Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents

Article abstract of DOI:10.1016/j.ejmech.2016.10.026

A small library of 7-pyrrolo[3,2-f]quinolinones was obtained by introducing benzoyl, sulfonyl and carbamoyl side chains at the 3-N position, and their cytotoxicity against a panel of leukemic and solid tumor cell lines was evaluated. Most of them showed high antiproliferative activity with GI₅₀s ranging from micro-to sub-nanomolar values, and these values correlated well with the inhibitory activities of the compounds against tubulin polymerization. Based on a recently proposed colchicine bind site inhibitors (CBSIs) pharmacophore, the interactions of the novel 7-PPyQs at the colchicine domain were rationalized. The most active compounds (4a and 4b) did not induce significant cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. In particular, 4a was a potent inducer of apoptosis in both the HeLa and Jurkat cell lines. On the other hand, the sulfonyl derivative 4b exhibited a lower potency in comparison with 4a. With both compounds, induction of apoptosis was associated with dissipation of the mitochondrial transmembrane potential and production of reactive oxygen species, suggesting that cells treated with the compounds followed the intrinsic pathway of apoptosis.

Full text of DOI:10.1016/j.ejmech.2016.10.026

Accepted Manuscript
Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-
pyrroloquinolinone 3-amide derivatives as potent antimitotic agents
Davide Carta, Roberta Bortolozzi, Mattia Sturlese, Veronica Salmaso, Ernest Hamel,
Giuseppe Basso, Laura Calderan, Luigi Quintieri, Stefano Moro, Giampietro Viola,
Maria Grazia Ferlin
PII:
S0223-5234(16)30888-1
10.1016/j.ejmech.2016.10.026
EJMECH 8991
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 September 2016
Revised Date: 10 October 2016
Accepted Date: 13 October 2016
Please cite this article as: D. Carta, R. Bortolozzi, M. Sturlese, V. Salmaso, E. Hamel, G. Basso, L.
Calderan, L. Quintieri, S. Moro, G. Viola, M.G. Ferlin, Synthesis, structure-activity relationships and
biological evaluation of 7-phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.026.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis, structure-activity relationships and biological evaluation of 7-phenyl-
pyrroloquinolinone 3-amide derivatives as potent antimitotic agents.
1
2
1
1
3
Davide Carta , Roberta Bortolozzi , Mattia Sturlese , Veronica Salmaso , Ernest Hamel , Giuseppe
2
1
1
1
2
Basso , Laura Calderan , Luigi Quintieri , Stefano Moro , Giampietro Viola , Maria Grazia
1
*
Ferlin .

ACCEPTED MANUSCRIPT
Synthesis, structure-activity relationships and biological evaluation of 7-
phenyl-pyrroloquinolinone 3-amide derivatives as potent antimitotic agents
1
2
1
1
3
Davide Carta , Roberta Bortolozzi , Mattia Sturlese , Veronica Salmaso , Ernest Hamel ,
2
1
1
1
2
Giuseppe Basso , Laura Calderan , Luigi Quintieri , Stefano Moro , Giampietro Viola , Maria
1
*
Grazia Ferlin
1
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131
Padova, Italy.
2
Department of Woman’s and Child’s Health, University of Padova, Laboratory of
Oncohematology, 35128 Padova, Italy.
3
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, Frederick National Laboratory for Cancer Research, National
Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.
1

ACCEPTED MANUSCRIPT
Abstract
A small library of 7-pyrrolo[3,2-f]quinolinones was obtained by introducing benzoyl, sulfonyl
and carbamoyl side chains at the 3-N position, and their cytotoxicity against a panel of
leukemic and solid tumor cell lines was evaluated. Most of them showed high
antiproliferative activity with GI50s ranging from micro- to sub-nanomolar values, and these
values correlated well with the inhibitory activities of the compounds against tubulin
polymerization. Based on a recently proposed colchicine bind site inhibitors (CBSIs)
pharmacophore, the interactions of the novel 7-PPyQs at the colchicine domain were
rationalized. The most active compounds (4a and 4b) did not induce significant cell death in
normal human lymphocytes, suggesting that the compounds may be selective against cancer
cells. In particular, 4a was a potent inducer of apoptosis in both the HeLa and Jurkat cell
lines. On the other hand, the sulfonyl derivative 4b exhibited a lower potency in comparison
with 4a. With both compounds, induction of apoptosis was associated with dissipation of the
mitochondrial transmembrane potential and production of reactive oxygen species, suggesting
that cells treated with the compounds followed the intrinsic pathway of apoptosis.
Keywords. Microtubules, phenylpyrroloquinolinone, tubulin, apoptosis, molecular docking,
structure-activity relationships.
2

ACCEPTED MANUSCRIPT
1. Introduction
Drugs interfering with microtubules (MTs) represent a class of compounds of great interest in
the area of anticancer therapy. MTs are an essential component of the cellular cytoskeleton, as
they regulate and participate in a variety of cellular functions that include motility,
morphology, intracellular transport, signal transduction, and cell division [1]. MTs are
composed of α/β tubulin heterodimers that have polymerized into cylindrical structures, and,
therefore, both natural and synthetic agents able to interfere with tubulin polymerization or
depolymerization, thereby altering MT dynamics, continue to attract considerable attention in
the field of chemotherapeutic research [2]. Not only are there clinically used natural and
semisynthetic antimitotics (such as, paclitaxel, other taxanes, eribulin, ixabepilone and vinca
akaloids), but there is also a large number of structurally dissimilar small molecules with high
affinity for the colchicine site on tubulin. These compounds are able to inhibit the
proliferation of a wide variety of human cancer cells [3]. Moreover, these agents can also
affect the tumor endothelial vasculature required for the growth of tumor mass. These types of
tubulin inhibitors might provide new therapeutic approaches to treat cancers and overcome
limitations of existing tubulin interactive drugs [4]. In the last decade, we have been
developing phenylpyrroloquinolinone (PPyQ) derivatives that show interesting in vitro and in
vivo antitumor activity. Both 2-PPyQs and 7-PPyQs act as tubulin polymerization inhibitors
by binding at the colchicine site in β-tubulin [5,6]. Although less cytotoxic, the 2-PPyQ
compounds were also found to exhibit interesting in vitro and in vivo antiangiogenic
properties [7]. The more cytotoxic 7-PPyQ derivatives showed very remarkable in vitro
biological properties and good antitumor activity in vivo. In particular, some 7-PPyQs,
characterized by alkyl substitutions at the pyrrole nitrogen, showed increased cytotoxicity
with nanomolar GI50 values, and these compounds overcame the resistance observed with the
3

ACCEPTED MANUSCRIPT
clinically used agents vincristine and taxol [8,9]. In the latter series, the 3N-cyclopropyl
methyl 7-PPyQ derivative MG 2477 (Figure 1, 10), was taken as lead compound due to its
very strong cytotoxicity (nanomolar range GI50s) and its potent interaction with tubulin. Its
activities as an inhibitor of tubulin polymerization and of colchicine binding to tubulin were
similar to those of the reference compound combretastatin A-4: 0.90 µM assembly IC50 and
83% inhibition of colchicine binding for compound 10 versus values of 1.1 and 99%,
respectively, for CA-4 [10, 11]. Compound 10 was also demonstrated to induce autophagy in
the A549 cell line [12]. Very recently, in an effort to produce additional highly active
compounds, numerous related analogues were designed, synthesized and studied, resulting in
the discovery of a potent 3N-acyl derivative MG 2603 (Figure 1, 11) showing low nanomolar
GI50 values. Compound 11, too, showed an anti-tubulin mechanism profile similar to that of
10, but it was also able to inhibit a number of kinases involved in tumor progression.
Moreover, 11 showed reduced toxicity in non tumor cell lines and synergized with
conventional chemotherapeutic agents in inhibiting leukemia cell proliferation [13].
Driven by the SARs we have collected during the development of the PPyQs [14] (Figure 1)
and remembering the recent results with the 3N-acyl 7-PPyQ derivative, the aim of the
present work was the design, synthesis and evaluation of novel analogues substituted at the
pyrrole N with acyl, sulfonyl and carbamoyl side chains. In designing the novel derivatives,
we preserved the structural elements crucial for the best antiproliferative activity, such as the
[
3,2-f] geometry of the pyrroloquinoline core, the un-substituted phenyl ring at the 7 and the
carbonyl group at the 9 position, without any other substitutions except for the 3 position
Figure 1). Biological investigations included cellular cytotoxicity, tubulin inhibition assays
(
and an apoptosis assay, together with docking simulations in the colchicine site of β-tubulin.
4

ACCEPTED MANUSCRIPT
This allowed us to obtain more knowledge on the key substitutions at the pyrrole N for
effective interactions at the colchicine site.
2
2
. Results and discussion
.1 Chemistry
Scheme 1 shows the route to 7-PPyQs bearing a side chain bound to the pyrrole N via a
carbonyl group according to the previously reported general synthesis to 7-PPyQs [7]. The
starting commercial 5-nitroindole was reacted with various acyl and sulfonyl chlorides in
order to obtain directly the 3N-substituted indole derivatives 1a-d. Compound 1e was
prepared by means of a one-pot procedure consisting first of activation of 5-nitroindole with
p-nitrophenylchloroformate to give the reactive 3-p-nitrophenylcarbamate intermediate and
then reaction with cyclopropylamine (19% yield). The next reduction step of 5-nitro- to 5-
.
aminoindole intermediates was accomplished by a chemical procedure with SnCl 2H O, 37%
2
2
HCl in methanol at reflux to give indole derivatives 2a-d. In the case of 1e, the reduction did
not produce any corresponding amino compound. While, by a catalytic procedure with H and
2
C/Pd 10% in EtOAc/EtOH at atmospheric pressure, indoline compounds 5a, b, d and e were
obtained. Note that by the above chemical method aminoindole derivative 2a was obtained in
poor yields due to the formation of a mixture of various chloro-derivatives (not shown).
Therefore the synthesis to 4a did not proceed beyond the c step. Aminoindole derivatives 2b-
d were then condensed with ethyl benzoyl acetate to provide the eneamine intermediates 3b-d
to be then thermally cyclized into the final 7-PPyQs 4b-d. In the same way, indolines 5a, b, d
and e gave eneamine derivatives 6a, b, d and e with benzoyl acetate. However, when these
were submitted to thermal cyclization in diphenyl ether, 6a, b and d gave a mixture of
isomeric compounds angular 7a, b, d and linear 8a, b, d, whereas derivative 6e did not react.
5

ACCEPTED MANUSCRIPT
It is worth emphasizing that, by the catalytic procedure at the reaction conditions used here,
we never observed the formation either of aminoindolines or the subsequent cyclization to
linear tricyclic compounds.
Scheme 2 shows an alternative method to obtain the final compounds 4a and 4e, which could
not be prepared by the route shown in the Scheme 1. The previously described 7-PPyQ 9 [8],
available in our laboratory, was submitted to an acylation reaction with benzoyl chloride.
After a laborious purification procedure, 7-PPyQ 4a was obtained in a 37% yield. Compound
4e was obtained by the same one pot procedure described above, consisting of the reaction of
7-PPyQ 9 to give the intermediate 3-p-nitrophenylcarbamate, which was not isolated,
followed by reaction with cyclopropylamine (30% yield).
2.2 Biological evaluation
2.2.1 In vitro antiproliferative activities and SAR analysis.
On the basis of previous biological activity data on 7-PPyQs and docking simulations of 11
into the colchicine site of tubulin [13], the new compounds were designed to obtain additional
SAR information by modifying the nature and size of substituents at the 3 position of the 7-
PPyQ tricycle. Evaluation of antiproliferative activities of 4a-e, 7a,b, 7d, and 8a,b was
performed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay against a panel of 11 human tumor cell lines (CCRF-CEM, HL-60, RS4;11, Jurkat,
SEM, MV4;11, THP-1, HeLa, A549, HT-29, MCF-7). GI50 values, the concentrations that
inhibit cell growth by 50%, are presented in Table 1. Most of the novel 7-PPyQs possessed
antiproliferative activity, inhibiting cell growth with nanomolar to micromolar GI50 values,
except for the linear 1,2-dihydro 8b (GI50>10000 nM) having a methanesulfonyl group at the
3
position. In contrast, its angular isomer 7b showed GI50 values in a high nanomolar range,
6

ACCEPTED MANUSCRIPT
demonstrating that for partially hydrogenated pyrroloquinolinones the [3,2-f] geometry is also
preferred for cytotoxic activity as it was for fully aromatic compounds. Comparable behavior
was also observed for the benzamidic derivatives 7a and 8a, although not as dramatic: the
linear 8a showed GI50 values in the micromolar, while the angular compound 7b had IC50
values in the sub-micromolar range. Moreover, from the data presented in Table 1, it is
evident that the angular, fully aromatic 7-PPyQs 4a,b,d were more cytotoxic than the
corresponding hydrogenated analogues 7a,b,d. This was most remarkable for the pair 4a and
7a, with the former having GI50 values in the 0.1-10 nM range and the latter GI50 values in the
250-2650 nM range. Of particular note were the low nanomolar and sub-nanomolar GI50
values obtained with the series 4a-4e, in both the leukemic and solid tumor cell lines. Overall,
the most active of the new compounds was the benzamidic derivative 4a, with subnanomolar
concentrations in five of the eleven cell lines, with slightly lower GI50 values in the solid
tumor lines (GI50s 0.2, 0.1 and 0.2 nM in the HeLa, HT-29 and MCF-7 cells, respectively).
Previously, we had observed that 7-PPyQs were more cytotoxic against leukemic cells. Thus,
the preferential activity of 4a against solid tumor cell lines is worth emphasizing. The same
relative activity against the solid tumor cell lines was also observed for sulfamidic derivatives
4b-d, but not for the ureidic derivative 4e. We also note that there did not appear to be a
significant steric hindrance factor among the compounds evaluated here, with similar
antiproliferative activities observed among the series 4a-4e and the previously evaluated
compound 11.
We conclude that the 7-PPyQ derivatives 4a-e, chemically modified at 3 position with
substitutions such as carbonyl, sulfonyl and carbamoyl groups, maintained strong cytotoxicity
against tumor cell lines. These substitutions were made to further explore the SARs, and they
have confirmed what was observed previously with carbonyl compound 11. Substitutions
7

ACCEPTED MANUSCRIPT
with oxygenated groups, although of diverse nature and volume, confer very high
antiproliferative activity on 7-PPyQs. Due to their broad spectrum of potent activity, 4a and
4b were selected for further biological investigations on mechanism of action.
2.2.2 Evaluation of cytotoxicity in human non-cancer cells.
To obtain a preliminary indication of the cytotoxic potential of these derivatives in normal
human cells, the two most active compounds (4a and 4b) were evaluated in vitro against
peripheral blood lymphocytes (PBL) from healthy donors (Table 2). Compound 4a showed a
GI50 of 28 µM in quiescent lymphocytes, while in the presence of the mitogenic stimulus
phytohematoaglutinin (PHA), the GI50 decreased to about 15 µM. Notably, this value was
almost 1000-2000 times higher than that observed against the lymphoblastic cell lines CCRF-
CEM and Jurkat. These results indicate that 4a has a significant effect in rapidly proliferating
cells but not in quiescent cells, as previously observed for other antimitotic derivatives
developed by our group [13]. Compound 4b was completely inactive in both quiescent and
proliferating lymphocytes.
2.2.3 Inhibition of tubulin polymerization and colchicine binding.
To evaluate the tubulin interaction properties of compounds 4a-e, we investigated their effects
3
on inhibition of tubulin polymerization and the binding of [ H]colchicine to tubulin (Table 3)
[
15,16]. For comparison, CA-4 and 3c were examined in contemporaneous experiments as
references compounds. Among the test compounds, 4a strongly inhibited tubulin assembly
assay with an IC50 of 0.89 µM, a value that was lower than that obtained for the reference
compound CA-4 (IC50=1.2 µM). Compounds 4b and 4c showed an IC50 similar to that of CA-
4
while 4d and 4e were less effective than the reference compound (IC =2.2-2.4 µM). These
50
8

ACCEPTED MANUSCRIPT
results correlate well with the growth inhibitory effects exhibited by the test compounds,
indicating that their antiproliferative activity derives from an interaction with tubulin.
In the colchicine studies, compound 4a was the most active inhibitor of the binding of
3
[
H]colchicine to its domain on tubulin, with 70% inhibition occurring with this derivative at
5
µM. Nevertheless, 4a was less potent than CA-4 in this assay. In these experiments CA-4
inhibited colchicine binding by 98% at 5 µM. The other investigated compounds (4b-e)
showed weaker inhibitory activity, with less than 50% inhibition of colchicine binding to
tubulin.
2.2.4 Computational studies
Docking studies were carried out to investigate the binding mode of the novel inhibitors with
the aim of interpreting experimental affinity data. A relevant number of experimentally
derived complex structures of colchicine binding site inhibitors (CBSI) were recently
deposited in the Protein Data Bank (PDB) [17]. Interestingly, the superposition of the
different crystal structures reveals a significant variability in the sidechains of the residues
belonging to the colchicine site depending on the chemical nature of the ligand, as shown in
SI_Figure 1 (see Supplementary Material). Moreover, the resolution of the crystal structures
spanned a broad range (2.19-3.75 Å). As a consequence of this heterogeneity, we carried out a
benchmark study, using the DockBench tool [18], to identify the most accurate docking
model among 14 different ones and to determine which protein conformation was most
appropriate to model our analogues. The benchmark study on the self-docking procedure was
performed on 14 tubulin–CBSI complexes from the PDB as listed in table SI_Table 1. The
benchmark results, summarized in SI_Figure 2, revealed that several protocols showed a good
ability to reproduce the experimental complex geometries for most of the experimental
9

ACCEPTED MANUSCRIPT
structures. Among them, GOLD software coupled to the PLP/goldscore/chemscore scoring
function, gave the best results. In particular, GOLD protocols returned accurate predictions
for the complete dataset. As a consequence of the overall good performance of the
benchmark, we focused our attention on the identification of the most suitable
crystallographic protein structure for the docking simulation of the PPyQ class of compounds.
This step was crucial because of the variety of different sidechain orientations for certain
residues in the colchicine site (such as βGlu200, βCys239, βLeu248, and βLeu255, as shown
in SI_Figure 1. We have addressed this critical issue by comparing the shape similarity and
the pharmacophoric determinants conservation between the ligands present in the complexes
(summarized in SI_Table 1) and our representative compound, 4a. Plinabulin (PDB ID:
5C8Y) showed the highest shape similarity according Tversky coefficient ( > 0.7 ) and, more
notably, the plinabulin key moieties for the tubulin interaction are nicely conserved as shown
in Figure 2, Panel A. Not surprisingly, all analogues showed a common binding mode similar
to that of plinabulin, as shown by SI_Video 1. As depicted in Figure 2 (Panel B), the
diketopiperazinic core of plinabulin is mimicked by the pyrroloquinolinone core, maintaining
the key hydrogen bond interaction with the backbone of βVal236 as anticipated by the shape-
based superposition (plinabulin and 4a, Figure 2, Panel A). In addition to the conserved
hydrogen bond, the pyrroloquinolinone scaffold guarantees strong hydrophobic interactions
with βLeu253, βAla314 and βIle368. The phenyl ring in position 7 reproduced the same
scheme of interaction to the benzylidene moiety of plinabulin through hydrophobic
interactions with residues βPhe167, βTyr200, and βLeu250. The substituents at the N-pyrrole
were placed in the pocket formed by residues: βLys350, βThr351, βAla314, βAla352 and, for
the more bulky substituents, also αThr179. Notably, this binding mode is compatible with a
competitive mechanism of action at the colchicine site. The 1,2-dihydro derivatives showed
1
0

ACCEPTED MANUSCRIPT
minor differences in their orientation. The main difference is the reduction in the interaction
strength with βVal236, in particular for the linear isomers (8a and 8b) (SI_Video 1).
To evaluate if molecular docking was also able to explain the differences in the inhibition of
colchicine binding by compounds 4a-e, we performed a per-residue analysis along the series,
in which the contribution for each residue belonging to the binding site is computed for each
synthesized compound (Figure 2, Panel C). In particular, we measured the electrostatic
interaction energy and score, taking into account hydrophobic interactions. In SI_Video 1 is
reported the per-residue analysis for all the analogs in Table 1, including the reference
compound 11. The resulting heatmap suggests a very similar pattern of interaction for all the
compounds. In this context, the narrow differences in colchicine binding and tubulin
assembly inhibition are difficult to rationalize. The higher inhibition in the colchicine binding
of 4a among the 4a-4e derivatives could be ascribed to the orientation of the benzamidic
moiety that is directed between the βLys350 and αThr179 residues, while in the 4b-4e
analogues the N substituent assumes a slightly different orientation. The differences in the cell
growth inhibition data are difficult to rationalize only with tubulin docking, possibly because
of the involvement of other targets [13]. A clearer interpretation can be made for compound
8b, since its inhibition of tumor cell growth was negligible, and this analogue showed poor
scores in molecular docking primarily because of a poor interaction with βVal236, as shown
in the per residue analysis (SI_Video 1).
2.2.5 Compounds 4a and 4b induce mitotic arrest of the cell cycle
To investigate whether compounds 4a and 4b affected cell cycle progression, we evaluated by
flow cytometry the effect of different concentrations of compounds after a 24 h of treatment
1
1

ACCEPTED MANUSCRIPT
of HeLa and Jurkat cells. As shown in Figure 3, compound 4a caused a significant G2/M
arrest in a concentration-dependent manner in both cell lines, with a rise in G2/M cells
occurring at a concentration of 50 nM, while at the highest concentration (100 nM) more than
50% of the cells were arrested in G2/M. In the HeLa cells, the G2/M block was accompanied
by a significant reduction of both G1 and S phase cells, suggesting that cell proliferation is
impaired. For compound 4b, we observed a similar behavior but less marked as compared
with 4a, in good agreement with the respective IC50 values found in the tubulin
polymerization assay.
2.2.6 Compounds 4a and 4b induce apoptosis through the mitochondrial death pathway
To evaluate the mode of cell death induced by 4a and 4b, we performed the annexin-
V/propidium iodide (PI) assay by flow cytometry. Staining with annexin-V and with PI
allows discrimination between live cells (annexin-V-/PI-), early apoptotic cells (annexin-
V+/PI-), late apoptotic cells (annexin-V+/PI+) and necrotic cells (annexin-V-/PI+). The
experiments were carried out both in Hela and Jurkat cells. As shown in Figures 4 and 5,
compounds 4a and 4b induce a significant time- and concentration-dependent increase of
apoptotic cells in both cell lines. In particular, after 24 h we found a significant accumulation
of early apoptotic (annexin-V+/PI-) cells starting from the lower concentrations and an
increase of late apoptotic cells (annexin-V+/PI+) after 48 h treatments, indicating that the
compounds trigger cells to a massive apoptotic cell death. In good agreement with the
respective GI50 values (Table 1), compound 4a is the more potent inducer of apoptosis both in
Hela and Jurkat cells at the 50 nM concentration.
1
2

ACCEPTED MANUSCRIPT
Loss of mitochondrial transmembrane potential (ꢀψmt) and release of apoptogenic factor has
been described as an early event in the apoptotic process [19,20]. ∆ψmt was evaluated by flow
cytometry using the fluorescence of the dye JC-1. In normal conditions (high ∆ψmt), JC-1
displays a red fluorescence (590 nm), while mitochondrial depolarization is indicated by a
shift to green fluorescence (525 nm).
In both Hela and Jurkat cells, treatment with 4a and 4b induced a marked increase in the
percentage of cells with low ∆ψmt (Figure 6), and this occurred in a time- and concentration-
dependent fashion. Depolarization of mitochondrial potential leads to the induction of the
intrinsic pathway of apoptosis and is associated with the appearance of annexin-V positivity
in the treated cells, as shown above and indicating the cells are in an early apoptotic stage. In
fact, the disruption of ∆ψmt and the intrinsic activation of apoptosis are characteristic of
antimitotic drugs and have been observed with both microtubule stabilizing and destabilizing
agents in different cell types. It is also well known that mitochondrial potential impairment
and the resulting damage to mitochondrial function induce generation of reactive oxygen
species (ROS) [21,22]. Superoxide anion is produced by mitochondria due to a shift from the
2
normal 4-electron reduction of O to a 1-electron reduction when cytochrome c is released
from mitochondria upon apoptosis [23, 24].
2
Using dichlorodihydrofluorescein diacetate (H -DCFDA), which is oxidized to the
fluorescent compound dichlorofluorescein (DCF) upon ROS induction [23], we measured
ROS production after treatment with compounds 4a and 4b. As shown in Figure 7 (Panels B
and D), the two compounds induced the production of large amounts of ROS in comparison
with control cells. This was observed in both the Jurkat and HeLa cells, in good agreement
with the dissipation of ∆ψmt described above.
1
3

ACCEPTED MANUSCRIPT
2.2.7 Metabolic stability of 4a in human liver microsomes.
Liver microsomal oxidation and hydrolysis represent major routes of drug metabolism in
mammals, including humans [25]. In vitro studies were therefore carried out to get
preliminary information on the stability of compound 4a to oxidative and hydrolytic
metabolism by human liver microsomes. As shown in Figure 8 (panel A), compound 4a (10
µM) was relatively stable in human liver microsomes (1 mg/mL) with more than 60%
compound remaining after 60 min incubation at 37°C. Interestingly, compound 4a
disappearance was not influenced by the presence of NADPH (Figure 8, panel A), a cofactor
for both cytochrome P450- and flavin monooxygenase-mediated oxidations [25], and was
accompanied by formation of a fluorescent metabolite whose retention time corresponded
exactly to that of authentic compound 9 (panel B). Collectively, these findings indicate that
compound 4a is partially susceptible to microsomal enzyme hydrolysis and that this
catabolism produce compound 9 which retain a significant antiproliferative activity as
previously demonstrated [8].
3. Conclusion
With the aim to further explore the SARs of the 7-PPyQ class of compounds, we examined
the effects of various oxygenated functionalities at the 3N position. A small series of novel
derivatives was synthesized, and their antiproliferative activities and with their mechanism of
action were investigated. The chemical series included both angular and linear compounds
and fully aromatic and partially hydrogenated derivatives. Most compounds had significant
antiproliferative activity, inhibiting cell growth with nanomolar to micromolar GI50 values,
thus confirming that, in general, oxygenated substitutions at the 3 position improve
cytotoxicity. In the series, the [3,2-f] angular geometry once again was required for obtaining
1
4

ACCEPTED MANUSCRIPT
potent cytotoxic compounds, and this [3,2-f] configuration was present both in the fully
aromatic benzoyl 4a and the methanesulfonyl 4b. Compound 4a was the most active of the
new agents, even having sub-nanomolar GI50s in some of the cell lines studied. Thus,
compound 4a was even more cytotoxic than the previously described compound 11.
Moreover, 4a had a significant effect only in rapidly proliferating cells but not in quiescent
and proliferating lymphocytes. Investigations on the mechanism of action of 4a confirmed
that it was a strong inhibitor of tubulin polymerization, as were previously described 7-PPyQ
derivatives. Both in the Hela and Jurkat cell lines, 4a was more effective than 4b in blocking
2
the cell cycle at the G M phase, inducing apoptosis through the mitochondrial death pathway
with production of ROS. In agreement with the experimental results obtained in this work,
docking simulations suggested that the synthesized inhibitors had high affinity for the
colchicine site of tubulin, with interactions with the binding site most similar to those
observed with the known inhibitor plinabulin. The 1,2-dihydro derivative 8b was slightly
penalized by affecting the geometry of the hydrogen bond with βVal236.
Additionally, in vitro metabolic stability studies indicated that a human liver microsomes
esterases can catalyze the cleavage of the amide bond of 4a, leading to formation of an active
metabolite, namely compound 9. These findings may be valuable for future in vivo studies.
4. Experimental section
4.1 Chemistry
Melting points were determined on a Buchi M-560 capillary melting point apparatus and are
uncorrected. Infrared spectra were recorded on a PerkinElmer 1760 FTIR spectrometer with
-1
KBr pressed disks and on a Varian ATR FTIR; all values are expressed in cm . UV−vis
1
5

ACCEPTED MANUSCRIPT
1
spectra were recorded on a Thermo Helyos α spectrometer. H NMR spectra were determined
on Bruker 300 and 400 MHz spectrometers, with the solvents indicated; chemical shifts are
reported in δ (ppm) downfield from tetramethylsilane as internal reference. Coupling
constants are given in Hertz. In the case of multiplets, chemical shifts were measured starting
from the approximate center. Integrals were satisfactorily in line with those expected on the
basis of compound structure. Elemental analyses were performed in the Microanalytical
Laboratory, Department of Pharmaceutical Sciences, University of Padova, on a PerkinElmer
C, H, N elemental analyzer model 240B, and analyses indicated by the symbols of the
elements were within ±0.4% of the theoretical values. Analytical data are presented in detail
for each final compound in the Supporting Information. Mass spectra were obtained on a Mat
112 Varian Mat Bremen (70 eV) mass spectrometer and on an Applied Biosystems Mariner
System 5220 LC/MS (nozzle potential 140 eV). Column flash chromatography was
performed on Merck silica gel (250−400 mesh ASTM); chemical reactions were monitored
by analytical thin-layer chromatography (TLC) on Merck silica gel 60 F- 254 glass plates.
Solutions were concentrated on a rotary evaporator under reduced pressure. Starting materials
were purchased from Sigma-Aldrich and Alfa Aesar, and solvents were from Carlo Erba,
Fluka and Lab-Scan. DMSO was obtained anhydrous by distillation under vacuum and stored
on molecular sieves.
The purity of new tested compounds was checked by HPLC using the instrument HPLC
VARIAN ProStar model 210, with detector DAD VARIAN ProStar 335. The analysis was
performed with a flow of 1 mL/min, a C-18 column of dimensions 250 mm × 4.6 mm, a
particle size of 5 ꢁm, and a loop of 10 ꢁL. The detector was set at 300 nm. The mobile phase
2
consisted of phase A (Milli-Q H O, 18.0 Mꢂ, TFA 0.05%) and phase B (95% MeCN, 5%
1
6

ACCEPTED MANUSCRIPT
phase A). Gradient elution was performed as reported: 0 min, % B = 10; 0−20 min, % B = 90;
25 min, % B = 90; 26 min, % B = 10; 31 min, % B = 10.
4.1.1 General procedure for the synthesis of 1N-substituted nitroindoles (1a−e)
As a typical procedure, the synthesis of 1-benzoyl-5-nitro-1H-indole 1a is described in detail.
Into a two-necked 50 mL round-bottomed flask, 0.666 g (27.7 mmol, 3 eq.) of NaH, 60%
dispersion in mineral oil, was placed and washed with toluene (3 × 10 mL). With stirring, a
solution of commercial 5-nitroindole, 1.50 g (9.25 mmol, 1 eq.) in 5 mL of anhydrous DMF,
was dropped into the flask, and the initial yellow color changed to red with the formation of
H gas. After 30 min at room temperature, a solution of benzoyl chloride, 3.21 mL (27.7
2
mmol, d=1.21 g/mL, 3 eq.) in 3 mL dry DMF, was added, and the reaction mixture was
stirred for 2 h. The reaction was monitored by TLC analysis (eluent toluene/n-Hex/EtOAc,
1:1:1). At the end of the reaction, 25 mL of water was added, and the solvent was evaporated
under reduced pressure, leaving a residue, which was extracted with EtOAc (3 × 50 mL). The
organic phase, washed with water and dried over anhydrous Na SO , was concentrated under
2
4
vacuum giving a crude yellow solid (2.446 g). This crude product was purified with a silica
gel chromatographic column (3 x 35 cm, 230−400 mesh, eluent toluene/n-Hex/EtOAc, 1:1:1,
yielding 2.345 g of a pure yellow solid.
4
.1.1.1 1-Benzoyl-5-nitro-1H-indole (1a). Yield: 94.9%. R = 0.83 (eluent toluene/n-
f
1
Hex/EtOAc, 1:1:1); H NMR (400 MHz, DMSO-d ) δ = 8.67 (d, J=2.25 Hz, 1H, H-4), 8.43
6
(
d, J=9.12 Hz, 1H, H-7), 7.74 (m, 1H, H-4’), 8.28 (dd, J=9.12 Hz and 2.37 Hz, 1H, H-6), 7.82
m, 2H, H-2’ and -H-6’), 7.64 (m, 2H, H-3’ and H-5’), 7.62 (d, J=3.64 Hz, 1H, H-2), 7.00
(
13
ppm (dd, J=3.78 Hz and J=0.60 Hz, 1H, H-3); C NMR (75 MHz, DMSO-d
6
) δ = 109.74 (C-
3),113.19 (C-6), 117.05 (C-7), 120.55 (C-4), 129.69 (C-2’ and C-6’), 130.27 (C-3’ and C-5’),
1
7

ACCEPTED MANUSCRIPT
1
1
2
31.56 (C-4’), 132.39 (C-2), 133.55 (C-3a), 133.94 (C-7a), 139.43 (C-1’), 144.66 (C-5),
+
+
69.24 ppm (C=O); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C H N O ,
15
11
2
3
67.0779; found, 267.0787.
4.1.1.2 1-Methanesulfonyl-5-nitro-1H-indole (1b). Compound 1b was prepared as for
compound 1a by reacting 0.888 g of NaH 60% (37.03 mmol, 3 eq.) and 2 g (12.34 m mol) of
-nitroindole dissolved in 5 mL of DMF and 2.86 mL of methanesulfonyl chloride (37.03
5
mmol, d=1.48 g/mL, 3 eq.). Reaction time: 2 h (TLC, eluent EtOAc/n-Hex, 2:1). 2.564 g of a
solid bright yellow solid was obtained, and this was used in the next synthetic step without
1
further purification. Yield: 91.9%. R
f
= 0.54 (eluent EtOAc/n-Hex, 2:1); H NMR (400 MHz,
DMSO-d ) δ = 8.67 (d, J=2.20 Hz, 1H, H-4), 8.25 (dd, J=9.14 Hz and J=2.30 Hz, 1H, H-6),
6
8.05 (d, J=9.16 Hz, 1H, H-7), 7.85 (d, J=3.68 Hz, 1H, H-2), 7.08 (dd, J=7.08 Hz and J=0.72
1
3
Hz, 1H, H-3), 3.60 ppm (s, 3H, SO
2
CH
3
); C NMR (101 MHz, DMSO-d
6
) δ = 42.07
2 3
(SO CH ), 109.22 (C-3), 114.08 (C-7), 119.81 (C-6), 130.33 (C-2), 130.58 (C-3a), 137.71 (C-
+
+
7
2
a), 143.96 ppm (C-5); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C
9
H
9
N
2
O
4
S ,
41.0283; found, 241.0236.
4.1.1.3 1-(4-methylbenzenesulfonyl)-5-nitro-1H-indole (1c). Compound 1c was prepared as
for compound 1a by reacting 0.444 g of NaH 60% (18.51 mmol, 3 eq.) and 1 g (6.17 mmol)
of 5-nitroindole dissolved in 5 mL of DMF and 5.53 g of p-toluenesulfonyl chloride (18.51
mmol, 3 eq.). Reaction time: 2 h (TLC, eluent n-Hex/EtOAc, 9:1). 1.786 g of a solid bright
orange solid was obtained which was used in the next synthetic step without any additional
1
further purification. Yield: 91.6%. R
DMSO-d
f
= 0.76 (eluent EtOAc/n-Hex, 9:1); H NMR (400 MHz,
6
) δ = 8.59 (d, J=1.88 Hz, 1H, H-4), 8.21 (dd, J=9.18 Hz and J=2.26 Hz, 1H, H-6),
1
8

ACCEPTED MANUSCRIPT
8.15 (dt, J=9.18 Hz and J=0.98 Hz, 1H, H-7), 8.07 (d, J=3.72 Hz, 1H, H-2), 7.94 (m,
AA’BB’, J=8.44 Hz, J=2.11 Hz and J=1.83 Hz, 2H, H-2’ and H-6’), 7.43 (m, AA’BB’,
J=8.58 Hz and J=0.62 Hz, 2H, H-3’ and H-5’), 7.07 (dd, J=3.70 Hz and J=0.66 Hz), 1H, H-3),
1
3
2
1
3
3 6 3
.23 ppm (s, 3H, -CH ); C NMR (75 MHz, DMSO-d ) δ = 21.91 (-CH ), 110.93 (C-3),
14.54 (C-7), 118.85 (C-4), 120.55 (C-6), 127.77 (C-2’ and C-6’), 130.90 (C-2), 131.34 (C-
a), 131.37 (C-3’ and C-5’), 134.59 (C-4’), 137.81 (C-7a), 144.61 (C-5), 147.09 ppm (C-1’);
+
+
HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C H N O S , 317.0596; found,
15
13
2
4
317.0585.
4.1.1.4 5-Nitro-1-[4-(trifluoromethyl)benzenesulfonyl]-1H-indole (1d). Compound 1d was
prepared as described for compound 1a by reacting 0.670 g of NaH 60% (27.75 mmol, 3 eq.)
and 1.5 g (9.25 mmol) of 5-nitroindole dissolved in 5 mL of DMF and 3.39 g of 4-
(trifluoromethyl)benzenesulfonyl chloride (13.87 mmol, 1.5 eq.). Reaction time: 1 h (TLC,
eluent n-Hex/EtOAc, 9:1). 2.845 g of a solid bright yellowish powdery solid was obtained,
f
and this was used in the next synthetic step without further purification. Yield: 82.7%. R =
1
0
.79 (eluent EtOAc/n-Hex/toluene, 1:1:1); H NMR (400 MHz, DMSO-d
6
) δ = 8.60 (d,
J=2.12 Hz, 1H, H-4), 8.29 (m, AA’BB’, J=8.28 Hz, 2H, H-2’ and H-6’), 8.22 (dd, J=9.20 Hz
and J=2.16 Hz, 1H, H-6), 8.18 (d, J=9.16 Hz, 1H, H-7), 8.13 (d, J=3.72 Hz, 1H, H-2), 8.01
1
3
(
m, AA’BB’, J=8.44 Hz, 2H, H-3’ and H-5’), 7.13 ppm (d, J=3.72 Hz, 1H, H-1); C NMR
75 MHz, DMSO-d ) δ = 111.29 (C-3), 114.17 (C-7), 118.59 (C-4), 120.49 (C-6), 123.41 (q,
), 127.8250 (q, J=3.65 Hz, C-3’ and C-5’), 128.46 (C-2’ and C-6’), 130.44
C-2), 131.12 (C-3a), 134.84 (q, J=32.69 Hz, C-4’), 137.42 (C-7a), 140.53 (C-1’), 144.46
(
6
J=273.30 Hz, -CF
3
(
+
+
10 3 2 4
ppm (C-5); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C15H F N O S , 371.0313;
found, 371.0309.
1
9

ACCEPTED MANUSCRIPT
4.1.1.5 N-cyclopropyl-5-nitro-1H-indole-1-carboxamide (1e). To a stirred slurry of NaH
(
0.630 g of a 60% mineral oil dispersion, 26.27 mmol, 3 eq.) in THF (25 mL) at 0 °C, under
, was cautiously added 5-nitroindole (1.42 g, 8.75 mmol, 1eq.) previously dissolved in THF
5 mL). The reaction mixture was stirred at 0 °C for 60 min, then transferred, via cannula, to a
N
2
(
solution of 4-nitrophenyl chloroformate (2.11 g, 10.5 mmol, 1.2 eq.) in THF (8.5 mL). The
resultant reaction mixture was stirred at ambient temperature for 15 h (TLC, eluent n-
Hex/EtOAc, 2:1), prior to removal of the solvent by concentration in vacuo. The residue
2
obtained was suspended in EtOAc (100 mL), then filtered and washed with EtOAc and Et O
to give 2.515 g (87.6%) of a pale yellow solid. The resulting activated carbamate 5-nitro-1-(4-
nitrophenoxycarbonyl)indole was immediately used as follows: a 2.0 M solution of
cyclopropylamine (0.382 mL, 5.48 mmol, d=0.824 g/mL, 8 eq.) in THF (2.75 mL) was added
to a solution of 5-nitro-1-(4-nitrophenoxycarbonyl)indole (0.245 g, 0.686 mmol, 1eq.) in THF
(5 mL). The resultant reaction mixture was stirred at ambient temperature for 4 h and
monitored by TLC (eluent n-Hex/EtOAc, 2:1), prior to removal of the solvent by
concentration to dryness in vacuo. The residue obtained was partitioned between EtOAc (100
mL) and H O (100 mL). The layers were separated, and the aqueous phase was extracted with
2
EtOAc (2 x 30 mL). The combined organic extracts were washed with sat’d NaHCO
mL), brine and finally dried over NaSO and concentrated in vacuo to give a yellow solid
which was suspended in Et O (35 mL), filtered and washed with Et
.066 g of a pale yellow solid. Yield: 39.1%. R
3
(100
4
2
2
O (2 x 20 mL) to give
1
0
f
= 0.26 (eluent n-Hex/EtOAc, 2:1); H NMR
(
400 MHz, DMSO-d ) δ= 8.59 (d, J=2.25 Hz, 1H, H-4), 8.50 (d, J=2.31 Hz, 1H, NH), 8.39 (d,
6
J=9.18 Hz, 1H, H-7), 8.15 (dd, J=9.21 Hz and J=2.40 Hz, 1H, H-6), 8.02 (d, J=3.89 Hz, 1H,
H-2), 6.92 (dd, J=3.69 Hz and J=0.55, 1H, H-3), 2.80 (sex J=3.12 Hz, 1H, NH-CH), 0.8-0.6
2
0

ACCEPTED MANUSCRIPT
1
3
ppm (m, 4H, -CH
2 2 6 2 2
CH -); C NMR (75 MHz, DMSO-d ) δ = 6.75 (-CH CH -), 22.75 (NCH-
CH CH -), 104.32 (C-3), 112.16 (C-7), 116.74 (C-6), 117.62 (C-4), 127.30 (C-2), 129.55 (C-
2
2
+
3
a),135.38 (C-7a), 140.95 (C-5), 160.11 ppm (C=O); HRMS (ESI-MS, 140 eV): m/z [M+H ]
+
calculated for C12
H
12
N
3
O
3
, 246.0879; found, 246.0871.
4.1.2 General procedure for the synthesis of 1N-substituted aminoindoles 2a−d
As a typical procedure, the synthesis of 1-benzoyl-5-amino-1H-indole 2a is described in
detail. Into a two-necked 50 mL round-bottomed flask, 3.241 g of 1-benzoyl-5-nitro-1H-
indole (1a) (12.17 mmol, 1 eq.), 10.986 g of SnCl
2 2
·2H O (48.68 mmol, 4 eq.), 2 mL of HCl
37% and 30 mL of methanol were added. The reaction mixture was refluxed for 3 h, and the
reaction progress was monitored by TLC (n-Hex/EtOAc, 1:1). At the end, the solvent was
evaporated, the residue was taken up with aqueous NaOH 20% (20 mL), and the resulting
suspension was extracted with diethyl ether (4 × 50 mL). The combined extracts, washed with
2 4
brine and treated with anhydrous Na SO , were evaporated to dryness on a rotary evaporator
to yield 1.536 g of a semisolid yellow product, made up of three different reaction products.
In order to obtain the desired pure 1-benzoyl-5-amino-1H-indole, the raw powder was
purified in a silica gel chromatographic column ( 3 x 28 cm, 230−400 mesh, eluent n-
Hex/EtOAc, 1:1, yielding 0.267 g of a pure yellow solid.
f
4.1.2.1 1-benzoyl-5-amino-1H-indole (2a). Yield: 19.5%. R = 0.33 (eluent n-Hex/EtOAc,
1
1
:1); H NMR (400 MHz, DMSO-d
6
) δ = 7.98 (d, J=8.73 Hz, 1H, H-7), 7.70 (m, 2H, H-2’ and
H-6’), 7.66 (m, 1H, H-4’), 7.59 (m, 2H, H-3’ and H-5’), 7.16 (d, J=3.72 Hz, 1H, H-3), 6.75
(d, J=2.07 Hz, 1H, H-4), 6.65 (dd, J=8.74 Hz and J=2.20 Hz, 1H, H-6), 6.51 (d, J=3.42Hz,
13
1
2 6
H, H-2), 5.05 ppm (s, 2H, NH ); C NMR (75 MHz, DMSO-d ) δ = 101.99 (C-3), 113.14
2
1

ACCEPTED MANUSCRIPT
(
(
(
C-7), 115.82 (C-6), 118.34 (C-4), 128.44 (C-2’ and C-6’), 129.04 (C-3’ and C-5’), 129.47
C-4’), 131.83 (C-1’), 133.84 (C-2), 134.72 (C-3a), 137.46 (C-7a), 144.04 (C-5), 172.01 ppm
+
+
13 2
C=O); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C15H N O , 237.1028; found,
237.1031.
4.1.2.2 1-methanesulfonyl-5-amino-1H-indole (2b). Compound 2b was prepared as described
for compound 2a by reacting 1 g (4.16 mmol, 1 eq.) of the appropriate 5-nitroindole
derivative 1b and 4.69 g of SnCl
brown solid. Yield: 80.6%; R
DMSO-d
2
·2H
2
O (20.80 mmol, 5 eq.), obtaining 0.963 g of a slightly
1
f
= 0.37 (eluent n-Hex/EtOAc, 1:1); H NMR (400 MHz,
6
) δ = 7.48 (dt, J=8.76 Hz and J=0.66 Hz, 1H, H-7), 7.34 (d, J=3.96 Hz, 1H, H-2),
.75 (dd, J=2.20 Hz and J=0.50 Hz, 1H, H-4), 6.67 (dd, J=8.76 Hz and J=2.24 Hz, 1H, H-6),
.59 (dd, J=3.64 Hz and J=0.75 Hz, 1H, H-3), 4.95 (bs, 2H, NH ), 3.24 ppm (s, 3H, SO CH );
6
6
2
2
3
1
3
C NMR (101 MHz, DMSO-d
6 2 3
) δ = 41.06 (SO CH ), 105.02 (C-4), 108.96 (C-3), 113.92 (C-
7), 114.21 (C-6), 127.28 (C-3a), 127.82 (C-2), 132.35 (C-7a), 145.95 ppm (C-5); HRMS
+
+
(
9 11 2 2
ESI-MS, 140 eV): m/z [M+H ] calculated for C H N O S , 211.0541; found, 211.0542.
4.1.2.3 1-[4-(trifluoromethyl)benzenesulfonyl]-5-amino-1H-indole (2d). Compound 2d was
prepared as described for compound 2a by reacting 1.25 g (3.37 mmol, 1 eq.) of the
appropriate 5-nitroindole derivative 1d and 3.80 g of SnCl ·2H O (16.87 mmol, 5 eq.),
= 0.30 (eluent n-
2
2
obtaining 1.160 g of a slightly brown solid. Yield: 99.9%; R
f
1
Hex/EtOAc/toluene, 1:1:1); H-NMR (400 MHz, DMSO-d
6
)= δ: 7.77 (m, J=8.40 Hz, J=1.98
and J=1.76 Hz, 2H, H-3’ and H-5’), 7.67 (d, J=8.76 Hz, 1H, H-7), 7.58 (d, J=3.60 Hz, 1H, H-
), 7.30 (m, J=8.12 Hz, 2H, H-2’ and H-6’), 6.77 (d, J=2.04 Hz, 1H, H-4), 6.73 (dd, J=8.72
Hz and J=2.09 Hz, 1H, H-6), 6.61 (dd, J=3.64 Hz and J= 0.60 Hz, 1H, H-3), 4.98 ppm (bs,
2
13
2
2 6
H, -NH ); C NMR (101 MHz, DMSO-d ) δ : 105.92 (C-4), 110.02 (C-3), 114.18 (C-7),
2
2

ACCEPTED MANUSCRIPT
114.51 (C-6), 124.55 (q, J=245.01 Hz, CF
3
), 127.00 (C-3’ and C-5’), 127.50 (C-2), 127.81
(C-3a), 130.45 (C-2’ and C-6’), 133.83 (q, J= 33.01 Hz, C-4’), 134.69 (C-7a), 143.98 (C-5),
+
+
1
3
4
45.87 ppm (C-1’); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C15
H
12
F
3
N
2
O
2
S ,
41.0572; found, 341.0569.
.1.3 General procedure for the synthesis of 1N-substituted aminoindoles 2c, 5a, 5b, 5d, 5e.
As a typical procedure, the synthesis of 1-(4-methylbenzenesulfonyl)-5-amino-1H-indole 2c
is described in detail. Into a three-necked flask of 500 mL, previously dried in an oven, about
0
.300 g of C/Pd 10% and approximately 60 mL of EtOAc were placed. After connecting the
flask to an elastomer balloon containing H gas, the mixture was stirred at room temperature
for 1 h in order to saturate the suspension of C/Pd with H . Then, 1.9 g (6.00 mmol) of the
2
2
appropriate 5-nitroindole derivative 1c in 15 mL of EtOAc was added dropwise to the
suspension, and the mixture was stirred under H at atmospheric pressure and heated by
2
means of an oil bath at 50−60 °C for 15 h, monitoring the progress of the reaction by TLC
analysis (EtOAc/n-Hex, 9:1). At the end of the reaction, the mixture was filtered, and the
solution was concentrated to dryness on a rotary evaporator to give 1.680 g of semisolid dark
purple sticky product.
f
4.1.3.1 1-(4-methylbenzenesulfonyl)-5-amino-1H-indole (2c). Yield: 97.8%. R = 0.76 (eluent
1
n-Hex/EtOAc, 9:1); H NMR (400 MHz, DMSO-d
6
) δ = 7.77 (m, AA’BB’, J=8.40 Hz, J=1.98
Hz and J=1.76 Hz, 2H, H-2’ and H‐6’), 7.67 (d, J=8.76 Hz, 1H, H-7), 7.58 (d, J=3.60 Hz, 1H,
H-2), 7.30 (m, AA’BB’, J=8.12 Hz, 2H, H-3’ and H-5’), 6.77 (d, J=2.04 Hz, 1H, H‐4), 6.73
(dd, J=8.72 Hz and J=2.09 Hz, 1H, H‐6), 6.61 (dd, J=3.64 Hz and J=0.60 Hz, 1H, H-3), 4.95
13
(
bs, 2H, -NH
2 3 6 3
), 2.26 ppm (s, 3H, -CH ); C NMR (101 MHz, DMSO-d ) δ = 21.40 (-CH ),
105.92 (C-4), 110.02 (C-3), 114.18 (C-7), 114.51 (C-6), 127.00 (C-2’ and C-6’), 127.50 (C-
2
3

ACCEPTED MANUSCRIPT
2), 127.81 (C‐3a), 130.45 (C‐3’ and C-5’), 132.27 (C-4’), 134.69 (C-7a), 143.98 (C‐5), 145.87
+
+
ppm (C‐1’); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C15
15 2 2
H N O S , 287.0854;
found, 287.0851.
4
.1.3.2 1-benzoyl-2,3-dihydro-5-amino-1H-indole (5a). Yield: 98.8%. R
f
= 0.54 (eluent n-
1
Hex/EtOAc, 5:4); H NMR (400 MHz, DMSO-d ) δ = 7.94 (d, J=8.40, 1H, H-6), 7.49 (m, 6H,
6
H-7, H-2’, H-3’, H-4’, H-5’ and H-6’), 6.48 (d, J=1.89, 1H, H-4), 4.98 (bs, 2H, -NH
2
), 3.87 (t,
-3); C NMR (101 MHz, DMSO-d ) δ =
2
), 110.98 (C-4), 113.94 (C-6), 115.98 (C-7), 128.24 (C-
13
J=8.15 Hz, 2H, H
2
-2), 2.93 ppm (t, J=8.14, 2H, H
), 51.24 (N-CH CH
2
6
29.27 (N-CH
2
CH
2
2
2’ and C-6’), 128.99 (C-3’ and C-5’), 129.32 (C-4’), 132.37 (C-3a), 134.23 (C-7a), 135.54
+
(
C-1’), 143.82 (C-5), 166.83 ppm (C=O); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated
+
for C15
H
15
N
2
O , 239.1184; found, 239.1179.
4
.1.3.3 1-methanesulfonyl-2,3-dihydro-5-amino-1H-indole (5b). Yield: 94.8%. R
f
= 0.15
) δ = 6.95 (d, J=8.48 Hz, 1H, H-7),
.50 (m, J=2.37 Hz, 1H, H-4), 6.39 (dd, J=8.50 Hz and J=2.34 Hz, 1H, H-6), 4.91 (bs, 2H, -
NH ), 3.82 (t, J=8.24 Hz, 2H, N-CH CH ), 2.96 (t, J=8.20 Hz, 2H, N-CH CH ), 2.81 ppm (s,
); C NMR (101 MHz, DMSO-d ) δ = 28.30 (N-CH CH ), 33.48 (SO CH ),
0.39 (N-CH CH ), 111.26 (C-4), 112.92 (C-6), 115.57 (C-7), 131.88 (C-3a), 133.66 (C-7a),
1
(eluent n-Hex/EtOAc, 2:1); H NMR (400 MHz, DMSO-d
6
6
2
2
2
2
2
13
3
5
1
2
H, SO
2
CH
3
6
2
2
2
3
2
2
+
+
9 13 2 2
46.17 ppm (C-5); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C H N O S ,
13.0698; found, 213.0663.
4
9
7
.1.3.4 1-[4-(trifluoromethyl)benzenesulfonyl]-2,3-dihydro-5-amino-1H-indole (5d). Yield:
1
8.2%. R
f
= 0.51 (eluent n-Hex/EtOAc/toluene, 1:1:1); H-NMR (400 MHz, DMSO-d
6
) δ =
.90 (m, J=8.98 Hz, 2H, H-3’ and H-5’), 7.40 (m, J=8.98 Hz, 2H, H-2’ and H-6’), 7.19 (d,
J=8.52 Hz, 1H, H-7), 6.41 (dd, J=8.50 Hz and J=2.26 Hz, 1H, H-6), 6.33 (d, J=2.16 Hz, 1H,
2
4

ACCEPTED MANUSCRIPT
H-4), 4.99 (s, 2H, -NH
2
), 3.85 (t, J=8.11 Hz, 2H, H
C NMR (101 MHz, DMSO-d6) δ = 28.17 (C-2), 50.69 (C-3), 111.00 (C-4), 113.13 (C-6),
16.93 (C-7), 126.27 (q, J=3.80 Hz, C-3’ and C-5’), 127.35 (q, J=269.56 Hz, CF ), 128.57 (C-
’ and C-6’), 132.44 (C-3a), 133.35 (q, J=32.32 Hz, C-4’), 134.44 (C-7a), 140.59 (C-1’),
2 2
-3), 2.56 ppm (t, J=8.10 Hz, 2H, H -2);
1
3
1
2
1
3
3
+
+
46.93 ppm (C-5).; HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C15
H
14
F
3
N
2
O
2
S ,
41.0572; found, 341.0569.
4.1.3.5 N-cyclopropyl-2,3-dihydro-5-amino-1H-indole-1-carboxamide (5e). Yield: 91.2%. R
f
1
=
0.23 (eluent n-Hex/EtOAc, 2:1); H NMR (400 MHz, DMSO-d
6
) δ= 7.56 (d, J=8.43 Hz,
1
2
1
=
H, H-7), 6.46 (m, 2H, H-4 and NH), 6.35 (dd, J=8.44 Hz and J=2.32 Hz, 1H, H-6), 4.63 (s,
H, -NH ), 3.77 (t, J=8.58 Hz, 2H, H -2), 2.99 (t, J=8.53 Hz, 2H, H -3), 2.60 (sex, J=3.46 Hz,
2
2
2
13
H, NH-CH-CH CH -), 0.6-0.4 ppm (m, 4H, -CH CH -); C NMR (101 MHz, DMSO-d ) δ
2
2
2
2
6
6.42 (-CH
2
CH
2
-), 23.47 (C-3), 27.89 (C-2), 46.89 (NH-CH-CH
2
CH
2
-), 111.27 (C-4),
112.39 (C-6), 115.04 ppm (C-7), 130.32 (C-3a), 133.83 (C-7a), 143.82 (C-5), 162.35 ppm
+
+
(
16 3
C=O); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C12H N O , 218.1293; found,
218.1245.
4.1.4 General procedure for the synthesis of acrylate derivatives 3b−d and 6a, 6b, 6d and 6e.
As a typical procedure, the synthesis of (E,Z)-Ethyl 3-(1-(methanesulfonyl)-1H-indol-5-
ylamino)-3-phenylacrylate 3b is described in detail. In a 100 mL round-bottomed flask, 1.4 g
(6.66 mmol, 1 eq.) of 1-methanesulfonyl-5-amino-1H-indole 2a in 25 mL of absolute ethanol
was condensed with 1.73 mL (9.99 mmol; d=1.11 g/mL, 1.5 eq.) of commercial ethyl
benzoylacetate and 0.5 mL of glacial acetic acid in the presence of 100 mg of Drierite
4
(anhydrous CaSO ). The mixture was refluxed for about 24 h, the reaction being monitored
2
5

ACCEPTED MANUSCRIPT
by TLC analysis (eluent n-Hex/EtOAc, 2:1). Even though the reaction was not complete after
24 h, the mixture was cooled and filtered to remove the Drierite; the resulting solution was
evaporated to dryness under vacuum and the residue (2.420 g) purified by silica gel
chromatography (3 x 35 cm, 230−400 mesh, eluent n-Hex/EtOAc, 2:1) to yield 1.54 g of a
deep yellow powdery solid.
4
6
1
.1.4.1(E,Z)-Ethyl 3-(1-(methanesulfonyl)-1H-indol-5-ylamino)-3-phenylacrylate (3b). Yield:
1
0.2%. R = 0.65 (eluent n-Hex/EtOAc, 2:1); H NMR (400 MHz, DMSO-d ) δ = 10.25 (s,
f
6
H, NH), 7.58 (d, J=8.80 Hz, 1H, H-7), 7.49 (d, J=3.68 Hz, 1H, H-2), 7.36 (m, 5H, 2’-,3’-,4’-
,
5’-,6’-H), 7.02 (d, J=2.16 Hz, 1H, H-4), 6.84 (dd, J=8.82 Hz and 2.18 Hz, 1H, H-6), 6.62 (dd,
J=3.68 Hz and J=0.72 Hz, 1H, H-3), 4.94 (s, 1H, C=C-H), 4.15 (q, J=7.09 Hz, 2H,
13
OCH
2 3 2 3 2 3
CH ), 3.38 (s, 3H, SO CH ), 1.24 ppm (t, J=7.10 Hz, 3H, OCH CH ); C NMR (101
MHz, DMSO-d ) δ = 14.76 (OCH CH ), 41.35 (SO CH ), 59.21 (OCH CH ), 90.70 (C=C-H),
6
2
3
2
3
2
3
1
6
1
09.09 (C-3), 113.26 (C-7), 115.26 (C-4), 120.62 (C-6), 127.80 (C-2), 128.45 (C-2’ and C-
’), 128.94 (C-3’ and C-5’), 130.00 (C-4’), 130.77 (C-3a), 131.07 (C-1’), 135.80 (C-7a),
36.22 (C-5), 159.49 (C=C-H), 169.45 ppm (COO-CH
2
CH
S , 385.1222; found, 385.1213.
.1.4.2 (E,Z)-Ethyl 3-(1-(p-toluenesulfonyl)-1H-indol-5-ylamino)-3-phenylacrylate (3c).
3
); HRMS (ESI-MS, 140 eV): m/z
+
+
[
21 2 4
M+H ] calculated for C20H N O
4
Compound 3c was prepared as described for compound 3b by reacting 3.177 g (11.10 mmol)
of the appropriate 5-aminoindole derivative 2c, obtaining after column chromatography 2.336
f
g of a brownish sticky semisolid product. Yield: 45.7%. R = 0.62 (eluent n-Hex/EtOAc, 2:1);
1
H NMR (400 MHz, DMSO-d
6
) δ = 10.18 (s, 1H, NH), 7.79 (m, AA’BB’, J=8.32 Hz, 2H, H-
2’ and H-6’), 7.69 (d, J=3.64 Hz, 1H, H-2), 7.65 (d, J=8.84 Hz, 1H, H-7), 7.35 (m, AA’BB’,
J= 8.61 Hz, 2H, H-3’ and H-5’), 7.31 (m, 5H, H-2’’, H-3’’,H -4’’, H-5’’ and H-6’’), 6.92 (d,
J=1.88 Hz, 1H, H-4), 6.78 (dd, J=8.61 Hz and J=1.82 Hz, 1H, H-6), 6.61 (d, J=3.68 Hz, 1H,
2
6

ACCEPTED MANUSCRIPT
2 3 3
H-3), 4.92 (s, 1H, C=C-H), 4.12 (q, J=7.14 Hz, 2H, -OCH CH ), 2.31 (s, 3H, -CH ), 1.22 ppm
13
(
t, J=7.17 Hz, 3H, -OCH CH ); C NMR (101 MHz, DMSO-d ) δ = 14.85 (-OCH CH ),
2
3
6
2
3
21.46 (-CH
3
), 59.34 (-OCH
2
3
CH ), 90.99 (C=C-H), 109.72 (C-3), 113.65 (C-7), 115.30 (C-4),
120.87 (C-6), 127.13 (C-2’ and C-6’), 128.24 (C-2), 128.53 (C-2’’ and C-6’’), 129.03 (C-3’’
and C-5’’), 130.11 (C-4’’), 130.65 (C-3’ and C-5’), 130.91 (C-3a), 131.26 (C-1’’), 134.53 (C-
’), 135.85 (C-7a), 136.69 (C-5), 145.95 (C-1’), 159.39 (C=C-H), 169.48 ppm
4
+
+
(
COOCH CH ); HRMS (ESI-MS, 140 eV): m/z [M+H ] calculated for C H N O S ,
2 3 26 25 2 4
461.1535; found, 461.1539.
4.1.4.3
(E,Z)-Ethyl-3-(1-(4-(trifluoromethyl)benzenesulfonyl)-1H-indol-5-ylamino)-3-
phenylacrylate (3d). Compound 3c was prepared as described for compound 3b by reacting
.160 g (3.41 mmol) of the appropriate 5-aminoindole derivative 2d, obtaining after column
1
f
chromatography 1.020 g of a yellow powdery solid. Yield: 58.1%. R = 0.78 (eluent n-
1
Hex/EtOAc, 8:2); H NMR (400 MHz,DMSO-d
6
)= δ: 10.18 (s, 1H, NH), 7.79 (m, AA’BB’,
J=8.32 Hz, 2H, H-2’ and H-6’), 7.69 (d, J=3.64 Hz, 1H, H-2), 7.65 (d, J=8.84 Hz, 1H, H-7),
.35 (m, AA’BB’, J= 8.61 Hz, 2H, H-3’ and H-5’), 7.31 (m, 5H, H-2’’, H-3’’,H -4’’, H-5’’
and H-6’’), 6.92 (d, J=1.88 Hz, 1H, H-4), 6.78 (dd, J=8.61 Hz and J=1.82 Hz, 1H, H-6), 6.61
7
(
d, J=3.68 Hz, 1H, H-3), 4.92 (s, 1H, C=C-H), 4.12 (q, J=7.14 Hz, 2H, -OCH
2
CH
) δ = 14.84 (-OCH
), 90.90 (C=C-H), 109.71 (C-3), 113.64 (C-7), 115.36 (C-4), 120.89 (C-6),
27.11 (C-2’ and C-6’), 128.28 (C-2), 128.52 (C-2’’ and C-6’’), 128.99 (q, J=269.28 Hz,
3
), 1.22 ppm
13
(t, J=7.17 Hz, 3H, -OCH
2
CH
3
); C NMR (101 MHz, DMSO-d
6
2
CH ),
3
5
2 3
9.37 (-OCH CH
1
CF ), 129.01 (q, J=3.85 Hz, C-3’’ and C-5’’), 130.65 (C-3’ and C-5’), 130.91 (C-3a), 131.26
3
(C-1’’), 133.28 (q, J=32.35 Hz, C-4’’) 134.53 (C-4’), 135.85 (C-7a), 136.69 (C-5), 145.95 (C-
2
7

ACCEPTED MANUSCRIPT
+
1
2 3
’), 159.39 (C=C-H), 170.01 ppm (COOCH CH ); HRMS (ESI-MS, 140 eV): m/z [M+H ]
+
calculated for C H F N O S , 515.1252; found, 515.12.49.
26
22
3
2
4
4.1.4.4 (E,Z)-Ethyl 3-(1-(benzoyl)-2,3-dihydro-1H-indol-5-ylamino)-3-phenylacrylate (6a).
Compound 5a was prepared as described for compound 3b by reacting 1.96 g (8.22 mmol) of
the appropriate 5-aminoindole derivative 5a, obtaining after column chromatography 0.940 g
1
of a brown viscous oil. Yield: 27.7%. R = 0.70 (eluent n-Hex/EtOAc/toluene, 1:1:1); H
f
NMR (400 MHz, DMSO-d
H, H-4’), 7.60 (m, 1H, H-4’’), 7.60-7.32 (m, 8H, H-2”, -3”, -5”, -6” and H-3’, -5’, and H-6,
H-7), 6.72 (d, J=1.32 Hz, 1H, H-4), 4.90 (s, 1H, C=CH-), 4.14 (q, J=7.08 Hz, 2H, -OCH CH
.92 (t, J=8.23 Hz, 2H, H-3), 2.90 (t, J=8.32 Hz, 2H, H-2), 1.20 ppm (t, J=7.09 Hz, 3H, -
6
) δ = 10.14 (s, 1H, NH), 7.95 (m, 2H, H-2’ and H-6’), 7.68 (m,
1
2
3
)
3
1
3
CH CH ); C NMR (101 MHz, DMSO-d ) δ = 15.24 (-OCH CH ), 29.23 (C-3), 52.26 (C-2),
2
3
6
2
3
2 3
58.92 (-OCH CH ), 91.07 (C=C-H), 114.23 (C-7), 116.32 (C-4), 119.28 (C-6), 127.11 (C-2’
and C-6’), 128.52 (C-2’’ and C-6’’), 129.91 (C-3’’ and C-5’’), 130.23 (C-3’ and C-5’),
1
30.91 (C-3a), 131.18 (C-1’’), 133.84 (C-4’’) 134.82 (C-4’), 135.75 (C-7a), 136.82 (C-5),
46.23 (C-1’), 158.83 (C=C-H), 165.24 (NC=O), 171.91 ppm (COOCH CH ); HRMS (ESI-
1
2
3
+
+
3
MS, 140 eV): m/z [M+H ] calculated for C H N O , 413.1865; found, 413.1859.
26
25
2
4
.1.4.5
phenylacrylate (6b). Compound 6b was prepared as described for compound 3b by reacting
.460 g (2.16 mmol) of the appropriate 5-aminoindole derivative 5b, obtaining after column
(E,Z)-Ethyl
3-(1-(methanesulfonyl)-2,3-dihydro-1H-indol-5-ylamino)-3-
0
chromatography 0.540 g of a brown sticky oil. Yield: 64.8%. R = 0.37 (eluent n-Hex/EtOAc,
f
1
2
:1); H NMR (400 MHz, DMSO-d
6
) δ = 10.11 (s, 1H, NH), 7.38 (m, 1H, H-4’), 7.34 (m, 4H,
H-2’, -3’, -5’ and 6’), 6.96 (d, J=8.56 Hz, 1H, H-7), 6.72 (d, J=2.20 Hz, 1H, H-4), 6.52 (dd,
2
8