Novel 3-Substituted 7-Phenylpyrrolo[3,2-f]quinolin-9(6H)-ones as Single Entities with Multitarget Antiproliferative Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b00805

A series of chemically modified 7-phenylpyrrolo[3,2-f]quinolinones was synthesized and evaluated as anticancer agents. Among them, the most cytotoxic (subnanomolar GI₅₀ values) amidic derivative 5f was shown to act as an inhibitor of tubulin polymerization (IC₅₀, 0.99 μM) by binding to the colchicine site with high affinity. Moreover, 5f induced cell cycle arrest in the G2/M phase of the cell cycle in a concentration dependent manner, followed by caspase-dependent apoptotic cell death. Compound 5f also showed lower toxicity in nontumoral cells, suggesting selectivity toward cancer cells. Additional experiments revealed that 5f inhibited the enzymatic activity of multiple kinases, including AURKA, FLT3, GSK3A, MAP3K, MEK, RSK2, RSK4, PLK4, ULK1, and JAK1. Computational studies showed that 5f can be properly accommodated in the colchicine binding site of tubulin as well as in the ATP binding clefts of all examined kinases. Our data indicate that the excellent antiproliferative profile of 5f may be derived from its interactions with multiple cellular targets.

Full text of DOI:10.1021/acs.jmedchem.5b00805

Article
pubs.acs.org/jmc
Novel 3‑Substituted 7‑Phenylpyrrolo[3,2‑f ]quinolin-9(6H)‑ones as
Single Entities with Multitarget Antiproliferative Activity
Davide Carta,†,∥ Roberta Bortolozzi,‡,∥ Ernest Hamel,§ Giuseppe Basso,‡ Stefano Moro,†
Giampietro Viola,‡ and Maria Grazia Ferlin*,†
†Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
‡Laboratory of Oncohematology, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, Italy
§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,
United States
S
* Supporting Information
ABSTRACT: A series of chemically modified 7-phenylpyrrolo[3,2-f ]quinolinones was synthesized and evaluated as anticancer
agents. Among them, the most cytotoxic (subnanomolar GI50 values) amidic derivative 5f was shown to act as an inhibitor of
tubulin polymerization (IC50, 0.99 μM) by binding to the colchicine site with high affinity. Moreover, 5f induced cell cycle arrest
in the G2/M phase of the cell cycle in a concentration dependent manner, followed by caspase-dependent apoptotic cell death.
Compound 5f also showed lower toxicity in nontumoral cells, suggesting selectivity toward cancer cells. Additional experiments
revealed that 5f inhibited the enzymatic activity of multiple kinases, including AURKA, FLT3, GSK3A, MAP3K, MEK, RSK2,
RSK4, PLK4, ULK1, and JAK1. Computational studies showed that 5f can be properly accommodated in the colchicine binding
site of tubulin as well as in the ATP binding clefts of all examined kinases. Our data indicate that the excellent antiproliferative
profile of 5f may be derived from its interactions with multiple cellular targets.
of tumor mass. These types of tubulin inhibitors might provide
new therapeutic approaches to treat cancers and overcome
limitations of existing tubulin interactive drugs.3
Our previously described 2- and 7-phenylpyrrolo-
quinolinones (2- and 7-PPyQs) (Figure 1) are a class of
antiproliferative agents acting as tubulin polymerization
inhibitors by binding at the colchicine site of β-tubulin.4,5
Members of the less cytotoxic 2-PPyQ family were also found
to exhibit interesting in vitro and in vivo antiangiogenic
properties.6
INTRODUCTION
■
Cancer is a leading cause of disease worldwide, accounting for
12.7 million new cases every year, and this number is expected
to rise to 26 million by 2030. Considering the impact on human
health and economics, cancer presents a major challenge to the
scientific world, and there is a necessity to discover new agents
for the treatment of this disease. Most of the drugs in
preclinical development are represented by small molecules. In
2013, for example, 9 out of 10 new cancer drugs launched on
the market were small molecules.1
The first generation of 7-PPyQs showed interesting in vitro
biological properties. Some derivatives had micromolar GI50
values and good antitumor activity in vivo.5 The second
generation, characterized by alkyl substitutions at the pyrrolic
nitrogen, showed increased cytotoxicity with nanomolar GI50
values, and these compounds overcame cross resistance
observed with the clinically used agents vincristine and
paclitaxel.7 In the latter series, the 3N-cyclopropylmethyl 7-
Currently, combined anticancer therapies or multitargeted
drugs are preferred over traditional cytotoxic treatment, with
the aim to overcome resistance and toxicity drawbacks. These
events often prevent successful treatment and are responsible
for reduced survival times.2 In the field of chemotherapeutics,
the widely used antitubulin agents exhibit significant cytotox-
icity by inhibiting microtubule dynamics. There are several
structurally dissimilar small molecules with high affinity for the
colchicine site on tubulin able to inhibit the proliferation of a
wide variety of human cancer cells. Moreover, these agents also
affect the tumor endothelial vasculature required for the growth
Received: May 27, 2015
© XXXX American Chemical Society
A
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designed, synthesized, and evaluated in cellular cytotoxicity and
tubulin inhibition assays, resulting in the discovery of potent
amidic derivatives. Furthermore, our recent study indicated that
22 is highly effective in reducing cell viability, and the reduced
survival of A549 cells is associated with an initial autophagy that
may be mediated by inhibition of the PI3K/Akt/mTOR
pathway.9 This pathway plays a variety of physiological roles,
including regulation of cell growth and proliferation, and is
perhaps the most frequently disregulated pathway in human
cancers conferring aggressiveness and poor prognosis.10,11
These findings prompted us to determine whether the new
7-PPyQ derivatives also interfere with the PI3K/Akt pathway
and exert kinase inhibitory activity.
RESULTS AND DISCUSSION
■
Figure 1. General chemical structures of 2-PPyQs, 7-PPyQs, the
previous 7-PPyQs 19−22, and the known antitubulin agents colchicine
and combretastatin A4.
Chemistry. The general method leading to the novel 3-
substituted 7-PPyQ compounds was previously reported7 and
consists of four steps as described in Scheme 1A. First,
commercially available 5-nitroindole (1) was subjected to an N-
alkylation or acylation reaction using appropriate halogenated
compounds to obtain the nitroindole derivatives 2a−f. The
same reaction conditions were used with all chloro and bromo
compounds and with ethyl acrylate in anhydrous DMF in the
presence of NaH at room temperature, resulting in high yields
of the reaction products. The catalytic reduction (Pd/C 10%,
H2 at atmospheric pressure, ethyl acetate) of intermediates 2a−
d gave aminoindoles 3a−d in almost quantitative yields. In the
case of 2f (and for 2e not shown) the catalytic procedure
furnished the partially reduced derivative 6f as described in
Scheme 1B. To obtain fully aromatic aminoindoles 3e and 3f, a
chemical reduction with SnCl2 in methanol at reflux was
necessary but resulted in lower final yields. The condensation
PPyQ derivative (22, MG 2477) was taken as lead compound
due to its very strong cytotoxicity (nanomolar range GI50
values) 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 (CA-4, Figure 1) (0.90 μM assembly IC50
and 83% inhibition of colchicine binding for 22 versus values of
1.1 μM and 99%, respectively, for CA-4).7
In an effort to produce additional highly active compounds,
numerous related analogues were synthesized, and the effects of
a variety of substitutions at diverse positions of the 3H-
pyrrolo[3,2-f ]quinolin-9-one nucleus (PyQ) were evaluated.8
In the present study, a number of new 7-PPyQ derivatives were
a
Scheme 1. Synthetic Routes to PPyQs 5a−f (A) and 8f (B)
a
Reagents and conditions: (a) NaH 60%, bromoethyl alcohol, cyclopropylmethyl bromide, 3-bromopropionitrile, ethyl chloroacetate,
cyclopropylcarbonyl chloride, n-propionyl chloride, anhydrous DMF, rt, 3 h, 47−99%; (b) H2, Pd/C 10%, ethyl acetate, 50 °C, 4−14 h, 90−
98%; (c) SnCl2/HCl, CH3OH, reflux, 1.5 h, 47−80%; (d) ethyl benzoylacetate or ethyl dimethoxybenzoylacetate, absolute ethanol, reflux 15 h, 37−
87%; (e) diphenyl ether, 250 °C, 10−15 min, 30−76%.
B
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reactions of aminoindoles 3a and 3c−f with ethyl benzoylace-
tate and of 3b with ethyl dimethoxybenzoylacetate were carried
out in absolute ethanol at reflux and yielded the acrylate
derivatives 4a−f as crude material, which first had to be purified
by silica gel column chromatography and then submitted to
thermal cyclization in boiling diphenyl ether (250 °C) to obtain
5a−f. Cyclization products 5a−f were further purified by
recrystallization or column chromatography, with their purity
verified by HPLC (>95%). In Scheme 1B, 6f obtained by
means of catalytic reduction of the nitro compound 2f was
reacted with ethyl benzoylacetate (7f) and thermally cyclized to
the partially hydrogenated 7-PPyQ 8f.
intermediate quinolinium iodide, which was not isolated but
yielded 13 by treatment of the reaction mixture with 1 N
NaOH at reflux.12 The exact structures with the precise
alkylation or acylation sites in 9−13 were obtained by 1D and
2D NMR HMBC experiments (Figures 2−4 in Supporting
Information).
As shown in Scheme 2C, the ethyl 3N-propanoate 218 was
reduced with LiAlH4 in anhydrous THF to yield the
corresponding alcohol 14. Alternatively, 21 underwent alkaline
hydrolysis in 1 N NaOH in methanol to provide the
corresponding carboxylic acid 15. Similarly, 5d yielded acid
16 (Scheme 2D).
As shown in Scheme 2, the chemical transformations of the
As shown in Scheme 2E, the 3N-propanenitrile 5c, prepared
as described in Scheme 1, was reduced with NaBH4 in the
presence CoCl2 catalyst in methanol and in the presence of an
equimolar quantity of di-tert-butyl dicarbonate. This somewhat
laborious procedure13 was superior to others14,15 because the
tBoc protected 7-PPyQ amine 17 was obtained as a solid
product, which was more easily isolated from the reaction
mixture than the free amine. Compound 17 was dissolved in an
ethyl acetate/methanol mixture, and excess HCl dry gas was
bubbled through the mixture to yield the hydrochloride
compound 18 as a beige powder. 1D and 2D NMR
spectrometry and absorption spectroscopy indicated that
compound 18 was in the form of 9-hydroxypyrroloquinoline
(Figures 7−10 in Supporting Information). We speculate that
the hydroxylic form was obtained because of the treatment with
HCl gas (see Experimental Section). This was confirmed
through an investigation of the behavior of compound 18 in
aqueous solution as a function of pH. On the basis of 1H NMR
and UV−vis spectra, it was observed that the two quinolinonic
and hydroxyquinolinic tautomers interconvert in a pH
dependent way: above pH 4 the keto tautomer predominates,
while below pH 2 the enolic one is dominant (Figure 28 in
Supporting Information). In the pH range 4−9, the spectra did
not change, indicating that the compound was in the keto
tautomer. Thus, at physiologic pH all 7-PPyQs, including
compound 18, are in the keto tautomer, with a carbonyl group
at position 9 as previously proposed from the SAR findings.8
Biological Characterization. Antiproliferative Activity in
Cellular Assays and SAR Analysis. On the basis of previous
biological activity data on 7-PPyQs7,10 and docking simulations
of 22 in the colchicine site of tubulin,9 the new compounds
were designed to obtain additional SAR information by
modifying the nature and size of substituents at the 3, 6, 7,
and 9 positions of the PyQ tricycle. Evaluation of
antiproliferative activities of 5a−f, 8−16, and 18 was performed
with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay16 against a panel of five solid tumor
(HeLa, A549, HT-29, MCF-7, OVCAR-3) and nine leukemia
cell lines (MOLT-3, CCRF-CEM, HL-60, K562, RS4;11,
Jurkat, SEM, MV4;11, THP-1). 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 ethers 10 and 11 and the acids 15 and
16. Of particular note were the subnanomolar GI50 values
obtained with 5f, but also with 5e and 12, in selected leukemic
cell lines.
two previously reported 3-ethyl 7-PPyQs (19),6 ethyl 3N-
a
Scheme 2. Synthesis of 7-PPyQs 9−18
a
Reagents and conditions: (a) CH3I or Br(CH2)3CH3,
BrCH2C6H5NO2, NaH, DMF, rt, 2 h, 64−74%; (b) C6H5CH2OCOCl,
NaH, DMF, 0 °C, 2 h, 96%; (c) CH3I, reflux; (d) NaOH 1 N, reflux,
18%; (e) LiAlH4, anhydrous THF, rt, 3 h, 70%; (f) NaOH 1 N,
methanol, reflux, 2 h, 85%; (g) NaBH4, CoCl2·6H2O, di-tert-butyl
dicarbonate, methanol, 10 h, 77%; (h) HCl dry gas, ethyl acetate, 58%.
propanoate (21)7 and of some 7-PPyQs described in Scheme 1
are shown. In Scheme 2A, 19, when reacted with CH3I, C4H9Br
or NO2C6H5CH2Br in anhydrous DMF and in the presence of
NaH, yielded the respective mixed ethers 9−11 as the only
reaction products, bearing the alkoxylic function in the 9
position. Similarly, 19 reacted with benzylchloroformate and
NaH in methanol at 0 °C to furnish the 9-substituted carbonate
ester 12 as the only reaction product. In Scheme 2B, in order to
obtain compound 13, the 6N-methyl derivative of 19,
important for our SAR analysis, the 9-methoxy-PPyQ 9 was
refluxed with excess CH3I for 2 h. This reaction provided the
Overall, the most active of the new compounds was 5f
(average GI50, 15 nM). It frequently had lower GI50 values than
the previously described compounds 19−22. Three additional
compounds also had average GI50 values of <150 nM: 5e (22
C
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Table 1. Growth Inhibitory Activity of PPyQ derivatives 5a−f, 8f, 9−16, and 18−22 for Human Cancer Solid Tumor (A) and
Leukemic Cell Lines (B)
a
(A) GI50 (nM)
compd
HeLa
A549
HT-29
MCF-7
OVCAR-3
5a
5b
5c
5d
5e
5f
1563 135
2856 736
191 72
7756 1126
7456 3601
928 125
236 16
>10000
256 31
>10000
9863 1236
>10000
356 61
34589 119
82 21
2863 61
326 17
>10000
2456 231
4785 432
5
1
2
78
9
4
12
1
8
0.5
8
3
1
1
12
5
0.6
0.4
5
71 12
8f
38 0.5
111 51
>10000
145 78
45
15
19
8
243 51
9
9
1456 623
>10000
1863 921
>10000
32
9
10
11
12
13
14
15
16
18
>10000
>10000
>10000
>10000
>10000
>10000
>10000
15
1
21
5
18
923 221
48
8
211 20
1656 632
95 16
>10000
9
2
936 41
175 12
5563 55
>10000
463 65
311 125
>10000
>10000
1072 119
32 15
nd
1236 258
38
7
8
>10000
>10000
>10000
2136 369
32 11
nd
>10000
>10000
551
11
2
5
1569 62
32 12
389 112
45 11
b
19
8
b
20
0.9
5
1
2
1
c
21
452 102
21 12
nd
492 85
61
589 189
42 15
nd
b
22
21
2
1
14
8
a
(B) GI50 (nM)
compd
MOLT-3
CCRF-CEM
HL-60
K562
1
RS4;11
Jurkat
SEM
MV4;11
THP-1
5a
5b
5c
5d
5e
5f
252 53
5850 2301
131 33
201 67
5.1 1.3
2.3 1.4
145 52
163 44
>10000
>10000
8.5 3.2
145 21.2
21.5 11.6
>10000
>10000
515 45
nd
4705 2122
>10000
254 11
9
74 32
845 19
161 33
452 61
92 12
311 42
2850 918
4632 126
1324 351
274 42
2415 152
2.5 0.5
6936 432
275 171
83 42
2450 323
1905 832
2211 453
0.4 0.2
1986 232
323
2305 1123
392 161
>10000
nd
6
49
9
5213 1265
nd
263 105
1650 221
>10000
2.1 0.9
1
0.5
9
2
36
19
2
8
125 32
74 25
355 72
34 12
>10000
17
4
2.0 0.6
6
2
0.1 0.05
0.3 0.03
0.4 0.1
8f
232 35
115 42
>10000
172
27
5
82 10
21
5
3
1
14
18
3
2
19
23
2
6
513 112
37 13
>10000
9
3
9
0.1
10
11
12
13
14
15
16
18
19
20
21
22
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
18
5
3
0.5
145 21
29
3
0.8
0.5 0.2
231 61
0.4 0.06
1
0.1
623 212
14
4
0.8
1
0.6
526 123
405 78
>10000
>10000
2423 581
nd
1142 532
78 12
>10000
>10000
nd
30
8
2
1569 521
78 12
>10000
>10000
492 35
nd
821 236
408 51
>10000
>10000
271 18
nd
6
8
2
3
3
>10000
>10000
>10000
>10000
>10000
158 51
nd
2375 372
951 51
0.5 0.02
nd
7756 1231
82 11
>10000
482 75
0.5 0.2
0.9 0.3
0.5 0.01
0.5 0.03
1
0.1
2
0.3
nd
nd
nd
0.6 0.4
nd
nd
nd
nd
nd
2.0 0.1
6.1 0. 5
196 61
0.4
2
2
0.4
0.5
nd
nd
nd
nd
nd
1
nd
nd
nd
a
GI50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean SEM from the dose−
b
c
response curves of at least three independent experiments. nd not determined. Referencef 7. Reference 8.
nM), 12 (22 nM), and 14 (120 nM). GI50 values below 100
nM in at least three cells lines were also observed with
compounds 5c, 8f, and 9. In an attempt to razionalize
structure−antiproliferative activity relationships, the 7-PPyQs
mentioned here and the corresponding previously described
active 3-alkyl derivatives were ordered into four subsets on the
bases of length, chemical nature, and size of the various
substitutions, as depicted in Scheme 3. The first three subsets
were selected by taking in consideration the substitutions at
position 3, in comparison with previously described potent 3N-
alkyl substituted 7-PPyQs, compounds 19 (3-ethyl), 20 (3-
propyl), and 22 (3-cyclopropylmethyl).7 The fourth subset
includes compounds obtained by modifying the 9 position of
compound 19 (9-CO) to confirm the requirement for the
carbonyl as a hydrogen bond acceptor in the active site on
tubulin. Furthermore, the 6 position of 19 was methylated to
gain SAR information on the 6 position and the phenyl at the 7
position was replaced by a dimethoxyphenyl moiety to
determine the importance of the size and nature of the aryl
substituent at that position.
From a comparison of the first two subsets and their GI50
values (Table 1), cytotoxicity changes with the lipophilic
character of the chain at the 3 position: the shorter and more
D
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conclude that the bulkier substituent led to a compound with a
weaker interaction with tubulin. This is in agreement with what
was previously observed with the series of 3-unsubstituted 7-
PPyQs.5
Scheme 3. Subsets of 7-PPyQ Derivatives Prearranged for
the Structure−Antiproliferative Activity Relationships
Discussion
We therefore conclude that the 7-PPyQ molecule tolerates
very few chemical modifications and that for significant
cytotoxicity only the 3-N position allows for a limited variety
of substitutions. Substituents at this position require appro-
priate characteristics of size and polarity, and 3N-acylation
yielded the highly active amidic compounds 5e and 5f. Due to
its broad spectrum of potent antiproliferative activity, 5f was
selected for further biological investigations on mechanism of
action and for comparison with 22.
Evaluation of Antiproliferative Activity of 5f in Nontumor
Cells. We investigated the effect of 5f and of 22 in human
lymphocytes and human umbilical vein endothelial cells
(HUVECs), isolated from healthy donors. As shown in Table
2, in both unstimulated and mitogen-activated lymphocytes, the
Table 2. Cytotoxicity of 5f in Human Noncancer Cells
a
GI50 (μM)
cell line
5f
>100
22
b
PBLresting
77.6 20.0
5.0 0.9
c
PBLPHA
95.7 8.9
polar is the chain, the weaker is the cytotoxicity, with the acids
15 and 16 being completely inactive.
HUVEC
22.1 10.9
7.8 0.55
a
Compound concentration required to reduce cell growth by 50%.
Values are the mean SEM for three separate experiments. PBL not
stimulated with PHA. PBL stimulated with PHA.
In the third subset, an important chemical modification was
introduced, consisting of an amidic connection between the
indole ring and the side chain, to yield the amidic derivatives 5e
and 5f. This chemical modification provided compounds with
potent antiproliferative activities, comparable with the activity
obtained with parent 22. As noted above, these two
compounds, along with compound 12, had the strongest
antiproliferative activity. The fourth subset includes analogues
of compound 19 designed and synthesized in order to evaluate
substitutions at the 9 position. Previously, we had stressed the
essential role of the carbonyl moiety for the antitubulin activity
of 7-PPyQs in comparison with the corresponding
phenylpyrroloquinoline lacking this moiety,7 but we had not
evaluated the effect of other substituents at position 9. The
substituents in 9, 10, and 11, lacking a carbonyl moiety, had
reduced or minimal antiproliferative activity as compared with
19. Moreover, as expected, the GI50 progressively decreased
with steric hindrance in comparing the methyl derivative 9 with
10 and 11 with still bulkier groups. However, the bulkiest 9-
benzyl carbonate derivative 12 was found to be a highly
cytotoxic agent because it probably decomposed to 19 in the
intracellular environment. Indeed, it is stable as a solid, while in
dilute aqueous solution it quickly decomposes to the
corresponding phenylpyrroloquinolinone (as proved by
HPLC). Moreover, this degradation is in agreement with the
data obtained with tubulin (Table 3) that show compound 12
to be as strong an inhibitor as its parent compound 19.
To obtain data regarding the 6 position of the 7-PPyQs, we
synthesized and evaluated compound 13 by attaching a
methylic group at the quinolinic nitrogen of 19. This led to a
compound with reduced antiproliferative activity with respect
to the unsubstituted parent compound 19. Finally, in order to
get further information about the size and nature of the pocket
in tubulin with which the phenyl at position 7 interacts, 5b was
designed and synthesized. In this compound the unsubstituted
7-phenyl group of 19 was replaced by a dimethoxyphenyl
group. This compound was relatively inactive, leading us to
b
c
two compounds showed low toxicity as compared with their
potent activity in cancer cells. Somewhat greater cytotoxicity
was observed in HUVECs when incubated with both
compounds. In both cases the GI50 values of 7.8 (22) and
22.1 (5f) μM are significantly higher than the values obtained
in the panel of tumor cells. These results suggest that both
compounds may have a preferential selectivity toward cancer
cells, especially 5f.
5f Binds to the Colchicine Site of Tubulin and Inhibits
Tubulin Assembly. In previous studies we found that the
antiproliferative activity of other 7-PPyQs resulted from an
interaction with tubulin at the colchicine site.17−20 Thus, the
compounds of this new series were evaluated for their
inhibition of tubulin polymerization and for their ability to
inhibit [3H]colchicine binding to tubulin. For comparison, CA-
4, a drug candidate in clinical trials in its phosphorylated form,
was evaluated as a reference compound in simultaneous
experiments (Table 3). CA-4 is a well described, highly potent
competitive inhibitor of the binding of colchicine to tubulin.17
In the assembly assay,18 compound 5f was found to be the most
active of the new agents as an inhibitor of tubulin polymer-
ization, with an IC50 (0.99 μM) comparable with that of both
22 (0.90 μM) and CA-4 (1.20 μM). In the [3H]colchichine
binding assay,19 compound 5f was the most active new
derivative (69% inhibition at 5 μM), although this activity was
somewhat less than that observed with both 22 and CA-4 (83%
and 99% inhibition, respectively, at 5 μM). There was good
correlation between inhibition of tubulin polymerization,
inhibition of colchicine binding, and antiproliferative activity
of the new derivatives, in particular since 5f had the highest
activity in all three assays. Note that compound 12 behaved
similarly to 19, which is in a good correlation with
antiproliferative activity data. Of the compounds examined
E
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tubulin heterodimer. Not surprisingly, the accommodation of 5f
in the colchicine cleft is similar to that previously described for
22.9 Also in this case, docking simulations showed that, similar
to colchicine and 22, 5f also can be accommodated in the same
hydrophobic cleft, adopting an energetically stable conforma-
tion. Moreover, the most stable conformation of 5f reproduced
the scheme of chemical interactions (predominantly through
hydrophobic interactions with Val181, Ala250, Cys241, Ala316,
Val318, and Ile378 of the β-subunit) observed for colchicine,
together with a similar interaction with the bordering T5 loop
of α-tubulin, demonstrating a plausible competitive mechanism
of action at the colchicine site.
5f Induces Cell Cycle Arrest at the G2/M Phase of the Cell
Cycle. The effect of 5f on cell cycle progression was examined
by flow cytometry in the HeLa, Jurkat, and RS4;11 cell lines
after a 24 h treatment with different concentrations of the agent
(Figure 3). For all cell lines, the treatment resulted in the
accumulation of cells in the G2/M phase of the cell cycle. In
particular, the increase of G2/M cells began at 30 nM and was
concentration dependent until a plateau was reached at 60 nM.
In the three cell lines, G2/M arrest was accompanied by a
concomitant reduction in the proportion of cells in the G1
Table 3. Inhibition of Tubulin Polymerization and
Colchicine Binding by Compounds 5a−f, 8f, 9, 12−22, and
CA-4
inhibition of tubulin assembly
inhibition of colchicine binding
bc
ac
,
,
compd
IC50 SD (μM)
inhibition SD (%)
5a
5b
5c
5d
5e
5f
2.9 0.1
>20
11
nd
20
nd
52
69
30
nd
77
61
4
1
1.3 0.05
6.6 0.3
0.99 0.1
0.99 0.1
2.6 0.2
5.7 0.7
0.74 0.07
1.8 0.3
1.2 0.1
8.0 0.1
>20
5
3
4
8f
9
12
13
14
15
16
18
2
1
40 0.9
nd
nd
>20
nd
d
19
0.57 0.02
0.58 0.05
0.75 0.04
0.90 003
1.2 0.1
73 0.7
d
20
77
75
3
4
e
21
d
22
83 0.5
CA-4
99 0.06
a
Inhibition of tubulin polymerization. Tubulin was at 10 μM.
b
c
Inhibition of [3H]colchicine binding. Tubulin and colchicine were
at 1 and 5 μM, respectively, and the tested compounds were at 5 μM.
d
e
nd, not determined. Reference 7. Reference 8.
with tubulin, somewhat decreased activity was observed with 13
and 14 with respect to 5f. These compounds inhibited
assembly with IC50 values of 1.8 and 1.2 μM, respectively.
These compounds were also less active as inhibitors of
colchichine binding than 5f and CA-4, 61% and 40% inhibition,
respectively.
As previously described for 22, to further examine the
competitive binding of 5f with colchicine in the active site of
the 1SA0 structure, computer-based docking of 5f was
performed using the MOE-Dock program.21,22 Figure 2 depicts
the binding mode of 5f in the presumptive colchicine site. In
particular, the colchicine site is mostly buried in the
intermediate domain of the β-subunit, even if colchicine can
also interact with loop T5 of the neighboring α-subunit (Figure
2), consistent with the observation that colchicine stabilizes the
Figure 3. Percentage of cells in each phase of the cell cycle in HeLa
(A), RS4;11 (B), and Jurkat (C) cells treated with 5f at the indicated
concentrations for 24 h. Cells were fixed and labeled with PI and
analyzed by flow cytometry as described in the Experimental Section.
Figure 2. Comparison of the crystallographic structure of colchicine
(in green) in complex with tubulin (Protein Data Bank code 1SA0)
and the energetically most favorable pose of 5f (in pink) obtained by
molecular docking simulation. Hydrogen atoms are omitted.
Data are represented as the mean
SEM of three independent
experiments.
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phase. Only in HeLa cells was a significant decrease of S phase
cells observed.
with 5f at either 0.1 or 1 μM caused no significant variations in
cyclin B expression after a 24 h treatment. However, we
observed a marked decrease in the expression of the
phosphorylated form of cdc2 (Tyr15) and that of cdc25C, in
particular with 1 μM 5f. These data, along with accumulation of
cells in the G2/M phase, suggest that 5f-induced G2/M arrest
was not due to defects in G2/M regulatory proteins but rather
is closely linked with acceleration of entry into mitosis.
In addition to the analysis of proteins that control cell cycle
checkpoints, we also examined the phosphorylation of the
tumor suppressor p53 at Ser15 after treating Jurkat cells with
5f. It is well-known that prolonged mitotic arrest induces DNA
damage and, consequently, the phosphorylation of p53 at Ser15
that leads to p53 stabilization and accumulation.26,27 As shown
in Figure 4, we detected a concentration-dependent marked
increase of p53-Ser15 that is particularly evident with 1 μM 5f.
At the same time, we also observed a marked increase in the
phosphorylation of histone γH2A.X at Ser139, which is an early
sensitive indicator of DNA damage.28
We next studied the association between 5f-induced G2/M
arrest and alterations in the expression of proteins that regulate
cell division. The cdc2/cyclin B complex controls both entry
into and exit from mitosis. Phosphorylation of cdc2 on Tyr15
and phosphorylation of cdc25C phosphatase on Ser216
negatively regulate the activation of the cdc2/cyclin B complex.
Thus, dephosphorylation of these proteins is needed to activate
the cdc2/cyclin B complex. Cdc25C is a major phosphatase
that dephosphorylates the site on cdc2 and autodephosphor-
ylates itself. Phosphorylation of cdc25C directly stimulates both
its phosphatase and autophosphatase activities, a condition
necessary to activate cdc2/cyclin B on entry of cells into
mitosis.23−25 As shown in Figure 4, in Jurkat cells treatment
5f Induces Apoptotic Cell Death. To characterize the mode
of cell death induced by 5f, biparametric flow cytometric
analysis with annexin-V/propidium iodide (PI) was performed
on different cell lines. PI binds to DNA and is permeable only
in dead, nonapoptotic cells, while annexin-V has high affinity
for phosphatidylserine (PS) that is exposed only on the outer
membrane leaflet of apoptotic cells. Thus, the dual staining
permits quantitation of live cells (annexin-V−/PI−), early
apoptotic cells (annexin-V+/PI−), late apoptotic cells (annex-
in-V+/PI+), and necrotic cells (annexin-V−/PI+). As shown in
Figure 5, 5f induced an increase in the annexin V+ fraction, and
thus of apoptosis, in a concentration dependent manner in
HeLa cells (Figure 5A) and in leukemic cell lines such as Jurkat
(Figure 5B), RS4;11 (Figure 5C), and K562 (Figure 5D).
Figure 4. Effect of 5f on G2/M regulatory proteins and DNA-damage
marker proteins. Jurkat cells were treated for 24 h with the indicated
concentration of 5f. The cells were harvested and lysed for the
detection of cyclin B, p-cdc2Tyr15, cdc25C, H2A.XSer139, and p53Ser15
expression by Western blot analysis. To confirm equal protein loading,
each membrane was stripped and reprobed with an anti-β-actin
antibody.
Figure 5. Flow cytometric analysis of apoptotic cells after treatment of HeLa (A), Jurkat (B), RS4;11 (C), and K562 (D) cells with 5f at the
indicated concentrations after incubation for 24 h. The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow
cytometry. Data are represented as the mean SEM of three independent experiments.
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To evaluate which caspases were involved in the apoptotic
cell death induced by 5f, we analyzed Jurkat cell protein extracts
by immunoblot. After a 24 h treatment with 5f, we observed the
cleavage of activator caspases 2, 8, and 9 (Figure 6A).
Moreover, we observed also the activation of the effector
caspase-3 and the subsequent cleavage of its substrate PARP.
attractive pharmacological tool for induction of Mcl-1
depletion.33,34
Survivin is a member of IAP family (inhibitor of apoptosis
protein).35 In general, the IAPs function through direct
interactions to inhibit the activity of several caspases, including
caspase-3, caspase-7, and caspase-9, and IAPs thereby inhibit
the processing and activation of these enzymes.36 We found
that survivin was phosphorylated on Thr32 following treatment
with 5f. This effect is consistent with cell cycle arrest in mitosis
and is shared by various antimitotic drugs.37,38
5f Induces Inhibition of the PI3K/Akt/mTOR Pathway.
Recently, we identified 22, a structural analogue of 5f, as a
potent antimitotic compound that might interfere with the
PI3K/Akt/mTOR pathway.9 Therefore, we evaluated the effect
of compound 5f on this pathway in Jurkat cells, which are a
PTEN-null, T-cell acute lymphoblastic leukemia cell line. As
shown in Figure 7, 5f induced the reduction of the p85
Figure 7. Western blot analysis of PI3K/AKT/mTOR after treatment
of Jurkat cells with 5f at the indicated concentrations for 24 h. To
confirm equal protein loading, each membrane was stripped and
reprobed with an anti-β-actin antibody.
Figure 6. (A). Western blot analysis of caspase-8, caspase-9, caspase-2,
caspase-3, and PARP after a 24 h treatment of Jurkat cells with 5f at
the indicated concentrations. (B) Western blot analysis of Bcl-XL, Bcl-
2, survivinThr32, and Mcl-1 after treatment of Jurkat cells with 5f at the
indicated concentrations for 24 h. To confirm equal protein loading,
each membrane was stripped and reprobed with an anti-β-actin
antibody.
regulatory subunit of PI3K and caused the decrease of the
phosphorylated (active) forms of mTOR (Ser2448) and AKT
(Ser473, Thr308). 5f treatment also induced a decrease in the
phosphorylation of mTOR downstream targets such as S6
kinase (Ser240/244), 4E-BP1 (Ser65), and GSK3 (Ser21).
5f Inhibits Enzymatic Activity of Different Protein Kinases.
Because of the significant effect of 5f on the PI3K/Akt/mTOR
pathway, we performed DiscoveRx KINOMEscan39 profiles on
a panel of 386 distinct human protein kinases at 1 μM 5f
(Figure 8). The percent inhibition at this concentration is
represented with red dots (Figure 8A) that, depending on their
size, indicate the residual activity of the kinase.
With this screening platform, we found that 5f at 1 μM
induced over 50% inhibition of 23 protein kinases (Figure 8B).
We found that 5f did not directly inhibit PI3K, Akt, and
mTOR. However, among the inhibited kinases were AURKA,
FLT3, GSK3A, MAP3K, MEK4, RSK2, RSK4, PLK4, ULK1,
and JAK2 that in cells could interact with the PI3K/Akt/
mTOR pathway. These results suggest that suppression of
these kinases could contribute to the high cytotoxicity caused
by 5f.
The intracellular apoptotic program can be modulated by the
expression of Bcl-2 family proteins that include both
proapoptotic and antiapoptotic members. Bcl-2 family proteins
are the main regulators of the mitochondrial apoptotic pathway
through the control of mitochondrial membrane permeability
and release of cytochrome c.29 It is well-known that cancer cells
express high levels of Bcl antiapoptotic proteins such as Bcl-2,
Bcl-XL, Mcl-1, Bcl-w, Boo/Diva, Bcl-B, and NR-13 and that
microtubule targeting agents induce the downregulation of the
Bcl-2 antiapoptotic protein, thus promoting apoptosis.30,31 As
shown in Figure 6B in Jurkat cells, after a 24 h incubation,
compound 5f induced a significant decrease in Bcl-2, Bcl-XL,
and Mcl-1 expression at the lower concentration (0.1 μM)
examined. These results are in agreement with recent studies
that point out the importance of Mcl-1 in cell death induced by
antitubulin agents.32 In addition, Mcl-1 appears to play a major
role in cancer cell survival, especially in leukemia cells. Thus,
this observation with 5f suggestes that the agent could be an
To better understand the apparently unpredictable behavior
of 22 and 5f as protein kinase inhibitors, a computer-based
automated docking of both compounds was performed as
described in detail in the Experimental Section. For this
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Figure 8. (A) TREEspot interaction maps: kinases found to bind 5f are marked with red circles, where larger circles indicate higher affinity binding
and the side percentages indicate residual kinase activity. (B) Histogram of the inhibitory activity of 5f on kinases inhibited more than 50%.
computational study, we selected from among the kinases that
were inhibited more than 50% (Figure 8B) and whose
crystallographic structures were deposited in the Protein Data
Bank. As reported in detail in the Experimental Section, the
ATP binding sites of five protein kinases (RSK2, PLK4, FLT3,
JAK1, and GSK3)40−42 were used to explore the binding of
both 22 and 5f. We found that these 7-phenylpyrrolo-
quinolinones could be readily accommodated in the ATP
binding clefts of all selected protein kinases but that the
ligand−kinase stabilization energy associated with the binding
of 5f was much higher (around 4−9 kcal/mol higher) as
compared with 22. In particular, the experimental inhibitory
activity shown by 5f can be attributed to the presence of the
amide moiety at the 3-position through the formation of a
strong H-bonding interaction with the side chain of amino acids
located inside the ATP binding cavity (Figure 9). In Table 4 are
Figure 9. Energetically most favorable pose of 5f (in pink) in the ATP
binding side of selected protein kinases.
summarized the most relevant binding features of 5f with the
five selected protein kinases.
5f Synergizes with Conventional Chemotherapeutic
Table 4. Relevant Binding Features of 5f against Five
Agents in Inhibiting Leukemia Cell Proliferation. Since 5f
Selected Protein Kinases
induced a strong decrease in cancer cell proliferation, we
a
evaluated whether 5f had promise when used in combination
with commonly used chemotherapeutics in leukemia treatment.
To this end, three different leukemia cell lines, two
glucocorticoid-resistant (Jurkat, THP-1) and one glucocorti-
coid-sensitive (MV4;11) were treated for 48 h with 5f in
combination with selected chemotherapeutic agents (i.e.,
dexamethasone, daunorubicin, and Ara-C) used frequently to
treat leukemic patients. Compound 5f was combined with
different drugs at a fixed molar combination ratio, and cell
viability was analyzed by the MTT assay (Figure 10 and
Supporting Information Figure 29).
protein
kinase
crucial H-bonding interaction with amide
Δ(ΔEinter)
IV→5f
moiety in 3-position of 5f
(kcal/mol)
RSK2
PLK4
FLT3
JAK1
GSK3
Thr210 (dCO/HO ≅ 2.8 Å)
Lys41 (dCO/HN ≅ 3.1 Å)
Lys644 (dCO/HN ≅ 2.1 Å)
Lys908 (dCO/HN ≅ 3.0 Å)
Lys85 (dCO/HN ≅ 2.2 Å)
−6.5
−4.5
−9.0
−5.0
−6.0
a
Δ(ΔEinter
)
= [(ΔEinter)IV] − [(ΔEinter)5f].
IV→5f
As described above, 5f alone had significant cytotoxicity
when used as a single agent (Table 1). When used in
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Table 5. Compound 5f Synergizes with Drugs Used in
Leukemia Therapy
a
b
b
b
CI
Dex + 5f (10:1) Dauno + 5f (10:1) AraC + 5f (100:1)
Jurkat Cell Line
CI (GI50)
CI (GI75)
CI (GI90)
0.67
0.81
0.97
0.21
11.9
0.79
14.7
0.45
1.0
MV4;11 Cell Line
CI (GI50)
CI (GI75)
CI (GI90)
0.016
0.06
0.25
0.22
0.09
0.38
1.8
0.17
0.14
THP-1 Cell Line
CI (GI50)
CI (GI75)
CI (GI90)
0.22
0.17
0.13
0.17
0.17
0.17
0.14
0.15
0.15
a
b
Combination indexes evaluated at different points. Molar combina-
tion ratios.
in antiproliferative potency occur at the 3N position. However,
substituents at this position must show suitable size and
polarity properties in order to preserve the strong cytotoxicity
of the most active compounds. This was observed, for example,
with compounds 20, 5a, and 14 (Table 1), in which the
potency decreased in the following order: 20 (3N-propyl, low
nanomolar GI50) > 14 (3N-propyl alcohol, high nanomolar
GI50) > 5a (3N-ethyl alcohol, high micromolar GI50).
Compound 15 (N-propanoic acid) and compound 16 (N-
ethanoic acid) were both inactive. Among all compounds
reported here, the amidic derivatives 5e and 5f, obtained by
acylation of the pyrrolic N, showed very high cytotoxicity.
Importantly, when tested in human noncancer cell lines, 22
and especially 5f showed very low toxicity compared with
tumor cell lines (Table 2).
All 7-PPyQs examined share the same primary mechanism of
action, inhibition of tubulin polymerization by binding in the
colchicine site (Table 3). In general, their potency as
antitubulin agents correlated well with their potency as
antiproliferative agents. The effect of 5f on cell cycle
progression was examined with three cell lines (Figure 2),
and the agent caused G2/M arrest. We showed alterations in
expression of proteins that regulate cell division (Figure 3).
These studies indicated that 5f at 1 μM induced G2/M arrest
not because of defects in G2/M regulatory proteins but rather
because there was an acceleration of entry into mitosis (Figure
4). As observed in some cancer cell lines, in a concentration
dependent manner, 5f, like 22, caused cell death by apoptosis
involving activator caspases 2, 8, and 9, activation of the effector
caspase-3, and subsequent cleavage of PARP. Moreover, as with
other microtubule targeting agents, at 0.1 μM in Jurkat cells, 5f
induced downregulation of the antiapoptotic proteins Bcl-2,
Bcl-XL, and particularly Mcl-1, which is a key factor governing
cell survival and often overexpressed in cancer cells. Also, as
noted with various antimitotic drugs, survivin was found to be
phosphorylated upon treatment with 5f, additional evidence of
cell cycle arrest in mitosis (Figure 5).
Figure 10. Effect of 5f treatment alone and in combination with
different chemotherapic drugs in MV4;11 cells. Cells were treated at
the indicated concentrations and at a fixed combination ratio, and
viability was assessed by the MTT test after a 48 h incubation. Data are
expressed as the mean SEM of three independent experiments.
combination with chemotherapeutic drugs, we observed a
synergistic increase in cytotoxicity, as demonstrated by
combination index (CI) values obtained by the analytic method
of Chou and Talalay.43−45 As summarized in Table 5, in which
CI values calculated at the GI50, GI75, and GI90 values are
shown, in almost all cases, but not with Jurkat cells for Ara-C
treatment, 5f and the selected chemotherapeutic drugs acted in
a synergistic fashion (CI < 1). Moreover, in the glucocorticoid
resistant cells, Jurkat and THP-1, 5f was able to restore
glucocorticoid sensitivity, suggesting that 5f might be able to
optimize the efficacy of existing therapies for leukemia.
CONCLUSION
Another interesting finding of our work is that 5f modulates
the PI3K/Akt/mTOR pathway which plays a major role in
tumorigenesis and progression, since it is often hyperactivated
in many types of tumors. Although this observation was already
described by our group in A549 cell lines with compound 22, in
this work we show this effect was maintained also in PTEN null
cell line Jurkat. In this context it is worthwhile to note that
■
A series of new 7-PPyQs was efficiently synthesized and
biologically evaluated as anticancer agents. With respect to the
previously reported analogues, the new compounds were
further modified at the 3, 6, 7, and 9 positions. Our findings
reported here show that the only chemical modifications
tolerated by the 7-PPyQ pharmacophore without a major loss
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(9.25 mmol) in 5 mL of anhydrous DMF, was dropped into the flask,
and the initial yellow color changed to red with the formation of H2
gas. After 30 min at room temperature, a solution of 2-bromoethanol,
0.981 mL (13.87 mmol; d = 1.763 g/mL) in 1 mL dry DMF, was
added, and the reaction mixture was left to stir for 24 h. The reaction
was monitored by TLC analysis (eluent toluene/n-hexane/ethyl
acetate, 1:1:0.3). 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 ethyl acetate (3 × 50 mL). The
organic phase, washed with water and dried over anhydrous Na2SO4,
was concentrated under vacuum giving a crude yellow solid (1.898 g).
This crude product was purified with a silica gel chromatographic
column (d 3 cm, l 35 cm, 230−400 mesh, eluent ethyl acetate/n-
hexane, 7:3), yielding 1.587 g of a pure yellow solid.
PTEN mutations are associated with bad prognosis and that
PTEN null tumors are particularly aggressive due to the strong
activation of PI3K/Akt/mTOR pathway. Thus, the therapeutic
potential of 5f with respect to other antimitotic compounds
could be strongly enhanced and extended to different kinds of
tumor.
By a KINOMEscan analysis with 1 μM 5f, we found greater
than 50% inhibition of 23 kinases (Figure 8B), but there was no
direct inhibitory effect on the enzymatic activity on PI3K, Akt,
and mTOR. Therefore, our results indicated that a simple
chemical modification made by linking an acyl side chain to the
pyrrolic nitrogen endowed this compound class with a kinase
inhibiting activity and that suppression of the activity of
selected kinases might contribute to the high cytotoxicity of 5f.
Finally, 5f sensitized leukemia cell lines to the action of
frequently used chemotherapeutic drugs, leading to cell death
in a synergistic way. This effect was observed with agents with
different mechanisms of action. In particular, we observed that
5f was able to restore the activity of dexamethasone in
glucocorticoid resistant cells (Jurkat and THP-1). If confirmed
in planned in vivo studies, this would be a valuable property of
5f.
2-(5-Nitro-1H-indol-1-yl)ethanol (2a). Yield 82.2%; Rf = 0.31
(ethyl acetate/n-hexane 7:3); mp = 82 °C; 1H NMR (400 MHz,
DMSO-d6) δ = 3.86 (q, 2H, J = 5.23 Hz, CH2-CH2-OH), 4.44 (t, 2H, J
= 5.23 Hz, CH2-CH2-OH), 5.07 (t, 1H, J = 5.23 Hz, OH), 6.87 (dd,
1H, J = 3.18 and J = 0.59 Hz, 3-H), 7.76 (d, 1H, J = 3.18 Hz, 2-H),
7.82 (d, 1H, J = 9.15 Hz, 7-H), 8.15 (dd, 1H, J = 9.15 and J = 2.30 Hz,
6-H), 8.70 (d, 1H, J = 2.30 Hz, 4-H); 13C NMR (101 MHz, DMSO-
d6) δ = 49.08 (NCH2CH2OH), 61.07 (NCH2CH2OH), 103.24 (3-C),
112.36 (7-C), 116.93 (6-C), 119.24 (4-C), 134.12 (2-C), 135.22 (3a-
C), 138.24 (7a-C), 143.16 ppm (5-C). HRMS (ESI-MS, 140 eV): m/z
+
[M + H+] calculated for C10H11N2O3 , 207.2054; found, 207.1987.
1-(Cyclopropylmethyl)-4-nitro-1H-indole (2b). Compound 2b
was prepared as for compound 2a, and the analytical data are reported
in ref 7.
EXPERIMENTAL SECTION
■
Materials and Methods. 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 on a Thermo
3-(5-Nitro-1H-indol-1-yl)propanenitrile (2c). Compound 2c
was prepared as for compound 2a by reacting 0.720 g of NaH 60%
(30 mmol) and 1.621 g (10.0 mmol) of 5-nitroindole 1 dissolved in 5
mL of DMF and 1.24 mL of 3-bromopropionitrile (15 mmol, d =
1.615 g/mL). Reaction time 5 h (TLC ethyl acetate/n-hexane/toluene,
1:1:1). A solid product (2.564 g) was obtained. Yield 99%; Rf = 0.27
(toluene/n-hexane/ethyl acetate, 1:1:1); 1H NMR (400 MHz, DMSO-
d6) δ = 3.10 (t, 2H, J = 6.60 Hz, NCH2CH2CN), 4.61 (t, 2H, J = 6.52
Hz, NCH2CH2CN), 6.81 (dd, 1H, J = 3.24 Hz and J = 0.8 Hz, 3-H),
7.72 (d, 1H, J = 3.31 Hz, 2-H), 7.83 (dt, 1H, J = 9.13 Hz and J = 0.7
Hz, 7-H), 8.06 (ddd, 1H, J = 9.08 Hz, J = 2.29 Hz and J = 0.25 Hz, 6-
H), 8.59 ppm (dd, 1H, J = 2.24 Hz and J = 0.25 Hz, 4-H); 13C NMR
(101 MHz, DMSO-d6) δ = 19.08 (NCH2CH2CN), 42.07
(NCH2CH2CN), 104.76 (3-C), 111.06 (7-C), 117.03 (6-C), 118.04
(4-C), 119.10 (CN), 132.76 (2-C), 132.76 (3a-C), 139.07 (7a-C),
141,48 ppm (5-C). HRMS (ESI-MS, 140 eV): m/z [M + H+]
1
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 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 μm, and a loop of 10 μL. The detector was set at 300 nm. The
mobile phase consisted of phase A (Milli-Q H2O, 18.0 MΩ, TFA
0.05%) and phase B (95% MeCN, 5% phase A). The gradient elution
was performed as reported: 0 min, % B = 10; 0−20 min, % B = 90;
20−25 min, % B = 90; 25−26 min, % B = 10; 26−31 min, % B = 10.
General Procedure for the Synthesis of 1N-Substituted
Nitroindoles (2a−f). As a typical procedure, the synthesis of 2-(5-
nitro-1H-indol-1-yl)ethanol 2a is described in detail. Into a two-
necked 50 mL round-bottomed flask, 0.888 g (37 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 1, 1.50 g
+
calculated for C11H10N3O2 , 216.2155; found, 216.1290.
Ethyl 2-(5-Nitro-1H-indol-1-yl)acetate (2d). Compound 2d was
prepared as for compound 2a by reacting 0.445 g of NaH 60% (18.51
mmol) and 1.00 g (6.17 mmol) of 5-nitroindole 1 dissolved in 10 mL
of toluene and 1.5456 g (9.25 mmol, d = 1.506 g/L) of bromoethyl
acetate in 5 mL toluene. Reaction time was 4 h by TLC analysis (ethyl
acetate/n-hexane/toluene, 1:1:1). After extraction, 1.400 g of the crude
material was obtained, which was chromatographed on a silica gel
column (d = 3 cm, l = 30 cm, 230−400 mesh, ethyl acetate/n-hexane/
toluene, 1:1:1) giving 0.665 g of a pure bright white solid. Yield 47.5%;
mp = 52−53 °C; Rf = 0.63 (eluent ethyl acetate/n-hexane/toluene,
1:1:1); 1H NMR (300 MHz, DMSO-d6) δ = 1.21 (t, 3H, J = 7.05 Hz,
-OCH2CH3), 4.17 (q, 2H, J = 7.05 Hz, -OCH2CH3), 5.26 (s, 2H,
NCH2), 6.78 (dd, 1H, J = 3.24 Hz and J = 0.76 Hz, 3-H), 7.61 (d, 1H,
J = 3.24 Hz, 2-H), 7.64 (d, 1H, J = 9.15 Hz and J = 0.76 Hz, 7-H), 8.04
(dd, 1H, J = 9.15 Hz and J = 2.28 Hz, 6-H), 8.58 ppm (d, 1H, J = 2.09
Hz, 4-H); 13C NMR (75 MHz, DMSO-d6)
δ = 16.83
(NCH2COOCH2CH3), 47.15 (NCH2COOCH2CH3), 64.34
(NCH2COOCH2CH3), 102.02 (3-C), 111.66 (7-C), 117.74 (6-C),
120.00 (4-C), 132.64 (2-C), 134.73 (3a-C), 137.98 (7a-C), 145.05 (5-
C), 167.72 ppm (NCH2COOCH2CH3). HRMS (ESI-MS, 140 eV):
+
m/z [M + H+] calculated for C12H13N2O4 , 249.2421; found,
249.1497.
1-(5-Nitro-1H-indol-1-yl)propan-1-one (2e). Compound 2e
was prepared as for compound 2a by reacting 0.444 g of NaH 60%
(18.50 mmol) and 1.00 g (6.17 mmol) of commercial 5-nitroindole
K
DOI: 10.1021/acs.jmedchem.5b00805
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Journal of Medicinal Chemistry
Article
(1) dissolved in 5 mL of anhydrous DMF and 0.808 mL (9.25 mmol, d
= 1.059 g/mL) of propionyl chloride dissolved in 1 mL of anhydrous
DMF. On addition of the propionyl chloride solution, a white
precipitate formed. After about 1 h, the solution was cooled (ice bath)
and treated with 15 mL of water to quench the excess NaH. The
precipitate formed was collected by filtration under vacuum, washed
several times with water, and desiccated under vacuum, yielding 0.920
g of a white crystalline compound. Yield 68%; Rf = 0.63 (ethyl acetate/
n-hexane/toluene, 1:1:1); mp = 177 °C; 1H NMR (400 MHz, DMSO-
d6) δ = 1.19 (t, 3H, J = 7.25 Hz, C(O)CH2CH3), 3.13 (q, 2H, J = 7.25
Hz, C(O)CH2CH3), 6.97 (d, 1H, J = 3.34 Hz, 3-H), 8.14 (d, 1H, J =
3.34 Hz, 2-H), 8.20 (dd, 1H, J = 9.29 Hz and J = 2.25 Hz, 6-H), 8.52
(dt, 1H, J = 9.29 Hz, 7-H), 8.58 ppm (d, 1H, J = 2.25 Hz, 4-H); 13C
NMR (101 MHz, DMSO-d6) δ = 8.75 (C(O)CH2CH3), 28.97 (C(O)
CH2CH3), 109.15 (3-C), 116.63 (7-C), 117.50 (4-C), 120.11 (6-C),
130.27 (2-C), 130.41 (3a-C), 138.41 (7a-C), 143.80 (5-C), 173.73
ppm (NC(O)). HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated
nitroindole derivative 2c, obtaining 1.842 g of a brown oil. Yield 97%;
Rf = 0.11 (ethyl acetate/n-hexane/toluene, 1:1:1); 1H NMR (400
MHz, DMSO-d6) δ = 2.95 (t, 2H, J = 6.88 Hz, NCH2CH2CN), 4.36
(t, 2H, J = 6.88 Hz, NCH2CH2CN), 4.52 (s, 2H, NH2), 6.17 (dd, 1H,
J = 3.12 Hz and J = 0.85 Hz, 3-H), 6.55 (ddd, 1H, J = 8.73 Hz, J = 2.13
Hz and J = 0.33 Hz, 6-H), 6.70 (dd, 1H, J = 2.12 Hz and J = 0.61 Hz,
4-H), 7.21 (d, 1H, J = 3.12 Hz, 2-H), 7.23 ppm (dt, 1H, J = 8.74 Hz
and J = 0.73 Hz, 7-H); 13C NMR (101 MHz, DMSO-d6) δ = 19.05
(NCH2CH2CN), 41.70 (NCH2CH2CN), 100.29 (3-C), 104.03 (4-C),
110.32 (7-C), 112.33 (6-C), 119.44 (CN), 128.45 (2-C), 129.70 (3a-
C), 129.75 (7a-C), 142.19 ppm (5-C). HRMS (ESI-MS, 140 eV): m/z
+
[M + H+] calculated for C11H12N3 , 186.1031; found, 186.1012.
Ethyl 2-(5-Amino-1H-indol-1-yl)acetate (3d). Compound 3d
was prepared as for compound 3c by reacting 0.500 g of nitroindole
derivative 2d (2.01 mmol), obtaining 0.396 g of a violet solid. Yield
1
90%; Rf = 0.43 (n-hexane/ethyl acetate, 1:1); H NMR (300 MHz,
DMSO-d6) δ = 1.21 (t, 3H, J = 7.05 Hz, CH2CH3), 4.13 (q, 2H, J =
7.05 Hz, CH2CH3), 4.50 (bs, 2H, NH2), 4.95 (s, 2H, CH2), 6.15 (dd,
1H, J = 3.05 Hz and J = 0.76 Hz, 3-H), 6.50 (dd, 1H, J = 8.58 Hz and J
= 2.09 Hz, 6-H), 6.67 (d, 1H, J = 2.09 Hz and J = 0.52 Hz, 4-H), 7.02
(d, 1H, J = 8.77 Hz and J = 0.76 Hz, 7-H), 7.11 ppm (d, 1H, J = 3.05
+
for C11H11N2O3 , 219.1261; found, 219.1291.
Cyclopropyl(5-nitro-1H-indol-1-yl)methanone (2f). Com-
pound 2f was prepared as for compound 2a by reacting 0.445 g of
NaH 60% (18.51 mmol) and 1.012 g (6.24 mmol) of 5-nitroindole 1
dissolved in 10 mL DMF and 1.10 mL of cylopropanecarbonyl
chloride (12.20 mmol, d = 1.152 g/mL). Reaction time was 3 h,
monitoring by TLC (ethyl acetate/n-hexane/toluene, 1:1:1). After
extraction, 0.860 g of crude reaction product, a golden solid (70%),
was obtained, which was chromatographed on a silica gel column (d =
3 cm, l = 35 cm, 230−400 mesh, ethyl acetate/n-hexane/toluene,
1:1:1) to yield 0.752 g of pure compound 2f. Yield 52%; Rf = 0.62
Hz, 2-H); 13C NMR (75 MHz, DMSO-d6)
δ = 15.22
(NCH2COOCH2CH3), 45.53 (NCH2COOCH2CH3), 64.22
(NCH2COOCH2CH3), 101.20 (3-C), 104.73 (4-C), 112.74 (7-C),
114.01 (6-C), 126.06 (2-C), 127.94 (3a-C), 130.02 (7a-C), 143.06 (5-
C), 168.25 ppm (NCH2COOCH2CH3). HRMS (ESI-MS, 140 eV):
+
m/z [M + H+] calculated for C12H15N2O2 , 219.2592; found,
219.2537.
1
(ethyl acetate/n-hexane/toluene, 1:1:1); mp = 45−48 °C; H NMR
(5-Aminoindolin-1-yl)(cyclopropyl)methanone (6f). Com-
pound 6f was prepared as for compound 3a by reacting 3.27 mmol
(0.759 g) of 5-nitroindole 2f at room temperature, obtaining 0.653 g
of a slightly pink solid. Yield 98%; Rf = 0.37 (ethyl acetate/n-hexane,
8:2); mp = 159 °C; 1H NMR (300 MHz, DMSO-d6) δ = 0.80 (m, 4H,
CH2-CH2), 1.85 (m, 1H, CH), 3.04 (t, 2H, J = 8.01 Hz, 2-H2), 4.18 (t,
2H, J = 8.01 Hz, 3-H2), 4.81 (bs, 2H, NH2), 6.30 (dd, 1H, J = 8.58 Hz
and J = 2.09 Hz, 6-H), 6.45 (s, 1H, 4-H), 6.71 ppm (d, 1H, J = 8.58
Hz, 7-H); 13C NMR (75 MHz, DMSO-d6) δ = 8.26 (CH2CH2), 14.02
(CH), 28.66 (3-C), 48.96 (2-C), 103.76 (4-C), 111.22 (7-C), 115.92
(6-C), 125.76 (3a-C), 127.42 (7a-C), 141.01 (5-C), 170.44 ppm
(NC(O)CHCH2CH2). HRMS (ESI-MS, 140 eV): m/z [M + H+]
calculated for C12H15N2O+, 203.2598; found, 203.2524.
(400 MHz, DMSO-d6) δ = 1.17 (m, 4H, CH2-CH2), 2.77 (m, 1H,
CH), 7.05 (dd, 1H, J = 3.81 Hz and J = 0.76 Hz, 3-H), 8.20 (dd, 1H, J
= 9.15 Hz and J = 2.48 Hz, 6-H), 8.47 (d, 1H, J = 3.18 Hz, 2-H), 8.50
(d, 1H, J = 9.15 Hz, 7-H), 8.61 ppm (d, 1H, J = 2.48 Hz, 4-H); 13C
NMR (101 MHz, DMSO-d6) δ = 15.14 (CH2CH2), 26.34 (CH),
101.99 (3-C), 113.14 (7-C), 115.82 (6-C), 118.34 (4-C), 133.84 (2-
C), 134.72 (3a-C), 137.46 (7a-C), 144.04 (5-C), 172.01 ppm
(NC(O)CHCH2CH2). HRMS (ESI-MS, 140 eV): m/z [M + H+]
+
calculated for C12H11N2O3 , 231.2268; found, 231.1382.
General Procedure for the Synthesis of 1N-Substituted
Aminoindoles 3a−d and 6f. As a typical procedure, the synthesis of
5-aminoindole 3a 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 ethyl acetate were placed. After connecting
the flask to an elastomer balloon containing hydrogen gas, the mixture
was stirred at room temperature for 1 h in order to saturate the
suspension of C/Pd with hydrogen. Then, 7.70 mmol (1.587 g) of
nitroindole derivative 2a in 15 mL of ethyl acetate was added dropwise
to the suspension, and the mixture was stirred under hydrogen at
atmospheric pressure and heated by means of an oil bath at 50−60 °C
for 15 h, monitoring the progress of the reaction by TLC analysis
(ethyl acetate/n-hexane, 7:3). At the end of the reaction, the mixture
was filtered, and the solution was concentrated to dryness on a
rotavapor to give 1.234 g of semisolid dark purple product.
General Procedure for the Synthesis of 5-Aminoindoles 3e
and 3f. As a typical procedure, the synthesis of 5-aminoindole 3e is
described in detail. Into a two-necked 50 mL round-bottomed flask,
0.350 g of 1-(5-nitro-1H-indol-1-yl)propan-1-one (2e) (1.60 mmol),
1.809 g of SnCl2·2H2O (8.02 mmol), and 25 mL of methanol were
added. The reaction mixture was refluxed for 3 h, and the reaction
progress was monitored by TLC (n-hexane/ethyl acetate, 2:8). 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 anhydrous Na2SO4, were evaporated to dryness on a
rotary evaporator to yield 0.240 g of a semisolid yellow product.
1-(5-Amino-1H-indol-1-yl)propan-1-one (3e). Yield 80%; mp =
2-(5-Amino-1H-indol-1-yl)ethanol (3a). Yield 91%; mp = 124
1
1
°C; Rf = 0.20 (ethyl acetate/n-hexane, 7:3). H NMR (400 MHz,
132 °C; Rf = 0.27 (ethyl acetate/n-hexane/toluene, 1:1:1). H NMR
DMSO-d6) δ = 4.55 (q, 2H, J = 5.67 Hz, CH2CH2OH), 4.92 (bs, 2H,
NH2), 4.96 (t, 2H, J = 5.67 Hz, CH2CH2OH), 5.80 (t, 1H, J = 5.67
Hz, OH), 7.06 (dd, 1H, J = 3.25 Hz and J = 0.41 Hz, 3-H), 7.62 (dd,
1H, J = 8.79 Hz and J = 2.13 Hz, 6-H), 7.87 (d, 1H, J = 2.13 Hz, 4-H),
7.93 (d, 1H, J = 3.25 Hz, 2-H), 8.04 ppm (d, 1H, J = 8.79 Hz, 7-H);
13C NMR (101 MHz, DMSO-d6) δ = 47.34 (NCH2CH2OH), 60.77
(NCH2CH2OH), 100.34 (3-C), 105.72 (4-C), 111.34 (7-C), 113.75
(6-C), 127.96 (2-C), 128.14 (3a-C), 129.94 (7a-C), 142.90 ppm (5-
C). HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated for
C10H13N2O+, 177.2225, found:177.1565.
1-(Cyclopropylmethyl)-4-amino-1H-indole (3b). Compound
3b was prepared as for compound 3a, and the analytical data are
reported in ref 7.
3-(5-Amino-1H-indol-1-yl)propanenitrile (3c). Compound 3c
was prepared as for compound 3a by reacting 2.853 g (13.26 mmol) of
(400 MHz, DMSO-d6) δ = 1.16 (t, 3H, J = 7.33 Hz, C(O)CH2CH3),
2.96 (q, 2H, J = 7.33 Hz, C(O)CH2CH3), 4.91 (bs, 2H, NH2), 6.17
(dd, 1H, J = 3.12 Hz and J = 0.85 Hz, 3-H), 6.55 (ddd, 1H, J = 8.74
Hz, J = 2.13 Hz and J = 0.33 Hz, 6-H), 6.70 (dd, 1H, J = 2.13 Hz and J
= 0.61 Hz, 4-H), 7.21 (d, 1H, J = 3.12 Hz, 2-H), 7.23 ppm (dt, 1H, J =
8.74 Hz and J = 0.85 Hz, 7-H); 13C NMR (101 MHz, DMSO-d6) δ =
9.36 (C(O)CH2CH3), 28.45 (C(O)CH2CH3), 100.29 (3-C), 104.03
(4-C), 110.31 (7-C), 112.33 (6-C), 128.44 (2-C), 129.70 (3a-C),
129.75 (7a-C), 142.19 (5-C), 174.80 ppm (NC(O)). HRMS (ESI-MS,
140 eV): m/z [M + H+] calculated for C11H13N2O+, 189.2332; found,
189.2388.
Cyclopropyl(5-amino-1H-indol-1-yl)methanone (3f). Com-
pound 3f was prepared as for compound 3e by reacting 0.500 g of
of 5-nitroindole 2f (2.15 mmol), 2.9 g of SnCl2·2H2O (12.85 mmol),
and 30 mL of methanol to yield 0.205 g of a pearly white solid. Yield
L
DOI: 10.1021/acs.jmedchem.5b00805
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Journal of Medicinal Chemistry
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1
47%. Rf = 0.70 (n-hexane/ethyl acetate, 2:8); H NMR (300 MHz,
DMSO-d6) δ = 1.03 (m, 4H, CH2-CH2), 2.60 (m, 1H, CH), 4.91 (s,
2H, NH2), 6.54 (dd, 1H, J = 3.62 Hz and J = 0.57 Hz, 3-H), 6.57 (dd,
1H, J = 8.77 Hz and J = 2.09 Hz, 6-H), 6.71 (d, 1H, J = 2.09 Hz, 4-H),
7.98 (d, 1H, J = 3.24 Hz, 2-H), 8.00 ppm (dd, 1H, J = 8.77 Hz and J =
0.57 Hz, 7-H); 13C NMR (75 MHz, DMSO-d6) δ = 15.95 (CH2CH2),
28.44 (CH), 101.15 (3-C), 104.33 (4-C), 112.77 (7-C), 113.24 (6-C),
128.06 (2-C), 128.94 (3a-C), 129.74 (7a-C), 142.52 (5-C), 171.61
ppm (NC(O)CHCH2CH2). HRMS (ESI-MS, 140 eV): m/z [M + H+]
calculated for C12H13N2O+, 201.2439; found, 201.2482.
by reacting 2.148 g (11.60 mmol) of 5-aminoindole derivative 3c and
3.01 mL (17.7 mmol) of ethyl benzoylacetate, obtaining after column
chromatography (d = 3 cm, l = 30 cm, 230−400 mesh, eluent n-
hexane/ethyl acetate, 1:1) 2.54 g of a dark red solid. Yield 71%; Rf =
0.72 (n-hexane/ethyl acetate, 1:1); 1H NMR (400 MHz, DMSO-d6) δ
= 1.24 (t, 3H, J = 6.92 Hz, CHCOOCH2CH3), 2.97 (t, 2H, J = 6.34
Hz, NCH2CH2CN), 4.14 (q, 2H, J = 6.92 Hz,, CHCOOCH2CH3),
4.39 (t, 2H, J = 6.34 Hz, NCH2CH2CN), 4.85 (s, 1H,
CHCOOCH2CH3), 6.28 (dd, 1H, J = 3.19 Hz and J = 0.69 Hz, 3-
H), 6.66 (dd, 1H, J = 8.71 Hz and J = 2.08 Hz, 6-H), 6.98 (m, 1H, J =
2.08 Hz, 4-H), 7.29 (m, 1H, 2-H), 7.29 (m, 1H, 4′-H), 7.35 (m, 1H, 7-
H), 7.37 (m, 2H, 2′-H and 6′-H), 7.35 (m, 2H, 3′-H and 5′-H), 10.27
ppm (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) δ = 14.94
(CHCOOCH2CH3), 19.01 (NCH2CH2CN), 41.71 (NCH2CH2CN),
59.14 (CHCOOCH2CH3), 89.12 (CHCOOCH2CH3), 101.68 (3-C),
110.44 (7-C), 115.25 (4-C), 118.75 (6-C), 119.29 (CN), 128.64 (3a-
C), 128.89 (2′-C and 6′-C), 128.59 (5′-C and 3′-C), 129.85 (4′-C),
129.87 (2-C), 132.93 (7a-C), 133.06 (1′-C), 136.31 (5-C), 160.50
(NHCCH), 169.79 ppm (CHCOOCH2CH3). HRMS (ESI-MS, 140
General Procedure for the Synthesis of Acrylate Derivatives
4a−f and 7f. As a typical procedure, the synthesis of acrylate
derivative 4a is described in detail. In a 100 mL round-bottomed flask,
1.234 g (7.00 mmol) of 3-substituted aminoindole 3a in 25 mL of
absolute ethanol was condensed with 1.82 mL (10.5 mmol; d = 1.11 g/
mL) of commercial ethyl benzoylacetate and 0.5 mL of glacial acetic
acid in the presence of 100 mg of Drierite. The mixture was refluxed
for about 24 h, the reaction being monitored by TLC analysis (n-
hexane/ethyl acetate, 3:7). 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 (d = 3 cm, l =
35 cm, 230−400 mesh, eluent n-hexane/ethyl acetate, 3:7) to yield
1.47 g of a semisolid yellow product.
+
eV): m/z [M + H+] calculated for C22H22N3O2 , 360.4284; found,
360.4242.
(E,Z)-Ethyl 3-(1-((Ethoxycarbonyl)methyl)-1H-indol-5-ylami-
no)-3-phenylacrylate (4d). Compound 4d was prepared as for
compound 4a by reacting 0.800 g (3.66 mmol) of 5-aminoindole 3d,
to yield after silica gel column chromatography (d = 2.5 cm, l = 30 cm,
230−400 mesh, eluent n-hexane/ethyl acetate, 1:1) 1.024 g of a
semisolid red product. Yield 70%; Rf = 0.54 (n-hexane/ethyl acetate,
(E,Z)-Ethyl 3-(1-(2-Hydroxyethyl)-1H-indol-5-ylamino)-3-
phenylacrylate (4a). Yield 60%; Rf = 0.58 (n-hexane/ethyl acetate,
3:7); H NMR (300 MHz, DMSO-d6) δ = 1.11 (t, 3H, J = 7.05 Hz,
1
1
CH2CH3), 3.89 (q, 2H, J = 7.05 Hz, CH2CH2OH), 4.13 (q, 2H, J =
7.05 Hz, CH2CH3), 4.61 (t, 2H, J = 7.05 Hz, CH2CH2OH), 4.83 (s,
1H, CH), 5.15 (t, 1H, J = 7.05 Hz, OH), 7.16 (d, 1H, J = 3.05 Hz, 3-
H), 7.67 (m, 5H, 2′-H, 3′-H, 4′-H, 5′-H, 6′-H), 7.71 (dd, 1H, J = 8.77
and J = 2.10 Hz, 6-H), 8.02 (d, 1H, J = 2.10 Hz, 4-H), 8.07 (d, 1H, J =
3.05 Hz, 2-H), 8.13 (d, 1H, J = 8.77 Hz, 7-H), 10.25 ppm (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6) δ = 14.75 (CHCOOCH2CH3),
46.94 (NCH2CH2OH), 60.24 (CHCOOCH2CH3), 61.56
(NCH2CH2OH), 89.44 (CHCOOCH2CH3), 102.62 (3-C), 110.75
(7-C), 114.73 (4-C), 118.12 (6-C), 127.22 (3a-C), 128.94 (2′-C and
6′-C), 129.44 (5′-C and 3′-C), 129.87 (4′-C), 130.44 (2-C), 132.03
(7a-C), 133.76 (1′-C), 137.42 (5-C), 159.92 (NHCCH), 169.84 ppm
(CHCOOCH2CH3). HRMS (ESI-MS, 140 eV): m/z [M + H+]
1:1); H NMR (300 MHz, DMSO-d6) δ = 1.11 (t, 3H, J = 7.05 Hz,
CH2CH3), 1.16 (t, 3H, J = 7.05 Hz, CH2CH3), 4.01 (q, 2H, J = 7.05
Hz, CH2CH3), 4.13 (q, 2H, J = 7.05 Hz, CH2CH3), 4.83 (s, 1H, CH),
4.95 (s, 2H, CH2) 6.20 (d, 1H, J = 3.05 Hz, 3-H), 6.60 (dd, 1H, J =
8.77 Hz and J = 2.10 Hz, 6-H), 6.97 (d, 1H, J = 2.10 Hz, 4-H), 7.30
(m, 7H, 2-H, 7-H, 2′-H, 3′-H, 4′-H, 5′-H, and 6′-H), 10.25 ppm (s,
1H, NH); 13C NMR (75 MHz, DMSO-d6)
δ = 14.33
(CHCOOCH2CH3), 15.64 (NCH2COOCH2CH3), 46.55
(NCH2COOCH2CH3), 60.24 (CHCOOCH2CH3), 61.15
(NCH2COOCH2CH3), 88.95(CHCOOCH2CH3), 102.36 (3-C),
110.82 (7-C), 114.46 (4-C), 118.25 (6-C), 127.16 (3a-C),
128.77(2′-C and 6′-C), 129.41 (5′-C and 3′-C), 129.82 (4′-C),
130.11 (2-C), 132.09 (7a-C), 133.24 (1′-C), 137.77 (5-C), 160.14
(NHCCH), 168.88 (CHCOOCH2 CH3 ), 169.76 ppm
(NCH2COOCH2CH3);HRMS (ESI-MS, 140 eV): m/z [M + H+]
+
calculated for C21H23N2O3 , 351.4184; found, 351.4194.
(E,Z)-Ethyl 3-(1-(Cyclopropylmethyl)-1H-indol-5-ylamino)-3-
(3,4-dimethoxyphenyl)acrylate (4b). Compound 4b was prepared
as for compound 4a by reacting 0.500 g (2.68 mmol) of 5-aminoindole
3b, obtaining after column chromatography 0.387 g of a semisolid
+
calculated for C23H25N2O4 , 393.4551; found, 393.4577.
(E,Z)-Ethyl 3-(1-Propionyl-1H-indol-5-ylamino)-3-phenyla-
crylate (4e). Compound 4e was prepared as for compound 4a by
reacting 0.240 g (1.28 mmol) of 5-aminoindole 3e and 0.332 mL of
ethyl benzoyl acetate (1.92 mmol, d = 1.11 g/mL). An amount of
0.525 g of a crude product was obtained, and this was purified by silica
gel column chromatography (d = 2 cm, l = 15 cm, 230−400 mesh,
eluent n-hexane/ethyl acetate/toluene, 1:1:1), giving 0.250 g of a
semisolid yellow product. Yield 53%; Rf = 0.72 (ethyl acetate/n-
1
product. Yield 87%; Rf = 0.76 and Rf = 0.70 (ethyl acetate); H NMR
(300 MHz, DMSO-d6) including NMR signals of both isomers δ =
0.32 (dd, 2H, J = 4.95 Hz and J = 1.14 Hz, CH2-CH2), 0.46 (dd, 2H, J
= 8.20 Hz and J = 1.90 Hz, CH2-CH2), 1.17 (t, 3H, J = 7.05 Hz,
CH2CH3), 1.23 (t, 3H, J = 7.05 Hz, CH2CH3), 1.29 (m, 1H, CH), 3.54
and 3.68 (s, 6H, -OCH3), 3.81 and 3.85 (s, 6H, -OCH3), 4.09 (m, 2H,
J = 6.29 Hz, CH2CH3), 4.13 (m, 2H, J = 7.05 Hz, CH2CH3), 4.88 (s,
1H, CH), 6.10 and 6.25 (dd, 2H, J = 2.86 Hz and J = 0.76 Hz, J = 3.05
Hz, J = 0.57 Hz, 3-H), 6.51 (dd, 2H, J = 9.15 Hz and J = 2.09 Hz, 6-
H), 6.64 (dd, 2H, J = 8.39 and J = 1.90 Hz, 6′-H), 6.82 (d, 1H, J = 8.96
Hz, 5′-H), 7.02 (d, 1H, J = 0.90 Hz, 2′-H), 7.12 (d, 1H, J = 8.58 Hz, 7-
H), 7.18 (dd, 2H, J = 6.29 Hz and J = 3.05 Hz, 4-H), 7.31 and 7.36 (d,
2H, J = 8.77 Hz and J = 3.05 Hz, 5-H), 7.44 (d, 1H, J = 2.09 Hz, 2-H),
7.61 (dd, 1H, J = 8.58 Hz and J = 2.28 Hz, 6-H), 10.19 ppm (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6) δ = 8.24 (CH2CH2), 13.07
(CH), 14.34 (CHCOOCH2CH3), 60.24 (CHCOOCH2CH3), 62.04
(OCH3), 62.61 (OCH3), 88.78 (CHCOOCH2CH3), 101.91 (3-C),
111.77 (7-C), 114.97 (4-C), 115.85 (2′-C), 117.24 (6-C), 119.82 (5′-
C), 122.45 (6′-C), 126.92 (3a-C), 130.44 (2-C), 132.03 (7a-C),
135.62 (1′-C), 137.23 (5-C), 160.44 (NHCCH), 163.03 (3′-C),
164.24 (4′-C), 169.84 ppm (CHCOOCH2CH3). HRMS (ESI-MS,
1
hexane/toluene, 1:1:1); H NMR (400 MHz, DMSO-d6) δ = 1.16 (t,
3H, J = 7.33 Hz, C(O)CH2CH3), 1.24 (t, 3H, J = 6.92 Hz,
COOCH2CH3), 2.96 (q, 2H, J = 7.33 Hz, C(O)CH2CH3), 4.14 (q,
2H, J = 6.92 Hz, COOCH2CH3), 4.85 (s, 1H, CH), 6.28 (dd, 1H, J =
3.19 Hz and J = 0.69 Hz, 3-H), 6.66 (dd, 1H, J = 8.70 Hz and J = 2.08
Hz, 6-H), 6.97 (m, 1H, J = 2.08 Hz, 4-H), 7.28 (d, 1H, J = 3.19 Hz, 2-
H), 7.29 (m, 1H, 4′-H), 7.34 (m, 2H, 3′-H and 5′-H), 7.35 (dd, 1H, J
= 8.70 Hz and J = 0.69 Hz, 7-H), 7.37 (m, 2H, 2′-H and 6′-H), 10.26
ppm (bs, 1H, NH); 13C NMR (101 MHz, DMSO-d6) δ = 9.36
(C(O)CH2CH3), 14.94 (COOCH2CH3), 28.45 (C(O)CH2CH3),
59.14 (COOCH2CH3), 89.11 (CH), 101.68 (3-C), 110.44 (7-C),
115.25 (4-C), 118.76 (6-C), 128.59 (3′-C and 5′-C), 128.64 (3a-C),
128.89 (2′-C and 6′-C), 129.84 (4′-C), 129.87 (2-C), 132.93 (7a-C),
133.06 (1′-C), 136.31 (5-C), 160.51 (C-CH), 169.79
(COOCH2CH3), 174.80 ppm (COCH2CH3). HRMS (ESI-MS, 140
+
+
140 eV): m/z [M + H+] calculated for C25H29N2O4 , 421.5082; found,
eV): m/z [M + H+] calculated for C22H23N2O3 , 363.4291; found,
421.5095.
363.4273.
(E,Z)-Ethyl 3-(1-(2-Cyanoethyl)-1H-indol-5-ylamino)-3-phe-
nylacrylate (4c). Compound 4c was prepared as for compound 4a
(E,Z)-Ethyl 3-(1-Cyclopropylmethanone-1H-indol-5-ylami-
no)-3-phenylacrylate (4f). Compound 4f was prepared as for
M
DOI: 10.1021/acs.jmedchem.5b00805
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
compound 4a by reacting 0.750 g (3.74 mmol) of 5-aminoindole
derivative 3f with ethyl benzoylacetate (1.72 mL, 5.61 mmol). An
amount of 1.150 g of crude product was obtained, and this was
chromatographed on a silica gel column (d = 2.5 cm, l = 30 cm, 230−
400 mesh, eluent ethyl acetate/n-hexane, 4:6) yielding 0.694 g of a
1610, 1470, 1055 cm−1. UV−vis (MeOH): λmax (ε) = 206 (1.435), 270
(1.341), 345 nm (0.642); λmin (ε) = 195 (0.453), 247 (0.600), 314 nm
(0.344). HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated for
C19H17N2O2 , 305.1290; found, 305.1298. RP-C18-HPLC: 97.24%, tR
= 10.02 min.
+
1
yellow oil. Yield 49%; Rf = 0.76 (ethyl acetate/n-hexane, 4:6); H
3-(Cyclopropylmethyl)-7-(3,4-dimethoxyphenyl)-3H-
pyrrolo[3,2-f ]quinolin-9(6H)-one (5b). Compound 5b was pre-
pared as for compound 5a by reacting 1.5 g (3.56 mmol) of
dimethoxyphenylacrylate derivative 4b. An amount of 0.801 g of a
rough dark green solid was obtained, and this was chromatographed
on a silica gel column (d = 2.5 cm, l = 30 cm, 230−400 mesh, eluent
ethyl acetate) yielding 0.450 g of a green solid. Yield 35%; mp = 291
NMR (300 MHz, DMSO-d6) δ = 1.05 (m, 4H, CH2CH2), 1.17 (t, 3H,
J = 7.05 Hz, CH2CH3), 2.64 (m, 1H, CH), 4.13 (q, 2H, J = 7.05 Hz,
CH2CH3), 4.92 (s, 1H, CH), 6.58 (d, 1H, J = 3.62 Hz, 3-H), 6.73 (dd,
1H, J = 8.84 Hz and J = 2.01 Hz, 6-H), 6.99 (d, 1H, J = 2.01 Hz, 4-H),
7.32 (m, 3H, 3′-H, 5′-H and 4′-H), 7.94 (m, 2H, J = 7.43 Hz, 2′-H and
6′-H), 8.01 (d, 1H, J = 8.77 Hz, 7-H), 8.11 (d, 1H, J = 3.81 Hz, 2-H),
10.24 ppm (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ = 14.75
(CHCOOCH2CH3), 15.36 (CH2CH2), 28.44 (CH), 61.04
(CHCOOCH2CH3), 89.27 (CHCOOCH2CH3), 102.94 (3-C),
110.35 (7-C), 114.68 (4-C), 118.24 (6-C), 127.36 (3a-C), 128.83
(2′-C and 6′-C), 129.26 (5′-C and 3′-C), 129.65 (4′-C), 130.25 (2-C),
132.25 (7a-C), 133.78 (1′-C), 137.89 (5-C), 160.44 (NHCCH),
169.44 (CHCOOCH2CH3), 171.91 ppm (NC(O)CHCH2CH2).
HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated for
1
°C; Rf = 0.83 (blue fluorescent spot, ethyl acetate); H NMR (300
MHz, DMSO-d6) δ = 0.41 (m, 2H, CH2-CH2), 0.53 (m, 2H, CH2-
CH2), 1.26 (m, 1H, CH), 3.85 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3),
4.15 (d, 2H, CH2), 6.37 (bs, 1H, 8-H), 7.14 (d, 1H, J = 8.21 Hz, 5-H),
7.56 (m, 2H, 1-H and 5′-H), 7.86 (m, 3H, 2-H, 2′-H and 6′-H), 7.91
(d, 1H, J = 8.92 Hz, 4-H), 11.50 ppm (bs, 1H, NH); 13C NMR (75
MHz, DMSO-d6) δ = 4.87 (CH2CH2), 5.28 (CH2CH2), 8.83
(CHCH2CH2), 56.16 (-OCH3), 61.52 (-OCH3), 63.51 (−CH2-)
102.74 (1-C), 106.44 (8-C), 108.21 (5-C), 112.11 (4-C), 116.81 (9a-
C), 124.14 (9b-C), 124.86 (4′-C), 125.43 (3′-C), 125.98 (2′-C),
126.14 (2-C), 130.42 (1′-C), 134.21 (3a-C), 135.15 (5a-C), 147.13
(6′-C), 148.56 (7-C), 153.31 (5′-C), 175.23 ppm (9-C). IR (ATR
+
C23H23N2O3 , 375.4398; found, 375.4356.
(E,Z)-Ethyl 3-(1-Cyclopropylmethanone-1H-indolin-5-ylami-
no)-3-phenylacrylate (7f). Compound 7 was prepared as for
compound 4a by reacting 0.653 g (3.22 mmol) of 5-aminoindole
derivative 2f and 0.836 mL diethyl benzoylacetate (4.83 mmol). An
amount of 0.956 g of crude product was obtained, and this was purified
by silica gel column chromatography (d = 2.5 cm, l = 35 cm, 230−400
mesh, eluent ethyl acetate/n-hexane, 3:7), giving 0.459 g of a yellow
solid. Yield 37%. Rf = 0.79 (ethyl acetate/n-hexane, 8:2); mp = 133 °C;
1H NMR (300 MHz, DMSO-d6) δ = 0.80 (m, 4H, CH2CH2), 1.17 (t,
ZnSe): ν
λmax (ε) = 221 (0.717), 293 (0.670), 343 nm (0.320); λmin (ε) = 258
̃
= 3180, 2950, 2880, 1615, 1490 cm−1. UV−vis (MeOH):
(0.226), 318 nm (0.172); fluorescence (MeOH), λexc = 300 nm, λems
=
451 nm. HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated for
+
C23H23N2O3 , 375.1709; found, 375.1715. RP-C18-HPLC: 99.82%, tR
= 12.53 min.
3H, J = 6.95 Hz, CH2CH3), 2.64 (m, 1H, CH-COOCH2CH3), 3.18 (t,
2H, J = 7.84 Hz, 3-H2), 4.13 (q, 2H, J = 6.95 Hz, CH2CH3), 4.29 (t,
2H, J = 7.84 Hz, 2-H2), 6.73 (dd, 2H, J = 8.84 Hz and J = 2.01 Hz, 6-
H), 6.99 (d, 1H, J = 2.01 Hz, 4-H), 7.32 (m, 3H, 3′-H, 5′-H and 4′-H),
7.94 (m, 2H, 2′-H and 6′-H), 8.01 (d, 1H, J = 8.84 Hz, 7-H), 10.25
ppm (bs, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ = 8.44
(CH2CH2), 13.96 (CH), 14.26 (CHCOOCH2CH3), 28.02 (3-C),
48.47 (2-C), 60.84 (CHCOOCH2CH3), 89.72 (CHCOOCH2CH3),
110.26 (7-C), 114.75 (4-C), 118.23 (6-C), 127.75 (3a-C), 128.34 (2′-
C and 6′-C), 129.34 (5′-C and 3′-C), 129.75 (4′-C), 132.11 (7a-C),
133.87 (1′-C), 137.23 (5-C), 160.86 (NHCCH), 170.07
(CHCOOCH2CH3), 172.51 ppm (NC(O)CHCH2CH2). HRMS
3-(9-Oxo-7-phenyl-6H-pyrrolo[3,2-f ]quinolin-3(9H)-yl)-
propanenitrile (5c). Compound 5c was prepared as for compound
5a by reacting 2.54 g (7.06 mmol) of phenylacrylate derivative 4c. An
amount of 1.680 g of a yellow solid product was obtained. Yield 76%;
Rf = 0.72 (blue fluorescent spot, ethyl acetate/methanol, 8:2); 1H
NMR (400 MHz, DMSO-d6) δ = 3.10 (t, 2H, J = 6.51 Hz,
NCH2CH2CN), 4.63 (t, 2H, J = 6.51 Hz, NCH2CH2CN), 6.40 (s, 1H,
8-H), 7.50 (m, 1H, 4′-H), 7.56 (m, 1H, 2-H), 7.57 (m, 2H, 3′-H and
5′-H), 7.60 (m, 1H, 5-H), 7.61 (m, 1H, 1-H), 7.86 (m, 2H, 2′-H and
6′-H), 8.00 (d, 1H, J = 8.64 Hz, 4-H), 11.69 ppm (bs, 1H, NH); 13C
NMR (101 MHz, DMSO-d6) δ = 19.55 (NCH2CH2CN), 41.85
(NCH2CH2CN), 104.81 (1-C), 108.72 (8-C), 113.06 (5-C), 116.04
(4-C), 118.26 (9a-C), 119.27 (CN), 123.90 (9b-C), 127.83 (2′-C and
6′-C), 129.16 (4′-C), 129.44 (3′-C and 5′-C), 130.51 (2-C), 131.63
(5a-C), 135.00 (7-C), 137.04 (3a-C), 137.04 (1′-C), 178.38 ppm (9-
C). IR (KBr): υ = 3424, 2247, 1506 cm−1. UV−vis (MeOH): λmax (ε)
= 204 (0.608), 269 (0.378), 336 nm (0.179); λmin (ε) = 245 (0.183),
311 nm (0.101). HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated
+
+
(ESI-MS, 140 eV): m/z [M + H] calculated for C23H25N2O3 ,
377.4557; found, 377.4547.
General Procedure for the Synthesis of Phenylpyrrolo-
quinolinones 5a−f and 8f. As a typical procedure, the synthesis of
the phenylpyrroloquinolinone derivative 5a is described in detail. In a
two-necked round-bottomed flask, 50 mL of diphenyl ether was heated
to boiling. An amount of 1.47 g (4.2 mmol) of acrylate derivative 4a
was then added portionwise, and the resulting mixture was refluxed for
15 min. After cooling to room temperature, an amount of 25 mL of
diethyl ether was added, and the mixture was left for 12 h. Then the
separated precipitate was collected by filtration and washed many
times with diethyl ether. The product (0.945 g) was purified by silica
gel column chromatography (d = 2.5, l = 30, 230−400 mesh, eluent
ethyl acetate/methanol, 9:1), obtaining 0.511 g of a slightly brown
product.
3-(2-Hydroxyethyl)-7-phenyl-3H-pyrrolo[3,2-f ]quinolin-
9(6H)-one (5a). Yield 40%; Rf = 0.32 (blue fluorescent spot,
dichloromethane/methanol, 9:1); mp = 161 °C; 1H NMR (300 MHz,
DMSO-d6) δ = 3.76 (m, 2H, CH2CH2OH), 4.34 (t, 2H, J = 5.62 Hz,
CH2CH2OH), 4.94 (bs, 1H, OH), 6.47 (s, 1H, 8-H), 7.49 (d, 1H, J =
2.94 Hz, 2-H), 7.53 (m, 1H, 1-H), 7.58 (m, 4H, 5-H, 3′-H, 4′-H and
5′-H), 7.78 (m, 2H, 2′-H and 6′-H), 7.90 (dd, 1H, J = 9.02 Hz and J =
0.46 Hz, 4-H), 11.74 ppm (bs, 1H, NH); 13C NMR (75 MHz, DMSO-
d6) δ = 49.00 (CH2CH2OH), 61.22 (CH2CH2OH), 103.98 (1-C),
108.22 (8-C), 112.83 (5-C), 116.38 (4-C), 117.84 (9a-C), 123.38 (9b-
C), 127.80 (6′-C and 2′-C), 129.42 (3′-C and 5′-C), 129.72 (2-C),
130.42 (4′-C), 132.16 (1′-C), 132.18 (3a-C), 137.11 (5a-C), 149.63
(7-C), 178.86 ppm (9-C). IR (ATR ZnSe): ν = 3150, 2900, 2850,
for C20H16N3O+, 314.1293; found, 314.1303. RP-C18 HPLC: tR
=
11.09 min, 98.18%.
Ethyl 2-(9-Oxo-7-phenyl-6H-pyrrolo[3,2-f ]quinolin-3(9H)-
yl)acetate (5d). Compound 5d was prepared as for compound 5a
by reacting 1.024 g (3.15 mmol) of phenylacrylate derivative 4d. An
amount of 0.634 g of a beige solid was obtained, and this was purified
by silica gel column chromatography (d = 2.5, l = 35, 230−400 mesh,
eluent ethyl acetate) to yield 0.380 g of a golden beige solid. Yield
30%; mp = 107 °C; Rf = 0.27 (fluorescent blue spot, ethyl acetate); 1H
NMR (300 MHz, DMSO-d6) δ = 1.63 (t, 3H, J = 7.02 Hz, CH2CH3),
4.20 (q, 2H, J = 7.02 Hz, CH2CH3), 4.95 (s, 2H, CH2), 6.79 (d, 1H, J
= 1.38 Hz, 8-H), 7.87 (d, 1H, J = 3.01 Hz, 1-H), 7.99 (m, 5H, 4′-H, 5′-
H, 3′-H, 4-H and 2-H), 8.21 (d, 1H, J = 9.06 Hz, 5-H), 8.26 (m, 2H,
2′-H and 6′-H), 11.66 ppm (bs, 1H, NH); 13C NMR (75 MHz,
DMSO-d6) δ = 14.15 (CH2CH3), 61.85 (CH2CH3), 68.34 (−CH2-),
102.71 (1-C), 107.62 (8-C), 111.06 (5-C), 114.04 (4-C), 117.26 (9a-
C), 124.17 (9b-C), 128.33 (2′-C and 6′-C), 130.15 (4′-C), 130.84 (3′-
C and 5′-C), 134.56 (2-C), 135.67 (5a-C), 138.00 (7-C), 138.54 (3a-
C), 139.94 (1′-C), 178.68 ppm (9-C). IR (KBr): ν = 3500, 1650, 1500
cm−1. UV−vis (MeOH): λmax (ε) = 203 (0.700), 265 (0.840), 339 nm
(0.360), λmin (ε) = 246 (0.650), 314 nm (0.250). HRMS (ESI-MS, 140
N
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Journal of Medicinal Chemistry
Article
+
eV): m/z [M + H+] calculated for C21H19N2O3 , 347.1396; found,
General Procedure for Synthesis of 9-Alkoxyphenylpyrrolo-
quinolinones 9−11. As a typical procedure, the synthesis of 9-
methoxyl derivative 9 is described in detail. Into a two-necked 50 mL
flask 0.0625 g (2.60 mmol) of NaH 60% was placed and washed three
times with toluene and finally suspended in 15 mL of anhydrous THF
under a nitrogen atmosphere. Then, to the cooled suspension, 0.150 g
(0.520 mmol) of starting material 19 (MG 2474)7 was added. The
reaction mixture changed from yellow to slightly brown while
developing white fumes (hydrogen) and was stirred for 45 min.
Next, 0.01 mL (1.56 mmol, d = 2.28 g/mL) of CH3I in 1 mL of
anhydrous THF was introduced, and the resulting mixture was stirred
at room temperature. The progress of the reaction was monitored by
TLC analysis (ethyl acetate/methanol, 9:1). After 12 h, the reaction
mixture was treated with 20 mL of water, neutralized with 3 N HCl
and extracted with ethyl acetate (3 × 50 mL). The combined extracts
were dried with anhydrous Na2SO4, and the solvent was removed on a
rotavapor to yield 0.156 g of a dark dense liquid. The crude reaction
product was purified by silica gel column chromatography (d = 2.5, l =
25 cm, 230−400 mesh, eluent ethyl acetate/methanol, 9:1) to yield
0.100 g of a white solid.
347.1372. RP-C18-HPLC: 98.84%, tR = 10.07 min.
7-Phenyl-3-propionyl-3H-pyrrolo[3,2-f ]quinolin-9(6H)-one
(5e). Compound 5e was prepared as for compound 5a by reacting
0.250 g (0.689 mmol) of phenylacrylate derivative 4e. An amount of
0.130 g of a solid product was obtained, and this was recrystallized
from methanol giving 0.065 g of a slightly brown solid. Yield 30%; mp
= 289 °C (with decomposition); Rf = 0.73 (blue fluorescent spot, ethyl
1
acetate/methanol, 8:2); H NMR (400 MHz, DMSO-d6) δ = 1.21 (t,
3H, J = 7.23 Hz, C(O)CH2CH3), 3.15 (q, 2H, J = 7.23 Hz, CH2), 6.42
(s, 1H, 8-H), 7.59 (m, 3H, 3′-H, 4′-H and 5′-H), 7.75 (d, 1H, J = 9.06
Hz, 5-H), 7.84 (m, 1H, 1-H), 7.86 (m, 2H, 2′-Hand6′-H), 8.03 (d, 1H,
J = 3.61 Hz, 2-H), 8.69 (d, 1H, J = 9.06 Hz, 4-H), 11.84 ppm (bs, 1H,
NH); 13C NMR (101 MHz, DMSO-d6) δ = 9.03 (CH2CH3), 28.87
(CH2CH3), 109.01 (8-C), 109.93 (1-C), 116.03 (5-C), 117.78 (9a-C),
120.77 (4-C), 126.75 (9b-C), 127.43 (2-C), 127.91 (2′-C and 6′-C),
129.45 (3′-C and 5′-C), 130.73 (4′-C), 131.03 (3a-C), 134.77 (1′-C),
138.60 (5a-C), 149.10 (7-C), 173.03 (C(O)CH2CH3), 178.51 ppm
(9-C). IR (ATR ZnSe): ν = 2937, 1701, 1627, 1461 cm−1. UV−vis
(MeOH): λmax (ε) = 203 (0.374), 272 (0.445), 326 nm (0.165); λmin
(ε)= 243 (0.183), 306 nm (0.112); fluorescence (MeOH), λexc = 272
nm, λems = 413 nm. HRMS (ESI-MS, 140 eV): m/z [M + H+]
3-Ethyl-9-methoxy-7-phenyl-3H-pyrrolo[3,2-f ]quinoline (9).
Yield 64%; mp = 248 °C; Rf = 0.88 (green fluorescent spot, ethyl
1
+
acetate/methanol, 9:1); H NMR (400 MHz, DMSO-d6) δ = 1.42 (t,
calculated for C20H17N2O2 , 317.1290; found, 317.1301. RP-C18-
3H, J = 7.21 Hz, CH2CH3), 4.23 (s, 3H, OCH3), 4.37 (q, 2H, J = 7.21
Hz, CH2CH3), 7.20 (dd, 1H, J = 3.00 and J = 0.69 Hz), 7.44 (m, 1H,
4′-H), 7.54 (m, 3H, 2-H, 3′-H, 5′-H), 7.60 (s, 1H, 8-H), 7.74 (d, 1H, J
= 9.08 Hz, 5-H), 7.96 (dd, 1H, J = 9.08 Hz and J = 0.69 Hz, 4-H), 8.29
ppm (dd, 2H, J = 8.34 Hz and J = 1.31 Hz, 6′-H, 2′-H); 13C NMR (75
MHz, DMSO-d6) δ = 15.48 (CH2CH3), 40.14 (CH2CH3), 55.39
(OCH3), 104.38 (C-1), 113.35 (C-8), 114.75 (C-9a), 118.09 (C-4),
119.08 (C-5), 122.11 (C-9b), 126.28 (C-2), 126.48 (C-2′ and C-6′),
128.28 (C-3′ and C-5′), 130.93 (C-4′), 139.16 (C-3a), 139.45 (C-1′),
145.33 (C-5a), 153.17 (C-7), 162.66 ppm (C-9). UV−vis (MeOH):
λmax= 218 (0.984), 265 (0.525), 351 nm (0.367); λmin= 247 (0.634),
310 nm (0.145). HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated
for C20H19N2O+, 303.1497; found, 303.1491. RP-C18-HPLC: 99.02%,
tR = 15.93 min.
HPLC: 97.23%, tR = 12.97 min.
3-(Cyclopropylmethanone)-7-phenyl-H-pyrrolo[3,2-f ]-
quinolin-9(6H)-one (5f). Compound 5f was prepared as for
compound 5a by reacting 0.650 g (2.13 mmol) of phenylacrylate
derivative 4f. An amount of 0.430 g of a solid product was obtained,
and this was recrystallized from ethyl acetate giving 0.266 g of a gray
solid. Yield 46%; mp = 298 °C; Rf = 0.62 (chloroform/methanol, 9:1);
1H NMR (300 MHz, DMSO-d6) δ = 1.15 (m, 4H, CH2CH2), 2.79 (m,
1H, CH), 6.41 (s, 1H, 8-H), 7.71 (m, 3H, 3′-H, 4′-H and 5′-H), 7.83
(d, 1H, J = 3.46 Hz, 1-H), 7.97 (m, 3H, 5-H, 2′-H, 6′-H), 8.44 (d, 1H,
J = 3.46 Hz, 2-H), 8.65 (d, 1H, J = 8.82 Hz, 4-H), 11.94 ppm (bs, 1H,
NH); 13C NMR (75 MHz, DMSO-d6) δ = 12.45 (CH2CH2), 13.05
(CH2CH2), 107.94 (8-C), 108.75 (1-C), 115.54 (5-C), 116.40 (9a-C),
119.65 (4-C), 125.65 (9b-C), 126.71 (2-C), 127.15 (2′-C and 6′-C),
128.32 (3′-C and 5′-C), 129.50 (4′-C), 130.83 (3a-C), 132.91 (1′-C),
137.56 (5a-C), 148.40 (7-C), 172.44 (C(O)CHCH2CH2), 177.31
ppm (9-C). IR (ATR ZnSe): υ = 3350, 3085, 2940, 1724, 1654, 1455
cm−1. UV−vis (MeOH): λmax (ε) = 203 (0.748), 274 (0.880), 325 nm
(0.340); λmin (ε)= 243 (0.122), 306 nm (0.168); fluorescence
(MeOH), λexc = 300 nm, λems = 412 nm. HRMS (ESI-MS, 140 eV):
3-Ethyl-7-phenyl-9-butoxy-3H-pyrrolo[3,2-f ]quinoline (10).
Compound 10 was prepared as for compound 9 by reacting 0.300 g
(1.04 mmol) of compound 22 dissolved in 5 mL of anhydrous DMF
and 0.17 mL (1.56 mmol; d = 1.27 g/mL) of BrC4H9 in 1 mL of di-
DMF. An amount of 0.348 g of a dark brown solid was obtained, and
this was recrystallized from methanol giving 0.259 g of a slightly brown
solid. Yield 72%; mp = 120 °C; Rf = 0.87 (green fluorescent spot, ethyl
+
m/z [M + H+] calculated for C21H17N2O2 , 329.1290; found,
329.1297. RP-C18 HPLC: tR = 12.93 min, 98.13%.
1
acetate/methanol, 8:2); H NMR (300 MHz, DMSO-d6) δ = 1.03 (t,
3H, J = 7.52 Hz, CH2CH2CH2CH3), 1.41 (t, 3H, J = 7.23 Hz,
CH2CH3), 1.63 (sextet, 2H, J = 7.52 Hz, CH2CH2CH2CH3), 1.98
(quintuplet, 2H, J = 7.52 Hz and J = 6.15 Hz, CH2CH2CH2CH3), 4.34
(q, 2H, J = 7.23 Hz, CH2CH3), 4.44 (t, 2H, J = 6.15 Hz,
CH2CH2CH2CH3), 7.23 (d, 1H, J = 2.81 Hz, 1-H), 7.46 (m, 1H,
4′-H), 7.54 (d, 1H, J = 2.81 Hz, 2-H), 7.54 (m, 3H, 2′-H, 3′-H, 5′-H),
7.60 (s, 1H, 8-H), 7.75 (d, 1H, J = 9.09 Hz, 5-H), 7.94 (d, 1H, J = 9.09
Hz, 4-H), 8.30 ppm (m, 1H, 6′-H); 13C NMR (75 MHz, DMSO-d6) δ
3-(Cyclopropylmethanone)-2,3-dihydro-7-phenyl-1H-
pyrrolo[3,2-f ]quinolin-9(6H)-one (8f). Compound 8f was prepared
as for compound 5a by reacting 0.350 g (0.94 mmol) of phenylacrylate
derivative 7f. An amount of 0.202 g of a yellow solid product was
obtained, and this was purified by silica gel column chromatography (d
= 2.5, l = 30, 230−400 mesh, eluent ethyl acetate/methanol, 8:2)
yielding 0.120 g of a slightly yellow solid. Yield 30%; mp = 317 °C
(with decomposition); Rf = 0.67 (blue fluorescent spot, ethyl acetate/
=
14.22 (CH2CH2 CH2CH3), 16.46 (CH2 CH3 ), 19.54
1
methanol, 8:2); H NMR (400 MHz, DMSO-d6) δ = 0.88 (m, 4H,
(CH2CH2CH2CH3), 31.26 (CH2CH2CH2CH3), 41.10 (CH2CH3),
68.45 (CH2CH2CH2CH3), 99.25 (C-8), 105.16 (C-1), 114.36 (C-9a),
115.64 (C-4), 120.17 (C-9b), 123.17 (C-4 and C-5), 127.29 (C-2′ and
C-6′), 129.01 (C-3′ and C-5′), 129.21 (C-4′), 131.94 (C-3a), 140.19
(C-1′), 146.41 (C-5a), 154.14 (C-7), 162.96 ppm (C-9). UV−vis
(MeOH): λmax (ε) = 210 (1.011), 239 (0.743), 284 nm (1.610); λmin
(ε)= 229 (0.663), 248 nm (0.591). IR (ATR ZnSe): 2953−2869
(CH2, CH3), 1524−1469 (CC), 1230 (C−O) cm−1; fluorescence
(MeOH), λexc = 284 nm, λem = 514.92 nm. HRMS (ESI-MS, 140 eV):
m/z [M + H]+ calculated for C23H25N2O+, 345.1967; found, 345.1997.
RP-C18 HPLC: 99.84%, tR = 17.60 min.
9-(Benzyloxy)-3-ethyl-7-phenyl-3H-pyrrolo[3,2-f ]quinoline
(11). Compound 11 was prepared as for compound 9 by reacting
0.150 g (0.520 mmol) of compound 22 dissolved in 5 mL of
anhydrous DMF and 0.360 g (1.560 mmol) of 4-nitrobenzylbromide
dissolved in 2−3 mL of anhydrous DMF. An amount of 0.164 g of a
CH2CH2), 1.95 (m, 1H, CH), 3.77 (t, 2H, J = 8.34 Hz, 1-H2), 4.36 (t,
2H, J = 8.34 Hz, 2-H2), 6.22 (d, 1H, J = 1.82 Hz, 8-H), 7.58 (m, 4H, 5-
H, 3′-H, 4′-H, and 5′-H), 7.81 (m, 2H, 2′-H and 6′-H), 8.41 (d, 1H, J
= 8.49 Hz, 4-H), 11.75 ppm (bs, 1H, NH); 13C NMR (101 MHz,
DMSO-d6) δ = 8.25 (CH2CH2), 13.68 (CH), 29.69 (1-C), 49.06 (2-
C), 107.58 (8-C), 118.08 (5-C), 120.78 (4-C), 122.38 (9a-C), 127.68
(2′-C and 6′-C), 129.43 (3′-C and 5′-C), 130.27 (9b-C), 130.83 (4-
C), 134.57 (1′-C), 138.00 (3a-C), 139.96 (5a-C), 149.63 (7-C),
171.43 (C(O)CHCH2CH2), 178.86 (9-C). IR (ATR ZnSe): υ = 2960,
2920, 1630, 1590, 1440 cm−1. UV−vis (MeOH): λmax (ε) = 204
(0.992), 279 (1.557), 352 nm (0.281); λmin (ε)= 195 (0.322), 237
(0.443), 336 nm (0.225); fluorescence (MeOH), λexc = 277 nm, λems
=
460 nm. HRMS (ESI-MS, 140 eV): m/z [M + H+] calculated for
+
C21H19N2O2 , 331.1441; found, 331.1423. RP-C18 HPLC: tR = 11.56
min, 96.33%.
O
DOI: 10.1021/acs.jmedchem.5b00805
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dark brown solid product was obtained, and this was purified by silica
gel column chromatography (d = 2.5, l = 25 cm, 230−400 mesh,
eluent ethyl acetate/methanol, 9:1) giving 0.132 g of a brown solid.
Yield 74%; mp = 131−134 °C; Rf = 0.76 (green fluorescent spot, ethyl
acetate/methanol, 8:2); 1H NMR (300 MHz, CDCl3) δ = 1.50 (t, 3H,
J = 7.25 Hz, CH2CH3), 4.25 (q, 2H, J = 7.31 Hz, CH2CH3), 5.96 (s,
2H, -CH2-), 7.15 (d, 1H, J = 3.01 Hz, 1-H), 7.23 (d, 1H, J = 3.02 Hz,
2-H), 7.51 (m, 3H, 3′-H, 4′-H, and 5′-H), 7.61 (d, 2H, J = 8.55 Hz, 2″-
H and 6″-H), 7.84 (d, 1H, J = 9.09 Hz, 5-H), 7.93 (s, 1H, 8-H), 8.15
(d, 1H, J = 9.03 Hz, 4-H), 8.27 (dd, 2H, J = 7.17 Hz and J = 3.23 Hz,
2′-H and 6′-H), 8.35 ppm (m, 3H, J = 8.77 Hz, 3″-H, 4″-H, and 5″-
H); 13C NMR (75 MHz, CDCl3) δ = 15.31 (CH2CH3), 41.04
(CH2CH3), 45.96 (−CH2-), 104.05 (C-1), 110.02 (C-8), 115.18 (C-
4), 115.52 (C-5), 118.45 (C-9a), 126.10 (C-9b), 127.96 (C-2), 128.39
(C-2″ and C-6″), 128.43 (C-2′ and C-6′), 128.53 (C-3″ and C-5″),
128.75 (C-3′ and C-5′), 129.23 (C-4″), 129.54 (C-4′), 131.97 (C-3a),
134.28 (C-1″), 134.81 (C-1′), 152.33 (C-5a), 154.26 (C-7), 164.72
(C-9), 170.94 ppm (−COO). UV−vis (MeOH): λmax (ε) = 205
(0.925), 239 (0.524), 281 nm (0.295); λmin (ε) = 231 (0.628), 247 nm
(0.263). HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated for
C26H23N2O+ = 379.1810; found, 379.1805. RP-C18-HPLC: 96.54%, tR
= 16.74 min.
NCH2CH3), 3.67 (s, 3H, N(quinoline)-CH3), 4.37 (q, 2H, J = 7.20
Hz, NCH2CH3), 6.04 (s, 1H, H-8), 7.57 (m, 1H, H-2), 7.57 (m, 5H,
H-2′, H-3′, H-4′, H-5′, H-6′), 7.59 (d, 1H, J = 9.35 Hz, H-5), 7.67 (dd,
1H, J = 2.92 Hz and J = 0.72 Hz, H-1), 8.00 ppm (dd, 1H, J = 9.35 Hz
and J = 0.70 Hz, H-4); 13C NMR (101 MHz, DMSO-d6) δ = 16.37
(NCH2CH3), 38.69 (N(quinoline)-CH3), 41.02 (NCH2CH3), 104.38
(C-1), 110.96 (C-5), 112.59 (C-8), 115.94 (C-4), 119.63 (C-9a),
124.30 (C-2), 124.30 (C-9b), 129.15 (C-3′ and C-5′), 129.24 (C-2′
and C-6′), 129.45 (C-4′), 131.65 (C-1′), 136.68 (C-5a), 138.05 (C-
3a), 177.16 ppm (C-9). IR (KBr): 3447.02 (CO), 2924.41 (CH3,
CH2CH3), 1582.55 (CC), 1450.86 (CH3) cm−1. UV−vis (MeOH):
λmax (ε) = 351 (0.415), 264 (0.746), 219 nm (0.99); λmin (ε) = 306.35
(0.138), 246 nm (0.467). HRMS (ESI-MS, 140 eV): m/z [M + H+]
calculated for C20H19N2O+, 303.1497; found, 303.1500. RP-C18-
HPLC: 98.77%, tR = 11.49 min.
3-(3-Hydroxypropyl)-7-phenyl-3H-pyrrolo[3,2-f ]quinolin-
9(6H)-one (14). Into a 100 mL dry two-necked flask, 0.084 g (2.22
mmol) of LiAlH4 and 15 mL of anhydrous THF were placed. After
stirring the suspension for 2−3 min under a nitrogen atmosphere,
0.200 g (0.562 mmol) of pyrroloquinolinone derivative 21 dissolved in
15 mL of anhydrous THF was added and the mixture stirred for 2 h at
room temperature under a nitrogen atmosphere. The reaction was
monitored by TLC (ethyl acetate/n-hexane, 8:2). At the end of the
reaction, excess LiAlH4 was deactivated by adding 10 mL of saturated
NH4Cl in water. After filtering the suspension, the filtrate was
concentrated to dryness on a rotavapor, yielding a dark brown residue.
The latter was treated with water and extracted with ethyl acetate (3 ×
50 mL). The combined extracts were washed several times with water
and dried with sodium sulfate. The solvent was removed in a
rotavapor, giving a dark yellow solid (0.156 g). Finally, the reaction
product was purified with a silica gel chromatography column (d = 3
cm, l = 30 cm, 230−400 mesh, eluent ethyl acetate/n-hexane, 8:2) to
give 0.123 g of a pale-yellow solid. Yield 70%; Rf = 0.45 (blue
Synthesis of Benzyl 3-Ethyl-7-phenyl-3H-pyrrolo[3,2-f ]-
quinolin-9-ylcarbonate (12). Into a two-necked 50 mL flask
0.0625 g (2.60 mmol) of NaH 60% was placed and washed at least
three times with toluene and finally suspended in 15 mL of anhydrous
THF under a nitrogen atmosphere. Then, to the ice-bath cooled
suspension, 0.150 g (0.520 mmol) of the starting material 197 was
added. The reaction mixture changed from yellow to slightly brown
while developing white fumes (hydrogen) and was stirred for 45 min.
Next, 0.15 mL (1.04 mmol, d = 1.195 g/(mL) of benzylchloroformate
in 2 mL of anhydrous THF was introduced, and the mixture was
stirred at 0 °C. The progress of the reaction was monitored by TLC
analysis (ethyl acetate/methanol, 9:1). After 3 h, the reaction mixture
was treated with 15 mL of water, neutralized with 3 N HCl, and
extracted with ethyl acetate (3 × 50 mL). The combined extracts were
dried with anhydrous Na2SO4, and the solvent was removed on a
rotavapor to yield a dark yellow semisolid residue that was
recrystallized from methanol giving 0.212 g of a yellow solid. Yield
96%; mp = 215−218 °C; Rf = 0.89 (blue fluorescent spot, ethyl
acetate/methanol, 9:1); 1H NMR (300 MHz, CDCl3) δ = 1.53 (t, 3H,
J = 7.30 Hz, CH2CH3), 4.31 (q, 2H, J = 7.31 Hz, CH2CH3), 5.40 (s,
2H, CH2-Benz), 7.13 (d, 1H, J = 3.05 Hz, 1-H), 7.23 (d, 1H, J = 3.06
Hz, 2-H), 7.40 (m, 3H, 3″-H, 4″-H, and 5″-H), 7.46 (d, 2H, J = 8.57
Hz, 6″-H and 2″-H), 7.51 (m, 3H, 3′-H, 4′-H, and 5′-H), 7.80 (d, 1H,
J = 8.02 Hz, 5-H), 7.94 (s, 1H, 8-H), 8.05 (d, 1H, J = 8.05 Hz, 4-H),
8.18 ppm (m, 2H, J = 7.19 Hz and J = 3.21 Hz, 6′-H and 2′-H); 13C
NMR (75 MHz, CDCl3) δ = 15.62 (CH2CH3), 41.08 (CH2CH3),
69.37 (CH2-Benz), 104.05 (C-1), 110.02 (C-8), 115.18 (C-4), 115.52
(C-5), 118.45 (C-9a), 126.10 (C-9b), 127.09 (C-2), 127.96 (C-2″ and
C-6″), 128.18 (C-2′ and C-6′), 128.22 (C-3″ and C-5″), 128.27 (C-3′
and C-5′), 129.39 (C-4′), 129.43 (C-4″), 131.97 (C-3a), 134.28 (C-
1″), 134.81 (C-1′), 154.26 (C-5a), 154.37 (C-7), 164.72 (C-9), 178.24
ppm (-OCOO-). UV−vis (MeOH): λmax (ε) = 207 (0.458), 284
(0.273), 339 nm (0.101); λmin (ε) = 250 (0.317), 316 nm (0.082).
1
fluorescent spot, ethyl acetate/n-hexane, 8:2); mp = 213-215 °C; H
NMR (300 MHz, DMSO-d6) δ = 1.94 (m, 2H, J = 6.51 Hz,
NCH2CH2CH2OH), 3.38 (t, 2H, J = 6.13 Hz, NCH2CH2CH2OH),
4.38 (t, 2H, J = 6.89 Hz, NCH2CH2CH2OH), 5.47 (bs, 1H, OH), 6.70
(s, 1H, 8-H), 7.53 (m, 2H, J = 2.92 Hz, 1-H and 2-H), 7.61 (m, 3H, 3′-
H, 4′-H and 5′-H), 7.71 (d, 1H, J = 8.98 Hz, 5-H), 7.90 (m, 2H, J =
7.54 Hz and J = 2.31 Hz, 6′-H and 2′-H), 8.01 (d, 1H, J = 9.00 Hz, 4-
H), 12.46 ppm (bs, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ =
33.41 (NCH2CH2CH2OH), 42.68 (NCH2CH2CH2OH), 57.60
(NCH2CH2CH2OH), 103.78 (C-1), 116.56 (C-8), 118.53 (C-4),
122.10 (C-5), 123.36 (C-9a), 127.51 (C-9b), 127.51 (C-2), 128.97 (C-
2′ and C-6′), 129.16 (C-3′ and C-5′), 130.23 (C-4′), 131.49 (C-3a),
134.19 (C-1′), 148.29 (C-5a), 156.56 (C-7), 178.74 ppm (C-9). UV−
vis (MeOH): λmax (ε) = 205 (0.925), 271 (0.714), 346 nm (0.451);
λmin = 248 (0.613), 314 nm (0.514). HRMS (ESI-MS, 140 eV): m/z
+
[M + H]+ calculated for C20H19N2O2 = 319.1447; found, 319.1472.
RP-C18-HPLC: 98.78%, tR = 10.25 min.
3-(9-Oxo-7-phenyl-6H-pyrrolo[3,2-f ]quinolin-3(9H)-yl)-
propanoic Acid (15). Into a 50 mL two-necked flask, a solution of
0.200 g (0562 mmol) of pyrroloquinolinone derivative II (MG 2540)
in 15 mL of methanol was placed, and 3 mL of 20% NaOH in water
was added. The mixture was stirred at 60−70 °C for about 2 h, and the
reaction was monitored by TLC (chloroform/methanol, 9:1). At the
end of the reaction, the solution was cooled to room temperature, and
the solvent was removed on a rotavapor, yielding a dark yellow solid.
The dry residue was treated with water and then extracted with ethyl
acetate (2 × 50 mL) to remove unreacted compounds. The aqueous
phase was then acidified to pH 3−4 with 37% HCl, and immediately a
yellow precipitate formed. The precipitate was collected, washed
several times with water, and dried under vacuum to yield 0.157 g of
an intensely yellow solid. Yield 85%; Rf = 0.20 (blue fluorescent spot,
chloroform/methanol, 9:1); mp = 280 °C; 1H NMR (300 MHz,
DMSO-d6) δ = 2.80 (t, 2H, J = 6.76 Hz, NCH2CH2COOH), 4.52 (t,
2H, J = 6.76 Hz, NCH2CH2COOH), 6.54 (s, 1H, 8-H), 7.51 (dd, 2H,
J = 2.98 Hz, 2-H and 1-H), 7.58 (m, 3H, 3′-H, 4′-H and 5′-H), 7.63
(d, 1H, J = 8.96 Hz, 5-H), 7.86 (dd, 2H, J = 7.71 Hz and J = 2.01 Hz,
6′-H and 2′-H), 7.96 (d, 1H, J = 9.00 Hz, 4-H), 12.46 ppm (bs, 1H,
+
HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated for C27H23N2O3 ,
423.1709; found, 423.1705. RP-C18-HPLC: 98.52%, tR = 17.04 min.
3-Ethyl-6-methyl-7-phenyl-3H-pyrrolo[3,2-f ]quinolin-9(6H)-
one (13). In a 100 mL flask, 0.175 g (0.58 mmol) of 9-methoxy-3-
ethyl-7-phenyl-3H-pyrrolo[3,2-f ]quinoline (9) was dissolved in 3 mL
of CH3I. The solution was maintained at reflux for 12 h, and the
reaction was monitored by TLC (ethyl acetate/methanol, 8:2) until
the starting material 9 disappeared and a new yellow fluorescent spot
appeared. Then, 5 mL of 1.5 M NaOH in water was added to the
reaction mixture and heating at reflux resumed for 2 h. After cooling,
the mixture was extracted with ethyl acetate (4 × 50 mL), and the
combined extracts were dried with sodium sulfate and filtered. The
solvent was evaporated, giving 0.031 g of a pure yellow solid. Yield
18%; Rf = 0.86 (blue fluorescent spot, ethyl acetate/methanol, 8:2); 1H
NMR (400 MHz, DMSO-d6) δ = 1.42 (t, 3H, J = 7.20 Hz,
P
DOI: 10.1021/acs.jmedchem.5b00805
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
NH), (no signal for the carboxylic acid proton); 13C NMR (75 MHz,
calculated for C25H28N3O3 = 418.2131; found, 418.2137. RP-C18-
HPLC: 96.55%, tR = 10.76 min.
D M S O - d 6 )
δ = 3 4 . 6 0 ( N C H 2 C H 2 C O O H ) , 4 1 . 2 0
3-(3-Aminopropyl)-7-phenyl-3H-pyrrolo[3,2-f ]quinolin-9-ol
hydrochloride (18). In a 100 mL flask, 0.644 g (1.54 mmol) of
pyrroloquinolinone derivative 17 was dissolved in 55 mL of an ethyl
acetate/ethanol 2:1 mixture, and dry HCl gas was bubbled into the
solution until a yellow precipitate formed. The mixture was left at 0−4
°C overnight, and the resulting precipitate was collected and dried
under vacuum to yield 0.413 g of a powdery beige solid. Yield: 58%; Rf
= 0.20 (blue fluorescent spot, chloroform/methanol, 8:2); mp = 194
(NCH2CH2COOH), 103.43 (C-1), 112.40 (C-8), 115.44 (C-5),
122.10 (C-4), 123.24 (C-9a), 127.41 (C-9b), 127.47 (C-2), 128.77 (C-
2′ and C-6′), 129.06 (C-3′ and C-5′), 129.97 (C-4′), 130.13 (C-3a),
131.35 (C-1′), 134.22 (C-5a), 147.44 (C-7), 167.35 (COOH), 171.87
ppm (C-9). UV−vis (MeOH): λmax (ε) = 206 (0.981), 271 (0.613),
347 nm (0.451); λmin (ε) = 247 (0.714), 314 nm (0.581). HRMS (ESI-
MS, 140 eV): m/z [M + H]+ calculated for C20H17N2O3+ = 333.1239;
found, 333.1265. RP-C18-HPLC: 97.98%, tR = 9.17 min.
1
°C; H NMR (400 MHz, DMSO-d6) δ = 2.16 (quintuplet, 2H, J =
2-(9-Oxo-7-phenyl-6H-pyrrolo[3,2-f ]quinolin-3(9H)-yl)acetic
Acid (16). Into a 50 mL two-necked flask, 0.150 g (0.43 mmol) of
pyrroloquinolinone derivative 5d in 8 mL of methanol was place, and
3 mL of 20% NaOH in water was added. The mixture was stirred at
60−70 °C for about 2 h, and the reaction was monitored by TLC
(chloroform/methanol, 9:1). At the end of the reaction, the solution
was cooled to room temperature, and the solvent was removed on a
rotavapor, yielding a greyish-white solid (0.120 g). The residue was
treated with water and then extracted with ethyl acetate (2 × 50 mL)
to remove unreacted compounds. The aqueous phase was then
acidified to pH 3−4 with 37% HCl, and immediately a white
precipitate formed. The precipitate was collected, washed several times
with water, and dried under vacuum, giving 0.085 g of a white
crystalline product. Yield 63%; Rf = 0.18 (chloroform/methanol, 7:3);
6 . 8 4 H z , N C H 2 C H 2 C H 2 N H3 + C l − ) , 2 . 7 7 ( m , 2 H ,
NCH2CH2 CH2 NH3+ Cl− ), 4.58 (t, 2H,
J = 6.86 Hz,
+
NCH2CH2CH2NH3 Cl−), 7.42 (dd, 1H, J = 2.99 Hz and J = 0.65
Hz, H-1), 7.67 (s, 1H, H-8), 7.70 (m, 1H, H-4′), 7.71 (m, 2H, H-3′
and H-5′), 7.86 (d, 1H, J = 3.21 Hz, H-2), 8.02 (m, 2H, H-2′ and H-
+
6′), 8.17 (d, 1H, J = 9.19 Hz, H-5), 8.19 (m, 3H, NH3 ), 8.46 (d, 1H, J
= 6.86 Hz, H-4), 14.70 ppm (s, 1H, OH); 13C NMR (101 MHz,
DMSO-d6)
δ =
28.94 (NCH2CH2CH2NH3+Cl−), 36.78
+
+
(NCH2CH2CH2NH3 Cl−), 43.82 (N-CH2CH2CH2NH3 Cl−), 105.19
(C-8), 105.40 (C-1), 113.22 (C-5), 114.10 (C-9a), 120.13 (C-4),
120.68 (C-9b), 129.05 (C-2′ and C-6′), 129.83 (C-3′ and C-5′),
130.95 (C-2), 132.12 (C-4′), 132.58 (C-3a), 132.61 (C-1′), 137.87
(C-5a), 151.15 (C-7), 169.98 ppm (CO). IR (KBr): 3411.56 (OH),
1618 (CC) cm−1. UV−vis (MeOH): λmax (ε) = 338 (0.081), 270
(0.162), 204 nm (0.169); λmin (ε) = 312 nm (0.046), 246 nm (0.079);
fluorescence (H2O), λexc = 215.00 nm, λem = 430.00 nm. HRMS (ESI-
MS, 140 eV): m/z [M + H]+ calculated for C20H20N3O+ = 318.1606;
found, 318.1641, m/(2z) [M + 2H]2+ calculated for C20H21N3O2+
159.5837; found, 159.5801. RP-C18-HPLC: 99.57%, tR = 8.96 min.
Biology. Cell Lines and Growth Inhibition Assays. The human
cell lines were the following: cervix carcinoma (HeLa), adenocarci-
nomic alveolar basal epithelial cells (A549), colon adenocarcinoma
(HT-29), breast adenocarcinoma (MCF-7), ovarian carcinoma
(OVCAR-3), acute T-cell lymphoblastic leukemia (MOLT-3, Jurkat,
and CCRF-CEM), chronic myelogenous leukemia cells (K562), B-cell
leukemia (RS4;11and SEM), acute myelocytic leukemia cells
(MV4;11), and acute monocytic leukemia cells (THP-1). All cell
lines were purchased from the American Type Culture Collection.
Leukemic cell lines were cultured in RPMI-1640 (Gibco, Milano,
Italy), while solid tumor cell lines were cultured in DMEM (Gibco,
Milano, Italy). Both media were supplemented with 10% fetal bovine
serum (FBS), glutamine (2 mM), penicillin (100 U/mL), and
streptomycin (100 μg/mL; all media components were from Life
Technologies, Italy) and maintained at 37 °C in a humidified
atmosphere with 5% CO2. The cytotoxic activity of the compounds
was determined using a standard MTT based colorimetric assay
(Sigma-Aldrich, Milano, Italy). Briefly, cells were seeded at a density of
8 × 103 cells/well in 96-well microtiter plates. After 24 h, cells were
exposed to various concentrations of the test compounds. After
different times, cell survival was determined by the addition of an
MTT solution as described previously.16 The GI50 value is defined as
the compound concentration required to inhibit cell proliferation by
50%.
1
mp = 273 °C; H NMR (300 MHz, DMSO-d6) δ = 5.67 (s, 2H,
CH2COOH), 6.79 (d, 1H, J = 1.38 Hz, 8-H), 7.87 (d, 1H, J = 3.01 Hz,
1-H), 7.99 (m, 5H, 3′-H, 5′-H, 4′-H, 4-H and 2-H), 8.21 (d, 1H, J =
9.06 Hz, 5-H), 8.26 (m, 2H, 6′-H and 2′-H), 11.66 (bs, 1H, NH),
13.10 ppm (bs, 1H, COOH); 13C NMR (75 MHz, DMSO-d6) δ =
55.72 (NCH2COOH), 104.33 (C-1), 113.30 (C-5), 116.34 (C-8),
123.00 (C-4), 124.14 (C-9a), 126.31 (C-9b), 128.31 (C-2), 128.67 (C-
2′ and C-6′), 129.67 (C-3′ and C-5′), 130.87 (C-3a), 131.03 (C-4′),
132.15 (C-1′), 135.12 (C-5a), 148.34 (C-7), 168.32 (COOH), 174.12
ppm (C-9). UV−vis (MeOH): λmax (ε) = 226 (0.815), 268 (0.613),
346 nm (0.481); λmin (ε) = 249 (0.724), 315 nm (0.523). HRMS (ESI-
+
MS, 140 eV): m/z [M + H]+ calculated for C19H15N2O3 319.1083,
found 319.1060. RP-C18-HPLC: 98.12%, tR = 8.74 min.
tert-Butyl 3-(9-Oxo-7-phenyl-6H-pyrrolo[3,2-f ]quinolin-
3(9H)-yl)propylcarbamate (17). In a 100 mL flask, 0.626 g of
pyrroloquinolinone derivative 5c was dissolved in hot MeOH. The
solution was cooled to 0 °C, and 1.310 g (6 mmol) of di-tert-butyl
dicarbonate and 0.110 g (0.4 mmol) of CoCl2·6H2O were added. To
the resulting suspension 0.757 g (20 mmol), NaBH4 was added
portionwise, with development of hydrogen and formation of a black
precipitate. The mixture was stirred for 12 h at room temperature, and
the formation of the reaction product was monitored by TLC (ethyl
acetate/methanol, 8:2). Finally, 0.8 mL (2 mmol, d = 0.955 g/mL) of
diethylenetriamine was added, and the mixture was dried on a
rotavapor. The resulting clear solid was dissolved in 50 mL of ethyl
acetate and the solution washed with a saturated solution of NaHCO3
(3 × 25 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated to dryness, yielding 0.644 g of a light brown solid.
1
Yield 77%; Rf = 0.79 (ethyl acetate/methanol, 8:2); H NMR (400
MHz, DMSO-d6) δ = 1.37 (s, 3H, OC(CH3)3), 1.39 (s, 6H,
PBL from healthy donors were obtained by separation on a
Lymphoprep (Fresenius KABI Norge AS) gradient. After extensive
washing, cells were resuspended (1.0 × 106 cells/mL) in RPMI-1640
with 10% FBS and incubated overnight. For cytotoxicity evaluations in
proliferating PBL cultures, nonadherent cells were resuspended at 5 ×
105 cells/mL in growth medium, containing 2.5 μg/mL PHA (Irvine
Scientific). Different concentrations of the test compounds were
added, and viability was determined 72 h later by the MTT test. For
cytotoxicity evaluations in resting PBL cultures, nonadherent cells
were resuspended (5 × 105 cells/mL) and treated for 72 h with the
test compounds, as described above.
HUVECs were prepared from human umbilical cord veins, as
previously described.29 The adherent cells were maintained in M200
medium supplemented with low serum growth supplement containing
FBS, hydrocortisone, human epithelial growth factor (hEGF), basic
fibroblast growth factor (bFGF), heparin, gentamycin/amphotericin
OC(CH3 )3 ), 1.89 (quintuplet, 2H,
NCH2CH2CH2NHCO), 2.91 (m, 2H, J = 6.66 Hz and J = 5.88 Hz,
NCH2 CH2 CH2 NHCO), 4.29 (t, 2H, 6.93 Hz,
J = 6.942 Hz,
J
=
NCH2CH2CH2NHCO), 6.38 (d, 1H, J = 1.78 Hz, H-8), 6.97 (t,
1H, J = 5.05 Hz, NHCO), 7.50 (d, 1H, J = 2.87 Hz, H-2), 7.55 (d, 1H,
J = 2.77 Hz, H-1), 7.58 (m, 4H, H-3′, H-4′, H-5′, and H-5), 7.85 (m,
2H, H-2′ and H-6′), 7.87 (d, 1H, J = 9.04 Hz, H-4), 11.64 ppm (s, 1H,
NH); 13C NMR (101 MHz, DMSO-d6) δ = 28.68 (OC(CH3)3), 28.72
(OC(CH3 )3 ), 31. 03 (NCH2 CH2 CH2 NHCO), 37. 96
(NCH2CH2CH2NHCO), 43.84 (NCH2CH2CH2NHCO), 104.02
(C-1), 108.63 (C-8), 112.71 (C-5), 118.31 (C-9a), 123.57 (C-9b),
127.81 (C-2′ and C-6′), 129.38 (C-2), 129.43 (C-3′ and C-5′), 130.46
(C-4′), 131.70 (C-3a), 135.04 (C-1′), 136.82 (C-5a), 147.71 (C-7),
156.10 (NHCOO), 178.37 ppm (C-9). IR (KBr): 3461.11 (CO),
1653 (CONH) cm−1. HRMS (ESI-MS, 140 eV): m/z [M + H]+
Q
DOI: 10.1021/acs.jmedchem.5b00805
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Journal of Medicinal Chemistry
Article
(Life Technologies, Monza, Italy). Once confluent, the cells were
detached by trypsin−EDTA solution and used in experiments from the
first to sixth passages.
implemented in the MOE program. To minimize contacts between
hydrogen atoms, the structures were subjected to Amber99 force
field47 minimization until the rms of the conjugate gradient was <0.1
kcal mol−1 Å−1, keeping the heavy atoms fixed at their crystallographic
positions.
Effects on Tubulin Polymerization and on Colchicine
Binding to Tubulin. To evaluate the effect of the compounds on
tubulin assembly in vitro,17 varying concentrations of compounds were
preincubated with 10 μM bovine brain tubulin in glutamate buffer at
30 °C and then cooled to 0 °C. After addition of 0.4 mM GTP, the
mixtures were transferred to 0 °C cuvettes in a recording
spectrophotometer and warmed to 30 °C. Tubulin assembly was
followed turbidimetrically at 350 nm. The IC50 was defined as the
compound concentration that inhibited the extent of assembly by 50%
after a 20 min incubation. The ability of the test compounds to inhibit
colchicine binding to tubulin was measured as described19 except that
the reaction mixtures contained 1 μM tubulin, 5 μM [3H]colchicine,
and 5 μM test compound.
Molecular Docking Protocol. All 3-substituted 7-PPyQs were built
using the “Builder” module of MOE, and each compound was docked
into the presumptive binding sites (colchicine or ATP binding sites)
using flexible MOE-Dock methodology. The purpose of MOE-Dock is
to search for favorable binding configurations between a small, flexible
ligand and a rigid macromolecular target. Searching is conducted
within a user-specified 3D docking box, using the “Tabu search”48
protocol and the MMFF94 force field.49 Charges for ligands were
imported from the MOPAC program50 output files. MOE-Dock
performs a user-specified number of independent docking runs (50 in
the present case) and writes the resulting conformations and their
energies to a molecular database file. The resulting ligand/protein
complexes were subjected to MMFF94 all-atom energy minimization
until the rms of conjugate gradient was <0.1 kcal mol−1 Å−1. GB/SA
approximation51 has been used to model the electrostatic contribution
to the free energy of solvation in a continuum solvent model. The
interaction energy values were calculated as the energy of the complex
minus the energy of the ligand minus the energy of the protein:
Analysis of Cell Cycle Distribution. 5 ×105 cells in exponential
growth were treated with different concentrations of test compound
for different times. After the incubation, cells were collected,
centrifuged, and fixed with ice-cold ethanol (70%) and stained with
PI as described previously.45
Externalization of PS. Surface exposure of PS on apoptotic cells
was measured by flow cytometry with a Coulter Cytomics FC500
(Beckmann Coulter, USA) instrument by adding annexin-V-FITC and
PI to cells according to the manufacturer′s instructions (annexin-V
Fluos, Roche Diagnostic, Monza, Italy).
ΔEinter = Ecomplex − (EL + Eprotein
)
Western Blot Analysis. Cells were treated with test compounds
and, after different times, collected, centrifuged, and washed with ice
cold phosphate buffered saline. The pellet was then resuspended in
lysis buffer as described.25 The protein concentration in the
supernatant was determined using the BCA protein assay (Pierce,
Milano, Italy). Equal amounts of protein (10−20 μg) were resolved
using SDS−PAGE gel electrophoresis (Criterion precast Tris-HCl gel,
Biorad Laboratories, Milano, Italy) and transferred to PVDF Hybond-
p membranes (GE Healthcare, Milano, Italy). Membranes were
blocked with 2% ECL-blocking solution (GE Healthcare, Milano,
Italy) for 2 h, with rotation at room temperature. Membranes were
then incubated with primary antibodies against Bcl-2, p53, PARP,
procaspase-9, procaspase-8, procaspase-2, Mcl-1, Bcl-XL (Cell Signal-
ing, Milano, Italy), β-actin (Sigma-Aldrich, Milano, Italy), and caspase-
3 (Novus Biologicals, Milano, Italy) overnight. Membranes were next
incubated with peroxidase-conjugated secondary antibodies (Invitro-
gen, Milano, Italy) for 60 min. All membranes were visualized using
ECL Select (GE Healthcare, Milano, Italy) and exposed to Hyperfilm
MP (GE Healthcare, Milano, Italy). To ensure equal protein loading,
each membrane was stripped and reprobed with anti-β-actin antibody.
Drug Combination Studies. Cell proliferation was assessed by
the MTT assay after treatment. Equal concentrations of cells were
plated in triplicate in a 96-well plate and treated for 48 h using scalar
dilutions of 5f, combined with cytarabine (Aractyn, Pfizer),
daunorubicin (Pfizer), and dexamethasone (Sigma-Aldrich) at fixed
combination ratios. The effectiveness of various drug combinations
was analyzed by the Calcusyn, version 2.1 software (Biosoft). The
combination index (CI) was calculated according to the Chou−Talalay
method. A combination index of 1 indicates an additive effect of the
two drugs. Combination index values less than 1 indicate synergy, and
combination index values greater than 1 indicate antagonism.
Molecular Docking Simulations. Target Structures. The three-
dimensional structures of tubulin in complex with colchicine as well as
all selected protein kinases were retrieved from the Protein Data Bank
(www.rcsb.org).21 The collected PDB structures are summarized in
Table 2 in Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00805.
1
Complete H and 13C NMR signals assignment and
structure elucidation for compound 10; 1H NMR
spectrum (400 MHz, DMSO-d6) of compound 10;
13C{1H} NMR spectrum (101 MHz, DMSO-d6) of
compound 10; 1H−1H 2D COSY and 1H−13C 2D
HMBC NMR correlation tables (DMSO-d6) of com-
pound 10; HRMS (ESI-MS, 140 eV) spectrum of
compound 10; HPLC trace of compound 10; complete
1H and 13C NMR signals assignment and structure
1
elucidation for compound 18; H NMR spectrum (400
MHz, DMSO-d6) of compound 18; 13C{1H} NMR
spectrum (101 MHz, DMSO-d6) of compound 18;
1H−1H 2D COSY NMR correlation table (DMSO-d6) of
compound 18; HRMS (ESI-MS, 140 eV) spectrum of
compound 18; HPLC trace of compound 18; HRMS
(ESI, 140 eV) spectra and HPLC traces of compounds
5a−f, 8f, 9, 11−17; behavior of compound 18 in
aqueous solution at various pH values; elemental analysis
of all final compounds 5a−f, 8f, and 9−18; combination
cytotoxicity of 5f and daunorubucine (Dauno), dexa-
methasone (Dex), cytarabine (Ara-C); dose−response
curves of Jurkat (A) and THP1 (B) to 5f; Table S2 of the
collected PDB structures and the corresponding
references; Table S3 of simplified molecular-input line-
entry for all final compounds 5a−f, 8f, and 9−18 (PDF)
Molecular formula strings (CSV)
The assessment of crystallographic structure quality has been
performed with the Structure Preparation tool of the Molecular
Operation Environment (MOE, version 2014.09) program.46 Critical
structural issues (such as missing or poorly resolved atomic data,
anomalous topological properties present in amino acids units as well
as anomalous bonding patterns of non amino acids units) were fixed
when necessary. Hydrogen atoms were added and their appropriate
protonation state was fixed using the Protonate3D tool as
Accession Codes
PDB codes are the following: 1SA0, 4EL9, 4JXF, 1RJB, 4E4N,
4AFJ.
R
DOI: 10.1021/acs.jmedchem.5b00805
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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AUTHOR INFORMATION
Corresponding Author
*E-mail: mariagrazia.ferlin@unipd.it. Fax: (+39) 0498275366.
Phone: (+39) 0498271603.
■
(10) Markman, B.; Dienstmann, R.; Tabernero, J. Targeting the
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∥D.A. and R.B. contributed equally to this work.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The synthetic work coordinated by M.G.F. was carried out with
financial support from the University of Padova, Italy, and the
Italian Ministry for University and Research (MIUR), Rome,
Italy. The molecular modeling work coordinated by S.M. was
carried out with financial support from the University of
Padova, Italy, and the Italian Ministry for University and
Research (MIUR), Rome, Italy. S.M. is also very grateful to the
Chemical Computing Group for its scientific and technical
partnership. G.V., R.B. and G.B. thank the Fondazione Cariparo
by the “Progetto Ricerca Pediatrica”. The content is solely the
responsibility of the authors and does not necessarily reflect the
official views of the National Institutes of Health.
ABBREVIATIONS USED
■
PPyQ, phenylpyrroloquinolinone; CA-4, combretastatin A-4;
PyQ, 3H-pyrrolo[3,2-f ]quinolin-9-one; ATR, attenuated total
reflectance; MOE, Molecular Operating Environment; FBS,
fetal bovine serum; PI, propidium iodide; PS, phosphatidylser-
ine; HUVEC, human umbilical vein endothelial cell; PBL,
peripheral blood lymphocyte; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide
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