist mainly in an inactive state, however,
recognition of their respective ligands enables them to bind
6
,7
The initial response of the innate immune system to invading
ATP and, in turn, to trigger signaling cascades that ultimately
bring about an inflammatory response, typified by proinflamma-
tory cytokine and chemokine secretion as well as secretion of
infectious agents is based on recognition of their distinct molecular
patterns through specific pattern recognition receptors. The
response to bacterial peptidoglycan components is mediated
mainly by the cytoplasmic nucleotide-binding oligomerization
domain (NOD)-like receptors, NOD1 and NOD2.1 These receptors
are structurally very similar as they belong to the AAA+ (ATPases
associated with various cellular activities) ATPase family of
1
,2,8
antimicrobial peptides.
The dipeptide D-Glu-meso-DAP (iE-
DAP) is the key sequence necessary and sufficient for ligand recog-
,2
9–11
nition and consequent triggering of NOD1 activation
NOD2 is activated by muramyl dipeptide (MDP).
while
1
2,13
The down-
stream signaling pathways of NODs are tightly controlled by
3
,4
8
proteins.
Both possess a tripartite structure composed of a
numerous accessory proteins. Both pathways lead to activation
C-terminal sensor domain comprising leucine-rich repeats and a
centrally-located nucleotide-binding domain, and differ solely in
the arrangement of their N-terminal effector domains that consist
of either one (NOD1) or two (NOD2) caspase activation and recruit-
of nuclear factor
jB (NF-jB) and mitogen-associated protein
kinases (MAPKs) while also playing important roles in apoptosis
8
,14–16
and autophagy.
Although the role of NODs in the induction of inflammatory
processes is still largely unknown, dysregulation of their function
caused either by single nucleotide polymorphisms or by other
mutations in their encoding genes, has been linked to numerous
5
ment domains.
8
,14,15
Abbreviations: C12-iE-DAP, lauroyl-
iE-DAP, -glutamyl-meso-diaminopimelic acid; IL, interleukin; MAPK, mitogen-
activated protein kinase; NF- B, nuclear factor B; NOD, nucleotide-binding
c
-
D
-glutamyl-meso-diaminopimelic acid;
inflammatory disorders that affect NF-
j
B activity.
Similarly,
c-D
overexpression of both NOD1 and NOD2 has been reported to elicit
j
j
8,14,15
NF-j
B activation.
Furthermore, ligand-elicited overactivation
oligomerization domain protein; PAMP, pathogen-associated molecular pattern;
RA, residual activity; RIP2, receptor-interacting serine–threonine protein kinase;
SAR, structure–activity relationship; SEAP, secreted embryonic alkaline
phosphatase.
of NOD1 has been associated with the progression of multiple scle-
1
7
18
19
rosis, coronary arteritis, ocular inflammation, fetal inflamma-
2
0
21,22
tion and adipose tissue inflammation.
In addition, activation
⇑
Corresponding author. Tel.: +386 1 4769 574; fax: + 386 1 4258 031.
E-mail address: ziga.jakopin@ffa.uni-lj.si ( Zˇ . Jakopin).
of NOD1 has recently been reported to augment cancer cell
http://dx.doi.org/10.1016/j.bmc.2016.08.044
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

2
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2
3
metastatic potential. Compared to NOD2, which is expressed
mostly in professional immune cells,24 NOD1 has a much wider tis-
2
5
sue distribution suggesting a broader role in inflammation. Con-
trolled suppression of NOD1 activity could therefore constitute a
novel approach to treating inflammatory and autoimmune dis-
eases which involve NOD1 in their pathogenesis. Targeting the
up-stream pathways responsible for NF-jB signaling would be
expected to leave most innate immunity defense mechanisms
intact. In addition, novel NOD1 inhibitors would provide chemical
probes that could enable further elucidation of the role of this pro-
tein in a variety of inflammatory processes.
Relatively little has been reported on the structural require-
ments for achieving NOD1 agonistic activity, apart from the reports
of Agnihotri and Jakopin.2
6,27
On the other hand, three distinct
chemical classes of selective small molecule NOD1 inhibitors, 2-
aminobenzimidazoles, purine-2,6-diones, and quinazolininones
have thus far been identified via a high-throughput screening
2
8–31
campaign.
-aminobenzimidazole class, has been found to selectively inhibit
NOD1-dependent NF-
B activation.31 The exact mechanism of
action is not known, although it has been shown to interact
Noditinib-1 (ML-130), that belongs to the
2
j
Figure 1. The design of novel NOD1 inhibitors based on structures of 1 (Noditinib-
1) and 2.
3
2
directly with NOD1 protein without affecting the binding of ATP.
Our group has been heavily engaged in the design, synthesis
and evaluation of NOD1 and NOD2 agonists.27,33–37 Here, however,
we report the results of focusing on the discovery of novel inhibi-
tors targeting NOD1 protein. Making use of the existing knowledge
of the SAR of known NOD1 inhibitors, we have designed and syn-
thesized new compounds based on an indole scaffold employing
the »scaffold hopping« approach. The NOD1-overexpressing HEK-
Blue cell line served as a model for studying the ability of these
a privileged structure and is present in a number of biologically
active compounds. Replacement of the benzimidazole scaffold
39
with an indole has already proved to be successful in increasing
the potency of HCV NS5B polymerase inhibitors, based on greater
40
lipophilicity of the scaffold, thus facilitating cell permeation.
Although SAR analysis of the benzimidazole series has shown that
28,31
the presence of an NH
2
group at 2-position is essential,
we
compounds to suppress the C12-iE-DAP (lauroyl-
c-D
-glutamyl-
replaced it with various functionalities in order to further explore
the chemical space. In addition, various substituents (R) were
introduced at position 4 of the phenyl moiety, in order to shed light
on the effect of the stereoelectronic nature of the substituent on
bioactivity and selectivity. Finally, sulfonyl, carbonyl and methy-
lene linkers were used in order to determine the effect of the nat-
ure of the spacer X on the bioactivity and selectivity, since it has
already been demonstrated that the sulfonyl group leads to the
most active compounds of the series and is also essential for their
meso-diaminopimelic acid)-induced activation of NF-
j
B by inhibi-
tion of NOD1. An analogous assay, utilizing NOD2-overexpressing
HEK-Blue cells and measuring inhibition of NOD2-dependent acti-
vation upon stimulation with MDP, was used as a counterscreen to
determine NOD1 versus NOD2 selectivity profiles of selected
compounds.
2
2
. Results
2
8,31
selectivity.
.1. Design
2
.2. Chemistry
The structure of the actual binding site of the reference NOD1
inhibitor Noditinib-1 (1) in the molecular target is still unknown,
so novel NOD1 inhibitors cannot be designed using structure-
based design, the structure–activity relationship (SAR) of known
NOD1 inhibitors thus provides the only possible basis for rational
drug design. Given the potent NOD1 inhibitory activity of 1 and
the balanced NOD1- and NOD2-inhibitory activities of compound
Initially, the synthesis was attempted of compounds 8 and 12,
that are counterparts to their benzimidazole reference compounds
1 and 2. The synthesis of 1-arylsulfonyl-indole derivative 8 is
depicted in Scheme 1. First, the carboxylic group of indole-2-
carboxylic acid was protected in the form of ethyl ester 3, followed
by reaction with p-toluenesulfonyl chloride in THF in the presence
of potassium tert-butoxide and a catalytic amount of 18-crown-6
2, these compounds were identified as interesting starting points
4
1
for the design of novel inhibitors. This enters an unexplored region
of chemical space and involves an in-depth exploration of their
SAR. Since no information regarding their binding modes is avail-
able, the ligand-based design was employed to investigate the
effect of the following structural changes (shown in Fig. 1): (i)
replacement of the central benzimidazole scaffold with an indole
core (Fig. 1, in blue); (ii) substitution, at position 2, of the indole
heterocycle (Fig. 1, Y); (iii) substitution at position 4 of the phenyl
moiety (Fig. 1, R) and (iv) variation of the linker connecting the
phenyl and indole moieties (Fig. 1, X).
to obtain compound 4. The hydrazide 5 was then obtained by
reacting compound 4 with hydrazine hydrate. Treatment of hydra-
zide 5 with a NaNO and HCl mixture gave the acyl azide which, on
2
heating in toluene, underwent a Curtius rearrangement to the cor-
responding isocyanate that, in turn, reacted with tert-butanol to
4
2,43
afford the desired tert-butyl carbamate 7.
This procedure, how-
ever, produced poor yields and an alternative synthetic method
was therefore adopted. Alkaline hydrolysis of the ester 4 furnished
4
1
the corresponding acid 6, which was transformed directly into
tert-butyl carbamate 7 in a convenient one-pot reaction, via a
Curtius rearrangement reaction employing diphenyl phosphorazi-
»
Scaffold hopping« is an approach in drug design, in which the
4
4,45
core framework of a molecule is replaced by another scaffold, with
the aim to improve the drug-like/physico-chemical properties of
the parent molecule or of finding similar potent compounds that
exist in novel chemical space.38 The indole scaffold is considered
date (DPPA) as described.
In the final step, acidolytic removal
of the N-protecting group was attempted by reacting the carba-
mate 7 with 4 M HCl in dioxane. Although the reaction yielded
the desired product 8, the latter proved to be unstable.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
H
3
C
3
H C
O
O
O
O
S
N
S
N
H
N
H
N
O
(i)
O
(ii)
O
(iii)
O
OH
OEt
OEt
OH
3
4
6
(vi)
(iv)
H
3
C
3
H C
3
H C
O
O
O
O
S
N
S
N
(v)
O
O
S
N
(vii)
O
NHBoc
+Cl-
2
NH
NHNH
2
5
7
8
t
2
Scheme 1. Synthesis of the 1-arylsulfonyl-indole derivative 8. Reagents and conditions: (i) SOCl , EtOH, reflux; (ii) p-toluenesulfonyl chloride, BuOK, 18-crown-6, THF;
t
t
+
(
iii) 2 M KOH, EtOH; (iv) DPPA, Et
3
N, 4 Å molecular sieves, BuOH, 82 °C; (v) 4 M HCl, dioxane; (vi) NH
2
NH
2
 H
2 2
O, EtOH; (vii) NaNO /H , then BuOH, toluene, 110 °C.
N-benzylated analog 12 was synthesized as shown in Scheme 2.
N-alkylation of the starting ethyl indole-2-carboxylate (3), in the
Several other derivatives of N-benzylated 2-iminoindolines
(compounds 33–38) and of N-sulfonylated ethyl indole-2-carboxy-
lates (compounds 39–43) were synthesized (Scheme 3). The start-
ing ethyl indole-2-carboxylate (3) was first N-alkylated with
4
6
presence of Cs
2
CO
3
,
was followed by alkaline hydrolysis of the
ester 9, yielding N-benzylated indole-2-carboxylic acid 10. Subse-
quent Curtius rearrangement of the latter, employing the success-
fully established DPPA procedure, gave the tert-butyl carbamate 11
in good yield.
In the final step, N-Boc deprotection was achieved by reacting
the carbamate 11 with 4 M HCl in dioxane, affording the desired
product 12 in the indoline tautomeric form.
46
appropriate benzyl bromides in the presence of Cs
2
CO
3
,
affording
the desired alkylated products 13–18 in excellent yields.
Compound 19, incorporating a cyclopropyl moiety, was obtained
from the corresponding bromo-derivative 18 via a modified Suzuki
4
7
cross-coupling reaction.
The obtained ethyl esters 13–19
were subjected to alkaline hydrolysis, giving the N-benzylated
Cl
Cl
Cl
Cl
H
N
O
(i)
N
O
(ii)
(iii)
N
O
N
(iv)
N
NH2+Cl-
NHBoc
OEt
OEt
OH
3
9
10
11
12
Scheme 2. Synthesis of the N-benzylated compound 12. Reagents and conditions: (i) 4-chlorobenzyl bromide, Cs CO
2
3
3
, acetonitrile, 60 °C; (ii) 2 M KOH, EtOH; (iii) DPPA, Et N,
t
4
Å molecular sieves, BuOH, 82 °C; (iv) 4 M HCl, dioxane.
R
1
R
1
R1
R1
H
N
O
(i)
N
O
(ii)
N
O
(iii)
N
(iv)
N
NH2+Cl-
NHBoc
OEt
OEt
OH
3
1
3 (R = H)
20 (R = H)
27
33
(R
1
1
(R
1
1
1
1
1
1
= H)
1
= H)
1
1
1
1
1
1
4 (R
1
1
= F)
= Me)
21 (R = F)
28 (R = Me)
1
34 (R = Me)
1
5 (R
22 (R = Me)
29 (R = iPr)
1
35 (R = iPr)
(
v)
1
6 (R = iPr)
23 (R = iPr)
30
36
1
1
1
1
(R
= CF
3
)
(R
1 3
= CF )
7 (R
8 (R
= CF
= Br)
3
)
24 (R = CF )
31 (R = Br)
37 (R = Br)
1
3
1
25 (R = Br)
1
32 (R = cPr)
1
38(R = cPr)
9 (R = cPr)
26 (R = cPr)
1
1
R
2
O
O
S
N
O
OEt
3
4
4
4
4
9 (R = F)
2
0 (R
1 (R
2
2
= MeO)
= NO
2
)
2 (R = Cl)
2
2 3
3 (R = CF )
Scheme 3. Synthesis of N-benzylated 2-iminoindolines (compounds 33–38) and N-sulfonylated ethyl indole-2-carboxylates (compounds 39–43). Reagents and conditions:
t
(
i) p-substituted benzyl bromide, Cs
2 3 3
CO , acetonitrile, 60 °C, except for compound 19; (ii) 2 M KOH, EtOH; (iii) DPPA, Et N, 4 Å molecular sieves, BuOH, 82 °C; (iv) 4 M HCl,
t
dioxane; (v) p-substituted arylsulfonyl chloride, BuOK, 18-crown-6, THF.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

4
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
indole-2-carboxylic acids 20–26.48 Subsequent Curtius rearrange-
ment of these acids employing the established DPPA procedure,
afforded the corresponding carbamates 27–32.
Acidolytic removal of the N-Boc protecting group in the final
step, using 4 M HCl in dioxane, yielded the final 2-iminoindolines
2.3. Biological characterization
2.3.1. Evaluation of cytotoxicity of synthesized compounds
An MTS assay in which the proliferation rates of HEK-Blue
NOD1 cells were measured in the presence of Noditinib-1 and of
the synthesized potential NOD1 inhibitors 4–19 and 33–45 was
employed to screen these compounds for potential cytotoxicity.
Cells were treated for 24 h with the compound of interest at con-
3
3–38. The N-arylsulfonylindole derivatives 39–43 were obtained
simply using the well established procedure by sulfonylating ethyl
indole-2-carboxylate (3) with the corresponding arylsulfonyl chlo-
ride in the presence of potassium tert-butoxide.
centrations of up to 25 lM. Comparison of the resulting metabolic
Further analogs of 8 were synthesized (Scheme 4). As described
in Scheme 1, ethyl indole-2-carboxylate (3) was sulfonylated with
p-toluenesulfonylchloride, followed by alkaline hydrolysis of the
resulting ester to afford 6. The latter was then converted to its car-
activities with that of the untreated control showed that all com-
pounds were well tolerated by HEK-Blue NOD1 cells, since their
residual metabolic activities did not fall below 80% at the maxi-
mum concentration tested (Fig. 2).
4
9
2 4 2 3
boxamide counterpart 45 using the [(Boc )O]-(NH ) CO system.
The carbonyl linker-incorporating congener of 8, compound 44,
was synthesized by acylation with p-toluoyl chloride in the pres-
ence of potassium tert-butoxide and 18-crown-6 in THF.41
The structures of all synthesized compounds were fully charac-
terized by spectroscopy. The presence of individual signals of the
2.3.2. Evaluation of NOD1-inhibitory activity of synthesized
compounds
These potential NOD1 inhibitors, together with Noditinib-1 (1),
were examined for their effectiveness using the HEK-Blue assay.
The cell line HEK-Blue NOD1 stably overexpresses the human
1
amino group protons in the H NMR spectra of N-benzylated prod-
ucts attests to the considerable double-bond character of the bond
NOD1 gene and an NF-
phosphatase (SEAP) reporter gene. Recognition of an NOD1 agonist
triggers a signaling pathway leading to the activation of NF- B and
jB-inducible secreted embryonic alkaline
of the exocyclic nitrogen atom to the
a
-carbon atom, as a conse-
j
5
0
quence of which these protons become non-equivalent. This
observation confirmed that 2-aminoindole salts existed in the
imine form. Finally, since compound 8 proved unstable, we
addressed the stability of other compounds belonging to the
the production of SEAP. During these experiments, the cells were
preincubated for 1 h with the newly synthesized potential NOD1
inhibitors and with Noditinib-1 at concentrations of 25 lM. They
were then stimulated for 18 h with 100 nM C12-iE-DAP, followed
by spectrophotometric determination of SEAP activity in the super-
natant (Fig. 3).
The positive control, a known NOD1 agonist C12-iE-DAP,
increased NF-jB transcriptional activity significantly relative to
2
2
-iminoindoline structural class. It should be noted, that the
-aminoindole bases are very sensitive to atmospheric oxygen
51
and are known to undergo autoxidation, whereas their salts are
stable under these conditions.5
0,52–54
Our compounds, although
initially in the form of hydrochlorides, would be expected to be
deprotonated under the conditions of the biological assays
employed (pH ꢀ7.2), rendering them prone to autoxidation. Thus,
to understand the stability of the free base, it was prepared from 12
by liberating it from the salt by treatment with sodium hydrogen-
carbonate and subsequent extraction to ether. Its presence was
that in untreated cells, whereas Noditinib-1 (1) completely abol-
ished this activity to its basal level (residual activity (RA) = 9%).
The derivatives 4–7 belonging to the N-sulfonylated set of com-
pounds are characterized by the presence of different groups at
position 2 of the N-sulfonylated indole heterocycle. From this set
only the ethyl ester 4 (RA = 24%) and hydrazide 5 (RA = 68%) inhib-
1
confirmed by H NMR spectroscopy immediately after the basifica-
ited the C12-iE-DAP-induced increase in NF-jB transcriptional
tion. The solution of free base 12b (see Supporting information)
was then left to stand in air for 3 weeks, its 1H NMR spectrum
being recorded at the final time-point. The spectra showed that
the synthesized 2-iminoindolines are stable towards autoxidation
activity, by 76% and 32%, respectively. The free acid analog 6 did
not exhibit any NOD1 inhibitory activity, most probably due to
its unfavorable cell permeation ability—the pH conditions under
which the assay is executed render the compound completely ion-
ized. Similarly, the tert-butyl carbamate 7 was completely devoid
(
see Supporting information).
H
3
C
3
H C
O
O
O
O
S
N
H
N
S
N
O
(iii)
O
(i), (ii)
O
OEt
NH2
OH
6
45
(iv)
3
H C
O
N
O
OEt
44
t
2
Scheme 4. Synthesis of compounds 44 and 45. Reagents and conditions: (i) p-toluenesulfonyl chloride, K BuO, 18-crown-6, THF; (ii) 2 M KOH, EtOH; (iii) Py, Boc O,
t
(
4 2 3
NH ) CO , THF, rt; (iv) p-toluoyl chloride, BuOK, 18-crown-6, THF.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 2. Proliferation rates, expressed as metabolic activities, of HEK-Blue cells after 24 h treatment with the synthesized compounds and Noditinib-1 (25
l
M). The data are
means of three independent experiments ± SD.
prepared and screened for NOD1-inhibitory activity in order to
further explore the chemical space of the N-sulfonyl series. In this
second set of compounds, we aimed to assess the effect of the size
of different substituents positioned at the 4-site by comparing the
obtained results to that for the parent compound 4 (p-methyl)
(
see Fig. 4). The 4-fluoro analog of the latter, compound 39, inhibited
NOD1-dependent activity (RA = 24%) to a degree similar to that of
the parent compound 4 (RA = 24%), whereas the 4-chloro analog
4
2 (RA = 30%) decreased the NOD1 activity by 70%. Replacement
of the methyl group by a trifluoromethyl bioisosteric moiety,
however, giving compound 43, led to a slightly smaller decrease
in activity (RA = 37%). The 4-methoxy analog 40 (RA = 36%) acted
similarly. Surprisingly, compound 41, which bears a bulkier 4-nitro
substituent, inhibited NOD1 activation effectively (RA = 27%), its
results being on par with those of 4 and 39. These results
demonstrate a remarkable tolerance of diverse 4-substituents in
the N-sulfonylated series of compounds for NOD1 inhibitory
activity.
Figure 3. Effects of synthesized compounds 4–7, 9–12 and Noditinib-1 (1) on the
inhibition of C12-iE-DAP-induced NF- B transcriptional activity. Secreted embry-
onic alkaline phosphatase (SEAP) activity in HEK-Blue cells was measured after
their exposure to the NOD1 inhibitors (25 M) for 1 h followed by stimulation with
C12-iE-DAP (100 nM) for 18 h. Columns represent means of duplicates of three
j
l
*
**
independent experiments. Error bars indicate ± SD; p <0.05 and p <0.01 vs C12-iE-
DAP-treated cells.
of NOD1 inhibitory activity. Thus, the presence of the
2
-carboxyethyl function is evidently pertinent for the bioactivity
of this structural type of compound.
Results obtained from the N-benzylated series were interesting,
the introduction of the 4-chlorobenzyl moiety resulting in
compound 9 which inhibited the C12-iE-DAP-induced increase in
NF-jB transcriptional activity by 71% (RA = 29%). In addition,
within this series the 2-iminoindoline 12 was also identified as a
moderate inhibitor of NOD1-dependent activity (RA = 44%). Simi-
larly to what was observed in the N-sulfonyl series, the free acid
1
0 and tert-butyl carbamate 11 displayed no activity whatsoever.
These results thus indicate that the presence of the 2-carboxyethyl
moiety and, to a minor extent, of the 2-imino substituent, is essen-
tial for the desired bioactivity of compounds of this structural type.
Given the promising inhibitory activity in the preliminary assay
of hit compound 4 (RA = 24%), further compounds incorporating
differentially para-substituted benzenesulfonyl moieties were
Figure 4. Inhibition of C12-iE-DAP-induced NF-
compounds 4 and 39–43. Secreted embryonic alkaline phosphatase (SEAP) activity
was measured in HEK-Blue cells after pretreatment with NOD1 inhibitors (25 M)
for 1 h followed by stimulation with C12-iE-DAP (100 nM) for 18 h. Columns
represent means of duplicates of three independent experiments. Error bars
indicate ± SD; **p <0.01 vs C12-iE-DAP-treated cells.
jB transcriptional activity by
l
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

6
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
The N-alkylated compound 9 exemplified another good starting
point for further optimization, due to its promising inhibitory
activity observed in the preliminary assay (RA = 29%). We therefore
explored the SAR of the N-benzylated series of compounds,
employing the well established bioisosteric replacement approach.
By introducing classical and nonclassical bioisosteric replacements
of the chlorine atom at the 4-position, we probed the novel chem-
ical space with the aim of finding an optimal balance of steric,
hydrophobic, electronic and hydrogen-bonding properties. The
set of novel synthesized compounds 13–19 includes six com-
pounds with different bioisosteric replacements for the chlorine
atom and one compound in which the chlorine atom was simply
removed. These bioisosteres of 9 were then screened for their
NOD1-inhibitory activity (Fig. 5).
Replacement of the 4-Cl substituent with its fluorine counter-
part, resulted in a significantly lower NOD1 inhibitory activity as
exemplified by 14 (RA = 53%). A similarly lower value of activity
was observed in the case of the removal of chlorine atom (com-
pound 13 (RA = 58%)). On the other hand, compound 15, with a
methyl group at the 4-position, was the best of the series, exhibit-
ing a high NOD1-inhibitory ability (RA = 24%). Introduction of the
trifluoromethyl bioisosteric moiety, however, resulted in a lower
degree of inhibition (compound 17 (RA = 46%)). Analogous results
were obtained for the 4-iPr analog 16 (RA = 42%), the 4-Br analog
Figure 6. Inhibition of C12-iE-DAP-induced NF-jB transcriptional activity by
compounds 12, 33–38, 44 and 45. Secreted embryonic alkaline phosphatase (SEAP)
activity was measured in HEK-Blue cells after pretreatment with NOD1 inhibitors
(
25
lM) followed by the addition of C12-iE-DAP (100 nM) and a subsequent
incubation for 18 h. Columns represent means of duplicates of three independent
*
**
experiments. Error bars indicate ± SD; p <0.05 and p <0.01 vs C12-iE-DAP-treated
cells.
NOD1-inhibitory activities of compounds
4 (sulfonyl linker;
RA = 24%), 44 (carbonyl linker; RA = 55%) and 15 (methylene lin-
ker; RA = 24%) showed that the nature of the linker is another
important factor determining NOD1 inhibitory activity. While
compounds 4 and 15, that incorporate the sulfonyl and methylene
linkers, displayed similar NOD1-inhibitory capacity, introduction
of a carbonyl linker (compound 44) significantly reduced NOD1-
inhibitory activity. Finally, the fact that the free acid analog of 4,
compound 6, exhibited no NOD1-inhibitory activity was presumed
also to be the result of its unfavorable cell permeation capacity,
being completely ionized under the pH of the assay. Replacement
of the carboxylic acid function of 6 by a carboxamide moiety gave
compound 45, that displayed only weak NOD1-inhibitory activity
(RA = 76%) further reinforcing the presumption.
1
8 (RA = 45%) and the 4-cyclopropyl analog 19 (RA = 39%).
The SAR of the N-benzylated 2-iminoindoline structural type of
compound, exemplified by hit compound 12 (RA = 44%), was next
explored, using the bioisosteric replacement approach described
above. Six new compounds 33–38 were prepared, five of them
incorporating different bioisosteric replacements for the chlorine
atom and one in which the chlorine atom was removed. As
expected, bioisosteric replacement of the chlorine substituent
resulted in complete loss of inhibitory activity for all but one of
the compounds (Fig. 6).
The only exception was compound 33, lacking a substituent at
4
-position, which still retained weak NOD1-inhibitory activity
(
RA = 72%), although less than that of the parent compound. These
results indicate that the chlorine atom is important for the activity
of this series of compounds, since its replacement rendered the
compounds 34–38 inactive.
The effect of the linker connecting the indole moiety to the
aromatic ring was investigated. Comparison of the exhibited
2.3.3. Determination of IC50 of selected compounds and their
selectivity versus NOD2
The new group of inhibitors was further investigated by select-
ing certain compounds for additional experiments in which their
IC50 and dose dependence were determined (listed in Table 1).
An analogous assay on the HEK-Blue NOD2 cell line was used as
a counterscreen, in terms of ascertaining the compounds’ selectiv-
ity profile, by measuring the effect on NOD2-dependent activation
of NF-
obtained with experiments carried out at a single concentration
of 25 M. The NOD1 and NOD2 inhibition effects of all the com-
jB. The dose-dependence results corroborated those
l
pounds tested are dose-dependent, following a non-linear semilog-
arithmic model. The reference NOD1 inhibitor, Noditinib-1 (1) (IC50
(
NOD1) = 0.771 lM; IC50 (NOD2) = 54.9 lM) exhibits a submicro-
molar IC50 on NOD1 and is ꢀ75-fold selective towards NOD2. These
results are in agreement with previously reported activities.31
Importantly, these results reveal the nonselective nature of the
indole scaffold in terms of inhibition of NOD1 and NOD2. The
chemical class of N-sulfonylated indoles possessing the 2-ethoxy-
carbonyl moiety emerged as the most potent of the series, display-
ing similar potencies in the micromolar range toward NOD1 and
NOD2. Compound
4 (IC50 (NOD1) = 5.74 lM; IC50 (NOD2) =
6
.45 M) was identified as the best of the series, possessing
l
Figure 5. Effects of compounds 9 and 13–19 on the inhibition of C12-iE-DAP-
NOD1- and NOD2-inhibitory activities in the lower micromolar
range. These results show that compound 4 is 7-fold less potent
than Noditinib-1 (1) in terms of NOD1 inhibition and completely
devoid of selective activity for NOD1 or NOD2 as opposed to Nodi-
tinib-1 (1). Further, the results provide some indication as to how
the stereoelectronic nature of the substituent R at the 4-position of
induced NF-
SEAP) activity was measured in HEK-Blue cells after pretreatment with NOD1
inhibitors (25 M) followed by the addition of C12-iE-DAP (100 nM) and
jB transcriptional activity. Secreted embryonic alkaline phosphatase
(
l
a
subsequent incubation for 18 h. Columns represent means of duplicates of three
independent experiments. Error bars indicate ± SD; *p <0.05 and **p <0.01 versus
C12-iE-DAP-treated cells.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Table 1
C12-iE-DAP (a synthetic NOD1 agonist) and MDP (NOD2 agonist)
were obtained from InvivoGen, Inc., (San Diego, CA); LPS was from
Sigma. The NOD1 antagonist, Noditinib-1 (ML-130), was synthe-
Inhibition of NOD1 and NOD2 by selected compounds
H
3
C
R
Cl
3
1
sized as described. Analytical TLC was performed on Merck 60
F254 silica gel plates (0.25 mm), using visualization with ultravio-
let light and ninhydrin. Column chromatography was carried out
on silica gel 60 (particle size 240–400 mesh). Melting points were
determined on a Reichert hot stage microscope and are uncor-
O
S
N
X
N
O
O
N
NH
2
NH
2
+Cl-
N
OEt
1
13
rected. H and C NMR spectra were recorded at 400 MHz and
00 MHz, respectively, on a Bruker AVANCE III spectrometer in
DMSO-d , CDCl , or MeOH-d solution, with TMS as the internal
1
(Noditinib-1)
compounds 4,15,39,41,42,44
12
1
6
3
4
Compd
R
X
IC50 NOD1 (
lM)
IC50 NOD2 (lM)
standard. Spectra were assigned using gradient COSY and HSQC
experiments. IR spectra were recorded on a Perkin–Elmer 1600
FT-IR spectrometer. Mass spectra were obtained using a VG-Ana-
lytical Autospec Q mass spectrometer. HPLC analyses were per-
formed on an Agilent Technologies HP 1100 instrument with
G1365B UV–VIS detector (210, 220 or 254 nm), using a Luna C18
column (4.6 Â 150 mm) at a flow rate of 1 mL/min. The eluant
was a mixture of 0.1% TFA in water (A) and acetonitrile (B). Method
A: Gradient was 50% B to 80% B in 30 min; Method B: Gradient was
30% B to 80% B in 32 min; Method C: Gradient was 30% B to 80% B
in 30 min. The purity of all pharmacologically investigated com-
pounds was >95% as determined by RP-HPLC.
1
4
—
CH
F
NO
Cl
—
0.771
5.74
9.22
15.0
19.8
16.0
7.78
15.7
54.9
6.45
12.6
16.9
23.4
24.6
9.04
>25
3
SO
SO
SO
SO
CO
CH
—
2
2
2
2
39
41
42
44
15
12
2
CH
CH
—
3
3
2
the phenyl moiety affects the bioactivity. Introduction of bulkier
groups possessing electron-withdrawing properties have been
shown, here and elsewhere,31 to decrease inhibitory potency on
both targets, as exemplified by the 4-chloro analog 42 (IC50
(
NOD1) = 19.8
lM; IC50 (NOD2) = 23.4
l
M) and the 4-nitro analog
4.2. General procedures
4
1 (IC50 (NOD1) = 15.0
l
M; IC50 (NOD2) = 16.9 l
M). On the other
hand, replacement of the 4-methyl group by a 4-fluoro group
4.2.1. N-sulfonylation of indole derivatives
resulted in NOD-inhibitory activities (compound 39; IC50 (NOD1)
Ethyl indole-2-carboxylate (3) (5 mmol) was dissolved in THF
(15 mL); potassium tert-butoxide (7.5 mmol) was then added and
the mixture stirred for 15 min at room temperature. Subsequently,
the corresponding sulfonyl chloride (5.5 mmol) was added to the
reaction mixture which was heated overnight at reflux. Upon com-
pletion of the reaction, which was monitored by TLC, the mixture
was concentrated in vacuo and the residue was dissolved in
dichloromethane (80 mL). The organic phase was washed with
=
9.22 lM; IC50 (NOD2) = 12.6 lM) similar to those of the parent
compound 4. In general, the presence of an electron-donating
group on the phenyl moiety resulted in compounds with higher
potencies. The nature of the linker type is also seen to be important
in determining the NOD1- and NOD2-inhibitory activities. While
the sulfonyl- and methylene-incorporating analogs 4 and 15
displayed similar activities in NOD1 and NOD2 inhibition, their
carbonyl-incorporating congener 44 exhibited ꢀ3-fold lower
potencies against the two targets. Finally, the only representative
of the 2-iminoindoline class, compound 12, exhibited an IC50 of
water (2 Â 30 mL), dried with anhydrous Na
2 4
SO then concen-
trated in vacuo. The residue was purified by flash silica gel column
chromatography (gradient elution; starting eluent: hexane/ethyl
acetate 6:1 v/v) to afford pure N-sulfonylated compounds.
1
5.7
mary, compounds 4 and 15 exhibit balanced dual activities of less
than 10 M on the two targets. Our SAR exploratory campaign has
lM on NOD1, whereas its NOD2 IC50 was >25 lM. In sum-
l
4.2.2. N-alkylation of indole derivatives
thus resulted in the identification of dual NOD1 and NOD2 inhibi-
tors based on a novel scaffold.
Ethyl indole-2-carboxylate (3) (5.0 mmol) was first dissolved in
acetonitrile (40 mL); cesium carbonate (7.5 mmol) was then added
and the mixture stirred for 15 min at room temperature. Subse-
quently, the corresponding alkyl bromide (5.5 mmol) was added
to the reaction mixture which was heated overnight at reflux.
Upon completion of the reaction, which was monitored by TLC,
the mixture was concentrated in vacuo and the residue dissolved
in dichloromethane (80 mL). The organic phase was washed with
3
. Conclusions
In conclusion, therapeutic targeting of NOD1 is still largely
unexplored due to the lack of information regarding the underlying
mechanisms of NOD1 activation and its regulation. A new class of
2 4
water (2 Â 30 mL), dried with anhydrous Na SO and then concen-
2
-ethoxycarbonylindoles has here been identified as providing
trated in vacuo to afford sufficiently pure N-alkylated products.
potent dual inhibitors of NOD1/2-induced NF- B transcriptional
j
activation. This ligand-based work has established a deeper under-
standing of the structure–activity relationship with regard to the
NOD1- and NOD2-inhibitory activities. The synthesized com-
pounds described here provide not only new chemical tools for fur-
ther development of NOD1 and NOD2 inhibitors but also valuable
research tools for elucidating the role of these proteins in NOD-
related diseases as well as in normal host-defense mechanisms.
4
.2.3. Alkaline hydrolysis
To a solution of ethyl ester (2 mmol) in ethanol was added 1 M
KOH (4 mL) and the mixture stirred for 24 h at room temperature.
Upon completion of the reaction which was monitored by TLC, the
ethanol was evaporated from the mixture and the remaining water
phase acidified with 1 M HCl to pH ꢀ3. The obtained precipitate
was filtered off and dried at 75 °C in an oven to afford compounds.
4
4
. Experimental section
4.2.4. Curtius rearrangement
To a solution of carboxylic acid (1 mmol) in tert-butanol were
added pulverized 4 Å molecular sieves (100 mg). The mixture
was stirred at 30 °C. After 20 min triethylamine (1.5 equiv;
1.5 mmol) and diphenylphosphorazidate (1.5 equiv; 1.5 mmol)
were added and the mixture was heated overnight at 82 °C. Upon
.1. General
Chemicals were obtained from Acros, Aldrich Chemical Co.,
Molekula and Fluka, and used without further purification.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

8
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
completion of the reaction, the mixture was concentrated in vacuo
and the residue purified by flash silica gel column chromatography
7.32–7.34 (m, 2H, indole-H), 7.43–7.45 (m, 1H, indole-H), 7.85 (d,
1H, J = 7.8 Hz, Ar–H), 8.21 (d, 2H, J = 7.8 Hz, Ar–H), 8.35 (s, 1H,
1
3
(
gradient elution; starting eluent: hexane/ethyl acetate 6:1 v/v) to
Ar–H), 11.76 (s, 1H, –COOH) ppm. C NMR (DMSO-d
d = 21.03, 114.90, 115.45, 122.68, 124.24, 126.78, 127.09, 128.15,
29.85, 132.99, 134.45, 136.90, 145.34, 162.11 ppm. MS (ESI):
6
, 100 MHz):
afford compounds.
1
+
4
.2.5. Acidolytic cleavage of the Boc protecting group
m/z (%) = 316.1 (M+H) . IR (ATR):
m
= 2841, 1703, 1369, 1203,
À1
To an ice-chilled stirred mixture of tert-butyl carbamates
1177, 1151, 1130, 1086, 816, 744, 672, 624, 570, 560 cm . HPLC
(
0.3 mmol) in dioxane (5 mL) 4 M HCl in dioxane was added and
R 4
(254 nm): 100%, t = 15.69 min. HRMS Calcd for C16H14NO S m/z:
+
the mixture allowed to warm to room temperature. After 3 h the
reaction was complete and the solvent was evaporated in vacuo.
The residue was washed three times with diethyl ether giving suf-
ficiently pure products.
316.0644 (M+H) , found 316.0641. Anal. Calcd for C16
4
H13NO S
(%): C, 60.94; H, 4.16; N, 4.44. Found: C, 60.66; H, 3.82; N, 4.51.
4.3.5. tert-Butyl (1-(4-toluenesulfonyl)-1H-indole-2-yl)carba-
mate (7)
4
4
.3. Characterization of compounds
Synthesized from 6 (1 mmol) according to the General proce-
dure for Curtius rearrangement. Pink crystals, yield: 193 mg
1
.3.1. Ethyl 1H-indole-2-carboxylate (3)
Synthesized from indole-2-carboxylic acid according to the stan-
(50%); mp/°C.
(–CH
H
NMR (CDCl
3
, 400 MHz): d = 1.49 (s, 9H,
3
)
3
), 2.26 (s, 2H, Ar–CH
3
), 6.67 (s, 1H, –NH-CO), 7.08–7.12
dard procedure for acid-catalyzed esterification (150 mmol). Yellow
(m, 3H, indole-H and Ar–H), 7.24–7.26 (m, 1H, indole-H), 7.55 (d,
amorphous solid, yield: 24,7 g (87%); mp 116–120 °C; lit. 122–
2H, J = 8.4 Hz, indole-H), 7.90–7.92 (m, 1H, Ar–H), 8.45 (s, 1H,
1
13
1
4
7
7
25 °C. H NMR (CDCl
.44 (q, 2H, J = 7.2 Hz, –CH
.26–7.27 (m, 1H, indole-H), 7.33–7.37 (m, 1H, indole-H),
.44–7.46 (m, 1H, indole-H), 7.71–7.73 (m, 1H, indole-H), 8.92
3
, 400 MHz): d = 1.45 (t, 3H, J = 7.2 Hz, –CH
3
),
Ar–H) ppm.
6
C NMR (DMSO-d , 100 MHz): d = 20.96, 27.93,
2
CH ), 7.16–7.20 (m, 1H, indole-H),
3
80.10, 102.94, 114.30, 120.49, 123.91, 124.01, 126.52, 128.84,
129.97, 133.13, 134.06, 145.39, 152.89 ppm. MS (ESI): m/z (%) =
+
387.1 (M+H) . IR (ATR):
m
= 3384, 2977, 1733, 1597, 1513, 1453,
+
(
(
s, 1H, indole-NH) ppm. MS (ESI): m/z (%) = 190.1 (M+H) . IR
ATR):
1336, 1297, 1243, 1213, 1144, 1086, 1019, 923, 850, 797, 743,
À1
m
= 3307, 1686, 1525, 1381, 1339, 1307, 1247, 1200, 1144,
702, 633, 630, 571 cm . HPLC (254 nm): 97.7%, t
HRMS Calcd for
387.1376.
R
= 30.97 min.
À1
+
1
020, 820, 771, 743, 663, 607, 580 cm
.
HRMS Calcd for
20 23 2 4
C H N O S m/z: 387.1379 (M+H) , found
+
11 2
C H12NO m/z: 190.0868 (M+H) , found 190.0865.
4
.3.2. Ethyl 1-(4-toluenesulfonyl)-1H-indole-2-carboxylate (4)55
Synthesized from 3 (5 mmol) according to the General proce-
4.3.6. 1-(4-Toluenesulfonyl)-indoline-2-imminium chloride (8)
Synthesized from 7 (0.5 mmol) according to the General proce-
dure for N-sulfonylation. Orange crystals, yield: 1,27 g (74%); mp
dure for acidolysis. Orange solidified oil, yield: 146 mg (90%); mp/
1
1
9
–
7
5–97 °C. H NMR (CDCl
CH ), 2.40 (s, 3H, Ar–CH
.27–7.29 (m, 4H, indole-H and Ar–H), 7.34–7,35 (m, 1H, indole-
3
, 400 MHz): d = 1.42 (t, 3H, J = 7.2 Hz,
°C. H NMR (CDCl
indoline-CH
2
3
, 400 MHz): d = 2.43 (s, 3H, –Ar–CH
), 5.71–5.73 (m, 1H, indole-H), 6.81–6.83 (m, 3H,
indole-H), 7.89–7.92 (m, 4H, Ar–H), 8.89 (s, 1H, indole-NH ) ppm.
3
), 3.23 (s, 2H,
3
3
), 4.43 (q, 2H, J = 7.2 Hz, –CH –CH ),
2
3
2
+
H), 7.42–7.44 (m, 1H, indole-H), 7.57–7.59 (m, 1H, Ar–H), 7.92 (d,
H, J = 8.4 Hz, Ar–H), 8.12–8.14 (m, H, Ar–H) ppm. 13C NMR
DMSO-d , 100 MHz): d = 13.86, 21.00, 61.77, 114.91, 116.43,
MS (ESI): m/z (%) = 288.1 (MÀCl+H) . IR (ATR):
m
= 2961, 2359,
À1
2
2341, 1660, 1406, 1327, 1258, 1012, 791, 668, 560 cm . HRMS
+
(
6
Calcd for C15
H
16
N
2
O
2
S m/z: 288.0932 (MÀCl+H) , found 288.0938.
1
1
22.86, 124.43, 126.95, 127.15, 128.07, 129.93, 131.48, 134.15,
36.95, 145.51, 160.86 ppm. MS (ESI): m/z (%) = 344.1 (M+H) . IR
+
4.3.7. Ethyl 1-(4-chlorobenzyl)-1H-indole-2-carboxylate (9)57
Synthesized from 3 (5 mmol) according to the General proce-
(
1
ATR):
m
= 1729, 1366, 1337, 1264, 1191, 1175, 1147, 1129, 1092,
À1
042, 1017, 816, 758, 743, 676, 652, 579, 555 cm . HPLC
dure for N-alkylation. Yellow crystals, yield: 1,21 g (77%); mp
1
(
3
254 nm): 100%, t
44.0957 (M+H) , found 344.0954.
R
= 24.66 min. HRMS Calcd for C18
4
H18NO S m/z:
98–100 °C. H NMR (CDCl
3
, 400 MHz): d = 1.29 (t, 3H, J = 7.2 Hz,
CH ), 5.85 (s, 2H, Ar–CH ), 7.03
d, 2H, J = 8.0 Hz, Ar–H), 7.17 (t, 1H, J = 8.0 Hz, Ar–H), 7.31–7.39
(m, 4H, indole-H and Ar–H), 7.58 (dd, 1H, J = 7.2 Hz, J = 0.4 Hz,
+
–CH
3
), 4.29 (q, 2H, J = 7.2 Hz, –CH
2
3
2
(
4
.3.3. Ethyl 1-(4-toluenesulfonyl)-1H-indole-2-hydrazide (5)
Synthesized from 4 (2 mmol) according to the following proce-
13
indole-H), 7.76 (d, 1H, J = 8.0 Hz, indole-H) ppm.
C NMR
dure for hydrazinolysis. Orange crystals, yield: 546 mg (83%); mp
(DMSO-d , 100 MHz): d = 14.07, 46.49, 60.48, 110.77, 111.19,
6
1
9
7–100 °C.
Ar–CH ), 4.56–4.59 (m, 2H, –NH–NH
.26–7.28 (m, 1H, indole-H), 7.37–7.40 (m, 3H, indole-H),
.62–7.64 (m, 1H, Ar–H), 7.94–7.97 (m, 1H, Ar–H), 8.06 (d, 2H,
) ppm. 13C NMR (DMSO-
, 100 MHz): d = 21.03, 69.75, 111.53, 114.23, 122.04, 123.86,
25.73, 127.42, 128.29, 129.80, 134.55, 135.52, 145.32,
H
NMR (DMSO-d
6
,
400 MHz): d = 2.34 (s, 3H,
120.92, 122.53, 125.35, 125.52, 127.05, 128.08, 128.45, 131.60,
+
3
2
), 6.94 (s, 1H, indole-H),
137.51, 138.95, 161.16 ppm. MS (ESI): m/z (%) = 314.1 (M+H) . IR
7
7
(ATR):
m
= 1709, 1517 1488, 1456, 1410, 1259, 1219, 1182, 1139,
À1
1117, 1012, 825, 798, 760, 742 cm . HPLC (254 nm): 95.1%,
J = 6.2 Hz, Ar–H), 9.96 (s, 1H, –NH–NH
2
t
R
= 30.90 min. HRMS Calcd for
C
18
H
17ClNO
2
m/z: 314.0948
+
d
1
1
1
1
9
6
(M+H) , found 314.0951.
+
4.3.8. 1-(4-Chlorobenzyl)-1H-indole-2-carboxylic acid (10)57
Synthesized from 9 (3 mmol) according to the General proce-
60.51 ppm. MS (ESI): m/z (%) = 330.1 (M+H) . IR (ATR):
m = 2964,
676, 1544, 1483, 1446, 1368, 1342, 1259, 1174, 1149, 1087,
À1
015, 926, 869, 796, 747, 669, 631, 581 cm . HPLC (254 nm):
dure for alkaline hydrolysis. White crystals, yield: 846 mg (99%);
1
9.5%, t
R
= 7.79 min. HRMS Calcd for C16
H
16
N
3
O
3
S m/z: 330.0912
S Â 0.65C
%): C, 57.83; H, 5.30; N, 11.69. Found: C, 57.76; H, 5.26; N, 11.71.
mp 198–202 °C. H NMR (DMSO-d
6
, 400 MHz): d = 5.87 (s, 2H,
+
(
M+H) , found 330.0918. Anal. Calcd for C16
H
15
N
3
O
3
H
2 6
O
Ar–CH
7.34 (m, 4H, indole-H and Ar–H), 7.53–7.56 (m, 1H, indole-H),
.70–7.72 (m, 1H, indole-H), 13.00 (s, 1H, –COOH) ppm. 13C NMR
(DMSO-d 100 MHz): d = 14.39, 56.35, 56.76, 62.27, 69.94,
2
), 7.03–7.05 (m, 2H, Ar–H), 7.12–7.15 (m, 1H, Ar–H), 7.28–
(
7
4
.3.4. 1-(4-Toluenesulfonyl)-1H-indole-2-carboxylic acid (6)56
Synthesized from 5 (1 mmol) according to the General proce-
6
,
115.20, 115.35, 116.20, 116.56, 123.31, 124.84, 127.55, 128.57,
128.75, 129.99, 131.96, 133.94, 137.30, 161.49, 164.30,
dure for alkaline hydrolysis. Pink crystals, yield: 274 mg (87%);
1
À
mp 137–141 °C. H NMR (DMSO-d
6
, 400 MHz): d = 2.33 (s, 3H,
166.51 ppm. MS (ESI): m/z (%) = 284.0 (MÀH) . IR (ATR):
Ar–CH ), 6.38 (s, 1H, indole-H), 7.14–7.17 (m, 1H, indole-H),
3
m = 2690, 1675, 1520, 1485, 1459, 1438, 1269, 1206, 1164, 1183,
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
À1
1
089, 1014, 898, 840, 825, 803, 747, 582, 564 cm . HPLC
4.3.13. Ethyl 1-(4-methylbenzyl)-1H-indole-2-carboxylate (15)
(
254 nm): 97.7%, t
R
= 20.40 min. HRMS Calcd for C16
H
11ClNO
2
Synthesized from 3 (5 mmol) according to the General proce-
dure for N-alkylation. Pale yellow oil, yield: 1.14 g (78%); mp/°C.
À
m/z: 284.0478 (MÀH) , found 284.0490.
1
H NMR (CDCl
), 4.29 (q, 2H, J = 7.2 Hz, –CH
), 6.92 (d, 2H, J = 8.4 Hz, Ar–H), 7.06 (d, 2H, J = 7.6 Hz, Ar–H),
3
, 400 MHz): d = 1.29 (t, 3H, J = 7.2 Hz, –CH
3
), 2.21
4
.3.9. tert-Butyl (1-(4-chlorobenzyl)-1H-indole-2-yl)carbamate
11)
Synthesized from 10 (1 mmol) according to the General proce-
dure for Curtius rearrangement. Orange solidified oil, yield:
(s, 3H, Ar–CH
CH
3
2
CH ), 5.81 (s, 2H, Ar–
3
(
2
7.13 (dt, 1H, J = 8.0 Hz, J = 0.8 Hz, Ar–H), 7.30 (dt, 1H, J = 8.0 Hz,
J = 1.2 Hz, Ar–H), 7.36 (d, 1H, J = 0.8 Hz, Ar–H), 7.56 (dd, 1H,
1
1
9
7
7
71 mg (48%); mp/°C. H NMR (DMSO-d
6
, 400 MHz): d = 1.34 (s,
J = 8.4 Hz, J = 0.8 Hz, Ar–H), 7.71 (d, 1H, J = 8.0 Hz, Ar–H) ppm. MS
+
H, (–CH ), 5.36 (s, 2H, Ar–CH ), 6.36 (s, 1H, indole-H), 6.97–
3
)
3
2
(ESI): m/z (%) = 294.1 (M+H) . IR (ATR):
m
= 2980, 1704, 1516,
.05 (m, 2H, Ar–H), 7.32–7.36 (m, 4H, indole-H and Ar–H), 7.46–
1480, 1456, 1410, 1376, 1351, 1319, 1245, 1183, 1163, 1138,
À1
.52 (m, 2H, indole-H), 9.36 (s, 1H, –NH–CO–) ppm. MS (ESI): m/z
1118, 1093, 1025, 1012, 799, 767, 748 cm . HPLC (254 nm):
+
(
%) = 357.1 (M+H) . IR (ATR):
m
= 2910, 2166, 1684, 1588, 1486,
97.3%, t
(M+H) , found 294.1491.
R
= 30.40 min. HRMS Calcd for C19
2
H20NO m/z: 294.1494
+
1
1
368, 1335, 1298, 1269, 1202, 1180, 1156, 1093, 1057, 1024,
010, 940, 762, 686, 597, 557 cm . HRMS Calcd for C20
À1
H
22ClN
H
2
O
2
+
m/z: 357.1370 (M+H) , found 357.1361. Anal. Calcd for C20
O (%): C, 67.32; H, 5.93; N, 7.85. Found: C, 67.22; H, 5.72; N, 8.15.
2
21ClN
2
-
4.3.14. Ethyl 1-(4-isopropylbenzyl)-1H-indole-2-carboxylate (16)
Synthesized from 3 (5 mmol) according to the General proce-
dure for N-alkylation. Beige crystals, yield: 1.32 g (82%); mp
1
4
.3.10. 1-(4-Chlorobenzyl)-indoline-2-imminium chloride (12)
Synthesized from 11 (0.5 mmol) according to the General pro-
56–60 °C. H NMR (CDCl
3
, 400 MHz): d = 1.12 (d, 6H, J = 6.8 Hz,
), 2.76–2.83 (m, 1H, (CH
), 5.82 (s, 2H, Ar–CH ), 6.95
3
(CH )
2
CH), 1.29 (t, 3H, J = 7.2 Hz, –CH
CH), 4.29 (q, 2H, J = 7.2 Hz, –CH
(d, 2H, J = 8.0 Hz, Ar–H), 7.11–7.16 (m, 3H, Ar–H), 7.31 (dt, 1H,
J = 7.6 Hz, J = 0.8 Hz, Ar–H), 7.36 (d, 1H, J = 0.4 Hz, Ar–H), 7.58 (d,
1H, J = 8.0 Hz, Ar–H), 7.72 (d, 1H, J = 8.0 Hz, Ar–H) ppm. MS (ESI):
m/z (%) = 322.2 (M+H) . IR (ATR):
1477, 1459, 1441, 1414, 1351, 1319, 1276, 1261, 1246, 1189,
1164, 1138, 1122, 1098, 1050, 1026, 1010, 813, 766, 746 cm
HPLC (254 nm): 93.6%, t = 29.42 min. HRMS Calcd for C21
m/z: 322.1807 (M+H) , found 322.1798.
3
3 2
) -
cedure for acidolysis. White amorphous solid, yield: 57 mg (39%);
2
CH
3
2
1
mp 128–129 °C. H NMR (DMSO-d
doline-CH –), 5.31 (s, 2H, Ar–CH
.23 (t, 1H, J = 7.4 Hz, indoline-H), 7,33 (t, 1H, J = 7.4 Hz, indoline-
H), 7.40 (d, 2H, J = 8.8 Hz, Ar–H), 7.41–7.52 (m, 3H, Ar–H and
indoline-H), 10.07 (s, 1H, indoline-NH ), 10.49 (s, 1H, indol–NH–
, 100 MHz): d = 35.92, 45.04,
6
, 400 MHz): d = 4.37 (s, 2H, –in-
2
2
), 7.14 (d, 1H, J = 7.6 Hz, Ar–H),
7
+
m = 2956, 2929, 1699, 1514,
2
1
3
À1
CH
2
–Ar) ppm. C NMR (DMSO-d
6
.
1
1
11.01, 124.53, 124.75, 126.26, 127.95, 128.72, 129.11, 132.58,
32.93, 143.30, 170.25 ppm. MS (ESI): m/z (%) = 257.0 (MÀClÀH) .
R
H24NO
2
À
+
IR (ATR):
m
= 2910, 1692, 1604, 1494, 1465, 1434, 1368, 1098, 885,
À1
8
12, 799, 749, 663, 642, 598, 560 cm . HPLC (254 nm): 97.4%,
4.3.15. Ethyl 1-(4-trifluoromethylbenzyl)-1H-indole-2-carboxy-
5
9
t
R
= 6.91 min. HRMS Calcd for
C
15
H13ClN
2
m/z: 223.1235
(%): C,
late (17)
À
(
6
MÀClÀH) , found 223.1231. Anal. Calcd for C15
H
14Cl
2
N
2
Synthesized from 3 (5 mmol) according to the General proce-
dure for N-alkylation. White crystals, yield: 1.2 g (66%); mp 48–
2 °C. H NMR (CDCl
4.27 (q, 2H, J = 7.2 Hz, –CH
1.45; H, 4.81; N, 9.55. Found: C, 61.70; H, 4.52; N, 9.35.
1
5
3
, 400 MHz): d = 1.27 (t, 3H, J = 7.2 Hz, –CH
3
),
4
.3.11. Ethyl 1-benzyl-1H-indole-2-carboxylate (13)57
2
CH ), 5.95 (s, 2H, Ar–CH ), 7.15–7.19
3
2
Synthesized from 3 (5 mmol) according to the General proce-
dure for N-alkylation. Yellow crystals, yield: 1.06 g (76%); mp
(m, 3H, Ar–H), 7.33 (dt, 1H, J = 6.8 Hz, J = 1.2 Hz, indole-H), 7.41
(d, 1H, J = 0.8 Hz, indole-H), 7.57 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz,
indole-H), 7.65 (d, 2H, J = 8.0 Hz, indole-H), 7.75 (d, 1H, J = 8.0 Hz,
1
5
3–55 °C. H NMR (CDCl
3
, 400 MHz): d = 1.39 (t, 3H, J = 7.2 Hz,
CH ), 5.88 (s, 2H, Ar–CH ),
+
–
CH ), 4.36 (q, 2H, J = 7,2 Hz, –CH
3
2
3
2
indole-H) ppm. MS (ESI): m/z (%) = 348.1 (M+H) . IR (ATR):
7
.07–7.08 (m, 2H, Ar–H), 7.17–7.40 (m, 6H, indole-H and Ar–H),
m = 2981, 2359, 2337, 1703, 1620, 1519, 1482, 1459, 1414, 1355,
7
.43 (d, 1H, J = 0.8 Hz, indole-H), 7.73–7.75 (m, 1H, indole-H)
1321, 1261, 1200, 1159, 1141, 1106, 1065, 1018, 900, 821, 742,
1
3
À1
ppm.
C NMR (DMSO-d
6
, 100 MHz): d = 14.07, 47.05, 60.42,
754, 731, 642 cm . HPLC (254 nm): 100%, t
Calcd for C19H F NO m/z: 348.1211 (M+H) , found 348.1205.
17 3 2
R
= 29.26 min. HRMS
+
1
1
=
1
10.63, 111.30, 120.80, 122.47, 125.22, 125.51, 126.18, 127.00,
27.18, 128.44, 138.45, 139.03, 161.22 ppm. MS (ESI): m/z (%)
+
280.1 (M+H) . IR (ATR):
m
= 1704, 1513, 1457, 1350, 1315, 1251,
4.3.16. Ethyl 1-(4-bromobenzyl)-1H-indole-2-carboxylate (18)
Synthesized from 3 (5 mmol) according to the General proce-
À1
191, 1136, 1095, 1025, 811, 743, 423, 964, 605, 593, 569 cm
= 27.94 min. HRMS Calcd for C18
m/z: 280.1338 (M+H) , found 280.1339.
.
HPLC (254 nm): 100%, t
R
H18NO
2
dure for N-alkylation. Pale yellow crystals, yield: 1.6 g (87%); mp
+
1
94–97 °C. H NMR (CDCl
3
, 400 MHz): d = 1.28 (t, 3H, J = 7.2 Hz,
CH ), 5.82 (s, 2H, Ar–CH ), 6.94
–
CH
3
), 4.28 (q, 2H, J = 7.2 Hz, –CH
2
3
2
4
.3.12. Ethyl 1-(4-fluorobenzyl)-1H-indole-2-carboxylate (14)58
Synthesized from 3 (5 mmol) according to the General proce-
(d, 2H, J = 8.4 Hz, Ar–H), 7.15 (t, 1H, J = 8.0 Hz, Ar–H), 7.32 (dd,
1H, J = 7.2 Hz, J = 1.2 Hz, indole-H), 7.38 (d, 1H, J = 0.8 Hz, indole-
H), 7.47 (d, 2H, J = 8.04 Hz, indole-H), 7.57 (dd, 1H, J = 8.8 Hz,
dure for N-alkylation. Yellow crystals, yield: 1.17 g (79%); mp
1
78–80 °C. H NMR (CDCl
–CH ), 4.30 (q, 2H, J = 7.2 Hz, –CH
7.09–7.16 (m, 5H, indole-H and Ar–H), 7.31–7.35 (m, 1H, indole-
3
, 400 MHz): d = 1.29 (t, 3H, J = 7.2 Hz,
J = 0.8 Hz, indole-H), 7.73 (d, 1H, J = 8.0 Hz, indole-H) ppm. MS
+
3
2
CH ), 5.84 (s, 2H, Ar–CH ),
3
2
(ESI): m/z (%) = 358.0 (M+H) . IR (ATR):
m
= 2982, 1709, 1517,
1481, 1457, 1440, 1410, 1352, 1259, 1249, 1196, 1181, 1163,
À1
H), 7.38 (d, 1H, J = 0.8 Hz, indole-H), 7.59–7.62 (m, 1H, indole-H),
.72–7.74 (m, 1H, indole-H) ppm. 13C NMR (DMSO-d
, 100 MHz):
d = 14.08, 46.38, 60.47, 110.74, 111.26, 115.14, 115.35, 120.87,
1141, 1120, 1096, 1069, 1027, 1009, 826, 794, 748, 740 cm . HPLC
7
6
R 2
(254 nm): 100%, t = 29.56 min. HRMS Calcd for C18H17BrNO m/z:
+
358.0443 (M+H) , found 358.0453.
1
1
22.51, 125.30, 125.52, 127.04, 128.25, 128.33, 134.59, 134.62,
38.92, 159.98, 161.21, 162.39 ppm. MS (ESI): m/z (%) = 298.1 (M
4.3.17. Ethyl 1-(4-cyclopropylbenzyl)-1H-indole-2-carboxylate
(19)
+
+
1
5
C
H) . IR (ATR):
m
= 1675, 1501, 1456, 1365, 1311, 1248, 1145,
088, 1053, 1013, 936, 887, 827, 791, 736, 685, 632, 600, 574,
Synthesized from 18 according to the following procedure for
À1
47
55 cm . HPLC (254 nm): 100%, t
R
= 27.99 min. HRMS Calcd for
Suzuki cross-coupling. To a stirred solution of 18 (3 mmol) in a
+
18
H
17FNO
2
m/z: 298.1243 (M+H) , found 298.1238.
mixture of toluen (10 mL) and water (600 lL), cyclopropylboronic
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

1
0
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
acid (3.9 mmol), tricyclohexylphosphine (0.3 mmol) and K
3
PO
4
4.3.21. 1-(4-Isopropylbenzyl)-1H-indole-2-carboxylic acid (23)
Synthesized from 16 (3 mmol) according to the General proce-
(
10.5 mmol) were added and the mixture was bubbled with argon
for 5 min. After removing the dissolved oxygen Pd(OAc)
2
dure for alkaline hydrolysis. Beige amorphous solid, yield:
1
(
0.15 mmol) was added and the mixture was heated to 100 °C for
783 mg (89%); mp 218–221 °C. H NMR (DMSO-d
6
, 400 MHz):
CH),
), 6.97 (d, 2H, J = 8.4 Hz, Ar–H), 7.11–7.15 (m,
three h and then cooled to room temperature. Water (30 mL) was
added to the reaction mixture, then it was extracted with ethyl
acetate (2 Â 40 mL). The organic phase was washed with brine
3 2 3 2
d = 1.14 (d, 6H, J = 7.2 Hz, (CH ) CH), 2.77–2.84 (m, 1H, (CH )
5.84 (s, 2H, Ar–CH
2
3H, Ar–H), 7.29 (dt, 1H, J = 7.2 Hz, J = 1.2 Hz, Ar–H), 7.32 (d, 1H,
J = 0.8 Hz, Ar–H), 7.56 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H), 7.70
(
2 4
30 mL), dried with anhydrous Na SO and then concentrated in
vacuo to afford the crude residue. This product was additionally
purified using flash column chromatography (gradient elution;
starting eluent: hexane/ethyl acetate 9:1 v/v) to afford sufficiently
pure 19 as pale yellow oil, yield: 651 mg (68%); mp/. 1H NMR
(d, 1H, J = 8.0 Hz, Ar–H), 12.99 (s, 1H, –COOH) ppm. MS (ESI): m/z
À
(%) = 292.1 (MÀH) . IR (ATR):
m = 2960, 2870, 1678, 1522, 1484,
1456, 1445, 1428, 1349, 1266, 1247, 1197, 1136, 745, 735,
À1
À
727 cm . HRMS Calcd for C19
H18NO
2
m/z: 292.1338 (MÀH) ,
(
(
1
CDCl
m, 2H, –CH
H, (CH CH), 4.30 (q, 2H, J = 7.2 Hz, –CH
CH ), 6.92 (d, 2H, J = 8.4 Hz, Ar–H), 6.96 (d, 2H, J = 8.0 Hz, Ar–H),
3
, 400 MHz): d = 0.57–0.60 (m, 2H, –CH
CH –), 1.31 (t, 3H, J = 7.2 Hz, –CH
CH ), 5.81 (s, 2H, Ar–
2
CH
2
–), 0.86–0.91
found 292.1331.
2
2
3
), 1.81–1.85 (m,
2
)
2
2
3
4.3.22. 1-(4-Trifluoromethylbenzyl)-1H-indole-2-carboxylic
5
9
2
acid (24)
7
.14 (t, 1H, J = 7.6 Hz, Ar–H), 7.31 (t, 1H, J = 8.0 Hz, Ar–H), 7.37 (s,
Synthesized from 17 (3 mmol) according to the General proce-
1
H, Ar–H), 7.58 (d, 1H, J = 8.8 Hz, Ar–H), 7.72 (d, 1H, J = 8.0 Hz,
dure for alkaline hydrolysis. Pale yellow amorphous solid, yield:
+
1
Ar–H) ppm. MS (ESI): m/z (%) = 320.2 (M+H) . IR (ATR):
m
= 2981,
948 mg (99%); mp 180–184 °C. H NMR (DMSO-d
6
, 400 MHz):
2
1
1
9
603, 2360, 2342, 1705, 1673, 1614, 1518, 1482, 1457, 1442,
2
d = 5.97 (s, 2H, Ar–CH ), 6.97 (d, 2H, J = 8.8 Hz, Ar–H), 7.14 (dt,
429, 1408, 1350, 1319, 1246, 1187, 1162, 1137, 1120, 1093,
1H, J = 7.6 Hz, J = 1.2 Hz, Ar–H), 7.18 (d, 2H, J = 7.6 Hz, Ar–H), 7.29
(dt, 1H, J = 7.6 Hz, J = 1.2 Hz, Ar–H), 7.35 (d, 1H, J = 0.8 Hz, Ar–H),
7.53 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H), 7.64 (d, 2H, J = 8.0 Hz,
À1
072, 1046, 1011, 897, 801, 765, 746 cm . HPLC (254 nm):
R 2
5.5%, t = 29.58 min. HRMS Calcd for C21H22NO m/z: 320.1651
+
(
M+H) , found 320.1646.
Ar–H), 7.72 (d, 1H, J = 8.0 Hz, Ar–H), 13.03 (s, 1H, –COOH) ppm.
À
MS (ESI): m/z (%) = 318.1 (MÀH) . IR (ATR):
m = 2879, 2605, 2526,
4
.3.18. 1-Benzyl-1H-indole-2-carboxylic acid (20)57
2362, 2343, 1671, 1619, 1520, 1484, 1458, 1446, 1429, 1352,
Synthesized from 13 (3 mmol) according to the General proce-
1321, 1265, 1203, 1160, 1139, 1109, 1065, 1015, 920, 897, 851,
À1
dure for alkaline hydrolysis. White crystals, yield: 723 mg (96%);
835, 822, 771, 752, 738, 647 cm . HRMS Calcd for C17
11
H F
3
NO
2
1
À
mp 198–200 °C. H NMR (DMSO-d
6
, 400 MHz): d = 5.89 (s, 2H,
m/z: 318.0742 (MÀH) , found 318.0748.
Ar–CH ), 7.02–7.04 (m, 2H, Ar–H), 7.11–7.15 (m, 1H, Ar–H), 7.18–
2
7
7
.22 (m, 1H, indole-H), 7.24–7.29 (m, 3H, indole-1H and Ar–H),
.30–7.33 (m, 1H, indole-H), 7.54 (dd, 1H, J = 8.6 Hz, J = 0.8 Hz,
4.3.23. 1-(4-Bromobenzyl)-1H-indole-2-carboxylic acid (25)
Synthesized from 18 (3 mmol) according to the General proce-
indole-H), 7.70–7.72 (m, 1H, indole-H), 12.99 (s, 1H, –COOH)
dure for alkaline hydrolysis. Dirty white amorphous solid, yield:
1
3
1
ppm. C NMR (DMSO-d
6
, 100 MHz): d = 46.84, 110.33, 111.26,
908 mg (92%); mp 176–180 °C. H NMR (DMSO-d
6
, 400 MHz):
1
1
20.60, 122.33, 124.84, 125.58, 126.25, 126.95, 128.21, 128.42,
38.64, 138.88, 162.91 ppm. MS (ESI): m/z (%) = 252.1 (M+H) . IR
2
d = 5.84 (s, 2H, Ar–CH ), 6.97 (d, 2H, J = 8.8 Hz, Ar–H), 7.13 (dt,
+
1H, J = 7.6 Hz, J = 0.8 Hz, Ar–H), 7.29 (dt, 1H, J = 7.6 Hz, J = 1.2 Hz,
Ar–H), 7.33 (d, 1H, J = 8.4 Hz, Ar–H), 7.47 (d, 2H, J = 8.4 Hz, Ar–H),
7.53 (dd, 1H, J = 8.4 Hz, J = 1.2 Hz, Ar–H), 7.71 (d, 1H, J = 7.6 Hz,
(
ATR):
m
= 2870, 1665, 1514, 1484, 1456, 1423, 1354, 1322, 1266,
À1
1
202, 1133, 885, 816, 745, 732, 719, 694, 583, 567 cm . HRMS
+
Calcd for C16
H14NO
2
m/z: 252.1025 (M+H) , found 252.1015.
Ar–H), 13.02 (s, 1H, –COOH) ppm. MS (ESI): m/z (%) = 328.0
À
(
MÀH) . IR (ATR):
m = 2929, 2604, 2360, 2342, 1672, 1615, 1519,
4
.3.19. 1-(4-Fluorobenzyl)-1H-indole-2-carboxylic acid (21)58
1483, 1459, 1440, 1429, 1321, 1266, 1207, 1164, 1138, 1072,
1011, 826, 800, 747, 736 cm . HRMS Calcd for C16
327.9973 (MÀH) , found 327.9979.
À1
Synthesized from 14 (3 mmol) according to the General proce-
dure for alkaline hydrolysis. White crystals, yield: 702 mg (87%);
H
11BrNO
2
m/z:
À
1
mp 176–180 °C. H NMR (DMSO-d
Ar–CH ), 7.09–7.15 (m, 5H, indole-1H and Ar–H), 7.28–7.33 (m,
H, indole-1H and Ar–H), 7.58 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz,
6
, 400 MHz): d = 5.86 (s, 2H,
2
4.3.24. 1-(4-Cyclopropylbenzyl)-1H-indole-2-carboxylic acid (26)
Synthesized from 19 (3 mmol) according to the General proce-
2
indole-H), 7.71 (d, 1H, J = 7.8 Hz, indole-H), 13.01 (s, 1H, –COOH)
dure for alkaline hydrolysis. White crystals, yield: 274 mg (94%);
À
1
ppm. MS (ESI): m/z (%) = 268.1 (MÀH) . IR (ATR):
m
= 2528, 1674,
mp 182–186 °C. H NMR (DMSO-d
6
, 400 MHz): d = 1.14 (d, 6H,
CH), 5.84 (s, 2H,
), 6.97 (d, 2H, J = 8.4 Hz, Ar–H), 7.11–7.15 (m, 3H, Ar–H),
1
1
607, 1512, 1483, 1460, 1445, 1425, 1354, 1318, 1268, 1206,
3 2 3 2
J = 7.2 Hz, (CH ) CH), 2.77–2.84 (m, 1H, (CH )
À1
17, 1134, 1091, 1016, 898, 826, 805, 768, 739, 615, 573 cm
.
Ar–CH
2
HPLC (254 nm): 100%, t
m/z: 268.0774 (MÀH) , found 268.0786.
R
= 18.04 min. HRMS Calcd for C16
H
11FNO
2
7.29 (dt, 1H, J = 7.2 Hz, J = 1.2 Hz, Ar–H), 7.32 (d, 1H, J = 0.8 Hz,
Ar–H), 7.56 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H), 7.70 (d, 1H,
À
J = 8.0 Hz, Ar–H), 12.99 (s, 1H, –COOH) ppm. MS (ESI): m/z (%)
.3.20. 1-(4-Methylbenzyl)-1H-indole-2-carboxylic acid (22)60
Synthesized from 15 (3 mmol) according to the General proce-
dure for alkaline hydrolysis. Beige solid, yield: 724 mg (91%); mp
= 290.1 (MÀH) . IR (ATR):
À
m = 3021, 2600, 2530, 1670, 1615,
4
1518, 1483, 1459, 1442, 1428, 1357, 1321, 1270, 1199, 1165,
1138, 1102, 1047, 1017, 897, 846, 826, 807, 766, 747, 733 cm
HRMS Calcd for
290.1189.
À1
.
1
À
1
59–163 °C. H NMR (DMSO-d
6
, 400 MHz): d = 2.21 (s, 3H, Ar–
C
19
H
16NO
2
m/z: 290.1181 (MÀH) , found
CH ), 5.82 (s, 2H, Ar–CH ), 6.93 (d, 2H, J = 8.4 Hz, Ar–H), 7.05 (d,
3
2
2
7
H, J = 7.6 Hz, Ar–H), 7.11 (dt, 1H, J = 8.0 Hz, J = 0.8 Hz, Ar–H),
.27 (dt, 1H, J = 8.0 Hz, J = 1.2 Hz, Ar–H), 7.30 (d, 1H, J = 0.8 Hz,
4.3.25. tert-Butyl (1-benzyl-1H-indole-2-yl)carbamate (27)
Ar–H), 7.52 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H), 7.68 (d, 1H,
Synthesized from 20 (1 mmol) according to the General proce-
J = 8.0 Hz, Ar–H), 12.97 (s, 1H, –COOH) ppm. MS (ESI): m/z (%)
dure for Curtius rearrangement. Brown amorphous solid, yield:
À
1
=
1
1
C
264.1 (MÀH) . IR (ATR):
m
= 2938, 2603, 2361, 2343, 1673,
242 mg (75%); mp 147–151 °C. H NMR (DMSO-d
6
, 400 MHz):
614, 1522, 1483, 1456, 1443, 1426, 1354, 1323, 1269, 1206,
d = 1.44 (s, 9H, (–CH
3
)
3 2
), 5.37 (s, 2H, Ar–CH ), 6.35 (s, 1H, indole-
À1
139, 1118, 895, 820, 789, 749, 733 cm . HRMS Calcd for
H), 6.96–7.04 (m, 4H, Ar–H), 7.18–7.22 (m, 4H, indole-H), 7.44–
À
13
17
H15NO
2
m/z: 264.1025 (MÀH) , found 264.1030.
7.46 (m, 1H, indole-H), 9.36 (s, 1H, –NH–CO–) ppm. C NMR
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
+
(
1
1
DMSO-d
6
,
100 MHz): d = 27.96, 44.95, 79.52, 93.42, 109.81,
Calcd for C21
Anal. Calcd for C21
7.16. Found: C, 64.18; H, 5.50; N, 7.49.
H
22
F
3
N
2
O
F
2
m/z: 391.1633 (M+H) , found 391.1631.
19.41, 119.45, 120.37, 126.48, 126.91, 126.99, 128.37, 133.64,
34.40, 138.04, 153.40 ppm. MS (ESI): m/z (%) = 323.2 (M+H) . IR
H
21
3
N
2
O
2
 0.05H O (%): C, 64.46; H, 5.44; N,
2
+
(
1
ATR):
m
= 3284, 1693, 1553, 1460, 1395, 1369, 1334, 1313, 1148,
À1
060, 775, 746, 732, 709, 692 cm . HRMS Calcd for C20
H
23
N
2
O
2
4.3.29. tert-Butyl (1-(4-bromobenzyl)-1H-indole-2-
yl)carbamate (31)
+
m/z: 323.1760 (M+H) , found 323.1747. Anal. Calcd for
C
6
20
H
22
N
2
O
2
(%): C, 74.51; H, 6.88; N, 8.69. Found: C, 74.34; H,
Synthesized from 25 (1 mmol) according to the General proce-
.65; N, 8.56.
dure for Curtius rearrangement. Beige amorphous solid, yield:
1
2
44 mg (61%); mp 132–136 °C. H NMR (DMSO-d
6
, 400 MHz):
4
.3.26. tert-Butyl (1-(4-methylbenzyl)-1H-indole-2-yl)carbamate
d = 1.43 (s, 9H, (–CH
3
)
3
2
), 5.34 (s, 2H, Ar–CH ), 6.35 (s, 1H,
(
28)
Synthesized from 22 (1 mmol) according to the General proce-
dure for Curtius rearrangement. Brown amorphous solid, yield:
indole-H), 6.95–7.01 (m, 4H, Ar–H), 7.26 (d, 1H, J = 8.4 Hz, Ar–H),
7.32–7.35 (m, 1H, Ar–H), 7.44–7.48 (m, 3H, Ar–H), 9.34
(s, 1H, –NH–CO–) ppm. C NMR (DMSO-d , 100 MHz): d = 27.94,
6
1
3
1
1
95 mg (58%); mp 147–151 °C. H NMR (DMSO-d
d = 1.44 (s, 9H, (–CH ), 2.22 (s, 3H, Ar–CH ), 5.30 (s, 2H, Ar–CH
.32 (s, 1H, indole-H), 6.93–7.01 (m, 4H, Ar–H), 7.06 (d, 2H,
J = 8.0 Hz, Ar–H), 7.26 (dd, 1H, J = 8.0 Hz, J = 0.8 Hz, Ar–H), 7.33
6
, 400 MHz):
44.38, 79.58, 109.73, 119.48, 119.58, 120.06, 120.51, 128.67,
3
)
3
3
2
),
130.41, 131.26, 131.37, 131.56, 133.59, 134.29, 137.51,
+
6
153.37 ppm. MS (ESI): m/z (%) = 401.1 (M+H) . IR (ATR):
m
= 3221,
3136, 3080, 3019, 2979, 2928, 2360, 2342, 2170, 1685, 1578, 1561,
1510, 1487, 1463, 1442, 1392, 1368, 1335, 1313, 1259, 1216, 1152,
(
(
dd, 1H, J = 8.4 Hz, J = 1.2 Hz, Ar–H), 7.42–7.51 (m, 2H, Ar–H), 9.33
s, 1H, –NH–CO–) ppm. 13C NMR (DMSO-d
, 100 MHz): d = 20.57,
7.96, 44.74, 79.51, 109.86, 119.38, 120.31, 126.53, 128.90,
33.60, 134.34, 134.98, 136.12, 153.41 ppm. MS (ESI): m/z (%)
6
1106, 1070, 1058, 1011, 996, 968, 943, 918, 845, 806, 778, 763,
À1
2
1
=
2
1
1
6
744, 727, 688, 670, 645 cm . HRMS Calcd for C20
H
22BrN
2 2
O m/z:
+
401.0865 (M+H) , found 401.0874. Anal. Calcd for C20
H21BrN O
2 2
+
337.2 (M+H) . IR (ATR):
m
= 3258, 3174, 3050, 3018, 2981, 2927,
(%): C, 59.86; H, 5.27; N, 6.98. Found: C, 59.78; H, 4.97; N, 7.02.
360, 2342, 2168, 1692, 1612, 1590, 1578, 1554, 1514, 1481,
462, 1393, 1369, 1333, 1312, 1251, 1204, 1181, 1150, 1107,
060, 1011, 965, 909, 891, 838, 810, 772, 761, 751, 730, 688, 658,
4.3.30. tert-Butyl (1-(4-cyclopropylbenzyl)-1H-indole-2-
yl)carbamate (32)
À1
+
44 cm . HRMS Calcd for C21
H
25
N
H
2
O
2
m/z: 337.1916 (M+H) ,
 0.25CH Cl (%): C,
1.36; H, 6.90; N, 7.83. Found: C, 71.70; H, 6.42; N, 8.29.
Synthesized from 26 (1 mmol) according to the General proce-
found 337.1912. Anal. Calcd for C21
7
24
N
2
O
2
2
2
dure for Curtius rearrangement. Ochre amorphous solid, yield:
1
203 mg (56%); mp 123–127 °C. H NMR (DMSO-d
6
, 400 MHz):
CH –),
CH), 5.28 (s, 2H,
), 6.31 (s, 1H, indole-H), 6.92–7.45 (m, 8H, Ar–H), 9.34 (s,
d = 0.55–0.57 (m, 2H, –CH
1.42 (s, 9H, (–CH ), 1.80–1.82 (m, 1H, (CH
Ar–CH
6
1H, –NH–CO–) ppm. C NMR (DMSO-d , 100 MHz): d = 9.27,
2
CH
2
–), 0.85–0.91 (m, 2H, –CH
2
2
4
.3.27. tert-Butyl(1-(4-isopropylbenzyl)-1H-indole-2-yl)carbamate
3
)
3
2 2
)
(
29)
Synthesized from 23 (1 mmol) according to the General proce-
dure for Curtius rearrangement. Brown amorphous solid, yield:
2
1
3
14.67, 27.97, 44.69, 79.53, 107.11, 109.85, 109.90, 119.38, 120.32,
125.32, 126.52, 126.89, 130.41, 133.61, 134.36, 134.90, 142.49,
1
1
86 mg (51%); mp 127–131 °C. H NMR (DMSO-d
d = 1.13 (d, 6H, J = 7.2 Hz, (CH CH), 1.43 (s, 9H, (–CH
.76–2.83 (m, 1H, (CH CH), 5.31 (s, 2H, Ar–CH ), 6.32 (s, 1H,
indole-H), 6.94–7.02 (m, 4H, Ar–H), 7.12 (d, 2H, J = 8.0 Hz, Ar–H),
6
, 400 MHz):
+
3
)
2
)
3 3
),
153.39, 166.07 ppm. MS (ESI): m/z (%) = 363.2 (M+H) . IR (ATR):
2
3
)
2
2
m = 3217, 3135, 3080, 3009, 2976, 2927, 2360, 2341, 2170, 1687,
1613, 1578, 1559, 1504, 1463, 1443, 1399, 1364, 1336, 1316,
1254, 1215, 1152, 1106, 1059, 1046, 1015, 995, 966, 941, 920,
901, 889, 845, 831, 812, 798, 777, 763, 745, 729, 712, 667, 623,
7
1
2
1
+
2
1
1
7
C
.28–7.35 (m, 1H, Ar–H), 7.44 (d, 1H, J = 7.2 Hz, Ar–H), 9.35 (s,
H, –NH–CO–) ppm. 13C NMR (DMSO-d
6
, 100 MHz): d = 23.81,
À1
+
7.96, 33.02, 79.51, 109.83, 119.39, 125.25, 126.27, 126.51,
606 cm . HRMS Calcd for C23
H
27
N
2
O
H
2
m/z: 363.2073 (M+H) ,
O (%): C,
34.35, 135.42, 147.13. 153.39 ppm. MS (ESI): m/z (%) = 365.2 (M
found 363.2068. Anal. Calcd for C23
73.47; H, 7.37; N, 7.45. Found: C, 73.09; H, 6.94; N, 7.72.
26
N
2
O
2
 0.75H
2
+
H) . IR (ATR):
m
= 3222, 3140, 3050, 2979, 2956, 2928, 2866,
360, 2342, 2169, 1689, 1613, 1580, 1560, 1511, 1489, 1465
449, 1395, 1372, 1354, 1336, 1316, 1259, 1182, 1153, 1105,
4.3.31. 1-Benzyl-indoline-2-imminium chloride (33)
062, 1053, 1015, 997, 966, 941, 920, 892, 842, 815, 778, 765,
Synthesized from 27 (0.5 mmol) according to the General pro-
À1
46, 734, 717, 688, 669, 630, 607 cm
.
HRMS Calcd for
cedure for acidolysis. White amorphous solid, yield: 106 mg
+
1
23
H
29
N
2
O
2
m/z: 365.2229 (M+H) , found 365.2219. Anal. Calcd
 0.3H O (%): C, 74.68; H, 7.79; N, 7.57. Found: C,
(82%); mp 247–250 °C. H NMR (DMSO-d
6
, 400 MHz): d = 4.38 (s,
for C23
7
H
28
N
2
O
2
2
2H, –indoline-CH
2
–), 5.38 (s, 2H, Ar–CH
2
), 7.14 (d, 1H, J = 8.0 Hz,
4.05; H, 7.04; N, 7.46.
Ar–H), 7.19–7.21 (m, 1H, indoline-H), 7.33–7.37 (m, 6H, indoline-
H and Ar–H), 7.49 (d, 1H, J = 7.2 Hz, indoline-H), 10.36 (s, 1H,
1
3
4
.3.28. tert-Butyl (1-(4-trifluoromethylbenzyl)-1H-indole-2-yl)-
2 2 6
–NH ), 10.71 (s, 1H, –NH ) ppm. C NMR (DMSO-d , 100 MHz):
carbamate (30)
d = 35.89, 45.57, 111.10, 124.54, 124.72, 126.23, 127.03, 127.95,
Synthesized from 24 (1 mmol) according to the General proce-
128.81, 133.74, 143.41, 170.23 ppm. MS (ESI): m/z (%) = 223.1
+
dure for Curtius rearrangement. Beige amorphous solid, yield:
(MÀCl) . IR (ATR):
m
= 2943, 1681, 1601, 1493, 1468, 1443, 1376,
1
À1
2
15 mg (55%); mp 146–150 °C. H NMR (DMSO-d
6
, 400 MHz):
), 6.38 (s, 1H, indole-
H), 6.98–7.04 (m, 2H, Ar–H), 7.17 (d, 2H, J = 8.0 Hz, Ar–H), 7.26–
.28 (m, 1H, Ar–H), 7.32–7.35 (m, 1H, Ar–H), 7.46–7.51 (m, 2H,
Ar–H), 7.65 (d, 2H, J = 8.0 Hz, Ar–H), 9.36 (s, 1H, –NH–CO–) ppm.
891, 746, 731, 709, 694, 662, 580 cm . HPLC (254 nm): 100%,
+
d = 1.40 (s, 9H, (–CH
3
)
3
), 5.47 (s, 2H, Ar–CH
2
t
R
= 4.24 min. HRMS Calcd for C15
H
15
N
2
m/z: 223.1235 (MÀCl) ,
found 223.1231. Anal. Calcd for
68.66; H, 5.80; N, 10.68. Found: C, 68.95; H, 5.40; N, 10.57.
C H
15 15
N
2
Cl  0.1HCl (%): C,
7
1
3
C NMR (DMSO-d
6
, 100 MHz): d = 27.89, 44.60, 79.57, 109.66,
4.3.32. 1-(4-Methylbenzyl)-indoline-2-imminium chloride (34)
1
1
19.54, 119.67, 120.61, 125.30, 125.34, 127.05, 129.66, 130.40,
Synthesized from 28 (0.5 mmol) according to the General pro-
33.66, 134.33, 142.95, 153.34 ppm. MS (ESI): m/z (%) = 391.2 (M
cedure for acidolysis. Dark brown amorphous solid, yield: 59 mg
+
1
+
1
1
9
H) . IR (ATR):
m
= 3293, 2989, 2972, 2934, 2361, 2342, 2170,
(43%); mp 70–74 °C. H NMR (DMSO-d
6
, 400 MHz): d = 2.27 (s,
–), 5.25 (s, 2H, Ar–CH ),
701, 1620, 1586, 1561, 1514, 1489, 1458, 1422, 1394, 1369,
360, 1325, 1264, 1247, 1211, 1152, 1117, 1067, 1018, 999, 967,
3H, Ar–CH
3
), 4.36 (s, 2H, –indoline-CH
2
2
6.93 (d, 1H, J = 7.6 Hz, Ar–H), 7.06 (d, 1H, J = 7.6 Hz, Ar–H), 7.13
(d, 1H, J = 7.6 Hz, Ar–H), 7.19 (d, 2H, J = 7.6 Hz, Ar–H), 7.26 (d, 2H,
À1
20, 829, 815, 801, 769, 742, 729, 719, 684, 643, 619 cm . HRMS
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

1
2
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
J = 7.6 Hz, Ar–H), 7.30–7.34 (m, 1H, Ar–H), 7.49 (dd, 1H, J = 7.2 Hz,
(35%); mp 62–66 °C. 1H NMR (DMSO-d
(m, 2H, –CH CH –), 0.91–0.96 (m, 2H, –CH
1H, (CH CH), 4.36 (s, 2H, –indoline-CH –), 5.28 (s, 2H, Ar–CH
2
6
, 400 MHz): d = 0.62–0.66
CH –), 1.86–1.93 (m,
),
1
3
J = 0.8 Hz, Ar–H), 10.04 (s, 1H, –NH
NMR (DMSO-d , 100 MHz): d = 37.81, 44.83, 58.03, 103.47, 111.61,
26.93, 127.01, 127.60, 128.98, 129.37, 143.76, 147.43,
2
), 10.46 (s, 1H, –NH
2
) ppm.
C
2
2
2
2
6
2
)
2
2
1
1
m
7.08 (d, 2H, J = 8.4 Hz, Ar–H), 7.16 (d, 1H, J = 7.6 Hz, Ar–H), 7.21
(dt, 1H, J = 7.6 Hz, J = 0.8 Hz, Ar–H), 7.26 (d, 2H, J = 8.4 Hz, Ar–H),
7.33 (dt, 1H, J = 8.0 Hz, J = 1.2 Hz, Ar–H), 7.49 (d, 1H, J = 6.8 Hz,
+
69.41 ppm. MS (ESI): m/z (%) = 237.1 (MÀCl) . IR (ATR):
= 2973, 2922, 2169, 1698, 1589, 1515, 1482, 1463, 1392, 1365,
2 2
), 10.59 (s, 1H, –NH
) ppm. 13C NMR
1
6
C
264, 1203, 1179, 1155, 1111, 1058, 1022, 1010, 965, 780, 739,
Ar–H), 10.22 (s, 1H, –NH
(DMSO-d , 100 MHz): d = 10.01, 36.36, 44.69, 111.65, 125.02,
125.20, 126.18, 126.70, 127.62, 128.44, 131.01, 143.89, 144.18,
À1
88 cm . HPLC (254 nm): 94.1%, t
R
= 6.72 min. HRMS Calcd for
6
+
16
H
17
N
2
m/z: 237.1392 (M-Cl) , found 237.1388.
+
1
m
70.58 ppm. MS (ESI): m/z (%) = 263.2 (MÀCl) . IR (ATR):
4
.3.33. 1-(4-Isopropylbenzyl)-indoline-2-imminium chloride (35)
= 2973, 2926, 2169, 1698, 1605, 1590, 1487, 1463, 1392,
1366, 1261, 1204, 1153, 1111, 1010, 966, 781, 739, 687 cm
À1
Synthesized from 29 (0.5 mmol) according to the General pro-
.
cedure for acidolysis. Brown amorphous solid, yield: 86 mg
HPLC (254 nm): 91.3%, t = 8.82 min. HRMS Calcd for C18
R
H
19
N
2
1
+
(
57%); mp 82–86 °C. H NMR (DMSO-d
6
, 400 MHz): d = 1.17 (d,
CH), 2.81–2.90 (m, 1H, (CH CH), 4.37 (s,
–), 5.31 (s, 2H, Ar–CH ), 6.95–7.00 (m, 1H, Ar–
m/z: 263.1548 (MÀCl) , found 263.1541. Anal. Calcd for
6
H, J = 7.2 Hz, (CH
3
)
2
3
)
2
C
18
H
14ClN
2
 0.3HCl (%): C, 69.80; H, 6.28; N, 9.04. Found:
2
H, –indoline-CH
2
2
C, 70.12; H, 5.60; N, 8.87.
H), 7.12–7.36 (m, 6H, Ar–H), 7.50 (d, 1H, J = 7.6 Hz, Ar–H), 10.27
s, 1H, –NH ), 10.63 (s, 1H, –NH
) ppm. 13C NMR (DMSO-d
00 MHz): d = 24.28, 33.57, 36.35, 45.80, 111.62, 125.21, 126.71,
(
2
2
6
,
4.3.37. Ethyl 1-((4-fluorophenyl)sulfonyl)-1H-indole-2-carboxy-
late (39)
1
1
1
m
1
27.20, 127.63, 128.47, 131.62, 143.94, 148.70, 161.46,
Synthesized from 3 (5 mmol) according to the General proce-
+
70.58 ppm. MS (ESI): m/z (%) = 265.2 (MÀCl) . IR (ATR):
dure for N-sulfonylation. Orange crystals, yield: 1.00 g (58%);
1
= 2960, 2929, 2169, 1698, 1610, 1514, 1481, 1464, 1420, 1365,
mp 95–98 °C.
J = 7.2 Hz, –CH
H
NMR (CDCl
3
,
400 MHz): d = 1.43 (t, 3H,
CH ), 7.15–7.20 (m,
À1
242, 1206, 1157, 1112, 1056, 1019, 965, 739, 689 cm . HPLC
3
), 4.43 (q, 2H, J = 7.2 Hz, –CH
2
3
(
2
254 nm): 94.7%, t
65.1705 (MÀCl) , found 265.1712.
R
= 10.59 min. HRMS Calcd for C15
H13ClN
2
m/z:
3H, indole-H and Ar–H), 7.32–7.34 (m, 1H, indole-H), 7.45–7.47
(m, 1H, indole-H), 7.59–7.60 (m, 1H, Ar–H), 8.09–8.11 (m, 3H,
+
1
3
Ar–H) ppm.
6
C NMR (DMSO-d , 100 MHz): d = 13.85, 61.80,
4
.3.34. 1-(4-Trifluoromethylbenzyl)-indoline-2-imminium
114.91, 116.78, 116.98, 117.01, 123.00, 124.54, 127.36, 127.98,
130.29, 130.39, 131.29, 133.51, 133.54, 137.06, 160.67, 163.99,
166.52 ppm. MS (ESI): m/z (%) = 348.1 (M+H) . IR (ATR):
chloride (36)
+
Synthesized from 30 (0.5 mmol) according to the General pro-
cedure for acidolysis. Beige amorphous solid, yield: 65 mg (40%);
m
= 1726, 1589, 1495, 1366, 1337, 1265, 1176, 1149, 1127, 1088,
1
À1
mp 87–91 °C. H NMR (DMSO-d
6
, 400 MHz): d = 4.39 (s, 2H, –indo-
), 7.14 (d, 1H, J = 8.0 Hz, Ar–H), 7.22
t, 1H, J = 7.2 Hz, Ar–H), 7.33 (t, 1H, J = 7.6 Hz, Ar–H), 7.52 (d, 1H,
J = 7.2 Hz, Ar–H), 7.61 (d, 2H, J = 8.0 Hz, Ar–H), 7.77 (d, 2H,
J = 8.0 Hz, Ar–H), 10.44 (s, 1H, –NH ), 10.74 (s, 1H, –NH ) ppm.
, 100 MHz): d = 36.46, 45.79, 111.40, 125.12,
920, 861, 846, 818, 757, 711, 682, 655, 579, 554 cm . HPLC
(254 nm): 100%, t
z: 348.0706 (M+H) , found 348.0706. Anal. Calcd for C17 14FNO S
(%): C, 58.78; H, 4.06; N, 4.03. Found: C, 58.88; H, 3.62; N,
4.05.
line-CH
2
–), 5.51 (s, 2H, Ar–CH
2
R
= 23.62 min. HRMS Calcd for C17
+
H15FNO
4
S m/
(
H
4
2
2
1
3
C NMR (DMSO-d
6
1
1
m
1
25.29, 126.12, 126.16, 126.74, 128.31, 128.52, 139.19, 143.81,
4.3.38. Ethyl 1-((4-methoxyphenyl)sulfonyl)-1H-indole-2-car-
boxylate (40)
+
71.00 ppm. MS (ESI): m/z (%) = 291.1 (MÀCl) . IR (ATR):
= 2978, 2173, 1701, 1481, 1464, 1420, 1393, 1367, 1323, 1247,
Synthesized from 3 (5 mmol) according to the General proce-
À1
159, 1121, 1066, 1018, 968, 741 cm . HPLC (254 nm): 97.9%,
dure for N-sulfonylation. Yellow crystals, yield: 1.38 g (77%); mp
+
1
t
R
= 8.62 min. HRMS Calcd for C16
H
14
F
3
N
H
2
m/z: 291.1109 (MÀCl) ,
95–97 °C. H NMR (CDCl
3
, 400 MHz): d = 1.42 (t, 3H, J = 7.2 Hz,
), 4.43 (q, 2H, J = 7.2 Hz, –CH –CH ),
found 291.1104. Anal. Calcd for C16
5
14
F
3
N
2
 0.1HCl (%): C,
–CH
3
), 2.40 (s, 3H, Ar–CH
3
2
3
8.17; H, 4.30; N, 8.48. Found: C, 58.18; H, 4.06; N, 8.32.
7.27–7,29 (m, 4H, indole-H and Ar–H), 7.34–7,35 (m, 1H, indole-
H), 7.42–7.44 (m, 1H, indole-H), 7.57–7.59 (m, 1H, Ar–H), 7.93–
13
4
.3.35. 1-(4-Bromobenzyl)-indoline-2-imminium chloride (37)
7.85 (m, 2H, Ar–H), 8.12–8.14 (m, H, Ar–H) ppm.
C NMR
Synthesized from 31 (0.5 mmol) according to the General pro-
(DMSO-d , 100 MHz): d = 14.39, 56.35, 62.28, 115.21, 115.35,
6
cedure for acidolysis. Light brown amorphous solid, yield: 77 mg
116.57, 123.32, 124.85, 127.56, 128.57, 128.74, 129.99, 131.96,
137.29, 161.49, 164.30 ppm. MS (ESI): m/z (%) = 360.1 (M+H) . IR
1
+
(
46%); mp 78–82 °C. H NMR (DMSO-d
H, –indoline-CH –), 5.33 (s, 2H, Ar–CH
Ar–H), 7.21 (t, 1H, J = 7.6 Hz, Ar–H), 7.30–7.35 (m, 3H, Ar–H),
.49 (d, 1H, J = 7.6 Hz, Ar–H), 7.59 (d, 2H, J = 8.4 Hz, Ar–H), 10.28
s, 1H, –NH ), 10.63 (s, 1H, –NH
) ppm. 13C NMR (DMSO-d
6
, 400 MHz): d = 4.35 (s,
2
2
2
), 7.13 (d, 2H, J = 8.0 Hz,
(ATR):
m
= 1722, 1594, 1497, 1365, 1336, 1311, 1265, 1149, 1087,
À1
1017, 838, 804, 745, 677, 629 cm . HPLC (254 nm): 100%,
7
R 5
t = 23.13 min. HRMS Calcd for C18H18NO S m/z: 360.0906 (M
+
(
2
2
6
,
+H) , found 360.0905.
1
1
1
m
1
6
C
00 MHz): d = 36.43, 45.54, 111.51, 114.46, 121.63, 125.10,
25.25, 126.73, 128.48, 129.83, 132.17, 133.74, 143.79,
4.3.39. Ethyl 1-((4-nitrophenyl)sulfonyl)-1H-indole-2-carboxy-
late (41)
+
70.85 ppm. MS (ESI): m/z (%) = 301.0 (MÀCl) . IR (ATR):
= 2968, 2924, 2169, 1697, 1604, 1591, 1566, 14871463, 1392,
Synthesized from 3 (5 mmol) according to the General proce-
366, 1261, 1204, 1152, 1112, 1070, 1010, 966, 781, 739,
dure for N-sulfonylation. Yellow crystals, yield: 1.3 g (68%); mp
À1
1
87 cm . HPLC (254 nm): 96.1%, t
R
= 7.50 min. HRMS Calcd for
m/z: 301.0340 (MÀCl) , found 301.0333. Anal. Calcd
14BrClN
 0.25HCl (%): C, 51.96; H, 4.14; N, 8.08. Found:
C, 52.27; H, 3.87; N, 7.69.
104–108 °C. H NMR (CDCl
3
, 400 MHz): d = 1.39 (t, 3H, J = 7.2 Hz,
–CH ), 7.31–7.35 (m, 1H,
+
15
H
14BrN
H
2
–CH
3
), 4.38 (q, 2H, J = 7.2 Hz, –CH
2
3
for C15
2
Ar–H), 7.47–7.51 (m, 1H, Ar–H), 7.61 (td, 1H, J = 7.6 Hz, J = 1.2 Hz,
Ar–H), 8.15 (dd, 1H, J = 8.8 Hz, J = 0.8 Hz, Ar–H), 8.23 (d, 2H,
J = 9.2 Hz, Ar–H), 8.32 (d, 2H, J = 9.2 Hz, Ar–H) ppm. 13C NMR
4
(
.3.36. 1-(4-Cyclopropylbenzyl)-indoline-2-imminium chloride
38)
Synthesized from 32 (0.5 mmol) according to the General pro-
cedure for acidolysis. Light brown amorphous solid, yield: 52 mg
3
(CDCl , 100 MHz): d = 14.16, 62.13, 115.20, 118.09, 122.96,
124.16, 124.71, 127.73, 128.11, 128.71, 131.47, 138.40, 144.38,
+
150.49, 160.76 ppm. MS (ESI): m/z (%) = 375.1 (M+H) . IR (ATR):
m
= 1726, 1536, 1352, 1334, 1178, 1151, 1128, 1089, 1039, 1014,
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
À1
8
50, 740, 683, 662, 615 cm . HPLC (254 nm): 100%, t
R
= 24.04 min.
4.3.43. Ethyl 1-(4-toluenesulfonyl)-1H-indole-2-carboxamide (45)
Synthesized from 6 (1 mmol) according to the following amida-
+
HRMS Calcd for m/z: 375.0651 (M+H) , found
17 15 2 6
C H N O S
3
75.0659.
tion procedure. White crystals, yield: 182 mg (58%); mp 218–
1
2
20 °C. H NMR (DMSO-d
6
, 400 MHz): d = 2.34 (s, 3H, Ar–CH
3
),
4
.3.40. Ethyl 1-((4-chlorophenyl)sulfonyl)-1H-indole-2-
6.98 (s, 1H, Ar–H), 7.27 (t, 1H, J = 7.2 Hz, Ar–H), 7.37–7.40 (m,
3H, Ar–H), 7.61 (d, 1H, J = 7.6 Hz, Ar–H), 7.72 (s, 1H, Ar–H), 7.94
(dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H), 8.02 (d, 1H, J = 8.4 Hz, Ar–H),
8.22 (s, 1H, Ar–H) ppm. C NMR (DMSO-d , 100 MHz): d = 21.52,
6
111.72, 114.91, 122.52, 124.37, 126.18, 127.88, 128.89, 130.25,
6
1
carboxylate (42)
Synthesized from 3 (5 mmol) according to the General proce-
1
3
dure for N-sulfonylation. Brown amorphous solid, yield: 1.1 g
1
(
63%); mp 86–90 °C. H NMR (CDCl
3
, 400 MHz): d = 1.42 (t, 3H,
–CH ), 7.22 (d, 1H,
J = 0.8 Hz, Ar–H), 7.31 (dt, 1H, J = 8.0 Hz, J = 0.8 Hz, Ar–H), 7.44–
J = 7.2 Hz, –CH
3
), 4.43 (q, 2H, J = 7.2 Hz, –CH
2
3
135.13, 136.17, 136.76, 145.69, 163.20 ppm. MS (ESI): m/z (%)
+
= 315.1 (M+H) . IR (ATR):
m = 3426, 3146, 1697, 1671, 1595, 1552,
7
1
.48 (m, 3H, Ar–H), 7.34–7,35 (m, 1H, indole-H), 7.42–7.44 (m,
H, indole-H), 7.59 (d, 1H, J = 7.6 Hz, Ar–H), 8.02 (d, 2H,
1450, 1395, 1361, 1307, 1234, 1187, 1169, 1139, 1086, 1015,
À1
1012, 910, 841, 808, 746, 703, 675, 660, 632 cm
.
HPLC
S m/
J = 7.6 Hz, Ar–H), 8.13 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H) ppm.
(254 nm): 99.5%, t
R
= 11.94 min. HRMS Calcd for C16
H
15
N
2
O
3
1
3
+
3
C NMR (CDCl , 100 MHz): d = 14.17, 62.05, 115.27, 117.23,
z: 315.0803 (M+H) , found 315.0801.
1
1
m
7
22.70, 124.37, 127.28, 128.19, 128.89, 129.28, 131.71, 137.07,
38.15, 161.15 ppm. MS (ESI): m/z (%) = 364.0 (M+H) . IR (ATR):
+
4.4. Cell culture (HEK-Blue NOD1 and NOD2 cells)
= 1625, 1477, 1396, 1319, 1249, 1195, 1176, 1149, 1095, 1011,
À1
57, 664, 620, 574 cm . HPLC (254 nm): 100%, t
R
= 26.58 min.
HEK-Blue NOD1 and NOD2 cells (Invivogen) were cultured in
DMEM medium (Sigma) with 10 % heat-deactivated FBS (Gibco),
+
4
HRMS Calcd for C17H15NO SCl m/z: 364.0410 (M+H) , found
3
64.0415.
2 mM
streptomycin (Sigma), 4.5 g glucose (Sigma) and 100
mocin (Invivogen) for 2 passages. All subsequent passages were
cultured in the medium supplemented with 30 g/mL Blasticidin
(Invivogen) and 100 g/mL Zeocin (Invivogen). The experiments
L
-glutamine (Sigma), 50 U/mL penicillin (Sigma), 50 ng/mL
l
g/mL Nor-
4
2
.3.41. Ethyl 1-((4-trifluoromethylphenyl)sulfonyl)-1H-indole-
-carboxylate (43)
Synthesized from 3 (5 mmol) according to the General proce-
l
l
dure for N-sulfonylation. Ochre amorphous solid, yield: 1.6 g
were carried out on passages 7–12.
1
(
80%); mp 62–64 °C. H NMR (CDCl
J = 7.2 Hz, –CH ), 4.43 (q, 2H, J = 7.2 Hz, –CH
J = 0.8 Hz, Ar–H), 7.32 (dt, 1H, J = 8.0 Hz, J = 0.8 Hz, Ar–H), 7.48
dt, 1H, J = 8.8 Hz, J = 1.2 Hz, Ar–H), 7.59 (dd, 1H, J = 7.6 Hz,
J = 0.8 Hz, Ar–H), 7.76 (d, 2H, J = 8.4 Hz, Ar–H), 8.19 (dd, 1H,
3
, 400 MHz): d = 1.41 (t, 3H,
3
2
–CH ), 7.27 (d, 1H,
3
4.5. Metabolic activity assay
(
The tested compounds were dissolved in DMSO and further
diluted in culture medium to a desired final concentration, so that
the final concentration of DMSO did not exceed 0.1%. HEK-Blue
1
3
J = 8.4 Hz, J = 0.8 Hz, Ar–H), 8.24 (d, 2H, J = 8.8 Hz, Ar–H) ppm.
NMR (CDCl , 100 MHz): d = 14.08, 62.06, 115.23, 117.61, 122.83,
24.50, 126.10, 126.14, 126.18, 127.49, 127.98, 128.16, 128.42,
31.63, 138.30, 142.32, 160.96 ppm. MS (ESI): m/z (%) = 398.1 (M
C
4
3
NOD1 cells (1 Â 10 cells/well) were seeded in triplicates on 96
1
1
+
1
6
C
well plates in 100 lL culture medium and treated with 25 lM con-
centration of each compound or with the corresponding volume of
vehicle—0.1% DMSO (control cells). After 24 h, metabolic activity
was measured using the CellTiter 96Ò Aqueous One Solution Cell
Proliferation Assay (Promega, Madison/WI, USA), in accordance
with the manufacturer’s instructions. Three biological replicates
were performed for each experiment.
+
H) . IR (ATR): m = 3306, 1720, 1685, 1527, 1367, 1340, 1311,
248, 1172, 1140, 1061, 1016, 918, 841, 771, 736, 714, 665,
À1
03 cm . HPLC (254 nm): 100%, t
R
= 27.66 min. HRMS Calcd for
+
18 4 3
H15NO SF m/z: 398.0674 (M+H) , found 398.0682.
4
.3.42. Ethyl 1-(4-toluoyl)-1H-indole-2-carboxylate (44)
Synthesized from 3 according to the following procedure for N-
TM
4.6. Measurement of NF-
Detection)
jB transcriptional activity (HEK-Blue
acylation. Ethyl indole-2-carboxylate (5 mmol) was first dissolved
in THF (15 mL) then potassium tert-butoxide (7.5 mmol) was
added and the mixture was stirred for 15 min at room tempera-
ture. Subsequently, toluoyl chloride (5.5 mmol) was added to the
reaction mixture which was heated overnight at reflux. Upon com-
pletion of the reaction, which was monitored by TLC, the mixture
was concentrated in vacuo and the residue was dissolved in
dichloromethane (80 mL). The organic phase was washed with
The tested compounds were dissolved in DMSO and further
diluted in culture medium to a desired final concentration, so that
the final concentration of DMSO did not exceed 0.1%. For screening
5
for potential NOD1 inhibitors, HEK-Blue NOD1 cells (5 Â 10 cells/
mL) were assayed in duplicate for NF-jB transcriptional activity
after treatment with 25
1 h, followed by the addition of 100 nM C12-iE-DAP and subsequent
incubation for 18 h (37 °C, 5% CO , 100% humidity). The control cells
lM NOD1 inhibitors or Noditinib-1 for
water (2 Â 30 mL), dried with anhydrous Na
2 4
SO and then concen-
trated in vacuo. The residue was purified by flash silica gel column
chromatography (gradient elution; starting eluent: hexane/ethyl
2
(NT) were only treated with the corresponding volume of vehicle—
acetate 6:1 v/v) to afford pure 44. White amorphous solid, yield:
0.1% DMSO. For determining IC50, HEK-Blue NOD1 and NOD2 cells
1
5
9
37 mg (61%); mp/°C. H NMR (CDCl
3
, 400 MHz): d = 1.14 (t, 3H,
), 4.04 (q, 2H, J = 7.2 Hz,
), 7.27–7.33 (m, 3H, Ar–H), 7.39–7.43 (m, 2H, Ar–H),
.65 (d, 2H, J = 8.4 Hz, Ar–H), 7.73 (dd, 1H, J = 8.0 Hz, J = 1.2 Hz,
(5 Â 10 cells/mL) were assayed in duplicates for NF-
j
B transcrip-
M NOD1
inhibitors or Noditinib-1, followed by the addition of 100 nM C12-
iE-DAP or 2 M MDP, respectively, and subsequent incubation for
J = 7.2 Hz, –CH
–
7
3
), 2.44 (s, 3H, Ar–CH
3
tional activity after 1 h pre-treatment with 0.625–25 l
2 3
CH –CH
l
1
3
Ar–H), 7.77 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Ar–H) ppm. C NMR
CDCl , 100 MHz): d = 13.92, 21.76, 61.31, 113.96, 115.31, 122.53,
23.29, 127.03, 127.19, 129.49, 129.64, 131.06, 132.77, 138.82,
18 h. SEAP activity was determined spectrophotometrically as
absorbance at 620 nm on a Tecan Safire 2 microplate reader (Read-
ing, UK). Assays were performed in three biological replicates.
(
1
1
3
+
44.23, 161.06, 169.11 ppm. MS (ESI): m/z (%) = 308.1 (M+H) . IR
(
1
ATR):
m
= 1714, 1608, 1541, 1444, 1371, 1306, 1229, 1200, 1154,
4.7. Statistics
À1
093, 1015, 957, 913, 888, 832, 790, 750, 605 cm . HPLC
(
3
254 nm): 100%, t
08.1287 (M+H) , found 308.1278.
R
= 26.52 min. HRMS Calcd for C19
H18NO
3
m/z:
All experiments were performed at least twice, with average
values expressed as means ± SD. IC50 values of NOD1/2 inhibition
+
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044

1
4
K. Ke cˇ ek Plešec et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
were calculated by a non-linear regression model using Graph Pad
Prism 6 software. Statistical significance was determined with the
Dunnet multiple comparison test. Differences were considered sig-
nificant for p <0.05 and highly significant for p <0.01.
26. Agnihotri, G.; Ukani, R.; Malladi, S. S.; Warshakoon, H. J.; Balakrishna, R.; Wang,
X.; David, S. A. J. Med. Chem. 2011, 54, 1490.
2
7. Jakopin, Zˇ .; Gobec, M.; Kodela, J.; Hazdovac, T.; Mlinari cˇ -Raš cˇ an, I.; Sollner, M.
Eur. J. Med. Chem. 2013, 69, 232.
28. Magnuson, G.; Khan, P.; Yuan, H.; Brown, B.; Divlianska, D. B.; Stonich, D.;
Peddibhotla, S.; Su, Y.; Dad, S.; Sergienko, E.; Chung, T. D. Y.; Roth, G. P.; Wimer,
C.; Diaz, P.; Correa, R. G.; Reed, J. C. High Throughput Screening Assays for NOD1
Inhibitors—Probe 1; Probe Reports from the NIH Molecular Libraries Program:
Bethesda (MD), 2010.
Acknowledgments
2
9. Magnuson, G.; Khan, P.; Yuan, H.; Brown, B.; Divlianska, D. B.; Stonich, D.;
Peddibhotla, S.; Su, Y.; Dad, S.; Sergienko, E.; Chung, T. D. Y.; Roth, G. P.; Wimer,
C.; Diaz, P.; Correa, R. G.; Reed, J. C. High Throughput Screening Assays for NOD1
Inhibitors—Probe 2; Probe Reports from the NIH Molecular Libraries Program:
Bethesda (MD), 2010.
This work was supported by the Slovenian Research Agency
grant (0787-P208). The authors thank Roger Pain for proofreading
the manuscript.
3
3
0. Rickard, D. J.; Sehon, C. A.; Kasparcova, V.; Kallal, L. A.; Haile, P. A.; Zeng, X.;
Montoute, M. N.; Poore, D. D.; Li, H.; Wu, Z.; Eidam, P. M.; Emery, J. G.; Marquis,
R. W.; Gough, P. J.; Bertin, J. PLoS One 2014, 9, e96737.
1. Khan, P. M.; Correa, R. G.; Divlianska, D. B.; Peddibhotla, S.; Sessions, E. H.;
Magnuson, G.; Brown, B.; Suyama, E.; Yuan, H.; Mangravita-Novo, A.;
Vicchiarelli, M.; Su, Y.; Vasile, S.; Smith, L. H.; Diaz, P. W.; Reed, J. C.; Roth, G.
P. ACS Med. Chem. Lett. 2011, 2, 780.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.08.044.
3
2. Correa, R. G.; Khan, P. M.; Askari, N.; Zhai, D.; Gerlic, M.; Brown, B.; Magnuson,
References and notes
G.; Spreafico, R.; Albani, S.; Sergienko, E.; Diaz, P. W.; Roth, G. P.; Reed, J. C.
Chem. Biol. 2011, 18, 825.
1
2
.
.
Caruso, R.; Warner, N.; Inohara, N.; Nuñez, G. Immunity 2014, 41, 898.
Philpott, D. J.; Sorbara, M. T.; Robertson, S. J.; Croitoru, K.; Girardin, S. E. Nat.
Rev. Immunol. 2014, 14, 9.
Hanson, P. I.; Whiteheart, S. W. Nat. Rev. Mol. Cell Biol. 2005, 6, 519.
Ammelburg, M.; Frickey, T.; Lupas, A. N. J. Struct. Biol. 2006, 156, 2.
Proell, M.; Riedl, S. J.; Fritz, J. H.; Rojas, A. M.; Schwarzenbacher, R. PLoS One
3
3
3. Jakopin, Zˇ .; Corsini, E.; Gobec, M.; Mlinari cˇ -Raš cˇ an, I.; Sollner, M. Eur. J. Med.
Chem. 2011, 46, 3762.
4. Jakopin, Zˇ .; Gobec, M.; Mlinari cˇ -Raš cˇ an, I.; Sollner, M. J. Med. Chem. 2012, 55,
3.
4.
5.
6478.
3
3
3
3
3
4
5. Jakopin, Zˇ . Tetrahedron Lett. 2015, 56, 504.
6. Jakopin, Zˇ . Curr. Med. Chem. 2013, 20, 2068.
2
008, 3, e2119.
7. Gobec, M.; Mlinari cˇ -Raš cˇ an, I.; Sollner, M. Eur. J. Med. Chem. 2016, 116, 1.
8. Langdon, S. R.; Ertl, P.; Brown, N. Mol. Inf. 2010, 29, 366.
9. de Sa Alvers, F. R.; Barreiro, E. J. Mini Rev. Med. Chem. 2009, 9, 782.
0. Beaulieu, P. L.; Gillard, J.; Bykowski, D.; Brochu, C.; Dansereau, N.; Duceppe, J.
S.; Hache, B.; Jakalian, A.; Lagace, L.; LaPlante, S.; McMercher, G.; Moreau, E.;
Perreault, S.; Stammers, T.; Thauvette, L.; Warrington, J.; Kukolj, G. Bioorg. Med.
Chem. Lett. 2006, 16, 4987.
1. Silvestri, R.; De Martino, G.; La Regina, G.; Artico, M.; Massa, S.; Vargiu, L.;
Mura, M.; Loi, A. G.; Marceddu, T.; La Colla, P. J. Med. Chem. 2003, 46, 2482.
2. Lebel, H.; Leogane, O. Org. Lett. 2005, 7, 4107.
3. Katritzky, A. R.; Widyan, K.; Kirichenko, K. J. Org. Chem. 2007, 72, 5802.
4. Jia, G.; Lown, J. W. Bioorg. Med. Chem. 2000, 8, 1607.
6
.
Mo, J.; Boyle, J. P.; Howard, C. B.; Monie, T. P.; Davis, B. K.; Duncan, J. A. J. Biol.
Chem. 2012, 287, 23057.
Askari, N.; Correa, R. G.; Zhai, D.; Reed, J. C. J. Biotechnol. 2012, 157, 75.
Jakopin, Zˇ . J. Med. Chem. 2014, 57, 6897.
Girardin, S. E.; Boneca, I. G.; Carneiro, L. A. M.; Antignac, A.; Jéhanno, M.; Viala,
J.; Tedin, K.; Taha, M. K.; Labigne, A.; Zähringer, U.; Coyle, A. J.; DiStefano, P. S.;
Bertin, J.; Sansonetti, P. J.; Philpott, D. J. Science 2003, 300, 1584.
7.
8.
9.
4
1
0. Chamaillard, M.; Hashimoto, M.; Horie, Y.; Masumoto, J.; Qiu, S.; Saab, L.;
Ogura, Y.; Kawasaki, A.; Fukase, K.; Kusumoto, S.; Valvano, M. A.; Foster, S. J.;
Mak, T. W.; Nuñez, G.; Inohara, N. Nat. Immunol. 2003, 4, 702.
4
4
4
4
1
1. Girardin, S. E.; Jéhanno, M.; Mengin-Lecreulx, D.; Sansonetti, P. J.; Alzari, P. M.;
Philpott, D. J. J. Biol. Chem. 2005, 280, 38648.
5. Murakami, Y.; Watanabe, T.; Sakai, M.; Yokoyama, Y. Chem. Pharm. Bull. 1988,
1
2. a) Girardin, S. E.; Travassos, L. H.; Hervé, M.; Blanot, D.; Boneca, I. G.; Philpott,
D. J.; Sansonetti, P. J.; Mengin-Lecreulx, D. J. Biol. Chem. 2003, 278, 41702; b)
Inohara, N.; Ogura, Y.; Fontalba, A.; Gutierrez, O.; Pons, F.; Crespo, J.; Fukase, K.;
Inamura, S.; Kusumoto, S.; Hashimoto, M.; Foster, S. J.; Moran, A. P.; Fernandez-
Luna, J. L.; Nuñez, G. J. Biol. Chem. 2003, 278, 5509.
3
6, 3732.
4
4
4
6. Fink, D. M. Synlett 2004, 2394.
7. Wallace, D. J.; Chen, C. Tetrahedron Lett. 2002, 43, 6987.
8. Kettle, J. G.; Faull, A. W.; Barker, A. J.; Davies, D. H.; Stone, M. A. Bioorg. Med.
Chem. Lett. 2004, 14, 405.
9. Pozdnev, V. F. Tetrahedron Lett. 1995, 36, 7115.
0. Golubeva, G. A.; Portnov, Y. N.; Kost, A. N. Chem. Heterocycl. Compd. 1973, 9,
1
3. Dzierzbicka, K.; Wardowska, A.; Trzonkowski, P. Curr. Med. Chem. 2011, 18,
4
5
2
438.
1
1
1
4. Correa, R. G.; Milutinovic, S.; Reed, J. C. Biosci. Rep. 2012, 608, 597.
5. Moreira, L. O.; Zamboni, D. S. Front. Immunol. 2012, 3, 328.
471.
5
5
1. Nakagawa, M.; Yamaguchi, H.; Hino, T. Tetrahedron Lett. 1970, 11, 4035.
2. Mangette, J. E.; Chen, X.; Krishnamoorthy, R.; Vellekoop, A. S.; Csakai, A. J.;
Camara, F.; Paquette, W. D.; Wang, H.-J.; Takahashi, H.; Fleck, R.; Roth, G. P.
Tetrahedron Lett. 2011, 52, 1292.
6. Travassos, L. H.; Carneiro, L. A.; Ramjeet, M.; Hussey, S.; Kim, Y. G.; Magalhães,
J. G.; Yuan, L.; Soares, F.; Chea, E.; Le Bourhis, L.; Boneca, I. G.; Allaoui, A.; Jones,
N. L.; Nuñez, G.; Girardin, S. E.; Philpott, D. J. Nat. Immunol. 2010, 11, 55.
7. Shaw, P. J.; Barr, M. J.; Lukens, J. R.; McGargill, M. A.; Chi, H.; Mak, T. W.;
Kanneganti, T. D. Immunity 2011, 34, 75.
1
1
5
3. Hino, T.; Nakagawa, M.; Hashizume, T.; Yamaji, N.; Miwa, Y. Tetrahedron Lett.
1970, 11, 2205.
8. Nishio, H.; Kanno, S.; Onoyama, S.; Ikeda, K.; Tanaka, T.; Kusuhara, K.; Fujimoto,
Y.; Fukase, K.; Sueishi, K.; Hara, T. Arterioscler. Thromb. Vasc. Biol. 2011, 31,
5
4. Kost, A. N.; Portnov, Y. N.; Golubeva, G. A.; Popova, A. G.; Mushket, B. Chem.
Heterocycl. Compd. 1980, 16, 917.
5. Sayyad, M.; Nanaji, Y.; Ghorai, M. K. J. Org. Chem. 2015, 80, 12659.
6. Takeda, T.; Harada, S.; Nishida, A. Org. Lett. 2015, 17, 5184.
7. Sindac, J. A.; Barraza, S. J.; Dobry, C. J.; Xiang, J.; Blakely, P. K.; Irani, D. N.; Keep,
R. F.; Miller, D. J.; Larsen, S. D. J. Med. Chem. 2013, 56, 9222.
8. Breteche, A.; Duflos, M.; Dassonville, A.; Nourrisson, M.-R.; Brelet, J.; Le Baut,
G.; Grimaud, N.; Petit, J.-Y. J. Enzyme Inhib. Med. Chem. 2002, 17, 415.
9. Hopkins, C. R.; O’Neil, S. V.; Laufersweiler, M. C.; Wang, Y.; Pokross, M.; Mekel,
M.; Evdokimov, A.; Walter, R.; Kontoyianni, M.; Petrey, M. E.; Sabatakos, G.;
Roesgen, J. T.; Richardson, E.; Demuth, T. P., Jr. Bioorg. Med. Chem. Lett. 2006, 16,
1
093.
5
5
5
1
2
2
9. Rosenzweig, H. L.; Galster, K. T.; Planck, S. R.; Rosenbaum, J. T. Invest.
Ophthalmol. Visual Sci. 2009, 50, 1746.
0. Cardenas, I.; Mulla, M. J.; Myrtolli, L.; Sfakianaki, A. K.; Norwitz, E. R.; Tadesse,
S.; Guller, S.; Abrahamas, V. M. J. Immunol. 2011, 187, 980.
1. Schertzer, J. D.; Tamrakar, A. K.; Magalhaes, J. G.; Pereira, S.; Bilan, P. J.;
Fullerton, M. D.; Liu, Z.; Steinberg, G. R.; Giacca, A.; Philpott, D. J.; Klip, A.
Diabetes 2011, 60, 2206.
5
5
2
2
2
2
2. Zhao, L.; Hu, P.; Zhou, Y.; Purohit, J.; Hwang, D. Am. J. Physiol. Endocrinol. Metab.
2
011, 301, E587.
5
659.
0. Elliott, J. D.; Leber, J. D.; Thompson, S. K.; Halbert, S. M., PCT Int. Appl. 1996, WO
618393 A1 19960620.
3. Jiang, H.; Najmeh, S.; Berube, J.; Leone, A.; Savage, P.; Giannias, B.; Bourdeau, F.;
Rousseau, S.; Park, M.; Ferri, L. E. Cancer Res. 2015, 75, 3162.
4. Ogura, Y.; Inohara, N.; Benito, A.; Chen, F. F.; Yamaoka, S.; Nuñez, G. J. Biol.
Chem. 2001, 276, 4812.
5. Inohara, N.; Koseki, T.; del Peso, L.; Hu, Y.; Yee, C.; Chen, S.; Carrio, R.; Merino,
J.; Liu, D.; Ni, J.; Núñez, G. J. Biol. Chem. 1999, 274, 14560.
6
6
9
1. Ragno, R.; Coluccia, A.; La Regina, G.; De Martino, G.; Piscitelli, F.; Lavecchia, A.;
Novellino, E.; Bergamini, A.; Ciaprini, C.; Sinistro, A.; Maga, G.; Crespan, E.;
Artico, M.; Silvestri, R. J. Med. Chem. 2006, 49, 3172.
Please cite this article in press as: Ke cˇ ek Plešec, K.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.044