Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones: Synthesis, biological activity and SARs

Article abstract of DOI:10.1016/j.ejmech.2017.11.038

Earlier studies had confirmed that the 7-phenylpyrroloquinolinone (7-PPyQ) nucleus was an important scaffold for new chemotherapeutic drugs targeting microtubules. For wide-ranging SARs, a series of derivatives were synthesized through a robust procedure. For comparison with the reference 3-ethyl-7-PPyQ 31, the angular geometry and substituents at the 3 and 7 positions were varied to explore interactions inside the colchicine site of tubulin. Of the new compounds synthesized, potent cytotoxicity (low and sub-nanomolar GI₅₀ values) was observed with 21 and 24, both more potent than 31, in both leukemic and solid tumor cell lines. Neither compound 21 nor 24 induced significant cell death in normal human lymphocytes, suggesting that the compounds may be selectively active against cancer cells. In particular, 24 was a potent inducer of apoptosis in the A549 and HeLa cell lines. With both compounds, induction of apoptosis was associated with dissipation of the mitochondrial transmembrane potential and production of reactive oxygen species, indicating that cells treated with the compounds followed the intrinsic pathway of apoptosis. Moreover, immunoblot analysis revealed that compound 24 even at 50 nM reduced the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1. Finally, molecular docking studies of the newly synthesized compounds demonstrate that active pyrroloquinolinone derivatives strongly bind in the colchicine site of β-tubulin.

Full text of DOI:10.1016/j.ejmech.2017.11.038

Accepted Manuscript
Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones: Synthesis,
biological activity and SARs
Roberta Bortolozzi, Elena Mattiuzzo, Matteo Dal Pra, Mattia Sturlese, Stefano Moro,
Ernest Hamel, Davide Carta, Giampietro Viola, Maria Grazia Ferlin
PII:
S0223-5234(17)30941-8
10.1016/j.ejmech.2017.11.038
EJMECH 9913
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2017
Revised Date: 15 November 2017
Accepted Date: 17 November 2017
Please cite this article as: R. Bortolozzi, E. Mattiuzzo, M.D. Pra, M. Sturlese, S. Moro, E. Hamel, D.
Carta, G. Viola, M.G. Ferlin, Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones:
Synthesis, biological activity and SARs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.038.
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ACCEPTED MANUSCRIPT
Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones:
synthesis, biological activity and SARs.
1
*
1*
2
2
2
Roberta Bortolozzi , Elena Mattiuzzo , Matteo Dal Pra , Mattia Sturlese , Stefano Moro , Ernest
3
2
1
2
Hamel , Davide Carta , Giampietro Viola , Maria Grazia Ferlin

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Targeting tubulin polymerization by novel 7-aryl-pyrroloquinolinones:
synthesis, biological activity and SARs.
1
*
1*
2
2
2
Roberta Bortolozzi , Elena Mattiuzzo , Matteo Dal Pra , Mattia Sturlese , Stefano Moro , Ernest
3
2
1
2
Hamel , Davide Carta , Giampietro Viola , Maria Grazia Ferlin
1
Department of Woman’s and Child’s Health, University of Padova, Laboratory of
Oncohematology, 35128 Padova, Italy.
2
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131
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.
*equal contributing authors
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Abstract
Earlier studies had confirmed that the 7-phenylpyrroloquinolinone (7-PPyQ) nucleus was an
important scaffold for new chemotherapeutic drugs targeting microtubules. For wide-ranging SARs,
a series of derivatives were synthesized through a robust procedure. For comparison with the
reference 3-ethyl-7-PPyQ 31, the angular geometry and substituents at the 3 and 7 positions were
varied to explore interactions inside the colchicine site of tubulin. Of the new compounds
synthesized, potent cytotoxicity (low and sub-nanomolar GI values) was observed with 21 and 24,
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both more potent than 31, in both leukemic and solid tumor cell lines. Neither compound 21 nor 24
induced significant cell death in normal human lymphocytes, suggesting that the compounds may
be selectively active against cancer cells. In particular, 24 was a potent inducer of apoptosis in the
A549 and HeLa cell lines. With both compounds, induction of apoptosis was associated with
dissipation of the mitochondrial transmembrane potential and production of reactive oxygen
species, indicating that cells treated with the compounds followed the intrinsic pathway of
apoptosis. Moreover, immunoblot analysis revealed that compound 24 even at 50 nM reduced the
expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1. Finally, molecular docking studies of
the newly synthesized compounds demonstrate that active pyrroloquinolinone derivatives strongly
bind in the colchicine site of β-tubulin.
Keywords. Microtubules, phenylpyrroloquinolinone, tubulin, apoptosis, molecular docking,
structure-activity relationships.
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1
. Introduction
A major difference between cancer cells and many healthy cells is that transformed cells often
divide much faster. The microtubule (MT) cytoskeleton is highly dynamic in mitosis, and therefore
dividing cells are particularly susceptible to agents that target tubulin [1]. Colchicine site inhibitors
exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation
[
2]. Despite significant structural diversity, colchicine site inhibitors are believed to act by a
common mechanism via binding to the colchicine site on tubulin. Among them, we can include 7-
phenyl-pyrroloquinolinones (7-PPyQs) and, especially, the 3-substituted derivatives bind to the site
with high affinity, inhibiting microtubule assembly and therefore producing a strong
antiproliferative effect. Their activities are similar to those of the reference compound
combretastatin A-4 [3-9]. The more cytotoxic 7-PPyQ derivatives showed remarkably in vitro
biological properties and good antitumor activity in vivo [3]. Some 7-PPyQs, characterized by alkyl
substituents at the pyrrole nitrogen, show increased cytotoxicity with nanomolar and sub-nanomolar
GI50 values and overcome the resistance observed with the clinically used drugs vincristine and
taxol [4].In an effort to produce additional highly active compounds, numerous related analogs were
designed, synthesized and studied, resulting in the discovery of potent 3N-acyl derivatives showing
low nanomolar GI50 values and an anti-tubulin mechanism profile similar to that previously
observed [8]. Moreover, the most recent 3-benzoyl-7-PPyQ is even more cytotoxic than the initially
reported compounds. Moreover, the 3-benzoyl-7-PPyQ has minimal toxicity for nontumor cell
lines, good in vitro metabolic stability and synergizes with conventional chemotherapeutic agents in
inhibiting leukemia cell proliferation [9]. Through a tubulin docking study with 7-PPyQ amide
derivatives at the colchicine site, we observed a hydrogen bond involving the 6-NH and strong
hydrophobic interactions due to the pyrrolo[3,2-f]quinolinone scaffold and to the phenyl ring at
position 7. The substituents at the 3N-pyrrole were placed in a pocket formed by numerous amino
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acid residues. This binding mode is compatible with a competitive mechanism of action at the
colchicine site.
In the present study, we enrich the available library of pyrroloquinolinone derivatives to obtain
greater knowledge of SARs for further development of PPyQs and to understand better how PPyQs
are accommodated in the colchicine site pocket.
The reference compound, 3-ethyl-7-phenyl-3H-pyrrolo[3-2-f]quinolin-9-one (31, Figure 1) was our
start point for chemical modifications [4]. Compound 31 has the elements determined to be crucial
for anti-tubulin activity, such as the [3,2-f] configuration, the 7-phenyl and 9-carbonyl groups and
the ethyl group at position 3. All these elements were important for good antiproliferative effects.
This study explores the role of larger 3 and 7 substituents and various scaffold geometries. Among
our most interesting findings were that placement of a naphthalene moiety at the 7 position, a 3N-n-
octadecyl alkyl chain and [3,2-g] and [3,2-h] geometries led to enhanced cytotoxicity. The synthesis
and biological activities of the new compounds are summarized in this report.
2
2
. Results and discussion
.1 Chemistry
The 4-step method leading to 7-PPyQ compounds was previously described [4] (Scheme 1). First,
commercially available 5-, 6- or 7-nitroindoles were subjected to an N-alkylation reaction using
appropriate halogenated compounds to obtain the nitroindole derivatives 1-4. The same reaction
conditions were used with the chloro- and bromo-compounds in anhydrous DMF in the presence of
NaH, at room temperature with the ethyl bromide and 50° C with the n-octadecyl chloride, resulting
in high yields of the reaction products. The catalytic reduction (Pd/C 10%, H at atmospheric
2
pressure, ethyl acetate) of intermediates 1-4 gave the corresponding aminoindoles 5-8 in almost
quantitative yields. The non-commercially available β-ketoesters 9-12 were prepared following a
method reported in the literature [10] by reacting the appropriate methyl-aryl ketone and diethyl
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carbonate in the presence of NaH, in anhydrous dioxane at 80° C for 6 h, with good yields. Next, in
order to obtain the enamine derivatives 13-18 as condensation products of the reactions between the
β
-ketoesters and aminoindoles, compounds 5 and 6 were reacted with commercial ethyl
acetoacetate, benzoyl acetate and the prepared compounds 9-12. All the reactions were carried out
in absolute ethanol at reflux in the presence of a catalytic amount of acetic acid, yielding the
acrylate derivatives as crude material 13-18. These had to be purified by silica gel column
chromatography before being submitted to thermal cyclization in boiling diphenyl ether (250 °C) to
obtain the final products 19-24. Using the same reaction conditions, enamines 25 and 28 were also
obtained by reacting 3-ethyl-6-aminoindole (7) and 3-ethyl-7-amino-indole (8) with benzoyl
acetate. These compounds were also purified before being cyclized to the tricyclic products. In the
case of enamine 25, an un-resolvable mixture of two cyclized compounds was obtained. By NMR
spectroscopy it was possible to identify compound 26 as a [2,3-f] angular and 27 as a [3,2-g] linear
PPyQ. In the same way, from enamine 28 a mixture was obtained that was separated giving the two
compounds in the scheme: the angular [3,2-h] PPyQ 29 and the [1,4]diazepin-indole derivative 30.
All cyclization products were purified by re-crystallization from a suitable solvent or by flash
chromatography, with their purity verified by HPLC (>95%).
2.2 Biological evaluation
2.2.1 In vitro antiproliferative activities and SAR analysis
The new compounds were designed to obtain additional SAR information by modifying the size of
substituents at the 3 and 7 position of the aryl-PyQ tricycle, as well as the angular geometry.
Evaluation of antiproliferative activities of 19-24, 26-27, 29 and 30 was performed with the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against a panel of 7 human
tumor cell lines (HeLa, A549, HT-29, MDA-MB-231, RS4;11, Jurkat, and Kasumi-1). GI values,
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0
the concentrations that inhibit cell growth by 50%, are presented in Table 1. As reference
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compound CA-4 was also added. From the cytotoxicity data reported in the Table, we can make
some relevant remarks on SARs on the changed structural elements with respect to reference
compound 31 (Figure 1). Regarding the 3-substituent on compounds 19 and 20, replacing the ethyl
group of 31 with a bulky linear alkyl chain (18 C) caused a massive loss of antiproliferative activity
in all cell lines examined. This behavior was expected for 19, which lacks the crucial phenyl in the
7
position, but this loss of activity was unexpected for 20, which has the 7-phenyl group. In past
studies [4], very high cytotoxicity was found for compounds with side chains up to 5 C at position 3
with a 7-phenyl. Compounds 21-24, all bearing a 3-ethyl chain as in compound 31, had nanomolar
GI s, with compounds 21 and 24 all being especially active. These compounds all have even
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bulkier aryl 7-substituents than the 7-phenyl group, with the active 21 and 24 having, respectively,
an α-naphthyland a benzodioxole substituent. In general, for angular [3,2-f] PPyQs such bulky
groups are well tolerated. Of particular note, 21 and 24 had greater activity than 31, in both the
leukemic and solid tumor cell lines. In contrast, compound 22 with a 7-β-naphthyl substituent was
27-1430-fold less cytotoxic than compound 21, depending on the cell line. In part, this must
indicate a less favorable fit into the colchicine site relative to 21. Cytotoxicity again increased with
compound 23, which had a 7-tetrahydronaphthyl substituent. Concerning the third structural
element taken into consideration in this study, the configuration of the tricycle scaffold, the GI s
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obtained with compounds 26, 27 and 29 clearly indicate that the geometries [f] and [g] of 26 and 27,
respectively, produce at least one highly cytotoxic compound (nanomolar GI s), while the [h]
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geometry does not (compound 29). Compound 30, a diazepine-indole derivative, was also tested,
and it was completely inactive (GI s > 10000 nM).
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2.2.2 Evaluation of cytotoxicity of compounds 21 and 24 in human non-cancer cells
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To obtain a preliminary indication of the cytotoxic potential of these derivatives in normal human
cells, two of the most active compounds (21 and 24) were evaluated in vitro against peripheral
blood lymphocytes (PBL) from healthy donors (Table 2). Compound 21 was completely inactive in
quiescent lymphocytes (GI >100 µM), while in the presence of the mitogenic stimulus
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phytohematoaglutinin (PHA), the GI was about 25.7 µM. Notably, this value was almost 10000-
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3
1
0000 times higher than that observed against the lymphoblastic cell lines Jurkat and CEM (Tables
and 3). These results indicate that 21 has a modest inhibitory effect in rapidly proliferating non-
cancer cells but not in quiescent cells, as previously observed for other antimitotic derivatives
developed by our group [8,9]. Analogous behavior was observed for compound 24, which showed a
GI of 45.3 µM in quiescent lymphocytes, while, in the presence of (PHA, the GI decreased to
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50
about 23.0 µM.
2.2.3 Effect of compounds21 and 24 on multidrug resistant cells
To investigate whether these derivatives are substrates of drug efflux pumps, two of the most active
Vbl-100
compounds (21 and 24) were tested against CEM
cells that are a multidrug-resistant line
selected against vinblastine [4] and that overexpress P-glycoprotein (P-gp). This membrane protein
acts as a drug efflux pump and exhibits resistance to a wide variety of structurally unrelated
anticancer drugs and other compounds. As shown in Table 3, both compounds exhibited cytotoxic
Vbl100
activity in the CEM
cell line that was even higher than their activity against the parental line.
Thus, these derivatives are not substrates for P-gp.
2.2.4 Inhibition of tubulin polymerization and colchicine binding
To evaluate the tubulin interaction properties of compounds 21, 23, 24 and of the mixture 26-27, we
investigated their effects on the inhibition of tubulin polymerization and on the binding of
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3
[
H]colchicine to tubulin (Table 4) [12,13]. For comparison, CA-4 was examined in
contemporaneous experiments as a reference compound along with compound 31. Among the test
compounds, 21 and 24 strongly inhibited tubulin assembly with IC below 1 µM (0.99 and 0.84
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0
µM, respectively) but slightly higher than that obtained for the reference compounds CA-4
IC =0.64 µM) and 31 (IC =0.57 µM). Compound 23 also inhibited tubulin assembly with a low
(
5
0
50
IC value of 1.1 µM, while the mixture 26-27 had less activity, with an IC of 6.2 µM. The PPyQ
5
0
50
3
compounds all inhibited the binding of [ H]colchicine to tubulin, with the best activity occurring
with 21 and 24, but none approached CA-4 in its potency as an inhibitor of colchicine binding.
These results with tubulin correlate well with the growth inhibitory effects exhibited by compounds
21, 23 and 24, indicating that their antiproliferative activity derives mostly from an interaction with
tubulin. For the mixture 26-27, the correlation between cytotoxicity and inhibitory effects on
tubulin is perhaps not as good and could indicate there is an additional mechanism of action for this
compound.
2.2.5 Influence of test compounds 21, 23 and 24 on the cell cycle
The effect of compounds 21, 23 and 24 on cell cycle progression was examined by flow cytometry
in Hela cells (Figure 2). After a 24 h treatment, all compounds induced a G2/M arrest, although
compound 21 showed a more modest accumulation in G2/M cells than occurred with the other
compounds. In contrast, compounds 23 and 24 induced a greater G2/M block even at lower
concentrations. A concomitant reduction of both the S and G1 phases was also observed.
We also studied the association between 24-induced G2/M arrest and alterations in G2/M regulatory
protein expression in HeLa cells. As shown in Figure 3, compound 24 caused, in a time- and
concentration-dependent manner, an increase in cyclin B1 expression after 24 and 48 h, indicating
an activation of the mitotic checkpoint following drug exposure. This effect was confirmed by a
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reduction in the expression of phosphatase cdc25c at 24 h, followed by a disappearance in its
expression at 48 h. This was associated with the appearance of slower migrating forms of
phosphatase cdc25c indicative of cdc25c phosphorylation. The phosphorylation of cdc25c directly
stimulates its phosphatase activity, and this is necessary to activate cdc2/cyclin B on entry into
mitosis [14,15].We also observed a decrease of the phosphorylated form of cdc2 kinase, in
particular after the 48 h treatment.
2.2.6 Compounds 21, 23 and 24 induce apoptosis in different cell lines
To evaluate the mode of cell death induced by test compounds, we performed a bi-parametric
cytofluorimetric analysis using propidium iodide (PI) and annexin-V-FITC, which stain DNA and
phosphatidylserine (PS) residues, respectively. We used two cell lines, Hela and A549, in which we
evaluated the effects of compounds 21, 23 and 24. In additional experiments we have also evaluated
the apoptotic response of CA-4, as reference compounds, in HeLa cells. As shown in Figure 4, the
three compounds had different potencies. In both cell lines, the compounds induced apoptosis in a
time and concentration dependent manner, with compounds 21 and 24 being the most active
compounds in good agreement with the cytotoxicity data. To note, that CA-4 induce a strong
apoptotic response at the concentration of 0.1 µM in HeLa cells in well agreement with previous
reports [16,17]
2.2.7 Compound 24 induced mitochondrial depolarization and reactive oxygen species (ROS)
production
Mitochondria play an essential role in the propagation of apoptosis [18,19]. It is well established
that, at an early stage, apoptotic stimuli alter the mitochondrial transmembrane potential (∆ψmt).
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∆
ψ was monitored by the fluorescence of the dye JC-1 [20]. HeLa cells treated with compound 24
mt
(0.1-1.0 µM) showed a time-dependent increase in the percentage of cells with low ∆ψmt (Figure 4,
Panel A). The depolarization of the mitochondrial membrane is associated with the appearance of
annexin-V positivity in the treated cells when they are in an early apoptotic stage. In fact, the
dissipation of ∆ψmt is characteristic of apoptosis and has been observed with both microtubule
stabilizing and destabilizing agents, including other derivatives, in different cell types [21-24]. It is
well known that mitochondrial membrane depolarization is associated with the mitochondrial
production of ROS [25,26]. Therefore, we investigated whether ROS production increased after
treatment with the test compounds. We utilized the fluorescence indicator 2,7-
dichlorodihydrofluorescein diacetate (H -DCFDA) [27]. As shown in Figure 4 (Panel B) compound
2
24 induced significant production of ROS starting at 12-24 h of treatment at 1 µM, in good
agreement with the mitochondrial depolarization described above.
2.2.8 Compound 24 induced PARP activation and caused a decrease in the expression of anti-
apoptotic proteins
As shown in Figure 5, compound 24 in HeLa cells caused a concentration and time-dependent
cleavage of poly (ADP-Ribose) polymerase (PARP), confirming its pro-apoptotic activity.
We also investigated the expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1. Bcl-2 play
a major role in controlling apoptosis through the regulation of mitochondrial processes and the
release of mitochondrial proapoptotic molecules that are important for the cell death pathway [28-
30]. Our results (Figure 6) showed that the expression of the anti-apoptotic protein Bcl-2 was
decreased starting after a 24 h treatment at both concentration used (0.1 and 1.0 µM). The decrease
in expression of Mcl-1 was even greater.
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2.3 Computational Studies
Molecular docking studies were carried out to investigate the possible binding mode of the novel
inhibitors with the aim of interpreting the biological data. Recently, we reported a study that
identified two fundamental aspects of the interaction of 7-PPyQ derivatives with tubulin that
enhanced our ability to obtain reliable docking results: which protein conformation is most suitable
to accommodate the 7-PPyQ scaffold and which docking protocol performs best in reproducing the
experimental structure of the complexes associated with that protein conformation [9]. Taking
advantage of these findings, we docked eight of our derivatives, 19-24 and 29-30. As with the
previously reported 7-PPyQ derivatives and, by analogy, with plinabulin [31], we observed that the
most potent compounds 21, 23, and 24 occupied the colchicine site by establishing and maintaining
a key hydrogen bond interaction with the backbone of βVal236 mediated by the pyrroloquinolinone
scaffold. The fused-ring system, in addition, guaranteed strong hydrophobic interactions with
βLeu253, βAla314, and βIle368. The substituents in position 7 established hydrophobic interactions
with residues βPhe167, βTyr200, and βLeu250. The alkyl substituents at the N-pyrrole were placed
in the pocket formed by residues βLys350, βThr351, βAla314, βAla352, and βThr179. Among the
newly synthesized compounds, most of the small structural differences that caused strong
divergences in activity are nicely explained by molecular docking (Video-S1). Compounds 21 and
22 only differ in the position of the conjugation of the naphthyl group in alpha and in beta position,
respectively. While 21 preserves the typical interaction scheme, and, in particular, the hydrogen
bond with βVal236, compound 22 fails to achieve a similar interaction (Figure 7). More subtle
structural differences, as with compounds 22-24, are more difficult to rationalize, even in terms of
the shape of their substituent at position 7 (Figure 7). While the most active derivative 24 is able,
through the benzodioxole group to establish a further hydrogen bond with βGln134 in addition to
the typical hydrogen mediated by the pyrroloquinolinone core with βVal236 (Figure 7), both 22 and
23 fail to display the usual interaction with βVal236. The differences between the naphthyl and
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tetrahydronaphthyl substituents are minimal, but these differences abolish the activity of 22.
However, it is difficult to ascribe the diverse biological effects to such small structural difference.
Derivatives with a bulky substituent at the N-pyrrole apparently are able to retain the interaction
pattern that occurs with the most potent compounds, 21, 23, and 24. Docking results suggest that
such derivatives could theoretically be accommodated in the colchicine site and reproduce the
binding mode of the most potent derivatives. The long linear alkyl chain (18 carbon atoms)
characterizing analogs 19 and 20 may protrude between the beta-strand 373-381 and the alpha helix
2
52-259 and interact with the α-tubulin subunit. However, such a hydrophobic group would
drastically affect the physicochemical properties of these compounds. The logP reaches values of
.15 and 7.69 for 19 and 20, respectively (Table S1), suggesting a plausible sequestration by the
membrane. Molecular docking also revealed that alternative scaffold geometries like the angular
3,2-h] PPyQ 29 and the [1,4]diazepin-indole derivative 30 were not able to reproduce the
7
[
interaction pattern of the most potent compounds (per-residue interaction heat map profile, Video
S1).
3. Conclusion
Continuing our research aimed to expand our understanding of the SAR data of PPyQs, we
synthesized a small library of analogs by chemically modifying three structural elements:
substituents on the 3N pyrrole (19 and 20), varying the aryl group at the 7 position (21-24) and the
[
3,2-f] angular geometry (26-29). Evaluation of antiproliferative activity by the MTT test indicated
that extensive elongation of the alkyl chain bound to 3N sharply decreased cytotoxicity (19 and 20),
while enlargement of the aryl moiety at position 7 could increase cytotoxicity (21, 23 and 24) . The
un-separable mixture of compounds 26 and 27 showed that [f] and/or [g] angular geometries,
respectively, could result in high cytotoxic activity, while compound 29, with [h] angular geometry
did not. Compounds 21, 23 and 24 were the most cytotoxic compounds (nanomolar GI s) among
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the newly synthesized compounds, and they acted by an apoptotic mechanism and were excellent
inhibitors of tubulin polymerization through an interaction at the colchicine site.
In addition, compounds 21 and 24 showed only modest effects in a rapidly proliferating noncancer
cell and were completely inactive in quiescent cells. Both derivatives were not substrates for the
Pgp drug efflux pump, as shown by their excellent cytotoxic activity against the multidrug-resistant
Vbl100
CEM
cell line. Compound 24 also induced mitochondrial depolarization and ROS production,
PARP activation and decreased expression of anti-apoptotic proteins. The molecular docking
studies of all derivatives showed that they could bind into the colchicine site in a manner similar to
that of previously studied PPyQs [9]. Furthermore, the molecular docking studies provided good
explanations for the effects of different substituents on the PPyQ nucleus. In particular, the
molecular docking study revealed that the most active derivative 24 (sub-nanomolar GI s) is able
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0
through the benzodioxole group to establish an additional hydrogen bond with β-tubulin. Taking
into account all of our results presented here, from a SAR point of view, compound 24 represents
enhancement of the pharmacodynamics optimization process of the antimitotic PPyQ class, and it
therefore merits in vivo evaluation.
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
potassium bromide pressed disks; all values are expressed in cm−1. UV−vis spectra were recorded
1
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 centre. Integrals were
1
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satisfactorily in line with those expected based on compound structure. Mass spectra were obtained
on a Mat 112 Varian Mat Bremen (70 eV) mass spectrometer and 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. Microwave assisted
reactions were performed on a CEM Discover® monomode reactor with a built-in infrared sensor
assisted-temperature monitoring and automatic power control; all reactions were performed in
closed devices under pressure control. Solutions were concentrated on a rotary evaporator under
reduced pressure. 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 x 4.6 mm, a particle
size of 5 mm, and a loop of 10 µL. The detector was set at 300 nm. The mobile phase consisted of
phase A (Milli-Q H O, 18.0 MU, TFA 0.05%) and phase B (95% MeCN, 5% phase A). Gradient
2
elution was performed as reported: 0 min, % B = 10; 20 min, % B = 90; 25 min, % B = 90; 26 min,
%
B = 10; 31 min, % B = 10.
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 over molecular sieves.
4
.1.1 General Procedure for the Synthesis of 1N-Substituted Nitroindoles (1-4). As a typical
procedure, the synthesis of 5-nitro-1-octadecyl-1H-indole 1 is described in detail. Into a two-necked
00 mL round-bottomed flask, 0.333 g (13.88 mmol) of NaH, 60% dispersion in mineral oil, was
placed and washed with toluene (3 × 10 mL). With stirring, a solution of commercial 5-nitroindole,
.750 g (4.625 mmol), in 5 mL of anhydrous DMF, was dropped into the flask, and the initial
1
0
yellow color changed to red with the formation of H gas. After 40 min at room temperature, the
2
mixture was cooled to 0 °C, and 2.36 mL (6.94 mmol, d=0.849 g/mL) of 1-chlorooctadecane was
1
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dropped into the flask and 0.050 g of NaI were added to the mixture. The reaction was monitored by
TLC analysis (eluent toluene/n-hexane/ethyl acetate, 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 that was
extracted with ethyl acetate (3 × 50 mL). The organic phase, washed with water and brine and dried
over anhydrous Na SO , was concentrated under vacuum giving a crude yellow solid (2.522 g).
2
4
This crude product was purified with a silica gel chromatographic column (d 3 cm, l 35 cm, 230-
400 mesh, eluent n-hexane/toluene, 1:1), yielding 1.618 g of a pure yellow powder.
1
4
.1.1.1 5-nitro-1-octadecyl-1H-indole (1). Yield: 84.5 %; Rf: 0.48 (n-hexane/toluene, 1:1); H NMR
(
400 MHz, CDCl ): δ 0.90 (t, J = 6.86 Hz, 3 H, N-CH CH (CH ) CH ), 1.26-1.33 (m, 30 H, N-
3 2 2 2 15 3
CH CH (CH ) CH ), 1.87 (quin, J = 7.12 Hz, 2H, N-CH CH (CH ) CH ), 4.17 (t, J = 7.16 Hz,
2
2
2 15
3
2
2
2 15
3
2
H, N-CH CH (CH ) CH ), 6.69 (dd, J= 3.22, 0.74 Hz, 1H, H-3), 7.26 (d, J = 3.24 Hz, 1H, H-2),
2 2 2 15 3
7.37 (d, J = 9.08 Hz, 1H, H-7), 8.13 (dd, J=9.10, 2.22 Hz, 1H, H-6), 8.61 (d, J = 2.24 Hz, 1H, H-4)
13
ppm;
C
NMR (101 MHz, CDCl ): δ 14.11 (N-CH CH (CH ) CH ), 22.68 (N-
3 2 2 2 15 3
CH CH (CH ) CH ), 26.89 (N-CH CH (CH ) CH ), 29.15 (N-CH CH (CH ) CH ), 29.35 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.43 (N-CH CH (CH ) CH ), 29.50 (N-CH CH (CH ) CH ), 29.57 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.62 (N-CH CH (CH ) CH ), 29.65 (N-CH CH (CH ) CH ), 29.68 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 30.21 (N-CH CH (CH ) CH ), 31.92 (N-CH CH (CH ) CH ), 46.92 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 103.85 (C-3), 109.18 (C-7), 117.13 (C-6), 118.27 (C-4), 127.69 (C-3a),
2
2
2 15
3
130.92 (C-2), 138.81 (C-7a), 141.50 (C-5) ppm.
4.1.1.2 1-ethyl-5-nitro-1H-indole (2). Compound 2 was prepared as for compound 1 by reacting
commercial 5-nitroindole (1.50 g, 9.25 mmol), NaH (0.666 g, 27.75 mmol), and bromoethane (1.04
mL, 13.88 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis
(eluent toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted
to yield 1.649 g of yellow solid product. Yield: 93.7%; Rf: 0.67 (toluene/n-hexane/ethyl acetate,
1
5

ACCEPTED MANUSCRIPT
1
1
:1:1); H NMR (300 MHz, DMSO-d ): δ 0.55 (t, J = 7.1 Hz, 3H, CH ), 3.47 (q, J = 7.1 Hz, 2H,
6
3
CH ), 5.93 (d, J = 3.3 Hz, 1H, H-2), 6.87 (m, 2H, H-7 e H-3), 7.20 (dd, J = 9.0, 2.1 Hz, 1H, H-6),
2
7.74 (d, J = 2.1 Hz, 1H, H-4) ppm.
4.1.1.3 1-ethyl-6-nitro-1H-indole (3). Compound 3 was prepared as for compound 1 by reacting
commercial 6-nitroindole (1.00 g, 6.17 mmol), NaH (0.444 g, 18.50 mmol), and bromoethane (0.69
mL, 9.25 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis (eluent
toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted to yield
1
1
.110 g of yellow solid. Yield: 94.5 %; Rf: 0.75 (toluene/n-hexane/ethyl, 1:1:1); H NMR (300
MHz, CDCl ): δ 1.45 (t, J = 7.29 Hz, 3H, CH CH ), 4.20 (q, J = 7.30 Hz, 2H, CH CH ), 6.52 (dd, J
3
2
3
2
3
=
3.07 Hz e J = 0.82 Hz, 1H, H-3), 7.34 (d, J = 3.09 Hz, 1H, H-2), 7.58 (d, J = 8.73 Hz, 1H, H-4),
1
3
7
.93 (dd, J = 8.79 Hz e J = 2.04 Hz, 1H, H-5), 8.27 ppm (d, J = 1.95 Hz, 1H, H-7); C NMR (75
MHz, CDCl ): δ 15.60 (CH CH ), 41.53 (CH CH ), 102.28 (C-3), 106.40 (C-7), 114.85 (C-5),
3
2
3
2
3
120.80 (C-4), 132.83 (C-2), 133.41 (C-3a), 134.27 (C-7a), 142.85 (C-6) ppm.
4.1.1.4 1-ethyl-7-nitro-1H-indole (4). Compound 4 was prepared as for compound 1 by reacting
commercial 7-nitroindole (1.00 g, 6.17 mmol), NaH (0.444 g, 18.50 mmol), and bromoethane (0.69
mL, 9.25 mmol) in DMF at room temperature. The reaction was monitored by TLC analysis (eluent
toluene/n-hexane/ethyl acetate, 1:1:1). At the end of the reaction, the mixture was extracted to yield
1
1
.103 g of yellow solid. Yield: 94.0 %; Rf: 0.82 (toluene/n-hexane/ethyl acetate, 1:1:1); H NMR
(
300 MHz, CDCl ): δ 1.33 (t, J = 7.23 Hz, 3H, CH CH ), 4.30 (q, J = 7.21 Hz, 2H, CH CH ), 6.67
3 2 3 2 3
(d, J = 3.24 Hz, 1H, H-3), 7.14 (t, J = 7.84 Hz, 1H, H-5), 7.22 (d, J = 3.24 Hz, 1H, H-2), 7.82 (dd, J
1
3
=
7.84, 0.82 Hz, 1H, H-4), 7.87 (dd, J = 7.78, 1.00 Hz, 1H, H-6) ppm; C NMR (75 MHz CDCl ):
3
δ 16.01 (CH CH ), 44.83 (CH CH ), 103.01 (C-3), 118.40 (C-5), 119.78 (C-4), 126.13 (C-3a),
2
3
2
3
127.13 (C-6), 132.00 (C-2), 133.83 (C-7a), 136.87 (C-7) ppm.
1
6

ACCEPTED MANUSCRIPT
4.1.2 General Procedure for the Synthesis of 1N-Substituted Nitroindoles (5-8). As a typical
procedure, the synthesis of 1-octadecyl-1H-indol-5-amine 5 is described in detail. Into a two-necked
flask, previously dried in an oven, about 0.300 g of Pd/C 10% and approximately 50 mL of ethyl
acetate were placed. After connecting the flask to an elastomer balloon containing H , the mixture
2
was stirred at room temperature for 1 h in order to saturate the suspension of Pd/C with H . Then,
2
1.00 g (2.41 mmol) of nitroindole derivative 1 in 15 mL of ethyl acetate was added dropwise to the
suspension, and the mixture was stirred under H at atmospheric pressure and heated by means of
2
an oil bath at 50-60 °C, monitoring the progress of the reaction by TLC analysis (toluene/n-hexane,
1:1). At the end of the reaction, the mixture was filtered through a celite pad, and the solution was
concentrated under vacuum to give 0.784 g of semisolid dark purple product.
1
4
.1.2.1 1-octadecyl-1H-indol-5-amine (5). Yield: 84.8 %; Rf: 0.12 (n-hexane:toluene, 1:1); H NMR
(
400 MHz, CDCl ): δ 0.90 (t, J = 6.78 Hz, 3H, N-CH CH (CH ) CH ), 1.26-1.31 (m, 30H, N-
3
2
2
2 15
3
CH CH (CH ) CH ), 1.82 (quin, J = 7.06 Hz, 2H, N-CH CH (CH ) CH ), 4.05 (t, J = 7.12 Hz,
2
2
2 15
3
2
2
2 15
3
2
6
H, N-CH CH (CH ) CH ), 6.31 (d, J = 3.04 Hz, 1H, H-3), 6.72 (dd, J = 8.58, 2.22 Hz, 1H, H-6),
2 2 2 15 3
.98 (d, J = 2.16 Hz, 1H, H-4), 7.03 (d, J = 3.04 Hz, 1H, H-2), 7.17 ppm (d, J = 8.60 Hz, 1H, H-7);
1
3
C NMR (101 MHz, CDCl3): δ 14.11 (N-CH CH (CH ) CH ), 22.69 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2
2
4
2
7.01 (N-CH CH (CH ) CH ), 29.26 (N-CH CH (CH ) CH ), 29.36 (N-CH CH (CH ) CH ),
2 2 2 15 3 2 2 2 15 3 2 2 2 15 3
9.50 (N-CH CH (CH ) CH ), 29.57 (N-CH CH (CH ) CH ), 29.61 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
9.64 (N-CH CH (CH ) CH ), 29.66 (N-CH CH (CH ) CH ), 29.67 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
9.69 (N-CH CH (CH ) CH ), 30.26 (N-CH CH (CH ) CH ), 31.93 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
6.50 (N-CH CH (CH ) CH ), 99.52 (C-3), 106.21 (C-4), 109.93 (C-7), 112.48 (C-6), 128.21 (C-
2
2
2 15
3
), 129.39 (C-3a), 131.29 (C-7a), 138.36 (C-5) ppm.
4.1.2.2 1-ethyl-1H-indol-5-amine (6). Compound 6 was prepared as for compound 5 by reacting
1.65 g of compound 2 (8.67 mmol), obtaining 1.36 g of a semisolid purple compound. Yield: 97.9
1
7

ACCEPTED MANUSCRIPT
1
%
; Rf: 0.28 (toluene/n-hexane/ethyl, 1:1:1); H NMR (300 MHz, DMSO-d ): δ 1.55 (t, J = 7.3 Hz,
6
3
H, CH ), 4.06 (q, J = 7.3 Hz, 2H, CH ), 4.48 (s br, 2H, NH ), 6.15 (d, J = 3.05 Hz, 1H, H-2), 6.54
3 2 2
(dd, J = 8.5, 2.28 Hz, 1H, H-6), 6.69 (d, J = 2.28 Hz, 1H, H-4), 7.16 (m, 2H, H-3 e H-7) ppm.
4.1.2.3 1-ethyl-1H-indol-6-amine (7). Compound 7 was prepared as for compound 5 by reacting
1.45 g of compound 3 (7.61 mmol), obtaining 1.26 g of a semisolid purple compound. Yield: 99.8
1
%
; Rf: 0.28 (toluene/n-hexane/ethyl acetate, 1:1:1); H NMR (300 MHz, CDCl ): δ 1.33 (t, J = 7.26
3
Hz, 3H, CH CH ), 3.42 (s, 2H, NH ), 3.95 (q, J = 7.26 Hz, 2H, CH CH ), 6.28 (d, J = 3.00 Hz, 1H,
2
3
2
2
3
H-3), 6.47 (dd, J = 8.29, 2.95 Hz, 1H, H-5), 6.54 (d, J = 1.94 Hz, 1H, H-7), 6.83 (d, J = 3.29 Hz,
1
3
1
4
2
H, H-2), 7.31 ppm (d, J = 8.31 Hz, 1H, H-4); C NMR (75 MHz, CDCl ): δ 15.30 (CH CH ),
3 2 3
0.80 (CH CH ), 94.95 (C-7), 100.94 (C-3), 110.29 (C-5), 121.57 (C-4), 122.03 (C-3a), 125.14 (C-
2
3
), 136.91 (C-7a), 141.69 (C-6) ppm.
4.1.2.4 1-ethyl-1H-indol-7-amine (8). Compound 8 was prepared as for compound 5 by reacting
1.43 g of compound 3 (7.64 mmol), obtaining 1.34 g of a semisolid purple compound. Yield: 99.8
1
%
; Rf: 0.48 (toluene/n-hexane/ethyl acetate, 1:1:1); H NMR (300 MHz, CDCl ): δ 1.51 (t, J = 7.23
3
Hz, 3H, CH CH ), 3.72 (s, 2H, NH ), 4.43 (q, J = 7.21 Hz, 2H, CH CH ), 6.45 (d, J = 3.09 Hz, 1H,
2
3
2
2
3
H-3), 6.53 (dd, J = 7.36, 0.88 Hz, 1H, H-4), 6.93 (dd, J = 7.83, 7.44 Hz, 1H, H-5), 7.01 (d, J = 3.12
1
3
Hz, 1H, H-2), 7.17 ppm (dd, J = 7.92, 1.05 Hz, 1H, H-6); C NMR (75 MHz CDCl ): δ 18.13
3
(
CH CH ), 43.87 (CH CH ), 101.64 (C-3), 110.20 (C-4), 113.23 (C-6), 120.19 (C-5), 126.52 (C-
2 3 2 3
3a), 128.74 (C-2), 131.19 (C-7a), 132.14 (C-7) ppm.
4.1.3 General Procedure for the Synthesis of Aryl Ethyl Acetate Derivatives 9-12. As a typical
procedure, the preparation of aryl ethyl acetate derivative 9 is described in detail. 2.36 g (98.33
mmol) of NaH 60% dispersion in mineral oil was placed in a 100 mL round-bottomed flask and
washed with toluene to remove oil. Then, 7.12 mL (59.75 mmol, d=0.975 g/mL) of diethyl
1
8

ACCEPTED MANUSCRIPT
carbonate and 20 mL of anhydrous dioxane were added. The mixture was heated to 80 °C with
stirring and then 4.46 mL (29.37 mmol, d=1.120 g/mL) of 1-acetyl naphthalene was added
dropwise. The mixture was stirred at 80 °C for 15 h and monitored by TLC analysis (eluent ethyl
acetate/petroleum ether, 1:5). The reaction mixture was allowed to cool to room temperature, and
25 mL of water was added. The water layer was separated and extracted twice with ethyl acetate.
The organic layers were combined and washed with water and brine, dried over Na SO , filtered,
2
4
and evaporated to dryness, giving 5.73 g of a yellow oil. The crude product was used for the next
reaction step without further purification.
4.1.3.1 Ethyl 3-(naphthalen-5-yl)-3-oxopropanoate (9). Yield: 80.52%; Rf: 0.68 (EtOAc:petroleum
1
ether, 1:5); H-NMR (DMSO-d )= δ 1.11 (t, J = 7.09 Hz, 3H, OCH CH ), 4.08 (q, J = 7.09 Hz, 2H,
6
2
3
OCH CH ), 4.30 (s, 2H, COCH CO), 7.68 (m, 3H, H-3, H-6, H-7), 8.03 (m, J = 8.84 Hz, 1H, H-8),
2
3
2
8.18 (m, J = 8.23 Hz, 2H, H-4, H-5), 8.57 (d, J = 8.39 Hz, 1H, H-2).
4.1.3.2 Ethyl 3-(naphthalen-6-yl)-3-oxopropanoate (10). Compound 10 was prepared as for
compound 9 by reacting 2.5 g of commercial 2-acetyl-naphthalene (14.69 mmol) with 3.55 mL
29.31 mmol) of diethyl carbonate and 1.17 g (48.79 mmol) of NaH in 20 mL of dioxane to yield
.45 g of crude product. This was purified by column chromatography (d 3 cm, l 35 cm, 230-400
(
2
mesh, eluent ethyl acetate/petroleum ether, 1:5), yielding 1.429 g of a yellow solid. Yield: 40.1 %;
1
Rf: 0.55 (EtOAc:petroleum ether, 1:5); H NMR (300 MHz, CDCl ): δ 1.19 (t, J = 7.14 Hz, 3H,
3
OCH CH ) 4.05 (s, 2H, OCH O), 4.16 (q, J = 7.13 Hz, 2H, OCH CH ), 7.49-7.58 (m, 2H, H-6' e H-
2
3
2
2
3
7
’), 7.81 (d, J = 8.58 Hz, 1H, H-5'), 7.84 (d, J = 8.49 Hz, 1H, H-4'), 7.90 (d, J = 7.98 Hz, 1H, H-8'),
.94 (dd, J=8.68, 1.78 Hz, 1H, H-3'), 8.38 (d, J = 1.26 Hz, 1H, H-1’). Keto-enol ratio 81:19.
7
4.1.3.3 Ethyl 3-(1,2,3,4-tetrahydronaphthalen-7-yl)-3-oxopropanoate (11). Compound 11 was
prepared as for compound 9 by reacting 0.945 mL of commercial 6-acetyl-1,2,3,4-
1
9

ACCEPTED MANUSCRIPT
tetrahydronaphthalene (5.74 mmol) with 1.39 mL (11.48 mmol) of diethyl carbonate and 0.457 g
(19.06 mmol) of NaH in 20 mL of dioxane to yield 0.694 g of crude product. This was purified by
column chromatography (d 3 cm, l 35 cm, 230-400 mesh, eluent ethyl acetate/petroleum ether, 1:5),
1
yielding 0.351 g of a yellow solid. Yield: 24.8 %; Rf: 0.78 (EtOAc:petroleum ether, 1:5); H NMR
(
300 MHz, CDCl ): δ 1.25 (t, J = 7.15 Hz, 3H, OCH CH ), 1.81 (m, 4H, H-6' e H-7’), 2.81 (m, 4H,
3 2 3
H-5' e H-8'), 3.94 (s, 2H, OCH O), 4.20 (q, J = 7.13 Hz, 2H, OCH CH ), 7.14 (d, J = 8.61 Hz, 1H,
2
2
3
H-4'), 7.63 (dd, J=7.18, 1.87 Hz, 1H, H-3'), 7.65 (m, 1H, H-1'). Keto-enol ratio 90:10.
4.1.3.4 Ethyl 3-(benzo[d][1,3]dioxol-6-yl)-3-oxopropanoate (12). Compound 12 was prepared as
for compound 9 by reacting 1.642 g of commercial 1-(benzo[d][1,3]dioxol-6-yl)ethanone (10
mmol) with 2.42 mL (20 mmol) of diethyl carbonate and 0.672 g (28 mmol) of NaH in 20 mL of
THF to yield 2.162 g of a yellow oil. The crude product is used for the next reaction step without
1
further purification. Yield: 91.5 %; Rf: 0.40 (EtOAc:petroleum ether, 1:5); H NMR (300 MHz,
CDCl ): δ 1.25 (t, J = 7.14 Hz, 3H, OCH CH ), 3.90 (OCCH CO), 4.19 (q, J = 7.14 Hz, 2H,
3
2
3
2
OCH CH ), 6.04 (COCH OC), 6.84 (d, J = 8.16 Hz, 1H, H-4'), 7.41 (d, J = 1.74 Hz, 1H, H-1'), 7.51
2
3
2
(dd, J = 8.17, 1.78 Hz, 1H, H-3'). Keto-enol ratio 95:5.
4.1.4 General Procedure for the Synthesis of Acrylate Derivatives 13-18, 25 and 28. As a typical
procedure, the synthesis of acrylate derivative 13 is described in detail. In a 100 mL round-
bottomed flask, 0.786 g (2.045 mmol) of 3-substituted aminoindole 5 in 10 mL of absolute ethanol
was condensed with 0.388 mL (3.07 mmol; d = 1.029 g/ mL) of commercial ethyl acetoacetate and
0.5 mL of glacial acetic acid in the presence of 100 mg of drierite. The mixture was refluxed for
about 48 h, the reaction being monitored by TLC analysis (n-hexane/ethyl acetate, 8:2). At the end
of the reaction, the mixture was cooled and filtered to remove the drierite. The resulting solution
was evaporated to dryness under vacuum and the residue (1.039 g) purified by silica gel
chromatography (d = 3 cm, l = 35 cm, 230−400 mesh, eluent n-hexane/ethyl acetate, 8:2) to yield
2
0

ACCEPTED MANUSCRIPT
0.770 g of a semisolid brown product.
4.1.4.1 (E,Z)-ethyl 3-(1-octadecyl-1H-indol-5-ylamino)but-2-enoate (13). Yield: 75.8%; Rf: 0.80 (n-
1
hexane/ethyl acetate, 8:2); H NMR (400 MHz, CDCl ): δ 0.90 (t, J = 6.85 Hz, 3H, N-
3
CH CH (CH ) CH ), 1.27-1.33 (m, 30H, N-CH CH (CH ) CH ), 1.31 (t, J = 7.10 Hz, 3H,
2
2
2 15
3
2
2
2 15
3
COOCH CH ), 1.85 (quin, J = 6.88 Hz, 2H, N-CH CH (CH ) CH ), 1.93 (s, 3H, NHCCH ) 4.11 (t,
2
3
2
2
2 15
3
3
J = 7.20 Hz, 2H, N-CH CH (CH ) CH ), 4.18 (q, J = 7.13 Hz, 2H, COOCH CH ), 4.67 (s, 1H,
2
2
2 15
3
2
3
NHCCH), 6.46 (d, J = 3.08 Hz,1H, H-3), 6.97 (dd, J=8.56, 1.96 Hz, 1H, H-6), 7.13 (d, J = 3.12 Hz,
1
3
1
H, H-2), 7.28 (d, J = 8.56 Hz, 1H, H-7), 7.37 (d, J = 1.92 Hz, 1H, H-4) 10.28 ppm (s, 1H, NH); C
NMR (101 MHz, CDCl ): δ 14.11 (N-CH CH (CH ) CH ), 14.67 (COOCH CH ), 20.25
3
2
2
2 15
3
2
3
(
NHCCH3) 20.25 (N-CH CH (CH ) CH ), 22.68 (N-CH CH (CH ) CH ), 27.00 (N-
2 2 2 15 3 2 2 2 15 3
CH CH (CH ) CH ), 29.23 (N-CH CH (CH ) CH ), 29.25 (NCH CH (CH ) CH ), 29.35 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.49 (N-CH CH (CH ) CH ), 29.56 (N-CH CH (CH ) CH ), 29.61 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.65 (NCH CH (CH ) CH ), 29.67 (N-CH CH (CH ) CH ), 29.69 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 30.11 (N-CH CH (CH ) CH ), 30.26 (N-CH CH (CH ) CH ), 31.92
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
(
NCH CH (CH ) CH ), 46.62 (N-CH CH (CH ) CH ), 58.50 (COOCH CH ), 83.81 (NHCCH),
2 2 2 15 3 2 2 2 15 3 2 3
1
7
00.89 (C-3), 109.51 (C-7), 118.05 (C-4), 120.38 (C-6), 128.61 (C-3a), 128.83 (C-2), 131.16 (C-
a), 134.21 (C-5), 160.94 (NHCCH ), 170.57 (COOCH CH ) ppm.
3
2
3
4.1.4.2 (E,Z)-ethyl 3-(1-octadecyl-1H-indol-5-ylamino)-3-phenylacrylate (14). Compound 14 was
prepared as for compound 13 by reacting 0.974 mL of commercial ethyl benzoylacetate (5.07
mmol, d = 1.11 g/mL) with 1.301 g (3.38 mmol) of previously prepared compound 5, yielding
2.328 g of crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35
cm, 230−400 mesh, eluent n-hexane/ethyl acetate, 8:2) to yield 0.405 g of a semisolid brown
1
product. Yield: 21.7%; Rf: 0.77 (n-hexane/ethyl acetate, 8:2); H NMR (300 MHz, CDCl ): δ 0.80
3
(
t, J = 6.70 Hz, 3H, N-CH CH (CH ) CH ), 1.18-1.16 (m, 30H, N-CH CH (CH ) CH ), 1.24 (t, J
2 2 2 15 3 2 2 2 15 3
2
1

ACCEPTED MANUSCRIPT
=
7.10 Hz, 3H, COOCH CH ) 1.68 (quin, J = 6.75 Hz, 2H, N-CH CH (CH ) CH ), 3.91 (t, J =
2
3
2
2
2 15
3
7
.20 Hz, 2H, NCH CH (CH ) CH ), 4.13 (q, J = 7.11 Hz, 2H, COOCH CH ), 4.85 (s, 1H,
2 2 2 15 3 2 3
NHCCH), 6.19 (d, J = 3.03 Hz, 1H, H-3), 6.53 (dd, J=8.68, 2.11 Hz, 1H, H-6), 6.92 (d, J = 1.72 Hz,
H, H-4), 6.93 (d, J = 3.12 Hz, 1H, H-2), 6.95 (d, J = 9.00 Hz, 1H, H-7), 7.13 (m, 1H, H-4'), 7.16
1
1
3
(
m, 2H, H-3' e H-5’), 7.28 (m, 2H, H-2’ e H-6’), 10.15 (s, 1H, NH) ppm. C NMR (75 MHz,
CDCl ): δ 14.10 (N-CH CH (CH ) CH ), 14.67 (COOCH CH ), 20.22 (N-CH CH (CH ) CH ),
3
2
2
2 15
3
2
3
2
2
2 15
3
2
2
2
2
3
2.98 (N-CH CH (CH ) CH ), 27.06 (NCH CH (CH ) CH ), 29.12 (N-CH CH (CH ) CH ),
2 2 2 15 3 2 2 2 15 3 2 2 2 15 3
9.34 (N-CH CH (CH ) CH ), 29.56 (N-CH CH (CH ) CH ), 29.67 (NCH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
9.76 (N-CH CH (CH ) CH ), 29.82 (N-CH CH (CH ) CH ), 29.84 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
9.91 (N-CH CH (CH ) CH ), 30.23 (NCH CH ( CH ) CH ), 30.67 (N-CH CH (CH ) CH ),
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
1.45 (N-CH CH (CH ) CH ), 46.16 (N-CH CH (CH ) CH ), 57.27 (COOCH CH ), 84.18
2
2
2 15
3
2
2
2 15
3
2
3
(NHCCH), 104.28 (C-3), 113.28 (C-4), 114.27 (C-7), 116.28 (C-6), 124.82 (C-3a), 127.82 (C-2),
1
5
28.36 (C-2’ e C-6’), 129.27 (C-3’ e C-5’) 130.17 (C-4’), 130.28 (C-7a), 131.01 (C-1’), 139.82 (C-
), 154.85 (NHCCH), 173.82 (COOCH CH ) ppm.
2
3
4.1.4.3 (E,Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(naphthalen-1-yl)acrylate (15). Compound 15
was prepared as for compound 13 by reacting 1.815 g (7.49 mmol) of compound 9 with 1 g (6.24
mmol) of previously prepared compound 6, giving 2.461 g of crude product, which was purified by
silica gel column chromatography (d = 3 cm, l = 35 cm, 230−400 mesh, eluent n-hexane/ethyl
acetate/toluene, 1:1:1) to yield 0.879 g of a semisolid brown product. Yield: 36.6%; Rf: 0.82 (n- /
1
acetate, 8:2); H NMR (400 MHz, DMSO-d ): δ 1.21 (t, J = 7.16 Hz, 3H, COOCH CH ), 1.41 (t, J
6
2
3
=
7.20 Hz, 3H, NCH CH ), 4.19 (q, J = 7.14 Hz, 2H, COOCH CH ), 4.35 (q, J = 7.22 Hz, 2H,
2 3 2 3
NCH CH ), 4.92 (s, 1H, NHCCH), 7.24 (d, J = 2.12 Hz, 1H, H-5), 7.43 (dd, J = 8.81 Hz, 1H, H-6),
2
3
7.59 (m, 1H, H-3), 7.85 (m, 1H, H-2), 7.90 (dd, J = 8.95, 0.64 Hz, 1H, H-7), 8.08 (m, J = 8.56 Hz e
1
3
J=2.14 Hz, 1H, H-8'), 8.13 (m, J=8.23 Hz, 1H, H-2'), 11.93 ppm (s, 1H, NH); C NMR (101 MHz,
DMSO-d6): δ 16.39 (NHCH CH ), 17.04 (COOCH CH ), 41.03 (NCH CH ), 60.04
2
3
2
3
2
3
2
2

ACCEPTED MANUSCRIPT
(
COOCH CH ), 88.94 (NHCCH), 104.10 (C-3), 112.53 (C-6), 115.98 (C-7), 118.31 (C-4), 123.64
2
3
(C-3a), 125.30 (C-2), 125.87 (C-aro), 126.94 (Caro), 127.64 (C-aro), 127.77 (C-aro), 128.64 (C-
aro), 128.92 (C-8'), 130.12 (C-2'), 131.06 (C-8'a), 131.46 (C-4'), 132.44 (C-7a), 133.61 (C-1'),
147.37 (C-5), 153.12 (NHCCH), 170.24 (CO).
4.1.4.4 (E,Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(naphthalen-3-yl)acrylate (16). 16 was
prepared as for compound 13 by reacting 1 g (4.13mmol) of compound 10 with 0.440 g (2.75
mmol) of previously prepared compound 6, giving 1.567 g of crude product, which was purified by
silica gel column chromatography (d = 3 cm, l = 35 cm, 230−400 mesh, eluent n- /ethyl acetate 8:2)
to yield 0.692 g of a semisolid orange product. Yield: 65.5%; Rf: 0.44 (n-hexane/ethyl acetate, 8:2);
1
H NMR (300 MHz, CDCl ): δ 1.19 (t, J = 7.12 Hz, 3H, NCH CH ), 1.30 (t, J = 7.26 Hz, 3H,
3
2
3
COOCH CH ), 3.95 (q, J = 7.27 Hz, 2H, NCH CH ), 4.16 (q, J =7.12 Hz, 2H, COOCH CH ), 4.98
2
3
2
3
2
3
(s, 1H, NHCCH), 6.17 (dd, J = 3.09, 0.51 Hz, 1H, H-3), 6.58 (dd, J = 8.73, 2.07 Hz, 1H, H-6), 7.00
(dd, J=1.47 Hz, 1H, H-4), 7.24 (dd, J = 8.53, 1.69 Hz, 1H, H-3’), 7.38 (m, 1H, H-7’), 7.39 (d, J =
2
1
.97 Hz, 1H, H-2), 7.46 (m, 1H, H-6), 7.84 (d, J = 8.49 Hz, 1H, H-7), 7.92 (m, 1H, H-5’), 7.93 (m,
13
H, H-8’), 8.38 (s, 1H, H-1'), 10.39 (s, 1H, NH) ppm. C NMR (75 MHz, CDCl ): δ 15.92
3
(
NCH CH ), 16.38 (COOCH CH ), 41.38 (NCH CH ), 59.27 (COOCH CH ), 90.28 (NHCCH),
2 3 2 3 2 3 2 3
1
1
8
01.27 (C-3), 109.27 (C-4), 115.88 (C-7), 116.27 (C-6), 126.93 (C-5'), 126.95 (C-6'), 127.27 (C-3’),
27.37 (C-2), 127.82 (C-7'), 127.82 (C-8'), 129.73 (C-4'), 129.81 (C-3a), 132.81 (C-7a), 133.82 (C-
'a), 133.98 (C-1'), 134.82 (C-4'a), 135.62 (C-2’), 142.73 (C-5), 149.12 (NHCCH), 175.98 (CO)
ppm.
4.1.4.5 (E,Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(1,2,3,4-tetrahydronaphthalen-7-yl)acrylate
(17). Compound 17 was prepared as for compound 13 by reacting 0.373 g (1.51 mmol) of
compound 11 with 0.201 g (1.26 mmol) of previously prepared compound 6, giving 0.535 g of
crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm,
2
3

ACCEPTED MANUSCRIPT
230−400 mesh, eluent n-hexane/ethyl acetate 8:2) to yield 0.337 g of a semisolid brown-orange
1
product. Yield: 68.8%; Rf: 0.54 (n-hexane/ethyl acetate, 8:2); H NMR (300 MHz, CDCl ): δ 1.19
3
(
t, J = 7.14 Hz, 3H, NCH CH ), 1.34 (t, J = 7.27 Hz, 3H, COOCH CH ), 1.67 (m, 4H, H-6' e H-7'),
2 3 2 3
2
.60 (m, 4H, H-5' e H-8’), 4.00 (q, J = 7.28 Hz, 2H, NCH CH ), 4.13 (q, J = 7.11 Hz, 2H,
2 3
COOCH CH ), 4.83 (s, 1H, NHCCH), 6.23 (dd, J=3.07, 0.58 Hz, 1H, H-3), 6.57 (dd, J = 8.67, 2.10
2
3
Hz, 1H, H-6), 6.76 (d, J = 7.95 Hz, 1H, H-7), 6.97 (d, J = 2.91 Hz, 1H, H-1'), 7.07 (d, J = 8.55 Hz,
H, H-3'), 7.42 (d, J = 1.56 Hz, 1H, H-4'), 7.57 (m, 1H, H-4'), 7.57 (m, 1H, H-2), 10.27 (s, 1H, NH)
1
13
ppm; C NMR (75 MHz, CDCl ): δ 15.92 (NCH CH ), 15.93 (COOCH CH ), 24.96 (C-6' e C-7'),
3
2
3
2
3
3
1
4
0.04 (C-5' e C-8'), 40.72 (NCH CH ), 61.38 (COOCH CH ), 89.97 (NHCCH), 102.83 (C-3),
2 3 2 3
08.38 (C-4), 116.23 (C-6), 117.28 (C-7), 124.28 (C-3a), 124.82 (C-3'), 126.28 (C-1'), 127.28 (C-
'), 127.73 (C-2), 132.73 (C-7a), 135.28 (C-4'a), 136.93 (C-8'a), 139.83 (C-1'), 142.82 (C-5), 156.38
(NHCCH), 175.81 (CO) ppm.
4.1.4.6 (E,Z)-ethyl 3-(1-ethyl-1H-indol-5-ylamino)-3-(benzo[d][1,3]dioxol-6-yl)acrylate (18).
Compound 18 was prepared as for compound 13 by reacting 1.130 g (4.79 mmol) of compound 12
with 0.639 g (3.99 mmol) of previously prepared compound 6, giving 1.761 g of crude product,
which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230−400 mesh,
eluent n-hexane/ethyl acetate 8:2) to yield 0.558 g of a semisolid brown product. Yield: 16.1%; Rf:
1
0
.67 (n-hexane/ethyl acetate, 8:2); H NMR (300 MHz, CDCl ): δ 1.26 (t, J = 7.18 Hz, 3H,
3
NCH CH ), 1.37 (t, J = 7.23 Hz, 3H, COOCH CH ), 4.08 (q, J = 7.28 Hz, 2H, NCH CH ), 4.33 (q,
2
3
2
3
2
3
J = 7.13 Hz, 2H, COOCH CH ), 4.89 (s, 1H, NHCCH), 6.05 (s, 2H, OCH O), 6.31 (d, J=3.09 Hz,
2
3
2
1H, H-3), 6.65 (d, J = 8.10 Hz, 1H, H-7), 6.86 (d, J = 8.19 Hz, 1H, H-4'), 6.89 (dd, J = 8.09, 1.71
Hz, 1H, H-6), 7.02 (d, J = 2.01 Hz, 1H, H-4), 7.05 (d, J = 3.15 Hz, 1H, H-2), 7.43 (d, J = 1.69 Hz,
13
1
H, H-1'), 7.53 (dd, J=8.17, 1.78 Hz, 1H, H-3'), 10.28 (s, 1H, NH) ppm; C NMR (75 MHz,
CDCl ): δ 15.93 (NCH CH ), 16.38 (COOCH CH ), 41.28 (NCH CH ), 61.28 (COOCH CH ),
3
2
3
2
3
2
3
2
3
9
0.03 (NHCCH), 100.24 (OCH O), 104.28 (C-3), 108.28 (C-4), 110.28 (C-4'), 115.38 (C-7), 116.28
2
2
4

ACCEPTED MANUSCRIPT
(C-6), 122.36 (C-3), 122.38 (C-3a), 124.73 (C-1), 127.82 (C-2), 135.47 (C-1'), 136.38 (C-7a),
142.38 (C-5), 147.28 (C-4'b), 148.37 (C-4a'), 157.38 (NHCCH), 174.28 (CO) ppm.
4.1.4.7 (E,Z)-ethyl 3-(1-ethyl-1H-indol-6-ylamino)-3-phenylacrylate (25). Compound 25 was
prepared as for compound 13 by reacting 2.27 g of commercial ethyl benzoylacetate (11.79 mmol, d
1.11 g/mL) with 1.26 g (7.86 mmol) of previously prepared compound 7, giving 2.943 g of crude
=
product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm, 230−400
mesh, eluent n-hexane/ethyl acetate 6:4) to yield 2.069 g of a brown product. Yield: 78.7%; Rf: 0.87
1
(
n-hexane/ethyl acetate, 6:4); H NMR (300 MHz, CDCl ): δ 1.10 (t, J = 7.26 Hz, 3H, NCH CH ),
3
2
3
1
2
1
7
1
4
5
.25 (t, J = 7.12 Hz, 3H, COOCH CH ), 3.78 (q, J = 7.27 Hz, 2H, NCH CH ), 4.14 (q, J = 7.11 Hz,
2 3 2 3
H, COOCH CH ), 4.89 (s, 1H, NHCCH) 6.26 (dd, J=3.12, 0.75 Hz, 1H, H-3), 6.45 (d, J = 1.30 Hz,
2
3
H, H-7), 6.51 (dd, J = 8.40, 1.92 Hz, 1H, H-5), 6.88 (d, J = 3.12 Hz, 1H, H-2), 7.16 (m, 1H, H-4'),
.19 (m, 2H, H-3' e H-5’), 7.26 (d, J = 8.37 Hz, 1H, H-4), 7.30 (m, J = 8.77 Hz, 2H, H-2' e H-6’),
1
3
0.39 (s, 1H, NH) ppm; C NMR (75 MHz, CDCl ): δ 14.61 (NCH CH ), 15.10 (COOCH CH ),
3
2
3
2
3
0.91 (NCH CH ), 59.19 (COOCH CH ), 89.51 (NHCCH), 100.74 (C-3), 103.76 (C-7), 115.86 (C-
2
3
2
3
), 120.77 (C-4), 124.87 (C-3a), 127.10 (C-2), 128.29 (C-2' e C-6’), 128.39 (C-3' e C-5’), 129.11
C-4’), 134.72 (C-1’), 135.51 (C-7a), 136.45 (C-6), 159.84 (NHCCH), 170.34 (CO) ppm.
(
4.1.4.8 (E,Z)-ethyl 3-(1-ethyl-1H-indol-7-ylamino)-3-phenylacrylate (28). Compound 28 was
prepared as for compound 13 by reacting 2.418 g of commercial ethyl benzoylacetate (12.59 mmol,
d = 1.11 g/mL) with 1.35 g (8.39 mmol) of previously prepared compound 8, giving 3.166 g of
crude product, which was purified by silica gel column chromatography (d = 3 cm, l = 35 cm,
2
4
3
4
30−400 mesh, eluent n-hexane/ethyl acetate 6:4) to yield 1.242 g of a brown product. Yield:
1
4.3%; Rf: 0.84 (n-hexane/ethyl acetate, 6:4); H NMR (300 MHz, CDCl ): δ 1.28 (t, J = 7.14 Hz,
3
H, NCH CH ), 1.50 (q, J = 7.15 Hz, 2H, COOCH CH ), 4.24 (q, J = 7.15 Hz, 2H, COOCH CH ),
2
3
2
3
2
3
.42 (q, J = 7.23 Hz, 2H, NCH CH ), 5.13 (s, 1H, NHCCH), 6.44 (d, J = 3.12 Hz, 1H, H-3), 6.52
2
3
2
5

ACCEPTED MANUSCRIPT
(dd, J = 7.35, 0.72 Hz, 1H, H-4), 6.91 (t, J = 7.63 Hz, 1H, H-5), 7.00 (d, J = 3.12 Hz, 1H, H-2), 7.16
(dd, J = 7.92, 0.87 Hz, 1H, H-6), 7.51 (m, J = 7.80 Hz, 2H, H-3' e H-5'), 7.62 (m, J=7.46, 2.05 Hz,
1
3
1
H, H-4’), 7.97 (m, J =8.55 Hz, 2H, H-2' e H-6’), 10.62 ppm (s, 1H, NH); C NMR (75 MHz,
CDCl ): δ 14.09 (NCH CH ), 18.11 (COOCH CH ), 43.86 (NCH CH ), 61.52 (COOCH CH ),
3
2
3
2
3
2
3
2
3
90.31 (NHCCH), 101.63 (C-3), 110.19 (C-4), 120.18 (C-5) 126.06 (C-3a), 128.52 (C-6) 128.56 (C-
2), 128.72 (C-4’), 128.72 (C-6), 128.80 (C-3’ e C-5’), 133.78 (C-1’), 135.94 (C-7a), 135.99 (C-7),
161.15 (NHCCH), 173.23 (CO) ppm.
4.1.5 General Procedure for the Synthesis of Phenylpyrroloquinolinones 19-24, 26, 27 and 29. As a
typical procedure, the synthesis of the phenylpyrroloquinolinone derivative 19 is described in detail.
In a two-necked round-bottomed flask, 30 mL of diphenyl ether was heated to boiling. Acrylate
derivative 13 (0.758 g, 1.53 mmol) was then added portionwise, and the resulting mixture was
refluxed for 15 min. After cooling to room temperature, 25 mL of diethyl ether was added, and the
mixture was left for 12 h. The separated precipitate was collected by filtration and washed many
times with diethyl ether. The crude product (0.534 g) was purified by flash column chromatography
(
eluent CHCl /methanol, 9:1), obtaining 0.165 g of a white powder.
3
4.1.5.1 7-methyl-3-octadecyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (19). Yield: 23.6%; Rf: 0.59
1
(
blue fluorescent spot, CHCl /methanol, 9:1); mp = 140°C; H NMR (400 MHz, CDCl ): δ 0.89 (t, J
3
3
=
=
6.84 Hz, 3H, N-CH CH (CH ) CH ), 1.31 - 1.25 (m, 30H, N-CH CH (CH ) CH ), 1.86 (quin, J
2 2 2 15 3 2 2 2 15 3
6.64 Hz, 2H, NCH CH (CH ) CH ), 2.45 (s, 3H, NHCCH ), 4.21 (t, J = 7.06 Hz, 2H,
2
2
2 15
3
3
NCH CH (CH ) CH ), 6.39 (s, 1H, H-8), 7.28 (d, J = 2.90 Hz, 1H, H-2), 7.63 (d, J = 9.00 Hz, 1H,
2
2
2 15
3
1
3
H-4), 7.69 (d, J = 8.92 Hz, 1H, H-5), 7.83 (d, J = 2.88 Hz, 1H, H-1), 12.22 (s, 1H, NH) ppm; C
NMR (101 MHz, CDCl ): δ 14.00 (N-CH CH (CH ) CH ), 19.67 (NHCCH ), 22.48 (N-
3
2
2
2 15
3
3
CH CH (CH ) CH ), 24.99 (N-CH CH (CH ) CH ), 26.74 (NCH CH (CH ) CH ), 29.03 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.14 (N-CH CH (CH ) CH ), 29.29 (N-CH CH (CH ) CH ), 29.35 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
2
6

ACCEPTED MANUSCRIPT
CH CH (CH ) CH ), 29.40 (NCH CH (CH ) CH ), 29.44 (N-CH CH (CH ) CH ), 29.48 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 30.56 (N-CH CH (CH ) CH ), 31.71 (N-CH CH (CH ) CH ), 46.53
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
(
NCH CH (CH ) CH ), 104.29 (C-1), 108.73 (C-8), 111.88 (C-5), 115.48 (C-4), 116.69 (C-9a),
2 2 2 15 3
122.92 (C-9b), 128.32 (C-2), 131.55 (C-3a), 136.44 (C-5a), 147.80 (C-7), 182.01 (C-9) ppm; IR
(KBr): ν= 3391.59 (NH), 2919.43 (aliphatic C-H), 2847.80 (aliphatic C-H), 1637.16 (C=O),
-
1
1
3
1
2
522.64 (C=C) cm ; UV-Vis (H O): 231.18 nm (A = 203.78 mAU), 275.70 nm (A = 93.22 mAU),
2
51.08 nm (45.03 mAU); fluorescence (MeOH): λexc = 350 nm, λems = 416 nm; HRMS (ESI-MS,
+
+
40 eV): m/z [M+H] calculated for C H N O , 451.3688; found, 451.3769; RP-C18 HPLC: t =
3
0
47
2
R
6.96 min, 99.9%.
4.1.5.2 3-octadecyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (20). Compound 20 was
prepared as described for compound 19 by reacting 0.406 g (7.3 mmol) of the appropriate
phenylacrylate derivative 14 to yield 0.389 g of a raw solid, which was purified by flash column
chromatography (eluent CHCl /methanol, 95:5) to yield 0.175 g of a grey powder. Yield: 47.1%; Rf:
3
1
0
0
.38 (blue fluorescent spot, CHCl /methanol, 95:5); mp = 156°C; H NMR (300 MHz, CDCl ): δ
3
3
.89 (t, J = 6.72 Hz, 3H, N-CH CH (CH ) CH ), 1.25-1.32 (m, 30H, N-CH CH (CH ) CH ), 1.88
2
2
2 15
3
2
2
2 15
3
(
quin, J = 6.67 Hz, 2H, NCH CH (CH ) CH ), 4.24 (t, J = 7.15 Hz, 2H, N-CH CH (CH ) CH ),
2 2 2 15 3 2 2 2 15 3
6
7
1
.65 (s br, 1H, H-8), 7.31 (m, 3H, H-3’, H-4’ e H-5’), 7.33 (d, J = 3.00 Hz, 1H, H-2), 7.65 (d, J =
.29 Hz, 1H, H-4), 7.67 (m, 2H, H-2’ e H-6'), 7.68 (d, J = 9.00 Hz, 1H, H-5), 7.84 (d, J = 2.91 Hz,
1
3
H, H-1), ppm. C NMR (75 MHz, CDCl ): δ 14.02 (N-CH CH (CH ) CH ), 22.51
3
2
2
2 15
3
(
NCH CH (CH ) CH ), 26.77 (N-CH CH (CH ) CH ), 29.07 (N-CH CH (CH ) CH ), 29.17 (N-
2 2 2 15 3 2 2 2 15 3 2 2 2 15 3
CH CH (CH ) CH ), 29.18 (N-CH CH (CH ) CH ), 29.33 (NCH CH (CH ) CH ), 29.34 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.38 (N-CH CH (CH ) CH ), 29.43 (N-CH CH (CH ) CH ), 29.46 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 29.50 (N-CH CH (CH ) CH ), 30.60 (NCH CH (CH ) CH ), 31.73 (N-
2
2
2 15
3
2
2
2 15
3
2
2
2 15
3
CH CH (CH ) CH ), 46.56 (N-CH CH (CH ) CH ), 104.42 (C-1), 108.28 (C-8), 112.66 (C-4),
2
2
2 15
3
2
2
2 15
3
115.48 (C-5), 117.75 (C-9a), 123.16 (C-9b), 127.10 (C-2’), 127.60 (C-6'), 128.31 (C-3’), 128.74 (C-
2
7

ACCEPTED MANUSCRIPT
5’), 129.92 (C-2), 131.66 (C-4’), 134.75 (C-1’), 136.97 (C-3a), 139.18 (C-5a), 148.33 (C-7), 176.28
(C-9) ppm; IR (KBr): ν= 3403.42 (NH), 3090 (aliphatic C-H), 2903.12 (aliphatic C-H), 1651.15
-1
(
C=O), 1513.68 (C=C) cm ; UV-Vis (H O): 225.3 nm (A = 200.81 mAU), 276.10 nm (A = 197.23
2
mAU), 353.05 nm (76.28 mAU); fluorescence (MeOH): λexc = 350 nm, λems = 458 nm; HRMS
+
+
(
ESI-MS, 140 eV): m/z [M+H] calculated for C H N O , 513.3845; found, 513.3945; RP-C18
35 49 2
HPLC: t = 31.711 min, 98.02%.
R
4.1.5.3 3-ethyl-7-(naphthalen-1-yl)-3H-pyrrolo[3,2-f]quinolin-9(6H)-one (21). Compound 21 was
prepared as described for compound 19 by reacting 0.877 g (2.28 mmol) of the appropriate
phenylacrylate derivative 15 to yield 0.150 g of a yellow powder. Yield: 19.4 %; Rf: 0.66 (light blue
1
fluorescent spot, CHCl /methanol, 9:1); mp = 219°C; H NMR (400 MHz, DMSO-d ): δ 1.41 (t, J =
3
6
7
8
1
8
4
.20 Hz, 3H, NCH CH ), 4.34 (q, J = 7.22 Hz, 2H, NCH CH ), 6.20 (s br, 1H, H-8), 7.43 (dd, J =
2 3 2 3
.81 Hz, 1H, H-5), 7.52-7.75 (m, 5H, H-3', H-4', H-5', H-6' and H-7'), 7.59 (m, 1H, H-1) 7.85 (m,
H, H-2), 7.90 (dd, J = 8.95, 0.64 Hz, 1H, H-4), 8.08 (m, J = 8.56, 2.14 Hz, 1H, H-8'), 8.13 (m, J =
1
3
.23 Hz, 1H, H-2'), 12.01 (s br, 1H, NH) ppm; C NMR (101 MHz, DMSO-d ): δ 16.39 (NCH H ),
6
2
3
1.03 (NCH CH ), 104.10 (C-1), 111.58 (C-8), 112.53 (C-5), 115.98 (C-4), 118.31 (C-9a), 123.64
2
3
(
C-9b), 125.30 (C-2), 125.87 (aromatic-C), 126.94 (aromatic-C), 127.64 (aromatic-C), 127.77
aromatic-C), 128.64 (aromatic-C), 128.92 (C-8'), 130.12 (C-2'), 131.06 (C-8'a), 131.46 (C-4'a),
(
1
3
32.44 (C-3a), 133.61 (C-1'), 133.81 (C-7), 147.37 (C-5a), 178.15 (CO) ppm; IR (KBr): ν =
-
1
436.75 (NH), 3024 (aliphatic C-H), 2974.66 (aliphatic C-H), 1608.39 (C=O), 1511.10 (C=C) cm ;
UV-Vis (H O): 292 nm (A = 899 mAU), 340 nm (A = 501 mAU); fluorescence (MeOH): λexc = 350
2
+
+
nm, λems = 479 nm; HRMS (ESI-MS, 140 eV): m/z [M+H] calculated for C H N O , 339.1492;
23
19
2
found, 339.1540; RP-C18 HPLC: t = 14.34 min, 95.13%.
R
4.1.5.4 3-ethyl-7-(naphthalen-3-yl)-3H-pyrrolo[3,2-f]quinoline-9(6H)-one (22). Compound 22 was
prepared as described for compound 19 by reacting 0.297 g (0.77 mmol) of the appropriate
2
8