Synthesis and biological activity of novel mono-indole and mono-benzofuran inhibitors of bacterial transcription initiation complex formation

Article abstract of DOI:10.1016/j.bmc.2015.02.037

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and σ⁷⁰/σ^A factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-σ⁷⁰/σ^A interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

Full text of DOI:10.1016/j.bmc.2015.02.037

Bioorganic & Medicinal Chemistry 23 (2015) 1763–1775
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of novel mono-indole and
mono-benzofuran inhibitors of bacterial transcription initiation
complex formation
Marcin Mielczarek a, Ruth V. Thomas a, Cong Ma c, Hakan Kandemir a,e, Xiao Yang c, Mohan Bhadbhade a,
David StC. Black a, Renate Griffith d, Peter J. Lewis c, Naresh Kumar a,b,
⇑
a School of Chemistry, UNSW Australia, Sydney, NSW 2052, Australia
b Australian Centre for Nanomedicine, University of New South Wales, Sydney 2052, Australia
c School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
d School of Medical Sciences, Department of Pharmacology, UNSW Australia, Sydney, NSW 2052, Australia
e School of Chemistry, Faculty of Art and Science, Namik Kemal University, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of
several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein–pro-
Received 9 January 2015
Revised 12 February 2015
Accepted 17 February 2015
Available online 25 February 2015
tein interaction between RNA polymerase core and r70 A factors in bacteria. In this study, the reaction of
/r
indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines
afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and
7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel
Keywords:
molecules were found to inhibit the RNA polymerase–r70 A interaction as measured by ELISA, and also
/r
Mono-indoles
Mono-benzofurans
RNA polymerase
inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure–activity
relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the
hydrophilic–hydrophobic balance is an important determinant of biological activity.
Ó 2015 Elsevier Ltd. All rights reserved.
r70 rA factors
/
Antibacterial activity
Structure–activity relationship (SAR)
1. Introduction
development of novel classes of antibiotics and the elucidation of
their molecular targets have to be urgently pursued.29
Antibiotics have been of fundamental importance in con-
temporary medicine and pharmacy since the discovery of peni-
cillin, an antibacterial agent from the fungus Penicillium notatum
by Alexander Fleming in 1928.1,2 However, as a result of their
excessive and sometimes unjustified use, bacterial strains resistant
to these drugs have rapidly emerged.3–25 The loss of potency of
existing antibiotics against resistant strains has been exacerbated
by the continually decreasing numbers of new antimicrobial com-
pounds being brought to the market.26–28
Current antibacterial drug discovery is largely focused on the
derivatization of existing classes of antibiotics. However, such
compounds may be prone to inactivation by existing bacterial
resistance mechanisms. Therefore, in order to combat the rise
and spread of antibiotic resistant strains, the discovery and
Bacterial RNA polymerase (RNAP) is an attractive target for
novel antibacterial agents. Several classes of antibiotics have been
discovered that inhibit the activity of RNAP via different molecular
mechanisms, and some of these molecules are important compo-
nents of antibacterial therapies.30–39 However, only a few classes
of antibacterial agents targeting bacterial transcription initiation
complex formation, and in particular the b0-CH-r70 r2A.2 protein–
/
protein interaction, have been discovered.2,32,34,35,40 Synthetic mole-
cules 1, 2 and 3 have been found to efficiently inhibit transcription
initiation in bacteria (Fig. 1).34,35
Bacterial transcription is a sequential process comprising three
main stages: initiation, elongation and termination.41 There are
two forms of bacterial RNAP: core and holoenzyme.41 Both enzyme
forms are catalytically active, but only the holoenzyme formed by
the association of the core enzyme with
r factors is able to recog-
nize DNA promoter sequences and initiate transcription with
appropriate specificity and efficiency.41–48 The RNAP core enzyme
⇑
Corresponding author. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141.
E-mail address: n.kumar@unsw.edu.au (N. Kumar).
consists of five subunits: a2 dimer, b, b0 and x 32,41
r factors are
.
http://dx.doi.org/10.1016/j.bmc.2015.02.037
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1764
M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
O
penetration through the outer membrane in Gram-negative bac-
teria or the cell wall in Gram-positive bacteria were encountered.2
Given that the indole nucleus has been discovered to be a bioactive
moiety against bacterial transcription initiation complex forma-
tion, we were interested in developing smaller compounds based
on indole or the related benzofuran scaffolds that could circumvent
the problems associated with the larger bis-indoles, while retain-
ing potent antibacterial activity.
OH
S
O
1
OH
O
S
N
S
N
O
3
HO
O
In this paper, we report the synthesis and evaluation of the anti-
bacterial activity of a library of low molecular weight molecules
active against both Gram-positive and Gram-negative bacteria,
and present our structure–activity relationship (SAR) studies on
H
N
S
O
N
S
S
these novel inhibitors of the essential b0-CH-r70 r2A.2 protein–
/
N
2
O
protein interaction.
2. Results and discussion
Figure 1. Synthetic molecules inhibiting transcription initiation.
2.1. Synthesis of starting materials
unique to bacteria and the closely related r70 factors in Gram-
negative bacteria, and rA factors in Gram-positive bacteria are
essential and required for the transcription of the majority of
expressed genes.2 The RNAP holoenzyme is formed via protein–
protein interaction between two highly conserved motifs: the b0-
In order to produce a library of mono-indole- and mono-
benzofuran-based amides, hydrazides, glyoxyloylamides and gly-
oxyloylhydrazides, we employed well-established procedures that
had previously been developed in our group for the synthesis of the
CH region of RNAP core and region 2.2 of the r70
/
rA factor.49
biologically active bis-indoles
4 and 5
(Fig. 2).2 3-Aryl-4,6-
Since the regions of the r70
/
rA factor family that interact with
dimethoxyindoles 6a–b,56 4,6-dimethoxy-3-methylindole 6c56
and 4,6-dimethoxy-2,3-dimethylindole 6d57 were synthesized
according to previously reported methods. These indoles were sub-
sequently reacted with trichloroacetyl chloride and oxalyl chloride
to yield a variety of derivatives, including 7-trichloroacetylindoles
7a–b, 2-trichloroacetylindoles 8a–c, indole-7-glyoxyloyl chlorides
9a–b and indole-2-glyoxyloyl chlorides 10a–b (Scheme 1).2
Following another well-established method developed by our
group, 4,6-dimethoxy-3-phenylbenzofuran 1158 was synthesized
and subsequently reacted with oxalyl chloride to afford benzo-
furan-2-glyoxyloyl chloride 12. Additionally, indole 13 was treated
the RNAP core enzyme are highly conserved across different bac-
teria species,32,42,44,48,50,51 molecules capable of inhibiting the b0-
CH-r70 rA2.2 interaction would be expected to exhibit broad spec-
/
trum antibacterial activity. Therefore, the design and development
of such novel drug candidates, potentially unique both in terms of
their mechanism of antibacterial activity as well as in the structure
of their chemical scaffolds, could help to combat bacterial infections
that are resistant to currently used antibiotics.
Our previous research aimed at the identification of potential
inhibitors of the b0-CH-r70 r2A.2 protein–protein interaction resulted
/
in the synthesis of a library of novel bis-indole compounds capable
of efficiently disrupting this interaction in
a
cell-free assay.2
Modelling studies based on a Bacillus subtilis RNAP homology
model52 and isothermal titration calorimetry experiments40 proved
that the bis-indoles inhibited transcription initiation by binding to
the b0-CH region of the RNAP core enzyme. Interestingly, many of
the synthesized molecules were found to exclusively inhibit the
growth of Escherichia coli,2 allowing them to potentially be used as
non-conventional antibacterial agents against Gram-negative bac-
teria.53–55 Compounds 4 and 5 were the most potent inhibitors of
E. coli and B. subtilis growth, respectively (Fig. 2).2
OMe
OMe
R
R
CCl3
O
+
N
N
MeO
MeO
H
H
O
CCl3
R
8a
8b
8c
4-BrC6H4
4-ClC6H4
Me
R
7a
4-BrC6H4
7b 4-ClC6H4
a
Since the bis-indoles synthesized in our previous studies were
relatively large, issues related to solubility as well as limited
R1
OMe
R2
R1
R2
N
H
MeO
6a 4-BrC6H4
H
H
H
6b
6c Me
6d
4-ClC6H4
Cl
b
Me
Me
OMe
R1
OMe
OMe
R
O
NH
N
R2
+
MeO
H
N
N
Cl
MeO
O
MeO
H
H
O
NH
HN
NH
O
O
O
R
O
O
HN
Cl
10a 4-BrC6H4
10b 4-ClC6H4
O
R1
R2
Me
OMe
HN
O
9a 4-BrC6H4
H
9b
9c Me
4-ClC6H4
H
Me
N
H
MeO
4
5
Scheme 1. Reagents and conditions: (a) CCl3COCl (3 equiv), anhydrous 1,2-
dichloroethane, 80 °C, 3.5 h, 20–37% (7a–b), 10–23% (8a–c); (b) oxalyl chloride
(3 equiv), anhydrous diethyl ether, 0 °C ? rt, 3 h, 30–33% (9a–b), 35–43% (10a–b).
Figure 2. Bis-indole inhibitors of the interaction between RNAP core and r70 rA in
/
bacteria.

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
1765
with oxalyl chloride, yielding indole-3-glyoxyloyl chloride 1459
(Scheme 2).
R1
R1
OMe
OMe
O
O
R2
R2
N
N
MeO
O
MeO
H
H
2.2. Synthesis of glyoxyloylamides and glyoxyloylhydrazides
O
R1
R2
a
R3
R1
When indole-2-glyoxyloyl chlorides 10a–b, indole-3-glyoxyloyl
chloride 14 and benzofuran-2-glyoxyloyl chloride 12 were reacted
with a variety of excess amines, the corresponding indole-2-gly-
oxyloylamides 15a–l, indole-3-glyoxyloylamides 17a–d and
15a
15b
15c
15d
15e
4-BrC6H4
4-ClC6H4
4-ClC6H4
R2
R3
a
a
b
b
c
e
a
b
d
h
h
i
a
b
16a
16b
16c
4-BrC6H4
4-ClC6H4
4-BrC6H4
H
H
H
H
H
H
Me
Me
Me
H
4-BrC6H4
4-BrC6H4
Me
c
d
benzofuran-2-glyoxyloylamides
18a–d,
respectively,
were
16d 4-ClC6H4
15f
a
b
c
d
e
synthesized in 10–55% yield (Fig. 3).
16e
16f
16g Me
16h
16i
16j
16k
16l
4-ClC6H4
4-BrC6H4
15g Me
The range of amines used included secondary heterocyclic ami-
nes such as piperidine, morpholine, pyrrolidine, N-methyl-piper-
azine, N-benzylpiperazine and N-(pyridin-2-yl)piperazine, and
primary aliphatic amines such as ethyl glycinate. The reactions
were carried out in anhydrous diethyl ether at room temperature
for 3–6 h or in anhydrous 1,2-dichloroethane at 60–70 °C for
3 h. Indole-3-glyoxyloylamides 17a–b have been previously
reported.60
15h
15i
15j
15k
15l
Me
Me
Me
Me
4-ClC6H4
Me
Me
f
4-BrC6H4
4-ClC6H4
4-BrC6H4
g
H
H
H
H
R
O
16m 4-ClC6H4
16n 4-BrC6H4
i
O
j
In a similar manner, indole-7-glyoxyloylamides 16a–m and
indole-7-glyoxyloylhydrazides 16n–o were produced in 27–88%
yield when the indole-7-glyoxyloyl chlorides 9a–c were treated
with excess secondary heterocyclic amines, primary aromatic ami-
nes such as aniline and p-toluidine, or phenylhydrazine in anhy-
drous diethyl ether at room temperature for 1–6 h (Fig. 3). An
ORTEP diagram61 showing one of the four independent molecules
for 16a is presented in Figure 4.
16o
H
j
4-ClC6H4
N
H
R
a
b
d
e
R
a
b
d
OMe
17a
17b
17c
17d
18a
O
18b
18c
18d
O
R
O
MeO
e
Figure 3. Glyoxyloylamides and glyoxyloylhydrazides; a = piperidin-1-yl, b = mor-
pholin-1-yl, c = pyrrolidin-1-yl, d = N-(pyridin-2-yl)piperazin-1-yl, e = N-ben-
zylpiperazin-1-yl, f = N-methylpiperazin-1-yl, g = ethyl 2-aminoacetyl, h = anilinyl,
i = toluidinyl, j = phenylhydrazinyl.
2.3. Synthesis of carboxamides and carboxhydrazides
The indole-2-carboxamide 19 was synthesized in 69% yield
from the reaction between 2-trichloroacetylindole 8b and morpho-
line in anhydrous acetonitrile at room temperature for 9 h (Fig. 5).
Similarly, the reaction of 7-trichloroacetylindoles 7a–b with ani-
line and p-toluidine in anhydrous acetonitrile at reflux for 12 h
afforded the respective indole-7-carboxamides 20a–c in 51–60%
yield. Reactions of 7-trichloroacetylindoles 7a–b and phenylhydra-
zine in anhydrous acetonitrile at room temperature for 3 h resulted
in formation of indole-7-carboxhydrazides 20d–e in 61% yield
(Fig. 5).
inhibition was evaluated at ca. 200 lM compound concentration
using two representative bacterial species, B. subtilis (Gram-positive)
and E. coli (Gram-negative), and is expressed as a% inhibition of the
negative control (bacterial growth in the absence of the inhibitor).
Some of the molecules precipitated out of the solution when diluted
to ca. 200 lM concentration with the growth medium (Table 1).
Molecules 15l, 16c, 16l–m, 16o and 20d–e showed significant
(P20%) and exclusive inhibition of E. coli growth, with compounds
15l and 16c being the most potent selective inhibitors of E. coli
growth (53% and 59% inhibition, respectively). On the other hand,
molecule 16j showed 36% inhibition of B. subtilis growth and 33%
inhibition of E. coli growth, indicating its potential as a broad spec-
trum antibacterial agent. Compounds 15c, 16g–h and 18b affected
the exponential phase of B. subtilis growth, while molecules 16c–d
inhibited the exponential phase of E. coli growth.
2.4. Evaluation of biological activity
The library of thirty-nine synthesized compounds was then
evaluated for antibacterial activity. The inhibition of the interaction
between r70
ined by ELISA at 15
as a % inhibition of the negative control (the interaction between
rA2.2 and b0-CH in the absence of the inhibitor). Bacterial growth
/
rA2.2 and the b0-CH region of the core RNAP was exam-
l
M concentration of compound and is expressed
For compounds 16c and 18b further experiments were per-
formed to demonstrate concentration–response behaviour. ELISA
r70
/
experiments in the concentration range 2–500
determination of IC50 values for these two compounds
(33.9 3.9 M for 16c and 71.6 3.0 M for 18b). Inhibition of
bacterial growth was also tested for these two compounds at con-
centrations of 13–1600 (growth curves shown in
Supplementary material). 16c inhibited E. coli growth at concentra-
tions of >100 M. Similarly, for 18b, inhibition of B. subtilis growth
was observed at concentrations of >100 M.
lM allowed
l
l
OMe
OMe
lM
a
O
O
O
Cl
MeO
MeO
l
O
11
12
l
Molecules 16e, 17d and 18b were the most potent inhibitors of
B. subtilis growth, with growth inhibition values of 60%, 70% and
70% at 200 lM, respectively. However, since these compounds
Cl
O
O
a
exhibited relatively low or no activity in the ELISA, their mecha-
N
H
N
nism of antibacterial activity might not involve the inhibition of
H
the b0-CH-r70 r2A.2 interaction. The same applies, but to a smaller
/
13
14
extent, to molecules 16d, 16e and 19 with respect to inhibition of
E. coli growth.
Scheme 2. Reagents and conditions: (a) oxalyl chloride (3 equiv), anhydrous
diethyl ether, 0 °C ? rt, 3 h, 92% (14).

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M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
Table 1
b0-CH-r70 rA2.2 binding inhibitory activity and anti-bacterial activity of the synthe-
/
sized compounds.
Compound b0-CH-r70
/
rA2.2 binding inhibition at
Growth inhibition at
15
l
M by ELISA (%)
200
lM (%)
Bacillus
subtilis
Escherichia
coli
15a
15b
15c
15d
15e
15f
15g
15h
15i
39
NA
27
NA
25
17
33
44
48
46
44
42
32
NA
31
NA
NA
8
22a
NA
37a,b
NA
19
NA
2
NA
14
7a
18
37a
NA
38
NA
2
NA
38
16
NA
53a
26
NA
59b
38b
23
6
24
NA
13
33
23
29
20
24a
20a
9
4
4
6
6
10
42
13
12
13
23
21
15j
15k
15l
7
NA
NAa
14
16a
16b
16c
16d
16e
16f
16g
16h
16i
NA
NA
NA
60c
NA
57b
42b
12
39
7
27
34
23
16j
16k
16l
36
26
ND
37
34
26
37
39
27
21
29
41
NA
49
ND
ND
44
60
NA
NA
10a
NAa
12
16m
16n
16o
17a
17b
17c
17d
18b
18c
19
20a
20b
20c
20d
20e
NA
10
70
70b
3
NA
7
36
13
NA
NA
Figure 4. An ORTEP diagram showing one of the four independent molecules for
16a (thermal ellipsoids are drawn at 50% probability level).
OMe
OMe
O
R1
R1
a
b
c
R2
O
Precipitation at ca. 200
Affects exponential phase of bacterial growth.
Staphylococcus aureus, NA: no activity, ND: not determined.
lM.
N
N
MeO
MeO
H
H
R2
R1
R1
R2
a
19
4-ClC6H4
negative bacteria might account for the differences between the
inhibition of the b0-CH-r70 r2A.2 interaction and the inhibition of
R2
b
/
20a
4-BrC6H4
bacterial growth observed for some molecules. For example, com-
20b 4-BrC6H4
c
c
20c
20d
20e
4-ClC6H4
pounds 15f–k containing a Me substituent at position 3 of the indole
ring inhibited the b0-CH-r70 r2A.2 interaction with potency compara-
/
4-BrC6H4
4-ClC6H4
d
d
ble to their structural analogues containing a C6H5, 4-BrC6H4 or 4-
ClC6H4 substituent at the same position, but showed a dramatic
decrease or even a complete loss of bacterial growth inhibitory activ-
ity. However, compound 15i having a N-(pyridin-2-yl)piperazin-1-yl
substituent and compound 15j having a N-benzylpiperazin-1-yl sub-
stituent in a side chain at position 2 of the indole ring still exhibited
low or moderate ability to inhibit bacterial growth. We hypothesize
that the analogues 15i–j containing relatively bulky and lipophilic
substituents had superior ability to cross the outer membrane of
Gram-negative bacteria and the cell wall of Gram-positive bacteria
compared to analogues containing smaller and less lipophilic sub-
stituents, such as a piperidin-1-yl (15f), morpholin-1-yl (15g), pyrro-
lidin-1-yl (15h) and N-methylpiperazin-1-yl (15k). In compounds
15i–j, the decrease in molecular lipophilicity resulting from replace-
ment of an aryl substituent at position 3 of the indole ring by the
smaller Me substituent is compensated by incorporation of bulky
and lipophilic substituents in a side chain at position 2 of the indole
Figure 5. Amides and hydrazides; a = morpholin-1-yl, b = anilinyl, c = toluidinyl,
d = phenylhydrazinyl.
2.5. Structure–activity relationship (SAR) studies
The ability of the compounds to inhibit the b0-CH-r70 r2A.2 inter-
/
action can be predicted by mapping the compounds onto a pharma-
cophore developed earlier.40 This pharmacophore describes the
interactions the
r
factor forms with the b0-CH region and was devel-
oped based on the B. subtilis RNAP homology model and mutagenesis
experiments. Compounds which fit this pharmacophore well, map-
ping all features, have >80% chance of activity (>20% inhibition at
15
l
M) in the ELISA assay.
The ability of the compounds to cross the cell walls of
Gram-positive bacteria and the outer membranes of Gram-

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
1767
ring. This hypothesis is further strengthened by calculating clogP
values for these compounds in the uncharged state, with 15i–j
exhibiting the highest values (3.2 and 3.7) in the series.
complex formation in bacteria. Inhibition of the interaction
between the b0-CH region of the core RNAP and r70 rA2.2 was evalu-
/
ated in a cell-free system by ELISA. The compounds were also exam-
ined as potential inhibitors of B. subtilis and E. coli growth in culture.
Compared with the previously reported bis-indoles that gene-
In general, it was observed that an optimal clogP range exists
for growth inhibition of E. coli. For clogP values below 3 no signifi-
cant activity is observed and most activity is observed in the clogP
range of 3–4, although some compounds with high clogP values
show >25% growth inhibition (15e, 16a, 16j, 16l). This is consistent
with the hypothesis that the compounds cross into E. coli by pas-
sive transport through the outer membranes, with the reason for
the upper limit of clogP related to a lack of aqueous solubility.
This was observed experimentally for 15a, 15c, 15l, 16n, and
16o. Judging by the predicted solubilities, this could have affected
the growth inhibitory activities of a further 24 of the tested com-
pounds, which all had low to very low predicted solubilities (for
the neutral form).
Some compounds can be protonated to a noticeable extent at
piperazine ‘non-amide’ nitrogen with predicted pKa values of 5.6
or higher given in square brackets (15e [5.6], 15i [5.6], 15j [7.5],
15k [9.8], 16f [8.1], 16i [5.6], 17c [5.6], 17d [8.1], 18c [5.6], 18d
[8.1]). This obviously improves solubility, however, the charged
forms are expected to have limited ability to cross bacterial
membranes.
rally exhibited high inhibition of the b0-CH-r70 r2A.2 interaction,2
/
the mono-indoles and mono-benzofurans synthesized in this work
showed less potent activity. On one hand, the smaller mono-indoles
and mono-benzofurans might have better cellular permeability than
the larger bis-indoles, but on the other hand, the larger molecules
might fit better into the binding site at the b0-CH-r70 r2A.2 interface.
/
However, the decrease in the ability of the monomeric compounds to
inhibit the formation of transcription initiation assembly did not
result in a significant decrease in their antibacterial ability, as evi-
denced by the fact that the mono-indoles and mono-benzofurans
were able to inhibit bacterial growth with potency comparable to
the bis-indoles. On the other hand, in contrast to the bis-indoles
which preferentially inhibited E. coli growth,2 many of the mono-
meric molecules were found to inhibit the growth of both B. subtilis
and E. coli. Six compounds were found to be exclusive inhibitors of
E. coli growth, while only one compound 16h inhibited B. subtilis
growth exclusively. This observation indicates that the mono-indole
scaffolds could potentially be developed as either broad spectrum
antibacterial agents, or as selective inhibitors against Gram-negative
bacteria. The ability to inhibit Gram-negative bacteria is especially
significant in light of the rapid emergence of resistance in Gram-
negative strains such as E. coli, Klebsiella pneumoniae, Salmonella
spp., Proteus mirabilis and Pseudomonas aeruginosa to currently used
antibiotics.53–55
Interestingly, compounds in general show lower activity against
B. subtilis, and no optimal clogP range was observed. Calculated
logP, pKa and aqueous solubility values for all the molecules are
presented in Supplementary material.
The glyoxyloylamides 16a, 16c, 16j and 16l, and the glyoxy-
loylhydrazide 16n bearing the larger but less electron-withdraw-
ing 4-BrC6H4 substituent at position 3 of the indole ring were
Structure–activity relationship studies carried out on the library
of mono-indole compounds suggested that both molecular size
and hydrophilicity–lipophilicity balance have to be considered
when designing antibacterial agents, in order to generate com-
pounds capable of crossing the outer membrane in Gram-negative
bacteria and the cell wall in Gram-positive bacteria whilst retain-
ing appropriately high solubility.
found to possess greater potency against both the b0-CH-r70 r2A.2
/
interaction and bacterial growth compared to the corresponding
analogues 16b, 16d, 16k, 16m and 16o that possess the smaller
but more electron-withdrawing 4-ClC6H4 substituent at the same
position.
Since a 4-BrC6H4 substituent is more bulky than a 4-ClC6H4 sub-
stituent, its incorporation into a compound results in an increase in
overall lipophilicity, as confirmed by the clogP values, that might
enhance the ability of the molecule in crossing the outer mem-
brane of Gram-negative bacteria and the cell wall of Gram-positive
bacteria. Therefore, the increased bacterial growth inhibition of the
analogues bearing the 4-BrC6H4 substituent might be a result of
not only an increased ability of the molecules to inhibit the b0-
When compared to the bis-indoles synthesized in the previous
work, the monomeric compounds developed in the present study
showed not only an enhancement of solubility, but also retention
of significant antibacterial activity in terms of both inhibition of
the b0-CH-r70 r2A.2 interaction and reduction of bacterial growth in
/
culture. Therefore, future research will be aimed at the design and
synthesis of monomeric inhibitors of bacterial transcription initia-
tion complex formation.
CH-r70
resulting in higher intracellular compound concentration for inter-
rupting the b0-CH-r70 r2A.2 interaction within cells.
/
rA2.2 interaction, but also an increased cellular permeability
/
4. Experimental
Interestingly, this trend was the reverse of that observed for the
previously reported bis-indole inhibitors, where the replacement
of a 4-ClC6H4 substituent by a 4-BrC6H4 substituent resulted in
4.1. Materials and measurement methods
decreased inhibition of the b0-CH-r70 r2A.2 interaction and reduced
/
Trichloroacetyl chloride was synthesized from trichloroacetic
acid according to a slightly modified literature procedure.62 All
other reagents and solvents were purchased from Ajax Finechem,
Alfa Aesar, Chem-Supply and Sigma–Aldrich. No further purification
was performed for commercial chemicals. Anhydrous acetonitrile
and anhydrous diethyl ether were obtained from a PureSolv MD
Solvent Purification System available in the School of Chemistry
at UNSW. 1H NMR and 13C NMR spectra were recorded in CDCl3
and DMSO-d6 on a Bruker Avance III 300 MHz spectrometer in
the Nuclear Magnetic Resonance Facility in the Mark Wainwright
Analytical Centre at UNSW and internally calibrated to the solvent
peaks. Chemical shifts (d) were given in parts per million (ppm).
Splitting patterns were reported as a singlet (s), broad singlet (br
s), doublet (d) and multiplet (m), and the observed coupling
constants (J) provided in Hertz (Hz). Peaks were assigned where
possible. Infrared spectra were acquired on a Thermo Nicolet
bacterial growth inhibitory activity.2
One possible explanation for this might be that the bis-indole
inhibitors, being relatively large, had more difficulties in crossing
the outer membrane or cell wall of bacterial cells. Therefore,
replacement of the 4-BrC6H4 substituent by the less bulky 4-
ClC6H4 substituent in the bis-indoles might have enhanced their
cellular permeability as a result of a reduction in molecular size,
thereby increasing their biological activity.
3. Conclusions
A library of forty-one mono-indole- and mono-benzofuran-
based glyoxyloylamides, glyoxyloylhydrazides, carboxamides and
carboxhydrazides was synthesized, fully characterized and
screened for its potential ability to inhibit transcription initiation

1768
M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
Avatar 370 FT-IR spectrophotometer using KBr discs. The
p-toluidine or phenylhydrazine and the reaction mixture stirred
at room temperature for 1 h. The resulting precipitate was filtered
out, washed with water and air-dried to afford the respective gly-
oxyloylamide or glyoxyloylhydrazide as a yellow solid in 27–41%
yield.
wavenumbers (mmax) related to the transmittance minima were
reported in cmꢀ1. Ultraviolet–visible light spectra were recorded
using a Varian Cary 100 Bio UV–visible spectrophotometer. The
absorption maxima (kmax) in nm were reported together with the
molar absorptivities (e). HPLC quality tetrahydrofuran was used
as the solvent for UV–vis measurements. High resolution mass
spectra were acquired using a Thermo Scientific LTQ Orbitrap XL
LC–MS mass spectrometer (electrospray ionization mode) in the
Bioanalytical Mass Spectrometry Facility in the Mark Wainwright
Analytical Centre at UNSW. Masses found for hydrogen adducts
([M+H]+) and sodium adducts ([M+Na]+) were reported with
accuracy to four decimal places. Required masses were calculated
using Xcalibur software installed on the spectrometer. Melting
points were measured using a Mel-Temp melting point apparatus
and are uncorrected. Both gravity column chromatography and
flash column chromatography were performed using Grace
Davisil LC60A 40–63 micron silica gel. Preparative thin layer chro-
matography was carried out using 3 mm ꢁ 200 mm ꢁ 200 mm
glass plates coated with Merck Kieselgel 60 GF254 silica gel.
Reaction progress was monitored by thin-layer chromatography
using Merck TLC Silica gel 60 F254 aluminium sheets and
detection by short and long wavelength ultraviolet light.
Temperature quoted as 0 °C was achieved with a cooling bath of
ice-water.
4.2.2.1. 2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N-phenylacetamide (16j). Yellow solid (38%); mp 286–
288 °C; 1H NMR (300 MHz, DMSO-d6): d 3.82 (s, 3H, OMe), 3.94
(s, 3H, OMe), 6.50 (s, 1H, H5), 7.06–7.16 (m, 1H, ArH), 7.26 (d,
J = 2.5 Hz, 1H, H2), 7.32–7.43 (m, 2H, ArH), 7.44–7.59 (m, 4H,
ArH), 7.64–7.75 (m, 2H, ArH), 10.50 (s, 1H, NH), 11.62 (d,
J = 2.1 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 55.8, 57.6
(OMe), 89.0 (C5), 101.2 (C7), 110.1 (C3), 116.4, 118.9 (ArC),
119.5, 123.5 (ArCH), 123.9 (C2), 128.9, 130.5, 131.1 (ArCH),
134.6, 137.1, 138.8, 161.0, 161.7 (ArC), 166.3, 188.0 (C@O); IR
(KBr): mmax 3330 (NH), 1655 (C@O), 1585 (C@O), 1530, 1326,
1215,
1118 cmꢀ1
;
UV–vis
(THF):
kmax
219 nm
(e
27,900 cmꢀ1 Mꢀ1), 238 (47,700), 331 (20,300); HRMS (+ESI): found
m/z 479.0594 ([M+H]+), [C24H19BrN2O4H]+ requires m/z 479.0606
(monoisotopic mass).
4.2.2.2. 2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N-phenylacetamide (16k). Yellow solid (28%); mp 286–
289 °C; 1H NMR (300 MHz, DMSO-d6): d 3.82 (s, 3H, OMe), 3.94
(s, 3H, OMe), 6.50 (s, 1H, H5), 7.06–7.16 (m, 1H, ArH), 7.26 (d,
J = 2.5 Hz, 1H, H2), 7.32–7.45 (m, 4H, ArH), 7.50–7.61 (m, 2H,
ArH), 7.65–7.74 (m, 2H, ArH), 10.50 (s, 1H, NH), 11.61 (d,
J = 2.1 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 55.8, 57.6
(OMe), 89.0 (C5), 101.2 (C7), 110.2 (C3), 116.3 (ArC), 119.5, 123.5
(ArCH), 123.9 (C2), 127.6, 128.9 (ArCH), 130.4 (ArC), 130.7
(ArCH), 134.3, 137.1, 138.8, 161.0, 161.7 (ArC), 166.4, 188.0
(C@O); IR (KBr): mmax 3323 (NH), 1655 (C@O), 1585 (C@O), 1535,
4.2. Synthetic chemistry details
4.2.1. Starting materials
The well-established methods developed in our group were
utilized to synthesize 3-aryl-4,6-dimethoxyindoles 6a–b,56
4,6-dimethoxy-3-methylindole 6c,56 4,6-dimethoxy-2,3-dimethyl-
indole 6d57 and 4,6-dimethoxy-3-phenylbenzofuran 1158 which
were subsequently reacted with trichloroacetyl chloride and
oxalyl chloride to afford respective 2-trichloroacetylindoles 8a–c2
and 7-trichloroacetylindoles 7a–b,2 indole-2-glyoxyloyl chlorides
10a–b2 and indole-7-glyoxyloyl chlorides 9a–b.2
4,6-Dimethoxy-2,3-dimethylindole-7-glyoxyloyl chloride 9c
and 4,6-dimethoxy-3-phenylbenzofuran-2-glyoxyloyl chloride 12
were synthesized following the general procedure for the synthesis
of glyoxyloyl chlorides2 and purified by gravity column
chromatography.
1326, 1216, 1119, 1091 cmꢀ1; UV–vis (THF): kmax 234 nm (
e
54,000 cmꢀ1 Mꢀ1), 252 (52,700), 337 (24,500); HRMS (+ESI): found
m/z 435.1107 ([M+H]+), [C24H19ClN2O4H]+ requires m/z 435.1112
(monoisotopic mass).
4.2.2.3. 2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N-(p-tolyl)acetamide (16l). Yellow solid (41%); mp 275–
277 °C. Found: C, 60.84; H, 4.29; N, 5.73; C25H21BrN2O4 requires:
C, 60.86; H, 4.29; N, 5.68); 1H NMR (300 MHz, DMSO-d6): d 2.29
(s, 3H, Me), 3.81 (s, 3H, OMe), 3.94 (s, 3H, OMe), 6.50 (s, 1H, H5),
7.13–7.22 (m, 2H, ArH), 7.26 (d, J = 2.5 Hz, 1H, H2), 7.43–7.66 (m,
6H, ArH), 10.40 (br s, 1H, NH), 11.61 (d, J = 2.2 Hz, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 20.5 (Me), 55.8, 57.6 (OMe), 89.0 (C5),
101.2 (C7), 110.1 (C3), 116.4, 118.9 (ArC), 119.5 (ArCH), 123.8
(C2), 129.3, 130.5, 131.1 (ArCH), 132.5, 134.6, 136.3, 137.2, 160.9,
161.7 (ArC), 166.2, 188.1 (C@O); IR (KBr): mmax 3332 (NH), 1652
(C@O), 1588 (C@O), 1536, 1325, 1215, 1094 cmꢀ1; UV–vis (THF):
Indole-3-glyoxyloyl chloride 14 was prepared according to the
literature procedure.59
4.2.1.1. 4,6-Dimethoxy-2,3-dimethylindole-7-glyoxyloyl chlo-
ride (9c). Dark green solid (55%); mp 126–130 °C (decomp.);
1H NMR (300 MHz, CDCl3): d 2.29 (s, 6H, Me), 3.94 (s, 3H, OMe),
4.00 (s, 3H, OMe), 6.06 (s, 1H, H5), 9.73 (br s, 1H, NH); 13C NMR
(75 MHz, CDCl3): d 10.5, 11.2 (Me), 55.8, 56.8 (OMe), 86.6 (C5),
97.5 (C7), 108.2 (C3), 114.1 (C3a), 129.6 (C2), 136.8 (C7a), 161.4
(C6), 163.3 (C4), 165.2, 179.4 (C@O).
kmax 203 nm (
e
67,000 cmꢀ1 Mꢀ1), 253 (36,200), 336 (16,000);
HRMS (+ESI): found m/z 493.0756 ([M+H]+), [C25H21BrN2O4H]+
requires m/z 493.0763 (monoisotopic mass).
4.2.1.2. 4,6-Dimethoxy-3-phenylbenzofuran-2-glyoxyloyl chlo-
ride (12). Bright green solid (64%); mp 171–173 °C (decomp.);
1H NMR (300 MHz, CDCl3): d 3.67 (s, 3H, OMe), 3.88 (s, 3H,
OMe), 6.28 (d, J = 1.8 Hz, 1H, H5), 6.69 (d, J = 1.8 Hz, 1H, H7),
7.34–7.58 (m, 5H, ArH); 13C NMR (75 MHz, CDCl3): d 55.7, 56.1
(OMe), 87.9 (C5), 96.1 (C7), 112.1 (C3a), 127.6, 129.2 (ArCH),
130.3 (ArC), 130.4 (ArCH), 136.2 (C3), 143.5 (C2), 157.0 (C4),
158.8 (C7a), 164.3 (C6), 165.6, 174.7 (C@O).
4.2.2.4. 2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N-(p-tolyl)acetamide (16m). Yellow solid (27%); mp 275–
277 °C. Found: C, 66.90; H, 4.67; N, 6.26; C25H21ClN2O4 requires:
C, 66.89; H, 4.72; N, 6.24; 1H NMR (300 MHz, DMSO-d6): d 2.29
(s, 3H, Me), 3.81 (s, 3H, OMe), 3.94 (s, 3H, OMe), 6.50 (s, 1H, H5),
7.17 (d, J = 8.3 Hz, 2H, ArH), 7.25 (d, J = 2.4 Hz, 1H, H2), 7.34–7.47
(m, 2H, ArH), 7.49–7.65 (m, 4H, ArH), 10.39 (s, 1H, NH), 11.60 (d,
J = 1.5 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 20.5 (Me),
55.8, 57.6 (OMe), 89.0 (C5), 101.2 (C7), 110.1 (C3), 116.3 (ArC),
119.5 (ArCH), 123.8 (C2), 127.6, 129.2 (ArCH), 130.4 (ArC), 130.7
4.2.2. General procedure for the synthesis of glyoxyloylamides
16j–m and glyoxyloylhydrazides 16n–o
To a solution of the respective glyoxyloyl chloride (1 mmol) in
anhydrous diethyl ether (10–20 mL) was added excess aniline,

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
1769
(ArCH), 132.5, 134.3, 136.3, 137.1, 160.9, 161.7 (ArC), 166.2, 188.1
4.2.3.1. 1-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (15a). Bright yellow solid; mp
175–178 °C; 1H NMR (300 MHz, DMSO-d6): d 1.17–1.41 (m, 4H,
CH2), 1.41–1.57 (m, 2H, CH2), 2.90 (t, J = 4.8 Hz, 2H, CH2), 3.04 (t,
J = 4.8 Hz, 2H, CH2), 3.56 (s, 3H, OMe), 3.80 (s, 3H, OMe), 6.15 (d,
J = 1.8 Hz, H5), 6.51 (d, J = 1.8 Hz, H7), 7.18–7.31 (m, 2H, ArH),
7.46–7.58 (m, 2H, ArH), 11.93 (br s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 23.6, 24.2, 25.1, 40.5, 46.1 (CH2), 55.2, 55.4 (OMe),
86.2 (C5), 93.3 (C7), 112.5, 120.8, 125.4, 127.8 (ArC), 129.3
(ArCH), 132.3 (ArC), 133.0 (ArCH), 139.8, 156.2, 161.0 (ArC),
164.0, 182.8 (C@O); IR (KBr): mmax 3320 (NH), 2935, 2854, 1647
(C@O), 1600 (C@O), 1573 (C@O), 1521, 1482, 1297, 1251, 1220,
1163, 1127, 1074, 1003, 815, 771 cmꢀ1; UV–vis (THF): kmax
(C@O); IR (KBr): mmax 3336 (NH), 1653 (C@O), 1585 (C@O), 1536,
1326, 1216, 1093 cmꢀ1
;
UV–vis (THF): kmax 203 nm
(e
63,500 cmꢀ1 Mꢀ1), 253 (40,100), 336 (17,200); HRMS (+ESI): found
m/z 449.1263 ([M+H]+), [C25H21ClN2O4H]+ requires m/z 449.1268
(monoisotopic mass).
4.2.2.5. 2-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N0-phenylacetohydrazide (16n). Yellow solid (31%); mp
248–250 °C; 1H NMR (300 MHz, DMSO-d6): d 3.89 (s, 3H, OMe),
3.93 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.98 (s, 3H, OMe), 6.47 (s,
1H, H5), 6.51 (s, 1H, H5), 6.69–7.64 (m, 20H, H2, ArH), 7.74 (s,
1H, NH), 7.90 (d, J = 2.5 Hz, 1H, NH), 9.54 (s, 1H, NH), 10.13 (d,
J = 2.5 Hz, 1H, NH), 11.50 (d, J = 2.0 Hz, 1H, NH), 11.58 (d,
J = 2.0 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 55.8, 55.8,
57.0, 57.2 (OMe), 88.4, 88.8 (C5), 101.2, 101.4 (C7), 109.8, 110.0
(C3), 112.4, 113.5 (ArCH), 116.1, 116.3 (ArC), 118.6 (ArCH), 118.8,
118.9 (ArC), 119.6 (ArCH), 123.6, 123.7 (C2), 128.5, 128.7, 130.5,
130.5, 131.1, 131.1 (ArCH), 134.6, 134.7, 136.8, 137.1, 148.4,
149.3, 160.5, 160.9, 161.4, 161.8 (ArC), 167.7, 172.0, 188.9, 189.0
(C@O) (mixture of two rotamers in solid state due to hydrogen
bonding); IR (KBr): mmax 3318 (NH), 3284, 1695 (C@O), 1578
210 nm (e
63,800 cmꢀ1 Mꢀ1), 261 (27,800), 336 (23,500); HRMS
(+ESI): found m/z 493.0729 ([M+Na]+), [C23H23BrN2O4Na]+ requires
m/z 493.0733 (monoisotopic mass).
4.2.3.2. 1-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (15b). Green-yellow solid
(55%); mp 181–183 °C (decomp.); 1H NMR (300 MHz, DMSO-d6):
d 1.15–1.56 (m, 6H, CH2), 2.89 (t, J = 4.8 Hz, 2H, CH2), 3.04 (t,
J = 4.8 Hz, 2H, CH2), 3.55 (s, 3H, OMe), 3.80 (s, 3H, OMe), 6.14 (d,
J = 1.9 Hz, 1H, H5), 6.50 (d, J = 1.9 Hz, 1H, H7), 7.23–7.33 (m, 2H,
ArH), 7.33–7.43 (m, 2H, ArH), 11.92 (br s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d 23.6, 24.2, 25.1, 40.5, 46.1 (CH2), 55.2,
55.4 (OMe), 86.2 (C7), 93.3 (C5), 112.6 (C3), 125.4 (C2), 126.4
(ArCH), 127.8, 131.9, 132.1 (ArC), 132.7 (ArCH), 139.8, 156.2,
160.9 (ArC), 164.0, 182.8 (C@O); IR (KBr): mmax 3322 (NH), 2935,
2855, 1650 (C@O), 1628 (C@O), 1601 (C@O), 1573 (C@O), 1522,
1484, 1298, 1251, 1220, 1164, 1127, 816, 771 cmꢀ1; UV–vis
(C@O), 1345, 1218, 1087 cmꢀ1; UV–vis (THF): kmax 203 nm (
e
67,900 cmꢀ1 Mꢀ1), 232 (41,900), 337 (17,500); HRMS (+ESI): found
m/z 494.0710 ([M+H]+), [C24H20BrN3O4H]+ requires m/z 494.0715
(monoisotopic mass).
4.2.2.6. 2-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
oxo-N0-phenylacetohydrazide (16o). Yellow solid (30%); mp
247–249 °C. Found: C, 63.89; H, 4.41; N, 9.38; C24H20ClN3O4
requires: C, 64.07; H, 4.48; N, 9.34; 1H NMR (300 MHz, DMSO-
d6): d 3.89 (s, 3H, OMe), 3.93 (s, 3H, OMe), 3.95 (s, 3H, OMe),
3.98 (s, 3H, OMe), 6.47 (s, 1H, H5), 6.51 (s, 1H, H5), 6.70–7.62
(m, 20H, H2, ArH), 7.73 (s, 1H, NH), 7.90 (d, J = 2.4 Hz, 1H, NH),
9.53 (s, 1H, NH), 10.13 (d, J = 2.4 Hz, 1H, NH), 11.49 (d, J = 2.2 Hz,
1H, NH), 11.57 (d, J = 2.2 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-
d6): d 55.8, 55.8, 57.0, 57.2 (OMe), 88.4, 88.8 (C5), 101.2, 101.4
(C7), 109.9, 110.0 (C3), 112.4, 113.5 (ArCH), 116.1, 116.3 (ArC),
118.6, 119.6 (ArCH), 123.6, 123.8 (C2), 127.5, 127.6, 128.5, 128.7
(ArCH), 130.3, 130.4 (ArC), 130.7, 130.7 (ArCH), 134.3, 134.3,
136.8, 137.1, 148.4, 149.3, 160.5, 160.9, 161.3, 161.8 (ArC), 167.7,
172.0, 188.9, 189.0 (C@O) (mixture of two rotamers in solid state
due to hydrogen bonding); IR (KBr): mmax 3314 (NH), 3284, 1693
(THF): kmax 213 nm
(e
25,800 cmꢀ1 Mꢀ1), 261 (18,600), 336
(18,600); HRMS (+ESI): found m/z 449.1241 ([M+Na]+),
[C23H23ClN2O4Na]+ requires m/z 449.1239 (monoisotopic mass).
4.2.3.3. 1-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(morpholin-1-yl)ethane-1,2-dione (15c). Pale yellow solid
(82%); mp 208–210 °C; 1H NMR (300 MHz, DMSO-d6): d 2.93 (t,
J = 4.4 Hz, 2H, CH2), 3.11 (t, J = 4.4 Hz, 2H, CH2), 3.28–3.48 (m, 4H,
CH2), 3.56 (s, 3H, OMe), 3.80 (s, 3H, OMe), 6.15 (d, J = 1.8 Hz, H5),
6.51 (d, J = 1.8 Hz, H7), 7.28–7.37 (m, 2H, ArH), 7.37–7.47 (m, 2H,
ArH), 11.97 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 40.2,
45.4 (CH2), 55.2, 55.4 (OMe), 65.1, 65.4 (CH2), 86.2 (C5), 93.4
(C7), 112.6, 125.7 (ArC), 126.5 (ArCH), 127.8, 131.9, 132.2 (ArC),
132.8 (ArCH), 140.0, 156.3, 161.1 (ArC), 164.4, 182.1 (C@O); IR
(KBr): mmax 3304 (NH), 2967, 2933, 2862, 1644 (C@O), 1603
(C@O), 1577 (C@O), 1524, 1488, 1434, 1384, 1294, 1274, 1240,
1216, 1153, 1138, 1113, 1090, 1013, 818, 767 cmꢀ1; UV–vis
(C@O), 1581 (C@O), 1345, 1219, 1184, 1089 cmꢀ1
(THF): kmax 203 nm
63,200 cmꢀ1 Mꢀ1), 231 (37,100), 253
; UV–vis
(
e
(28,000), 337 (15,400); HRMS (+ESI): found m/z 450.1212
([M+H]+), [C24H20ClN3O4H]+ requires m/z 450.1221 (monoisotopic
mass).
(THF): kmax 210 nm
(e
63,100 cmꢀ1 Mꢀ1), 261 (33,400), 339
(18,400); HRMS (+ESI): found m/z 451.1027 ([M+Na]+),
[C22H21ClN2O5Na]+ requires m/z 451.1031 (monoisotopic mass).
4.2.3. General procedure for the synthesis of glyoxyloylamides
15a–e, 15l, 16a–i, 17a–d and 18a–d
4.2.3.4. 1-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
To a solution or suspension of the respective glyoxyloyl chloride
(0.3–2.3 mmol) in anhydrous diethyl ether (10–30 mL) was added
excess amine (3–5 equiv) and the reaction mixture stirred at room
temperature for 3–6 h. The resulting precipitate was filtered out,
washed with diethyl ether (3 ꢁ 5 mL), 10% aqueous HCl solution
(3 ꢁ 5 mL), water (3 ꢁ 5 mL) and air-dried to afford the respective
glyoxyloylamide as a white, pink, yellow, orange or green solid in
30–88% yield. Note 1: additional recrystallization from isopropanol
(5 mL) was performed for compounds 16a–b and 16d. Note 2:
crude product 15d was precipitated from the reaction mixture by
addition of 10% aqueous HCl solution (30 mL), filtered out (after
separation of the aqueous phase from the organic phase), washed
with water (3 ꢁ 5 mL) and air-dried.
(pyrrolidin-1-yl)ethane-1,2-dione
(15d). Grey-green
solid
(30%); mp 236–238 °C (decomp.); 1H NMR (300 MHz, DMSO-d6):
d 1.52–1.79 (m, 4H, CH2), 2.75 (t, J = 6.8 Hz, 2H, CH2), 3.15 (t,
J = 6.8 Hz, 2H, CH2), 3.56 (s, 3H, OMe), 3.80 (s, 3H, OMe), 6.15 (d,
J = 1.9 Hz, 1H, H5), 6.50 (d, J = 1.9 Hz, 1H, H7), 7.15–7.29 (m, 2H,
ArH), 7.42–7.57 (m, 2H, ArH), 11.94 (s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d 23.2, 25.2, 44.4, 45.6 (CH2), 55.2, 55.4
(OMe), 86.2 (C7), 93.3 (C5), 112.4 (C3), 120.7 (ArC), 125.6 (C2),
127.4 (ArC), 129.4 (ArCH), 132.5 (ArC), 132.8 (ArCH), 139.9,
156.2, 161.0 (ArC), 163.9, 182.9 (C@O); IR (KBr): mmax 3329 (NH),
2958, 2882, 1642 (C@O), 1629 (C@O), 1615 (C@O), 1577 (C@O),
1521, 1483, 1452, 1268, 1207, 1154, 1137, 1011, 814, 698 cmꢀ1
UV–vis (THF): kmax 213 nm (
48,600 cmꢀ1 Mꢀ1), 261 (20,700),
;
e

1770
M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
336 (18,800); HRMS (+ESI): found m/z 479.0578 ([M+Na]+),
[C22H21BrN2O4Na]+ requires m/z 479.0577 (monoisotopic mass).
(C5), 102.1 (C7), 110.8 (C3), 118.0 (ArC), 122.1 (C2), 127.9, 130.8
(ArCH), 132.1, 134.0, 138.8, 161.8, 162.1 (ArC), 167.5, 190.4
(C@O); IR (KBr): mmax 3395 (NH), 2936, 2857, 1625 (C@O), 1583
(C@O), 1561 (C@O), 1537, 1467, 1448, 1351, 1323, 1297, 1253,
1216, 1184, 1119, 1091, 1011, 798, 763 cmꢀ1; UV–vis (THF): kmax
4.2.3.5. 1-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-2-yl)-2-
(4-(pyridin-2-yl)piperazin-1-yl)ethane-1,2-dione (15e). White
solid; mp 273–277 °C; 1H NMR (300 MHz, DMSO-d6): d 3.03 (br
s, 2H, CH2), 3.21 (t, J = 4.5 Hz, 2H, CH2), 3.28–3.46 (m, 4H, CH2),
3.56 (s, 3H, OMe), 3.81 (s, 3H, OMe), 6.15 (d, J = 1.9 Hz, H5), 6.51
(d, J = 1.9 Hz, H7), 6.64–6.72 (m, 1H, H5 pyridine), 6.78, 6.81 (2t,
J = 0.75 Hz, 1H, H3 pyridine), 7.20–7.31 (m, 2H, ArH), 7.43–7.51
(m, 2H, ArH), 7.51–7.61 (m, 1H, H4 pyridine), 8.07–8.20 (m, 1H,
H6 pyridine), 11.99 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d
39.6, 43.5, 44.1, 44.6 (CH2), 55.3, 55.4 (OMe), 86.2 (C5), 93.4 (C7),
107.3 (C3 pyridine), 112.5 (ArC), 113.5 (C5 pyridine), 120.9,
125.7, 127.8 (ArC), 129.4 (ArCH), 132.3 (ArC), 133.1 (ArCH), 137.7
(C4 pyridine), 140.0 (ArC), 147.6 (C6 pyridine), 156.3 (ArC), 158.4
(C2 pyridine), 161.1 (ArC), 164.5, 182.2 (C@O); IR (KBr): mmax
3411 (NH), 3148 (NH), 3109 (NH), 3012, 2959, 2935, 2841, 1644
(C@O), 1614 (C@O), 1575 (C@O), 1488, 1435, 1240, 1204, 1157,
211 nm (
e
30,300 cmꢀ1 Mꢀ1), 232 (28,400), 253 (34,000), 268
(24,800), 329 (21,300); HRMS (+ESI): found m/z 449.1238
([M+Na]+), [C23H23ClN2O4Na]+ requires m/z 449.1239 (monoiso-
topic mass).
4.2.3.9. 1-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
(morpholin-1-yl)ethane-1,2-dione (16c). Pale yellow solid; mp
247–250 °C; 1H NMR (300 MHz, DMSO-d6): d 3.25 (t, J = 4.5 Hz, 2H,
CH2), 3.49–3.63 (m, 4H, CH2), 3.69 (t, J = 4.5 Hz, 2H, CH2), 3.95 (s,
3H, OMe), 3.96 (s, 3H, OMe), 6.50 (s, 1H, H5), 7.22 (d, J = 2.5 Hz,
H2), 7.42–7.58 (m, 4H, ArH), 11.58 (d, J = 2.1 Hz, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 40.6, 45.4 (CH2), 55.9, 57.2 (OMe),
65.6, 65.8 (CH2), 88.5 (C5), 101.3 (C7), 110.0, 116.4, 118.9 (ArC),
123.8 (C2), 130.5, 131.1 (ArCH), 134.6, 136.9, 161.2, 161.5 (ArC),
167.1, 188.7 (C@O); IR (KBr): mmax 3292 (NH), 2956, 2922, 2853,
1633 (C@O), 1582 (C@O), 1557, 1530, 1348, 1275, 1214, 1178,
1134, 1008, 810, 770 cmꢀ1
; UV–vis (THF): kmax 214 nm (e
23,800 cmꢀ1 Mꢀ1), 254 (28,400), 337 (18,300); HRMS (+ESI): found
m/z 571.0945 ([M+Na]+), [C27H25BrN4O4Na]+ requires m/z
571.0951 (monoisotopic mass).
1112, 1024, 796, 763 cmꢀ1
; UV–vis (THF): kmax 210 nm (e
61,800 cmꢀ1 Mꢀ1), 255 (35,600), 331 (16,900); HRMS (+ESI): found
m/z 495.0521 ([M+Na]+), [C22H21BrN2O5Na]+ requires m/z
495.0526 (monoisotopic mass).
4.2.3.6. Ethyl (2-(3-(4-chlorophenyl)-4,6-dimethoxy-1H-indol-
2-yl)-2-oxoacetyl)glycinate (15l). Dark orange solid; mp 154–
156 °C; 1H NMR (300 MHz, DMSO-d6): d 1.19 (t, J = 7.1 Hz, 3H,
CH2CH3), 3.58 (s, 3H, OMe), 3.60 (d, J = 5.9 Hz, 2H, CH2), 3.80 (s,
3H, OMe), 4.10 (q, J = 7.1 Hz, CH2CH3), 6.15 (d, J = 1.9 Hz, H5), 6.63
(d, J = 1.9 Hz, H7), 7.27–7.39 (m, 4H, ArH), 9.04 (t, J = 5.9 Hz, 1H,
NH), 11.88 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 14.0
(CH2CH3), 40.2 (CH2), 55.2, 55.3 (OMe), 60.6 (CH2CH3), 86.6 (C5),
93.3 (C7), 112.3, 126.2 (ArC), 126.5 (ArCH), 127.1, 131.6 (ArC),
132.5 (ArCH), 133.1, 139.7, 156.0, 160.8 (ArC), 164.9, 168.8, 179.3
(C@O); IR (KBr): mmax 3386, 3318 (NH), 2970, 2938, 2839, 1747
(C@O), 1672 (C@O), 1617 (C@O), 1575 (C@O), 1510, 1479, 1384,
1311, 1259, 1207, 1157, 1131, 1089, 998, 815 cmꢀ1; UV–vis (THF):
4.2.3.10. 1-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-
2-(morpholin-1-yl)ethane-1,2-dione (16d). Pale yellow solid
(76%); mp 239–241 °C; 1H NMR (300 MHz, CDCl3): d 3.39 (t,
J = 4.5 Hz, 2H, CH2), 3.67 (t, J = 4.5 Hz, 2H, CH2), 3.70–3.76 (m, 2H,
CH2), 3.77–3.84 (m, 2H, CH2), 3.92 (s, 3H, OMe), 3.98 (s, 3H,
OMe), 6.20 (s, 1H, H5), 7.09 (d, J = 2.3 Hz, 1H, H2), 7.28–7.37 (m,
2H, ArH), 7.40–7.52 (m, 2H, ArH), 10.69 (br s, 1H, NH); 13C NMR
(75 MHz, CDCl3): d 41.3, 46.0 (CH2), 55.7, 57.3 (OMe), 66.4, 66.7
(CH2), 87.8 (C5), 102.0 (C7), 110.9 (C3), 118.2 (ArC), 122.2 (C2),
127.9, 130.8 (ArCH), 132.1, 133.8, 138.6, 162.1, 162.1 (ArC),
167.8, 189.5 (C@O); IR (KBr): mmax 3377 (NH), 2974, 2906, 2852,
1635 (C@O), 1585 (C@O), 1559 (C@O), 1535, 1513, 1467, 1351,
1323, 1293, 1272, 1212, 1183, 1158, 1110, 1093, 1024, 973, 798,
kmax 212 nm (
e
64,600 cmꢀ1 Mꢀ1), 262 (34,800), 359 (16,600);
HRMS (+ESI): found m/z 467.0969 ([M+Na]+), [C22H21ClN2O6Na]+
requires m/z 467.0980 (monoisotopic mass).
764 cmꢀ1; UV–vis (THF): kmax 211 nm (
e
6,900 cmꢀ1 Mꢀ1), 235
(10,600), 254 (17,700), 270 (15,000), 332 (13,500); HRMS (+ESI):
found m/z 451.1025 ([M+Na]+), [C22H21ClN2O5Na]+ requires m/z
451.1031 (monoisotopic mass).
4.2.3.7. 1-(3-(4-Bromophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (16a). Pale yellow solid
(65%); mp 236–238 °C; 1H NMR (300 MHz, CDCl3): d 1.44–1.61
(m, 2H, CH2), 1.61–1.74 (m, 4H, CH2), 3.32 (t, J = 5.5 Hz, 2H, CH2),
3.66 (br s, 2H, CH2), 3.91 (s, 3H, OMe), 3.95 (s, 3H, OMe), 6.19 (s,
1H, H5), 7.09 (d, J = 2.3 Hz, 1H, H2), 7.34–7.52 (m, 4H, ArH),
10.75 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 24.7, 25.4,
25.7, 41.8, 46.8 (CH2), 55.6, 57.1 (OMe), 87.8 (C5), 102.0 (C7),
110.7 (C3), 118.0, 120.2 (ArC), 122.1 (C2), 130.8, 131.2 (ArCH),
134.4, 138.7, 161.7, 162.1 (ArC), 167.5, 190.4 (C@O); IR (KBr): mmax
3389 (NH), 2930, 2855, 1637 (C@O), 1581 (C@O), 1560 (C@O),
1537, 1514, 1467, 1347, 1323, 1296, 1251, 1217, 1185, 1156,
4.2.3.11. 1-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-
2-(pyrrolidin-1-yl)ethane-1,2-dione (16e). Pale yellow solid
(83%); mp decomp. >170 °C; 1H NMR (300 MHz, CDCl3): d 1.85–
2.04 (m, 4H, CH2), 3.38 (t, J = 6.5 Hz, 2H, CH2), 3.62 (t, J = 6.5 Hz,
2H, CH2), 3.91 (s, 3H, OMe), 3.94 (s, 3H, OMe), 6.18 (s, 1H, H5),
7.09 (d, J = 2.3 Hz, 1H, H2), 7.28–7.36 (m, 2H, ArH), 7.44–7.51 (m,
2H, ArH), 10.70 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 24.5,
26.0, 45.1, 46.4 (CH2), 55.6, 57.5 (OMe), 87.8 (C5), 101.5 (C7),
110.9 (C3), 118.1 (ArC), 122.1 (C2), 127.9, 130.8 (ArCH), 132.1,
133.9, 138.8, 161.8, 162.2 (ArC), 167.7, 190.2 (C@O); IR (KBr): mmax
3385 (NH), 2968, 2878, 1626 (C@O), 1578 (C@O), 1556 (C@O),
1119, 1091, 1008, 798, 763 cmꢀ1; UV–vis (THF): kmax 211 nm (
e
13,600 cmꢀ1 Mꢀ1), 235 (15,500), 254 (20,900), 271 (16,900), 329
(15,600); HRMS (+ESI): found m/z 493.0730 ([M+Na]+),
[C23H23BrN2O4Na]+ requires m/z 493.0733 (monoisotopic mass).
1537, 1465, 1452, 1350, 1274, 1262, 1217, 1093, 979, 800 cmꢀ1
;
UV–vis (THF): kmax 211 nm (
e
32,100 cmꢀ1 Mꢀ1), 253 (24,200),
330 (14,800); HRMS (+ESI): found m/z 435.1091 ([M+Na]+),
[C22H21ClN2O4Na]+ requires m/z 435.1082 (monoisotopic mass).
4.2.3.8. 1-(3-(4-Chlorophenyl)-4,6-dimethoxy-1H-indol-7-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (16b). Pale yellow solid
(88%); mp 226–228 °C; 1H NMR (300 MHz, CDCl3): d 1.43–1.61
(m, 2H, CH2), 1.61–1.80 (m, 4H, CH2), 3.32 (t, J = 5.5 Hz, 2H, CH2),
3.66 (br s, 2H, CH2), 3.92 (s, 3H, OMe), 3.95 (s, 3H, OMe), 6.20 (s,
1H, H5), 7.09 (d, J = 2.3 Hz, 1H, H2), 7.28–7.36 (m, 2H, ArH),
7.42–7.52 (m, 2H, ArH), 10.74 (br s, 1H, NH); 13C NMR (75 MHz,
CDCl3): d 24.7, 25.4, 25.7, 41.8, 46.8 (CH2), 55.6, 57.2 (OMe), 87.8
4.2.3.12. 1-(4-Benzylpiperazin-1-yl)-2-(3-(4-bromophenyl)-4,6-
dimethoxy-1H-indol-7-yl)ethane-1,2-dione (16f). Pale yellow
solid; mp 196–198 °C; 1H NMR (300 MHz, DMSO-d6): d 2.34 (t,
J = 4.1 Hz, 2H, CH2), 2.46 (t, J = 4.1 Hz, 2H, CH2), 3.25 (t, J = 4.1 Hz,
2H, CH2), 3.53 (s, 2H, CH2 benzyl), 3.56 (t, J = 4.1 Hz, 2H, CH2), 3.88
(s, 3H, OMe), 3.94 (s, 3H, OMe), 6.47 (s, 1H, H5), 7.22 (d, J = 2.6 Hz,

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
1771
H2), 7.23–7.39 (m, 5H, ArH), 7.41–7.57 (m, 4H, ArH), 11.57 (d,
J = 2.3 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 40.2, 45.0,
51.7, 51.8 (CH2), 55.9, 57.0 (OMe), 61.9 (CH2 benzyl), 88.4 (C5),
101.3 (C7), 109.9, 116.3, 118.9 (ArC), 123.7 (C2), 127.1, 128.2,
129.0, 130.5, 131.1 (ArCH), 134.6, 136.9, 137.5, 161.1, 161.5 (ArC),
166.8, 188.9 (C@O); IR (KBr): mmax 3407 (NH), 2938, 2811, 1636
(C@O), 1578 (C@O), 1558, 1349, 1217 cmꢀ1; UV–vis (THF): kmax
4.2.3.16. 1-(1H-Indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione
(17a).
White solid; mp 182–185 °C; 1H NMR (300 MHz,
DMSO-d6): d 1.33–1.51 (m, 2H, CH2), 1.51–1.75 (m, 4H, CH2),
3.22–3.32 (m, 2H, CH2), 3.49–3.67 (m, 2H, CH2), 7.19–7.38 (m,
2H, ArH), 7.50–7.58 (m, 1H, ArH), 8.05–8.25 (m, 2H, ArH), 12.28
(br s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d 23.9, 25.0, 25.9,
41.2, 46.4 (CH2), 112.6 (ArCH), 113.1 (ArC), 120.9, 122.5, 123.5
(ArCH), 124.8 (ArC), 136.8 (ArCH), 136.9 (ArC), 165.7, 186.8
(C@O); IR (KBr): mmax 3152 (NH), 2920, 2857, 1612 (C@O), 1583
(C@O), 1519, 1498, 1462, 1444, 1270, 1244, 1161, 1128, 752,
210 nm (e
100,500 cmꢀ1 Mꢀ1), 253 (39,900), 318 (32,400); HRMS
(+ESI): found m/z 584.1160 ([M+Na]+), [C29H28BrN3O4Na]+ requires
m/z 584.1155 (monoisotopic mass).
738 cmꢀ1; UV–vis (THF): kmax 214 nm ( 23,300 cmꢀ1 Mꢀ1), 245
e
(12,100), 264 (10,000), 302 (14,700); HRMS (+ESI): found m/z
257.1291 ([M+H]+), [C15H16N2O2H]+ requires m/z 257.1285
(monoisotopic mass).
4.2.3.13. 1-(4,6-Dimethoxy-2,3-dimethyl-1H-indol-7-yl)-2-(piper-
idin-1-yl)ethane-1,2-dione (16g). Yellow solid; mp 206–209 °C;
1H NMR (300 MHz, DMSO-d6): d 1.36–1.50 (m, 2H, CH2), 1.50–1.71
(m, 4H, CH2), 2.24 (s, 3H, Me), 2.25 (s, 3H, Me), 3.17 (t, J = 5.3 Hz,
2H, CH2), 3.50 (t, J = 5.3 Hz, 2H, CH2), 3.86 (s, 3H, OMe), 3.98 (s,
3H, OMe), 6.34 (s, 1H, H5), 10.82 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 10.4, 10.7 (Me), 24.0, 24.9, 25.1, 40.7, 45.9 (CH2),
55.9, 56.9 (OMe), 87.4 (C5), 101.3 (C7), 105.8 (C3), 113.1 (C3a),
130.0 (C2), 135.4 (C7a), 160.4 (C6), 160.6 (C4), 166.9, 189.2 (C@O);
IR (KBr): mmax 3532 (NH), 3356 (NH), 2922, 2849, 1637 (C@O),
1596 (C@O), 1561 (C@O), 1451, 1360, 1307, 1251, 1220, 1181,
4.2.3.17. 1-(1H-Indol-3-yl)-2-(morpholin-1-yl)ethane-1,2-dione
(17b).
Pale pink solid; mp 185–187 °C; 1H NMR (300 MHz,
DMSO-d6): d 3.24–3.45 (m, 2H, CH2), 3.47–3.58 (m, 2H, CH2),
3.58–3.67 (m, 2H, CH2), 3.67–3.83 (m, 2H, CH2), 7.15–7.42 (m,
2H, ArH), 7.45–7.66 (m, 1H, ArH), 8.06–8.18 (m, 1H, ArH), 8.21 (s,
1H, ArH), 12.34 (br s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d
41.0, 46.0, 65.9, 66.2 (CH2), 112.7 (ArCH), 113.1 (ArC), 120.9,
122.6, 123.6 (ArCH), 124.9, 137.0 (ArC), 137.3 (ArCH), 165.9,
186.0 (C@O); IR (KBr): mmax 3157 (NH), 2977, 2925, 2860, 1613
(C@O), 1584 (C@O), 1519, 1443, 1242, 1157, 1113, 964, 783,
1150, 1123, 990, 787 cmꢀ1
; UV–vis (THF): kmax 210 nm (e
35,200 cmꢀ1 Mꢀ1), 255 (27,000), 329 (11,600); HRMS (+ESI): found
m/z 367.1619 ([M+Na]+), [C19H24N2O4Na]+ requires m/z 367.1628
(monoisotopic mass).
744 cmꢀ1; UV–vis (THF): kmax 212 nm ( 26,000 cmꢀ1 Mꢀ1), 247
e
(13,000), 265 (11,100), 305 (15,700); HRMS (+ESI): found m/z
281.0904 ([M+Na]+), [C14H14N2O3Na]+ requires m/z 281.0897
(monoisotopic mass).
4.2.3.14. 1-(4,6-Dimethoxy-2,3-dimethyl-1H-indol-7-yl)-2-(mor-
pholin-1-yl)ethane-1,2-dione (16h). Yellow solid; mp 277–
280 °C (decomp.); 1H NMR (300 MHz, DMSO-d6): d 2.23 (s, 3H,
Me), 2.25 (s, 3H, Me), 3.21 (t, J = 4.6 Hz, 2H, CH2), 3.48–3.61 (m,
4H, CH2), 3.68 (t, J = 4.6 Hz, 2H, CH2), 3.90 (s, 3H, OMe), 3.98 (s,
3H, OMe), 6.35 (s, 1H, H5), 10.83 (s, 1H, NH); 13C NMR (75 MHz,
DMSO-d6): d 10.4, 10.7 (Me), 40.5, 45.4 (CH2), 55.9, 57.0 (OMe),
65.6, 65.8 (CH2), 87.4 (C5), 101.2 (C7), 105.9 (C3), 113.2 (C3a),
130.1 (C2), 135.3 (C7a), 160.4 (C6), 160.9 (C4), 167.3, 188.5 (C@O);
IR (KBr): mmax 3363 (NH), 2915, 2853, 1652 (C@O), 1616, 1597
(C@O), 1561 (C@O), 1439, 1360, 1307, 1269, 1226, 1206, 1173,
4.2.3.18.
yl)ethane-1,2-dione (17c).
1-(1H-Indol-3-yl)-2-(4-(pyridin-2-yl)piperazin-1-
White solid; mp 246–251 °C; 1H
NMR (300 MHz, DMSO-d6): d 3.39–3.58 (m, 4H, CH2), 3.61–3.82
(m, 4H, CH2), 6.63–6.73 (m, 1H, H5 pyridine), 6.82, 6.85 (2t,
J = 0.75 Hz, 1H, H3 pyridine), 7.21–7.38 (m, 2H, ArH), 7.49–7.65
(m, 2H, H4 pyridine, ArH indole), 8.08–8.20 (m, 2H, H6 pyridine,
ArH indole), 8.22 (s, 1H, ArH), 12.31 (br s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d 40.3, 44.2, 44.9, 45.1 (CH2), 107.4 (C3 pyri-
dine), 112.7 (ArCH), 113.1 (ArC), 113.5 (C5 pyridine), 120.9,
122.6, 123.6 (ArCH), 124.9, 137.0 (ArC), 137.3 (C4 pyridine),
137.7 (ArCH), 147.6 (C6 pyridine), 158.6 (C2 pyridine), 165.9,
186.2 (C@O); IR (KBr): mmax 3433 (NH), 3139 (NH), 3111 (NH),
2920, 1614 (C@O), 1591 (C@O), 1482, 1435, 1282, 1242, 1157,
1149, 1131, 1117, 1016, 988, 789 cmꢀ1
; UV–vis (THF): kmax
210 nm (e
29,200 cmꢀ1 Mꢀ1), 253 (25,600), 331 (12,600); HRMS
(+ESI): found m/z 369.1414 ([M+Na]+), [C18H22N2O5Na]+ requires
m/z 369.1421 (monoisotopic mass).
957, 782, 742 cmꢀ1
;
UV–vis (THF): kmax 213 nm
(e
4.2.3.15.
1-(4,6-Dimethoxy-2,3-dimethyl-1H-indol-7-yl)-2-(4-
25,600 cmꢀ1 Mꢀ1), 250 (36,700), 305 (22,200); HRMS (+ESI): found
m/z 335.1496 ([M+H]+), [C19H18N4O2H]+ requires m/z 335.1503
(monoisotopic mass).
(pyridin-2-yl)piperazin-1-yl)ethane-1,2-dione (16i). Pale yellow
solid; mp 264–266 °C (decomp.); 1H NMR (300 MHz, DMSO-d6):
d 2.24 (s, 3H, Me), 2.26 (s, 3H, Me), 3.27–3.37 (m, 2H, CH2),
3.44–3.52 (m, 2H, CH2), 3.56–3.71 (m, 4H, CH2), 3.85 (s, 3H,
OMe), 3.98 (s, 3H, OMe), 6.34 (s, 1H, H5), 6.64–6.72 (m, 1H, H5
pyridine), 6.83, 6.86 (2s, 1H, H3 pyridine), 7.50–7.63 (m, 1H, H4
pyridine), 8.07–8.21 (m, 1H, H6 pyridine), 10.86 (s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 10.4, 10.7 (Me), 39.9, 44.2, 44.3, 44.6
(CH2), 55.9, 57.0 (OMe), 87.5 (C5), 101.3 (C7), 105.9 (C3), 107.4
(C3 pyridine), 113.2 (C3a), 113.5 (C5 pyridine), 130.1 (C2), 135.4
(C7a), 137.7 (C4 pyridine), 147.6 (C6 pyridine), 158.7 (C2 pyridine),
160.5 (C6), 160.9 (C4), 167.3, 188.7 (C@O); IR (KBr): mmax 3415
(NH), 2924, 2852, 1650 (C@O), 1617, 1591 (C@O), 1557 (C@O),
1478, 1435, 1384, 1356, 1309, 1248, 1220, 1184, 1149, 1126,
4.2.3.19.
1,2-dione (17d).
1-(4-Benzylpiperazin-1-yl)-2-(1H-Indol-3-yl)ethane-
White solid; mp 175–178 °C; 1H NMR
(300 MHz, DMSO-d6): d 2.32 (t, J = 4.9 Hz, 2H, CH2), 2.46 (t,
J = 4.9 Hz, 2H, CH2), 3.33 (t, J = 4.9 Hz, 2H, CH2), 3.49 (s, 2H, CH2
benzyl), 3.62 (t, J = 4.9 Hz, 2H, CH2), 7.19–7.41 (m, 7H, ArH),
7.49–7.60 (m, 1H, ArH), 8.05–8.22 (m, 2H, ArH), 12.30 (br s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6): d 40.6, 45.6, 52.0, 52.7 (CH2),
61.8 (CH2 benzyl), 112.7 (ArCH), 113.1 (ArC), 120.9, 122.5, 123.6
(ArCH), 124.8 (ArC), 127.1, 128.2, 128.9 (ArCH), 136.9 (ArC),
137.0 (ArCH), 137.7 (ArC), 165.8, 186.2 (C@O); IR (KBr): mmax
3152 (NH), 3113 (NH), 3026, 2943, 2919, 2813, 2768, 1613
(C@O), 1584 (C@O), 1519, 1463, 1442, 1255, 1241, 1156, 1142,
1013, 984, 801, 776 cmꢀ1
; UV–vis (THF): kmax 211 nm (e
19,600 cmꢀ1 Mꢀ1), 252 (35,600), 329 (11,100); HRMS (+ESI): found
m/z 445.1838 ([M+Na]+), [C23H26N4O4Na]+ requires m/z 445.1846
(monoisotopic mass).
998, 781, 775, 756, 746 cmꢀ1; UV–vis (THF): kmax 212 nm (
25,600 cmꢀ1 Mꢀ1), 246 (9,800), 265 (8,400), 304 (12,800); HRMS
e

1772
M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
(+ESI): found m/z 348.1710 ([M+H]+), [C21H21N3O2H]+ requires m/z
348.1707 (monoisotopic mass).
156.4, 157.3, 163.4 (ArC), 163.4, 180.3 (C@O); IR (KBr): mmax 3496,
2937, 2811, 1735, 1652 (C@O), 1613 (C@O), 1590 (C@O), 1551,
1504, 1445, 1366, 1321, 1221, 1202, 1152, 1117, 998, 745 cmꢀ1
;
4.2.3.20.
1-(4,6-Dimethoxy-3-phenylbenzofuran-2-yl)-2-
UV–vis (THF): kmax 210 nm (e
42,700 cmꢀ1 Mꢀ1), 254 (19,600), 346
(piperidin-1-yl)ethane-1,2-dione (18a). Pale pink solid; mp
104–108 °C; 1H NMR (300 MHz, DMSO-d6): d 1.26–1.45 (m, 4H,
CH2), 1.45–1.64 (m, 2H, CH2), 3.07 (t, J = 4.9 Hz, 2H, CH2), 3.14
(t, J = 5.3 Hz, 2H, CH2), 3.62 (s, 3H, OMe), 3.87 (s, 3H, OMe), 6.46
(d, J = 1.9 Hz, 1H, H5), 6.96 (d, J = 1.9 Hz, 1H, H7), 7.35–7.52 (m,
5H, ArH); 13C NMR (75 MHz, DMSO-d6): d 23.7, 24.5, 25.3, 40.9,
46.2 (CH2), 55.8, 56.1 (OMe), 88.2 (C5), 96.0 (C7), 111.1 (ArC),
127.0, 128.5 (ArCH), 129.6 (ArC), 130.3 (ArCH), 132.3, 143.6,
156.4, 157.3, 163.4 (ArC), 163.4, 180.8 (C@O); IR (KBr): mmax
3464, 2939, 2858, 1640 (C@O), 1619 (C@O), 1594 (C@O), 1556,
1507, 1447, 1322, 1289, 1251, 1223, 1204, 1153, 1116, 1057,
(21,400); HRMS (+ESI): found m/z 507.1883 ([M+Na]+),
[C29H28N2O5Na]+ requires m/z 507.1890 (monoisotopic mass).
4.2.4. General procedure for the synthesis of carboxamides 19
and 20a–c, and carboxhydrazides 20d–e
To a solution of the respective trichloroacetylindole (0.3–
0.6 mmol) in anhydrous acetonitrile (20 mL) was added excess ani-
line, p-toluidine (25–95 equiv), phenylhydrazine (3 equiv) or mor-
pholine (5 equiv) followed by triethylamine as a catalyst (5 drops)
and the reaction mixture stirred at room temperature for 3–9 h
(phenylhydrazine, morpholine) or heated at reflux overnight (ani-
line, p-toluidine). The solvent was evaporated under reduced pres-
sure and the residue was treated with diethyl ether (10 mL). The
resulting solid was filtered out and air-dried to afford the respec-
tive carboxamide or carboxyhydrazide as a white or yellow solid
in 51–69% yield. Note 1: additional recrystallization from methanol
was performed for compounds 20d–e. Note 2: product 19 was pre-
cipitated from the reaction mixture by addition of water (30 mL),
filtered out, washed with water (3 ꢁ 5 mL) and air-dried.
813, 750 cmꢀ1; UV–vis (THF): kmax 210 nm ( 26,100 cmꢀ1 Mꢀ1),
e
254 (18,600), 345 (22,100); HRMS (+ESI): found m/z 394.1654
([M+H]+), [C23H23NO5H]+ requires m/z 394.1649 (monoisotopic
mass).
4.2.3.21. 1-(4,6-Dimethoxy-3-phenylbenzofuran-2-yl)-2-(mor-
pholin-1-yl)ethane-1,2-dione (18b). Creamy white solid; mp
134–136 °C; 1H NMR (300 MHz, DMSO-d6): d 3.08 (t, J = 4.6 Hz,
2H, CH2), 3.22 (t, J = 4.6 Hz, 2H, CH2), 3.37–3.51 (m, 4H, CH2),
3.63 (s, 3H, OMe), 3.87 (s, 3H, OMe), 6.46 (d, J = 1.9 Hz, 1H, H5),
6.97 (d, J = 1.9 Hz, 1H, H7), 7.34–7.59 (m, 5H, ArH); 13C NMR
(75 MHz, DMSO-d6): d 40.5, 45.5 (CH2), 55.8, 56.1 (OMe), 65.3,
65.6 (CH2), 88.2 (C5), 96.0 (C7), 111.1 (ArC), 127.1, 128.6 (ArCH),
129.6 (ArC), 130.4 (ArCH), 132.8, 143.6, 156.4, 157.4, 163.5 (ArC),
163.7, 180.1 (C@O); IR (KBr): mmax 3444, 3120, 3059, 2957, 2913,
2856, 1642 (C@O), 1594 (C@O), 1552, 1509, 1464, 1443, 1322,
1272, 1232, 1204, 1152, 1115, 1008, 808 cmꢀ1; UV–vis (THF): kmax
4.2.4.1. (3-(4-Chlorophenyl)-4,6-dimethoxy-1H-Indol-2-yl)(mor-
pholino)methanone (19).
Pale yellow solid (69%); mp decomp.
>250 °C; 1H NMR (300 MHz, CDCl3): d 3.29 (br s, 8H, CH2), 3.72 (s,
3H, OMe), 3.83 (s, 3H, OMe), 6.20 (d, J = 1.8 Hz, 1H, H5), 6.47 (d,
J = 1.8 Hz, 1H, H7), 7.33–7.44 (m, 4H, ArH), 9.27 (br s, 1H, NH); 13C
NMR (75 MHz, CDCl3): d 55.2, 55.7 (OMe), 66.2 (CH2), 86.5 (C7),
93.1 (C5), 110.8 (C3), 117.3 (ArC), 124.6 (C2), 128.0, 132.4 (ArCH),
133.1, 133.4, 138.0, 155.7, 159.2 (ArC), 164.3 (C@O); IR (KBr): mmax
3276 (NH), 2959, 2930, 2849, 1625 (C@O), 1585 (C@O), 1536, 1457,
1270, 1230, 1208, 1151, 1134, 1115, 862, 823, 809, 750 cmꢀ1; UV–
210 nm (e
29,500 cmꢀ1 Mꢀ1), 254 (19,300), 347 (22,300); HRMS
(+ESI): found m/z 418.1258 ([M+Na]+), [C22H21NO6Na]+ requires
m/z 418.1261 (monoisotopic mass).
vis (THF): kmax 222 nm (
e
31,600 cmꢀ1 Mꢀ1), 252 (34,100), 305
(15,900); HRMS (+ESI): found m/z 423.1086 ([M+Na]+),
[C21H21ClN2O4Na]+ requires m/z 423.1082 (monoisotopic mass).
4.2.3.22.
1-(4,6-Dimethoxy-3-phenylbenzofuran-2-yl)-2-(4-
(pyridin-2-yl)piperazin-1-yl)ethane-1,2-dione (18c). Pale pink
solid; mp 162–164 °C; 1H NMR (300 MHz, DMSO-d6): d 3.18 (t,
J = 4.5 Hz, 2H, CH2), 3.26–3.50 (m, 6H, CH2), 3.62 (s, 3H, OMe),
3.87 (s, 3H, OMe), 6.46 (d, J = 1.8 Hz, 1H, H5), 6.63–6.72 (m, 1H,
H5 pyridine), 6.78, 6.81 (2s, 1H, H3 pyridine), 6.97 (d, J = 1.8 Hz,
1H, H7), 7.31–7.50 (m, 5H, ArH), 7.50–7.60 (m, 1H, H4 pyridine),
8.04–8.20 (m, 1H, H6 pyridine); 13C NMR (75 MHz, DMSO-d6): d
40.0, 43.7, 44.3, 44.7 (CH2), 55.8, 56.1 (OMe), 88.2 (C5), 96.0 (C7),
107.3 (C3 pyridine), 111.1 (ArC), 113.5 (C5 pyridine), 127.1, 128.6
(ArCH), 129.6 (ArC), 130.4 (ArCH), 132.8 (ArC), 137.7 (C4 pyridine),
143.7 (ArC), 147.6 (C6 pyridine), 156.4, 157.4 (ArC), 158.4 (C2 pyr-
idine), 163.5 (ArC), 163.7, 180.2 (C@O); IR (KBr): mmax 3448, 3060,
3011, 2970, 2932, 2830, 1635 (C@O), 1590 (C@O), 1557, 1479,
1436, 1383, 1316, 1282, 1233, 1203, 1153, 1116, 981, 773,
4.2.4.2.
dole-7-carboxamide (20a).
3-(4-Bromophenyl)-4,6-dimethoxy-N-phenyl-1H-in-
White solid (60%); mp 226–
228 °C; 1H NMR (300 MHz, CDCl3): d 3.90 (s, 3H, OMe), 4.13 (s,
3H, OMe), 6.31 (s, 1H, H5), 7.10–7.19 (m, 2H, H2, ArH), 7.34–
7.43 (m, 2H, ArH), 7.43–7.53 (m, 4H, ArH), 7.63–7.72 (m, 2H,
ArH), 10.05 (s, 1H, NH), 11.23 (br s, 1H, NH); 13C NMR (75 MHz,
CDCl3): d 55.4, 57.4 (OMe), 87.9 (C5), 98.3 (C7), 111.4 (C3),
117.0, 119.9 (ArC), 120.8 (ArCH), 122.4 (C2), 124.1, 129.1, 130.8,
131.2 (ArCH), 134.9, 138.6, 139.7, 156.7, 157.8 (ArC), 165.7
(C@O); IR (KBr): mmax 3384 (NH), 1644 (C@O), 1591 (C@O),
1542, 1442, 1313, 1249, 975, 756 cmꢀ1; UV–vis (THF): kmax
246 nm (e
35,400 cmꢀ1 Mꢀ1), 279 (22,500), 330 (23,900); HRMS
(+ESI): found m/z 451.0649 ([M+H]+), [C23H19BrN2O3H]+ requires
m/z 451.0657 (monoisotopic mass).
753 cmꢀ1; UV–vis (THF): kmax 210 nm ( 44,600 cmꢀ1 Mꢀ1), 253
e
(41,300), 346 (21,500); HRMS (+ESI): found m/z 494.1685
([M+Na]+), [C27H25N3O5Na]+ requires m/z 494.1686 (monoisotopic
mass).
4.2.4.3.
3-(4-Bromophenyl)-4,6-dimethoxy-N-(p-tolyl)-1H-in-
dole-7-carboxamide (20b). White solid (51%); mp 239–241 °C;
1H NMR (300 MHz, DMSO-d6): d 2.29 (s, 3H, Me), 3.91 (s, 3H,
OMe), 4.12 (s, 3H, OMe), 6.56 (s, 1H, H5), 7.13–7.22 (m, 2H, ArH),
7.25 (d, J = 2.5 Hz, 1H, H2), 7.43–7.57 (m, 4H, ArH), 7.63–7.73 (m,
2H, ArH), 10.07 (s, 1H, NH), 11.50 (d, J = 1.7 Hz, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d 20.5 (Me), 55.5, 57.3 (OMe), 88.4
(C5), 98.2 (C7), 110.2 (C3), 115.4, 118.5 (ArC), 120.1 (ArCH),
123.6 (C2), 129.1, 130.4, 131.0 (ArCH), 132.3, 135.1, 136.3, 138.0,
156.1, 156.9 (ArC), 164.2 (C@O); IR (KBr): mmax 3336 (NH), 1635
(C@O), 1587 (C@O), 1535, 1311, 1241, 978 cmꢀ1; UV–vis (THF):
4.2.3.23. 1-(4-Benzylpiperazin-1-yl)-2-(4,6-dimethoxy-3-phenyl-
benzofuran-2-yl)ethane-1,2-dione (18d). Pale yellow solid; mp
108–110 °C; 1H NMR (300 MHz, DMSO-d6): d 2.20 (t, J = 4.6 Hz,
2H, CH2), 2.26 (t, J = 4.6 Hz, 2H, CH2), 3.09 (t, J = 4.6 Hz, 2H, CH2),
3.19 (t, J = 4.6 Hz, 2H, CH2), 3.46 (s, 2H, CH2 benzyl), 3.62 (s, 3H,
OMe), 3.87 (s, 3H, OMe), 6.45 (d, J = 1.8 Hz, 1H, H5), 6.95 (d,
J = 1.8 Hz, 1H, H7), 7.21–7.51 (m, 10H, ArH); 13C NMR (75 MHz,
DMSO-d6): d 40.2, 45.2, 51.2, 51.8 (CH2), 55.8, 56.1 (OMe), 61.6
(CH2 benzyl), 88.2 (C5), 96.0 (C7), 111.1 (ArC), 127.1, 127.1, 128.2,
128.6, 128.9 (ArCH), 129.5 (ArC), 130.3 (ArCH), 132.6, 137.5, 143.6,
kmax 247 nm (
e
40,900 cmꢀ1 Mꢀ1), 238 (25,700), 331 (29,300);

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
1773
HRMS (+ESI): found m/z 465.0808 ([M+H]+), [C24H21BrN2O3H]+
requires m/z 465.0814 (monoisotopic mass).
chromatography (4.0–5.5 cm ꢁ 3.5 cm silica gel column filling,
ethyl acetate/n-hexane 40%/60% (v/v) ? 50%/50% (v/v) ? 60%/40%
(v/v)) afforded the respective glyoxyloylamides as yellow or brown
solids in 10–54% yield.
4.2.4.4. 3-(4-Chlorophenyl)-4,6-dimethoxy-N-(p-tolyl)-1H-in-
dole-7-carboxamide (20c).
White solid (56%); mp 234–
Note: 1.3 equiv of N-benzylpiperazine and N-(pyridin-2-
yl)piperazine, 2.0 equiv of N-methylpiperazine and pyrrolidine,
and 5.0 equiv of morpholine and piperidine were used, respec-
tively. Additional purification by preparative thin layer chro-
matography (ethyl acetate/n-hexane 35%/65% (v/v)) was
performed for compound 15h in order to obtain an analytically
pure product.
236 °C; 1H NMR (300 MHz, DMSO-d6): d 2.29 (s, 3H, Me), 3.91
(s, 3H, OMe), 4.12 (s, 3H, OMe), 6.55 (s, 1H, H5), 7.13–7.21 (m,
2H, ArH), 7.24 (d, J = 2.5 Hz, 1H, H2), 7.34–7.43 (m, 2H, ArH),
7.49–7.59 (m, 2H, ArH), 7.63–7.73 (m, 2H, ArH), 10.07 (s, 1H,
NH), 11.50 (d, J = 1.6 Hz, 1H, NH); 13C NMR (75 MHz, DMSO-d6):
d 20.5 (Me), 55.4, 57.3 (OMe), 88.4 (C5), 98.2 (C7), 110.3 (C3),
115.3 (ArC), 120.1 (ArCH), 123.6 (C2), 127.5, 129.1 (ArCH), 130.0
(ArC), 130.7 (ArCH), 132.3, 134.7, 136.3, 138.0, 156.1, 156.9
(ArC), 164.2 (C@O); IR (KBr): mmax 3336 (NH), 1636 (C@O), 1592
(C@O), 1529, 1311, 1242, 978 cmꢀ1; UV–vis (THF): kmax 247 nm
4.2.5.1. 1-(4,6-Dimethoxy-3-methyl-1H-indol-2-yl)-2-(piperi-
din-1-yl)ethane-1,2-dione (15f). Pale yellow solid (42%); mp
215–218 °C; 1H NMR (300 MHz, CDCl3): d 1.50–1.62 (m, 2H,
CH2), 1.62–1.74 (m, 4H, CH2), 2.67 (s, 3H, Me), 3.39 (t, J = 5.4 Hz,
2H, CH2), 3.68 (t, J = 5.4 Hz, 2H, CH2), 3.82 (s, 3H, OMe), 3.86 (s,
3H, OMe), 6.07 (d, J = 1.8 Hz, 1H, H5), 6.31 (d, J = 1.8 Hz, 1H, H7),
9.15 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 12.0 (Me), 24.6,
25.4, 26.3, 42.3, 47.2 (CH2), 55.4, 55.7 (OMe), 85.8 (C5), 93.0
(C7), 114.6 (C3a), 126.3 (C2), 128.0 (C3), 140.3 (C7a), 158.0 (C6),
162.1 (C4), 166.2, 181.5 (C@O); IR (KBr): mmax 3443 (NH), 3243
(NH), 2936, 2857, 1630 (C@O), 1598 (C@O), 1576 (C@O), 1524,
1439, 1291, 1252, 1215, 1147, 802, 771 cmꢀ1; UV–vis (THF): kmax
(e
35,700 cmꢀ1 Mꢀ1), 282 (21,800), 330 (24,700); HRMS (+ESI):
found m/z 421.1312 ([M+H]+), [C24H21ClN2O3H]+ requires m/z
421.1319 (monoisotopic mass).
4.2.4.5. 3-(4-Bromophenyl)-4,6-dimethoxy-N0-phenyl-1H-indole-
7-carbohydrazide (20d). Yellow solid (61%); mp 248–250 °C; 1H
NMR (300 MHz, DMSO-d6): d 3.91 (s, 3H, OMe), 4.09 (s, 3H, OMe),
6.54 (s, 1H, H5), 6.67–6.76 (m, 1H, ArH), 6.78–6.88 (m, 2H, ArH),
7.10–7.23 (m, 3H, H2, ArH), 7.41–7.57 (m, 4H, ArH), 7.91 (d,
J = 3.1 Hz, 1H, NH), 9.76 (d, J = 3.1 Hz, 1H, NH), 11.38 (d, J = 2.1 Hz,
1H, NH); 13C NMR (75 MHz, DMSO-d6): d 55.4, 56.9 (OMe), 88.1
(C5), 96.5 (C7), 110.0 (C3), 112.4 (ArCH), 115.3 (ArC), 118.47
(ArCH), 118.48 (ArC), 123.4 (C2), 128.7, 130.4, 131.0 (ArCH), 135.1,
138.2, 149.8, 156.6, 156.9 (ArC), 166.5 (C@O); IR (KBr): mmax 3346
(NH), 3315, 1641 (C@O), 1591 (C@O), 1540, 1343, 1270, 1218,
212 nm (e
43,500 cmꢀ1 Mꢀ1), 253 (20,900), 336 (30,900); HRMS
(+ESI): found m/z 353.1468 ([M+Na]+), [C18H22N2O4Na]+ requires
m/z 353.1472 (monoisotopic mass).
4.2.5.2. 1-(4,6-Dimethoxy-3-methyl-1H-indol-2-yl)-2-(mor-
pholin-1-yl)ethane-1,2-dione (15g). Pale yellow solid (54%);
mp 201–204 °C; 1H NMR (300 MHz, CDCl3): d 2.68 (s, 3H, Me),
3.41–3.58 (m, 2H, CH2), 3.63–3.71 (m, 2H, CH2), 3.72–3.80 (m,
4H, CH2), 3.81 (s, 3H, OMe), 3.86 (s, 3H, OMe), 6.07 (d,
J = 1.8 Hz, 1H, H5), 6.29 (d, J = 1.8 Hz, 1H, H7), 9.22 (br s, 1H,
NH); 13C NMR (75 MHz, CDCl3): d 12.1 (Me), 41.8, 46.5 (CH2),
55.4, 55.7 (OMe), 66.6, 66.9 (CH2), 85.8 (C5), 93.2 (C7), 114.6
(C3a), 127.0 (C2), 128.0 (C3), 140.5 (C7a), 158.1 (C6), 162.4
(C4), 166.2, 180.3 (C@O); IR (KBr): mmax 3355 (NH), 2974, 2915,
2862, 1642 (C@O), 1579 (C@O), 1526, 1433, 1383, 1291, 1275,
1234, 1213, 1155, 1132, 1103, 987, 810, 752 cmꢀ1; UV–vis
976 cmꢀ1; UV–vis (THF): kmax 202 nm ( 56,300 cmꢀ1 Mꢀ1), 238
e
(42,400), 280 (22,100); HRMS (+ESI): found m/z 466.0759 ([M+H]+),
[C23H20BrN3O3H]+ requires m/z 466.0766 (monoisotopic mass).
4.2.4.6.
3-(4-Chlorophenyl)-4,6-dimethoxy-N0-phenyl-1H-in-
dole-7-carbohydrazide (20e). White solid; mp 240–243 °C
(decomp.); 1H NMR (300 MHz, DMSO-d6): d 3.91 (s, 3H, OMe),
4.09 (s, 3H, OMe), 6.54 (s, 1H, H5), 6.71 (t, J = 7.2 Hz, 1H, ArH),
6.78–6.91 (m, 2H, ArH), 7.07–7.28 (m, 3H, H2, ArH), 7.32–7.45 (m,
2H, ArH), 7.47–7.60 (m, 2H, ArH), 7.91 (d, J = 2.9 Hz, 1H, NH), 9.76
(d, J = 2.9 Hz, 1H, NH), 11.38 (d, J = 1.6 Hz, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d 55.4, 56.9 (OMe), 88.1 (C5), 96.5 (C7),
110.1 (C3), 112.4 (ArCH), 115.3 (ArC), 118.5 (ArCH), 123.4 (C2),
127.5, 128.7 (ArCH), 130.0 (ArC), 130.6 (ArCH), 134.7, 138.2,
149.9, 156.6, 156.9 (ArC), 166.5 (C@O); IR (KBr): mmax 3359 (NH),
(THF): kmax 212 nm (
e
36,900 cmꢀ1 Mꢀ1), 254 (17,400), 337
(22,200); HRMS (+ESI): found m/z 355.1263 ([M+Na]+),
[C17H20N2O5Na]+ requires m/z 355.1264 (monoisotopic mass).
4.2.5.3.
1-(4,6-Dimethoxy-3-methyl-1H-indol-2-yl)-2-(pyrro-
1645 (C@O), 1593 (C@O), 1536, 1345, 1215, 1102, 978, 754 cmꢀ1
;
lidin-1-yl)ethane-1,2-dione (15h). Pale brown solid (10%); mp
159–161 °C; 1H NMR (300 MHz, CDCl3): d 1.94 (s, 4H, CH2), 2.72
(s, 3H, Me), 3.44–3.74 (m, 4H, CH2), 3.82 (s, 3H, OMe), 3.87 (s,
3H, OMe), 6.07 (s, 1H, H5), 6.29 (s, 1H, H7), 9.64 (br s, 1H, NH);
13C NMR (75 MHz, CDCl3): d 12.3 (Me), 24.0, 26.4, 46.2, 47.6
(CH2), 55.4, 55.7 (OMe), 85.8 (C5), 92.9 (C7), 114.5 (C3a), 127.3
(C2), 128.0 (C3), 139.9 (C7a), 158.0 (C6), 162.0 (C4), 165.2 (C@O),
second C@O signal missing; IR (KBr): mmax 3421 (NH), 3233 (NH),
2965, 2937, 2878, 2836, 1633 (C@O), 1596 (C@O), 1577 (C@O),
UV–vis (THF): kmax 242 nm (
e
37,700 cmꢀ1 Mꢀ1), 284 (16,100),
295 (17,000); HRMS (+ESI): found m/z 422.1262 ([M+H]+),
[C23H20ClN3O3H]+ requires m/z 422.1266 (monoisotopic mass).
4.2.5. General procedure for the synthesis of glyoxyloylamides
15f–k
To a solution of 4,6-dimethoxy-3-methylindole in anhydrous
1,2-dichloroethane (5 mL) prepared in a three-neck round-bottom
flask and cooled to 0 °C was added oxalyl chloride (1.5 equiv) in
one batch via syringe and the resulting mixture stirred under a
nitrogen atmosphere for 30 min. The respective secondary hetero-
cyclic amine (1.3–5.0 equiv) was added dropwise to a generated
suspension of 4,6-dimethoxy-3-methylindole-2-glyo-xyloyl chlo-
ride and the resulting reaction mixture heated at 60–70 °C under
a nitrogen atmosphere for 3 h. After cooling to room temperature
and quenching with water (15 mL) the organic phase was sepa-
rated and the aqueous phase extracted with ethyl acetate
(3 ꢁ 5 mL). Extracts were combined with the organic phase and
dried over anhydrous sodium sulfate. The solution was mixed with
silica gel and the solvents were removed in vacuo. Flash
1524, 1420, 1384, 1326, 1271, 1220, 1154, 1131, 1079, 802 cmꢀ1
;
UV–vis (THF): kmax 212 nm (
e
31,300 cmꢀ1 Mꢀ1), 256 (15,300),
339 (17,100); HRMS (+ESI): found m/z 339.1310 ([M+Na]+),
[C17H20N2O4Na]+ requires m/z 339.1315 (monoisotopic mass).
4.2.5.4. 1-(4,6-Dimethoxy-3-methyl-1H-indol-2-yl)-2-(4-(pyri-
din-2-yl)piperazin-1-yl)ethane-1,2-dione (15i). Brown solid
(30%); mp 187–189 °C; 1H NMR (300 MHz, CDCl3): d 2.68 (s, 3H,
Me), 3.41–3.74 (m, 6H, CH2), 3.81, 3.85 (2s, 8H, CH2, OMe), 6.06
(s, 1H, H5), 6.30 (s, 1H, H7), 6.52–6.83 (m, 2H, H5, H3 pyridine),
7.50 (t, J = 6.7 Hz, 1H, H4 pyridine), 8.18 (s, 1H, H6 pyridine),
9.27 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 12.1 (Me), 41.2,

1774
M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
45.0, 45.6, 45.8 (CH2), 55.4, 55.7 (OMe), 85.8 (C5), 93.1 (C7), 107.5
(C3 pyridine), 114.3 (C5 pyridine), 114.6 (C3a), 126.9 (C2), 128.0
(C3), 137.9 (C4 pyridine), 140.5 (C7a), 148.1 (C6 pyridine), 158.1
(C2 pyridine), 159.0 (C6), 162.3 (C4), 166.4, 180.6 (C@O); IR
(KBr): mmax 3415 (NH), 3308 (NH), 2934, 2838, 1639 (C@O), 1605
(C@O), 1588 (C@O), 1560 (C@O), 1523, 1476, 1434, 1381, 1296,
1231, 1213, 1159, 1129, 982, 776 cmꢀ1; UV–vis (THF): kmax
addition of 100 l
l TMB substrate system (3,30,5,50-tetramethyl-
benzidine liquid substrate system for ELISA, Sigma–Aldrich) to
each well. The plate was incubated with shaking at 600 rpm in a
FLUOstar Optima plate reader (BMG Labtech) at room temperature
for 6 min prior to measurement of the absorbance at 600 nm.
Samples were tested in triplicate and the absorbance of each
sample was compared to the control without exposure to
compounds to calculate absolute inhibition percentages.
213 nm (e
33,500 cmꢀ1 Mꢀ1), 252 (31,400), 336 (20,100); HRMS
(+ESI): found m/z 431.1689 ([M+Na]+), [C22H24N4O4Na]+ requires
m/z 431.1690 (monoisotopic mass).
4.3.2. Growth inhibition assay
Compounds were dissolved at 50 mM in DMSO and then
4.2.5.5. 1-(4-Benzylpiperazin-1-yl)-2-(4,6-dimethoxy-3-methyl-
diluted to 200
individual wells in a 96-well plate. E. coli DH5
cells were grown at 37 °C in 5 ml LB with shaking until the
OD600 reached 0.6–0.7, and 5 l of the culture was added to each
well. The plate was incubated in a FLUOstar Optima plate reader
(BMG Labtech) at 37 °C shaking at 600 rpm. The OD600 of the
culture was taken every 10 min over a 16 h period using LB as
the blank. Samples were tested in triplicate and the growth
pattern of each sample was compared to cells exposed to equal
amounts of DMSO.
l
M in 100
l
l of Luria–Bertani (LB) medium into
1H-indol-2-yl)ethane-1,2-dione (15j).
Pale yellow solid (41%);
a
or B. subtilis 168
mp 142–144 °C; 1H NMR (300 MHz, CDCl3): d 2.44 (t, J = 4.0 Hz,
2H, CH2), 2.53 (t, J = 4.0 Hz, 2H, CH2), 2.67 (s, 3H, Me), 3.47 (t,
J = 4.0 Hz, 2H, CH2), 3.52 (s, 2H, CH2 benzyl), 3.76 (t, J = 4.0 Hz,
2H, CH2), 3.81 (s, 3H, OMe), 3.86 (s, 3H, OMe), 6.07 (s, 1H, H5),
6.30 (s, 1H, H7), 7.17–7.47 (m, 5H, ArH), 9.27 (s, 1H, NH); 13C
NMR (75 MHz, CDCl3): d 12.0 (Me), 41.4, 46.1, 52.3, 53.0 (CH2),
55.3, 55.6 (OMe), 62.9 (CH2 benzyl), 85.9 (C5), 93.0 (C7), 114.5
(C3a), 126.6 (C2), 127.4 (ArCH phenyl), 128.0 (C3), 128.4 (ArCH
phenyl), 129.2 (ArCH phenyl), 137.5 (ArC phenyl), 140.5 (C7a),
158.0 (C6), 162.1 (C4), 166.2, 181.0 (C@O); IR (KBr): mmax 3421
(NH), 3309 (NH), 2938, 2803, 1646 (C@O), 1608 (C@O), 1575
l
4.4. Prediction of molecular properties
(C@O), 1522, 1437, 1241, 1215, 1152, 1130, 989, 811 cmꢀ1
;
Molecular properties of uncharged compounds were predicted
using Discovery Studio (DS) software, version 4.0 (accel-rys.com).
Pharmacophore mapping was also performed in DS using default
parameters as described previously.40
UV–vis (THF): kmax 212 nm (
e
37,600 cmꢀ1 Mꢀ1), 253 (14,100),
335 (18,100); HRMS (+ESI): found m/z 422.2073 ([M+H]+),
[C24H27N3O4H]+ requires m/z 422.2074 (monoisotopic mass).
DS uses the following methods for these predictions: a Ghose–
Crippen algorithm for clogP,63 a method by Cheng and Merz for
aqueous solubility64 and a proprietary method for pKa estimation
based on reproducing the standard values for amino acids.65
4.2.5.6. 1-(4,6-Dimethoxy-3-methyl-1H-indol-2-yl)-2-(4-methyl-
piperazin-1-yl)ethane-1,2-dione (15k). Bright yellow solid (39%);
mp 192–194 °C; 1H NMR (300 MHz, CDCl3): d 2.29 (s, 3H, N-Me),
2.37 (t, J = 4.4 Hz, 2H, CH2), 2.48 (t, J = 4.4 Hz, 2H, CH2), 2.65 (s, 3H,
Me), 3.46 (t, J = 4.4 Hz, 2H, CH2), 3.76 (t, J = 4.4 Hz, 2H, CH2), 3.78
(s, 3H, OMe), 3.84 (s, 3H, OMe), 6.04 (s, 1H, H5), 6.29 (s, 1H, H7),
9.44 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3): d 12.0 (Me), 41.3,
45.9 (CH2), 46.1 (N-Me), 54.3, 54.9 (CH2), 55.3, 55.6 (OMe), 85.8
(C5), 93.0 (C7), 114.5 (C3a), 126.7 (C2), 128.0 (C3), 140.6 (C7a),
158.0 (C6), 162.2 (C4), 166.2, 180.9 (C@O); IR (KBr): mmax 3343
(NH), 2970, 2938, 2857, 2800, 1641 (C@O), 1601 (C@O), 1578
4.5. Crystallographic methods
A suitable single crystal of compound 16a was selected under
the polarizing microscope (Leica M165Z), mounted on a Micro-
Mount (MiTeGen, USA) consisting of a thin polymer tip with a
wicking aperture. The X-ray diffraction measurements were car-
ried out on a Bruker kappa-II CCD diffractometer at 150 K by using
(C@O), 1522, 1448, 1284, 1254, 1212, 1150, 990, 810, 771 cmꢀ1
;
IlS
Incoatec Microfocus Source with Mo-K
a
radiation
UV–vis (THF): kmax 213 nm (
e
32,500 cmꢀ1 Mꢀ1), 253 (15,600),
(k = 0.710723 Å). The single crystal, mounted on the goniometer
using cryo loops for intensity measurements, was coated with
paraffin oil and then quickly transferred to the cold stream using
an Oxford Cryo stream attachment.
337 (21,100); HRMS (+ESI): found m/z 368.1576 ([M+Na]+),
[C18H23N3O4Na]+ requires m/z 368.1581 (monoisotopic mass).
4.3. Biological assays
During orientation matrix refinement, it became clear that
these crystals show peculiar imperfection, as seen from the frames
grabbed of reciprocal lattice views down a⁄ and b⁄ axis. The lattice
rows along c⁄ show a slight modulation (upper figure, shown in
Supplementary material) that results in non-exact superposition
of lattice points while looking down b⁄ axis (lower figure, shown
in Supplementary material). Several crystals were selected, but
all exhibited the same behaviour.
4.3.1. ELISA
Full-length rA was overproduced, purified25 and diluted to 250
nM in phosphate buffered saline (PBS). 100 ll of the solution was
added into NUNC Maxisorp™ microtitre plate wells, followed by
overnight incubation at 4 °C. The wells were washed three times
with 300 ll of PBS and blocked by incubating with 300 ll of 1%
(w/v) BSA in PBS at room temperature for 2 h. After blocking, plates
were washed three times with wash buffer (PBS, 0.05% (v/v)
Tween-20). 400 nM purified GST tagged RNAPb0 subunit frag-
Symmetry related absorption corrections using the program
SADABS66 were applied and the data corrected for Lorentz and
polarisation effects using Bruker APEX2 software.67 The structure
was solved by Direct methods and the full-matrix least-square
refinement was carried out using SHELXL.68 The non-hydrogen
atoms were refined anisotropically. The asymmetric unit contained
four independent molecules in the asymmetric unit. After includ-
ing all the non-H atoms and H-atoms, the final difference Fourier
contained peaks ꢂ6 e Åꢀ3 near (ꢂ2 Å away) from each of the four
Br atoms. These could not be explained as any disordered (for
example, water molecules) atoms. Also, the temperature factor of
one of the carbon atoms C18B was non-positive definite during
the refinement, which was restrained with the commands DELU
ment26 in 50
ll PBS was mixed with 30 lM compounds in 50 ll
PBS at 37 °C for 10 min, and then added to wells followed by
incubation at room temperature for 1 h. Wells were washed three
times with 300 ll of PBS/Tween-20 wash buffer, 100 ll of rabbit
anti-GST primary antibody (1:2000 in PBS) was added to each well
and the wells were incubated at room temperature for 1 h. Wells
were washed three times with 300 ll of PBS/Tween-20 wash buf-
fer. HRP-conjugated goat-anti-rabbit secondary antibody (1:2000
in PBS) was added to each well and the wells were incubated at
room temperature for 1 h. Interactions were detected by the

M. Mielczarek et al. / Bioorg. Med. Chem. 23 (2015) 1763–1775
17. Barbosa, T. M.; Levy, S. B. Drug Resist. Update. 2000, 3, 303.
1775
and SIMU. The two alerts at level A arising due to the above fea-
tures (residual electron density peaks and anisotropy of C18B)
should be attributed to the modulated lattice of these crystals.
Although, this effect allowed us to proceed with the structure solu-
tion and refinement to the present level of accuracy.
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1H NMR, 13C NMR and HRMS-ESI spectra and a table of molecu-
lar properties for all the compounds, additional software-gener-
ated figures related to the crystal structure of molecule 16a,
diagrams showing RNAP holoenzyme formation inhibition and
bacterial growth inhibition curves for compounds 16c and 18b.
A cif file describing the crystal structure of compound 16a
(CCDC deposition number: 1022650).
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Acknowledgments
This work was supported by a Project Grant from the National
Health and Medical Research Council (NHMRC) Australia to N.K.,
P.L. and R.G. [NHMRC Grant APP1008014].
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M.M. is thankful to UNSW Australia for
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We are thankful to the Nuclear Magnetic Resonance Facility and
the Bioanalytical Mass Spectrometry Facility in the Mark
Wainwright Analytical Centre at UNSW Australia for an opportu-
nity of using their measurement instruments.
Determination of IC50 values for compounds 16c and 18b
performed by Dr Samuel K. Kutty is greatly appreciated.
Supplementary data
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These data include MOL files and InChiKeys of the most important
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