Synthesis and in Vitro Evaluation of Novel 5-Nitroindole Derivatives as c-Myc G-Quadruplex Binders with Anticancer Activity

Article abstract of DOI:10.1002/cmdc.202000835

Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.

Full text of DOI:10.1002/cmdc.202000835

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Synthesis and in Vitro Evaluation of Novel 5-Nitroindole
Derivatives as c-Myc G-Quadruplex Binders with Anticancer
Activity
Vijaykumar D. Nimbarte,[a] Julia Wirmer-Bartoschek,[a] Santosh L. Gande,[a, b]
Islam Alshamleh,[a] Marcel Seibert,[c] Hamid Reza Nasiri,[a] Frank Schnütgen,[b, c, d]
Hubert Serve,[b, c, d] and Harald Schwalbe*[a, b, d]
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Lead-optimization strategies for compounds targeting c-Myc G-
quadruplex (G4) DNA are being pursued to develop anticancer
drugs. Here, we investigate the structure-activity- relationship
(SAR) of a newly synthesized series of molecules based on the
pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA.
Our synthesized series allows modulation of flexible elements
with a structurally preserved scaffold. Biological and biophysical
analyses illustrate that substituted 5-nitroindole scaffolds bind
to the c-Myc promoter G-quadruplex. These compounds down-
regulate c-Myc expression and induce cell-cycle arrest in the
sub-G1/G1 phase in cancer cells. They further increase the
concentration of intracellular reactive oxygen species. NMR
spectra show that three of the newly synthesized compounds
interact with the terminal G-quartets (5’- and 3’-ends) in a 2:1
stoichiometry.
Introduction
by small molecules leads to down-regulation of the expression
of their respective genes.[10–13] Up to 80% of all solid tumours
(including gastrointestinal, ovarian and breast tumours) over-
express c-Myc.[14] It has been proposed that the G4 present in
NHE III1 in the c-Myc gene is crucial for transcriptional
silencing.[15,16] This element is comprised of 27 nucleotides
containing five G-tracts of which G-tracts 2, 3, 4 and 5 form a
parallel G4 as the major conformation. In biophysical studies
therefore a shortened version (Pu22) is used to facilitate
investigations (Figure 1).[17] It was shown that G4-stabilizing
ligands binding to Pu22 can downregulate c-Myc
transcription.[15] Further, the c-Myc protein-dependent prolifer-
ation can be inhibited, leading to inhibition of cancer cell
growth.[18]
In addition, redox homeostasis is essential for the main-
tenance of diverse cellular processes. Cancer cells have higher
levels of reactive oxygen species (ROS) than normal cells as
result of hypermetabolism. Recently, anticancer therapies that
induce oxidative stress by increasing ROS and/or inhibiting
antioxidant processes have received significant attention. The
DNA G-quadruplexes (G4) are noncanonical DNA structures,
found within guanine-rich sequences.[1] The basic structural unit
is the G-tetrad, which is stabilized by the association of four
guanines into
a
cyclic Hoogsteen hydrogen-bonding
arrangement.[2,3] Till date, more than 716000 G4-forming
sequences have been identified in the human genomic DNA by
G4-sequencing techniques.[4] G4 are implied in the regulation of
human genes:[4–6] their formation is significantly associated with
tumour suppressors and somatic copy number alterations
related to cancer development.[7] They are found in the
promoter regions of oncogenes including c-Myc, c-KIT, BCL-2,
and in telomeres.[8,9] G4 structures are interesting targets in
cancer drug discovery. G4 stabilization in oncogene promoters
[a] V. D. Nimbarte, Dr. Julia Wirmer-Bartoschek, Dr. S. L. Gande, I. Alshamleh,
Dr. H. R. Nasiri, Prof. Dr. H. Schwalbe
Institute for Organic Chemistry and Chemical Biology
Center for Biomolecular Magnetic Resonance (BMRZ)
Goethe University Frankfurt
Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
E-mail: schwalbe@nmr.uni-frankfurt.de
[b] Dr. S. L. Gande, Dr. F. Schnütgen, Prof. Dr. H. Serve, Prof. Dr. H. Schwalbe
German Cancer Research Center and German Cancer Consortium
Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)
[c] M. Seibert, Dr. F. Schnütgen, Prof. Dr. H. Serve
Department of Medicine 2, Hematology/Oncology
University Hospital Frankfurt, Goethe University
Theodor-Stern-Kai 7, 60596 Frankfurt am Main (Germany)
[d] Dr. F. Schnütgen, Prof. Dr. H. Serve, Prof. Dr. H. Schwalbe
Frankfurt Cancer Institute (FCI)
Theodor-Stern-Kai 7, 60596 Frankfurt am Main (Germany)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000835
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
Figure 1. DNA sequence of the human Myc gene promoter. The G4-forming
region NHE III1 sequence is shown, a schematic structure of the P22
sequence is shown on the right.
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acceleration of accumulative ROS disrupts redox homeostasis
and causes severe damage in cancer cells.[19]
fluorescence screening of series of substituted heterocycles.
Our findings were validated using fluorescence intensity
titrations, microscale thermophoresis (MST)-based analyses, and
subsequent evaluation in cellular assays and structural studies
by NMR spectroscopy.
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A number of different ligands have been developed to
target G4 DNAs including indolylmethyleneindanone, indeno-
pyrimidine and bisbenzimidazole carboxamide derivatives of
naphthyridine
and
phenanthroline,[20,21]
indoles,[22]
7-
azaindoles,[23] 1H-indazol-3-yl,[24] benzothiazole,[25] imidazo[1,5-a]
pyridine,[26] 2,6-diaminopyrimidin-4-ol,[27] 1H-pyrazolo[4,3-d]
pyrimidin-7-amine,[28] morpholino,[28] bis-indoles,[29,30] 2-hydroxy-
naphthalene-1,4-dione,[31]1,4-dihydroxyanthracene-9,10-
dione,[32] benzofuran and piperonal, which were derived from
several alkaloids.[33]
Lead identification strategy: High-throughput screening
Our investigations started from the parent compound 1-methyl-
1H-indol-5-amine (3 in Scheme 1)[34] and an initial library
comprised of 129 structurally and chemically diverse commer-
cially available fragments containing at least one aromatic
moiety (Table S1 in the Supporting Information fragment no.
A1–A129). Compounds in this fragment family followed
principal criteria for fragment libraries (Figure S14, Tables S5–
S7).[35–37]
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Herein, we describe pyrrolidine-substituted 5-nitroindole
compounds as a new class of G4 ligands that bind to the c-Myc
promoter G4 sequence. These new compounds induce c-Myc
downregulation both, at transcription and translation level
along with the generation of reactive oxygen species inducing
antiproliferative effects in the cancerous cells. This novel
pharmacophore was identified and improved by iterative
The initial screening against the c-Myc Pu22 DNA was
conducted by a fluorescence intercalator displacement (FID)
Scheme 1. A) NaH, DMF, RT, 1 h, CH3I, RT, 8 h, 95%; B) Pd/C, H2, MeOH, RT, 3 h, 52%; C) K2CO3, DMF, 1,3-dibromopropane and 1,2-dibromoethane, RT, 4 h,
°
65%; D) dry ACN, pyrrolidine, reflux, 4 h, 32%; E) POCl3, DMF, 0 C-RT, 1 h, 46%; F) NaBH4, MeOH, substituted amine, RT, 3 h, 72%; G) K2CO3, DMF, RT, 3 h, 62%;
°
°
H) POCl3, DMF, 0 C-RT, 1 h, 30%; I) NaBH4, MeOH, substituted amine, RT, 3 h, 56%, J) Pd/C, H2, MeOH, RT, 3 h, 52%; K) NaN3, DMF, 80 C, reflux, 4 h, 48%; L)
°
POCl3, DMF, 0 C-RT, 1 h, 35%; M) NaBH4, MeOH, corresponding amine, RT, 3 h, 45%; N) Pd/C, H2, MeOH, RT, 3 h, 23%.
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assay[38] that utilizes thiazole orange (TO) as fluorescent dye. TO
is a well-validated probe for screening G4-binding fragment
molecules.[39] It is highly fluorescent when bound. Its
fluorescence is quenched after displacement (λex =501 nm,
intermediate 4a.[41] In the next step, 4a was protected with
pyrrolidine to obtain 5.The intermediate 5 was then treated
with the Vilsmeier reagent to generate carbaldehyde 6 and to
facilitate the one pot in-situ generation of 7. In route III,
intermediate 8 was obtained in 60% yield via Vilsmeier-Haack
reaction starting from 5-nitro-1H-indole (1). This reaction was a
key step to generate conjugates 9–13 in 56% yields in a one
pot in-situ reaction of aldehydes with substituted amines in
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4
5
6
7
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9
λem =539 nm). Using this assay, we identified fragment 9a (see
Scheme 1<xschr1) and a general trend towards the fused 5-
and 6-membered heterocycles as suitable scaffolds (Figures S2
and S3).
presence of NaBH4 as
a reducing agent. Pd/C-catalysed
hydrogenation[40] of the nitro group in 9 to the amine in 9a and
for the conversion of 5 to 5a was applied. During the
development of these synthetic strategies, we found that the
substituted 5-aminoindole derivatives are potentially unstable
against air oxidation.
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Synthetic strategy for lead optimization
To further validate the findings of the screening of these
commercially available fragments and to gain deeper insight
into the structure-activity-relationship, several other indoles, 5-
nitroindoles, 5-aminoindoles, 7-azaindole and indazole deriva-
tives were synthesized and tested (a total of 52 new
compounds). We developed feasible synthetic strategies to
generate drug-like fragments derived from 5-nitroindole
(Schemes 1 and 2), from indole (Schemes 3 and S5) and from
methylmethanamine derivatives (Scheme S4)
In route 1 of synthetic Scheme 1, scaffold 1-methyl-1H-
indol-5-amine (3) was obtained in 60% yield by Pd/C-catalysed
hydrogenation of 2, which is a nucleophilic substitution product
of nitro-indole 1.[40] In route II, intermediates 4a and b were
generated via a nucleophilic substitution reaction of 1 with 1,3-
dibromopropane and 1,2-dibromoethane, respectively, which
generates the 5-nitroindole dimer 17a as a by-product of
In addition to this, the indole derivatives 16 and 16a were
synthesized via route IV in a three-step synthesis starting from
intermediate 4a. In a first step,4a was allowed to react with
NaN3 in DMF to form the azide substituted derivative 14 in a
nucleophilic substitution reaction in 60% yield. Precursor 14
was then treated with the Vilsmeier reagent to obtain
carbaldehyde 15 in 35% yield. Conjugates 16 and 16a were
obtained from carbaldehyde 15 by one pot in-situ reaction of
the corresponding substituted amine with aldehyde group in
presence of NaBH4 in methanol media with 45% yield. In
Scheme 2, 2-propyne-substituted nitro-indoles 21a and 21b
were prepared via substitution reaction of propargyl/butargyl
bromide with 5-nitroindole (1) to obtain intermediates19a and
19b. Compound 20 could be synthesized from 19a in a
Vilsmeier–Haack reaction.
In addition to above in Scheme 3 several other aldehydes
(2a, 23, 24 and 25) were used to prepare conjugates 3a, b,
23a, b, e, 24a–c, e and 25b, d, e by one pot in-situ reaction of
substituted amine with aldehyde in presence of NaBH4 in
methanol media with 56% yield. In addition, 7-azaindole
derivatives and 1-(3-(pyrrolidin-1-yl) propyl)-1H-indole were
synthesized as described in Scheme 4 and Scheme 5 discussed
in the Supporting Information.
Scheme 2. A) K2CO3, DMF, 3-bromo-1-propyne/4-bromobut-1-yne, RT, 4 h,
°
86–88%; B) POCl3, DMF, 0 C-RT, 1 h, 58%; C) NaBH4, MeOH, substituted
amine, RT, 3 h, 38%
Evaluation of expanded hit series
The 52 expanded and in-house synthesized compounds were
subjected to additional rounds of FID screening (Figure S4). The
affinity of the 12 best fragments 9a, 9, 12, 7, 5, 5a, b, 24b, 25b,
25, 23b and A4 (Figures 2 and S5) along with parent fragment
3[34] were further investigated in the TO assay.[42] Indole
containing compounds in general bind better than compounds
containing another heterocycle such as 24b, 25b, 25e and
23b. Within the indoles, 5-nitro indoles 12, 7 and 5 as well as 5-
aminoindoles 3, 9a and 5a bind with DC50 values lower than
10 μM. Substitution on the fifth position of the indole seems to
be important for the activity: the 5-nitro-indole compound 9
binds more weakly than the corresponding 5-aminoindole
compound 9a (aminogramine), while the unsubstituted indole
compound 5b binds more weakly than the corresponding nitro
or amine substituted compounds 5 and 5a, respectively.
Further, substitution of position 2 reduces affinity as can be
°
°
Scheme 3. A) POCl3, DMF, À 10 C to RT, reflux, 100 C, 1 h, 66%; B) NaBH4,
MeOH, substituted amine, RT, 3 h, 46%
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Figure 3. c-Myc-ligand interactions observed by 1H NMR spectra (imino and
aromatic region). a) Comparison of interactions of c-Myc with different
fragments at a [DNA]/[ligand] ratio of 1:4. Signals clearly belonging to the
respective ligand are indicated by asterisks. b) Interactions of fragment 7
with c-Myc at different DNA/ligands ratios. a+b) assignments (17) of imino
signals and clearly separated aromatic protons of loop and tail residues are
indicated; grey boxes indicate signals of minor conformations; experimental
conditions: 0.1 mM c-Myc in 25 mM KPi, 70 mM KCl, pH 7, 0.025 mM DSS and
10% [D6]DMSO.
Figure 2. a) Chemical structure of best hits from initial FID experiments.
b) Dose-response curves representing DC50 and KD results from FID titrations,
MST measurements and FAM experiments for compound 7 of best hits from
initial FID experiments. c) DC50 and KD results from FID titrations, MST
measurements and FAM experiments. Errors are within 5%.
(5b) the selectivity towards the c-Myc G4 compared to the
duplex DNA (Kd duplex/Kd c-Myc) is reduced to the 6.55 (Figure S7).
Taken all three biophysical screens, we found that the
amine and nitro functional group on the fifth position of central
indole core plays a crucial role in improvement of G4 binding.
In addition to these, the introduction of methylene bridged
pyrrolidine at the third position along with the protection of N-
indole functional group plays crucial role in further improve-
ment of binding affinities to c-Myc G4.
observed by the comparison of compound
3
with
compound A4.
Microscale thermophoresis (MST)[43] and fluorescence bind-
ing titrations were used to further evaluate the binding affinities
of the indole compounds (3, 5, 7, 9a, 9,12, A4, 5a and 5b) to c-
Myc G4 (Figure 2b and c). Consistent with the FID measure-
ments, comparison of compounds 5 (KMDST =1.42 μM), 5a (KMDST
=
2.87 μM) and 5b (KMDST =15.1 μM) differing only at the fifth
position suggests that nitro and amino functionalities at the
fifth position of the indole heterocycle are beneficial for the
binding. Most promising affinities with KMDST of 1.32 and
1.42 μM; KFDAM of 2.4 and 2.83 μM were obtained for compounds
5 and 7, respectively, both of which contain a methylene
bridged pyrrolidine substitution at the first position of the
indole skeleton, while compound 12 bearing the same side
chain only at the third position of the indole has an
approximately threefold higher KD in both methods. Further-
more, based on MST assays, we observed that the 5-nitroindole
compounds (5 and 7) with methylene bridged pyrrolidine side-
chains are distinctly selective for c-Myc G4 over the duplex DNA
NMR characterization of binding
The interactions of 3, 5, 7, 9a and 12 with c-Myc were
characterized by NMR spectroscopy. Figure 3a shows NMR
spectra of the DNA-ligand complexes at a ratio of 1:4 ([DNA]/
[ligand]). In the spectrum of the DNA alone, signals (indicated
by grey boxes) of minor conformations of the DNA are well
visible. These signals disappear upon addition of ligand,
indicating the stabilization of the major conformation upon
binding. For ligands 3 and 9a, only minor chemical shift
perturbations and weak line broadening are observed corre-
sponding to weak binding in fast exchange regime.
with Kd duplex/Kd
ranging from 24.12–29.23 (Figure S7 and
c-MYC
Table S2). While in the case of the 5-aminoindole compound
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This differs drastically for ligand 12 where severe line
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3
4
5
6
7
8
broadening in combination with chemical shift changes are
nicely visible, for example, for G13 of the DNA and indicate
stronger interactions in the intermediate exchange regime.
Strong chemical shift perturbations are observed for 5 and even
more so for 7 coupled to only slight line broadening, allowing a
detailed examination of the DNA-ligand interactions as shown
for 7 in Figure 3b.
Already at a [DNA]/[ligand] ratio of 1:1, signals of minor
conformations have disappeared and shifting of a significant
number of signals rather than appearance of new signals
indicate binding in fast exchange. Most prominent chemical
shift perturbations with values (positive and negative) around
0.06 ppm at ratio 1:1 are observed for imino-signals of G19,
G15 and G6, located at the 3’-tetrad of G4 (see also Figure 5a).
Perturbations between 0.034 and 0.045 ppm are observed for
the imino-signals of G4, G8 and G13, while all other perturba-
tions are in magnitude smaller than 0.022 ppm. G4, G8 and G13
belong to the 5’-tetrad. These findings indicate that the ligand
binds to both, the 3’ and the 5’ tetrad with slight preference for
the 3’-tetrad. This trend is also seen at a concentration ratio of
1:2 where CSPs for the respective 6 imino-signals are between
0.68 and 1.2 ppm in magnitude while they are less than
0.037 ppm for the other iminos. Binding and structural
rearrangement of residues in loop and capping structures can
be followed by analysing the aromatic NMR resonances. Signals
of A12, A22, T1, T20 and A21 are reasonably well resolved to
allow the assessment of conformational changes upon binding.
As A12 does not shift upon binding the loop does not change
its environment upon binding. This is different for T1, T20, A21
and A22. These residues located in the capping structures (see
Figure 5a) are perturbed significantly upon binding indicating
rearrangement of the capping structures to accommodate the
ligands. This rearrangement is also seen in the literature for c-
Myc,[44] as well as for other G4s.[45]
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Figure 4. Cell cytotoxicity assays for tested ligands in HeLa cells: a) Dose-
response and IC50values derived for tested ligands in HeLa cells after 72 h of
treatment. b) Western blot analysis (see also Figure S6) and qRT-PCR show
transcriptional downregulation of the c-Myc gene, by tested ligands at
defined concentrations. c) Hoechst staining-mediated cell cycle analysis of
HeLa cells treated with fragments 5, 7 and 12 at 6 μM concentrations for 24
and 48 h (see also Figure S7 and Tables S2 and S3). Error bars represent the
standard error of the mean calculated from three replicates.
0.73 μM respectively (Table 1 and Figure 4a). To determine the
potential role of ROS in the anticancer activity of 5-nitroindole
compounds, we also detect the effects of N-acetyl cysteine
(NAC) after treatment of HeLa cells with compound 7 (Fig-
ure S12). The viability of HeLa cells was 44.6�2.5% after 5 μM
of compound 7 treatment, which was reversed to be 77.8�
1.9% by the 1-h pretreatment with 2.5 mM NAC.
In agreement with the above NMR investigations the
binding stoichiometry of 2 ligands per DNA was determined by
additional fluorescence measurements for fragment 7 (Fig-
ure S8).
The above findings suggest that substituted 5-nitro indole
compounds (5 and 7) could improve the antiproliferative
activity due to generation of reactive oxygen species along with
c-Myc stabilization in cancerous cells. However, we cannot rule
out that these antiproliferative activities against the HeLa cells
could be due to several other multiple targets. Interestingly,
most promising compounds 5 and 7 did not significantly inhibit
the growth of normal kidney epithelial cells (NKE) even at 5, 10
and 30 μM, respectively (Figure S13). We further analysed the
functional effects of the substituted 5-nitroindole compounds 5,
7, 12, 3 and 9a on the transcription and translation of the c-Myc
gene. Immuno blot results showed that the compounds 5 and
12 reduced the expression of c-Myc protein levels in HeLa cells
relative to untreated cells, by approximately 30 and 50% when
treated with 3 and 10 μM, respectively, and similarly for
compound 7, by 43% at 3 μM and 65% at10 μM. The
expression of control house-keeping gene, GAPDH was not
affected by the tested ligands (Figure S10). Furthermore, at the
In vitro characterization of DNA-ligand interaction
The above biophysical and structural results show that the
optimized ligands have a high binding affinity for the c-Myc
promoter G-quadruplexes. To investigate the effect of these
indole fragments on cancer cell proliferation, all identified hits
from high throughput FID screening (3, 9a, 9, 12, 7, 5, 5a, 5b,
24b, 25b, 25e, 23b and A4) were analysed in vitro by cell
viability assays using HeLa cells[46] (Figure 4a). Substituted 5-
nitroindoles derivatives have been found to show broad-
spectrum anticancer activities against different cancer cell
lines.[47] The anti-proliferation effects of substituted 5-nitro-
indole derivatives against human cancer HeLa cells were further
investigated by Alamar blue assay, as shown in Figure 4a.
Among all the ligands, 5 and 7 inhibited cell proliferation most
efficiently with an IC50 value of 5.08�0.91 μM and 5.89�
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transcriptional level by qRT-PCR, compound 5 showed a more
1
2
3
4
5
prominent decrease in the expression of c-Myc mRNA at 10 μM
in comparison to compound 7 and 12 (Figure 4b). Interestingly,
the effect of the compounds on c-MYC expression is more
apparent on the mRNA level than on the protein level. We
suspect that the effect appears earlier on the mRNA level than
on the protein level.
6
7
To investigate the effect of ligands (5, 7 and 12) on the cell
cycle in HeLa cells, we performed Hoechst-staining-mediated
cell cycle analysis (Figure 4c and Tables S3 and S4). Upon
exposure to fragments 5 and 7 at 6 μM concentration for 24 h,
cell cycle arrest was observed with fewer replicating cells
undergoing DNA replication (S/G2/M phase), and the effect is
not clear for compound 12. After 48 h of treatment, HeLa cells
showed prominent cell cycle arrest and cell death (sub-G1) with
significant increase in the sub G1 population to 70.9 and 85.7%,
respectively when treated with fragments 5 and 7, and these
effects were not observed for cells treated with compound 12.
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Discussion and Conclusion
In our investigations, we set out to identify new binders to
target the c-Myc promoter G-quadruplex starting from a
previously identified compounds via high throughput FID
screening of commercially obtained compounds. We identified
the 5-nitro indole core as a lead structure and created a library
of new scaffolds based on fragment expansion to improve the
total polar surface area of the novel compounds exploiting the
chemical nature of methylene bridged pyrrolidine-substituted
5-nitroindole conjugates, which may aid in the electrostatic
binding and contributes to hydrogen binding interactions of
the fragments to G4 base pairs. Several synthetic strategies
were reported to optimize and generate best hits starting from
indoles, 5-nitroindoles and methylmethanamine derivatives.
After screening all compounds applying an FID assay, several
biophysical investigations were performed to evaluate the
structure-activity relationships of the 13 best fragments (Fig-
ure 5b). We found that either an amino or a nitro-group at the
fifth position of the central indole core is critical for binding,
while protection of the N-indole at the first position plays a
significant role in improved G4 binding. In addition to above
Figure 5. a) Binding of compound 7 by NMR mapped onto the structure of
free c-Myc (PDB ID: 1XAV). Shifting imino resonances of guanines in the
tetrads are coloured in red, assigned loop and tail resonances are indicated
in black (not shifting) and light red (shifting). b) SAR found in this
investigation.
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10 mL of ethanol was dropped into a suspension of 10% Pd/C
(12.5 mg) saturated with H2 in ethanol (20 mL). The mixture was
stirred at room temperature for 3 h, according to TLC analysis
(MeOH/DCM 1:3). The catalyst was recovered and the filtrate was
evaporated under reduced pressure to dryness to give the
corresponding amino-indoles.
inclusion of a methylene bridged pyrrolidine side chain at the
third position of the indole core is crucial for G4 binding and
with KD/DC50 values in the same magnitudes, large differences
in the binding are seen by investigations using NMR-spectro-
scopy.
1
2
3
4
5
6
7
8
9
In biological investigations, significant differences were
observed in between the compounds selected from the high
throughput screening. Conjugates 5 and 7 with the inclusion of
the imino linked pyrrolidine side chain at the third position and
N-indole protections with methylene bridged pyrrolidines have
better IC50 values and cell arrest potential. In comparisons,
substituted 5-amino indoles along with the unprotected N-
indole 5-nitro compound 12 with imino linked pyrrolidine side
chain at the third position is found to be ineffective against the
cancerous cells. In addition, compound 5 and 7 exhibited
improved c-Myc binding compared to that of the previously
reported parent compound (3). Furthermore, in vitro biological
evaluations we found that c-Myc mRNA and protein level is
significantly downregulated by the treatment of 5-nitro indole
compounds (5, 7 and 12). In vitro cellular studies with FACS
analysis revealed that upon treatment with compounds 5 and 7
at 6 μM prominent sub-G1/G1 phase arrest was observed in the
cell cycle. One explanation for the relative supremacy of the 5-
nitro indole compounds compared to the 5-amino indole
compounds in the cell assays could be the smaller long-term
stability of the substituted amino-indoles. Based on our
investigations, we could conclude that these antiproliferative
effects against cancerous cells might be due to the c-Myc
downregulation at both transcription and translation level
along with the generation of reactive oxygen species by nitro
group of the most active compounds.
Generalized procedure for intermediates 6, 8, 15, 2a and 20 with
Vilsmeier-Haack reaction (A): POCl3 (1.2 mL, 9.25 mmol, 1.5 equiv.)
as reaction solvent was added in dropwise manner to the dried
°
DMF (10 mL) under inert condition at 0 C. This mixture was allowed
to stir at room temperature for 1 hour followed with the addition of
reactant (1 equiv.) 1, 5, 14, 19a, and 1a. The reaction mixture was
then allowed to react for 1 hour by stirring at room temperature.
The reaction mixture was then quenched with equal amount of ice
water and 50% NaOH was added until a pH of 9.
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Generalized procedure for the condensation of substituted
carboxaldehyde and corresponding amines (B) to generate (7, 9,
10, 11, 12, 13, 16, 16a, 21a, 21b, 3a, 3b, 23a, b, e, 24a–c, e and
25b, d, e): Substituted carboxaldehydes 6, 8, 15, 2a, 20, 23, 24 and
25 (1 equiv.) and substituted amines (3 equiv.) were dissolved in
50 mL of dry EtOH: CH3CN (1:1). The resulting mixture stirred for
2 h at room temperature and then the solvent was concentrated
under reduced pressure. The resultant residue was dissolved in
20 mL EtOH and then NaBH4 (10 equiv.) was added to it in portion
wise. The reaction mixture is allowed to stir for 24 h at room
temperature, and then the excess of NaBH4 was filtered off and the
solvent was evaporated to dryness. The resultant solid was treated
with deionized water and extracted with CH2Cl2 (3×50 mL). The
organic phase was evaporated under reduced pressure.
Generalized procedure for making HCl salt (C): The solid was
dissolved in EtOH/dioxane (1:3) and precipitated with aqueous HCl
37% to obtain its hydrochloride salt. The precipitate was filtered
and recrystallized with MeOH to give the desired product.
Generalized procedure for nucleophilic substitution of alkynes
(19a and 19b): 5-Nitro-1H-indole (2.01 g, 12.3 mmol) has added to
a solution of anhydrous KOH (0.692 g, 12.3 mmol) in DMF (100 mL)
at room temperature for 30 min. followed with the addition of 3-
bromoprop-1-yne and 4-bromobut-1-yne (4 equiv.) and reaction
mixture was allowed to stir overnight.
Experimental Section
Chemistry: All solvents and reagents were purified by standard
techniques or used as supplied from commercial sources (Sigma-
Aldrich, unless stated otherwise). All reactions were generally
carried out under inert atmosphere unless otherwise noted. TLC
was performed on Kieselgel 60 F254 plates, and spots were
visualized under UV light. Products were purified by flash
chromatography on silica gel (100–200 mesh). 1H NMR spectra were
recorded on 600 MHz instruments at 298 K. 13C NMR spectra were
recorded at 151 MHz with proton decoupling. Chemical shifts are
reported in parts per million (ppm) and are referred to the residual
solvent peak. The following notations are used: singlet (s); doublet
(d); triplet (t); quartet (q); multiplet (m); broad (br). Coupling
constants are quoted in Hertz and are denoted as J. Mass spectra
were recorded on a Micromass Q-Tof (ESI) spectrometer. Purity of
the key target compounds were detected by HPLC system [Waters
e2695 (alliance), Column: GRACE-C18 (250 mm×4.6 mm×5 mm),
Mobile phase: 1% TFA buffer and ACN and methanol, Flow rate:
1 mL/min] consisting of low-pressure gradient pump plus auto
sampler and Photo Diode Array (PDA) detector. The output signal
was monitored and processed using Empower 2 software.
1-Methyl-5-amino-1H-indole (3): A solution of a 1-methyl-5-nitro-
1H-indole (2) (5.87 mmol) is treated according to protocol A. Yield
°
96%; brown solid. Rf =0.44 (eluent MeOH/DCM 1:3) 0.44; Mp 87 C;
1H NMR (600 MHz, [D6]DMSO): δ=7.09 (dd, J=9.2, 5.8 Hz, 2H), 6.68
(s, 1H), 6.54 (d, J=4.5 Hz, 1H), 6.10 (d, J=2.9 Hz, 1H), 4.45 (bs, 2H),
3.66 (s, 3H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=211.30, 141.55,
130.56, 129.22, 111.82, 109.77, 103.59, 98.77, 32.65 ppm; HRMS: m/z
calcd for C9H10N2 [M+H]+ 146.19, found 146.19.
5-Nitro-1-(3-bromopropyl)-1H-Indole
(4a):
5-Nitro-1H-indole
(2.01 g, 12.3 mmol) was added to a solution of anhydrous KOH
(0.692 g, 12.3 mmol) in DMF (100 mL) at room temperature for
30 min, followed by the addition of 1,3-dibromopropane (3.77 mL,
37.0 mmol). The reaction mixture was then allowed to stir over-
night. Solvent was removed under reduced pressure, and the crude
product was purified on silica as a stationary phase; Yield 46% as
1
°
yellow crystals; Rf =0.65 (eluent EA/n-Hex 1:4); Mp 102 C. H NMR
(600 MHz, CDCl3): δ=8.58 (d, J=2.2 Hz, 1H), 8.04 (d, J=9.1 Hz, 1H),
7.71 (d, J=9.1 Hz, 1H), 7.67 (d, J=3.2 Hz, 1H), 6.78 (d, J=3.1 Hz,
1H), 4.40 (t, J=6.5 Hz, 2H), 3.28–3.35 (m, 2H), 2.33–2.44 (m, 2H)
ppm; 13C NMR (151 MHz, CDCl3): δ=141.79, 138.84, 131.22, 127.92,
118.42, 117.52, 109.33, 104.54, 44.53, 32.70, 30.05 ppm; ESI-MS: m/z
282.0 [M+H]; HRMS: m/z calcd for C11H11BrN2O2 [M+H]+ 282.0,
found 282.0.
Declaration of purity: All final compounds were equal to or more
1
than 95% pure by HPLC (for key target compounds) and H NMR
(600 MHz and 400 MHz) for the synthesized compounds.
Generalized procedure for the reduction of nitro to amine (3, 5a,
9a and 11a): a solution of a nitro conjugates (2, 5, 9 and 11) in
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1-(2-Bromoethyl)-5-nitro-1H-indole (4b): 5-Nitro-1H-indole (2.01 g,
12.3 mmol) was added to a solution of anhydrous KOH (0.692 g,
12.3 mmol) in DMF (100 mL) at room temperature for 30 min,
followed by the addition of 1, 2-dibromoethane (3.77 mL,
37.0 mmol). The reaction mixture was then allowed to stir over-
night. Solvent was removed under reduced pressure, and the crude
111.96, 53.28, 51.95, 44.88, 28.32, 23.08 ppm. HRMS: m/z calcd for
C22H33N5O2 [M+Na]+ 422.25.
1
2
3
4
5
6
7
8
9
5-Nitroindole-3-carboxaldehyde (8): The crude product was ob-
tained from 5-nitroindole (1) by the Vilsmeier-Haack general
protocol. The product was purified on silica as a stationary phase;
with 1:4 MeOH/DCM. Yield 85% as yellow amorphous solid; Rf =
product was purified on silica as a stationary phase; Yield 46% as
0.55 (eluent MeOH/DCM 1:4) 0.55; M.p: 109–111 C; 1H NMR
°
1
°
yellow crystals; Rf =0.65 (eluent EA/n-Hex 1:4); Mp 112–114 C. H
(600 MHz, CDCl3): δ=9.64 (s, 1H), 8.69 (d, J=7.8 Hz, 1H), 7.73 (m,
2H), 7.18 (d, J=6 Hz, 1H), 2.50–2.62 (br, 1H) ppm; 13C NMR
(151 MHz, CDCl3): δ=183.97, 138.90, 118.13, 117.38, 111.91 ppm;
HRMS (ESI): m/z calcd for C9H6N2O3: 190.04, found: 191.09.
NMR (600 MHz, CDCl3): δ=8.60 (d, J=1.9 Hz, 1H), 8.18–8.10 (m, 1H),
7.37 (d, J=9.1 Hz, 1H), 7.31 (d, J=3.2 Hz, 1H), 6.72 (d, J=3.2 Hz,
1H), 4.61–4.57 (t, J=6.5 Hz, 2H), 3.68 (t, J=6.9 Hz, 2H) ppm; 13C
NMR (151 MHz, CDCl3): δ=142.07, 138.72, 131.15, 128.19, 118.50,
117.70, 107.70, 109.06, 104.85, 48.15, 29.73 ppm; ESI-MS: m/z 269.09
[M+H]; HRMS: m/z calcd for C10H9BrN2O2 [M+H]+ 269.09, found
269.09.
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N,N-Dimethyl-1-(5-nitro-1H-indol-3-yl)methanamine (9): 5-Nitroin-
dole-3-carboxaldehyde (8) (1 equiv.) and 3 equivalents of dimeth-
ylamine (40% aqueous, 60 mL) allowed to react according to the
protocol B. The crude product is obtained as yellow crystalline solid
and further purified on silica as a stationary phase with DCM/MeOH
(9:1) over silica gel column. Yield 60% as yellow solid; Rf =0.45
1,3-Bis(5-nitro-1H-indol-1-yl)propane (17a): Yield 46% as yellow
crystals; Rf =0.85 (eluent EA/n-Hex 1:4); Mp 112–114 C. 1H NMR
°
(600 MHz, CDCl3): δ=8.50 (t, J=5.0 Hz, 2H), 8.09 (dd, J=9.1, 2.0 Hz,
2H), 7.51 (d, J=3.4 Hz, 2H), 7.43–7.39 (m, 2H), 7.20–7.12 (m, 2H),
6.73 (d, J=3.4 Hz, 2H), 5.27 (dd, J=15.7, 7.1 Hz, 2H), 4.91 (d, J=
8.9 Hz, 2H) ppm. 13C NMR (151 MHz, CDCl3): δ=30.05, 32.70, 44.53,
104.54, 109.33, 117.52, 118.42, 127.92, 131.22, 138.84, 141.79 ppm;
ESI-MS: m/z 364.35 [M+H]+; HRMS: m/z calcd for C19H16N4O4 [M+
H]+ 364.35, found 364.35.
(eluent MeOH/DCM 1:4) 0.45; Mp 114–116 C; 1H NMR (600 MHz,
°
[D6]DMSO): δ=11.71 (s, 1H), 8.62 (d, J=5.9 Hz, 1H), 8.00 (dt, J=
11.6, 4.2 Hz, 2H), 7.56–7.51 (m, 1H), 3.68 (s, 2H), 2.22 (s, 6H) ppm. 13
C
NMR (151 MHz, DMSO-d6): δ=140.09, 139.47, 132.50, 128.31,
126.81, 116.34, 111.59, 69.59, 54.07, 44.65 ppm; HRMS (ESI): m/z
calcd for C11H13N3O2: 219.10, found: 219.10.
N,N-Dimethyl-1-(5-amino-1H-indol-3-yl)methanamine (9a): Com-
pound 9 was treated with protocol A. The crude product was
purified with DCM/MeOH (9:1) over silica gel column. (10a) Yield
5-Nitro-1-(3-(pyrrolidin-1-yl) propyl)-1H-indole (5): Purified 5-nitro-
1-(3-bromopropyl)-1H-indole (4a; 0.24 g, 0.88 mmol) was dissolved
in dry ACN (6 mL/mmol) and to this pyrrolidine (3–10 equiv.) was
added, and the mixture was refluxed for 3–4 h. Solvent was
concentrated under reduced pressure. The crude product was
purified by column chromatography (eluent MeOH/DCM 1:3); using
°
96%; Brown solid. Rf =0.41(eluent MeOH/DCM 1:3); Mp 87–89 C;
1H NMR (600 MHz, [D6]DMSO): δ=10.38 (s, 1H), 7.04–6.99 (m, 2H),
6.76 (s, 1H), 6.47 (dd, J=8.6, 4.9 Hz, 1H), 3.31–3.32 (b, 2H) 4.41 (s,
2H), 2.12 (s, 6H) ppm;13C NMR (151 MHz, [D6]DMSO): δ=211.06,
140.62, 129.95, 128.50, 124.07, 111.63, 111.20, 110.40, 102.17, 54.50,
44.83 ppm; HRMS (ESI): m/z calcd for C11H15N3: 189.13 found: 189.13.
silica as a stationary phase; Yield 36% as yellow viscous solid; Rf
1
°
(eluent MeOH/ DCM 1:4) 0.35; Mp 112–114 C. H NMR (600 MHz,
[D6]DMSO): δ=8.58 (d, J=2.2 Hz, 1H), 8.05 (dd, J=9.1, 2.3 Hz, 1H),
7.74 (d, J=9.1 Hz, 1H), 7.68 (d, J=3.1 Hz, 1H), 6.79 (d, J=3.2 Hz,
1H), 4.36 (t, J=7.0 Hz, 2H), 3.03 (s, 4H), 2.92 (s, 2H), 2.08–2.14 (br,
2H), 1.85 (bs, 4H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=140.79,
138.63, 132.38, 127.37, 117.58, 116.40, 110.34, 103.91, 53.31, 51.60,
43.43, 26.93, 22.71 ppm; ESI-MS: m/z 273.33 [M+H]+; HRMS: m/z
calcd for C15H19N3O2 [M+H]+ 273.15, found 273.15.
N1,N1-Dimethyl-N2-((5-nitro-1H-indol-3-yl)methyl)ethane-1,2-dia-
mine (10): 5-Nitroindole-3-carboxaldehyde (8; 6 g, 62.5 mmol) and
N1,N1-dimethylethane-1,2-diamine (40% aqueous, 60 mL) were
allowed to react according to the protocol B. Yield 36%, yellow
solid; Rf =0.23 (Eluent MeOH/DCM 1:3); Mp 162–164 C; 1H
°
NMR:(600 MHz, CDCl3): δ=11.68 (s, 1H), 8.53 (dd, J=8.6, 3.6 Hz, 1H),
8.02–7.92 (m, 1H), 7.57–7.45 (m, 2H), 3.77 (s, 2H), 3.46 (bs, 1H), 2.54
(t, J=4.6 Hz, 2H), 2.46 (t, J=6.3 Hz, 2H), 2.06 (s, 6H) ppm; 13C NMR:
(151 MHz, CDCl3): δ=140.27, 139.42, 128.91, 126.30, 116.41, 116.42,
116.10, 112.11, 51.00, 46.25, 45.35, 43.05, 11.35 ppm; HRMS (ESI): m/
z calcd for C13H18N4O2: 262.14, found: [M+H]+ =263.14.
5-Nitro-1-(3-(pyrrolidin-1-yl)
propyl)-1H-indole-3-carbaldehyde
(6): The crude product is obtained from 5-nitro-1-(3-(pyrrolidin-1-yl)
propyl)-1H-indole (5) by Vilsmeier-Haack general protocol. Further
purified on silica as a stationary phase with 1:4 MeOH/DCM. Yield
56% as yellow solid; Rf =0.25 (eluent MeOH/DCM 1:4); Mp 132–
1
°
134 C. H NMR (600 MHz, [D6]DMSO): δ=9.99 (s, 1H), 8.93 (s, 1H),
N1,N1-Diethyl-N2-((5-nitro-1H-indol-3-yl)methyl)ethane-1,2-dia-
mine (11): 5-Nitroindole-3-carboxaldehyde (8) (6 g, 62.5 mmol) and
N1,N1-diethylethane-1,2-diamine (40% aqueous, 60 mL) were al-
lowed to react according to the protocol B. Yield 36%, yellow solid;
8.60 (s, 1H), 8.18 (d, J=9.0 Hz, 1H), 7.88 (d, J=9.1 Hz, 1H), 4.41 (t,
J=6.7 Hz, 2H), 3.40 (s, 4H), 2.37–2.40 (br, 2H), 1.98–2.00 (br, 2H),
1.67 (br, 4H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=185.08,
143.87, 143.40, 140.06, 123.87, 119.06–118.89, 118.35, 117.11,
111.96, 53.28, 51.95, 44.88, 28.32, 23.08 ppm; HRMS: m/z calcd for
C16H19N3O3 [M+H]+ 301.14, found 301.14.
Rf =0.23 (eluent MeOH/DCM 1:3) 0.23; Mp 152–154 C; 1H NMR
°
(600 MHz, [D6]DMSO): δ=11.84 (s, 1H), 8.67 (s, 1H), 7.97 (d, J=
8.0 Hz, 1H), 7.70 (s, 1H), 7.62–7.47 (m, 1H), 4.09 (s, 2H), 3.78 (s, 1H),
2.73 (m, 2H), 2.57 (s, 2H), 2.41 (t, J=17.3 Hz, 4H), 1.01–0.82 (m, 6H)
ppm. 13C NMR (151 MHz, [D6]DMSO): δ=140.12, 139.57, 127.96,
126.76, 116.94, 116.83, 116.38, 115.11, 111.58, 57.01, 50.58, 49.05,
44.99 ppm; HRMS (ESI): m/z calcd for C15H22N4O2: 290.17, found=
290.17.
N-((5-Nitro-1-(3-(pyrrolidin-1-yl)propyl)-1H-indol-3-yl)methyl)-2-
(pyrrolidin-1-yl)ethanamine (7): In
carboxaldehyde-5-nitro-1-[3-(1-pyrrolidinyl)propyl]-1H-Indole
a
round bottom flask, 8-
(6)
(6 g, 0.0199 mol) and 2-(pyrrolidin-1-yl)ethanamine (4.54 g,
0.039 mol) were reacted according to protocol B. The product was
purified on silica as a stationary phase with 1:4 MeOH/DCM. Yield
46% as yellow viscous solid; Rf (eluent MeOH/DCM 1:4) 0.25; 1H
NMR (600 MHz, [D6]DMSO): δ=8.63 (d, J=2.3 Hz, 1H), 8.03 (dt, J=
9.1, 2.1 Hz, 1H), 7.67–7.63 (m, 1H), 7.53 (s, 1H), 4.31–4.23 (m, 2H),
3.92 (bs, 1H), 2.66 (dd, J=13.0, 6.4 Hz, 2H), 2.35 (dd, J=18.3, 2.7 Hz,
9H), 2.31–2.23 (m, 3H), 1.96–1.87 (m, 2H), 1.72–1.65 (m, 6H), 1.66–
1.56 (m, 4H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=185.08,
143.87, 143.40, 140.06, 123.87, 119.06–118.89, 118.35, 117.11,
N-((5-Nitro-1H-indol-3-yl)methyl)-2-(pyrrolidin-1-yl)ethanamine
(12): 5-Nitroindole-3-carboxaldehyde (8; 6 g, 62.5 mmol) and 2-
(pyrrolidin-1-yl)ethanamine (40% aqueous, 60 mL) were allowed to
react according to the protocol B. Yield 26%, yellow solid; Rf =0.13
(eluent MeOH/DCM 1:3) 0.13; Mp 162–164 C. 1H NMR (600 MHz,
°
CDCl3): δ=11.70 (bs, 1H), 8.67 (t, J=9.1 Hz, 1H), 8.05–7.97 (m, 1H),
7.58–7.53 (m, 2H), 4.01 (s, 2H), 3.48 (bs, 1H), 2.73 (t, J=6.4 Hz, 2H),
2.58 (t, J=6.4 Hz, 2H), 2.49–2.40 (m, 4H), 1.71–1.62 (m, 4H) ppm; 13
C
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NMR (151 MHz, CDCl3): δ=140.11, 139.61, 127.82, 126.18, 116.83,
116.19, 115.60, 111.86, 54.35, 53.43, 46.61, 43.35, 26.46, 22.85 ppm;
HRMS (ESI): m/z calcd for C15H20N4O2: 288.16, found=288.16.
N,N-Dimethyl-N’-[[5-nitro-1-(3-Azidopropyl)-1H-indole]methyl]-
1,2-Ethanediamine (16a): Compound 8 (1 g, 3.663 mmol, 1 equiv.)
and about 3 equiv. of N1,N1-diethylethane-1,2-diamine ( 1.284 g,
11.07 mmol) were allowed to react according to generalized
procedure B. Crude product was then further purified with DCM/
MeOH (9:1) over silica gel column. Yield 46%, yellow solid; Rf =
1
2
3
4
5
6
7
8
9
N1-(2-Aminoethyl)-N2-(2-((2-(((5-nitro-1H-indol-3-yl)methyl)
amino)ethyl)amino)ethyl)ethane-1,2-diamine (13): Yield 46%, Yel-
low solid.1H NMR (600 MHz, D2O): δ=8.59 (s, 1H), 8.00 (d, J=9.2 Hz,
1H), 7.67 (d, J=3.8 Hz, 1H), 7.48 (d, J=9.1 Hz, 1H), 4.58–4.40 (m,
2H), 3.68 (s, 2H), 3.62–2.73 (m, 19H), 1.10 (t, J=7.1 Hz, 2H) ppm. 13C
NMR (151 MHz, D2O): δ=141.10, 131.11, 125.63, 117.90, 115.39,
112.0, 65.0, 50.77, 43.0, 43.06, 42.34, 35.27, 16.84 ppm; HRMS (ESI):
calcd for C17H29N7O2 363.24; found 363.24.
0.33(eluent MeOH/DCM 1:3); Mp 162–164 C; 1H NMR (600 MHz,
°
[D6]DMSO): δ=8.63 (d, J=2.3 Hz, 1H), 8.60 (d, J=2.3 Hz, 1H), 8.03
(dt, J=9.1, 2.1 Hz, 2H), 7.66 (dd, J=9.1, 4.7 Hz, 2H), 7.55 (d, J=
5.6 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H), 4.35–4.21 (m, 4H), 3.92 (s, 2H),
2.71–2.60 (m, 3H), 2.35 (dd, J=18.3, 2.7 Hz, 2H), 2.24 (dd, J=14.1,
7.7 Hz, 2H), 2.02–1.87 (m, 2H), 1.76–1.49 (m, 4H) ppm. 13C NMR
(151 MHz, [D6]DMSO): δ=140.11, 139.22, 130.92, 130.39, 126.30,
116.70, 116.47, 116.12, 114.59, 110.20, 85.45, 55.25, 53.42, 53.03,
52.79, 51.84, 47.46, 43.62, 30.81, 28.75, 24.19, 23.06 ppm; HRMS
(ESI): calcd for C18H27N7O2 373.22; found 374.22.
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1-(3-(Pyrrolidin-1-yl)propyl)-1H-indol-5-amine (5a): 5-nitro-1-(3-
(pyrrolidin-1-yl)propyl)-1H-indole (5) was treated as per the Gener-
alized procedure for the reduction of nitro to amine; The crude
product is brown in colour and highly unstable in nature so we
could not purify it further; Yield 23% as yellow viscous solid; Rf =
5-Nitro-1-(prop-2-yn-1-yl)-1H-indole (19a): Yield 86%, yellow solid;
1
0.25 (eluent MeOH/DCM 1:4) 0.25; H NMR (600 MHz, [D6]DMSO):
1
°
Rf =0.46 (eluent EA/cyclo 1:3); M.p: 113–115 C; H NMR (600 MHz,
δ=7.11–7.09 (m, 2H), 6.69 (s, 1H), 6.54–6.56 (d, J=7.0 Hz, 1H), 6.09–
6.11(d, J=7.3 Hz, 1H), 4.46 (br, 2H), 4.34–4.37 (t, J=6.5 Hz, 2H),
3.04–3.08 (br, 6H), 2.11–2.14 (m, 2H), 1.86–191 (m, 4H) ppm; 13C
NMR (151 MHz, [D6]DMSO) δ140.79, 138.63, 132.38, 127.37, 117.58,
116.40, 110.34, 103.91, 53.31, 51.60, 43.43, 26.93, 141.73, 137.73,
125.53 125.42, 117.85, 115.52, 112.19, 54.16, 54.54, 45.86, 43.37,
26.61, 23.31 ppm; ESI-MS: m/z 244.17 [M+H]+.
CDCl3): δ=8.57 (d, J=2.2 Hz, 1H), 8.17–8.11 (m, 1H), 7.43 (d, J=
9.1 Hz, 1H), 7.37 (d, J=3.3 Hz, 1H), 6.71 (d, J=3.3 Hz, 1H), 4.93 (d,
J=2.6 Hz, 2H), 2.48 (t, J=2.6 Hz, 1H) ppm; 13C NMR (151 MHz,
CDCl3): δ=142.04, 138.04, 130.46, 128.18, 118.25, 117.63, 109.37,
104.64, 76.33, 74.60, 36.27 ppm; HRMS (ESI): calcd for C11H8N2O2
200.06; found 201.06.
1-(But-3-yn-1-yl)-5-nitro-1H-indole (19b): Yield 88%, yellow solid;
1-(3-Azidopropyl)-5-nitro-1H-indole (14): To a stirring mixture of 4
(1 g, 3.225 mmol, 1 equiv.) in 15 mL Dry DMF about 3 equiv. of
sodium azide (0.6386 g, 9.667 mmol) were added and allowed to
reflux for 3–5 h. Followed by quenching with ice water and
extraction with ethyl acetate. Organic extract was then concen-
trated under reduced pressure, and the crude product was purified
on silica as a stationary phase with cyclohexane/ethyl acetate (9:1)
1
°
Rf =0.39(eluent EA/cyclo 1:3); M.p: 113–115 C; H NMR (600 MHz,
[D6]DMSO): δ=8.52 (t, J=2.7 Hz, 1H), 8.08–8.03 (m, 1H), 7.32 (d, J=
9.1 Hz, 1H), 7.25 (t, J=2.7 Hz, 1H), 6.62 (d, J=3.0 Hz, 1H), 4.28 (q,
J=6.9 Hz, 2H), 2.65 (tt, J=5.4, 2.7 Hz, 2H), 1.98 (dt, J=5.3, 2.7 Hz,
1H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=131.54, 128.56, 118.1,
118.09, 109.41, 105.17, 80.2, 72.14, 46.39, 21.12 ppm; HRMS (ESI):
calcd for C12H10N2O2 214.07; found 215.07.
over silica gel column. Yield 56%, yellow solid; Rf =(eluent EA/cyclo
1
°
1:3) 0.63; Mp 92–94 C. H NMR (600 MHz, [D6]DMSO): δ=10.00 (s,
5-Nitro-1-(prop-2-yn-1-yl)-1H-indole-3-carbaldehyde (20): The
crude product was purified on silica as a stationary phase; with 1:4
EA/CH. The product was a yellow solid. 1H NMR (600 MHz, [D6]
DMSO): δ=10.01 (s, 1H), 8.93 (t, J=6.6 Hz, 1H), 8.61 (s, 1H), 8.23
(dd, J=9.1, 2.4 Hz, 1H), 7.87 (d, J=9.1 Hz, 1H), 5.33 (d, J=2.5 Hz,
2H), 3.61 (t, J=2.5 Hz, 1H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=
185.49, 143.43, 142.92, 139.37, 123.94, 118.99, 118.41, 117.24,
112.08, 77.32, 36.41 ppm.
1H), 8.94 (d, J=2.1 Hz, 1H), 8.62 (s, 1H), 8.19 (dd, J=9.1, 2.3 Hz, 1H),
7.90 (d, J=9.1 Hz, 1H), 4.43 (t, J=7.0 Hz, 2H), 3.40 (t, J=6.6 Hz, 2H),
2.07–2.11 (br, 2H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=185.13,
146.04–144.33, 143.35, 139.92, 123.97, 118.75, 118.26, 117.17,
111.90, 47.89, 44.23, 28.53 ppm; HRMS (ESI): calcd for C11H11N5O2
245.09; found [M+H]+ 246.09.
1-(3-Azidopropyl)-5-nitro-1H-indole-3-carbaldehyde (15): The
crude product was further purified with Cyclohexane: Ethyl acetate
(9:1) over silica gel column. Yield 66%, yellow solid; Rf =0.31(eluent
N,N-Dimethyl-1-(5-nitro-1-(prop-2-yn-1-yl)-1H-indol-3-yl)
methanamine (21a): 5-nitro-1-(prop-2-yn-1-yl)-1H-indole-3-carbal-
dehyde and 3 equivalent of dimethylamine (40% aqueous, 60 mL)
were allowed to react according to the protocol B. 1H NMR
(600 MHz, [D6]DMSO): δ=8.61–8.59 (d, J=7.1 Hz, 1H), 8.11 (dd, J=
9.1, 2.2 Hz, 1H), 7.72 (s, 1H), 7.75 (d, J=9.1 Hz, 1H), 5.17 (d, J=
2.4 Hz, 2H), 4.29 (s, 2H), 3.61 (bs, 1H), 2.17–2.18 (m, 6H) ppm; 13C
NMR (151 MHz, [D6]DMSO): δ=141.08, 139.16, 131.14, 127.62,
117.15, 116.99, 116.92, 115.37, 110.96, 78.78, 76.28, 53.96, 44.96,
35.93 ppm; HRMS (ESI): calcd for C14H15N3O2 257.12; found 258.12.
EA/cyclo 1:3); Mp 102–104 C; 1H NMR (600 MHz, [D6]DMSO): δ=
°
10.00 (s, 1H), 8.94 (d, J=2.1 Hz, 1H), 8.62 (s, 1H), 8.19 (dd, J=9.1,
2.3 Hz, 1H), 7.90 (d, J=9.1 Hz, 1H), 4.43 (t, J=7.0 Hz, 2H), 3.40 (t, J=
6.6 Hz, 2H), 2.07–2.11 (br, 2H) ppm; 13C NMR (151 MHz, [D6]DMSO):
δ=185.13, 146.04, 144.33, 143.35, 139.92, 123.97, 118.75, 118.26,
117.17, 111.90, 47.89, 44.23, 28.53 ppm; HRMS (ESI): calcd for
C12H11N5O3 273.09; found 273.09.
1-(1-(3-Azidopropyl)-5-nitro-1H-indol-3-yl)-N,N-dimeth-
ylmethanamine (16): Compound 8 (1 g, 3.663 mmol, 1 equiv.) and
N1,N1-Diethyl-N2-((5-nitro-1-(prop-2-yn-1-yl)-1H-indol-3-yl)
methyl)ethane-1,2-diamine (21b): 5-nitro-1-(prop-2-yn-1-yl)-1H-
indole-3-carbaldehyde and N1,N1-diethylethane-1,2-diamine (40%
aqueous, 60 mL) were allowed to react according to the protocol B.
1H NMR (400 MHz, [D6]DMSO): δ=8.83 (d, J=7.1 Hz, 1H), 8.13 (dd,
J=9.1, 2.2 Hz, 1H), 7.82 (s, 1H), 7.75 (d, J=9.1 Hz, 1H), 5.25 (d, J=
6.4 Hz, 2H), 4.29 (s, 2H), 3.61 (bs, 2H), 3.01–2.93 (m, 2H), 2.84–2.74
(m, 2H), 2.67 (q, J=7.1 Hz, 4H), 1.05–0.96 (m, 6H) ppm; 13C NMR
(151 MHz, [D6]DMSO): δ=140.81, 138.29, 132.66, 126.62, 117.03,
116.61, 110.87, 110.69, 78.7, 76.22, 48.79, 46.14, 43.39, 41.20, 35.69,
10.11 ppm. HRMS (ESI): calcd for C18H24N4O2328.19; found 329.19.
3 equiv.
of
N1,
N1-dimethylethane-1,2-diamine
(1.284 g,
11.070 mmol) were allowed to react according to generalized
procedure B. Crude product was then further purified with DCM/
MeOH (9:1) over silica gel column. Yield 26%, yellow solid; Rf =
0.43(eluent MeOH/DCM 1:3) 0.43; Mp 102–104 C. 1H NMR
°
(600 MHz, [D6]DMSO): δ=8.66 (d, J=1.8 Hz, 1H), 8.03 (dd, J=9.1,
2.1 Hz, 1H), 7.67 (d, J=9.1 Hz, 1H), 7.56 (s, 1H), 4.29 (t, J=6.9 Hz,
2H), 3.95 (s, 2H), 3.30–3.32 (bs, 4H), 2.64 (t, J=6.4 Hz, 2H), 2.44 (q,
J=7.1 Hz, 2H), 1.99–2.03 (br, 2H) ppm; 13C NMR (151 MHz, [D6]
DMSO): δ=140.35, 139.09, 126.55, 116.58, 110.24, 51.79, 47.98,
46.34, 43.59, 43.09, 28.90, 11.61 ppm. HRMS (ESI): calcd for
C14H18N6O2 302.15; found 302.15.
1H-Indole-3-carbaldehyde (2a): The crude product was obtained
from 1H-indole (5) by Vilsmeier-Haack general protocol A. Crude
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product was purified with DCM/MeOH (9:1) over silica gel column.
N1-((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)-N2-(2-((2-aminoethyl)
1
1
2
3
4
5
6
7
8
9
Yield 66%, White crystalline solid; Rf (eluent MeOH/DCM 1:3) 0.33;
amino)ethyl)ethane-1,2-diamine (24c): Yield 36%, white solid; H
NMR (600 MHz, D2O): δ=8.76 (d, J=12.4 Hz, 1H), 8.40 (d, J=
15.1 Hz, 1H), 7.90 (s, 1H), 7.61 (d, J=6.3 Hz, 1H), 4.61 (dd, J=
12.5 Hz, 13.2 Hz, 6H), 3.63–2.47 (m, 14H) ppm; HRMS (ESI): calcd for
C14H24N6 276.21; found [M+H]+ 277.21.
1
°
M.p: 123–125 C; H NMR (600 MHz, CDCl3): δ=9.60 (s, 1H), 7.83 (d,
J=6 Hz, 2H), 7.53 (d, J=6 Hz, 1H), 7.10 (d, J=6 Hz, 1H), 6.89–6.84
(m, 2H) ppm; 13C NMR (151 MHz, CDCl3): δ=184.11, 136.63, 122.92,
121.57, 120.63, 111.50 ppm. (ESI): m/z calcd for C9H7NO:145.10,
found: 146.02.
N-((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)-2-(pyrrolidin-1-yl)
1
1-(1H-Indol-3-yl)-N, N-dimethylmethanamine (3a): Yield 46%,
ethanamine (24e): Yield 46%, white solid; H NMR (600 MHz, [D6]
°
yellow solid; Rf =0.43(eluent MeOH/DCM 1:3) 0.43; M.p: 143–145 C.
DMSO): δ=11.44 (bs, 1H), 8.19 (dd, J=6.6, 3.8 Hz, 1H), 8.00 (dd, J=
8.5, 7.6 Hz, 1H), 7.92–7.85 (m, 1H), 7.41–7.31 (m, 1H), 7.08–6.92 (m,
1H), 3.85 (s, 2H), 3.72 (s, 2H), 2.67–2.61 (m, 2H), 2.46–2.33 (m, 2H),
2.31–2.23 (m, 2H), 1.70–1.53 (m, 4H) ppm; 13C NMR (151 MHz, [D6]
DMSO): δ=148.80, 142.41, 127.45, 126.94, 124.88, 123.80, 119.57,
119.17, 114.83, 112.83, 112.87, 111.47, 85.47, 55.30, 53.42, 49.62,
22.71 ppm; HRMS (ESI): calcd for C14H20N4 244.17; found [M+H]+
245.17.
1H NMR (600 MHz, [D6]DMSO): δ=10.92 (s, 1H), 7.61 (d, J=7.9 Hz,
1H), 7.36 (d, J=8.1 Hz, 1H), 7.21 (t, J=5.1 Hz, 1H), 7.08 (t, J=7.5 Hz,
1H), 6.98 (t, J=7.4 Hz, 1H), 3.55 (s, 2H), 2.17 (s, 6H) ppm; 13C NMR
(151 MHz, [D6]DMSO): δ=136.32, 127.52, 124.38, 120.85, 118.98,
118.29, 111.50, 111.24, 54.36, 44.82 ppm. HRMS: m/z calcd for
C11H14N2 [M+H]+ 174.12, found 174.12.
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N1-((1H-Indol-3-yl)methyl)-N2-(2-((2-((2-aminoethyl)amino)ethyl)
amino)ethyl)ethane-1,2-diamine (3b): Yield 46%, white solid; 1H
NMR (600 MHz, D2O): δ=10.62 (s, 1H), 7.78 (d, J=7.88 Hz, 1H), 7.61
(d, J=2.48 Hz, 1H), 7.58 (s, 1H), 7.33 (t, J=7.34 Hz, 1H), 7.26 (t, J=
7.38 Hz, 1H), 4.57 (s, 2H), 3.39–3.48 (m, 22H) ppm; 13C NMR
(151 MHz, D2O): δ=42.20, 42.93, 43.77, 43.92, 44.24, 103.58, 112.32,
118.01, 120.29, 122.62, 126.13, 128.20, 136.19 ppm; HRMS (ESI):
calcd for C17H30N6 318.26; found [M+H]+ 319.26.
N1-(2-Aminoethyl)-N2-(2-((2-((benzo[d][1,3]dioxol-5-ylmethyl)
amino)ethyl)amino)ethyl)ethane-1,2-diamine (25b): Yield 46%,
1
white solid; H NMR (600 MHz, D2O): δ=6.91 (dd, J=12.5, 6.4 Hz,
4H), 5.96 (s, 2H), 4.25–4.05 (m, 2H), 3.59–2.73 (m, 21H) ppm; 13C
NMR (151 MHz, D2O): δ=147.80, 138.45, 133.80, 131.13, 124.69,
116.42, 105.97, 51.84, 44.65, 43.51, 42.46, 41.57, 40.07, 35.20,
18.06 ppm; HRMS (ESI): calcd for C16H29N5O2 323.23; found [M+H]+
324.23.
1-(1H-Indazol-3-yl)-N,N-dimethylmethanamine (23a): Yield 36%,
°
white solid; Rf =0.51(eluent MeOH/DCM 1:3) 0.51; M.p: 143–145 C;
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2, N2-dimethylethane-1,2-
diamine (25d): Yield 56%, white solid; Rf (eluent MeOH/DCM 1:3)
1H NMR (600 MHz, [D6]DMSO): δ=12.93 (d, J=12.7 Hz, 1H), 8.00 (s,
1H), 7.59 (s, 1H), 7.47 (d, J=8.6 Hz, 1H), 7.29 (d, J=8.6 Hz, 1H), 3.44
(s, 2H), 2.12 (s, 6H) ppm; 13C NMR (151 MHz, [D6]DMSO): δ=211.08,
133.18, 130.77, 127.53, 122.53, 119.87, 109.83, 63.28, 44.66 ppm;
HRMS (ESI): calcd for C10H13N3 175.11; found [M+H]+ 176.11.
0.13; M.p: 103–105 C. 1H NMR (600 MHz, [D6]DMSO): δ=6.91 (s,
°
1H), 6.79 (dd, J=12.5, 7.9 Hz, 2H), 5.97 (s, 1H), 3.63 (s, 2H), 2.54–2.56
(m, 2H), 2.33 (t, J=6.4 Hz, 4H), 2.11 (s, 6H) ppm; 13C NMR (151 MHz,
[D6]DMSO): δ=147.23, 145.69, 134.46, 120.98, 108.32, 107.66,
100.51, 58.51, 52.48, 45.85, 45.08 ppm; HRMS (ESI): calcd for
C12H18N2O2 222.14; found 222.14.
N1-((1H-Indazol-3-yl)methyl)-N2-(2-((2-((2-aminoethyl)amino)
ethyl)amino)ethyl)ethane-1,2-diamine (23b): Yield 66%, white
solid; 1H NMR (600 MHz, D2O): δ=8.14 (d, J=6.0 Hz, 1H), 7.92 (s,
1H), 7.65 (t, J=7.6 Hz, 1H), 7.46 (d, J=7.8 Hz, 1H), 4.39 (s, 2H), 3.56–
2.68 (m, 23H) ppm; 13C NMR (151 MHz, D2O): δ=140.22, 134.64,
128.54, 123.62, 122.79, 51.97, 43.52, 42.90, 42.30, 42.24 ppm. HRMS
(ESI): calcd for C16H29N7 319.25; found [M+H]+ 320.25.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-2-(pyrrolidin-1-yl)ethanamine
(25e): Yield 46%, white solid; 1H NMR (600 MHz, D2O): δ=7.05–6.92
(m, 4H), 6.04 (s, 2H), 4.26 (s, 2H), 3.57 (dt, J=14.6, 6.6 Hz, 8H), 2.12
(s, 4H) ppm; 13C NMR (151 MHz, D2O): δ=148.02, 147.67, 124.35,
123.55, 117.89, 114.96, 108.80, 109.14, 101.74, 54.83, 51.53, 49.47,
41.94, 22.39; HRMS (ESI): calcd for C14H20N2O2 248.15; found [M+
H]+ 249.15.
N-((1H-Indazol-3-yl)methyl)-2-(pyrrolidin-1-yl)ethanamine (23e):
Yield 56%, white solid; Rf =0.27(eluent MeOH/DCM 1:3); M.p: 173–
1
°
175 C. H NMR (600 MHz, [D6]DMSO): δ=7.92 (s, 1H), 7.56 (s, 1H),
N1-((5-Amino-1H-indol-3-yl)methyl)-N2, N2-diethylethane-1, 2-di-
amine (11a): Brown (crude); 1H NMR (600 MHz, [D6]DMSO): δ=
10.32 (s, 1H), 7.03 (d, J=3.8 Hz, 1H), 7.00 (d, J=2.3 Hz, 2H), 6.71 (d,
J=1.9 Hz, 1H), 6.46 (dd, J=8.5, 2.0 Hz, 2H), 3.71 (s, 2H), 3.17 (d, J=
4.9 Hz, 2H), 2.64–2.55 (m, 3H), 2.48–2.39 (m, 4H), 0.95–0.88 (m, 6H);
11a is unstable in nature and hence degrades very quickly, so it is
not possible to do further analysis.
7.39 (d, J=8.6 Hz, 1H), 7.25 (d, J=8.6 Hz, 1H), 3.70 (d, J=5.7 Hz,
2H), 2.50 (s, 2H), 2.48–2.38 (m, 2H), 2.37–2.22 (m, 2H), 1.90 (d, J=
5.8 Hz, 4H), 1.65–1.48 (m, 4H) ppm; 13C NMR (151 MHz, [D6]DMSO):
δ=132.60, 126.55, 118.69, 109.46, 55.33, 55.39, 53.70, 53.25, 53.16,
47.53, 23.07 ppm. HRMS (ESI): calcd for C14H20N4 244.17; found [M+
H]+ 245.17.
N,N-Dimethyl-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)methanamine
Please refer to the Supporting Information for the general
procedure and synthetic Scheme 4.
(24a): Yield 56%, white solid; Rf =0.13 (eluent MeOH/DCM 1:3);
1
°
M.p: 143–145 C. H NMR (600 MHz, [D6]DMSO): δ=11.39 (bs, 1H),
DNAs: The c-Myc DNA (TGAGGGTGGGTAGGGTGGGTAA) was pur-
chased by Eurofins, Germany as HPSF (High Purity Salt Free)
purified oligos and further purified by HPLC, and stock solutions
(100 μM) were made by resuspending the DNA in molecular
8.22–8.17 (m, 1H), 8.00 (dd, J=6.3, 5.2 Hz, 1H), 7.34 (s, 1H), 7.06–
7.02 (m, 1H), 3.55 (s, 2H), 2.16 (d, J=4.3 Hz, 6H) ppm;13C NMR
(151 MHz, [D6]DMSO): δ=185.17, 148.80, 145.17, 142.64, 127.37,
124.83, 119.78, 118.43, 115.43, 114.72, 110.20, 54.14, 44.33; HRMS
(ESI): calcd for C10H13N3 175.11; found [M+H]+ 176.11.
°
biology grade water and quantified by A260 at 95 C, using ɛ 260
values as provided by the manufacturers, before the DNAs were
°
N1-((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)-N2-(2-((2-((2-aminoeth-
yl)amino)ethyl)amino)ethyl)ethane-1,2-diamine (24b): Yield 36%,
white solid; 1H NMR (600 MHz, D2O): δ=8.74 (dd, J=8.0, 7.8 Hz,
1H), 8.41 (m, 1H), 8.02–7.78 (m, 1H), 7.58 (t, J=6.7 Hz, 1H), 4.56 (d,
J=9.3 Hz, 2H), 3.76–2.72 (m, 23H) ppm; 13C NMR (151 MHz, D2O):
δ=138.22, 137.01, 133.41, 131.41, 131.98, 124.50, 116.58, 105.39,
52.57, 51.88, 50.33, 48.99, 44.65, 43.65, 43.46; HRMS (ESI): calcd for
C16H29N7 319.25; found [M+H]+ 320.26.
aliquoted and stored at À 20 C. All samples were freshly prepared
prior to each experiment, the Myc DNA was dissolved in the
respective buffer heated at 90 C for 5 min, and slowly cooled down
to room temperature. All other reagents were purchased from
°
Sigma-Aldrich unless otherwise stated.
Fragment screening with thiazole displacement assays: The FID
assay was performed using the procedure described earlier.[39]
Dissociation constant (KD) for TO binding to c-Myc22-merhas been
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determined and reported in (see the Supporting Information and
Figure S1) using the following conditions 0.25 μM DNA, 0.5 μM
Thiazole Orange, 10% DMSO, 20 mM Na caco, 140 mM KCl, pH 7
(25 μL/well). All fragment molecules were 95% pure and obtained
from commercial sources or were synthesized in house. For assay
optimization sufficient negative and positive controls were used,
DMSO only (10% v/v) wells, which contained no small molecule,
were used as a negative control, while positive control wells
consisted target DNA and the intercalator TO.
silapentane-5-sulfonate (DSS) was used as internal reference. Water-
gate W5 pulse sequence with gradients (37) was used for water
suppression.
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Cells and culture conditions: HeLa cells (human cervical cancer
cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM,
Gibco® Life Technologies) supplemented with 10% foetal bovine
serum (PAN Biotech), and 1% penicillin/streptomycin (Invitrogen).
NKE cells were cultured in RPMI 1640 (GIBCO) with 10% FBS. HeLa
cells were maintained in 25 or 75 cm2 flasks in a humidified
°
For screening, 1.25 μL of each fragment from its original 100 mM
DMSO stock plate was transferred to a 384 well assay plate (low
volume, flat bottom black NBS treated, Corning 3820) with each
384 well plate containing 182 fragments with negative and positive
controls. 23.75 μL of the annealed c-Myc oligo containing 0.25 μM
DNA, 0.5 μM Thiazole Orange, 20 mM Na cacodylate, 140 mM KCl,
pH 7 were added to the ligands and the plate was incubated for
30 min at room temperature. The fluorescent measurements were
atmosphere containing 5% CO2, at 37 C. For cell passaging, biotase
(Invitrogen) was used for detaching the cells and the standard
Neubauer chamber for counting the cells and seeding accordingly.
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Cell proliferation assay: HeLa cells were used for performing the
cell viability assays. A day before the assay cells were seeded in a
96 well plate (Nunclon 96 Flat Bottom Transparent), with a density
of 1000 cells per well in 100 μL. Ligand dilutions were prepared in
°
DMSO, vortexed and stored in À 20 C dilution range varied
°
taken at 25 C using an excitation filter of 510 nm and an emission
between 0, 3, 10, 30, 100, 300, 1000, 3000, 9000, 30000, 100000 nM
depending upon the potency of the respective fragments. The
working concentrations were set up in culture medium and added
to the cells, to a final DMSO concentration of 0.1%. Cells treated
with the fragments were placed back in the 5% CO2 incubator.
After 3 days of fragment treatment cell viability assay was
performed using Alamar Blue reagent (Thermo Scientific) following
the instructions of the manufacturer. The fluorescence signal of the
Alamar Blue was measured with infinite 200 Pro Micro Plate Reader
(Tecan i-control) with the excitation wavelength 540 nm and
emission wavelength 590 nm. Ligand treatments were performed
in triplicates and were repeated three times. The dose-dependent
cytotoxicity on cells was evaluated in comparison to the DMSO
control. To scavenge the ROS produced by substituted 5-nitro-
indoles, HeLa cells were pretreated with 2.5 mM NAC for 1 h and
then treated with 5-nitroindoles for 72 h. After compound treat-
ment, Alamar Blue reagent (10 μL, 5 mg/mL) was then added into
each well for 4 h incubation. Absorbance were then taken to
estimate the cell viability.
filter of 540 nm using an Infinite 200 Pro Micro Plate Reader (Tecan
i-control). Experiments were performed in triplicate and were
repeated three times. The ligands were ranked according to their
TO displacement effect and those fragments showing�95%
displacement were subjected to a dose response, under the original
screening conditions Titration Scheme of c-Myc: c(TO)=0.5 μM; c(c-
Myc)=0,5 μM; c(DMSO)=10%; c (ligand)=256, 128, 64, 32, 16, 8, 4,
2, 1, 0.5, 0.25, 0.125, and 0.0625 μM; RT; 10% DMSO, 20 mM Na
cacodylate, 140 mM KCl, pH 7.The 50% displacement value (DC50),
were calculated for ligands as described in the supplementary.
Microscale thermophoresis (MST) assay: The fluorescently labelled
oligonucleotides (FAMÀ Pu22, 5’-TGAGGGTGGGTAGGGTGGGTAA-3’
and FAM-duplex, 5’-CGCGCGCGTTTTCGCGCGCG-3’, was diluted
from stock to the required concentration (10 μM) in 10 mM Tris-HCl
buffer, pH 7.4, in the presence of 150 mM KCl. Then the mix was
°
annealed by heating at 95 C for 5 min, gradually cooled to room
°
temperature, and incubated at 4 C overnight. The concentration of
fragments was varied from 0 to 20 μM for G-Quadruplex sequences
and 0 to 200 μM for tested the KD of duplex DNA. A 12-point
dilution series was prepared for each DNA. After incubation, the
samples were loaded into MST standard-treated glass capillaries,
and MST analysis was performed using a Monolith NT.115 instru-
ment (Nano Temper).
The percentage of viable cells was calculated by the following
equation:
Absorbance of treated cells
Absorbance of untreated cells
% viable cells ¼
� 100
Fluorescence intensityy titration FAM-labelled Myc (pu22): 5’-
°
TGAGGGTGGGTAGGGTGGGTAA-3’ was heated at 95 C for 3 min,
The final IC50 values were calculated by using the GraphPad Prism
6.0 software. In the GraphPad Prism® equation, log-transformed
concentration values and the effect data were fitted to a four-
parameter logistic equation. The original, % control, or % inhibition
data are represented by Y along with their minimal (min) and
maximal (max) values. The inhibitor concentration is represented by
X, IC50 is the concentration at 50% maximal value, and Hill Slope is
the slope factor.
allowed to cool to RT, and diluted to 50 nM in 10 mM Tris·HCl
buffer, pH 7.4, in the presence of 150 mM KCl. Compound was
added as a solution both in buffer containing 2–3% DMSO, and the
sample was allowed to equilibrate for 10 min. Fluorescence
intensity spectra were recorded at RT using an Infinite 200 Pro
Micro Plate Reader (Tecan i-control). Fluorescence intensity was
recorded at an excitation wavelength of 645 nm, with the resulting
emission spectrum recorded from 650 to 800 nm, and the
fluorescence intensity at the emission maximum was used in all
calculations. The concentration of fragments was varied from 0 to
20 μM for G-Quadruplex DNA.
ðmax À minÞ
Y ¼ min þ
� 100
1 þ 10ððXÀ logIC50Þ�Hill slope
NMR: All NMR measurements were recorded at 600 MHz at 298 K.
Samples contained 0.1 mM c-Myc in 25 mM KPi, 70 mM KCl, pH 7,
0.025 mM DSS and 10% [D6]DMSO. Due to poor solubility of the
fragments in the absence of DNA, a single sample for each DNA:
fragment ratio was prepared to keep the DMSO concentration
constant. For concentration the titration, the DNA was provided as
a 100 μM solution in 25 mM Tris·HCl buffer (pH 7.4) with 100 mM
KCl in 10% [D6]DMSO/90% H2O. Small amounts of the ligand stock
solution in 100% [D6]DMSO were added directly into the NMR tube
(10% [D6]DMSO at the end of the titration). 2,2-Dimethyl-2-
c-Myc expression and immuno blotting: for monitoring the
expression levels of c-Myc after ligand treatment, Western blot
analysis was performed. Cells treated for 24 h with different
fragments in varying concentrations were harvested with biotase,
and suspended in PBS buffer containing EDTA free protease
inhibitor. Followed by cell lysis with ultrasonicator and quantifica-
tion of the cell lysate with Roti-Quant. Equal amounts of the cell
lysates were loaded onto the pre-casted NuPAGE 4–12% Bis-Tris
Gels and with the MES SDS running buffer (50 mM MES, 50 mM Tris
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base, 0.1% SDS, 1 mM EDTA, pH 7.3) gel electrophoresis was
performed. Proteins on the gel were transferred onto the PVDF
membrane (activated by brief incubation in methanol) for immuno-
blotting. After the protein transfer, the membrane was blocked for
an hour in 5% non-fat milk in TBS buffer. The blot was then probed
either with c-Myc rabbit monoclonal antibody (1:2000, #5605, Cell
Signaling) in 1X TBS with 5% BSA, in 1X TBS with 5% BSA or
GAPDH monoclonal antibody in 1X PBS with 5% non-fat dried milk
adherent cells by trypsin treatment), washed with PBS and trans-
ferred in a FACS tube. The unstained cells serve as FACS control
(negative signal).
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Statistics: All results are representative of three independent
experiments and the data presented are expressed as mean �SD.
Statistical analysis was performed using Student’s t-test except that
the Young’s modulus is performed using Kruskal-Waillis test, and p
<0.05 was regarded as statistically significant.
°
and incubated overnight at 4 C, on a gel rocking platform shaker.
To get rid off the unbound primary antibody, the membrane was
washed three times with PBST (PBS with 0.05% Tween 20) for
10 min/wash. Further, the membrane was probed either with
horsera dish peroxidase (HRP)-conjugated affinipure goat anti-
rabbit IgG (H+L) sera in PBS (1:5000, Dianova) or with HRP-
conjugated goat anti-mouse secondary antibody in PBS (1:5000,
Dianova) respectively, for 1 h. Followed by three washing steps
with PBS Tween (0.05%, 10 min/wash). Subsequently, the mem-
brane was developed with PierceTM ECL chemiluminescent HRP
substrate, and the chemiluminescence signals were detected using
digital Lumi-imager (Roche). Relative intensities of the ECL signals
were then determined using Image J software.
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Acknowledgements
We wish to acknowledge Alix Tröster, Dr. Irene Bessi, Dr. Sridhar
Sreeramulu for stimulating discussions and to Elke Stirnal and
Kerstin Witt for analytical for HPLC purification. Funding was
provided by EU ITN-projects: Hyperpol and HaemMetabolome,
DFG-Normalverfahren G-quadruplex and the state of Hesse for
BMRZ. This work was additionally supported by the Deutsche
Forschungsgemeinschaft (SFB1177; SCHN1166/4-1 (to F.S.). Open
access funding enabled and organized by Projekt DEAL.
Reverse transcription polymerase chain reaction (RT-PCR). Total
RNA was isolated after 24 h ligand treatment using TRIzol reagent
(Invitrogen, Life Technologies) according to the manufacturer’s
instructions. RNA was quantified, and 1 μg of RNA was used for
cDNA preparation using Verso cDNA synthesis kit. The relative
transcript expression level for genes was measured by quantitative
real-time PCR using SYBR Green-based method. ΔCt values were
calculated by the difference in threshold cycles (Ct) between test
and control samples. 18 s rRNA gene was used as an internal
control for normalizing the cDNA concentration of each sample.
Primers used for monitoring the gene expressions are as follows:
Conflict of Interest
The authors declare no conflict of interest.
Keywords: c-Myc
· G-quadruplex binders · nitroindoles ·
oncogene promoters, reactive oxygen species, structure-activity
relationships
c-Myc forward : 50-CTGCGACGAGGAGGAGGACT-30
c-Myc reverse : 50-GGCAGCAGCTCGAATTTCTT-30
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Manuscript received: October 24, 2020
Revised manuscript received: January 26, 2021
Accepted manuscript online: January 28, 2021
Version of record online: ■■■, ■■■■
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V. D. Nimbarte, Dr. Julia Wirmer-
Bartoschek, Dr. S. L. Gande, I.
Alshamleh, M. Seibert, Dr. H. R.
Nasiri, Dr. F. Schnütgen, Prof. Dr. H.
Serve, Prof. Dr. H. Schwalbe*
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Targeting DNA-G-quadruplexes:
Fragment extension of indole
fragments resulted in the synthesis of
a series of pyrrolidine-substituted 5-
nitroindole derivatives that bind to
the c-Myc G-quadruplex DNA in a 2:1
stoichiometry. We could show that
the compounds downregulate c-Myc
expression and induce cell-cycle arrest
in cancer cells.
Synthesis and in Vitro Evaluation
of Novel 5-Nitroindole Derivatives
as c-Myc G-Quadruplex Binders
with Anticancer Activity